Monica MONTANARI - personale UniMoRe

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Monica MONTANARI

Personale tecnico amministrativo
Dipartimento di Scienze della Vita sede ex-Scienze Biomediche

Pubblicazioni

2021 - Magnesium favors the capacity of vitamin d3 to induce the monocyte differentiation of u937 cells [Articolo su rivista]
Parenti, S.; Sandoni, L.; Montanari, M.; Zanocco-Marani, T.; Anesi, A.; Iotti, S.; Manfredini, R.; Frassineti, C.; Davalli, P.; Grande, A.
abstract

The hematopoietic U937 cells are able to differentiate into monocytes, macrophages, or osteoclasts when stimulated, respectively, with vitamin D3 (VD3), phorbol 12-myristate 13-acetate (PMA) or PMA plus VD3. We have previously demonstrated that magnesium (Mg) strongly potentiates the osteoclastic differentiation of U937 cells. In this study, we investigated whether such an effect may be ascribed to a capacity of Mg to modulate the monocyte differentiation of U937 cells and/or to an ability of Mg and VD3 to act directly and independently on the early phases of the osteoclastic differentiation. To address this issue, we subjected U937 cells to an individual and combined treatment with Mg and VD3 and then we analyzed, by flow cytometry and quantitative real-time polymerase chain reaction, the expression of a number of genes related to the early phases of the differentiation pathways under consideration. The results obtained indicated that Mg favors the monocyte differentiation of U937 cells induced by VD3 and at the same time, Mg contrasts the inhibitory effect that VD3 exerts on the osteoclastic differentiation in the absence of PMA. The crucial and articulated role played by Mg in diverse pathways of the osteoclastic differentiation of U973 cells is emphasized.

2020 - 20-OH-ecdysone and extracellular ATP are not pro-autophagic factors for the lepidopteran fat bodycell line, IPLB-LdFB [Abstract in Atti di Convegno]
Ferrari, A; Fiorino, R; Montanari, M; Accorsi, A; Simonini, R; de Eguileor, M; Malagoli, D
abstract

2020 - Calpain Activation Is the Major Cause of Cell Death in Photoreceptors Expressing a Rhodopsin Misfolding Mutation [Articolo su rivista]
Comitato, A.; Schiroli, D.; Montanari, M.; Marigo, V.
abstract

The majority of mutations in rhodopsin (RHO) cause misfolding of the protein and has been linked to degeneration of photoreceptor cells in the retina. A lot of attention has been set on targeting ER stress for the development of new therapies for inherited retinal degeneration caused by mutations in the RHO gene. Nevertheless, the cell death pathway activated by RHO misfolded protein is still debated. In this study, we analyzed the retina of the knock-in mouse expressing the P23H misfolded mutant RHO. We found persistent unfolded protein response (UPR) during degeneration. Interestingly, long-term stimulation of the PERK branch of ER stress had a protective effect by phosphorylating nuclear factor erythroid 2–related factor 2 (NRF2) transcription factor, associated with antioxidant responses. Otherwise, we provide evidence that increased intracellular calcium and activation of calpains strongly correlated with rod photoreceptor cell death. By blocking calpain activity, we significantly decreased the activation of caspase-7 and apoptosis-inducing factor (AIF), two cell death effectors, and cell demise, and effectively protected the retina from degeneration caused by the P23H dominant mutation in RHO.

2020 - Chitosan/heparin polyelectrolyte complexes as ion-paring approach to encapsulate heparin in orally administrable SLN: In vitro evaluation [Articolo su rivista]
Maretti, E.; Pavan, B.; Rustichelli, C.; Montanari, M.; Dalpiaz, A.; Iannuccelli, V.; Leo, E.
abstract

Enhancing oral bioavailability of hydrophilic drugs by encapsulation in lipid-based nanocarriers, including Solid Lipid Nanoparticles (SLN), has been well documented. In this work, high molecular weight heparin was “insolubilized” by an “ion-paring” approach, forming Chitosan/Heparin Polyelectrolyte Complexes (PEC) to promote its encapsulation in SLN. Hybrid PEC-SLN, heparin-loaded SLN (H-SLN) as well as naked PEC were prepared and characterized regarding size, Z potential, morphology, drug loading and drug release. Physicochemical characterization of the nanoparticles was also performed by differential scanning calorimetry (DSC), and Fourier Transform Infra-Red (FTIR) analysis. FITC-labeled naked PEC along with Nile Red labeled PEC-SLN were assessed on CaCo-2 cells to study cytotoxicity as well as cell internalization ability by cytometric and confocal analysis. Transepithelial electrical resistance (TEER) was measured on NCM460 cell monolayers to evaluate whether chitosan may induce a modification of tight junctions’ integrity at epithelial level. Results showed that the minimum size of PEC (around 170 nm) was at pH 5.5 with a positive surface charge and after encapsulation in SLN produced hybrid PEC-SLN with a size of about 370 nm and a negative zeta potential. In comparison to both H-SLN and naked PEC, PEC-SLN were able to achieve a pH-controlled drug release and showed on CaCo-2 cells low toxicity and rapid internalization. Finally, TEER measurements highlighted that the hybrid nanocarriers were internalized without interference in the membrane resistance. Therefore, PEC-SLN could be considered valuable candidate for further in vivo investigations about the systemic bioavailability of oral heparin.

2018 - Pigment epithelium-derived factor hinders photoreceptor cell death by reducing intracellular calcium in the degenerating retina [Articolo su rivista]
Comitato, Antonella; Subramanian, Preeti; Turchiano, Giandomenico; Montanari, Monica; Becerra, S. Patricia; Marigo, Valeria
abstract

Calcium ions play a critical role in neuronal cell death. Pigment epithelium-derived factor (PEDF) is a promising neuroprotective protein for photoreceptor cells but the mechanisms mediating its effects against retinal degeneration are still not well characterized. We addressed this question in the rd1 degenerating mouse retina that bears a mutation in the Pde6b gene encoding one subunit of the phosphodiesterase enzyme. Loss of phosphodiesterase activity in rod photoreceptor cells increases cyclic guanosine monophosphate (cGMP) levels leading to a rise in intracellular calcium. Short-term treatments with recombinant human PEDF protein decreased intracellular calcium in photoreceptors in vivo. Taking advantage of calcium pump blockers, we defined that PEDF signaling acts on PMCA calcium pumps to lower intracellular calcium. PEDF restrained cell death pathways activated by high calcium levels and engaging calpains, BAX and AIF. The neurotrophic effects were mediated by the PEDF receptor (PEDF-R), encoded by the PNPLA2 gene. Finally, peptides containing the neurotrophic domain of PEDF targeted these same cell death pathways in vivo. The findings reveal rescue from death of degenerating photoreceptor cells by a PEDF-mediated preservation of intracellular calcium homeostasis.

2017 - Self-Assembled Lipid Nanoparticles for Oral Delivery of Heparin-Coated Iron Oxide Nanoparticles for Theranostic Purposes [Articolo su rivista]
Truzzi, Eleonora; Bongio, Chiara; Sacchetti, Francesca; Maretti, Eleonora; Montanari, Monica; Iannuccelli, Valentina; Vismara, Elena; Leo, Eliana Grazia
abstract

Recently, solid lipid nanoparticles (SLNs) have attracted increasing attention owing to their potential as an oral delivery system, promoting intestinal absorption in the lymphatic circulation which plays a role in disseminating metastatic cancer cells and infectious agents throughout the body. SLN features can be exploited for the oral delivery of theranostics. Therefore, the aim of this work was to design and characterise self-assembled lipid nanoparticles (SALNs) to encapsulate and stabilise iron oxide nanoparticles non-covalently coated with heparin (Fe@hepa) as a model of a theranostic tool. SALNs were characterised for physico-chemical properties (particle size, surface charge, encapsulation efficiency, in vitro stability, and heparin leakage), as well as in vitro cytotoxicity by methyl thiazole tetrazolium (MTT) assay and cell internalisation in CaCo-2, a cell line model used as an indirect indication of intestinal lymphatic absorption. SALNs of about 180 nm, which are stable in suspension and have a high encapsulation efficiency (>90%) were obtained. SALNs were able to stabilise the heparin coating of Fe@hepa, which are typically unstable in physiological environments. Moreover, SALNs-Fe@hepa showed no cytotoxicity, although their ability to be internalised into CaCo-2 cells was highlighted by confocal microscopy analysis. Therefore, the results indicated that SALNs can be considered as a promising tool to orally deliver theranostic Fe@hepa into the lymphatic circulation, although further in vivo studies are needed to comprehend further potential applications.

2016 - A novel 2,3-benzodiazepine-4-one derivative AMPA antagonist inhibits G2/M transition and induces apoptosis in human leukemia Jurkat T cell line [Articolo su rivista]
Parenti, Sandra; Casagrande, Giacomo; Montanari, Monica; Espahbodinia, M.; Ettari, R.; Grande, Alexis; Corsi, Lorenzo
abstract

It has been shown that the antagonism of glutamate receptors activity was able inhibit proliferation and induce apoptosis in several neuronal and non-neuronal cancer cell lines. In addition, it has been shown that glutamate might facilitate the spread and growth of leukemia T cells through interactions with AMPA receptors. The aim of the present study was to investigate the modulation of cell cycle elicited by a novel 2,3-benzodiazepine-4- one non-competitive AMPA antagonist derivative in the human leukemia Jurkat T cells. Our results indicated that the 1-(4-amino-3,5-dimethylphenyl)-3,5-dihydro-7,8-ethylenedioxy-4 h-2,3- benzodiazepin-4-one, named 1 g, exerted a significant growth inhibition of leukemia Jurkat T cells in a time and dose dependent manner, arresting the transition of G2/M phase through activation of Myt-1. The molecule also induced apoptosis through the enhanced expression of the pro-apoptotic p53, and the inhibition of Bcl-2, and Bcl-xl, followed by the activation of caspase-3. The results suggested that compound 1 g might act mostly as a cytostatic rather than cytotoxic compound. Al- though further studies are necessary, in order to identify others specific pathways involved in the activity of the present molecule, the presented results identified a novel molecule acting on specific G2/M checkpoint reg- ulation pathway. Finally, our data suggest that compound 1 g might be a good molecule for future development in the cancer research

2016 - Dominant and recessive mutations in rhodopsin activate different cell death pathways [Articolo su rivista]
Comitato, Antonella; DI SALVO, MARIA TERESA; Turchiano, Giandomenico; Montanari, Monica; Sakami, Sanae; Palczewski, Krzysztof; Marigo, Valeria
abstract

Mutations in rhodopsin (RHO) are a common cause of retinal dystrophy and can be transmitted by dominant or recessive inheritance. Clinical symptoms caused by dominant and recessive mutations in patients and animal models are very similar but the molecular mechanisms leading to retinal degeneration may differ. We characterized three murine models of retina degeneration caused by either Rho loss of function or expression of the P23H dominant mutation in Rho. Rho loss of function is characterized by activation of calpains and apoptosis-inducing factor (Aif) in dying photoreceptors. Retinas bearing the P23H dominant mutations activate both the calpain-Aif cell death pathway and ER-stress responses that together contribute to photoreceptor cell demise. In vivo treatment with the calpastatin peptide, a calpain inhibitor, was strongly neuroprotective in mice lacking Rho while photoreceptor survival in retinas expressing the P23H dominant mutation was more affected by treatment with salubrinal, an inhibitor of the ER-stress pathway. The further reduction of photoreceptor cell demise by co-treatment with calpastatin and salubrinal suggests co-activation of the calpain and ER-stress death pathways in mice bearing dominant mutations in the Rho gene.

2015 - In vitro behaviour of hybrid lipid/chitosan nanoparticles for the oral delivery of heparin [Abstract in Atti di Convegno]
Sacchetti, Francesca; Raffaella, Aracri; Maretti, Eleonora; Iannuccelli, Valentina; Montanari, Monica; Barbara, Pavan; Alessandro, Dalpiaz; Leo, Eliana Grazia
abstract

2015 - Monocyte-macrophage differentiation of acute myeloid leukemia cell lines by small molecules identified through interrogation of the Connectivity Map database [Articolo su rivista]
Manzotti, Gloria; Parenti, Sandra; Ferrari, Giovanna; Soliera, Angela Rachele; Cattelani, Sara; Montanari, Monica; Grande, Alexis; Calabretta, Bruno; Ertel, Adam; Cavalli, Daniel
abstract

The transcription factor C/EBPα is required for granulocytic differentiation of normal myeloid progenitors and is frequently inactivated in acute myeloid leukemia (AML) cells. Ectopic expression of C/EBPα in AML cells suppresses proliferation and induces differentiation suggesting that restoring C/EBPα expression/activity in AML cells could be therapeutically useful. Unfortunately, current approaches of gene or protein delivery in leukemic cells are unsatisfactory. However, "drug repurposing" is becoming a very attractive strategy to identify potential new uses for existing drugs. In this study, we assessed the biological effects of candidate C/EBPα-mimetics identified by interrogation of the Connectivity Map database. We found that amantadine, an antiviral and anti-Parkinson agent, induced a monocyte-macrophage-like differentiation of HL60, U937, Kasumi-1 myeloid leukemia cell lines, as indicated by morphology and differentiation antigen expression, when used in combination with suboptimal concentration of all trans retinoic acid (ATRA) or Vit D3. The effect of amantadine depends, in part, on increased activity of the vitamin D receptor (VDR), since it induced VDR expression and amantadine-dependent monocyte-macrophage differentiation of HL60 cells was blocked by expression of dominant-negative VDR. These results reveal a new function for amantadine and support the concept that screening of the Connectivity Map database can identify small molecules that mimic the effect of transcription factors required for myelo-monocytic differentiation.

2014 - In vitro evaluation on CaCo-2 cells of hybrid lipid/chitosan nanoparticles for the oral delivery of heparin. [Abstract in Atti di Convegno]
Sacchetti, Francesca; Aracri, Raffaella; Maretti, Eleonora; Iannuccelli, Valentina; Montanari, Monica; Leo, Eliana Grazia
abstract

Enhanced oral bioavailability of poorly aqueous soluble drugs encapsulated in a number of lipid-based formulations, including emulsions, micellar systems, self-emulsifying drug delivery systems, liposomes and solid lipid nanoparticles (SLN) via lymphatic delivery has been documented (1). In the present work, SLN were designed for the oral delivery of heparin in order to take advantage from the lymphatic intestinal transport pathway. In order to improve the incorporation of a high hydrophilic compound in a lipid matrix, heparin was “insolubilized” by the coupling with chitosan. In this aim we have developed chitosan/heparin Polyelectrolyte complexes (PEC). Such as systems are able to complex stably heparin (up to pH < 6.8) (2) and after pelletization by centrifugation were embedded in SLN obtaining a hybrid system lipid/chitosan nanoparticles (PEC-SLN). Since no in-vitro lymphoid tissue is currently available, CaCo-2 cell monolayer could be considered an alternative in vitro model to be used as a screening tool before animal studies are undertaken (1). Aim of the work In this work naked PEC, hybrid PEC-SLN as well as heparin-loaded SLN (Hep-SLN) were characterized as regard as the size, zeta potential, morphological characteristics, drug loading and in vitro drug release. Moreover FITC labeled PEC along with Red Nile labeled PEC-SLN and empty SLN were evaluated on CaCo-2 cell line in order to study their cytotoxicity by MTT test and their cell internalization ability by cytometric and confocal analysis. Experimentals Size and zeta potential were measured by Zetasizer Nano ZS (Malvern), morphological characteristics by SEM-FEI (SEM, Nova NanoSEM 450, Fei) and by AFM (Park Autoprobe Atomic Force Microscope (Park Instruments) Intenalization extent was visualized by confocal laser scanning microscopy (CLSM) (Leica DM IRE2) Results and discussion Results demonstrated that PEC size is highly influenced by pH, being 322 ±30 nm at the optimal pH 6.5. PEC-SLN and Hep-SLN displayed a size between 150 and 400 nm and a zeta potential from -20 to -36 mV. The drug loading was higher for PEC-SLN respect to Hep-SLN (50.4 ± 6 and 32.4 ± 4 UI/100 mg, respectively) while the heparin release rate was faster for Hep-SLN than for PEC-SLN, achieving a percentage of heparin released of 100% and 20%, respectively, in 6 h in simulated intestinal fluid. These data indicate that heparin was not well encapsulated in the Hep-SLN while after conjugation with chitosan stable heparin encapsulation was achieved in SLN. Finally naked PEC labeled with FITC along with PEC-SLN labeled with Red Nile was evaluated in vitro on CaCo-2 cells. MTT test showed that all the samples were poor cytotoxic even for long incubation time (overnight). Internalization data showed that PEC complexes (A) achieved a poor internalization level in the cells while PEC-SLN (B) have proved to be able to entry CaCo-2 cells in a time-dependent manner. Conclusions PEC-SLN being able to enter in the CaCo-2 cells unlike the naked PEC, probably due to their lipidic nature, can be considered promising carrier to be further studied as promoter for the lymphatic intestinal transport of heparin

2014 - Lipid-based microparticles for TB inhaled therapy: physical properties and cell internalization [Abstract in Atti di Convegno]
Maretti, Eleonora; Rossi, Tiziana; Leo, Eliana Grazia; Montanari, Monica; Romagnoli, Marcello; Sacchetti, Francesca; Iannuccelli, Valentina
abstract

Tuberculosis (TB) disease is caused by Mycobacterium tuberculosis that survives and replicates within human alveolar macrophages and is characterized by a long chronic stage of infection and progressive pathology mainly compromising (90% of cases) the respiratory system. Current TB therapies have exploited conventional routes of administration, such as oral or intramuscular, based on high and frequent dosages to maintain the drug therapeutic concentration in infection site because of poor drug permeability, poor drug bioavailability and pre-systemic clearance. An alternative acceptable therapy to systemic treatments involves inhalation route delivering the drug directly to the desired site, enabling a rapid onset of the action and avoiding the long period of the current treatment and the first-pass metabolism, as well as the use of high doses of drug resulting in drug resistance onset and in severe side effects on other organs. Inhaled TB therapy can presuppose the development of micro- or nanoparticles acting as drug carriers toward the alveolar region in the deepest lung so inducing the endocytosis process of alveolar macrophages being many antimicrobials difficult to cross cell membranes (1-3). Lipid-based particulate systems have been poorly investigated for TB inhaled therapy (4) though they were generally recognized as safe, poor liable to swell upon contact with the moisture located into the lungs and, consequently, to release the drug before the target site. Among the lipid-based particulate systems, Solid Lipid Microparticles (SLM), constituted by a solid lipid core stabilized by a surfactant at the surface, exhibit several favourable properties as production without organic solvents and long-term stability. In the present study, SLM loaded with rifampicin, a first-line anti-TB drug, were developed by the melt emulsification technique and evaluated in a perspective of an inhaled therapy for the treatment of TB infection. The lipid-based microparticles designed as rifampicin carrier showed features proper to be delivered from a DPI device, to deposit onto alveolar epithelium and to be internalized by macrophages in which Mycobacterium tuberculosis resides.

2013 - Design flexibility influencing the in vitro behavior of cationic SLN as a nonviral gene vector [Articolo su rivista]
Vighi, Eleonora; Montanari, Monica; Hanuskova, Miriam; Iannuccelli, Valentina; Coppi, Gilberto; Leo, Eliana Grazia
abstract

In this paper SLN were prepared using stearic acid as main lipid component, stearylamine as cationic agent and protamine as transfection promoter and adding phosphatidylcholine (PC), cholesterol (Chol) or both to obtain three different multicomponent SLN (SLN-PC, SLN-Chol and SLN-PC-Chol, respectively). Cytotoxicity and transfection efficiency of the obtained SLN:pDNA complexes were evaluated on three different immortalized cell lines: COS-I (African green monkey kidney cell line), HepG2 (human hepatocellular liver carcinoma cell line) and Na1300 (murine neuroblastoma cell line). Samples were characterized for the exact quantitative composition, particle size, morphology, zeta potential and pDNA binding ability. All the three SLN samples were about 250-300nm in size with a positive zeta potential, whereas SLN:pDNA complexes were about 300-400nm in size with a less positive zeta potential, depending on the SLN composition. Concerning the cell tolerance, the three samples showed a level of cytotoxicity lower than that of the positive control polyethylenimine (PEI), regardless of the cell lines. The best transfection performance was observed for SLN-PC-Chol on COS-I cells while a transfection level lower than PEI was observed on HepG2 cells, regardless the SLN composition. On Na1300 cells, SLN-Chol showed a double efficiency with respect to PEI. Comparing these results to those obtained with the same kind of SLN without PC and/or Chol, it is possible to conclude that the addition of Chol and/or PC to the composition of cationic SLN modify the cell tolerance and the transfection efficiency of the gene vector in a manner strictly dependent on the cell type and the internalization pathways.

2013 - The Orosomucoid1 protein is involved in the vitamin D – mediated macrophage de-activation process [Articolo su rivista]
Gemelli, Claudia; Martello, Andrea; Montanari, Monica; ZANOCCO MARANI, Tommaso; Salsi, Valentina; Zappavigna, Vincenzo; Parenti, Sandra; Vignudelli, Tatiana; Selmi, Tommaso; Ferrari, Sergio; Grande, Alexis
abstract

Orosomucoid 1 (ORM1), also named Alpha 1 acid glycoprotein A (AGP-A), is an abundant plasma protein characterized by anti-inflammatory and immune-modulating properties. The present study was designed to identify a possible correlation between ORM1 and Vitamin D3 (1,25(OH)2D3), a hormone exerting a widespread effect on cell proliferation, differentiation and regulation of the immune system. In particular, the data described here indicated that ORM1 is a 1,25(OH)2D3 primary response gene, characterized by the presence of a VDRE element inside the 1kb sequence of its proximal promoter region. This finding was demonstrated with gene expression studies, Chromatin Immunoprecipitation and luciferase transactivation experiments and confirmed by VDR full length and dominant negative over-expression. In addition, several experiments carried out in human normal monocytes demonstrated that the 1,25(OH)2D3 - VDR – ORM1 pathway plays a functional role inside the macrophage de-activation process and that ORM1 may be considered as a signaling molecule involved in the maintenance of tissue homeostasis and remodeling.

2012 - The role of protamine amount in the transfection performance of cationic SLN designed as a gene nanocarrier [Articolo su rivista]
Vighi, Eleonora; Montanari, Monica; Ruozi, Barbara; Iannuccelli, Valentina; Leo, Eliana Grazia
abstract

Cationic solid lipid nanoparticles (SLN) have been recently proposed as non-viral vectors in systemic gene therapy. The aim of this study was to evaluate the effect of the protamine amount used as the transfection promoter in SLN-mediated gene delivery. Three protamine-SLN samples (Pro25, Pro100, and Pro200) prepared by adding increasing amounts of protamine were characterized for their size, zeta potential, and protamine loading level. The samples were evaluated for pDNA complexation ability by gel-electrophoresis analysis and for cytotoxicity and transfection efficiency by using different cell lines (COS-I, HepG2, and Na1300). The size of SLN was ~230 nm and only Pro200 showed few particle aggregates. Unlike the Pro25 sample with the lowest protamine loading level, the others SLN samples (Pro100 and Pro200) exhibited a good ability in complexing pDNA. A cell-line dependent cytotoxicity lower than that of the positive control PEI (polyethilenimmine) was observed for all the SLN. Among these, only Pro100, having an intermediate amount of protamine, appeared able to promote pDNA cell transfer, especially in a neuronal cell line (Na1300). In conclusion, the amount of protamine as the transfection promoter in SLN affects not only the gene delivery ability of SLN but also their capacity to transfer genes efficiently to specific cell types.

2011 - Alpha – 1 – acid glycoprotein-A is a new VDR transcriptional target involved in monocyte differentiation and activation processes. [Poster]
Gemelli, Claudia; Martello, Andrea; Montanari, Monica; Parenti, Sandra; Vignudelli, Tatiana; Selmi, Tommaso; ZANOCCO MARANI, Tommaso; Ferrari, Sergio; Grande, Alexis
abstract

Orosomucoid 1 (ORM1), also named Alpha 1 acid glycoprotein A (AGP-A), is an abundant plasma protein characterized by anti-inflammatory and immune-modulating properties. The present study was designed to identify a possible correlation between ORM1 and Vitamin D3 (1,25(OH)2D3), a hormone exerting a widespread effect on cell proliferation, differentiation and regulation of the immune system. In particular, the data described here indicated that ORM1 is a 1,25(OH)2D3 primary response gene, characterized by the presence of a VDRE element inside the 1kb sequence of its proximal promoter region. This finding was demonstrated with gene expression studies, Chromatin Immunoprecipitation and luciferase transactivation experiments and confirmed by VDR full length and dominant negative over-expression. In addition, several experiments carried out in human normal monocytes demonstrated that the 1,25(OH)2D3--VDR--ORM1 pathway plays a functional role inside the macrophage de-activation process and that ORM1 may be considered as a signaling molecule involved in the maintenance of tissue homeostasis and remodeling.

2011 - Characterization of the cell growth inhibitory effects of a novel DNA-intercalating bipyridyl-thiourea-Pt(II) complex in cisplatin-sensitive and-resistant human ovarian cancer cells [Articolo su rivista]
Marverti, Gaetano; Ligabue, Alessio; Montanari, Monica; Guerrieri, Davide; M., Cusumano; M. L., Di Pietro; L., Troiano; DI VONO, Elena; S., Iotti; G., Farruggia; F., Wolf; Monti, Maria Giuseppina; Frassineti, Chiara
abstract

The cellular effects of a novel DNA-intercalatingagent, the bipyridyl complex of platinum(II) with diphenylthiourea, [Pt(bipy)(Ph2-tu)2]Cl2, has been analyzed in thecisplatin (cDDP)—sensitive human ovarian carcinoma cellline, 2008, and its—resistant variant, C13* cells, in which thehighest accumulation and cytotoxicity was found among sixrelated bipyridyl thiourea complexes. We also show here thatthis complex causes reactive oxygen species to form andinhibits topoisomerase II activity to a greater extent in thesensitive than in the resistant line. The impairment of thisenzyme led to DNA damage, as shown by the comet assay.As a consequence, cell cycle distribution has also beengreatly perturbed in both lines. Morphological analysisrevealed deep cellular derangement with the presence of cellular masses, together with increased membrane permeability and depolarization of the mitochondrial membrane. Some of these effects, sometimes differentially evident between the two cell lines, might also be related to the decrease of total cell magnesium content caused by this thiourea complex both in sensitive and resistant cells, though the basal content of this ion was higher in the cDDP-resistant line. Altogether these results suggest that this compound exerts its cytotoxicity by mechanisms partly mediated by the resistance phenotype. In particular, cDDP-sensitive cells were affected mostly by impairing topoisomerase II activity and by increasing membrane permeability and the formation of reactive oxygen species; conversely, mitochondrial impairment appeared to play the most important role in the action of complex F in resistant cells.

2011 - Microparticulate polyelectrolyte complexes for gentamicin transport across intestinal epithelial [Articolo su rivista]
Iannuccelli, Valentina; Montanari, Monica; Bertelli, Davide; Pellati, Federica; Coppi, Gilberto
abstract

Polysaccharide microparticles for the oral administration of gentamicin were designed in order to obtainan increased drug absorption by means of microparticle transport across the intestinal epithelia. Alginate/chitosan microparticles with a size of ∼ 2 μm were developed by spray-drying a water solution containingthe drug complexed with the polyanionic alginate and subsequent alginate cross-linking process bycalcium ions and chitosan. The pre-formulation study, performed by changing the concentration of bothcross-linkers, led to the selection of the most suitable formulation which was assayed for its capacity to be translocated across intestinal epithelia, via both M cells contained in Follicle Associated Epithelium (FAE) ofPeyer’s patches and enterocytes of the mucosal epithelium. An ex vivo perfusion technique of rabbit andrat intestinal tissues containing Peyer’s patches combined with an in vitro method by using Caco-2 cellmonolayers demonstrated the microparticulate carrier ability to be taken up by both M cells and enterocytes.However, only the endocytosis by M cells appeared to provide the microparticle transport from theepithelium toward deeper sub-epithelial regions.

2011 - Structural investigation and intracellular trafficking of a novel multicomposite cationic solid lipid nanoparticle platform as a pDNA carrier [Articolo su rivista]
Vighi, Eleonora; Leo, Eliana Grazia; Montanari, Monica; Mucci, Adele; Hanuskova, Miriam; Iannuccelli, Valentina
abstract

Background: The ability to efficiently cross cellular barriers and accomplish high-level transgene expression is a critical challenge to broad application of nonviral vectors, such as cationic solid lipid nanoparticles (SLN).Aims: This study aims to design and characterize in vitro multicomposite SLN as a novel platform for pDNA delivery.Results/Discussion: The distribution of each component (stearic acid, stearylamine, phosphatidylcholine, cholesterol, protamine and Pluronic F68) in the SLN matrix was studied by electron spectroscopy for chemical analysis and NMR in order to establish its influence on SLN cytotoxicity and transfection efficiency. Multicomposite SLN mediated the expression of enhanced green fluorescent protein in a way comparable with the positive control,but inducing a lower cytotoxicity. Moreover, the carrier exhibited the ability to enter the nucleoli, probably as a result of the synergic action of the nuclear localization signal of protamine and the flexibility of the lipid matrix owing to the phosphatidylcholine. Conclusion: The multicomposite SLN showed good transfection efficiency and negligible cytotoxicity, both crucial factors for an efficient gene-delivery system. Considering the fact that nucleolihave emerged in recent years as important targets in many fields, this novel carrier could have significant future therapy involvements whenever there is a requirement to overcome subcellular barriers. However, further work needs to be carried out in order to fully characterize the formulation, to elucidate where alternative colloidal structures might exist and play a role in obtaining the results presented.

2010 - Cellular uptake and toxicity of microparticles in a perspective of polymyxin B oral administration [Articolo su rivista]
Coppi, Gilberto; Montanari, Monica; Rossi, Tiziana; Bondi, Moreno; Iannuccelli, Valentina
abstract

Alginate/chitosan microparticles for the targeting of Polymyxin B to M-cells were assayed for transport ability by enterocytes. Caco-2 cells model, combined with confocal microscopy, showed that microparticles were endocytosed. Furthermore, microparticles maintained the biological activity of the antibiotic and decreased the antibiotic cytotoxicity against Vero cells cultures. Therefore, simultaneous pathways via both M-cells and enterocytes could be proposed for such a microparticulate carrier.

2010 - Mesalazine inhibits the beta-catenin signalling pathway acting through the upregulation of mu-protocadherin gene in colo-rectal cancer cells [Articolo su rivista]
Parenti, Sandra; Ferrarini, F; Zini, Roberta; Montanari, Monica; Losi, Lorena; Canovi, B; Ferrari, Sergio; Grande, Alexis
abstract

BACKGROUND: Several reports indicate that mesalazine (5-aminosalicylic acid, 5-ASA) is a promising candidate for the chemoprevention of colo-rectal cancer because of its ability to reach the purpose avoiding the unwanted side effects usually associated with prolonged administration of nonsteroidal anti-inflammatory drugs. This activity of 5-ASA is probably the consequence of a number of effects determined on colo-rectal cancer cells, consisting of reduced proliferation, increased apoptosis and activation of cell cycle checkpoints and DNA repair processes. A recent observation has suggested that inhibition of beta-catenin signalling could induce these cellular effects. AIM: To characterize better the capacity of 5-ASA to inhibit the beta-catenin signalling pathway. METHODS: Genes belonging to the beta-catenin signalling pathway were analysed in colo-rectal cancer cell lines treated with 5-ASA using a combination of laboratory assays that are able to detect their phenotypic expression and functional activity. RESULTS: The results obtained indicated that 5-ASA induces the expression of a protein called mu-protocadherin that belongs to the cadherin superfamily and is able to sequester beta-catenin on the plasmatic membrane of treated cells hampering its function. CONCLUSION: These findings suggest that mu-protocadherin might be employed as a biological marker to monitor the chemopreventive efficacy of 5-ASA.

2010 - Nuclear localization of cationic solid lipid nanoparticles containing Protamine as transfection promoter [Articolo su rivista]
Vighi, Eleonora; Montanari, Monica; Ruozi, Barbara; Tosi, Giovanni; Magli, Alessandro; Leo, Eliana Grazia
abstract

Protamine has attracted much attention as DNA condenser and nuclear transfer enhancer although theexcess of hydrophilicity and the strong DNA pack restrain its potentialities. In order to overcome this lim-itation, we added Protamine in the composition of solid lipid nanoparticles (SLN-Protamine) and we com-pared this carrier with the same kind of SLN containing Esterquat 1 instead of Protamine (SLN-EQ1).Carriers cytotoxicity was assessed on COS-I cells evaluating the cell cycle by propidium iodide test, whilethe transfection efficiency was studied using pEGFP as plasmid model. The cell penetrating activity ofProtamine inside the lipid vectors was evaluated studying cell internalization by confocal microscopyusing Red Nile-labeled carriers. SLN-Protamine:pDNA showed a mean diameter five-times smaller thanthe size of SLN-EQ1:pDNA and a remarkably lesser cytotoxicity. Transfection by SLN-Protamine:pDNAwas seven-times more effective compared with the Protamine:pDNA polyplexes while no transfectioncapacity was observed for SLN-EQ1:pDNA complexes due to their inability to be internalized owing totheir larger dimension. Red Nile-SLN-Protamine were localized in endocytic-like vesicles into the nuclearmembrane suggesting the inclusion of Protamine in nano-lipophilic systems may enhance the reductionin the complex dimensions, the nuclear pDNA translocation and the pDNA release in the cell

2010 - The role of protamine in the gene delivery by cationic SLN on different cell lines [Abstract in Atti di Convegno]
Vighi, Eleonora; Montanari, Monica; Ruozi, Barbara; Battini, Renata; Leo, Eliana Grazia
abstract

In this study we produced cationic SLN using stearicacid, stearylamine and protamine sulfate astransfection promoter, in order to improve SLN transfection capacity without the use of adjuvantsubstances. In fact, protamine is a cationic smallprotein with high arginine content that is FDAapproved for the parenteral administration. Since protamine is a nuclear proteinthat helps DNA packaging in sperm cells, it is also used as transfection acceleratorin the gene delivery.Therefore, in this work we optimized the protamineamount in SLN formulation in order to evaluate therole of this protein in the transfection activity ofcationic SLN.

2010 - pDNA condensation capacity and in vitro gene delivery properties of cationic solid lipid nanoparticles [Articolo su rivista]
Vighi, Eleonora; Ruozi, Barbara; Montanari, Monica; Battini, Renata; Leo, Eliana Grazia
abstract

Cationic solid lipid nanoparticles (SLN) are promising nonviral gene delivery carriers suitable for systemic administration. The objective of this study was to investigate the relationship between the composition of cationic SLN and their ability to condense plasmid DNA (pDNA) and to transfer it in neuroblastoma cells. The SLN were prepared by using stearic acid and stearylamine as lipid core along with Esterquart 1 (EQ1) or Protamine obtaining two samples (SLN-EQ1 and SLN-Protamine, respectively). The cationic SLN were freeze-dried after preparation and their physical–chemical properties, including the surface composition and the transfection efficiency were investigated. The results showed that the two samples had similar size, zeta potential and pDNA binding properties but SLN-Protamine were able to condense pDNA more efficaciously than SLN-EQ1 forming smaller and less positive complexes. SLN-Protamine:pDNA complexes demonstrated to be less cytotoxic and more efficient in the transfection of Na1300 cell line than SLN-EQ1:pDNA. These findings were attributed to the different surface composition of the two samples and in particular to the localization of the Protamine on the surface of the particle while EQ1 in the lipid core. In conclusion the results here suggest that not only the z-potential but also the surface composition may affect the pDNA condensation proprieties and thus the transfection efficiency of nonviral gene nanocarriers.

2009 - Cationic solid lipid nanoparticles containing Protamine as transfection vector on neuroblastoma cell line [Relazione in Atti di Convegno]
Vighi, Eleonora; Ruozi, Barbara; Tosi, Giovanni; Battini, Renata; Montanari, Monica; Leo, Eliana Grazia
abstract

Sono stati presentati i risultati di studi effettuati in vitro sull'efficienza di transfezione di SLN allestite con diversi componenti tra i quali protamina.

2009 - Development of new formulations of cationic solid lipid nanoparticles (SLNs) for the drug delivery to the brain [Relazione in Atti di Convegno]
Vighi, Eleonora; Ruozi, Barbara; Montanari, Monica; Leo, Eliana Grazia
abstract

Development of new formulations of cationic solid lipid nanoparticles (SLNs) for the drug delivery to the brain

2009 - Flow cytometry and live confocal analysis for the evaluation of the uptake and intracellular distribution of FITC-ODN into HaCaT cells [Articolo su rivista]
Ruozi, Barbara; Montanari, Monica; Vighi, Eleonora; Tosi, Giovanni; Tombesi, Andrea; Battini, Renata; Restani, Cinzia; Leo, Eliana Grazia; Forni, Flavio; Vandelli, Maria Angela
abstract

In this study the mechanism of the internalisation and the cellular distribution of 5’ fluorescein conjugated PS-ODN (FITC-ODN) after transfection with different mixed lipidic vesicles/oligo complexes (lipoplexes) have been investigated. Mixed lipidic vesicles were prepared with one of the most used cationic lipid (DOTAP) and different amount of a cholic acid (UDCA) to release the oligo into HaCaT cells. Using flow cytometry, the cellular uptake of the oligo was studied with and without different inhibitors able to block selectively the different pathways involved in the internalisation mechanism. The intracellular distribution of the oligo was analysed by confocal laser scanning microscopy (CLSM) treating the cells with the lipoplexes and directly observing without any fixing procedure. To better carry out the co-localization studies, fluorescent labelled markers, specific for the different cellular compartments, were co-incubated with FITC-ODN.The different lipidic vesicles affect the internalisation mechanism of FITC-ODN. After using the inhibitors, the uptake of complexes involved a different internalization mechanism. The live CLSM analysis demonstrated that, after 1h from the complex incubation, the oligo was transferred into cells and localized into the endosomes; after 24 h, oligo was intracellularly localized close to the nuclear structure in a punctuate pattern. However, the results from fusion experiments showed also a binding of a quite amount of oligo with the cell membranes.

2009 - Formulazione e caratterizzazione di nanoparticelle polimeriche per la somministrazione orale di eparina [Abstract in Atti di Convegno]
Vighi, Eleonora; Ruozi, Barbara; Montanari, Monica; Leo, Eliana Grazia
abstract

Nanoparticelle polimeriche per la somministrazione di eparina. studi di caratterizzazione tecnologica ed uptake in CACO-2

2008 - Intestinal uptake of Polymyxin B microparticles [Abstract in Atti di Convegno]
Coppi, Gilberto; Montanari, Monica; Mattioli, Federico; Iannuccelli, Valentina
abstract

Introduction Polymyxin B (PMB) is a cationic peptidic antibiotic used extensively for parenteral and topical treatment of Gram-negative infections. However, PMB therapy is associated with considerable toxicity, mainly nephrotoxicity and neurotoxicity. The oral route for PMB administration has not been considered owing to the pH value and proteolytic degradation in the gastrointestinal tract, as well as the negligible absorption through the intestinal absorption, probably owing to its cationic charge. Therefore, to achieve a PMB oral administration perspective, providing less toxicity and cationic charge, calcium alginate/chitosan microparticles were designed. The developed microparticles, previously characterized in vitro and in vivo, showed clear advantages in terms of reduced PMB toxicity and prolonged systemic antibacterial effects [1-3]. To examine the microparticle uptake process, in the present study, cell internalization capacity of fluorescent microparticles loaded with PMB was evaluated by means of Caco-2 cell lines and quantified by cytofluorimetry. Moreover, microparticle location inside the cells was revealed by confocal microscopy. Experimental methods Microparticle preparation The microparticle preparation and the in vitro characterisation were previously described [1]. Shortly, fluorescent calcium alginate/chitosan (CaA/CHT/FITC) microparticles were prepared by spray-drying a 0.5% water solution of NaA/PMB/FITC (3:1:0.01) and by crosslinking the microparticles with calcium chloride and chitosan. The obtained crosslinked microparticles were characterised for morphology and size, fluorescence, FITC release in simulated intestinal fluid, PMB content, PMB in vitro release and microbiological activity preservation. Caco-2 internalization Caco-2 cells were cultured at 37°C in Dulbecco’s Modified Eagle’s Minimal Essential Medium (DMEM) with 10% FBS (Foetal Bovine Serum) and 1% nonessential aminoacids. Cells were seeded 48 h before the incubation with the microparticle suspension. Fluorescent FITC crosslinked microparticles suspended in water (10 mg/5 ml) and diluted (1:1 or 1:2) in DMEM, were applied to the cell monolayer and then incubated at either 4°C or 37°C for 5 h or overnight. Cells were washed twice with Phosphate Buffer Saline (PBS) and then collected by trypsinization for the cytometry analysis or alternatively fixed for confocal microscopy. Flowcytometry evaluation of intracellular uptake was performed by a Coulter Epics XL flow cytometer equipped with 488 nm argon laser. FITC fluorescent cells were expressed as a percentage of the total cell population. Results and discussion As previously observed [1, 2], the obtained CaA/CHT/FITC microparticles exhibited a good water dispersibility, a nearly spherical shape with a wrinkled surface (Figure 1) and a size ranging from 0.5 to 2.5 µm (mean size 0.78 ± 0.55), being about 75% of population less than 1 µm. Moreover, microparticles were still fluorescent following a 4 h release assay in simulated intestinal fluid, as observed by epifluorescence videomicroscopy (Figure 2). Figure 1 – SEM image of CaA/CHT/FITC microparticles Figure 2 - Epifluorescence microscopy image of CaA/CHT/FITC microparticles The antibiotic entrapped inside the microparticles (loading level 11.86 ± 0.70% w/w, encapsulation efficiency of about 47%) which was found associated to the alginate chain by an electrostatic interaction and maintaining its microbiological activity, was released negligibly in simulated gastric fluid at pH 3.0 and gradually in simulated intestinal fluid. Uptake and absorption of CaA/CHT microparticles in Caco-2 monolayers were evaluated by cytofluorimetry and confocal microscopy. Fluorescence microscopy revealed that the microparticles were indeed attached to Caco-2 cells following 5 h incubation. Reducing the temperatu

2008 - The vitamin D3/Hox-A10 pathway supports MafB function during the monocyte differentiation of human CD34+ hemopoietic progenitors [Articolo su rivista]
Gemelli, Claudia; Orlandi, Claudia; ZANOCCO MARANI, Tommaso; Martello, A; Vignudelli, Tatiana; Ferrari, Francesco; Montanari, Monica; Parenti, Sandra; Testa, Anna; Grande, Alexis; Ferrari, Sergio
abstract

Although a considerable number of reports indicate an involvement of the Hox-A10 gene in the molecular control of hemopoiesis, the conclusions of such studies are quite controversial given that they support, in some cases, a role in the stimulation of stem cell self-renewal and myeloid progenitor expansion, whereas in others they implicate this transcription factor in the induction of monocyte-macrophage differentiation. To clarify this issue, we analyzed the biological effects and the transcriptome changes determined in human primary CD34+ hemopoietic progenitors by retroviral transduction of a full-length Hox-A10 cDNA. The results obtained clearly indicated that this homeogene is an inducer of monocyte differentiation, at least partly acting through the up-regulation of the MafB gene, recently identified as the master regulator of such a maturation pathway. By using a combined approach based on computational analysis, EMSA experiments, and luciferase assays, we were able to demonstrate the presence of a Hox-A10-binding site in the promoter region of the MafB gene, which suggested the likely molecular mechanism underlying the observed effect. Stimulation of the same cells with the vitamin D3 monocyte differentiation inducer resulted in a clear increase of Hox-A10 and MafB transcripts, indicating the existence of a precise transactivation cascade involving vitamin D3 receptor, Hox-A10, and MafB transcription factors. Altogether, these data allow one to conclude that the vitamin D3/Hox-A10 pathway supports MafB function during the induction of monocyte differentiation.

2007 - Characterization of cationic SLN/protamine complex as a non-viral transfer vector [Abstract in Atti di Convegno]
Vighi, Eleonora; Ruozi, Barbara; Tosi, Giovanni; Montanari, Monica; Leo, Eliana Grazia
abstract

Characterization of cationic SLN/protamine complex as a non-viral transfer vector

2007 - Dotap/Udca vesicles: novel approach in oligonucleotide delivery [Articolo su rivista]
Ruozi, Barbara; Battini, Renata; Montanari, Monica; Mucci, Adele; Tosi, Giovanni; Forni, Flavio; Vandelli, Maria Angela
abstract

The relatively hydrophilic bile acid, ursodeoxycholic acid (UDCA), was used as additive to DOTAP cationic liposomes to evaluate the effect on the cellular uptake of an oligonucleotide. Nuclear magnetic resonance studies were applied to estimate the relative amount of incorporated UDCA into the lipidic bilayers. The interaction between the new formulations and 5’fluorescein conjugated 29-mer phosphorothioate oligonucleotide (PS-ODN) was studied using gel electrophoresis experiments. Besides, DOTAP or DOTAP/UDCA vesicles (MixVes; DOTAP/UDCA 1:0.25, 1:0.5, 1:1 and 1:2 molar ratio) were complexed with PS-ODN and tested after transfections the cellular uptake and the localization of the oligo in HaCaT cell line by the use of cytofluorimetric and confocal microscopic analysis. DOTAP lipid formulated in presence of defined amount of UDCA forms more stable, flexible and active MixVes. In particular, the MixVes at 1:0.25 and 1:0.5 molar ratio increase and modify the cellular uptake of PS-ODN if compared with DOTAP liposomes 3 h after the transfection studies. Besides, the in vitro data suggest that these new formulations are not toxic.

2007 - Flow Cytometry and live confocal analysis in the evaluation of uptake and intracellular distribution of FITC-ODN into HaCaT cells [Abstract in Atti di Convegno]
Ruozi, Barbara; Tosi, Giovanni; Tombesi, Andrea; Montanari, Monica; Leo, Eliana Grazia; Restani, Cinzia; Forni, Flavio; Vandelli, Maria Angela
abstract

Flow Cytometry and live confocal analysis in the evaluation of uptake and intracellular distribution of FITC-ODN into HaCaT cells

2007 - Nuove formulazioni liposomiali per la veicolazione di materiale genico in cellule cheratinocito simili [Abstract in Atti di Convegno]
Ruozi, Barbara; Montanari, Monica; Tosi, Giovanni; Mucci, Adele; Vighi, Eleonora; Battini, Renata; Leo, Eliana Grazia; Forni, Flavio; Vandelli, Maria Angela
abstract

Nuove formulazioni liposomiali per la veicolazione di materiale genico in cellule cheratinocito simili

2007 - Re-dispersible cationic solid lipid nanoparticles (SLNs) freeze-dried without cryoprotectors: Characterization and ability to bind the pEGFP-plasmid [Articolo su rivista]
Vighi, Eleonora; Ruozi, Barbara; Montanari, Monica; Battini, Renata; Leo, Eliana Grazia
abstract

Cationic solid lipid nanoparticles (SLNs) have recently been suggested for non-viral gene delivery as a promising alternative to the liposomes. The aim of this study was to investigate the possibility to obtain re-dispersible cationic SLNs after a freeze-drying process in the absence of lyo- and/or cryoprotectors. The physical-chemical characteristics of cationic SLNs and their ability to bind gene material were investigated before and after the freeze-drying. To perform this study three samples of cationic SLNs, based on stearic acid, Compritol or cetylpalmitate, were prepared and characterized by PCS (photon correlation spectroscopy) and AFM (atomic force microscopy). The results indicated that solely the re-dispersed sample of stearic acid (SLN-SA) became very similar in terms of size and morphology to the fresh prepared sample, although it displayed a sensible reduction of the zeta potential (from 39.2 to 23.3 mV). By both the DSC (differential scanning calorimetry) and the ESCA (electron spectroscopy for chemical analysis) determinations, the reduction of the zeta potential was ascribed to the loss of the cationic lipids from the particle surface due to the rearrangement of the stearic acid lattice after the freeze-drying. Finally, the gel electrophoresis analysis demonstrated that SLN-SA re-suspended in PBS are unable to complex the DNA, while the SLN-SA re-dispersed in water displayed the same ability to bind DNA as the fresh prepared sample. We can conclude that cationic SLNs, based on stearic acid, retain the ability to complex DNA even after the freeze-drying in the absence of lyo- or cryoprotectors; thus, the powder form of this sample represents an attractive candidate to be investigated as in vivo DNA vector formulation.

2006 - IDENTIFICATION OF A MOLECULAR SIGNATURE PREDICTIVE OF REFRACTORINESS IN ACUTE MYELOID LEUKEMIA [Abstract in Atti di Convegno]
Tenedini, Elena; Tagliafico, Enrico; Manfredini, Rossella; Ferrari, Francesco; Roncaglia, Enrica; Fantoni, Luca; Grande, Alexis; Parenti, Sandra; ZANOCCO MARANI, Tommaso; Gemelli, Claudia; Tatiana Vignudelli, Tatiana; Montanari, Monica; Zini, Roberta; Salati, Simona; Bianchi, Elisa; Bicciato, Silvio; Ferrari, Sergio
abstract

Acute Myeloid Leukemia (AML) blast cells are immature committed myeloid cells unable to spontaneously undergo terminal maturation, characterized by heterogeneous sensitivity to natural differentiation inducers. No data are available so far by which infer the AML’s response to differentiating therapy. Thus, we have initially profiled by GeneChip arrays the gene expression of several AML cell lines: they derived by the original blast cell populations and are still characterized by the same immunophenotype, retain a different sensitivity or resistance to All-Trans Retinoic-Acid (ATRA) and Vitamin-D3 (VD) and never undergo spontaneously terminal maturation. Here we show that differences exist by which predict the cell line differentiation fate. Next we constructed a signature able to predict resistance or sensitivity to the differentiation induction and tested it, using a TaqMan platform, for its capability to predict the in-vitro response of 28 VD or ATRA treated AML blast cell populations. Finally, by a meta-analysis of public available microarray data we demonstrated that our signature of 11 genes, among them is particularly intriguing the presence of Meis1 and ID3, that was formerly designed to identify differentiation therapy resistant populations, turned out to be a good classifier for clusters of patients known to have poor prognostic significance.

2006 - Identification of a molecular signature predictive of sensitivity to differentiation induction in acute myeloid leukemia [Articolo su rivista]
Tagliafico, Enrico; Tenedini, Elena; Manfredini, Rossella; Grande, Alexis; Ferrari, F.; Roncaglia, Enrica; Bicciato, Silvio; Zini, Roberta; Salati, Simona; Bianchi, Elisa; Gemelli, Claudia; Montanari, Monica; Vignudelli, Tatiana; ZANOCCO MARANI, Tommaso; Parenti, Sandra; Paolucci, Paolo; Martinelli, G.; Piccaluga, P. P.; Baccarani, M.; Specchia, G.; Torelli, U.; Ferrari, Sergio
abstract

Acute myeloid leukemia (AML) blasts are immature committed myeloid cells unable to spontaneously undergo terminal maturation, and characterized by heterogeneous sensitivity to natural differentiation inducers. Here, we show a molecular signature predicting the resistance or sensitivity of six myeloid cell lines to differentiation induced in vitro with retinoic acid or vitamin D. The identified signature was further validated by TaqMan assay for the prediction of response to an in vitro differentiation assay performed on 28 freshly isolated AML blast populations. The TaqMan assay successfully predicts the in vitro resistance or responsiveness of AML blasts to differentiation inducers. Furthermore, performing a meta-analysis of publicly available microarray data sets, we also show the accuracy of our prediction on known phenotypes and suggest that our signature could become useful for the identification of patients eligible for new therapeutic strategies.

2006 - Solid lipid nanoparticles: a new cationic lipid matrix composition for gene transfer, [Abstract in Atti di Convegno]
Vighi, Eleonora; Ruozi, Barbara; Montanari, Monica; Battini, Renata; Forni, Flavio; Leo, Eliana Grazia
abstract

Solid lipid nanoparticles: a new cationic lipid matrix composition for gene transfer

2006 - Tfe3 expression is closely associated to macrophage terminal differentiation of human hematopoietic myeloid precursors. [Articolo su rivista]
ZANOCCO MARANI, Tommaso; Vignudelli, Tatiana; Gemelli, Claudia; Pirondi, Sara; Testa, Anna; Montanari, Monica; Parenti, Sandra; Tenedini, Elena; Grande, Alexis; Ferrari, Sergio
abstract

The MItf-Tfe family of basic helix–loop–helix leucine zipper (bHLH-Zip) transcription factors encodes four family members: MItf, Tfe3, TfeB and TfeC. In vitro, each protein of the family binds DNA in a homo- or heterodimeric form with other family members. Tfe3 is involved in chromosomal translocations recurrent in different tumors and it has been demonstrated, by in vivo studies, that it plays, redundantly with MItf, an important role in the process of osteoclast formation, in particular during the transition from mono-nucleated to multi-nucleated osteoclasts. Since mono-nucleated osteoclasts derive from macrophages we investigated whether Tfe3 might play a role upstream during hematopoietic differentiation. Here we show that Tfe3 is able to induce mono-macrophagic differentiation of U937 cells, in association with a decrease of cell proliferation and an increase of apoptosis. We also show that Tfe3 does not act physiologically during commitment of CD34+ hematopoietic stem cells (HSCs), since it is not able to direct HSCs toward a specific lineage as observed by clonogenic assay, but is a strong actor of terminal differentiation since it allows human primary myeloblasts' maturation toward the macrophage lineage.

2006 - Virally mediated MafB transduction induces the monocyte commitment of human CD34+ hematopoietic stem/progenitor cells [Articolo su rivista]
Gemelli, Claudia; Montanari, Monica; Tenedini, Elena; ZANOCCO MARANI, Tommaso; Vignudelli, Tatiana; Siena, Michela; Zini, Roberta; Salati, Simona; Tagliafico, Enrico; Manfredini, Rossella; Specchia, G; Grande, Alexis; Ferrari, Sergio
abstract

Upregulation of specific transcription factors is a generally accepted mechanism to explain the commitment of hematopoietic stem cells along precise maturation lineages. Based on this premise, transduction of primary hematopoietic stem/progenitor cells with viral vectors containing the investigated transcription factors appears as a suitable experimental model to identify such regulators. Although MafB transcription factor is believed to play a role in the regulation of monocytic commitment, no demonstration is, to date, available supporting this function in normal human hematopoiesis. To address this issue, we retrovirally transduced cord blood CD34+ hematopoietic progenitors with a MafB cDNA. Immunophenotypic and morphological analysis of transduced cells demonstrated the induction of a remarkable monomacrophage differentiation. Microarray analysis confirmed these findings and disclosed the upregulation of macrophage-related transcription factors belonging to the AP-1, MAF, PPAR and MiT families. Altogether our data allow to conclude that MafB is a key regulator of human monocytopoiesis.

2005 - Correlation between differentiation plasticity and mRNA expression profiling of CD34+-derived CD14- and CD14+ human normal myeloid precursors [Articolo su rivista]
Montanari, Monica; Gemelli, Claudia; Tenedini, Elena; ZANOCCO MARANI, Tommaso; Vignudelli, T; Siena, M; Zini, Roberta; Salati, Simona; Chiossi, G; Tagliafico, Enrico; Manfredini, Rossella; Grande, Alexis; Ferrari, Sergio
abstract

In spite of their apparently restricted differentiation potentiality, hematopoietic precursors are plastic cells able to trans-differentiate from a maturation lineage to another. To better characterize this differentiation plasticity, we purified CD14- and CD14+ myeloid precursors generated by 'in vitro' culture of human CD34+ hematopoietic progenitors. Morphological analysis of the investigated cell populations indicated that, as expected, they consisted of granulocyte and monocyte precursors, respectively. Treatment with differentiation inducers revealed that CD14- cells were bipotent granulo-monocyte precursors, while CD14+ cells appeared univocally committed to a terminal macrophage maturation. Flow cytometry analysis demonstrated that the conversion of granulocyte precursors to the mono-macrophage maturation lineage occurs through a differentiation transition in which the granulocyte-related myeloperoxidase enzyme and the monocyte-specific CD14 antigen are co-expressed. Expression profiling evidenced that the observed trans-differentiation process was accompanied by a remarkable upregulation of the monocyte-related MafB transcription factor.

2005 - IDENTIFICATION OF A MOLECULAR SIGNATURE PREDICTIVE OF REFRACTORINESS IN ACUTE MYELOID LEUKEMIA [Abstract in Atti di Convegno]
Tagliafico, Enrico; Tenedini, Elena; Manfredini, Rossella; Ferrari, Sergio; Roncaglia, Enrica; Fantoni, Luca; Grande, Alexis; Parenti, Sandra; ZANOCCO MARANI, Tommaso; Gemelli, Claudia; Vignudelli, Tatiana; Montanari, Monica; Zini, Roberta; Salati, Simona; Bianchi, Elisa; Bicciato, Silvio; Specchia, Giorgina; Martinelli, Giovanni; Baccarani, Michele; Piccaluga, Pier Paolo; Torelli, Umberto; Ferrari, Sergio
abstract

Acute Myeloid Leukemia (AML) blast cells are immature committed myeloid cells unable to spontaneously undergo terminal maturation, characterized by heterogeneous sensitivity to natural differentiation inducers. No data are available so far by which infer the AML’s response to differentiating therapy. Thus, we have initially profiled by GeneChip arrays the gene expression of several AML cell lines: they derived by the original blast cell populations and are still characterized by the same immunophenotype, retain a different sensitivity or resistance to ATRA and VD and never undergo spontaneously terminal maturation. Here we show that differences exist by which predict the cell line differentiation fate. Next we constructed a signature able to predict resistance or sensitivity to the differentiation induction and tested it, using a TaqMan platform, for its capability to predict the in-vitro response of 28 VD or ATRA treated AML blast cell populations. Finally, by a meta-analysis of public available microarray data we demonstrated that our signature, that was formerly designed to identify differentiation therapy resistant populations, turned out to be a good classifier for clusters of patients with citogenetically and molecularly defined lesions that are known to have poor prognostic significance.

2005 - The kinetic status of hematopoietic stem cell subpopulations underlies a differential expression of genes involved in self-renewal, commitment, and engraftment. [Articolo su rivista]
Manfredini, Rossella; Zini, Roberta; Salati, Simona; Siena, M; Tenedini, Elena; Tagliafico, Enrico; Montanari, Monica; ZANOCCO MARANI, Tommaso; Gemelli, Claudia; Vignudelli, T; Grande, A; Fogli, M; Rossi, L; Fagioli, Me; Catani, L; Lemoli, Rm; Ferrari, Sergio
abstract

The gene expression profile of CD34(-) hematopoietic stem cells (HSCs) and the correlations with their biological properties are still poorly understood. To address this issue, we used the DNA microarray technology to compare the expression profiles of different peripheral blood hemopoietic stem/progenitor cell subsets, lineage-negative (Lin(-)) CD34(-), Lin(-)CD34(+), and Lin(+)CD34(+) cells. The analysis of gene categories differentially expressed shows that the expression of CD34 is associated with cell cycle entry and metabolic activation, such as DNA, RNA, and protein synthesis. Moreover, the significant upregulation in CD34(-) cells of pathways inhibiting HSC proliferation induces a strong differential expression of cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and growth-arrest genes. According to the expression of their receptors and transducers, interleukin (IL)-10 and IL-17 showed an inhibitory effect on the clonogenic activity of CD34(-) cells. Conversely, CD34(+) cells were sensitive to the mitogenic stimulus of thrombopoietin. Furthermore, CD34(-) cells express preferentially genes related to neural, epithelial, and muscle differentiation. The analysis of transcription factor expression shows that the CD34 induction results in the upregulation of genes related to self-renewal and lineage commitment. The preferential expression in CD34(+) cells of genes supporting the HSC mobilization and homing to the bone marrow, such as chemokine receptors and integrins, gives the molecular basis for the higher engraftment capacity of CD34(+) cells. Thus, the different kinetic status of CD34(-) and CD34(+) cells, detailed by molecular and functional analysis, significantly influences their biological behavior

2004 - Polyamine depletion switches the form of 2-deoxy-D-ribose-induced cell death from apoptosis to necrosis in HL-60 cells. [Articolo su rivista]
Monti, Maria Giuseppina; Ghiaroni, Stefania; Marverti, Gaetano; Montanari, Monica; Moruzzi, Maria Stella
abstract

Our previous studies demonstrated that intracellular polyamine depletion blocked HL-60 cell apoptosis triggered by exposure to 2-deoxy- -ribose (dRib). Here, we have characterized the intracellular events underlying the apoptotic effects of dRib and the involvement of polyamines in these effects. Treatment of HL-60 cells with dRib induces loss of mitochondrial transmembrane potential, radical oxygen species production, intracellular glutathione depletion and translocation of Bax from cytosol to membranes. These effects are followed by cell death. However, the mode of cell death caused by dRib depends on intracellular levels of polyamines. -Rib-treated cells with normal polyamine levels, progressing through the G1 into the S and G2/M phases, undergo apoptosis, while in polyamine-depleted cells, being blocked at the G1 phase, cell death mechanisms are switched to necrosis. The present study points to a relationship between the cell cycle distribution and the mode of cell death, and suggests that the level of intracellular spermidine, essential to cell cycle progression, may determine whether a cell dies by apoptosis or necrosis in response to a death stimulus.

2003 - Development of an IL-6 antagonist peptide that induces apoptosis in IL-6 dependent 7TD1 cells. [Articolo su rivista]
Manfredini, Rossella; Tenedini, Elena; M., Siena; Tagliafico, Enrico; Montanari, Monica; Grande, Alexis; ZANOCCO MARANI, Tommaso; C., Poligani; Zini, Roberta; A., Bergamaschi; DE RIENZO, Francesca; DE BENEDETTI, Pier Giuseppe; Menziani, Maria Cristina; Ferrari, Sergio
abstract

Interleukin-6 (IL-6) is a pleiotropic cytokine involved in the regulation of proliferation and differentiation of hematopoietic cells and in the pathogenesis of many diseases, including multiple myeloma. This study pursues a way to interfere with IL-6 pathway in an attempt to modulate its biological activity. Here we describe the rational design and biological evaluation of peptides able to antagonize the murine IL-6 activity by interfering with IL-6 Receptor alpha in 7TD1 cells, a IL-6-dependent B-cell line. Of the peptide tested, only Guess 4a is capable of interfering with IL-6 transducing pathway, therefore inducing growth arrest and apoptosis of 7TD1 cells.

2002 - Gene Expression profile of Vitamin D3 treated HL60 cells shows a phenotypic but not a complete functional conversion to monocytes [Abstract in Atti di Convegno]
Tenedini, Elena; Bergamaschi, A; Manfredini, Rossella; Percudani, R; Siena, M; ZANOCCO MARANI, Tommaso; Grande, Alexis; Montanari, Monica; Gemelli, Claudia; Torelli, U; Ferrari, Sergio; Tagliafico, Enrico
abstract

Acute Myeloid leukemia blast cells are characterized by their inability to proceed spontaneously toward terminal differentiation. To tackle this problem we have studied the changes occurring in the gene expression profile during the differentiation of HL60 cells treated with VD using the Affymetrix GeneChip technology and we have compared the molecular phenotype of VD induced cells to that of CD14+ pheripheral monocytes.

2002 - Gene expression Profile of Human Myeloid Cells [Abstract in Atti di Convegno]
Siena, M; Manfredini, R; Bergamaschi, A; Tenedini, Elena; Tagliafico, Enrico; ZANOCCO MARANI, Tommaso; Montanari, Monica; Gemelli, Claudia; Grande, Alexis; Ferrari, Sergio
abstract

Array technologies have made it possible to monitor simultaneously the expression pattern of thousands of genes. Working on normal human hempoietic stem cells it is possible to evaluate their gene expression profile, changes in gene expression occurring in their early commitment phase and to compare the gene expression profiling with normallly differentiated myeloid cells, i.e. granulocytes and monocytes.

2002 - Requirement of the coiled coil domains of p92c-Fes for nuclear translocation in myeloid cells upon induction of differentiation [Abstract in Atti di Convegno]
ZANOCCO MARANI, Tommaso; Siena, M; Tagliafico, Enrico; Manfredini, Rossella; Tenedini, Elena; Montanari, Monica; Grande, Alexis; Gemelli, Claudia; Ferrari, Sergio
abstract

The non-receptor tyrosine kinase Fes is expressed in hematopoietic progenitors, differentiated myeloid cells and other cell types, such as vascular endothelial cells and neuroblastoma cell lines. To further clarify this point we performed confocal microscopy and western blot experiments on myeloid cell lines and COS1 cells. In myeloid cells the treatment with differentiation inducing agents such as ATRA, PMA and VD is followed by an increase of Fes abundance in the nuclear compartment. The active form of Fes is phosphorylated on residue 713 and is present into the nucleus while treated cells with the tyrosine kinase inhibitor Genistein clearly showed that phosphorylation is not a required event in order to Fes to translocate to the nucleus.

1999 - Induction of a functional vitamin D receptor in all-trans-retinoic acid- induced monocytic differentiation of M2-type leukemic blast cells [Articolo su rivista]
Manfredini, R.; Trevisan, F.; Grande, A.; Tagliafico, E.; Montanari, M.; Lemoli, R.; Visani, G.; Tura, S.; Ferrari, S.; Ferrari, S.
abstract

Different types of acute myeloid leukemia blast cells were induced to differentiate in vitro with all-trans-retinoic acid (ATRA) and vitamin D3 (VD). M0/M1 leukemic cells are not sensitive to differentiating agents, whereas M3 leukemic cells are induced to undergo granulocytic differentiation after ATRA treatment but are not sensitive to VD. M2 leukemic blast cells behave differently because they undergo monocytic differentiation with both the differentiation inducers. To gain some insight into the maturation of M2- type leukemic cells, we studied the molecular mechanisms underlying monocytic differentiation induced by ATRA and VD in spontaneous M2 blast cells as well as in Kasumi-1 cells (an acute myeloid leukemia M2-type cell line). Our results indicate that ATRA as well as VD efficiently increases the nuclear abundance of VD receptor (VDR) and promotes monocytic differentiation. VDR is functionally active in ATRA-treated Kasumi-1 cells because it efficiently heterodimerizes with retinoid X receptor, binds to a DR3-type vitamin D- responsive element, and activates the transcription of a vitamin D-responsive element-regulated reporter gene. Consistent with these findings, VD- responsive genes are induced by ATRA treatment of Kasumi-1 cells, suggesting that the genetic program underlying monocytic differentiation is activated. The molecular mechanism by which ATRA increases the nuclear abundance of a functional VDR is still unknown, but our data clearly indicate that the M2 leukemic cell context is only permissive of monocytic differentiation.

Università degli studi di Modena e Reggio Emilia

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