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A phase II study was conducted to ascertain whether sequential exposure to decitabine followed by rapamycin, an mTOR (mechanistic target of rapamycin) inhibitor would result in better responses than decitabine alone. Newly diagnosed acute myelogenous leukemia (AML) patients who were >65 years old and not eligible for intensive induction regimens or patients with relapsed or refractory AML received 10 days of decitabine followed by 12 days of rapamycin in cycle 1 and 5 days of decitabine followed by 17 days of rapamycin in subsequent cycles. The composite complete remission rate (CR) was 33 % (CR plus CR with incomplete count recovery). Median overall survival was 7.7 months in newly diagnosed elderly AML patients and 6.6 months in relapsed/refractory AML patients. Twenty-four evaluable patients were enrolled, and the study did not meet its primary endpoint of demonstrating a significant improvement in composite CR rate with the combination as compared to an established historical CR rate of 25 % with decitabine alone. Despite that, the survival rates in relapsed/refractory cases appear comparable to what is reported with other salvage regimens, and no significant patterns of non-hematologic toxicity were noted. 50 % of subjects in the de novo group achieved a composite CR which is significantly higher (p = 0.02) than the rate of 25 % with decitabine alone. This trial is registered at clinical trials.gov as NCT02109744.

Bone marrow mesenchymal stromal cells (MSCs) constitute one of the important components of the hematopoietic microenvironmental niche. In vivo studies have shown that depletion of marrow MSCs resulted in reduction of hematopoietic stem cell content, and there is in vitro evidence that marrow MSCs are able to support leukemia progenitor cell proliferation and survival and provide resistance to cytotoxic therapies. How MSCs from leukemia marrow differ from normal counterparts and how they are influenced by the presence of leukemia stem and progenitor cells are still incompletely understood. In this work, we compared normal donor (ND) and acute myelogenous leukemia (AML) derived MSCs and found that AML-MSCs had increased adipogenic potential with improved ability to support survival of leukemia progenitor cells. To identify underlying changes, RNA-Seq analysis was performed. Gene ontology and pathway analysis revealed adipogenesis to be among the set of altered biological pathways dysregulated in AML-MSCs as compared with ND-MSCs. Expression of both SOX9 and EGR2 was decreased in AML-MSCs as compared with ND-MSCs. Increasing expression of SOX9 decreased adipogenic potential of AML-MSCs and decreased their ability to support AML progenitor cells. These findings suggest that AML-MSCs possess adipogenic potential which may enhance support of leukemia progenitor cells.

We have previously shown that regulatory T cells (Tregs) infiltrating follicular lymphoma lymph nodes are quantitatively and qualitatively different than those infiltrating normal and reactive nodes. To gain insight into how such Treg populations differ, we performed RNA sequence (RNAseq) analyses on flow sorted Tregs from all three sources. We identify several molecules that could contribute to the observed increased suppressive capacity of follicular lymphoma nodal tregs, including upregulation of CTLA-4, IL-10, and GITR, all confirmed by protein expression. In addition, we identify, and confirm functionally, a novel mechanism by which Tregs target to and accumulate within a human tumor microenvironment, through the down regulation of S1PR1, SELL (L-selectin) and CCR7, potentially resulting in greater lymph node retention. In addition we identify and confirm functionally the upregulation of the chemokine receptor CXCR5 as well as the secretion of the chemokines CXCL13 and IL-16 demonstrating the unique ability of the follicular derived Tregs to localize and accumulate within not only the malignant lymph node, but also localize and accumulate within the malignant B cell follicle itself. Such findings offer significant new insights into how follicular lymphoma nodal Tregs may contribute to the biology of follicular lymphoma and identify several novel therapeutic targets.

A novel strain of H1N1 influenza A virus (pH1N1) emerged in 2009, causing a worldwide pandemic. Several studies suggest that this virus is antigenically more closely related to human influenza viruses that circulated prior to 1957 than viruses of more recent seasonal influenza varieties. The extent to which individuals who are naïve to the 2009 pH1N1 virus carry cross-reactive CD8+ T cells is not known, but a certain degree of reactivity would be expected since there is substantial conservation among the internal proteins of the virus. In the present study, we examined the production of multiple cytokines in response to virus from CD8+ T cells in healthy adult subjects, between 18 and 50 years of age (born post 1957), who had no evidence of exposure to the 2009 pH1N1 virus, and had blood collected prior to the emergence of the pandemic in April of 2009. Human peripheral blood mononuclear cells (PBMCs) were stimulated in vitro with a panel of live viruses, and assayed by intracellular cytokine staining and flow cytometry. Although results were variable, most subjects exhibited cytokine positive CD8+ T cells in response to pH1N1. Cytokine producing cells were predominantly single positive (IL2, IFNγ, or TNFα); triple-cytokine producing cells were relatively rare. This result suggests that although many adults carry cross-reactive T cells against the emergent pandemic virus, these cells are in a functionally limited state, possibly because these subjects have not had recent exposure to either seasonal or pandemic influenza strains.

We aim to define the role of Kupffer cells in intrahepatic antigen presentation, using the selective delivery of antigen to Kupffer cells rather than other populations of liver antigen-presenting cells. To achieve this we developed a novel antigen delivery system that can target antigens to macrophages, based on a galactosylated low-density lipoprotein nano-scale platform. Antigen was delivered via the galactose particle receptor (GPr), internalized, degraded and presented to T cells. The conjugation of fluoresceinated ovalbumin (FLUO-OVA) and lactobionic acid with LDL resulted in a substantially increased uptake of FLUO-OVA by murine macrophage-like ANA1 cells in preference to NIH-3T3 cells, and by primary peritoneal macrophages in preference to primary hepatic stellate cells. Such preferential uptake led to enhanced proliferation of OVA specific T cells, showing that the galactosylated LDL nano-scale platform is a successful antigen carrier, targeting antigen to macrophages but not to all categories of antigen presenting cells. This system will allow targeted delivery of antigen to macrophages in the liver and elsewhere, addressing the question of the role of Kupffer cells in liver immunology. It may also be an effective way of delivering drugs or vaccines directly at macrophages.

After activation, populations of antigen-specific T cells flow between sites of antigen expression, local lymphoid structures and other lymphoid and non-lymphoid organs. In this study, we documented the in vivo dynamics of a CD8(+) T cell response to antigen delivered using herpes simplex virus amplicon vectors and revealed several unexpected features. First, the T cells localized to the site of vector injection, as well as the draining lymph node within 24-48 h. Second, the major site to which T cells later redistributed were intra-abdominal lymphoid organs, including milky spots, mesenteric and lumbar lymph nodes. We determined the relationship between bioluminescent signal and antigen-specific T cell numbers in various lymphoid organs, and concluded that bioluminescent signal is a valid surrogate measure of T cell abundance in superficial lymph nodes, but not in deeper structures such as the spleen.

In addition to participation in homing, egress, and transmigration of hematopoietic cells, marrow endothelium also contributes to cell proliferation and survival. Endothelial cells from multiple vascular beds are able to prevent spontaneous or therapy-induced apoptosis in acute myelogenous leukemia (AML) blasts. Marrow-derived endothelial cells from leukemia patients have not been well-characterized, and in this work, endothelial cells were purified from marrow aspirates from normal subjects or from newly diagnosed AML patients to compare these cells phenotypically and functionally. By reverse transcription polymerase chain reaction, these cells express CD31, Tie-2, vascular endothelial growth factor (VEGF), and endothelial nitric oxide synthase (eNOS), supporting endothelial origin. They take up acetyl low-density lipoprotein and are able to form tubular structures. Culture of AML cells with endothelial cells from both normal and AML subjects supported adhesion, transmigration, and leukemia colony-forming unit outgrowth. RNA-sequencing analysis revealed 130 genes significantly up- or downregulated in AML-derived endothelial cells as compared with those derived from normal marrow. The genes differentially expressed (p < 0.001) were included in biological function categories involving cancer, cell development, cell growth and proliferation, cell signaling, inflammatory response, and cell death and survival. Further pathway analysis revealed upregulation of c-Fos and genes involved in chemotaxis such as CXCL16. AML-derived endothelial cells are similar in phenotype and function to their normal marrow-derived counterparts, but genomic analysis suggests a differential signature with altered expression of genes, which could play a role in leukemogenesis or leukemia cell maintenance in the marrow microenvironment.

The oxidative burst in circulating polymorphonuclear leukocytes (PMN) plays a fundamental role in pulmonary defense and injury. Flow cytometric techniques have been developed for quantitation of oxidative burst activity at the single cell level using 2',7'-dichlorofluorescin (DCFH). However, the specific reactive oxidant species being measured using this method are not clearly defined. Isolated human PMN were loaded with DCFH diacetate, stimulated with phorbol myristate acetate (PMA) in the presence or absence of specific reagents, and analyzed using flow cytometry. Addition of PMA resulted in a 90-fold increase in the fluorescence intensity of DCFH-loaded neutrophils (p.01). Inhibition of NADPH oxidase activity using a calmodulin antagonist (W-13) decreased PMA-induced DCFH oxidation by 70% (p.05). Inhibition of nitric oxide synthase using N(G)-monomethyl-L-arginine (NMMA) did not significantly reduce DCFH oxidation, and did not alter the action of W-13. Addition of superoxide dismutase (SOD) had no effect, but catalase, with or without SOD, suppressed DCFH oxidation by 90% (p.01). These data suggest that hydrogen peroxide, and not NO, is primarily responsible for the PMA-induced oxidation of DCFH in human PMN under these conditions.

Background: Bone marrow mesenchymal stem cells (BMSCs) constitute one of the important cellular components of the hematopoietic microenvironmental niche. These cells are capable of differentiation into osteoprogenitors which comprise part of the endosteal niche. In vivo studies have shown that depletion of BMSCs resulted in reduction of hematopoietic stem cell content, and there is direct in vitro evidence that BMSCs are able to support both normal and leukemia progenitor cell proliferation and survival and contribute to leukemia cell resistance to cytotoxic therapies. Whether BMSCs from leukemia marrow differ from normal counterparts and how they contribute to and are influenced by the leukemia environmental niche are still incompletely understood. In this work we sought to compare normal and AML-derived BMSCs and to define the propensity of BMSCs from leukemia patients for osteogenic or adipogenic differentiation. Methods: BMSCs were isolated from marrow aspirates of normal donor and AML patients using standard methodologies. Donors were age-matched to the leukemia patients to the extent possible in each comparative experiment. Such cells met the definition of MSCs in that they were adherent and expressed CD73, CD90, CD44, CD117 and CD105 and had osteoblastic and adipogenic differentiation capabilities. All comparisons were done at comparable passage number between samples. Results: BMSCs from AML donors demonstrated irregular vs. spindle shaped morphology as compared with BMSCs from younger donors To determine if we could detect a gene signature difference between AML and normal BMSCs which would predict adipogenic propensity, RNA-Seq analysis was performed on AML and normal donor specimens(n=3 in each group), all from subjects >50 years of age using CuffDiff (v2.0.2). This identified 88 genes differentially expressed between the two groups. A heat map was generated using Euclidean distance hierarchical clustering of gene expression values from individual samples. This readily grouped AML vs. normal samples. Pathway analysis using Ingenuity Pathway Analysis (IPA) predicted dysregulation of four canonical pathways in AML-BMSCs as compared to normal BMSCs. These included 1) Role of tissue factor in cancer, 2) Airway pathology in COPD, 3) Oleate biosynthesis, and 4) Adipogenesis. The last two pathways are consistent with the biological observation of enhanced adipogenesis in AML-derived BMSCs. IPA analysis proposed a model of altered adipogenic differentiation in AML-BMSCs attributable to lower expression of two key regulatory genes, SOX9 and EGR2. Reduced expression of SOX9 and EGR2 in AML BMSCs as compared to normal BMSCs was validated by qRT-PCR and western blot analysis. SOX9 is reported to contribute to the commitment of MSCs to adipogenic phenotype through negative influence on expression of key transcription factors in adipogenesis, and ERG2 downregulation is required in some systems for adipocyte lineage commitment. Conclusion: Understanding the role that adipogenic MSCs play during leukemia evolution and treatment could offer insight into pathogenesis and potential therapies for these disorders. Disclosures Liesveld: Onconova: Other: Data safety monitoring board; Astex: Honoraria; glycomimetics: Research Funding.

T lymphocytes of the CD8+ class are critical in delivering cytotoxic function and in controlling viral and intracellular infections. These cells are "helped" by T lymphocytes of the CD4+ class, which facilitate their activation, clonal expansion, full differentiation and the persistence of memory. In this study we investigated the impact of CD4+ T cells on the location of CD8+ T cells, using antibody-mediated CD4+ T cell depletion and imaging the antigen-driven redistribution of bioluminescent CD8+ T cells in living mice. We documented that CD4+ T cells influence the biodistribution of CD8+ T cells, favoring their localization to abdominal lymph nodes. Flow cytometric analysis revealed that this was associated with an increase in the expression of specific integrins. The presence of CD4+ T cells at the time of initial CD8+ T cell activation also influences their biodistribution in the memory phase. Based on these results, we propose the model that one of the functions of CD4+ T cell "help" is to program the homing potential of CD8+ T cells.

Data on 60-Hz electric field (EF) induced reduction in growth rate of plant roots have strongly supported the hypothesis that the effect is related to an EF-induced transmembrane potential (Vim). An investigation was undertaken to determine if this hypothesis is also applicable to 60-Hz EF-induced reductions in growth rate of mammalian cells in vitro. Human lymphoblastic (RPMI 1788) and human carcinoma (HeLa) cells were selected for study, the former having a relatively small diameter (11.2 microns), and the latter having a relatively large diameter (15.4 microns). The 60-Hz EFs ranged from 430-1200 V/m in the culture medium. The growth rate of RPMI 1788 cells after 4-days was depressed by about 42% at a 60-Hz EF of 1000-1200 V/m with a response threshold occurring at 950 V/m; the Vim at the response threshold was 8 mV. There was no 60-Hz EF-induced effect on HeLa cell growth rate of a Vim of 8 mV (60-Hz EF = 700 V/m); a statistically significant effect was achieved at Vim of 11 mV (950 V/m). The data support the hypothesis that above a threshold 60-Hz EF, Vim acts as the initial signal leading to growth rate reductions.

The immune system depends on the extensive proliferation of rare Ag-specific precursor T lymphocytes, followed by their differentiation, the delivery of effector function, and finally death by apoptosis. T cells that lack the E2F-1 transcription factor, which is activated as cells pass the restriction point and enter S phase, show defects in activation-induced cell death. We now report that E2F-1 increases the activity of an apoptotic pathway that is important in murine primary T cells. Thus, E2F-1 promotes the transcription of Bid, a molecule that links death receptor signaling to the activation of apoptotic mechanisms in mitochondria. It also promotes the transcription of caspase-8, the enzyme that cleaves and activates Bid. Enforced expression of Bid can partially restore apoptosis in E2F-1-deficient T cells. Thus, E2F-1 integrates cell cycle progression with apoptosis.

This study examined the effects of nitrogen dioxide (NO2) exposure on airway inflammation, blood cells, and antiviral respiratory defense. Twenty-one healthy volunteers were exposed on separate occasions to air and 0.6 and 1.5 ppm NO2for 3 h with intermittent moderate exercise. Phlebotomy and bronchoscopy were performed 3.5 h after each exposure, and recovered cells were challenged with respiratory viruses in vitro. Blood studies revealed a 4.1% NO2dose-related decrease in hematocrit ( P = 0.003). Circulating total lymphocytes ( P = 0.024) and T lymphocytes ( P = 0.049) decreased with NO2exposure. Exposure to NO2increased the blood lymphocyte CD4+-to-CD8+ratio from 1.74 ± 0.11 to 1.85 ± 0.12 in males but decreased it from 1.88 ± 0.19 to 1.78 ± 0.19 in females ( P < 0.001 for gender difference). Polymorphonuclear leukocytes in bronchial lavage increased with NO2exposure ( P = 0.003). Bronchial epithelial cells obtained after exposure to 1.5 ppm NO2released 40% more lactate dehydrogenase after challenge with respiratory syncytial virus than with air exposure ( P = 0.024). In healthy subjects, exposures to NO2at levels found indoors cause mild airway inflammation, effects on blood cells, and increased susceptibility of airway epithelial cells to injury from respiratory viruses.

An investigation was undertaken to define a 60-Hz electric field exposure system which would affect a eukaryotic cell system while mitigating a potential thermal rise. The biological effectiveness of pulsed 60-Hz electric fields on a cell system of defined sensitivity to continuous-wave 60-Hz electric fields was sought. Roots of garden pea (Pisum sativum L.) were exposed to pulsed trains of 60-Hz, 430 V/m electric fields. The "on" time was constant at 1 s and the "off" time varied. The repetitive on:off regimens used were 1:20, 1:50, 1:100, 1:130, 1:200, and 1:300. With continuous or 1:20 pulsed fields the growth response was equivalent (representing a 60% depression in root growth rate). The severity of the growth effect diminished as the off time increased; for the 1:100 regimen, the relative growth rate was depressed by about 30%; for the 1:300 regimen, the relative growth rate was equal to that of the controls.