Tolerogenic IDO1+CD83- Langerhans Cells in Sentinel Lymph Nodes of Patients with Melanoma.

Langerhans cells (LCs) are crucial regulators of anti-cancer immune responses. Cancer, however, can alter DCs functions leading to tolerance. The enzyme indoleamine 2,3-dioxygenase (IDO1) plays a crucial role in this process. In sentinel lymph nodes (SLNs) of patients with melanoma, LCs show phenotypical and functional alterations favoring tolerance. Herein we aimed to investigate IDO1 expression in SLN LCs from patients with melanoma. We showed by immunofluorescence analysis that a portion of Langerin+ LCs, located in the SLN T cell-rich area, displayed the typical dendritic morphology and expressed IDO1. There was no significant difference in the expression of IDO between SLN with or without metastases. Double IDO1/CD83 staining identified four LCs subsets: real mature IDO1−CD83+ LCs; real immature IDO1−CD83− LCs; tolerogenic mature IDO1+CD83+ LCs; tolerogenic immature IDO1+CD83− LCs. The latter subset was significantly increased in metastatic SLNs as compared to negative ones (p < 0.05), and in SLN LCs of patients with mitotic rate (MR) > 1 in primary melanoma, as compared to MR ≤ 1 (p < 0.05). Finally, immature SLN LCs, after in vitro stimulation by inflammatory cytokines, acquired a maturation profile by CD83 up-regulation. These results provide new input for immunotherapeutic approaches targeting in vivo LC of patients with melanoma.

Establishment and characterization of a new spontaneously immortalized ER-/PR-/HER2+ human breast cancer cell line, DHSF-BR16.

Invasive ductal carcinoma (IDC) constitutes the most frequent malignant cancer endangering women's health. In this study, a new spontaneously immortalized breast cancer cell line, DHSF-BR16 cells, was isolated from the primary IDC of a 74-years old female patient, treated with neoadjuvant chemotherapy and disease-free 5-years after adjuvant chemotherapy. Primary breast cancer tissue surgically removed was classified as ER-/PR-/HER2+, and the same phenotype was maintained by DHSF-BR16 cells. We examined DHSF-BR16 cell morphology and relevant biological and molecular markers, as well as their response to anticancer drugs commonly used for breast cancer treatment. MCF-7 cells were used for comparison purposes. The DHSF-BR16 cells showed the ability to form spheroids and migrate. Furthermore, DHSF-BR16 cells showed a mixed stemness phenotype (i.e. CD44+/CD24-/low), high levels of cytokeratin 7, moderate levels of cytokeratin 8 and 18, EpCAM and E-Cadh. Transcriptome analysis showed 2071 differentially expressed genes between DHSF-BR16 and MCF-7 cells (logFC > 2, p-adj < 0.01). Several genes were highly upregulated or downregulated in the new cell line (log2 scale fold change magnitude within - 9.6 to + 12.13). A spontaneous immortalization signature, mainly represented by extracellular exosomes-, plasma membrane- and endoplasmic reticulum membrane pathways (GO database) as well as by metabolic pathways (KEGG database) was observed in DHSF-BR16 cells. Also, these cells were more resistant to anthracyclines compared with MCF-7 cells. Overall, DHSF-BR16 cell line represents a relevant model useful to investigate cancer biology, to identify both novel prognostic and drug response predictive biomarkers as well as to assess new therapeutic strategies.

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, ß2-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 Breslow's 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); ß2-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.

The chemokine (C-C motif) ligand protein synthesis inhibitor bindarit prevents cytoskeletal rearrangement and contraction of human mesangial cells.

Intraglomerular mesangial cells (MCs) maintain structural and functional integrity of renal glomerular microcirculation and homeostasis of mesangial matrix. Following different types of injury, MCs change their phenotype upregulating the expression of α-smooth muscle actin (α-SMA), changing contractile abilities and increasing the production of matrix proteins, chemokines and cytokines. CCL2 is a chemokine known to be involved in the pathogenesis of renal diseases. Its glomerular upregulation correlates with the extent of renal damage. Bindarit is an indazolic derivative endowed with anti-inflammatory activity when tested in experimental diseases. It selectively inhibits the synthesis of inflammatory C-C chemokines including CCL2, CCL7 and CCL8. This work aims to analyse bindarit effects on ET1-, AngII- and TGFß-induced mesangial cell dysfunction. Bindarit significantly reduced AngII-, ET1- and TGFß-induced α-SMA upregulation. In a collagen contraction assay, bindarit reduced AngII-, ET1- and TGFß-induced HRMC contraction. Within 3-6h stimulation, vinculin organization and phosphorylation was significantly impaired by bindarit in AngII-, ET1- and TGFß-stimulated cells without any effect on F-actin distribution. Conversely, p38 phosphorylation was not significantly inhibited by bindarit. Our data strengthen the importance of CCL2 on ET-1, AngII- and TGFß-induced mesangial cell dysfunction, adding new insights into the cellular mechanisms responsible of bindarit protective effects in human MC dysfunction.

Dendritic cells recruitment in melanoma metastasis treated by electrochemotherapy.

Electrochemotherapy (ECT) is a novel treatment for recurrent or in-transit unresectable melanoma metastases based on the administration of anti-neoplastic drugs followed by cancer cell electroporation. Whether ECT can also induce anti-tumour immunity is unclear. We addressed this issue investigating the presence of dendritic cells (DCs) in the inflammatory infiltrate of ECT-treated lesions. Biopsies from melanoma patients (n = 9) were taken before ECT (T0), at d7 and d14 after treatment and studied by immunofluorescence with DCs-related antibodies. Epidermal Langerin(+) Langerhans cells (LCs) were the most represented subset before treatment. ECT induced a significant reduction in epidermal LCs number at d7 (p < 0.001), while they were completely replaced at d14. Similarly, the few LCs observed intermingled with metastatic melanoma cells at T0 decreased after treatment (p < 0.001), suggesting an ECT-induced activation of LCs. Consistently, at d1 after ECT (n = 3 patients), LCs were found to express CCR7, which mediates LCs migration to regional lymph nodes, and CD83, the typical DCs maturation marker. In contrast, plasmacytoid DCs (pDCs) were not present at T0, but significantly increased after ECT both in melanoma metastasis (p < 0.001) and perilesionally (p < 0.05). Similarly, CD1c(+) dermal DCs (dDCs), observed in low number before ECT, strongly increased at d7 and even more at d14 (p < 0.05 and p < 0.001, respectively). Notably, some dDCs expressed CD83. These data suggest that ECT promotes LCs migration from the tumour to draining lymph nodes and pDCs and dDCs recruitment at the site of the lesion. These findings may help to design new strategies of in situ DCs vaccination in cancer patients.

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.

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.

Ultrastructural and functional alterations of mitochondria in perilesional vitiligo skin.

BACKGROUND: Vitiligo is a chronic acquired hypomelanotic disorder affecting 0.5-2% of the world's 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.

Induction of CD83+CD14+ nondendritic antigen-presenting cells by exposure of monocytes to IFN-alpha.

IFN-alpha 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-alpha effects on PBMC in terms of dendritic cell (DC) differentiation, as PBMC are exposed to high IFN-alpha levels during treatment of infections and cancers. We show that in vitro IFN-alpha exposure induced rapid and strong up-regulation of the DC-maturation markers CD80, CD86, and CD83 in bulk PBMC. Consistently, IFN-alpha induced up-regulation of these molecules on purified monocytes within 24 h. Up-regulation of CD80 and CD83 expression was IFN-alpha concentration-dependent. In contrast to GM-CSF + IL-4-generated DCs, most of the IFN-alpha-challenged CD83(+) cells coexpressed the monocyte marker CD14. Despite a typical mature DC immunophenotype, IFN-alpha-treated monocytes conserved phagocytic activity and never acquired a dendritic morphology. In mixed lymphocyte reactions IFN-alpha-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-alpha-treated monocytes. At variance with untreated or GM-CSF + IL-4-exposed monocytes, those challenged with IFN-alpha showed long-lasting STAT-1 phosphorylation. Remarkably, CD83(+)CD14(+) cells were present in varicella skin lesions in close contact with IFN-alpha-producing cells. The present findings suggest that IFN-alpha alone promptly generates nondendritic APCs able to stimulate memory immune responses. This may represent an additional mode of action of IFN-alpha in vivo.

Plasmacytoid dendritic cells represent a major dendritic cell subset in sentinel lymph nodes of melanoma patients and accumulate in metastatic nodes.

Plasmacytoid dendritic cells (pDC) represent the main source of interferon-alpha, a cytokine with antitumor activity. However, in vitro studies point to pDC as a key subset for induction of tolerance. Herein, we investigated pDC in sentinel lymph nodes (SLN) of melanoma patients. We report that pDC were constantly found in SLN and represented, with Langerhans cells, the most frequent dendritic cell subset. Their frequency in positive (with metastasis) SLN was significantly higher than in negative (without metastasis) SLN. PDC were observed in the T cell-rich areas of lymph nodes, particularly around high endothelial venules and, in metastatic nodes, they accumulated in close vicinity with melanoma nests. Finally, pDC capability to produce interferon-alpha in situ was impaired. Consistently, pDC expressed CD86, but neither CD80 nor CD83, suggesting a not complete activation in melanoma-draining lymph nodes. These results are consistent with the hypothesis of a tolerogenic role played by pDC in tumor immunology.

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.