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Samuele PEPPOLONI

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

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Pubblicazioni

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 - 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.

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

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

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

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

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

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

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 - 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 - 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 - 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.

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 - 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 - 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 - 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 - 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

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

2015 - CANDIDA ALBICANS HYPHAL DEVELOPMENT AND BIOFILM FORMATION/PERSISTENCE ARE DIFFERENTIALLY AFFECTED BY MOUTHWASHES DEPENDING UPON THEIR COMPOSITION. [Abstract in Atti di Convegno]
Paulone, Simona; Malavasi, G.; Neglia, Rachele Giovanna; Orsi, Carlotta Francesca; Peppoloni, Samuele; Ardizzoni, Andrea; Cermelli, Claudio; Blasi, Elisabetta
abstract

Introduction: Oral candidiasis is a frequent opportunistic fungal infection, occurring especially in susceptible individuals. This pathology, mainly associated with Candida albicans species, may be prevented by a good oral hygiene, the daily use of toothbrush and mouthwashes. Among several virulence factors, C. albicans has the ability to switch from yeast-to-hyphal forms and to produce biofilm, thus contrasting antimicrobial agents and host immune defences as well [1-3]. The aim of this study is to investigate the susceptibility of C. albicans, in terms of growth, hyphal formation and biofilm production/persistence, to mouthwashes with different composition. Materials and Methods: Candida albicans SC5314 and 7 commercial mouthwashes have been employed: 3 with 0.2% chlorhexidine digluconate; 1 with 0.06% chlorhexidine digluconate and 250 ppm F- sodium fluoride; 3 with 125-250ppm Fsodium, amino and/or stannous fluoride. The effects of the mouthwashes on C. albicans were assessed by crystal violet, tetrazolium salt reduction assays and morphological analysis by microscopy. By using four different protocols, combining different concentrations and different contact times, the mouthwashes were tested against: 1) C. albicans growth on Muller-Hinton agar, as assessed by disk diffusion assay; 2) hyphal formation and biofilm production by yeast cells, cultured in RPMI + 10% FBS; 3) early pre-formed (24 h-old) Candida biofilm and 4) mature (48 h-old) Candida biofilm. Results: The highest anti-Candida activity was consistently exhibited by the chlorhexidine digluconate- containing mouthwashes, irrespective of the protocols employed. Morphological and functional impairments occurred and fungal survival/ growth were impaired as well; the effects strictly depended on both the dilution employed and the time of contact. Discussion and Conclusions: Both C. albicans hyphal development and biofilm formation/persistence are affected by mouthwashes, provided that they contain chlorhexidine digluconate. Thus, special attention should be used when choosing mouthwashes for prevention and/or treatment of Candida-associated oral pathologies. 1. Ramage G, et al. Eukaryot Cell 2005; 4: 633-8. 2. Orsi CF, et al. Microb Pathog 2014; 69-70: 20-7 3. Orsi CF, et al. J Biol Regul Homeost Agents 2014; 28: 743-52.

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

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

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

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

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

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

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

2013 - 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 - 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 - Immunogenic Properties of Streptococcus agalactiae FbsA Fragments [Articolo su rivista]
Papasergi, S; Lanza Cariccio, V; Pietrocola, G; Domina, M; D'Aliberti, D; Trunfio, Mg; Signorino, G; Peppoloni, Samuele; Biondo, C; Mancuso, G; Midiri, A; Rindi, S; Teti, G; Speziale, P; Felici, F; Beninati, C.
abstract

Several species of Gram-positive bacteria can avidly bind soluble and surface-associated fibrinogen (Fng), a property that is considered important in the pathogenesis of human infections. To gain insights into the mechanism by which group B Streptococcus (GBS), a frequent neonatal pathogen, interacts with Fng, we have screened two phage displayed genomic GBS libraries. All of the Fng-binding phage clones contained inserts encoding fragments of FbsA, a protein displaying multiple repeats. Since the functional role of this protein is only partially understood, representative fragments were recombinantly expressed and analyzed for Fng binding affinity and ability to induce immune protection against GBS infection. Maternal immunization with 6pGST, a fragment containing five repeats, significantly protected mouse pups against lethal GBS challenge and these protective effects could be recapitulated by administration of anti-6pGST serum from adult animals. Notably, a monoclonal antibody that was capable of neutralizing Fng binding by 6pGST, but not a non-neutralizing antibody, could significantly protect pups against lethal GBS challenge. These data suggest that FbsA-Fng interaction promotes GBS pathogenesis and that blocking such interaction is a viable strategy to prevent or treat GBS infections.

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

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

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

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

2013 - The 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 - 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 - 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 - IDENTIFICATION OF FIBRINOGEN BINDING PROTEIN FRAGMENTS USING GENOMIC PHAGE DISPLAY LIBRARIES OF GROUP B STREPTOCOCCUS [Poster]
Domina, Maria; Lanza Cariccio, Veronica; Papasergi, Salvatore; Mancuso, Giuseppe; Pietrocola, Giampiero; Rindi, Simonetta; Colombari, Bruna; Teti, Giuseppe; Peppoloni, Samuele; Speziale, Pietro; Felici, Franco; Beninati, Concetta
abstract

Obiective: The ability to bind fibrinogen (Fng) is a virulence determinant for a diverse group of bacterial pathogens. Group B streptococcus (GBS) is an important cause of sepsis and invasive infections. The objective of the present work was to obtain novel insights regarding the mechanism of Fng binding by GBS. Methods: We constructed two genomic libraries of GBS using, respectively, the COH-1 and 2603 R/V strains. These libraries were selected using Fng-coated beads as bait. For virulence studies neonatal (24-hour-old) BALB/c mice (Charles River) were inoculated s.c. with a LD90 (60 CFU/pup) of the wild type serotype III GBS strain or with a fbsA defective mutant. Results: After one round of selection of the COH-1 library approximately 10% of the clones strongly reacted against Fng by immunoblot of phage plaques. The percentage of Fng-binding clones rose to 100% after 3 rounds of selection. All reactive clones contained 2-5 repeats of fibrinogen binding protein A (FbsA), a previously described factor shown to mediate adherence and invasion of human epithelial cells. We further show here that FbsA is an important virulence factor, as evidenced by a decreased ability of the fbsA mutant to cause infection. Experiments are underway to characterize the protective activities of FbsA after active immunization with the protein fragments identified by phage display libraries. Moreover we have developed an anti-FbsA neutralizing monoclonal antibody that is being tested for its ability to passively protect neonatal mice from GBS infection. Conclusion:Our data confirm and extend previous studies indicating that FbsA is an important Fng binding factor. Moreover we show that FbsA is an essential virulence factor in vivo.

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 - Protective activity of a protein fragment identified by screening of a phage display genomic library of Streptococcus pneumoniae [Poster]
Cardaci, Angela; Domina, Maria; Lanza Cariccio, Veronica; Galbo, Roberta; Midiri, Angelina; Mandanici, Francesca; Peppoloni, Samuele; Felici, Franco; Teti, Giuseppe; Beninati, Concetta
abstract

Objective: Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The pathogenicity of pneumococci has been attributed to various virulence factors, most of which are located on its surface. The present study was undertaken to identify novel virulence factors and antigens with immunoprotective activity by using a phage display genomic library of Streptococcus pneumoniae. Methods. A lambda phage display library from the D39 S. pneumoniae strain was selected using sera from patients convalescing from invasive pneumococcal disease. For virulence studies, adult BALB/c mice (Charles River) were inoculated i.v. with the wild type D39 strain or with an equal number of CFUs from a mutant lacking spr1875 (Δspr1875). Results. Using one a convalescent serum, we selected from the phage display library an insert encoding for a fragment designated as R4, 161 amino acid in length, whose sequence matched ORF spr1875 D39 genome. This ORF encoded for a putative surface protein with a signal peptide and a LysM domain, which is found in a number of important virulence factors. To evaluate whether the spr1875 protein is involved in pneumococcal pathogenicity we constructed the Δspr1875. Using 7 x 106 CFUs for challenge, all mice inoculated with D39 died in six days, while 80% of the mice challenged with Δspr1875 survived. The R4 fragment was capable of inducing significant, albeit partial, protection in mice inoculated with a wide variety of virulent streptococcal strains. Conclusion. We have identified a novel virulence factor of S. pneumoniae that showed protective activity in a mouse model of systemic pneumococcal disease. Further studies are needed to characterize the biological activities of this antigen.

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 - RUOLO DI PRODOTTI GENICI IDENTIFICATI MEDIANTE LIBRERIE LAMBDA-DISPLAY DA GENOMA COMPLETO DI PNEUMOCOCCO NELL’INTERAZIONE IN VITRO CON CELLULE MICROGLIALI ED EPITELIALI [Poster]
Peppoloni, Samuele; Colombari, Bruna; Manca, Lidia; Teti, Giuseppe; Speziale, Pietro; Felici, Franco; Beninati, Concetta
abstract

Streptococcus pneumoniae è un batterio responsabile di polmoniti, batteriemie, otiti medie e meningiti. In questo studio, mediante l’uso dei mutanti isogenici Δspr0075, Δspr1370 e Δspr1875 del ceppo acapsulato DP1004, abbiamo valutato il ruolo di queste proteine nell’interazione tra pneumococco e cellule microgliali ed epiteliali. Lo screening iniziale di una libreria genomica phage-display di S. pneumoniae con sieri di pazienti infettati ci ha permesso di identificare cloni fagici che portano epitopi B diversi. Tra questi sono stati isolati 6 frammenti antigenici corrispondenti a 3 proteine, mai identificate finora, codificate dalle ORFs spr0075, spr1370 e spr1875 della sequenza genomica del ceppo R6. Mediante modello di infezione in vitro abbiamo saggiato la suscettibilità dei suddetti mutanti alla fagocitosi, e la loro sopravvivenza all’interno delle cellule microgliali BV2. I risultati ottenuti dimostrano che tutti i ceppi sono efficientemente internalizzati; in particolare, i livelli di fagocitosi di Δspr0075 sono più bassi di quelli osservati con gli altri ceppi. Inoltre, la sopravvivenza dei mutanti all’interno della microglia è significativamente diversa: le CFU residue dei ceppi Δspr1370 e Δspr1875 sono rispettivamente più elevate e più basse di quelle osservate con Δspr0075 e DP1004. Mediante indagini al microscopio è stata infine valutata l’interazione dei diversi mutanti di pneumococco con le cellule epiteliali umane BEAS-2B, A549 e HEp-2. In tutti i casi si osserva una significativa riduzione della capacità di aderire alle cellule epiteliali in questione. Nel complesso, questi risultati indicano che le proteine identificate sono coinvolte nell’interazione di pneumococco con la microglia e le cellule epiteliali. In particolare, la proteina codificata dall’ORF spr1875 sembra svolgere un ruolo importante nella virulenza del batterio, in quanto il mutante Δspr1875 è più suscettibile del DP1004 all’attività antimicrobica microglia-mediata; tali evidenze sono in accordo con i risultati ottenuti in un modello murino, in cui il mutante capsulato Δspr1875 risulta essere meno virulento del ceppo wt D39 (Beninati C. et al., XVIII Lancefield International Symposium 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.

2010 - Plasminogen- and fibronectin-binding protein B is involved in the adherence of Streptococcus pneumoniae to human epithelial cells [Articolo su rivista]
S., Papasergi; M., Garibaldi; G., Tuscano; G., Signorino; S., Ricci; Peppoloni, Samuele; I., Pernice; C., Lo Passo; G., Teti; F., Felici; C., Beninati
abstract

Streptococcus pneumoniae is a major cause of morbidity andmortality worldwide. The ability of this bacterium to adhere toepithelial cells is considered as an essential early step in colonization and infection. By screening a whole genome phage display library with sera from infected patients, we previously identified three antigenic fragments matching open reading framespr0075 of the strain R6 genome. This locus encodes for an120-kDa protein, herein referred to as plasminogen- andfibronectin-binding protein B (PfbB), which displays an LPXTGcell wall anchoring motif and six repetitive domains. In thisstudy, by using isogenic pfbB-deleted mutants of the encapsulatedD39 and of the unencapsulated DP1004 type 2 pneumococcalstrains, we show that PfbB is involved in S. pneumoniaeadherence to various epithelial respiratory tract cell lines. Ourdata suggest that PfbB directly mediates bacterial adhesion,because fluorescent beads coated with the recombinant PfbBsp17 fragment (encompassing one of the six repetitive domainsand the C-terminal region) efficiently bound to epithelial cells.Mutants lacking PfbB bound to fibronectin and plasminogenconsiderably less efficiently than wild type bacteria, whereassp17-coated beads specifically bound to both of these substrates.Taken together, our data suggest that, by directly interactingwith fibronectin, PfbB significantly increases the abilityof S. pneumoniae to adhere to human epithelial cells.

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

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

2008 - 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 - 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 - 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 - Safety and immunogencity of an intramuscular Helicobacter pylori vaccine in noninfected volunteers: a phase I study [Articolo su rivista]
Malfertheiner, P.; Schultze, V.; Rosenkranz, B.; Kaufmann, S. H.; Ulrichs, T.; Novicki, D.; Norelli, F.; Contorni, M.; Peppoloni, Samuele; Berti, D.; Tornese, D.; Ganju, J.; Palla, E.; Rappuoli, R.; Scharschmidt, B. F.; Del Giudice, G.
abstract

Introduction: Helicobacter pylori infection is among the most common human infections and the major risk factor for peptic disease and gastric cancer. Immunization with vaccines containing the H pylori vacuolating cytotoxin A (VacA), cytotoxin-associated antigen (CagA), and neutrophil-activating protein(NAP), alone or in combination, have been shown to prevent experimental infection in animals. Aim: We sought to study the safety and immunogenicity of a vaccine consisting of recombinant VacA, CagA, and NAP given intramuscularly with aluminium hydroxide as an adjuvant to noninfected healthy subjects.Methods: This controlled, single-blind Phase I study randomized 57 H pylori-negative volunteers into 7 study arms exploring 2 dosages (10 and 25 g) of each antigen and 3 schedules (0, 1, 2 weeks; 0, 1, 2 months; and 0, 1, 4 months) versus alum controls. All participants were followed for 5 months. Thirty-six subjectsreceived a booster vaccination 18–24 months after the completion of the primary vaccination. Results: Local and systemic adverse reactions were mild and similar in placebo and vaccine recipients on the monthly schedules. All subjects responded to 1 or 2 of the antigens and 86% of all vaccines mounted immunoglobulinG antibody responses to all 3 antigens. Vaccinees exhibitedan antigen-specific cellular response. Vaccination 18–24 months later elicited anamnestic antibody and cellular responses. Conclusions: This intramuscular H pylori vaccine demonstrated satisfactory safety and immunogenicity, produced antigen-specific T-cell memory, and, therefore, warrants further clinical study.

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 - 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 - 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 - Discovery of novel Streptococcus pneumoniae antigens by screening a whole genome lambda-display library [Abstract in Atti di Convegno]
Peppoloni, Samuele; E., Beghetto; N., Gargano; S. Ricci, S.; G., Garufi; M. R., Oggioni; G., Pozzi; F., Felici
abstract

Discovery of novel Streptococcus pneumoniae antigens by screening a whole genome lambda-display library

2006 - Discovery of novel Streptococcus pneumoniae antigens by screening a whole-genome λ-display library [Articolo su rivista]
E., Beghetto; N., Gargano; S., Ricci; G., Garufi; Peppoloni, Samuele; F., Montagnani; M., Oggioni; G., Pozzi; F., Felici
abstract

Streptococcus pneumoniae is a causative agent of otitis media, pneumonia, meningitis and sepsis in humans. For the development of effective vaccines able to prevent pneumococcal infection, characterization of bacterial antigens involved in host immune response is crucial. In order to identify pneumococcal proteins recognized by host antibody response, we created an S. pneumoniae D39 genome library, displayed on lambda bacteriophage. The screening of such a library, with sera either from infected individuals or mice immunized with the S. pneumoniae D39 strain, allowed identification of phage clones carrying S. pneumoniae B-cell epitopes. Epitope-containing fragments within the families of the histidine-triad proteins (PhtE, PhtD), the choline-binding proteins (PspA, CbpD) and zinc metalloproteinase B (ZmpB) were identified. Moreover, library screening also allowed the isolation of phage clones carrying three distinct antigenic regions of a hypothetical pneumococcal protein, encoded by the ORF spr0075 in the R6 strain genome sequence. In this work, Spr0075 is first identified as an expressed S. pneumoniae gene product, having an antigenic function during infection.

2006 - Identificazione di nuovi antigeni proteici di Sterptococcus pneumoniae mediante screening di una libreria genomica lambda display [Abstract in Atti di Convegno]
Peppoloni, Samuele; E., Beghetto; N., Gargano; S., Ricci; G., Garufi; F., Montagnani; M., Oggioni; G., Pozzi; F., Felici
abstract

Identificazione di nuovi antigeni proteici di Sterptococcus pneumonaie mediante screening di una libreria genomica lambda display

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 - Nuovi antigeni pneumococcici e immunità [Abstract in Atti di Convegno]
Peppoloni, Samuele
abstract

Nuovi antigeni pneumococcici e immunità

2005 - Protective immune responses to meningococcal C conjugate vaccine after intranasal immunization of mice with the LTK63 mutant plus chitosan or trimethyl chitosan chloride as novel delivery platform [Articolo su rivista]
B. C., Baudner; J. C., Verhoef; M. M., Giuliani; Peppoloni, Samuele; R., Rappuoli; G., Del Giudice; H. E., Junginger
abstract

Chitosan and its derivative N-trimethyl chitosan chloride (TMC), given as microparticles or powder suspensions, and the non-toxic mucosal adjuvant LTK63, were evaluated for intranasal immunization with the group C meningococcal conjugated vaccine (CRM-MenC). Mice immunized intranasally with CRM-MenC formulated with chitosan or TMC and the LTK63 mutant, showed high titers of serum and mucosal antibodies specific for the MenC polysaccharide. Neither significant differences were observed between microparticle formulations and powder suspensions nor when LTK63 was pre-associated to the delivery system or not. The bactericidal activity measured in serum of mice immunized intranasally with the conjugated vaccine formulated with the delivery systems and the LT mutant was superior to the activity in serum of mice immunized subcutaneously. Importantly, intranasal but not parenteral immunization, induced bactericidal antibodies at the nasal level, when formulated with both delivery system and adjuvant.

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

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 - 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 - Safety and immunogenicity in animals of subunit influenza vaccine given intranasally with mutants of Escherichia coli heat-labile enterotoxin (LT) [Articolo su rivista]
Baudner, Barbara C; Peppoloni, Samuele; Ruggiero, Paolo; Contorni, Mario; Morandi, Maurizio; Pizza, Mariagrazia; Podda, Audino; Rappuoli, Rino; Del Giudice, Giuseppe
abstract

Mucosal delivery of inactivated vaccine against respiratory diseases offers several advantages over the traditional intramuscular injection. This requires the use of a potent adjuvant and/or of delivery systems to obtain strong local and systemic immune responses. Site-directed mutagenesis has allowed the generation of mutants such as LTK63 (Ser to Lys substitution at position 63 in the A subunit) and LTR72 (Ala to Arg at position 72) with abolished or strongly reduced toxicity while still retaining strong mucosal adjuvanticity. Both of them have been used to formulate a subunit influenza vaccine, administered intranasally together with nanoparticles and were shown to be able to potentiate its immunogenicity. These vaccine formulations were totally safe in different animal models. © 2004, Elsevier B.V.

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.

2004 - Therapeutic vaccination against Helicobacter pylori in the beagle dog experimental model: Safety, immunogenicity, and efficacy [Articolo su rivista]
Rossi, G.; Ruggiero, P.; Peppoloni, Samuele; Pancotto, L.; Fortuna, D.; Lauretti, L.; Volpini, G.; Mancianti, S.; Corazza, M.; Taccini, E.; Rappuoli, R.; DEL GIUDICE, G.
abstract

Helicobacter pylori is a gram-negative bacterium that colonizes the human gastric mucosa causing gastritis and peptic ulcer and increasing the risk of gastric cancer. The efficacy of current antibiotic-based therapies can be limited by problems of patient compliance and increasing antibiotic resistance; the vaccine approach can overcome these limits. The present study describes the therapeutic vaccination of experimentally H. pyloriinfectedbeagle dogs, an animal model that reproduces several aspects of the human infection with H. pylori. The vaccine consisted of three recombinant H. pylori antigens, CagA, VacA, and NAP, formulated at different doses (10, 25, or 50 g each) with alum and administered intramuscularly either weekly or monthly. No adverse effects were observed after vaccination and a good immunoglobulin G response was generated against each of the three antigens. Bacterial colonization and gastritis were decreased after the completion of the vaccination cycle, especially in the case of the monthly immunization schedule. In conclusion, therapeutic vaccination in the beagle dog model was safe and immunogenic and was able to limit H. pylori colonization and the related gastric pathology.

2003 - Enhanced mucosal and systemic immune responses to Helicobacter pylori antigens through mucosal priming followed by systemic boosting immunizaztion [Articolo su rivista]
M., Vajdy; M., Singh; M., Ugozzoli; M., Briones; E., Soenawan; L., Cuadra; J., Kazzaz; P., Ruggiero; Peppoloni, Samuele; F., Norelli; G., DEL GIUDICE; D., O'Hagan
abstract

It is estimated that Helicobacter pylori infects the stomachs of over 50% of the world's population and if not treated may cause chronic gastritis, peptic ulcer disease, gastric adenocarcinoma and gastric B-cell lymphoma. The aim of this study was to enhance the mucosal and systemic immune responses against the H. pylori antigens cytotoxin-associated gene A (CagA) and neutrophil-activating protein (NAP), through combinations of mucosal and systemic immunizations in female BALB/c mice. We found that oral or intranasal (i.n.) followed by i.m. immunizations induced significantly higher serum titres against NAP and CagA compared to i.n. alone, oral alone, i.m. alone, i.m. followed by i.n. or i.m. followed by oral immunizations. However, only oral followed by i.m. immunizations induced anti-NAP antibody-secreting cells in the stomach. Moreover, mucosal immunizations alone or in combination with i.m., but not i.m. immunizations alone, induced mucosal immunoglobulin A (IgA) responses in faeces. Any single route or combination of immunization routes with NAP and CagA preferentially induced antigen-specific splenic interleukin-4-secreting cells and far fewer interferon-γ-secreting cells in the spleen. Moreover, i.n. immunizations alone or in combination with i.m. immunizations induced predominantly serum IgG1 and far less serum IgG2a. Importantly, we found that while both i.n. and i.m. recall immunizations induced similar levels of serum antibody responses, mucosal IgA responses in faeces were only achieved through i.n. recall immunization. Collectively, our data show that mucosal followed by systemic immunization significantly enhanced local and systemic immune responses and that i.n. recall immunization is required to induce both mucosal and systemic memory type responses.

2003 - Mutants of Escherichia coli heat-labile enterotoxin as safe and strong adjuvants for intranasal delivery of vaccines. [Articolo su rivista]
Peppoloni, Samuele; P., Ruggiero; M., Contorni; M., Morandi; M., Pizza; R., Rappuoli; A., Podda; G., Del Giudice
abstract

Cholera toxin and Escherichia coli heat-labile enterotoxin are powerful mucosal adjuvants but their high toxicity hampers their use in humans. Site-directed mutagenesis has allowed the generation of several cholera toxin and E. coli heat-labile enterotoxin mutants with abolished or strongly reduced toxicity that still retain strong mucosal adjuvanticity. Among them, LTK63 (Ser to Lys substitution at position 63 in the A subunit) is completely nontoxic and LTR72 (Ala to Arg at position 72) retains a very low residual enzymatic activity. Both of them have been shown to be safe and effective in enhancing the immunogenicity of intranasally coadministered vaccines, also resulting in protective responses in several animal models. Clinical grade preparations of these mutants have now been produced, tested in animals and proven to be totally safe. Indeed, they did not induce any inflammatory event in the respiratory tract nor, more importantly, in the olfactory bulbs and in the meninges. The fully nontoxic LTK63 mutant has now been successfully tested in human volunteers with a trivalent subunit influenza vaccine.

2003 - The quest for a vaccine against Helicobacter pylori: how to move from mouse to man? [Articolo su rivista]
Ruggiero, P.; Peppoloni, Samuele; Rappuoli, R.; DEL GIUDICE, G.
abstract

Several lines of evidence from experimental animal models of infection have clearly demonstrated the feasibility of a prophylactic and therapeutic vaccine against Helicobacter pylori. However, comparatively few clinical studies have been carried out to evaluate whether the positive results obtained in animals can be reproduced in humans. The preliminary results obtained with single component, mucosally delivered vaccines have shown very limited results thus far. Very good immunogenicity and safety profiles are now being obtained with parenterally delivered, aluminium hydroxide-adjuvanted multicomponent candidate vaccines. For sure, better vaccine formulations, better antigen preparation(s), better adjuvants, and better delivery systems have to be designed and tested for safety and immunogenicity. These studies are also needed for deciphering those aspects of the effector immune responses that correlate with protection against H. pylori infection and disease.

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 - New strategies for the prevention and treatment of Helicobacter pylori infection [Articolo su rivista]
Ruggiero, P.; Peppoloni, Samuele; Berti, D.; Rappuoli, R.; DEL GIUDICE, G.
abstract

Helicobacter pylori infects the stomach of > 50% of the human population worldwide, with higher prevalence in the developing countries. A strict correlation between H. pylori infection and gastroduodenal diseases has been demonstrated, including gastritis, peptic ulcer and gastric cancer. Current therapies against H. pylori consist of an antisecretory plus antibiotics. These therapies are effective in 80 - 90% of the cases; presently, no alternative therapies have been shown to give comparable or better results. There are two main reasons for therapy failure: poor compliance, which results in cure discontinuation, and antibiotic resistance. To overcome the drawbacks inherent to any antibiotic therapy, a prophylactic vaccine seems to be the most reasonable approach. Vaccines have been developed based on data obtained in animal models, a number of which are currently in Phase I clinical trials, in some cases giving encouraging data for safety and immunogenicity. In the absence of any immunological correlate of protection against H. pylori, it will be possible to evaluate the efficacy of these vaccines only in large Phase III clinical trials.

2000 - A mucosal vaccine against diphtheria: formulation of cross reacting material (CRM197) of diphtheria toxin with chitosan enhances local and systemic antibody and Th2 response following nasal delivery [Articolo su rivista]
Mcneela, E.; O'Connor, D.; JABBAL GILL, I.; Illum, L.; Davis, S.; Pizza, M.; Peppoloni, Samuele; Rappuoli, R.; Mills, K.
abstract

The development of new generation vaccines against diphtheria is dependent on the identification of antigens and routes of immunization that are capable of stimulating immune responses similar to, or greater than, those obtained with the parenterally-delivered toxoid vaccine, while reducing the adverse effects that have been associated with the traditional vaccine. In this study, we examined the cellular and humoral immune responses in mice generated after both parenteral and mucosal immunizations with cross-reacting material (CRM(197)) of diphtheria toxin. We found that both native and mildly formaldehyde-treated CRM(197) and conventional diphtheria toxoid (DT) induced mixed Th1/Th2 responses and similar levels of anti-DT serum IgG following parenteral immunization. In contrast, CRM(197) preparations were poorly immunogenic when administered intranasally in solution. However, formulation of the antigens with chitosan significantly enhanced their immunogenicity, inducing high levels of antigen-specific IgG, secretory IgA, toxin-neutralizing antibodies and T cell responses, predominately of Th2 subtype. Furthermore, intranasal immunization with CRM(197) and chitosan induced protective antibodies against the toxin in a guinea pig passive challenge model. We also found that priming parenterally with DT in alum and boosting intranasally with CRM(197) was a very effective method of immunization in mice, capable of inducing high levels of anti-DT IgG and neutralizing antibodies in the serum and secretory IgA in the respiratory tract. Our findings suggest that boosting intranasally with CRM(197) antigen may be very effective in adolescents or adults who have previously been parenterally immunized with a conventional diphtheria toxoid vaccine.

2000 - Immunogenicity of the B monomer of Escherichia coli heat-Labile Toxin expressed on the surface of the Streptococcus gordonii [Articolo su rivista]
RICCI, S.; MEDAGLINI, B.; RUSH, C.; MARCELLO, A.; PEPPOLONI, Samuele; MANGANELLI, R.; PALU, G.; POZZI, G.
abstract

The B monomer of the Escherichia coli heat-labile toxin (LTB) was expressed on the surface of the human oral commensal bacterium Streptococcus gordonii. Recombinant bacteria expressing LTB were used to immunize BALB/c mice subcutaneously and intragastrically. The LTB monomer expressed on the streptococcal surface proved to be highly immunogenic, as LTB-specific immunoglobulin G (IgG) serum titers of 140,000 were induced after systemic immunization. Most significantly, these antibodies were capable of neutralizing the enterotoxin in a cell neutralization assay. Following mucosal delivery, antigen-specific IgA antibodies were found in feces and antigen-specific IgG antibodies were found in sera. Analysis of serum IgG subclasses showed a clear predominance of IgG1 when recombinant bacteria were inoculated subcutaneously, while a prevalence of IgG2a was observed upon intragastric delivery, suggesting, in this case, the recruitment of a Th1 type of immune response.

2000 - Physicochemical characterisation of the pertussis vaccine. [Articolo su rivista]
Ceccarini, C.; Contorni, M.; D'Ascenzi, S.; Gallo, E.; Maffei, M.; Mannucci, D.; Marsili, I.; Magagnoli, C.; Peppoloni, Samuele; Rappuoli, R.; Ravenscroft, N.; Ricci, S.
abstract

The characterisation of an acellular pertussis vaccine composed of a genetically modified pertussis toxin, filamentous haemagglutinin and pertactin is described. The three antigens are submitted to a mild treatment with formaldehyde in the presence of lysine before their use in vaccine formulation. Characterisation is performed by amino acid analysis, SDS-PAGE, analytical size exclusion chromatography and, in the case of pertactin, isoelectrofocusing. The effect of some variables on pertactin formaldehyde treatment has been studied by means of isoelectrofocusing and mouse immunogenicity.

1997 - Acellular pertussis vaccines composed of genetically detoxified pertussis toxin induce long-lasting humoral and cellular responses in adults. [Articolo su rivista]
DI TOMMASO, A.; Bartalini, M.; Peppoloni, Samuele; Podda, A.; Rappuoli, R.; DE MAGISTRIS, M.
abstract

New generation pertussis vaccines, containing only purified Bordetella pertussis antigens, have been proven safe, immunogenic and efficacious. They have, however, raised new questions regarding the mechanism of protection from whooping cough and the duration of the immune response following vaccination. In addition to the antibody (Ab) titer, the level of pertussis toxin (PT) neutralizing antibodies may be very important in protection and the role of cell-ediated immunity needs to be defined. We have previously reported the safety and immunogenicity results of two phase I trials in adult volunteers with two acellular pertussis vaccines containing genetically detoxified PT alone or in combination with filamentous hemagglutinin (FHA) and 69K protein. In this work, we present the results of a long term follow-up study of the immune response in the same vaccinees. We evaluated the Ab response, the PT neutralizing titer and the peripheral blood T cell response up to 4 years following vaccination. Our results show that in adults the level of antibodies to PT, FHA and 69K and the PT neutralizing titers slightly decline between 2.5 and 12 months after the last vaccine dose, but they remain high in the following 2-4 years, showing levels 10-100 times higher than pre-vaccination values. The T cell responses were more heterogeneous among vaccinees but they did not show any significant decline throughout the period monitored.

1995 - Acellular pertussis vaccine composed of genetically inactivated pertussis toxin [Articolo su rivista]
Peppoloni, Samuele; Pizza, M.; DE MAGISTRIS, M.; Bartoloni, A.; Rappuoli, R.
abstract

Whooping cough, an acute respiratory disease affecting over sixty million infants, can be prevented by vaccination. The vaccine currently used, composed of killed bacterial cells, however, has been associated with many side effects. An improved vaccine against the disease should contain pertussis toxin (PT), a major virulent factor of Bordetella pertussis (B. pertussis). In order to be included in the vaccine, PT needs to be detoxified and the chemical methods used so far are not completely satisfactory, since they give a product with reduced immunogenicity and possible residual toxicity. To avoid this problem, we have used recombinant DNA technologies to clone the PT gene, express it in bacteria, map the B and T cell epitopes of the molecule and identify the amino acids that are important for the enzymatic activity and toxicity. Based on this information, the gene coding for PT was mutated to produce an inactive protein. This genetically modified PT was non toxic, highly immunogenic and able to protect mice from intracerebral challenge with virulent B. pertussis. The mutant was included as a main component of an acellular pertussis vaccine which has been shown in numerous clinical trials to be more safe and immunogenic than the old cellular vaccine.

1994 - Comparative study of a whole-cell pertussis vaccine and a recombinant acellular pertussis vaccine. [Articolo su rivista]
Podda, A.; CARAPELLA DE LUCA, E.; Contu, B.; Furlan, R.; Maida, A.; Moiraghi, A.; Stramare, D.; Titone, L.; Uxa, F.; DI PISA, F.; Peppoloni, Samuele; Nencioni, L.; RAPPUOLI R., AND THE ITALIAN MULTICENTER GROUP FOR THE STUDY OF RECOMBINANT ACELLULAR PERTUSSIS VACCINE
abstract

The safety and immunogenicity of an acellular pertussis vaccine containing the genetically detoxified pertussis toxin PT-9K/129G, filamentous hemagglutinin, and pertactin, together with diphtheria and tetanus toxoids, were compared with those of a whole-cell pertussis component-diphtheria-tetanus vaccine. Four hundred eighty infants were enrolled into this prospective, multicenter, double-blind study. Each infant was randomly given three doses of one of the two vaccines at 2, 4, and 6 months of age. Both local and systemic adverse reactions, reported within 48 hours and 7 days of each injection, were less frequent after the acellular vaccine than after the whole-cell vaccine. The enzyme-linked immunosorbent assay titers to pertussis toxin, filamentous hemagglutinin, and pertactin, as well as the pertussis toxin-neutralizing titer measured by the Chinese hamster ovary cell assay, were significantly higher after the acellular vaccine was given. Both vaccines induced adequate levels of anti-diphtheria and anti-tetanus antibodies. We conclude that the recombinant acellular pertussis vaccine produces fewer reactions than the whole-cell vaccine and provides a high antibody response against the antigens of Bordetella pertussis involved in bacterial adhesion and systemic toxic effects.

1994 - Probing the structure-activity relationship of E.coli LT-A by site directed mutagenesis [Articolo su rivista]
Pizza, M. G.; Domenighini, M.; Hol, W.; Giannelli, V.; Fontana, M. R.; Giuliani, M. M.; Magagnoli, C.; Peppoloni, Samuele; Manetti, R.; Rappuoli, R.
abstract

Computer analysis of the crystallographic structure of the A subunit of Escherichia coli heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site-directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53-->Glu or Asp, Ser-63-->Lys, Val-97-->Lys, Tyr-104-->Lys or Asp, and Ser-114-->Lys or Glu. This group also included mutations in amino acids such as Arg-7, Glu-110 and Glu-112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu-41-->Phe, Ala-45-->Tyr or Glu, Val-53-->Tyr, Val-60-->Gly, Ser-68-->Pro, His-70-->Pro, Val-97-->Tyr and Ser-114-->Tyr. The third group contained those molecules that maintained a wild-type level of toxicity in spite of the mutations introduced: Arg-54-->Lys or Ala, Tyr-59-->Met, Ser-68-->Lys, Ala-72-->Arg, His or Asp and Arg-192-->Asn. The results provide a further understanding of the structure-function of the active site and new, non-toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.

1993 - Immunogenicity of an acellular pertussis vaccine composed of genetically inactivated pertussis toxin combined with FHA and pertactin in infants and children [Articolo su rivista]
Podda, A.; CARAPELLA DE LUCA, E.; Titone, L.; Casadei, A. M.; Cascio, A.; Bartalini, M.; Volpini, G.; Peppoloni, Samuele; Marsili, I.; Nencioni, L. AND RAPPUOLI R.
abstract

We studied the immunogenicity of an acellular pertussis vaccine composed of genetically detoxified pertussis toxin (PT-9K/129G), filamentous haemagglutinin, and a 69-kilodalton protein, pertactin, in 30 children aged 12 to 24 months and in 80 infants aged 2 to 4 months. A significant increase of the neutralizing titer and of the titers against pertussis toxin, filamentous hemagglutinin, and pertactin, as determined by enzyme-linked immunosorbent assay, was achieved after three doses of vaccine in all the children; a significant increase of these antibody titers was obtained in 100%, 96.1%, 93.5%, and 98.7% of the infants, respectively.

1992 - Acellular pertussis vaccine composed of genetically inactivated pertussis toxin: safety and immunogenicity in 12- to 24- and 2- to 4-month-old children. [Articolo su rivista]
Podda, A.; CARAPELLA DE LUCA, E.; Titone, L.; Casadei, A. M.; Cascio, A.; Peppoloni, Samuele; Volpini, G.; Marsili, I.; Nencioni, L. AND RAPPUOLI R.
abstract

To determine whether a nontoxic derivative of pertussis toxin obtained by recombinant DNA technology, PT-9K/129G, is a good candidate for a new pertussis vaccine, we examined the safety and the immunogenicity in children of a vaccine containing 15 micrograms of PT-9K/129G protein and 0.5 mg of aluminum hydroxide per dose. Fifty-three children 12 to 24 months of age and 21 infants aged 2 to 4 months were injected with two and three doses, respectively. The vaccine did not induce significant local or systemic reactions and elicited an increase of antibody titer in more than 98% of the children. The geometric mean of the toxin-neutralizing titers increased after each dose and was 85 units in children given two doses and 196 units in those given three doses. Two children who had detectable antibody levels before the first immunization had a high response (greater than 320 units) to the first vaccine dose. The findings suggest that PT-9K/129G is a promising antigen to be included in the development of acellular pertussis vaccines.

1991 - Immunostimulation by a partially retro-inverso tuftsin analogue containing Thr 1 (NHCO) (R,S) Lys 2 modification [Articolo su rivista]
Verdini, A. S.; Pinori, M.; Viscomi, G. C.; Pileri, P.; Peppoloni, Samuele; Silvestri, S. AND NENCIONI L.
abstract

The tuftsin retro-inverso analogue H-Thr psi[NHCO](R,S)Lys-Pro-Arg-OH was synthesized through a novel procedure for the high-yield incorporation of isolated retro-inverso bonds into peptide chains and the use of the new Meldrum's acid derivative (CH3)2C(OCO)2CH(CH2)4NHCOCF3 followed by its efficient coupling in solution to trimethylsilylated H-D-Thr(t-Bu)NH2. Closely related peptide impurities were eliminated both from the crude final peptide and the fully protected tetrapeptide amide precursor via ion-exchange and reversed-phase displacement chromatography, respectively. The tuftsin retro-inverso analogue proved to be completely resistant to enzymatic degradation in vitro, either against isolated aminopeptidases or human plasma proteolytic enzymes. When administered either orally or intravenously, it was significantly more active than normal tuftsin in increasing the number of specific antibody secreting cells in spleen of mice immunized with sheep erythrocytes. Furthermore, the analogue exerted an enhanced stimulatory effect on the cytotoxic activity of splenocytes against YAC-1 tumor cells. Finally, retro-inverso-tuftsin was about 10-fold more potent than the native peptide in reducing rat adjuvant arthritis. The resistance of the retro-inverso analogue to peptidases might explain the increased in vivo activities and allows its further immunopharmacological characterization.

1991 - Lymphokine secretion and cytotoxic activity of human CD4+ T cell clones against Bordetella pertussis [Articolo su rivista]
Peppoloni, Samuele; Nencioni, L.; DI TOMMASO, A.; Tagliabue, A.; Parronchi, P.; Romagnani, S.; Rappuoli, R. AND DE MAGISTRIS M. T.
abstract

Human CD4+ T-cell clones specific for pertussis toxin and other Bordetella pertussis antigens have been tested for their cytotoxic activity, lymphokine production, and capacity to induce immunoglobulin synthesis. Clones specific for the S1 subunit of pertussis toxin were cytotoxic for autologous Epstein-Barr virus-transformed B cells, which had been pulsed with the native antigen, the recombinant S1 subunit of pertussis toxin, or synthetic peptides derived from the S1 amino acid sequence. The killing of antigen-pulsed target cells was class II restricted. All of the T-cell clones produced mostly interleukin-2 and gamma interferon and assisted allogeneic B cells in the production of immunoglobulins M and G but not immunoglobulin E. The potential in vivo role of the cytotoxic activity of these clones is discussed.

1991 - Phase I clinical trial of an acellular pertussis vaccine composed of genetically detoxified pertussis toxin combined with FHA and 69 K [Articolo su rivista]
Podda, A.; Nencioni, L.; Marsili, I.; Peppoloni, Samuele; Volpini, G.; Donati, D.; DI TOMMASO, A.; Magistris, M. T. AND RAPPUOLI R.
abstract

An acellular pertussis vaccine composed of genetically detoxified pertussis toxin (PT-9K/129G), filamentous haemagglutinin (FHA) and pertactin (69 kDa protein) was evaluated in adult volunteers, in double blind, versus placebo. No fever was reported in either group. Mild local reactions were reported after injection of both vaccine and placebo. After the first dose a marked increase in antibodies to PT, FHA and 69 kDa protein was seen in vaccinated subjects with the exception of one who responded well to PT and FHA but did not show a humoral response to the 69 kDa protein. All vaccinees acquired cellular immunity against the three antigens. No significant variation was observed in the humoral or cellular responses after the second dose.

1991 - Properties of the pertussis toxin mutant PT-9K/129G after formaldehyde treatment. [Articolo su rivista]
Nencioni, L.; Volpini, G.; Peppoloni, Samuele; Bugnoli, M.; DE MAGISTRIS, M. T.; Marsili, I. AND RAPPUOLI R.
abstract

Formaldehyde treatment is a method routinely used to detoxify diphtheria, tetanus, and pertussis toxins as well as other molecules suitable for vaccine production. To investigate whether chemical detoxification alters the immunological properties of vaccine components, we have treated the pertussis toxin mutant PT-9K/129G with formaldehyde and tested the properties of the resulting molecules. Very low concentrations of formaldehyde stabilize the molecule without affecting the physicochemical and immunological parameters. Increasing doses of formaldehyde abolish the mitogenic and hemagglutinating activities of PT-9K/129G. At the same time, the molecule loses the ability to be recognized by a monoclonal antibody specific for a major protective epitope on the S1 subunit of pertussis toxin and its affinity for anti-pertussis toxin polyclonal antibodies is also reduced. In marked contrast, the ability of PT-9K/129G to be recognized by human T-cell clones is not affected by Formalin treatment. In vivo, the formaldehyde-treated molecules induce amounts of specific antibodies comparable with those of untreated molecules but significantly lower levels of toxin-neutralizing antibodies. Furthermore, the formaldehyde-treated molecules also show a reduced protective activity in the intracerebral challenge assay.

1990 - Metabolic, humoral and cellular responses in adult volunteers immunized with the genetically inactivated pertussis toxin mutant PT-9K/129G [Articolo su rivista]
Podda, A.; Nencioni, L.; DE MAGISTRIS, M. T.; DI TOMMASO, A.; Bossu', P.; Nuti, S.; Pileri, P.; Peppoloni, Samuele; Bugnoli, M.; Ruggiero, P.; Marsili, I.; Derrico, A.; Tagliabue, A. AND RAPPUOLI R.
abstract

PT-9K/129G, a nontoxic mutant of pertussis toxin (PT) obtained by genetic manipulation, has been shown in animal models to be a promising candidate for new vaccines against whooping cough. To assess the safety and the immunogenicity of PT-9K/129G in humans, a pilot study has been performed in adult volunteers. The protein was found to be safe, capable of inducing high titers of toxin-neutralizing antibodies, and capable of generating immunological memory. In fact, vaccination caused an increase of cell-mediated response to PT, PT-9K/129G, S1 subunit, and B oligomer, indicating that memory T cells are induced by the vaccine. Since PT-9K/129G is mitogenic for T lymphocytes in vitro, it was investigated whether this activity is also present in vivo. No variation was observed in the proportion of T cells (CD3+), T helper cells (CD4+), and cytotoxic T cells (CD8+), as well as in that of other lymphoid populations, by FACS analysis. Interestingly, no thorough correlation was found between humoral and cellular responses. In one case, a very high cellular response was present in absence of detectable antibodies, suggesting that the antibody response, which is the only parameter measured in most clinical trials, may not give a complete picture of the response induced by a vaccine.

1989 - A short synthetic peptide fragment of human interleukin-1 beta increases both human and murine natural killer activity [Articolo su rivista]
Peppoloni, Samuele; Bossu', P.; Boraschi, D.; Tagliabue, A.
abstract

The effect of a short synthetic fragment of human interleukin-1 beta (hu IL-1 beta) on natural killer (NK) activity was examined. Peripheral-blood mononuclear cells (PBMC) from normal donors showed a significant increase in NK activity against K562 leukemia cells after preincubation for 18 h with the IL-1 peptide. A similar augmentation was not observed after culturing the cells in the presence of hu IL-1 beta. The increase in tumor cell lysis could not be ascribed to a cytolytic activity of the synthetic fragment on target cells, since the peptide caused no direct lysis of various tumor cell lines. Although the peptide enhanced NK cytotoxicity of PBMC, highly purified large granular lymphocytes were not susceptible to its stimulatory effect. The addition to the cultures of antibodies to human interleukin-2 (hu IL-2) completely blocked the peptide-induced boost of NK cytotoxicity, suggesting that IL-2 is mainly involved in the activation process. The ability of the IL-1 peptide to increase NK activity was further confirmed in vivo in the mouse. Cytotoxicity against YAC-1 lymphoma cells, which was very low in the spleen of untreated BALB/c mice, was in fact significantly increased after a single inoculation of the peptide. These data thus indicate that a short synthetic peptide fragment of hu IL-1 beta is able to increase both human and murine NK activity.

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.

1987 - In vivo resistance of secondary antitumor immune response to cyclophosphamide: effects on T cell subsets. [Articolo su rivista]
Peppoloni, Samuele; Mathieson, B. J.; Herberman, R. B.; Overton, R. W.; Gorelik, E.
abstract

We have analyzed the effects of high doses of cyclophosphamide (Cy) on primary and secondary antitumor immune response against immunogenic (tum-) variants of Lewis lung carcinoma (3LL) treated in vitro with UV light. Normal mice and mice previously immunized with tum- clones wer inoculated i.p. with Cy (200 mg/kg body weight) and 24 h later challenged intrafootpad with tum- or parental 3LL cells. Cy treatment suppressed the primary immune response of normal animals and allowed the growth of tum- cells. In contrast, Cy-treated immune mice rejected the tumor challenge. The in vivo treatment with Cy decreased the total number of lymphoid cells in the spleens, as well as the proportion of B lymphocytes; however, it increased the percentage of both Lyt2+ and L3T4+ lymphocytes. Thus, the immunosuppressive effects of Cy on the primary antitumor response could not be attributed to elimination of major T lymphocyte subpopulations. Although the treatment of immune mice with Cy did not significantly impair their antitumor resistance, nor the proportion of Lyt2+ and L3T4+ lymphocytes in their spleens, the in vitro generation of cytotoxic T lymphocytes (CTL) was markedly reduced. After Cy treatment, the proliferative ability of spleen cells in response to interleukin-2 (IL-2) was substantially impaired. Using monoclonal antibodies to the IL-2 receptor, we found that Cy-treated T lymphocytes failed to fully express the IL-2 receptor following in vitro stimulation with irradiated tumor cells. In line with these findings, the in vitro generation of CTL was not restored by addition of recombinant IL-2 to the cultures. In vivo experiments using purified functional subsets of immune T cells showed that Lyt1+, but not Lyt2+ lymphocytes were able to transfer antitumor immunity in normal irradiated recipients. Therefore, since Ly1+ T lymphocytes were responsible for the antitumor resistance in vivo, the Cy-induced impairment of CTL generation did not affect the ability of immune mice to reject a secondary tumor challenge.

1987 - Lewis lung carcinoma (3LL) cells treated in vitro with ultraviolet radiation show reduced metastatic ability due to an augmented immunogenicity [Articolo su rivista]
Peppoloni, Samuele; Herberman, R. B.; Gorelik, E.
abstract

The metastatic ability of 3LL tumor following in vitro irradiation with ultraviolet (u.v.) light was studied. Tumor cells were exposed to two courses of u.v.-irradiation (3LL X 2u.v. cells) and after two weeks of culture they were inoculated intravenously (i.v.) into syngeneic mice. These cells produced significantly fewer pulmonary metastases than the untreated population. In addition, intrafootpad (i.f.p.) injections of 3LL X 2u.v. cells into immunocompetent animals induced tumors only in 40 per cent of recipients. Interestingly, in normal mice with progressively growing 3LL X 2u.v. tumors, the formation of spontaneous pulmonary metastases was prevented, whereas metastatic foci were observed in 70 per cent of the nude recipients. The metastatic properties of u.v.-treated tumor cells were further analysed by using individual clones with varying immunogenicity. We found that variants with augmented immunogenicity also showed a parallel decrease in metastatic potential. Studies on H-2 antigen expression in different clones revealed that immunogenic and low metastatic variants expressed levels of H-2 antigens higher than the tumorigenic and metastatic clones. Finally, by using cyclophosphamide (Cy) treatment and adoptive transfer of immune spleen cells were able to eradicate macroscopic 3LL pulmonary metastasis. These results demonstrate that the decrease of metastatic ability in u.v.-treated cells was mainly due to an increase in their immunogenicity and H-2 antigen expression.

1986 - Increase in H-2 antigen expression and immunogenicity of BL6 melanoma cells treated with N-methyl-N'-nitronitrosoguanidine [Articolo su rivista]
Gorelik, E.; Peppoloni, Samuele; Overton, R.; Herberman, R. B.
abstract

Treatment of the BL6 melanoma cells in vitro with N-methyl-N'-nitronitrosoguanidine dramatically increased their expression of H-2Kb and H-2Db antigens as well as beta 2-microglobulin but not Class 2 major histocompatibility complex antigens. The treated tumor cells also became immunogenic and were rejected in 70% of syngeneic C57BL/6 recipients, whereas these tumor cells produced progressively growing tumors in 100% of irradiated (550 R) or nude mice. In contrast to the effects of N-methyl-N'-nitronitrosoguanidine treatment, no influence of H-2 antigen expression or tumorigenicity was found when BL6 melanoma cells were treated with 5-azacytidine, phorbol myristate acetate, 5-bromodeoxyuridine, theophylline, or 6-thioguanine. H-2 antigen expression and the tumorigenic properties of 48 individual clones derived from BL6T2 melanoma line and 15 clones from the original BL6 melanoma were investigated. No H-2 antigens were found on the cell surface of the parental BL6 clones, whereas all tum- clones from the BL6T2 line expressed high levels of H-2 antigens. Although four of six tested tum+ clones had high levels of H-2b antigen expression similar to that of tum- clones, they were nonimmunogenic. These data indicate that an increase in major histocompatibility complex antigen expression is essential but not sufficient for the immunogenicity of tumor cells. This conclusion was also supported by the results of interferon treatment of BL6 melanoma cells: this induced an increase in the expression of beta 2-microglobulin and Class 1 H-2b antigens but not an increase in their immunogenicity. Detection of tumor-associated transplantation antigens on the melanoma cells also appeared to be dependent on the level of expression of H-2 antigens. Although tum+ clones grew in normal mice, immune mice were able to prevent the growth of tum+ clones with high levels of H-2 antigens. However, immune mice only partially inhibited the growth of the parental BL6 melanoma or tum+ clones which have low expression of H-2 antigens.

1986 - Induction of highly immunogenic variants of Lewis lung carcinoma tumor by ultraviolet irradiation. [Articolo su rivista]
Peppoloni, Samuele; Herberman, R. B.; Gorelik, E.
abstract

This study was undertaken to determine whether in vitro treatment of Lewis lung carcinoma (3LL) cells with ultraviolet (UV) radiation could increase their immunogenicity. Tumor cells were irradiated with UV light from a germicidal lamp (254 nm; UV-C) at a dose of 720 J/sq m. After 2 weeks of culture, the surviving cell population was cloned by limiting dilution. Cell suspensions of each clone were injected intrafootpad in C57BL/6 mice at a dose of 2.5 X 10(5) cells per mouse. Eighty independent clones were tested. Fifty-one clones showed decreased tumorigenicity and failed to grow in 20 to 95% of immunocompetent mice, whereas they produced tumors in 100% of irradiated (550 R) and athymic nude mice. These clones were designated "tum-" (nontumorigenic) clones. In contrast, all 25 clones selected from the untreated parental 3LL induced progressively growing tumors in 100% of the mice. After two courses of UV treatment, the uncloned 3LL population was rejected in 45% of inoculated mice. Mice rejecting an inoculum of a tum- clone were completely resistant to subsequent challenge with higher doses of the same or unrelated tum- clones. This resistance was fully expressed even after irradiation of immune mice with 550 R. Mice immune to a tum- clone also were able to prevent the growth of various tum+ clones or untreated 3LL tumor cells. When tum- and tum+ clone cells were simultaneously inoculated intrafootpad in opposite legs, rejection of tum- clone resulted also in the prevention of the growth of tum+ clone. Spleen cells of immune mice caused rapid elimination of radiolabeled 3LL tumor cells from the place of their inoculation (intrafootpad) and prevented tumor growth. In an in vitro cytotoxic assay, spleen cells after in vivo and in vitro immunization with tum- clones demonstrated high cytotoxic activity against various tum+ clones and parental 3LL cells, as well as against tum- clones. In addition, parental 3LL tumor cells and tum- cells were similarly able to inhibit cytotoxic activity in the cold target inhibition assay. However, in contrast to tum- cells, 3LL cells were less efficient in in vitro restimulation of cytotoxic activity of immune spleen cells. Therefore, these data suggest that tum-, tum+, and parental 3LL cells share a common antigenic specificity, which is not immunogenic in 3LL cells. UV treatment presumably converted the antigenic determinants present in the 3LL cells into an immunogenic form.