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Claudio CERMELLI

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

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


2021 - In vitro virucidal efficacy of a dry steam disinfection system against Human Coronavirus, Human Influenza Virus, and Echovirus [Articolo su rivista]
Marchesi, Isabella; Sala, Arianna; Frezza, Giuseppina; Paduano, Stefania; Turchi, Sara; Bargellini, Annalisa; Borella, Paola; Cermelli, Claudio
abstract

This in vitro study was aimed to assess the efficacy of dry steam in inactivating Human Coronavirus OC43 (HCoV-OC43) as surrogate of SARS-CoV-2, Human Influenza Virus A/H1N1/ WSN/33 and Echovirus 7 on stainless steel, polypropylene, and cotton. The virus models were chosen on the basis of their transmission route and environmental resistance. Tests were carried out under a laminar flow cabinet, where two panels of each material were contaminated with a viral suspension. The inocula were left to dry and then the virus on untreated panel (control) was collected by swabbing in order to determine the initial titer. The other panel was treated using a professional vacuum cleaner equipped with a dry steam generator. Dry steam is generated in a boiler where tap water is heated up to 155 °C at 5.5 bar pressure and then during the passage along the flexible hose the temperature decreases to a value between 100 C and 110 C at the output. The dry steam was applied for four sec with a window wiper on metal and plastic panels or a brush covered by a microfiber cap on cotton, simulating the steam application during routine cleaning. After the treatment, infectious virus possibly remained on the surface was collected following the same swabbing procedure applied for controls. HCoV-OC43 and Echovirus 7 were titrated by end-point method on HCT-8 line cells and Vero cells, respectively, while Human Influenza Virus was quantified by plaque reduction assay on MDCK cells. Dry steam resulted effective against the three viruses on all tested materials, achieving a mean Log10 reduction factor >=4 in viral titer of treated samples compared with controls according to UNI EN 14476:2019. Thus, dry steam may be proposed as an ease to use, effective, fast, and nontoxic alternative to chemicals for surface disinfection without damaging materials. Therefore, this device could be employed not only in healthcare facilities but also in occupational, domestic, and community settings, with advantages for environment and human health.


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 long-standing history of Corynebacterium parvum, immunity, and viruses [Articolo su rivista]
Palmieri, B.; Vadala, M.; Roncati, L.; Garelli, A.; Scandone, F.; Bondi, M.; Cermelli, C.
abstract

We report a review of all the experimental and clinical studies performed in the last 60 years on the antiviral activity of inactivated Corynebacterium parvum (Cutibacterium acnes). This bacterium has been originally investigated and used for its oncolytic properties linked to immunomodulating activity, but the interest to successfully prevent and treat bacterial, fungal, and viral infections and lethality, uprising the innate immunity barriers produced many experimental models and very few clinical studies. The dramatic defenseless situation due to impending CoViD-19 pandemic claims to exhume and highlight this aspecific strategy in preventive and therapeutic settings; as a matter of fact, no new or mutated virus can potentially escape to this strong innate immune surveillance strengthened by adequate C. parvum protocols.


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

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


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

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


2017 - Apoptosis and inflammatory response in human astrocytes are induced by a transmissible cytotoxic agent of neurological origin [Articolo su rivista]
Beretti, Francesca; Ardizzoni, Andrea; Cermelli, Claudio; Guida, Marianna; Maraldi, Tullia; Pietrosemoli, P; Paulone, Simona; De Pol, Anto; Blasi, Elisabetta; Portolani, Marinella
abstract

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


2017 - Herpes Simplex Virus-1 entrapped in Candida albicans biofilm displays decreased sensitivity to antivirals and UVA1 laser treatment [Articolo su rivista]
Ascione, Cristian; Sala, Arianna; Mazaheri-Tehrani, Elham; Paulone, Simona; Palmieri, Beniamino; Blasi, Elisabetta; Cermelli, Claudio
abstract

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


2017 - 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 - Chronic and recurrent benign lymphadenopathy without constitutional symptoms associated with human herpesvirus-6B reactivation [Articolo su rivista]
Forghieri, Fabio; Luppi, Mario; Barozzi, Patrizia; Riva, Giovanni; Monica, Morselli; Bigliardi, Sara; Quadrelli, Chiara; Vallerini, Daniela; Maccaferri, Monica; Coluccio, Valeria; Paolini, Ambra; Colaci, Elisabetta; Goretta, Bonacorsi; Maiorana, Antonino; Sara, Tagliazucchi; Fabio, Rumpianesi; Mattioli, Francesco; Presutti, Livio; Gelmini, Roberta; Cermelli, Claudio; Giulio, Rossi; Patrizia, Comoli; Marasca, Roberto; Narni, Franco; Potenza, Leonardo
abstract

Chronic/recurrent behaviour may be encountered in some distinct atypical or malignant lymphoproliferations, while recurrences are not generally observed in reactive/benign lymphadenopathies. We retrospectively anal- ysed a consecutive series of 486 human immunodeficiency virus-negative adults, who underwent lymphadenectomy. Neoplastic and benign/reactive histopathological pictures were documented in 299 (61 5%) and 187 (38 5%) cases, respectively. Of note, seven of the 111 (6 3%) patients with benign lymphadenopathy without well-defined aetiology, showed chronic/ recurrent behaviour, without constitutional symptoms. Enlarged lymph nodes were round in shape and hypoechoic, mimicking lymphoma. Reac- tive follicular hyperplasia and paracortical expansion were observed. Human herpesvirus (HHV)-6B positive staining in follicular dendritic cells (FDCs) was documented in all seven patients. Serological, molecular and immunological examinations suggested HHV-6B reactivation. Among the remaining 104 cases with reactive lymphoid hyperplasia in the absence of well-known aetiology and without recurrences, positivity for HHV-6B on FDCs was found in three cases, whereas in seven further patients, a scanty positivity was documented in rare, scattered cells in inter-follicular regions. Immunohistochemistry for HHV-6A and HHV-6B was invariably negative on 134 lymph nodes, with either benign pictures with known aetiology or malignant lymphoproliferative disorders, tested as further controls. Future studies are warranted to investigate a potential association between HHV- 6B reactivation and chronic/recurrent benign lymphadenopathy.


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.


2014 - Herpes simplex virus-1 entrapped in Candida albicans biofilm displays decreased sensitivity to antivirals. [Poster]
Cermelli, Claudio; MAZAHERI TEHRANI, Elham; Sala, A.; Orsi, Carlotta Francesca; Neglia, Rachele Giovanna; Blasi, Elisabetta
abstract

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


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

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


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

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


2012 - Chronic/relapsing lymphadenopathy associated with HHV-6B infection: a new benign clinico-pathologic entity occurring in immunocompetent individuals [Abstract in Rivista]
Forghieri, Fabio; Potenza, Leonardo; Barozzi, Patrizia; Vallerini, Daniela; Riva, Giovanni; Zanetti, E; Quadrelli, C; Morselli, M; Leonardi, G; Maccaferri, M; Paolini, Ambra; Coluccio, Valeria; Colaci, Elisabetta; Pedrazzi, Letizia; Fantuzzi, Valeria; Bigliardi, Sara; Soci, Francesco; Bonacorsi, G; Zaldini, P; Rossi, G; Milani, M; Rivasi, Francesco; Gennari, W; Pecorari, M; Grottola, Antonella; Tagliazucchi, S; Rumpianesi, F; Mattioli, F; Presutti, Livio; Franzoni, Chiara; Gelmini, Roberta; Saviano, Massimo; Cermelli, Claudio; Marasca, Roberto; Narni, Franco; Luppi, Mario
abstract

Background. HHV-6 DNA sequences were disclosed in lymph node (LN) tis- sues of several patients with lymphoid malignancies, but a direct major role of HHV-6 in lymphoid malignant transformation has so far not been confirmed. In contrast, active HHV-6 infection has been associated to either infectious mononucleosis-like syndrome or acute lymphadenitis occurring in febrilepatients with systemic symptoms, or to Rosai-Dorfman disease in which viral antigens have been detected by immunohistochimical (IHC) analyses in both histiocytes and follicular dendritic cells (FDCs). Methods. We have retrospec- tively analyzed clinical and pathological data of 365 adult patients, consecutive- ly observed at our Institution over a period of 5 years (2006-2010), because of enlarged superficial lymph nodes and subsequently undergoing lymphadenec- tomy. In the benign/reactive cases in which well-recognized etiologies have been excluded, an involvement of HHV-6 active infection or reactivation was investigated by molecular and immunohistochemical examinations. Results. Malignant disorders, namely malignant lymphoproliferative disorders or solid cancer metastases, were found in 227 cases (62%), whereas in 138 cases (38%) benign/reactive pictures were documented on lymph node examination. Among these latter cases, a well-recognized etiology was demonstrated in 84 patients (61%), while in 54 cases (39%), a well-defined non-malignant reactive/infectious cause could not be documented. Immunohistochemical analyses resulted negative for both HHV-6A and HHV-6B in 38 of these latter lymph nodes (70%). In 7 patients (13%), a scattered, scanty and aspecific pos- itivity for HHV-6B late protein was documented in rare interfollicular plasma cells and histiocytes. Surprisingly, in 9 patients (17%), immunohistochemical analyses showed HHV-6B positive staining of FDCs, together with scattered positivity of interfollicular cells. These 9 HIV-negative adult patients (median age 42 years, range 18-76 years), with either localized or generalized LAP, were observed for a median follow-up of 38 months (range 28-166). Of note, six of them presented with recurrent LAP (one to 3 recurrences), without evolving into lymphoma. A common LN histological pattern at presentation showed florid fol- licular hyperplasia with concurrent mild paracortical expansion. Three cases also showed features consistent with PTGC. Constitutional symptoms were absent in all patients. The IHC reactions for both HHV-6A and HHV-6B, per- formed on further control cases, represented by 131 LN tissues from patients with either benign LAP induced by other known etiologies or lymphoma, were invariably negative. Serology was positive for both IgM and IgG with high avid- ity suggesting viral reactivation/reinfection. However, the molecular analyses failed to detect HHV-6 viremias in cell-free-serum samples of all the 9 patients with positive HHV-6B IHC staining, while positivity for HHV-6B DNA was dis- closed by PCR analyses in 7 out of the 7 LN tissues studied. Conclusions. We show for the first time that local reactivation/infection of HHV-6B should be con- sidered among the possible causes of chronic/relapsing benign LAP in immuno- competent individuals. IHC is the method of choice for investigating the pres- ence of HHV-6 infection in such cases. HHV-6B may indirectly modulate and trigger the proliferation of lymphocytes, by locally affecting FDCs and LN microenvironment. FDCs may indeed be involved in presenting HHV-6B anti- gens to other immune cells, mainly cortical B lymphocytes.


2012 - In vitro interactions between viruses and Candida biofilm. [Poster]
Mazaheri, E; Sala, A.; Orsi, Carlotta Francesca; Neglia, Rachele Giovanna; Blasi, Elisabetta; Cermelli, Claudio
abstract

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


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

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


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

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


2010 - Funghi e virus nelle infezioni miste: storie di sinergie e di contrasti [Abstract in Atti di Convegno]
Cermelli, Claudio
abstract

I casi clinici di infezioni polimicrobiche riportati in letteratura sono in continuo aumento, da un lato per il sempre più alto numero di individui suscettibili, dall’altro per il significativo incremento nella sensibilità delle tecniche di indagine, che consentono di diagnosticare casi prima non rilevabili. Gli eventi biomolecolari che caratterizzano l’interazione patogeno-patogeno, consentendo l’instaurarsi di una infezione mista ed eventualmente influenzando il suo decorso, rimangono scarsamente studiati, soprattutto quando sono coinvolti microrganismi filogeneticamente distanti. Il macrofago ha un ruolo fondamentale nella difesa contro i microrganismi, sia come effettore antimicrobico diretto, sia come elemento in grado di polarizzare la risposta immunitaria adattativa. I meccanismi che stanno alla base della reattività macrofagica nel corso di infezioni singole sono stati oggetto di studi approfonditi; diversamente, le possibili conseguenze di un’infezione mista sul macrofago e le eventuali interazioni, sinergistiche o antagonistiche, tra patogeni notoriamente macrofagotropi costituiscono un campo di indagine ancora scarsamente esplorato. Da alcuni anni, il nostro gruppo di ricerca indaga questo aspetto, con l’obiettivo di meglio comprendere i meccanismi che controllano la patogenesi delle infezioni miste da miceti e virus e la concomitante immunoreattività dell’ospite. In particolare, è stato messo a punto ed impiegato un modello in vitro di infezione doppia in cui macrofagi umani sono stati dapprima esposti ad un Herpesvirus (Herpes Simplex Virus-1, virus di Epstein-Barr, Herpesvirus Umano-6) e poi infettati con funghi opportunisti patogeni (Cryptococcus neoformans oppure Candida albicans). Successivamente, i macrofagi così trattati sono stati valutati per reattività antifungina, in termini di fagocitosi, killing microbico e pattern di secrezione, così come per espressione di molecole di superficie e per profilo d’espressione genica. I dati ottenuti finora dimostrano una profonda alterazione dell’immunoreattività macrofagica in risposta all’infezione da Herpesvirus, con un aumento della fagocitosi associato ad una diminuzione del killing verso il fungo. Anche l’espressione genica e i dati relativi al pattern di secrezione citochinica e delle molecole di superficie indicano una condizione di iporesponsività da parte del macrofago esposto ad Herpesvirus. Questi dati in vitro dimostrano un’interazione sinergica tra patogeni a svantaggio dell’ospite, suggerendo che nel paziente un’infezione erpetica attiva possa rappresentare un ulteriore fattore di rischio nei riguardi delle micosi opportunistiche.


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

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


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

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


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

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


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

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


2009 - Growth of Epstein-Barr Virus on monocytes and its effects on immune functions. [Abstract in Atti di Convegno]
Cuoghi, A.; Orsi, Carlotta Francesca; Blasi, Elisabetta; Cermelli, Claudio
abstract

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


2009 - “Microbiologia medica” Volume 3 della nuova edizione italiana sulla settima edizione di Prescott, Harley and Klein’s “Microbiology” . [Traduzione di Libro]
Neglia, Rachele Giovanna; Cermelli, Claudio
abstract

Manuale di Microbiologia medica


2008 - EFFECT OF EUCALYPTUS ESSENTIAL OIL ON RESPIRATORY BACTERIA AND VIRUSES [Articolo su rivista]
Cermelli, Claudio; Anna, Fabio; Fabio, Giuliana; Quaglio, Giampaola
abstract

The activity of Eucalyptus globulus essentialoil was determined for 120 isolates of Streptococcus pyogenes,20 isolates of S. pneumoniae, 40 isolates of S.agalactiae, 20 isolates of Staphylococcus aureus, 40 isolatesof Haemophilus influenzae, 30 isolates of H.parainfluenzae, 10 isolates of Klebsiella pneumoniae, 10isolates of Stenotrophomonas maltophilia and two viruses,a strain of adenovirus and a strain of mumps virus, allobtained from clinical specimens of patients with respiratorytract infections. The cytotoxicity was evaluated onVERO cells by the MTT test. The antibacterial activity wasevaluated by the Kirby Bauer paper method, minimuminhibitory concentration, and minimum bactericidal concentration.H. influenzae, parainfluenzae, and S.maltophilia were the most susceptible, followed by S.pneumoniae. The antiviral activity, assessed by means ofvirus yield experiments titered by the end-point dilutionmethod for adenovirus, and by plaque reduction assay formumps virus, disclosed only a mild activity on mumpsvirus.


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

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


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

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


2008 - Relation between psychoneuroendocrine profile in stressful conditions and antibodies to herpesvirus 6 and 7 [Articolo su rivista]
R., Vivoli; Rovesti, Sergio; Borella, Paola; Cermelli, Claudio
abstract

The aim of the present study is to evaluate whether the individual susceptibility to infectious disease is influenced by the psychological profile of cadets exposed to stressful events associated with military lifestyle in academy and if the neuroendocrine responses to stressful events is related with humoral immunity estimated by measuring antibody titres to human herpesvirus (HHV-6)7 (HHV-7) and to what extent it is influenced by personality traits. It has been observed that cadets with lower psychoaptitude scores (1-2) have a significant higher susceptibility to infectious disease (x2=7.95; p=0.019) compared to subjects with higher scores. A positive relationship between cortisol and antibody titers to HHV-6 (r=0.304; p=0.024) it has been found. It can be interesting to observe that antibody titers on HHV-6 are also related to psychoaptitude profile (r=0.239; p=0.044). The antibody titers to HHV-7 are negatively related to the 5 scales of BFQ and in particular with subdimension Co (cordiality) of BFQ (r=0.401; p=0.002). The survey carried out on over 1,500 cadets of the Military Academy of Modena shows that the susceptibility to infectious diseases during the first six months of admission to the Academy seem to be influenced by the psychoaptitudinal profile. The finding of a positive relationship between serum cortisol and antibody vs HHV-6 suggests that the impairment of the immune system linked to circulatory cortisol levels may induce a reactivation of a latent herpesvirus 6 with related increase of antibody titers.


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

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


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

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


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

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


2007 - Encephaloradiculomyelitis associated to HHV-7 and CMV co-infection in immunocompetent host [Articolo su rivista]
Ginanneschi, F; Donati, D; Moschettini, D; Dominici, F; Cermelli, Claudio; Rossi, A.
abstract

An active co-infection with CMV and HHV-7 has been never described in immunocompetent patients. The authors describe a case of encephaloradiculomyelitis in an immunocompetent man. Polymerase chain reaction (PCR) performed on cerebrospinal fluid (CSF) showed positivity for DNA of Cytomegalovirus (CMV) and Herpes-virus type 7 (HHV-7), whereas the same test applied on peripheral blood mononuclear cells gave negative result. These results are highly supportive of an infection of the central and peripheral nervous systems, caused by CMV and HHV7. Such viral co-infection has only been described in immune-depressed patients with CMV disease, in which HHV-7 was supposed to act as a cofactor, enhancing clinical manifestations. The same mechanism is presumably responsible for the development of encephaloradiculomyelitis clinical signs in the present case. This is the second case in which DNA of HHV-7 has been found in the CSF of an adult immunocompetent patient. This novel observation suggests that the search for viral DNA in the CSF should be performed also in immunocompetent patients. (c) 2006 Published by Elsevier B.V.


2007 - HSV-1 induces dysregulation of monocyte anticandida functions [Abstract in Atti di Convegno]
Cermelli, Claudio; Orsi, Carlotta Francesca; Fantoni, L.; Lugli, E.; Blasi, Elisabetta
abstract

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


2007 - Screening of the antibacterial effects of a variety of essential oils on the microrganims responsible for respiratory infections [Articolo su rivista]
Fabio, A; Cermelli, Claudio; Fabio, Giuliana; Nicoletti, P; Quaglio, Giampaola
abstract

The aim of this study was to examine the cytotoxicity and the antibacterial effects of a variety of essential oils on major respiratory tract pathogens. The cytotoxicity of 13 essential oils was evaluated on Vero cells. The antibacterial activity was evaluated by the Kirby Bauer paper method, minimum inhibitory and bactericidal concentration against Streptococcus pyogenes, agalactiae, pneumoniae and Klebsiella pneumoniae, Haemophilus influenzae, Staphylococcus aureus and Stenotrophomonas maltophilia isolated from clinical specimens. The antibiotic sensitivity of these isolates was examined. Some oils showed inhibition of bacterial growth against most of the organisms examined. Cinnamon and thyme showed the strongest action followed by clove. The results reported in this paper indicate that thyme can be considered as a potential antimicrobial agent for the treatment of some respiratory tract infections in man.


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

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


2006 - Beretti F., Cermelli C., Cenacchi V., Orsi C., Blasi E., Portolani M. [Abstract in Atti di Convegno]
Beretti, Francesca; Cermelli, Claudio; Cenacchi, V.; Orsi, Carlotta Francesca; Blasi, Elisabetta; Portolani, Marinella
abstract

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


2006 - Case report: primary infection by human herpesvirus 6 variant a with the onset of myelitis. [Articolo su rivista]
Portolani, Marinella; M., Pecorari; W. F., Gennari; Beretti, Francesca; Sabbatini, Anna Maria Teresa; Casolari, Chiara; F., Rumpianesi; Cermelli, Claudio; M., Santini; F., Mori
abstract

A case of primary infection by human herpesvirus 6 (HHV-6) variant A in a 54-year-old woman, which occurred at the same time as the onset of encephalomyelitis, is reported. The correlation between the two events is discussed. It is speculated that, during the early phase of the infection, the HHV-6 spread to the central nervous system and triggered a pathogenic process that initially developed without symptoms. When the neurological disorders appeared, HHV-6 had already established a latent state: only the virus carried by infected blood cells was detected in the cerebrospinal fluid.


2006 - HSV-1 induces macrophage activation and dysregulation in monocytes [Abstract in Atti di Convegno]
Cermelli, Claudio; Cenacchi, V.; Beretti, Francesca; Blasi, Elisabetta
abstract

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


2006 - Human herpesvirus 6 (HHV-6) U94/REP protein inhibits betaherpesvirus replication [Articolo su rivista]
Caselli, E; Bracci, A; Galvan, M; Boni, M; Rotola, A; Bergamini, C; Cermelli, Claudio; Dal Monte, P; Gompels, Ua; Cassai, E; Di Luca, D.
abstract

Human herpesvirus 6 (HHV-6) is the only human herpesvirus encoding U94/rep, homologue to the parvovirus non-structural gene rep68/78. Results to date suggest that HHV-6 U94/rep might regulate viral gene expression and have a role in viral latency. To determine the effect of U94/ REP upon viral replication, the protein was produced. The purified U94/REP retained the characteristic immunological features. It was internalized and localized in the nucleus of human cells, showing marked inhibitory activity on the replication of HHV-6 (both variants A and 13). The effect of U94/REP was dose-dependent and sensitive to treatment with single-stranded but not double-stranded DNA. U94/REP inhibited the replication of other betaherpesviruses, HHV-7 and human cytomegalovirus, but had no effect on herpes simplex virus. These results confirm the action of U94/rep latency gene in the regulation of HHV-6 replication with implications for co-reactivations and latency of human betaherpesviruses.


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

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


2006 - Human herpesvirus-6 dysregulates monocyte-mediated anticryptococcal defences [Articolo su rivista]
Cermelli, Claudio; Cenacchi, Valeria; Beretti, Francesca; Pezzini, Francesco; D., Di Luca; Blasi, Elisabetta
abstract

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


2006 - Human herpesvirus-6B active infection associated with relapsing bilateral anterior optic neuritis [Articolo su rivista]
Moschettini, D; Franceschini, R; Vaccaro, Nm; Cermelli, Claudio; Pezzini, Francesco; Balestrieri, M; Cerase, A; Bartalini, S; Ulivelli, M; Tosi, Gm; Donati, D.
abstract

Background and objectives: Human herpesvirus-6 (HHV-6) is the causative agent of exanthem subitum. Both HHV-6 variants, A and B, have been associated with central nervous system (CNS) diseases, suggesting a wide neuropathogenic potential. We describe a case of recurrent bilateral anterior optic neuritis with HHV-6 active infection associated with clinical relapses. Case report: A 23-year old woman presented with progressive visual impairment, bilateral papillitis and painful ocular movements. Nested polymerase chain reaction (PCR) for DNA viruses, HHV-6 variant specific real time quantitative PCR, serological analysis and retrotranscription PCR (RT-PCR) for HHV-6 mRNA transcripts were performed. Nested PCR in PBMC and CSF samples was negative for all viruses but positive for HHV-6 DNA, subtyped as HHV-6B. The disease had a relapsing/remitting course. During relapses PBMC samples remained positive for HHV-6 DNA, and HHV-6 active infection was confirmed by the presence of anti-HHV-6 IgM and of HHV-6 U27 mRNA transcript. High viremia levels and relapses were overlapping. After the last relapse, the patient was successfully treated with gancyclovir. Conclusions: The case reported here suggests a possible association of HHV-6 in bilateral optic neuritis. HHV-6 could be monitored when bilateral optic neuritis is identified, in order to establish an appropriate antiviral therapy. (c) 2006 Elsevier B.V. All rights reserved.


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.


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 - Antiviral and virucide activity of propolis [Abstract in Atti di Convegno]
Cermelli, Claudio; Beretti, Francesca; Cenacchi, V.; Quaglio, Giampaola; Portolani, Marinella
abstract

Not available


2005 - Cell culture isolation of a transmissible cytotoxicity from a human sample of cerebrospinal fluid [Articolo su rivista]
Portolani, Marinella; Beretti, Francesca; Cermelli, Claudio; Am, Bartoletti; P., Pietrosemoli; Bargellini, Annalisa; DE POL, Anto; Rossini, Gian Paolo
abstract

We investigated a transmissible cytotoxicity isolated in VERO cell cultures from a sample of cerebrospinal fluid (CSF) drawn from a woman with ischemic brain injury. Amorphous aggregates formed by subunities of similar to 11nm of diameter were detected in ultracentrifugates from partially purified cytotoxic cell preparations in the absence of virion-like particles which might justify the trasmissibility of this cytotoxic activity. Results of chemico-physical studies provided indications on the presence in the CSF of two protease-resistant acidic glycoproteins of about 39 and 27 kDa, respectively. The conformational change of a proteinic molecule may associate with particular properties such as tendency to aggregation, resistance to proteolysis, cytotoxicity. Considering that these same properties are shared by proteins present in the CSF sample under study, a hypothesis to pursue is that the CSF inoculum we isolated contained misfolded proteins formed in vivo following the ischemic injury of brain tissue. As far as the in vitro transmissibility of the cytotoxic activity, this could take place following the reproduction of the alterations of those proteins, independently of the original cause(s) which have fostered their formation in vivo.


2005 - recurrent bilateral optic neuritis associated with active infection by Human Herpesvirus 6B [Abstract in Atti di Convegno]
Moscehttini, D; Vaccaro, Nm; Celeste, Ag; Cermelli, Claudio; Pezzini, F; Franceschini, R; Donati, D.
abstract

Not available


2004 - Antiviral activity of propolis [Abstract in Atti di Convegno]
Cermelli, Claudio; Cenacchi, V; Bergamini, F; Lonetti, I; Beretti, Francesca; Quaglio, Giampaola
abstract

Propolis, a natural product of beehives, comprises a complex of chemicals, the most important being flavonoids, which are known to havw antiviral activities. Alcohol solutions of 5 different commercial products were tested in vitro against HSV-1, CoxB5, Adenovirus, Mumps virus.All the propolis preparations under study showed antiviral activity against at least some of the virus tested, although with different efficacy.


2004 - Comparison of culture and immunoassay for detection of Escherichia coli O157 in raw minced meat and hamburger [Articolo su rivista]
Fabio, Giuliana; Cermelli, Claudio; H., Nanni; Quaglio, Giampaola
abstract

Verocytotoxin-producing Escherichia coli O157 (VTEC) is an important food-borne pathogen of humans. The serious complications of VTEC infection and the established reservoir of VTEC in cattle used for mass food production are a public health concern. In this study 500 samples of hamburger and minced meat were examined for presence of E. coli O157. For E. coli detection, Tryptic Soy Broth supplemented (with novobiocin and bile salts) and Sorbitol Mc Conkey agar were used; an automated rapid enzyme linked fluorescent immunoassay (VIDAS E. coli O157) was also evaluated. E. coli O157 was found in 5 samples of hamburger, 2 strains were found to be positive for verocytotoxin production on Vero cells.PMID: 15164632 [PubMed - indexed for MEDLINE]


2004 - Diagnostica delle gastroenteriti virali [Abstract in Rivista]
Cermelli, Claudio
abstract

Vengono illustrati gli agenti eziologici principali della gastroenteriti virali (rotavirus, norovirus, adenovirus, astrovirus), le loro caratteristiche epidemiologiche e i metodi diagnostici


2004 - Indagine sulla eziologia di infezioni a prevalente trasmissione alimentare. [Articolo su rivista]
Fabio, Giuliana; Cermelli, Claudio; Fabio, A.; Casolari, Chiara; Quaglio, P.
abstract

not available


2004 - Synthesis and antiviral activity of new benzothiadiazine dioxide derivates [Articolo su rivista]
Tait, Annalisa; Luppi, Amedeo; Cermelli, Claudio
abstract

In continuing our search in the benzothiadiazine field, we have synthesized new alkylated 2,1,3-BTD and 1,2,4-BTD derivatives. The procedure of alkylation is led via Mitsunobu reaction with good yields. The compounds were tested for their antiviral activity versus some viral strain (ADV, HHV-6, Cox-B5 and H-CMV) and most of them are resulted active at micromolar level. Except compound 1a that presents a high cytotoxicity (maximum non toxic concentration was 3μM), all the other molecules prepared are poorly cytotoxic (maximum non toxic concentrations were >25μM).


2003 - Detection of human herpesvirus-6 in mesial temporal lobe epilepsy surgical brain resections [Articolo su rivista]
Donati, D; Akhyani, N; Fogdell Hahn, A; Cermelli, Claudio; Cassiani Ingoni, R; Vortmeyer, A; Heiss, Jd; Cogen, P; Gaillard, Wd; Sato, S; Theodore, Wh; Jacobson, S.
abstract

Background: Human herpesvirus-6 (HHV-6), a ubiquitous beta-herpesvirus, is the causative agent of roseola infantum and has been associated with a number of neurologic disorders including seizures, encephalitis/meningitis, and multiple sclerosis. Although the role of HHV-6 in human CNS disease remains to be fully defined, a number of studies have suggested that the CNS can be a site for persistent HHV-6 infection. Objective: To characterize the extent and distribution of HHV-6 in human glial cells from surgical brain resections of patients with mesial temporal lobe epilepsy (MTLE). Method: Brain samples from eight patients with MTLE and seven patients with neocortical epilepsy (NE) undergoing surgical resection were quantitatively analyzed for the presence of HHV-6 DNA using a virus-specific real-time PCR assay. HHV-6 expression was also characterized by western blot analysis and in situ immunohistochemistry (IHC). In addition, HHV-6-reactive cells were analyzed for expression of glial fibrillary acidic protein (GFAP) by double immunofluorescence. Results: DNA obtained from four of eight patients with MTLE had significantly elevated levels of HHV-6 as quantified by real-time PCR. HHV-6 was not amplified in any of the seven patients with NE undergoing surgery. The highest levels of HHV-6 were demonstrated in hippocampal sections (up to 23,079 copies/10(6) cells) and subtyped as HHV-6B. Expression of HHV-6 was confirmed by western blot analysis and IHC. HHV-6 was co-localized to GFAP-positive cells that morphologically appeared to be astrocytes. Conclusions: HHV-6B is present in brain specimens from a subset of patients with MTLE and localized to astrocytes in the absence of inflammation. The amplification of HHV-6 from hippocampal and temporal lobe astrocytes of MTLE warrants further investigation into the possible role of HHV-6 in the development of MTLE.


2003 - High frequency of human herpesvirus 6 DNA in multiple sclerosis plaques isolated by laser microdissection [Articolo su rivista]
Cermelli, C.; Berti, R.; Soldan, S. S.; Mayne, M.; D'Ambrosia, J. M.; Ludwin, S. K.; Jacobson, S.
abstract

The frequency of human herpesvirus 6 (HHV-6) DNA was assessed in autopsy material from multiple sclerosis (MS) plaques and normal-appearing white matter (NAWM) from brains of persons with MS, healthy brains, and brains of persons with other neurologic diseases. Specific areas from formalin-fixed, paraffin-embedded brain tissue samples were isolated by laser microscope. DNA was extracted from laser microdissected brain material, and HHV-6 genomic sequences were amplified by nested polymerase chain reaction. We analyzed 44 NAWM samples and 64 MS plaques from 13 patients with MS, 46 samples from 13 patients with non-MS neurologic disorders, and 41 samples from 12 healthy control brains. Of the 44 NAWM samples, 7 (15.9%) were positive for HHV-6 DNA sequences, versus 37 (57.8%) of 64 MS plaques (P<.0005). HHV-6 DNA was detected in 10 (21.7%) of 46 samples from patients with non-MS neurologic disorders and in 11 (26.8%) of 41 samples from patients without known neurologic disease. Although the frequency of HHV-6 DNA did not differ significantly by sample type, HHV-6 DNA was significantly more common in MS plaques, suggesting that HHV-6 may play a role in MS pathogenesis.


2003 - Risk of sporadic amyotrophic lateral sclerosis associated with seropositivity for herpesviruses and echovirus-7. [Articolo su rivista]
Cermelli, Claudio; Vinceti, Marco; Beretti, Francesca; Pietrini, V.; Nacci, G.; Pietrosemoli, P.; Bartoletti, A.; Guidetti, D.; Sola, P.; Bergomi, Margherita; Vivoli, Gianfranco; Portolani, Marinella
abstract

We examined the association between risk of sporadic amyotrophic lateral sclerosis (ALS) and seroprevalence of antibodies to echovirus-7 (echo-7) and herpesviruses 6, 7, and 8 through a population-based case-control study. We enrolled in a northern Italy area 20 newly diagnosed ALS cases and 20 referents. Risk of ALS was higher in subjects seropositive for echo-7 when we used the immunofluorescent assay, while little increase was noted with the neutralization test. Considering the different characteristics of these two serological assays, these results suggest an association between disease risk and infection with enterovirus (EV) family members (not specifically echo-7). ALS risk was slightly associated with seropositivity of human herpesvirus-6 (odds ratio: 3.2; p = 0.102) and more strongly with human herpesvirus-8 seropositivity (odds ratio: 8.4; p = 0.064), though these point estimates were statistically unstable due to the limited number of observed cases. The findings of this study warrant further investigation in larger studies of the possible etiologic role of EV or herpesvirus infection in sporadic ALS.


2002 - Detection of Antibodies Directed against Human Herpesvirus 6 U94/REP in Sera of Patients Affected by Multiple Sclerosis [Articolo su rivista]
Caselli, E; Boni, M; Bracci, A; Rotola, A; Cermelli, Claudio; Castellazzi, M; DI LUCA, D; Cassai, E.
abstract

The association between human herpesvirus 6 (HHV-6) and multiple sclerosis (MS) is controversial. In fact, it is difficult to establish a causative role of HHV-6, due to the high prevalence of latently infected individuals in the healthy population. Therefore, the presence of virus sequences in tissue biopsy does not support a viral role, and serological assays do not show significant differences between MS patients and control populations. The only viral gene expressed during latency is U94/rep. Therefore, we have developed a serological assay for the detection of antibodies specifically directed against U94/REP protein. Different populations were analyzed by enzyme-linked immunosorbent assay, including healthy controls, MS patients, and subjects with diseases unrelated to HHV-6 infection, including other neurological diseases. The results show statistically significant differences (P > 0.01) between MS patients and control groups, both in antibody prevalence (87 and 43.9%, respectively) and in geometric mean titer (1:515 and 1:190, respectively). The detection of antibodies specific for HHV-6 U94/REP shows that the immune system is exposed to this antigen during natural infection. The higher prevalence and higher titers of antibodies to U94/REP suggest that MS patients and control groups might experience different exposures to HHV-6.


2002 - Evaluation of a vero cell toxicity test to detect EHEC infection [Articolo su rivista]
Cermelli, Claudio; Fabio, G; Casolari, C; Quaglio, P.
abstract

An epidemiologic investigation was carried out in Modena (Italy) to evaluate the prevalence of faecal VEROtoxin (FVT) in diarrhoeal stool specimens. One thousand and sixty-six stool specimens, submitted to the Clinical Microbiology Laboratory of the University Hospital of Modena, were collected and faecal filtrates tested for neutralizable cytotoxin by a toxicity test on VERO cells. Cytopathic effect on VERO cells was produced by 301 stool specimens (28%); neutralizable VT was detected in 40 (13%) out of 301 positive samples (3.7% of 1066 specimens). The prevalent FVT type was VT2 (50%), followed by VT1 (32.5%) and VT1+2 (17.5%). We evaluated an assay that detects both VTs directly from stool specimens to demonstrate that enterohemorrhagic strains (EHEC) should be considerated a causative agent of sporadic non-bloody diarrhoea. Our results suggest that toxin neutralization assay is a sensitive and specific technique and may be used as an alternative method to diagnose diarrhoeal infections caused by EHEC.


2002 - Selenite inhibition of Coxsackie virus B5 replication: implications on the etiology of Keshan disease. [Articolo su rivista]
Cermelli, Claudio; Vinceti, Marco; Scaltriti, E.; Bazzani, E.; Beretti, Francesca; Vivoli, Gianfranco; Portolani, Marinella
abstract

Keshan disease is a cardiomyopathy of unknown origin reported in some areas of China. Because of epidemiologic features, this disease was ascribed to an infectious agent, likely a Coxsackie virus, but it has also been thought to depend on selenium deficiency, mainly because selenite is effective in its prophylaxis. We examined the hypothesis that pharmacological activity of selenite on Coxsackie virus growth was associated with prevention of Keshan disease. We studied the antiviral effects of three selenium compounds on Coxsackie virus B5 replication: five μM selenite reduced viral replication, whilst 10 μM selenate and selenomethionine did not exhibit any antiviral activity. The inhibitory activity of selenite on viral replication was due to its toxicity following its interaction with thiols, as that activity could be blocked by dithiothreitol, a sulfhydryl-protecting agent known to reverse several toxic effect of selenite. Zinc, another inhibitor of selenite toxicity, also counteracted the antiviral effect of selenite. The selenium compounds showed only limited activity against herpes simplex 1 virus and IHD strain of vaccinia virus. A direct inhibitory effect of selenite on Coxsackie virus replication might explain the efficacy demonstrated by this compound in the prophylaxis of Keshan disease.


2001 - Antiviral and antiproliferative activity in vitro of some new benzimidazole derivatives [Articolo su rivista]
M., Castelli; M., Malagoli; L., Lupo; Rossi, Tiziana; Casolari, Chiara; Cermelli, Claudio; A., Zanca; Baggio, Giosuè Gabriele
abstract

The antiviral and antiproliferative activity of new compounds having n-benzenesulphony 1-2 (2 or 3-pyridylethyl) benzimidazole as a base structure were studied in vitro. Their antitumour activity against human chronic myeloid leukaemia cells was evaluated and compared with that of equimolar doses of daunorubicin. Only compound 7a, with the presence of both the pyridyl moiety bound at the ethylenic bridge in C-2 of benzimidazole and the nitro-group in the benzene ring, displays a selective antiproliferative effect against certain leukaemia cells and a good antiviral activity especially towards the Coxsackie B5 virus. However, it should be noted that, in the case of hydroxybenzyl-benzimidazole, resistance also builds up to compound 7a, the Coxsackie B5 virus developing resistance to it after about ten runs. Cytotoxicity tests show that many of these substances are well tolerated by the VERO cells. The mechanism of action is still unclear.


2001 - Antiviral and antiproliferative activity in vitro of some new benzimidazole derivatives. [Articolo su rivista]
Castelli, M; Malagoli, M; Lupo, L; ROSSI RICCOMI, T; Casolari, C; Cermelli, Claudio; Zanca, ; Baggio, G.
abstract

The antiviral and antiproliferative activity of new compounds having n-benzenesulphony 1-2 (2 or 3-pyridylethyl) benzimidazole as a base structure were studied in vitro. Their antitumour activity against human chronic myeloid leukaemia cells was evaluated and compared with that of equimolar doses of daunorubicin. Only compound 7a, with the presence of both the pyridyl moiety bound at the ethylenic bridge in C-2 of benzimidazole and the nitro-group in the benzene ring, displays a selective antiproliferative effect against certain leukaemia cells and a good antiviral activity especially towards the Coxsackie B5 virus. However, it should be noted that, in the case of hydroxybenzyl-benzimidazole, resistance also builds up to compound 7a, the Coxsackie B5 virus developing resistance to it after about ten runs. Cytotoxicity tests show that many of these substances are well tolerated by the VERO cells. The mechanism of action is still unclear.


2001 - Gene expression profile of herpesvirus-infected T cells obtained using immunomicroarrays: induction of proinflammatory mechanisms. [Articolo su rivista]
Mayne, M.; Cheadle, C.; Soldan, S. S. S.; Cermelli, Claudio; Yamano, Y.; Akhyani, N.; Nagel, J. E.; Taub, D. D.; Becker, K. G.; Jacobson, S.
abstract

Herpesvirus infections can frequently lead to acute inflammation, yet the mechanisms regulating this event remain poorly understood. In order to determine some of the immunological mechanisms regulated by human herpesvirus infections, we studied the gene expression profile of lymphocytes infected with human herpesvirus 6 (HHV-6) by using a novel immunomicroarray. Our nylon-based immunomicroarray contained more than 1,150 immune response-related genes and was highly consistent between experiments. Experimentally, we found that independently of the HHV-6 strain used to infect T cells, multiple proinflammatory genes were increased and anti-inflammatory genes were decreased at the mRNA and protein levels. HHV-6 strains A and B increased expression of the genes for interleukin-18 (IL-18), the IL-2 receptor, members of the tumor necrosis factor alpha superfamily receptors, mitogen-activated protein kinase, and Janus kinase signaling proteins. As reported previously, CD4 protein levels were also increased significantly. Specific type 2 cytokines, including IL-10, its receptor, and IL-14, were downregulated by HHV-6 infection and, interestingly, amyloid precursor proteins and type 1 and 2 presenilins. Thus, T cells respond to HHV-6 infection by inducing a type 1 immune response that may play a significant role in the development and progression of diseases associated with HHV-6, including pediatric, hematologic, transplant, and neurologic disorders.


2000 - Cytotoxicity and probable mechanism of action of sulphimidazole [Articolo su rivista]
CASTELLI, M; MALAGOLI, M; LUPO, L; ROFFIA, S; PAOLUCCI, F; CERMELLI, Claudio; ZANCA, A; BAGGIO, G.
abstract

Sulphimidazole (1-methyl-2((4-aminophenyl)-sulphonyl)-amino-5-nitroimidazole) is a new compound in which a p-aminobenzenesulphonamide radical has been attached at position 2 of the 5-nitroimidazole ring. It possesses a useful spectrum of activity in vitro against various anaerobic microorganisms and its action against aerobic and facultative bacteria is synergically enhanced in association with trimethoprim. In the present study, we determined the cytotoxicity in vitro of sulphimidazole and trimethoprim, both alone and in combination, and analysed the viability of Vero cells and the protein content of their cell lysate in the presence of increasing concentrations of these drugs. Also, in order to verify the hypothesis that the action of sulphimidazole against aerobic and facultative bacteria is mediated by the sulphonamide component of the molecule, while that against anaerobic bacteria depends on the action of the nitro group of the 5-nitroimidazole ring, we studied the mechanism of action of the new compound both indirectly, by means of microbiological techniques, and directly, by determining its oxidoreduction potential with respect to that of metronidazole. The results show that sulphimidazole is only slightly toxic in vitro for Vero cells, either alone or in association with trimethoprim, and that the combination of the two functional groups in a single molecule not only maintains its structure-activity relationship intact but also broadens its antibacterial spectrum.


2000 - Viruses and Multiple Sclerosis [Articolo su rivista]
CERMELLI, Claudio; JACOBSON, S.
abstract

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system whose cause is still unknown. Many findings suggest an infectious etiology or, at least, that infectious agents in association with host genetic factors may play a role in the pathogenesis of this disease. Accumulating data, including animal models, human models of virus induced demyelination, epidemiologic, and laboratory findings, demonstrate that viruses and host genetic factors can interact to cause immune-mediated demyelination. While many viruses have been postulated as a possible cause of MS, to date, no "MS virus" has been definitively shown to be associated with this disease. Alternatively, ubiquitous viruses are being considered as the environmental "triggers" that have been postulated to be involved in the MS disease process. We will focus on recent studies with human herpesvirus 6 and MS as how a common virus may be associated with this disorder in a subset of infected individuals.


1999 - Epstein-Barr virus DNA in the cerebrospinal fluid of patients with human immunodeficiency virus infection and central nervous system disorders [Articolo su rivista]
Portolani, Marinella; Cermelli, Claudio; M., Meacci; P., Pietrosemoli; Sabbatini, Anna Maria Teresa; Mc, Cerri; Guaraldi, Giovanni; B., De Rienzo
abstract

Routine search for herpesvirus types 1-5 by nested polymerase chain reaction revealed Epstein-Barr virus (EBV) DNA in the cerebrospinal fluid (CSF) of ten out of seventy-nine patients with human immunodeficiency virus (HIV) infection and central nervous system (CNS) disorders not associated with the presence of primary CNS lymphomas. One out of the ten CSF samples was positive for EBV DNA only, six were also positive for microbial agents of recognised neurological pathogenicity while the remaining three samples had a high content of HIV p24 Ag. When six available CSF samples out of the ten EBV DNA positive specimens were investigated for an intrathecal EBV antibody response, all six samples proved EBV antibody-free. The concurrent detection of neurotropic infectious agents and the absence of EBV antibodies in the CSF contribute to the uncertainty on the role of EBV in the neurological illness of the patients studied. One hypothesis considered is that the presence of EBV DNA in the CSF of a large fraction of the ten patients under study is an incidental event associated with EBV reactivation in the host's peripheral blood monocytes, but not related to the genesis of neurological disorders.


1999 - Herpesvirus DNA is frequently detected in liver tissue from hepatitis C patients [Articolo su rivista]
Cermelli, Claudio; M., Concari; P., Pietrosemoli; M., Meacci; Sabbatini, Anna Maria Teresa; A., Divincenzo; Carubbi, Francesca; Loria, Paola; A., Bagni; Carulli, Nicola; Portolani, Marinella
abstract

Background and aims: Herpesviruses infect the liver and cause minor hepatitis. Our aim is to verify the presence of herpesviruses in the liver from hepatitis C patients and the possible influence of these viruses in the liver disease. Methods: We searched for herpesvirus DNA in liver biopsies from patients with hepatitis C and from a control group without hepatitis by means of nested polymerase chain reaction. Serological investigations were carried out as well. Results: Thirty-four liver specimens from hepatitis C patients were examined, 12 of which (35.3%) were positive for at least one herpesvirus DNA, whereas among the 19 control specimens only two were positive (10.5%; P = 0.049). Liver biopsies from seven patients, three with acute hepatitis of unknown origin, three with non-alcoholic steatohepatitis and one with autoimmune hepatitis were also investigated and three positive samples were found. Conclusions: The prevalence of herpesvirus DNA was found higher in patients with hepatitis C than in individuals without hepatitis. The influence of herpesviruses on the clinical course of hepatitis C is considered. (C) 1999 Elsevier Science B.V. All rights reserved.


1999 - Prevalenza degli herpesvirus nel tessuto epatico da soggetti con epatite C [Articolo su rivista]
Cermelli, C.; Concari, M.; Pietrosemoli, P.; Meacci, M.; Sabbatini, A. M. T.; Loria, P.; Divincenzo, A.; Portolani, M.
abstract

We report the results of a study on the search for herpesvirus DNA in liver biopsy from patients with chronic hepatitis C and from a control group without hepatitis. Thirty-four liver samples from hepatitis C patients were examined, 12 of which (35.3%) were positive for at least one herpesvirus DNA, whereas among the 19 control liver samples only 2 were positive (10.5%; p= 0.049). Liver biopsies from 7 patients, 3 with acute hepatitis of unknown origin, 3 with non alcoholic steatoheapatitis and 1 with autoimmune hepatitis were investigated too and three positive samples were found. The prevalence of herpesvirus DNA higher in patients with hepatitis C than in individuals without hepatitis is discussed and the possible influence of herpesviruses both on the clinical course and on the therapy of hepatitis C is considered.


1998 - Detection of Epstein-Barr virus DNA in cerebrospinal fluid from immunocompetent individuals with brain disorders [Articolo su rivista]
Portolani, Marinella; P., Pietrosemoli; M., Meacci; Sabbatini, Anna Maria Teresa; M., Pecorari; G., Mantovani; Cermelli, Claudio
abstract

Fifty four cerebrospinal fluid samples obtained from as many immunocomponent patients with disorders of the central nervous system were investigated for the presence of herpesvirus DNA by nested polymerase chain reaction in order to determine an etiological diagnosis. Four of these samples proved positive for the presence of Epstein-Barr virus DNA (7.4%). The result of this diagnostic study is reported to draw insiders' attention to the possible presence of EBV in cerebrospinal fluid from patients with central nervous system diseases.


1998 - Epstein-Barr virus DNA in cerebrospinal fluid from an immunocompetent man with herpes simplex virus encephalitis. [Articolo su rivista]
Portolani, Marinella; Sabbatini, Anna Maria Teresa; M., Meacci; P., Pietrosemoli; Cermelli, Claudio; P., Lunghi; F., Golinelli; R., Stacca
abstract

Herpes simplex virus 1 meningo-encephalitis was ascertained in a 63-year-old immunocompetent man. To determine the duration of the persistence of herpesvirus DNA in the central nervous system, the cerebrospinal fluid was periodically monitored by polymerase chain reaction for 53 days. In addition to HSV-1, Epstein - Barr virus DNA was detected in the cerebrospinal fluid 9 days after disease onset. The possible meaning of the Epstein-Barr virus DNA finding is discussed.


1998 - Human herpesvirus-7 DNA in cerebrospinal fluid. [Articolo su rivista]
Portolani, Marinella; S., Leoni; A., Guerra; Cermelli, Claudio; Mantovani, Gabriella; P., Pietrosemoli; M., Meacci; Sabbatini, Anna Maria Teresa; M., Pecorari
abstract

An infection by HHV-7 with presence of virus DNA in the spinal fluid was ascertained in a twenty five month old boy with an epileptic syndrome shortly after birth. The child was frequently admitted to hospital due to his basal disease and frequent bacterial infections. In the occasion of one of these hospital admissions for bacterial infections in conjunctiva, spleen and a lung, virological investigations were also carried out disclosing the presence of HHV-7 DNA in a sample of spinal fluid and of serum and the absence of DNAs from the other herpesviruses. The patient's monitoring for HHV-7 showed the presence of HHV-7 DNA in a sample of serum and in various samples of peripheral blood lymphocytes and saliva, but not in the cerebrospinal fluid sample successive to that positive for HHV-7 DNA. Forty seven days before the diagnosis of HHV-7 infection, the patient received a human gamma-globulin therapy which obscured the humoral response mounted against the virus by the host, so, whether the HHV-7 presence in the central nervous system was associated with a primary or a reactivated infection remained uncertain. The absence of detectable HHV-7 serum IgM antibody, however, suggests the HHV-7 DNA finding on the occasion of a virus reactivation rather than a primary infection. The virological data suggest that HHV-7 may have possibly reached the central nervous system in the course of a viremia. The absence of HHV-6 and HHV-7 antibodies, present in the patient's serum at a high level, support the integrity of the blood-brain barrier. A possible pathogenetic role of HHV-7 in the child's disease seems unlikely, since the epileptic syndrome was pre-existing.


1997 - An outbreak of infantile aseptic meningitis caused by Echo 30 [Articolo su rivista]
Sabbatini, A. M. T.; Pietrosemoli, P.; Mantovani, G.; Corassori, L.; Mattei, F.; Guidi, B.; Borghi, A.; Squadrini, F.; Cermelli, C.; Portolani, M.
abstract

This paper describes an outbreak of infantile aseptic meningitis which occurred in an Apennine village during the summer 1993. It was possible to assess the etiology of the syndrome by means of virological (virus isolation, identification by hemagglutination test and typing by Lim BenyeshMelnick antiserum) and serological (complement fixation, immune-electromicroscopy, neutralisation test) methods. Three viral isolates (one from the pharyngeal and one from the faecal swab of a patient and one from the cerebrospinal fluid sample of a another patient) were obtained which were identified as Echovirus 30. All 9 patients showed an antibody response against ECHO 30.


1996 - Growth of human herpesvirus 6 in HepG2 cells [Articolo su rivista]
Cermelli, Claudio; M., Concari; Carubbi, Francesca; Fabio, Giuliana; Sabbatini, Anna Maria Teresa; M., Pecorari; P., Pietrosemoli; M., Meacci; E., Guicciardi; Carulli, Nicola; Portolani, Marinella
abstract

HepG2 cells, a well differentiated liver cell line, were shown to be permissive for both human herpesvirus 6 (HHV-6) A and B strains by three independent methods of analysis: detection of viral antigens, viral DNA sequences and infectious virus. HepG2 cell infection with HHV-6 resulted in functional damage as shown by the increased release in the culture medium of some hepatocyte markers. Cells surviving the acute infection were serially passaged without showing cytopathic effect, but, some months later, HHV-6 DNA was still present in the cells and virus induction with a phorbol ester was successful. A possible pathogenetic role of HHV-6 in liver diseases is discussed. Experiments of HepG2 infection with human herpesvirus 7 (HHV-7) were also carried out. The lack of an efficient virus replication suggested a difficulty for HHV-7 to infect hepatic cells.


1995 - A three year follow-up of low grade squamous intraepithelial lesions: Persistence of lesions is significantly influenced by the presence of high risk papillomaviruses [Articolo su rivista]
Ghirardini, C.; Raisi, O.; Camerlo, M.; Prosperi, L.; Cermelli, C.
abstract

A cohort of 921 patients with low-grade squamous intraepithelial lesions (LG-SILs) was studied every six months for a period of 3 years by cytology and colposcopy. Regression of the initial cell injury to a normal cytology was observed in 585/921 cases (63.5%), while cytological abnormalities were still present at the same or higher grade, after three years, in 336/921 cases (36.5%). A sample of 122 Papanicolaou (Pap}stained cervical smears from the cohort of 921 LG-SILs were processed by an in situ hybridization (ISH) assay to search for the presence of DNA of human papillomaviruses (HPVs) considered at high oncogenic risk: 16/18 and 31/35/51. HPV DNA typization was positive for 47/122 lesions (38.5%). Among these 47 lesions associated with high risk HPV DNA, 12 (25.5%) healed and 35 (74.5%) persisted. Of the 75 HPV DNA negative lesions, 46 (61.3%) healed and 29 (38.7%) persisted. The results are discussed in order to define a correct protocol of LG-SIL follow-up.


1995 - SENDAI VIRUS AND HERPES-VIRUS TYPE-1 INDUCE APOPTOSIS IN HUMAN PERIPHERAL-BLOOD MONONUCLEAR-CELLS [Articolo su rivista]
Tropea, F; Troiano, L; Monti, D; Lovato, E; Malorni, W; Rainaldi, G; Mattana, P; Viscomi, G; Ingletti, Mc; Portolani, Marinella; Cermelli, Claudio; Cossarizza, Andrea; Franceschi, C.
abstract

Recent reports suggest that several viruses, besides human immunodeficiency virus, induce apoptosis in infected cells, We report here that Sendai virus or Herpes simplex virus type 1 (HSV-1), two potent inducers of interferon-alpha, caused cell death in a consistent number of human peripheral blood mononuclear cells, A careful analysis of infected cells by different techniques, such as optical and electron microscopy, DNA agarose gel electrophoresis, and cytofluorimetric analysis of DNA content, showed that cell death was of apoptotic type, Sendai virus was more apoptogenic than HSV-1, and it was further studied to understand the mechanism(s) by which it induced apoptosis, Physical (uv and heat) and chemical (beta-propiolactone) inactivation reduced or abolished the apoptogenic power of Sendai virus, The use of a novel technique, which allows the study of mitochondrial membrane potential (MMP) in intact cells by flow cytometry, showed that a decrease of MMP is concomitant with the appearance of the hypodiploid peak. These results suggest that Sendai virus and HSV-1 can be added to the list of viruses causing apoptosis, which appears to be a general mechanism occurring during viral infection.


1994 - IgG antibodies to papillomavirus genus-antigens in adult men with cancer of the urinary bladder [Articolo su rivista]
Mantovani, G.; Cermelli, C.; Malagoli, M.; Provvisionato, A.; Ferrari, P.; Castagnetti, M.; Portolani, M.
abstract

Forty-five sera from men with bladder cancer were examined in a micro solid-phase enzyme-linked immunosorbent assay (ELISA) and in a Western-blotting (WB) assay for the presence of IgG antibodies to papillomavirus (PV) genus-antigens of bovine origin. The ELISA detected PV antibodies in 75.6% of cancer patients. This antibody frequency was significantly higher than that found in both healthy males (22.7%) and patients with urological disorders (24%). A similar correlation among the PV antibody frequencies of the three groups was found with WB assay: 60% of the neoplastic group showed PV antibodies versus 17.3% in healthy males and 32.6% in non-neoplastic patients. Within the same group, 78% to 87% sera showed the same reactivity to both assays. Of these concordant sera, PV positive sera were 55.6% in cancer patients, 13.3% in healthy adults and 19.6% in patients with urological disorders. ELISA PV antibody level in the cancer group was higher than in each of the two control groups. The meaning of the humoral response to PV genus-antigens in men with bladder cancer is discussed.


1992 - SYNTHESIS AND BIOLOGICAL-ACTIVITY OF NEW HETEROCYCLIC STRUCTURES - [1,3]THIAZINO[2,3-I]PURINE, THIAZOLO[3,2-C] ]1,2,3]TRIAZOLO[4,5-E]PYRIMIDINE AND [1,2,3]TRIAZOLO [4', 5'/4,5] PYRIMIDO[6,1-B][1,3]THIAZINE [Articolo su rivista]
Rinaldi, M; Pecorari, P; Costantino, Luca; Provvisionato, A; Malagoli, M; Cermelli, Claudio
abstract

Some derivatives of thiazolo[3,2-c]pyrimidine, pyrimido[6,1-b][1,3]thiazine, thiazolo[2,3-i]purine, [1,3]thiazino[2,3-i]purine, thiazolo[3,2-c][1,2,3]triazolo[4,5-e]pyrimidine and [1,2,3]triazolo[4,5':4,5]pyrimido[6,1-b][1,3]thiazine were prepared. The compounds were tested for antimicrobial and antimycotic activity on a number of strains, namely, Escherichia coli, Proteus vulgaris, P. mirabilis, Pseudomonas aeruginosa, Salmonella sp., Staphylococcus aureus, Streptococcus faecalis, Bacillus subtilis, Sarcina lutea, Candida albicans, Aspergillus sp., and for antiviral activity on Herpes simplex virus, Type 1 (HSV-1), Vescicular stomatitis virus (VSV) and Coxsackievirus B5 (CoxB5). The compounds proved to be devoid of activity against viruses, mycetes and gram-negative bacteria, while some of them exhibited a modest activity against gram-positive bacterial strains.


1991 - SYNTHESIS AND BIOLOGICAL-ACTIVITY OF PYRIMIDO[2,1-B][1,3]THIAZINE, [1,3]THIAZINO[3,2-A]PURINE AND [1,2,3]TRIAZOLO[4,5-D][1,3]THIAZINO[3,2-A]PYRIMIDINE DERIVATIVES AND THIAZOLE ANALOGS [Articolo su rivista]
Pecorari, P; Rinaldi, M; Costantino, Luca; Provvisionato, A; Cermelli, Claudio; Portolani, Marinella
abstract

Some series of thiazolo[3,2-a]pyrimidine, pyrimido[2,1-b] [1,3]thiazine, thiazolo[3,2-a]purine, [1,3]thiazino[3,2-a]purine, thiazolo[3,2-a][1,2,3]triazolo[4,5-d]pyrimidine and [1,2,3]triazolo[4,5-d][1,3]thiazino[3,2-a]pyrimidine derivatives, variously functionalized, were prepared. The compounds were tested for antimicrobial and antimycotic activity on a number of strains, namely: E. coli, Proteus vulgaris, P. mirabilis, Pseudomanas aeruginosa, Salmonella sp., Staphylococcus aureus, S. faecalis, Bacillus subtilis, Sarcina lutea, Candida albicans, C. tropicalis, Aspergillus sp., and for antiviral activity on Herpes simplex virus Type 1, Vesicular stomatitis virus and Coxsackievirus B5. The compounds proved to be devoid of activity against viruses and gram-negative bacteria, while some of them exhibited modest activity against gram-positive bacterial strains.