58 research outputs found

    Expression of CD38 in human neuroblastoma SH-SY5Y cells.

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    Human CD38 antigen is a 42–45 kDa type II transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long C-terminal extracellular region. It is widely expressed in different cell types including thymocytes, activated T cells, and terminally differentiated B cells (plasma cells) and it is involved in cellular proliferation and adhesion. CD38 acts as an ectocyclase that converts NAD+ to the Ca2+-releasing second messenger cyclic ADP-ribose (cADPR). It has been also demonstrated that increased extracellular levels of NAD+ and cADPR are involved in inflammatory diseases and in cellular damage, such as ischemia. In the present study, we have characterized the expression of CD38 in human neuroblastoma SH-SY5Y cell line. All-trans-retinoic acid (ATRA) treatment was used to induce cell differentiation. Our results indicate that: a) even if SH-SY5Y cells have a negative phenotype express CD38 at nuclear level, ATRA treatment does not influence this pattern; b) CD38 localizing to the nucleus may co-localize with p80-coilin positive nuclear-coiled bodies; c) purified nuclei, by Western blot determinations using anti-CD38 antibodies, display a band with a molecular mass of −42 kDa; d) SH-SY5Y cells show nuclear ADP-ribosyl cyclase due to CD38 activity; e) the basal level of CD38 mRNA shows a time-dependent increase after treatment with ATRA. These results suggest that the presence of constitutive fully functional CD38 in the SH-SY5Y nucleus has some important implications for intracellular generation of cADP-ribose and subsequent nucleoplasmic calcium release

    Loss of nuclear BAP1 protein expression is a marker of poor prognosis in patients with clear cell renal cell carcinoma

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    BAP1 is a gene situated on chromosome 3p in a region that is deleted in over 90% of Renal Cell Carcinomas (RCCs) (1,2). In the present study we studied BAP1 immunohistochemical expression in a large series of conventional clear cell RCCs (ccRCCs) treated with radical nephrectomy and we assessed the prognostic value of their expression in terms of patients survival at long-term follow-up. 154 consecutive patients with ccRCC were selected from a prospective database and considered for the study purpose; all patients were treated with radical nephrectomy and lymphadenectomy at our Institute of Urology between 1983 and 1985. The features considered in this study were tumor size, grade and stage, vascular and capsular invasion, incidence of metastasis and patient specific survival; all these parameters were correlated with immunohistochemical cytoplasmic and nuclear expression of BPA-1 in tumoral tissue. Median follow-up was 196.18 months (range 5 to 274); median survival was 125.34 months (range 5 to 274 months). We found that nuclear BAP1 expression showed a high frequency of loss in tumoral cells; nuclear BAP1 negative tumors had higher tumor size, higher Fuhrman grade, and higher stage, a greater amount of vascular and capsular invasion and a higher incidence of metastases. We have demonstrated that nuclear BAP1 expression is a marker of prognosis in ccRCC, having an impact on cancer-specific survival. The clinical importance for BAP1 will be realized with the identification and application of targeted therapies and with individualized approaches in the adjuvant and/or metastatic setting

    IGF1 and IGF1/VEGF cross-talk on human mesenchymal stromal cells (hMSCs): the role of stem cell sources in bone healing

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    Repair of skeletal defects remains a considerable biomedical problem. One of the major obstacles of the different tested strategies still remains the vascularization of engineered scaffolds. To this aim we have examined the ability of IGF1, alone or in association with VEGF, to modulate Periosteum Derived Progenitor Cells (PDPCs) (Ferretti et al., 2012) and Skin-Mesenchymal Stromal Cells (S-MSCs) (Orciani and Di Primio, 2013) osteoblastic or endothelial commitment. A selected gene panel for endothelial and osteoblastic differentiation as well as genes that can affect MAPK and PI3K/AKT signalling pathways were investigated. Our results showed a different commitment of PDPCs and S-MSCs under growth factor stimulation: the former are induced towards an osteoblastic differentiation, whilst the latter seem to be brought to an endothelial phenotype. This commitment is also related to a diverse MAPK or PI3K/AKT signalling pathway activation. Our results open intriguing perspective for the development of innovative bone tissue engineering approaches based on associated angiogenesis and osteogenesis. Further investigations are however necessary to gain insight on the real cross-talk between proliferation and differentiation in adult stem cells. The work was supported by Italian FIRB (RBAP10MLK7) and PRIN (2010J8RYS7) project grants

    The sign of psoriasis in mesenchymal stem cells of the skin

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    Psoriasis is a chronic inflammatory and immune-mediated skin disease, characterized by epidermal hyperproliferation, abnormal keratinocyte differentiation and angiogenesis, whose skin lesions are promoted by exogenous and endogenous factors. The cutaneous and systemic over-expression of several pro-inflammatory cytokines, observed in the initiation, maintenance and recurrence of skin lesions, is known to be caused also by reactive oxygen species (ROS). For this reason it has been postulated that ROS production and compromised function of antioxidant system may be involved in the pathogenesis of psoriasis. Some typical features of psoriasis, like growth rate, expression of VEGF and iNOS as well as the production of VEGF and nitric oxide (NO) and some antioxidant responses, have already been extensively evaluated in differentiated cells of psoriatic skin, but no indications are still available about, the mesenchymal stem, that may be isolated from skin (S-MSCs)

    Bone regeneration strategies in the elderly: the role of ageing and replicative senescence in periosteal-derived stem cells

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    Periosteum contains resident progenitor cells (PDPCs) representing an attractive alternative source of mesenchymal stem cells (MSCs) for skeletal tissue engineering approaches based on cell recruitment (1). Increased in life expectancy point out the necessity for customized strategies to restore bone loss due to trauma and/or disease in elderly. Aim of the present research was the evaluation of the ageing impact on PDPCs isolated from differently aged subjects. Moreover, since long-term culture could lead MSCs to senescence, the effects of culture expansion method on young PDPC through sequential serial passages were examined. Age-related increase of p53 expression and impairment in proliferating capacity were observed; those findings were strictly related to nitric oxide (NO) release. Moreover, qRT-PCR analysis showed a greater expression of genes involved in bone remodelling in elderly donors. As far as replicative in vitro expansion was concerned, we observed that later PDPC passages exhibited the typical “replicative senescence” features (i.e. flattened and enlarged morphology, prolonged population doubling time and increased SA-βgal activity). In these cells, p16 rather than p53 seemed to be involved in senescence processes. Similarly to the elderly, the decrease in proliferating ability of in vitro senescent PDPCs was concomitant with a higher NO production, and the changes in the expression of genes involved in bone resorption and RANKL/OPG ratio were superimposable. Interestingly, the relationship between NO release and ageing could represent a cutting edge “replicative senescence index” as emerged by our System Biology approach. In conclusion, our findings suggest that in vivo cell ageing and in vitro subculturing must be taken into account when testing regenerative tissue strategies that use progenitor cells. Indeed, cells (e.g. MSCs and PDPCs) from the earliest subculture passages could be useful to validate any bone tissue engineering strategies, whilst the later ones could be used to test in vitro scaffolds for regenerative medicine approaches in elderly.This work was supported by grants from MIUR (Project PRIN 2010, MIND-2010J8RYS7)

    Age-related changes in human periosteal derived stem cells: a matter for effective bone regeneration strategies

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    Possible age-related changes in Mesenchymal Stem Cells (MSCs) are of great inter- est in view of their use for regenerative medicine approaches also in the elderly. Con- sidering the primary role of periosteum in bone biology and to acquire data for a cell- based therapy stimulating graft osseointegration, we tried to identify specific aging markers or pattern of expression in human periosteal precursor cells (PDPCs). To this aim periosteal tissue was obtained from differently aged healthy subjects, gender matched with a mean age of 16, 28, 63 and 92 years. Immunohistochemical detection of Ki67 and p53, Nitric Oxide (NO) production and qRT- PCR of a selected gene pan- el for early osteoblastic differentiation (bmp2 and runx2) and bone remodelling (IL-6, RANKL and OPG) were evaluated. Our data evidenced that both Ki67 and p53 rep- resent striking markers of cell-cycle arrest in these cells and that their expression cor- relates with NO production. In addition, age affects genes involved in bone remodel- ling, with a significant increase in IL-6 mRNA expression as well as in RANKL/OPG ratio. As far as NO release is concerned, our data showed higher levels in PDPCs iso- lated from the elderly and a good correlation with the immunohistochemical analysis Moreover, mathematical modelling, in silico simulations and biochemical experiments were combined to investigate about possible underlying quantitative correlations. A clear one-term exponential relationship emerged from a comparison of involved marker trends against age of donors concerning measured NO concentration / Ki67 ratio. This analytical approach confirmed Ki67 as a senescence marker to be focused on. We believe that this study, taking into account age-related changes in human PDPCs, opens up new regenerative medicine strategies for aged bone and/or bone metabolic diseases

    Analysis of tight junctions in placentas affected by chorioamnionitis: in vivo and in vitro analysis

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    The human placenta and fetal membranes provide a barrier regulating the transfer of materials between the mother and the developing fetus throughout gestation. Chorioamnionitis is an important risk factor for preterm delivery that is associated with high perinatal morbidity and mortality. Chorioamnionitis is the term applied to infections of the placenta and membranes resulting in high concentrations of IL- 1beta, IL-6, IL-8 and TGF-beta in the amniotic fluid (D’Alquen et al., 2005). With progression of inflammation, immune cells penetrate blood vessels and infiltrate the umbilical cord, resulting in funisitis (Romero and Mazor, 1988). In normal conditions the two important physical entities in endothelial/epithelial paracellular clefts are adherens junctions and tight junctions. Tight junction governs the paracellular movement of water, solutes and immune cells, through the intercellular space creating a boundary between the apical and basolateral sides of cellular barriers (Gruenheid and Finlay, 2003). We have evaluated the localization of tight junctions studying the Zonula Occludens-1 (ZO-1) and Occludin expressions as well as the localization of adherent junctions, testing the expression of VE-cadherin and beta-catenin in placentas from normal gestations, from preterm idiopathic deliveries and from chorioamnionitis by immunohistochemistry. In addition, we have evaluated the mRNAs by real time PCR, the protein levels of these molecules by Western blot analysis in placental tissues, and to better clarify the action of some cytokines on occludin we performed in vitro analysis of HUVEC cultures. Our more striking result is the decrease of occludin expression in placentas from chorioamnionitis and an evident action of the cytokines on this molecule

    Cellule endoteliali di cordone ombelicale umano: studi delle caratteristiche morfologiche e funzionali

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    Dottorato di ricerca in citomorfologia. 8. ciclo. Coordinatore C. RizzoliConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

    3D CT in the demonstration of ossified stylohyoid complex: a pictorial essay.

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    Abstract Ossification of the stylohyoid complex may be discovered during radiological examination of the cervical spine or at dental panoramic examination. Sometimes it is correlated with atypical facial pain. We report a case in which computed tomography demonstrated the ossified styloid chain with articulations using 3D bony surface reconstruction. -------------------------------------------------------------------------------
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