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.

Seagrass collapse due to synergistic stressors is not anticipated by phenological changes.

Seagrasses are globally declining and often their loss is due to synergies among stressors. We investigated the interactive effects of eutrophication and burial on the Mediterranean seagrass, Posidonia oceanica. A field experiment was conducted to estimate whether shoot survival depends on the interactive effects of three levels of intensity of both stressors and to identify early changes in plants (i.e., morphological, physiological and biochemical, and expression of stress-related genes) that may serve to detect signals of imminent shoot density collapse. Sediment burial and nutrient enrichment produced interactive effects on P. oceanica shoot survival, as high nutrient levels had the potential to accelerate the regression of the seagrass exposed to high burial (HB). After 11 weeks, HB in combination with either high or medium nutrient enrichment caused a shoot loss of about 60%. Changes in morphology were poor predictors of the seagrass decline. Likewise, few biochemical variables were associated with P. oceanica survival (the phenolics, ORAC and leaf δ34S). In contrast, the expression of target genes had the highest correlation with plant survival: photosynthetic genes (ATPa, psbD and psbA) were up-regulated in response to high burial, while carbon metabolism genes (CA-chl, PGK and GADPH) were down-regulated. Therefore, die-offs due to high sedimentation rate in eutrophic areas can only be anticipated by altered expression of stress-related genes that may warn the imminent seagrass collapse. Management of local stressors, such as nutrient pollution, may enhance seagrass resilience in the face of the intensification of extreme climate events, such as floods.

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.

Artificial soft sediment resuspension and high density opportunistic macroalgal mat fragmentation as method for increasing sediment zoobenthic assemblage diversity in a eutrophic lagoon.

Superficial soft sediment resuspension and partial fragmentation of high density opportunistic macroalgal mats were investigated by boat to determine the impact on zoobenthic assemblages in a eutrophic Mediterranean lagoon. Sediment resuspension was used to oxidise superficial organic sediments as a method to counteract the effects of eutrophication. Likewise, artificial decay of macroalgal mat was calculated to reduce a permanent source of sediment organic matter. An area of 9ha was disturbed (zone D) and two other areas of the same size were left undisturbed (zones U). We measured chemical-physical variables, estimated algal biomass and sedimentary organic matter, and conducted qualitative and quantitative determinations of the zoobenthic species detected in sediment and among algal mats. The results showed a constant major reduction in labile organic matter (LOM) and algal biomass in D, whereas values in U remained stable or increased. In the three zones, however, bare patches of lagoon bed increased in size, either by direct effect of the boats in D or by anaerobic decay of the algal mass in U. Zoobenthic assemblages in algal mats reduced the number of species in D, probably due to the sharp reduction in biomass, but remained stable in U, whereas in all three areas abundance increased. Sediment zoobenthic assemblages increased the number of species in D, as expected, due to drastic reduction in LOM, whereas values in U remained stable and again abundance increased in all three zones. In conclusion, we confirmed that reduction of sediment organic load enabled an increase in the number of species, while the algal mats proved to be an important substrate in the lagoon environment for zoobenthic assemblages, especially when mat alternated with bare intermat areas of lagoon bed. Sediment resuspension is confirmed as a management criterion for counteracting the effects of eutrophication and improving the biodiversity of zoobenthic assemblages in eutrophic lagoon environments.

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.

Suitability of the ALien Biotic IndEX (ALEX) for assessing invasion of macroalgae across different Mediterranean habitats.

The ALien Biotic IndEX (ALEX) has been recently proposed to evaluate biological invasions in soft-bottom macro-invertebrate assemblages. The present paper proposes the use of ALEX in sessile assemblages of Mediterranean hard bottom habitats and tests it along gradients of invasion. For five invasive macroalgae a variable number of case studies per each of four habitats were examined from the available data sets. For each case study samples were attributed to four levels of invasion depending on the abundance of the invading macroalgae. Results showed that the application of ALEX to sessile assemblages of hard bottoms allows to qualify the level of invasion along the considered gradients. Moreover, the decline of index values matched the impact of invasion on species number of the assemblages. Results also suggest that the concurrent use of ALEX and indices of benthic quality status can be a valuable tool to assess biopollution in hard bottom habitats.

A new approach to macroalgal bloom control in eutrophic, shallow-water, coastal areas.

In summer 2012, an experiment was conducted in a shallow eutrophic lagoon with poor water exchange to determine the consequences of harvesting algae on the algal mat itself, which was traversed and repeatedly disturbed by large harvester boats. Four areas with high macroalgal density, measuring half a hectare each, were selected. Two were subjected to frequent disturbance of the algal mat and sediment (12 two-hour operations over a 38-day period) and the other two were left undisturbed as control. The following variables were determined: 1) water column physical chemistry and nutrients; 2) redox potential, nutrients and organic load in sediments; 3) C, N and P content of algal thalli; 4) macroalgal biomass. In 2013, a further experiment was conducted on a larger scale. Biomass was estimated in a high-density mat measuring 235 ha, where macroalgae were harvested and stirred up by four harvesting boats, and in two high-density mats measuring 150 and 120 ha, left undisturbed as control (9.15, 9.92 and 3.68 kg/m(2), respectively). In the first experiment, no significant changes were observed in the water column. In sediment the main variation was a significant reduction in labile organic matter in the disturbed areas and a significant increase mainly in refractory organic matter in the undisturbed areas. Biomass showed a significant drastic reduction in disturbed areas and substantial stability in undisturbed areas. In the large-scale experiment, the biomass of the disturbed mat declined by about 63%, only 6.5% of which was due to harvesting. On the other hand, the undisturbed mat with higher density underwent a natural decline in biomass of about 23% and the other increased by about 50%. These results demonstrate that disturbance of high-density mat in shallow water by boats can cause decay of the mat.

Physico-chemical and nutrient variable stratifications in the water column and in macroalgal thalli as a result of high biomass mats in a non-tidal shallow-water lagoon.

A field study to check parameter stratification during high density growth of four opportunistic macroalgae was carried out in Orbetello lagoon (Italy). The effects of macroalgal masses were compared with a seagrass meadow and two lagoon areas with bare bottoms as controls for pH, temperature, dissolved oxygen, salinity, nitrite, nitrate ammonium and orthophosphate. The nutrient content of thalli and sediment redox were measured. Macroalgae showed differences in stratification of thalli nutrient content. Mat with low density and high volume produced stratifications in the water column, but it did not produce nutrient release by sediment. In contrast, high density and low and high volume mats led to sharp falls in dissolved oxygen, with negative values of sediment Eh and anoxic trigger processes that presumably led to release of sediment nutrients. This depended on thallus type: heavy thalli compacted the mat and light ones distributed more widely in the water column.

Assessment of resuspended matter and redistribution of macronutrient elements produced by boat disturbance in a eutrophic lagoon.

Harvesting of macroalgae by specially equipped boats in a shallow eutrophic lagoon produces evident sediment resuspension. To outline the environmental effects of this disturbance, we examined the quantity of fall-out and the distances travelled by sediment and macronutrients from the source of boat disturbance. Resuspended sediment fall-out (RSFO) was trapped at different distances from the boat path to determine total dry weight, total nitrogen (TN), total carbon (TC), total organic carbon (TOC), total sulphur (TS) and total phosphorus (TP). The data was analysed by principal components analysis (PCA) and linear discriminant analysis (LDA) on PCA factors. Fall-out of C, N, S and P from the plume of resuspended sediment indicated significant re-arrangement of these nutrients: RSFO dry weight and S content decreased with distance from the boat path, whereas TP increased and was the variable responsible for most discrimination at 100 m. The mass of resuspended matter was relatively large, indicating that the boats considerably reshuffle lagoon sediment.

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.

Spread of Alsidium corallinum C. Ag. in a Tyrrhenian eutrophic lagoon dominated by opportunistic macroalgae.

In 2007, the Rhodophyceae Alsidium corallinum C. Ag., a marine taxon, bloomed in the eutrophic lagoon of Orbetello (Tuscany, Italy) for the first time, becoming the dominant species in spring and summer. In November, its biomass collapsed. The hypothesis examined in this study is that the bloom expressed a relatively low eutrophic level of the ecosystem after intense disposal of accumulated sedimentary organic matter (OM) by dystrophic processes in the two years preceding the bloom. To verify the hypothesis, we compared water physical-chemical variables, sediment redox (Eh) and OM, and standing crops of macroalgae and seagrass from the database of routine monitoring between 2005 and 2008. We also used dissolved nutrient data obtained in 2007 and 2008, as well as data on chlorophyll and total suspended matter in the water column during the microalgal bloom of 2007, and C, N and P content in thalli of the Chlorophycea Chaetomorpha linum and the Rhodophyceae Gracilariopsis longissima and A. corallinum obtained in 2007. In 2007, unusually low values of dissolved inorganic nitrogen (DIN) were recorded. Combined with stable values of soluble reactive phosphorus (SRPs), low DIN led to a reduction of about one order of magnitude in the DIN:SRP atomic ratio with respect to the past and to 2008. G. longissima accumulated C, N and P more than the other species and A. corallinum proved to be less demanding. Sediment OM was lower in the autumn of years characterized by dystrophy, confirming that summer dystrophic events coincided with maximum energy dissipation in this ecosystem. However, as soon as OM and DIN values increased (2008), the vegetation shifted towards blooms of G. longissima and C. linum, while A. corallinum almost disappeared. The results sustain the hypothesis that the bloom of A. corallinum was due to a decline in DIN that limited G. longissima, and to intense turbidity of the water caused by microphytes that developed after the dystrophic event of summer 2006. The latter probably limited the development of C. linum, which could only develop at the edges of the lagoon.

Threats to macroalgal coralligenous assemblages in the Mediterranean Sea.

Coralligenous habitat is one of the most important coastal systems of the Mediterranean Sea. This paper evaluates the main threats to macroalgal coralligenous habitats through a review of the relevant literature. Sedimentation, nutrient enrichment and biological invasions can cause severe alterations in the structure of coralligenous assemblages due to the regression of perennial structuring species and increases in populations of ephemeral algae. Under pristine conditions, mechanical disturbance seems to be easily mitigated by the recovery capability of coralligenous assemblages; however, such disturbances seriously affect coralligenous structure if they occur in concert with other stressors. Important synergetic effects among all the studied anthropogenic disturbances are also highlighted. The main consequences of the considered stressors are the loss of the complexity of macroalgal assemblages and the deterioration of both alpha and beta diversity.

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.

Effects of nutrient enrichment on macroalgal coralligenous assemblages.

Effects of eutrophication on marine ecosystems have been widely studied, even if both the effects on deep subtidal rocky assemblages and response of different successional stages to nutrients impact are still not clear. In this context, the study aimed to evaluate the effects of nutrient enrichment on Mediterranean macroalgal assemblages associated with coralligenous habitat. A manipulative field experiment was carried out by supplying both mature and early successional stages of assemblages with nutrients. A total of 62 macroalgal species were identified. Multivariate and univariate analyses showed that the structure of both mature and early successional macroalgal assemblages of coralligenous significantly varied between areas treated with nutrients and not treated areas. Moreover, differences were stronger when macroalgal assemblages were in the early successional stage than in the mature one. Results highlighted the role played by nutrients in determining the structure of macroalgal coralligenous assemblages, furthermore suggesting possible synergetic effects with other kinds of disturbances.