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Elisabetta BLASI

Professore Ordinario
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

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 - 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 - Attenuation of Pseudomonas aeruginosa Virulence by Pomegranate Peel Extract [Articolo su rivista]
Peppoloni, S.; Colombari, B.; Tagliazucchi, D.; Odorici, A.; Ventrucci, C.; Meto, A.; Blasi, E.
abstract

Pseudomonas aeruginosa is an opportunistic pathogen often responsible for biofilm-associated infections. The high adhesion of bacterial cells onto biotic/abiotic surfaces is followed by production of an extracellular polysaccharidic matrix and formation of a sessile community (the biofilm) by the release of specific quorum-sensing molecules, named autoinducers (AI). When the concentrations of AI reach a threshold level, they induce the expression of many virulence genes, including those involved in biofilm formation, motility, pyoverdine and pyocyanin release. P. aeruginosa embedded into biofilm becomes resistant to both conventional drugs and the host’s immune response. Accordingly, biofilm-associated infections are a major clinical problem underlining the need for new antimicrobial therapies. In this study, we evaluated the effects of pomegranate peel extract (PomeGr) in vitro on P. aeruginosa growth and biofilm formation; moreover, the release of four AI was assessed. The phenolic profile of PomeGr, exposed or not to bacteria, was determined by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) analysis. We found that bacterial growth, biofilm production and AI release were impaired upon PomeGr treatment. In addition, the PomeGr phenolic content was also markedly hampered following incubation with bacterial cells. In particular, punicalagin, punicalin, pedunculagin, granatin, di-(HHDP-galloyl-hexoside) pentoside and their isomers were highly consumed. Overall, these results provide novel insights on the ability of PomeGr to attenuate P. aeruginosa virulence; moreover, the AI impairment and the observed consumption of specific phenolic compounds may offer new tools in designing innovative therapeutic approaches against bacterial infections.

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 - Effectiveness of Two Different Fluoride-Based Agents in the Treatment of Dentin Hypersensitivity: A Prospective Clinical Trial [Articolo su rivista]
Qeli, E.; Toti, C.; Odorici, A.; Blasi, E.; Tragaj, E.; Tepedino, M.; Masedu, F.; Kacani, G.; Hysi, D.; Meto, A.; Fiorillo, L.; Meto, A.
abstract

Hyperesthesia is related to increased sensitivity of dental tissues to mechanical, chemical and thermal stimuli. The aim of this prospective clinical trial was to compare the effectiveness of a calcium-fluoride-forming agent (Tiefenfluorid®, Humanchemie GmbH, Alfeld, Germany) with that of a fluoride varnish (Enamelast™, Ultradent Inc., Cologne, Germany) in the treatment of dental hyperesthesia in adult patients. In total, 176 individuals (106 females and 70 males, aged 18–59 years old) diagnosed with dental hyperesthesia (DH) were enrolled. The main clinical symptoms were hyperesthesia from coldness and sweetness during chewing; the types of clinical lesions were also determined and recorded. The patients were selected randomly and divided into two groups: (i) the first group of 96 patients was treated with Tiefenfluorid® applied in three appointments at 7-day intervals; (ii) the second group of 80 patients was treated with Enamelast™, applied seven times at 7-day intervals. All the patients were recalled 7 days, 14 days, 1 month, 3 months, and 6 months from the last application. At the baseline and during every follow-up visit, the DH was measured with a pulp tester. A random intercept/random slope model was used to evaluate the effect of the treatment, at various times with respect to the initial diagnosis. Within the limits of the present study, Tiefenfluorid® was more effective than Enamelast™ against DH in that it provided long-lasting results, with a significant improvement still detected at the latest 6-month follow-up.

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 - Novel Options to Counteract Oral Biofilm Formation: In Vitro Evidence [Articolo su rivista]
Odorici, A.; Colombari, B.; Bellini, P.; Meto, A.; Venturelli, I.; Blasi, E.
abstract

Biofilm production on biotic and abiotic surfaces is crucial in the pathogenesis of most infections, particularly those occurring in the oral cavity. Its prevention and/or control may greatly facilitate the management of patients with oral diseases. Here, the antibiofilm activity of a biomimetic hydroxyapatite and a natural compound, MicroRepair (MicroR) and pomegranate (PomeGr), respectively, was assessed. By luminescence/fluorescence-based assays, Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus) and Candida albicans (C. albicans) were tested for biofilm production in the presence of MicroR and/or PomeGr. We found that both MicroR and PomeGr could affected biofilm production; however, the efficacy of the two, given alone or in combination, varied according to the microbial agent considered. These data open to clinical studies aimed at defining the most efficacious protocols to counteract oral biofilm-associated infections.

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

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

2022 - 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 - Copper–calcium hydroxide and permanent electrophoretic current for treatment of apical periodontitis [Articolo su rivista]
Meto, A.; Droboniku, E.; Blasi, E.; Colombari, B.; Tragaj, E.; Cervino, G.; Fiorillo, L.; Meto, A.
abstract

Endodontic failure has been and continues to be a problem for endodontics-specialists. Complicated anatomy, numerous foramens, and accessory canals are an environment for microorganisms to infect the teeth. The purpose of the present work was to evaluate the regeneration of copper–calcium hydroxide (Cupral)-endodontically treated teeth diagnosed with apical periodontitis using an electrophoresis technique. In total, 132 patients, aging from 19 to 65 years old, underwent endodontic treatment mono-and multi-radicular teeth, with complicated canals from January 2019 to June 2020. The patients were divided into two groups: (i) the control group—which included 54 patients (n = 62 teeth) receiving endodontic paste (Calcipast + 1) and, as final filling, the AHPlus™ cement—and (ii) the Cupral group, which included 78 patients (n = 80 teeth) receiving Cupral paste plus the electrophoretic current and, as final filling, the Atacamit-alkaline cement. The clinical cases were periodically observed along an 18-month follow-up period via radiography. Data were expressed as focal size of the lesions (mean ± standard error (SEM) of all the radiographic outcomes) observed in each group at each interval point. Statistical analysis was performed using the Student’s t-test that allowed us to compare the control and Cupral groups; the statistical significance was set at p < 0.05 and p < 0.01, where the latter was highly significant. Before treatments, the focal sizes were 4.8 mm and 4.95 mm for control and Cupral-treated groups, respectively. After 6 months, the mean focal sizes were 3.9 mm and 2.14 mm for the control and Cupral groups, respectively. After 12 months, in the control group, the mean focal size was measured at 2.8 mm, while, in Cupral group, the lesion size decreased down to 0.31 mm and a highly dynamic regeneration of the destructive focal-bone occurred. After 18 months, the lesions were further significantly reduced in the control group (mean values of 2.62 mm), while they were barely detectable in the Cupral group (0.2 mm). In conclusion, we provide initial evidence that the Cupral-electrophoresis methodology is effective in treating destructive periodontitis of teeth with problematic canals up to 18 months, thus allowing teeth preservation.

2021 - EDTA and Taurolidine Affect Pseudomonas aeruginosa Virulence In Vitro-Impairment of Secretory Profile and Biofilm Production onto Peritoneal Dialysis Catheters [Articolo su rivista]
Colombari, B.; Alfano, G.; Gamberini, C.; Cappelli, G.; Blasi, E.
abstract

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 for treatment of biofilm-harboring peritoneal catheters. This study investigated the effects of two lock solutions, EDTA and taurolidine, on an in vitro model of Pseudomonas aeruginosa biofilm-related peritoneal catheter infection. Silicone peritoneal catheters were incubated for 24 h with a bioluminescent strain of P. aeruginosa. Then, serial dilutions of taurolidine and/or EDTA were applied (for 24 h) once or twice onto the contaminated catheters, and P. aeruginosa viability/persistence were evaluated in real time up to 120 h using a Fluoroskan reader. On selected supernatants, high-performance liquid chromatography mass spectrometry (HPLC-MS) analysis was performed to measure the production of autoinducers (AI), phenazines, and pyocyianines. Taurolidine alone or in combination with EDTA caused a significant decrease of bacterial load and biofilm persistence on the contaminated catheters. The treatment did not lead to the sterilization of the devices, yet it resulted in a substantial destructuration of the catheter-associated P. aeruginosa biofilm. HPLC-MS analysis showed that the treatment of biofilm-harboring catheters with taurolidine and EDTA also affected the secretory activity of the pathogen. EDTA and taurolidine affect P. aeruginosa biofilm produced on peritoneal catheters and profoundly compromise the microbial secretory profile. Future studies are needed to establish whether such lock solutions can be used to render peritoneal catheterrelated infections more susceptible to antibiotic treatment.

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 - Evaluation of antimicrobial effect of air-polishing treatments and their influence on human dental pulp stem cells seeded on titanium disks [Articolo su rivista]
Di Tinco, R.; Bertani, G.; Pisciotta, A.; Bertoni, L.; Bertacchini, J.; Colombari, B.; Conserva, E.; Blasi, E.; Consolo, U.; Carnevale, G.
abstract

Dental implants are one of the most frequently used treatment options for tooth replacement, and titanium is the metal of choice due to its demonstrated superiority in resisting corrosion, lack of allergic reactions and mechanical strength. Surface roughness of titanium implants favors the osseointegration process; nevertheless, its topography may provide a suitable substrate for bacterial biofilm deposition, causing peri-implantitis and leading to implant failure. Subgingival prophylaxis treatments with cleansing powders aimed to remove the bacterial accumulation are under investigation. Two different air-polishing powders-glycine and tagatose-were assayed for their cleaning and antimicrobial potential against a Pseudomonas biofilm and for their effects on human dental pulp stem cells (hDPSCs), seeded on sandblasted titanium disks. Immunofluorescence analyses were carried out to evaluate cell adhesion, proliferation, stemness and osteogenic differentiation. The results demonstrate that both the powders have a great in vitro cleaning potential in the early period and do not show any negative effects during hDPSCs osteogenic differentiation process, suggesting their suitability for enhancing the biocompatibility of titanium implants. Our data suggest that the evaluated cleansing systems reduce microbial contamination and allow us to propose tagatose as an adequate alternative to the gold standard glycine for the air-polishing prophylaxis treatment.

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.

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

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

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

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

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

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

2020 - Propolis affects Pseudomonas aeruginosa Growth, Biofilm Formation, Phenazines and eDNA Release: Potential Involvement of Polyphenols [Articolo su rivista]
Meto, Aida; Colombari, Bruna; Meto, Agron; Boaretto, Giorgia; Pinetti, Diego; Marchetti, Lucia; Benvenuti, Stefania; Pellati, Federica; Blasi, Elisabetta
abstract

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen responsible for a wide range of clinical conditions, from mild infections to life-threatening nosocomial biofilm-associated diseases, which are particularly severe in susceptible individuals. The aim of this in vitro study was to assess the effects of an Albanian propolis on several virulence-related factors of P. aeruginosa, such as growth ability, biofilm formation, extracellular DNA (eDNA) release and phenazine production. To this end, propolis was processed using three different solvents and the extracted polyphenolic compounds were identified by means of High Performance Liquid Chromatography coupled to–Mass spectrometry (HPLC-MS) analysis. As assessed by a bioluminescence-based assay, among the three propolis extracts, the ethanol (EtOH) extract was the most effective in inhibiting both microbial growth and biofilm formation, followed by propylene glycol (PG) and polyethylene glycol 400 (PEG 400) propolis extracts. Also, Pseudomonas exposure to propolis EtOH extract caused a decrease in eDNA release and phenazine production. Interestingly, caffeic acid phenethyl ester (CAPE) and quercetin decreased upon propolis EtOH extract exposure to bacteria. Overall, our data add new insights on the anti-microbial properties of a natural compound, such as propolis against P. aeruginosa. The potential implications of these findings will be discussed.

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

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

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

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

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

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

2019 - Anti-Candida albicans germ tube antibodies reduce in vitro growth and biofilm formation of C. albicans [Articolo su rivista]
Carrano, Giulia; Paulone, Simona; Lainz, Lucía; Sevilla, María-Jesús; Blasi, Elisabetta; Moragues, María-Dolores
abstract

Background: Invasive candidiasis by Candida albicans is associated with high morbidity and mortality, due in part to the late implementation of an appropriate antifungal therapy hindered by the lack of an early diagnosis. Aims: We aimed to evaluate the in vitro antifungal activity of the antibodies against C. albicans germ tubes (CAGTA) raised in a rabbit model of candidemia. Methods: We measured the effect of CAGTA activity by colorimetric XTT and crystal violet assays, and colony forming units count, both on C. albicans planktonic cells and during the course of biofilm formation and maturation. Viability and cell morphology were assessed by optical, fluorescent or scanning electron microscopy. Results: CAGTA ≥50 μg/ml caused a strong inhibition of C. albicans blastospores growth, and DiBAC fluorescent staining evidenced a fungicidal activity. Moreover, electron microscopy images revealed that CAGTA induced morphological alterations of the surface of C. albicans germ tubes grown free as well as in biofilm. Interestingly, CAGTA ≥80 μg/ml reduced the amount of C. albicans biofilm, and this effect started at the initial adhesion stage of the biofilm formation, during the first 90 min. Conclusions: This is the first report showing that CAGTA reduce C. albicans growth, and impair its metabolic activity and ability to form biofilm in vitro. The antigens recognized by CAGTA could be the basis for the development of immunization protocols that might protect against Candida infections.

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

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

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

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

2019 - Corrigendum to “Performance of Candida albicans germ tube antibodies (CAGTA) and its association with (1 → 3)-β-D-glucan (BDG) for diagnosis of invasive candidiasis (IC)” (Diagnostic Microbiology & Infectious Disease (2019) 93(1) (39–43), (S073288931830230X), (10.1016/j.diagmicrobio.2018.07.007)) [Articolo su rivista]
Pini, P.; Colombari, B.; Marchi, E.; Castagnoli, A.; Venturelli, C.; Sarti, M.; Blasi, E.
abstract

In the article “Performance of Candida albicans germ tube antibodies (CAGTA) and its association with (1 → 3)-β-D-glucan (BDG) for diagnosis of invasive candidiasis (IC),” the authors’ names are listed incorrectly. The correct names are as follows: Pietro Pini, Bruna Colombari, Enrico Marchi, Anna Castagnoli, Claudia Venturelli, Mario Sarti, Elisabetta Blasi.

2019 - Differential efficacy of two dental implant decontamination techniques in reducing microbial biofilm and re-growth onto titanium disks in vitro [Articolo su rivista]
Meto, A.; Conserva, E.; Liccardi, F.; Colombari, B.; Consolo, U.; Blasi, E.
abstract

Dental implants are crucial therapeutic devices for successful substitution of missing teeth. Failure cases are mainly pathogen-associated events, allowing clinical progression toward peri-mucositis or peri-implantitis. The aim of this study was to compare the performance of two mechanical decontamination systems, Nickel-Titanium brush (Brush) and Air-Polishing system with 40 μm bicarbonate powder (BIC-40), by means of a novel bioluminescence-based model that measures microbial load in real time. Briefly, 30 disks were contaminated using the bioluminescent Pseudomonas aeruginosa strain (BLI-P. aeruginosa), treated with Brush (30 s rounds, for 90 s) or BIC-40 (30 s, at 5 mm distance) procedure, and then assessed for microbial load, particularly, biofilm removal and re-growth. Our results showed that Brush and BIC-40 treatment reduced microbial load of about 1 and more than 3 logs, respectively. Furthermore, microbial re-growth onto Brush-treated disks rapidly occurred, while BIC-40-treated disks were slowly recolonized, reaching levels of microbial load consistently below those observed with the controls. In conclusion, we provide evidence on the good performance of BIC-40 as titanium device-decontamination system, the clinical implication for such findings will be discussed.

2019 - Efficacy of a Copper-Calcium-Hydroxide Solution in Reducing Microbial Plaque on Orthodontic Clear Aligners: A Case Report [Articolo su rivista]
Meto, A.; Colombari, B.; Castagnoli, A.; Sarti, M.; Denti, L.; Blasi, E.
abstract

The aim of this study was to investigate the ability of a copper-calcium-hydroxide-based compound to remove microbial plaque naturally produced onto orthodontic clear aligners. A commercially available dental paste, named Cupral, based on copper-calcium-hydroxide, was used. A healthy volunteer (female, 32 years old), undergoing orthodontic treatment with thermoplastic clear aligners was enrolled. By conventional/confocal microscopy and colony-forming unit (CFU) assay, 2-week used aligners were examined for microbial plaque, prior and following exposure to Cupral. Confocal microscopy revealed abundant plaque irregularly distributed onto the aligner surface. Following Cupral treatment, a drastic decrease occurred in plaque thickness and matrix presence. As assessed by the CFU assay, total microbial load approached 10 9 CFUs/aligner, with slight differences in aerobiosis and anaerobiosis culture conditions; six macroscopically different types of colonies were detected and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Following Cupral treatment, microbial load dropped to undetectable levels, irrespectively of the conditions considered. Exposure of clear aligners to Cupral results in the elimination of contaminating microorganisms; the antimicrobial activity is retained up to 1.25% concentration. Overall, our data describe a novel use of Cupral, a copper-calcium-hydroxide-based compound, in daily hygiene practices with promising results.

2019 - Evaluation of Biological Response of STRO-1/c-Kit Enriched Human Dental Pulp Stem Cells to Titanium Surfaces Treated with Two Different Cleaning Systems. [Articolo su rivista]
Conserva, E; Pisciotta, A; Bertoni, L; Bertani, Giulia; Meto, A; Colombari, B; Blasi, E; Bellini, P; de Pol, A; Consolo, U; Carnevale, G.
abstract

Peri-implantitis-an infection caused by bacterial deposition of biofilm-is a common complication in dentistry which may lead to implant loss. Several decontamination procedures have been investigated to identify the optimal approach being capable to remove the bacterial biofilm without modifying the implant surface properties. Our study evaluated whether two different systems-Ni-Ti Brushes (Brush) and Air-Polishing with 40 µm bicarbonate powder (Bic40)-might alter the physical/chemical features of two different titanium surfaces-machined (MCH) and Ca++ nanostructured (NCA)-and whether these decontamination systems may affect the biological properties of human STRO-1+/c-Kit+ dental pulp stem cells (hDPSCs) as well as the bacterial ability to produce biofilm. Cell morphology, proliferation and stemness markers were analysed in hDPSCs grown on both surfaces, before and after the decontamination treatments. Our findings highlighted that Bic40 treatment either maintained the surface characteristics of both implants and allowed hDPSCs to proliferate and preserve their stemness properties. Moreover, Bic40 treatment proved effective in removing bacterial biofilm from both titanium surfaces and consistently limited the biofilm re-growth. In conclusion, our data suggest that Bic40 treatment may operatively clean smooth and rough surfaces without altering their properties and, consequently, offer favourable conditions for reparative cells to hold their biological properties.

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

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

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

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

2019 - 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.

2019 - Performance of Candida albicans germ tube antibodies (CAGTA) and its association with (1 → 3)-β-D-glucan (BDG) for diagnosis of invasive candidiasis (IC) [Articolo su rivista]
Pini, Pietro; Colombari, Bruna; Marchi, Enrico; Castagnoli, Anna; Claudia, Venturelli; Mario, Sarti; Blasi, Elisabetta
abstract

Invasive candidiasis (IC) plays an important role as severe infection. Elder population, immunocompromised individuals, and intensive care unit (ICU) patients, especially when exposed to major surgery, are the most affected. IC diagnosis and treatment are difficult because of the absence of pathognomonic signs and symptoms. In addition, culture-based examination (gold standard) is known to have low sensitivity and long time to report. All these often lead to unnecessary and costly empirical antifungal therapies, burdened also by the onset of drug resistance and serious side effects for the patient. To partially overcome these problems, in recent years, novel noncultural markers have been investigated with the aim of easily and rapidly achieving an early diagnosis of IC. Such novel markers include the pan-fungal antigen (1 → 3)-β-D-glucan (BDG) and the anti–Candida albicans germ tube antibodies (CAGTAs). We retrospectively analyzed the presence of CAGTA on −80 °C stored serum samples, where the level of BDG had been previously assessed in a prospective study conducted in the Azienda Ospedaliero–Universitaria Policlinic of Modena (Pini et al. Infection 44:223–233, 2016). In particular, we selected 29 samples from proven IC episodes and 28 from non-IC cases. The 29 IC samples had been diagnosed as infections by C. albicans (n = 16), C. glabrata (n = 8), C. parapsilosis (n = 1), C. pelliculosa (n = 1), and C. tropicalis (n = 1), while 2 samples had intrasurgery biopsies positive for yeast (compatible with Candida spp.). The 28 control samples (non-IC) included 9 sera with positive blood cultures [E. faecium (n = 5), S. pneumoniae (n = 2), P. aeruginosa + A. baumannii (n = 2)] and 19 negative blood cultures. The CAGTA immunofluorescence assay was performed using 1:40, 1:80, 1:160, and 1:320 dilutions (reference dilution, as indicated by the manufacturer). According to the protocol, the samples were evaluated by the operator-dependent optical reading based on immunofluorescence positive/negative samples. In parallel, with the aim of standardizing the reading, the fluorescence images were captured, and the data were expressed as arbitrary fluorescence units (AFU). Finally, the results were interpreted as positive or negative using a cutoff provided by receiver operating characteristic (ROC) curves (Youden index). The traditional operator-dependent optical reading and the AFU measuring protocol provided comparable information with respect to the processed samples since IC and non-IC sera were correctly identified by the 2 CAGTA reading strategies in most of the cases. Interestingly, the AFU reading enabled a semiquantitative evaluation of the samples and an objective interpretation of the results. Based on the cutoff value, the AFU-based CAGTA procedure demonstrated a sensitivity of 52% and a specificity of 89%, while BDG showed a sensitivity of 90% and a specificity of 75%; the overall accuracy was 70% and 83% for CAGTA and BDG, respectively. The association of the 2 markers greatly increased both sensitivity and accuracy to 97% and 84%, respectively. As expected, when excluding non–C. albicans episodes, the sensitivity of CAGTA increased from 52% to 86%; moreover, with the exclusion of the non–deep-seated episodes, the sensitivity of CAGTA increased to 67% and reached 100% for C. albicans deep-seated candidiasis. Finally, when evaluating the influence of colonization, BDG demonstrated the most drastic decrease in specificity that dropped from 88% in noncolonized to 58% in colonized patients. With the exception of non–C. albicans episodes, CAGTA is a good marker of IC, particularly in the presence of deep-seated candidiasis. The performance of CAGTA greatly increases when used in combination with BDG.

2019 - Prognostic Potential of the Panfungal Marker (1 → 3)-β-D-Glucan in Invasive Mycoses Patients [Articolo su rivista]
Pini, P; Venturelli, C; Girardis, M; Forghieri, F; Blasi, E
abstract

We analyze the prognostic potential of (1 → 3)-β-D-glucan (BG) levels in predicting clinical outcomes in patients with invasive fungal infections, on a population undergoing 253 episodes (177 with positive and 76 with negative outcome). Using linear regression analysis, we assessed the prognostic potential of kinetically evaluated BG levels and we found an overall sensitivity and specificity of 68 and 82%, respectively. Moreover, using an interpretative algorithm based on two distinct cutoff values, we were able to predict the outcome in 84% of the studied population with a diagnostic accuracy of 82%.

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

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

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

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

2018 - Dr. Luigi (Gigi) Varesio: A memorial [Articolo su rivista]
Young, H.; Blasi, E.; Cox, G.; Carla Bosco, M.
abstract

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 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 - 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 - Herpes Simplex Virus-1 entrapped in Candida albicans biofilm displays decreased sensitivity to antivirals and UVA1 laser treatment [Articolo su rivista]
Ascione, Cristian; Sala, Arianna; Mazaheri-Tehrani, Elham; Paulone, Simona; Palmieri, Beniamino; Blasi, Elisabetta; Cermelli, Claudio
abstract

Abstract Background: Recently, we published data suggesting a mutualistic relationship between HSV-1 and Candida. albicans; in particular: (a) HSV-1 infected macrophages are inhibited in their anti-Candida effector function and (b) Candida biofilm protects HSV-1 from inactivation. The present in vitro study is aimed at testing the effects of Candida biofilm on HSV-1 sensitivity to pharmacological and physical stress, such as antiviral drugs (acyclovir and foscarnet) and laser UVA1 irradiation. We also investigated whether fungus growth pattern, either sessile or planktonic, influences HSV-1 sensitivity to antivirals. Methods: Mature Candida biofilms were exposed to HSV-1 and then irradiated with laser light (UVA1, 355 λ). In another set of experiments, mature Candida biofilm were co-cultured with HSV-1 infected VERO cells in the presence of different concentrations of acyclovir or foscarnet. In both protocols, controls unexposed to laser or drugs were included. The viral yield of treated and untreated samples was evaluated by end-point titration. To evaluate whether this protective effect might occur in relation with a different growth pattern, HSV-1 infected cells were co-cultured with either sessile or planktonic forms of Candida and then assessed for susceptibility to antiviral drugs. Results: UVA1 irradiation caused a 2 Log reduction of virus yield in the control cultures whereas the reduction was only 1 Log with Candida biofilm, regardless to the laser dose applied to the experimental samples (50 or 100 J/cm2). The presence of biofilm increased the IC90 from 18.4–25.6 J/cm2. Acyclovir caused a 2.3 Log reduction of virus yield in the control cultures whereas with Candida biofilm the reduction was only 0.5 Log; foscarnet determined a reduction of 1.4 Log in the controls and 0.2 Log in biofilm cultures. Consequently, the ICs50 for acyclovir and foscarnet increased by 4- and 12-folds, respectively, compared to controls. When HSV-1 was exposed to either sessile or planktonic fungal cells, the antiviral treatments caused approximately the same weak reduction of virus yield. Conclusions: These data demonstrate that: (1) HSV-1 encompassed in Candida biofilm is protected from inactivation by physical (laser) and pharmacological (acyclovir or foscarnet) treatments; (2) the drug antiviral activity is reduced at a similar extent for both sessile or planktonic Candida.

2017 - Pneumocystis jirovecii genotyping: experience in a tertiary-care hospital in Northern Italy [Articolo su rivista]
Pini, Pietro; Orsi, Carlotta F; La Regina, Annunziata; Peppoloni, Samuele; Berrilli, Federica; Blasi, Elisabetta; Di Cave, David
abstract

Respiratory samples from Pneumocystis jirovecii pneumonia (PJP) cases collected at a tertiary-care university hospital in Modena were analyzed for the presence of specific polymorphisms in the mitochondrial large subunit ribosomal RNA (mtLSU-rRNA). Retrospectively, 57 cases were selected in a six-year period and 34 out of the 57 processed BAL samples returned PCR positive results, thus allowing further molecular analysis. The following P.jirovecii genotype distribution was observed: genotype 3 (50%), genotype 2 (23%), genotype 1 (18%), genotypes 1 or 4 (9%). These data add novel insights on P.jirovecii epidemiology, investigating a previously unstudied area of Northern Italy. A peculiar local distribution is highlighted with respect to other areas within the national panorama, thus encouraging further in-depth studies in an attempt to better understand the overall situation concerning P.jirovecii genotype circulation.

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

2016 - Evaluation of serum (1 → 3)-β-D-glucan clinical performance: kinetic assessment, comparison with galactomannan and evaluation of confounding factors [Articolo su rivista]
Pini, Pietro; Bettua, C; Orsi, Carlotta Francesca; Venturelli, C; Forghieri, F; Bigliardi, S; Faglioni, L; Luppi, Fabrizio; Serio, L; Codeluppi, M; Luppi, Mario; Mussini, Cristina; Girardis, Massimo; Blasi, Elisabetta
abstract

Purpose We investigated the clinical performance of (1 → 3)-β-d-glucan (BG), as an early marker of invasive fungal infections (IFI), in different clinical settings. Methods BG serum levels were assessed by Fungitell (Associates of Cape Cod, Inc), in parallel with galactomannan (GM) when requested by clinicians. By a prospective monocentric study, 270 episodes at risk or with suspect of IFI were enrolled, namely 58 proven-probable invasive aspergillosis (IA), 27 proven invasive candidiasis (IC), 11 possible IC, 16 P.jirovecii pneumonia (PJP), 4 episodes of other IFI and 154 non-IFI controls. Results We found that (a) the BG overall sensitivity, specificity, positive predictive value and negative predictive value (NPV) were 87.9, 80.5, 76.7 and 89.9 %, respectively; (b) the highest sensitivity was found in the IC groups, followed by PJP, IA and other IFI groups; (c) an association was observed between BG kinetics and patients outcome; (d) in the IA episodes, the combination of BG or GM vs GM alone increased sensitivity from 60.0 to 83.3 % in the haematological patients; (e) false-positive BG results were related to Gram-negative infections or infusion of polyclonal IgM-enriched immunoglobulins, where high levels of BG were indeed detected. Conclusion Besides strengthening its overall good clinical performance, we provide evidence that serum BG correlates with clinical outcome and that, once used in combination with GM, BG allows to enhance IFI diagnosis rate. The high sensitivity and NPV, observed in the Intensive Care Unit setting, open to BG validation as a marker for assessment of antifungal treatment.

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

2015 - Clinical performance of a commercial real-time PCR assay for Aspergillus DNA detection in serum samples from high-risk patients: comparison with a galactomannan enzyme immunoassay [Articolo su rivista]
Pini, Pietro; Orsi, Carlotta Francesca; Luppi, Fabrizio; Girardis, Massimo; Blasi, Elisabetta
abstract

We investigated the clinical performance of a polymerase chain reaction (PCR)-based commercial platform, the Myconostica MycAssay™ Aspergillus (MAP), for fungal DNA detection in the serum of patients at risk of invasive aspergillosis (IA). Sixty-four hospitalized patients were prospectively enrolled and a total of 71 different episodes were investigated (30 episodes were clinically/microbiologically classified as IA and 41 as control episodes). When MAP was compared to the galactomannan (GM) assay, no significant differences were found in terms of sensitivity (46.7 % vs. 50.0 %), specificity (97.6 % vs. 95.1 %), positive predictive value (PPV) (93.3 % vs. 88.2 %), and negative predictive value (NPV) (71.4 % vs. 72.2 %). The corresponding areas under the curve (AUC) of the receiver operating characteristic (ROC) curves were also superimposable. Overall, because of the good agreement between the two assays and considering the high specificity and PPV of the MAP, we suggest the use of this PCR-based platform as a second-level examination for the evaluation of clinically undefined cases where culture or GM have provided positive results.

2015 - Detection of Pneumocystis jirovecii and Aspergillus spp. DNa in bronchoalveolar lavage fluids by commercial real-time PCr assays: comparison with conventional diagnostic tests [Articolo su rivista]
Orsi, Carlotta Francesca; Bettua, Clotilde; Pini, Pietro; Venturelli, Claudia; La Regina, Annunziata; Morace, Giulia; Luppi, Mario; Forghieri, Fabio; Bigliardi, Sara; Luppi, Fabrizio; Codeluppi, Mauro; Girardis, Massimo; Blasi, Elisabetta
abstract

The present study employed two commercial real-time PCR kits, MycAssay™ Pneumocystis (PJ-PCR) and MycAssay™ Aspergillus (ASP-PCR), for the search of fungal DNA on 44 bronchoalveolar lavage (BAL) fluids from patients at risk of invasive fungal disease. Operationally, on the basis of clinical diagnosis and according to the European Organization for Research and Treatment Cancer/Mycoses Study Group (EORTC/MSG) criteria, patients were clustered in 3 groups: a P. jirovecii pneumonia (PCP) group, an invasive aspergillosis (IA) group and a control (CTRL) group, consisting of 8, 10 and 24 patients, respectively. The results were compared to those obtained with conventional diagnostic assays, including BAL culture, galactomannan-ELISA (GM) and immunofluorescence (IF). The PJ-PCR assay returned a sensitivity and specificity of 100% and 94.4%, respectively. The ASP-PCR assay showed a sensitivity and specificity of 80% and 97.1%. When compared to the culture assay, the ASP-PCR showed enhanced sensitivity, and a good level of agreement (kappa = 0.63) was observed between ASP-PCR and GM assays. Overall, our data emphasize the diagnostic usefulness of the two commercial real-time PCR assays, especially in high-risk patients where timing is critical and a low fungal burden may hamper correct and prompt diagnosis by conventional tests.

2015 - Trichphyton violaceum and T. soudanese: re-emerging pathogens in Italy, 2005-2013 [Articolo su rivista]
Farina, C.; Fazii, P.; Imberti, G.; Lombardi, G.; Passera, M.; Andreoni, S.; Manso, E.; Arosio, M.; Vailati, F.; Bruno, G.; Mattei, R.; Perin, S.; Marini, F.; Blasi, Elisabetta; Conte, M.; Savio, C.; Zavarise, G.; Pediatria, U. O.; Cavanna, C.; Carpi, D.; Saletti, A.; Sanna, S.
abstract

Dermatomycoses due to Trichophyton violaceum are described in Mediterranean Countries, North Africa and in the Horn of Africa where T. soudanense is present too, but it was rare until few years ago in Italy. Aim of the present study was to evaluate an Italian multicenter 9 year (2005-2013) experience concerning these re-emerging pathogens. Fifty three fungal strains were sent from clinical laboratories to the Medical Mycology Committee (CoSM) - Italian Association of Clinical Microbiology (AMCLI) for mycological confirmation. Strains were identified as T. violaceum (23) and T. soudanense (30) by phenotypic and genotypic methods. These dermatophytes present epidemiological (high rate of inter-human transmission, high risk among adopted children coming from countries of either the Horn of Africa or Sub-Saharan Africa also in outbreaks of tinea capitis) and clinical peculiarities (reduced alopecia, presence of exudative lesions) confirming the originality of these “imported” dermatophyte infections

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 - Herpes simplex virus-1 entrapped in Candida albicans biofilm displays decreased sensitivity to antivirals. [Poster]
Cermelli, Claudio; MAZAHERI TEHRANI, Elham; Sala, A.; Orsi, Carlotta Francesca; Neglia, Rachele Giovanna; Blasi, Elisabetta
abstract

BACKGROUND AND AIM Biofilms represent a serious clinical problem because of the increased resistance of biofilm-associated organisms to antimicrobial agents and the potential for these organisms to cause infections in patients with indwelling medical devices. The presence of some pathogenic viruses in water biofilms underlines the ability of viruses to attach and cling to biofilms retaining their infectivity. Recently we demonstrated that, in vitro, human pathogenic viruses, including HSV-1, can be encompassed in C. albicans biofilm. This biofilm is responsible for severe device-related disseminated infections causing invasive candidemias with a very high rate of mortality. The aim of this in vitro study was to ascertain whether encompassment of Herpes Simplex Virus type 1 (HSV-1) in Candida biofilm impacts virus sensitivity to acyclovir and foscarnet. METHODS VERO cells were infected with HSV-1 and added to mature Candida biofilms in the presence and the absence of acyclovir or foscarnet. After 24h incubation, the amount of infectious virus embedded in biofilm was titrated on VERO cells. Similarly, different drug scalar concentrations were tested in order to determine the inhibiting dose 50 (ID50). In order to evaluate whether the impact on drug antiviral activity is related to the presence of biofilm matrix or to the steric obstruction of the hyphal mass, the efficacy of antiviral drugs were also tested comparing the virus inhibition growth in the presence of Candida biofilm with that obtained in cultures of the same candida strain grown in planktonic hyphal form. RESULTS Acyclovir 50 µM caused a 2,3 Log reduction (99.5%) of virus yield in the control cultures whereas in the presence of Candida biofilm the reduction was only 05 Log (68.5%); foscarnet determined a reduction of 1.4 Log (96%) in the controls and 0.2 Log (36.9%) in biofilm cultures. IDs50 for acyclovir were 5.4µM and 22.6 µM in the absence and in the presence of Candida biofim respectively; for foscarnet IDs50 were 54 µM and 661 µM, respectively. DISCUSSION Encompassment of HSV1-infected cells within the biofilm causes a dramatic decrease in antiviral efficacy of ayclovir and foscarnet. We can speculate that circulating HSV-1 infected cells might be retained in the biofilm and, later on, released as either single cells or within biofilm small fragments. Therefore, Candida biofilm on medical devices may be a source of viral infections with a reduced drug sensitivity.

2014 - Human pathogenic viruses are retained in and released by Candida albicans biofilm in vitro [Articolo su rivista]
Mazaheritehrani, Elham; Sala, Arianna; Orsi, Carlotta Francesca; Neglia, Rachele Giovanna; Morace, Giulia; Blasi, Elisabetta; Cermelli, Claudio
abstract

Candida albicans is the most prevalent human fungal pathogen associated with biofilm formation on indwelling medical devices. Under this form, Candida represents an infectious reservoir difficult to eradicate and possibly responsible for systemic, often lethal infections. Currently, no information is available on the occurrence and persistence of pathogenic viruses within C. albicans biofilm. Therefore, the aim of this study was to investigate whether Herpes Simplex Virus type 1 (HSV-1) and Coxsackievirus type B5 (CVB5) can be encompassed in Candida biofilm, retain their infectivity and then be released. Thus, cell-free virus inocula or HSV-1-infected cells were added to 24 h-old fungal biofilm in tissue culture plates; 48 h later, the biofilm was detached by washing and energetic scratching and the presence of virus in the rescued material was end-point titrated on VERO cells. Planktonic Candida cultures and samples containing only medium were run in parallel as controls. We found that both HSV-1 and CVB5 free virus particles, as well as HSV-1 infected cells remain embedded in the biofilm retaining their infectivity. As a second step, the influence of biofilm on virus sensitivity to sodium hypochlorite and to specific neutralizing antibodies was investigated. The results showed that virus encompassment in fungal biofilm reduces virus sensitivity to chemical inactivation but does not affect antibody neutralization. Overall, these data provide the first in vitro evidence that viruses can be encompassed within Candida biofilm and then be released. Thus, it may be speculated that Candida biofilm can be a reservoir of viruses too, posing a further health risk.

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

2014 - Routine use of a protease zymogen-based colorimetric assay for the detection of Beta-glucan and its role in clinical practice [Articolo su rivista]
Farina, Claudio; Lombardi, Gianluigi; Andreoni, Stefano; Manso, Esther; Perin, Silvana; Panellis, Dimitrios; Fazii, Paolo; Conte, Marco; Sanna, Silvana; Pini, Pietro; Blasi, Elisabetta
abstract

The detection of Aspergillus antigen (galactomannan) is considered a reliable marker for the diagnosis of invasive aspergillosis (IA), yet the sensibility and specificity of the assays commonly employed in routine are not optimal. The aim of the present study was to investigate whether the detection of another panfungal antigen, the (1,3)-b-D-glucan could have an auxiliary role in the identification of patients with IA. The study was carried out on 63 sera belonging to patients who had been screened for galactomannan, according to the clinical suspect of IA. Our data show that the positive galactomannan results were not confirmed by positive (1,3)-b-D-glucan results in patients receiving therapy with beta-lactam antibiotics associated with tazobactam, whereas in all the other cases, with the exception of four, the results of the (1,3)-b-D-glucan test were confirmatory of the galactomannan results.

2013 - Candida albicans biofilm can retain and release Human Herpes simplex virus type 1 in vitro [Abstract in Rivista]
Mazaheri, E.; Sala, A.; Orsi, Carlotta Francesca; Blasi, Elisabetta; Cermelli, Claudio
abstract

Microorganisms universally attach to surfaces and produce extracellular polymeric saccharides (EPS), resulting in the formation of a biofilm. Biofilms can pose a serious problem for public health because of the increased resistance of biofilm-associated organisms to antimicrobial agents and the potential for these organisms to cause infections in patients with indwelling medical devices. Moreover, involvement of enteric viruses with a variety of biofilms has been reported, although very little is known about this phenomenon. The presence of some pathogenic viruses in water biofilms underlines the ability of viruses to attach and cling to biofilms retaining their infectivity. No information is available so far on interactions between pathogenic viruses and Candida albicans biofilm. This biofilm is responsible for severe device-related disseminated infections causing invasive candidemias with a very high rate of mortality. The aim of this in vitro study was to ascertain whether Herpes Simplex Virus type 1 (HSV-1) can be encompassed in Candida biofilm produced in cell culture plates and/or on silicone and PVC catheters. HSV-1 was added to mature biofilms and the amount of infectious virus embedded in biofilm matrix detached by washing and energetic scratching was titrated on VERO cells 24-48 h later. Experiments with planktonic Candida were carried out in parallel, as well as in the absence of Candida. According to our results, free virus particles of HSV-1, as well as HSV-1 infected cells, remain embedded in Candida biofilm on tissue cell culture plates as well as on both types of catheter with a significantly higher load than in the presence of planktonic Candida or in the negative controls. These results provide the first evidence that infectious viruses, after being entrapped in Candida biofilms, can retain their infectivity and be released posing a health risk for patients with implanted medical devices. Interactions between HSV-1 embedded in Candida biofilm and disinfectants as well as neutralizing antibodies and drugs are discussed.

2013 - Contribution of different pneumococcal virulence factors to experimental meningitis in mice [Articolo su rivista]
Ricci, S; Gerlini, A; Pammolli, A; Chiavolini, D; Braione, V; Tripodi, Sa; Colombari, Bruna; Blasi, Elisabetta; Oggioni, Mr; Peppoloni, Samuele; Pozzi, G.
abstract

BACKGROUND: Pneumococcal meningitis (PM) is a life-threatening disease with a high case-fatality rate and elevated risk for serious neurological sequelae. In this study, we investigated the contribution of three major virulence factors of Streptococcus pneumoniae, the capsule, pneumococcal surface protein A (PspA) and C (PspC), to the pathogenesis of experimental PM. METHODS: Mice were challenged by the intracranic route with the serotype 4 TIGR4 strain (wt) and three isogenic mutants devoid of PspA, PspC, and the capsule. Survival, bacterial counts, and brain histology were carried out. To study the interaction between S. pneumoniae mutants and microglia, phagocytosis and survival experiments were performed using the BV2 mouse microglial cell line. RESULTS: Virulence of the PspC mutant was comparable to that of TIGR4. In contrast, survival of animals challenged with the PspA mutant was significantly increased compared with the wt, and the mutant was also impaired at replicating in the brain and blood of infected mice. Brain histology indicated that all strains, except for the unencapsulated mutant, caused PM. Analysis of inflammation and damage in the brain of mice infected with TIGR4 or its unencapsulated mutant demonstrated that the rough strain was unable to induce inflammation and neuronal injury, even at high challenge doses. Results with BV2 cells showed no differences in phagocytic uptake between wt and mutants. In survival assays, however, the PspA mutant showed significantly reduced survival in microglia compared with the wt. CONCLUSIONS: PspA may contribute to PM pathogenesis possibly by interacting with microglia at early infection stages, while PspC had limited importance in the disease. The rough mutant did not cause brain inflammation, neuronal damage or mouse death, strengthening the key role of the capsule in PM.

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 GENOTIPO HWP1 DI CANDIDA ALBICANS INFLUENZA LA SUSCETTIBILITÀ FUNGINA ALLE CELLULE MICROGLIALI, MA NON LA PRODUZIONE DI BIOFILM [Poster]
Orsi, Carlotta Francesca; Borghi, Elisa; Colombari, Bruna; Neglia, Rachele Giovanna; Quaglino, Daniela; Morace, Giulia; Blasi, Elisabetta
abstract

Introduzione. Il gene HWP1 di Candida albicans (Ca) codifica per una proteina della parete cellulare fungina coinvolta nella crescita ifale e nell’adesione del lievito alle cellule epiteliali; il ruolo di questa proteina nella patogenesi della candidosi non è ancora completamente chiarito. Il presente studio, mediante l’impiego di ceppi d’isolamento clinico di Ca e ceppi di laboratorio isogenici per HWP1, intende valutare il ruolo del genotipo HWP1 nella formazione del biofilm e nell’interazione in vitro tra Ca e le cellule microgliali (immunoeffettore cerebrale). Materiali e metodi. Sei ceppi clinici di Ca (3 eterozigoti e 3 omozigoti per il gene HWP1, indicati rispettivamente con gli acronimi H/h e H/H) e 2 ceppi di laboratorio (DAY286, wildtype per HWP1, H/H-wt, e FJS24, suo mutante isogenico HWP1-deleto, H/H-KO), sono stati studiati in vitro per valutare: a) la capacità di formare biofilm, b) i livelli di espressione del gene HWP1, c) l’ultrastruttura e d) la suscettibilità alle cellule microgliali, sia in termini di inibizione della formazione del biofilm che di induzione di una risposta secretoria. Risultati. Confrontando tra loro i ceppi clinici, i genotipi H/h hanno dimostrato una ridotta capacità di formare biofilm e una maggiore suscettibilità al danno mediato dalla cellula microgliale; a differenza della controparte H/H, i genotipi H/h hanno anche esibito una ridotta capacità a stimolare la produzione di ossido nitrico e di TNFα da parte delle cellule microgliali. A seguito dell’esposizione alle cellule microgliali, i ceppi H/H, ma non quelli H/h, hanno mostrato aumentati livelli di mRNA HWP1 specifico. Confrontando i 2 ceppi isogenici per HWP1, il genotipo H/H-KO ha mostrato una aumentata suscettibilità al danno mediato dalla microglia rispetto al ceppo H/H-wt, mentre il biofilm è stato prodotto in misura comparabile da parte di entrambi i ceppi. Inoltre, la microscopia elettronica a trasmissione ha rivelato differenze tra i due ceppi in termini di spessore della parete fungina, presenza di strutture superficiali adesine-simili e granuli intracitoplasmatici; tali differenze sono mantenute anche in seguito all'esposizione alle cellule microgliali. Conclusioni. Questi dati indicano che il genotipo HWP1 di Ca influenza la suscettibilità delle cellule fungine alla microglia, ma non ha effetto sulla capacità del fungo di formare biofilm.

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 - MARKER INNOVATIVI NELLA DIAGNOSTICA DELLE MICOSI INVASIVE [Poster]
Blasi, Elisabetta
abstract

L’incidenza delle infezioni fungine invasive (IFI) è in continuo aumento nei pazienti critici, quali gli onco-ematologici o i trapiantati, i soggetti comunque sottoposti a terapia immunosoppressiva o i pazienti con AIDS. Una diagnosi rapida ed un trattamento tempestivo delle IFI sono cruciali, in quanto ne condizionano la prognosi riducendo significativamente la mortalità. L’approccio diagnostico convenzionale, basato sull’esame colturale, attualmente considerato il gold standard, è poco sensibile e/o tardivo nel fornire risultati utili, mentre le indagini istopatologiche, sebbene dirimenti, sono problematiche e spesso difficilmente praticabili. Le indagini molecolari, basate sulla ricerca di biomarker specifici (acidi nucleici/antigeni fungini o anticorpi serici), offrono indubbi vantaggi in termini di rapidità, sensibilità e specificità. Inoltre, grazie alla non invasività delle indagini richieste, la ricerca di tali biomarker può essere effettuata in modo seriale, consentendo il monitoraggio del paziente critico, al fine di stabilire la tempistica di un eventuale trattamento pre-emptive e/o verificare l’efficacia della terapia. In particolare, il saggio per la ricerca di β-D-glucano (marker panfungino) ha un valore predittivo negativo pari al 100%, mentre la sua negativizzazione nel soggetto sottoposto a trattamento è indice di successo terapeutico. La presenza del galattomannano, così come la rilevazione di una glicoproteina extracellulare di Aspergillus, mediante test immuno-cromatografico recentemente descritto, hanno un elevato valore predittivo. La presenza simultanea dell’antigene mannano e degli anticorpi anti-mannano (marker genere-specifici) ha una sensibilità particolarmente elevata nei pazienti critici (esclusi gli immunodepressi) e precede in modo significativo la positivizzazione dell’emocoltura. I test molecolari, basati sull’amplificazione di acidi nucleici fungini, rappresentano degli strumenti diagnostici molto rapidi e soprattutto sensibili, nei casi in cui l’iter convenzionale fornisce risultati ripetutamente negativi. Infine, per quanto concerne il contributo della diagnostica sierologica nell’identificazione dei soggetti con IFI, è stato messo a punto un saggio in immunofluorescenza per la ricerca di anticorpi verso il tubulo germinativo di Candida albicans (CAGTA) ed un test miniaturizzato e multi-parametrico, basato sulla tecnologia del microarray di proteine, per la determinazione quantitativa della risposta anticorpale nei confronti di 11 antigeni diversi di C. albicans; in tutti i casi, sensibilità (77-89%) e specificità (91-100%) si sono dimostrate particolarmente elevate. Recentemente, l’approccio basato sul microarray di proteine è stato applicato con successo anche nella diagnostica delle micosi invasive da funghi dimorfi, consentendo l’identificazione rapida di soggetti affetti da istoplasmosi e coccidioidomicosi d’importazione. Nel complesso, sebbene l’efficacia diagnostico-clinica dei metodi non-colturali resti ancora poco definita, il loro impiego è fortemente auspicabile soprattutto alle luce del fatto che essi consentono indagini multidirezionali e multi-parametriche, cruciali nell’ottimizzazione del percorso diagnostico delle IFI.

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 - In vitro interactions between viruses and Candida biofilm. [Poster]
Mazaheri, E; Sala, A.; Orsi, Carlotta Francesca; Neglia, Rachele Giovanna; Blasi, Elisabetta; Cermelli, Claudio
abstract

BACKGROUND Candida albicans is known as one of the major cause of infections related to biofilms forming on medical devices such as catheters, artifical vales, prostheses which become a source invasive candidiasis with a high mortality rates (30-50%). So far, only few studies investigated the interactions between human pathogenic viruses and biofilms, mainly focused on water biofilms. To our knowledge, there are no studies on the interplay between biofilms in humans and viruses. In this study, we studied whether Herpes Simplex Virus type 1 (HSV-1) and Coxsackievirus type B5 (CoxB5) can be encompassed in Candida biofilm, retaining their infectivity, and then be released. Moreover, we investigated the ability of Candida biofilm to hold non adhering HSV-1 infected cells within the matrix. METHODS Candida albicans biofilms were grown in tissue culture microplates and then exposed to HSV-1 or CoxB5 for 48h: after deep washing and energetic scapring of the wells to remove the matrix, the residual presence of virus was end-point titrated on VERO cells. In parallel, wells with a strain of non-biofilm producer Candida albicans (planktonic) and negative controls with only medium were processed at the same way. Alternatively, Candida biofilms were exposed to non adhering HSV-1-infected cells and then, after washing and scraping, the number of living cells attached to the biofilms and virus titer were determined. RESULTS AND DISCUSSION Both free virus particles of HSV-1 and CoxB5 and HSV-1 infected cells remained embedded in the biofilmf with a significantly higher load than in the presence of planktonic Candida or in the negative controls. Candida biofilm can be a reservoir for viruses.

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 - Performance of two commercial real-time PCR assays for the detection of Aspergillus and Pneumocystis DNA in bronchoalveolar lavage fluid samples from critical care patients. [Articolo su rivista]
Orsi, C. F.; Gennari, W.; Venturelli, C.; La Regina, A.; Pecorari, M.; Righi, E.; Machetti, M.; Blasi, E.
abstract

This article investigates the performance of 2 commercial real-time polymerase chain reaction (PCR) assays, MycAssay™ Aspergillus (MycAspAssay) and MycAssay™ Pneumocystis (MycPCPAssay), on the ABI 7300 platform for the detection of Aspergillus (Asp) or Pneumocystis jirovecii (Pj) DNA in bronchoalveolar lavage (BAL) samples from 20 patients. Operationally, patients enrolled were clustered into 3 groups: invasive aspergillosis group (IA, 7 patients), Pj pneumonia group (PCP, 8 patients), and negative control group (5patients). All the IA patients were MycAspAssay positive, whereas 12 non-IA patients returned negative PCR results. Furthermore, 7 of 8 PCP patients were MycPCPAssay positive, while 9 non-PCP patients were PCR negative. In conclusion, these data provide an early indication of the effectiveness of both the MycAspAssay and MycPCPAssay on the ABI 7300 platform for the detection of either Asp or Pj DNA in BAL from patients with deep fungal infections.

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 - Immunobiological characterization of genetic products, identified from whole genome lambda-display libraries of Pneumococcus [Poster]
Peppoloni, Samuele; Colombari, Bruna; Blasi, Elisabetta; Garibaldi, Manuela; Pernice, Ida; Manca, Lidia; Lo Passo, Carla; Speziale, Pietro; Papasergi, Salvatore; Teti, Giuseppe; Felici, Franco; Beninati, Concetta
abstract

Objectives: Streptococcus pneumoniae is an important cause of morbidity and mortality worldwide, including meningitis. Here, we employed the isogenic Δspr0075-, Δspr1370- and Δspr1875-knock-out mutants of the DP1004 pneumococcal strain to investigate the role of these proteins in the interaction with microglia, the resident brain macrophages. Methods: By screening a whole genome phage display library with sera from patients, we identified clones carrying pneumococcal B-cell epitopes. Among these, we isolated six antigenic fragments of sequences matching three previously unidentified proteins, encoded by the ORFs spr0075, spr1370 and spr1875 of R6 strain genomic sequence. By using an in vitro infection model and a gentamicin protection assay we evaluated, respectively, the ability of microglial BV2 cells to phagocytose and kill the isogenic mutants and the survival of these bacteria inside the microglia. Results: All strains were efficiently internalized by BV2 cells; yet the levels of phagocytosis obtained with Δspr0075 strain were lower than those observed with the other groups. The survival of the deleted strains inside the microglia was significantly different. The residual CFU of Δspr1370 and Δspr1875 mutants were, indeed, respectively, higher and lower, than those observed with parental DP1004. Moreover, preliminary results indicate that a similar trend is also observed in a bactericidal assay using BV2 as effector cells. Conclusion: These findings indicate that the proteins encoded by spr1370 and spr1875 loci are involved in interaction between S. pneumoniae and microglia.

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 - Interaction between Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium subspecies paratuberculosis with the enteric glia and microglial cells [Articolo su rivista]
S., Cannas; P., Molicotti; A., Bua; D., Usai; L. A., Sechi; A. M., Scanu; Blasi, Elisabetta; S., Zanetti
abstract

We investigated the interaction of Mycobacterium avium subspecies paratuberculosis, M. bovis and M. tuberculosis and different glial cells (enteric glial and microglial cells) in order to evaluate the infecting ability of these microorganisms and the effects produced on these cells, such as the evaluation of cytokines expression.Our experiments demonstrated the adhesion of M. paratuberculosis to the enteroglial cells and the induction of IL-1A and IL-6 expression; M. tuberculosis and M. bovis showed a good adhesive capability to the enteric cell line with the expression of the following cytokines: IL-1A and IL-1B, TNF−α, G-CSF and GM-CSF; M. bovis induced the expression of IL-6 too.The experiment performed with the microglial cells confirmed the results obtained with the enteroglial cells after the infection with M. tuberculosis and M. bovis, whereas M. paratuberculosisstimulated the production of IL-1A and IL-1B. Enteroglial and microglial cells, could be the target of pathogenic mycobacteria and, even if present in different locations (Enteric Nervous System and Central Nervous System), show to have similar mechanism of immunomodulation.

2011 - POLIMORFISMO DEL GENE HWP1 IN CANDIDA ALBICANS: POSSIBILE RUOLO NELLA PRODUZIONE DI BIOFILM E NELL’INTERAZIONE CON IL MACROFAGO [Poster]
Orsi, Carlotta Francesca; Borghi, Elisa; Colombari, Bruna; Neglia, Rachele Giovanna; Morace, Giulia; Blasi, Elisabetta
abstract

Introduzione: Candida albicans (Ca) è frequentemente responsabile di candidosi profonde, soprattutto nei portatori di cateteri od altri dispositivi medici, sulla cui superficie spesso si riscontra la comparsa di biofilm (1). La formazione di biofilm, a cui si associa la maggior resistenza di Ca ai farmaci antifungini così come l’aumentata virulenza (2), procede secondo una complessa serie di eventi, finemente controllati che coinvolgono numerosi geni tra cui HWP1 (Hyphal Wall Protein 1), codificante per una proteina essenziale per la crescita ifale. Il presente studio intende valutare il ruolo del biofilm ed in particolare del genopito HWP1 nell’interazione in vitro tra Ca e macrofago. Materiali e metodi: isolati clinici di Ca recentemente identificati come eterozigoti ed omozigoti (3 per ciascun assetto genetico) per il gene HWP1 (3), sono stati studiati in vitro per a) capacità di produrre biofilm, in presenza o assenza di siero fetale bovino e/o di CO2, e b) suscettibilità alle cellule microgliali BV2 (4); le indagini hanno comportato la quantificazione della produzione di biofilm mediante colorazione con cristal violetto o XTT, e valutazione della reattività del macrofago mediante dosaggio nei surnatanti di citochine, lattato deidrogenasi, ecc. Risultati: i ceppi saggiati hanno dimostrato a) aumentata produzione di biofilm in presenza di siero e di CO2 in tutti i ceppi, ma in modo più evidente negli isolati omozigoti; b) maggiore resistenza verso la microglia, nel caso dei ceppi omozigoti per HWP1 e c) produzione più elevata di TNFα in macrofagi esposti ai ceppi omozigoti. Conclusioni: questi dati indicano che la produzione di biofilm conferisce a Ca una maggiore resistenza alla reattività della cellula microgliale; la differenza osservata tra ceppi genotipicamente diversi per HWP1 suggerisce un ruolo importante di questo gene, oltre che nella crescita delle ife, anche nella interazione con le cellule dell’immunità innata. (1) Douglas, L. J. (2003). Trends Microbiol 11: 30–36 (2) Katragkou A et al. (2010). JID, 15:1941-1949 (3) Borghi et al. (2011). 21st ECCMID, Milano (4) Blasi E et al. (1991). J Neuroimmunol 32: 49-57

2011 - RICERCA DI DNA DI Pneumocystis jirovecii NEL LIQUIDO DI LAVAGGIO BRONCOALVEOLARE MEDIANTE IL TEST IN REAL-TIME PCR MYCASSAY™ PNEUMOCYSTIS [Poster]
Orsi, Carlotta Francesca; Gennari, William; Venturelli, Claudia; La Regina, Annunziata; Bertazzoni, Giorgia; Machetti, Marco; Blasi, Elisabetta
abstract

Introduzione: Pneumocystis jirovecii (Pj) è un fungo opportunista patogeno responsabile di gravi malattie respiratorie nell'ospite immunocompromesso. La diagnosi di polmonite da Pj (PCP) si basa su prove radiologiche e sulla rilevazione microscopica di antigeni mediante immunofluorescenza (IF); se non diagnosticata e/o non trattata tempestivamente, la malattia può portare a insufficienza respiratoria e morte. Il presente studio retrospettivo intende valutare l’efficacia del test in real-time PCR (MycAssay Pneumocystis, MycPcpAssay, Myconostica) su piattaforma ABI PRISM 7300 per la rilevazione di DNA fungino in campioni di BAL provenienti da pazienti con diagnosi di PCP. Metodi: il DNA, estratto mediante MycXtra (Myconostica), da 20 campioni di BAL da 8 pazienti con diagnosi clinica di PCP, da 7 pazienti con diagnosi clinica di aspergillosi invasiva (AI) e da 5 controlli negativi, è stato amplificato con il kit MycPcpAssay. I risultati della real-time PCR sono stati confrontati con i risultati ottenuti con i test diagnostici di IF (Merifluor Pneumocystis, Meridian) e di biologia molecolare Pj Alert kit (Nanogen) in uso presso il laboratorio. Risultati: dei 20 campioni analizzati, 12 sono risultati MycPcpAssay-positivi per Pj: 8 campioni appartengono al gruppo di pazienti con diagnosi di PCP e 4 campioni appartengono al gruppo con diagnosi di AI. In particolare, la positività di 3 di 4 soggetti con diagnosi di AI è stata confermata da Pj Alert kit; nessuno dei 5 soggetti appartenenti al gruppo di controllo è risultato positivo mediante PCR. Questi dati sono stati confrontati con i risultati ottenuti in IF: 6 pazienti IF positivi erano anche PCR positivi; 5 pazienti IF negativi erano PCR positivi e un paziente IF positivo era PCR negativo sia col test MycPcpAssay che col Pj Alert kit. Conclusioni: questi dati, che devono essere considerati preliminari al fine di una validazione del kit MycAssay Pneumocystis su piattaforma ABI PRISM 7300, forniscono una prima indicazione dell’efficacia del kit per la rilevazione del DNA di Pj in BAL di pazienti con sospetta PCP.

2011 - RUOLO DELLA SUPEROSSIDO DISMUTASI DI Enterococcus faecalis SULLA REATTIVITA’ DELLA MICROGLIA IN VITRO [Poster]
Pellegrino, MARIA TERESA; Colombari, Bruna; Manca, Lidia; Giard, Jean Christophe; Hartke, Axel; Sanguinetti, Maurizio; Posteraro, Brunella; Fadda, Giovanni; Blasi, Elisabetta; Peppoloni, Samuele
abstract

Introduzione Enterococcus faecalis è un batterio responsabile di gravi infezioni nosocomiali del tratto urinario, endocarditi e sepsi, a cui segue, seppur raramente, la meningite con una mortalità del 21%. Mediante l’uso di ceppi batterici knock-out (ko), abbiamo recentemente dimostrato (1) che l’assenza di MnSOD rende il batterio più suscettibile al killing intracellulare della microglia. In questo studio, è stato valutato il ruolo della MnSOD sulla reattività della cellula microgliale all’infezione con E. faecalis, analizzando l’espressione dei recettori purinergici P2Y6 e P2Y12, l’induzione di NFkB ed il rilascio di citochine proinfiammatorie e ossido nitrico (NO). Metodi L’espressione di P2Y6 e P2Y12 in cellule microgliali BV2 incubate con i diversi ceppi batterici è stata analizzata mediante indagini citofluorimetriche. Il dosaggio di citochine (IL-1 β, TNF-α e MIP-1 α.) e di NO nei surnatanti è stato eseguito rispettivamente mediante saggio Elisa e reattivo di Griess; la determinazione di NFkB in lisati cellulari è stata effettuata mediante kit “Nuclear Extract Kit” e “TransAMtm NFkB”. Risultati I dati ottenuti mostrano un aumento di P2Y6 e una diminuzione di P2Y12 in cellule microgliali infettate con il mutante MnSOD-ko, rispetto a cellule esposte al ceppo parentale JH2-2. Nelle stesse cellule, si osserva una maggiore induzione di NFkB ed una più elevata produzione di citochine proinfiammatorie e di NO. Conclusioni Questi risultati forniscono una prima evidenza sul ruolo immunomodulatorio della MnSOD di E. faecalis nei confronti della cellula microgliale. La resistenza di E. faecalis al killing intracellulare mediato dalla microglia, osservata in precedenza (1), può essere pertanto spiegata con la ridotta attivazione della cellula microgliale in presenza di MnSOD batterica. 1) Peppoloni et al., Microbiology, 2011

2011 - Role of the (Mn)superoxide dismutase of Enterococcus faecalis in the in vitro interaction with microglia [Articolo su rivista]
Peppoloni, Samuele; B., Posteraro; Colombari, Bruna; Manca, Lidia; A., Hartke; J. C., Giard; M., Sanguinetti; G., Fadda; Blasi, Elisabetta
abstract

Enterococcus faecalis is a significant human pathogen worldwide and is responsible for severenosocomial and community-acquired infections. Although enterococcal meningitis is rare,mortality is considerable, reaching 21 %. Nevertheless, the pathogenetic mechanisms of thisinfection remain poorly understood, even though the ability of E. faecalis to avoid or survivephagocytic attack in vivo may be very important during the infection process. We previouslyshowed that the manganese-cofactored superoxide dismutase (MnSOD) SodA of E. faecalis wasimplicated in oxidative stress responses and, interestingly, in the survival within mouse peritonealmacrophages using an in vivo–in vitro infection model. In the present study, we investigated therole of MnSOD in the interaction of E. faecalis with microglia, the brain-resident macrophages. Byusing an in vitro infection model, murine microglial cells were challenged in parallel with the wildtypestrain JH2-2 and its isogenic sodA deletion mutant. While both strains were phagocytosedby microglia efficiently and to a similar extent, the DsodA mutant was found to be significantlymore susceptible to microglial killing than JH2-2, as assessed by the antimicrobial protectionassay. In addition, a significantly higher percentage of acidic DsodA-containing phagosomes wasfound and these also underwent enhanced maturation as determined by the expression ofendolysosomal markers. In conclusion, these results show that the MnSOD of E. faecaliscontributes to survival of the bacterium in microglial cells by influencing their antimicrobial activity,and this could even be important for intracellular killing in neutrophils and thus for E. faecalispathogenesis.

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 - Candida metapsilosis as the least virulent member of the 'C. parapsilosis' complex [Articolo su rivista]
Orsi, Carlotta Francesca; Colombari, Bruna; Blasi, Elisabetta
abstract

Results of recent molecular studies have provided evidence of three distinct species within the Candida parapsilosis complex, namely Candida parapsilosis, Candida orthopsilosis and Candida metapsilosis. While there are initial data pertaining to the virulence of these Candida species with respect to reconstituted epidermal and oral epithelial tissues, there have been no studies, as of yet, on their interaction with immune cells. Employing an in vitro infection model using microglial cells, we investigated the pathogenetic potential of different isolates of each of these three species. We show that C. metapsilosis isolates are more susceptible to microglia-mediated antifungal activity, as compared with those of C. parapsilosis and C. orthopsilosis. Interestingly, C. metapsilosis isolates are also phagocytosed to a lower extent, but the yeast-containing phagosomes exhibit the highest degree of acidification in comparison with the phagosomes containing C. parapsilosis or C. orthopsilosis. Furthermore, when assessing microglia secretory response to infection, comparable high levels of MIP-1α and little or no TNF-α production are observed with all of these Candida species. Finally, unlike C. metapsilosis infected cells, microglial cells infected with C. parapsilosis and C. orthopsilosis release high and time-dependent levels of lactate dehydrogenase (LDH). Overall, these findings point to C. metapsilosis as the least virulent member of the ‘C. parapsilosis’ complex.

2010 - The encapsulated strain TIGR4 of Streptococcus pneumoniae is phagocytosed but is resistant to intracellular killing by mouse microglia [Articolo su rivista]
Peppoloni, Samuele; S., Ricci; Orsi, Carlotta Francesca; Colombari, Bruna; M. M., De Santi; Messinò, Massimino; G., Fabio; Zanardi, Alessio; Righi, Elena; V., Braione; S., Tripodi; D., Chiavolini; M., Cintorino; Zoli, Michele; M. R., Oggioni; Blasi, Elisabetta; G., Pozzi
abstract

The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae as it confers resistance to phagocytosis. The encapsulated serotype 4 TIGR4 strain was shown to be efficiently phagocytosed by the mouse microglial cell line BV2, whereas the type 3 HB565 strain resisted phagocytosis. Comparing survival after uptake of TIGR4 or its unencapsulated derivative FP23 in gentamicin protection and phagolysosome maturation assays, it was shown that TIGR4 was protected from intracellular killing. Pneumococcal capsular genes were up-regulated in intracellular TIGR4 bacteria recovered from microglial cells. Actual presence of bacteria inside BV2 cells was confirmed by transmission electron microscopy (TEM) for both TIGR4 and FP23 strains, but typical phagosomes/phagolysosomes were detected only in cells infected with the unencapsulated strain. In a mouse model of meningitis based on intracranic inoculation of pneumococci, TIGR4 caused lethal meningitis with an LD(50) of 2 × 10(2) CFU, whereas the LD(50) for the unencapsulated FP23 was greater than 10(7) CFU. Phagocytosis of TIGR4 by microglia was also demonstrated by TEM and immunohistochemistry on brain samples from infected mice. The results indicate that encapsulation does not protect the TIGR4 strain from phagocytosis by microglia, while it affords resistance to intracellular killing.

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 - Growth of Epstein-Barr Virus on monocytes and its effects on immune functions. [Abstract in Atti di Convegno]
Cuoghi, A.; Orsi, Carlotta Francesca; Blasi, Elisabetta; Cermelli, Claudio
abstract

EBV is the etiologic agent of infectious mononucleosis. It is also associated with different malignancies and other diseases including emophagocytic syndrome, a severe disorder in which erithrocytes are phagocytated by macrophages and B cells. Moreover, EBV has been postulated to be involved in the pathogenesis of multiple sclerosis. We investigated the ability of EBV to infect a monocytic human cell line (THP-1) in vitro and the effects of virus growth on the monocytic immune functions. The EBV chronically infected B cell line B95.8, stimulated with phorbol and butyrrate, was used as a source of virus. The growth of EBV on THP-1 virus was documented by IFA for viral antigens and quantification of viral DNA by RealTime PCR TaqMan. EBV infected THP-1 disclosed augmented phagocytosis of Candida albicans as assessed by a phagocytosis assay performed with a double fluorescence staining which allows to distinguish internalized fungi from those not ingested. Moreover, EBV infected THP-1 also displayed an increased ability to kill cells of a human oligodendrocytes line (MO3.1). Clones of chronically infected THP-1 cells have been obtained which are presently being characterized.Our results, showing alteration of monocytic immune functions induced by EBVinfection, support a pathogenetic role of this virus in emophagocytic syndrome and in multiple sclerosis.

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 - Bordetella [Capitolo/Saggio]
Blasi, Elisabetta; Neglia, Rachele Giovanna; Peppoloni, Samuele
abstract

Descrizione del genere Bordetella: classificazione, meccanismi patogenetici, manifestazioni cliniche, diagnosi di laboratorio, epidemiologia, terapia e profilassi.

2008 - Brucella [Capitolo/Saggio]
Blasi, Elisabetta; Neglia, Rachele Giovanna; Peppoloni, Samuele
abstract

Descrizione del genere Brucella: classificazione, meccanismi patogenetici, manifestazioni cliniche, diagnosi di laboratorio, epidemiologia, terapia e profilassi.

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 - EFFETTI DELLA TOSSINA DI ORIGINE ALGALE YESSOTOSSINA (YTX) SULLA ATTIVITA’ DEL MACROFAGO [Abstract in Atti di Convegno]
Orsi, Carlotta Francesca; Colombari, Bruna; Rossini, Gian Paolo; Callegari, Federica; Martino, Antonio; Blasi, Elisabetta; Peppoloni, Samuele
abstract

La yessotossina (YTX) è un composto polietere solfatato isolato per la prima volta dalla ghiandola digestiva del mollusco bivalve Patinopecten yessonsis e prodotto da diverse specie di alghe. La prima dimostrazione della presenza di YTX in mitili dell’Adriatico risale al giugno del 1995. Inizialmente inclusa nel gruppo delle tossine diarroiche, la YTX mostra bassissima tossicità in seguito a somministrazione per via orale e non è in grado di provocare diarrea nell’uomo. Esperimenti condotti nel topo hanno dimostrato che la tossina è letale in seguito ad inoculazione intraperitoneo, mentre la sua tossicità acuta per os è discussa. Evidenze ottenute in vitro hanno indicato che l’esposizione di cellule di origine umana o murina a YTX induce alterazioni cellulari con conseguente morte, frequentemente per apoptosi. Studi successivi hanno dimostrato che YTX modifica le strutture responsabili dell’adesione cellula-cellula con accumulo di un frammento di caderina E, alterandone la sua interazione con le catenine, cui segue una forte perdita di adesione cellulare. In questo studio abbiamo valutato gli effetti della YTX sul macrofago. I risultati ottenuti dimostrano che l’esposizione di cellule macrofagiche J774 a YTX (1 nM per 36 ore) ne riduce fortemente la vitalità in vitro. Il trattamento per periodi più brevi (12 ore) non è letale per la cellula, ma ne altera significativamente la funzionalità diminuendone la capacità di fagocitare in vitro cellule di Candida albicans. Questo fenomeno potrebbe coinvolgere alterazioni al citoscheletro della cellula macrofagica, in quanto l’organizzazione di F-actina, rilevata mediante microscopia a fluorescenza, risulta essere marcatamente modificata in cellule esposte a YTX. Studi paralleli dimostrano che cellule J774 trattate con YTX ed esposte a Candida hanno una marcata inibizione nella maturazione del fagosoma, in seguito ad ingestione dei lieviti. Al contrario, cellule esposte a YTX producono livelli di citochine (TNF-α, MIP-1α e MIP-2) significativamente più elevati di quelli osservati con cellule non trattate. Nel loro insieme i risultati di questo studio indicano che YTX svolge un ruolo immunosoppressore nei confronti della cellula macrofagica.

2008 - Gene expression profiling on monocytes displaying Herpes Simplex virus 1-induced dysregulation of antifungal defences. [Abstract in Atti di Convegno]
Cermelli, Claudio; Cuoghi, A.; Orsi, Carlotta Francesca; Fantoni, L.; Blasi, Elisabetta
abstract

BACKGROUND: Epstein Barr Virus (EBV) is responsible of many oncologic and non-oncologic diseases, including hemophagocytic syndrome in which red blood cells are phagocytosed by macrophages and B cells. EBV is reported to infect mainly B lymphocytes and epithelial cells; virus can be detected also in rare monocytes/macrophages, but its efficient replication in these cells has not been demonstrated.MATERIALS AND METHODS: EBV was obtained from P3RH1 cells stimulated with phorbol and butirate. A human monocytic cell line (THP-1) and an EBV negative B cell line (BJA-B) were infected with P3HR1- EBV strain: virus growth was monitored by IFA with a monoclonal antibody and by Real-Time PCR TaqMan. The effects of virus replication on phagocytosis of Candida albicans were evaluated by means of a double stain immunofluorescence assay. Intracellular killing of phagocytosed candida cells was measured by the colony forming unit inhibition assay.RESULTS AND CONCLUSION EBV can productively infect THP-1 monocyte cell line. EBV infection results in a significant increase in macrophage phagocytic activity versus Candida albicans as well as alteration of intracellular killing. Also BJA-B cells were stimulated in their phagocytic activity by EBV infection

2008 - Haemophilus [Capitolo/Saggio]
Blasi, Elisabetta; Neglia, Rachele Giovanna; Peppoloni, Samuele
abstract

Descrizione del genere Haemophilus: classificazione, meccanismi patogenetici, manifestazioni cliniche, diagnosi di laboratorio, epidemiologia, terapia e profilassi.

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 - HSV-1 induces dysregulation of monocyte anticandida functions [Abstract in Atti di Convegno]
Cermelli, Claudio; Orsi, Carlotta Francesca; Fantoni, L.; Lugli, E.; Blasi, Elisabetta
abstract

Clinical cases of double infections by fungi and viruses are increasing, especially in immunocompromised hosts. To date, the biomolecular events characterizing the outcome of polymicrobic diseases remain poorly investigated: little is known on the mutual interactions occurring between pathogens and on their concomitant, either synergistic or antagonistic, effects. 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, was infected with HSV-1 and then exposed to Candida albicans. The effects of HSV-1 infection on macrophages were measured as capability to alter macrophage-mediated effector functions, namely phagocytosis and killing of Candida, and as gene and protein expression by FACS and RNA microarrays. Phagocytosis of Candida by THP-1 cells was significantly increased when macrophages were infected with HSV-1. Conversely, antifungal activity was impaired in HSV-1 infected macrophages at 6 hours after infection with Candida. FACS analysis of protein expression revealed a significant downregulation of TLR2 and TLR4, important molecules involved in fungi recognition and increase in the number of apoptotic cells. Gene expression profile disclosed a huge decrease in gene presence in HSV-1 infected macrophage (40% of gene presence in uninfected cells vs 20% in infected cells). The analysis of gene clusters showed a downregulation of genes involved in opsonized phagocytosis and intracellular killing, of many adhesion molecules and of TLR2; on the contrary, several lectin receptor genes (involved in Candida adhesion and phagocytosis) and apoptosis genes were significantly up-regulated.

2007 - Identification and characterization of an aspartyl protease from Cryptococcus neoformans [Articolo su rivista]
Pinti, Marcello; Orsi, Carlotta Francesca; Gibellini, Lara; Esposito, Roberto; Cossarizza, Andrea; Blasi, Elisabetta; Peppoloni, Samuele; Mussini, Cristina
abstract

Abstract Cryptococcosis, caused by Cryptococcus neoformans, is an invasive infection often occurring in AIDS patients. Potent therapy against HIV, which includes protease inhibitors (PIs), has beneficial effects also on opportunistic infections by pathogens such as C. neoformans and C. albicans. PIs inhibit growth of C. albicans by affecting the activity of its aspartyl proteases. We identified, cloned and sequenced a cDNA from C. neoformans encoding for a putative aspartyl protease (CnAP1), and the corresponding genomic region. The gene cnap1 codifies for a protein of 505 aa, with a canonical aspartyl protease structure. We purified the recombinant protein and analyzed its activity in the presence of PIs (Indinavir, Lopinavir, Ritonavir), but did not evidence any inhibition of protease activity. The transcriptional level of cnap1 in C. neoformans is constant in different media. The absence of any inhibition activity by PIs suggests that other targets for PIs might exist in C. neoformans.

2007 - Interaction of pneumococcal surface structures with microglia [Abstract in Atti di Convegno]
S., Ricci; Messinò, Massimino; V., Braione; Colombari, Bruna; M., De Santi; E., Beghetto; G., Tuscano; Fabio, Giuliana; F., Iannelli; M., Cintorino; F., Felici; M. R., Oggioni; Blasi, Elisabetta; G., Pozzi; Peppoloni, Samuele
abstract

Interaction of pneumococcal surface structures with microglia

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 - Adaptive response of microglial cells to in vitro infection by Candida albicans isolates with different genomic backgrounds [Articolo su rivista]
Neglia, Rachele Giovanna; Colombari, Bruna; Peppoloni, Samuele; Orsi, Carlotta Francesca; A., Tavanti; S., Senesi; Blasi, Elisabetta
abstract

It has been recently demonstrated that Candida albicans isolates with distinct genomic backgrounds (namely, b and c genotypes) press different susceptibility to antifungal activity by human monocytes in vitro. We show here that, although comparable in their ability to undergo dimorphic transition and in susceptibility to phagocytosis by microglial cells, the b and c isolates show striking differences in terms of intracellular survival. Only the c genotype resists indeed to intracellular killing and eventually replicates inside microglial cells, that in turn respond to fungal infection, preferentially towards the c genotype, with nuclear factor-kappa B (NF-kappa B) activation and increased Mip1 alpha production. These data indicate that C albicans-microglial cell interaction is strictly dependent upon fungal genotype, strengthening the potential significance of genotyping as prognostic parameter in clinical infections by C albicans. (c) 2006 Elsevier Ltd. All rights reserved.

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 - Beretti F., Cermelli C., Cenacchi V., Orsi C., Blasi E., Portolani M. [Abstract in Atti di Convegno]
Beretti, Francesca; Cermelli, Claudio; Cenacchi, V.; Orsi, Carlotta Francesca; Blasi, Elisabetta; Portolani, Marinella
abstract

Microglia activation occurs during brain injury, ischemia and several neurological disorders. Moreover, microglial cells are highly dynamic structures also during the “resting” state in vivo, releasing neurotrophic factors and/or pro- and anti-inflammatory cytokines. Recently, we obtained a transmissible cytotoxic activity (TCA) from the cerebrospinal fluid of a patient with brain ischemia. We demonstrated that this TCA is associated with one or 2 protein(s), that supposedly underwent misfolding, and causes apoptotic cytotoxicity in a variety of cell lines. In this work, we studied microglia response to this TCA. The murine brain macrophage cell line RR4 was stimulated with TCA. Its response was evaluated as phagocytosis and antifungal activity against C. Albicans, and as secretion pattern, measuring MIP-1and TNF- by commercial sandwich ELISAs, nitric oxide (NO) by Griess reaction and phosphorylation of p38 by Fast Activated Cell-based ELISA.Microglia stimulated with TCA showed an increase in phagocytosis of C. albicans. On the contrary, the macrophage capability to kill the ingested fungi was diminished. The analysis of soluble factors secreted by microglial cells in response to TCA demonstrated an increase in MIP-1TNF- NO and an activation of p38 MAP kinase. These results suggest an initial microglia activation induced by TCA leading to an increase in candida ingestion not followed by killing of the fungus. Activation of p38 MAP kinase could suggest the induction of a signaling cascade leading to TCA production which in turn could cause failure in the antifungal activity. Moreover, MIP-1a, NO and TNF-a secretion by microglia cells and induction of apoptosis could provide an in vitro model for the cell events associated with brain ischemia.

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 - HSV-1 induces macrophage activation and dysregulation in monocytes [Abstract in Atti di Convegno]
Cermelli, Claudio; Cenacchi, V.; Beretti, Francesca; Blasi, Elisabetta
abstract

Clinical cases of double infections by fungi and viruses are increasing, especially in immunocompromised hosts. To date, the biomolecular events that characterize the outcome of polymicrobic diseases remain poorly investigated and little is known on the mutual interactions occurring between pathogens. In order to investigate the interplay between microrganisms co-infecting macrophagic cells, we recently set up an in vitro model in which a monocytic cell line was infected with human herpesvirus 6 and C. neoformans. In the present work, we used an similar model to understand the molecular mechanisms underlying interactions between herpes simplex virus 1 (HSV-1) and C. albicans. The monocytic cell line THP-1 was infected with HSV-1 and, after an overnight incubation, cells were exposed to C. albicans. The cell response to the viral infection was evaluated as phagocytosis of C. albicans and killing of the ingested fungus. Moreover, a number of activation markers (CD38, CD69, CD95) and adhesion molecules (CD54, TLR-2, CD11b, CD106) was evaluated by FACS analysis in THP-1 cells. THP-1 cells infected with HSV-1 showed increased phagocytosis of C. albicans but reduced killing capability, suggesting that in the course of a double infection macrophages could contribute to yeast dissemination. Activation markers (CD38 and CD69) were found down expressed by FACS analysis in HSV-1-infected THP-1 cells, accounting for the failure in the antifungal activity.

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 - Human herpesvirus-6 dysregulates monocyte-mediated anticryptococcal defences [Articolo su rivista]
Cermelli, Claudio; Cenacchi, Valeria; Beretti, Francesca; Pezzini, Francesco; D., Di Luca; Blasi, Elisabetta
abstract

In order to investigate the interplay occurring between pathogens in the course of double infections, an in vitro model was set up in which the monocytic cell line THP-1 was 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, it was shown that unlike THP-1 cells exposed to cell-free virus inocula, THP-1 exposed to HHV-6-producing lymphocytes exhibited augmented phagocytosis against Cn. The phenomenon occurred after 24 In of monocyte/lymphocyte co-culture and was independent of direct cell-to-cell contact. Moreover, in the presence of HHV-6, THP-1 cells expressed enhanced secretory responses but reduced capability to counteract fungal infection: the enhanced ingestion by monocytes was 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 - Interaction of leptospires with murine microglial cells [Articolo su rivista]
M., Cinco; R., Domenis; S., Perticarari; G., Presani; A., Marangoni; Blasi, Elisabetta
abstract

Leptospires, the agents of leptospirosis, exert tropism for the central nervous system, in the course of mammal infection. We evaluated the interaction between murine microglial cells and strains of pathogenic L. interrogans leptospires and non-pathogenic L. biflexa leptospires, mainly by flow cytometric assays. In the absence of opsonic conditions microglia are capable of ingesting - even quite slowly - the spirochetes and killing the non-pathogenic strain. The adhesion to microglia, which is quick and relevant for all the strains, does not involve the CR3 integrin receptor. These findings suggest that the murine microglia - in non opsonic conditions in vitro - do not effectively clear the pathogenic leptospires.

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 - Risposta differenziale della cellula di microglia ad isolati clinici di Candida albicans con differente genotipo [Abstract in Atti di Convegno]
Neglia, Rachele Giovanna; Colombari, Bruna; Peppoloni, Samuele; Orsi, Carlotta Francesca; A., Tavanti; S., Senesi; Blasi, Elisabetta
abstract

Risposta differenziale della cellula di microglia ad isolati clinici di Candida albicans con differente genotipo

2006 - The TIGR4 strain of Streptococcus pneumoniae is phagocytosed but resists intracellular killing by microglia in experimental pneumococcal meningitis [Abstract in Atti di Convegno]
S., Ricci; Peppoloni, Samuele; S., Tripodi; D., Chaivolini; R., Parigi; V., Braione; Zanardi, Alessio; M., Messinò; F., Iannelli; M., De Santi; M., Cintorino; M. R., Oggioni; Zoli, Michele; Blasi, Elisabetta; G., Pozzi
abstract

The TIGR4 strain of Streptococcus pneumoniae is phagocytosed but resists intracellular killing by microglia in experimental pneumococcal meningitis

2006 - The lack of Pneumococcal surface protein C (PspC) increases the susceptibility of Streptococcus pneumoniae to the killing by microglial cells [Articolo su rivista]
Peppoloni, Samuele; Colombari, Bruna; Neglia, Rachele Giovanna; Quaglino, Daniela; F., Iannelli; Mr, Oggioni; G., Pozzi; Blasi, Elisabetta
abstract

Microglial cells, the resident phagocytes in the brain, share many phenotypical and functional characteristics with peripheral macrophages, suggesting that they may participate in an innate immune response against microorganisms invading the central nervous system (CNS). In this study, we demonstrate that the microglial cells constitutively exhibit antibacterial activity in vitro against Streptococcus pneumoniae. By using a Pneumococcal surface protein C (PspC)-deleted strain and its wild-type counterpart, we found that the extent of such an activity is significantly influenced by the presence of a PspC molecule on the bacterial surface. The PspC- mutant FP20 is indeed more susceptible than the PspC+ strain HB565 to microglial killing. Interestingly, this phenomenon is observed when using a medium supplemented with heat-inactivated foetal bovine serum (FBS). Electron microscopy studies indicate that the microglial cells interact more efficiently with PspC- than with PspC+ pneumococci. Moreover, upon infection with the PspC- mutant, microglial cells produce levels of TNF-alpha, MIP-2, IL-10 and nitric oxide, significantly higher than those observed with PspC+ bacteria. These findings indicate that the lack of PspC significantly enhances the susceptibility of S. pneumoniae to both bactericidal activity and secretory response by the microglial cells, suggesting that this molecule may play an important role in the invasion of CNS by pneumococcus.

2005 - Alterazione delle difese monocito-mediate nei confronti di Cryptococcus neoformans in presenza dell’Herpesvirus umano 6 [Abstract in Atti di Convegno]
Cenacchi, Valeria; Cermelli, Claudio; A. M., Argentieri; Beretti, Francesca; Peppoloni, Samuele; Neglia, Rachele Giovanna; Blasi, Elisabetta
abstract

Alterazione delle difese monocito-mediate nei confronti di Cryptococcus neoformans in presenza dell’Herpesvirus umano 6Introduzione: I casi clinici di infezioni miste sono in aumento, specialmente in ospiti immunocompromessi, ma gli eventi biomolecolari che caratterizzano la patogenesi delle malattie polimicrobiche sono ancora scarsamente conosciuti. In un modello in vitro di infezione mista da Cryptococcus neoformans ed herpesvirus umano 6 (HHV-6), entrambi altamente neurotropi e causa di gravi patologie a livello del sistema nervoso centrale nel paziente immunocompromesso, abbiamo valutato le alterazioni funzionali del macrofago esposto alla doppia infezione.Materiali e Metodi: Cellule monocitiche THP-1 sono state esposte ad HHV-6 variante A (U1102), attraverso la co-coltura con cellule linfocitarie (linea JJHAN) infettate produttivamente (THP-1/JJHANHHV-6) o con cellule di controllo (THP-1/JJHANMOCK) per tempi diversi; successivamente, le co-colture sono state esposte a C. neoformans (isolato clinico ottenuto dal liquor di un paziente con meningite) e quindi saggiate per risposta secretoria (test ELISA) ed attività antimicrobica mediante valutazione della fagocitosi (allestimento di citopreparati opportunamente colorati) e dell’attività anticriptococcica (test di inibizione delle unità formanti colonia). Risultati: Rispetto ai controlli THP-1/JJHANMOCK, le co-colture THP-1/JJHANHHV-6 mostrano a) significativa produzione di IL-12 e IFN- che aumentano ulteriormente dopo infezione con C. neoformans; b) aumentata fagocitosi nei riguardi C. neoformans; c) scarsa ed invariata attività di killing a tempi brevi e d) ridotta capacità di contenere la crescita fungina a tempi successivi.Conclusioni: La presenza di HHV-6 altera la funzionalità macrofagica a vantaggio di C. neoformans facilitandone la localizzazione intracellulare e promuovendone la replicazione.(Fondi PRIN-2003).

2005 - Alterazione delle difese monocito-mediate nei confronti di miceti in presenza di virus erpetici [Abstract in Atti di Convegno]
Cermelli, Claudio; Cenacchi, Valeria; Pezzini, Francesco; Peppoloni, Samuele; Neglia, Rachele Giovanna; Blasi, Elisabetta
abstract

Alterazione delle difese monocito-mediate nei confronti di miceti in presenza di virus erpetici

2005 - Biological importance of the two Toll-like receptors, TLR2 and TLR4, in macrophage response to infection with Candida albicans [Articolo su rivista]
Blasi, Elisabetta; Mucci, Anna; Neglia, Rachele Giovanna; Pezzini, Francesco; Colombari, Bruna; D., Radzioch; Cossarizza, Andrea; E., Lugli; G., Volpini; G., Del Giudice; Peppoloni, Samuele
abstract

The aim of this study was to assess the role of TLR2, TLR4 and MyD88 accessory molecule in the effector and secretory response of macrophages to viable microbial agents. Using TLR-deleted macrophage cell lines generated from the bone marrow of genetically engineered mice (TLR4 gene-deficient, MyD88- and TLR2-knockout mice) and wild-type control mice, we found that TLR2-deleted macrophages exhibit increased ability to contain Candida albicans infection compared to TLR2+/+ counterpart. In contrast, both MyD88-/- and TLR4-/- macrophages retain levels of functional activity comparable to that of the respective wild-type MyD88+/+ and TLR4+/+ controls. The difference in anticandidal effector functions observed between TLR2-/- and TLR2+/+ macrophages is abrogated upon opsonization of the fungal target and interestingly is not observed when using other microbial targets, such as Streptococcus pneumoniae and Helicobacter pylori. When tested for secretory response to C albicans, TLR2-deleted macrophages show a pattern of cytokine production similar to that of TLR2+/+ controls. Finally, flow cytometry analysis reveals that TLR2-deleted macrophages express only TLR4, while, as expected, TLR2+/+ macrophages are both TLR2 and TLR4 positive; in no cases, modulation of such markers occurs in macrophages exposed to C albicans infection. In conclusion, these data indicate that TLR2 and TLR4 have different biological relevance, in which TLR2 but not TLR4, is involved in the accomplishment of macrophage-mediated anticandidal activity, while the secretory response to C albicans appears to be TLR4 but not TLR2-dependent. (c) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

2005 - La presenza della capsula è necessaria per lo sviluppo di meningite pneumococcica nel topo [Abstract in Atti di Convegno]
S., Ricci S; D., Chiavolini; S., Tripodi; R., Parigi; Peppoloni, Samuele; F., Iannelli; V., Braione; M., Cintorino; M. R., Oggioni; Blasi, Elisabetta; G., Pozzi
abstract

La presenza della capsula è necessaria per lo sviluppo di meningite pneumococcica nel topo

2005 - RUOLO BIOLOGICO DI DUE RECETTORI TOLL-SIMILI, TLR2 E TLR4, NELLA RISPOSTA MACROFAGICA A CANDIDA ALBICANS [Abstract in Atti di Convegno]
Pezzini, Francesco; A., Mucci; Neglia, Rachele Giovanna; Colombari, Bruna; Cossarizza, Andrea; Lugli, Enrico; G., Del Giudice; Peppoloni, Samuele; Blasi, Elisabetta
abstract

RUOLO BIOLOGICO DI DUE RECETTORI TOLL-SIMILI, TLR2 E TLR4, NELLA RISPOSTA MACROFAGICA A CANDIDA ALBICANSIntroduzione: I TLR sono una classe di recettori coinvolti nel riconoscimento di agenti microbici da parte di cellule dell’immunità innata e nella conseguente risposta effettrice. La loro funzione si è rivelata complessa e profondamente diversificata in relazione anche al microrganismo con cui entrano in contatto. L’obiettivo di questo studio è stato valutare il ruolo delle molecole TLR2, TLR4 e MyD88 nella risposta effettrice e secretoria del macrofago nei riguardi di C. albicans.Materiali e metodi: Linee macrofagiche generate dal midollo osseo di topi geneticamente modificati (TLR2-/-, TLR4-/-, MyD88-/-) e dai rispettivi controlli (TLR2+/+, TLR4+/+, MyD88+/+) sono state saggiate per attività fagocitica, attività antimicrobica e produzione di citochine dopo infezione con C. albicans. Risultati: I macrofagi TLR2-/- mostrano una fagocitosi significativamente aumentata e una maggiore capacità di contenere l’infezione da C. albicans rispetto alla controparte TLR2+/+. In contrasto, sia i macrofagi TLR4-/- che quelli MyD88-/- mantengono livelli di attività funzionale sostanzialmente invariati rispetto ai controlli. La differenza nelle funzioni effettrici anticandida osservata tra macrofagi TLR2-/- e TLR2+/+ viene annullata con l’opsonizzazione del micete e non viene osservata nei riguardi di altri microrganismi. In risposta all’ infezione con C. albicans, i macrofagi TLR2-/- producono livelli di citochine comparabili a quelli dei controlli TLR2+/+. Infine, l’analisi in citofluorimetria a flusso rivela che, come atteso, i macrofagi TLR2-/- esprimono solo il TLR4, mentre i macrofagi TLR2+/+ sono positivi sia al TLR2 che al TLR4; in nessun caso, vi è modulazione di questi markers nei macrofagi esposti all’infezione con C. albicans.Conclusioni: Questi dati indicano che il TLR2 e il TLR4 hanno differente ruolo biologico; in particolare, il TLR2, e non il TLR4, è coinvolto nell’espletamento dell’attività anticandida da parte del macrofago, mentre la risposta secretoria sembra essere dipendente dal TLR4 e non dal TLR2.

2005 - Ruolo della proteina pneumococcica di superficie PspC nel l’interazione in vitro di Streptococcus pneumoniae con le cellule microgliali [Abstract in Atti di Convegno]
Peppoloni, Samuele; Colombari, Bruna; Neglia, Rachele Giovanna; Quaglino, Daniela; Martino, Antonio; F., Iannelli; M. R., Oggioni; G., Pozzi; Blasi, Elisabetta
abstract

Ruolo della proteina pneumococcica di superficie PspC nel l’interazione in vitro di Streptococcus pneumoniae con le cellule microglialiIntroduzione: Le cellule microgliali, i principali fagociti residenti del distretto cerebrale, possiedono caratteristiche fenotipiche e funzionali tipiche dei macrofagi e perciò si pensa possano svolgere un ruolo importante nell’immunità innata contro le infezioni cerebrali. Streptococcus pneumoniae è responsabile di gravi malattie, quali polmonite, batteremia e meningite. Tra i suoi fattori di virulenza c’è PspC, una molecola di adesione presente nel 75% circa dei pneumococchi, che oltre a svolgere un ruolo fondamentale nel processo di colonizzazione, interferisce con i processi di attivazione del complemento, in quanto lega il fattore H e previene così l’opsonofagocitosi. In questo studio, abbiamo valutato il ruolo di PspC nell’interazione di S. pneumoniae con la cellula microgliale. Materiali e Metodi: mediante un modello di infezione in vitro della linea microgliale BV2 con i ceppi batterici HB565 ed il suo mutante isogenico PspC- F20, abbiamo valutato la morfologia, l’attività antipneumococcica e secretoria della microglia. Risultati: le cellule microgliali BV2 mostrano una marcata attività antibatterica nei confronti di entrambi i ceppi; tuttavia, esse legano ed uccidono in maniera più efficiente i batteri mutanti PspC- rispetto a quelli del ceppo parentale HB565, anche in assenza di complemento. Infine, cellule BV2 stimolate con il ceppo PspC- producono livelli di citochine, quali TNF-α, MIP-2, IL-10 e ossido nitrico più elevati di quelli osservati in seguito a stimolazione con il ceppo selvaggio.Conclusioni: l’assenza di PspC aumenta sia l’attività battericida che la risposta secretoria delle cellule microgliali, suggerendo che questa molecola possa giocare un ruolo importante nell’insorgenza della meningite pneumococcica.

2005 - The Pneumococcal surface protein C (PspC) influences the susceptibility of Streptococcus pneumoniae to microglia [Abstract in Atti di Convegno]
Peppoloni, Samuele; Colombari, Bruna; Quaglino, Daniela; Neglia, Rachele Giovanna; Martino, Antonio; F., Iannelli; M. R., Oggioni; S., Ricci; G., Pozzi; Blasi, Elisabetta
abstract

The Pneumococcal surface protein C (PspC) influences the susceptibility of Streptococcus pneumoniae to microglia

2005 - The polisaccharyde capsule is necessary for the development of fatal pneumococcal meningitis in mice [Abstract in Atti di Convegno]
S., Ricci; D., Chiavolini; S., Tripodi; Peppoloni, Samuele; R., Parigi; F., Iannelli; M. R., Oggioni; M., Cintorino; Blasi, Elisabetta; G., Pozzi
abstract

The polisaccharyde capsule is necessary for the development of fatal pneumococcal meningitis in mice

2005 - Valutazione sperimentale della contaminazione microbica dei materiali condizionanti per protesi rimovibile, valutazione preliminare [Abstract in Rivista]
Bortolini, Sergio; Natali, Alfredo; Santi, S.; Blasi, Elisabetta; Consolo, Ugo
abstract

Vedasi l'allegato

2004 - La proteina di superficie PspC di pneumococco conferisce resistenza al killing in vitro di Streptococcus pneumoniae da parte di cellule microgliali in maniera indipendente dal complemento [Abstract in Atti di Convegno]
Peppoloni, Samuele; Colombari, Bruna; Quaglino, Daniela; Neglia, Rachele Giovanna; Martino, Antonio; F., Iannelli; S., Ricci; M. R., Oggioni; G., Pozzi; Blasi, Elisabetta
abstract

La proteina di superficie PspC di pneumococco conferisce resistenza al killing in vitro di Streptococcus pneumoniae da parte di cellule microgliali in maniera indipendente dal complemento. In assenza di complemento le cellule microgliali BV2 uccidono efficientemente S. pneumoniae in vitro e la presenza di PspC ne riduce la capacità battericida, così come quella secretoria. PcpC svolge quindi un ruolo patogenetico importante nell'interazione diretta tra il pneumococco e la microglia

2004 - Method for inducing experimental pneumococcal meningitis in outbred mice [Articolo su rivista]
Chiavolini, D; Tripodi, S; Parigi, R; Oggioni, M; Blasi, Elisabetta; Cintorino, M; Pozzi, G; Ricci, S.
abstract

In the present work, we describe in detail a simple, reproducible and efficient method to induce pneumococcal meningitis in outbred mice by using the intracranial subarachnoidal route of infection. Bacteria were injected into the subarachnoid space through a soft point located 3.5 mm rostral from the bregma. The model was tested with several doses of pneumococci of three capsular serotypes (2, 3 and 4), and mice survival was recorded. Lethal doses killing 50 % of animals infected with type 2, 3 and 4 S. pneumoniae were 3.2 × 10, 2.9 × 10 and 1.9 × 102 colony forming units, respectively. Characterisation of the disease caused by the type 4 strain showed that in moribund mice systemic dissemination of pneumococci to blood and spleen occurred. Histological analysis of the brain of animals infected with type 4 S. pneumoniae proved the induction of meningitis closely resembling the disease in humans. The proposed method for inducing pneumococcal meningitis in outbred mice is easy-to-perform, fast, cost-effective, and reproducible, irrespective of the serotype of pneumococci used.

2004 - Metodologia per l'induzione di meningite sperimentale da Streptococcus pneumoniae in topi outbred [Abstract in Atti di Convegno]
S., Ricci; D., Chiavolini; S., Tripodi; R., Parigi; M. R., Oggioni; Peppoloni, Samuele; Blasi, Elisabetta; M., Cintorino; G., Pozzi
abstract

Metodologia per l'induzione di meningite sperimentale da Streptococcus pneumoniae in topi outbred

2004 - Ruolo del sierotipo capsulare nella suscettibilità di Streptococcus pneumoniae alla cellula microgliale [Abstract in Atti di Convegno]
Peppoloni, Samuele; Neglia, Rachele Giovanna; Colombari, Bruna; V., Fantoni; Quaglio, Giampaola; D., Chiavolini; D., Medaglini; F., Iannelli; M. R., Oggioni; S., Ricci; G., Pozzi; Blasi, Elisabetta
abstract

Ruolo del sierotipo capsulare nella suscettibilità di Streptococcus pneumoniae alla cellula microgliale

2004 - The human immunodeficiency virus (HIV) protease inhibitor indinavir directly affects the opportunistic fungal pathogen Cryptococcus neoformans [Articolo su rivista]
Blasi, Elisabetta; Colombari, Bruna; Orsi, Carlotta Francesca; Pinti, Marcello; Troiano, L.; Cossarizza, Andrea; Esposito, Roberto; Peppoloni, Samuele; Mussini, Cristina; Neglia, Rachele Giovanna
abstract

Highly active antiretroviral therapy (HAART), that includes human immunodeficiency virus (HIV) protease inhibitors (PIs), has been remarkably efficacious including against some opportunistic infections. In this report we investigated the effect(s) of the PI indinavir on protease activity by Cryptococcus neoformans, an opportunistic fungal pathogen responsible for recurrent meningoencephalitis in AIDS patients. Indinavir was also tested for potential effects on other parameters, such as fungal viability, growth ability and susceptibility to immune effector cells. It was found that indinavir impaired cryptococcal protease activity in a time- and dose-dependent fashion. The phenomenon was similarly detectable in ATCC/laboratory strains and clinical isolates. C neoformans growth rate was also significantly reduced upon exposure to indinavir, while fungal viability was not affected and mitochondrial toxicity not detected. Furthermore, as assessed by an in vitro infection model, indinavir significantly and consistently augmented C neoformans susceptibility to microglial cell-mediated phagocytosis and killing. Overall, by providing the first evidence that indinavir directly affects C neoformans, these data add new in vitro insights on the wide-spectrum efficacy of PIs, further arguing for the clinical relevance of HAART against opportunistic infections in AIDS. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

2003 - Antifungal activity of macrophages engineered to produce IFNgamma: inducibility by picolinic acid [Articolo su rivista]
Mucci, A; Varesio, L; Neglia, Rachele Giovanna; Colombari, B; Pastorino, S; Blasi, Elisabetta
abstract

Macrophages are important antimicrobial effectors, whose efficacy is greatly enhanced by interferon-gamma (IFNgamma). We recently engineered a mouse macrophage cell line to express the IFNgamma gene in a inducible manner. Such macrophages, Mphi10, include a construct containing the IFNgamma gene under the control of the synthetic promoter HRE3x-Tk. Picolinic acid (PA) is a catabolite of tryptophan, known to exert costimulatory activities on macrophages and expected to act on transcriptional elements within HRE3x-Tk promoter. Since evidence exists on the efficacy of engineered macrophages as carriers of therapeutic genes against tumors, we tested Mphi10, under basal conditions and following exposure to PA, as IFNgamma-producing cells in in vitro models of fungal infection. We found that Mphi10 constitutively exhibited anticryptococcal and anticandidal activity, low but detectable levels of IFNgamma mRNA and undetectable levels of nitric oxide synthase (iNOS) transcripts. Treatment with PA caused time-dependent enhancement of antifungal activity. The phenomenon was associated with the induction of both IFNgamma and iNOS gene expression and was followed by IFNgamma and NO production. The effect of the Mphi10-produced IFNgamma on other cells was also investigated by a transwell co-culture system. A major enhancement of phagocytosis and antifungal activity was observed in BV2 microglial cells that had been co-cultured with Mphi10. Such an increase was only evident when Mphi10 had been pretreated with PA and was abrogated by concomitant addition of anti-IFNgamma antibodies. In conclusion, we show that Mphi10 respond to PA with the production of IFNgamma, which retains the ability to induce antifungal activity in the producing macrophages as well as in other macrophage populations. The potential use of Mphi10 as vectors for therapeutic genes in infectious diseases is discussed

2003 - CRIPTOCOCCOSI CEREBRALE RECIDIVANTE: ACQUISIZIONE DI FENOTIPO FUNGINO A RIDOTTA SUSCETTIBILITA’ NEI RIGUARDI DI CELLULE MICROGLIALI [Articolo su rivista]
Neglia, Rachele Giovanna; Mucci, Anna; Colombari, Bruna; F., Baldelli; Blasi, Elisabetta
abstract

Sono stati studiati i caratteri fenotipici di tre isolati seriali di Cryptococcus neoformans ottenuti da un paziente con episodi multipli di criptococcosi cerebrale: gli isolati 1525 e 1526 da sangue e liquor (primo episodio) ed il 1782 da liquor (recidiva dopo 3 anni) sono stati saggiati per suscettibilità agli immunoeffettori cerebrali, impiegando la linea microgliale murina BV2. Indagini precedenti avevano dimostrato che i due isolati da liquor (1526 e 1782) erano geneticamente identici, implicando che la recidiva era stata causata da una persistenza/riattivazione dello stesso stipite. In questo studio, i tre isolati hanno mostrato una diversa sensibilità sia alla fagocitosi che all'uccisione; in particolare, il 1782 è risultato di gran lunga il più resistente. L'impiego di criptococchi melanizzati ha rivelato un ulteriore potenziamento di questa capacità di resistenza, soprattutto per il 1782 in cui si è osservato anche un recupero massivo del potenziale replicativo. Nessuno degli isolati si è dimostrato capace di indurre produzione apprezzabile di citochine (IL-6 e TNFα ), mentre tutti sono risultati ugualmente efficaci nell'inibire la risposta secretoria della microglia all'endotossina. Nel complesso, questi dati possono essere interpretati alla luce di un processo di "microevoluzione" per cui C.neoformans, modificando il proprio assetto fenotipico, si è garantito la sopravvivenza/persistenza nell'organismo ospite e la possibilita' quindi di riattivarsi provocando una recidiva clinicamente manifesta.

2002 - Antibody-dependent macrophage-mediated activity against Helicobacter pylori in the absence of complement [Articolo su rivista]
Peppoloni, Samuele; S., Mancianti; G., Volpini; S., Nuti; P., Ruggiero; R., Rappuoli; Blasi, Elisabetta; G., Del Giudice
abstract

Helicobacter pylori is a Gram-negative bacterium, which chronically infects the stomach. Little is known about the immune mechanisms limiting the spread of infection and/or contributing to protection after experimental immunization. In this study, we investigated the hypothesis that specific antibodies and host cells cooperate in the immunity against H. pylori. Antibody-dependent cellular activity against H. pylori was assessed using specific immune serum, or purified IgG, in an in vitro assay, with peritoneal cells as effector cells. The natural antibacterial activity of peritoneal cells was significantly augmented by H. pylori-specific antibodies in a dose-dependent manner. A novel finding was that this killing effect did not require functional complement. Most of the bactericidal activity was associated with cells that were adherent, DX5-, CD19-, CD11c, Thy-1.2(-), CD11b(+) and CD16/32(+), indicating that the main effector population was represented by macrophages. Similar antibacterial killing was obtained with the macrophage cell line GG2EE. Cytochalasin D significantly impaired this antibacterial activity, suggesting that phagocytosis plays a major role in the antibody-mediated H. pylori killing.

2002 - Differential microbial clearance and immunoresponse of Balb/c (Nramp1 susceptible) and DBA2 (Nramp1 resistant) mice intracerebrally infected with Mycobacterium bovis BCG (BCG). [Articolo su rivista]
Mazzolla, R; Puliti, M; Barluzzi, R; Neglia, Rachele Giovanna; Bistoni, F; Barbolini, Giuseppe; Blasi, Elisabetta
abstract

In mice, the gene encoding Nramp1 (natural resistance-associated protein 1) exists in two allelic forms, differing for a point mutation.According to Nramp1 genotype, extensive literature documents a clear-cut distinction of inbred strains in two non-overlapping groups that phenotypically express resistance (Nramp1r) and susceptibility (Nramp1s) to systemic infections. Here, we provide evidence that Nramp1r (DBA/2) and Nramp1s (Balb/c) mice differently handle intracerebral infection with Mycobacterium bovis BCG. Distinct trends of microbial clearance from the brain and also different patterns of local immune responses occur, thus arguing on the involvement of Nramp1 gene product on the accomplishment of cerebral anti-mycobacterial defenses.

2002 - Iron overload exacerbates experimental meningoencephalitis by Cryptococcus neoformans [Articolo su rivista]
R., Barluzzi; S., Saleppico; A., Nocentini; J. R., Boelaert; Neglia, Rachele Giovanna; F., Bistoni; Blasi, Elisabetta
abstract

This study was aimed at investigating the effects of iron overload on the onset and outcome of cerebral cryptococcosis. To this purpose, iron dextran-administered mice were intracerebrally challenged with virulent melanogenic and avirulent non-melanogenic strains of Cryptococcus neoformans. The results shown here provide the first evidence that iron overload exacerbates the outcome of cryptococcal meningoencephalitis, irrespective of the fungal strain employed; pathogen colonization of the brain is facilitated, local cytokine response is delayed and/or prevented.

2001 - Evidence of microevolution in a clinical case of recurrent Cryptococcus neoformans meningoencephalitis [Articolo su rivista]
Blasi, Elisabetta; A., Brozzetti; D., Francisci; Neglia, Rachele Giovanna; G., Cardinali; F., Bistoni; V., Vidotto; F., Baldelli
abstract

The aim of this study was to examine three serial isolates of Cryptococcus neoformans from a patient with AIDS for genotypical and phenotypical characteristics. The isolates were obtained during a first episode of cryptococcosis (simultaneous sampling of blood and cerebrospinal fluid) and after a relapse 3 years later (sampling of cerebrospinal fluid). Pulsed-field gel electrophoresis and random amplification of polymorphic DNA revealed that the blood isolate 1525 (first episode) was different from the two cerebrospinal fluid isolates (1526, first episode; 1782, relapse), yet the cerebrospinal fluid isolates were indistinguishable from each other regardless of the analysis performed. Phenotypical studies showed that isolate 1782 had significantly improved resistance to phagocytosis and killing by monocytes and polymorphonuclear cells and an altered efficacy in evoking cytokine response (augmentation of tumour necrosis factor-alpha, interleukin [IL]-1 beta, IL-10, and interferon-gamma, decrease of IL-12). Interestingly, capsule size and antifungal drug resistance remained unchanged, while production of phospholipase and protease. was consistently enhanced in the 1782 isolate with respect to the 1525 and 1526 isolates. In conclusion, in serial Cryptococcus neoformans isolates from a patient with AIDS, phenotypical changes but not molecular changes were documented, thus supporting the role of microevolution as a pathogenetic mechanism(s) for persistence/reactivation of fungal organisms.

2001 - Experimental results on chloroquine and AIDS-related opportunistic infections [Articolo su rivista]
Boelaert, Jr; Appelberg, R; Gomes, Ms; Blasi, Elisabetta; Mazzolla, R; Grosset, J; Lounis, N; Soteriadou, K; Thiakaki, M; Taramelli, D; Tognazioli, C.
abstract

Apart from its antiretroviral effects, chloroquine (CQ) has other properties that may be of interest in setting of HIV infection. First, it is an anti-inflammatory drug and, second, it may help to prevent several AIDS-related opportunistic infections. Increasing experimental data demonstrate the ability of CQ to affect microbial growth both in vitro and in vivo, trough mechanisms only partially elucidated. This strongly suggests its use in combination with other drugs, as terapeutical agents in HIV positive patients, especially in countries with limited resources.

2001 - Nramp1 gene affects selective early steps in macrophage-mediated anti-cryptococcal defense [Articolo su rivista]
Blasi, Elisabetta; Colombari, Bruna; Mucci, Anna; Cossarizza, Andrea; Radzioch, D; Boelaert, Jr; Neglia, Rachele Giovanna
abstract

Cryptococcus neoformans is an opportunistic fungus responsible for severe and often recurrent meningoencephalitis in immunodepressed patients. Initial evidence suggests that C. neoformans is a facultative intracellular pathogen; however, the strategies by which C. neoformans undergoes survival and eventually proliferation have not been elucidated. We investigated the role of Nrampl gene in macrophage-mediated anti-cryptococcal defense. Using cell lines expressing the functional, mutated or knockout gene, it was established that Nramp1 (1) is not involved in the phagocytic event, (2) influences anti-cryptococcal activity in the early steps but not at later times, and (3) is unrelated to the biomolecular pathways through which C. neoformans impairs macrophage secretory response. Although the functional role of Nramp1 is still far from being elucidated, the present data add insight into its involvement in macrophage-mediated antimicrobial defense, particularly in the initial steps allowing C. neoformans growth inhibition

2001 - S100B expression in and effects on microglia [Articolo su rivista]
Adami, C; Sorci, G; Blasi, Elisabetta; Agneletti, Al; Bistoni, F; Donato, R.
abstract

We evaluated the intracellular and extracellular biological role of S100B protein with respect to microglia. S100B, which belongs to the multigenic family of Ca2+-binding proteins, is abundant in astrocytes where it is found diffusely in the cytoplasm and is associated with membranes and cytoskeleton constituents. S100B protein is also secreted by astrocytes and acts on these cells to stimulate nitric oxide secretion in an autocrine manner. However, little is known about the relationship between S100B and microglia. To address this issue, we used primary microglia from newborn rat cortex and the BV-2 microglial cell line, a well-established cell model for the study of microglial properties. S100B expression was assessed by immunofluorescence in primary microglia and by RT-PCR, Western blotting, and immunofluorescence in BV-2 cells. S100B was found in microglia in the form of a filamentous network as well as diffusely in the cytoplasm and associated with intracellular membranes. S100B relocated around phagosomes during BV-2 phagocytosis of opsonized Cryptococcus neoformans. Furthermore, interferon-gamma (IFN-gamma) treatment caused cell shape changes and redistribution of S100B, and downregulation of S100B mRNA expression in BV-2 cells. Treatment of BV-2 cells with nanomolar to micromolar amounts of S100B resulted in increased IFN-gamma -induced expression of inducible nitric oxide synthase mRNA as well as nitric oxide secretion. Taken together, these data suggest a possible role for S100B in the accomplishment/regulation of microglial cell functions.

2000 - Establishment of protective immunity against cerebral cryptococcosis by means of an avirulent, non melanogenic Cryptococcus neoformans strain [Articolo su rivista]
R., Barluzzi; A., Brozzetti; G., Mariucci; M., Tantucci; Neglia, Rachele Giovanna; F., Bistoni; Blasi, Elisabetta
abstract

The opportunistic fungal pathogen, Cryptococcus neoformans, shows a marked predilection for the central nervous system (CNS). This can be partially explained by its ability to synthesize melanin starting from the catecholamines, highly concentrated at the CNS level. Two cryptococcal strains, the avirulent non-melanogenic strain Sb26 and the virulent melanogenic revertant strain Sb26Rev, were used in a murine model of intracerebral (i.c.) infection, in order to evaluate their virulence and immunomodulating properties at the cerebral level. We found that, unlike Sb26Rev, Sb26 i.c. infection was never lethal regardless of the challenging dose. Sb26Rev infection resulted in massive CNS tissue damage, associated with little or no cytokine response, as established by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Differently, Sb26 infection failed to alter CNS structure, while inducing IL-12 p40, TNF-alpha, IL-1 beta, IFN-gamma and iNOS specific-gene expression as well as IL-12, TNF-alpha and IL-1 beta cytokine production. Interestingly, all Sb26 infected mice survived a subsequent lethal, challenge with Sb26Rev. The phenomenon was associated with enhanced IL-12, TNF-alpha and IL-1 beta production and was strictly specific, as shown by heterologous challenges and delayed type of hypersensitivity assay. Overall, we provide evidence that protective immunity against cerebral cryptococcosis is established by means of an avirulent strain of C. neoformans. (C) 2000 Elsevier Science B.V. All rights reserved.

2000 - Inducible expression of the long pentraxin PTX3 in the central nervous system [Articolo su rivista]
Polentarutti, N.; Bottazzi, B.; Di Santo, E.; Blasi, Elisabetta; Agnello, D.; Ghezzi, P.; Introna, M.; Bartfai, T.; Richards, G.; Mantovani, A.
abstract

PTX3 is a prototypic long pentraxin consisting of a C terminal 203-amino acid pentraxin-like domain coupled with an N-terminal 178-amino acid unrelated portion. PTX3 is induced by primary proinflammatory signals in various cell types, most prominently macrophages and endothelial cells. Other long pentraxins, such as murine or rat neuronal pentraxin 1 (NP1) and human neuronal pentraxin 2 (NPTX2), are expressed in the central nervous system (CNS). The present study was designed to investigate whether PTX3 is expressed in the brain and to define the structures and cells involved. Intracerebroventricular (i.c.v.), but not i.v., injection of LPS induced high levels of PTX3 mRNA in the mouse brain. In contrast NP1 is constitutively expressed in the murine CNS and is not modulated by LPS administration. I.c.v. IL-1β was also a potent inducer of PTX3 expression in the CNS, whereas TNFα was substantially less effective and IL-6 induced a barely detectable signal. Central administration of LPS and IL-1 induced PTX3 also in the periphery (heart), whereas the reverse did not occur. Expression of PTX3 was also observed in the brain of mice infected with Candida albicans (C. albicans) or Cryptococcus neoformans. (C. neoformans). The kinetics of PTX3 gene induction were consistently different between C. albicans- and C. neoformans-infected mice, according to the diverse outcome of the CNS immune reaction. In situ hybridization revealed that i.c.v. injection of LPS induced a strong PTX3 expression in presumptive glial cells, in the white matter (corpus callosum, fimbria) and meningeal pia mater as well as in dentate gyrus hilus and granule cells. No constitutive expression of PTX3 was detected. Central expression of PTX3 may amplify mechanisms of innate resistance and damage in the CNS. The possibility of a direct interaction of PTX3 with neuronal cells, as suggested for NPTX2, remains to be explored.

1999 - Cryptococcosis and smoking: The potential role of iron [Articolo su rivista]
Boelaert, Jr; Blasi, Elisabetta
abstract

Smoking, a hazard for the general population, may be a major risk factor in HIV infection. Smoking is known to upregulate HIV replication, enhance the risk of accelerated atherosclerosis, etc.Moreover, the smoking-induced pulmonary iron loading may predispose HIV-infected persons to an increased risk of infection by a large variety of obligatory and facultative intracellular pathogens. HIV-infected persons should therefore be strongly advised to refrain from smoking.

1999 - Differential effector and secretory functions of microglial cell lines derived from BCG-resistant and -susceptible congenic mouse strains [Articolo su rivista]
M., Puliti; R., Mazzolla; A., Brozzetti; Neglia, Rachele Giovanna; D., Radzioch; F., Bistoni; Blasi, Elisabetta
abstract

Using congenic strains of mice susceptible (bcg(s)) or resistant (bcg(r)) to BCG, murine microglial cell lines, RR4.R (BCG-resistant) and RR8.S (BCG-susceptible), were established in vitro. Comparative studies revealed that, although phagocytic to a similar extent, RR4.R cells were more active than RR8.S cells in terms of antimycobacterial activity. Interestingly, cells of resistant genotype secreted more nitric oxide, TNF-alpha and IL-12, but less IL-6, than susceptible cells, when stimulated with IFN-gamma alone or in combination with lipolysaccharide. Nevertheless, no significant differences were observed between the two cell lines in terms of IL-1 beta or IL-10 secretion, or on assessment of cytokine production following exposure to a massive dose of Lipopolysaccharide. Overall, these data provide the first evidence that resistant/susceptible genotype influences antimycobacterial activity, NO and cytokine production in microglial cells, the prototype of cerebral macrophages. (C) 1999 Elsevier Science B.V. All rights reserved.

1999 - Differential effects of iron load on basal and interferon-gamma plus lipopolysaccharide enhance anticryptococcal activity by the murine microglial cell line BV-2 [Articolo su rivista]
Saleppico, S; Boelaert, Jr; Sale, Fo; Mazzolla, R; Morucci, P; Bistoni, F; Blasi, Elisabetta
abstract

Here we evaluated the influence of intracellular iron levels on the constitutive and interferon (IFN)-gamma plus lipopolysaccharide (LPS) enhanced anticryptococcal activity by the murine microglial cell line BV-2. We demonstrated chat iron loading via ferric nitrilotriacetate (FeNTA) resulted in a significant increase in the constitutive levels of anticryptococcal activity, while the enhancing effects by IFN-gamma plus LPS were prevented. Accordingly, a major increase was observed in the levels of thiobarbituric reactive substance (TBARS) produced upon iron loading under basal conditions, whereas IFN-gamma plus LPS treatment, that per se did not affect TEARS production, prevented by about 50% the enhancement otherwise occurring in response to iron loading. The potential involvement of multiple effector system and their relation to intracellular iron will be discussed. (C) 1999 Elsevier Science B.V. All rights reserved.

1999 - Tetanus toxin impairs accessory and secretory functions in interferon-gamma-treated murine macrophages [Articolo su rivista]
Pitzurra, L; Adami, C; Sevilla, M; Polonelli, L; Bistoni, F; Blasi, Elisabetta
abstract

Tetanus neurotoxin (TT), a product of microbial origin, acts as a zinc endopeptidase on vesicle-associated membrane proteins (VAMP). We have demonstrated that TT displays inhibitory effects on secretory and accessory functions in the murine macrophage (M phi) cell line GG2EE, Nitric oxide (NO) secretion was decreased when interferon (IFN)-gamma-pretreated GG2EE M phi s were coincubated with a fungal costimulus (SMP200) and TT, When heat-inactivated TT was used this effect was not evident, The TT-mediated phenomenon was dose-dependent and specific since, under the same experimental conditions, it did not affect interleukin-6 or tumor necrosis factor-alpha secretion. Furthermore, IFN-gamma-induced major histocompatibility complex class II molecule expression and GG2EE accessory function, assessed by SMP200-stimulated lymphoproliferation, were also inhibited by TT. Such inhibition was incomplete, in line with our previous results showing that TT partially cleaves VAMP proteins in murine M phi.

1998 - Glycosaminoglycan profile in macrophages exposed to Candida albicans and interleukins [Articolo su rivista]
M., Bodo; Blasi, Elisabetta; E., Becchetti; M., Giammarioli; C., Conte; S., Bellocchio; T., Baroni; C., Bellucci; F., Bistoni
abstract

Glycosaminoglycans (GAG), are extracellular matrix macromolecules that affect the phagocytic properties of macrophages. In order to assess whether the interaction between macrophages and Candida albicans (iCa) provokes changes in the phenotype, we analyzed the GAG profiles in two macrophage lines, ANA-1 (from murine bone-marrow) and BV-2 (from murine brain). We also investigated GAG modulation by interleukin-1alpha (IL-1alpha) and interleukin-6 (IL-6). During iCa treatment and even after the addition of ILs, ANA-1 accumulated less total GAG compared to controls. IL-1 treatment, combined with iCa exposure, induced a decrease in heparan sulfate and chondroitin sulfate chains, and an increase in the hyaluronic acid percentage. IL-6 treatment, with or without iCa, decreased the hyaluronic acid/sulfated GAG ratio. The GAG pattern in BV-2 appears to be different to ANA-1 and iCa exposure does not induce any difference in total GAG. The inhibitory effect induced by ILs on GAG synthesis is less than that observed in ANA-1 and the GAG elution profile is modulated to a lesser extent by treatment with ILs and/or iCa compared to the ANA-1. We suggest that the observed changes in the expression of the individual GAG classes may be responsible for the macrophage functional heterogeneity.

1998 - Role of the capsule in microglial cell-Cryptococcus neoformans interaction: impairment of antifungal activity but not of secretory functions [Articolo su rivista]
Barluzzi, R; Brozzetti, A; Delfino, D; Bistoni, F; Blasi, Elisabetta
abstract

Using two isogenic strains of Cryptococcus neoformans, we studied the influence of the capsule in C. neoformans microglial-cell interaction. We demonstrate that the acapsular mutant yeasts (CAP67) are more susceptible to phagocytosis and killing than encapsulated yeasts (B3501) by the murine microglial cells, BV-2. RT-PCR analysis showed that the pattern of gene transcripts for tumour necrosis factor alpha (TNF-a), interleukin (IL)-1 beta, IL-6, IL-12p40 and granulocyte macrophage colony stimulating factor re:mains unchanged following BV-2 cell infection with CAP67 or B3501 yeasts. Moreover, no induction of TNF-alpha secretion occurs in BV-2 cells infected with either B3501 or CAP67 yeasts or exposed to glucuronoxylomannan (GXM) or galactoxylomannan (GalXM). Nevertheless, lipopolysaccharide-induced TNF-alpha secretion is downregulated by cell infection with B3501 or CAP67 yeasts or exposure to GXM or GalXM. Overall, by means of a continuous cell line, it appears that the C. neoformans capsule is detrimental to microglial cell antifungal activity, while no effect can be attributed to the capsule as trend of cytokine gene expression and TNF-alpha secretion.

1997 - A low virulent strain of Candida albicans enhances brain anticryptococcal defenses: characterization of the local immune reaction by RT-PCR and histochemical analysis [Articolo su rivista]
R., Barluzzi; R., Mazzolla; A., Brozzetti; M., Puliti; G., Mariucci; P., Mosci; F., Bistoni; Blasi, Elisabetta
abstract

Here we studied the involvement of PCA-2, a low-virulent strain of Candida albicans known to act as a potent stimulating agent in the development of cryptococcal meningoencephalitis. To this purpose, mice received saline or PCA-2 intracerebrally 7 days before lethal local challenge with Cryptococcus neoformans. We found that, following C, neoformans challenge, PCA-2-treated but not saline-treated mice exhibited (a) delayed brain colonization, (b) enhanced median survival times, (c) massive local immune reaction consisting of abundant astrocytes, microglial and inflammatory cells, and (d) a peculiar trend of cytokine gene expression, including high steady-state levels of interleukin (IL)-1 beta and tumor necrosis factor alpha transcripts, fluctuating levels of interferon gamma and inducible nitric oxide synthase mRNA and lately detectable IL-6 gene expression, PCA-2-mediated immunostimulating properties were partially impaired by aminoguanidine or pentoxifylline treatment, further strengthening the conclusion that soluble mediators, including proinflammatory cytokines and nitric oxide, are important defense elements against cryptococcal meningoencephalitis.

1997 - Enhanced resistance to Cryptococcus neoformans infection induced by chloroquine in a murine model of meningoencephalitis [Articolo su rivista]
Mazzolla, R; Barluzzi, R; Brozzetti, A; Boelaert, Jr; Luna, T; Saleppico, S; Bistoni, F; Blasi, Elisabetta
abstract

Although the pathogenesis of cerebral cryptococcosis is poorly understood, local immune cells, such as microglia and astrocytes, likely play a critical role in containing infection, Chloroquine (CQ) is a weak base that accumulates within acidic vacuoles and increases their pH. Consequently, proteolytic activity of lysosomal enzymes and intracellular iron release/availability are impaired, resulting in decreased availability of nutrients crucial to microorganism survival and growth in the host, We found that CQ enhances BV2 microglial-cell-mediated anticryptococcal activity in vitro, The phenomenon is (i) evident when both unopsonized and opsonized microorganisms are used and (ii) mimicked by NH4Cl, another weak base, and by bafilomycin A(1), an inhibitor of vacuolar-type H+-ATPases. In vivo, intracerebral administration of CQ before lethal local challenge with Cryptococcus neoformans results in a significant augmentation of median survival time and a marked reduction of yeast growth in the brain and is associated with the enhancement of local interleukin 1 beta (IL-1 beta) and 1L-6 mRNA transcripts, Overall, these results provide the first evidence that CQ enhances anticryptococcal host defenses.

1997 - Immunology and pathogenesis of infections of the central nervous system(CNS) [Articolo su rivista]
Saleppico, S.; Barluzzi, R.; Mazzolla, R.; Puliti, M.; Blasi, E.
abstract

The central nervous system (CNS) has long been regarded as an immunologically privileged site for the presence of blood-brain and blood- cerebrospinal fluid barriers. Nevertheless, experimental evidence indicates that, under physiological conditions, minimal amounts of blood-derived immune cells exist within the brain and cooperate with the resident immune elements, such as microglia and astrocyte, to the surveillance of the district. Following microbial invasion, both blood-derived and local effector systems synergize against the pathogen. The timing and entity of such reaction is crucial, allowing the clearance of the pathogen or rather contributing to the extent of sometimes irreversible brain tissue damage.

1997 - Potent antifungal effects of a new derivative of partricin A in a murine model of cerebral cryptococcosis [Articolo su rivista]
Luna, T; Mazzolla, R; Romano, G; Blasi, Elisabetta
abstract

A new member of the polyene family, N-dimethylaminoacetyl-partricin A 2-dimethylaminoethylamide diaspartate (SPA),was investigated and was found to be more effective than amphotericin B (i) in vivo by enhancing mouse resistance to cryptococcal meningoencephalitis and (ii) in vitro by potentiating the anticryptococcal activity of murine microglial cells.

1996 - Biomolecular events involved in the establishment of brain anticandidal resistance [Articolo su rivista]
Mazzolla, R.; Barluzzi, R.; Puliti, M.; Saleppico, S.; Mosci, P.; Bistoni, F.; Blasi, Elisabetta
abstract

Using a murine model, we have demonstrated the establishment of cerebral resistance to local lethal challenge with Candida albicans strain CA-6, by previous intracerebral (i.c.) infection with the low-virulent strain PCA-2. Here we show that i.c. infection with PCA-2 is effective in drastically reducing brain colonization following secondary infection with CA-6. As assessed by colony forming unit assay and histopathological analysis, microbial counts are impaired, granuloma formation and hyphal growth are also reduced in brains of PCA-2- and CA-B-infected mice with respect to CA-6-challenged mice. Furthermore, using PCR studies, we found that, while PCA-2 (i.e. healing infection) induces transient cytokine gene expression in the mouse brain, CA-6 lethal challenge results in long-lasting (until mouse death) high levels of all cytokine gene transcripts assessed. Finally, brains from mice that will resist CA-6 challenge, because of previous infection with PCA-2, also exhibit a transient induction of all cytokine genes. Only IL-1 beta remains highly expressed at all time-points tested. Overall, these results provide evidence that healing and non-healing C. albicans i.c. infections differ in the immune reaction(s) locally evoked, at least in terms of cytokine gene expression, strongly suggesting cytokine involvement in the establishment of brain anticandidal resistance.

1996 - Candida albicans stress mannoprotein, SMP 200, enhances tumor necrosis factor secretion in the murine macrophage cell line ANA-1 [Articolo su rivista]
L., Pitzurra; L., Polonelli; C., Cantelli; M., Gerloni; J., Ponton; J., Bikandi; Blasi, Elisabetta
abstract

Tumour necrosis factor (TNF) secretory activity has been studied in the murine macrophage cell line ANA-1 following in vitro exposure to Candida albicans 200 kDa stress mannoprotein (SMP200). Treatment of ANA-1 murine macrophages with 200 kDa stress mannoprotein results in increased TNF secretion. The phenomenon is (i) dose- and time-dependent, (ii) abrogated by 200 kDa stress mannoprotein preincubation with a specific monoclonal antibody, and (iii) dependent on intact murine macrophage Ca2+/calmodulin-dependent protein kinase function.

1996 - Iron regulates microglial cell-mediated secretory and effector functions [Articolo su rivista]
Saleppico, S.; Mazzolla, R.; Boelaert, J. R.; Puliti, M.; Barluzzi, R.; Bistoni, F.; Blasi, Elisabetta
abstract

Iron homeostasis and macrophage physiology are tightly intertwined. In the present study, we evaluated the influence of iron loading on the constitutive and interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS)-induced functional and secretory properties of microglial cells, using the in vitro established murine cell line BV-2. We demonstrate that iron augments the basal and IFN-gamma plus LPS-enhanced anti-Candida albicans activity exerted by BV-2 cells and that the phenomenon occurs with no enhancement of phagocytic activity. Furthermore, when the secretory properties of IFN-gamma plus LPS-treated BV-2 cells were assessed, we found that tumor necrosis factor remains unchanged while nitric oxide production is significantly reduced in iron-loaded cells. The addition of the iron chelator deferiprone (L1) reverts the effects of iron on BV-2 functional and secretory properties. These data suggest that iron differently affects secretory and effector functions of BV-2 microglial cells, thus implying that iron interferes with murine microglial cell physiology

1996 - Tetanus toxin-sensitive VAMP-related proteins are present in murine macrophages [Articolo su rivista]
Pitzurra, L; Rossetto, O; Chimienti, Ar; Blasi, Elisabetta; Bistoni, F.
abstract

The light chain of tetanus neurotoxin (TeTx) is a zinc endopeptidase specific for VAMP/synaptobrevin (VAMP), a 120-amino-acid integral protein previously described in the small synaptic vesicles of neuronal cells. TeTx has been shown to be active also on nonneuronal cells. By SDS-PAGE and quantitative immunoblotting on proteins derived from murine macrophages (M phi) exposed to TeTx, we have shown that: (1) VAMP-related proteins are also present in M phi and (2) such proteins are sensitive to TeTx proteolytic cleavage. The demonstration that TeTx acts on VAMP-related proteins also in M phi offers a new and useful tool for molecular studies on M phi exocytosis.

1995 - Biomolecular Events Involved in AnticryptococcalResistance in the Brain [Articolo su rivista]
Blasi, Elisabetta; Barluzzi, R; Mazzolla, R; Pitzurra, L; Puliti, M; Saleppico, S; Bistonif,
abstract

We have recently shown that intracerebral (i.c.) administration of heat-killed Cryptococcus neoformans (HCN) enhances mouse resistance to a subsequent local challenge with lethal doses of viable yeast cells. Here we show that i.c. administration of HCN is also effective in significantly delaying brain colonization of mice intravenously infected with viable C. neoformans. PCR analysis revealed that interleukin 6 (IL-6) and IL-1 beta gene expression occurs in brain of HCN-treated mice but not in brains of saline-treated controls. In contrast, no differences are observed in terms of tumor necrosis factor alpha and IL-1 alpha gene transcripts, which are slightly and highly detectable, respectively, in saline-treated mice and which remain such also following HCN treatment. Furthermore, i.c. administration of exogenous IL-6 or IL-1 beta, but not tumor necrosis factor alpha, before local challenge with viable C. neoformans results in significantly reduced microbial counts in the brain and blood and in increased mouse survival. Taken together, these observations provide initial evidence that brain anticryptococcal resistance involves elicitation of a local cytokine response, involving primarily IL-6 and IL-1 beta

1995 - Differential susceptibility of yeast and hyphal forms of Candida albicans to macrophage-derived nitrogen-containing compounds [Articolo su rivista]
Blasi, Elisabetta; L., Pitzurra; M., Puliti; Ar, Chimienti; R., Mazzolla; R., Barluzzi; F., Bistoni
abstract

Candida albicans is a dimorphic Fungus capable of transition from the yeast form (Y-Candida) to the hyphal form (H-Candida). Both Y-Candida and H-Candida are known to be growth inhibited by murine macrophages (M phi) in vitro. In the present report, we demonstrate that M phi exposed to interferon gamma (IFN-gamma) plus lipopolysaccharide (LPS) show enhanced anti-Y-Candida and anti-H-Candida activities. To further investigate the phenomenon, Y-Candida and H-Candida were assessed for susceptibilities to M phi-derived supernatants. Only the growth of H-Candida, and not that of Y-Candida, is impaired by cell-free supernatants from M phi, treated with IFN-gamma plus LPS. In contrast, no H-Candida growth inhibition occurs when supernatants from M phi exposed to IFN-gamma plus LPS in the presence of N-G-monomethyl-L-arginine, an inhibitor of nitric oxide (NO) synthesis, are employed. Finally, supernatants from M phi incubated with sodium nitroprusside, an NO-generating agent, also show anti-H-Candida activity, In conclusion, these results indicate that H-Candida but not Y-Candida is susceptible to extracellular antifungal mechanisms employed by M phi, which likely involve stable nitrogen-containing compounds.

1995 - Differential susceptibility of yeast and hyphal forms of Candida albicans to proteolytic activity of macrophages [Articolo su rivista]
Blasi, E.; Pitzurra, L.; Chimienti, A. R.; Mazzolla, R.; Puliti, M.; Barluzzi, R.; Bistoni, F.
abstract

The dimorphic transition of Candida albicans from the yeast (Y-Candida) to the hyphal (H-Candida) form is a complex event whose relevance in fungal pathogenicity is still poorly understood. Using a cloned macrophage (Mφ) cell line (ANA-1), we have previously shown that a Mφ can discriminate between the two fungal forms, eliciting different secretory responses. In the present study, we investigated the susceptibility of Y-Candida and H-Candida to Mφ proteolytic activity. In particular, sodium dodecyl sulfate- polyacrylamide gel electrophoresis and Western blot (immunoblot) techniques were employed to analyze the patterns of lyticase proteinaceous extracts from cell walls of Y-Candida and H-Candida which had been unexposed or exposed to ANA-1 Mφs for 3 h. Silver staining allowed detection of a complex protein pattern in both forms of C. albicans, qualitatively and quantitatively differing from each other, mainly at molecular masses below 106 kDa. Western blot staining with anti-C. albicans mannan antibodies and convalescent-phase sera of mice previously infected systemically or intracerebrally with C. albicans showed that, after contact with Mφs, Y-Candida but not H-Candida proteinaceous cell wall components are profoundly modified, with substantial reduction and/or disappearance of many bands. Our experimental approach provides initial insights into the differential susceptibility of Y-Candida and H-Candida to the proteolytic activity of Mφs.

1995 - Influence of the Bcg locus on macrophage response to the dimorphic fungus Candida albicans [Articolo su rivista]
M., Puliti; D., Radzioch; R., Mazzolla; R., Barluzzi; F., Bistoni; Blasi, Elisabetta
abstract

The Bcg/Tty/Lsh gene (candidate Nramp) controls natural resistance to several parasites, such as Mycobacterium bovis, Leishmania donovani, and Salmonella typhimurium. Using two macrophage (M phi) cell lines (B10R and B10S) derived from mouse strains congenic at Beg, we found that M phi s from resistant mice (B10R M phi s) act more effectively against the two morphogenetic forms of the dimorphic fungus Candida albicans compared with M phi s from susceptible mice (B10S M phi s). Moreover, when assessed for tumor necrosis factor secretion in response to the hyphal form of C. albicans, B10R M phi s are significantly more effective at expressing this secretory function than are B10S M phi s, closely resembling the trend of response to lipopolysaccharide. Overall, these results provide insight into the influence of the Beg locus on the M phi response to C. albicans.

1995 - ROLE OF NITRIC-OXIDE AND MELANOGENESIS IN THE ACCOMPLISHMENT OF ANTICRYPTOCOCCAL ACTIVITY BY THE BV-2 MICROGLIAL CELL-LINE [Articolo su rivista]
Blasi, Elisabetta; Barluzzi, R; Mazzolla, R; Tancini, B; Saleppico, S; Puliti, M; Pitzurra, L; Bistoni, F.
abstract

In the present paper, we investigated the involvement of cryptococcal melanogenesis and macrophage nitric oxide (NO) production in the accomplishment of anticryptococcal activity by microglial effector cells, using the murine cell line BV-2. We demonstrate that the constitutive levels of anticryptococcal activity exerted by BV-2 cells is significantly enhanced upon interferon gamma plus lipopolysaccharide treatment. The phenomenon, which occurs with no enhancement of phagocytic activity, is associated with the production of high levels of NO and is abolished by addition of N-G-monomethyl-L-arginine. Comparable patterns of results are observed employing either unopsonized or opsonized microbial targets, the latter microorganisms being markedly more susceptible to BV-2 cell antimicrobial activity. Furthermore, melanization of Cryptococcus neoformans significantly reduces its susceptibility to BV-2 antimicrobial activity, regardless of the fact that activated macrophages or opsonized microorganisms have been employed. In conclusion, our results provide evidence that NO-dependent events are involved in the fulfillment of anticryptococcal activity by activated microglial cells and that fungal melanization is a precious escamotage through which C. neoformans overcomes host defenses.

1994 - Anticryptococcal Resistance in the Mouse Brain: Beneficial Effects ofLocal Administration of Heat-Inactivated Yeast Cells [Articolo su rivista]
Blasi, Elisabetta; Mazzolla, R; Barluzzi, R; Mosci, P; Bistoni, F.
abstract

Using a murine model, we have previously shown that brain resistance to local infection with opportunistic fungi is affected by manipulation of the host myelomonocytic compartment. Here, we demonstrate thatintracerebral administration of heat-inactivated Cryptococcus neoformans (H-CN) yeast cells results in a consistent enhancement of mouse survival to subsequent local challenge with lethal doses of C. neoformans. The phenomenon, more pronounced upon double H-CN treatment, is associated with (i) massive local inflammatoryresponse, (ii) reduced growth of the fungus within the brain, and (iii) induction of delayed-type hypersensitivity. Moreover, H-CN treatment confers protection against local heterologous challenges. Our data provide initial evidence that intracerebral administration of H-CN results in the establishment of aspecific and specific immune responses; the mechanisms of elicitation and relative contributions to host antimicrobial resistance remain to be elucidated.

1994 - Comparative studies on functional and secretory properties of macrophage cell lines derived from different anatomical sites [Articolo su rivista]
Blasi, E.; Puliti, M.; Pitzurra, L.; Barluzzi, R.; Mazzolla, R.; Adami, C.; Cox, G. W.; Bistoni, F.
abstract

In the present study, we compared four macrophage (Mφ) cell lines from different anatomical origins for functional and secretory activities against the two morphogenetic forms of the fungus Candida albicans. We show that all the cell lines actively phagocytize the yeast and exert antimicrobial activity against both forms o3 Candida, although Mφ of microglial origin are the most effective. When assessed for secretory properties, microglial Mφ exhibit a peculiar patten with respect to other Mφ populations under either basal or stimulated conditions. In particular, only microglial Mφ fail to respond to the hyphal form of the fungus (H-Candida), which instead acts as a potent tumor necrosis factor inducer in the other Mφ cell lines. When exposed to H-Candida, microglial Mφ are indistinguishable from other Mφ in their ability to modulate specific surface adhesion molecules. In addition to strengthening the knowledge on functional heterogeneity among Mφ, our data provide evidence on the peculiar behavior of microglial Mφ. To what extent Mφ heterogeneity may be related to tissue homeostasis is discussed. © 1994.

1994 - Different events involved in the induction of macrophage tumor necrosis factor by Candida albicans and lipopolysaccharide. [Articolo su rivista]
Blasi, Elisabetta; Pitzurra, L; Puliti, M; Mazzolla, R; Barluzzi, R; Saleppico, S; Bistoni, F.
abstract

Using an in vitro experimental model, we have recently demonstrated that Candida albicans in its hyphal form (H-Candida), similarly to lipopolysaccharide (LPS), enhances tumor necrosis factor (TNF) secretory response in the cloned macrophage (M phi) population ANA-1. Here we show that H-Candida and LPS each differ in their requirements for intact protein kinase functions, susceptibility to 0.4-microns micropore-size membranes, and sensitivity to polymyxin B. These results, together with the synergistic effect occurring between H-Candida and LPS in inducing TNF response, indicate the existence of different receptor(s) and/or signal-transduction pathway(s) through which the two stimuli act.

1994 - Heterogeneous secretory response of phagocytes from different anatomical districts to the dimorphic fungus Candida albicans. [Articolo su rivista]
Blasi, Elisabetta; Puliti, M; Pitzurra, L; Bartoli, A; Bistoni, F.
abstract

In the present study, we have examined the ability of phagocytes from different anatomical districts to discriminate between the two morphogenetic forms of Candida albicans. We have demonstrated that resident peritoneal macrophages (RP-M phi) and thioglycollate-elicited peritoneal macrophages (TP-M phi) were able to distinguish between the hyphal (H-Candida) and the yeast (Y-Candida) form of the fungus, since TNF production was observed only upon exposure of RP-M phi and TP-M phi to H-Candida. In contrast, splenic macrophages (S-M phi), bone marrow-derived macrophages (BM-M phi) and polymorphonuclear neutrophils (PMN) did not discriminate between the two forms because S-M phi and PMN produced TNF regardless of the morphogenetic status of the fungus, while BM-M phi did not. Under the same experimental conditions, we failed to observe IL-1 production from any of the phagocytic cell populations examined, with the exception of PMN. This implies that the interaction between phagocytes and C. albicans triggers differential secretory responses depending upon the morphogenetic status of the fungus and the anatomical localization of the immune cells.

1994 - Pattern of cytokine gene expression in brains of mice protected by picolinic acid against lethal intracerebral infection with Candida albicans. [Articolo su rivista]
Blasi, Elisabetta; Bartoli, A; Barluzzi, R; Mazzolla, R; Bistoni, F.
abstract

Recently, we demonstrated that intracerebral (i.c.) administration of picolinic acid (PLA) confers protection against a lethal local challenge with the opportunistic pathogen Candida albicans. By histopathological studies, we show here that mice receiving PLA treatment survive challenge and no evidence of fungal invasion is found within the brain compartment. In contrast, PLA-untreated mice succumb to infection within 7-10 days and show massive brain colonization with extensive granulomatous reaction. By PCR analysis, we show that, unlike naive brains, PLA-treated brains show transient activation of TNF alpha, IL-1 beta and IL-6 genes. C. albicans infection results in high levels of all cytokine transcripts, the phenomenon being long-lasting in PLA-untreated brains, while gradually declining in PLA-treated brains. The only exception is IL-1 beta, whose levels remain high at the latest time-points tested, also in PLA-treated brains. Finally, IL-1 alpha, constitutively detectable in naive brains, is slightly enhanced by C. albicans challenge, regardless of prior treatment. These findings, together with the knowledge that PLA is a potent co-stimulus for macrophages, suggest the involvement of cytokine circuits, likely of macrophage origin, in anti-Candida resistance established by PLA at the cerebral level

1994 - Systemic infection with Herpes bovis virus 2 evokes a biphasic immune response in the mouse [Articolo su rivista]
Puliti, M.; Cenci, E.; Vecchiarelli, A.; Blasi, E.; Merletti, L.
abstract

We evaluated the effects of systemic infection by Herpes bovis virus 2 (HBV-2) on a murine experimental system. We provide evidence that such infection is lethal for the immunocompromised but not for the immunocompetent mouse in which a biphasic immune response is elicited. In particular, 1 day post-infection, we observed a rapid transient depression induced by the virus, as documented by a decrease in peripheral leukocyte counts, mitogenic spleen cell response and resistance to a secondary microbial challenge. Later, HBV-2 infection boosted cytokine secretion and enhanced antimicrobial and antitumoral activities by the splenic district. In conclusion, our experimental model discloses some immunological aspects underlying the complex host-virus interaction.

1994 - Tumor Necrosis Factor as an Autocrine and ParacrineSignal Controlling the Macrophage SecretoryResponse to Candida albicans [Articolo su rivista]
Blasi, Elisabetta; L., Pitzurra; A., Bartoli; M., Puliti; F., Bistoni
abstract

We have previously demonstrated that the hyphal form of Candida albicans (H-Candida), but not the yeast form (Y-Candida), acts as a macrophage-stimulating agent. The early response (1 to 3 h) of the macrophage cell line ANA-1 to H-Candida results in enhanced tumor necrosis factor (TNF) transcription and production.Here we show that when coincubation times are prolonged (3 to 24 h), Y-Candida also exhibits stimulatory properties. This phenomenon has been ascribed to the occurrence of the dimorphic transition, as demonstrated by microscopic evaluation of the cultures and by experiments in which both killed Y-Candida and the agerminative strain C. albicans PCA-2 failed to induce cytokine production. TNF produced in response to H-Candida acts as an autocrine and paracrine signal controlling the macrophage secretory response to C. albicans. In fact, addition of anti-TNF polyclonal antibodies to the coculture of ANA-1 macrophages and H-Candida results in a marked and time-dependent decrease of TNF transcript levels. Moreover, pretreatmentof macrophages with recombinant TNF for 3 h enhances TNF and induces interleukin-1 production in response to both forms of Candida, while pretreatment for 18 h renders macrophages refractory to any stimuli.Interestingly, the kinetics of interleukin-1 transcription and secretion in response to H-Candida are delayed with respect to those of TNF. Overall, these data indicate that TNF, produced by macrophages in response to H-Candida, regulates its own production as well as that of other soluble factors, thus suggesting that this cytokine plays multiple roles in the immune mechanisms involved in Candida infection.

1993 - Differential Host Susceptibility to Intracerebral Infections with Candida albicans and Cryptococcus neoformans [Articolo su rivista]
Blasi, Elisabetta; R., Barluzzi; R., Mazzolla; F., Bistoni
abstract

To investigate the immune defense mechanisms employed against fungi in the brain, mice were experimentally infected by intracerebral inoculation of Candida albicans or Cryptococcus neoformans. Parameters such as median survival time and numbers of yeast cells in the brains were assessed for naive and immunomodulated mice. We found that no mice survived either C. albicans or C. neoformans challenge at doses of .106 yeast cells per mouse. However, when the inoculum size was decreased ('105 yeast cells per mouse), C. albicans was no longer lethal (100%S survival), whereas 100 and 70%o of the mice still succumbed to challenge doses of 104 and 103 C. neoformans yeast cells, respectively. Pharmacological manipulation and transfer experiments revealed that the myelomonocytic compartment had a minor role against C. neoformans but was deeply involved in thecontrol of intracerebral C. albicans infection. By counting the number of yeast cells in the brains of naive and immunomodulated animals, we established that, unlike C. albicans, C. neoformans remained essentially in the brain, where massive colonization and damage occurred whether naive or immunomodulated defensemechanisms were employed by the host. Overall, these data suggest that the differential role of the myelomonocytic compartment, together with the diverse tropisms of the two fungi, can explain the different development and outcome of intracerebral C. albicans and C. neoformans infections.

1993 - Protective effect of picolinic acid on mice intracerebrally infected with lethal doses of Candida albicans [Articolo su rivista]
Blasi, E.; Mazzolla, R.; Pitzurra, L.; Barluzzi, R.; Bistoni, F.
abstract

We have studied the effects of picolinic acid (PLA), a product of tryptophan degradation, on mouse susceptibility to intracerebral infection with Candida albicans. We show that intraperitoneal administration of PLA significantly enhances the median survival time of mice inoculated with the lethal challenge. Furthermore, intracerebral administration of this agent induces a protective state against the local lethal infection, the phenomenon depending upon the administration schedule and doses of PLA employed. According to survival data, yeast growth in the brain as well as yeast colonization of the kidneys are drastically reduced in PLA-treated mice compared with those for untreated controls. Northern (RNA) blot analysis of brain tissues demonstrates that mRNA levels specific for tumor necrosis factor and interleukin 1 are augmented and induced, respectively, after inoculation of PLA. These results indicate that PLA has a protective effect likely involving elicitation of a cytokine response in vivo against fungal infections.

1993 - Tetanus toxin selectively impairs anti-tumoral but not anti-microbial macrophage-mediated effector functions [Articolo su rivista]
Pitzurra, L.; Puliti, M.; Burhan Fuad, M. A.; Bistoni, F.; Blasi, E.
abstract

The present study was designed to establish the susceptibility of macrophage-mediated effector functions to tetanus toxin (TT). Using the murine macrophage cell line, GG2EE, generated in vitro by v-raf/v-myc oncogenes, we have previously provided evidence that TT selectively inhibits interferon gamma (IFN-γ), but not basal, lysozyme activity. Here we show that while neither phagocytic nor candidacidal activities are affected by TT treatment, antitumoral activity is significantly impaired after exposure to TT. This phenomenon, which is dose-dependent, is fully ascribed to the holotoxin, as heat inactivated TT, C or A-B fragments result ineffective. Furthermore, C but not A-B fragment competes with TT in abrogating its inhibitory effects. Overall, these data indicate that TT is not a broad-spectrum, down-regulating signal on macrophage-mediated functions, thus implying that its toxic action is exerted on specific molecular targets. © 1993.

1993 - Toxic Effects of Tetanus Toxin on GG2EE Macrophages:Prevention of Gamma Interferon-Mediated Upregulationof Lysozyme-Specific mRNA Levels [Articolo su rivista]
L., Pitzurra; Blasi, Elisabetta; M., Puliti; F., Bistoni
abstract

By using a nonneuronal cell system, evidence has previously been provided that tetanus toxin (TT) intoxication occurs in macrophages, impairing their secretory activity as well as their antitumoral activity. Inparticular, both secreted and total lysozyme (LZM) activities are reduced by TT treatment, provided that GG2EE macrophages have been preexposed to gamma interferon (IFN-y). In an attempt to provide insight into the molecular mechanisms underlying this phenomenon, we focused our attention on the levels of LZM-specifictranscripts. GG2EE macrophages preexposed to IFN-y exhibited augmented levels of LZM-specific mRNA.Such an effect was detected 1 h after removal of IFN-y, peaked at 3 h, and gradually decreased with time in culture. Exposure of IFN-y-pretreated GG2EE macrophages to TT resulted in the prevention of theIFN-y-mediated upregulation of LZM mRNA levels. The phenomenon was mediated by the holotoxin (>1 .g/ml) and abrogated by preexposure of the macrophages to the C fragment of IT. Protein kinase C (PKC) and Ca2+-calmodulin-dependent PK were likely involved in the IFN-ymediated upregulation of LZM mRNA levels and biological activity, as assessed by PK inhibitors. Furthermore, PK inhibitors mimicked TT in impairing LZM activity of GG2EE macrophages, thus suggesting that impairment of PKC and/or the Ca2+-calmodulin-dependent PK pathway(s) may be one of the events involved in ITT intoxication ofmacrophages.

1992 - An immortalized cell line expresses properties of activated microglial cells [Articolo su rivista]
Bocchini, V.; Mazzolla, R.; Barluzzi, R.; Blasi, E.; Sick, P.; Kettenmann, H.
abstract

Murine cultured microglial cells were immortalized after infection with a v‐raf/v‐myc recombinant retro‐virus. This immortalized cell line (BV‐2) shares properties with body macrophages with respect to the antigen profile, their phagocytic capacity and antimicrobial activity. BV‐2 cells are not constitu‐tively able to kill tumor cells in vitro, but acquire antitumor activity following an increase in [Ca++]i. BV‐2 cells, like microglial cells, are however, distinct from peripheral macrophages by their expression of inwardly rectifying K+ cannels in concert with a Jack in outwardly rectifying K+ channels and the formation of spineous processes. The BV‐2 cell line thus represents a suitable model for in vitro studies of activated microglial cells. Copyright © 1992 Wiley‐Liss, Inc.

1992 - Calcium ionophore A-23187 inhibits the secretion of β-hexosaminidase from the GG2EE mouse macrophage cell line [Articolo su rivista]
Beccari, T.; Datti, A.; Orlacchio, A.; Farinelli, S.; Blasi, E.
abstract

Secretion of the lysosomal enzyme β-N-acetylhexosaminidase is inhibited by calcium ionophore A-23187 in the GG2EE macrophage cell line. Such inhibition is time and dose dependent. Calcium ionophore A-23187 treatment causes a change in the pattern of hexosaminidase isoenzymes detectable in the cell extract, as assessed by DEAE-cellulose chromatography. In particular, control cells show two hexosaminidase isoenzymes corresponding to hexosaminidase A and B, whereas cells treated with calcium ionophore A-23187 express a third isoenzyme form with properties similar to hexosaminidase S.

1992 - Candida albicans hyphal form enhances tumor necrosis factor mRNA levels and protein secretion in murine ANA-1 macrophages. [Articolo su rivista]
Blasi, Elisabetta; Pitzurra, L; Puliti, M; Bartoli, A; Bistoni, F.
abstract

We have demonstrated that Candida albicans in its hyphal form (H-Candida) acts as a stimulating agent in the cloned macrophage population ANA-1. Both tumor necrosis factor (TNF) mRNA levels and secreted biological activity augment in ANA-1 macrophages exposed to H-Candida. Such effects are observed at an effector-to-target cell ratio of 1:1 and occur after 1 and 3 hr of coincubation, respectively. The phenomenon is independent of the metabolic status of the fungus, since viable as well as heat-killed H-Candida are comparable in inducing TNF mRNA levels. The extent and kinetics of H-Candida-mediated effects are similar to those observed following exposure of ANA-1 macrophages to lipopolysaccharide (LPS). This implies that C. albicans in its hyphal form is a potent macrophage modulator; whether it acts through the same mechanism(s) as LPS remains to be elucidated

1992 - Early Differential Molecular Response of a Macrophage Cell Line toYeast and Hyphal Forms of Candida albicans [Articolo su rivista]
Blasi, Elisabetta; L., Pitzurra; M., Puliti; L., Lanfrancone; F., Bistoni
abstract

The dimorphic transition of Candida albicans from the yeast (Y-Candida) to the hyphal (H-Candida) form is a complex event; the relevance of this transition in fungal pathogenicity is still poorly understood. By using a cloned macrophage cell line (ANA-1), we questioned whether the interaction between macrophages and Y-Candida or H-Candida could affect specific cell functions, i.e., tumor necrosis factor and lysozyme production. We found that ANA-1 macrophages selectively responded to H-Candida with increased tumor necrosis factor and downregulated lysozyme, as assessed by measurement of relative mRNA levels and secreted biological activities. The H-Candida-mediated effects were (i) dependent upon the ratio between ANA-1 macrophages and H-Candida, (ii) detectable after 1 h of coincubation, and (iii) accomplished without fungal ingestion. Conversely, Y-Candida, which was found inside the ANA-1 macrophages, did not affect tumor necrosis factor and lysozyme production, nor did it prevent the macrophage response to other stimuli. Overall, these results indicate that a macrophage can distinguish between Y-Candida and H-Candida and that only the latter is able to modulate specific functions. H-Candida is recognized and probably processed as an extracellular target. The possible implication of macrophages as autocrine and paracrine regulatory cells during Candida infections is discussed.

1992 - Experimental Model of Intracerebral Infection withCryptococcus neoformans: Roles of Phagocytesand Opsonization [Articolo su rivista]
Blasi, Elisabetta; R., Barluzzi; R., Mazzolla; P., Mosci; F., Bistoni
abstract

A murine model of intracerebral (i.c.) infection with Cryptococcus neoformans in which naive mice receiving an i.c. fungal inoculation developed a severe disease has been established. The effect was strictly dependent on the number of microorganisms injected and evolved as lethal meningoencephalitis. Murine susceptibility to i.c.infection with C. neoformans was enhanced by treatment with chloroquine and colchicine, agents known to greatly affect the host phagocytic compartment. Furthermore, the life spans of both naive and drug-treated mice were significantly augmented when opsonized fungi were injected. Therefore, phagocyte-mediated mechanisms are likely involved in local resistance to i.c. infection with C. neoformans. Further support for this conclusion was supplied by in vitro data showing that microglial cells were proficient anticryptococcal effectors,provided opsonized microorganisms were used.

1992 - Inhibition of proliferation of retrovirus-immortalized macrophages by LPS and IFN-γ: Possible autocrine down-regulation of cell growth by induction of IL1 and TNF [Articolo su rivista]
Ayroldi, E.; Blasi, E.; Varesio, L.; Wiltrout, R. H.
abstract

The GG2EE macrophage tumor cell line was previously established by immortalization of C3H/HeJ mouse bone marrow cells with the J2 retrovirus which contains the v-myc and v-raf oncogenes. Studies on the control of GGZEE cell proliferation in vitro have recently been performed. We observed that the combination of 5-25 U/ml recombinant mouse interferon-γ (rmIFN-γ) plus 0.03 - 0.3 μg/ml lipopolysaccharide (LPS) markedly inhibited the proliferation of GG2EE cells (by >95%)in vitro, while either agent alone inhibited only by <40% and 0-10%, respectively. Subsequent studies established that biologically active ILI-like (2-4 U/ml) and TNF α-like (50-100 U/ml) activities were released into the supernatants of LPS-treated GG2EE cells. The combination of IFN-γ + LPS induced more (6-8 U/ml) ILI release. These results suggested that the inhibition of proliferation of GG2EE cells by IFN-γ + LPS could have been mediated in part by cytokines produced by the cells themselves. rhIL1 α at a concentration of 10 U/ml inhibited GG2EE proliferation by 25-30%, while rmIFN-γ (25 U/ml) + rhIL1 α (10 U/ml) inhibited proliferation by 98%. Thus, 10 U/ml rhIL1 α could completely replace LPS in the LPS + rmIFN-γ combination. Further, the combination of low doses of rhIL1 α (0.1 to 1 U/ml) plus rmTNF α (250 U/ml), which together inhibited proliferation by <20% synergized with doses of 5 to 25 U/ml rmIFN-γ to inhibit proliferation of GG2EE cells by 98-99%. These results suggest that cytokines produced by the cells themselves can synergize with rmIFN-γ to inhibit the oncogene-driven proliferation of GG2EE cells. © 1992 Kluwer Academic Publishers.

1992 - Reliability of in vitro models for studies on immune functions by normal and immunomodulated phagocytes [Articolo su rivista]
Bistoni, F.; Blasi, E.; Vecchiarelli, A.; Mazzolla, R.; Cenci, E.; Puliti, M.; Sbaraglia, G.
abstract

1991 - A rapid Candida albicans hyphal‐form growth inhibition assay: determination of myelomonocytic‐mediated antifungal activity: Schnellverfahren zur Messung der Wachstumshemmung von Candida albicans durch Myelomonozyten‐vermittelte antifungale Aktivität [Articolo su rivista]
Scaringi, L.; Blasi, E.; Cornacchione, P.; Bietta, C.; Bistoni, F.
abstract

Summary. An in vitro microassay for the measurement of Candida albicans hyphal‐form growth inhibition by myelomonocytic cells is described. The assay is rapid, easy‐to‐perform and objective. A Candida strain capable of in vitro dimorphic transition from yeast to hyphal form has been employed. The assay is based on the incorporation of 3H‐glu‐cose by the fungus, the effect being dependent upon the time of pulse, size of the inoculum and concentration of radiolabelled metabolite. In particular, C. albicans hyphal form, obtained by a 3 h incubation in vitro in the presence of 10% fetal calf serum, is co‐incubated with the effector cells. A pulse with 3H‐glucose in water is then performed and the radioactivity incorporated by the residual Candida is taken as an indication of hyphal growth. We found that polymorphonuclear cells, peritoneal macrophages and the cloned GG2EE macrophage cell line significantly inhibited hyphal growth, the effects being time and effector‐to‐target cell ratio dependent. Zusammenfassung. Es wird ein Mikroverfahren in vitro zur Messung der Wachstumshemmung von Candida albicans in der Myzelphase durch Mye‐lomonozyten beschrieben. Der Ansatz ist schnell, leicht in der Ausführung und liefert objektive Re‐sultate. Hierzu wurde ein Candida‐Stamm verwen‐det mit der Fähigkeit, in vitro den dimorphen Pha‐senwechsel von der Hefe‐ zur Myzelphase zu durch‐laufen. Der Ansatz basiert auf dem 3H‐Glucose‐Ein‐bau durch den Pilz. Dieser Einbau ist abhängig von der Expositionszeit, der Inoculum‐Größe und der Konzentration des radioaktiv markierten Metabo‐liten. Hierzu wird die C. albicans‐Myzelphase, er‐halten nach 3 h Bebrütung in vitro in der Gegen‐wart von 10%igem fötalem Kälberserum, mit den Effektorzellen ko‐inkubiert. Dann werden die Hefen der 3H‐Glucose in Wasser ausgesetzt, und die ein‐gebaute Radioaktivität in den Candida‐Zellen wird als Indikator des Hyphenwachstums angesehen. Unsere Ergebnisse zeigen, daß polymorphonukleäre Zellen, Peritonealmakrophagen und die geklonte GG2EE‐Makrophagen‐Zellinie das Hyphenwachs‐tum in Abhängigkeit von der Expositionszeit und dem Effektor/Zielzell‐Verhältnis signifikant hem‐men. Copyright © 1991, Wiley Blackwell. All rights reserved

1991 - Fungicidal activity of Candida albicans-induced murine lymphokine-activated killer cells against C. albicans hyphae in vitro [Articolo su rivista]
Scaringi, L.; Blasi, E.; Rosati, E.; Marconi, P.; Bistoni, F.
abstract

Multiple intraperitoneal injections of inactivated Candida albicans cells resulted in the generation of cytotoxic peritoneal cells with phenotypical and functional properties similar to in vitro-generated lymphokine-activated killer (LAK) cells. Using an in vitro [3H]glucose uptake assay, C. albicans-induced LAK-like (CA-LAK) cells exhibited high levels of anti-hyphal activity, the effects being effector to target cell (E:T) ratio- and time-dependent. Maximal levels of anti-C. albicans activity (approximately 60%) were observed after 4 h and at E:T ≥ 300:1. Similar patterns of anti-C. albicans activity were exerted by in vivo-activated natural killer (NK) cells, in vitro interleukin-2- (IL-2) generated LAK cells and polymorphonuclear cells. The anti-hyphal activity of CA-LAK cells was enriched by separation on a Percoll gradient, F2 and F3 fractions retaining most of the activity. Experiments using immunodepressed animals demonstrated that the in vivo lethality of the C. albicans hyphal form is significantly affected by in vitro pre-exposure to CA-LAK cells. While control mice receiving C. albicans alone had a median survival time of 2 d, mice receiving C. albicans pre-exposed to CA-LAK cells (E:T = 300:1) had a median survival time of 15 d. Overall, the susceptibility of the C. albicans hyphal form to CA-LAK cells suggests that C. albicans-induced effectors might play a significant role as a second-line defence mechanism against the C. albicans hyphal form.

1991 - Intracerebral transfer of an in vitro established microglial cell line: local induction of a protective state against lethal challenge with Candida albicans. [Articolo su rivista]
Blasi, Elisabetta; Mazzolla, R.; Barluzzi, R.; Mosci, P.; Bartoli, A.; Bistoni, F.
abstract

An in vitro generated BV-2 microglial cell line has been transferred intracerebrally into syngeneic immunocompetent mice prior to local challenge with Candida albicans. The transfer resulted in the establishment of local protection against a lethal dose of C. albicans, which was accompanied by an impairment of yeast growth in the brain and kidneys. Upon histological examination of brain sections from BV-2 cell-pretreated mice, it was found that the size and number of granulomas was reduced as compared to untreated controls receiving Candida alone. These observations provide direct evidence that microglia play a crucial role in the local defense against intracerebral infections.

1991 - Microglial cell-mediated anti-Candida activity: temperature, ions, protein kinase C as crucial elements. [Articolo su rivista]
Blasi, Elisabetta; Mazzolla, R; Barluzzi, R; Bistoni, F.
abstract

An in vitro established microglial cell line, BV-2, constitutively exhibits high levels of anti-Candida activity. To elucidate the cascade of events leading to the accomplishment of such activity, we studied its dependence on temperature and ion availability. The role of protein kinases has also been studied by the specific inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) and N-(2-guanidinoethyl)-5-isoquinoline sulfonamide hydrochloride (HA 1004). We found that (a) the BV-2 cell/Candida conjugate formation is a discrete step, temperature-, ion- and protein kinase-independent; (b) the phagocytic event, which is protein kinase-independent, is significantly impaired by temperature decrease and ion deprivation; (c) the fulfillment of anti-Candida effects is strictly dependent upon temperature, ion availability and functional protein kinase. Functional protein kinase C, but not other kinases, is required for the accomplishment of anti-Candida activity, which, in fact, is selectively abrogated by H7 but not HA. Furthermore, protein kinase C activators, such as 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1-oleoyl-2-acetyl glycerol (OAG), consistently potentiate BV-2 cell-mediated anti-Candida activity, the phenomena being dose-dependent. These results indicate that the multistep events leading a microglial cell to express anti-Candida activity can be dissected and differentiated for biochemical and biological demands, the latest along the cascade being the most demanding steps.

1990 - A rapid objective immunofluorescence microassay application for detection of surface and intracellular antigents [Articolo su rivista]
Pitzurra, L.; Blasi, E.; Bartoli, A.; Marconi, P.; Bistoni, F.
abstract

An indirect immunofluorescence microassay, which permits automated reading, has been employed for simple, rapid and objective detection of surface and intracellular antigens. Initially, the cells, spun in microplates, are fixed with glutaraldehyde (0.25% v/v in PBS). Following fixation, the cells can be stored at 4°C for up to 2 weeks before being used in the immunofluorescence microassay. The fixed cells are then stained according to standard procedures using appropriate first and fluorescein-conjugated second antibodies. An automated and quantitative evaluation of the fluorescence intensity of the cell samples was achieved using the Titertek Fluoroskan II automatic reader. This microassay was shown to be suitable for the detection of the surface MAC1 antigen and intracellular v-myc protein in the GG2EE macrophage cell line. © 1990.

1990 - Augmentation of GG2EE macrophage cell line-mediated anti-Candida activity by gamma interferon, tumor necrosis factor and interleukin 1. [Articolo su rivista]
Blasi, Elisabetta; Farinelli, S.; Varesio, L.; Bistoni, F.
abstract

The expression of anti-Candida activity in the GG2EE macrophage cell line, generated by immortalization of fresh bone marrow with v-raf and v-myc oncogenes, was studied. GG2EE cells spontaneously inhibited the growth of an agerminative mutant of Candida albicans in vitro. The anti-Candida activity was maximal after 8 h of coculture and was proportional to the effector-to-target ratio. Gamma interferon (IFN-gamma), interleukin-1 (IL-1), and tumor necrosis factor (TNF) all significantly enhanced the anti-Candida activity of GG2EE cells. In contrast, IL-3, IL-4, and colony-stimulating factor 1 were ineffective. The augmentation of anti-Candida activity was not always concomitant with enhancement of phagocytosis, since IFN-gamma and colony-stimulating factor 1, but not IL-1 or TNF, augmented the phagocytic ability of GG2EE cells. Furthermore, the augmentation of anti-Candida activity in GG2EE cells did not correlate with the acquisition of antitumor activity. In fact, none of the cytokines alone were able to induce antitumor activity in GG2EE cells, which, however, could be activated to a tumoricidal stage by IFN-gamma plus heat-killed Listeria monocytogenes. These findings demonstrate that GG2EE cells exhibit spontaneous anti-Candida activity and that such activity is enhanced by TNF, IL-1, and IFN-gamma.

1990 - Differential susceptibility of yeast- and hyphal-forms of Candida albicans to various natural effector cells [Articolo su rivista]
Scaringi, L.; Cornacchione, P.; Bietta, C.; Bistoni, F.; Blasi, E.
abstract

1990 - Gamma Interferon‐Induced Specific Binding of Tetanus Toxin on the GG2EE Macrophage Cell Line [Articolo su rivista]
Blasi, E.; Pitzurra, L.; Fuad, A. M. B.; Marconi, P.; Bistoni, F.
abstract

We have recently demonstrated that tetanus toxin (TT) selectively inhibited gamma interferon‐(IFN‐γ)‐induced, but not basal, lysozyme activity in the GG2EE macrophage cell line. By an indirect immunofluorescence technique, we show that tetanus toxin, as well as the BIIb tetanus toxin‐derived fragment, selectively binds to IFN‐γ‐treated cells but fails to bind to control cells. Moreover, BIIb fragment and TT share the same binding sites, as demonstrated by competition/ displacement experiments. These data indicate that IFN‐γ pretreatment results in the acquisition of tetanus toxin binding sites on GG2EE cells by a mechanism(s) yet to be identified. Copyright © 1990, Wiley Blackwell. All rights reserved

1990 - Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus. [Articolo su rivista]
Blasi, Elisabetta; Barluzzi, R.; Bocchini, V.; Mazzolla, R.; Bistoni, F.
abstract

A murine cell line (BV-2) has been generated by infecting primary microglial cell cultures with a v-raf/v-myc oncogene carrying retrovirus (J2). BV-2 cells expressed nonspecific esterase activity, phagocytic ability and lacked peroxidase activity. Such cells secreted lysozyme and, following appropriate stimulation, also interleukin 1 and tumor necrosis factor. Furthermore, BV-2 cells exhibited spontaneous anti-Candida activity and acquired tumoricidal activity upon treatment with interferon-gamma. Phenotypically, BV-2 cells resulted positive for MAC1 and MAC2 antigens, and negative for MAC3, glial fibrillary acidic protein (GFAP) and galactocerebroside (GC) antigens. Since BV-2 cells retain most of the morphological, phenotypical and functional properties described for freshly isolated microglial cells, we can conclude that J2 virus infection has resulted in the immortalization of active microglial cells

1990 - Inhibition of interferon gamma induced lysozyme activity by tetanus toxin in the GG2EE macrophage cell line: role of TT-derived fragments and gangliosides [Articolo su rivista]
Pitzurra, L.; Burham, A. F.; Marconi, P.; Blasi, E.
abstract

1990 - Picolinic acid, a catabolite of tryptophan, as the second signal in the activation of IFN-gamma primed macrophages. [Articolo su rivista]
Varesio, L.; Clayton, M.; Blasi, Elisabetta; Ruffman, R.; Radzioch, D.
abstract

We have studied the effects of picolinic acid, a product of tryptophan degradation, on the activation of mouse peritoneal macrophages (M phi). Picolinic acid acts synergistically with IFN-gamma in activating M phi from C57BL/6 mice. Moreover, M phi from C3H/HeJ mice and C3H/HeN that do not become cytotoxic in response to IFN-gamma alone could be fully activated by exposure to picolinate plus IFN-gamma. These results indicate that picolinic acid is a potent costimulator of M phi activation that functions as a second signal. Inasmuch as we have previously demonstrated that the activation of cytotoxic M phi correlates with specific changes in ribosomal RNA (rRNA), we investigated whether picolinic acid could modify M phi RNA metabolism. Picolinic acid inhibited the synthesis of total M phi RNA, the accumulation of newly synthesized 28S rRNA, and augmented the steady state levels of rRNA precursors (pre-rRNA). These changes in RNA metabolism were similar to those previously described in murine M phi activated in vitro or in vivo to express tumoricidal activity. These results demonstrate that picolinic acid is a potent, biologic M phi second signal, suggest that the changes in rRNA are causally connected with the expression of tumoricidal activity, and suggest the existance of an autocrine effect mediated by picolinic acid.

1989 - Antimicrobial activity of a macrophage cell line generated in vitro with v-raf/v-myc oncogenes [Articolo su rivista]
Blasi, E.; Farinelli, S.; Bartoli, A.; Varesio, L.; Bistoni, F.
abstract

1989 - Generation of macrophage cell line from fresh bone marrow cells with a myc/raf recombinant retrovirus [Articolo su rivista]
Blasi, E.; Radzioch, D.; Merletti, L.; Varesio, L.
abstract

We have studied the effects of infection of fresh murine bone marrow (BM) cells by recombinant retroviruses carrying v-raf and v-myc oncogenes, either alone or in combination. Viruses containing v-raf or v-myc alone failed to induce BM proliferation in 24 out of 27 experiments performed so far, only the J2 virus containing both v-raf and v-myc oncogenes induced BM proliferation. Exogenous growth factors (GF) were not required to sustain the mitogenic effect of J2 virus. Infection with retroviruses carrying only v-raf or v-myc did not induce BM cell growth, indicating that co-expression of the two oncogenes was needed to provide the mitogenic signal(s) for BM proliferation. The kinetics of growth of the J2 virus-infected cells (J2 cells) were characteristically biphasic. The initial burst of proliferation was always followed by a quiescent phase culminating in cell death, which could not be reversed by addition of exogenous GF. In contrast, active proliferation of the quiescent monolayers could be restored by addition of dextran-based beads to the cultures, showing that the growth arrest of J2 cells was a reversible process. J2 cells actively growing in the presence of CT-beads could be expanded and cloned and subsequently grew continuously independent of the CT-beads. Eighteen clones obtained from different infections were all macrophages (M phi) by morphological criteria and all of them expressed the same membrane phenotype compatible with M phi, demonstrating that J2 virus infection leads to immortalization of the same BM-derived monocytic subpopulation. When injected in vivo, J2 cells produced histiocytic tumors in nude mice, but did not grow in immunocompetent syngeneic mice. The cells induced to proliferate in vitro in response to J2 virus infection appeared to be limited to the BM compartment, since spleen cells, thymocytes, peritoneal M phi and liver large granular lymphocytes did not grow in vitro in response to J2 virus. The immortalization of BM cells by J2 virus infection represents a novel reproducible experimental system to deliberately generate M phi lines, which proliferate in response to viral oncogenes and do not require exogenous GF to initiate or to sustain their continuous proliferation.

1989 - Heterogeneity of hematopoietic cells immortalized by v-myc/v-raf recombinant retrovirus infection of bone marrow or fetal liver [Articolo su rivista]
Cox, G. W.; Mathieson, B. J.; Gandino, L.; Blasi, E.; Radzioch, D.; Varesio, L.
abstract

The J2 recombinant retrovirus expressing v-myc/v-raf (also known as MYC/RAF1) immortalized macro-phages from the bone marrow of lipopolysaccharide-responsive mouse strains, producing the ANA-1 cell line from C57BL/6 mice and the INF-3A cell line from C3H/HeN mice. In contrast, J2 recombinant retrovirus infection of the fetal liver from C57BL/6-Ly-5a mice immortalized a cell line (GGD) that did not exhibit the characteristics of mature macrophages. The GGD cell line was classified as leuko-cytic on the basis of its expression of the Ly-6B.2, FcγR, and Ly-5.2 antigens. Our results indicate that the J2 recombinant retrovirus selectively immortalizes macrophages from the bone marrow of C57BL/6 and C3H/HeN mice but immortalizes cells without definitive macrophage characteristics from murine fetal liver under the same culture conditions. [J Natl Cancer Inst 81: 1492-1496, 1989] © 1989 Oxford University Press.

1989 - Protective immunity induced by low-virulence Candida albicans: Cytokine production in the development of the anti-infectious state [Articolo su rivista]
Vecchiarelli, A.; Cenci, E.; Puliti, M.; Blasi, E.; Puccetti, P.; Cassone, A.; Bistoni, F.
abstract

A low-virulence, agerminative strain of Candida albicans (PCA-2) is able to confer a high degree of nonspecific protection against subsequent challenge with highly virulent microorganisms in mice. In an attempt to better define the effect of PCA-2 vaccination on the immune system and the nature of the mechanisms involved in this protective state, we evaluated the pattern and kinetics of production of selected cytokines in PCA-2-treated mice. Thus, granulocyte/monocyte colony-stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin 1 (IL-1) were measured in the sera and spleen cell supernatants of vaccinated mice. In both cases, high levels of CSF, TNF, IL-1, and IFN were found 6 hr after PCA-2 infection and persisted for many days. There was always a correlation between the ability of PCA-2 to induce antimicrobial protection in vivo and its ability to cause cytokine production in vitro. Supernatants of splenocyte cultures from PCA-2-infected animals possessed macrophage-activating activity, as measured in microbiological assays. These data suggest an important involvement of cytokines in the nonspecific anti-infectious immunity induced by PCA-2, and also suggest a crucial role for IL-1 as an endogenous adjuvant in the initiation of the immune response to PCA-2. © 1989.

1989 - Selective inhibition of cytokine-induced lysozyme activity by tetanus toxin in the GG2EE macrophage cell line. [Articolo su rivista]
Pitzurra, L.; Marconi, P.; Bistoni, F.; Blasi, Elisabetta
abstract

This study was designed to evaluate the effects of tetanus toxin (TT) on lysozyme (LZM) activity by the GG2EE macrophage cell line. GG2EE cells spontaneously produced low amounts of LZM, which were mostly secreted into the culture medium. Upon treatment with various cytokines, GG2EE cells exhibited altered LZM activity. In particular, exposure of GG2EE cells to alpha/beta interferon (IFN-alpha/beta) reduced LZM activity, as opposed to treatment with gamma interferon (IFN-gamma) or colony-stimulating factor 1, which potentiated LZM activity. Spontaneous LZM activity of GG2EE cells was not susceptible to TT action; in contrast, when IFN-gamma- or colony-stimulating factor 1-susceptible cells were treated with TT, a significant reduction on LZM activity was observed. The TT inhibitory effect was dose dependent and manifested only after a 6-h incubation of GG2EE cells with TT. Treatment of GG2EE cells with heat-inactivated TT as well as Ibc- and B-IIb-TT-derived fragments was found to be ineffective, while pretreatment with B-IIb- but not with Ibc-TT-derived fragment abrogated the TT effect. Overall, these data indicate the existence of a specific TT-GG2EE cell interaction, leading to selective inhibition of cytokine-induced LZM activity.

1989 - Tumor necrosis factor-alpha secretion by splenocytes of Candida albicans infected mice [Articolo su rivista]
Vecchiarelli, A.; Puliti, M.; Blasi, E.; Bistoni, F.
abstract

1988 - In vitro proliferation of human large granular lymphocytes with v-raf/v-myc recombinant retrovirus. [Articolo su rivista]
Peppoloni, Samuele; Blasi, Elisabetta; Ortaldo, J. R.; Rapp, U. R.; Riccardi, C.; Varesio, L.
abstract

The effect of infection with a retrovirus carrying v-raf/v-myc oncogenes (J2 virus) on the in vitro proliferation of human large granular lymphocytes (LGL) was investigated. LGL infected with J2 virus (J2LGL), unlike uninfected cells, grew with a proliferation peak eight days after infection. Such cells retained the morphology and functional properties typical of LGL. Furthermore, 5% of J2LGL produced virus the day after infection, whereas non-virus production was detectable five days later. These data indicate that J2 virus provides a transient mitogenic signal for LGL.

1988 - Inhibition of retroviral mRNA expression in the murine macrophage cell line GG2EE by biologic response modifiers [Articolo su rivista]
Blasi, Elisabetta; Radzioch, D.; Varesio, L.
abstract

We immortalized the GG2EE macrophage (M phi) cell line by infection of freshly isolated bone marrow cells with the recombinant J2 retrovirus carrying v-raf and v-myc oncogenes. We investigated the expression of J2 virus mRNA in relationship with the proliferative ability and tumoricidal activity of GG2EE cells exposed to biologic response modifiers (BRM). Calcium ionophore (Ca2+I), picolinic acid (PA), or IFN-gamma were employed to activate GG2EE cells. Each BRM was due to inhibit the proliferation of GG2EE cells in a dose-dependent manner, whereas only Ca2+I or the combined treatment with PA plus IFN-gamma induced tumoricidal GG2EE cells. J2 virus mRNA expression was not affected by PA or IFN-gamma, but it was dramatically decreased by Ca2+I or PA plus IFN-gamma. These results indicated that the expression of J2 mRNA can be inhibited in GG2EE cells by appropriate BRM such as Ca2+I or IFN-gamma plus PA. In contrast, the expression of 2'5'-oligoadenylate synthetase mRNA was augmented to similar levels by treatment of the GG2EE cells with IFN-gamma alone or in combination with PA. The down-regulation of J2 mRNA expression was not associated with the antiproliferative activity of the BRM but rather with their ability to induce tumoricidal activity. These results suggest that the process of activation of tumoricidal macrophages also triggers a mechanism(s) of resistance to viral mRNA expression. Moreover, the finding that IFN-gamma or PA inhibit cell proliferation but not J2 mRNA expression indicates that the intracellular targets of these BRM are intact, independent from and unaffected by J2 virus expression.

1988 - Tumor formation by a murine macrophage cell line immortalized in vitro by v-raf and v-myc oncogenes [Articolo su rivista]
Blasi, E.; Varesio, L.; Wiltrout, R. H.
abstract

Murine bone marrow cells immortalized in vitro by the J2 recombinant retrovirus bearing the v-raf and v-myc oncogenes have the functional and phenotypic characteristics of macrophages. The present study was designed to determine whether these cells are tumorigenic in athymic or euthymic mice. One cloned cell line (GG2EE), that had been previously derived and characterized was used for this purpose. The results demonstrated that GG2EE cells were tumorigenic in allogeneic athymic BALB/c mice at doses of 1×104 to 1×107 cells per mouse regardless of the route administration. All mice utlimately died of progressive tumor growth. Conversely, the GG2EE cells were nontumorigenic or transiently tumorigenic in syngeneic euthymic C3H/HeJ mice. Further studies in BALB/c athymic mice demonstrated that the GG2EE cells were directly tumorigenic since ascites tumors (GG2EE-V) that developed expressed the H-2k surface phenotype of the injected GG2EE cells, excluding the possibility that the J2 virus constitutively produced by GG2EE cells caused in vivo transformation and therefore tumors of host cell origin. The in vivo passaged cells continued to express the M1/69, MAC-1, MAC-2, F4/80, Fc receptor and Ly5.1 antigens characterically expressed on the parental line. Biological properties including interferon-γ-induced Ia expression, phagocytosis, and activation for cytotoxicity were also retained following in vivo passage. These results demonstrated that J2 virus-immortalized GG2EE cells were directly tumorigenic in athymic mice in vivo and that the macrophage phenotype was maintained in these neoplastic cells. These observations suggest that this tumor model may be valuable for the study of macrophage function as well as therapeutic approaches to oncogen-expressing retrovirus-induced tumors. © 1988 Springer-Verlag.

1987 - A murine macrophage cell line, immortalized by v-raf and v-myc oncogenes, exhibits normal macrophage differentiated functions [Articolo su rivista]
Blasi, Elisabetta; Radzioch, D.; Durum, S. K.; Varesio, L.
abstract

In vitro immortalized cell lines with the morphology and phenotype of mature macrophages (M phi) have been generated by infecting freshly isolated bone marrow cells from C3H/HeJ mice with a recombinant retrovirus carrying v-raf and v-myc oncogenes. All of the clones obtained had M phi-like phenotypes, and one such clone, GG2EE, has been compared to normal M phi to ascertain the effects of immortalization on the expression of the biological functions of the lines. GG2EE cells expressed cytotoxic activity against L5178Y, P815 or RL male 1 target cells in response to stimulation with interferon-gamma (IFN-gamma) and heat-killed Listeria monocytogenes; in contrast, they failed to kill YAC-1 target cells. GG2EE cells did not constitutively express I-A or I-E antigens; nevertheless, I region-coded antigens could be induced by IFN-gamma treatment. GG2EE cells produced interleukin 1 upon stimulation with a T cell-derived lymphokine; they were weakly phagocytic, yet became highly phagocytic following IFN-gamma treatment. Since c-fos mRNA is augmented in peritoneal exudate M phi by protein kinase C activators but not by IFN-gamma, we evaluated the effects of calcium ionophore, phorbol myristate acetate, L-alpha-1-oleoyl-2-acetoyl-sn-3 glycerol (OAG) and IFN-gamma on the levels of c-fos mRNA in GG2EE cells. We found that calcium ionophore, PMA and OAG stimulation enhanced the expression of c-fos mRNA, but IFN-gamma treatment did not. The kinetics of c-fos induction in GG2EE cells were also comparable to those observed in peritoneal exudate M phi. Overall, the GG2EE cell line has the same biological properties as normal tissue M phi. Because it is capable of both constitutive and inducible M phi-like functions, this cell line provides a valuable tool for studying the molecular mechanisms controlling induction and/or expression of biological activities in M phi. It is striking that a cell line immortalized in vitro by two oncogenes, v-raf and v-myc, behaves, according to the criteria mentioned above, like a normal M phi population.

1987 - Daunomycin and aracytin change c-myc oncogene transcripts in human erythroleukaemic cell line during cellular differentiation [Articolo su rivista]
Tonini, G. P.; Parodi, M. T.; Blasi, E.; Gronberg, A.; Varesio, L.
abstract

1987 - Erythroid differentiation and modulation of c-myc expression induced by antineoplastic drugs in the human leukemic cell line k562 [Articolo su rivista]
Tonini, G. P.; Radzioch, D.; Gronberg, A.; Clayton, M.; Blasi, E.; Benetton, G.; Varesio, L.
abstract

The human leukemia cell line K562 expresses constitutively high levels of c-myc mRNA and can be induced to differentiate along the erythroid lineage. Treatment of K562 cells with the antineoplastic drugs 1-β-d-arabinofuranosylcytosine and daunomycin causes differentiation into hemoglobin-producing cells. The differentiation process is associated with an early block of cellular proliferation occurring during the first 24 h of treatment. RNA synthesis is progressively reduced to 20 to 30% of the control levels after 3 days of exposure to the drugs. Dot and Northern blot analyses were performed to evaluate the levels of c-myc or globin mRNA during the differentiation of K562. Daunomycin and I-β-D-arab-inofuranosylcytosine, despite their distinct chemical nature, induced similar modulation of mRNA levels. Globin mRNA did not change during the first 24 h of culture and began to increase after 48 h of treatment with drugs, reflecting the kinetic of appearance of hemoglobin-producing cells. In contrast, a transient decrease of c-myc mRNA was observed after the first 24 h of drug treatment, followed by a return to normal levels of c-myc mRNA after 48 h of treatment. Thus, the expression of c-myc mRNA in K562 did not reflect their proliferative activity nor their stage of differentiation. We speculate that the transient down-regulation of c-myc mRNA may be an initial event in the erythroid differentiation of K562. © 1987, American Association for Cancer Research. All rights reserved.

1987 - IL 1 induction by murine T cell clones: Detection of an IL 1-inducing lymphokine [Articolo su rivista]
Takacs, L.; Berzofsky, J. A.; York-Jolley, J.; Akahoshi, T.; Blasi, E.; Durum, S. K.
abstract

T cell activation is widely believed to depend on interleukin 1 (IL 1) provided by antigen (Ag)-presenting cells (APC). Because IL 1 is not a constitutive product of APC, we examined the features of its production during the interaction of murine T cell clones and APC. We observed that IL 1 was detectable in supernatants of most myoglobin-specific T cell clones grown in APC and Ag. Two of these T cell clones induced exceptionally high levels of IL 1 in their supernatants, and these same clones demonstrated the unusual restriction to I-E(k), which is a low responder type for sperm whale myoglobin. One of these clones was characterized additionally as to the mechanism of IL 1 induction. This clone rapidly stimulated IL 1 production in the APC population (detectable at 4 hr of co-culture) or in macrophages (MΦ) or a MΦ-like cell line. IL 1 induction was Ag dependent and H-2 restricted. Induction was radioresistant, both on the part of the T cell and of the IL 1 producer. The IL 1-induction process was attributable to a lymphokine produced by the T cell clone. This lymphokine was distinct form IFN-γ, TNF and CSF-1 and may account for a principal mechanism of T→APC signalling. The induced IL 1 was the same in size, co-mitogenicity, and pyrogenicity as lipopolysaccharide-induced IL 1.

1987 - Regulation of bone marrow cell survival in short-term cultures: A new macrophage function [Articolo su rivista]
Blasi, E.; Back, T. C.; Stull, S. W.; Varesio, L.
abstract

The involvement of macrophages (Mφ) in the regulation of bone marrow (BM) cell survival in short-term cultures was studied. We developed a system to measure the survival of fresh BM cells in vitro, by evaluating 111indium (111In) release from prelabeled BM cells. 111In release was proportional to cell death and inversely related to the number of trypan blue excluding cells. Upon 24 hr of culture in conventional medium, more than 50% of BM cells died. In order to investigate whether BM cell death could be reduced by coculture with other cell types, 111In-labeled BM cells were incubated for 24 hr with peritoneal Mφ, thymocytes (THY), or polymorphonuclear cells (PMN) and then assayed for their survival. We found that coculture of BM cells with Mφ dramatically increased BM survival, whereas THY or PMN consistently failed to enhance BM survival. The ability to promote BM cell survival, here designated nurse activity, represented a novel function of Mφ and was further characterized. The stage of activation of Mφ did not influence their nurse activity, since Mφ elicited in vivo by proteose-peptone, thioglycollate, or Bacillus Calmette-Guérin, as well as resident Mφ unstimulated or activated in vitro with lipopolysaccharide, equally sustained survival of BM cells. BM-derived Mø (adherent cells from BM cultures maintained in 20% L-cell-conditioned medium for 14 days) were equally effective in exerting nurse activity. Moreover, nurse activity was also exerted across the histocompatibility barriers. Supernatants from Mφ cultures or killed Mφ were ineffective. We propose that the nurse effect of Mφ on BM is a primitive function that may play an important role in the development of the hemopoietic system. © 1987.

1986 - Comparison of five short-term assays that measure nonspecific cytotoxicity mediated to tumor cells by activated macrophages [Articolo su rivista]
Russell, S. W.; Pace, J. L.; Varesio, L.; Akporiaye, E.; Blasi, E.; Celado, A.; Schreiber, R. D.; Schultz, R. M.; Stevenson, A. P.; Stewart, C. C.
abstract

Five different short term assays (<48 h) used to measure macrophage-mediated, nonspecific cytotoxicity were compared under similar conditions in the same laboratory using the same reagents. The purpose was to determine the extent to which results were comparable. Three of the assays were dependent on the release of a radioisotope to measure cytotoxicity, one was dependent on cell counting, and the last was dependent on flow cytometric quantification of remaining viable tumor target cells after they had been exposed to macrophages. The variables examined were the following: a) three different populations of macrophages; b) four different kinds of target cells; c) two types of radioisotopes; and d) two different agents that trigger the expression of cytolytic activity by primed macrophages. Recombinant gamma interferon was used as the priming agent in all the experiments. There was unexpectedly good agreement between the results of the various assays. No differences were found among the different macrophage populations, the isotopes or the triggering agents. Perhaps the most important finding was that differences in target cell susceptibility to killing by activated macrophages, which were apparent in assays of less than 24 h duration, disappeared when the same kinds of targets were compared in assays of greater than 40 h duration. The results of this study are an important first step toward standardizing the way in which macrophage-mediated, nonspecific cytotoxicity is measured in short-term assays, laboratory to laboratory.

1985 - A recombinant retrovirus carrying MYC and RAF oncogenes selectively transforms bone marrow (BM)-derived macrophages (MO) [Relazione in Atti di Convegno]
Blasi, E.; Rapp, U.; Borchert, P.
abstract

1985 - Generation of murine macrophage (MΦ) cell lines by infection of bone marrow cells (BM) with recombinant retroviruses carrying myc and raf oncogenes [Articolo su rivista]
Blasi, E.; Rapp, U.; Borchert, P.
abstract

1985 - Modulation of polymorphonucleate-mediated cytotoxicity against Candida albicans by thymosin α1 [Articolo su rivista]
Bistoni, F.; Baccarini, M.; Blasi, E.; Riccardi, C.; Marconi, P.; Garaci, E.
abstract

In the present work we analyze the effects of thymosin α1 treatment on the number and the candidacidal activity of murine polymorphonuclear leukocytes. The data we obtained showed that the treatment with thymosin α1 (100 μg/Kg s.c.) 10, 8, 6, 4 and 2 days before the assay may result in a significant numerical augmentation of circulating polymorphonucleates in the peripheral blood, as well as of their candidacidal activity when measured in vitro in both a 4-h cytotoxicity assay and a CFU inhibition assay against Candida albicans microorganisms. On the other hand, a single dose of thymosin α1 (500 μg/Kg s.c.) 3 days before the assay resulted in a significant decrease of the candidacidal activity of mouse polymorphonucleates. The data are discussed with regard to the immunomodulating capacity of thymison α1 and to our previously reported observation concerning the ability of the drug to modulate the resistance against systemic Candida albicans infection.

1985 - Selective immortalization of murine macrophages from fresh bone marrow by a raf/myc recombinant murine retrovirus [Articolo su rivista]
Blasi, Elisabetta; Mathieson, B. J.; Varesio, L.; Cleveland, J. L.; Borchert, P. A.; Rapp, U. R.
abstract

Myeloid precursors can be grown in vitro in the presence of specific growth factors; however, their expansion is limited by a competing process of terminal differentiation. Proto-oncogenes seem to be involved in cellular proliferation and/or differentiation and may also play a role in the myelopoietic process. Murine myeloid precursors which are grown in vitro with growth factors respond with augmented self-renewal upon infection with recombinant retroviruses carrying the v-myc or v-src oncogenes, suggesting a synergism or complementation between some viral oncogenes (v-onc) and certain growth factors. We now show that the combination of two v-onc genes (raf and myc) induces the selective proliferation of monocytic cells from fresh murine bone marrow (BM) in the absence of a specific growth factor supplement. Depending on the culture conditions these cells can either differentiate and cease to proliferate or grow continuously, thus mimicking the alternative pathways that can be followed by committed BM stem cells in vivo.

1985 - The strain of mouse and assay conditions influence whether MuIFN-γ primes or activates macrophages for tumor cell killing [Articolo su rivista]
Pace, J. L.; Varesio, L.; Russell, S. W.; Blasi, E.
abstract

Investigators from two different laboratories have compared several variables in the short-term macrophage-mediated cytotoxicity assays used by each group to study the role of MuIFN-γ in macrophage activation. The findings suggest that the capacity of MuIFN-γ to activate macrophages without the need for a second triggering stimulus is related to assay conditions and, most especially, the strain of mouse used to provide the macrophages.

1984 - Induction of cytotoxic macrophages by γ-interferon (γIFN) is associated with intracellular accumulation of the methyl donor S-adenosyl-methionine (SAM) [Articolo su rivista]
Bonvini, E.; Blasi, E.; Hoffman, T.; Varesio, L.
abstract

1984 - Potent Activation of Mouse Macrophages by Recombinant lnterferon-γ [Articolo su rivista]
Varesio, L.; Blasi, E.; Thurman, G. B.; Wiltrout, R. H.; Herberman, R. B.; Talmadge, J. E.
abstract

The ability of recombinant interferon-γ (IFN-γ ) to activate mouse macrophages was investigated. The use of recombinant IFN-γ has the advantage of being devoid of contaminating lymphokines. Two preparations of IFN-7 were utilized, one which was not glycosylated and which was highly purified from Escherichia coli and another which was glycosylated and which was from transfected COS-7 monkey cells. Both preparations of recombinant IFN-γ activated murine macrophages to kill lymphoma and melanoma tumor targets, suggesting that glycosylate of the protein or the presence of other mammalian proteins is not essential for activation. Significant levels of cytolytic activity were induced from IFN-γ (1 to 10 units/ml). This activity was undiminished by treatment of the IFN-γ preparations with poly-mixin B at doses which neutralized endotoxin (50 μ g/ml). Similarly, IFN-γ, at low concentrations, induced an inhibition of migration by macrophages. Based on antiviral activity, IFN-γ was shown to be 100 to 1000 times more potent than was IFN-β as a macrophage-activating agent. Taken together, these results demonstrate that murine IFN-γ is a macrophage-activating factor which is effective at physiological concentrations. Of particular interest is the observation that the nonglycosylated E. coli-derived IFN-γ is active and therefore may be of value for therapeutic studies, since it can be easily produced in large amounts. © 1984, American Association for Cancer Research. All rights reserved.

1984 - Requirement for protein synthesis for induction of macrophage tumoricidal activity by IFN-α and IFN-β but not by IFN-γ [Articolo su rivista]
Blasi, E.; Herberman, R. B.; Varesio, L.
abstract

Peritoneal mouse macrophages elicited by proteose-peptone (pMΦ) were treated in vitro with IFN-α, IFN-β, or IFN-γ in the presence or absence of cycloheximide (Cy), a reversible inhibitor of protein synthesis, and were assayed for cytolytic activity against tumor cells. Inhibition of protein synthesis during treatment of pMΦ with IFN-α or IFN-β prevented the development of cytotoxic activity. In contrast, IFN-γ was fully capable of inducing cytotoxic pMΦ in the presence of Cy. Moreover, pMΦ treated with mixtures of IFN in the presence of Cy were activated for cytotoxicity only by IFN-γ together with IFN-α or IFN-β, but not by IFN-α plus IFN-β. These results indicate that the activation of pMΦ by IFN-γ is independent of new protein synthesis, whereas the activation of pMΦ by IFN-α and/or IFN-β requires active protein synthesis, suggesting that the mechanism of induction of cytotoxic pMΦ by IFN-γ differs from that by the other types of IFN.

1984 - Role of protein synthesis in the activation of cytotoxic mouse macrophages by lymphokines [Articolo su rivista]
Blasi, E.; Varesio, L.
abstract

The role of protein synthesis during the activation of macrophages (Mφ) by lymphokines (LK) was studied. Peritoneal murine macrophages elicited by proteose-peptone (pMφ) were activated with LK (supernatants from normal mouse spleen cells pulsed with concanavalin A) and tested for cytotoxicity in an 18 hr assay against 111In-labeled L5178Y lymphoma target cells. Reversible (cycloheximide and puromycin) or poorly reversible (emetine and pactamycin) inhibitors of protein synthesis were added during activation, and their effects on pMφ-mediated cytotoxicity and pMφ protein synthesis were measured. Minimal concentrations of inhibitors, reducing the rate of protein synthesis by more than 90% without toxic effects on macrophages, were chosen. Exposure of pMφ to LK for 2 to 18 hr in the presence of reversible inhibitors of protein synthesis did not affect the induction of cytolytic activity, indicating that protein synthesis was not required during the activation period. In contrast, activation of macrophages for 2 hr in the presence of poorly reversible inhibitors of protein synthesis resulted in a considerable reduction of cytolytic activity. The impairment of cytotoxic activity was also evident when pMφ were treated with such drugs during the first 2 hr of an 18 hr exposure to LK or when LK-activated macrophages were treated for 2 hr with the drugs before the addition of the targets. These results demonstrate that active protein synthesis is not required during the exposure of pMφ to LK, but that new proteins have to be synthesized to allow the expression of the cytotoxic activity in LK-activated pMφ. © 1984.

1983 - Comparison between natural reactivity (NR) against Candida albicans and natural killer (NK) activity against YAC-1 tumor cells [Articolo su rivista]
Bistoni, F.; Baccarini, M.; Blasi, E.; Riccardi, C.; Favalli, C.
abstract

Cytotoxic activity against Candida albicans was measured in vitro in a 4-h 51Cr-release assay. The levels of reactivity correlated well with the number of polymorphonuclear cells in the effector population, being augmented by the enrichment of polymorphonuclear granulocutes. To exclude the possible role of contaminating natural killer cells, natural killer activity against tumour cells was compared with natural reactivity against Candida albicans in vitro. The findings indicate that there are many differences between these reactivities including organ and strain distribution, age dependency, adherence to nylon, and susceptibility to modulation by immuno-adjuvants and to treatment with anti-Thy 1.2 antiserum plus complement. These data further define in vitro polymorphonucleate-mediated cytotoxicity against Candida albicans on the basis of the above-mentioned criteria and clearly demonstrate that this in vitro reactivity could not be due to the presence of contaminating natural killer cells in the effector cell population.

1983 - Correlation Between In Vivo and In Vitro Studies ofModulation of Resistance to Experimental Candida albicansInfection by Cyclophosphamide in Mice [Articolo su rivista]
F., Bistoni; M., Baccarini; Blasi, Elisabetta; P., Marconi; P., Puccetti; E., Garaci
abstract

Mice receiving a single injection of cyclophosphamide (150 mg/kg) 1 to 6 days before inoculation with viable Candida albicans showed an increased susceptibility to the challenge accompanied by a reduction in peripheral blood polymorphonuclear leukocytes and lymphocytes as well as in spleen cellularity. Several immunological in vitro functions also appeared to be dramatically depressed. Most of these hematological and functional parameters returned to control valuesby day 9 after cyclophosphamide administration, at a time when resistance to C. albicans infection appeared to be unchanged. However, when exposure to cyclophosphamide occurred 12 to 21 days before inoculation with the live yeast, enhanced resistance was observed with the majority of the animals surviving challenge. To gain some insight into the mechanisms underlying this late increasein resistance to C. albicans infection after cyclophosphamide administration, we analyzed a series of immunological functions, including the in vitro candidacidal activity of polymorphonuclear neutrophils and plastic-adherent and nonadherentspleen cells as well as the activity of natural killer cells and alloreactive Tlymphocytes. The results show that a numerical rebound of blood polymorphonuclear neutrophils and the appearance of a highly candidacidal cell population in the spleen may be among the factors underlying the late increase in resistance to C. albicans after administration of cyclophosphamide.

1983 - Phagocytic killing of candida albicans by different murine effector cells [Articolo su rivista]
Baccarini, M.; Blasi, E.; Puccetti, P.; Bistoni, F.
abstract

Three major phagocytic populations in the mouse were tested in vitro for killing of Candida albicans by means of 51Cr release assay: early inflammatory peritoneal polymorphonuclear cells (PMN), unfractionated or adherent spleen cells and resident peritoneal macrophages (PEC). Considerable candidacidal activity was found in the early inflammatory neutrophil and adherent spleen cell populations. On the contrary, only limited activity was found to be associated with resident peritoneal macrophages. The phagocytic killing apparently involved multiple mechanisms. © 1983 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

1982 - A radiolabel release microassay for phagocytic killing of Candida albicans [Articolo su rivista]
Bistoni, F.; Baccarini, M.; Blasi, E.; Puccetti, P.; Marconi, P.
abstract

The chromium release technique for quantifying intracellular killing of radiolabelled Candida albicans particles was exploited in a microassay in which murine and human phagocytes acted as effectors under peculiarly simple conditions. At appropriate effector: target ratios and with a 4 h incubation, up to 50% specific chromium release could be detected in the supernatant with no need for opsonization or lysis of phagocytes. This simple microassay permits easy-to-perform, simultaneous testing of a variety of different phagocytes even if only available in limited amounts, and provides an objective measurement of intracellular killing of Candida albicans. © 1982.

1981 - Cell wall skeletons of Candida albicans and micrococci as antitumor immunoadjuvants [Articolo su rivista]
Favalli, C.; Baccarini, M.; Blasi, E.; Bevilacqua, R.; Rinaldi, C.; Mastroviti, F.
abstract