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Alessandra MARCONI
Professore Associato
Dipartimento Chirurgico, Medico, Odontoiatrico e di Scienze Morfologiche con interesse Trapiantologico, Oncologico e di Medicina Rigenerativa
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Pubblicazioni
2024
- 3D human foreskin model for testing topical formulations of sildenafil citrate
[Articolo su rivista]
Magnano, Greta Camilla; Quadri, Marika; Palazzo, Elisabetta; Lotti, Roberta; Loschi, Francesca; Dall'Acqua, Stefano; Abrami, Michela; Larese Filon, Francesca; Marconi, Alessandra; Hasa, Dritan
abstract
: Sildenafil citrate is an approved drug used for the treatment of erectile dysfunction and premature ejaculation. Despite a widespread application, sildenafil citrate shows numerous adverse cardiovascular effects in high-risk patients. Local transdermal drug delivery of this drug is therefore being explored as an interesting and noninvasive alternative administration method that avoids adverse effects arised from peak plasma drug concentrations. Although human and animal skin represents the most reliable models to perform penetration studies, they involve a series of ethical issues and restrictions. For these reasons new in vitro approaches based on artificially reconstructed human skin or "human skin equivalents" are being developed as possible alternatives for transdermal testing. There is little information, however, on the efficiency of such new in vitro methods on cutaneous penetration of active ingredients. The objective of the current study was to investigate the sildenafil citrate loaded in three commercial transdermal vehicles using 3D full-thickness skin equivalent and compare the results with the permeability experiments using porcine skin. Our results demonstrated that, while the formulation plays an imperative role in an appropriate dermal uptake of sildenafil citrate, the D coefficient results obtained by using the 3D skin equivalent are comparable to those obtained by using the porcine skin when a simple drug suspension is applied (1.17 × 10-10 ± 0.92 × 10-10 cm2/s vs 3.5 × 102 ± 3.3 × 102 cm2/s), suggesting that in such case, this 3D skin model can be a valid alternative for ex-vivo skin absorption experiments.
2023
- Blocking soluble Fas Ligand ameliorates pemphigus: PC111 efficacy in ex-vivo human pemphigus models
[Articolo su rivista]
Lotti, Roberta; Hundt, Jennifer E; Ludwig, Ralf J; Bennett, Brydon; Amato, Antonino; Marconi, Alessandra; Pincelli, Carlo
abstract
: Pemphigus is a life-threatening, chronic, autoimmune bullous disease affecting both the skin and the mucous membranes. Based on the mainstream concept that blister formation occurs upon binding of autoantibodies to their antigen proteins (desmoglein1, DSG1 and desmoglein3, DSG3), current therapies mostly aim to suppress the immune system. To avoid the severe side effects associated with the chronic use of immunosuppressive treatments, we have developed PC111, a fully human monoclonal antibody targeting human Fas ligand (FasL). We have provided a number of in vitro and in vivo evidences showing that soluble FasL induces keratinocyte apoptosis followed by acantholysis. An anti-murine FasL prevents blister formation in the pemphigus neonatal mouse model. To confirm the mechanism of action (MoA) and the efficacy of PC111 in a human pemphigus context, we used the keratinocyte dissociation assay and two independent Human Skin Organ Cultures (HSOC) pemphigus models. PC111 reduced acantholysis in vitro, as shown by the dose-dependent reduction of fragments in the monolayer cultures. In the first HSOC model, normal human skin was subcutaneously injected with a scFv antibody fragment directed against DSG1 and DSG3, resulting in a severe acantholysis (70-100%) after 24 hours. PC111 inhibited blister formation to around 50% of control. In the second model, normal human skin was injected with a mixture of pemphigus patients' autoantibodies resulting in a less severe acantholysis (20-30%). PC111 significantly suppressed blister formation to more than 75% up to 72 hours. These results confirm PC111 MoA and demonstrates the efficacy of the anti-FasL antibody also in a pemphigus setting.
2023
- CD271 activation prevents low to high-risk progression of cutaneous squamous cell carcinoma and improves therapy outcomes
[Articolo su rivista]
Quadri, Marika; Tiso, Natascia; Musmeci, Francesco; I Morasso, Maria; R Brooks, Stephen; REGGIANI BONETTI, Luca; Panini, Rossana; Lotti, Roberta; Marconi, Alessandra; Pincelli, Carlo; Palazzo, Elisabetta
abstract
2022
- Activation of cGMP-Dependent Protein Kinase Restricts Melanoma Growth and Invasion by Interfering with the EGF/EGFR Pathway
[Articolo su rivista]
Quadri, M.; Comitato, A.; Palazzo, E.; Tiso, N.; Rentsch, A.; Pellacani, G.; Marconi, A.; Marigo, V.
abstract
Drug resistance mechanisms still characterize metastatic melanoma, despite the new treatments that have been recently developed. Targeting of the cGMP/protein kinase G pathway is emerging as a therapeutic approach in cancer research. In this study, we evaluated the anticancer effects of two polymeric-linked dimeric cGMP analogs able to bind and activate protein kinase G, called protein kinase G activators (PAs) 4 and 5. PA5 was identified as the most effective compound on melanoma cell lines as well as on patient-derived metastatic melanoma cells cultured as three-dimensional spheroids and in a zebrafish melanoma model. PA5 was able to significantly reduce cell viability, size, and invasion of melanoma spheroids. Importantly, PA5 showed a tumor-specific outcome because no toxic effect was observed in healthy melanocytes exposed to the cGMP analog. We defined that by triggering protein kinase G, PA5 interfered with the EGF pathway as shown by lower EGFR phosphorylation and reduction of activated, phosphorylated forms of protein kinase B and extracellular signal‒regulated kinase 1/2 in melanoma cells. Finally, PA5 significantly reduced the metastatic process in zebrafish. These studies open future perspectives for the cGMP analog PA5 as a potential therapeutic strategy for melanoma.
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.
2022
- Investigating Cutaneous Squamous Cell Carcinoma in vitro and in vivo: Novel 3D Tools and Animal Models
[Articolo su rivista]
Quadri, Marika; Marconi, Alessandra; Sandhu, Simran K; Kiss, Alexi; Efimova, Tatiana; Palazzo, Elisabetta
abstract
Cutaneous Squamous Cell Carcinoma (cSCC) represents the second most common type of skin cancer, which incidence is continuously increasing worldwide. Given its high frequency, cSCC represents a major public health problem. Therefore, to provide the best patients' care, it is necessary having a detailed understanding of the molecular processes underlying cSCC development, progression, and invasion. Extensive efforts have been made in developing new models allowing to study the molecular pathogenesis of solid tumors, including cSCC tumors. Traditionally, in vitro studies were performed with cells grown in a two-dimensional context, which, however, does not represent the complexity of tumor in vivo. In the recent years, new in vitro models have been developed aiming to mimic the three-dimensionality (3D) of the tumor, allowing the evaluation of tumor cell-cell and tumor-microenvironment interaction in an in vivo-like setting. These models include spheroids, organotypic cultures, skin reconstructs and organoids. Although 3D models demonstrate high potential to enhance the overall knowledge in cancer research, they lack systemic components which may be solved only by using animal models. Zebrafish is emerging as an alternative xenotransplant model in cancer research, offering a high-throughput approach for drug screening and real-time in vivo imaging to study cell invasion. Moreover, several categories of mouse models were developed for pre-clinical purpose, including xeno- and syngeneic transplantation models, autochthonous models of chemically or UV-induced skin squamous carcinogenesis, and genetically engineered mouse models (GEMMs) of cSCC. These models have been instrumental in examining the molecular mechanisms of cSCC and drug response in an in vivo setting. The present review proposes an overview of in vitro, particularly 3D, and in vivo models and their application in cutaneous SCC research.
2022
- Isolation of an "early" transit amplifying keratinocyte population in human epidermis: a role for the low affinity neurotrophin receptor CD271
[Articolo su rivista]
Lotti, Roberta; Palazzo, Elisabetta; Quadri, Marika; Dumas, Marc; Schnebert, Sylvianne; Biondini, Diego; Bianchini, Maria Anastasia; Nizard, Carine; Pincelli, Carlo; Marconi, Alessandra
abstract
In the interfollicular epidermis (IFE), stem cells (KSC) generate transit amplifying (TA) cells that, after symmetric divisions, produce differentiating daughters. Here, we isolated and characterized the highly proliferative interfollicular epidermal basal cell population "early" TA (ETA) cells, based on their capacity to adhere to type IV collagen. Proliferation and colony-forming efficiency in ETA cells are lower than in KSC but higher than in "late" TA (LTA). Stemness, proliferation, and differentiation markers confirmed that ETA cells display a unique phenotype. Skin reconstructs derived from ETA cells present different features (epidermal thickness, Ki67, and Survivin expression), as compared to skin equivalents generated from either KSC or LTA cells. The low-affinity neurotrophin receptor CD271, which regulates the KSC to TA cell transition in the human epidermis through an on/off switch control mechanism, is predominantly expressed in ETA cells. Skin equivalents generated from siRNA CD271 ETA cells display a more proliferative and less differentiated phenotype, as compared to mock-derived reconstructs. Consistently, CD271 overexpression in LTA cells generates a more proliferative skin equivalent than mock LTA cells. Finally, the CD271 level declines with cellular senescence, while it induces a delay in p16INK4 expression. We conclude that ETA cells represent the first KSC progenitor with exclusive features. CD271 identifies and modulates ETA cells, thus participating in the early differentiation and regenerative capacity of the human epidermis.
2021
- A Novel Multi-Action Emollient Plus Cream Improves Skin Barrier Function in Patients with Atopic Dermatitis: In vitro and Clinical Evidence
[Articolo su rivista]
Quadri, Marika; Lotti, Roberta; Bonzano, Laura; Ciardo, Silvana; Guanti, Mario Bruno; Pellacani, Giovanni; Pincelli, Carlo; Marconi, Alessandra
abstract
Emollients capable of restoring the skin barrier function would extend their role beyond basic maintenance therapy in atopic dermatitis (AD).
2021
- Promoter Methylation Leads to Decreased ZFP36 Expression and Deregulated NLRP3 Inflammasome Activation in Psoriatic Fibroblasts
[Articolo su rivista]
Bertesi, M.; Fantini, S.; Alecci, C.; Lotti, R.; Martello, A.; Parenti, S.; Carretta, C.; Marconi, A.; Grande, A.; Pincelli, C.; Zanocco Marani, T.
abstract
The mRNA-destabilizing protein tristetraprolin (TTP), encoded by the ZFP36 gene, is known to be able to end inflammatory responses by directly targeting and destabilizing mRNAs encoding pro-inflammatory cytokines. We analyzed its role in psoriasis, a disease characterized by chronic inflammation. We observed that TTP is downregulated in fibroblasts deriving from psoriasis patients compared to those deriving from healthy individuals and that psoriatic fibroblasts exhibit abnormal inflammasome activity compared to their physiological counterpart. This phenomenon depends on TTP downregulation. In fact, following restoration, TTP is capable of directly targeting for degradation NLRP3 mRNA, thereby drastically decreasing inflammasome activation. Moreover, we provide evidence that ZFP36 undergoes methylation in psoriasis, by virtue of the presence of long stretches of CpG dinucleotides both in the promoter and the coding region. Besides confirming that a perturbation of TTP expression might underlie the pathogenesis of psoriasis, we suggest that deregulated inflammasome activity might play a role in the disease alongside deregulated cytokine expression.
2021
- Specific activation of the CD271 intracellular domain in combination with chemotherapy or targeted therapy inhibits melanoma progression
[Articolo su rivista]
Saltari, Annalisa; Dzung, Andreas; Quadri, Marika; Tiso, Natascia; Facchinello, Nicola; Hernandez-Barranco, Alberto; Garcia-Silva, Susana; Nogués, Laura; Stoffel, Corinne Isabelle; Cheng, Phil F; Turko, Patrick; Eichhoff, Ossia M; Truzzi, Francesca; Marconi, Alessandra; Pincelli, Carlo; Peinado, Héctor; Dummer, Reinhard; Levesque, Mitchell P
abstract
CD271 (NGFR) is a neurotrophin receptor that belongs to the tumor necrosis receptor (TNFR) family. Upon ligand binding, CD271 can mediate either survival or cell death. While the role of CD271 as a marker of tumor-initiating cells is still a matter of debate, its role in melanoma progression has been well documented. Moreover, CD271 has been shown to be upregulated after exposure to both chemotherapy and targeted therapy. In this study, we demonstrate that activation of CD271 by a short β-amyloid-derived peptide (Aβ(25-35)) in combination with either chemotherapy or MAPK inhibitors induces apoptosis in 2D and 3D cultures of 8 melanoma cell lines. This combinatorial treatment significantly reduced metastasis in a zebrafish xenograft model and led to significantly decreased tumor volume in mice. Administration of Aβ(25-35) in ex vivo tumors from immunotherapy- and targeted therapy-resistant patients significantly reduced proliferation of melanoma cells, showing that activation of CD271 can overcome drug resistance. Aβ(25-35) was specific to CD271-expressing cells and induced CD271 cleavage and phosphorylation of JNK (pJNK). The direct protein-protein interaction of pJNK with CD271 led to PARP1 cleavage, p53 and caspase activation, and pJNK-dependent cell death. Aβ(25-35) also mediated mitochondrial reactive oxygen species (mROS) accumulation, which induced CD271 overexpression. Finally, CD271 upregulation inhibited mROS production, revealing the presence of a negative feedback loop in mROS regulation. These results indicate that targeting CD271 can activate cell death pathways to inhibit melanoma progression and potentially overcome resistance to targeted therapy.
2020
- Wnt/CTNNB1 signal transduction pathway inhibits the expression of ZFP36 in squamous cell carcinoma, by inducing transcriptional repressors SNAI1, SLUG and TWIST
[Articolo su rivista]
Zanfi, E. D.; Fantini, S.; Lotti, R.; Bertesi, M.; Marconi, A.; Grande, A.; Manfredini, R.; Pincelli, C.; Zanocco-Marani, T.
abstract
The Wnt/CTNNB1 pathway is often deregulated in epithelial tumors. The ZFP36 gene, encoding the mRNA binding protein Tristetraprolin (TTP), is downregulated in several cancers, where it has been described to behave as a tumor suppressor. By this report, we show that Wnt/CTNNB1 pathway is constitutively activated, and ZFP36 expression is downregulated in Squamous Cell Carcinoma (SCC) cell lines compared to normal keratinocytes. Moreover, we suggest that the decrease of ZFP36 expression might depend on the activity of transcriptional repressors SNAI1, SLUG and TWIST, whose expression is induced by Wnt/CTNNB1, highlighting a potential regulatory mechanism underlying ZFP36 downregulation in epithelial cancers.
2019
- Apremilast Normalizes Gene Expression of Inflammatory Mediators in Human Keratinocytes and Reduces Antigen-Induced Atopic Dermatitis in Mice
[Articolo su rivista]
Schafer, Peter H; Adams, Mary; Horan, Gerald; Truzzi, Francesca; Marconi, Alessandra; Pincelli, Carlo
abstract
Apremilast, an oral phosphodiesterase (PDE) 4 inhibitor, has demonstrated efficacy in psoriasis, while its efficacy in atopic dermatitis (AD) was found to be modest. AD is a chronic inflammatory skin disease associated with activation of T helper (Th) 2 and Th17 immunity and a compromised epidermal barrier.
2019
- Development of a Desmocollin-3 Active Mouse Model Recapitulating Human Atypical Pemphigus
[Articolo su rivista]
Lotti, Roberta; Atene, Claudio Giacinto; Marconi, Alessandra; Di Rocco, Giulia; Reggiani Bonetti, L; Zanocco Marani, Tommaso; Pincelli, Carlo
abstract
Pemphigus vulgaris (PV) is a life-threatening mucocutaneous autoimmune blistering disease. It is often associated with autoantibodies to the desmosomal adhesion proteins Desmoglein 3 (DSG3) and Desmoglein 1 (DSG1). Recently, auto-antigens, such as desmocollins and others have been described in PV and in atypical pemphigus forms such as Pemphigus Herpetiformis (PH), Pemphigus Vegetans (PVeg), and Paraneoplastic Pemphigus (PP). Desmocollins belong to a cadherin subfamily that provides structure to the desmosomes and play an important role in cell-to-cell adhesion. In order to verify the pathogenic activity of anti-Desmocollin 3 (DSC3) antibodies, we developed an active disease model of pemphigus expressing anti-DSC3 autoantibodies or antiDSC3 and anti-DSG3 antibodies. This approach included the adoptive transfer of DSC3 and/or DSG3 lymphocytes to Rag2(-/-) immunodeficient mice that express DSC3 and DSG3. Our results show that the presence of anti-DSC3 auto-antibodies is sufficient to determine the appearance of a pathological phenotype relatable to pemphigus, but with features not completely super-imposable to those observed in the DSG3 active model, suggesting that the DSC3 active model might mimic the atypical pemphigus. Moreover, the presence of both anti-DSC3 and anti-DSG3 antibodies determines a more severe phenotype and a slower response to prednisolone. In conclusion, we have developed an adult DSC3 pemphigus mouse model that differs from the DSG3 model and supports the concept that antigens other than desmogleins may be responsible for different phenotypes in human pemphigus.
2019
- Do DLX3 and CD271 Protect Human Keratinocytes from Squamous Tumor Development?
[Articolo su rivista]
Palazzo, Elisabetta; Marconi, Alessandra; Pincelli, Carlo; Morasso, Maria I
abstract
Well-regulated epidermal homeostasis depends on the function of different classes of factors, such as transcription regulators and receptors. Alterations in this homeostatic balance may lead to the development of cutaneous squamous tumorigenesis. The homeobox transcription factor DLX3 is determinant for a p53-dependent regulation of epidermal differentiation and modulates skin carcinogenesis. The maintenance of skin homeostasis also involves the action of neurotrophins (NTs) and their receptors, Trk and CD271. While Trk receptor overexpression is a hallmark of cancer, there are conflicting data on CD271 expression and function in cutaneous SCC (cSCC). Previous studies have reported NT receptors expression in head and neck SSC (HNSCC). We show that CD271 is expressed at low levels in primary cSCC cells and the number of CD271+ cells correlates with cell cohesion in SCC spheroids. In normal epidermis, CD271 is expressed in proliferative progenitor cells and DLX3 in terminally differentiated keratinocytes. Brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) increase DLX3 expression. In the absence of a functional BDNF receptor TrkB in keratinocytes, we hypothesize that the BDNF-dependent DLX3 response could be mediated via CD271. Altogether, our results support a putative CD271-DLX3 connection in keratinocytes, which might be crucial to preventing squamous skin cancer.
2018
- Progress in melanoma modeling in vitro
[Articolo su rivista]
Marconi, Alessandra; Quadri, Marika; Saltari, Annalisa; Pincelli, Carlo
abstract
Melanoma is one of the most studied neoplasia, although laboratory techniques used for investigating this tumor are not fully reliable. Animal models may not predict the human response due to differences in skin physiology and immunity. In addition, international guidelines recommend to develop processes that contribute to the reduction, refinement and replacement of animals for experiments (3Rs). Adherent cell culture has been widely used for the study of melanoma to obtain important information regarding melanoma biology. Nonetheless, these cells grow in adhesion on the culture substrate which differs considerably from the situation in vivo. Melanoma grows in a 3D spatial conformation where cells are subjected to a heterogeneous exposure to oxygen and nutrient. In addition, cell-cell and cell-matrix interaction play a crucial role in the pathobiology of the tumor as well as in the response to therapeutic agents. To better study melanoma new techniques, including spherical models, tumorospheres, and melanoma skin equivalents have been developed. These 3D models allow to study tumors in a microenvironment that is more close to the in vivo situation, and are less expensive and time consuming than animal studies. This review will also describe the new technologies applied to skin reconstructs such as organ-on-a-chip that allows skin perfusion through microfluidic platforms. 3D in vitro models, based on the new technologies, are becoming more sophisticated, representing at a great extent the in vivo situation, the "perfect" model that will allow less involvement of animals up to their complete replacement, is still far from being achieved. This article is protected by copyright. All rights reserved.
2018
- Soluble Fas Ligand is essential for blister formation in Pemphigus
[Articolo su rivista]
Lotti, Roberta; Shu, En; Petrachi, Tiziana; Marconi, Alessandra; Palazzo, Elisabetta; Quadri, Marika; Lin, Ann; O’Reillyan, Lorraine A.; Pincelli, Carlo
abstract
Pemphigus is a blistering disease characterized by pemphigus autoantibodies (PVIgG)
directed mostly against desmogleins (Dsgs), resulting in the loss of keratinocyte adhesion
(acantholysis). Yet, the mechanisms underlying blister formation remain to be
clarified. We have shown previously that anti-Fas ligand (FasL) antibody (Ab) prevents
PVIgG-induced caspase-8 activation and Dsg cleavage in human keratinocytes, and
that sera from pemphigus patients contain abnormally increased levels of FasL. Here,
we demonstrate that recombinant FasL induces the activation of caspases prior to Dsg
degradation, and anti-FasL Ab prevents acantholysis in cultured keratinocytes. Moreover,
the silencing of FasL reduces PVIgG-induced caspase-8 activation and Dsg3 cleavage.
Following injection of PVIgG into mice, FasL is upregulated at 1–3 h and is followed by
caspase-8-mediated keratinocyte apoptosis, before blister formation. The administration
of anti-FasL Ab after PVIgG injection blocks blister formation in mice. Furthermore, we
injected PVIgG into two different gene-targeted mutant mice that selectively lack either
secreted soluble FasL (sFasL), FasLΔs/Δs mice, or the membrane-bound form of FasL
(mFasL), FasLΔm/Δm mice. After PVIgG treatment, blisters are only visible in FasLΔm/Δm animals,
lacking mFasL, but still producing sFasL, similar to wild-type (C57BL/6) animals.
By contrast, a significant decrease in the relative acantholytic area is observed in the
FasLΔs/Δs animals. These results demonstrate that soluble FasL plays a crucial role in the
mechanisms of blister formation, and blockade of FasL could be an effective therapeutic
approach for pemphigus.
2016
- CD271 Down-Regulation Promotes Melanoma Progression and Invasion in Three-Dimensional Models and in Zebrafish
[Articolo su rivista]
Saltari, Annalisa; Truzzi, Francesca; Quadri, Marika; Lotti, Roberta; Palazzo, Elisabetta; Grisendi, Giulia; Tiso, Natascia; Marconi, Alessandra; Pincelli, Carlo
abstract
CD271 is a neurotrophin receptor variably expressed in melanoma. Although contradictory data are reported on its role as a marker of tumor-initiating cells, little is known about its function in tumor progression. CD271 expression was higher in spheroids derived from freshly isolated cells of primary melanomas and in primary WM115 and WM793-B cell lines, and it decreased during progression to advanced stages in cells isolated from metastatic melanomas and in metastatic WM266-4 and 1205Lu cell lines. Moreover, CD271 was scarcely detected in the highly invasive spheroids (SKMEL28 and 1205Lu). CD271, originally expressed in the epidermis of skin reconstructs, disappeared when melanoma started to invade the dermis. SKMEL8 CD271(-) cells showed greater proliferation and invasiveness in vitro and were associated with a higher number of metastases in zebrafish compared with CD271(+) cells. CD271 silencing in WM115 induced a more aggressive phenotype in vitro and in vivo. On the contrary, CD271 overexpression in SKMEL28 cells reduced invasion in vitro, and CD271 overexpressing 1205Lu cells was associated with a lower percentage of metastases in zebrafish. A reduced cell-cell adhesion was also observed in the absence of CD271. Taken together, these results indicate that CD271 loss is critical for melanoma progression and metastasis.
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
- Phosphodiesterase 4 in inflammatory diseases: Effects of apremilast in psoriatic blood and in dermal myofibroblasts through the PDE4/CD271 complex
[Articolo su rivista]
Schafer, Peter H; Truzzi, Francesca; Parton, Anastasia; Wu, Lei; Kosek, Jolanta; Zhang, Ling Hua; Horan, Gerald; Saltari, Annalisa; Quadri, Marika; Lotti, Roberta; Marconi, Alessandra; Pincelli, Carlo
abstract
Phosphodiesterases 4 (PDE4) act as proinflammatory enzymes via degradation of cAMP, whereas PDE4 inhibitors play an anti-inflammatory role in vitro and in vivo. In particular, apremilast has been recently approved for the treatment of psoriasis and psoriatic arthritis. However, little is known on the expression pattern of PDE4 in psoriasis. We report that PDE4B and PDE4D mRNA are overexpressed in peripheral blood mononuclear cells (PBMC) from psoriasis, as compared with normal controls, while apremilast reduces PBMC production of a number of pro-inflammatory cytokines and increases the levels of anti-inflammatory mediators. PDE4 expression is up-regulated in psoriatic dermis as compared with normal skin, with particular regard to fibroblasts. This is confirmed in vitro, where both dermal fibroblasts (DF) and, to a greater extent, myofibroblasts (DM) express all PDE4 isoforms at the mRNA and protein level. Because PDE4 interacts with the nerve growth factor (NGF) receptor CD271 in lung fibroblasts, we evaluated the relationship and function of PDE4 and CD271 in normal human skin fibroblasts. All PDE4 isoforms co-immunoprecipitate with CD271 in DM, while apremilast inhibits apoptosis induced by β-amyloid, a CD271 ligand, in DM. Furthermore, apremilast significantly reduces NGF- and transforming growth factor-β1 (TGF-β1)-induced fibroblast migration, and inhibits DF differentiation into DM mediated by NGF or TGF-β1. Finally, in DM, apremilast significantly reduces cAMP degradation induced by treatment with β-amyloid. Taken together, these results indicate that PDE4 play an important role in psoriasis. In addition, the study reveals that the PDE4/CD271 complex could be important in modulating fibroblast functions.
2016
- Survivin modulates squamous cell carcinoma-derived stem-like cell proliferation, viability and tumor formation in vivo
[Articolo su rivista]
Lotti, Roberta; Palazzo, Elisabetta; Petrachi, Tiziana; Dallaglio, Katiuscia; Saltari, Annalisa; Truzzi, Francesca; Quadri, Marika; Puviani, Mario; Maiorana, Antonino; Marconi, Alessandra; Pincelli, Carlo
abstract
Squamous Cell Carcinoma-derived Stem-like Cells (SCC-SC) originate from alterations in keratinocyte stem cells (KSC) gene expression and sustain tumor development, invasion and recurrence. Since survivin, a KSC marker, is highly expressed in SCC-SC, we evaluate its role in SCC-SC cell growth and SCC models. Survivin silencing by siRNA decreases clonal growth of SCC keratinocytes and viability of total, rapidly adhering (RAD) and non-RAD (NRAD) cells from primary SCC. Similarly, survivin silencing reduces the expression of stem cell markers (OCT4, NOTCH1, CD133, β1-integrin), while it increases the level of differentiation markers (K10, involucrin). Moreover, survivin silencing improves the malignant phenotype of SCC 3D-reconstruct, as demonstrated by reduced epidermal thickness, lower Ki-67 positive cell number, and decreased expression of MMP9 and psoriasin. Furthermore, survivin depletion by siRNA in RasG12V-IκBα-derived tumors leads to smaller tumor formation characterized by lower mitotic index and reduced expression of the tumor-associated marker HIF1α, VEGF and CD51. Therefore, our results indicate survivin as a key gene in regulating SCC cancer stem cell formation and cSCC development.
2015
- CD271 Mediates Stem Cells to Early Progeny Transition in Human Epidermis
[Articolo su rivista]
Truzzi, Francesca; Saltari, Annalisa; Palazzo, Elisabetta; Lotti, Roberta; Petrachi, Tiziana; Dallaglio, Katiuscia; Gemelli, Claudia; Grisendi, Giulia; Dominici, Massimo; Pincelli, Carlo; Marconi, Alessandra
abstract
CD271 is the low-affinity neurotrophin (p75NTR) receptor that belongs to the tumor necrosis factor receptor superfamily. Because in human epidermis, CD271 is predominantly expressed in transit-amplifying (TA) cells, we evaluated the role of this receptor in keratinocyte differentiation and in the transition from keratinocyte stem cells (KSCs) to progeny. Calcium induced an upregulation of CD271 in subconfluent keratinocytes, which was prevented by CD271 small interfering RNA. Furthermore, CD271 overexpression provoked the switch of KSCs to TA cells, whereas silencing CD271 induced TA cells to revert to a KSC phenotype, as shown by the expression of β1-integrin and by the increased clonogenic ability. CD271(+) keratinocytes sorted from freshly isolated TA cells expressed more survivin and keratin 15 (K15) compared with CD271(-) cells and displayed a higher proliferative capacity. Early differentiation markers and K15 were more expressed in the skin equivalent generated from CD271(+) TA than from those derived from CD271(-) TA cells. By contrast, late differentiation markers were more expressed in skin equivalents from CD271(-) than in reconstructs from CD271(+) TA cells. Finally, skin equivalents originated from CD271(-) TA cells displayed a psoriatic phenotype. These results indicate that CD271 is critical for keratinocyte differentiation and regulates the transition from KSCs to TA cells.
2015
- Hypoxia-Inducible Factor-1α and CD271 inversely correlate with melanoma invasiveness
[Articolo su rivista]
Marconi, Alessandra; Borroni, Riccardo Giovanni; Truzzi, Francesca; Longo, Caterina; Pistoni, Francesca; Pellacani, Giovanni; Pincelli, Carlo
abstract
Melanoma is characterized, among other features, by microenvironmental factors and by an altered apoptotic machinery. Melanoma cell response to a hypoxic environment is transcriptionally regulated by the Hypoxia-Inducible Factor (HIF)-1α. p75 neurotrophin receptor (p75(NTR) ), also called CD271, mediates apoptosis in several cell systems. The purpose of this study was to analyze the expression of HIF-1α and CD271 in melanomas at different phases of progression, as evaluated by histology and reflectance confocal microscopy (RCM). By RCM, 41.67% tumors were characterized by the presence of a population of dendritic and pleomorphic cells (D+P), corresponding to in situ melanoma; 25% exhibited a predominantly round-cell (RN) proliferation with histologic features of superficial melanoma, and 33.33% showed the presence of cells aggregated in nests (DN), typical of invasive melanoma. HIF-1α was scarcely detected in D+P and in RN melanomas, while it was highly expressed in DN tumors. By contrast, CD271 positive cells were mostly detected in D+P population, and barely observed in the other subtypes. This work demonstrates that CD271 expression inversely correlates with hypoxia in melanoma, and that the two markers may be used in the future as diagnostic/prognostic tools for this neoplasm.
2015
- Notch cooperates with survivin to maintain stemness and to stimulate proliferation in human keratinocytes during ageing
[Articolo su rivista]
Palazzo, Elisabetta; Morandi, Paolo; Lotti, Roberta; Saltari, Annalisa; Truzzi, Francesca; Schnebert, Sylvianne; Dumas, Marc; Marconi, Alessandra; Pincelli, Carlo
abstract
The Notch signaling pathway orchestrates cell fate by either inducing cell differentiation or maintaining cells in an undifferentiated state. This study aims to evaluate Notch expression and function in normal human keratinocytes. Notch1 is expressed in all epidermal layers, though to a different degree of intensity, with a dramatic decrease during ageing. Notch1 intracellular domain (N1ICD) levels are decreased during transit from keratinocyte stem cells (KSC) to transit amplifying (TA) cells, mimicking survivin expression in samples from donors of all ages. Calcium markedly reduces N1ICD levels in keratinocytes. N1ICD overexpression induces the up-regulation of survivin and the down-regulation of keratin 10 and involucrin, while increasing the S phase of the cell cycle. On the other hand, Notch1 inhibition (DAPT) dose-dependently decreases survivin, stimulates differentiation, and reduces keratinocyte proliferation in samples from donors of all ages. Silencing Notch downgrades survivin and increases keratin 10. In addition, Notch1 inhibition decreases survivin levels and proliferation both in KSC and TA cells. Finally, while survivin overexpression decreases keratinocyte differentiation and increases N1ICD expression both in KSC and TA cells, silencing survivin results in N1ICD down-regulation and an increase in differentiation markers. These results suggest that the Notch1/survivin crosstalk contributes to the maintenance of stemness in human keratinocytes
2015
- Organ culture and Reflectance Confocal Microscopy as new integrated tools for barrier rescue studies in inflammatory skin diseases
[Articolo su rivista]
Petrachi, Tiziana; Lotti, Roberta; Palazzo, Elisabetta; Truzzi, Francesca; Saltari, Annalisa; Morandi, Paolo; Ciardo, Silvana; Pellacani, Giovanni; Pincelli, Carlo; Marconi, Alessandra
abstract
Here we present a new integrated approach to understand skin barrier recovery after physical (tape stripping, TS) or chemical (SDS) injury by combining human skin organ culture and Reflectance Confocal Microscopy (RCM). TS in vitro produced a complete removal of stratum corneum and lipids, a drastic decrease of structural and adhesion proteins, and an increase in cell proliferation. Epidermal recovery with either proliferation or differentiation rescue was observed after 18 hours, with no apoptotic cell detection. On the other hand, when skin organ cultures were exposed to 2% SDS, cellular junctions were disrupted and the expression of late differentiation markers decreased. Junctions repair was detected 24 hours after treatment, with the restoration of epidermal integrity. Both models (TP or SDS) showed the induction of immune-inflammatory markers, such as psoriasin, keratin 16, and the increase in Langerhans cell number. RCM confirmed all the morphological and structural features presented by the organ cultures, thus making this technique fast and easily applicable in the context of dermatological research. These results indicate that combination of skin organ models and RCM can be successfully used for the study of barrier perturbation in skin diseases, for toxicology tests, and for evaluating novel therapies.
2014
- Expression of nuclear survivin in normal skin and squamous cell carcinoma: a possible role in tumor invasion
[Articolo su rivista]
Dallaglio, Katiuscia; Petrachi, Tiziana; Marconi, Alessandra; Truzzi, Francesca; Lotti, Roberta; Saltari, Annalisa; Morandi, Paolo; M., Puviani; Maiorana, Antonino; Pincelli, Carlo
abstract
Background: Survivin is detected in few adult normal cells and it is highly expressed in cancer. Nuclear survivin facilitates cell cycle entry, while the mitochondrial pool protects cells from apoptosis. Survivin is overexpressed in keratinocyte stem cells (KSC) and protects them from apoptosis.
Methods: As KSC are at the origin of squamous cell carcinoma (SCC), we evaluated survivin expression in normal and cancerous skin in vivo by immunohistochemistry and western blotting. HaCaT cells overexpressing survivin and wound-healing assay are used. Anova and Student-T tests are used for statistical analysis.
Results: Survivin is localized both in the cytoplasm and in the nucleus of normal adult and young keratinocytes. Nuclear survivin is detected in one every 10/11 basal keratinocytes. When present in suprabasal cells, nuclear survivin is co-expressed with K10, but not with K15 or p75-neurotrophin-receptor (p75NTR), a transit amplifying cell marker. Nuclear, but not cytoplasmic survivin expression dramatically increases in actinic keratosis and in SCC in situ, as compared to normal epidermis, and it is highest in poorly differentiated SCC. In SCC tumors, nuclear survivin-positive cells are mainly K10/p75NTR-negative and K15-positive. In poorly differentiated tumors, survivin mostly localizes in the deep infiltrating areas. When overexpressed in keratinocytes, survivin increases cell migration.
Conclusion: High survivin expression and the subcellular localization of survivin correlate with keratinocyte differentiation and are associated with undifferentiated and more invasive SCC phenotype.
2014
- New modulated genes in psoriasis-derived keratinocyte subpopulations.
[Abstract in Rivista]
Lotti, Roberta; Tenedini, Elena; Fabiano, A; Truzzi, Francesca; Saltari, Annalisa; Morandi, Paolo; Marconi, Alessandra; Pincelli, Carlo
abstract
T cells play a crucial role in the pathogenesis of psoriasis, recent data emphasize the key role of keratinocytes that, by carrying intrinsic alterations, could determine the formation of skin lesions resembling psoriasis, without the participation of T-cell derived cytokines. In particular, transit amplifying (TA) cells, the stem cell progeny, seems to be responsible for the psoriatic phenotype. The aim of this study was to analyze the role of keratinocytes sub-populations in the pathogenesis of psoriasis. We analyzed the gene expression profile (GEP) of human keratinocyte sub-populations (stem, “early” TA (ETA) and “late” TA (LTA) cells), derived from healthy skin, lesional and non-lesional psoriatic skin. The total RNA samples, extracted from keratinocyte subpopulations immediately after separation, were hybridized onto the Affymetrix human U133 plus 2.0 GeneChip Array. We identified a small number of up-regulated genes (12 probe sets, corresponding to 8 genes) in keratinocyte subpopulations derived from lesional psoriasis vs. healthy and non-lesional psoriasis. We confirmed the increased expression levels of TCN1, S100A7A, KYNU, SERPINB13, FOXE1, but solely due to the keratinocyte component. We identified for the first time the up-regulation of TMEM171, CLEC7A and NDRG4, which seems to correlate with the pathophysiology of psoriasis. Moreover, GEP analysis of lesional psoriasis sub-populations, as compared to the non-lesional psoriatic counterpart revealed the modulation of 17 probe sets, corresponding to 13 genes. Among these genes, we recognized for the first time the up-regulation of IL13RA1, CCDC109B and CD47. These results indicate the importance of keratinocyte compartment in psoriasis, opening the way to the study of new genes potentially critical in the pathogenesis of psoriasis.
2013
- E-FABP induces differentiation in normal human keratinocytes and modulates the differentiation process in psoriatic keratinocytes in vitro.
[Articolo su rivista]
Dallaglio, Katiuscia; Marconi, Alessandra; Truzzi, Francesca; Lotti, Roberta; Palazzo, Elisabetta; Petrachi, Tiziana; Saltari, Annalisa; Coppini, Maurizio; Pincelli, Carlo
abstract
Epidermal fatty acid-binding protein (E-FABP) is a lipid carrier, originally discovered in human epidermis. We show that E-FABP is almost exclusively expressed in postmitotic (PM) keratinocytes, corresponding to its localization in the highest suprabasal layers, while it is barely expressed in keratinocyte stem cells (KSC) and transit amplifying (TA) keratinocytes. Transfection of normal human keratinocytes with recombinant (r) E-FABP induces overexpression of K10 and involucrin. On the other hand, E-FABP inhibition by siRNA downregulates K10 and involucrin expression in normal keratinocytes through NF-κB and JNK signalling pathways. E-FABP is highly expressed in psoriatic epidermis, and it is mainly localized in stratum spinosum. Psoriatic PM keratinocytes overexpress E-FABP as compared to the same population in normal epidermis. E-FABP inhibition in psoriatic keratinocytes markedly reduces differentiation, while it upregulates psoriatic markers such as survivin and K16. However, under high-calcium conditions, E-FABP silencing downregulates K10 and involucrin, while survivin and K16 expression is completely abolished. These data strongly indicate that E-FABP plays an important role in keratinocyte differentiation. Moreover, E-FABP modulates differentiation in psoriatic keratinocytes.
2013
- Isolation and Characterization of Squamous Cell Carcinoma-Derived Stem-like Cells: Role in Tumor Formation
[Articolo su rivista]
Dallaglio, Katiuscia; Petrachi, Tiziana; Marconi, Alessandra; Truzzi, Francesca; Lotti, Roberta; Saltari, Annalisa; Morandi, Paolo; M., Puviani; Maiorana, Antonino; D. R., Roop; Pincelli, Carlo
abstract
In human epidermis, keratinocyte stem cells (KSC) are characterized by high levels of β1-integrin, resulting in the rapid adhesion to type IV collagen. Since epithelial tumors originate from KSC, we evaluated the features of rapidly adhering (RAD) keratinocytes derived from primary human squamous cell carcinoma of the skin (cSCC).
RAD cells expressed higher levels of survivin, a KSC marker, as compared to non-rapidly adhering (NRAD) cells. Moreover, RAD cells proliferated to a greater extent and were more efficient in forming colonies than NRAD cells. RAD cells also migrated significantly
better than NRAD cells. When seeded in a silicone chamber and grafted onto the back skin of NOD SCID mice, RAD cells formed tumors 2–4 fold bigger than those derived from NRAD cells. In tumors derived from RAD cells, the mitotic index was significantly higher
than in those derived from NRAD cells, while Ki-67 and survivin expression were more pronounced in RAD tumors. This study suggests that SCC RAD stem cells play a critical role in the formation and development of epithelial tumors.
2013
- Keratinocyte Stem Cells: Biology and Clinical Applications
[Capitolo/Saggio]
Pincelli, Carlo; Marconi, Alessandra
abstract
Human epidermis represents a large reservoir of stem cells that continue to self-renew throughout life. Stem cells are essential for skin regeneration and for repair after wounding. They allow long-term culture of keratinocytes that produce large sheets of epidermis to cover extensive burns, thus being lifesaving for these patients. Furthermore, stem cells can be expanded in culture, genetically modified to correct the gene deficiency in genetic skin diseases. This chapter will describe the most recent data on stem cell biology and the potential medical applications of these cells.
2013
- Targeted Gene Addition in Human Epithelial
Stem Cells by Zinc-finger Nuclease-mediated
Homologous Recombination
[Articolo su rivista]
Coluccio, Andrea; Miselli, Francesca; Angelo, Lombardo; Marconi, Alessandra; Guidantonio Malagoli, Tagliazucchi; Manuel A., Gonçalves; Pincelli, Carlo; Giulietta, Maruggi; Marcela Del, Rio; Luigi, Naldini; Fernando, Larcher; Mavilio, Fulvio; Recchia, Alessandra
abstract
Preclinical and clinical studies showed that autologous
transplantation of epidermis derived from genetically
modified epithelial stem cells (EpSCs) leads to long-term
correction of inherited skin adhesion defects. These studies
were based on potentially genotoxic retroviral vectors.
We developed an alternative gene transfer strategy aimed
at targeting a “safe harbor” locus, the adeno-associated
virus integration site 1 (AAVS1), by zinc-finger nuclease
(ZFN)-induced homologous recombination (HR). Delivery
of AAVS1-specific ZFNs and a GFP-expressing HR cassette
by integration-defective lentiviral (LV) vectors (IDLVs) or
adenoviral (Ad) vectors resulted in targeted gene addition
with an efficiency of >20% in a human keratinocyte
cell line, >10% in immortalized keratinocytes, and <1%
in primary keratinocytes. Deep sequencing of the AAVS1
locus showed that ZFN-induced double-strand breaks are
mostly repaired by nonhomologous end joining (NHEJ)
in primary cells, indicating that poor induction of the
HR-dependent DNA repair pathway may be a significant
limitation for targeted gene integration. Skin equivalents
derived from unselected keratinocyte cultures coinfected
with a GFP-IDLV and a ZFN-Ad vector were grafted
onto immunodeficient mice. GFP-positive clones were
observed in all grafts up to 18 weeks post-transplantation.
By histological and molecular analysis, we were able
to demonstrate highly efficient targeting of the AAVS1
locus in human repopulating EpSCs.
2012
- A novel human psoriatic skin reconstruct: The role of transit amplifying keratinocytes and of p75 neurotrophin receptor
[Abstract in Rivista]
Truzzi, Francesca; Marconi, Alessandra; Lotti, Roberta; Dallaglio, Katiuscia; Palazzo, Elisabetta; Petrachi, Tiziana; Saltari, Annalisa; Pincelli, Carlo
abstract
.
2012
- Apoptotic pathways in the pathogenesis of pemphigus: targets for new therapies
[Articolo su rivista]
Lotti, Roberta; Marconi, Alessandra; Pincelli, Carlo
abstract
Pemphigus is a group of rare autoimmune blistering diseases of the skin in which autoantibodies to desmosome cadherins, desmogleins, induce loss of cell-cell adhesion (acantholysis). In addition to steric hindrance and activation of intracellular phosphorylation cascade signaling pathways, apoptosis has been suggested to contribute to the mechanism by which pathogenic IgG induces acantholysis. We review the literature examining the role of apoptosis in pemphigus. Current data recognize a central role of apoptosis in the mechanisms of blister induction. In particular, here we stress the key role of FasL in pemphigus, as it is able to first induce apoptosis, then acantholysis. Being pro-apoptotic molecules important in blister formation, they could represent new specific targets for pemphigus treatment.
2012
- Changes in survivin subcellular localization correlates with different stages of differentiation in normal and cancerous skin
[Abstract in Rivista]
K., Dallaglio; Petrachi, Tiziana; Marconi, Alessandra; Morandi, Paolo; Maiorana, Antonino; Pincelli, Carlo
abstract
.
2012
- E-FABP induces differentiation in normal and psoriatic keratinocytes
[Abstract in Rivista]
Dallaglio, Katiuscia; Marconi, Alessandra; Truzzi, Francesca; Lotti, Roberta; Palazzo, Elisabetta; Petrachi, Tiziana; Saltari, Annalisa; Pincelli, Carlo
abstract
.
2012
- PC111: a monoclonal anti-Fas Ligand antibody for the treatment of pemphigus
[Abstract in Rivista]
Lotti, Roberta; Marconi, Alessandra; D., Katiuscia; Truzzi, Francesca; Petrachi, Tiziana; Pincelli, Carlo
abstract
.
2012
- Role of neurotrophins on dermal fibroblast survival and differentiation
[Articolo su rivista]
Palazzo, Elisabetta; Marconi, Alessandra; Truzzi, Francesca; Dallaglio, Katiuscia; Petrachi, Tiziana; P., Humbert; S., Schnebert; E., Perrier; M., Dumas; Pincelli, Carlo
abstract
Neurotrophins (NTs) belong to a family of growth factors that play a critical role in the control of skin homeostasis. NTs act through the low-affinity receptor p75NTR and the high-affinity receptors TrkA, TrkB and TrkC. Here we show that dermal fibroblasts (DF) and myofibroblasts (DM) synthesize and secrete all NTs and express NT receptors. NTs induce differentiation of DF into DM, as shown by the expression of α-SMA protein. The Trk inhibitor K252a, TrkA/Fc, TrkB/Fc or TrkC/Fc chimera prevents DF and DM proliferation. In addition, p75NTR siRNA inhibits DF proliferation, indicating that both NT receptors mediate DF proliferation induced by endogenous NTs. Autocrine NTs also induce DF migration through p75NTR and Trk, as either silencing of p75NTR or Trk/Fc chimeras prevent this effect, in absence of exogenous NTs. Finally, NGF or BDNF statistically increase the tensile strength in a dose dependent manner, as measured in a collagen gel through the GlaSbox device. Taken together, these results indicate that NTs exert a critical role on fibroblast and could be involved in tissue remodelling and wound healing
2012
- Survivin: a dual player in healthy and diseased skin
[Articolo su rivista]
Dallaglio, Katiuscia; Marconi, Alessandra; Pincelli, Carlo
abstract
Survivin belongs to the inhibitor of apoptosis (IAP) protein family, and, in addition to the antiapoptotic functions, it also regulates the cell cycle. The survivin gene generates five major isoforms with diverse and opposite functions. Survivin is highly expressed in cancer and in few normal adult tissues, including skin. It is mostly detected in the nucleus of keratinocyte stem cells (KSCs), but it is also expressed in melanocytes and fibroblasts. Survivin isoforms are differentially detected in subpopulations of human keratinocytes, exerting contrasting activities. Survivin has an important role in the regulation of cell cycle in keratinocytes, and it protects these cells from anoikis and UV-induced apoptosis. In melanoma, survivin is abundantly expressed, and its subcellular localization varies depending upon tumor thickness and invasiveness. Survivin overexpression has been shown in squamous cell carcinoma (SCC), and it is also involved in UVB-induced carcinogenesis. The presence of survivin both in the nucleus and in the cytoplasm throughout the epidermal layers of psoriatic lesions suggests the involvement of this protein in the keratinocyte alterations typical of this disease. Additional studies on the expression of survivin isoforms and their subcellular localization in relation to function will confirm the key role of survivin in the skin and will open the field to new therapeutic strategies for many cutaneous conditions
2012
- The p75 neurotrophin receptor triggers the keratinocyte stem-transit amplifying cell transition in normal human epidermis
[Abstract in Rivista]
Marconi, Alessandra; Truzzi, Francesca; Lotti, Roberta; Dallaglio, Katiuscia; Palazzo, Elisabetta; Petrachi, Tiziana; Saltari, Annalisa; Pincelli, Carlo
abstract
.
2011
- A novel human psoriatic skin equivalent: the importance of transit amplifying keratinocytes
[Abstract in Rivista]
Lotti, Roberta; Truzzi, Francesca; Marconi, Alessandra; Pincelli, Carlo
abstract
Psoriasis is an immune-mediated disease of the skin characterized
by keratinocyte hyperproliferation, altered differentiation
and resistance to apoptosis. Histologically, psoriasis is represented
by augmented thickness of the epidermal compartment
of the skin, acanthosis and intense immune cell infiltrate. Psoriasis
is thought to be determined by both genetic and environmental
components. Psoriasis has long been considered
only an immunocyte-mediated disease, but recent data demonstrate
the important role of keratinocytes in triggering the
disease. Here we sought to develop an in vitro reconstructed skin model that would display the phenotypic and molecular
characteristics of psoriatic epidermis in a controlled manner
and in the absence of immune cells, providing a tool for the
study of keratinocyte biology and allowing the screening of
antipsoriatic drugs. Human skin equivalents were generated in
the following way: the dermal compartment was a mixture of
rat-tail collagen I and human fibroblasts either from healthy
adult skin or from patients with psoriasis. Given the importance
of transit amplifying (TA) cells in the pathogenesis of
psoriasis, we used either TA cells or stem cells isolated from
healthy human skin for the epidermal compartment. As a control,
we also analysed skin equivalents raised from the total
keratinocyte population. The combination of psoriatic fibroblasts
and normal TA cells produced the best psoriatic
phenotype, with a statistically significant increase of epidermal
thickness, areas of acanthosis and expression of psoriatic markers,
such as S100A7/psoriasin, K16 and phospho-Stat3
(Tyr705). Moreover, given the absence of p75NTR in psoriatic
epidermis and the apoptotic role of p75NTR receptor
mainly expressed by TA keratinocytes, we set up skin reconstructs
with either p75NTR-positive or p75NTR-depleted TA
cells. Skin reconstructs assembled with p75NTR-positive TA
keratinocytes were almost negative for psoriatic markers. By
contrast, skin reconstructs generated from p75NTR-depleted
TA displayed a more psoriasiform phenotype. These results
demonstrate the central role of TA cells in psoriasis together
with the importance of psoriatic fibroblasts in triggering the
psoriatic modifications.
2011
- Neurotrophins in healthy and diseased skin
[Articolo su rivista]
Truzzi, Francesca; Marconi, Alessandra; Pincelli, Carlo
abstract
Neurotrophins (NT) belong to a family of structurally andfunctionally related proteins that, depending on the tissuecontext and the receptors involved, promote either neuronalcell survival and differentiation or cell death. NT, and inparticular NGF, were first identified as neurotrophic factorssupporting the synthesis and development of sensory neuronsin the central and peripheral nervous system. it is now widelyaccepted that NT also act as growth factors in non-neuronalcells, including the skin. in the skin, most cell types are ableto secrete and/or to respond to stimulation by NT, creatinga unique network of molecular signaling in the cutaneousmicroenvironment. Moreover, many skin diseases have beenassociated with an involvement of a number of neural factorsincluding NT, but less attention has been given to the role ofNT as growth factors in the development of skin pathologies.This review summarizes currently data on the expression andfunction of NT and their receptors in several cell types in theskin. Moreover it focuses on the role of the skin NT network intwo cutaneous conditions, melanoma and psoriasis where NTare clearly involved.
2011
- Notch protein expression changes in human skin during ageing, keratinocyte differentiation and UVB-exposure
[Abstract in Rivista]
Marconi, Alessandra; Palazzo, Elisabetta; Dallaglio, Katiuscia; Truzzi, Francesca; Lotti, Roberta; Petrachi, Tiziana; Pincelli, Carlo
abstract
Epidermal integrity is guaranteed by the presence of keratinocyte stem cells (KSCs) and the correct balance between cell proliferation, differentiation and apoptosis. The microenviroment or “niche” where KSCs reside has a key role in the regulation of these processes and comprise all the factors expressed or released by skin cells. During ageing, epidermal niche changes determine the behavior of KSCs and their progeny (or transit amplifying (TA) cells). Notch proteins (Notch-1, -2, -3, -4) comprise a family of surface receptors which are implicated in maintaining epidermal homeostasis and, for this reason, they are fundamental for the comprehension of the mechanism within epidermal niche in normal condition and during photoageing.
First, we have collected skin samples from different age donors: young (Y-under 20 years), adult (A-between 20 and 50 years) and old (O-over 50 years). We have shown that CK15 and CK10 expression don’t change with age, while there is a reduction in Ki67-positive cells and a increasing in the number of epidermal layers expressing involucrin. In culture, keratinocytes present a reduction in proliferation and in CFE in direct ratio to age. In particular, we have shown that TA cells are eventually the most involved.
In skin biopsies, Notch-1 expression shown a reduction with age, while Notch-2 presents a different localization. In keratinocyte cell culture, Notch-1 decrease with age, while Notch-2 expression seems to be higher Y cells and constant in A and O cells. Notch-1 and Notch-2 are mainly expressed by TA cells and both proteins present a cytoplasmic localization in KSCs and TA cells, except for Notch-1 which is also present in the nucleus of TA cells. Moreover, both proteins are mainly expressed and activated after stimulus with calcium. Notch-1 activation decrease in confluent and more differentiated keratinocytes from different age, while its expression present a reduction in subconfluent cells and a increase in confluent cells in direct ratio to age. Notch-2 is mainly expressed and activated with confluence in all age class. Furthermore, Notch-1 is strongly activated after stimulus with UVB75 in Y keratinocyte. In addition, it is re-activated in O keratinocytes after stimulus with UVB5 and it is not expressed in more aged keratinocytes with respect to Y ones. Notch-2 is mainly expressed after stimulus with UVB5.
This data confirm the role of Notch proteins within epidermal niche and their possible involvement in the mechanisms of photoaging.
2011
- Notch-1 and Notch-2 modulate keratinocyte stem cell viability and differentiation during skin ageing and UVB exposure
[Abstract in Rivista]
Palazzo, Elisabetta; Marconi, Alessandra; M., Dumas; S., Schnebert; Dallaglio, Katiuscia; Truzzi, Francesca; Lotti, Roberta; Petrachi, Tiziana; Pincelli, Carlo
abstract
Notch are a family of surface receptors implicated in maintaining epidermal homeostasis. In
the epidermis, CK15 and CK10 expression does not change with age, while there is a reduction
in Ki67-positive cells and an increased involucrin expression. In culture, keratinocytes display
reduced proliferation and a lower colony forming efficiency, as a function of age. Transit
amplifying cells appear more affected than stem keratinocytes by ageing. In epidermal sections,
Notch-1 expression shows a reduction with age, while Notch-2 is located in the upper layers in
Y (under 20-years), in all layers in A (between 20 and 60-years), and predominantly in the basal
layer in O (over 60-years). In cultures, Notch-1 activation decreases with age, while Notch-2
seems to be more activated in Y cells than in A and O cells. Notch-1 and Notch-2 are mainly
expressed in the cytoplasm of TA cells, Notch-1 being present also in the nucleus. Notch protein
inhibition reduces keratinocyte stem cell viability, possibly through survivin downregulation.
Inhibiting Notch-1 also induces G1 arrest in keratinocytes at all ages. Notch proteins are activated
by calcium, while Notch-1 activation decreases in more differentiated keratinocytes. On the
other hand, Notch-2 is activated at cell confluence, in all age groups. Furthermore, Notch-1
is up-regulated upon UVB irradiation (75 mJ/cm2) in Y keratinocytes and is re-activated in O
keratinocytes after UVB (5 mJ/cm2). Taken together, these data confirm the role of Notch proteins
within epidermal niche and their possible involvement in the mechanisms of photoaging.
2011
- The p75 neurotrophin receptor acts as a "switch on-off" protein in early transit amplifying differentiation
[Abstract in Rivista]
Truzzi, Francesca; Marconi, Alessandra; Lotti, Roberta; Dallaglio, Katiuscia; Palazzo, Elisabetta; Petrachi, Tiziana; Saltari, Annalisa
abstract
p75 neurotrophin receptor (p75NTR), expressed in transit amplifying (TA) cells, mediates
neurotrophin (NT) signals alone or in combination with the high affinity receptor Trk. In the
present work, we studied the role of p75NTR during keratinocyte differentiation. p75NTR
was up-regulated during keratinocyte differentiation in cell culture system both with serum
and with calcium treatment. When p75NTR was silenced, calcium treatment failed to induce
differentiation in subconfluent keratinocytes. In human skin, p75NTR is only expressed in the
basal keratinocyte layer and is confined to a MIB-1 negative cell population. p75NTR+ cells,
isolated by magnetic cell sorting, were less differentiated and proliferated less than p75NTR–
cells in vitro, which in turn include keratinocyte stem cells (KSC), as shown by western blotting
and confocal analysis. p75NTR+ keratinocytes, isolated from TA cells, expressed more survivin
and CK15, while displayed less CK10 than p75NTR- TA. Colony forming efficiency and long term
proliferation analysis revealed that p75NTR+ TA yielded a higher number of cells than p75NTRTA,
suggesting that p75NTR+ cells are early TA cells. p75NTR retroviral infection of KSC induced
a more differentiated phenotype, with the same features of TA cells. Finally, skin reconstructs
originated from TA p75NTR- cells generated a hyperproliferative phenotype. These results suggest
that p75NTR+ keratinocytes represent a subpopulation of TA cells, directly derived from stem
cells that just started the differentiation process. p75NTR may act as a “switch on-off” protein in
stem-TA transition, initiating keratinocyte differentiation.
2011
- The p75 neurotrophin receptor acts as a "switch on-off" protein in keratinocyte differentiation
[Abstract in Rivista]
Palazzo, Elisabetta; Truzzi, Francesca; Marconi, Alessandra; Lotti, Roberta; Dallaglio, Katiuscia; Petrachi, Tiziana; Pincelli, Carlo
abstract
p75 neurotrophin receptor (p75NTR) mediates neurotrophin (NT) signals alone or in combination with the high affinity receptor Trk, also in cell settings outside the nervous system. In the present work, we studied the role of p75NTR during keratinocyte differentiation. p75NTR protein was up-regulated after serum treatment in both confluent and post-confluent keratinocytes, together with the expression of cytokeratin 10 (CK10) and involucrin. Moreover, calcium treatment of subconfluent keratinocytes induced the expression of involucrin, CK10, and the up-regulation of p75NTR, both at the mRNA and at the protein level. Western blotting and confocal analysis showed that p75NTR positive (+) keratinocytes isolated by magnetic cell sorting, expressed less CK10 and CK15, as compared to p75NTR negative (-) cells. p75NTR+ keratinocytes formed a lower number of colonies and proliferated to a lesser extent, as compared to p75NTR- cells. p75NTR is only expressed in the basal keratinocyte layer and is confined to a MIB-1 negative transit amplifying (TA) cell population. p75NTR+ keratinocytes, isolated from TA cells, expressed more survivin and CK15, while displayed less CK10 than p75NTR- TA. Colony forming efficiency was higher in p75NTR+ TA than in p75NTR- TA cells. Moreover, long term proliferation analysis revealed that p75NTR+ TA yielded a higher number of cells than p75NTR- TA. p75NTR retroviral infection of stem cells induced a more differentiated phenotype, with the same features of TA cells. On the other hand, when p75NTR was silenced, calcium treatment failed to induce differentiation in subconfluent keratinocytes, as shown by the absence of involucrin expression. These results suggest that p75NTR+ keratinocytes represent a subpopulation of TA cells, directly derived from stem cells that just started the differentiation process. p75NTR may act as a “switch on-off” protein in stem-TA transition initiating keratinocyte differentiation.
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
- A previously unreported function of beta1B integrin isoform in caspase-8-dependent integrin-mediated keratinocyte death
[Articolo su rivista]
Lotti, Roberta; Marconi, Alessandra; Truzzi, Francesca; Dallaglio, Katiuscia; Gemelli, Claudia; R. G., Borroni; Palazzo, Elisabetta; Pincelli, Carlo
abstract
Integrins regulate adhesive cell-matrix interactions and mediate survival signals. On the other hand, unligated or free cytoplasmic fragments of integrins induce apoptosis in many cell types (integrin-mediated death). We have previously shown that b1 integrins expression protects keratinocyte stem cells from anoikis, while the role of the b1B integrin isoform has never been clarified. Here we report that suspended keratinocytes undergo apoptosis via the activation of caspase-8, independently of Fas/Fas Ligand system. Indeed, anti-b1 integrin neutralizing antibodies induced apoptosis in short-hairpin-RNA-Fas-Associated-Death-Domain treated cells. Moreover, before and during suspension, caspase-8 directly associated with b1 integrin, that in turn internalized and progressively degraded, shedding the cytoplasmic domain. b1B was expressed only in the cytoplasm in a perinuclear fashion and remained unaltered during suspension. At 24 hrs, as b1A located close to the nucleus, b1B co-localized with b1A and co-immunoprecipitated with caspase-8. Caspase-8 was activated earlier in b1B integrin transfected keratinocytes, and these cells underwent a higher rate of apoptosis than mock cells. By contrast, caspase-8 was not activated in siRNA b1B transfected cells. These results indicate that when b1A is unligated, b1B is responsible for “integrin-mediated death” in human keratinocytes.
2010
- E-FABP mediates human keratinocyte differentiation
[Abstract in Rivista]
Dallaglio, Katiuscia; Marconi, Alessandra; Truzzi, Francesca; Lotti, Roberta; Palazzo, Elisabetta; T., Petrachi; T., Bertalot; Pincelli, Carlo
abstract
.
2010
- Keratinocyte Stem Cells: friends and foes
[Articolo su rivista]
Pincelli, Carlo; Marconi, Alessandra
abstract
Skin and its appendages provide a protective barrier against the assaults of the environment. To perform its role, epidermis undergoes an ongoing renewal through a balance of proliferation and differentiation/apoptosis called homeostasis. Keratinocyte stem cells reside in a special microenvironment called niche in basal epidermis, adult hair follicle and sebaceous glands. While a definite marker has yet to be detected, data raised part in humans and part in the mouse system, point to a critical role of stem and its progeny transit amplifying cells in epidermal homeostasis. Stem cells are protected from apoptosis and are long-resident in adult epidermis. This renders them more prone to be the origin of skin cancer. In this review, we will outline the main features of adult stem cells in mouse and humans and discuss their fate in relation to differentiation, apoptosis and cancer.
2010
- Neurotrophins stimulate human fibroblast differentiation, migration and contractile strenght
[Abstract in Rivista]
Dallaglio, Katiuscia; Palazzo, Elisabetta; Marconi, Alessandra; C., Viennet; M., Dumas; P., Humbert; Pincelli, Carlo
abstract
.
2010
- The p75 neurotrophin receptor is involved in early keratinocyte differentiation
[Abstract in Rivista]
Truzzi, Francesca; Marconi, Alessandra; Lotti, Roberta; Dallaglio, Katiuscia; Palazzo, Elisabetta; T., Petrachi; Pincelli, Carlo
abstract
.
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
.
2009
- Anti-Fas Ligand neutralizing antibodies prevent acantholysis both in vitro and in vivo
[Abstract in Rivista]
Marconi, Alessandra; Lotti, Roberta; Truzzi, Francesca; Dallaglio, Katiuscia; Borroni, Riccardo; Palazzo, Elisabetta; Y., Kitajima; Pincelli, Carlo
abstract
.
2009
- Apoptosis precedes desmoglein cleavage and keratinocyte dissociation in pemphigus: anti-Fas Ligand neutralizing antibodies prevent acantholysis both in vitro and in vivo
[Abstract in Rivista]
Lotti, Roberta; E., Shu; Truzzi, Francesca; Palazzo, Elisabetta; Marconi, Alessandra; Y., Kitajima; Pincelli, Carlo
abstract
.
2009
- Endogenous survivin modulates survival and proliferation in normal human keratinocytes and during UVB irradiation
[Abstract in Rivista]
Marconi, Alessandra; Dallaglio, Katiuscia; Palazzo, Elisabetta; M., Dumas; Truzzi, Francesca; Lotti, Roberta; F., Bonte; Pincelli, Carlo
abstract
.
2009
- Endogenous survivin modulates survival andproliferation in UVB-treated human keratinocytes
[Articolo su rivista]
Dallaglio, Katiuscia; Palazzo, Elisabetta; Marconi, Alessandra; M., Dumas; Truzzi, Francesca; Lotti, Roberta; F., Bontè; Pincelli, Carlo
abstract
Survivin is a bi-functional member of inhibitor ofapoptosis protein family, as it is able to both inhibit apoptosisand to regulate cell cycle. We investigated the role of survivin inhuman keratinocytes under normal conditions and during UVBirradiation. Survivin siRNA decreases proliferation and inducesapoptosis in human keratinocytes, in a mode consistent with themitotic catastrophe. Low doses UVB increase survivin expressionat earlier times, while high doses down-regulate survivin level.Low doses UVB induce cell cycle arrest in G2 ⁄ M, while highdoses UVB cause apoptosis. Moreover, overexpression of survivinprotects keratinocytes from UVB-induced apoptosis, and silencingof survivin renders keratinocytes more susceptible to UVBinducedcell death. Finally, survivin siRNA increases UVB-inducedreduction of cell proliferation. Taken together, these resultsindicate that survivin plays a critical role in epidermalhomeostasis in normal conditions and during UVB exposure, withpossible implication in skin carcinogenesis.
2009
- Neurotrophins and their receptors stimulate fibroblast differentiation, migration and tensile strength
[Abstract in Rivista]
Palazzo, Elisabetta; Marconi, Alessandra; Truzzi, Francesca; Lotti, Roberta; Dallaglio, Katiuscia; Borroni, Riccardo; M., Dumas; Pincelli, Carlo
abstract
.
2008
- Defective p75 neurotrophin receptor in transit amplifying cells renders psoriatic keratinocytes more resistant to apoptosis
[Abstract in Rivista]
Truzzi, Francesca; Marconi, Alessandra; Lotti, Roberta; Dallaglio, Katiuscia; Giannetti, Alberto; Pincelli, Carlo
abstract
.
2008
- Inhibition of keratinocyte proliferation with a novel compound for the topical treatment of psoriasis and dermatitis
[Abstract in Rivista]
L., Bertarione Rava Rossa; S., Traversa; V., Mainero; Pincelli, Carlo; Marconi, Alessandra
abstract
.
2008
- Neurothrophins and their receptors stimulate melanoma cell proliferation and migration
[Abstract in Rivista]
Truzzi, Francesca; Marconi, Alessandra; Lotti, Roberta; Dallaglio, Katiuscia; Borroni, Riccardo; L., French; Pincelli, Carlo
abstract
.
2008
- Neurotrophins and their receptors stimulate melanoma cell proliferation and migration
[Articolo su rivista]
Truzzi, Francesca; Marconi, Alessandra; Lotti, Roberta; Dallaglio, Katiuscia; L. E., French; B. L., Hempstead; Pincelli, Carlo
abstract
Melanoma is a highly aggressive skin tumor that originates in the epidermis from melanocytes. As melanocytes share with the nervous system a common neuroectodermal origin and express all neurotrophins (NTs), we evaluated the expression and function of NTs and their receptors in melanoma. We report that primary and metastatic melanoma cell lines synthesize and secrete all NTs. Moreover, melanoma cells express the low-affinity (p75NTR) and the high-affinity tyrosine kinase NT receptors (Trk). The inhibition of Trk receptors by either K252a or Trk/Fc chimeras prevents proliferation, indicating that autocrine NTs are responsible for this effect. NT-3, NT-4, and nerve growth factor (NGF) induce cell migration, with a stronger effect on metastatic cell lines. Transfection with p75NTR small interfering RNA (p75NTRsiRNA) or treatment with K252a inhibits NT-induced melanoma cell migration, indicating that both the low- and high-affinity NT receptors mediate this effect. All melanoma cell lines express the p75NTR coreceptor sortilin by which proNGF stimulates migration in melanoma cells, but not in cells transfected with p75NTRsiRNA. These results indicate that NTs, through their receptors, play a critical role in the progression of melanoma.
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
.
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.
2008
- Pin Cell
[Spin Off]
Pincelli, Carlo; Marconi, Alessandra
abstract
PinCell is a biotech company founded in October 2008 as an academic spin-off of the University of Modena and Reggio Emilia by initiative of Prof. Carlo Pincelli and Dr. Alessandra Marconi.
The Mission is to research and develop novel molecules that will increase the therapeutic options for neoplastic and chronic inflammatory skin diseases.
The research and development process is focused on drug discovery, drug physico-chemical characterization, preclinical pharmacology and toxicology studies and early phase clinical trials on healthy volunteers (phase I)
PinCell directs its discoveries to other biotechnological companies or to pharmaceutical industries in need for novel ideas for drug development or looking for products at advanced stage of development requiring rapid commercial transfer.
2008
- Remedies for pemphigus containing anti Fas ligand antibodies
[Brevetto]
Pincelli, Carlo; Marconi, Alessandra
abstract
The present invention refers to the use of FasL antagonists, e.g. of humanized antibodies directed against human Fas ligands (also named CD95L or Apo1L and hereinafter abbreviated as FasL) for the prevention and/or treatment of skin diseases associated with keratinocytes acantholysis, particularly for the prevention and/or treatment of pemphigus.
2008
- Survivin siRNA causes cell cycle arrest in G2/M and increases UVB-induced apoptosis in human keratinocytes
[Abstract in Rivista]
Dallaglio, Katiuscia; Marconi, Alessandra; Lotti, Roberta; Truzzi, Francesca; M., Dumas; F., Bonte; Pincelli, Carlo
abstract
.
2007
- A novel topical treatment of psoriasis: inhibition of NGF-induced keratinocyte proliferation
[Abstract in Rivista]
L., Bertarione Rava Rossa; S., Traversa; V., Mainero; D., Barone; C., Oderda; S., Fumero; Pincelli, Carlo; Marconi, Alessandra
abstract
.
2007
- CT327: a novel mini-PEGylated compound for topical treatment of psoriasis
[Abstract in Rivista]
L., Bertarione Rava Rossa; S., Traversa; V., Mainero; D., Barone; C., Oderda; S., Fumero; Pincelli, Carlo; Marconi, Alessandra
abstract
.
2007
- CT327: a novel topical treatment of psoriasis and dermatitis
[Abstract in Rivista]
L., Bertarione Rava Rossa; S., Traversa; V., Mainero; D., Barone; C., Oderda; S., Fumero; Pincelli, Carlo; Marconi, Alessandra
abstract
.
2007
- Carboxyfullerenes localize within mitochondria and prevent the UVB-induced intrinsic apoptotic pathway
[Articolo su rivista]
F., Chirico; C., Fumelli; Marconi, Alessandra; A., Tinari; E., Straface; W., Malorni; R., Pellicciari; Pincelli, Carlo
abstract
Carboxyfullerenes (CF) act as free radical scavengers in many cell settings and prevent apoptosis in vitro and in vivo. CF protect normal human keratinocytes from UVB-induced apoptosis, although the mechanisms underlying this effect remain to be clarified. Double-staining confocal laser microscopy revealed that CF penetrate the cell and colocalize with cytokeratin-18 within cytoplasm. This localization was confirmed by transmission electron microscopy that showed CF intermingled with keratin filaments. Moreover, double-staining with the mitochondrial marker anti-F1-ATPase antibody demonstrated that CF are expressed in mitochondria. Transmission electron microscopy confirmed that CF actually localize within mitochondria. Then, normal human keratinocytes were UVB-irradiated in the presence or absence of CF at different doses. CF protected keratinocytes from apoptosis induced by reactive oxygen species. CF scavenging effect is associated with a partial blockade of the UVB-induced intrinsic apoptotic pathway by down-modulating caspase-9 activation and cytochrome c release, and by inhibiting the down-regulation of the inhibitor of apoptosis proteins (IAP) survivin, livin, IAP-1 and IAP-2. Finally, CF prevented the cleavage of Bid, up-regulation of Bad and down-regulation of Mcl-1 induced by UVB. Taken together, these results indicate that CF penetrate human keratinocytes, localize within mitochondria where they act both by scavenging free radicals and by protecting cells from apoptosis.
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 (NTR) mediates chemotherapy-induced apoptosis in melanoma cells
[Abstract in Rivista]
Truzzi, Francesca; Marconi, Alessandra; Lotti, Roberta; Dallaglio, Katiuscia; Borroni, Riccardo; Pincelli, Carlo
abstract
.
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
.
2007
- Remedies for pemphigus containing anti Fas Ligand antibodies
[Brevetto]
Pincelli, Carlo; Marconi, Alessandra
abstract
Disclosure of the use of FasL antagonist, e.g. of humanized antibodies directed against human Fas Ligands (also named CD95L or Apo1L and hereinafter abbreviated as FasL) for the prevention and/or treatment of skin diseases associated with keratinocytes acantholysis, particularly for the prevention and/or treatment of pemphigus
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.
2007
- Survivin modulates cell cycle and interferes with UV-induced apoptosis in human keratinocytes
[Abstract in Rivista]
Dallaglio, Katiuscia; Marconi, Alessandra; M., Dumas; F., Bonte; Lotti, Roberta; Truzzi, Francesca; Gemelli, Claudia; Pincelli, Carlo
abstract
.
2007
- p75 neurotrophin receptor (NTR) is up-regulated by chemotherapy and induces apoptosis in melanoma cells
[Abstract in Rivista]
Marconi, Alessandra; Truzzi, Francesca; Lotti, Roberta; Dallaglio, Katiuscia; Pincelli, Carlo
abstract
.
2006
- Anoikis is triggered by the association between beta 1B integrin and caspase-8 in human keratinocytes
[Abstract in Rivista]
Lotti, Roberta; Marconi, Alessandra; Dallaglio, Katiuscia; Truzzi, Francesca; Pincelli, Carlo
abstract
.
2006
- Expression and function of neurotrophins and their receptors in human melanocytes.
[Articolo su rivista]
Marconi, Alessandra; Panza, Mc; Bonnet Duquennoy, M; Lazou, K; Kurfurst, R; Truzzi, Francesca; Lotti, Roberta; DE SANTIS, Giorgio; Dumas, M; Bonté, F; Pincelli, Carlo
abstract
Melanocytes and cells of the nervous system are of common ectodermal origin and neurotrophins (NT) have been shown to be released by human keratinocytes. We investigated the expression and function of NT [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, NT-4/-5] and their receptors in human melanocytes. Human melanocytes produce all NT in different amounts, whereas they only release NT-4. NT-4 release is downregulated, whereas NT-3 is upregulated by ultraviolet (UVB) irradiation. Melanocytes treated with phorbol 12-myristate 13-acetate (PMA) express TrkA and TrkB, but not TrkC. NT fail to stimulate melanocyte proliferation, whereas they stimulate the synthesis of tyrosinase and tyrosinase-related protein-1 (TRP-1). Finally, NT-3, NT-4 and NGF increase melanin production. Taken together, these results demonstrate an intriguing interaction between melanocytes and the nervous system. We speculate that NT could be considered the target of therapy for disorders of skin pigmentation.
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
.
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
.
2006
- Neurotrophin modulate melanogenesis in human melanocytes
[Abstract in Rivista]
C., Lazou; Marconi, Alessandra; C., Marteau; M., Bonnet Duquennoy; R., Kurfurst; M., Dumas; F., Bonte; Pincelli, Carlo
abstract
.
2006
- Neurotrophins (NT) modulate survival and chemotaxis in metastatic and primary melanoma cell lines through their high-affinity receptors Trk
[Abstract in Rivista]
Truzzi, Francesca; Marconi, Alessandra; Lotti, Roberta; Dallaglio, Katiuscia; M., Pignatti; L., French; Pincelli, Carlo
abstract
.
2006
- Neurotrophins (NT) modulate survival and chemotaxis in metastatic and primary melanoma cell lines through their high-affinity receptors Trk
[Abstract in Rivista]
Truzzi, Francesca; Marconi, Alessandra; Lotti, Roberta; Dallaglio, Katiuscia; M., Pignatti; L., French; Pincelli, Carlo
abstract
.
2006
- Neurotrophins in skin biology and pathology
[Articolo su rivista]
V. A., Botchkarev; M., Yaar; E. M. J., Peters; S. P., Raychaudhuri; N. V., Botchkareva; Marconi, Alessandra; S. K., Raychaudhuri; R., Paus; Pincelli, Carlo
abstract
Neurotrophins (NTs) belong to a family of growth factors, which control the development, maintenance, and apoptotic death of neurons and also fulfill multiple regulatory functions outside the nervous system. Biological effects induced by NTs strongly depend on the pattern of NT receptor/ co-receptors expression in target cells, as well as on the set of intracellular adaptor molecules that link NT signalling to distinct biochemical pathways. In this review, we summarize data on the molecular mechanisms underlying the involvement of NTs in the control of non- neuronal functions in normal skin ( e. g. keratinocyte proliferation, melanocyte development and apoptosis, hair growth). We also review the data on the role for NTs and their receptors in a number of pathological skin conditions ( stress- induced hair loss, psoriasis, atopic dermatitis). Although additional efforts are required to fully understand mechanisms underlying the involvement of NTs and their receptors in controlling functions of normal and pathologically altered skin cells, substantial evidence suggests that modulation of NT signalling by NTs receptor agonists/ antagonists may be developed as intervention modalities in distinct skin and hair growth pathologies.
2006
- Survivin identifies keratinocyte stem cells and it is down-regulated by anti beta 1 integrin during anoikis
[Abstract in Rivista]
Dallaglio, Katiuscia; Lotti, Roberta; Marconi, Alessandra; Truzzi, Francesca; M., Dumas; F., Bonte; Pincelli, Carlo
abstract
.
2006
- p75 Neurotrophin Receptor (p75NTR) signals apoptosis either alone or in association with its co-receptor sortilin in human keratinocytes
[Abstract in Rivista]
Truzzi, Francesca; Marconi, Alessandra; Atzei, Paola; Lotti, Roberta; Dallaglio, Katiuscia; Pincelli, Carlo
abstract
.
2005
- Beta 1 integrin associates with caspase-8 and triggers anoikis in human keratinocytes
[Abstract in Rivista]
Lotti, Roberta; Marconi, Alessandra; Panza, Maria Cristina; Pincelli, Carlo
abstract
.
2005
- Blockade of beta 1 integrin directly activates caspase-8-triggered extrinsic apoptotic pathway in human keratinocytes
[Abstract in Rivista]
Lotti, Roberta; Marconi, Alessandra; Panza, Maria Cristina; Gemelli, Claudia; M., Leverkus; Giannetti, Alberto; Pincelli, Carlo
abstract
.
2005
- Brain-derived neurotrophic factor (BDNF) and pro-nerve growth factor (NGF) induce apoptosis in human keratinocytes through the p75 neurotrophin receptor
[Abstract in Rivista]
Panza, Maria Cristina; Atzei, Paola; Truzzi, Francesca; Lotti, Roberta; Marconi, Alessandra; Pincelli, Carlo
abstract
.
2005
- Evaluation of different serological tests for the diagnosis of pemphigus
[Abstract in Rivista]
Vaschieri, Cristina; Marconi, Alessandra; Pincelli, Carlo; Giannetti, Alberto
abstract
.
2005
- POLYMER CONJUGATES OF K-252A AND DERIVATIVES THEREOF
[Brevetto]
Traversa, S.; Bagnod, R.; Barone, D.; Beratarione Rava Rossa, L.; Fumero, S.; Mainero, V.; Marconi, A.; Oderda, C.; Pincelli, C.; Lorenzetto, C.; Beccaria, L.
abstract
The present invention relates to novel polymer conjugates of K-252a and derivatives thereof and to their use for the preparation of a pharmaceutical composition useful for the prevention, alleviation and treatment of kinase-associated pathologies. In particular, the present invention relates to the prevention, alleviation and treatment of HMGB1-associated pathologies. In a particular aspect, the invention relates to the use of the novel polymer conjugates of K-252a and derivatives thereof in the preparation of a pharmaceutical composition useful for the prevention, alleviation and treatment of neurological disorders, neuropathies and neurodegenerative disorders of the central and peripheral nervous system.; In a further preferred aspect, the invention relates to the use of the polymer conjugates in the preparation of a pharmaceutical composition useful for the prevention, alleviation and treatment of dermal pathologies, in particular dermal pathologies associated with an excessive keratinocyte proliferation, in particular psoriasis. In a still further aspect, the invention relates to the use of the polymer conjugates in the prevention, alleviation and treatment of NGF-related pain. More specifically, the present invention relates to a polymer conjugate of K-252a and derivatives thereof, wherein the polymer is polyethylene glycol or methoxy-polyethylene glycol formula.
2005
- Phenotype and function of a keratinocyte subpopulation enriched in stem cells.
[Abstract in Rivista]
Marconi, Alessandra; Panza, Maria Cristina; Lotti, Roberta; M., Dumas; F., Bontè; Pincelli, Carlo
abstract
.
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
.
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
.
2004
- Blockade of beta(1) integrin activates caspase-8 apoptotic pathway without Fas/FasL modulation in human keratinocytes
[Abstract in Rivista]
Marconi, Alessandra; Lotti, Roberta; Panza, Maria Cristina; T., Wachter; M., Leverkus; Giannetti, Alberto; Pincelli, Carlo
abstract
.
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
.
2004
- Effect of NGF on matrix metalloproteinases and tissue inhibitors of metalloproteinases produced by human dermal fibroblasts and myofibroblasts
[Abstract in Rivista]
C., Gondran; M., Dumas; Marconi, Alessandra; Truzzi, Francesca; Atzei, Paola; F., Bonte; Pincelli, Carlo; S., Schnebert
abstract
.
2004
- FLICE/caspase-8 activation triggers anoikis induced by beta(1)-integrin blockade in human keratinocytes
[Abstract in Rivista]
Marconi, Alessandra; Fila, Chiara; Panza, Maria Cristina; R., Tiberio; T., Wacter; M., Leverkus; Pincelli, Carlo
abstract
.
2004
- FLICE/caspase-8 activation triggers anoikis induced by beta(1)-integrin blockade in human keratinocytes
[Articolo su rivista]
Marconi, Alessandra; P., Atzei; C., Panza; C., Fila; R., Tiberio; Truzzi, Francesca; T., Wachter; M., Leverkus; Pincelli, Carlo
abstract
Beta(1)-integrin protects keratinocyte stem cells (KSC) from cell-detachment apoptosis ('anoikis'). Here we show that caspase-8 active protein is detected in both young transit amplifying (TA) cells and TA cells, but not in KSC. On suspension, caspases are activated earlier in young TA than in KSC, whereas anti-beta(1)-integrin neutralizing antibody accelerates caspase activation in both KSC and young TA. Caspases 8 and 10 are the first caspases to be activated whereas caspase-8 inhibitor zIETD-fmk delays the activation of Bid, caspase-9 and caspase-3. However, the caspase-9 inhibitor zLEDH-fmk does not block the activation of caspase-8, Bid, caspase-10 and caspase-3. Moreover, caspase-8, but not caspase-9 inhibitor partially prevents keratinocyte anoikis. As FLIP inhibits caspase-8 processing, we retrovirally infected HaCaT keratinocytes with c-FLIPL. Anti-beta(1)-integrin fails to activate caspase-8, Bid, caspase-9 and to induce the release of cytochrome c in c-FLIPL overexpressing keratinocytes. Finally, overexpression of c-FLIPL partially prevents anoikis in both suspended and anti-beta(1) integrin-treated cells. Taken together, these results indicate that the extrinsic apoptotic pathway triggered by caspase-8 predominates in keratinocyte anoikis. However, the release of cytochrome c and the later activation of caspase-9 seem to suggest that the intrinsic mitochondrial pathway may intervene as a positive feedback loop of caspase activation.
2004
- Nerve growth factor receptors modulates apoptosis in melanoma cell lines
[Abstract in Rivista]
Truzzi, Francesca; Marconi, Alessandra; Atzei, Paola; M., Pignatti; L., French; Pincelli, Carlo
abstract
.
2004
- The neurotrophin network in human skin
[Abstract in Rivista]
Marconi, Alessandra; M., Dumas; Fila, Chiara; Truzzi, Francesca; Atzei, Paola; M., Pignatti; F., Bonte; Giannetti, Alberto; Pincelli, Carlo
abstract
.
2004
- The p75 neurotrophin receptor signals apoptosis in a subpopulation of human keratinocytes
[Abstract in Rivista]
Atzei, Paola; Marconi, Alessandra; M., Pignatti; Truzzi, Francesca; Giannetti, Alberto; Pincelli, Carlo
abstract
.
2003
- Disruption of beta1 integrin induces keratinocyte stem cell anoikis through the extrinsic apoptotic pathway
[Abstract in Rivista]
Marconi, Alessandra; C., Fila; R., Tiberio; Giannetti, Alberto; Pincelli, Carlo
abstract
.
2003
- Expression and function of neurotrophins and their receptors in cultured human keratinocytes
[Articolo su rivista]
Marconi, Alessandra; M., Terracina; C., Fila; J., Franchi; F., Bontè; G., Romagnoli; R., Maurelli; C. M., Failla; M., Dumas; Pincelli, Carlo
abstract
Whereas nerve growth factor has been extensively studied in human keratinocytes, little is known on the role of other members of the neurotrophin family. We investigated the expression and function of neurotrophins and neurotrophin receptors in cultured human keratinocytes. We demonstrated by reverse transcription-polymerase chain reaction that keratinocytes synthesize neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5. These cells also express tyrosinase kinase A and C, the nerve growth factor and neuro-trophin-3 high-affinity receptors, respectively. On the other hand, only the truncated extracellular isoform of tyrosinase kinase B, the high-affinity brain-derived neurotrophic factor and neurotrophin-4/5 receptor, is detected in keratinocytes. Moreover, neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5 proteins are secreted by human keratinocytes at low levels. Keratinocyte stem cells synthesize the highest amounts of nerve growth factor, while they secrete higher levels of nerve growth factor as compared with transit amplifying cells. Neurotrophin-3 stimulates keratinocyte proliferation, where brain-derived neurotrophic factor or neurotrophin-4/5 does not exert any effect on keratinocyte proliferation. Addition of neurotrophin-3 slightly upregulates the secretion of nerve growth factor, whereas nerve growth factor strongly augments neurotrophin-3 release. Ultraviolet B irradiation downregulates nerve growth factor, whereas it augments neurotrophin-3 and neurotrophin-4/5 protein levels. Ultraviolet A irradiation increases the level of neurotrophin-3, whereas it does not exert any effect on the other neurotrophins. Finally, neurotrophins other than nerve growth factor fail to protect human keratinocytes from ultraviolet B-induced apoptosis. This work delineates a functional neurotrophin network, which may contribute to epidermal homeostasis.
2003
- Expression and function of neurotrophins in human keratinocytes
[Abstract in Rivista]
C., Fila; Marconi, Alessandra; M., Dumas; D., Baracchi; J., Franchi; F., Bonté; Pincelli, Carlo
abstract
.
2003
- Fas ligand in pemphigus sera induces keratinocyte apoptosis through the activation of caspase-8
[Articolo su rivista]
M., Puviani; Marconi, Alessandra; E., Cozzani; Pincelli, Carlo
abstract
The Fas/Fas ligand system triggers the extrinsic apoptotic pathway and is involved in several inflammatory conditions, also at the skin level. The Fas/Fas ligand cell death pathway plays a major role in anoikis, a type of apoptosis characterized by cell detachment. As pemphigus is characterized by loss of cell to cell adhesion, we evaluated the role of anoikis and Fas ligand in this bullous disease. We report that, in suprabasal epidermis from perilesional pemphigus skin, most keratinocytes are apoptotic. Moreover, Fas ligand levels are markedly increased in sera from pemphigus patients, whereas they are undetectable in sera from patients undergoing steroid treatment. Sera from untreated patients but not from patients under steroids induce keratinocyte apoptosis. Pemphigus-sera-induced cell death is partially inhibited by pretreatment with anti-Fas ligand antibodies and by incubation with caspase-8 inhibitor Z-IETD-FMK. Finally, caspase-8 is activated in keratinocytes provided with sera from pemphigus patients, whereas cleavage is partially blocked by pretreatment of sera with anti-Fas ligand antibody. These results suggest that increased Fas ligand in pemphigus sera is responsible for keratinocyte apoptosis, which occurs through the activation of a caspase-8-driven extrinsic apoptotic pathway.
2003
- The p75 neurotrophin receptor modulates apoptosis and differentiation in a subpopulation of human keratinocytes
[Abstract in Rivista]
D., Baracchi; Marconi, Alessandra; M., Pignatti; C., Fila; Pincelli, Carlo
abstract
.
2002
- Caspase-8-activated signal predominates in keratinocyte anoikis
[Abstract in Rivista]
C., Fila; Marconi, Alessandra; S., Krajewski; J. C., Reed; Giannetti, Alberto; Pincelli, Carlo
abstract
.
2002
- Keratinocytes enriched for stem cells are protected from anoikis via an integrin signaling pathway in a Bcl-2 dependent manner
[Articolo su rivista]
R., Tiberio; Marconi, Alessandra; C., Fila; C., Fumelli; M., Pignatti; S., Krajewski; Giannetti, Alberto; J. C., Reed; Pincelli, Carlo
abstract
Because inhibition of integrin signaling induces apoptosis, we investigated whether keratinocytes expressing beta1 and alpha6beta4 integrins (enriched for stem cells) are protected from cell death. Keratinocytes rapidly adhering to type IV collagen expressed highest levels of beta1 and alpha6beta4 and of the anti-apoptotic stem cell marker p63. Apoptotic cells were significantly higher in slowly adhering than in rapidly adhering keratinocytes. Anti-beta1 integrin caused a significant increase in apoptotic cells, while it decreased Bcl-2 levels in stem keratinocytes. Bax and Bad proteins were higher in slowly adhering than in rapidly adhering cells. By contrast, Bcl-2, Bcl-x and Mcl-1 proteins were highest in rapidly adhering keratinocytes and nearly absent in slowly adhering cells. After addition of anti-beta1 integrin, the apoptotic rate was significantly higher in HaCaT cells not expressing Bcl-2 than in controls. These results indicate that keratinocytes enriched for stem cells are protected from apoptosis via beta1 integrin, in a Bcl-2 dependent manner. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
2002
- Pemphigus sera, expressing high levels of Fas ligand, induces keratinocyte apoptosis
[Abstract in Rivista]
Puviani, Mario; Marconi, Alessandra; Fila, Chiara; E., Cozzani; Giannetti, Alberto; Pincelli, Carlo
abstract
.
2002
- The low-affinity neurotrophin receptor p75 mediates the proapoptotic activity of brain-derived neurotrophic factor in keratinocytes
[Abstract in Rivista]
Marconi, Alessandra; Fila, Chiara; M., Pignatti; Giannetti, Alberto; Pincelli, Carlo
abstract
.
2002
- The p75 neurotrophin receptor mediates spontaneous and UV-induced apoptosis in keratinocytes
[Abstract in Rivista]
Marconi, Alessandra; M., Pignatti; Fila, Chiara; Puviani, Mario; Giannetti, Alberto; Pincelli, Carlo
abstract
.
2001
- Stem keratinocytes are protected from cell death via an integrin signaling pathway, in a bcl-2-dependent manner
[Abstract in Rivista]
R., Tiberio; Marconi, Alessandra; D., Ottani; S., Krajewski; J., Reed; Giannetti, Alberto; Pincelli, Carlo
abstract
.
2001
- The antiapoptotic bcl-2 proteins are highest and caspases are not activated in basal keratinocytes expressing high levels of p63
[Abstract in Rivista]
Marconi, Alessandra; C., Fila; S., Bertellini; M., Pignatti; S., Krajewski; J. C., Reed; Giannetti, Alberto; Pincelli, Carlo
abstract
.
2001
- The low-affinity neurotrophin receptor (p75) mediates apoptosis in human keratinocytes
[Abstract in Rivista]
Marconi, Alessandra; C., Fila; S., Bertellini; M., Pignatti; Garuti, Cinzia; Giannetti, Alberto; Pincelli, Carlo
abstract
.
2000
- Autocrine nerve growth factor in human keratinocytes
[Articolo su rivista]
Pincelli, Carlo; Marconi, Alessandra
abstract
Biologically active nerve growth factor (NGF) is synthesised and released by proliferating normal human keratinocytes. NGF up-regulates the expression of NGF mRNA in keratinocytes. Keratinocytes express both the low (p75)- and the high-affinity (TrkA) NGF-receptors, which are located in the basal layer of the epidermis. K252, a specific inhibitor of trk phosphorylation, blocks NGF-induced keratinocyte proliferation, in absence of exogenous NGF. Normal keratinocytes over-expressing TrkA proliferate better than control transfectants, while the NGF mimicking anti-Trk antibody induces an increased keratinocyte proliferation in Trk over-expressing cells as compared to mock transfected keratinocytes. In addition, NGF over-expressing keratinocytes proliferate better than mock transfected cells. K252, by blocking TrkA phosphorylation, induces apoptosis in normal keratinocytes, but not in keratinocytes over-expressing bcl-2. Further-more, NGF transfected keratinocytes are protected from UV-B-induced keratinocyte apoptosis, by maintaining constant levels of Bcl-2 and Bcl-x(L). Taken together these results support the concept of an autocrine survival system sustained by NGF and its high-affinity receptor in human keratinocytes. Because NGF and Trk levels are highly expressed in psoriasis, one could speculate that NGF autocrine system plays a role in the mechanisms associated with this and other hyperproliferative skin conditions, including cancer. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.
2000
- Carboxyfullerenes protect human keratinocytes from ultraviolet-B-induced apoptosis
[Articolo su rivista]
C., Fumelli; Marconi, Alessandra; S., Salvioli; E., Straface; W., Malorni; A. M., Offidani; R., Pellicciari; G., Schettini; Giannetti, Alberto; D., Monti; C., Franceschi; Pincelli, Carlo
abstract
Carboxyfullerene, a water-soluble carboxylic acid derivative of a fullerene, which acts as a free-radical scavenger, was investigated as a protective agent against ultraviolet-light-induced damage in human keratinocytes. First, we demonstrate that carboxyfullerene is not cytotoxic for these cells. In addition, this compound significantly reduces the ultraviolet-B-induced inhibition of keratinocyte proliferation and protects keratinocytes from apoptosis caused by ultraviolet B irradiation in a time- and dose-dependent fashion. Furthermore, the percentage of cells with depolarized mitochondria is significantly lower in ultraviolet-B-irradiated keratinocytes pretreated with carboxyfullerene than in cells provided with diluent alone. Carboxyfullerene also protects human keratinocytes from apoptosis induced by exposure to deoxy-D-ribose, a sugar that causes cell death through a pathway involving oxidative stress. On the other hand, ultraviolet B downregulates bcl-2 levels in human keratinocytes, and carboxyfullerene fails to prevent this effect. These results suggest that carboxy- fullerene protects human keratinocytes from ultraviolet B damage possibly via a mechanism interfering with the generation of reactive oxygen species from depolarized mitochondria without the involvement of bcl-2.
2000
- Keratinocyte nerve growth factor: more than just a neurotrophin
[Relazione in Atti di Convegno]
Pincelli, Carlo; Marconi, Alessandra
abstract
.
2000
- The free-radical scavengers carboxyfullerens protect human keratinocytes from UVB-induced apoptosis
[Abstract in Rivista]
C., Fumelli; Marconi, Alessandra; Giannetti, Alberto; Pincelli, Carlo
abstract
.
1999
- Keratinocytes that adhere most rapidly to type IV collagen and display characteristics of stem cells are protected from apoptosis
[Abstract in Rivista]
Marconi, Alessandra; R., Tiberio; C., Fumelli; Giannetti, Alberto; Pincelli, Carlo
abstract
.
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.
1999
- The neurotrophin family in human keratinocytes
[Abstract in Rivista]
Marconi, Alessandra; M., Terracina; C. M., Failla; G., Romagnoli; R., Maurelli; Pincelli, Carlo; Giannetti, Alberto; G., Zambruno
abstract
.
1998
- Carboxyfullerens reduce apoptotic death induced in cultured human keratinocytes by ultraviolet radiation.
[Abstract in Rivista]
C., Fumelli; Marconi, Alessandra; A., Offidani; D., Monti; R., Pellicciari; Giannetti, Alberto; C., Franceschi; Pincelli, Carlo
abstract
.
1998
- Keratinocytes that adhere most rapidly to type IV collagen and express high levels beta 1 integrin do not undergo apoptosis.
[Abstract in Rivista]
R., Tiberio; Marconi, Alessandra; Giannetti, Alberto; Pincelli, Carlo
abstract
.
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
.
1997
- 1,25-dihydroxyvitamin D-3, transforming growth factor beta 1, calcium, and ultraviolet B radiation induce apoptosis in cultured human keratinocytes
[Articolo su rivista]
Benassi, Luisa; D., Ottani; F., Fantini; Marconi, Alessandra; C., Chiodino; Giannetti, Alberto; Pincelli, Carlo
abstract
Apoptosis is a cellular process of self-directed suicide that plays a key role during morphogenesis and in the maintenance of homeostasis in continuously renewing tissues, Currently, apoptosis is detected mainly by gel electrophoresis of fragmented DNA and by typical ultrastructural features such as cell shrinkage and chromatin condensation, Recently, an in situ technique was developed that allows the detection of the apoptotic process in cells and the quantitation of apoptosis in cell populations, We applied this technique to evaluate the apoptotic process in cultured normal human keratinocytes under basic conditions and after stimulation with factors and agents that are presumed but have never been proved to induce apoptosis in these cells, Apoptosis was analyzed after stimulation with 1,25-dihydroxyvitamin D-3[1,25(OH)(2)D-3], transforming growth factor beta 1 (TGF beta 1), calcium, UVB, or tumor necrosis factor alpha (TNF alpha), All these factors except TNF alpha induced apoptosis in human keratinocytes. Whereas UVB and calcium were good apoptogenic stimuli at 6 and 24 h, respectively, the vitamin D derivative and TGF beta 1 induced apoptosis after 5 and 6 d in culture, Apoptosis was also established by DNA fragmentation and electron microscopy. Finally, TUNEL technique showed that the number of apoptotic cells increases slightly (5-10%) from 24 to 144 h even in untreated keratinocytes, Our studies indicate that factors normally involved in the regulation of cell growth and differentiation can also control apoptosis.
1997
- HaCat keratinocytes overexpressing bcl-2 are resistant to apoptosis induced by inhibition of autocrine nerve growth factor activity.
[Abstract in Rivista]
Pincelli, Carlo; A., Haake; D., Ottani; Benassi, Luisa; Marconi, Alessandra; R., Polakowska; Giannetti, Alberto
abstract
.
1997
- Human keratinocytes overexpressing nerve growth factor are resistant to spontaneous and UV-induced apoptosis.
[Abstract in Rivista]
Marconi, Alessandra; D., Ottani; Giannetti, Alberto; Pincelli, Carlo
abstract
.
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
.
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
.
1995
- Detection Of Circular And Integrated Forms Of Hiv-1 Dna In Epidermal Langerhans Cells Of Aids Patients
[Abstract in Rivista]
G., Zambruno; A., Cimarelli; Marconi, Alessandra; C., Angelinduclos; M., Sala; Giannetti, Alberto; U., Bertazzoni
abstract
.
1995
- Discontinuous distribution of HIV-1 quasispecies in epidermal Langerhans cells of an AIDS patient and evidence for double infection.
[Relazione in Atti di Convegno]
M., Sala; G., Zambruno; J. P., Vartanian; Marconi, Alessandra; Giannetti, Alberto; U., Bertazzoni; S., Wain Hobson
abstract
In the present study HIV-1 quasispecies were analysed in Langerhans cells derived from eight spli-thickness skin patches obtained from clinically normal skin taken soon after autopsy from an AIDS patients (coded RI). RI was an Italian drug user who had been HIV-1 Ab positive from 1986. It is not possible to formally conclude that patient RI was infected twice. RI could have been infected once by blood from a doubly infected individual. However double infection clearly must have occurred at some point. A number of independent experimental studies and phylogenetic analysis provides strong evidence for double infection. That detection is so infrequent might reflect the fact that a second infection most probably would occur in the face of HIV-specific immune response, so restricting viral replication.
1995
- In vitro infection of epidermal Langerhans cells with human immunodeficiency virus type 1
[Relazione in Atti di Convegno]
G., Zambruno; G., Girolomoni; M. C., Re; E., Ramazzotti; Marconi, Alessandra; G., Furlini; M., Vignoli; M., La Placa; Giannetti, Alberto
abstract
Infection of Langerhans cells (LC) by HIV is relevant for several reason. LC of mucosal epithelia may be among the first cells to be infected following mucosal HIV-1 exposure. Secondly, LC may serve as a reservoir for continued infection of CD4+T cells, especially in lymph nodes where epidermal LC migrate following antigenic activation. In this study we have shown that LC can be efficiently infected in vitro with HIV and can transmit the infection to other susceptible target cells.
1995
- In-Vitro Infection Of Human Epidermal Langerhans Cells With Hiv-1
[Articolo su rivista]
E., Ramazzotti; Marconi, Alessandra; M. C., Re; G., Girolomoni; G., Cenacchi; M., Vignoli; G., Zambruno; G., Furlini; M., La Placa; Giannetti, Alberto
abstract
Epidermal Langerhans' cells (LC) from human immunodeficiency virus type-1 (HIV-1)-infected patients harbour HIV-1 proviral DNA and RNA. In the present study, we investigated whether LG from epidermis of normal, HIV-seronegative subjects could be infected in vitro with HIV-1. Epidermal cells (EC) spontaneously detached from epidermal sheet cultures were enriched for LG (10-25% of CD1a(+)/CD4(+) cells), deprived of contaminating T cells and then incubated with HIV-1(IIIB). After 24 hr, purified LC and LC-depleted EG fractions were obtained by immunomagnetic separation. Polymerase chain reaction (PGR) analysis showed the presence of HTV-1 proviral DNA (gag) only in purified LC. In addition, LC-enriched EC, purified LG, LC-depleted EG or the non-permissive cell line, TF-1, the latter having being previously challenged with HIV-1(IIIB) for the same length of time as the EC, were co-cultivated with C8166 cells, and the co-cultures assessed for the presence of HIV DNA by PCR. Go-cultures of C8166 cells with purified LC or LC-enriched EG previously exposed to HIV-1(IIIB) exhibited a time-dependent increase in HIV proviral DNA. In contrast, PCR analysis of C8166 cells co-cultured with either LC-depleted EC or TF-1 cells gave negative results. Finally, C8166 cells co-cultured with HIV-infected LC formed syncytia, showed membrane budding and released numerous retroviral particles. The results indicate that LC from normal subjects can be infected in vitro with HIV and can transmit infection to myeloid cells. This in vitro model may help in understanding the regulation of HIV infection of LC.
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
- Cultured Normal Human Keratinocytes Secrete Stem-Cell Factor And Express C-Kit Receptor
[Abstract in Rivista]
G., Zambruno; Marconi, Alessandra; Manfredini, Rossella; D., Ottani; M., Pizzanelli; Giannetti, Alberto; G., Girolomoni
abstract
nd
1994
- Epidermal Langerhans Cells From An Aids Patient Display A Nonhomogeneous Spatial-Distribution Of Hiv-1 Quasi-Species
[Abstract in Rivista]
M., Sala; G., Zambruno; J. P., Vartanian; Marconi, Alessandra; Giannetti, Alberto; U., Bertazzoni; S., Wain Hobson
abstract
nd
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
.
1994
- Hydroperoxide metabolism in cultured normal human Keratinocytes a spin trapping study
[Capitolo/Saggio]
Giannetti, Alberto; Marconi, Alessandra; G., Zambruno; Iannone, Anna; Tomasi, Aldo
abstract
.
1994
- In-Vitro Infection Of Epidermal Langerhans Cells With Human-Immunodeficiency-Virus Type-1
[Abstract in Rivista]
G., Girolomoni; G., Zambruno; M. C. Re E., Ramazzotti; Marconi, Alessandra; G., Furlini; M., Vignoli; Giannetti, Alberto; M., La placa
abstract
.
1994
- Quantitation By Competitive Pcr Of Hiv-1 Proviral Dna In Epidermal Langerhans Cells Of Hiv-Infected Patients
[Articolo su rivista]
A., Cimarelli; G., Zambruno; Marconi, Alessandra; G., Girolomoni; U., Bertazzoni; Giannetti, Alberto
abstract
Langerhans cells (LC) belong to the dendritic cell family and represent the principal antigen presenting cells populating squamous epithelia. We have reported the presence of human immunodeficiency virus Type 1 (HIV-1) proviral DNA and RNA in purified LC from the epidermis of seropositive patients. The aim of this study was to quantify HIV-1 proviral DNA in LC of infected patients using a competitive polymerase chain reaction (PCR) assay. Bulk epidermal cell (EC) suspensions were obtained from the skin of nine AIDS patients and six seronegative subjects. Purified LC and LC-depleted EC were prepared by immunomagnetic separation using an anti-CD1a monoclonal antibody. LC preparations did not contain T cells, as assessed by reverse transcription PCR analysis of the T cell receptor beta-chain gene (C region). In addition, no CD14(+) cells could be detected in LC fractions by immunostaining of cytospin preparations. To quantify HIV-1 DNA, a new competitive PCR system was devised using SK145/150 as primers (gag) and a competitor plasmid DNA with a modified sequence (209 instead of 142 bp). The number of HIV-1 DNA copies found in the LC of AIDS patients ranged from 107 to 3,645/10(5) LC. In contrast, LC-depleted EC from the same subjects were all negative. The results indicate that in AIDS patients the frequency of infected LC is comparable to that reported for peripheral blood CD4(+) T cells.
1994
- Spatial Discontinuities In Human-Immunodeficiency-Virus Type-1 Quasi-Species Derived From Epidermal Langerhans Cells Of A Patient With Aids And Evidence For Double Infection
[Articolo su rivista]
M., Sala; G., Zambruno; J. P., Vartanian; Marconi, Alessandra; U., Bertazzoni; S., Wain Hobson
abstract
A nonhomogeneous spatial distribution of human immunodeficiency virus type 1 quasispecies was observed for epidermal Langerhans cells purified from skin patches taken from a patient with AIDS soon after death. Each patch presented a unique collection of sequences, distinct from those of juxtaposed patches or those derived from the other leg. Infection of Langerhans cells by virus from underlying T cells in the dermis might explain this partition. The analysis revealed the presence of two distinct cocirculating viral strains, indicating double infection.
1994
- Transforming Growth-Factor (TGF)-Beta(1) Enhances Alpha(5)Beta(1) And Alpha(V)Beta(5) Integrin Expression And Migration Of Normal Human Keratinocytes
[Abstract in Rivista]
G., Zambruno; Marconi, Alessandra; A., Melchiori; P. C., Marchisio; Giannetti, Alberto; DE LUCA, Michele
abstract
nd
1993
- Detection Of Hiv-1 Rna In Epidermal Langerhans Cells Of Hiv-1 Infected Patients
[Abstract in Rivista]
G., Girolomoni; G., Zambruno; A., Cimarelli; Marconi, Alessandra; M., Negroni; U., Bertazzoni; Giannetti, Alberto
abstract
.
1993
- Direct Detection Of Hiv-1 Rna In Epidermal Langerhans Cells Of Hiv-Infected Patients
[Articolo su rivista]
Giannetti, Alberto; G., Zambruno; A., Cimarelli; Marconi, Alessandra; M., Negroni; G., Girolomoni; U., Bertazzoni
abstract
Human Langerhans cells (LC) are bone marrow-derived, HLA-DR+, CD1a+, and CD4+ dendritic antigen-presenting cells found in stratified squamous epithelia. As other members of the dendritic leukocyte family, to which they belong, LC have been reported as targets for HIV-1 infection. The aim of the present study was to investigate whether HIV-1 RNA is expressed in epidermal LC of HIV-1-infected patients. Bulk epidermal cell (EC) suspensions were prepared from skin of nine recently deceased AIDS patients and 11 seronegative controls. Purified LC (94 +/- 4% HLA-DR+ cells with no CD3+ cells, as assessed by flow microfluorimetry analysis) and LC-depleted EC were obtained by immunomagnetic separation using an anti-CD1a monoclonal antibody. Samples were analyzed for the presence of HIV-1 RNA by reverse transcription of a spliced mRNA region of the tat gene, followed by polymerase chain reaction amplification. HIV-1-spliced RNA was detected in LC from 6 of 9 patients examined, whereas LC-depleted EC fractions from the same patients were all negative. The results indicate that epidermal LC from HIV-seropositive patients actively transcribe HIV-1 proviral DNA, further supporting the hypothesis that HIV productively infected LC could serve as a reservoir of the virus in the epidermis and as a source for the infection of T lymphocytes.
1993
- Distinctive Integrin Expression In The Newly Forming Epidermis During Wound-Healing In Humans
[Articolo su rivista]
A., Cavani; G., Zambruno; Marconi, Alessandra; V., Manca; M., Marchetti; Giannetti, Alberto
abstract
The integrin receptor family plays a fundamental role in mediating cell attachment to a variety of extracellular matrix molecules. In normal human epidermis, the alpha2beta1, alpha3beta1, alpha6beta4, and alpha(v)beta5 integrin heterodimers are expressed and appear largely confined to the basal cell layer. In the present study, beta1, beta4, and alpha(v) integrin expression in the epidermis during wound healing in humans was examined. Punch biopsies were performed on healthy volunteers. At daily intervals up to day 8, and at days 11, 14, 21, and 28, the wound site was surgically removed. Using immunofluorescence microscopy, several modifications of the integrin expression pattern were observed on migrating keratinocytes during the re-epithelialization phase of the wound-healing process: i) alpha(v) expression was strongly enhanced and polarized at the basal pole of basal keratinocytes; ii) among the beta1 integrins, alpha3beta1, was overexpressed and distributed over the entire basal keratinocyte membrane and a weak alpha5beta1 reactivity became evident; and iii) alpha6beta4 was detected as a linear staining along the newly forming dermal-epidermal junction. Moreover, both during the re-epithelialization phase and during the first 2 weeks after wound closure, alpha3, alpha6, alpha(v) beta1, and beta4 were no longer confined to the basal layer, as in normal epidermis, but were also found on several suprabasal cell layers. These results suggest that alpha(v)beta5, alpha3beta1, and alpha5beta1 may be the main integrin receptors mediating keratinocyte spreading and migration over the provisional matrix of the wound bed.
1993
- Free-Radical Production During Metabolism Of Organic Hydroperoxides By Normal Human Keratinocytes
[Articolo su rivista]
Iannone, Anna; Marconi, Alessandra; G., Zambruno; Giannetti, Alberto; V., Vannini; Tomasi, Aldo
abstract
Evidence of a relationship between tumor production induced by various organic (hydro)peroxides and free radical formation has been shown in cultured murine keratinocytes and human skin-tumor cell line. In the present study the bioactivation of cumene hydroperoxide, t-butyl-hydroperoxide, and benzoyl peroxide via one-electron oxidation or reduction was compared in freshly isolated and in cultured normal human keratinocytes. The formation of methyl free radicals during the metabolism of cumene and t-butyl-hydroperoxide was shown by the electron spin resonance-spin trapping technique. Radical formation increased under hypoxic conditions. An intracellular activation site was demonstrated by the use of two spin-trapping agents, the hydrophilic, membrane-impermeable, 3,5-dibromo-4-nitrosobenzenesulfonic acid and the lipophilic, membrane-permeable alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone. At 30 min incubation and 25 mM concentration, hydroperoxides exhibited cytotoxicity, as indicated by trypan blue exclusion and lactate dehydrogenase release assay; free radicals were concurrently trapped. Hydroperoxides at a lower concentration (1 mM) did not significantly affect cell viability. However, free radical production was still detected using a membrane-permeable spin trap. The incubation of keratinocytes with benzoyl peroxide did not show any peroxide-dependent radical adduct. No significant differences in bioactivation capability were demonstrated between freshly isolated and cultured human keratinocytes. The results indicate that cultured human keratinocytes can be used as a model system for the study of the metabolic activation to free radical intermediates of toxic and carcinogenic compounds in the epidermis.
1993
- Phosphatidylserine Enhances The Ability Of Epidermal Langerhans Cells To Induce Contact Hypersensitivity
[Articolo su rivista]
G., Girolomoni; S., Pastore; V., Zacchi; A., Cavani; Marconi, Alessandra; Giannetti, Alberto
abstract
Phosphatidylserine (PS) modulates several immune functions in vitro, including T cell activation, antibody and cytokine production, and macrophage growth. In the present work we studied the effects of PS on the induction of contact hypersensitivity (CH) in mice. BALB/c mice painted with PS (9.4-75 mg/kg) and with a sensitizing dose of DNFB or oxazolone on the same skin site exhibited a dose-dependent augmentation of CH reactions to either DNFB (>60%) or oxazolone (>35%), respectively. Bovine brain PS-enriched phospholipid mixture, lyso-PS, and dipalmitoyl-PS also induced similar enhanced CH responses, whereas phosphatidylglycerols had no effect. Increased CH was observed only when PS was applied from 2 days before to 12 h after DNFB. Immunization of naive syngeneic mice with skin grafts that were treated with PS and DNFB also led to enhanced (>50%) CH responses. In addition, immunization by iv injection of epidermal cell suspensions enriched for Langerhans cells (LC) or of purified LC that were treated with PS (1-100 muM, 30 min, 37-degrees-C), and then modified in vitro with DNBS (1 mg/ml, 30 min, 37-degrees-C) led to increased (>30-75%) CH responses in recipient syngeneic animals. Finally, adoptive transfer of DNFB-immune lymph node cells obtained from mice that were treated with PS induced augmented CH responses in recipient animals. The results suggest that PS is capable of up-regulating the induction of CH in mice by stimulating the APC function of epidermal LC.
1992
- Integrin expression during human wound healing
[Abstract in Rivista]
A., Cavani; G., Zambruno; V., Manca; Marconi, Alessandra; Giannetti, Alberto
abstract
nd
1991
- Detection Of Hiv-1 In Epidermal Langerhans Cells Of Hiv-Infected Patients Using The Polymerase Chain-Reaction
[Articolo su rivista]
G., Zambruno; L., Mori; Marconi, Alessandra; N., Mongiardo; B., De Rienzo; U., Bertazzoni; Giannetti, Alberto
abstract
Langerhans cells (LC) are bone marrow - derived, HLA-DR+, CD1a+, dendritic antigen-presenting cells found in stratified squamous epithelia. Within resident epidermal cells (EC), LC are the only cells expressing the CD4 antigen and are, therefore, a possible target for human immunodeficiency virus (HIV) infection. To date, conflicting results have been reported on the in vivo infection of LC by HIV. The aim of the present study was to investigate the presence of HIV-1 proviral DNA in epidermal LC of HIV-1 - infected patients. EC suspensions were prepared from clinically normal skin of nine seropositive patients. Purified LC and LC-depleted EC were obtained by immunomagnetic separation and analyzed for the presence of HIV-1 proviral DNA by the polymerase chain reaction using primer pairs from different conserved regions (env and gag) of the HIV-1 genome. HIV-1 proviral DNA was detected in LC from seven of nine patients. LC-depleted EC fractions from the same nine patients were all negative, with the exception of one case. Altogether these results demonstrate that epidermal LC are infected by HIV-1 and constitute the only resident cell type in the epidermis harboring the virus. Further studies are, however, needed to demonstrate HIV replication in LC and to elucidate the functional role of LC in this infection.
1990
- Free radical detection during xenobiotics metabolism by human keratinocytes
[Capitolo/Saggio]
Giannetti, Alberto; Marconi, Alessandra; M. L., Santantonio; G., Zambruno; Iannone, Anna; Tomasi, Aldo
abstract
.