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Elena ROSSI

Personale tecnico amministrativo
Dipartimento Chirurgico, Medico, Odontoiatrico e di Scienze Morfologiche con interesse Trapiantologico, Oncologico e di Medicina Rigenerativa

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Pubblicazioni

2023 - Obesity and Wound Healing: Focus on Mesenchymal Stem Cells [Articolo su rivista]
Alma, Antonio; Marconi, Guya Diletta; Rossi, Elena; Magnoni, Cristina; Paganelli, Alessia
abstract

Chronic wounds represent nowadays a major challenge for both clinicians and researchers in the regenerative setting. Obesity represents one of the major comorbidities in patients affected by chronic ulcers and therefore diverse studies aimed at assessing possible links between these two morbid conditions are currently ongoing. In particular, adipose tissue has recently been described as having metabolic and endocrine functions rather than serving as a mere fat storage deposit. In this setting, adipose-derived stem cells, a peculiar subset of mesenchymal stromal/stem cells (MSCs) located in adipose tissue, have been demonstrated to possess regenerative and immunological functions with a key role in regulating both adipocyte function and skin regeneration. The aim of the present review is to give an overview of the most recent findings on wound healing, with a special focus on adipose tissue biology and obesity.

2023 - Wound Healing after Acellular Dermal Substitute Positioning in Dermato-Oncological Surgery: A Prospective Comparative Study [Articolo su rivista]
Paganelli, Alessia; Naselli, Andrea Giovanni; Bertoni, Laura; Rossi, Elena; Azzoni, Paola; Pisciotta, Alessandra; Cesinaro, Anna Maria; Benassi, Luisa; Kaleci, Shaniko; Garbarino, Federico; Ferrari, Barbara; Fiorentini, Chiara; Reggiani, Camilla; Magnoni, Cristina
abstract

Background: MatriDerm and Integra are both widely used collagenic acellular dermal matrices (ADMs) in the surgical setting, with similar characteristics in terms of healing time and clinical indication. The aim of the present study is to compare the two ADMs in terms of clinical and histological results in the setting of dermato-oncological surgery. Methods: Ten consecutive patients with medical indications to undergo surgical excision of skin cancers were treated with a 2-step procedure at our Dermatologic Surgery Unit. Immediately after tumor removal, both ADMs were positioned on the wound bed, one adjacent to the other. Closure through split-thickness skin grafting was performed after approximately 3 weeks. Conventional histology, immunostaining and ELISA assay were performed on cutaneous samples at different timepoints. Results: No significant differences were detected in terms of either final clinical outcomes or in extracellular matrix content of the neoformed dermis. However, Matriderm was observed to induce scar retraction more frequently. In contrast, Integra was shown to carry higher infectious risk and to be more slowly reabsorbed into the wound bed. Sometimes foreign body-like granulomatous reactions were also observed, especially in Integra samples. Conclusions: Even in the presence of subtle differences between the ADMs, comparable global outcomes were demonstrated after dermato-oncological surgery.

2022 - Mesenchymal stromal cells promote the proliferation of basal stem cells and efficient epithelization in organotypic models of wound healing [Articolo su rivista]
Paganelli, A.; Benassi, L.; Rossi, E.; Tarentini, E.; Magnoni, C.
abstract

Adipose derived mesenchymal stromal cells (ADSCs) represent a fascinating tool in the scenario of wound healing and regenerative medicine. Recent data already demonstrated that ADSCs could exert a stimulatory action on epithelial cells through secretion of soluble factors. The aim of the present study was to assess how ADSCs guide wound re-epithelization in vitro in the presence of keratinocytes. We used an organotypic model of wound healing and we seeded keratinocytes on a ADSC-induced dermal matrix. Conventional hematoxylin–eosin stain and immunohistochemistry staining for Ki67, p63 and pan-keratins were performed at different timepoints. Histological sections of organotypic cultures showed complete coverage of the ADSC-induced matrix by keratinocytes. Proliferation of basal stem cells was found to be the main mechanism responsible for epithelization of the dermis. In conclusion, ADSC do not only stimulate dermal regeneration through collagen deposition but also promote epithelization.

2022 - The Dark Side of Adipose-Derived Mesenchymal Stromal Cells in Cutaneous Oncology: Roles, Expectations, and Potential Pitfalls [Articolo su rivista]
Paganelli, Alessia; Rossi, Elena; Magnoni, Cristina
abstract

Adipose-derived stromal cells (ADSCs) have well-established regenerative and immunomodulatory properties. For such reasons, ADSCs are currently under investigation for their use in the setting of both regenerative medicine and autoimmune diseases. As per dermatological disorders, mesenchymal stromal cell (MSC)-based strategies represent potential therapeutic tools not only for chronic ulcers and wound healing, but also for immune-mediated dermatoses. However, a growing body of research has been focusing on the role of MSCs in human cancers, due to the potential oncological risk of using MSC-based strategies linked to their antiapoptotic, proangiogenic, and immunosuppressive properties. In the dermatological setting, ADSCs have shown not only to promote melanoma growth and invasiveness, but also to induce drug resistance. In contrast, genetically modified ADSCs have been demonstrated to efficiently target therapies at tumor sites, due to their migratory properties and their peculiar tropism for cancer microenvironment. The present review briefly summarizes the findings published so far on the use of ADSCs in the dermato-oncological setting, with the majority of data being available for melanoma.

2022 - Use of confocal microscopy imaging for in vitro assessment of adipose-derived mesenchymal stromal cells seeding on acellular dermal matrices: 3D reconstruction based on collagen autofluorescence [Articolo su rivista]
Paganelli, A.; Tarentini, E.; Benassi, L.; Scelfo, D.; Pisciotta, A.; Rossi, E.; Magnoni, C.
abstract

Background: Both mesenchymal stromal cells (MSCs) and acellular dermal matrices (ADMs) represent fascinating therapeutic tools in the wound healing scenario. Strategies aimed at combining these two treatment modalities are currently under investigation. Moreover, scarcity of quantitative, nondestructive techniques for quality assessment of engineered tissues poses great limitations in regenerative medicine and collagen autofluorescence-based imaging techniques are acquiring great importance in this setting. Objective: Our goals were to assess the in vitro interactions between ADSCs and ADMs and to analyze extracellular-matrix production. Methods: Adipose-derived MSCs (ADSC) were plated on 8-mm punch biopsies of a commercially available ADM (Integra®). Conventional histology with hematoxylin-eosin staining, environmental scanning electron microscopy, and confocal-laser scanning microscopy were used to obtain imaging of ADSC-seeded ADMs. Collagen production by ADSCs was quantified by mean fluorescence intensity (MFI), expressed in terms of positive pixels/field, obtained through ImageJ software processing of three-dimensional projections from confocal scanning images. Control conditions included: fibroblast-seeded ADM, ADSC- and fibroblast-induced scaffolds, and Integra® alone. Results: ADSCs were efficiently seeded on Integra® and were perfectly incorporated in the pores of the scaffold. Collagen production was revealed to be significantly higher when ADSCs were seeded on ADM rather than in all other control conditions. Collagen autofluorescence was efficiently used as a surrogate marker of ECM production. Conclusions: Combined therapies based on MSCs and collagenic ADMs are promising therapeutic options for chronic wounds. Not only ADSCs can be efficiently seeded on ADMs, but ADMs also seem to potentiate their regenerative properties, as highlightable from fluorescence confocal imaging.

2020 - Extracellular matrix deposition by adipose-derived stem cells and fibroblasts: a comparative study [Articolo su rivista]
Paganelli, A.; Benassi, L.; Rossi, E.; Magnoni, C.
abstract

Cell-based strategies are today widely studied as possible therapies for wound healing. In this setting, fibroblasts play a key role since they are the main dermal cellular component and are responsible for extracellular matrix secretion. Several works report on the possibility of using fibroblast-derived extracellular matrix scaffolds for wound healing in skin injuries. While fibroblast-based substitutes have already been intensively studied by other groups, we focused our attention on the possibility of creating an adipose-derived stem cell (ADSC)-induced dermal scaffold for wound healing. ADSCs are a particular subset of mesenchymal stem cells present in the stromal vascular fraction of the adipose tissue. The aim of our work was to compare the ability of ADSCs and fibroblast to produce in vitro a scaffolding material, both in terms of collagen and fibronectin production. ADSCs turned out to be capable of efficiently producing a collagen and fibronectin-containing dermal matrix upon stimulation with ascorbic acid. We observed fibronectin and collagen production by ADSCs to be even more abundant when compared to fibroblasts’. Our results support the use of ADSC-induced sheets instead of fibroblast-based dermal substitutes as wound-healing strategies in full-thickness skin injuries.

2020 - Synergic effect of plasma exeresis and non–cross‐linked low and high molecular weight hyaluronic acid to improve neck skin laxities [Articolo su rivista]
Paganelli, Alessia; Mandel, Victor Desmond; Pellacani, Giovanni; Rossi, Elena
abstract

Background: Many therapeutic options are today available for neck aging, but little evidence exists about the efficacy of combining such procedures. Nonsurgical treatment of neck laxities and wrinkles is often preferred by patients, and combined strategies are nowadays emerging as the standard of care. Both plasma exeresis and hyaluronic acid (HA) injection are two emerging techniques in this setting. Aims: To investigate the synergic effect of plasma exeresis and non-cross-linked HA injection, in stabilized hybrid complex of low and high molecular weight, in terms of both tolerability (assessed using VAS scale of pain) and improvement of neck skin laxities, according to the GAIS score assigned by patients and clinicians. Patients/methods: Ten consecutive patients with signs of neck skin laxities (≥ type 3 according to the Glogau wrinkle scale) aged between 35 and 65 years were enrolled in our study. Two treatment sessions were performed. During the first session, both plasma exeresis and HA injection were performed. Patients were re-evauated after 30 days, and HA injection in the wrinkles of the neck was repeated. After 30 days from the second treatment session, a follow-up visit was performed to assess global efficacy of the two-step combined treatment and to monitor eventual long-term side effects. Results: A GAIS score of 1 or 2 was present in 90% of the treated cases, according to both patients and clinicians. Mean VAS value for pain was 2.4/10. Minor side effects such as erythema and/or edema were transient and completely resolved. No major adverse events were observed. Conclusions: We strongly encourage the combined treatment with plasma exeresis and non-cross-linked HA injection for its promising remodeling effects in the field of neck rejuvenation.

2019 - Favre-Racouchot disease: systematic review and possible therapeutic strategies [Articolo su rivista]
Paganelli, Alessia; Mandel, Victor Desmond; Kaleci, Shaniko; Pellacani, Giovanni; Rossi, Elena
abstract

Favre-Racouchot disease (FRD) is a relatively common dermatosis induced by chronic sun exposure. FRD is clinically and histologically characterized by the presence of both comedones and cysts in the context of an elastotic degeneration of the dermis. Those lesions are mainly located in the face, especially in the malar and periorbital areas. Smoking and radiotherapy seem to play a role in the pathogenesis together with UV exposure. The disease affects predominantly the aged population and seems to be a problem of mainly aesthetic concern. No official guidelines are available for the treatment of FRD; most common therapeutic strategies are represented by topical retinoids and laser treatments. The goal of our review was to identify the principal clinical and epidemiological characteristics of FRD and to analyse all the therapeutic strategies available. We also focused our attention on the follow-up of this particular dermatosis. Our aim was therefore to suggest alternative possible strategies for both the treatment and the follow-up of these patients. Our data support the efficacy of carbon dioxide laser and topical retinoids in the setting of FRD, but we also suggest considering alternative treatments, such as plasma exeresis. We also recommend planning both a short-term and a long-term follow-up visit, as the majority of relapses is observed after 10-12 months.

2018 - Favre-Racouchot syndrome: report of a case treated by plasma exeresis [Articolo su rivista]
Rossi, Elena; Paganelli, Alessia; Mandel, Victor Desmond; Pellacani, Giovanni
abstract

[No abstract available]

2018 - Identifying the factors that influence surgeon's compliance with excisional margins of non-melanoma skin cancer [Articolo su rivista]
Cautela, Jennifer Marchetti; Mannocci, Alice; Reggiani, Camilla; Persechino, Flavia; Ferrari, Federica; Rossi, Elena; Passini, Erika; Bellini, Pierantonio; Meleti, Marco; WERTZBERGER ROWAN, Sara; Magnoni, Cristina
abstract

The rising incidence of Non Melanoma Skin Cancers (NMSC) leads to a high number of surgical procedures worldwide. The strict compliance with international guidelines with regard to excisional margins may help decrease the number of re-excision procedures and reduce the risk of NMSC recurrence. The aim of this study was to investigate the prevalence of excisional margins as recommended by the European Academy of Dermatology and Venereology (EADV) and the European Dermatology Forum (EDF) guidelines, and the factors (demographic or clinical) that influence surgeons' compliance with these guidelines.This was a prevalence study looking at surgical excisions of NMSCs performed over a period of 2 years (2011-2012). A sample size of 1669 patients was considered. Definition of excisional margins recommended by the international guidelines (EADV and EDF) were used as point of reference for the analysis. Tumor and histologic specimen size were calculated ex vivo by 5 different pathologists. The size of skin specimens was measured with a major axis and a minor axis. The same was done for the tumor present on the skin specimens. The differences between the major and minor axes of surgical specimen and tumor were calculated. These differences were subsequently divided by two, hypothesizing that the lesion had the same distance from the margins of the surgical specimen. The differences obtained were named "Delta", the formulas applied being the following: Delta major = (major axis specimen-major axis tumor)/2; Delta minor = (minor axis specimen -minor axis tumor)/2.Results show a significant statistical difference, associated with factors such as: Age of the patient, anatomical localization of the tumor, histological diagnosis, and surgeons' experience.The identification of these factors sheds light on clinicians' practice and decision-making regarding excisional margins. Hopefully a higher level of adherence to the guidelines can be achieved in the future.

2018 - Plasma exeresi per l’acne attiva: efficacia clinica e documentazione microscopica in vivo mediante microscopio confocale a riflettanza [Relazione in Atti di Convegno]
Mandel, Victor Desmond; Rossi, Elena
abstract

[Abstract non disponibile]

2018 - Plasma exeresis for active acne vulgaris: Clinical and in vivo microscopic documentation of treatment efficacy by means of reflectance confocal microscopy [Articolo su rivista]
Rossi, Elena; Mandel, Victor Desmond; Paganelli, Alessia; Farnetani, Francesca; Pellacani, Giovanni
abstract

[No abstract available]

2018 - Plasma Exeresis Treatment for Epidermoid Cysts: A Minimal Scarring Technique [Articolo su rivista]
Rossi, Elena; Paganelli, Alessia; Mandel, Victor Desmond; Pellacani, Giovanni
abstract

Background: Epidermoid cysts are cutaneous benign tumors commonly seen in young or middle-aged adults. Plasma exeresis is an innovative technique for several skin conditions: it causes ionization of the atmospheric gas between the proximal tip of the device and the tissue to be treated, creating sublimation of the tissue. Objective: To remove the cyst with a novel technique that allows a good cosmetic result. Materials and methods: Patients with clinical diagnosis of at least one epidermal cyst, aged between 18 and 70 years were enrolled. A standardized procedure was used. After administration of topical and sometimes local anesthesia (for cysts bigger than 1 cm), a tiny hole was created with plasma exeresis. The content of the cyst was then extruded and Micro Hartman Alligator Ear Forceps pulled out the loosened capsule. Results: Twenty patients aged between 18 and 68 years were enrolled: 11 males (55%) and 9 females (45%). Twenty-eight cysts were successfully removed. The diameter ranged from 3 to 24 mm. No side effects were observed. The scar measured not more than 3 mm. Conclusion: This study suggests that plasma exeresis could represent a good and safe option to remove noninfected cysts on cosmetic areas, although further study is required.

2018 - The smart approach: feasibility of lentigo maligna superficial margin assessment with hand-held reflectance confocal microscopy technology [Articolo su rivista]
Pellacani, G.; De Carvalho, N.; Ciardo, S.; Ferrari, Beatrice; Cesinaro, A. M.; Farnetani, F.; Bassoli, S.; Guitera, P.; Star, P.; Rawson, R.; Rossi, E.; Magnoni, C.; Gualdi, G.; Longo, C.; Scope, A.
abstract

Background: Lentigo maligna may be challenging to clear surgically. Objective: To evaluate feasibility of using superficial skin cuts as RCM imaging anchors for attaining negative surgical margins in lentigo maligna. Methods: Included patients presented with lentigo maligna near cosmetically sensitive facial structures. We evaluated, with hand-held-RCM, microscopic clearance of melanoma beyond its dermoscopically detected edges. Evaluated margins were annotated using shallow skin cuts. If a margin was positive at ‘first-step’ RCM evaluation, we sequentially advanced the margin radially outward at that segment by 2-mm intervals until an RCM-negative margin was identified. Prior to final surgical excision, we placed sutures at the outmost skin cuts to allow comparison of RCM and histopathological margin assessments. Primary outcome measure was histopathological verification that RCM-negative margins were clear of melanoma. Results: The study included 126 first-step margin evaluations in 23 patients, median age 70 years (range: 43–91). Seventeen patients (74%) had primary in-situ melanoma and six (26%) invasive melanoma, mean thickness 0.3 mm (range 0.2–0.4 mm). Six cases (26%) showed complete negative RCM margins on ‘first-step’, 11 (48%) were negative at ‘second-step’, and four (17%) at ‘third-step’. In two additional cases (9%), margins clearance could not be determined via RCM due to widespread dendritic cells proliferation. The RCM-negative margins in all 21 cases proved clear of melanoma on histopathology. Of the 15 cases that returned at 1-year follow-up, none showed any residual melanoma on dermoscopic and RCM examinations. Interobserver reproducibility showed fair agreement between bedside RCM reader and blinded remote-site reader, with Spearman's rho of 0.48 and Cohen's kappa of 0.43; using bedside reader as reference, the remote reader's sensitivity was 92% and specificity 57% in positive margin detection. Conclusions: Margin mapping of lentigo maligna with hand-held-RCM, using superficial skin cuts, appears feasible. This approach needs validation by larger studies.

2017 - Synergic effect of buccal fat pad pedicled flap and dermal acellular matrix for large cheek defect [Poster]
Rossi, Elena; Salgarelli, Attilio Carlo; Mandel, Victor Desmond; Magnoni, Cristina
abstract

Introduction & Objectives: Reconstruction of large defects of the upper cheek defects still remains a challenge for the surgeon, than can apply different techniques. We present a new method involving the use of a dermal regeneration template to achieve an improved, faster healing of pedicled buccal fat flap in a 75-years-old woman affected by melanoma of the upper-middle cheek. The tumor involved soft tissue, zygomatic arch and periocular fact. Material & Methods: The choice of the surgical technique consisted first in the creation of a buccal fat pad to restore the important lack of tissue over the underlying bones, then in the positioning of a dermal acellular matrix. Three weeks later, once the neodermal formation was finished, a split thickness graft was placed. Results: This is a not yet described association that represents a good surgical option for the restoration of large cheeck defects that allows good functional and cosmetic result in older patient when minimal surgical invasion and operative duration are necessary because of a patient’s general condition. The post-operative course with this surgical technique was regular and a good functional result was achieved. Conclusions: This technique provides an adequate functional coverage, a restoration of soft tissue lacking and an acceptable cosmetic result without ectropion.

2016 - Effect of diets supplemented with different conjugated linoleic acid (CLA) isomers on protein expression in C57/BL6 mice [Articolo su rivista]
DELLA CASA, Lara; Rossi, Elena; Romanelli, Claudia; Gibellini, Lara; Iannone, Anna
abstract

The individual genetic variations, as a response to diet, have recently caught the attention of several researchers. In addition, there is also a trend to assume food containing beneficial substances, or to supplement food with specific compounds. Among these, there is the conjugated linoleic acid (CLA), which has been demonstrated to reduce fat mass and to increase lean mass, even though its mechanism of action is still not known. We investigated the effect of CLA isomers (CLA c9,t11 and CLA t10,c12) on the proteomic profile of liver, adipose tissue, and muscle of mouse, with the aim of verifying the presence of a modification in fat and lean mass, and to explore the mechanism of action.

2013 - Ovarian cancer: can proteomics give new insights for therapy and diagnosis? [Articolo su rivista]
Toss, Angela; DE MATTEIS, Elisabetta; Rossi, Elena; Casa, L. D.; Iannone, Anna; Federico, Massimo; Cortesi, Laura
abstract

The study of the ovarian proteomic profile represents a new frontier in ovarian cancer research, since this approach is able to enlighten the wide variety of post-translational events (such as glycosylation and phosphorylation). Due to the possibility of analyzing thousands of proteins, which could be simultaneously altered, comparative proteomics represent a promising model of possible biomarker discovery for ovarian cancer detection and monitoring. Moreover, defining signaling pathways in ovarian cancer cells through proteomic analysis offers the opportunity to design novel drugs and to optimize the use of molecularly targeted agents against crucial and biologically active pathways. Proteomic techniques provide more information about different histological types of ovarian cancer, cell growth and progression, genes related to tumor microenvironment and specific molecular targets predictive of response to chemotherapy than sequencing or microarrays. Estimates of specificity with proteomics are less consistent, but suggest a new role for combinations of biomarkers in early ovarian cancer diagnosis, such as the OVA1 test. Finally, the definition of the proteomic profiles in ovarian cancer would be accurate and effective in identifying which pathways are differentially altered, defining the most effective therapeutic regimen and eventually improving health outcomes.

2012 - Conjugated linoleic acid alters lipid metabolism in hepatocytes [Abstract in Atti di Convegno]
Iannone, Anna; Rossi, Elena; DELLA CASA, Lara; Barchetti, Andrea; Battistini, Nino Carlo
abstract

not available

2012 - Conjugated linoleic acid isomers modulate protein expression profile in rat hepatocytes [Articolo su rivista]
Rossi, Elena; DELLA CASA, Lara; S., Piana; Iannone, Anna
abstract

Conjugated linoleic acid (CLA) is a polyunsaturatedfatty acid, which has been recently proven to beeffective in reducing body fat mass, but brings as a sideeffect, the liver enlargement due to an increased lipidcontent. The in vivo lipogenic activity has been suggestedto be due to the reduction in fat mass and to the consequentmetabolism of blood glucose to fatty acid in the liver ratherthan in the adipose tissue. We investigated the ability ofCLA to directly induce steatosis by modulating theexpression pattern of hepatic proteins involved in lipidmetabolism. To avoid interferences derived from CLAmetabolism by other tissues, we used the in vitro model offreshly isolated rat hepatocytes incubated in the presence ofdifferent CLA isomers. The direct effect of CLA on lipidaccumulation in hepatocytes was demonstrated by thealtered expression pattern of several proteins involved inlipid metabolism, as assessed by two-dimensional gelelectrophoresis and confirmed by Western blotting analysis.The CLA isomer c9,t11 was most effective in modulatingthe protein expression profile.

2011 - Protein expression patterns associated with advanced stage ovarian cancer [Articolo su rivista]
Cortesi, L.; Rossi, Elena; DELLA CASA, Lara; Barchetti, Andrea; Nicoli, A.; Piana, S.; Abrate, M.; LA SALA, Giovanni Battista; Federico, Massimo; Iannone, Anna
abstract

This is a comparative proteomic study on biopsies from patients with ovarian cancer to identify potential diagnostic/prognostic biomarkers in both healthy and tumor tissue, interstitial fluid (normal interstitial fluid and tumoral interstitial fluid and peritoneal effusion. Protein expression/identification was evaluated by 2-DE and MS analysis: six proteins showed differential expression in tumoral interstitial fluid and tumor tissue compared to normal interstitial fluid and healthy tissue: five were found to be downregulated and identified as galectin 3, glutathione S-transferase A-2, retinol binding protein 1, phosphatidylethanolamine-binding protein and annexin 5, while the calgranulin, was significantly upregulated in all pathological samples, including the ascitic fluid. Validation of S100A8 overexpression in carcinoma tissue was obtained by immunohistochemistry. To our knowledge, this is the first study to report an over-expression of calgranulin by 2-DE associated with MS/MS analysis on surgical biopsy. The reduced expression of galectin 3 and retinol binding protein 1 in cystic fluid and serum of patients with early stage disease is confirmed in this study. The results highlight alterations in proteins that control cell-cycle progression and apoptosis, as well as factors that modulate the activity of signal transduction pathways. Moreover, this study suggests that calgranulin expression may be used as a diagnostic and/or prognostic biomarker.

2009 - Elongation factor 1 alpha interacts with phospho-Akt in breast cancer cells and regulates their proliferation, survival and motility [Articolo su rivista]
Pecorari, Luisa; Marin, O.; Silvestri, C.; Candini, Olivia; Rossi, Elena; Guerzoni, Clara; Cattelani, Sara; Mariani, S. A.; Corradini, Francesca; Ferrari, Giovanna; Cortesi, L.; Bussolari, Rita; Raschellà, G.; Federico, Massimo; Calabretta, Bruno
abstract

BACKGROUND: Akt/PKB is a serine/threonine kinase that has attracted much attention because of its central role in regulating cell proliferation, survival, motility and angiogenesis. Activation of Akt in breast cancer portends aggressive tumour behaviour, resistance to hormone-, chemo-, and radiotherapy-induced apoptosis and it is correlated with decreased overall survival. Recent studies have identified novel tumor-specific substrates of Akt that may provide new diagnostic and prognostic markers and serve as therapeutic targets. This study was undertaken to identify pAkt-interacting proteins and to assess their biological roles in breast cancer cells. RESULTS: We confirmed that one of the pAkt interacting proteins is the Elongation Factor EF1alpha. EF1alpha contains a putative Akt phosphorylation site, but is not phosphorylated by pAkt1 or pAkt2, suggesting that it may function as a modulator of pAkt activity. Indeed, downregulation of EF1alpha expression by siRNAs led to markedly decreased expression of pAkt1 and to less extent of pAkt2 and was associated with reduced proliferation, survival and invasion of HCC1937 cells. Proliferation and survival was further reduced by combining EF1alpha siRNAs with specific pAkt inhibitors whereas EF1alpha downregulation slightly attenuated the decreased invasion induced by Akt inhibitors. CONCLUSION: We show here that EF1alpha is a pAkt-interacting protein which regulates pAkt levels. Since EF1alpha is often overexpressed in breast cancer, the consequences of EF1alpha increased levels for proliferation, survival and invasion will likely depend on the relative concentration of Akt1 and Akt2.

2009 - Identification of protein clusters predictive of response to chemotherapy in breast cancer patients [Articolo su rivista]
L., Cortesi; Barchetti, Andrea; DE MATTEIS, Elisabetta; Rossi, Elena; DELLA CASA, Lara; Marcheselli, Luigi; Tazzioli, Giovanni; M. G., Lazzaretti; G., Ficarra; Federico, Massimo; Iannone, Anna
abstract

An attempt for the identification of potential biomarkers predictive of response to chemotherapy (CHT) in breast cancer patients has been performed by the use of two-dimensional electrophoresis and mass spectrometry analysis. Since growth and progression of tumor cells depend also on stromal factors in the microenvironment, we choose to investigate the proteins secreted in Tumor Interstitial Fluid (TIF) and in Normal Interstitial Fluids (NIF). One-hundred and twenty-two proteins have been analyzed and a comparison was also made between the proteomic profile of responders versus nonresponders to CHT. At baseline, proteins isolated in TIF and NIF of all the 28 patients show significant differences in expression. Two clusters of proteins, differentially expressed in TIF with respect to NIF were found. Most significant is the decreased expression in TIF of CRYAB. In the protein metabolism group, also FIBB was found decreased. Some proteins involved in energy pathways were overexpressed (PGAM-1, ALDO A, PGK1, G3Pcn), while some other were down-regulated (CAH2, G3Pdx, PRDX6, TPIS). The same trend was observed for signal transduction proteins, with 14-3-3-Z overexpressed, and ANXA2 and PEBP 1 down-regulated. Moreover, an analysis has been conducted comparing protein expression in interstitial fluids of responders and nonresponders, irrespective of TIF or NIF source. This analysis lead us to identify two clusters of proteins with a modified expression, which might be predictive of response to CHT. In responders, an increase in expression of LDHA, G3Pdx, PGK1sx (energy pathways), VIME (cell growth and maintenance) and 14-3-3-Z (signal transduction), coupled with a decreased expression of TPIS, CAH 2, G3Psx, PGK 1dx (energy pathways), TBB5 (cell growth and maintenance), LDHB and FIBB (protein metabolism), was found. We observed that CHT modifies the expression of these cluster proteins since, after treatment, their expression in TIF of responder is generally decreased. Patients not responding to CHT show an unchanged expression pattern in TIF, with the exception of protein 14-3-3-Z, which is overexpressed, and a decreased expression in NIF of several cluster proteins. In conclusion, the identification of protein clusters associated with response to CHT might be important for predicting the efficacy of a specific antineoplastic drug and for the development of less empiric strategies in choosing the therapy to be prescribed to the single patient

2008 - Proteomic analysis of early urinary biomarkers of renal changes in type 2 diabetic patients [Articolo su rivista]
Bellei, Elisa; Rossi, Elena; Lucchi, L; Uggeri, S; Albertazzi, Alberto; Tomasi, Aldo; Iannone, Anna
abstract

Diabetic nephropathy (DN) is a complication associated with diabetes, leading to end-stage renal disease (ESRD). Despite significant progress in understanding DN, the cellular mechanisms leading to the renal damage are incompletely defined. In this study, with the aim to identify urine biomarkers for the early renal alterations in type 2 diabetes mellitus (T2D), we performed urinary proteomic analysis of 10 normoalbuminuric patients with T2D, 12 patients with type 2 DN (T2DN), and 12 healthy subjects. Proteins were separated by 2-DE and identified with ESI-Q-TOF MS/MS. Comparing the patients proteomic profiles with those of normal subjects, we identified 11 gradually differently changed proteins. The decreased proteins were the prostatic add phosphatase precursor, the ribonuclease and the kallikrem-3. Eight proteins were progressively increased in both patients groups: transthyretin precursor, Ig K chain C region, Ig K chain V-II region Cum, Ig K-chain V-III region SIE, carbonic anhydrase 1, plasma retinol-binding protein, β-2-microglobulin precursor, β-2-glycoprotein 1. The proteomic analysis allowed us to identify several increased urinary proteins, not only in T2DN but also in T2D patients in which the microalbuminuria was in the normal range. These patterns of urinary proteins might represent a potential tool for a better understanding of diabetic renal damage.

2006 - Caratterizzazione di proteine nel tessuto e nel liquido interstiziale di neoplasie della mammella [Abstract in Atti di Convegno]
Barchetti, A; Cortesi, L; Rossi, E; Bellei, E; Tomasi, A; Iannone, A; Federico, M.
abstract

La classificazione dei tumori della mammella si basa attualmente sui parametri clinici e sulla morfologia cellulare; l’analisi immunoistochimica utilizza un numero ristretto di fattori prognostici e predittivi correlati a diverse funzioni cellulari come la proliferazione, l’angiogenesi, l’invasione e la metastatizzazione. Questi parametri si limitano a classificare i pazienti in sottogruppi con differente prognosi mentre l’uso di nuove tecnologie, quali lo studio delle proteine attraverso elettroforesi bidimensionale (2D), potrebbe portare ad un approccio terapeutico individualizzato e ad una ottimizzazione del trattamento. Lo scopo del nostro studio è quello di individuare nuovi markers nell’ambito dei tumori invasivi della mammella confrontando il profilo di espressione proteica di tessuto normale, patologico, di TIF (tumoral interstitial fluid) e di NIF (normal interstitial fluid). Nel microambiente tumorale si concentrano infatti un gran numero di molecole in parte secrete, in parte trasportate da vescicole di membrana o liberate in seguito a morte cellulare. Abbiamo ottenuto 8 biopsie mammarie da pazienti affette da carcinoma duttale infiltrante (G3) e carcinoma lobulare infiltrante (G2/3). L’indagine è stata condotta utilizzando circa 200 mg di tessuto mammario di carcinoma duttale e di tessuto adiacente sano. I frammenti di biopsia, sospesi in tampone PBS, sono stati incubati in CO2 5% a 37°C per un’ora e successivamente centrifugati 20 min. a 3000g. Da questa coltura è stato recuperato e analizzato il surnatante contenente le proteine rilasciate dal tessuto. Successivamente l’estrazione proteica è stata effettuata anche sul pellet di tessuto bioptico polverizzato in azoto e sonicato in tampone di lisi. Per ciascuna analisi 100ug di proteine sono state sottoposte ad elettroforesi 2D e visualizzate con metodica silver staining. Gli spots di interesse sono stati caratterizzati con la tecnica HPLC-ESI-MS/MS in seguito a purificazione e digestione con tripsina. L’analisi preliminare delle mappe bidimensionali attraverso il programma PDQuest (BIO-RAD) ha evidenziato spots proteici la cui intensità variava significativamente nei campioni tumorali. Da segnalare un’aumentata espressione dell’oncogene mitogeno DJ-1(PARK 7), coinvolto in pathways di trasduzione del segnale Ras dipendenti e delle proteine LYPA (Acyl-protein thioesterase) e LDHB (lactate dehydrogenase B) la cui overespressione è stata segnalata in linee cellulari di tumore mammario resistenti al trattamento anti-estrogenico. È stato inoltre riscontrato un aumentato livello di PSA1 (Prostatic Specific Antigen), una proteasi serinica associata in letteratura ad un basso indice di proliferazione e ad una prognosi favorevole. L’analisi e il confronto di un maggiore numero di casi permetterà di chiarire il significato e di ottenere informazioni specifiche sul pattern di espressione genica dei differenti stadi e istotipi del carcinoma mammario. Inoltre il confronto con pattern proteici sierici potrebbe essere di aiuto nell’identificazione di nuovi e predittivi marker ematici di neoplasia.

2006 - Markers urinari di nefropatia diabetica [Poster]
Bellei, E; Rossi, E; Lucchi, L; Uggeri, S; Albertazzi, A; Tomasi, A; Iannone, A
abstract

Il diabete, come riportato ampiamente in letteratura, è una delle principali cause di insufficienza renale in stadio terminale (ESRD). Nonostante i progressi raggiunti nella comprensione della nefropatia diabetica, i meccanismi patogenetici che inducono il danno non sono ancora ben definiti. Alcuni studi hanno evidenziato come l’infiammazione cronica e i danni ossidativi ad essa correlati contribuiscano in modo determinante alle complicanze osservate in questi pazienti. Si può inoltre ipotizzare la presenza di modificazioni post-traduzionali di alcune proteine, che potrebbero essere impiegate come biomarkers prognostici e predittivi di malattia. Profili proteomici altamente specifici sono stati caratterizzati per varie patologie, soprattutto tumorali, ma indagini su profili proteomici nella nefropatia diabetica umana non sono tuttora presenti in letteratura. In questo studio abbiamo confrontato pattern proteici di soggetti sani con quelli di pazienti con nefropatia diabetica, cercando di individuare quadri proteici caratteristici dei diversi stadi di patogenesi, nonché modifiche post-traduzionali peculiari o mutazioni correlate alla patologia in esame. Partendo da liquidi biologici facilmente reperibili e maneggiabili come le urine, è possibile infatti valutare, mediante elettroforesi bidimensionale (2-DE), la funzionalità e i differenti livelli di espressione di molecole urinarie. Sono stati selezionati 20 pazienti con diabete di tipo II associato a nefropatia (valori di creatininemia > 1,5 mg/dl e presenza di microalbuminuria). Alle urine, immediatamente dopo la raccolta, è stato aggiunto un cocktail di inibitori delle proteasi. Una aliquota di ciascun campione è stata precipitata in acetone a -20°C, centrifugata e il pellet risospeso in un opportuno tampone. Dopo aver misurato la concentrazione proteica totale tramite un kit commerciale, 10 microgrammi di proteine sono stati utilizzati per l’analisi bidimensionale. I gel ottenuti sono stati colorati con metodica Silver stain massa-compatibile, gli spots di interesse trattati con tripsina e i peptidi estratti identificati tramite spettrometria ESI-LC- MS/MS. I risultati preliminari hanno evidenziato un aumento dei livelli delle seguenti proteine nelle urine dei pazienti diabetici nefropatici: albumina, transferrina e immunoglobuline G (IgG), che dalla letteratura risultano essere indici di danno glomerulare; alpha-1-microglobulina e alpha-1-antitripsina, indicatori di alterata funzionalità tubulare. Inoltre altre tre glicoproteine (zinc-alpha-2-glicoprotein, alpha-1-acid-glicoprotein e leucine-rich-alpha-2-glicoprotein), risultano quantitativamente aumentate. La presenza invece di altri due indicatori di danno tubulare, ovvero beta-2-microglobulina (b-2MG) e retinol binding protein (RBP), oltre alla proteina di membrana perlecan, è stata riscontrata solamente nelle urine dei pazienti e non in quelle dei soggetti di controllo. In conclusione, la 2-DE accoppiata alla spettrometria di massa potrebbe rappresentare un metodo rapido, sensibile e non-invasivo per identificare markers precoci di patologia renale.

2006 - Ricerca di biomarkers nel tumore ovarico mediante analisi proteomica [Abstract in Atti di Convegno]
Rossi, E; Cortesi, L; Barchetti, A; Bellei, E; Monari, E; Tomasi, A; Iannone, A; Abrate, M; Federico, M.
abstract

Il carcinoma ovarico rappresenta la quarta causa di morte nel sesso femminile in Europa e negli Stati Uniti. Nell’80 % dei casi infatti, la diagnosi avviene in uno stadio avanzato poiché la maggior parte delle neoplasie ovariche rimane clinicamente asintomatica negli stadi I e II. La sola terapia chirurgica assicura una sopravvivenza a cinque anni del 90 % delle pazienti diagnosticate allo stadio I, percentuale che si riduce al 35% negli stadi più avanzati. Il nostro obbiettivo è quello di utilizzare l’elettroforesi bidimensionale (2DE) e la spettrometria di massa per identificare e caratterizzare nuovi specifici biomarkers tumorali ovarici che permettano di effettuare una diagnosi precoce, di monitorare la progressione tumorale e di impostare una terapia mirata. Le indagini preliminari sono state effettuate su campioni bioptici di tessuto ovarico sano e tumorale, e su liquido proveniente da lavaggio peritoneale. Le proteine sono state estratte da circa 100 mg di tessuto congelato in azoto liquido, polverizzato in un mortaio e risospeso in tampone di lisi contenente urea, tiourea, CHAPS, Tris-HCl, ampholine pH 3-10 e inibitori delle proteasi. Dopo incubazione a temperatura ambiente per un’ora, i campioni sono stati sonicati e centrifugati. Il pellet, ottenuto dopo precipitazione in acetone freddo, è stato risospeso in un buffer di reidratazione (urea, tiourea, CHAPS, DTT e ampholine) e il contenuto proteico totale misurato con il metodo Bradford. L’isoelettrofocalizzazione (IEF) è stata effettuata caricando 80 g di proteine totali su IPG strips a range di pH 3-10 non lineare, fino al raggiungimento di 70000 Vh finali. Le proteine focalizzate sono state ridotte con DTT e alchilate con Iodoacetamide in tampone di equilibrazione (Urea, Tris-HCl, Glicerolo, SDS, Blu di Bromofenolo) prima della separazione elettroforetica su gels di poliacrilamide (SDS–PAGE), a concentrazione omogenea 10% e a gradiente 8-16%. I gel sono stati infine colorati con metodica Silver-staining massa-compatibile. L’analisi con il programma PDQuest ha evidenziato la presenza di numerosi spot proteici differenzialmente espressi nei campioni tumorali rispetto ai controlli sani; la spettrometria di massa ESI-Q-TOF-MS/MS porterà all’identificazione delle proteine di interesse. Per quanto riguarda il liquido derivato da lavaggio peritoneale, prima dell’analisi 2DE le aliquote prelevate sono state concentrate con filtri Amicon aventi cut-off di PM 5 KDa. Parallelamente alla 2DE, sui campioni tissutali è stata effettuata anche l’analisi con tecnologia SELDI-TOF (Surface Enhanced Laser Desorption Ionisation-Time of Flight). I risultati ottenuti hanno evidenziato variazioni significative dell’espressione di numerose proteine, soprattutto a basso peso molecolare (<20 KDa). Lo scopo dello studio ha la finalità di giungere all’identificazione di biomarcatori specifici del tumore ovarico che possono essere evidenziati anche nel liquido peritoneale ed eventualmente nel siero, fungendo da fattori prognostici della malattia ed anche predittivi di risposta al trattamento con derivati del platino.

2006 - Ricerca di markers diagnostici, prognostici e predittivi in carcinomi mammari attraverso analisi proteomica [Abstract in Atti di Convegno]
Pecorari, L; Silvestri, C; Candini, O; Rossi, E; Bellei, E; Bussolari, R; Iannone, A; Federico, M; Cortesi, L; Calabretta, B.
abstract

Le basi molecolari dei tumori sporadici della mammella sono solo in parte conosciute: un comune meccanismo molecolare, che è stato riportato in circa il 75% dei casi, è l’attivazione della proteina oncogenica ad attività Serin/Treonin chinasica AKT. Una delle principali funzioni di pAKT (la cui forma attiva è fosforilata) è quella di promuovere la sopravvivenza cellulare mediata da fattori di crescita bloccando i meccanismi intrinseci dell’apoptosi. Recentemente numerosi studi si sono concentrati sull’approfondimento del ruolo dell’oncoproteina AKT nel carcinoma mammario; la conclusione comune a questi studi è che si possano ottenere benefici clinici rilevanti dall’appropriata combinazione terapeutica tra chemioterapici convenzionali e inibitori specifici di pAKT. Sarebbe inoltre di notevole importanza chiarire in che modo la valutazione dell’attività di pAKT possa essere interpretata ai fini diagnostici, prognostici e predittivi in pazienti con carcinoma mammario. Questo progetto di ricerca si propone di approfondire questi aspetti utilizzando un approccio basato su tecniche di analisi proteomica. Su diversi tipi di linee continue di carcinoma mammario con diverse caratteristiche e diverso grado di aggressività (ErbB2+ / ErbB2- / BRCA1 mutato / BRCA1 w.t.) sono stati eseguiti e sono tuttora in corso, esperimenti d’inibizione/deplezione della proteina pAkt. In particolare dalla linea SKBr3 è stato possibile ottenere estratti proteici contenenti una discreta quota di pAKT; inoltre esperimenti di immunoprecipitazione selettiva hanno consentito di isolare gli interattomi di pAKT e di AKT non fosforilata. Questi immunoprecipitati sono poi stati sottoposti ad elettroforesi bidimensionale e gli spot indicativi di proteine varianti (associate quindi ad una forma rispetto all’altra) analizzati tramite spettrometria di massa. Attualmente una sola nuova potenziale proteina cliente, Elongation Factor 1 alfa (EF1a), è stata identificata ed è in via di caratterizzazione. Per approfondire il ruolo di questa proteina (nota per essere iperespressa in diversi tipi di carcinoma tra i quali l’ovarico, il mammario e il prostatico) sono stati condotti studi di “RNAinterference” che hanno permesso di asserire che in cellule di carcinoma mammario la riduzione dell’espressione di EF1a (livelli diminuiti del 60%) porta ad una riduzione del 30% della clonogenicità. Sembra inoltre che diminuiti livelli d’espressione di EF1 associati a trattamenti a base di 17AAG (inibitore selettivo dell’attività di HSP90 che causa forti riduzioni ai livelli di pAKT) abbiano un effetto sinergico nella riduzione della proliferazione cellulare. Parallelamente a questi studi, stiamo programmando un’analisi globale di tutte le proteine che manifestano una espressione differenziale dopo l’inibizione di pAKT (globale, perché non si riferisce alle sole proteine dell’interattoma, ma all’estratto totale). Questo verrà realizzato tramite confronto diretto delle mappe proteiche ottenute dopo elettroforesi bidimensionale degli estratti totali (trattamento inibitorio specifico vs. controllo) individuando e identificando gli “spots” che rappresentano proteine la cui espressione varia a seguito dell’inibizione dell’attività chinasica di pAKT. La scoperta di nuove proteine substrato della attività chinasica di pAKT e l’approfondimento dei meccanismi biologici e molecolari da essa regolati, rappresentano un importante punto di partenza per la progettazione di strategie terapeutiche ottimali e per la validazione di nuovi markers diagnostici, prognostici o predittivi in situazioni di carcinoma mammario.

2006 - 2-D proteomic analysis of urine in diabetic patients with nephropathy [Abstract in Rivista]
Tomasi, Aldo; L., Lucchi; Uggeri, Simona; Bellei, Elisa; Rossi, Elena; Albertazzi, Alberto; Iannone, Anna
abstract

Mortality and morbidity associated with diabetes is diabetic nephropathy. We carried out a research project on the identification of specific proteomic profiles in patients with diabetic nephropathy. The rationale for this study is based on studies demonstrating that chronic inflammation and oxidative stress determine the complications observed in diabetic patients. Such a pathogenesis will determine post-translational protein modification, bringing to the identification of prognostic and predictive markers. Recent advances in proteomic profiling technologies have allowed preliminary profiling. Various specific proteomic profiles have been described for various pathologies, in particular tumors. Studies on proteomic profiling in diabetic nephropathy have not been performed yet. We analyzed easily available biological samples such as urine to evaluate the expression and function levels of urinary molecules that could become diagnostic, prognostic and predictive markers of disease.

1993 - Overexpression of c-kit in a leukemic cell population carrying a trisomy 4 and its relationship with the proliferative capacity [Articolo su rivista]
Ferrari, S.; Grande, A.; Zucchini, P.; Manfredini, R.; Tagliafico, E.; Rossi, E.; Temperani, P.; Torelli, G.; Emilia, G.; Torelli, U.
abstract

The expression of c-kit and its ligand, the stem cell factor (SCF), was studied in five cases of acute myeloid leukemia. One of these had a trisomy of chromosome 4, where the c-kit oncogene is located. In this case, the c-kit oncogene was overexpressed, but matched by a low expression of its ligand, SCF. The molecular evaluation of the growth rate by c-myc and the histone H3 expression indicated that the growth fraction of this cell population was very low. In one of the other leukemic cell populations studied, characterized by a low expression of c-kit and an elevated expression of the SCF, the growth fraction was also very low. Our results suggest that at least for some receptor oncogenes, the simple overexpression cannot be taken as an indication that the oncogene is involved in the deregulation of cell proliferation. © 1993 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

1990 - Differential effects of c-myb and c-fes antisense oligodeoxynucleotides on granulocytic differentiation of human myeloid leukemia HL60 cells [Articolo su rivista]
Ferrari, S.; Donelli, A.; Manfredini, R.; Sarti, M.; Roncaglia, R.; Tagliafico, E.; Rossi, E.; Torelli, G.; Torelli, U.
abstract

To gain some insight into the role of c-myb and c-fes in myeloid differentiation, the authors have analyzed the ability of HL60 cells to differentiate in response to several different inducers after inhibition of c-myb and c-fes function. This function has been inhibited almost completely by using deoxynucleotides complementary to two 18-nucleotide sequences of c-myb and c-fes encoding mRNA. After 5 days in culture, in several separate experiments with different oligomer preparations, more than 90% growth inhibition was observed in c-myb antisense-treated HL60 cells. At this time, independent of the differentiation inducer used, c-myb antisense-treated HL60 cells differentiate only along the monocytic pathway, whereas in sense oligomer-treated cultures, retinoic acid and dimethyl sulfoxide induced granulocytic differentiation. No perturbation of the HL60 cell growth was observed after 5 days of treatment with antisense c-fes oligomer. However, induction to granulocytic differentiation by retinoic acid and dimethyl sulfoxide resulted in progressive cell death, whereas monocytic differentiation by other differentiation inducers was only marginally affected. These results suggest that granulocytic, unlike monocytic, differentiation requires c-myb-conditioned proliferation and the activity of the protein encoded by c-fes.

1990 - Noncoordinated Expression of S6, S11, and S14 Ribosomal Protein Genes in Leukemic Blast Cells [Articolo su rivista]
Ferrari, S.; Manfredini, R.; Tagliafico, E.; Rossi, E.; Donelli, A.; Torelli, G.; Torelli, U.
abstract

The steady state levels of mRNAs codying for the ribosomal proteins S6, Sil, and SI4 have been evaluated in quiescent and proliferating human fibroblasts and in resting and proliferating human peripheral blood mononuclear cells. It was found that the amounts of ribosomal protein mRN A are very similar and are not increased by serum or mitogen stimulation. The constitutive expression of these genes appears to be coordinately regulated and it is not modified after protein synthesis inhibition by cycloheximide. The ribosomal protein mRNA was also assayed in 15 different populations of human leukemic blast cells. In these populations the abundance of each ribosomal protein mRNA is remarkably different from the other. The results of our present experiments indicate that the expression of the three ribosomal protein genes undergoes independent noncoordinated changes in the large majority of the leukemic populations studied. © 1990, American Association for Cancer Research. All rights reserved.