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Sample records for activated m2 phenotype

  1. Monocyte Differentiation towards Protumor Activity Does Not Correlate with M1 or M2 Phenotypes

    PubMed Central

    Chimal-Ramírez, G. Karina; Espinoza-Sánchez, Nancy Adriana; Chávez-Sánchez, Luis; Arriaga-Pizano, Lourdes

    2016-01-01

    Macrophages facilitate breast cancer progression. Macrophages were initially classified as M1 or M2 based on their distinct metabolic programs and then expanded to include antitumoral (M1) and protumoral (M2) activities. However, it is still uncertain what markers define the pro- and antitumoral phenotypes and what conditions lead to their formation. In this study, monocytic cell lines and primary monocytes were subjected to commonly reported protocols of M1/M2 polarization and conditions known to engage monocytes into protumoral functions. The results showed that only IDO enzyme and CD86 M1 markers were upregulated correlating with M1 polarization. TNF-α, CCR7, IL-10, arginase I, CD36, and CD163 were expressed indistinguishably from M1 or M2 polarization. Similarly, protumoral engaging resulted in upregulation of both M1 and M2 markers, with conditioned media from the most aggressive breast cancer cell line promoting the greatest changes. In spite of the mixed phenotype, M1-polarized macrophages exhibited the highest expression/secretion of inflammatory mediators, many of which have previously been associated with breast cancer aggressiveness. These data argue that although the existence of protumoral macrophages is unquestionable, their associated phenotypes and the precise conditions driving their formation are still unclear, and those conditions may need both M1 and M2 stimuli. PMID:27376091

  2. Proinflammatory-activated glioma cells induce a switch in microglial polarization and activation status, from a predominant M2b phenotype to a mixture of M1 and M2a/B polarized cells

    PubMed Central

    Lisi, Lucia; Stigliano, Egidio; Lauriola, Libero; Navarra, Pierluigi; Russo, Cinzia Dello

    2014-01-01

    Malignant gliomas are primary brain tumors characterized by morphological and genetic complexities, as well as diffuse infiltration into normal brain parenchyma. Within gliomas, microglia/macrophages represent the largest tumor-infiltrating cell population, contributing by at least one-third to the total tumor mass. Bi-directional interactions between glioma cells and microglia may therefore play an important role on tumor growth and biology. In the present study, we have characterized the influence of glioma-soluble factors on microglial function, comparing the effects of media harvested under basal conditions with those of media obtained after inducing a pro-inflammatory activation state in glioma cells. We found that microglial cells undergo a different pattern of activation depending on the stimulus; in the presence of activated glioma-derived factors, i.e. a condition mimicking the late stage of pathology, microglia presents as a mixture of polarization phenotypes (M1 and M2a/b), with up-regulation of iNOS (inducible nitric oxide synthase), ARG (arginase) and IL (interleukine)-10. At variance, microglia exposed to basal glioma-derived factors, i.e. a condition resembling the early stage of pathology, shows a more specific pattern of activation, with increased M2b polarization status and up-regulation of IL-10 only. As far as viability and cell proliferation are concerned, both LI-CM [LPS (lipopolysaccharide)–IFNγ (interferon γ) conditioned media] and C-CM (control-conditioned media) induce similar effects on microglial morphology. Finally, in human glioma tissue obtained from surgical resection of patients with IV grade glioblastoma, we detected a significant amount of CD68 positive cells, which is a marker of macrophage/microglial phagocytic activity, suggesting that in vitro findings presented here might have a relevance in the human pathology as well. PMID:24689533

  3. The human tissue-biomaterial interface: a role for PPARγ-dependent glucocorticoid receptor activation in regulating the CD163+ M2 macrophage phenotype.

    PubMed

    Bullers, Samuel J; Baker, Simon C; Ingham, Eileen; Southgate, Jennifer

    2014-09-01

    In vivo studies of implanted acellular biological scaffolds in experimental animals have shown constructive remodeling mediated by anti-inflammatory macrophages. Little is known about the human macrophage response to such biomaterials, or the nature of the signaling mechanisms that govern the macrophage phenotype in this environment. The cellular events at the interface of a tissue and implanted decellularized biomaterial were examined by establishing a novel ex vivo tissue culture model in which surgically excised human urinary tract tissue was combined with porcine acellular bladder matrix (PABM). Evaluation of the tissue-biomaterial interface showed a time-dependent infiltration of the biomaterial by CD68(+) CD80(-) macrophages. The migration of CD68(+) cells from the tissue to the interface was accompanied by maturation to a CD163(hi) phenotype, suggesting that factor(s) associated with the biomaterial or the wound edge was/were responsible for the active recruitment and polarization of local macrophages. Glucocorticoid receptor (GR) and peroxisome proliferator activated receptor gamma (PPARγ) signaling was investigated as candidate pathways for integrating inflammatory responses; both showed intense nuclear labeling in interface macrophages. GR and PPARγ activation polarized peripheral blood-derived macrophages from a default M1 (CD80(+)) toward an M2 (CD163(+)) phenotype, but PPARγ signaling predominated, as its antagonism blocked any GR-mediated effect. Seeding on PABM was effective at polarizing peripheral blood-derived macrophages from a default CD80(+) phenotype on glass to a CD80(-) phenotype, with intense nuclear localization of PPARγ. These results endorse in vivo observations that the infiltration of decellularized biological scaffolds, exemplified here by PABM, is pioneered by macrophages. Thus, it appears that natural factors present in PABM are involved in the active recruitment and polarization of macrophages to a CD163(+) phenotype, with

  4. The Human Tissue–Biomaterial Interface: A Role for PPARγ-Dependent Glucocorticoid Receptor Activation in Regulating the CD163+ M2 Macrophage Phenotype

    PubMed Central

    Bullers, Samuel J.; Baker, Simon C.; Ingham, Eileen

    2014-01-01

    In vivo studies of implanted acellular biological scaffolds in experimental animals have shown constructive remodeling mediated by anti-inflammatory macrophages. Little is known about the human macrophage response to such biomaterials, or the nature of the signaling mechanisms that govern the macrophage phenotype in this environment. The cellular events at the interface of a tissue and implanted decellularized biomaterial were examined by establishing a novel ex vivo tissue culture model in which surgically excised human urinary tract tissue was combined with porcine acellular bladder matrix (PABM). Evaluation of the tissue–biomaterial interface showed a time-dependent infiltration of the biomaterial by CD68+ CD80− macrophages. The migration of CD68+ cells from the tissue to the interface was accompanied by maturation to a CD163hi phenotype, suggesting that factor(s) associated with the biomaterial or the wound edge was/were responsible for the active recruitment and polarization of local macrophages. Glucocorticoid receptor (GR) and peroxisome proliferator activated receptor gamma (PPARγ) signaling was investigated as candidate pathways for integrating inflammatory responses; both showed intense nuclear labeling in interface macrophages. GR and PPARγ activation polarized peripheral blood-derived macrophages from a default M1 (CD80+) toward an M2 (CD163+) phenotype, but PPARγ signaling predominated, as its antagonism blocked any GR-mediated effect. Seeding on PABM was effective at polarizing peripheral blood-derived macrophages from a default CD80+ phenotype on glass to a CD80− phenotype, with intense nuclear localization of PPARγ. These results endorse in vivo observations that the infiltration of decellularized biological scaffolds, exemplified here by PABM, is pioneered by macrophages. Thus, it appears that natural factors present in PABM are involved in the active recruitment and polarization of macrophages to a CD163+ phenotype, with activation of

  5. Cervical Cancer Cell Supernatants Induce a Phenotypic Switch from U937-Derived Macrophage-Activated M1 State into M2-Like Suppressor Phenotype with Change in Toll-Like Receptor Profile

    PubMed Central

    Sánchez-Reyes, Karina; Bravo-Cuellar, Alejandro; Hernández-Flores, Georgina; Lerma-Díaz, José Manuel; Jave-Suárez, Luis Felipe; Gómez-Lomelí, Paulina; de Celis, Ruth; Aguilar-Lemarroy, Adriana; Domínguez-Rodríguez, Jorge Ramiro; Ortiz-Lazareno, Pablo Cesar

    2014-01-01

    Cervical cancer (CC) is the second most common cancer among women worldwide. Infection with human papillomavirus (HPV) is the main risk factor for developing CC. Macrophages are important immune effector cells; they can be differentiated into two phenotypes, identified as M1 (classically activated) and M2 (alternatively activated). Macrophage polarization exerts profound effects on the Toll-like receptor (TLR) profile. In this study, we evaluated whether the supernatant of human CC cells HeLa, SiHa, and C-33A induces a shift of M1 macrophage toward M2 macrophage in U937-derived macrophages. Results. The results showed that soluble factors secreted by CC cells induce a change in the immunophenotype of macrophages from macrophage M1 into macrophage M2. U937-derived macrophages M1 released proinflammatory cytokines and nitric oxide; however, when these cells were treated with the supernatant of CC cell lines, we observed a turnover of M1 toward M2. These cells increased CD163 and IL-10 expression. The expression of TLR-3, -7, and -9 is increased when the macrophages were treated with the supernatant of CC cells. Conclusions. Our result strongly suggests that CC cells may, through the secretion of soluble factors, induce a change of immunophenotype M1 into M2 macrophages. PMID:25309919

  6. The Phenotypic and Functional Features of Human M2 Macrophages Generated Under Low Serum Conditions.

    PubMed

    Sakhno, L V; Shevela, E Ya; Tikhonova, M A; Ostanin, A A; Chernykh, E R

    2016-02-01

    The phenotypic and functional features of human M2 macrophages, in particular, their immunosuppressive activity, can considerably vary depending on M2 polarizing stimulus. This study was aimed at the investigation of cytokine production and pro-apoptogenic/inhibitory molecule expression in macrophages generated with GM-CSF using either standard conditions (M1) or deficiency of serum/growth factors (M2-LS cells). In contrast to M1, M2-LS cells were characterized by an enhanced content of CD206(+), B7-H1(+), FasL(+) and TRAIL(+) cells along with a decreased production of IFN-γ, IL-5, IL-6, IL-13, TNF-α, IL-17 and MCP-1. In addition, M2-LS exhibited a lower T cell stimulatory activity in MLC that was associated with the higher numbers of apoptotic and the lower numbers of proliferating T cells. B7-H1 plays a key role in M2-LS-mediated cytotoxic effects as the neutralization of B7-H1 reduces the apoptosis-inducing activity of M2-LS, while the blocking of CD206 and TRAIL reduces the cytostatic activity of M2 macrophages. PMID:26678544

  7. [Nle4, D-Phe7]-α-MSH Inhibits Toll-Like Receptor (TLR)2- and TLR4-Induced Microglial Activation and Promotes a M2-Like Phenotype

    PubMed Central

    Carniglia, Lila; Ramírez, Delia; Durand, Daniela; Saba, Julieta; Caruso, Carla; Lasaga, Mercedes

    2016-01-01

    α-melanocyte stimulating hormone (α-MSH) is an anti-inflammatory peptide, proved to be beneficial in many neuroinflammatory disorders acting through melanocortin receptor 4 (MC4R). We previously determined that rat microglial cells express MC4R and that NDP-MSH, an analog of α-MSH, induces PPAR-γ expression and IL-10 release in these cells. Given the great importance of modulation of glial activation in neuroinflammatory disorders, we tested the ability of NDP-MSH to shape microglial phenotype and to modulate Toll-like receptor (TLR)-mediated inflammatory responses. Primary rat cultured microglia were stimulated with NDP-MSH followed by the TLR2 agonist Pam3CSK4 or the TLR4 agonist LPS. NDP-MSH alone induced expression of the M2a/M2c marker Ag1 and reduced expression of the M2b marker Il-4rα and of the LPS receptor Tlr4. Nuclear translocation of NF-κB subunits p65 and c-Rel was induced by LPS and these effects were partially prevented by NDP-MSH. NDP-MSH reduced LPS- and Pam3CSK4-induced TNF-α release but did not affect TLR-induced IL-10 release. Also, NDP-MSH inhibited TLR2-induced HMGB1 translocation from nucleus to cytoplasm and TLR2-induced phagocytic activity. Our data show that NDP-MSH inhibits TLR2- and TLR4-mediated proinflammatory mechanisms and promotes microglial M2-like polarization, supporting melanocortins as useful tools for shaping microglial activation towards an alternative immunomodulatory phenotype. PMID:27359332

  8. Azithromycin protects mice against ischemic stroke injury by promoting macrophage transition towards M2 phenotype.

    PubMed

    Amantea, Diana; Certo, Michelangelo; Petrelli, Francesco; Tassorelli, Cristina; Micieli, Giuseppe; Corasaniti, Maria Tiziana; Puccetti, Paolo; Fallarino, Francesca; Bagetta, Giacinto

    2016-01-01

    To develop novel and effective treatments for ischemic stroke, we investigated the neuroprotective effects of the macrolide antibiotic azithromycin in a mouse model system of transient middle cerebral artery occlusion. Intraperitoneal administration of azithromycin significantly reduced blood-brain barrier damage and cerebral infiltration of myeloid cells, including neutrophils and inflammatory macrophages. These effects resulted in a dose-dependent reduction of cerebral ischemic damage, and in a remarkable amelioration of neurological deficits up to 7 days after the insult. Neuroprotection was associated with increased arginase activity in peritoneal exudate cells, which was followed by the detection of Ym1- and arginase I-immunopositive M2 macrophages in the ischemic area at 24-48 h of reperfusion. Pharmacological inhibition of peritoneal arginase activity counteracted azithromycin-induced neuroprotection, pointing to a major role for drug-induced polarization of migratory macrophages towards a protective, non-inflammatory M2 phenotype. PMID:26518285

  9. EXTENDED CULTURE OF MACROPHAGES FROM DIFFERENT SOURCES AND MATURATION RESULTS IN A COMMON M2 PHENOTYPE

    PubMed Central

    Chamberlain, Lisa M.; Holt-Casper, Dolly; Gonzalez-Juarrero, Mercedes; Grainger, David W.

    2015-01-01

    Inflammatory responses to biomaterials heavily influence the environment surrounding implanted devices, often producing foreign body reactions. The macrophage is a key immunomodulatory cell type consistently associated with implanted biomaterials and routinely employed in short term in vitro cell studies of biomaterials aiming to reproduce host responses. Inconsistencies within these studies, including differently sourced cells, different durations of culture, and assessment of different activation markers, lead to many conflicting results in vitro that confound consistency and conclusions. We hypothesize that different experimentally popular monocyte-macrophage cell types have intrinsic in vitro culture-specific differences that yield conflicting results. Recent studies demonstrate changes in cultured macrophage cytokine expression over time, leading to the hypothesis that changes in macrophage phenotype also occur in response to extended culture. Here, macrophage cells of different transformed and primary-derived origins were cultured for 21 days on model polymer biomaterials. Cell type-based differences in morphology and cytokine/chemokine expression as well as changes in cell surface biomarkers associated with differentiation stage, activation state, and adhesion were compared. Results reflect consistent macrophage development towards an M2 phenotype via up-regulation of the macrophage mannose receptor for all cell types following 21-day extended culture. Significantly, implanted biomaterials experiencing the foreign body response and encapsulation in vivo often elicit a shift towards an analogous M2 macrophage phenotype. In vitro “default” of macrophage cultures, regardless of lineage, to this M2 state in the presence of biomaterials at long culture periods is not recognized but has important implications to in vitro modeling of in vivo host response. PMID:25684281

  10. Extended culture of macrophages from different sources and maturation results in a common M2 phenotype.

    PubMed

    Chamberlain, Lisa M; Holt-Casper, Dolly; Gonzalez-Juarrero, Mercedes; Grainger, David W

    2015-09-01

    Inflammatory responses to biomaterials heavily influence the environment surrounding implanted devices, often producing foreign-body reactions. The macrophage is a key immunomodulatory cell type consistently associated with implanted biomaterials and routinely used in short-term in vitro cell studies of biomaterials aiming to reproduce host responses. Inconsistencies within these studies, including differently sourced cells, different durations of culture, and assessment of different activation markers, lead to many conflicting results in vitro that confound consistency and conclusions. We hypothesize that different experimentally popular monocyte-macrophage cell types have intrinsic in vitro culture-specific differences that yield conflicting results. Recent studies demonstrate changes in cultured macrophage cytokine expression over time, leading to the hypothesis that changes in macrophage phenotype also occur in response to extended culture. Here, macrophage cells of different transformed and primary-derived origins were cultured for 21 days on model polymer biomaterials. Cell type-based differences in morphology and cytokine/chemokine expression as well as changes in cell surface biomarkers associated with differentiation stage, activation state, and adhesion were compared. Results reflect consistent macrophage development toward an M2 phenotype via up-regulation of the macrophage mannose receptor for all cell types following 21-day extended culture. Significantly, implanted biomaterials experiencing the foreign-body response and encapsulation in vivo often elicit a shift toward an analogous M2 macrophage phenotype. In vitro "default" of macrophage cultures, regardless of lineage, to this M2 state in the presence of biomaterials at long culture periods is not recognized, but has important implications to in vitro modeling of in vivo host response. PMID:25684281

  11. Baicalin ameliorates experimental inflammatory bowel disease through polarization of macrophages to an M2 phenotype.

    PubMed

    Zhu, Wei; Jin, Zaishun; Yu, Jianbo; Liang, Jun; Yang, Qingdong; Li, Fujuan; Shi, Xuekui; Zhu, Xiaodong; Zhang, Xiaoli

    2016-06-01

    Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders of the intestinal tract. Baicalin, originally isolated from the root of the Chinese herb Huangqin (Scutellaria baicalensis Georgi) and its main active ingredient, has a protective effect against inflammatory responses in several diseases. The present study investigated the effects of baicalin on macrophage polarization and its therapeutic role in IBD. Murine peritoneal macrophages and mice with colitis were treated with baicalin. Macrophage subset distribution, M1 and M2 macrophage-associated mRNA expression, and interferon regulatory factor 4 and 5 (IRF4 and IRF5) expression were analyzed. siRNA transfection into mouse peritoneal macrophages was utilized to suppress IRF4. Fluorescence-activated cell sorting, western blot, and real-time PCR analyses were performed. Baicalin (50μM) limited lipopolysaccharide (LPS)-induced M1 macrophage polarization; decreased LPS-induced tumor necrosis factor α, interleukin (IL)-23, and IRF5 expression; and increased IL-10, arginase-1 (Arg-1), and IRF4 expression. siRNA-mediated IRF4 silencing significantly impaired baicalin activity. Furthermore, pretreatment with baicalin (100mg/kg) in mice with dextran sodium sulfate (DSS)-induced colitis ameliorated the severity of colitis and significantly decreased the disease activity index (baicalin group, 3.33±0.52 vs. DSS group, 5.67±1.03). Baicalin (100mg/kg) also repressed IRF5 protein expression and promoted IRF4 protein expression in the lamina propria mononuclear cells, and induced macrophage polarization to the M2 phenotype. In summary, our results showed that baicalin upregulates IRF4 protein expression and reverses LPS-induced macrophage subset redistribution. Thus, baicalin alleviates DSS-induced colitis by modulating macrophage polarization to the M2 phenotype. PMID:27039210

  12. Anthocyanin effects on microglia M1/M2 phenotype: Consequence on neuronal fractalkine expression.

    PubMed

    Meireles, Manuela; Marques, Cláudia; Norberto, Sónia; Santos, Paulo; Fernandes, Iva; Mateus, Nuno; Faria, Ana; Calhau, Conceição

    2016-05-15

    Microglia mediate multiple aspects of neuroinflammation, including cytotoxicity, repair, regeneration, and immunosuppression due to their ability to acquire diverse activation states, or phenotypes. Modulation of microglial phenotype or microglia-neuron crosstalk can be an appealing neurotherapeutic strategy. Anthocyanins are a class of flavonoids found e.g., in berries that has been attracting interest due to its neuroprotective potential. However, there are no data clarifying the impact of anthocyanins on microglial phenotype or on microglia-neuron crosstalk (CX3CR1/CX3CL1). N9 microglia cell line was treated with 1μM cyanidin (Cy), cyanidin-3-glucose (Cy3glc) and a methylated form of cyanidin-3-glucose (Met-Cy3glc) in basal conditions and with LPS/IL-4 stimulation. SH-SY5Y cell line was treated with the conditioned medium of microglia and with the anthocyanins alone. At basal conditions, microglia treatment with anthocyanins for 24h induced a less pro-inflammatory profile. Decreased TNF-α mRNA expression was induced either by Cy and Met-Cy3glc. LPS markedly increase IL-6 mRNA expression, which was lowered by Cy3glc. IL-1β LPS-induced expression was reverted by Cy. Cy increased CX3CL1 mRNA expression in SH-SY5Y comparing either with control or LPS. Anthocyanins and metabolites were not able to shift microglia to an M2 strict phenotype however they did interact with microglia biology. There was an attenuation of M1 phenotype and increase of neuronal expression of CX3CL1 mRNA. Understanding how flavonoids modulate microglia-neuron crosstalk can open new directions for future nutritional interventions. PMID:26965567

  13. Fasciola hepatica tegumental antigens indirectly induce an M2 macrophage-like phenotype in vivo.

    PubMed

    Adams, P N; Aldridge, A; Vukman, K V; Donnelly, S; O'Neill, S M

    2014-10-01

    The M2 subset of macrophages has a critical role to play in host tissue repair, tissue fibrosis and modulation of adaptive immunity during helminth infection. Infection with the helminth, Fasciola hepatica, is associated with M2 macrophages in its mammalian host, and this response is mimicked by its excretory-secretory products (FhES). The tegumental coat of F. hepatica (FhTeg) is another major source of immune-modulatory molecules; we have previously shown that FhTeg can modulate the activity of both dendritic cells and mast cells inhibiting their ability to prime a Th1 immune response. Here, we report that FhTeg does not induce Th2 immune responses but can induce M2-like phenotype in vivo that modulates cytokine production from CD4(+) cells in response to anti-CD3 stimulation. FhTeg induces a RELMα expressing macrophage population in vitro, while in vivo, the expression of Arg1 and Ym-1/2 but not RELMα in FhTeg-stimulated macrophages was STAT6 dependent. To support this finding, FhTeg induces RELMα expression in vivo prior to the induction of IL-13. FhTeg can induce IL-13-producing peritoneal macrophages following intraperitoneal injection This study highlights the important role of FhTeg as an immune-modulatory source during F. hepatica infection and sheds further light on helminth-macrophage interactions. PMID:25039932

  14. Pomegranate juice polyphenols induce a phenotypic switch in macrophage polarization favoring a M2 anti-inflammatory state.

    PubMed

    Aharoni, Saar; Lati, Yoni; Aviram, Michael; Fuhrman, Bianca

    2015-01-01

    It was documented that pomegranate has anti-inflammatory effects. In this study, we investigated a direct effect of pomegranate juice (PJ) and its polyphenols on macrophage inflammatory phenotype. In vitro, PJ and its major polyphenols dose-dependently attenuated macrophage response to M1 proinflammatory activation in J774.A1 macrophage-like cell line. This was evidenced by a significant decrease in TNFα and IL-6 secretion in response to stimulation by IFNγ and Lipopolysaccharide. In addition, PJ and punicalagin dose-dependently promoted the macrophages toward a M2 anti-inflammatory phenotype, as determined by a significant increase in the spontaneous secretion of IL-10. In mice, supplementation with dietary PJ substantially inhibited the M2 to M1 macrophage phenotypic shift associated with age, toward a favorable anti-inflammatory M2 phenotype. This effect was also reflected in the mice atherosclerotic plaques, as evaluated by the distinct expression of arginase isoforms. PJ consumption inhibited the increment of arginase II (Arg II, M1) mRNA expression during aging, and maintained the levels of Arg I (M2) expression similar to those in young mice aorta. This study demonstrates, for the first time, that pomegranate polyphenols directly suppress macrophage inflammatory responses and promote M1 to M2 switch in macrophage phenotype. Furthermore, this study indicates that PJ consumption may inhibit the progressive proinflammatory state in the aorta along atherosclerosis development with aging, due to a switch in macrophage phenotype from proinflammatory M1 to anti-inflammatory M2. PMID:25650983

  15. From Monocytes to M1/M2 Macrophages: Phenotypical vs. Functional Differentiation

    PubMed Central

    Italiani, Paola; Boraschi, Diana

    2014-01-01

    Studies on monocyte and macrophage biology and differentiation have revealed the pleiotropic activities of these cells. Macrophages are tissue sentinels that maintain tissue integrity by eliminating/repairing damaged cells and matrices. In this M2-like mode, they can also promote tumor growth. Conversely, M1-like macrophages are key effector cells for the elimination of pathogens, virally infected, and cancer cells. Macrophage differentiation from monocytes occurs in the tissue in concomitance with the acquisition of a functional phenotype that depends on microenvironmental signals, thereby accounting for the many and apparently opposed macrophage functions. Many questions arise. When monocytes differentiate into macrophages in a tissue (concomitantly adopting a specific functional program, M1 or M2), do they all die during the inflammatory reaction, or do some of them survive? Do those that survive become quiescent tissue macrophages, able to react as naïve cells to a new challenge? Or, do monocyte-derived tissue macrophages conserve a “memory” of their past inflammatory activation? This review will address some of these important questions under the general framework of the role of monocytes and macrophages in the initiation, development, resolution, and chronicization of inflammation. PMID:25368618

  16. Role of the tumor suppressor ARF in macrophage polarization: Enhancement of the M2 phenotype in ARF-deficient mice.

    PubMed

    Herranz, Sandra; Través, Paqui G; Luque, Alfonso; Hortelano, Sonsoles

    2012-11-01

    The ARF locus is frequently inactivated in human cancer. The oncosuppressor ARF has indeed been described as a general sensor for different situation of cellular stress. We have previously demonstrated that ARF deficiency severely impairs inflammatory responses in vitro and in vivo, establishing a role for ARF in the regulation of innate immunity. The aim of the present work was to get further insights into the immune functions of ARF and to evaluate its possible contribution to the polarization of macrophages toward the M1 or M2 phenotype. Our results demonstrate that resting Arf(-/-) macrophages express high levels of Ym1 and Fizz-1, two typical markers of alternatively-activated macrophages (M2). Additionally, Arf(-/-) peritoneal macrophages showed an impaired response to lipopolysaccharide (a classical inducer of M1 polaryzation) and a reduced production of pro-inflammatory cytokines/chemokines. Moreover, upon stimulation with interleukin-4 (IL-4), an inducer of the M2 phenotype, well established M2 markers such as Fizz-1, Ym1 and arginase-1 were upregulated in Arf(-/-) as compared with wild type macrophages. Accordingly, the cytokine and chemokine profile associated with the M2 phenotype was significantly overexpressed in Arf(-/-) macrophages responding to IL-4. In addition, multiple pro-angiogenic factors such as VEGF and MMP-9 were also increased. In summary, these results indicate that ARF contributes to the polarization and functional plasticity of macrophages. PMID:23243586

  17. Expression of area-specific M2-macrophage phenotype by recruited rat monocytes in duct-ligation pancreatitis.

    PubMed

    Yu, Enqiao; Goto, Mataro; Ueta, Hisashi; Kitazawa, Yusuke; Sawanobori, Yasushi; Kariya, Taro; Sasaki, Masaru; Matsuno, Kenjiro

    2016-06-01

    Acute pancreatitis remains a disease of uncertain pathogenesis and no established specific therapy. Previously, we found a predominant increase and active proliferation of macrophages in the inflamed tissues of a rat duct-ligation pancreatitis model. To analyze the origin and possible role of these macrophages, we investigated their in situ cellular kinetics in a rat model of duct-ligation pancreatitis using a recently established method of multicolor immunostaining for macrophage markers and for proliferating cells with ethynyl deoxyuridine. To detect monocyte-derived macrophages, green fluorescent protein-transgenic (GFP(+)) leukocytes were transferred to monocyte-depleted recipients. In the inflamed pancreas, infiltrating macrophages were mainly two phenotypes, CD68(+)CD163(-) round cells and CD68(+)CD163(+) large polygonal cells, both of which showed active proliferation. In the interlobular area, the proportions of CD68(+)CD163(low) and CD68(+)CD163(high) cells increased over time. Most expressed the M2-macrophage markers CD206 and arginase 1. In contrast, in the interacinar area, CD68(+) cells did not upregulate CD163 and CD206, but ~30 % of them expressed the M1 marker nitric oxide synthase 2 on day 4. GFP(+)-recruited cells were primarily CD68(+)CD163(-) monocytes on day 1 and showed phenotypic changes similar to those of the monocyte non-depleted groups. In conclusion, infiltrating macrophages mostly formed two distinct subpopulations in different areas: monocyte-derived macrophages with the M2 phenotype in the interlobular area or non-M2 phenotype in the interacinar area. Involvement of resident macrophages might be minor in this model. These results are the first demonstration of an upregulated M2 phenotype in rat inflammatory monocytes, which may promote tissue repair. PMID:26860866

  18. Understanding the Mysterious M2 Macrophage through Activation Markers and Effector Mechanisms

    PubMed Central

    Rőszer, Tamás

    2015-01-01

    The alternatively activated or M2 macrophages are immune cells with high phenotypic heterogeneity and are governing functions at the interface of immunity, tissue homeostasis, metabolism, and endocrine signaling. Today the M2 macrophages are identified based on the expression pattern of a set of M2 markers. These markers are transmembrane glycoproteins, scavenger receptors, enzymes, growth factors, hormones, cytokines, and cytokine receptors with diverse and often yet unexplored functions. This review discusses whether these M2 markers can be reliably used to identify M2 macrophages and define their functional subdivisions. Also, it provides an update on the novel signals of the tissue environment and the neuroendocrine system which shape the M2 activation. The possible evolutionary roots of the M2 macrophage functions are also discussed. PMID:26089604

  19. Regulation of Macrophage Motility by the Water Channel Aquaporin-1: Crucial Role of M0/M2 Phenotype Switch

    PubMed Central

    Tyteca, Donatienne; Nishino, Tomoya; Debaix, Huguette; Van Der Smissen, Patrick; N'Kuli, Francisca; Hoffmann, Delia; Cnops, Yvette; Rabolli, Virginie; van Loo, Geert; Beyaert, Rudi; Huaux, François; Devuyst, Olivier; Courtoy, Pierre J.

    2015-01-01

    The water channel aquaporin-1 (AQP1) promotes migration of many cell types. Although AQP1 is expressed in macrophages, its potential role in macrophage motility, particularly in relation with phenotype polarization, remains unknown. We here addressed these issues in peritoneal macrophages isolated from AQP1-deficient mice, either undifferentiated (M0) or stimulated with LPS to orientate towards pro-inflammatory phenotype (classical macrophage activation; M1). In non-stimulated macrophages, ablation of AQP1 (like inhibition by HgCl2) increased by 2–3 fold spontaneous migration in a Src/PI3K/Rac-dependent manner. This correlated with cell elongation and formation of lamellipodia/ruffles, resulting in membrane lipid and F4/80 recruitment to the leading edge. This indicated that AQP1 normally suppresses migration of resting macrophages, as opposed to other cell types. Resting Aqp1-/- macrophages exhibited CD206 redistribution into ruffles and increased arginase activity like IL4/IL13 (alternative macrophage activation; M2), indicating a M0-M2 shift. In contrast, upon M1 orientation by LPS in vitro or peritoneal inflammation in vivo, migration of Aqp1-/- macrophages was reduced. Taken together, these data indicate that AQP1 oppositely regulates macrophage migration, depending on stimulation or not by LPS, and that macrophage phenotypic and migratory changes may be regulated independently of external cues. PMID:25719758

  20. Regulation of macrophage motility by the water channel aquaporin-1: crucial role of M0/M2 phenotype switch.

    PubMed

    Tyteca, Donatienne; Nishino, Tomoya; Debaix, Huguette; Van Der Smissen, Patrick; N'Kuli, Francisca; Hoffmann, Delia; Cnops, Yvette; Rabolli, Virginie; van Loo, Geert; Beyaert, Rudi; Huaux, François; Devuyst, Olivier; Courtoy, Pierre J

    2015-01-01

    The water channel aquaporin-1 (AQP1) promotes migration of many cell types. Although AQP1 is expressed in macrophages, its potential role in macrophage motility, particularly in relation with phenotype polarization, remains unknown. We here addressed these issues in peritoneal macrophages isolated from AQP1-deficient mice, either undifferentiated (M0) or stimulated with LPS to orientate towards pro-inflammatory phenotype (classical macrophage activation; M1). In non-stimulated macrophages, ablation of AQP1 (like inhibition by HgCl2) increased by 2-3 fold spontaneous migration in a Src/PI3K/Rac-dependent manner. This correlated with cell elongation and formation of lamellipodia/ruffles, resulting in membrane lipid and F4/80 recruitment to the leading edge. This indicated that AQP1 normally suppresses migration of resting macrophages, as opposed to other cell types. Resting Aqp1-/- macrophages exhibited CD206 redistribution into ruffles and increased arginase activity like IL4/IL13 (alternative macrophage activation; M2), indicating a M0-M2 shift. In contrast, upon M1 orientation by LPS in vitro or peritoneal inflammation in vivo, migration of Aqp1-/- macrophages was reduced. Taken together, these data indicate that AQP1 oppositely regulates macrophage migration, depending on stimulation or not by LPS, and that macrophage phenotypic and migratory changes may be regulated independently of external cues. PMID:25719758

  1. Macrophages in Langerhans cell histiocytosis are differentiated toward M2 phenotype: their possible involvement in pathological processes.

    PubMed

    Ohnishi, Koji; Komohara, Yoshihiro; Sakashita, Naomi; Iyama, Ken-Ichi; Murayama, Toshihiko; Takeya, Motohiro

    2010-01-01

    Although numerous macrophages are found in the lesions of Langerhans cell histiocytosis (LCH), their activation phenotypes and their roles in the disease process have not been clarified. Paraffin-embedded LCH samples were examined on immunohistochemistry and it was found that CD163 can be used to distinguish infiltrated macrophages from neoplastic Langerhans cells (LC). The number of CD163-positve macrophages was positively correlated with the number of multinucleated giant cells (MGC), indicating that most MGC are derived from infiltrated macrophages. A significant number of CD163-positive macrophages were positive for interleukin (IL)-10 and phospho-signal transducer and activator of transcription-3 (pSTAT3), an IL-10-induced signal transduction molecule. This indicates that these macrophages are polarized to anti-inflammatory macrophages of M2 phenotype. Tumor-derived macrophage-colony-stimulating factor (M-CSF) was considered to responsible for inducing M2 differentiation of infiltrated macrophages. The number of CD163-positive macrophages in different cases of LCH varied, and interestingly the density of CD163-positive macrophages was inversely correlated with the Ki-67-positivity of LC. Although the underlying mechanism is not fully elucidated, macrophage-derived IL-10 was considered to be involved in the suppression of tumor cell proliferation via activation of STAT3. PMID:20055949

  2. Bioinformatics approach to evaluate differential gene expression of M1/M2 macrophage phenotypes and antioxidant genes in atherosclerosis.

    PubMed

    da Rocha, Ricardo Fagundes; De Bastiani, Marco Antônio; Klamt, Fábio

    2014-11-01

    Atherosclerosis is a pro-inflammatory process intrinsically related to systemic redox impairments. Macrophages play a major role on disease development. The specific involvement of classically activated, M1 (pro-inflammatory), or the alternatively activated, M2 (anti-inflammatory), on plaque formation and disease progression are still not established. Thus, based on meta-data analysis of public micro-array datasets, we compared differential gene expression levels of the human antioxidant genes (HAG) and M1/M2 genes between early and advanced human atherosclerotic plaques, and among peripheric macrophages (with or without foam cells induction by oxidized low density lipoprotein, oxLDL) from healthy and atherosclerotic subjects. Two independent datasets, GSE28829 and GSE9874, were selected from gene expression omnibus (http://www.ncbi.nlm.nih.gov/geo/) repository. Functional interactions were obtained with STRING (http://string-db.org/) and Medusa (http://coot.embl.de/medusa/). Statistical analysis was performed with ViaComplex(®) (http://lief.if.ufrgs.br/pub/biosoftwares/viacomplex/) and gene score enrichment analysis (http://www.broadinstitute.org/gsea/index.jsp). Bootstrap analysis demonstrated that the activity (expression) of HAG and M1 gene sets were significantly increased in advance compared to early atherosclerotic plaque. Increased expressions of HAG, M1, and M2 gene sets were found in peripheric macrophages from atherosclerotic subjects compared to peripheric macrophages from healthy subjects, while only M1 gene set was increased in foam cells from atherosclerotic subjects compared to foam cells from healthy subjects. However, M1 gene set was decreased in foam cells from healthy subjects compared to peripheric macrophages from healthy subjects, while no differences were found in foam cells from atherosclerotic subjects compared to peripheric macrophages from atherosclerotic subjects. Our data suggest that, different to cancer, in atherosclerosis there is

  3. Bipolar/rod-shaped microglia are proliferating microglia with distinct M1/M2 phenotypes.

    PubMed

    Tam, Wing Yip; Ma, Chi Him Eddie

    2014-01-01

    Microglia are generally considered the resident immune cells in the central nervous system (CNS) that regulate the primary events of neuroinflammatory responses. Microglia also play key roles in repair and neurodegeneration of the CNS after injury. Recent studies showed that trains of bipolar/rod-shaped microglia align end-to-end along the CNS injury site during the initial recovery phase. However, the cellular characteristics of bipolar/rod-shaped microglia remain largely unknown. Here, we established a highly reproducible in vitro culture model system to enrich and characterize bipolar/rod-shaped microglia by simply generating multiple scratches on a poly-d-lysine/laminin-coated culture dish. Trains of bipolar/rod-shaped microglia formed and aligned along the scratches in a manner that morphologically resembled microglial trains observed in injured brain. These bipolar/rod-shaped microglia were highly proliferative and expressed various M1/M2 markers. Further analysis revealed that these bipolar/rod-shaped microglia quickly transformed into amoeboid microglia within 30 minutes of lipopolysaccharide treatment, leading to the upregulation of pro-inflammatory cytokine gene expression and the activation of Jak/Stat. In summary, our culture system provides a model to further characterize this highly dynamic cell type. We suggest that bipolar/rod-shaped microglia are crucial for repairing the damaged CNS and that the molecular mechanisms underlying their morphological changes may serve as therapeutic biomarkers. PMID:25452009

  4. Telmisartan prevention of LPS-induced microglia activation involves M2 microglia polarization via CaMKKβ-dependent AMPK activation.

    PubMed

    Xu, Yuan; Xu, Yazhou; Wang, Yurong; Wang, Yunjie; He, Ling; Jiang, Zhenzhou; Huang, Zhangjian; Liao, Hong; Li, Jia; Saavedra, Juan M; Zhang, Luyong; Pang, Tao

    2015-11-01

    Brain inflammation plays an important role in the pathophysiology of many psychiatric and neurological diseases. During brain inflammation, microglia cells are activated, producing neurotoxic molecules and neurotrophic factors depending on their pro-inflammatory M1 and anti-inflammatory M2 phenotypes. It has been demonstrated that Angiotensin II type 1 receptor blockers (ARBs) ameliorate brain inflammation and reduce M1 microglia activation. The ARB telmisartan suppresses glutamate-induced upregulation of inflammatory genes in cultured primary neurons. We wished to clarify whether telmisartan, in addition, prevents microglia activation through polarization to an anti-inflammatory M2 phenotype. We found that telmisartan promoted M2 polarization and reduced M1 polarization in LPS-stimulated BV2 and primary microglia cells, effects partially dependent on PPARγ activation. The promoting effects of telmisartan on M2 polarization, were attenuated by an AMP-activated protein kinase (AMPK) inhibitor or AMPK knockdown, indicating that AMPK activation participates on telmisartan effects. Moreover, in LPS-stimulated BV2 cells, telmisartan enhancement of M2 gene expression was prevented by the inhibitor STO-609 and siRNA of calmodulin-dependent protein kinase kinase β (CaMKKβ), an upstream kinase of AMPK. Furthermore, telmisartan enhanced brain AMPK activation and M2 gene expression in a mouse model of LPS-induced neuroinflammation. In addition, telmisartan reduced the LPS-induced sickness behavior in this in vivo model, and this effect was prevented by prior administration of an AMPK inhibitor. Our results indicate that telmisartan can be considered as a novel AMPK activator, suppressing microglia activation by promoting M2 polarization. Telmisartan may provide a novel, safe therapeutic approach to treat brain disorders associated with enhanced inflammation. PMID:26188187

  5. Parkin Regulates the Activity of Pyruvate Kinase M2*

    PubMed Central

    Liu, Kun; Li, Fanzhou; Han, Haichao; Chen, Yue; Mao, Zebin; Luo, Jianyuan; Zhao, Yingming; Zheng, Bin; Gu, Wei; Zhao, Wenhui

    2016-01-01

    Parkin, a ubiquitin E3 ligase, is mutated in most cases of autosomal recessive early onset Parkinson disease. It was discovered that Parkin is also mutated in glioblastoma and other human malignancies and that it inhibits tumor cell growth. Here, we identified pyruvate kinase M2 (PKM2) as a unique substrate for parkin through biochemical purification. We found that parkin interacts with PKM2 both in vitro and in vivo, and this interaction dramatically increases during glucose starvation. Ubiquitylation of PKM2 by parkin does not affect its stability but decreases its enzymatic activity. Parkin regulates the glycolysis pathway and affects the cell metabolism. Our studies revealed the novel important roles of parkin in tumor cell metabolism and provided new insight for therapy of Parkinson disease. PMID:26975375

  6. Parkin Regulates the Activity of Pyruvate Kinase M2.

    PubMed

    Liu, Kun; Li, Fanzhou; Han, Haichao; Chen, Yue; Mao, Zebin; Luo, Jianyuan; Zhao, Yingming; Zheng, Bin; Gu, Wei; Zhao, Wenhui

    2016-05-01

    Parkin, a ubiquitin E3 ligase, is mutated in most cases of autosomal recessive early onset Parkinson disease. It was discovered that Parkin is also mutated in glioblastoma and other human malignancies and that it inhibits tumor cell growth. Here, we identified pyruvate kinase M2 (PKM2) as a unique substrate for parkin through biochemical purification. We found that parkin interacts with PKM2 both in vitro and in vivo, and this interaction dramatically increases during glucose starvation. Ubiquitylation of PKM2 by parkin does not affect its stability but decreases its enzymatic activity. Parkin regulates the glycolysis pathway and affects the cell metabolism. Our studies revealed the novel important roles of parkin in tumor cell metabolism and provided new insight for therapy of Parkinson disease. PMID:26975375

  7. Pyropia yezoensis glycoprotein promotes the M1 to M2 macrophage phenotypic switch via the STAT3 and STAT6 transcription factors.

    PubMed

    Choi, Jeong-Wook; Kwon, Mi-Jin; Kim, In-Hye; Kim, Young-Min; Lee, Min-Kyeong; Nam, Taek-Jeong

    2016-08-01

    Macrophage polarization has been well documented. Macrophages can aquire two phenotypes, the pro-inflammatory M1 phenotype, and the anti-inflammatory and wound healing M2 phenotype. The M1 macrophage phenotype has been linked to metabolic disease and is also associated with cancer-related inflammation. Of note, macrophage polarization can be influenced by the extracellular environment. In the current study, we examined the effects of Pyropia yezoensis glycoprotein (PYGP) on M1 to M2 macrophage polarization in lipopolysaccharide (LPS)-stimulated macrophages. RAW 264.7 macrophages stimulated with LPS exhibited an upregulated expression of pro-inflammatory mediators, namely of the M1 markers, nitric oxide (NO), reactive oxygen species (ROS), interleukin (IL)-6, IL-1β, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and nitric oxide synthase‑2 (NOS-2). Treatment with PYGP inhibited the production of M1 markers and increased arginase 1 (ARG1), chitinase-like 3 (Chil3; also known as Ym1), resistin like beta (RETNLB; also known as FIZZ1), IL-10, CD163, CD206, peroxisome proliferator-activated receptor γ (PPARγ) and Krüppel-like factor 4 (KLF4) M2 marker gene expression. The signal transducer and activator of transcription (STAT)3 and STAT6 transcription factors were phosphorylated following treatment with PYGP. However, the silencing of STAT3 and STAT6 using siRNA in the macrophages decreased ARG1, Ym1 and FIZZ1 M2 marker gene expression in spite of treatment of PYGP. These findings suggest that PYGP exerts anti-inflammatory effects by regulating the M1 to M2 phenotypic switch through STAT3 and STAT6. Thus, PYGP may have potential for use as a natural remedy for inflammatory diseases. PMID:27353313

  8. Identification of a 6-Cytokine Prognostic Signature in Patients with Primary Glioblastoma Harboring M2 Microglia/Macrophage Phenotype Relevance

    PubMed Central

    Li, Mingyang; Zhang, Chuanbao; Wang, Zheng; Hu, Huimin; Liu, Yanwei; Li, Qingbin; Wen, Jinchong; Sun, Bo; Wang, Xiaofeng; Jiang, Tao; Jiang, Chuanlu

    2015-01-01

    Background Glioblastomas (GBM) are comprised of a heterogeneous population of tumor cells, immune cells, and extracellular matrix. Interactions among these different cell types and pro-/anti-inflammatory cytokines may promote tumor development and progression. Aims The objective of this study was to develop a cytokine-related gene signature to improve outcome prediction for patients with primary GBM. Methods Here, we used Cox regression and risk-score analysis to develop a cytokine-related gene signature in primary GBMs from the whole transcriptome sequencing profile of the Chinese Glioma Genome Atlas (CGGA) database (n=105). We also examined differences in immune cell phenotype and immune factor expression between the high-risk and low-risk groups. Results Cytokine-related genes were ranked based on their ability to predict survival in the CGGA database. The six genes showing the strongest predictive value were CXCL10, IL17R, CCR2, IL17B, IL10RB, and CCL2. Patients with a high-risk score had poor overall survival and progression-free survival. Additionally, the high-risk group was characterized by increased mRNA expression of M2 microglia/macrophage markers and elevated levels of IL10 and TGFβ1. Conclusion The six cytokine-related gene signature is sufficient to predict survival and to identify a subgroup of primary GBM exhibiting the M2 cell phenotype. PMID:25978454

  9. A novel small-molecule agonist of PPAR-γ potentiates an anti-inflammatory M2 glial phenotype.

    PubMed

    Song, Gyun Jee; Nam, Youngpyo; Jo, Myungjin; Jung, Myungsu; Koo, Ja Young; Cho, Wansang; Koh, Minseob; Park, Seung Bum; Suk, Kyoungho

    2016-10-01

    Neuroinflammation is a key process for many neurodegenerative diseases. Activated microglia and astrocytes play an essential role in neuroinflammation by producing nitric oxide (NO), inflammatory cytokines, chemokines, and neurotoxins. Therefore, targeting glia-mediated neuroinflammation using small-molecules is a potential therapeutic strategy. In this study, we performed a phenotypic screen using microglia cell-based assay to identify a hit compound containing N-carbamoylated urethane moiety (SNU-BP), which inhibits lipopolysaccharide (LPS)-induced NO production in microglia. SNU-BP inhibited pro-inflammatory cytokines and inducible nitric oxide synthase in LPS-stimulated microglia, and potentiated interleukin-4-induced arginase-1 expression. PPAR-γ was identified as a molecular target of SNU-BP. The PPAR response element reporter assay revealed that SNU-BP specifically activated PPAR-γ, but not PPAR-δ or -α, confirming that PPAR-γ is the target protein of SNU-BP. The anti-inflammatory effect of SNU-BP was attenuated by genetic and pharmacological inhibition of PPAR-γ. In addition, SNU-BP induced an anti-inflammatory phenotype in astrocytes as well, by inhibiting pro-inflammatory NO and TNF-α, while increasing anti-inflammatory genes, such as arginase-1 and Ym-1. Finally, SNU-BP exhibited an anti-inflammatory effect in the LPS-injected mouse brain, demonstrating a protective potential for neuroinflammatory diseases. PMID:27288982

  10. Inhibition of Sprouty2 polarizes macrophages toward an M2 phenotype by stimulation with interferon γ and Porphyromonas gingivalis lipopolysaccharide.

    PubMed

    Atomura, Ryo; Sanui, Terukazu; Fukuda, Takao; Tanaka, Urara; Toyoda, Kyosuke; Taketomi, Takaharu; Yamamichi, Kensuke; Akiyama, Hajime; Nishimura, Fusanori

    2016-03-01

    Periodontitis is a chronic inflammatory disorder caused by specific bacteria residing in the biofilm, particularly Porphyromonas gingivalis (Pg). Sprouty2 (Spry2) functions as a negative regulator of the fibroblast growth factor (FGF) signaling pathway. We previously demonstrated that sequestration of Spry2 induced proliferation and osteogenesis in osteoblastic cells by basic FGF (bFGF) and epidermal growth factor (EGF) stimulation in vitro, but diminished cell proliferation in gingival epithelial cells. In addition, Spry2 knockdown in combination with bFGF and EGF stimulation increases periodontal ligament cell proliferation and migration accompanied by prevention of osteoblastic differentiation. In this study, we investigated the mechanisms through which Spry2 depletion by interferon (IFN) γ and Pg lipopolysaccharide (LPS) stimulation affected the physiology of macrophages in vitro. Transfection of macrophages with Spry2 small-interfering RNA (siRNA) promoted the expression of genes characteristic of M2 alternative activated macrophages, induced interleukin (IL)-10 expression, and enhanced arginase activity, even in cells stimulated with IFNγ and Pg LPS. In addition, we found that phosphoinositide 3-kinase (PI3K) and AKT activation by Spry2 downregulation enhanced efferocytosis of apoptotic cells by increasing Rac1 activation and decreasing nuclear factor kappa B (NFκB) p65 phosphorylation but not signal transducer and activator of transcription 1 (STAT1) phosphorylation. Collectively, our results suggested that topical administration of Spry2 inhibitors may efficiently resolve inflammation in periodontal disease as macrophage-based anti-inflammatory immunotherapy and may create a suitable environment for periodontal wound healing. These in vitro findings provide a molecular basis for new therapeutic approaches in periodontal tissue regeneration. PMID:27042307

  11. A humanized anti-M2 scFv shows protective in vitro activity against influenza

    SciTech Connect

    Bradbury, Andrew M; Velappan, Nileena; Schmidt, Jurgen G

    2008-01-01

    M2 is one of the most conserved influenza proteins, and has been widely prospected as a potential universal vaccine target, with protection predominantly mediated by antibodies. In this paper we describe the creation of a humanized single chain Fv from 14C2, a potent monoclonal antibody against M2. We show that the humanized scFv demonstrates similar activity to the parental mAb: it is able to recognize M2 in its native context on cell surfaces and is able to show protective in vitro activity against influenza, and so represents a potential lead antibody candidate for universal prophylactic or therapeutic intervention in influenza.

  12. Graft-Infiltrating Macrophages Adopt an M2 Phenotype and Are Inhibited by Purinergic Receptor P2X7 Antagonist in Chronic Rejection.

    PubMed

    Wu, C; Zhao, Y; Xiao, X; Fan, Y; Kloc, M; Liu, W; Ghobrial, R M; Lan, P; He, X; Li, X C

    2016-09-01

    Macrophages exhibit diverse phenotypes and functions; they are also a major cell type infiltrating chronically rejected allografts. The exact phenotypes and roles of macrophages in chronic graft loss remain poorly defined. In the present study, we used a mouse heart transplant model to examine macrophages in chronic allograft rejection. We found that treatment of C57BL/6 mice with CTLA4 immunoglobulin fusion protein (CTLA4-Ig) prevented acute rejection of a Balb/c heart allograft but allowed chronic rejection to develop over time, characterized by prominent neointima formation in the graft. There was extensive macrophage infiltration in the chronically rejected allografts, and the graft-infiltrating macrophages expressed markers associated with M2 cells but not M1 cells. In an in vitro system in which macrophages were polarized into either M1 or M2 cells, we screened phenotypic differences between M1 and M2 cells and identified purinergic receptor P2X7 (P2x7r), an adenosine triphosphate (ATP)-gated ion channel protein that was preferentially expressed by M2 cells. We further showed that blocking the P2x7r using oxidized ATP (oATP) inhibited M2 induction in a dose-dependent fashion in vitro. Moreover, treatment of C57BL/6 recipients with the P2x7r antagonist oATP, in addition to CTLA4-Ig treatment, inhibited graft-infiltrating M2 cells, prevented transplant vasculopathy, and induced long-term heart allografts survival. These findings highlight the importance of the P2x7r-M2 axis in chronic rejection and establish P2x7r as a potential therapeutic target in suppression of chronic rejection. PMID:27575724

  13. Intratumoral Delivery of IL-21 Overcomes Anti-Her2/Neu Resistance through Shifting Tumor-Associated Macrophages from M2 to M1 Phenotype.

    PubMed

    Xu, Meng; Liu, Mingyue; Du, Xuexiang; Li, Sirui; Li, Hang; Li, Xiaozhu; Li, Ying; Wang, Yang; Qin, Zhihai; Fu, Yang-Xin; Wang, Shengdian

    2015-05-15

    Tumor resistance is a major hurdle to anti-Her2/neu Ab-based cancer therapy. Current strategies to overcome tumor resistance focus on tumor cell-intrinsic resistance. However, the extrinsic mechanisms, especially the tumor microenvironment, also play important roles in modulating the therapeutic response and resistance of the Ab. In this study, we demonstrate that tumor progression is highly associated with TAMs with immune-suppressive M2 phenotypes, and deletion of TAMs markedly enhanced the therapeutic effects of anti-Her2/neu Ab in a HER2/neu-dependent breast cancer cell TUBO model. Tumor local delivery of IL-21 can skew TAM polarization away from the M2 phenotype to a tumor-inhibiting M1 phenotype, which rapidly stimulates T cell responses against tumor and dramatically promotes the therapeutic effect of anti-Her2 Ab. Skewing of TAM polarization by IL-21 relies substantially on direct action of IL-21 on TAMs rather than stimulation of T and NK cells. Thus, our findings identify the abundant TAMs as a major extrinsic barrier for anti-Her2/neu Ab therapy and present a novel approach to combat this extrinsic resistance by tumor local delivery of IL-21 to skew TAM polarization. This study offers a therapeutic strategy to modulate the tumor microenvironment to overcome tumor-extrinsic resistance. PMID:25876763

  14. IL-10 is required for polarization of macrophages to M2-like phenotype by mycobacterial DnaK (heat shock protein 70).

    PubMed

    Lopes, Rafael L; Borges, Thiago J; Zanin, Rafael F; Bonorino, Cristina

    2016-09-01

    Macrophages are key cells in the innate immune system. They phagocytose pathogens and cellular debris, promote inflammation, and have important roles in tumor immunity. Depending on the microenvironment, macrophages can polarize to M1 (inflammatory) or M2 (anti-inflammatory) phenotypes. Extracellular DnaK (the bacterial ortholog of the mammalian Hsp70) from Mycobacterium tuberculosis (Mtb) was described to exert immune modulatory roles in an IL-10 dependent manner. We have previously observed that endotoxin-free DnaK can polarize macrophages to an M2-like phenotype. However, the mechanisms that underlie this polarization need to be further investigated. IL-10 has been described to promote macrophage polarization, so we investigated the involvement of this cytokine in macrophages stimulated with extracellular DnaK. IL-10 was required to induce the expression of M2 markers - Ym1 and Fizz, when macrophages were treated with DnaK. Blockade of IL-10R also impaired DnaK induced polarization, demonstrating the requirement of the IL-10/IL-10R signaling pathway in this polarization. DnaK was able to induce TGF-β mRNA in treated macrophages in an IL-10 dependent manner. However, protein TGF-β could not be detected in culture supernatants. Finally, using an in vivo allogeneic melanoma model, we observed that DnaK-treated macrophages can promote tumor growth in an IL-10-dependent manner. Our results indicate that the IL-10/IL-10R axis is required for DnaK-induced M2-like polarization in murine macrophages. PMID:27337694

  15. Conditioned media from macrophages of M1, but not M2 phenotype, inhibit the proliferation of the colon cancer cell lines HT-29 and CACO-2

    PubMed Central

    ENGSTRÖM, ALEXANDER; ERLANDSSON, ANN; DELBRO, DICK; WIJKANDER, JONNY

    2014-01-01

    Solid tumors are infiltrated by stroma cells including macrophages and these cells can affect tumor growth, metastasis and angiogenesis. We have investigated the effects of conditioned media (CM) from different macrophages on the proliferation of the colon cancer cell lines HT-29 and CACO-2. CM from THP-1 macrophages and monocyte-derived human macrophages of the M1 phenotype, but not the M2 phenotype, inhibited proliferation of the tumor cells in a dose-dependent manner. Lipopolysaccaharide and interferon γ was used for differentiation of macrophages towards the M1 phenotype and CM were generated both during differentiation (M1DIFF) and after differentiation (M1). M1 and M1DIFF CM as well as THP-1 macrophage CM resulted in cell cycle arrest in HT-29 cells with a decrease of cells in S phase and an increase in G2/M phase. Treatment of HT-29 cells with M1DIFF, but not M1 or THP-1 macrophage CM, resulted in apoptosis of about 20% of the tumor cells and this was accompanied by lack of recovery of cell growth after removal of CM and subsequent culture in fresh media. A protein array was used to identify cytokines released from M1 and M2 macrophages. Among the cytokines released by M1 macrophages, tumor necrosis factor α and CXCL9 were tested by direct addition to HT-29 cells, but neither affected proliferation. Our results indicate that M1 macrophages inhibit colon cancer cell growth and have the potential of contributing to reducing tumor growth in vivo. PMID:24296981

  16. In vitro evaluation of inhibitory effect of nuclear factor-kappaB activity by small interfering RNA on pro-tumor characteristics of M2-like macrophages.

    PubMed

    Kono, Yusuke; Kawakami, Shigeru; Higuchi, Yuriko; Yamashita, Fumiyoshi; Hashida, Mitsuru

    2014-01-01

    Tumor-associated macrophages (TAMs) have an alternatively activated macrophage phenotype (M2) and promote cancer cell proliferation, angiogenesis and metastasis. Nuclear factor-kappaB (NF-κB) is one of the master regulators of macrophage polarization. Here, we investigated the effect of inhibition of NF-κB activity by small interfering RNA (siRNA) on the pro-tumor response of macrophages located in the tumor microenvironment in vitro. We used mouse peritoneal macrophages cultured in conditioned medium from colon-26 cancer cells as an in vitro TAM model (M2-like macrophages). Transfection of NF-κB (p50) siRNA into M2-like macrophages resulted in a significant decrease in the secretion of interleukin (IL)-10 (a T helper 2 (Th2) cytokine) and a significant increase of T helper 1 (Th1) cytokine production (IL-12, tumor necrosis factor-α, and IL-6). Furthermore, vascular endothelial growth factor production and matrix metalloproteinase-9 mRNA expression in M2-like macrophages were suppressed by inhibition of NF-κB expression with NF-κB (p50) siRNA. In addition, there was a reduction of arginase mRNA expression and increase in nitric oxide production. The cytokine secretion profiles of macrophages cultured in conditioned medium from either B16BL6 or PAN-02 cancer cells were also converted from M2 to classically activated (M1) macrophages by transfection of NF-κB (p50) siRNA. These results suggest that inhibition of NF-κB activity in M2-like macrophages alters their phenotype toward M1. PMID:24141263

  17. Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

    SciTech Connect

    Anastasiou, Dimitrios; Yu, Yimin; Israelsen, William J.; Jiang, Jian-Kang; Boxer, Matthew B.; Hong, Bum Soo; Tempel, Wolfram; Dimov, Svetoslav; Shen, Min; Jha, Abhishek; Yang, Hua; Mattaini, Katherine R.; Metallo, Christian M.; Fiske, Brian P.; Courtney, Kevin D.; Malstrom, Scott; Khan, Tahsin M.; Kung, Charles; Skoumbourdis, Amanda P.; Veith, Henrike; Southall, Noel; Walsh, Martin J.; Brimacombe, Kyle R.; Leister, William; Lunt, Sophia Y.; Johnson, Zachary R.; Yen, Katharine E.; Kunii, Kaiko; Davidson, Shawn M.; Christofk, Heather R.; Austin, Christopher P.; Inglese, James; Harris, Marian H.; Asara, John M.; Stephanopoulos, Gregory; Salituro, Francesco G.; Jin, Shengfang; Dang, Lenny; Auld, Douglas S.; Park, Hee-Won; Cantley, Lewis C.; Thomas, Craig J.; Vander Heiden, Matthew G.

    2012-08-26

    Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. This data supports the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.

  18. Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

    PubMed Central

    Anastasiou, Dimitrios; Yu, Yimin; Israelsen, William J.; Jiang, Jian-kang; Boxer, Matthew B.; Hong, Bum Soo; Tempel, Wolfram; Dimov, Svetoslav; Shen, Min; Jha, Abhishek; Yang, Hua; Mattaini, Katherine R.; Metallo, Christian M.; Fiske, Brian P.; Courtney, Kevin D.; Malstrom, Scott; Khan, Tahsin M.; Kung, Charles; Skoumbourdis, Amanda P.; Veith, Henrike; Southall, Noel; Walsh, Martin J.; Brimacombe, Kyle R.; Leister, William; Lunt, Sophia Y.; Johnson, Zachary R.; Yen, Katharine E.; Kunii, Kaiko; Davidson, Shawn M.; Christofk, Heather R.; Austin, Christopher P.; Inglese, James; Harris, Marian H.; Asara, John M.; Stephanopoulos, Gregory; Salituro, Francesco G.; Jin, Shengfang; Dang, Lenny; Auld, Douglas S.; Park, Hee-Won; Cantley, Lewis C.; Thomas, Craig J.; Vander Heiden, Matthew G.

    2012-01-01

    Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. PKM2 interaction with phosphotyrosine-containing proteins inhibits enzyme activity and increases availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small molecule PKM2 activators inhibit growth of xenograft tumors. Structural studies reveal that small molecule activators bind PKM2 at the subunit interaction interface, a site distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small molecule activation of PKM2 can interfere with anabolic metabolism. PMID:22922757

  19. Pyruvate kinase M2 regulates Hif-1α activity and IL-1β induction and is a critical determinant of the warburg effect in LPS-activated macrophages.

    PubMed

    Palsson-McDermott, Eva M; Curtis, Anne M; Goel, Gautam; Lauterbach, Mario A R; Sheedy, Frederick J; Gleeson, Laura E; van den Bosch, Mirjam W M; Quinn, Susan R; Domingo-Fernandez, Raquel; Johnston, Daniel G W; Jiang, Jian-Kang; Jiang, Jain-Kang; Israelsen, William J; Keane, Joseph; Thomas, Craig; Clish, Clary; Vander Heiden, Matthew; Vanden Heiden, Matthew; Xavier, Ramnik J; O'Neill, Luke A J

    2015-01-01

    Macrophages activated by the TLR4 agonist LPS undergo dramatic changes in their metabolic activity. We here show that LPS induces expression of the key metabolic regulator Pyruvate Kinase M2 (PKM2). Activation of PKM2 using two well-characterized small molecules, DASA-58 and TEPP-46, inhibited LPS-induced Hif-1α and IL-1β, as well as the expression of a range of other Hif-1α-dependent genes. Activation of PKM2 attenuated an LPS-induced proinflammatory M1 macrophage phenotype while promoting traits typical of an M2 macrophage. We show that LPS-induced PKM2 enters into a complex with Hif-1α, which can directly bind to the IL-1β promoter, an event that is inhibited by activation of PKM2. Both compounds inhibited LPS-induced glycolytic reprogramming and succinate production. Finally, activation of PKM2 by TEPP-46 in vivo inhibited LPS and Salmonella typhimurium-induced IL-1β production, while boosting production of IL-10. PKM2 is therefore a critical determinant of macrophage activation by LPS, promoting the inflammatory response. PMID:25565206

  20. Pyruvate Kinase M2 Activates mTORC1 by Phosphorylating AKT1S1.

    PubMed

    He, Chang-Liang; Bian, Yang-Yang; Xue, Yu; Liu, Ze-Xian; Zhou, Kai-Qiang; Yao, Cui-Fang; Lin, Yan; Zou, Han-Fa; Luo, Fang-Xiu; Qu, Yuan-Yuan; Zhao, Jian-Yuan; Ye, Ming-Liang; Zhao, Shi-Min; Xu, Wei

    2016-01-01

    In cancer cells, the mammalian target of rapamycin complex 1 (mTORC1) that requires hormonal and nutrient signals for its activation, is constitutively activated. We found that overexpression of pyruvate kinase M2 (PKM2) activates mTORC1 signaling through phosphorylating mTORC1 inhibitor AKT1 substrate 1 (AKT1S1). An unbiased quantitative phosphoproteomic survey identified 974 PKM2 substrates, including serine202 and serine203 (S202/203) of AKT1S1, in the proteome of renal cell carcinoma (RCC). Phosphorylation of S202/203 of AKT1S1 by PKM2 released AKT1S1 from raptor and facilitated its binding to 14-3-3, resulted in hormonal- and nutrient-signals independent activation of mTORC1 signaling and led accelerated oncogenic growth and autophagy inhibition in cancer cells. Decreasing S202/203 phosphorylation by TEPP-46 treatment reversed these effects. In RCCs and breast cancers, PKM2 overexpression was correlated with elevated S202/203 phosphorylation, activated mTORC1 and inhibited autophagy. Our results provided the first phosphorylome of PKM2 and revealed a constitutive mTORC1 activating mechanism in cancer cells. PMID:26876154

  1. Pyruvate Kinase M2 Activates mTORC1 by Phosphorylating AKT1S1

    PubMed Central

    He, Chang-Liang; Bian, Yang-Yang; Xue, Yu; Liu, Ze-Xian; Zhou, Kai-Qiang; Yao, Cui-Fang; Lin, Yan; Zou, Han-Fa; Luo, Fang-Xiu; Qu, Yuan-Yuan; Zhao, Jian-Yuan; Ye, Ming-Liang; Zhao, Shi-Min; Xu, Wei

    2016-01-01

    In cancer cells, the mammalian target of rapamycin complex 1 (mTORC1) that requires hormonal and nutrient signals for its activation, is constitutively activated. We found that overexpression of pyruvate kinase M2 (PKM2) activates mTORC1 signaling through phosphorylating mTORC1 inhibitor AKT1 substrate 1 (AKT1S1). An unbiased quantitative phosphoproteomic survey identified 974 PKM2 substrates, including serine202 and serine203 (S202/203) of AKT1S1, in the proteome of renal cell carcinoma (RCC). Phosphorylation of S202/203 of AKT1S1 by PKM2 released AKT1S1 from raptor and facilitated its binding to 14-3-3, resulted in hormonal- and nutrient-signals independent activation of mTORC1 signaling and led accelerated oncogenic growth and autophagy inhibition in cancer cells. Decreasing S202/203 phosphorylation by TEPP-46 treatment reversed these effects. In RCCs and breast cancers, PKM2 overexpression was correlated with elevated S202/203 phosphorylation, activated mTORC1 and inhibited autophagy. Our results provided the first phosphorylome of PKM2 and revealed a constitutive mTORC1 activating mechanism in cancer cells. PMID:26876154

  2. Expression of the Homeobox Gene HOXA9 in Ovarian Cancer Induces Peritoneal Macrophages to Acquire an M2 Tumor-Promoting Phenotype

    PubMed Central

    Ko, Song Yi; Ladanyi, Andras; Lengyel, Ernst; Naora, Honami

    2015-01-01

    Tumor-associated macrophages (TAMs) exhibit an M2 macrophage phenotype that suppresses anti-tumor immune responses and often correlates with poor outcomes in patients with cancer. Patients with ovarian cancer frequently present with peritoneal carcinomatosis, but the mechanisms that induce naïve peritoneal macrophages into TAMs are poorly understood. In this study, we found an increased abundance of TAMs in mouse i.p. xenograft models of ovarian cancer that expressed HOXA9, a homeobox gene that is associated with poor prognosis in patients with ovarian cancer. HOXA9 expression in ovarian cancer cells stimulated chemotaxis of peritoneal macrophages and induced macrophages to acquire TAM-like features. These features included induction of the M2 markers, CD163 and CD206, and the immunosuppressive cytokines, IL-10 and chemokine ligand 17, and down-regulation of the immunostimulatory cytokine, IL-12. HOXA9-mediated induction of TAMs was primarily due to the combinatorial effects of HOXA9-induced, tumor-derived transforming growth factor-β2 and chemokine ligand 2 levels. High HOXA9 expression in clinical specimens of ovarian cancer was strongly associated with increased abundance of TAMs and intratumoral T-regulatory cells and decreased abundance of CD8+ tumor-infiltrating lymphocytes. Levels of immunosuppressive cytokines were also elevated in ascites fluid of patients with tumors that highly expressed HOXA9. HOXA9 may, therefore, stimulate ovarian cancer progression by promoting an immunosuppressive microenvironment via paracrine effects on peritoneal macrophages. PMID:24332016

  3. Tumor hypoxia enhances non-small cell lung cancer metastasis by selectively promoting macrophage M2 polarization through the activation of ERK signaling

    PubMed Central

    Ma, Shenglin; Dong, Rong; Meng, Wen; Ying, Meidan; Weng, Qinjie; Chen, Zibo; Ma, Jian; Fang, Qingxia; He, Qiaojun; Yang, Bo

    2014-01-01

    Hypoxia is a common phenomenon occurring in the majority of human tumors and has been proved to play an important role in tumor progression. However, it remains unclear that whether the action of hypoxia on macrophages is a main driving force of hypoxia-mediated aggressive tumor behaviors. In the present study, we observe that high density of M2 macrophages is associated with metastasis in adenocarcinoma Non-Small Cell Lung Cancer (NSCLC) patients. By applying the in vivo hypoxia model, the results suggest that intermittent hypoxia significantly promotes the metastasis of Lewis lung carcinoma (LLC), accompanied with more CD209+ macrophages infiltrated in primary tumor tissue. More intriguingly, by skewing macrophages polarization away from the M1- to a tumor-promoting M2-like phenotype, hypoxia and IL-6 cooperate to enhance the LLC metastasis both in vitro and in vivo. In addition, we also demonstrate that skewing of macrophage M2 polarization by hypoxia relies substantially on activation of ERK signaling. Collectively, these observations unveil a novel tumor hypoxia concept involving the macrophage phenotype shift and provide direct evidence for lung cancer intervention through modulating the phenotype of macrophages. PMID:25313135

  4. Niacin and olive oil promote skewing to the M2 phenotype in bone marrow-derived macrophages of mice with metabolic syndrome.

    PubMed

    Montserrat-de la Paz, Sergio; Naranjo, Maria C; Lopez, Sergio; Abia, Rocio; Muriana, Francisco J G; Bermudez, Beatriz

    2016-05-18

    Metabolic syndrome (MetS) is associated with obesity, dyslipemia, type 2 diabetes and chronic low-grade inflammation. The aim of this study was to determine the role of high-fat low-cholesterol diets (HFLCDs) rich in SFAs (HFLCD-SFAs), MUFAs (HFLCD-MUFAs) or MUFAs plus omega-3 long-chain PUFAs (HFLCD-PUFAs) on polarisation and inflammatory potential in bone marrow-derived macrophages (BMDMs) from niacin (NA)-treated Lep(ob/ob)LDLR(-/-) mice. Animals fed with HFLCD-SFAs had increased weight and serum triglycerides, and their BMDMs accumulated triglycerides over the animals fed with HFLCD-MUFAs or -PUFAs. Furthermore, BMDMs from animals fed with HFLCD-SFAs were polarised towards the M1 phenotype with functional competence to produce pro-inflammatory cytokines, whereas BMDMs from animals fed with HFLCD-MUFAs or -PUFAs were skewed to the anti-inflammatory M2 phenotype. These findings open opportunities for developing novel nutritional strategies with olive oil as the most important dietary source of MUFAs (notably oleic acid) to prevent development and progression of metabolic complications in the NA-treated MetS. PMID:27116638

  5. M2L4 coordination capsules with tunable anticancer activity upon guest encapsulation.

    PubMed

    Ahmedova, Anife; Mihaylova, Rositsa; Momekova, Denitsa; Shestakova, Pavletta; Stoykova, Silviya; Zaharieva, Joana; Yamashina, Masahiro; Momekov, Georgi; Akita, Munetaka; Yoshizawa, Michito

    2016-08-16

    Metallosupramolecular cages and capsules have gained increasing popularity as both molecular containers and anticancer agents. For successful combination of these properties a thorough analysis of the effect of guest encapsulation on the host's cytotoxic properties is highly required. Here we report on the cytotoxicity modulation of Pt(ii) and Pd(ii)-linked M2L4 coordination capsules upon encapsulation of guest molecules such as pyrene and caffeine. The anticancer activity of the capsules against various human cancer cells (HT-29, T-24, HL-60 and its resistant counterparts HL-60/Dox and HL-60/CDDP) significantly altered upon the guest encapsulation. The encapsulation of pyrene molecules causes a decrease in the cytotoxicity of the Pt(ii) capsule, which is stronger than that of the Pd(ii) capsule. The cytotoxicities of the caffeine containing capsules are lower than that of the empty capsules (except for HL-60), but still superior to cisplatin under the same conditions. The observed trends in the anticancer activity of the capsules and their host-guest complexes correlate with their different stabilities toward glutathione, estimated by NMR-based kinetic experiments. Mechanistic insights into the observed cytotoxicities are obtained by fluorescence microscopy imaging of tumor cells treated with the capsules and their pyrene complexes. The data suggest the glutathione-triggered disassembly of the capsular structures as a potential activation pathway for their cytotoxicities. PMID:27488015

  6. Inhibitory and Activating Effects of Some Flavonoid Derivatives on Human Pyruvate Kinase Isoenzyme M2.

    PubMed

    Adem, Sevki; Aslan, Abdulselam; Ahmed, Ishtiaq; Krohn, Karsten; Guler, Caglar; Comaklı, Veysel; Demirdag, Ramazan; Kuzu, Muslum

    2016-02-01

    Pyruvate kinase isoenzyme M2 (PKM2) is expressed excessively in many different cancer types and it plays an important role in the control of glucose metabolism. Thus, it is evaluated as an important target in the development of medication for cancer. The flavonoids comprise a large group of natural products with variable phenolic structures and occur mainly in plants. They are of great interest due to their biological properties. In this study, the effects of various flavonoid derivatives on the PKM2 enzyme activity were analyzed in vitro. The flavonoid derivatives 1 and 2 showed inhibition effect with IC50 values of <60 μM. IC50 values of compounds 3-8 were calculated as 134, 415, 145, 163, 295 μM, and 3.5 mM, respectively. The molecules 9-12 showed an activation effect with values of AC50 of less than 90 μM. The IC50 values of the derivatives 13-17 were calculated as 115, 150, 200, 221, and 275 μM, respectively. The results show that catechin derivatives can be probably used as lead compounds for the design of PKM2 enzyme activators and inhibitors. PMID:26708302

  7. Nitroxyl (HNO) reduces endothelial and monocyte activation and promotes M2 macrophage polarization.

    PubMed

    Andrews, Karen L; Sampson, Amanda K; Irvine, Jennifer C; Shihata, Waled A; Michell, Danielle L; Lumsden, Natalie G; Lim, Chloe; Huet, Olivier; Drummond, Grant R; Kemp-Harper, Barbara K; Chin-Dusting, Jaye P F

    2016-09-01

    Nitroxyl anion (HNO) donors are currently being assessed for their therapeutic utility in several cardiovascular disorders including heart failure. Here, we examine their effect on factors that precede atherosclerosis including endothelial cell and monocyte activation, leucocyte adhesion to the endothelium and macrophage polarization. Similar to the NO donor glyceryl trinitrate (GTN), the HNO donors Angeli's salt (AS) and isopropylamine NONOate (IPA/NO) decreased leucocyte adhesion to activated human umbilical vein endothelial cells (HUVECs) and mouse isolated aorta. This reduction in adhesion was accompanied by a reduction in intercellular adhesion molecule-1 (ICAM-1) and the cytokines monocyte chemoattractant protein 1 (MCP-1) and interleukin 6 (IL-6) which was inhibitor of nuclear factor κB (NFκB) α (IκBα)- and subsequently NFκB-dependent. Intriguingly, the effects of AS on leucocyte adhesion, like those on vasodilation, were found to not be susceptible to pharmacological tolerance, unlike those observed with GTN. As well, HNO reduces monocyte activation and promotes polarization of M2 macrophages. Taken together, our data demonstrate that HNO donors can reduce factors that are associated with and which precede atherosclerosis and may thus be useful therapeutically. Furthermore, since the effects of the HNO donors were not subject to tolerance, this confers an additional advantage over NO donors. PMID:27231254

  8. Phenotypic, functional, and plasticity features of classical and alternatively activated human macrophages.

    PubMed

    Tarique, Abdullah A; Logan, Jayden; Thomas, Emma; Holt, Patrick G; Sly, Peter D; Fantino, Emmanuelle

    2015-11-01

    Macrophages are dynamic cells that mature under the influence of signals from the local microenvironment into either classically (M1) or alternatively (M2) activated macrophages with specific functional and phenotypic properties. Although the phenotypic identification of M1 and M2 macrophages is well established in mice, this is less clear for human macrophages. In addition, the persistence and reversibility of polarized human phenotypes is not well established. Human peripheral blood monocytes were differentiated into uncommitted macrophages (M0) and then polarized to M1 and M2 phenotypes using LPS/IFN-γ and IL-4/IL-13, respectively. M1 and M2 were identified as CD64(+)CD80(+) and CD11b(+)CD209(+), respectively, by flow cytometry. Polarized M1 cells secreted IP-10, IFN-γ, IL-8, TNF-α, IL-1β, and RANTES, whereas M2 cells secreted IL-13, CCL17, and CCL18. Functionally, M2 cells were highly endocytic. In cytokine-deficient medium, the polarized macrophages reverted back to the M0 state within 12 days. If previously polarized macrophages were given the alternative polarizing stimulus after 6 days of resting in cytokine-deficient medium, a switch in polarization was seen (i.e., M1 macrophages switched to M2 and expressed CD11b(+)CD209(+) and vice versa). In summary, we report phenotypic identification of human M1 and M2 macrophages, their functional characteristics, and their ability to be reprogrammed given the appropriate stimuli. PMID:25870903

  9. Activation and proton transport mechanism in influenza A M2 channel.

    PubMed

    Wei, Chenyu; Pohorille, Andrew

    2013-11-01

    Molecular dynamics trajectories 2 μs in length have been generated for the pH-activated, tetrameric M2 proton channel of the influenza A virus in all protonation states of the pH sensor located at the His(37) tetrad. All simulated structures are in very good agreement with high-resolution structures. Changes in the channel caused by progressive protonation of His(37) provide insight into the mechanism of proton transport. The channel is closed at both His(37) and Trp(41) sites in the singly and doubly protonated states, but it opens at Trp(41) upon further protonation. Anions access the charged His(37) and by doing so stabilize the protonated states of the channel. The narrow opening at the His(37) site, further blocked by anions, is inconsistent with the water-wire mechanism of proton transport. Instead, conformational interconversions of His(37) correlated with hydrogen bonding to water molecules indicate that these residues shuttle protons in high-protonation states. Hydrogen bonds between charged and uncharged histidines are rare. The valve at Val(27) remains on average quite narrow in all protonation states but fluctuates sufficiently to support water and proton transport. A proton transport mechanism in which the channel, depending on pH, opens at either the histidine or valine gate is only partially supported by the simulations. PMID:24209848

  10. Purinergic signaling during macrophage differentiation results in M2 alternative activated macrophages.

    PubMed

    Barberà-Cremades, Maria; Baroja-Mazo, Alberto; Pelegrín, Pablo

    2016-02-01

    Macrophages represent a highly heterogenic cell population of the innate immune system, with important roles in the initiation and resolution of the inflammatory response. Purinergic signaling regulates both M1 and M2 macrophage function at different levels by controlling the secretion of cytokines, phagocytosis, and the production of reactive oxygen species. We found that extracellular nucleotides arrest macrophage differentiation from bone marrow precursors via adenosine and P2 receptors. This results in a mature macrophage with increased expression of M2, but not M1, genes. Similar to adenosine and ATP, macrophage growth arrested with LPS treatment resulted in an increase of the M2-related marker Ym1. Recombinant Ym1 was able to affect macrophage proliferation and could, potentially, be involved in the arrest of macrophage growth during hematopoiesis. PMID:26382298

  11. Noble metal nanoparticle-induced oxidative stress modulates tumor associated macrophages (TAMs) from an M2 to M1 phenotype: An in vitro approach.

    PubMed

    Pal, Ramkrishna; Chakraborty, Biswajit; Nath, Anupam; Singh, Leichombam Mohindro; Ali, Mohammed; Rahman, Dewan Shahidur; Ghosh, Sujit Kumar; Basu, Abhishek; Bhattacharya, Sudin; Baral, Rathindranath; Sengupta, Mahuya

    2016-09-01

    Diagnosis of cancer and photothermal therapy using optoelectronic properties of noble metal nanoparticles (NPs) has established a new therapeutic approach for treating cancer. Here we address the intrinsic properties of noble metal NPs (gold and silver) as well as the mechanism of their potential antitumor activity. For this, the study addresses the functional characterization of tumor associated macrophages (TAMs) isolated from murine fibrosarcoma induced by a chemical carcinogen, 3-methylcholanthrene (MCA). We have previously shown antitumor activity of both gold nanoparticles (AuNPs) and silver nanoparticle (AgNPs) in vivo in a murine fibrosarcoma model. In the present study, it has been seen that AuNPs and AgNPs modulate the reactive oxygen species (ROS) and reactive nitrogen species (RNS) production, suppressing the antioxidant system of cells (TAMs). Moreover, the antioxidant-mimetic action of these NPs maintain the ROS and RNS levels in TAMs which act as second messengers to activate the proinflammatory signaling cascades. Thus, while there is a downregulation of tumor necrosis factor-α (TNF-α) and Interleukin-10 (IL-10) in the TAMs, the proinflammatory cytokine Interleukin-12 (IL-12) is upregulated resulting in a polarization of TAMs from M2 (anti-inflammatory) to M1 (pro-inflammatory) nature. PMID:27344639

  12. Silencing MicroRNA-155 Attenuates Cardiac Injury and Dysfunction in Viral Myocarditis via Promotion of M2 Phenotype Polarization of Macrophages.

    PubMed

    Zhang, Yingying; Zhang, Mengying; Li, Xueqin; Tang, Zongsheng; Wang, Xiangmin; Zhong, Min; Suo, Qifeng; Zhang, Yao; Lv, Kun

    2016-01-01

    Macrophage infiltration is a hallmark feature of viral myocarditis. As studies have shown that microRNA-155 regulates the differentiation of macrophages, we aimed to investigate the role of microRNA-155 in VM. We report that silencing microRNA-155 protects mice from coxsackievirus B3 induced myocarditis. We found that microRNA-155 expression was upregulated and localized primarily in heart-infiltrating macrophages and CD4(+) T lymphocytes during acute myocarditis. In contrast with wildtype (WT) mice, microRNA-155(-/-) mice developed attenuated viral myocarditis, which was characterized by decreased cardiac inflammation and decreased intracardiac CD45(+) leukocytes. Hearts of microRNA-155(-/-) mice expressed decreased levels of the IFN-γ and increased levels of the cytokines IL-4 and IL-13. Although total CD4(+) and regulatory T cells were unchanged in miR-155(-/-) spleen proportionally, the activation of T cells and CD4(+) T cell proliferation in miR-155(-/-) mice were significantly decreased. Beyond the acute phase, microRNA-15(5-/-) mice had reduced mortality and improved cardiac function during 5 weeks of follow-up. Moreover, silencing microRNA-155 led to increased levels of alternatively-activated macrophages (M2) and decreased levels of classically-activated macrophages (M1) in the heart. Combined, our studies suggest that microRNA-155 confers susceptibility to viral myocarditis by affecting macrophage polarization, and thus may be a potential therapeutic target for viral myocarditis. PMID:26931072

  13. Silencing MicroRNA-155 Attenuates Cardiac Injury and Dysfunction in Viral Myocarditis via Promotion of M2 Phenotype Polarization of Macrophages

    PubMed Central

    Zhang, Yingying; Zhang, Mengying; Li, Xueqin; Tang, Zongsheng; Wang, Xiangmin; Zhong, Min; Suo, Qifeng; Zhang, Yao; Lv, Kun

    2016-01-01

    Macrophage infiltration is a hallmark feature of viral myocarditis. As studies have shown that microRNA-155 regulates the differentiation of macrophages, we aimed to investigate the role of microRNA-155 in VM. We report that silencing microRNA-155 protects mice from coxsackievirus B3 induced myocarditis. We found that microRNA-155 expression was upregulated and localized primarily in heart-infiltrating macrophages and CD4+ T lymphocytes during acute myocarditis. In contrast with wildtype (WT) mice, microRNA-155−/− mice developed attenuated viral myocarditis, which was characterized by decreased cardiac inflammation and decreased intracardiac CD45+ leukocytes. Hearts of microRNA-155−/− mice expressed decreased levels of the IFN-γ and increased levels of the cytokines IL-4 and IL-13. Although total CD4+ and regulatory T cells were unchanged in miR-155−/− spleen proportionally, the activation of T cells and CD4+ T cell proliferation in miR-155−/− mice were significantly decreased. Beyond the acute phase, microRNA-155−/− mice had reduced mortality and improved cardiac function during 5 weeks of follow-up. Moreover, silencing microRNA-155 led to increased levels of alternatively-activated macrophages (M2) and decreased levels of classically-activated macrophages (M1) in the heart. Combined, our studies suggest that microRNA-155 confers susceptibility to viral myocarditis by affecting macrophage polarization, and thus may be a potential therapeutic target for viral myocarditis. PMID:26931072

  14. Interleukin-10 conjugated electrospun polycaprolactone (PCL) nanofibre scaffolds for promoting alternatively activated (M2) macrophages around the peripheral nerve in vivo.

    PubMed

    Potas, Jason R; Haque, Farhia; Maclean, Francesca L; Nisbet, David R

    2015-05-01

    Macrophages play a key role in tissue regeneration following peripheral nerve injury by preparing the surrounding parenchyma for regeneration, however, they can be damaging if the response is excessive. Interleukin 10 (IL-10) is a cytokine that promotes macrophages toward an anti-inflammatory/wound healing state (M2 phenotype). The bioactive half-life of IL-10 is dependent on the cellular microenvironment and ranges from minutes to hours in vivo. Our objective was to extend the in vivo bioavailability and bioactivity of IL-10 by attaching the protein onto nanofibrous scaffolds and demonstrating increased expression levels of M2 macrophages when placed around healthy intact peripheral nerves. IL-10 was adsorbed and covalently bound to electrospun poly(ε-caprolactone) (PCL) nanofibrous scaffolds. In vivo bioavailability and bioactivity of IL-10 was confirmed by wrapping IL-10 conjugated nanofibres around the sciatic nerves of Wistar rats and quantifying M2 macrophages immunohistochemically double labelled with ED1 and either arginase 1 or CD206. IL-10 remained immobilised to PCL scaffolds for more than 120 days when stored in phosphate buffered saline at room temperature and for up to 14d ays when implanted around the sciatic nerve. IL-10 conjugated nanofibres successfully induced macrophage polarisation towards the M2 activated state within the scaffold material as well as the adjacent tissue surrounding the nerve. PCL biofunctionalised nanofibres are useful for manipulating the cellular microenvironment. Materials such as these could potentially lead to new therapeutic strategies for nervous tissue injuries as well as provide novel investigative tools for biological research. PMID:25837415

  15. Anxiolytic activity of pyridoindole derivatives SMe1EC2 and SMe1M2: behavioral analysis using rat model

    PubMed Central

    Sedláčková, Natália; Ponechalová, Veronika; Ujházy, Eduard; Dubovický, Michal; Mach, Mojmír

    2011-01-01

    Anxiety and mood disorders have become very significant affections in the last decades. According to WHO at least one mental disease occurred per year in 27% of EU inhabitants (more than 82 mil. people). It is estimated that by 2020, depression will be the main cause of morbidity in the developed countries. These circumstances call for research for new prospective drugs with anxiolytic and antidepressive properties exhibiting no toxicity and withdrawal effect and possessing beneficial properties, like antioxidant and/or neuroprotective effects. The aim of this study was to obtain information about psychopharmacological properties of pyridoindole derivatives SMe1EC2 and SMe1M2, using non-invasive behavioral methods in rats. The battery of ethological tests (open field, elevated plus-maze, light/dark box exploration, forced swim test) was used to obtain information about anxiolytic and antidepressant activity of the pyridoindole derivatives. The substances were administered intraperitoneally 30 minutes before the tests at doses of 1, 10 and 25 mg/kg. In the behavioral tests, SMe1EC2 was found to exert anxiolytic activity in elevated plus maze with no affection of locomotor activity. The highest dose of SMe1M2 increased the time spent in the lit part of the Light/Dark box, however this result was influenced by inhibition of motor activity of the rats. Similar findings were observed also in elevated plus-maze, although these results were not statistically significant. In conclusion, from the results of our study it is evident that both pyridoindoles acted on the CNS. In the highest dose, SMe1M2 was found to possess rather sedative than anxiolytic or antidepressant activity. PMID:22319256

  16. M2 priority screening system for near-term activities: Project documentation. Final report December 11, 1992--May 31, 1994

    SciTech Connect

    1993-08-12

    From May through August, 1993, the M-2 Group within M Division at LANL conducted with the support of the LANL Integration and Coordination Office (ICO) and Applied Decision Analysis, Inc. (ADA), whose purpose was to develop a system for setting priorities among activities. This phase of the project concentrated on prioritizing near-tenn activities (i.e., activities that must be conducted in the next six months) necessary for setting up this new group. Potential future project phases will concentrate on developing a tool for setting priorities and developing annual budgets for the group`s operations. The priority screening system designed to address the near-term problem was developed, applied in a series of meeting with the group managers, and used as an aid in the assignment of tasks to group members. The model was intended and used as a practical tool for documenting and explaining decisions about near-term priorities, and not as a substitute for M-2 management judgment and decision-making processes.

  17. Epigenetic Control of Macrophage Shape Transition towards an Atypical Elongated Phenotype by Histone Deacetylase Activity.

    PubMed

    Cabanel, Mariana; Brand, Camila; Oliveira-Nunes, Maria Cecilia; Cabral-Piccin, Mariela Pires; Lopes, Marcela Freitas; Brito, Jose Marques; de Oliveira, Felipe Leite; El-Cheikh, Marcia Cury; Carneiro, Katia

    2015-01-01

    Inflammatory chronic pathologies are complex processes characterized by an imbalance between the resolution of the inflammatory phase and the establishment of tissue repair. The main players in these inflammatory pathologies are bone marrow derived monocytes (BMDMs). However, how monocyte differentiation is modulated to give rise to specific macrophage subpopulations (M1 or M2) that may either maintain the chronic inflammatory process or lead to wound healing is still unclear. Considering that inhibitors of Histone Deacetylase (HDAC) have an anti-inflammatory activity, we asked whether this enzyme would play a role on monocyte differentiation into M1 or M2 phenotype and in the cell shape transition that follows. We then induced murine bone marrow progenitors into monocyte/macrophage differentiation pathway using media containing GM-CSF and the HDAC blocker, Trichostatin A (TSA). We found that the pharmacological inhibition of HDAC activity led to a shape transition from the typical macrophage pancake-like shape into an elongated morphology, which was correlated to a mixed M1/M2 profile of cytokine and chemokine secretion. Our results present, for the first time, that HDAC activity acts as a regulator of macrophage differentiation in the absence of lymphocyte stimuli. We propose that HDAC activity down regulates macrophage plasticity favoring the pro-inflammatory phenotype. PMID:26196676

  18. Adsorption of Reactive Red M-2BE dye from water solutions by multi-walled carbon nanotubes and activated carbon.

    PubMed

    Machado, Fernando M; Bergmann, Carlos P; Fernandes, Thais H M; Lima, Eder C; Royer, Betina; Calvete, Tatiana; Fagan, Solange B

    2011-09-15

    Multi-walled carbon nanotubes and powdered activated carbon were used as adsorbents for the successful removal of Reactive Red M-2BE textile dye from aqueous solutions. The adsorbents were characterised by infrared spectroscopy, N(2) adsorption/desorption isotherms and scanning electron microscopy. The effects of pH, shaking time and temperature on adsorption capacity were studied. In the acidic pH region (pH 2.0), the adsorption of the dye was favourable using both adsorbents. The contact time to obtain equilibrium at 298K was fixed at 1h for both adsorbents. The activation energy of the adsorption process was evaluated from 298 to 323K for both adsorbents. The Avrami fractional-order kinetic model provided the best fit to the experimental data compared with pseudo-first-order or pseudo-second-order kinetic adsorption models. For Reactive Red M-2BE dye, the equilibrium data were best fitted to the Liu isotherm model. Simulated dyehouse effluents were used to check the applicability of the proposed adsorbents for effluent treatment. PMID:21724329

  19. Sub-THz and Hα Activity during the Preflare and Main Phases of a GOES Class M2 Event

    NASA Astrophysics Data System (ADS)

    Kaufmann, Pierre; Marcon, Rogério; Guillermo Giménez de Castro, C.; White, Stephen M.; Raulin, Jean-Pierre; Correia, Emilia; Olavo Fernandes, Luis; de Souza, Rodney V.; Godoy, Rodolfo; Marun, Adolfo; Pereyra, Pablo

    2011-12-01

    Radio and optical observations of the evolution of flare-associated phenomena have shown an initial and rapid burst at 0.4 THz only followed subsequently by a localized chromospheric heating producing an Hα brightening with later heating of the whole active region. A major instability occurred several minutes later producing one impulsive burst at microwaves only, associated with an M2.0 GOES X-ray flare that exhibited the main Hα brightening at the same site as the first flash.The possible association between long-enduring time profiles at soft X-rays, microwaves, Hα, and sub-THz wavelengths is discussed. In the decay phase, the Hα movie shows a disrupting magnetic arch structure ejecting dark, presumably chromospheric, material upward. The time sequence of events suggests genuine interdependent and possibly non-thermal instabilities triggering phenomena, with concurrent active region plasma heating and material ejection.

  20. Myelin alters the inflammatory phenotype of macrophages by activating PPARs

    PubMed Central

    2013-01-01

    Background Foamy macrophages, containing myelin degradation products, are abundantly found in active multiple sclerosis (MS) lesions. Recent studies have described an altered phenotype of macrophages after myelin internalization. However, mechanisms by which myelin affects the phenotype of macrophages and how this phenotype influences lesion progression remain unclear. Results We demonstrate that myelin as well as phosphatidylserine (PS), a phospholipid found in myelin, reduce nitric oxide production by macrophages through activation of peroxisome proliferator-activated receptor β/δ (PPARβ/δ). Furthermore, uptake of PS by macrophages, after intravenous injection of PS-containing liposomes (PSLs), suppresses the production of inflammatory mediators and ameliorates experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The protective effect of PSLs in EAE animals is associated with a reduced immune cell infiltration into the central nervous system and decreased splenic cognate antigen specific proliferation. Interestingly, PPARβ/δ is activated in foamy macrophages in active MS lesions, indicating that myelin also activates PPARβ/δ in macrophages in the human brain. Conclusion Our data show that myelin modulates the phenotype of macrophages by PPAR activation, which may subsequently dampen MS lesion progression. Moreover, our results suggest that myelin-derived PS mediates PPARβ/δ activation in macrophages after myelin uptake. The immunoregulatory impact of naturally-occurring myelin lipids may hold promise for future MS therapeutics. PMID:24252308

  1. Jacalin-Activated Macrophages Exhibit an Antitumor Phenotype

    PubMed Central

    Danella Polli, Cláudia; Pereira Ruas, Luciana; Chain Veronez, Luciana; Herrero Geraldino, Thais; Rossetto de Morais, Fabiana; Roque-Barreira, Maria Cristina; Pereira-da-Silva, Gabriela

    2016-01-01

    Tumor-associated macrophages (TAMs) have an ambiguous and complex role in the carcinogenic process, since these cells can be polarized into different phenotypes (proinflammatory, antitumor cells or anti-inflammatory, protumor cells) by the tumor microenvironment. Given that the interactions between tumor cells and TAMs involve several players, a better understanding of the function and regulation of TAMs is crucial to interfere with their differentiation in attempts to skew TAM polarization into cells with a proinflammatory antitumor phenotype. In this study, we investigated the modulation of macrophage tumoricidal activities by the lectin jacalin. Jacalin bound to macrophage surface and induced the expression and/or release of mainly proinflammatory cytokines via NF-κB signaling, as well as increased iNOS mRNA expression, suggesting that the lectin polarizes macrophages toward the antitumor phenotype. Therefore, tumoricidal activities of jacalin-stimulated macrophages were evaluated. High rates of tumor cell (human colon, HT-29, and breast, MCF-7, cells) apoptosis were observed upon incubation with supernatants from jacalin-stimulated macrophages. Taken together, these results indicate that jacalin, by exerting a proinflammatory activity, can direct macrophages to an antitumor phenotype. Deep knowledge of the regulation of TAM functions is essential for the development of innovative anticancer strategies. PMID:27119077

  2. Jacalin-Activated Macrophages Exhibit an Antitumor Phenotype.

    PubMed

    Danella Polli, Cláudia; Pereira Ruas, Luciana; Chain Veronez, Luciana; Herrero Geraldino, Thais; Rossetto de Morais, Fabiana; Roque-Barreira, Maria Cristina; Pereira-da-Silva, Gabriela

    2016-01-01

    Tumor-associated macrophages (TAMs) have an ambiguous and complex role in the carcinogenic process, since these cells can be polarized into different phenotypes (proinflammatory, antitumor cells or anti-inflammatory, protumor cells) by the tumor microenvironment. Given that the interactions between tumor cells and TAMs involve several players, a better understanding of the function and regulation of TAMs is crucial to interfere with their differentiation in attempts to skew TAM polarization into cells with a proinflammatory antitumor phenotype. In this study, we investigated the modulation of macrophage tumoricidal activities by the lectin jacalin. Jacalin bound to macrophage surface and induced the expression and/or release of mainly proinflammatory cytokines via NF-κB signaling, as well as increased iNOS mRNA expression, suggesting that the lectin polarizes macrophages toward the antitumor phenotype. Therefore, tumoricidal activities of jacalin-stimulated macrophages were evaluated. High rates of tumor cell (human colon, HT-29, and breast, MCF-7, cells) apoptosis were observed upon incubation with supernatants from jacalin-stimulated macrophages. Taken together, these results indicate that jacalin, by exerting a proinflammatory activity, can direct macrophages to an antitumor phenotype. Deep knowledge of the regulation of TAM functions is essential for the development of innovative anticancer strategies. PMID:27119077

  3. Synthesis, characterization, and hydrogen storage enhancement of M2(BDC)2dabco with palladium-doped activated carbon.

    PubMed

    Lin, Kuen-Song; Adhikari, Abhijit Krishna; Chang, Kai-Che; Chiang, Chao-Lung; Wang, Chieh-Hung

    2014-04-01

    Two bi-organic type metal organic frameworks (MOFs) such as Co2(BDC)2dabco and Zn2(BDC)2 dabco have been synthesized by hydrothermal method and characterized along with hydrogen adsorption. The hydrogen adsorption capacity of these MOFs was increased after doping by palladium-activated carbon. Co2(BDC)2dabco has cracked and folded thin film like surface while Zn2(BDC)2dabco has a brick-typed cubic structure with particle size about 10-15 microm identified by FE-SEM. The XRD patterns represents that both MOFs have the well crystalline structure. Nitrogen adsorption isotherms show that both structures have Type I adsorption isotherm with the BET specific surface area of 1,390 and 1,433 m2 g(-1) for Co2(BDC)2dabco and Zn2(BDC)2dabco, respectively. Pristine Co2(BDC)2dabco and Zn2(BDC)2dabco can store about 0.22 and 0.25 wt.% of H2 measured at 298 K and 32 bar. This capacity was greatly enhanced by doping palladium-activated carbon to 0.31 and 0.41 wt.%, respectively. Moreover, both structures were also characterized by XANES/EXAFS. EXAFS spectra indicate that Co2(BDC)2dabco has the Co--O bond distance of 2.030 A with the coordination number of 4.2 while Zn2(BDC)2dabco has 2.015 angstroms bond distance of Zn--O with the coordination number of 3.4. PMID:24734682

  4. Macrophage phenotypes in atherosclerosis.

    PubMed

    Colin, Sophie; Chinetti-Gbaguidi, Giulia; Staels, Bart

    2014-11-01

    Initiation and progression of atherosclerosis depend on local inflammation and accumulation of lipids in the vascular wall. Although many cells are involved in the development and progression of atherosclerosis, macrophages are fundamental contributors. For nearly a decade, the phenotypic heterogeneity and plasticity of macrophages has been studied. In atherosclerotic lesions, macrophages are submitted to a large variety of micro-environmental signals, such as oxidized lipids and cytokines, which influence the phenotypic polarization and activation of macrophages resulting in a dynamic plasticity. The macrophage phenotype spectrum is characterized, at the extremes, by the classical M1 macrophages induced by T-helper 1 (Th-1) cytokines and by the alternative M2 macrophages induced by Th-2 cytokines. M2 macrophages can be further classified into M2a, M2b, M2c, and M2d subtypes. More recently, additional plaque-specific macrophage phenotypes have been identified, termed as Mox, Mhem, and M4. Understanding the mechanisms and functional consequences of the phenotypic heterogeneity of macrophages will contribute to determine their potential role in lesion development and plaque stability. Furthermore, research on macrophage plasticity could lead to novel therapeutic approaches to counteract cardiovascular diseases such as atherosclerosis. The present review summarizes our current knowledge on macrophage subsets in atherosclerotic plaques and mechanism behind the modulation of the macrophage phenotype. PMID:25319333

  5. E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2

    SciTech Connect

    Fang, Zejun; Gong, Chaoju; Liu, Hong; Zhang, Xiaomin; Mei, Lingming; Song, Mintao; Qiu, Lanlan; Luo, Shuchai; Zhu, Zhihua; Zhang, Ronghui; Gu, Hongqian; Chen, Xiang

    2015-08-21

    As the ribonucleotide reductase small subunit, the high expression of ribonucleotide reductase small subunit M2 (RRM2) induces cancer and contributes to tumor growth and invasion. In several colorectal cancer (CRC) cell lines, we found that the expression levels of RRM2 were closely related to the transcription factor E2F1. Mechanistic studies were conducted to determine the molecular basis. Ectopic overexpression of E2F1 promoted RRM2 transactivation while knockdown of E2F1 reduced the levels of RRM2 mRNA and protein. To further investigate the roles of RRM2 which was activated by E2F1 in CRC, CCK-8 assay and EdU incorporation assay were performed. Overexpression of E2F1 promoted cell proliferation in CRC cells, which was blocked by RRM2 knockdown attenuation. In the migration and invasion tests, overexpression of E2F1 enhanced the migration and invasion of CRC cells which was abrogated by silencing RRM2. Besides, overexpression of RRM2 reversed the effects of E2F1 knockdown partially in CRC cells. Examination of clinical CRC specimens demonstrated that both RRM2 and E2F1 were elevated in most cancer tissues compared to the paired normal tissues. Further analysis showed that the protein expression levels of E2F1 and RRM2 were parallel with each other and positively correlated with lymph node metastasis (LNM), TNM stage and distant metastasis. Consistently, the patients with low E2F1 and RRM2 levels have a better prognosis than those with high levels. Therefore, we suggest that E2F1 can promote CRC proliferation, migration, invasion and metastasis by regulating RRM2 transactivation. Understanding the role of E2F1 in activating RRM2 transcription will help to explain the relationship between E2F1 and RRM2 in CRC and provide a novel predictive marker for diagnosis and prognosis of the disease. - Highlights: • E2F1 promotes RRM2 transactivation in CRC cells. • E2F1 promotes the proliferation of CRC cells by activating RRM2. • E2F1 promotes the migration and

  6. The Conserved Lys-95 Charged Residue Cluster Is Critical for the Homodimerization and Enzyme Activity of Human Ribonucleotide Reductase Small Subunit M2*

    PubMed Central

    Chen, Xinhuan; Xu, Zhijian; Zhang, Lingna; Liu, Hongchuan; Liu, Xia; Lou, Meng; Zhu, Lijun; Huang, Bingding; Yang, Cai-Guang; Zhu, Weiliang; Shao, Jimin

    2014-01-01

    Ribonucleotide reductase (RR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides for DNA synthesis. Human RR small subunit M2 exists in a homodimer form. However, the importance of the dimer form to the enzyme and the related mechanism remain unclear. In this study, we tried to identify the interfacial residues that may mediate the assembly of M2 homodimer by computational alanine scanning based on the x-ray crystal structure. Co-immunoprecipitation, size exclusion chromatography, and RR activity assays showed that the K95E mutation in M2 resulted in dimer disassembly and enzyme activity inhibition. In comparison, the charge-exchanging double mutation of K95E and E98K recovered the dimerization and activity. Structural comparisons suggested that a conserved cluster of charged residues, including Lys-95, Glu-98, Glu-105, and Glu-174, at the interface may function as an ionic lock for M2 homodimer. Although the measurements of the radical and iron contents showed that the monomer (the K95E mutant) was capable of generating the diiron and tyrosyl radical cofactor, co-immunoprecipitation and competitive enzyme inhibition assays indicated that the disassembly of M2 dimer reduced its interaction with the large subunit M1. In addition, the immunofluorescent and fusion protein-fluorescent imaging analyses showed that the dissociation of M2 dimer altered its subcellular localization. Finally, the transfection of the wild-type M2 but not the K95E mutant rescued the G1/S phase cell cycle arrest and cell growth inhibition caused by the siRNA knockdown of M2. Thus, the conserved Lys-95 charged residue cluster is critical for human RR M2 homodimerization, which is indispensable to constitute an active holoenzyme and function in cells. PMID:24253041

  7. The activation of Wnt signaling by a STAT6-dependent macrophage phenotype promotes mucosal repair in murine IBD.

    PubMed

    Cosín-Roger, J; Ortiz-Masiá, D; Calatayud, S; Hernández, C; Esplugues, J V; Barrachina, M D

    2016-07-01

    The complete repair of the mucosa constitutes a key goal in inflammatory bowel disease (IBD) treatment. The Wnt signaling pathway mediates mucosal repair and M2 macrophages that coordinate efficient healing have been related to Wnt ligand expression. Signal transducer and activator of transcription 6 (STAT6) mediates M2 polarization in vitro and we hypothesize that a STAT6-dependent macrophage phenotype mediates mucosal repair in acute murine colitis by activating the Wnt signaling pathway. Our results reveal an impaired mucosal expression of M2 macrophage-associated genes and delayed wound healing in STAT6(-/-) mice treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS). These mice also exhibited decreased mucosal expression of Wnt2b, Wnt7b, and Wnt10a, diminished protein levels of nuclear β-catenin that is mainly located in crypts adjacent to damage, and reduced mRNA expression of two Wnt/β-catenin target molecules Lgr5 and c-Myc when compared with wild-type (WT) mice. Murine peritoneal macrophages treated with interleukin-4 (IL-4) and polarized toward an M2a phenotype overexpressed Wnt2b, Wnt7b, and Wnt10a in a STAT6-dependent manner. Administration of a Wnt agonist as well as transfer of properly polarized M2a macrophages to STAT6(-/-) mice activated the Wnt signaling pathway in the damaged mucosa and accelerated wound healing. Our results demonstrate that a STAT6-dependent macrophage phenotype promotes mucosal repair in TNBS-treated mice through activation of the Wnt signaling pathway. PMID:26601901

  8. Structure-activity relationships of new analogues of arecaidine propargyl ester at muscarinic M1 and M2 receptor subtypes.

    PubMed Central

    Moser, U.; Lambrecht, G.; Wagner, M.; Wess, J.; Mutschler, E.

    1989-01-01

    1. The potency of arecaidine propargyl ester (APE) and of several analogues containing a modified ester side chain has been assessed at M1 and M2 muscarinic receptor subtypes. APE was shown to act as a potent agonist at ganglionic M1 receptors in the pithed rat, at M2 receptors in guinea-pig isolated atria (-log EC50 = 8.22) and ileum (-log EC50 = 7.77). 2. The arecaidine 2-butynyl and 2-pentynyl esters were approximately equipotent with APE at M1 and M2 receptors, whereas the 2-hexynyl derivative was found to be less potent than APE in atria (-log EC50 = 6.80) and ileum (-log EC50 = 6.70) by about one order of magnitude. The 2-heptynyl and 3-phenyl propargyl esters exhibited no agonist actions in atria and ileum. 3. Shifting the triple bond from the 2 to the 3 position and introducing a bulky group at position 1 of the ester side chain of APE and analogues resulted in competitive antagonists (pA2 ranging from 4.9 to 7.3). 4. APE and its 2-butynyl analogue showed some agonistic selectivity for cardiac M2 receptors (potency ratio, ileum/atria = 2.8 and 4.6 respectively). All antagonists in this series of compounds were not selective in terms of affinity since their pA2 values at cardiac and ileal M2 receptors were similar (potency ratios, ileum/atria = 0.4 to 1.2). PMID:2924082

  9. Novel Markers to Delineate Murine M1 and M2 Macrophages

    PubMed Central

    Jablonski, Kyle A.; Amici, Stephanie A.; Webb, Lindsay M.; Ruiz-Rosado, Juan de Dios; Popovich, Phillip G.; Partida-Sanchez, Santiago; Guerau-de-Arellano, Mireia

    2015-01-01

    Classically (M1) and alternatively activated (M2) macrophages exhibit distinct phenotypes and functions. It has been difficult to dissect macrophage phenotypes in vivo, where a spectrum of macrophage phenotypes exists, and also in vitro, where low or non-selective M2 marker protein expression is observed. To provide a foundation for the complexity of in vivo macrophage phenotypes, we performed a comprehensive analysis of the transcriptional signature of murine M0, M1 and M2 macrophages and identified genes common or exclusive to either subset. We validated by real-time PCR an M1-exclusive pattern of expression for CD38, G-protein coupled receptor 18 (Gpr18) and Formyl peptide receptor 2 (Fpr2) whereas Early growth response protein 2 (Egr2) and c-Myc were M2-exclusive. We further confirmed these data by flow cytometry and show that M1 and M2 macrophages can be distinguished by their relative expression of CD38 and Egr2. Egr2 labeled more M2 macrophages (~70%) than the canonical M2 macrophage marker Arginase-1, which labels 24% of M2 macrophages. Conversely, CD38 labeled most (71%) in vitro M1 macrophages. In vivo, a similar CD38+ population greatly increased after LPS exposure. Overall, this work defines exclusive and common M1 and M2 signatures and provides novel and improved tools to distinguish M1 and M2 murine macrophages. PMID:26699615

  10. Chronic Trypanosoma cruzi infection potentiates adipose tissue macrophage polarization toward an anti-inflammatory M2 phenotype and contributes to diabetes progression in a diet-induced obesity model

    PubMed Central

    Cabalén, María E.; Cabral, María F.; Sanmarco, Liliana M.; Andrada, Marta C.; Onofrio, Luisina I.; Ponce, Nicolás E.; Aoki, María P.; Gea, Susana; Cano, Roxana C.

    2016-01-01

    Chronic obesity and Chagas disease (caused by the protozoan Trypanosoma cruzi) represent serious public health concerns. The interrelation between parasite infection, adipose tissue, immune system and metabolism in an obesogenic context, has not been entirely explored. A novel diet-induced obesity model (DIO) was developed in C57BL/6 wild type mice to examine the effect of chronic infection (DIO+I) on metabolic parameters and on obesity-related disorders. Dyslipidemia, hyperleptinemia, and cardiac/hepatic steatosis were strongly developed in DIO mice. Strikingly, although these metabolic alterations were collectively improved by infection, plasmatic apoB100 levels remain significantly increased in DIO+I, suggesting the presence of pro-atherogenic small and dense LDL particles. Moreover, acute insulin resistance followed by chronic hyperglycemia with hypoinsulinemia was found, evidencing an infection-related-diabetes progression. These lipid and glucose metabolic changes seemed to be highly dependent on TLR4 expression since TLR4−/− mice were protected from obesity and its complications. Notably, chronic infection promoted a strong increase in MCP-1 producing macrophages with a M2 (F4/80+CD11c-CD206+) phenotype associated to oxidative stress in visceral adipose tissue of DIO+I mice. Importantly, infection reduced lipid content but intensified inflammatory infiltrates in target tissues. Thus, parasite persistence in an obesogenic environment and the resulting host immunometabolic dysregulation may contribute to diabetes/atherosclerosis progression. PMID:26921251

  11. The combination of glutamate receptor antagonist MK-801 with tamoxifen and its active metabolites potentiates their antiproliferative activity in mouse melanoma K1735-M2 cells.

    PubMed

    Ribeiro, Mariana P C; Nunes-Correia, Isabel; Santos, Armanda E; Custódio, José B A

    2014-02-15

    Recent reports suggest that N-methyl-d-aspartate receptor (NMDAR) blockade by MK-801 decreases tumor growth. Thus, we investigated whether other ionotropic glutamate receptor (iGluR) antagonists were also able to modulate the proliferation of melanoma cells. On the other hand, the antiestrogen tamoxifen (TAM) decreases the proliferation of melanoma cells, and is included in combined therapies for melanoma. As the efficacy of TAM is limited by its metabolism, we investigated the effects of the NMDAR antagonist MK-801 in combination with TAM and its active metabolites, 4-hydroxytamoxifen (OHTAM) and endoxifen (EDX). The NMDAR blockers MK-801 and memantine decreased mouse melanoma K1735-M2 cell proliferation. In contrast, the NMDAR competitive antagonist APV and the AMPA and kainate receptor antagonist NBQX did not affect cell proliferation, suggesting that among the iGluR antagonists only the NMDAR channel blockers inhibit melanoma cell proliferation. The combination of antiestrogens with MK-801 potentiated their individual effects on cell biomass due to diminished cell proliferation, since it decreased the cell number and DNA synthesis without increasing cell death. Importantly, TAM metabolites combined with MK-801 promoted cell cycle arrest in G1. Therefore, the data obtained suggest that the activity of MK-801 and antiestrogens in K1735-M2 cells is greatly enhanced when used in combination. PMID:24240127

  12. Pharmacological Characterization of the Spectrum of Antiviral Activity and Genetic Barrier to Drug Resistance of M2-S31N Channel Blockers.

    PubMed

    Ma, Chunlong; Zhang, Jiantao; Wang, Jun

    2016-09-01

    Adamantanes (amantadine and rimantadine) are one of the two classes of Food and Drug Administration-approved antiviral drugs used for the prevention and treatment of influenza A virus infections. They inhibit viral replication by blocking the wild-type (WT) M2 proton channel, thus preventing viral uncoating. However, their use was discontinued due to widespread drug resistance. Among a handful of drug-resistant mutants, M2-S31N is the predominant mutation and persists in more than 95% of currently circulating influenza A strains. We recently designed two classes of M2-S31N inhibitors, S31N-specific inhibitors and S31N/WT dual inhibitors, which are represented by N-[(5-cyclopropyl-1,2-oxazol-3-yl)methyl]adamantan-1-amine (WJ379) and N-[(5-bromothiophen-2-yl)methyl]adamantan-1-amine (BC035), respectively. However, their antiviral activities against currently circulating influenza A viruses and their genetic barrier to drug resistance are unknown. In this report, we evaluated the therapeutic potential of these two classes of M2-S31N inhibitors (WJ379 and BC035) by profiling their antiviral efficacy against multidrug-resistant influenza A viruses, in vitro drug resistance barrier, and synergistic effect with oseltamivir. We found that M2-S31N inhibitors were active against several influenza A viruses that are resistant to one or both classes of Food and Drug Administration-approved anti-influenza drugs. In addition, M2-S31N inhibitors display a higher in vitro genetic barrier to drug resistance than amantadine. The antiviral effect of WJ379 was also synergistic with oseltamivir carboxylate. Overall, these results reaffirm that M2-S31N inhibitors are promising antiviral drug candidates that warrant further development. PMID:27385729

  13. Discovery of 3-(trifluoromethyl)-1H-pyrazole-5-carboxamide activators of the M2 isoform of pyruvate kinase (PKM2).

    PubMed

    Xu, Yong; Liu, Xiao-Hui; Saunders, Michael; Pearce, Scott; Foulks, Jason M; Parnell, K Mark; Clifford, Adrianne; Nix, Rebecca N; Bullough, Jeremy; Hendrickson, Thomas F; Wright, Kevin; McCullar, Michael V; Kanner, Steven B; Ho, Koc-Kan

    2014-01-15

    Activators of the pyruvate kinase M2 (PKM2) are currently attracting significant interest as potential anticancer therapies. They may achieve a novel antiproliferation response in cancer cells through modulation of the classic 'Warburg effect' characteristic of aberrant metabolism. In this Letter, we describe the optimization of a weakly active screening hit to a structurally novel series of small molecule 3-(trifluoromethyl)-1H-pyrazole-5-carboxamides as potent PKM2 activators. PMID:24374270

  14. The combination of glutamate receptor antagonist MK-801 with tamoxifen and its active metabolites potentiates their antiproliferative activity in mouse melanoma K1735-M2 cells

    SciTech Connect

    Ribeiro, Mariana P.C.; Nunes-Correia, Isabel; Santos, Armanda E.; Custódio, José B.A.

    2014-02-15

    Recent reports suggest that N-methyl-D-aspartate receptor (NMDAR) blockade by MK-801 decreases tumor growth. Thus, we investigated whether other ionotropic glutamate receptor (iGluR) antagonists were also able to modulate the proliferation of melanoma cells. On the other hand, the antiestrogen tamoxifen (TAM) decreases the proliferation of melanoma cells, and is included in combined therapies for melanoma. As the efficacy of TAM is limited by its metabolism, we investigated the effects of the NMDAR antagonist MK-801 in combination with TAM and its active metabolites, 4-hydroxytamoxifen (OHTAM) and endoxifen (EDX). The NMDAR blockers MK-801 and memantine decreased mouse melanoma K1735-M2 cell proliferation. In contrast, the NMDAR competitive antagonist APV and the AMPA and kainate receptor antagonist NBQX did not affect cell proliferation, suggesting that among the iGluR antagonists only the NMDAR channel blockers inhibit melanoma cell proliferation. The combination of antiestrogens with MK-801 potentiated their individual effects on cell biomass due to diminished cell proliferation, since it decreased the cell number and DNA synthesis without increasing cell death. Importantly, TAM metabolites combined with MK-801 promoted cell cycle arrest in G1. Therefore, the data obtained suggest that the activity of MK-801 and antiestrogens in K1735-M2 cells is greatly enhanced when used in combination. - Highlights: • MK-801 and memantine decrease melanoma cell proliferation. • The combination of MK-801 with antiestrogens inhibits melanoma cell proliferation. • These combinations greatly enhance the effects of the compounds individually. • MK-801 combined with tamoxifen active metabolites induces cell cycle arrest in G1. • The combination of MK-801 and antiestrogens is an innovative strategy for melanoma.

  15. RAD001 (everolimus) attenuates experimental autoimmune neuritis by inhibiting the mTOR pathway, elevating Akt activity and polarizing M2 macrophages.

    PubMed

    Han, Ranran; Gao, Juan; Zhai, Hui; Xiao, Jinting; Ding, Ya'nan; Hao, Junwei

    2016-06-01

    Guillain-Barre' syndrome (GBS) is an acute, postinfectious, immune-mediated, demyelinating disease of peripheral nerves and nerve roots. As a classical animal model of GBS, experimental autoimmune neuritis (EAN) has become well-accepted. Additionally, the potent immune modulation exerted by mammalian target of rapamycin (mTOR) inhibitors has been used to treat cancers and showed beneficial effects. Here we demonstrate that the mTOR inhibitor RAD001 (everolimus) protected rats from the symptoms of EAN, as shown by decreased paralysis, diminished inflammatory cell infiltration, reductions in demyelination of peripheral nerves and improved nerve conduction. Furthermore, RAD001 shifted macrophage polarization toward the protective M2 phenotype and modified the inflammatory milieu by downregulating the production of pro-inflammatory cytokines including IFN-γ and IL-17as well as upregulating the release of anti-inflammatory cytokines such as IL-4 and TGF-β. Amounts of the mTOR downstream targets p-P70S6K and p-4E-BP1 in sciatic nerves decreased, whereas the level of its upstream protein p-Akt was elevated. This demonstrated that RAD001 inhibited the mTOR pathway and encouraged the expression of p-Akt, which led to M2 macrophage polarization, thus improved the outcome of EAN in rats. Consequently, RAD001 exhibits strong potential as a therapeutic strategy for ameliorating peripheral poly-neuropathy. PMID:27063582

  16. Mast Cell Phenotype, Location, and Activation in Severe Asthma

    PubMed Central

    Balzar, Silvana; Fajt, Merritt L.; Comhair, Suzy A. A.; Erzurum, Serpil C.; Bleecker, Eugene; Busse, William W.; Castro, Mario; Gaston, Benjamin; Israel, Elliot; Schwartz, Lawrence B.; Curran-Everett, Douglas; Moore, Charity G.; Wenzel, Sally E.

    2011-01-01

    Rationale: Severe asthma (SA) remains poorly understood. Mast cells (MC) are implicated in asthma pathogenesis, but it remains unknown how their phenotype, location, and activation relate to asthma severity. Objectives: To compare MC-related markers measured in bronchoscopically obtained samples with clinically relevant parameters between normal subjects and subjects with asthma to clarify their pathobiologic importance. Methods: Endobronchial biopsies, epithelial brushings, and bronchoalveolar lavage were obtained from subjects with asthma and normal subjects from the Severe Asthma Research Program (N = 199). Tryptase, chymase, and carboxypeptidase A (CPA)3 were used to identify total MC (MCTot) and the MCTC subset (MCs positive for both tryptase and chymase) using immunostaining and quantitative real-time polymerase chain reaction. Lavage was analyzed for tryptase and prostaglandin D2 (PGD2) by ELISA. Measurements and Main Results: Submucosal MCTot (tryptase-positive by immunostaining) numbers were highest in “mild asthma/no inhaled corticosteroid (ICS) therapy” subjects and decreased with greater asthma severity (P = 0.002). In contrast, MCTC (chymase-positive by immunostaining) were the predominant (MCTC/MCTot > 50%) MC phenotype in SA (overall P = 0.005). Epithelial MCTot were also highest in mild asthma/no ICS, but were not lower in SA. Instead, they persisted and were predominantly MCTC. Epithelial CPA3 and tryptase mRNA supported the immunostaining data (overall P = 0.008 and P = 0.02, respectively). Lavage PGD2 was higher in SA than in other steroid-treated groups (overall P = 0.02), whereas tryptase did not differentiate the groups. In statistical models, PGD2 and MCTC/MCTot predicted SA. Conclusions: Severe asthma is associated with a predominance of MCTC in the airway submucosa and epithelium. Activation of those MCTC may contribute to the increases in PGD2 levels. The data suggest an altered and active MC population contributes to SA pathology

  17. Cyclooxygenase-2 inhibition attenuates hypoxic cancer cells induced m2-polarization of macrophages.

    PubMed

    Dubey, P; Shrivastava, R; Tripathi, C; Jain, N K; Tewari, B N; Lone, M-U-D; Baghel, K S; Kumar, V; Misra, S; Bhadauria, S; Bhatt, M L B

    2014-01-01

    Tumor-associated macrophages (TAMs), represent a major subpopulation of tumor infiltrating immune cells. These alternatively activated M2-polarized macrophages are well known for their pro-tumor functions. Owing to their established role in potentiating tumor-neovasculogenesis and metastasis, TAMs have emerged as promising target for anti-cancer immunotherapy. One of the key TAMs related phenomenon that is amenable to therapeutic intervention is their phenotype switching into alternatively activated M2-polarized macrophages. Hindering macrophage polarization towards a pro-tumor M2 phenotype, or better still reprogramming the M2 like TAMs towards M1 subtype is being considered a beneficial anti-cancer strategy. Hypoxic tumor milieu has been proposed as one of the most plausible factor governing M2-polarization of macrophages. We recently demonstrated that hypoxic tumor cells imparted a pro—angiogenic M2 skewed phenotype to macrophages. Furthermore, sizeable body of data indicates for participation of cyclooxygenase-2 (COX-2) in macrophage polarization. Concordantly, inhibition of COX-2 is associated with impaired macrophage polarization. Prompted by this in the current study we decided to explore if inhibition of COX-2 activity via chemical inhibitors may prevent hypoxic cancer cell induced M2-polarization of macrophages. We observed that treatment with Flunixin meglumine, an established preferential inhibitor of COX-2 activity markedly inhibited hypoxic cancer cell induced of M2-polarization of macrophages thereby indicating for usage of COX-2 inhibition as possible anti-cancer treatment modality. PMID:25210855

  18. Higenamine promotes M2 macrophage activation and reduces Hmgb1 production through HO-1 induction in a murine model of spinal cord injury.

    PubMed

    Zhang, Zhenyu; Li, Mingchao; Wang, Yan; Wu, Jian; Li, Jiaping

    2014-12-01

    Spinal cord injury (SCI) is considered to be primarily associated with loss of motor function and leads to the activation of diverse cellular mechanisms in the central nervous system to attempt to repair the damaged spinal cord tissue. Higenamine (HG) (1-[(4-hydroxyphenyl) methyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol), an active ingredient of Aconiti Lateralis Radix Praeparata, has been traditionally used as a heart stimulant and anti-inflammatory agent in oriental countries. However, the function and related mechanism of HG on SCI have never been investigated. In our current study, HG treatment displayed increased myelin sparring and enhanced spinal cord repair process. The numbers of CD4(+) T cells, CD8(+) T cells, Ly6G(+) neutrophils and CD11b(+) macrophages were all significantly lower in the HG-treated group than that in the control group after SCI. HG administration increased the expression of IL-4 and IL-10 and promoted M2 macrophage activation. Significantly reduced Hmgb1 expression was also observed in HG-treated mice with SCI. Furthermore, HG treatment promoted HO-1 production. The increased number of M2 macrophages, decreased expression of Hmgb1 and promoted locomotor recovery induced by HG were all reversed with additional HO-1 inhibitor treatment. In conclusion, HG promotes M2 macrophage activation and reduces Hmgb1 expression dependent on HO-1 induction and then promotes locomotor function after SCI. PMID:25445960

  19. Enantioselective Synthesis of Optically Active Polymeric Homo- and Bimetallic Oxalate-Bridged Networks [M(2)(ox)(3)](n)().

    PubMed

    Andrés, Román; Gruselle, Michel; Malézieux, Bernard; Verdaguer, Michel; Vaissermann, Jacqueline

    1999-10-18

    The synthesis of the enantiomeric forms of the two- and three-dimensional polymers: {[NBu(4)][MnCr(ox)(3)]}(n)() (1) (Bu = n-butyl, ox = oxalate), {[M(bpy)(3))][LiCr(ox)(3)]}(n)() (M = Ni (2), Ru (4)) (bpy = 2,2'-bipyridine), {[M(bpy)(3))][Mn(2)(ox)(3)]}(n)() (M = Ni (3), Ru (5)) using resolved [Cr(ox)(3)](3)(-) and [M(bpy)(3)](2+) (M = Ni, Ru) species as chiral building blocks, and their structural characterization are reported. The optical activity of these systems arises from the helical chirality of the tris-chelated subunits with Delta or Lambda configurations. Bimetallic two-dimensional optically active network 1 results from the stacking of similar metallo-oxalate honeycomblike layers containing [Cr(ox)(3)](3)(-) units of the same chirality. The assembly of homochiral species leads to optically active three-dimensional 3-connected 10-gon nets 2-5. Solid state circular dichroism (CD) measurements demonstrate the enantiomeric character of the obtained polymers. Absolute configurations of the metal centers have been assigned according to circular dichroism and X-ray diffraction data. Enantiomerically pure single crystals of the two enantiomeric forms, {[Ru(Delta)(bpy)(3))][Li(Delta)Cr(Delta)(ox)(3)]H(2)O}(n)() (4Delta) and {[Ru(Lambda)(bpy)(3))][Li(Lambda)Cr(Lambda)(ox)(3)]H(2)O}(n)() (4Lambda), have been obtained and the structures determined by X-ray diffraction studies (crystals data: cubic system, space group P2(1)3, a (Å) = 15.293(8) (4Delta), 15.289(2) (4Lambda), Z = 4). PMID:11671185

  20. Fractional M2-branes

    NASA Astrophysics Data System (ADS)

    Aharony, Ofer; Bergman, Oren; Jafferis, Daniel Louis

    2008-11-01

    We consider two generalizations of the Script N = 6 superconformal Chern-Simons-matter theories with gauge group U(N) × U(N). The first generalization is to Script N = 6 superconformal U(M) × U(N) theories, and the second to Script N = 5 superconformal O(2M) × USp(2N) and O(2M+1) × USp(2N) theories. These theories are conjectured to describe M2-branes probing C4/Zk in the unitary case, and C4/{\\widehat{D}}k in the orthogonal/symplectic case, together with a discrete flux, which can be interpreted as |M-N| fractional M2-branes localized at the orbifold singularity. The classical theories with these gauge groups have been constructed before; in this paper we focus on some quantum aspects of these theories, and on a detailed description of their M theory and type IIA string theory duals.

  1. SUB-THz AND H{alpha} ACTIVITY DURING THE PREFLARE AND MAIN PHASES OF A GOES CLASS M2 EVENT

    SciTech Connect

    Kaufmann, Pierre; Gimenez de Castro, C. Guillermo; Raulin, Jean-Pierre; Correia, Emilia; Fernandes, Luis Olavo; De Souza, Rodney V.; Marcon, Rogerio; White, Stephen M.; Godoy, Rodolfo; Marun, Adolfo; Pereyra, Pablo

    2011-12-01

    Radio and optical observations of the evolution of flare-associated phenomena have shown an initial and rapid burst at 0.4 THz only followed subsequently by a localized chromospheric heating producing an H{alpha} brightening with later heating of the whole active region. A major instability occurred several minutes later producing one impulsive burst at microwaves only, associated with an M2.0 GOES X-ray flare that exhibited the main H{alpha} brightening at the same site as the first flash.The possible association between long-enduring time profiles at soft X-rays, microwaves, H{alpha}, and sub-THz wavelengths is discussed. In the decay phase, the H{alpha} movie shows a disrupting magnetic arch structure ejecting dark, presumably chromospheric, material upward. The time sequence of events suggests genuine interdependent and possibly non-thermal instabilities triggering phenomena, with concurrent active region plasma heating and material ejection.

  2. Direct measurements of oscillatory glycolysis in pancreatic islet β-cells using novel fluorescence resonance energy transfer (FRET) biosensors for pyruvate kinase M2 activity.

    PubMed

    Merrins, Matthew J; Van Dyke, Aaron R; Mapp, Anna K; Rizzo, Mark A; Satin, Leslie S

    2013-11-15

    Pulses of insulin released from pancreatic β-cells maintain blood glucose in a narrow range, although the source of these pulses is unclear. We and others have proposed that positive feedback mediated by the glycolytic enzyme phosphofructokinase-1 (PFK1) enables β-cells to generate metabolic oscillations via autocatalytic activation by its product fructose 1,6-bisphosphate (FBP). Although much indirect evidence has accumulated in favor of this hypothesis, a direct measurement of oscillating glycolytic intermediates has been lacking. To probe glycolysis directly, we engineered a family of inter- and intramolecular FRET biosensors based on the glycolytic enzyme pyruvate kinase M2 (PKAR; pyruvate kinase activity reporter), which multimerizes and is activated upon binding FBP. When introduced into Min6 β-cells, PKAR FRET efficiency increased rapidly in response to glucose. Importantly, however, metabolites entering downstream of PFK1 (glyceraldehyde, pyruvate, and ketoisocaproate) failed to activate PKAR, consistent with sensor activation by FBP; the dependence of PKAR on FBP was further confirmed using purified sensor in vitro. Using a novel imaging modality for monitoring mitochondrial flavin fluorescence in mouse islets, we show that slow oscillations in mitochondrial redox potential stimulated by 10 mm glucose are in phase with glycolytic efflux through PKM2, measured simultaneously from neighboring islet β-cells expressing PKAR. These results indicate that PKM2 activity in β-cells is oscillatory and are consistent with pulsatile PFK1 being the mediator of slow glycolytic oscillations. PMID:24100037

  3. An HPLC-MS/MS method for simultaneous determination of the active metabolites of febuxostat (67M-1, 67M-2 and 67M-4) in human plasma.

    PubMed

    Xie, Huiru; Wang, Zhijun; Deng, Ke; Jiang, Xuehua; Wang, Ling; Lv, Guoyu

    2014-11-01

    An HPLC-MS/MS method for simultaneously determination of the active metabolites (67M-1, 67M-2 and 67M-4) in human plasma using clopidogrel as the internal standard was developed and validated. The compounds were extracted by protein precipitation using acetonitrile and separated using a C8 column by a gradient elution with the mobile phase consisting of acetonitrile (containing 0.1% formic acid) and 0.1% formic acid. Quantification was performed using multiple reaction monitoring in positive mode with m/z transitions of 333.1-261.0, 333.1-261.0, 347.0-261.0 and 322.2-184.1 for 67M-1, 67M-2, 67M-4 and clopidogrel (Internal Standard), respectively. This method was validated in terms of specificity, linearity, precision, accuracy, and stability. The lower limit of quantification of this method was 0.5 ng/mL and the calibration curve was linear over the concentration range of 0.5-150 ng/mL. The intra- and inter-run precision was less than 11.67% and 8.64%, respectively, with the accuracy between 98.33% and 108.38%. The samples were stable under all the tested conditions. This method has been successfully applied to the pharmacokinetic study of febuxostat in healthy Chinese volunteers following oral administration of 40 mg and 80 mg febuxostat. PMID:25222745

  4. IL4I1 Is a Novel Regulator of M2 Macrophage Polarization That Can Inhibit T Cell Activation via L-Tryptophan and Arginine Depletion and IL-10 Production

    PubMed Central

    Yue, Yinpu; Huang, Wei; Liang, Jingjing; Guo, Jing; Ji, Jian; Yao, Yunliang; Zheng, Mingzhu; Cai, Zhijian; Lu, Linrong; Wang, Jianli

    2015-01-01

    Interleukin 4-induced gene-1 (IL4I1) was initially described as an early IL-4-inducible gene in B cells. IL4I1 protein can inhibit T cell proliferation by releasing its enzymatic catabolite, H2O2, and this effect is associated with transient down-regulation of T cell CD3 receptor-zeta (TCRζ) expression. Herein, we show that IL4I1 contributes to the regulation of macrophage programming. We found that expression of IL4I1 increased during bone marrow-derived macrophage (BMDM) differentiation, expression of IL4I1 is much higher in primary macrophages than monocytes, and IL4I1 expression in BMDMs could be induced by Th1 and Th2 cytokines in two different patterns. Gene expression analysis revealed that overexpression of IL4I1 drove the expression of M2 markers (Fizz1, Arg1, YM-1, MR) and inhibited the expression of M1-associated cytokines. Conversely, knockdown of IL4I1 by siRNA resulted in opposite effects, and also attenuated STAT-3 and STAT-6 phosphorylation. Furthermore, IL4I1 produced by macrophages catalyzed L-tryptophan degradation, while levo-1-methyl-tryptophan (L-1-MT), but not dextro-1-methyl-tryptophan, partially rescued IL4I1-dependent inhibition of T cell activation. Other inhibitors, such as diphenylene iodonium (DPI), an anti-IL-10Rα blocking antibody, and a nitric oxide synthase inhibitor, NG-monomethyl-L-arginine, also had this effect. Overall, our findings indicate that IL4I1 promotes an enhanced M2 functional phenotype, which is most likely associated with the phosphorylation of STAT-6 and STAT-3. Moreover, DPI, L-1-MT, NG-monomethyl-L-arginine, and anti-IL-10Rα blocking antibody were all found to be effective IL4I1 inhibitors in vitro. PMID:26599209

  5. Succinyl-5-aminoimidazole-4-carboxamide-1-ribose 5'-Phosphate (SAICAR) Activates Pyruvate Kinase Isoform M2 (PKM2) in Its Dimeric Form.

    PubMed

    Yan, Ming; Chakravarthy, Srinivas; Tokuda, Joshua M; Pollack, Lois; Bowman, Gregory D; Lee, Young-Sam

    2016-08-23

    Human pyruvate kinase isoform M2 (PKM2) is a glycolytic enzyme isoform implicated in cancer. Malignant cancer cells have higher levels of dimeric PKM2, which is regarded as an inactive form of tetrameric pyruvate kinase. This perceived inactivity has fueled controversy about how the dimeric form of pyruvate kinase might contribute to cancer. Here we investigate enzymatic properties of PKM2(G415R), a variant derived from a cancer patient, which we show by size-exclusion chromatography and small-angle X-ray scattering to be a dimer that cannot form a tetramer in solution. Although PKM2(G415R) binds to fructose 1,6-bisphosphate (FBP), unlike the wild type this PKM2 variant shows no activation by FBP. In contrast, PKM2(G415R) is activated by succinyl-5-aminoimidazole-4-carboxamide-1-ribose 5'-phosphate (SAICAR), an endogenous metabolite that we previously showed correlates with an increased level of cell proliferation and promotes protein kinase activity of PKM2. Our results demonstrate an important and unexpected enzymatic activity of the PKM2 dimer that likely has a key role in cancer progression. PMID:27481063

  6. Lysophosphatidylcholine perpetuates macrophage polarization toward classically activated phenotype in inflammation.

    PubMed

    Qin, Xiaofei; Qiu, Chunguang; Zhao, Luosha

    2014-01-01

    Pro-inflammatory macrophages are involved in vascular inflammation and serve as the major effector cells in the pathophysiology of atherosclerosis. Phosphatidylcholine (PC) is a major phospholipid moiety affixed to oxidized low-density lipoprotein (oxLDL) and thought to play important roles in the development of atherosclerosis. In this study we described that a bioactive lipid derivative, lysophosphatidylcholine (lysoPC), generated from hydrolysis of the PC moiety of oxidized LDL, promoted and stabilized a strong M1 phenotype in macrophage polarization. Another derivative, 9-hydroxyoctadecadienoic acid (9-HODE), did not show the similar biological function. Blockade of G protein coupled receptor, G2A, which mediates the signal transduction of lysoPC, diminished the effects of lysoPC on the macrophage polarization toward M1 phenotype. The results provide insights into the new mechanism on how oxidized LDL participates in tissue inflammation in atherosclerosis. PMID:24841857

  7. Serum Stability and Affinity Optimization of an M2 Macrophage-Targeting Peptide (M2pep).

    PubMed

    Ngambenjawong, Chayanon; Gustafson, Heather H; Pineda, Julio M; Kacherovsky, Nataly A; Cieslewicz, Maryelise; Pun, Suzie H

    2016-01-01

    Tumor associated macrophages (TAMs) are a major stromal component of the tumor microenvironment in several cancers. TAMs are a potential target for adjuvant cancer therapies due to their established roles in promoting proliferation of cancer cells, angiogenesis, and metastasis. We previously discovered an M2 macrophage-targeting peptide (M2pep) which was successfully used to target and deliver a pro-apoptotic KLA peptide to M2-like TAMs in a CT-26 colon carcinoma model. However, the effectiveness of in vivo TAM-targeting using M2pep is limited by its poor serum stability and low binding affinity. In this study, we synthesized M2pep derivatives with the goals of increasing serum stability and binding affinity. Serum stability evaluation of M2pepBiotin confirmed its rapid degradation attributed to exolytic cleavage from the N-terminus and endolytic cleavages at the W10/W11 and S16/K17 sites. N-terminal acetylation of M2pepBiotin protected the peptide against the exolytic degradation while W10w and K(17,18,19)k substitutions were able to effectively protect endolytic degradation at their respective cleavage sites. However, no tested amino acid changes at the W10 position resulted in both protease resistance at that site and retention of binding activity. Therefore, cyclization of M2pep was investigated. Cyclized M2pep better resisted serum degradation without compromising binding activity to M2 macrophages. During the serum stability optimization process, we also discovered that K9R and W10Y substitutions significantly enhanced binding affinity of M2pep. In an in vitro binding study of different M2pep analogs pre-incubated in mouse serum, cyclic M2pep with K9R and W10Y modifications (cyclic M2pep(RY)) retained the highest binding activity to M2 macrophages over time due to its improved serum stability. Finally, we evaluated the in vivo accumulation of sulfo-Cy5-labeled M2pep and cyclic M2pep(RY) in both the CT-26 and 4T1 breast carcinoma models. Cyclic M2pep

  8. Serum Stability and Affinity Optimization of an M2 Macrophage-Targeting Peptide (M2pep)

    PubMed Central

    Ngambenjawong, Chayanon; Gustafson, Heather H.; Pineda, Julio M.; Kacherovsky, Nataly A.; Cieslewicz, Maryelise; Pun, Suzie H.

    2016-01-01

    Tumor associated macrophages (TAMs) are a major stromal component of the tumor microenvironment in several cancers. TAMs are a potential target for adjuvant cancer therapies due to their established roles in promoting proliferation of cancer cells, angiogenesis, and metastasis. We previously discovered an M2 macrophage-targeting peptide (M2pep) which was successfully used to target and deliver a pro-apoptotic KLA peptide to M2-like TAMs in a CT-26 colon carcinoma model. However, the effectiveness of in vivo TAM-targeting using M2pep is limited by its poor serum stability and low binding affinity. In this study, we synthesized M2pep derivatives with the goals of increasing serum stability and binding affinity. Serum stability evaluation of M2pepBiotin confirmed its rapid degradation attributed to exolytic cleavage from the N-terminus and endolytic cleavages at the W10/W11 and S16/K17 sites. N-terminal acetylation of M2pepBiotin protected the peptide against the exolytic degradation while W10w and K(17,18,19)k substitutions were able to effectively protect endolytic degradation at their respective cleavage sites. However, no tested amino acid changes at the W10 position resulted in both protease resistance at that site and retention of binding activity. Therefore, cyclization of M2pep was investigated. Cyclized M2pep better resisted serum degradation without compromising binding activity to M2 macrophages. During the serum stability optimization process, we also discovered that K9R and W10Y substitutions significantly enhanced binding affinity of M2pep. In an in vitro binding study of different M2pep analogs pre-incubated in mouse serum, cyclic M2pep with K9R and W10Y modifications (cyclic M2pep(RY)) retained the highest binding activity to M2 macrophages over time due to its improved serum stability. Finally, we evaluated the in vivo accumulation of sulfo-Cy5-labeled M2pep and cyclic M2pep(RY) in both the CT-26 and 4T1 breast carcinoma models. Cyclic M2pep

  9. Induction of M2 regulatory macrophages through the β2 adrenergic receptor with protection during endotoxemia and acute lung injury

    PubMed Central

    Grailer, Jamison J.; Haggadone, Mikel D.; Sarma, J. Vidya; Zetoune, Firas S.; Ward, Peter A.

    2014-01-01

    Main drivers of acute inflammation are macrophages, which are known to have receptors for catecholamines. Based on their function, macrophages are broadly categorized as either M1 (pro-inflammatory) or M2 phenotypes (anti-inflammatory). In this study, we investigated catecholamine-induced alterations in the phenotype of activated macrophages. In the presence of lipopolysaccharide (LPS), mouse peritoneal macrophages acquired an M1 phenotype. However, the co-presence of LPS and either epinephrine or norepinephrine resulted in a strong M2 phenotype including high levels of arginase-1 and interleukin (IL) -10, and reduced expression of M1 markers. Furthermore, epinephrine enhanced macrophage phagocytosis and promoted type 2 T cell responses in vitro, which are known features of M2 macrophages. Analysis of M2 subtype-specific markers indicated that LPS and catecholamine co-treated macrophages were not alternatively activated, but of the regulatory macrophage subtype. Interestingly, catecholamines signaled through the β2 adrenergic receptor, but not the canonical cAMP/protein kinase A signaling pathway. Instead, the M2 pathway required an intact phosphoinositol 3-kinase pathway. Blockade of the β2 adrenergic receptor reduced survival and enhanced injury during mouse models of endotoxemia and LPS-induced acute lung injury, respectively. These results demonstrate a role for the β2 adrenergic receptor in promoting the M2 macrophage phenotype. PMID:24642449

  10. Alteration of O-GlcNAcylation affects serine phosphorylation and regulates gene expression and activity of pyruvate kinase M2 in colorectal cancer cells.

    PubMed

    Chaiyawat, Parunya; Chokchaichamnankit, Daranee; Lirdprapamongkol, Kriengsak; Srisomsap, Chantragan; Svasti, Jisnuson; Champattanachai, Voraratt

    2015-10-01

    O-GlcNAcylation is a dynamic post-translational modification that has extensive crosstalk with phosphorylation either at the same or adjacent sites of various proteins. We have previously reported that O-GlcNAcylation level was increased in primary breast and colorectal cancer, but the interplay of the two modifications remains unclear. Therefore, we explored crosstalk of the modifications by RNA interference against O-GlcNAc transferase (OGT) in colorectal cancer cells. Two-dimensional immunoblotting and mass spectrometric analysis showed that the levels of O-GlcNAc and serine phosphorylation of many proteins including serine hydroxymethyltransferase, cytokeratin-8, pyruvate kinase M2 (PKM2), heterogeneous nuclear ribonucleoprotein L, and lamin-B1, were reduced in siOGT cells compared to siScramble cells. In HT29 cells, immunoprecipitated PKM2 revealed decreased O-GlcNAc and serine phosphorylation levels after siOGT knockdown, but increased levels after treatment with Thiamet-G, an inhibitor of O-GlcNAcase (OGA). In addition, when global O-GlcNAcylation was enhanced by treating cells with Thiamet-G, PKM2 expression level was upregulated, but PKM2-specific activity was decreased. On the other hand, in OGT knockdown cells, PKM2 expression level was downregulated, but PKM2-specific activity was increased. Moreover, the metastatic colorectal cancer cells, SW620, had more O-GlcNAc-PKM2 and showed lower PKM2-specific activity compared to the non-metastatic colorectal cancer SW480 cells. These results suggested roles of O-GlcNAcylation in modulating serine phosphorylation, as well as in regulating PKM2 activity and expression. Interfering levels of O-GlcNAcylation of PKM2 may be a novel target in controlling cancer metabolism and tumorigenesis of colorectal cancer. PMID:26252736

  11. Nucleation process of an M2 earthquake in a deep gold mine in South Africa inferred from on-fault foreshock activity

    NASA Astrophysics Data System (ADS)

    Yabe, Y.; Nakatani, M.; Naoi, M.; Philipp, J.; Janssen, C.; Kawakata, H.; Dresen, G. H.; Ogasawara, H.

    2014-12-01

    We observed foreshock activity of an Mw2.2 earthquake (the mainshock) that occurred in a gabbroic dyke at a depth of about 3.3 km from the surface in a deep gold mine in South Africa. Foreshock activity, selectively occurring on a plane on which the mainshock would occur, lasted for at least six months until the mainshock. Rock samples in the mainshock source region were recovered by drilling afterward. Indication of ancient hydrothermal alteration on the rupture plane of the mainshock suggests that the foreshock activity occurred on a pre-existing weakness, probably a healed joint, to nucleate the mainshock. The foreshocks during the three months leading up to the mainshock concentrated to three clusters (F1-F3), which, we interpreted, represent the nucleation at multiple sites. The temporal variation in the foreshock activity in the three months can be well explained by the temporal variation of the stressing state in the source region of the mainshock due to nearby mining. One of these clusters (cluster F2) showed an accelerated activity from about 10 days before the mainshock, while activity over the entire foreshock area was rather constant. The foreshock sources in the final 41 hours, during which the stress state was constant, migrated from F2 to F1 that neighbored to the mainshock hypocenter, suggesting coalescence of the two nuclei. The occurrence of mainshock was 0.4-2.3 days earlier than the time expected from an extrapolation of the accelerated foreshock activity in F2. The nucleation of mainshock may have been advanced to the criticality for dynamic instability in a stepwise manner upon the coalescence of nuclei.While the heterogeneity of geological structures obscures the straightforward manifestation of self-driven quasi-static nucleation, the present careful analysis suggests that some essence of such nucleation as known from the fracture theory and laboratory experiments was caught in the pre-M2 AE data on a natural joint at a depth of 3.3 km.

  12. PPARγ Ligands Switched High Fat Diet-Induced Macrophage M2b Polarization toward M2a Thereby Improving Intestinal Candida Elimination

    PubMed Central

    Olagnier, David; Bernad, José; Perez, Laurence; Burcelin, Rémy; Valentin, Alexis; Auwerx, Johan; Pipy, Bernard; Coste, Agnès

    2010-01-01

    Obesity is associated with a chronic low-grade inflammation that predisposes to insulin resistance and the development of type 2 diabetes. In this metabolic context, gastrointestinal (GI) candidiasis is common. We recently demonstrated that the PPARγ ligand rosiglitazone promotes the clearance of Candida albicans through the activation of alternative M2 macrophage polarization. Here, we evaluated the impact of high fat diet (HFD)-induced obesity and the effect of rosiglitazone (PPARγ ligand) or WY14643 (PPARα ligand) both on the phenotypic M1/M2 polarization of peritoneal and cecal tissue macrophages and on the outcome of GI candidiasis. We demonstrated that the peritoneal macrophages and the cell types present in the cecal tissue from HF fed mice present a M2b polarization (TNF-αhigh, IL-10high, MR, Dectin-1). Interestingly, rosiglitazone induces a phenotypic M2b-to-M2a (TNF-αlow, IL-10low, MRhigh, Dectin-1high) switch of peritoneal macrophages and of the cells present in the cecal tissue. The incapacity of WY14643 to switch this polarization toward M2a state, strongly suggests the specific involvement of PPARγ in this mechanism. We showed that in insulin resistant mice, M2b polarization of macrophages present on the site of infection is associated with an increased susceptibility to GI candidiasis, whereas M2a polarization after rosiglitazone treatment favours the GI fungal elimination independently of reduced blood glucose. In conclusion, our data demonstrate a dual benefit of PPARγ ligands because they promote mucosal defence mechanisms against GI candidiasis through M2a macrophage polarization while regulating blood glucose level. PMID:20877467

  13. Inhibition of Microglia Activation as a Phenotypic Assay in Early Drug Discovery

    PubMed Central

    Figuera-Losada, Mariana; Rojas, Camilo; Slusher, Barbara S.

    2014-01-01

    Complex biological processes such as inflammation, cell death, migration, proliferation, and the release of biologically active molecules can be used as outcomes in phenotypic assays during early stages of drug discovery. Although target-based approaches have been widely used over the past decades, a disproportionate number of first-in-class drugs have been identified using phenotypic screening. This review details phenotypic assays based on inhibition of microglial activation and their utility in primary and secondary screening, target validation, and pathway elucidation. The role of microglia, both in normal as well as in pathological conditions such as chronic neurodegenerative diseases, is reviewed. Methodologies to assess microglia activation in vitro are discussed in detail, and classes of therapeutic drugs known to decrease the proinflammatory and cytotoxic responses of activated microglia are appraised, including inhibitors of glutaminase, cystine/glutamate antiporter, nuclear factor κB, and mitogen-activated protein kinases. PMID:23945875

  14. KIT oncogene inhibition drives intratumoral macrophage M2 polarization.

    PubMed

    Cavnar, Michael J; Zeng, Shan; Kim, Teresa S; Sorenson, Eric C; Ocuin, Lee M; Balachandran, Vinod P; Seifert, Adrian M; Greer, Jonathan B; Popow, Rachel; Crawley, Megan H; Cohen, Noah A; Green, Benjamin L; Rossi, Ferdinand; Besmer, Peter; Antonescu, Cristina R; DeMatteo, Ronald P

    2013-12-16

    Tumor-associated macrophages (TAMs) are a major component of the cancer microenvironment. Modulation of TAMs is under intense investigation because they are thought to be nearly always of the M2 subtype, which supports tumor growth. Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and typically results from an activating mutation in the KIT oncogene. Using a spontaneous mouse model of GIST and 57 freshly procured human GISTs, we discovered that TAMs displayed an M1-like phenotype and function at baseline. In both mice and humans, the KIT oncoprotein inhibitor imatinib polarized TAMs to become M2-like, a process which involved TAM interaction with apoptotic tumor cells leading to the induction of CCAAT/enhancer binding protein (C/EBP) transcription factors. In human GISTs that eventually developed resistance to imatinib, TAMs reverted to an M1-like phenotype and had a similar gene expression profile as TAMs from untreated human GISTs. Therefore, TAM polarization depends on tumor cell oncogene activity and has important implications for immunotherapeutic strategies in human cancers. PMID:24323358

  15. Aftershock activity of a M2 earthquake in a deep South African gold mine - spatial distribution and magnitude-frequency relation

    NASA Astrophysics Data System (ADS)

    Naoi, M. M.; Nakatani, M.; Kwiatek, G.; Plenkers, K.; Yabe, Y.

    2009-12-01

    An earthquake of M 2.1 occurred on December 27, 2007 in a deep South African gold mine (Yabe et al., 2008). It occurred within a sensitive high frequency seismic network consisting of eight high frequency AE sensors (up to 200 kHz) and a tri-axial accelerometer (up to 25 kHz). Within 150 hours following the earthquake, our AE network detected more than 20,000 events within 250 m of the center of the network. We have located aftershocks assuming homogeneous medium (Fig. a), based on their manually-picked arrival times of P and S waves. This aftershock seismicity can be clearly separated into five clusters. Each sequence obeyed Omori ‘s law and had the similar p-value (p ~ 1.3). The cluster A in Fig. a is very planar. More than 90 % aftershocks of the cluster are within a 3 m thickness while the cluster has a lateral dimension of ~100m x 100m. The density of aftershocks normal to the planar cluster follows an exponential distribution with about 0.6 m characteristic length. The distribution of the cluster A coincides with one of the nodal planes of the main shock estimated by the waveform inversion. Hence, cluster A is thought to delineate the main rupture. Clusters B to E coincide with the edge of mining cavity or background seismicity recognized before the mainshock. Remarkable off-fault aftershock activities occurred only in these four areas. We have determined moment magnitude (Mw) of 17,350 earthquakes using AE waveforms (Mw > -5.4). As AE sensors have complex frequency characteristics, we use the amplitude in a narrow frequency band (2 - 4 kHz). Directivity of the AE sensor (~20 db) is corrected by comparison with the accelerometer record. Absolute magnitude has been given by an empirical relationship between AE amplitude and Mw determined by the spectral level of the accelerometer record. Mw determination from accelerometer record was done for ~ 0.5 % of aftershocks detected by AE sensors. Moment magnitudes of these selected earthquakes resulted in values

  16. Endoplasmic Reticulum Stress Controls M2 Macrophage Differentiation and Foam Cell Formation*

    PubMed Central

    Oh, Jisu; Riek, Amy E.; Weng, Sherry; Petty, Marvin; Kim, David; Colonna, Marco; Cella, Marina; Bernal-Mizrachi, Carlos

    2012-01-01

    Macrophages are essential in atherosclerosis progression, but regulation of the M1 versus M2 phenotype and their role in cholesterol deposition are unclear. We demonstrate that endoplasmic reticulum (ER) stress is a key regulator of macrophage differentiation and cholesterol deposition. Macrophages from diabetic patients were classically or alternatively stimulated and then exposed to oxidized LDL. Alternative stimulation into M2 macrophages lead to increased foam cell formation by inducing scavenger receptor CD36 and SR-A1 expression. ER stress induced by alternative stimulation was necessary to generate the M2 phenotype through JNK activation and increased PPARγ expression. The absence of CD36 or SR-A1 signaling independently of modified cholesterol uptake decreased ER stress and prevented the M2 differentiation typically induced by alternative stimulation. Moreover, suppression of ER stress shifted differentiated M2 macrophages toward an M1 phenotype and subsequently suppressed foam cell formation by increasing HDL- and apoA-1-induced cholesterol efflux indicating suppression of macrophage ER stress as a potential therapy for atherosclerosis. PMID:22356914

  17. Tumourigenic phenotypes of human melanoma cell lines in nude mice determined by an active antitumour mechanism.

    PubMed Central

    Jacubovich, R.; Cabrillat, H.; Gerlier, D.; Bailly, M.; Doré, J. F.

    1985-01-01

    Ten human melanoma cell lines (HMCL) were tested for their ability to grow subcutaneously in nude mice. Using a standard inoculum, the HMCL could be characterized by their highly, fairly or poorly xenografting phenotype. These phenotypes were stable and the phenotype of one HMCL was recovered within cell clones derived from it. The role of nude mice natural defences in the expression of HMCL xenografting phenotypes was studied. Sublethal whole body irradiation and silica pretreatment of recipients enabled poorly tumourigenic HMCL to grow in most animals without affecting their splenic NK activity. Admixture of BCG or MDP encapsulated in liposomes with highly tumourigenic HMCL resulted in the abrogation of tumour growth in naive nude mice. The long lasting abrogating of NK activity in vivo by treatment with anti-asialo-GM1 anti-serum did not enhance the growth of a poorly tumourigenic HMCL. The HMCL were found to be resistant to in vitro murine NK activity. These results showed that the expression of the HMCL xenografting phenotypes could be controlled by the nude mice natural defences. NK cells did not seem to be largely involved whereas macrophages might be good candidates as anti-xenograft effectors. PMID:3882112

  18. Effect of rTsP53 on the M1/M2 activation of bone-marrow derived macrophage in vitro

    PubMed Central

    Chen, Zhibin; Li, Fan; Yang, Wen; Liang, Yanbing; Tang, Hao; Li, Zhenyu; Wu, Jingguo; Liang, Huaping; Ma, Zhongfu

    2015-01-01

    We investigated that if rTsP53 could be used to activate bone-marrow derived macrophage (BMDM) into M2 macrophage and stop M1 macrophage activation. After 72 h incubation in blank culture medium, cells with PE-CCR7 (-) and FITC-CD206 (-) was extracted and its mean proportion was 92.30 ± 0.22%. With the stimulation of 20 μg/ml IFN-γ for 72 h, cells with PE-CCR7 (+) was extracted and its mean proportion was 16.24 ± 0.82%. With the stimulation of IL-3/IL-14 (both 10 μg/ml) for 72 h, cells with FICT-CD206 (+) was extracted and its mean proportion was 87.32 ± 4.29%. Co-incubation with different dose of rTsP53 (0.001 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, 2 μg/ml, 5 μg/ml, 10 μg/ml, respectively) for 72 h, FITC-CD206 (+) macrophage was extracted. The mean proportion in each group was 1.09 ± 0.22%, 2.13 ± 0.13%, 4.91 ± 0.07%, 5.48 ± 0.29%, 9.81 ± 0.06%, 12.83 ± 0.55%, 17.87 ± 0.02%, respectively. The dose of rTsP53 was significantly positive correlated to the proportion of FITC-CD206 (+) macrophage. Co-incubation with 20 μg/ml IFN-γ and 5 μg/ml rTsP53 for 72 h, cells with PE-CCR7 (+) was extracted and its mean proportion was 10.60 ± 0.19%. Compared to that of mere co-incubation with IFN-γ, there was significant difference between the two groups. ELISA showed that Th1 cytokines’ (IFN-γ, IL-6 and TNF-α) level decreased in the culture medium supernatant of BMDM co-incubated with rTsP53. There was negative correlation between the Th1 cytokines’ level and the dose of rTsP53. Both Th2 cytokines (IL-4 and IL-13) and regulatory cytokines in the culture medium increased. There was positive correlation between the Th2 cytokines’ level and the dose of rTsP53. There was also positive correlation between the regulatory cytokines’ level and the dose of rTsP53. Compared to that of BMDM co-incubated with IFN-γ, levels of TNF-α and IL-6 were significant lower than that of BMDM co-incubated with both IFN-γ and rTsP53 (both P < 0.05), while the levels

  19. 3-Azatetracyclo[5.2.1.15,8.01,5]undecane derivatives: from wild-type inhibitors of the M2 ion channel of influenza A virus to derivatives with potent activity against the V27A mutant

    PubMed Central

    Rey-Carrizo, Matias; Torres, Eva; Ma, Chunlong; Barniol-Xicota, Marta; Wang, Jun; Wu, Yibing; Naesens, Lieve; DeGrado, William F.; Lamb, Robert A.; Pinto, Lawrence H.; Vázquez, Santiago

    2013-01-01

    We have synthesized and characterized a series of compounds containing the 3-azatetracyclo[5.2.1.15,8.01,5]undecane scaffold designed as analogs of amantadine, an inhibitor of the M2 proton channel of influenza A virus. Inhibition of the wild-type (wt) M2 channel and the amantadine-resistant A/M2-S31N and A/M2-V27A mutant ion channels were measured in Xenopus oocytes using two-electrode voltage clamp (TEV) assays. Most of the novel compounds inhibited the wt ion channel in the low micromolar range. Of note, several compounds inhibited the A/M2 V27A mutant ion channel, one of them with submicromolar IC50. None of the compounds was found to inhibit the S31N mutant ion channel. The antiviral activity of three novel dual wt and A/M2-V27A channels inhibitors was confirmed by influenza virus yield assays. PMID:24237039

  20. Activity and Structural Comparisons of Solution Associating and Monomeric Channel-Forming Peptides Derived from the Glycine Receptor M2 Segment

    PubMed Central

    Cook, Gabriel A.; Prakash, Om; Zhang, Ke; Shank, Lalida P.; Takeguchi, Wade A.; Robbins, Ashley; Gong, Yu-Xi; Iwamoto, Takeo; Schultz, Bruce D.; Tomich, John M.

    2004-01-01

    A number of channel-forming peptides derived from the second transmembrane (TM) segment (M2) of the glycine receptor α1 subunit (M2GlyR), including the 22-residue sequence NK4-M2GlyR p22 wild type (WT) (KKKKPARVGLGITTVLTMTTQS), induce anion permeation across epithelial cell monolayers. In vitro assays suggest that this peptide or related sequences might function as a candidate for ion channel replacement therapy in treating channelopathies such as cystic fibrosis (CF). The wild-type sequence forms soluble associations in water that diminish its efficacy. Introduction of a single substitution S22W at the C-terminus, NK4-M2GlyR p22 S22W, eliminates the formation of higher molecular weight associations in solution. The S22W peptide also reduces the concentration of peptide required for half-maximal anion transport induced across Madin-Darby canine kidney cells (MDCK) monolayers. A combination of 2D double quantum filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating frame nuclear Overhauser effect spectroscopy (ROESY) data were recorded for both the associating WT and nonassociating S22W peptides and used to compare the primary structures and to assign the secondary structures. High-resolution structural studies were recorded in the solvent system (40% 2,2,2-Trifluoroethanol (TFE)/water), which gave the largest structural difference between the two peptides. Nuclear Overhauser effect crosspeak intensity provided interproton distances and the torsion angles were measured by spin-spin coupling constants. These constraints were put into the DYANA modeling program to generate a group of structures. These studies yielded energy-minimized structures for this mixed solvent environment. Structure for both peptides is confined to the 15-residue transmembrane segments. The energy-minimized structure for the WT peptide shows a partially helical extended structure. The S22W peptide

  1. Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells.

    PubMed

    Berndt, Alexander; Büttner, Robert; Gühne, Stefanie; Gleinig, Anna; Richter, Petra; Chen, Yuan; Franz, Marcus; Liebmann, Claus

    2014-04-01

    Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients' outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCMTGF, FCMPDGF) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCMB). FCMTGF stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCMTGF≫FCMPDGF induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCMTGF>FCMPDGF) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. PMID:24394543

  2. Rac2 Controls Tumor Growth, Metastasis and M1-M2 Macrophage Differentiation In Vivo

    PubMed Central

    Joshi, Shweta; Singh, Alok R.; Zulcic, Muamera; Bao, Lei; Messer, Karen; Ideker, Trey; Dutkowski, Janusz; Durden, Donald L.

    2014-01-01

    Although it is well-established that the macrophage M1 to M2 transition plays a role in tumor progression, the molecular basis for this process remains incompletely understood. Herein, we demonstrate that the small GTPase, Rac2 controls macrophage M1 to M2 differentiation and the metastatic phenotype in vivo. Using a genetic approach, combined with syngeneic and orthotopic tumor models we demonstrate that Rac2-/- mice display a marked defect in tumor growth, angiogenesis and metastasis. Microarray, RT-PCR and metabolomic analysis on bone marrow derived macrophages isolated from the Rac2-/- mice identify an important role for Rac2 in M2 macrophage differentiation. Furthermore, we define a novel molecular mechanism by which signals transmitted from the extracellular matrix via the α4β1 integrin and MCSF receptor lead to the activation of Rac2 and potentially regulate macrophage M2 differentiation. Collectively, our findings demonstrate a macrophage autonomous process by which the Rac2 GTPase is activated downstream of the α4β1 integrin and the MCSF receptor to control tumor growth, metastasis and macrophage differentiation into the M2 phenotype. Finally, using gene expression and metabolomic data from our Rac2-/- model, and information related to M1-M2 macrophage differentiation curated from the literature we executed a systems biologic analysis of hierarchical protein-protein interaction networks in an effort to develop an iterative interactome map which will predict additional mechanisms by which Rac2 may coordinately control macrophage M1 to M2 differentiation and metastasis. PMID:24770346

  3. Effect of Insulin Analogues on Insulin/IGF1 Hybrid Receptors: Increased Activation by Glargine but Not by Its Metabolites M1 and M2

    PubMed Central

    Pierre-Eugene, Cécile; Pagesy, Patrick; Nguyen, Tuyet Thu; Neuillé, Marion; Tschank, Georg; Tennagels, Norbert; Hampe, Cornelia; Issad, Tarik

    2012-01-01

    Background In diabetic patients, the pharmacokinetics of injected human insulin does not permit optimal control of glycemia. Fast and slow acting insulin analogues have been developed, but they may have adverse properties, such as increased mitogenic or anti-apoptotic signaling. Insulin/IGF1 hybrid receptors (IR/IGF1R), present in most tissues, have been proposed to transmit biological effects close to those of IGF1R. However, the study of hybrid receptors is difficult because of the presence of IR and IGF1R homodimers. Our objective was to perform the first study on the pharmacological properties of the five marketed insulin analogues towards IR/IGF1R hybrids. Methodology To study the effect of insulin analogues on IR/IGF1R hybrids, we used our previously developed Bioluminescence Resonance Energy Transfer (BRET) assay that permits specific analysis of the pharmacological properties of hybrid receptors. Moreover, we have developed a new, highly sensitive BRET-based assay to monitor phophatidylinositol-3 phosphate (PIP3) production in living cells. Using this assay, we performed a detailed pharmacological analysis of PIP3 production induced by IGF1, insulin and insulin analogues in living breast cancer-derived MCF-7 and MDA-MB231 cells. Results Among the five insulin analogues tested, only glargine stimulated IR/IGF1R hybrids with an EC50 that was significantly lower than insulin and close to that of IGF1. Glargine more efficiently stimulated PIP3 production in MCF-7 cells but not in MDA-MB231 cells as compared to insulin. In contrast, glargine metabolites M1 and M2 showed lower potency for hybrid receptors stimulation, PIP3 production, Akt and Erk1/2 phosphorylation and DNA synthesis in MCF-7 cells, compared to insulin. Conclusion Glargine, possibly acting through IR/IGF1R hybrids, displays higher potency, whereas its metabolites M1 and M2 display lower potency than insulin for the stimulation of proliferative/anti-apoptotic pathways in MCF-7 cells. PMID:22848683

  4. Crosstalk among electrical activity, trophic factors and morphogenetic proteins in the regulation of neurotransmitter phenotype specification.

    PubMed

    Borodinsky, Laura N; Belgacem, Yesser H

    2016-04-01

    Morphogenetic proteins are responsible for patterning the embryonic nervous system by enabling cell proliferation that will populate all the neural structures and by specifying neural progenitors that imprint different identities in differentiating neurons. The adoption of specific neurotransmitter phenotypes is crucial for the progression of neuronal differentiation, enabling neurons to connect with each other and with target tissues. Preliminary neurotransmitter specification originates from morphogen-driven neural progenitor specification through the combinatorial expression of transcription factors according to morphogen concentration gradients, which progressively restrict the identity that born neurons adopt. However, neurotransmitter phenotype is not immutable, instead trophic factors released from target tissues and environmental stimuli change expression of neurotransmitter-synthesizing enzymes and specific vesicular transporters modifying neuronal neurotransmitter identity. Here we review studies identifying the mechanisms of catecholaminergic, GABAergic, glutamatergic, cholinergic and serotonergic early specification and of the plasticity of these neurotransmitter phenotypes during development and in the adult nervous system. The emergence of spontaneous electrical activity in developing neurons recruits morphogenetic proteins in the process of neurotransmitter phenotype plasticity, which ultimately equips the nervous system and the whole organism with adaptability for optimal performance in a changing environment. PMID:26686293

  5. M2-F1 cockpit

    NASA Technical Reports Server (NTRS)

    1963-01-01

    This photo shows the cockpit configuration of the M2-F1 wingless lifting body. With a top speed of about 120 knots, the M2-F1 had a simple instrument panel. Besides the panel itself, the ribs of the wooden shell (left) and the control stick (center) are also visible. The wingless, lifting body aircraft design was initially conceived as a means of landing an aircraft horizontally after atmospheric reentry. The absence of wings would make the extreme heat of re-entry less damaging to the vehicle. In 1962, Dryden management approved a program to build a lightweight, unpowered lifting body as a prototype to flight test the wingless concept. It would look like a 'flying bathtub,' and was designated the M2-F1, the 'M' referring to 'manned' and 'F' referring to 'flight' version. It featured a plywood shell placed over a tubular steel frame crafted at Dryden. Construction was completed in 1963. The first flight tests of the M2-F1 were over Rogers Dry Lake at the end of a tow rope attached to a hopped-up Pontiac convertible driven at speeds up to about 120 mph. This vehicle needed to be able to tow the M2-F1 on the Rogers Dry Lakebed adjacent to NASA's Flight Research Center (FRC) at a minimum speed of 100 miles per hour. To do that, it had to handle the 400-pound pull of the M2-F1. Walter 'Whitey' Whiteside, who was a retired Air Force maintenance officer working in the FRC's Flight Operations Division, was a dirt-bike rider and hot-rodder. Together with Boyden 'Bud' Bearce in the Procurement and Supply Branch of the FRC, Whitey acquired a Pontiac Catalina convertible with the largest engine available. He took the car to Bill Straup's renowned hot-rod shop near Long Beach for modification. With a special gearbox and racing slicks, the Pontiac could tow the 1,000-pound M2-F1 110 miles per hour in 30 seconds. It proved adequate for the roughly 400 car tows that got the M2-F1 airborne to prove it could fly safely and to train pilots before they were towed behind a C-47

  6. Anti-influenza M2e antibody

    DOEpatents

    Bradbury, Andrew M.

    2013-04-16

    Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

  7. Anti-influenza M2e antibody

    DOEpatents

    Bradbury, Andrew M.

    2011-12-20

    Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

  8. Serine protease activity and residual LEKTI expression determine phenotype in Netherton syndrome.

    PubMed

    Hachem, Jean-Pierre; Wagberg, Fredrik; Schmuth, Matthias; Crumrine, Debra; Lissens, Willy; Jayakumar, Arumugam; Houben, Evi; Mauro, Theodora M; Leonardsson, Göran; Brattsand, Maria; Egelrud, Torbjorn; Roseeuw, Diane; Clayman, Gary L; Feingold, Kenneth R; Williams, Mary L; Elias, Peter M

    2006-07-01

    Mutations in the SPINK5 gene encoding the serine protease (SP) inhibitor, lymphoepithelial-Kazal-type 5 inhibitor (LEKTI), cause Netherton syndrome (NS), a life-threatening disease, owing to proteolysis of the stratum corneum (SC). We assessed here the basis for phenotypic variations in nine patients with "mild", "moderate", and "severe" NS. The magnitude of SP activation correlated with both the barrier defect and clinical severity, and inversely with residual LEKTI expression. LEKTI co-localizes within the SC with kallikreins 5 and 7 and inhibits both SP. The permeability barrier abnormality in NS was further linked to SC thinning and proteolysis of two lipid hydrolases (beta-glucocerebrosidase and acidic sphingomyelinase), with resultant disorganization of extracellular lamellar membranes. SC attenuation correlated with phenotype-dependent, SP activation, and loss of corneodesmosomes, owing to desmoglein (DSG)1 and desmocollin (DSC)1 degradation. Although excess SP activity extended into the nucleated layers in NS, degrading desmosomal mid-line structures with loss of DSG1/DSC1, the integrity of the nucleated epidermis appears to be maintained by compensatory upregulation of DSG3/DSC3. Maintenance of sufficient permeability barrier function for survival correlated with a compensatory acceleration of lamellar body secretion, providing a partial permeability barrier in NS. These studies provide a mechanistic basis for phenotypic variations in NS, and describe compensatory mechanisms that permit survival of NS patients in the face of unrelenting SP attack. PMID:16601670

  9. Behavioral Analysis of Dopaminergic Activation in Zebrafish and Rats Reveals Similar Phenotypes.

    PubMed

    Ek, Fredrik; Malo, Marcus; Åberg Andersson, Madelene; Wedding, Christoffer; Kronborg, Joel; Svensson, Peder; Waters, Susanna; Petersson, Per; Olsson, Roger

    2016-05-18

    Zebrafish is emerging as a complement to mammals in behavioral studies; however, there is a lack of comparative studies with rodents and humans to establish the zebrafish as a predictive translational model. Here we present a detailed phenotype evaluation of zebrafish larvae, measuring 300-3000 variables and analyzing them using multivariate analysis to identify the most important ones for further evaluations. The dopamine agonist apomorphine has previously been shown to have a complex U-shaped dose-response relationship in the variable distance traveled. In this study, we focused on breaking down distance traveled into more detailed behavioral phenotypes for both zebrafish and rats and identified in the multivariate analysis low and high dose phenotypes with characteristic behavioral features. Further analysis of single parameters also identified an increased activity at the lowest concentration indicative of a U-shaped dose-response. Apomorphine increased the distance of each swim movement (bout) at both high and low doses, but the underlying behavior of this increase is different; at high dose, both bout duration and frequency increased whereas bout max speed was higher at low dose. Larvae also displayed differences in place preference. The low dose phenotype spent more time in the center, indicative of an anxiolytic effect, while the high-dose phenotype had a wall preference. These dose-dependent effects corroborated findings in a parallel rat study and previous observations in humans. The translational value of pharmacological zebrafish studies was further evaluated by comparing the amino acid sequence of the dopamine receptors (D1-D4), between zebrafish, rats and humans. Humans and zebrafish share 100% of the amino acids in the binding site for D1 and D3 whereas D2 and D4 receptors share 85-95%. Molecular modeling of dopamine D2 and D4 receptors indicated that nonconserved amino acids have limited influence on important ligand-receptor interactions. PMID

  10. DPPH free radical scavenging activity and phenotypic difference in hepatoprotective plant (Silybum marianum L.).

    PubMed

    Ahmad, Nisar; Fazal, Hina; Abbasi, Bilal Haider; Anwar, Shazma; Basir, Abdul

    2013-06-01

    Silybum marianum L. is medicinally important for its active principle component silymarin. Silymarin regenerates damaged hepatic tissues. On the basis of such regenerative properties, the radical scavenging activity (1,1-diphenyl-2-picrylhydrazyl (DPPH)) of different tissues and the phenotypic difference of the hepatoprotective species, S. marianum L. were evaluated. There was less phenotypic difference in purple and white varieties of S. marianum. Assay of the antioxidant potential of different parts of the plant revealed that significantly higher activity (78.2%) was observed in seeds of the purple flowering plant than seeds of white flowering plant (49%) after different time intervals. Young leaves collected from white flowering plant exhibit 64.8% activity, which is higher than the purple flowering plant (55.1%). Significantly, same activity was observed in mature leaves of white (52%) and purple flowering plants (50%). The main stem collected from both the varieties exhibits similar activity from 50 to 52%. A 67.2% activity was recorded for mature roots of white flowering plant followed by roots of the purple variety (65%). The present study revealed that seeds and roots of both the varieties scavenge and detoxify more DPPH free radicals than other plant parts and can be used as a source of natural antioxidants and food additives. PMID:22362017

  11. Biomechanical activation of vascular endothelium as a determinant of its functional phenotype

    PubMed Central

    García-Cardeña, Guillermo; Comander, Jason; Anderson, Keith R.; Blackman, Brett R.; Gimbrone, Michael A.

    2001-01-01

    One of the striking features of vascular endothelium, the single-cell-thick lining of the cardiovascular system, is its phenotypic plasticity. Various pathophysiologic factors, such as cytokines, growth factors, hormones, and metabolic products, can modulate its functional phenotype in health and disease. In addition to these humoral stimuli, endothelial cells respond to their biomechanical environment, although the functional implications of this biomechanical paradigm of activation have not been fully explored. Here we describe a high-throughput genomic analysis of modulation of gene expression observed in cultured human endothelial cells exposed to two well defined biomechanical stimuli—a steady laminar shear stress and a turbulent shear stress of equivalent spatial and temporal average intensity. Comparison of the transcriptional activity of 11,397 unique genes revealed distinctive patterns of up- and down-regulation associated with each type of stimulus. Cluster analyses of transcriptional profiling data were coupled with other molecular and cell biological techniques to examine whether these global patterns of biomechanical activation are translated into distinct functional phenotypes. Confocal immunofluorescence microscopy of structural and contractile proteins revealed the formation of a complex apical cytoskeleton in response to laminar shear stress. Cell cycle analysis documented different effects of laminar and turbulent shear stresses on cell proliferation. Thus, endothelial cells have the capacity to discriminate among specific biomechanical forces and to translate these input stimuli into distinctive phenotypes. The demonstration that hemodynamically derived stimuli can be strong modulators of endothelial gene expression has important implications for our understanding of the mechanisms of vascular homeostasis and atherogenesis. PMID:11296290

  12. A systematic analysis of recombination activity and genotype-phenotype correlation in human recombination-activating gene 1 deficiency

    PubMed Central

    Lee, Yu Nee; Frugoni, Francesco; Dobbs, Kerry; Walter, Jolan E.; Giliani, Silvia; Gennery, Andrew R.; Al-Herz, Waleed; Haddad, Elie; LeDeist, Francoise; Bleesing, Jack H.; Henderson, Lauren A.; Pai, Sung-Yun; Nelson, Robert P.; El-Ghoneimy, Dalia H.; El-Feky, Reem A.; Reda, Shereen M.; Hossny, Elham; Soler-Palacin, Pere; Fuleihan, Ramsay L.; Patel, Niraj C.; Massaad, Michel J.; Geha, Raif S.; Puck, Jennifer M.; Palma, Paolo; Cancrini, Caterina; Chen, Karin; Vihinen, Mauno; Alt, Frederick W.; Notarangelo, Luigi D.

    2014-01-01

    Background The recombination-activating gene (RAG) 1/2 proteins play a critical role in the development of T and B cells by initiating the VDJ recombination process that leads to generation of a broad T-cell receptor (TCR) and B-cell receptor repertoire. Pathogenic mutations in the RAG1/2 genes result in various forms of primary immunodeficiency, ranging from T−B− severe combined immune deficiency to delayed-onset disease with granuloma formation, autoimmunity, or both. It is not clear what contributes to such heterogeneity of phenotypes. Objective We sought to investigate the molecular basis for phenotypic diversity presented in patients with various RAG1 mutations. Methods We have developed a flow cytometry–based assay that allows analysis of RAG recombination activity based on green fluorescent protein expression and have assessed the induction of the Ighc locus rearrangements in mouse Rag1−/− pro-B cells reconstituted with wild-type or mutant human RAG1 (hRAG1) using deep sequencing technology. Results Here we demonstrate correlation between defective recombination activity of hRAG1 mutant proteins and severity of the clinical and immunologic phenotype and provide insights on the molecular mechanisms accounting for such phenotypic diversity. Conclusions Using a sensitive assay to measure the RAG1 activity level of 79 mutations in a physiologic setting, we demonstrate correlation between recombination activity of RAG1 mutants and the severity of clinical presentation and show that RAG1 mutants can induce specific abnormalities of the VDJ recombination process. PMID:24290284

  13. Nucleation process of an M2 earthquake in a deep gold mine in South Africa inferred from on-fault foreshock activity

    NASA Astrophysics Data System (ADS)

    Yabe, Yasuo; Nakatani, Masao; Naoi, Makoto; Philipp, Joachim; Janssen, Christoph; Watanabe, Takayoshi; Katsura, Taishi; Kawakata, Hironori; Georg, Dresen; Ogasawara, Hiroshi

    2015-08-01

    Using a network of sensitive high-frequency acoustic emission sensors, we observed foreshock activity of an Mw 2.2 earthquake (main shock) in a deep gold mine in South Africa. Foreshock activity, which selectively occurred on a part of the rupture plane of the forthcoming main shock, lasted for at least 6 months until the main shock. Rock samples recovered from the main shock source region showed evidence of ancient hydrothermal alteration on the main shock rupture plane, suggesting that the foreshock activity occurred on a preexisting weakness. The foreshocks during 3 months before the main shock were concentrated in three clusters (F1-F3), which we interpret as representing localized preslip at multiple sites. While the location of mining area, the source of stress perturbations, changed with time, the locations of foreshock clusters did not change, suggesting that the preslip patches were controlled by strength heterogeneity rather than stress distribution. Activity over the entire foreshock area was generally constant, but the largest cluster (F2) showed accelerated activity starting at least 7 days before the main shock, while mining stress did not increase in this period. The main shock initiated at a point close to F1, away from F2. All the six foreshocks during the final 41 h occurred in F1 and F2 and in-between. These suggest that in the last stage of the preparation process of the main shock, preslip patches interacted with each other through the stress concentration ahead of the expanding preslip patch (F2), which should be the only driving force of the preparation process under the constant external load.

  14. Paraoxonase 1 activity and phenotype distribution in premenopausal and postmenopausal women

    PubMed Central

    Butorac, Dražan; Ćelap, Ivana; Kačkov, Sanja; Robić, Vera; Miletić, Tomislav; Meštrić, Zlata Flegar; Hulina, Andrea; Kuna, Krunoslav; Grubišić, Tihana Žanić; Rajković, Marija Grdić

    2014-01-01

    Introduction Postmenopausal women have higher risk of cardiovascular disease. One of the contributing factors could be reduced activity of anti-atherogenic enzyme paraoxonase 1 (PON1). The aim of this study was to examine differences in the lipid status, paraoxonase and arylesterase PON1 activities and PON1 phenotype in women with regular menstrual cycle and in postmenopausal women. Materials and methods: The study included 51 women in reproductive age (25 in follicular and 26 in luteal phase of the menstrual cycle) and 23 women in postmenopause. Lipid parameters in sera were determined using original reagents and according to manufacturer protocol. PON1 activity in serum was assessed by spectrophotometric method with substrates: paraoxon and phenylacetate. PON1 phenotype was determined by double substrate method. Results: Compared to the women in follicular and luteal phase, postmenopausal women have significantly higher concentration of triglyceride [0.9 (0.7–1.3), 0.7 (0.6–1.0) vs. 1.5 (0.9–1.7) mmol/L; P = 0.002], cholesterol [5.10 (4.78–6.10), 5.05 (4.70–5.40) vs. 6.30 (5.73–7.23) mmol/L; P < 0.001], LDL [3.00 (2.56–3.63), 3.00 (2.70–3.70) vs. 3.90 (3.23–4.50) mmol/L; P < 0.001], and apolipoprotein B [0.88 (0.75–1.00), 0.79 (0.68–1.00) vs. 1.07 (0.90–1.24) mmol/L; P = 0.002]. PON1 basal [104 (66–260), 106 (63–250) vs. 93 (71–165) U/L; P = 0.847] and salt-stimulated paraoxonase activity [210 (131–462), 211 (120–442) vs. 180 (139–296) U/L; P = 0.857] as well as arylesterase activity [74 (63–82), 70 (54–91) vs. 70 (60–81) kU/L; P = 0.906] and PON1 phenotype (P = 0.810) were not different in the study groups. Conclusion: There are no differences in PON1 activity and PON1 phenotype between women with regular menstrual cycle and postmenopausal women. PMID:24969921

  15. Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells

    SciTech Connect

    Berndt, Alexander; Büttner, Robert; Gühne, Stefanie; Gleinig, Anna; Richter, Petra; Chen, Yuan; Franz, Marcus; Liebmann, Claus

    2014-04-01

    Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients’ outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCM{sub TGF}, FCM{sub PDGF}) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCM{sub B}). FCM{sub TGF} stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCM{sub TGF}≫FCM{sub PDGF} induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCM{sub TGF}>FCM{sub PDGF}) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. - Highlights: • A cell culture model for cancer associated fibroblasts is described. • The mutual interaction with OSCC cells leads to up-regulation of EGFR in tumour cells. • mCAF induces EGFR downstream signalling with increased proliferation in OSCC. • Erk activation is associated with protein interaction with vimentin

  16. Linking trait-based phenotypes to prefrontal cortex activation during inhibitory control.

    PubMed

    Rodrigo, Achala H; Di Domenico, Stefano I; Graves, Bryanna; Lam, Jaeger; Ayaz, Hasan; Bagby, R Michael; Ruocco, Anthony C

    2016-01-01

    Inhibitory control is subserved in part by discrete regions of the prefrontal cortex whose functionality may be altered according to specific trait-based phenotypes. Using a unified model of normal range personality traits, we examined activation within lateral and medial aspects of the prefrontal cortex during a manual go/no-go task. Evoked hemodynamic oxygenation within the prefrontal cortex was measured in 106 adults using a 16-channel continuous-wave functional near-infrared spectroscopy system. Within lateral regions of the prefrontal cortex, greater activation was associated with higher trait levels of extraversion, agreeableness and conscientiousness, and lower neuroticism. Higher agreeableness was also related to more activation in the medial prefrontal cortex during inhibitory control. These results suggest that personality traits reflecting greater emotional stability, extraversion, agreeableness and conscientiousness may be associated with more efficient recruitment of control processes subserved by lateral regions of the prefrontal cortex. These findings highlight key links between trait-based phenotypes and neural activation patterns in the prefrontal cortex underlying inhibitory control. PMID:26163672

  17. Lactate Activates HIF-1 in Oxidative but Not in Warburg-Phenotype Human Tumor Cells

    PubMed Central

    De Saedeleer, Christophe J.; Copetti, Tamara; Porporato, Paolo E.; Verrax, Julien

    2012-01-01

    Cancer can be envisioned as a metabolic disease driven by pressure selection and intercellular cooperativeness. Together with anaerobic glycolysis, the Warburg effect, formally corresponding to uncoupling glycolysis from oxidative phosphorylation, directly participates in cancer aggressiveness, supporting both tumor progression and dissemination. The transcription factor hypoxia-inducible factor-1 (HIF-1) is a key contributor to glycolysis. It stimulates the expression of glycolytic transporters and enzymes supporting high rate of glycolysis. In this study, we addressed the reverse possibility of a metabolic control of HIF-1 in tumor cells. We report that lactate, the end-product of glycolysis, inhibits prolylhydroxylase 2 activity and activates HIF-1 in normoxic oxidative tumor cells but not in Warburg-phenotype tumor cells which also expressed lower basal levels of HIF-1α. These data were confirmed using genotypically matched oxidative and mitochondria-depleted glycolytic tumor cells as well as several different wild-type human tumor cell lines of either metabolic phenotype. Lactate activates HIF-1 and triggers tumor angiogenesis and tumor growth in vivo, an activity that we found to be under the specific upstream control of the lactate transporter monocarboxylate transporter 1 (MCT1) expressed in tumor cells. Because MCT1 also gates lactate-fueled tumor cell respiration and mediates pro-angiogenic lactate signaling in endothelial cells, MCT1 inhibition is confirmed as an attractive anticancer strategy in which a single drug may target multiple tumor-promoting pathways. PMID:23082126

  18. Modeling multiple M2-branes

    SciTech Connect

    Bagger, Jonathan; Lambert, Neil

    2007-02-15

    We investigate the worldvolume theory that describes N coincident M2-branes ending on an M5-brane. We argue that the fields that describe the transverse spacetime coordinates take values in a nonassociative algebra. We postulate a set of supersymmetry transformations and find that they close into a novel gauge symmetry. We propose a three-dimensional N=2 supersymmetric action to describe the truncation of the full theory to the scalar and spinor fields, and show how a Basu-Harvey fuzzy funnel arises as the Bogomol'nyi-Prasad-Sommerfield solution to this theory.

  19. Phenotypic Diversity Caused by Differential RpoS Activity among Environmental Escherichia coli Isolates▿†

    PubMed Central

    Chiang, Sarah M.; Dong, Tao; Edge, Thomas A.; Schellhorn, Herb E.

    2011-01-01

    Enteric bacteria deposited into the environment by animal hosts are subject to diverse selective pressures. These pressures may act on phenotypic differences in bacterial populations and select adaptive mutations for survival in stress. As a model to study phenotypic diversity in environmental bacteria, we examined mutations of the stress response sigma factor, RpoS, in environmental Escherichia coli isolates. A total of 2,040 isolates from urban beaches and nearby fecal pollution sources on Lake Ontario (Canada) were screened for RpoS function by examining growth on succinate and catalase activity, two RpoS-dependent phenotypes. The rpoS sequence was determined for 45 isolates, including all candidate RpoS mutants, and of these, six isolates were confirmed as mutants with the complete loss of RpoS function. Similarly to laboratory strains, the RpoS expression of these environmental isolates was stationary phase dependent. However, the expression of RpoS regulon members KatE and AppA had differing levels of expression in several environmental isolates compared to those in laboratory strains. Furthermore, after plating rpoS+ isolates on succinate, RpoS mutants could be readily selected from environmental E. coli. Naturally isolated and succinate-selected RpoS mutants had lower generation times on poor carbon sources and lower stress resistance than their rpoS+ isogenic parental strains. These results show that RpoS mutants are present in the environment (with a frequency of 0.003 among isolates) and that, similarly to laboratory and pathogenic strains, growth on poor carbon sources selects for rpoS mutations in environmental E. coli. RpoS selection may be an important determinant of phenotypic diversification and, hence, the survival of E. coli in the environment. PMID:21948830

  20. Extensive macrophage accumulation in young and old Niemann-Pick C1 model mice involves the alternative, M2, activation pathway and inhibition of macrophage apoptosis.

    PubMed

    Deutsch, Gail; Muralidhar, Akshay; Le, Ellen; Borbon, Ivan A; Erickson, Robert P

    2016-03-10

    We have studied the pathophysiology of lung disease which occurs in two mouse models of Niemann-Pick C1 disease. We utilized Npc1(-/-) mice transgenic for normal gene expression in glia or neurons and glia at ages several fold the usual and a mouse model of the juvenile form of NPC1, a point mutation, at one age to confirm some findings. Lung weights, as per cent of body weight, increase much more than liver and spleen weights. Although pulmonary function parameters only vary for hysteresis between young and older Npc1(-/-) mice, they are markedly different than those found in normal control mice. Cholesterol accumulation continued in the older mice but sphingosine-1-phosphate was not increased. Bronchoalveolar lavage (BAL) showed a massive increase (26×) in the number of macrophages. Histologic examination from the older, transgenic Npc1(-/-) mice showed small foci of alveolar proteinosis and evidence of hemorrhage, as well as dense macrophage accumulation. A large subset of macrophages was immunopositive for Fizz1 or arginase-1, markers of the alternative activation pathway, while no Fizz1 or arginase-1 positive macrophages were found in wild-type mice. The percentage of marker positive macrophages was relatively stable at 5-10% at various ages and within the 2 transgenic models. Phosphohistone H3 and Ki67 showed low levels of proliferation of these macrophages. Apoptosis was prominent within lung capillary endothelial cells, but limited within macrophages. Thus, activation of the alternative pathway is involved in Niemann-Pick C1 associated pulmonary macrophage accumulation, with low proliferation of these cells balanced by low levels of apoptosis. PMID:26707209

  1. Quantitative changes in tumor-associated M2 macrophages characterize cholangiocarcinoma and their association with metastasis.

    PubMed

    Thanee, Malinee; Loilome, Watcharin; Techasen, Anchalee; Namwat, Nisana; Boonmars, Thidarut; Pairojkul, Chawalit; Yongvanit, Puangrat

    2015-01-01

    The tumor microenvironment (TME) includes numerous non-neoplastic cells such as leukocytes and fibroblasts that surround the neoplasm and influence its growth. Tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) are documented as key players in facilitating cancer appearance and progression. Alteration of the macrophage (CD68, CD163) and fibroblast (α-SMA, FSP-1) cells in Opisthorchis viverrini (Ov)-induced cholangiocarcinoma (CCA) was here assessed using liver tissues from an established hamster model and from 43 human cases using immunohistochemistry. We further investigated whether M2-activated TAMs influence CCA cell migration ability by wound healing assay and Western blot analysis. Macrophages and fibroblasts change their phenotypes to M2-TAMs (CD68+, CD163+) and CAFs (α-SMA+, FSP-1+), respectively in the early stages of carcinogenesis. Interestingly, a high density of the M2-TAMs CCA in patients is significantly associated with the presence of extrahepatic metastases (p=0.021). Similarly, CD163+ CCA cells are correlated with metastases (p=0.002), and they may be representative of an epithelial-to-mesenchymal transition (EMT) with increased metastatic activity. We further showed that M2-TAM conditioned medium can induce CCA cell migration as well as increase N-cadherin expression (mesenchymal marker). The present work revealed that significant TME changes occur at an early stage of Ov-induced carcinogenesis and that M2-TAMs are key factors contributing to CCA metastasis, possibly via EMT processes. PMID:25854403

  2. Distribution, activation and tryptase/chymase phenotype of mast cells in the rheumatoid lesion.

    PubMed Central

    Tetlow, L C; Woolley, D E

    1995-01-01

    OBJECTIVE--To determine the distribution, activation, and tryptase/chymase phenotype of mast cells (MCs) in the rheumatoid lesion. METHODS--MC tryptase and chymase were studied by immunohistochemistry using monoclonal antibodies and examination by brightfield, interference, and fluorescent microscopy. Thirty four specimens of cartilage-pannus junction and 26 specimens of rheumatoid synovium, all derived from knee surgery, were examined. RESULTS--MCs were identified in all specimens examined, but their distribution and local concentrations varied, both within and between specimens. As a proportion of total synovial cells, there were more MCs in fibrous synovial tissues than in those with active inflammatory cell infiltrations; MCs usually showed a peripheral distribution around lymphocytic/mononuclear cell infiltrations. Most cartilage-pannus specimens demonstrated local concentrations of MCs at, or close to, sites of cartilage erosion, a significant proportion of which showed extracellular tryptase indicative of MC degranulation. MC degranulation was often associated with localised oedema and disruption of the stromal matrix. Two MC phenotypes were identified: one population contained tryptase alone (MCT) whilst another contained both tryptase and chymase (MCTC). The ratio MCT:MCTC approximated 8:1. CONCLUSIONS--This histological study demonstrated that local concentrations of MCs and their activation/degranulation are commonly observed in the rheumatoid lesion, and especially at sites of cartilage erosion. Such observations add weight to the concept that MCs contribute to the processes of inflammation, matrix degradation and tissue remodelling. Images PMID:7668897

  3. Activated Leukocyte Cell Adhesion Molecule (ALCAM or CD166) Modulates Bone Phenotype and Hematopoiesis

    PubMed Central

    Hooker, R. Adam; Chitteti, Brahmananda R.; Egan, Patrick H.; Cheng, Ying-Hua; Himes, Evan R.; Meijome, Tomas; Srour, Edward F.; Fuchs, Robyn K.; Kacena, Melissa A.

    2015-01-01

    Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166), is expressed on osteoblasts (OB) and hematopoietic stem cells (HSC) residing in the hematopoietic niche, and may have important regulatory roles in bone formation. Because HSC numbers are reduced 77% in CD166−/− mice, we hypothesized that changes in bone phenotype and consequently the endosteal niche may partially be responsible for this alteration. Therefore, we investigated bone phenotype and OB function in CD166−/− mice. Although osteoclastic measures were not affected by loss of CD166, CD166−/− mice exhibited a modest increase in trabecular bone fraction (42%), and increases in osteoid deposition (72%), OB number (60%), and bone formation rate (152%). Cortical bone geometry was altered in CD166−/− mice resulting in up to 81% and 49% increases in stiffness and ultimate force, respectively. CD166−/− OB displayed elevated alkaline phosphatase (ALP) activity and mineralization, and increased mRNA expression of Fra 1, ALP, and osteocalcin. Overall, CD166−/− mice displayed modestly elevated trabecular bone volume fraction with increased OB numbers and deposition of osteoid, and increased OB differentiation in vitro, possibly suggesting more mature OB are secreting more osteoid. This may explain the decline in HSC number in vivo because immature OB are mainly responsible for hematopoiesis enhancing activity. PMID:25730656

  4. EMT phenotype is induced by increased Src kinase activity via Src-mediated caspase-8 phosphorylation.

    PubMed

    Zhao, Yang; Li, XiaoJun; Sun, XiangFei; Zhang, YunFeng; Ren, Hong

    2012-01-01

    Caspase-8 governs multiple cell responses to the microenvironmental cues. However, its integration of "death-life" signalings remains elusive. In our study, the role of caspase-8-Src is well-established as a promoter for migration or metastasis in Casp8(+)Src(+) A549/H226 cells in vivo and in vitro. In particular for nude mice models, mice implanted with Casp8(+)Src(+) A459/H226 cells remarkably increased spontaneous tumor metastatic burden with a significant survival disadvantage. Additionally, we detect that Src-mediated caspase-8 phosphorylation stimulates Src phosphorylation at Tyr-416 via the linkage of Src SH2 domain with phosph-Tyr-380 site of caspase-8. In turn, activated Src can efficiently induce epithelial-mesenchymal transition (EMT) phenotypic features to promote tumor cells metastasis. Surprisingly, RXDLL motif deletion in the DEDa of caspase-8 attenuates tumor cell migration or metastasis via impairing the recruitment of caspase-8 into the cellular periphery where activated Src is subject to caspase-8 phosphorylation. Together, a simple model is that the peripherization of caspase-8 is well-poised to facilitate Src-mediated caspase-8 phosphrylation at Tyr-380, then binding of phospho-Tyr380 of caspase-8 to Src SH2 domain may maintain Src in an active conformation to induce EMT phenotype, a key step toward cancer metastasis. PMID:22508042

  5. Alternatively activated macrophages derived from monocytes and tissue macrophages are phenotypically and functionally distinct

    PubMed Central

    Gundra, Uma Mahesh; Girgis, Natasha M.; Ruckerl, Dominik; Jenkins, Stephen; Ward, Lauren N.; Kurtz, Zachary D.; Wiens, Kirsten E.; Tang, Mei San; Basu-Roy, Upal; Mansukhani, Alka; Allen, Judith E.

    2014-01-01

    Macrophages adopt an alternatively activated phenotype (AAMs) when activated by the interleukin-4receptor(R)α. AAMs can be derived either from proliferation of tissue resident macrophages or recruited inflammatory monocytes, but it is not known whether these different sources generate AAMs that are phenotypically and functionally distinct. By transcriptional profiling analysis, we show here that, although both monocyte and tissue-derived AAMs expressed high levels of Arg1, Chi3l3, and Retnla, only monocyte-derived AAMs up-regulated Raldh2 and PD-L2. Monocyte-derived AAMs were also CX3CR1-green fluorescent protein (GFP)high and expressed CD206, whereas tissue-derived AAMs were CX3CR1-GFP and CD206 negative. Monocyte-derived AAMs had high levels of aldehyde dehydrogenase activity and promoted the differentiation of FoxP3+ cells from naïve CD4+ cells via production of retinoic acid. In contrast, tissue-derived AAMs expressed high levels of uncoupling protein 1. Hence monocyte-derived AAM have properties associated with immune regulation, and the different physiological properties associated with AAM function may depend on the distinct lineage of these cells. PMID:24695852

  6. Basophil phenotypes in chronic idiopathic urticaria in relation to disease activity and autoantibodies.

    PubMed

    Eckman, John A; Hamilton, Robert G; Gober, Laura M; Sterba, Patricia M; Saini, Sarbjit S

    2008-08-01

    Potentially pathogenic IgG autoantibodies to IgE or its receptor, Fc epsilonRIalpha, have been detected in approximately 40% of chronic idiopathic urticaria (CIU) patients. CIU patients' basophils display distinct altered Fc epsilonRIalpha-mediated degranulation. CIU patients with basophil histamine release in response to polyclonal goat anti-human IgE > or = 10% are classified as CIU responders (CIU-R) and < 10% are CIU non-responders (CIU-NR). We compared the presence of autoantibodies to basophil degranulation phenotypes and to disease status (active or inactive). Sera were collected from non-CIU subjects and CIU subjects who participated in a longitudinal study of disease severity and had defined basophil degranulation phenotypes. Immunoenzymetric assays (IEMA) quantified IgG anti-Fc epsilonRIalpha and anti-IgE. IgG anti-Fc epsilonRIalpha antibody was detected in 57% of CIU-R (n=35), 55% of CIU-NR (n=29), and 57% of non-CIU subjects (n=23), whereas IgG anti-IgE was present in 43% of CIU-R, 45% of CIU-NR, and 30% of non-CIU subjects. Both the autoantibody levels and the functional basophil phenotype remained stable in subjects with active disease (n=16), whereas there was an enhancement in basophil function as subjects evolved into a state of remission (n=6), which appears independent of the presence of autoantibody. IEMAs detected a similar frequency of autoantibodies in CIU-R, CIU-NR, and non-CIU subjects. Basophil function may be independent of IEMA-detected autoantibodies. PMID:18356810

  7. Integrated phenotypic and activity-based profiling links Ces3 to obesity and diabetes

    PubMed Central

    Dominguez, Eduardo; Galmozzi, Andrea; Chang, Jae Won; Hsu, Ku-Lung; Pawlak, Joanna; Li, Weiwei; Godio, Cristina; Thomas, Jason; Partida, David; Niessen, Sherry; O'Brien, Paul E.; Russell, Aaron P.; Watt, Matthew J.; Nomura, Daniel K.; Cravatt, Benjamin F.; Saez, Enrique

    2014-01-01

    Phenotypic screening is making a comeback in drug discovery as the maturation of chemical proteomics methods has facilitated target identification for bioactive small molecules. A limitation of these approaches is that time-consuming genetic methods or other means is often required to determine the biologically relevant target(s) from among multiple protein-compound interactions that are typically detected. Here, we have combined phenotypic screening of a directed small-molecule library with competitive activity-based protein profiling to map and functionally characterize the targets of screening hits. Using this approach, we identify carboxylesterase 3 (Ces3 or Ces1d) as a primary molecular target of bioactive compounds that promote lipid storage in adipocytes. We further show that Ces3 activity is dramatically elevated during adipocyte differentiation. Treatment of two mouse models of obesity-diabetes with a Ces3 inhibitor ameliorates multiple features of metabolic syndrome, illustrating the power of the described strategy to accelerate the identification and pharmacologic validation of new therapeutic targets. PMID:24362705

  8. Evaluation of Proposed In Vivo Probe Substrates and Inhibitors for Phenotyping Transporter Activity in Humans.

    PubMed

    Momper, Jeremiah D; Tsunoda, Shirley M; Ma, Joseph D

    2016-07-01

    Drug transporters are present in various tissues and have a significant role in drug absorption, distribution, and elimination. The International Transporter Consortium has identified 7 transporters of increasing importance from evidence of clinically significant transporter-mediated drug-drug interactions. The transporters are P-glycoprotein, breast cancer resistance protein, organic anion transporting polypeptide (OATP) 1B1, OATP1B3, organic cation transporter 2, organic anion transporters (OAT) 1, and OAT3. Decision trees were created based on in vitro experiments to determine whether an in vivo transporter-mediated drug-drug interaction study is needed. Phenotyping is a methodology that evaluates real-time in vivo transporter activity, whereby changes in a probe substrate or probe inhibitor reflect alternations in the activity of the specified transporter. In vivo probe substrates and/or probe inhibitors have been proposed for each aforementioned transporter. In vitro findings and animal models provide the strongest evidence regarding probe specificity. However, such findings have not conclusively correlated with human phenotyping studies. Furthermore, the extent of contribution from multiple transporters in probe disposition complicates the ability to discern if study findings are the result of a specific transporter and thus provide a recommendation for a preferred probe for a drug transporter. PMID:27385182

  9. Type I interferons produced by dendritic cells promote their phenotypic and functional activation.

    PubMed

    Montoya, Maria; Schiavoni, Giovanna; Mattei, Fabrizio; Gresser, Ion; Belardelli, Filippo; Borrow, Persephone; Tough, David F

    2002-05-01

    Resting dendritic cells (DCs) are resident in most tissues and can be activated by environmental stimuli to mature into potent antigen-presenting cells. One important stimulus for DC activation is infection; DCs can be triggered through receptors that recognize microbial components directly or by contact with infection-induced cytokines. We show here that murine DCs undergo phenotypic maturation upon exposure to type I interferons (type I IFNs) in vivo or in vitro. Moreover, DCs either derived from bone marrow cells in vitro or isolated from the spleens of normal animals express IFN-alpha and IFN-beta, suggesting that type I IFNs can act in an autocrine manner to activate DCs. Consistent with this idea, the ability to respond to type I IFN was required for the generation of fully activated DCs from bone marrow precursors, as DCs derived from the bone marrow of mice lacking a functional receptor for type I IFN had reduced expression of costimulatory and adhesion molecules and a diminished ability to stimulate naive T-cell proliferation compared with DCs derived from control bone marrow. Furthermore, the addition of neutralizing anti-IFN-alpha/beta antibody to purified splenic DCs in vitro partially blocked the "spontaneous" activation of these cells, inhibiting the up-regulation of costimulatory molecules, secretion of IFN-gamma, and T-cell stimulatory activity. These results show that DCs both secrete and respond to type I IFN, identifying type I interferons as autocrine DC activators. PMID:11964292

  10. The mechanisms of HSC activation and epigenetic regulation of HSCs phenotypes

    PubMed Central

    Page, Agata; Mann, Derek A.; Mann, Jelena

    2016-01-01

    Epigenetics is a dynamically expanding field of science entailing numerous regulatory mechanisms controlling changes of gene expression in response to environmental factors. Over the recent years there has been a great interest in epigenetic marks as a potential diagnostic and prognostic tool or future target for treatment of various human diseases. There is an increasing body of published research to suggest that epigenetic events regulate progression of chronic liver disease. Experimental manipulation of epigenetic signatures such as DNA methylation, histone acetylation / methylation and the activities of proteins that either annotate or interpret these epigenetic marks can have profound effects on the activation and phenotype of HSC, key cells responsible for onset and progression of liver fibrosis. This review presents recent advances in epigenetic alterations, which could provide mechanistic insight into the pathogenesis of chronic liver disease and provide novel clinical applications.

  11. Xuebijing Injection Promotes M2 Polarization of Macrophages and Improves Survival Rate in Septic Mice

    PubMed Central

    Liu, Yan-Cun; Yao, Feng-Hua; Chai, Yan-Fen; Dong, Ning; Sheng, Zhi-Yong; Yao, Yong-Ming

    2015-01-01

    Xuebijing (XBJ) injection, a concoction of several Chinese herbs, has been widely used as an immunomodulator for the treatment of severe sepsis in China. However, the precise mechanisms responsible for its efficacy have not been fully elucidated. In our study, we determined the flow cytometry markers (F4/80, CD11c, and CD206), the levels of secreted cytokines (TNF-α, IL-6, and IL-10), and the expression of specific proteins of M2 (Ym1, Fizz1, and Arg1) to assess macrophage polarization. Treatment with XBJ lowered M1 associated cytokine levels and increased the level of M2 associated cytokine level. The percentage of M2 phenotype cells of XBJ group was much higher than that of the control group. Expressions of phosphorylated Janus kinase 1 (JAK1) and signal transducer and activator of transcription 6 (STAT6) were markedly enhanced after the administration of XBJ; on the other hand, the M2 associated cytokines and proteins were decreased following treatment with JAK1 or STAT6 inhibitor. In addition, the treatment of XBJ significantly improved the survival rate of septic mice. These studies demonstrate that XBJ can markedly promote M2 polarization and improve the survival rate of septic mice, thereby contributing to therapeutic effect in the treatment of septic complications. PMID:26064161

  12. Retention of sedentary obese visceral white adipose tissue phenotype with intermittent physical activity despite reduced adiposity.

    PubMed

    Wainright, Katherine S; Fleming, Nicholas J; Rowles, Joe L; Welly, Rebecca J; Zidon, Terese M; Park, Young-Min; Gaines, T'Keaya L; Scroggins, Rebecca J; Anderson-Baucum, Emily K; Hasty, Alyssa H; Vieira-Potter, Victoria J; Padilla, Jaume

    2015-09-01

    Regular physical activity is effective in reducing visceral white adipose tissue (AT) inflammation and oxidative stress, and these changes are commonly associated with reduced adiposity. However, the impact of multiple periods of physical activity, intercalated by periods of inactivity, i.e., intermittent physical activity, on markers of AT inflammation and oxidative stress is unknown. In the present study, 5-wk-old male C57BL/6 mice were randomized into three groups (n = 10/group): sedentary, regular physical activity, and intermittent physical activity, for 24 wk. All animals were singly housed and fed a diet containing 45% kcal from fat. Regularly active mice had access to voluntary running wheels throughout the study period, whereas intermittently active mice had access to running wheels for 3-wk intervals (i.e., 3 wk on/3 wk off) throughout the study. At death, regular and intermittent physical activity was associated with similar reductions in visceral AT mass (approximately -24%, P < 0.05) relative to sedentary. However, regularly, but not intermittently, active mice exhibited decreased expression of visceral AT genes related to inflammation (e.g., monocyte chemoattractant protein 1), immune cell infiltration (e.g., CD68, CD11c, F4/80, CD11b/CD18), oxidative stress (e.g., p47 phagocyte oxidase), and endoplasmic reticulum stress (e.g., CCAAT enhancer-binding protein homologous protein; all P < 0.05). Furthermore, regular, but not intermittent, physical activity was associated with a trend toward improvement in glucose tolerance (P = 0.059). Collectively, these findings suggest that intermittent physical activity over a prolonged period of time may lead to a reduction in adiposity but with retention of a sedentary obese white AT and metabolic phenotype. PMID:26180183

  13. M2 polarization enhances silica nanoparticle uptake by macrophages.

    PubMed

    Hoppstädter, Jessica; Seif, Michelle; Dembek, Anna; Cavelius, Christian; Huwer, Hanno; Kraegeloh, Annette; Kiemer, Alexandra K

    2015-01-01

    While silica nanoparticles have enabled numerous industrial and medical applications, their toxicological safety requires further evaluation. Macrophages are the major cell population responsible for nanoparticle clearance in vivo. The prevailing macrophage phenotype largely depends on the local immune status of the host. Whereas M1-polarized macrophages are considered as pro-inflammatory macrophages involved in host defense, M2 macrophages exhibit anti-inflammatory and wound-healing properties, but also promote tumor growth. We employed different models of M1 and M2 polarization: granulocyte-macrophage colony-stimulating factor/lipopolysaccharide (LPS)/interferon (IFN)-γ was used to generate primary human M1 cells and macrophage colony-stimulating factor (M-CSF)/interleukin (IL)-10 to differentiate M2 monocyte-derived macrophages (MDM). PMA-differentiated THP-1 cells were polarized towards an M1 type by LPS/IFN-γ and towards M2 by IL-10. Uptake of fluorescent silica nanoparticles (Ø26 and 41 nm) and microparticles (Ø1.75 μm) was quantified. At the concentration used (50 μg/ml), silica nanoparticles did not influence cell viability as assessed by MTT assay. Nanoparticle uptake was enhanced in M2-polarized primary human MDM compared with M1 cells, as shown by flow cytometric and microscopic approaches. In contrast, the uptake of microparticles did not differ between M1 and M2 phenotypes. M2 polarization was also associated with increased nanoparticle uptake in the macrophage-like THP-1 cell line. In accordance, in vivo polarized M2-like primary human tumor-associated macrophages obtained from lung tumors took up more nanoparticles than M1-like alveolar macrophages isolated from the surrounding lung tissue. In summary, our data indicate that the M2 polarization of macrophages promotes nanoparticle internalization. Therefore, the phenotypical differences between macrophage subsets should be taken into consideration in future investigations on nanosafety, but

  14. M2 polarization enhances silica nanoparticle uptake by macrophages

    PubMed Central

    Hoppstädter, Jessica; Seif, Michelle; Dembek, Anna; Cavelius, Christian; Huwer, Hanno; Kraegeloh, Annette; Kiemer, Alexandra K.

    2015-01-01

    While silica nanoparticles have enabled numerous industrial and medical applications, their toxicological safety requires further evaluation. Macrophages are the major cell population responsible for nanoparticle clearance in vivo. The prevailing macrophage phenotype largely depends on the local immune status of the host. Whereas M1-polarized macrophages are considered as pro-inflammatory macrophages involved in host defense, M2 macrophages exhibit anti-inflammatory and wound-healing properties, but also promote tumor growth. We employed different models of M1 and M2 polarization: granulocyte-macrophage colony-stimulating factor/lipopolysaccharide (LPS)/interferon (IFN)-γ was used to generate primary human M1 cells and macrophage colony-stimulating factor (M-CSF)/interleukin (IL)-10 to differentiate M2 monocyte-derived macrophages (MDM). PMA-differentiated THP-1 cells were polarized towards an M1 type by LPS/IFN-γ and towards M2 by IL-10. Uptake of fluorescent silica nanoparticles (Ø26 and 41 nm) and microparticles (Ø1.75 μm) was quantified. At the concentration used (50 μg/ml), silica nanoparticles did not influence cell viability as assessed by MTT assay. Nanoparticle uptake was enhanced in M2-polarized primary human MDM compared with M1 cells, as shown by flow cytometric and microscopic approaches. In contrast, the uptake of microparticles did not differ between M1 and M2 phenotypes. M2 polarization was also associated with increased nanoparticle uptake in the macrophage-like THP-1 cell line. In accordance, in vivo polarized M2-like primary human tumor-associated macrophages obtained from lung tumors took up more nanoparticles than M1-like alveolar macrophages isolated from the surrounding lung tissue. In summary, our data indicate that the M2 polarization of macrophages promotes nanoparticle internalization. Therefore, the phenotypical differences between macrophage subsets should be taken into consideration in future investigations on nanosafety, but

  15. 5-Azacytidine Promotes an Inhibitory T-Cell Phenotype and Impairs Immune Mediated Antileukemic Activity

    PubMed Central

    Stübig, Thomas; Luetkens, Tim; Hildebrandt, York; Atanackovic, Djordje; Binder, Thomas M. C.; Fehse, Boris; Kröger, Nicolaus

    2014-01-01

    Demethylating agent, 5-Azacytidine (5-Aza), has been shown to be active in treatment of myeloid malignancies. 5-Aza enhances anticancer immunity, by increasing expression of tumor-associated antigens. However, the impact of 5-Aza immune responses remains poorly understood. Here, T-cell mediated tumor immunity effects of 5-Aza, are investigated in vitro and in vivo. T-cells from healthy donors were treated with 5-Aza and analyzed by qRT-PCR and flow cytometry for changes in gene expression and phenotype. Functionality was assessed by a tumor lysis assay. Peripheral blood from patients treated with 5-Aza after alloSCT was monitored for changes in T-cell subpopulations. 5-Aza treatment resulted in a decrease in CD8+ T-cells, whereas CD4+ T-cells increased. Furthermore, numbers of IFN-γ+ T-helper 1 cells (Th1) were reduced, while Treg-cells showed substantial increase. Additionally, CD8+ T-cells exhibited limited killing capacity against leukemic target cells. In vivo data confirm the increase of Treg compartment, while CD8+ T-effector cell numbers were reduced. 5-Aza treatment results in a shift from cytotoxic to regulatory T-cells with a functional phenotype and a major reduction in proinflammatory Th1-cells, indicating a strong inhibition of tumor-specific T-cell immunity by 5-Aza. PMID:24757283

  16. 5-azacytidine promotes an inhibitory T-cell phenotype and impairs immune mediated antileukemic activity.

    PubMed

    Stübig, Thomas; Badbaran, Anita; Luetkens, Tim; Hildebrandt, York; Atanackovic, Djordje; Binder, Thomas M C; Fehse, Boris; Kröger, Nicolaus

    2014-01-01

    Demethylating agent, 5-Azacytidine (5-Aza), has been shown to be active in treatment of myeloid malignancies. 5-Aza enhances anticancer immunity, by increasing expression of tumor-associated antigens. However, the impact of 5-Aza immune responses remains poorly understood. Here, T-cell mediated tumor immunity effects of 5-Aza, are investigated in vitro and in vivo. T-cells from healthy donors were treated with 5-Aza and analyzed by qRT-PCR and flow cytometry for changes in gene expression and phenotype. Functionality was assessed by a tumor lysis assay. Peripheral blood from patients treated with 5-Aza after alloSCT was monitored for changes in T-cell subpopulations. 5-Aza treatment resulted in a decrease in CD8+ T-cells, whereas CD4+ T-cells increased. Furthermore, numbers of IFN-γ + T-helper 1 cells (Th1) were reduced, while Treg-cells showed substantial increase. Additionally, CD8+ T-cells exhibited limited killing capacity against leukemic target cells. In vivo data confirm the increase of Treg compartment, while CD8+ T-effector cell numbers were reduced. 5-Aza treatment results in a shift from cytotoxic to regulatory T-cells with a functional phenotype and a major reduction in proinflammatory Th1-cells, indicating a strong inhibition of tumor-specific T-cell immunity by 5-Aza. PMID:24757283

  17. GABA-mediated regulation of the activity-dependent olfactory bulb dopaminergic phenotype

    PubMed Central

    Akiba, Yosuke; Sasaki, Hayato; Huerta, Patricio T.; Estevez, Alvaro G.; Baker, Harriet; Cave, John W.

    2009-01-01

    Gamma-amino-butyric acid (GABA) regulates the proliferation and migration of olfactory bulb (OB) interneuron progenitors derived from the subventricular zone (SVZ), but the role of GABA in the differentiation of these progenitors has been largely unexplored. This study examined the role of GABA in the differentiation of OB dopaminergic interneurons using neonatal forebrain organotypic slice cultures prepared from transgenic mice expressing GFP under the control of the tyrosine hydroxylase (Th) gene promoter (ThGFP). KCl-mediated depolarization of the slices induced ThGFP expression. The addition of GABA to the depolarized slices further increased GFP fluorescence by inducing ThGFP expression in an additional set of periglomerular cells. These findings showed that GABA promoted differentiation of SVZ-derived OB dopaminergic interneurons and suggested that GABA indirectly regulated Th expression and OB dopaminergic neuron differentiation through an acceleration of the maturation rate for the dopaminergic progenitors. Additional studies revealed that the effect of GABA on ThGFP expression required activation of L- and P/Q-type Ca+2 channels as well as GABAA and GABAB receptors. These voltage-gated Ca+2 channels and GABA receptors have previously been shown to be required for the co-expressed GABAergic phenotype in the OB interneurons. Together, these findings suggest that Th expression and the differentiation of OB dopaminergic interneurons are coupled to the co-expressed GABAergic phenotype, and demonstrate a novel role for GABA in neurogenesis. PMID:19301430

  18. Neisseria gonorrhoeae Modulates Immunity by Polarizing Human Macrophages to a M2 Profile.

    PubMed

    Ortiz, María Carolina; Lefimil, Claudia; Rodas, Paula I; Vernal, Rolando; Lopez, Mercedes; Acuña-Castillo, Claudio; Imarai, Mónica; Escobar, Alejandro

    2015-01-01

    Current data suggest that Neisseria gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and antigen-presenting cells. The present report is focused on gonococcus evasion mechanism on macrophages (MФ) and its impact in the subsequent immune response. In response to various signals MФ may undergo classical-M1 (M1-MФ) or alternative-M2 (M2-MФ) activation. Until now there are no reports of the gonococcus effects on human MФ polarization. We assessed the phagocytic ability of monocyte-derived MФ (MDM) upon gonococcal infection by immunofluorescence and gentamicin protection experiments. Then, we evaluated cytokine profile and M1/M2 specific-surface markers on MФ challenged with N. gonorrhoeae and their proliferative effect on T cells. Our findings lead us to suggest N. gonorrhoeae stimulates a M2-MФ phenotype in which some of the M2b and none of the M1-MФ-associated markers are induced. Interestingly, N. gonorrhoeae exposure leads to upregulation of a Programmed Death Ligand 1 (PD-L1), widely known as an immunosuppressive molecule. Moreover, functional results showed that N. gonorrhoeae-treated MФ are unable to induce proliferation of human T-cells, suggesting a more likely regulatory phenotype. Taken together, our data show that N. gonorroheae interferes with MФ polarization. This study has important implications for understanding the mechanisms of clearance versus long-term persistence of N. gonorroheae infection and might be applicable for the development of new therapeutic strategies. PMID:26125939

  19. Neisseria gonorrhoeae Modulates Immunity by Polarizing Human Macrophages to a M2 Profile

    PubMed Central

    Ortiz, María Carolina; Lefimil, Claudia; Rodas, Paula I.; Vernal, Rolando; Lopez, Mercedes; Acuña-Castillo, Claudio; Imarai, Mónica; Escobar, Alejandro

    2015-01-01

    Current data suggest that Neisseria gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and antigen-presenting cells. The present report is focused on gonococcus evasion mechanism on macrophages (MФ) and its impact in the subsequent immune response. In response to various signals MФ may undergo classical-M1 (M1-MФ) or alternative-M2 (M2-MФ) activation. Until now there are no reports of the gonococcus effects on human MФ polarization. We assessed the phagocytic ability of monocyte-derived MФ (MDM) upon gonococcal infection by immunofluorescence and gentamicin protection experiments. Then, we evaluated cytokine profile and M1/M2 specific-surface markers on MФ challenged with N. gonorrhoeae and their proliferative effect on T cells. Our findings lead us to suggest N. gonorrhoeae stimulates a M2-MФ phenotype in which some of the M2b and none of the M1-MФ-associated markers are induced. Interestingly, N. gonorrhoeae exposure leads to upregulation of a Programmed Death Ligand 1 (PD-L1), widely known as an immunosuppressive molecule. Moreover, functional results showed that N. gonorrhoeae-treated MФ are unable to induce proliferation of human T-cells, suggesting a more likely regulatory phenotype. Taken together, our data show that N. gonorroheae interferes with MФ polarization. This study has important implications for understanding the mechanisms of clearance versus long-term persistence of N. gonorroheae infection and might be applicable for the development of new therapeutic strategies. PMID:26125939

  20. A monolithic glass chip for active single-cell sorting based on mechanical phenotyping.

    PubMed

    Faigle, Christoph; Lautenschläger, Franziska; Whyte, Graeme; Homewood, Philip; Martín-Badosa, Estela; Guck, Jochen

    2015-03-01

    The mechanical properties of biological cells have long been considered as inherent markers of biological function and disease. However, the screening and active sorting of heterogeneous populations based on serial single-cell mechanical measurements has not been demonstrated. Here we present a novel monolithic glass chip for combined fluorescence detection and mechanical phenotyping using an optical stretcher. A new design and manufacturing process, involving the bonding of two asymmetrically etched glass plates, combines exact optical fiber alignment, low laser damage threshold and high imaging quality with the possibility of several microfluidic inlet and outlet channels. We show the utility of such a custom-built optical stretcher glass chip by measuring and sorting single cells in a heterogeneous population based on their different mechanical properties and verify sorting accuracy by simultaneous fluorescence detection. This offers new possibilities of exact characterization and sorting of small populations based on rheological properties for biological and biomedical applications. PMID:25537986

  1. Antimicrobial Activity of Gallium Protoporphyrin IX against Acinetobacter baumannii Strains Displaying Different Antibiotic Resistance Phenotypes

    PubMed Central

    Arivett, Brock A.; Fiester, Steven E.; Ohneck, Emily J.; Penwell, William F.; Kaufman, Cynthia M.; Relich, Ryan F.

    2015-01-01

    A paucity of effective, currently available antibiotics and a lull in antibiotic development pose significant challenges for treatment of patients with multidrug-resistant (MDR) Acinetobacter baumannii infections. Thus, novel therapeutic strategies must be evaluated to meet the demands of treatment of these often life-threatening infections. Accordingly, we examined the antibiotic activity of gallium protoporphyrin IX (Ga-PPIX) against a collection of A. baumannii strains, including nonmilitary and military strains and strains representing different clonal lineages and isolates classified as susceptible or MDR. Susceptibility testing demonstrated that Ga-PPIX inhibits the growth of all tested strains when cultured in cation-adjusted Mueller-Hinton broth, with a MIC of 20 μg/ml. This concentration significantly reduced bacterial viability, while 40 μg/ml killed all cells of the A. baumannii ATCC 19606T and ACICU MDR isolate after 24-h incubation. Recovery of ATCC 19606T and ACICU strains from infected A549 human alveolar epithelial monolayers was also decreased when the medium was supplemented with Ga-PPIX, particularly at a 40-μg/ml concentration. Similarly, the coinjection of bacteria with Ga-PPIX increased the survival of Galleria mellonella larvae infected with ATCC 19606T or ACICU. Ga-PPIX was cytotoxic only when monolayers or larvae were exposed to concentrations 16-fold and 1,250-fold higher than those showing antibacterial activity, respectively. These results indicate that Ga-PPIX could be a viable therapeutic option for treatment of recalcitrant A. baumannii infections regardless of the resistance phenotype, clone lineage, time and site of isolation of strains causing these infections and their iron uptake phenotypes or the iron content of the media. PMID:26416873

  2. The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer.

    PubMed

    Hartung, Anne-Mette; Swensen, Jeff; Uriz, Inaki E; Lapin, Morten; Kristjansdottir, Karen; Petersen, Ulrika S S; Bang, Jeanne Mari V; Guerra, Barbara; Andersen, Henriette Skovgaard; Dobrowolski, Steven F; Carey, John C; Yu, Ping; Vaughn, Cecily; Calhoun, Amy; Larsen, Martin R; Dyrskjøt, Lars; Stevenson, David A; Andresen, Brage S

    2016-05-01

    Costello syndrome (CS) may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE) and creation of an Exonic Splicing Silencer (ESS). We show that this vulnerability of HRAS exon 2 is caused by a weak 3' splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO) that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping. PMID:27195699

  3. The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer

    PubMed Central

    Kristjansdottir, Karen; Petersen, Ulrika S. S.; Bang, Jeanne Mari V.; Guerra, Barbara; Andersen, Henriette Skovgaard; Dobrowolski, Steven F.; Carey, John C.; Yu, Ping; Calhoun, Amy; Larsen, Martin R.; Dyrskjøt, Lars; Stevenson, David A.; Andresen, Brage S.

    2016-01-01

    Costello syndrome (CS) may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE) and creation of an Exonic Splicing Silencer (ESS). We show that this vulnerability of HRAS exon 2 is caused by a weak 3’ splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO) that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping. PMID:27195699

  4. Pyruvate kinase M2 at a glance

    PubMed Central

    Yang, Weiwei; Lu, Zhimin

    2015-01-01

    Reprogrammed metabolism is a key feature of cancer cells. The pyruvate kinase M2 (PKM2) isoform, which is commonly upregulated in many human cancers, has been recently shown to play a crucial role in metabolism reprogramming, gene transcription and cell cycle progression. In this Cell Science at a glance article and accompanying poster, we provide a brief overview of recent advances in understanding the mechanisms underlying the regulation of PKM2 expression, enzymatic activity, metabolic functions and subcellular location. We highlight the instrumental role of the non-metabolic functions of PKM2 in tumorigenesis and evaluate the potential to target PKM2 for cancer treatment. PMID:25770102

  5. XPD Helicase Structures and Activities: Insights into the Cancer and Aging Phenotypes from XPD Mutations

    SciTech Connect

    Tainer, John; Fan, Li; Fuss, Jill O.; Cheng, Quen J.; Arvai, Andrew S.; Hammel, Michal; Roberts, Victoria A.; Cooper, Priscilla K.; Tainer, John A.

    2008-06-02

    Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

  6. XPD Helicase Structures And Activities: Insights Into the Cancer And Aging Phenotypes From XPD Mutations

    SciTech Connect

    Fan, L.; Fuss, J.O.; Cheng, Q.J.; Arvai, A.S.; Hammel, M.; Roberts, V.A.; Cooper, P.K.; Tainer, J.A.

    2009-05-18

    Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

  7. Impaired Peroxisome Proliferator-activated Receptor-γ Contributes to Phenotypic Modulation of Vascular Smooth Muscle Cells during Hypertension*

    PubMed Central

    Zhang, Lili; Xie, Peng; Wang, Jingzhou; Yang, Qingwu; Fang, Chuanqin; Zhou, Shuang; Li, Jingcheng

    2010-01-01

    The phenotypic modulation of vascular smooth muscle cells (VSMCs) plays a pivotal role in hypertension-induced vascular changes including vascular remodeling. The precise mechanisms underlying VSMC phenotypic modulation remain elusive. Here we test the role of peroxisome proliferator-activated receptor (PPAR)-γ in the VSMC phenotypic modulation during hypertension. Both spontaneously hypertensive rat (SHR) aortas and SHR-derived VSMCs exhibited reduced PPAR-γ expression and excessive VSMC phenotypic modulation identified by reduced contractile proteins, α-smooth muscle actin (α-SMA) and smooth muscle 22α (SM22α), and enhanced proliferation and migration. PPAR-γ overexpression rescued the expression of α-SMA and SM22α, and inhibited the proliferation and migration in SHR-derived VSMCs. In contrast, PPAR-γ silencing exerted the opposite effect. Activating PPAR-γ using rosiglitazone in vivo up-regulated aortic α-SMA and SM22α expression and attenuated aortic remodeling in SHRs. Increased activation of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling was observed in SHR-derived VSMCs. PI3K inhibitor LY294002 rescued the impaired expression of contractile proteins, and inhibited proliferation and migration in VSMCs from SHRs, whereas constitutively active PI3K mutant had the opposite effect. Overexpression or silencing of PPAR-γ inhibited or excited PI3K/Akt activity, respectively. LY294002 counteracted the PPAR-γ silencing induced proliferation and migration in SHR-derived VSMCs, whereas active PI3K mutant had the opposite effect. In contrast, reduced proliferation and migration by PPAR-γ overexpression were reversed by the active PI3K mutant, and further inhibited by LY294002. We conclude that PPAR-γ inhibits VSMC phenotypic modulation through inhibiting PI3K/Akt signaling. Impaired PPAR-γ expression is responsible for VSMC phenotypic modulation during hypertension. These findings highlight an attractive therapeutic target for

  8. M1 and M2 macrophage proteolytic and angiogenic profile analysis in atherosclerotic patients reveals a distinctive profile in type 2 diabetes.

    PubMed

    Roma-Lavisse, Charlotte; Tagzirt, Madjid; Zawadzki, Christophe; Lorenzi, Rodrigo; Vincentelli, André; Haulon, Stephan; Juthier, Francis; Rauch, Antoine; Corseaux, Delphine; Staels, Bart; Jude, Brigitte; Van Belle, Eric; Susen, Sophie; Chinetti-Gbaguidi, Giulia; Dupont, Annabelle

    2015-07-01

    This study aimed to investigate atherosclerotic mediators' expression levels in M1 and M2 macrophages and to focus on the influence of diabetes on M1/M2 profiles. Macrophages from 36 atherosclerotic patients (19 diabetics and 17 non-diabetics) were cultured with interleukin-1β (IL-1β) or IL-4 to induce M1 or M2 phenotype, respectively. The atherosclerotic mediators' expression was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed that M1 and M2 macrophages differentially expressed mediators involved in proteolysis and angiogenesis processes. The proteolytic balance (matrix metalloproteinase-9 (MMP-9)/tissue inhibitor of metalloproteinase-1 (TIMP-1), MMP-9/plasminogen activator inhibitor-1 (PAI-1) and MMP-9/tissue factor pathway inhibitor-2 (TFPI-2) ratios) was higher in M1 versus M2, whereas M2 macrophages presented higher angiogenesis properties (increased vascular endothelial growth factor/TFPI-2 and tissue factor/TFPI-2 ratios). Moreover, M1 macrophages from diabetics displayed more important proangiogenic and proteolytic activities than non-diabetics. This study reveals that M1 and M2 macrophages could differentially modulate major atherosclerosis-related pathological processes. Moreover, M1 macrophages from diabetics display a deleterious phenotype that could explain the higher plaque vulnerability observed in these subjects. PMID:25966737

  9. Phenotype Specific Analyses Reveal Distinct Regulatory Mechanism for Chronically Activated p53

    PubMed Central

    Cairns, Jonathan M.; Menon, Suraj; Pérez-Mancera, Pedro A.; Tomimatsu, Kosuke; Bermejo-Rodriguez, Camino; Ito, Yoko; Chandra, Tamir; Narita, Masako; Lyons, Scott K.; Lynch, Andy G.; Kimura, Hiroshi; Ohbayashi, Tetsuya; Tavaré, Simon; Narita, Masashi

    2015-01-01

    The downstream functions of the DNA binding tumor suppressor p53 vary depending on the cellular context, and persistent p53 activation has recently been implicated in tumor suppression and senescence. However, genome-wide information about p53-target gene regulation has been derived mostly from acute genotoxic conditions. Using ChIP-seq and expression data, we have found distinct p53 binding profiles between acutely activated (through DNA damage) and chronically activated (in senescent or pro-apoptotic conditions) p53. Compared to the classical ‘acute’ p53 binding profile, ‘chronic’ p53 peaks were closely associated with CpG-islands. Furthermore, the chronic CpG-island binding of p53 conferred distinct expression patterns between senescent and pro-apoptotic conditions. Using the p53 targets seen in the chronic conditions together with external high-throughput datasets, we have built p53 networks that revealed extensive self-regulatory ‘p53 hubs’ where p53 and many p53 targets can physically interact with each other. Integrating these results with public clinical datasets identified the cancer-associated lipogenic enzyme, SCD, which we found to be directly repressed by p53 through the CpG-island promoter, providing a mechanistic link between p53 and the ‘lipogenic phenotype’, a hallmark of cancer. Our data reveal distinct phenotype associations of chronic p53 targets that underlie specific gene regulatory mechanisms. PMID:25790137

  10. Fluctuation of Rac1 activity is associated with the phenotypic and transcriptional heterogeneity of glioma cells.

    PubMed

    Yukinaga, Hiroko; Shionyu, Clara; Hirata, Eishu; Ui-Tei, Kumiko; Nagashima, Takeshi; Kondo, Shinji; Okada-Hatakeyama, Mariko; Naoki, Honda; Matsuda, Michiyuki

    2014-04-15

    Phenotypic heterogeneity of cancer cells is caused not only by genetic and epigenetic alterations but also by stochastic variation of intracellular signaling molecules. Using cells that stably express Förster resonance energy transfer (FRET) biosensors, we show here a correlation between a temporal fluctuation in the activity of Rac1 and the invasive properties of C6 glioma cells. By using long-term time-lapse imaging, we found that Rac1 activity in C6 glioma cells fluctuated over a timescale that was substantially longer than that of the replication cycle. Because the relative level of Rac1 activity in each cell was unaffected by a suspension-adhesion procedure, we were able to sort C6 glioma cells according to the levels of Rac1 activity, yielding Rac1(high) and Rac1(low) cells. The Rac1(high) cells invaded more efficiently than did Rac1(low) cells in a Matrigel invasion assay. We assessed the transcriptional profiles of Rac1(high) and Rac1(low) cells and performed gene ontology analysis. Among the 14 genes that were most associated with the term 'membrane' (membrane-related genes) in Rac1(high) cells, we identified four genes that were associated with glioma invasion and Rac1 activity by using siRNA knockdown experiments. Among the transcription factors upregulated in Rac1(high) cells, Egr2 was found to positively regulate expression of the four membrane-related invasion-associated genes. The identified signaling network might cause the fluctuations in Rac1 activity and the heterogeneity in the invasive capacity of glioma cells. PMID:24522191

  11. Active Notch1 Confers a Transformed Phenotype to Primary Human Melanocytes

    PubMed Central

    Pinnix, Chelsea C.; Lee, John T.; Liu, Zhao-Jun; McDaid, Ronan; Balint, Klara; Beverly, Levi J.; Brafford, Patricia A.; Xiao, Min; Himes, Benjamin; Zabierowski, Susan E.; Yashiro-Ohtani, Yumi; Nathanson, Katherine L.; Bengston, Ana; Pollock, Pamela M.; Weeraratna, Ashani T.; Nickoloff, Brian J.; Pear, Warren S.; Capobianco, Anthony J.; Herlyn, Meenhard

    2009-01-01

    The importance of MAPK signaling in melanoma is underscored by the prevalence of activating mutations in N-Ras and B-Raf; yet, clinical development of inhibitors of this pathway has been largely ineffective, suggesting that alternative oncogenes may also promote melanoma. Notch is an interesting candidate that has only been correlated with melanoma development and progression; a thorough assessment of tumor-initiating effects of activated Notch on human melanocytes would clarify the mounting correlative evidence and perhaps identify a novel target for an otherwise untreatable disease. Analysis of a substantial panel of cell lines and patient lesions demonstrated that Notch activity is significantly higher in melanomas than their non-transformed counterparts. The use of a constitutively-active, truncated Notch transgene construct (NIC) was exploited to determine if Notch activation is a ‘driving’ event in melanocytic transformation or instead a ‘passenger’ event associated with melanoma progression. NIC-infected melanocytes displayed increased proliferative capacity and biological features more reminiscent of melanoma such as dysregulated cell adhesion and migration. Gene expression analyses supported these observations and aided in the identification of MCAM, an adhesion molecule associated with acquisition of the malignant phenotype, as a direct target of Notch transactivation. NIC-positive melanocytes grew at clonal density, proliferated in limiting media conditions, and also exhibited anchorage-independent growth suggesting that Notch, alone, is a transforming oncogene in human melanocytes, a phenomenon not previously described for any melanoma oncogene; this new information yields valuable insight into the basic epidemiology of melanoma and launches a realm of possibilities for drug intervention in this deadly disease. PMID:19549918

  12. Expansion of highly activated invariant natural killer T cells with altered phenotype in acute dengue infection.

    PubMed

    Kamaladasa, A; Wickramasinghe, N; Adikari, T N; Gomes, L; Shyamali, N L A; Salio, M; Cerundolo, V; Ogg, G S; Malavige, G Neelika

    2016-08-01

    Invariant natural killer T (iNKT) cells are capable of rapid activation and production of cytokines upon recognition of antigenic lipids presented by CD1d molecules. They have been shown to play a significant role in many viral infections and were observed to be highly activated in patients with acute dengue infection. In order to characterize further their role in dengue infection, we investigated the proportion of iNKT cells and their phenotype in adult patients with acute dengue infection. The functionality of iNKT cells in patients was investigated by both interferon (IFN)-γ and interleukin (IL)-4 ex-vivo enzyme-linked immunospot (ELISPOT) assays following stimulation with alpha-galactosyl-ceramide (αGalCer). We found that circulating iNKT cell proportions were significantly higher (P = 0·03) in patients with acute dengue when compared to healthy individuals and were predominantly of the CD4(+) subset. iNKT cells of patients with acute dengue had reduced proportions expressing CD8α and CD161 when compared to healthy individuals. The iNKT cells of patients were highly activated and iNKT activation correlated significantly with dengue virus-specific immunoglobulin (Ig)G antibody levels. iNKT cells expressing Bcl-6 (P = 0·0003) and both Bcl-6 and inducible T cell co-stimulator (ICOS) (P = 0·006) were increased significantly in patients when compared to healthy individuals. Therefore, our data suggest that in acute dengue infection there is an expansion of highly activated CD4(+) iNKT cells, with reduced expression of CD161 markers. PMID:26874822

  13. Refined mapping of the G[sub M2] activator protein (GM2A) locus to 5q31. 3-q33. 1, distal to the spinal muscular atrophy locus

    SciTech Connect

    Heng, H.H.Q.; Xie, B.; Shi, X.M.; Tsui, L.C.; Mahuran, D.J. )

    1993-11-01

    The G[sub M2] activator locus (GM2A) had previously been considered as a candidate gene for some forms of spinal muscular atrophy (SMA; mapped to 5q11.2-q13.3). It was eliminated as a possible candidate because PCR-based mapping failed to localize the gene to chromosome 5, as was previously reported using an ELISA-based methodology. However, the authors demonstrated that the PCR primers used preferentially amplified a processed pseudogene (GM2AP) that was mapped to chromosome 3 and that GM2A was located on chromosome 5. In this report, they reconsider the candidacy of GM2A by refining its localization on chromosome 5 using fluorescence in situ hybridization. They localize GM2A to 5q31.3-q33.1; thus, it is not a candidate gene for SMA. 11 refs., 2 figs.

  14. Aldehyde dehydrogenase activity selects for the holoclone phenotype in prostate cancer cells

    SciTech Connect

    Doherty, R.E.; Haywood-Small, S.L.; Sisley, K.; Cross, N.A.

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Isolated ALDH{sup Hi} PC3 cells preferentially form primitive holoclone-type colonies. Black-Right-Pointing-Pointer Primitive holoclone colonies are predominantly ALDH{sup Lo} but contain rare ALDH{sup Hi} cells. Black-Right-Pointing-Pointer Holoclone-forming cells are not restricted to the ALDH{sup Hi} population. Black-Right-Pointing-Pointer ALDH phenotypic plasticity occurs in PC3 cells (ALDH{sup Lo} to ALDH{sup Hi} and vice versa). Black-Right-Pointing-Pointer ALDH{sup Hi} cells are observed but very rare in PC3 spheroids grown in stem cell medium. -- Abstract: Aldehyde dehydrogenase 1 (ALDH) activity is considered to be a marker of cancer stem cells (CSCs) in many tumour models, since these cells are more proliferative and tumourigenic than ALDH{sup Lo} cells in experimental models. However it is unclear whether all CSC-like cells are within the ALDH{sup Hi} population, or whether all ALDH{sup Hi} cells are highly proliferative and tumourigenic. The ability to establish a stem cell hierarchy in vitro, whereby sub-populations of cells have differing proliferative and differentiation capacities, is an alternate indication of the presence of stem cell-like populations within cell lines. In this study, we have examined the interaction between ALDH status and the ability to establish a stem cell hierarchy in PC3 prostate cancer cells. We demonstrate that PC3 cells contain a stem cell hierarchy, and isolation of ALDH{sup Hi} cells enriches for the most primitive holoclone population, however holoclone formation is not restricted to ALDH{sup Hi} cells. In addition, we show that ALDH activity undergoes phenotypic plasticity, since the ALDH{sup Lo} population can develop ALDH{sup Hi} populations comparable to parental cells within 2 weeks in culture. Furthermore, we show that the majority of ALDH{sup Hi} cells are found within the least primitive paraclone population, which is circumvented by culturing PC3 cells as spheroids in

  15. A study of the relationship between clinical phenotypes and plasma iduronate-2-sulfatase enzyme activities in Hunter syndrome patients

    PubMed Central

    Lee, Ok Jeong; Kim, Su-Jin; Sohn, Young Bae; Park, Hyung-Doo; Lee, Soo-Youn; Kim, Chi-Hwa; Ko, Ah-Ra; Yook, Yeon-Joo; Lee, Su-Jin; Park, Sung Won; Kim, Se-Hwa; Cho, Sung-Yoon; Kwon, Eun-Kyung; Han, Sun Ju

    2012-01-01

    Purpose Mucopolysaccharidosis type II (MPS II or Hunter syndrome) is a rare lysosomal storage disorder caused by iduronate-2-sulfatase (IDS) deficiency. MPS II causes a wide phenotypic spectrum of symptoms ranging from mild to severe. IDS activity, which is measured in leukocyte pellets or fibroblasts, was reported to be related to clinical phenotype by Sukegawa-Hayasaka et al. Measurement of residual plasma IDS activity using a fluorometric assay is simpler than conventional measurements using skin fibroblasts or peripheral blood mononuclear cells. This is the first study to describe the relationship between plasma IDS activity and clinical phenotype of MPS II. Methods We hypothesized that residual plasma IDS activity is related to clinical phenotype. We classified 43 Hunter syndrome patients as having attenuated or severe disease types based on clinical characteristics, especially intellectual and cognitive status. There were 27 patients with the severe type and 16 with the attenuated type. Plasma IDS activity was measured by a fluorometric enzyme assay using 4-methylumbelliferyl-α-iduronate 2-sulphate. Results Plasma IDS activity in patients with the severe type was significantly lower than that in patients with the attenuated type (P=0.006). The optimal cut-off value of plasma IDS activity for distinguishing the severe type from the attenuated type was 0.63 nmol·4 hr-1·mL-1. This value had 88.2% sensitivity, 65.4% specificity, and an area under receiver-operator characteristics (ROC) curve of 0.768 (ROC curve analysis; P=0.003). Conclusion These results show that the mild phenotype may be related to residual lysosomal enzyme activity. PMID:22474463

  16. Polarized M2 macrophages in dogs with visceral leishmaniasis.

    PubMed

    Moreira, Pamela Rodrigues Reina; Fernando, Filipe Santos; Montassier, Hélio José; André, Marcos Rogério; de Oliveira Vasconcelos, Rosemeri

    2016-08-15

    The objective of the present study was to analyze the skin (nasal surface and ear regions), lymph nodes (popliteal and pre-scapular), spleen and liver of dogs with visceral leishmaniasis (VL), in order to investigate the relationship between the parasite load measured as DNA copy number of Alpha gene of DNA polymerase of Leishmania infantum by quantitative PCR and the number of M2 macrophages by immunohistochemistry. A set of 29 naturally infected dogs from an endemic area for VL were sampled and another set of six dogs negative for VL and from a non-endemic area were analyzed as the control group (C). The spleen presented the highest number of Leishmania DNA copies, with significant differences between the groups G1 and G2 (with and without skin lesions, respectively). The M2 phenotype immunostaining predominated among the macrophages in granulomas and inflammatory infiltrates of samples from the skin, lymph nodes and spleens examined. The presence of M2 macrophages in dogs from infected group differed significantly from the control group, in all organs analyzed, excepted liver. The highest proportion of M2 macrophages coincided with the highest parasitism loads found in more susceptible organs of VL dogs, even in the skin, considered a more resistant organ, while the liver showed low parasitism load and low immunostaining for M2 macrophages with no significant differences between infected and negative groups. It was concluded that the predominance of M2 phenotype in VL dogs favored the multiplication of Leishmania infantum in organs of dogs that are more susceptible to Leishmania infection, as skin, lymph nodes and spleen. PMID:27514887

  17. Genetic contributions to circadian activity rhythm and sleep pattern phenotypes in pedigrees segregating for severe bipolar disorder.

    PubMed

    Pagani, Lucia; St Clair, Patricia A; Teshiba, Terri M; Service, Susan K; Fears, Scott C; Araya, Carmen; Araya, Xinia; Bejarano, Julio; Ramirez, Margarita; Castrillón, Gabriel; Gomez-Makhinson, Juliana; Lopez, Maria C; Montoya, Gabriel; Montoya, Claudia P; Aldana, Ileana; Navarro, Linda; Freimer, Daniel G; Safaie, Brian; Keung, Lap-Woon; Greenspan, Kiefer; Chou, Katty; Escobar, Javier I; Ospina-Duque, Jorge; Kremeyer, Barbara; Ruiz-Linares, Andres; Cantor, Rita M; Lopez-Jaramillo, Carlos; Macaya, Gabriel; Molina, Julio; Reus, Victor I; Sabatti, Chiara; Bearden, Carrie E; Takahashi, Joseph S; Freimer, Nelson B

    2016-02-01

    Abnormalities in sleep and circadian rhythms are central features of bipolar disorder (BP), often persisting between episodes. We report here, to our knowledge, the first systematic analysis of circadian rhythm activity in pedigrees segregating severe BP (BP-I). By analyzing actigraphy data obtained from members of 26 Costa Rican and Colombian pedigrees [136 euthymic (i.e., interepisode) BP-I individuals and 422 non-BP-I relatives], we delineated 73 phenotypes, of which 49 demonstrated significant heritability and 13 showed significant trait-like association with BP-I. All BP-I-associated traits related to activity level, with BP-I individuals consistently demonstrating lower activity levels than their non-BP-I relatives. We analyzed all 49 heritable phenotypes using genetic linkage analysis, with special emphasis on phenotypes judged to have the strongest impact on the biology underlying BP. We identified a locus for interdaily stability of activity, at a threshold exceeding genome-wide significance, on chromosome 12pter, a region that also showed pleiotropic linkage to two additional activity phenotypes. PMID:26712028

  18. Cells transformed by murine herpesvirus 68 (MHV-68) release compounds with transforming and transformed phenotype suppressing activity resembling growth factors.

    PubMed

    Šupolíková, M; Staňová, A Vojs; Kúdelová, M; Marák, J; Zelník, V; Golais, F

    2015-12-01

    In this study, we investigated the medium of three cell lines transformed with murine herpesvirus 68 (MHV-68) in vitro and in vivo, 68/HDF, 68/NIH3T3, and S11E, for the presence of compounds resembling growth factors of some herpesviruses which have displayed transforming and transformed phenotype suppressing activity in normal and tumor cells. When any of spent medium was added to cell culture we observed the onset of transformed phenotype in baby hamster kidney cells (BHK-21) cells and transformed phenotype suppressing activity in tumor human epithelial cells (HeLa). In media tested, we have identified the presence of putative growth factor related to MHV-68 (MHGF-68). Its bivalent properties have been blocked entirely by antisera against MHV-68 and two monoclonal antibodies against glycoprotein B (gB) of MHV-68 suggesting viral origin of MHGF-68. The results of initial efforts to separate MHGF-68 on FPLC Sephadex G15 column in the absence of salts revealed the loss of its transforming activity but transformed phenotype suppressing activity retained. On the other hand, the use of methanol-water mobile phase on RP-HPLC C18 column allowed separation of MHGF-68 to two compounds. Both separated fractions, had only the transforming activity to normal cells. Further experiments exploring the nature and the structure of hitherto unknown MHGF-68 are now in the progress to characterize its molecular and biological properties. PMID:26666191

  19. Genetic contributions to circadian activity rhythm and sleep pattern phenotypes in pedigrees segregating for severe bipolar disorder

    PubMed Central

    Pagani, Lucia; St. Clair, Patricia A.; Teshiba, Terri M.; Service, Susan K.; Fears, Scott C.; Araya, Carmen; Araya, Xinia; Bejarano, Julio; Ramirez, Margarita; Castrillón, Gabriel; Gomez-Makhinson, Juliana; Lopez, Maria C.; Montoya, Gabriel; Montoya, Claudia P.; Aldana, Ileana; Navarro, Linda; Freimer, Daniel G.; Safaie, Brian; Keung, Lap-Woon; Greenspan, Kiefer; Chou, Katty; Escobar, Javier I.; Ospina-Duque, Jorge; Kremeyer, Barbara; Ruiz-Linares, Andres; Cantor, Rita M.; Lopez-Jaramillo, Carlos; Macaya, Gabriel; Molina, Julio; Reus, Victor I.; Sabatti, Chiara; Bearden, Carrie E.; Takahashi, Joseph S.; Freimer, Nelson B.

    2016-01-01

    Abnormalities in sleep and circadian rhythms are central features of bipolar disorder (BP), often persisting between episodes. We report here, to our knowledge, the first systematic analysis of circadian rhythm activity in pedigrees segregating severe BP (BP-I). By analyzing actigraphy data obtained from members of 26 Costa Rican and Colombian pedigrees [136 euthymic (i.e., interepisode) BP-I individuals and 422 non–BP-I relatives], we delineated 73 phenotypes, of which 49 demonstrated significant heritability and 13 showed significant trait-like association with BP-I. All BP-I–associated traits related to activity level, with BP-I individuals consistently demonstrating lower activity levels than their non–BP-I relatives. We analyzed all 49 heritable phenotypes using genetic linkage analysis, with special emphasis on phenotypes judged to have the strongest impact on the biology underlying BP. We identified a locus for interdaily stability of activity, at a threshold exceeding genome-wide significance, on chromosome 12pter, a region that also showed pleiotropic linkage to two additional activity phenotypes. PMID:26712028

  20. Transgenic mice with a constitutively active aryl hydrocarbon receptor display a gender-specific bone phenotype.

    PubMed

    Wejheden, Carolina; Brunnberg, Sara; Larsson, Sune; Lind, P Monica; Lind, Pia M; Andersson, Göran; Hanberg, Annika

    2010-03-01

    Bone tissue homeostasis is governed by hormones, growth factors, and cytokines and can be distorted by environmental pollutants, such as ligands to the aryl hydrocarbon receptor (AhR). A transgenic mouse expressing a constitutively active aryl hydrocarbon receptor (CA-AhR), mimicking continuous low-dose exposure to AhR ligands, was used to explore potential long-term effects of these ligands on bone. The density, content, and dimensions of cortical and trabecular bone, as well as physical properties, were significantly altered in female transgenic mice, while almost no alterations were detected in males. Osteoclast volume density and serum level of C-telopeptide of type I collagen (CTX), reflecting osteoclast activity, were both increased by approximately 60% in female CA-AhR mice, while serum tartrate-resistant acid phosphatase (TRAP) 5b, reflecting osteoclast numbers, was unchanged. Subsequently, the resorption index (CTX/TRAP 5b) was increased by 90%, indicating increased osteoclast activity in female CA-AhR. Moreover, the protein level of the osteoclast collagenase cathepsin K was increased by 40% in bone extracts of female CA-AhR mice. The messenger RNA expression of several osteoclast- and osteoblast-associated genes was altered in female transgenic mice but not in males. Notably, early markers for osteoclast and osteoblast differentiation were normal, while the expression of functional markers of osteoclasts and osteoblasts were reduced. In conclusion, a low continuous activation of the AhR leads to a skeletal phenotype with increased bone resorption associated with more ductile bones in females but not in males. The results indicate the presence of an interaction between the AhR and a female-specific mechanism implicated in inhibition of osteoclast development and function. Female bone tissue appears more susceptible to dioxins and other AhR ligands than male bone tissue. PMID:19934163

  1. Ovarian cancer stem-like cells elicit the polarization of M2 macrophages.

    PubMed

    Zhang, Qing; Cai, Da-Jun; Li, Bin

    2015-06-01

    Ovarian cancer is a life‑threatening disease in females worldwide. The polarization of macrophages is crucial in oncogenesis and the development of ovarian cancer. Increasing evidence has supported the correlation between ovarian cancer stem‑like cells (OCSCs) and macrophages, however, whether OCSCs can affect the polarization of macrophages and the underlying mechanisms involved remain to be elucidated. To examine the interplay between OCSCs and macrophages, a co‑culture system was used to detect the effect of OCSCs on macrophage polarization. The expression of cluster of differentiation 206+ and the secretion of interleukin‑10 were significantly increased and the production of tumor necrosis factor‑α was suppressed, confirming macrophage polarization to M2 macrophages. Further investigation of the macrophages in a Transwell culture system with OCSCs revealed polarization to the M2 macrophages to a similar extent, indicating that the cytokines of the OCSCs, rather than direct cell‑cell contact, are important for the polarization of M2 macrophages. Furthermore, the expression levels of chemokine (C‑C motif) ligand (CCL)2, cyclooxygenase (COX)‑2 and prostaglandin E2 (PGE2) were increased in the Transwell system and the inhibition of COX‑2, but not CCL2, significantly decreased the polarization of the M2 macrophages. In addition, mechanistic analysis revealed the importance of the COX‑2/PGE2 pathway in OCSCs to activate Janus kinase (JAK) signaling in macrophages to elicit M2 polarization. These findings provided the first evidence, to the best of our knowledge, that OCSCs are capable of altering macrophages into the M2 phenotype via the overexpression of COX‑2 and the increased production of PGE2 cytokines and that the JAK signaling pathway in macrophages is important for this alteration. The present study provided evidence supporting possible molecular targets for cancer treatment. PMID:25672286

  2. Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice.

    PubMed

    Park, Hyo Jin; Oh, Ji Hoon; Ha, Sang-Jun

    2016-01-01

    Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4(+) T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-γ, TNF-α, and IL-2. It has been demonstrated that Treg cells' ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from naïve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells. PMID:27404802

  3. Antibacterial activities and structure-activity relationships of a panel of 48 compounds from Kenyan plants against multidrug resistant phenotypes.

    PubMed

    Omosa, Leonidah K; Midiwo, Jacob O; Mbaveng, Armelle T; Tankeo, Simplice B; Seukep, Jackson A; Voukeng, Igor K; Dzotam, Joachim K; Isemeki, John; Derese, Solomon; Omolle, Ruth A; Efferth, Thomas; Kuete, Victor

    2016-01-01

    In the current study forty eight compounds belonging to anthraquinones, naphthoquinones, benzoquinones, flavonoids (chalcones and polymethoxylated flavones) and diterpenoids (clerodanes and kauranes) were explored for their antimicrobial potential against a panel of sensitive and multi-drug resistant Gram-negative and Gram-positive bacteria. The minimal inhibitory concentration (MIC) determinations on the tested bacteria were conducted using modified rapid INT colorimetric assay. To evaluate the role of efflux pumps in the susceptibility of Gram-negative bacteria to the most active compounds, they were tested in the presence of phenylalanine arginine β-naphthylamide (PAβN) (at 30 µg/mL) against selected multidrug resistance (MDR) bacteria. The anthraquinone, emodin, naphthaquinone, plumbagin and the benzoquinone, rapanone were active against methicillin resistant Staphylococcus aureus (MRSA) strains of bacteria with MIC values ranging from 2 to 128 μg/mL. The structure activity relationships of benzoquinones against the MDR Gram-negative phenotype showed antibacterial activities increasing with increase in side chain length. In the chalcone series the presence of a hydroxyl group at C3' together with a methoxy group and a second hydroxyl group in meta orientation in ring B of the chalcone skeleton appeared to be necessary for minimal activities against MRSA. In most cases, the optimal potential of the active compounds were not attained as they were extruded by bacterial efflux pumps. However, the presence of the PAβN significantly increased the antibacterial activities of emodin against Gram-negative MDR E. coli AG102, 100ATet; K. pneumoniae KP55 and KP63 by >4-64 g/mL. The antibacterial activities were substantially enhanced and were higher than those of the standard drug, chloramphenicol. These data clearly demonstrate that the active compounds, having the necessary pharmacophores for antibacterial activities, including some quinones and chalcones are

  4. M2 Polarization of Monocytes-Macrophages Is a Hallmark of Indian Post Kala-Azar Dermal Leishmaniasis.

    PubMed

    Mukhopadhyay, Debanjan; Mukherjee, Shibabrata; Roy, Susmita; Dalton, Jane E; Kundu, Sunanda; Sarkar, Avijit; Das, Nilay K; Kaye, Paul M; Chatterjee, Mitali

    2015-10-01

    The high level of functional diversity and plasticity in monocytes/macrophages has been defined within in vitro systems as M1 (classically activated), M2 (alternatively activated) and deactivated macrophages, of which the latter two subtypes are associated with suppression of cell mediated immunity, that confers susceptibility to intracellular infection. Although the Leishmania parasite modulates macrophage functions to ensure its survival, what remains an unanswered yet pertinent question is whether these macrophages are deactivated or alternatively activated. This study aimed to characterize the functional plasticity and polarization of monocytes/macrophages and delineate their importance in the immunopathogenesis of Post kala-azar dermal leishmaniasis (PKDL), a chronic dermatosis of human leishmaniasis. Monocytes from PKDL patients showed a decreased expression of TLR-2/4, along with an attenuated generation of reactive oxidative/nitrosative species. At disease presentation, an increased mRNA expression of classical M2 markers CD206, ARG1 and PPARG in monocytes and lesional macrophages indicated M2 polarization of macrophages which was corroborated by increased expression of CD206 and arginase-1. Furthermore, altered vitamin D signaling was a key feature in PKDL, as disease presentation was associated with raised plasma levels of monohydroxylated vitamin D3 and vitamin D3- associated genes, features of M2 polarization. Taken together, in PKDL, monocyte/macrophage subsets appear to be alternatively activated, a phenotype that might sustain disease chronicity. Importantly, repolarization of these monocytes to M1 by antileishmanial drugs suggests that switching from M2 to M1 phenotype might represent a therapeutic opportunity, worthy of future pharmacological consideration. PMID:26496711

  5. MicroRNA-720 suppresses M2 macrophage polarization by targeting GATA3

    PubMed Central

    Zhong, Yan; Yi, Chun

    2016-01-01

    Macrophages are highly plastic cells with the ability to differentiate into both M1- and M2-polarized phenotypes. As a distinct M2-polarized population, tumour-associated macrophages (TAMs) promote tumorigenesis owing to their pro-angiogenic and immune-suppressive functions in tumour microenvironment. In the present study, we found that the microRNA-720 (miR-720) was down-regulated in TAMs isolated from breast carcinomas and M2-polarization macrophages. Overexpression of miR-720 attenuated M2 phenotype expression and thus inhibited M2 polarization. We further identified GATA binding protein 3 (GATA3), a transcriptional factor that plays an important role in M2 macrophage polarization, was the downstream target of miR-720. Ectopic expression of GATA3 restored the M2 phenotype in miR-720 overexpressed macrophages. Importantly, overexpression of miR-720 inhibited pro-migration behaviour and phagocytic ability of M2-polarized macrophages. Thus, our data suggest that miR-720 plays an important role in regulating M2 macrophage polarization and function. PMID:27354564

  6. Behavioral Phenotype of Fmr1 Knock-Out Mice during Active Phase in an Altered Light/Dark Cycle123

    PubMed Central

    Saré, R. Michelle

    2016-01-01

    Abstract Fragile X syndrome (FXS) is the most commonly inherited form of intellectual disability and is a disorder that is also highly associated with autism. FXS occurs as a result of an expanded CGG repeat sequence leading to transcriptional silencing. In an animal model of FXS in which Fmr1 is knocked out (Fmr1 KO), many physical, physiological, and behavioral characteristics of the human disease are recapitulated. Prior characterization of the mouse model was conducted during the day, the inactive phase of the circadian cycle. Circadian rhythms are an important contributor to behavior and may play a role in the study of disease phenotype. Moreover, changes in the parameters of circadian rhythm are known to occur in FXS animal models. We conducted an investigation of key behavioral phenotypes in Fmr1 KO mice during their active phase. We report that phase did not alter the Fmr1 KO phenotype in open field activity, anxiety, and learning and memory. There was a slight effect of phase on social behavior as measured by time in chamber, but not by time spent sniffing. Our data strengthen the existing data characterizing the phenotype of Fmr1 KO mice, indicating that it is independent of circadian phase. PMID:27294193

  7. Dectin-1 Activation by a Natural Product β-Glucan Converts Immunosuppressive Macrophages into an M1-like Phenotype.

    PubMed

    Liu, Min; Luo, Fengling; Ding, Chuanlin; Albeituni, Sabrin; Hu, Xiaoling; Ma, Yunfeng; Cai, Yihua; McNally, Lacey; Sanders, Mary Ann; Jain, Dharamvir; Kloecker, Goetz; Bousamra, Michael; Zhang, Huang-ge; Higashi, Richard M; Lane, Andrew N; Fan, Teresa W-M; Yan, Jun

    2015-11-15

    Tumor-associated macrophages (TAM) with an alternatively activated phenotype have been linked to tumor-elicited inflammation, immunosuppression, and resistance to chemotherapies in cancer, thus representing an attractive target for an effective cancer immunotherapy. In this study, we demonstrate that particulate yeast-derived β-glucan, a natural polysaccharide compound, converts polarized alternatively activated macrophages or immunosuppressive TAM into a classically activated phenotype with potent immunostimulating activity. This process is associated with macrophage metabolic reprograming with enhanced glycolysis, Krebs cycle, and glutamine utilization. In addition, particulate β-glucan converts immunosuppressive TAM via the C-type lectin receptor dectin-1-induced spleen tyrosine kinase-Card9-Erk pathway. Further in vivo studies show that oral particulate β-glucan treatment significantly delays tumor growth, which is associated with in vivo TAM phenotype conversion and enhanced effector T cell activation. Mice injected with particulate β-glucan-treated TAM mixed with tumor cells have significantly reduced tumor burden with less blood vascular vessels compared with those with TAM plus tumor cell injection. In addition, macrophage depletion significantly reduced the therapeutic efficacy of particulate β-glucan in tumor-bearing mice. These findings have established a new paradigm for macrophage polarization and immunosuppressive TAM conversion and shed light on the action mode of β-glucan treatment in cancer. PMID:26453753

  8. Drug Repositioning by Kernel-Based Integration of Molecular Structure, Molecular Activity, and Phenotype Data

    PubMed Central

    Wang, Yongcui; Chen, Shilong; Deng, Naiyang; Wang, Yong

    2013-01-01

    Computational inference of novel therapeutic values for existing drugs, i.e., drug repositioning, offers the great prospect for faster and low-risk drug development. Previous researches have indicated that chemical structures, target proteins, and side-effects could provide rich information in drug similarity assessment and further disease similarity. However, each single data source is important in its own way and data integration holds the great promise to reposition drug more accurately. Here, we propose a new method for drug repositioning, PreDR (Predict Drug Repositioning), to integrate molecular structure, molecular activity, and phenotype data. Specifically, we characterize drug by profiling in chemical structure, target protein, and side-effects space, and define a kernel function to correlate drugs with diseases. Then we train a support vector machine (SVM) to computationally predict novel drug-disease interactions. PreDR is validated on a well-established drug-disease network with 1,933 interactions among 593 drugs and 313 diseases. By cross-validation, we find that chemical structure, drug target, and side-effects information are all predictive for drug-disease relationships. More experimentally observed drug-disease interactions can be revealed by integrating these three data sources. Comparison with existing methods demonstrates that PreDR is competitive both in accuracy and coverage. Follow-up database search and pathway analysis indicate that our new predictions are worthy of further experimental validation. Particularly several novel predictions are supported by clinical trials databases and this shows the significant prospects of PreDR in future drug treatment. In conclusion, our new method, PreDR, can serve as a useful tool in drug discovery to efficiently identify novel drug-disease interactions. In addition, our heterogeneous data integration framework can be applied to other problems. PMID:24244318

  9. Unprimed, M1 and M2 Macrophages Differentially Interact with Porphyromonas gingivalis

    PubMed Central

    Lenzo, Jason C.; Fong, Shao B.; Reynolds, Eric C.

    2016-01-01

    Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Tissue macrophages are amongst the first immune cells to respond to bacteria and depending on the cytokine profile at the infection site, macrophages are primed to react to infection in different ways. Priming of naive macrophages with IFN-γ produces a classical pro-inflammatory, antibacterial M1 macrophage after TLR ligation, whereas priming with IL-4 induces an anti-inflammatory tissue-repair M2 phenotype. Previous work has shown that M1 are preferentially generated in gingival tissue following infection with P. gingivalis. However, few studies have investigated the interactions of macrophage subsets with P. gingivalis cells. The aim of this study was to determine the ability of naive, M1 and M2 macrophages to phagocytose P. gingivalis and investigate how this interaction affects both the bacterial cell and the macrophage. M1 and M2 macrophages were both found to have enhanced phagocytic capacity compared with that of naive macrophages, however only the naive and M1 macrophages were able to produce a respiratory burst in order to clear the bacteria from the phagosome. P. gingivalis was found to persist in naive and M2, but not M1 macrophages for 24 hours. Phagocytosis of P. gingivalis also induced high levels of TNF-α, IL-12 and iNOS in M1 macrophages, but not in naive or M2 macrophages. Furthermore, infection of macrophages with P. gingivalis at high bacteria to macrophage ratios, while inducing an inflammatory response, was also found to be deleterious to macrophage longevity, with high levels of apoptotic cell death found in macrophages after infection. The activation of M1 macrophages observed in this study may contribute to the initiation and maintenance of a pro-inflammatory state during chronic periodontitis. PMID:27383471

  10. MMP28 promotes macrophage polarization toward M2 cells and augments pulmonary fibrosis

    PubMed Central

    Gharib, Sina A.; Johnston, Laura K.; Huizar, Isham; Birkland, Timothy P.; Hanson, Josiah; Wang, Ying; Parks, William C.; Manicone, Anne M.

    2014-01-01

    Members of the MMP family function in various processes of innate immunity, particularly in controlling important steps in leukocyte trafficking and activation. MMP28 (epilysin) is a member of this family of proteinases, and we have found that MMP28 is expressed by macrophages and regulates their recruitment to the lung. We hypothesized that MMP28 regulates other key macrophage responses, such as macrophage polarization. Furthermore, we hypothesized that these MMP28-dependent changes in macrophage polarization would alter fibrotic responses in the lung. We examined the gene expression changes in WT and Mmp28−/− BMDMs, stimulated with LPS or IL-4/IL-13 to promote M1 and M2 cells, respectively. We also collected macrophages from the lungs of Pseudomonas aeruginosa-exposed WT and Mmp28−/− mice to evaluate changes in macrophage polarization. Lastly, we evaluated the macrophage polarization phenotypes during bleomycin-induced pulmonary fibrosis in WT and Mmp28−/− mice and assessed mice for differences in weight loss and total collagen levels. We found that MMP28 dampens proinflammatory macrophage function and promots M2 programming. In both in vivo models, we found deficits in M2 polarization in Mmp28−/− mice. In bleomycin-induced lung injury, these changes were associated with reduced fibrosis. MMP28 is an important regulator of macrophage polarization, promoting M2 function. Loss of MMP28 results in reduced M2 polarization and protection from bleomycin-induced fibrosis. These findings highlight a novel role for MMP28 in macrophage biology and pulmonary disease. PMID:23964118

  11. TLR4 and DC-SIGN receptors recognized Mycobacterium scrofulaceum promoting semi-activated phenotype on bone marrow dendritic cells.

    PubMed

    Cruz-Aguilar, Marisa; Castillo-Rodal, Antonia I; Schcolnik-Cabrera, Alejandro; Bonifaz, Laura C; Molina, Gabriela; López-Vidal, Yolanda

    2016-07-01

    Nontuberculous mycobacteria (NTM) are recognized as emerging pathogens and their immune regulatory mechanisms are not well described yet. From them, Mycobacterium avium is known to be a weak activator of dendritic cells (DCs) that impairs the response induced by BCG vaccine. However, whether other NTM such as Mycobacterium scrofulaceum may modulate the activation of DCs, has not been extensively studied. Here, we exposed bone marrow-derived DCs (BMDCs) to M. scrofulaceum and we analyzed the effect on the activation of DCs. We found that M. scrofulaceum has a comparable ability to induce a semi-mature DC phenotype, which was produced by its interaction with DC-SIGN and TLR4 receptors in a synergic effect. BMDCs exposed to M. scrofulaceum showed high expression of PD-L2 and production of IL-10, as well as low levels of co-stimulatory molecules and pro-inflammatory cytokines. In addition to immunophenotype induced on DCs, changes in morphology, re-organization of cytoskeleton and decreased migratory capacity are consistent with a semi-mature phenotype. However, unlike other pathogenic mycobacteria, the DC-semi-mature phenotype induced by M. scrofulaceum was reversed after re-exposure to BCG, suggesting that modulation mechanisms of DC-activation used by M. scrofulaceum are different to other known pathogenic mycobacteria. This is the first report about the immunophenotypic characterization of DC stimulated by M. scrofulaceum. PMID:27450002

  12. Present and Future of M2M

    NASA Astrophysics Data System (ADS)

    Ono, Satoru; Watanabe, Takashi

    In recent years, the rapid progress in the development of hardware and software technologies enables tiny and low cost information devices hereinafter referred to as Machine to be widely available. M2M (Machine to Machine) has been of much attention where many tiny machines are connected to each other through networks with minimal human intervention to provide smooth and intelligent management. M2M is a promising core technology providing timely, flexible, efficient and comprehensive service at low cost. M2M has wide variety of applications including energy management system, environmental monitoring system, intelligent transport system, industrial automation system and other applications. M2M consists of terminals and networks that connect them. In this paper, we mainly focus on M2M networking and mention the future direction of the technology.

  13. Relationships Between RNA Polymerase II Activity and Spt Elongation Factors to Spt- Phenotype and Growth in Saccharomyces cerevisiae.

    PubMed

    Cui, Ping; Jin, Huiyan; Vutukuru, Manjula Ramya; Kaplan, Craig D

    2016-01-01

    The interplay between adjacent transcription units can result in transcription-dependent alterations in chromatin structure or recruitment of factors that determine transcription outcomes, including the generation of intragenic or other cryptic transcripts derived from cryptic promoters. Mutations in a number of genes in Saccharomyces cerevisiae confer both cryptic intragenic transcription and the Suppressor of Ty (Spt(-)) phenotype for the lys2-128∂ allele of the LYS2 gene. Mutants that suppress lys2-128∂ allow transcription from a normally inactive Ty1 ∂ promoter, conferring a LYS(+) phenotype. The arrangement of transcription units at lys2-128∂ is reminiscent of genes containing cryptic promoters within their open reading frames. We set out to examine the relationship between RNA Polymerase II (Pol II) activity, functions of Spt elongation factors, and cryptic transcription because of the previous observation that increased-activity Pol II alleles confer an Spt(-) phenotype. We identify both cooperating and antagonistic genetic interactions between Pol II alleles and alleles of elongation factors SPT4, SPT5, and SPT6 We find that cryptic transcription at FLO8 and STE11 is distinct from that at lys2-128∂, though all show sensitivity to reduction in Pol II activity, especially the expression of lys2-128∂ found in Spt(-) mutants. We determine that the lys2-128∂ Spt(-) phenotypes for spt6-1004 and increased activity rpo21/rpb1 alleles each require transcription from the LYS2 promoter. Furthermore, we identify the Ty1 transcription start site (TSS) within the ∂ element as the position of Spt(-) transcription in tested Spt(-) mutants. PMID:27261007

  14. Relationships Between RNA Polymerase II Activity and Spt Elongation Factors to Spt- Phenotype and Growth in Saccharomyces cerevisiae

    PubMed Central

    Cui, Ping; Jin, Huiyan; Vutukuru, Manjula Ramya; Kaplan, Craig D.

    2016-01-01

    The interplay between adjacent transcription units can result in transcription-dependent alterations in chromatin structure or recruitment of factors that determine transcription outcomes, including the generation of intragenic or other cryptic transcripts derived from cryptic promoters. Mutations in a number of genes in Saccharomyces cerevisiae confer both cryptic intragenic transcription and the Suppressor of Ty (Spt-) phenotype for the lys2-128∂ allele of the LYS2 gene. Mutants that suppress lys2-128∂ allow transcription from a normally inactive Ty1 ∂ promoter, conferring a LYS+ phenotype. The arrangement of transcription units at lys2-128∂ is reminiscent of genes containing cryptic promoters within their open reading frames. We set out to examine the relationship between RNA Polymerase II (Pol II) activity, functions of Spt elongation factors, and cryptic transcription because of the previous observation that increased-activity Pol II alleles confer an Spt- phenotype. We identify both cooperating and antagonistic genetic interactions between Pol II alleles and alleles of elongation factors SPT4, SPT5, and SPT6. We find that cryptic transcription at FLO8 and STE11 is distinct from that at lys2-128∂, though all show sensitivity to reduction in Pol II activity, especially the expression of lys2-128∂ found in Spt- mutants. We determine that the lys2-128∂ Spt- phenotypes for spt6-1004 and increased activity rpo21/rpb1 alleles each require transcription from the LYS2 promoter. Furthermore, we identify the Ty1 transcription start site (TSS) within the ∂ element as the position of Spt- transcription in tested Spt- mutants. PMID:27261007

  15. Wound administration of M2-polarized macrophages does not improve murine cutaneous healing responses.

    PubMed

    Jetten, Nadine; Roumans, Nadia; Gijbels, Marion J; Romano, Andrea; Post, Mark J; de Winther, Menno P J; van der Hulst, Rene R W J; Xanthoulea, Sofia

    2014-01-01

    Macrophages play a crucial role in all stages of cutaneous wound healing responses and dysregulation of macrophage function can result in derailed wound repair. The phenotype of macrophages is influenced by the wound microenvironment and evolves during healing from a more pro-inflammatory (M1) profile in early stages, to a less inflammatory pro-healing (M2) phenotype in later stages of repair. The aim of the current study was to investigate the potential of exogenous administration of M2 macrophages to promote wound healing in an experimental mouse model of cutaneous injury. Bone marrow derived macrophages were stimulated in-vitro with IL-4 or IL-10 to obtain two different subsets of M2-polarized cells, M2a or M2c respectively. Polarized macrophages were injected into full-thickness excisional skin wounds of either C57BL/6 or diabetic db/db mice. Control groups were injected with non-polarized (M0) macrophages or saline. Our data indicate that despite M2 macrophages exhibit an anti-inflammatory phenotype in-vitro, they do not improve wound closure in wild type mice while they delay healing in diabetic mice. Examination of wounds on day 15 post-injury indicated delayed re-epithelialization and persistence of neutrophils in M2 macrophage treated diabetic wounds. Therefore, topical application of ex-vivo generated M2 macrophages is not beneficial and contraindicated for cell therapy of skin wounds. PMID:25068282

  16. Wound Administration of M2-Polarized Macrophages Does Not Improve Murine Cutaneous Healing Responses

    PubMed Central

    Jetten, Nadine; Roumans, Nadia; Gijbels, Marion J.; Romano, Andrea; Post, Mark J.; de Winther, Menno P. J.; van der Hulst, Rene R. W. J.; Xanthoulea, Sofia

    2014-01-01

    Macrophages play a crucial role in all stages of cutaneous wound healing responses and dysregulation of macrophage function can result in derailed wound repair. The phenotype of macrophages is influenced by the wound microenvironment and evolves during healing from a more pro-inflammatory (M1) profile in early stages, to a less inflammatory pro-healing (M2) phenotype in later stages of repair. The aim of the current study was to investigate the potential of exogenous administration of M2 macrophages to promote wound healing in an experimental mouse model of cutaneous injury. Bone marrow derived macrophages were stimulated in-vitro with IL-4 or IL-10 to obtain two different subsets of M2-polarized cells, M2a or M2c respectively. Polarized macrophages were injected into full-thickness excisional skin wounds of either C57BL/6 or diabetic db/db mice. Control groups were injected with non-polarized (M0) macrophages or saline. Our data indicate that despite M2 macrophages exhibit an anti-inflammatory phenotype in-vitro, they do not improve wound closure in wild type mice while they delay healing in diabetic mice. Examination of wounds on day 15 post-injury indicated delayed re-epithelialization and persistence of neutrophils in M2 macrophage treated diabetic wounds. Therefore, topical application of ex-vivo generated M2 macrophages is not beneficial and contraindicated for cell therapy of skin wounds. PMID:25068282

  17. Lipid-laden cells differentially distributed in the aging brain are functionally active and correspond to distinct phenotypes.

    PubMed

    Shimabukuro, Marilia Kimie; Langhi, Larissa Gutman Paranhos; Cordeiro, Ingrid; Brito, José M; Batista, Claudia Maria de Castro; Mattson, Mark P; Mello Coelho, Valeria de

    2016-01-01

    We characterized cerebral Oil Red O-positive lipid-laden cells (LLC) of aging mice evaluating their distribution, morphology, density, functional activities and inflammatory phenotype. We identified LLC in meningeal, cortical and neurogenic brain regions. The density of cerebral LLC increased with age. LLC presenting small lipid droplets were visualized adjacent to blood vessels or deeper in the brain cortical and striatal parenchyma of aging mice. LLC with larger droplets were asymmetrically distributed in the cerebral ventricle walls, mainly located in the lateral wall. We also found that LLC in the subventricular region co-expressed beclin-1 or LC3, markers for autophagosome or autophagolysosome formation, and perilipin (PLIN), a lipid droplet-associated protein, suggesting lipophagic activity. Some cerebral LLC exhibited β galactosidase activity indicating a senescence phenotype. Moreover, we detected production of the pro-inflammatory cytokine TNF-α in cortical PLIN(+) LLC. Some cortical NeuN(+) neurons, GFAP(+) glia limitans astrocytes, Iba-1(+) microglia and S100β(+) ependymal cells expressed PLIN in the aging brain. Our findings suggest that cerebral LLC exhibit distinct cellular phenotypes and may participate in the age-associated neuroinflammatory processes. PMID:27029648

  18. Lipid-laden cells differentially distributed in the aging brain are functionally active and correspond to distinct phenotypes

    PubMed Central

    Shimabukuro, Marilia Kimie; Langhi, Larissa Gutman Paranhos; Cordeiro, Ingrid; Brito, José M.; Batista, Claudia Maria de Castro; Mattson, Mark P.; de Mello Coelho, Valeria

    2016-01-01

    We characterized cerebral Oil Red O-positive lipid-laden cells (LLC) of aging mice evaluating their distribution, morphology, density, functional activities and inflammatory phenotype. We identified LLC in meningeal, cortical and neurogenic brain regions. The density of cerebral LLC increased with age. LLC presenting small lipid droplets were visualized adjacent to blood vessels or deeper in the brain cortical and striatal parenchyma of aging mice. LLC with larger droplets were asymmetrically distributed in the cerebral ventricle walls, mainly located in the lateral wall. We also found that LLC in the subventricular region co-expressed beclin-1 or LC3, markers for autophagosome or autophagolysosome formation, and perilipin (PLIN), a lipid droplet-associated protein, suggesting lipophagic activity. Some cerebral LLC exhibited β galactosidase activity indicating a senescence phenotype. Moreover, we detected production of the pro-inflammatory cytokine TNF-α in cortical PLIN+ LLC. Some cortical NeuN+ neurons, GFAP+ glia limitans astrocytes, Iba-1+ microglia and S100β+ ependymal cells expressed PLIN in the aging brain. Our findings suggest that cerebral LLC exhibit distinct cellular phenotypes and may participate in the age-associated neuroinflammatory processes. PMID:27029648

  19. Parthenolide Relieves Pain and Promotes M2 Microglia/Macrophage Polarization in Rat Model of Neuropathy

    PubMed Central

    Popiolek-Barczyk, Katarzyna; Kolosowska, Natalia; Makuch, Wioletta; Rojewska, Ewelina; Jurga, Agnieszka M.; Pilat, Dominika

    2015-01-01

    Neuropathic pain treatment remains a challenge because pathomechanism is not fully understood. It is believed that glial activation and increased spinal nociceptive factors are crucial for neuropathy. We investigated the effect of parthenolide (PTL) on the chronic constriction injury to the sciatic nerve (CCI)-induced neuropathy in rat. We analyzed spinal changes in glial markers and M1 and M2 polarization factors, as well as intracellular signaling pathways. PTL (5 µg; i.t.) was preemptively and then daily administered for 7 days after CCI. PTL attenuated the allodynia and hyperalgesia and increased the protein level of IBA1 (a microglial/macrophage marker) but did not change GFAP (an astrocyte marker) on day 7 after CCI. PTL reduced the protein level of M1 (IL-1β, IL-18, and iNOS) and enhanced M2 (IL-10, TIMP1) factors. In addition, it downregulated the phosphorylated form of NF-κB, p38MAPK, and ERK1/2 protein level and upregulated STAT3. In primary microglial cell culture we have shown that IL-1β, IL-18, iNOS, IL-6, IL-10, and TIMP1 are of microglial origin. Summing up, PTL directly or indirectly attenuates neuropathy symptoms and promotes M2 microglia/macrophages polarization. We suggest that neuropathic pain therapies should be shifted from blanketed microglia/macrophage suppression toward maintenance of the balance between neuroprotective and neurotoxic microglia/macrophage phenotypes. PMID:26090236

  20. Arylamine N-acetyltransferase (NAT2) mutations and their allelic linkage in unrelated caucasian individuals: Correlation with phenotypic activity

    SciTech Connect

    Cascorbi, I.; Drakoulis, N.; Brockmoeller, J.

    1995-09-01

    The polymorphic arylamine N-acetyltransferase (NAT2; EC2.3.1.5) is supposed to be a susceptibility factor for several drug side effects and certain malignancies. A group of 844 unrelated German subjects was genotyped for their acetylation type, and 563 of them were also phenotyped. Seven mutations of the NAT2 gene were evaluated by allele-specific PCR (mutation 341C to T) and PCR-RFLP for mutations at nt positions 191, 282, 481, 590, 803, and 857. From the mutation pattern eight different alleles, including the wild type coding for rapid acetylation and seven alleles coding for slow phenotype, were determined. Four hundred ninety-seven subjects had a genotype of slow acetylation (58.9%; 95% confidence limits 55.5%-62.2%). Phenotypic acetylation capacity was expressed as the ratio of 5-acetylamino-6-formylamino-3-methyluracil and 1-methylxanthine in urine after caffeine intake. Some 6.7% of the cases deviated in genotype and phenotype, but sequencing DNA of these probands revealed no new mutations. Furthermore, linkage pattern of the mutations was always confirmed, as tested in 533 subjects. In vivo acetylation capacity of homozygous wild-type subjects (NAT2{sup *}4/{sup *}4) was significantly higher than in heterozygous genotypes (P = .001). All mutant alleles showed low in vivo acetylation capacities, including the previously not-yet-defined alleles {sup *}5A, {sup *}5C, and {sup *}13. Moreover, distinct slow genotypes differed significantly among each other, as reflected in lower acetylation capacity of {sup *}6A, {sup *}7B, and {sup *}13 alleles than the group of {sup *}5 alleles. The study demonstrated differential phenotypic activity of various NAT2 genes and gives a solid basis for clinical and molecular-epidemiological investigations. 34 refs., 4 figs., 7 tabs.

  1. Spreading Depression Requires Microglia and is Decreased by their M2a Polarization from Environmental Enrichment

    PubMed Central

    Pusic, Kae M.; Pusic, Aya D.; Kemme, Jordan; Kraig, Richard P.

    2014-01-01

    Microglia play an important role in fine-tuning neuronal activity. In part, this involves their production of tumor necrosis factor alpha (TNFα), which increases neuronal excitability. Excessive synaptic activity is necessary to initiate spreading depression (SD). Increased microglial production of pro-inflammatory cytokines promotes initiation of SD, which, when recurrent, may play a role in conversion of episodic to high frequency and chronic migraine. Previous work shows that this potentiation of SD occurs through increased microglial production of TNFα and reactive oxygen species, both of which are associated with an M1-skewed microglial population. Hence, we explored the role of microglia and their M1 polarization in SD initiation. Selective ablation of microglia from rat hippocampal slice cultures confirmed that microglia are essential for initiation of SD. Application of minocycline to dampen M1 signaling led to increased SD threshold. In addition, we found that SD threshold was increased in rats exposed to environmental enrichment. These rats had increased neocortical levels of interleukin-11 (IL-11), which decreases TNFα signaling and polarized microglia to an M2a-dominant phenotype. M2a microglia reduce pro-inflammatory signaling and increase production of anti-inflammatory cytokines, and therefore may protect against SD. Nasal administration of IL-11 to mimic effects of environmental enrichment likewise increased M2a polarization and increased SD threshold, an effect also seen in vitro. Similarly, application of conditioned medium from M2a polarized primary microglia to slice cultures also increased SD threshold. Thus, microglia and their polarization state play an essential role in SD initiation, and perhaps by extension migraine with aura and migraine. PMID:24723305

  2. Induction of Murine Macrophage M2 Polarization by Cigarette Smoke Extract via the JAK2/STAT3 Pathway

    PubMed Central

    Shi, Hengfei; Chen, Guopu; Dong, Ping; Zhang, Weiyun

    2014-01-01

    Cigarette smoking is a major pathogenic factor in lung cancer. Macrophages play an important role in host defense and adaptive immunity. These cells display diverse phenotypes for performing different functions. M2 type macrophages usually exhibit immunosuppressive and tumor-promoting characteristics. Although macrophage polarization toward the M2 phenotype has been observed in the lungs of cigarette smokers, the molecular basis of the process remains unclear. In this study, we evaluated the possible mechanisms for the polarization of mouse macrophages that are induced by cigarette smoking (CS) or cigarette smoke extract (CSE). The results showed that exposure to CSE suppressed the production of reactive oxygen species (ROS) and nitric oxide (NO) and down-regulated the phagocytic ability of Ana-1 cells. The CD163 expressions on the surface of macrophages from different sources were significantly increased in in vivo and in vitro studies. The M1 macrophage cytokines TNF-α, IL-12p40 and enzyme iNOS decreased in the culture supernatant, and their mRNA levels decreased depending on the time and concentration of CSE. In contrast, the M2 phenotype macrophage cytokines IL-10, IL-6, TGF-β1 and TGF-β2 were up-regulated. Moreover, phosphorylation of JAK2 and STAT3 was observed after the Ana-1 cells were treated with CSE. In addition, pretreating the Ana-1 cells with the STAT3 phosphorylation inhibitor WP1066 inhibited the CSE-induced CD163 expression, increased the mRNA level of IL-10 and significantly decreased the mRNA level of IL-12. In conclusion, we demonstrated that the M2 polarization of macrophages induced by CS could be mediated through JAK2/STAT3 pathway activation. PMID:25198511

  3. Microbial metabolite butyrate facilitates M2 macrophage polarization and function

    PubMed Central

    Ji, Jian; Shu, Dingming; Zheng, Mingzhu; Wang, Jie; Luo, Chenglong; Wang, Yan; Guo, Fuyou; Zou, Xian; Lv, Xiaohui; Li, Ying; Liu, Tianfei; Qu, Hao

    2016-01-01

    Metabolites from intestinal microbes modulate the mucosal immune system by regulating the polarization and expansion of T cells. Whether the microbial metabolites influence macrophage polarization, however, is poorly understood. Here, we show that the large bowel microbial fermentation product, butyrate, facilitates M2 macrophage polarization, in vitro and in vivo. The supernatant from butyrate-treated M2 macrophage increased the migration and enhanced the wound closure rate of MLE-12 cells. Butyrate attenuated intestinal inflammation in mice with dextran sulfate sodium (DSS)-induced colitis, with a significant increase in colonic expression of the M2 macrophage-associated protein, Arg1. M2 macrophage treated with butyrate, had increased activation of the H3K9/STAT6 signaling pathway, suggesting a mechanism for butyrate facilitated M2 macrophage polarization. Collectively, our study indicated that commensal microbe-derived butyrate is a novel activator of STAT6-mediated transcription through H3K9 acetylation driving M2 macrophage polarization, and delineated new insights into the immune interplay underlying inflammatory bowel disease. PMID:27094081

  4. Microbial metabolite butyrate facilitates M2 macrophage polarization and function.

    PubMed

    Ji, Jian; Shu, Dingming; Zheng, Mingzhu; Wang, Jie; Luo, Chenglong; Wang, Yan; Guo, Fuyou; Zou, Xian; Lv, Xiaohui; Li, Ying; Liu, Tianfei; Qu, Hao

    2016-01-01

    Metabolites from intestinal microbes modulate the mucosal immune system by regulating the polarization and expansion of T cells. Whether the microbial metabolites influence macrophage polarization, however, is poorly understood. Here, we show that the large bowel microbial fermentation product, butyrate, facilitates M2 macrophage polarization, in vitro and in vivo. The supernatant from butyrate-treated M2 macrophage increased the migration and enhanced the wound closure rate of MLE-12 cells. Butyrate attenuated intestinal inflammation in mice with dextran sulfate sodium (DSS)-induced colitis, with a significant increase in colonic expression of the M2 macrophage-associated protein, Arg1. M2 macrophage treated with butyrate, had increased activation of the H3K9/STAT6 signaling pathway, suggesting a mechanism for butyrate facilitated M2 macrophage polarization. Collectively, our study indicated that commensal microbe-derived butyrate is a novel activator of STAT6-mediated transcription through H3K9 acetylation driving M2 macrophage polarization, and delineated new insights into the immune interplay underlying inflammatory bowel disease. PMID:27094081

  5. p21-activated kinase 1 determines stem-like phenotype and sunitinib resistance via NF-κB/IL-6 activation in renal cell carcinoma

    PubMed Central

    Zhu, Y; Liu, H; Xu, L; An, H; Liu, W; Liu, Y; Lin, Z; Xu, J

    2015-01-01

    The p21-activated kinase 1 (PAK1), a serine/threonine kinase that orchestrates cytoskeletal remodeling and cell motility, has been shown to function as downstream node for various oncogenic signaling pathways to promote cell proliferation, regulate apoptosis and accelerate mitotic abnormalities, resulting in tumor formation and invasiveness. Although alterations in PAK1 expression and activity have been detected in various human malignancies, its potential biological and clinical significance in renal cell carcinoma (RCC) remains obscure. In this study, we found increased PAK1 and phosphorylated PAK1 levels in tumor tissues according to TNM stage progression. Elevated phosphorylated PAK1 levels associated with progressive features and indicated unfavorable overall survival (OS) as an independent adverse prognosticator for patients with RCC. Moreover, PAK1 kinase activation with constitutive active PAK1 mutant T423E promoted growth, colony formation, migration, invasion and stem-like phenotype of RCC cells, and vice versa, in PAK1 inhibition by PAK1 kinase inactivation with specific PAK1 shRNA, dead kinase PAK1 mutant K299R or allosteric inhibitor IPA3. Stem-like phenotype due to sunitinib administration via increased PAK1 kinase activation could be ameliorated by PAK1 shRNA, PAK1 mutant K299R and IPA3. Furthermore, nuclear factor-κB (NF-κB)/interleukin-6 (IL-6) activation was found to be responsible for PAK1-mediated stem-like phenotype following sunitinib treatment. Both IL-6 neutralizing antibody and IPA3 administration enhanced tumor growth inhibition effect of sunitinib treatment on RCC cells in vitro and in vivo. Our results unraveled that oncogenic activation of PAK1 defines an important mechanism for maintaining stem-like phenotype and sunitinib resistance through NF-κB/IL-6 activation in RCC, lending PAK1-mediated NF-κB/IL-6 activation considerable appeal as novel pharmacological therapeutic targets against sunitinib resistance. PMID:25675297

  6. Lineage-specific STAT5 target gene activation in hematopoietic progenitor cells predicts the FLT3(+)-mediated leukemic phenotype.

    PubMed

    Müller, T A; Grundler, R; Istvanffy, R; Rudelius, M; Hennighausen, L; Illert, A L; Duyster, J

    2016-08-01

    Mutations that activate FMS-like tyrosine kinase 3 (FLT3) are frequent occurrences in acute myeloid leukemia. Two distinct types of mutations have been described: internal duplication of the juxtamembranous domain (ITD) and point mutations of the tyrosine kinase domain (TKD). Although both mutations lead to constitutive FLT3 signaling, only FLT3-ITD strongly activates signal transducer and activator of transcription 5 (STAT5). In a murine transplantation model, FLT3-ITD induces a myeloproliferative neoplasm, whereas FLT3-TKD leads to a lymphoid malignancy with significantly longer latency. Here we report that the presence of STAT5 is critical for the development of a myeloproliferative disease by FLT3-ITD in mice. Deletion of Stat5 in FLT3-ITD-induced leukemogenesis leads not only to a significantly longer survival (82 vs 27 days) of the diseased mice, but also to an immunophenotype switch with expansion of the lymphoid cell compartment. Interestingly, we were able to show differential STAT5 activation in FLT3-ITD(+) myeloid and lymphoid murine progenitors. STAT5 target genes such as Oncostatin M were highly expressed in FLT3-ITD(+) myeloid but not in FLT3-ITD(+) lymphoid progenitor cells. Strikingly, FLT3-TKD expression in combination with Oncostatin M is sufficient to reverse the phenotype to a myeloproliferative disease in FLT3-TKD mice. Thus, lineage-specific STAT5 activation in hematopoietic progenitor cells predicts the FLT3(+)-mediated leukemic phenotype in mice. PMID:27046463

  7. Treatment with Recombinant Trichinella spiralis Cathepsin B-like Protein Ameliorates Intestinal Ischemia/Reperfusion Injury in Mice by Promoting a Switch from M1 to M2 Macrophages.

    PubMed

    Liu, Wei-Feng; Wen, Shi-Hong; Zhan, Jian-Hua; Li, Yun-Sheng; Shen, Jian-Tong; Yang, Wen-Jing; Zhou, Xing-Wang; Liu, Ke-Xuan

    2015-07-01

    Intestinal ischemia/reperfusion (I/R) injury, in which macrophages play a key role, can cause high morbidity and mortality. The switch from classically (M1) to alternatively (M2) activated macrophages, which is dependent on the activation of STAT6 signaling, has been shown to protect organs from I/R injuries. In the current study, the effects of recombinant Trichinella spiralis cathepsin B-like protein (rTsCPB) on intestinal I/R injury and the potential mechanism related to macrophage phenotypes switch were investigated. In a mouse I/R model undergoing 60-min intestinal ischemia followed by 2-h or 7-d reperfusion, we demonstrated that intestinal I/R caused significant intestinal injury and induced a switch from M2 to M1 macrophages, evidenced by a decrease in levels of M2 markers (arginase-1 and found in inflammatory zone protein), an increase in levels of M1 markers (inducible NO synthase and CCR7), and a decrease in the ratio of M2/M1 macrophages. RTsCPB reversed intestinal I/R-induced M2-M1 transition and promoted M1-M2 phenotype switch evidenced by a significant decrease in M1 markers, an increase in M2 markers, and the ratio of M2/M1 macrophages. Meanwhile, rTsCPB significantly ameliorated intestinal injury and improved intestinal function and survival rate of animals, accompanied by a decrease in neutrophil infiltration and an increase in cell proliferation in the intestine. However, a selective STAT6 inhibitor, AS1517499, reversed the protective effects of rTsCPB by inhibiting M1 to M2 transition. These findings suggest that intestinal I/R injury causes a switch from M2 to M1 macrophages and that rTsCPB ameliorates intestinal injury by promoting STAT6-dependent M1 to M2 transition. PMID:25987744

  8. Revisiting the Endocytosis of the M2 Muscarinic Acetylcholine Receptor

    PubMed Central

    Ockenga, Wymke; Tikkanen, Ritva

    2015-01-01

    The agonist-induced endocytosis of the muscarinic acetylcholine receptor M2 is different from that of the other members of the muscarinic receptor family. The uptake of the M2 receptor involves the adapter proteins of the β-arrestin family and the small GTPase ADP-ribosylation factor 6. However, it has remained inconclusive if M2 endocytosis is dependent on clathrin or the large GTPase dynamin. We here show by means of knocking down the clathrin heavy chain that M2 uptake upon agonist stimulation requires clathrin. The expression of various dominant-negative dynamin-2 mutants and the use of chemical inhibitors of dynamin function revealed that dynamin expression and membrane localization as such appear to be necessary for M2 endocytosis, whereas dynamin GTPase activity is not required for this process. Based on the data from the present and from previous studies, we propose that M2 endocytosis takes place by means of an atypical clathrin-mediated pathway that may involve a specific subset of clathrin-coated pits/vesicles. PMID:25985102

  9. Gut dysbiosis promotes M2 macrophage polarization and allergic airway inflammation via fungi-induced PGE2

    PubMed Central

    Kim, Yun-Gi; Udayanga, Kankanam Gamage Sanath; Totsuka, Naoya; Weinberg, Jason B.; Núñez, Gabriel; Shibuya, Akira

    2014-01-01

    SUMMARY Although imbalances in gut microbiota composition, or “dysbiosis”, are associated with many diseases, the effects of gut dysbiosis on host systemic physiology are less well characterized. We report that gut dysbiosis induced by antibiotic (Abx)-treatment promotes allergic airway inflammation by shifting macrophage polarization in the lung toward the alternatively activated M2 phenotype. Adoptive transfer of alveolar macrophages derived from Abx-treated mice was sufficient to increase allergic airway inflammation. Abx-treatment resulted in the overgrowth of a commensal fungal Candida species in the gut and increased plasma concentrations of prostaglandin E2 (PGE2), which induced M2 macrophage polarization in the lung. Suppression of PGE2 synthesis by the cyclooxygenase inhibitors aspirin and celecoxib suppressed M2 macrophage polarization and decreased allergic airway inflammatory cell infiltration in Abx-treated mice. Thus, Abx-treatment can cause overgrowth of particular fungal species in the gut and promote M2 macrophage activation at distant sites to influence systemic responses including allergic inflammation. PMID:24439901

  10. Functional and phenotypic characteristics of alternative activation induced in human monocytes by interleukin-4 or the parasitic nematode Brugia malayi.

    PubMed

    Semnani, Roshanak Tolouei; Mahapatra, Lily; Moore, Vanessa; Sanprasert, Vivornpun; Nutman, Thomas B

    2011-10-01

    Human monocytes from patients with patent filarial infections are studded with filarial antigen and express markers associated with alternative activation of macrophages (MΦ). To explore the role of filaria-derived parasite antigen in differentiation of human monocytes, cells were exposed to microfilariae (mf) of Brugia malayi, and their phenotypic and functional characteristics were compared with those of monocytes exposed to factors known to generate either alternatively (interleukin-4 [IL-4]) or classically (macrophage colony-stimulating factor [MCSF]) activated MΦ. IL-4 upregulated mRNA expression of CCL13, CCL15, CCL17, CCL18, CCL22, CLEC10A, MRC1, CADH1, CD274, and CD273 associated with alternative activation of MΦ but not arginase 1. IL-4-cultured monocytes had a diminished ability to promote proliferation of both CD4(+) and CD8(+) T cells compared to that of unexposed monocytes. Similar to results with IL-4, exposure of monocytes to live mf induced upregulation of CCL15, CCL17, CCL18, CCL22, CD274, and CD273 and downregulation of Toll-like receptor 3 (TLR3), TLR5, and TLR7. In contrast to results with MCSF-cultured monocytes, exposure of monocytes to mf resulted in significant inhibition of the phagocytic ability of these cells to the same degree as that seen with IL-4. Our data suggest that short exposure of human monocytes to IL-4 induces a phenotypic characteristic of alternative activation and that secreted filarial products skew monocytes similarly. PMID:21788379

  11. Activation of endogenous human stem cell-associated retroviruses (SCARs) and therapy-resistant phenotypes of malignant tumors.

    PubMed

    Glinsky, Gennadi V

    2016-07-01

    Recent reports revealed consistent activation of specific endogenous retroviral elements in human preimplantation embryos and embryonic stem cells. Activity of stem cell associated retroviruses (SCARs) has been implicated in seeding thousands of human-specific regulatory sequences in the hESC genome. Activation of specific SCARs has been demonstrated in patients diagnosed with multiple types of cancer, autoimmune diseases, and neurodegenerative disorders, and appears associated with clinically lethal therapy resistant death-from-cancer phenotypes in a sub-set of cancer patients diagnosed with different types of malignant tumors. A hallmark feature of human-specific SCAR integration sites is deletions of ancestral DNA. Analysis of human-specific genetic loci of SCARs' stemness networks in tumor samples of TCGA cohorts representing 29 cancer types suggests that this approach may facilitate identification of pan-cancer genomic signatures of clinically-lethal disease defined by the presence of somatic non-silent mutations, gene-level copy number changes, and transcripts and proteins' expression of SCAR-regulated host genes. Present analyses indicate that multiple lines of strong circumstantial evidence support the hypothesis that activation of SCARs' networks may play an important role in cancer progression and metastasis, perhaps contributing to the emergence of clinically-lethal therapy-resistant death-from-cancer phenotypes. PMID:27084523

  12. 1,25-Dihydroxyvitamin D3 Promotes High Glucose-Induced M1 Macrophage Switching to M2 via the VDR-PPARγ Signaling Pathway

    PubMed Central

    Zhang, Xiaoliang; Zhou, Min; Guo, Yinfeng; Song, Zhixia; Liu, Bicheng

    2015-01-01

    Macrophages, especially their activation state, are closely related to the progression of diabetic nephropathy. Classically activated macrophages (M1) are proinflammatory effectors, while alternatively activated macrophages (M2) exhibit anti-inflammatory properties. 1,25-Dihydroxyvitamin D3 has renoprotective roles that extend beyond the regulation of mineral metabolism, and PPARγ, a nuclear receptor, is essential for macrophage polarization. The present study investigates the effect of 1,25-dihydroxyvitamin D3 on macrophage activation state and its underlying mechanism in RAW264.7 cells. We find that, under high glucose conditions, RAW264.7 macrophages tend to switch to the M1 phenotype, expressing higher iNOS and proinflammatory cytokines, including TNFα and IL-12. While 1,25-dihydroxyvitamin D3 significantly inhibited M1 activation, it enhanced M2 macrophage activation; namely, it upregulated the expression of MR, Arg-1, and the anti-inflammatory cytokine IL-10 but downregulated the M1 markers. However, the above effects of 1,25-dihydroxyvitamin D3 were abolished when the expression of VDR and PPARγ was inhibited by VDR siRNA and a PPARγ antagonist. In addition, PPARγ was also decreased upon treatment with VDR siRNA. The above results demonstrate that active vitamin D promoted M1 phenotype switching to M2 via the VDR-PPARγ pathway. PMID:25961000

  13. Cathepsin S-mediated autophagic flux in tumor-associated macrophages accelerate tumor development by promoting M2 polarization

    PubMed Central

    2014-01-01

    Background Tumor-associated macrophages (TAMs) are the major component of tumor-infiltrating leukocytes. TAMs are heterogeneous, with distinct phenotypes influenced by the microenvironment surrounding tumor tissues, but relatively little is known about the key molecular in these cells that contribute to malignant phenotypes. Autophagic activity is a critical factor in tumor development that contributes to enhancing cellular fitness and survival in the hostile tumor microenvironment. However, the molecular basis and relations between autophagy and TAMs polarization remain unclear. Methods Cathepsin S (Cat S) expression was analyzed in human colon carcinoma and normal colon tissues. In vivo effects were evaluated using PancO2 subcutaneous tumor model and SL4 hepatic metastasis model. Immunofluorescence staining, flow cytometry and real-time PCR were done to examine TAMs polarization. Western blotting assay, transmission electron microscopy, mCherry-GFP-LC3 transfection and DQ-BSA degradation assays were carried out to determine its role in regulating autophagy. Results In the present study, we showed that the enhanced expression of Cat S correlated with the severity of histologic grade as well as clinical stage, metastasis, and recurrence, which are known indicators of a relatively poor prognosis of human colon carcinoma. Cat S knockout led to decreased tumor growth and metastasis. Moreover, Cat S knockout inhibited M2 macrophage polarization during tumor development. We further demonstrated that Cat S was required for not only autophagic flux but also the fusion processes of autophagosomes and lysosomes in TAMs. Importantly, we found that Cat S contributed to tumor development by regulating the M2 phenotype of TAMs through the activation of autophagy. Conclusions These results indicated that Cat S-mediated autophagic flux is an important mechanism for inducing M2-type polarization of TAMs, which leads to tumor development. These data provide strong evidence for a

  14. M2-F1 in flight

    NASA Technical Reports Server (NTRS)

    1965-01-01

    The M2-F1 Lifting Body is seen here under tow, high above Rogers Dry Lake near the Flight Research Center (later redesignated the Dryden Flight Research Center), Edwards, California. R. Dale Reed effectively advocated the project with the support of NASA research pilot Milt Thompson. Together, they gained the support of Flight Research Center Director Paul Bikle. After a six-month feasibility study, Bikle gave approval in the fall of 1962 for the M2-F1 to be built. The wingless, lifting body aircraft design was initially concieved as a means of landing an aircraft horizontally after atmospheric reentry. The absence of wings would make the extreme heat of re-entry less damaging to the vehicle. In 1962, Flight Research Center management approved a program to build a lightweight, unpowered lifting body as a prototype to flight test the wingless concept. It would look like a 'flying bathtub,' and was designated the M2-F1, the 'M' referring to 'manned' and 'F' referring to 'flight' version. It featured a plywood shell placed over a tubular steel frame crafted at Dryden. Construction was completed in 1963. The first flight tests of the M2-F1 were over Rogers Dry Lake at the end of a tow rope attached to a hopped-up Pontiac convertible driven at speeds up to about 120 mph. These initial tests produced enough flight data about the M2-F1 to proceed with flights behind a NASA C-47 tow plane at greater altitudes. The C-47 took the craft to an altitude of 12,000 where free flights back to Rogers Dry Lake began. Pilot for the first series of flights of the M2-F1 was NASA research pilot Milt Thompson. Typical glide flights with the M2-F1 lasted about two minutes and reached speeds of 110 to l20 mph. More than 400 ground tows and 77 aircraft tow flights were carried out with the M2-F1. The success of Dryden's M2-F1 program led to NASA's development and construction of two heavyweight lifting bodies based on studies at NASA's Ames and Langley research centers--the M2-F2 and the HL

  15. Major histocompatibility complex-unrestricted cytolytic activity of human T cells: analysis of precursor frequency and effector phenotype

    SciTech Connect

    Patel, S.S.; Thiele, D.L.; Lipsky, P.E.

    1987-12-01

    The frequency and phenotype of human T cells that mediate major histocompatibility complex (MHC)-unrestricted cytolysis were analyzed. T cell clones were generated by culturing adherent cell-depleted peripheral blood mononuclear cells at a density of 0.3 cell/well with phytohemagglutinin, recombinant interleukin 2 (rIL-2), and irradiated autologous peripheral blood mononuclear cells and/or Epstein-Barr virus-transformed lymphoblastoid cell lines. All of the 198 clones generated by this method were T cells (CD2/sup +/, CD3/sup +/, CD4/sup +/ or CD2/sup +/, CD3/sup +/, CD8/sup +/) that possessed potent lytic activity against K562, an erythroleukemia line sensitive to lysis by human natural killer cells, and Cur, a renal carcinoma cell line resistant to human natural killer activity. Cytolysis, measured by /sup 51/Cr release, was MHC-unrestricted, since the clones were able to lyse MHC class I or class II negative targets, as well as MHC class I and class II negative targets. Although the clones produced tissue necrosis factor/lymphotoxin-like molecules, lysis of Cur of K562 was not mediated by a soluble factor secreted by the clones. These data indicate that the capacity for MHC-unrestricted tumoricidal activity and expression of NKH1 and CD11b, but not CD 16, are properties common to all or nearly all human peripheral blood-derived T cell clones regardless of CD4 or CD8 phenotype.

  16. M2-F1 In Tow Flight

    NASA Technical Reports Server (NTRS)

    1964-01-01

    The M2-F1 lifting body is seen here under tow at the Flight Research Center (later redesignated the Dryden Flight Research Center), Edwards, California. The wingless, lifting body aircraft design was initially conceived as a means of landing an aircraft horizontally after atmospheric reentry. The absence of wings would make the extreme heat of re-entry less damaging to the vehicle. In 1962, Dryden management approved a program to build a lightweight, unpowered lifting body as a prototype to flight test the wingless concept. It would look like a 'flying bathtub,' and was designated the M2-F1, the 'M' referring to 'manned' and 'F' referring to 'flight' version. It featured a plywood shell placed over a tubular steel frame crafted at Dryden. Construction was completed in 1963. The first flight tests of the M2-F1 were over Rogers Dry Lake at the end of a tow rope attached to a hopped-up Pontiac convertible driven at speeds up to about 120 mph. These initial tests produced enough flight data about the M2-F1 to proceed with flights behind a NASA C-47 tow plane at greater altitudes. The C-47 took the craft to an altitude of 12,000 feet where free flights back to Rogers Dry Lake began.

  17. M2-F1 simulator cockpit

    NASA Technical Reports Server (NTRS)

    1963-01-01

    This early simulator of the M2-F1 lifting body was used for pilot training, to test landing techniques before the first ground tow attempts, and to test new control configurations after the first tow attempts and wind-tunnel tests. The M2-F1 simulator was limited in some ways by its analog simulator. It had only limited visual display for the pilot, as well. The wingless, lifting body aircraft design was initially conceived as a means of landing an aircraft horizontally after atmospheric reentry. The absence of wings would make the extreme heat of re-entry less damaging to the vehicle. In 1962, Dryden management approved a program to build a lightweight, unpowered lifting body as a prototype to flight test the wingless concept. It would look like a 'flying bathtub,' and was designated the M2-F1, the 'M' referring to 'manned' and 'F' referring to 'flight' version. It featured a plywood shell placed over a tubular steel frame crafted at Dryden. Construction was completed in 1963. The first flight tests of the M2-F1 were over Rogers Dry Lake at the end of a tow rope attached to a hopped-up Pontiac convertible driven at speeds up to about 120 mph. This vehicle needed to be able to tow the M2-F1 on the Rogers Dry Lakebed adjacent to NASA's Flight Research Center (FRC) at a minimum speed of 100 miles per hour. To do that, it had to handle the 400-pound pull of the M2-F1. Walter 'Whitey' Whiteside, who was a retired Air Force maintenance officer working in the FRC's Flight Operations Division, was a dirt-bike rider and hot-rodder. Together with Boyden 'Bud' Bearce in the Procurement and Supply Branch of the FRC, Whitey acquired a Pontiac Catalina convertible with the largest engine available. He took the car to Bill Straup's renowned hot-rod shop near Long Beach for modification. With a special gearbox and racing slicks, the Pontiac could tow the 1,000-pound M2-F1 110 miles per hour in 30 seconds. It proved adequate for the roughly 400 car tows that got the M2-F1 airborne

  18. M2-F1 in flight

    NASA Technical Reports Server (NTRS)

    1964-01-01

    The M2-F1 Lifting Body is seen here under tow by an unseen C-47 at the NASA Flight Research Center (later redesignated the Dryden Flight Research Center), Edwards, California. The low-cost vehicle was the first piloted lifting body to be test flown. The lifting-body concept originated in the mid-1950s at the National Advisory Committee for Aeronautics' Ames Aeronautical Laboratory, Mountain View California. By February 1962, a series of possible shapes had been developed, and R. Dale Reed was working to gain support for a research vehicle. The wingless, lifting body aircraft design was initially concieved as a means of landing an aircraft horizontally after atmospheric reentry. The absence of wings would make the extreme heat of re-entry less damaging to the vehicle. In 1962, Dryden management approved a program to build a lightweight, unpowered lifting body as a prototype to flight test the wingless concept. It would look like a 'flying bathtub,' and was designated the M2-F1, the 'M' referring to 'manned' and 'F' referring to 'flight' version. It featured a plywood shell placed over a tubular steel frame crafted at Dryden. Construction was completed in 1963. The first flight tests of the M2-F1 were over Rogers Dry Lake at the end of a tow rope attached to a hopped-up Pontiac convertible driven at speeds up to about 120 mph. These initial tests produced enough flight data about the M2-F1 to proceed with flights behind a NASA C-47 tow plane at greater altitudes. The C-47 took the craft to an altitude of 12,000 where free flights back to Rogers Dry Lake began. Pilot for the first series of flights of the M2-F1 was NASA research pilot Milt Thompson. Typical glide flights with the M2-F1 lasted about two minutes and reached speeds of 110 to l20 mph. More than 400 ground tows and 77 aircraft tow flights were carried out with the M2-F1. The success of Dryden's M2-F1 program led to NASA's development and construction of two heavyweight lifting bodies based on studies at

  19. Nodal promotes invasive phenotypes via a Mitogen Activated Protein Kinase-dependent pathway

    PubMed Central

    Quail, DF; Zhang, G; Findlay, SD; Hess, DA; Postovit, LM

    2016-01-01

    The progression of cancer from localized to invasive disease is requisite for metastasis, and is often characterized by epithelial-to-mesenchymal transition (EMT) and alterations in cellular adhesion and migration. Studies have shown that this transition is associated with an up-regulation of embryonic stem cell-associated genes, resulting in a dedifferentiated phenotype and poor patient prognosis. Nodal is an embryonic factor that plays a critical role in promoting early invasive events during development. Nodal is silenced as stem cells differentiate; however, it re-emerges in adult life during placentation and mammary gland development, and is aberrantly expressed in many cancers. Here, we show that Nodal over-expression, in poorly-invasive breast cancer and choriocarcinoma cells, causes increased invasion and migration in vitro. Furthermore, we show that Nodal over-expression in these epithelial cancer types induces an EMT-like event concomitant with the internalization of E-Cadherin. This ability of Nodal to promote cellular invasion and EMT-like phenomena is dependent upon the phosphorylation of ERK1/2. Since Nodal normally signals through SMADs, these findings lend insight into an alternative pathway that is hijacked by this protein in cancer. To evaluate the clinical implications of our results, we show that Nodal inhibition reduces liver tumor burden in a model of spontaneous breast cancer metastasis in vivo, and that Nodal loss-of-function in aggressive breast cancer lines results in a decrease in invasive phenotypes. Our results demonstrate that Nodal is involved in promoting invasion in multiple cellular contexts, and that Nodal inhibition may be useful as a therapeutic target for patients with progressive disease. PMID:23334323

  20. Phenotypic and metabolic investigation of a CSF-1R kinase receptor inhibitor (BLZ945) and its pharmacologically active metabolite.

    PubMed

    Krauser, Joel A; Jin, Yi; Walles, Markus; Pfaar, Ulrike; Sutton, James; Wiesmann, Marion; Graf, Daniel; Pflimlin-Fritschy, Veronique; Wolf, Thierry; Camenisch, Gian; Swart, Piet

    2015-02-01

    1. 4-[2((1R,2R)-2-Hydroxycyclohexylamino)-benzothiazol-6-yloxyl]-pyridine-2-carboxylic acid methylamide (BLZ945) is a small molecule inhibitor of CSF-1R kinase activity within osteoclasts designed to prevent skeletal related events in metastatic disease. Key metabolites were enzymatically and structurally characterized to understand the metabolic fate of BLZ945 and pharmacological implications. The relative intrinsic clearances for metabolites were derived from in vitro studies using human hepatocytes, microsomes and phenotyped with recombinant P450 enzymes. 2. Formation of a pharmacologically active metabolite (M9) was observed in human hepatocytes. The M9 metabolite is a structural isomer (diastereomer) of BLZ945 and is about 4-fold less potent. This isomer was enzymatically formed via P450 oxidation of the BLZ945 hydroxyl group, followed by aldo-keto reduction to the alcohol (M9). 3. Two reaction phenotyping approaches based on fractional clearances were applied to BLZ945 using hepatocytes and liver microsomes. The fraction metabolized (fm) or contribution ratio was determined for each metabolic reaction type (oxidation, glucuronidation or isomerization) as well as for each metabolite. The results quantitatively illustrate contribution ratios of the involved enzymes and pathways, e.g. the isomerization to metabolite M9 accounted for 24% intrinsic clearance in human hepatocytes. In summary, contribution ratios for the Phase I and Phase II pathways can be determined in hepatocytes. PMID:25180976

  1. Antibacterial activities of Beilschmiedia obscura and six other Cameroonian medicinal plants against multi-drug resistant Gram-negative phenotypes

    PubMed Central

    2014-01-01

    Background The rapid spread of bacteria expressing multi-drug resistance propels the search for new antibacterial agents. The present study was designed to evaluate the antibacterial activities of the methanol extracts from Beilschmiedia obscura and six other Cameroonian plants against a panel of twenty nine Gram-negative bacteria including Multi-drug resistant (MDR) phenotypes. Methods The phytochemical investigations of the extracts were carried out according to the standard methods and the liquid micro-dilution assay was used for all antibacterial assays. Results Phytochemical analysis showed the presence of alkaloids in all studied extracts. Other chemical classes of secondary metabolites such as anthocyanines, anthraquinones flavonoids, saponins, tannins, sterols and triterpenes were selectively detected in the extracts. The extract from the fruits of Beilschmiedia obscura, Pachypodanthium staudtii leaves and Peperomia fernandopoiana (whole plant) displayed the best spectrum of activity with MIC values ranging from 16 to 1024 μg/mL against at least 65% and above of the tested bacteria. The extract from Beilschmiedia obscura was the most active with MIC values below 100 μg/mL against ten of the tested bacteria. This extract also showed MBC values below 1024 μg/mL against 55.17% of the studied microorganisms. Phenylalanine arginine β-naphthylamide (PAβN) significantly modulated the activities of extracts from the leaves and fruits of Pachypodanthium staudtii and Beilschmiedia obscura respectively, by increasing their inhibitory activity against Klebsiella pneumoniae KP55 strain at least four fold. Conclusion The overall results of the present investigation provide information for the possible use of the methanol extracts of the studied plant species, especially B. obscura to fight infectious diseases caused by Gram-negative bacteria including MDR phenotypes. PMID:25023038

  2. Active maternal phenotype is established before breeding and leads offspring to align growth trajectory outcomes and reflex ontogeny.

    PubMed

    Santana Muniz, Gisélia; Beserra, Renata; da Silva, Giselle de Paula; Fragoso, Jéssica; Lira, Allan de Oliveira; Nascimento, Elizabeth; Manhães de Castro, Raul; Leandro, Carol Góis

    2014-04-22

    The main goals of this study were to classify dams according to the level of voluntary physical activity before breeding and during pregnancy/lactation and to evaluate the effects on growth trajectory and reflex ontogenesis of offspring. Voluntary physical activity was ranked by traveled distance, time and daily estimated calorie burned. Thirty-five female Wistar rats were classified as control (C, n=5), inactive (I, n=10), active (A, n=8) and very active (VA, n=12). During 30d before breeding, traveled distance, average speed, time and calorie burned were daily recorded for active and very active groups. Traveled distance was recorded each 2h every day of adaptation. Body weight, food intake and fasting glycemia were measured throughout the experiment. During lactation, litters were evaluated in terms of physical features and reflex ontogeny. VA showed a progressive increase in the traveled distance and time while A dams presented constant values. VA rats showed lower body weight and higher food intake. During pregnancy, both groups performed less than 1km/day. Pups from A and VA dams showed higher lateral-lateral axis of the skull, longitudinal axis, tail length, and anticipation of the pavilion and auditory canal opening, and erupting incisors. I, A and VA groups showed a delay of righting, cliff aversion and vibrissae placing reflexes. In conclusion, active maternal phenotype is established before breeding allowing mothers to fit ecological and influencing growth trajectory outcomes and reflex ontogeny of the offspring based on matrilineal experience. PMID:24560842

  3. What is $$\\Delta m^2_{ee}$$ ?

    DOE PAGESBeta

    Parke, Stephen

    2016-03-09

    Here, the current short baseline reactor experiments, Daya Bay and RENO (Double Chooz) have measured (or are capable of measuring) an effective Δm2 associated with the atmospheric oscillation scale of 0.5 km/MeV in electron antineutrino disappearance. In this paper, I compare and contrast the different definitions of such an effective Δm2 and argue that the simple, L/E independent definition given by Δmee2≡cos2θ12Δm312+sin2θ12Δm322, i.e. “the νe weighted average of Δm312 and Δm322,” is superior to all other definitions and is useful for both short baseline experiments mentioned above and for the future medium baseline experiments JUNO and RENO-50.

  4. M2-F1 in Tow

    NASA Technical Reports Server (NTRS)

    1964-01-01

    The M2-F1 lifting body is seen here being towed behind a C-47 at the Flight Research Center (later redesignated the Dryden Flight Research Center), Edwards, California. The wingless, lifting body aircraft design was initially conceived as a means of landing an aircraft horizontally after atmospheric re-entry. The absence of wings would make the extreme heat of re-entry less damaging to the vehicle.

  5. NAD(+)-dependent activation of Sirt1 corrects the phenotype in a mouse model of mitochondrial disease.

    PubMed

    Cerutti, Raffaele; Pirinen, Eija; Lamperti, Costanza; Marchet, Silvia; Sauve, Anthony A; Li, Wei; Leoni, Valerio; Schon, Eric A; Dantzer, Françoise; Auwerx, Johan; Viscomi, Carlo; Zeviani, Massimo

    2014-06-01

    Mitochondrial disorders are highly heterogeneous conditions characterized by defects of the mitochondrial respiratory chain. Pharmacological activation of mitochondrial biogenesis has been proposed as an effective means to correct the biochemical defects and ameliorate the clinical phenotype in these severely disabling, often fatal, disorders. Pathways related to mitochondrial biogenesis are targets of Sirtuin1, a NAD(+)-dependent protein deacetylase. As NAD(+) boosts the activity of Sirtuin1 and other sirtuins, intracellular levels of NAD(+) play a key role in the homeostatic control of mitochondrial function by the metabolic status of the cell. We show here that supplementation with nicotinamide riboside, a natural NAD(+) precursor, or reduction of NAD(+) consumption by inhibiting the poly(ADP-ribose) polymerases, leads to marked improvement of the respiratory chain defect and exercise intolerance of the Sco2 knockout/knockin mouse, a mitochondrial disease model characterized by impaired cytochrome c oxidase biogenesis. This strategy is potentially translatable into therapy of mitochondrial disorders in humans. PMID:24814483

  6. Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene: correlation between sulfate transport activity and chondrodysplasia phenotype.

    PubMed

    Karniski, L P

    2001-07-01

    The diastrophic dysplasia sulfate transporter (DTDST) gene encodes a transmembrane protein that transports sulfate into chondrocytes to maintain adequate sulfation of proteoglycans. Mutations in this gene are responsible for four recessively inherited chondrodysplasias that include diastrophic dysplasia, multiple epiphyseal dysplasia, atelosteogenesis type 2 and achondrogenesis 1B (ACG-1B). To determine whether the DTDST mutations found in individuals with these chondrodysplasias differ functionally from each other, we compared the sulfate transport activity of 11 reported DTDST mutations. Five mutations, G255E, Delta a1751, L483P, R178X and N425D, had minimal sulfate transport function following expression in Xenopus laevis oocytes. Two mutations, Delta V340 and R279W, transported sulfate at rates of 17 and 32%, respectively, of wild-type DTDST. Four mutations, A715V, C653S, Q454P and G678V, had rates of sulfate transport nearly equal to that of wild-type DTDST. Transport kinetics were not different among the four mutations with near-normal sulfate transport function and wild-type DTDST. When the sulfate transport function of the different DTDST mutations are grouped according to the general phenotypes, individuals with the most severe form, ACG-1B, tend to be homozygous for null mutations, individuals with the moderately severe atelosteogenesis type 2 have at least one allele with a loss-of-function mutation, and individuals with the mildest forms are typically homozygous for mutations with residual sulfate transport function. However, in the X.laevis oocyte expression system, the correlation between residual transport function and the severity of phenotype was not absolute, suggesting that factors in addition to the intrinsic sulfate transport properties of the DTDST protein may influence the phenotype in individuals with DTDST mutations. PMID:11448940

  7. Influence of autologous dendritic cells on cytokine-induced killer cell proliferation, cell phenotype and antitumor activity in vitro

    PubMed Central

    Cao, Jingsong; Chen, Cong; Wang, Yuhuan; Chen, Xuecheng; Chen, Zeying; Luo, Xiaoling

    2016-01-01

    Dendritic cell (DCs) are essential antigen processing and presentation cells that play a key role in the immune response. In this study, DCs were co-cultured with cytokine-induced killer cells (DC-CIKs) in vitro to detect changes in cell proliferation, cell phenotype and cell cytotoxicity. The results revealed that the DCs were suitable for co-culture with CIKs at day 7, and that cell quantity of DC-CIKs was lower than that of CIKs until day 11, but it was significantly improved to 1.17-fold that of CIKs at day 13. Flow cytometry was used to detect the cell phenotype of CIKs and DC-CIKs. Compared with CIKs at day 13, the percentage of CD3+, CD3+CD4+, CD3+CD8+ and CD3+CD56+ T cells in DC-CIKs was significantly improved 1.02, 1.79, 1.26 and 2.44-fold, respectively. In addition, trypan blue staining analysis demonstrated that the cell viability of CIKs and DC-CIKs was 96% and 98%, respectively. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis verified that CIK and DC-CIK cytotoxicity in Hela cells was 58% and 80%, respectively, with a significant difference. Taken together, our results indicate that the cell proliferation, cell phenotype and antitumor activity of CIKs were all enhanced following co-culture with DCs in vitro. These results are likely to be useful for DC-CIK application in antitumor therapies. PMID:27602134

  8. Antibacterial activities of selected Cameroonian spices and their synergistic effects with antibiotics against multidrug-resistant phenotypes

    PubMed Central

    2011-01-01

    Background The emergence of multi-drug resistant (MDR) phenotypes is a major public health problem today in the treatment of bacterial infections. The present study was designed to evaluate the antibacterial activities of the methanol extracts of eleven Cameroonian spices on a panel of twenty nine Gram negative bacteria including MDR strains. Methods The phytochemical analysis of the extracts was carried out by standard tests meanwhile the liquid micro-broth dilution was used for all antimicrobial assays. Results Phytochemical analysis showed the presence of alkaloids, phenols and tannins in all plants extracts. The results of the antibacterial assays indicated that all tested extracts exert antibacterial activities, with the minimum inhibitory concentration (MIC) values varying from 32 to 1024 μg/ml. The extracts from Dichrostachys glomerata, Beilschmiedia cinnamomea, Aframomum citratum, Piper capense, Echinops giganteus, Fagara xanthoxyloïdes and Olax subscorpioïdea were the most active. In the presence of efflux pump inhibitor, PAßN, the activity of the extract from D. glomerata significantly increased on 69.2% of the tested MDR bacteria. At MIC/5, synergistic effects were noted with the extract of D. glomerata on 75% of the tested bacteria for chloramphenicol (CHL), tetracycline (TET) and norfloxacin (NOR). With B. cinnamomea synergy were observed on 62.5% of the studied MDR bacteria with CHL, cefepime (FEP), NOR and ciprofloxacin (CIP) and 75% with erythromycin (ERY). Conclusion The overall results provide information for the possible use of the studied extracts of the spices in the control of bacterial infections involving MDR phenotypes. PMID:22044718

  9. Phenotypic characterization of Salmonella isolated from food production environments associated with low-water activity foods.

    PubMed

    Finn, Sarah; Hinton, Jay C D; McClure, Peter; Amézquita, Aléjandro; Martins, Marta; Fanning, Séamus

    2013-09-01

    Salmonella can survive for extended periods of time in low-moisture environments posing a challenge for modern food production. This dangerous pathogen must be controlled throughout the production chain with a minimal risk of dissemination. Limited information is currently available describing the behavior and characteristics of this important zoonotic foodborne bacterium in low-moisture food production environments and in food. In our study, the phenotypes related to low-moisture survival of 46 Salmonella isolates were examined. Most of the isolates in the collection could form biofilms under defined laboratory conditions, with 57% being positive for curli fimbriae production and 75% of the collection positive for cellulose production, which are both linked with stronger biofilm formation. Biocides in the factory environment to manage hygiene were found to be most effective against planktonic cells but less so when the same bacteria were surface dried or present as a biofilm. Cellulose-producing isolates were better survivors when exposed to a biocide compared with cellulose-negative isolates. Examination of Salmonella growth of these 18 serotypes in NaCl, KCl, and glycerol found that glycerol was the least inhibitory of these three humectants. We identified a significant correlation between the ability to survive in glycerol and the ability to survive in KCl and biofilm formation, which may be important for food safety and the protection of public health. PMID:23992493

  10. A Role for Diminished GABA Transporter Activity in the Cortical Discharge Phenotype of MeCP2-Deficient Mice.

    PubMed

    Zhang, Liang; Wither, Robert G; Lang, Min; Wu, Chiping; Sidorova-Darmos, Elena; Netchev, Hristo; Matolcsy, Catherine B; Snead, Orlando Carter; Eubanks, James H

    2016-05-01

    Cortical network hyper-excitability is a common phenotype in mouse models lacking the transcriptional regulator methyl-CPG-binding protein 2 (MeCP2). Here, we implicate enhanced GABAB receptor activity stemming from diminished cortical expression of the GABA transporter GAT-1 in the genesis of this network hyper-excitability. We found that administering the activity-dependent GABAB receptor allosteric modulator GS-39783 to female Mecp2(+/-) mice at doses producing no effect in wild-type mice strongly potentiated their basal rates of spontaneous cortical discharge activity. Consistently, administering the GABAB receptor antagonist CGP-35348 significantly decreased basal discharge activity in these mice. Expression analysis revealed that while GABAB or extra-synaptic GABAA receptor prevalence is preserved in the MeCP2-deficient cortex, the expression of GAT-1 is significantly reduced from wild-type levels. This decrease in GAT-1 expression is consequential, as low doses of the GAT-1 inhibitor NO-711 that had no effects in wild-type mice strongly exacerbated cortical discharge activity in female Mecp2(+/-) mice. Taken together, these data indicate that the absence of MeCP2 leads to decreased cortical levels of the GAT-1 GABA transporter, which facilitates cortical network hyper-excitability in MeCP2-deficient mice by increasing the activity of cortical GABAB receptors. PMID:26499511

  11. M2-F1 in flight

    NASA Technical Reports Server (NTRS)

    1963-01-01

    This 25-second clip shows Milt Thompson being towed in the M2-F1 behind a C-47 aircraft. The M2-F1 lifting body, dubbed the 'flying bathtub' by the media, was the precursor of a remarkable series of wingless flying vehicles that contributed data used in the Space Shuttles, the X-33 Advanced Technology Demonstrator for the next century's Reusable Launch Vehicle, and the X-38 Technology Demonstrator for crew return from the International Space Station. Based on the ideas and basic design of Alfred J. Eggers and others at the Ames Aeronautical Laboratory (now the Ames Research Center), Mountain View, California, in the mid-1950's, the M2-F1 was built in 1962-63 over a four-month period for a cost of only about $30,000, plus an additional $8,000-$10,000 for an ejection seat. Engineers and technicians at the NASA Flight Research Center (now NASA Dryden) kept costs low by designing and fabricating it partly in-house, with the plywood shell constructed by a local sailplane builder. Someone at the time estimated that it would have cost a major aircraft company $150,000 to build the same vehicle. Unlike the later lifting bodies, the M2-F1 was unpowered and was initially towed by a souped-up Pontiac convertible until it was airborne. Later a C-47 took over the towing duties. Flown by such famous research pilots as Milt Thompson, Bruce Peterson, Chuck Yeager, and Bill Dana, the lightweight flying bathtub demonstrated that a wingless vehicle shaped for reentry into the Earth's atmosphere from space could be flown and landed safely. Flown from 1963 to 1966, the lightweight M2-F1 paved the way for the heavyweight M2-F2, M2-F3, HL-10, X-24A, and X-24B lifting bodies that flew under rocket power after launch from a B-52 mothership. The heavyweights flew from 1966 to 1975, demonstrating the viability and versatility of the wingless configuration and the ability of a vehicle with low lift-over-drag characteristics to fly to high altitudes and then to land precisely with their rocket

  12. Current Concept and Update of the Macrophage Plasticity Concept: Intracellular Mechanisms of Reprogramming and M3 Macrophage “Switch” Phenotype

    PubMed Central

    Malyshev, Igor; Malyshev, Yuri

    2015-01-01

    Macrophages play a key role in immunity. In this review, we consider the traditional notion of macrophage plasticity, data that do not fit into existing concepts, and a hypothesis for existence of a new switch macrophage phenotype. Depending on the microenvironment, macrophages can reprogram their phenotype toward the proinflammatory M1 phenotype or toward the anti-inflammatory M2 phenotype. Macrophage reprogramming involves well-coordinated changes in activities of signalling and posttranslational mechanisms. Macrophage reprogramming is provided by JNK-, PI3K/Akt-, Notch-, JAK/STAT-, TGF-β-, TLR/NF-κB-, and hypoxia-dependent pathways. Posttranscriptional regulation is based on micro-mRNA. We have hypothesized that, in addition to the M1 and M2 phenotypes, an M3 switch phenotype exists. This switch phenotype responds to proinflammatory stimuli with reprogramming towards the anti-inflammatory M2 phenotype or, contrarily, it responds to anti-inflammatory stimuli with reprogramming towards the proinflammatory M1 phenotype. We have found signs of such a switch phenotype in lung diseases. Understanding the mechanisms of macrophage reprogramming will assist in the selection of new therapeutic targets for correction of impaired immunity. PMID:26366410

  13. C1GALT1 Promotes Invasive Phenotypes of Hepatocellular Carcinoma Cells by Modulating Integrin β1 Glycosylation and Activity

    PubMed Central

    Liu, Chiung-Hui; Hu, Rey-Heng; Huang, Miao-Juei; Lai, I-Rue; Chen, Chia-Hua; Lai, Hong-Shiee; Wu, Yao-Ming; Huang, Min-Chuan

    2014-01-01

    Cancer cell invasion and metastasis are the primary causes of treatment failure and death in hepatocellular carcinoma (HCC). We previously reported that core 1 β1,3-galactosyltransferase (C1GALT1) is frequently overexpressed in HCC tumors and its expression is associated with advanced tumor stage, metastasis, and poor survival. However, the underlying mechanisms of C1GALT1 in HCC malignancy remain unclear. In this study, we found that overexpression of C1GALT1 enhanced HCC cell adhesion to extracellular matrix (ECM) proteins, migration, and invasion, whereas RNAi-mediated knockdown of C1GALT1 suppressed these phenotypes. The promoting effect of C1GALT1 on the metastasis of HCC cells was demonstrated in a mouse xenograft model. Mechanistic investigations showed that the C1GALT1-enhanced phenotypic changes in HCC cells were significantly suppressed by anti-integrin β1 blocking antibody. Moreover, C1GALT1 was able to modify O-glycans on integrin β1 and regulate integrin β1 activity as well as its downstream signaling. These results suggest that C1GALT1 could enhance HCC invasiveness through integrin β1 and provide novel insights into the roles of O-glycosylation in HCC metastasis. PMID:25089569

  14. Shaping the Murine Macrophage Phenotype: IL-4 and cAMP Synergistically Activate the Arginase I Promoter

    PubMed Central

    Sheldon, Kathryn E.; Shandilya, Harish; Kepka-Lenhart, Diane; Poljakovic, Mirjana; Ghosh, Arundhati; Morris, Sidney M.

    2013-01-01

    Arginase I is a marker of murine M2 macrophages and is highly expressed in many inflammatory diseases. The basis for high arginase I expression in macrophages in vivo is incompletely understood but likely reflects integrated responses to combinations of stimuli. Our objective was to elucidate mechanisms involved in modulating arginase I induction by IL-4, the prototypical activator of M2 macrophages. IL-4 and 8-bromo-cAMP (8-Br-cAMP) individually induce arginase I, but together they rapidly and synergistically induce arginase I mRNA, protein, and promoter activity in murine macrophage cells. Arginase I induction by IL-4 requires binding of the transcription factors STAT6 and C/EBPβ to the IL-4 response element of the arginase I gene. Chromatin immunoprecipitation (ChIP) showed that the synergistic response involves binding of both transcription factors to the IL-4 response element at levels significantly greater than in response to IL-4 alone. The results suggest that C/EBPβ is a limiting factor for the level of STAT6 bound to the IL-4 response element. The enhanced binding in the synergistic response was not due to increased expression of either STAT6 or C/EBPβ but correlated primarily with increased nuclear abundance of C/EBPβ. Our findings also suggest that induction of arginase I expression is stochastic; i.e., differences in induction reflect differences in probability of transcriptional activation and not simply differences in rate of transcription. Results of the present study also may be useful for understanding mechanisms underlying regulated expression of other genes in macrophages and other myeloid-derived cells in health and disease. PMID:23913966

  15. NK Cells of Kidney Transplant Recipients Display an Activated Phenotype that Is Influenced by Immunosuppression and Pathological Staging

    PubMed Central

    Daemen, Kerstin; Keil, Jana; Stevanovic-Meyer, Maja; Lehner, Frank; Haller, Hermann; Blume, Cornelia; Falk, Christine S.

    2015-01-01

    To explore phenotype and function of NK cells in kidney transplant recipients, we investigated the peripheral NK cell repertoire, capacity to respond to various stimuli and impact of immunosuppressive drugs on NK cell activity in kidney transplant recipients. CD56dim NK cells of kidney transplanted patients displayed an activated phenotype characterized by significantly decreased surface expression of CD16 (p=0.0003), CD226 (p<0.0001), CD161 (p=0.0139) and simultaneously increased expression of activation markers like HLA-DR (p=0.0011) and CD25 (p=0.0015). Upon in vitro stimulation via Ca++-dependent signals, down-modulation of CD16 was associated with induction of interferon (IFN)-γ expression. CD16 modulation and secretion of NFAT-dependent cytokines such as IFN-γ, TNF-α, IL-10 and IL-31 were significantly suppressed by treatment of isolated NK cells with calcineurin inhibitors but not with mTOR inhibitors. In kidney transplant recipients, IFN-γ production was retained in response to HLA class I-negative target cells and to non-specific stimuli, respectively. However, secretion of other cytokines like IL-13, IL-17, IL-22 and IL-31 was significantly reduced compared to healthy donors. In contrast to suppression of cytokine expression at the transcriptional level, cytotoxin release, i.e. perforin, granzyme A/B, was not affected by immunosuppression in vitro and in vivo in patients as well as in healthy donors. Thus, immunosuppressive treatment affects NK cell function at the level of NFAT-dependent gene expression whereby calcineurin inhibitors primarily impair cytokine secretion while mTOR inhibitors have only marginal effects. Taken together, NK cells may serve as indicators for immunosuppression and may facilitate a personalized adjustment of immunosuppressive medication in kidney transplant recipients. PMID:26147651

  16. M2 Muscarinic acetylcholine receptor modulates rat airway smooth muscle cell proliferation

    PubMed Central

    2013-01-01

    Airways chronic inflammatory conditions in asthma and COPD are characterized by tissue remodeling, being smooth muscle hyperplasia, the most important feature. Non-neuronal and neuronal Acetylcholine acting on muscarinic receptors (MAChRs) has been postulated as determinant of tissue remodeling in asthma and COPD by promoting proliferation and phenotypic changes of airway smooth muscle cells (ASMC). The objective was to evaluate proliferative responses to muscarinic agonist as carbamylcholine (Cch) and to identify the MAchR subtype involved. ASMC were isolated from tracheal fragments of Sprague–Dawley rats by enzymatic digestion. Proliferation assays were performed by MTS-PMS method. Viability was confirmed by trypan blue exclusion method. Mitogens as, epidermal growth factor (EGF), Tumor necrosis factor-alpha (TNF-α) and fetal bovine serum (FBS) increased ASMC proliferation (p < 0.05, n = 5). Cch alone increased ASMC proliferation at 24 and 48 hrs. However, combination of Cch with other mitogens exhibited a dual effect, synergistic proliferation effect in the presence of EGF (5 ng/mL) and 5% FBS and inhibiting the proliferation induced by 10% FBS, EGF (10 ng/mL) and TNF-α (10 ng/mL). To determine the MAChR subtype involved in these biological responses, a titration curve of selective muscarinic antagonists were performed. The Cch stimulatory and inhibitory effects on ASCM proliferation was blocked by AF-DX-116 (M2AChR selective antagonist), in greater proportion than 4-DAMP (M3AChR selective antagonist), suggesting that the modulation of muscarinic agonist-induced proliferation is M2AChR mediated responses. Thus, M2AChR can activate multiple signal transduction systems and mediate both effects on ASMC proliferation depending on the plethora and variable airway microenvironments existing in asthma and COPD. PMID:24377382

  17. Using a PyMOL Activity to Reinforce the Connection between Genotype and Phenotype in an Undergraduate Genetics Laboratory

    PubMed Central

    Simmons, Alexandra D.; Nguyen, Thao K. T.; Follis, Jack L.; Ribes-Zamora, Albert

    2014-01-01

    With the purpose of developing an activity that would help clarify genetic concepts related to the connection between genotype and phenotype and the nature of mutations, we designed a three hour teaching module using the PyMol software. The activity starts with two pre-laboratory assignments, one to learn how to use PyMol and the other to read about a specific protein or protein family. During the laboratory students are given instructions where and how to find additional information on a specific disease and its causal mutations in order to prepare a 10-minute, in-class presentation. Using a post activity, anonymous quiz, we found a statistically significant different grade distribution in students that participated in the PyMol activity relative to a control group. We also found a significant improvement in the student’s comprehension when answering questions regarding the nature of mutations and protein structure. This demonstrates the utility of this simulation activity as a vehicle to improve student’s understanding of specific key genetic concepts. PMID:25461967

  18. Antibacterial Activities of Selected Cameroonian Plants and Their Synergistic Effects with Antibiotics against Bacteria Expressing MDR Phenotypes

    PubMed Central

    Lacmata, Stephen T.; Kuete, Victor; Dzoyem, Jean P.; Tankeo, Simplice B.; Teke, Gerald Ngo; Kuiate, Jules R.; Pages, Jean-Marie

    2012-01-01

    The present work was designed to assess the antibacterial properties of the methanol extracts of some Cameroonian medicinal plants and the effect of their associations with currently used antibiotics on multidrug resistant (MDR) Gram-negative bacteria overexpressing active efflux pumps. The antibacterial activities of twelve methanol extracts of medicinal plants were evaluated using broth microdilution. The results of this test showed that three extracts Garcinia lucida with the minimal inhibitory concentrations (MIC) varying from 128 to 512 μg/mL, Garcinia kola (MIC of 256 to 1024 μg/mL), and Picralima nitida (MIC of 128 to 1024 μg/mL) were active on all the twenty-nine studied bacteria including MDR phenotypes. The association of phenylalanine arginine β-naphthylamide (PAβN or efflux pumps inhibitor) to different extracts did not modify their activities. At the concentration of MIC/2 and MIC/5, the extracts of P. nitida and G. kola improved the antibacterial activities of some commonly used antibiotics suggesting their synergistic effects with the tested antibiotics. The results of this study suggest that the tested plant extracts and mostly those from P. nitida, G. lucida and G. kola could be used alone or in association with common antibiotics in the fight of bacterial infections involving MDR strains. PMID:22474511

  19. The Carbapenem Inactivation Method (CIM), a Simple and Low-Cost Alternative for the Carba NP Test to Assess Phenotypic Carbapenemase Activity in Gram-Negative Rods

    PubMed Central

    van der Zwaluw, Kim; de Haan, Angela; Pluister, Gerlinde N.; Bootsma, Hester J.; de Neeling, Albert J.; Schouls, Leo M.

    2015-01-01

    A new phenotypic test, called the Carbapenem Inactivation Method (CIM), was developed to detect carbapenemase activity in Gram-negative rods within eight hours. This method showed high concordance with results obtained by PCR to detect genes coding for the carbapenemases KPC, NDM, OXA-48, VIM, IMP and OXA-23. It allows reliable detection of carbapenemase activity encoded by various genes in species of Enterobacteriaceae (e.g., Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae), but also in non-fermenters Pseudomonas aeruginosa and Acinetobacter baumannii. The CIM was shown to be a cost-effective and highly robust phenotypic screening method that can reliably detect carbapenemase activity. PMID:25798828

  20. Differentiation and activation phenotypes of lung T lymphocytes differ from those of circulating T lymphocytes.

    PubMed Central

    Davidson, B L; Faust, J; Pessano, S; Daniele, R P; Rovera, G

    1985-01-01

    We used dual laser two-color flow cytometry to compare the expression of surface markers associated with activation and with differentiation in lung and peripheral blood T lymphocytes from normal subjects. T cell subsets, defined based on their reactivity with monoclonal antibodies (MAb) OKT3, OKT4, and OKT8, were analyzed for expression of activation antigens as detected by MAbs to the interleukin-2 receptor, the transferrin receptor, and HLA-DR determinants. Whereas circulating T lymphocytes expressed the three activation antigens at low levels, and the total of T4+ and T8+ cells always approximated the number of T3+ cells, lung T lymphocytes of the T3+, T4+, and T8+ populations expressed the activation antigens at variable levels in combinations not seen in circulating lymphocytes, and the sum of T4+ and T8+ cells always exceeded the T3+ total. A proportion of T4+T8+ cells was detected in lung lymphocytes. PMID:3926821

  1. Mixed-phenotype acute leukemia (MPAL) exhibits frequent mutations in DNMT3A and activated signaling genes.

    PubMed

    Eckstein, Olive S; Wang, Linghua; Punia, Jyotinder N; Kornblau, Steven M; Andreeff, Michael; Wheeler, David A; Goodell, Margaret A; Rau, Rachel E

    2016-08-01

    Mixed-phenotype acute leukemia (MPAL) is a heterogeneous group of poor-prognosis leukemias with immunophenotypic features of at least two cell lineages. The full spectrum of genetic mutations in this rare disease has not been elucidated, limiting our understanding of disease pathogenesis and our ability to devise targeted therapeutic strategies. Here, we sought to define the mutational landscape of MPAL by performing whole-exome sequencing on samples from 23 adult and pediatric MPAL patients. We identified frequent mutations of epigenetic modifiers, most notably mutations of DNMT3A, in 33% of adult MPAL patients. Mutations of activated signaling pathways, tumor suppressors, and transcription factors were also frequent. Importantly, many of the identified mutations are potentially therapeutically targetable, with agents currently available or in various stages of clinical development. Therefore, the mutational spectrum that we have identified provides potential biological insights and is likely to have clinical relevance for patients with this poor-prognosis disease. PMID:27208809

  2. The transglutaminase type 2 and pyruvate kinase isoenzyme M2 interplay in autophagy regulation.

    PubMed

    Altuntas, Sara; Rossin, Federica; Marsella, Claudia; D'Eletto, Manuela; Diaz-Hidalgo, Laura; Farrace, Maria Grazia; Campanella, Michelangelo; Antonioli, Manuela; Fimia, Gian Maria; Piacentini, Mauro

    2015-12-29

    Autophagy is a self-degradative physiological process by which the cell removes worn-out or damaged components. Constant at basal level it may become highly active in response to cellular stress. The type 2 transglutaminase (TG2), which accumulates under stressful cell conditions, plays an important role in the regulation of autophagy and cells lacking this enzyme display impaired autophagy/mitophagy and a consequent shift their metabolism to glycolysis. To further define the molecular partners of TG2 involved in these cellular processes, we analysed the TG2 interactome under normal and starved conditions discovering that TG2 interacts with various proteins belonging to different functional categories. Herein we show that TG2 interacts with pyruvate kinase M2 (PKM2), a rate limiting enzyme of glycolysis which is responsible for maintaining a glycolytic phenotype in malignant cells and displays non metabolic functions, including transcriptional co-activation and protein kinase activity. Interestingly, the ablation of PKM2 led to the decrease of intracellular TG2's transamidating activity paralleled by an increase of its tyrosine phosphorylation. Along with this, a significant decrease of ULK1 and Beclin1 was also recorded, thus suggesting a block in the upstream regulation of autophagosome formation. These data suggest that the PKM2/TG2 interplay plays an important role in the regulation of autophagy in particular under cellular stressful conditions such as those displayed by cancer cells. PMID:26702927

  3. The transglutaminase type 2 and pyruvate kinase isoenzyme M2 interplay in autophagy regulation

    PubMed Central

    Altuntas, Sara; Rossin, Federica; Marsella, Claudia; D'Eletto, Manuela; Hidalgo, Laura Diaz; Farrace, Maria Grazia; Campanella, Michelangelo; Antonioli, Manuela; Fimia, Gian Maria; Piacentini, Mauro

    2015-01-01

    Autophagy is a self-degradative physiological process by which the cell removes worn-out or damaged components. Constant at basal level it may become highly active in response to cellular stress. The type 2 transglutaminase (TG2), which accumulates under stressful cell conditions, plays an important role in the regulation of autophagy and cells lacking this enzyme display impaired autophagy/mitophagy and a consequent shift their metabolism to glycolysis. To further define the molecular partners of TG2 involved in these cellular processes, we analysed the TG2 interactome under normal and starved conditions discovering that TG2 interacts with various proteins belonging to different functional categories. Herein we show that TG2 interacts with pyruvate kinase M2 (PKM2), a rate limiting enzyme of glycolysis which is responsible for maintaining a glycolytic phenotype in malignant cells and displays non metabolic functions, including transcriptional co-activation and protein kinase activity. Interestingly, the ablation of PKM2 led to the decrease of intracellular TG2's transamidating activity paralleled by an increase of its tyrosine phosphorylation. Along with this, a significant decrease of ULK1 and Beclin1 was also recorded, thus suggesting a block in the upstream regulation of autophagosome formation. These data suggest that the PKM2/TG2 interplay plays an important role in the regulation of autophagy in particular under cellular stressful conditions such as those displayed by cancer cells. PMID:26702927

  4. Functional characterizations of venom phenotypes in the eastern diamondback rattlesnake (Crotalus adamanteus) and evidence for expression-driven divergence in toxic activities among populations.

    PubMed

    Margres, Mark J; Walls, Robert; Suntravat, Montamas; Lucena, Sara; Sánchez, Elda E; Rokyta, Darin R

    2016-09-01

    Phenotypes frequently vary across and within species. The connection between specific phenotypic effects and function, however, is less understood despite being essential to our understanding of the adaptive process. Snake venoms are ideal for identifying functionally important phenotypic variation because venom variation is common, and venoms can be functionally characterized through simple assays and toxicity measurements. Previous work with the eastern diamondback rattlesnake (Crotalus adamanteus) used multivariate statistical approaches to identify six unique venom phenotypes. We functionally characterized hemolytic, gelatinase, fibrinogenolytic, and coagulant activity for all six phenotypes, as well as one additional venom, to determine if the statistically significant differences in toxin expression levels previously documented corresponded to differences in venom activity. In general, statistical differences in toxin expression predicted the identified functional differences, or lack thereof, in toxic activity, demonstrating that the statistical approach used to characterize C. adamanteus venoms was a fair representation of biologically meaningful differences. Minor differences in activity not accounted for by the statistical model may be the result of amino-acid differences and/or post-translational modifications, but overall we were able to link variation in protein expression levels to variation in function as predicted by multivariate statistical approaches. PMID:27179420

  5. Somatic Mutations in CCK2R Alter Receptor Activity that Promote Oncogenic Phenotypes

    PubMed Central

    Willard, Melinda D.; Lajiness, Mary E.; Wulur, Isabella H.; Feng, Bo; Swearingen, Michelle L.; Uhlik, Mark T.; Kinzler, Kenneth W.; Velculescu, Victor E.; Sjöblom, Tobias; Markowitz, Sanford D.; Powell, Steven M.; Vogelstein, Bert; Barber, Thomas D.

    2013-01-01

    The roles of cholecystokinin 2 receptor (CCK2R) in numerous physiologic processes in the gastrointestinal tract and central nervous system are ‘well documented. There has been some evidence that CCK2R alterations play a role in cancers, but the functional significance of these alterations for tumorigenesis is unknown. We have identified six mutations in CCK2R among a panel of 140 colorectal cancers and 44 gastric cancers. We show that these mutations increase receptor activity, activate multiple downstream signaling pathways, increase cell migration, and promote angiogenesis. Our findings suggest that somatic mutations in CCK2R may promote tumorigenesis through deregulated receptor activity and highlight the importance of evaluating CCK2R inhibitors to block both the normal and mutant forms of the receptor. PMID:22516348

  6. Activity Suppression Behavior Phenotype in SULT4A1 Frameshift Mutant Zebrafish.

    PubMed

    Crittenden, Frank; Thomas, Holly R; Parant, John M; Falany, Charles N

    2015-07-01

    Since its identification in 2000, sulfotransferase (SULT) 4A1 has presented an enigma to the field of cytosolic SULT biology. SULT4A1 is exclusively expressed in neural tissue, is highly conserved, and has been identified in every vertebrate studied to date. Despite this singular level of conservation, no substrate or function for SULT4A1 has been identified. Previous studies demonstrated that SULT4A1 does not bind the obligate sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate, yet SULT4A1 is classified as a SULT superfamily member based on sequence and structural similarities to the other SULTs. In this study, transcription activator-like effector nucleases were used to generate heritable mutations in the SULT4A1 gene of zebrafish. The mutation (SULT4A1(Δ8)) consists of an 8-nucleotide deletion within the second exon of the gene, resulting in a frameshift mutation and premature stop codon after 132 AA. During early adulthood, casual observations were made that mutant zebrafish were exhibiting excessively sedentary behavior during the day. These observations were inconsistent with published reports on activity in zebrafish that are largely diurnal organisms and are highly active during the day. Thus, a decrease in activity during the day represents an abnormal behavior and warranted further systematic analysis. EthoVision video tracking software was used to monitor activity levels in wild-type (WT) and SULT4A1(Δ8/Δ8) fish over 48 hours of a normal light/dark cycle. SULT4A1(Δ8/Δ8) fish were shown to exhibit increased inactivity bout length and frequency as well as a general decrease in daytime activity levels when compared with their WT counterparts. PMID:25934576

  7. Activity Suppression Behavior Phenotype in SULT4A1 Frameshift Mutant Zebrafish

    PubMed Central

    Crittenden, Frank; Thomas, Holly R.; Parant, John M.

    2015-01-01

    Since its identification in 2000, sulfotransferase (SULT) 4A1 has presented an enigma to the field of cytosolic SULT biology. SULT4A1 is exclusively expressed in neural tissue, is highly conserved, and has been identified in every vertebrate studied to date. Despite this singular level of conservation, no substrate or function for SULT4A1 has been identified. Previous studies demonstrated that SULT4A1 does not bind the obligate sulfate donor, 3′-phosphoadenosine-5′-phosphosulfate, yet SULT4A1 is classified as a SULT superfamily member based on sequence and structural similarities to the other SULTs. In this study, transcription activator-like effector nucleases were used to generate heritable mutations in the SULT4A1 gene of zebrafish. The mutation (SULT4A1Δ8) consists of an 8-nucleotide deletion within the second exon of the gene, resulting in a frameshift mutation and premature stop codon after 132 AA. During early adulthood, casual observations were made that mutant zebrafish were exhibiting excessively sedentary behavior during the day. These observations were inconsistent with published reports on activity in zebrafish that are largely diurnal organisms and are highly active during the day. Thus, a decrease in activity during the day represents an abnormal behavior and warranted further systematic analysis. EthoVision video tracking software was used to monitor activity levels in wild-type (WT) and SULT4A1Δ8/Δ8 fish over 48 hours of a normal light/dark cycle. SULT4A1Δ8/Δ8 fish were shown to exhibit increased inactivity bout length and frequency as well as a general decrease in daytime activity levels when compared with their WT counterparts. PMID:25934576

  8. Phenotypic plasticity of post-fire activity and thermal biology of a free-ranging small mammal.

    PubMed

    Stawski, Clare; Körtner, Gerhard; Nowack, Julia; Geiser, Fritz

    2016-05-15

    Ecosystems can change rapidly and sometimes irreversibly due to a number of anthropogenic and natural factors, such as deforestation and fire. How individual animals exposed to such changes respond behaviourally and physiologically is poorly understood. We quantified the phenotypic plasticity of activity patterns and torpor use - a highly efficient energy conservation mechanism - in brown antechinus (Antechinus stuartii), a small Australian marsupial mammal. We compared groups in densely vegetated forest areas (pre-fire and control) with a group in a burned, open habitat (post-fire). Activity and torpor patterns differed among groups and sexes. Females in the post-fire group spent significantly less time active than the other groups, both during the day and night. However, in males only daytime activity declined in the post-fire group, although overall activity was also reduced on cold days in males for all groups. The reduction in total or diurnal activity in the post-fire group was made energetically possible by a ~3.4-fold and ~2.2-fold increase in the proportion of time females and males, respectively, used torpor in comparison to that in the pre-fire and control groups. Overall, likely due to reproductive needs, torpor was more pronounced in females than in males, but low ambient temperatures increased torpor bout duration in both sexes. Importantly, for both male and female antechinus and likely other small mammals, predator avoidance and energy conservation - achieved by reduced activity and increased torpor use - appear to be vital for post-fire survival where ground cover and refuges have been obliterated. PMID:27001165

  9. ADAM17 promotes breast cancer cell malignant phenotype through EGFR-PI3K-AKT activation

    PubMed Central

    Zheng, Xuguang; Jiang, Feng; Katakowski, Mark; Zhang, Zheng Gang; Lu, Qing-e; Chopp, Michael

    2009-01-01

    A disintegrin and metalloproteinase-17 (ADAM17) is involved in proteolytic ectodomain shedding of several membrane-bound growth factors and cytokines. The expression and activity of ADAM17 increase under some pathological conditions such as stroke and glioma. ADAM17 promotes neural progenitor cell migration and contributes to stroke-induced neurogenesis after stroke and brain tumor growth and invasion. In the present study, we sought to elucidate whether ADAM17 contributes to breast cancer progression and its mechanisms. To this end, we examined the role of ADAM17 in the proliferation, invasion, and tube formation of MDA-MB-231 breast cancer cells in vitro. Stable transfection of the MDA-MB-231 cell line with either a plasmid for over-expression of human ADAM17, or a siRNA to ADAM17 was employed in this study to establish high or low ADAM17 expression in breast cancer cells, respectively. For study of mechanism, the ADAM17 inhibitor TAPI-2 and the PI3K-AKT inhibitor LY294002 were used to counteract high ADAM17 expression or the activated PI3K-AKT pathway. Proliferation of MDA-MB-231 breast cancer cells were tested by MTT, Bromodeoxyuridine incorporation assay, growth curve, and sulforhodamine B assay. Matrigel invasion assays were used to assess the ability of MDA-MB-231 cells to penetrate the Extra Cellular Matrix. A Matrigel tube formation assay was performed to test capillary tube formation ability. EGFR-PI3K-Akt pathway activation in MDA-MB-231 cells under different ADAM17 expression levels were tested by Western blot and ELISA. Our data show that ADAM17 promotes the MDA-MB-231 malignant phenotype by increased proliferation, invasion and angiogenesis. TGF-α, VEGF secretion and VEGF expression was increasing by ADAM17 and counteracted by ADAM17 siRNA, TAPI-2, and LY294002 in MDA-MB-231 cells. ADAM17 activated, whereas ADAM17 siRNA, TAPI-2, and LY294002 deactivated the EGFR-PI3K-AKT signal pathway, which correlated with MDA-MB-231 cell malignant phenotype

  10. Sexual dimorphism of brain aromatase activity in medaka: induction of a female phenotype by estradiol.

    PubMed Central

    Melo, A C; Ramsdell, J S

    2001-01-01

    In this study we identified sex-dependent dimorphism of brain aromatase in the teleost medaka and examined its regulation by sex steriods. We first investigated differential distribution of brain aromatase activity in sexually mature male and female medaka in serial coronal sections of the brain and identified the hypothalamic nuclei contained in each section using the brain atlas of medaka. In the brain of male medaka, high levels of activity are localized in sections containing the preoptic (POA) and suprachiasmatic nuclei (SC) (63-75 fmol/hr) and low levels in the nuclei periventricular dorsalis (HD), ventralis (HV), and caudalis (Hc), nuclei diffusus of lobulus inferiores (NDIL), and nuclei tuberi anteriores (TA) and posteriores (TP) (< 25 fmol/hr). In the brain of female medaka high aromatase activity is localized in sections containing the HD, HV, Hc, NDIL, TA, and TP (85-80 fmol/hr) and highly variable levels in the POA and SC (23-70 fmol/hr). The concentration and time dependency of the exposure of male medaka to estradiol on the total brain aromatase activity and morphologic sex characteristics were determined next. Estradiol increased the activity of brain aromatase in a concentration-dependent manner at 2.5 and 25 microg/L, but the increase was lower at higher concentrations of the hormone. The effect was time dependent, gradually increasing up to the fifth day of exposure, after which it reached a plateau. Estradiol induction of brain aromatase analyzed using Lineweaver-Burke plots of saturation assays revealed a non-first-order reaction. The results indicate that a positive feedback mechanism regulates brain aromatase and imply that the sexual dimorphic distribution of aromatase may be highly sensitive to physiologic cues and environmental perturbations in fish. PMID:11333187

  11. Oxysterol mixture and, in particular, 27-hydroxycholesterol drive M2 polarization of human macrophages.

    PubMed

    Marengo, Barbara; Bellora, Francesca; Ricciarelli, Roberta; De Ciucis, Chiara; Furfaro, AnnaLisa; Leardi, Riccardo; Colla, Renata; Pacini, Davide; Traverso, Nicola; Moretta, Alessandro; Pronzato, Maria Adelaide; Bottino, Cristina; Domenicotti, Cinzia

    2016-01-01

    Macrophages play a crucial role in atherosclerosis progression. Classically activated M1 macrophages have been found in rupture-prone atherosclerotic plaques whereas alternatively activated macrophages, M2, localize in stable plaque. Macrophage accumulation of cholesterol and of its oxidized derivatives (oxysterols) leads to the formation of foam cells, a hallmark of atherosclerotic lesions. In this study, the effects of oxysterols in determining the functional polarization of human macrophages were investigated. Monocytes, purified from peripheral blood mononuclear cells of healthy donors, were differentiated into macrophages (M0) and treated with an oxysterol mixture, cholesterol, or ethanol, every 4 H for a total of 4, 8, and 12 H. The administration of the compounds was repeated in order to maintain the levels of oxysterols constant throughout the treatment. Compared with ethanol treatment, the oxysterol mixture decreased the surface expression of CD36 and CD204 scavenger receptors and reduced the amount of reactive oxygen species whereas it did not affect either cell viability or matrix metalloprotease-9 activity. Moreover, the oxysterol mixture increased the expression of both liver X receptor α and ATP-binding cassette transporter 1. An enhanced secretion of the immunoregulatory cytokine IL-10 accompanied these events. The results supported the hypothesis that the constant levels of oxysterols and, in particular, of 27-hydroxycholesterol stimulate macrophage polarization toward the M2 immunomodulatory functional phenotype, contributing to the stabilization of atherosclerotic plaques. © 2015 The Authors BioFactors published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 42(1):80-92, 2016. PMID:26669587

  12. Exoplanets in the M2K Survey

    NASA Astrophysics Data System (ADS)

    Boyajian, Tabetha; Fischer, Debra; Gaidos, Eric; Giguere, Matt

    2013-07-01

    Late type stars are ideal targets for the detection of low-mass planets residing in habitable zones. In such systems, not only is the stellar noise a minimum, but the lower stellar mass affords larger reflex velocities and the lower stellar luminosity moves the habitable zone inward. The M2K program is a high precision Doppler survey monitoring a couple hundred late-type stars over the past few years in search for such important exoplanetary systems. We present updated orbits of known exoplanet systems and newly detected exoplanet systems that have resulted from this program. We also advertise the Planethunters.org "Guest Scientist" program as well as our survey to measure stellar diameters and temperatures with long baseline optical interferometry.

  13. LOSA-M2 aerosol Raman lidar

    SciTech Connect

    Balin, Yu S; Bairashin, G S; Kokhanenko, G P; Penner, I E; Samoilova, S V

    2011-10-31

    The scanning LOSA-M2 aerosol Raman lidar, which is aimed at probing atmosphere at wavelengths of 532 and 1064 nm, is described. The backscattered light is received simultaneously in two regimes: analogue and photon-counting. Along with the signals of elastic light scattering at the initial wavelengths, a 607-nm Raman signal from molecular nitrogen is also recorded. It is shown that the height range of atmosphere probing can be expanded from the near-Earth layer to stratosphere using two (near- and far-field) receiving telescopes, and analogue and photon-counting lidar signals can be combined into one signal. Examples of natural measurements of aerosol stratification in atmosphere along vertical and horizontal paths during the expeditions to the Gobi Desert (Mongolia) and Lake Baikal areas are presented.

  14. Perivascular M2 Macrophages Stimulate Tumor Relapse after Chemotherapy

    PubMed Central

    Hughes, Russell; Qian, Bin-Zhi; Rowan, Charlotte; Muthana, Munitta; Keklikoglou, Ioanna; Olson, Oakley C.; Tazzyman, Simon; Danson, Sarah; Addison, Christina; Clemons, Mark; Gonzalez-Angulo, Ana Maria; Joyce, Johanna A.; De Palma, Michele; Pollard, Jeffrey W.; Lewis, Claire E.

    2016-01-01

    Tumor relapse after chemotherapy-induced regression is a major clinical problem, because it often involves inoperable metastatic disease. Tumor-associated macrophages (TAM) are known to limit the cytotoxic effects of chemotherapy in preclinical models of cancer. Here, we report that an alternatively activated (M2) subpopulation of TAMs (MRC1+TIE2HiCXCR4Hi) accumulate around blood vessels in tumors after chemotherapy, where they promote tumor revascularization and relapse, in part, via VEGF-A release. A similar perivascular, M2-related TAM subset was present in human breast carcinomas and bone metastases after chemotherapy. Although a small proportion of M2 TAMs were also present in hypoxic tumor areas, when we genetically ablated their ability to respond to hypoxia via hypoxia-inducible factors 1 and 2, tumor relapse was unaffected. TAMs were the predominant cells expressing immunoreactive CXCR4 in chemotherapy-treated mouse tumors, with the highest levels expressed by MRC1+ TAMs clustering around the tumor vasculature. Furthermore, the primary CXCR4 ligand, CXCL12, was upregulated in these perivascular sites after chemotherapy, where it was selectively chemotactic for MRC1+ TAMs. Interestingly, HMOX-1, a marker of oxidative stress, was also upregulated in perivascular areas after chemotherapy. This enzyme generates carbon monoxide from the breakdown of heme, a gas known to upregulate CXCL12. Finally, pharmacologic blockade of CXCR4 selectively reduced M2-related TAMs after chemotherapy, especially those in direct contact with blood vessels, thereby reducing tumor revascularization and regrowth. Our studies rationalize a strategy to leverage chemotherapeutic efficacy by selectively targeting this perivascular, relapse-promoting M2-related TAM cell population. PMID:26269531

  15. Reporter Phage and Breath Tests: Emerging Phenotypic Assays for Diagnosing Active Tuberculosis, Antibiotic Resistance, and Treatment Efficacy

    PubMed Central

    Jain, Paras; Thaler, David S.; Maiga, Mamoudou; Timmins, Graham S.; Bishai, William R.; Hatfull, Graham F.; Larsen, Michelle H.; Jacobs, William R.

    2011-01-01

    The rapid and accurate diagnosis of active tuberculosis (TB) and its drug susceptibility remain a challenge. Phenotypic assays allow determination of antibiotic susceptibilities even if sequence data are not available or informative. We review 2 emerging diagnostic approaches, reporter phage and breath tests, both of which assay mycobacterial metabolism. The reporter phage signal, Green fluorescent protein (GFP) or β-galactosidase, indicates transcription and translation inside the recipient bacilli and its attenuation by antibiotics. Different breath tests assay, (1) exhaled antigen 85, (2) mycobacterial urease activity, and (3) detection by trained rats of disease-specific odor in sputum, have also been developed. When compared with culture, reporter phage assays shorten the time for initial diagnosis of drug susceptibility by several days. Both reporter phage and breath tests have promise as early markers to determine the efficacy of treatment. While sputum often remains smear and Mycobacterium tuberculosis DNA positive early in the course of efficacious antituberculous treatment, we predict that both breath and phage tests will rapidly become negative. If this hypothesis proves correct, phage assays and breath tests could become important surrogate markers in early bactericidal activity (EBA) studies of new antibiotics. PMID:21996696

  16. Phenotypic assessment of the ovicidal activity of monepantel and monepantel sulfone on gastro-intestinal nematode eggs.

    PubMed

    Bartley, D J; Meslé, M; Donegan, H; Devin, L; Morrison, A A

    2016-04-15

    The in vitro ovicidal activity of the amino acetonitrile derivative, monepantel (MPTL) and its active metabolite monepantel sulfone (MPTL-SO2) were assessed against a number of commercially important nematode species of ruminants, namely Teladorsagia circumcincta, Haemonchus contortus and Trichostrongylus axei. An egg hatch test (EHT) was used to make the assessment of both drug sensitive and drug resistant isolates. Both MPTL and MPTL-SO2 showed moderate ovicidal activity in vitro against all of the species examined, although species specific differences as measured by inhibitory concentration were observed. Analysis of the drug sensitive isolates showed H. contortus to be the most sensitive to both MPTL and MPTL-SO2 (ED50 1.7 and 2.7μg/ml respectively) followed by T. circumcincta (ED50 2.1 and 2.7μg/ml respectively) followed by T. axei (ED50 68.7 and 60.1μg/ml respectively). Overall the EHT results would suggest no "global" in vitro discriminatory dose for detection of MPTL resistance is likely to be achievable, using the egg hatch test, due to large inherent variability observed between species. The test identified a dose dependent increase in MPTL and MPTL-SO2 sensitivity in two MPTL resistant T. circumcincta isolates and therefore offers to be a promising tool for the phenotypic characterisation of MPTL sensitivity, allowing exploration into the mechanisms involved in selection and development of MPTL resistance. PMID:26995727

  17. Effects of early-onset voluntary exercise on adult physical activity and associated phenotypes in mice.

    PubMed

    Acosta, Wendy; Meek, Thomas H; Schutz, Heidi; Dlugosz, Elizabeth M; Vu, Kim T; Garland, Theodore

    2015-10-01

    The purpose of this study was to evaluate the effects of early-life exercise on adult physical activity (wheel running, home-cage activity), body mass, food consumption, and circulating leptin levels in males from four replicate lines of mice selectively bred for high voluntary wheel running (High Runner or HR) and their four non-selected control (C) lines. Half of the mice were given wheel access shortly after weaning for three consecutive weeks. Wheel access was then removed for 52 days, followed by two weeks of adult wheel access for all mice. A blood sample taken prior to adult wheel testing was analyzed for circulating leptin concentration. Early-life wheel access significantly increased adult voluntary exercise on wheels during the first week of the second period of wheel access, for both HR and C mice, and HR ran more than C mice. During this same time period, activity in the home cages was not affected by early-age wheel access, and did not differ statistically between HR and C mice. Throughout the study, all mice with early wheel access had lower body masses than their sedentary counterparts, and HR mice had lower body masses than C mice. With wheel access, HR mice also ate significantly more than C mice. Early-life wheel access increased plasma leptin levels (adjusted statistically for fat-pad mass as a covariate) in C mice, but decreased them in HR mice. At sacrifice, early-life exercise had no statistically significant effects on visceral fat pad, heart (ventricle), liver or spleen masses (all adjusted statistically for variation in body mass). Results support the hypothesis that early-age exercise in mice can have at least transitory positive effects on adult levels of voluntary exercise, in addition to reducing body mass, and may be relevant for the public policy debates concerning the importance of physical education for children. PMID:26079567

  18. Clarification of the phenotypic characteristics and anti-tumor activity of Hedyotis diffusa.

    PubMed

    Lee, Hong-Zin; Bau, Da-Tian; Kuo, Chao-Lin; Tsai, Ru-Yin; Chen, Yu-Chang; Chang, Yu-Hao

    2011-01-01

    Hedyotis diffusa Willd. (Rubiaceae) is an important folk herb used to prevent and cure hepatitis and liver cancer in Taiwan. For differentiation of H. diffusa from counterfeits, macroscopic and microscopic characters of H. diffusa, H. corymbosa and H. tenelliflora were examined in this study. According to Trypan blue exclusion assay and Western blot analysis, H. diffusa had a significant inhibition of cell growth and induction of cell apoptosis in COLO 205 (colon cancer), Hep 3B (hepatocellular carcinoma) and H460 (lung cancer) cell lines. This study also used high-performance liquid chromatography (HPLC) to determine the quality control of H. diffusa. The HPLC data showed that ursolic and oleanolic acid are the components of the H. diffusa, consisting of approximately 4.66-4.80% and 1.86-1.96%, respectively. Our study also demonstrated that ursolic acid has significant anti-tumor activity in COLO 205, Hep 3B and H460 cancer cells. PMID:21213409

  19. Childhood-onset systemic lupus erythematosus in Singapore: clinical phenotypes, disease activity, damage, and autoantibody profiles.

    PubMed

    Tan, J H T; Hoh, S F; Win, M T M; Chan, Y H; Das, L; Arkachaisri, T

    2015-08-01

    Childhood-onset systemic lupus erythematosus (cSLE) is a multisystem autoimmune disease characterized by immune dysregulation affecting patients less than 18 years old. One-fifth of SLE cases are diagnosed during childhood. cSLE presents differently from adults and has a more severe and aggressive course. We describe the clinical and antibody profiles in our cSLE Singapore cohort. All cSLE patients who satisfied the 1997 American College of Rheumatology diagnostic criteria were captured in our lupus registry from January 2009 to January 2014. Data including demographic, cumulative clinical, serologic data, and damage indices were collected. Adjusted mean SLEDAI-2K (AMS) was used to summarize disease activity over multiple visits. Cluster analysis using non-hierarchical K-means procedure was performed on eight selected antibodies. The 64 patients (female:male ratio 5:1; Chinese 45.3%, Malay 28.1%, Indian 9.4%, and other races 17.2%) had a mean onset age of 11.5 years (range 2.1-16.7) and mean age at diagnosis was 11.9 years (range 2.6-18.0). Our study demonstrated differences in clinical manifestations for which hematologic involvement was the most common manifestation with less renal disease and uncommon neurologic manifestation as compared to other cSLE cohorts reported in our region. Antibody clusters were identified in our cohort but their clinical association/discrimination and outcome prediction required further validation study. Outcomes of our cohort in regard to disease activity after therapy and organ damages were comparable if not better to other cSLE cohorts elsewhere. Steroid-related damage, including symptomatic multifocal avascular necrosis and cataract, were not uncommon locally. Infection remains the major cause of death for the continent. Nevertheless, the five year survival rate of our cohort (98.4%) was high. PMID:25926055

  20. Global Phenotypic Characterization of Effects of Fluoroquinolone Resistance Selection on the Metabolic Activities and Drug Susceptibilities of Clostridium perfringens Strains

    PubMed Central

    Park, Miseon

    2014-01-01

    Fluoroquinolone resistance affects toxin production of Clostridium perfringens strains differently. To investigate the effect of fluoroquinolone resistance selection on global changes in metabolic activities and drug susceptibilities, four C. perfringens strains and their norfloxacin-, ciprofloxacin-, and gatifloxacin-resistant mutants were compared in nearly 2000 assays, using phenotype microarray plates. Variations among mutant strains resulting from resistance selection were observed in all aspects of metabolism. Carbon utilization, pH range, osmotic tolerance, and chemical sensitivity of resistant strains were affected differently in the resistant mutants depending on both the bacterial genotype and the fluoroquinolone to which the bacterium was resistant. The susceptibilities to gentamicin and erythromycin of all resistant mutants except one increased, but some resistant strains were less susceptible to amoxicillin, cefoxitin, ceftriaxone, chloramphenicol, and metronidazole than their wild types. Sensitivity to ethidium bromide decreased in some resistant mutants and increased in others. Microarray analysis of two gatifloxacin-resistant mutants showed changes in metabolic activities that were correlated with altered expression of various genes. Both the chemical structures of fluoroquinolones and the genomic makeup of the wild types influenced the changes found in resistant mutants, which may explain some inconsistent reports of the effects of therapeutic use of fluoroquinolones on clinical isolates of bacteria. PMID:25587280

  1. Global Phenotypic Characterization of Effects of Fluoroquinolone Resistance Selection on the Metabolic Activities and Drug Susceptibilities of Clostridium perfringens Strains.

    PubMed

    Park, Miseon; Rafii, Fatemeh

    2014-01-01

    Fluoroquinolone resistance affects toxin production of Clostridium perfringens strains differently. To investigate the effect of fluoroquinolone resistance selection on global changes in metabolic activities and drug susceptibilities, four C. perfringens strains and their norfloxacin-, ciprofloxacin-, and gatifloxacin-resistant mutants were compared in nearly 2000 assays, using phenotype microarray plates. Variations among mutant strains resulting from resistance selection were observed in all aspects of metabolism. Carbon utilization, pH range, osmotic tolerance, and chemical sensitivity of resistant strains were affected differently in the resistant mutants depending on both the bacterial genotype and the fluoroquinolone to which the bacterium was resistant. The susceptibilities to gentamicin and erythromycin of all resistant mutants except one increased, but some resistant strains were less susceptible to amoxicillin, cefoxitin, ceftriaxone, chloramphenicol, and metronidazole than their wild types. Sensitivity to ethidium bromide decreased in some resistant mutants and increased in others. Microarray analysis of two gatifloxacin-resistant mutants showed changes in metabolic activities that were correlated with altered expression of various genes. Both the chemical structures of fluoroquinolones and the genomic makeup of the wild types influenced the changes found in resistant mutants, which may explain some inconsistent reports of the effects of therapeutic use of fluoroquinolones on clinical isolates of bacteria. PMID:25587280

  2. RasGRP3, a Ras activator, contributes to signaling and the tumorigenic phenotype in human melanoma

    PubMed Central

    Li, Luowei; Kedei, Noemi; Tóth, Zsuzsanna E.; Czap, Alexandra; Velasquez, Julia F.; Mihova, Daniela; Michalowski, Aleksandra M.; Yuspa, Stuart H.; Blumberg, Peter M.

    2014-01-01

    RasGRP3, an activator for H-Ras, R-Ras and Rap1/2, has emerged as an important mediator of signaling downstream from receptor coupled phosphoinositide turnover in B and T cells. Here, we report that RasGRP3 showed a high level of expression in multiple human melanoma cell lines as well as in a subset of human melanoma tissue samples. Suppression of endogenous RasGRP3 expression in these melanoma cell lines reduced Ras-GTP formation as well as c-Met expression and Akt phosphorylation downstream from HGF or EGF stimulation. RasGRP3 suppression also inhibited cell proliferation and reduced both colony formation in soft agar and xenograft tumor growth in immunodeficient mice, demonstrating the importance of RasGRP3 for the transformed phenotype of the melanoma cells. Reciprocally, overexpression of RasGRP3 in human primary melanocytes altered cellular morphology, markedly enhanced cell proliferation, and rendered the cells tumorigenic in a mouse xenograft model. Suppression of RasGRP3 expression in these cells inhibited downstream RasGRP3 responses and suppressed cell growth, confirming the functional role of RasGRP3 in the altered behavior of these cells. The identification of the role of RasGRP3 in melanoma highlights its importance, as a Ras activator, in the phosphoinositide signaling pathway in human melanoma and provides a new potential therapeutic target. PMID:21602881

  3. Predominance of M2-polarized macrophages in bladder cancer affects angiogenesis, tumor grade and invasiveness

    PubMed Central

    TAKEUCHI, HISASHI; TANAKA, MICHIO; TANAKA, AYAKO; TSUNEMI, AKISA; YAMAMOTO, HIDENOBU

    2016-01-01

    Tumor-associated macrophages (TAMs) often assume an immunoregulatory M2 phenotype. Thus, the aim of the present study was to clarify the correlation of vascularity and TAMs, in particular the M2 phenotype in the stroma and tumor areas, with the clinical and pathological outcomes of patients with bladder cancer. The TAM counts and microvessel counts (MVCs) were determined immunohistochemically in 21 patients with bladder cancer. The number of infiltrating TAMs was measured using immunohistochemistry with anti-cluster of differentiation (CD)68 and anti-CD163 antibodies, to identify a macrophage lineage marker and an M2-polarized-specific cell surface receptor, respectively. CD68+ and CD163+ macrophages were evaluated in the stroma and tumor areas, and areas with a high density of infiltrating cell spots were counted. MVCs were determined using immunohistochemistry with anti-CD34 antibodies. The results revealed that the higher ratio of CD163+/CD68+ macrophages in the stroma, tumor and total tumor tissues were correlated with a higher stage and grade (P<0.05). In addition, the low ratio of CD68+/CD34+ microvessels was correlated with a higher stage (P<0.05). There was also a positive correlation between TAMs and MVC (r2=0.25; P<0.05). These results suggest that the TAM polarized M2 phenotype affects microvessels, pathological outcome, tumor grade and invasiveness. PMID:27123124

  4. Protease activated receptor-1 inhibits the Maspin tumor-suppressor gene to determine the melanoma metastatic phenotype

    PubMed Central

    Villares, Gabriel J.; Zigler, Maya; Dobroff, Andrey S.; Wang, Hua; Song, Renduo; Melnikova, Vladislava O.; Huang, Li; Braeuer, Russell R.; Bar-Eli, Menashe

    2011-01-01

    The thrombin receptor protease activated receptor-1 (PAR-1) is overexpressed in metastatic melanoma cell lines and tumor specimens. Previously, we demonstrated a significant reduction in tumor growth and experimental lung metastasis after PAR-1 silencing via systemic delivery of siRNA encapsulated into nanoliposomes. Gene expression profiling identified a 40-fold increase in expression of Maspin in PAR-1–silenced metastatic melanoma cell lines. Maspin promoter activity was significantly increased after PAR-1 silencing, suggesting that PAR1 negatively regulates Maspin at the transcriptional level. ChIP analyses revealed that PAR-1 decreases binding of Ets-1 and c-Jun transcription factors to the Maspin promoter, both known to activate Maspin transcription. PAR-1 silencing did not affect Ets-1 or c-Jun expression; rather it resulted in increased expression of the chromatin remodeling complex CBP/p300, as well as decreased activity of the CBP/p300 inhibitor p38, resulting in increased binding of Ets-1 and c-Jun to the Maspin promoter and higher Maspin expression. Functionally, Maspin expression reduced the invasive capability of melanoma cells after PAR-1 silencing, which was abrogated after rescuing with PAR-1. Furthermore, tumor growth and experimental lung metastasis was significantly decreased after expressing Maspin in a metastatic melanoma cell line. Moreover, silencing Maspin in PAR-1–silenced cells reverted the inhibition of tumor growth and experimental lung metastasis. Herein, we demonstrate a mechanism by which PAR-1 negatively regulates the expression of the Maspin tumor-suppressor gene in the acquisition of the metastatic melanoma phenotype, thus attributing an alternative function to PAR-1 other than coagulation. PMID:21187389

  5. Protease activated receptor-1 inhibits the Maspin tumor-suppressor gene to determine the melanoma metastatic phenotype.

    PubMed

    Villares, Gabriel J; Zigler, Maya; Dobroff, Andrey S; Wang, Hua; Song, Renduo; Melnikova, Vladislava O; Huang, Li; Braeuer, Russell R; Bar-Eli, Menashe

    2011-01-11

    The thrombin receptor protease activated receptor-1 (PAR-1) is overexpressed in metastatic melanoma cell lines and tumor specimens. Previously, we demonstrated a significant reduction in tumor growth and experimental lung metastasis after PAR-1 silencing via systemic delivery of siRNA encapsulated into nanoliposomes. Gene expression profiling identified a 40-fold increase in expression of Maspin in PAR-1-silenced metastatic melanoma cell lines. Maspin promoter activity was significantly increased after PAR-1 silencing, suggesting that PAR1 negatively regulates Maspin at the transcriptional level. ChIP analyses revealed that PAR-1 decreases binding of Ets-1 and c-Jun transcription factors to the Maspin promoter, both known to activate Maspin transcription. PAR-1 silencing did not affect Ets-1 or c-Jun expression; rather it resulted in increased expression of the chromatin remodeling complex CBP/p300, as well as decreased activity of the CBP/p300 inhibitor p38, resulting in increased binding of Ets-1 and c-Jun to the Maspin promoter and higher Maspin expression. Functionally, Maspin expression reduced the invasive capability of melanoma cells after PAR-1 silencing, which was abrogated after rescuing with PAR-1. Furthermore, tumor growth and experimental lung metastasis was significantly decreased after expressing Maspin in a metastatic melanoma cell line. Moreover, silencing Maspin in PAR-1-silenced cells reverted the inhibition of tumor growth and experimental lung metastasis. Herein, we demonstrate a mechanism by which PAR-1 negatively regulates the expression of the Maspin tumor-suppressor gene in the acquisition of the metastatic melanoma phenotype, thus attributing an alternative function to PAR-1 other than coagulation. PMID:21187389

  6. Progeny from irradiated colorectal cancer cells acquire an EMT-like phenotype and activate Wnt/β-catenin pathway.

    PubMed

    Bastos, Lilian Gonçalves dos Reis; de Marcondes, Priscila Guimarães; de-Freitas-Junior, Julio Cesar Madureira; Leve, Fernanda; Mencalha, André Luiz; de Souza, Waldemir Fernandes; de Araujo, Wallace Martins; Tanaka, Marcelo Neves; Abdelhay, Eliana Saul Furquim Werneck; Morgado-Díaz, José Andrés

    2014-12-01

    Radiotherapy remains a major approach to adjuvant therapy for patients with advanced colorectal cancer, however, the fractionation schedules frequently allow for the repopulation of surviving tumors cells, neoplastic progression, and subsequent metastasis. The aim of the present study was to analyze the transgenerational effects induced by radiation and evaluate whether it could increase the malignant features on the progeny derived from irradiated parental colorectal cancer cells, Caco-2, HT-29, and HCT-116. The progeny of these cells displayed a differential radioresistance as seen by clonogenic and caspase activation assay and had a direct correlation with survivin expression as observed by immunoblotting. Immunofluorescence showed that the most radioresistant progenies had an aberrant morphology, disturbance of the cell-cell adhesion contacts, disorganization of the actin cytoskeleton, and vimentin filaments. Only the progeny derived from intermediary radioresistant cells, HT-29, reduced the E-cadherin expression and overexpressed β-catenin and vimentin with increased cell migration, invasion, and metalloprotease activation as seen by immunoblotting, wound healing, invasion, and metalloprotease activity assay. We also observed that this most aggressive progeny increased the Wnt/β-catenin-dependent TCF/LEF activity and underwent an upregulation of mesenchymal markers and downregulation of E-cadherin, as determined by qRT-PCR. Our results showed that the intermediate radioresistant cells can generate more aggressive cellular progeny with the EMT-like phenotype. The Wnt/β-catenin pathway may constitute an important target for new adjuvant treatment schedules with radiotherapy, with the goal of reducing the migratory and invasive potential of the remaining cells after treatment. PMID:25103643

  7. Estrogen-related receptor gamma regulates dopaminergic neuronal phenotype by activating GSK3β/NFAT signaling in SH-SY5Y cells.

    PubMed

    Lim, Juhee; Choi, Hueng-Sik; Choi, Hyun Jin

    2015-05-01

    The orphan nuclear receptor estrogen-related receptor gamma (ERRγ) is highly expressed in the nervous system during embryogenesis and in adult brains, but its physiological role in neuronal development remains unknown. In this study, we evaluated the relevance of ERRγ in regulating dopaminergic (DAergic) phenotype and the corresponding signaling pathway. We used retinoic acid (RA) to differentiate human neuroblastoma SH-SY5Y cells. RA induced neurite outgrowth of SH-SY5Y cells with an increase in DAergic neuron-like properties, including up-regulation of tyrosine hydroxylase, dopamine transporter, and vesicular monoamine transporter 2. ERRγ, but not ERRα, was up-regulated by RA, and participated in RA effect on SH-SY5Y cells. ERRγ over-expression enhanced mature DAergic neuronal phenotype with neurite outgrowth as with RA treatment; and RA-induced increase in DAergic phenotype was attenuated by silencing ERRγ expression. ERRγ appears to have a crucial role in morphological and functional regulation of cells that is selective for DAergic neurons. Polo-like kinase 2 was up-regulated in ERRγ-over-expressing SH-SY5Y cells, which was involved in phosphorylation of glycogen synthase kinase 3β and resulting downstream activation of nuclear factor of activated T cells. The likely involvement of ERRγ in regulating the DAergic neuronal phenotype makes this orphan nuclear receptor a novel target for understanding DAergic neuronal differentiation. We propose the relevance of estrogen-related receptor gamma (ERRγ) in regulating dopaminergic neuronal phenotype: ERRγ is up-regulated by retinoic acid in SH-SY5Y cells, and enhances dopaminergic phenotypes and induces neurite outgrowth; Polo-like kinase 2 (PLK2) and glycogen synthase kinase 3 beta/nuclear factor of activated T cells (GSK3β/NFAT) signaling are responsible for the ERRγ effect. Our findings provide the first insights into the role of ERRγ in the brain, as a novel approach toward understanding

  8. Surface modification of nanoparticles enables selective evasion of phagocytic clearance by distinct macrophage phenotypes

    NASA Astrophysics Data System (ADS)

    Qie, Yaqing; Yuan, Hengfeng; von Roemeling, Christina A.; Chen, Yuanxin; Liu, Xiujie; Shih, Kevin D.; Knight, Joshua A.; Tun, Han W.; Wharen, Robert E.; Jiang, Wen; Kim, Betty Y. S.

    2016-05-01

    Nanomedicine is a burgeoning industry but an understanding of the interaction of nanomaterials with the immune system is critical for clinical translation. Macrophages play a fundamental role in the immune system by engulfing foreign particulates such as nanoparticles. When activated, macrophages form distinct phenotypic populations with unique immune functions, however the mechanism by which these polarized macrophages react to nanoparticles is unclear. Furthermore, strategies to selectively evade activated macrophage subpopulations are lacking. Here we demonstrate that stimulated macrophages possess higher phagocytic activities and that classically activated (M1) macrophages exhibit greater phagocytic capacity than alternatively activated (M2) macrophages. We show that modification of nanoparticles with polyethylene-glycol results in decreased clearance by all macrophage phenotypes, but importantly, coating nanoparticles with CD47 preferentially lowers phagocytic activity by the M1 phenotype. These results suggest that bio-inspired nanoparticle surface design may enable evasion of specific components of the immune system and provide a rational approach for developing immune tolerant nanomedicines.

  9. Influenza M2 Transmembrane Domain Senses Membrane Heterogeneity and Enhances Membrane Curvature.

    PubMed

    Ho, Chian Sing; Khadka, Nawal K; She, Fengyu; Cai, Jianfeng; Pan, Jianjun

    2016-07-01

    Targeting host cell membranes by M2 of influenza A virus is important for virus invasion and replication. We study the transmembrane domain of M2 (M2TM) interacting with mica-supported planar bilayers and free-standing giant unilamellar vesicles (GUVs). Using solution atomic force microscopy (AFM), we show that the size of M2TM oligomers is dependent on lipid composition. The addition of M2TM to lipid bilayers containing liquid-ordered (Lo) and liquid-disordered (Ld) phases reveals that M2TM preferentially partitions into the Ld phase; phase-dependent partitioning results in a larger rigidity of the Ld phase. We next use fluorescence microscopy to study the effects of M2TM on phase-coexisting GUVs. In particular, M2TM is found to increase GUVs' miscibility transition temperature Tmix. The augmented thermodynamic stability can be accounted for by considering an enhanced energy barrier of lipid mixing between coexisting phases. Our GUV study also shows that M2TM can elicit an array of vesicle shapes mimicking virus budding. M2TM enhanced membrane curvature is consistent with our AFM data, which show altered membrane rigidity and consequently line tension at domain edges. Together, our results highlight that in addition to conducting protons, M2TM can actively regulate membrane heterogeneity and augment membrane curvature. PMID:27285399

  10. Mitochondrial Ultrastructural Alterations and Declined M2 Receptor Density Were Involved in Cardiac Dysfunction in Rats after Long Term Treatment with Autoantibodies against M2 Muscarinic Receptor

    PubMed Central

    Wang, Jin; Wang, Li; Wu, Ye; Wang, Jie; Lv, Tingting; Liu, Huirong

    2015-01-01

    Background Previous studies showed that autoantibodies (M2-AA) against the second extracellular loop of M2 muscarinic receptor (M2AChR-el2) from dilated cardiomyopathy (DCM) serum could induce DCM-like morphological changes in mice hearts. However, the effects of M2-AA on the cardiac function during the process of DCM and the potential mechanisms are not fully known. The present study was designed to dynamically observe the cardiac function, mitochondrial changes, and M2 receptor binding characteristics in rats long-term stimulated with M2-AA in vivo. Methods M2-AA-positive model was established by actively immunizing healthy male Wistar rats with synthetic M2AChR-el2 peptide for 18 months. Meanwhile, vehicle group rats were administrated with physiological saline. The change of mitochondrial membrane potential (ΔΨm) was detected by radionuclide imaging. The ultrastructure of mitochondria was observed under electron microscopy. The M2 receptor binding characteristics were determined by radioactive ligand binding assay. Results After immunization for 12 months, compared with vehicle group, M2AChR-el2-immunized rats showed decreased myocardial contractility and cardiac diastolic function evidenced by declined maximal rate of rise of ventricular pressure and increased left ventricular end-diastolic pressure, respectively. Additionally, mitochondrial swelling and vacuolation were observed. At 18 months, M2AChR-el2-immunized rats manifested significant decreased cardiac systolic and diastolic function and pathological changes such as enlargement of right ventricular cavity and wall thinning; and the mitochondrial damage was aggravated. Furthermore, the M2 receptor maximum binding capacity (Bmax) of the M2AChR-el2-immunized rats significantly decreased, while the M2 receptor dissociation constant (Kd) was increased. Conclusions Our study suggested that long-term stimulation with M2-AA leaded to the ventricular dilatation and gradual deterioration of cardiac dysfunction

  11. AHCC Activation and Selection of Human Lymphocytes via Genotypic and Phenotypic Changes to an Adherent Cell Type: A Possible Novel Mechanism of T Cell Activation

    PubMed Central

    Olamigoke, Loretta; Mansoor, Elvedina; Mann, Vivek; Ellis, Ivory; Okoro, Elvis; Wakame, Koji; Fuji, Hajime; Kulkarni, Anil; Francoise Doursout, Marie; Sundaresan, Alamelu

    2015-01-01

    Active Hexose Correlated Compound (AHCC) is a fermented mushroom extract and immune supplement that has been used to treat a wide range of health conditions. It helps in augmentation of the natural immune response and affects immune cell activation and outcomes. The goal of this project was to study and understand the role and mechanisms of AHCC supplementation in the prevention of immunosuppression through T cell activation. The method described here involves “in vitro” culturing of lymphocytes, exposing them to different concentrations of AHCC (0 μg/mL, 50 μg/mL, 100 μg/mL, 250 μg/mL, and 500 μg/mL) at 0 hours. Interestingly, clumping and aggregation of the cells were seen between 24 and 72 hours of incubation. The cells lay down extracellular matrix, which become adherent, and phenotypical changes from small rounded lymphocytes to large macrophage-like, spindle shaped, elongated, fibroblast-like cells even beyond 360 hours were observed. These are probably translated from genotypic changes in the cells since the cells propagate for at least 3 to 6 generations (present observations). RNA isolated was subjected to gene array analysis. We hypothesize that cell adhesion is an activation and survival pathway in lymphocytes and this could be the mechanism of AHCC activation in human lymphocytes. PMID:26788109

  12. AHCC Activation and Selection of Human Lymphocytes via Genotypic and Phenotypic Changes to an Adherent Cell Type: A Possible Novel Mechanism of T Cell Activation.

    PubMed

    Olamigoke, Loretta; Mansoor, Elvedina; Mann, Vivek; Ellis, Ivory; Okoro, Elvis; Wakame, Koji; Fuji, Hajime; Kulkarni, Anil; Francoise Doursout, Marie; Sundaresan, Alamelu

    2015-01-01

    Active Hexose Correlated Compound (AHCC) is a fermented mushroom extract and immune supplement that has been used to treat a wide range of health conditions. It helps in augmentation of the natural immune response and affects immune cell activation and outcomes. The goal of this project was to study and understand the role and mechanisms of AHCC supplementation in the prevention of immunosuppression through T cell activation. The method described here involves "in vitro" culturing of lymphocytes, exposing them to different concentrations of AHCC (0 μg/mL, 50 μg/mL, 100 μg/mL, 250 μg/mL, and 500 μg/mL) at 0 hours. Interestingly, clumping and aggregation of the cells were seen between 24 and 72 hours of incubation. The cells lay down extracellular matrix, which become adherent, and phenotypical changes from small rounded lymphocytes to large macrophage-like, spindle shaped, elongated, fibroblast-like cells even beyond 360 hours were observed. These are probably translated from genotypic changes in the cells since the cells propagate for at least 3 to 6 generations (present observations). RNA isolated was subjected to gene array analysis. We hypothesize that cell adhesion is an activation and survival pathway in lymphocytes and this could be the mechanism of AHCC activation in human lymphocytes. PMID:26788109

  13. Phenotypic assays identify azoramide as a small-molecule modulator of the unfolded protein response with antidiabetic activity.

    PubMed

    Fu, Suneng; Yalcin, Abdullah; Lee, Grace Y; Li, Ping; Fan, Jason; Arruda, Ana Paula; Pers, Benedicte M; Yilmaz, Mustafa; Eguchi, Kosei; Hotamisligil, Gökhan S

    2015-06-17

    The endoplasmic reticulum (ER) plays a critical role in protein, lipid, and glucose metabolism as well as cellular calcium signaling and homeostasis. Perturbation of ER function and chronic ER stress are associated with many pathologies ranging from diabetes and neurodegenerative diseases to cancer and inflammation. Although ER targeting shows therapeutic promise in preclinical models of obesity and other pathologies, the available chemical entities generally lack the specificity and other pharmacological properties required for effective clinical translation. To overcome these challenges and identify new potential therapeutic candidates, we first designed and chemically and genetically validated two high-throughput functional screening systems that independently measure the free chaperone content and protein-folding capacity of the ER. With these quantitative platforms, we characterized a small-molecule compound, azoramide, that improves ER protein-folding ability and activates ER chaperone capacity to protect cells against ER stress in multiple systems. This compound also exhibited potent antidiabetic efficacy in two independent mouse models of obesity by improving insulin sensitivity and pancreatic β cell function. Together, these results demonstrate the utility of this functional, phenotypic assay platform for ER-targeted drug discovery and provide proof of principle for the notion that specific ER modulators can be potential drug candidates for type 2 diabetes. PMID:26084805

  14. Phenotyping of UGT1A1 Activity Using Raltegravir Predicts Pharmacokinetics and Toxicity of Irinotecan in FOLFIRI

    PubMed Central

    Lee, Lawrence Soon-U; Seng, Kok-Yong; Wang, Ling-Zhi; Yong, Wei-Peng; Hee, Kim-Hor; Soh, Thomas I.; Wong, Andrea; Cheong, Pei F.; Soong, Richie; Sapari, Nur S.; Soo, Ross; Fan, Lu; Lee, Soo-Chin; Goh, Boon C.

    2016-01-01

    Background Irinotecan toxicity correlates with UGT1A1 activity. We explored whether phenotyping UGT1A1 using a probe approach works better than current genotyping methods. Methods Twenty-four Asian cancer patients received irinotecan as part of the FOLFIRI regimen. Subjects took raltegravir 400 mg orally and intravenous midazolam 1 mg. Pharmacokinetic analyses were performed using WinNonLin and NONMEM. Genomic DNA was isolated and screened for the known genetic variants in UGT1A1 and CYP3A4/5. Results SN-38G/SN-38 AUC ratio correlated well with Raltegravir glucuronide/ Raltegravir AUC ratio (r = 0.784 p<0.01). Midazolam clearance correlated well with irinotecan clearance (r = 0.563 p<0.01). SN-38 AUC correlated well with Log10Nadir Absolute Neutrophil Count (ANC) (r = -0.397 p<0.05). Significant correlation was found between nadir ANC and formation rate constant of raltegravir glucuronide (r = 0.598, P<0.005), but not UGT1A1 genotype. Conclusion Raltegravir glucuronide formation is a good predictor of nadir ANC, and can predict neutropenia in East Asian patients. Prospective studies with dose adjustments should be done to develop raltegravir as a probe to optimize irinotecan therapy. Trial Registration Clinicaltrials.gov NCT00808184 PMID:26808671

  15. Association of the epithelial-to-mesenchymal transition phenotype with responsiveness to the p21-activated kinase inhibitor, PF-3758309, in colon cancer models

    PubMed Central

    Pitts, Todd M.; Kulikowski, Gillian N.; Tan, Aik-Choon; Murray, Brion W.; Arcaroli, John J.; Tentler, John J.; Spreafico, Anna; Selby, Heather M.; Kachaeva, Maria I.; McPhillips, Kelly L.; Britt, Blair C.; Bradshaw-Pierce, Erica L.; Messersmith, Wells A.; Varella-Garcia, Marileila; Eckhardt, S. Gail

    2013-01-01

    The p21-activated kinase (PAK) family of serine/threonine kinases, which are overexpressed in several cancer types, are critical mediators of cell survival, motility, mitosis, transcription, and translation. In the study presented here, we utilized a panel of colorectal cancer (CRC) cell lines to identify potential biomarkers of sensitivity or resistance that may be used to individualize therapy to the PAK inhibitor PF-03758309. We observed a wide range of proliferative responses in the CRC cell lines exposed to PF-03758309, this response was recapitulated in other phenotypic assays such as anchorage-independent growth, three-dimensional (3D) tumor spheroid formation, and migration. Interestingly, we observed that cells most sensitive to PF-03758309 exhibited up-regulation of genes associated with a mesenchymal phenotype (CALD1, VIM, ZEB1) and cells more resistant had an up-regulation of genes associated with an epithelial phenotype (CLDN2, CDH1, CLDN3, CDH17) allowing us to derive an epithelial-to-mesenchymal transition (EMT) gene signature for this agent. We assessed the functional role of EMT-associated genes in mediating responsiveness to PF-3758309, by targeting known genes and transcriptional regulators of EMT. We observed that suppression of genes associated with the mesenchymal phenotype conferred resistance to PF-3758309, in vitro and in vivo. These results indicate that PAK inhibition is associated with a unique response phenotype in CRC and that further studies should be conducted to facilitate both patient selection and rational combination strategies with these agents. PMID:23543898

  16. The full expression of the ity phenotype in ityr mice requires C3 activation by Salmonella lipopolysaccharide.

    PubMed Central

    Nishikawa, F; Yoshikawa, S; Harada, H; Kita, M; Kita, E

    1998-01-01

    Our previous study has shown that the rapid and sufficient activation of complement by Salmonella lipopolysaccharide occurs in genetically resistant (Ityr) A/J mice. To assess whether the level of complement activation by a virulent strain of Salmonella typhimurium regulates the level of murine natural resistance, we compared levels of serum complement activation by S. typhimurium and kinetics of serum-opsonized S. typhimurium grown in macrophages using several strains of resistant (Ityr) and susceptible (Itys) mice. Itys macrophages killed intracellular S. typhimurium to the same extent as did Ityr macrophages when the pathogen was opsonized with Ityr serum. Opsonization of S. typhimurium with Itys serum reduced intracellular killing activity in Ityr macrophages to the same level as seen with Itys macrophages. Incubation of S. typhimurium with 25% Mg2+ EGTA (5 mm MgCl2-3 mm ethylene glycol-bis (beta-aminotheyl either)-N,N,N',N'-tetraacetic acid)-chelated Ityr serum resulted in higher levels of C3 deposition onto the surface of this bacteria, C3b generation and also C3 consumption, compared with that with Mg2+ EGTA-chelated Itys serum. Opsonization of S. typhimurium with A/J serum prior to infection increased early resistance in Itys mice. Infection with a virulent strain of S. typhimurium induced the expression of interleukin-10 (IL-10) mRNA at higher levels in C57BL/6 mice than in A/J mice. However, opsonization of S. typhimurium with A/J serum decreased bacterial growth in the spleen of C57BL/6 mice to the same level as observed for A/J mice in association with decreased expression levels of IL-10 mRNA. Moreover, administration of anti-C3 antibodies reduced the resistance of A/J mice in association with a decrease in serum levels of C3. These results indicate that the high level of complement activation via the alternative pathway in Ityr serum by a virulent strain of S. typhimurium reduces the virulence of this pathogen, which may contribute to the full

  17. HDL Induces the Expression of the M2 Macrophage Markers Arginase 1 and Fizz-1 in a STAT6-Dependent Process

    PubMed Central

    Fisher, Edward A.

    2013-01-01

    Our lab has previously shown in a mouse model that normalization of a low HDL level achieves atherosclerotic plaque regression. This included the shift from a pro (“M1”) to an anti-inflammatory (“M2”) phenotypic state of plaque macrophages. Whether HDL can directly cause this phenotypic change and, if so, what the signaling mechanism is, were explored in the present studies. Murine primary macrophages treated with HDL showed increased gene expression for the M2 markers Arginase-1 (Arg-1) and Fizz-1, which are classically induced by IL-4. HDL was able to potentiate the IL-4-induced changes in Arg-1, and tended to do the same for Fizz-1, while suppressing the expression of inflammatory genes in response to IFNγ. The effects of either IL-4 or HDL were suppressed when macrophages were from STAT6-/- mice, but inhibitor studies suggested differential utilization of JAK isoforms by IL-4 and HDL to activate STAT6 by phosphorylation. Overall, our results describe a new function of HDL, namely its ability to directly enrich macrophages in markers of the M2, anti-inflammatory, state in a process requiring STAT6. PMID:23991225

  18. Therapeutic Metformin/AMPK Activation Promotes the Angiogenic Phenotype in the ERα Negative MDA-MB-435 Breast Cancer Model

    PubMed Central

    Phoenix, Kathryn N.; Vumbaca, Frank; Claffey, Kevin P.

    2008-01-01

    Metformin, a first line treatment for type 2 diabetes, has been implicated as a potential anti-neoplastic agent for breast cancers as well as other cancers. Metformin is known to work in part through the activation of AMP-dependent kinase (AMPK). AMPK is a key regulator of cellular energy homeostasis, especially under stress conditions where biosynthetic pathways are blocked by the phosphorylation of downstream AMPK substrates. Stimulation of AMPK by metformin resulted in a significant repression of cell proliferation and active MAPK1/2 in both estrogen receptor α (ERα) negative (MDA-MB-231, MDA-MB-435) and positive (MCF-7, T47D) human breast cancer cell lines. However, when ERα negative MDA-MB-435 cells were treated with metformin, they demonstrated increased expression of vascular endothelial growth factor (VEGF) in an AMPK dependent manner; while the ERα positive MCF-7 cells did not. Systemic therapy with metformin was tested for efficacy in an orthotopic model of ERα negative breast cancer performed in athymic nude mice. Surprisingly, metformin therapy significantly improved tumorigenic progression as compared to untreated controls. The metformin-treated group showed increased VEGF expression, intratumoral microvascular density and reduced necrosis. Metformin treatment was sufficient, however, to reduce systemic IGF-1 and the proliferation rate of tumor cells in vascularized regions. The data presented here suggests that, although metformin significantly represses breast cancer cell growth in vitro, the efficacy with respect to its therapeutic application for ERα negative breast cancer lesions in vivo may result in promotion of the angiogenic phenotype and increased tumorigenic progression. PMID:18256928

  19. Dopamine induces growth inhibition and vascular normalization through reprogramming M2-polarized macrophages in rat C6 glioma

    SciTech Connect

    Qin, Tian; Wang, Chenlong; Chen, Xuewei; Duan, Chenfan; Zhang, Xiaoyan; Zhang, Jing; Chai, Hongyan; Tang, Tian; Chen, Honglei; Yue, Jiang; Li, Ying; Yang, Jing

    2015-07-15

    Dopamine (DA), a monoamine catecholamine neurotransmitter with antiangiogenic activity, stabilizes tumor vessels in colon, prostate and ovarian cancers, thus increases chemotherapeutic efficacy. Here, in the rat C6 glioma models, we investigated the vascular normalization effects of DA and its mechanisms of action. DA (25, 50 mg/kg) inhibited tumor growth, while a precursor of DA (levodopa) prolonged the survival time of rats bearing orthotopic C6 glioma. DA improved tumor perfusion, with significant effects from day 3, and a higher level at days 5 to 7. In addition, DA decreased microvessel density and hypoxia-inducible factor-1α expression in tumor tissues, while increasing the coverage of pericyte. Conversely, an antagonist of dopamine receptor 2 (DR2) (eticlopride) but not DR1 (butaclamol) abrogated DA-induced tumor regression and vascular normalization. Furthermore, DA improved the delivery and efficacy of temozolomide therapy. Importantly, DA increased representative M1 markers (iNOS, CXCL9, etc.), while decreasing M2 markers (CD206, arginase-1, etc.). Depletion of macrophages by clodronate or zoledronic acid attenuated the effects of DA. Notably, DA treatment induced M2-to-M1 polarization in RAW264.7 cells and mouse peritoneal macrophages, and enhanced the migration of pericyte-like cells (10T1/2), which was reversed by eticlopride or DR2-siRNA. Such changes were accompanied by the downregulation of VEGF/VEGFR2 signaling. In summary, DA induces growth inhibition and vascular normalization through reprogramming M2-polarized macrophages. Thus, targeting the tumor microvasculature by DA represents a promising strategy for human glioma therapy. - Highlights: • Dopamine induces tumor growth inhibition and vascular normalization in rat C6 glioma. • Dopamine switches macrophage phenotype from M2 to M1. • Dopamine-induced vascular normalization is mediated by macrophage polarization. • Dopamine is a promising agent targeting the microvasculature in tumor

  20. p-21 activated kinase 4 (PAK4) maintains stem cell-like phenotypes in pancreatic cancer cells through activation of STAT3 signaling.

    PubMed

    Tyagi, Nikhil; Marimuthu, Saravanakumar; Bhardwaj, Arun; Deshmukh, Sachin K; Srivastava, Sanjeev K; Singh, Ajay P; McClellan, Steven; Carter, James E; Singh, Seema

    2016-01-28

    Pancreatic cancer (PC) remains a highly lethal malignancy due to its unusual chemoresistance and high aggressiveness. A subpopulation of pancreatic tumor cells, known as cancer stem cells (CSCs), is considered responsible not only for tumor-maintenance, but also for its widespread metastasis and therapeutic failure. Here we investigated the role of p-21 activated kinase 4 (PAK4) in driving PC stemness properties. Our data demonstrate that triple-positive (CD24(+)/CD44(+)/EpCAM(+)) subpopulation of pancreatic CSCs exhibits greater level of PAK4 as compared to triple-negative (CD24(-)/CD44(-)/EpCAM(-)) cells. Moreover, PAK4 silencing in PC cells leads to diminished fraction of CD24, CD44, and EpCAM positive cells. Furthermore, we show that PAK4-silenced PC cells exhibit decreased sphere-forming ability and increased chemosensitivity to gemcitabine toxicity. PAK4 expression is also associated with enhanced levels of stemness-associated transcription factors (Oct4/Nanog/Sox2 and KLF4). Furthermore, our data show decreased nuclear accumulation and transcriptional activity of STAT3 in PAK4-silenced PC cells and restitution of its activity leads to restoration of stem cell phenotypes. Together, our findings deliver first experimental evidence for the involvement of PAK4 in PC stemness and support its clinical utility as a novel therapeutic target in PC. PMID:26546043

  1. Flavocoxid, a dual inhibitor of cyclooxygenase and 5-lipoxygenase, blunts pro-inflammatory phenotype activation in endotoxin-stimulated macrophages

    PubMed Central

    Altavilla, D; Squadrito, F; Bitto, A; Polito, F; Burnett, BP; Di Stefano, V; Minutoli, L

    2009-01-01

    Background and purpose: The flavonoids, baicalin and catechin, from Scutellaria baicalensis and Acacia catechu, respectively, have been used for various clinical applications. Flavocoxid is a mixed extract containing baicalin and catechin, and acts as a dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (LOX) enzymes. The anti-inflammatory activity, measured by protein and gene expression of inflammatory markers, of flavocoxid in rat peritoneal macrophages stimulated with Salmonella enteritidis lipopolysaccharide (LPS) was investigated. Experimental approach: LPS-stimulated (1 µg·mL−1) peritoneal rat macrophages were co-incubated with different concentrations of flavocoxid (32–128 µg·mL−1) or RPMI medium for different incubation times. Inducible COX-2, 5-LOX, inducible nitric oxide synthase (iNOS) and inhibitory protein κB-α (IκB-α) levels were evaluated by Western blot analysis. Nuclear factor κB (NF-κB) binding activity was investigated by electrophoretic mobility shift assay. Tumour necrosis factor-α (TNF-α) gene and protein expression were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. Finally, malondialdehyde (MDA) and nitrite levels in macrophage supernatants were evaluated. Key results: LPS stimulation induced a pro-inflammatory phenotype in rat peritoneal macrophages. Flavocoxid (128 µg·mL−1) significantly inhibited COX-2 (LPS = 18 ± 2.1; flavocoxid = 3.8 ± 0.9 integrated intensity), 5-LOX (LPS = 20 ± 3.8; flavocoxid = 3.1 ± 0.8 integrated intensity) and iNOS expression (LPS = 15 ± 1.1; flavocoxid = 4.1 ± 0.4 integrated intensity), but did not modify COX-1 expression. PGE2 and LTB4 levels in culture supernatants were consequently decreased. Flavocoxid also prevented the loss of IκB-α protein (LPS = 1.9 ± 0.2; flavocoxid = 7.2 ± 1.6 integrated intensity), blunted increased NF-κB binding activity (LPS = 9.2 ± 2; flavocoxid = 2.4 ± 0.7 integrated intensity) and the

  2. Cigarette Smoke and the Induction of Urokinase Plasminogen Activator Receptor In Vivo: Selective Contribution of Isoforms to Bronchial Epithelial Phenotype.

    PubMed

    Portelli, Michael A; Stewart, Ceri E; Hall, Ian P; Brightling, Christopher E; Sayers, Ian

    2015-08-01

    The urokinase plasminogen activator receptor (uPAR) gene (PLAUR) has been identified as an asthma susceptibility gene, with polymorphisms within that gene being associated with baseline lung function, lung function decline, and lung function in a smoking population. Soluble cleaved uPAR (scuPAR), a molecule identified as a marker of increased morbidity and mortality in a number of diseases, has been shown to be elevated in the airways of patients with asthma and in patients with chronic obstructive pulmonary disease. However, the functionality of soluble receptor isoforms and their relationship with an important initiator for obstructive lung disease, cigarette smoke, remains undefined. In this study, we set out to determine the effect of cigarette smoke on soluble uPAR isoforms, its regulatory pathway and the resultant effect on bronchial epithelial cell function. We identified a positive association between cigarette pack-years and uPAR expression in the airway bronchial epithelium of biopsies from patients with asthma (n = 27; P = 0.0485). In vitro, cigarette smoke promoted cleavage of uPAR from the surface of bronchial epithelial cells (1.5× induction; P < 0.0001) and induced the soluble spliced isoform through changes in messenger RNA expression (∼2× change; P < 0.001), driven by loss of endogenous 3' untranslated region suppression. Elevated expression of the soluble isoforms resulted in a proremodeling cell phenotype, characterized by increased proliferation and matrix metalloproteinase-9 expression in primary bronchial epithelial cells. This suggests that cigarette smoke elevates soluble receptor isoforms in bronchial epithelial cells through direct (cleavage) and indirect (messenger RNA expression) means. These findings provide further insight into how cigarette smoke may influence changes in the airways of importance to airway remodeling and obstructive lung disease progression. PMID:25490122

  3. EGFR activating mutations correlate with a Fanconi anemia-like cellular phenotype that includes PARP inhibitor sensitivity

    PubMed Central

    Pfäffle, Heike N.; Wang, Meng; Gheorghiu, Liliana; Ferraiolo, Natalie; Greninger, Patricia; Borgmann, Kerstin; Settleman, Jeffrey; Benes, Cyril H.; Sequist, Lecia V.; Zou, Lee; Willers, Henning

    2013-01-01

    In lung cancer patients whose tumors harbor activating mutations in the epidermal growth factor receptor (EGFR), increased responses to platinum-based chemotherapies are seen compared to wild-type cancers. However, the mechanisms underlying this association have remained elusive. Here, we describe a cellular phenotype of crosslinker sensitivity in a subset of EGFR-mutant lung cancer cell lines that is reminiscent of the defects seen in cells impaired in the Fanconi Anemia pathway, including a pronounced G2/M cell-cycle arrest and chromosomal radial formation. We identified a defect downstream of FANCD2 at the level of recruitment of FAN1 nuclease and DNA interstrand crosslink (ICL) unhooking. The effect of EGFR mutation was epistatic with FANCD2. Consistent with the known role of FANCD2 in promoting RAD51 foci formation and homologous recombination repair (HRR), EGFR-mutant cells also exhibited an impaired RAD51 foci response to ICLs, but not to DNA double-strand breaks. EGFR kinase inhibition affected RAD51 foci formation neither in EGFR mutant nor wild-type cells. In contrast, EGFR depletion or overexpression of mutant EGFR in wild-type cells suppressed RAD51 foci, suggesting an EGFR kinase-independent regulation of DNA repair. Interestingly, EGFR-mutant cells treated with the PARP inhibitor olaparib also displayed decreased FAN1 foci induction, coupled with a putative block in a late HRR step. As a result, EGFR-mutant lung cancer cells exhibited olaparib sensitivity in-vitro and in-vivo. Our findings provide insight into the mechanisms of cisplatin and PARP inhibitor sensitivity of EGFR-mutant cells, yielding potential therapeutic opportunities for further treatment individualization in this genetically defined subset of lung cancer. PMID:23966292

  4. Phenotyping studies to assess the effects of phytopharmaceuticals on in vivo activity of main human cytochrome p450 enzymes.

    PubMed

    Zadoyan, Gregor; Fuhr, Uwe

    2012-09-01

    The extensive use of herbal drugs and their multiple components and modes of action suggests that they may also cause drug interactions by changing the activity of human cytochrome P450 enzymes. The purpose of the present review is to present the available data for the top 14 herbal drug sales in the U. S. Studies describing the effects of herbal drugs on phenotyping substrates for individual CYPs were identified by a comprehensive MEDLINE search. Drugs included Allium sativum (Liliaceae), Echinacea purpurea (Asteraceae), Serenoa repens (Arecaceae), Ginkgo biloba (Ginkgoaceae), Vaccinium macrocarpon (Ericaceae), Glycine max (Fabaceae), Panax ginseng (Araliaceae), Actea racemosa (Ranunculaceae), Hypericum perforatum (Hypericaceae), Silybum marianum (Asteraceae), Camellia sinensis (Theaceae), Valeriana officinalis (Valerianaceae), Piper methysticum (Piperaceae), and Hydrastis canadensis (Ranunculaceae) preparations. We identified 70 clinical studies in 69 publications. The majority of the herbal drugs appeared to have no clear effects on most of the CYPs examined. If there was an effect, there was mild inhibition in almost all cases, as seen with garlic or kava effects on CYP2E1 and with soybean components on CYP1A2. The most pronounced effects were induction of CYP3A and other CYPs by St. John's wort and the inhibitory effect of goldenseal on CYP3A and CYP2D6, both being borderline between mild and moderate in magnitude. With the exceptions of St.John's wort and goldenseal, the information currently available suggests that concomitant intake of the herbal drugs addressed here is not a major risk for drugs that are metabolized by CYPs. PMID:22588833

  5. Metformin prevents cancer metastasis by inhibiting M2-like polarization of tumor associated macrophages

    PubMed Central

    Ding, Ling; Liang, Guikai; Yao, Zhangting; Zhang, Jieqiong; Liu, Ruiyang; Chen, Huihui; Zhou, Yulu; Wu, Honghai; Yang, Bo; He, Qiaojun

    2015-01-01

    Accumulated evidence suggests that M2-like polarized tumor associated macrophages (TAMs) plays an important role in cancer progression and metastasis, establishing TAMs, especially M2-like TAMs as an appealing target for therapy intervention. Here we found that metformin significantly suppressed IL-13 induced M2-like polarization of macrophages, as illustrated by reduced expression of CD206, down-regulation of M2 marker mRNAs, and inhibition of M2-like macrophages promoted migration of cancer cells and endothelial cells. Metformin triggered AMPKα1 activation in macrophage and silencing of AMPKα1 partially abrogated the inhibitory effect of metformin in IL-13 induced M2-like polarization. Administration of AICAR, another activator of AMPK, also blocked the M2-like polarization of macrophages. Metformin greatly reduced the number of metastases of Lewis lung cancer without affecting tumor growth. In tumor tissues, the percentage of M2-like macrophage was decreased and the area of pericyte-coated vessels was increased. Further, the anti-metastatic effect of metformin was abolished when the animals were treated with macrophages eliminating agent clodronate liposome. These findings suggest that metformin is able to block the M2-like polarization of macrophages partially through AMPKα1, which plays an important role in metformin inhibited metastasis of Lewis lung cancer. PMID:26497364

  6. β-Adrenergic-stimulated macrophages: Comprehensive localization in the M1-M2 spectrum.

    PubMed

    Lamkin, Donald M; Ho, Hsin-Yun; Ong, Tiffany H; Kawanishi, Carly K; Stoffers, Victoria L; Ahlawat, Nivedita; Ma, Jeffrey C Y; Arevalo, Jesusa M G; Cole, Steve W; Sloan, Erica K

    2016-10-01

    β-Adrenergic signaling can regulate macrophage involvement in several diseases and often produces anti-inflammatory properties in macrophages, which are similar to M2 properties in a dichotomous M1 vs. M2 macrophage taxonomy. However, it is not clear that β-adrenergic-stimulated macrophages may be classified strictly as M2. In this in vitro study, we utilized recently published criteria and transcriptome-wide bioinformatics methods to map the relative polarity of murine β-adrenergic-stimulated macrophages within a wider M1-M2 spectrum. Results show that β-adrenergic-stimulated macrophages did not fit entirely into any one pre-defined category of the M1-M2 spectrum but did express genes that are representative of some M2 side categories. Moreover, transcript origin analysis of genome-wide transcriptional profiles located β-adrenergic-stimulated macrophages firmly on the M2 side of the M1-M2 spectrum and found active suppression of M1 side gene transcripts. The signal transduction pathways involved were mapped through blocking experiments and bioinformatics analysis of transcription factor binding motifs. M2-promoting effects were mediated specifically through β2-adrenergic receptors and were associated with CREB, C/EBPβ, and ATF transcription factor pathways but not with established M1-M2 STAT pathways. Thus, β-adrenergic-signaling induces a macrophage transcriptome that locates on the M2 side of the M1-M2 spectrum but likely accomplishes this effect through a signaling pathway that is atypical for M2-spectrum macrophages. PMID:27485040

  7. Juvenile-onset G(M2)-gangliosidosis in an African-American child with nystagmus.

    PubMed

    Paciorkowski, Alex R; Sathe, Swati; Zeng, Bei-Jin; Torres, Paola; Rosengren, Sally S; Kolodny, Edwin

    2008-04-01

    G(M2)-gangliosidosis is a neurodegenerative lysosomal disease with several clinical variants. We describe a 2-year-old black child with juvenile-onset disease, who presented with abnormal eye movements and cherry-red spots of the maculae. Mutation analysis of the HEXA gene revealed the patient to be a compound heterozygote (M1V/Y37N). The M1V mutation was previously described in an African-American child with acute infantile G(M2)-gangliosidosis. The Y37N mutation is novel. This combination of mutations is consistent with juvenile-onset disease, and provides further evidence for the association of the M1V mutation with individuals of black ancestry. The presence of oculomotor abnormalities is an unusual finding in this form of G(M2)-gangliosidosis, and adds to the phenotypic spectrum. PMID:18358410

  8. Motion-to-Energy (M2E) Power Generation Technology

    ScienceCinema

    INL

    2009-09-01

    INL researchers developed M2E, a new technology that converts motion to energy. M2E uses an innovative, optimized microgenerator with power management circuitry that kinetically charges mobile batteries from natural motion such as walking.

  9. Interleukin-36 potently stimulates human M2 macrophages, Langerhans cells and keratinocytes to produce pro-inflammatory cytokines.

    PubMed

    Dietrich, Damien; Martin, Praxedis; Flacher, Vincent; Sun, Yu; Jarrossay, David; Brembilla, Nicolo; Mueller, Christopher; Arnett, Heather A; Palmer, Gaby; Towne, Jennifer; Gabay, Cem

    2016-08-01

    Interleukin (IL)-36 cytokines belong to the IL-1 family and include three agonists, IL-36 α, β and γ and one inhibitor, IL-36 receptor antagonist (IL-36Ra). IL-36 and IL-1 (α and β) activate similar intracellular pathways via their related heterodimeric receptors, IL-36R/IL-1RAcP and IL-1R1/IL-1RAcP, respectively. However, excessive IL-36 versus IL-1 signaling induces different phenotypes in humans, which may be related to differential expression of their respective receptors. We examined the expression of IL-36R, IL-1R1 and IL-1RAcP mRNA in human peripheral blood, tonsil and skin immune cells by RT-qPCR. Monocyte-derived dendritic cells (MDDC), M0, M1 or M2-polarized macrophages, primary keratinocytes, dermal macrophages and Langerhans cells (LC) were stimulated with IL-1β or IL-36β. Cytokine production was assessed by RT-qPCR and immunoassays. The highest levels of IL-36R mRNA were found in skin-derived keratinocytes, LC, dermal macrophages and dermal CD1a(+) DC. In the blood and in tonsils, IL-36R mRNA was predominantly found in myeloid cells. By contrast, IL-1R1 mRNA was detected in almost all cell types with higher levels in tonsil and skin compared to peripheral blood immune cells. IL-36β was as potent as IL-1β in stimulating M2 macrophages, keratinocytes and LC, less potent than IL-1β in stimulating M0 macrophages and MDDC, and exerted no effects in M1 and dermal macrophages. Levels of IL-1Ra diminished the ability of M2 macrophages to respond to IL-1. Taken together, these data are consistent with the association of excessive IL-36 signaling with an inflammatory skin phenotype and identify human LC and M2 macrophages as new IL-36 target cells. PMID:27259168

  10. 26 CFR 1.401(m)-2 - ACP test.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... determined under § 1.401(m)-2(b)(2)(iv) (as it appeared in the April 1, 2007, edition of 26 CFR part 1). (E... determined under § 1.401(m)-2(b)(2)(vi) (as it appeared in the April 1, 2007, edition of 26 CFR Part 1). If... 26 Internal Revenue 5 2010-04-01 2010-04-01 false ACP test. 1.401(m)-2 Section 1.401(m)-2...

  11. A strong loss-of-function mutation in RAN1 results in constitutive activation of the ethylene response pathway as well as a rosette-lethal phenotype

    NASA Technical Reports Server (NTRS)

    Woeste, K. E.; Kieber, J. J.; Evans, M. L. (Principal Investigator)

    2000-01-01

    A recessive mutation was identified that constitutively activated the ethylene response pathway in Arabidopsis and resulted in a rosette-lethal phenotype. Positional cloning of the gene corresponding to this mutation revealed that it was allelic to responsive to antagonist1 (ran1), a mutation that causes seedlings to respond in a positive manner to what is normally a competitive inhibitor of ethylene binding. In contrast to the previously identified ran1-1 and ran1-2 alleles that are morphologically indistinguishable from wild-type plants, this ran1-3 allele results in a rosette-lethal phenotype. The predicted protein encoded by the RAN1 gene is similar to the Wilson and Menkes disease proteins and yeast Ccc2 protein, which are integral membrane cation-transporting P-type ATPases involved in copper trafficking. Genetic epistasis analysis indicated that RAN1 acts upstream of mutations in the ethylene receptor gene family. However, the rosette-lethal phenotype of ran1-3 was not suppressed by ethylene-insensitive mutants, suggesting that this mutation also affects a non-ethylene-dependent pathway regulating cell expansion. The phenotype of ran1-3 mutants is similar to loss-of-function ethylene receptor mutants, suggesting that RAN1 may be required to form functional ethylene receptors. Furthermore, these results suggest that copper is required not only for ethylene binding but also for the signaling function of the ethylene receptors.

  12. M2K Planet Search: Spectroscopic Screening and Transit Photometry

    NASA Astrophysics Data System (ADS)

    Mann, Andrew; Gaidos, E.; Fischer, D.; Lepine, S.

    2010-10-01

    The M2K project is a search for planets orbiting nearby early M and late K dwarf drawn from the SUPERBLINK catalog. M and K dwarfs are highly attractive targets for finding low-mass and habitable planets because (1) close-in planets are more likely to orbit within their habitable zone, (2) planets orbiting them induce a larger Doppler signal and have deeper transits than similar planets around F, G, and early K type stars, (3) planet formation models predict they hold an abundance of super-Earth sized planets, and (4) they represent the vast majority of the stars close enough for direct imaging techniques. In spite of this, only 10% of late K and early M dwarfs are being monitored by current Doppler surveys. As part of the M2K project we have obtained low-resolution spectra for more than 2000 of our sample of 10,000 M and K dwarfs. We vet our sample by screening these stars for high metallicity and low chromospheric activity. We search for transits on targets showing high RMS Doppler signal and photometry candidates provided by SuperWASP project. By using "snapshot” photometry have been able to achieve sub-millimag photometry on numerous transit targets in the same night. With further follow-up observations we will be able to detect planets smaller than 10 Earth masses.

  13. Hepatic Localization of Macrophage Phenotypes during Fibrogenesis and Resolution of Fibrosis in Mice and Humans

    PubMed Central

    Beljaars, Leonie; Schippers, Marlies; Reker-Smit, Catharina; Martinez, Fernando O.; Helming, Laura; Poelstra, Klaas; Melgert, Barbro N.

    2014-01-01

    Macrophages have been found to both promote liver fibrosis and contribute to its resolution by acquiring different phenotypes based on signals from the micro-environment. The best-characterized phenotypes in the macrophage spectrum are labeled M1 (classically activated) and M2 (alternatively activated). Until now the in situ localization of these phenotypes in diseased livers is poorly described. In this study, we therefore aimed to localize and quantify M1- and M2-dominant macrophages in diseased mouse and human livers. The scarred collagen-rich areas in cirrhotic human livers and in CCl4-damaged mouse livers contained many macrophages. Though total numbers of macrophages were higher in fibrotic livers, the number of parenchymal CD68-positive macrophages was significantly lower as compared to normal. Scar-associated macrophages were further characterized as either M1-dominant (IRF-5 and interleukin-12) or M2-dominant (CD206, transglutaminase-2, and YM-1) and significantly higher numbers of both of these were detected in diseased livers as compared to healthy human and mouse livers. Interestingly, in mouse, livers undergoing resolution of fibrosis, the total number of CD68+ macrophages was significantly lower compared to their fibrotic counterparts. M2-dominant (YM-1) macrophages were almost completely gone in livers undergoing resolution, while numbers of M1-dominant (IRF-5) macrophages were almost unchanged and the proteolytic activity (MMP9) increased. In conclusion, this study shows the distribution of macrophage subsets in livers of both human and murine origin. The presence of M1- and M2-dominant macrophages side by side in fibrotic lesions suggests that both are involved in fibrotic responses, while the persistence of M1-dominant macrophages during resolution may indicate their importance in regression of fibrosis. This study emphasizes that immunohistochemical detection of M1/M2-dominant macrophages provides valuable information in addition to widely used

  14. Identifying functional microRNAs in macrophages with polarized phenotypes.

    PubMed

    Graff, Joel W; Dickson, Anne M; Clay, Gwendolyn; McCaffrey, Anton P; Wilson, Mary E

    2012-06-22

    Macrophages respond to external stimuli with rapid changes in expression of many genes. Different combinations of external stimuli lead to distinct polarized activation patterns, resulting in a spectrum of possible macrophage activation phenotypes. MicroRNAs (miRNAs) are small, noncoding RNAs that can repress the expression of many target genes. We hypothesized that miRNAs play a role in macrophage polarization. miRNA expression profiles were determined in monocyte-derived macrophages (MDMs) incubated in conditions causing activation toward M1, M2a, M2b, or M2c phenotypes. One miRNA guide strand and seven miRNA passenger strands were significantly altered. Changes were confirmed in MDMs from six separate donors. The amplitude of miRNA expression changes in MDMs was smaller than described studies of monocytes responding to inflammatory stimuli. Further investigation revealed this correlated with higher basal miRNA expression in MDMs compared with monocytes. The regulation of M1- and M2b-responsive miRNAs (miR-27a, miR-29b, miR-125a, miR-146a, miR-155, and miR-222) was similar in differentiated THP-1 cells and primary MDMs. Studies in this model revealed cross-talk between IFNγ- and LPS-associated pathways regulating miRNA expression. Furthermore, expression of M1-associated transcripts was increased in THP-1 cells transfected with mimics of miR-29b, miR-125a-5p, or miR-155. The apparent inflammatory property of miR-29b and miR-125a-5p can be at least partially explained by repression of TNFAIP3, a negative regulator of NF-κB signaling. Overall, these data suggest miRNAs can contribute to changes in macrophage gene expression that occur in different exogenous activating conditions. PMID:22549785

  15. M2/M1 ratio of tumor associated macrophages and PPAR-gamma expression in uveal melanomas with class 1 and class 2 molecular profiles.

    PubMed

    Herwig, Martina C; Bergstrom, Chris; Wells, Jill R; Höller, Tobias; Grossniklaus, Hans E

    2013-02-01

    Macrophages have been found to be negative predictors of outcome in patients with uveal melanoma. In particular, recent studies point toward a disease-progressing role of proangiogenic M2 macrophages in melanomas with monosomy 3. Although most studies implicate a protective effect of PPAR-gamma activation in tumors, PPAR-gamma has also been shown to promote the polarization of M1 macrophages toward the M2 phenotype. The purpose of this investigation was first, to characterize the phenotype of tumor infiltrating macrophages and second, to study PPAR-gamma expression in uveal melanomas with molecular gene expression profile as prognostic predictors for patients' outcome. Twenty specimens from patients with uveal melanoma were analyzed for clinical and histologic tumor characteristics. The molecular RNA profile (class 1 or class 2) was commercially determined. Using immunohistochemical techniques, the specimens were dual labeled for CD68 and CD163. CD68 + CD163- M1 macrophages and CD68 + CD163+ M2 macrophages were analyzed in ten high power fields sparing macrophage-poor areas and a mean value was calculated for each tumor. The tumors were immunostained for von Willebrand factor and the micro vascular density (MVD) was analyzed according to Foss. To assess the proliferative rate of each tumor, Ki67 expression was evaluated in ten high power fields followed by calculation of a mean value. Expression of PPAR-gamma was evaluated using a score from 0 (no staining) to 3 (tumor entirely stained). Statistical analysis and a respective correlation were made between histologic characteristics, molecular profile, type of tumor infiltrating macrophages (M1 vs. M2), MVD, proliferative rate, and PPAR-gamma expression. Our results showed a correlation between the ratio of M2/M1 macrophages and the molecular profile with a ratio of approximately 1 corresponding to molecular class 1 and a ratio of approximately 2 corresponding to molecular class 2 (p = 0.01). The ratio of M2/M1

  16. Hepatocellular carcinomas promote tumor-associated macrophage M2-polarization via increased B7-H3 expression.

    PubMed

    Kang, Fu-Biao; Wang, Ling; Li, Dong; Zhang, Yin-Ge; Sun, Dian-Xing

    2015-01-01

    B7 family members are aberrantly expressed on the human hepatocellular carcinoma (HCC) cell surface, and induce local and systemic immunosuppression. Tumor-associated macrophages (TAMs) are a significant immune cell subpopulation in HCC and may be induced to express co-inhibitory molecules including B7 homologue 3 (B7-H3). In the present study, 79.3% of the HCC tissue samples showed high expression of B7-H3 which was positively correlated with the number of TAMs in the evaluated cancer tissues. Furthermore, high levels of TAMs or B7-H3 were associated with a shorter survival time of the HCC patients. In vitro, B7-H3 expression was upregulated at both the mRNA and protein levels in phorbol 12-myristate 13-acetate (PMA)-induced THP-1 cells cocultured with HepG2 cells in a Transwell system. In addition, B7-H3 promoted PMA-induced THP-1 cells to differentiate into the M2 phenotype, with evidence of increases in arginase 1 (Arg1), vascular endothelial cell growth factor (VEGF) and macrophage-derived chemokine (CCL22) mRNA following coculture with HepG2 cells. However, this phenomenon was abrogated through knockdown of B7-H3 by RNA interference or by blocking the signal transducer and activator of trans-cription 3 (STAT3) signaling pathway. Overall, these results suggest that the B7-H3-mediated STAT3 signaling pathway is an important mechanism for inducing M2-type polarization of TAMs, which accelerates HCC development. Our findings may support the development of novel therapeutic strategies for HCC patients through the skewing of the TAM phenotype by targeting the B7-H3 signaling pathway. PMID:25370943

  17. In silico functional dissection of saturation mutagenesis: Interpreting the relationship between phenotypes and changes in protein stability, interactions and activity

    PubMed Central

    Pires, Douglas E. V.; Chen, Jing; Blundell, Tom L.; Ascher, David B.

    2016-01-01

    Despite interest in associating polymorphisms with clinical or experimental phenotypes, functional interpretation of mutation data has lagged behind generation of data from modern high-throughput techniques and the accurate prediction of the molecular impact of a mutation remains a non-trivial task. We present here an integrated knowledge-driven computational workflow designed to evaluate the effects of experimental and disease missense mutations on protein structure and interactions. We exemplify its application with analyses of saturation mutagenesis of DBR1 and Gal4 and show that the experimental phenotypes for over 80% of the mutations correlate well with predicted effects of mutations on protein stability and RNA binding affinity. We also show that analysis of mutations in VHL using our workflow provides valuable insights into the effects of mutations, and their links to the risk of developing renal carcinoma. Taken together the analyses of the three examples demonstrate that structural bioinformatics tools, when applied in a systematic, integrated way, can rapidly analyse a given system to provide a powerful approach for predicting structural and functional effects of thousands of mutations in order to reveal molecular mechanisms leading to a phenotype. Missense or non-synonymous mutations are nucleotide substitutions that alter the amino acid sequence of a protein. Their effects can range from modifying transcription, translation, processing and splicing, localization, changing stability of the protein, altering its dynamics or interactions with other proteins, nucleic acids and ligands, including small molecules and metal ions. The advent of high-throughput techniques including sequencing and saturation mutagenesis has provided large amounts of phenotypic data linked to mutations. However, one of the hurdles has been understanding and quantifying the effects of a particular mutation, and how they translate into a given phenotype. One approach to overcome

  18. Tailor-Made Zinc-Finger Transcription Factors Activate FLO11 Gene Expression with Phenotypic Consequences in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Shieh, Jia-Ching; Cheng, Yu-Che; Su, Mao-Chang; Moore, Michael; Choo, Yen; Klug, Aaron

    2007-01-01

    Cys2His2 zinc fingers are eukaryotic DNA-binding motifs, capable of distinguishing different DNA sequences, and are suitable for engineering artificial transcription factors. In this work, we used the budding yeast Saccharomyces cerevisiae to study the ability of tailor-made zinc finger proteins to activate the expression of the FLO11 gene, with phenotypic consequences. Two three-finger peptides were identified, recognizing sites from the 5′ UTR of the FLO11 gene with nanomolar DNA-binding affinity. The three-finger domains and their combined six-finger motif, recognizing an 18-bp site, were fused to the activation domain of VP16 or VP64. These transcription factor constructs retained their DNA-binding ability, with the six-finger ones being the highest in affinity. However, when expressed in haploid yeast cells, only one three-finger recombinant transcription factor was able to activate the expression of FLO11 efficiently. Unlike in the wild-type, cells with such transcriptional activation displayed invasive growth and biofilm formation, without any requirement for glucose depletion. The VP16 and VP64 domains appeared to act equally well in the activation of FLO11 expression, with comparable effects in phenotypic alteration. We conclude that the functional activity of tailor-made transcription factors in cells is not easily predicted by the in vitro DNA-binding activity. PMID:17710146

  19. AMP-Activated Kinase (AMPK) Activation by AICAR in Human White Adipocytes Derived from Pericardial White Adipose Tissue Stem Cells Induces a Partial Beige-Like Phenotype

    PubMed Central

    Abdul-Rahman, Omar; Kristóf, Endre; Doan-Xuan, Quang-Minh; Vida, András; Nagy, Lilla; Horváth, Ambrus; Simon, József; Maros, Tamás; Szentkirályi, István; Palotás, Lehel; Debreceni, Tamás; Csizmadia, Péter; Szerafin, Tamás; Fodor, Tamás; Szántó, Magdolna; Tóth, Attila; Kiss, Borbála; Bacsó, Zsolt; Bai, Péter

    2016-01-01

    Beige adipocytes are special cells situated in the white adipose tissue. Beige adipocytes, lacking thermogenic cues, morphologically look quite similar to regular white adipocytes, but with a markedly different response to adrenalin. White adipocytes respond to adrenergic stimuli by enhancing lipolysis, while in beige adipocytes adrenalin induces mitochondrial biogenesis too. A key step in the differentiation and function of beige adipocytes is the deacetylation of peroxisome proliferator-activated receptor (PPARγ) by SIRT1 and the consequent mitochondrial biogenesis. AMP-activated protein kinase (AMPK) is an upstream activator of SIRT1, therefore we set out to investigate the role of AMPK in beige adipocyte differentiation using human adipose-derived mesenchymal stem cells (hADMSCs) from pericardial adipose tissue. hADMSCs were differentiated to white and beige adipocytes and the differentiation medium of the white adipocytes was supplemented with 100 μM [(2R,3S,4R,5R)-5-(4-Carbamoyl-5-aminoimidazol-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate (AICAR), a known activator of AMPK. The activation of AMPK with AICAR led to the appearance of beige-like morphological properties in differentiated white adipocytes. Namely, smaller lipid droplets appeared in AICAR-treated white adipocytes in a similar fashion as in beige cells. Moreover, in AICAR-treated white adipocytes the mitochondrial network was more fused than in white adipocytes; a fused mitochondrial system was characteristic to beige adipocytes. Despite the morphological similarities between AICAR-treated white adipocytes and beige cells, functionally AICAR-treated white adipocytes were similar to white adipocytes. We were unable to detect increases in basal or cAMP-induced oxygen consumption rate (a marker of mitochondrial biogenesis) when comparing control and AICAR-treated white adipocytes. Similarly, markers of beige adipocytes such as TBX1, UCP1, CIDEA, PRDM16 and TMEM26 remained the same when

  20. Differential partial activation phenotype and production of tumour necrosis factor-α by conventional dendritic cells in response to lipopolysaccharide in HIV+ viraemic subjects and HIV+ controllers

    PubMed Central

    Camacho-Sandoval, R; Del Río Estrada, P M; Rivero-Arrieta, A; Reyes-Terán, G; Bonifaz, L C

    2014-01-01

    HIV+ subjects are reported to have increased soluble CD14 (sCD14) in plasma, an indicator of microbial translocation. We evaluated if microbial translocation has a differential impact on the activation and function of conventional dendritic cells (cDC) from viraemic HIV+ subjects and HIV+ controllers (CTs). The HIV+ subjects were classified into two groups according to their plasma viral load (pVL): CT and viraemic. Subjects without HIV were included as controls (HIV–). The frequencies and phenotypes of cDC from these subjects were evaluated by multi-parameter flow cytometry. In addition, peripheral blood mononuclear cells (PBMCs) were stimulated with lipopolysaccharide (LPS) or single-stranded RNA40 (ssRNA40), the phenotype of the cDC and the intracellular production of tumour necrosis factor (TNF)-α by the cDC were evaluated by flow cytometry. We observed a partial activation phenotype for the cDC in the viraemic subjects and CTs ex vivo and after LPS activation, which showed differences in the expression of CD40 and CD86. Furthermore, in response to LPS the cDC from the viraemic subjects produced more TNF-α compared to the cDC from CTs. Interestingly, the percentage of TNF-α+ cDC was found to be correlated positively with the pVL. The partial activation of cDC and the over-production of TNF-α in response to LPS in viraemic HIV+ subjects might be related to the increased chronic activation observed in these subjects. In contrast, cDC from CTs seem to have a regulated response to LPS, indicating that they respond differently to chronic immune activation. These results may have implications in the development of HIV therapies and vaccines using DC. PMID:25130456

  1. M2b Monocytes Provoke Bacterial Pneumonia and Gut Bacteria-Associated Sepsis in Alcoholics.

    PubMed

    Tsuchimoto, Yusuke; Asai, Akira; Tsuda, Yasuhiro; Ito, Ichiaki; Nishiguchi, Tomoki; Garcia, Melanie C; Suzuki, Sumihiro; Kobayashi, Makiko; Higuchi, Kazuhide; Suzuki, Fujio

    2015-12-01

    Chronic alcohol consumption markedly impairs host antibacterial defense against opportunistic infections. γ-irradiated NOD-SCID IL-2Rγ(null) mice inoculated with nonalcoholic PBMCs (control PBMC chimeras) resisted Klebsiella pneumonia and gut bacteria-associated sepsis, whereas the chimeras created with alcoholic PBMCs (alcoholic PBMC chimeras) were very susceptible to these infections. M1 monocytes (IL-12(+)IL-10(-)CD163(-)CD14(+) cells), major effector cells in antibacterial innate immunity, were not induced by a bacterial Ag in alcoholic PBMC cultures, and M2b monocytes (CCL1(+)CD163(+)CD14(+) cells), which predominated in alcoholic PBMCs, were shown to be inhibitor cells on the Ag-stimulated monocyte conversion from quiescent monocytes to M1 monocytes. CCL1, which functions to maintain M2b macrophage properties, was produced by M2b monocytes isolated from alcoholic PBMCs. These M2b monocytes reverted to quiescent monocytes (IL-12(-)IL-10(-)CCL1(-)CD163(-)CD14(+) cells) in cultures supplemented with CCL1 antisense oligodeoxynucleotide, and the subsequent quiescent monocytes easily converted to M1 monocytes under bacterial Ag stimulation. Alcoholic PBMC chimeras treated with CCL1 antisense oligodeoxynucleotide were resistant against pulmonary infection by K. pneumoniae and sepsis stemming from enterococcal translocation. These results indicate that a majority of monocytes polarize to an M2b phenotype in association with alcohol abuse, and this polarization contributes to the increased susceptibility of alcoholics to gut and lung infections. Bacterial pneumonia and gut bacteria-associated sepsis, frequently seen in alcoholics, can be controlled through the polarization of macrophage phenotypes. PMID:26525287

  2. Murine Gammaherpesvirus M2 Protein Induction of IRF4 via the NFAT Pathway Leads to IL-10 Expression in B Cells

    PubMed Central

    Rangaswamy, Udaya S.; Speck, Samuel H.

    2014-01-01

    Reactivation of the gammaherpesviruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) from latently infected B cells has been linked to plasma cell differentiation. We have previously shown that the MHV68 M2 protein is important for virus reactivation from B cells and, when expressed alone in primary murine B cells, can drive B cell differentiation towards a pre-plasma cell phenotype. In addition, expression of M2 in primary murine B cells leads to secretion of high levels of IL-10 along with enhanced proliferation and survival. Furthermore, the absence of M2 in vivo leads to a defect in the appearance of MHV68 infected plasma cells in the spleen at the peak of MHV68 latency. Here, employing an inducible B cell expression system, we have determined that M2 activates the NFAT pathway in a Src kinase-dependent manner – leading to induction of the plasma cell-associated transcription factor, Interferon Regulatory Factor-4 (IRF4). Furthermore, we show that expression of IRF4 alone in a B cell line up-regulates IL-10 expression in culture supernatants, revealing a novel role for IRF4 in B cell induced IL-10. Consistent with the latter observation, we show that IRF4 can regulate the IL-10 promoter in B cells. In primary murine B cells, addition of cyclosporine (CsA) resulted in a significant decrease in M2-induced IL-10 levels as well as IRF4 expression, emphasizing the importance of the NFAT pathway in M2- mediated induction of IL-10. Together, these studies argue in favor of a model wherein M2 activation of the NFAT pathway initiates events leading to increased levels of IRF4 – a key player in plasma cell differentiation – which in turn triggers IL-10 expression. In the context of previous findings, the data presented here provides insights into how M2 facilitates plasma cell differentiation and subsequent virus reactivation. PMID:24391506

  3. A base substitution in the promoter associated with the human haptoglobin 2-1 modified phenotype decreases transcriptional activity and responsiveness to interleukin-6 in human hepatoma cells

    SciTech Connect

    Grant, D.J.; Maeda, N. )

    1993-05-01

    An A-to-C base substitution at nucleotide position -61 in the promoter region of the human haptoglobin gene (Hp) has been shown to be strongly associated with the haptoglobin 2-1 modified (Hp2-1mod) phenotype. In order to investigate whether this base substitution is the cause of reduced expression of the Hp[sup 2] allele relative to the Hp[sup 1] allele in individuals with the Hp2-1mod phenotype, the authors used the chloramphenicol acetyl transferase (CAT) expression system to evaluate promoter function. In HepG2 cells, which normally express their endogenous haptoglobin genes, CAT plasmid constructs with the -61C base change in the promoter had about 10-fold-lower transcriptional activity after transfection than did the Hp control construct. The -61C substitution also rendered the construct unresponsive to treatment by interleukin-6 after transfection into Hep3B2 cells, which normally do not express haptoglobin but do so in response to stimulation by acute-phase reactants. In addition, two base substitutions, T to A and A to G, at positions -104 and -55G, respectively, in the promoter region of the Hp[sup 1] allele, are also associated with the Hp2-1mod phenotype. CAT constructs with both substitutions (-104A-55G) and with one substitution (-55G) showed activity similar to that in the Hp control when transfected into both HepG2 and Hep3B2 cells, although interleukin-6 induction was less than with the Hp control construct. These results further support the hypothesis that the Hp2-1mod phenotype results, in part, from the -61C mutation in the promoter region of the Hp[sup 2] gene.

  4. Evaluation of cytochrome P450 2C9 activity in normal, healthy, adult Western Indian population by both phenotyping and genotyping

    PubMed Central

    Swar, Balkrishna D.; Bendkhale, Shital R.; Rupawala, Abbas; Sridharan, Kannan; Gogtay, Nithya J.; Thatte, Urmila M.; Kshirsagar, Nilima A.

    2016-01-01

    Objectives: Cytochrome P450 2C9 (CYP2C9) is a member of cytochrome P450 (CYP) family that accounts for nearly 18% of the total CYP protein content in the human liver microsomes and catalyzes almost 15–20% of the drugs. Considering the paucity of data on the polymorphisms of CYP2C9 in Western Indian population, the present study was conducted to evaluate the prevalence of CYP2C9 polymorphisms (*1, *2 and *3) and correlate it with the activity using flurbiprofen (FLB) as a probe drug. Materials and Methods: A 100 mg FLB capsule was administered to 298 healthy adult participants. Venous blood samples were analyzed at 2 h postdose for the estimation of FLB and 4-hydroxy FLB. Metabolic ratio (MR) was calculated to determine the extent of poor metabolizer (PM) and rapid metabolizer status using probit plot. Genotyping of CYP2C9 polymorphism was performed using polymerase chain reaction-restriction fragment length polymorphism technique. Results: Of the total 298 participants, phenotype was assessable in 288 and genotype was performed in 289 participants. The median (range) MR of the study population was 6.6 (1.65–66.05). Five participants were found to be PMs by phenotype. Of the total 289 participants, 209 (72.3%) (66.7, 77.2) had CYP2C9*1/*1, 25 (8.7%) (5.8, 12.7) with CYP2C9*1/*2, 55 (19%) (14.8, 24.1) had CYP2C9*1/*3, 3 (1%) (0.3, 3.3) had CYP2C9*2/*3 genotype. A significant association between phenotype and genotype was observed. Conclusion: To conclude, the present study found significant association of CYP2C9 activity by both phenotype and genotype and these findings have to be corroborated in different kinds of patients. PMID:27298492

  5. Environmental and Genetic Activation of Hypothalamic BDNF Modulates T-cell Immunity to Exert an Anticancer Phenotype.

    PubMed

    Xiao, Run; Bergin, Stephen M; Huang, Wei; Slater, Andrew M; Liu, Xianglan; Judd, Ryan T; Lin, En-Ju D; Widstrom, Kyle J; Scoville, Steven D; Yu, Jianhua; Caligiuri, Michael A; Cao, Lei

    2016-06-01

    Macroenvironmental factors, including a patient's physical and social environment, play a role in cancer risk and progression. Our previous studies show that living in an enriched environment (EE) providing complex stimuli confers an anticancer phenotype in mice mediated, in part by a specific neuroendocrine axis, with brain-derived neurotrophic factor (BDNF) as the key brain mediator. Here, we investigated how an EE modulated T-cell immunity and its role in the EE-induced anticancer effects. Our data demonstrated that CD8 T cells were required to mediate the anticancer effects of an EE in an orthotropic model of melanoma. In secondary lymphoid tissue (SLT), an EE induced early changes in the phenotype of T-cell populations, characterized by a decrease in the ratio of CD4 T helper to CD8 cytotoxic T lymphocytes (CTL). Overexpression of hypothalamic BDNF reproduced EE-induced T-cell phenotypes in SLT, whereas knockdown of hypothalamic BDNF inhibited EE-induced immune modulation in SLT. Both propranolol and mifepristone blocked the EE-associated modulation of CTLs in SLT, suggesting that both the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis were involved. Our results demonstrated that enhanced anticancer effect of an EE was mediated at least in part through modulation of T-cell immunity and provided support to the emerging concept of manipulating a single gene in the brain to improve cancer immunotherapy. Cancer Immunol Res; 4(6); 488-97. ©2016 AACR. PMID:27045020

  6. Activation of bile acid signaling improves metabolic phenotypes in high-fat diet-induced obese mice.

    PubMed

    Pierre, Joseph F; Martinez, Kristina B; Ye, Honggang; Nadimpalli, Anuradha; Morton, Timothy C; Yang, Jinghui; Wang, Qiang; Patno, Noelle; Chang, Eugene B; Yin, Deng Ping

    2016-08-01

    The metabolic benefits induced by gastric bypass, currently the most effective treatment for morbid obesity, are associated with bile acid (BA) delivery to the distal intestine. However, mechanistic insights into BA signaling in the mediation of metabolic benefits remain an area of study. The bile diversion () mouse model, in which the gallbladder is anastomosed to the distal jejunum, was used to test the specific role of BA in the regulation of glucose and lipid homeostasis. Metabolic phenotype, including body weight and composition, glucose tolerance, energy expenditure, thermogenesis genes, total BA and BA composition in the circulation and portal vein, and gut microbiota were examined. BD improves the metabolic phenotype, which is in accord with increased circulating primary BAs and regulation of enterohormones. BD-induced hypertrophy of the proximal intestine in the absence of BA was reversed by BA oral gavage, but without influencing BD metabolic benefits. BD-enhanced energy expenditure was associated with elevated TGR5, D2, and thermogenic genes, including UCP1, PRDM16, PGC-1α, PGC-1β, and PDGFRα in epididymal white adipose tissue (WAT) and inguinal WAT, but not in brown adipose tissue. BD resulted in an altered gut microbiota profile (i.e., Firmicutes bacteria were decreased, Bacteroidetes were increased, and Akkermansia was positively correlated with higher levels of circulating primary BAs). Our study demonstrates that enhancement of BA signaling regulates glucose and lipid homeostasis, promotes thermogenesis, and modulates the gut microbiota, which collectively resulted in an improved metabolic phenotype. PMID:27340128

  7. Alcohol-Induced miR-27a Regulates Differentiation and M2 Macrophage Polarization of Normal Human Monocytes

    PubMed Central

    Saha, Banishree; Bruneau, Johanna C.; Kodys, Karen; Szabo, Gyongyi

    2015-01-01

    Alcohol abuse is a leading cause of liver disease characterized by liver inflammation, fatty liver, alcoholic hepatitis, or liver cirrhosis. Immunomodulatory effects of alcohol on monocytes and macrophages contribute to alcoholic liver disease. Alcohol use, an independent risk factor for progression of hepatitis C virus (HCV) infection–mediated liver disease, impairs host defense and alters cytokine production and monocyte/macrophage activation. We hypothesized that alcohol and HCV have synergistic effects on the phenotype and function of monocytes. Our data show that acute alcohol binge drinking in healthy volunteers results in increased frequency of CD16+ and CD68+ and M2-type (CD206+, dendritic cell [DC]-SIGN+–expressing and IL-10–secreting) circulating CD14+ monocytes. Expression of HCV-induced CD68 and M2 markers (CD206 and DC-SIGN) in normal monocytes was further enhanced in the presence of alcohol. The levels of microRNA (miR)-27a was significantly upregulated in monocytes cultured in the presence of alcohol or alcohol and HCV as compared with HCV alone. The functional role of miR-27a in macrophage polarization was demonstrated by transfecting monocytes with an miR-27a inhibitor that resulted in reduced alcohol- and HCV- mediated monocyte activation (CD14 and CD68 expression), polarization (CD206 and DC-SIGN expression), and IL-10 secretion. Over-expression of miR-27a in monocytes enhanced IL-10 secretion via activation of the ERK signaling pathway. We found that miR-27a promoted ERK phosphorylation by downregulating the expression of ERK inhibitor sprouty2 in monocytes. Thus, we identified that sprouty2 is a target of miR-27a in human monocytes. In summary, our study demonstrates the regulatory role of miR-27a in alcohol-induced monocyte activation and polarization. PMID:25716995

  8. Adoptive transfer of M2 macrophages promotes locomotor recovery in adult rats after spinal cord injury.

    PubMed

    Ma, Shan-Feng; Chen, Yue-Juan; Zhang, Jing-Xing; Shen, Lin; Wang, Rui; Zhou, Jian-Sheng; Hu, Jian-Guo; Lü, He-Zuo

    2015-03-01

    Classically activated pro-inflammatory (M1) and alternatively activated anti-inflammatory (M2) macrophages populate the local microenvironment after spinal cord injury (SCI). The former type is neurotoxic while the latter has positive effects on neuroregeneration and is less toxic. In addition, while the M1 macrophage response is rapidly induced and sustained, M2 induction is transient. A promising strategy for the repair of SCI is to increase the fraction of M2 cells and prolong their residence time. This study investigated the effect of M2 macrophages induced from bone marrow-derived macrophages on the local microenvironment and their possible role in neuroprotection after SCI. M2 macrophages produced anti-inflammatory cytokines such as interleukin (IL)-10 and transforming growth factor β and infiltrated into the injured spinal cord, stimulated M2 and helper T (Th)2 cells, and produced high levels of IL-10 and -13 at the site of injury. M2 cell transfer decreased spinal cord lesion volume and resulted in increased myelination of axons and preservation of neurons. This was accompanied by significant locomotor improvement as revealed by Basso, Beattie and Bresnahan locomotor rating scale, grid walk and footprint analyses. These results indicate that M2 adoptive transfer has beneficial effects for the injured spinal cord, in which the increased number of M2 macrophages causes a shift in the immunological response from Th1- to Th2-dominated through the production of anti-inflammatory cytokines, which in turn induces the polarization of local microglia and/or macrophages to the M2 subtype, and creates a local microenvironment that is conducive to the rescue of residual myelin and neurons and preservation of neuronal function. PMID:25476600

  9. Disease Phenotype, Activity and Clinical Course Prediction Based on C-Reactive Protein Levels at Diagnosis in Patients with Crohn’s Disease: Results from the CONNECT Study

    PubMed Central

    Kwon, Jee Hye; Im, Jong Pil; Ye, Byong Duk; Cheon, Jae Hee; Jang, Hyun Joo; Lee, Kang Moon; Kim, You Sun; Kim, Sang Wook; Kim, Young Ho; Song, Geun Am; Han, Dong Soo; Kim, Won Ho; Kim, Joo Sung

    2016-01-01

    Background/Aims C-reactive protein (CRP) is an easily measured index of disease activity, but its ability to predict clinical course is controversial. We therefore designed a study to determine whether the CRP level at Crohn’s disease (CD) diagnosis is a valuable indicator of the disease phenotype, activity, and clinical course. Methods We retrospectively analyzed 705 CD patients from 32 institutions. The patients were classified into two groups according to CRP level. The patients’ demographic and clinical characteristics and their use of immunosuppressive or biological agents were recorded. Disease location and behavior, hospitalization, and surgery were analyzed. Results A high CRP was associated with younger age, steroid use, colonic or ileocolonic location, high CD activity index, and active inflammation at colonoscopy (p<0.001). As the disease progressed, patients with high CRP were more likely to exhibit strictures (p=0.027). There were significant differences in the use of 5-aminosalicylic acid, antibiotics, corticosteroids, azathioprine, and infliximab (p<0.001, p<0.001, p<0.001, p<0.001, and p=0.023, respectively). Hospitalization was also more frequent in patients with high CRP. Conclusions The CRP level at diagnosis is useful for evaluating the phenotype, activity, and clinical course of CD. Closer follow-up strategies, with early aggressive treatment, could be considered for patients with high CRP. PMID:27021506

  10. Assessing the contribution of thrombospondin-4 induction and ATF6α activation to endoplasmic reticulum expansion and phenotypic modulation in bladder outlet obstruction.

    PubMed

    Krawczyk, Katarzyna K; Ekman, Mari; Rippe, Catarina; Grossi, Mario; Nilsson, Bengt-Olof; Albinsson, Sebastian; Uvelius, Bengt; Swärd, Karl

    2016-01-01

    Phenotypic modulation of smooth muscle cells is a hallmark of disease. The associated expansion of endoplasmic reticulum (ER) volume remains unexplained. Thrombospondin-4 was recently found to promote ATF6α activation leading to ER expansion. Using bladder outlet obstruction as a paradigm for phenotypic modulation, we tested if thrombospondin-4 is induced in association with ATF6α activation and ER expansion. Thrombospondin-4 was induced and ATF6α was activated after outlet obstruction in rodents. Increased abundance of spliced of Xbp1, another ER-stress sensor, and induction of Atf4 and Creb3l2 was also seen. Downstream of ATF6α, Calr, Manf, Sdf2l1 and Pdi increased as did ER size, whereas contractile markers were reduced. Overexpression of ATF6α, but not of thrombospondin-4, increased Calr, Manf, Sdf2l1 and Pdi and caused ER expansion, but the contractile markers were inert. Knockout of thrombospondin-4 neither affected bladder growth nor expression of ATF6α target genes, and repression of contractile markers was the same, even if ATF6α activation was curtailed. Increases of Xbp1s, Atf4 and Creb3l2 were similar. Our findings demonstrate reciprocal regulation of the unfolded protein response, including ATF6α activation and ER expansion, and reduced contractile differentiation in bladder outlet obstruction occurring independently of thrombospondin-4, which however is a sensitive indicator of obstruction. PMID:27581066

  11. Assessing the contribution of thrombospondin-4 induction and ATF6α activation to endoplasmic reticulum expansion and phenotypic modulation in bladder outlet obstruction

    PubMed Central

    Krawczyk, Katarzyna K.; Ekman, Mari; Rippe, Catarina; Grossi, Mario; Nilsson, Bengt-Olof; Albinsson, Sebastian; Uvelius, Bengt; Swärd, Karl

    2016-01-01

    Phenotypic modulation of smooth muscle cells is a hallmark of disease. The associated expansion of endoplasmic reticulum (ER) volume remains unexplained. Thrombospondin-4 was recently found to promote ATF6α activation leading to ER expansion. Using bladder outlet obstruction as a paradigm for phenotypic modulation, we tested if thrombospondin-4 is induced in association with ATF6α activation and ER expansion. Thrombospondin-4 was induced and ATF6α was activated after outlet obstruction in rodents. Increased abundance of spliced of Xbp1, another ER-stress sensor, and induction of Atf4 and Creb3l2 was also seen. Downstream of ATF6α, Calr, Manf, Sdf2l1 and Pdi increased as did ER size, whereas contractile markers were reduced. Overexpression of ATF6α, but not of thrombospondin-4, increased Calr, Manf, Sdf2l1 and Pdi and caused ER expansion, but the contractile markers were inert. Knockout of thrombospondin-4 neither affected bladder growth nor expression of ATF6α target genes, and repression of contractile markers was the same, even if ATF6α activation was curtailed. Increases of Xbp1s, Atf4 and Creb3l2 were similar. Our findings demonstrate reciprocal regulation of the unfolded protein response, including ATF6α activation and ER expansion, and reduced contractile differentiation in bladder outlet obstruction occurring independently of thrombospondin-4, which however is a sensitive indicator of obstruction. PMID:27581066

  12. Identification of a farnesol analog as a Ras function inhibitor using both an in vivo Ras activation sensor and a phenotypic screening approach

    PubMed Central

    Srinivasan, Kamalakkannan; Subramanian, Thangaiah; Spielmann, H. Peter

    2013-01-01

    Mutations in Ras isoforms such as K-Ras, N-Ras, and H-Ras contribute to roughly 85, 15, and 1 % of human cancers, respectively. Proper membrane targeting of these Ras isoforms, a prerequisite for Ras activity, requires farnesylation or geranylgeranylation at the C-terminal CAAX box. We devised an in vivo screening strategy based on monitoring Ras activation and phenotypic physiological outputs for assaying synthetic Ras function inhibitors (RFI). Ras activity was visualized by the trans-location of RBDRaf1-GFP to activated Ras at the plasma membrane. By using this strategy, we screened one synthetic farnesyl substrate analog (AGOH) along with nine putative inhibitors and found that only m-CN-AGOH inhibited Ras activation. Phenotypic analysis of starving cells could be used to monitor polarization, motility, and the inability of these treated cells to aggregate properly during fruiting body formation. Incorporation of AGOH and m-CN-AGOH to cellular proteins was detected by western blot. These screening assays can be incorporated into a high throughput screening format using Dictyostelium discoideum and automated microscopy to determine effective RFIs. These RFI candidates can then be further tested in mammalian systems. PMID:24194124

  13. Combined 17β-Estradiol with TCDD Promotes M2 Polarization of Macrophages in the Endometriotic Milieu with Aid of the Interaction between Endometrial Stromal Cells and Macrophages.

    PubMed

    Wang, Yun; Chen, Hong; Wang, NingLing; Guo, HaiYan; Fu, Yonglun; Xue, Songguo; Ai, Ai; Lyu, Qifeng; Kuang, Yanping

    2015-01-01

    The goal of this study is to elucidate the effects of 17β-estradiol and TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) on macrophage phenotypes in the endometriotic milieu. Co-culture of endometrial stromal cells (ESCs) and U937 cells (macrophage cell line) was performed to simulate the endometriotic milieu and to determine the effects of 17β-estradiol and/or TCDD on IL10, IL12 production and HLA-DR, CD86 expression by U937 macrophages. We found that combining 17β-estradiol with TCDD has a synergistic effect on inducing M2 activation when macrophages are co-cultured with ESCs. Moreover, the combination of 17β-estradiol and TCDD significantly enhanced STAT3 and P38 phosphorylation in macrophages. Differentiation of M2 macrophages induced by 17β-estradiol and TCDD were effectively abrogated by STAT3 and P38MAPK inhibitors, but not by ERK1/2 and JNK inhibitors. In conclusion, 17β-estradiol and TCDD in the ectopic milieu may lead to the development of endometriosis by inducing M2 polarization of macrophages through activation of the STAT3 and P38MAPK pathways. PMID:25950905

  14. Combined 17β-Estradiol with TCDD Promotes M2 Polarization of Macrophages in the Endometriotic Milieu with Aid of the Interaction between Endometrial Stromal Cells and Macrophages

    PubMed Central

    Wang, NingLing; Guo, HaiYan; Fu, Yonglun; Xue, Songguo; Ai, Ai; Lyu, Qifeng; Kuang, Yanping

    2015-01-01

    The goal of this study is to elucidate the effects of 17β-estradiol and TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) on macrophage phenotypes in the endometriotic milieu. Co-culture of endometrial stromal cells (ESCs) and U937 cells (macrophage cell line) was performed to simulate the endometriotic milieu and to determine the effects of 17β-estradiol and/or TCDD on IL10, IL12 production and HLA-DR, CD86 expression by U937 macrophages. We found that combining 17β-estradiol with TCDD has a synergistic effect on inducing M2 activation when macrophages are co-cultured with ESCs. Moreover, the combination of 17β-estradiol and TCDD significantly enhanced STAT3 and P38 phosphorylation in macrophages. Differentiation of M2 macrophages induced by 17β-estradiol and TCDD were effectively abrogated by STAT3 and P38MAPK inhibitors, but not by ERK1/2 and JNK inhibitors. In conclusion, 17β-estradiol and TCDD in the ectopic milieu may lead to the development of endometriosis by inducing M2 polarization of macrophages through activation of the STAT3 and P38MAPK pathways. PMID:25950905

  15. EHR Big Data Deep Phenotyping

    PubMed Central

    Lenert, L.; Lopez-Campos, G.

    2014-01-01

    Summary Objectives Given the quickening speed of discovery of variant disease drivers from combined patient genotype and phenotype data, the objective is to provide methodology using big data technology to support the definition of deep phenotypes in medical records. Methods As the vast stores of genomic information increase with next generation sequencing, the importance of deep phenotyping increases. The growth of genomic data and adoption of Electronic Health Records (EHR) in medicine provides a unique opportunity to integrate phenotype and genotype data into medical records. The method by which collections of clinical findings and other health related data are leveraged to form meaningful phenotypes is an active area of research. Longitudinal data stored in EHRs provide a wealth of information that can be used to construct phenotypes of patients. We focus on a practical problem around data integration for deep phenotype identification within EHR data. The use of big data approaches are described that enable scalable markup of EHR events that can be used for semantic and temporal similarity analysis to support the identification of phenotype and genotype relationships. Conclusions Stead and colleagues’ 2005 concept of using light standards to increase the productivity of software systems by riding on the wave of hardware/processing power is described as a harbinger for designing future healthcare systems. The big data solution, using flexible markup, provides a route to improved utilization of processing power for organizing patient records in genotype and phenotype research. PMID:25123744

  16. Potent Neutralization of Influenza A Virus by a Single-Domain Antibody Blocking M2 Ion Channel Protein

    PubMed Central

    Wei, Guowei; Meng, Weixu; Guo, Haijiang; Pan, Weiqi; Liu, Jinsong; Peng, Tao; Chen, Ling; Chen, Chang-You

    2011-01-01

    Influenza A virus poses serious health threat to humans. Neutralizing antibodies against the highly conserved M2 ion channel is thought to offer broad protection against influenza A viruses. Here, we screened synthetic Camel single-domain antibody (VHH) libraries against native M2 ion channel protein. One of the isolated VHHs, M2-7A, specifically bound to M2-expressed cell membrane as well as influenza A virion, inhibited replication of both amantadine-sensitive and resistant influenza A viruses in vitro, and protected mice from a lethal influenza virus challenge. Moreover, M2-7A showed blocking activity for proton influx through M2 ion channel. These pieces of evidence collectively demonstrate for the first time that a neutralizing antibody against M2 with broad specificity is achievable, and M2-7A may have potential for cross protection against a number of variants and subtypes of influenza A viruses. PMID:22164266

  17. A Constitutively Active Gαi3 Protein Corrects the Abnormal Retinal Pigment Epithelium Phenotype of Oa1−/− mice

    PubMed Central

    Young, Alejandra; Wang, Ying; Ahmedli, Novruz B.; Jiang, Meisheng; Farber, Debora B.

    2013-01-01

    Purpose Ocular Albinism type 1 (OA1) is a disease caused by mutations in the OA1 gene and characterized by the presence of macromelanosomes in the retinal pigment epithelium (RPE) as well as abnormal crossing of the optic axons at the optic chiasm. We showed in our previous studies in mice that Oa1 activates specifically Gαi3 in its signaling pathway and thus, hypothesized that a constitutively active Gαi3 in the RPE of Oa1−/− mice might keep on the Oa1 signaling cascade and prevent the formation of macromelanosomes. To test this hypothesis, we have generated transgenic mice that carry the constitutively active Gαi3 (Q204L) protein in the RPE of Oa1−/− mice and are now reporting the effects that the transgene produced on the Oa1−/− RPE phenotype. Methods Transgenic mice carrying RPE-specific expression of the constitutively active Gαi3 (Q204L) were generated by injecting fertilized eggs of Oa1−/− females with a lentivirus containing the Gαi3 (Q204L) cDNA. PCR, Southern blots, Western blots and confocal microscopy were used to confirm the presence of the transgene in the RPE of positive transgenic mice. Morphometrical analyses were performed using electron microscopy to compare the size and number of melanosomes per RPE area in putative Oa1−/−, Gαi3 (Q204L) transgenic mice with those of wild-type NCrl and Oa1−/− mice. Results We found a correlation between the presence of the constitutively active Gαi3 (Q204L) transgene and the rescue of the normal phenotype of RPE melanosomes in Oa1−/−, Gαi3 (Q204L) mice. These mice have higher density of melanosomes per RPE area and a larger number of small melanosomes than Oa1−/− mice, and their RPE phenotype is similar to that of wild-type mice. Conclusions Our results show that a constitutively active Gαi3 protein can by-pass the lack of Oa1 protein in Oa1−/− mice and consequently rescue the RPE melanosomal phenotype. PMID:24098784

  18. Functional and Phenotypic Characteristics of Alternative Activation Induced in Human Monocytes by Interleukin-4 or the Parasitic Nematode Brugia malayi ▿ †

    PubMed Central

    Semnani, Roshanak Tolouei; Mahapatra, Lily; Moore, Vanessa; Sanprasert, Vivornpun; Nutman, Thomas B.

    2011-01-01

    Human monocytes from patients with patent filarial infections are studded with filarial antigen and express markers associated with alternative activation of macrophages (MΦ). To explore the role of filaria-derived parasite antigen in differentiation of human monocytes, cells were exposed to microfilariae (mf) of Brugia malayi, and their phenotypic and functional characteristics were compared with those of monocytes exposed to factors known to generate either alternatively (interleukin-4 [IL-4]) or classically (macrophage colony-stimulating factor [MCSF]) activated MΦ. IL-4 upregulated mRNA expression of CCL13, CCL15, CCL17, CCL18, CCL22, CLEC10A, MRC1, CADH1, CD274, and CD273 associated with alternative activation of MΦ but not arginase 1. IL-4-cultured monocytes had a diminished ability to promote proliferation of both CD4+ and CD8+ T cells compared to that of unexposed monocytes. Similar to results with IL-4, exposure of monocytes to live mf induced upregulation of CCL15, CCL17, CCL18, CCL22, CD274, and CD273 and downregulation of Toll-like receptor 3 (TLR3), TLR5, and TLR7. In contrast to results with MCSF-cultured monocytes, exposure of monocytes to mf resulted in significant inhibition of the phagocytic ability of these cells to the same degree as that seen with IL-4. Our data suggest that short exposure of human monocytes to IL-4 induces a phenotypic characteristic of alternative activation and that secreted filarial products skew monocytes similarly. PMID:21788379

  19. Refinement of the prediction of N-acetyltransferase 2 (NAT2) phenotypes with respect to enzyme activity and urinary bladder cancer risk.

    PubMed

    Selinski, Silvia; Blaszkewicz, Meinolf; Ickstadt, Katja; Hengstler, Jan G; Golka, Klaus

    2013-12-01

    Polymorphisms of N-acetyltransferase 2 (NAT2) are well known to modify urinary bladder cancer risk as well as efficacy and toxicity of pharmaceuticals via reduction in the enzyme's acetylation capacity. Nevertheless, the discussion about optimal NAT2 phenotype prediction, particularly differentiation between different degrees of slow acetylation, is still controversial. Therefore, we investigated the impact of single nucleotide polymorphisms and their haplotypes on slow acetylation in vivo and on bladder cancer risk. For this purpose, we used a study cohort of 1,712 bladder cancer cases and 2,020 controls genotyped for NAT2 by RFLP-PCR and for the tagSNP rs1495741 by TaqMan(®) assay. A subgroup of 344 individuals was phenotyped by the caffeine test in vivo. We identified an 'ultra-slow' acetylator phenotype based on combined *6A/*6A, *6A/*7B and *7B/*7B genotypes containing the homozygous minor alleles of C282T (rs1041983, *6A, *7B) and G590A (rs1799930, *6A). 'Ultra-slow' acetylators have significantly about 32 and 46 % lower activities of caffeine metabolism compared with other slow acetylators and with the *5B/*5B genotypes, respectively (P < 0.01, both). The 'ultra-slow' genotype showed an association with bladder cancer risk in the univariate analysis (OR = 1.31, P = 0.012) and a trend adjusted for age, gender and smoking habits (OR = 1.22, P = 0.082). In contrast, slow acetylators in general were not associated with bladder cancer risk, neither in the univariate (OR = 1.02, P = 0.78) nor in the adjusted (OR = 0.98, P = 0.77) analysis. In conclusion, this study suggests that NAT2 phenotype prediction should be refined by consideration of an 'ultra-slow' acetylation genotype. PMID:24221535

  20. Macrophages in spinal cord injury: phenotypic and functional change from exposure to myelin debris

    PubMed Central

    Wang, Xi; Cao, Kai; Sun, Xin; Chen, Yongxiong; Duan, Zhaoxia; Sun, Li; Guo, Lei; Bai, Paul; Sun, Dongming; Fan, Jianqing; He, Xijing; Young, Wise; Ren, Yi

    2014-01-01

    Macrophage activation and persistent inflammation contribute to the pathological process of spinal cord injury (SCI). It was reported that M2 macrophages were induced at 3–7 days after SCI but M2 markers were reduced or eliminated after 1 week. By contrast, M1 macrophage response is rapidly induced and then maintained at injured spinal cord. However, factors that modulate macrophage phenotype and function are poorly understood. We developed a model to distinguished bone marrow derived macrophages (BMDMs) from residential microglia and explored how BMDMs change their phenotype and functions in response to the lesion-related factors in injured spinal cord. Infiltrating BMDMs expressing higher Mac-2 and lower CX3CR1 migrate to the epicenter of injury, while microglia expressing lower Mac-2 but higher CX3CR1 distribute to the edges of lesion. Myelin debris at the lesion site switches BMDMs from M2 phenotype towards M1-like phenotype. Myelin debris activate ATP-binding cassette transporter A1 (ABCA1) for cholesterol efflux in response to myelin debris loading in vitro. However, this homeostatic mechanism in injured site is overwhelmed, leading to the development of foamy macrophages and lipid plaque in the lesion site. The persistence of these cells indicates a pro-inflammatory environment, associated with enhanced neurotoxicity and impaired wound healing. These foamy macrophages have poor capacity to phagocytose apoptotic neutrophils resulting in uningested neutrophils releasing their toxic contents and further tissue damage. In conclusion, these data demonstrate for the first time that myelin debris generated in injured spinal cord modulates macrophage activation. Lipid accumulation following macrophage phenotype switch contributes to SCI pathology. PMID:25452166

  1. Pyruvate kinase M2 is a phosphotyrosine-binding protein

    SciTech Connect

    Christofk, H.R.; Vander Heiden, M.G.; Wu, N.; Asara, J.M.; Cantley, L.C.

    2008-06-03

    Growth factors stimulate cells to take up excess nutrients and to use them for anabolic processes. The biochemical mechanism by which this is accomplished is not fully understood but it is initiated by phosphorylation of signalling proteins on tyrosine residues. Using a novel proteomic screen for phosphotyrosine-binding proteins, we have made the observation that an enzyme involved in glycolysis, the human M2 (fetal) isoform of pyruvate kinase (PKM2), binds directly and selectively to tyrosine-phosphorylated peptides. We show that binding of phosphotyrosine peptides to PKM2 results in release of the allosteric activator fructose-1,6-bisphosphate, leading to inhibition of PKM2 enzymatic activity. We also provide evidence that this regulation of PKM2 by phosphotyrosine signalling diverts glucose metabolites from energy production to anabolic processes when cells are stimulated by certain growth factors. Collectively, our results indicate that expression of this phosphotyrosine-binding form of pyruvate kinase is critical for rapid growth in cancer cells.

  2. Human pyruvate kinase M2: a multifunctional protein.

    PubMed

    Gupta, Vibhor; Bamezai, Rameshwar N K

    2010-11-01

    Glycolysis, a central metabolic pathway, harbors evolutionary conserved enzymes that modulate and potentially shift the cellular metabolism on requirement. Pyruvate kinase, which catalyzes the last but rate-limiting step of glycolysis, is expressed in four isozymic forms, depending on the tissue requirement. M2 isoform (PKM2) is exclusively expressed in embryonic and adult dividing/tumor cells. This tetrameric allosterically regulated isoform is intrinsically designed to downregulate its activity by subunit dissociation (into dimer), which results in partial inhibition of glycolysis at the last step. This accumulates all upstream glycolytic intermediates as an anabolic feed for synthesis of lipids and nucleic acids, whereas reassociation of PKM2 into active tetramer replenishes the normal catabolism as a feedback after cell division. In addition, involvement of this enzyme in a variety of pathways, protein-protein interactions, and nuclear transport suggests its potential to perform multiple nonglycolytic functions with diverse implications, although multidimensional role of this protein is as yet not fully explored. This review aims to provide an overview of the involvement of PKM2 in various physiological pathways with possible functional implications. PMID:20857498

  3. 26 CFR 1.401(m)-2 - ACP test.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... determined under § 1.401(m)-2(b)(2)(iv) (as it appeared in the April 1, 2007, edition of 26 CFR part 1). (E... determined under § 1.401(m)-2(b)(2)(vi) (as it appeared in the April 1, 2007, edition of 26 CFR Part 1)....

  4. Effect of communicating genetic and phenotypic risk for type 2 diabetes in combination with lifestyle advice on objectively measured physical activity: protocol of a randomised controlled trial

    PubMed Central

    2012-01-01

    Background Type 2 diabetes (T2D) is associated with increased risk of morbidity and premature mortality. Among those at high risk, incidence can be halved through healthy changes in behaviour. Information about genetic and phenotypic risk of T2D is now widely available. Whether such information motivates behaviour change is unknown. We aim to assess the effects of communicating genetic and phenotypic risk of T2D on risk-reducing health behaviours, anxiety, and other cognitive and emotional theory-based antecedents of behaviour change. Methods In a parallel group, open randomised controlled trial, approximately 580 adults born between 1950 and 1975 will be recruited from the on-going population-based, observational Fenland Study (Cambridgeshire, UK). Eligible participants will have undergone clinical, anthropometric, and psychosocial measurements, been genotyped for 23 single-nucleotide polymorphisms associated with T2D, and worn a combined heart rate monitor and accelerometer (Actiheart®) continuously for six days and nights to assess physical activity. Participants are randomised to receive either standard lifestyle advice alone (control group), or in combination with a genetic or a phenotypic risk estimate for T2D (intervention groups). The primary outcome is objectively measured physical activity. Secondary outcomes include self-reported diet, self-reported weight, intention to be physically active and to engage in a healthy diet, anxiety, diabetes-related worry, self-rated health, and other cognitive and emotional outcomes. Follow-up occurs eight weeks post-intervention. Values at follow-up, adjusted for baseline, will be compared between randomised groups. Discussion This study will provide much needed evidence on the effects of providing information about the genetic and phenotypic risk of T2D. Importantly, it will be among the first to examine the impact of genetic risk information using a randomised controlled trial design, a population-based sample, and

  5. Call Off the Dog(ma): M1/M2 Polarization Is Concurrent following Traumatic Brain Injury

    PubMed Central

    Morganti, Josh M.; Riparip, Lara-Kirstie; Rosi, Susanna

    2016-01-01

    Following the primary mechanical impact, traumatic brain injury (TBI) induces the simultaneous production of a variety of pro- and anti-inflammatory molecular mediators. Given the variety of cell types and their requisite expression of cognate receptors this creates a highly complex inflammatory milieu. Increasingly in neurotrauma research there has been an effort to define injury-induced inflammatory responses within the context of in vitro defined macrophage polarization phenotypes, known as “M1” and “M2”. Herein, we expand upon our previous work in a rodent model of TBI to show that the categorization of inflammatory response cannot be so easily delineated using this nomenclature. Specifically, we show that TBI elicited a wide spectrum of concurrent expression responses within both pro- and anti-inflammatory arms. Moreover, we show that the cells principally responsible for the production of these inflammatory mediators, microglia/macrophages, simultaneously express both “M1” and “M2phenotypic markers. Overall, these data align with recent reports suggesting that microglia/macrophages cannot adequately switch to a polarized “M1-only” or “M2-only” phenotype, but display a mixed phenotype due to the complex signaling events surrounding them. PMID:26808663

  6. Call Off the Dog(ma): M1/M2 Polarization Is Concurrent following Traumatic Brain Injury.

    PubMed

    Morganti, Josh M; Riparip, Lara-Kirstie; Rosi, Susanna

    2016-01-01

    Following the primary mechanical impact, traumatic brain injury (TBI) induces the simultaneous production of a variety of pro- and anti-inflammatory molecular mediators. Given the variety of cell types and their requisite expression of cognate receptors this creates a highly complex inflammatory milieu. Increasingly in neurotrauma research there has been an effort to define injury-induced inflammatory responses within the context of in vitro defined macrophage polarization phenotypes, known as "M1" and "M2". Herein, we expand upon our previous work in a rodent model of TBI to show that the categorization of inflammatory response cannot be so easily delineated using this nomenclature. Specifically, we show that TBI elicited a wide spectrum of concurrent expression responses within both pro- and anti-inflammatory arms. Moreover, we show that the cells principally responsible for the production of these inflammatory mediators, microglia/macrophages, simultaneously express both "M1" and "M2" phenotypic markers. Overall, these data align with recent reports suggesting that microglia/macrophages cannot adequately switch to a polarized "M1-only" or "M2-only" phenotype, but display a mixed phenotype due to the complex signaling events surrounding them. PMID:26808663

  7. A double feedback loop mediated by microRNA-23a/27a/24-2 regulates M1 versus M2 macrophage polarization and thus regulates cancer progression

    PubMed Central

    Ma, Sisi; Liu, Min; Xu, Zhenbiao; Li, Yanshuang; Guo, Hui; Ge, Yehua; Liu, Yanxin; Zheng, Dexian; Shi, Juan

    2016-01-01

    In response to microenvironmental signals, macrophages undergo different types of activation, including the “classic” pro-inflammatory phenotype (also called M1) and the “alternative” anti-inflammatory phenotype (also called M2). Macrophage polarized activation has profound effects on immune and inflammatory responses, but mechanisms underlying the various types of macrophage is still in its infancy. In this study, we reported that M1-type stimulation could down-regulate miR-23a/27a/24-2 cluster transcription through the binding of NF-κB to this cluster's promoter and that miR-23a in turn activated the NF-κB pathway by targeting A20 and thus promoted the production of pro-inflammatory cytokines. Furthermore, STAT6 occupied the miR-23a/27a/24-2 cluster promoter and activated their transcription in IL-4-stimulated macrophages. In addition, miR-23a in turn suppressed the JAK1/STAT-6 pathway and reduced the production of M2 type cytokines by targeting JAK1 and STAT-6 directly, while miR-27a showed the same phenotype by targeting IRF4 and PPAR-γ. The miR-23a/27a/24-2 cluster was shown to be significantly decreased in TAMs of breast cancer patients, and macrophages overexpressing the miR-23a/27a/24-2 cluster inhibited tumor growth in vivo. Taken together, these data integrated microRNA expression and function into macrophage polarization networks and identified a double feedback loop consisting of the miR-23a/27a/24-2 cluster and the key regulators of the M1 and M2 macrophage polarization pathway. Moreover, miR-23a/27a/24-2 regulates the polarization of tumor-associated macrophages and thus promotes cancer progression. PMID:26540574

  8. A double feedback loop mediated by microRNA-23a/27a/24-2 regulates M1 versus M2 macrophage polarization and thus regulates cancer progression.

    PubMed

    Ma, Sisi; Liu, Min; Xu, Zhenbiao; Li, Yanshuang; Guo, Hui; Ge, Yehua; Liu, Yanxin; Zheng, Dexian; Shi, Juan

    2016-03-22

    In response to microenvironmental signals, macrophages undergo different types of activation, including the "classic" pro-inflammatory phenotype (also called M1) and the "alternative" anti-inflammatory phenotype (also called M2). Macrophage polarized activation has profound effects on immune and inflammatory responses, but mechanisms underlying the various types of macrophage is still in its infancy. In this study, we reported that M1-type stimulation could down-regulate miR-23a/27a/24-2 cluster transcription through the binding of NF-κB to this cluster's promoter and that miR-23a in turn activated the NF-κB pathway by targeting A20 and thus promoted the production of pro-inflammatory cytokines. Furthermore, STAT6 occupied the miR-23a/27a/24-2 cluster promoter and activated their transcription in IL-4-stimulated macrophages. In addition, miR-23a in turn suppressed the JAK1/STAT-6 pathway and reduced the production of M2 type cytokines by targeting JAK1 and STAT-6 directly, while miR-27a showed the same phenotype by targeting IRF4 and PPAR-γ. The miR-23a/27a/24-2 cluster was shown to be significantly decreased in TAMs of breast cancer patients, and macrophages overexpressing the miR-23a/27a/24-2 cluster inhibited tumor growth in vivo. Taken together, these data integrated microRNA expression and function into macrophage polarization networks and identified a double feedback loop consisting of the miR-23a/27a/24-2 cluster and the key regulators of the M1 and M2 macrophage polarization pathway. Moreover, miR-23a/27a/24-2 regulates the polarization of tumor-associated macrophages and thus promotes cancer progression. PMID:26540574

  9. The M2 macrophages induce autophagic vascular disorder and promote mouse sensitivity to urethane-related lung carcinogenesis.

    PubMed

    Li, G-G; Guo, Z-Z; Ma, X-F; Cao, N; Geng, S-N; Zheng, Y-Q; Meng, M-J; Lin, H-H; Han, G; Du, G-J

    2016-06-01

    Tumor vessels are known to be abnormal, with typically aberrant, leaky and disordered vessels. Here, we investigated whether polarized macrophage phenotypes are involved in tumor abnormal angiogenesis and what is its mechanism. We found that there was no difference in chemotaxis of polarized M1 and M2 macrophages to lewis lung carcinoma (LLC) cells and that either M1 or M2 macrophage-conditioned media had no effect on LLC cell proliferation. Unexpectedly, the M2 but not M1 macrophage-conditioned media promoted the proliferation of human umbilical vein endothelial cells (HUVECs) and simultaneously increased endothelial cell permeability in vitro and angiogenic index in the chick embryo chorioallantoic membrane (CAM). The treatment with M2 but not M1 macrophage-conditioned media increased autophagosomes as well as microtubule-associated protein light chain 3B (LC3-B) expression (a robust marker of autophagosomes) but decreased p62 protein expression (a selective autophagy substrate) in HUVECs, the treatment with chloroquine that blocked autophagy abrogated the abnormal angiogenic efficacy of M2 macrophage-conditioned media. These results were confirmed in urethane-induced lung carcinogenic progression. Urethane-induced lung carcinogenesis led to more M2 macrophage phenotype and increased abnormal angiogenesis concomitant with the upregulation of LC3-B and the downregulation of p62. Clodronate liposome-induced macrophage depletion, chloroquine-induced autophagic prevention or salvianolic acid B-induced vascular protection decreased abnormal angiogenesis and lung carcinogenesis. In addition, we found that the tendency of age-related M2 macrophage polarization also promoted vascular permeability and carcinogenesis in urethane carcinogenic progression. These findings indicate that the M2 macrophages induce autophagic vascular disorder to promote lung cancer progression, and the autophagy improvement represents an efficacious strategy for abnormal angiogenesis and cancer

  10. Hexa-D-Arginine treatment increases 7B2•PC2 activity in hyp-mouse osteoblasts and rescues the HYP phenotype

    PubMed Central

    Yuan, Baozhi; Feng, Jian Q.; Bowman, Stephen; Liu, Ying; Blank, Robert D.; Lindberg, Iris; Drezner, Marc K.

    2012-01-01

    Inactivating mutations of PHEX/Phex underlie disease in patients with X-linked hypophosphatemia (XLH) and the hyp-mouse, a murine homologue of the human disorder. Although increased serum FGF-23 underlies the HYP phenotype, the mechanism(s) by which PHEX mutations inhibit FGF-23 degradation and/or enhance production remains unknown. Here we show that treatment of wild type mice with the proprotein convertase (PC) inhibitor, Decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone, increases serum FGF-23 and produces the HYP phenotype. Since PC2 is uniquely co-localized with PHEX in osteoblasts/bone, we examined if PC2 regulates PHEX-dependent FGF-23 cleavage and production. Transfection of murine osteoblasts with PC2 and its chaperone protein 7B2 cleaved FGF-23, while Signe1 (7B2) RNAi transfection, which limited 7B2 protein production, decreased FGF-23 degradation and increased Fgf-23 mRNA and protein. The mechanism by which decreased 7B2•PC2 activity influences Fgf-23 mRNA was linked to reduced conversion of proBMP1 to active BMP1, which resulted in limited cleavage of DMP1, and consequent increased Fgf-23 mRNA. The significance of decreased 7B2•PC2 activity in XLH was confirmed by studies of hyp-mouse bone, which revealed significantly decreased Sgne1 (7B2) mRNA and 7B2 protein, and limited cleavage of proPC2 to active PC2. The expected downstream effects of these changes included decreased FGF-23 cleavage and increased FGF-23 synthesis, secondary to decreased BMP1-mediated degradation of DMP1. Subsequent Hexa-D-Arginine treatment of hyp-mice enhanced bone 7B2•PC2 activity, normalized FGF-23 degradation and production, and rescued the HYP phenotype. These data suggest decreased PHEX-dependent 7B2•PC2 activity is central to the pathogenesis of XLH. PMID:22886699

  11. The stay-green phenotype of TaNAM-RNAi wheat plants is associated with maintenance of chloroplast structure and high enzymatic antioxidant activity.

    PubMed

    Checovich, Mariana L; Galatro, Andrea; Moriconi, Jorge I; Simontacchi, Marcela; Dubcovsky, Jorge; Santa-María, Guillermo E

    2016-07-01

    TaNAM transcription factors play an important role in controlling senescence, which in turn, influences the delivery of nitrogen, iron and other elements to the grain of wheat (Triticum aestivum) plants, thus contributing to grain nutritional value. While lack or diminished expression of TaNAMs determines a stay-green phenotype, the precise effect of these factors on chloroplast structure has not been studied. In this work we focused on the events undergone by chloroplasts in two wheat lines having either control or diminished TaNAM expression due to RNA interference (RNAi). It was found that in RNAi plants maintenance of chlorophyll levels and maximal photochemical efficiency of photosystem II were associated with lack of chloroplast dismantling. Flow cytometer studies and electron microscope analysis showed that RNAi plants conserved organelle ultrastructure and complexity. It was also found that senescence in control plants was accompanied by a low leaf enzymatic antioxidant activity. Lack of chloroplast dismantling in RNAi plants was associated with maintenance of protein and iron concentration in the flag leaf, the opposite being observed in control plants. These data provide a structural basis for the observation that down regulation of TaNAMs confers a functional stay-green phenotype and indicate that the low export of iron and nitrogen from the flag leaf of these plants is concomitant, within the developmental window studied, with lack of chloroplast degradation and high enzymatic antioxidant activity. PMID:27061370

  12. Association of hp1181 and hp1184 Genes With the Active Efflux Phenotype in Multidrug-Resistant Isolates of Helicobacter pylori

    PubMed Central

    Falsafi, Tahereh; Ehsani, Azadeh; Attaran, Bahareh; Niknam, Vahid

    2016-01-01

    Background During the last decades the rate of multidrug resistance among clinical Helicobacter pylori isolates has increased. Active pumping out of the drugs may be an important mechanism for multidrug resistance in H. pylori strains. Objectives The aim of this study was to evaluate the association of two H. pylori efflux-genes, hp1181 and hp1184 with the active-efflux phenotype in MDR clinical-strains of H. pylori. Materials and Methods Minimal inhibitory concentration (MIC) and drug accumulation for β-lactames, Tetracycline (TET), Erythromycin (ERY), Metronidazole (MTZ), Ciprofloxacin (CIP) and Ethidium Bromide (EtBr) was performed in the presence and absence of carbonyl cyanide M-Chlorophenyl Hydrazone (CCCP). Presence of hp1181 and hp1184 genes was detected by the polymerase chain reaction (PCR). RT-PCR was performed to compare expression of efflux genes by MDR strains, demonstrating active efflux with the strains without active efflux. Results Two- to four-fold decrease in minimum inhibitory concentration (MIC) and two-fold increase in accumulation were observed for EtBr in the presence of CCCP for 67% (8) of 12 MDR strains. With CCCP, two- to four-fold decrease in MIC and 1.4- to 1.8-fold increase in the accumulation of β-lactames, TET, CIP and MTZ were obtained for 42% (5) of the MDR strains. Six, five and three of the 12 MDR strains amplified hp1184, hp1181, and both of them, respectively. The RT-PCR product for expression of hp1181 by MDR strains was approximately 100 bp shorter than that of the 26695 susceptible standard strain. Conclusions Expression of the genes hp1184 and hp1181 are associated with the specific active efflux of EtBr and non-related antibiotics, respectively. For displaying these phenotypes, a post-transcriptional regulation step may be required. PMID:27303615

  13. The regulatory peptide pidotimod facilitates M2 macrophage polarization and its function.

    PubMed

    Hu, Shenglan; Fu, Xudong; Fu, Aikun; Du, Wei; Ji, Jian; Li, Weifen

    2014-05-01

    Pidotimod is a synthetic dipeptide with biological and immunological activity in innate immune responses. It has been reported that pidotimod could promote functional maturation of dendritic cells, but little is known about the regulation of macrophages. Recent studies have demonstrated that M1 or M2 polarized macrophages are of great importance for responses to microorganism infection or host mediators. The aim of this study was to determine the effectiveness of pidotimod on mouse bone marrow-derived macrophage polarization and its function. The results showed that pidotimod had no influence on M1-polarized macrophage. While interestingly, a significant increase of M2 marker gene expression (Arg1, Fizz1, Ym1, MR) was observed (p < 0.01) in IL-4-induced M2 macrophage treated with pidotimod. In addition, cell surface expression of mannose receptor was dramatically enhanced using fluorescence activated cell sorter (FACS) analysis. Furthermore, the function of M2 macrophage was also determinated. The results showed that the supernatant of pidotimod-treated M2 macrophage could increase the migration (p < 0.05) and enhance the wound closure rate (p < 0.05) of MLE-12 cells. Collectively, it could be concluded that pidotimod significantly facilitated IL-4-induced M2 macrophage polarization and improves its function. PMID:24481486

  14. Theoretical Assessment of 178m2Hf De-Excitation

    SciTech Connect

    Hartouni, E P; Chen, M; Descalle, M A; Escher, J E; Loshak, A; Navratil, P; Ormand, W E; Pruet, J; Thompson, I J; Wang, T F

    2008-10-06

    This document contains a comprehensive literature review in support of the theoretical assessment of the {sup 178m2}Hf de-excitation, as well as a rigorous description of controlled energy release from an isomeric nuclear state.

  15. Update on the role of alternatively activated macrophages in asthma

    PubMed Central

    Jiang, Zhilong; Zhu, Lei

    2016-01-01

    Lung macrophages link innate and adaptive immune responses during allergic airway inflammatory responses. Alveolar macrophages (AMs) and interstitial macrophages are two different phenotypes that differentially exert immunological function under physiological and pathological conditions. Exposure to pathogen induces polarization of AM cells into classically activated macrophages (M1 cells) and alternatively activated macrophages (M2 cells). M1 cells dominantly express proinflammatory cytokines such as TNF-α and IL-1 β and induce lung inflammation and tissue damage. M2 cells are further divided into M2a and M2c subsets. M2a cells dominantly produce allergic cytokines IL-4 and IL-13, but M2c cells dominantly produce anti-inflammatory cytokine IL-10. M2a and M2c cells are differently involved in initiation, inflammation resolution, and tissue remodeling in the different stages of asthma. Microenvironment dynamically influences polarization of AM cells. Cytokines, chemokines, and immune-regulatory cells interplay and affect the balance between the polarization of M1 and M2 cells, subsequently influencing disease progression. Thus, modulation of AM phenotypes through molecular intervention has therapeutic potential in the treatment of asthma and other allergic inflammatory diseases. This review updated recent advances in polarization and functional specialization of these macrophage subtypes with emphasis on modulation of polarization of M2 cells in asthma of human subjects and animal models. PMID:27350756

  16. Fibroblasts from phenotypically normal palmar fascia exhibit molecular profiles highly similar to fibroblasts from active disease in Dupuytren's Contracture

    PubMed Central

    2012-01-01

    Background Dupuytren's contracture (DC) is a fibroproliferative disorder characterized by the progressive development of a scar-like collagen-rich cord that affects the palmar fascia of the hand and leads to digital flexion contractures. DC is most commonly treated by surgical resection of the diseased tissue, but has a high reported recurrence rate ranging from 27% to 80%. We sought to determine if the transcriptomic profiles of fibroblasts derived from DC-affected palmar fascia, adjacent phenotypically normal palmar fascia, and non-DC palmar fascial tissues might provide mechanistic clues to understanding the puzzle of disease predisposition and recurrence in DC. Methods To achieve this, total RNA was obtained from fibroblasts derived from primary DC-affected palmar fascia, patient-matched unaffected palmar fascia, and palmar fascia from non-DC patients undergoing carpal tunnel release (6 patients in each group). These cells were grown on a type-1 collagen substrate (to better mimic their in vivo environments). Microarray analyses were subsequently performed using Illumina BeadChip arrays to compare the transcriptomic profiles of these three cell populations. Data were analyzed using Significance Analysis of Microarrays (SAM v3.02), hierarchical clustering, concordance mapping and Venn diagram. Results We found that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected fascia of DC patients exhibited a much greater overlap than fibroblasts derived from the palmar fascia of patients undergoing carpal tunnel release. Quantitative real time RT-PCR confirmed the differential expression of select genes validating the microarray data analyses. These data are consistent with the hypothesis that predisposition and recurrence in DC may stem, at least in part, from intrinsic similarities in the basal gene expression of diseased and phenotypically unaffected palmar fascia fibroblasts. These data also demonstrate that a collagen

  17. Up-regulation of Store-operated Ca2+ Entry and Nuclear Factor of Activated T Cells Promote the Acinar Phenotype of the Primary Human Salivary Gland Cells.

    PubMed

    Jang, Shyh-Ing; Ong, Hwei Ling; Liu, Xibao; Alevizos, Ilias; Ambudkar, Indu S

    2016-04-15

    The signaling pathways involved in the generation and maintenance of exocrine gland acinar cells have not yet been established. Primary human salivary gland epithelial cells, derived from salivary gland biopsies, acquired an acinar-like phenotype when the [Ca(2+)] in the serum-free medium (keratinocyte growth medium, KGM) was increased from 0.05 mm (KGM-L) to 1.2 mm (KGM-H). Here we examined the mechanism underlying this Ca(2+)-dependent generation of the acinar cell phenotype. Compared with cells in KGM-L, those in KGM-H display enhancement of Orai1, STIM1, STIM2, and nuclear factor of activated T cells 1 (NFAT1) expression together with an increase in store-operated Ca(2+) entry (SOCE), SOCE-dependent nuclear translocation of pGFP-NFAT1, and NFAT-dependent but not NFκB-dependent gene expression. Importantly, AQP5, an acinar-specific protein critical for function, is up-regulated in KGM-H via SOCE/NFAT-dependent gene expression. We identified critical NFAT binding motifs in the AQP5 promoter that are involved in Ca(2+)-dependent up-regulation of AQP5. These important findings reveal that the Ca(2+)-induced switch of salivary epithelial cells to an acinar-like phenotype involves remodeling of SOCE and NFAT signaling, which together control the expression of proteins critically relevant for acinar cell function. Our data provide a novel strategy for generating and maintaining acinar cells in culture. PMID:26903518

  18. M2SG: mapping human disease-related genetic variants to protein sequences and genomic loci

    PubMed Central

    Ji, Renkai; Cong, Qian; Li, Wenlin; Grishin, Nick V.

    2013-01-01

    Summary: Online Mendelian Inheritance in Man (OMIM) is a manually curated compendium of human genetic variants and the corresponding phenotypes, mostly human diseases. Instead of directly documenting the native sequences for gene entries, OMIM links its entries to protein and DNA sequences in other databases. However, because of the existence of gene isoforms and errors in OMIM records, mapping a specific OMIM mutation to its corresponding protein sequence is not trivial. Combining computer programs and extensive manual curation of OMIM full-text descriptions and original literature, we mapped 98% of OMIM amino acid substitutions (AASs) and all SwissProt Variant (SwissVar) disease-related AASs to reference sequences and confidently mapped 99.96% of all AASs to the genomic loci. Based on the results, we developed an online database and interactive web server (M2SG) to (i) retrieve the mapped OMIM and SwissVar variants for a given protein sequence; and (ii) obtain related proteins and mutations for an input disease phenotype. This database will be useful for analyzing sequences, understanding the effect of mutations, identifying important genetic variations and designing experiments on a protein of interest. Availability and implementation: The database and web server are freely available at http://prodata.swmed.edu/M2S/mut2seq.cgi. Contact: grishin@chop.swmed.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24002112

  19. A Bacteriophage Lambda-Based Genetic Screen for Characterization of the Activity and Phenotype of the Human Immunodeficiency Virus Type 1 Protease

    PubMed Central

    Martínez, Miguel-Angel; Cabana, Marta; Parera, Mariona; Gutierrez, Arantxa; Esté, José A.; Clotet, Bonaventura

    2000-01-01

    Human immunodeficiency virus type 1 (HIV-1) resistance to antiretroviral drugs is the main cause of patient treatment failure. Despite the problems associated with interpretation of HIV-1 resistance testing, resistance monitoring should help in the rational design of initial or rescue antiretroviral therapies. It has previously been shown that the activity of the HIV-1 protease can be monitored by using a bacteriophage lambda-based genetic assay. This genetic screening system is based on the bacteriophage lambda regulatory circuit in which the viral repressor cI is specifically cleaved to initiate the lysogenic to lytic switch. We have adapted this simple lambda-based genetic assay for the analysis of the activities and phenotypes of different HIV-1 proteases. Lambda phages that encode HIV-1 proteases either from laboratory strains (strain HXB2) or from clinical samples are inhibited in a dose-dependent manner by the HIV-1 protease inhibitors indinavir, ritonavir, saquinavir, and nelfinavir. Distinct susceptibilities to different drugs were also detected among phages that encode HIV-1 proteases carrying different resistance mutations, further demonstrating the specificity of this assay. Differences in proteolytic processing activity can also be directly monitored with this genetic screen system since two phage populations compete in culture with each other until one phage outgrows the other. In summary, we present here a simple, safe, and rapid genetic screening system that may be used to predict the activities and phenotypes of HIV-1 proteases in the course of viral infection and antiretroviral therapy. This assay responds appropriately to well-known HIV-1 protease inhibitors and can be used to search for new protease inhibitors. PMID:10770741

  20. Comprehensive Behavioral Phenotyping of Ts65Dn Mouse Model of Down Syndrome: Activation of β1-Adrenergic Receptor by Xamoterol as a Potential Cognitive Enhancer

    PubMed Central

    Faizi, Mehrdad; Bader, Patrick L.; Tun, Christine; Encarnacion, Angelo; Kleschevnikov, Alexander; Belichenko, Pavel; Saw, Nay; Priestley, Matthew; Tsien, Richard W; Mobley, William C; Shamloo, Mehrdad

    2012-01-01

    Down Syndrome (DS) is the most prevalent form of mental retardation caused by genetic abnormalities in humans. This has been successfully modeled in mice to generate the Ts65Dn mouse, a genetic model of DS. This transgenic mouse model shares a number of physical and functional abnormalities with people with DS, including changes in the structure and function of neuronal circuits. Significant abnormalities in noradrenergic (NE-ergic) afferents from the locus coeruleus to the hippocampus, as well as deficits in NE-ergic neurotransmission are detected in these animals. In the current study we characterized in detail the behavioral phenotype of Ts65Dn mice, in addition to using pharmacological tools for identification of target receptors mediating the learning and memory deficits observed in this model of DS. We undertook a comprehensive approach to mouse phenotyping using a battery of standard and novel tests encompassing: i) locomotion (Activity Chamber, PhenoTyper, and CatWalk), ii) learning and memory (spontaneous alternation, delayed matching-to-place water maze, fear conditioning, and Intellicage), and iii) social behavior. Ts65Dn mice showed increased locomotor activity in novel and home cage environments. There were significant and reproducible deficits in learning and memory tests including spontaneous alternation, delayed matching-to-place water maze, Intellicage place avoidance and contextual fear conditioning. Although Ts65Dn mice showed no deficit in sociability in the 3-chamber test, a marked impairment in social memory was detected. Xamoterol, a β1-adrenergic receptor (β1-ADR) agonist, effectively restored the memory deficit in contextual fear conditioning, spontaneous alternation and novel object recognition. These behavioral improvements were reversed by betaxolol, a selective β1-ADR antagonist. In conclusion, our results demonstrate that this mouse model of Down Syndrome display cognitive deficits which is mediated by imbalance in noradrenergic

  1. Intranodular clusters of activated cells with T follicular helper phenotype in nodular lymphocyte predominant Hodgkin lymphoma: a pilot study of 32 cases from Finland.

    PubMed

    Nathwani, Bharat N; Vornanen, Martine; Winkelmann, Ria; Kansal, Rina; Doering, Claudia; Hartmann, Sylvia; Hansmann, Martin L

    2013-09-01

    In nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), little is known about the presence of intranodular clusters of cytologically activated lymphoid cells producing a moth-eaten pattern histologically. This pilot study of 32 NLPHL cases from Finland ascertained (1) the frequency of the intranodular clusters of activated lymphoid cells, (2) the immunophenotype of the activated cells, (3) the size and immunophenotype of the rosetting cells, and (4) the clinical significance of the activated cells. Histologically, intranodular clusters of activated cells produced a moth-eaten pattern in 100% (32 cases; subtle in 62.5%, overt in 37.5%). In immunostains, activated cells in subtle clusters (20 cases) were very difficult to identify. Twelve cases had overt clusters of activated cells, which were positive with CD3, CD4, PD1, CXCL13 (T follicular helper [T(FH)] phenotype), but rarely with Ki-67 and BCL2. Most activated rosetting cells had the same immunophenotype as the nonrosetting cells, except for CXCL13. Clinical presentation for all 32 Finnish patients was distinctive: 97% men, 97% with peripheral lymphadenopathy and 35.5% with stage III/IV disease. Only 22% relapsed; 97% were in remission. There was no significant clinical difference between cases with overt and subtle clusters. Intranodular activated TFH cells in NLPHL appeared to be nonproliferating and not long-living, and they were not associated with any adverse clinical outcome. Although most activated cells were TFH cells, it seemed that they were unable to increase the number of malignant cells. The pathogenetic role of the intranodular activated TFH and the small T cells in NLPHL needs further investigation. PMID:23684509

  2. A novel bispecific EGFR/Met antibody blocks tumor-promoting phenotypic effects induced by resistance to EGFR inhibition and has potent antitumor activity.

    PubMed

    Castoldi, R; Ecker, V; Wiehle, L; Majety, M; Busl-Schuller, R; Asmussen, M; Nopora, A; Jucknischke, U; Osl, F; Kobold, S; Scheuer, W; Venturi, M; Klein, C; Niederfellner, G; Sustmann, C

    2013-12-12

    Simultaneous targeting of epidermal growth factor receptor (EGFR) and Met in cancer therapy is under pre-clinical and clinical evaluation. Here, we report the finding that treatment with EGFR inhibitors of various tumor cells, when stimulated with hepatocyte growth factor (HGF) and EGF, results in transient upregulation of phosphorylated AKT. Furthermore, EGFR inhibition in this setting stimulates a pro-invasive phenotype as assessed in Matrigel-based assays. Simultaneous treatment with AKT and EGFR inhibitors abrogates this invasive growth, hence functionally linking signaling and phenotype. This observation implies that during treatment of tumors a balanced ratio of EGFR and Met inhibition is required. To address this, we designed a bispecific antibody targeting EGFR and Met, which has the advantage of a fixed 2:1 stoichiometry. This bispecific antibody inhibits proliferation in tumor cell cultures and co-cultures with fibroblasts in an additive manner compared with treatment with both single agents. In addition, cell migration assays reveal a higher potency of the bispecific antibody in comparison with the antibodies' combination at low doses. We demonstrate that the bispecific antibody inhibits invasive growth, which is specifically observed with cetuximab. Finally, the bispecific antibody potently inhibits tumor growth in a non-small cell lung cancer xenograft model bearing a strong autocrine HGF-loop. Together, our findings strongly support a combination treatment of EGFR and Met inhibitors and further evaluation of resistance mechanisms to EGFR inhibition in the context of active Met signaling. PMID:23812422

  3. Novel spirothiazamenthane inhibitors of the influenza A M2 proton channel.

    PubMed

    Arns, Steve; Balgi, Aruna D; Shimizu, Yoko; Pfeifer, Tom A; Kumar, Nag; Shidmoossavee, Fahimeh S; Sun, Sharon; Tai, Sheldon S-H; Agafitei, Olga; Jaquith, James B; Bourque, Elyse; Niikura, Masahiro; Roberge, Michel

    2016-09-14

    The development of treatments for influenza that inhibit the M2 proton channel without being susceptible to the widespread resistance mechanisms associated with the adamantanes is an ongoing challenge. Using a yeast high-throughput yeast growth restoration assay designed to identify M2 channel inhibitors, a single screening hit was uncovered. This compound (3), whose structure was incorrectly identified in the literature, is an inhibitor with similar potency to amantadine against WT M2. A library of derivatives of 3 was prepared and activity against WT M2 and the two principal mutant strains (V27A and S31N) was assessed in the yeast assay. The best compounds were further evaluated in an antiviral plaque reduction assay using engineered WT, V27A and S31N M2 influenza A strains with otherwise identical genetic background. Compound 63 was found to inhibit all three virus strains in this cell based antiviral assay at micromolar concentrations, possibly through a mechanism other than M2 inhibition. PMID:27187859

  4. βν Integrin Inhibits Chronic and High Level Activation of JNK to Repress Senescence Phenotypes in Drosophila Adult Midgut

    PubMed Central

    Okumura, Takashi; Takeda, Koji; Taniguchi, Kiichiro; Adachi-Yamada, Takashi

    2014-01-01

    Proper control of adult stem cells including their proliferation and differentiation is crucial in maintaining homeostasis of well-organized tissues/organs throughout an organism's life. The Drosophila adult midgut has intestinal stem cells (ISCs), which have been exploited as a simple model system to investigate mechanisms controlling adult tissue homeostasis. Here, we found that a viable mutant of βν integrin (βint-ν), encoding one of two Drosophila integrin β subunits, showed a short midgut and abnormal multilayered epithelia accompanied by an increase in ISC proliferation and misdifferentiation defects. The increase in ISC proliferation and misdifferentiation was due to frequent ISC duplication expanding a pool of ISCs, which was caused by depression of the Notch signalling, and up-regulation of unpaired (upd), a gene encoding an extracellular ligand in the JAK/STAT signalling pathway. In addition, we observed that abnormally high accumulation of filamentous actin (F-actin) was caused in the βint-ν mutant enterocytes. Furthermore, the defects were rescued by suppressing c-Jun N-terminal kinase (JNK) signalling, which was up-regulated in a manner correlated with the defect levels in the above-mentioned βint-ν mutant phenotype. These symptoms observed in young βint-ν mutant midgut were very similar to those in the aged midgut in wild type. Our results suggested that βint-ν has a novel function for the Drosophila adult midgut homeostasis under normal conditions and provided a new insight into possible age-related diseases caused by latent abnormality of an integrin function. PMID:24586740

  5. Phenotype transition of CD4{sup +} T cells from CD45RA to CD45RO is accompanied by cell activation and proliferation

    SciTech Connect

    Johannisson, A.; Festin, R.

    1995-04-01

    An investigation of proliferation and activation events in subsets of human CD4{sup +} cells, defined by their expression of CD45RA and CD45RO, is reported. A single-laser based assay for the study of multiple surface antigens and two-parameter cell cycle analysis was used for sorting of and subsequent analysis of proliferation in CD4{sup +}CD45RA{sup +}CD45RO{sup {minus}}, CD4{sup +}CD45RA{sup {minus}}CD45RO{sup +} subsets and phenotypically intermediate stages. After labelling with BrdUrd, cells were sorted with flow cytometry on the basis of light-scattering properties and staining with anti-CD45RA, anti-CD45RO, and anti-CD4 markers. Sorted cells were double stained with anti-BrdUrd-antibodies and PI, and the frequencies of proliferating cells were determined. After 48 h, the highest rate of proliferation was found among cells with a phenotype intermediate between CD4{sup +}CD45RA{sup +}CD45RO{sup {minus}} and CD4{sup +}CD45RA{sup {minus}}CD45RO{sup +}. After 72 h of culture, the situation was changed insofar as the point of highest proliferation had shifted towards the CD4{sup +}CD45RA{sup {minus}}CD45RO{sup +} population. These findings were further corroborated by four-color staining with anti-CD4, anti-CD45RA, anti-CD45RO, and Hoechst 33342. This indicates that the phenotype transition is accompanied by cell proliferation. The correlated temporal expression of antigens related to activation (HLA-DR, CD25, CD69, CD71) and cell adhesion (CD11a, CD54, L-selectin) in each of the different subsets was also investigated. All the activation markers CD25, CD69, and CD71 show a more heterogeneous pattern of expression among the CD4{sup +} CD45RA{sup {minus}}CD45RO{sup +} cells than the CD4{sup +} CD45RA{sup +}CD45RO{sup {minus}} cells, indicating a subpopulation of CD4{sup +}CD45RA{sup {minus}}CD45RO{sup +} cells responding more slowly to the mitogenic stimulation. 32 refs., 8 figs.

  6. PTEN inhibits macrophage polarization from M1 to M2 through CCL2 and VEGF-A reduction and NHERF-1 synergism.

    PubMed

    Li, Ning; Qin, Junfang; Lan, Lan; Zhang, Hongyao; Liu, Fang; Wu, Zhaozhen; Ni, Hong; Wang, Yue

    2015-01-01

    PTEN has been studied in several tumor models as a tumor suppressor. In this study, we explored the role of PTEN in the inhibition state of polarized M2 subtype of macrophage in tumor microenvironment (TME) and the underlying mechanisms. To elucidate the potential effect in TME, RAW 264.7 macrophages and 4T1 mouse breast cancer cells were co-cultured to reconstruct tumor microenvironment. After PTEN was down-regulated with shRNA, the expression of CCL2 and VEGF-A, which are definited to promote the formation of M2 macrophages, have a dramatically increase on the level of both gene and protein in co-cultured RAW 264.7 macrophages. And at the same time, NHERF-1 (Na(+)/H(+) exchanger regulating factor-1), another tumor suppressor has a similar tendency to PTEN. Q-PCR and WB results suggested that PTEN and NHERF-1 were consistent with one another no matter at mRNA or protein level when exposed to the same stimulus. Coimmunoprecipitation and immunofluorescence techniques confirmed that PTEN and NHERF-1 were coprecipitated, and NHERF-1 protein expression was properly reduced with rCCL2 effect. In addition, cell immunofluorescence images revealed a profound transferance, in co-cultured RAW 264.7 macrophages, an up-regulation of NHERF-1 could promote the PTEN marked expression on the cell membrane, and this form for the interaction was not negligible. These observations illustrate PTEN with a certain synergy of NHERF-1, as well as down-regulation of CCL2 suppressing M2 macrophage transformation pathway. The results suggest that the activation of PTEN and NHERF-1 may impede the evolution of macrophages beyond the M1 into M2 phenotype in tumor microenvironment. PMID:25756512

  7. Anatomy of a Discovery: M1 and M2 Macrophages

    PubMed Central

    Mills, Charles Dudley

    2015-01-01

    M1 and M2 macrophage-type responses kill or repair in vivo. The unique ability of macrophages to make these polar opposite type of responses provides primary host protection and maintains tissue homeostasis throughout the animal kingdom. In humans and other higher animals, M1 and M2-type macrophage responses also initiate and direct T cells/adaptive immunity to provide additional protection such as Th1 (cytotoxic) or Th2 (antibody-mediated) type responses. Hence, macrophages were renamed M1 and M2 to indicate the central role of macrophages/innate immunity in immune systems. These findings indicate that the long held notion that adaptive immunity controls innate immunity was backward: a sea change in understanding how immune responses occur. The clinical impact of M1/kill and M2/repair responses is immense playing pivotal roles in curing (or causing) many diseases including infections, cancer, autoimmunity, and atherosclerosis. How M1/M2 came to be is an interesting story that, like life, involved Direction, Determination, Discouragement, and Discovery. PMID:25999950

  8. Temporal phenotypic features distinguish polarized macrophages in vitro.

    PubMed

    Melton, David W; McManus, Linda M; Gelfond, Jonathan A L; Shireman, Paula K

    2015-05-01

    Macrophages are important in vascular inflammation and environmental factors influence macrophage plasticity. Macrophage transitions into pro-inflammatory (M1) or anti-inflammatory (M2) states have been defined predominately by measuring cytokines in culture media (CM). However, temporal relationships between cellular and secreted cytokines have not been established. We measured phenotypic markers and cytokines in cellular and CM of murine bone marrow-derived macrophages at multiple time points following stimulation with IFN-γ + LPS (M1), IL-4 (M2a) or IL-10 (M2c). Cytokines/proteins in M1-polarized macrophages exhibited two distinct temporal patterns; an early (0.5-3 h), transient increase in cellular cytokines (GM-CSF, KC-GRO, MIP-2, IP-10 and MIP-1β) and a delayed (3-6 h) response that was more sustained [IL-3, regulated on activation normal T cell expressed and secreted (RANTES), and tissue inhibitor of metalloproteinases 1 (TIMP-1)]. M2a-related cytokine/cell markers (IGF-1, Fizz1 and Ym1) were progressively (3-24 h) increased post-stimulation. In addition, novel patterns were observed. First, and unexpectedly, cellular pro-inflammatory chemokines, MCP-1 and MCP-3 but not MCP-5, were comparably increased in M1 and M2a macrophages. Second, Vegfr1 mRNA was decreased in M1 and increased in M2a macrophages. Finally, VEGF-A was increased in the CM of M1 cultures and strikingly reduced in M2a coinciding with increased Vegfr1 expression, suggesting decreased VEGF-A in M2a CM was secondary to increased soluble VEGFR1. In conclusion, macrophage cytokine production and marker expression were temporally regulated and relative levels compared across polarizing conditions were highly dependent upon the timing and location (cellular versus CM) of the sample collection. For most cytokines, cellular production preceded increases in the CM suggesting that cellular regulatory pathways should be studied within 6 h of stimulation. The divergent polarization-dependent expression

  9. Temporal Phenotypic Features Distinguish Polarized Macrophages In Vitro

    PubMed Central

    Melton, David W.; McManus, Linda M.; Gelfond, Jonathan A.L.; Shireman, Paula K.

    2015-01-01

    Macrophages are important in vascular inflammation and environmental factors influence macrophage plasticity. Macrophage transitions into pro-inflammatory (M1) or anti-inflammatory (M2) states have been defined predominately by measuring cytokines in culture media (CM). However, temporal relationships between cellular and secreted cytokines have not been established. We measured phenotypic markers and cytokines in cellular and CM of murine bone marrow-derived macrophages at multiple time points following stimulation with IFN-γ+LPS (M1), IL-4 (M2a), or IL-10 (M2c). Cytokines/proteins in M1-polarized macrophages exhibited two distinct temporal patterns; an early (0.5–3 hr), transient increase in cellular cytokines (GM-CSF, KC-GRO, MIP-2, IP-10 and MIP-1β) and a delayed (3–6 hrs) response that was more sustained [IL-3, regulated on activation normal T cell expressed and secreted (RANTES), and tissue inhibitor of metalloproteinases 1 (TIMP-1)]. M2a-related cytokine/cell markers (IGF-1, Fizz1, and Ym1) were progressively (3–24 hrs) increased post-stimulation. Additionally, novel patterns were observed. First, and unexpectedly, cellular pro-inflammatory chemokines, MCP-1 and MCP-3 but not MCP-5, were comparably increased in M1 and M2a macrophages. Second, Vegfr1 mRNA was decreased in M1 and increased in M2a macrophages. Finally, VEGF-A was increased in the CM of M1 cultures and strikingly reduced in M2a coinciding with increased Vegfr1 expression, suggesting decreased VEGF-A in M2a CM was secondary to increased soluble VEGFR1. In conclusion, macrophage cytokine production and marker expression were temporally regulated and relative levels compared across polarizing conditions were highly dependent upon the timing and location (cellular vs. CM) of the sample collection. For most cytokines, cellular production preceded increases in the CM suggesting that cellular regulatory pathways should be studied within 6 hours of stimulation. The divergent polarization

  10. M2-F1 in flight on tow line

    NASA Technical Reports Server (NTRS)

    1964-01-01

    The M2-F1 Lifting Body is seen here under tow at the Flight Research Center (later redesignated the Dryden Flight Research Center), Edwards, California. The wingless, lifting-body aircraft design was initially concieved as a means of landing an aircraft horizontally after atmospheric reentry. The absence of wings would make the extreme heat of re-entry less damaging to the vehicle. In 1962, Flight Research Center management approved a program to build a lightweight, unpowered lifting body as a prototype to flight test the wingless concept. It would look like a 'flying bathtub,' and was designated the M2-F1, the 'M' referring to 'manned' and 'F' referring to 'flight' version. It featured a plywood shell placed over a tubular steel frame crafted at Dryden. Construction was completed in 1963. The M2-F1 project had limited goals. They were to show that a piloted lifting body could be built, that it could not only fly but be controlled in flight, and that it could make a successful landing. While the M2-F1 did prove the concept, with a wooden fuselage and fixed landing gear, it was far from an operational spacecraft. The next step in the lifting-body development was to build a heavyweight, rocket-powered vehicle that was more like an operational lifting body, albeit one without the thermal protection system that would be needed for reentry into the atmosphere from space at near-orbital speeds. The first flight tests of the M2-F1 were over Rogers Dry Lake at the end of a tow rope attached to a hopped-up Pontiac convertible driven at speeds up to about 120 mph. These initial tests produced enough flight data about the M2-F1 to proceed with flights behind a NASA C-47 tow plane at greater altitudes. The C-47 took the craft to an altitude of 12,000 where free flights back to Rogers Dry Lake began. Pilot for the first series of flights of the M2-F1 was NASA research pilot Milt Thompson. Typical glide flights with the M2-F1 lasted about two minutes and reached speeds of 110 to

  11. Operational guidance for using DOT-6M/2R packaging

    SciTech Connect

    Kelly, D.L.; Hummer, J.H.

    1994-03-01

    The purpose of this paper is to describe a new US Department of Energy (DOE), Transportation Management Division task to create a US Department of Transportation (DOT) Specification 6M/2R packaging configuration user`s guide. The need for a user`s guide was identified because the DOT-6M/2R packaging configuration is widely used by DOE site contractors, and DOE receives many questions about the approved packaging configurations. Currently, two DOE organizations have the authority to approve new DOT-6M/2R configurations. For Defense Programs, the Transportation and Packaging Safety Division (EH-332) administers the program. For Environmental Restoration and Waste Management, the Transportation Management Division (EM-261) administers the program.

  12. M2-F1 on lakebed with pilot Milt Thompson

    NASA Technical Reports Server (NTRS)

    1963-01-01

    NASA Flight Research Pilot Milt Thompson, shown here on the lakebed with the M2-F1 lifting body, was an early backer of R. Dale Reed's lifting-body proposal. He urged Flight Research Center director Paul Bikle to approve the M2-F1's construction. Thompson also made the first glide flights in both the M2-F1 and its successor, the heavyweight M2-F2. The wingless, lifting body aircraft design was initially conceived as a means of landing an aircraft horizontally after atmospheric reentry. The absence of wings would make the extreme heat of re-entry less damaging to the vehicle. In 1962, NASA Flight Research Center (later Dryden Flight Research Center, Edwards, CA) management approved a program to build a lightweight, unpowered lifting body as a prototype to flight test the wingless concept. It would look like a 'flying bathtub,' and was designated the M2-F1, the 'M' referring to 'manned' and 'F' referring to 'flight' version. It featured a plywood shell placed over a tubular steel frame crafted at Dryden. Construction was completed in 1963. The first flight tests of the M2-F1 were over Rogers Dry Lake at the end of a tow rope attached to a hopped-up Pontiac convertible driven at speeds up to about 120 mph. This vehicle needed to be able to tow the M2-F1 on the Rogers Dry Lakebed adjacent to NASA's Flight Research Center (FRC) at a minimum speed of 100 miles per hour. To do that, it had to handle the 400-pound pull of the M2-F1. Walter 'Whitey' Whiteside, who was a retired Air Force maintenance officer working in the FRC's Flight Operations Division, was a dirt-bike rider and hot-rodder. Together with Boyden 'Bud' Bearce in the Procurement and Supply Branch of the FRC, Whitey acquired a Pontiac Catalina convertible with the largest engine available. He took the car to Bill Straup's renowned hot-rod shop near Long Beach for modification. With a special gearbox and racing slicks, the Pontiac could tow the 1,000-pound M2-F1 110 miles per hour in 30 seconds. It proved

  13. AS04-adjuvanted virus-like particles containing multiple M2 extracellular domains of influenza virus confer improved protection.

    PubMed

    Lee, Yu-Na; Kim, Min-Chul; Lee, Young-Tae; Hwang, Hye Suk; Cho, Min-Kyoung; Lee, Jong Seok; Ko, Eun-Ju; Kwon, Young-Man; Kang, Sang-Moo

    2014-07-31

    The ectodomain of matrix protein 2 (M2e) of influenza virus is suggested to be a rational target for a universal influenza A vaccine. However, there are some concerns that M2e vaccines might not be highly effective in the general population with diverse genetic backgrounds. Here we examined the immunogenicity and protective efficacy of the baculovirus-derived virus-like particles containing multiple M2e (M2eVLP) with AS04 adjuvant in a C57BL/6 mouse strain (H-2(b)). M2eVLP vaccine induced significant levels of M2e-specific IgG in C57BL/6 mice after vaccination. Furthermore, M2eVLP adjuvanted with AS04 was more effective than M2eVLP alone in conferring protection as well as in inducing recall humoral and T cell responses specific for M2e after lethal influenza virus challenge. A mechanistic study provides evidence that activation of dendritic cells by the toll-like receptor 4 agonist MPL in the AS04 adjuvant was associated with interferon-γ producing CD4 T cell responses. Our results suggest that AS04 adjuvanted M2eVLP vaccines have the potential to improve cross-protection. PMID:24951867

  14. Cytokinin Activity of cis-Zeatin and Phenotypic Alterations Induced by Overexpression of Putative cis-Zeatin-O-glucosyltransferase in Rice1[C][W][OA

    PubMed Central

    Kudo, Toru; Makita, Nobue; Kojima, Mikiko; Tokunaga, Hiroki; Sakakibara, Hitoshi

    2012-01-01

    cis-Zeatin (cZ) is generally regarded as a cytokinin with little or no activity, compared with the highly active trans-zeatin (tZ). Although recent studies suggested possible roles for cZ, its physiological significance remains unclear. In our studies with rice (Oryza sativa), cZ inhibited seminal root elongation and up-regulated cytokinin-inducible genes, and its activities were comparable to those of tZ. Tracer experiments showed that exogenously supplied cZ-riboside was mainly converted into cZ derivatives but scarcely into tZ derivatives, indicating that isomerizations of cZ derivatives into tZ derivatives are a minor pathway in rice cytokinin metabolism. We identified three putative cZ-O-glucosyltransferases (cZOGT1, cZOGT2, and cZOGT3) in rice. The cZOGTs preferentially catalyzed O-glucosylation of cZ and cZ-riboside rather than tZ and tZ-riboside in vitro. Transgenic rice lines ectopically overexpressing the cZOGT1 and cZOGT2 genes exhibited short-shoot phenotypes, delay of leaf senescence, and decrease in crown root number, while cZOGT3 overexpressor lines did not show shortened shoots. These results propose that cZ activity has a physiological impact on the growth and development of rice. PMID:22811434

  15. M2-F1 ejection seat test at South Edwards

    NASA Technical Reports Server (NTRS)

    1963-01-01

    The M2-F1 was fitted with an ejection seat before the airtow flights began. The project selected the seat used in the T-37 as modified by the Weber Company to use a rocket rather than a ballistic charge for ejection. To test the ejection seat, the Flight Research Center's Dick Klein constructed a plywood mockup of the M2-F1's top deck and canopy. On the first firings, the test was unsuccessful, but on the final test the dummy in the seat landed safely. The M2-F1 ejection seat was later used in the two Lunar Landing Research Vehicles and the three Lunar Landing Training Vehicles. Three of them crashed, but in each case the pilot ejected from the vehicle successfully. The wingless, lifting body aircraft design was initially conceived as a means of landing an aircraft horizontally after atmospheric reentry. The absence of wings would make the extreme heat of re-entry less damaging to the vehicle. In 1962, Dryden management approved a program to build a lightweight, unpowered lifting body as a prototype to flight test the wingless concept. It would look like a 'flying bathtub,' and was designated the M2-F1, the 'M' referring to 'manned' and 'F' referring to 'flight' version. It featured a plywood shell placed over a tubular steel frame crafted at Dryden. Construction was completed in 1963. The first flight tests of the M2-F1 were over Rogers Dry Lake at the end of a tow rope attached to a hopped-up Pontiac convertible driven at speeds up to about 120 mph. This vehicle needed to be able to tow the M2-F1 on the Rogers Dry Lakebed adjacent to NASA's Flight Research Center (FRC) at a minimum speed of 100 miles per hour. To do that, it had to handle the 400-pound pull of the M2-F1. Walter 'Whitey' Whiteside, who was a retired Air Force maintenance officer working in the FRC's Flight Operations Division, was a dirt-bike rider and hot-rodder. Together with Boyden 'Bud' Bearce in the Procurement and Supply Branch of the FRC, Whitey acquired a Pontiac Catalina convertible with

  16. Increase in a distinct pulmonary macrophage subset possessing an antigen-presenting cell phenotype and in vitro APC activity following silica exposure

    SciTech Connect

    Migliaccio, Christopher T. . E-mail: christopher.migliaccio@umontana.edu; Hamilton, Raymond F.; Holian, Andrij

    2005-06-01

    Silica inhalation results in chronic lung inflammation and fibrosis. While the role of the alveolar macrophage (AM) is considered key to the effects of silica on lung pathology, the etiology is not completely understood. Evidence suggests an increase in antigen presenting cell (APC) activity as a contributing factor to this process, as well as potential roles for both AM and interstitial macrophages (IM) in silicosis. In order to study the effects of crystalline silica on the APC activity of pulmonary macrophages, mice were exposed intranasally and changes in pulmonary macrophage populations were assessed using flow cytometry. Following intranasal instillation of silica, a significant increase in the APC activity of AM was observed, as well as a significant increase in a subset of IM expressing classic APC markers (MHC class II, CD11c). In addition, an in vitro system using bone marrow-derived macrophages (BMDM) was generated to assess the effects of silica on the APC activity of macrophages in vitro. Data using BMDM in the in vitro APC assay demonstrated a significant increase in APC activity following silica exposure, but not following exposure to saline or a control particle (TiO{sub 2}). Using a combination of in vivo and in vitro experiments, the current study describes a significant increase in an interstitial macrophage subset with an APC phenotype, as well as an increase in the APC activity of both AM and BMDM, as a direct result of exposure to crystalline silica. These studies suggest a specific mechanism, macrophage subset activation, by which crystalline silica exposure results in chronic pulmonary inflammation and, eventually, fibrosis.

  17. M2e-Based Universal Influenza A Vaccines

    PubMed Central

    Deng, Lei; Cho, Ki Joon; Fiers, Walter; Saelens, Xavier

    2015-01-01

    The successful isolation of a human influenza virus in 1933 was soon followed by the first attempts to develop an influenza vaccine. Nowadays, vaccination is still the most effective method to prevent human influenza disease. However, licensed influenza vaccines offer protection against antigenically matching viruses, and the composition of these vaccines needs to be updated nearly every year. Vaccines that target conserved epitopes of influenza viruses would in principle not require such updating and would probably have a considerable positive impact on global human health in case of a pandemic outbreak. The extracellular domain of Matrix 2 (M2e) protein is an evolutionarily conserved region in influenza A viruses and a promising epitope for designing a universal influenza vaccine. Here we review the seminal and recent studies that focused on M2e as a vaccine antigen. We address the mechanism of action and the clinical development of M2e-vaccines. Finally, we try to foresee how M2e-based vaccines could be implemented clinically in the future. PMID:26344949

  18. M2-F1 in hangar with Pontiac tow vehicle

    NASA Technical Reports Server (NTRS)

    1963-01-01

    The M2-F1 Lifting Body is seen here in a hangar with its hotrod Pontiac convertible tow vehicle at the Flight Research Center (later the Dryden Flight Research Center), Edwards, California. The car was a 1963 Pontiac Catalina convertible, fitted with a 421-cubic-inch tripower engine like those being run at the Daytona 500 auto race. The vehicle also had a four-speed transmission and a heavy-duty suspension and cooling system. A roll bar was also added and the passenger seat turned around so an observer could watch the M2-F1 while it was being towed. The rear seat was removed and a second, side-facing seat installed. The lifting-body team used the Pontiac for all the ground-tow flights over the next three years. The wingless, lifting body aircraft design was initially conceived as a means of landing an aircraft horizontally after atmospheric reentry. The absence of wings would make the extreme heat of re-entry less damaging to the vehicle. In 1962, Dryden management approved a program to build a lightweight, unpowered lifting body as a prototype to flight test the wingless concept. It would look like a 'flying bathtub,' and was designated the M2-F1, the 'M' referring to 'manned' and 'F' referring to 'flight' version. It featured a plywood shell placed over a tubular steel frame crafted at Dryden. Construction was completed in 1963. The first flight tests of the M2-F1 were over Rogers Dry Lake at the end of a tow rope attached to a hopped-up Pontiac convertible driven at speeds up to about 120 mph. This vehicle needed to be able to tow the M2-F1 on the Rogers Dry Lakebed adjacent to NASA's Flight Research Center (FRC) at a minimum speed of 100 miles per hour. To do that, it had to handle the 400-pound pull of the M2-F1. Walter 'Whitey' Whiteside, who was a retired Air Force maintenance officer working in the FRC's Flight Operations Division, was a dirt-bike rider and hot-rodder. Together with Boyden 'Bud' Bearce in the Procurement and Supply Branch of the FRC, Whitey

  19. Increased PTP1B expression and phosphatase activity in colorectal cancer results in a more invasive phenotype and worse patient outcome

    PubMed Central

    Hoekstra, Elmer; Das, Asha M.; Swets, Marloes; Cao, Wanlu; van der Woude, C. Janneke; Bruno, Marco J.; Peppelenbosch, Maikel P.; Kuppen, Peter J.K.; ten Hagen, Timo L.M.; Fuhler, Gwenny M.

    2016-01-01

    Cell signaling is dependent on the balance between phosphorylation of proteins by kinases and dephosphorylation by phosphatases. This balance if often disrupted in colorectal cancer (CRC), leading to increased cell proliferation and invasion. For many years research has focused on the role of kinases as potential oncogenes in cancer, while phosphatases were commonly assumed to be tumor suppressive. However, this dogma is currently changing as phosphatases have also been shown to induce cancer growth. One of these phosphatases is protein tyrosine phosphatase 1B (PTP1B). Here we report that the expression of PTP1B is increased in colorectal cancer as compared to normal tissue, and that the intrinsic enzymatic activity of the protein is also enhanced. This suggests a role for PTP1B phosphatase activity in CRC formation and progression. Furthermore, we found that increased PTP1B expression is correlated to a worse patient survival and is an independent prognostic marker for overall survival and disease free survival. Knocking down PTP1B in CRC cell lines results in a less invasive phenotype with lower adhesion, migration and proliferation capabilities. Together, these results suggest that inhibition of PTP1B activity is a promising new target in the treatment of colorectal cancer and the prevention of metastasis. PMID:26942883

  20. Phenotypic debrisoquine 4-hydroxylase activity among extensive metabolizers is unrelated to genotype as determined by the Xba-I restriction fragment length polymorphism.

    PubMed Central

    Turgeon, J; Evans, W E; Relling, M V; Wilkinson, G R; Roden, D M

    1991-01-01

    1. The major pathway for 4-hydroxylation of debrisoquine in man is polymorphic and under genetic control. More than 90% of subjects (extensive metabolizers, EMs) have active debrisoquine 4-hydroxylase (cytochrome P450IID6) while in the remainder (poor metabolizers, PMs), cytochrome P450IID6 activity is greatly impaired. 2. Within the EM group, cytochrome P450IID6-mediated metabolism of a range of substrates varies widely. Some of this intra-phenotype non-uniformity may be explained by the presence of two subsets of subjects with different genotypes (heterozygotes and homozygotes). 3. Cytochrome P450IID6 substrates have not differentiated between these two genotypes. However, a restriction fragment length polymorphism (RFLP) which identifies mutant alleles of cytochrome P450IID6 locus has been described and can definitively assign genotype in some heterozygous EM subjects. 4. In this study, we used RFLP analysis and encainide as a model substrate to determine if non-uniformity in cytochrome P450IID6 activity among EMs is related to genotype. We tested the hypothesis that heterozygotes exhibit intermediate metabolic activity and that homozygous dominants exhibit the highest activity. We proposed encainide as a useful substrate for this purpose since cytochrome P450IID6 catalyzes not only its biotransformation to O-desmethyl encainide (ODE) but also the subsequent metabolism of ODE to 3-methoxy-O-desmethyl encainide (MODE). 5. A single 50 mg oral dose of encainide was administered to 139 normal volunteers and 14 PMs were identified. Urinary ratios among encainide, ODE and MODE in the remaining 125 EM subjects revealed a wide range of cytochrome P450IID6 activity. However, Southern blotting of genomic DNA digested with XbaI identified obligate heterozygotes in both extremes of all ratio distributions.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 2 PMID:1685663

  1. Internal steel structure of M2-F1

    NASA Technical Reports Server (NTRS)

    1963-01-01

    The internal steel structure for the M2-F1 was built at the Flight Research Center (predecessor of the Dryden Flight Research Center, Edwards, CA) in a section of the calibration hangar dubbed 'Wright Bicycle Shop.' Visible are the stick, rudder pedals, and ejection seat. The external wooden shell was attached to the steel structure. The wingless, lifting body aircraft design was initially conceived as a means of landing an aircraft horizontally after atmospheric reentry. The absence of wings would make the extreme heat of re-entry less damaging to the vehicle. In 1962, Dryden management approved a program to build a lightweight, unpowered lifting body as a prototype to flight test the wingless concept. It would look like a 'flying bathtub,' and was designated the M2-F1, the 'M' referring to 'manned' and 'F' referring to 'flight' version. It featured a plywood shell placed over a tubular steel frame crafted at Dryden. Construction was completed in 1963. The first flight tests of the M2-F1 were over Rogers Dry Lake at the end of a tow rope attached to a hopped-up Pontiac convertible driven at speeds up to about 120 mph. This vehicle needed to be able to tow the M2-F1 on the Rogers Dry Lakebed adjacent to NASA's Flight Research Center (FRC) at a minimum speed of 100 miles per hour. To do that, it had to handle the 400-pound pull of the M2-F1. Walter 'Whitey' Whiteside, who was a retired Air Force maintenance officer working in the FRC's Flight Operations Division, was a dirt-bike rider and hot-rodder. Together with Boyden 'Bud' Bearce in the Procurement and Supply Branch of the FRC, Whitey acquired a Pontiac Catalina convertible with the largest engine available. He took the car to Bill Straup's renowned hot-rod shop near Long Beach for modification. With a special gearbox and racing slicks, the Pontiac could tow the 1,000-pound M2-F1 110 miles per hour in 30 seconds. It proved adequate for the roughly 400 car tows that got the M2-F1 airborne to prove it could fly

  2. Antibacterial activity and genotypic-phenotypic characteristics of bacteriocin-producing Bacillus subtilis KKU213: potential as a probiotic strain.

    PubMed

    Khochamit, Nalisa; Siripornadulsil, Surasak; Sukon, Peerapol; Siripornadulsil, Wilailak

    2015-01-01

    The antimicrobial activity and probiotic properties of Bacillus subtilis strain KKU213, isolated from local soil, were investigated. The cell-free supernatant (CFS) of a KKU213 culture containing crude bacteriocins exhibited inhibitory effects on Gram-positive bacteria, including Bacillus cereus, Listeria monocytogenes, Micrococcus luteus, and Staphylococcus aureus. The antibacterial activity of the CFS precipitated with 40% ammonium sulfate (AS) remained even after treatment at 60 and 100 °C, at pH 4 and 10 and with proteolytic enzymes, detergents and heavy metals. When analyzed by SDS-PAGE and overlaid with the indicator strains B. cereus and S. aureus, the 40% AS precipitate exhibited inhibitory activity on proteins smaller than 10 kDa. However, proteins larger than 25 kDa and smaller than 10 kDa were still observed on a native protein gel. Purified subtilosin A was prepared by Amberlite XAD-16 bead extraction and HPLC and analyzed by Nano-LC-QTOF-MS. Its molecular mass was found to be 3.4 kDa, and it retained its antibacterial activity. These results are consistent with the detection of the anti-listerial subtilosin A gene of the sbo/alb cluster in the KKU213 strain, which is 100% identical to that of B. subtilis subsp. subtilis 168. In addition to stable and cyclic subtilosin A, a mixture of many extracellular antibacterial peptides was also detected in the KKU213 culture. The KKU213 strain produced extracellular amylase, cellulase, lipase and protease, is highly acid-resistant (pH 2) when cultured in inulin and promotes health and reduces infection of intestinally colonized broiler chickens. Therefore, we propose that bacteriocin-producing B. subtilis KKU213 could be used as a potential probiotic strain or protective culture. PMID:25440998

  3. The thyroid cancer PAX8-PPARG fusion protein activates Wnt/TCF-responsive cells that have a transformed phenotype.

    PubMed

    Vu-Phan, Dang; Grachtchouk, Vladimir; Yu, Jingcheng; Colby, Lesley A; Wicha, Max S; Koenig, Ronald J

    2013-10-01

    A chromosomal translocation results in the production of a paired box 8-peroxisome proliferator-activated receptor gamma (PAX8-PPARG) fusion protein (PPFP) in ∼35% of follicular thyroid carcinomas. To examine the role of PPFP in thyroid oncogenesis, the fusion protein was stably expressed in the non-transformed rat thyroid cell line PCCL3. PPFP conferred on PCCL3 cells the ability to invade through Matrigel and to form colonies in anchorage-independent conditions. PPFP also increased the fraction of cells with Wnt/TCF-responsive green fluorescent protein reporter gene expression. This Wnt/TCF-activated population was enriched for colony-forming and invading cells. These actions of PPFP required a functional PPARG DNA binding domain (DBD) within PPFP and were further stimulated by PPARG agonists. These data indicate that PPFP, through its PPARG DBD, induces Wnt/TCF pathway activation in a subpopulation of cells, and these cells have properties of cellular transformation including increased invasiveness and anchorage-independent growth. PMID:24025583

  4. Diverse Bone Morphogenetic Protein Expression Profiles and Smad Pathway Activation in Different Phenotypes of Experimental Canine Mammary Tumors

    PubMed Central

    Wensman, Helena; Heldin, Nils-Erik; Pejler, Gunnar; Hellmén, Eva

    2009-01-01

    Background BMPs are currently receiving attention for their role in tumorigenesis and tumor progression. Currently, most BMP expression studies are performed on carcinomas, and not much is known about the situation in sarcomas. Methodology/Principal Findings We have investigated the BMP expression profiles and Smad activation in clones from different spontaneous canine mammary tumors. Spindle cell tumor and osteosarcoma clones expressed high levels of BMPs, in particular BMP-2, -4 and -6. Clones from a scirrhous carcinoma expressed much lower BMP levels. The various clones formed different tumor types in nude mice but only clones that expressed high levels of BMP-6 gave bone formation. Phosphorylated Smad-1/5, located in the nucleus, was detected in tumors derived from clones expressing high levels of BMPs, indicating an active BMP signaling pathway and BMP-2 stimulation of mammary tumor cell clones in vitro resulted in activation of the Smad-1/5 pathway. In contrast BMP-2 stimulation did not induce phosphorylation of the non-Smad pathway p38 MAPK. Interestingly, an increased level of the BMP-antagonist chordin-like 1 was detected after BMP stimulation of non-bone forming clones. Conclusions/Significance We conclude that the specific BMP expression repertoire differs substantially between different types of mammary tumors and that BMP-6 expression most probably has a biological role in bone formation of canine mammary tumors. PMID:19771160

  5. M2-F2 flight preparation and launch

    NASA Technical Reports Server (NTRS)

    1969-01-01

    This movie clip runs about 27 seconds and shows the cockpit canopy close-out by the ground crew, the aircraft hanging from the NB-52B wing pylon, and the M2-F2 being dropped away from the mothership. A fleet of lifting bodies flown at the NASA Flight Research Center (FRC), Edwards, California, from 1963 to l975 demonstrated the ability of pilots to maneuver (in the atmosphere) and safely land a wingless vehicle. These lifting bodies were basically designed so they could fly back to Earth from space and be landed like an aircraft at a pre-determined site. They served as precursors of today's Space Shuttle, the X-33, and the X-38, providing technical and operational engineering data that shaped all three space vehicles. (In 1976 NASA renamed the FRC as the NASA Dryden Flight Research Center (DFRC) in honor of Hugh L. Dryden.) In 1962, FRC Director Paul Bikle approved a program to build a lightweight, unpowered lifting body as a prototype to flight test the wingless concept. It would look like a 'flying bathtub,' and was designated the M2-F1. Built by Gus Briegleb, a sailplane builder from El Mirage, California, it featured a plywood shell, placed over a tubular steel frame crafted at the FRC. Construction was completed in 1963. The success of Dryden's M2-F1 program led to NASA's development and construction of two heavyweight lifting bodies based on studies at NASA Ames Research Center and NASA and Langley Research Center -- the M2-F2 and the HL-10, both built by the Northrop Corporation, Los Angeles, California. The 'M' refers to 'manned' and 'F' refers to 'flight' version. 'HL' comes from 'horizontal landing' and '10' is for the tenth lifting body model to be investigated by Langley. The first flight of the M2-F2 -- which looked much like the M2-F1 -- occurred on July 12, 1966. Thompson was the pilot. By then, the same B-52 used to air launch the famed X-15 rocket research aircraft had been modified to also carry the lifting bodies into the air and Thompson was

  6. Differential Binding of Rimantadine Enantiomers to Influenza A M2 Proton Channel.

    PubMed

    Wright, Anna K; Batsomboon, Paratchata; Dai, Jian; Hung, Ivan; Zhou, Huan-Xiang; Dudley, Gregory B; Cross, Timothy A

    2016-02-10

    Rimantadine hydrochloride (α-methyl-1-adamantane-methalamine hydrochloride) is a chiral compound which exerts antiviral activity against the influenza A virus by inhibiting proton conductance of the M2 ion channel. In complex with M2, rimantadine has always been characterized as a racemic mixture. Here, we report the novel enantioselective synthesis of deuterium-labeled (R)- and (S)-rimantadine and the characterization of their protein-ligand interactions using solid-state NMR. Isotropic chemical shift changes strongly support differential binding of the enantiomers to the proton channel. Position restrained simulations satisfying distance restraints from (13)C-(2)H rotational-echo double-resonance NMR show marked differences in the hydrogen-bonding pattern of the two enantiomers at the binding site. Together these results suggest a complex set of interactions between (R)-rimantadine and the M2 proton channel, leading to a higher stability for this enantiomer of the drug in the channel pore. PMID:26804976

  7. ICAM-1 suppresses tumor metastasis by inhibiting macrophage M2 polarization through blockade of efferocytosis.

    PubMed

    Yang, M; Liu, J; Piao, C; Shao, J; Du, J

    2015-01-01

    Efficient clearance of apoptotic cells (efferocytosis) can profoundly influence tumor-specific immunity. Tumor-associated macrophages are M2-polarized macrophages that promote key processes in tumor progression. Efferocytosis stimulates M2 macrophage polarization and contributes to cancer metastasis, but the signaling mechanism underlying this process is unclear. Intercellular cell adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein member of the immunoglobulin superfamily, which has been implicated in mediating cell-cell interaction and outside-in cell signaling during the immune response. We report that ICAM-1 expression is inversely associated with macrophage infiltration and the metastasis index in human colon tumors by combining Oncomine database analysis and immunohistochemistry for ICAM-1. Using a colon cancer liver metastasis model in ICAM-1-deficient (ICAM-1(-/-)) mice and their wild-type littermates, we found that loss of ICAM-1 accelerated liver metastasis of colon carcinoma cells. Moreover, ICAM-1 deficiency increased M2 macrophage polarization during tumor progression. We further demonstrated that ICAM-1 deficiency in macrophages led to promotion of efferocytosis of apoptotic tumor cells through activation of the phosphatidylinositol 3 kinase/Akt signaling pathway. More importantly, coculture of ICAM-1(-/-) macrophages with apoptotic cancer cells resulted in an increase of M2-like macrophages, which was blocked by an efferocytosis inhibitor. Our findings demonstrate a novel role for ICAM-1 in suppressing M2 macrophage polarization via downregulation of efferocytosis in the tumor microenvironment, thereby inhibiting metastatic tumor progression. PMID:26068788

  8. Identification of benzopyrone as a common structural feature in compounds with anti-inflammatory activity in a zebrafish phenotypic screen

    PubMed Central

    Robertson, Anne L.; Ogryzko, Nikolay V.; Henry, Katherine M.; Loynes, Catherine A.; Foulkes, Matthew J.; Meloni, Marco M.; Wang, Xingang; Ford, Christopher; Jackson, Malcolm; Ingham, Philip W.; Wilson, Heather L.; Farrow, Stuart N.; Solari, Roberto; Flower, Roderick J.; Jones, Simon; Whyte, Moira K. B.

    2016-01-01

    ABSTRACT Neutrophils are essential for host defence and are recruited to sites of inflammation in response to tissue injury or infection. For inflammation to resolve, these cells must be cleared efficiently and in a controlled manner, either by apoptosis or reverse migration. If the inflammatory response is not well-regulated, persistent neutrophils can cause damage to host tissues and contribute to the pathogenesis of chronic inflammatory diseases, which respond poorly to current treatments. It is therefore important to develop drug discovery strategies that can identify new therapeutics specifically targeting neutrophils, either by promoting their clearance or by preventing their recruitment. Our recent in vivo chemical genetic screen for accelerators of inflammation resolution identified a subset of compounds sharing a common chemical signature, the bicyclic benzopyrone rings. Here, we further investigate the mechanisms of action of the most active of this chemical series, isopimpinellin, in our zebrafish model of neutrophilic inflammation. We found that this compound targets both the recruitment and resolution phases of the inflammatory response. Neutrophil migration towards a site of injury is reduced by isopimpinellin and this occurs as a result of PI3K inhibition. We also show that isopimpinellin induces neutrophil apoptosis to drive inflammation resolution in vivo using a new zebrafish reporter line detecting in vivo neutrophil caspase-3 activity and allowing quantification of flux through the apoptotic pathway in real time. Finally, our studies reveal that clinically available ‘cromones’ are structurally related to isopimpinellin and have previously undescribed pro-resolution activity in vivo. These findings could have implications for the therapeutic use of benzopyrones in inflammatory disease. PMID:27079522

  9. Identification of benzopyrone as a common structural feature in compounds with anti-inflammatory activity in a zebrafish phenotypic screen.

    PubMed

    Robertson, Anne L; Ogryzko, Nikolay V; Henry, Katherine M; Loynes, Catherine A; Foulkes, Matthew J; Meloni, Marco M; Wang, Xingang; Ford, Christopher; Jackson, Malcolm; Ingham, Philip W; Wilson, Heather L; Farrow, Stuart N; Solari, Roberto; Flower, Roderick J; Jones, Simon; Whyte, Moira K B; Renshaw, Stephen A

    2016-06-01

    Neutrophils are essential for host defence and are recruited to sites of inflammation in response to tissue injury or infection. For inflammation to resolve, these cells must be cleared efficiently and in a controlled manner, either by apoptosis or reverse migration. If the inflammatory response is not well-regulated, persistent neutrophils can cause damage to host tissues and contribute to the pathogenesis of chronic inflammatory diseases, which respond poorly to current treatments. It is therefore important to develop drug discovery strategies that can identify new therapeutics specifically targeting neutrophils, either by promoting their clearance or by preventing their recruitment. Our recent in vivo chemical genetic screen for accelerators of inflammation resolution identified a subset of compounds sharing a common chemical signature, the bicyclic benzopyrone rings. Here, we further investigate the mechanisms of action of the most active of this chemical series, isopimpinellin, in our zebrafish model of neutrophilic inflammation. We found that this compound targets both the recruitment and resolution phases of the inflammatory response. Neutrophil migration towards a site of injury is reduced by isopimpinellin and this occurs as a result of PI3K inhibition. We also show that isopimpinellin induces neutrophil apoptosis to drive inflammation resolution in vivo using a new zebrafish reporter line detecting in vivo neutrophil caspase-3 activity and allowing quantification of flux through the apoptotic pathway in real time. Finally, our studies reveal that clinically available 'cromones' are structurally related to isopimpinellin and have previously undescribed pro-resolution activity in vivo These findings could have implications for the therapeutic use of benzopyrones in inflammatory disease. PMID:27079522

  10. A comparative phenotypical analysis of rheumatoid nodules and rheumatoid synovium with special reference to adhesion molecules and activation markers

    PubMed Central

    Elewaut, D.; De Keyser, F.; De Wever, N.; Baeten, D.; Van Damme, N.; Verbruggen, G.; Cuvelier, C.; Veys, E.

    1998-01-01

    OBJECTIVES—(1)To analyse the in situ expression of adhesion molecules in rheumatoid nodules. (2) To compare the endothelial expression of adhesion molecules in synovial tissue and subcutaneous nodules obtained from the same patients. (3) To compare the expression of adhesion molecules and activation markers on T cell lines from nodules and synovium.
METHODS—(1) Immunohistochemical analysis by APAAP technique of E selectin, CD44, ICAM-1, PECAM-1, and VCAM-1 was performed on 10 rheumatoid nodules from seven patients with rheumatoid arthritis (RA); nodules and synovium were simultaneously analysed from three patients. (2) T cell lines were generated from RA nodules (n=7) and synovium (n=7) by interleukin 2 expansion, and subsequently characterised by flow cytometry for surface expression of αEβ7, α4β7, CD44, L selectin, LFA-1a, PECAM-1, and CD30.
RESULTS—(1) In rheumatoid nodules, the palisading layer strongly stains for ICAM-1 and PECAM-1, but less pronounced for CD44. VCAM-1 staining was usually negative. ICAM-1 is upregulated in the vessels surrounding the central zone of fibrinoid necrosis. The immunohistological picture in different nodules derived from the same patient was similar. (2) The endothelial expression of adhesion molecules is comparable in RA nodules and synovium on an individual level, except for E selectin, which is overexpressed in nodule endothelium. (3) T cell lines from nodules and synovium display similar adhesion molecule profiles. However, the expression of CD30, a T cell activation marker linked with Th2 subsets, is higher in nodules compared with synovium.
CONCLUSION—These data support a recirculation hypothesis of T cells between articular and extra-articular manifestations in RA, although the activation state of the T cells in each of these localisations may differ.

 Keywords: T cells; adhesion molecules; rheumatoid nodules; rheumatoid synovium PMID:9797554

  11. Tyrosine 129 of the Murine Gammaherpesvirus M2 Protein Is Critical for M2 Function In Vivo

    PubMed Central

    Rangaswamy, Udaya S.; O’Flaherty, Brigid M.; Speck, Samuel H.

    2014-01-01

    A common strategy shared by all known gammaherpesviruses is their ability to establish a latent infection in lymphocytes – predominantly in B cells. In immunocompromised patients, such as transplant recipients or AIDS patients, gammaherpesvirus infections can lead to the development of lymphoproliferative disease and lymphoid malignancies. The human gamma-herpesviruses, EBV and KSHV, encode proteins that are capable of modulating the host immune signaling machinery, thereby subverting host immune responses. Murine gamma-herpesvirus 68 (MHV68) infection of laboratory strains of mice has proven to be useful small-animal model that shares important pathogenic strategies with the human gamma-herpesviruses. The MHV68 M2 protein is known to manipulate B cell signaling and, dependent on route and dose of virus inoculation, plays a role in both the establishment of latency and virus reactivation. M2 contains two tyrosines that are targets for phosphorylation, and have been shown to interact with the B cell signaling machinery. Here we describe in vitro and in vivo studies of M2 mutants which reveals that while both tyrosines Y120 and Y129 are required for M2 induction of IL-10 expression from primary murine B cells in vitro, only Y129 is critical for reactivation from latency and plasma cell differentiation in vivo. PMID:25122496

  12. Autism phenotypes in ZnT3 null mice: Involvement of zinc dyshomeostasis, MMP-9 activation and BDNF upregulation

    PubMed Central

    Yoo, Min Heui; Kim, Tae-Youn; Yoon, Young Hee; Koh, Jae-Young

    2016-01-01

    To investigate the role of synaptic zinc in the ASD pathogenesis, we examined zinc transporter 3 (ZnT3) null mice. At 4–5 weeks of age, male but not female ZnT3 null mice exhibited autistic-like behaviors. Cortical volume and neurite density were significantly greater in male ZnT3 null mice than in WT mice. In male ZnT3 null mice, consistent with enhanced neurotrophic stimuli, the level of BDNF as well as activity of MMP-9 was increased. Consistent with known roles for MMPs in BDNF upregulation, 2.5-week treatment with minocycline, an MMP inhibitor, significantly attenuated BDNF levels as well as megalencephaly and autistic-like behaviors. Although the ZnT3 null state removed synaptic zinc, it rather increased free zinc in the cytosol of brain cells, which appeared to increase MMP-9 activity and BDNF levels. The present results suggest that zinc dyshomeostasis during the critical period of brain development may be a possible contributing mechanism for ASD. PMID:27352957

  13. Adiponectin Enhances Cold-Induced Browning of Subcutaneous Adipose Tissue via Promoting M2 Macrophage Proliferation.

    PubMed

    Hui, Xiaoyan; Gu, Ping; Zhang, Jialiang; Nie, Tao; Pan, Yong; Wu, Donghai; Feng, Tianshi; Zhong, Cheng; Wang, Yu; Lam, Karen S L; Xu, Aimin

    2015-08-01

    Adiponectin is an abundant adipokine with pleiotropic protective effects against a cluster of obesity-related cardiometabolic disorders. However, its role in adaptive thermogenesis has scarcely been explored. Here we showed that chronic cold exposure led to a markedly elevated production of adiponectin in adipocytes of subcutaneous white adipose tissue (scWAT), which in turn bound to M2 macrophages in the stromal vascular fraction. Chronic cold exposure-induced accumulation of M2 macrophages, activation of beige cells, and thermogenic program were markedly impaired in scWAT of adiponectin knockout (ADN KO) mice, whereas these impairments were reversed by replenishment with adiponectin. Mechanistically, adiponectin was recruited to the cell surface of M2 macrophages via its binding partner T-cadherin and promoted the cell proliferation by activation of Akt, consequently leading to beige cell activation. These findings uncover adiponectin as a key efferent signal for cold-induced adaptive thermogenesis by mediating the crosstalk between adipocytes and M2 macrophages in scWAT. PMID:26166748

  14. Loss of desmocollin-2 confers a tumorigenic phenotype to colonic epithelial cells through activation of Akt/β-catenin signaling

    PubMed Central

    Kolegraff, Keli; Nava, Porfirio; Helms, My N.; Parkos, Charles A.; Nusrat, Asma

    2011-01-01

    Desmocollin-2 (Dsc2) and desmoglein-2 (Dsg2) are transmembrane cell adhesion proteins of desmosomes. Reduced expression of Dsc2 has been reported in colorectal carcinomas, suggesting that Dsc2 may play a role in the development and/or progression of colorectal cancer. However, no studies have examined the mechanistic contribution of Dsc2 deficiency to tumorigenesis. Here we report that loss of Dsc2 promotes cell proliferation and enables tumor growth in vivo through the activation of Akt/β-catenin signaling. Inhibition of Akt prevented the increase in β-catenin–dependent transcription and proliferation following Dsc2 knockdown and attenuated the in vivo growth of Dsc2-deficient cells. Taken together, our results provide evidence that loss of Dsc2 contributes to the growth of colorectal cancer cells and highlight a novel mechanism by which the desmosomal cadherins regulate β-catenin signaling. PMID:21325624

  15. LRH-1 mediates anti-inflammatory and antifungal phenotype of IL-13-activated macrophages through the PPARγ ligand synthesis

    PubMed Central

    Lefèvre, Lise; Authier, Hélène; Stein, Sokrates; Majorel, Clarisse; Couderc, Bettina; Dardenne, Christophe; Eddine, Mohamad Ala; Meunier, Etienne; Bernad, José; Valentin, Alexis; Pipy, Bernard; Schoonjans, Kristina; Coste, Agnès

    2015-01-01

    Liver receptor homologue-1 (LRH-1) is a nuclear receptor involved in the repression of inflammatory processes in the hepatointestinal tract. Here we report that LRH-1 is expressed in macrophages and induced by the Th2 cytokine IL-13 via a mechanism involving STAT6. We show that loss-of-function of LRH-1 in macrophages impedes IL-13-induced macrophage polarization due to impaired generation of 15-HETE PPARγ ligands. The incapacity to generate 15-HETE metabolites is at least partially caused by the compromised regulation of CYP1A1 and CYP1B1. Mice with LRH-1-deficient macrophages are, furthermore, highly susceptible to gastrointestinal and systemic Candida albicans infection. Altogether, these results identify LRH-1 as a critical component of the anti-inflammatory and fungicidal response of alternatively activated macrophages that acts upstream from the IL-13-induced 15-HETE/PPARγ axis. PMID:25873311

  16. A polarizing question: do M1 and M2 microglia exist?

    PubMed

    Ransohoff, Richard M

    2016-07-26

    Microglial research has entered a fertile, dynamic phase characterized by novel technologies including two-photon imaging, whole-genome transcriptomic and epigenomic analysis with complementary bioinformatics, unbiased proteomics, cytometry by time of flight (CyTOF; Fluidigm) cytometry, and complex high-content experimental models including slice culture and zebrafish. Against this vivid background of newly emerging data, investigators will encounter in the microglial research literature a body of published work using the terminology of macrophage polarization, most commonly into the M1 and M2 phenotypes. It is the assertion of this opinion piece that microglial polarization has not been established by research findings. Rather, the adoption of this schema was undertaken in an attempt to simplify data interpretation at a time when the ontogeny and functional significance of microglia had not yet been characterized. Now, terminology suggesting established meaningful pathways of microglial polarization hinders rather than aids research progress and should be discarded. PMID:27459405

  17. Adipose tissue and vascular phenotypic modulation by voluntary physical activity and dietary restriction in obese insulin-resistant OLETF rats

    PubMed Central

    Crissey, Jacqueline M.; Jenkins, Nathan T.; Lansford, Kasey A.; Thorne, Pamela K.; Bayless, David S.; Vieira-Potter, Victoria J.; Rector, R. Scott; Thyfault, John P.; Laughlin, M. Harold

    2014-01-01

    Adipose tissue (AT)-derived cytokines are proposed to contribute to obesity-associated vascular insulin resistance. We tested the hypothesis that voluntary physical activity and diet restriction-induced maintenance of body weight would both result in decreased AT inflammation and concomitant improvements in insulin-stimulated vascular relaxation in the hyperphagic, obese Otsuka Long-Evans Tokushima fatty (OLETF) rat. Rats (aged 12 wk) were randomly assigned to sedentary (SED; n = 10), wheel running (WR; n = 10), or diet restriction (DR; n = 10; fed 70% of SED) for 8 wk. WR and DR rats exhibited markedly lower adiposity (7.1 ± 0.4 and 15.7 ± 1.1% body fat, respectively) relative to SED (27 ± 1.2% body fat), as well as improved blood lipid profiles and systemic markers of insulin resistance. Reduced adiposity in both WR and DR was associated with decreased AT mRNA expression of inflammatory genes (e.g., MCP-1, TNF-α, and IL-6) and markers of immune cell infiltration (e.g., CD8, CD11c, and F4/80). The extent of these effects were most pronounced in visceral AT compared with subcutaneous and periaortic AT. Markers of inflammation in brown AT were upregulated with WR but not DR. In periaortic AT, WR- and DR-induced reductions in expression and secretion of cytokines were accompanied with a more atheroprotective gene expression profile in the adjacent aortic wall. WR, but not DR, resulted in greater insulin-stimulated relaxation in the aorta; an effect that was, in part, mediated by a decrease in insulin-induced endothelin-1 activation in WR aorta. Collectively, we show in OLETF rats that lower adiposity leads to less AT and aortic inflammation, as well as an exercise-specific improvement in insulin-stimulated vasorelaxation. PMID:24523340

  18. Phenotypic and functional characteristics of active suppressor cells against IFN-gamma production in PHA-stimulated cord blood lymphocytes

    SciTech Connect

    Seki, H.; Taga, K.; Matsuda, A.; Uwadana, N.; Hasui, M.; Miyawaki, T.; Taniguchi, N.

    1986-11-15

    Cord blood mononuclear cells (MNC) were defective in their ability to produce interferon-gamma (IFN-gamma) on stimulation with phytohemagglutinin (PHA) or recombinant interleukin 2, whereas cord MNC could induce comparable amounts of IFN-gamma with adult controls on stimulation with a streptococcal preparation, OK-432. Moreover, irradiation of cord MNC with 1500 rad before PHA stimulation could restore the IFN-gamma production. Kinetic studies indicated that such augmentation of IFN-gamma production by irradiation was evident when cord MNC were irradiated before or by 12 hr of PHA-stimulated culture. But irradiation after 18 hr or more of PHA stimulation did not exert any significant augmentation on IFN-gamma production by cord MNC. It seemed most likely that the ability of IFN-gamma production is already mature at birth, but radiosensitive suppressor effectors on IFN-gamma production are activated within cord MNC at an early stage of PHA stimulation, resulting in poor IFN-gamma production by cord MNC. PHA-induced IFN-gamma production by OKT3+, OKT4+, and OKT8- cord cells were markedly enhanced by irradiation with 1,500 rad before the culture. Coculture experiments disclosed that cord OKT4+ cells, but not OKT4- cells, when prestimulated with PHA for 24 hr, exerted active suppression on PHA-induced IFN-gamma production by adult MNC in a dose-dependent manner. These results suggested that radiosensitive suppressor effectors on IFN-gamma production were induced within the OKT4+ T cell subset of cord MNC on PHA stimulation.

  19. Combinatorial phenotypic screen uncovers unrecognized family of extended thiourea inhibitors with copper-dependent anti-staphylococcal activity.

    PubMed

    Dalecki, Alex G; Malalasekera, Aruni P; Schaaf, Kaitlyn; Kutsch, Olaf; Bossmann, Stefan H; Wolschendorf, Frank

    2016-04-01

    The continuous rise of multi-drug resistant pathogenic bacteria has become a significant challenge for the health care system. In particular, novel drugs to treat infections of methicillin-resistant Staphylococcus aureus strains (MRSA) are needed, but traditional drug discovery campaigns have largely failed to deliver clinically suitable antibiotics. More than simply new drugs, new drug discovery approaches are needed to combat bacterial resistance. The recently described phenomenon of copper-dependent inhibitors has galvanized research exploring the use of metal-coordinating molecules to harness copper's natural antibacterial properties for therapeutic purposes. Here, we describe the results of the first concerted screening effort to identify copper-dependent inhibitors of Staphylococcus aureus. A standard library of 10 000 compounds was assayed for anti-staphylococcal activity, with hits defined as those compounds with a strict copper-dependent inhibitory activity. A total of 53 copper-dependent hit molecules were uncovered, similar to the copper independent hit rate of a traditionally executed campaign conducted in parallel on the same library. Most prominent was a hit family with an extended thiourea core structure, termed the NNSN motif. This motif resulted in copper-dependent and copper-specific S. aureus inhibition, while simultaneously being well tolerated by eukaryotic cells. Importantly, we could demonstrate that copper binding by the NNSN motif is highly unusual and likely responsible for the promising biological qualities of these compounds. A subsequent chemoinformatic meta-analysis of the ChEMBL chemical database confirmed the NNSNs as an unrecognized staphylococcal inhibitor, despite the family's presence in many chemical screening libraries. Thus, our copper-biased screen has proven able to discover inhibitors within previously screened libraries, offering a mechanism to reinvigorate exhausted molecular collections. PMID:26935206

  20. IUE observations of the 'Butterfly' Nebula M2-9

    NASA Technical Reports Server (NTRS)

    Feibelman, W. A.

    1984-01-01

    IUE observations of the peculiar 'Butterfy' nebula M2-9 indicate that it is not a normal planetary nebula. The ultraviolet spectrum is characterized by few emission lines and a weak continuum. Mg II 2800 A is the strongest emission line present and may be indicative of a binary nucleus. Lines of N v, Q I, N III, N IV, Si III, and C III are seen, but C IV and O III are conspicuous by their absence. T(e) = 10,250 + or - 400 K was determined for the core. Nitrogen in the core is found to be overabundant by about a factor of 5 over the solar value. M2-9 may be an object in the early stages of becoming a planetary nebula.

  1. Activation of p53 in Down Syndrome and in the Ts65Dn Mouse Brain is Associated with a Pro-Apoptotic Phenotype.

    PubMed

    Tramutola, Antonella; Pupo, Gilda; Di Domenico, Fabio; Barone, Eugenio; Arena, Andrea; Lanzillotta, Chiara; Broekaart, Diede; Blarzino, Carla; Head, Elizabeth; Butterfield, D Allan; Perluigi, Marzia

    2016-03-01

    Down syndrome (DS) is the most common genetic cause of intellectual disability, resulting from trisomy of chromosome 21. The main feature of DS neuropathology includes early onset of Alzheimer's disease (AD), with deposition of senile plaques and tangles. We hypothesized that apoptosis may be activated in the presence of AD neuropathology in DS, thus we measured proteins associated with upstream and downstream pathways of p53 in the frontal cortex from DS cases with and without AD pathology and from Ts65Dn mice, at different ages. We observed increased acetylation and phosphorylation of p53, coupled to reduced MDM2/p53 complex level and lower levels of SIRT1. Activation of p53 was associated with a number of targets (BAX, PARP1, caspase-3, p21, heat shock proteins, and PGC1α) that were modulated in both DS and DS/AD compared with age-matched controls. In particular, the most relevant changes (increased p-p53 and acetyl-p53 and reduced formation of MDM2/p53 complex) were found to be modified only in the presence of AD pathology in DS. In addition, a similar pattern of alterations in the p53 pathway was found in Ts65Dn mice. These results suggest that p53 may integrate different signals, which can result in a pro-apoptotic-phenotype contributing to AD neuropathology in people with DS. PMID:26967221

  2. ACTIVATION OF p53 IN DOWN SYNDROME AND IN THE Ts65Dn MOUSE BRAIN IS ASSOCIATED WITH A PRO-APOPTOTIC PHENOTYPE

    PubMed Central

    Di Domenico, Fabio; Barone, Eugenio; Arena, Andrea; Lanzillotta, Chiara; Brokeaart, Diede; Blarzino, Carla; Head, Elizabeth; Butterfield, D Allan; Perluigi, Marzia

    2016-01-01

    Down Syndrome (DS) is the most common genetic cause of intellectual disability resulting from trisomy of chromosome 21. The main feature of DS neuropathology includes early onset of Alzheimer's disease, with deposition of senile plaques and tangles. We hypothesized that apoptosis may be activated in the presence of AD neuropathology in DS, thus we measured proteins associated with upstream and downstream pathways of p53 in the frontal cortex from DS cases with and without AD pathology and from Ts65Dn mice, at different ages. We observed increased acetylation and phosphorylation of p53, coupled to reduced MDM2-mediated ubiquitination and lower levels of SIRT1. Activation of p53 was associated with a number of down-stream targets (bax, PARP1, caspase-3, heat shock proteins and PGC1α) that were modulated in both DS and DS/AD compared with age-matched controls. In particular, the most relevant changes (increased p-p53, acetyl-p53 and reduced formation of MDM2/p53 complex) were found to be modified only in the presence of AD pathology in DS. In addition, a similar pattern of alterations in the p53 pathway were found in Ts65Dn mice. These results suggest that p53 may integrate different signals, which can result in a pro-apoptotic-phenotype contributing to AD neuropathology in people with DS. PMID:26967221

  3. Activating transcription factor 4 mediates a multidrug resistance phenotype of esophageal squamous cell carcinoma cells through transactivation of STAT3 expression.

    PubMed

    Zhu, Hongwu; Chen, Xiong; Chen, Bin; Chen, Bei; Fan, Jianyong; Song, Weibing; Xie, Ziying; Jiang, Dan; Li, Qiuqiong; Zhou, Meihua; Sun, Dayong; Zhao, Yagang

    2014-11-01

    Multidrug resistance (MDR) is a major challenge to the clinical treatment of esophageal cancer. The stress response gene activating transcription factor 4 (ATF4) is involved in homeostasis and cellular protection. However, relatively little is known about the expression and function of ATF4 in esophageal squamous cell carcinoma (ESCC) MDR. In this study, we investigate the potential role and mechanisms of ATF4 in ESCC MDR. We demonstrated that overexpression of ATF4 promotes the MDR phenotype in ESCC cells, while depletion of ATF4 in the MDR ESCC cell line induces drug re-sensitization. We also demonstrated that ATF4 transactivates STAT3 expression by directly binding to the signal transducers and activators of transcription 3 (STAT3) promoter, resulting in MDR in ESCC cells. Significantly, inhibition of STAT3 by small interfering RNA (siRNA) or a selective inhibitor (JSI-124) reintroduces therapeutic sensitivity. In addition, increased Bcl-2, survivin, and MRP1 expression levels were observed in ATF4-overexpressing cells. In conclusion, ATF4 may promote MDR in ESCC cells through the up-regulation of STAT3 expression, and thus is an attractive therapeutic target to combat therapeutic resistance in ESCC. PMID:25130172

  4. M2 world ocean tide from tide gauge measurements

    SciTech Connect

    Francis, O.; Mazzega, P. )

    1991-06-01

    An empirical model of the M2 oceanic tide has been computed form the harmonic constants of a subset of deep sea and coastal tide gauge measurements. The optimal interpolation of these data based on inverse theory' uses a priori covariance functions deduced from a global hydrodynamical model. The inverse solution, produced with its associated error maps and samples of error spectra, is surprisingly good when compared to in situ data and to a hydrodynamical model.

  5. M2 World Ocean tide from tide gauge measurements

    NASA Astrophysics Data System (ADS)

    Francis, O.; Mazzega, P.

    An empirical model of the M2 oceanic tide has been computed from the harmonic constants of a subset of deep sea and coastal tide gauge measurements. The optimal interpolation of these data based on “inverse theory” uses a priori covariance functions deduced from a global hydrodynamical model. The inverse solution, produced with its associated error maps and samples of error spectra, is surprisingly good when compared to in situ data and to a hydrodynamical model.

  6. 26 CFR 1.401(m)-2 - ACP test.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... satisfy the ACP test, but not in excess of 100%. In this case, an increase in the match rate from 50% to... under the ACP test only to the extent they do not exceed 25.00% of compensation. In this case, all of... determined under § 1.401(m)-2(b)(2)(iv) (as it appeared in the April 1, 2007, edition of 26 CFR part 1)....

  7. Biochemical characterization and functional analysis of the POU transcription factor POU-M2 of Bombyx mori.

    PubMed

    Liu, Lina; Li, Yu; Wang, Yejing; Zhao, Peng; Wei, Shuguang; Li, Zhenzhen; Chang, Huaipu; He, Huawei

    2016-05-01

    POU-M2 is a homeodomain transcription factor which plays important roles in the development and silk synthesis of Bombyx mori. In this study, we expressed, purified and characterized POU-M2 and studied its transcription regulation on fibroin heavy chain gene of Bombyx mori. Gel filtration showed POU-M2 existed as a dimer in solution. Far-UV circular dichroism spectra indicated POU-M2 had a well-defined α-helix structure and the α-helix content was about 26.4%. The thermal unfolding transition of POU-M2 was a cooperative process. Tm, ΔH and ΔS were 45.15±0.2°C, 138.4±0.5KJ/mol and 0.4349±0.04KJ/(mol·K), respectively. Western blotting analysis indicated the expression level of POU-M2 increased slightly from day 3 to day 7 of the fifth instar larvae in the posterior silk gland. POU-M2 was positioned in the nucleus of cells. The luciferase reporter assay demonstrated POU-M2 could stimulate the promoter activity of fibroin heavy chain gene, and the activation effect was dependent on the amount of POU-M2. Our study suggested POU-M2 may be involved in the transcriptional regulation of fibroin heavy chain gene. These findings expand toward a better understanding of the structure of POU-M2 and its function in silk synthesis of Bombyx mori. PMID:26854886

  8. Characterization of the N-glycans of recombinant bee venom hyaluronidase (Api m 2) expressed in insect cells.

    PubMed

    Soldatova, Lyudmila N; Tsai, Chaoming; Dobrovolskaia, Ekaterina; Marković-Housley, Zora; Slater, Jay E

    2007-01-01

    Honeybee venom hyaluronidase (Api m 2) is a major glycoprotein allergen. Previous studies have indicated that recombinant Api m 2 expressed in insect cells has enzyme activity and IgE binding comparable with that of native Api m 2. In contrast, Api m 2 expressed in Escherichia coli does not. In this study, we characterized the carbohydrate side chains of Api m 2 expressed in insect cells, and compared our data with the established carbohydrate structure of native Api m 2. We assessed both the monosaccharide and the oligosaccharide content of recombinant Api m 2 using fluorophore-assisted carbohydrate electrophoresis and HPLC. To identify the amino acid residues at which glycosylation occurs, we digested recombinant Api m 2 with endoproteinase Glu-C and identified the fragments that contained carbohydrate by specific staining. Recombinant Api m 2 expressed in insect cells contains N-acetylglucosamine, mannose, and fucose, as well as trace amounts of glucose and galactose, and the oligosaccharide analysis is consistent with heterogeneous oligosaccharide chains consisting of two to seven monosaccharides. No sialic acid or N-acetylgalactosamine were detected. These results are similar to published data for native Api m 2, although some monosaccharide components appear to be absent in the recombinant protein. Analysis of proteolytic digests indicates that of the four candidate N-glycosylation sites, carbohydrate chains are attached at asparagines 115 and 263. Recombinant Api m 2 expressed in insect cells has enzymic activity and IgE binding comparable with the native protein, and its carbohydrate composition is very similar. PMID:17479607

  9. Photoinducing the hidden M2 phase in VO2

    NASA Astrophysics Data System (ADS)

    Walko, D. A.; Smith, R. K.; Wen, Haidan; Dichiara, A. D.; Jeong, Jaewoo; Samant, Mahensh G.; Parkin, Stuart S. P.

    We used time-resolved x-ray diffraction to study photoinduced structural phase transitions in a 170-nm-thick VO2 film grown on sapphire (1,0,-1,0). Heating the unstrained film from room temperature induces the well-known phase transition from the monoclinic (M1) phase directly to the high-temperature tetragonal rutile (R) phase. In contrast, upon ultrafast optical excitation, the phase transition depends strongly on the laser intensity. At low fluences, the film is partially transformed into the monoclinic M2 phase, a phase which generally is observed only in doped or strained materials. Above a threshold at higher fluences, a small portion of the film is transformed into the M2 phase, decaying on a time scale of a few nanoseconds, while the majority of the film is transformed into the R phase which can persist for tens of nanoseconds. We further discuss the effects of laser wavelength on the efficiency of producing the M2 phase. Work at the Advanced Photon Source supported by DOE Contract No. DE-AC02-06CH11357.

  10. State-of-the-art Model M-2 Maintenance System

    SciTech Connect

    Herndon, J.N.; Martin, H.L.; Satterlee, P.E. Jr.; Jelatis, D.G.; Jennrich, C.E.

    1984-04-01

    The Model M-2 Maintenance System is part of an ongoing program within the Consolidated Fuel Reprocessing Program (CFRP) at Oak Ridge National Laboratory (ORNL) to improve remote manipulation technology for future nuclear fuel reprocessing and other remote applications. Techniques, equipment, and guidelines which can improve the efficiency of remote maintenance are being developed. The Model M-2 Maintenance System, installed in the Integrated Equipment Test (IET) Facility at ORNL, provides a complete, integrated remote maintenance system for the demonstration and development of remote maintenance techniques. The system comprises a pair of force-reflecting servomanipulator arms, television viewing, lighting, and auxiliary lifting capabilities, thereby allowing manlike maintenance operations to be executed remotely within the remote cell mockup area in the IET. The Model M-2 Maintenance System incorporates an upgraded version of the proven Central Research Laboratories' Model M servomanipulator. Included are state-of-the-art brushless dc servomotors for improved performance, remotely removable wrist assemblies, geared azimuth drive, and a distributed microprocessor-based digital control system. 5 references, 8 figures.

  11. Urine bactericidal activity against Escherichia coli isolates exhibiting different resistance phenotypes/genotypes in an in vitro pharmacodynamic model simulating urine concentrations obtained after oral administration of a 400-milligram single dose of cefditoren-pivoxil.

    PubMed

    Sevillano, David; Aguilar, Lorenzo; Alou, Luis; Giménez, María-José; Torrico, Martha; González, Natalia; Cafini, Fabio; Relaño, María-Teresa; Coronel, Pilar; Prieto, José

    2008-03-01

    Activity of simulated cefditoren urinary concentrations was determined against seven Escherichia coli isolates. Bactericidal activity was obtained from 4 to 24 h against TEM-1 (penicillinase production/hyperproduction), TEM-34 (IRT-6), and TEM-116 (extended-spectrum beta-lactamase [ESBL]) and from 6 to 8 h against SHV/TEM-116 (ESBL) but never against SHV/TEM-1 (ESBL). Extension of bactericidal activity depended on the resistance genotype/phenotype tested. PMID:18160517

  12. Urine Bactericidal Activity against Escherichia coli Isolates Exhibiting Different Resistance Phenotypes/Genotypes in an In Vitro Pharmacodynamic Model Simulating Urine Concentrations Obtained after Oral Administration of a 400-Milligram Single Dose of Cefditoren-Pivoxil▿

    PubMed Central

    Sevillano, David; Aguilar, Lorenzo; Alou, Luis; Giménez, María-José; Torrico, Martha; González, Natalia; Cafini, Fabio; Relaño, María-Teresa; Coronel, Pilar; Prieto, José

    2008-01-01

    Activity of simulated cefditoren urinary concentrations was determined against seven Escherichia coli isolates. Bactericidal activity was obtained from 4 to 24 h against TEM-1 (penicillinase production/hyperproduction), TEM-34 (IRT-6), and TEM-116 (extended-spectrum beta-lactamase [ESBL]) and from 6 to 8 h against SHV/TEM-116 (ESBL) but never against SHV/TEM-1 (ESBL). Extension of bactericidal activity depended on the resistance genotype/phenotype tested. PMID:18160517

  13. Primary oocyte transcriptional activation of aqp1ab by the nuclear progestin receptor determines the pelagic egg phenotype of marine teleosts.

    PubMed

    Zapater, Cinta; Chauvigné, François; Tingaud-Sequeira, Angèle; Finn, Roderick Nigel; Cerdà, Joan

    2013-05-15

    In marine teleosts, the aqp1ab water channel plays a vital role in the development of the pelagic egg phenotype. However, the developmental control of aqp1ab activation during oogenesis remains to be established. Here, we report the isolation of the 5'-flanking region of the teleost gilthead seabream aqp1ab gene, in which we identify conserved cis-regulatory elements for the binding of the nuclear progestin receptor (Pgr) and members of the Sox family of transcription factors. Subcellular localization studies indicated that the Pgr, as well as sox3 and -8b transcripts, are co-expressed in seabream oogonia, whereas in meiosis-arrested primary growth (pre-vitellogenic) oocytes, when aqp1ab mRNA and protein are first synthesized, the Pgr appears to be completely translocated from the ooplasm into the nucleus. By contrast, sox9b is highly expressed in more advanced oocytes, coinciding with a strong depletion of aqp1ab transcripts in the oocyte. Functional characterization of wild-type and mutated aqp1ab promoter constructs, using mammalian cells and Xenopus laevis oocytes, demonstrated that aqp1ab transcription is initiated by the Pgr, which is activated by the progestin 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P), the natural ligand of the seabream Pgr. In vitro incubation of seabream primary ovarian explants with the follicle-stimulating hormone or 17,20β-P confirmed that progestin-activated Pgr enhanced Aqp1ab synthesis via the aqp1ab promoter. However, transactivation assays in heterologous systems showed that Sox transcription factors can potentially modulate this mechanism. These data uncover the existence of an endocrine pathway involved in the early activation of a water channel necessary for egg formation in marine teleosts. PMID:23499660

  14. Disabled homolog 2 controls macrophage phenotypic polarization and adipose tissue inflammation

    PubMed Central

    Adamson, Samantha E.; Moravec, Radim; Senthivinayagam, Subramanian; Montgomery, Garren; Chen, Wenshu; Han, Jenny; Sharma, Poonam R.; Mullins, Garrett R.; Gorski, Stacey A.; Cooper, Jonathan A.; Kadl, Alexandra; Enfield, Kyle; Braciale, Thomas J.; Harris, Thurl E.

    2016-01-01

    Acute and chronic tissue injury results in the generation of a myriad of environmental cues that macrophages respond to by changing their phenotype and function. This phenotypic regulation is critical for controlling tissue inflammation and resolution. Here, we have identified the adaptor protein disabled homolog 2 (DAB2) as a regulator of phenotypic switching in macrophages. Dab2 expression was upregulated in M2 macrophages and suppressed in M1 macrophages isolated from both mice and humans, and genetic deletion of Dab2 predisposed macrophages to adopt a proinflammatory M1 phenotype. In mice with myeloid cell–specific deletion of Dab2 (Dab2fl/fl Lysm-Cre), treatment with sublethal doses of LPS resulted in increased proinflammatory gene expression and macrophage activation. Moreover, chronic high-fat feeding exacerbated adipose tissue inflammation, M1 polarization of adipose tissue macrophages, and the development of insulin resistance in DAB2-deficient animals compared with controls. Mutational analyses revealed that DAB2 interacts with TNF receptor–associated factor 6 (TRAF6) and attenuates IκB kinase β–dependent (IKKβ-dependent) phosphorylation of Ser536 in the transactivation domain of NF-κB p65. Together, these findings reveal that DAB2 is critical for controlling inflammatory signaling during phenotypic polarization of macrophages and suggest that manipulation of DAB2 expression and function may hold therapeutic potential for the treatment of acute and chronic inflammatory disorders. PMID:26927671

  15. Disabled homolog 2 controls macrophage phenotypic polarization and adipose tissue inflammation.

    PubMed

    Adamson, Samantha E; Griffiths, Rachael; Moravec, Radim; Senthivinayagam, Subramanian; Montgomery, Garren; Chen, Wenshu; Han, Jenny; Sharma, Poonam R; Mullins, Garrett R; Gorski, Stacey A; Cooper, Jonathan A; Kadl, Alexandra; Enfield, Kyle; Braciale, Thomas J; Harris, Thurl E; Leitinger, Norbert

    2016-04-01

    Acute and chronic tissue injury results in the generation of a myriad of environmental cues that macrophages respond to by changing their phenotype and function. This phenotypic regulation is critical for controlling tissue inflammation and resolution. Here, we have identified the adaptor protein disabled homolog 2 (DAB2) as a regulator of phenotypic switching in macrophages. Dab2 expression was upregulated in M2 macrophages and suppressed in M1 macrophages isolated from both mice and humans, and genetic deletion of Dab2 predisposed macrophages to adopt a proinflammatory M1 phenotype. In mice with myeloid cell-specific deletion of Dab2 (Dab2fl/fl Lysm-Cre), treatment with sublethal doses of LPS resulted in increased proinflammatory gene expression and macrophage activation. Moreover, chronic high-fat feeding exacerbated adipose tissue inflammation, M1 polarization of adipose tissue macrophages, and the development of insulin resistance in DAB2-deficient animals compared with controls. Mutational analyses revealed that DAB2 interacts with TNF receptor-associated factor 6 (TRAF6) and attenuates IκB kinase β-dependent (IKKβ-dependent) phosphorylation of Ser536 in the transactivation domain of NF-κB p65. Together, these findings reveal that DAB2 is critical for controlling inflammatory signaling during phenotypic polarization of macrophages and suggest that manipulation of DAB2 expression and function may hold therapeutic potential for the treatment of acute and chronic inflammatory disorders. PMID:26927671

  16. Phenotyping of leukocytes and granulocyte and monocyte phagocytic activity in the peripheral blood and uterus of cows with endometritis.

    PubMed

    Brodzki, P; Kostro, K; Brodzki, A; Lisiecka, U; Kurek, L; Marczuk, J

    2014-08-01

    This study was a comparative evaluation of selected immunological parameters in peripheral blood and uterine wash samples from cows with a normal postpartum period compared with cows with endometritis. We aimed to determine the usefulness of these parameters in monitoring the puerperium. In total, 40 cows were included in the study: 20 had endometritis (experimental group), and 20 did not have uterine inflammation (control group). Animals were chosen on the basis of cytological and bacteriological test results. The tests were conducted 5, 22, and 40 days postpartum. In both groups, flow cytometric analysis of the surface molecules CD4, CD8, CD21, CD25, and CD14 in the peripheral blood and uterine washings was performed. Granulocyte and monocyte phagocytic activity was determined using a commercial Phagotest kit that was adapted for flow cytometry. The percentage of phagocytic granulocytes and monocytes in both the peripheral blood and the uterine washings was significantly lower for cows in the experimental group compared with the control group (P < 0.01). A significant decrease (P < 0.01) in the percentage of CD4+, CD25+, CD14+, and CD4 + CD25(high) leukocyte subpopulations was also observed in the peripheral blood of cows with endometritis. A significant decrease (P < 0.01) in CD21+ lymphocytes and an increase in CD8+ lymphocytes was detected in uterine washings. The results of this work indicate that cell immunity dysfunction may be the main factor causing advanced inflammation of the uterus in endometritis. Knowledge of the immunological mechanisms observed in cows with endometritis might aid in choosing the correct immunomodulating agent-based adjuvant therapy. PMID:24857644

  17. Variants of the Ankyrin Repeat Domain 6 Gene (ANKRD6) and Muscle and Physical Activity Phenotypes Among European-Derived American Adults

    PubMed Central

    Van Deveire, Katherine N.; Scranton, Sarah K.; Kostek, Mathew A.; Angelopoulos, Theodore J.; Clarkson, Priscilla M.; Gordon, Paul M.; Moyna, Niall M.; Visich, Paul S.; Zoeller, Robert F.; Thompson, Paul D.; Devaney, Joseph M.; Gordish-Dressman, Heather; Hoffman, Eric P.; Maresh, Carl M.; Pescatello, Linda S.

    2014-01-01

    Ankyrin repeat domain 6 (ANKRD6) is a ubiquitous protein that associates with early development in mammals and is highly expressed in the brain, spinal cord, and heart of humans. We examined the role of 8 ANKRD6 single-nucleotide polymorphisms (SNPs) on muscle performance and habitual physical activity (PA). Single-nucleotide polymorphisms were 545 T>A (rs9362667), 485 M>L (rs61736690), 233 T>M (rs2273238), 128 I>L (rs3748085), 631 P>L (rs61739327), 122 Q>E (rs16881983), 197805 G>A (rs9344950), and 710 L>X (NOVEL). This study consisted of 922 healthy, untrained, European-derived American men (n = 376, 23.6 ± 0.3 years, 25.0 ± 0.2 kg·m−2) and women (n = 546, 23.2 ± 0.2 years, 24.0 ± 0.2 kg·m−2). Muscle strength (maximum voluntary contraction [MVC] and 1 repetition maximum [1RM]) and size (cross-sectional area [CSA]) were assessed before and after 12 weeks of unilateral resistance training (RT). A subsample (n = 536, 23.4 ± 0.2 years, 24.6 ± 0.2 kg·m−2) completed the Paffenbarger Physical Activity Questionnaire. Associations among ANKRD6 genotypes and muscle phenotypes were tested with repeated measure analysis of covariance (ANCOVA) and PA phenotypes with multivariate ANCOVA, with age and body mass index as covariates. ANKRD6 122 Q>E was associated with increased baseline biceps CSA. ANKRD6 545 A>T and ANKRD6 710 L>X were associated with increased 1RM and MVC in response to RT, respectively. ANKRD6 631 P>L was associated with increased biceps CSA response to RT and time spent in moderate-intensity PA among the total sample and women. ANKRD6 genetic variants were associated with the muscle size and strength response to RT and habitual PA levels. Further research is needed to validate our results and explore mechanisms for the associations we observed. PMID:22580979

  18. M2-F1 under tow across lakebed by car

    NASA Technical Reports Server (NTRS)

    1963-01-01

    This 20-second clip shows the M2-F1 being towed by the Pontiac across Rogers Dry Lakebed. The M2-F1 lifting body, dubbed the 'flying bathtub' by the media, was the precursor of a remarkable series of wingless flying vehicles that contributed data used in the Space Shuttles, the X-33 Advanced Technology Demonstrator for the next century's Reusable Launch Vehicle, and the X-38 Technology Demonstrator for crew return from the International Space Station. Based on the ideas and basic design of Alfred J. Eggers and others at the Ames Aeronautical Laboratory (now the Ames Research Center), Mountain View, California, in the mid-1950's, the M2-F1 was built in 1962-63 over a four-month period for a cost of only about $30,000, plus an additional $8,000-$10,000 for an ejection seat. Engineers and technicians at the NASA Flight Research Center (now NASA Dryden) kept costs low by designing and fabricating it partly in-house, with the plywood shell constructed by a local sailplane builder. Someone at the time estimated that it would have cost a major aircraft company $150,000 to build the same vehicle. Unlike the later lifting bodies, the M2-F1 was unpowered and was initially towed by a souped-up Pontiac convertible until it was airborne. Later a C-47 took over the towing duties. Flown by such famous research pilots as Milt Thompson, Bruce Peterson, Chuck Yeager, and Bill Dana, the lightweight flying bathtub demonstrated that a wingless vehicle shaped for reentry into the Earth's atmosphere from space could be flown and landed safely. Flown from 1963 to 1966, the lightweight M2-F1 paved the way for the heavyweight M2-F2, M2`F3, HL-10, X-24A, and X-24B lifting bodies that flew under rocket power after launch from a B-52 mothership. The heavyweights flew from 1966 to 1975, demonstrating the viability and versatility of the wingless configuration and the ability of a vehicle with low lift-over-drag characteristics to fly to high altitudes and then to land precisely with their

  19. Retinal Targets ALDH Positive Cancer Stem Cell and Alters the Phenotype of Highly Metastatic Osteosarcoma Cells

    PubMed Central

    Mu, Xiaodong; Patel, Stuti; Mektepbayeva, Damel; Mahjoub, Adel; Huard, Johnny; Weiss, Kurt

    2015-01-01

    Aldehyde dehydrogenase (ALDH) is a cancer stem cell marker. Retinoic acid has antitumor properties, including the induction of apoptosis and inhibition of proliferation. Retinal, the precursor of retinoic acid, can be oxidized to retinoic acid by dehydrogenases, including ALDH. We hypothesized that retinal could potentially be transformed to retinoic acid with higher efficiency by cancer stem cells, due to the higher ALDH activity. We previously observed that ALDH activity is greater in highly metastatic K7M2 osteosarcoma (OS) cells than in nonmetastatic K12 OS cells. We also demonstrated that ALDH activity correlates with clinical metastases in bone sarcoma patients, suggesting that ALDH may be a therapeutic target specific to cells with high metastatic potential. Our current results demonstrated that retinal preferentially affected the phenotypes of ALDH-high K7M2 cells in contrast to ALDH-low K12 cells, which could be mediated by the more efficient transformation of retinal to retinoic acid by ALDH in K7M2 cells. Retinal treatment of highly metastatic K7M2 cells decreased their proliferation, invasion capacity, and resistance to oxidative stress. Retinal altered the expression of metastasis-related genes. These observations indicate that retinal may be used to specifically target metastatic cancer stem cells in OS. PMID:26819566

  20. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

    SciTech Connect

    Haga, Kazuko; Kruse, Andrew C.; Asada, Hidetsugu; Yurugi-Kobayashi, Takami; Shiroishi, Mitsunori; Zhang, Cheng; Weis, William I.; Okada, Tetsuji; Kobilka, Brian K.; Haga, Tatsuya; Kobayashi, Takuya

    2012-03-15

    The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

  1. Chop Deficiency Protects Mice Against Bleomycin-induced Pulmonary Fibrosis by Attenuating M2 Macrophage Production.

    PubMed

    Yao, Yingying; Wang, Yi; Zhang, Zhijun; He, Long; Zhu, Jianghui; Zhang, Meng; He, Xiaoyu; Cheng, Zhenshun; Ao, Qilin; Cao, Yong; Yang, Ping; Su, Yunchao; Zhao, Jianping; Zhang, Shu; Yu, Qilin; Ning, Qin; Xiang, Xudong; Xiong, Weining; Wang, Cong-Yi; Xu, Yongjian

    2016-05-01

    C/EBP homologous protein (Chop) has been shown to have altered expression in patients with idiopathic pulmonary fibrosis (IPF), but its exact role in IPF pathoaetiology has not been fully addressed. Studies conducted in patients with IPF and Chop(-/-) mice have dissected the role of Chop and endoplasmic reticulum (ER) stress in pulmonary fibrosis pathogenesis. The effect of Chop deficiency on macrophage polarization and related signalling pathways were investigated to identify the underlying mechanisms. Patients with IPF and mice with bleomycin (BLM)-induced pulmonary fibrosis were affected by the altered Chop expression and ER stress. In particular, Chop deficiency protected mice against BLM-induced lung injury and fibrosis. Loss of Chop significantly attenuated transforming growth factor β (TGF-β) production and reduced M2 macrophage infiltration in the lung following BLM induction. Mechanistic studies showed that Chop deficiency repressed the M2 program in macrophages, which then attenuated TGF-β secretion. Specifically, loss of Chop promoted the expression of suppressors of cytokine signaling 1 and suppressors of cytokine signaling 3, and through which Chop deficiency repressed signal transducer and activator of transcription 6/peroxisome proliferator-activated receptor gamma signaling, the essential pathway for the M2 program in macrophages. Together, our data support the idea that Chop and ER stress are implicated in IPF pathoaetiology, involving at least the induction and differentiation of M2 macrophages. PMID:26883801

  2. Molecular Mechanisms That Influence the Macrophage M1–M2 Polarization Balance

    PubMed Central

    Wang, Nan; Liang, Hongwei; Zen, Ke

    2014-01-01

    As an essential component of innate immunity, macrophages have multiple functions in both inhibiting or promoting cell proliferation and tissue repair. Diversity and plasticity are hallmarks of macrophages. Classical M1 and alternative M2 activation of macrophages, mirroring the Th1–Th2 polarization of T cells, represent two extremes of a dynamic changing state of macrophage activation. M1-type macrophages release cytokines that inhibit the proliferation of surrounding cells and damage contiguous tissue, and M2-type macrophages release cytokines that promote the proliferation of contiguous cells and tissue repair. M1–M2 polarization of macrophage is a tightly controlled process entailing a set of signaling pathways, transcriptional and posttranscriptional regulatory networks. An imbalance of macrophage M1–M2 polarization is often associated with various diseases or inflammatory conditions. Therefore, identification of the molecules associated with the dynamic changes of macrophage polarization and understanding their interactions is crucial for elucidating the molecular basis of disease progression and designing novel macrophage-mediated therapeutic strategies. PMID:25506346

  3. Peptide-MHC-I from Endogenous Antigen Outnumber Those from Exogenous Antigen, Irrespective of APC Phenotype or Activation

    PubMed Central

    Sei, Janet J.; Haskett, Scott; Kaminsky, Lauren W.; Lin, Eugene; Truckenmiller, Mary E.; Bellone, Clifford J.; Buller, R. Mark; Norbury, Christopher C.

    2015-01-01

    Naïve anti-viral CD8+ T cells (TCD8+) are activated by the presence of peptide-MHC Class I complexes (pMHC-I) on the surface of professional antigen presenting cells (pAPC). Increasing the number of pMHC-I in vivo can increase the number of responding TCD8+. Antigen can be presented directly or indirectly (cross presentation) from virus-infected and uninfected cells, respectively. Here we determined the relative importance of these two antigen presenting pathways in mousepox, a natural disease of the mouse caused by the poxvirus, ectromelia (ECTV). We demonstrated that ECTV infected several pAPC types (macrophages, B cells, and dendritic cells (DC), including DC subsets), which directly presented pMHC-I to naïve TCD8+ with similar efficiencies in vitro. We also provided evidence that these same cell-types presented antigen in vivo, as they form contacts with antigen-specific TCD8+. Importantly, the number of pMHC-I on infected pAPC (direct presentation) vastly outnumbered those on uninfected cells (cross presentation), where presentation only occurred in a specialized subset of DC. In addition, prior maturation of DC failed to enhance antigen presentation, but markedly inhibited ECTV infection of DC. These results suggest that direct antigen presentation is the dominant pathway in mice during mousepox. In a broader context, these findings indicate that if a virus infects a pAPC then the presentation by that cell is likely to dominate over cross presentation as the most effective mode of generating large quantities of pMHC-I is on the surface of pAPC that endogenously express antigens. Recent trends in vaccine design have focused upon the introduction of exogenous antigens into the MHC Class I processing pathway (cross presentation) in specific pAPC populations. However, use of a pantropic viral vector that targets pAPC to express antigen endogenously likely represents a more effective vaccine strategy than the targeting of exogenous antigen to a limiting p

  4. Peptide-MHC-I from Endogenous Antigen Outnumber Those from Exogenous Antigen, Irrespective of APC Phenotype or Activation.

    PubMed

    Sei, Janet J; Haskett, Scott; Kaminsky, Lauren W; Lin, Eugene; Truckenmiller, Mary E; Bellone, Clifford J; Buller, R Mark; Norbury, Christopher C

    2015-06-01

    Naïve anti-viral CD8+ T cells (TCD8+) are activated by the presence of peptide-MHC Class I complexes (pMHC-I) on the surface of professional antigen presenting cells (pAPC). Increasing the number of pMHC-I in vivo can increase the number of responding TCD8+. Antigen can be presented directly or indirectly (cross presentation) from virus-infected and uninfected cells, respectively. Here we determined the relative importance of these two antigen presenting pathways in mousepox, a natural disease of the mouse caused by the poxvirus, ectromelia (ECTV). We demonstrated that ECTV infected several pAPC types (macrophages, B cells, and dendritic cells (DC), including DC subsets), which directly presented pMHC-I to naïve TCD8+ with similar efficiencies in vitro. We also provided evidence that these same cell-types presented antigen in vivo, as they form contacts with antigen-specific TCD8+. Importantly, the number of pMHC-I on infected pAPC (direct presentation) vastly outnumbered those on uninfected cells (cross presentation), where presentation only occurred in a specialized subset of DC. In addition, prior maturation of DC failed to enhance antigen presentation, but markedly inhibited ECTV infection of DC. These results suggest that direct antigen presentation is the dominant pathway in mice during mousepox. In a broader context, these findings indicate that if a virus infects a pAPC then the presentation by that cell is likely to dominate over cross presentation as the most effective mode of generating large quantities of pMHC-I is on the surface of pAPC that endogenously express antigens. Recent trends in vaccine design have focused upon the introduction of exogenous antigens into the MHC Class I processing pathway (cross presentation) in specific pAPC populations. However, use of a pantropic viral vector that targets pAPC to express antigen endogenously likely represents a more effective vaccine strategy than the targeting of exogenous antigen to a limiting p

  5. Regulation of Notch 1 signaling in THP-1 cells enhances M2 macrophage differentiation.

    PubMed

    Singla, Reetu D; Wang, Jing; Singla, Dinender K

    2014-12-01

    Macrophage polarization is emerging as an important area of research for the development of novel therapeutics to treat inflammatory diseases. Within the current study, the role of Notch1R in macrophage differentiation was investigated as well as downstream effects in THP-1 monocytes cultured in "inflammation mimicry" media. Interference of Notch signaling was achieved using either the pharmaceutical inhibitor DAPT or Notch1R small interfering RNA (siRNA), and Notch1R expression, macrophage phenotypes, and anti- and proinflammatory cytokine expression were evaluated. Data presented show that Notch1R expression on M1 macrophages as well as M1 macrophage differentiation is significantly elevated during cellular stress (P < 0.05). However, under identical culture conditions, interference to Notch signaling via Notch1R inhibition mitigated these results as well as promoted M2 macrophage differentiation. Moreover, when subjected to cellular stress, macrophage secretion of proinflammatory cytokines was significantly heightened (P < 0.05). Importantly, Notch1R inhibition not only diminished proinflammatory cytokine secretion but also enhanced anti-inflammatory protein release (P < 0.05). Our data suggest that Notch1R plays a pivotal role in M1 macrophage differentiation and heightened inflammatory responses. Therefore, we conclude that inhibition of Notch1R and subsequent downstream signaling enhances monocyte to M2 polarized macrophage outcomes and promotes anti-inflammatory mediation during cellular stress. PMID:25260616

  6. IL-33 Contributes to Schistosoma japonicum-induced Hepatic Pathology through Induction of M2 Macrophages

    PubMed Central

    Peng, Hui; Zhang, Qixian; Li, Xiaojuan; Liu, Zhen; Shen, Jia; Sun, Rui; Wei, Jie; Zhao, Jia; Wu, Xiaoying; Feng, Feng; Zhong, Shuping; Sun, Xi; Wu, Zhongdao

    2016-01-01

    Interleukin (IL)-33 is involved in T helper (Th)2-biased immune responses in mice infected with Schistosoma, but the precise mechanism remains to be elucidated. Herein, we investigated the role of IL-33 and its receptor ST2L in hepatic granuloma pathology induced by Schistosoma japonicum infection. We found that IL-33 induced the increased production of IL-5 and IL-13 from splenocytes and liver mononuclear cells (MNCs) of infected mice. The infected mice developed significantly higher number of ST2L-expressing cells in spleen and liver. Most of the ST2L-expressing cells in liver were F4/80+ macrophages, indicating the key role of macrophages in the response to IL-33. However, the liver MNCs in male-only worm infection had a poor response to IL-33, though elevated serum IL-33 was observed. ST2L+F4/80+ cells were lower in male-only worm infection than that of mixed infection. IL-33 and soluble egg antigen (SEA) upregulated ST2L expression on macrophages in vitro and ST2L-expressing macrophage displayed MHCII-CD11b+M2 phenotype. Macrophage deletion significantly attenuated IL-33-induced type 2 immunity and egg granuloma formation during S. japonicum infection. These data demonstrate that IL-33 contributes to hepatic granuloma pathology through induction of M2 macrophages during S. japonicum infection. PMID:27445267

  7. A Comparative Analysis of Synthetic Quorum Sensing Modulators in Pseudomonas aeruginosa: New Insights into Mechanism, Active Efflux Susceptibility, Phenotypic Response, and Next-Generation Ligand Design

    PubMed Central

    2015-01-01

    Quorum sensing (QS) is a chemical signaling mechanism that allows bacterial populations to coordinate gene expression in response to social and environmental cues. Many bacterial pathogens use QS to initiate infection at high cell densities. Over the past two decades, chemical antagonists of QS in pathogenic bacteria have attracted substantial interest for use both as tools to further elucidate QS mechanisms and, with further development, potential anti-infective agents. Considerable recent research has been devoted to the design of small molecules capable of modulating the LasR QS receptor in the opportunistic pathogen Pseudomonas aeruginosa. These molecules hold significant promise in a range of contexts; however, as most compounds have been developed independently, comparative activity data for these compounds are scarce. Moreover, the mechanisms by which the bulk of these compounds act are largely unknown. This paucity of data has stalled the choice of an optimal chemical scaffold for further advancement. Herein, we submit the best-characterized LasR modulators to standardized cell-based reporter and QS phenotypic assays in P. aeruginosa, and we report the first comprehensive set of comparative LasR activity data for these compounds. Our experiments uncovered multiple interesting mechanistic phenomena (including a potential alternative QS-modulatory ligand binding site/partner) that provide new, and unexpected, insights into the modes by which many of these LasR ligands act. The lead compounds, data trends, and mechanistic insights reported here will significantly aid the design of new small molecule QS inhibitors and activators in P. aeruginosa, and in other bacteria, with enhanced potencies and defined modes of action. PMID:26491787

  8. Association of gene polymorphisms encoding dopaminergic system components and platelet MAO-B activity with alcohol dependence and alcohol dependence-related phenotypes.

    PubMed

    Nedic Erjavec, Gordana; Nenadic Sviglin, Korona; Nikolac Perkovic, Matea; Muck-Seler, Dorotea; Jovanovic, Tanja; Pivac, Nela

    2014-10-01

    The present study aimed to evaluate the association of alcohol dependence and alcohol dependence-related phenotypes with platelet monoamine oxidase type B (MAO-B) activity, Val108/158Met of catechol-o-methyltransferase (COMT), variable number of tandem repeats (VNTR) in the third exon of dopamine receptor D4 (DRD4) gene, VNTR in the 3'-untranslated region of dopamine transporter (DAT) gene, -1021C/T of dopamine beta-hydroxylase (DBH) and MAO-B intron 13 polymorphisms. The study included 1270 Caucasian men and women of Croatian origin: 690 patients with alcohol dependence and 580 healthy controls. Patients with alcohol dependence were subdivided according to the presence or absence of withdrawal symptoms, aggressive behavior, severity of alcohol dependence, delirium tremens, comorbid depression, suicidal behavior, lifetime suicide attempt and early/late onset of alcohol abuse. The results, corrected for multiple testing, revealed increased platelet MAO-B activity in patients with alcohol dependence, subdivided into those with or without alcohol-related liver diseases, compared to control subjects (P<0.001). In addition, we found an increased frequency of the COMT Met/Met genotype among suicidal (P=0.002) and patients who attempted suicide (P<0.001) and an increased frequency of COMT Val/Val genotype in patients with an early onset of alcohol dependence (P=0.004). This study provides data from a sample of ethnically homogeneous unrelated Caucasian subjects for future meta-analyses and suggests that the increased platelet MAO-B activity might be used as independent peripheral indicator of alcohol dependence, while COMT Val108/158Met polymorphism is associated with increased suicidality and early onset of alcohol dependence. PMID:25035107

  9. Anti-CD47 Treatment Stimulates Phagocytosis of Glioblastoma by M1 and M2 Polarized Macrophages and Promotes M1 Polarized Macrophages In Vivo.

    PubMed

    Zhang, Michael; Hutter, Gregor; Kahn, Suzana A; Azad, Tej D; Gholamin, Sharareh; Xu, Chelsea Y; Liu, Jie; Achrol, Achal S; Richard, Chase; Sommerkamp, Pia; Schoen, Matthew Kenneth; McCracken, Melissa N; Majeti, Ravi; Weissman, Irving; Mitra, Siddhartha S; Cheshier, Samuel H

    2016-01-01

    Tumor-associated macrophages (TAMs) represent an important cellular subset within the glioblastoma (WHO grade IV) microenvironment and are a potential therapeutic target. TAMs display a continuum of different polarization states between antitumorigenic M1 and protumorigenic M2 phenotypes, with a lower M1/M2 ratio correlating with worse prognosis. Here, we investigated the effect of macrophage polarization on anti-CD47 antibody-mediated phagocytosis of human glioblastoma cells in vitro, as well as the effect of anti-CD47 on the distribution of M1 versus M2 macrophages within human glioblastoma cells grown in mouse xenografts. Bone marrow-derived mouse macrophages and peripheral blood-derived human macrophages were polarized in vitro toward M1 or M2 phenotypes and verified by flow cytometry. Primary human glioblastoma cell lines were offered as targets to mouse and human M1 or M2 polarized macrophages in vitro. The addition of an anti-CD47 monoclonal antibody led to enhanced tumor-cell phagocytosis by mouse and human M1 and M2 macrophages. In both cases, the anti-CD47-induced phagocytosis by M1 was more prominent than that for M2. Dissected tumors from human glioblastoma xenografted within NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice and treated with anti-CD47 showed a significant increase of M1 macrophages within the tumor. These data show that anti-CD47 treatment leads to enhanced tumor cell phagocytosis by both M1 and M2 macrophage subtypes with a higher phagocytosis rate by M1 macrophages. Furthermore, these data demonstrate that anti-CD47 treatment alone can shift the phenotype of macrophages toward the M1 subtype in vivo. PMID:27092773

  10. Anti-CD47 Treatment Stimulates Phagocytosis of Glioblastoma by M1 and M2 Polarized Macrophages and Promotes M1 Polarized Macrophages In Vivo

    PubMed Central

    Kahn, Suzana A.; Azad, Tej D.; Gholamin, Sharareh; Xu, Chelsea Y.; Liu, Jie; Achrol, Achal S.; Richard, Chase; Sommerkamp, Pia; Schoen, Matthew Kenneth; McCracken, Melissa N.; Majeti, Ravi; Weissman, Irving; Mitra, Siddhartha S.; Cheshier, Samuel H.

    2016-01-01

    Tumor-associated macrophages (TAMs) represent an important cellular subset within the glioblastoma (WHO grade IV) microenvironment and are a potential therapeutic target. TAMs display a continuum of different polarization states between antitumorigenic M1 and protumorigenic M2 phenotypes, with a lower M1/M2 ratio correlating with worse prognosis. Here, we investigated the effect of macrophage polarization on anti-CD47 antibody-mediated phagocytosis of human glioblastoma cells in vitro, as well as the effect of anti-CD47 on the distribution of M1 versus M2 macrophages within human glioblastoma cells grown in mouse xenografts. Bone marrow-derived mouse macrophages and peripheral blood-derived human macrophages were polarized in vitro toward M1 or M2 phenotypes and verified by flow cytometry. Primary human glioblastoma cell lines were offered as targets to mouse and human M1 or M2 polarized macrophages in vitro. The addition of an anti-CD47 monoclonal antibody led to enhanced tumor-cell phagocytosis by mouse and human M1 and M2 macrophages. In both cases, the anti-CD47-induced phagocytosis by M1 was more prominent than that for M2. Dissected tumors from human glioblastoma xenografted within NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice and treated with anti-CD47 showed a significant increase of M1 macrophages within the tumor. These data show that anti-CD47 treatment leads to enhanced tumor cell phagocytosis by both M1 and M2 macrophage subtypes with a higher phagocytosis rate by M1 macrophages. Furthermore, these data demonstrate that anti-CD47 treatment alone can shift the phenotype of macrophages toward the M1 subtype in vivo. PMID:27092773

  11. Localization of the M2 muscarinic cholinergic receptor in dendrites, cholinergic terminals, and noncholinergic terminals in the rat basolateral amygdala: An ultrastructural analysis.

    PubMed

    Muller, Jay F; Mascagni, Franco; Zaric, Violeta; Mott, David D; McDonald, Alexander J

    2016-08-15

    Activation of M2 muscarinic receptors (M2Rs) in the rat anterior basolateral nucleus (BLa) is critical for the consolidation of memories of emotionally arousing events. The present investigation used immunocytochemistry at the electron microscopic level to determine which structures in the BLa express M2Rs. In addition, dual localization of M2R and the vesicular acetylcholine transporter protein (VAChT), a marker for cholinergic axons, was performed to determine whether M2R is an autoreceptor in cholinergic axons innervating the BLa. M2R immunoreactivity (M2R-ir) was absent from the perikarya of pyramidal neurons, with the exception of the Golgi complex, but was dense in the proximal dendrites and axon initial segments emanating from these neurons. Most perikarya of nonpyramidal neurons were also M2R-negative. About 95% of dendritic shafts and 60% of dendritic spines were M2 immunoreactive (M2R(+) ). Some M2R(+) dendrites had spines, suggesting that they belonged to pyramidal cells, whereas others had morphological features typical of nonpyramidal neurons. M2R-ir was also seen in axon terminals, most of which formed asymmetrical synapses. The main targets of M2R(+) terminals forming asymmetrical (putative excitatory) synapses were dendritic spines, most of which were M2R(+) . The main targets of M2R(+) terminals forming symmetrical (putative inhibitory or neuromodulatory) synapses were unlabeled perikarya and M2R(+) dendritic shafts. M2R-ir was also seen in VAChT(+) cholinergic terminals, indicating a possible autoreceptor role. These findings suggest that M2R-mediated mechanisms in the BLa are very complex, involving postsynaptic effects in dendrites as well as regulating release of glutamate, γ-aminobutyric acid, and acetylcholine from presynaptic axon terminals. J. Comp. Neurol. 524:2400-2417, 2016. © 2016 Wiley Periodicals, Inc. PMID:26779591

  12. Induced Differentiation of Human Myeloid Leukemia Cells into M2 Macrophages by Combined Treatment with Retinoic Acid and 1α,25-Dihydroxyvitamin D3

    PubMed Central

    Takahashi, Hiromichi; Hatta, Yoshihiro; Iriyama, Noriyoshi; Hasegawa, Yuichiro; Uchida, Hikaru; Nakagawa, Masaru; Makishima, Makoto; Takeuchi, Jin; Takei, Masami

    2014-01-01

    Retinoids and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) induce differentiation of myeloid leukemia cells into granulocyte and macrophage lineages, respectively. All-trans retinoic acid (ATRA), which is effective in the treatment of acute promyelocytic leukemia, can induce differentiation of other types of myeloid leukemia cells, and combined treatment with retinoid and 1,25(OH)2D3 effectively enhances the differentiation of leukemia cells into macrophage-like cells. Recent work has classified macrophages into M1 and M2 types. In this study, we investigated the effect of combined treatment with retinoid and 1,25(OH)2D3 on differentiation of myeloid leukemia THP-1 and HL60 cells. 9-cis Retinoic acid (9cRA) plus 1,25(OH)2D3 inhibited proliferation of THP-1 and HL60 cells and increased myeloid differentiation markers including nitroblue tetrazolium reducing activity and expression of CD14 and CD11b. ATRA and the synthetic retinoic acid receptor agonist Am80 exhibited similar effects in combination with 1,25(OH)2D3 but less effectively than 9cRA, while the retinoid X receptor agonist HX630 was not effective. 9cRA plus 1,25(OH)2D3 effectively increased expression of M2 macrophage marker genes, such as CD163, ARG1 and IL10, increased surface CD163 expression, and induced interleukin-10 secretion in myeloid leukemia cells, while 9cRA alone had weaker effects on these phenotypes and 1,25(OH)2D3 was not effective. Taken together, our results demonstrate selective induction of M2 macrophage markers in human myeloid leukemia cells by combined treatment with 9cRA and 1,25(OH)2D3. PMID:25409436

  13. Glioma-Associated Microglia/Macrophages Display an Expression Profile Different from M1 and M2 Polarization and Highly Express Gpnmb and Spp1

    PubMed Central

    Szulzewsky, Frank; Pelz, Andreas; Feng, Xi; Synowitz, Michael; Markovic, Darko; Langmann, Thomas; Holtman, Inge R.; Wang, Xi; Eggen, Bart J. L.; Boddeke, Hendrikus W. G. M.; Hambardzumyan, Dolores; Wolf, Susanne A.; Kettenmann, Helmut

    2015-01-01

    Malignant glioma belong to the most aggressive neoplasms in humans with no successful treatment available. Patients suffering from glioblastoma multiforme (GBM), the highest-grade glioma, have an average survival time of only around one year after diagnosis. Both microglia and peripheral macrophages/monocytes accumulate within and around glioma, but fail to exert effective anti-tumor activity and even support tumor growth. Here we use microarray analysis to compare the expression profiles of glioma-associated microglia/macrophages and naive control cells. Samples were generated from CD11b+ MACS-isolated cells from naïve and GL261-implanted C57BL/6 mouse brains. Around 1000 genes were more than 2-fold up- or downregulated in glioma-associated microglia/macrophages when compared to control cells. A comparison with published data sets of M1, M2a,b,c-polarized macrophages revealed a gene expression pattern that has only partial overlap with any of the M1 or M2 gene expression patterns. Samples for the qRT-PCR validation of selected M1 and M2a,b,c-specific genes were generated from two different glioma mouse models and isolated by flow cytometry to distinguish between resident microglia and invading macrophages. We confirmed in both models the unique glioma-associated microglia/macrophage phenotype including a mixture of M1 and M2a,b,c-specific genes. To validate the expression of these genes in human we MACS-isolated CD11b+ microglia/macrophages from GBM, lower grade brain tumors and control specimens. Apart from the M1/M2 gene analysis, we demonstrate that the expression of Gpnmb and Spp1 is highly upregulated in both murine and human glioma-associated microglia/macrophages. High expression of these genes has been associated with poor prognosis in human GBM, as indicated by patient survival data linked to gene expression data. We also show that microglia/macrophages are the predominant source of these transcripts in murine and human GBM. Our findings provide new

  14. Precipitation Kinetics of M2C Carbide in Severely Ausformed 13Co-8Ni Secondary Hardening Steels

    NASA Astrophysics Data System (ADS)

    Cho, Ki Sub; Park, Sung Soo; Kim, Hong Kyu; Song, Young Beum; Kwon, Hoon

    2015-04-01

    With continuous heating calorimetric data as a basis, the kinetics of M2C formation during isothermal aging was modeled in severely ausformed 13Co-8Ni steels using the Johnson-Mehl-Avrami theory coupled with a variation of effective activation energy with respect to the degree of transformation. These results were compared with small-angle neutron scattering measurements and discussed in terms of variations in the thermodynamic and kinetic behavior of M2C precipitation. In particular, the M2C carbides in the deformed samples contained more Fe content compared with the non-deformed samples. As this can be ascribed to the ausforming effect increasing the driving force for M2C nucleation, it consequently leads to the decrease of the growth/coarsening rate for M2C carbides at over-aged conditions.

  15. Antitubercular specific activity of ibuprofen and the other 2-arylpropanoic acids using the HT-SPOTi whole-cell phenotypic assay

    PubMed Central

    Guzman, Juan D; Evangelopoulos, Dimitrios; Gupta, Antima; Birchall, Kristian; Mwaigwisya, Solomon; Saxty, Barbara; McHugh, Timothy D; Gibbons, Simon; Malkinson, John; Bhakta, Sanjib

    2013-01-01

    Objectives Lead antituberculosis (anti-TB) molecules with novel mechanisms of action are urgently required to fuel the anti-TB drug discovery pipeline. The aim of this study was to validate the use of the high-throughput spot culture growth inhibition (HT-SPOTi) assay for screening libraries of compounds against Mycobacterium tuberculosis and to study the inhibitory effect of ibuprofen (IBP) and the other 2-arylpropanoic acids on the growth inhibition of M tuberculosis and other mycobacterial species. Methods The HT-SPOTi method was validated not only with known drugs but also with a library of 47 confirmed anti-TB active compounds published in the ChEMBL database. Three over-the-counter non-steroidal anti-inflammatory drugs were also included in the screening. The 2-arylpropanoic acids, including IBP, were comprehensively evaluated against phenotypically and physiologically different strains of mycobacteria, and their cytotoxicity was determined against murine RAW264.7 macrophages. Furthermore, a comparative bioinformatic analysis was employed to propose a potential mycobacterial target. Results IBP showed antitubercular properties while carprofen was the most potent among the 2-arylpropanoic class. A 3,5-dinitro-IBP derivative was found to be more potent than IBP but equally selective. Other synthetic derivatives of IBP were less active, and the free carboxylic acid of IBP seems to be essential for its anti-TB activity. IBP, carprofen and the 3,5-dinitro-IBP derivative exhibited activity against multidrug-resistant isolates and stationary phase bacilli. On the basis of the human targets of the 2-arylpropanoic analgesics, the protein initiation factor infB (Rv2839c) of M tuberculosis was proposed as a potential molecular target. Conclusions The HT-SPOTi method can be employed reliably and reproducibly to screen the antimicrobial potency of different compounds. IBP demonstrated specific antitubercular activity, while carprofen was the most selective agent among the

  16. SDiff gauge theory and the M2 condensate

    NASA Astrophysics Data System (ADS)

    Bandos, Igor A.; Townsend, Paul K.

    2009-02-01

    We develop a general formalism for the construction, in D-dimensional Minkowski space, of gauge theories for which the gauge group is the infinite-dimensional group SDiffn of volume-preserving diffeomorphisms of some closed n-dimensional manifold. We then focus on the D = 3 SDiff3 superconformal gauge theory describing a condensate of M2-branes; in particular, we derive its Script N = 8 superfield equations from a pure-spinor superspace action, and we describe its relationship to the D = 3 SDiff2 super-Yang-Mills theory describing a condensate of D2-branes.

  17. Surface modification of nanoparticles enables selective evasion of phagocytic clearance by distinct macrophage phenotypes.

    PubMed

    Qie, Yaqing; Yuan, Hengfeng; von Roemeling, Christina A; Chen, Yuanxin; Liu, Xiujie; Shih, Kevin D; Knight, Joshua A; Tun, Han W; Wharen, Robert E; Jiang, Wen; Kim, Betty Y S

    2016-01-01

    Nanomedicine is a burgeoning industry but an understanding of the interaction of nanomaterials with the immune system is critical for clinical translation. Macrophages play a fundamental role in the immune system by engulfing foreign particulates such as nanoparticles. When activated, macrophages form distinct phenotypic populations with unique immune functions, however the mechanism by which these polarized macrophages react to nanoparticles is unclear. Furthermore, strategies to selectively evade activated macrophage subpopulations are lacking. Here we demonstrate that stimulated macrophages possess higher phagocytic activities and that classically activated (M1) macrophages exhibit greater phagocytic capacity than alternatively activated (M2) macrophages. We show that modification of nanoparticles with polyethylene-glycol results in decreased clearance by all macrophage phenotypes, but importantly, coating nanoparticles with CD47 preferentially lowers phagocytic activity by the M1 phenotype. These results suggest that bio-inspired nanoparticle surface design may enable evasion of specific components of the immune system and provide a rational approach for developing immune tolerant nanomedicines. PMID:27197045

  18. Surface modification of nanoparticles enables selective evasion of phagocytic clearance by distinct macrophage phenotypes

    PubMed Central

    Qie, Yaqing; Yuan, Hengfeng; von Roemeling, Christina A.; Chen, Yuanxin; Liu, Xiujie; Shih, Kevin D.; Knight, Joshua A.; Tun, Han W.; Wharen, Robert E.; Jiang, Wen; Kim, Betty Y.S.

    2016-01-01

    Nanomedicine is a burgeoning industry but an understanding of the interaction of nanomaterials with the immune system is critical for clinical translation. Macrophages play a fundamental role in the immune system by engulfing foreign particulates such as nanoparticles. When activated, macrophages form distinct phenotypic populations with unique immune functions, however the mechanism by which these polarized macrophages react to nanoparticles is unclear. Furthermore, strategies to selectively evade activated macrophage subpopulations are lacking. Here we demonstrate that stimulated macrophages possess higher phagocytic activities and that classically activated (M1) macrophages exhibit greater phagocytic capacity than alternatively activated (M2) macrophages. We show that modification of nanoparticles with polyethylene-glycol results in decreased clearance by all macrophage phenotypes, but importantly, coating nanoparticles with CD47 preferentially lowers phagocytic activity by the M1 phenotype. These results suggest that bio-inspired nanoparticle surface design may enable evasion of specific components of the immune system and provide a rational approach for developing immune tolerant nanomedicines. PMID:27197045

  19. Chemical rescue of histidine selectivity filter mutants of the M2 ion channel of influenza A virus.

    PubMed

    Venkataraman, Padmavati; Lamb, Robert A; Pinto, Lawrence H

    2005-06-01

    The influenza virus M2 proton-selective ion channel activity facilitates virus uncoating, a process that occurs in the acidic environment of the endosome. The M2 channel causes acidification of the interior of the virus particle, which results in viral protein-protein dissociation. The M2 protein is a homotetramer that contains in its aqueous pore a histidine residue (His-37) that acts as a selectivity filter and a tryptophan residue (Trp-41) that acts as a channel gate. Substitution of His-37 modifies M2 ion channel properties drastically. However, the results of such experiments are difficult to interpret because substitution of His-37 could cause gross structural changes to the channel pore. We described here experiments in which partial or, in some cases, full rescue of specific M2 ion channel properties of His-37 substitution mutants was achieved by addition of imidazole to the bathing medium. Chemical rescue was demonstrated for three histidine substitution mutant ion channels (M2-H37G, M2-H37S, and M2-H37T) and for two double mutants in which the Trp-41 channel gate was also mutated (H37G/W41Y and H37G/W41A). Currents of the M2-H37G mutant ion channel were inhibited by Cu(II), which has been shown to coordinate with His-37 in the wild-type channel. Chemical rescue was very specific for imidazole. Buffer molecules that were neutral when protonated (4-morpholineethanesulfonic acid and 3-morpholino-2-hydroxypropanesulfonic acid) did not rescue ion channel activity of the M2-H37G mutant ion channel, but 1-methylimidazole did provide partial rescue of function. These results were consistent with a model for proton transport through the pore of the wild-type channel in which the imidazole side chain of His-37 acted as an intermediate proton acceptor/donor group. PMID:15784624

  20. M2 Proton Channel: Toward a Model of a Primitive Proton Pump

    NASA Astrophysics Data System (ADS)

    Wei, Chenyu; Pohorille, Andrew

    2015-06-01

    Transmembrane proton transfer was essential to early cellular systems in order to transduce energy for metabolic functions. The reliable, efficient and controlled generation of proton gradients became possible only with the emergence of active proton pumps. On the basis of features shared by most modern proton pumps we identify the essential mechanistic steps in active proton transport. Further, we discuss the mechanism of action of a small, transmembrane M2 proton channel from influenza A virus as a model for proton transport in protocells. The M2 channel is a 94-residue long, α-helical tetramer that is activated at low pH and exhibits high selectivity and directionality. A shorter construct, built of transmembrane fragments that are only 24 amino acids in length, exhibits very similar proton transport properties. Molecular dynamics simulations on the microsecond time-scale carried out for the M2 channel provided atomic level details on the activation of the channel in response to protonation of the histidine residue, His37. The pathway of proton conduction is mediated by His37, which accepts and donates protons at different interconverting conformation states when pH is lower than 6.5. The Val27 and Trp41 gates and the salt bridge between Asp44 and Arg45 further enhance the directionality of proton transport. It is argued that the architecture and the mechanism of action similar to that found in the M2 channel might have been the perfect starting point for evolution towards the earliest proton pumps, indicating that active proton transport could have readily emerged from simple, passive proton channels.

  1. Agonists with supraphysiological efficacy at the muscarinic M2 ACh receptor

    PubMed Central

    Schrage, R; Seemann, WK; Klöckner, J; Dallanoce, C; Racké, K; Kostenis, E; De Amici, M; Holzgrabe, U; Mohr, K

    2013-01-01

    Background and Purpose Artificial agonists may have higher efficacy for receptor activation than the physiological agonist. Until now, such ‘superagonism’ has rarely been reported for GPCRs. Iperoxo is an extremely potent muscarinic receptor agonist. We hypothesized that iperoxo is a ‘superagonist’. Experimental Approach Signalling of iperoxo and newly synthesized structural analogues was compared with that of ACh at label-free M2 muscarinic receptors applying whole cell dynamic mass redistribution, measurement of G-protein activation, evaluation of cell surface agonist binding and computation of operational efficacies. Key Results In CHO-hM2 cells, iperoxo significantly exceeds ACh in Gi/Gs signalling competence. In the orthosteric loss-of-function mutant M2-Y1043.33A, the maximum effect of iperoxo is hardly compromised in contrast to ACh. ‘Superagonism’ is preserved in the physiological cellular context of MRC-5 human lung fibroblasts. Structure–signalling relationships including iperoxo derivatives with either modified positively charged head group or altered tail suggest that ‘superagonism’ of iperoxo is mechanistically based on parallel activation of the receptor protein via two orthosteric interaction points. Conclusion and Implications Supraphysiological agonist efficacy at muscarinic M2 ACh receptors is demonstrated for the first time. In addition, a possible underlying molecular mechanism of GPCR ‘superagonism’ is provided. We suggest that iperoxo-like orthosteric GPCR activation is a new avenue towards a novel class of receptor activators. Linked Article This article is commented on by Langmead and Christopoulos, pp. 353–356 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12142 PMID:23062057

  2. M2 proton channel: toward a model of a primitive proton pump.

    PubMed

    Wei, Chenyu; Pohorille, Andrew

    2015-06-01

    Transmembrane proton transfer was essential to early cellular systems in order to transduce energy for metabolic functions. The reliable, efficient and controlled generation of proton gradients became possible only with the emergence of active proton pumps. On the basis of features shared by most modern proton pumps we identify the essential mechanistic steps in active proton transport. Further, we discuss the mechanism of action of a small, transmembrane M2 proton channel from influenza A virus as a model for proton transport in protocells. The M2 channel is a 94-residue long, α-helical tetramer that is activated at low pH and exhibits high selectivity and directionality. A shorter construct, built of transmembrane fragments that are only 24 amino acids in length, exhibits very similar proton transport properties. Molecular dynamics simulations on the microsecond time-scale carried out for the M2 channel provided atomic level details on the activation of the channel in response to protonation of the histidine residue, His37. The pathway of proton conduction is mediated by His37, which accepts and donates protons at different interconverting conformation states when pH is lower than 6.5. The Val27 and Trp41 gates and the salt bridge between Asp44 and Arg45 further enhance the directionality of proton transport. It is argued that the architecture and the mechanism of action similar to that found in the M2 channel might have been the perfect starting point for evolution towards the earliest proton pumps, indicating that active proton transport could have readily emerged from simple, passive proton channels. PMID:25777465

  3. Macrophage reprogramming: influence of latex beads with various functional groups on macrophage phenotype and phagocytic uptake in vitro.

    PubMed

    Akilbekova, Dana; Philiph, Rachel; Graham, Austin; Bratlie, Kaitlin M

    2015-01-01

    Macrophages play a crucial role in initiating immune responses with various functions ranging from wound healing to antimicrobial actions. The type of biomaterial is suggested to influence macrophage phenotype. Here, we show that exposing M1- and M2-activated macrophages to polystyrene latex beads bearing different functional groups can alter secretion profiles, providing a possible method for altering the course of the host response. Macrophages were stimulated with either lipopolysaccharide or interleukin (IL) 4 and cultured for 24 h with 10 different latex beads. Proinflammatory cytokines (tumor necrosis factor α, monocyte chemotactic protein 1) and nitrite served as markers for the M1 phenotype and proangiogenic cytokine (IL-10) and arginase activity for M2 cells. The ability of the macrophages to phagocytize Escherichia coli particles and water contact angles of the polymers were also assessed. Different patterns of cytokine expression and phagocytosis activity were induced by the various particles. Particles did not polarize the cells toward one specific phenotype versus another, but rather induced changes in both pro- and anti-inflammatory markers. Our results suggest a dependence of pro- and anti-inflammatory cytokines and phagocytic activities on material type and cytokine stimuli. These data also illustrate how biomaterials can be exploited to alter host responses for drug delivery and tissue engineering applications. PMID:24639060

  4. MULTIWAVELENGTH PHOTOMETRY IN THE GLOBULAR CLUSTER M2

    SciTech Connect

    Dalessandro, E.; Lanzoni, B.; Ferraro, F.R.; Beccari, G.; Schiavon, R.; Rood, R.T.

    2009-06-15

    We present a multiwavelength photometric analysis of the globular cluster M2. The data set has been obtained by combining high-resolution (Hubble Space Telescope/WFPC2 and ACS) and wide-field (Galaxy Evolution Explorer) space observations and ground-based (MEGACAM-CFHT, EMMI-NTT) images. The photometric sample covers the entire cluster extension from the very central regions up to the tidal radius and beyond. It allows an accurate determination of the cluster center of gravity and other structural parameters derived from the star count density profile. Moreover, we study the Blue Straggler Star (BSS) population and its radial distribution. A total of 123 BSSs have been selected, and their radial distribution has been found to be bimodal (highly peaked in the center, decreasing at intermediate radii, and rising outward), as already found in a number of other clusters. The radial position of the minimum of the BSS distribution is consistent with the radius of avoidance caused by the dynamical friction of massive (1.2 M {sub sun}) objects over the cluster age. We also searched for gradients in the red giant branch (RGB) and the asymptotic giant branch (AGB) populations. At the 2{sigma} level, we found an overabundance of AGB stars within the core radius and confirmed the result of Sohn et al. that the central region of M2 is bluer than the outer part. We show that the latter is due to a deficit of very luminous RGB stars in the central region.

  5. The iron dispersion of the globular cluster M2, revised

    NASA Astrophysics Data System (ADS)

    Lardo, C.; Mucciarelli, A.; Bastian, N.

    2016-03-01

    M2 has been claimed to possess three distinct stellar components that are enhanced in iron relative to each other. We use equivalent width measurements from 14 red giant branch stars from which Yong et al. detect a ˜0.8 dex wide, trimodal iron distribution to redetermine the metallicity of the cluster. In contrast to Yong et al., which derive atmospheric parameters following only the classical spectroscopic approach, we perform the chemical analysis using three different methods to constrain effective temperatures and surface gravities. When atmospheric parameters are derived spectroscopically, we measure a trimodal metallicity distribution, that well resembles that by Yong et al. We find that the metallicity distribution from Fe II lines strongly differs from the distribution obtained from Fe I features when photometric gravities are adopted. The Fe I distribution mimics the metallicity distribution obtained using spectroscopic parameters, while the Fe II shows the presence of only two stellar groups with metallicity [Fe/H] ≃ -1.5 and -1.1 dex, which are internally homogeneous in iron. This finding, when coupled with the high-resolution photometric evidence, demonstrates that M2 is composed by a dominant population (˜99 per cent) homogeneous in iron and a minority component (˜1 per cent) enriched in iron with respect to the main cluster population.

  6. Marginal fluctuations as instantons on M2/D2-branes

    NASA Astrophysics Data System (ADS)

    Naghdi, M.

    2014-03-01

    We introduce some (anti-) M/D-branes through turning on the corresponding field strengths of the 11- and 10-dimensional supergravity theories over spaces, where we use and for the internal spaces. Indeed, when we add M2/D2-branes on the same directions with the near horizon branes of the Aharony-Bergman-Jafferis-Maldacena model, all symmetries and supersymmetries are preserved trivially. In this case, we obtain a localized object just in the horizon. This normalizable bulk massless scalar mode is a singlet of and , and it agrees with a marginal boundary operator of the conformal dimension of . However, after performing a special conformal transformation, we see that the solution is localized in the Euclideanized space and is attributable to the included anti-M2/D2-branes, which are also necessary to ensure that there is no back-reaction. The resultant theory now breaks all supersymmetries to , while the other symmetries are so preserved. The dual boundary operator is then set up from the skew-whiffing of the representations and for the supercharges and scalars, respectively, while the fermions remain fixed in of the original theory. Besides, we also address another alternate bulk to boundary matching procedure through turning on one of the gauge fields of the full gauge group along the same lines with a similar situation to the one faced in the AdS/CFT correspondence. The latter approach covers the difficulty already faced with in the bulk-boundary matching procedure for as well.

  7. The iron dispersion of the globular cluster M2, revised

    PubMed Central

    Lardo, C.; Mucciarelli, A.; Bastian, N.

    2016-01-01

    M2 has been claimed to possess three distinct stellar components that are enhanced in iron relative to each other. We use equivalent width measurements from 14 red giant branch stars from which Yong et al. detect a ∼0.8 dex wide, trimodal iron distribution to redetermine the metallicity of the cluster. In contrast to Yong et al., which derive atmospheric parameters following only the classical spectroscopic approach, we perform the chemical analysis using three different methods to constrain effective temperatures and surface gravities. When atmospheric parameters are derived spectroscopically, we measure a trimodal metallicity distribution, that well resembles that by Yong et al. We find that the metallicity distribution from Fe ii lines strongly differs from the distribution obtained from Fe i features when photometric gravities are adopted. The Fe i distribution mimics the metallicity distribution obtained using spectroscopic parameters, while the Fe ii shows the presence of only two stellar groups with metallicity [Fe/H] ≃ −1.5 and −1.1 dex, which are internally homogeneous in iron. This finding, when coupled with the high-resolution photometric evidence, demonstrates that M2 is composed by a dominant population (∼99 per cent) homogeneous in iron and a minority component (∼1 per cent) enriched in iron with respect to the main cluster population. PMID:27274701

  8. Control of Viremia Enables Acquisition of Resting Memory B Cells with Age and Normalization of Activated B Cell Phenotypes in HIV-Infected Children.

    PubMed

    Muema, Daniel M; Macharia, Gladys N; Hassan, Amin S; Mwaringa, Shalton M; Fegan, Greg W; Berkley, James A; Nduati, Eunice W; Urban, Britta C

    2015-08-01

    HIV affects the function of all lymphocyte populations, including B cells. Phenotypic and functional defects of B cells in HIV-infected adults have been well characterized, but defects in children have not been studied to the same extent. We determined the proportion of B cell subsets and frequencies of Ag-specific memory B cells in peripheral blood from HIV-infected children and healthy controls, using flow cytometry and B cell ELISPOT, respectively. In addition, we measured the quantities and avidities of plasma Abs against various Ags by ELISA. We also determined plasma levels of BAFF and expression of BAFF receptors on B cells. Children with high HIV viremia had increased proportions of activated mature B cells, tissue-like memory B cells and plasmablasts, and low proportions of naive B cells when compared with community controls and children with low HIV viremia, similar to adults infected with HIV. HIV-infected groups had lower proportions of resting memory B cells than did community controls. Notably, high HIV viremia prevented the age-dependent accumulation of class-switched resting memory B cells. HIV-infected children, regardless of the level of viremia, showed lower quantities and avidities of IgG and lower frequencies of memory B cells against Expanded Program on Immunization vaccines. The HIV-infected children had an altered BAFF profile that could have affected their B cell compartment. Therefore, B cell defects in HIV-infected children are similar to those seen in HIV-infected adults. However, control of HIV viremia is associated with normalization of activated B cell subsets and allows age-dependent accumulation of resting memory B cells. PMID:26116511

  9. Complex Disease–, Gene–, and Drug–Drug Interactions: Impacts of Renal Function, CYP2D6 Phenotype, and OCT2 Activity on Veliparib Pharmacokinetics

    PubMed Central

    Li, Jing; Kim, Seongho; Sha, Xianyi; Wiegand, Richard; Wu, Jianmei; LoRusso, Patricia

    2014-01-01

    Purpose Veliparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, undergoes renal excretion and liver metabolism. This study quantitatively assessed the interactions of veliparib with metabolizing enzyme (CYP2D6) and transporter (OCT2) in disease settings (renal impairment). Experimental Design Veliparib in vitro metabolism was examined in human liver microsomes and recombinant enzymes carrying wild-type CYP2D6 or functional defect variants (CYP2D6*10 and *4). Plasma pharmacokinetics were evaluated in 27 patients with cancer. A parent–metabolite joint population model was developed to characterize veliparib and metabolite (M8) pharmacokinetics and to identify patient factors influencing veliparib disposition. A physiologically based pharmacokinetic model integrated with a mechanistic kidney module was developed to quantitatively predict the individual and combined effects of renal function, CYP2D6 phenotype, and OCT2 activity on veliparib pharmacokinetics. Results In vitro intrinsic clearance of CYP2D6.1 and CYP2D6.10 for veliparib metabolism were 0.055 and 0.017 μL/min/pmol CYP, respectively. Population mean values for veliparib oral clearance and M8 clearance were 13.3 and 8.6 L/h, respectively. Creatinine clearance was identified as the significant covariate on veliparib oral clearance. Moderate renal impairment, CYP2D6 poor metabolizer, and co-administration of OCT2 inhibitor (cimetidine) increased veliparib steady-state exposure by 80%, 20%, and 30%, respectively. These factors collectively led to >2-fold increase in veliparib exposure. Conclusions Renal function (creatinine clearance) is a significant predictor for veliparib exposure in patients with cancer. Although a single factor (i.e., renal impairment, CYP2D6 deficiency, and reduced OCT2 activity) shows a moderate impact, they collectively could result in a significant and potentially clinically relevant increase in veliparib exposure. PMID:24947923

  10. Progression of Osteosarcoma from a Non-Metastatic to a Metastatic Phenotype Is Causally Associated with Activation of an Autocrine and Paracrine uPA Axis

    PubMed Central

    Endo-Munoz, Liliana; Cai, Na; Cumming, Andrew; Macklin, Rebecca; Merida de Long, Lilia; Topkas, Eleni; Mukhopadhyay, Pamela; Hill, Michelle; Saunders, Nicholas A

    2015-01-01

    Pulmonary metastasis is the major untreatable complication of osteosarcoma (OS) resulting in 10–20% long-term survival. The factors and pathways regulating these processes remain unclear, yet their identification is crucial in order to find new therapeutic targets. In this study we used a multi-omics approach to identify molecules in metastatic and non-metastatic OS cells that may contribute to OS metastasis, followed by validation in vitro and in vivo. We found elevated levels of the urokinase plasminogen activator (uPA) and of the uPA receptor (uPAR) exclusively in metastatic OS cells. uPA was secreted in soluble form and as part of the protein cargo of OS-secreted extracellular vesicles, including exosomes. In addition, in the tumour microenvironment, uPA was expressed and secreted by bone marrow cells (BMC), and OS- and BMC-derived uPA significantly and specifically stimulated migration of metastatic OS cells via uPA-dependent signaling pathways. Silencing of uPAR in metastatic OS cells abrogated the migratory response to uPA in vitro and decreased metastasis in vivo. Finally, a novel small-molecule inhibitor of uPA significantly (P = 0.0004) inhibited metastasis in an orthotopic mouse model of OS. Thus, we show for the first time that malignant conversion of OS cells to a metastatic phenotype is defined by activation of the uPA/uPAR axis in both an autocrine and paracrine fashion. Furthermore, metastasis is driven by changes in OS cells as well as in the microenvironment. Finally, our data show that pharmacological inhibition of the uPA/uPAR axis with a novel small-molecule inhibitor can prevent the emergence of metastatic foci. PMID:26317203

  11. Progression of Osteosarcoma from a Non-Metastatic to a Metastatic Phenotype Is Causally Associated with Activation of an Autocrine and Paracrine uPA Axis.

    PubMed

    Endo-Munoz, Liliana; Cai, Na; Cumming, Andrew; Macklin, Rebecca; Merida de Long, Lilia; Topkas, Eleni; Mukhopadhyay, Pamela; Hill, Michelle; Saunders, Nicholas A

    2015-01-01

    Pulmonary metastasis is the major untreatable complication of osteosarcoma (OS) resulting in 10-20% long-term survival. The factors and pathways regulating these processes remain unclear, yet their identification is crucial in order to find new therapeutic targets. In this study we used a multi-omics approach to identify molecules in metastatic and non-metastatic OS cells that may contribute to OS metastasis, followed by validation in vitro and in vivo. We found elevated levels of the urokinase plasminogen activator (uPA) and of the uPA receptor (uPAR) exclusively in metastatic OS cells. uPA was secreted in soluble form and as part of the protein cargo of OS-secreted extracellular vesicles, including exosomes. In addition, in the tumour microenvironment, uPA was expressed and secreted by bone marrow cells (BMC), and OS- and BMC-derived uPA significantly and specifically stimulated migration of metastatic OS cells via uPA-dependent signaling pathways. Silencing of uPAR in metastatic OS cells abrogated the migratory response to uPA in vitro and decreased metastasis in vivo. Finally, a novel small-molecule inhibitor of uPA significantly (P = 0.0004) inhibited metastasis in an orthotopic mouse model of OS. Thus, we show for the first time that malignant conversion of OS cells to a metastatic phenotype is defined by activation of the uPA/uPAR axis in both an autocrine and paracrine fashion. Furthermore, metastasis is driven by changes in OS cells as well as in the microenvironment. Finally, our data show that pharmacological inhibition of the uPA/uPAR axis with a novel small-molecule inhibitor can prevent the emergence of metastatic foci. PMID:26317203

  12. Control of Viremia Enables Acquisition of Resting Memory B Cells with Age and Normalization of Activated B Cell Phenotypes in HIV-Infected Children

    PubMed Central

    Muema, Daniel M.; Macharia, Gladys N.; Hassan, Amin S.; Mwaringa, Shalton M.; Fegan, Greg W.; Berkley, James A.; Urban, Britta C.

    2015-01-01

    HIV affects the function of all lymphocyte populations, including B cells. Phenotypic and functional defects of B cells in HIV-infected adults have been well characterized, but defects in children have not been studied to the same extent. We determined the proportion of B cell subsets and frequencies of Ag-specific memory B cells in peripheral blood from HIV-infected children and healthy controls, using flow cytometry and B cell ELISPOT, respectively. In addition, we measured the quantities and avidities of plasma Abs against various Ags by ELISA. We also determined plasma levels of BAFF and expression of BAFF receptors on B cells. Children with high HIV viremia had increased proportions of activated mature B cells, tissue-like memory B cells and plasmablasts, and low proportions of naive B cells when compared with community controls and children with low HIV viremia, similar to adults infected with HIV. HIV-infected groups had lower proportions of resting memory B cells than did community controls. Notably, high HIV viremia prevented the age-dependent accumulation of class-switched resting memory B cells. HIV-infected children, regardless of the level of viremia, showed lower quantities and avidities of IgG and lower frequencies of memory B cells against Expanded Program on Immunization vaccines. The HIV-infected children had an altered BAFF profile that could have affected their B cell compartment. Therefore, B cell defects in HIV-infected children are similar to those seen in HIV-infected adults. However, control of HIV viremia is associated with normalization of activated B cell subsets and allows age-dependent accumulation of resting memory B cells. PMID:26116511

  13. Bone morphogenetic protein 7 polarizes THP-1 cells into M2 macrophages.

    PubMed

    Rocher, Crystal; Singla, Reetu; Singal, Pawan K; Parthasarathy, Sampath; Singla, Dinender K

    2012-07-01

    It was hypothesized that monocyte treatment with bone morphogenetic protein 7 (BMP7) would significantly enhance monocyte polarization into M2 macrophages as well as increasing the levels of anti-inflammatory cytokines. In a cell culture system using monocytes (human acute monocytic leukemia cell line THP-1), we studied the effects of BMP7 on monocytes polarizing into M2 macrophages. The data demonstrate that THP-1 cells contain a BMP type II receptor (BMPR2), and that its activation is significantly (p < 0.05) increased following treatment with BMP7. Furthermore, there was an increase of M2 macrophages, BMPR2, and anti-inflammatory cytokines interleukin (IL)-10 and IL-1ra compared with the respective controls. Moreover, treatment with BMP7 caused a significant (p < 0.05) decrease in the levels of pro-inflammatory c