Andrea ARDIZZONI - personale UniMoRe

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Andrea ARDIZZONI

Personale tecnico amministrativo
sede ex-Scienze di Sanità Pubblica Dipartimento Chirurgico, Medico, Odontoiatrico e di Scienze Morfologiche con interesse Trapiantologico, Oncologico e di Medicina Rigenerativa
Ricercatore t.d. art. 24 c. 3 lett. B
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

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 - 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 - Correlating the physico-chemical properties of two conventional glazed porcelain stoneware tiles in relation to cleanability and sanitization [Articolo su rivista]
CEDILLO GONZALEZ, ERIKA IVETH; Chierici, Paolo; Buttazzo, Marta; Siligardi, Cristina; Blasi, Elisabetta; Ardizzoni, Andrea
abstract

Keeping surfaces clean can reduce the spread of infections. In particular, to decrease the potential for SARS CoV-2 contamination, performing disinfection of high-touching surfaces. Several ceramic tiles and porcelain stoneware tiles with antimicrobial properties are already available on the market. However, the widespread use of antimicrobial glazed stoneware tiles may require to replace the ceramic surfaces already present in many buildings. The unfeasibility of such replacement can be due to both product durability (lifetime of a tile is usually long) and/or monetary restrictions. Furthermore, as porcelain stoneware per se does not have antimicrobial activity, these materials are fabricated by adding chemical agents able to provide antimicrobial properties. This approach requires a compatibility between the antimicrobial agents and the glaze formulation, as well as a careful control of the firing cycle and the final properties of the ceramic products. It follows that the final cost of antimicrobial tiles is not competitive with that of conventional tiles. In the latter, the persistence of potential pathogens on the surfaces is a crucial problem to face: the longer a pathogen survives on a surface, the longer it may be a source of transmission and thus endanger susceptible subjects. In this work, bacteria's capacity to adhere and to be effectively removed from two conventional glazed porcelain stoneware tiles (under dirty and clean conditions) was investigated. Two different glazes were tested, one mainly glassy (glossy) and the other mainly crystalline (matt). The sanitization procedures were carried out by chemical and chemo-mechanical procedures. The results showed that chemo-mechanical sanitization was the most effective, and the best results could be obtained on the stoneware tiles coated with the mainly glassy glaze, with the lowest porosity and the lower roughness values and water contact angles, especially under clean conditions.

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.

2022 - Does cleanability lead to hygienic ceramic tiles? Investigation of the correlation between the cleanability of ceramic glazes and their antibacterial activity [Abstract in Atti di Convegno]
Cedillo-Gonzalez, Ei; Ariza-Tarazona, Mc; Ardizzoni, A; Blasi, E; Siligardi, C
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 - Sanitization of different pocelain stoneware tiles after bacterial contamination [Abstract in Atti di Convegno]
Cedillo-Gonzalez, Ei; Ardizzoni, A; Blasi, E; Siligardi, C
abstract

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 - 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.

2019 - Antiviral Activity of Synthetic Peptides Derived from Physiological Proteins [Articolo su rivista]
Sala, Arianna; Ardizzoni, Andrea; Ciociola, Tecla; Magliani, Walter; Conti, Stefania; Blasi, Elisabetta; Cermelli, Claudio
abstract

Background/Aims: New antivirals are needed to supplement or replace currently used drugs. The aim of this study was to evaluate the antiviral activity of synthetic peptides derived from physiological proteins. Methods: Vero cell monolayers were infected with herpes simplex virus 1, vesicular stomatitis virus, adenovirus, and coxsackievirus B5 strains in the presence of different concentrations of the selected peptides and viral yield was determined by plaque reduction assays to evaluate the antiviral activity of the peptides. Virucidal activity was evaluated by determining the residual infectivity of viral suspensions treated for 1 h with the peptides at the same concentrations as in the viral yield assays. Results: Among the investigated peptides, the killer peptide proved to exert a considerable antiviral activity against herpes simplex virus, attributable to a direct effect on virus particles, while its derivative K10S showed to be effective against the four investigated virus strains only at the highest concentration tested, yet, the inhibitory effects were only partial. Conclusion: Overall, initial evidence is provided on the antiviral activity of several peptides, as well as of their derivatives. Further investigation is warranted to ascertain the mechanism of action in order to develop new potential antiviral drugs.

2019 - Longitudinal Survey of Fungi in the Human Gut: ITS Profiling, Phenotyping, and Colonization [Articolo su rivista]
Raimondi, Stefano; Amaretti, Alberto; Gozzoli, Caterina; Simone, Marta; Righini, Lucia; Candeliere, Francesco; Brun, Paola; Ardizzoni, Andrea; Colombari, Bruna; Paulone, Simona; Castagliuolo, Ignazio; Cavalieri, Duccio; Blasi, Elisabetta; Rossi, Maddalena; Peppoloni, Samuele
abstract

The fungal component of the intestinal microbiota of eight healthy subjects was studied over 12 months using metagenome survey and culture-based approaches. Aspergillus, Candida, Debaryomyces, Malassezia, Penicillium, Pichia, and Saccharomyces were the most recurrent and/or dominant fungal genera, according to metagenomic analysis. The biodiversity of fungal communities was lower and characterized by greater unevenness, when compared to bacterial microbiome. The dissimilarities both among subjects and over the time within the same subject suggested that most of the fungi passed through the gastro-intestinal tract (GIT) without becoming stable colonizers. Certain genera, such as Aspergillus and Penicillium, were isolated in a minority of cases, although they recurred abundantly and frequently in the metagenomics survey, likely being environmental or food-borne fungi that do not inhabit the GIT. Candida genus was recurrently detected. Candida albicans isolates dominated among the cultivable mycobiota and longitudinally persisted, likely as commensals inhabiting the intestine or regularly reaching it from Candida-colonized districts, such as the oral cavity. Other putative colonizers belonged to Candida zeylanoides, Geotrichum candidum, and Rhodotorula mucilaginosa, with persisting biotypes being identified. Phenotyping of fungal isolates indicated that C. albicans adhered to human epithelial cells more efficiently and produced greater amounts of biofilm in vitro than non-albicans Candida (NAC) and non-Candida fungi (NCF). The C. albicans isolates also induced the highest release of HBD-2 by human epithelial cells, further differing from NAC and NCF. Nine representative isolates were administered to mice to evaluate the ability to colonize the intestine. Only two out of three C. albicans strains persisted in stools of animals 2 weeks after the end of the oral administration, whereas NAC and NCF did not. These results confirm the allochthonous nature of most the intestinal fungi, while C. albicans appears to be commonly involved in stable colonization. A combination of specific genetic features in the microbe and in the host likely allow colonization from fungi normally present solely as passengers. It remains to be established if other species identified as potential colonizers, in addition to Candida, are true inhabitants of the GIT or rather reach the intestine spreading from other body districts.

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.

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 - Apoptosis and inflammatory response in human astrocytes are induced by a transmissible cytotoxic agent of neurological origin [Articolo su rivista]
Beretti, Francesca; Ardizzoni, Andrea; Cermelli, Claudio; Guida, Marianna; Maraldi, Tullia; Pietrosemoli, P; Paulone, Simona; De Pol, Anto; Blasi, Elisabetta; Portolani, Marinella
abstract

We demonstrated the presence of an in vitro transmissible cytotoxic agent (TCA) in the cerebrospinal fluid (CSF) of patients with different acute neurological diseases. The nature of this agent is still a matter of study since repeated attempts have failed to identify it as a conventional infectious agent. Here, we describe the mechanisms through which TCA affects human astrocytes, demonstrating:a late apoptotic process, mediated by caspases 9 and 3 activation, involving the Bcl2-Bak-axis;an early and late p38 MAPK activation;an interference with the IL-8 and MCP-1 secretory response. These in vitro data provide initial evidence of TCA involvement as a pro-apoptotic and pro-inflammatory signal, directly affecting astrocytic behavior. The implications of these findings in certain neurological diseases will be discussed.

2017 - Candida albicans survival, growth and biofilm formation are differently affected by mouthwashes: an in vitro study [Articolo su rivista]
Paulone, Simona; Malavasi, G; Ardizzoni, Andrea; Orsi, Carlotta Francesca; Peppoloni, Samuele; Neglia, Rachele Giovanna; Blasi, Elisabetta
abstract

Candida albicans is the most common cause of oral mycoses. The aim of the present study was to investigate in vitro the susceptibility of C. albicans to mouthwashes, in terms of growth, survival and biofilm formation.Candida albicans, laboratory strain SC5314, and 7 commercial mouthwashes were employed: 3 with 0.2% chlorhexidine digluconate; 1 with 0.06% chlorhexidine digluconate and 250 ppm F sodium fluoride; 3 with fluorine-containing molecules. None of the mouthwashes contained ethanol in their formulations. The anti-Candida effects of the mouthwashes were assessed by disk diffusion, crystal violet and XTT assays. By using five protocols combining different dilutions and contact times the mouthwashes were tested against:1) C. albicans growth;2) biofilm formation;3) survival of fungal cells in early, developing and mature Candida biofilm.Chlorhexidine digluconate-containing mouthwashes consistently exhibited the highest anti-Candida activity, irrespective of the protocols employed. Fungal growth, biofilm formation and survival of Candida cells within biofilm were impaired, the effects strictly depending on both the dilution employed and the time of contact.These in vitro studies provide evidence that mouthwashes exert anti-Candida activity against both planktonic and biofilm fungal structures, but to a different extent depending on their composition. This suggests special caution in the choice of mouthwashes for oral hygiene, whether aimed at prevention or treatment of oral candidiasis.

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 - Human biliary tree stem/progenitor cells immunomodulation: Role of hepatocyte growth factor [Articolo su rivista]
Maraldi, Tullia; Guida, Marianna; Beretti, Francesca; Resca, Elisa; Carpino, Guido; Cardinale, Vincenzo; Gentile, Raffaele; Ardizzoni, Andrea; Murgia, Alba; Alvaro, Domenico; Gaudio, Eugenio; DE POL, Anto
abstract

Aim: Human biliary tree stem/progenitor cells (hBTSC) are multipotent epithelial stem cells with the potential for allogenic transplant in liver, biliary tree, and pancreatic diseases. Human mesenchymal stem cells, but also epithelial stem cells, are able to modulate immune responses with different types of secretion molecules. Methods: The initial aim of the present study was to develop for the first time a culture protocol in order to expand hBTSC invitro through passages, allowing to maintain a similar stem cell and secretome profile. Furthermore, we investigated the secretome profile of the hBTSC to assess the production of molecules capable of affecting immune feedback. Results: We found that hepatocyte growth factor produced by hBTSC exerts its cytoprotective role inducing apoptosis in human immune cells, such as lymphocytes. Conclusions: The present study, therefore, supports the hypothesis that hBTSC can be useful for the purpose of regenerative medicine, as they can be banked and expanded, and they can secrete immunoregulatory factors.

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.

2016 - Effectiveness of the antibody-derived killer peptide on Candida albicans biofilm [Poster]
Paulone, Simona; Orsi, Carlotta Francesca; Ardizzoni, Andrea; Polonelli, Luciano; Magliani, Walter; Conti, Stefania; Posteraro, Brunella; Sanguinetti, Maurizio; Cermelli, Claudio; Peppoloni, Samuele; Blasi, Elisabetta
abstract

2015 - C. albicans with different genomic background reveal diverse host adaptation and differential processing by phagocytes [Abstract in Atti di Convegno]
Rizzetto, L; Di Paola, M; Colombari, B; De Filippo, C; Ardizzoni, A; Bernà, L; Tocci, N; Lionetti, P; Blasi, E; Cavalieri, D; Peppoloni, S
abstract

2015 - CANDIDA ALBICANS HYPHAL DEVELOPMENT AND BIOFILM FORMATION/PERSISTENCE ARE DIFFERENTIALLY AFFECTED BY MOUTHWASHES DEPENDING UPON THEIR COMPOSITION. [Abstract in Atti di Convegno]
Paulone, Simona; Malavasi, G.; Neglia, Rachele Giovanna; Orsi, Carlotta Francesca; Peppoloni, Samuele; Ardizzoni, Andrea; Cermelli, Claudio; 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, the daily use of toothbrush and mouthwashes. Among several virulence factors, C. albicans has the ability to switch from yeast-to-hyphal forms and to produce biofilm, thus contrasting antimicrobial agents and host immune defences as well [1-3]. The aim of this study is to investigate the susceptibility of C. albicans, in terms of growth, hyphal formation and biofilm production/persistence, to mouthwashes with different composition. Materials and Methods: Candida albicans SC5314 and 7 commercial mouthwashes have been employed: 3 with 0.2% chlorhexidine digluconate; 1 with 0.06% chlorhexidine digluconate and 250 ppm F- sodium fluoride; 3 with 125-250ppm Fsodium, amino and/or stannous fluoride. The effects of the mouthwashes on C. albicans were assessed by crystal violet, tetrazolium salt reduction assays and morphological analysis by microscopy. By using four different protocols, combining different concentrations and different contact times, the mouthwashes were tested against: 1) C. albicans growth on Muller-Hinton agar, as assessed by disk diffusion assay; 2) hyphal formation and biofilm production by yeast cells, cultured in RPMI + 10% FBS; 3) early pre-formed (24 h-old) Candida biofilm and 4) mature (48 h-old) Candida biofilm. Results: The highest anti-Candida activity was consistently exhibited by the chlorhexidine digluconate- containing mouthwashes, irrespective of the protocols employed. Morphological and functional impairments occurred and fungal survival/ growth were impaired as well; the effects strictly depended on both the dilution employed and the time of contact. Discussion and Conclusions: Both C. albicans hyphal development and biofilm formation/persistence are affected by mouthwashes, provided that they contain chlorhexidine digluconate. Thus, special attention should be used when choosing mouthwashes for prevention and/or treatment of Candida-associated oral pathologies. 1. Ramage G, et al. Eukaryot Cell 2005; 4: 633-8. 2. Orsi CF, et al. Microb Pathog 2014; 69-70: 20-7 3. Orsi CF, et al. J Biol Regul Homeost Agents 2014; 28: 743-52.

2015 - Candida albicans isolates with different genomic backgrounds show diverse host adaptation and differential susceptibility to microgial cell-mediated defenses [Poster]
Peppoloni, S; Blasi, E; Rizzetto, L; Di Paola, M; Paulone, S; Colombari, B; Ardizzoni, A; Tocci, N; Lionetti, P; De Filippo, C; Bernà, L; Cavalieri, D
abstract

2014 - An antibody reactivity-based assay for diagnosis of invasive candidiasis using protein array [Articolo su rivista]
Ardizzoni, Andrea; Posteraro, Brunella; Baschieri, MARIA CRISTINA; Bugli, Francesca; Sáez Rosòn, Arantza; Manca, Lidia; Cacaci, Margherita; Paroni Sterbini, Francesco; De Waure, Chiara; Sevilla, Maria Jesus; Peppoloni, Samuele; Sanguinetti, Maurizio; Moragues, Maria Dolores; Blasi, Elisabetta
abstract

The increased incidence of invasive candidiasis and of patients at risk requires early diagnosis and treatment to improve prognosis and survival. The aim of this study was to set up a ten-protein array-based immunoassay to assess the IgG antibody responses against ten well-known immunogenic C. albicans proteins (Bgl2, Eno1, Pgk1, Pdc11, Fba1, Adh1, Als3, Hwp1, Hsp90 and Grp2) in 51 patients with invasive candidiasis (IC) and in 38 culture-negative controls (non-IC). Antibody levels were higher against Bgl2, Eno1, Pgk1, Als3, Hwp1 and Grp2, than against Adh1, Pdc11, Fba1 and Hsp90, irrespectively of the patient group considered. Moreover, the IgG levels against Bgl2, Eno1, Pgk1 and Grp2 were significantly higher in IC than in non-IC patients. Furthermore, the ROC curves generated by the analysis of the antibody responses against Bgl2, Grp2 and Pgk1 displayed AUC values above 0.7, thus discriminating IC and non-IC patients. According to these results, the employment of the microarray immunoassay (a rapid, sensitive and multiparametric system), in parallel with conventional diagnostics, can help to spot IC patients. This ultimately will allow to initiate an early, focused and optimized antifungal therapy.

2014 - Differential efficacy of endodontic obturation procedures: an ex vivo study [Articolo su rivista]
Ardizzoni, Andrea; Generali, Luigi; Righi, Elena; Baschieri, MARIA CRISTINA; Cavani, Francesco; Manca, Lidia; Lugli, Eleonora; Migliarese, Luigi; Blasi, Elisabetta; Neglia, Rachele Giovanna
abstract

By means of a double-chamber model, different root canal filling materials and procedures were compared. Briefly, the root canals of single-rooted human teeth, extracted for periodontal reasons, were instrumented and obturated by gutta-percha/Pulp Canal Sealer EWT (PCS) or by Resilon, in association with different sealers (Real Seal, RelyX Unicem or Meta). Obturation was achieved by traditional continuous wave of condensation technique (TCWCT), a modified version of it (MCWCT), or single cone technique (SCT). The obturated roots, inserted in a double-chamber model, were sterilized by gamma irradiation. Next, Enterococcus faecalis was added to the upper chamber and the specimens were incubated at 37 °C for up to 120 days; the development of turbidity in the lower chambers' broths indicated bacterial leakage through the obturated root canals. The kinetics of leakage were analyzed in different groups by means of Kaplan-Meier statistics and compared by log-rank test. The results showed that root canals obturated with either gutta-percha/PCS using the MCWCT, Resilon/Real Seal SCT or Resilon/RelyX Unicem using the TCWCT displayed significantly better performance than the remaining groups (p < 0.01). Histological evaluation, performed to investigate microbial localization inside specimens, confirmed that this parameter varied according to the obturation procedures and materials employed. This ex vivo study indicates that gutta-percha/PCS, if used with the MCWCT, is as effective as Resilon when coupled to Real Seal with the SCT or, interestingly, to RelyX Unicem with the TCWCT. These data suggest that further improvement of the currently employed root canal filling procedures is achievable, depending on both the filling materials and the technique employed, thus encouraging clinical studies in this direction.

2014 - Immunoreactivity of microglial cells to in vitro infection by Candida albicans isolates with different genomic backgrounds [Poster]
Peppoloni, S; Colombari, B; Ardizzoni, A; Rizzetto, L; De Filippo, C; Di Paola, M; Bernà, L; Cavalieri, D; Blasi, E
abstract

2014 - Impact of Candida albicans hyphal wall protein 1 (HWP1) genotype on biofilm production and fungal susceptibility to microglial cells [Articolo su rivista]
Orsi, Carlotta Francesca; Borghi, Elisa; Colombari, Bruna; Neglia, Rachele Giovanna; Quaglino, Daniela; Ardizzoni, Andrea; Morace, Giulia; Blasi, Elisabetta
abstract

The hyphal wall protein 1 (HWP1) gene of Candida albicans encodes for a fungal cell wall protein, required for hyphal development and yeast adhesion to epithelial cells; yet, its role in pathogenesis remains largely unknown. In the present study, we analyzed two C. albicans laboratory strains, the DAY286 (HWP1/HWP1) and the null mutant FJS24 (hwp1/hwp1) and six clinical isolates [3 harbouring the homozygous HWP1 gene (HWP1/HWP1) and 3 the heterologous gene (HWP1/hwp1)]. Biofilm production, fungal HWP1 mRNA levels and ultrastructural morphology were investigated; also, the susceptibility of these strains to microglial cells was evaluated, in terms of fungal damage and immune cell-mediated secretory response. When comparing the two laboratory strains, biofilm was produced to a similar extent independently on the genetic background, while the susceptibility to microglial cell-mediated damage was higher in the hwp1/hwp1 mutant than in the HWP1/HWP1 counterpart. Also, transmission electron microscopy revealed differences between the two in terms of abundance in surface adhesin-like structures, fungal cell wall shape and intracellular granules. When comparing the clinical isolates grouped according to their HWP1 genotype, reduced biofilm production and increased susceptibility to microglial cell-mediated damage occurred in the HWP1/hwp1 isolates with respect to the HWP1/HWP1 counterparts; furthermore, upon exposure to microglial cells, the HWP1/HWP1 isolates, but not the HWP1/hwp1 counterpart, showed enhanced HWP1 mRNA levels. Finally, both laboratory and clinical isolates exhibited reduced ability to stimulate TNFα and nitric oxide production by microglial cells in the case of heterozygous or null mutant HWP1 genotype. Overall, these data indicate that C. albicans HWP1 genotype influences pathogen morphological structure as well as its interaction with microglial cells, while fungal biofilm production results unaffected, thus arguing on its role as virulence factor that directly affects host mediated defences.

2014 - Indagini multidirezionali nella diagnosi di infezione fungina invasiva: ricerca di beta-glucano e rilevazione di profili anticorpali specifici [Abstract in Atti di Convegno]
Pini, P; Ardizzoni, A; Orsi, Cf; Bettua, C; Venturelli, C; Girardis, M; Luppi, M; Codeluppi, M; Peppoloni, S; Posteraro, B; Sanguinetti, M; Saez-Roson, A; Moragues, Md; Blasi, E
abstract

2014 - Inhibitory effects of different lactobacilli on Candida albicans hyphal formation and biofilm development. [Articolo su rivista]
Orsi, Carlotta Francesca; Sabia, Carla; Ardizzoni, Andrea; Colombari, Bruna; Neglia, Rachele Giovanna; Peppoloni, Samuele; Morace, G; Blasi, Elisabetta
abstract

The aim of this study was to investigate the effects of different species of Lactobacilli on hyphal formation and biofilm development by the opportunistic fungal pathogen Candida albicans. We employed 4 different Lactobacillus species, namely L. rhamnosus, L. acidophilus, L. plantarum and L. reuteri, and 2 C. albicans strains, the reference DAY286 and its isogenic hwp1/hwp1 mutant, the FJS24 strain. As assessed by morphological analysis and quantitative colorimetric assays, Lactobacillus crude filtrate supernatant fluids (CFSF) affected Candida, impairing both hyphal formation and biofilm production. The CFSF-mediated phenomenon occurred in a dilution- and time-dependent manner and was consistently observed, irrespective of the C. albicans HWP1 genotype.

2014 - Initial geno-phenotypic characterization of colon mycobiota from healthy donors [Poster]
Rossi, M; Colombari, B; Ardizzoni, A; Raimondi, S; Gozzoli, C; Simone, M; Orsi, Cf; Neglia, Rg; Amaretti, A; Peppoloni, S; Cermelli, C; Blasi, E
abstract

2013 - DIAGNOSI SIEROLOGICA, MEDIANTE MYCOARRAY, DI UN CASO DI ISTOPLASMOSI DA IMPORTAZIONE IN UN TURISTA ITALIANO DI RITORNO DAL BRASILE [Poster]
Ardizzoni, Andrea; Baschieri, MARIA CRISTINA; Manca, Lidia; Salvadori, Caterina; Marinacci, Ginevra; Farina, Claudio; Viale, Pierluigi; Blasi, Elisabetta
abstract

Introduzione L’istoplasmosi, infezione granulomatosa provocata dal fungo dimorfo Histoplasma capsulatum, viene contratta per inalazione dei conidi in aree dove tale fungo è endemico. Date le manifestazioni cliniche aspecifiche, la diagnosi di laboratorio è estremamente importante per confermare il sospetto di malattia, soprattutto in aree come la nostra, in cui i casi, sebbene rari, possono essere sia da importazione che autoctoni. Recentemente, è stato messo a punto il “mycoarray”, un saggio multiparametrico rapido, in grado di rilevare anticorpi specifici nei confronti di Histoplasma ed altri funghi dimorfi (1). Metodi Estratti antigenici da funghi dimorfi (H. capsulatum, C. immitis, B. dermatitidis, P. brasiliensis) sono stati deposti su vetrini microarray mediante un sistema robotizzato ad alta precisione. I vetrini sono stati cimentati con il siero del paziente e la formazione degli immunocomplessi è stata rivelata utilizzando anticorpi secondari marcati in fluorescenza. Dopo lettura, mediante un apposito scanner, il segnale è stato quantificato ed analizzato. Risultati Il paziente (maschio italiano di 30 anni, di ritorno da un viaggio “on-the-road” in Brasile), era stato ricoverato per una febbre persistente che non rispondeva a terapia antibiotica empirica. A seguito della presenza di infiltrati e di linfoadenopatia mediastinica, rivelati mediante TAC del torace, è stata condotta una biopsia linfonodale suggestiva di infezione fungina da dimorfi. L’analisi del siero mediante mycoarray ha mostrato una elevata sieroreattività, IgG e IgM, nei confronti degli antigeni di H. capsulatum. Nessuna reattività è invece stata evidenziata nei confronti degli antigeni da altri funghi dimorfi (2). Conclusioni Grazie alle sue peculiarità (miniaturizzazione, multiparametricità e rapidità di esecuzione) il mycoarray si rivela uno strumento prezioso per facilitare la diagnosi rapida di micosi primitive, specialmente in paesi non endemici. Ringraziamenti Lavoro in parte supportato da MIUR, PRIN-200985J87J Bibliografia (1) Ardizzoni et al., (2011) New Microbiol, 34:307-16. (2) Ardizzoni et al., (2013) JTM, 20:336-9

2013 - Diagnostic utility of a microarray based on 11 proteins of Candida albicans for the diagnosis of invasive candidiasis [Abstract in Rivista]
Sáez Rosón, A; Ardizzoni, Andrea; Baschieri, MARIA CRISTINA; Bugli, F; Paroni Sterbini, F; Orsi, Carlotta Francesca; Manca, Lidia; Sanguinetti, M; Posteraro, B; Cuétara, Ms; Olazabal, I; García Ruiz, Jc; Peppoloni, Samuele; Blasi, Elisabetta; Moragues, Md
abstract

Objective. The purpose of this study was to set up a protein microarray test based on 11 proteins of C. albicans for the detection of IgG antibodies in sera from patients with invasive candidiasis (IC) and determine its diagnostic utility. Methods and patients. The microarray was set up with 10 C. albicans recombinant proteins: Eno1, Als3-N, Hwp1-N, Eno1-RM, Bgl2, Grp, Pgk1, Fba, Pdc, Adh1, and an enolase purified from a dithiothreitol-extract of cell walls from C. albicans mycelial phase (CW-Eno). Antigens, IgG standard curve, and controls were printed onto glass slides using computer controlled high speed robotics. The arrays were processed with sera from IC and control patients and then fluorescence labeled secondary antibodies were added. The signal captured by each spot was detected by a laser scan reader and quantified. When possible, the cut-off values werecalculated as mean mass of antibody detected in the control group sera plus 2 times the standard deviation. Twenty three sera from 15 patients with proven IC due to C. albicans and 33 sera from 28 patients at risk for IC but without clinical or microbiological data confirming a fungal infection (control group) were studied. Results. The performance of the microarray assay for each antigen is shown in Table 1. The best results were obtained with CW-Eno, showing a sensitivity and a specificity of 80 and 96.43%, respectively. Pgk1, Grp and Eno1 exhibited lower sensitivity values (33-47%), but their specificity reached values equal or greater than 93% Also, for all the other antigens, the specificity was very high with values above 96%. Furthermore, we clustered those antigens which separately had returned the best sensitivity. As shown in Table 2, the clustering raised the sensitivity up to 100%, while the specificity ranged between 85 and dropped to 89% . Conclusions. The detection of serum IgG antibodies against proteins of C. albicans by a protein microarray exhibited moderate diagnostic utility values when assessed independently, being CW-Eno the main exception. However, when considering the microarray results either as a whole or as clusters of selected proteins (CW-Eno, Pgk1, Eno1 and Grp; or CW-eno and Pgk1), the system proved to be an efficient diagnostic tool for IC. Further studies with a larger number of patients are needed to confirm the results of the present study.

2013 - IL MYCOARRAY: UN TEST RAPIDO PER LA DIAGNOSI SIEROLOGICA DI MICOSI ENDEMICHE [Poster]
Ardizzoni, Andrea; Baschieri, MARIA CRISTINA; Manca, Lidia; Meacci, Marisa; Venturelli, Claudia; Farina, Claudio; Blasi, Elisabetta
abstract

Introduzione Le micosi da funghi dimorfi, estremamente rare in Europa, sono rappresentate da casi di importazione, con l’eccezione della istoplasmosi per la quale sono stati segnalati anche casi autoctoni. La bassa frequenza, in combinazione con l’aspecificità delle manifestazioni cliniche, rende difficile una diagnosi rapida. Scopo del presente studio è la valutazione dell’utilizzo del saggio mycoarray (1) come strumento multiparametrico e rapido, a supporto della sierologia convenzionale, nella diagnosi di laboratorio delle micosi endemiche. Metodi Antigeni fungini (H. capsulatum, C. immitis, B. dermatitidis e P. brasiliensis), diluizioni scalari di anticorpi IgG e IgM e controlli sono stati deposti su vetrini microarray per mezzo di un sistema robotizzato ad alta precisione. I vetrini così ottenuti sono stati cimentati col siero, opportunamente diluito, e successivamente con anticorpi secondari marcati con fluorofori per rivelare l’avvenuta formazione degli immunocomplessi. Il segnale è stato acquisito mediante uno scanner, quantificato ed analizzato per mezzo di un apposito software. Risultati Il mycoarray ha mostrato elevate sensibilità e specificità: un primo studio retrospettivo, condotto su sieri da pazienti con diagnosi certa di istoplasmosi o di coccidioidomicosi, ha fornito risultati consistenti con i dati clinici e di laboratorio, acquisiti con la diagnostica di routine. Indagini su ulteriori campioni da casi clinici “sospetti”, analizzati con il mycoarray, hanno fornito risultati originali utili per la diagnosi definitiva di micosi endemica. Conclusioni Il mycoarray presenta una serie di peculiarità (miniaturizzazione, multiparametricità e rapidità di esecuzione) che lo rendono estremamente utile come strumento di laboratorio a sussidio del percorso convenzionale nella diagnosi di micosi primitive, specialmente in zone a bassa endemicità. Ringraziamenti Lavoro in parte supportato da MIUR, PRIN-200985J87J Bibliografia (1) Ardizzoni et al., (2011) New Microbiol, 34:307-16.

2013 - LA PROTEINA SPR1875 DI STREPTOCOCCUS PNEUMONIAE PROTEGGE IL BATTERIO DAL KILLING DELLA MICROGLIA [Poster]
Peppoloni, Samuele; Colombari, Bruna; Beninati, Concetta; Felici, Franco; Teti, Giuseppe; Speziale, Pietro; Ricci, Susanna; Ardizzoni, Andrea; Manca, Lidia; Blasi, Elisabetta
abstract

Introduzione. Streptococcus pneumoniae è un’importante causa di morbilità e mortalità nel mondo. Per lo sviluppo di vaccini efficaci nel prevenire le malattie pneumococciche è cruciale l’identificazione e la caratterizzazione degli antigeni batterici coinvolti nella risposta immunitaria dell’ospite. Nel presente lavoro, abbiamo utilizzato il ceppo acapsulato DP1004 ed il suo mutante isogenico knock-out per spr1875 al fine di studiare il ruolo della proteina Spr1875 nell’interazione di S. pneumoniae con la microglia. Materiali e metodi. Mediante screening di una libreria genomica phage-display con sieri di pazienti convalescenti sono stati identificati cloni con epitopi B pneumococcici. Tra questi, è stato isolato un frammento, denominato R4, codificato dalla ORF spr1875. Per valutare il ruolo di Spr1875 nella patogenicità di S. pneumoniae è stato costruito un ceppo mutante privo della proteina. Mediante l’uso di un modello di infezione in vitro ed un saggio di protezione agli antibiotici abbiamo valutato la capacità delle cellule microgliali BV2 di fagocitare ed uccidere il mutante spr1875-ko, nonché la sua sopravvivenza all’interno delle cellule BV2, rispetto al ceppo parentale DP1004. Risultati. Entrambi i ceppi erano fagocitati efficientemente ed in modo simile dalla microglia. Tuttavia, la sopravvivenza del ceppo mutante spr1875-ko all’interno delle BV2 era significativamente più bassa di quella osservata con il ceppo selvaggio. In accordo, la percentuale di fagolisosomi acidi in cellule microgliali contenenti batteri spr1875-ko era marcatamente più elevata di quella registrata in cellule infettate con DP1004. Inoltre, erano osservate differenze significative tra i due ceppi anche in termini di suscettibilità al killing della microglia. Conclusioni. Questi risultati indicano che l’antigene Spr1875 protegge il batterio dal killing mediato dalla microglia, suggerendo che questa proteina possa giocare un importante ruolo nella patogenesi della meningite pneumococcica.

2013 - RICERCA DI MARCATORI PER LA DIAGNOSI DI INFEZIONE FUNGINA INVASIVAE (IFI) MEDIANTE APPROCCIO DIAGNOSTICO COMBINATO [Poster]
Bettua, Clotilde; Ardizzoni, Andrea; Orsi, Carlotta Francesca; Pini, Pietro; Venturelli, Claudia; Girardis, Massimo; Peppoloni, Samuele; Posteraro, Brunella; Sanguinetti, Maurizio; Moragues, Maria Dolores; Blasi, Elisabetta
abstract

Introduzione L'incidenza di IFI ed in particolare delle candidiasi nei pazienti ricoverati in terapia intensiva è in costante aumento ed è associata ad un’elevata mortalità. Evidenze recenti sottolineano l’importanza di indagini multidirezionali e multi-parametriche ai fini di una diagnosi più rapida e precoce possibile. Scopo del presente studio è valutare l’efficacia di un approccio combinato che prevede la ricerca dell’antigene beta-glucano e di anticorpi specifici verso antigeni selezionati di Candida. Metodi 18 pazienti (Reparto di Terapia Intensiva, AOU-Policlinico Modena) sono stati arruolati e suddivisi in due gruppi: gruppo IFI (pazienti con aspergillosi o candidiasi invasiva proven/probable o con diagnosi clinica di pneumocistosi polmonare) e gruppo non-IFI (soggetti ospedalizzati con diagnosi diversa da IFI). In totale, sono stati saggiati 42 sieri con il saggio panfungino (1-3)-β-D-glucano (BG, Fungitell ®) e mediante un saggio sierologico “home-made” basato su microarray proteico per la determinazione quantitativa della risposta anticorpale nei confronti di 11 antigeni di Candida albicans. Risultati Il saggio BG ha mostrato sensibilità, specificità, valore predittivo positivo e negativo di 100%, 85,7%, 88,9 % e 100% rispettivamente; l’analisi delle curve ROC ha restituito una AUC di 0.857 (95% CI: 0.577-1). Il saggio sierologico ha rilevato nei pazienti con emocoltura positiva, una risposta anticorpale nei confronti di 3 antigeni (Bgl2, Pgk1 e Grp2), recentemente descritti come marker diagnostici di candidasi invasiva (Ardizzoni et al., 2013 – submitted). Nessuna risposta anticorpale significativa è invece emersa dall’analisi del siero di pazienti con probabile/possibile IFI e di pazienti non-IFI. Conclusioni La combinazione dei risultati del BG assay e del saggio sierologico in microarray, condotti parallelamente all’emocoltura, può fornire indicazioni diagnostiche e prognostiche aggiuntive in pazienti con provata o sospetta candidiasi invasiva. Riteniamo che questo approccio diagnostico combinato possa rivelarsi particolarmente utile soprattutto nei pazienti con emocoltura negativa.

2013 - The Mycoarray as an aid for diagnosis of an imported case of Histoplasmosis in an Italian traveler returning from Brasil [Articolo su rivista]
Ardizzoni, Andrea; Baschieri, Maria Cristina; Manca, Lidia; Salvadori, Caterina; Marinacci, Ginevra; Farina, Claudio; Viale, Pierluigi; Blasi, Elisabetta
abstract

We describe an imported case of Histoplasmosis, whose serological profile was established by means of a protein-based microarray platform, the recently described mycoarray. Because of its peculiarities, such a novel tool greatly facilitates the rapid and multiparametric assessment of patients' serological status and lends itself to be employed as an aid in the diagnosis of primary mycoses, especially in non endemic countries.

2013 - The Spr1875 protein confers resistance to the microglia-mediated killing of Streptococcus pneumoniae [Articolo su rivista]
Peppoloni, Samuele; Colombari, Bruna; Beninati, C; Felici, F; Teti, G; Speziale, P; Ricci, S; Ardizzoni, Andrea; Manca, Lidia; Blasi, Elisabetta
abstract

By screening a whole-genome λ-display library of Streptococcus pneumoniae, we have previously identified a novel surface protein, named Spr1875, that exhibited immunogenic properties and was closely related to pneumococcal virulence. In the present study, we investigated the role of the Spr1875 antigen in the interaction of S. pneumoniae with microglia, the resident brain macrophages. By using an in vitro infection model, the BV2 microglial cell line was challenged with the S. pneumoniae strain DP1004 and its isogenic spr1875-deleted mutant (Δspr1875). Both strains were phagocytosed by microglia efficiently and to a similar extent; however, the DP1004 strain was more resistant than the Δspr1875 mutant to the intracellular killing, as assessed by antibiotic protection and phagosome maturation assays. Moreover, significant differences between the two strains were also observed in terms of susceptibility to microglia-mediated killing. Taken together, these results indicate that S. pneumoniae-microglial cell interplay is influenced by the presence of Spr1875, suggesting that this protein may play a role in the pathogenesis of pneumococcal meningitis.

2013 - The Spr1875 protein of Streptococcus pneumoniae protects the bacterium from microglia-mediated killing [Poster]
Peppoloni, Samuele; Colombari, Bruna; Beninati, Concetta; Felici, Franco; Teti, Giuseppe; Speziale, Pietro; Ardizzoni, Andrea; Manca, Lidia; Blasi, Elisabetta
abstract

Objectives: S. pneumoniae is a major cause of morbidity and mortality worldwide. For the development of vaccines effective in preventing pneumococcal infection identification/characterization of bacterial antigens involved in host immune response is crucial. In this study, we employed the unencapsulated DP1004 strain and its isogenic spr1875-ko mutant to investigate the role of the Spr1875 protein in the interaction of S. pneumoniae with microglia, the resident brain macrophages. Methods: by screening a whole genome phage display library with sera from convalescent patients we identified clones carrying pneumococcal B-cell epitopes. Among these, we isolated an antigenic fragment, designated R4, encoded by the ORF spr1875 in the R6 strain genomic sequence. To evaluate whether Spr1875 is involved in pneumococcal pathogenicity a mutant strain lacking this protein was constructed. Here, by using an in vitro infection model and an antibiotic protection assay, we investigated the ability of microglial BV2 cells to phagocytose and kill the spr1875-ko mutant, as well as the survival of these bacteria inside BV2 cells. Results: both strains were internalized by microglia efficiently and to a similar extent. However, the survival of the spr1875-ko strain inside the cells was significantly lower than that observed with the parental DP1004. Accordingly, the percentage of acidic phagosomes in BV2 cells bearing spr1875-ko pneumococci was markedly higher than that containing DP1004 bacteria. A different susceptibility between the two strains was also observed when S. pneumoniae-microglia interaction was assessed by a bactericidal assay. Conclusion: these findings indicate that the Spr1875 antigen confers resistance to microglia-mediated killing of S. pneumoniae, suggesting that this protein may play an important role in the pathogenesis of pneumococcal meningitis.

2012 - IL MICROARRAY PROTEICO NELL’ANALISI DEI LIQUIDI AMNIOTICI: RICERCA DI MARKER PRECOCI DI INFIAMMAZIONE E/O INFEZIONE CORRELABILI AL PARTO PRETERMINE [Abstract in Atti di Convegno]
Manca, Lidia; Ardizzoni, Andrea; Baschieri, MARIA CRISTINA; Capodanno, Francesco; Soncini, Emanuele; Peppoloni, Samuele; LA SALA, Giovanni Battista; Blasi, Elisabetta
abstract

40° Congresso della SIM, Riccione 7-10 ottobre, 2012

2012 - PROFILO DELLA RISPOSTA ANTICORPALE A CANDIDA ALBICANS IN DIFFERENTI CATEGORIE DI PAZIENTI A RISCHIO DI INFEZIONI FUNGINE INVASIVE [Poster]
Baschieri, MARIA CRISTINA; Ardizzoni, Andrea; Manca, Lidia; Saez Roson, Arantza; Bugli, Francesca; Paroni Sterbini, Francesco; Orsi, Carlotta Francesca; Sanguinetti, Maurizio; Posteraro, Brunella; Moragues, Maria Dolores; Blasi, Elisabetta
abstract

L’aumento continuo nel numero e nella tipologia dei pazienti immunocompromessi comporta un continuo incremento dei casi di infezioni fungine invasive (IFI), il cui esito infausto è spesso ascrivibile al ritardo nella diagnosi e conseguentemente all’impossibilità di instaurare una precoce ed appropriata terapia antifungina. La candidosi invasiva (IC), particolarmente frequente, riveste un ruolo di primaria importanza tra le IFI, visto anche l’elevato tasso di mortalità (30-50%). I metodi comunemente impiegati nella diagnosi di IFI sono spesso caratterizzati da lunghi tempi di indagine oltre che da scarsa sensibilità e specificità. Grazie all’impiego di nuove tecnologie particolarmente versatili e molto sensibili (es: microarray proteici), studi recenti hanno riproposto la sierologia come percorso diagnostico rilevante nella identificazione rapida e precoce di IC. In particolare, sono state fornite le prime evidenze circa la presenza di specifici marker immunologici in grado di portare alla distinzione tra pazienti affetti da IC e soggetti di controllo. Alla luce di questi dati, abbiamo messo a punto un microarray proteico per la rilevazione dei titoli anticorpali IgG nei confronti di 10 diversi antigeni ricombinanti di C. albicans (Adh, Als-3, Bgl-2, Fba, Grp, Hwp-1, Pdc, Pgk e due diverse forme ricombinanti di Eno-1) e di 1 antigene purificato (Eno-1) dalla parete. Mediante questo chip, vengono saggiati sieri da diverse categorie di soggetti (pazienti affetti da IC, pazienti ospedalizzati per patologie non IFI e soggetti sani di controllo). I dati finora ottenuti mostrano come i livelli di anticorpi nei confronti di alcuni antigeni, in particolare Eno-1, Grp e Pgk, siano più alti nei pazienti con IC rispetto alle altre categorie di soggetti considerati. Una volta acquisiti sufficienti dati, verranno fatte valutazioni di tipo statistico e sarà quindi possibile stabilire l’efficacia di questo nuovo percorso di indagine, sia nell’individuazione che nel monitoraggio di soggetti con e/o a rischio di IFI.

2012 - Protein microarrays on midtrimester amniotic fluids: a novel approach for the diagnosis of early intrauterine inflammation related to preterm delivery [Articolo su rivista]
La Sala, Giovanni Battista; Ardizzoni, Andrea; Capodanno, F; Manca, Lidia; Baschieri, Maria Cristina; Soncini, E; Peppoloni, Samuele; Blasi, Elisabetta
abstract

Novel technologies that allow simultaneous assessment of multiple biomarkers provide new and promising diagnostic/prognostic approaches. By protein microarrays, here we analyzed amniotic fluids (AF) from 50 women with preterm delivery (PTD) and 50 control women, who delivered at term. In detail, cytokines, chemokines, matrix metalloproteinases and antigen-specific antibodies were assessed. The AF analysis showed significant differences between women with preterm and term delivery in the levels of IL-1alpha, IL-1beta, IL-4, IL-6, IL-8, MCP-1, IFN-gamma and anti-HSV2 IgG. No significant differences were observed in the levels of TNF-alpha, MMP-2, MMP-9 and specific IgG for seven vertically transmitted pathogens. In conclusion, we demonstrated the feasibility of protein microarrays in the diagnosis of early intrauterine inflammation. The significant association between the increased levels of certain cytokines and preterm delivery argues on their relevance as early pathogenetic markers for identification of risk patients.

2011 - Acido ialuronico: valutazione della interazione in vitro con batteri e miceti [Abstract in Atti di Convegno]
Palmieri, Beniamino; Iannitti, Tommaso; Ardizzoni, Andrea; Caratozzolo, Manuela; Blasi, Elisabetta; Neglia, Rachele Giovanna
abstract

2011 - DETECTION OF ANTIGEN-SPECIFIC ANTIBODIES IN SERUM AND FOLLICULAR FLUIDS BY PROTEIN MICROARRAYS. [Poster]
Ardizzoni, Andrea; Baschieri, MARIA CRISTINA; Manca, Lidia; Capodanno, Francesco; LA SALA, Giovanni Battista; Blasi, Elisabetta
abstract

Background. Wide spectrum serological screening of women prior to or during pregnancy may greatly help in preventing vertically transmitted infections (VTI) and their severe consequences to the foetus/newborn. Protein microarrays, made up by spotting many antigens onto a restricted area of a microscope slide, allow to detect, in one shot, specific antibodies against a wide range of antigenic specificities. Objectives. To detect, in paired serum and follicular fluid (FF) samples, antigen-specific antibodies against vertically transmitted pathogens by protein microarrays; to investigate the potential relation between antibody profiles and pregnancy outcome. Methods. Serum and FF paired samples were collected from 102 women undergoing in vitro fertilization (IVF). Antigens, human antibodies and controls were spotted in an orderly manner by high speed robotics. Microarrays were processed with serum or FF and the occurred immunocomplexes were revealed by fluorescently-labelled secondary antibodies. The fluorescent signals were read by a laser scanner and quantified by a dedicated software. Conclusions. Antigen-specific antibodies can be effectively detected in serum and FF by microarray. The presence or absence of certain antigen-specific antibodies is significantly related to clinical parameters such as the number of inseminated good quality oocytes or the number of successful embryo transfers. These results encourage protein microarrays employment for wide spectrum investigations in diagnosing VTI; in particular, the use of biological matrices other than serum may help addressing yet unravelled questions.

2011 - Detection of follicular fluid and serum antibodies by protein microarrays in women undergoing in vitro fertilization treatment. [Articolo su rivista]
Ardizzoni, Andrea; Manca, Lidia; Capodanno, F; Baschieri, MARIA CRISTINA; Rondini, I; Peppoloni, Samuele; Righi, Elena; LA SALA, Giovanni Battista; Blasi, Elisabetta
abstract

A protein microarray serological assay was used to assess the antibody profile of 102women subjected to in vitro fertilization treatment. The studies were conducted on pairs of serum and follicular fluid samples, collected from each woman on the same day at the time of oocyte recovery. The samples, stored as frozen aliquotes, were assessed by both microarray and ELISA. Follicular fluids and sera were screened to detect the presence of specific IgG and IgM antibodies against seven vertically transmitted pathogens. The IgG reactivityof follicular fluids closely mirrored that of serum in all the patients and for all the antigens, with an agreement of more than 85%. IgM antibodies were undetectable in follicular fluids. The antibody patterns were subsequently related to the biological and clinical outcomesof in vitro fertilization cycles. The results showed that varicella zoster virus (VZV) IgG positive women and cytomegalovirus (CMV) IgG negative women had on average a higher number of inseminated, good quality oocytes compared to VZV IgG negative and CMV IgG positive women. In addition, the rate of successful embryo transfers was significantly higher in Toxoplasma gondii IgG negative women than in their positive counterparts. Overall, the microarray was proven to be a suitable tool for detecting analytes in follicular fluids, therefore supporting its application in a wide spectrum of investigations.

2011 - I MICROARRAY PROTEICI: NUOVI STRUMENTI DI INDAGINE NELLA DIAGNOSTICA DELLE INFEZIONI DA PATOGENI DEL COMPLESSO TORCH. [Poster]
Ardizzoni, Andrea; Manca, Lidia; Baschieri, MARIA CRISTINA; Capodanno, Francesco; LA SALA, Giovanni Battista; Blasi, Elisabetta
abstract

Lo screening sierologico su larga scala, effettuato prima o durante la gravidanza, fornisce un mezzo efficace per prevenire le infezioni a trasmissione verticale (ITV) e le loro gravi conseguenze per il prodotto del concepimento. Utilizzando un microarray proteico da noi messo a punto (1), abbiamo voluto determinare i livelli di anticorpi antigene-specifici nei confronti di patogeni a trasmissione verticale nel siero, nei liquidi follicolari (LF) e nei liquidi amniotici (LA) di pazienti gravide sottoposte a IVF, al fine di correlare profili anticorpali specifici con l’esito della gravidanza. Sieri e LF sono stati prelevati da 102 pazienti sottoposte a trattamenti di procreazione medicalmente assistita. LA sono stati ottenuti da 100 pazienti di cui 50 con parto pretermine e 50 con parto a termine. I microarray, allestiti con antigeni, anticorpi e controlli di segnale, come precedentemente descritto (1), sono stati processati con siero, LF o LA e la formazione degli immunocomplessi è stata rivelata mediante anticorpi secondari marcati in fluorescenza. I segnali sono stati letti con uno scanner contenente laser a differenti lunghezze d’onda e successivamente quantificati ed analizzati da un software dedicato. Il saggio ha consentito di rivelare in maniera efficace IgG antigene-specifiche in tutte le matrici saggiate, mentre anticorpi IgM sono stati rilevati solamente nel siero. In particolare, la presenza o assenza di determinati anticorpi IgG nelle coppie di campioni siero/LF provenienti dalla stessa paziente è risultata significativamente correlata a parametri clinici, quali il numero di oociti inseminati di buona qualità o il numero di trasferimenti embrionali avvenuti con successo (2). L’analisi dei profili anticorpali sui LA nel secondo gruppo di 100 pazienti è tuttora in corso. Su questo gruppo di pazienti, inoltre, si sta impiegando un microarray commerciale per valutare la presenza di citochine e/o chemochine da correlare eventualmente con il quadro clinico della paziente e con l’esito della gravidanza (parto a termine o pretermine). Risultati preliminari hanno consentito di dimostrare l’efficacia dell’approccio, evidenziando la presenza di livelli significativi di alcune citochine nei fluidi amniotici. Sono in corso indagini per determinare il contenuto di citochine in tutti i LA oggetto di studio. I dati finora ottenuti sostengono fortemente l’utilizzo del microarray proteico in indagini ad ampio spettro nella diagnosi di ITV; inoltre, l’utilizzo di matrici biologiche differenti dal siero può fornire un valido contributo per la risoluzione di quesiti diagnostici ancora irrisolti. (1) Ardizzoni et al., Eur J Clin Microbiol Infect Dis, 2009. (2) Ardizzoni et al., J Reprod Immunol, 2011.

2011 - I MICROARRAY PROTEICI: POSSIBILI SCENARI NELLA DIAGNOSTICA DELLE MICOSI INVASIVE [Poster]
Blasi, Elisabetta; Baschieri, MARIA CRISTINA; Ardizzoni, Andrea
abstract

I microarray proteici sono comunemente distinguibili in due tipi, uno dedicato a stabilire la presenza e la quantità di una proteina o di un anticorpo specifico presenti in una determinata matrice biologica (“abundance-based microarrays”) e l’altro a valutarne la funzione (“function-based microarrays”) (1). Per quanto attiene alla prima categoria, sono stati descritti sistemi a cattura molto simili a quelli utilizzati nei saggi ELISA che prevedono l’immobilizzazione su un supporto solido di molecole di cattura. Tali molecole possono essere anticorpi, soprattutto monoclonali, per la rilevazione quali-quantitativa di analiti specifici, oppure antigeni purificati/ricombinati per la determinazione di specifici titoli anticorpali. Ad oggi, questo tipo di microarray ha trovato un grosso impiego nel campo della proteomica, nella ricerca di nuovi farmaci e nella identificazione di marcatori di malattia, principalmente nell’ambito delle infezioni virali e dei tumori. Le caratteristiche dei saggi diagnostici su piattaforma microarray, quali l’elevata sensibilità, la multiparametricità, la miniaturizzazione e la possibilità di automazione, hanno portato alla messa a punto di piattaforme innovative, particolarmente utili anche per la loro duttilità. L’utilizzo dei microarray proteici in diagnostica ha portato inoltre a riconsiderare la sierologia come strumento di indagine potenzialmente utile nella diagnosi di micosi opportunistiche invasive e nella valutazione dell’efficacia di terapie antifungine. In un lavoro recentemente pubblicato, è stato identificato un gruppo di 13 antigeni di Candida albicans che consente di discriminare pazienti con candidemia, da soggetti sani o colonizzati; sempre gli stessi autori hanno descritto un altro gruppo di 33 antigeni, che permette di distinguere sierologicamente pazienti in fase acuta di malattia da pazienti convalescenti (2). Dall’altra parte, nella diagnosi di micosi primitive da patogeni quali Histoplasma capsulatum e Coccidioides immitis, in cui la sierologia riveste un ruolo di primo piano, l’impiego del microarray proteico ha portato ad accorpare in un unico chip antigeni dei diversi patogeni (3). In particolare, il saggio messo a punto consente di identificare simultaneamente soggetti positivi per uno o più patogeni, sulla base dei livelli di IgM e/o IgG specifiche. Similmente, sono stati riconosciuti come marcatori di polmonite gli alti livelli di anticorpi sierici verso la proteina Msg1 di Pneumocystis jirovecii (4), mentre la comparsa di citochine proinfiammatorie e della proteina C-reattiva sono risultate preziosi marcatori sierologici precoci di aspergillosi invasiva (5). Nel complesso, visto l’incremento nel numero sia di micosi opportunistiche in soggetti particolarmente difficili sia di micosi primitive (in passato inusuali, ora più frequenti dato l’aumento dei flussi turistici e migratori), i contributi forniti dalle nuove tecnologie saranno fondamentali per comprendere meglio il quadro clinico associato alla micosi invasiva, grazie ad un metodo diagnostico veloce e multiparametrico. Questo aspetto risulterà particolarmente interessante in quanto consentirà di valutare contemporaneamente tipi diversi di parametri che includono non solo il patogeno ed i suoi prodotti, ma anche l’ospite con la sua peculiare reattività antimicrobica, sia innata che adattativa. (1) LaBaer and Ramachandran, Curr Opin Chem Biol, 2005 (2) Mochon et al., Plos Pathogens, 2010 (3) Ardizzoni et al., New Microbiol, 2011 (4) Djawe et al., Plos One, 2010 (5) Chai et al., J. Infect Dis, 2010

2011 - In vitro evaluation of antiviral and virucidal activity of a high molecular weight hyaluronic acid. [Articolo su rivista]
Cermelli, Claudio; Cuoghi, A; Scuri, M; Bettua, C; Neglia, Rachele Giovanna; Ardizzoni, Andrea; Blasi, Elisabetta; Iannitti, T; Palmieri, Beniamino
abstract

BACKGROUND: hyaluronic acid (HA), a non-sulphated glycosaminoglycan, is present in synovial fluid, vitreous humour serum and many connective tissues. Pharmaceutical preparations of HA are used in clinical practice for wound healing, joint pain, kerato-conjunctivitis, asthma, mouth care, oesophageal-reflux, and gastritis. Moreover, it is used as a filler to counteract ageing and facial lipoatrophy. Our study aims at investigating the in vitro antiviral activity of a high molecular weight HA.METHODS: the MTT test was used to rule out the potential toxic effects of HA on the different cell lines used in the antiviral assays. The antiviral activity of HA against Coxsackievirus B5, Herpes simplex virus-1, Mumps virus, Adenovirus-5, Influenza Virus A/H1N1, Human Herpesvirus-6, Porcine Parvovirus, Porcine Reproductive and Respiratory Syndrome Virus was assessed by virus yield assays.RESULTS: the most effective inhibition was observed against Coxsackievirus B5, with 3Log reduction of the virus yield at 4mg/ml, and a reduction of 3.5Log and 2Log, at 2mg/ml and 1mg/ml, respectively: the selectivity index was 16. Mumps virus was highly inhibited too showing a reduction of 1.7Log at 1mg/ml and 1Log at 4mg/ml and 2mg/ml (selectivity index = 12). The selectivity index for Influenza Virus was 12 with the highest inhibition (1Log) observed at 4mg/ml. Herpes simplex virus-1 and Porcine Parvovirus were mildly inhibited, whereas no antiviral activity was observed with respect to Adenovirus-5, Human Herpesvirus-6, Porcine Reproductive and Respiratory Syndrome Virus. No HA virucidal activity was ever observed against any of the viruses tested. Kinetic experiments showed that both Coxsackievirus B5 and Herpes simplex virus-1 replication were consistently inhibited, not influenced by the time of HA addition, during the virus replication cycle.CONCLUSIONS: the spectrum of the antiviral activity exhibited by HA against both RNA and DNA viruses, known to have different structures (with or without envelope) and replication strategies, suggests a non specific mechanism of action, probably involving cell membrane-virus interaction steps. The results of the kinetic experiments support this hypothesis.

2011 - Influence of hyaluronic acid on bacterial and fungal species, including clinically relevant opportunistic pathogens. [Articolo su rivista]
Ardizzoni, Andrea; Neglia, Rachele Giovanna; Baschieri, MARIA CRISTINA; Cermelli, Claudio; Caratozzolo, M; Righi, Elena; Palmieri, Beniamino; Blasi, Elisabetta
abstract

Hyaluronic acid (HA) has several clinical applications (aesthetic surgery, dermatology, orthopaedics and ophtalmology). Following recent evidence, suggesting antimicrobial and antiviral properties for HA, we investigated its effects on 15 ATCC strains, representative ofclinically relevant bacterial and fungal species. The in vitro system employed allowed to assess optical density of broth cultures as a measure of microbial load in a time-dependent manner. The results showed that different microbial species and, sometimes, different strains belonging to the same species, are differently affected by HA. In particular, staphylococci, enterococci, Streptococcus mutans, twoEscherichia coli strains, Pseudomonas aeruginosa, Candida glabrata and C. parapsilosis displayed a HA dosedependent growth inhibition; no HA effects were detected in E. coli ATCC 13768 and C. albicans; S. sanguinis was favoured by the highest HA dose. Therefore, the influence of HA on bacteria and fungi warrants further studies aimedat better establishing its relevance in clinical applications.

2011 - The protein “mycoarray”: a novel serological assay for the laboratory diagnosis of primitive endemic mycoses [Articolo su rivista]
Ardizzoni, Andrea; Baschieri, MARIA CRISTINA; Manca, Lidia; Orsi, Carlotta Francesca; C., Venturelli; M., Meacci; Peppoloni, Samuele; C., Farina; Blasi, Elisabetta
abstract

A protein microarray containing fungal antigens (the “mycoarray”) has been set up to provide rapid and appropriateserodiagnosis of primitive endemic mycoses, an important cause of morbidity and mortality in an increasingly highnumber of patients. The mycoarray consists of three antigen extracts (histoplasmin, coccidioidin and Coccidioides “TP”)and antibody dilution curves were spotted on microarray slides. The arrays were processed with coccidioidomycosisand histoplasmosis patients’ sera or with control sera and the occurring immunocomplexes were detected by indirectimmunofluorescence. In agreement with clinical and microbiological diagnosis, the results distinguished betweenhistoplasmosis and coccidioidomycosis patients. In addition, the assay could clearly discriminate between IgM andIgG antibody reactivity. No reactivity was ever observed in the arrays processed with negative control sera. Therefore,this pilot study demonstrates that the “mycoarray” is sensitive and specific enough to discriminate between healthyindividuals and patients with histoplasmosis or coccidioidomycosis. Because of miniaturization and multiparametricity,the new assay cuts costs and processing time. Thus, once clinically validated and implemented as a large-scale array,the “mycoarray” will be ready to be applied to the daily clinical practice.

2011 - VALUTAZIONE DEI PROFILI IgG E IgM NEI CONFRONTI DI PATOGENI A TRASMISSIONE VERTICALE IN DIVERSE MATRICI BIOLOGICHE MEDIANTE MICROARRAY PROTEICO [Poster]
Ardizzoni, Andrea; Manca, Lidia; Baschieri, MARIA CRISTINA; Capodanno, Francesco; LA SALA, Giovanni Battista; Blasi, Elisabetta
abstract

Lo screening sierologico su larga scala, effettuato prima o durante la gravidanza, fornisce un mezzo efficace per prevenire infezioni a trasmissione verticale (ITV) e le loro gravi conseguenze per il prodotto del concepimento. Utilizzando un microarray proteico da noi messo a punto (1), abbiamo voluto determinare i livelli di anticorpi antigene-specifici nei confronti di patogeni a trasmissione verticale nel siero, nei fluidi follicolari (FF) e nei fluidi amniotici (FA) di pazienti gravide, al fine di correlare profili anticorpali specifici con l’esito della gravidanza. Siero e FF sono stati prelevati da 102 pazienti sottoposte a trattamenti di procreazione medicalmente assistita. FA sono stati ottenuti da 100 pazienti di cui 50 con parto pretermine e 50 con gravidanza a termine. I microarray, allestiti con antigeni, anticorpi e controlli di segnale, come precedentemente descritto (1) sono stati processati con siero, FF o FA e la formazione degli immunocomplessi è stata rivelata mediante anticorpi secondari marcati in fluorescenza. I segnali sono stati letti con uno scanner contenente laser a differente lunghezza d’onda e successivamente quantificati ed analizzati da un software dedicato. Il saggio ha consentito di rivelare in maniera efficace anticorpi antigene-specifici in tutte le matrici saggiate. In particolare, la presenza o assenza di determinati anticorpi nelle coppie di campioni siero/FF provenienti dalla stessa paziente è risultata significativamente correlata a determinati parametri clinici, quali il numero di oociti inseminati di buona qualità o il numero di trasferimenti embrionali avvenuti con successo. L’analisi dei profili anticorpali sui FA del secondo gruppo di 100 pazienti è tuttora in corso. I dati ottenuti incoraggiano l’utilizzo del microarray proteico per indagini ad ampio spettro nella diagnosi di ITV; inoltre, l’utilizzo di matrici biologiche differenti dal siero può fornire un valido contributo per la risoluzione di problemi ancora aperti. (1) Ardizzoni et al., Eur J Clin Microbiol Infect Dis, 2009.

2010 - Sealing ability of gutta-percha in root canals obturated by a modified continuous wave of condensation technique. [Abstract in Rivista]
Generali, Luigi; Mancuso, R.; Travaglini, Domenico; Ardizzoni, Andrea; Giannetti, Luca; Bertoldi, Carlo; Ambu, E.
abstract

Vedi allegato

2010 - The protein “mycoarray”: a novel immunoassay for the serological diagnosis of primitive invasive mycoses [Poster]
Ardizzoni, Andrea; Baschieri, MARIA CRISTINA; Manca, Lidia; Farina, Claudio; Cermelli, Claudio; Meacci, Marisa; Venturelli, Claudia; Blasi, Elisabetta
abstract

Objectives. Invasive fungal infections are an important cause of morbidity and mortality in an increasingly higher number of patients, also because of difficulties in providing a rapid and appropriate diagnosis. In some cases, detection of a specific antibody response is a crucial diagnostic tool; however, the available serological assays often provide qualitative results only, their sensitivity and specificity are poor and long time procedures are required. In addition, patients who suffer from an invasive mycosis may have multiple infections likely underestimated by conventional diagnostic approaches. In order to couple the serology of primitive invasive mycoses to the protein microarray technology, a “mycoarray” assay has been designed and set up.Methods. Four antigen extracts (histoplasmin, coccidioidin, Coccidioides “TP” antigen and aspergillin) and the appropriate controls were spotted in various conditions onto a restricted area of a microscope slide. The printed slides were then incubated with immune sera produced in goat against each single antigen or, subsequently, with human sera (6 from patients affected by primitive invasive mycoses and 7 from healthy individuals). The occurring immunocomplexes were detected by indirect immunofluorescence.Results. The pilot experiments, conducted using the goat immune sera, allowed to establish the optimal spotting conditions for each antigen in terms of both spotting buffer and extracts’ dilution. The “mycoarrays”, obtained by spotting all the fungal antigens with the best condition, were then processed with sera either from patients or control subjects. The reactivity observed in the arrays processed with the patients’ samples was in agreement with the clinical and microbiological diagnosis; no reactivity was ever observed in the arrays processed with the negative control sera.Conclusions. The “protein mycoarray” is sensitive enough to discriminate between healthy individuals and patients affected by histoplasmosis or coccidioidomycosis. This novel diagnostic tool, because of its intrinsic features, miniaturization and multiparametricity, can contribute to cut out costs and to shorten times-to-results, with the potentiality to be included in the daily clinical practice in the near future.

2010 - Wide spectrum activity of a high molecular weight Hyaluronic Acid. [Abstract in Rivista]
Cermelli, Claudio; Cuoghi, A.; Scuri, M.; Bettua, C.; Ardizzoni, Andrea; Neglia, Rachele Giovanna; Blasi, Elisabetta
abstract

Background Hyaluronic acid (HA), a non-sulphated glycosaminoglycan, is present in synovial fluid, vitreous humour and many connective tissues. Pharmaceutical preparations of HA are used in clinical practice for wound healing, joint pain, kerato-conjunctivitis, asthma, mouth care, oesophageal-reflux, and gastritis. Moreover, it is used as a filler to counteract aging and facial lypoatrophy. Here we investigated the antiviral activity in vitro of a high molecular weight (1,8KD) HA used in aesthetic medicine.Methods The MTT test was used to investigate the cytotoxicity of HA on the different cell lines used for virus growth: VERO, MDCK, PK15, JJHAN, MARC145. With the aim of investigating whether HA may affect cell membrane stabilization, experiments were carried out in which VERO cells were pre-treated with HA and then exposed to two lysis solutions, HCl and Triton X-100. The antiviral activity of HA was assessed by viral yield assays against Coxsackievirus B5 (COXB5), Herpes simplex virus-1 (HSV-1), Mumps virus (MV), Adenovirus (ADV), Influenza Virus (WSN33-AH1N1), Human Herpesvirus-6 (HHV-6), Porcine Parvovirus (PPV), Porcine Respiratory Reproductive Syndrome Virus (PRRSV). The virucide activity was assessed by exposing the different viral inocula to HA for 30’ at room T° and then their residual infectivity was titrated on cells. Time course experiments were carried out with COXB5 and HSV-1 within a replication single cycle by adding. HA at different time points.Results The MTT test showed that HI displayed no significant toxicity up to 4mg/ml. In lysis experiments, HA was able to protect VERO cells from lysis by both TritonX-100 and HCl. HSV-1, COXB5, MV, WSN33, PPV were inhibited by HA, the most effective inhibition being observed against COXB5 (3,5 Log reduction) and MV (1.7Log reduction). No virucidal activity by HA was ever observed against any of the viruses tested. Kinetic experiments showed that both COXB5 and HSV-1 were consistently inhibited independently upon the time of HA addition during the virus replication cycle. Conclusions The wide spectrum of antiviral activity exhibited by HA against both RNA and DNA viruses, known to have different structures (with or without envelope) and replication strategies, suggests a non specific mechanism of action, likely involving cell membrane-virus interaction steps either in virus entry or exit. The results of the kinetic experiments and of the cell protection from lysis support this hypothesis.

2010 - Yessotoxin inhibits phagocytic activity of macrophages. [Articolo su rivista]
Orsi, Carlotta Francesca; Colombari, Bruna; Callegari, Federica; Todaro, Mary Antonio Donatello; Ardizzoni, Andrea; Rossini, Gian Paolo; Blasi, Elisabetta; Peppoloni, Samuele
abstract

Yessotoxin (YTX) is a sulphated polyether compound produced by some species of dinoflagellate algae, that can be accumulated in bivalve mollusks and ingested by humans upon eating contaminated shellfish. Experiments in mice have demonstrated the lethal effect of YTX after intraperitoneal injection, whereas its oral administration has only limited acute toxicity, coupled with an alteration of plasma membrane protein turnover in the colon of the animals. In vitro studies have shown that this effect is due to the inhibition of endocytosis induced by the toxin. In this work, we investigated the effects of YTX on phagocytosis by using the J774 macrophage cell line. We found that macrophages exposed to 10 or 1nM YTX display a reduced phagocytic activity against Candida albicans; moreover, phagosome maturation is also inhibited in these cells. Such results were confirmed with resident peritoneal macrophages from normal mice. The inhibition of both phagocytosis and phagosome maturation likely involves cytoskeletal alterations, since a striking rearrangement of the F-actin organization occurs in YTX-treated J774 macrophages. Surprisingly, YTX also enhances cytokine production (TNF-alpha, MIP-1alpha and MIP-2) by J774 macrophages. Overall, our results show that low doses of YTX significantly affect both effector and secretory functions of macrophages.

2009 - A protein microarray immunoassay for the serological evaluation of the antibody response in vertically transmitted infections. [Articolo su rivista]
Ardizzoni, Andrea; B., Capuccini; Baschieri, MARIA CRISTINA; Orsi, Carlotta Francesca; F., Rumpianesi; Peppoloni, Samuele; Cermelli, Claudio; M., Meacci; A., Crisanti; P., Steensgaard; Blasi, Elisabetta
abstract

The detection of specific serum antibodies is mainly achieved by enzyme-linked immunosorbent assay (ELISA). Here, we describe the setting up of a microarray-based serological assay to screen for IgG and IgM against vertically transmitted pathogens (Toxoplasma gondii, rubella virus, cytomegalovirus, herpes simplex virus types 1 and 2, varicella zoster virus, Chlamydia trachomatis). The test, accommodated onto a restricted area of a microscope slide, consists of: (a) the immobilization of antigens and human IgG and IgM antibody dilution curves, laid down in an orderly manner; (b) addition of serum samples; (c) detection of antigen–serum antibodies complexes by indirect immunofluorescence. The IgG and IgM curves provide an internal calibration system for the interpolation of the signals from the single antigens. The test was optimized in terms of spotting conditions and processing protocol. The detection limit was 400 fg for the IgG assay and 40 fg for the IgM assay; the analytical specificity was >98%. The clinical sensitivity returned an average value of 78%, the clinical specificity was >96%, the predictive values were >73%, and the efficiency was >88%. The results obtained make this test a promising tool, suitable for introduction in the clinical diagnostic routine of vertically transmitted infections, in parallel (and in future as an alternative) to ELISA.

2009 - An in vitro and ex vivo study on two antibiotic-based endodontic irrigants: a challenge to sodium hypochlorite [Articolo su rivista]
Ardizzoni, Andrea; Blasi, Elisabetta; C., Rimoldi; L., Giardino; E., Ambu; Righi, Elena; Neglia, Rachele Giovanna
abstract

Amongst the bacterial species which most often cause endodontic failures, Enterococcus faecalis is the most important. This study compared the effectiveness of sodium hypochlorite and two new generation antibiotic-based endodontic irrigants, Tetraclean and MTAD. By means of an in vitro agar dilution assay, we show that both Tetraclean and MTAD are 100% effective against 54 clinical isolates at dilutions up to 1:256 and 1:1048, respectively, whereas sodium hypochlorite completely loses its effectiveness when diluted more than 32 times. The bactericidal effect of both Tetraclean and MTAD can be ascribed not just to their antibiotic component per se, but also to a synergistic effect among the several ingredients included in the formulations. Moreover, by an ex vivo model of teeth extracted and experimentally infected with E. faecalis ATCC 29212, we show that both the antibiotic-based endodontic irrigants are effective in eliminating bacterial cells in 93 to 100% of the test samples. The results of these pre-clinical studies strongly supporta wider use of this new group of endodontic irrigants in daily clinical practice.

2009 - Antiviral activity of Hyaluronic Acid [Abstract in Atti di Convegno]
Cuoghi, A.; Orsi, Carlotta Francesca; Ardizzoni, Andrea; Neglia, Rachele Giovanna; Blasi, Elisabetta; Cermelli, Claudio
abstract

Hyaluronic acid is a non-sulfated glycosaminoglycan widely distributed in connective tissues. It has several medical applications including ophtalmic surgery, treatment of osteoarthitis and atopic dermatitis. Moreover, ialuronic acid gels are used in regenerative medicine for wound healing and are injected for filling soft tissues defectes such as facial wrinkles or lipoatrophy in lipodistrophic patients. In order to ascertain whether an antimicrobial activity is associated with the tissue regeneration ability, we investigated the in vitro antiviral and virucidal activity of a mid molecular size ialuronic acid gel used as facial filling and for wound healing.MTT test on VERO cells was used to assess the toxicity of the product which resulted devoid of any cytotoxicity at the concentrations (4mg/ml) used for medical purposes.By means of virus yield assays, we evaluated the ability of this type of ialuronic acid to inhibit the growth of herpes simplex vitus type 1, mumps virus, coxsackievirus B5 and adenovirus. The virucidal activity on the same viruses was assessed treating viral preparations with ialuronic acid for 10 minutes at room temperature and then testing the residual infectivity of these preparations. The results obtained show that ialuronic acid can inhibit the growth of HSV-1 and mumps virus (1 Log reduction at least).

2009 - Gene expression profiling of monocytes displaying herpes simplex virus 1 induced dysregulation of antifungal defences [Articolo su rivista]
Cermelli, Claudio; Orsi, Carlotta Francesca; A., Cuoghi; Ardizzoni, Andrea; Tagliafico, Enrico; Neglia, Rachele Giovanna; Peppoloni, Samuele; Blasi, Elisabetta
abstract

Recently, we showed that herpes simplex virus 1 (HSV-1)-infected monocytes have altered antifungal defences, in particular they show augmented phagocytosis of Candida albicans followed by a failure of the intracellular killing of the ingested fungi. On the basis of these functional data, comparative studies were carried out on the gene expression profile of cells infected with HSV-1 and/or C. albicans in order to investigate the molecular mechanisms underlying such virus-induced dysfunction. Affymetrix GeneChip technology was used to evaluate the cell transcription pattern, focusing on genes involved in phagocytosis, fungal adhesion, antimicrobial activity and apoptosis. The results indicated there was: (a) prevalent inhibition of opsonin-mediated phagocytosis, (b) upregulation of several pathways of antibody- and complement-independent phagocytosis, (c) inhibition of macrophage activation, (d) marked dysregulation of oxidative burst, (e) induction of apoptosis.

2009 - I microarray proteici: una sfida da raccogliere [Relazione in Atti di Convegno]
Blasi, Elisabetta; Ardizzoni, Andrea
abstract

2009 - Il microarray proteico nella sierodiagnosi delle infezioni a trasmissione verticale [Relazione in Atti di Convegno]
Ardizzoni, Andrea; Baschieri, MARIA CRISTINA; Manca, Lidia; Blasi, Elisabetta
abstract

2009 - Sierodiagnosi di infezioni a trasmissione verticale mediante microarray proteico [Poster]
Ardizzoni, Andrea; Baschieri, MARIA CRISTINA; Manca, Lidia; Cuoghi, Alessandro; Cermelli, Claudio; Peppoloni, Samuele; Blasi, Elisabetta
abstract

2009 - The ABC transporter-encoding gene AFR1 affects the resistance of Cryptococcus neoformans to microglia-mediated antifungal activity by delaying phagosomal maturation. [Articolo su rivista]
Orsi, Carlotta Francesca; Colombari, Bruna; Ardizzoni, Andrea; Peppoloni, Samuele; Neglia, Rachele Giovanna; Posteraro, B; Morace, G; Fadda, G; Blasi, Elisabetta
abstract

The pathogenic yeast Cryptococcus neoformans has evolved several strategies to survive within phagocytes. Recently, it has been demonstrated that upregulation of the ATP binding cassette transporter-encoding gene antifungal resistance 1 (AFR1) is important not only for determining the resistance of C. neoformans to fluconazole but also in influencing fungal virulence. In the present study, we showed that the fluconazole-resistant AFR1-overexpressing mutant strain was not sensitive to microglia-mediated anticryptococcal activity, as compared with the fluconazole-susceptible isogenic strains, the wild type and the afr1Delta mutant. Interestingly, although the three strains were phagocytosed to a similar extent, reduced acidification and delayed maturation were observed in phagosomes containing the AFR1-overexpressing strain with respect to the others. These findings provide the first evidence that upregulation of the AFR1 gene affects C. neoformans-microglia interplay, adding insights to the complexity of cryptococcal virulence and to its unexpected link with azole resistance.

2008 - Caratterizzazione immunobiologica di nuovi antigeni proteici di Streptococcus pneumoniae selezionati mediante "phage display" [Abstract in Atti di Convegno]
Peppoloni, Samuele; F., Felici; S., Ricci; M., Oggioni; G., Pozzi; Ardizzoni, Andrea; Colombari, Bruna; Orsi, Carlotta Francesca; Messinò, Massimino; C., Lo Passo; I., Pernice; G., Teti; A., Ruggeri; C., Beninati
abstract

Caratterizzazione immunobiologica di nuovi antigeni proteici di Streptococcus pneumoniae selezionati mediante "phage display"

2008 - Comparative in vitro and ex vivo studies on the bactericidal activity of Tetraclean, a new generation endodontic irrigant, and sodium hypochlorite [Articolo su rivista]
Neglia, Rachele Giovanna; Ardizzoni, Andrea; L., Giardino; E., Ambu; S., Grazi; S., Calignano; C., Rimoldi; Righi, Elena; Blasi, Elisabetta
abstract

The aim of this study was to compare the efficacy of a new generation endodontic irrigant, Tetraclean, to the widely used sodium hypochlorite. Tetraclean combines a powerful detergent effect with a strong antimicrobial efficacy, whereas sodium hypochlorite has several drawbacks and is sometimes ineffective in preventing microbial-mediated endodontic failure. The bactericidal activity of both irrigants against Enterococcus faecalis, the most commonly isolated species from root canals of teeth with post-treatment disease, was assessed i) in vitro, according to the European Standard lines for the evaluation of the bactericidal activity of chemical disinfectants, and ii) with an ex vivo model of extracted and decoronated human teeth, infected with E. faecalis and subsequently irrigated with either of the irrigants. Both irrigants display very similar bactericidal activity against E. faecalis in vitro. However, the ex vivo model shows that only in the teeth irrigated with Tetraclean did the bacterial burden gradually drop until no bacteria were detectable a few days post-irrigation. Vice versa, in the teeth irrigated with sodium hypochlorite, the drop in the bacterial burden was rapid but temporary and most of the teeth were colonized again by 48 hours post-irrigation.

2008 - Herpes simplex virus type 1 dysregulates anti-fungal defenses preventing monocyte activation and downregulating toll-like receptor-2 [Articolo su rivista]
Cermelli, Claudio; Orsi, Carlotta Francesca; Ardizzoni, Andrea; Lugli, Enrico; Cenacchi, Valeria; Cossarizza, Andrea; Blasi, Elisabetta
abstract

We investigated the interplay occurring between pathogens in the course of dual infections, using an in vitro model in which the THP-1 monocytic cell line is first infected with HSV-1 and then exposed to Ca or Cn. These three pathogens share some pathogenic features: they cause opportunistic infections, target macrophages and are neurotropic. Here, we show that HSV-1-infected THP-1 cells exhibited augmented phagocytosis against the two opportunistic fungi but reduced capability to counteract fungal infection: the better ingestion by monocytes was followed by facilitated fungal survival and replication. Reduced IL-12 production was also observed. Cytofluorimetric analysis showed that HSV-1-infected monocytes exhibit: (i) downregulated TLR-2 and TLR-4, critical structures in fungal recognition; (ii) reduced expression of CD38 and CD69, known to be important markers of monocyte activation; and (iii) enhanced expression of apoptosis and necrosis markers, in the absence of altered cell proliferation. Overall, these findings imply that HSV-1 infection prevents monocyte activation, thus leading to a significant dysfunction of the monocyte-mediated anti-Candida response; HSV-1 induced apoptosis and necrosis of monocytes further contribute to this impairment.

2008 - Il gene AFR1 di Cryptococcus neoformans codificante per un trasportatore ABC, altera la resistenza del patogeno alla microglia: ritardo nella maturazione dei fagosomi. [Abstract in Atti di Convegno]
Orsi, Carlotta Francesca; Colombari, Bruna; Ardizzoni, Andrea; Peppoloni, Samuele; Neglia, Rachele Giovanna; B., Posteraro; G., Morace; G., Fadda; Blasi, Elisabetta
abstract

Introduzione: Cryptococcus neoformans (Cn) è un fungo opportunista patogeno dotato di spiccato neurotropismo suscettibile almeno in vitro alle difese mediate dalla microglia (1). Recentemente, è stato dimostrato come l’iperespressione del gene AFR1 conferisce a Cn sia la resistenza al fluconazolo (2) che un’aumentata virulenza (3). Il presente studio intende valutare il ruolo del fenotipo AFR1 nell’interazione tra Cn e l’immunoeffettore cerebrale.Materiali e metodi: l’ isolato clinico BPY22 e due suoi mutanti in cui il gene AFR1 è stato deleto per mutagenesi inserzionale (ceppo BPY444) o reintrodotto sotto il controllo del promotore forte gpd1 (ceppo BPY445) (3), sono stati impiegati in saggi di infezione in cellule di microglia (1) in vitro e quindi comparati per i) suscettibilità a fagocitosi e al killing e ii) maturazione fagosomiale. Risultati: i ceppi saggiati hanno dimostrato: a) simile suscettibilità alla fagocitosi, b) diversa sensibilità al killing e c) diversa maturazione dei fagosomi; in particolare, il processo di maturazione è significativamente ritardato (ridotta acidificazione e comparsa di marcatori tardivi quali Rab7, Rab9 e LAMP2) nei vacuoli di fagocitosi contenenti il ceppo che iperesprime il gene AFR1. Conclusioni: questi dati definiscono il ruolo del gene AFR1 nella resistenza di Cn alla cellula microgliale dimostrandone la capacità di interferire con l’acidificazione e la maturazione del fagosoma e di conseguenza con la sopravvivenza intracellulare del patogeno.

2008 - Il microarray proteico: peculiarità, vantaggi e limiti [Relazione in Atti di Convegno]
Blasi, Elisabetta; Ardizzoni, Andrea
abstract

2008 - Microarray proteici: applicazioni nella diagnostica prenatale [Relazione in Atti di Convegno]
Ardizzoni, Andrea; Capuccini, Barbara; Baschieri, MARIA CRISTINA; Crisanti, Andrea; Gallucci, Giuseppina; Manzoni, Angelo; Meacci, Marisa; Rumpianesi, Fabio; Blasi, Elisabetta
abstract

2007 - A transmissible cytotoxic activity isolated from a patient with brain ischemia causes microglial cell activation and dysfunction. [Articolo su rivista]
Beretti, Francesca; Cenacchi, Valeria; Portolani, Marinella; Ardizzoni, Andrea; Blasi, Elisabetta; Cermelli, Claudio
abstract

Microglial cell activation occurs during brain injury, ischemia, and in several neurologic disorders. Recently, we isolated a transmissible cytotoxic activity (TCA) from the cerebrospinal fluid of a patient with brain ischemia. Such a TCA, associated with one or more protein(s) that supposedly had undergone in vivo misfolding, causes apoptosis in vitro in different cell lines, including microglial cells. The TCA producing cells and the potential in vivo role of such cytotoxic activity remains to be elucidated. Here, we investigated the in vitro effects of TCA on microglial cell immune functions.2. The murine microglial cell line RR4 was exposed to TCA, and then its response was evaluated as: (a) phagocytosis and antifungal activity against Candida albicans; (b) secretory pattern; and (c) levels of p38 phosphorylation.3. Unlike mock-treated controls, microglial cells exposed to TCA showed an increase in phagocytic activity. Unexpectedly, their capability to kill the ingested fungi significantly diminished. Moreover, TCA-treated cells produced amounts of macrophage inflammatory protein 1-alpha, tumor necrosis factor-alpha, and nitric oxide significantly higher than mock-treated cells. Finally, phosphorylation of p38 mitogen-activated protein kinase (MAPK) was detected in TCA-treated but not in mock-treated controls as early as 30 min after treatment.4. Overall, these results indicate that TCA causes a rapid molecular response in microglial cells, by the time, leading to an intriguing effector and secretory dysfunction.

2007 - ALTERATA REATTIVITA’ MACROFAGICA IN CORSO DI INFEZIONE MISTA DA VIRUS E FUNGHI:VALUTAZIONI FUNZIONALI, CITOFLUORIMETRICHE E DI ESPRESSIONE GENICA [Abstract in Atti di Convegno]
Cermelli, Claudio; Peppoloni, Samuele; Ardizzoni, Andrea; Tagliafico, Enrico; Blasi, Elisabetta
abstract

Base: I casi clinici di infezioni miste da funghi e virus sono in aumento, soprattutto negli ospiti immunocompromessi.Ciononostante, gli eventi biomolecolari che caratterizzano l’andamento di infezioni polimicrobiche sono tuttora poco conosciuti:scarse sono le conoscenze sulle interazioni che si verificano tra i patogeni e sui derivanti effetti, sinergistici o antagonistici.Nell’ambito del presente lavoro, abbiamo indagato sulla reattività macrofagica nel corso di infezioni miste, sostenute davirus HSV-1 e funghi opportunisti patogeni.Metodi: Sulla base di recenti studi (Cermelli C. et al., 2006), cellule THP-1 infettate per 18 ore con HSV-1 venivano esposte aCandida albicans o Cryptococcus neoformans e quindi saggiate per fagitosi, killing (CFU), marker fenotipici (citofluorimetria)ed espressione genica (microarray di RNA).Risultati: La fagocitosi di entrambi i miceti risulta significativamente aumentata nei monociti infettati da HSV-1 mentre l’attivitàantifungina è diminuita (significativa sopravvivenza e replicazione intracellulare dei due miceti). Al citofluorimetro, celluleTHP-1 infettate mostrano a) significativa downregolazione di TLR2 e TLR4, importanti molecole coinvolte nel riconoscimentodei miceti; b) ridotta espressione di CD38 e CD69, marker di attivazione cellulare; 3) aumento dei marker di apoptosi enecrosi. Il profilo di espressione genica indica un drastico calo (circa il 50%) nella quantità di geni espressi e una modulazionedell’espressione dei geni che comunque restano accesi nelle cellule infettate da HSV-1 rispetto ai controlli (> 7.500 genisovra- o sotto-espressi di almeno 3 volte). In particolare, l’analisi genica per cluster mostra uno spegnimento dei geni coinvoltinella fagocitosi opsonizzata e un aumento dell’espressione di quelli associati alla fagocitosi non opsonizzata. I geni di TLR2and TLR4 risultano downregolati così come molti geni coinvolti nel killing intracellulare.Conclusioni: Questi dati dimostrano che HSV-1 è in grado di alterare la funzione del macrofago fino a renderlo inerme o addiritturapromotore della sopravvivenza e della replicazione del fungo, sottolineando la possibilità di effetti sinergici in vivo nelcorso di infezioni miste.

2007 - APPLICAZIONE DEI MICROARRAY PROTEICI NELLA DIAGNOSTICA AVANZATA DI PATOLOGIEMICROBICHE E VIRALI [Abstract in Atti di Convegno]
Ardizzoni, Andrea; Peppoloni, Samuele; Cermelli, Claudio; Marisa, Meacci; Andrea, Crisanti; Francesca, Baldracchini; Angelo, Manzoni; Franca, Santoro; Giuseppina, Gallucci; Blasi, Elisabetta
abstract

L’immobilizzazione di matrici microscopiche (microarray) di acidi nucleici su substrato solido ha rappresentato un passo avanticruciale per lo sviluppo di saggi multiparametrici che consentissero l’analisi dei livelli di espressione genica di un organismo.Tuttavia l’analisi dell’espressione genica non fornisce indicazioni sull’abbondanza e funzione di biomolecole fondamentali nelladefinizione di un fenotipo e/o nell’evoluzione di un processo patogenetico. Pertanto sono stati messi a punto microarray contenentiproteine, anticorpi, lipidi, carboidrati con applicazioni in ricerca e in diagnostica. In questa ottica nel presente lavoro, ci siè proposti di mettere a punto un sistema di diagnostica avanzata, basato sulla tecnologia del microarray proteico, per la determinazionesimultanea e multiparametrica di IgG e IgM specifiche nei confronti di antigeni microbici importanti nella diagnosticaprenatale. Microarray di IgG e IgM umane ed antigeni microbici vengono deposti su vetrini da microscopio con superficiechimicamente reattiva per mezzo di un sistema robotizzato ad alta precisione. I microarray cosí prodotti vengono incubati consiero e successivamente con anticorpi marcati con fluorofori per la rilevazione del segnale. I vetrini vengono infine analizzaticon uno strumento che combina microscopia laser confocale e ricostruzione digitale dell’immagine. L’intensitá del segnale incorrispondenza degli antigeni viene quantificata utilizzando come riferimento le curve di calibrazione generate depositando sulvetrino quantitá decrescenti di IgG e IgM. Esperimenti di validazione hanno messo in evidenza come il saggio immunologicoper la rilevazione delle IgG dirette contro gli antigeni del complesso ToRCH avesse sensibilitá di 0.5 μg/mL e una precisionetra 1,7% e 14,6% per tutti gli antigeni analizzati. Utilizzando campioni di siero provenienti da pazienti, si è ottenuta una eccellenteconcordanza tra microarray ed ELISA che sottolinea l’efficacia del sistema microarray. Considerati i notevoli vantaggi intermini di costo e convenienza, riteniamo che la tecnologia del microarray proteico possa essere, in un prossimo futuro, introdottacome sistema di routine nei laboratori di analisi.

2007 - NF-kB activation and p38 phosphorilation in microglial cells infected with Leptospira or exposed to partially purified leptospiral lipoproteins [Articolo su rivista]
Blasi, Elisabetta; Ardizzoni, Andrea; Colombari, Bruna; Neglia, Rachele Giovanna; Baschieri, MARIA CRISTINA; Peppoloni, Samuele; M., Cinco
abstract

Recently, we have shown a differential susceptibility of non-pathogenic vs. pathogenic leptospires to phagocytosis and killing by microglial cells. Although all ingested to some extent, only the pathogenic strains survived intracellularly while the non-pathogenic ones were killed in a time-dependent manner. By the same infection model, here we demonstrate that microglial cells respond to Leptospira infection with a time- and dose-dependent induction of molecular signals (p38 phosphorilation and NF-kB activation) and the production of soluble factors (cytokines and nitric oxide). Such bio-molecular response is predominantly observed against the pathogenic Leptospira; the phenomenon is reproduced by leptospiral lipoproteins and, to a lower extent, by leptospiral-derived LPS. These data provide initial evidence that Leptospira affects microglial cell response in a different manner depending upon the virulence of the infecting strain; specific bacterial components happen to be involved in the induction of such pathogen-induced immune response.

2006 - Applicazioni del microarray proteico nella diagnosi diretta per la sierotipizzazione di agenti patogeni [Abstract in Atti di Convegno]
F., Baldracchini; Ardizzoni, Andrea; Blasi, Elisabetta; W., Low; Casolari, Chiara; Neglia, Rachele Giovanna; T., Bacarese Hamilton; Peppoloni, Samuele; Cermelli, Claudio; A., Crisanti
abstract

Applicazioni del microarray proteico nella diagnosi diretta per la sierotipizzazione di agenti patogeni

2006 - COMPARAZIONE DELLA EFFICACIA ANTIBATTERICA DI TETRACLEAN, UN IRRIGANTE ENDODONTICO DI NUOVA GENERAZIONE, CON IPOCLORITO DI SODIO. [Abstract in Atti di Convegno]
Ardizzoni, Andrea; Neglia, Rachele Giovanna; L., Giardino; E., Ambu; Grazi, Silvia; S., Calignano; C., Rimoldi; Peppoloni, Samuele; Blasi, Elisabetta
abstract

COMPARAZIONE DELLA EFFICACIA ANTIBATTERICA DI TETRACLEAN, UN IRRIGANTE ENDODONTICO DI NUOVA GENERAZIONE, CON IPOCLORITO DI SODIO.A. Ardizzoni1, R. Neglia1, L. Giardino2, E. Ambu3, S. Grazi1, S. Calignano1, C. Rimoldi1, S. Peppoloni1, E. Blasi1.1Dipartimento di Scienze di Sanità Pubblica, Università di Modena e Reggio Emilia; 2Dipartimento di Periodontologia, Università di Brescia; 3Dipartimento di Neuroscienze, Testa, Collo e Riabilitazione, Unità Operativa di Odontoiatria e Chirurgia Maxillo-Facciale, Università di Modena e Reggio Emilia.L’efficacia di un irrigante endodontico consiste nella sua capacità di penetrare in aree difficili da raggiungere, come i canalicoli dentinali, e di uccidere i microrganismi ivi residenti, causando un danno minimo ai tessuti dell’ospite. Pertanto, la scelta di un irrigante endodontico dovrebbe assicurare un adeguato effetto detergente ed una completa disinfezione dei canali endodontici e dei tubuli dentinali. Lo scopo di questo lavoro è di confrontare l’efficacia antibatterica di un irrigante endodontico di nuova generazione, Tetraclean®, con quella di ipoclorito di sodio al 5,25%. Quest’ultimo, nonostante oggi venga ampiamente utilizzato nella pratica clinica endodontica, presenta molti svantaggi e a volte risulta inefficace nel prevenire fallimenti endodontici associati a colonizzazione microbica dei canali endodontici e dei tubuli dentinali. Esperimenti condotti in vitro hanno mostrato come entrambi gli irriganti testati esercitino attività battericida simile nei confronti di Enterococcus faecalis, il patogeno più frequentemente causa di infezioni a carico del sistema radicolo-canalicolare. Tuttavia, test condotti ex vivo su denti estratti, sterilizzati e re-infettati con E. faecalis e successivamente irrigati con l’uno o l’altro degli irriganti, hanno messo in luce come solo nei denti trattati con Tetraclean® la carica batterica diminuisce gradualmente, finchè a partire da alcuni giorni dopo l’irrigazione non sono più determinabili cellule batteriche vitali. Al contrario, nei denti irrigati con sodio ipoclorito, l’abbattimento della carica batterica è più rapido, ma solo transitorio e la maggior parte dei denti viene ricolonizzata a partire da 48 ore dopo l’irrigazione. Questi risultati indicano come Tetraclean® abbia tutte le potenzialità per l’impiego, come irrigante di nuova generazione, nella pratica clinica quotidiana.

2006 - Human herpesvirus-6 dysregulates monocyte-ediated anticryptococcal defences. [Abstract in Atti di Convegno]
Blasi, Elisabetta; Cenacchi, V; Beretti, Francesca; Pezzini, F; Argentieri, A; Ardizzoni, Andrea; Neglia, Rachele Giovanna; Orsi, Carlotta Francesca; Di Luca, D; Cermelli, Claudio
abstract

In order to investigate the interplay occurring between pathogens in the course of double infections, we set up an in vitro model in which the monocytic cell line, THP-1, is exposed to Cryptococcus neoformans (Cn) and Human Herpesvirus 6 (HHV-6). Cn and HHV-6, both highly neurotropic, can cause serious diseases of the central nervous system and have monocytes, among other cell types, as target cells, causing alteration of their secretion pattern. Here, we show that, unlike THP-1 cells exposed to cell-free virus inocula, THP-1 exposed to HHV-6 producing lymphocytes exhibit augmented phagocytosis against Cn. The phenomenon occurs after 24 hours of monocyte/lymphocyte co-culture and it is independent of direct cell-to-cell contact. Moreover, in the presence of HHV-6, THP-1 cells express enhanced secretory responses but reduced capability to counteract fungal infection: the better ingestion by monocytes is followed by facilitated fungal survival and replication. These data provide initial in vitro evidence that HHV-6 may dysregulate monocyte-mediated anticryptococcal defences with an overall pro-cryptococcus result.

2006 - In vitro and ex vivo studies on the antibacterial efficacy of sodium hypochlorite and two new generation endodontic irrigants, Tetraclean® and MTAD, in comparison with sodium hypochlorite. [Relazione in Atti di Convegno]
C., Rimoldi; Ardizzoni, Andrea; Neglia, Rachele Giovanna; Blasi, Elisabetta; Generali, Luigi; E., Ambu
abstract

The aim of this work was to compare the efficacy of two endodontic iorrigants of new generation, Tetraclean and MTAD. Their antimicrobial effectiveness was assessed by in vitro and in vivo studies. Sodium hypochlorite was included as standard reference irrigant.

2006 - MICROARRAY PROTEICI NELLA DIAGNOSI DIRETTA: SIEROTIPIZZAZIONE DI AGENTI PATOGENI [Abstract in Atti di Convegno]
F., Baldracchini; Ardizzoni, Andrea; Blasi, Elisabetta; W., Low; Casolari, Chiara; Neglia, Rachele Giovanna; T., Bacarese Hamilton; Peppoloni, Samuele; Cermelli, Claudio; A., Crisanti
abstract

MICROARRAY PROTEICI NELLA DIAGNOSI DIRETTA: SIEROTIPIZZAZIONE DI AGENTI PATOGENI.F. Baldracchini1, A. Ardizzoni2, E. Blasi2, W. Low1, C. Casolari3 ,R. Neglia2 , T.Bacarese-Hamilton1, S. Peppoloni2, C. Cermelli2, A. Crisanti1.1Department of Biological Sciences, Section of Infection and Immunity, Imperial College – London (U.K. ). 2Dipartimento di Scienze di Sanità Pubblica, Università di Modena e Reggio Emilia. 3Dipartimento Integrato dei Servizi Diagnostici e di Laboratorio, Università di Modena e Reggio Emilia.La tecnologia del microarray è diventata uno strumento cruciale per la diagnostica e la ricerca. Nell’ultimo decennio microarray proteici e di anticorpi hanno trovato numerose applicazioni in campo diagnostico, clinico e di laboratorio offrendo diversi vantaggi verso i metodi tradizionali. Lo scopo di questo progetto è sviluppare un saggio che come substrato di cattura ha un array di anticorpi specifici per la tipizzazione di batteri responsabili di varie patologie cliniche. La metodica di laboratorio oggi più comunemente utilizzata per la sierotipizzazione batterica, è il test di agglutinazione diretta, spesso complementato da tipizzazione fagica, ELISA e Western Blot. Nonostante il loro impiego routinario, queste tecniche risultano inadeguate quando si richiedano determinazioni rapide, quantitative, o multiparametriche a costi contenuti. La tecnologia microarray offre la possibilità di superare queste limitazioni. In quest’ottica, il presente studio ha valutato l’impiego del microarray proteico per la tipizzazione delle Salmonelle. Brevemente, anticorpi specifici per uno o più sierotipi di Salmonella vengono deposti su vetrini da laboratorio chimicamente attivati. Gli array così prodotti vengono incubati con i sierotipi di Salmonella precedentemente inattivati per fissazione chimica o mediante calore. L’immunocomplesso, risultante dal legame tra l’anticorpo e i rispettivi antigeni batterici, viene rivelato mediante immunofluorescenza indiretta. I vetrini vengono infine sottoposti a scansione mediante un sistema che utilizza microscopia laser confocale abbinata a ricostruzione digitale dell’immagine. I risultati preliminari di questo studio indicano che: 1) gli antisieri sono correttamente deposti sul vetrino e mantengono la loro specifica reattività; 2) i batteri, anche dopo fissazione, mantengono intatte le loro caratteristiche antigeniche e vengono riconosciuti dagli anticorpi diretti contro l’antigene somatico. Sono in corso indagini per ottimizzare i parametri tecnici concernenti soprattutto il protocollo di processazione. Questo studio pilota fornirà la procedura per la sierotipizzazione delle Salmonella mediante microarray proteici e porrà le basi per espandere l’impiego di tale metodologia alla identificazione di altri agenti patogeni clinicamente rilevanti.

2006 - Protein microarrays in allergy diagnosis [Abstract in Atti di Convegno]
Elviri, L; Mazzoleni, G; Ardizzoni, A; Low, W; Careri, M; Bacarese-Hamilton, T; Crisanti, A
abstract

2005 - Allergen microarrays [Capitolo/Saggio]
Bacarese-Hamilton, Tito; Gray, Julian; Ardizzoni, Andrea; Crisanti, Andrea
abstract

Allergy affects more than 25% of Western populations (1) and is estimated to be the sixth leading cause of chronic disease in the United States and Western Europe. The complexity of the condition is such that hundreds of common allergens have been described, and in order to maximize diagnostic efficiency there is an urgent clinical requirement for assays to provide multiple-allergen determination in a timely and cost-effective manner. Miniaturized immunoassays that utilize protein microarray technology now offer the possibility of circumventing most of the current limitations in the serodiagnosis of allergic disease. The heterogeneous nature of allergens presents many challenges in all aspects of developing such arrays, from immobilization of the capture molecule to detection of the bound ligand. In addition, there is no simple method of protein amplification (such as PCR for nucleic acids), and stabilization is yet a further major consideration. Notwithstanding these challenges, protein microarrays have been developed for the serodiagnosis of allergies and other complex clinical conditions. These assays exhibit good analytical and clinical performance and deliver significant advantages in convenience and cost compared with traditional ELISA test formats. This chapter details the techniques employed in the construction and processing of an allergen array specific for the serodiagnosis of allergic disease. An overview of protein microarray technology is provided and the principles that underpin the suitability for use of this technology in the identification and measurement of particular proteins in patient sera (serum profiling) are discussed.

2005 - Microarray proteici per la sierodiagnosi di patologie infettive [Relazione in Atti di Convegno]
Ardizzoni, Andrea; Bacarese-Hamilton, Tito; Crisanti, Andrea
abstract

2005 - Protein microarrays for the serodiagnosis of clinical conditions [Relazione in Atti di Convegno]
Ardizzoni, Andrea; Bacarese-Hamilton, Tito; Crisanti, Andrea
abstract

2004 - Protein arrays for serodiagnosis of disease [Capitolo/Saggio]
Bacarese-Hamilton, Tito; Ardizzoni, Andrea; Gray, Julian; Crisanti, Andrea
abstract

Protein microarrays offer the possibility to circumvent most of the current limitations in the serodiagnosis of allergy, autoimmune, and infectious disease by allowing the simultaneous, multiparametric determination of specific subclasses of antibodies directed against many pathogenic antigens. Microarray immunoassays have been developed with these characteristics. A first-generation assay, for the serodiagnosis of infectious disease, allows the determination of IgG and IgM antibodies to various viral and bacterial antigens. In addition, a second-generation assay, designed for the serodiagnosis of allergic disease, permits the determination of IgE antibodies to various allergens implicated in allergic disease. Slides printed with antibody dilution curves and antigen are first incubated with serum samples and then subsequently with secondary antibodies. For detection of human IgG and IgM, fluorescently labeled secondary antibodies are employed. However, because of low-level concentrations of circulating IgE antibodies, a more sensitive protocol is required for human IgE detection. Here, fluorescence is delivered via the coupling of the secondary antibody to tyramide signal-amplification reagentry. Human IgG, IgM, or IgE bound to the printed antigens can then be revealed by confocal scanning microscopy and quantified with internal calibration curves. Generation of analytical and clinical data have demonstrated that the microarray test format provides equivalent performance to enzyme-linked immunosorbent assay (ELISA) tests and offers a significant advantage in convenience and cost when compared to traditional test formats.

2004 - Serodiagnosis of infectious diseases with antigen microarrays [Articolo su rivista]
Bacarese-Hamilton, T; Mezzasoma, L; Ardizzoni, A; Bistoni, F; Crisanti, A
abstract

Aims: To generate protein microarrays by printing microbial antigens on slides to enable the simultaneous determination in human sera of antibodies directed against Toxoplasma gondii, rubella virus, cytomegalovirus and herpes simplex virus (HSV) types 1 and 2. Methods and Results: Antigens were printed on activated glass slides using high-speed robotics. The slides were incubated with serum samples and subsequently with fluorescently labelled secondary antibodies. Human IgG and IgM bound to the printed antigens were detected using confocal scanning microscopy and quantified with internal calibration curves. The microarray assay could detect as little as 0Æ5 pg of both IgG and IgM bound onto the glass surface. Precision profiles ranged from 1Æ7 to 18Æ5% for all the antigens. Microarrays and commercial ELISAs were utilized to detect serum antibodies against the ToRCH antigens in a panel of characterized human sera. Overall >80% concordance was obtained between microarray and ELISA kits in the classification of sera. Conclusions: These results indicate that the microarray is a suitable assay format for the serodiagnosis of infectious diseases. Significance and Impact of Study: Antigen microarrays can be optimized for clinical use, their performance is equivalent to ELISA but they offer significant advantages in throughput, convenience and cost.

2003 - Antigen microarrays for serodiagnosis of infectious diseases [Relazione in Atti di Convegno]
Bacarese-Hamilton, Tito; Ardizzoni, Andrea; Crisanti, Andrea
abstract

2003 - High performance microarray immunoassays for serodiagnosis of infectious disease and allergies [Poster]
Bacarese-Hamilton, Tito; Ardizzoni, Andrea; Gray, Julian; Crisanti, Andrea
abstract

2002 - Detection of allergen-specific IgE on microarrays by use of signal amplification techniques [Articolo su rivista]
Bacarese-Hamilton, Tito; Mezzasoma, Letizia; Ingham, Colin; Ardizzoni, Andrea; Rossi, Ruggero; Bistoni, Francesco; Crisanti, Andrea
abstract

2001 - Osteocyte Lacunar Size–Lamellar Thickness Relationships in Human Secondary Osteons [Articolo su rivista]
Ardizzoni, Andrea
abstract

Previous investigations carried out in our laboratory on secondary osteons have shown that osteocyte lacunae decrease in size from the cement line towards the Haversian canal, and lamellar bone is made up of alternating nonosteocytic dense lamellae and osteocytic loose lamellae, all having an interwoven texture of collagen fibers. Such alternation of acellular and cellular lamellae was hypothesized to depend on osteocyte recruitment from osteogenic laminae in successive layers, assuming that the loose lamellae form because of alignment and fusion of the periosteocytic loosely arranged collagen fibers. In order to discover whether a correlation really exists between osteocyte lacunar size and lamellar thickness, as would be expected if the above-mentioned hypothesis were true, both these parameters were measured in completed secondary osteons in relation to their distance from the Haversian canal. The size of osteocyte lacunae was measured under light microscopy on undecalcified drymounted ground section of tibial compact bone from three adult males and three adult females not affected by metabolic bone disease. The measurement of the thickness of bony lamellae was carried out on the same samples under scanning electron microscopy. Statistical analyses of the results showed that the decrease in size of osteocytic lacunae from the outer to the inner osteonal wall is paralleled by a decrease in thickness of osteocytic loose lamellae. The fact that acellular dense lamellae do not follow such a decremental pattern, but remain of the same thickness throughout the osteonic wall, corroborates Marotti’s view that the formation of lamellar bone depends on the orderly distribution of the osteocytes in alternating planes. The topographical distribution of osteocyte lacunar size and lamellar thickness is briefly discussed in relation to secondary osteon mechanical function.

2001 - Osteocyte-bone lining cell system responds to cyclic loading in a dose dependent manner [Abstract in Rivista]
Rubinacci, A.; Covini, M.; Bisogni, C.; Villa, I.; Galli, M.; Palumbo, Carla; Ferretti, Marzia; Ardizzoni, Andrea; Marotti, Gastone
abstract

The mutual interaction between osteocyte-bone lining cell system (OBLCS) and the surrounding bone extracellular fluid which feels the continuous network of lacuno-canalicular microcavities plays a role in mechanotransduction. The study shows that OBLCS responds to cyclic loading depending on the applied loading parameters in a dose- dependent manner.

2001 - Osteocyte-osteoclast morphological relationships and the putative role of osteocyte in bone remodeling [Articolo su rivista]
Palumbo, Carla; Ferretti, Marzia; A., Ardizzoni; Zaffe, Davide; Marotti, Gastone
abstract

An osteocyte lacunae differential count under the light microscope (LM) (1-lacunae with live osteocytes, 2-empty lacunae and lacunae with degenerating osteocytes) was carried out outside the reversal lines of osteonic lamellar bone from various mammals and man to evaluate the possibility of osteocyte survival where osteoclast resorption had occurred. The polarized light microscope (PLM) was used to establish the curvature of bony lamellae outside the convexity of reversal lines: concave lamellae indicate osteocytes reabsorbed on their vascular side where they radiate long vascular dendrites; convex lamellae indicate bone resorption on the osteocyte mineral side, radiating short dendrites. In all samples it was found that: a) about 60% of osteocytes outside the reversal lines were live; b) the percentage of alive osteocytes close to reversal lines is higher when they are attacked on their mineral side. The present data support our view that surviving osteocytes, particularly those attacked from their mineral side, might intervene in the final phase of bone resorption (osteoclast inhibition?). The fact that under the transmission electron microscope (TEM) intercellular contacts were never observed between osteocytes and osteoclasts indicates that if a modulation should occur between these two cellular types it could take place by a paracrine route only. The putative role of the cells of the osteogenic system, particularly osteocytes, in the bone remodeling cycle is also discussed.

2001 - Osteocyte-osteoclast morphological relationships and the putative role of osteocytes in bone remodeling. [Articolo su rivista]
Palumbo, Carla; Ferretti, Marzia; Ardizzoni, Andrea; Zaffe, Davide; Marotti, Gastone
abstract

An osteocyte lacunae differential count under the light microscope (LM) (1-lacunae with live osteocytes, 2-empty lacunae and lacunae with degenerating osteocytes) was carried out outside the reversal lines of osteonic lamellar bone from various mammals and man to evaluate the possibility of osteocyte survival where osteoclast resorption had occurred. The polarized light microscope (PLM) was used to establish the curvature of bony lamellae outside the convexity of reversal lines: concave lamellae indicate osteocytes reabsorbed on their vascular side where they radiate long vascular dendrites; convex lamellae indicate bone resorption on the osteocyte mineral side, radiating short dendrites. In all samples it was found that: a) about 60\% of osteocytes outside the reversal lines were live; b) the percentage of alive osteocytes close to reversal lines is higher when they are attacked on their mineral side. The present data support our view that surviving osteocytes, particularly those attacked from their mineral side, might intervene in the final phase of bone resorption (osteoclast inhibition?). The fact that under the transmission electron microscope (TEM) intercellular contacts were never observed between osteocytes and osteoclasts indicates that if a modulation should occur between these two cellular types it could take place by a paracrine route only. The putative role of the cells of the osteogenic system, particularly osteocytes, in the bone remodeling cycle is also discussed.

2001 - The osteocyte as transducer of mechanical strains into biological signals [Abstract in Atti di Convegno]
Marotti, G; Palumbo, C; Ferretti, M; Ardizzoni, A; Rubinacci, A; Covini, M; Dondi Benelli, F; Villa, I; Galli, E
abstract

2000 - Histomorphometric analysis of osteoclast effect on osteocyte viability [Abstract in Atti di Convegno]
Palumbo, Carla; Ferretti, Marzia; Zaffe, Davide; Ardizzoni, Andrea; Marotti, Gastone
abstract

2000 - Osteocyte lacunar size-lamellar thickness relationships [Relazione in Atti di Convegno]
Ardizzoni, Andrea; Muglia, Maria Antonietta; Marotti, Gastone
abstract

2000 - The role of osteocyte-bone lining cell system as a transducer and modulator of strain-related bone signals [Abstract in Atti di Convegno]
Rubinacci, A; Covini, M; Dondi Benelli, F; Villa, I; Galli, M; Palumbo, C; Ferretti, M; Ardizzoni, A; Marotti, G
abstract

2000 - Transduction of mechanical strain into biological message at the bone cellular level: preliminary report [Abstract in Atti di Convegno]
Marotti, G; Palumbo, C; Ferretti, M; Ardizzoni, A; Rubinacci, A; Covini, M; Dondi Benelli, F; Villa, I; Galli, E
abstract

1999 - Osteocyte size and lamellar thickness in human secondary osteons: preliminary data [Abstract in Atti di Convegno]
Ardizzoni, Andrea; Muglia, Maria Antonietta; Marotti, Gastone
abstract

1998 - Density and elasticity of trabecular bone tissue influence independently the us propagation: an in vitro study [Abstract in Atti di Convegno]
De Terlizzi, F; Battista, S; Cadossi, R; Canè, V; Ardizzoni, A
abstract

1998 - Influence of bone tissue density and elasticity on ultrasound propagation: an in vitro study [Abstract in Atti di Convegno]
De Terlizzi, F; Battista, S; Canè, V; Ardizzoni, A; Cadossi, R
abstract

1998 - Influenza della densità e dell'elasticità del tessuto osseo sulla propagazione degli ultrasuoni: studio in vitro [Abstract in Atti di Convegno]
De Terlizzi, F; Canè, V; Ardizzoni, A; Cadossi, R
abstract

1997 - A SEM study on osteocyte proptoplasm by a new technical procedure: preliminary observations [Abstract in Atti di Convegno]
Muglia, Maria Antonietta; Ardizzoni, Andrea
abstract

1997 - JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess ΔΨ changes in intact cells: Implications for studies on mitochondrial functionality during apoptosis [Articolo su rivista]
Salvioli, S.; Ardizzoni, A.; Franceschi, C.; Cossarizza, A.
abstract

The sensitivity and specificity of three fluorescent probes used for cytofluorimetric analysis of mitochondrial membrane potential (ΔΨ) were studied in the U937 human cell line. First, the role of plasmamembrane in influencing the binding of the probes to mitochondria has been investigated. The depolarization of plasmamembrane with high doses of extracellular KCl had no immediate effects on the loading of JC-1, DiOC6(3) and rhodamine 123 (R123). However, after a few hours of culture in the presence of KCl, significant changes were observed only in cells stained with DiOC6(3). Second, a comparative study was performed concerning the effects of agents capable of collapsing ΔΨ. While adding FCCP to cell cultures resulted in consistent changes in the fluorescence emission of both JC-1 and DiOC6(3) - but not of R123 - only cells stained with JC-1 responded to valinomycin. On the whole, our data indicate that JC-1 is a reliable probe for analyzing ΔΨ changes with flow cytometry, while the others shown a lower sensitivity (R123), or a non-coherent behaviour, due to a high sensitivity to changes in plasmamembrane potential [DiOC6(3)]. These data cast some doubts on those studies that, using fluorescent probes that have a low sensitivity to ΔΨ, hypothesized that the fall in ΔΨ is one of the early events, if not one of the main causes, of apoptosis.

1995 - Experimentally induced microfractures in human lamellar bone [Abstract in Atti di Convegno]
Muglia, Maria Antonietta; Ardizzoni, Andrea
abstract

Università degli studi di Modena e Reggio Emilia

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