Nuova ricerca

Paola SENA

Ricercatore Universitario
Dipartimento Chirurgico, Medico, Odontoiatrico e di Scienze Morfologiche con interesse Trapiantologico, Oncologico e di Medicina Rigenerativa

Home | Curriculum(pdf) | Didattica |


Pubblicazioni

2023 - Flow-dependent shear stress affects the biological properties of pericyte-like cells isolated from human dental pulp [Articolo su rivista]
Bertani, Giulia; Di Tinco, Rosanna; Bertoni, Laura; Orlandi, Giulia; Pisciotta, Alessandra; Rosa, Roberto; Rigamonti, Luca; Signore, Michele; Bertacchini, Jessika; Sena, Paola; De Biasi, Sara; Villa, Erica; Carnevale, Gianluca
abstract

Background: Human dental pulp stem cells represent a mesenchymal stem cell niche localized in the perivascular area of dental pulp and are characterized by low immunogenicity and immunomodulatory/anti-inflammatory properties. Pericytes, mural cells surrounding the endothelium of small vessels, regulate numerous functions including vessel growth, stabilization and permeability. It is well established that pericytes have a tight cross talk with endothelial cells in neoangiogenesis and vessel stabilization, which are regulated by different factors, i.e., microenvironment and flow-dependent shear stress. The aim of this study was to evaluate the effects of a pulsatile unidirectional flow in the presence or not of an inflammatory microenvironment on the biological properties of pericyte-like cells isolated from human dental pulp (hDPSCs). Methods: Human DPSCs were cultured under both static and dynamic conditions with or without pre-activated peripheral blood mononuclear cells (PBMCs). Pulsatile unidirectional flow shear stress was generated by using a specific peristaltic pump. The angiogenic potential and inflammatory properties of hDPSCs were evaluated through reverse phase protein microarrays (RPPA), confocal immunofluorescence and western blot analyses. Results: Our data showed that hDPSCs expressed the typical endothelial markers, which were up-regulated after endothelial induction, and were able to form tube-like structures. RPPA analyses revealed that these properties were modulated when a pulsatile unidirectional flow shear stress was applied to hDPSCs. Stem cells also revealed a downregulation of the immune-modulatory molecule PD-L1, in parallel with an up-regulation of the pro-inflammatory molecule NF-kB. Immune-modulatory properties of hDPSCs were also reduced after culture under flow-dependent shear stress and exposure to an inflammatory microenvironment. This evidence was strengthened by the detection of up-regulated levels of expression of pro-inflammatory cytokines in PBMCs. Conclusions: In conclusion, the application of a pulsatile unidirectional flow shear stress induced a modulation of immunomodulatory/inflammatory properties of dental pulp pericyte-like cells.

2023 - Human dental pulp stem cells (hDPSCs) promote the lipofibroblast transition in the early stage of a fibro-inflammatory process [Articolo su rivista]
Pisciotta, Alessandra; DI TINCO, Rosanna; Bertani, Giulia; Orlandi, Giulia; Bertoni, Laura; Pignatti, Elisa; Orciani, Monia; Sena, Paola; Bertacchini, Jessika; Salvarani, Carlo; Carnevale, Gianluca
abstract

Introduction: In autoimmune diseases, particularly in systemic sclerosis and chronic periaortitis, a strict correlation between chronic inflammation and fibrosis exists. Since the currently used drugs prove mostly effective in suppressing inflammation, a better comprehension of the molecular mechanisms exerted by cell types implicated in fibro-inflammation is needed to develop novel therapeutic strategies. Mesenchymal stromal/stem cells (MSCs) are being matter of deep investigation to unveil their role in the evolution of fibrogenetic process. Several findings pointed out the controversial implication of MSCs in these events, with reports lining at a beneficial effect exerted by external MSCs and others highlighting a direct contribution of resident MSCs in fibrosis progression. Human dental pulp stem cells (hDPSCs) have demonstrated to hold promise as potential therapeutic tools due to their immunomodulatory properties, which strongly support their contribution to tissue regeneration. Methods: Our present study evaluated hDPSCs response to a fibro-inflammatory microenvironment, mimicked in vitro by a transwell co-culture system with human dermal fibroblasts, at early and late culture passages, in presence of TGF-β1, a master promoter of fibrogenesis. Results and Discussion: We observed that hDPSCs, exposed to acute fibro-inflammatory stimuli, promote a myofibroblast-to-lipofibroblast transition, likely based on BMP2 dependent pathways. Conversely, when a chronic fibro-inflammatory microenvironment is generated, hDPSCs reduce their anti-fibrotic effect and acquire a pro-fibrotic phenotype. These data provide the basis for further investigations on the response of hDPSCs to varying fibro-inflammatory conditions.

2022 - Colon cancer in a 12-year-old girl with hypertriglyceridemia [Articolo su rivista]
Pedroni, Monica; Leon, Maurizio Ponz de; Bonetti, Luca Reggiani; Viel, Alessandra; Noto, Davide; Nascimbeni, Fabio; Sena, Paola; Roncucci, Luca
abstract

2022 - DIFFERENZE DI GENERE NELLA COMORBILITÀ TRA SINTOMATOLOGIA ANSIOSO-DEPRESSIVA, SINDROME METABOLICA E ADENOMI COLORETTALI [Abstract in Atti di Convegno]
Rioli, Giulia; Bonamici, Caterina; Mancini, Stefano; Mattei, Giorgio; Alboni, Silvia; Sena, Paola; Roncucci, Luca; Fiore, Gianluca; Pingani, Luca; Ferrari, Silvia; Galeazzi, Gian Maria
abstract

2022 - Differenze di genere nella comorbilità tra sintomatologia ansioso-depressiva, sindrome metabolica e adenomi colorettali [Abstract in Rivista]
Rioli, G.; Bonamici, C.; Mancini, S.; Mattei, G.; Alboni, S.; Sena, P.; Roncucci, L.; Fiore, G.; Pingani, L.; Ferrari, S.; Galeazzi, G. M.
abstract

2022 - Expression of Autophagic and Inflammatory Markers in Normal Mucosa of Individuals with Colorectal Adenomas: A Cross Sectional Study among Italian Outpatients Undergoing Colonoscopy [Articolo su rivista]
Sena, Paola; Mancini, Stefano; Pedroni, Monica; Reggiani Bonetti, Luca; Carnevale, Gianluca; Roncucci, Luca
abstract

: Colorectal cancer (CRC) ranks among the three most common cancers in terms of both cancer incidence and cancer-related deaths in Western industrialized countries. Lifetime risk of colorectal cancer may reach 6% of the population living in developed countries. In the current era of personalized medicine, CRC is no longer considered as a single entity. In more recent years many studies have described the distinct differences in epidemiology, pathogenesis, genetic and epigenetic alterations, molecular pathways and outcome depending on the anatomical site. The aim of our study is to assess in a multidimensional model the association between metabolic status and inflammatory and autophagic changes in the normal colorectal mucosa classified as right-sided, left-sided and rectum, and the presence of adenomas. One hundred and sixteen patients undergoing colonoscopy were recruited and underwent a complete serum lipid profile, immunofluorescence analysis of colonic biopsies for MAPLC3 and myeloperoxidase expression, matched with clinical and anthropometric characteristics. Presence of adenomas correlated with cholesterol (total and LDL) levels, IL-6 levels, and MAPLC3 tissue expression, especially in the right colon. In conclusion, serum IL-6 amount and autophagic markers could be good predictors of the presence of colorectal adenomas.

2022 - Gender differences in Anxious-depressive symptomatology, Metabolic Syndrome and Colorectal Adenomas among outpatients undergoing colonoscopy: a cross-sectional study according to a PNEI perspective [Articolo su rivista]
Rioli, Giulia; Mattei, Giorgio; Bonamici, Caterina; Mancini, Stefano; Alboni, Silvia; Cannazza, Giuseppe; Sena, Paola; Roncucci, Luca; Pingani, Luca; Ferrari, Silvia; Galeazzi, Gian Maria
abstract

2022 - IL RUOLO DELL’INFIAMMAZIONE SISTEMICA CRONICA E DELLA VIA METABOLICA DELLE CHINURENINE NELLA COMORBIDITÀ TRA ANSIA, DEPRESSIONE E SINDROME METABOLICA: RISULTATI DI UNO STUDIO CROSS-SECTIONAL [Abstract in Atti di Convegno]
Rioli, Giulia; Bonamici, Caterina; Macini, Stefano; Mattei, Giorgio; Alboni, Silvia; Sena, Paola; Roncucci, Luca; Fiore, Gianluca; Pingani, Luca; Ferrari, Silvia; Galeazzi, Gian Maria
abstract

2022 - Il ruolo dell'infiammazione sistemica cronica e della via metabolica delle chinurenine nella comorbidilità tra ansia, depressione e sindrome metabolica: risultati di uno studio cross-sectional [Abstract in Rivista]
Rioli, G.; Bonamici, C.; Macini, S.; Mattei, G.; Alboni, S.; Sena, P.; Roncucci, L.; Fiore, G.; Pingani, L.; Ferrari, S.; Galeazzi, G. M.
abstract

2021 - Autoimmunity profiles as prognostic indicators in patients with colorectal cancer versus those with cancer at other sites: A prospective study [Articolo su rivista]
Sena, P.; Mancini, S.; Bertacchini, J.; Carnevale, G.; Pedroni, M.; Roncucci, L.
abstract

Colorectal cancer represents a paradigmatic model of inflammatory carcinogenesis accom-panied by the production of several kinds of tumor-associated autoantibodies (TAABs). The specific aim of this study is to define the clinical impact of the presence of non-specific circulating TAABs in a cohort of cancer patients and to establish whether significant differences were present between colorectal cancer and cancers at other sites. For this aim a prospective study was developed and a five-year survival analysis performed. Indirect immunofluorescence on rat tissues for non-organ specific autoantibodies (NOSAs: liver-kidney-stomach), on rat colon substrates (colon-related autoantibodies, CAAs) and on HEp-2 cell lines was performed. NOSA positivity was more frequent in patients with colorectal cancer than in those with cancer at other sites. Survival analysis demonstrated a significantly worse prognosis in cancer patients positive for TAABs. CAA positivity is a predictor of survival, independently from the presence of comorbidities, and HEp-2 reactivity was a strong predictor of survival in a stepwise Cox-regression model, including stage at diagnosis. Overall overproduction of TAABs is associated with advanced oncological disease, the presence of metastasis, and poorer prognosis of cancer patients.

2021 - Preliminary results of a multidisciplinary italian study adopting a psycho-neuro-endocrine-immunological (Pnei) approach to the study of colorectal adenomas [Articolo su rivista]
Mancini, S.; Alboni, S.; Mattei, G.; Rioli, G.; Sena, P.; Marchi, M.; Sacchetti, A.; Boarino, V.; Roncucci, L.; Galeazzi, G. M.; Ferrari, S.
abstract

Background and aim of the work: Colorectal mucosal precancerous lesions, metabolic syndrome (MetS) and psychiatric disorders may share a common low-grade local and systemic inflammation. Aim is to report on preliminary data concerning a research adopting a psycho-neuro-endocrine-immune (PNEI) approach to study outpatients undergoing colonoscopy. Methods: A sample of patients undergoing colonosco-py was cross-sectionally investigated. Data on colorectal adenomas, MetS, early atherosclerosis, anxious-de-pressive symptoms, personality traits, and inflammatory markers were statistically analyzed. Results: Sixty-two patients were recruited (female 50%, mean age: 60.8±9.4 years). The prevalence of adenomas and MetS was respectively of 45.2% and 41.9%. Anxiety and depressive symptoms were detected in 16 (32.7%) and 9 (18.4%) subjects, respectively. The presence of adenomas positively correlated with male sex (p=0.01), age (p<0.01), IL-6 (p=0.03), hsCRP (p=0.04), and MetS (p=0.03); it was also associated with hsCRP concentra-tion (aOR=3.81, p=0.03). Conclusions: Proinflammatory atherogenic status, psychological traits, increased mucosal inflammation, and metabolic parameters may share a common a pathogenic mechanism, worth studying.

2021 - Role of PD-L1 in licensing immunoregulatory function of dental pulp mesenchymal stem cells [Articolo su rivista]
Di Tinco, R.; Bertani, G.; Pisciotta, A.; Bertoni, L.; Pignatti, E.; Maccaferri, M.; Bertacchini, J.; Sena, P.; Vallarola, A.; Tupler, R.; Croci, S.; Bonacini, M.; Salvarani, C.; Carnevale, G.
abstract

Background: Dental pulp stem cells (DPSCs) are low immunogenic and hold immunomodulatory properties that, along with their well-established multi-potency, might enhance their potential application in autoimmune and inflammatory diseases. The present study focused on the ability of DPSCs to modulate the inflammatory microenvironment through PD1/PD-L1 pathway. Methods: Inflammatory microenvironment was created in vitro by the activation of T cells isolated from healthy donors and rheumatoid arthritis (RA) patients with anti-CD3 and anti-CD28 antibodies. Direct and indirect co-cultures between DPSCs and PBMCs were carried out to evaluate the activation of immunomodulatory checkpoints in DPSCs and the inflammatory pattern in PBMCs. Results: Our data suggest that the inflammatory stimuli trigger DPSCs immunoregulatory functions that can be exerted by both direct and indirect contact. As demonstrated by using a selective PD-L1 inhibitor, DPSCs were able to activate compensatory pathways targeting to orchestrate the inflammatory process by modulating pro-inflammatory cytokines in pre-activated T lymphocytes. The involvement of PD-L1 mechanism was also observed in autologous inflammatory status (pulpitis) and after direct exposure to pre-activated T cells from RA patients suggesting that immunomodulatory/anti-inflammatory properties are strictly related to their stemness status. Conclusions: Our findings point out that the communication with the inflammatory microenvironment is essential in licensing their immunomodulatory properties.

2021 - The Combination of AHCC and ETAS Decreases Migration of Colorectal Cancer Cells, and Reduces the Expression of LGR5 and Notch1 Genes in Cancer Stem Cells: A Novel Potential Approach for Integrative Medicine [Articolo su rivista]
Paganelli, F.; Chiarini, F.; Palmieri, A.; Martinelli, M.; Sena, P.; Bertacchini, J.; Roncucci, L.; Cappellini, A.; Martelli, A. M.; Bonucci, M.; Fiorentini, C.; Ferri, I. H.
abstract

The AHCC standardized extract of cultured Lentinula edodes mycelia, and the standardized extract of Asparagus officinalis stem, trademarked as ETAS, are well known supplements with im-munomodulatory and anticancer potential. Several reports have described their therapeutic effects, including antioxidant and anticancer activity and improvement of immune response. In this study we aimed at investigating the effects of a combination of AHCC and ETAS on colorectal cancer cells and biopsies from healthy donors to assess the possible use in patients with colorectal cancer. Our results showed that the combination of AHCC and ETAS was synergistic in inducing a significant decrease in cancer cell growth, compared with single agents. Moreover, the combined treatment induced a significant increase in apoptosis, sparing colonocytes from healthy donors, and was able to induce a strong reduction in migration potential, accompanied by a significant modulation of proteins involved in invasiveness. Finally, combined treatment was able to significantly downregulate LGR5 and Notch1 in SW620 cancer stem cell (CSC) colonospheres. Overall, these findings support the potential therapeutic benefits of the AHCC and ETAS combinatorial treatment for patients with colorectal cancer.

2020 - Gonadotrophins modulate cell death-related genes expression in human endometrium [Articolo su rivista]
Sacchi, S.; Sena, P.; Addabbo, C.; Cuttone, E.; La Marca, A.
abstract

Gonadotrophins exert their functions by binding follicle-stimulating hormone receptor (FSHR) or luteinizing hormone and human chorionic gonadotropin receptor (LHCGR) present on endometrium. Within ovaries, FSH induces autophagy and apoptosis of granulosa cells leading to atresia of non-growing follicles, whereas hCG and LH have anti-apoptotic functions. Endometrial cells express functioning gonadotrophin receptors. The objective of this study was to analyze the effect of gonadotrophins on physiology and endometrial cells survival. Collected endometria were incubated for 48 or 72 h with 100 ng/mL of recombinant human FSH (rhFSH), recombinant human LH (rhLH) or highly purified hCG (HPhCG) alone or combined. Controls omitted gonadotrophins. The effect of gonadotrophins on cytochrome P450 family 11 subfamily A polypeptide 1 (CYP11A1), hypoxia inducible factor 1α (HIF1A), and cell-death-related genes expression was evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Immunohistochemistry for microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B) and apoptotic protease activating factor 1 (APAF-1) was performed. Gonadotrophins are able to modulate the endometrial cells survival. FSH induced autophagy and apoptosis by increasing the relative expression of MAP1LC3B and FAS receptor. In FSH-treated samples, expression of apoptosis marker APAF-1 was detected and co-localized on autophagic cells. hCG and LH does not modulate the expression of cell-death-related genes while the up-regulation of pro-proliferative epiregulin gene was observed. When combined with FSH, hCG and LH prevent autophagy and apoptosis FSH-induced. Different gonadotrophins specifically affect endometrial cells viability differently: FSH promotes autophagy and apoptosis while LH and hCG alone or combined with rhFSH does not.

2019 - Deep learning techniques for detecting preneoplastic and neoplastic lesions in human colorectal histological images [Articolo su rivista]
Sena, P.; Fioresi, R.; Faglioni, F.; Losi, L.; Faglioni, G.; Roncucci, L.
abstract

Trained pathologists base colorectal cancer identification on the visual interpretation of microscope images. However, image labeling is not always straightforward and this repetitive task is prone to mistakes due to human distraction. Significant efforts are underway to develop informative tools to assist pathologists and decrease the burden and frequency of errors. The present study proposes a deep learning approach to recognize four different stages of cancerous tissue development, including normal mucosa, early preneoplastic lesion, adenoma and cancer. A dataset of human colon tissue images collected and labeled over a 10-year period by a team of pathologists was partitioned into three sets. These were used to train, validate and test the neural network, comprising several convolutional and a few linear layers. The approach used in the present study is ‘direct’; it labels raw images and bypasses the segmentation step. An overall accuracy of >95% was achieved, with the majority of mislabeling referring to a near category. Tests on an external dataset with a different resolution yielded accuracies >80%. The present study demonstrated that the neural network, when properly trained, can provide fast, accurate and reproducible labeling for colon cancer images, with the potential to significantly improve the quality and speed of medical diagnoses.

2018 - Evidence for expression and functionality of FSH and LH/hCG receptors in human endometrium [Articolo su rivista]
Sacchi, Sandro; Sena, Paola; Degli Esposti, Chiara; Lui, Jessica; La Marca, Antonio
abstract

Purpose: Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) mediate intracellular functions by binding their specific protein G-coupled gonadotrophin receptor, respectively FSH receptor (FSHR) and LH/choriogonadotrophin receptor (LHCGR). Whereas the expression of FSHR and LHCGR in mammals was considered gonad-specific and cell-specific, studies identified gonadotrophin receptors in human female extragonadal reproductive tissues. This study aims to demonstrate that gonadotrophin receptors are expressed in endometrium and mediates intracellular functions. Methods: Collected endometria (n = 12) from healthy patients (mean age of 36 ± 6) were primary cultured for 24 h. The presence of gonadotrophin receptors was evaluated by RT-PCR followed by the sequencing of the resulted amplicons and by immunohistochemistry in original samples. Endometrial primary cultures were treated with increasing concentration (range 0–100 ng/ml) of either recombinant human LH (rhLH) or recombinant human FSH (rhFSH). Endometria controls had gonadotrophin replaced by the same volume of the culture medium. In gonadotrophin-treated samples, it was evaluated the intracellular cyclic adenosine monophosphate (cAMP) content by enzymatic immunoassay and the expression of steroidogenic genes by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). Results: The sequencing of the RT-PCR amplicons confirmed the presence of both gonadotrophin receptors and immunohistochemistry localized them on the membrane of endometrial glands cells throughout the glandular epithelium. The gonadotrophin-receptor complex was able to increase the intracellular cAMP in a dose-response and time-course manner and to induce steroidogenic genes expression. Conclusion: This study demonstrates that both gonadotrophin receptors are expressed along the glandular epithelium of endometria and they mediate the effects of gonadotrophins on intracellular functions.

2018 - Medical Database for Detecting Neoplastic Lesions in Human Colorectal Cancer with Deep Learning [Articolo su rivista]
Fioresi, Rita; Faglioni, Francesco; Sena, Paola
abstract

Medical databases are fundamental for developing new techniques for early detection of neoplastic cells. They are however difficult to obtain, since the labelling of the images is often operator dependent, requires specialized skills and the written informed consent of the patient. The variability of structures in biological tissue poses a challenge to both manual and automated analysis of histopathology slides. Although some authors showed moderate to good agreement among expert pathologists, and satisfactory results on their intra-observer reliability, other studies found that even experienced pathologists frequently disagree on tissue classification, which may lead to the conclusion that solely using expert scoring as gold standard for histopathological assessment could be insufficient. Hence, there is a growing demand for robust computational methods in order to increase reproducibility of diagnoses. In this note we present a database containing images of preneoplastic and neoplastic colorectal tissues and in a forthcoming paper we will describe our proposed DL algorithm to classify them into the following categories: normal mucosa, early preneoplastic lesions, adenomas, cancer.

2018 - Metformin induces apoptosis and alters cellular responses to oxidative stress in Ht29 colon cancer cells: Preliminary findings [Articolo su rivista]
Sena, P; Mancini, S; Benincasa, M; Mariani, Francesco; Palumbo, C; Roncucci, L
abstract

Accumulating evidence suggests that metformin, used as an antidiabetic drug, possesses anti-cancer properties. Metformin reduced the incidence and growth of experimental tumors in vivo. In a randomized clinical trial among nondiabetic patients, metformin treatment significantly decreased the number of aberrant crypt foci compared to the untreated group with a follow-up of 1 month. In our study, HT29 cells were treated with graded concentrations of metformin, 10 mM/25 mM/50 mM for 24/48 h. We performed immunofluorescence experiments by means of confocal microscopy and western blot analysis to evaluate a panel of factors involved in apoptotic/autophagic processes and oxidative stress response. Moreover, HT29 cells treated with metformin were analyzed by a flow cytometry assay to detect the cell apoptotic rate. The results demonstrate that metformin exerts growth inhibitory effects on cultured HT29 cells by increasing both apoptosis and autophagy; moreover, it affects the survival of cultured cells inhibiting the transcriptional activation of Nuclear factor E2-related factor 2 (NRF-2) and nuclear factor-kappa B (NF-κB). The effects of metformin on HT29 cells were dose- and time-dependent. These results are very intriguing since metformin is emerging as a multi-faceted drug: It has a good safety profile and is associated with low cost and might be a promising candidate for the prevention or the treatment of colorectal cancer.

2018 - Muscle-to-bone crosstalk: the Wnt/-catenin pathway is a candidate mechanism mediating the signalling between C2C12 muscle cells and 2T3 osteoblasts [Abstract in Rivista]
Magaro', MARIA SARA; Bertacchini, Jessika; Poti', Francesco; Checchi, Marta; Benincasa, Marta; Sena, Paola; Palumbo, Carla
abstract

The study aims to determine whether myokines can potentially regulate osteogenesis,

2017 - An Italian observational study on subclinical cardiovascular risk factors and depressive symptomatology. A suggestion for the potential utility of a sinergic cardio-psychiatric perspective [Abstract in Rivista]
Tassi, S.; Rioli, Giulia; Mattei, Giorgio; Mancini, Stefano; Alboni, Silvia; Roncucci, Luca; Sena, Paola; Mariani, Francesco; Marchi, Mattia; Fabbrizi, Andrea; Feltri, L.; Visentini, Chiara; Pollutri, Gabriella; Artoni, Cecilia; Saraceni, Serena; Galli, Giacomo; Spiga, Giulia; Minarini, Alessandro; Perrone, Daniela; Galletti, Martina; Giambalvo, Nina; Montardi, Giulia; Galeazzi, Gian Maria; Ferrari, Silvia
abstract

Introduction Growing evidence has been collected over the complex, intertwined pathophysiological connection among subclinical cardiovascular (CV) disease, i.e. atherosclerosis, systemic low pro-inflammatory states and psychiatric disorders/symptomatology (anxiety, depression), with controversial results. Aim Aim of this study was to investigate the possible link between subclinical CV risk factors (atherosclerosis), depressive symptoms, and inflammation. Methods Cross-sectional study. Inclusion criteria: outpatients aged ≥40 years, attending colonoscopy after positive faecal occult blood test, negative medical history for cancer. Collected data: blood pressure, glycaemia, lipid profile, waist circumference, BMI, PCR (C reactive protein), LPS (bacterial lipopolysaccharide), ultrasound carotid intima-media thickness (c-IMT). Psychometric tests: HADS, TCI, IMSA, SF36. Statistical analysis performed with STATA13. Results The 54 patients enrolled were equally distributed by gender. CV risk factors were common in the study population, with 33 patients (61.11%) with hypertension, 14 (25.93%) with hyperglycaemia, 20 (37.4%) with hypertriglyceridemia, 19 (35.19%) with low HDL and 64.81% with overweight. High levels of PCR were found in 24 subjects (44.44%). Right c-IMT was increased in 26.41% of the sample, and 11.32% had an atheromatous plaque. Left c-IMT was increased in 24.53% of patients, with a plaque in 7.55% of them. Clinically relevant depressive symptoms were found in the 18.87% of the sample and were statistically significantly associated with PCR (OR = 28.63; P = 0.01). Conclusions Evidence contributing to the so-called “inflammation theory” of depression and supporting the association between mood and CV disorders was here collected, supporting the need for a multidisciplinary approach to the diagnosis and treatment of such conditions, assuming a clinically-translated PNEI (psycho-neuro-endocrino-immunological) perspective.

2017 - Myeloperoxidase expression in human colonic mucosa is related to systemic oxidative balance in healthy subjects [Articolo su rivista]
Mancini, Stefano; Mariani, Francesco; Sena, Paola; Benincasa, Marta; Roncucci, Luca
abstract

Objectives: To improve understanding of the preclinical stage of colonic inflammation by exploring the existence of a link between early inflammatory changes in the colonic mucosa and the systemic redox balance. Methods: Clinical characteristics, a fasting blood draw, and mucosal biopsies from the right, left, and sigmoid-rectum colonic tracts collected from 28 healthy individuals (14/14 males/females) who underwent colonoscopy. Myeloperoxidase (MPO) positive cells infiltrating colonic mucosa specimens were assessed by immunohistochemistry, and patients divided into high or low MPO expressing cells/optical field groups (MPOhighor MPOlow, respectively).The systemic oxidative balance has been studied through derived-Reactive Oxygen Metabolites (d-ROMs), Biological Antioxidant Potential (BAP), and Lipoperoxide-cholesterol Oxidizing (LP-CHOLOX) tests on serum. Results: MPOhighpatients demonstrated an increased systemic oxidative stress compared to MPOlowindividuals (P = 0.035), especially when MPO is referred to the left-sided colonic mucosa (P = 0.007). MPOlowsubjects in the sigmoid-rectum showed a significant higher antioxidant capacity in the serum (P < 0.02). Sex-specific differences in MPO expression (male and female: 4.6 ± 3.2 and 2.6 ± 1.5 MPO-positive cells/optical field, respectively, P = 0.044), and a decreasing gradient in MPO expression moving from the cecum to the rectum (ascendant, descendant, and sigmoid-rectum: 3.7 ± 2.8, 3.1 ± 1.7, and 1.4 ± 0.5, respectively, P = 0.012) were also found and discussed. Discussion: The study is the first demonstrating a connection between systemic redox balance and MPO expression in the colonic mucosa, according to the colonic tract and patient gender. Further research evaluating the MPO expression in the human colon and its relationship with pathological conditions could benefit from these results.

2017 - Myeloperoxidase-positive cell infiltration of normal colorectal mucosa is related to body fatness and is predictive of adenoma occurrence [Articolo su rivista]
Mariani, F.; Boarino, V.; Bertani, A.; Merighi, A.; Pedroni, M.; Rossi, G.; Mancini, S.; Sena, P.; Benatti, P.; Roncucci, L.
abstract

Body fatness is a risk factor for colorectal cancer, and promotes an inflammatory environment. Indeed, inflammation in normal colorectal mucosa may be a factor linking body fatness to colorectal carcinogenesis. In this study, we evaluated myeloperoxidase (MPO)-positive cells infiltration of normal colorectal mucosa as a marker of cancer-promoting inflammation in overweight and obese subjects. One hundred and three subjects with normal colonoscopy entered the study. Waist circumference (WC) and body mass index (BMI) were measured, and MPO-positive cells on histological sections of biopsies of normal colorectal mucosa were counted under a light microscope. The occurrence of adenomas was then evaluated on follow-up colonoscopies. Mean MPO-positive cell count (±s.e.m.) was higher in subject with a WC equal or above the obesity cutoff values according to gender (2.63±0.20 vs 2.06±0.18, P=0.03), and in subjects with BMI equal or above 25 kg m ' 2 (2.54±0.18 vs 1.97±0.20, P=0.03). A Cox proportional hazard model showed that mean MPO-positive cell count in normal colorectal mucosa was the only factor independently related to occurrence of adenomas in follow-up colonoscopies. Though preliminary, these results show that MPO-positive cell infiltration in normal colorectal mucosa is related with body fatness, as evaluated by WC and BMI, and it may be considered a useful and simple marker to estimate adenoma occurrence risk.

2017 - Prevalence of metabolic syndrome and of symptoms of anxiety and depression in patients undergoing colonoscopy [Abstract in Rivista]
Marchi, Mattia; Alboni, Silvia; Fabbrizi, Andrea; Feltri, L.; Galli, Giacomo; Guicciardi, Alessia; Mancini, Stefano; Mattei, Giorgio; Minarini, Alessandro; Perrone, Daniela; Rioli, Giulia; Roncucci, Luca; Sena, Paola; Ferrari, Silvia
abstract

Introduction Metabolic syndrome (MetS) is defined by metabolic and cardio-vascular impairments and is frequently associated with anxiety and depressive disorders. Both MetS and anxiety-depressive syndromes feature similar systemic inflammatory alterations. Inflammation of the large bowel is also a key factor for the development of colorectal cancer (CRC). Objective To measure the prevalence of MetS and symptoms of anxiety and depression among patients undergoing colonoscopy. Methods Cross-sectional study. Patients undergoing colonoscopy aged 40 or more, with negative history for neoplasia or inflammatory bowel disease, were enrolled. Data collected: colonoscopy outcome, presence/absence of MetS (IDF and ATP III criteria), presence/absence of depressive and anxiety symptoms assessed with HADS. Results The sample was made up of 53 patients (female 24, 45.3%). Mean age was 60.66 ± 9.08. At least one adenoma was found to 23 patients (43.3%). Prevalence of MetS ranged from 34% to 36% (ATP III and IDF criteria, respectively). Prevalence of depressive and anxiety symptoms was 20% and 33%, respectively. Conclusion Prevalence of MetS, anxiety and depressive symptoms among patients undergoing colonoscopy was higher than in the general population.

2017 - Scleral ossicles as natural biomaterials on which vascular-like network is promoted from Mouse Aortic Endothelial cells (MAECs): preliminary results [Abstract in Rivista]
Checchi, Marta; Grisendi, Giulia; Bertacchini, Jessika; Magaro', MARIA SARA; Ferretti, Marzia; Benincasa, Marta; Sena, Paola; Cavani, Francesco; Palumbo, Carla
abstract

When a severe fracture is difficult to self-recovered, it is defined as “critical-size” bone defect. Till now, many efforts have been made by the tissue engineering (TE) to generate scaffolds suitable for recovering of this type of fracture, but the main obstacle remains the lack of an appropriate vascularization of the scaffolds. In the field of the regenerative medicine, the TE has developed many different biomaterials, with various features and peculiar functions, to be used in combination with cells and growth factors, in the generation of specialized constructs. Our proposal of natural scaffolds useful to obtain complex constructs concerns peculiar bony chips extracted from the eye bulb of adult chickens: the scleral ossicles (SOs). This proposed model is interesting because once SOs reach the definitive size in the adult animal, they are devoted only to mechanical stereotyped stress for their lifetime so that the activation of the bone remodelling should be avoided and, to do this, the osteocytes undergo massive apoptosis, making the ossicles like decellularized bones [1]. The novelty of our proposal is that the scaffolds do not require surface treatment (like further matrix deposition on the SO surface) since they are characterized, like all bones, by the well-known organic components such as type I-collagen fibres, proteoglycans and glycoproteins. The latter, for example, play the role of adhesion proteins and therefore can mediate the adhesion of the endothelial cells that should develop the vascular network. Our final goal is to obtain an in vitro 3D-vascularized natural constructs, from scaffolds easily available in nature to use in vivo for the healing of “critical-size” bone defeats. Previously [2] we identified the best preparation methods to obtain suitable SO surface for cell culture. Recently, we have performed a series of in vitro experiments to test the biocompatibility properties of the support; then, cell adhesion tests, viability and proliferation assay were carried out. Further, we tried to induce a vascular-like network organization of Mouse Aortic Endothelial Cells (MAECs) directly on the SOs surface, stimulating the cells with a known angiogenic factor, the Vascular Endothelial Growth Factor (VEGF), getting encouraging preliminary results.

2016 - PRELIMINARY OBSERVATIONS ON SCLERAL OSSICLES IN PERFORMING FUNCTIONALIZED 3D VASCULARIZED SCAFFOLDS FOR "CRITICAL_SIZE" BONE DEFECT HEALING [Abstract in Rivista]
Checchi, Marta; Smargiassi, Alberto; Ferretti, Marzia; Sena, Paola; Benincasa, Marta; Cavani, Francesco; Sola, Marco; Ranieri, Antonio; Stefania, Mitola; Palumbo, Carla
abstract

PRELIMINARY OBSERVATIONS ON SCLERAL OSSICLES IN PERFORMING FUNCTIONALIZED 3D VASCULARIZED SCAFFOLDS FOR "CRITICAL_SIZE" BONE DEFECT HEALING

2016 - Prognostic significance of the presence of circulating non-orga specific autoantibodies in a cohort of cancer patients: A five-year follow-up study [Abstract in Rivista]
Mancini, Stefano; Mariani, Francesco; Sena, Paola; Muratori, Paolo; Muratori, Luigi; Degli Esposti, Claudia; Roncucci, Luca
abstract

Serum non-organ specific autoantibodies are indicative of a worse prognosis in cancer patients

2016 - Subclinical inflammation of colorectal mucosa is related to systemic oxidative balance in healthy adult subjects [Abstract in Rivista]
Mancini, Stefano; Mariani, Francesco; Sena, Paola; Benincasa, Marta; Roncucci, Luca
abstract

Oxidative balance is related to myeloperroxidase-positive cell count in normal colorectal mucosa

2016 - The Proteasome Inhibitor Bortezomib Maintains Osteocyte Viability in Multiple Myeloma Patients by Reducing Both Apoptosis and Autophagy: A New Function for Proteasome Inhibitors [Articolo su rivista]
Toscani, Denise; Palumbo, Carla; Dalla Palma, Benedetta; Ferretti, Marzia; Bolzoni, Marina; Marchica, Valentina; Sena, Paola; Martella, Eugenia; Mancini, Cristina; Ferri, Valentina; Costa, Federica; Accardi, Fabrizio; Craviotto, Luisa; Aversa, Franco; Giuliani, Nicola
abstract

Multiple myeloma (MM) is characterized by severely imbalanced bone remodeling. In this study, we investigated the potential effect of proteasome inhibitors (PIs), a class of drugs known to stimulate bone formation, on the mechanisms involved in osteocyte death induced byMMcells. First, we performed a histological analysis of osteocyte viability on bone biopsies on a cohort of 37MMpatients with symptomatic disease. A significantly higher number of viable osteocytes was detected in patients treated with a bortezomib (BOR)-based regimen compared with those treated without BOR. Interestingly, both osteocyte autophagy and apoptosis were affected in vivo by BOR treatment. Thereafter, we checked the in vitro effect of BOR to understand the mechanisms whereby BOR maintains osteocyte viability in bone fromMM patients. We found that osteocyte and preosteocyte autophagic death was triggered during coculturing with MM cells. Our evaluation was conducted by analyzing either autophagy markers microtubule-associated protein light chain 3 beta (LC3B) and SQSTM1/sequestome 1 (p62) levels, or the cell ultrastructure by transmission electron microscopy. PIs were found to increase the basal levels of LC3 expression in the osteocytes while blunting the myeloma-induced osteocyte death. PIs also reduced the autophagic death of osteocytes induced by high-dose dexamethasone (DEX) and potentiated the anabolic effect of PTH(1-34). Our data identify osteocyte autophagy as a new potential target in MM bone disease and support the use of PIs to maintain osteocyte viability and improve bone integrity in MM patients.

2016 - The anti-Müllerian hormone (AMH) acts as a gatekeeper of ovarian steroidogenesis inhibiting the granulosa cell response to both FSH and LH [Articolo su rivista]
Sacchi, Sandro; D'Ippolito, Giovanni; Sena, Paola; Marsella, Tiziana; Tagliasacchi, Daniela; Maggi, Elena; Argento, Cindy; Tirelli, Alessandra; Giulini, Simone; La Marca, Antonio
abstract

Anti Müllerian Hormone (AMH) has a negative and inhibitory role in many functions of human granulosa-lutein cells (hGCs) including notoriously the reduction of the aromatase CYP19A1 expression induced by follicle-stimulating hormone (FSH). No data have been provided on the possible role of AMH in modulating the response to luteinizing hormone (LH) (alone or combined with FSH) as well as its effect on other enzymes involved in steroidogenesis including aromatase P450scc. The aim of this study was to investigate the role of AMH as regulator of the basal and stimulated steroids production by hGCs.

2015 - Autophagy is upregulated during colorectal carcinogenesis, and in DNA microsatellite stable carcinomas [Articolo su rivista]
Sena, Paola; Mariani, Francesco; Mancini, Stefano; Benincasa, Marta; Magnani, Giulia; Pedroni, Monica; Palumbo, Carla; Roncucci, Luca
abstract

Cancer cells are exposed to a wide range of stress sources, such as nutrient deprivation and hypoxia, as well as cytotoxic chemotherapy and radiotherapy. Certain forms of stress can also promote survival activating the metabolic autophagy pathway in cancer cells. Autophagy is dramatically increased in cancer cells. In these conditions, it is becoming evident that autophagy protects cells, by providing an alternative energy source and by eliminating dysfunctional organelles or proteins. Its role in tumorigenesis is more controversial and both the presence and the absence of autophagy have been implicated. Autophagy is known to be associated with the poor outcome of patients with various types of cancers, and its effectiveness as a prognostic marker in colorectal cancer was demonstrated by several studies. The inhibition of autophagy may be a potential therapeutic target in colorectal cancer. In vitro experiments have shown that the inhibition of autophagy increases 5-FU-induced apoptosis. There are two trials currently investigating the addition of chloroquine to 5-FU-based chemotherapy and bevacizumab. In the present study, we evaluated the expression of LC3B-II in samples of human colorectal microadenomas (i.e., dysplastic aberrant crypt foci) and carcinomas compared to normal mucosa. Furthermore, the expression pattern of LC3B-II was assessed in carcinomas classified as DNA microsatellite stable (MSS) and unstable (MSI). Thus, immunofluorescence techniques coupled with confocal microscopy and immunoblot experiments were performed. The results clearly showed a significant increase in expression of the autophagic key factor in microadenomas and carcinomas with respect to normal mucosa. In MSS carcinomas, the level of LC3B-II expression was higher than that in the MSI carcinomas.

2015 - Effects of PTH(1-34) during fracture healing after experimental bone drilling in rat femur: novel aspects [Abstract in Rivista]
Smargiassi, Alberto; Ferretti, Marzia; Cavani, Francesco; Sena, Paola; Benincasa, Marta; Palumbo, Carla
abstract

The study concerns the role of PTH(1-34) during bone lesion repair. 3-month-old male Sprague-Dawley rats, in which trans-cortical holes were drilled at femur middiaphysis, were divided in groups with/without Teriparatide administration (40g/ Kg/day), and sacrificed at different times (10, 28, 45 days). In 2002 (1) we demonstrated the occurrence of two successive bone forming processes during both skeletal organogenesis and bone repair, i.e. static (SO) and dynamic (DO) osteogenesis: the former (due to stationary osteoblasts, haphazardly grouped in cords) producing preliminary bad quality trabecular bone, the latter (due to typical polarized osteoblasts organized in ordered movable laminae) producing mechanically valid bone tissue. In brief, the primary function of SO is to provide a rigid scaffold, containing osteocytes (i.e. mechano-sensors), to DO-osteoblastic laminae; therefore, in DO mechanical factors can play a crucial role in transduction of mechanical stresses into biological signals. In the present work, histomorphometric analysis showed that, already after 10 days from drilling, notwithstanding the holes are temporarily filled by the same amount of newly-formed trabecular bone (produced by SO) independently from the treatment, the number of movable osteoblast laminae (typical of DO), covering the trabecular surface, is statistically higher in animals submitted to PTH(1-34) administration than in the control ones; this suggests that the mere effect of Teriparatide is to anticipate the occurrence of dynamic osteogenesis involved in the production of good quality bone more suitable to loading. These findings are also supported by the higher values of microhardness as well as the more ordered-fibered texture (observed by polarized light) in treated animals with respect to control ones that strongly indicates the qualitative (instead of quantitative) effect of PTH (1-34) in improving bone healing. The present investigation could be of crucial importance in further translational clinical research in humans to define the best therapeutic strategies in recovering skeletal lesions, particularly in terms of time of administration of PTH(1-34).

2015 - Melanocortins promote neurogenesis and counteract cognitive decline in a transgenic mouse model of moderate Alzheimer’s disease [Abstract in Rivista]
Neri, Laura; Canalini, Fabrizio; Calevro, Anita; Ottani, Alessandra; Vandini, Eleonora; Sena, Paola; Zaffe, Davide; Giuliani, Daniela; Guarini, Salvatore
abstract

Alzheimer's disease (AD), both sporadic and genetic, is a chronic disorder characterized by activation of the amyloid/tau cascade in the hippocampus and isocortex. Besides neuroprotective approaches, also neurorestorative strategies for AD are under intensive investigations. [1] The melanocortin system consists of endogenous neuropeptides of the adrenocorticotropin/melanocyte-stimulating hormone (ACTH/MSH) family, acting via five different metabotropic melanocortin receptor subtypes (MC1-MC5). Melanocortins also induce neuroprotection associated with long-lasting functional recovery and counteraction of cognitive decline, as found in acute experimental neurodegenerative conditions and more recently in a chronic neurodegenerative disease as AD. [2] Further, these endogenous peptides have been by us reported to stimulate neurogenesis in an acute neurodegenerative disorder as ischemic stroke. [3] Here we investigated the possible neuroprotective and neurogenic effect of melanocortins in AD with a medium level of severity by using 24 week-old (at the start of the study) APPSwe transgenic mice (Tg2576). METHODS: Tg2576 mice were treated (once daily on days 1-50) with a nanomolar dose of the melanocortin analog [Nle4,D-Phe7]α-melanocyte-stimulating hormone (NDP-α-MSH). Animals were prepared for 5-bromo-2’-deoxyuridine (BrdU) labeling of proliferating cells at days 1-11 of the study, and histological and immunohistochemical studies of the brain were performed for the assessment of neurogenesis. Further, the mouse ability to learn and recall was evaluated by means of the Morris water-maze test at the twenty-seventh week (starting 14 days after the first BrdU injection) and thirty-first week of age. Within 90 min the end of the last behavioural test (day 50 of the study; 31 week-old mice) animals were killed and the brains were removed and processed for histological examination. The whole hippocampi were dissected from brains of some animals to perform western blot analysis of the Zif268 protein (Zif268 protein is transiently expressed after synaptic activation). All values were analyzed by means of two-way repeated measures ANOVA (behavioral data) or one-way ANOVA (all other data), both followed by the Student-Newman-Keuls’ test. A value of p < 0.05 was considered significant. RESULTS: Treatment of Tg2576 mice with the melanocortin analog [Nle4,D-Phe7]α-melanocyte-stimulating hormone (NDP-α-MSH) reduced cerebral cortex/hippocampus level of Aβ deposit (p < 0.001), increased hippocampus Zif268 expression (p <0.001), improved brain histological picture and cognitive functions (p <0.001), relative to saline-treated Tg2576 animals, and no signs of toxicity were recorded. Further, immunohistochemical examination of the hippocampus on day 50 (end of the study) showed, in the dentate gyrus of NDP-α-MSH-treated Tg2576 mice, a very elevated number of BrdU immunoreactive cells colocalized with NeuN (indicator of mature neurons) and Zif268 (indicator of functionally integrated neurons), in comparison with saline-treated Tg2576 animals (p <0.001); no newly formed astrocytes were found. Animal pretreatment (before each administration of NDP-α-MSH) with the selective melanocortin MC4 receptor antagonist HS024 prevented all favourable effects of NDP-α-MSH (p <0.001). CONCLUSIONS: Our data suggest that MC4 receptor-stimulating melanocortins are able to counteract cognitive decline in experimental AD not only by affording neuroprotection, but also by inducing intense neurogenesis. These agents could be candidates for an innovative and safe strategy to counteract AD progression in humans.

2015 - NDP-α-MSH induces intense neurogenesis and cognitive recovery in Alzheimer transgenic mice through activation of melanocortin MC4 receptors [Articolo su rivista]
Giuliani, Daniela; Neri, Laura; Canalini, Fabrizio; Calevro, Anita; Ottani, Alessandra; Vandini, Eleonora; Sena, Paola; Zaffe, Davide; Guarini, Salvatore
abstract

Melanocortins exert neuroprotection in a variety of experimental neurodegenerative disorders, including Alzheimer's disease (AD). Further, in previous research we showed that these endogenous peptides stimulate neurogenesis in an acute neurodegenerative disorder such as ischemic stroke. In the present research, we investigated the potential neurogenic effect of melanocortins in AD using APPSwe transgenic mice (Tg2576). To this purpose, 24week-old animals were prepared for 5-bromo-2'-deoxyuridine (BrdU) labeling of proliferating cells on days 1-11 of the study. Treatment of Tg2576 mice with nanomolar doses of the melanocortin analog [Nle(4),D-Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH), administered once daily from day 1 to 50, improved brain histology and cognitive functions relative to saline-treated Tg2576 animals. No signs of toxicity were observed. Immunohistochemical examination of the hippocampus at the end of the study (day 50) showed that NDP-α-MSH-treated Tg2576 mice had a greater number of BrdU immunoreactive cells colocalized with NeuN (an indicator of mature neurons) and Zif268 (an indicator of functionally integrated neurons) in the dentate gyrus, relative to saline-treated Tg2576 animals; no newly formed astrocytes were found. Animal pretreatment with selective melanocortin MC4 receptor antagonist HS024 before each NDP-α-MSH administration prevented all the beneficial effects of the peptide. The present data indicate that MC4 receptor stimulation by a melanocortin prevents cognitive decline in experimental AD, this effect being associated not only with neuroprotection but also with an intense neurogenesis. MC4 receptor agonists could be innovative and safe candidates to counteract AD progression in humans.

2014 - Effect of PTH (1-34) on trabecular bone of rat vertebral body in induced-biochemical osteoporosis by calcium- deprived diet [Abstract in Rivista]
Ferretti, Marzia; Cavani, Francesco; Smargiassi, Alberto; Sena, Paola; Benincasa, Marta; Palumbo, Carla
abstract

Rats fed calcium-deprived diet were used as experimental model for studying bone modelling alterations during biochemical osteoporosis and recovery of bone loss. Such model is suitable to evaluate the possible effects exerted by PTH(1-34) in preventing as well as in recovering metabolic osteoporosis. Three-month-old Sprague Dawley male rats were divided in different groups: some fed normal diet or calcium-deprived diet with/without 40µg/Kg/day PTH(1-34), provided by Eli Lilly-USA, for 4 weeks and some with restoration of normal diet with/without PTH (1-34) for further 4 weeks. To evaluate the occurrence of osteogenesis during the first 4 weeks of the experimental period, rats received three labels of bone deposition at 1st, 20th and 27th day (and then were sacrificed); during the successive 4 weeks (in which those rats previously fed with calcium-deprived diet had restoration of normal diet), animals received three labels of bone deposition at 1st, 7th and 14th day. Histomorphometrical analyses were performed on cortical and trabecular bone taken from the central level of the 5th lumbar vertebral body, transversely sectioned. The results showed that differences among the groups were observed mainly in trabecular bone with respect to cortical one, thus underlining the different role of the two types of bone architecture in mineral and skeletal homeostasis. Concerning trabecular bone, the observations showed that administration of PTH (1-34) during calcium-deprived diet and/or during the restoration of normal diet induces higher deposition of trabecular bone with respect to that recorded in rats that never received PTH(1-34), neither during calcium-deprived diet nor during restoration of normal diet. Since increments of trabecular bone are detectable only after the period of diet restoration (but not before), the authors suggest that a chronic administration of PTH (1-34) is necessary to achieve appreciable results on bone mass recovery.

2014 - Increased expression of autophagy-related proteins in human colorectal cancer development, and correlation with DNA Microsatellite Stable and Unstable [Abstract in Rivista]
Sena, Paola; Mancini, Stefano; Mariani, Francesco; Pedroni, Monica; Magnani, Giulia; Benincasa, Marta; Roncucci, Luca
abstract

Autophagy is upregulated during human colorectal carcinogenesis

2014 - Induction of altered cellular response to oxydative stress in HT29 colon cancer cells treated with metformin [Abstract in Rivista]
Sena, Paola; Mancini, Stefano; Mariani, Francesco; Pedroni, Monica; Benincasa, Marta; Roncucci, Luca
abstract

Metformin induces oxydative stress in HT29 colon cancer cells

2014 - Inflammatory pathways in the early steps of colorectal cancer development [Articolo su rivista]
Mariani, Francesco; Sena, Paola; Roncucci, Luca
abstract

Colorectal cancer is a major cause of cancer-related death in many countries. Colorectal carcinogenesis is a stepwise process which, from normal mucosa leads to malignancy. Many factors have been shown to influence this process, however, at present, several points remain obscure. In recent years some hypotheses have been considered on the mechanisms involved in cancer development, expecially in its early stages. Tissue injury resulting from infectious, mechanical, or chemical agents may elicit a chronic immune response resulting in cellular proliferation and regeneration. Chronic inflammation of the large bowel (as in inflammatory bowel diseases), has been associated with the subsequent development of colorectal cancer. In this review we examine the inflammatory pathways involved in the early steps of carcinogenesis, with particular emphasis on colorectal. Firstly, we describe cells and proteins recently suggested as central in the mechanism leading to tumor development. Macrophages and neutrophils are among the cells mostly involved in these processes and proteins, as cyclooxygenases and resolvins, are crucial in these inflammatory pathways. Indeed, the activation of these pathways establishes an oxidative and anaerobic microenvironment with DNA damage to epithelial cells, and shifting from an aerobic to an anaerobic metabolism. Many cellular mechanisms, such as proliferation, apoptosis, and autophagy are altered causing failure to control normal mucosa repair and renewal.

2014 - Melanocortins protect against brain damage and counteract cognitive decline in a transgenic mouse model of moderate Alzheimer׳s disease. [Articolo su rivista]
Giuliani, Daniela; Galantucci, M; Neri, L; Canalini, F; Calevro, Anita; Bitto, A; Ottani, Alessandra; Vandini, E; Sena, Paola; Sandrini, Maurizio; Squadrito, F; Zaffe, Davide; Guarini, Salvatore
abstract

We previously reported that melanocortins induce neuroprotection in experimental acute and chronic neurodegenerative conditions, including Alzheimer׳s disease (AD) of mild severity. Here we investigated whether melanocortins afford neuroprotection and counteract cognitive decline in AD with a medium level of severity by using 24 week-old (at the start of the study) APPSwe transgenic mice (Tg2576). Saline-treated (days 1-50) control Tg2576 mice showed an impairment in spatial learning and memory, associated (at day 50, end of the study) with hippocampus at low levels of the synaptic activity-dependent gene Zif268, relevant brain changes such as cerebral cortex/hippocampus increased level of β-amyloid (Aβ) deposit, and neuronal loss, in comparison with wild-type animals. Treatment of Tg2576 mice (once daily at days 1-50) with a nanomolar dose of the melanocortin analog [Nle4,D-Phe7]α-melanocyte-stimulating hormone (NDP-α-MSH) reduced cerebral cortex/hippocampus level of Aβ deposit, decreased neuronal loss, increased hippocampus Zif268 expression and improved cognitive functions, relative to saline-treated Tg2576 mice. Pharmacological blockade of melanocortin MC4 receptors with the MC4 receptor antagonist HS024 prevented all favorable effects of NDP-α-MSH. Our data indicate that MC4 receptor-stimulating melanocortins are able to counteract cognitive decline in experimental AD of medium severity through induction of neuroprotection and improvement of synaptic transmission. After further studies, these agents could gain a role as disease modifying therapeutics for AD.

2014 - Morphological and quantitative analysis of BCL6 expression in human colorectal carcinogenesis. [Articolo su rivista]
Sena, Paola; Mariani, Francesco; Benincasa, Marta; PONZ DE LEON, Maurizio; Di Gregorio, C; Mancini, Stefano; Cavani, Francesco; Smargiassi, Alberto; Palumbo, Carla; Roncucci, Luca
abstract

The aim of the present study was to determine whether BCL6 is expressed during malignant transformation of the large bowel and to assess whether, and to what extent, immunoreactivity is related to the different stages of neoplastic progression. Samples of normal colorectal mucosa (n=22), microadenomas (n=22) and colorectal cancer (n=22), were analyzed by immunohistochemistry, immunofluorescence coupled with confocal microscopy and western blotting. Our results clearly outlined the marked increase occurring in both intensity and density of BCL6 protein expression in the normal mucosa-microadenoma-carcinoma sequence. Immunohistochemistry and immunofluorescence analyses showed that BCL6 is expressed at low levels in normal mucosa and increases in microadenoma and in cancer with statistical significance. These results were confirmed by western blotting data. The increasing expression of BCL6 in human colorectal cancer development suggests the involvement of BCL6 in tumor progression, from the earliest stages of carcinogenesis with significant increase in cancer. The enhanced understanding of the biological role of BCL6, previously shown to exert a key role in lymphomagenesis, may lead to a re-evaluation of this protein and may highlight the importance of performing further studies in order to identify novel therapeutic targets for colorectal cancer.

2014 - Oral surgery biomaterials: analyses of Al2O3-treated titanium surfaces tested with fibroblast and osteocyte cell lines [Abstract in Rivista]
Smargiassi, Alberto; Ferretti, Marzia; Cavani, Francesco; Sena, Paola; Benincasa, Marta; Zaffe, Davide; Facciani, Valentino; Gabrel, Ivano; Palumbo, Carla
abstract

Two different cell lines - MLO-Y4 (murine osteocytes) and 293 (human fibroblasts) - cultured for 48 hours in standard media were used to analyse engineered bio-materials (i.e. Al2O3 shot-peened titanium surfaces). Distribution, density and expression of adhesion molecules (fibronectin and vitronectin) were evaluated under scanning electron microscope (SEM) and confocal microscope (CM) as previously described [1]. The engineered biomaterial surfaces showed under SEM irregular morphology displaying variously-shaped spicules, obtained by shooting different-in-size particles of Al2O3 against the scaffolds of biomaterial. DAPI and fluorochrome-conjugated antibodies were used to highlight nuclei, fibronectin and vitronectin, under CM; cell distribution was analysed after Gold-Palladium sputtering of samples by SEM. Both SEM and CM observations showed better outcome in terms of cell adhesion and distribution in treated titanium surfaces with respect to the untreated ones. The results obtained clearly showed that this kind of surface-treated titanium, used to manufacture devices for dental implantology: i) is very suitable for cell colonization, essential prerequisite for the best osseointegration, and ii) represents an excellent solution for the development of further engineered implants with the target to obtain recovery of dental function stable over time. Further studies on these Al2O3 shot-peened-titanium surfaces, both in vitro and in vivo, will be needed to obtain accurate definition of better biomaterial outcome, also after additional treatments. References [1] Palumbo et al. (2013) Immunocytochemical and structural comparative study of committed versus multipotent stem cells cultured with different biomaterials. Micron 47: 1–9.

2014 - Up-regulation of the chemo-attractive receptor ChemR23 and occurrence of apoptosis in human chondrocytes isolated from fractured calcaneal osteochondral fragments [Articolo su rivista]
Sena, Paola; Manfredini, Giuseppe; Benincasa, Marta; Mariani, Francesco; Smargiassi, Alberto; Catani, Fabio; Palumbo, Carla
abstract

To study the expression level of a panel of pro/anti-apoptotic factors and inflammation-related receptors in chondral fragments from patients undergoing surgical treatment for intra-articular calcaneal fractures, cartilage fragments were retrieved from calcaneal fractures of 20 patients subjected to surgical treatment. Primary cultures were performed using chondral fragments from fractured and control patients. Chondrocyte cultures from each patient of the fractured and control groups were subjected to immunofluorescence staining and quantitatively analyzed under confocal microscopy. Proteins extracted from the cultured chondrocytes taken from the fractured and control groups were processed for Western blot experiments and densitometric analysis. The percentage of apoptotic cells was determined using the cleaved PARP-1 antibody. The proportion of labelled cells was 35% for fractured specimens, compared with 7% for control samples. Quantification of caspase-3 active and Bcl-2 proteins in chondrocyte cultures showed a significant increase of the apoptotic process in fractured specimens compared with control ones. Fractured chondrocytes were positively stained for ChemR23 with statistically significant differences with respect to control samples. Densitometric evaluation of the immunoreactive bands confirmed these observations. Human articular chondrocytes obtained from patients with intra-articular calcaneal fractures express higher levels of pivotal pro-apoptotic factors, and of the chemoattractive receptor ChemR23, compared with control cultures. On the basis of these observations, the authors hypothesize that consistent prolonged chondrocyte death, associated with the persistence of high levels of proinflammatory factors, could enhance the deterioration of cartilage tissue with consequent development of post-traumatic arthritis following intra-articular bone fracture.

2013 - Biodegradable device applied in flatfoot surgery: Comparative studies between clinical and technological aspects of removed screws [Articolo su rivista]
Ruozi, Barbara; Belletti, Daniela; Giuseppe, Manfredini; Tonelli, Massimo; Paola, Sena; Vandelli, Maria Angela; Forni, Flavio; Tosi, Giovanni
abstract

Poly-L-lactide (PLLA) is one of the most used polymers for biomedical application; its use in sutures and other implants has been widely investigated. Although the knowledge of PLLA biodegradation and biocompatibility features is deep, PLLA screws used to correct the flat foot deformity have deserved attention since they are not degraded inmost of cases after a long period of years (3–7) fromthe implantation. In this article, a clinical and radiological evaluation (NMR, histological and clinical outcomes) on patients was correlatedwith physico-chemical characterization (by SEM, DSC, GPC and XRD analysis at different temperatures) on both native and patientrecovered screws together with the theoretical degradation processes of PLLA-based implants. The data demonstrated the need for crossing the biodegradation and bioabsorption of the polymer with the characteristics of both the device (geometry, structure and fabrication process) and the implantation site.

2013 - Chondrocyte expression of apoptotic and pro-inflammatory factors in the development of post- traumatic arthritis in humans [Abstract in Rivista]
Sena, Paola; Benincasa, Marta; Cavani, Francesco; Ferretti, Marzia; Smargiassi, Alberto; Manfredini, Giuseppe; Palumbo, Carla
abstract

The development of post-traumatic arthritis following intra-articular fracture remains an important unsolved clinical problem. The possibility that extensive chondrocyte apoptosis occurs following intra-articular fracture, thus contributing to the development of post-traumatic arthritis, has received increasing attention [1]. It has been demonstrated the existence of a direct correlation between the rate of apoptosis and the severity of osteoarthritis [2]. Pharmacologic inhibitors of enzymes involved in apoptosis have been explored as potential therapeutic agents [3]. In the present study we aimed to deepen the characterization of apoptotic mediators, expressed by chondrocytes, involved in human post-traumatic arthritis following intra-articular fracture and the possible implication of pro-inflammatory receptors in arthritis. The expression of a panel of pro/anti apoptotic factors (Caspase-3, PARP-1, BCL2) and inflammation-related receptors (ChemR23) were analysed in chondrocytes from patients undergoing surgery for intra-articular calcaneal fractures. The factors were investigated by immunofluorescence coupled with confocal analysis and western blotting, followed by densitometric evaluation of chondrocyte cultures harvested from patients with intra-articular fractures compared with control ones. The results clearly demonstrated that a statistically significant difference exists in the expression of pro/anti apoptotic factors and ChemR23 between fractured and control patients. In conclusion our data suggest that increased chondrocyte death, occurring after cartilage injury together with inflammatory process, could play a pivotal role in the onset of arthritic disease. References [1]. Hembree W.C. et al. (2007) Viability and apoptosis of human chondrocytes in osteochon-dral fragments following joint trauma. J Bone Joint Surg Br 89(10): 1388-95. [2] Kim H.A. et al. (2000) Apoptotic chondrocyte death in human osteoarthritis. J Rheumatol 27: 455–462. [3] D'Lima D. et al. (2006) Caspase inhibitors reduce severity of cartilage lesions in experi-mental osteoarthritis. Arthritis Rheum 54(6): 1814-1821.

2013 - Immunocytochemical and structural comparative study of committed versus multipotent stem cells cultured with different biomaterials. [Articolo su rivista]
Palumbo, Carla; Baldini, Andrea; Cavani, Francesco; Sena, Paola; Benincasa, Marta; Ferretti, Marzia; Zaffe, Davide
abstract

The aim of this work was the comparison of the behavior of committed (human osteoblast cells - hOB - from bone biopsies) versus multipotent (human dental pulp stem cells - hDPSC - from extracted teeth) cells, cultured on shot-peened titanium surfaces, since the kind of cell model considered has been shown to be relevant in techniques widely used in studies on composition/morphology of biomaterial surfaces. The titanium surface morphology, with different roughness, and the behavior of cells were analyzed by confocal microscope (CM), scanning electron microscope (SEM) and X-ray microanalysis. The best results, in terms of hOB adhesion/distribution, were highlighted by both CM and SEM in cultured plates having 20-mum-depth cavities. On the contrary, CM and SEM results highlighted the hDPSC growth regardless the different surface morphology, arranged in overlapped layers due to their high proliferation rate, showing their unfitness in biomaterial surface test. Nevertheless, hDPSC cultured inside 3D-matrices reproduced an osteocyte-like three-dimensional network, potentially useful in the repair of critical size bone defects. The behavior of the two cell models suggests a different use in biomaterial cell cultures: committed osteoblast cells could be appropriate in selecting the best surfaces to improve osseointegration, while multipotent cells could be suitable to obtain in vitro osteocyte-like network for regenerative medicine. The originality of the present work consists in studying for the first time two different cell models (committed versus multipotent) compared in parallel different biomaterial cultures, thus suggesting distinct targets for each cellular model. Copyright 2012 Elsevier Ltd. All rights reserved.

2013 - Induced Biochemical osteoporosis: Effects of 1-month calcium–deprived diet on rat bone remodelling with/without contemporary administration of PTH(1-34) [Abstract in Rivista]
Ferretti, Marzia; Cavani, Francesco; Sena, Paola; Benincasa, Marta; Smargiassi, Alberto; Palumbo, Carla
abstract

It is known that rats fed calcium-deprived diet develop osteoporosis due to en-hanced bone resorption secondary to parathyroid overactivity resulting from nutritional hypocalcemia. Therefore, rats provide a good experimental animal model for studying bone remodelling alterations during biochemical osteoporosis. This preliminary study is performed in 3 month-old Sprague Dawley male rats, divided into 4 groups (5 rats each): 1) base line, 2) normal diet for 4 weeks, 3) calcium-deprived diet for 4 weeks; 4) calcium-deprived diet for 4 weeks plus contemporary administration of PTH(1-34) 40µg/kg/day. Three labels of osteogenesis were performed at 1st , 20th and 27th day of experimental period in order to evaluate bone formation during animal treatment. His-tomorphometrical analyses were performed on cortical bone of femoral diaphyses, as well as on trabecular bone of distal femoral metaphyses, both transversely sectioned. The preliminary results showed that at femur mid-diaphyseal level the diet induced a reduction of cortical bone area (even if not significant) with enlargement of the medul-lary canal due to endosteal resorption, while periosteal neo-deposition is similar in all groups and particularly abundant in those periosteal regions mainly devoted in answering the mechanical demands. PTH(1-34) treatment seems to reduce endosteal resorption only in those surfaces where periosteal mechanical loading are less consistent. Conversely, PTH(1-34) treatment doesn't seem to affect osteoblast activity. Moreover, in distal femoral metaphyses, diet induced osteoclast activity, with a decrease in the amount of trabecular bone volume, confirming that this architecture is mainly devoted in answering the metabolic demands. The novelty of the proposed model Is the contemporary administration of PTH(1-34) together with calcium deprived diet to evaluate induced-biochemical osteoporosis. This model seems a good starting point for successive studies in order to study bone alterations during unbalanced calcium metabolism frequently occurring in aging and to define time and manner of bone mass recovery.

2013 - Myeloma-Induced Osteocyte Death Was Blunted By Proteasome Inhibitors Through The Modulation Of Autophagy [Poster]
Toscani, Denise; Palumbo, Carla; Palma, Benedetta Dalla; Bolzon, Marina; Ferretti, Marzia; Sena, Paola; Guasco, Daniela; Martella, Eugenia; Aversa, Franco; Giuliani, Nicola
abstract

Osteocytes are critical in the maintenance of bone integrity regulating bone remodeling through the cell death and autophagy, a cellular process stress-induced to prolong cell survival but when induced excessively can cause cell death. Recently we have demonstrated that an increased osteocyte death is involved in multiple myeloma (MM)-induced osteolysis. However the mechanisms involved in this process as well as the effect of the proteasome inhibitors able to stimulate bone formation are not known and have been investigated in this study. Firstly the effect of the proteasome inhibitors BOR and MG262 on osteocyte viability was evaluated in vitro in murine osteocytic cell line MLO-Y4 and in the human pre-osteocytic one HOB-01. Both cell lines were co-coltured for 48 hours in the presence or absence of the human myeloma cell lines (HMCLs) RPMI8226 and JJN3, placed in a traswell insert. The treatment for 12-24 hours with (BOR) (2nM) and MG262 (10nM) significantly blunted MLO-Y4 and HOB-01 cell death. In addition, dexamethasone (DEX)-induced MLO-Y4 apoptosis, obtained at high doses (10-5-10-6 M), was reduced by the treatment with proteasome inhibitors. Interestingly, we found that PTH short-term treatment potentiated the in vitro effects of proteasome inhibitors on DEX-induced osteocyte death. To evaluate the presence of autophagy in osteocytes, we checked the expression of the autophagic marker LC3 both by confocal microscopy and western blot analysis in the co-colture system with MLO-Y4 and RPMI-8226. Prevalence of autophagic cell death and in a lesser extent apoptosis was observed in this system. BOR increased the basal level of LC3 indicating a pro-survival and protective function of autophagy against the BOR-induce stress. On the contrary, when cells undergo to a stronger stress such as in the presence of HMCLs or by treatment with high dose of DEX we found that both proteasome inhibitors BOR and MG262 blocked autophagic cell death in osteocytes. To translate our in vitro evidence in a clinical perspective, thereafter we performed a histological evaluation on bone biopsies of a cohort of 37 newly diagnosis MM patients 31 of them with symptomatic MM and 6 with smoldering MM (SMM). The 55% of patients with MM have evidence of osteolytic lesions at the X-rays survey. Bone biopsies were obtained at the diagnosis and after an average time of 12 months of treatment or observation. Osteocyte viability was evaluated in a total of 500 lacunae per histological sections. A significant increase of the number of viable osteocytes was demonstrated in MM patients treated with BOR-based regimen as compared to those treated without BOR (% median increase: +6% vs. +1.30%; p=0.017). Patients treated with BOR alone showed the highest increase of osteocyte viability, as compared to those either treated without BOR (+11.6% vs. +1.3%, p=0.0019) or treated with BOR plus DEX (+11.6% vs. +4.4%, p=0.01). A reduction of both osteocyte apoptosis and autophagy was demonstrated by TUNEL assays and confocal microscopy. On the other hand, any significant difference was not observed in patients treated with Thalidomide (THAL) or Immunomodulatory drugs (IMiDs) than in those untreated with these drugs (p= 0.7). A multiple regression non-parametric analysis showed that BOR had a significant positive impact on osteocyte viability (p=0.042) whereas THAL/IMiDs as well as Zoledronic acid (ZOL) treatments have not (p=0.2). BOR also counterbalanced the negative effect of DEX treatment (p=0.035). Our data suggest that proteasome inhibitors blunted osteocyte cell death induced by MM cells and DEX through the modulation of the autophagy supporting their use to improve bone integrity in MM patients.

2013 - PLZF expression during colorectal cancer development and in normal colorectal mucosa according to body size, as marker of colorectal cancer risk. [Articolo su rivista]
Mariani, Francesco; Sena, Paola; Magnani, Giulia; Mancini, Stefano; Palumbo, Carla; PONZ DE LEON, Maurizio; Roncucci, Luca
abstract

Promyelocytic leukemia zinc finger protein (PLZF) is a protein involved in various signaling, growth regulatory, and differentiation pathways, including development/function of some T cells. Here, we aimed at the detection of PLZF during colorectal carcinogenesis, using immunofluorescence, and at the evaluation of the colocalization of PLZF with CD2 and CD56 positive cells (T, γδ, NK, and NKT cells), using confocal-microscopy, along colorectal carcinogenesis, since its earliest stages, that is, dysplastic aberrant crypt foci (ACF). Furthermore, we analyzed PLZF in the normal colonic mucosa (NM) according to anthropometric parameters of the subject. NM exhibited strong CD56 fluorescent staining. This infiltration was lost in both ACF and colorectal carcinoma (CRC), while PLZF presence increased from NM to ACF and CRC. Strong association was found between CD56+ colonic mucosa cell infiltration and body mass index. Interestingly, an increased stromal PLZF-reactivity was present in NM of obese subjects. This study shows that overexpression of PLZF and exclusion of NK cells in dysplastic microenvironment are very early events in the stepwise sequence leading to CRC and that lower levels of CD56+ cells in NM, together with increased levels of PLZF+ cells, can be a reflection of colon cancer risk due to obesity.

2013 - Th Inducing POZ-Kruppel Factor (ThPOK) Is a Key Regulator of the Immune Response since the Early Steps of Colorectal Carcinogenesis [Articolo su rivista]
Mariani, Francesco; Sena, Paola; Pedroni, Monica; Benatti, Piero; Manni, Paola; Di Gregorio, C; Manenti, Antonio; Palumbo, Carla; PONZ DE LEON, Maurizio; Roncucci, Luca
abstract

We purposed to evaluate the role of Th inducing POZ-Kruppel Factor (ThPOK), a transcriptional regulator of T cell fate, in tumour-induced immune system plasticity in colorectal carcinogenesis. The amounts of CD4+, CD8+ and CD56+ and ThPOK+ cells infiltrate in normal colorectal mucosa (NM), in dysplastic aberrant crypt foci (microadenomas, MA), the earliest detectable lesions in colorectal carcinogenesis, and in colorectal carcinomas (CRC), were measured, and the colocalization of ThPOK with the above-mentioned markers of immune cells was evaluated using confocal microscopy. Interestingly, ThPOK showed a prominent increase since MA. A strong colocalization of ThPOK with CD4 both in NM and in MA was observed, weaker in carcinomas. Surprisingly, there was a peak in the colocalization levels of ThPOK with CD8 in MA, which was evident, although to a lesser extent, in carcinomas, too. In conclusion, according to the data of the present study, ThPOK may be considered a central regulator of the earliest events in the immune system during colorectal cancer development, decreasing the immune response against cancer cells.

2012 - APPLICATION OF POLY-L-LACTIDE SCREWS IN FLAT FOOT SURGERY: HISTOLOGICAL AND RADIOLOGICAL ASPECTS OF BIO-ABSORPTION OF DEGRADABLE DEVICES. [Articolo su rivista]
Sena, Paola; Manfredini, G.; Barbieri, Claudia; Mariani, Francesco; Tosi, Giovanni; Ruozi, Barbara; Ferretti, Marzia; Marzona, Laura; Palumbo, Carla
abstract

The flat foot in childhood is a condition frequently observed in orthopedic practice but it is still debated when and in which patients surgical corrective treatment is appropriate; recently, the application of poly-L-lactic-acid (PLLA) screws was proposed. The present study investigates a group of 33 patients treated with PLLA expansion endorthesis in order to evaluate the deformity correction. Clinical and radiological outcomes in patients were correlated with: a) morphological characterization of screws both before and after being removed from patients, when necessary; b) histological and bio-molecular evaluation of degradation processes of the implants, focusing attention on the correlation between the cellular cohort involved in inflammatory reaction and the bio-absorption degree of PLLA screws. Deformity correction was mostly achieved, with minimal need of screw removal; the results obtained clearly show the occurrence of chronic rather than acute inflammation in removed screw specimens.At the histological level, after biomaterial implantation, the sequence of events occurring in the surrounding tissues ultimately ends in the formation of foreign body giant cells (FBGCs) at the tissue/material interface; but the mechanisms which influence the fate of screw implants, i.e. the resolution of acute inflammation rather than the progression towards chronic inflammation, are of crucial importance for biodegradable materials like “polylactic acid”. In fact, the FBGC response ensures a long-term mechanism which eliminates the foreign material from the body, but at the same time the implications of prolonged FBGC responses, which generate negative side effects, could significantly impede the healing progress.

2012 - Matrix metalloproteinases 15 and 19 are stromal regulators of colorectal cancer development from the early stages [Articolo su rivista]
Sena, Paola; Mariani, Francesco; Marzona, Laura; Benincasa, Marta; PONZ DE LEON, Maurizio; Palumbo, Carla; Roncucci, Luca
abstract

Matrix metalloproteinases (MMPs) have been well characterized for their ability to degrade extracellular matrix proteins and, thus, they have been studied to elucidate their involvement in both tumor development and progression. In the present study, attention was focused on MMP-15 and MMP-19, two less known members of the MMP family. The expression profile of MMP-15 and -19 was assayed in samples of normal colorectal mucosa, microadenomas and cancer using confocal analysis, western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Both qRT-PCR and western blotting showed that MMP-15 and MMP-19 appeared to be upregulated during colorectal tumorigenesis, with different expression patterns: MMP-15 expression level increases from normal mucosa to microadenomas, with a reduced level in cancer with respect to microadenomas; the semiquantitative immunofluorescence analysis showed a stromal localization of this protein in the early phases of neoplastic transformation. Increasing amount of MMP-19 mRNA and protein levels were observed in the progression of colonic lesions; MMP-19 staining increased in the normal mucosa-microadenoma-carcinoma sequence. Such different expression patterns, are probably due to the different roles played in colorectal tumorigenesis by these two molecules. Conflicting data on the role of these proteins in tumor progression have been reported, thus, an improved understandingof the biological roles of MMPs, in particular the lesser known members such as MMP-15 and 19, in colorectal cancer may lead to a re-evaluation of the use of MMP inhibitors and suggests the need of integrated translational studies on MMP expression patterns.

2012 - Osteocyte apoptosis and absence of bone remodeling in human auditory ossicles and scleral ossicles of lower vertebrates: a mere coincidence or linked processes? [Articolo su rivista]
Palumbo, Carla; Cavani, Francesco; Sena, Paola; Benincasa, Marta; Ferretti, Marzia
abstract

Considering the pivotal role as bone mechanosensors ascribed to osteocytes in bone adaptation to mechanical strains, the present study analyzed whether a correlation exists between osteocyte apoptosis and bone remodeling in peculiar bones, such as human auditory ossicles and scleral ossicles of lower vertebrates, which have been shown to undergo substantial osteocyte death and trivial or no bone turnover after cessation of growth. The investigation was performed with a morphological approach under LM (by means of an in situ end-labeling technique) and TEM. The results show that a large amount of osteocyte apoptosis takes place in both auditory and scleral ossicles after they reach their final size. Additionally, no morphological signs of bone remodeling were observed. These facts suggest that (1) bone remodeling is not necessarily triggered by osteocyte death, at least in these ossicles, and (2) bone remodeling does not need to mechanically adapt auditory and scleral ossicles since they appear to be continuously submitted to stereotyped stresses and strains; on the contrary, during the resorption phase, bone remodeling might severely impair the mechanical resistance of extremely small bony segments. Thus, osteocyte apoptosis could represent a programmed process devoted to make stable, when needed, bone structure and mechanical resistance.

2012 - Overweight, inflammation of normal colorectal mucosa, and cancer risk [Abstract in Rivista]
Roncucci, Luca; Mariani, Francesco; Sena, Paola; Palumbo, Carla; Pedroni, Monica
abstract

Myeloperoxidase-positive cell infiltration of normal colorectal mucosa is related to body fatness and colorectal cancer risk.

2012 - Proposed roles of the immune response regulator-ThPOK in human colorectal cancer progression [Abstract in Rivista]
Sena, Paola; Mariani, Francesco; Roncucci, Luca; Benincasa, Marta; PONZ DE LEON, Maurizio; Palumbo, Carla
abstract

Solid tumours are commonly infiltrated by several immune cells [1-3]. In cancer, immune cells play conflicting roles with both the potentials to eliminate or to promote malignancy. In contrast to infiltration of cells responsible for chronic inflammation, the presence of high numbers of lymphocytes, especially T cells, has been reported to be important as indicator of good prognosis in many types of cancer [4-7]. The thorough knowledge of both manners and pathways with which tumors are able to evade immune-mediated attack, once established, is therefore of crucial importance. The strategies to escape anti-tumor immune responses include the limited priming or differentiation of antitumor T cells and the role of tumor microenvironment in order to prevent infiltration or activation of effector phase functions. We proposed to evaluate the role of Th inducing POZ-Kruppel Factor (ThPOK), a transcriptional regulator of T cell fate, in tumour-induced immune system plasticity during colorectal carcinogenesis. Data were collected on the amounts of CD4+, CD8+ and CD56+ as well as on ThPOK+ cells infiltrated in normal colorectal mucosa (NM), in dysplastic aberrant crypt foci (microadenomas, MA, the earliest detectable lesions in colorectal carcinogenesis) and in colorectal carcinomas (CRC); moreover, the colocalization of ThPOK with the above-mentioned markers of immune cells was evaluated using confocal microscopy. Interestingly, ThPOK showed a prominent increase since MA. A strong colocalization of ThPOK with CD4 both in NM and in MA was observed, weaker in carcinomas. Surprisingly, there was a peak in the colocalization levels of ThPOK with CD8 in MA, which was evident, although to a lesser extent, also in carcinomas. In conclusion, according to the data of the present study, ThPOK may be considered a central regulator of the earliest events in the immune system during colorectal cancer development. The novelty of the present study is the proposed role of ThPOK in influencing the immune response against cancer cells. References [1] Dunn et al. (2004) The immunobiology of cancer immunosurveillance and immunoediting. Immunity 21: 137-148. [2] Knaapen et al. (2006) Neutrophils and respiratory tract DNA damage and mutagenesis: a review. Muta-genesis 21: 225-236. [3] Coussens and Werb (2002) Inflammation and cancer. Nature 420: 860-867. [4] Watt and House (1978) Colonic carcinoma: a quantitative assessment of lymphocyte infiltration at the periphery of colonic tumors related to prognosis. Cancer 41: 279-282. [5] Galon et al. (2006) Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 313: 1960-1964. [6] Pagès et al. (2009) In situ cytotoxic and memory T cells predict outcome in patients with early-stage colorectal cancer. J Clin Oncol 27: 5944-5951. [7] Mlecnik et al. (2010) Biomolecular network reconstruction identifies T-cell homing factors associated with survival in colorectal. Gastroenterology 138: 1429-1434.

2012 - Role of osteocyte apoptosis in peculiar ossicles of the hearing sense organ: preliminary observations on hearing loss and osteoporosis [Abstract in Rivista]
Palumbo, Carla; Presutti, Livio; Genovese, Elisabetta; Cavani, Francesco; Sena, Paola; Benincasa, Marta; Ferretti, Marzia
abstract

Starting point of the present study is the osteocyte role in bone remodelling that allows bone adaptation to mechanical load [1-3]. Bone remodelling has been investigated in relation to the occurrence of apoptosis [4] to understand if and how the process of programmed cell death interferes with bone turnover. In 1998, in a study on human middle ear, Marotti et al. [5] demonstrated that: 1) over 40% of osteo-cytes are dead within the 2nd year of age (but the authors were not able to demonstrate if osteocyte death occurred by degeneration or apoptosis); 2) bone remodelling occurs only occasionally. Recently [6], we showed that: 1) osteocytes of human auditory ossicles die by apoptosis; 2) also osteocytes located inside scleral ossicles of lower vertebrate eye (reptiles and birds) phylogenetically so far from human auditory ossicles are widely affected by apoptosis (about 60%); 3) in scleral ossicles bone turnover never occur. It is to be noted that both auditory ossicles of human ear and scleral ossicles of vertebrate eye are peculiar bony segments continuously submitted to stereotyped stresses and strains, with specialized func-tions: the first are involved in sound wave transmission and the latter protect the eyeball against deformation during the movement and have a role in visual accomodation, providing attachment for the ciliary muscles. In both cases, bone remodelling might severely impair, by resorption, the mechanical resistance of these extremely small specialized bony segments. Thus, we suggested that in auditory and scleral ossicles, submitted to stereotyped loading for all life, bone mechanical adaptation is not needed and osteocyte programmed death could represent the mechanism to avoid bone remodelling and to make stable, when necessary, bone structure and mechanical resistance. More recently, to confirm this hypothesis, clinical data were collected from a cohort of patients aged 55-85 years affected by hearing loss. The main target of the present study is to exclude any correlation between hearing loss and osteoporosis. During osteoporosis, unbalanced bone turnover causes the bone depletion in skeletal segments; such condition, in the peculiar ossicles of human middle ear, should imply hearing impairment. Our preliminary observations indicate, instead, that osteoporotic patients do not show higher percentage of hearing loss with respect to non osteoporotic ones. This evidence is ascribable to osteocyte apoptosis of auditory ossicles that avoid bone remodelling, thus assuring the integrity of such bony segments also in osteoporotic conditions. References [1] Turner (1991) Omeostatic control of bone structure: an application of feed-bach theory. Bone 12: 203-217. [2] Turner and Forwood (1995) What role does the osteocyte network play in bone adaptation? Bone 16: 283-285. [3] Marotti (1996) The structure of bone tissue and the cellular control oftheir deposition. IJAE 101(4): 26-79. [4] Noble et al. (1997) Identification of apoptotic changes in osteocytes in normal and pathological human bone. Bone 20: 273-282. [5] Marotti et al. (1998) Morphometric investigation on osteocytes in human auditory ossicles. Ann Anat 180: 449-453. [6] Palumbo et al. (2012) Osteocyte apoptosis in human auditory ossicles and scleral ossicles of lower ver-tebrates: a mere coincidence or linked processes? Calcif. Tissue Int. 90: 211-218.

2012 - Structural and histomorphometric evaluations of ferutinin effects on the uterus of ovariectomized rats during osteoporosis treatment [Articolo su rivista]
Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Benincasa, Marta; Sena, Paola; Carnevale, Gianluca; Zavatti, Manuela; Vittoria Di, Viesti; Zanoli, Paola; Palumbo, Carla
abstract

Aims: The effects of chronic administration of Ferutinin (phytoestrogen found in the plants of genus Ferula),compared with those elicited by estradiol benzoate, were evaluated, following ovariectomy, on the uterus ofovariectomized rats as regard weight, size, structure and histomorphometry.Main methods: The experimental study included 40 female Sprague–Dawley rats, assigned to two different protocols,i.e. preventive and recovering. In the preventive protocol, ferutinin (2 mg/kg/day)was orally administeredfor 30 days, starting from the day after ovariectomy; in the recovering protocol, ferutinin was administered, atthe same dosage, for 30 days starting fromthe 60th day after ovariectomy, when osteoporosiswas clearly established.Its effects were compared with those of estradiol benzoate (1.5 μg per rat twice a week, subcutaneouslyinjected) vs. vehicle-treated ovariectomized controls and vehicle-treated sham-operated controls. Uteri were removed,weighed and analysed under both the structural and histomorphometrical points of view.Key findings: Our data show that ferutinin acts, similarly to estradiol benzoate, on the uterus stimulating endometrialand myometrial hypertrophy; this notwithstanding, the phytoestrogen ferutinin, in contrast to estrogentreatment, appears to increase apoptosis in uterine luminal and glandular epithelia.Significance: Ferutinin, used in osteoporosis treatment primarily for bonemass recovering, seems in linewith aneventual protective function against uterine carcinoma, unlike estrogens so far employed in hormone replacementtherapy (HRT).

2011 - EXPRESSION PROFILES OF MATRIX METALLOPROTEINASES 15 AND 19 IN HUMAN COLORECTAL CARCINOGENESIS [Abstract in Rivista]
Sena, Paola; Mariani, Francesco; Marzona, Laura; Roncucci, Luca; Palumbo, Carla
abstract

INTRODUCTION: The matrix metalloproteinases (MMPs) have been well characterized for their ability to degrade extracellular matrix proteins and thus they have been extensively studied to elucidate their involvement in both tumour development and progression. In the present study the attention were focused on two members of MMP family, i.e. MMP-15 and MMP-19.METHODS: The expression profile of MMP-15 and 19 were assayed from samples of normal mucosa, microadenomas and cancer using confocal analysis, Western blotting and quantitative reverse transcription polymerase chain reaction (Q-PCR).RESULTS: The semiquantitative immunofluorescence analysis showed that MMP-15 expression level increases from normal mucosa to microadenomas, with a reduced level in cancer respect to microadenomas, while MMP-19 staining increases in normal mucosa-microadenoma-carcinoma sequence.Both Q-PCR and Western blot methods correlate with immunofluorescence behaviour of MMP-15 and 19, showing a significantly higher expression of MMP-15 in microadenomas compared to normal mucosa, and indicating an increasingly amount of MMP-19 levels in the progression of colon lesions.CONCLUSIONS: MMP-15 and 19 appear to be up-regulated during tumorigenesis, with different expression patterns, that are probably due to the different roles played by these two molecules. The literature up to now reported show conflicting data regarding the role of these proteins in tumor progression so that the improved understanding of the biological roles of MMPs in colorectal cancer should lead to a re-evaluation of the use of MMP inhibitors and highlight the importance of integrated translational studies on the MMP expression patterns.

2011 - Effect of pulsed electromagnetic fields (PEMFs) on condrogenic phenotype maintenance of MSCs in presence of pro-inflammatory cytochines: preliminary results [Abstract in Rivista]
Palumbo, Carla; Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Benincasa, Marta; Sena, Paola; Gian Luigi, Sacchetti; Stefania, Setti; Cadossi, Ruggero
abstract

The aim of the present study was to evaluate in vitro the effects of PEMFs on maintenance over time of chondrocyte phenotype of conditioned Mesenchimal Stem Cells (MSCs), in presence of pro-inflammatory cytokines (IL-ß1). MSCs, taken from bone marrow, were pellet-cultured in medium conditioning towards the chondrogenic lineage. Two targets were pursued: the first was to standardize the method to obtain chondrocyte pellets in terms of type/amount of withdrawal, time/degree of differentiation and amount of extracellular matrix production; the second was to extend over time chondrocyte differentiation, checking the phenotype maintenance, after adding pro-inflammatory IL-ß1 cytokine in culture medium with/without the application of PEMFs (device provided by IGEA-Carpi). The pellets obtained were coltured for different times (21, 28, 34 days), verifying the presence of type-II collagen (as index of chondrocyte differentiation) both by means of TEM analysis and immunoreaction. The best differentiation was obtained after 28 days of culture; in such pellets the studies were performed in triplicate for 15 days, identifying four experimental conditions: 1) without IL-b1 and PEMFs; 2) with IL-b1, without PEMFs; 3) without IL-b1 and with PEMFs; 4) with IL-b1 and PEMFs. The parameters of applied PEMFs were 1.7mT and 75Hz, and the time of application was 4 hours/day. Medium was changed every 3-4 days and stored for the evaluations of PGE2 (indicative of inflammation) and proteoglycans (indicative of chondrogenic differentiation). At the end of the experiment, each pellet was fixed with paraformaldehyde 4% and embedded in paraffin; sections (5 µm thick) were obtained and stained with Toluidin Blue in order to evaluate metachromasia. The results indicate that: 1) only the pellets treated with IL-b1 without PEMFs did not show metachromasia, indicanting a chondrocyte de-differentiation towards fibroblastic phenotype; 2) only in pellets treated with IL-b1 and with PEMF application, after about 12 days of treatment the amount of PGE2 in medium decreases (31%) while the proteoglycan production slightly increases (2%).In conclusion, if the results will be confirmed, pulsed electromagnetic fields could be proposed in preventing chondrocyte de-differentiation due to inflammation induced by IL-ß1; this with the final aim to integrate regenerative medicine techniques to apply in the healing of joint cartilage lesions with bio-physic energy devices, in order to obtain a stable-in-time recovery of physiologic function of articular surfaces that suffered a severe injury.

2011 - INTERACTION OF BIOPHYSIC STIMULI ON CONDROGENIC DIFFERENTIATION OF MSCs: PRELIMINARY RESULTS ON THE EVALUATION OF ANTI-INFLAMMATORY EFFECT OF ELECTROMAGNETIC FIELDS [Abstract in Rivista]
Palumbo, Carla; Ferretti, Marzia; Bertoni, Laura; Cavani, Francesco; Benincasa, Marta; Taronna, ANGELO PIO; Sena, Paola; Setti, S.; Cadossi, Ruggero
abstract

The aim of the present study is to investigate in vitro chondrocyte-like cells treated with electromagnetic fields to evaluate over time maintenance of chondrocyte phenotype, in presence of pro-inflammatory cytokines (IL-1ß). Mesenchimal Stem Cells taken from bone marrow were cultured (in pellet) in medium conditioning towards the chondrogenic lineage. The targets are firstly to standardize the method to obtain chondrocyte pellets in terms of a) type/amount of withdrawal, b) time/degree of differentiation, and c) amount of extracellular matrix production; secondly, to extend over time chondrocyte differentiation, checking the phenotype maintenance, after adding pro-inflammatory cytokines in culture medium with/without the application of electromagnetic fields (device provided by IGEA-Carpi). The pellets obtained were coltured for different times (21, 28, 34 days), verifying the presence of type 2 collagen (index of chondrocyte differentiation). The best differentiation was obtained after 28 days of culture. In such pellets, after inflammatory induction and application of electromagnetic field (1.7mT, 75Hz) for 15 days, the observations showed that after about 12 days of treatment the amount of PGE2 in medium decreases (31%) while the proteoglycan production slightly increases (2%). In conclusion, electromagnetic fields could be proposed (if the results will be confirmed) in preventing chondrocyte de-differentiation due to inflammation induced by IL-1ß, to integrate regenerative medicine techniques in the healing of cartilage lesions.

2011 - Quantification and localization of matrix metalloproteinases (MMP15 and MMP19) in human colorectal carcinogenesis. [Abstract in Rivista]
Sena, Paola; Mariani, Francesco; Marzona, Laura; Roncucci, Luca; Palumbo, Carla
abstract

Matrix metalloproteinases (MMPs) are capable of degrading all kinds of extracellular matrix proteins, but they also can process a number of other bioactive molecules. They are well known to be involved in the cleavage of cell surface receptors, the release of apoptotic ligands (as FAS ligand) and chemokine/cytokine activation/de-activation. MMPs are also thought to play a pivotal role on cell processes like proliferation, migration (adhesion/dispersion), differentiation, apoptosis and host defence; thus they have been extensively studied to elucidate their involvement in both tumour development and progression. In the present study the attention was focused on two members of MMP family, i.e. MMP-15 and MMP-19, not jet well investigated as far as their role is concerned in the onset of colorectal neoplastic pathologies.The expression profile of MMP-15 and MMP-19 was assayed from samples of: a) normal mucosa, b) microadenomas and c) cancer, using confocal analysis, Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Western blot and qRT-PCR showed that MMP-15 expression level increases from normal mucosa to microadenomas, with a reduced level in cancer respect to microadenomas. The semiquantitative immunofluorescence analysis correlate with these data showing a localization exclusively at the stromal level of this protein, suggestive of an important role of stromal compartment, especially in the early phases of neoplastic transformation.Increasingly amount of MMP-19 mRNA and protein levels were instead recorded in the progression of colon lesions both in epithelial and stromal compartments. MMP-19 staining increases in parallel to normal mucosa ® microadenoma® carcinoma sequence; in particular this protein was showed to be expressed at the epithelial level only at the end of the sequence, indicating an intriguing epithelial involvement of MMP-19 production only in the late stages of carcinogenesis.In conclusion, MMP-15 and MMP-19 appear to be up-regulated during tumorogenesis, with different expression patterns, which in turn are probably due to the different roles played by these two molecules. The results reported up to now in literature show conflicting data regarding the specific role of these proteins in tumour progression so that the improved understanding of the biological roles of MMPs in colorectal cancer suggests a re-evaluation of the use of MMP inhibitors and highlights the importance of integrated translational studies on the MMP expression patterns.

2011 - RGB method in immunofluorescence investigations on stem cells [Articolo su rivista]
Riccio, Massimo; E., Resca; Bertoni, Laura; Cavani, Francesco; Sena, Paola; Ferretti, Marzia; Baldini, Andrea; Palumbo, Carla; DE POL, Anto
abstract

Colour is not related to a particular discipline, but it is transversely present in many circles and inalmost all the aspects of life. It has a special value in art, but also as far as other disciplines areconcerned, like the sciences, the colour is at the basis of some of their intrinsic significances and it oftenneeded to allow the interpretation of some of their phenomena as well. As regards the development ofcell biology knowledge, colour acquired more and more importance in revealing the observations of theresearchers. A field in which the methods based on the colours are particularly employed is theimmunofluorescence, used to identify specific proteins in cells and tissues. These techniques combinethe fluorochrome properties with specific molecules, i.e. antibodies, directed against particularsubstances to investigate, for example a specific protein. In single immunofluorescence analysis, thesignal from an excited fluorochrome corresponds to a particular protein. In multiple immunofluorescenceanalysis, two or more signals are simultaneously detected to show the localization of differentproteins on the same sample. The three primary colours red, green and blue were currently assigned tothe signals from immunofluorescence-processed samples and visualized by the RGB method. In thepresent work, different examples of RGB applications in immunocytochemical investigations areshowed: the first concerns the multiple analysis of three markers, localized in different loci of the cellplasma membrane; the second is related to the co-localization of two signals in the same site of specificsubcellular structures. In this case the secondary colours, obtained by overlapping the primary ones,demonstrate the specific co-presence of two proteins in the same site. With the present paper, theauthors wish to underline the relevant role of colours also in those areas in which colours are the meansnot the end.

2010 - Altered Expression of Apoptosis Biomarkers in Human Colorectal Microadenomas [Articolo su rivista]
Sena, Paola; Roncucci, Luca; Marzona, Laura; Mariani, Francesco; Maffei, Stefania; Manenti, Antonio; DE POL, Anto
abstract

Human colorectal microadenomas are considered the earliest detectable premalignant lesions in the colon. They can be identified as aggregates of enlarged crypts with thicker epithelial linings and elongated luminal openings on the colonic mucosal surface after methylene blue staining and observation under a dissecting microscope. Multiple lines of evidence suggest that a central role in neoplastic development is played by the inhibition of apoptosis, followed by disruption of DNA repair. Understanding the early mechanisms of colorectal carcinogenesis may help develop new approaches of colorectal cancer prevention and treatment. The aim of the present study was to quantify poly-ADP ribose polymerase 1 (PARP-1)-positive cells and to evaluate apoptotic control mechanisms through Caspase-3 active and Bcl-2 protein expression in human microadenomas and in normal colorectal mucosa using immunofluorescence techniques coupled with confocal microscopy and immunoblot experiments. The mean percentage of PARP-1-positive epithelial cells was 3.0 +/- 0.37% (SD) and 15.67 +/- 0.40% in microadenoma and in normal mucosa, respectively. Proteins involved in programmed cell death were differently expressed in microadenoma and in normal mucosa. Indeed, by semiquantitative immunoflourescence analysis, confirmed byWestern blot, microadenoma showed high levels of Caspase-3 active and low levels of Bcl-2 expression, whereas the opposite was true for normal colorectal mucosa. In the stroma of normal colorectal mucosa, fibroblast-like cells and neutrophils were the cells that underwent apoptosis to a greater extent. In conclusion, malfunction of the control mechanisms of programmed cell death seems present in the early stages of colorectal cancer development. Cancer Epidemiol Biomarkers Prev; 19(2); 351-7. (C) 2010 AACR.

2010 - Altered expression of apoptosis biomarkers in human colorectal microadenomas (Cancer Epidemiology, Biomarkers & Prevention (2010) 19, (351-357)) [Articolo su rivista]
Sena, P.; Roncucci, L.; Marzona, L.; Mariani, F.; Maffei, S.; Manenti, A.; De Pol, A.
abstract

2009 - Cyclooxygenase-2 and Hypoxia-Inducible Factor-1 alpha protein expression is related to inflammation, and up-regulated since the early steps of colorectal carcinogenesis [Articolo su rivista]
Mariani, Francesco; Sena, Paola; Marzona, Laura; Riccio, Massimo; Fano, Rita Adriana; Manni, Paola; C., Di Gregorio; A., Pezzi; PONZ DE LEON, Maurizio; Monni, Sebastiano Graziano; DE POL, Anto; Roncucci, Luca
abstract

Chronic mucosal inflammation is considered a risk factor for colorectal cancer. Neutrophils are a major source of oxidants, whereas cyclooxygenase 2 (COX-2) and Hypoxia Inducible Factor-1 alpha (HIF-1 alpha) protein expression levels are increased in inflammatory and malignant lesions. The main purpose of the present study was to evaluate myeloperoxidase (MPO) positive cell infiltration, COX-2 and HIF-1 alpha protein expression in colorectal carcinogenesis, especially in its early phases, using immunohistochemistry and immunofluorescence confocal microscopy techniques. MPO, COX-2 and HIF-1 alpha proteins were expressed at higher rates in the normal colorectal mucosa of patients with inflammatory bowel diseases and colorectal tumours than in patients with normal colonoscopy. A gradual increase in COX-2 and HIF-1 alpha protein expression was observed in dysplastic aberrant crypt foci, adenomas and carcinomas, showing a strong relation to dysplasia. In conclusion, the present study supports the hypothesis of a key role of inflammation in malignant transformation of colorectal mucosa. The evaluation of some early markers related to inflammation in the mucosa of the large bowel may serve as potential tool for prognosis and therapeutic strategies. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

2009 - Human MATER localization in specific cell domains of oocytes and follicular cells [Articolo su rivista]
Sena, Paola; Riccio, Massimo; Marzona, Laura; A., Nicoli; T., Marsella; Marmiroli, Sandra; Bertacchini, Jessika; Fano, Rita Adriana; LA SALA, Giovanni Battista; DE POL, Anto
abstract

MATER (Maternal Antigen That Embryos Require) is an oocyte-specific protein dependent on the maternal genome and required for early embryonic development. The gene products expressed in oocytes play important roles in folliculogenesis, fertilization and pre-implantation development. The aim of this study was to characterize the localization and distribution pattern of the human MATER protein during follicular development and after ovulation, to determine its functional role. Immunocytochemistry experiments coupled with confocal and electron microscopy analysis were carried out to determine the ultrastructural localization of MATER in human ovarian tissue and in isolated oocytes, obtained during IVF procedures. Human cumulus cells were cultured, with or without cycloheximide, to confirm endogenous biosynthesis of the protein. Human MATER is detectable at the onset of the follicular maturation process, suggesting this protein has a role at earlier stages in the human compared with other mammalian species. The presence of MATER is specific to the oocyte and follicular cells that, during maturation, are spatially and functionally associated with the oocyte. The nuclear, nucleolar and mitochondrial localization hints at a possible role in RNA processing and the metabolic activity of the cell.

2009 - MATER protein as substrate of PKCepsilon in human cumulus cells. [Articolo su rivista]
Maraldi, Tullia; Riccio, Massimo; Sena, Paola; Marzona, Laura; A., Nicoli; Marca, A. L.; Marmiroli, Sandra; Bertacchini, Jessika; LA SALA, Giovanni Battista; DE POL, Anto
abstract

High activity of the phosphoinositide 3-kinase/Akt pathway in cumulus cells plays an important role in FSH regulation of cell function and Protein Kinase C epsilon (PKCepsilon) collaborates with these signalling pathways to regulate cell proliferation. Relevant roles in follicular development are played by Maternal Antigen That Embryos Require (MATER) that is a cumulus cell- and oocyte-specific protein dependent on the maternal genome. We recently demonstrated that human MATER localizes at specific domains of oocytes and, for the first time, also in cumulus cells. MATER contains a carboxy-terminal leucine-rich repeat domain involved in protein-protein interactions regulating different cellular functions. Here we investigated the functional role of MATER. Thus, we performed coimmunoprecipitation experiments using HEK293T cells expressing human MATER; a similar approach was then followed in human cumulus/follicular cells. In MATER(+)HEK293T cells, we observed that this protein acts as a phosphorylation substrate of PKCepsilon. Western blot experiments indicate that, unlike oocytes, human cumulus cells express PKCepsilon. Immunoprecipitation and confocal analysis suggest for the first time that MATER protein interacts with this protein kinase in cumulus cells under physiological conditions. Since PKCepsilon is known to collaborate with antiapoptotic signalling pathways, this suggests a novel mechanism for the function of MATER in follicular maturation.

2009 - Multipotent stem cells in vitro differentiation on 3-D scaffolds. [Abstract in Rivista]
Palumbo, Carla; Riccio, M.; Resca, E.; Maraldi, Tullia; Bertoni, Laura; Sena, Paola; DE POL, Anto
abstract

Goal of the study is to obtain cell/scaffold complexes to use in regenerative medicine.

2008 - Human ovarian tissue cryopreservation: effect of sucrose concentration on morphological features after thawing [Articolo su rivista]
Marsella, Tiziana; Sena, Paola; Xella, Susanna; LA MARCA, Antonio; Giulini, Simone; DE POL, Anto; Volpe, Annibale; Marzona, Laura
abstract

Recent improvements in techniques in clinical assisted reproduction have led to an increased interest in the cryopreservation of human ovarian tissue as a way of preserving fertility and ovarian steroidogenic activity in young cancer patients. Acceptable follicular survival in frozen-thawed human ovarian tissue has generally been reported. Since a 0.3 mol/l sucrose concentration in cryopreservation solutions evidently increases human oocyte survival after cryopreservation, the aim of this study was to observe the effect of sucrose concentrations of 0.2 mol/l and 0.3 mol/l on human ovarian tissue survival after thawing. Ovarian cortical slices from 10 patients, 22-36 years of age, were cryopreserved slowly using 0.2 mol/l or 0.3 mol/l sucrose with 1,2-propanediol (1.5 mol/l) as the cryoprotectants. Light and electron microscopy were used for the histological analyses. Results showed that both treatments produced an increase in damaged cells; however, the use of 0.3 mol/l sucrose showed a smaller percentage of damaged germ cells than 0.2 mol/l sucrose, and therefore was less detrimental to the thawed ovarian tissue. However as the damage occurred principally in the stroma and follicular cells rather than in the oocytes, the suitability of these cryopreservation protocols must be further evaluated prior to considering the use of stored ovarian cortex for autografting after thawing.

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.

2008 - Two peculiar conditions following a coma: A clinical case of heterotopic ossification concomitant with keloid formation [Articolo su rivista]
Palumbo, Carla; Ferretti, Marzia; Pierluigi, Bonucci; Sena, Paola; Bertoni, Laura; Cavani, Francesco; Andrea, Celli; Rovesta, Claudio
abstract

The etiology and formation pattern of heterotopic ossifications (HO) are still unknown. They occur in soft tissues in which bone does not normally form, near one or more proximal joints. In this article, the authors report a peculiar case of a 31-year-old patient affected by scapulo-humeral ankylosis that occurred about 6 months after a coma, in which two unusual concomitant conditions were observed: HO formation in the scapulo-humeral region and the development of keloids during wound repair. The scapulo-humeral ankylosis was resolved surgically with the removal of the HO, which was then studied morphologically to understand its formation pattern. By light microscopy and transmission electron microscopy, it was observed that heterotopic bone displays the normal microscopic structure of primary bone, in which two types of bone tissue were recognized, i.e., woven-fibered bone, deeply located and produced first, and lamellar bone. This suggests that the pattern of HO formation retraces the ontogenetic steps that normally occur during intramembranous ossification. The authors also discuss the peculiar concomitance of HO formation and keloid development, speculating that, although they are different conditions localized in dissimilar regions, they might be hypothetically triggered by a common event, such as the release of factors likely issued during the coma status.

2006 - Subcellular localization of beta-catenin and APC proteins in colorectal preneoplastic and neoplastic lesions RID B-2583-2012 [Articolo su rivista]
Sena, Paola; Saviano, Massimo; Monni, Sebastiano Graziano; Losi, Lorena; Roncucci, Luca; Marzona, Laura; DE POL, Anto
abstract

Adenomatous polyposis coli (APC) is a tumor suppressor gene whose main function is the destabilization of beta-catenin, a key effector of the Wnt signaling pathway. This gene is defective in familial adenomatous polyposis (FAP), a dominantly inherited disease, but inactivation of APC has been reported also in most sporadic colorectal tumors and it is considered an early event in colorectal tumorigenesis. The aim of the present study was to evaluate the intracellular ultrastructural distribution of beta-catenin and APC proteins in epithelial cells of normal colorectal mucosa, aberrant crypt foci (ACF, an early premalignant lesion) and cancer. We used the immunogold electron microscopic method to identify both proteins. Normal colonic epithelial cells showed a strong membranous expression of beta-catenin and lacked cytoplasmic and nuclear expression. Normal cells showed APC localization pattern characterized by diffuse nuclear expression and along the plasma membrane. In ACF and in carcinoma an absent or reduced membranous expression of beta-catenin was associated with an increased nuclear and cytoplasmatic expression. In aberrant crypt foci and carcinoma, APC was evident inside the nucleus and at the level of cell-cell junctions, but it was decreased in the cytoplasm. This method allowed the accurate localization of proteins of the Writ signaling pathway in the early steps of colorectal carcinogenesis. The similar pattern of subcellular distribution of APC and beta-catenin in dysplastic ACF and colorectal cancer suggests that ACF are precursor lesions of sporadic and FAP-associated colorectal carcinoma. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

2004 - Developmental expression and subcellular localization of mouse MATER, an oocyte-specific protein essential for early development [Articolo su rivista]
Zb, Tong; L., Gold; DE POL, Anto; K., Vanevski; H., Dorward; Sena, Paola; Palumbo, Carla; Ca, Bondy; Lm, Nelson
abstract

We reported previously that Mater is a maternal effect gene that is required for early embryonic development beyond the two-cell stage in mice. Here we show the expressional profile of Mater and its protein during oogenesis and embryogenesis as well as its subcellular localization in oocytes. Mater mRNA was detectable earliest in oocytes of type 2 follicles, whereas MATER protein appeared earliest in oocytes of type 3a primary follicles. Both mRNA and protein accumulated during oocyte growth. In situ hybridization showed that Mater mRNA appeared progressively less abundant in oocytes beyond type 5a primary follicles. By ribonuclease protection assay, Mater mRNA was abundant in germinal vesicle oocytes, but was undetectable in all stages of preimplantation embryos. In contrast, the protein persisted throughout preimplantation development. Immunogold electron microscopic analysis revealed that MATER was located in oocyte mitochondria and nucleoli, and close to nuclear pores. Taken together, our data indicate that Mater gene transcription and protein translation are active during oogenesis, but appear inactive during early embryogenesis. Thus, Mater and its protein are expressed in a manner typical of maternal effect genes. The presence of MATER protein in mitochondria and nucleoli suggests that it may participate in both cytoplasmic and nuclear events during early development.

2001 - Effects of estrogens and oxytocin on the development of neonatal mammalian ovary [Articolo su rivista]
Marzona, Laura; R., Arletti; Benelli, Augusta; Sena, Paola; DE POL, Anto
abstract

The preservation and death of germ cells in the neonatal mammalian ovary are linked with the presence of hormones. Estrogens and oxytocin are present at birth in all mammalian vertebrates. The aim of this study was to examine their role in the development of the neonatal ovary and also in the preservation and death of germ cells in the neonatal period: apoptotic phenomena play a fundamental role in the control of their number. Female neonatal mice were treated at birth with estradiol monobenzoate or oxytocin and sacrificed after 5 days. The ovaries were sectioned in toto into semi-thin sections, in order to calculate their volume. Thin sections were also carried out to verify, under the transmission electron microscope (T.E.M.), the cells in apoptosis. The ovaries treated with the greater concentration of estradiol monobenzoate showed a volume that was significantly greater than that of the controls and a reduction of germ cells in apoptosis. The ovaries treated with oxytocin at all degrees of concentration had a volume significantly less than the controls and they also had a higher number of germ cells in apoptosis.

2001 - Influence of estrogens and oxytocin on germ cells death in the neonatal mammalian ovary [Articolo su rivista]
DE POL, Anto; Benelli, Augusta; Arletti, R.; Cavazzuti, E.; Sena, Paola; Vaccina, F.; Marzona, Laura
abstract

During mammalian oogenesis, some processes involve proliferation and others drastic reduction of germ cells. This study reports on the role played by two hormones, estradiol monobenzoate and oxytocin, in the control of the number of germ cells in the neonatal mouse ovary. Female neonatal mice were treated with doses ranging between 0.1 and 1 microg/mouse of estradiol monobenzoate or oxytocin and sacrificed at 5 days of postnatal age. The results showed that in the animals treated with estrogen, follicular development was more advanced than that of controls. Further the number of germ cells in apoptosis was drastically reduced. In the animals treated with oxytocin, the follicular development was arrested at the stage of primary follicles. In addition, the number of apoptotic germ cells increased if compared with that of the controls.

1998 - Apoptosis in different stages of human oogenesis. [Articolo su rivista]
De Pol, Anto; Marzona, Laura; Vaccina, F.; Negro, R.; Sena, Paola; Forabosco, A.
abstract

Apoptosis is an active form of cell death characterized by a series of morphological changes that become particularly evident at the ultrastructural level. The majority of ovarian germ cells undergo degeneration during prenatal and reproductive life and only in recent studies has it been demonstred that this drop is due to an apoptotic process. We evaluated this process during human oogenesis in prenatal life and we studied the ultrastructural changes that occur in apoptosis in various phases of the meiotic process. From our observations it is clear that apoptosis involves two main phases of the meiotic process: an earlier one concerning the oogonia and oocytes in the preleptotene stage, and a later one that mainly concerns the oocytes in the pachytene stage.