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Francesca BERETTI

Personale tecnico amministrativo
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede Policlinico

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Pubblicazioni

2023 - Effect of the Enrichment in c-Kit Stem Cell Potential of Foetal Human Amniotic Fluid Cells: Characterization from Single Cell Analysis to the Secretome Content [Articolo su rivista]
Casciaro, Francesca; Beretti, Francesca; Gatti, Martina; Persico, Giuseppe; Bertucci, Emma; Giorgio, Marco; Maraldi, Tullia
abstract

Human amniotic fluid cells (hAFSCs) are a fascinating foetal cell-type that have important stem cell characteristics; however, they are a heterogeneous population that ranges from totally differentiated or progenitor cells to highly multipotent stem cells. There is no single approach to isolating the stem cell component, but the selection of a subpopulation of hAFSCs expressing c-Kit is widely employed, while a deep characterization of the two populations is still lacking. Here we performed single-cell and bulk RNAseq analysis to compare the gene expression profiles of adherent amniotic fluid cells and their subpopulation c-Kit . Information deriving from this high throughput technology on the transcriptome was then confirmed for specific targets with protein expression experiments and functional analysis. In particular, transcriptome profiling identified changes in cellular distribution among the different clusters that correlated with significant differential expression in pathways related to stemness, proliferation, and cell cycle checkpoints. These differences were validated by RT-PCR, immunofluorescence, WB, and cell cycle assays. Interestingly, the two populations produced secretomes with different immune-modulating and pro-regenerative potentials. Indeed, the presence of TGF, HGF, IDO was higher in EVs deriving from c-Kit+ cells, unlike IL- 6. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting c-Kit positive fractions with higher potential in regenerative medicine applications.

2023 - Human Neuromuscular Junction on a Chip: Impact of Amniotic Fluid Stem Cell Extracellular Vesicles on Muscle Atrophy and NMJ Integrity [Articolo su rivista]
Gatti, Martina; Dittlau, Katarina Stoklund; Beretti, Francesca; Yedigaryan, Laura; Zavatti, Manuela; Cortelli, Pietro; Palumbo, Carla; Bertucci, Emma; Van Den Bosch, Ludo; Sampaolesi, Maurilio; Maraldi, Tullia
abstract

: Neuromuscular junctions (NMJs) are specialized synapses, crucial for the communication between spinal motor neurons (MNs) and skeletal muscle. NMJs become vulnerable in degenerative diseases, such as muscle atrophy, where the crosstalk between the different cell populations fails, and the regenerative ability of the entire tissue is hampered. How skeletal muscle sends retrograde signals to MNs through NMJs represents an intriguing field of research, and the role of oxidative stress and its sources remain poorly understood. Recent works demonstrate the myofiber regeneration potential of stem cells, including amniotic fluid stem cells (AFSC), and secreted extracellular vesicles (EVs) as cell-free therapy. To study NMJ perturbations during muscle atrophy, we generated an MN/myotube co-culture system through XonaTM microfluidic devices, and muscle atrophy was induced in vitro by Dexamethasone (Dexa). After atrophy induction, we treated muscle and MN compartments with AFSC-derived EVs (AFSC-EVs) to investigate their regenerative and anti-oxidative potential in counteracting NMJ alterations. We found that the presence of EVs reduced morphological and functional in vitro defects induced by Dexa. Interestingly, oxidative stress, occurring in atrophic myotubes and thus involving neurites as well, was prevented by EV treatment. Here, we provided and validated a fluidically isolated system represented by microfluidic devices for studying human MN and myotube interactions in healthy and Dexa-induced atrophic conditions-allowing the isolation of subcellular compartments for region-specific analyses-and demonstrated the efficacy of AFSC-EVs in counteracting NMJ perturbations.

2022 - Evidence for mitochondrial Lonp1 expression in the nucleus [Articolo su rivista]
Gibellini, Lara; Borella, Rebecca; De Gaetano, Anna; Zanini, Giada; Tartaro, Domenico Lo; Carnevale, Gianluca; Beretti, Francesca; Losi, Lorena; De Biasi, Sara; Nasi, Milena; Forcato, Mattia; Cossarizza, Andrea; Pinti, Marcello
abstract

The coordinated communication between the mitochondria and nucleus is essential for cellular activities. Nonetheless, the pathways involved in this crosstalk are scarcely understood. The protease Lonp1 was previously believed to be exclusively located in the mitochondria, with an important role in mitochondrial morphology, mtDNA maintenance, and cellular metabolism, in both normal and neoplastic cells. However, we recently detected Lonp1 in the nuclear, where as much as 22% of all cellular Lonp1 can be found. Nuclear localization is detectable under all conditions, but the amount is dependent on a response to heat shock (HS). Lonp1 in the nucleus interacts with heat shock factor 1 (HSF1) and modulates the HS response. These findings reveal a novel extramitochondrial function for Lonp1 in response to stress.

2022 - Exosomes Derived from Human Amniotic Fluid Mesenchymal Stem Cells Preserve Microglia and Neuron Cells from Aβ [Articolo su rivista]
Zavatti, M.; Gatti, M.; Beretti, F.; Palumbo, C.; Maraldi, T.
abstract

Background: Neuroinflammation is involved in neuronal cell death that occurs in neurodegenerative diseases such as Alzheimer’s disease (AD). Microglia play important roles in regulating the brain amyloid beta (Aβ) levels, so immunomodulatory properties exerted by mesenchymal stem cells may be exploited to treat this pathology. The evidence suggests that the mechanism of action of human amniotic fluid stem cells (hAFSCs) is through their secretome, which includes exosomes (exo). Methods: We examined the effect of exosomes derived from human amniotic fluid stem cells (hAFSCs-exo) on activated BV-2 microglia cells by lipopolysaccharide (LPS) as a neuroinflammation model. To investigate the exo effect on the interplay between AD neurons and microglia, SH-SY5Y neuroblastoma cells treated with Aβ were exposed to a conditioned medium (CM) obtained from activated BV-2 or co-culture systems. Results: We found that the upregulation of the markers of pro-inflammatory microglia was prevented when exposed to hAFSC-exo whereas the markers of the anti-inflammatory macrophage phenotype were not affected. Interestingly, the hAFSC-exo pretreatment significantly inhibited the oxidative stress rise and apoptosis occurring in the neurons in presence of both microglia and Aβ. Conclusion: We demonstrated that hAFSC-exo mitigated an inflammatory injury caused by microglia and significantly recovered the neurotoxicity, suggesting that hAFSC-exo may be a potential therapeutic agent for inflammation-related neurological conditions, including AD.

2021 - Amniotic fluid stem cell-derived extracellular vesicles counteract steroid-induced osteoporosis in vitro [Articolo su rivista]
Gatti, M.; Beretti, F.; Zavatti, M.; Bertucci, E.; Luz, S. R.; Palumbo, C.; Maraldi, T.
abstract

Background—Osteoporosis is characterized by defects in both quality and quantity of bone tissue, which imply high susceptibility to fractures with limitations of autonomy. Current therapies for osteoporosis are mostly concentrated on how to inhibit bone resorption but give serious adverse effects. Therefore, more effective and safer therapies are needed that even encourage bone formation. Here we examined the effect of extracellular vesicles secreted by human amniotic fluid stem cells (AFSC) (AFSC-EV) on a model of osteoporosis in vitro. Methods—human AFSC-EV were added to the culture medium of a human pre-osteoblast cell line (HOB) induced to differentiate, and then treated with dexamethasone as osteoporosis inducer. Aspects of differentiation and viability were assessed by immunofluorescence, Western blot, mass spectrometry, and histological as-says. Since steroids induce oxidative stress, the levels of reactive oxygen species and of redox related proteins were evaluated. Results—AFSC-EV were able to ameliorate the differentiation ability of HOB both in the case of pre-osteoblasts and when the differentiation process was affected by dexa-methasone. Moreover, the viability was increased and parallelly apoptotic markers were reduced. The presence of EV positively modulated the redox unbalance due to dexamethasone. Conclusion— these findings demonstrated that EV from hAFSC have the ability to recover precursor cell potential and delay local bone loss in steroid-related osteoporosis.

2021 - The Interplay between HGF/c-met Axis and Nox4 in BRAF Mutated Melanoma [Articolo su rivista]
Beretti, Francesca; Farnetani, Francesca; Reggiani Bonetti, Luca; Fabbiani, Luca; Zavatti, Manuela; Maiorana, Antonino; Pellacani, Giovanni; Maraldi, Tullia
abstract

Melanoma is the leading cause of death due to cutaneous malignancy and its incidence is on the rise. Several signaling pathways, including receptor tyrosine kinases, have a role in the development and progression of melanocytic lesions and malignant melanoma. Among those, the hepatocyte growth factor (HGF)/c-met axis is emerging as a critical player because it can play a role in drug resistance. Indeed, 50% of melanoma patients present BRAF mutations, however, all responders develop resistance to the inhibitors typically within one year of treatment. Interestingly, BRAF inhibitors induce reactive oxygen species (ROS) in melanoma cells, therefore, the aim of this study was to investigate a possible interplay between HGF/c-met and ROS sources, such as NADPH oxidases (Nox).

2021 - Unravelling Heterogeneity of Amplified Human Amniotic Fluid Stem Cells Sub-Populations [Articolo su rivista]
Casciaro, Francesca; Zia, Silvia; Forcato, Mattia; Zavatti, Manuela; Beretti, Francesca; Bertucci, Emma; Zattoni, Andrea; Reschiglian, Pierluigi; Alviano, Francesco; Bonsi, Laura; Follo, Matilde Yung; Demaria, Marco; Roda, Barbara; Maraldi, Tullia
abstract

Human amniotic fluid stem cells (hAFSCs) are broadly multipotent immature progenitor cells with high self-renewal and no tumorigenic properties. These cells, even amplified, present very variable morphology, density, intracellular composition and stemness potential, and this heterogeneity can hinder their characterization and potential use in regenerative medicine. Celector® (Stem Sel ltd.) is a new technology that exploits the Non-Equilibrium Earth Gravity Assisted Field Flow Fractionation principles to characterize and label-free sort stem cells based on their solely physical characteristics without any manipulation. Viable cells are collected and used for further studies or direct applications. In order to understand the intrapopulation heterogeneity, various fractions of hAFSCs were isolated using the Celector® profile and live imaging feature. The gene expression profile of each fraction was analysed using whole-transcriptome sequencing (RNAseq). Gene Set Enrichment Analysis identified significant differential expression in pathways related to Stemness, DNA repair, E2F targets, G2M checkpoint, hypoxia, EM transition, mTORC1 signalling, Unfold Protein Response and p53 signalling. These differences were validated by RT-PCR, immunofluorescence and differentiation assays. Interestingly, the different fractions showed distinct and unique stemness properties. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting certain cellular fractions with the highest potential to use in regenerative medicine.

2020 - Comparison of the therapeutic effect of amniotic fluid stem cells and their exosomes on monoiodoacetate-induced animal model of osteoarthritis [Articolo su rivista]
Zavatti, M.; Beretti, F.; Casciaro, F.; Bertucci, E.; Maraldi, T.
abstract

The cartilage tissue engineering associated with stem cell-related therapies is becoming very interesting since adult articular cartilage has limited intrinsic capacity for regeneration upon injury. Amniotic fluid stem cells (AFSC) have been shown to produce exosomes with growth factors and immunomodulating molecules that could stop tissue degradation and induce cartilage repair. Based on this state of the art, the main aim of this study was to explore the efficacy of the secreted exosomes, compared to their AFSC source, in MIA-induced animal model of osteoarthritis mimicking a chronic and degenerative process, where inflammation is also involved and lead to irreversible joint damage. Exosomes, obtained by the use of a commercial kit, prior to the injection in animal knee joints, were characterized for the presence of typical markers and HGF, TGFβ, and IDO. Then, analyses were performed by histology, immunohistochemistry, and behavioral scoring up to 3 weeks after the treatment. Exosome-treated defects showed enhanced pain tolerance level and improved histological scores than the AFSC-treated defects. Indeed by 3 weeks, TGFβ-rich exosome samples induced an almost complete restoration of cartilage with good surface regularity and with the characteristic of hyaline cartilage. Moreover, cells positive for resolving macrophage marker were more easily detectable into exosome-treated joints. Therefore, a modulating role for exosomes on macrophage polarization is conceivable, as demonstrated also by experiments performed on THP1 macrophages. In conclusion, this study demonstrates for the first time the efficacy of human AFSC exosomes in counteract cartilage damage, showing a positive correlation with their TGFβ content.

2020 - Oxidative stress in Alzheimer's disease: In vitro therapeutic effect of amniotic fluid stem cells extracellular vesicles [Articolo su rivista]
Gatti, M.; Zavatti, M.; Beretti, F.; Giuliani, D.; Vandini, E.; Ottani, A.; Bertucci, E.; Maraldi, T.
abstract

Alzheimer's disease (AD) is characterized by abnormal protein aggregation, deposition of extracellular β-amyloid proteins (Aβ), besides an increase of oxidative stress. Amniotic fluid stem cells (AFSCs) should have a therapeutic potential for neurodegenerative disorders, mainly through a paracrine effect mediated by extracellular vesicles (EV). Here, we examined the effect of EV derived from human AFSCs (AFSC-EV) on the disease phenotypes in an AD neuron primary culture. We observed a positive effect of AFSC-EV on neuron morphology, viability, and Aβ and phospho-Tau levels. This could be due to the apoptotic and autophagic pathway modulation derived from the decrease in oxidative stress. Indeed, reactive oxygen species (ROS) were reduced, while GSH levels were enhanced. This modulation could be ascribed to the presence of ROS regulating enzymes, such as SOD1 present into the AFSC-EV themselves. This study describes the ROS-modulating effects of extracellular vesicles alone, apart from their deriving stem cell, in an AD in vitro model, proposing AFSC-EV as a therapeutic tool to stop the progression of AD.

2020 - Prolonged hypoxia delays aging and preserves functionality of human amniotic fluid stem cells [Articolo su rivista]
Casciaro, F.; Borghesan, M.; Beretti, F.; Zavatti, M.; Bertucci, E.; Follo, M. Y.; Maraldi, T.; Demaria, M.
abstract

Human amniotic fluid stem cells (hAFSCs) are an emerging tool in regenerative medicine because they have the ability to differentiate into various lineages and efficiently improve tissue regeneration with no risk of tumorigenesis. Although hAFSCs are easily isolated from the amniotic fluid, their expansion ex vivo is limited by a quick exhaustion which impairs replicative potential and differentiation capacity. In this study, we evaluate various aging features of hAFSCs cultured at different oxygen concentrations. We show that low oxygen (1% O2) extends stemness and proliferative features, and delays induction of senescence-associated markers. Hypoxic hAFSCs activate a metabolic shift and increase resistance to pro-apoptotic stimuli. Moreover, we observe that cells at low oxygen remain capable of osteogenesis for prolonged periods of time, suggesting a more youthful phenotype. Together, these data demonstrate that low oxygen concentrations might improve the generation of functional hAFSCs for therapeutic use by delaying the onset of cellular aging.

2019 - A new approach for the separation, characterization and testing of potential prionoid protein aggregates through hollow-fiber flow field-flow fractionation and multi-angle light scattering [Articolo su rivista]
Marassi, Valentina; Beretti, Francesca; Roda, Barbara; Alessandrini, Andrea; Facci, Paolo; Maraldi, Tullia; Zattoni, Andrea; Reschiglian, Pierluigi; Portolani, Marinella
abstract

Protein misfolding and aggregation are the common mechanisms in a variety of aggregation-dependent diseases. The compromised proteins often assemble into toxic, accumulating amyloid-like structures of various lengths and their toxicity can also be transferred both in vivo and in vitro a prion-like behavior. The characterization of protein interactions, degradation and conformational dynamics in biological systems still represents an analytical challenge in the prion-like protein comprehension. In our work, we investigated the nature of a transferable cytotoxic agent, presumably a misfolded protein, through the coupling of a multi-detector, non-destructive separation platform based on hollow-fiber flow field-flow fractionation with imaging and downstream in vitro tests. After purification with ion exchange chromatography, the transferable cytotoxic agentwas analyzed with Atomic Force Microscopy and statistical analysis, showing that the concentration of protein dimers and low n-oligomer forms was higher in the cytotoxic sample than in the control preparation. To assess whether the presence of these species was the actual toxic and/or self-propagating factor, we employed HF5 fractionation, with UV and Multi-Angle Light Scattering detection, to define proteins molar mass distribution and abundance, and fractionate the sample into size-homogeneous fractions. These fractions were then tested individually in vitro to investigate the direct correlation with cytotoxicity. Only the later-eluted fraction, which contains high-molar mass aggregates, proved to be toxic onto cell cultures. Moreover, it was observed that the selective transfer of toxicity also occurs for one lower-mass fraction, suggesting that two different mechanisms, acute and later induced toxicity, are in place. These results strongly encourage the efficacy of this platform to enable the identification of protein toxicants.

2019 - Clusterin enhances AKT2-mediated motility of normal and cancer prostate cells through a PTEN and PHLPP1 circuit [Articolo su rivista]
Bertacchini, Jessika; Mediani, Laura; Beretti, Francesca; Guida, Marianna; Ghalali, Aram; Brugnoli, Federica; Bertagnolo, Valeria; Petricoin, Emanuel; Poti, Francesco; Arioli, Jessica; Anselmi, Laura; Bari, Alessia; Mccubrey, James; Martelli, Alberto M.; Cocco, Lucio; Capitani, Silvano; Marmiroli, Sandra
abstract

Clusterin (CLU) is a chaperone-like protein with multiple functions. sCLU is frequently upregulated in prostate tumor cells after chemo- or radiotherapy and after surgical or pharmacological castration. Moreover, CLU has been documented to modulate the cellular homolog of murine thymoma virus akt8 oncogene (AKT) activity. Here, we investigated how CLU overexpression influences phosphatidylinositol 3′-kinase (PI3K)/AKT signaling in human normal and cancer epithelial prostate cells. Human prostate cells stably transfected with CLU were broadly profiled by reverse phase protein array (RPPA), with particular emphasis on the PI3K/AKT pathway. The effect of CLU overexpression on normal and cancer cell motility was also tested. Our results clearly indicate that CLU overexpression enhances phosphorylation of AKT restricted to isoform 2. Mechanistically, this can be explained by the finding that the phosphatase PH domain leucine-rich repeat-containing protein phosphatase 1 (PHLPP1), known to dephosphorylate AKT2 at S474, is markedly downregulated by CLU, whereas miR-190, a negative regulator of PHLPP1, is upregulated. Moreover, we found that phosphatase and tensin homolog (PTEN) was heavily phosphorylated at the inhibitory site S380, contributing to the hyperactivation of AKT signaling. By keeping AKT2 phosphorylation high, CLU dramatically enhances the migratory behavior of prostate epithelial cell lines with different migratory and invasive phenotypes, namely prostate normal epithelial 1A (PNT1A) and prostatic carcinoma 3 (PC3) cells. Altogether, our results unravel for the first time a circuit by which CLU can switch a low migration phenotype toward a high migration phenotype, through miR-190-dependent downmodulation of PHLPP1 expression and, in turn, stabilization of AKT2 phosphorylation.

2019 - Comparing two visualization protocols for tomosynthesis in screening: specificity and sensitivity of slabs versus planes plus slabs [Articolo su rivista]
Iotti, V.; Giorgi Rossi, P.; Nitrosi, A.; Ravaioli, S.; Vacondio, R.; Campari, C.; Marchesi, V.; Ragazzi, M.; Bertolini, M.; Besutti, G.; Mori, C. A.; Pattacini, P.; Coriani, C.; Pescarolo, M.; Stefanelli, G.; Tondelli, G.; Beretti, F.; Caffarri, S.; Paterlini, L.; Canovi, L.; Colli, M.; Boschini, M.; Cavuto, S.; Braglia, L.
abstract

Objectives: Tomosynthesis (DBT) has proven to be more sensitive than digital mammography, but it requires longer reading time. We retrospectively compared accuracy and reading times of a simplified protocol with 1-cm-thick slabs versus a standard protocol of slabs + 1-mm-spaced planes, both integrated with synthetic 2D. Methods: We randomly selected 894 DBTs (including 12 cancers) from the experimental arm of the RETomo trial. DBTs were read by two radiologists to estimate specificity. A second set of 24 cancers (8 also present in the first set) mixed within 276 negative DBTs was read by two radiologists. In total, 28 cancers with 64 readings were used to estimate sensitivity. Radiologists read with both protocols separated by a 3-month washout. Only women that were positive at the screening reading were assessed. Variance was estimated taking into account repeated measures. Results: Sensitivity was 82.8% (53/64, 95% confidence interval (95% CI) 67.2–92.2) and 90.6% (95% CI 80.2–95.8) with simplified and standard protocols, respectively. In the random screening setting, specificity was 97.9% (1727/1764, 95% CI 97.1–98.5) and 96.3% (95% CI 95.3–97.1), respectively. Inter-reader agreement was 0.68 and 0.54 with simplified and standard protocols, respectively. Median reading times with simplified protocol were 20% to 30% shorter than with standard protocol. Conclusions: A simplified protocol reduced reading time and false positives but may have a negative impact on sensitivity. Key Points: • The adoption of digital breast tomosynthesis (DBT) in screening, more sensitive than mammography, could be limited by its potential effect on the radiologists’ workload, i.e., increased reading time and fatigue. • A DBT simplified protocol with slab only, compared to a standard protocol (slab plus planes) both integrated with synthetic 2D, reduced time and false positives but had a negative impact on sensitivity.

2019 - Influence of selenium on the emergence of neuro tubule defects in a neuron-like cell line and its implications for amyotrophic lateral sclerosis [Articolo su rivista]
Maraldi, T.; Beretti, F.; Anselmi, L.; Franchin, C.; Arrigoni, G.; Braglia, L.; Mandrioli, J.; Vinceti, M.; Marmiroli, S.
abstract

Impairment of the axonal transport system mediated by intracellular microtubules (MTs) is known to be a major drawback in neurodegenerative processes. Due to a growing interest on the neurotoxic effects of selenium in environmental health, our study aimed to assess the relationship between selenium and MTs perturbation, that may favour disease onset over a genetic predisposition to amyotrophic lateral sclerosis. We treated a neuron-like cell line with sodium selenite, sodium selenate and seleno-methionine and observed that the whole cytoskeleton was affected. We then investigated the protein interactome of cells overexpressing αTubulin-4A (TUBA4A) and found that selenium increases the interaction of TUBA4A with DNA- and RNA-binding proteins. TUBA4A ubiquitination and glutathionylation were also observed, possibly due to a selenium-dependent increase of ROS, leading to perturbation and degradation of MTs. Remarkably, the TUBA4A mutants R320C and A383 T, previously described in ALS patients, showed the same post-translational modifications to a similar extent. In conclusion this study gives insights into a specific mechanism characterizing selenium neurotoxicity.

2019 - Melanoma types by in vivo reflectance confocal microscopy correlated with protein and molecular genetic alterations: A pilot study [Articolo su rivista]
Beretti, F.; Bertoni, L.; Farnetani, F.; Pellegrini, C.; Gorelli, G.; Cesinaro, A. M.; Reggiani Bonetti, L.; Di Nardo, L.; Kaleci, S.; Chester, J.; Longo, C.; Massi, D.; Fargnoli, M. C.; Pellacani, G.
abstract

Cutaneous melanoma (CM) is one of the most prevalent skin cancers, which lacks both a prognostic marker and a specific and lasting treatment, due to the complexity of the disease and heterogeneity of patients. Reflectance confocal microscopy (RCM) in vivo analysis is a versatile approach offering immediate morphological information, enabling the identification of four primary cutaneous RCM CM types. Whether RCM CM types are associated with a specific protein and molecular genetic profiles at the tissue level remains unclear. The current pilot study was designed to identify potential correlations between RCM CM types and specific biological characteristics, combining immunohistochemistry (IHC) and molecular analyses. Eighty primary CMs evaluated at patient bedside with RCM (type 1 [19, 24%], type 2 [12, 15%], type 3 [7, 9%] and type 4 [42, 52%]) were retrospectively evaluated by IHC stains (CD271, CD20, CD31, cyclin D1), fluorescence in situ hybridization FISH for MYC gain and CDKN2A loss and molecular analysis for somatic mutations (BRAF, NRAS and KIT). RCM CM types correlated with markers of stemness property, density of intra-tumoral lymphocytic B infiltrate and cyclin D1 expression, while no significant association was found with blood vessel density nor molecular findings. RCM CM types show a different marker profile expression, suggestive of a progression and an increase in aggressiveness, according to RCM morphologies.

2018 - Amniotic fluid stem cell exosomes: Therapeutic perspective [Articolo su rivista]
Beretti, Francesca; Zavatti, Manuela; Casciaro, Francesca; Comitini, Giuseppina; Franchi, Fabrizia; Barbieri, Veronica; La Sala, Giovanni B.; Maraldi, Tullia
abstract

It is widely accepted that the therapeutic potential of stem cells can be largely mediated by paracrine factors, also included into exosomes. Thus, stem cell-derived exosomes represent a major therapeutic option in regenerative medicine avoiding, if compared to stem cells graft, abnormal differentiation and tumor formation. Exosomes derived from mesenchymal stem cells (MSC) induce damaged tissue repair, and can also exert immunomodulatory effects on the differentiation, activation and function of different lymphocytes. Therefore, MSC exosomes can be considered as a potential treatment for inflammatory diseases and also an ideal candidate for allogeneic therapy due to their low immunogenicity. Amniotic fluid stem cells (AFSCs) are broadly multipotent, can be expanded in culture, and can be easily cryopreserved in cellular banks. In this study, morphology, phenotype, and protein content of exosomes released into amniotic fluid in vivo and from AFSC during in vitro culture (conditioned medium) were examined. We found that AFSC-derived exosomes present different molecules than amniotic fluid ones, some of them involved in immunomodulation, such transforming growth factor beta and hepatic growth factors. The immunomodulatory effect of AFSC's exosomes on peripheral blood mononuclear cells stimulated with phytohemagglutinin was compared to that of the supernatant produced by such conditioned media deprived of exosomes. We present evidence that the principal effect of AFSC conditioned media (without exosomes) is the induction of apoptosis in lymphocytes, whereas exposure to AFSC-derived exosomes decreases the lymphocyte's proliferation, supporting the hypothesis that the entire secretome of stem cells differently affects immune-response. © 2017 BioFactors, 44(2):158–167, 2018.

2018 - Nuclear Nox4 interaction with prelamin A is associated with nuclear redox control of stem cell aging [Articolo su rivista]
Casciaro, Francesca; Beretti, Francesca; Zavatti, Manuela; McCubrey, James A.; Ratti, Stefano; Marmiroli, Sandra; Follo, Matilde Y.; Maraldi, Tullia
abstract

Mesenchymal stem cells have emerged as an important tool that can be used for tissue regeneration thanks to their easy preparation, differentiation potential and immunomodulatory activity. However, an extensive culture of stem cells in vitro prior to clinical use can lead to oxidative stress that can modulate different stem cells properties, such as self-renewal, proliferation, differentiation and senescence. The aim of this study was to investigate the aging process occurring during in vitro expansion of stem cells, obtained from amniotic fluids (AFSC) at similar gestational age. The analysis of 21 AFSC samples allowed to classify them in groups with different levels of stemness properties. In summary, the expression of pluripotency genes and the proliferation rate were inversely correlated with the content of reactive oxygen species (ROS), DNA damage signs and the onset premature aging markers, including accumulation of prelamin A, the lamin A immature form. Interestingly, a specific source of ROS, the NADPH oxidase isoform 4 (Nox4), can localize into PML nuclear bodies (PML-NB), where it associates to prelamin A. Besides, Nox4 post translational modification, involved in PML-NB localization, is linked to its degradation pathway, as it is also for prelamin A, thus possibly modulating the premature aging phenotype occurrence.

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 - Clusterin enhances migration and invasion of prostate cancer cells through an isoform-specific Akt2/miR-190/PHLPP1 circuit. [Abstract in Rivista]
Jessika Bertacchini, Marianna Guida; Mediani, Laura; Aram, Ghalali; Poti', Francesco; Arioli, Jessica; Federica, Brugnoli; Valeria, Bertagnolo; Beretti, Francesca; Lucio, Cocco; Capitani, Silvano; Palumbo, Carla; Marmiroli, Sandra
abstract

During prostate cancer progression cancer cells undergo a variety of molecular alterations that lead to the acquisition of uncontrolled growth properties. One such set of molecular alterations is mediated by the PI3K/Akt signaling pathway. Here, we describe a regulatory system that modulates the phosphoinosited 3-kinase/Akt (PI3K/Akt) pathway downstream of secreted Clusterin (sCLU) in normal and cancer prostate cells. The overexpression of sCLU is very frequent in prostate cancer, and can lead to Akt-activation. This prompted us to investigate how sCLU overexpression influences PI3K/Akt signaling network in a study model represented by human epithelial prostate PNT1A cells stably transfected with sCLU or with empty vector alone. We found that CLU cells show a marked differential phosphorylation of several members of the PI3K/Akt cascade, and in particular of Akt2. Moreover, we found that the phosphatase PHLPP1, known to dephosphorylate Akt2 at S473, is severely downregulated in CLU compared to MOCK cells. We thus investigated whether sCLU alters PHLPP1 protein stability or expression. Our results indicate that sCLU indeed stimulates PHLPP1 degradation by β-TrCP. Interestingly, we further demonstrated that sCLU alters also PHLPP1 through the negative regulator miR-190. Next, because sCLU has been reported to inhibit or to stimulate the aggressive behavior of cancer cells depending on the cell model, we investigated the effects of CLU overexpression or addition of recombinant Clusterin to the medium on cell migration and invasion in PNT1A cell line, which is not expected to display an invasive phenotype, and in the cancer prostate epithelial cell lines LNCaP and PC3. The result was extremely clear: not only CLU overexpression gives PNT1A cells the same behavior of wild-type PC3 cells, but also increases the migration and invasion index of all the above cell models by two to four times, compared to controls. As a confirmation, in the same model silencing of Clusterin abrogates migration of CLU cells. Next, the effect of Akt single-isoform silencing on cell migration was explored. While silencing of Akt1 affected migration only slightly, silencing of Akt2 prevented migration of both MOCK and CLU cells completely. The same result was obtained by pharmacological inhibition of Akt2. All together our results, clearly demonstrate for the first time that Clusterin can switch the low migration phenotype of normal prostate cells towards a high migration phenotype through the modulation of the expression of the PHLPP1 and, in turn, the activity of Akt2.

2017 - DEVELOPMENT OF A NOVEL METHOD FOR AMNIOTIC FLUID STEM CELL STORAGE [Articolo su rivista]
Zavatti, Manuela; Beretti, Francesca; Casciaro, Francesca; Comitini, Giuseppina; Franchi, Fabrizia; Barbieri, Veronica; Bertoni, Laura; DE POL, Anto; LA SALA, Giovanni Battista; Maraldi, Tullia
abstract

Background - Current procedures for collection of human Amniotic Fluid Stem Cells (hAFSCs) imply that amniotic fluid cells were cultured in flask for two weeks, than can be devoted to research purpose. However, hAFSCs could be retrieved directly from a small amount of amniotic fluid that can be obtained at the time of diagnostic amniocentesis. The aim of the study was to verify if a direct freezing of amniotic fluid cells is able to maintain and / or improve the potential of the sub-population of stem cells. Methods - We compared the potential of the hAFSCs depending on the moment in which they are frozen, cells obtained directly from amniotic fluid aspiration (D samples) and cells cultured in flask before freezing (C samples). Colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, senescence, apoptosis, and differentiation potential of C and D samples were compared. Results - hAFSCs isolated from D samples expressed MSC markers until later passages, had a good proliferation rate, and exhibited differentiation capacity similar to hAFSCs of C samples. Interestingly, the direct freezing induce a higher concentration of cells positive for pluripotency stem cell markers, without teratoma formation in vivo. Conclusions - This study suggests that minimal processing may be adequate for the banking of amniotic fluid cells, avoiding in vitro passages before the storage and exposure to high oxygen concentration affecting stem cell properties. This technique might be a reasonable approach in terms of costs and for the process of accreditation in GMP for a stem cell bank.

2017 - Human biliary tree stem/progenitor cells immunomodulation: Role of hepatocyte growth factor [Articolo su rivista]
Maraldi, Tullia; Guida, Marianna; Beretti, Francesca; Resca, Elisa; Carpino, Guido; Cardinale, Vincenzo; Gentile, Raffaele; Ardizzoni, Andrea; Murgia, Alba; Alvaro, Domenico; Gaudio, Eugenio; DE POL, Anto
abstract

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

2016 - Estrogen receptor signaling in the ferutinin-induced osteoblastic differentiation of human amniotic fluid stem cells [Articolo su rivista]
Zavatti, Manuela; Guida, M; Maraldi, Tullia; Beretti, Francesca; Bertoni, Laura; LA SALA, Giovanni Battista; DE POL, Anto
abstract

Ferutinin is a diaucane sesquiterpene with a high estrogenic activity. Since ferutinin is able to enhance osteoblastic differentiation of human amniotic fluid stem cells (hAFSCs), the aim of this study was to evaluate the role of the estrogen receptors α (ERα) and G-protein coupled receptor 30 (GPR30) in ferutinin-mediated osteoblastic differentiation. Moreover, it was investigated if MEK/ERK and PI3K/Akt signaling pathways are involved in ferutinin-induced effects.

2016 - NADPH oxidase-4 and MATER expressions in granulosa cells: Relationships with ovarian aging [Articolo su rivista]
Maraldi, Tullia; Resca, Elisa; Nicoli, Alessia; Beretti, Francesca; Zavatti, Manuela; Capodanno, Francesco; Morini, Daria; Palomba, Stefano; LA SALA, Giovanni Battista; DE POL, Anto
abstract

Aims Relevant roles in follicular development and ovulation are played by maternal antigen that embryos require (MATER), product of a maternal effect gene, and by reactive oxygen species (ROS), indispensable for the induction of ovulatory genes. At the moment, the relationship between these two biological systems and their involvement in the ovarian aging have not been still clarified. The aim of the current experimental study was to analyse the age-related changes of the MATER and NOX proteins. Materials and methods MATER and ROS homeostasis was studied in granulosa cells (GCs) and cumulus cells (CCs) of infertile patients who undergone oocyte retrieval for in vitro fertilization cycles using Western blot and confocal immunofluorescence analysis. Samples were obtained from subjects with age ≥ 40 years (cases) and with age ≤ 37 years (controls). Key findings The expression pattern of MATER and NOX observed in GCs was not different from that observed in CCs. High levels of both proteins were detected in the control samples. A significant lower expression of both MATER and NOX4 was observed in the case versus control samples. Significance The expression of MATER and NOX4 proteins are closely related to the follicular development and ovulation with particular regard for ovarian aging.

2015 - Critical-size bone defect repair using amniotic fluid stem cell/collagen constructs: Effect of oral ferutinin treatment in rats [Articolo su rivista]
Zavatti, Manuela; Bertoni, Laura; Maraldi, Tullia; Resca, Elisa; Beretti, Francesca; Guida, Marianna; La Sala, Giovanni Battista; De Pol, Anto
abstract

Aims: This study aims to evaluate the bone regeneration in a rat calvarias critical size bone defect treated with a construct consisting of collagen type I and human amniotic fluid stem cells (AFSCs) after oral administration of phytoestrogen ferutinin. Main methods: In 12 week old male rats (n = 10), we performed two symmetric full-thickness cranial defects on each parietal region, and a scaffold was implanted into each cranial defect. The rats were divided into four groups: 1) collagen scaffold, 2) collagen scaffold + ferutinin at a dose of 2 mg/kg/5 mL, 3) collagen scaffold + AFSCs, and 4) collagen scaffold + AFSCs + ferutinin. The rats were sacrificed after 4 weeks, and the calvariae were removed, fixed, embedded in paraffin and cut into 7 pm thick sections. Histomorphometric measures, immunohistochemical and immunofluorescence analyses were performed on the paraffin sections. Key findings: The histomorphometric analysis on H&E stained sections showed a significant increase in the regenerated area of the 4th group compared with the other groups. Immunohistochemistly performed with a human anti-mitochondrial antibody showed the presence of AFSCs 4 weeks after the transplant. Immunofluorescence analysis revealed the presence of osteocalcin and estrogen receptors (ER alpha. and GPR30) in all-groups, with a greater expression of all markers in samples where the scaffold was treated with AFSCs and the rats were orally administered ferutinin. Significance: Our results demonstrated that the oral administration of ferutinin is able to improve the bone regeneration of critical-size bone defects in vivo that is obtained with collagen-AFSCs constructs.

2015 - Enrichment in c-Kit improved differentiation potential of amniotic membrane progenitor/stem cells [Articolo su rivista]
Resca, Elisa; Zavatti, Manuela; Maraldi, Tullia; Bertoni, Laura; Beretti, Francesca; Guida, Marianna; La Sala, Giovanni Battista; Guillot, P. V; David, A. L; Sebire, N. J; De Pol, Anto; De Coppi, P.
abstract

Introduction Human term placenta has attracted increasing attention as an alternative source of stem cells for regenerative medicine since it is accessible without ethical objections. The amniotic membrane (AM) contains at least two stem cell types from different embryological origins: ectodermal amniotic epithelial stem cells, and mesodermal mesenchymal stromal cells. Among the second group we studied the characteristics of amniotic mesenchymal cells (AMC) versus the ones enriched for the commonly used surface marker c-Kit (amniotic progenitor/stem cells-ASC), a stem cell factor receptor with crucial functions in a variety of biological systems and presents in early progenitors of different origin, as been already demonstrated in the enriched chorionic stem cells. Methods After isolation, cells from the amniotic membranes (amniotic cells-AC) were selected for c-Kit (ASC) and compared these cells with c-Kit unselected (AMC), evaluating the expression of other stem cell markers (Oct-4, Tra-1-81, SSEA-4), CD271 and Slug. Results Immunofluorescence analysis showed that ASC cells exhibited greater stem cell marker expression and included more CD271 and Slug positive cells. This was consistent with the interpretation that c-Kit enriched AC show greater stemness capacity compared to c-Kit unselected AMC. Discussion AMC and ASC can both differentiate into various cell types including adipogenic, osteogenic, chondrogenic, neurogenic and hepatic lineages, but the enrichment in c-Kit improved stemness and differentiation potential of ASC.

2015 - Role of hepatocyte growth factor in the immunomodulation potential of amniotic fluid stem cells [Articolo su rivista]
Maraldi, Tullia; Beretti, Francesca; Guida, Marianna; Zavatti, Manuela; De Pol, Anto
abstract

Human amniotic fluid stem cells (hAFSCs) may be useful for regenerative medicine because of their potential to differentiate into all three germ layers and to modulate immune response with different types of secretion molecules. This last issue has not been completely elucidated. The aim of this study was to investigate the secretome profile of the hAFSC, focusing on the role of hepatocyte growth factor (HGF) in immunoregulation through short and long cocultures with human peripheral blood mononuclear cells. We found that HGF produced by hAFSCs exerts a cytoprotective role, inducing an increase in caspase-dependent apoptosis in human immune cells. This study provides evidence supporting the hypothesis that amniotic fluid is an ideal source of stem cells for expansion and banking properties for therapeutic use. hAFSCs not only are less immunogenic but also can secrete immunoregulatory factors that may be useful in autoimmune diseases or allogenic implants. SIGNIFICANCE: New information about the secretome pattern is reported in this paper. Human amniotic fluid stem cells (hAFSCs) possess immunomodulatory properties involving hepatocyte growth factor production. hAFSCs could be used in immunotherapies and might be able to avoid allogenic rejection

2014 - Human amniotic fluid stem cells: neural differentiation in vitro and in vivo [Articolo su rivista]
Maraldi, Tullia; Bertoni, Laura; Riccio, Massimo; Zavatti, Manuela; Carnevale, Gianluca; Resca, Elisa; Guida, Marianna; Beretti, Francesca; LA SALA, Giovanni Battista; DE POL, Anto
abstract

The successful integration of stem cells after their implantation into the brain has become a central issue in modern neuroscience. In this study, we test the neural differentiation potential of c-Kit(+)/Oct-4(+) human amniotic fluid stem cells (hAFSCs) in vitro and their survival and integration in vivo. hAFSCs were induced towards neural differentiation and specific markers (GFAP, β-III tubulin, CNPase, MAP2, NeuN, synapsines, S100, PMP22) were detected by immunofluorescence and Western blot analysis. Glial proteins were expressed as early as 2 weeks after the initial differentiation stimulus, whereas neuronal markers started to appear from the third week of differentiation under culturing conditions of high cell density. This timeline suggested that glial cells possessed a promoting role in the differentiation of hAFSCs towards a neuronal fate. hAFSCs were then implanted into the lateral ventricle of the brain of 1-day-old rats, since neuronal development occurs up to 1 month after birth in this animal model. Our data showed that hAFSCs survived for up to 6 weeks post-implantation, were integrated into various areas of the central nervous system and migrated away from the graft giving rise to mature neurons and oligodendrocytes. We conclude that hAFSCs are able to differentiate and integrate into nervous tissue during development in vivo.

2014 - Nuclear Nox4-derived reactive oxygen species in myelodysplastic syndromes [Articolo su rivista]
Guida, Marianna; Maraldi, Tullia; Beretti, Francesca; Follo, Matilde Y; Manzoli, Lucia; DE POL, Anto
abstract

A role for intracellular ROS production has been recently implicated in the pathogenesis and progression of a wide variety of neoplasias. ROS sources, such as NAD(P)H oxidase (Nox) complexes, are frequently activated in AML (acute myeloid leukemia) blasts and strongly contribute to their proliferation, survival, and drug resistance. Myelodysplastic syndromes (MDS) comprise a heterogeneous group of disorders characterized by ineffective hematopoiesis, with an increased propensity to develop AML. The molecular basis for MDS progression is unknown, but a key element in MDS disease progression is the genomic instability. NADPH oxidases are now recognized to have specific subcellular localizations, this targeting to specific compartments for localized ROS production. Local Nox-dependent ROS production in the nucleus may contribute to the regulation of redox-dependent cell growth, differentiation, senescence, DNA damage, and apoptosis. We observed that Nox1, 2, and 4 isoforms and p22phox and Rac1 subunits are expressed in MDS/AML cell lines and MDS samples, also in the nuclear fractions. Interestingly, Nox4 interacts with ERK and Akt1 within nuclear speckle domain, suggesting that Nox4 could be involved in regulating gene expression and splicing factor activity. These data contribute to the elucidation of the molecular mechanisms used by nuclear ROS to drive MDS evolution to AML.

2014 - Sirtuin 3 interacts with Lon protease and regulates its acetylation status. [Articolo su rivista]
Gibellini, Lara; Pinti, Marcello; Beretti, Francesca; Pierri, Cl; Onofrio, A; Riccio, Massimo; Carnevale, Gianluca; DE BIASI, Sara; Nasi, Milena; Torelli, F; Boraldi, Federica; DE POL, Anto; Cossarizza, Andrea
abstract

Lon is a mitochondrial protease that degrades oxidized damaged proteins, assists protein folding and participates in maintaining mitochondrial DNA levels. Changes in Lon mRNA levels, protein levels and activity are not always directly correlated, suggesting that Lon could be regulated at post translational level. We found that Lon and SIRT3, the most important mitochondrial sirtuin, colocalize and coimmunoprecipitate in breast cancer cells, and silencing or inhibition of Lon did not alter SIRT3 levels. Silencing of SIRT3 increased the levels of Lon protein and of its acetylation, suggesting that Lon is a target of SIRT3, likely at K917.

2013 - Human amniotic fluid-derived and dental pulp-derived stem cells seeded into collagen scaffold repair critical-size bone defects promoting vascularization. [Articolo su rivista]
Maraldi, Tullia; Riccio, Massimo; Pisciotta, Alessandra; Zavatti, Manuela; Carnevale, Gianluca; Beretti, Francesca; LA SALA, Giovanni Battista; A., Motta; DE POL, Anto
abstract

INTRODUCTION: The main aim of this study is to evaluate potential human stem cells, such as dental pulp stem cells (DPSC) and amniotic fluid stem cells (AFSC), combined with collagen scaffold, to reconstruct critical size cranial bone defects in animal model. METHODS: We performed two symmetric full-thickness cranial defects on each parietal region of rats and we replenished them with collagen scaffolds with or without stem cells already seeded into and addressed towards osteogenic lineage in vitro. After 4 and 8 weeks cranial tissue samples were taken for histological and immunofluorescence analysis. RESULTS: We observed a new bone formation in all the samples but the most relevant difference in defect correction were shown by stem cell-collagen samples at 4 weeks after implant, suggesting a faster regeneration ability of the combined constructs. The presence of human cells in the newly-formed bone was confirmed by confocal analysis with an antibody directed to a human mitochondrial protein. Furthermore, human cells were found to be an essential part of new vessel formation in the scaffold. CONCLUSIONS: All these data confirmed the strong potential of bioengineered constructs of stem cell-collagen scaffold for correcting large cranial defects in animal model and highlighting the role of stem cells in neo vascularization during skeletal defect reconstruction.

2013 - In vitro differentiation into insulin-producing β-cells of stem cells isolated from human amniotic fluid and dental pulp. [Articolo su rivista]
Carnevale, Gianluca; Riccio, Massimo; Pisciotta, Alessandra; Beretti, Francesca; Maraldi, Tullia; Zavatti, Manuela; Cavallini, Gian Maria; LA SALA, Giovanni Battista; Ferrari, Adriano; DE POL, Anto
abstract

AIM: To investigate the ability of human amniotic fluid stem cells and human dental pulp stem cells to differentiate into insulin-producing cells. METHODS: Human amniotic fluid stem cells and human dental pulp stem cells were induced to differentiate into pancreatic β-cells by a multistep protocol. Islet-like structures were assessed in differentiated human amniotic fluid stem cells and human dental pulp stem cells after 21 days of culture by dithizone staining. Pancreatic and duodenal homebox-1, insulin and Glut-2 expression were detected by immunofluorescence and confocal microscopy. Insulin secreted from differentiated cells was tested with SELDI-TOF MS and by enzyme-linked immunosorbent assay. RESULTS: Human amniotic fluid stem cells and human dental pulp stem cells, after 7 days of differentiation started to form islet-like structures that became evident after 14 days of induction. SELDI-TOF MS analysis, revealed the presence of insulin in the media of differentiated cells at day 14, further confirmed by enzyme-linked immunosorbent assay after 7, 14 and 21 days. Both stem cell types expressed, after differentiation, pancreatic and duodenal homebox-1, insulin and Glut-2 and were positively stained by dithizone. Either the cytosol to nucleus translocation of pancreatic and duodenal homebox-1, either the expression of insulin, are regulated by glucose concentration changes. Day 21 islet-like structures derived from both human amniotic fluid stem cells and human dental pulp stem cell release insulin in a glucose-dependent manner. CONCLUSION: The present study demonstrates the ability of human amniotic fluid stem cells and human dental pulp stem cell to differentiate into insulin-producing cells, offering a non-pancreatic, low-invasive source of cells for islet regeneration.

2013 - Inhibition of nuclear nox4 activity by plumbagin: effect on proliferative capacity in human amniotic stem cells. [Articolo su rivista]
Guida, Marianna; Maraldi, Tullia; Resca, Elisa; Beretti, Francesca; Zavatti, Manuela; Bertoni, Laura; LA SALA, Giovanni Battista; DE POL, Anto
abstract

Human amniotic fluid stem cells (AFSC) with multilineage differentiation potential are novel source for cell therapy. However, in vitro expansion leads to senescence affecting differentiation and proliferative capacities. Reactive oxygen species (ROS) have been involved in the regulation of stem cell pluripotency, proliferation, and differentiation. Redox-regulated signal transduction is coordinated by spatially controlled production of ROS within subcellular compartments. NAD(P)H oxidase family, in particular Nox4, has been known to produce ROS in the nucleus; however, the mechanisms and the meaning of this function remain largely unknown. In the present study, we show that Nox4 nuclear expression (nNox4) increases during culture passages up to cell cycle arrest and the serum starvation causes the same effect. With the decrease of Nox4 activity, obtained with plumbagin, a decline of nuclear ROS production and of DNA damage occurs. Moreover, plumbagin exposure reduces the binding between nNox4 and nucleoskeleton components, as Matrin 3. The same effect was observed also for the binding with phospho-ERK, although nuclear ERK and P-ERK are unchanged. Taken together, we suggest that nNox4 regulation may have important pathophysiologic effects in stem cell proliferation through modulation of nuclear signaling and DNA damage.

2013 - Skin aging: in vivo microscopic assessment of epidermal and dermal changes by means of confocal microscopy [Articolo su rivista]
Longo, Caterina; Casari, Alice; Beretti, Francesca; Cesinaro, Anna Maria; Pellacani, Giovanni
abstract

BACKGROUND: Skin aging is thought to be a complex biological process that is traditionally classified as intrinsic and extrinsic aging. Several clinical score and instrumental devices have been applied to obtain a precise assessment of skin aging. Among them, confocal microscopy has emerged as a new technique capable of assessing cytoarchitectural changes with a nearly histopathologic resolution. OBJECTIVE: We sought to determine the microscopic skin changes occurring on the face in different age groups by means of confocal microscopy. METHODS: The skin of the cheek in 63 volunteers belonging to distinct age groups was analyzed by confocal microscopy. In 4 cases, routine histopathology was performed on site-matched surplus areas from routine excisions for obtaining a better comparison with confocal findings. RESULTS: Young skin was characterized by regular polygonal keratinocytes and thin reticulated collagen fibers. With aging, more irregularly shaped keratinocytes and areas with unevenly distributed pigmentation and increased compactness of collagen fibers were observed. In the elderly, thinning of the epidermis, marked keratinocyte alterations, and huddles of collagen and curled fibers, corresponding to elastosis, were present. A side-by-side correlation between confocal descriptors and histopathologic aspects has been provided in a few cases. LIMITATIONS: Reticular dermal changes cannot be assessed because of the limited depth laser penetration. CONCLUSIONS: Confocal microscopy was successfully applied to identify in vivo skin changes occurring in aged skin at both the epidermal and dermal levels at histopathologic resolution. This offers the possibility to test cosmetic product efficacy and to identify early signs of sun damage.

2013 - The protein kinase Akt/PKB regulates both prelamin A degradation and Lmna gene expression [Articolo su rivista]
Bertacchini, Jessika; Beretti, Francesca; Vittoria, Cenni; Guida, Marianna; Federica, Gibellini; Mediani, Laura; Oriano, Marin; Nadir M., Maraldi; DE POL, Anto; Giovanna, Lattanzi; Lucio, Cocco; Marmiroli, Sandra
abstract

The serine/threonine kinase Akt/PKB is a major signaling hub integrating metabolic, survival, growth and cell cycle regulatory signals. The definition of the phospho-motif cipher driving phosphorylation by Akt led to the identification of hundreds of putative substrates, and it is therefore pivotal to name those whose phosphorylation by Akt is of consequence to biological processes. The Lmna gene products lamin A/C and their precursor prelamin A (collectively called A-type lamins) are type V intermediate filaments proteins forming a filamentous meshwork, the lamina, underneath the inner nuclear membrane, for nuclear envelope structures organization and interphase chromatin anchoring. In our previous work we reported that A-type lamins are phosphorylated by Akt at S301 and S404 in physiological conditions, and are therefore bona fide substrates of Akt. We describe here that Akt phosphorylation at S404 targets the precursor prelamin A for degradation. We further demonstrate that Akt regulates also Lmna transcription. All together, our study unveils a previously unknown function of Akt in the control of prelamin A stability and expression. Moreover, given the large number of diseases related to prelamin A, our findings represent a further important step bridging basic A-type lamins physiology to therapeutic approaches for lamin A-linked disorders.

2012 - Electron microscope study on a transmissible cytotoxic factor isolated from cerebrospinal fluid of neurological patients: analysis of its structure and of its effects on various types of cell cultures [Articolo su rivista]
Guerra, Deanna; Ronchetti, Ivonne; Beretti, Francesca; Bartoletti, Anna Maria; Pietrosemoli, Paola; Gelormini, M. S; Malagolini, N; Muscatello, Umberto; Portolani, Marinella
abstract

In previous studies, the present group showed that a factor, present in the cerebrospinal fluid of seven neuro-patients, was capable of inducing cell damages on cell cultures of epithelial cells (Vero), glial cells (DG54-MG) and human primary lymphocytes. The cytotoxicity, once induced, could be transmitted to fresh cell cultures using crude preparations obtained from the cytotoxic cell cultures.

2012 - Human serum promotes osteogenic differentiation of Human Dental Pulp Stem Cells in vitro and in vivo. [Articolo su rivista]
Pisciotta, Alessandra; Riccio, Massimo; Carnevale, Gianluca; Beretti, Francesca; Gibellini, Lara; Maraldi, Tullia; Cavallini, Gian Maria; Ferrari, Adriano; Bruzzesi, G; DE POL, Anto
abstract

Human dental pulp is a promising alternative source of stem cells for cell-based tissue engineering in regenerative medicine, for the easily recruitment with low invasivity for the patient and for the self-renewal and differentiation potential of cells. So far, in vitro culture of mesenchymal stem cells is usually based on supplementing culture and differentiation media with foetal calf serum (FCS). FCS is known to contain a great quantity of growth factors, and thus to promote cell attachment on plastic surface as well as expansion and differentiation. Nevertheless, FCS as an animal origin supplement may represent a potential means for disease transmission besides leading to a xenogenic immune response. Therefore, a significant interest is focused on investigating alternative supplements, in order to obtain a sufficient cell number for clinical application, avoiding the inconvenients of FCS use. In our study we have demonstrated that human serum (HS) is a suitable alternative to FCS, indeed its addition to culture medium induces a high hDPSCs proliferation rate and improves the in vitro osteogenic differentiation. Furthermore, hDPSCs-collagen constructs, pre-differentiated with HS-medium in vitro for 10 days, when implanted in immunocompromised rats, are able to restore critical size parietal bone defects. Therefore these data indicate that HS is a valid substitute for FCS to culture and differentiate in vitro hDPSCs in order to obtain a successful bone regeneration in vivo.

2012 - In vivo confocal microscopy for detection and grading of dysplastic nevi: a pilot study [Articolo su rivista]
Pellacani, Giovanni; Farnetani, Francesca; Gonzalez, G; Longo, Caterina; Cesinaro, Am; Casari, Alice; Beretti, Francesca; Seidenari, Stefania; Gill, M.
abstract

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2011 - A novel biomarker harvesting nanotechnology identifies Bak as a candidate melanoma biomarker in serum. [Articolo su rivista]
Longo, Caterina; G., Gambara; V., Espina; A., Luchini; B., Bishop; A. S., Patanarut; E. F., Petricoin; Beretti, Francesca; B., Ferrari; E., Garaci; DE POL, Anto; Pellacani, Giovanni; L. A., Liotta
abstract

Melanoma represents only 4\% of all skin cancers, but nearly 80\% of skin cancer deaths. This manuscript applies several new measurement technologies with the purpose of elucidating molecular signatures of melanoma aggressiveness.We sought to determine whether low-abundant serum proteins related to apoptotic pathways could be measured and correlated with defined melanoma subtypes. Hydrogel core shell nanoparticles, a new technology capable of selectively entrapping low molecular weight proteins and protecting them from enzymatic degradation, were used to capture candidate serum biomarkers. Biomarker levels were correlated with confocal microscopy, thereby representing a combination of new technologies for in vivo histologic documentation.Among a panel of analyzed serum proteins, Bak was differentially expressed between nevi and melanomas. Melanomas with higher Bak serum levels exhibited more pronounced junctional activity on confocal imaging, whereas lesions with 'sparse' dermal nests had weak Bak expression.Our study links serum proteome analysis with confocal microscopic clinical in vivo histologic classification of melanomas. Bak has not been previously measured in serum. Bak differential expression among melanoma subtypes confirms the importance of the apoptotic pathway as a contributor to melanoma aggressiveness.

2011 - Ankrd2/ARPP is a novel Akt2 specific substrate andregulates myogenic differentiation upon cellular exposure to H(2)O(2). [Articolo su rivista]
Cenni, Vittoria; Bavelloni, A; Beretti, Francesca; Tagliavini, F; Manzoli, L; Lattanzi, G; Maraldi, Nm; Cocco, L; Marmiroli, Sandra
abstract

Activation of Akt-mediated signaling pathways is crucial for survival, differentiation, and regeneration of muscle cells. A proteomic-based search for novel substrates of Akt was therefore undertaken in C(2)C(12) murine muscle cells exploiting protein characterization databases in combination with an anti-phospho-Akt substrate antibody. A Scansite database search predicted Ankrd2 (Ankyrin repeat domain protein 2, also known as ARPP) as a novel substrate of Akt. In vitro and in vivo studies confirmed that Akt phosphorylates Ankrd2 at Ser-99. Moreover, by kinase assay with recombinant Akt1 and Akt2, as well as by single-isoform silencing, we demonstrated that Ankrd2 is a specific substrate of Akt2. Ankrd2 is typically found in skeletal muscle cells, where it mediates the transcriptional response to stress conditions. In an attempt to investigate the physiological implications of Ankrd2 phosphorylation by Akt2, we found that oxidative stress induced by H(2)O(2) triggers this phosphorylation. Moreover, the forced expression of a phosphorylation-defective mutant form of Ankrd2 in C(2)C(12) myoblasts promoted a faster differentiation program, implicating Akt-dependent phosphorylation at Ser-99 in the negative regulation of myogenesis in response to stress conditions.

2011 - De novo melanoma and melanoma arising from pre-existing nevus: In vivo morphologic differences as evaluated by confocal microscopy [Articolo su rivista]
Longo, Caterina; Rito, Cintia; Beretti, Francesca; Cesinaro, Anna Maria; Piñeiro Maceira, Juan; Seidenari, Stefania; Pellacani, Giovanni
abstract

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2011 - Neutralization by human serum samples of a transmissible agent isolated from the cerebrospinal fluid of neurological patients. [Articolo su rivista]
Beretti, Francesca; P., Pietrosemoli; A. M., Bartoletti; M., Gerolmini; Bellei, Elisa; Tomasi, Aldo; Portolani, Marinella
abstract

A transmissible cytotoxic agent thought to be associated with one or more misfolded protein(s) was found in severalcerebrospinal fluid (CSF) samples from neurological patients. Since some experiments carried out to identify this unusualinfectious factor showed the block of its propagation by rabbit gammaglobulins (IgGs), the search for such anactivity by human IgGs was programmed. Neutralizing assays carried out using human sera as IgGs source showeda blocking property displayed by: twenty serum samples from as many patients with a diagnosis of acute infection,two of ten sera from healthy subjects and four serum samples from patients with lupus erythematous (SLE). Whenneutralizing sera were tested on cell cultures in immunofluorescence assays for the serum ability to label specific protein(s), similar fluorescent pictures resulted in treated and control cells. On the other hand, the SLE serum samplesdisclosed a granulosity of the nuclear material of cytotoxic cells in accordance with the DNA apoptotic laddering reportedin previous papers. Oxidative disorders, as suggested by the immunoblotting analysis of the antioxidant enzymesMn-superoxide dismutase (SOD2) and heme-oxygenase 1 (HO-1), point to an alteration of the oxidative pathwayamong the causes of the DNA damage induced by the cytotoxic transmissible agent under study.

2011 - Pigmented nodular Basal cell carcinomas in differential diagnosis with nodular melanomas: confocal microscopy as a reliable tool for in vivo histologic diagnosis. [Articolo su rivista]
Casari, Alice; Pellacani, Giovanni; Seidenari, Stefania; A. M., Cesinaro; Beretti, Francesca; P., Pepe; Longo, Caterina
abstract

Nodular basal cell carcinoma, especially when pigmented, can be in differential diagnosis with nodular melanomas, clinically and dermoscopically. Reflectance confocal microscopy is a relatively new imaging technique that permits to evaluate in vivo skin tumors with a nearly histological resolution. Here, we present four cases of challenging nodular lesions where confocal microscopy was able to clarify the diagnosis.

2009 - A-type lamins and signaling: the PI 3-kinase/Akt pathway moves forward [Articolo su rivista]
Marmiroli, Sandra; Bertacchini, Jessika; Beretti, Francesca; V., Cenni; M., Guida; DE POL, Anto; N. M., Maraldi; G., Lattanzi
abstract

Lamin A/C is a nuclear lamina constituent mutated in a number of human inherited disorders collectively referred to as laminopathies. The occurrence and significance of lamin A/C interplay with signaling molecules is an old question, suggested by pioneer studies performed in vitro. However, this relevant question has remained substantially unanswered, until data obtained in cellular and organismal models of laminopathies have indicated two main aspects of lamin A function. The first aspect is that lamins establish functional interactions with different protein platforms, the second aspect is that lamin A/C activity and altered function may elicit different effects in different cells and tissue types and even in different districts of the same tissue. Both these observations strongly suggest that signaling mechanisms targeting lamin A/C or its binding partners may regulate such a plastic behavior. A number of very recent data show involvement of kinases, as Akt and Erk, or phosphatases, as PP1 and PP2, in lamin A-linked cellular mechanisms. Moreover, altered activation of signaling in laminopathies and rescue of the pathological phenotype in animal models by inhibitors of signaling pathways, strongly suggest that signaling effectors related to lamin A/C may be implicated in the pathogenesis of laminopathies and may represent targets of therapeutic intervention. In face of such an open perspective of basic and applied research, we review current evidence of lamin A/C interplay with signaling molecules, with particular emphasis on the lamin A-Akt interaction and on the biological significance of their relationship.

2008 - Lamin A Ser404 Is a Nuclear Target of Akt Phosphorylation in C2C12 Cells [Articolo su rivista]
Vittoria, Cenni; Bertacchini, Jessika; Beretti, Francesca; Giovanna, Lattanzi; Alberto, Bavelloni; Riccio, Massimo; Maria, Ruzzene; Oriano, Marin; Giorgio, Arrigoni; Veena, Parnaik; Manfred, Wehnert; Nadir M., Maraldi; DE POL, Anto; Lucio, Cocco; Marmiroli, Sandra
abstract

Akt/PKB is a central activator of multiple signaling pathways coupled with a large number of stimuli. Although both localization and activity of Akt in the nuclear compartment are well-documented, most Akt substrates identified so far are located in the cytoplasm, while nuclear substrates have remained elusive. A proteomic-based search for nuclear substrates of Akt was undertaken, exploiting 2D-electrophoresis/MS in combination with an anti-Akt phosphosubstrate antibody. This analysis indicated lamin A/C as a putative substrate of Akt in C2C12 cells. In vitro phosphorylation of endogenous lamin A/C by recombinant Akt further validated this result. Moreover, by phosphopeptide analysis and point mutation, we established that lamin A/C is phosphorylated by Akt at Ser404, in an evolutionary conserved Akt motif. To delve deeper into this, we raised an antibody against the lamin A Ser404 phosphopeptide which allowed us to determine that phosphorylation of lamin A Ser404 is triggered by the well-known Akt activator insulin, and is therefore to be regarded as a physiological response. Remarkably, expression of S404A lamin A in primary cells from healthy tissue caused the nuclear abnormalities that are a hallmark of Emery-Dreifuss muscular dystrophy (EDMD) cells. Indeed, it is known that mutations at several sites in lamin A/C cause autosomal dominant EDMD. Very importantly, we show here that Akt failed to phosphorylate lamin A/C in primary cells from an EDMD-2 patient with lamin A/C mutated in the Akt consensus motif. Together, our data demonstrate that lamin A/C is a novel signaling target of Akt, and implicate Akt phosphorylation of lamin A/C in the correct function of the nuclear lamina.

2008 - Propagation of a transmissible cytotoxic activity on cultures of human peripheral blood lymphocytes [Articolo su rivista]
Marinella, Portolani; Beretti, Francesca; Anna M., Bartoletti; Paola, Pietrosemoli; Stefano, Salvioli; Claudio, Franceschi; Sena, Paola; DE POL, Anto
abstract

Positive results were attained when human peripheral blood lymphocytes (PBLs) were investigated for their ability to propagate a transmissible cytotoxic activity (TCA) isolated on VERO cell cultures from a sample of cerebrospinal fluid (CSF) drawn from a woman with ischemic brain injury. In consideration of this finding it can be assumed that "in vivo" blood lymphocytes contributed to give rise to the TCA detected "in vitro" in the CSF inoculum.

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.

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 - Cerebrospinal fluid samples from patients with various neurological disorders have a transmissible cytotoxic activity with similar properties [Articolo su rivista]
Beretti, Francesca; Pietrosemoli, Paola; Bartoletti, Anna Maria; Rondini, Ilaria; Portolani, Marinella
abstract

When inoculated into cell cultures to search for cytopathic viruses, six out of 384 cerebrospinal fluid (CSF) samples from patients with different neurological disorders proved to have a transmissible cytotoxic activity (TCA) not correlated to a conventional infectious agents. Properties shown by a TCA previously detected in the CSF sample of a patient with brain ischemia (Portolani et al., 2005) were shared by each of the newly isolated TCAs. We conclude that independently of the neurological clinical picture shown by the patient, the TCA detected in the CSF samples under study could have the same origin.

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

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

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

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

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

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

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 - 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 - Post-mortem diagnosis of encephalitis in a 75-year-old man associated with human herpesvirus-6 variant A [Articolo su rivista]
Portolani, Marinella; Tamassia, Mg; Gennari, W.; Pecorari, M.; Beretti, Francesca; Alu', M.; Maiorana, Antonino; Migaldi, Mario
abstract

Abstract An HHV-6 variant A infection is described in a 75 year-old man in association with meningoencephalitis identified at autopsy. The patient presented with fever and anorexia, then he developed altered consciousness, motor weakness, progressive lethargy, and coma, and died 21 days after hospital admission. Histopathological examination showed perivascular lymphocytic infiltrates in the central nervous system (CNS). Serum and cerebral spinal fluid (CSF) samples drawn from the patient were tested for viruses by a nested polymerase chain reaction (nPCR). HHV-6 primers A and C [Aubin et al., [1991]: J Clin Microb 29: 367-372] and HS6AE and HS6AF from [Dewhurst et al. ([1993]): J Clin Microb 31: 416-418] disclosed a 750 bp genomic product of HHV-6 in both types of biological samples. Restricted site analysis showed that the HHV-6 DNA amplified belonged to the variant A of the virus. Short sequences of HHV-6 DNA could also be detected in the DNA extracted from formalin-fixed, paraffin-embedded sections of CNS tissues by use of one (GM5 and GM6) of three pairs of HHV-6 primers that were selected. Immunohistochemical examination of brain sections, employing a specific monoclonal antibody directed against the HHV-6 gp 102 protein, detected the viral antigen in neurons and glial cells. J. Med. Virol. 77:244-248, 2005. © 2005 Wiley-Liss, Inc.

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 - Non-specific febrile illness by Coxsackievirus A16 in a 6-day-old newborn. [Articolo su rivista]
Portolani, M; Bartoletti, Am; Pietrosemoli, P; Beretti, Francesca; Della Casa Muttini, E; Roversi, Mf; Livio, L; Ferrari, Fabrizio
abstract

A case of non-specific febrile illness by Coxsackievirus A 16 (CA 16) in a 6-day-old newborn whose mother had developed hand, foot and mouth disease manifestations 2 days after delivery is reported. Notwithstanding the concurrence of negative circumstances like the presence of the enterovirus genome in the cerebrospinal fluid, absence of type specific neutralizing antibody and a few days of life, the newborn recovered 3 days after the onset of fever. This case, also characterized by the absence of mucosal and cutaneous erythematous manifestations typical of CA16 infection, draws attention to the possibility that non-specific febrile illness in newborns usually suspected of bacterial origin may have an enteroviral cause.

2004 - Search for viruses in the CSF of patients with acute neurological disorders: possible involvement of beta-and gamma-herpesviruses [Articolo su rivista]
Portolani, Marinella; Cartelli, Domenico; Beretti, Francesca; Tamassia, Maria Grazia; Gennari, William; Pecorari, Monica; Casolari, Chiara
abstract

In the years 1999-2001, 868 samples of cerebrospinal fluid (CSF) from as many patients with acute neurological manifestations of suspected viral origin were analysed for the presence of viruses at the Centre for the Diagnosis of Viral Diseases of the University of Modena and Reggio Emilia. Neurological patients included 788 immunocompetent subjects and 80 patients with impaired immunity due to human immunodeficiency virus (HIV) seropositivity. Of the CSF samples, 125 (15.8%) were positive for one or more viruses among the immunocompetent patients, whereas 33 (41.1%) were positive among the HIV cohort. DNA and RNA viruses were detected in the first group of CSF samples whereas only DNA viruses were found in the second group. In immunocompetent patients the frequency of enteroviruses prevailed over that of other RNA virus families (p = 0.001) and that of herpesviruses over the frequency of other DNA virus families (p = 0.001). Among herpesvirus members, the Epstein-Barr gamma-herpesvirus prevailed on alpha-herpesviruses in each of the two groups of patients (p = 0.05 in the immunocompetent group and p = 0.006 in HIV-positive patients). The clinical relevance both of this virus and of beta-herpesviruses as a cause of neurological disorders is discussed.

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 - 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.

2002 - Severe encephalopathy associated with reactivated human herpesvirus 6 in a six year-old immunocompetent child. [Articolo su rivista]
Portolani, Marinella; M., Pecorari; W., Gennari; Sabbatini, Anna Maria Teresa; M., Meacci; Beretti, Francesca; G., Frigieri; P., Bergonzini
abstract

When a six year-old immunocompetent child affected by encephalitis was subjected to virological studies, human herpesvirus 6 variant B2 resulted to be the cause of illness. Laboratory diagnosis based on the finding of human herpesvirus 6 genome in the cerebrospinal fluid of the patient both at the beginning of the disease and on the occasion of a relapse which occurred forty days after the patient's hospital discharge. The presence of high-avidity IgG to human herpesvirus 6 detected in the patient's serum at the time of the first hospital admission proved that he had suffered from a past infection by human herpesvirus 6. In the consequence of this, the human herpesvirus 6 DNA finding in the patient's cerebrospinal fluid was to ascribed to virus reactivation. In the light of virological and serological results, the clinical case described underlines the ability of human herpesvirus 6 to cause neurological disorders not only during primary infections but also during infections supported by rescued virus.

2002 - Symptomatic infections by toscana virus in the Modena province in the triennium 1999-2001. [Articolo su rivista]
Portolani, Marinella; Sabbatini, Anna Maria Teresa; Beretti, Francesca; W., Gennari; M. G., Tamassia; M., Pecorari
abstract

We report nine cases of clinical manifestations by Toscana virus (TOSV) diagnosed at the Centre for the Diagnosis of Viral Diseases of the University of Modena and Reggio Emilia, in the inhabitants of Modena province (Emilia Romagna region), in the triennium 1999-2001. TOSV was involved in six cases of meningitis, two of meningoencephalitis and one case of febrile erythema without meningeal manifestations. Six TOSV-infected patients had stayed for a few days in a different area of the Tuscany region before the onset of clinical manifestations so, only three cases of infections by TOSV had an autochthonous origin.

2001 - Case of fatal encephalitis by HHV-6 variant A [Articolo su rivista]
Portolani, Marinella; M., Pecorari; Mg, Tamassia; W., Gennari; Beretti, Francesca; Guaraldi, Giovanni
abstract

A fatal case is reported of encephalitis in an 85-year-old man caused by the human herpesvirus 6 variant A. The virological diagnosis was. based on the findings of the virus variant genomic sequences both in the cerebrospinal fluid and serum of the patient. Moreover, virus replication in nervous tissue was suggested by a viral load higher in the cerebrospinal fluid than in the peripheral blood. The association of a central nervous system infection with the A variant of human herpesvirus 6 is interesting because of the difficulty in establishing a pathological role for this virus strain. Epstein-Barr virus DNA was detected in the patient´s cerebrospinal fluid in association with human herpesvirus 6 DNA. The presence of the Epstein-Barr virus genomic sequences in the cerebro-spinal fluid was considered to be unimportant clinically.

2001 - Human herpesvirus 8 DNA in the cerebrospinal fluid of a patient with sensory impairment [Articolo su rivista]
Portolani, Marinella; Sabbatini, Anna Maria Teresa; W., Gennari; Beretti, Francesca; G., Greco; M., Santangelo
abstract

This note reports the finding of human herpes virus 8 DNA in the cerebrospinal fluid of a woman with sensory impairment correlated with a basal disease suspected to be multiple sclerosis.

2001 - Outbreak of aseptic meningitis by ECHO 4: Prevalence of clinical cases among adults [Articolo su rivista]
Portolani, Marinella; M., Pecorari; P., Pietrosemoli; A., Bartoletti; Sabbatini, Anna Maria Teresa; M., Meacci; W., Gennari; E., Bazzani; Beretti, Francesca; Guaraldi, Giovanni
abstract

Twenty five cases of meningitis occurred in urban areas surrounding a city (Modena) in Northern Italy, in the period May-July 1999. When the patients were admitted to the Infectious Diseases Division of the University of Modena and Reggio Emilia Hospital and studied by virological and serological methods, the meningitis proved to have an enteroviral origin and enterovirus ECHO 4 type was responsible for all cases of illness. An epidemiological characteristic of the enteroviral meninigitis outbreak was the adult age in 23 out of the 25 patients (mean age 24.50 +/- 7.84years). The monthly distribution of the aseptic meningitis cases was the following: five cases occurred in May, 13 in June and seven in July. The origin of the spread of the virus infection and the reason for its sudden end remained unknown. The unusual drop in temperature which occurred in the geographic area involved in the aseptic meningitis outbreak at the beginning of August could have interfered with the slowdown in virus circulation.