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The identification of discrete neutrophil populations, as well as the characterization of their immunoregulatory properties, is an emerging topic under extensive investigation. In such regard, the presence of circulating CD66b+ neutrophil populations, exerting either immunosuppressive or proinflammatory functions, has been described in several acute and chronic inflammatory conditions. However, due to the lack of specific markers, the precise phenotype and maturation status of these neutrophil populations remain unclear. Herein, we report that CD10, also known as common acute lymphoblastic leukemia antigen, neutral endopeptidase, or enkephalinase, can be used as a marker that, within heterogeneous populations of circulating CD66b+ neutrophils present in inflammatory conditions, clearly distinguishes the mature from the immature ones. Accordingly, we observed that the previously described immunosuppressive neutrophil population that appears in the circulation of granulocyte colony-stimulating factor (G-CSF)-treated donors (GDs) consists of mature CD66b+CD10+ neutrophils displaying an activated phenotype. These neutrophils inhibit proliferation and interferon γ (IFNγ) production by T cells via a CD18-mediated contact-dependent arginase 1 release. By contrast, we found that immature CD66b+CD10- neutrophils, also present in GDs, display an immature morphology, promote T-cell survival, and enhance proliferation and IFNγ production by T cells. Altogether, our findings uncover that in GDs, circulating mature and immature neutrophils, distinguished by their differential CD10 expression, exert opposite immunoregulatory properties. Therefore, CD10 might be used as a phenotypic marker discriminating mature neutrophils from immature neutrophil populations present in patients with acute or chronic inflammatory conditions, as well as facilitating their isolation, to better define their specific immunoregulatory properties.

Scientific Reports 7: Article number: 40037; published online: 04 January 2017; updated: 22 May 2018 The original version of this Article contained an error in Figure 7A, where the immunoblot of flotillin from HeLa was a duplicate of the immunoblot of flotillin from HeLa-LAI. This error has been corrected in the HTML and PDF versions of the Article and the figure has been appropriately delineated.

TNF-like ligand 1A (TL1A) and its unique receptor death receptor 3 (DR3) acts as broad T-cell costimulator involved in regulatory mechanisms of adaptive immune response under physiological and pathological settings. Moreover, we have recently shown that TL1A negatively regulates B-cell proliferation. Despite increasing interest on the TL1A/DR3-axis functions, very little is known on its expression and role in leukemia. In this study, we investigated the expression and function of TL1A/DR3 axis in chronic lymphocytic leukemia (CLL). DR3 was differentially expressed in activated CLL cells and predominantly detected in patients with early clinical stage disease. Soluble TL1A has been revealed in the sera of CLL patients where higher TL1A levels were associated with early stage disease. T cells, monocytes and leukemic B cells have been identified as major sources of TL1A in CLL. The relevance of these findings has been sustained by functional data showing that exogenous TL1A reduces CLL proliferation induced by stimulation of the B cell receptor. Overall, these data document the expression of the TL1A/DR3 axis in early-stage CLL. They also identify a novel function for TL1A as a negative regulator of leukemic cell proliferation that may influence the CLL physiopathology and clinical outcome at an early-stage disease.

Human blood dendritic cells (DCs) include three main distinct subsets, namely the CD1c+ and CD141+ myeloid DCs (mDCs) and the CD303+ plasmacytoid DCs (pDCs). More recently, a population of slan/M-DC8+ cells, also known as "slanDCs", has been described in blood and detected even in inflamed secondary lymphoid organs and non-lymphoid tissues. Nevertheless, hallmarks of slan/M-DC8+ cells in tissues are poorly defined. Herein, we report a detailed characterization of the phenotype and function of slan/M-DC8+ cells present in human tonsils. We found that tonsil slan/M-DC8+ cells represent a unique DC cell population, distinct from their circulating counterpart and also from all other tonsil DC and monocyte/macrophage subsets. Phenotypically, slan/M-DC8+ cells in tonsils display a CD11c+HLA-DR+CD14+CD11bdim/negCD16dim/negCX3CR1dim/neg marker repertoire, while functionally they exhibit an efficient antigen presentation capacity and a constitutive secretion of TNFα. Notably, such DC phenotype and functions are substantially reproduced by culturing blood slan/M-DC8+ cells in tonsil-derived conditioned medium (TDCM), further supporting the hypothesis of a full DC-like differentiation program occurring within the tonsil microenvironment. Taken together, our data suggest that blood slan/M-DC8+ cells are immediate precursors of a previously unrecognizedcompetent DC subset in tonsils, and pave the way for further characterization of slan/M-DC8+ cells in other tissues.

HLA-C has been demonstrated to associate with HIV-1 envelope glycoprotein (Env). Virions lacking HLA-C have reduced infectivity and increased susceptibility to neutralizing antibodies. Like all others MHC-I molecules, HLA-C requires β2-microglobulin (β2m) for appropriate folding and expression on the cell membrane but this association is weaker, thus generating HLA-C free-chains on the cell surface. In this study, we deepen the understanding of HLA-C and Env association by showing that HIV-1 specifically increases the amount of HLA-C free chains, not bound to β2m, on the membrane of infected cells. The association between Env and HLA-C takes place at the cell membrane requiring β2m to occur. We report that the enhanced infectivity conferred to HIV-1 by HLA-C specifically involves HLA-C free chain molecules that have been correctly assembled with β2m. HIV-1 Env-pseudotyped viruses produced in the absence of β2m are less infectious than those produced in the presence of β2m. We hypothesize that the conformation and surface expression of HLA-C molecules could be a discriminant for the association with Env. Binding stability to β2m may confer to HLA-C the ability to preferentially act either as a conventional immune-competent molecule or as an accessory molecule involved in HIV-1 infectivity.

Human SH-SY5Y neuroblastoma cells are widely utilized in in vitro studies to dissect out pathogenetic mechanisms of neurodegenerative disorders. These cells are considered as neuronal precursors and differentiate into more mature neuronal phenotypes under selected growth conditions. In this study, in order to decipher the pathways and cellular processes underlying neuroblastoma cell differentiation in vitro, we performed systematic transcriptomic (RNA-seq) and bioinformatic analysis of SH-SY5Y cells differentiated according to a two-step paradigm: retinoic acid treatment followed by enriched neurobasal medium. Categorization of 1989 differentially expressed genes (DEGs) identified in differentiated cells functionally linked them to changes in cell morphology including remodelling of plasma membrane and cytoskeleton, and neuritogenesis. Seventy-three DEGs were assigned to axonal guidance signalling pathway, and the expression of selected gene products such as neurotrophin receptors, the functionally related SLITRK6, and semaphorins, was validated by immunoblotting. Along with these findings, the differentiated cells exhibited an ability to elongate longer axonal process as assessed by the neuronal cytoskeletal markers biochemical characterization and morphometric evaluation. Recognition of molecular events occurring in differentiated SH-SY5Y cells is critical to accurately interpret the cellular responses to specific stimuli in studies on disease pathogenesis.

The anti-inflammatory actions of IL-4 are well established through earlier findings. However, the exact mechanism it uses to downregulate the pro-inflammatory cytokine production through monocytes and macrophages is poorly understood. In this study, we examined the effect of IL-4 in the induction of 11β-HSD1 in the two main classes of monocytes, CD14++ CD16- (CD14) and CD14+ CD16+ (CD16). Peripheral Blood Mononuclear Cells (PBMCs) were isolated from 17 healthy donors and were sorted into CD14 and CD16 subpopulations using cell sorting. Effect of IL-4 on 11β-HSD1-enzyme activity was measured in sorted and unsorted monocytes using Homogeneous Time-Resolved Fluorescence (HTRF) and M1/M2 polarization analysis was performed by flow cytometry. Our results indicate that CD14 cells are the major source of 11β-HSD1 enzyme after IL-4 stimulation and that M2 phenotype is not a pre-requisite for its synthesis.

Resveratrol, a polyphenolic-stilbene, has received increased attention in the last decade due to its wide range of biological activities. Beta(β)-thalassemias are inherited red cell disorders, found worldwide, characterized by ineffective erythropoiesis and red cell oxidative damage with reduced survival. We evaluated the effects of low-dose-resveratrol (5 μM) on in vitro human erythroid differentiation of CD34(+) from normal and β-thalassemic subjects. We found that resveratrol induces accelerated erythroid-maturation, resulting in the reduction of colony-forming units of erythroid cells and increased intermediate and late erythroblasts. In sorted colony-forming units of erythroid cells resveratrol activates Forkhead-box-class-O3, decreases Akt activity and up-regulates anti-oxidant enzymes as catalase. In an in vivo murine model for β-thalassemia, resveratrol (2.4 mg/kg) reduces ineffective erythropoiesis, increases hemoglobin levels, reduces reticulocyte count and ameliorates red cell survival. In both wild-type and β-thalassemic mice, resveratrol up-regulates scavenging enzymes such as catalase and peroxiredoxin-2 through Forkhead-box-class-O3 activation. These data indicate that resveratrol inhibits Akt resulting in FoxO3 activation with upregulation of cytoprotective systems enabling the pathological erythroid precursors to resist the oxidative damage and continue to differentiate. Our data suggest that the dual effect of resveratrol on erythropoiesis through activation of FoxO3 transcriptional factor combined with the amelioration of oxidative stress in circulating red cells may be considered as a potential novel therapeutic strategy in treating β-thalassemia.

The anti-inflammatory actions of IL-4 are well established through earlier findings. However, the exact mechanism it uses to downregulate the pro-inflammatory cytokine production through monocytes and macrophages is poorly understood. In this study, we examined the effect of IL-4 in the induction of 11β-HSD1 in the two main classes of monocytes, CD14

The identification of discrete neutrophil populations, as well as the characterization of their immunoregulatory properties, is an emerging topic under extensive investigation. In such regard, the presence of circulating CD66b

// Alessandra Micheletti 1 , Giulia Finotti 1 , Federica Calzetti 1 , Silvia Lonardi 2 , Elisa Zoratti 3 , Mattia Bugatti 2 , Stefania Stefini 4 , William Vermi 2,5 and Marco A. Cassatella 1 1 Department of Medicine, Section of General Pathology, University of Verona, Verona, Italy 2 Department of Molecular and Translational Medicine, Section of Pathology, University of Brescia, Brescia, Italy 3 Applied Research on Cancer-Network (ARC-NET), University of Verona, Verona, Italy 4 Unit of Pediatric Otorhinolaryngology, Spedali Civili di Brescia, Brescia, Italy 5 Department of Pathology and Immunology, Washington University School of Medicine, Saint Louis, Missouri, USA Correspondence to: Marco A. Cassatella, email: // Keywords : slan/M-DC8+ cells, dendritic cells, monocytes, tonsil, differentiation, Immunology and Microbiology Section, Immune response, Immunity Received : September 10, 2015 Accepted : November 22, 2015 Published : December 18, 2015 Abstract Human blood dendritic cells (DCs) include three main distinct subsets, namely the CD1c + and CD141 + myeloid DCs (mDCs) and the CD303 + plasmacytoid DCs (pDCs). More recently, a population of slan/M-DC8 + cells, also known as “slanDCs”, has been described in blood and detected even in inflamed secondary lymphoid organs and non-lymphoid tissues. Nevertheless, hallmarks of slan/M-DC8 + cells in tissues are poorly defined. Herein, we report a detailed characterization of the phenotype and function of slan/M-DC8 + cells present in human tonsils. We found that tonsil slan/M-DC8 + cells represent a unique DC cell population, distinct from their circulating counterpart and also from all other tonsil DC and monocyte/macrophage subsets. Phenotypically, slan/M-DC8 + cells in tonsils display a CD11c + HLA-DR + CD14 + CD11b dim/neg CD16 dim/neg CX3CR1 dim/neg marker repertoire, while functionally they exhibit an efficient antigen presentation capacity and a constitutive secretion of TNFα. Notably, such DC phenotype and functions are substantially reproduced by culturing blood slan/M-DC8 + cells in tonsil-derived conditioned medium (TDCM), further supporting the hypothesis of a full DC-like differentiation program occurring within the tonsil microenvironment. Taken together, our data suggest that blood slan/M-DC8 + cells are immediate precursors of a previously unrecognizedcompetent DC subset in tonsils, and pave the way for further characterization of slan/M-DC8 + cells in other tissues.

Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed when metastatic events have occurred. Cancer stem cells (CSCs) play an important role in tumor initiation, metastasis, chemoresistance and relapse. A growing number of studies have suggested that CSCs exist in a dynamic equilibrium with more differentiated cancer cells via a bi‑directional regeneration that is dependent on the environmental stimuli. In this investigation, we obtain, by using a selective medium, PDAC CSCs from five out of nine PDAC cell lines, endowed with different tumorsphere‑forming ability. PDAC CSCs were generally more resistant to the action of five anticancer drugs than parental cell lines and were characterized by an increased expression of EpCAM and CD44v6, typical stem cell surface markers, and a decreased expression of E‑cadherin, the main marker of the epithelial state. PDAC CSCs were able to re‑differentiate into parental cells once cultured in parental growth condition, as demonstrated by re‑acquisition of the epithelial morphology, the decreased expression levels of EpCAM and CD44v6 and the increased sensitivity to anticancer drugs. Finally, PDAC CSCs injected into nude mice developed a larger subcutaneous tumor mass and showed a higher metastatic activity compared to parental cells. The present study demonstrates the ability to obtain CSCs from several PDAC cell lines and that these cells are differentially resistant to various anticancer agents. This variability renders them a model of great importance to deeply understand pancreatic adenocarcinoma biology, to discover new biomarkers and to screen new therapeutic compounds.

A human bone marrow-derived mesenchymal stromal cell (MSCs) and cord blood-derived CD34+ stem cell co-culture system was set up in order to evaluate the proliferative and differentiative effects induced by MSCs on CD34+ stem cells, and the reciprocal influences on gene expression profiles. After 10 days of co-culture, non-adherent (SN-fraction) and adherent (AD-fraction) CD34+ stem cells were collected and analysed separately. In the presence of MSCs, a significant increase in CD34+ cell number was observed (fold increase = 14.68), mostly in the SN-fraction (fold increase = 13.20). This was combined with a significant increase in CD34+ cell differentiation towards the BFU-E colonies and with a decrease in the CFU-GM. These observations were confirmed by microarray analysis. Through gene set enrichment analysis (GSEA), we noted a significant enrichment in genes involved in heme metabolism (e.g. LAMP2, CLCN3, BMP2K), mitotic spindle formation and proliferation (e.g. PALLD, SOS1, CCNA1) and TGF-beta signalling (e.g. ID1) and a down-modulation of genes participating in myeloid and lymphoid differentiation (e.g. PCGF2) in the co-cultured CD34+ stem cells. On the other hand, a significant enrichment in genes involved in oxygen-level response (e.g. TNFAIP3, SLC2A3, KLF6) and angiogenesis (e.g. VEGFA, IGF1, ID1) was found in the co-cultured MSCs. Taken together, our results suggest that MSCs can exert a priming effect on CD34+ stem cells, regulating their proliferation and erythroid differentiation. In turn, CD34+ stem cells seem to be able to polarise the BM-niche towards the vascular compartment by modulating molecular pathways related to hypoxia and angiogenesis.

Summary The role of dendritic cells (DCs) and macrophages in allogeneic haematopoietic stem cell transplant (HSCT) is critical in determining the extent of graft-versus-host response. The goal of this study was to analyse slanDCs, a subset of human proinflammatory DCs, in haematopoietic stem cell (HSC) sources, as well as to evaluate their 1-year kinetics of reconstitution, origin and functional capacities in peripheral blood (PB) and bone marrow (BM) of patients who have undergone HSCT, and their presence in graft-versus-host disease (GVHD) tissue specimens. slanDCs were also compared to myeloid (m)DCs, plasmacytoid (p)DCs and monocytes in HSC sources and in patients' PB and BM throughout reconstitution. slanDCs accounted for all HSC sources. In patients' PB and BM, slanDCs were identified from day +21, showing median frequencies comparable to healthy donors, donor origin and kinetics of recovery similar to mDCs, pDCs, and monocytes. Under cyclosporin treatment, slanDCs displayed a normal pattern of maturation, and maintained an efficient chemotactic activity and capacity of releasing tumour necrosis factor (TNF)-α upon lipopolysaccharide (LPS) stimulation. None the less, they were almost undetectable in GVHD tissue specimens, being present only in intestinal acute GVHD samples. slanDCs reconstitute early, being donor-derived and functionally competent. The absence of slanDCs from most of the GVHD-targeted tissue specimens seems to rule out the direct participation of these cells in the majority of the local reactions characterizing GVHD.

In both type 1 and type 2 diabetes, increased production of cytokines on autoimmune or metabolic basis is supposed to trigger an inflammatory process leading to dysfunction and death of pancreatic beta-cells. Therefore, anti-inflammatory pharmacological approaches aimed at blocking cytokine signalling pathways and consequent cytotoxicity in beta-cells are highly advisable. Based on previous evidence of cytokine antagonistic effects in other cell types, we explored the protective action of Hypericum perforatum (St-John's-wort) extract and its component hyperforin against cytokine-induced functional impairment and apoptosis in the INS-1E beta-cell line, searching for the underlying mechanisms. The results showed that either St-John's-wort extract or hyperforin (at 1-3 microM) prevented cytokine-induced impairment in glucose-stimulated insulin secretion and protected cells against apoptosis in a dose-dependent fashion. Inducible-NO-synthase expression was also potently hindered by the vegetal compounds. Interestingly, cytokine-induced activations of the signal-transducer-and-activator-of-transcription-1 (STAT-1) and the nuclear-factor-kappaB (NF-kappaB) were both down-regulated by SJW extract or HPF (range 0.5-5 microM) when evaluated by electrophoretic-mobility-shift-assay. Other transcription factors (CBF-1, SP-1) were unaffected. Components of SJW extract other than HPF were much less effective in down-regulating cytokine signalling. Significantly, inhibition of cytokine-elicited STAT-1 and NF-kappaB activation was confirmed in isolated rat and human islets incubated in the presence of these vegetal compounds. In conclusion, St-John's-wort extract and hyperforin are non-peptidyl compounds which, at low concentrations, target key mechanisms of cytokine-induced beta-cell injury, thereby improving beta-cell function and survival. Thus, they are potentially valuable for the prevention or limitation of beta-cell loss in diabetes.

To assess whether NLR pyrin domain-containing protein 3 (NLRP3) inflammasome, a multiprotein complex that mediates the activation of caspase-1 (CASP-1) and pro-inflammatory cytokines IL-18 and IL-1β, could be involved in the chronic inflammatory state observed in chronic kidney disease patients undergoing hemodialysis treatment (CKD-HD), we employed several biomolecular techniques including RT-PCR, western blot, FACS analysis, confocal microscopy and microarray. Interestingly, peripheral blood mononuclear cells from 15 CKD-HD patients showed higher mRNA levels of NLRP3, CASP-1, ASC, IL-1β, IL-18 and P2X7 receptor compared to 15 healthy subjects. Western blotting analysis confirmed the above results. In particular, active forms of CASP-1, IL1-β and IL-18 resulted significantly up-regulated in CKD-HD versus controls. Additionally, elevated mitochondrial ROS level, colocalization of NLRP3/ASC/mitochondria in peripheral blood mononuclear cells from CKD-HD patients and down-regulation of CASP-1, IL1-β and IL-18 protein levels in immune-cells of CKD-HD patients stimulated with LPS/ATP in presence of mitoTEMPO, inhibitor of mitochondrial ROS production, suggested a possible role of this organelle in the aforementioned CKD-associated inflammasome activation. Then, microarray analysis confirmed, in an independent microarray study cohort, that NLRP3 and CASP-1, along with other inflammasome-related genes, were up-regulated in 17 CKD-HD patients and they were able to clearly discriminate these patients from 5 healthy subjects. All together these data showed, for the first time, that NLRP3 inflammasome was activated in uremic patients undergoing dialysis treatment and they suggested that this unphysiological condition could be possibly induced by mitochondrial dysfunction.

Tumor necrosis factor (TNF)-like cytokine 1A (TL1A) is a member of the TNF superfamily, expressed on dendritic cells, macrophages, lymphocytes, and endothelial cells. It is the only described ligand for death receptor 3 (DR3), a death-domain containing receptor of the TNF-receptor superfamily, mainly expressed on lymphocytes, natural killer (NK) cells, and NK-T cells. TL1A–DR3 interaction results in co-stimulatory signaling for activated T cells, leading to amplification of inflammation and immune responses, correlated with greater pathogenicity in diverse autoimmune diseases. In contrast, in activated B cells the TL1A/DR3 system exerts inhibitory effect on cell proliferation, suggesting that TL1A may also have modulatory and homeostatic functions on B-cell expansion. Chronic lymphocytic leukemia (CLL) is the leukemia with the highest incidence among adults in Western countries. It is well documented that several elements within the tumor microenvironment, including antigens, cytokines, adhesion molecules, and surface receptors, play a fundamental role in supporting the growth of CLL. In contrast, little is known on regulatory mechanisms of CLL growth. In this study, we have investigated the possible regulatory role of the TL1A/DR3 system in B cells from CLL patients. CLL patients from the Hematology Unit at the University Hospital of Verona (Italy) were included in this study (n=37). Disease characteristics and demographic variables were collected on all patients. Purified B cells were obtained by negative selection. DR3 expression was measured on peripheral blood B cells by flow cytometry and western blot at baseline and following B cell receptor (BCR) stimulation with anti-human IgM. DR3 expression was confirmed on lymph-node CLL specimens by immunofluorescence. Metabolic activity of CLL B cells was analyzed by MTT assay. Apoptosis was analyzed by Annexin V assay. TL1A serum levels in CLL patients were measured by ELISA. Baseline analysis in vitro showed that DR3 was expressed at low levels in CLL B cells. Cell activation through stimulation of the BCR induced a significant increase of DR3 expression in a fraction of CLL B cells (p In summary, this study shows that B-CLL metabolic activity can be reduced through the activity of the TL1A/DR3 system, in the presence of the BCR stimulation. Furthermore, TL1A and DR3 levels are higher in patients with early-stage disease. Taken together, these findings suggest that the TL1A/DR3 system in vivo, in the presence of antigen stimulation, may modulate leukemic cell metabolism in early-stage CLL, thus influencing the clinical course of disease. Disclosures No relevant conflicts of interest to declare.

Abstract 3191 Resveratrol is a polyphenolic stilbene with anti-oxidant, anti-inflammatory and anti-tumoral bioactivities. High concentrations of resveratrol (50 μM) have been reported to induce HbF synthesis in an in vitro model of normal and beta-thalassemic erythropoiesis (Fibach E. Int J Mol Med 2012; Rodrigue CM. BJH 2001) and to improve erythropoiesis in a mouse model for Fanconi Anemia (Zhang Q. Blood 2010). Beta thalassemia (b-thal) is characterized by ineffective erythropoiesis and increased cellular oxidative stress. We studied the effects of resveratrol (5 μM) on erythropoiesis in vitro from peripheral CD34+ cells of healthy and b-thal subjects. Erythroid maturation was evaluated at 7, 9, 11 and 14 days of culture by cytofluorimetric analysis using the CD71-GPA-CD36 strategy that allows to separate CFU-E, Pro-E, Int-E and Late-Erythroblasts (Merryweather-Clarke AT. Blood 2011). Resveratrol reduced cell growth in both cell types, with a reduction of CFU-E, increased Int-E at day 7 and 9, and increased Int-E and Late-E at 11 and 14 days. The early maturation of erythroid progenitors was confirmed by morphological analysis of the cells. We sorted CFU-E cells (at 7 days) from resveratrol treated and untreated cells and analyzed the cell cycle, cyclinD1 and p21 expression. In both cell types resveratrol induced increased frequency of S-G2/M cells compared to untreated cells with increased p21 levels, suggesting decreased cycling of CFU-E with increased maturation of erythroblasts. No changes of gamma chain mRNA levels were present in cells treated with resveratrol (5 μM). Since FOXO3 is a key regulator of erythroid redox required for normal erythroid maturation (Marinkovic D. JCI 2007), FOXO3 expression and activity was assessed in sorted CFU (7day) and Int-E (11 day) with and without resveratrol. FOXO3a mRNA levels were increased in resveratrol treated cells in both sorted cell populations. We used nuclear localization as a surrogate assay for FOXO3a activity and found resveratrol increased the overall expression of FOXO3 protein in the nucleus without impacting significantly the nuclear/cytoplasmic ratio. Interestingly, resveratrol did not appear to modify FOXO1 expression or subcellular localization. These results suggest that resveratrol enhances specifically expression of FOXO3 in human erythroblasts. Dietary resveratrol supplementation (2.4 mg/Kg) was studied in wild-type and Hbb3th+/− mice (2 months of age) for 6 months. In resveratrol Hbb3th+/− treated mice increased Hb levels (8.3±0.6 vs 10.3±0.5 g/dL, n=12; P Disclosures: Cappellini: Novartis Pharmaceuticals: Honoraria, Research Funding.