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Cristina VASCHIERI

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

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Pubblicazioni

2022 - Dermoscopy, confocal microscopy and optical coherence tomography features of main inflammatory and autoimmune skin diseases: A systematic review [Articolo su rivista]
Guida, S.; Longhitano, S.; Ardigo, M.; Pampena, R.; Ciardo, S.; Bigi, L.; Mandel, V. D.; Vaschieri, C.; Manfredini, M.; Pezzini, C.; Arginelli, F.; Farnetani, F.; Zerbinati, N.; Longo, C.; Pellacani, G.
abstract

Background/Objectives: Non-invasive skin imaging features of main skin inflammatory and autoimmune diseases have been reported, although a comprehensive review of their correlation with histopathologic features is currently lacking. Therefore, the aim of this paper was to review the correlation of dermoscopic, reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) criteria of main inflammatory and autoimmune skin diseases with their corresponding histopathologic criteria correlation. Methods: Studies on human subjects affected by main inflammatory and autoimmune diseases, defining the correlation of dermoscopic, RCM or OCT with histopathologic criteria, were included in the review. Five groups of diseases were identified and described: psoriasiform, spongiotic and interface dermatitis, bullous diseases and scleroderma. Results: Psoriasiform dermatitis was typified by white scales, corresponding to hyperkeratosis, and vessels, observed with RCM and OCT. Spongiosis, corresponding to dark areas within the epidermis with RCM and OCT, was the main feature of spongiotic dermatitis. Interface dermatitis was characterised by dermoepidermal junction obscuration. Blisters, typical of bullous diseases, were visualised as dark areas with RCM and OCT while scleroderma lesions were characterised by dermoscopic fibrotic beams, related to dermal thickness variations, with specific OCT and histopathologic correlations. Conclusions: Although the role of RCM and OCT has yet to be defined in clinical practice, non-invasive skin imaging shows promising results on inflammatory and autoimmune skin diseases, due to the correlation with histopathologic features.

2022 - In Vivo Melanoma Cell Morphology Reflects Molecular Signature and Tumor Aggressiveness [Articolo su rivista]
Marconi, Alessandra; Quadri, Marika; Farnetani, Francesca; Ciardo, Silvana; Palazzo, Elisabetta; Lotti, Roberta; Cesinaro, Anna Maria; Fabbiani, Luca; Vaschieri, Cristina; Puviani, Mario; Magnoni, Cristina; Kaleci, Shaniko; Pincelli, Carlo; Pellacani, Giovanni
abstract

Melanoma is the deadliest type of skin cancer, characterized by high cellular heterogeneity which contributes to therapy resistance and unpredictable disease outcome. Recently, by correlating Reflectance-Confocal-Microscopy (RCM) morphology with histopathological type, we identified four distinct melanoma-subtypes: dendritic-cell (DC), round-cell (RC), dermal-nest (DN), and combined-type (CT) melanomas. In the present study, each RCM-melanoma subtype expressed a specific biomolecular profile and biological behavior in vitro. Markers of tumor aggressiveness, including Ki67, MERTK, nestin and stemness markers, were highest in the most invasive CT and DN melanomas, as compared to DC and RC. This was also confirmed in multicellular tumor spheroids. Transcriptomic analysis showed a modulation of cancer progression-associated genes from DC to CT melanomas. The switch from E- to N-cadherin expression proved the epithelial-to-mesenchymal transition from DC to CT subtypes. The DN melanoma was predominantly located in the dermis, as also shown in skin reconstructs. It displayed a unique behavior and a molecular profile associated with a high degree of aggressiveness. Altogether, our results demonstrate that each RCM-melanoma subtype has a distinct biological and gene expression profile, related to tumor aggressiveness, confirming that RCM can be a dependable tool for in vivo detecting different types of melanoma and for early diagnostic screening.

2019 - A comparative dermoscopic and reflectance confocal microscopy study of naevi and melanoma with negative pigment network [Articolo su rivista]
Farnetani, Francesca; Scope, Alon; Mazzoni, Laura; Mandel, Victor Desmond; Manfredini, Marco; Magi, Serena; Vaschieri, Cristina; Kaleci, Shaniko; Longo, Caterina; Ciardo, Silvana; Stanganelli, Ignazio; Pellacani, Giovanni
abstract

Background: Negative pigment network (NPN) is a dermoscopic structure observed more frequently among melanomas than naevi. Precise tissue correlates of NPN are still elusive. Objective: To describe the reflectance confocal microscopy (RCM) findings underlying NPN in melanocytic neoplasms. Methods: We retrospectively identified all melanocytic neoplasms displaying dermoscopic NPN that were imaged with RCM and subsequently biopsied between 2011 and 2015. Images from study lesions (n = 50) were evaluated for dermoscopic and RCM Criteria. Histopathological correlational study was performed in a subset of cases (n = 15). Results: The study data set consisted of 21 melanomas (42%) and 29 naevi (58%). Melanomas showed more frequently irregularly shaped globules than naevi (62% vs. 28%, P = 0.03); NPN also tended to be more asymmetrically located among melanomas (86%) than naevi (62%), albeit not significant (P = 0.06). Under RCM, we observed three patterns of dermal papillae (DP): (i) 'Dark DP' - whereby DP were devoid of nests and often surrounded by a junctional proliferation as thick-Rings - this pattern was less common among melanomas (n = 10, 48%) than naevi (n = 23, 79%, P = 0.02); (ii) 'Bulging DP' - whereby junctional nests of melanocytes protrude into the DP, often in association with junctional proliferation as Meshwork - with comparable frequency among melanomas (n = 12, 57%) and naevi (n = 23, 79%, P = 0.09) and (iii) 'Expanded DP' - whereby junctional and/or dermal nests filled and expanded the DP, often in association with dermal-epidermal junction (DEJ) Clod pattern - seen more commonly among melanomas (n = 15, 71%) than naevi (n = 6, 21%, P < 0.001). Dermoscopy-RCM correlation and comparison to histopathological findings show that the hypo-pigmented lines of NPN correlate with broadened epidermal retes, which often show overlying surface dells and wedge-shaped hypergranulosis, while the pigmented globules of NPN correlate with a predominantly-junctiona of melanocytes along and between the elongated retes. Conclusions: Dermoscopic NPN correlates with three DEJ RCM patterns with differing frequency between naevi and melanomas.

2019 - Effects of topical methotrexate loaded gold nanoparticle in cutaneous inflammatory mouse model [Articolo su rivista]
Fratoddi, I.; Benassi, L.; Botti, Eleonora; Vaschieri, C.; Venditti, I.; Bessar, H.; Samir, M. A.; Azzoni, P.; Magnoni, C.; Costanzo, A.; Casagrande, V.; Federici, M.; Bianchi, L.; Pellacani, G.
abstract

Gold nanoparticles functionalized with 3-mercapto-1 -propansulfonate (AuNPs-3MPS) have been prepared and loaded with Methotrexate (MTX), an immunosuppressive agent used in the systemic treatment of moderate-severe inflammatory diseases. The effects of the AuNPs-3MPS@MTX topically administered in vitro on skin model and in vivo on imiquimod-induced psoriasis-like mice model, have been studied. Clinical response, epidermal thickness, cell proliferation rate and inflammation were tested. AuNPs-3MPS@MTX treated mice showed a decreasing of scaling and erythema score, reduction of epidermal thickness, parakeratosis and hyperkeratosis, compared to AuNPs3MPS treated mice. Immunnhistocheinistry analysis staining displayed that Ki67, K6 CD3 and CD8 stainings were reduced in AuNPs3MPS@MTX treated mice. Blood evaluation showed no differences in blood count and in ALT and AST levels before and after AuNPs3MPS or AuNPs-3MPS@MTX treatment. Topical AuNPs-3MPS@MTX treatment is able to induce a reduction of keratinocytes hyperproliferation, epidermal thickness and also inflammatory infiltrate in vivo on imiquimod-induced psoriasis like mice model. Published by Elsevier Inc.

2019 - In vitro Engineering of a Skin Substitute Based on Adipose-Derived Stem Cells [Articolo su rivista]
Paganelli, A.; Benassi, L.; Pastar, I.; Pellegrini, M.; Azzoni, P.; Vaschieri, C.; Pisciotta, A.; Carnevale, G.; Pellacani, G.; Magnoni, C.
abstract

In the field of wound healing, stem cell-based strategies are gaining importance for their regenerative potential. Adipose-derived stem cells (ADSCs) are a particular subset of mesenchymal stem cells present in the stromal-vascular fraction of the adipose tissue, today considered very attractive for their relative abundance and accessibility in the human body. However, ADSCs are still not routinely used in normal clinical practice. Several studies have also reported ADSC transplantation in association with biomaterials in an attempt to enhance the local retention and growth rate of the cells. The aim of our study was to evaluate the ability of ADSCs to build a dermal scaffold to be potentially used as a dermal substitute in the field of wound healing, with optimal biocompatibility and mechanical properties. ADSCs were defined as CD90-, CD73-, and CD105-positive cells. ADSCs turned out to be capable of secreting all the main components of the extracellular matrix (ECM) upon stimulation, thus efficiently producing a collagen and fibronectin-containing dermal matrix. We also checked whether the ADSC-produced dermal scaffold could be seeded with keratinocytes. The scaffolding material directly produced by ADSCs has several advantages when compared to the commercially available ones: it is easily obtained from the patients and it is 100% biocompatible and supports cell-ECM interaction. Moreover, it represents a possible powerful therapeutic tool for patients with chronic ulcers since it appears to be potentially grafted with keratinocytes layers, thus bypassing the classical two-step grafting procedure.

2019 - Scouting Sigma Receptor Ligands As New Tools For The Treatment Of Neurodegenerative Diseases And Cancer [Relazione in Atti di Convegno]
Sorbi, C.; Linciano, P.; Tait, A.; Atene, C. G.; Guglielmo, L.; Di Rocco, G.; Ronsisvalle, S.; Denora, N.; Imbriano, C.; Rigillo, G.; Fossa, P.; Cichero, E.; Benassi, L.; Vaschieri, C.; Marocchi, F.; Lanfrancone, M. L.; Brasili, L.; Franchini, S.
abstract

Sigma receptors (Rs) are nowadays recognized as an unique class of membrane receptors divided into two subtypes, R and R. Rs regulate a number of physiological functions and their role has been evaluated in many disorders. Deficits in R are associated with neurodegeneration while their activation may represent a valuable strategy for the treatment of a number of neurodegenerative disorders. Moreover, R is overexpressed in a variety of cancer cells and selective R antagonists are reported to modulate cancer cell viability.1 R are also highly expressed proliferating tumors. R agonists are giving promising results in preclinical studies for the treatment of resistant or hardly treatable tumors and R ligands have been proposed as biomarkers for tumors proliferation.2 However, the identification of potent and selective ligands and the comprehension of the chemical features behind agonism/antagonism still remain a primary challenge in this field. With this aim, following a ligand-based approach, a library of over 120 ligands have been designed and synthesized over the years, by combining different substituted five-membered heterocyclic rings with appropriate R pharmacophoric amines. Compounds were tested for R and R affinity showing Ki values in the micromolar / sub-nanomolar range, with a selectivity mainly shifted toward the R. A detailed SAR, supported by molecular modelling, was drawn up. The intrinsic activity was determined in vivo for the most promising molecules. According to their profile, R agonists were tested for neuroprotection, whereas R antagonists / R agonists for anticancer activity. Preliminary results in SH-SY5Y neuroblastoma cells showed the ability of some compounds to protect neuronal cells from death induced by four toxicity models. Cell viability assays were performed on different cancer cell lines to assess the anti-proliferative potential of selected molecules. In particular, dose and time dependent treatments were done on prostate cancer cells, which express higher levels of both R and R compared to normal samples. Similarly, we assessed the effect of the compounds on melanoma cells: BS148, a potent and selective R agonist, showed anti-proliferative activity on immortalized and PDX (metastatic melanoma patient-derived xenografts) cell lines.3 Confocal microscopy studies with BS148 fluorescent probe revealed the internalization of BS148 within melanoma cells, with a cytoplasmatic localization, mostly in the perinuclear region, according to R distribution. Finally, to verify whether TMEM97 / R mediates BS148-antiproliferative activity, we stably overexpressed the TMEM97 gene in HeLa cells: TMEM97-Hela were more sensitive to BS148 anti-proliferative activity compared to control cells, which express endogenous R levels. Taken together, these results support the idea that R is an innovative target in cancer, paving the way for improved tools for cancer diagnosis, monitoring and therapy.

2018 - Reflectance confocal microscopy and optical coherence tomography for the diagnosis of bullous pemphigoid and pemphigus and surrounding sub-clinical lesions [Relazione in Atti di Convegno]
Mandel, Victor Desmond; Cinotti, Elisa; Benati, Elisa; Labeille, Bruno; Ciardo, Silvana; Vaschieri, Cristina; Cambazard, Frederic; Perrot, Jean-Luc; Pellacani, Giovanni
abstract

Background: Diagnosis of bullous pemphigoid (BP) and pemphigus is based on clinical features, histology, immunofluorescence and laboratory data. Objectives: To evaluate features of BP and pemphigus at reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) in order to provide a rapid non-invasive bed-side diagnosis. Secondary objective was to evaluate the detectability of clinically non-visible lesions. Methods: This was an observational, retrospective, multicentre study in which patients with suspicious lesions for BP or pemphigus underwent clinical assessment, RCM, OCT, blood tests and skin biopsy for histological and direct immunofluorescence examinations from January 2014 to December 2015. A total of 72 lesions in 24 selected patients were evaluated. Additionally, apparently unaffected skin at two different distances [near (1-2 cm) and far (2-3 cm)] from each lesion was examined in order to test sub-clinical lesion detectability. Instrument and acquisition procedures are described elsewhere. Results: RCM was able to detect sub-epidermal and intra-epidermal blisters respectively in 75% and 50% of the patients affected by BP and pemphigus. At OCT the exact blister level was identified in all patients. Acantholytic cells were observed only at RCM in pemphigus (62.5%). Fibrin deposition inside the blisters was only found in BP, evidenced both at RCM and OCT. Among patients with BP, subclinical blisters were detected in 9 (9.4%) clinically healthy skin, while among patients with pemphigus were observed in 10 (20.8%) apparently unaffected skin. Conclusions: RCM and/or OCT provide useful information for a rapid diagnosis of BP and pemphigus and for the identification of biopsy site. Combined use of RCM and OCT is optimal because associates the higher resolution of RCM with the greater penetration depth of OCT. OCT could be an optimal tool for treatment monitoring, especially in the cases of sub-clinical lesions.

2018 - Reflectance confocal microscopy and optical coherence tomography for the diagnosis of bullous pemphigoid and pemphigus and surrounding subclinical lesions [Articolo su rivista]
Mandel, Victor Desmond; Cinotti, Elisa; Benati, Elisa; Labeille, Bruno; Ciardo, Silvana; Vaschieri, Cristina; Cambazard, Frederic; Perrot, Jean-Luc; Pellacani, Giovanni
abstract

Background: Diagnosis of bullous pemphigoid (BP) and pemphigus is based on clinical features, histology, immunofluorescence and laboratory data. Objectives: To evaluate features of BP and pemphigus at reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) in order to provide a rapid non-invasive bed-side diagnosis. Secondary objective was to evaluate the detectability of clinically non-visible lesions. Methods: This was an observational, retrospective, multicentre study in which patients with suspicious lesions for BP or pemphigus underwent clinical assessment, RCM, OCT, blood tests and skin biopsy for histological and direct immunofluorescence examinations from January 2014 to December 2015. A total of 72 lesions in 24 selected patients were evaluated. Additionally, apparently unaffected skin at two different distances [near (1-2 cm) and far (2-3 cm)] from each lesion was examined to test subclinical lesion detectability. Results: RCM was able to detect subepidermal and intra-epidermal blisters, respectively, in 75% and 50% of the patients affected by BP and pemphigus. At OCT, the exact blister level was identified in all patients. Acantholytic cells were observed only at RCM in pemphigus (62.5%). Fibrin deposition inside the blisters was only found in BP, evidenced both at RCM and OCT. Among patients with BP, subclinical blisters were detected in nine (9.4%) clinically healthy skin, while among patients with pemphigus were observed in 10 (20.8%) apparently unaffected skin. Conclusion: RCM and/or OCT provide useful information for a rapid diagnosis of BP and pemphigus and for the identification of biopsy site. Combined use of RCM and OCT is optimal because associates the higher resolution of RCM with the greater penetration depth of OCT. OCT could be an optimal tool for treatment monitoring, especially in the cases of subclinical lesions. However, histopathologic and immunologic examinations remain the gold standard for establishing the final diagnosis.

2017 - Reflectance confocal microscopy and optical coherence tomography for the diagnosis of bullous pemphigoid and pemphigus [Poster]
Mandel, Victor Desmond; Cinotti, Elisa; Benati, Elisa; Labeille, Bruno; Ciardo, Silvana; Vaschieri, Cristina; Cambazard, Frederic; Perrot, Jean-Luc; Pellacani, Giovanni
abstract

Introduction & Objectives: Bullous pemphigoid (BP) and pemphigus (P) are autoimmune diseases characterized by the presence of blisters on the skin and/or the mucous membranes. The diagnosis of these bullous diseases is based on a combination of criteria encompassing clinical features, histology, immunofluorescence and laboratory data. The aim of this study was to evaluate features of BP and P at reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) in order to provide a rapid non-invasive bed-side diagnosis. Secondary objective was to evaluate the detectability of clinically non-visible lesions. Material & Methods: This was an observational, retrospective, multicentre study (University of Modena, Italy and University of Saint-Etienne, France) in which patients with suspicious lesions for BP or P underwent clinical assessment, RCM, OCT, blood tests and skin biopsy for histological and direct immunofluorescence examinations. A total of 72 lesions in 24 patients (16 with PB and 8 with P) were evaluated. Apparently unaffected skin was examined in order to test sub-clinical lesion detectability in all patients. Data analysis was performed from January 2014 to December 2015. Results: RCM was able to detect sub-epidermal and intra-epidermal blisters respectively in 75% and 50% of the patients affected by BP and P. At OCT the exact blister level was identified in all BP and P cases’. Acantholytic cells were observed only at RCM in P (62.5%). Fibrin deposition inside the blisters was only found in PB, evidenced both at RCM and OCT. Subclinical bullae were revealed on clinically healthy skin at OCT in some cases of BP and P. Conclusions: RCM and/or OCT can assist the clinician in providing rapid information through a non-invasive procedure for a rapid diagnosis of BP and P. Combined use of RCM and OCT for a real-time examination of the skin lesions associates the higher resolution of RCM with the greater penetration depth in cross-sectional view of OCT, providing in vivo quasi-histologic information.

2017 - Structure-Activity Relationship within a new series of σ1 and σ2 receptor ligands: identification of a novel σ2R agonist (BS148) with selective toxicity against metastatic melanoma [Articolo su rivista]
Franchini, Silvia; Sorbi, Claudia; Battisti, Umberto Maria; Tait, Annalisa; Bencheva, Leda Ivanova; Cichero, Elena; Fossa, Paola; Cilia, Antonio; Prezzavento, Orazio; Ronsisvalle, Simone; Aricò, Giuseppina; Benassi, Luisa; Vaschieri, Cristina; Azzoni, Paola; Magnoni, Cristina; Brasili, Livio
abstract

A new series of spiro-cyclic sigma ligands were prepared and studied. Most were found to have a high affinity and selectivity for σ1R; compounds 7b, 15b and 16a were shown to be σ1R agonists, while 16b was proven to be the only σ1R antagonist. Only 16a (BS148) exhibited σ2R selectivity and was able to inhibit the growth of metastatic malignant melanoma cell lines, whilst not affecting normal human melanocytes. BS148’s anti-proliferative activity suggests a σ2R agonist profile. Further, preliminary investigations indicate that, at least a part, the mechanism of metastatic malignant melanoma cell death induced by BS148 is due to apoptosis.

2017 - Un inusuale pemfigo localizzato [Poster]
Mandel, Victor Desmond; Farnetani, Francesca; Vaschieri, Cristina; Manfredini, Marco; Cesinaro, Anna Maria; Giusti, Francesca; Pellacani, Giovanni
abstract

Si presenta il caso di un uomo di 74 anni giunto alla nostra osservazione per la comparsa da circa 4 mesi di una lesione squamo-crostosa del dorso del naso suggestiva per un epitelioma e con pattern dermoscopico aspecifico. Veniva eseguita la microscopia laser confocale e la tomografia a coerenza ottica che mostravano inaspettate caratteristiche di pemfigo. Per confermare tale ipotesi venivano eseguiti ulteriori accertamenti. L’esame istologico evidenziava acantolisi a livello degli strati spinoso e granuloso, mentre l’immunofluorescenza diretta rivelava depositi di IgG e C3 in tutto lo spessore dell’epidermide. Anticorpi intercellulari soprabasali (1:40) sono stati evidenziati dall’immunofluorescenza indiretta. Infine, l’immunoblot mostrava la presenza nel siero di anticorpi diretti contro gli antigeni 130 kDa e 160 kDa, successivamente identificati come anticorpi diretti contro la desmogleina 1 e 3 mediante sistemi colorimetrici AP. Tali dati deponevano per una forma mista di pemfigo con caratteristiche sia della forma volgare che di quella foliacea.

2017 - Uso della microscopia laser confocale e della tomografia a coerenza ottica nella diagnosi del pemfigoide bolloso e del pemfigo [Poster]
Mandel, Victor Desmond; Cinotti, Elisa; Benati, Elisa; Ciardo, Silvana; Vaschieri, Cristina; Labeille, Bruno; Cambazard, Frederic; Perrot, Jean-Luc; Pellacani, Giovanni
abstract

Il pemfigoide bolloso (PB) ed il pemfigo (P) sono malattie autoimmuni caratterizzate dalla formazione di bolle sulla pelle e/o sulle mucose. La diagnosi di queste patologie viene posta sulla base della valutazione clinica, dei dati di laboratorio, dell’istologia, dell’immunofluorescenza diretta ed indiretta. Nel nostro studio osservazionale multicentrico sono stati valutati con la microscopia laser confocale (RCM) e con la tomografia a coerenza ottica (OCT) di 16 pazienti con PB e di 8 con P sia la cute apparentemente sana che tre lesioni bollose. L’OCT è risultato essere più preciso del RCM nella definizione della localizzazione delle bolle e nell’identificazione delle lesioni subcliniche. Grazie al potere risolutivo maggiore, l’RCM è in grado di visualizzare strutture a risoluzione quasi istologica e pertanto è risultato essere capace di valutare più parametri rispetto all’OCT, dimostrando di essere un ottimo strumento nella diagnosi differenziale con altre patologie bollose come ad esempio quelle di natura virale.

2016 - Electrochemotherapy induces apoptotic death in melanoma metastases: A histologic and immunohistochemical investigation [Articolo su rivista]
Bigi, Laura; Galdo, Giovanna; Cesinaro, Anna Maria; Vaschieri, Cristina; Marconi, Alessandra; Pincelli, Carlo; Fantini, Fabrizio
abstract

Background: Electrochemotherapy (ECT) is increasingly used in the treatment of primary and secondary skin tumors, but little is known about the pathologic mechanism responsible for tumor cell destruction in humans. Knowledge of detailed mechanism of host response after ECT may improve the treatment efficacy related to patient selection and technique refinements. Aim: The aim of the study was to investigate the histopathology and mechanism of cell death after ECT in cutaneous melanoma metastases. Methods: Skin biopsy specimens were sequentially obtained after ECT of cutaneous melanoma metastases, during a follow-up period of 2 months. Results from histologic evaluation and immunohistochemical characterization of the inflammatory infiltrate (CD3, CD4, CD8, CD56, Granzyme-B) were compared with a panel of apoptosis-related markers. Main outcome measures: Evidence of the mechanism of tumor cell damage, identification of histological and immunohistochemical signs of apoptosis and/or necrosis underlining a possible time course of tumor destruction and inflammatory reaction after ECT. Results: Early signs of epidermal degeneration, an increase of the inflammatory infiltrate, and initial tumor cell morphological changes were already detected 10 min after ECT. The cell damage progression, as demonstrated by histological and immunohistochemical evidence using apoptotic markers (TUNEL and caspase-3 staining), reached a climax 3 days after treatment, to continue until 10 days after. Scarring fibrosis and complete absence of tumor cells were observed in the late biopsy specimens. A rich inflammatory infiltrate with a prevalence of T-cytotoxic CD3/CD8-positive cells was detected 3 h after ECT and was still appreciable 3 months later. Conclusion: This study attempts to define the time course and characteristics of tumor response to ECT. The observations suggest both a direct necrotic cell damage and a rapid activation of apoptotic mechanisms that occur in the early phases of the cutaneous reaction to ECT. A persistent immune response of T-cytotoxic lymphocytes could possibly explain the long-term local tumor control.

2016 - Functionalized gold nanoparticles for topical delivery of methotrexate for the possible treatment of psoriasis [Articolo su rivista]
Bessar, Hagar; Venditti, Iole; Benassi, Luisa; Vaschieri, Cristina; Azzoni, Paola; Pellacani, Giovanni; Magnoni, Cristina; Botti, Elisabetta; Casagrande, Viviana; Federici, Massimo; Costanzo, Antonio; Fontana, Laura; Testa, Giovanna; Mostafa, Fawzia Farag; Ibrahim, Samia Ali; Russo, Maria Vittoria; Fratoddi, Ilaria
abstract

Gold nanoparticles (AuNPs) represent an effective choice for topical drug delivery systems thanks to their small size, general non-toxicity, ease of functionalization and high surface to volume ratio. Even if systemic, methotrexate still plays an important role in psoriasis treatment: its topical use shows insufficient percutaneus penetration owing to limited passive diffusion, high molecular weight and dissociation at physiological pH. The aim of our study was to design a new drug delivery nanocarrier for Methotrexate and to improve its solubility, stability and biodistribution. AuNPs were on purpose prepared with a hydrophilic stabilizing layer, in order to improve the colloidal stability in water. Water-soluble gold nanoparticles functionalized by sodium 3-mercapto-1-propansulfonate (Au-3MPS) were prepared and loaded with methotrexate (MTX). The loading efficiency of MTX on Au-3MPS was assessed in the range 70-80%, with a fast release (80% in one hour). The release was studied up to 24h reaching the value of 95%. The Au-3MPS@MTX conjugate was fully characterized by spectroscopic techniques (UV-vis, FTIR) and DLS. Preliminary toxicity tests in the presence of keratinocytes monolayers allowed to assess that the used Au-3MPS are not toxic. The conjugate was then topically used on C57BL/6 mouse normal skin in order to trace the absorption behavior. STEM images clearly revealed the distribution of gold nanoparticles inside the cells. In vitro studies showed that Methotrexate conjugated with Au-3MPS is much more efficient than Methotrexate alone. Moreover, DL50, based on MTT analysis, is 20 folds reduced at 48 h, by the presence of nanoparticles conjugation. UV-vis spectra for in vivo tracing of the conjugate on bare mouse skin after 24h of application, show increased delivery of Methotrexate in the epidermis and dermis using Au-3MPS@MTX conjugate, compared to MTX alone. Moreover we observed absence of the Au-3MPS in the dermis and in the epidermis, suggesting that these layers of the skin do not retain the nanoparticles. Based on our data, we found that the novel Au-3MPS@MTX conjugate is an effective non-toxic carrier for the satisfactory percutaneous absorption of Methotrexate and could help in possible topical treatment of psoriasis.

2016 - Pemphigus with features of both vulgaris and foliaceus variants localized to the nose [Articolo su rivista]
Mandel, Victor Desmond; Farnetani, Francesca; Vaschieri, Cristina; Manfredini, Marco; Cesinaro, Anna Maria; Giusti, Francesca; Pellacani, Giovanni
abstract

We report the case of a 74-year-old man affected by an unusual variant of pemphigus. He presented with a crusty and scaly lesion of the nose. We performed reflectance confocal microscopy and optical coherence tomography on the lesion, which suggested an unexpected diagnosis of pemphigus. Therefore, to confirm our diagnostic suspicions, we executed indirect immunofluorescence and two biopsies, one for histopathological examination and one for direct immunofluorescence. Histopathological evaluation showed acantholysis with formation of clefts in the granular and spinous layers of the epidermis. Direct immunofluorescence revealed immunoglobulin G and C3 deposit to the full thickness of the epidermis. Indirect immunofluorescence showed intercellular antibodies at a titer of 1:40 in the suprabasal epidermis. The immunoblot analysis using epidermal extract revealed the presence of circulating antibodies directed to 130- and 160-kDa antigens in the patient's serum. These two antigens were evidenced from nitrocellulose membrane with colorimetric AP systems, which highlighted the presence of autoantibodies against desmoglein (Dsg)1 and Dsg3 (sodium dodecylsulfate polyacrylamide gel electrophoresis). We also performed an enzyme-linked immunoassay. All these findings suggested that this patient's pemphigus had features of both vulgaris and foliaceus variants.

2011 - p75 neurotrophin receptor mediates apoptosis in transit-amplifying cells and its overexpression restores cell death in psoriatic keratinocytes. [Articolo su rivista]
Truzzi, Francesca; Marconi, Alessandra; P., Atzei; M. C., Panza; Lotti, Roberta; Dallaglio, Katiuscia; R., Tiberio; Palazzo, Elisabetta; Vaschieri, Cristina; Pincelli, Carlo
abstract

p75 neurotrophin receptor (p75NTR) belongs to the TNF-receptor superfamily and signals apoptosis in many cell settings. In human epidermis, p75NTR is mostly confined to the transit-amplifying (TA) sub-population of basal keratinocytes. Brain-derived neurotrophic factor (BDNF) or neurotrophin-4 (NT-4), which signals through p75NTR, induces keratinocyte apoptosis, whereas β-amyloid, a ligand for p75NTR, triggers caspase-3 activation to a greater extent in p75NTR transfected cells. Moreover, p75NTR co-immunoprecipitates with NRAGE, induces the phosphorylation of c-Jun N-terminal kinase (JNK) and reduces nuclear factor kappa B (NF-κB) DNA-binding activity. p75NTR also mediates pro-NGF-induced keratinocyte apoptosis through its co-receptor sortilin. Furthermore, BDNF or β-amyloid cause cell death in TA, but not in keratinocyte stem cells (KSCs) or in p75NTR silenced TA cells. p75NTR is absent in lesional psoriatic skin and p75NTR levels are significantly lower in psoriatic than in normal TA keratinocytes. The rate of apoptosis in psoriatic TA cells is significantly lower than in normal TA cells. BDNF or β-amyloid fail to induce apoptosis in psoriatic TA cells, and p75NTR retroviral infection restores BDNF- or β-amyloid-induced apoptosis in psoriatic keratinocytes. These results demonstrate that p75NTR has a pro-apoptotic role in keratinocytes and is involved in the maintenance of epidermal homeostasis.

2010 - p75NTR mediates apoptosis in transit amplifying (TA) cells and its overexpression resores cell death in psoriatic keratinocytes [Abstract in Rivista]
Truzzi, Francesca; Marconi, Alessandra; Lotti, Roberta; Dallaglio, Katiuscia; Palazzo, Elisabetta; Borroni, Riccardo; Vaschieri, Cristina; R., Tiberio; Pincelli, Carlo
abstract

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2008 - P75 neurotrophin receptor restores defective apoptosis in psoriatic keratinocytes: a role for transit amplifying cells [Abstract in Rivista]
Marconi, Alessandra; Truzzi, Francesca; Lotti, Roberta; Dallaglio, Katiuscia; Borroni, Riccardo; Vaschieri, Cristina; R., Tiberio; Pincelli, Carlo
abstract

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2008 - Pathologic changes after photodynamic therapy for basal cell carcinoma and Bowen disease: A histologic and immunohistochemical investigation [Articolo su rivista]
F., Fantini; A., Greco; A. M., Cesinaro; T., Surrenti; K., Peris; Vaschieri, Cristina; Marconi, Alessandra; Giannetti, Alberto; Pincelli, Carlo
abstract

Objective: To investigate the in vivo reactions and themechanisms of cell death after photodynamic therapy(PDT) for cutaneous carcinomas. Photodynamic therapyis a new treatment modality for nonmelanoma skin cancers.Its effects on target tissue have been well investigatedin vitro, where apoptosis appears to be the maineffector mechanism, but its effects remain undefined invivo.Design: Skin biopsy specimens were obtained sequentiallyafter PDT for basal cell carcinoma and in situ squamouscell carcinoma (Bowen disease). Evidence from routinehistologic evaluation was compared with a panel ofapoptosis-related (TUNEL [terminal deoxynucleotidyltransferase-mediated biotin–deoxyuridine triphosphatenick-end labeling], caspase-3, and Bcl-2) and inflammatory(CD4, CD8, CD20, CD68, and CD56) markers. Weused electron microscopy to evaluate cell damage at theultrastructural level.Main Outcome Measures: Evidence of the mechanismsof tumor cell damage after PDT, detection of histologicand/or immunohistochemical signs of apoptosis,and time course of the tumor destruction andinflammatory reaction.Results: Early epidermal damage and an acute dermalinflammatory response were detected 15 minutes afterPDT. In basal cell carcinoma, nodule damage progressedfrom scant apoptotic cells seen at the dermalepithelialjunction to massive destruction seen after 1 and2 days. The periphery of the basaloid nodules consistentlyshowed earlier and predominant damage, as demonstratedby the perfect coincidence of histologic and immunohistochemicalevidence with apoptotic markers(TUNEL and caspase-3 staining). Fibrosis and lentigolikechanges were seen in late biopsy specimens.Conclusions: This study defines the time course and characteristicsof the skin tumor response to PDT. Taken together,our observations suggest that direct damage tocancer cells is the main effector mechanism leading toPDT response. The involvement of apoptosis is demonstratedby the simultaneous appearance of histologic, immunohistochemical,and ultrastructural markers that occurin the early phases of the cutaneous reaction to PDT.These observations could help to develop future refinementsof the PDT technique.

2007 - Fas ligand and pemphigus sera induce desmoglein cleavage and apoptosis in human keratinocytes [Abstract in Rivista]
Lotti, Roberta; Marconi, Alessandra; Truzzi, Francesca; Dallaglio, Katiuscia; Vaschieri, Cristina; Pincelli, Carlo
abstract

Pemphigus is an autoimmune bullous disease characterized by loss of adhesion of keratinocytes that round up in a process known as acantholysis. While the molecular mechanisms underlying acantholysis are still unclear, it is well known that cell detachment is often associated with apoptosis. We have previously detected apoptotic keratinocytes in perilesional, yet undetached, pemphigus skin. Moreover, we have observed that Fas ligand (FasL) levels are significantly higher in untreated pemphigus sera (more than 0.1 ng/ml) than in controls. As pemphigus sera induce apoptosis in cultured human keratinocytes, we wanted to analyze the apoptotic mechanisms in pemphigus. We first confirmed that pemphigus lesions contain apoptotic keratinocytes, by showing caspase-3 activation. In addition, while Fas receptor (FasR) is expressed in the basal and partially in the suprabasal layers in pemphigus vulgaris (PV), it is detected throughout the epidermal layers in muco-cutaneous pemphigus. Furthermore, pemphigus sera-induced keratinocyte apoptosis is partially prevented by pretreatment with either caspase-8 inhibitor or anti-FasL neutralizing antibody. Moreover, caspase-8 activation induced by untreated pemphigus sera is partially inhibited by anti-FasL antibody. Untreated pemphigus sera induce the cleavage of desmoglein 1 and 3 (dsg 1, 3). Moreover, recombinant FasL dose-dependently cleaves dsg 1 and 3 (0.1, 10, 100 ng/ml). Finally, caspase-8 inihibitor prevents dsg cleavage, strongly indicating the critical role of FasL in the pathogenesis of pemphigus. In particular, high levels of FasL, contained in pemphigus sera exert a dual activity, by both inducing keratinocyte apoptosis and dsg cleavage.

2007 - P75 neurotrophin receptor exerts pro-apoptotic functions in human keratinocytes: a role in psoriasis [Abstract in Rivista]
Marconi, Alessandra; Truzzi, Francesca; Lotti, Roberta; Dallaglio, Katiuscia; Vaschieri, Cristina; R., Tiberio; Pincelli, Carlo
abstract

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2007 - Survivin identifies keratinocyte stem cells and is downregulated by anti-β1 integrin during anoikis [Articolo su rivista]
Marconi, Alessandra; Dallaglio, Katiuscia; Lotti, Roberta; Vaschieri, Cristina; Truzzi, Francesca; F., Fantini; Pincelli, Carlo
abstract

Survivin belongs to the family of inhibitor of apoptosis proteins and is involved in regulation of cell death as well as cell division. Here, we show that wild-type (WT) survivin is expressed in a subpopulation of basal keratinocytes in normal human skin at the cytoplasmic level. WT survivin is highly expressed in keratinocyte stem cells (KSCs), whereas its mRNA level decreases in transit amplifying (TA) cells and disappears in postmitotic (PM) cells. Likewise, WT survivin protein is expressed in KSCs, almost undetectable in TA cells, and absent in PM cells. Real time polymerase chain reaction demonstrates that the putative antiapoptotic isoforms survivin-2B and survivin-Delta Ex3 are expressed at the highest levels in KSCs, whereas they tend to decrease in TA cells and disappear in PM cells. On the contrary, the putative proapoptotic variants of survivin, survivin-3B, and survivin-2 alpha tend to be high in PM and TA cells and are almost absent in KSCs. By confocal microscopy, survivin is predominantly expressed at the nuclear level in KSCs, which proliferate significantly better than TA cells, which, in turn, express mostly cytosolic WT survivin. Blocking beta 1 integrin signal downregulates WT survivin mRNA and protein expression and induces apoptosis (anoikis) in KSCs. On the other hand, inhibition of beta 1 integrin upregulates mRNA expression of survivin-2 alpha. Taken together, these results indicate that survivin identifies human KSCs. Expression of nuclear survivin could reflect the different behavior between KSCs in vitro and in vivo, in terms of proliferation. Finally, survivin could be part of the niche protection by preventing anoikis in KSCs.

2006 - Fas ligand and pemphigus sera induce cleavage of desmogleins and apoptosis in human keratinocytes [Abstract in Rivista]
Marconi, Alessandra; Truzzi, Francesca; Lotti, Roberta; Vaschieri, Cristina; Dallaglio, Katiuscia; Giannetti, Alberto; Pincelli, Carlo
abstract

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2006 - Imiquimod modulates the expression of survivin, bcl-2 and Ki67 in skin tumors [Abstract in Rivista]
Marconi, Alessandra; Dallaglio, Katiuscia; Lotti, Roberta; Vaschieri, Cristina; K., Peris; Pincelli, Carlo
abstract

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2005 - Evaluation of different serological tests for the diagnosis of pemphigus [Abstract in Rivista]
Vaschieri, Cristina; Marconi, Alessandra; Pincelli, Carlo; Giannetti, Alberto
abstract

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2005 - Survivin is overexpressed in keratinocyte stem cells and is down-regulated by pro-apoptotic agents [Abstract in Rivista]
Marconi, Alessandra; Panza, Maria Cristina; Lotti, Roberta; Vaschieri, Cristina; K., Peris; Pincelli, Carlo
abstract

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2005 - p75 neurotrophin receptor co-localizes with sortilin and induces apoptosis in a subpopulation of human keratinocytes [Abstract in Rivista]
Atzei, Paola; Marconi, Alessandra; Panza, Maria Cristina; Vaschieri, Cristina; Truzzi, Francesca; J. C., Reed; Pincelli, Carlo
abstract

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2004 - Carboxyfullerenes localize at the intracellular level and prevents caspase cascade induced by UVB in human keratinocytes [Abstract in Rivista]
C., Fumelli; Marconi, Alessandra; M., Pignatti; Vaschieri, Cristina; Giannetti, Alberto; Pincelli, Carlo
abstract

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2001 - Il glutatione endogeno regola l'attivazione della n-smase nel processo di apoptosi indotta dai raggi UVB nei cheratinociti umani normali in coltura. [Abstract in Atti di Convegno]
Magnoni, Cristina; Euclidi, Emanuela; Bertazzoni, Giorgia; Benassi, Luisa; Vaschieri, Cristina; Seidenari, Stefania; Giannetti, Alberto
abstract

I raggi UVB sono le radiazioni maggiormente responsabili degli effetti fotobiologici a livello cutaneo. Come è noto dalla letteratura, i raggi UVB inducono apoptosi nei cheratinociti umani normali, ma i segnali ed i meccanismi che danno inizio alla risposta apoptotica sono ancora in parte da definire. Diversi studi hanno evidenziato che i ceramici, una particolare classe di sfingolipidi, sono implicati nella traduzione dei segnali intracellulari partecipando a diversi processi quali la modulazione del ciclo cellulare, l’induzione del processo di differenziazione e/o del fenomeno apoptotico. I ceramici intracellulari possono venire sintetizzati attraverso varie vie e una di queste implica l’attivazione di particolari enzimi, le sfingomielinasi acida e neutra, che idrolizzano la sfingomielina in fosfocolina e ceramide. Recenti studi in vitro su linee cellulari leucemiche hanno dimostrato che il glutatione (GSH), molecola ad attività antiossidante, inibisce in maniera dose-dipendente l’attivazione della sfingomielinasi neutra (N-SMase). Con il presente studio riportiamo che il pre-trattamento con GSH esogeno inibisce l’attivazione della N-SMase indotta da UVB, riduce la generazione di ceramici intracellulari e blocca parzialmente il processo apoptotico. Cheratinociti umani normali provenienti da interventi di chirurgia plastica sono stati coltivati su fibroblasti 3T3 trattati con mitomicina in terreno di coltura Dulbecco ed Ham F12 addizionato del 10% di siero di vitello fetale. A subconfluenza le cellule sono state pre-trattate con GSH 5mM, per 2h a 37°C e successivamente irradiate con UVB alla dose di 75mJ/ cm2. A tempi diversi le cellule sono state raccolte per valutare l’ attività enzimatica della N-SMase, la produzione di ceramici intracellulari, tramite cromatografia su strato sottile e il clivaggio della proteina poli(ADP-ribosio) polimerasi (PARP), come marker di apoptosi, tramite western-blot. L’esposizione ai raggi UVB induce attivazione della SMase neutra a 15 minuti con picco a 30 minuti dallo stimolo con UVB e idrolisi della sfingomielina con rapida comparsa di ceramici. L’aumento dei ceramici è risultato significativamente evidente già dopo 15 minuti dall’irradiazione UV e si mantiene fino a 24 ore. Il pre-trattamento con GSH inibisce, in modo significativo, l’attivazione in vitro della N-SMase e la produzione di ceramici a tutti i tempi studiati. L’analisi in western-blotting ha evidenziato il clivaggio di PARP a 16 ore dall’esposizione ai raggi UVB, mentre nei campioni protrattati con GSH non si osserva tale clivaggio evidenziando in questo modo un’interferenza con la trasduzione del segnale apoptotico. I nostri dati suggeriscono che il glutatione endogeno sia uno dei fattori che regolano l’attivazione della N-SMase nel processo di apoptosi indotta dai raggi UVB nei cheratinociti umani normali in coltura.

2001 - UVB-radiation induces neutral and acidic sphingomyelinases in cultured normal human keratinocytes. [Abstract in Atti di Convegno]
Euclidi, Emanuela; Magnoni, Cristina; Benassi, Luisa; Bertazzoni, Giorgia; Vaschieri, Cristina; Giannetti, Alberto; Seidenari, Stefania
abstract

The sphingomyelin pathway is an ubiquitous, evolutionary conserved signaling system which transduces extracellular signal into the cell. During the last year increasing evidence has shown that sphingolipids may play a role in intracellular signal transduction. The action of a ligand binding to a surface receptor results in early activation of the enzyme sphingomyelinase (SMases). SMase causes hydrolysis of membrane sphingomyelin (SM) and generation of ceramide. Activation of sphingomyelin cycle has been linked to various extracellular agents such as TNF-α and ionizing radiation. Activation of these targets is followed by three major cellular responses: cell growth arrest, induction of cell differentiation and/or induction of programmed cell death or apoptosis. The aim of the present study is to investigate whether activation of SMases and generation of ceramide could be induced by UVB radiation in normal human keratinocytes. (NHK).NHK were cultivated with mitomycin-treated 3T3 cells in Dulbecco’s modified Eagle’s medium/Ham’s F12 medium and, at preconfluency were irradiated with a UVB dose of 75mJ/cm2.At different times after UVB irradiation, cells were harvested for lipid extraction and for in vitro measurement of neutral and acidic SMaese enzymatic activity.Exposure to UVB radiation results in a rapid in vivo sphingomyelin hydrolysis and generation of ceramide as measured by TLC analysis. The ceramide accumulation starts at 15 min after UV exposure and progressively increased up to 24h.In vitro measurement of neutral SMases activity from UVB-treated NHK extracts, using labelled C14 sphingomyelin as substrate, shows that acidic SMase peaks 15 minutes after exposure to UVB, while neutral SMase peaks at 30 minutes. TUNEL shows apoptotic cells in normal human keratinocytes after UVB radiation with a peak at 48 h. These data indicate that UVB can act on cellular membranes inducing sphingomyelin hydrolysis and ceramide production through both neutral and acidic SMases.

2000 - L'esposizione ai raggi UVB induce l'attivazione delle sfingomielinasi neutra e acida in cheratinociti umani normali in coltura. [Abstract in Atti di Convegno]
Euclidi, Emanuela; Magnoni, Cristina; A., Di Nardo; Benassi, Luisa; Bertazzoni, Giorgia; Vaschieri, Cristina; Seidenari, Stefania; Giannetti, Alberto
abstract

Il pathway della sfingomielina, ubiquitario e conservato dal punto di vista evoluzionistico, partecipa alla trasduzione del segnale dall’esterno all’interno della cellula. La sfingomielina (SM) è uno sfingolipide concentrato preferenzialmente nella porzione esterna della membrana citoplasmatica delle cellule di mammifero, ma presente anche nella parte interna della membrana in piccola quota dove sembra essere implicata nei processi di trasduzione del segnale. In questo sistema, lo stimolo di alcuni recettori presenti sulla superficie cellulare attiva una famiglia di enzimi specifici, denominati sfingomielinasi, che idrolizzano la SM generando fosfocolina e ceramide.Due tipi di sfingomielinasi (SMasi) sono coinvolte in questo processo ed agiscono a pH neutro o acido. La SMasi neutra idrolizza la sfingomilelina nell’ambiente extracellulare, mentre la SMasi acida agisce principalmente all’interno dei lisosomi.E’ stato osservato in diverse linee cellulari che uno dei primi eventi del processo apoptotico indotto da TNF-, o da radiazioni ionizzanti, é rappresentato da una rapida attivazione di entrambe le SMasi con conseguente produzione di ceramidi .Inoltre dati recenti dimostrano che il trattamento con analoghi dei ceramidi esogeni é sufficiente per l’ induzione dell’apoptosi nei cheratinociti umani normali.Lo scopo del presente studio è quello di valutare il ruolo delle SMasi neutra e acida e dei ceramidi nel segnale intracellulare generato dai raggi UVB in cheratinociti umani normali.Cheratinociti umani normali provenienti da interventi di chirurgia plastica sono stati coltivati su fibroblasti 3T3 mitomicinati con terreno di coltura Dulbecco ed Ham F12 addizionato del 10% di siero di vitello fetale. A subconfluenza le cellule sono state irradiate con UVB alla dose di 100mJ/ cm2 e raccolte a tempi per il dosaggio dei lipidi intracellulari e per il dosaggio in vitro dell’ attività enzimatica delle sfingomielinasi.Il dosaggio dei lipidi intracellulari tramite TLC ha evidenziato che la esposizione a UVB induce rapida idrolisi della sfingomielina e aumento della quota di ceramidi. L’aumento dei ceramidi é risultato significativamente evidente dopo 15 minuti dall’esposizione con UVB con incremento successivo fino alle 24h. Il dosaggio delle attività enzimatiche è stato effettuato usando come substrato sfingomielina marcata con C14. La attività della SMasi acida mostra un picco a 15 minuti dalla esposizione ai raggi UVB, mentre per la SMase neutra il picco era presente a 30 minuti.Dopo 48h il numero di cellule apoptotiche erano significativamente alte rispetto al controllo.Questi dati, indicano che i raggi ultravioletti possono agire sulla membrana cellulare inducendo idrolisi della SM e conseguente produzione di ceramidi attraverso la attivazione sia della SMasi acida che di quella neutra.

2000 - Sintesi e rilascio di Interleuchina 1 alfa in seguito all'esposizione di sodio lauril solfato in cheratinociti umani normali. [Abstract in Atti di Convegno]
Bertazzoni, Giorgia; Benassi, Luisa; Magnoni, Cristina; A., Di Nardo; Euclidi, Emanuela; Vaschieri, Cristina; Giannetti, Alberto; Seidenari, Stefania
abstract

La risposta cellulare agli irritanti si accompagna alla produzione ed al rilascio di numerosi mediatori infiammatori tra cui l’interleuchina 1alfa(IL-1alfa). Il rilascio di questa citochina può quindi essere studiato come marker di tossicità acuta indotta dai detergenti.Lo scopo di questo lavoro è stato valutare la sintesi e il rilascio di IL-1alfa da parte di cheratinociti umani normali in coltura dopo esposizione a Sodio Lauril Solfato (SLS).I cheratinociti, estratti da cute proveniente da interventi di chirurgia plastica, sono stati messi in coltura con un mezzo definito privo di siero. A subconfluenza, sono stati trattati con concentrazioni di SLS variabili da 0,00001 a 0,01%. Dopo un’ora il mezzo di coltura è stato cambiato e a tempi differenti sono stati raccolti i sovranatanti per l’esecuzione del test ELISA e i lisati cellulari per l’analisi in Western Blot e Northern Blot.Dai risultati ottenuti è emerso che l’esposizione a SLS induce il rilascio di IL-1alfa nel sovranatante in modo dose-dipendente, raggiungendo il valore massimo alla concentrazione di 0,0025%, (corrispondente all’IC50). Il rilascio di IL-1alfa è risultato inoltre aumentato già dopo 15 min dal trattamento con valori massimi alle 24 ore.I livelli intracellulari di IL-1alfa diminuiscono nella prima ora dopo il trattamento, per poi aumentare e raggiungere valori significativamente alti a partire dalle 12 ore.La cinetica della sintesi di mRNA mostra un andamento bifasico, evidenziando un aumento immediato al termine dell’esposizione all’SLS e un secondo picco a 12 ore.

1999 - Nerve growth factor protects human keratinocytes from ultraviolet-B-induced apoptosis [Articolo su rivista]
Marconi, Alessandra; Vaschieri, Cristina; S., Zanoli; Giannetti, Alberto; Pincelli, Carlo
abstract

Ultraviolet radiation is a potent inducer of apoptosis, whereas autocrine nerve growth factor protects human keratinocytes from programmed cell death. To evaluate the role of nerve growth factor in the mechanisms of ultraviolet B-induced apoptosis, cultured human keratinocytes were ultraviolet B irradiated following pretreatment with K252, a specific inhibitor of the tyrosine kinase high-affinity nerve growth factor receptor. Here we report that the addition of K252 significantly enhanced keratinocyte apoptosis. We then transfected normal human keratinocytes with pNUT-hNGF, Nerve growth factor overexpressing keratinocytes secreted the highest amounts of nerve growth factor in culture supernatants, were more viable, and had a higher rate of proliferation than mock-transfected cells. Whereas ultraviolet B radiation downregulated nerve growth factor mRNA and protein as well as the tyrosine kinase high-affinity nerve growth factor receptor in normal keratinocytes, it failed to do so in nerve growth factor-transfected cells. Moreover, nerve growth factor overexpressing keratinocytes were partially resistant to apoptosis induced by increasing doses of ultraviolet B at 24 and 48 h. These results indicate that downregulation of nerve growth factor function plays an important part in the mechanisms of ultraviolet B-induced apoptosis in human keratinocytes. In addition, ultraviolet B caused a decrease in BCL-2 and BCL-x(L) expression in mock-transfected keratinocytes, but not in nerve growth factor overexpressing cells. Finally, nerve growth factor prevented the cleavage of the enzyme poly(ADP-ribose) polymerase induced in human keratinocytes by ultraviolet B, These results are consistent with a model whereby the autocrine nerve growth factor protects human keratinocytes from ultraviolet B-induced apoptosis by maintaining constant levels of BCL-2 and BCL-x(L), which in turn might block caspase activation.

1998 - The role of nerve growth factor in ultraviolet radiation-induced cell cycle arrest and apoptosis of normal human keratinocytes. [Abstract in Rivista]
Marconi, Alessandra; Vaschieri, Cristina; Giannetti, Alberto; Pincelli, Carlo
abstract

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1997 - Ultraviolet B-induced down-regulation of nerve growth factor and its high-affinity receptor (trk) mRNAs in human keratinocytes. [Abstract in Rivista]
Marconi, Alessandra; C., Chiodino; Vaschieri, Cristina; Giannetti, Alberto; Pincelli, Carlo
abstract

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1996 - Ultraviolet B radiations down regulate nerve growth factor mRNA and release in human keratinocytes. [Abstract in Rivista]
Marconi, Alessandra; C., Chiodino; Vaschieri, Cristina; Giannetti, Alberto; Pincelli, Carlo
abstract

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1995 - Transforming Growth-Factor-Beta-1 Modulates Beta-1 And Beta-5 Integrin Receptors And Induces The De-Novo Expression Of The Alpha-V-Beta-6 Heterodimer In Normal Human Keratinocytes - Implications For Wound-Healing. [Articolo su rivista]
G., Zambruno; P. C., Marchisio; Marconi, Alessandra; Vaschieri, Cristina; A., Melchiori; Giannetti, Alberto; DE LUCA, Michele
abstract

The molecular mechanism underlying the promotion of wound healing by TGF-beta 1 is incompletely understood. We report that TGF-beta 1 regulates the regenerative/migratory phenotype of normal human keratinocytes by modulating their integrin receptor repertoire. In growing keratinocyte colonies but not in fully stratified cultured epidermis, TGF-beta 1: (a) strongly upregulates the expression of the fibronectin receptor alpha 5 beta 1, the vitronectin receptor alpha v beta 5, and the collagen receptor alpha 2 beta 1 by differentially modulating the synthesis of their alpha and beta subunits; (b) downregulates the multifunctional alpha 3 beta 1 heterodimer; (c) induces the de novo expression and surface exposure of the alpha v beta 6 fibronectin receptor; (d) stimulates keratinocyte migration toward fibronectin and vitronectin; (e) induces a marked perturbation of the general mechanism of polarized domain sorting of both beta 1 and beta 4 dimers; and (f) causes a pericellular redistribution of alpha v beta 5. These data suggest that alpha 5 beta 1, alpha v beta 6, and alpha v beta 5, not routinely used by keratinocytes resting on an intact basement membrane, act as ''emergency'' receptors, and uncover at least one of the molecular mechanisms responsible for the peculiar integrin expression in healing human wounds. Indeed, TGF-beta 1 reproduces the integrin expression pattern of keratinocytes located at the injury site, particularly of cells in the migrating epithelial tongue at the leading edge of the wound. Since these keratinocytes are inhibited in their proliferative capacity, these data might account for the apparent paradox of a TGF-beta 1-dependent stimulation of epidermal wound healing associated with a growth inhibitory effect on epithelial cells.

1994 - Expression, regulation and function of B7 costimulatory molecule on human epidermal Langerhans cells. [Articolo su rivista]
G., Girolomoni; V., Zacchi; Marconi, Alessandra; Vaschieri, Cristina; G., Zambruno
abstract

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