High-density Neutrophils Research Articles

C3a elicits unique migratory responses in immature low-density neutrophils.

Neutrophils represent the immune system's first line of defense and are rapidly recruited into inflamed tissue. In cancer associated inflammation, phenotypic heterogeneity has been ascribed to this cell type, whereby neutrophils can manifest anti- or pro-metastatic functions depending on the cellular/micro-environmental context. Here, we demonstrate that pro-metastatic immature low-density neutrophils (iLDNs) more efficiently accumulate in the livers of mice bearing metastatic lesions compared with anti-metastatic mature high-density neutrophils (HDNs). Transcriptomic analyses reveal enrichment of a migration signature in iLDNs relative to HDNs. We find that conditioned media derived from liver-metastatic breast cancer cells, but not lung-metastatic variants, specifically induces chemotaxis of iLDNs and not HDNs. Chemotactic responses are due to increased surface expression of C3aR in iLDNs relative to HDNs. In addition, we detect elevated secretion of cancer-cell derived C3a from liver-metastatic versus lung-metastatic breast cancer cells. Perturbation of C3a/C3aR signaling axis with either a small molecule inhibitor, SB290157, or reducing the levels of secreted C3a from liver-metastatic breast cancer cells by short hairpin RNAs, can abrogate the chemotactic response of iLDNs both in vitro and in vivo, respectively. Together, these data reveal novel mechanisms through which iLDNs prefentially accumulate in liver tissue harboring metastases in response to tumor-derived C3a secreted from the liver-aggressive 4T1 breast cancer cells.

Circulating neutrophil subsets in advanced lung cancer patients exhibit unique immune signature and relate to prognosis.

The accumulation of circulating low-density neutrophils (LDN) has been described in cancer patients and associated with tumor-supportive properties, as opposed to the high-density neutrophils (HDN). Here we aimed to evaluate the clinical significance of circulating LDN in lung cancer patients, and further assessed its diagnostic vs prognostic value. Using mass cytometry (CyTOF), we identified major subpopulations within the circulating LDN/HDN subsets and determined phenotypic modulations of these subsets along tumor progression. LDN were highly enriched in the low-density (LD) fraction of advanced lung cancer patients (median 7.0%; range 0.2%-80%, n=64), but not in early stage patients (0.7%; 0.05%-6%; n=35), healthy individuals (0.8%; 0%-3.5%; n=15), or stable chronic obstructive pulmonary disease (COPD) patients (1.2%; 0.3%-7.4%, n=13). Elevated LDN (>10%) remarkably related with poorer prognosis in late stage patients. We identified three main neutrophil subsets which proportions are markedly modified in cancer patients, with CD66b+ /CD10low /CXCR4+ /PDL1inter subset almost exclusively found in advanced lung cancer patients. We found substantial variability in subsets between patients, and demonstrated that HDN and LDN retain a degree of inherent spontaneous plasticity. Deep phenotypic characterization of cancer-related circulating neutrophils and their modulation along tumor progression is an important advancement in understanding the role of myeloid cells in lung cancer.

ROLE OF CELLS OF INNATE IMMUNITY IN THE DEVELOPMENT OF MALIGNANT TUMORS OF THE BRAIN

This review presents data on changes in the activity of cells of innate and acquired immunity, namely leukocytes, neutrophils, platelets and lymphocytes in various malignant human tumors, including brain tumors. It was shown that against the background of immunosuppression of specific immunity, especially antitumor reactions, which are caused by factors such as prostaglandin E2, TGF-β, indolamine-2,3-dioxigenase (IDO) and interleukin (IL) -10, which leads to a decrease the sensitivity of T cells to proinflammatory signals and the ineffectiveness of the presentation of tumor antigens to immune cells, activation and polarization of innate immunity cells, namely neutrophils, macrophages and platelets, occurs.
 Macrophages are important immune cells of the microenvironment in a tumor site that change their phenotype from M1 cells with antitumor activity to M2, which enhance tumor growth. The release of metalloprotheasis from platelet α -granules destroys the components of the extracellular matrix, increases the ability of cancer cells to pass through the endothelial barrier, penetrate the parenchyma and create metastatic tissue damage. Previously, neutrophils were mainly considered as cells of the body’s first line of defense, mainly with antimicrobial functions, but now they are regarded as cells with tumor-stimulating, “protumorogenic” activity, since in many types of cancer an increased level of neutron is determined with a reduced content of lymphocytes in the peripheral blood and this is associated with a poor prognosis of the disease. The review analyzes the hypothesis that there are three subpopulations of neutrophils in cancer: normal high density neutrophils, immature low density neutrophils (G-MDSC) and large mature low density neutrophils. These types of cells have different functions, for example, neutrophils with high density are antitumor, and with low density - cells that can stimulate tumor growth. ]. Neutrophils realize their activity through molecules such as neutrophilic elastase (NE), cathepsin, arginase 1 (ARG1), matrix metalloproteinase-9 (MMP-9). Multidirectional changes in the parts of the immune system depend on the histogenesis and degree of malignancy of the tumors and indicate differentiated use immunotropic drugs in cancer patients, some should suppress the activity of innate immunity cells, others stimulate the acquired immune response.

Plasticity of High-Density Neutrophils in Multiple Myeloma is Associated with Increased Autophagy Via STAT3.

In both monoclonal gammopathy of uncertain significance (MGUS) and multiple myeloma (MM) patients, immune functions are variably impaired, and there is a high risk of bacterial infections. Neutrophils are the most abundant circulating leukocytes and constitute the first line of host defense. Since little is known about the contribution of autophagy in the neutrophil function of MGUS and MM patients, we investigated the basal autophagy flux in freshly sorted neutrophils of patients and tested the plastic response of healthy neutrophils to soluble factors of MM. In freshly sorted high-density neutrophils obtained from patients with MGUS and MM or healthy subjects, we found a progressive autophagy trigger associated with soluble factors circulating in both peripheral blood and bone marrow, associated with increased IFNγ and pSTAT3S727. In normal high-density neutrophils, the formation of acidic vesicular organelles, a morphological characteristic of autophagy, could be induced after exposure for three hours with myeloma conditioned media or MM sera, an effect associated with increased phosphorylation of STAT3-pS727 and reverted by treatment with pan-JAK2 inhibitor ruxolitinib. Taken together, our data suggest that soluble factors in MM can trigger contemporary JAK2 signaling and autophagy in neutrophils, targetable with ruxolitinib.

Immature Low-Density Neutrophils Exhibit Metabolic Flexibility that Facilitates Breast Cancer Liver Metastasis

Neutrophils are phenotypically heterogeneous and exert either anti- or pro-metastatic functions. We show that cancer-cell-derived G-CSF is necessary, but not sufficient, to mobilize immature low-density neutrophils (iLDNs) that promote liver metastasis. In contrast, mature high-density neutrophils inhibit the formation of liver metastases. Transcriptomic and metabolomic analyses of high- and low-density neutrophils reveal engagement of numerous metabolic pathways specifically in low-density neutrophils. iLDNs exhibit enhanced global bioenergetic capacity, through their ability to engage mitochondrial-dependent ATP production, and remain capable of executing pro-metastatic neutrophil functions, including NETosis, under nutrient-deprived conditions. We demonstrate that NETosis is an important neutrophil function that promotes breast cancer liver metastasis. iLDNs rely on the catabolism of glutamate and proline to support mitochondrial-dependent metabolism in the absence of glucose, which enables sustained NETosis. These data reveal that distinct pro-metastatic neutrophil populations exhibit a high degree of metabolic flexibility, which facilitates the formation of liver metastases.

Staphylococcus aureus drives expansion of low-density neutrophils in diabetic mice

Diabetic individuals are at considerable risk for invasive infection by Staphylococcus aureus, however, the mechanisms underlying this enhanced susceptibility to infection are unclear. We observed increased mortality following i.v. S. aureus infection in diabetic mice compared with nondiabetic controls, correlating with increased numbers of low-density neutrophils (LDNs) and neutrophil extracellular traps (NETs). LDNs have been implicated in the inflammatory pathology of diseases such as lupus, given their release of large amounts of NETs. Our goal was to describe what drives LDN increases during S. aureus infection in the diabetic host and mechanisms that promote increased NET production by LDNs. LDN development is dependent on TGF-β, which we found to be more activated in the diabetic host. Neutralization of TGF-β, or the TGF-β-activating integrin αvβ8, reduced LDN numbers and improved survival during S. aureus infection. Targeting S. aureus directly with MEDI4893*, an α toxin-neutralizing monoclonal antibody, blocked TGF-β activation, reduced LDNs and NETs, and significantly improved survival. A comparison of gene and protein expression in high-density neutrophils and LDNs identified increased GPCRs and elevated phosphatase and tensin homolog (PTEN) in the LDN subset. Inhibition of PTEN improved the survival of infected diabetic mice. Our data identify a population of neutrophils in infected diabetic mice that correlated with decreased survival and increased NET production and describe 3 therapeutic targets, a bacterial target and 2 host proteins, that prevented NET production and improved survival.

Understanding the Multifaceted Role of Neutrophils in Cancer and Autoimmune Diseases.

Neutrophils are one of the first immune cell types that are recruited to injury and infection site. As a vital component of the immune system, neutrophils are heterogeneous immune cells known to have phagocytic property and function in inflammation. Recent studies revealed that neutrophils play dual roles in tumor initiation, development, and progression. The multifunctional roles of neutrophils in diseases are mainly due to their production of different effector molecules under different conditions. N1 and N2 neutrophils or high density neutrophils (HDNs) and low density neutrophils (LDNs) have been used to distinguish neutrophils subpopulations with pro- vs. anti-tumor activity, respectively. Indeed, N1 and N2 neutrophils also represent immunostimulating and immunosuppressive subsets, respectively, in cancer. The emerging studies support their multifaceted roles in autoimmune diseases. Although such subsets are rarely identified in autoimmune diseases, some unique subsets of neutrophils, including low density granulocytes (LDGs) and CD177+ neutrophils, have been reported. Given the heterogeneity and functional plasticity of neutrophils, it is necessary to understand the phenotypical and functional features of neutrophils in disease status. In this article, we review the multifaceted activates of neutrophils in cancer and autoimmune diseases, which may support new classification of neutrophils to help understand their important functions in immune homeostasis and pathologies.

Tryptophan Shortage Due to IDO-1 Expressed by High-Density Neutrophils Induce Immune-Suppression and Plasma Cell Fitness in Multiple Myeloma

Tryptophan Shortage Due to IDO-1 Expressed by High-Density Neutrophils Induce Immune-Suppression and Plasma Cell Fitness in Multiple Myeloma

Abstract 609: Pro-atherosclerotic Low-density Neutrophils Are Present in Hypercholesterolemia

Introduction: Elevated cholesterol levels and increased accumulation of myeloid cells are key risk factors in the development of atherosclerosis. Whilst there is significant understanding of the impact of hypercholesterolemia on monocyte function, less is known about the impact of cholesterol on neutrophils. Neutrophils contain the necessary machinery to take up, synthesize, efflux and esterify cholesterol and unique neutrophil phenotypes have been shown in various pathologies, including low density neutrophils (LDNs) described in inflammatory diseases. Aim: Determine if cholesterol loading of neutrophils occurs in hypercholesterolemia and characterize the associated phenotypic and functional consequences. Methods: Patients (n=13) were a mix of familial hypercholesterolemia and mixed dyslipidemia all with total cholesterol > 7.0 mmol/L. Age and sex matched healthy donors were used as controls. Normal and low density neutrophils were isolated using a percoll density protocol and further purified by FACS selecting for CD66b + CD15 + cells. Results: LDNs were only present in hypercholesterolemic patients, accounting for 12.29.6% of the cells in the lower density percoll fraction (p<0.01 vs control). LDNs were morphologically similar to mature high density neutrophils and stained positive for neutral lipid. Functionally, LDNs secreted significantly more extracellular traps (NETs) in response to PMA treatment relative to normal density neutrophils isolated from the same patients or healthy controls (p<0.005). Cholesterol loaded neutrophils were more adhesive to endothelial cells compared to controls (p<0.005). Increased NETosis and neutrophil adhesion have been demonstrated to be atherogenic. Phagocytosis was increased in LDNs vs controls (p<0.05). These functional changes could all be replicated in vitro via treatment of neutrophils from healthy subjects with cholesterol or oxLDL. Conclusion: Hypercholesterolemia results in cholesterol loading and a pro-atherosclerotic phenotype in neutrophils. Our data provides evidence that this cholesterol loading occurs in the circulation. More work is now being carried out to further characterize the phenotype of LDNs and mechanisms of maturation during hypercholesterolemia.

Phenotypic and clinical characterization of low density neutrophils in patients with advanced lung adenocarcinoma.

PurposeAn immunosuppressive subgroup of neutrophils, low density neutrophils (LDNs) was reported to be closely related to several diseases. This study was designed to explore the association between LDNs and advanced lung adenocarcinoma, as well as potential mechanisms.ResultsThe expression levels of surface CD molecules on LDNs were different from high density neutrophils (HDNs), consistent with previous studies. The ratio of LDNs/HDNs, rather than the percentage of LDNs in peripheral blood mononuclear cells (PBMCs), was significantly higher in lung adenocarcinoma patients than healthy controls. It was also observed that the ratio decreased when patients received anti-cancer treatments, and increased when disease relapsed. Patients harboring positive epidermal growth factor receptor (EGFR) mutation had significantly higher ratios. Both the ratio and the percentage showed positive correlation with CD8+ T cells. Although significantly increased TGF-β was detected in lung adenocarcinoma patients, relationship between TGF-β and LDNs was not obvious.Materials and MethodsLDNs and HDNs levels of peripheral blood from 52 lung adenocarcinoma patients and 13 healthy controls were determined by flow cytometry. Lymphocytes and cytokines were also detected.ConclusionsTwo kinds of neutrophils with different phenotypes were identified in lung adenocarcinoma patients. Besides, we found the existence of high ratio of LDNs/HDNs in these patients, which is related to disease prognosis, EGFR mutation and bad immune status.

Immune Suppression By High-Density Neutrophils Predicts Short Progression-Free Survival in Multiple Myeloma

Background and Objective:Our previous work showed that neutrophils sedimenting on top of red cells after density gradients, defined as high-density neutrophils (HDN) are emerging as pro-tumoral players with immunesuppressive properties, by depleting the surrounding microenvironment from arginine (arg) and tryptophan (trp) through increased expression of arginase (Arg-1) and 2-3 indoleamine deoxygenase (IDO), but little is known about their clinical relevance in Multiple Myeloma (MM).Experimental design: We integrated two distinct unbiased approaches. First, we assessed gene expression profiles (GEP) and functional evaluation of HDN sorted from 60 newly diagnosed MM patients, 30 patients with MGUS, and 30 healthy subjects.. Second, we adopted ultra-high performance liquid and gas chromatography followed by mass spectrometry (UHPLC/GC-MS) on an independent series of 167 samples of bone marrow (BM) and peripheral plasma collected ad hocfrom 125 individuals, comprising newly diagnosed MM, MGUS, smoldering MM, and age-matched healthy volunteers (n=29). Progression-free survival in newly diagnosed MM patients was correlated respectively to HDN functional and expression features or metabolic profiles in the two series.Results:GEP analyses disclosed that, compared to MGUS and HD, MM-HDN showed increased expression of Arg-1, IDO-1 and the angiogenic factor PROK-2/BV8 , confirmed by qRT-PCR and immunofluorescence. Indeed, MM-HDN (but not MGUS- and HD-HDN) revealed immunosuppressive activity in co-cultures with allogeneic T-cells, reverted by the selective Arg-1 inhibitor nor-NOHA.In addition, circulating Arg-1 in serum was higher in MM (169.5 ± 17.6) than MGUS (93.2 ± 10.4, p=0.0028) and healthy subjects (70.1 ± 16.5 ng/ml, p=0.0017). In MM patients there was a progressive increase from ISS stage I through III (p=0.014). High Arg-1 in MM patients’ sera at diagnosis predicted clinical outcome after first line therapy, being associated to shorter progression-free survival (respectively 22.4 months versus unreached median, p=0.01).Metabolic profiling independently identified the same immunosuppressive Arg-1-urea-arginine (arg) and tryptophan (trp)-kinurenine-IDO pathways, with arg and trp significantly reduced along MM evolution. Lower levels of arg were associated to shorter PFS (37.2 versus 17.6 months, p=0.023).We then tested if aminoacid depletion (arg and trp) favors PCs growth. When exposed to trp-free or arg-free medium, human MM cell lines expressed more CHOP, p62, m-TOR and Blimp-1 (markers respectively of autophagy induction and immunoglobulin production); conversely, p62 silencing reduced Blimp-1, confirming the important role of p62-dependent mechanism in supporting PCs fitness.Conclusion: In MM, HDN are immunosuppressive due to increased Arg-1 and IDO expression. We propose the arginine/Arg-1 axis as a novel potential biomarker for MM prognosis. DisclosuresCiceri:MolMed SpA: Consultancy.

Contribution of High-Density Neutrophils to Multiple Myeloma Microenvironment Dysregulation

Background and objective: Multiple Myeloma (MM) is a plasma cell malignancy with a well documented immune dysfunction. Previous reports in murine models and our previous work in MM patients, showed that myeloid cells, both granulocyte-myeloid derived suppressor cells (G-MDSC) and neutrophils sedimenting on top of red cells after density gradients, defined as high-density neutrophils (HDN) are increased and exert an immunosuppressive activity. Moreover, we showed that myeloid precursors could be activate and acquire G-MDSC phenotype if cultures in presence of MM mesenchymal cells.Thus, we characterized functionally HDN to gauge their contribution in immunesuppression, angiogenesis and bone resorption in MM microenvironment.Methods:In HDN freshly-isolated from a series of 60 newly-diagnosed MM, 30 smoldering MM and 30 MGUS we investigated by RT-PCR, flow-cytometry, western-blot and immunofluorescence genes and proteins related to immunesuppression (Arg-1, IDO), angiogenesis (BV8, VEGF, MMP9) and bone resorption (IL1-b, MMP9, CHI3L).Results:First, MM-HDN exhibited an increased expression of immunesuppressive molecules Arg-1 and IDO compared to MGUS and healthy subjects (25.5 vs 6.2 vs 1 fold changes in gene expression, p=0.003), confirmed by functional assay of enzymatic activity, immunostaining and WB, positively correlated with advanced disease.Second, MM-HDN showed increased expression of BV8 and MMP9 (p=0.01, p=0.05) but not VEGF, as confirmed by immunofluorescence. In line with this observation, by using migration assay (CIM plate) MM-HDN induced neo-vasculogenesis in vitro by forming more and aberrant tubes than healthy or MGUS-HDN, thus to disclosure a novel proangiogenic mechanism driven by BV8.Third, MM-HDN showed increased expression of IL1-b, MMP9, CHI3L, associated to increased bone resorption activity, evaluated by dentin-disk assay. The same findings could be obtained treating bone matrix with MM-HDN-conditioned media, suggesting the crucial role of molecules released by MM-HDN but no from MGUS-HDN.Conclusion: taken together, our observations disclosure the contribution of aberrantly activated HDN in MM clinical features, contributing to immunosuppression, angiogenesis and bone resorption. DisclosuresNo relevant conflicts of interest to declare.

Arginase 1 Is a Marker of Myeloid-Mediated Immunosuppression with Prognostic Meaning in Classic Hodgkin Lymphoma

Purpose: Neutrophilia is hallmark of classic Hodgkin Lymphoma (cHL), but its precise characterization remains elusive. We aimed at investigating the immunosuppressive role of high-density neutrophils in HL.Experimental design: First, N-HL function was evaluated in vitro, showing increased arginase (Arg-1) expression and activity compared to healthy subjects. Second, we measured serum level of Arg-1 (s-Arg-1) by ELISA in two independent, training (N=40) and validation (N=78) sets.Results: s-Arg-1 was higher in patients with advanced stage (p=0.045), B-symptoms (p=0.0048) and a positive FDG-PET scan after two cycles of chemotherapy (PET-2, p=0.012). Baseline levels of s-Arg-1 >200 ng/mL resulted in 92% sensitivity and 56% specificity to predict a positive PET-2.Patients showing s-Arg-1 levels > 200 ng/mL had a shorter progression free survival (PFS). In multivariate analysis, PET-2 and s-Arg-1 at diagnosis were the only statistically significant prognostic variables related to PFS (respectively p=0.0004 and p=0.012).Moving from PET-2 status and s-Arg-1 level we constructed a prognostic score to predict long-term treatment outcome: low s-Arg-1 and negative PET-2 scan (score 0, N=63), with a 3-Y PFS of 89.5%; either positive PET-2 or high s-Arg-1 (score 1, N=46) with 3-Y PFS of 67.6%, and both positive markers (score 2, N=9) with a 3-Y PFS of 37% (p=0.0004).Conclusions: We conclude that N-HL are immunosuppressive through increased Arg-1 expression, a potential novel potential biomarker for HL prognosis. DisclosuresVetro:MLL Munich Leukemia Laboratory: Employment. Chiarenza:Gilead: Consultancy; Janssen: Consultancy; Roche: Consultancy.

The prognostic value of the myeloid-mediated immunosuppression marker Arginase-1 in classic Hodgkin lymphoma.

PurposeNeutrophilia is hallmark of classic Hodgkin Lymphoma (cHL), but its precise characterization remains elusive. We aimed at investigating the immunosuppressive role of high-density neutrophils in HL.Experimental designFirst, N-HL function was evaluated in vitro, showing increased arginase (Arg-1) expression and activity compared to healthy subjects. Second, we measured serum level of Arg-1 (s-Arg-1) by ELISA in two independent, training (N = 40) and validation (N = 78) sets.Resultss-Arg-1 was higher in patients with advanced stage (p = 0.045), B-symptoms (p = 0.0048) and a positive FDG-PET scan after two cycles of chemotherapy (PET-2, p = 0.012). Baseline levels of s-Arg-1 > 200 ng/mL resulted in 92% sensitivity and 56% specificity to predict a positive PET-2.Patients showing s-Arg-1 levels > 200 ng/mL had a shorter progression free survival (PFS). In multivariate analysis, PET-2 and s-Arg-1 at diagnosis were the only statistically significant prognostic variables related to PFS (respectively p = 0.0004 and p = 0.012).Moving from PET-2 status and s-Arg-1 level we constructed a prognostic score to predict long-term treatment outcome: low s-Arg-1 and negative PET-2 scan (score 0, N = 63), with a 3-Y PFS of 89.5%; either positive PET-2 or high s-Arg-1 (score 1, N = 46) with 3-Y PFS of 67.6%, and both positive markers (score 2, N = 9) with a 3-Y PFS of 37% (p = 0.0004).ConclusionsWe conclude that N-HL are immunosuppressive through increased Arg-1 expression, a novel potential biomarker for HL prognosis.

High-Density Neutrophils are Immunosuppressive in Multiple Myeloma Due to Increased Arginase-1, Predictor of Short Progression Free Survival

High-Density Neutrophils are Immunosuppressive in Multiple Myeloma Due to Increased Arginase-1, Predictor of Short Progression Free Survival

The Prognostic Value of the Myeloid-Mediated Immunosuppression Marker Arginase-1 in Classic Hodgkin Lymphoma

// Alessandra Romano 1 , Nunziatina Laura Parrinello 1 , Calogero Vetro 1 , Daniele Tibullo 1 , Cesarina Giallongo 1 , Piera La Cava 1 , Annalisa Chiarenza 1 , Giovanna Motta 1 , Anastasia L. Caruso 1 , Loredana Villari 2 , Claudio Tripodo 3 , Sebastiano Cosentino 4 , Massimo Ippolito 4 , Ugo Consoli 5 , Andrea Gallamini 6 , Stefano Pileri 7 , Francesco Di Raimondo 1 1 Division of Hematology, AOU “Policlinico - Vittorio Emanuele”, University of Catania, Catania, Italy 2 Division of Pathology, AOU “Policlinico - Vittorio Emanuele”, Catania, Italy 3 Tumor Immunology Unit, Department of Health Science, University of Palermo, Palermo, Italy 4 Nuclear Medicine Center, Azienda Ospedaliera Cannizzaro, Catania, Italy 5 Division of Hematology, ARNAS Garibaldi, Catania, Italy 6 Research, Innovation and Statistics Department. A. Lacassagne Cancer Centre, Nice, France 7 Unita di Diagnosi Emolinfopatologica, IEO, Milano, Italy Correspondence to: Alessandra Romano, email: sandrina.romano@gmail.com Francesco Di Raimondo, email: diraimon@unict.it Keywords: Arginase-1, Hodgkin lymphoma, PET-2 Received: June 30, 2016 Accepted: September 05, 2016 Published: September 14, 2016 ABSTRACT Purpose: Neutrophilia is hallmark of classic Hodgkin Lymphoma (cHL), but its precise characterization remains elusive. We aimed at investigating the immunosuppressive role of high-density neutrophils in HL. Experimental design: First, N-HL function was evaluated in vitro , showing increased arginase (Arg-1) expression and activity compared to healthy subjects. Second, we measured serum level of Arg-1 (s-Arg-1) by ELISA in two independent, training ( N = 40) and validation ( N = 78) sets. Results: s-Arg-1 was higher in patients with advanced stage ( p = 0.045), B-symptoms ( p = 0.0048) and a positive FDG-PET scan after two cycles of chemotherapy (PET-2, p = 0.012). Baseline levels of s-Arg-1 > 200 ng/mL resulted in 92% sensitivity and 56% specificity to predict a positive PET-2. Patients showing s-Arg-1 levels > 200 ng/mL had a shorter progression free survival (PFS). In multivariate analysis, PET-2 and s-Arg-1 at diagnosis were the only statistically significant prognostic variables related to PFS (respectively p = 0.0004 and p = 0.012). Moving from PET-2 status and s-Arg-1 level we constructed a prognostic score to predict long-term treatment outcome: low s-Arg-1 and negative PET-2 scan (score 0, N = 63), with a 3-Y PFS of 89.5%; either positive PET-2 or high s-Arg-1 (score 1, N = 46) with 3-Y PFS of 67.6%, and both positive markers (score 2, N = 9) with a 3-Y PFS of 37% ( p = 0.0004). Conclusions: We conclude that N-HL are immunosuppressive through increased Arg-1 expression, a novel potential biomarker for HL prognosis.

Isolation and Characterization of Low- vs. High-Density Neutrophils in Cancer.

Neutrophils are the most abundant of all white blood cells in the human circulation and serve as the first line of defense against microbial infections. Traditionally, neutrophils were viewed as a homogeneous population of myeloid cells. However, in recent years accumulating evidence has suggested that neutrophils are heterogeneous and that distinct neutrophil subsets may play very different roles. Here, we describe the methodology for isolation of high- and low-density neutrophils from the murine and human circulation using a density gradient and antibody based enrichment. We further describe the methodology for functional characterization of these different neutrophil subsets in the context of cancer.

Phenotypic Diversity and Plasticity in Circulating Neutrophil Subpopulations in Cancer

Controversy surrounds neutrophil function in cancer because neutrophils were shown to provide both pro- and antitumor functions. We identified a heterogeneous subset of low-density neutrophils (LDNs) that appear transiently in self-resolving inflammation but accumulate continuously with cancer progression. LDNs display impaired neutrophil function and immunosuppressive properties, characteristics that are in stark contrast to those of mature, high-density neutrophils (HDNs). LDNs consist of both immature myeloid-derived suppressor cells (MDSCs) and mature cells that are derived from HDNs in a TGF-β-dependent mechanism. Our findings identify three distinct populations of circulating neutrophils and challenge the concept that mature neutrophils have limited plasticity. Furthermore, our findings provide a mechanistic explanation to mitigate the controversy surrounding neutrophil function in cancer.

Long-term air pollution exposure and risk factors for cardiovascular diseases among the elderly in Taiwan

ObjectivesTo investigate changes in blood pressure, blood lipids, blood sugar and haematological markers of inflammation associated with changes in long-term exposure to ambient air pollutants.MethodsWe conducted secondary analyses of data...