Paola Di Gennaro

Cell Renewing in Neuroblastoma: Electrophysiological and Immunocytochemical Characterization of Stem Cells and Derivatives

We explored the stem cell compartment of the SH‐SY5Y neuroblastoma (NB) clone and its development by a novel approach, integrating clonal and immunocytochemical investigations with patch‐clamp measurements of ion currents simultaneously expressed on single cells. The currents selected were the triad IHERG, IKDR, INa, normally expressed at varying mutual ratios during development of neural crest stem cells, from which NB derives upon neoplastic transformation. These ratios could be used as electrophysiological clusters of differentiation (ECDs), identifying otherwise indistinguishable stages in maturation. Subcloning procedures allowed the isolation of highly clonogenic substrate‐adherent (S‐type) cells that proved to be p75‐ and nestinpositive and were characterized by a nude electrophysiological profile (ECDS0). These cells expressed negligible levels of the triad and manifested the capacity of generating the two following lineages: first, a terminally differentiating, smooth muscular lineage, positive for calponin and smooth muscle actin, whose electrophysiological profile is characterized by a progressive diminution of IHERG, the increase of IKDR and INa, and the acquisition of IKIR (ECDS2); second, a neuronal abortive pathway (NF‐68 positive), characterized by a variable expression of IHERG and IKDR and a low expression of INa (ECDNS). This population manifested a vigorous amplification, monopolizing the stem cell compartment at the expense of the smooth muscular lineage to such an extent that neuronal‐like (N‐type) cells must be continuously removed if the latter are to develop.

Ultrastructural and functional alterations of mitochondria in perilesional vitiligo skin

BACKGROUND Vitiligo is a chronic acquired hypomelanotic disorder affecting 0.5-2% of the worlds population. The two major pathogenetic hypotheses are focused on immune-mediated or toxic-mediated cell damage primarily directed at melanocytes. Recent experimental data underline the complex interactions that exist between melanocytes and other cells found in the skin. OBJECTIVE Among these cells, keratinocytes are able to influence both the survival and the functional activity of melanocytes. In order to gain insights into the involvement of different types of epidermic cells in the pathogenesis of vitiligo, we have performed an ultrastructural study on lesional, perilesional and normal skin from 12 patients. All these patients suffered from non-segmental vitiligo, with a similar clinical history in terms of lesion extension and duration of the disease. METHODS We have therefore grown cultures of keratinocytes from lesional, perilesional and healthy skin, evaluating the presence of oxidative damage and apoptotic markers in the cells. RESULTS Taken together, our results indicate that keratinocytes from perilesional skin show features of damaged cells. CONCLUSION Our data, besides considering the achromic patch as the terminal event of a chain of biological processes that take place in the perilesional skin, highlight keratinocytes as having an important role in the development of vitiligo.

Stromal fibroblasts synergize with hypoxic oxidative stress to enhance melanoma aggressiveness

On the basis of recent advances indicating a key role of microenvironment for tumor progression, we investigated the role of fibroblasts, macrophages and hypoxia, for primary melanoma aggressiveness. Our data indicate a key role of hypoxia in stromal reactivity, acting on both myofibroblasts and machrophages differentiation. Hypoxic myofibroblasts are more active than macrophages in inducing melanoma invasiveness and exploit their oxidative stress due to hypoxia to secrete soluble factors favouring melanoma invasion and chemotaxis. We underscore the key role of microenviroment on melanoma malignancy, highlighting reactive fibroblasts, intratumoral hypoxia and oxidative stress as promising targets for melanoma antimetastatic strategies.

Induction of CD83+CD14+ Nondendritic Antigen-Presenting Cells by Exposure of Monocytes to IFN-α

IFN-α is a well-known agent for treatment of viral and malignant diseases. It has several modes of actions, including direct influence on the immune system. We investigated IFN-α effects on PBMC in terms of dendritic cell (DC) differentiation, as PBMC are exposed to high IFN-α levels during treatment of infections and cancers. We show that in vitro IFN-α exposure induced rapid and strong up-regulation of the DC-maturation markers CD80, CD86, and CD83 in bulk PBMC. Consistently, IFN-α induced up-regulation of these molecules on purified monocytes within 24 h. Up-regulation of CD80 and CD83 expression was IFN-α concentration-dependent. In contrast to GM-CSF + IL-4-generated DCs, most of the IFN-α-challenged CD83+ cells coexpressed the monocyte marker CD14. Despite a typical mature DC immunophenotype, IFN-α-treated monocytes conserved phagocytic activity and never acquired a dendritic morphology. In mixed lymphocyte reactions IFN-α-treated monocytes were less potent than GM-CSF + IL-4-generated DCs but significantly more potent than untreated monocytes to induce T cell proliferation in bulk PBMC. However, only GM-CSF + IL-4-generated DCs were able to induce a significant proliferation of naive CD4+ T cells. Notably, autologous memory CD4+ T cells proliferated when exposed to tetanus toxoid-pulsed IFN-α-treated monocytes. At variance with untreated or GM-CSF + IL-4-exposed monocytes, those challenged with IFN-α showed long-lasting STAT-1 phosphorylation. Remarkably, CD83+CD14+ cells were present in varicella skin lesions in close contact with IFN-α-producing cells. The present findings suggest that IFN-α alone promptly generates nondendritic APCs able to stimulate memory immune responses. This may represent an additional mode of action of IFN-α in vivo.

Enhancing anti-melanoma immunity by electrochemotherapy and in vivo dendritic-cell activation

Combining electrochemotherapy with dendritic cell-based immunotherapy is a promising strategy against human metastatic melanoma that deserves to be clinically assessed. While electrochemotherapy induces a rapid regression of metastases, immunotherapy generates systemic anticancer immunity, contributes to eradicate the tumor and maintains an immunological memory to control relapse.

Human Langerhans cells are immature in melanoma sentinel lymph nodes

To the editor: The specific function of Langerhans cells (LCs) in the skin immune system is controversial and a matter of intense debate.[1][1] As epidermal LCs are potential targets for cancer immunotherapy, the topic is of primary importance.[2][2] Recently, van de Ven et al showed that LCs are

IDO and CD83 expression in human epidermal Langerhans cells

Langerhans cells (LCs) are a subset of dendritic cells (DCs) that side within epidermis and mucosae in immature state, self-antigens [3]. Furthermore von Bubnoff et al. [4] reported that mature IFN-g stimulated-LCs transiently expressed indoleamine 2,3-dioxygenase (IDO), a potent tolerogenic enzyme that induces immune peripheral tolerance [5], which is also involved in tumorinduced tolerance [6]. Therefore, IDO expression by LCs might indicate a possible regulatory/inhibitory function [4]. Resident LCs represent a promising target for cancer immunoth LC

Circulating dendritic cell subsets in psoriatic patients before and after biologic therapy.

Dear Editor, Psoriasis is an immune-mediated skin disease affecting approximately 2% of the population; its pathogenesis is characterized by a complex interplay of cells and cytokines. Recently, attention has focused on two main subpopulations of dendritic cells (DC), myeloid DC (mDC) and plasmacytoid DC (pDC), which represent a critical link between the innate and the adaptive immune systems. In the inflamed psoriatic dermis, there is an increase in mDC, probably derived from circulating DC precursors which migrate into the skin in response to chemotactic signals and synthesize high levels of pro-inflammatory cytokines (e.g. interleukin [IL]-12 and IL-23). pDC have been identified in the lesional and in the non-lesional psoriatic skin. Activated pDC produce interferon (IFN)-a in the early phase of the development of a psoriasis lesion with a strong amplification of a pathogenetic role of T lymphocytes. The antimicrobial peptide LL37 is expressed in high concentrations in psoriatic skin and through its binding with self-DNA is a potent activator of pDC. In a previous work, we have reported a reduction of both mDC and pDC in psoriatic lesions upon treatment with biologic therapies, but to date no studies have compared blood DC before and upon biologic treatment. Our purpose was to monitor the blood DC compartment in psoriatic patients before and during biologic therapy with infliximab (an anti-tumor necrosis factor-a molecule). Our analysis could better focus the activity of this drug on the number and phenotype of DC. Eight patients (four male and four female; aged 44–80 years, mean 46.25 years) affected by severe psoriasis (Psoriasis Area and Severity Index [PASI] score >10) treated at the PsoCare center of the Dermatological Unit of Florence University, were evaluated before and after 6 months of infliximab therapy (Shering-Plough, Levallois-Perret, France), given at a dose of 3 mg ⁄ kg i.v. then every 8 weeks. In addition, four healthy controls (matched with patients for sex and age) were included in the study. The study was approved by the local ethics committee, and all patients gave informed consent. Peripheral blood mononuclear cells were isolated from 3 mL of peripheral blood by density gradient centrifugation with Lymphoprep (Axis-Shield, Oslo, Norway) and washed in phosphatebuffered saline (PBS; EuroClone, Whetherby, UK). Multiple cell staining was performed with anti-BDCA-1-FITC conjugated (AD5-8E7, IgG2a) and BDCA-2-PE (AC144, IgG1) from Miltenyi Biotec (Bergisch, Gladbach, Germany), anti-HLA-DR-Pe ⁄ Cy7 (L243, IgG2a) from BioLegend (San Diego, CA, USA) and CD86Pe ⁄ Cy5 (2331 FUN-1, IG1) from BD-Pharmingen (San Diego, CA, USA) for 30 min at 4 C. All monoclonal antibodies dilutions and wash steps were done in PBS (EuroClone) containing 2% FCS (EuroClone) and 2 mm ⁄ L ethylenediamine tetraacetic acid (Sigma, Milan, Italy). Isotype-matched antibodies were used as negative controls. Cells were acquired using a FACSCanto flow cytometer (Becton-Dickinson, San Jose, CA, USA). The mDC and pDC were identified by the concomitant HLA-DR expression and mutually exclusive membrane expression of BDCA-1 or BDCA-2, respectively. The expression of CD86 required for T-cell activation was also analyzed. Statistical analysis was performed using Microsoft Excel software. The mean ± standard deviation of the percentage of DC and their subsets were calculated. Student’s t-test was used to compare the DC populations identified. Correlations were considered significant at P < 0.05. We detected both mDC and pDC in peripheral blood of untreated psoriatic patients and controls. The proportions of DC subsets are given in Table 1. Interestingly, psoriatic peripheral blood was characterized by a decreased number of both mDC and pDC subsets: mDC constituted 3.2 ± 0.3% of total peripheral blood mononuclear cells in untreated psoriatic patients versus 6.8 ± 4.5% in the healthy control group (P < 0.05) and pDC constituted

High Antigen Processing Machinery component expression in Langerhans cells from melanoma patients’ sentinel lymph nodes

Langerhans cells (LCs) from melanoma patients sentinel lymph nodes (SLN) are poor T cell activators mostly due to an immature immunophenotype. However Antigen Presenting Machinery (APM) role is unknown. We investigated HLA-class I APM components (Delta, LMP-7/10, TAP-1, Calnexin, Tapasin, β2-microglobulin and HLA-A,B,C) in LCs from healthy donors skin and melanoma patients SLN. APM component levels were low in immature epidermal LCs and significantly increased after maturation (p<0.05); their levels were significantly high in SLN LCs (p<0.01). APM component expression correlated with melanoma Breslows thickness and SLN metastases: HLA-A,B,C level was significantly lower in SLN LCs from thick lesions patients compared with those from thin/intermediate lesions (p<0.05); β2-microglobulin level was significantly higher in positive SLN LCs compared to negative ones (p<0.05). Functionally, SLN LCs did not phagocytose exogenous antigens. These findings extend LCs knowledge indicating that they are not fully impaired by melanoma, contributing to design new LCs-based therapeutic approaches.