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Eva PERICOLINI

Professore Associato
sede ex-Scienze di Sanità Pubblica Dipartimento Chirurgico, Medico, Odontoiatrico e di Scienze Morfologiche con interesse Trapiantologico, Oncologico e di Medicina Rigenerativa

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Pubblicazioni

- Zinc treatment prevents neutrophil recruitment and inflammation by blocking Candida albicans Pra1 during vulvovaginal candidiasis. [Poster]
Roselletti, Elena; Pericolini, Eva; De Seta, Francesco; Comar, Manola; Usher and Duncan Wilson, J.
abstract

Vulvovaginal candidiasis (VVC) affects around 75% of all women at least once in their life and 8% develop a recurrent form (rVVC), that substantially reduces the quality of life. In this vaginal disease, Candida albicans triggers a non-protective influx of neutrophils, which are unable to resolve the infection and results in aggressive local inflammation and symptomatic disease. Pharmacological control of VVC episodes and rVVC, although possible with maintenance antifungal therapy, remains problematic and does not eliminate the risk of both future re-infection and development of antifungal resistance. The aim of our study was to investigate the role of the zincophore Pra1 in VVC immunopathology and its modulation with zinc. Pra1 is a secreted C. albicans zinc binding protein released during zinc limitation and used by the fungus to forage for this essential micronutrient from the environment. In vitro tissue culture systems, a mouse model of vaginal candidiasis and vaginal samples from patients with VVC/rVVC were used to evaluate the expression and role of PRA1 in VVC immunopathology. In these systems, the modulation of Pra1 and the associated inflammation were also evaluated by the direct administration of a low amount of zinc. Our results show that C. albicans Pra1 directly induced neutrophil migration and inflammation. Interestingly, Candida glabrata, which has lost the PRA1 gene during its evolution fails to recruit neutrophils and heterologous expression of the PRA1 gene C. glabrata promoted neutrophil chemotaxis. We found that Pra1 is expressed by C. albicans at both neutral and acidic pH during vaginal epithelial infection and that expression was repressed by the addition of zinc. Robust PRA1 expression was also observed in clinical vaginal samples and a strong correlation between PRA1 expression and levels of the neutrophil-activating cytokine IL-8 was found. In an experimental murine model of VVC, PRA1 is expressed and the deletion of C. albicans PRA1 abrogated inflammation, including neutrophil recruitment, IL-1β and MIP-2 production, without affecting fungal burden. Zinc treatment of C. albicans wild type infected mice downregulated PRA1 expression and prevented inflammatory cytokine production and neutrophil infiltration. These data demonstrate that the zincophore Pra1 is expressed during VVC and can act as a potent neutrophil chemoattractant molecule, driving inflammation in a self-amplifying inflammatory cycle. For this reason, Pra1 can be considered a perfect target for blocking the symptoms of VVC and we demonstrate that his can be achieved with low levels of zinc supplementation.

2024 - An Untargeted Metabolomic Analysis of Lacticaseibacillus (L.) rhamnosus, Lactobacillus (L.) acidophilus, Lactiplantibacillus (L.) plantarum and Limosilactobacillus (L.) reuteri Reveals an Upregulated Production of Inosine from L. rhamnosus [Articolo su rivista]
Spaggiari, Luca; Pedretti, Natalia; Ricchi, Francesco; Pinetti, Diego; Campisciano, Giuseppina; De Seta, Francesco; Comar, Manola; Kenno, Samyr; Ardizzoni, Andrea; Pericolini, Eva
abstract

Lactic acid bacteria are considered an inexhaustible source of bioactive compounds; indeed, products from their metabolism are known to have immunomodulatory and anti-inflammatory activity. Recently, we demonstrated that Cell-Free Supernatants (CFS) obtained from Lactobacillus (L.) acidophilus, Lactiplantibacillus (L.) plantarum, Lacticaseibacillus (L.) rhamnosus, and Limosilactobacillus (L.) reuteri can impair Candida pathogenic potential in an in vitro model of epithelial vaginal infection. This effect could be ascribed to a direct effect of living lactic acid bacteria on Candida virulence and to the production of metabolites that are able to impair fungal virulence. In the present work, stemming from these data, we deepened our knowledge of CFS from these four lactic acid bacteria by performing a metabolomic analysis to better characterize their composition. By using an untargeted metabolomic approach, we detected consistent differences in the metabolites produced by these four different lactic acid bacteria. Interestingly, L. rhamnosus and L. acidophilus showed the most peculiar metabolic profiles. Specifically, after a hierarchical clustering analysis, L. rhamnosus and L. acidophilus showed specific areas of significantly overexpressed metabolites that strongly differed from the same areas in other lactic acid bacteria. From the overexpressed compounds in these areas, inosine from L. rhamnosus returned with the best identification profile. This molecule has been described as having antioxidant, anti-inflammatory, anti-infective, and neuroprotective properties. The biological significance of its overproduction by L. rhamnosus might be important in its probiotic and/or postbiotic activity.

2024 - Cell-Free Supernatant from a Strain of Bacillus siamensis Isolated from the Skin Showed a Broad Spectrum of Antimicrobial Activity [Articolo su rivista]
Pedretti, Natalia; Iseppi, Ramona; Condò, Carla; Spaggiari, Luca; Messi, Patrizia; Pericolini, Eva; DI CERBO, Alessandro; Ardizzoni, Andrea; Sabia, Carla
abstract

In recent years, the search for new compounds with antibacterial activity has drastically increased due to the spread of antibiotic-resistant microorganisms. In this study, we analyzed Cell-Free Supernatant (CFS) from Bacillus siamensis, assessing its potential antimicrobial activity against some of the main pathogenic microorganisms of human interest. To achieve this goal, we exploited the natural antagonism of skin-colonizing bacteria and their ability to produce compounds with antimicrobial activity. Biochemical and molecular methods were used to identify 247 strains isolated from the skin. Among these, we found that CFS from a strain of Bacillus siamensis (that we named CPAY1) showed significant antimicrobial activity against Staphylococcus aureus, Enterococcus faecalis, Streptococcus agalactiae, and Candida spp. In this study, we gathered information on CFS’s antimicrobial activity and on its sensitivity to chemical–physical parameters. Time–kill studies were performed; anti-biofilm activity, antibiotic resistance, and plasmid presence were also investigated. The antimicrobial compounds included in the CFS showed resistance to the proteolytic enzymes and were heat stable. The production of antimicrobial compounds started after 4 h of culture (20 AU/mL). CPAY1 CFS showed antimicrobial activity after 7 h of bacteria co-culture. The anti-biofilm activity of the CPAY1 CFS against all the tested strains was also remarkable. B. siamensis CPAY1 did not reveal the presence of a plasmid and showed susceptibility to all the antibiotics tested.

2023 - A New Phenotype in Candida-Epithelial Cell Interaction Distinguishes Colonization- versus Vulvovaginal Candidiasis- Associated Strains [Articolo su rivista]
Sala, Arianna; Ardizzoni, Andrea; Spaggiari, Luca; Vaidya, Nikhil; van der Schaaf, Jane; Rizzato, Cosmeri; Cermelli, Claudio; Mogavero, Selene; Krüger, Thomas; Himmel, Maximilian; Kniemeyer, Olaf; Brakhage, Axel A.; King, Benjamin L.; Lupetti, Antonella; Comar, Manola; de Seta, Francesco; Tavanti, Arianna; Blasi, Elisabetta; Wheeler, Robert T.; Pericolini, Eva
abstract

Vulvovaginal candidiasis (VVC) affects nearly 3/4 of women during their lifetime, and its symptoms seriously reduce quality of life. Although Candida albicans is a common commensal, it is unknown if VVC results from a switch from a commensal to pathogenic state, if only some strains can cause VVC, and/or if there is displacement of commensal strains with more pathogenic strains. We studied a set of VVC and colonizing C. albicans strains to identify consistent in vitro phenotypes associated with one group or the other. We find that the strains do not differ in overall genetic profile or behavior in culture media (i.e., multilocus sequence type [MLST] profile, rate of growth, and filamen- tation), but they show strikingly different behaviors during their interactions with vaginal epithelial cells. Epithelial infections with VVC-derived strains yielded stronger fungal prolif- eration and shedding of fungi and epithelial cells. Transcriptome sequencing (RNA-seq) analysis of representative epithelial cell infections with selected pathogenic or commensal isolates identified several differentially activated epithelial signaling pathways, including the integrin, ferroptosis, and type I interferon pathways; the latter has been implicated in damage protection. Strikingly, inhibition of type I interferon signaling selectively increases fungal shedding of strains in the colonizing cohort, suggesting that increased shedding correlates with lower interferon pathway activation. These data suggest that VVC strains may intrinsically have enhanced pathogenic potential via differential elicitation of epithelial responses, including the type I interferon pathway. Therefore, it may eventually be possible to evaluate pathogenic potential in vitro to refine VVC diagnosis.

2023 - A new strategy to prevent biofilm and clot formation in medical devices: the use of atmospheric non-thermal plasma assisted deposition of silver-based nanostructured coatings [Articolo su rivista]
Gallingani, T; Resca, E; Dominici, M; Gavioli, G; Laurita, R; Liguori, A; Mari, G; Ortolani, L; Pericolini, E; Sala, A; Laghi, G; Petrachi, T; Arnauld, Gf; Accorsi, L; Rizzoli, R; Colombo, V; Gherardi, M; Veronesi, E
abstract

In industrialized countries, health care associated infections, the fourth leading cause of dis- ease, are a major health issue. At least half of all cases of nosocomial infections are associ- ated with medical devices. Antibacterial coatings arise as an important approach to restrict the nosocomial infection rate without side effects and the development of antibiotic resis- tance. Beside nosocomial infections, clot formation affects cardiovascular medical devices and central venous catheters implants. In order to reduce and prevent such infection, we develop a plasma-assisted process for the deposition of nanostructured functional coatings on flat substrates and mini catheters. Silver nanoparticles (Ag NPs) are synthesized exploit- ing in-flight plasma-droplet reactions and are embedded in an organic coating deposited through hexamethyldisiloxane (HMDSO) plasma assisted polymerization. Coating stability upon liquid immersion and ethylene oxide (EtO) sterilization is assessed through chemical and morphological analysis carried out by means of Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). In the perspective of future clinical appli- cation, an in vitro analysis of anti-biofilm effect has been done. Moreover, we employed a murine model of catheter-associated infection which further highlighted the performance of Ag nanostructured films in counteract biofilm formation. The anti-clot performances coupled by haemo- and cytocompatibility assays have also been performed.

2023 - Anti-Candida and Anti-Inflammatory Properties of a Vaginal Gel Formulation: Novel Data Concerning Vaginal Infection and Dysbiosis [Articolo su rivista]
Spaggiari, Luca; Squartini Ramos, Gianfranco B.; Squartini Ramos, Caterina A.; Ardizzoni, Andrea; Pedretti, Natalia; Blasi, Elisabetta; De Seta, Francesco; Pericolini, Eva
abstract

Vaginal ecosystem is a unique environment where, in physiological conditions, lactobacilli dominate. However, pathogenic microbial species responsible for vaginitis and vaginosis can also harbor vaginal microbiota. To extend the data published by De Seta and Larsen in Pathogens (2021), we analyzed here both the anti-Candida and anti-inflammatory properties of the vaginal gel formulation, Respecta® Balance Gel (RBG), commercialized as an adjuvant to treat vaginitis and vaginosis. We evaluated its activity by an in vitro model where a monolayer of A-431 vaginal epithelial cells was infected by Candida albicans in the presence of RBG or the placebo formulation (pRBG). Specifically, we tested the RBG capacity to counteract C. albicans virulence factors and their anti-inflammatory properties. Our results show that, unlike the placebo, RBG reduces C. albicans adhesion, its capacity to form hyphae and C. albicans-induced vaginal cell damage. Interestingly, both RBG and pRBG reduce LPS-induced IL-8 secretion (with RBG being the most effective), demonstrating that also the placebo retains anti-inflammatory properties. From our experimental approach, we highlighted the possible role of farnesol on such effects, but we would like to point out that lactic acid, polydextrose and glycogen too must be relevant in the actual application. In summary, our results show that RBG impairs C. albicans virulence and is able to reduce the inflammation in the vaginal environment, ultimately allowing the establishment of a balanced vaginal ecosystem.

2023 - Candidalysin is a key player in activating vaginal cell ROS in an in vitro infection model [Poster]
Spaggiari, Luca; Ardizzoni, Andrea; Squartini-Ramos, Caterina; Squartini-Ramos, Gianfranco; Ricchi, Francesco; Blasi, Elisabetta; De Seta, Francesco; Pericolini, Eva
abstract

2023 - In vivo colonization and pathogenic potential of C. africana [Poster]
Kosmala, Daria; Sertour, Natacha; Martins, Ricardo; Leibundgut-Landmann, Salomé; Spaggiari, Luca; Ardizzoni, Andrea; Pericolini, Eva; Bougnoux, Marie-Elisabeth; D'Enfert, Christophe; Legrand, Mélanie
abstract

2023 - The pathogenic and colonization potential of Candida africana [Articolo su rivista]
Kosmala, Daria; Sertour, Natacha; Ricardo Fróis Martins, ; Spaggiari, Luca; Ardizzoni, Andrea; LeibundGut-Landmann, Salomé; Pericolini, Eva; Bougnoux, Marie-Elisabeth; D'Enfert, Christophe; Legrand, Mélanie
abstract

The Candida albicans population displays high genetic diversity illustrated by 18-well differentiated genetic clusters. Cluster 13, also known as Candida africana, is an outlying cluster and includes strains first described as atypical C. albicans isolates of vaginal origin, showing apparent tropism for the female genital tract. In our study, we combined in vitro, and in vivo models to explore the colonization and pathogenic potential of C. africana. We report that C. africana has similar fitness to C. albicans when it comes to colonization of the oral and vaginal mucosa, however it has decreased fitness in gastro-intestinal colonization and systemic infection. Interestingly, despite high population homogeneity, our in vitro data highlighted for the first time a variability in terms of growth rate, biofilm formation and filamentation properties between C. africana strains. Overall, our data lays the foundations for exploring specific features of C. africana that might contribute to its apparent niche restriction.

2023 - The Propionibacterium spp. extract P40 reduces Candida albicans-induced damage to vaginal epithelial cells and increases mitochondrial response to Candida albicans infection in vitro [Poster]
Ricchi, F; Ardizzoni, A; Blasi, E; De Seta, F; Pericolini, E
abstract

2023 - The Propionibacterium spp. extract reduces Candida albicans-induced damage to vaginal epithelial cells and increases mitochondrial response to Candida albicans infection in vitro [Poster]
Ricchi, Francesco; Ardizzoni, Andrea; Blasi, Elisabetta; DE SETA, Francesco; Pericolini, Eva
abstract

Introduction. Bacterial lysates are prepared by inactivated microorganisms and are extensively employed in clinical settings as immunomodulants and to improve mucosal immunity. However, despite their extensive clinical use, their effects on the host are only partially known. The Propionibacterium spp. extract (PE) is a bacterial lysate included as an active compound in a gel formulation used to treat the symptoms of vulvovaginal candidiasis. Here, we analyzed its possible beneficial effects in an in vitro model of vaginal epithelial cells infected with Candida. Materials and Methods. Initially, we analyzed the PE effects on C. albicans and C. parapsilosis growth by the microdilution method. We then assessed the capacity of PE to reduce C. albicans-induced damage of vaginal epithelial cells through the quantification of lactate-dehydrogenase released by damaged cells in the growth medium. Moreover, in order to test the capacity of the PE to modulate epithelial mitochondrial activity, we evaluated Reactive-Oxygen-Species (ROS) production by the infected epithelial cells, stimulated or not with PE. This was kinetically monitored through the analysis of emitted fluorescence, after addition of the MitoSOX Red probe. Results. Our results show that PE did not affect directly microbial growth. In addition, the epithelial cells stimulation with PE reduced C. albicans-induced cell damage. Moreover, the treatment with PE increased the epithelial cells mitochondrial activity in response to C. albicans infection in vitro. Discussion and Conclusions. Taken together, our results show that PE increases ROS production by epithelial cells in response to C. albicans infection. Therefore, our results suggest that the increased mitochondrial activity induced by PE, could protect epithelial cells against the damage induced by C. albicans infection.

2023 - Zinc prevents vaginal candidiasis by inhibiting expression of an inflammatory fungal protein [Articolo su rivista]
Roselletti, E.; Pericolini, E.; Nore, A.; Takacs, P.; Kozma, B.; Sala, A.; De Seta, F.; Comar, M.; Usher, J.; Brown, G. D.; Wilson, D.
abstract

Candida causes an estimated half-billion cases of vulvovaginal candidiasis (VVC) every year. VVC is most commonly caused by Candida albicans, which, in this setting, triggers nonprotective neutrophil infiltration, aggressive local inflammation, and symptomatic disease. Despite its prevalence, little is known about the molecular mechanisms underpinning the immunopathology of this fungal infection. In this study, we describe the molecular determinant of VVC immunopathology and a potentially straightforward way to prevent disease. In response to zinc limitation, C. albicans releases a trace mineral binding molecule called Pra1 (pH-regulated antigen). Here, we show that the PRA1 gene is strongly up-regulated during vaginal infections and that its expression positively correlated with proinflammatory cytokine concentrations in women. Genetic deletion of PRA1 prevented vaginal inflammation in mice, and application of a zinc solution down-regulated expression of the gene and also blocked immunopathology. We also show that treatment of women suffering from recurrent VVC with a zinc gel prevented reinfections. We have therefore identified a key mediator of symptomatic VVC, giving us an opportunity to develop a range of preventative measures for combatting this disease.

2022 - Editorial: Women in fungal pathogenesis 2021 [Articolo su rivista]
Gelli, Angie; Nobile, Clarissa J.; Pericolini, Eva; Wellington, Melanie
abstract

2022 - Effects of benzydamine and mouthwashes containing benzydamine on Candida albicans adhesion, biofilm formation, regrowth, and persistence [Articolo su rivista]
Ardizzoni, A.; Boaretto, G.; Pericolini, E.; Pinetti, D.; Capezzone de Joannon, A.; Durando, L.; Ragni, L.; Blasi, E.
abstract

Objectives To assess the effects of benzydamine and mouthwashes (MoWs) containing benzydamine on different stages of Candida albicans biofilm: adhesion, formation, persistence, and regrowth (if perturbed). Materialsandmethods C.albicansCA1398,carryingthebioluminescenceACT1p-gLUC59fusionproduct,wasemployed. Fungal cells were exposed for 1′, 5′, or 15′ to 4 different benzydamine concentrations (0.075 to 0.6%) to 2 mouthwashes (MoWs) containing benzydamine and to a placebo MoW (without benzydamine). Treated cells were tested for adhesion (90 min) and biofilm formation (24-h assay). Next, 24- and 48-h-old biofilms were exposed to benzydamine and MoWs to assess regrowth and persistence, respectively. The effects of benzydamine, MoWs containing benzydamine, and placebo on different biofilm stages were quantified by bioluminescence assay and by the production of quorum sensing (QS) molecules. Results Benzydamine and MoWs containing benzydamine impaired C. albicans ability to adhere and form biofilm, counter- acted C. albicans persistence and regrowth, and impaired a 48-h-old biofilm. Some of these effects paralleled with alterations in QS molecule secretion. Conclusions Our results show for the first time that benzydamine and MoWs containing benzydamine impair C. albicans capacity to form biofilm and counteract biofilm persistence and regrowth. Clinical relevance Benzydamine and MoWs containing benzydamine capacity to affect C. albicans biofilm provides an interesting tool to prevent and treat oral candidiasis. Likely, restraining C. albicans colonization through daily oral hygiene may counteract colonization and persistence by other critical oral pathogens, such as Streptococcus mutans, whose increased virulence has been linked to the presence of C. albicans biofilm.

2022 - In vitro analysis of epithelial tolerability and anti-Candida effect of a new lactic acid-based vaginal gel formulation [Poster]
Spaggiari, Luca; Squartini, GIANFRANCO R.; Ardizzoni, Andrea; DE SETA, Francesco; Blasi, Elisabetta; Pericolini, Eva
abstract

INTRODUCTION. Vulvovaginal candidiasis (VVC) is the most prevalent vaginal infection in adult women. It is mainly caused by Candida albicans, and it affects 75% of healthy women at least once during their reproductive age; 5-10% of such women have recurrent episodes (RVVC), with more of 4 episodes of acute VVC per year. Symptoms of VVC include itching, burning, swelling and redness of the vaginal mucosa with white vaginal discharge. The urinary system can also be affected, with pain and burning when urinating. This condition seriously damages the well-being and the life quality of the affected women. Since Candida is a commensal fungus of the vaginal mucosa of healthy women, the main question is how the fungus can switch from harmless component of the vaginal microbiota to virulent pathogen. In this work we analyzed the capacity of lactic acid-based vaginal gel formulation Respecta® Balance Gel (RBG) to counteract C. albicans virulence after epithelial cells infection in vitro. MATERIALS AND METHODS. For the establishment of the in vitro infection model, we used a monolayer of the A-431 vaginal epithelial cell line and two different strains of C. albicans (strain SC5314 and the bioluminescent strain gLUC59). Dose-dependent experiments were performed to test the epithelial tolerability to RBG (IHS srl, Biofarma Group) by monitoring lactate-dehydrogenase (LDH) release from damaged cells. The capacity of RGB to counteract Candida-induced epithelial damage were analysed by monitoring LDH release from cells. Fungal growth and adhesion capacity during vaginal epithelial cells infection in the presence of RGB were evaluated by quantify the Relative Luminescent Units (RLU) and CFU counts, respectively. RESULTS. Our results show that, at dilution 1:150, RGB is well tolerated by the vaginal epithelium and consequently we used this dose for the subsequent experiments. RBG was able to significantly reduce (by 65%) C. albicans-induced damage of vaginal epithelial cells. This effect was accompanied with the capacity of RGB to significantly reduce Candida adhesion to the epithelium (adhesion reduction by 34%). Intriguingly, no inhibition of fungal growth was observed after 24h of infection in the presence of RGB in our experimental conditions. DISCUSSION AND CONCLUSIONS. Our results show that RGB significantly reduce C. albicans-induced damage of vaginal epithelial cells. One of the mechanisms underlying this effect is the inhibition of C. albicans adhesion to the vaginal epithelial cells, which may prevent Candida from penetrating and damaging epithelial cells, hence counteract Candida virulence. Collectively our preliminary results suggest that RBG can strengthen the VVC therapy favoring the establishment of an ecosystem that prevent Candida virulence.

2022 - Lactobacillus acidophilus, L. plantarum, L. rhamnosus, and L. reuteri Cell-Free Supernatants Inhibit Candida parapsilosis Pathogenic Potential upon Infection of Vaginal Epithelial Cells Monolayer and in a Transwell Coculture System In Vitro [Articolo su rivista]
Spaggiari, L.; Sala, A.; Ardizzoni, A.; De Seta, F.; Singh, D. K.; Gacser, A.; Blasi, E.; Pericolini, E.
abstract

Vulvovaginal candidiasis (VVC) is a common clinical condition with symptoms and signs of vaginal inflammation in the presence of Candida species. At least one episode of VVC is experienced in up to 75% of women in the reproductive age group during their lifetime, and 5-8% of such women suffer from the chronic form. Most cases of VVC are still caused by C. albicans; however, the incidence of VVC cases by non-albicans (NAC) species, such as C. parapsilosisis, is continuously increasing. Despite the prevalence of VVC from NAC, to date little is known on these species, and almost nothing on the mechanisms that trigger the VVC. Lactobacillus spp. are the most represented microorganisms in the vaginal microbiota of healthy women. Here, cell-free supernatants (CFS) obtained from L. acidophilus, L. plantarum, L. rhamnosus, L. reuteri were assessed for their effect on C. parapsilosis virulence traits. Moreover, we assessed if such effect persists even after removal of the CFS (CFS-preincubation effect). Moreover, a transwell co-culture system was employed, by which the relevant antifungal effect was shown to be attributable to the compounds released by Lactobacilli. Our results suggests that Lactobacilli can work: a) by reducing C. parapsilosis virulence traits, as indicated by the reduced fungal proliferation, viability and metabolic activity and b) by improving epithelial resistance to the fungus. Overall, these data suggest that, in the context of vaginal microbiota, the Lactobacilli may play a role in preventing the onset of mucosal C. parapsilosis infection.

2022 - Pomegranate extract affects fungal biofilm production: consumption of phenolic compounds and alteration of fungal autoinducers release [Articolo su rivista]
Colombari, B; Tagliazucchi, D; Odorici, A; Pericolini, E; Foltran, I; Pinetti, D; Meto, A; Peppoloni, S; Blasi, E.
abstract

Candida albicans expresses numerous virulence factors that contribute to pathogenesis, including its dimorphic transition and even biofilm formation, through the release of specific quorum sensing molecules, such as the autoinducers (AI) tyrosol and farnesol. In particular, once organized as biofilm, Candida cells can elude conventional antifungal therapies and the host’s immune defenses as well. Accordingly, biofilm-associated infections become a major clinical challenge underlining the need of innovative antimicrobial approaches. The aim of this in vitro study was to assess the effects of pomegranate peel extract (PomeGr) on C. albicans growth and biofilm formation; in addition, the release of tyrosol and farnesol was investigated. The phenolic profile of PomeGr was assessed by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) analysis before and after exposure to C. albicans. Here, we showed that fungal growth, biofilm formation and AI release were altered by PomeGr treatment. Moreover, the phenolic content of PomeGr was substantially hampered upon exposure to fungal cells; particularly pedunculagin, punicalin, punicalagin, granatin, di-(HHDP-galloyl-hexoside)-pentoside and their isomers as well as ellagic acid–hexoside appeared highly consumed, suggesting their role as bioactive molecules against Candida. Overall, these new insights on the anti-Candida properties of PomeGr and its potential mechanisms of action may represent a relevant step in the design of novel therapeutic approaches against fungal infections.

2022 - The Candida albicans Pra1 zincophore promotes neutrophils recruitment and inflammation during vulvovaginal candidiasis [Poster]
Roselletti, E.; Pericolini, E.; Sala, A.; Comar, M.; De Seta, F.; Wilson, D.
abstract

The symptoms of infectious diseases are frequently caused by an over­zealous host immune response against the invading microorganism, rather than by the microorganism itself. This is the case for one of the most common mucosal infection, vulvovaginal candidiasis (VVC), where the yeast Candida albicans triggers a non‐protective influx of host immune cells, resulting in aggressive local inflammation and symptomatic disease. The aim of our study was to investigate the role of the zincophore Pra1 in VVC immunopathology. Pra1 is a secreted C. albicans zinc binding protein released during zinc limitation and used by the fungus to forage for this essential micronutrient from the environment. In vitro tissue culture systems, a murine model of experimental vaginal candidiasis and vaginal samples from VVC patients were used to evaluate the role of Pra1 in immunopathology during VVC. Our results show that Pra1 induced neutrophil migration, is expressed at both, neutral and acidic pH by C. albicans during infection of vaginal epithelial cells and the expression was repressed by the addition of zinc. Robust PRA1 expression was also found in clinical vaginal samples and a strong correlation between PRA1 expression and the neutrophil­activating cytokine IL­8 has been demonstrated. In an experimental murine model of VVC, deletion of C. albicans PRA1 abrogated inflammation without affecting fungal burden. These data demonstrate that the zincophore Pra1 is expressed during the VVC and can act as a potent neutrophil chemoattractant molecule, driving inflammation.

2021 - Compounds released from Lactobacillus (L.) acidophilus, L. plantarum, L. rhamnosus and L. reuteri inhibit Candida parapsilosis pathogenic potential after infection of vaginal epithelial cells in vitro. [Poster]
Spaggiari, Luca; Sala, Arianna; Ardizzoni, Andrea; Cermelli, Claudio; Peppoloni, Samuele; Blasi, Elisabetta; Pericolini, Eva
abstract

INTRODUCTION. Lactobacillus spp. are the most represented microorganisms in the vaginal microbiota of healthy women, where they provide a shelter against infections from several pathogens, such as the yeasts belonging to the genus Candida. The latter are responsible for the vulvovaginal candidiasis (VVC), a condition affecting up to 75% of women during their child-bearing age at least once in their lifetime. Moreover, 5-8% of such women develop the recurrent form of the disease (RVVC), consisting of at least 5 VVC episodes per year. Notwithstanding C. albicans is the main responsible of VVC cases, in the last decades, the incidence of VVC cases by non-albicans Candida (NAC) species has become prevalent, especially in some geographical areas. C. parapsilosis, in particular, has been reported to be second species most commonly isolated from women affected by VVC. However, little is known on this species, and on its role in the pathogenesis of VVC. MATERIALS AND METHODS. Cell-free supernatants (CFS) were obtained following an overnight culture of 4 different Lactobacilli species (L. acidophilus, L. plantarum, L. rhamnosus, L. reuteri). Lactobacilli-released compounds, contained in CFS, were assessed for their effect on several virulence factors of C. parapsilosis (strain CLIB214), such as growth rate, capacity to form pseudohyphae, capacity to adhere to a vaginal epithelium in vitro (A-431 cells monolayer) and to induce cell damage. The latter was evaluated by measuring lactate dehydrogenase (LDH) release from A431 cells. RESULTS. C. parapsilosis growth inhibition by L. acidophilus, L. plantarum and L. reuteri CFS was 47%, 55% and 52% respectively, whereas L. rhamnosus CFS effect was weaker (33% inhibition growth). All the Lactobacilli significantly inhibited C. parapsilosis adhesion to vaginal epithelial cells: upon incubation with CFS, only 5-7% of fungal cells adhered to epithelial cells, after 90 minutes incubation; differently, the adhesion of the control reached 19%. Interestingly, no effect on pseudohyphae formation by any of the CSF was ever observed. Finally, the C. parapsilosis-induced damage on A-431 cells was significantly reduced by the addition of the CSF. DISCUSSION AND CONCLUSIONS. Our results show that the investigated species of Lactobacilli release compounds capable to impair several C. parapsilosis virulence factors, such as growth rate and adhesion to vaginal epithelial cells; interestingly, while not affecting fungal capacity to form pseudohyphae, such compounds significantly reduce Candida-mediated epithelial damage.. These data suggest that, in the context of vaginal microbiota, these Lactobacilli species may play an important role in counteracting the onset of mucosal Candida infections.

2021 - EDTA and Taurolidine affect Pseudomonas aeruginosa virulence in vitro: impairment of secretory profile and biofilm production onto peritoneal dialysis catheters [Poster]
Colombari, Bruna; Gamberini, Christian; Alfano, Gaetano; Peppoloni, Samuele; Ardizzoni, Andrea; Pericolini, Eva; Cappelli, Gianni; Blasi, Elisabetta
abstract

Introduction: peritoneal catheter-associated biofilm infection is reported to be the main cause of refractory peritonitis in peritoneal dialysis patients. The application of antimicrobial lock therapy, based on results on central venous catheters, may be a promising option also for treatment of biofilm-harboring peritoneal catheters. In this study, we investigated the effects of two lock solutions, EDTA and Taurolidine, on an “in-vitro” model of Pseudomonas aeruginosa biofilm-related peritoneal catheter infection. Materials and Methods: silicon peritoneal catheters were incubated for 24 h with a bioluminescent strain of P. aeruginosa. After washing, serial concentrations of Taurolidine (0.5, 0.25 and 0.125 %) and EDTA (2.5, 0.75 and 0.25 %), either alone or in combination, were applied for 24 h, once or twice, onto the contaminated catheters and then P. aeruginosa viability/persistence was evaluated in real time up to 120 h, by a Fluoroskan reader. Moreover, on selected supernatants from biofilm treated or not with EDTA and/or Taurolidine, High-Performance Liquid Chromatography-Mass (HPLC) analysis was performed to measure phenazine and pyocianine production. Results: Taurolidine alone or in combination with EDTA caused a significant decrease of bacterial load and biofilm persistence onto the contaminated catheters. The lock solution treatment did not lead to the sterilization of the devices; yet, it resulted in a substantial destructuration of the peritoneal catheter-associated P. aeruginosa biofilm. Moreover, HPLC analysis showed that the treatment of biofilm-harboring catheters with EDTA and Taurolidine deeply affected the secretion of some key virulence-related molecules by P. aeruginosa, such as phenazines and pyocianines. Discussion and conclusions: EDTA and Taurolidine affect the formation and persistence of P. aeruginosa biofilm onto peritoneal catheters; moreover, also the secretion of P. aeruginosa virulence factors is profoundly compromised. Future studies are needed to establish whether such lock solutions can be used to render peritoneal catheter-related infections more susceptible to antibiotic treatment, thus avoiding/reducing the onset of the antibiotic resistance phenomena.

2021 - Host-microbe interaction in Candida mucosal infections: a complex balance (Interazione ospite-microrganismo nelle infezioni mucosali da Candida: un equilibrio complesso) [Relazione in Atti di Convegno]
Sala, Arianna; Ardizzoni, Andrea; Spaggiari, Luca; Cermelli, Claudio; Peppoloni, Samuele; Tavanti, Arianna; Rizzato, Cosmeri; Blasi, Elisabetta; Vaidya, Nikhil; VAN DER SCHAAF, Jane; WHEELER ROBERT, T.; Pericolini, Eva
abstract

INTRODUCTION. Vulvovaginal candidiasis (VVC) is a very common mucosal infection in women of childbearing age caused primarily by C. albicans, characterized by powerful inflammatory response associated with infiltration of non-protective neutrophils. C. albicans can also asymptomatically colonize the vaginal mucosa as part of the resident microbiota in healthy women. The loss of the epithelial tolerance triggers the onset of the disease with increased fungal burden and virulence. However, the tolerance threshold to C. albicans varies among women and the causes of such variability are still unknown. The scope of our work is to understand how epithelial cell tolerance to C. albicans breaks down, focusing on fungal-intrinsic factors and host-pathogen interaction. MATERIALS AND METHODS. We characterized several traits of C. albicans isolates obtained from symptomatic and asymptomatic women both in culture medium or after infection of vaginal epithelial cells A-431, to determine the intrinsic pathogenic potential of these strains as well as their pathogenic activity during the interaction with vaginal epithelial cells. We analyzed strains by Multi Locus Sequence Typing (MLST), sequencing of the gene encoding the candidalysin toxin, quantification of cell-wall epitope exposure, rate of fungal growth and propensity for filamentation. Moreover, C. albicans-induced damage of the epithelial cells was evaluated; IL-1beta production and C. albicans shedding together with exfoliated epithelial cells were also tested. RESULTS. The two sets of isolates from symptomatic and asymptomatic women did not differ in genetic profile or behaviour in culture media (i.e. MLST profile, rate of growth, filamentation) but they showed relevant differences when interacting with vaginal epithelial cells. Indeed, unlike the isolates from healthy colonized women, the VVC isolates showed a significantly greater propensity to induce C. albicans shedding in association with exfoliated epithelial cells. DISCUSSION AND CONCLUSIONS. Our results point towards the exclusion of the involvement of fungal intrinsic factors in host-C. albicans balance at mucosal level; rather, fungal traits that arise when interacting with the host correlate with the likelihood of symptomatic infection in VVC.

2021 - It takes two to tango: how a dysregulation of the innate immunity, coupled with Candida virulence, triggers VVC onset [Articolo su rivista]
Ardizzoni, A.; Wheeler, Rt; Pericolini, E.
abstract

Vulvovaginal candidiasis (VVC) is a symptomatic inflammation of the vagina mainly caused by C. albicans. Other species, such as C. parapsilosis, C. glabrata, C. tropicalis and C. krusei, are mainly associated to the recurrent form of the disease (RVVC), although with a lower frequency. In its yeast form, C. albicans is tolerated by the vaginal epithelium, but switching to the invasive hyphal form, co-regulated with the expression of genes encoding virulence factors such as Sap and candidalysin, allows for tissue damage. Vaginal epithelial cells play an important role by impairing C. albicans tissue invasion through several mechanisms such as epithelial shedding, secretion of mucin and strong interepithelial cell connections. However, morphotype switching coupled to increasing of the fungal burden can overcome the tolerance threshold and trigger an intense inflammatory response. Pathological inflammation is believed to be facilitated by an altered vaginal microbiome, i.e., Lactobacillus dysbiosis. Notwithstanding the damage caused by the fungus itself, the host response to the fungus plays an important role in the onset of VVC, exacerbating fungal-mediated damage. This response can be triggered by host PRR-fungal PAMP interaction and other more complex mechanisms (i.e., Sap-mediated NLRP3 activation and candidalysin), ultimately leading to strong neutrophil recruitment. However, recruited neutrophils appear to be ineffective at reducing fungal burden and invasion; therefore, they seem to contribute more to the symptoms associated with vaginitis than to protection against the disease. Recently, two aspects of the vulvovaginal environment have been found to associate with VVC and induce neutrophil anergy in vitro: perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) and heparan sulfate. Interestingly, CAGTA antibodies have also been found with higher frequency in VVC as compared to asymptomatic colonized women. This review highlights and discusses recent advances on understanding the VVC pathogenesis mechanisms as well as the role of host defenses during the disease.

2020 - Antibacterial Effects of MicroRepair®BIOMA-Based Toothpaste and Chewing Gum on Orthodontic Elastics Contaminated In Vitro with Saliva from Healthy Donors: A Pilot Study [Articolo su rivista]
Meto, Aida; Colombari, Bruna; Odorici, Alessandra; Giva, Lavinia Beatrice; Pericolini, Eva; La Regina, Annunziata; Blasi, Elisabetta
abstract

Several new products with innovative formulations are being proposed to facilitate oral care. Here, we evaluated the effects of a commercially available product, a toothpaste and chewing gum named Biorepair Peribioma, on oral microorganisms of healthy subjects. Saliva from six volunteers was collected during 20 min of mastication of a traditional gum (gum A) and the Biorepair Peribioma gum (gum P). Orthodontic elastics (OE) were in vitro contaminated with salivary samples, both A and P, and subsequently exposed or not to a Biorepair Peribioma toothpaste-conditioned supernatant (Tp-SUP). The salivary samples were tested for initial microbial load; hence, the contaminated OE were assessed for microbial growth, adhesion, biofilm formation and persistence; moreover, species identification was assessed. We found that the salivary samples A and P had similar microbial load; upon contamination, microbial adhesion onto the OE was detected to a lower extent when using saliva P with respect to saliva A. Microbial growth and biofilm formation, assessed at 24 h, remained at lower levels in OE exposed to saliva P, compared to saliva A. This difference between salivary samples A and P was confirmed when measuring biofilm persistence (48 h), while it was lost in terms of microbial re-growth (48 h). The Tp-SUP treatment drastically affected microbial load at 24 h and strongly impaired biofilm formation/persistence, in OE exposed to both salivary samples A and P. Finally, such treatment resulted in consistent overgrowth of Lactobacilli, bacterial species originally present both in the Biorepair Peribioma toothpaste and gum. In conclusion, by an in vitro pilot study, we show that the Biorepair Peribioma toothpaste and gum deeply affect oral microorganisms’ behavior, drastically impairing their ability to contaminate and produce plaque onto orthodontic devices.

2020 - Evaluation of benzydamine effects on Candida albicans adhesion, biofilm formation and persistence onto abiotic surfaces [Poster]
Ardizzoni, Andrea; Boaretto, Giorgia; Pericolini, Eva; CAPEZZONE DE JOANNON, Alessandra; Durando, Lucia; Ragni, Lorella; Sala, Arianna; Blasi, Elisabetta
abstract

Introduction. Candida albicans is the most abundant yeast colonizing the oral cavity. It behaves as an opportunistic pathogen, causing mucosal infections mainly in immunocompromised individuals; in addition, it is often associated to patients suffering from diabetes, oral cancer and terminally ill conditions. Benzydamine hydrochloride is a non-steroidal and anti-inflammatory agent. It has been included in the formulation of several mouthwashes because endowed with analgesic and anesthetic properties. Since benzydamine exerts antibacterial and antifungal activity in vitro, we assessed if this molecule could affect C. albicans virulence traits, such as adhesion, biofilm formation and persistence on abiotic surfaces. Materials and Methods. C. albicans CA1398, carrying the bioluminescence ACT1p-gLUC59 fusion product, was employed. Firstly, fungal cells were exposed for 1’, 5’, or 15’ to 4 different benzydamine concentrations (0.075%, 0.15%, 0.3% and 0.6%) and then tested for their capacity to adhere to plastic (90’ incubation) or to form a biofilm (24h assay). Secondly, 24 and 48h-old biofilms were exposed to the same concentrations of benzydamine and for the same times in order to assess biofilm persistence and regrowth. Benzydamine effects were quantified by measuring, in parallel, metabolically active fungal cells (bioluminescence assay) and viable cells (Colony Forming Units assay). Results. Benzydamine impaired ability to adhere to plastic and to form biofilm, in a dose-dependent fashion; such effects could be ascribed to a direct effect of benzydamine on Candida viability only when using the highest dosage. Moreover, benzydamine caused a dose-dependent decrement in the viability of Candida cells embedded in biofilm, no matter whether a 24h- or a 48h-old sessile community was tested. Discussion and Conclusions. Benzydamine not only impairs C. albicans biofilm formation, profoundly affecting the initial step of fungal cell adhesion to abiotic surfaces, but it is also able to counteract persistence and regrowth of a preformed biofilm. The capacity of benzydamine to affect C. albicans, a fungus responsible of oral diseases in several categories of susceptible subjects, makes this molecule a very interesting tool for both prevention and treatment of oral candidiasis. Studies employing benzydamine-containing mouthwashes will be carried out, in order to assess and compare the anti-Candida effects of different commercial products.

2020 - Perinuclear Anti-Neutrophil Cytoplasmic Antibodies (pANCA) Impair Neutrophil Candidacidal Activity and Are Increased in the Cellular Fraction of Vaginal Samples from Women with Vulvovaginal Candidiasis [Articolo su rivista]
Ardizzoni, Andrea; Sala, Arianna; Colombari, Bruna; Giva, Lavinia Beatrice; Cermelli, Claudio; Peppoloni, Samuele; Vecchiarelli, Anna; Roselletti, Elena; Blasi, Elisabetta; Wheeler, Robert T.; Pericolini, Eva
abstract

Vulvovaginal candidiasis (VVC) is primarily caused by Candida albicans and affects 75% of childbearing age women. Although C. albicans can colonize asymptomatically, disease is associated with an increased Candida burden, a loss of epithelial tolerance and a breakdown in vaginal microbiota homeostasis. VVC symptoms have been ascribed to a powerful inflammatory response associated with the infiltration of non-protective neutrophils (PMN). Here, we compared the immunological characteristics of vaginal fluids and cellular protein extracts obtained from 28 VVC women and from 23 healthy women colonized by Candida spp. We measured the levels of antibodies against fungal antigens and human autoantigens (anti-Saccharomyces cerevisiae antibodies (ASCA), C. albicans germ tube antibodies (CAGTAs) and perinuclear anti-neutrophil cytoplasmic antibodies (pANCA)), in addition to other immunological markers. Our results show that the pANCA levels detected in the cellular protein extracts from the vaginal fluids of symptomatic women were significantly higher than those obtained from healthy colonized women. Consistent with a potential physiologically relevant role for this pANCA, we found that specific anti-myeloperoxidase antibodies could completely neutralize the ex vivo killing capacity of polymorphonuclear cells. Collectively, this preliminary study suggests for the first time that pANCA are found in the pathogenic vaginal environment and can promptly impair neutrophil function against Candida, potentially preventing a protective response.

2020 - Prevention and treatment of autoimmune diseases with plant virus nanoparticles [Articolo su rivista]
Zampieri, Roberta; Brozzetti, Annalisa; Pericolini, Eva; Bartoloni, Elena; Gabrielli, Elena; Roselletti, Elena; Lomonosoff, George; Meshcheriakova, Yulia; Santi, Luca; Imperatori, Francesca; Merlin, Matilde; Tinazzi, Elisa; Dotta, Francesco; Nigi, Laura; Sebastiani, Guido; Pezzotti, Mario; Falorni, Alberto; Avesani, Linda
abstract

Plant viruses are natural, self-assembling nanostructures with versatile and genetically programmable shells, making them useful in diverse applications ranging from the development of new materials to diagnostics and therapeutics. Here, we describe the design and synthesis of plant virus nanoparticles displaying peptides associated with two different autoimmune diseases. Using animal models, we show that the recombinant nanoparticles can prevent autoimmune diabetes and ameliorate rheumatoid arthritis. In both cases, this effect is based on a strictly peptide-related mechanism in which the virus nanoparticle acts both as a peptide scaffold and as an adjuvant, showing an overlapping mechanism of action. This successful preclinical testing could pave the way for the development of plant viruses for the clinical treatment of human autoimmune diseases.

2020 - The β-Lactamase Inhibitor Boronic Acid Derivative SM23 as a New Anti-Pseudomonas aeruginosa Biofilm [Articolo su rivista]
Peppoloni, S.; Pericolini, E.; Colombari, B.; Pinetti, D.; Cermelli, C.; Fini, F.; Prati, F.; Caselli, E.; Blasi, Elisabetta
abstract

Pseudomonas aeruginosa is a Gram-negative nosocomial pathogen, often causative agent of severe device-related infections, given its great capacity to form biofilm. P. aeruginosa finely regulates the expression of numerous virulence factors, including biofilm production, by Quorum Sensing (QS), a cell-to-cell communication mechanism used by many bacteria. Selective inhibition of QS-controlled pathogenicity without affecting bacterial growth may represent a novel promising strategy to overcome the well-known and widespread drug resistance of P. aeruginosa. In this study, we investigated the effects of SM23, a boronic acid derivate specifically designed as β-lactamase inhibitor, on biofilm formation and virulence factors production by P. aeruginosa. Our results indicated that SM23: (1) inhibited biofilm development and production of several virulence factors, such as pyoverdine, elastase, and pyocyanin, without affecting bacterial growth; (2) decreased the levels of 3-oxo-C12-HSL and C4-HSL, two QS-related autoinducer molecules, in line with a dampened lasR/lasI system; (3) failed to bind to bacterial cells that had been preincubated with P. aeruginosa-conditioned medium; and (4) reduced both biofilm formation and pyoverdine production by P. aeruginosa onto endotracheal tubes, as assessed by a new in vitro model closely mimicking clinical settings. Taken together, our results indicate that, besides inhibiting β-lactamase, SM23 can also act as powerful inhibitor of P. aeruginosa biofilm, suggesting that it may have a potential application in the prevention and treatment of biofilm-associated P. aeruginosa infections.

2020 - The β-lactamase Inhibitor Boronic Acid SM23 as a new anti-Pseudomonas aeruginosa Biofilm Compound [Poster]
Peppoloni, Samuele; Pericolini, Eva; Colombari, Bruna; Pinetti, Diego; Cermelli, Claudio; Fini, Francesco; Prati, Fabio; Blasi, Elisabetta; Caselli, Emilia
abstract

BACKGROUND: Pseudomonas aeruginosa is a Gram-negative nosocomial pathogen, often causative agent of severe device-related infections, given its great ability to produce biofilm. P. aeruginosa finely regulates the expression of numerous virulence factors, including biofilm production, by Quorum Sensing (QS), an intercellular communication mechanism used by many bacteria. Biofilm formation can enhance bacterial resistance to antimicrobial agents due to a decreased penetration of the antibiotic and a reduced rate of bacterial cells in biofilm. Selective inhibition of biofilm formation may thus represent a novel promising strategy to overcome the well-known and widespread drug-resistance of P. aeruginosa. METHODS: In this study, we investigated the effects of SM23, a boronic acid derivate specifically designed as β-lactamase inhibitor, on biofilm formation and production of virulence factors, using the P. aeruginosa bioluminescent strain P1242. RESULTS: Our results indicated that SM23: a) inhibited the development of biofilm and the production of the virulence factors pyoverdine, elastase and pyocyanin, without affecting bacterial growth; b) decreased the levels of P. aeruginosa QS-related Autoinducers molecules 3-oxo-C12-HSL and C4-HSL by dampened lasR/lasI system gene expression in the biofilm; c) failed to bind to bacterial cells that had been preincubated with P. aeruginosa-conditioned medium; d) reduced both biofilm formation and pyoverdine production by P. aeruginosa onto endotracheal tubes, as assessed by a new in vitro model, closely mimicking the clinical settings. CONCLUSION: Taken together, our results indicate that, besides inhibiting β-lactamase, SM23 can also act as potent inhibitor of P. aeruginosa biofilm, suggesting that it may have a potential application in the prevention and treatment of biofilm-associated P. aeruginosa infections.

2020 - The β-lactamase Inhibitor Boronic Acid SM23 Inhibits Pseudomonas aeruginosa Biofilm Formation and Virulence Factor Production [Poster]
Peppoloni, Samuele; Pericolini, Eva; Colombari, Bruna; Pinetti, Diego; Cermelli, Claudio; Fini, Francesco; Prati, Fabio; Caselli, Emilia; Blasi, Elisabetta
abstract

Introduction: Pseudomonas aeruginosa is a Gram-negative nosocomial pathogen, often responsible of severe device-related infections, given its great ability to produce biofilm. P. aeruginosa finely regulates the expression of different virulence factors, including biofilm production, by Quorum Sensing (QS), an intercellular communication mechanism used by many bacteria. Biofilm formation enhances bacterial resistance to antimicrobial agents due to a decreased penetration of antibiotics and a reduced rate of growth of embedded bacteria. Thus, novel agents capable of selective inhibiting biofilm formation may represent a promising strategy to overcome the well-known and widespread drug-resistance of P. aeruginosa. Material and Methods: by using the bioluminescent P. aeruginosa strain P1242, we investigated the effects of SM23, a boronic acid derivative specifically designed as beta-lactamase inhibitor, on biofilm formation and virulence factor production by P. aeruginosa. Results: we found that SM23: a) inhibited both biofilm formation and production of the virulence factors, pyoverdine, elastase and pyocyanin, without affecting bacterial growth; b) decreased the levels of QS-related autoinducers molecules, namely 3-oxo-C12-HSL and C4-HSL, by reducing lasR/lasI system gene expression in the biofilm; c) failed to bind to bacterial cells that had been preincubated with P. aeruginosa-conditioned medium; d) reduced significantly P. aeruginosa biofilm and pyoverdine production on endotracheal tubes, an in vitro condition closely mimicking clinical settings. Discussion and Conclusions: taken together, our results indicate that, besides inhibiting beta-lactamase, the boronic acid SM23, can also act as potent inhibitor of P. aeruginosa virulence, by profoundly affecting biofilm and QS-related signals. These findings highlight potential application of this compound in the prevention and treatment of biofilm-associated P. aeruginosa infections.

2019 - Antimicrobial and antibiofilm efficacy of a copper/calcium hydroxide-based endodontic paste against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans [Articolo su rivista]
Meto, A.; Colombari, B.; Sala, A.; Pericolini, E.; Meto, A.; Peppoloni, S.; Blasi, Elisabetta
abstract

Endodontic biofilm is a microbial community, enclosed in a polymeric matrix of polysaccharide origin where are found pathogens, like bacteria and opportunistic fungi responsible for various endodontic pathologies. As clinical importance is the fact, that biofilm is extremely resistant to common intracanal irrigants, antimicrobial drugs and host immune responses. The aim of this study was to evaluate the in vitro efficacy of a Cu/CaOH2-based endodontic paste, against bacteria and fungi, such as Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. We found that such compound significantly reduced microbial replication time and cell growth. Moreover, biofilm formation and persistence were also affected; treated biofilms showed both a reduced number of cells and levels of released pyoverdine. This study provides the first evidence on effectiveness of this endodontic compound against microbial biofilms. Given its wide range of action, its use in prevention and treatment of the main oral biofilm-associated infections will be discussed.

2019 - Bioimmunological activities of Candida glabrata cellular mannan [Articolo su rivista]
Paulovicová, L; Paulovicová, E; Farkaš, P; Cížová, A; Bystrický, P; Jancinová, V; Turánek, J; Pericolini, E; Gabrielli, E; Vecchiarelli, A; Hrubiško, M.
abstract

Candida glabrata is a second most common human opportunistic pathogen which causes superficial but also life-threatening systemic candidiasis. According to the localization of mannans and mannoproteins in the outermost layer of the cell wall, mannan detection could be one of the first steps in the cell recognition of Candida cells by the host innate immune system. Mannans from the cell wall provide important immunomodulatory activities, compromising stimulation of cytokine production, induction of dendritic cells maturation and T-cell immunity. The model of DCs represents a promising tool to study immunomodulatory interventions throughout the vaccine development. Activated DCs induce, activate and polarize T-cell responses by expression of distinct maturation markers and cytokines regulating the adaptive immune responses. In addition, they are uniquely adept at decoding the fungus-associated information and translate it in qualitatively different T helper responses. We find out, that C. glabrata mannan is able to induce proliferation of splenocytes and to increase the production of TNF-α and IL-4. Next, increased the expression of co-stimulatory molecules CD80 and CD86 and the proportion of CD4+CD25+ and CD4+CD28+ T cells during in vitro stimulation of splenocytes.

2019 - Comparative analysis of commensal and pathogenic vulvovaginal clinical isolates of Candida albicans: hyphal morphology, molecular genetics and phylogenetic relationships [Poster]
Pericolini, E.; Sala, A.; Blasi, E.; Tavanti, A.; Rizzato, C.; Lupetti, A.; Van Der Schaaf, J.; Wheeler, Rt.
abstract

Introduction. Vulvovaginal candidiasis (VVC) is a common disorder affecting approximately 75% of women of childbearing age at least once during their life and it is caused primarily by Candida albicans. However, C. albicans is also a normal harmless commensal of the vaginal mucosa, hence a long-standing question is how the fungus switches from commensal state to a virulent pathogen. Studies performed in a murine vaginitis model suggest that host inflammatory processes drive the onset of symptomatic infection. Accordingly, our recent work on clinical samples from colonized and symptomatic women revealed different propensity to form hyphae. In addition, β-glucan, a fungal cell wall pro-inflammatory polysaccharide, was largely masked from immune recognition during vaginitis and enhanced β-glucan availability was only found in hyphae from symptomatic patients with a concomitant massive neutrophil infiltration. Materials and Methods. To further investigate any association between fungal virulence traits and VVC outcome, the following parameters were analyzed on commensal and clinically relevant isolates: MLST analysis, sequencing of the gene encoding the candidalysin toxin and percentage of hyphal fragments. Results. The results obtained so far suggest that none of these fungus-related parameters allow to discriminate between commensal and clinically relevant isolates. Discussion and Conclusions. Taken together, our preliminary data indicate that host-intrinsic mechanisms, rather than the fungus’ intrinsic virulence traits, may play a key role in the occurrence of VVC.

2019 - GILZ restrains neutrophil activation by inhibiting the MAPK pathway [Articolo su rivista]
Ricci, E; Ronchetti, S; Gabrielli, E; Pericolini, E; Gentili, M; Roselletti, E; Vecchiarelli, A; Riccardi, C.
abstract

Glucocorticoid-induced leucine zipper (GILZ) exerts anti-inflammatory effects on the immune cells. However, less is known about GILZ function in neutrophils. We aimed to define the specific role of GILZ in basal neutrophil activity during an inflammatory response. GILZ knockdown resulted in a persistent activation state of neutrophils, as evidenced by increased phagocytosis, killing activity, and oxidative burst in GILZ-knockout (KO) neutrophils. This enhanced response caused severe disease in a dinitrobenzene sulfonic acid (DNBS)-induced colitis model, where GILZ-KO mice had prominent granulocytic infiltrate and excessive inflammatory state. We used a Candida albicans intraperitoneal infection model to unravel the intracellular pathways affected by GILZ expression in activated neutrophils. GILZ-KO neutrophils had stronger ability to clear the infectious agent than the wild-type (WT) neutrophils, and there was more activation of the NOX2 (NADPH oxidase 2) and p47phox proteins, which are directly involved in oxidative burst. Similarly, the MAPK pathway components, that is, ERK and p38, which are involved in the oxidative burst pathway, were highly phosphorylated in GILZ-KO neutrophils. Evaluation of GILZ expression kinetics during C. albicans infection revealed down-regulation that correlated inversely with the state of neutrophil activation, which was evaluated as oxidative burst. Overall, our findings define GILZ as a regulator of neutrophil functions, as its expression contributes to limiting neutrophil activation by reducing the activation of the signaling pathways that control the basal neutrophil functions. Controlling GILZ expression could help regulate a continuous inflammatory state that can result in chronic inflammatory and autoimmune diseases.

2019 - Hyphal morphology, molecular genetics and phylogenetic relationships among commensal and pathogenic vulvovaginal isolates of Candida albicans [Poster]
Pericolini, E.; Sala, A.; Blasi, E.; Tavanti, A.; Rizzato, C.; Van Der Schaaf, J.; Wheeler, R. T.
abstract

Vaginal candidiasis is a common disorder in women of childbearing age, caused primarily by Candida albicans. Since C. albicans is a commensal fungus of the vaginal mucosa, a long-standing question is how the fungus switches from being a harmless commensal to a virulent pathogen. Clinical studies and murine vaginitis models suggest that host inflammatory processes drive the onset of symptomatic infection. In previous work with fresh clinical samples, we found that the pro-inflammatory cell wall polysaccharide β-glucan is largely masked from immune recognition during vulvovaginal infection. Enhanced β-glucan availability was only found in hyphae from symptomatic patients with strong neutrophil infiltration. There was high variability in levels of β-glucan exposure and hyphal morphology among colonizing and infection-associated isolates, and we reasoned that this could be explained by fungal-intrinsic factors and/or host-associated traits. We assayed several aspects of C. albicans isolated from symptomatic and asymptomatic individuals to determine any associations between fungal-intrinsic traits and virulence: MLST analysis, sequencing of the gene encoding the candidalysin toxin, and propensity to form hyphal cells. Preliminary results suggest that none of these indicators correlates with isolates causing symptomatic infection, indicating that host-intrinsic mechanisms may play the most important role in the occurrence of symptomatic infections.

2019 - Inhibition of Quorum Sensing-dependent biofilm formation and virulence factors in Pseudomonas aeruginosa by the boronic acid SM23 [Poster]
Peppoloni, S.; Pericolini, E.; Colombari, B.; Pinetti, D.; Fini, F.; Prati, F.; Blasi, E.; Caselli, E.
abstract

Introduction: Quorum sensing (QS) regulates the expression of virulence factors in P. aeruginosa. Inhibiting QS-controlled virulence factors without affecting the growth of P. aeruginosa may represent a promising strategy for overcoming its widespread and constantly increasing drug-resistance. In this study, we investigated the effects of SM23, a boronic acid, which was specifically designed as beta-lactamase inhibitor, on biofilm formation and virulence factor production by P. aeruginosa Material and Methods: the bioluminescent P. aeruginosa strain P1242 was employed. The effect of the boronic acid SM23 on P. aeruginosa were assessed by evaluating a) the biofilm formation and its morphology by crystal violet staining/bioluminescence and confocal microscopy and b) the production in cell supernatant of the virulence factors, pyoverdines and elastase. The pyoverdine release was assessed by measuring the fluorescence emission with a multi-well fluorescence plate reader and mass spectrometry, while the elastase activity was determined by the Ohman’s method, using the Elastin-Congo red as substrate. Finally a qRT-PCR was employed to study the SM23-induced changes in the expression of the QS genes lasI and lasR. Results: the SM23 significantly inhibited the development of biofilm and the production of virulence factors, as pyoverdines and elastase, without affecting bacterial growth. Preincubation of bacteria with P. aeruginosa-conditioned (24 h) medium completely prevented the binding of SM23 to the cells. By investigating the transcriptional changes related to QS, we found that Pseudomonas exposure to SM23 caused a notable decrease in the levels of lasI and lasR gene expression. Finally, the SM23 significantly reduced P. aeruginosa biofilm and pyoverdine production on endotracheal tubes, an in vitro condition closely mimicking clinical settings. Discussion and Conclusions: taken together, our results indicate that boronic acid SM23, besides inhibiting beta-lactamase, can also act as potent inhibitor of QS in P. aeruginosa, suggesting that it may have a potential application in the prevention and treatment of biofilm-associated P. aeruginosa infections.

2019 - Saccharomyces cerevisiae as a new therapeutic agent against oropharyngeal candidiasis [Poster]
Roselletti, Elena; Sabbatini, Samuele; Ballet, Nathalie; Perito, Stefano; Pericolini, Eva; Blasi, Elisabetta; Mosci, Paolo; CAYZEELE DECHERF, Amélie; Monari, Claudia; Vecchiarelli, Anna
abstract

Introduction. Oropharyngeal candidiasis is a common opportunistic mucosal infection of the oral cavity, mainly caused by an overgrowth of Candida albicans (C. albicans). This infection can inhibit nutritional intakes and strongly affect quality of life. To date, standard therapeutic strategies involving the administration of antifungal drugs can bring several side-effects, not least the emergence of drug-resistant strains. The purpose of this study is to investigate the effectiveness of Saccharomyces cerevisiae based probiotic (S.-cerevisiae) against oropharyngeal candidiasis. Materials and methods. C57BL/6J mice were immunosuppressed with subcutaneously injection of cortisone acetate. The mice were infected with bioluminescent (BLI) C. albicans and then treated sublingually with S. cerevisiae. In these mice fungal burden was imaged and quantified in the IVIS Lumina XRMS Imaging system. Gene expression of C. albicans virulence factors such as ALS3, SAP2 and SAP6 was examined. Histopathologic lesions of tongue were also evaluated. Results. Our results show that administration of S. cerevisiae in the oral cavity of C57BL/6J mice resulted in a protective effect against oropharyngeal candidiasis. This was related to: i) a decrease of C. albicans load in the oral cavity, esophagus, stomach and duodenum; ii) an early resolution of inflammatory process in the tongue; iii) a marked reduction of C. albicans virulence factors Discussion and Conclusions. These findings suggest that probiotic S. cerevisiae is able to positively reverse/attenuate the course of OPC infection.

2019 - Saccharomyces cerevisiae CNCM I-3856 as a New Therapeutic Agent Against Oropharyngeal Candidiasis [Articolo su rivista]
Roselletti, E.; Sabbatini, S.; Ballet, N.; Perito, S.; Pericolini, E.; Blasi, E.; Mosci, P.; Cayzeele Decherf, A.; Monari, C.; Vecchiarelli, Anna
abstract

Oropharyngeal candidiasis is a common opportunistic mucosal infection of the oral cavity, mainly caused by an overgrowth of Candida albicans. This infection can inhibit nutritional intakes and strongly affect quality of life. To date, standard therapeutic strategies involving the administration of antifungal drugs can bring several side effects, not least the emergence of drug-resistant strains. The purpose of this study is to investigate the effectiveness of Saccharomyces cerevisiae CNCM I-3856 (live or inactivated cells) against oropharyngeal candidiasis. Our results show that administration of S. cerevisiae CNCM I-3856 (live or inactivated cells) in the oral cavity of C57BL/6J mice resulted in a protective effect against oropharyngeal candidiasis. The strongest effect was obtained with live S. cerevisiae CNCM I-3856. This was related to: (1) a decrease in C. albicans load in the oral cavity, esophagus, stomach, and duodenum; (2) an early resolution of inflammatory process in the tongue; (3) a marked reduction in C. albicans virulence factors; and (4) a consistent increase in neutrophil antimicrobial capacity. These findings suggest that S. cerevisiae products are potentially beneficial in the treatment of oropharyngeal candidiasis.

2018 - Effects of Cupral® on the formation and persistence of microbial biofilms in vitro [Poster]
Meto, Aida; Colombari, Bruna; Pericolini, Eva; Peppoloni, Samuele; Blasi, Elisabetta
abstract

Introduction: endodontic biofilm is a microbial community, enclosed in a polymeric matrix of polysaccharide origin where are frequently found pathogenic microorganisms, such as Gram+, Gram- and opportunistic fungi, belonging to Candida spp, responsible for several endodontic pathologies. As clinical importance is the fact that biofilm is extremely resistant to common intra-canal irrigants, antimicrobial drugs and host immune defenses. The aim of this in vitro study was to evaluate the efficacy of Cupral® on planktonic forms of some pathogens, as well as to assess its ability to prevent and affect the formation/persistence of microbial biofilms. Materials and Methods: ATCC strains of S. aureus, P. aeruginosa and C. albicans were exposed to various concentrations of Cupral® (an antiseptic compound based on calcium and copper hydroxide, used in endodoncy) to investigate its antimicrobial efficacy. This activity has been evaluated in terms of microbial growth and cellular doubling time (optical density, colony forming units and doubling time assays), inhibition/persistence (crystal violet staining), viability of microbial cells embedded in the biofilms (live/dead stain) and pyoverdine production (fluorimetric assay). Finally, the morphology of Cupral®-treated biofilms was investigated by optical/confocal microscopy analysis. Results: the addition of Cupral® to microbial cultures, influences, in a significantly and dose-dependent manner, the doubling time and growth of microbial cultures. Cupral® antimicrobial activity was also assessed on biofilms formation and persistence with meaningful decreases of residual biomass (observed reductions of 47-94% for S. aureus, 28-95% for P. aeruginosa and 27-75 % for C. albicans). Cupral®-treated biofilms analyzed by optical and confocal microscopy revealed loss of typical sessile structure, with few scattered microbial cells and a reduced thickness. Finally, the addition of Cupral® reduced both the number of embedded alive cells in the biofilms and the levels of pyoverdine in the culture supernatants. Discussion and Conclusions: this pilot in vitro study provided the first evidences on Cupral® efficacy against microbial biofilms. The wide range of action (vs Gram+, Gram- and fungi) of Cupral® strongly suggests its use as compound in the prevention and treatment of main oral biofilm-associated infections.

2018 - Epitope unmasking in vulvovaginal candidiasis is associated with hyphal growth and neutrophilic infiltration [Articolo su rivista]
Pericolini, E.; Perito, S.; Castagnoli, A.; Gabrielli, E.; Mencacci, A.; Blasi, E.; Vecchiarelli, A.; T. Wheeler., R.
abstract

Vaginal candidiasis is a common disorder in women of childbearing age, caused primarily by the dimorphic fungus Candida albicans. Since C. albicans is a normal commensal of the vaginal mucosa, a long-standing question is how the fungus switches from being a harmless commensal to a virulent pathogen. Work with human subjects and in mouse disease models suggests that host inflammatory processes drive the onset of symptomatic infection. Fungal cell wall molecules can induce inflammation through activation of epithelial and immune receptors that trigger pro-inflammatory cytokines and chemokines, but pathogenic fungi can evade recognition by masking these molecules. Knowledge about which cell wall epitopes are available for immune recognition during human infection could implicate specific ligands and receptors in the symptoms of vaginal candidiasis. To address this important gap, we directly probed the surface of fungi present in fresh vaginal samples obtained both from women with symptomatic Candida vaginitis and from women that are colonized but asymptomatic. We find that the pro-inflammatory cell wall polysaccharide β-glucan is largely masked from immune recognition, especially on yeast. It is only exposed on a small percentage of hyphal cells, where it tends to co-localize with enhanced levels of chitin. Enhanced β-glucan availability is only found in symptomatic patients with strong neutrophil infiltration, implicating neutrophils as a possible driver of these cell wall changes. This is especially interesting because neutrophils were recently shown to be necessary and sufficient to provoke enhanced β-glucan exposure in C. albicans, accompanied by elevated immune responses. Taken together, our data suggest that the architecture of C. albicans cell wall can be altered by environmental stress during vaginal candidiasis.

2018 - Epitope unmasking in vulvovaginal candidiasis is associated with hyphal growth and neutrophilic infiltration. [Poster]
Pericolini, E.; Perito, S.; Castagnoli, A.; Gabrielli, E.; Mencacci, A.; Blasi, E.; Vecchiarelli, A.; Wheeler, R. T.
abstract

Objective: Vaginal candidiasis is a common disorder in women of childbearing age. Since Candida albicans is a normal commensal of the vaginal mucosa, a long-standing question is how the fungus switches from being a harmless commensal to a virulent pathogen. Work with human subjects and in mouse disease models suggests that host inflammatory processes drive the onset of symptomatic infection. The expression of Candida virulence traits, especially those related to hyphal growth, can induce a powerful inflammatory response in the vaginal mucosa that includes strong neutrophil recruitment. Fungal cell wall molecules can also induce inflammation through activation of epithelial and immune receptors that trigger pro-inflammatory cytokines and chemokines, but pathogenic fungi can evade recognition by masking these molecules. Knowledge about which cell wall epitopes are available for immune recognition during human infection could implicate specific ligands and receptors in the symptoms of vaginal candidiasis. Methods: To address this important gap, we have directly probed the surface of fungi present in fresh vaginal samples obtained both from women with symptomatic Candida vaginitis and from women that are colonized but asymptomatic. Results: We find that the pro-inflammatory cell wall polysaccharide β-glucan is largely masked from immune recognition, especially on yeast. It is only exposed on a small percentage of hyphal cells that also tend to have enhanced levels of chitin. Enhanced β-glucan availability is only found in symptomatic patients with strong neutrophil infiltration, implicating neutrophils as the driver of these cell wall changes. Conclusion: This is especially interesting because neutrophils were recently shown to be necessary and sufficient to provoke enhanced β-glucan exposure in C. albicans, which is associated with elevated immune responses, both in vitro and in mice. Taken together, our data suggest that the architecture of C. albicans cell wall is finely regulated during vaginal candidiasis and therefore could be a target for innovative therapies.

2018 - Genomic and phenotypic variation in morphogenetic networks of two Candida albicans isolates subtends their different pathogenic potential [Articolo su rivista]
Cavalieri, D.; Di Paola, M.; Rizzetto, Lisa; Tocci, N.; De Filippo, C.; Lionetti, P.; Ardizzoni, A.; Colombari, B.; Paulone, S.; Gut, I. G.; Luisa, Berná; Gut, M.; Blanc, J.; Kapushesky, M.; Pericolini, E.; Blasi, E.; Peppoloni, S.
abstract

The transition from commensalism to pathogenicity of Candida albicans reflects both the host inability to mount specific immune responses and the microorganism’s dimorphic switch efficiency. In this study, we used whole genome sequencing and microarray analysis to investigate the genomic determinants of the phenotypic changes observed in two C. albicans clinical isolates (YL1 and YQ2). In vitro experiments employing epithelial, microglial, and peripheral blood mononuclear cells were thus used to evaluate C. albicans isolates interaction with first line host defenses, measuring adhesion, susceptibility to phagocytosis, and induction of secretory responses. Moreover, a murine model of peritoneal infection was used to compare the in vivo pathogenic potential of the two isolates. Genome sequence and gene expression analysis of C. albicans YL1 and YQ2 showed significant changes in cellular pathways involved in environmental stress response, adhesion, filamentous growth, invasiveness, and dimorphic transition. This was in accordance with the observed marked phenotypic differences in biofilm production, dimorphic switch efficiency, cell adhesion, invasion, and survival to phagocyte-mediated host defenses. The mutations in key regulators of the hyphal growth pathway in the more virulent strain corresponded to an overall greater number of budding yeast cells released. Compared to YQ2, YL1 consistently showed enhanced pathogenic potential, since in vitro, it was less susceptible to ingestion by phagocytic cells and more efficient in invading epithelial cells, while in vivo YL1 was more effective than YQ2 in recruiting inflammatory cells, eliciting IL-1β response and eluding phagocytic cells. Overall, these results indicate an unexpected isolate-specific variation in pathways important for host invasion and colonization, showing how the genetic background of C. albicans may greatly affect its behavior both in vitro and in vivo. Based on this approach, we propose that the co-occurrence of changes in sequence and expression in genes and pathways driving dimorphic transition and pathogenicity reflects a selective balance between traits favoring dissemination of the pathogen and traits involved in host defense evasion. This study highlights the importance of investigating strain-level, rather than species level, differences, when determining fungal–host interactions and defining commensal or pathogen behavior.

2018 - In vitro effects of commercial mouthwashes on several virulence traits of Candida albicans, viridans streptococci and Enterococcus faecalis colonizing the oral cavity. [Articolo su rivista]
Ardizzoni, A; Pericolini, E; Paulone, S; Orsi, Cf; Castagnoli, A; Oliva, I; Strozzi, E; Blasi, E.
abstract

Oral microbiota consists of hundreds of different species of bacteria, fungi, protozoa and archaea, important for oral health. Oral mycoses, mostly affecting mucosae, are mainly caused by the opportunistic pathogen Candida albicans. They become relevant in denture-wearers elderly people, in diabetic patients, and in immunocompromised individuals. Differently, bacteria are responsible for other pathologies, such as dental caries, gingivitis and periodontitis, which affect even immune-competent individuals. An appropriate oral hygiene can avoid (or at least ameliorate) such pathologies: the regular and correct use of toothbrush, toothpaste and mouthwash helps prevent oral infections. Interestingly, little or no information is available on the effects (if any) of mouthwashes on the composition of oral microbiota in healthy individuals. Therefore, by means of in vitro models, we assessed the effects of alcohol-free commercial mouthwashes, with different composition (4 with chlorhexidine digluconate, 1 with fluoride, 1 with essential oils, 1 with cetylpyridinium chloride and 1 with triclosan), on several virulence traits of C. albicans, and a group of viridans streptococci, commonly colonizing the oral cavity. For the study here described, a reference strain of C. albicans and of streptococci isolates from pharyngeal swabs were used. Chlorhexidine digluconate- and cetylpyridinium chloride-containing mouthwashes were the most effective in impairing C. albicans capacity to adhere to both abiotic and biotic surfaces, to elicit proinflammatory cytokine secretion by oral epithelial cells and to escape intracellular killing by phagocytes. In addition, these same mouthwashes were effective in impairing biofilm formation by a group of viridans streptococci that, notoriously, cooperate with the cariogenic S. mutans, facilitating the establishment of biofilm by the latter. Differently, these mouthwashes were ineffective against other viridans streptococci that are natural competitors of S. mutans. Finally, by an in vitro model of mixed biofilm, we showed that mouthwashes-treated S. salivarius overall failed to impair C. albicans capacity to form a biofilm. In conclusion, the results described here suggest that chlorhexidine- and cetylpyridinium-containing mouthwashes may be effective in regulating microbial homeostasis of the oral cavity, by providing a positive balance for oral health. On the other side, chlorhexidine has several side effects that must be considered when prescribing mouthwashes containing this molecule.

2018 - MESSA A PUNTO DI UN SISTEMA INNOVATIVO PER MONITORARE IN TEMPO REALE LA FORMAZIONE DI BIOFILM DI PSEUDOMONAS AERUGINOSA SU TUBI ENDOTRACHEALI [Poster]
Sala, A.; Pericolini, E.; Colombari, B.; Ferretti, G.; Iseppi, R.; Ardizzoni, A.; Girardis, M.; Castagnoli, A.; Peppoloni, S.; Blasi, E.
abstract

INTRODUZIONE La maggior parte delle infezioni associate all’assistenza sono dovute alla capacità che molti patogeni hanno di produrre biofilm sui diversi dispositivi medici utilizzati. Ad esempio, i pazienti sottoposti a ventilazione assistita sono particolarmente a rischio di sviluppare infezioni respiratorie legate alla formazione di biofilm da parte di Pseudomonas aeruginosa su tubi endotracheali (TE), che evolvono spesso in polmoniti severe. La maggior parte delle attuali conoscenze relative alla formazione di tali biofilm sui dispositivi medici derivano da studi in vitro su micropiastre in polistirene o su materiali plastici. Tuttavia, i risultati che derivano da questi studi non rispecchiano pienamente ciò che accade a livello clinico, poiché la formazione del biofilm è fortemente influenzata da parametri quali la forma e la composizione dei materiali usati per produrre i TE, oltre che da fattori di virulenza microbici. In questo studio abbiamo messo a punto un sistema innovativo in vitro per monitorare in tempo reale la formazione di biofilm di P. aeruginosa su TE. METODI Tramite l’utilizzo di un ceppo batterico geneticamente modificato bioluminescente, è stato possibile monitorare in tempo reale la formazione di biofilm direttamente sui TE, attraverso la valutazione della bioluminescenza (BL). La validità di tale metodo innovativo è stata comparata a metodiche standard (cristal violetto e microscopia confocale). E’ stata inoltre valutata la percentuale di cellule vive/morte nel del biofilm formato sui TE, la produzione di pioverdina e la presenza di DNA extracellulare (in fluorescenza). RISULTATI Dimostriamo che: 1) P. aeruginosa è in grado di produrre biofilm su TE 2) il segnale di BL, emesso solo da cellule vitali, è proporzionale al numero di batteri rilevabili mediante conta delle unità formanti colonia, 3) la quantificazione del segnale consente di misurare il biofilm prodotto tenendo conto non solo del contributo dei fattori microbici ma anche della forma e del materiale di cui sono fatti i TE, 4) è possibile studiare la produzione di fattori di virulenza e l’attività metabolica dei batteri incorporati nel biofilm sui TE. CONCLUSIONI Il modello descritto è ad oggi il sistema in vitro che mima più da vicino quello che può accadere nei pazienti con infezioni TE-associate. Per tale motivo potrà avere un’immediata applicazione per lo screening e la valutazione dell’attività anti- biofilm di nuovi farmaci come anche di nuovi materiali per la produzione di dispositivi medici.

2018 - Real-time monitoring of Pseudomonas aeruginosa biofilm formation on endotracheal tubes in vitro [Articolo su rivista]
Pericolini, E.; Colombari, B.; Ferretti, Gianmarco; Iseppi, R.; Ardizzoni, A.; Girardis, M.; Sala, Arianna; Peppoloni, S.; Blasi, Elisabetta
abstract

BACKGROUND: Pseudomonas aeruginosa is an opportunistic bacterial pathogen responsible for both acute and chronic infections in humans. In particular, its ability to form biofilm, on biotic and abiotic surfaces, makes it particularly resistant to host's immune defenses and current antibiotic therapies as well. Innovative antimicrobial materials, like hydrogel, silver salts or nanoparticles have been used to cover new generation catheters with promising results. Nevertheless, biofilm remains a major health problem. For instance, biofilm produced onto endotracheal tubes (ETT) of ventilated patients plays a relevant role in the onset of ventilation-associated pneumonia. Most of our knowledge on Pseudomonas aeruginosa biofilm derives from in vitro studies carried out on abiotic surfaces, such as polystyrene microplates or plastic materials used for ETT manufacturing. However, these approaches often provide underestimated results since other parameters, in addition to bacterial features (i.e. shape and material composition of ETT) might strongly influence biofilm formation. RESULTS: We used an already established biofilm development assay on medically-relevant foreign devices (CVC catheters) by a stably transformed bioluminescent (BLI)-Pseudomonas aeruginosa strain, in order to follow up biofilm formation on ETT by bioluminescence detection. Our results demonstrated that it is possible: i) to monitor BLI-Pseudomonas aeruginosa biofilm development on ETT pieces in real-time, ii) to evaluate the three-dimensional structure of biofilm directly on ETT, iii) to assess metabolic behavior and the production of microbial virulence traits of bacteria embedded on ETT-biofilm. CONCLUSIONS: Overall, we were able to standardize a rapid and easy-to-perform in vitro model for real-time monitoring Pseudomonas aeruginosa biofilm formation directly onto ETT pieces, taking into account not only microbial factors, but also ETT shape and material. Our study provides a rapid method for future screening and validation of novel antimicrobial drugs as well as for the evaluation of novel biomaterials employed in the production of new classes of ETT.

2018 - Saccharomyces cerevisiae-based probiotic as novel anti-fungal and anti-inflammatory agent for therapy of vaginal candidiasis [Articolo su rivista]
Gabrielli, E.; Pericolini, E.; Ballet, N.; Roselletti, E.; Sabbatini, S.; Mosci, P.; Cayzeele Decherf, A.; Pélerin, F.; Perito, S.; Jüsten, P.; Vecchiarelli, A.
abstract

Previously we demonstrated that the treatment with live Saccharomyces cerevisiae exerts beneficial therapeutic effects against vaginal candidiasis. Here, we address potential mechanisms particularly examining the probiotic capacity to modulate both fungus and host-related factors. We show that the S. cerevisiae-based probiotic markedly affects the expression of virulence traits of Candida albicans such as aspartyl proteinases (SAPs) as well as hyphae-associated proteins Hwp1 and Ece1 in the vaginal cavity. On the host side, the probiotic suppression of the influx of neutrophils caused by the fungus into the vaginas of the mice is likely related to: (1) lower production of interleukin-8; and (2) inhibition of SAPs expression. However, these neutrophils displayed reactive oxygen species hyperproduction and increased killing activity as compared to the neutrophils of placebo-treated mice. There was no evidence of any cytotoxic effect by the probiotic, either when used in vivo on vaginal epithelial cell and organ architecture, or in in vitro in human vaginal epithelium. Inactivated yeast cells did not affect any of the factors above. In summary, the data suggest that the beneficial effect exerted by this S. cerevisiae-based probiotic is the result of its interference with the expression of fungus virulence factors coupled with the modulation of the inflammatory response of the host.

2017 - A synthetic killer peptide impairs Candida albicans biofilm formation and persistence in vitro [Abstract in Atti di Convegno]
Paulone, Simona; Ardizzoni, Andrea; Tavanti, Arianna; Piccinelli, Serena; Rizzato, Cosmeri; Lupetti, Antonella; Colombari, Bruna; Pericolini, Eva; Polonelli, Luciano; Magliani, Walter; Conti, Stefania; Posteraro, Brunella; Cermelli, Claudio; Blasi, Elisabetta; Peppoloni, Samuele
abstract

Introduction. Candida spp. colonize human skin and mucosae of healthy subjects, behaving as harmless commensals. Nevertheless, in susceptible patients (with medical devices or immunosuppressed), they behave as opportunistic pathogens also because of their capacity to form biofilms on mucosae or medical devices. Indeed, when embedded in a biofilm, Candida cells become more resistant to common disinfectants and most antifungal, including azoles. Thus, there is an urgent need to identify novel therapeutic molecules. Recently, several antibody-derived peptides have been shown to have antimicrobial, antiviral, immunomodulatory and antitumor activity both in vitro and in vivo. The aim of this study was to investigate the effect of a synthetic killer peptide (KP), on the formation and persistence of Candida biofilm. Materials and Methods. The reference strain C. albicans SC5314, two C. albicans fluconazole-resistant and two C. albicans fluconazole-susceptible isolates were employed. Together with a scrambled peptide, used as negative control, the KP (AKVTMTCSAS) was tested against Candida biofilm at different stage of development, by microscopy, crystal violet and tetrazolium salt reduction assays. Results. The KP strongly influenced the capacity of Candida albicans to form biofilm and significantly impairs preformed mature biofilm. KP treatment resulted in an increase in Candida oxidative stress response and membrane permeability; moreover, biofilm-related genes expression was markedly reduced. Similar inhibitory effects were observed against all the strains tested, irrespective of their resistance or susceptibility to the drug. Interestingly, the KP-mediated inhibitory effect was shown even against a catheter-associated C. albicans biofilm. Conclusions. These results provide the first evidence of the effects of KP against C. albicans biofilm, suggesting that this peptide may represent a novel potential molecule for treatment and prevention of biofilm-related C. albicans infections.

2017 - Chronic vaginal candidiasis is achievable in outbred CD-1 mice [Articolo su rivista]
Gabrielli, E.; Roselletti, E.; Pericolini, Eva; Sabbatini, S.; Vecchiarelli, A.; Cassone, A.
abstract

not available

2017 - Effects of different mouthwashes on biofilm formation by oral streptococci [Poster]
Ardizzoni, Andrea; Pericolini, Eva; Paulone, Simona; Castagnoli, Anna; Strozzi, Elena; Blasi, Elisabetta
abstract

Introduction. Oral microbiota is an extremely complex and dynamic system including bacteria, archea and fungi. Different locations of the oral cavity host a large variety of microbiota mostly organized as biofilm, characterized by spatial and temporal differences in their distribution. Several bacteria belonging to the genus Streptococcus act as early colonizers, binding to the adhesive pellicle and providing a substrate for the attachment of late colonizers bacteria. The latter, in turn, may be responsible of severe pathologies, such as carious lesions, gingivitis and periodontal lesions. Therefore, we evaluated the effects of commercial mouthwashes (MoWs) on biofilm (BF) formation/persistence of early colonizers Streptococci. Materials and methods. Fourteen isolates belonging to 5 different species of oral streptococci (S. salivarius, S. mitis/oralis, S. sanguinis, S. parasanguinis, S. vestibularis) and 3 isolates of Enterococcus faecalis were obtained from pharyngeal swabs and employed for the present study. All the bacteria were incubated for 1 minute with 6 commercial MoWs, 4 with (MoWs 1, 2, 3 and 7) and 2 without (MoWs 4 and 5) chlorhexidine digluconate (CHX), which is known to exert antibacterial and antifungal activity. Control groups of each strain were treated for 1 minute with PBS and used as negative controls. After treatment with MoWs or PBS, bacteria were seeded in 96-well plates and allowed to form BF for up to 48 hours. BF formation was assessed by crystal violet assay. The capacity to form BF was expressed as the optical density (OD) percentage of each MoW-treated strain, as compared to the OD of the PBS-treated strain, which was considered as 100%. Statistical analyses were carried out by one-way ANOVA test with Bonferroni post-hoc test. Results. CHX-containing MoWs were capable to inhibit BF formation in most of the cases. In detail, CHX-containing MoWs significantly reduced BF formation by all the S. salivarius and E. faecalis isolates, while only several S. parasanguinis, S. mitis/oralis, S. sanguinis and S. vestibularis isolates were affected. One of the 2 CHX-free MoWs was capable to significantly inhibit BF formation by S. salivarius and S. vestibularis, whereas the remaining CHX-free MoW did not prove to be effective in impairing BF formation by any of the bacterial isolates assessed. Discussion and conclusions. Similarly to Candida albicans, BF formation by oral streptococci is affected by MoWs, provided that they include CHX in their formulation. Since the streptococci used in the present study act as early colonizers in the multispecies microbial BF of the dental surface, special attention should be used when choosing MoWs for prevention and/or treatment of oral pathologies of microbial origin.

2017 - Effects of different mouthwashes on Candida albicans adhesion, susceptibility to phagocytic cells and capacity to elicit pro-inflammatory cytokine response [Poster]
Ardizzoni, Andrea; Pericolini, Eva; Paulone, Simona; Oliva, Ilaria; Orsi, Carlotta Francesca; Blasi, Elisabetta
abstract

Introduction. Oral candidiasis is a frequent opportunistic fungal infection, occurring especially in susceptible individuals. This pathology, mainly associated with Candida albicans species, may be prevented by a good oral hygiene, including the daily use of toothbrush and mouthwashes (MoWs). Among several virulence factors, C. albicans has the ability to adhere to epithelial surfaces, to avoid phagocytosis and/or intracellular killing and to elicit proinflammatory cytokines production. We have previously demonstrated that both C. albicans hyphal development and biofilm formation/persistence are affected by MoWs, provided that they contain chlorhexidine digluconate. Therefore, in this study we aim to expand our knowledge on MoWs effects by investigating the behaviour of MoWs-treated C. albicans, in terms of adhesion to both abiotic and biotic surfaces, susceptibility to phagocytosis and capacity to elicit pro-inflammatory immune responses. Materials and methods. C. albicans SC5314 and 6 commercial MoWs have been employed: 4 with and 2 without chlorhexidine digluconate (CHX), a component known to have antibacterial and antifungal activity. Adhesion was assessed by a bioluminescent strain of C. albicans SC5314; MoWs-treated and PBS-treated fungal cells were incubated in 96-well plates containing or not a monolayer of TR-146 oral epithelial cell line; after 60 min, plates were washed and the residual bioluminescent signal recorded. Susceptibility to phagocytosis was assessed by exposing MoWs-treated and PBS-treated C. albicans to phagocytic cell line BV2 (effector:target=1:2). Following 24 hours incubation of TR-146 cells with MoWs-treated and PBS-treated C. albicans, cytokine levels in supernatants were measured. Results. Adhesion of MoWs-treated C. albicans to abiotic surfaces was significantly lower than PBS-treated Candida. Adhesion of MoWs-treated C. albicans to TR-146 cells was significantly lower than PBS-treated Candida, in all but MoW 4. No differences could be highlighted in terms of susceptibility to phagocytosis (percent phagocytic cells and phagocytosis index) between MoWs-treated and PBS-treated Candida. On the contrary, significantly higher acidic phagolysosomes percentages were recorded from Candida treated with 4 out of 6 MoWs, with respect to PBS-treated fungi. Finally, Candida pretreatment with 4 out of 6 MoWs and 5 out of 6 MoWs impaired the production of IL-1alpha and IL-1beta respectively. Discussion and conclusions. C. albicans adhesion, susceptibility to phagocytosis and capacity to elicit pro-inflammatory cytokine response are affected by MoWs, especially those containing CHX. Thus, special attention should be used when choosing MoWs whether prevention and/or treatment of Candida-associated oral pathologies was intended.

2017 - Inflammatory response during human vaginal infection with Candida albicans [Poster]
Roselletti, Elena; Perito, Stefano; Gabrielli, Elena; Pericolini, Eva; Sabbatini, Samuele; Vecchiarelli, Anna
abstract

Introduction. The microbiological, pathological and clinical factors determining vaginal candidiasis and recurrent vaginal candidiasis have long been studied, particularly using rodent models. The validity of which for understanding the pathogenesis of disease in women has been questioned. The most prevalent agent is critically determined by activation of microbial and host factors leading to persistent vaginal inflammation coupled to the inability of the inflammatory cells to resolve the fungal infection. Here we studied the activation of inflammasome complex neutrophil-recruiting and activating cytokines in the vaginal secretion of women with clinically established vaginal candidiasis. Materials and methods. In human vaginal samples positive for C. albicans with vaginal candidiasis (n=20) and carriage (n=15), infiltration of neutrophils, inflammatory mediators such as IL-8 and IL-1β, activation of inflammasome complex and expression of aspartyl proteases (SAPs) were examined. Results. In vaginal swabs of patients with vaginal candidiasis we found: i) consistent recruitment of neutrophils; ii) appreciable level of IL-8 and IL-1β; iii) activation of inflammasome complex; iv) consistent expression of SAP2, SAP5 and SAP6. Conclusions. These results show that immunopathogenesis of vaginal candidiasis is mediated by local recruitment of neutrophils, inflammatory cytokines secretion and inflammasome activation that mirror the upregulation of SAP2, SAP5 and SAP6 gene expression.

2017 - NLRP3 inflammasome is a key player in human vulvovaginal disease caused by Candida albicans [Articolo su rivista]
Roselletti, Elena; Perito, Stefano; Gabrielli, Elena; Mencacci, Antonella; Pericolini, Eva; Sabbatini, Samuele; Cassone, Antonio; Vecchiarelli, Anna
abstract

The expression of host inflammatory and Candida albicans putative virulence factors was studied in women with vulvovaginal candidiasis (VVC; twenty) or colonized by the fungus but asymptomatic (carriers; fifteen) or non-colonized asymptomatic (ten subjects). Overexpression of genes encoding NLRP3 and caspase-1 inflammasome components sharply differentiated VVC patients from asymptomatic colonized or non-colonized women. Inflammasome expression was coupled with neutrophils recruitment in the vagina of VVC women and IL-1β and IL-8 production. Both cytokines were present, though to a lower concentration, also in the vaginal fluid of colonized and non-colonized women. Secretory aspartyl proteinases (SAPs) and hyphae associated genes HWP1 and ECE1 were upregulated in VVC but with some differences among infected women. The most overexpressed SAP gene was SAP2, that correlated with neutrophils accumulation. Our data provide clinical evidence that the intracytoplasmic activation of NLRP3 inflammasome complex plays a critical, pathogenesis relevant role in human VVC.

2017 - Role of the glucocorticoid-induced leucine zipper gene in dexamethasone-induced inhibition of mouse neutrophil migration via control of annexin A1 expression [Articolo su rivista]
Ricci, E; Ronchetti, S; Pericolini, Eva; Gabrielli, E; Cari, L; Gentili, M; Roselletti, E; Migliorati, G; Vecchiarelli, A; Riccardi, C.
abstract

The glucocorticoid-induced leucine zipper gene (GILZ) is a pivotal mediator of the anti-inflammatory effects of glucocorticoids (GCs) that are known to regulate the function of both adaptive and innate immunity cells. Our aim was to investigate the role of GILZ in GC-induced inhibition of neutrophil migration, as this role has not been investigated before. We found that GILZ expression was induced by dexamethasone (DEX), a synthetic GC, in neutrophils, and that it regulated migration of these cells into inflamed tissues under DEX treatment. Of note, inhibition of neutrophil migration was not observed in GILZ knockout mice with peritonitis that were treated by DEX. This was because DEX was unable to up-regulate annexin A1 (Anxa1) expression in the absence of GILZ. Furthermore, we showed that GILZ mediates Anxa1 induction by GC by transactivating Anxa1 expression at the promoter level via binding with the transcription factor, PU.1. The present findings shed light on the role of GILZ in the mechanism of induction of Anxa1 by GCs. As Anxa1 is an important protein for the resolution of inflammatory response, GILZ may represent a new pharmacologic target for treatment of inflammatory diseases.-Ricci, E., Ronchetti, S., Pericolini, E., Gabrielli, E., Cari, L., Gentili, M., Roselletti, E., Migliorati, G., Vecchiarelli, A., Riccardi, C. Role of the glucocorticoid-induced leucine zipper gene in dexamethasone-induced inhibition of mouse neutrophil migration via control of annexin A1 expression.

2017 - The synthetic killer peptide KP impairs Candida albicans biofilm in vitro [Articolo su rivista]
Paulone, Simona; Ardizzoni, Andrea; Tavanti, Arianna; Piccinelli, Serena; Rizzato, Cosmeri; Lupetti, Antonella; Colombari, Bruna; Pericolini, Eva; Polonelli, Luciano; Magliani, Walter; Conti, Stefania; Posteraro, Brunella; Cermelli, Claudio; Blasi, Elisabetta; Peppoloni, Samuele
abstract

Candida albicans is a commensal organism, commonly inhabiting mucosal surfaces of healthy individuals, as a part of the resident microbiota. However, in susceptible hosts, especially hospitalized and/or immunocompromised patients, it may cause a wide range of infections. The presence of abiotic substrates, such as central venous or urinary catheters, provides an additional niche for Candida attachment and persistence, particularly via biofilm development. Furthermore, Candida biofilm is poorly susceptible to most antifungals, including azoles. Here we investigated the effects of a synthetic killer peptide (KP), known to be active in vitro, ex vivo and/or in vivo against different pathogens, on C. albicans biofilm. Together with a scrambled peptide used as a negative control, KP was tested against Candida biofilm at different stages of development. A reference strain, two fluconazole-resistant and two fluconazole-susceptible C. albicans clinical isolates were used. KP-induced C. albicans oxidative stress response and membrane permeability were also analysed. Moreover, the effect of KP on transcriptional profiles of C. albicans genes involved in different stages of biofilm development, such as cell adhesion, hyphal development and extracellular matrix production, was evaluated. Our results clearly show that the treatment with KP strongly affected the capacity of C. albicans to form biofilm and significantly impairs preformed mature biofilm. KP treatment resulted in an increase in C. albicans oxidative stress response and membrane permeability; also, biofilm-related genes expression was significantly reduced. Comparable inhibitory effects were observed in all the strains employed, irrespective of their resistance or susceptibility to fluconazole. Finally, KP-mediated inhibitory effects were observed also against a catheter-associated C. albicans biofilm. This study provides the first evidence on the KP effectiveness against C. albicans biofilm, suggesting that KP may be considered as a potential novel tool for treatment and prevention of biofilm-related C. albicans infections.

2017 - Therapeutic activity of a Saccharomyces cerevisiae-based probiotic and inactivated whole yeast on vaginal candidiasis [Articolo su rivista]
Pericolini, Eva; Gabrielli, Elena; Ballet, Nathalie; Sabbatini, Samuele; Roselletti, Elena; Decherf Amélie, Cayzeele; Pélerin, Fanny; Luciano, Eugenio; Perito, Stefano; Jüsten, Peter; Vecchiarelli, Anna
abstract

Vulvovaginal candidiasis is the most prevalent vaginal infection worldwide and Candida albicans is its major agent. Vulvovaginal candidiasis is characterized by disruption of the vaginal microbiota composition, as happens following large spectrum antibiotic usage. Recent studies support the effectiveness of oral and local probiotic treatment for prevention of recurrent vulvovaginal candidiasis. Saccharomyces cerevisiae is a safe yeast used as, or for, the production of ingredients for human nutrition and health. Here, we demonstrate that vaginal administration of probiotic Saccharomyces cerevisiae live yeast (GI) and, in part, inactivated whole yeast Saccharomyces cerevisiae (IY), used as post-challenge therapeutics, was able to positively influence the course of vaginal candidiasis by accelerating the clearance of the fungus. This effect was likely due to multiple interactions of Saccharomyces cerevisiae with Candida albicans. Both live and inactivated yeasts induced coaggregation of Candida and consequently inhibited its adherence to epithelial cells. However, only the probiotic yeast was able to suppress some major virulence factors of Candida albicans such as the ability to switch from yeast to mycelial form and the capacity to express several aspartyl proteases. The effectiveness of live yeast was higher than that of inactivated whole yeast suggesting that the synergy between mechanical effects and biological effects were dominant over purely mechanical effects. The protection of epithelial cells to Candida-induced damage was also observed. Overall, our data show for the first time that Saccharomyces cerevisiae-based ingredients, particularly the living cells, can exert beneficial therapeutic effects on a widespread vaginal mucosal infection.

2016 - Gray phenotype: enhanced fitness strategy for Candida dubliniensis [Articolo su rivista]
Pericolini, Eva; Elena, Gabrielli
abstract

In this study Yue H. et al described for the first time the gray phenotype and tristable white-gray-opaque transitions in Candida dubliniensis. Here we discuss some intriguing aspects related to this virulence trait of Candida dubliniensis in comparison to Candida albicans and within the wider and complex phenotypic switch phenomenon so typical of the virulence program of these opportunistic pathogenic fungi. In particular, the relationship between the presence of gray phenotype and prevalence of Candida dubliniensis in the oral cavity of HIV-positive subjects is pointed out.

2016 - In vivo induction of neutrophils chemotaxis by secretory aspartyl proteinases of Candida albicans [Articolo su rivista]
Gabrielli, E.; Sabbatini, S.; Roselletti, E.; Kasper, L.; Perito, S.; Hube, B.; Cassone, A.; Vecchiarelli, A.; Pericolini, Eva
abstract

Secretory aspartyl proteinases (Saps) of Candida albicans are key virulence traits which cause inflammasome-dependent, aseptic inflammation in a mouse model of vaginitis. In this paper, neutrophil migration in response to Sap2, Sap6 and chemo-attractive products released from Sap-treated vaginal epithelium was measured in vitro, ex vivo and in vivo. Our results show that Sap2 and Sap6 induce neutrophil migration and production of potent chemoattractive chemokines such as IL-8 and MIP-2 by vaginal epithelial cells. Our data suggest that at least part of MIP-2 production depends upon IL-1β activity. The vaginal fluid of Candida-infected mice contained a heat-labile inhibitor of neutrophil candidacidal activity that was absent from the vaginal fluid of Sap-treated mice. Overall, our data provide additional information on the capacity of C. albicans Saps to cause aseptic vaginal inflammation and highlight the potential role of some chemokines released from vaginal epithelial cells in this phenomenon.

2016 - Peptides derived from the proteolysis of serum proteins and immunoglobulins induce IL-1beta production independently of inflammasome activation [Poster]
Roselletti, E.; Pericolini, Eva; Gabrielli, E.; Sabbatini, S.; Conti, S.; Ciociola, T.; Polonelli, L.; Vecchiarelli, A.
abstract

2016 - Secretory aspartyl proteinases of Candida albicans induce neutrophils chemotaxis [Poster]
Sabbatini, S.; Gabrielli, E.; Roselletti, E.; Perito, S.; Cassone, A.; Vecchiarelli, A; Pericolini, Eva
abstract

2016 - Therapeutic activity of Saccharomyces cerevisiae-based probiotic and inactivated whole yeast on vaginal candidiasis [Poster]
Pericolini, Eva; Gabrielli, E.; Ballet, N.; Sabbatini, S.; Roselletti, E.; Cayzeele Decherf, A.; Pèlerin, F.; Perito, S.; Justen, P.; Vecchiarelli, A.
abstract

2015 - A role for secretory aspartyl proteinase of Candida albicans in mouse vaginal inflammation and vaginal candidiasis [Abstract in Atti di Convegno]
Pericolini, Eva; Gabrielli, Elena; Roselletti, Elena; Luciano, Eugenio; Sabbatini, Samuele; Moser, Christian; Vecchiarelli, Anna; Cassone, Antonio
abstract

We sought for a role of secretory aspartyl proteinase 2, (Sap2), a major virulence trait of Candida albicans, in a murine experimental model of vaginal candidiasis. Injection of enzymatically active, full-length Sap2 into the mouse vagina caused local PMN (neutrophil) influx and accumulation of the inflammasome-dependent interleukin (IL)-1beta but not of inflammasome-independent TNFalpha. No inflammatory response was detected following treatment with a N-terminus-truncated, enzymatically-inactive tSap2. Anti-Sap2 antibodies, in particular Fab from a human combinatorial antibody library inhibited or abolished the inflammatory response, provided they were able, like Pepstatin A, a known Sap inhibitor, to inhibit Sap2 enzyme activity. The same antibodies and Pepstatin A also caused strong inhibition of neutrophil influx and cytokine production early during experimental vaginal infection by Candida albicans. Sap2 induced expression of activated caspase-1 in murine vaginal epithelial cells as well as in a human vaginal epithelial cell line and caspase-1 inhibition down-regulated IL-1beta and IL-18 production by the vaginal epithelial cells. Overall, the data demonstrate that Sap2 is a critical determinant of the acute inflammatory response by the epithelial cells in vaginal candidiasis, and support the notion that vaccine-induced or passively administered anti-Sap antibodies could control vaginal disease.

2015 - A role for secretory aspartyl proteinases of Candida albicans in mouse vaginal inflammation and vaginal candidiasis [Poster]
Pericolini, Eva; Gabrielli, E.; Roselletti, E.; Sabbatini, S.; Luciano, E.; Vecchiarelli, A.; Cassone, A.
abstract

2015 - Aspartyl proteinases of Candida albicans induce caspase-11: implication in promoting inflammatory response [Poster]
Gabrielli, Elena; Pericolini, Eva; Luciano, Eugenio; Sabbatini, Samuele; Roselletti, Elena; Perito, Stefano; Kasper, Lydia; Hube, Bernhard; Vecchiarelli, Anna
abstract

We recently demonstrated that the secreted aspartyl proteinases (Saps), Sap2 and Sap6, of Candida albicans have the potential to induce the canonical activation of NLRP3-inflammasome leading to the secretion of IL-1beta and IL-18 via caspase-1 activation. We also observed that the activation of caspase-1 is partially independent from the NLRP3 activation pathway. In this study, we examined whether Sap2 and Sap6 are also able to activate the noncanonical inflammasome pathway in murine macrophages. Our data show that both, Sap2 and Sap6, can activate caspase-11 through type I IFN production. Caspase-11 concurs to activate caspase-1 with subsequent increase of IL-1beta secretion. Endocytosis and internalization of Saps are required for the induction of type I IFN production, that is essential for induction of noncanonical inflammasome activation. Our study indicates a sophisticated interplay between caspase-1 and caspase-11 that connects canonical and noncanonical pathways of inflammasome activation in response to Candida albicans Saps.

2015 - Aspartyl proteinases of Candida albicans induce caspase-11: implication in promoting inflammatory response. [Abstract in Atti di Convegno]
Gabrielli, E.; Pericolini, Eva; Luciano, E.; Sabbatini, S.; Roselletti, E.; Perito, S.; Vecchiarelli, A.
abstract

2015 - Comparison between bioluminescence imaging technique and CFU count for the study of oropharyngeal candidiasis in mice [Articolo su rivista]
Gabrielli, E; Roselletti, E; Luciano, E; Sabbatini, S; Mosci, P; Pericolini, Eva
abstract

We recently described a bioluminescence in vivo imaging technique, representing a powerful tool to test the real-time progression of oropharyngeal candidiasis, hence potentially useful to evaluate the efficacy of antifungal therapies. In this study, the in vivo imaging technique was compared with CFU measurement of target organs (tongue, esophagus and stomach) for monitoring and quantifying oropharyngeal candidiasis. We have correlated these two analytical methods at different times post-infection using engineered, luminescent Candida albicans in mice rendered susceptible to oral candidiasis by cortisone-acetate. Scatter plots, Pearson correlation and Student's t test were used to compare the methods. We observed that the bioluminescence in vivo imaging technique was more reliable than CFU counts in detecting early infection of, and its extent in, the oral cavity of the mouse. This was also evident following the introduction of a variable such as treatment with fluconazole. The results described in this study could validate the bioluminescence in vivo imaging technique as a method to monitor and quantify oropharyngeal candidiasis and to assess early discovery of active compounds in vivo.

2015 - Experimental models of vaginal candidiasis and inflammation [Articolo su rivista]
Vecchiarelli, Anna; Gabrielli, Elena; Pericolini, Eva
abstract

There are at least two factors that play a key role in vulvovaginal candidiasis: the massive infiltration of neutrophils and extensive presence of hyphae in the vagina. Thus it is conceivable that the dynamic of inflammatory response starts with adherence of Candida to epithelial cells followed by release of S100A and inflammasome activation with IL-1β production and subsequent further increase of PMN migration and activation.

2015 - Induction of caspase-11 by aspartyl proteinases of Candida albicans and implication in promoting inflammatory response [Articolo su rivista]
Gabrielli, E; Pericolini, Eva; Luciano, E; Sabbatini, S; Roselletti, E; Perito, S; Kasper, L; Hube, B; Vecchiarelli, A.
abstract

We recently demonstrated that the secreted aspartyl proteinases (Saps), Sap2 and Sap6, of Candida albicans have the potential to induce the canonical activation of NLRP3-inflammasome leading to the secretion of IL-1β and IL-18 via caspase-1 activation. We also observed that the activation of caspase-1 is partially independent from the NLRP3 activation pathway. In this study, we examined whether Sap2 and Sap6 are also able to activate the noncanonical inflammasome pathway in murine macrophages. Our data show that both, Sap2 and Sap6, can activate caspase-11 through type I IFN production. Caspase-11 concurs to activate caspase-1 with subsequent increase of IL-1β secretion. Endocytosis and internalization of Saps are required for the induction of type I IFN production, that is essential for induction of noncanonical inflammasome activation. Our study indicates a sophisticated interplay between caspase-1 and caspase-11 that connects canonical and noncanonical pathways of inflammasome activation in response to C. albicans Saps.

2015 - Luciferase in vivo assay validation for oropharyngeal candidiasis in mice [Poster]
Roselletti, E.; Sabbatini, S.; Gabrielli, E.; Luciano, E.; Vecchiarelli, A.; Pericolini, Eva
abstract

2015 - Secretory Aspartyl Proteinases Cause Vaginitis and Can Mediate Vaginitis Caused by Candida albicans in Mice [Articolo su rivista]
Pericolini, Eva; Gabrielli, Elena; Amacker, Mario; Kasper, Lydia; Roselletti, Elena; Luciano, Eugenio; Sabbatini, Samuele; Kaeser, Matthias; Moser, Christian; Hube, Bernhard; Vecchiarelli, Anna; Cassone, Antonio
abstract

Vaginal inflammation (vaginitis) is the most common disease caused by the human-pathogenic fungus Candida albicans. Secretory aspartyl proteinases (Sap) are major virulence traits of C. albicans that have been suggested to play a role in vaginitis. To dissect the mechanisms by which Sap play this role, Sap2, a dominantly expressed member of the Sap family and a putative constituent of an anti-Candida vaccine, was used. Injection of full-length Sap2 into the mouse vagina caused local neutrophil influx and accumulation of the inflammasome-dependent interleukin-1β (IL-1β) but not of inflammasome-independent tumor necrosis factor alpha. Sap2 could be replaced by other Sap, while no inflammation was induced by the vaccine antigen, the N-terminal-truncated, enzymatically inactive tSap2. Anti-Sap2 antibodies, in particular Fab from a human combinatorial antibody library, inhibited or abolished the inflammatory response, provided the antibodies were able, like the Sap inhibitor Pepstatin A, to inhibit Sap enzyme activity. The same antibodies and Pepstatin A also inhibited neutrophil influx and cytokine production stimulated by C. albicans intravaginal injection, and a mutant strain lacking SAP1, SAP2, and SAP3 was unable to cause vaginal inflammation. Sap2 induced expression of activated caspase-1 in murine and human vaginal epithelial cells. Caspase-1 inhibition downregulated IL-1β and IL-18 production by vaginal epithelial cells, and blockade of the IL-1β receptor strongly reduced neutrophil influx. Overall, the data suggest that some Sap, particularly Sap2, are proinflammatory proteins in vivo and can mediate the inflammasome-dependent, acute inflammatory response of vaginal epithelial cells to C. albicans. These findings support the notion that vaccine-induced or passively administered anti-Sap antibodies could contribute to control vaginitis.

2014 - Evaluation of Anti-Candida Activity of Vitis vinifera L. Seed Extracts Obtained from Wine and Table Cultivars [Articolo su rivista]
Giovanna, Simonetti; Anna Rita, Santamaria; Felicia Diodata, D’Auria; Nadiamulinacci, ; Marzia, Innocenti; Francesca, Cecchini; Pericolini, Eva; Elena, Gabrielli; Simona, Panella; Donato, Antonacci; Anna Teresa, Palamara; Anna, Vecchiarelli; Gabriella, Pasqua
abstract

For the first time, grape seed extracts (GSEs), obtained from wine and table cultivars of Vitis vinifera L., cultured in experimental fields of Lazio and Puglia regions of Italy and grown in different agronomic conditions, have been tested on 43 Candida species strains.We demonstrated a significant correlation between the content of the flavan-3-ols in GSEs extracts, with a polymerization degree ≥4, and anti-Candida activity.Moreover, we demonstrated thatGSEs, obtained from plants cultured with reduced irrigation, showed a content of polymeric flavan-3-ols >250mg/gwith geometric mean MICvalues between 5.7 and 20.2 mg/L against Candida albicans reference strains. GSE, showing 573mg/g of polymeric flavan-3-ols, has been tested in an experimental murine model of vaginal candidiasis by using noninvasive in vivo imaging technique. The results pointed out a significant inhibition of Candida albicans load 5 days after challenge.These findings indicate that GSEs with high content of polymeric flavan-3-ols can be used in mucosal infection as vaginal candidiasis.

2014 - Functional Improvement of Regulatory T Cells From Rheumatoid Arthritis Subjects Induced by Capsular Polysaccharide Glucuronoxylomannogalactan [Articolo su rivista]
Pericolini, Eva; Elena, Gabrielli; Alessia, Alunno; Elena Bartoloni, Bocci; Stefano, Perito; Siu Kei, Chow; Elio, Cenci; Arturo, Casadevall; Roberto, Gerli; Anna, Vecchiarelli
abstract

Objective: Regulatory T cells (Treg) play a critical role in the prevention of autoimmunity, and the suppressive activity of these cells is impaired in rheumatoid arthritis (RA). The aim of the present study was to investigate function and properties of Treg of RA patients in response to purified polysaccharide glucuronoxylomannogalactan (GXMGal). Methods: Flow cytometry and western blot analysis were used to investigate the frequency, function and properties of Treg cells. Results: GXMGal was able to: i) induce strong increase of FOXP3 on CD4+ T cells without affecting the number of CD4+CD25+FOXP3+ Treg cells with parallel increase in the percentage of non-conventional CD4+CD25-FOXP3+ Treg cells; ii) increase intracellular levels of TGF-beta1 in CD4+CD25-FOXP3+ Treg cells and of IL-10 in both CD4+CD25+FOXP3+ and CD4+CD25-FOXP3+ Treg cells; iii) enhance the suppressive activity of CD4+CD25+FOXP3+ and CD4+CD25-FOXP3+ Treg cells in terms of inhibition of effector T cell activity and increased secretion of IL-10; iv) decrease Th1 response as demonstrated by inhibition of T-bet activation and down-regulation of IFN-gamma and IL-12p70 production; v) decrease Th17 differentiation by down-regulating pSTAT3 activation and IL-17A, IL-23, IL-21, IL-22 and IL-6 production. Conclusion: These data show that GXMGal improves Treg functions and increases the number and function of CD4+CD25-FOXP3+ Treg cells of RA patients. It is suggested that GXMGal may be potentially useful for restoring impaired Treg functions in autoimmune disorders and for developing Treg cell-based strategies for the treatment of these diseases.

2014 - Involvement of IL-17A in preventing the development of deep-seated candidiasis from oropharyngeal infection [Poster]
Elena, Gabrielli; Paolo, Mosci; Eugenio, Luciano; Stefano, Perito; Antonio, Cassone; Pericolini, Eva; Anna, Vecchiarelli
abstract

In this study we show that Il17a-/- mice develop invasive candidiasis from oropharyngeal infection whereas WT mice do not. By using an established murine model of oral candidiasis we document the spatial and temporal progression of fungal infection. The histological analysis of tissues in Il17a-/- mice showed massive infiltration of the fungus in the stomach and alterations of the gastrointestinal tract segments. Both increased permeability and mucosal ulcerations of the intestinal barrier are seen to favour Candida albicans dissemination which was quantified both in kidney and liver where typical candidal abscesses were detected. Neutrophil from Il17a-/- were as capable of phagocytosing the fungus comparable to that of WT mice, however, they showed decreased candidacidal ability. Our data implies that IL-17A is crucial for preventing the passage from mucosal to disseminated candidiasis. As such, our model may be suitable to study the mechanisms favouring Candida albicans translocation to internal organs.

2014 - Involvement of IL-17A in preventing the development of deep-seated candidiasis from oropharyngeal infection [Articolo su rivista]
Paolo, Mosci; Elena, Gabrielli; Eugenio, Luciano; Stefano, Perito; Antonio, Cassone; Pericolini, Eva; Anna, Vecchiarelli
abstract

In this study we show that corticosteroid-treated Il17a-/- mice develop invasive candidiasis from oropharyngeal infection whereas WT mice do not. By using an established murine model of oral candidiasis we document the spatial and temporal progression of fungal infection. The histological analysis of tissues in Il17a-/- mice showed massive infiltration of the fungus in the stomach and alterations of the gastrointestinal tract segments. Both increased permeability and mucosal ulcerations of the intestinal barrier are seen to favour Candida albicans dissemination which was quantified both in kidney and liver where typical candidal abscesses were detected. Neutrophils from Il17a-/- were as capable of phagocytosing the fungus comparable to that of WT mice, however, they showed decreased candidacidal ability. Our data implies that IL-17A is crucial for preventing the passage from mucosal to disseminated candidiasis. As such, our model may be suitable to study the mechanisms favouring Candida albicans translocation to internal organs.

2014 - Involvement of IL-17A in preventing the development of deep-seated candidiasis from oropharyngeal infection [Relazione in Atti di Convegno]
Elena, Gabrielli; Paolo, Mosci; Eugenio, Luciano; Stefano, Perito; Antonio, Cassone; Pericolini, Eva; Anna, Vecchiarelli
abstract

2014 - Purified microbial capsular polysaccharide Galactoxylomannan (GalXM) inhibits the effector T lymphocyte arm and potentiates the regulatory counterpart in rheumatoid arthritis [Poster]
Alunno, A; Pericolini, Eva; Gabrielli, E; Bistoni, O; Caterbi, S; Bartoloni, E; Terenzi, R; Valentini, E; La Paglia, G; Chow, Sk; Casadevall, A; Vecchiarelli, A; Gerli, R.
abstract

2014 - Secretory aspartyl-proteinases of Candida albicans contribute to severe inflammation observed in the course of vaginal candidiasis [Relazione in Atti di Convegno]
Pericolini, Eva; E., Gabrielli; E., Roselletti; E., Luciano; S., Sabbatini; A., Vecchiarelli; A., Cassone
abstract

Introduction. Candidiasis remains a notable health problem in subjects with underlying disease and several risk factors (diabetes, deep surgery, HIV infection, genetic predisposition and others) because of elevated mortality (candidemia and deep-seated infections), chronicity and recurrences in high prevalence clinical settings (oral and vaginal candidiasis), perceived increased risk of drug-resistance and lack of specific preventive measures such as vaccines. Within this framework, the high capacity of the major agent of candidiasis, Candida albicans (C. albicans), to express virulence factors causing inflammation and deceiving host immune system is remarkable and should constitute an important new concept/target to fight candidiasis by new tools providing an alternative or integration to the existing drugs. Secretory aspartyl-proteinases of C. albicans (SAP) are dominant virulence attributes of C. albicans and are considered to play an important role in the pathogenicity of this fungus. Materials and Methods. Using purified, enzymatically active, recombinant SAP, we demonstrated by western blot and flow cytometry analysis that SAP, including SAP2 and SAP6, are strong activators of NLRP3 inflammasome both in monocytes and epithelial cells. Results. The inflammasome response occurs via NLRP3 and caspase-1 activation which cleaves pro-IL1beta into secreted bioactive IL-1beta. The demonstration that proinflammatory cytokine production is induced by SAP through inflammasome activation implies new role for these virulence factors related to their structural features. Discussion and Conclusions. Particularly, inflammasome activation may contribute to severe inflammation and recruitment of neutrophils, a scenario characteristic in the pathology of vaginal candidiasis, during SAP production or exposure in the vaginal environment. Our data strongly suggest that SAP2 production may contribute to the excessive inflammatory response observed during C. albicans infections, which may be the hallmark of at least some Candida pathologies.

2014 - Secretory aspartyl-proteinases of Candida albicans contribute to severe inflammation observed in the course of vaginal candidiasis [Relazione in Atti di Convegno]
Pericolini, Eva; Elena, Gabrielli; Elena, Roselletti; Eugenio, Luciano; Samuele, Sabbatini; Anna, Vecchiarelli; Antonio, Cassone
abstract

Candidiasis remains a notable health problem in subjects with underlying disease and several risk factors (diabetes, deep surgery, HIV infection, genetic predisposition and others) because of elevated mortality (candidemia and deep-seated infections), chronicity and recurrences in high prevalence clinical settings (oral and vaginal candidiasis), perceived increased risk of drug-resistance and lack of specific preventive measures such as vaccines. Within this framework, the high capacity of the major agent of candidiasis, Candida albicans (C. albicans), to express virulence factors causing inflammation and deceiving host immune system is remarkable and should constitute an important new concept/target to fight candidiasis by new tools providing an alternative or integration to the existing drugs. Secretory aspartyl-proteinases of C. albicans (SAP) are dominant virulence attributes of C. albicans and are considered to play an important role in the pathogenicity of this fungus. Using purified, enzymatically active, recombinant SAP, we demonstrated that SAP, including SAP2 and SAP6, are strong activators of NLRP3 inflammasome both in monocytes and epithelial cells. This occurs via NLRP3 and caspase-1 activation which cleaves pro-IL1 into secreted bioactive IL-1. The demonstration that proinflammatory cytokine production is induced by SAP through inflammasome activation implies new role for these virulence factors related to their structural features. Particularly, inflammasome activation may contribute to severe inflammation and recruitment of neutrophils, a scenario characteristic in the pathology of vaginal candidiasis, during SAP production or exposure in the vaginal environment. Our data strongly suggest that SAP2 production may contribute to the excessive inflammatory response observed during C. albicans infections, which may be the hallmark of at least some Candida pathologies.

2013 - A novel bioluminescence mouse model for monitoring oropharyngeal candidiasis in mice [Articolo su rivista]
Mosci, P; Pericolini, Eva; Gabrielli, E; Kenno, S; Perito, S; Bistoni, F; D'Enfert, C; Vecchiarelli, A.
abstract

Oropharyngeal Candida albicans (C. albicans) infection usually occurs in patients with altered cell-mediated immune response. Many animal models have been developed for studying the pathogenesis of disease. Here we describe a new model for real-time monitoring of oral candidiasis. Mice were rendered susceptible to oral candidiasis by injection with cortisone acetate. Oral infection was performed by placing a swab saturated with genetically engineered bioluminescent strain of C. albicans sublingually. An in vivo imaging technique, exploiting stably trasformed C. albicans that costitutively express luciferase, was adopted. This novel longitudinal study represents a powerful tool to: (1) test real-time progression of infection, (2) identify the target site of C. albicans in specific organs, (3) evaluate the efficacy of antifungal therapies and (4) explore the spread of C. albicans from the local to systemic compartment in a new way.

2013 - A Purified Capsular Polysaccharide Markedly Inhibits Inflammatory Response during Endotoxic Shock [Articolo su rivista]
Piccioni, M; Monari, C; Kenno, S; Pericolini, Eva; Gabrielli, E; Pietrella, D; Perito, S; Bistoni, F; Kozel, Tr; Vecchiarelli, A.
abstract

Capsular material of the opportunistic fungus Cryptococcus neoformans is mainly composed of a polysaccharide named glucuronoxylomannan (GXM). In this study, the effects of GXM were analyzed in an in vivo experimental system of LPS-induced shock. Endotoxic shock was induced in mice by a single intraperitoneal injection of LPS from Escherichia coli. GXM treatment reduced the mortality of mice at early stages. Mice treated with LPS alone showed markedly increased plasma levels of TNF-α, IL-1β, and IL-6, whereas mice treated with GXM too showed significantly lower plasma levels of these cytokines. This effect was related to a marked suppression of Akt and IkBα activation. Importantly, the inhibitory effect of GXM on pro-inflammatory cytokine secretion was reproduced by treatment with Wortmannin, an inhibitor of the Akt transcription pathway. Our results indicate that GXM has a beneficial effect on endotoxic shock, resulting in a significant increase in rate of survival by dampening the hyper-inflammatory response.

2013 - Attività immunosoppressiva del polisaccaride microbico Galattoxilomannano [Relazione in Atti di Convegno]
Pericolini, Eva
abstract

2013 - Elucidating the immunological function of the Cryptococcus neoformans capsule [Articolo su rivista]
Anna, Vecchiarelli; Pericolini, Eva; Elena, Gabrielli; Samyr, Kenno; Stefano, Perito; Elio, Cenci; Claudia, Monari
abstract

The encapsulated fungal pathogen Cryptococcus neoformans represents a significant agent of life-threatening infections in immunocompromised subjects. A unique characteristic of Cryptococcus species is the presence of a polysaccharide capsule, which is essential for virulence and endows Cryptococcus with potent immunoregulatory properties. This review provides an overview of the immunological properties of the principal components of C. neoformans capsule.

2013 - Galactoxylomannan is a powerful immune system modulator able to rebalance Treg/Th17 ratio in rheumatoid arthritis [Poster]
Alunno, A; Pericolini, Eva; Bistoni, O; Gabrielli, E; Caterbi, S; Cenci, E; Bartoloni, E; Chow, Sk; Casadevall, A; Vecchiarelli, A; Gerli, R.
abstract

2013 - Secreted aspartic proteases of Candida albicans activate the NLRP3 inflammasome [Articolo su rivista]
Pietrella, D; Pandey, N; Gabrielli, E; Pericolini, Eva; Perito, S; Kasper, L; Bistoni, F; Cassone, A; Hube, B; Vecchiarelli, A.
abstract

In a recent report we demonstrated that distinct members of the secreted aspartic protease (Sap) family of Candida albicans are able to induce secretion of proinflammatory cytokines by human monocytes, independently of their proteolytic activity and specific pH optima. In particular, Sap2 and Sap6 potently induced IL-1β, TNF-α and IL-6 production. Here we demonstrate that Sap2 and Sap6 proteins trigger IL-1β and IL-18 production through inflammasome activation. This occurs via NLRP3 and caspase-1 activation, which cleaves pro-IL-1 into secreted bioactive IL-1β, a cytokine which was induced by Saps in monocytes, in monocyte-derived macrophages and in dendritic cells. Downregulation of NLRP3 by RNA interference strongly reduced the secretion of bioactive IL-1β. Inflammasome activation required Sap internalization via a clathrin-dependent mechanism, intracellular induction of K(+) efflux and ROS production. Inflammasome activation of monocytes induced by Sap2 and Sap6 differed from that induced by LPS-ATP in several aspects. Our data reveal novel immunoregulatory mechanisms of C. albicans and suggest that Saps contribute to the pathogenesis of candidiasis by fostering rather than evading host immunity.

2013 - The Microbial Capsular Polysaccharide Galactoxylomannan Inhibits IL-17A Production in Circulating T Cells from Rheumatoid Arthritis Patients [Articolo su rivista]
Pericolini, Eva; Alunno, A; Gabrielli, E; Bartoloni, E; Cenci, E; Chow, Sk; Bistoni, G; Casadevall, A; Gerli, R; Vecchiarelli, A.
abstract

The persistence of activated T cells in rheumatoid arthritis (RA) synovium may be attributable to increased homing, increased retention or a possible imbalance between cell proliferation and programmed cell death. Induction of apoptosis may represent a potential therapeutic approach. Galactoxylomannan (GalXM) from the opportunistic fungus Cryptococcus neoformans can interact with T cells and induce T-cell apoptosis through the inhibition of CD45 phosphatase activity. The aim of this study was to determine the effect of GalXM on circulating T cells from patients with RA and the underlying mechanisms. GalXM immunomodulating effect on apoptosis and signal transduction pathway involved in IL-17A production was evaluated on T cells. RA T-cell apoptosis, higher than that of control T cells, was further increased by GalXM through induction of caspase-3 activation. Activated T cells expressing the CD45RO molecule and producing IL-17A were the main target of GalXM-induced apoptosis. GalXM induced consistent impairment of IL-17A production and inhibition of STAT3, which was hyperactivated in RA. In conclusion, GalXM triggered apoptosis of activated memory T cells and interfered with IL-17A production in RA. These data suggest therapeutic targeting of deleterious Th17 cells in RA and other autoimmune diseases.

2013 - Un nuovo modello murino per monitorare in real-time la candidosi orofaringea [Abstract in Atti di Convegno]
E., Gabrielli; P., Mosci; S., Perito; S., Kenno; E., Luciano; Pericolini, Eva; A., Vecchiarelli
abstract

2012 - Antibody Constant Region Peptides Can Display Immunomodulatory Activity through Activation of the Dectin-1 Signalling Pathway [Articolo su rivista]
Gabrielli, E; Pericolini, Eva; Cenci, E; Monari, C; Magliani, W; Ciociola, T; Conti, S; Gatti, R; Bistoni, F; Polonelli, L; Vecchiarelli, A.
abstract

We previously reported that a synthetic peptide with sequence identical to a CDR of a mouse monoclonal antibody specific for difucosyl human blood group A exerted an immunomodulatory activity on murine macrophages. It was therapeutic against systemic candidiasis without possessing direct candidacidal properties. Here we demonstrate that a selected peptide, N10K, putatively deriving from the enzymatic cleavage of the constant region (Fc) of human IgG(1), is able to induce IL-6 secretion and pIkB-α activation. More importantly, it causes an up-regulation of Dectin-1 expression. This leads to an increased activation of β-glucan-induced pSyk, CARD9 and pIkB-α, and an increase in the production of pro-inflammatory cytokines such as IL-6, IL-12, IL-1β and TNF-α. The increased activation of this pathway coincides with an augmented phagocytosis of non opsonized Candida albicans cells by monocytes. The findings suggest that some Fc-peptides, potentially deriving from the proteolysis of immunoglobulins, may cause an unexpected immunoregulation in a way reminiscent of innate immunity molecules

2012 - Any antibody fragment might exert antifungal activity [Poster]
S., Conti; V., Magliani; T., Ciociola; E., Gabrielli; Pericolini, Eva; E., Cenci; S., Arancia; L., Polonelli
abstract

2012 - Immunomodulatory efficiency of Candida glabrata cell wall mannan [Abstract in Atti di Convegno]
Paulovikova, L.; Paulovikova, E.; Pericolini, Eva; Gabrielli, E. .; Vecchiarelli, A.
abstract

2012 - Mouse Strain-Dependent Differences in Estrogen Sensitivity During Vaginal Candidiasis [Articolo su rivista]
Mosci, P; Pietrella, D; Ricci, G; Pandey, N; Monari, C; Pericolini, Eva; Gabrielli, E; Perito, S; Bistoni, F; Vecchiarelli, A.
abstract

The animal models available for studying the immune response to genital tract infection require induction of a pseudo estrous state, usually achieved by administration of 17-β-estradiol. In our experimental model of vaginal candidiasis, under pseudo estrus, different strains of mice were used. We observed major differences in the clearance of Candida albicans infection among the different strains, ascribable to differing susceptibility to estradiol treatment. In the early phase of infection CD1, BALB/c, C57BL/6 albino and C57BL/6 mice were colonized to similar levels, while in the late phase of infection, BALB/c mice, which are considered genetically resistant to C. albicans infection, exhibited greater susceptibility to vaginal candidiasis than CD1 and C57BL/6 albino strains of mice. This was because estradiol induced "per se" enlarged and fluid-filled uteri, more pronounced in infected mice and consistently more evident in BALB/c and C57BL/6 mice than in CD1 mice. Unlike CD1, BALB/c and C57BL/6 mice showed a heavy fungal colonization of the uterus, even though C57BL/6 mice apparently cleared C. albicans from the vagina. The presence of C. albicans in the vagina and uterus was accompanied by a heavy bacterial load. Collectively these observations prompted us to carry out a careful analysis of estradiol effects in a mouse model of vaginal infection.

2012 - New approaches in the development of a vaccine for mucosal candidiasis: progress and challenges [Articolo su rivista]
Vecchiarelli, A; Pericolini, Eva; Gabrielli, E; Pietrella, D.
abstract

The commensal fungus Candida albicans causes mucosal candidiasis in the rapidly expanding number of immunocompromised patients. Mucosal candidiasis includes oropharyngeal, esophageal, gastrointestinal, and vaginal infections. Vulvovaginal candidiasis (VVC) and antimycotic-refractory recurrent VVC is a frequent problem in healthy childbearing women. Both these mucosal infections can affect the quality of life and finding new therapeutical and preventive approaches is a challenge. A vaccine against candidal infections would be a new important tool to prevent and/or cure mucosal candidiasis and would be of benefit to many patients. Several Candida antigens have been proposed as vaccine candidates including cell wall components and virulence factors. Here we discuss the recent progress and problems associated with vaccination against mucosal candidiasis.

2012 - Peptides of the constant region of antibodies display fungicidal activity [Articolo su rivista]
Polonelli, L; Ciociola, T; Magliani, W; Zanello, Pp; D'Adda, T; Galati, S; De Bernardis, F; Arancia, S; Gabrielli, E; Pericolini, Eva; Vecchiarelli, A; Arruda, Dc; Pinto, Mr; Travassos, Lr; Pertinhez, Ta; Spisni, A; Conti, S.
abstract

Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents.

2012 - The inflammasome plays a central role in inducing innate immune response to Candida albicans secreted aspartic proteases [Abstract in Atti di Convegno]
N., Pandey; D., Pietrella; L., Schild; Pericolini, Eva; E., Gabrielli; B., Hube; A., Vecchiarelli
abstract

2011 - A microbial polysaccharide promotes selective elimination of pathogenic T cells from peripheral blood of rheumatoid arthritis patients by apoptosis induction. [Abstract in Atti di Convegno]
Alunno, A.; Pericolini, Eva; Gabrielli, E.; Cenci, E.; Bartoloni Bocci, E.; Bistoni, O.; Chow, S. K.; Casadevall, A.; Vecchiarelli, A.; Gerli, R.
abstract

2011 - Cryptococcus neoformans galactoxylomannan is a potent negative immunomodulator, inspiring new approaches in anti-inflammatory immunotherapy [Articolo su rivista]
Vecchiarelli, A; Pericolini, Eva; Gabrielli, E; Chow, Sk; Bistoni, F; Cenci, E; Casadevall, A.
abstract

Cryptococcus neoformans is an opportunistic fungal pathogen responsible for life-threatening infections in immunocompromised individuals and occasionally in those with no known immune impairment. The fungus is endowed with several virulence factors, including capsular polysaccharides that play a key role in virulence. The capsule is composed of 90-95% glucuronoxylomannan (GXM), 5-8% galactoxylomannan (GalXM) and <1% mannoproteins. Capsular polysaccharides are shed into tissue where they produce many deleterious effects. Since GalXM has a smaller molecular mass, the molar concentration of GalXM in polysaccharide that is shed could exceed that of GXM in C. neoformans exopolysaccharides. Moreover, GalXM exhibits a number of unusual biologic properties both in vitro and in vivo. Here, we summarize the principal immunomodulatory effects of GalXM described during the last 20 years, particularly the mechanisms leading to induction of apoptosis in T lymphocytes, B lymphocytes and macrophages. Since the capacity of GalXM to induce widespread immune suppression is believed to contribute to the virulence of C. neoformans, this property might be exploited therapeutically to dampen the aberrant activation of immune cells during autoimmune disorders.

2011 - Immumodulatory effects of galactoxylomannan (GALXM) on T cells from patients with rheumatoid arthritis: increased apoptosis and reduced proliferation of effector CD3+ cells in vitro [Abstract in Atti di Convegno]
Alunno, A.; Pericolini, Eva; Gabrielli, E.; Cenci, E.; Bartoloni Bocci, E.; Bistoni, O.; Chow, S. K.; Casadevall, A.; Vecchiarelli, A.; Gerli, R.
abstract

2011 - Immunobiological properties of pathogenic Candida species natural cell wall derived polysaccharides and synthetically prepared oligosaccharides – protein conjugates. [Abstract in Atti di Convegno]
Lucia, Paulovicovà; Ema, Paulovicovà; Pericolini, Eva; Elena, Gabrielli; Alexander A., Karelin; Yury E., Tsvetkov; Nikolay E., Nifantiev; Vecchiarelli, A.
abstract

2010 - Role of CD45 signaling pathway in Galactoxylomannan-induced T cell damage [Articolo su rivista]
Pericolini, Eva; Gabrielli, E; Bistoni, F; Cenci, E; Perito, S; Chow, S; Riuzzi, F; Donato, R; Casadevall, A; A., Vecchiarelli
abstract

Previously, we reported that Galactoxylomannan (GalXM) activates the extrinsic and intrinsic apoptotic pathways through an interaction with the glycoreceptors on T cells. In this study we establish the role of the glycoreceptor CD45 in GalXM-induced T cell apoptosis, using CD45+/+ and CD452/2 cell lines, derived from BW5147 murine T cell lymphoma. Our results show that whereas CD45 expression is not required for GalXM association by the cells, it is essential for apoptosis induction. In CD45+/+ cells, CD45 triggering by GalXM reduces the activation of Lck, ZAP70 and Erk1/2. Conversely, in CD452/2 cells, Lck was hyperphosphorylated and did not show any modulation after GalXM stimulation. On the whole, our findings provide evidence that the negative regulation of Lck activation occurs via CD45 engagement. This appears to be related to the capacity of GalXM to antagonize T cell activation and induce T cell death. Overall this mechanism may be responsible for the immune paralysis that follows GalXM administration and could explain the powerful immunosuppression that accompanies cryptococcosis.

2009 - A microbial polysaccharide reduces the severity of rheumatoid arthritis by influencing Th17 differentiation and proinflammatory cytokines production [Articolo su rivista]
C., Monari; S., Bevilacqua; M., Piccioni; Pericolini, Eva; S., Perito; M., Calvitti; F., Bistoni; T. R., Kozel; A., Vecchiarelli
abstract

Rheumatoid arthritis (RA) is a chronic and debilitating autoimmune disease characterized by chronic joint inflammation with subsequent cartilage and bone destruction. RA is emerging as a model of IL-17-driven autoimmune inflammatory disease. IL-17 is a marker for Th17 cells, with its master regulator being the retinoic acid receptor-related orphan receptor (RORt) regulated by STAT3 signaling. Glucuronoxylomannan (GXM), a polysaccharide representing the main component of the capsular material of the opportunistic yeast Cryptococcus neoformans, exhibits potent immunosuppressive properties both in vitro and in vivo. The present study investigates the effects of GXM treatment on the progression of collagen-induced arthritis. GXM suppressed clinical signs of collagen-induced arthritis and blocked joint erosion progression. This effect was mediated by down-regulation of key cytokines involved in the pathogenesis of RA such as TNF- and IL-1, and upregulation of the inhibitory cytokine IL-10. Moreover, a reduction of IL-6 and TGF-, which inhibit Th17 differentiation with consequent decreased IL-17 production at the local and systemic level, was observed. The effect of GXM on Th17 differentiation mirrored the reduction in STAT3 activation and inhibition of RORt synthesis. Consequently, this work highlights the beneficial properties of an efficacious compound that could eventually be destined to the clinic.

2009 - A microbial polysaccharide reduces the severity of rheumatoid arthtritis via inhibition of Th17 differentiation [Abstract in Atti di Convegno]
Claudia, Monari; Sara, Bevilacqua; Miranda, Piccioni; Pericolini, Eva; Stefano, Perito; Mario, Calvitti; Francesco, Bistoni; Thomas R., Kozel; Anna, Vecchiarelli
abstract

2009 - Antibody complementarity-determining regions (CDRs): a bridge between adaptive and innate immunity [Articolo su rivista]
Gabrielli, E.; Pericolini, Eva; Cenci, E.; Ortelli, F.; Magliani, W.; Ciociola, T.; Bistoni, F.; Conti, S.; Vecchiarelli, A.; Polonelli, L.
abstract

BACKGROUND: It has been documented that, independently from the specificity of the native antibody (Ab) for a given antigen (Ag), complementarity determining regions (CDR)-related peptides may display differential antimicrobial, antiviral and antitumor activities. METHODOLOGY/PRINCIPAL FINDINGS: In this study we demonstrate that a synthetic peptide with sequence identical to V(H)CDR3 of a mouse monoclonal Ab (mAb) specific for difucosyl human blood group A is easily taken up by macrophages with subsequent stimulation of: i) proinflammatory cytokine production; ii) PI3K-Akt pathway and iii) TLR-4 expression. Significantly, V(H)CDR3 exerts therapeutic effect against systemic candidiasis without possessing direct candidacidal properties. CONCLUSIONS/SIGNIFICANCE: These results open a new scenario about the possibility that, beyond the half life of immunoglobulins, Ab fragments may effectively influence the antiinfective cellular immune response in a way reminiscent of regulatory peptides of innate immunity.

2009 - Involvement of glycoreceptors in galactoxylomannan-induced T cell death [Articolo su rivista]
Pericolini, Eva; Gabrielli, E.; Cenci, E.; De Jesus, M.; Bistoni, F.; Casadevall, A.; Vecchiarelli, A.
abstract

The major virulence factor of Cryptococcus neoformans is its capsular polysaccharide, which is also released into tissues. The shed polysaccharide is composed of glucuronoxylomannan, galactoxylomannan (GalXM), and mannoproteins. In a previous study, we demonstrated a direct interaction of purified soluble GalXM with T cells that induced their apoptosis. In this study, we focus on the mechanisms involved in the apoptotic effect of GalXM. In our experimental system, we analyzed the effect of GalXM on purified human T cells and Jurkat cells, a T cell line routinely used for apoptotic studies. Our results reveal that GalXM activates the extrinsic and intrinsic apoptotic pathways through the cleavage and recruitment of caspase-8. Caspase-8 elicits the downstream executioner caspase-3, caspase-6, and caspase-7 both directly and indirectly, via Bid cleavage and caspase-9 activation. These effects appeared to be primarily mediated by the interaction of GalXM with the glycoreceptors, which differed in human T and Jurkat cells. CD45 was primarily involved in Jurkat cells apoptosis while CD7 and CD43 mediated human T cell apoptosis. Our results highlight a new mechanism by which a microbial product can contribute to virulence through direct interaction with T cell glycoreceptors, thereby triggering lymphocyte apoptosis.

2009 - The GITRL-GITR system alters TLR-4 expression on DC during fungal infection [Articolo su rivista]
Vecchiarelli, A.; Pericolini, Eva; Gabrielli, E.; Agostini, M.; Bistoni, F.; Nocentini, G.; Cenci, E.; Riccardi, C.
abstract

The glucocorticoid-induced TNFR-related (GITR) protein is a member of the tumor necrosis factor receptor superfamily influencing natural and acquired immune response. GITR is activated by its ligand, GITRL, mainly expressed on antigen presenting cells. Previously, we demonstrated that GITR plays a role in regulating immune response to Candida albicans. Here we analyzed whether GITRL-GITR interaction influences the recognition of C. albicans by regulating the expression of pattern recognition receptors on splenic dendritic cells. Our report demonstrates that under physiological conditions and during candidiasis the GITRL-GITR system affects TLR-2 and TLR-4 expression on DC. These changes correlate with decrease in: MyD88 activation; CD80 and CD40 expression on DC; T cell activation response, including CD28 expression, IL-2 and IFN-gamma production. Our results point out that, during fungal infection, GITRL-GITR interaction modulates TLR-4 and TLR-2 expression, thereby altering the antigen presentation process, and suggesting a role of GITRL-GITR interaction in resistance against infectious diseases.

2008 - Indinavir influences biological function of dendritic cells and stimulates antifungal immunity [Articolo su rivista]
Pericolini, Eva; E., Cenci; E., Gabrielli; S., Perito; P., Mosci; F., Bistoni; A., Vecchiarelli
abstract

In this study, we analyzed the possibility that Indinavir (IDV), a well-known protease inhibitor (PI) used in highly active antiretroviral therapy, could affect immune response against the opportunistic fungus Cryptococcus neoformans. In particular, the quality of dendritic cell (DC) response was analyzed. The results reported here show that IDV treatment induces an expansion of DC with CD8alpha phenotype in spleens of infected hosts. Splenic CD11c+ DC expressed elevated costimulatory molecules such as CD40 and CD80, showed an increased expression of mRNA for proinflammatory cytokines, and secreted abundant IL-12. Integration of all aforementioned regulatory effects results in development of an efficient, T cell-protective response that reflects a consistent reduction in fungus colonization at a cerebral level. These results could help to elucidate the immunoregulatory activity of PI and point out the beneficial effects of IDV in regulating DC functions and antifungal activity. Therefore, although new PI are being introduced in the clinical setting, nevertheless, given its low cost and proven efficacy, IDV could still be considered a potential key compound in the treatment of HIV in resource-limited settings.

2008 - Studio degli effetti degli anticorpi monoclonali anti GXM per l'ottimizzazione della terapia della criptococcosi e valutazione del possibile utilizzo di farmaci inibitori delle proteasi, quali l'Indinavir, come anti-fungini. [Capitolo/Saggio]
Pericolini, Eva
abstract

2007 - Functional defect of natural immune system in an apparent immunocompetent patient with pulmonary cryptococcosis [Articolo su rivista]
Marroni, M.; Pericolini, Eva; Cenci, E.; Bistoni, F.; Vecchiarelli, A.
abstract

We report a case of pulmonary cryptococcosis in a 21-year-old Italian female smoker with no apparent immune disorder. In this study we demonstrated that: (i) patient's neutrophils and monocytes manifested a significant reduction of killing activity against Cryptococcus neoformans as well as Candida albicans; (ii) the suppression was more pronounced in monocytes than in neutrophils; (iii) neutrophils and monocytes showed a significant impairment of TNF-alpha, IL-1beta, and nitric oxide production. These results suggest that the apparent immunocompetent host with pulmonary cryptococcosis could have specific defects in natural immune system mechanisms.

2006 - A role for Cryptococcus neoformans galactoxylomannan in T cell apoptosis and function. [Poster]
E., Cenci; Pericolini, Eva; M., DE JESUS; F., Bistoni; A., Vecchiarelli; A., Casadevall
abstract

2006 - Cryptococcus neoformans capsular polysaccharide component Galactoxylomannan induces apoptosis of human T cells through activation of Caspase-8 [Articolo su rivista]
Pericolini, Eva; Cenci, E.; Monari, C.; De Jesus, M.; Bistoni, F.; Casadevall, A.; Vecchiarelli, A.
abstract

The major virulence factor of Cryptococcus neoformans is its polysaccharide capsule composed of glucuronoxylomannan (GXM), galactoxylomannan (GalXM) and mannoproteins. A variety of immunomodulating activities have been described for GXM and mannoproteins but little is known about possible interactions of GalXM with the immune system. In the present article, we investigate the effect of purified soluble GalXM on human T lymphocytes. The results indicate that, GalXM (i) can affect selected immune responses; (ii) causes significant impairment of T cell proliferation and increases interferon-gamma and interleukin-10 production; and (iii) induces apoptosis of T lymphocytes through activation of caspase-8 that terminates with fragmentation of DNA. These results are the first to suggest a role for GalXM in C. neoformans virulence by demonstrating that it can target human T cells, and that it may impair the development of an effective specific T cell response.

2006 - Indinavir-treated Cryptococcus neoformans promotes an efficient antifungal immune response in immunosuppressed hosts [Articolo su rivista]
Pericolini, Eva; Cenci, E.; Monari, C.; Perito, S.; Mosci, P.; Bistoni, G.; Vecchiarelli, A.
abstract

A previous paper demonstrated that indinavir affects the virulence of Cryptococcus neoformans, thereby rendering the fungus more susceptible to killing by natural effector cells. This study demonstrates that inoculation of immunosuppressed mice with C. neoformans previously exposed to indinavir, in comparison to untreated C. neoformans, results in: (i) a more pronounced secretion of interleukin-12 by splenic dendritic cells; (ii) reduction of CD14 and Fc gammaRs expression on splenic dendritic cells, and upregulation of CD86 and CD40 molecules; (iii) enhancement of interferon-y and interleukin-2 production by splenic T cells and increase of their proliferation in response to fungal antigens; and (iv) survival from an otherwise lethal challenge, correlated with a drastic decrease in colony forming units from brain and liver. In conclusion, these data indicate that indinavir interaction with C. neoformans could be beneficial because of its direct influence on fungal virulence and blunting of the deleterious effects exerted by C. neoformans on host immune response. Thus, indinavir could be crucial in addressing the outcome of cryptococcosis in immunocompromised hosts.

2006 - Microbial immune suppression mediated by direct engagement of inhibitory Fc receptor [Articolo su rivista]
Monari, C.; Kozel, T.; Paganelli, F.; Pericolini, Eva; Perito, S.; Bistoni, F.; Casadevall, A.; Vecchiarelli, A.
abstract

A microbial polysaccharide (glucuronoxylomannan (GXM)) exerts potent immunosuppression by direct engagement to immunoinhibitory receptor FcgammaRIIB. Activation of FcgammaRIIB by GXM leads to the recruitment and phosphorylation of SHIP that prevents IkappaBalpha activation. The FcgammaRIIB blockade inhibits GXM-induced IL-10 production and induces TNF-alpha secretion. GXM quenches LPS-induced TNF-alpha release via FcgammaRIIB. The addition of mAb to GXM reverses GXM-induced immunosuppression by shifting recognition from FcgammaRIIB to FcgammaRIIA. These findings indicate a novel mechanism by which microbial products can impair immune function through direct stimulation of an inhibitory receptor. Furthermore, our observations provide a new mechanism for the ability of specific Ab to reverse the immune inhibitory effects of certain microbial products.

2006 - Modulation of phenotype and function of dendritic cells by a therapeutic synthetic killer peptide. [Articolo su rivista]
Cenci, E.; Pericolini, Eva; Mencacci, A.; Conti, S.; Magliani, W.; Bistoni, F.; Polonelli, L.; Vecchiarelli, A.
abstract

The strong microbicidal effects of an engineered synthetic killer peptide (KP), which functionally mimics a fungal killer toxin, have been demonstrated extensively. Beta-glucan has been identified as a receptor for KP on fungal cell walls. Although the direct microbicidal and related therapeutic effects have been studied in depth, no information currently exists about the interaction of KP with immune cells. In this study, we exploited the possibility of KP binding to different murine immune cell populations. The results demonstrate that KP binds selectively to dendritic cells (DC) and to a lesser extent, to macrophages but not to lymphocytes and neutrophils; KP binding possibly occurs through major histocompatibility complex (MHC) class II, CD16/32, and cellular molecules recognized by anti-specific intercellular adhesion molecule-grabbing nonintegrin R1 antibodies; and KP modulates the expression of costimulatory and MHC molecules on DC and improves their capacity to induce lymphocyte proliferation. These findings provide evidence that this synthetic KP interacts selectively with DC and modulating their multiple functions, might also serve to improve the immune antimicrobial response.

2005 - Cryptococcus neoformans capsular glucuronoxylomannan induces expression of fas ligand in macrophages [Articolo su rivista]
Monari, C.; Pericolini, Eva; Bistoni, G.; Casadevall, A.; Kozel, T. R.; Vecchiarelli, A.
abstract

The major component of capsular material of Cryptococcus neoformans is glucuronoxylomannnan (GXM), a polysaccharide that exhibits potent immunosuppressive properties in vitro and in vivo. The results reported here show that 1) soluble purified GXM induces a prompt, long-lasting, and potent up-regulation of Fas ligand (FasL) on macrophages, 2) the up-regulation of FasL is related to induced synthesis and increased mobilization to the cellular surface, 3) this effect is largely mediated by interaction between GXM and TLR4, 4) FasL up-regulation occurs exclusively in GXM-loaded macrophages, 5) macrophages that show up-regulation of FasL induce apoptosis of activated T cells expressing Fas and Jurkat cells that constitutively express Fas, and 6) anti-Fas Abs rescue T cells from apoptosis induced by GXM. Collectively our results reveal novel aspects of the immunoregulatory properties of GXM and suggest that this nontoxic soluble compound could be used to dampen the immune response, to promote or accelerate the death receptor, and to fix FasL expression in a TLR/ligand-dependent manner. In the present study, we delineate potential new therapeutic applications for GXM that exploit death receptors as key molecular targets in regulating cell-mediated cytotoxicity, immune homeostasis, and the immunopathology of diseases.

2005 - Cryptococcus neoformans esposto ad Indinavir promuove un'efficiente risposta immune nell'ospite immunocompromesso [Relazione in Atti di Convegno]
Pericolini, Eva; E., Cenci; C., Monari; S., Perito; P., Mosci; F., Bistoni; A., Vecchiarelli
abstract

2005 - Glucuronoxylomannan, a microbial compound, regulates expression of costimulatory molecules and production of cytokines in macrophages [Articolo su rivista]
Monari, C.; Bistoni, F.; Casadevall, A.; Pericolini, Eva; Pietrella, D.; Kozel, T. R.; Vecchiarelli, A.
abstract

Glucuronoxylomannan (GXM) is a microbial compound that can modulate the immune response. We investigated (1) the receptors involved in uptake of GXM on monocyte-derived macrophages (MDMs) from healthy donors, (2) the effects of GXM on expression of specific receptors, (3) the effects of GXM mediated by pattern-recognition receptors, and (4) GXM modulation of MDM accessory and secretory functions. Cellular receptors involved in uptake of GXM included Fc gamma RII, CD18, Toll-like receptor (TLR) 4, and CD14. Some biological functions of MDMs were profoundly affected by treatment with GXM, resulting in (1) increased expression of CD40 and CD86 via perturbation of TLR4, (2) decreased expression of major histocompatibility complex class II, (3) induction of interleukin-10 but not of tumor necrosis factor-alpha, and (4) decreased lipopolysaccharide (LPS)-induced production of cytokines. GXM represents an attractive compound to limit inflammatory processes and induce an LPS-tolerant state.

2005 - Il Galattoxilomannano, componente della capsula polisaccaridica del Cryptococcus neoformans, induce apoptosi dei linfociti T umani tramite l'attivazione della caspasi-8 [Relazione in Atti di Convegno]
Pericolini, Eva; Elio, Cenci; Claudia, Monari; Francesco, Bistoni; Anna, Vecchiarelli
abstract

2005 - Il polisaccaride capsulare di C.neoformans induce l’espressione del FasL in macrofagi umani. [Abstract in Atti di Convegno]
C., Monari; Pericolini, Eva; F., Bistoni; A., Vecchiarelli
abstract

2005 - Influence of indinavir on virulence and growth of Cryptococcus neoformans [Articolo su rivista]
Monari, C.; Pericolini, Eva; Bistoni, G.; Cenci, E.; Bistoni, F.; Vecchiarelli, A.
abstract

Indinavir selectively inhibited production of some virulence factors of Cryptococcus neoformans, such as urease and protease, but not melanin and phospholipase; moreover, it interfered with capsule formation. These effects led to increased susceptibility of C. neoformans to intracellular killing by natural effector cells. Prolonged incubation with indinavir resulted in inhibition of fungal growth. Indinavir can attenuate the virulence of the fungus, thus augmenting its susceptibility to the antimicrobial activity of natural effector cells. The reduction in cryptococcal infections in human immunodeficiency virus-positive patients might also be related to the antifungal activity of highly active antiretroviral therapy.

2005 - The glucocorticoid-induced tumor necrosis factor receptor-related gene modulates the response to Candida albicans infection [Articolo su rivista]
Agostini, M; Cenci, E; Pericolini, Eva; Nocentini, G; Bistoni, G; Vecchiarelli, A; Riccardi, C.
abstract

The glucocorticoid-induced tumor necrosis factor (TNF) receptor-related gene (GITR; TNFRSF18) modulates immune response activating coaccessory signals in T cells and is expressed at high levels in CD4+CD25+ cells. Its ligand (GITRL) is expressed in antigen-presenting cells, where it is capable of promoting signaling. We investigated the role of GITR/GITRL interaction during disseminated candidiasis in GITR knockout (GITR-/-) mice. GITR-/- mice survived longer and had a significantly decreased yeast load in kidneys and brain compared to GITR+/+ mice. Since protective immunity to the fungus is mediated by antigen-specific T helper (Th) 1 cells, we studied in vitro cytokine production following infection. CD4+ T cells of GITR-/- mice demonstrated a more efficient Th1 polarization as suggested by a two- to threefold decreased production of interleukin- (IL-)4 and IL-10 and a four- to fivefold increased production of gamma interferon compared to GITR+/+ mice. This effect was not due to differences in lymphocyte and dendritic cell (DC) subpopulations in infected mice as demonstrated by flow cytometric studies. To verify whether DC activity was differently modulated, DCs were cocultured with CD4+ T cells in the presence of heat-inactivated Candida albicans. DCs, cocultured with GITR+/+ CD4+CD25+ cells produced a lower amount of IL-12 than DCs cocultured with GITR-/- CD4+CD25+ T cells. These results suggest that GITR regulates susceptibility to systemic candidiasis by negatively modulating IL-12 production and promoting polarization of CD4+ T cells towards Th2 by analogy with OX40, another TNF receptor superfamily member.

2004 - Indinavir influenza la virulenza di C. neoformans. [Abstract in Atti di Convegno]
C., Monari; Pericolini, Eva; A., Vecchiarelli
abstract

2004 - Indinavir influenza la virulenza di C. neoformans. [Abstract in Atti di Convegno]
C., Monari; Pericolini, Eva; E., Cenci; S., Perito; A., Vecchiarelli
abstract