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Sample records for cells elicits distinct

  1. Different Vaccine Vectors Delivering the Same Antigen Elicit CD8plus T Cell Responses with Distinct Clonotype and Epitope Specificity

    Energy Technology Data Exchange (ETDEWEB)

    M Honda; R Wang; W Kong; M Kanekiyo; Q Akahata; L Xu; K Matsuo; K Natarajan; H Robinson; et al.

    2011-12-31

    Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8{sup +} cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8{sup +} T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2D{sup d} better than the native sequences, and clones with distinct specificities were elicited by alternative vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8{sup +} T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.

  2. Different Vaccine Vectors Delivering the Same Antigen Elicit CD8+ T Cell Responses with Distinct Clonotype and Epitope Specificity

    Energy Technology Data Exchange (ETDEWEB)

    Honda, M.; Robinson, H.; Wang, R.; Kong, W.-P.; Kanekiyo, M.; Akahata, W.; Xu, L.; Matsuo, K.; Natarajan, K.; Asher, T. E.; Price, D. A.; Douek, D. C.; Margulies, D. H.; Nabel, G. J.

    2009-08-15

    Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8{sup +} cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8{sup +} T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2D{sup d} better than the native sequences, and clones with distinct specificities were elicited by alternative vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8{sup +} T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.

  3. Distinct gut-derived lactic acid bacteria elicit divergent dendritic cell-mediated NK cell responses

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen; Zeuthen, Louise Hjerrild; Christensen, Hanne;

    2007-01-01

    Lactic acid bacteria (LAB) are abundant in the gastrointestinal tract where they continuously regulate the immune system. NK cells are potently activated by dendritic cells (DCs) matured by inflammatory stimuli, and NK cells are present in the gut epithelium and in mesenteric lymph nodes, but it is...

  4. IL-25 simultaneously elicits distinct populations of innate lymphoid cells and multipotent progenitor type 2 (MPPtype2) cells

    OpenAIRE

    Saenz, Steven A.; Siracusa, Mark C.; Monticelli, Laurel A.; Ziegler, Carly G. K.; Kim, Brian S.; Brestoff, Jonathan R.; Peterson, Lance W.; Wherry, E. John; Goldrath, Ananda W; Bhandoola, Avinash; Artis, David

    2013-01-01

    The predominantly epithelial cell–derived cytokines IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) can promote CD4+ Th2 cell–dependent immunity, inflammation, and tissue repair at barrier surfaces through the induction of multiple innate immune cell populations. IL-25 and IL-33 were previously shown to elicit four innate cell populations, named natural helper cells, nuocytes, innate type 2 helper cells, and multipotent progenitor type 2 (MPPtype2) cells, now collectively termed group 2...

  5. Carbon black and titanium dioxide nanoparticles elicit distinct apoptotic pathways in bronchial epithelial cells

    OpenAIRE

    Baeza-Squiban Armelle; Fleury Jocelyne; Martens Johan A; Andreau Karine; Borot Marie-Caroline; Ferecatu Ioana; Thomassen Leen CJ; Hussain Salik; Marano Francelyne; Boland Sonja

    2010-01-01

    Abstract Background Increasing environmental and occupational exposures to nanoparticles (NPs) warrant deeper insight into the toxicological mechanisms induced by these materials. The present study was designed to characterize the cell death induced by carbon black (CB) and titanium dioxide (TiO2) NPs in bronchial epithelial cells (16HBE14o- cell line and primary cells) and to investigate the implicated molecular pathways. Results Detailed time course studies revealed that both CB (13 nm) and...

  6. Adoptive transfer of Tc1 or Tc17 cells elicits antitumor immunity against established melanoma through distinct mechanisms.

    Science.gov (United States)

    Yu, Yu; Cho, Hyun-Ii; Wang, Dapeng; Kaosaard, Kane; Anasetti, Claudio; Celis, Esteban; Yu, Xue-Zhong

    2013-02-15

    Adoptive cell transfer (ACT) of ex vivo-activated autologous tumor-reactive T cells is currently one of the most promising approaches for cancer immunotherapy. Recent studies provided some evidence that IL-17-producing CD8(+) (Tc17) cells may exhibit potent antitumor activity, but the specific mechanisms have not been completely defined. In this study, we used a murine melanoma lung-metastasis model and tested the therapeutic effects of gp100-specific polarized type I CD8(+) cytotoxic T (Tc1) or Tc17 cells combined with autologous bone marrow transplantation after total body irradiation. Bone marrow transplantation combined with ACT of antitumor (gp100-specific) Tc17 cells significantly suppressed the growth of established melanoma, whereas Tc1 cells induced long-term tumor regression. After ACT, Tc1 cells maintained their phenotype to produce IFN-γ, but not IL-17. However, although Tc17 cells largely preserved their ability to produce IL-17, a subset secreted IFN-γ or both IFN-γ and IL-17, indicating the plasticity of Tc17 cells in vivo. Furthermore, after ACT, the Tc17 cells had a long-lived effector T cell phenotype (CD127(hi)/KLRG-1(low)) as compared with Tc1 cells. Mechanistically, Tc1 cells mediated antitumor immunity primarily through the direct effect of IFN-γ on tumor cells. In contrast, despite the fact that some Tc17 cells also secreted IFN-γ, Tc17-mediated antitumor immunity was independent of the direct effects of IFN-γ on the tumor. Nevertheless, IFN-γ played a critical role by creating a microenvironment that promoted Tc17-mediated antitumor activity. Taken together, these studies demonstrate that both Tc1 and Tc17 cells can mediate effective antitumor immunity through distinct effector mechanisms, but Tc1 cells are superior to Tc17 cells in mediating tumor regression. PMID:23315072

  7. Gustatory stimuli representing different perceptual qualities elicit distinct patterns of neuropeptide secretion from taste buds

    OpenAIRE

    Geraedts, Maartje C. P.; Munger, Steven D.

    2013-01-01

    Taste stimuli that evoke different perceptual qualities (e.g., sweet, umami, bitter, sour, salty) are detected by dedicated subpopulations of taste bud cells that employ distinct combinations of sensory receptors and transduction molecules. Here, we report that taste stimuli also elicit unique patterns of neuropeptide secretion from taste buds that are correlated with those perceptual qualities. We measured tastant-dependent secretion of glucagon-like peptide-1 (GLP-1), glucagon and neuropept...

  8. Adoptive transfer of Tc1 or Tc17 cells elicits anti-tumor immunity against established melanoma through distinct mechanisms1

    OpenAIRE

    Yu, Yu; Cho, Hyun-II; Wang, Dapeng; Kaosaard, Kane; Anasetti, Claudio; Celis, Esteban; Yu, Xue-Zhong

    2013-01-01

    Adoptive cell transfer (ACT) of ex vivo activated autologous tumor-reactive T cells is currently one of the most promising approaches for cancer immunotherapy. Recent studies provided some evidence that Th17/Tc17 cells may exhibit potent anti-tumor activity, but the specific mechanisms have not been completely defined. In the present study, we used a murine melanoma lung-metastasis model and tested the therapeutic effects of gp100-specific polarized Tc1 or Tc17 cells combined with autologous ...

  9. Cancer-induced immunosuppression: IL-18-elicited immunoablative NK cells.

    Science.gov (United States)

    Terme, Magali; Ullrich, Evelyn; Aymeric, Laetitia; Meinhardt, Kathrin; Coudert, Jérôme D; Desbois, Mélanie; Ghiringhelli, François; Viaud, Sophie; Ryffel, Bernard; Yagita, Hideo; Chen, Lieping; Mécheri, Salaheddine; Kaplanski, Gilles; Prévost-Blondel, Armelle; Kato, Masashi; Schultze, Joachim L; Tartour, Eric; Kroemer, Guido; Degli-Esposti, Mariapia; Chaput, Nathalie; Zitvogel, Laurence

    2012-06-01

    During cancer development, a number of regulatory cell subsets and immunosuppressive cytokines subvert adaptive immune responses. Although it has been shown that tumor-derived interleukin (IL)-18 participates in the PD-1-dependent tumor progression in NK cell-controlled cancers, the mechanistic cues underlying this immunosuppression remain unknown. Here, we show that IL-18 converts a subset of Kit(-) (CD11b(-)) into Kit(+) natural killer (NK) cells, which accumulate in all lymphoid organs of tumor bearers and mediate immunoablative functions. Kit(+) NK cells overexpressed B7-H1/PD-L1, a ligand for PD-1. The adoptive transfer of Kit(+) NK cells promoted tumor growth in two pulmonary metastases tumor models and significantly reduced the dendritic and NK cell pools residing in lymphoid organs in a B7-H1-dependent manner. Neutralization of IL-18 by RNA interference in tumors or systemically by IL-18-binding protein dramatically reduced the accumulation of Kit(+)CD11b(-) NK cells in tumor bearers. Together, our findings show that IL-18 produced by tumor cells elicits Kit(+)CD11b(-) NK cells endowed with B7-H1-dependent immunoablative functions in mice. PMID:22427351

  10. Distinct ensembles of medial prefrontal cortex neurons are activated by threatening stimuli that elicit excitation vs. inhibition of movement.

    Science.gov (United States)

    Halladay, Lindsay R; Blair, Hugh T

    2015-08-01

    Neural circuits controlling defensive behavior were investigated by recording single units in medial prefrontal cortex (mPFC) and dorsolateral periaqueductal gray (dlPAG) while rats expressed conditioned fear responses to an auditory conditioned stimulus (CS; 20-s train of white noise pips) previously paired with an aversive unconditioned stimulus (US; 2-s train of periorbital shocks). The CS elicited conditioned movement inhibition (CMI; characterized by decreased movement speed and freezing) when rats had not recently encountered the US, whereas the CS elicited conditioned movement excitation (CME; characterized by increased movement speed and flight behavior) after recent US encounters. Many mPFC neurons were "strategy-selective" cells that changed their firing rates only when the CS elicited CME (15/71) or CMI (13/71) responses, whereas few mPFC cells (4/71) responded nonselectively to the CS during either response. By contrast, many dlPAG neurons (20/74) responded nonselectively to the CS, but most (40/74) were excited by the CS selectively during CME trials (and none during CMI trials). CME-selective neurons in dlPAG responded phasically after CS pips that elicited CME responses, whereas CME-selective neurons in mPFC showed tonically elevated activity before and after pips that evoked CME responses. These findings suggest that, at the time when the CS occurs, tonic firing rates of CME- and CMI-selective mPFC neurons may bias the rat's choice of whether to express CME vs. CMI responses, perhaps via projections to downstream structures (such as amygdala and PAG) that influence how sensory stimuli are mapped onto motor circuits that drive the expression of competing behaviors. PMID:25972588

  11. Induction of phenolic compounds in Hypericum perforatum L. cells by colletotrichum gloeosporioides elicitation

    OpenAIRE

    Conceição, Luís F. R.; Ferreres, Federico; Tavares, R. M.; Dias, Alberto Carlos Pires

    2006-01-01

    Changes in phenolic metabolism after elicitation with Colletotrichum gloeosporioides (CG) has been studied in Hypericum perforatum L. (HP) cell suspension cultures. Soluble phenolics were analysed by HPLC–DAD and HPLC–DAD–MS/MS. HP cultures elicited with the CG elicitor showed a significant increase in xanthone accumulation. Xanthone accumulation increased twelve fold when the cells were primed with methyl-jasmonate (MeJ) or salicylic acid (SA), before elicitation. HP cultures exposed only to...

  12. Diverse environmental stresses elicit distinct responses at the level of pre-mRNA processing in yeast.

    Science.gov (United States)

    Bergkessel, Megan; Whitworth, Gregg B; Guthrie, Christine

    2011-08-01

    Gene expression in eukaryotic cells is profoundly influenced by the post-transcriptional processing of mRNAs, including the splicing of introns in the nucleus and both nuclear and cytoplasmic degradation pathways. These processes have the potential to affect both the steady-state levels and the kinetics of changes to levels of intron-containing transcripts. Here we report the use of a splicing isoform-specific microarray platform to investigate the effects of diverse stress conditions on pre-mRNA processing. Interestingly, we find that diverse stresses cause distinct patterns of changes at this level. The responses we observed are most dramatic for the RPGs and can be categorized into three major classes. The first is characterized by accumulation of RPG pre-mRNA and is seen in multiple types of amino acid starvation regimes; the magnitude of splicing inhibition correlates with the severity of the stress. The second class is characterized by a rapid decrease in both pre- and mature RPG mRNA and is seen in many stresses that inactivate the TORC1 kinase complex. These decreases depend on nuclear turnover of the intron-containing pre-RNAs. The third class is characterized by a decrease in RPG pre-mRNA, with only a modest reduction in the mature species; this response is observed in hyperosmotic and cation-toxic stresses. We show that casein kinase 2 (CK2) makes important contributions to the changes in pre-mRNA processing, particularly for the first two classes of stress responses. In total, our data suggest that complex post-transcriptional programs cooperate to fine-tune expression of intron-containing transcripts in budding yeast. PMID:21697354

  13. Elicitation Phenolic Compounds in Cell Culture of Vitis vinifera L. by Phaeomoniella chlamydospora

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    Sák Martin

    2014-12-01

    Full Text Available The in vitro cell cultures of Vitis vinifera L. cv. St. Laurent were treated with two elicitors - synthetic methyl jasmonate and natural, prepared from grapevine plant infected with the Phaeomoniella chlamydospora, the agent causing the Esca disease of grapevine. Efficiency of phenolic compounds production after elicitation of cell culture was analysed immediately after treatment (15 min, 30 min, 60 min and later (after 24, 48, and 72 hours. The cell growth and content of phenolic compounds (+-catechin, (--epicatechin, p-coumaric acid, syringaldehyde, rutin, vanillic acid, and trans-resveratrol were analysed in cultivated cells as well as in cultivation medium. Pch-treatment increased production of total polyphenols the most significantly 15 min after the elicitation and in optimal time was 2.86 times higher than in nonelicited culture and 1.44 times higher than in MeJa induced cell culture.

  14. A Major Cell Surface Antigen of Coccidioides immitis Which Elicits Both Humoral and Cellular Immune Responses

    OpenAIRE

    Hung, Chiung-Yu; Ampel, Neil M.; Christian, Lara; Seshan, Kalpathi R.; Cole, Garry T.

    2000-01-01

    Multinucleate parasitic cells (spherules) of Coccidioides immitis isolates produce a membranous outer wall component (SOW) in vitro which has been reported to be reactive with antibody from patients with coccidioidal infection, elicits a potent proliferative response of murine immune T cells, and has immunoprotective capacity in a murine model of coccidioidomycosis. To identify the antigenic components of SOW, the crude wall material was first subjected to Triton X-114 extraction, and a water...

  15. Stabilization of F-actin prevents cAMP-elicited Cl- secretion in T84 cells.

    OpenAIRE

    Shapiro, M.; Matthews, J.; Hecht, G; Delp, C; Madara, J. L.

    1991-01-01

    T84 cells, a human intestinal epithelial cell line, serve as a model of electrogenic Cl- secretion. We find that cAMP-elicited Cl- secretion in T84 cells is accompanied by a marked redistribution of F-actin in the basolateral portion of the cell. To prevent this F-actin redistribution and thereby assess its importance to Cl- secretion, we have defined simple conditions under which this model epithelium can be loaded with nitrobenzoxadiazole (NBD)-phallicidin. This reagent binds F-actin with h...

  16. Human islets contain four distinct subtypes of β cells.

    Science.gov (United States)

    Dorrell, Craig; Schug, Jonathan; Canaday, Pamela S; Russ, Holger A; Tarlow, Branden D; Grompe, Maria T; Horton, Tamara; Hebrok, Matthias; Streeter, Philip R; Kaestner, Klaus H; Grompe, Markus

    2016-01-01

    Human pancreatic islets of Langerhans contain five distinct endocrine cell types, each producing a characteristic hormone. The dysfunction or loss of the insulin-producing β cells causes diabetes mellitus, a disease that harms millions. Until now, β cells were generally regarded as a single, homogenous cell population. Here we identify four antigenically distinct subtypes of human β cells, which we refer to as β1-4, and which are distinguished by differential expression of ST8SIA1 and CD9. These subpopulations are always present in normal adult islets and have diverse gene expression profiles and distinct basal and glucose-stimulated insulin secretion. Importantly, the β cell subtype distribution is profoundly altered in type 2 diabetes. These data suggest that this antigenically defined β cell heterogeneity is functionally and likely medically relevant. PMID:27399229

  17. Proteins differentially expressed in elicited cell suspension culture of Podophyllum hexandrum with enhanced podophyllotoxin content

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    Bhattacharyya Dipto

    2012-05-01

    Full Text Available Abstract Background Podophyllotoxin (PTOX, the precursor for semi-synthesis of cancer therapeutics like etoposide, teniposide and etophos, is primarily obtained from an endangered medicinal herb, Podophyllum hexandrum Royle. PTOX, a lignan is biosynthetically derived from the phenylpropanoid pathway. The aim of this study is to investigate changes in the P. hexandrum cell proteome potentially related to PTOX accumulation in response to methyl jasmonate (MeJA elicitation. High-resolution two-dimensional gel electrophoresis (2-DE followed by colloidal Coomassie staining and mass spectrometric analysis was used to detect statistically significant changes in cell’s proteome. Result The HPLC analysis showed approximately 7–8 fold change in accumulation of PTOX, in the 12day old cell suspension culture (i.e. after 9days of elicitation elicited with 100 μM MeJA as compared to the control. Using 2-DE a total of 233 spots was detected, out of which 105 spots were identified by MALDI TOF-TOF MS/MS. Data were subjected to functional annotation from a biological point of view through KEGG. The phenylpropanoid and monolignol pathway enzymes were identified, amongst these, chalcone synthase, polyphenol oxidase, caffeoyl CoA 3-O-methyltransferase, S-adenosyl-L-methionine-dependent methyltransferases, caffeic acid-O-methyl transferase etc. are noted as important. The relation of other differentially accumulated proteins with varied effects caused by elicitors on P. hexandrum cells namely stress and defense related protein, transcription and DNA replication and signaling are also discussed. Conclusions Elicitor-induced PTOX accumulation in P. hexandrum cell cultures provides a responsive model system to profile modulations in proteins related to phenylpropanoid/monolignol biosynthesis and other defense responses. Present findings form a baseline for future investigation on a non-sequenced medicinal herb P. hexandrum at molecular level.

  18. Antibodies to Interleukin-2 Elicit Selective T Cell Subset Potentiation through Distinct Conformational Mechanisms

    Czech Academy of Sciences Publication Activity Database

    Spangler, J.; Tomala, Jakub; Luca, V.C.; Jude, K.M.; Dong, S.; Ring, A.M.; Votavová, Petra; Pepper, M.; Kovář, Marek; Garcia, K.Ch.

    2015-01-01

    Roč. 42, č. 5 (2015), s. 815-825. ISSN 1074-7613 R&D Projects: GA ČR GA13-12885S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : IN-VIVO EXPANSION * AUTOIMMUNE-DISEASE * SMALL-MOLECULE Subject RIV: EC - Immunology Impact factor: 21.561, year: 2014

  19. Purification and cDNA Cloning of Isochorismate Synthase from Elicited Cell Cultures of Catharanthus roseus

    Science.gov (United States)

    van Tegelen, Léon J.P.; Moreno, Paolo R.H.; Croes, Anton F.; Verpoorte, Robert; Wullems, George J.

    1999-01-01

    Isochorismate is an important metabolite formed at the end of the shikimate pathway, which is involved in the synthesis of both primary and secondary metabolites. It is synthesized from chorismate in a reaction catalyzed by the enzyme isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to homogeneity from elicited Catharanthus roseus cell cultures. Two isoforms with an apparent molecular mass of 64 kD were purified and characterized. The Km values for chorismate were 558 and 319 μm for isoforms I and II, respectively. The isoforms were not inhibited by aromatic amino acids and required Mg2+ for enzyme activity. Polymerase chain reaction on a cDNA library from elicited C. roseus cells with a degenerated primer based on the sequence of an internal peptide from isoform II resulted in an amplification product that was used to screen the cDNA library. This led to the first isolation, to our knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD with an N-terminal chloroplast-targeting signal. The deduced amino acid sequence shares homology with bacterial ICS and also with anthranilate synthases from plants. Southern analysis indicates the existence of only one ICS gene in C. roseus. PMID:9952467

  20. Magnaporthe oryzae-Secreted Protein MSP1 Induces Cell Death and Elicits Defense Responses in Rice.

    Science.gov (United States)

    Wang, Yiming; Wu, Jingni; Kim, Sang Gon; Tsuda, Kenichi; Gupta, Ravi; Park, Sook-Young; Kim, Sun Tae; Kang, Kyu Young

    2016-04-01

    The Magnaporthe oryzae snodprot1 homolog (MSP1), secreted by M. oryzae, is a cerato-platanin family protein. msp1-knockout mutants have reduced virulence on barley leaves, indicating that MSP1 is required for the pathogenicity of rice blast fungus. To investigate the functional roles of MSP1 and its downstream signaling in rice, recombinant MSP1 was produced in Escherichia coli and was assayed for its functionality. Application of MSP1 triggered cell death and elicited defense responses in rice. MSP1 also induced H2O2 production and autophagic cell death in both suspension-cultured cells and rice leaves. One or more protein kinases triggered cell death, jasmonic acid and abscisic acid enhanced cell death, while salicylic acid suppressed it. We demonstrated that the secretion of MSP1 into the apoplast is a prerequisite for triggering cell death and activating defense-related gene expression. Furthermore, pretreatment of rice with a sublethal MSP1 concentration potentiated resistance to the pathogen. Taken together, our results showed that MSP1 induces a high degree of cell death in plants, which might be essential for its virulence. Moreover, rice can recognize MSP1, resulting in the induction of pathogen-associated molecular pattern-triggered immunity. PMID:26780420

  1. Timing the generation of distinct retinal cells by homeobox proteins.

    Directory of Open Access Journals (Sweden)

    Sarah Decembrini

    2006-09-01

    Full Text Available The reason why different types of vertebrate nerve cells are generated in a particular sequence is still poorly understood. In the vertebrate retina, homeobox genes play a crucial role in establishing different cell identities. Here we provide evidence of a cellular clock that sequentially activates distinct homeobox genes in embryonic retinal cells, linking the identity of a retinal cell to its time of generation. By in situ expression analysis, we found that the three Xenopus homeobox genes Xotx5b, Xvsx1, and Xotx2 are initially transcribed but not translated in early retinal progenitors. Their translation requires cell cycle progression and is sequentially activated in photoreceptors (Xotx5b and bipolar cells (Xvsx1 and Xotx2. Furthermore, by in vivo lipofection of "sensors" in which green fluorescent protein translation is under control of the 3' untranslated region (UTR, we found that the 3' UTRs of Xotx5b, Xvsx1, and Xotx2 are sufficient to drive a spatiotemporal pattern of translation matching that of the corresponding proteins and consistent with the time of generation of photoreceptors (Xotx5b and bipolar cells (Xvsx1 and Xotx2. The block of cell cycle progression of single early retinal progenitors impairs their differentiation as photoreceptors and bipolar cells, but is rescued by the lipofection of Xotx5b and Xvsx1 coding sequences, respectively. This is the first evidence to our knowledge that vertebrate homeobox proteins can work as effectors of a cellular clock to establish distinct cell identities.

  2. Vaginal immunization to elicit primary T-cell activation and dissemination.

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    Elena Pettini

    Full Text Available Primary T-cell activation at mucosal sites is of utmost importance for the development of vaccination strategies. T-cell priming after vaginal immunization, with ovalbumin and CpG oligodeoxynucleotide adjuvant as model vaccine formulation, was studied in vivo in hormone-synchronized mice and compared to the one induced by the nasal route. Twenty-four hours after both vaginal or nasal immunization, antigen-loaded dendritic cells were detected within the respective draining lymph nodes. Vaginal immunization elicited a strong recruitment of antigen-specific CD4(+ T cells into draining lymph nodes that was more rapid than the one observed following nasal immunization. T-cell clonal expansion was first detected in iliac lymph nodes, draining the genital tract, and proliferated T cells disseminated towards distal lymph nodes and spleen similarly to what observed following nasal immunization. T cells were indeed activated by the antigen encounter and acquired homing molecules essential to disseminate towards distal lymphoid organs as confirmed by the modulation of CD45RB, CD69, CD44 and CD62L marker expression. A multi-type Galton Watson branching process, previously used for in vitro analysis of T-cell proliferation, was applied to model in vivo CFSE proliferation data in draining lymph nodes 57 hours following immunization, in order to calculate the probabilistic decision of a cell to enter in division, rest in quiescence or migrate/die. The modelling analysis indicated that the probability of a cell to proliferate was higher following vaginal than nasal immunization. All together these data show that vaginal immunization, despite the absence of an organized mucosal associated inductive site in the genital tract, is very efficient in priming antigen-specific CD4(+ T cells and inducing their dissemination from draining lymph nodes towards distal lymphoid organs.

  3. Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine.

    Science.gov (United States)

    Ramanathan, Babu; Poh, Chit Laa; Kirk, Kristin; McBride, William John Hannan; Aaskov, John; Grollo, Lara

    2016-01-01

    Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine. PMID:27223692

  4. Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine.

    Directory of Open Access Journals (Sweden)

    Babu Ramanathan

    Full Text Available Dengue virus (DENV is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine.

  5. Distinct T cell signatures define subsets of patients with multiple sclerosis

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    Johnson, Mark C.; Pierson, Emily R.; Spieker, Andrew J.; Nielsen, A. Scott; Posso, Sylvia; Kita, Mariko; Buckner, Jane H.

    2016-01-01

    Objective: We investigated T cell responses to myelin proteins in the blood of healthy controls and 2 groups of patients with relapsing-remitting multiple sclerosis (RRMS) who exhibited lesions either predominantly in the brain or predominantly in the spinal cord in order to assess whether distinct neuroinflammatory patterns were associated with different myelin protein–specific T cell effector function profiles and whether these profiles differed from healthy controls. Methods: Peripheral blood mononuclear cells were obtained from patients with brain-predominant RRMS, patients with spinal cord–predominant RRMS, and age-matched healthy controls and analyzed by enzyme-linked immunosorbent spot assays to quantify interferon gamma–secreting (Th1) and interleukin 17–secreting (Th17) cells responding directly ex vivo to myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG). Results: Although MBP and MOG elicited different responses, patients with multiple sclerosis (MS) who had spinal cord–predominant lesions exhibited significantly higher Th17:Th1 ratios in response to both MBP and MOG compared to patients with brain-predominant MS. Incorporating the cytokine responses to both antigens into logistic regression models showed that these cytokine responses were able to provide good discrimination between patients with distinct neuroinflammatory patterns. Conclusions: Our findings suggest that the localization of lesions within the brain vs the spinal cord in patients with MS is associated with different effector T cell responses to myelin proteins. Further investigation of the relationship between T cell effector function, antigen specificities, and lesion sites may reveal features of pathogenic pathways that are distinct to patients with different neuroinflammatory patterns.

  6. [Gastric signet ring cell adenocarcinoma: A distinct entity].

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    Tabouret, Tessa; Dhooge, Marion; Rouquette, Alexandre; Brezault, Catherine; Beuvon, Frédéric; Chaussade, Stanislas; Coriat, Romain

    2014-04-01

    Gastric signet ring cell carcinoma (GSRC) is a distinct entity. Their incidence is increasing. The pathologist plays a central role in the identification of this entity. Diagnosis is based on an adenocarcinoma containing a majority of signet ring cells (above 50 %). The prognosis of GSRC is the same as gastric adenocarcinoma while GSRC appeared more aggressive. Signet ring cells present a low sensitivity to chemotherapy. This review aimed to discuss the histological, the prognostic and the therapeutic aspect of this entity. PMID:24440764

  7. Influence of fungal elicitation on glycyrrhizin production in transformed cell cultures of Abrus precatorius Linn

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    Vijai Singh Karwasara

    2011-01-01

    Full Text Available Background: Glycyrrhizin, obtained from Abrus precatorius (Indian liquorice, is a phytoconstituent of importance for pharmaceutical and food industries. Materials and Methods: High producing and fast growing cell lines of A. precatorius were developed by transformation with Agrobacterium tumefaciens for glycyrrhizin production. Its maximum transformation efficiency of 85% was obtained by infecting leaves with A. tumefaciens MTCC-431 supplemented with 50 μM acetosyringone. Thorough culture growth kinetics with sugar consumption profiles was established. Results: A twofold increase in glycyrrhizin productivity was obtained in transformed A. precatorius cell suspension cultures over the untransformed cultures. The fungal elicitors prepared from Aspergillus niger and Rhizopus stolonifer were tested at different concentrations to enhance glycyrrhizin production in transformed cell suspension cultures of A. precatorius. Maximum enhancement of 4.9- and 3.8-fold in glycyrrhizin contents, were obtained with A. niger (7.5% v/v and R. stolonifer (5.0% v/v, respectively, on the 5th day after elicitor treatment. Conclusion: This study indicates the prospective of the amalgamation of elicitation methodology with transformed cell cultures for the large-scale production of glycyrrhizin.

  8. Modification of Plasma Membrane Organization in Tobacco Cells Elicited by Cryptogein1[W

    Science.gov (United States)

    Gerbeau-Pissot, Patricia; Der, Christophe; Thomas, Dominique; Anca, Iulia-Andra; Grosjean, Kevin; Roche, Yann; Perrier-Cornet, Jean-Marie; Mongrand, Sébastien; Simon-Plas, Françoise

    2014-01-01

    Lipid mixtures within artificial membranes undergo a separation into liquid-disordered and liquid-ordered phases. However, the existence of this segregation into microscopic liquid-ordered phases has been difficult to prove in living cells, and the precise organization of the plasma membrane into such phases has not been elucidated in plant cells. We developed a multispectral confocal microscopy approach to generate ratiometric images of the plasma membrane surface of Bright Yellow 2 tobacco (Nicotiana tabacum) suspension cells labeled with an environment sensitive fluorescent probe. This allowed the in vivo characterization of the global level of order of this membrane, by which we could demonstrate that an increase in its proportion of ordered phases transiently occurred in the early steps of the signaling triggered by cryptogein and flagellin, two elicitors of plant defense reactions. The use of fluorescence recovery after photobleaching revealed an increase in plasma membrane fluidity induced by cryptogein, but not by flagellin. Moreover, we characterized the spatial distribution of liquid-ordered phases on the membrane of living plant cells and monitored their variations induced by cryptogein elicitation. We analyze these results in the context of plant defense signaling, discuss their meaning within the framework of the “membrane raft” hypothesis, and propose a new mechanism of signaling platform formation in response to elicitor treatment. PMID:24235133

  9. Modification of plasma membrane organization in tobacco cells elicited by cryptogein.

    Science.gov (United States)

    Gerbeau-Pissot, Patricia; Der, Christophe; Thomas, Dominique; Anca, Iulia-Andra; Grosjean, Kevin; Roche, Yann; Perrier-Cornet, Jean-Marie; Mongrand, Sébastien; Simon-Plas, Françoise

    2014-01-01

    Lipid mixtures within artificial membranes undergo a separation into liquid-disordered and liquid-ordered phases. However, the existence of this segregation into microscopic liquid-ordered phases has been difficult to prove in living cells, and the precise organization of the plasma membrane into such phases has not been elucidated in plant cells. We developed a multispectral confocal microscopy approach to generate ratiometric images of the plasma membrane surface of Bright Yellow 2 tobacco (Nicotiana tabacum) suspension cells labeled with an environment sensitive fluorescent probe. This allowed the in vivo characterization of the global level of order of this membrane, by which we could demonstrate that an increase in its proportion of ordered phases transiently occurred in the early steps of the signaling triggered by cryptogein and flagellin, two elicitors of plant defense reactions. The use of fluorescence recovery after photobleaching revealed an increase in plasma membrane fluidity induced by cryptogein, but not by flagellin. Moreover, we characterized the spatial distribution of liquid-ordered phases on the membrane of living plant cells and monitored their variations induced by cryptogein elicitation. We analyze these results in the context of plant defense signaling, discuss their meaning within the framework of the "membrane raft" hypothesis, and propose a new mechanism of signaling platform formation in response to elicitor treatment. PMID:24235133

  10. Recombinant yellow fever viruses elicit CD8+ T cell responses and protective immunity against Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    Raquel Tayar Nogueira

    Full Text Available Chagas' disease is a major public health problem affecting nearly 10 million in Latin America. Despite several experimental vaccines have shown to be immunogenic and protective in mouse models, there is not a current vaccine being licensed for humans or in clinical trial against T. cruzi infection. Towards this goal, we used the backbone of Yellow Fever (YF 17D virus, one of the most effective and well-established human vaccines, to express an immunogenic fragment derived from T. cruzi Amastigote Surface Protein 2 (ASP-2. The cDNA sequence of an ASP-2 fragment was inserted between E and NS1 genes of YF 17D virus through the construction of a recombinant heterologous cassette. The replication ability and genetic stability of recombinant YF virus (YF17D/ENS1/Tc was confirmed for at least six passages in Vero cells. Immunogenicity studies showed that YF17D/ENS1/Tc virus elicited neutralizing antibodies and gamma interferon (IFN-γ producing-cells against the YF virus. Also, it was able to prime a CD8(+ T cell directed against the transgenic T. cruzi epitope (TEWETGQI which expanded significantly as measured by T cell-specific production of IFN-γ before and after T. cruzi challenge. However, most important for the purposes of vaccine development was the fact that a more efficient protective response could be seen in mice challenged after vaccination with the YF viral formulation consisting of YF17D/ENS1/Tc and a YF17D recombinant virus expressing the TEWETGQI epitope at the NS2B-3 junction. The superior protective immunity observed might be due to an earlier priming of epitope-specific IFN-γ-producing T CD8(+ cells induced by vaccination with this viral formulation. Our results suggest that the use of viral formulations consisting of a mixture of recombinant YF 17D viruses may be a promising strategy to elicit protective immune responses against pathogens, in general.

  11. Distinct differentiation characteristics of individual human embryonic stem cell lines

    Directory of Open Access Journals (Sweden)

    Knuutila Sakari

    2006-08-01

    Full Text Available Abstract Background Individual differences between human embryonic stem cell (hESC lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61 from frozen-thawed human embryos and compare their individual differentiation characteristic. Results The cell lines were cultured either on human or mouse feeder cells. The cells grew significantly faster and could be passaged enzymatically only on mouse feeders. However, this was found to lead to chromosomal instability after prolonged culture. All hESC lines expressed the established markers of pluripotent cells as well as several primordial germ cell (PGC marker genes in a uniform manner. However, the cell lines showed distinct features in their spontaneous differentiation patterns. The embryoid body (EB formation frequency of FES 30 cell line was significantly lower than that of other lines and cells within the EBs differentiated less readily. Likewise, teratomas derived from FES 30 cells were constantly cystic and showed only minor solid tissue formation with a monotonous differentiation pattern as compared with the other lines. Conclusion hESC lines may differ substantially in their differentiation properties although they appear similar in the undifferentiated state.

  12. L-Amino Acids Elicit Diverse Response Patterns in Taste Sensory Cells: A Role for Multiple Receptors.

    Directory of Open Access Journals (Sweden)

    Shreoshi Pal Choudhuri

    Full Text Available Umami, the fifth basic taste, is elicited by the L-amino acid, glutamate. A unique characteristic of umami taste is the response potentiation by 5' ribonucleotide monophosphates, which are also capable of eliciting an umami taste. Initial reports using human embryonic kidney (HEK cells suggested that there is one broadly tuned receptor heterodimer, T1r1+T1r3, which detects L-glutamate and all other L-amino acids. However, there is growing evidence that multiple receptors detect glutamate in the oral cavity. While much is understood about glutamate transduction, the mechanisms for detecting the tastes of other L-amino acids are less well understood. We used calcium imaging of isolated taste sensory cells and taste cell clusters from the circumvallate and foliate papillae of C57BL/6J and T1r3 knockout mice to determine if other receptors might also be involved in detection of L-amino acids. Ratiometric imaging with Fura-2 was used to study calcium responses to monopotassium L-glutamate, L-serine, L-arginine, and L-glutamine, with and without inosine 5' monophosphate (IMP. The results of these experiments showed that the response patterns elicited by L-amino acids varied significantly across taste sensory cells. L-amino acids other than glutamate also elicited synergistic responses in a subset of taste sensory cells. Along with its role in synergism, IMP alone elicited a response in a large number of taste sensory cells. Our data indicate that synergistic and non-synergistic responses to L-amino acids and IMP are mediated by multiple receptors or possibly a receptor complex.

  13. Vibrio cholerae GbpA elicits necrotic cell death in intestinal cells.

    Science.gov (United States)

    Mandal, Sudipto; Chatterjee, Nabendu Sekhar

    2016-08-01

    Vibrio choleraeN-acetylglucosamine-binding protein GbpA is a secretory protein that facilitates the initial adherence of bacteria in the human intestine. Until now, considerable progress in the characterization of GbpA has been done, yet little is known about its role in host response. Our present studies demonstrated that GbpA at the amount secreted in the intestine resulted in decreased cell viability, altered cell morphology, disruption of cell membrane integrity and damage of cellular DNA indicating necrotic cell death. We observed that GbpA exposure leads to mitochondrial dysfunction, characterized by accumulation of reactive oxygen species (ROS), depolarization of mitochondrial membrane potential and depletion of ATP pool in host cells. Additionally, the intra-cellular ROS, accumulated in response to GbpA, were found to induce the migration of NF-κB from cytoplasm into nucleus in host cells. Taken together, these results prompted us to conclude that GbpA orchestrates a necrotic response in host cells which may have implications in immune response. PMID:27324251

  14. Frequency locking in hair cells: Distinguishing between distinct resonant mechanisms

    CERN Document Server

    Edri, Yuval; Yochelis, Arik

    2016-01-01

    The auditory system displays remarkable mechanical sensitivity and frequency discrimination. These attributes have been shown to rely on an amplification process, which requires biochemical feedback loops. In some systems, the active process was shown to lead to spontaneous oscillations of hair cell bundles. In the last decade, models that display proximity to an oscillatory onset (a.k.a. Hopf bifurcation) have gained increasing support due to many advantages in explaining the hearing phenomenology. Particularly, they exhibit resonant responses to distinct frequencies of incoming sound waves. Unlike previous studies, two types of driving forces are being examined: additive, in which the external forcing term does not couple directly on the systems observable (passive coupling), and parametric, in which the forcing term directly affects the observable and thus intrinsically modifies the systems properties (active coupling). By applying universal principles near the Hopf bifurcation onset, we find several funda...

  15. RMP Plays Distinct Roles in the Proliferation of Hepatocellular Carcinoma Cells and Normal Hepatic Cells

    OpenAIRE

    Yang, Sijun; Wang, Hongmin; Guo, Yunlan; Chen, Shaomu; Zhang, Mei-Yin; Shen, Jian; Yu, Huijun; Miao, Jingcheng; Wang, Hui-Yun; Wei, Wenxiang

    2013-01-01

    RMP has been shown to function in the transcription regulation through association with RNA polymerase (RNAP) II subunit RPB5. It also has been shown to be required for the proliferation of hepatocellular carcinoma (HCC) cells with an antiapoptotic property. In this article, we further demonstrate that RMP displays distinct features in HCC cells compared with normal hepatic cells. RMP expression is remarkably increased in various cancer cell lines including HCC cells when compared with normal...

  16. RMP Plays Distinct Roles in the Proliferation of Hepatocellular Carcinoma Cells and Normal Hepatic Cells

    Science.gov (United States)

    Yang, Sijun; Wang, Hongmin; Guo, Yunlan; Chen, Shaomu; Zhang, Mei-Yin; Shen, Jian; Yu, Huijun; Miao, Jingcheng; Wang, Hui-Yun; Wei, Wenxiang

    2013-01-01

    RMP has been shown to function in the transcription regulation through association with RNA polymerase (RNAP) II subunit RPB5. It also has been shown to be required for the proliferation of hepatocellular carcinoma (HCC) cells with an antiapoptotic property. In this article, we further demonstrate that RMP displays distinct features in HCC cells compared with normal hepatic cells. RMP expression is remarkably increased in various cancer cell lines including HCC cells when compared with normal cells. Depletion of RMP could inhibit the proliferation of HCC cells, but not the normal hepatic cells. RMP significantly prevented apoptosis of HCC cells in SMMC-7721 and HepG2, but had little effect on apoptosis in the normal hepatic cells. The mechanisms of RMP's distinct features rely on different responsive expressions of apoptosis factors induced by RMP in HCC and hepatic cells. Either overexpression or depletion of RMP significantly affected the expression of apoptosis factors in HCC cells. However, normal hepatic cells showed a tendency to resist RMP for the regulation of apoptosis. In the clinical samples, the increased expression of RMP in HCCs was also observed when compared with the matched non-tumor tissues from 30 HCC patients. The different expression levels of and distinct responses to RMP between HCC and hepatic cells suggest that RMP might serve as not only a biomarker for the diagnosis of HCC, but also a potential target for the HCC therapy. PMID:23847445

  17. Adenovirus vectors targeting distinct cell types in the retina.

    Science.gov (United States)

    Sweigard, J Harry; Cashman, Siobhan M; Kumar-Singh, Rajendra

    2010-04-01

    Purpose. Gene therapy for a number of retinal diseases necessitates efficient transduction of photoreceptor cells. Whereas adenovirus (Ad) serotype 5 (Ad5) does not transduce photoreceptors efficiently, previous studies have demonstrated improved photoreceptor transduction by Ad5 pseudotyped with Ad35 (Ad5/F35) or Ad37 (Ad5/F37) fiber or by the deletion of the RGD domain in the Ad5 penton base (Ad5DeltaRGD). However, each of these constructs contained a different transgene cassette, preventing the evaluation of the relative performance of these vectors, an important consideration before the use of these vectors in the clinic. The aim of this study was to evaluate these vectors in the retina and to attempt photoreceptor-specific transgene expression. Methods. Three Ad5-based vectors containing the same expression cassette were generated and injected into the subretinal space of adult mice. Eyes were analyzed for green fluorescence protein expression in flat-mounts, cross-sections, quantitative RT-PCR, and a modified stereological technique. A 257-bp fragment derived from the mouse opsin promoter was analyzed in the context of photoreceptor-specific transgene expression. Results. Each virus tested efficiently transduced the retinal pigment epithelium. The authors found no evidence that Ad5/F35 or Ad5/F37 transduced photoreceptors. Instead, they found that Ad5/F37 transduced Müller cells. Robust photoreceptor transduction by Ad5DeltaRGD was detected. Photoreceptor-specific transgene expression from the 257-bp mouse opsin promoter in the context of Ad5DeltaRGD vectors was found. Conclusions. Adenovirus vectors may be designed with tropism to distinct cell populations. Robust photoreceptor-specific transgene expression can be achieved in the context of Ad5DeltaRGD vectors. PMID:19892875

  18. Interdisciplinary Evaluation of Broadly-Reactive HLA Class II Restricted Epitopes Eliciting HIV-Specific CD4+T Cell Responses

    DEFF Research Database (Denmark)

    Buggert, M.; Norström, M.; Lundegaard, Claus; Lund, Ole; Nielsen, Morten; Karlsson, A. C.

    2011-01-01

    Background: CD4+ T cells orchestrate immune protection by ‘‘helping’’ other cells of our immune system to clear viral infections. It is well known that the preferential infection and depletion of CD4+ T cells contributes to hampered systemic T cell help following HIV infection. However, the...... functional and immunodominant discrepancies of CD4+ T cell responses targeting promiscuous MHC II restricted HIV epitopes remains poorly defined. Thus, utilization of interdisciplinary approaches might aid revealing broadly- reactive peptides eliciting CD4 + T cell responses. Methods: We utilized the novel...... bioinformatic prediction program NetMHCIIpan to select 64 optimized MHC II restricted epitopes located in the HIV Gag, Pol, Env, Nef and Tat regions. The epitopes were selected to cover the global diversity of the virus (multiple subtypes) and the human immune system(diverse MHC II types). Optimized...

  19. TCEB1-mutated Renal Cell Carcinoma: A Distinct Genomic and Morphologic Subtype

    OpenAIRE

    Hakimi, A. Ari; Tickoo, Satish K; Jacobsen, Anders; Sarungbam, Judy; Sfakianos, John P.; Sato, Yusuke; Morikawa, Teppei; Kume, Haruki; Fukayama, Masashi; Homma, Yukio; Chen, Ying-bei; Sankin, Alexander; Mano, Roy; Coleman, Jonathan A.; Russo, Paul

    2015-01-01

    Integrated sequencing analysis identified a group of tumors among clear cell renal cell carcinomas characterized by hotspot mutations in TCEB1 (a gene that contributes to the VHL complex to ubiquitinate hypoxia inducible factor). We analyzed 11 tumors from two distinct cohorts with TCEB1 mutations along with an expanded cohort to assess whether these should be considered an entity distinct from clear cell renal cell carcinoma and clear cell papillary renal cell carcinoma. All tumors were char...

  20. A Distinct Lung-Interstitium-Resident Memory CD8+ T Cell Subset Confers Enhanced Protection to Lower Respiratory Tract Infection

    Directory of Open Access Journals (Sweden)

    Pavlo Gilchuk

    2016-08-01

    Full Text Available The nature and anatomic location of the protective memory CD8+ T cell subset induced by intranasal vaccination remain poorly understood. We developed a vaccination model to assess the anatomic location of protective memory CD8+ T cells and their role in lower airway infections. Memory CD8+ T cells elicited by local intranasal, but not systemic, vaccination with an engineered non-replicative CD8+ T cell-targeted antigen confer enhanced protection to a lethal respiratory viral challenge. This protection depends on a distinct CXCR3LO resident memory CD8+ T (Trm cell population that preferentially localizes to the pulmonary interstitium. Because they are positioned close to the mucosa, where infection occurs, interstitial Trm cells act before inflammation can recruit circulating memory CD8+ T cells into the lung tissue. This results in a local protective immune response as early as 1 day post-infection. Hence, vaccine strategies that induce lung interstitial Trm cells may confer better protection against respiratory pathogens.

  1. Cancer Stem Cells: Distinct Entities or Dynamically Regulated Phenotypes?

    OpenAIRE

    Li, Yunqing; Laterra, John

    2012-01-01

    The origins of tumor propagating neoplastic stem-like cells (cancer stem cells, CSCs) and their relationship to the bulk population of tumor cells that lack stem-like tumor-propagating features(i.e. transit-amplifying cancer progenitor cells) remain unclear. Recent findings from multiple laboratories show that cancer progenitor cells have the capacity to dedifferentiate and acquire a stem-like phenotype in response to either genetic manipulation or environmental cues. These findings suggest t...

  2. CD4+CD45RBHi Interleukin-4 Defective T Cells Elicit Antral Gastritis and Duodenitis

    OpenAIRE

    Dohi, Taeko; Fujihashi, Kohtaro; Koga, Toshiya; Etani, Yuri; Yoshino, Naoto; Kawamura, Yuki I.; Mcghee, Jerry R

    2004-01-01

    We have analyzed the gastrointestinal inflammation which develops following adoptive transfer of IL-4 gene knockout (IL-4−/−) CD4+CD45RBHi (RBHi) T cells to severe combined immunodeficient (SCID) or to T cell-deficient, T cell receptor β and δ double knockout (TCR−/−) mice. Transfer of IL-4−/− RBHi T cells induced a similar type of colitis to that seen in SCID or TCR−/− recipients of wild-type (wt) RBHi T cells as reported previously. Interestingly, transfer of both wt and IL-4−/− RBHi T cell...

  3. The ethanol response gene Cab45 can modulate the impairment elicited by ethanol and ultraviolet in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Yunfeng Zhu; Quanli Wang; Wangru Xu; Sha Li

    2008-01-01

    High consumption of ethanolic beverages facilitates neurodegeneration,but the mechanism of this process still remained elusive.Suppression subtractive hybridization (SSH) is a technique for detection of rare transcripts.With SSH approach,we identified one ethanol response gene Cab45,which was down-regulated by ethanol with time-dependent manner in B104 cells.The full-length sequence of Cab45 gene was obtained by 5'-RACE (5'Rapid Amplification of cDNA Ends) for the first time in rat.Based on the sequence of deduced amino acid of rat Cab45,the alignment was conducted with its counterparts in different species and displayed a high conservation.Using different tissues in rat and cell lines,Cab45 was characterized by a ubiquitous expression and differentiation dependent down-regulation.Given that ethanol facilitates some cell differentiation,we hypothesize that Cab45 is involved in ethanol-mediated differentiation.With transient transfection,the function of Cab45 was investigated by up-regulation and down-regulation in PC12 cells.Ethanol treatment and UV exposure were conducted subsequently and cell proliferations were detected by MTT (Methyl Thiazolyl Tetrazolium) approach.It revealed that the up-regulation of Cab45 modulated the impairment elicited by ethanol and UV in transfected cells.As a member of new calcium binding protein family,the exact role of Cab45 still remains unclear.

  4. IL25 elicits a multipotent progenitor cell population that promotes TH2 cytokine responses

    Science.gov (United States)

    CD4+ T helper 2 (TH2) cells secrete interleukin (IL)4, IL5 and IL13, and are required for immunity to gastrointestinal helminth infections. However, TH2 cells also promote chronic inflammation associated with asthma and allergic disorders. The non-haematopoietic-cell-derived cytokines thymic stromal...

  5. Renshaw cells are inactive during motor inhibition elicited by the pontine microinjection of carbachol.

    Science.gov (United States)

    Morales, F R; Engelhardt, J K; Pereda, A E; Yamuy, J; Chase, M H

    1988-04-12

    The present study was undertaken to determine whether the postsynaptic inhibition of motoneurons that occurs following the pontine microinjection of carbachol in the decerebrate cat is due to the activity of Renshaw cells. Thirty-two out of 37 Renshaw cells (86%) were spontaneously active prior to the administration of carbachol, whereas only 2 out of 13 Renshaw cells (15%) discharged during carbachol-induced motor inhibition. In addition, discrete inhibitory synaptic potentials were observed in 33% of the Renshaw cells from which intracellular recordings were obtained after carbachol administration, indicating that these cells were actively inhibited. The finding that a population of Renshaw cells, which inhibit motoneurons, were themselves inhibited during a period of profound motoneuron inhibition was quite unexpected. These results support the conclusion that Renshaw cells are not the inhibitory interneurons that are responsible for the powerful inhibition of motoneurons that occurs following the pontine microinjection of carbachol. PMID:3380320

  6. Distinct metabolic responses of an ovarian cancer stem cell line

    OpenAIRE

    Kathleen A Vermeersch; Wang, Lijuan; McDonald, John F; Styczynski, Mark P.

    2014-01-01

    Background Cancer metabolism is emerging as an important focus area in cancer research. However, the in vitro cell culture conditions under which much cellular metabolism research is performed differ drastically from in vivo tumor conditions, which are characterized by variations in the levels of oxygen, nutrients like glucose, and other molecules like chemotherapeutics. Moreover, it is important to know how the diverse cell types in a tumor, including cancer stem cells that are believed to b...

  7. Characteristics of memory B cells elicited by a highly efficacious HPV vaccine in subjects with no pre-existing immunity.

    Science.gov (United States)

    Scherer, Erin M; Smith, Robin A; Simonich, Cassandra A; Niyonzima, Nixon; Carter, Joseph J; Galloway, Denise A

    2014-10-01

    Licensed human papillomavirus (HPV) vaccines provide near complete protection against the types of HPV that most commonly cause anogenital and oropharyngeal cancers (HPV 16 and 18) when administered to individuals naive to these types. These vaccines, like most other prophylactic vaccines, appear to protect by generating antibodies. However, almost nothing is known about the immunological memory that forms following HPV vaccination, which is required for long-term immunity. Here, we have identified and isolated HPV 16-specific memory B cells from female adolescents and young women who received the quadrivalent HPV vaccine in the absence of pre-existing immunity, using fluorescently conjugated HPV 16 pseudoviruses to label antigen receptors on the surface of memory B cells. Antibodies cloned and expressed from these singly sorted HPV 16-pseudovirus labeled memory B cells were predominantly IgG (>IgA>IgM), utilized diverse variable genes, and potently neutralized HPV 16 pseudoviruses in vitro despite possessing only average levels of somatic mutation. These findings suggest that the quadrivalent HPV vaccine provides an excellent model for studying the development of B cell memory; and, in the context of what is known about memory B cells elicited by influenza vaccination/infection, HIV-1 infection, or tetanus toxoid vaccination, indicates that extensive somatic hypermutation is not required to achieve potent vaccine-specific neutralizing antibody responses. PMID:25330199

  8. Mechanical stress is communicated between different cell types to elicit matrix remodeling

    OpenAIRE

    Swartz, M. A.; Tschumperlin, D. J.; Kamm, R.D.; Drazen, J M

    2001-01-01

    Tissue remodeling often reflects alterations in local mechanical conditions and manifests as an integrated response among the different cell types that share, and thus cooperatively manage, an extracellular matrix. Here we examine how two different cell types, one that undergoes the stress and the other that primarily remodels the matrix, might communicate a mechanical stress by using airway cells as a representative in vitro system. Normal stress is imposed on bro...

  9. 7α-Hydroxycholesterol Elicits TLR6-Mediated Expression of IL-23 in Monocytic Cells

    OpenAIRE

    Seo, Hyun Chul; Kim, Sun-Mi; Eo, Seong-Kug; Rhim, Byung-Yong; KIM, Koanhoi

    2015-01-01

    We investigated the question of whether 7-oxygenated cholesterol derivatives could affect inflammatory and/or immune responses in atherosclerosis by examining their effects on expression of IL-23 in monocytic cells. 7α-Hydroxycholesterol (7αOHChol) induced transcription of the TLR6 gene and elevated the level of cell surface TLR6 protein in THP-1 monocytic cells. Addition of an agonist of TLR6, FSL-1, to TLR6-expressing cells by treatment with 7αOHChol resulted in enhanced production of IL-23...

  10. Enhancement of vindoline and vinblastine production in suspension-cultured cells of Catharanthus roseus by artemisinic acid elicitation.

    Science.gov (United States)

    Liu, Jinwei; Zhu, Jianhua; Tang, Le; Wen, Wei; Lv, Shuangshuang; Yu, Rongmin

    2014-01-01

    Elicitation is an important strategy to improve production of secondary metabolites in vitro. Artemisinic acid was studied as a novel elicitor to enhance the yield of terpenoid indole alkaloids in the present paper. Our results demonstrated that the concentrations of vindoline and vinblastine were increased by sixfold and twofold, respectively, compared to those of the control group after treatment with artemisinic acid. To elucidate the underlying mechanism, we investigated the gene expression of four enzymes involved in the biosynthetic pathway of vinblastine in the suspension-cultured cells of Catharanthu sroseus. RT-PCR experiment showed that artemisinic acid was able to up-regulate the transcriptions of tryptophan decarboxylase, geraniol 10-hydroxylase, tabersonine 16-hydroxylase and deacetoxyvindoline 4-hydroxylase. PMID:23864440

  11. Netrin-1 Augments Chemokinesis in CD4+ T Cells In Vitro and Elicits a Proinflammatory Response In Vivo.

    Science.gov (United States)

    Boneschansker, Leo; Nakayama, Hironao; Eisenga, Michele; Wedel, Johannes; Klagsbrun, Michael; Irimia, Daniel; Briscoe, David M

    2016-08-15

    Netrin-1 is a neuronal guidance cue that regulates cellular activation, migration, and cytoskeleton rearrangement in multiple cell types. It is a chemotropic protein that is expressed within tissues and elicits both attractive and repulsive migratory responses. Netrin-1 has recently been found to modulate the immune response via the inhibition of neutrophil and macrophage migration. However, the ability of Netrin-1 to interact with lymphocytes and its in-depth effects on leukocyte migration are poorly understood. In this study, we profiled the mRNA and protein expression of known Netrin-1 receptors on human CD4(+) T cells. Neogenin, uncoordinated-5 (UNC5)A, and UNC5B were expressed at low levels in unstimulated cells, but they increased following mitogen-dependent activation. By immunofluorescence, we observed a cytoplasmic staining pattern of neogenin and UNC5A/B that also increased following activation. Using a novel microfluidic assay, we found that Netrin-1 stimulated bidirectional migration and enhanced the size of migratory subpopulations of mitogen-activated CD4(+) T cells, but it had no demonstrable effects on the migration of purified CD4(+)CD25(+)CD127(dim) T regulatory cells. Furthermore, using a short hairpin RNA knockdown approach, we observed that the promigratory effects of Netrin-1 on T effectors is dependent on its interactions with neogenin. In the humanized SCID mouse, local injection of Netrin-1 into skin enhanced inflammation and the number of neogenin-expressing CD3(+) T cell infiltrates. Neogenin was also observed on CD3(+) T cell infiltrates within human cardiac allograft biopsies with evidence of rejection. Collectively, our findings demonstrate that Netrin-1/neogenin interactions augment CD4(+) T cell chemokinesis and promote cellular infiltration in association with acute inflammation in vivo. PMID:27430720

  12. Dibenzanthracenes and benzochrysenes elicit both genotoxic and nongenotoxic events in rat liver 'stem-like' cells

    International Nuclear Information System (INIS)

    Polycyclic aromatic hydrocarbons (PAHs) with molecular weight 278 are a group of PAHs that are mostly not covered by the current monitoring programs, despite their relative abundance in environmental samples and possible carcinogenicity. Although benzo[g]chrysene (BgChry) and dibenz[a,h]anthracene (DBahA) have been for a long time studied as genotoxic, tumour-initiating compounds, little is known about the potential tumour-promoting effects of this group of PAHs. In the present study, we investigated their impact on activation of the aryl hydrocarbon receptor (AhR), induction of enzymes involved in metabolic activation of PAHs, disruption of cell cycle control in confluent cell population and inhibition of gap junctional intercellular communication (GJIC), using the rat liver epithelial cell line WB-F344 as a model of liver progenitor cells. We found that BgChry was the weakest inducer of the AhR-mediated activity, while relative potencies of benzo[b]chrysene (BbChry) and benzo[c]chrysene (BcChry) were comparable to the previously reported values for dibenzanthracenes. All compounds increased expression of cytochromes P450 1A1 and 1B1, and aldo-keto reductase 1C9. BgChry was found to induce high amounts of DNA adducts, which corresponded with induction of p53 phosphorylation at Ser15, apoptosis and accumulation of cells in S-phase of cell cycle, leading to a decrease in cell numbers. All other compounds were found to stimulate cell proliferation in contact-inhibited WB-F344 cells in a dose-dependent manner. We found that only BgChry, and to a lesser extent also BcChry, inhibited GJIC at high concentrations. Taken together, dibenzanthracenes and benzochrysenes, with exception of BgChry, seem to act primarily through deregulation of cell proliferation in liver epithelial cells, which is related to their relatively high AhR-mediated activity. The disruption of cell cycle control might contribute to their carcinogenic effects, as well as to carcinogenicity of complex

  13. Functional characteristics of neonatal rat β cells with distinct markers

    DEFF Research Database (Denmark)

    Martens, G A; Motté, E; Kramer, G; Stangé, G; Gaarn, L W; Hellemans, K; Nielsen, Jens Høiriis; Aerts, J M; Ling, Z; Pipeleers, D

    2014-01-01

    Neonatal β cells are considered developmentally immature and hence less glucose responsive. To study the acquisition of mature glucose responsiveness, we compared glucose-regulated redox state, insulin synthesis, and secretion of β cells purified from neonatal or 10-week-old rats with their...... had twofold lower expression of malate/aspartate-NADH shuttle and most glycolytic enzymes. Genome-wide profiling situated neonatal β cells at a developmental crossroad: they showed advanced endocrine differentiation when specifically analyzed for their mRNA/protein level of classical neuroendocrine...... high DLK1, but no association was found. In conclusion, the current study supports the importance of glycolytic NADH-shuttling in stimulus function coupling, presents basal hyperactivity as novel property of neonatal β cells, and provides potential markers to recognize intercellular developmental...

  14. Cellular memory of hypoxia elicits neuroblastoma metastasis and enables invasion by non-aggressive neighbouring cells.

    Science.gov (United States)

    Herrmann, A; Rice, M; Lévy, R; Pizer, B L; Losty, P D; Moss, D; Sée, V

    2015-01-01

    Therapies targeting cancer metastasis are challenging owing to the complexity of the metastatic process and the high number of effectors involved. Although tumour hypoxia has previously been associated with increased aggressiveness as well as resistance to radio- and chemotherapy, the understanding of a direct link between the level and duration of hypoxia and the individual steps involved in metastasis is still missing. Using live imaging in a chick embryo model, we have demonstrated that the exposure of neuroblastoma cells to 1% oxygen for 3 days was capable of (1) enabling cell migration towards blood vessels, (2) slowing down their velocity within blood vessels to facilitate extravasation and (3) promoting cell proliferation in primary and secondary sites. We have shown that cells do not have to be hypoxic anymore to exhibit these acquired capabilities as a long-term memory of prior hypoxic exposure is kept. Furthermore, non-hypoxic cells can be influenced by neighbouring hypoxic preconditioned cells and be entrained in the metastatic progression. The acquired aggressive phenotype relies on hypoxia-inducible factor (HIF)-dependent transcription of a number of genes involved in metastasis and can be impaired by HIF inhibition. Altogether, our results demonstrate the need to consider both temporal and spatial tumour heterogeneity because cells can 'remember' an earlier environment and share their acquired phenotype with their close neighbours. As a consequence, it is necessary to monitor the correct hypoxic markers to be able to predict the consequences of the cells' history on their behaviour and their potential response to therapies. PMID:25664931

  15. Human cytomegalovirus latency-associated proteins elicit immune-suppressive IL-10 producing CD4⁺ T cells.

    Directory of Open Access Journals (Sweden)

    Gavin M Mason

    Full Text Available Human cytomegalovirus (HCMV is a widely prevalent human herpesvirus, which, after primary infection, persists in the host for life. In healthy individuals, the virus is well controlled by the HCMV-specific T cell response. A key feature of this persistence, in the face of a normally robust host immune response, is the establishment of viral latency. In contrast to lytic infection, which is characterised by extensive viral gene expression and virus production, long-term latency in cells of the myeloid lineage is characterised by highly restricted expression of viral genes, including UL138 and LUNA. Here we report that both UL138 and LUNA-specific T cells were detectable directly ex vivo in healthy HCMV seropositive subjects and that this response is principally CD4⁺ T cell mediated. These UL138-specific CD4⁺ T cells are able to mediate MHC class II restricted cytotoxicity and, importantly, show IFNγ effector function in the context of both lytic and latent infection. Furthermore, in contrast to CDCD4⁺ T cells specific to antigens expressed solely during lytic infection, both the UL138 and LUNA-specific CD4⁺ T cell responses included CD4⁺ T cells that secreted the immunosuppressive cytokine cIL-10. We also show that cIL-10 expressing CD4⁺ T-cells are directed against latently expressed US28 and UL111A. Taken together, our data show that latency-associated gene products of HCMV generate CD4⁺ T cell responses in vivo, which are able to elicit effector function in response to both lytic and latently infected cells. Importantly and in contrast to CD4⁺ T cell populations, which recognise antigens solely expressed during lytic infection, include a subset of cells that secrete the immunosuppressive cytokine cIL-10. This suggests that HCMV skews the T cell responses to latency-associated antigens to one that is overall suppressive in order to sustain latent carriage in vivo.

  16. Epigenetic alterations differ in phenotypically distinct human neuroblastoma cell lines

    International Nuclear Information System (INIS)

    Epigenetic aberrations and a CpG island methylator phenotype have been shown to be associated with poor outcomes in children with neuroblastoma (NB). Seven cancer related genes (THBS-1, CASP8, HIN-1, TIG-1, BLU, SPARC, and HIC-1) that have been shown to have epigenetic changes in adult cancers and play important roles in the regulation of angiogenesis, tumor growth, and apoptosis were analyzed to investigate the role epigenetic alterations play in determining NB phenotype. Two NB cell lines (tumorigenic LA1-55n and non-tumorigenic LA1-5s) that differ in their ability to form colonies in soft agar and tumors in nude mice were used. Quantitative RNA expression analyses were performed on seven genes in LA1-5s, LA1-55n and 5-Aza-dC treated LA1-55n NB cell lines. The methylation status around THBS-1, HIN-1, TIG-1 and CASP8 promoters was examined using methylation specific PCR. Chromatin immunoprecipitation assay was used to examine histone modifications along the THBS-1 promoter. Luciferase assay was used to determine THBS-1 promoter activity. Cell proliferation assay was used to examine the effect of 5-Aza-dC on NB cell growth. The soft agar assay was used to determine the tumorigenicity. Promoter methylation values for THBS-1, HIN-1, TIG-1, and CASP8 were higher in LA1-55n cells compared to LA1-5s cells. Consistent with the promoter methylation status, lower levels of gene expression were detected in the LA1-55n cells. Histone marks associated with repressive chromatin states (H3K9Me3, H3K27Me3, and H3K4Me3) were identified in the THBS-1 promoter region in the LA1-55n cells, but not the LA1-5s cells. In contrast, the three histone codes associated with an active chromatin state (acetyl H3, acetyl H4, and H3K4Me3) were present in the THBS-1 promoter region in LA1-5s cells, but not the LA1-55n cells, suggesting that an accessible chromatin structure is important for THBS-1 expression. We also show that 5-Aza-dC treatment of LA1-55n cells alters the DNA methylation

  17. The effect of beta-interferon therapy on myelin basic protein-elicited CD4+ T cell proliferation and cytokine production in multiple sclerosis

    DEFF Research Database (Denmark)

    Hedegaard, Chris J; Krakauer, Martin; Bendtzen, Klaus; Sørensen, Per Soelberg; Sellebjerg, Finn; Nielsen, Claus H

    2008-01-01

    Interferon (IFN)-beta therapy has well-established clinical benefits in multiple sclerosis (MS), but the underlying modulation of cytokine responses to myelin self-antigens remains poorly understood. We analysed the CD4+ T cell proliferation and cytokine responses elicited by myelin basic protein...... (MBP) and a foreign recall antigen, tetanus toxoid (TT), in mononuclear cell cultures from fourteen MS patients undergoing IFN-beta therapy. The MBP-elicited IFN-gamma-, TNF-alpha- and IL-10 production decreased during therapy (p...

  18. Cutting edge: neuronal recognition by CD8 T cells elicits central diabetes insipidus.

    Science.gov (United States)

    Scheikl, Tanja; Pignolet, Béatrice; Dalard, Cécile; Desbois, Sabine; Raison, Danièle; Yamazaki, Masanori; Saoudi, Abdelhadi; Bauer, Jan; Lassmann, Hans; Hardin-Pouzet, Hélène; Liblau, Roland S

    2012-05-15

    An increasing number of neurologic diseases is associated with autoimmunity. The immune effectors contributing to the pathogenesis of such diseases are often unclear. To explore whether self-reactive CD8 T cells could attack CNS neurons in vivo, we generated a mouse model in which the influenza virus hemagglutinin (HA) is expressed specifically in CNS neurons. Transfer of cytotoxic anti-HA CD8 T cells induced an acute but reversible encephalomyelitis in HA-expressing recipient mice. Unexpectedly, diabetes insipidus developed in surviving animals. This robust phenotype was associated with preferential accumulation of cytotoxic CD8 T cells in the hypothalamus, upregulation of MHC class I molecules, and destruction of vasopressin-expressing neurons. IFN-γ production by the pathogenic CD8 T cells was necessary for MHC class I upregulation by hypothalamic neurons and their destruction. This novel mouse model, in combination with related human data, supports the concept that autoreactive CD8 T cells can trigger central diabetes insipidus. PMID:22504649

  19. Thiamine induced resistance to Plasmopara viticola in grapevine and elicited host-defense responses, including HR like-cell death.

    Science.gov (United States)

    Boubakri, Hatem; Wahab, Mohamed Ali; Chong, Julie; Bertsch, Christophe; Mliki, Ahmed; Soustre-Gacougnolle, Isabelle

    2012-08-01

    Recently, thiamine (VitaminB1) has been shown to induce resistance against Pseudomonas syringae in Arabidopsis plants through priming of defense responses. In this paper, we have demonstrated the efficiency of thiamine to induce resistance against downy mildew caused by the oomycete Plasmopara viticola in a susceptible Vitis vinifera cultivar "Chardonnay" under glasshouse controlled conditions by providing a dual mode of action involving direct antifungal activity and elicitation of host-defense responses. Thiamine-induced defense responses included the generation of hydrogen peroxide (H(2)O(2)) in both grapevine suspension cultured cells (SCC) and plant leaves, upregulation of an array of defense-related genes and the induction of other defense responses at subcellular level such as callose deposition in stomata cells, phenolic compounds accumulation and hypersensitive response (HR) like-cell death. Epifluorescence microscopy studies revealed dramatic changes in P. viticola individual developmental stages during its colonization of the intercellular space of the leaf mesophyll in thiamine-treated plants. Collectively, our report evidenced the efficiency of thiamine in the control of downy mildew in grapevine by direct and indirect effects, suggesting that thiamine could be an attractive alternative to chemical fungicides in disease management in vineyards. PMID:22698755

  20. Alarmin IL-33 elicits potent TB-specific cell-mediated responses.

    Science.gov (United States)

    Villarreal, Daniel O; Siefert, Rebekah J; Weiner, David B

    2015-01-01

    Tuberculosis (TB) still remains a major public health issue despite the current available vaccine for TB, Bacille Calmette Guerin (BCG). An effective vaccine against TB remains a top priority in the fight against this pandemic bacterial infection. Adequate protection against TB is associated with the development of TH1-type and CD8(+) T cell responses. One alarmin cytokine, interleukin 33 (IL-33), has now been implicated in the development of both CD4(+) TH1 and CD8(+) T cell immunity. In this study, we determined whether the administration of IL-33 as an adjuvant, encoded in a DNA plasmid, could enhance the immunogenicity of a TB DNA vaccine. We report that the co-immunization of IL-33 with a DNA vaccine expressing the Mycobacterium Tuberculosis (Mtb) antigen 85B (Ag85B) induced robust Ag85B-specific IFNγ responses by ELISpot compared to Ag85B alone. Furthermore, these enhanced responses were characterized by higher frequencies of Ag85B-specific, multifunctional CD4(+) and CD8(+) T cells. Vaccination with IL-33 also increased the ability of the Ag85B-specific CD8(+) T cells to undergo degranulation and to secrete IFNγ and TNFα cytokines. These finding highlights IL-33 as a promising adjuvant to significantly improve the immunogenicity of TB DNA vaccines and support further study of this effective vaccine strategy against TB. PMID:26091147

  1. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade.

    Science.gov (United States)

    McGranahan, Nicholas; Furness, Andrew J S; Rosenthal, Rachel; Ramskov, Sofie; Lyngaa, Rikke; Saini, Sunil Kumar; Jamal-Hanjani, Mariam; Wilson, Gareth A; Birkbak, Nicolai J; Hiley, Crispin T; Watkins, Thomas B K; Shafi, Seema; Murugaesu, Nirupa; Mitter, Richard; Akarca, Ayse U; Linares, Joseph; Marafioti, Teresa; Henry, Jake Y; Van Allen, Eliezer M; Miao, Diana; Schilling, Bastian; Schadendorf, Dirk; Garraway, Levi A; Makarov, Vladimir; Rizvi, Naiyer A; Snyder, Alexandra; Hellmann, Matthew D; Merghoub, Taha; Wolchok, Jedd D; Shukla, Sachet A; Wu, Catherine J; Peggs, Karl S; Chan, Timothy A; Hadrup, Sine R; Quezada, Sergio A; Swanton, Charles

    2016-03-25

    As tumors grow, they acquire mutations, some of which create neoantigens that influence the response of patients to immune checkpoint inhibitors. We explored the impact of neoantigen intratumor heterogeneity (ITH) on antitumor immunity. Through integrated analysis of ITH and neoantigen burden, we demonstrate a relationship between clonal neoantigen burden and overall survival in primary lung adenocarcinomas. CD8(+)tumor-infiltrating lymphocytes reactive to clonal neoantigens were identified in early-stage non-small cell lung cancer and expressed high levels of PD-1. Sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. T cells recognizing clonal neoantigens were detectable in patients with durable clinical benefit. Cytotoxic chemotherapy-induced subclonal neoantigens, contributing to an increased mutational load, were enriched in certain poor responders. These data suggest that neoantigen heterogeneity may influence immune surveillance and support therapeutic developments targeting clonal neoantigens. PMID:26940869

  2. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade

    Science.gov (United States)

    McGranahan, Nicholas; Furness, Andrew J. S.; Rosenthal, Rachel; Ramskov, Sofie; Lyngaa, Rikke; Saini, Sunil Kumar; Jamal-Hanjani, Mariam; Wilson, Gareth A.; Birkbak, Nicolai J.; Hiley, Crispin T.; Watkins, Thomas B. K.; Shafi, Seema; Murugaesu, Nirupa; Mitter, Richard; Akarca, Ayse U.; Linares, Joseph; Marafioti, Teresa; Henry, Jake Y.; Van Allen, Eliezer M.; Miao, Diana; Schilling, Bastian; Schadendorf, Dirk; Garraway, Levi A.; Makarov, Vladimir; Rizvi, Naiyer A.; Snyder, Alexandra; Hellmann, Matthew D.; Merghoub, Taha; Wolchok, Jedd D.; Shukla, Sachet A.; Wu, Catherine J.; Peggs, Karl S.; Chan, Timothy A.; Hadrup, Sine R.; Quezada, Sergio A.; Swanton, Charles

    2016-01-01

    As tumors grow, they acquire mutations, some of which create neoantigens that influence the response of patients to immune checkpoint inhibitors. We explored the impact of neoantigen intratumor heterogeneity (ITH) on antitumor immunity. Through integrated analysis of ITH and neoantigen burden, we demonstrate a relationship between clonal neoantigen burden and overall survival in primary lung adenocarcinomas. CD8+ tumor-infiltrating lymphocytes reactive to clonal neoantigens were identified in early-stage non–small cell lung cancer and expressed high levels of PD-1. Sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. T cells recognizing clonal neoantigens were detectable in patients with durable clinical benefit. Cytotoxic chemotherapy–induced subclonal neoantigens, contributing to an increased mutational load, were enriched in certain poor responders. These data suggest that neoantigen heterogeneity may influence immune surveillance and support therapeutic developments targeting clonal neoantigens. PMID:26940869

  3. Distinct regulatory functions of calpain 1 and 2 during neural stem cell self-renewal and differentiation.

    Directory of Open Access Journals (Sweden)

    Daniela M Santos

    Full Text Available Calpains are calcium regulated cysteine proteases that have been described in a wide range of cellular processes, including apoptosis, migration and cell cycle regulation. In addition, calpains have been implicated in differentiation, but their impact on neural differentiation requires further investigation. Here, we addressed the role of calpain 1 and calpain 2 in neural stem cell (NSC self-renewal and differentiation. We found that calpain inhibition using either the chemical inhibitor calpeptin or the endogenous calpain inhibitor calpastatin favored differentiation of NSCs. This effect was associated with significant changes in cell cycle-related proteins and may be regulated by calcium. Interestingly, calpain 1 and calpain 2 were found to play distinct roles in NSC fate decision. Calpain 1 expression levels were higher in self-renewing NSC and decreased with differentiation, while calpain 2 increased throughout differentiation. In addition, calpain 1 silencing resulted in increased levels of both neuronal and glial markers, β-III Tubulin and glial fibrillary acidic protein (GFAP. Calpain 2 silencing elicited decreased levels of GFAP. These results support a role for calpain 1 in repressing differentiation, thus maintaining a proliferative NSC pool, and suggest that calpain 2 is involved in glial differentiation.

  4. Elicitation, an Effective Strategy for the Biotechnological Production of Bioactive High-Added Value Compounds in Plant Cell Factories

    Directory of Open Access Journals (Sweden)

    Karla Ramirez-Estrada

    2016-02-01

    Full Text Available Plant in vitro cultures represent an attractive and cost-effective alternative to classical approaches to plant secondary metabolite (PSM production (the “Plant Cell Factory” concept. Among other advantages, they constitute the only sustainable and eco-friendly system to obtain complex chemical structures biosynthesized by rare or endangered plant species that resist domestication. For successful results, the biotechnological production of PSM requires an optimized system, for which elicitation has proved one of the most effective strategies. In plant cell cultures, an elicitor can be defined as a compound introduced in small concentrations to a living system to promote the biosynthesis of the target metabolite. Traditionally, elicitors have been classified in two types, abiotic or biotic, according to their chemical nature and exogenous or endogenous origin, and notably include yeast extract, methyl jasmonate, salicylic acid, vanadyl sulphate and chitosan. In this review, we summarize the enhancing effects of elicitors on the production of high-added value plant compounds such as taxanes, ginsenosides, aryltetralin lignans and other types of polyphenols, focusing particularly on the use of a new generation of elicitors such as coronatine and cyclodextrins.

  5. Delivery of antigenic candidates by a DNA/MVA heterologous approach elicits effector CD8+T cell mediated immunity against Trypanosoma cruzi

    OpenAIRE

    Gupta, Shivali; Garg, Nisha Jain

    2012-01-01

    In this study, we have characterized the immune mechanisms elicited by antigenic candidates, TcG2 and TcG4, delivered by a DNA-prime/MVA-boost approach, and evaluated the host responses to T. cruzi infection in C57BL/6 mice. Immunization of mice with antigenic candidates elicited antigen-specific, high-avidity, trypanolytic antibody response (IgG2b>IgG1) and CD8+T cells that exhibited type-1 cytolytic effector (CD8+CD107a+IFN-γ+Perforin+) phenotype. The extent of TcG2-dependent type 1 B and T...

  6. Cell-Demanded VEGF Release via Nanocapsules Elicits Different Receptor Activation Dynamics and Enhanced Angiogenesis.

    Science.gov (United States)

    Zhu, Suwei; Segura, Tatiana

    2016-06-01

    Although the delivery of vascular endothelial growth factor (VEGF) with extended release profiles has consistently shown beneficial therapeutic effects compared with bolus delivery, [Martino, M. M., F. Tortelli, M. Mochizuki, S. Traub, D. Ben-David, G. A. Kuhn, R. Muller, E. Livne, S. A. Eming, and J. A. Hubbell. Sci. Transl. Med. 3(100):100ra189, 2011; Martino, M. M., P. S. Briquez, A. Ranga, M. P. Lutolf, and J. A. Hubbell. Proc. Natl. Acad. Sci. USA. 110(12):4563-4568, 2013; Amiram, M., K. M. Luginbuhl, X. Li, M. N. Feinglos, and A. Chilkoti. Proc. Natl. Acad. Sci. USA. 110(8):2792-2797, 2013] it remains unclear if the reason is solely due to the physical availability and the reduced degradation of the protein. Here we studied the activation of VEGF receptor 2 (VR-2) by sustained released VEGF compared with bolus delivered VEGF to unveil that sustained delivery system alters the dynamics of receptor activation and affects the actions of cells between sprouting and proliferation. We utilized a protein nanocapsule delivery strategy that releases VEGF as mediated by extracellular proteases. These protein nanocapsules were synthesized through an aqueous assembly of a nanogel-peptide shell around the protein, leading to one to two proteins encapsulated per nanocapsule. Receptor activation studies revealed differential dynamics of receptor activation for slowly released VEGF compared with bolus delivered VEGF. As expected sustained released VEGF via nanocapsules resulted in enhanced vascular sprouting in vitro and in vivo. These studies demonstrate the physical presentation of VEGF, in this case of a slow release with time, can affect its molecular mechanism of actions and cause alterations in cellular responses and therapeutic outcomes. PMID:26940611

  7. A distinct subset of self-renewing human memory CD8+ T cells survives cytotoxic chemotherapy

    OpenAIRE

    Turtle, Cameron J.; Swanson, Hillary M.; Fujii, Nobuharu; Estey, Elihu H.; Riddell, Stanley R.

    2009-01-01

    The mechanisms that maintain human T cell memory during normal and perturbed homeostasis are not fully understood. The repeated induction of profound lymphocytopenia in patients undergoing multiple cycles of cytotoxic chemotherapy infrequently results in severe infections with viruses controlled by memory T cells, suggesting that some memory T cells survive chemotherapy and restore immunity. Here we identify a distinct subpopulation of memory CD8+ T cells with the ability to rapidly efflux an...

  8. Non-small cell lung cancer stem/progenitor cells are enriched in multiple distinct phenotypic subpopulations and exhibit plasticity

    OpenAIRE

    Akunuru, S; James Zhai, Q; Zheng, Y.

    2012-01-01

    Cancer stem cells (CSCs) represent a population of cancer cells that possess unique self-renewal and differentiation characteristics required for tumorigenesis and are resistant to chemotherapy-induced apoptosis. Lung CSCs can be enriched by several markers including drug-resistant side population (SP), CD133pos and ALDHhigh. Using human non-small cell lung adenocarcinoma cell lines and patient-derived primary tumor cells, we demonstrate that SP cells represent a subpopulation distinct from o...

  9. Cell-mediated immunity elicited by the blood stage malaria vaccine apical membrane antigen 1 in Malian adults: Results of a Phase I randomized trial

    OpenAIRE

    Lyke, Kirsten E; Daou, Modibo; DIARRA, ISSA; Kone, Abdoulaye; Kouriba, Bourema; Thera, Mohamadou A.; Dutta, Sheetij; Lanar, David E.; Heppner, D Gray; Doumbo, Ogobara K.; Plowe, Christopher V.; Sztein, Marcelo B.

    2009-01-01

    The development of a safe and effective malaria vaccine is impeded by the complexity of the Plasmodium life cycle. A vaccine that elicits both cell-mediated and humoral immune responses might be needed for protection against this multistage parasitic infection. Apical membrane antigen 1 (AMA-1) plays a key role in erythrocytic invasion but is also expressed in sporozoites and in late stage liver schizonts, where it may provide a target of protective cell-mediated immunity (CMI). A Phase 1 tri...

  10. Immediate dysfunction of vaccine-elicited CD8+ T cells primed in the absence of CD4+ T cells

    Science.gov (United States)

    Provine, Nicholas M.; Larocca, Rafael A.; Aid, Malika; Penaloza-MacMaster, Pablo; Badamchi-Zadeh, Alexander; Borducchi, Erica N.; Yates, Kathleen B.; Abbink, Peter; Kirilova, Marinela; Ng’ang’a, David; Bramson, Jonathan; Haining, W. Nicholas; Barouch, Dan H.

    2016-01-01

    CD4+ T cell help is critical for optimal CD8+ T cell memory differentiation and maintenance in many experimental systems. Additionally, many reports have identified reduced primary CD8+ T cell responses in the absence of CD4+ T cell help, which often coincides with reduced antigen or pathogen clearance. Here we demonstrate that absence of CD4+ T cells at the time of adenovirus vector immunization of mice led to immediate impairments in early CD8+ T cell functionality and differentiation. Unhelped CD8+ T cells exhibited a reduced effector phenotype, decreased ex vivo cytotoxicity, and decreased capacity to produce cytokines. This dysfunctional state was imprinted within 3 days of immunization. Unhelped CD8+ T cells expressed elevated levels of inhibitory receptors and exhibited transcriptomic exhaustion and anergy profiles by gene set enrichment analysis. Dysfunctional, impaired effector differentiation also occurred following immunization of CD4+ T cell-deficient mice with a poxvirus vector. This study demonstrates that following priming with viral vectors, CD4+ T cell help is required to promote both the expansion and acquisition of effector functions by CD8+ T cells, which is accomplished by preventing immediate dysfunction. PMID:27448585

  11. Epithelial basal cells are distinct from dendritic cells and macrophages in the mouse epididymis.

    Science.gov (United States)

    Shum, Winnie W; Smith, Tegan B; Cortez-Retamozo, Virna; Grigoryeva, Lubov S; Roy, Jeremy W; Hill, Eric; Pittet, Mikael J; Breton, Sylvie; Da Silva, Nicolas

    2014-05-01

    The epithelium that lines the epididymal duct establishes the optimal milieu in which spermatozoa mature, acquire motility, and are stored. This finely tuned environment also protects antigenic sperm against pathogens and autoimmunity, which are potential causes of transient or permanent infertility. The epididymal epithelium is pseudostratified and contains basal cells (BCs) that are located beneath other epithelial cells. Previous studies showed that in the mouse epididymis, BCs possess macrophage-like characteristics. However, we previously identified a dense population of cells belonging to the mononuclear phagocyte (MP) system (comprised of macrophages and dendritic cells) in the basal compartment of the mouse epididymis and showed that a subset of MPs express the macrophage marker F4/80. In the present study, we evaluate the distribution of BCs and MPs in the epididymis of transgenic CD11c-EYFP mice, in which EYFP is expressed exclusively in MPs, using antibodies against the BC marker keratin 5 (KRT5) and the macrophage marker F4/80. Immunofluorescence labeling for laminin, a basement membrane marker, showed that BCs and most MPs are located in the basal region of the epithelium. Confocal microscopy showed that in the initial segment, both BCs and MPs project intraepithelial extensions and establish a very intricate network. Flow cytometry experiments demonstrated that epididymal MPs and BCs are phenotypically distinct. BCs do not express F4/80, and MPs do not express KRT5. Therefore, despite their proximity and some morphological similarities with peritubular macrophages and dendritic cells, BCs do not belong to the MP system. PMID:24648397

  12. Distinct genetic alterations in small cell carcinoma from different anatomic sites

    OpenAIRE

    Zheng, Xiaoyong; Liu, Delong; Fallon, John T.; Zhong, Minghao

    2015-01-01

    Small cell carcinoma (SmCC) is a distinct clinicopathological entity first described in the lung. It represents approximately 15% of all bronchogenic carcinoma. Extrapulmonary small cell carcinoma (EPSmCC) morphologically indistinguishable from small cell lung cancer (SCLC) was first reported in 1930. Since its first description, EPSmCC has been reported in virtually all anatomical sites, including: gynecologic organs (ovary and cervix); genitourinary organs (urinary bladder and prostate); th...

  13. Distinct organ-specific metastatic potential of individual breast cancer cells and primary tumors

    OpenAIRE

    Minn, Andy J.; Kang, Yibin; Serganova, Inna; Gupta, Gaorav P.; Giri, Dilip D.; Doubrovin, Mikhail; Ponomarev, Vladimir; Gerald, William L; Blasberg, Ronald; Massagué, Joan

    2005-01-01

    We used bioluminescence imaging to reveal patterns of metastasis formation by human breast cancer cells in immunodeficient mice. Individual cells from a population established in culture from the pleural effusion of a breast cancer patient showed distinct patterns of organ-specific metastasis. Single-cell progenies derived from this population exhibited markedly different abilities to metastasize to the bone, lung, or adrenal medulla, which suggests that metastases to different organs have di...

  14. Genes and Gene Networks Involved in Sodium Fluoride-Elicited Cell Death Accompanying Endoplasmic Reticulum Stress in Oral Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Yoshiaki Tabuchi

    2014-05-01

    Full Text Available Here, to understand the molecular mechanisms underlying cell death induced by sodium fluoride (NaF, we analyzed gene expression patterns in rat oral epithelial ROE2 cells exposed to NaF using global-scale microarrays and bioinformatics tools. A relatively high concentration of NaF (2 mM induced cell death concomitant with decreases in mitochondrial membrane potential, chromatin condensation and caspase-3 activation. Using 980 probe sets, we identified 432 up-regulated and 548 down-regulated genes, that were differentially expressed by >2.5-fold in the cells treated with 2 mM of NaF and categorized them into 4 groups by K-means clustering. Ingenuity® pathway analysis revealed several gene networks from gene clusters. The gene networks Up-I and Up-II included many up-regulated genes that were mainly associated with the biological function of induction or prevention of cell death, respectively, such as Atf3, Ddit3 and Fos (for Up-I and Atf4 and Hspa5 (for Up-II. Interestingly, knockdown of Ddit3 and Hspa5 significantly increased and decreased the number of viable cells, respectively. Moreover, several endoplasmic reticulum (ER stress-related genes including, Ddit3, Atf4 and Hapa5, were observed in these gene networks. These findings will provide further insight into the molecular mechanisms of NaF-induced cell death accompanying ER stress in oral epithelial cells.

  15. 1‑O‑acetylbritannilactone combined with gemcitabine elicits growth inhibition and apoptosis in A549 human non‑small cell lung cancer cells.

    Science.gov (United States)

    Wang, Feng; Li, Hong; Qiao, Jian-Ou

    2015-10-01

    Non‑small‑cell lung cancer (NSCLC) accounts for ~85% of all lung cancer cases, with a 5‑year survival rate of britannica, a Chinese traditional medicine, has been demonstrated to have anticancer activity. In the present study, the antiproliferative and proapoptotic abilities of ABL alone or in combination with gemcitabine in a human NSCLC cell line were investigated. A549 cells were treated in vitro with ABL, gemcitabine, and a combination of ABL and gemcitabine for 72 h. The results demonstrated that ABL and gemcitabine inhibited cell growth and induced apoptosis of A549 cells. These effects were more potent following the combination of ABL and gemcitabine treatment than either agent alone. Furthermore, the signal transduction analysis revealed nuclear factor (NF)‑κB expression was significantly decreased by ABL and the combination treatment. The inhibitor nuclear factor κBα (IκBα) and Bax levels were upregulated whereas Bcl‑2 was substantially downregulated following treatment. The present findings suggest that ABL combined with gemcitabine elicits potent apoptosis of lung cancer cells and therefore, ABL has the potential to be developed as a chemotherapeutic agent. PMID:26151622

  16. KDM6B Elicits Cell Apoptosis by Promoting Nuclear Translocation of FOXO1 in Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Jun Ma

    2015-08-01

    Full Text Available Background/Aims: Non-small cell lung carcinoma (NSCLC is the most common type of lung cancer and the cause of most cancer-related deaths. The molecular mechanisms that are involved in NSCLC development are currently not well understood. Accumulating evidence shows that histone demethylases play important roles in the regulation of pathological developmental processes in many diseases, including various types of cancers. Methods: Mitochondrial membrane potential assays, migration and invasion assays, caspase-3 and caspase-9 activity assays and western blot analysis were used in this research. Results: We found that overexpression of KDM6B, a demethylase that acts on histone H3 at lysine 27 (H3K27, inhibited cell growth by initiating mitochondria-dependent apoptosis and by attenuating the invasion-metastasis cascade in NSCLC cells. Moreover, our results showed that KDM6B directly interacted with FOXO1 and that overexpression of KDM6B promoted nuclear accumulation of FOXO1. The effects of KDM6B on cell apoptosis and metastasis were weakened by knockdown of FOXO1 expression. On the contrary, knocking down expression of KDM6B inhibited cell apoptosis and promoted cell growth by mitigating the nuclear translocation of FOXO1 in NSCLC cells. Conclusions: These findings suggest that KDM6B may act in a pro-apoptotic role in NSCLC by causing the nuclear translocation of FOXO1.

  17. MeHg Developing Exposure Causes DNA Double-Strand Breaks and Elicits Cell Cycle Arrest in Spinal Cord Cells

    Directory of Open Access Journals (Sweden)

    Fabiana F. Ferreira

    2015-01-01

    Full Text Available The neurotoxicity caused by methylmercury (MeHg is well documented; however, the developmental neurotoxicity in spinal cord is still not fully understood. Here we investigated whether MeHg affects the spinal cord layers development. Chicken embryos at E3 were treated in ovo with 0.1 μg MeHg/50 μL saline solution and analyzed at E10. Thus, we performed immunostaining using anti-γ-H2A.X to recognize DNA double-strand breaks and antiphosphohistone H3, anti-p21, and anti-cyclin E to identify cells in proliferation and cell cycle proteins. Also, to identify neuronal cells, we used anti-NeuN and anti-βIII-tubulin antibodies. After the MeHg treatment, we observed the increase on γ-H2A.X in response to DNA damage. MeHg caused a decrease in the proliferating cells and in the thickness of spinal cord layers. Moreover, we verified that MeHg induced an increase in the number of p21-positive cells but did not change the cyclin E-positive cells. A significantly high number of TUNEL-positive cells indicating DNA fragmentation were observed in MeHg-treated embryos. Regarding the neuronal differentiation, MeHg induced a decrease in NeuN expression and did not change the expression of βIII-tubulin. These results showed that in ovo MeHg exposure alters spinal cord development by disturbing the cell proliferation and death, also interfering in early neuronal differentiation.

  18. MeHg Developing Exposure Causes DNA Double-Strand Breaks and Elicits Cell Cycle Arrest in Spinal Cord Cells

    Science.gov (United States)

    Ferreira, Fabiana F.; Ammar, Dib; Bourckhardt, Gilian F.; Kobus-Bianchini, Karoline; Müller, Yara M. R.; Nazari, Evelise M.

    2015-01-01

    The neurotoxicity caused by methylmercury (MeHg) is well documented; however, the developmental neurotoxicity in spinal cord is still not fully understood. Here we investigated whether MeHg affects the spinal cord layers development. Chicken embryos at E3 were treated in ovo with 0.1 μg MeHg/50 μL saline solution and analyzed at E10. Thus, we performed immunostaining using anti-γ-H2A.X to recognize DNA double-strand breaks and antiphosphohistone H3, anti-p21, and anti-cyclin E to identify cells in proliferation and cell cycle proteins. Also, to identify neuronal cells, we used anti-NeuN and anti-βIII-tubulin antibodies. After the MeHg treatment, we observed the increase on γ-H2A.X in response to DNA damage. MeHg caused a decrease in the proliferating cells and in the thickness of spinal cord layers. Moreover, we verified that MeHg induced an increase in the number of p21-positive cells but did not change the cyclin E-positive cells. A significantly high number of TUNEL-positive cells indicating DNA fragmentation were observed in MeHg-treated embryos. Regarding the neuronal differentiation, MeHg induced a decrease in NeuN expression and did not change the expression of βIII-tubulin. These results showed that in ovo MeHg exposure alters spinal cord development by disturbing the cell proliferation and death, also interfering in early neuronal differentiation. PMID:26793240

  19. Lactotransferrin-Cre reporter mice trace neutrophils, monocytes/macrophages and distinct subtypes of dendritic cells.

    Science.gov (United States)

    Kovacic, Boris; Hoelbl-Kovacic, Andrea; Fischhuber, Katrin M; Leitner, Nicole R; Gotthardt, Dagmar; Casanova, Emilio; Sexl, Veronika; Müller, Mathias

    2014-06-01

    Considerable effort has been expended to identify genes that account for myeloid lineage commitment and development. However, currently available non-invasive mouse models utilize myeloid-specific reporters that are significantly expressed in hematopoietic stem cells as well as lymphoid compartments. Here, we describe a myeloid-specific marker that is not shared by any other lineage. We show that lactotransferrin mRNA is expressed by Gr-1(+)/CD11b(+) cells in the bone marrow, as opposed to hematopoietic stem cells or any peripheral cell population. To follow the progeny of lactotransferrin-expressing bone marrow cells, we generated a mouse model in which a reporter gene is irreversibly activated from the lactotransferrin-promoter. We found that lactotransferrin-reporter labels a majority of neutrophils, monocytes, macrophages and distinct subtypes of dendritic cells, while excluding T, B, natural killer cells, interferon-producing killer dendritic cells, plasmacytoid dendritic cells, erythrocytes and eosinophils. Lactotransferrin-reporter(-) bone marrow cells retain lymphoid, erythroid and long-term repopulating potential, while lactotransferrin-reporter(+) bone marrow cells confer only myeloid, but not lymphoid potential. We conclude that lactotransferrin represents a late stage differentiation marker of neutrophils, macrophages and distinct subtypes of dendritic cells. PMID:24561791

  20. Administration of HPV DNA vaccine via electroporation elicits the strongest CD8+ T cell immune responses compared to intramuscular injection and intradermal gene gun delivery

    OpenAIRE

    Best, Simon R.; Peng, Shiwen; Juang, Chi-Mou; Hung, Chien-Fu; Hannaman, Drew; Saunders, John R.; Wu, T.-C.; Pai, Sara I.

    2009-01-01

    DNA vaccines are an attractive approach to eliciting antigen-specific immunity. Intracellular targeting of tumor antigens through its linkage to immunostimulatory molecules such as calreticulin (CRT) can improve antigen processing and presentation through the MHC Class I pathway and increase cytotoxic CD8+ T cell production. However, even with these enhancements, the efficacy of such immunotherapeutic strategies is dependent on the identification of an effective route and method of DNA admini...

  1. Human Lymphoid Tissues Harbor a Distinct CD69+CXCR6+ NK Cell Population.

    Science.gov (United States)

    Lugthart, Gertjan; Melsen, Janine E; Vervat, Carly; van Ostaijen-Ten Dam, Monique M; Corver, Willem E; Roelen, Dave L; van Bergen, Jeroen; van Tol, Maarten J D; Lankester, Arjan C; Schilham, Marco W

    2016-07-01

    Knowledge of human NK cells is based primarily on conventional CD56(bright) and CD56(dim) NK cells from blood. However, most cellular immune interactions occur in lymphoid organs. Based on the coexpression of CD69 and CXCR6, we identified a third major NK cell subset in lymphoid tissues. This population represents 30-60% of NK cells in marrow, spleen, and lymph node but is absent from blood. CD69(+)CXCR6(+) lymphoid tissue NK cells have an intermediate expression of CD56 and high expression of NKp46 and ICAM-1. In contrast to circulating NK cells, they have a bimodal expression of the activating receptor DNAX accessory molecule 1. CD69(+)CXCR6(+) NK cells do not express the early markers c-kit and IL-7Rα, nor killer cell Ig-like receptors or other late-differentiation markers. After cytokine stimulation, CD69(+)CXCR6(+) NK cells produce IFN-γ at levels comparable to CD56(dim) NK cells. They constitutively express perforin but require preactivation to express granzyme B and exert cytotoxicity. After hematopoietic stem cell transplantation, CD69(+)CXCR6(+) lymphoid tissue NK cells do not exhibit the hyperexpansion observed for both conventional NK cell populations. CD69(+)CXCR6(+) NK cells constitute a separate NK cell population with a distinct phenotype and function. The identification of this NK cell population in lymphoid tissues provides tools to further evaluate the cellular interactions and role of NK cells in human immunity. PMID:27226093

  2. Beige Adipocytes are a Distinct Type of Thermogenic Fat Cell in Mouse and Human

    OpenAIRE

    Wu, Jun; Boström, Pontus; Sparks, Lauren M; Ye, Li; Choi, Jang Hyun; Giang, An-Hoa; Khandekar, Melin; Nuutila, Pirjo; Schaart, Gert; Huang, Kexin; Tu, Hua; van Marken Lichtenbelt, Wouter D; Hoeks, Joris; Enerbäck, Sven; Schrauwen, Patrick

    2012-01-01

    Brown fat defends against hypothermia and obesity through thermogenesis mediated by mitochondrial UCP1. Recent data suggest that there are two distinct types of brown fat: classical brown fat derived from a myf-5 cellular lineage and UCP1-positive cells that emerge in white fat from a non-myf-5 lineage. Here we report the cloning of “beige” cells from murine white fat depots. Beige cells resemble white fat cells in having extremely low basal expression of UCP1, but like classical brown fat, t...

  3. MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage

    DEFF Research Database (Denmark)

    Gold, Marielle C.; McLaren, James E.; Reistetter, Joseph A.;

    2015-01-01

    line with this interpretation, MAIT cell clones with distinct TCRs responded differentially to a riboflavin metabolite. These results suggest that MAIT cells can discriminate between pathogen-derived ligands in a clonotype-dependent manner, providing a basis for adaptive memory via recruitment of...

  4. MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage

    DEFF Research Database (Denmark)

    Gold, Marielle C.; McLaren, James E.; Reistetter, Joseph A.;

    2014-01-01

    line with this interpretation, MAIT cell clones with distinct TCRs responded differentially to a riboflavin metabolite. These results suggest that MAIT cells can discriminate between pathogen-derived ligands in a clonotype-dependent manner, providing a basis for adaptive memory via recruitment of...

  5. CD8+ T-cells expressing interferon gamma or perforin play antagonistic roles in heart injury in experimental Trypanosoma cruzi-elicited cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    Jaline Coutinho Silverio

    Full Text Available In Chagas disease, CD8(+ T-cells are critical for the control of Trypanosoma cruzi during acute infection. Conversely, CD8(+ T-cell accumulation in the myocardium during chronic infection may cause tissue injury leading to chronic chagasic cardiomyopathy (CCC. Here we explored the role of CD8(+ T-cells in T. cruzi-elicited heart injury in C57BL/6 mice infected with the Colombian strain. Cardiomyocyte lesion evaluated by creatine kinase-MB isoenzyme activity levels in the serum and electrical abnormalities revealed by electrocardiogram were not associated with the intensity of heart parasitism and myocarditis in the chronic infection. Further, there was no association between heart injury and systemic anti-T. cruzi CD8(+ T-cell capacity to produce interferon-gamma (IFNγ and to perform specific cytotoxicity. Heart injury, however, paralleled accumulation of anti-T. cruzi cells in the cardiac tissue. In T. cruzi infection, most of the CD8(+ T-cells segregated into IFNγ(+ perforin (Pfn(neg or IFNγ(negPfn(+ cell populations. Colonization of the cardiac tissue by anti-T. cruzi CD8(+Pfn(+ cells paralleled the worsening of CCC. The adoptive cell transfer to T. cruzi-infected cd8(-/- recipients showed that the CD8(+ cells from infected ifnγ(-/-pfn(+/+ donors migrate towards the cardiac tissue to a greater extent and caused a more severe cardiomyocyte lesion than CD8(+ cells from ifnγ(+/+pfn(-/- donors. Moreover, the reconstitution of naïve cd8(-/- mice with CD8(+ cells from naïve ifnγ(+/+pfn(-/- donors ameliorated T. cruzi-elicited heart injury paralleled IFNγ(+ cells accumulation, whereas reconstitution with CD8(+ cells from naïve ifnγ(-/-pfn(+/+ donors led to an aggravation of the cardiomyocyte lesion, which was associated with the accumulation of Pfn(+ cells in the cardiac tissue. Our data support a possible antagonist effect of CD8(+Pfn(+ and CD8(+IFNγ(+ cells during CCC. CD8(+IFNγ(+ cells may exert a beneficial role, whereas CD8(+Pfn

  6. Gastrin: a distinct fate of neurogenin3 positive progenitor cells in the embryonic pancreas.

    Directory of Open Access Journals (Sweden)

    Yaron Suissa

    Full Text Available Neurogenin3(+ (Ngn3(+ progenitor cells in the developing pancreas give rise to five endocrine cell types secreting insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. Gastrin is a hormone produced primarily by G-cells in the stomach, where it functions to stimulate acid secretion by gastric parietal cells. Gastrin is expressed in the embryonic pancreas and is common in islet cell tumors, but the lineage and regulators of pancreatic gastrin(+ cells are not known. We report that gastrin is abundantly expressed in the embryonic pancreas and disappears soon after birth. Some gastrin(+ cells in the developing pancreas co-express glucagon, ghrelin or pancreatic polypeptide, but many gastrin(+ cells do not express any other islet hormone. Pancreatic gastrin(+ cells express the transcription factors Nkx6.1, Nkx2.2 and low levels of Pdx1, and derive from Ngn3(+ endocrine progenitor cells as shown by genetic lineage tracing. Using mice deficient for key transcription factors we show that gastrin expression depends on Ngn3, Nkx2.2, NeuroD1 and Arx, but not Pax4 or Pax6. Finally, gastrin expression is induced upon differentiation of human embryonic stem cells to pancreatic endocrine cells expressing insulin. Thus, gastrin(+ cells are a distinct endocrine cell type in the pancreas and an alternative fate of Ngn3+ cells.

  7. Do dental stem cells depict distinct characteristics? — Establishing their “phenotypic fingerprint”

    Science.gov (United States)

    Ponnaiyan, Deepa

    2014-01-01

    Dental tissues provide an alternate source of stem cells compared with bone marrow and have a similar potency as that of bone marrow derived mesenchymal stem cells. It has been established there are six types of dental stem cells: Dental pulp stem cells, stem cells from human exfoliated deciduous teeth, stem cells from apical papilla, periodontal ligament stem cells, dental follicle progenitor cells, oral periosteum stem cells and recently gingival connective tissue stem cells. Most of the dental tissues have a common developmental pathway; thus, it is relevant to understand whether stem cells derived from these closely related tissues are programmed differently. The present review analyzes whether stem cells form dental tissues depict distinct characteristics by gaining insight into differences in their immunophenotype. In addition, to explore the possibility of establishing a unique phenotypic fingerprint of these stem cells by identifying the unique markers that can be used to isolate these stem cells. This, in future will help in developing better techniques and markers for identification and utilization of these stem cells for regenerative therapy. PMID:24932185

  8. Distinct gene expression signatures in human embryonic stem cells differentiated towards definitive endoderm at single-cell level

    DEFF Research Database (Denmark)

    Norrman, Karin; Strömbeck, Anna; Semb, Henrik; Ståhlberg, Anders

    2013-01-01

    of anterior definitive endoderm (DE). Here, we differentiated human embryonic stem cells towards DE using three different activin A based treatments. Differentiation efficiencies were evaluated by gene expression profiling over time at cell population level. A panel of key markers was used to study...... were useful to monitor the temporal expression of genes involved in primitive streak formation and endoderm formation, while single-cell analysis allowed us to study cell culture heterogeneity and fingerprint individual cells. In addition, single-cell analysis revealed distinct gene expression patterns...

  9. Distinct P-glycoprotein precursors are overproduced in independently isolated drug-resistant cell lines.

    Science.gov (United States)

    Greenberger, L M; Lothstein, L; Williams, S S; Horwitz, S B

    1988-06-01

    A family of P-glycoproteins are overproduced in multidrug-resistant cells derived from the murine macrophage-like line J774.2. To determine whether individual family members are overproduced in response to different drugs, the P-glycoprotein precursors in several independently isolated cell lines, which were selected for resistance to vinblastine or taxol, were compared. Individual cell lines selected with vinblastine overproduced P-glycoprotein precursors of either 120 or 125 kDa. Taxol-selected cell lines overproduced either the 125-kDa precursor or both precursors simultaneously. Two similar but distinct peptide maps for the mature P-glycoproteins were observed. These maps corresponded to each precursor regardless of the drug used for selection. One vinblastine-resistant cell line switched from the 125- to the 120-kDa precursor when grown in increasing concentrations of drug. This change coincided with the overexpression of a distinct subset of mRNA species that code for P-glycoprotein. It is concluded that precursor expression is not drug-specific. These data suggest that individual overproduced P-glycoprotein family members are translated as distinct polypeptides. The results may help to explain the diversity in the multidrug-resistant phenotype. PMID:2897689

  10. Telomere dysfunction and cell survival: roles for distinctTIN2-containing complexes

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sahn-Ho; Davalos, Albert R.; Heo, Seok-Jin; Rodier, Francis; Beausejour, Christian; Kaminker, Patrick; Campisi, Judith

    2006-11-07

    Telomeres are maintained by three DNA binding proteins, TRF1, TRF2 and POT1, and several associated factors. One factor, TIN2, binds TRF1 and TRF2 directly and POT1 indirectly. These and two other proteins form a soluble complex that may be the core telomere-maintenance complex. It is not clear whether subcomplexes exist or function in vivo. Here, we provide evidence for two TIN2 subcomplexes with distinct functions in human cells. TIN2 ablation by RNA interference caused telomere uncapping and p53-independent cell death in all cells tested. However, we isolated two TIN2 complexes from cell lysates, each selectively sensitive to a TIN2 mutant (TIN2-13, TIN2-15C). In cells with wild-type p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere uncapping and eventual growth arrest. In cells lacking p53 function, TIN215C more than TIN2-13 caused genomic instability and cell death. Thus, TIN2 subcomplexes likely have distinct functions in telomere maintenance, and may provide selective targets for eliminating cells with mutant p53.

  11. EFFECT OF GAMMA IRRADIATION ON THE T CELL MEDIATED HYPERSENSITIVITY REACTION IN BALB/C MICE DISCREPANT EFFECT ON THE INDUCTION AND THE ELICITATION PHASE

    International Nuclear Information System (INIS)

    Gamma radiation is widely used in the treatment of malignant neoplasm, however, it deprives the host immune function which may retard tumor rejection by the immune response. The main purpose of the present study is to investigate the effect of gamma irradiation on the T cell hypersensitivity reaction in Balb/c mice. It aims also to elucidate the possible mechanisms of action of ionizing radiation on the phases of hypersensitivity reaction. Two groups of Balb/c mice, each of ten, were used in each experiment. The first group served as a control and the second group was exposed to 3 Gy gamma irradiation. The following parameters were determined: contact sensitivity reaction to oxazolone by mouse ear swelling response, local dendritic cell migration, local lymph node weight, local lymphocyte proliferation, circulating lymphocytes count, spleen and thymus cell count. The effect of gamma irradiation on the elicitation phase of contact sensitivity was also determined.Data from the present study showed that gamma irradiation caused a significant decrease of the mouse ear swelling response to 62 % of the control level, retarded dendritic cell migration and decreased lymph node weight to 45% of the control value. They also showed a significant decline in the lymphocyte proliferation in lymph node draining contact sensitization application. They also showed gamma irradiation-induced marked decline of thymocytes count while it slightly affected spleen cell count. Circulating lymphocytes were decreased significantly in gamma irradiated animals. Exposure to gamma irradiation enhanced the elicitation phase of contact sensitivity.In conclusion, data from the present study showed a negative impact on the induction phase and a positive effect of gamma irradiation on elicitation phase of contact sensitivity in Balb/c mice

  12. Dispersed cells represent a distinct stage in the transition from bacterial biofilm to planktonic lifestyles

    DEFF Research Database (Denmark)

    Chua, Song Lin; Liu, Yang; Yam, Joey Kuok Hoong;

    2014-01-01

    Bacteria assume distinct lifestyles during the planktonic and biofilm modes of growth. Increased levels of the intracellular messenger c-di-GMP determine the transition from planktonic to biofilm growth, while a reduction causes biofilm dispersal. It is generally assumed that cells dispersed from...... biofilms immediately go into the planktonic growth phase. Here we use single-nucleotide resolution transcriptomic analysis to show that the physiology of dispersed cells from Pseudomonas aeruginosa biofilms is highly different from those of planktonic and biofilm cells. In dispersed cells, the expression...... of the small regulatory RNAs RsmY and RsmZ is downregulated, whereas secretion genes are induced. Dispersed cells are highly virulent against macrophages and Caenorhabditis elegans compared with planktonic cells. In addition, they are highly sensitive towards iron stress, and the combination of a...

  13. Different mouse mast cell populations express various combinations of at least six distinct mast cell serine proteases.

    OpenAIRE

    Reynolds, D. S.; Stevens, R L; Lane, W S; Carr, M H; Austen, K. F.; Serafin, W E

    1990-01-01

    Mouse serosal mast cells (SMCs) and Kirsten sarcoma virus-immortalized mast cells store large amounts of mast cell carboxypeptidase A and serine proteases in their secretory granules. Secretory granule proteins from 2.6 x 10(6) purified SMCs were separated by NaDodSO4/PAGE, trans-blotted to poly(vinylidine difluoride) membranes, and subjected to amino-terminal amino acid sequencing. Four distinct mast cell serine proteases were identified. With mast cell carboxypeptidase A, these serine prote...

  14. Partitioning of genetically distinct cell populations in chimeric juveniles of the sponge Amphimedon queenslandica.

    Science.gov (United States)

    Gauthier, Marie; Degnan, Bernard M

    2008-01-01

    Natural chimerism, the fusion between genetically distinct conspecifics, is a process known to occur in various marine benthic invertebrates. Sponges (phylum Porifera) have proven to be a useful model to study the origin and evolution of allorecognition. Like some other invertebrates, they display an ontogenetic shift in their allorecognition response: genetically different individuals can fuse during early development, but, in most instances, not as adults. However, there is a limited understanding of the cellular organisation of sponge chimeras and the onset of this allorecognition response, which prevents integration of incompatible genotypes. Here we follow the behaviours and fates of cells derived from genetically distinct larvae of the demosponge Amphimedon queenslandica that have fused together at metamorphosis. By labelling individual larvae with different fluorescent dyes, we can follow cell movement in the postlarval chimeras. We observed that cells from the two individuals readily mixed for 2 weeks after the initial fusion. After that time, differently labelled cells began to sort into different postlarval cellular territories, with one lineage giving rise to choanocytes and the other to pinacocytes and cells of the mesohyl. These results suggest that a rapid ontogenetic shift in the allogeneic response of A. queenslandica occurs about 2 weeks after the initiation of metamorphosis and that the molecular basis of this response is also involved in creating differential cell affinities that underlie the construction of the sponge body plan. Compatible with this proposition is the observation that cells from postlarvae that are allowed to develop for 2 weeks before contact do not fuse and form a distinct boundary between genotypes. The successful chimeras remained stable for the duration of the experiment (3 weeks) raising the possibility that reproductive chimeras might persist in the natural environment, with a single genotype giving rise to germ cells

  15. Xanthomonas campestris cell-cell signalling molecule DSF (diffusible signal factor) elicits innate immunity in plants and is suppressed by the exopolysaccharide xanthan.

    Science.gov (United States)

    Kakkar, Akanksha; Nizampatnam, Narasimha Rao; Kondreddy, Anil; Pradhan, Binod Bihari; Chatterjee, Subhadeep

    2015-11-01

    Several secreted and surface-associated conserved microbial molecules are recognized by the host to mount the defence response. One such evolutionarily well-conserved bacterial process is the production of cell-cell signalling molecules which regulate production of multiple virulence functions by a process known as quorum sensing. Here it is shown that a bacterial fatty acid cell-cell signalling molecule, DSF (diffusible signal factor), elicits innate immunity in plants. The DSF family of signalling molecules are highly conserved among many phytopathogenic bacteria belonging to the genus Xanthomonas as well as in opportunistic animal pathogens. Using Arabidopsis, Nicotiana benthamiana, and rice as model systems, it is shown that DSF induces a hypersensitivity reaction (HR)-like response, programmed cell death, the accumulation of autofluorescent compounds, hydrogen peroxide production, and the expression of the PATHOGENESIS-RELATED1 (PR-1) gene. Furthermore, production of the DSF signalling molecule in Pseudomonas syringae, a non-DSF-producing plant pathogen, induces the innate immune response in the N. benthamiana host plant and also affects pathogen growth. By pre- and co-inoculation of DSF, it was demonstrated that the DSF-induced plant defence reduces disease severity and pathogen growth in the host plant. In this study, it was further demonstrated that wild-type Xanthomonas campestris suppresses the DSF-induced innate immunity by secreting xanthan, the main component of extracellular polysaccharide. The results indicate that plants have evolved to recognize a widely conserved bacterial communication system and may have played a role in the co-evolution of host recognition of the pathogen and the communication machinery. PMID:26248667

  16. Telomere dysfunction and cell survival: Roles for distinct TIN2-containing complexes

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sahn-ho; Davalos, Albert R.; Heo, Seok-Jin; Rodier, Francis; Zou, Ying; Beausejour, Christian; Kaminker, Patrick; Yannone, Steven M.; Campisi, Judith

    2007-10-02

    Telomeres are maintained by three DNA binding proteins (TRF1, TRF2 and POT1), and several associated factors. One factor, TIN2, binds TRF1 and TRF2 directly and POT1 indirectly. Along with two other proteins, TPP1 and hRap1, these form a soluble complex that may be the core telomere maintenance complex. It is not clear whether sub-complexes also exist in vivo. We provide evidence for two TIN2 sub-complexes with distinct functions in human cells. We isolated these two TIN2 sub-complexes from nuclear lysates of unperturbed cells and cells expressing TIN2 mutants TIN2-13, TIN2-15C, which cannot bind TRF2 or TRF1, respectively. In cells with wild-type p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere uncapping and eventual growth arrest. In cells lacking p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere dysfunction and cell death. Our findings suggest that distinct TIN2 complexes exist, and that TIN2-15C-sensitive subcomplexes are particularly important for cell survival in the absence of functional p53.

  17. W-cells in the cat retina: correlated morphological and physiological evidence for two distinct classes.

    Science.gov (United States)

    Stanford, L R

    1987-01-01

    Intracellular recording and iontophoresis of horseradish peroxidase were used to study the morphology of physiologically characterized W-cells in the cat retina. The recording experiments were performed in an in vivo preparation to allow the responses of these retinal ganglion cells to be compared with previous functional studies of these neurons. The physiological and morphological characteristics of 16 injected and recovered retinal W-cells were compared with similar data from 14 retinal X-cells injected in the same preparations. The soma sizes of retinal W-cells were found to fall into two distinct groups. The somata of the phasic W-cells, at every eccentricity, were smaller than the somata of tonic W-cells, with no overlap between the two distributions. Soma sizes of the tonic W-cells fell into the previously described "medium-sized" range of retinal ganglion cell soma sizes and were similar to, although slightly larger than, the soma sizes of physiologically identified beta- or X-cells. The dendritic arbors of all of the cells physiologically classified as tonic W-cells were similar. Every example of this type had four to five primary dendrites that branched a short distance from the soma to form a circular or cruciate dendritic arbor. The dendritic arrays of these cells were easily distinguishable from the compact dendritic arbors of the physiologically identified X-cells. The dendritic arbors of the phasic W-cells were much more heterogeneous, ranging from sparse, wide dendritic arbors to very compact dendritic arbors with many fine branches. No significant correlation was found between the extent of the dendritic arbor and the distance from the area centralis for either the tonic W-cells or the phasic W-cells. The axons of the tonic and phasic W-cells differed from one another and from X-cells on a number of different morphological and physiological measures. The intraretinal segments of the axons of the phasic W-cells had the smallest diameters of the

  18. Differences in TCDD-elicited gene expression profiles in human HepG2, mouse Hepa1c1c7 and rat H4IIE hepatoma cells

    Directory of Open Access Journals (Sweden)

    Burgoon Lyle D

    2011-04-01

    Full Text Available Abstract Background 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD is an environmental contaminant that elicits a broad spectrum of toxic effects in a species-specific manner. Current risk assessment practices routinely extrapolate results from in vivo and in vitro rodent models to assess human risk. In order to further investigate the species-specific responses elicited by TCDD, temporal gene expression responses in human HepG2, mouse Hepa1c1c7 and rat H4IIE cells were compared. Results Microarray analysis identified a core set of conserved gene expression responses across species consistent with the role of AhR in mediating adaptive metabolic responses. However, significant species-specific as well as species-divergent responses were identified. Computational analysis of the regulatory regions of species-specific and -divergent responses suggests that dioxin response elements (DREs are involved. These results are consistent with in vivo rat vs. mouse species-specific differential gene expression, and more comprehensive comparative DRE searches. Conclusions Comparative analysis of human HepG2, mouse Hepa1c1c7 and rat H4IIE TCDD-elicited gene expression responses is consistent with in vivo rat-mouse comparative gene expression studies, and more comprehensive comparative DRE searches, suggesting that AhR-mediated gene expression is species-specific.

  19. Melanopsin-expressing ganglion cells on macaque and human retinas form two morphologically distinct populations.

    Science.gov (United States)

    Liao, Hsi-Wen; Ren, Xiaozhi; Peterson, Beth B; Marshak, David W; Yau, King-Wai; Gamlin, Paul D; Dacey, Dennis M

    2016-10-01

    The long-term goal of this research is to understand how retinal ganglion cells that express the photopigment melanopsin, also known as OPN4, contribute to vision in humans and other primates. Here we report the results of anatomical studies using our polyclonal antibody specifically against human melanopsin that confirm and extend previous descriptions of melanopsin cells in primates. In macaque and human retina, two distinct populations of melanopsin cells were identified based on dendritic stratification in either the inner or the outer portion of the inner plexiform layer (IPL). Variation in dendritic field size and cell density with eccentricity was confirmed, and dendritic spines, a new feature of melanopsin cells, were described. The spines were the sites of input from DB6 diffuse bipolar cell axon terminals to the inner stratifying type of melanopsin cells. The outer stratifying melanopsin type received inputs from DB6 bipolar cells via a sparse outer axonal arbor. Outer stratifying melanopsin cells also received inputs from axon terminals of dopaminergic amacrine cells. On the outer stratifying melanopsin cells, ribbon synapses from bipolar cells and conventional synapses from amacrine cells were identified in electron microscopic immunolabeling experiments. Both inner and outer stratifying melanopsin cell types were retrogradely labeled following tracer injection in the lateral geniculate nucleus (LGN). In addition, a method for targeting melanopsin cells for intracellular injection using their intrinsic fluorescence was developed. This technique was used to demonstrate that melanopsin cells were tracer coupled to amacrine cells and would be applicable to electrophysiological experiments in the future. J. Comp. Neurol. 524:2845-2872, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc. PMID:26972791

  20. Modification of collagen IV by glucose or methylglyoxal alters distinct mesangial cell functions.

    Science.gov (United States)

    Pozzi, Ambra; Zent, Roy; Chetyrkin, Sergei; Borza, Corina; Bulus, Nada; Chuang, Peale; Chen, Dong; Hudson, Billy; Voziyan, Paul

    2009-10-01

    Diabetic nephropathy (DN) affects both glomerular cells and the extracellular matrix (ECM), yet the pathogenic mechanisms involving cell-matrix interactions are poorly understood. Glycation alters integrin-dependent cell-ECM interactions, and perturbation of these interactions results in severe renal pathology in diabetic animals. Here, we investigated how chemical modifications of the ECM by hyperglycemia and carbonyl stress, two major features of the diabetic milieu, affect mesangial cell functions. Incubation of collagen IV with pathophysiological levels of either the carbonyl compound methylglyoxal (MGO) or glucose resulted in modification of arginine or lysine residues, respectively. Mouse mesangial cells plated on MGO-modified collagen IV showed decreased adhesion and migration. Cells plated on glucose-modified collagen IV showed reduced proliferation and migration and increased collagen IV production. Inhibiting glucose-mediated oxidative modification of collagen IV lysine residues rescued the alterations in cell growth, migration, and collagen synthesis. We propose that diabetic ECM affects mesangial cell functions via two distinct mechanisms: modification of arginine residues by MGO inhibits cell adhesion, whereas oxidative modification of lysine residues by glucose inhibits cell proliferation and increases collagen IV production. These mechanisms may contribute to mesangial cell hypertrophy and matrix expansion in DN. PMID:19608705

  1. Precursors of murine B lymphocytes. Physical and functional characterization, and distinctions from myeloid stem cells

    International Nuclear Information System (INIS)

    The emergence of functional B cells was monitored in irradiated or unirradiated CBA/N recipients of either adult bone marrow or fetal liver from CBA/HT6T6 donors. The cells that are primarily responsible for the generation of B lymphocytes, at least during the first 6 weeks, are rapidly sedimenting, lack surface immunoglobulin, and are found in both the adult bone marrow and the fetal liver from day 12 onward. These pre-B cells are distinct from the colony-forming unit spleen (CFU-s). The pre-B cells are not homogeneous, however, as both physical and functional differences are found. These observations reinforce the thesis that committed progenitor cells for the humoral immune system are formed early in development and thereafter constitute the major precursor pool for the generation of B lymphocytes

  2. Two FOXP3(+)CD4(+) T cell subpopulations distinctly control the prognosis of colorectal cancers.

    Science.gov (United States)

    Saito, Takuro; Nishikawa, Hiroyoshi; Wada, Hisashi; Nagano, Yuji; Sugiyama, Daisuke; Atarashi, Koji; Maeda, Yuka; Hamaguchi, Masahide; Ohkura, Naganari; Sato, Eiichi; Nagase, Hirotsugu; Nishimura, Junichi; Yamamoto, Hirofumi; Takiguchi, Shuji; Tanoue, Takeshi; Suda, Wataru; Morita, Hidetoshi; Hattori, Masahira; Honda, Kenya; Mori, Masaki; Doki, Yuichiro; Sakaguchi, Shimon

    2016-06-01

    CD4(+) T cells that express the forkhead box P3 (FOXP3) transcription factor function as regulatory T (Treg) cells and hinder effective immune responses against cancer cells. Abundant Treg cell infiltration into tumors is associated with poor clinical outcomes in various types of cancers. However, the role of Treg cells is controversial in colorectal cancers (CRCs), in which FOXP3(+) T cell infiltration indicated better prognosis in some studies. Here we show that CRCs, which are commonly infiltrated by suppression-competent FOXP3(hi) Treg cells, can be classified into two types by the degree of additional infiltration of FOXP3(lo) nonsuppressive T cells. The latter, which are distinguished from FOXP3(+) Treg cells by non-expression of the naive T cell marker CD45RA and instability of FOXP3, secreted inflammatory cytokines. Indeed, CRCs with abundant infiltration of FOXP3(lo) T cells showed significantly better prognosis than those with predominantly FOXP3(hi) Treg cell infiltration. Development of such inflammatory FOXP3(lo) non-Treg cells may depend on secretion of interleukin (IL)-12 and transforming growth factor (TGF)-β by tissues and their presence was correlated with tumor invasion by intestinal bacteria, especially Fusobacterium nucleatum. Thus, functionally distinct subpopulations of tumor-infiltrating FOXP3(+) T cells contribute in opposing ways to determining CRC prognosis. Depletion of FOXP3(hi) Treg cells from tumor tissues, which would augment antitumor immunity, could thus be used as an effective treatment strategy for CRCs and other cancers, whereas strategies that locally increase the population of FOXP3(lo) non-Treg cells could be used to suppress or prevent tumor formation. PMID:27111280

  3. Clonal-Level Responses of Functionally Distinct Hematopoietic Stem Cells to Trophic Factors

    OpenAIRE

    Mallaney, Cates; Kothari, Alok; Martens, Andrew; Challen, Grant A.

    2013-01-01

    Recent findings from several groups have identified distinct classes of hematopoietic stem cells (HSCs) in the bone marrow, each with inherent functional biases in terms of their differentiation, self-renewal, proliferation and lifespan. It has previously been demonstrated that myeloid- and lymphoid-biased HSCs can be prospectively enriched based on their degree of Hoechst dye efflux. In the present study, we used differential Hoechst efflux to enrich lineage-biased HSC subtypes and analyzed ...

  4. Connectivity from OR37 expressing olfactory sensory neurons to distinct cell types in the hypothalamus

    Directory of Open Access Journals (Sweden)

    Andrea eBader

    2012-11-01

    Full Text Available Olfactory sensory neurons which express a member from the OR37 subfamily of odorant receptor genes are wired to the main olfactory bulb in a unique monoglomerular fashion; from these glomeruli an untypical connectivity into higher brain centers exists. In the present study we have investigated by DiI and transsynaptic tracing approaches how the connection pattern from these glomeruli into distinct hypothalamic nuclei is organized. The application of DiI onto the ventral domain of the bulb which harbors the OR37 glomeruli resulted in the labeling of fibers within the paraventricular and supraoptic nucleus of the hypothalamus; some of these fibers were covered with varicose-like structures. No DiI-labeled cell somata were detectable in these nuclei. The data indicate that projection neurons which originate in the OR37 region of the main olfactory bulb form direct connections into these nuclei. The cells that were labeled by the transsynaptic tracer WGA in these nuclei were further characterized. Their distribution pattern in the paraventricular nucleus was reminiscent of cells which produce distinct neuropeptides. Double labeling experiments confirmed that they contained vasopressin, but not the related neuropeptide oxytocin. Morphological analysis revealed that they comprise of magno- and parvocellular cells. A comparative investigation of the WGA-positive cells in the supraoptic nucleus demonstrated that these were vasopressin-positive, as well, whereas oxytocin-producing cells of this nucleus also contained no transsynaptic tracer. Together, the data demonstrate a connectivity from OR37 expressing sensory neurons to distinct hypothalamic neurons with the same neuropeptide content.

  5. Plumbagin elicits differential proteomic responses mainly involving cell cycle, apoptosis, autophagy, and epithelial-to-mesenchymal transition pathways in human prostate cancer PC-3 and DU145 cells

    Directory of Open Access Journals (Sweden)

    Qui JX

    2015-01-01

    critical role in the regulation of cell cycle, apoptosis, autophagy, epithelial to mesenchymal transition (EMT, and reactive oxygen species generation. The proteomic study showed substantial differences in response to PLB treatment between PC-3 and DU145 cells. PLB treatment significantly modulated the expression of critical proteins that regulate cell cycle, apoptosis, and EMT signaling pathways in PC-3 cells but not in DU145 cells. Consistently, our Western blotting analysis validated the bioinformatic and proteomic data and confirmed the modulating effects of PLB on important proteins that regulated cell cycle, apoptosis, autophagy, and EMT in PC-3 and DU145 cells. The data from the Western blot assay could not display significant differences between PC-3 and DU145 cells. These findings indicate that PLB elicits different proteomic responses in PC-3 and DU145 cells involving proteins and pathways that regulate cell cycle, apoptosis, autophagy, reactive oxygen species production, and antioxidation/oxidation homeostasis. This is the first systematic study with integrated computational, proteomic, and functional analyses revealing the networks of signaling pathways and differential proteomic responses to PLB treatment in prostate cancer cells. Quantitative proteomic analysis using SILAC represents an efficient and highly sensitive approach to identify the target networks of anticancer drugs like PLB, and the data may be used to discriminate the molecular and clinical subtypes, and to identify new therapeutic targets and biomarkers, for prostate cancer. Further studies are warranted to explore the potential of quantitative proteomic analysis in the identification of new targets and biomarkers for prostate cancer.Keywords: EMT, proteomics, SILAC

  6. The B-cell identity factor Pax5 regulates distinct transcriptional programmes in early and late B lymphopoiesis

    OpenAIRE

    Revilla-i-Domingo, Roger; Bilic, Ivan; Vilagos, Bojan; Tagoh, Hiromi; Ebert, Anja; Tamir, Ido M.; Smeenk, Leonie; Trupke, Johanna; Sommer, Andreas; Jaritz, Markus; Busslinger, Meinrad

    2012-01-01

    Genome-wide sequencing approaches reveal that the transcription factor Pax5 controls the identity and function of B cells by regulating the expression of distinct target genes in pro-B and mature B cells.

  7. A Two-Component DNA-Prime/Protein-Boost Vaccination Strategy for Eliciting Long-Term, Protective T Cell Immunity against Trypanosoma cruzi

    OpenAIRE

    Shivali Gupta; Garg, Nisha J.

    2015-01-01

    In this study, we evaluated the long-term efficacy of a two-component subunit vaccine against Trypanosoma cruzi infection. C57BL/6 mice were immunized with TcG2/TcG4 vaccine delivered by a DNA-prime/Protein-boost (D/P) approach and challenged with T. cruzi at 120 or 180 days post-vaccination (dpv). We examined whether vaccine-primed T cell immunity was capable of rapid expansion and intercepting the infecting T. cruzi. Our data showed that D/P vaccine elicited CD4+ (30-38%) and CD8+ (22-42%) ...

  8. DISTINCTIVE LOCALIZATION OF GROUP 3 LATE EMBRYOGENESIS ABUNDANT SYNTHESIZING CELLS DURING BRINE SHRIMP DEVELOPMENT.

    Science.gov (United States)

    Kim, Bo Yong; Song, Hwa Young; Kim, Mi Young; Lee, Bong Hee; Kim, Kyung Joo; Jo, Kyung Jin; Kim, Suhng Wook; Lee, Seung Gwan; Lee, Boo Hyung

    2015-07-01

    Despite numerous studies on late embryogenesis abundant (LEA) proteins, their functions, roles, and localizations during developmental stages in arthropods remain unknown. LEA proteins protect crucial proteins against osmotic stress during the development and growth of various organisms. Thus, in this study, fluorescence in situ hybridization was used to determine the crucial regions protected against osmotic stress as well as the distinctive localization of group 3 (G3) LEA(+) cells during brine shrimp development. Several cell types were found to synthesize G3 LEA RNA, including neurons, muscular cells, APH-1(+) cells, and renal cells. The G3 LEA(+) neuronal cell bodies outside of the mushroom body projected their axonal bundles to the central body, but those inside the mushroom body projected their axonal bundles toward the deutocerebrum without innervating the central body. The cell bodies inside the mushroom body received axons of the G3 LEA(+) sensory cells at the medial ventral cup of the nauplius eye. Several glands were found to synthesize G3 LEA RNA during the nauplius stages of brine shrimp, including the sinus, antennal I and II, salt, and three ectodermal glands. This study provides the first demonstration of the formation of G3 LEA(+) sinus glands at the emergence stages of brine shrimp. These results suggest that G3 LEA protein is synthesized in several cell types. In particular, specific glands play crucial roles during the emergence and nauplius stages of brine shrimp. PMID:25781424

  9. Interleukin-1 exerts distinct actions on different cell types of the brain in vitro

    Directory of Open Access Journals (Sweden)

    Ying An

    2011-01-01

    Full Text Available Ying An, Qun Chen, Ning QuanDepartment of Oral Biology, Ohio State University, Columbus, OH, USAAbstract: Interleukin-1 (IL-1 is a critical neuroinflammatory mediator in the central nervous system (CNS. In this study, we investigated the effect of IL-1 on inducing inflammation-related gene expression in three astrocyte, two microglial, and one brain endothelial cell line. Interleukin-1 beta (IL-1β is found to be produced by the two microglial cell lines constitutively, but these cells do not respond to IL-1β stimulation. The three astrocyte cell lines responded to IL-1ß stimulation by expressing MCP-1, CXCL-1, and VCAM-1, but different subtypes of astrocytes exhibited different expression profiles after IL-1β stimulation. The brain endothelial cells showed strongest response to IL-1β by producing MCP-1, CXCL-1, VCAM-1, ICAM-1, IL-6, and COX-2 mRNA. The induction of endothelial COX-2 mRNA is shown to be mediated by p38 MAPK pathway, whereas the induction of other genes is mediated by the NF-κB pathway. These results demonstrate that IL-1 exerts distinct cell type-specific action in CNS cells and suggest that IL-1-mediated neuroinflammation is the result of the summation of multiple responses from different cell types in the CNS to IL-1.Keywords: astrocyte, microglia, endothelial cells, signal transduction pathways, gene expression 

  10. Distinct Gene Regulatory Pathways for Human Innate versus Adaptive Lymphoid Cells.

    Science.gov (United States)

    Koues, Olivia I; Collins, Patrick L; Cella, Marina; Robinette, Michelle L; Porter, Sofia I; Pyfrom, Sarah C; Payton, Jacqueline E; Colonna, Marco; Oltz, Eugene M

    2016-05-19

    Innate lymphoid cells (ILCs) serve as sentinels in mucosal tissues, sensing release of soluble inflammatory mediators, rapidly communicating danger via cytokine secretion, and functioning as guardians of tissue homeostasis. Although ILCs have been extensively studied in model organisms, little is known about these "first responders" in humans, especially their lineage and functional kinships to cytokine-secreting T helper (Th) cell counterparts. Here, we report gene regulatory circuitries for four human ILC-Th counterparts derived from mucosal environments, revealing that each ILC subset diverges as a distinct lineage from Th and circulating natural killer cells but shares circuitry devoted to functional polarization with their Th counterparts. Super-enhancers demarcate cohorts of cell-identity genes in each lineage, uncovering new modes of regulation for signature cytokines, new molecules that likely impart important functions to ILCs, and potential mechanisms for autoimmune disease SNP associations within ILC-Th subsets. PMID:27156452

  11. Infrared signatures of Bacillus bacteria: Clear IR distinctions between sporulated and vegetative cells with chemical assignments

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, Timothy J.; Williams, Stephen D.; Valentine, Nancy B.; Su, Yin-Fong; Kreuzer-Martin, Helen W.; Wahl, Karen L.; Forrester, Joel B.

    2009-05-08

    This paper highlights the distinctions between the infrared (IR) absorption spectra of vegetative versus sporulated Bacillus bacteria. It is observed that there are unique signatures clearly associated with either the sporulated or the vegetative state, and that vegetative cells (and associated debris) can contribute to the spore spectra. A distinct feature at ~1739 cm-1 appears to be unique to vegetative cell spectra, and can also be used as an indicator of vegetative cells or cell debris in the spore spectra. The data indicate the band is caused by a phospholipid carbonyl bond and are consistent with, but do not prove it to be, either phosphatidyl ethanolamine (PE) or phosphatidyl glycerol (PG), the two major classes of phospholipids found in vegetative cells of Bacillus species. The endospore spectra show characteristic peaks at 1441, 1277, and 1015 cm-1 along with a distinct quartet of peaks at 766, 725, 701, and 659 cm-1. These are clearly associated with calcium dipicolinate trihydrate, CaDP•3H2O. We emphasize that the spore peaks, especially the quartet, arise from the calcium dipicolinate trihydrate and not from dipicolinic acid or other dipicolinate hydrate salts. The CaDP•3H2O vibrational peaks and the effects of hydration were studied using quantum chemistry in the PQS software package. The quartet is associated with many motions including contributions from the Ca2+ counterion and hydration waters including Ca-O-H bends, H2O-Ca-O torsions and O-C-O bends. The 1441 and 1015 cm-1 modes are planar pyridine modes with the 1441 mode primarily a ring C-N stretch and the 1015 mode primarily a ring C-C stretch.

  12. Infrared Signatures of Bacillus Bacteria: Clear IR Distinctions Between Sporulated and Vegetative Cells with Chemical Assighments

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, Timothy J.; Williams, Stephen D.; Valentine, Nancy B.; Su, Yin-Fong; Kreuzer-Martin, Helen W.; Wahl, Karen L.; Forrester, Joel B.

    2009-05-04

    This paper highlights the distinctions between the infrared (IR) absorption spectra of vegetative versus sporulated Bacillus bacteria. It is observed that there are unique signatures clearly associated with either the sporulated or the vegetative state, and that vegetative cells (and associated debris) can contribute to the spore spectra. A distinct feature at ~1739 cm-1 appears to be unique to vegetative cell spectra, and can also be used as an indicator of vegetative cells or cell debris in the spore spectra. The data indicate the band is caused by a phospholipid carbonyl bond and are consistent with, but do not prove it to be, either phosphatidyl ethanolamine (PE) or phosphatidyl glycerol (PG), the two major classes of phospholipids found in vegetative cells of Bacillus species. The endospore spectra show characteristic peaks at 1441, 1277, and 1015 cm-1 along with a distinct quartet of peaks at 766, 725, 701, and 659 cm-1. These are clearly associated with calcium dipicolinate trihydrate, CaDP•3H2O. We emphasize that the spore peaks, especially the quartet, arise from the calcium dipicolinate trihydrate and not from dipicolinic acid or other dipicolinate hydrate salts. The CaDP•3H2O vibrational peaks and the effects of hydration were studied using quantum chemistry in the PQS software package. The quartet is associated with many motions including contributions from the Ca2+ counterion and hydration waters including Ca-O-H bends, H2O-Ca-O torsions and O-C-O bends. The 1441 and 1015 cm-1 modes are planar pyridine modes with the 1441 mode primarily a ring C-N stretch and the 1015 mode primarily a ring C-C stretch.

  13. 3-Nitrofluoranthene (3-NF) but not 3-aminofluoranthene (3-AF) elicits apoptosis as well as programmed necrosis in Hepa1c1c7 cells

    International Nuclear Information System (INIS)

    In this study, we show that the environmental pollutant, 3-nitrofluoranthene (3-NF) but not its amine form, 3-aminofluoranthene (3-AF), induces apoptosis as well as regulated necrosis with necroptotic features in Hepa1c1c7 cells. Upon exposure to 3-NF, both typical apoptotic and necrotic cells were observed. A large number of the cells exhibited a characteristic partial nuclear chromatin condensation. Cycloheximide completely attenuated 3-NF-induced cell death. Activation of caspase-8, -9, and -3 were observed. Moreover, Z-VAD-FMK decreased the apoptotic cells, whereas the number of propidium iodide (PI)-positive cells with partial chromatin condensation was reduced by Nec-1, an inhibitor of receptor interacting protein (RIP-1). Cyp1a1, but not nitric oxide synthase (NOS), appears to be involved in activation of 3-NF to reactive metabolites. Increase in the number as well as size of lysosomes, myelinosomes, and activation of autophagy were also observed. 3-NF induced phosphorylation of ERK1/2, JNK and p38 MAPKs. Interestingly, while inhibitors of ERK1/2 and JNK reduced apoptotic as well as necrotic cell death, the p38 inhibitor, SB202190 reduced only the necrotic cell death. Taken together, 3-NF elicits both apoptosis and a caspase-independent programmed cell death (PCD) with autophagic characteristics. Conversely, with 3-AF, no apparent cytotoxic effects besides a reduction in cell proliferation was observed

  14. MicroRNAs define distinct human neuroblastoma cell phenotypes and regulate their differentiation and tumorigenicity

    International Nuclear Information System (INIS)

    Neuroblastoma (NB) is the most common extracranial solid tumor in children. NB tumors and derived cell lines are phenotypically heterogeneous. Cell lines are classified by phenotype, each having distinct differentiation and tumorigenic properties. The neuroblastic phenotype is tumorigenic, has neuronal features and includes stem cells (I-cells) and neuronal cells (N-cells). The non-neuronal phenotype (S-cell) comprises cells that are non-tumorigenic with features of glial/smooth muscle precursor cells. This study identified miRNAs associated with each distinct cell phenotypes and investigated their role in regulating associated differentiation and tumorigenic properties. A miRNA microarray was performed on the three cell phenotypes and expression verified by qRT-PCR. miRNAs specific for certain cell phenotypes were modulated using miRNA inhibitors or stable transfection. Neuronal differentiation was induced by RA; non-neuronal differentiation by BrdU. Changes in tumorigenicity were assayed by soft agar colony forming ability. N-myc binding to miR-375 promoter was assayed by chromatin-immunoprecipitation. Unsupervised hierarchical clustering of miRNA microarray data segregated neuroblastic and non-neuronal cell lines and showed that specific miRNAs define each phenotype. qRT-PCR validation confirmed that increased levels of miR-21, miR-221 and miR-335 are associated with the non-neuronal phenotype, whereas increased levels of miR-124 and miR-375 are exclusive to neuroblastic cells. Downregulation of miR-335 in non-neuronal cells modulates expression levels of HAND1 and JAG1, known modulators of neuronal differentiation. Overexpression of miR-124 in stem cells induces terminal neuronal differentiation with reduced malignancy. Expression of miR-375 is exclusive for N-myc-expressing neuroblastic cells and is regulated by N-myc. Moreover, miR-375 downregulates expression of the neuronal-specific RNA binding protein HuD. Thus, miRNAs define distinct NB cell phenotypes

  15. Overexpressed Genes/ESTs and Characterization of Distinct Amplicons on 17823 in Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ayse E. Erson

    2001-01-01

    Full Text Available 17823 is a frequent site of gene amplification in breast cancer. Several lines of evidence suggest the presence of multiple amplicons on 17823. To characterize distinct amplicons on 17823 and localize putative oncogenes, we screened genes and expressed sequence tags (ESTs in existing physical and radiation hybrid maps for amplification and overexpression in breast cancer cell lines by semiquantitative duplex PCR, semiquantitative duplex RT-PCR, Southern blot, Northern blot analyses. We identified two distinct amplicons on 17823, one including TBX2 and another proximal region including RPS6KB1 (PS6K and MUL. In addition to these previously reported overexpressed genes, we also identified amplification and overexpression of additional uncharacterized genes and ESTs, some of which suggest potential oncogenic activity. In conclusion, we have further defined two distinct regions of gene amplification and overexpression on 17823 with identification of new potential oncogene candidates. Based on the amplification and overexpression patterns of known and as of yet unrecognized genes on 17823, it is likely that some of these genes mapping to the discrete amplicons function as oncogenes and contribute to tumor progression in breast cancer cells.

  16. Ludic Elicitation: Using Games for Knowledge Elicitation

    Science.gov (United States)

    Cao, Yan

    2014-01-01

    Knowledge elicitation from human beings is important for many fields, such as decision support systems, risk communication, and customer preference studying. Traditional approaches include observations, questionnaires, structured and semi-structured interviews, and group discussions. Many publications have been studying different techniques for a…

  17. Accumulation of distinct prelamin A variants in human diploid fibroblasts differentially affects cell homeostasis

    Energy Technology Data Exchange (ETDEWEB)

    Candelario, Jose; Borrego, Stacey [Department of Molecular Microbiology and Immunology, Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033 (United States); Reddy, Sita, E-mail: sitaredd@usc.edu [Department of Biochemistry and Molecular Biology, Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033 (United States); Comai, Lucio, E-mail: comai@usc.edu [Department of Molecular Microbiology and Immunology, Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033 (United States)

    2011-02-01

    Lamin A is a component of the nuclear lamina that plays a major role in the structural organization and function of the nucleus. Lamin A is synthesized as a prelamin A precursor which undergoes four sequential post-translational modifications to generate mature lamin A. Significantly, a large number of point mutations in the LMNA gene cause a range of distinct human disorders collectively known as laminopathies. The mechanisms by which mutations in lamin A affect cell function and cause disease are unclear. Interestingly, recent studies have suggested that alterations in the normal lamin A pathway can contribute to cellular dysfunction. Specifically, we and others have shown, at the cellular level, that in the absence of mutations or altered splicing events, increased expression of wild-type prelamin A results in a growth defective phenotype that resembles that of cells expressing the mutant form of lamin A, termed progerin, associated with Hutchinson-Gilford Progeria syndrome (HGPS). Remarkably, the phenotypes of cells expressing elevated levels of wild-type prelamin A can be reversed by either treatment with farnesyltransferase inhibitors or overexpression of ZMPSTE24, a critical prelamin A processing enzyme, suggesting that minor increases in the steady-state levels of one or more prelamin A intermediates is sufficient to induce cellular toxicity. Here, to investigate the molecular basis of the lamin A pathway toxicity, we characterized the phenotypic changes occurring in cells expressing distinct prelamin A variants mimicking specific prelamin A processing intermediates. This analysis demonstrates that distinct prelamin A variants differentially affect cell growth, nuclear membrane morphology, nuclear distribution of lamin A and the fundamental process of transcription. Expression of prelamin A variants that are constitutively farnesylated induced the formation of lamin A aggregates and dramatic changes in nuclear membrane morphology, which led to reduced

  18. Mosaic vaccines elicit CD8+ T cell responses in monkeys that confer immune coverage of diverse HIV strains

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, Will [Los Alamos National Laboratory; Korber, Bette [Los Alamos National Laboratory

    2009-01-01

    Creation of a successful HIV vaccine will require the development of a strategy to generate cellular immunity with sufficient cross-clade breadth to deal with the extreme genetic diversity of the virus. Polyvalent mosaic immunogens derived from in silica recombination of natural strains of HIV are designed to induce cellular immune responses that maximally cover the sequence diversity of circulating virus isolates. Immunization of rhesus monkeys with plasmid DNA and recombinant vaccinia virus vaccine constructs expressing either consensus immunogens or polyvalent mosaic immunogens elicited a CD4+ T lymphocyte-biased response with comparably broad epitope-specific total T lymphocyte specificities. However, immunization with the mosaic immunogens induced HIV-specific CD8+ T lymphocyte responses with markedly greater depth and breadth. Therefore, the use of polyvalent mosaic immunogens is a promising strategy for a global vaccine for HIV.

  19. Experimental Measurements and Mathematical Modeling of Cytosolic Ca2+ Signatures upon Elicitation by Penta-N-acetylchitopentaose Oligosaccharides in Nicotiana tabacum Cell Cultures

    Directory of Open Access Journals (Sweden)

    Kalina Mrozek

    2013-11-01

    Full Text Available Plants have developed sophisticated recognition systems for different kinds of pathogens. Pathogen-associated molecular patterns (PAMPs can induce various defense mechanisms, e.g., the production of reactive oxygen species (ROS as an early event. Plant defense reactions are initiated by a signal transduction cascade involving the release of calcium ions (Ca2+ from both external and internal stores to the plant cytoplasm. This work focuses on the analysis of cytosolic Ca2+ signatures, experimentally and theoretically. Cytosolic Ca2+ signals were measured in Nicotiana tabacum plant cell cultures after elicitation with penta-N-acetylchitopentaose oligosaccharides (Ch5. In order to allow a mathematical simulation of the elicitor-triggered Ca2+ release, the Li and Rinzel model was adapted to the situation in plants. The main features of the Ca2+ response, like the specific shape of the Ca2+ transient and the dose-response relationship, could be reproduced very well. Repeated elicitation of the same cell culture revealed a refractory behavior with respect to the Ca2+ transients for this condition. Detailed analysis of the obtained data resulted in further modifications of the mathematical model, allowing a predictive simulation of Ch5-induced Ca2+ transients. The promising results may contribute to a deeper understanding of the underlying mechanisms governing plant defense.

  20. Interactions between cells with distinct mutations in c-MYC and Pten in prostate cancer.

    Directory of Open Access Journals (Sweden)

    Jongchan Kim

    2009-07-01

    Full Text Available In human somatic tumorigenesis, mutations are thought to arise sporadically in individual cells surrounded by unaffected cells. This contrasts with most current transgenic models where mutations are induced synchronously in entire cell populations. Here we have modeled sporadic oncogene activation using a transgenic mouse in which c-MYC is focally activated in prostate luminal epithelial cells. Focal c-MYC expression resulted in mild pathology, but prostate-specific deletion of a single allele of the Pten tumor suppressor gene cooperated with c-MYC to induce high grade prostatic intraepithelial neoplasia (HGPIN/cancer lesions. These lesions were in all cases associated with loss of Pten protein expression from the wild type allele. In the prostates of mice with concurrent homozygous deletion of Pten and focal c-MYC activation, double mutant (i.e. c-MYC+;Pten-null cells were of higher grade and proliferated faster than single mutant (Pten-null cells within the same glands. Consequently, double mutant cells outcompeted single mutant cells despite the presence of increased rates of apoptosis in the former. The p53 pathway was activated in Pten-deficient prostate cells and tissues, but c-MYC expression shifted the p53 response from senescence to apoptosis by repressing the p53 target gene p21(Cip1. We conclude that c-MYC overexpression and Pten deficiency cooperate to promote prostate tumorigenesis, but a p53-dependent apoptotic response may present a barrier to further progression. Our results highlight the utility of inducing mutations focally to model the competitive interactions between cell populations with distinct genetic alterations during tumorigenesis.

  1. Interdisciplinary Evaluation of Broadly-Reactive HLA Class II Restricted Epitopes Eliciting HIV-Specific CD4+T Cell Responses

    DEFF Research Database (Denmark)

    Buggert, M.; Norström, M.; Lundegaard, Claus; Lund, Ole; Nielsen, Morten; Karlsson, A. C.

    2011-01-01

    Background: CD4+ T cells orchestrate immune protection by ‘‘helping’’ other cells of our immune system to clear viral infections. It is well known that the preferential infection and depletion of CD4+ T cells contributes to hampered systemic T cell help following HIV infection. However, the funct...

  2. Distinct modulation of allergic T cell responses by subcutaneous versus sublingual allergen-specific immunotherapy

    DEFF Research Database (Denmark)

    Schulten, Véronique; Tripple, Victoria; Andersen, Kristian Aasbjerg; Backer, Vibeke; Lund, Gitte; Würtzen, Peter Adler; Sette, Alessandro; Peters, Bjoern

    2016-01-01

    mechanisms involved have not been fully explored. OBJECTIVE: To compare changes in the allergen-specific T cell response induced by subcutaneous versus sublingual administration of allergen-specific immunotherapy (AIT). METHODS: Grass pollen allergic patients were randomized into groups receiving either SCIT......: The most dominant immunological changes on a cellular level was a decrease in IL-5 in the SCIT group and a significant, transient increase of IL-10 observed after 10 months of treatment in both treated groups. The distinct routes of AIT administration may induce different immune-modulatory mechanisms...

  3. Functional melanocytes are readily reprogrammable from multilineage-differentiating stress-enduring (muse) cells, distinct stem cells in human fibroblasts.

    Science.gov (United States)

    Tsuchiyama, Kenichiro; Wakao, Shohei; Kuroda, Yasumasa; Ogura, Fumitaka; Nojima, Makoto; Sawaya, Natsue; Yamasaki, Kenshi; Aiba, Setsuya; Dezawa, Mari

    2013-10-01

    The induction of melanocytes from easily accessible stem cells has attracted attention for the treatment of melanocyte dysfunctions. We found that multilineage-differentiating stress-enduring (Muse) cells, a distinct stem cell type among human dermal fibroblasts, can be readily reprogrammed into functional melanocytes, whereas the remainder of the fibroblasts do not contribute to melanocyte differentiation. Muse cells can be isolated as cells positive for stage-specific embryonic antigen-3, a marker for undifferentiated human embryonic stem cells, and differentiate into cells representative of all three germ layers from a single cell, while also being nontumorigenic. The use of certain combinations of factors induces Muse cells to express melanocyte markers such as tyrosinase and microphthalmia-associated transcription factor and to show positivity for the 3,4-dihydroxy-L-phenylalanine reaction. When Muse cell-derived melanocytes were incorporated into three-dimensional (3D) cultured skin models, they localized themselves in the basal layer of the epidermis and produced melanin in the same manner as authentic melanocytes. They also maintained their melanin production even after the 3D cultured skin was transplanted to immunodeficient mice. This technique may be applicable to the efficient production of melanocytes from accessible human fibroblasts by using Muse cells, thereby contributing to autologous transplantation for melanocyte dysfunctions, such as vitiligo. PMID:23563197

  4. Temporal dynamics of distinct CA1 cell populations during unconscious state induced by ketamine.

    Directory of Open Access Journals (Sweden)

    Hui Kuang

    Full Text Available Ketamine is a widely used dissociative anesthetic which can induce some psychotic-like symptoms and memory deficits in some patients during the post-operative period. To understand its effects on neural population dynamics in the brain, we employed large-scale in vivo ensemble recording techniques to monitor the activity patterns of simultaneously recorded hippocampal CA1 pyramidal cells and various interneurons during several conscious and unconscious states such as awake rest, running, slow wave sleep, and ketamine-induced anesthesia. Our analyses reveal that ketamine induces distinct oscillatory dynamics not only in pyramidal cells but also in at least seven different types of CA1 interneurons including putative basket cells, chandelier cells, bistratified cells, and O-LM cells. These emergent unique oscillatory dynamics may very well reflect the intrinsic temporal relationships within the CA1 circuit. It is conceivable that systematic characterization of network dynamics may eventually lead to better understanding of how ketamine induces unconsciousness and consequently alters the conscious mind.

  5. Highly efficient derivation of ventricular cardiomyocytes from induced pluripotent stem cells with a distinct epigenetic signature

    Institute of Scientific and Technical Information of China (English)

    Huansheng Xu; Ibrahim J Domian; Erding Hu; Robert Willette; John Lepore; Alexander Meissner; Zhong Wang; Kenneth R Chien; B Alexander Yi; Hao Wu; Christoph Bock; Hongcang Gu; Kathy O Lui; Joo-Hye C Park; Ying Shao; Alyssa K Riley

    2012-01-01

    Cardiomyocytes derived from pluripotent stem cells can be applied in drug testing,disease modeling and cellbased therapy.However,without procardiogenic growth factors,the efficiency of cardiomyogenesis from pluripotent stem cells is usually low and the resulting cardiomyocyte population is heterogeneous.Here,we demonstrate that induced pluripotent stem cells (iPSCs) can be derived from murine ventricular myocytes (VMs),and consistent with other reports of iPSCs derived from various somatic cell types,VM-derived iPSCs (ViPSCs) exhibit a markedly higher propensity to spontaneously differentiate into beating cardiomyocytes as compared to genetically matched embryonic stem cells (ESCs) or iPSCs derived from tail-tip fibroblasts.Strikingly,the majority of ViPSC-derived cardiomyocytes display a ventricular phenotype.The enhanced ventricular myogenesis in ViPSCs is mediated via increased numbers of cardiovascular progenitors at early stages of differentiation.In order to investigate the mechanism of enhanced ventricular myogenesis from ViPSCs,we performed global gene expression and DNA methylation analysis,which revealed a distinct epigenetic signature that may be involved in specifying the VM fate in pluripotent stem cells.

  6. Sepsis Induces Hematopoietic Stem Cell Exhaustion and Myelosuppression through Distinct Contributions of TRIF and MYD88

    Directory of Open Access Journals (Sweden)

    Huajia Zhang

    2016-06-01

    Full Text Available Toll-like receptor 4 (TLR4 plays a central role in host responses to bacterial infection, but the precise mechanism(s by which its downstream signaling components coordinate the bone marrow response to sepsis is poorly understood. Using mice deficient in TLR4 downstream adapters MYD88 or TRIF, we demonstrate that both cell-autonomous and non-cell-autonomous MYD88 activation are major causes of myelosuppression during sepsis, while having a modest impact on hematopoietic stem cell (HSC functions. In contrast, cell-intrinsic TRIF activation severely compromises HSC self-renewal without directly affecting myeloid cells. Lipopolysaccharide-induced activation of MYD88 or TRIF contributes to cell-cycle activation of HSC and induces rapid and permanent changes in transcriptional programs, as indicated by persistent downregulation of Spi1 and CebpA expression after transplantation. Thus, distinct mechanisms downstream of TLR4 signaling mediate myelosuppression and HSC exhaustion during sepsis through unique effects of MyD88 and TRIF.

  7. Daptomycin resistance in enterococci is associated with distinct alterations of cell membrane phospholipid content.

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    Nagendra N Mishra

    Full Text Available BACKGROUND: The lipopeptide antibiotic, daptomycin (DAP interacts with the bacterial cell membrane (CM. Development of DAP resistance during therapy in a clinical strain of Enterococcus faecalis was associated with mutations in genes encoding enzymes involved in cell envelope homeostasis and phospholipid metabolism. Here we characterized changes in CM phospholipid profiles associated with development of DAP resistance in clinical enterococcal strains. METHODOLOGY: Using two clinical strain-pairs of DAP-susceptible and DAP-resistant E. faecalis (S613 vs. R712 and E. faecium (S447 vs. R446 recovered before and after DAP therapy, we compared four distinct CM profiles: phospholipid content, fatty acid composition, membrane fluidity and capacity to be permeabilized and/or depolarized by DAP. Additionally, we characterized the cell envelope of the E. faecium strain-pair by transmission electron microscopy and determined the relative cell surface charge of both strain-pairs. PRINCIPAL FINDINGS: Both E. faecalis and E. faecium mainly contained four major CM PLs: phosphatidylglycerol (PG, cardiolipin, lysyl-phosphatidylglycerol (L-PG and glycerolphospho-diglycodiacylglycerol (GP-DGDAG. In addition, E. faecalis CMs (but not E. faecium also contained: i phosphatidic acid; and ii two other unknown species of amino-containing PLs. Development of DAP resistance in both enterococcal species was associated with a significant decrease in CM fluidity and PG content, with a concomitant increase in GP-DGDAG. The strain-pairs did not differ in their outer CM translocation (flipping of amino-containing PLs. Fatty acid content did not change in the E. faecalis strain-pair, whereas a significant decrease in unsaturated fatty acids was observed in the DAP-resistant E. faecium isolate R446 (vs S447. Resistance to DAP in E. faecium was associated with distinct structural alterations of the cell envelope and cell wall thickening, as well as a decreased ability of DAP to

  8. Distinct roles of T-cell lymphopenia and the microbial flora for gastrointestinal and CNS autoimmunity.

    Science.gov (United States)

    Fischer, Henrike J; Witte, Ann-Kathrin; Walter, Lutz; Gröne, Hermann-Josef; van den Brandt, Jens; Reichardt, Holger M

    2016-05-01

    T-cell lymphopenia is a major risk factor for autoimmunity. Here we describe congenic Lewis (LEW) rats with a loss-of-function mutation in the Gimap5 gene, leading to a 92% reduction in peripheral T-cell numbers. Gimap5-deficient LEW rats developed eosinophilic autoimmune gastroenteritis accompanied by a 40-fold increase in IgE serum levels. This phenotype was ameliorated by antibiotic treatment, indicating a critical role of the microbial flora in the development of inflammatory bowel disease. Interestingly, Gimap5-deficient LEW rats showed strongly aggravated experimental autoimmune encephalomyelitis (EAE) after immunization with guinea pig myelin basic protein. This phenotype, however, persisted after antibiosis, confirming that the enhanced CNS autoimmune response in T-cell lymphopenic Gimap5-deficient LEW rats was unrelated to the composition of the microbial flora. Rather, it seems that it was caused by the 7-fold increase in the percentage of activated T cells producing IL-17 and IFN-γ, and the skewed T-cell receptor (TCR) repertoire, both of which were the result of T-cell lymphopenia and not affected by antibiosis. This notion was supported by the observation that adoptive T-cell transfer corrected the TCR repertoire and improved EAE. Collectively, our findings confirm a critical albeit differential role of T-cell lymphopenia in the susceptibility to organ-specific autoimmune responses.-Fischer, H. J., Witte, A.-K., Walter, L., Gröne, H.-J., van den Brandt, J., Reichardt, H. M. Distinct roles of T-cell lymphopenia and the microbial flora for gastrointestinal and CNS autoimmunity. PMID:26740263

  9. Activation of resting T cells: distinct roles of intact accessory cells, phorbol myristate acetate and interleukin 1

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    Davis, L.; Lipsky, P.E.

    1986-03-05

    The accessory cell (AC) signals involved in the activation of resting guinea pig T lymphocytes stimulated with mitogen (PHA), or the calcium ionophore, ionomycin (Ion) were examined. Activation of T cells was assessed by cell cycle analysis after acridine orange staining and /sup 3/H-thymidine incorporation. PHA-stimulated T cells depleted of all AC were unable to respond in the presence of phorbol myristate acetate (PMA), and/or interleukin 1 (IL 1). With suboptimal numbers of AC, PMA greatly augmented the number of T cells activated by PHA to enter and progress through the cell cycle, but only when present during the first few hours of culture. By contrast, IL 1 had little effect on the number of cells entering the cell cycle, although it enhanced S phase entry of the activated cells. IL 1 augmented DNA synthesis when added initially or later in culture. In contrast to the effects noted with PHA, PMA promoted activation and DNA synthesis of the majority of Ion stimulated cells in the complete absence of AC. IL 1 alone could not support Ion induced T cell activation although it enhanced T cell DNA synthesis in cultures stimulated by PMA and Ion. These studies indicate that intact AC, IL 1 and PMA-like signals play distinct roles in the progression of T cells through the initial cell cycle. Stimulation by Ion requires only PMA whereas PHA responses require intact AC and can be amplified by PMA. The major effect of IL 1 is to promote S phase entry of activated T cells.

  10. Molecular analysis of endothelial progenitor cell (EPC subtypes reveals two distinct cell populations with different identities

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    Simpson David A

    2010-05-01

    Full Text Available Abstract Background The term endothelial progenitor cells (EPCs is currently used to refer to cell populations which are quite dissimilar in terms of biological properties. This study provides a detailed molecular fingerprint for two EPC subtypes: early EPCs (eEPCs and outgrowth endothelial cells (OECs. Methods Human blood-derived eEPCs and OECs were characterised by using genome-wide transcriptional profiling, 2D protein electrophoresis, and electron microscopy. Comparative analysis at the transcript and protein level included monocytes and mature endothelial cells as reference cell types. Results Our data show that eEPCs and OECs have strikingly different gene expression signatures. Many highly expressed transcripts in eEPCs are haematopoietic specific (RUNX1, WAS, LYN with links to immunity and inflammation (TLRs, CD14, HLAs, whereas many transcripts involved in vascular development and angiogenesis-related signalling pathways (Tie2, eNOS, Ephrins are highly expressed in OECs. Comparative analysis with monocytes and mature endothelial cells clusters eEPCs with monocytes, while OECs segment with endothelial cells. Similarly, proteomic analysis revealed that 90% of spots identified by 2-D gel analysis are common between OECs and endothelial cells while eEPCs share 77% with monocytes. In line with the expression pattern of caveolins and cadherins identified by microarray analysis, ultrastructural evaluation highlighted the presence of caveolae and adherens junctions only in OECs. Conclusions This study provides evidence that eEPCs are haematopoietic cells with a molecular phenotype linked to monocytes; whereas OECs exhibit commitment to the endothelial lineage. These findings indicate that OECs might be an attractive cell candidate for inducing therapeutic angiogenesis, while eEPC should be used with caution because of their monocytic nature.

  11. Belief Elicitation in Experiments

    DEFF Research Database (Denmark)

    Blanco, Mariana; Engelmann, Dirk; Koch, Alexander;

    in the experiment. This raises two questions: (i) can we trust the existing belief elicitation results, (ii) can we avoid potential hedging confounds? Our results instill confidence regarding both issues. We propose an experimental design that eliminates hedging opportunities, and use this to test...... for the empirical relevance of hedging effects in the lab. We find no evidence for hedging, comparing the standard 'hedging-prone' belief elicitation treatment to a 'hedging-proof' design in a sequential prisoners' dilemma game. Our findings are strengthened by the absence of hedging even in an...... additional non-belief elicitation treatment using a financial investment frame, where hedging arguably would be most natural....

  12. Distinct Functions of Specialized Dendritic Cell Subsets in Atherosclerosis and the Road Ahead

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    Alma Zernecke

    2014-01-01

    Full Text Available Atherosclerotic vascular disease is modulated by immune mechanisms. Dendritic cells (DCs and T cells are present within atherosclerotic lesions and function as central players in the initiation and modulation of adaptive immune responses. In previous years, we have studied the functional contribution of distinct DC subsets in disease development, namely, that of CCL17-expressing DCs as well as that of plasmacytoid DCs that play specialized roles in disease development. This review focuses on important findings gathered in these studies and dissects the multifaceted contribution of CCL17-expressing DCs and pDCs to the pathogenesis of atherosclerosis. Furthermore, an outlook on future challenges faced when studying DCs in this detrimental disease are provided, and hurdles that will need to be overcome in order to enable a better understanding of the contribution of DCs to atherogenesis are discussed, a prerequisite for their therapeutic targeting in atherosclerosis.

  13. Spontaneously immortalised bovine mammary epithelial cells exhibit a distinct gene expression pattern from the breast cancer cells

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    Li Qianqian

    2010-10-01

    exhibit non-malignant transformation. Although this cell line displays altered patterns of gene expression, it is clearly distinct from malignant breast cancer cell line. It showed that co-inhibition of cellular senescence and mitochondrial apoptosis pathways coordinates BME65Cs cells immortalisation. Additionally, mechanisms other than gene mutation are likely to be involved in regulation of cellular functions. This study provides an insight into the relationship between cell senescence and immortalisation. BME65Cs cells will be useful in future studies of cellular senescence and tumorigenesis.

  14. Human embryonic stem cells and embryonal carcinoma cells have overlapping and distinct metabolic signatures.

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    Raed Abu Dawud

    Full Text Available While human embryonic stem cells (hESCs and human embryonal carcinoma cells (hECCs have been studied extensively at the levels of the genome, transcriptome, proteome and epigenome our knowledge of their corresponding metabolomes is limited. Here, we present the metabolic signatures of hESCs and hESCs obtained by untargeted gas chromatography coupled to mass spectrometry (GC-MS. Whilst some metabolites are common to both cell types, representing the self-renewal and house-keeping signatures, others were either higher (e.g., octadecenoic acid, glycerol-3-phosphate, 4-hydroxyproline or lower (e.g., glutamic acid, mannitol, malic acid, GABA in hESCs (H9 compared to hECCs (NTERA2, these represent cell type specific signatures. Further, our combined results of GC-MS and microarray based gene expression profiling of undifferentiated and OCT4-depleted hESCs are consistent with the Warburg effect which is increased glycolysis in embryonic cells and tumor cells in the presence of O(2 while oxidative phosphorylation (OXPHOS is impaired or even shut down. RNAi-based OCT4 knock down mediated differentiation resulted in the activation of the poised OXPHOS machinery by expressing missing key proteins such as NDUFC1, UQCRB and COX, increase in TCA cycle activity and decreased lactate metabolism. These results shed light on the metabolite layer of pluripotent stem cells and could potentially establish novel metabolic markers of self renewal and pluripotency.

  15. Distinct Tlr4-expressing cell compartments control neutrophilic and eosinophilic airway inflammation.

    Science.gov (United States)

    McAlees, J W; Whitehead, G S; Harley, I T W; Cappelletti, M; Rewerts, C L; Holdcroft, A M; Divanovic, S; Wills-Karp, M; Finkelman, F D; Karp, C L; Cook, D N

    2015-07-01

    Allergic asthma is a chronic, inflammatory lung disease. Some forms of allergic asthma are characterized by T helper type 2 (Th2)-driven eosinophilia, whereas others are distinguished by Th17-driven neutrophilia. Stimulation of Toll-like receptor 4 (TLR4) on hematopoietic and airway epithelial cells (AECs) contributes to the inflammatory response to lipopolysaccharide (LPS) and allergens, but the specific contribution of TLR4 in these cell compartments to airway inflammatory responses remains poorly understood. We used novel, conditionally mutant Tlr4(fl/fl) mice to define the relative contributions of AEC and hematopoietic cell Tlr4 expression to LPS- and allergen-induced airway inflammation. We found that Tlr4 expression by hematopoietic cells is critical for neutrophilic airway inflammation following LPS exposure and for Th17-driven neutrophilic responses to the house dust mite (HDM) lysates and ovalbumin (OVA). Conversely, Tlr4 expression by AECs was found to be important for robust eosinophilic airway inflammation following sensitization and challenge with these same allergens. Thus, Tlr4 expression by hematopoietic and airway epithelial cells controls distinct arms of the immune response to inhaled allergens. PMID:25465099

  16. Clonal-level responses of functionally distinct hematopoietic stem cells to trophic factors.

    Science.gov (United States)

    Mallaney, Cates; Kothari, Alok; Martens, Andrew; Challen, Grant A

    2014-04-01

    Recent findings from several groups have identified distinct classes of hematopoietic stem cells (HSCs) in the bone marrow, each with inherent functional biases in terms of their differentiation, self-renewal, proliferation, and lifespan. It has previously been demonstrated that myeloid- and lymphoid-biased HSCs can be prospectively enriched based on their degree of Hoechst dye efflux. In the present study, we used differential Hoechst efflux to enrich lineage-biased HSC subtypes and analyzed their functional potentials. Despite similar outputs in vitro, bone marrow transplantation assays revealed contrasting lineage differentiation in vivo. To stratify the molecular differences underlying these contrasting functional potentials at the clonal level, single-cell gene expression analysis was performed using the Fluidigm BioMark system and revealed dynamic expression of genes including Meis1, CEBP/α, Sfpi1, and Dnmt3a. Finally, single-cell gene expression analysis was used to unravel the opposing proliferative responses of lineage-biased HSCs to the growth factor TGF-β1, revealing a potential role for the cell cycle inhibitor Cdkn1c as molecular mediator. This work lends further credence to the concept of HSC heterogeneity, and it presents unprecedented molecular resolution of the HSC response to trophic factors using single-cell gene expression analysis. PMID:24373928

  17. Morphological Variability and Distinct Protein Profiles of Cultured and Endosymbiotic Symbiodinium cells Isolated from Exaiptasia pulchella

    Science.gov (United States)

    Pasaribu, Buntora; Weng, Li-Chi; Lin, I.-Ping; Camargo, Eddie; Tzen, Jason T. C.; Tsai, Ching-Hsiu; Ho, Shin-Lon; Lin, Mong-Rong; Wang, Li-Hsueh; Chen, Chii-Shiarng; Jiang, Pei-Luen

    2015-10-01

    Symbiodinium is a dinoflagellate that plays an important role in the physiology of the symbiotic relationships of Cnidarians such as corals and sea anemones. However, it is very difficult to cultivate free-living dinoflagellates after being isolated from the host, as they are very sensitive to environmental changes. How these symbiont cells are supported by the host tissue is still unclear. This study investigated the characteristics of Symbiodinium cells, particularly with respect to the morphological variability and distinct protein profiles of both cultured and endosymbiotic Symbiodinium which were freshly isolated from Exaiptasia pulchella. The response of the cellular morphology of freshly isolated Symbiodinium cells kept under a 12 h L:12 h D cycle to different temperatures was measured. Cellular proliferation was investigated by measuring the growth pattern of Symbiodinium cells, the results of which indicated that the growth was significantly reduced in response to the extreme temperatures. Proteomic analysis of freshly isolated Symbiodinium cells revealed twelve novel proteins that putatively included transcription translation factors, photosystem proteins, and proteins associated with energy and lipid metabolism, as well as defense response. The results of this study will bring more understandings to the mechanisms governing the endosymbiotic relationship between the cnidarians and dinoflagellates.

  18. Distinct angiotensin II receptor in primary cultures of glial cells from rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Raizada, M.K.; Phillips, M.I.; Crews, F.T.; Sumners, C.

    1987-07-01

    Angiotensin II (Ang-II) has profound effects on the brain. Receptors for Ang-II have been demonstrated on neurons, but no relationship between glial cells and Agn-II has been established. Glial cells (from the hypothalamus and brain stem of 1-day-old rat brains) in primary culture have been used to demonstrate the presence of specific Ang-II receptors. Binding of /sup 125/I-Ang-II to glial cultures was rapid, reversible, saturable, and specific for Ang-II. The rank order of potency of /sup 125/I-Ang-II binding was determined. Scatchard analysis revealed a homogeneous population of high-affinity binding sites with a B/sub max/ of 110 fmol/mg of protein. Light-microscopic autoradiography of /sup 125/I-Ang-II binding supported the kinetic data, documenting specific Ang-II receptors on the glial cells. Ang-II stimulated a dose-dependent hydrolysis of phosphatidylinositols in glial cells, an effect mediated by Ang-II receptors. However, Ang-II failed to influence (/sup 3/H) norepinephrine uptake, and catecholamines failed to regulate Ang-II receptors, effects that occur in neurons. These observations demonstrate the presence of specific Ang-II receptors on the glial cells in primary cultures derived from normotensive rat brain. The receptors are kinetically similar to, but functionally distinct from, the neuronal Ang-II receptors.

  19. Development of the Theta Comparative Cell Scoring Method to Quantify Diverse Phenotypic Responses Between Distinct Cell Types

    Science.gov (United States)

    Warchal, Scott J.; Dawson, John C.

    2016-01-01

    Abstract In this article, we have developed novel data visualization tools and a Theta comparative cell scoring (TCCS) method, which supports high-throughput in vitro pharmacogenomic studies across diverse cellular phenotypes measured by multiparametric high-content analysis. The TCCS method provides a univariate descriptor of divergent compound-induced phenotypic responses between distinct cell types, which can be used for correlation with genetic, epigenetic, and proteomic datasets to support the identification of biomarkers and further elucidate drug mechanism-of-action. Application of these methods to compound profiling across high-content assays incorporating well-characterized cells representing known molecular subtypes of disease supports the development of personalized healthcare strategies without prior knowledge of a drug target. We present proof-of-principle data quantifying distinct phenotypic response between eight breast cancer cells representing four disease subclasses. Application of the TCCS method together with new advances in next-generation sequencing, induced pluripotent stem cell technology, gene editing, and high-content phenotypic screening are well placed to advance the identification of predictive biomarkers and personalized medicine approaches across a broader range of disease types and therapeutic classes. PMID:27552144

  20. Induction of extracellular defense-related proteins in suspension cultured-cells of Daucus carota elicited with cyclodextrins and methyl jasmonate.

    Science.gov (United States)

    Sabater-Jara, Ana B; Almagro, Lorena; Pedreño, María A

    2014-04-01

    Suspension cultured-cells (SCC) of Daucus carota were used to evaluate the effect of methyl jasmonate and cyclodextrins, separately or in combination, on the induction of defense responses, particularly the accumulation of pathogenesis-related proteins. A comparative study of the extracellular proteome (secretome) between control and elicited carrot SCC pointed to the presence of amino acid sequences homologous to glycoproteins which have inhibitory activity against the cell-wall-degrading enzymes secreted by pathogens and/or are induced when carrot cells are exposed to a pathogen elicitor. Other amino acid sequences were homologous to Leucine-Rich Repeat domain-containing proteins, which play an essential role in defense against pathogens, as well as in the recognition of microorganisms, making them important players in the innate immunity of this plant. Also, some tryptic peptides were shown to be homologous to a thaumatin-like protein, showing high specificity to abiotic stress and to different reticuline oxidase-like proteins that displayed high levels of antifungal activity, suggesting that methyl jasmonate and cyclodextrins could play a role in mediating defense-related gene product expression in SCC of D. carota. Apart from these elicitor-inducible proteins, we observed the presence of PR-proteins in both control and elicited carrot SCC, suggesting that their expression is mainly constitutive. These PR-proteins are putative class IV chitinases, which also have inhibitory activity against pathogen growth and the class III peroxidases that participate in response to environmental stress (e.g. pathogen attack and oxidative), meaning that they are involved in defense responses triggered by both biotic and abiotic factors. PMID:24589476

  1. Searching in mother nature for anti-cancer activity: anti-proliferative and pro-apoptotic effect elicited by green barley on leukemia/lymphoma cells.

    Directory of Open Access Journals (Sweden)

    Elisa Robles-Escajeda

    Full Text Available Green barley extract (GB was investigated for possible anti-cancer activity by examining its anti-proliferative and pro-apoptotic properties on human leukemia/lymphoma cell lines. Our results indicate that GB exhibits selective anti-proliferative activity on a panel of leukemia/lymphoma cells in comparison to non-cancerous cells. Specifically, GB disrupted the cell-cycle progression within BJAB cells, as manifested by G2/M phase arrest and DNA fragmentation, and induced apoptosis, as evidenced by phosphatidylserine (PS translocation to the outer cytoplasmic membrane in two B-lineage leukemia/lymphoma cell lines. The pro-apoptotic effect of GB was found to be independent of mitochondrial depolarization, thus implicating extrinsic cell death pathways to exert its cytotoxicity. Indeed, GB elicited an increase of TNF-α production, caspase-8 and caspase-3 activation, and PARP-1 cleavage within pre-B acute lymphoblastic leukemia Nalm-6 cells. Moreover, caspase-8 and caspase-3 activation and PARP-1 cleavage were strongly inhibited/blocked by the addition of the specific caspase inhibitors Z-VAD-FMK and Ac-DEVD-CHO. Furthermore, intracellular signaling analyses determined that GB treatment enhanced constitutive activation of Lck and Src tyrosine kinases in Nalm-6 cells. Taken together, these findings indicate that GB induced preferential anti-proliferative and pro-apoptotic signals within B-lineage leukemia/lymphoma cells, as determined by the following biochemical hallmarks of apoptosis: PS externalization, enhanced release of TNF-α, caspase-8 and caspase-3 activation, PARP-1 cleavage and DNA fragmentation Our observations reveal that GB has potential as an anti-leukemia/lymphoma agent alone or in combination with standard cancer therapies and thus warrants further evaluation in vivo to support these findings.

  2. Interferon-beta induces distinct gene expression response patterns in human monocytes versus T cells.

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    Noa Henig

    Full Text Available BACKGROUND: Monocytes, which are key players in innate immunity, are outnumbered by neutrophils and lymphocytes among peripheral white blood cells. The cytokine interferon-β (IFN-β is widely used as an immunomodulatory drug for multiple sclerosis and its functional pathways in peripheral blood mononuclear cells (PBMCs have been previously described. The aim of the present study was to identify novel, cell-specific IFN-β functions and pathways in tumor necrosis factor (TNF-α-activated monocytes that may have been missed in studies using PBMCs. METHODOLOGY/PRINCIPAL FINDINGS: Whole genome gene expression profiles of human monocytes and T cells were compared following in vitro priming to TNF-α and overnight exposure to IFN-β. Statistical analyses of the gene expression data revealed a cell-type-specific change of 699 transcripts, 667 monocyte-specific transcripts, 21 T cell-specific transcripts and 11 transcripts with either a difference in the response direction or a difference in the magnitude of response. RT-PCR revealed a set of differentially expressed genes (DEGs, exhibiting responses to IFN-β that are modulated by TNF-α in monocytes, such as RIPK2 and CD83, but not in T cells or PBMCs. Known IFN-β promoter response elements, such as ISRE, were enriched in T cell DEGs but not in monocyte DEGs. The overall directionality of the gene expression regulation by IFN-β was different in T cells and monocytes, with up-regulation more prevalent in T cells, and a similar extent of up and down-regulation recorded in monocytes. CONCLUSIONS: By focusing on the response of distinct cell types and by evaluating the combined effects of two cytokines with pro and anti-inflammatory activities, we were able to present two new findings First, new IFN-β response pathways and genes, some of which were monocytes specific; second, a cell-specific modulation of the IFN-β response transcriptome by TNF-α.

  3. HCMV spread and cell tropism are determined by distinct virus populations.

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    Laura Scrivano

    Full Text Available Human cytomegalovirus (HCMV can infect many different cell types in vivo. Two gH/gL complexes are used for entry into cells. gH/gL/pUL(128,130,131A shows no selectivity for its host cell, whereas formation of a gH/gL/gO complex only restricts the tropism mainly to fibroblasts. Here, we describe that depending on the cell type in which virus replication takes place, virus carrying the gH/gL/pUL(128,130,131A complex is either released or retained cell-associated. We observed that virus spread in fibroblast cultures was predominantly supernatant-driven, whereas spread in endothelial cell (EC cultures was predominantly focal. This was due to properties of virus released from fibroblasts and EC. Fibroblasts released virus which could infect both fibroblasts and EC. In contrast, EC released virus which readily infected fibroblasts, but was barely able to infect EC. The EC infection capacities of virus released from fibroblasts or EC correlated with respectively high or low amounts of gH/gL/pUL(128,130,131A in virus particles. Moreover, we found that focal spread in EC cultures could be attributed to EC-tropic virus tightly associated with EC and not released into the supernatant. Preincubation of fibroblast-derived virus progeny with EC or beads coated with pUL131A-specific antibodies depleted the fraction that could infect EC, and left a fraction that could predominantly infect fibroblasts. These data strongly suggest that HCMV progeny is composed of distinct virus populations. EC specifically retain the EC-tropic population, whereas fibroblasts release EC-tropic and non EC-tropic virus. Our findings offer completely new views on how HCMV spread may be controlled by its host cells.

  4. Leishmania amazonensis promastigotes present two distinct modes of nucleus and kinetoplast segregation during cell cycle.

    Directory of Open Access Journals (Sweden)

    Marcelo Santos da Silva

    Full Text Available Here, we show the morphological events associated with organelle segregation and their timing in the cell cycle of a reference strain of Leishmania (L. amazonensis promastigotes, the main causative agent of Tegumentary leishmaniasis in the Americas. We show evidences that during the cell cycle, L. amazonensis promastigotes present two distinct modes of nucleus and kinetoplast segregation, which occur in different temporal order in different proportions of cells. We used DAPI-staining and EdU-labeling to monitor the segregation of DNA-containing organelles and DNA replication in wild-type parasites. The emergence of a new flagellum was observed using a specific monoclonal antibody. The results show that L. amazonensis cell cycle division is peculiar, with 65% of the dividing cells duplicating the kinetoplast before the nucleus, and the remaining 35% doing the opposite or duplicating both organelles concomitantly. In both cases, the new flagellum appeared during S to G2 phase in 1N1K cells and thus before the segregation of both DNA-containing organelles; however, we could not determine the exact timing of flagellar synthesis. Most of these results were confirmed by the synchronization of parasites using hydroxyurea. Altogether, our data show that during the cell cycle of L. amazonensis promastigotes, similarly to L. donovani, the segregation of nucleus and kinetoplast do not follow a specific order, especially when compared to other trypanosomatids, reinforcing the idea that this characteristic seems to be species-specific and may represent differences in cellular biology among members of the Leishmania genus.

  5. Distinct and conserved prominin-1/CD133-positive retinal cell populations identified across species.

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    József Jászai

    Full Text Available Besides being a marker of various somatic stem cells in mammals, prominin-1 (CD133 plays a role in maintaining the photoreceptor integrity since mutations in the PROM1 gene are linked with retinal degeneration. In spite of that, little information is available regarding its distribution in eyes of non-mammalian vertebrates endowed with high regenerative abilities. To address this subject, prominin-1 cognates were isolated from axolotl, zebrafish and chicken, and their retinal compartmentalization was investigated and compared to that of their mammalian orthologue. Interestingly, prominin-1 transcripts--except for the axolotl--were not strictly restricted to the outer nuclear layer (i.e., photoreceptor cells, but they also marked distinct subdivisions of the inner nuclear layer (INL. In zebrafish, where the prominin-1 gene is duplicated (i.e., prominin-1a and prominin-1b, a differential expression was noted for both paralogues within the INL being localized either to its vitreal or scleral subdivision, respectively. Interestingly, expression of prominin-1a within the former domain coincided with Pax-6-positive cells that are known to act as progenitors upon injury-induced retino-neurogenesis. A similar, but minute population of prominin-1-positive cells located at the vitreal side of the INL was also detected in developing and adult mice. In chicken, however, prominin-1-positive cells appeared to be aligned along the scleral side of the INL reminiscent of zebrafish prominin-1b. Taken together our data indicate that in addition to conserved expression of prominin-1 in photoreceptors, significant prominin-1-expressing non-photoreceptor retinal cell populations are present in the vertebrate eye that might represent potential sources of stem/progenitor cells for regenerative therapies.

  6. An oral recombinant Salmonella enterica serovar Typhimurium mutant elicits systemic antigen-specific CD8+ T cell cytokine responses in mice

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    Chin'ombe Nyasha

    2009-04-01

    Full Text Available Abstract Background The induction of antigen-specific CD8+ T cell cytokine responses against an attenuated, oral recombinant Salmonella enterica serovar Typhimurium vaccine expressing a green fluorescent protein (GFP model antigen was investigated. A GFP expression plasmid was constructed in which the gfp gene was fused in-frame with the 5' domain of the Escherichia coli β-galactosidase α-gene fragment with expression under the lac promoter. Groups of mice were orally immunized three times with the bacteria and systemic CD8+ T cell cytokine responses were evaluated. Results High level of the GFP model antigen was expressed by the recombinant Salmonella vaccine vector. Systemic GFP-specific CD8+ T cell cytokine (IFN-γ and IL-4 immune responses were detected after mice were orally vaccinated with the bacteria. It was shown that 226 net IFN-γ and 132 net IL-4 GFP-specific SFUs/10e6 splenocytes were formed in an ELISPOT assay. The level of IFN-γ produced by GFP peptide-stimulated cells was 65.2-fold above background (p Conclusion These results suggested that a high expressing recombinant Salmonella vaccine given orally to mice would elicit antigen-specific CD8+ T cell responses in the spleen. Salmonella bacteria may, therefore, be used as potential mucosal vaccine vectors.

  7. Targeting inhibitor of apoptosis proteins by Smac mimetic elicits cell death in poor prognostic subgroups of chronic lymphocytic leukemia.

    Science.gov (United States)

    Opel, Daniela; Schnaiter, Andrea; Dodier, Dagmar; Jovanovic, Marjana; Gerhardinger, Andreas; Idler, Irina; Mertens, Daniel; Bullinger, Lars; Stilgenbauer, Stephan; Fulda, Simone

    2015-12-15

    Inhibitor of apoptosis (IAP) proteins are highly expressed in chronic lymphocytic leukemia (CLL) cells and contribute to evasion of cell death and poor therapeutic response. Here, we report that Smac mimetic BV6 dose-dependently induces cell death in 28 of 51 (54%) investigated CLL samples, while B-cells from healthy donors are largely unaffected. Importantly, BV6 is significantly more effective in prognostic unfavorable cases with, e.g., non-mutated VH status and TP53 mutation than samples with unknown or favorable prognosis. The majority of cases with 17p deletion (10/12) and Fludarabine refractory cases respond to BV6, indicating that BV6 acts independently of p53. BV6 also triggers cell death under survival conditions mimicking the microenvironment, e.g., by adding CD40 ligand or conditioned medium. Gene expression profiling identifies cell death, NF-κB and redox signaling among the top pathways regulated by BV6 not only in CLL but also in core-binding factor (CBF) acute myeloid leukemia (AML). Consistently, BV6 stimulates production of reactive oxygen species (ROS), which are contributing to BV6-induced cell death, since antioxidants reduce cell death. While BV6 causes degradation of cellular inhibitor of apoptosis (cIAP)1 and cIAP2 and nuclear factor-kappaB (NF-κB) pathway activation in primary CLL samples, BV6 induces cell death independently of caspase activity, receptor-interacting protein (RIP)1 activity or tumor necrosis factor (TNF)α, as zVAD.fmk, necrostatin-1 or TNFα-blocking antibody Enbrel fail to inhibit cell death. Together, these novel insights into BV6-regulated cell death in CLL have important implications for developing new therapeutic strategies to overcome cell death resistance especially in poor prognostic CLL subgroups. PMID:26096065

  8. The mature activating natural killer cell immunologic synapse is formed in distinct stages.

    Science.gov (United States)

    Orange, Jordan S; Harris, K Eliza; Andzelm, Milena M; Valter, Markus M; Geha, Raif S; Strominger, Jack L

    2003-11-25

    Natural killer (NK) cells form a structure at their interface with a susceptible target cell called the activating NK cell immunologic synapse (NKIS). The mature activating NKIS contains a central and peripheral supramolecular activation cluster (SMAC), and includes polarized surface receptors, filamentous actin (F-actin) and perforin. Evaluation of the NKIS in human NK cells revealed CD2, CD11a, CD11b and F-actin in the peripheral SMAC (pSMAC) with perforin in the central SMAC. The accumulation of F-actin and surface receptors was rapid and depended on Wiskott-Aldrich syndrome protein-driven actin polymerization. The accumulation at and arrangement of these molecules in the pSMAC was not affected by microtubule depolymerization. The polarization of perforin, however was slower and required intact actin, Wiskott-Aldrich syndrome protein, and microtubule function. Thus the process of CD2, CD11a, CD11b, and F-actin accumulation in the pSMAC and perforin accumulation in the central SMAC of the NKIS are sequential processes with distinct cytoskeletal requirements. PMID:14612578

  9. Distinctive left-sided distribution of adrenergic-derived cells in the adult mouse heart.

    Directory of Open Access Journals (Sweden)

    Kingsley Osuala

    Full Text Available Adrenaline and noradrenaline are produced within the heart from neuronal and non-neuronal sources. These adrenergic hormones have profound effects on cardiovascular development and function, yet relatively little information is available about the specific tissue distribution of adrenergic cells within the adult heart. The purpose of the present study was to define the anatomical localization of cells derived from an adrenergic lineage within the adult heart. To accomplish this, we performed genetic fate-mapping experiments where mice with the cre-recombinase (Cre gene inserted into the phenylethanolamine-n-methyltransferase (Pnmt locus were cross-mated with homozygous Rosa26 reporter (R26R mice. Because Pnmt serves as a marker gene for adrenergic cells, offspring from these matings express the β-galactosidase (βGAL reporter gene in cells of an adrenergic lineage. βGAL expression was found throughout the adult mouse heart, but was predominantly (89% located in the left atrium (LA and ventricle (LV (p<0.001 compared to RA and RV, where many of these cells appeared to have cardiomyocyte-like morphological and structural characteristics. The staining pattern in the LA was diffuse, but the LV free wall displayed intermittent non-random staining that extended from the apex to the base of the heart, including heavy staining of the anterior papillary muscle along its perimeter. Three-dimensional computer-aided reconstruction of XGAL+ staining revealed distribution throughout the LA and LV, with specific finger-like projections apparent near the mid and apical regions of the LV free wall. These data indicate that adrenergic-derived cells display distinctive left-sided distribution patterns in the adult mouse heart.

  10. Rhein Elicits In Vitro Cytotoxicity in Primary Human Liver HL-7702 Cells by Inducing Apoptosis through Mitochondria-Mediated Pathway

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    Guy-Armel Bounda

    2015-01-01

    Full Text Available Objective. To study rhein-induced apoptosis signaling pathway and to investigate its molecular mechanisms in primary human hepatic cells. Results. Cell viability of HL-7702 cells treated with rhein showed significant decrease in dose-dependent manner. Following rhein treatment (25 μM, 50 μM, and 100 μM for 12 h, the detection of apoptotic cells was significantly analyzed by flow cytometry and nuclear morphological changes by Hoechst 33258, respectively. Fatty degeneration studies showed upregulation level of the relevant hepatic markers (P < 0.01. Caspase activities expressed significant upregulation of caspase-3, caspase-9, and caspase-8. Moreover, apoptotic cells by rhein were significantly inhibited by Z-LEHD-FMK and Z-DEVD-FMK, caspase-9 inhibitor, and caspase-3 inhibitor, respectively. Overproduction of reactive oxygen species, lipid peroxidation, and loss of mitochondrial membrane potential were detected by fluorometry. Additionally, NAC, a ROS scavenger, significantly attenuated rhein-induced oxidative damage in HL-7702 cells. Furthermore, real-time qPCR results showed significant upregulation of p53, PUMA, Apaf-1, and Casp-9 and Casp-3 mRNA, with no significant changes of Fas and Cytochrome-c. Immunoblotting revealed significant Cytochrome-c release from mitochondria into cytosol and no change in Fas expression. Conclusion. Taken together, these observations suggested that rhein could induce apoptosis in HL-7702 cells via mitochondria-mediated signal pathway with involvement of oxidative stress mechanism.

  11. Metabolic changes of salicylic acid-elicited Catharanthus roseus cell suspension cultures monitored by NMR-based metabolomics

    OpenAIRE

    Mustafa, Natali Rianika; Kim, Hye Kyong; Choi, Young Hae; Verpoorte, Robert

    2009-01-01

    The effect of salicylic acid (SA) on the metabolic profile of Catharanthus roseus suspension cells throughout a time course (0, 6, 12, 24, 48 and 72 h after treatment) was investigated using NMR spectroscopy and multivariate data analysis. When compared to control cell lines, SA-treated cells showed a high level of sugars (glucose and sucrose) up to 48 h after treatment, followed by a dynamic change in amino acids, phenylpropanoids, and tryptamine. Additionally, one compound—2,5-dihydroxybenz...

  12. Distinct centromere domain structures with separate functions demonstrated in live fission yeast cells.

    Science.gov (United States)

    Appelgren, Henrik; Kniola, Barbara; Ekwall, Karl

    2003-10-01

    Fission yeast (Saccharomyces pombe) centromere DNA is organized in a central core region flanked on either side by a region of outer repeat (otr) sequences. The otr region is known to be heterochromatic and bound by the Swi6 protein whereas the central core region contains an unusual chromatin structure involving the histone H3 variant Cnp1 (S. pombe CENP-A). The central core is the base for formation of the kinetochore structure whereas the flanking region is important for sister centromere cohesion. We have previously shown that the ultrastructural domain structure of S. pombe centromeres in interphase is similar to that of human centromeres. Here we demonstrate that S. pombe centromeres are organized in cytologically distinct domains even in mitosis. Fluorescence in situ hybridization of fixed metaphase cells revealed that the otr regions of the centromere were still held together by cohesion even after the sister kinetochores had separated. In live cells, the central cores and kinetochores of sister chromosomes could be distinguished from one another when they were subjected to mitotic tension. The function of the different centromeric domains was addressed. Transacting mutations affecting the kinetochore (nuf2) central core domain (mis6) and the heterochromatin domain (rik1) were analyzed in live cells. In interphase, both nuf2 and mis6 caused declustering of centromeres from the spindle pole body whereas centromere clustering was normal in rik1 despite an apparent decondensation defect. The declustering of centromeres in mis6 cells correlated with loss the Ndc80 kinetochore marker protein from the centromeres. Interestingly the declustered centromeres were still restricted to the nuclear periphery thus revealing a kinetochore-independent peripheral localization mechanism for heterochromatin. Time-lapse microscopy of live mis6 and nuf2-1 mutant cells in mitosis showed similar severe misaggregation phenotypes whereas the rik1 mutants showed a mild cohesion

  13. Distinct cytokines balance the development of regulatory T cells and interleukin-10-producing regulatory B cells

    Czech Academy of Sciences Publication Activity Database

    Holáň, Vladimír; Zajícová, Alena; Javorková, Eliška; Trošan, Peter; Chudičková, Milada; Pavlíková, M.; Krulová, Magdaléna

    2014-01-01

    Roč. 141, č. 4 (2014), s. 577-586. ISSN 0019-2805 R&D Projects: GA MZd(CZ) NT14102; GA ČR(CZ) GAP301/11/1568; GA ČR GAP304/11/0653 Grant ostatní: GA MŠk(CZ) MSM0021620858 Institutional support: RVO:68378041 Keywords : Autoregulation * B cells * Cytokines Subject RIV: FF - HEENT, Dentistry Impact factor: 3.795, year: 2014

  14. Intranasal Delivery of Recombinant Parvovirus-Like Particles Elicits Cytotoxic T-Cell and Neutralizing Antibody Responses

    OpenAIRE

    Sedlik, C.; Dridi, A.; Deriaud, E; Saron, M F; Rueda, P; Sarraseca, J.; Casal, J I; Leclerc, C.

    1999-01-01

    We previously demonstrated that chimeric porcine parvovirus-like particles (PPV:VLP) carrying heterologous epitopes, when injected intraperitoneally into mice without adjuvant, activate strong CD4+ and CD8+ T-cell responses specific for the foreign epitopes. In the present study, we investigated the immunogenicity of PPV:VLP carrying a CD8+ T-cell epitope from the lymphocytic choriomeningitis virus (LCMV) administered by mucosal routes. Mice immunized intranasally with recombinant PPV:VLP, in...

  15. Genetically Attenuated Plasmodium berghei Liver Stages Persist and Elicit Sterile Protection Primarily via CD8 T Cells

    OpenAIRE

    Mueller, Ann-Kristin; Deckert, Martina; Heiss, Kirsten; Goetz, Kristin; Matuschewski, Kai; Schlüter, Dirk

    2007-01-01

    Live-attenuated Plasmodium liver stages remain the only experimental model that confers complete sterile protection against malaria. Irradiation-attenuated Plasmodium parasites mediate protection primarily by CD8 T cells. In contrast, it is unknown how genetically attenuated liver stage parasites provide protection. Here, we show that immunization with uis3(−) sporozoites does not cause breakthrough infection in T and B-cell-deficient rag1−/− and IFN-γ−/− mice. However, protection was abolish...

  16. Metabolic analysis of elicited cell suspension cultures of Cannabis sativa L. by 1H-NMR spectroscopy

    OpenAIRE

    Peč, Jaroslav; Flores-Sanchez, Isvett Josefina; Choi, Young Hae; Verpoorte, Robert

    2010-01-01

    Abstract Cannabis sativa L. plants produce a diverse array of secondary metabolites. Cannabis cell cultures were treated with jasmonic acid (JA) and pectin as elicitors to evaluate their effect on metabolism from two cell lines using NMR spectroscopy and multivariate data analysis. According to principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA), the chloroform extract of the pectin-treated cultures were more different than control and JA-trea...

  17. Synergy between phorbol esters, 1-oleyl-2-acetylglycerol, urushiol, and calcium ionophore in eliciting aggregation of marine sponge cells.

    OpenAIRE

    Weissmann, G; Azaroff, L; Davidson, S.; Dunham, P

    1986-01-01

    Aggregation of marine sponge cells (Microciona prolifera) resembles stimulus-response coupling of higher organisms in which activation of protein kinase C and movements of intracellular Ca provide twin signals. We now report that activators of protein kinase C (phorbol esters) and ionomycin act synergistically to aggregate sponge cells. Surprisingly--since extracellular Ca is required for integrity of the species-specific aggregation factor--synergistic aggregation proceeded in the complete a...

  18. BMP and Hedgehog Regulate Distinct AGM Hematopoietic Stem Cells Ex Vivo.

    Science.gov (United States)

    Crisan, Mihaela; Solaimani Kartalaei, Parham; Neagu, Alex; Karkanpouna, Sofia; Yamada-Inagawa, Tomoko; Purini, Caterina; Vink, Chris S; van der Linden, Reinier; van Ijcken, Wilfred; Chuva de Sousa Lopes, Susana M; Monteiro, Rui; Mummery, Christine; Dzierzak, Elaine

    2016-03-01

    Hematopoietic stem cells (HSC), the self-renewing cells of the adult blood differentiation hierarchy, are generated during embryonic stages. The first HSCs are produced in the aorta-gonad-mesonephros (AGM) region of the embryo through endothelial to a hematopoietic transition. BMP4 and Hedgehog affect their production and expansion, but it is unknown whether they act to affect the same HSCs. In this study using the BRE GFP reporter mouse strain that identifies BMP/Smad-activated cells, we find that the AGM harbors two types of adult-repopulating HSCs upon explant culture: One type is BMP-activated and the other is a non-BMP-activated HSC type that is indirectly controlled by Hedgehog signaling through the VEGF pathway. Transcriptomic analyses demonstrate that the two HSC types express distinct but overlapping genetic programs. These results revealing the bifurcation in HSC types at early embryonic stages in the AGM explant model suggest that their development is dependent upon the signaling molecules in the microenvironment. PMID:26923823

  19. BMP and Hedgehog Regulate Distinct AGM Hematopoietic Stem Cells Ex Vivo

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    Mihaela Crisan

    2016-03-01

    Full Text Available Hematopoietic stem cells (HSC, the self-renewing cells of the adult blood differentiation hierarchy, are generated during embryonic stages. The first HSCs are produced in the aorta-gonad-mesonephros (AGM region of the embryo through endothelial to a hematopoietic transition. BMP4 and Hedgehog affect their production and expansion, but it is unknown whether they act to affect the same HSCs. In this study using the BRE GFP reporter mouse strain that identifies BMP/Smad-activated cells, we find that the AGM harbors two types of adult-repopulating HSCs upon explant culture: One type is BMP-activated and the other is a non-BMP-activated HSC type that is indirectly controlled by Hedgehog signaling through the VEGF pathway. Transcriptomic analyses demonstrate that the two HSC types express distinct but overlapping genetic programs. These results revealing the bifurcation in HSC types at early embryonic stages in the AGM explant model suggest that their development is dependent upon the signaling molecules in the microenvironment.

  20. Adipose stromal cells contain phenotypically distinct adipogenic progenitors derived from neural crest.

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    Yoshihiro Sowa

    Full Text Available Recent studies have shown that adipose-derived stromal/stem cells (ASCs contain phenotypically and functionally heterogeneous subpopulations of cells, but their developmental origin and their relative differentiation potential remain elusive. In the present study, we aimed at investigating how and to what extent the neural crest contributes to ASCs using Cre-loxP-mediated fate mapping. ASCs harvested from subcutaneous fat depots of either adult P0-Cre/or Wnt1-Cre/Floxed-reporter mice contained a few neural crest-derived ASCs (NCDASCs. This subpopulation of cells was successfully expanded in vitro under standard culture conditions and their growth rate was comparable to non-neural crest derivatives. Although NCDASCs were positive for several mesenchymal stem cell markers as non-neural crest derivatives, they exhibited a unique bipolar or multipolar morphology with higher expression of markers for both neural crest progenitors (p75NTR, Nestin, and Sox2 and preadipocytes (CD24, CD34, S100, Pref-1, GATA2, and C/EBP-delta. NCDASCs were able to differentiate into adipocytes with high efficiency but their osteogenic and chondrogenic potential was markedly attenuated, indicating their commitment to adipogenesis. In vivo, a very small proportion of adipocytes were originated from the neural crest. In addition, p75NTR-positive neural crest-derived cells were identified along the vessels within the subcutaneous adipose tissue, but they were negative for mural and endothelial markers. These results demonstrate that ASCs contain neural crest-derived adipocyte-restricted progenitors whose phenotype is distinct from that of non-neural crest derivatives.

  1. Distinct molecular forms of human T cell receptor gamma/delta detected on viable T cells by a monoclonal antibody

    OpenAIRE

    1988-01-01

    A second type of TCR molecule has been identified on human and murine T lymphocytes, which involves the protein products of the gamma and delta genes. T lymphocytes bearing this receptor may constitute a separate cell lineage with a distinct immune function. We have produced an mAb, which specifically detects human TCR-gamma/delta in native as well as denatured states, this in contrast to previously used anti-gamma chain peptide sera, which only reacted with denatured protein. The receptor oc...

  2. HPV-E7 Delivered by Engineered Exosomes Elicits a Protective CD8+ T Cell-Mediated Immune Response

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    Paola Di Bonito

    2015-03-01

    Full Text Available We developed an innovative strategy to induce a cytotoxic T cell (CTL immune response against protein antigens of choice. It relies on the production of exosomes, i.e., nanovesicles spontaneously released by all cell types. We engineered the upload of huge amounts of protein antigens upon fusion with an anchoring protein (i.e., HIV-1 Nefmut, which is an inactive protein incorporating in exosomes at high levels also when fused with foreign proteins. We compared the immunogenicity of engineered exosomes uploading human papillomavirus (HPV-E7 with that of lentiviral virus-like particles (VLPs incorporating equivalent amounts of the same antigen. These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs. It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G. Most important, we provide evidence that the inoculation in mouse of exosomes uploading HPV-E7 induces production of anti-HPV E7 CTLs, blocks the growth of syngeneic tumor cells inoculated after immunization, and controls the development of tumor cells inoculated before the exosome challenge. These results represent the proof-of-concept about both feasibility and efficacy of the Nefmut-based exosome platform for the induction of CD8+ T cell immunity.

  3. Polyclonal B-cell activation by Neisseria meningitidis capsular polysaccharides elicit antibodies protective against Trypanosoma cruzi infection in vitro.

    Science.gov (United States)

    Oliveira, T G; Milani, S R; Travassos, L R

    1996-01-01

    A hyperimmune rabbit antiserum against group C Neisseria meningitidis agglutinated and lysed Trypanosoma cruzi metacyclic trypomastigotes in a complement-mediated reaction. Immunization of rabbits with the purified polysaccharide C from N. meningitidis and of human volunteers with the AC-polysaccharide vaccine against meningitis also resulted in antibody production cross-reactive with T. cruzi infective forms. The rabbit antibodies bound to parasites, lysed metacyclic forms, and recognized several components from lysates of cell-derived trypomastigotes. The sera from six human volunteers reacted with cell-cultured trypomastigotes in vitro, lysed these forms, and recognized glycoconjugates migrating diffusely on the top of immunoblots. One serum also reacted with the isolated mucin-like glycoconjugate carrying the Ssp-3 epitope from cell-derived trypomastigotes, but treatment with sialidase did not abolish this reactivity. The anti-AC human antiserum also protected against HeLa cell infection and markedly decreased the number of parasites liberated after cell burst. The polyclonal response that resulted from human immunization with N. meningitidis polysaccharides A and C comprised trypanolytic antibodies that recognized nonsialylated epitopes expressed on infective forms of the parasite. It is suggested that human AC vaccination could be potentially helpful as an adjuvant to a specific immunotherapy of Chagas disease, developed with native or recombinant antigens of the parasite. PMID:8811466

  4. Redundant and antagonistic functions of galectin-1, -3, and -8 in the elicitation of T cell responses.

    Science.gov (United States)

    Tribulatti, María Virginia; Figini, María Gabriela; Carabelli, Julieta; Cattaneo, Valentina; Campetella, Oscar

    2012-04-01

    Galectins, a family of mammalian lectins, have emerged as key regulators of the immune response. We previously demonstrated that galectin (Gal)-8, from the tandem-repeat subgroup, exerts two well-defined effects on mouse naive peripheral CD4 T cells: Ag-specific costimulation and Ag-independent proliferation. These stimulatory signals on naive T cells have not been described for any other Gal. Therefore, we investigated whether Gal-1 and Gal-3, two prominent members of the Gal family, share the stimulatory effects exerted by Gal-8 on naive T cells. We found that Gal-1 costimulated Ag-specific T cell responses similarly to Gal-8, as evaluated in the DO11.10 TCR(OVA)-transgenic mouse model, by acting simultaneously on APCs and target CD4 T cells. In contrast, Gal-3 failed to costimulate Ag-specific T cell responses; moreover, it antagonized both Gal-1 and Gal-8 signals. We observed that both Gal-1 and Gal-3 were unable to induce Ag-independent proliferation; however, when two Gal-1 molecules were covalently fused, the resulting chimeric protein efficiently promoted proliferation. This finding indicates that Gal-1 might eventually induce proliferation and, moreover, stresses the requirement of a tandem-repeat structure. Remarkably, a single dose of recombinant Gal-1 or Gal-8 administered together with a suboptimal Ag dose to DO11.10 mice strengthened weak responses in vivo. Taken together, these findings argue for the participation of Gals in the initiation of the immune response and allow the postulation of these lectins as enhancers of borderline Ag responses, thus representing potential adjuvants for vaccine formulations. PMID:22357632

  5. The selective cytotoxicity elicited by phytochemical extract from Senecio graveolens (Asteraceae) on breast cancer cells is enhanced by hypoxia.

    Science.gov (United States)

    Echiburú-Chau, Carlos; Alfaro-Lira, Susana; Brown, Nelson; Salas, Cristian O; Cuellar, Mauricio; Santander, Javier; Ogalde, Juan Pablo; Rothhammer, Francisco

    2014-04-01

    Breast cancer is the second cause of cancer‑related deaths in woman and the incidence of the disease has increased worldwide, in part due to improvements in early detection. Several drugs with anticancer effects have been extracted from plants in the last 20 years, many of which are particularly effective against breast cancer cells. In particular, we have become interested in the ethanolic extract from Senecio graveolens (synonym of S. nutans), a plant commonly called Chachacoma, in an effort to isolate compounds that could demonstrate cytotoxic effects on breast cancer cells. Senecio (Asteraceae) is the largest gender in Chile comprising approximatly 200 species. These herbs inhabit areas over 3,500 meters above the sea level in the Andes Mountains. S. graveolens is commonly used by local communities for its medicinal properties, particularly its capacity to ameliorate high-altitude-associated sickness. The cytotoxic effect of the alcoholic extract from S. graveolens, as well as its most abundant compound 4-hydroxy-3-(3-methyl-2-butenyl)acetophenone, were tested in the breast cancer cell lines ZR-75-1, MCF-7 and MDA-MB‑231, and non-tumorigenic MCF-10F cells. We show that the phytochemical extract was able to induce cytotoxicity in cancer cells but not in MCF-10F. Importantly, this effect was enhanced under hypoxic conditions. However, 4-hydroxy-3-(3-methyl-2-butenyl)acetophenone, the main compound, did not by itself show an effective anticarcinogenic activity in comparison to the whole extract. Interestingly, the cytotoxic effect of the phytochemical extract was dependent on the basal MnSOD protein expression. Thus, cytotoxicity was increased when MnSOD levels were low, but resistance was evident when protein levels were high. Additionally, the crude extract seems to trigger cell death by a variety of processes, including autophagy, apoptosis and necrosis, in MCF-7 cells. In summary, S. graveolens extract possess anticancer activity displaying a specific

  6. Enhanced accumulation of phytosterols and phenolic compounds in cyclodextrin-elicited cell suspension culture of Daucus carota.

    Science.gov (United States)

    Miras-Moreno, Begoña; Almagro, Lorena; Pedreño, M A; Sabater-Jara, Ana Belén

    2016-09-01

    In this work, suspension-cultured cells of Daucus carota were used to evaluate the effect of β-cyclodextrins on the production of isoprenoid and phenolic compounds. The results showed that the phytosterols and phenolic compounds were accumulated in the extracellular medium (15100μgL(-1) and 477.46μgL(-1), respectively) in the presence of cyclodextrins. Unlike the phytosterol and phenolic compound content, β-carotene (1138.03μgL(-1)), lutein (25949.54μgL(-1)) and α-tocopherol (8063.82μgL(-1)) chlorophyll a (1625.13μgL(-1)) and b (9.958 (9958.33μgL(-1)) were mainly accumulated inside the cells. Therefore, cyclodextrins were able to induce the cytosolic mevalonate pathway, increasing the biosynthesis of phytosterols and phenolic compounds, and accumulate them outside the cells. However, in the absence of these cyclic oligosaccharidic elicitors, carrot cells mainly accumulated carotenoids through the methylerythritol 4-phosphate pathway. Therefore, the use of cyclodextrins would allow the extracellular accumulation of both phytosterols and phenolic compounds by diverting the carbon flux towards the cytosolic mevalonate/phenylpropanoid pathway. PMID:27457992

  7. Distinct and site-specific phosphorylation of the retinoblastoma protein at serine 612 in differentiated cells.

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    Takayuki Hattori

    Full Text Available The retinoblastoma susceptibility protein (pRB is a phosphoprotein that regulates cell cycle progression at the G1/S transition. In quiescent and early G1 cells, pRB predominantly exists in the active hypophosphorylated form. The cyclin/cyclin-dependent protein kinase complexes phosphorylate pRB at the late G1 phase to inactivate pRB. This event leads to the dissociation and activation of E2F family transcriptional factors. At least 12 serine/threonine residues in pRB are phosphorylated in vivo. Although there have been many reports describing bulk phosphorylation of pRB, detail research describing the function of each phosphorylation site remains unknown. Besides its G1/S inhibitory function, pRB is involved in differentiation, prevention of cell death and control of tissue fate. To uncover the function of phosphorylation of pRB in various cellular conditions, we have been investigating phosphorylation of each serine/threonine residue in pRB with site-specific phospho-serine/threonine antibodies. Here we demonstrate that pRB is specifically phosphorylated at Ser612 in differentiated cells in a known kinase-independent manner. We also found that pRB phosphorylated at Ser612 still associates with E2F-1 and tightly binds to nuclear structures including chromatin. Moreover, expression of the Ser612Ala mutant pRB failed to induce differentiation. The findings suggest that phosphorylation of Ser612 provides a distinct function that differs from the function of phosphorylation of other serine/threonine residues in pRB.

  8. Mapping the distinctive populations of lymphatic endothelial cells in different zones of human lymph nodes.

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    Saem Mul Park

    Full Text Available The lymphatic sinuses in human lymph nodes (LNs are crucial to LN function yet their structure remains poorly defined. Much of our current knowledge of lymphatic sinuses derives from rodent models, however human LNs differ substantially in their sinus structure, most notably due to the presence of trabeculae and trabecular lymphatic sinuses that rodent LNs lack. Lymphatic sinuses are bounded and traversed by lymphatic endothelial cells (LECs. A better understanding of LECs in human LNs is likely to improve our understanding of the regulation of cell trafficking within LNs, now an important therapeutic target, as well as disease processes that involve lymphatic sinuses. We therefore sought to map all the LECs within human LNs using multicolor immunofluorescence microscopy to visualize the distribution of a range of putative markers. PROX1 was the only marker that uniquely identified the LECs lining and traversing all the sinuses in human LNs. In contrast, LYVE1 and STAB2 were only expressed by LECs in the paracortical and medullary sinuses in the vast majority of LNs studied, whilst the subcapsular and trabecular sinuses lacked these molecules. These data highlight the existence of at least two distinctive populations of LECs within human LNs. Of the other LEC markers, we confirmed VEGFR3 was not specific for LECs, and CD144 and CD31 stained both LECs and blood vascular endothelial cells (BECs; in contrast, CD59 and CD105 stained BECs but not LECs. We also showed that antigen-presenting cells (APCs in the sinuses could be clearly distinguished from LECs by their expression of CD169, and their lack of expression of PROX1 and STAB2, or endothelial markers such as CD144. However, both LECs and sinus APCs were stained with DCN46, an antibody commonly used to detect CD209.

  9. Atorvastatin blocks increased l-type Ca2+ current and cell injury elicited by angiotensin II via inhibiting oxide stress.

    Science.gov (United States)

    Ma, Yanzhuo; Kong, Lingfeng; Qi, Shuying; Wang, Dongmei

    2016-04-01

    Thel-type Ca(2+)current (ICa,l) plays a crucial role in shaping action potential and is involved in cardiac arrhythmia. Statins have been demonstrated to contribute to anti-apoptotic and anti-arrhythmic effects in the heart. Here, we examined whether atorvastatin regulates theICa,land cell injury induced by angiotensin II (AngII) as well as the putative intracellular cascade responsible for the effects. Cultured neonatal rat ventricular myocytes were incubated with AngII for 24 h, and then cell injury and expression levels of Nox2/gp91(phox), p47(phox) ,and Cav1.2 were analyzed. In addition,ICa,lwas recorded using the whole-cell patch-clamp technique, and mechanisms of atorvastatin actions were also investigated. It was found that the number of apoptotic cardiomyocytes was increased and cell viability was significantly decreased after AngII administration. AngII also augmented the expressions of Nox2/gp91(phox)and p47(phox)compared with control cardiomyocytes. Exposure to AngII evokedICa,lin a voltage-dependent manner without affecting theI-Vrelationship. In addition, AngII enhanced membrane Cav1.2 expression. These effects were abolished in the presence of the reactive oxygen species (ROS) scavenger, manganese (III)-tetrakis 4-benzoic acid porphyrin [Mn(III)TBAP], or the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, atorvastatin. These results suggested that atorvastatin mediates cardioprotection against arrhythmias and cell injury by controlling the AngII-ROS cascade. PMID:26940997

  10. Heat stress causes spatially-distinct membrane re-modelling in K562 leukemia cells.

    Directory of Open Access Journals (Sweden)

    Gábor Balogh

    Full Text Available Cellular membranes respond rapidly to various environmental perturbations. Previously we showed that modulations in membrane fluidity achieved by heat stress (HS resulted in pronounced membrane organization alterations which could be intimately linked to the expression and cellular distribution of heat shock proteins. Here we examine heat-induced membrane changes using several visualisation methods. With Laurdan two-photon microscopy we demonstrate that, in contrast to the enhanced formation of ordered domains in surface membranes, the molecular disorder is significantly elevated within the internal membranes of cells preexposed to mild HS. These results were compared with those obtained by anisotropy, fluorescence lifetime and electron paramagnetic resonance measurements. All probes detected membrane changes upon HS. However, the structurally different probes revealed substantially distinct alterations in membrane heterogeneity. These data call attention to the careful interpretation of results obtained with only a single label. Subtle changes in membrane microstructure in the decision-making of thermal cell killing could have potential application in cancer therapy.

  11. Distinct TLR-mediated cytokine production and immunoglobulin secretion in human newborn naïve B cells.

    Science.gov (United States)

    Pettengill, Matthew A; van Haren, Simon D; Li, Ning; Dowling, David J; Bergelson, Ilana; Jans, Jop; Ferwerda, Gerben; Levy, Ofer

    2016-08-01

    Neonatal innate immunity is distinct from that of adults, which may contribute to increased susceptibility to infection and limit vaccine responses. B cells play critical roles in protection from infection and detect PAMPs via TLRs, that, when co-activated with CD40, can drive B-cell proliferation and Ab production. We characterized the expression of TLRs in circulating B cells from newborns and adults, and evaluated TLR- and CD40-mediated naïve B-cell class-switch recombination (CSR) and cytokine production. Gene expression levels of most TLRs was similar between newborn and adult B cells, except that newborn naïve B cells expressed more TLR9 than adult naïve B cells. Neonatal naïve B cells demonstrated impaired TLR2- and TLR7- but enhanced TLR9-mediated cytokine production. Significantly fewer newborn naïve B cells underwent CSR to produce IgG, an impairment also noted with IL-21 stimulation. Additionally, co-stimulation via CD40 and TLRs induced greater cytokine production in adult B cells. Thus, while newborn naïve B cells demonstrate adult-level expression of TLRs and CD40, the responses to stimulation of these receptors are distinct. Relatively high expression of TLR9 and impaired CD40-mediated Ig secretion contributes to distinct innate and adaptive immunity of human newborns and may inform novel approaches to early-life immunization. PMID:27252169

  12. Signals involved in T cell activation. II. Distinct roles of intact accessory cells, phorbol esters, and interleukin 1 in activation and cell cycle progression of resting T lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Davis, L.; Lipsky, P.E.

    1986-05-15

    The signals involved in the initiation of mitogen-induced activation of resting guinea pig T cells were examined. The combination of phytohemagglutinin (PHA) and 4..beta..-phorbol 12-myristate 13-acetate (PMA) stimulated DNA synthesis by accessory cell (AC)-depleted T cells cultured at high density, but the use of low density cultures indicated that intact AC were absolutely necessary for PHA-stimulated T cell DNA synthesis even in the presence of PMA, interleukin 1 (IL 1), or interleukin 2 (IL 2). In contrast, AC-depleted T cells were able to respond to the combination of the calcium ionophore, ionomycin, and PMA regardless of the cell density at which they were cultured. Results of cell cycle analysis support the conclusion that intact AC, IL 1, and a PMA-like signal play distinct roles in the progression of mitogen stimulated T cells through the first round of the cell cycle.

  13. The Immunodominance Change and Protection of CD4+ T-Cell Responses Elicited by an Envelope Protein Domain III-Based Tetravalent Dengue Vaccine in Mice.

    Directory of Open Access Journals (Sweden)

    Hsin-Wei Chen

    Full Text Available Dengue is the leading cause of mosquito-borne viral infections and no vaccine is available now. Envelope protein domain III (ED3 is the major target for the binding of dengue virus neutralizing antibodies; however, the ED3-specifc T-cell response is less well understood. To investigate the T-cell responses to four serotypes of dengue virus (DENV-1 to 4, we immunized mice using either a tetravalent ED3-based DNA or protein vaccine, or combined both as a DNA prime-protein boost strategy (prime-boost. A significant serotype-dependent IFN-γ or IL-4 response was observed in mice immunized with either the DNA or protein vaccine. The IFN-γ response was dominant to DENV-1 to 3, whereas the IL-4 response was dominant to DENV-4. Although the similar IgG titers for the four serotypes were observed in mice immunized with the tetravalent vaccines, the neutralizing antibody titers varied and followed the order of 2 = 3>1>4. Interestingly, the lower IFN-γ response to DENV-4 is attributable to the immunodominance change between two CD4+ T-cell epitopes; one T-cell epitope located at E349-363 of DENV-1 to 3 was more immunogenic than the DENV-4 epitope E313-327. Despite DENV-4 specific IFN-γ responses were suppressed by immunodominance change, either DENV-4-specific IFN-γ or neutralizing antibody responses were still recalled after DENV-4 challenge and contributed to virus clearance. Immunization with the prime-boost elicited both IFN-γ and neutralizing antibody responses and provided better protection than either DNA or protein immunization. Our findings shed light on how ED3-based tetravalent dengue vaccines sharpen host CD4 T-cell responses and contribute to protection against dengue virus.

  14. Heterologous prime-boost regimens with a recombinant chimpanzee adenoviral vector and adjuvanted F4 protein elicit polyfunctional HIV-1-specific T-Cell responses in macaques.

    Science.gov (United States)

    Lorin, Clarisse; Vanloubbeeck, Yannick; Baudart, Sébastien; Ska, Michaël; Bayat, Babak; Brauers, Geoffroy; Clarinval, Géraldine; Donner, Marie-Noëlle; Marchand, Martine; Koutsoukos, Marguerite; Mettens, Pascal; Cohen, Joe; Voss, Gerald

    2015-01-01

    HIV-1-specific CD4+ and CD8+ T lymphocytes are important for HIV-1 replication control. F4/AS01 consists of F4 recombinant fusion protein (containing clade B Gag/p24, Pol/RT, Nef and Gag/p17) formulated in AS01 Adjuvant System, and was shown to induce F4-specific polyfunctional CD4+ T-cell responses in humans. While replication-incompetent recombinant HIV-1/SIV antigen-expressing human adenoviral vectors can elicit high-frequency antigen-specific CD8+ T-cell responses, their use is hampered by widespread pre-existing immunity to human serotypes. Non-human adenovirus serotypes associated with lower prevalence may offer an alternative strategy. We evaluated the immunogenicity of AdC7-GRN ('A'), a recombinant chimpanzee adenovirus type 7 vector expressing clade B Gag, RT and Nef, and F4/AS01 ('P'), when delivered intramuscularly in homologous (PP or AA) and heterologous (AAPP or PPAA) prime-boost regimens, in macaques and mice. Vaccine-induced HIV-1-antigen-specific T cells in peripheral blood (macaques), liver, spleen, and intestinal and genital mucosa (mice) were characterized by intracellular cytokine staining. Vaccine-specific IgG antibodies (macaques) were detected using ELISA. In macaques, only the heterologous prime-boost regimens induced polyfunctional, persistent and balanced CD4+ and CD8+ T-cell responses specific to each HIV-1 vaccine antigen. AdC7-GRN priming increased the polyfunctionality of F4/AS01-induced CD4+ T cells. Approximately 50% of AdC7-GRN-induced memory CD8+ T cells exhibited an effector-memory phenotype. HIV-1-specific antibodies were detected with each regimen. In mice, antigen-specific CD4+ and CD8+ T-cell responses were detected in the mucosal and systemic anatomical compartments assessed. When administered in heterologous prime-boost regimens, AdC7-GRN and F4/AS01 candidate vaccines acted complementarily in inducing potent and persistent peripheral blood HIV-1-specific CD4+ and CD8+ T-cell responses and antibodies in macaques. Besides

  15. B7h-expressing dendritic cells and plasma B cells mediate distinct outcomes of ICOS costimulation in T cell-dependent antibody responses

    Directory of Open Access Journals (Sweden)

    Larimore Kevin

    2012-06-01

    Full Text Available Abstract Background The ICOS-B7h costimulatory receptor-ligand pair is required for germinal center formation, the production of isotype-switched antibodies, and antibody affinity maturation in response to T cell-dependent antigens. However, the potentially distinct roles of regulated B7h expression on B cells and dendritic cells in T cell-dependent antibody responses have not been defined. Results We generated transgenic mice with lineage-restricted B7h expression to assess the cell-type specific roles of B7h expression on B cells and dendritic cells in regulating T cell-dependent antibody responses. Our results show that endogenous B7h expression is reduced on B cells after activation in vitro and is also reduced in vivo on antibody-secreting plasma B cells in comparison to both naïve and germinal center B cells from which they are derived. Increasing the level of B7h expression on activated and plasma B cells in B-B7hTg mice led to an increase in the number of antibody-secreting plasma cells generated after immunization and a corresponding increase in the concentration of antigen-specific high affinity serum IgG antibodies of all isotypes, without affecting the number of responding germinal center B cells. In contrast, ICOS costimulation mediated by dendritic cells in DC-B7hTg mice contributed to germinal center formation and selectively increased IgG2a production without affecting the overall magnitude of antibody responses. Conclusions Using transgenic mice with lineage-restricted B7h expression, we have revealed distinct roles of ICOS costimulation mediated by dendritic cells and B cells in the regulation of T cell-dependent antibody responses.

  16. DNA methylation analysis reveals distinct methylation signatures in pediatric germ cell tumors

    International Nuclear Information System (INIS)

    Aberrant DNA methylation is a prominent feature of many cancers, and may be especially relevant in germ cell tumors (GCTs) due to the extensive epigenetic reprogramming that occurs in the germ line during normal development. We used the Illumina GoldenGate Cancer Methylation Panel to compare DNA methylation in the three main histologic subtypes of pediatric GCTs (germinoma, teratoma and yolk sac tumor (YST); N = 51) and used recursively partitioned mixture models (RPMM) to test associations between methylation pattern and tumor and demographic characteristics. We identified genes and pathways that were differentially methylated using generalized linear models and Ingenuity Pathway Analysis. We also measured global DNA methylation at LINE1 elements and evaluated methylation at selected imprinted loci using pyrosequencing. Methylation patterns differed by tumor histology, with 18/19 YSTs forming a distinct methylation class. Four pathways showed significant enrichment for YSTs, including a human embryonic stem cell pluripotency pathway. We identified 190 CpG loci with significant methylation differences in mature and immature teratomas (q < 0.05), including a number of CpGs in stem cell and pluripotency-related pathways. Both YST and germinoma showed significantly lower methylation at LINE1 elements compared with normal adjacent tissue while there was no difference between teratoma (mature and immature) and normal tissue. DNA methylation at imprinted loci differed significantly by tumor histology and location. Understanding methylation patterns may identify the developmental stage at which the GCT arose and the at-risk period when environmental exposures could be most harmful. Further, identification of relevant genetic pathways could lead to the development of new targets for therapy

  17. Mitochondrial DNA mutation-elicited oxidative stress, oxidative damage, and altered gene expression in cultured cells of patients with MERRF syndrome.

    Science.gov (United States)

    Wu, Shi-Bei; Ma, Yi-Shing; Wu, Yu-Ting; Chen, Yin-Chiu; Wei, Yau-Huei

    2010-06-01

    Myoclonic epilepsy and ragged-red fibers (MERRF) syndrome is a rare disorder characterized by myoclonus, muscle weakness, cerebellar ataxia, heart conduction block, and dementia. It has been documented that 80-90% of the patients with MERRF syndrome are caused by the A8344G mutation in the tRNA(Lys) gene of mitochondrial DNA (mtDNA). We and other investigators have reported that the mtDNA mutation results in not only inefficient generation of adenosine triphosphate but also increased production of reactive oxygen species (ROS) in cultured cells harboring A8344G mutation of mtDNA. In addition, we found an imbalance in the gene expression of antioxidant enzymes in the skin fibroblasts of MERRF patients. The mRNA, protein, and enzyme activity levels of manganese-superoxide dismutase were increased, but those of Cu,Zn-SOD, catalase, and glutathione peroxidase did not show significant changes. Recently, we showed that the excess ROS could damage voltage-dependent anion channel, prohibitin, Lon protease, and aconitase in the MERRF cells. Moreover, there was a dramatic increase in the gene expression and activity of matrix metalloproteinase 1, which may contribute to the cytoskeleton remodeling involved in the weakness and atrophy of muscle commonly seen in MERRF patients. Taken together, we suggest that mtDNA mutation-elicited oxidative stress, oxidative damage, and altered gene expression are involved in the pathogenesis and progression of MERRF syndrome. PMID:20411357

  18. Oral Delivery of a Novel Recombinant Streptococcus mitis Vector Elicits Robust Vaccine Antigen-Specific Oral Mucosal and Systemic Antibody Responses and T Cell Tolerance.

    Directory of Open Access Journals (Sweden)

    Emily Xie

    Full Text Available The pioneer human oral commensal bacterium Streptococcus mitis has unique biologic features that make it an attractive mucosal vaccine or therapeutic delivery vector. S. mitis is safe as a natural persistent colonizer of the mouth, throat and nasopharynx and the oral commensal bacterium is capable of inducing mucosal antibody responses. A recombinant S. mitis (rS. mitis that stably expresses HIV envelope protein was generated and tested in the germ-free mouse model to evaluate the potential usefulness of this vector as a mucosal vaccine against HIV. Oral vaccination led to the efficient and persistent bacterial colonization of the mouth and the induction of both salivary and systemic antibody responses. Interestingly, persistently colonized animals developed antigen-specific systemic T cell tolerance. Based on these findings we propose the use of rS. mitis vaccine vector for the induction of mucosal antibodies that will prevent the penetration of the mucosa by pathogens such as HIV. Moreover, the first demonstration of rS. mitis having the ability to elicit T cell tolerance suggest the potential use of rS. mitis as an immunotherapeutic vector to treat inflammatory, allergic and autoimmune diseases.

  19. Identification of Distinct Breast Cancer Stem Cell Populations Based on Single-Cell Analyses of Functionally Enriched Stem and Progenitor Pools

    OpenAIRE

    Nina Akrap; Daniel Andersson; Eva Bom; Pernilla Gregersson; Anders Ståhlberg; Göran Landberg

    2016-01-01

    Summary The identification of breast cancer cell subpopulations featuring truly malignant stem cell qualities is a challenge due to the complexity of the disease and lack of general markers. By combining extensive single-cell gene expression profiling with three functional strategies for cancer stem cell enrichment including anchorage-independent culture, hypoxia, and analyses of low-proliferative, label-retaining cells derived from mammospheres, we identified distinct stem cell clusters in b...

  20. Cutting Edge: Distinct Glycolytic and Lipid Oxidative Metabolic Programs Are Essential for Effector and Regulatory CD4+ T Cell Subsets

    OpenAIRE

    Ryan D Michalek; Gerriets, Valerie A.; Jacobs, Sarah R.; Macintyre, Andrew N.; MacIver, Nancie J.; Mason, Emily F.; Sullivan, Sarah A.; Nichols, Amanda G.; Rathmell, Jeffrey C.

    2011-01-01

    Stimulated CD4+ T lymphocytes can differentiate into effector T cell (Teff) or inducible regulatory T cell (Treg) subsets with specific immunological roles. We show that Teff and Treg require distinct metabolic programs to support these functions. Th1, Th2, and Th17 cells expressed high surface levels of the glucose transporter Glut1 and were highly glycolytic. Treg, in contrast, expressed low levels of Glut1 and had high lipid oxidation rates. Consistent with glycolysis and lipid oxidation p...

  1. In vitro propagation and cell cultures of memory tonic herb Evolvulus alsinoides: a best source for elicited production of scopoletin.

    Science.gov (United States)

    Naikawadi, Vikas Bandu; Ahire, Mahendra Laxman; Lahiri, Anindita; Nikam, Tukaram Dayaram

    2016-04-01

    Evolvulus alsinoides L. is used for preparation of 'Shankhapushpi', an important popular ayurvedic drug that contributes considerably to the improvement of memory power. The improvement is attributed to the presence of furanocoumarin scopoletin, a metabolite with a wide range of biological activities. This report describes, for the first time, an in vitro culture system for propagation and enhanced production of scopoletin. Different concentrations of auxins and cytokinins individually and in combination were used in Murashige and Skoog (MS) medium to induce shoot regeneration in cotyledonary nodal explants and callus formation in leaf explants. The best response was achieved in MS medium fortified with 5.0 μM 6-benzyladenine (BA) in which 96 % of cultures produced 7.6 ± 0.6 shoots per explant. Regenerated shoots were rooted on MS medium with 5.0 μM indole-3-acetic acid (IAA). Plantlets were successfully acclimatized and established in soil. MS medium fortified with 10 μM BA + 5.0 μM IAA showed maximum growth and accumulation of scopoletin in cell cultures. Cell cultures could be maintained over 24 months. The influences of auxins, cytokinins, organic acids, amino acids, and fungal-derived elicitors on production of scopoletin were studied. Presence of either L-arginine, sodium pyruvate, or yeast extract highly promoted scopoletin production as compared with control and achieved 75.02-, 72.13-, and 57.98-fold higher accumulation, respectively. The results presented herein have laid solid foundation for large-scale production of scopoletin and further investigation of its purification and utilization as a novel pharmaceutical drug. PMID:26621800

  2. Distinction Between Cell Proliferation and Apoptosis Signals Regulated by Brain-Derived Neurotrophic Factor in Human Periodontal Ligament Cells and Gingival Epithelial Cells.

    Science.gov (United States)

    Kashiwai, Kei; Kajiya, Mikihito; Matsuda, Shinji; Ouhara, Kazuhisa; Takeda, Katsuhiro; Takata, Takashi; Kitagawa, Masae; Fujita, Tsuyoshi; Shiba, Hideki; Kurihara, Hidemi

    2016-07-01

    Previously, we reported that brain-derived neurotrophic factor (BDNF) enhances periodontal tissue regeneration by inducing periodontal ligament cell proliferation in vivo. In addition, the down growth of gingival epithelial cells, which comprises a major obstacle to the regeneration, was not observed. However, the underlying molecular mechanism is still unclear. Therefore, this study aimed to investigate the effect of BDNF on cell proliferation and apoptosis in human periodontal ligament (HPL) cells and human gingival epithelial cells (OBA9 cells) and to explore the molecular mechanism in vitro. HPL cells dominantly expressed a BDNF receptor, TrkB, and BDNF increased cell proliferation and ERK phosphorylation. However, its proliferative effect was diminished by a MEK1/2 inhibitor (U0126) and TrkB siRNA transfection. Otherwise, OBA9 cells showed a higher expression level of p75, which is a pan-neurotrophin receptor, than that of HPL cells. BDNF facilitated not cell proliferation but cell apoptosis and JNK phosphorylation in OBA9 cells. A JNK inhibitor (SP600125) and p75 siRNA transfection attenuated the BDNF-induced cell apoptosis. Moreover, OBA9 cells pretreated with SP600125 or p75 siRNA showed cell proliferation by BDNF stimulation, though it was reduced by U0126 and TrkB siRNA. Interestingly, overexpression of p75 in HPL cells upregulated cell apoptosis and JNK phosphorylation by BDNF treatment. These results indicated that TrkB-ERK signaling regulates BDNF-induced cell proliferation, whereas p75-JNK signaling plays roles in cell apoptotic and cytostatic effect of BDNF. Overall, BDNF activates periodontal ligament cells proliferation and inhibits the gingival epithelial cells growth via the distinct pathway. J. Cell. Biochem. 117: 1543-1555, 2016. © 2015 Wiley Periodicals, Inc. PMID:26581032

  3. Distinct Morphology of Human T-Cell Leukemia Virus Type 1-Like Particles.

    Science.gov (United States)

    Maldonado, José O; Cao, Sheng; Zhang, Wei; Mansky, Louis M

    2016-01-01

    The Gag polyprotein is the main retroviral structural protein and is essential for the assembly and release of virus particles. In this study, we have analyzed the morphology and Gag stoichiometry of human T-cell leukemia virus type 1 (HTLV-1)-like particles and authentic, mature HTLV-1 particles by using cryogenic transmission electron microscopy (cryo-TEM) and scanning transmission electron microscopy (STEM). HTLV-1-like particles mimicked the morphology of immature authentic HTLV-1 virions. Importantly, we have observed for the first time that the morphology of these virus-like particles (VLPs) has the unique local feature of a flat Gag lattice that does not follow the curvature of the viral membrane, resulting in an enlarged distance between the Gag lattice and the viral membrane. Other morphological features that have been previously observed with other retroviruses include: (1) a Gag lattice with multiple discontinuities; (2) membrane regions associated with the Gag lattice that exhibited a string of bead-like densities at the inner leaflet; and (3) an arrangement of the Gag lattice resembling a railroad track. Measurement of the average size and mass of VLPs and authentic HTLV-1 particles suggested a consistent range of size and Gag copy numbers in these two groups of particles. The unique local flat Gag lattice morphological feature observed suggests that HTLV-1 Gag could be arranged in a lattice structure that is distinct from that of other retroviruses characterized to date. PMID:27187442

  4. A whole recombinant yeast-based therapeutic vaccine elicits HBV X, S and Core specific T cells in mice and activates human T cells recognizing epitopes linked to viral clearance.

    Directory of Open Access Journals (Sweden)

    Thomas H King

    Full Text Available Chronic hepatitis B infection (CHB is characterized by sub-optimal T cell responses to viral antigens. A therapeutic vaccine capable of restoring these immune responses could potentially improve HBsAg seroconversion rates in the setting of direct acting antiviral therapies. A yeast-based immunotherapy (Tarmogen platform was used to make a vaccine candidate expressing hepatitis B virus (HBV X, surface (S, and Core antigens (X-S-Core. Murine and human immunogenicity models were used to evaluate the type and magnitude of HBV-Ag specific T cell responses elicited by the vaccine. C57BL/6J, BALB/c, and HLA-A*0201 transgenic mice immunized with yeast expressing X-S-Core showed T cell responses to X, S and Core when evaluated by lymphocyte proliferation assay, ELISpot, intracellular cytokine staining (ICS, or tumor challenge assays. Both CD4+ and CD8+ T cell responses were observed. Human T cells transduced with HBc18-27 and HBs183-91 specific T cell receptors (TCRs produced interferon gamma (IFNγ following incubation with X-S-Core-pulsed dendritic cells (DCs. Furthermore, stimulation of peripheral blood mononuclear cells (PBMCs isolated from CHB patients or from HBV vaccine recipients with autologous DCs pulsed with X-S-Core or a related product (S-Core resulted in pronounced expansions of HBV Ag-specific T cells possessing a cytolytic phenotype. These data indicate that X-S-Core-expressing yeast elicit functional adaptive immune responses and supports the ongoing evaluation of this therapeutic vaccine in patients with CHB to enhance the induction of HBV-specific T cell responses.

  5. A Research on Distinctive Industries and Spatial Organization Model of Interbasin Cells

    OpenAIRE

    Feng Han

    2010-01-01

    In order to solve the pollution to water resources caused by Chinese industry, this paper proposes to select and develop distinctive industries based on industry performance, to establish a clustering model for the spatial organization of distinctive industries based on market behavior and to construct a network of industrial clusters with special characteristics.

  6. Automated Preferences Elicitation

    Czech Academy of Sciences Publication Activity Database

    Kárný, Miroslav; Guy, Tatiana Valentine

    Prague : Institute of Information Theory and Automation, 2011, s. 20-25. ISBN 978-80-903834-6-3. [The 2nd International Workshop od Decision Making with Multiple Imperfect Decision Makers. Held in Conjunction with the 25th Annual Conference on Neural Information Processing Systems (NIPS 2011). Sierra Nevada (ES), 16.12.2011-16.12.2011] R&D Projects: GA MŠk 1M0572; GA ČR GA102/08/0567 Institutional research plan: CEZ:AV0Z10750506 Keywords : elicitation * decision making * Bayesian decision making * fully probabilistic design Subject RIV: BB - Applied Statistics, Operational Research http://library.utia.cas.cz/separaty/2011/AS/karny-automated preferences elicitation.pdf

  7. Distinct population of highly malignant cells in a head and neck squamous cell carcinoma cell line established by xenograft model

    Directory of Open Access Journals (Sweden)

    Jan Chia-Ing

    2009-11-01

    Full Text Available Abstract The progression and metastasis of solid tumors, including head and neck squamous cell carcinoma (HNSCC, have been related to the behavior of a small subpopulation of cancer stem cells. Here, we have established a highly malignant HNSCC cell line, SASVO3, from primary tumors using three sequential rounds of xenotransplantation. SASVO3 possesses enhanced tumorigenic ability both in vitro and in vivo. Moreover, SASVO3 exhibits properties of cancer stem cells, including that increased the abilities of sphere-forming, the number of side population cells, the potential of transplanted tumor growth and elevated expression of the stem cell marker Bmi1. Injection of SASVO3 into the tail vein of nude mice resulted in lung metastases. These results are consistent with the postulate that the malignant and/or metastasis potential of HNSCC cells may reside in a stem-like subpopulation.

  8. Distinct Redox Profiles of Selected Human Prostate Carcinoma Cell Lines: Implications for Rational Design of Redox Therapy

    International Nuclear Information System (INIS)

    The effects of several cancer chemotherapeutic drugs and radiation are mediated, at least in part, by oxidative stress. To better understand this process, we analyzed certain biochemical properties affecting reduction-oxidation (redox) balance in normal prostate epithelial cells and several prostate cancer cell lines. Highly aggressive androgen-independent prostate cancer PC3 cells demonstrated significantly higher levels of total antioxidant capacity (AC) and intra- and extracellular glutathione (GSH)/glutathione disulfide (GSSG) ratios when compared with normal prostate epithelial PrEC cells. WPE1-NB26 cells, a prostate cancer cell line derived from immortalized RWPE1 human prostate epithelial cells, demonstrated significantly higher levels of total AC and intra- and extracellular GSH/GSSG ratios, but lower levels of intracellular reactive oxygen/nitrogen species and lipid peroxidation compared with RWPE1 cells. LNCaP-C4-2 cells, a more aggressive prostate cancer derived from less aggressive androgen-responsive LNCaP cells, exhibited higher levels of AC and extracellular GSH/GSSG ratio when compared to LNCaP cells. Specific cell types showed distinct cytotoxic responses to redox-modulating compounds. WPE1-NB26 cells were more sensitive to phenethyl isothiocyanate and tumor necrosis factor (TNF) than RWPE1 cells, while PC3 cells were more sensitive to TNF than PrEC cells. These results are consistent with the hypothesis that cancer cell redox state may modulate responses to redox-modulating therapeutic regimens

  9. Distinct Redox Profiles of Selected Human Prostate Carcinoma Cell Lines: Implications for Rational Design of Redox Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Chaiswing, Luksana [Department of Pathology and Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin, 1111 Highland Ave., WIMR 7168, Madison, WI 53705 (United States); Zhong, Weixiong; Oberley, Terry D., E-mail: toberley@wisc.edu [Department of Pathology and Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin, 1111 Highland Ave., WIMR 7168, Madison, WI 53705 (United States); Pathology and Laboratory Medicine Service, William S. Middleton Memorial Veterans Hospital, Rm A-35, 2500 Overlook Terrace, Madison, WI 53705 (United States)

    2011-09-13

    The effects of several cancer chemotherapeutic drugs and radiation are mediated, at least in part, by oxidative stress. To better understand this process, we analyzed certain biochemical properties affecting reduction-oxidation (redox) balance in normal prostate epithelial cells and several prostate cancer cell lines. Highly aggressive androgen-independent prostate cancer PC3 cells demonstrated significantly higher levels of total antioxidant capacity (AC) and intra- and extracellular glutathione (GSH)/glutathione disulfide (GSSG) ratios when compared with normal prostate epithelial PrEC cells. WPE1-NB26 cells, a prostate cancer cell line derived from immortalized RWPE1 human prostate epithelial cells, demonstrated significantly higher levels of total AC and intra- and extracellular GSH/GSSG ratios, but lower levels of intracellular reactive oxygen/nitrogen species and lipid peroxidation compared with RWPE1 cells. LNCaP-C4-2 cells, a more aggressive prostate cancer derived from less aggressive androgen-responsive LNCaP cells, exhibited higher levels of AC and extracellular GSH/GSSG ratio when compared to LNCaP cells. Specific cell types showed distinct cytotoxic responses to redox-modulating compounds. WPE1-NB26 cells were more sensitive to phenethyl isothiocyanate and tumor necrosis factor (TNF) than RWPE1 cells, while PC3 cells were more sensitive to TNF than PrEC cells. These results are consistent with the hypothesis that cancer cell redox state may modulate responses to redox-modulating therapeutic regimens.

  10. Distinct Redox Profiles of Selected Human Prostate Carcinoma Cell Lines: Implications for Rational Design of Redox Therapy

    Directory of Open Access Journals (Sweden)

    Luksana Chaiswing

    2011-09-01

    Full Text Available The effects of several cancer chemotherapeutic drugs and radiation are mediated, at least in part, by oxidative stress. To better understand this process, we analyzed certain biochemical properties affecting reduction-oxidation (redox balance in normal prostate epithelial cells and several prostate cancer cell lines. Highly aggressive androgen-independent prostate cancer PC3 cells demonstrated significantly higher levels of total antioxidant capacity (AC and intra- and extracellular glutathione (GSH/glutathione disulfide (GSSG ratios when compared with normal prostate epithelial PrEC cells. WPE1-NB26 cells, a prostate cancer cell line derived from immortalized RWPE1 human prostate epithelial cells, demonstrated significantly higher levels of total AC and intra- and extracellular GSH/GSSG ratios, but lower levels of intracellular reactive oxygen/nitrogen species and lipid peroxidation compared with RWPE1 cells. LNCaP-C4-2 cells, a more aggressive prostate cancer derived from less aggressive androgen-responsive LNCaP cells, exhibited higher levels of AC and extracellular GSH/GSSG ratio when compared to LNCaP cells. Specific cell types showed distinct cytotoxic responses to redox-modulating compounds. WPE1-NB26 cells were more sensitive to phenethyl isothiocyanate and tumor necrosis factor (TNF than RWPE1 cells, while PC3 cells were more sensitive to TNF than PrEC cells. These results are consistent with the hypothesis that cancer cell redox state may modulate responses to redox-modulating therapeutic regimens.

  11. T3 glycoprotein is functional although structurally distinct on human T-cell receptor γ T lymphocytes

    International Nuclear Information System (INIS)

    The T-cell receptor (TCR) γ gene product occurs in association with T3 (CD3) polypeptides on the surface of human T lymphocytes. TCR γ lymphocytes express arrays of T3 polypeptides distinct from those typically observed on TCR αβ lymphocytes. This report demonstrates that identical T3 γ, δ, and element of polypeptides are synthesized by TCR γ lymphocytes and TCR αβ lymphocytes. However, the processing of T3 δ oligosaccharides is distinct in the two cell types. This observation may suggest distinct quaternary structures of these receptor complexes. Despite these structural differences, the T3 molecule on TCR γ lymphocytes is functional. It is associated with and comodulates with TCR γ and it serves as a substrate from protein kinase C-mediated phosphorylation. Anti-T3 monoclonal antibodies induce a rapid increase in cytoplasmic free calcium, indicating that the receptor complex is involved in signal transduction and triggering of TCR γ lymphocytes

  12. Transcriptome comparison of distinct osteolineage subsets in the hematopoietic stem cell niche using a triple fluorescent transgenic mouse model.

    Science.gov (United States)

    Yu, Vionnie W C; Lymperi, Stefania; Ferraro, Francesca; Scadden, David T

    2015-09-01

    The bone marrow niche is recognized as a central player in maintaining and regulating the behavior of hematopoietic stem and progenitor cells. Specific gain-of and loss-of function experiments perturbing a range of osteolineage cells or their secreted proteins had been shown to affect stem cell maintenance (Calvi et al, 2003 [1]; Stier et al., 2005 [2]; Zhang et al., 2003 [3]; Nilsson et al., 2005 [4]; Greenbaum et al., 2013 [5]) and engraftment (Adam et al., 2006, 2009 [6,7]). We used specific in vivo cell deletion approaches to dissect the niche cell-parenchymal cell dependency in a complex bone marrow microenvironment. Endogenous deletion of osteocalcin-expressing (Ocn(+)) cells led to a loss of T immune cells (Yu et al., 2015 [8]. Ocn(+) cells express the Notch ligand DLL4 to communicate with T-competent progenitors, and thereby ensuring T precursor production and expression of chemotactic molecules on their cell surface for subsequent thymic seeding. In contrast, depletion of osterix-expressing (Osx(+)) osteoprogenitors led to reduced B immune cells. These distinct hematopoietic phenotypes suggest specific pairing of mesenchymal niche cells and parenchymal hematopoietic cells in the bone marrow to create unique functional units to support hematopoiesis. Here, we present the global gene expression profiles of these osteolineage subtypes utilizing a triple fluorescent transgenic mouse model (OsxCre(+);Rosa-mCh(+);Ocn:Topaz(+)) that labels Osx(+) cells red, Ocn(+) cells green, and Osx(+) Ocn(+) cells yellow. This system allows isolation of distinct osteolineage subsets within the same animal by flow cytometry. Array data that have been described in our study [8] are also publically available from NCBI Gene Expression Omnibus (GEO) with the accession number GSE66042. Differences in gene expression may correlate with functional difference in supporting hematopoiesis. PMID:26484277

  13. Evaluation of the ability of N-terminal fragment of lethal factor of Bacillus anthracis for delivery of Mycobacterium T cell antigen ESAT-6 into cytosol of antigen presenting cells to elicit effective cytotoxic T lymphocyte response

    International Nuclear Information System (INIS)

    We report the ability of N-terminal fragment of lethal factor of Bacillus anthracis to deliver genetically fused ESAT-6 (early secretory antigen target), a potent T cell antigen of Mycobacterium tuberculosis, into cytosol to elicit Cytotoxic T lymphocyte (CTL) response. In vitro Th1 cytokines data and CTL assay proved that efficient delivery of LFn.ESAT-6 occurs in cytosol, in the presence of protective antigen (PA), and leads to generation of effective CTL response. Since CTL response is essential for protection against intracellular pathogens and, it is well known that only single T cell epitope or single antigenic protein is not sufficient to elicit protective CTL response due to variation or polymorphism in MHC-I alleles among the individuals, we suggest that as a fusion protein LFn can be used to deliver multiepitopes of T cells or multiproteins which can generate effective CTLs against intracellular pathogens like M. tuberculosis. It can be used to enhance the protective efficacy of BCG vaccine

  14. Immunochemical characterization of inhibitory mouse cortical neurons: Three chemically distinct classes of inhibitory cells

    OpenAIRE

    Xu, Xiangmin; Roby, Keith D.; Edward M Callaway

    2010-01-01

    The cerebral cortex has diverse types of inhibitory neurons. In rat cortex, past research has shown that parvalbumin (PV), somatostatin (SOM), calretinin (CR), and cholecystokinin (CCK) label four distinct chemical classes of GABAergic interneurons. However, in contrast to rat cortex, previous studies indicate that there is significant co-localization of SOM and CR in mouse cortical inhibitory neurons. In the present study, we further characterized immunochemical distinctions among mouse inhi...

  15. Common and distinct signals specify the distribution of blood and vascular cell lineages in Xenopus laevis embryos.

    Science.gov (United States)

    Iraha, Fumie; Saito, Yoshinari; Yoshida, Keiko; Kawakami, Masatoki; Izutsu, Yumi; Daar, Ira Owen; Maéno, Mitsugu

    2002-10-01

    In an effort to elucidate the regulatory mechanisms that determine the fate of blood cells and vascular cells in the ventral blood island mesoderm, the embryonic expression of Xtie-2, a Xenopus homolog of the tie-2 receptor tyrosine kinase, was examined. Whole-mount in situ hybridization analysis revealed that Xtie-2 mRNA is expressed at the late tailbud stage within the regions where endothelial precursor cells exist. On the ventral side of embryos, Xtie-2-positive cells are predominantly present just outside the boundary of alpha-globin-positive cells, thus the expression pattern of these two markers seems mutually exclusive. Further experiments revealed that there is a consistent and strong correlation between the induction of Xtie-2 and alpha-globin expression in embryos and explant tissues. First, these two markers displayed overlapping expression in embryos ventralized by the removal of a "dorsal determinant" from the vegetal cytoplasm at the 1-cell stage. Second, expression of both Xtie-2 and alpha-globin were markedly induced in ectodermal explants (animal caps) from embryos co-injected with activin and bone morphogenetic protein (BMP)-4 RNA. Furthermore, both Xtie-2 and alpha-globin messages were strongly positive in dorsal marginal zone explants that had been injected with BMP-4 RNA. In contrast, however, there was a clear distinction in the localization of these two transcripts in embryos dorsalized by LiCl treatment. Distinct localization was also found in the ventral marginal zone (VMZ) explants. Using the VMZ explant system, we demonstrate a role of fibroblast growth factor (FGF) signaling in enhancing the vascular cell marker and reducing the blood cell marker. The present study suggests that the early steps of blood and vascular cell differentiation are regulated by a common BMP-4-dependent signaling; however, distinct factor(s) such as FGF are involved in different distribution of these two cell lineages. PMID:12392573

  16. Immunization with Ty21a live oral typhoid vaccine elicits cross-reactive multifunctional CD8+ T cell responses against Salmonella enterica serovar Typhi, S. Paratyphi A and S. Paratyphi B in humans

    Science.gov (United States)

    Wahid, Rezwanul; Fresnay, Stephanie; Levine, Myron M.; Sztein, Marcelo B.

    2015-01-01

    Previously we have extensively characterized Salmonella enterica serovar Typhi (S. Typhi)-specific cell-mediated immune responses (CMI) in volunteers orally immunized with the licensed Ty21a typhoid vaccine. In this study we measured Salmonella-specific multifunctional (MF) CD8+ T cell responses to further investigate whether Ty21a elicits cross reactive CMI against S. Paratyphi A and S. Paratyphi B, which also cause enteric fever. Ty21a elicited cross-reactive CMI against all three Salmonella serotypes were predominantly observed in CD8+ T effector/memory (TEM) and, to a lesser extent, in CD8+CD45RA+ TEM (TEMRA) subsets. These CD8+ T cell responses were largely mediated by MF cells co producing IFN-γ, MIP-1β and expressing CD107a with or without TNF-α. Significant proportions of Salmonella-specific MF cells expressed the gut homing molecule integrin α4β7. In most subjects similar MF responses were observed to S. Typhi and S. Paratyphi B, but not to S. Paratyphi A. These results suggest that Ty21a elicits MF CMI against Salmonella which could be critical in clearing the infection. Moreover, because S. Paratyphi A is a major public concern and Ty21a was shown in field studies not to afford cross-protection to S. Paratyphi A, these results will be important in developing a S. Typhi/S. Paratyphi A bivalent vaccine against enteric fevers. PMID:25872480

  17. All or none cell responses of Ca2+-dependent K channels elicited by calcium or lead in human red cells can be explained by heterogeneity of agonist distribution

    International Nuclear Information System (INIS)

    We have studied the all or none cell response of Ca2+-dependent K+ channels to added Ca in human red cells depleted of ATP by incubation with iodoacetate and inosine. A procedure was used which allows separation and differential analysis of responding and nonresponding cells. Responding (H for heavy) cells incubated in medium containing 5 mM K lose KCl and water and increase their density to the point of sinking on diethylphthalate (specific gravity = 1.12) on centrifugation. Nonresponding (L for light) cells do not lose KCl at all. There is no intermediate behavior. Increasing the Ca concentration in the medium increases the fraction of cells which become H. No differences in the sensitivity to Ca2+ of the individual K+ channels were detected in inside-out vesicles prepared either from H or from L cells. The Ca content of H cells was higher than that of L cells. Cells depleted of ATP by incubation with iodoacetate and inosine sustain pump-leak Ca fluxes of about 15 mumol/liter cells per hour. ATP seems to be resynthesized in these cells at the expense of cell 2,3-diphosphoglycerate stores at a rate of about 150 mumol/liter cells per hour. Inhibition of 2,3-diphosphoglycerate phosphatase by tetrathionate increased 6-8 times the measured rate of uptake of external 45Ca. This was accompanied by an increase in the fraction of H cells. All or none cell responses of Ca2+-dependent K channels have also been evidenced in intact human red cells on addition of Pb. They have the same characteristics as those in responding and nonresponding cells. The detailed study of the kinetics of Pb-induced shrinkage of red cells suspended in medium containing 5 mM K showed that changes of Pb concentration changed not only the fraction of H cells but also the rate of shrinkage of responding cells. H cells generated by Pb treatment contained significantly more lead than L cells

  18. Immunization against Clostridium perfringens cells elicits protection against Clostridium tetani in mouse model: identification of cross-reactive proteins using proteomic methodologies

    Directory of Open Access Journals (Sweden)

    Singh Lokendra

    2008-11-01

    Full Text Available Abstract Background Clostridium tetani and Clostridium perfringens are among the medically important clostridial pathogens causing diseases in man and animals. Several homologous open reading frames (ORFs have been identified in the genomes of the two pathogens by comparative genomic analysis. We tested a likelihood of extensive sharing of common epitopes between homologous proteins of these two medically important pathogens and the possibility of cross-protection using active immunization. Results Eight predominant cross-reactive spots were identified by mass spectrometry and had hits in the C. tetani E88 proteome with significant MOWSE scores. Most of the cross-reactive proteins of C. tetani shared 65–78% sequence similarity with their closest homologues in C. perfringens ATCC13124. Electron transfer flavoprotein beta-subunit (CT3 was the most abundant protein (43.3%, followed by methylaspartate ammonia-lyase (36.8% and 2-phosphoglycerate dehydratase (35.6%. All the proteins were predicted to be cytoplasmic by PSORT protein localization algorithm. Active immunization with C. perfringens whole cells elicited cross-protective immunity against C. tetani infection in a mouse model. Conclusion Most of the dominant cross-reactive proteins of C. tetani belonged to the cluster of orthologous group (COG functional category, either of posttranslational modification, protein turnover, and chaperones (O or energy production and conversion (C. The homologs of the identified proteins have been shown to play role in pathogenesis in other Gram-positive pathogenic bacteria. Our findings provide basis for the search of potential vaccine candidates with broader coverage, encompassing more than one pathogenic clostridial species.

  19. Functional Proteomics Screen Enables Enrichment of Distinct Cell Types from Human Pancreatic Islets

    OpenAIRE

    Revital Sharivkin; Walker, Michael D.; Yoav Soen

    2015-01-01

    The current world-wide epidemic of diabetes has prompted attempts to generate new sources of insulin-producing cells for cell replacement therapy. An inherent challenge in many of these strategies is the lack of cell-surface markers permitting isolation and characterization of specific cell types from differentiating stem cell populations. Here we introduce an iterative proteomics procedure allowing tag-free isolation of cell types based on their function. Our method detects and associates sp...

  20. Transplantable progenitors of natural killer cells are distinct from those of T and B lymphocytes.

    OpenAIRE

    Hackett, J; Bosma, G C; Bosma, M J; Bennett, M.; Kumar, V

    1986-01-01

    We have utilized a mouse mutant (C.B-17 scid) that lacks functional T and B lymphocytes to examine the relationship among transplantable progenitors of natural killer (NK) cells, T cells, and B cells. The NK-progenitor cells contained in the bone marrow were detected by their ability to generate mature NK cells, following transfer of bone marrow cells into NK cell-depleted and lethally irradiated mice. Regeneration of NK activity in the recipient mice was monitored by two different assays: th...

  1. Mmp1 and Mmp2 cooperatively induce Drosophila fat body cell dissociation with distinct roles

    OpenAIRE

    Jia, Qiangqiang; Liu, Yang; Liu, Hanhan; Li, Sheng

    2014-01-01

    During Drosophila metamorphosis, the single-cell layer of fat body tissues gradually dissociates into individual cells. Via a fat body-specific RNAi screen in this study, we found that two matrix metalloproteinases (MMPs), Mmp1 and Mmp2, are both required for fat body cell dissociation. As revealed through a series of cellular, biochemical, molecular, and genetic experiments, Mmp1 preferentially cleaves DE-cadherin-mediated cell-cell junctions, while Mmp2 preferentially degrades basement memb...

  2. Functional proteomics screen enables enrichment of distinct cell types from human pancreatic islets.

    Directory of Open Access Journals (Sweden)

    Revital Sharivkin

    Full Text Available The current world-wide epidemic of diabetes has prompted attempts to generate new sources of insulin-producing cells for cell replacement therapy. An inherent challenge in many of these strategies is the lack of cell-surface markers permitting isolation and characterization of specific cell types from differentiating stem cell populations. Here we introduce an iterative proteomics procedure allowing tag-free isolation of cell types based on their function. Our method detects and associates specific cell-surface markers with particular cell functionality by coupling cell capture on antibody arrays with immunofluorescent labeling. Using this approach in an iterative manner, we discovered marker combinations capable of enriching for discrete pancreatic cell subtypes from human islets of Langerhans: insulin-producing beta cells (CD9high/CD56+, glucagon-producing alpha cells (CD9-/CD56+ and trypsin-producing acinar cells (CD9-/CD56-. This strategy may assist future beta cell research and the development of diagnostic tools for diabetes. It can also be applied more generally for function-based purification of desired cell types from other limited and heterogeneous biological samples.

  3. Mycobacterial secretion systems ESX-1 and ESX-5 play distinct roles in host cell death and inflammasome activation

    KAUST Repository

    Abdallah, Abdallah

    2011-09-28

    During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5 - two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators - during the macrophage infection cycle. Using wild-type, ESX-1- and ESX-5-deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1b activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread. Copyright © 2011 by The American Association of Immunologists, Inc.

  4. Mycobacterial secretion systems ESX-1 and ESX-5 play distinct roles in host cell death and inflammasome activation.

    Science.gov (United States)

    Abdallah, Abdallah M; Bestebroer, Jovanka; Savage, Nigel D L; de Punder, Karin; van Zon, Maaike; Wilson, Louis; Korbee, Cees J; van der Sar, Astrid M; Ottenhoff, Tom H M; van der Wel, Nicole N; Bitter, Wilbert; Peters, Peter J

    2011-11-01

    During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5--two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators--during the macrophage infection cycle. Using wild-type, ESX-1- and ESX-5-deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1β activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread. PMID:21957139

  5. Correlation between circulating white blood cell counts and level of protective immune response against bovine viral diarrhea virus elicited by a modified live vaccine

    Science.gov (United States)

    Two trials (T1 and T2) were conducted to examine the range of responses elicited against bovine viral diarrhea virus (BVDV) by vaccination with modified live vaccine and to determine the level of response required for prevention of clinical disease. For T1, BVDV neutralizing (BVDV VN) titers were de...

  6. The B-cell identity factor Pax5 regulates distinct transcriptional programmes in early and late B lymphopoiesis

    Science.gov (United States)

    Revilla-i-Domingo, Roger; Bilic, Ivan; Vilagos, Bojan; Tagoh, Hiromi; Ebert, Anja; Tamir, Ido M; Smeenk, Leonie; Trupke, Johanna; Sommer, Andreas; Jaritz, Markus; Busslinger, Meinrad

    2012-01-01

    Pax5 controls the identity and development of B cells by repressing lineage-inappropriate genes and activating B-cell-specific genes. Here, we used genome-wide approaches to identify Pax5 target genes in pro-B and mature B cells. In these cell types, Pax5 bound to 40% of the cis-regulatory elements defined by mapping DNase I hypersensitive (DHS) sites, transcription start sites and histone modifications. Although Pax5 bound to 8000 target genes, it regulated only 4% of them in pro-B and mature B cells by inducing enhancers at activated genes and eliminating DHS sites at repressed genes. Pax5-regulated genes in pro-B cells account for 23% of all expression changes occurring between common lymphoid progenitors and committed pro-B cells, which identifies Pax5 as an important regulator of this developmental transition. Regulated Pax5 target genes minimally overlap in pro-B and mature B cells, which reflects massive expression changes between these cell types. Hence, Pax5 controls B-cell identity and function by regulating distinct target genes in early and late B lymphopoiesis. PMID:22669466

  7. Competition of two distinct actin networks for actin defines a bistable switch for cell polarization

    Science.gov (United States)

    Lomakin, Alexis J.; Lee, Kun-Chun; Han, Sangyoon J.; Bui, D A.; Davidson, Michael; Mogilner, Alex; Danuser, Gaudenz

    2015-01-01

    Symmetry-breaking polarization enables functional plasticity of cells and tissues and is yet not well understood. Here we show that epithelial cells, hard-wired to maintain a static morphology and to preserve tissue organization, can spontaneously switch to a migratory polarized phenotype upon relaxation of the actomyosin cytoskeleton. We find that myosin-II engages actin in the formation of cortical actomyosin bundles and thus makes it unavailable for deployment in the process of dendritic growth normally driving cell motility. At low contractility regimes epithelial cells polarize in a front-back manner due to emergence of actin retrograde flows powered by dendritic polymerization of actin. Coupled to cell movement, the flows transport myosin-II from the front to the back of the cell, where the motor locally “locks” actin in contractile bundles. This polarization mechanism could be employed by embryonic and cancer epithelial cells in microenvironments where high contractility-driven cell motion is inefficient. PMID:26414403

  8. TCR repertoire and Foxp3 expression define functionally distinct subsets of CD4+ Treg cells1

    OpenAIRE

    Kuczma, Michal; Pawlikowska, Iwona; Kopij, Magdalena; Podolsky, Robert; Rempala, Grzegorz A.; Kraj, Piotr

    2009-01-01

    Despite extensive research efforts to characterize peripheral regulatory T cells (Treg) expressing transcription factor Foxp3, their subset complexity, phenotypic characteristics, TCR repertoire and antigen specificities remain ambiguous. Here, we identify and define two subsets of peripheral Treg cells differing in Foxp3 expression level and TCR repertoires. Treg cells expressing a high level of Foxp3 and TCRs not utilized by naive CD4+ T cells present a stable suppressor phenotype and domin...

  9. Cells release subpopulations of exosomes with distinct molecular and biological properties

    NARCIS (Netherlands)

    Willms, Eduard; Johansson, Henrik J; Mäger, Imre; Lee, Yi; Blomberg, K Emelie M; Sadik, Mariam; Alaarg, Amr; Smith, C I Edvard; Lehtiö, Janne; El Andaloussi, Samir; Wood, Matthew J A; Vader, Pieter

    2016-01-01

    Cells release nano-sized membrane vesicles that are involved in intercellular communication by transferring biological information between cells. It is generally accepted that cells release at least three types of extracellular vesicles (EVs): apoptotic bodies, microvesicles and exosomes. While a wi

  10. Multiparameter Analysis of Human Bone Marrow Stromal Cells Identifies Distinct Immunomodulatory and Differentiation-Competent Subtypes

    NARCIS (Netherlands)

    S. James (Sally); J. Fox (James); F. Afsari (Farinaz); J. Lee (Jennifer); S. Clough (Sally); C. Knight (Charlotte); J. Ashmore (James); P. Ashton (Peter); O. Preham (Olivier); M.J. Hoogduijn (Martin); R.D.A.R. Ponzoni (Raquel De Almeida Rocha); Y. Hancock; M. Coles (Mark); P.G. Genever (Paul)

    2015-01-01

    textabstractBone marrow stromal cells (BMSCs, also called bone-marrow-derived mesenchymal stromal cells) provide hematopoietic support and immunoregulation and contain a stem cell fraction capable of skeletogenic differentiation. We used immortalized human BMSC clonal lines for multi-level analysis

  11. Naive and memory T cells show distinct pathways of lymphocyte recirculation

    OpenAIRE

    1990-01-01

    In this report, we have addressed two questions concerning immunological memory: the way in which naive and memory T cells recirculate through the body, and the intrinsic rate of division within the naive and memory populations. We identified naive and memory T cells in sheep by their cell surface phenotype and their ability to respond to recall antigen. Memory T cells were CD2hi, CD58hi, CD44hi, CD11ahi, and CD45R-, as pertains in man. T cells that crossed from blood to the tissues of the hi...

  12. Effects of an antimitotic drug on mechanical behaviours of the cytoskeleton in distinct grades of colon cancer cells.

    Science.gov (United States)

    Seyedpour, S M; Pachenari, M; Janmaleki, M; Alizadeh, M; Hosseinkhani, H

    2015-04-13

    Biomechanical behaviours of cells change during cancer progression due to alterations in the main cytoskeletal proteins. Microtubules play a vital role in mitosis and in supporting the integrity of the cell due to their ability to withstand high compressive loads. Accordingly, microtubule-targeting agents (MTAs) have become one of the most promising classes of drugs in cancer therapy. This study evaluated changes in visco-elastic parameters induced by an appropriate concentration of an antimitotic drug in two different grades of colon cancer cells. Actin microfilaments and microtubules contents in the cells were evaluated by Western blot analysis and fluorescence intensity calculation. Micropipette aspiration experiments showed that the MTA had distinct mechanical effects on different cell lines. The more aggressive the cells, the greater the reduction in elasticity and viscosity. Invasive cells had a higher initial instantaneous Young's modulus than primary cells, but this reduced to approximately one half of the values for primary cells after 48 h of drug treatment. A considerable association was seen between the changes in mechanical properties and the microtubule to F-actin microfilament content ratio, which decreased with MTA treatment. PMID:25678199

  13. hESC Differentiation toward an Autonomic Neuronal Cell Fate Depends on Distinct Cues from the Co-Patterning Vasculature

    Directory of Open Access Journals (Sweden)

    Lisette M. Acevedo

    2015-06-01

    Full Text Available To gain insight into the cellular and molecular cues that promote neurovascular co-patterning at the earliest stages of human embryogenesis, we developed a human embryonic stem cell model to mimic the developing epiblast. Contact of ectoderm-derived neural cells with mesoderm-derived vasculature is initiated via the neural crest (NC, not the neural tube (NT. Neurovascular co-patterning then ensues with specification of NC toward an autonomic fate requiring vascular endothelial cell (EC-secreted nitric oxide (NO and direct contact with vascular smooth muscle cells (VSMCs via T-cadherin-mediated homotypic interactions. Once a neurovascular template has been established, NT-derived central neurons then align themselves with the vasculature. Our findings reveal that, in early human development, the autonomic nervous system forms in response to distinct molecular cues from VSMCs and ECs, providing a model for how other developing lineages might coordinate their co-patterning.

  14. Characterization of two novel cell lines with distinct heterogeneity derived from a single human bile duct carcinoma.

    Directory of Open Access Journals (Sweden)

    Jinghan Wang

    Full Text Available BACKGROUND: Intratumoral heterogeneity reflects subclonal diversity and accounts for a variety of clinically defined phenotypes including the development of drug resistance and recurrence. However, intratumoral heterogeneity of bile duct carcinoma (BDC is rarely studied. METHODS: Two highly heterogeneous cell lines named EH-CA1a and EH-CA1b were established from a primary tumor tissue of a pathologically proven BDC. Distinct heterogeneity and underlying mechanisms of two cell lines in karyotype, colony formation, tumorgenicity, and sensitivity to chemoradiotherapy were intensively studied. RESULTS: Both cell lines showed typical morphology of cancer cells. EH-CA1a cells grew as free-floating aggregates, while EH-CA1b cells grew adherently as a monolayer. EH-CA1a cells had higher cloning efficiencies and were able to keep proliferating under hypoxic condition. Coincidentally, hypoxia-induced factor-1α (HIF1α and vascular endothelial growth factor (VEGF mRNA were significantly higher in EH-CA1a cells than in EH-CA1b cells. Both cell lines were tumorigenic in nude mouse, however, EH-CA1a cells showed more aggressive characteristics. Most importantly, the EH-CA1a cells showed much more resistance against radiation and chemotherapy with gemcitabine. Metastasis-related genes including matrix metalloproteinase 2 (MMP-2, MMP-9, epithelial-mesenchymal transition (EMT markers such as Vimentin, Snail, and Twist, are more highly expressed in EH-CA1a cells than in EH-CA1b cells. Moreover, the percentage of cells expressing cancer stem cell-like marker, CD133, in EH-CA1a cells is much higher than that in EH-CA1b cells. Moreover, knockdown of CD133 in both EH-CA1a and EH-CA1b cells significantly reduced their invasive potential and increased their sensitivities to radiation and gemcitabine, suggesting the differential expression of CD133 protein may partially account for the difference in malignancy between these two cancer cells. CONCLUSION: Establishment

  15. Members of the heat-shock protein 70 family promote cancer cell growth by distinct mechanisms

    DEFF Research Database (Denmark)

    Rohde, Mikkel; Daugaard, Mads; Jensen, Mette Hartvig;

    2005-01-01

    Whereas the stress-inducible heat-shock protein 70 (Hsp70) has gained plenty of attention as a putative target for tumor therapy, little is known about the role of other Hsp70 proteins in cancer. Here we present the first thorough analysis of the expression and function of the cytosolic Hsp70...... survival of tumorigenic as well as nontumorigenic cells depended on Hsc70. Cancer cells depleted for Hsp70 and Hsp70-2 displayed strikingly different morphologies (detached and round vs. flat senescent-like), cell cycle distributions (G2/M vs. G1 arrest) and gene expression profiles. Only Hsp70-2 depletion...... proteins in human cancer cells and identify Hsp70-2, a protein essential for spermatogenesis, as an important regulator of cancer cell growth. Targeted knock-down of the individual family members by RNA interference revealed that both Hsp70 and Hsp70-2 were required for cancer cell growth, whereas the...

  16. Edges of human embryonic stem cell colonies display distinct mechanical properties and differentiation potential

    OpenAIRE

    Rosowski, Kathryn A.; Mertz, Aaron F.; Samuel Norcross; Dufresne, Eric R.; Valerie Horsley

    2015-01-01

    In order to understand the mechanisms that guide cell fate decisions during early human development, we closely examined the differentiation process in adherent colonies of human embryonic stem cells (hESCs). Live imaging of the differentiation process reveals that cells on the outer edge of the undifferentiated colony begin to differentiate first and remain on the perimeter of the colony to eventually form a band of differentiation. Strikingly, this band is of constant width in all colonies,...

  17. Distinctions in sensitivity and repair of cells of children with some hereditary diseases

    Energy Technology Data Exchange (ETDEWEB)

    Zasukhina, G.D.; Barashnev, Yu.I.; Vasil' eva, I.M.; Sdirkova, N.I.; Semyachkina, A.N. (AN SSSR, Moscow. Inst. Obshchej Genetiki)

    A study was made of blood cell sensitivity of children with some hereditary diseases, to ..gamma..-radiation and 4-nitro-quinoline-1-oxide. Using the host cell reactivation and chromatographic methods we revealed the increase in the sensitivity to the above mentioned agents and inhibition of the repair function in cells of patients with the following diseases: Marfan's disease, histidinemia, osteogenesis imperfecta, Sylvere-Russelle, Laurence, Franchescetti, and Losch-Nychane syndromes.

  18. Distinctions in sensitivity and repair of cells of children with some hereditary diseases

    International Nuclear Information System (INIS)

    A study was made of blood cell sensitivity of children, with some hereditary diseases, to ν-radiation and 4-nitro-quinoline-1-oxide. Using the host cell reactivation and chromatographic methods we revealed the increase in the sensitivity to the above mentioned agents and inhibition of the repair function in cells of patients with the following diseases: Marfan's disease, histidinemia, osteogenesis imperfecta, Sylvere-Russelle, Laurence, Franchescetti, and Losch-Nychane syndromes

  19. Cells release subpopulations of exosomes with distinct molecular and biological properties

    OpenAIRE

    Eduard Willms; Johansson, Henrik J; Imre Mäger; Yi Lee; Blomberg, K. Emelie M.; Mariam Sadik; Amr Alaarg; C.I. Edvard Smith; Janne Lehtiö; Samir EL Andaloussi; Matthew J A Wood; Pieter Vader

    2016-01-01

    Cells release nano-sized membrane vesicles that are involved in intercellular communication by transferring biological information between cells. It is generally accepted that cells release at least three types of extracellular vesicles (EVs): apoptotic bodies, microvesicles and exosomes. While a wide range of putative biological functions have been attributed to exosomes, they are assumed to represent a homogenous population of EVs. We hypothesized the existence of subpopulations of exosomes...

  20. Brown adipose tissue harbors a distinct sub-population of regulatory T cells.

    Directory of Open Access Journals (Sweden)

    Dasa Medrikova

    Full Text Available Regulatory T (Treg cells are critical determinants of both immune responses and metabolic control. Here we show that systemic ablation of Treg cells compromised the adaptation of whole-body energy expenditure to cold exposure, correlating with impairment in thermogenic marker gene expression and massive invasion of pro-inflammatory macrophages in brown adipose tissue (BAT. Indeed, BAT harbored a unique sub-set of Treg cells characterized by a unique gene signature. As these Treg cells respond to BAT activation upon cold exposure, this study defines a BAT-specific Treg sub-set with direct implications for the regulation of energy homeostasis in response to environmental stress.

  1. Oral squamous cell carcinomas in age distinct population: A comparison of p53 immunoexpression

    OpenAIRE

    Akhilesh Chandra; Anil Singh; Bastian Thattil Sebastian; Archana Agnihotri; Ruchita Bali; Pushpendra Kumar Verma

    2013-01-01

    Aims: The study aimed to assess the diffused expression of p53 protein in patients with OSCC and its association with age at diagnosis (using 50 years as a cut point). Study Design: Ten normal oral mucosa and sixty OSCC lesions from age-distinct patient populations were immunohistochemically analyzed for the expression of p53 protein. Results: In OSCC cases, 31 out of total 60 cases (51.67%) showed positive expression for p53 protein and it was more common in older study group (56.67%) ...

  2. Monocyte-derived macrophages exhibit distinct and more restricted HIV-1 integration site repertoire than CD4(+) T cells.

    Science.gov (United States)

    Kok, Yik Lim; Vongrad, Valentina; Shilaih, Mohaned; Di Giallonardo, Francesca; Kuster, Herbert; Kouyos, Roger; Günthard, Huldrych F; Metzner, Karin J

    2016-01-01

    The host genetic landscape surrounding integrated HIV-1 has an impact on the fate of the provirus. Studies analysing HIV-1 integration sites in macrophages are scarce. We studied HIV-1 integration site patterns in monocyte-derived macrophages (MDMs) and activated CD4(+) T cells derived from seven antiretroviral therapy (ART)-treated HIV-1-infected individuals whose cells were infected ex vivo with autologous HIV-1 isolated during the acute phase of infection. A total of 1,484 unique HIV-1 integration sites were analysed. Their distribution in the human genome and genetic features, and the effects of HIV-1 integrase polymorphisms on the nucleotide selection specificity at these sites were indistinguishable between the two cell types, and among HIV-1 isolates. However, the repertoires of HIV-1-hosting gene clusters overlapped to a higher extent in MDMs than in CD4(+) T cells. The frequencies of HIV-1 integration events in genes encoding HIV-1-interacting proteins were also different between the two cell types. Lastly, HIV-1-hosting genes linked to clonal expansion of latently HIV-1-infected CD4(+) T cells were over-represented in gene hotspots identified in CD4(+) T cells but not in those identified in MDMs. Taken together, the repertoire of genes targeted by HIV-1 in MDMs is distinct from and more restricted than that of CD4(+) T cells. PMID:27067385

  3. Monocyte-derived macrophages exhibit distinct and more restricted HIV-1 integration site repertoire than CD4+ T cells

    Science.gov (United States)

    Kok, Yik Lim; Vongrad, Valentina; Shilaih, Mohaned; Di Giallonardo, Francesca; Kuster, Herbert; Kouyos, Roger; Günthard, Huldrych F.; Metzner, Karin J.

    2016-01-01

    The host genetic landscape surrounding integrated HIV-1 has an impact on the fate of the provirus. Studies analysing HIV-1 integration sites in macrophages are scarce. We studied HIV-1 integration site patterns in monocyte-derived macrophages (MDMs) and activated CD4+ T cells derived from seven antiretroviral therapy (ART)-treated HIV-1-infected individuals whose cells were infected ex vivo with autologous HIV-1 isolated during the acute phase of infection. A total of 1,484 unique HIV-1 integration sites were analysed. Their distribution in the human genome and genetic features, and the effects of HIV-1 integrase polymorphisms on the nucleotide selection specificity at these sites were indistinguishable between the two cell types, and among HIV-1 isolates. However, the repertoires of HIV-1-hosting gene clusters overlapped to a higher extent in MDMs than in CD4+ T cells. The frequencies of HIV-1 integration events in genes encoding HIV-1-interacting proteins were also different between the two cell types. Lastly, HIV-1-hosting genes linked to clonal expansion of latently HIV-1-infected CD4+ T cells were over-represented in gene hotspots identified in CD4+ T cells but not in those identified in MDMs. Taken together, the repertoire of genes targeted by HIV-1 in MDMs is distinct from and more restricted than that of CD4+ T cells. PMID:27067385

  4. The epidermis comprises autonomous compartments maintained by distinct stem cell populations

    DEFF Research Database (Denmark)

    Page, Mahalia E; Lombard, Patrick; Ng, Felicia;

    2013-01-01

    populations. In contrast, upon wounding, stem cell progeny from multiple compartments acquire lineage plasticity and make permanent contributions to regenerating tissue. We further show that oncogene activation in Lrig1(+ve) cells drives hyperplasia but requires auxiliary stimuli for tumor formation...

  5. Two distinct modes for propagation of histone PTMs across the cell cycle

    DEFF Research Database (Denmark)

    Alabert, Constance; Barth, Teresa K; Reverón-Gómez, Nazaret; Sidoli, Simone; Schmidt, Andreas; Jensen, Ole N; Imhof, Axel; Groth, Anja

    2015-01-01

    Epigenetic states defined by chromatin can be maintained through mitotic cell division. However, it remains unknown how histone-based information is transmitted. Here we combine nascent chromatin capture (NCC) and triple-SILAC (stable isotope labeling with amino acids in cell culture) labeling to...

  6. Interaction proteomics analysis of polycomb proteins defines distinct PRC1 complexes in mammalian cells

    DEFF Research Database (Denmark)

    Vandamme, Julien; Völkel, Pamela; Rosnoblet, Claire; Le Faou, Perrine; Angrand, Pierre-Olivier

    2011-01-01

    in order to identify interacting partners of CBX family proteins under the same experimental conditions. Our analysis identified with high confidence about 20 proteins co-eluted with CBX2 and CBX7 tagged proteins, about 40 with CBX4, and around 60 with CBX6 and CBX8. We provide evidences that the CBX...... family proteins are mutually exclusive and define distinct PRC1-like protein complexes. CBX proteins also interact with different efficiencies with the other PRC1 components. Among the novel CBX interacting partners, protein kinase 2 associates with all CBX-PRC1 protein complexes, whereas 14-3-3 proteins...... specifically bind to CBX4. 14-3-3 protein binding to CBX4 appears to modulate the interaction between CBX4 and the BMI1/PCGF components of PRC1, but has no effect on CBX4-RING1/RNF2 interaction. Finally, we suggest that differences in CBX protein interactions would account, at least in part, for distinct...

  7. Sets of RNA repeated tags and hybridization-sensitive fluorescent probes for distinct images of RNA in a living cell.

    Directory of Open Access Journals (Sweden)

    Takeshi Kubota

    Full Text Available BACKGROUND: Imaging the behavior of RNA in a living cell is a powerful means for understanding RNA functions and acquiring spatiotemporal information in a single cell. For more distinct RNA imaging in a living cell, a more effective chemical method to fluorescently label RNA is now required. In addition, development of the technology labeling with different colors for different RNA would make it easier to analyze plural RNA strands expressing in a cell. METHODOLOGY/PRINCIPAL FINDINGS: Tag technology for RNA imaging in a living cell has been developed based on the unique chemical functions of exciton-controlled hybridization-sensitive oligonucleotide (ECHO probes. Repetitions of selected 18-nucleotide RNA tags were incorporated into the mRNA 3'-UTR. Pairs with complementary ECHO probes exhibited hybridization-sensitive fluorescence emission for the mRNA expressed in a living cell. The mRNA in a nucleus was detected clearly as fluorescent puncta, and the images of the expression of two mRNAs were obtained independently and simultaneously with two orthogonal tag-probe pairs. CONCLUSIONS/SIGNIFICANCE: A compact and repeated label has been developed for RNA imaging in a living cell, based on the photochemistry of ECHO probes. The pairs of an 18-nt RNA tag and the complementary ECHO probes are highly thermostable, sequence-specifically emissive, and orthogonal to each other. The nucleotide length necessary for one tag sequence is much shorter compared with conventional tag technologies, resulting in easy preparation of the tag sequences with a larger number of repeats for more distinct RNA imaging.

  8. Several distinct properties of the IgE repertoire determine effector cell degranulation in response to allergen challenge

    DEFF Research Database (Denmark)

    Christensen, Lars Harder; Holm, Jens-Christian; Lund, Gitte; Riise, Erik; Lund, Kaare

    2008-01-01

    E affinity for allergen, and IgE clonality are all distinct properties of allergic patients' IgE repertoires. However, the inability to isolate individual IgE antibodies from allergic patients' sera presents a major barrier to understanding the importance of patient-specific IgE repertoires for the...... manifestation and severity of allergic symptoms. OBJECTIVE: We sought to investigate how individual properties of an IgE repertoire affect effector cell degranulation. METHODS: A panel of recombinant IgE (rIgE) antibodies specific for the major house dust mite allergen Der p 2 was developed and characterized in...... non-allergen-specific IgE, more even concentration of individual allergen-specific IgE clones, increased IgE affinity for allergen, and increased number of allergen epitopes recognized by the IgE repertoire (increased IgE clonality). CONCLUSION: This study demonstrates how distinct properties of the...

  9. Distinct Gut-Derived Bacteria Differentially Affect Three Types of Antigen-Presenting Cells and Impact on NK- and T-Cell Responses

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen; Hansen, Anne Marie Valentin; Frøkiær, Hanne

    previously been examined, but this study revealed that their effect on other kinds of APCs is markedly different. When APCs matured by different bacteria were added to either NK-cells or T-cells, different APCs combined with distinct strains of bacteria caused the production of varying amounts of cytokines...... through these mechanisms. The bacteria examined can potentially be used in tailored probiotic foods exploring their immunomodulatory properties....

  10. KIF7 Controls the Proliferation of Cells of the Respiratory Airway through Distinct Microtubule Dependent Mechanisms.

    Directory of Open Access Journals (Sweden)

    Garry L Coles

    2015-10-01

    Full Text Available The cell cycle must be tightly coordinated for proper control of embryonic development and for the long-term maintenance of organs such as the lung. There is emerging evidence that Kinesin family member 7 (Kif7 promotes Hedgehog (Hh signaling during embryonic development, and its misregulation contributes to diseases such as ciliopathies and cancer. Kif7 encodes a microtubule interacting protein that controls Hh signaling through regulation of microtubule dynamics within the primary cilium. However, whether Kif7 has a function in nonciliated cells remains largely unknown. The role Kif7 plays in basic cell biological processes like cell proliferation or cell cycle progression also remains to be elucidated. Here, we show that Kif7 is required for coordination of the cell cycle, and inactivation of this gene leads to increased cell proliferation in vivo and in vitro. Immunostaining and transmission electron microscopy experiments show that Kif7dda/dda mutant lungs are hyperproliferative and exhibit reduced alveolar epithelial cell differentiation. KIF7 depleted C3H10T1/2 fibroblasts and Kif7dda/dda mutant mouse embryonic fibroblasts have increased growth rates at high cellular densities, suggesting that Kif7 may function as a general regulator of cellular proliferation. We ascertained that in G1, Kif7 and microtubule dynamics regulate the expression and activity of several components of the cell cycle machinery known to control entry into S phase. Our data suggest that Kif7 may function to regulate the maintenance of the respiratory airway architecture by controlling cellular density, cell proliferation, and cycle exit through its role as a microtubule associated protein.

  11. Evidence of distinct tumour-propagating cell populations with different properties in primary human hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Federico Colombo

    Full Text Available BACKGROUND AND AIMS: Increasing evidence that a number of malignancies are characterised by tumour cell heterogeneity has recently been published, but there is still a lack of data concerning liver cancers. The aim of this study was to investigate and characterise tumour-propagating cell (TPC compartments within human hepatocellular carcinoma (HCC. METHODS: After long-term culture, we identified three morphologically different tumour cell populations in a single HCC specimen, and extensively characterised them by means of flow cytometry, fluorescence microscopy, karyotyping and microarray analyses, single cell cloning, and xenotransplantation in NOD/SCID/IL2Rγ/⁻ mice. RESULTS: The primary cell populations (hcc-1, -2 and -3 and two clones generated by means of limiting dilutions from hcc-1 (clone-1/7 and -1/8 differently expressed a number of tumour-associated stem cell markers, including EpCAM, CD49f, CD44, CD133, CD56, Thy-1, ALDH and CK19, and also showed different doubling times, drug resistance and tumorigenic potential. Moreover, we found that ALDH expression, in combination with CD44 or Thy-1 negativity or CD56 positivity identified subpopulations with a higher clonogenic potential within hcc-1, hcc-2 and hcc-3 primary cell populations, respectively. Karyotyping revealed the clonal evolution of the cell populations and clones within the primary tumour. Importantly, the primary tumour cell population with the greatest tumorigenic potential and drug resistance showed more chromosomal alterations than the others and contained clones with epithelial and mesenchymal features. CONCLUSIONS: Individual HCCs can harbor different self-renewing tumorigenic cell types expressing a variety of morphological and phenotypical markers, karyotypic evolution and different gene expression profiles. This suggests that the models of hepatic carcinogenesis should take into account TPC heterogeneity due to intratumour clonal evolution.

  12. Coilin phosphomutants disrupt Cajal body formation, reduce cell proliferation and produce a distinct coilin degradation product.

    Directory of Open Access Journals (Sweden)

    Zunamys I Carrero

    Full Text Available Coilin is a nuclear phosphoprotein that accumulates in Cajal bodies (CBs. CBs participate in ribonucleoprotein and telomerase biogenesis, and are often found in cells with high transcriptional demands such as neuronal and cancer cells, but can also be observed less frequently in other cell types such as fibroblasts. Many proteins enriched within the CB are phosphorylated, but it is not clear what role this modification has on the activity of these proteins in the CB. Coilin is considered to be the CB marker protein and is essential for proper CB formation and composition in mammalian cells. In order to characterize the role of coilin phosphorylation on CB formation, we evaluated various coilin phosphomutants using transient expression. Additionally, we generated inducible coilin phosphomutant cell lines that, when used in combination with endogenous coilin knockdown, allow for the expression of the phosphomutants at physiological levels. Transient expression of all coilin phosphomutants except the phosphonull mutant (OFF significantly reduces proliferation. Interestingly, a stable cell line induced to express the coilin S489D phosphomutant displays nucleolar accumulation of the mutant and generates a N-terminal degradation product; neither of which is observed upon transient expression. A N-terminal degradation product and nucleolar localization are also observed in a stable cell line induced to express a coilin phosphonull mutant (OFF. The nucleolar localization of the S489D and OFF coilin mutants observed in the stable cell lines is decreased when endogenous coilin is reduced. Furthermore, all the phosphomutant cells lines show a significant reduction in CB formation when compared to wild-type after endogenous coilin knockdown. Cell proliferation studies on these lines reveal that only wild-type coilin and the OFF mutant are sufficient to rescue the reduction in proliferation associated with endogenous coilin depletion. These results emphasize

  13. Endocrine disrupting chemicals affect the adipogenic differentiation of mesenchymal stem cells in distinct ontogenetic windows

    Energy Technology Data Exchange (ETDEWEB)

    Biemann, Ronald, E-mail: ronald.biemann@medizin.uni-halle.de [Department of Anatomy and Cell Biology, Martin Luther University, Faculty of Medicine, Halle (Germany); Navarrete Santos, Anne [Department of Anatomy and Cell Biology, Martin Luther University, Faculty of Medicine, Halle (Germany); Navarrete Santos, Alexander [Department of Cardiothoracic Surgery, Martin Luther University, Faculty of Medicine, Halle (Germany); Riemann, Dagmar [Department of Immunology, Martin Luther University, Faculty of Medicine, Halle (Germany); Knelangen, Julia [Department of Anatomy and Cell Biology, Martin Luther University, Faculty of Medicine, Halle (Germany); Blueher, Matthias [Department of Medicine, University of Leipzig, Leipzig (Germany); Koch, Holger [Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the Ruhr-University Bochum (IPA), Ruhr-University Bochum, Bochum (Germany); Fischer, Bernd [Department of Anatomy and Cell Biology, Martin Luther University, Faculty of Medicine, Halle (Germany)

    2012-01-13

    Highlights: Black-Right-Pointing-Pointer Endocrine disrupting chemicals affect adipogenesis in mesenchymal stem cells (MSC). Black-Right-Pointing-Pointer The adipogenic impact depends strongly on the window of exposure. Black-Right-Pointing-Pointer Bisphenol A reduces the potential of MSC to differentiate into adipocytes. Black-Right-Pointing-Pointer DEHP and TBT trigger the adipogenic differentiation of mesenchymal stem cells. Black-Right-Pointing-Pointer BPA, DEHP and TBT did not affect adipogenesis in embryonic stem cells. -- Abstract: Endocrine disrupting chemicals (EDC) like bisphenol A (BPA), bis(2-ethylhexyl)phthalate (DEHP) and tributyltin (TBT) are ubiquitously present in the environment and in human tissues. They bind to nuclear hormone receptors and affect cellular and developmental processes. In this study, we show that BPA, DEHP and TBT affect the adipogenic differentiation of murine mesenchymal stem cells (MSC, C3H/10T1/2) in a concentration-, stage- and compound-specific manner. C3H/10T1/2 cells and embryonic stem cells (CGR8) were exposed to BPA, DEHP or TBT at different stages of cell determination and differentiation (undifferentiated growth, adipogenic induction and terminal adipogenic differentiation). The final amount of differentiated adipocytes, cellular triglyceride content and mRNA expression of adipogenic marker genes (adiponectin, FABP4, PPAR{gamma}2, LPL) were quantified and compared with corresponding unexposed cells. BPA (10 {mu}M) decreased subsequent adipogenic differentiation of MSC, when cells were exposed during undifferentiated growth. In contrast, DEHP (100 {mu}M) during the hormonal induction period, and TBT (100 nM) in all investigated stages, enhanced adipogenesis. Importantly, exposure of undifferentiated murine embryonic stem cells did not show any effect of the investigated EDC on subsequent adipogenic differentiation.

  14. Immunization with Ty21a live oral typhoid vaccine elicits crossreactive multifunctional CD8+ T-cell responses against Salmonella enterica serovar Typhi, S. Paratyphi A, and S. Paratyphi B in humans.

    Science.gov (United States)

    Wahid, R; Fresnay, S; Levine, M M; Sztein, M B

    2015-11-01

    Previously we have extensively characterized Salmonella enterica serovar Typhi (S. Typhi)-specific cell-mediated immune (CMI) responses in volunteers orally immunized with the licensed Ty21a typhoid vaccine. In this study we measured Salmonella-specific multifunctional (MF) CD8+ T-cell responses to further investigate whether Ty21a elicits crossreactive CMI against S. Paratyphi A and S. Paratyphi B that also cause enteric fever. Ty21a-elicited crossreactive CMI responses against all three Salmonella serotypes were predominantly observed in CD8+ T effector/memory (T(EM)) and, to a lesser extent, in CD8+CD45RA+ T(EM) (T(EMRA)) subsets. These CD8+ T-cell responses were largely mediated by MF cells coproducing interferon-γ and macrophage inflammatory protein-1β and expressing CD107a with or without tumor necrosis factor-α. Significant proportions of Salmonella-specific MF cells expressed the gut-homing molecule integrin α4β7. In most subjects, similar MF responses were observed to S. Typhi and S. Paratyphi B, but not to S. Paratyphi A. These results suggest that Ty21a elicits MF CMI responses against Salmonella that could be critical in clearing the infection. Moreover, because S. Paratyphi A is a major public concern and Ty21a was shown in field studies not to afford cross-protection to S. Paratyphi A, these results will be important in developing a S. Typhi/S. Paratyphi A bivalent vaccine against enteric fevers. PMID:25872480

  15. Cell adhesion over two distinct surfaces varied with chemical and mechanical properties

    International Nuclear Information System (INIS)

    Chitosan is widely recognized as a natural and proper scaffold material; however, as a base substrate, it shows little promotion effect for the growth of cultured fibroblast cells. In this study, chitosan in a film form was prepared and used as a cell-culturing matrix, followed by patterning the evaporated Au upon it. Micro-scale Au clusters of ∼ 150 μm in diameter and ∼ 20 nm in thickness were then patterned and adhered upon the chitosan matrix. Physical and chemical properties of Au/chitosan were characterized. In particular, nano-indentation with dynamic contact module was applied to measure the nano-hardness of the tailored surfaces on Au/chitosan. Fibroblast cells were thereafter cultured on Au/chitosan. Experimental results demonstrated that as compared with the chitosan matrix, Au clusters and their boundary area exhibited favorable to promote cell adhesion, spreading, and growth. As well, nano-hardness on the boundary area of Au/chitosan significantly enhanced, while the cultured fibroblast cells aggregated upon Au clusters and the boundary area. In combination with the possible chemical and mechanical changes resulted by the evaporation of Au clusters upon the chitosan matrix, a selectively-enhanced Au/chitosan to promote fibroblast cells proliferation was created. Such design is anticipated for enabling a surface for scaffold materials with the cell-guidable function.

  16. Distinct effects of SIRT1 in cancer and stromal cells on tumor promotion.

    Science.gov (United States)

    Shin, Dong Hoon; Choi, Yong-Joon; Jin, Peng; Yoon, Haejin; Chun, Yang-Sook; Shin, Hyun-Woo; Kim, Ja-Eun; Park, Jong-Wan

    2016-04-26

    The lysyl deacetylase SIRT1 acts as a metabolic sensor in adjusting metabolic imbalance. To explore the role of SIRT1 in tumor-stroma interplay, we designed an in vivo tumor model using SIRT1-transgenic mice. B16F10 mouse melanoma grew more quickly in SIRT1-transgenic mice than in wild-type mice, whereas SIRT1-overexpressing one grew slowly in both mice. Of human tumors, SIRT1 expression in stromal fibroblasts was found to correlate with poor prognosis in ovarian cancer. B16F10 and human ovarian cancer (SKOV3 and SNU840) cells were more proliferative in co-culture with SIRT1-overexpressiong fibroblasts. In contrast, SIRT1 within cancer cells has a negative effect on cell proliferation. In conditioned media from SIRT1-overexpressing fibroblasts, matrix metalloproteinase-3 (MMP3) was identified in cytokine arrays to be secreted from fibroblasts SIRT1-dependently. Fibroblast-derived MMP3 stimulated cancer cell proliferation, and such a role of MMP3 was also demonstrated in cancer/fibroblast co-grafts. In conclusion, SIRT1 plays differential roles in cancer and stromal cells. SIRT1 in stromal cells promotes cancer growth by producing MMP3, whereas SIRT1 in cancer cells inhibits growth via an intracellular event. The present study provides a basis for setting new anticancer strategies targeting SIRT1. PMID:26992208

  17. Distinct roles of Eps8 in the maturation of cochlear and vestibular hair cells.

    Science.gov (United States)

    Tavazzani, Elisa; Spaiardi, Paolo; Zampini, Valeria; Contini, Donatella; Manca, Marco; Russo, Giancarlo; Prigioni, Ivo; Marcotti, Walter; Masetto, Sergio

    2016-07-22

    Several genetic mutations affecting the development and function of mammalian hair cells have been shown to cause deafness but not vestibular defects, most likely because vestibular deficits are sometimes centrally compensated. The study of hair cell physiology is thus a powerful direct approach to ascertain the functional status of the vestibular end organs. Deletion of Epidermal growth factor receptor pathway substrate 8 (Eps8), a gene involved in actin remodeling, has been shown to cause deafness in mice. While both inner and outer hair cells from Eps8 knockout (KO) mice showed abnormally short stereocilia, inner hair cells (IHCs) also failed to acquire mature-type ion channels. Despite the fact that Eps8 is also expressed in vestibular hair cells, Eps8 KO mice show no vestibular deficits. In the present study we have investigated the properties of vestibular Type I and Type II hair cells in Eps8-KO mice and compared them to those of cochlear IHCs. In the absence of Eps8, vestibular hair cells show normally long kinocilia, significantly shorter stereocilia and a normal pattern of basolateral voltage-dependent ion channels. We have also found that while vestibular hair cells from Eps8 KO mice show normal voltage responses to injected sinusoidal currents, which were used to mimic the mechanoelectrical transducer current, IHCs lose their ability to synchronize their responses to the stimulus. We conclude that the absence of Eps8 produces a weaker phenotype in vestibular hair cells compared to cochlear IHCs, since it affects the hair bundle morphology but not the basolateral membrane currents. This difference is likely to explain the absence of obvious vestibular dysfunction in Eps8 KO mice. PMID:27132230

  18. Association of Neisseria gonorrhoeae Opa(CEA with dendritic cells suppresses their ability to elicit an HIV-1-specific T cell memory response.

    Directory of Open Access Journals (Sweden)

    Qigui Yu

    Full Text Available Infection with Neisseria gonorrhoeae (N. gonorrhoeae can trigger an intense local inflammatory response at the site of infection, yet there is little specific immune response or development of immune memory. Gonococcal surface epitopes are known to undergo antigenic variation; however, this is unlikely to explain the weak immune response to infection since individuals can be re-infected by the same serotype. Previous studies have demonstrated that the colony opacity-associated (Opa proteins on the N. gonorrhoeae surface can bind human carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1 on CD4⁺ T cells to suppress T cell activation and proliferation. Interesting in this regard, N. gonorrhoeae infection is associated with impaired HIV-1 (human immunodeficiency virus type 1-specific cytotoxic T-lymphocyte (CTL responses and with transient increases in plasma viremia in HIV-1-infected patients, suggesting that N. gonorrhoeae may also subvert immune responses to co-pathogens. Since dendritic cells (DCs are professional antigen presenting cells (APCs that play a key role in the induction of an adaptive immune response, we investigated the effects of N. gonorrhoeae Opa proteins on human DC activation and function. While morphological changes reminiscent of DC maturation were evident upon N. gonorrhoeae infection, we observed a marked downregulation of DC maturation marker CD83 when the gonococci expressing CEACAM1-specific Opa(CEA, but not other Opa variants. Consistent with a gonococcal-induced defect in maturation, Opa(CEA binding to CEACAM1 reduced the DCs' capacity to stimulate an allogeneic T cell proliferative response. Moreover, Opa(CEA-expressing N. gonorrhoeae showed the potential to impair DC-dependent development of specific adaptive immunity, since infection with Opa(CEA-positive gonococci suppressed the ability of DCs to stimulate HIV-1-specific memory CTL responses. These results reveal a novel mechanism to explain

  19. Association of Neisseria gonorrhoeae Opa(CEA) with dendritic cells suppresses their ability to elicit an HIV-1-specific T cell memory response.

    Science.gov (United States)

    Yu, Qigui; Chow, Edith M C; McCaw, Shannon E; Hu, Ningjie; Byrd, Daniel; Amet, Tohti; Hu, Sishun; Ostrowski, Mario A; Gray-Owen, Scott D

    2013-01-01

    Infection with Neisseria gonorrhoeae (N. gonorrhoeae) can trigger an intense local inflammatory response at the site of infection, yet there is little specific immune response or development of immune memory. Gonococcal surface epitopes are known to undergo antigenic variation; however, this is unlikely to explain the weak immune response to infection since individuals can be re-infected by the same serotype. Previous studies have demonstrated that the colony opacity-associated (Opa) proteins on the N. gonorrhoeae surface can bind human carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) on CD4⁺ T cells to suppress T cell activation and proliferation. Interesting in this regard, N. gonorrhoeae infection is associated with impaired HIV-1 (human immunodeficiency virus type 1)-specific cytotoxic T-lymphocyte (CTL) responses and with transient increases in plasma viremia in HIV-1-infected patients, suggesting that N. gonorrhoeae may also subvert immune responses to co-pathogens. Since dendritic cells (DCs) are professional antigen presenting cells (APCs) that play a key role in the induction of an adaptive immune response, we investigated the effects of N. gonorrhoeae Opa proteins on human DC activation and function. While morphological changes reminiscent of DC maturation were evident upon N. gonorrhoeae infection, we observed a marked downregulation of DC maturation marker CD83 when the gonococci expressing CEACAM1-specific Opa(CEA), but not other Opa variants. Consistent with a gonococcal-induced defect in maturation, Opa(CEA) binding to CEACAM1 reduced the DCs' capacity to stimulate an allogeneic T cell proliferative response. Moreover, Opa(CEA)-expressing N. gonorrhoeae showed the potential to impair DC-dependent development of specific adaptive immunity, since infection with Opa(CEA)-positive gonococci suppressed the ability of DCs to stimulate HIV-1-specific memory CTL responses. These results reveal a novel mechanism to explain why

  20. Functionally distinct Gata3/Chd4 complexes coordinately establish T helper 2 (Th2) cell identity

    OpenAIRE

    Hosokawa, Hiroyuki; Tanaka, Tomoaki; Suzuki, Yutaka; Iwamura, Chiaki; Ohkubo, Shuichi; Endoh, Kanji; Kato, Miki; Endo, Yusuke; Onodera, Atsushi; Tumes, Damon John; Kanai, Akinori; Sugano, Sumio; Nakayama, Toshinori

    2013-01-01

    GATA binding protein 3 (Gata3) is a GATA family transcription factor that controls differentiation of naïve CD4 T cells into T helper 2 (Th2) cells. However, it is unknown how Gata3 simultaneously activates Th2-specific genes while repressing those of other Th lineages. Here we show that chromodomain helicase DNA-binding protein 4 (Chd4) forms a complex with Gata3 in Th2 cells that both activates Th2 cytokine transcription and represses the Th1 cytokine IFN-γ. We define a Gata3/Chd4/p300 tran...

  1. Distinct roles of CD4+ T cell subpopulations in retroviral immunity: lessons from the Friend virus mouse model

    Directory of Open Access Journals (Sweden)

    Kassiotis George

    2011-09-01

    Full Text Available Abstract It is well established that CD4+ T cells play an important role in immunity to infections with retroviruses such as HIV. However, in recent years CD4+ T cells have been subdivided into several distinct populations that are differentially regulated and perform widely varying functions. Thus, it is important to delineate the separate roles of these subsets, which range from direct antiviral activities to potent immunosuppression. In this review, we discuss contributions from the major CD4+ T cell subpopulations to retroviral immunity. Fundamental concepts obtained from studies on numerous viral infections are presented along with a more detailed analysis of studies on murine Friend virus. The relevance of these studies to HIV immunology and immunotherapy is reviewed.

  2. Pilot Study on the Use of DNA Priming Immunization to Enhance Y. pestis LcrV-Specific B Cell Responses Elicited by a Recombinant LcrV Protein Vaccine

    Directory of Open Access Journals (Sweden)

    Wei Li

    2013-12-01

    Full Text Available Recent studies indicate that DNA immunization is powerful in eliciting antigen-specific antibody responses in both animal and human studies. However, there is limited information on the mechanism of this effect. In particular, it is not known whether DNA immunization can also enhance the development of antigen-specific B cell development. In this report, a pilot study was conducted using plague LcrV immunogen as a model system to determine whether DNA immunization is able to enhance LcrV-specific B cell development in mice. Plague is an acute and often fatal infectious disease caused by Yersinia pestis (Y. pestis. Humoral immune responses provide critical protective immunity against plague. Previously, we demonstrated that a DNA vaccine expressing LcrV antigen can protect mice from lethal mucosal challenge. In the current study, we further evaluated whether the use of a DNA priming immunization is able to enhance the immunogenicity of a recombinant LcrV protein vaccine, and in particular, the development of LcrV-specific B cells. Our data indicate that DNA immunization was able to elicit high-level LcrV antibody responses when used alone or as part of a prime-boost immunization approach. Most significantly, DNA immunization was also able to increase the levels of LcrV-specific B cell development. The finding that DNA immunization can enhance antigen-specific B cell responses is highly significant and will help guide similar studies in other model antigen systems.

  3. Indolent small intestinal CD4+ T-cell lymphoma is a distinct entity with unique biologic and clinical features.

    Directory of Open Access Journals (Sweden)

    Elizabeth Margolskee

    Full Text Available Enteropathy-associated T-cell lymphomas (EATL are rare and generally aggressive types of peripheral T-cell lymphomas. Rare cases of primary, small intestinal CD4+ T-cell lymphomas with indolent behavior have been described, but are not well characterized. We describe morphologic, phenotypic, genomic and clinical features of 3 cases of indolent primary small intestinal CD4+ T-cell lymphomas. All patients presented with diarrhea and weight loss and were diagnosed with celiac disease refractory to a gluten free diet at referring institutions. Small intestinal biopsies showed crypt hyperplasia, villous atrophy and a dense lamina propria infiltrate of small-sized CD4+ T-cells often with CD7 downregulation or loss. Gastric and colonic involvement was also detected (n = 2 each. Persistent, clonal TCRβ gene rearrangement products were detected at multiple sites. SNP array analysis showed relative genomic stability, early in disease course, and non-recurrent genetic abnormalities, but complex changes were seen at disease transformation (n = 1. Two patients are alive with persistent disease (4.6 and 2.5 years post-diagnosis, despite immunomodulatory therapy; one died due to bowel perforation related to large cell transformation 11 years post-diagnosis. Unique pathobiologic features warrant designation of indolent small intestinal CD4+ T-cell lymphoma as a distinct entity, greater awareness of which would avoid misdiagnosis as EATL or an inflammatory disorder, especially celiac disease.

  4. Distinct expression levels and patterns of stem cell marker, aldehyde dehydrogenase isoform 1 (ALDH1, in human epithelial cancers.

    Directory of Open Access Journals (Sweden)

    Shan Deng

    Full Text Available Aldehyde dehydrogenase isoform 1 (ALDH1 has been proved useful for the identification of cancer stem cells. However, our knowledge of the expression and activity of ALDH1 in common epithelial cancers and their corresponding normal tissues is still largely absent. Therefore, we characterized ALDH1 expression in 24 types of normal tissues and a large collection of epithelial tumor specimens (six cancer types, n = 792 by immunohistochemical staining. Using the ALDEFUOR assay, ALDH1 activity was also examined in 16 primary tumor specimens and 43 established epithelial cancer cell lines. In addition, an ovarian cancer transgenic mouse model and 7 murine ovarian cancer cell lines were analyzed. We found that the expression levels and patterns of ALDH1 in epithelial cancers are remarkably distinct, and they correlate with their corresponding normal tissues. ALDH1 protein expression levels are positively correlated with ALDH1 enzymatic activity measured by ALDEFLUOR assay. Long-term in vitro culture doesn't significantly affect ALDH1 activity in epithelial tumor cells. Consistent with research on other cancers, we found that high ALDH1 expression is significantly associated with poor clinical outcomes in serous ovarian cancer patients (n = 439, p = 0.0036. Finally, ALDH(br tumor cells exhibit cancer stem cell properties and are resistant to chemotherapy. As a novel cancer stem cell marker, ALDH1 can be used for tumors whose corresponding normal tissues express ALDH1 in relatively restricted or limited levels such as breast, lung, ovarian or colon cancer.

  5. Influence of electrospun scaffolds prepared from distinct polymers on proliferation and viability of endothelial cells

    International Nuclear Information System (INIS)

    We compared electrospun nonwoven scaffolds from polylactic acid (PLA), polycaprolactone (PCL), and polyhydroxybutyrate/valerate (PHBV)/polycaprolactone (PHBV/PCL). The surface of PHBV/PCL and PCL scaffolds was highly porous and consisted of randomly distributed fibers, whilst the surface of PLA scaffolds consisted of thin straight fibers, which located more sparsely, forming large pores. Culture of EA.hy 926 endothelial cells on these scaffolds during 7 days and further fluorescent microscopy demonstrated that the surface of PHBV/PCL scaffolds was most favorable for efficient adhesion, proliferation, and viability of endothelial cells. The lowest proliferation rate and cell viability were detected on PLA scaffolds. Therefore, PHBV/PCL electrospun nonwoven scaffolds demonstrated the best results regarding endothelial cell proliferation and viability as compared to PCL and PLA scaffolds

  6. Influence of electrospun scaffolds prepared from distinct polymers on proliferation and viability of endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Matveeva, V. G., E-mail: matveeva-vg@mail.ru; Antonova, L. V., E-mail: antonova.la@mail.ru; Velikanova, E. A.; Sergeeva, E. A.; Krivkina, E. O.; Glushkova, T. V.; Kudryavtseva, Yu. A.; Barbarash, O. L.; Barbarash, L. S. [Research Institute for Complex Issues of Cardiovascular Diseases, Kemerovo, 650002 (Russian Federation)

    2015-10-27

    We compared electrospun nonwoven scaffolds from polylactic acid (PLA), polycaprolactone (PCL), and polyhydroxybutyrate/valerate (PHBV)/polycaprolactone (PHBV/PCL). The surface of PHBV/PCL and PCL scaffolds was highly porous and consisted of randomly distributed fibers, whilst the surface of PLA scaffolds consisted of thin straight fibers, which located more sparsely, forming large pores. Culture of EA.hy 926 endothelial cells on these scaffolds during 7 days and further fluorescent microscopy demonstrated that the surface of PHBV/PCL scaffolds was most favorable for efficient adhesion, proliferation, and viability of endothelial cells. The lowest proliferation rate and cell viability were detected on PLA scaffolds. Therefore, PHBV/PCL electrospun nonwoven scaffolds demonstrated the best results regarding endothelial cell proliferation and viability as compared to PCL and PLA scaffolds.

  7. Influence of electrospun scaffolds prepared from distinct polymers on proliferation and viability of endothelial cells

    Science.gov (United States)

    Matveeva, V. G.; Antonova, L. V.; Velikanova, E. A.; Sergeeva, E. A.; Krivkina, E. O.; Glushkova, T. V.; Kudryavtseva, Yu. A.; Barbarash, O. L.; Barbarash, L. S.

    2015-10-01

    We compared electrospun nonwoven scaffolds from polylactic acid (PLA), polycaprolactone (PCL), and polyhydroxybutyrate/valerate (PHBV)/polycaprolactone (PHBV/PCL). The surface of PHBV/PCL and PCL scaffolds was highly porous and consisted of randomly distributed fibers, whilst the surface of PLA scaffolds consisted of thin straight fibers, which located more sparsely, forming large pores. Culture of EA.hy 926 endothelial cells on these scaffolds during 7 days and further fluorescent microscopy demonstrated that the surface of PHBV/PCL scaffolds was most favorable for efficient adhesion, proliferation, and viability of endothelial cells. The lowest proliferation rate and cell viability were detected on PLA scaffolds. Therefore, PHBV/PCL electrospun nonwoven scaffolds demonstrated the best results regarding endothelial cell proliferation and viability as compared to PCL and PLA scaffolds.

  8. Cooperation between distinct viral variants promotes growth of H3N2 influenza in cell culture.

    Science.gov (United States)

    Xue, Katherine S; Hooper, Kathryn A; Ollodart, Anja R; Dingens, Adam S; Bloom, Jesse D

    2016-01-01

    RNA viruses rapidly diversify into quasispecies of related genotypes. This genetic diversity has long been known to facilitate adaptation, but recent studies have suggested that cooperation between variants might also increase population fitness. Here, we demonstrate strong cooperation between two H3N2 influenza variants that differ by a single mutation at residue 151 in neuraminidase, which normally mediates viral exit from host cells. Residue 151 is often annotated as an ambiguous amino acid in sequenced isolates, indicating mixed viral populations. We show that mixed populations grow better than either variant alone in cell culture. Pure populations of either variant generate the other through mutation and then stably maintain a mix of the two genotypes. We suggest that cooperation arises because mixed populations combine one variant's proficiency at cell entry with the other's proficiency at cell exit. Our work demonstrates a specific cooperative interaction between defined variants in a viral quasispecies. PMID:26978794

  9. Distinct Tlr4-expressing cell compartments control neutrophilic and eosinophilic airway inflammation

    OpenAIRE

    McAlees, Jaclyn W.; Whitehead, Gregory S.; Harley, Isaac T. W.; Cappelletti, Monica; Rewerts, Cheryl L.; Holdcroft, A. Maria; Divanovic, Senad; Wills-Karp, Marsha; Finkelman, Fred D.; Karp, Christopher L.; Cook, Donald N.

    2014-01-01

    Allergic asthma is a chronic, inflammatory lung disease. Some forms of allergic asthma are characterized by Th2-driven eosinophilia while others are distinguished by Th17-driven neutrophilia. Stimulation of Toll-like receptor 4 (TLR4) on hematopoietic and airway epithelial cells (AECs) contributes to the inflammatory response to lipopolysaccharide (LPS) and allergens, but the specific contribution of TLR4 in these cell compartments to airway inflammatory responses remains poorly understood. W...

  10. Distinct mathematical behavior of apoptotic versus non-apoptotic tumor cell death

    International Nuclear Information System (INIS)

    Purpose: The presence or absence of a p53-dependent apoptosis response has previously been shown to greatly influence radiosensitivity in tumor cells. Here, we examine clonogenic survival curves for two genetically related oncogene transformed cell lines differing in the presence or absence of p53 and apoptosis. Solid tumor radiosensitivity patterns have been previously described for these lines. Materials and Methods: Oncogene-transformed fibroblasts derived from E1A + Ras transfection of p53-wild-type or p53-null mouse embryonic fibroblasts were plated as single cells and irradiated at increasing radiation doses in single fractions from 1.5 to 11 Gy. Clonogenic cell survival assays were obtained. Survival data are fit to a linear-quadratic relationship: S = e-αD-βD2. Apoptosis was assessed and quantitated morphologically by staining with the fluorescent nuclear dye DAPI, by TUNEL assay for DNA fragmentation, and by measurement of apoptotic cysteine protease cleavage activity in cytosolic extracts. Results: Whereas radiation triggers massive apoptosis in the presence of p53, it produces no measurable DNA fragmentation, apoptotic cysteine protease cleavage activity, or morphological changes of apoptosis in the cells lacking p53. These contrasting mechanisms of death display dramatically different quantitative behavior: log-survival of apoptotic cells is linearly proportional to dose (S = e-αD), whereas survival of non-apoptotic (p53 null) is linear-quadratic with a significant quadratic contribution. The surviving fraction at 2 Gy (SF-2) for p53-null cells was 70% verses 12% for p53-intact cells. Conclusions: In this system, apoptosis appears to exhibit a dominance of single-event which produces a very high α/β ratio, and no significant shoulder; whereas non-apoptotic death in this system exhibits a comparatively small linear component, a low α/β ratio, and a larger shoulder

  11. Distinctive localization of antigen-presenting cells in human lymph nodes

    OpenAIRE

    Angel, Catherine E.; Chen, Chun-Jen J.; Horlacher, Oliver C.; Winkler, Sintia; John, Thomas; Browning, Judy; MacGregor, Duncan; Cebon, Jonathan; Dunbar, P. Rod

    2009-01-01

    Professional antigen-presenting cells (APCs) are sentinel cells of the immune system that present antigen to T lymphocytes and mediate an appropriate immune response. It is therefore surprising that knowledge of the professional APCs in human lymph nodes is limited. Using 3-color immunohistochemistry, we have identified APCs in human lymph nodes, excluding plasmacytoid APCs, that fall into 2 nonoverlapping classes: (1) CD209+ APCs, coexpressing combinations of CD206, CD14, and CD68, that occu...

  12. CB-07DISTINCT RADIATION RESPONSE OF SLOW-DIVIDING CANCER STEM CELLS

    OpenAIRE

    Deleyrolle, Loic; Nabilsi, Nancy; Griffith, Benjamin; Pasternack, Nick; Dajac, Kyle; Patel, Jaimin; Rohaus, Mark; Harding, Angus; Kladde, Michael; Steindler, Dennis; Reynolds, Brent; Siebzehnrubl, Florian

    2014-01-01

    Glioblastoma, the most frequent primary malignancy of the central nervous system, is almost universally fatal despite aggressive therapies, such as surgical resection, adjuvant radiation and chemotherapy, which remain largely palliative. With increasing evidence showing that glioblastoma cancer stem cells play an important role in tumor escape from conventional therapies and disease recurrence, the targeting of cancer stem cells with different therapeutic strategies provides new avenues of re...

  13. Adipose Stromal Cells Contain Phenotypically Distinct Adipogenic Progenitors Derived from Neural Crest

    OpenAIRE

    Yoshihiro Sowa; Tetsuya Imura; Toshiaki Numajiri; Kosuke Takeda; Yo Mabuchi; Yumi Matsuzaki; Kenichi Nishino

    2013-01-01

    Recent studies have shown that adipose-derived stromal/stem cells (ASCs) contain phenotypically and functionally heterogeneous subpopulations of cells, but their developmental origin and their relative differentiation potential remain elusive. In the present study, we aimed at investigating how and to what extent the neural crest contributes to ASCs using Cre-loxP-mediated fate mapping. ASCs harvested from subcutaneous fat depots of either adult P0-Cre/or Wnt1-Cre/Floxed-reporter mice contain...

  14. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17

    OpenAIRE

    Park, Heon; Li, Zhaoxia; Yang, Xuexian O.; Chang, Seon Hee; Nurieva, Roza; Wang, Yi-hong; Wang, Ying; Hood, Leroy; Zhu, Zhou; Tian, Qiang; Dong, Chen

    2005-01-01

    Interleukin 17 (IL-17) has been linked to autoimmune diseases, although its regulation and function have remained unclear. Here we have evaluated in vitro and in vivo the requirements for the differentiation of naive CD4 T cells into effector T helper cells that produce IL-17. This process required the costimulatory molecules CD28 and ICOS but was independent of the cytokines and transcription factors required for T helper type 1 or type 2 differentiation. Furthermore, both IL-4 and interfero...

  15. Dual Processing of FAT1 Cadherin Protein by Human Melanoma Cells Generates Distinct Protein Products*

    OpenAIRE

    Sadeqzadeh, Elham; de Bock, Charles E; Zhang, Xu Dong; Shipman, Kristy L.; Scott, Naomi M.; Song, Chaojun; Yeadon, Trina; Oliveira, Camila S.; Jin, Boquan; Hersey, Peter; Boyd, Andrew W.; Burns, Gordon F.; Thorne, Rick F.

    2011-01-01

    The giant cadherin FAT1 is one of four vertebrate orthologues of the Drosophila tumor suppressor fat. It engages in several functions, including cell polarity and migration, and in Hippo signaling during development. Homozygous deletions in oral cancer suggest that FAT1 may play a tumor suppressor role, although overexpression of FAT1 has been reported in some other cancers. Here we show using Northern blotting that human melanoma cell lines variably but universally express FAT1 and less comm...

  16. Distinct myeloid progenitor differentiation pathways identified through single cell RNA sequencing

    OpenAIRE

    Drissen, R; Buza-Vidas, N.; Woll, P.; Thongjuea, S.; Gambardella, A; Giustacchini, A.; E. Mancini; Zriwil, A.; Lutteropp], M; Grover, A.; Mead, A.; Sitnicka, E; Jacobsen, SE; Nerlov, C.

    2016-01-01

    According to current models for hematopoiesis, lymphoid-primed multi-potent progenitors (LMPPs; Lin– Sca-1+ c-Kit+ CD34+ Flt3hi) and common myeloid progenitors (CMPs; Lin– Sca- 1+ c-Kit+ CD34+ CD41hi) establish an early branch point for separate lineage commitment pathways from hematopoietic stem cells, with the notable exception that both pathways are proposed to generate all myeloid innate immune cell types through the same myeloidrestricted pre-granul...

  17. Human colonic goblet cells. Demonstration of distinct subpopulations defined by mucin-specific monoclonal antibodies.

    OpenAIRE

    Podolsky, D K; Fournier, D A; Lynch, K E

    1986-01-01

    We studied glycoprotein content of human colonic goblet cells, using a library of monoclonal antibodies (MAbs) directed against purified human colonic mucin (HCM). Using indirect immunofluorescence (IIF), we found that 17 of 23 anti-HCM MAbs stained some or all goblet cells of normal human colonic mucosa. We observed a variety of cellular staining patterns, including (a) diffuse (homogeneous) staining of intracellular mucin, (b) speckled (inhomogeneous) staining of mucin droplets, (c) periphe...

  18. SOX2 co-occupies distal enhancer elements with distinct POU factors in ESCs and NPCs to specify cell state.

    Directory of Open Access Journals (Sweden)

    Michael A Lodato

    Full Text Available SOX2 is a master regulator of both pluripotent embryonic stem cells (ESCs and multipotent neural progenitor cells (NPCs; however, we currently lack a detailed understanding of how SOX2 controls these distinct stem cell populations. Here we show by genome-wide analysis that, while SOX2 bound to a distinct set of gene promoters in ESCs and NPCs, the majority of regions coincided with unique distal enhancer elements, important cis-acting regulators of tissue-specific gene expression programs. Notably, SOX2 bound the same consensus DNA motif in both cell types, suggesting that additional factors contribute to target specificity. We found that, similar to its association with OCT4 (Pou5f1 in ESCs, the related POU family member BRN2 (Pou3f2 co-occupied a large set of putative distal enhancers with SOX2 in NPCs. Forced expression of BRN2 in ESCs led to functional recruitment of SOX2 to a subset of NPC-specific targets and to precocious differentiation toward a neural-like state. Further analysis of the bound sequences revealed differences in the distances of SOX and POU peaks in the two cell types and identified motifs for additional transcription factors. Together, these data suggest that SOX2 controls a larger network of genes than previously anticipated through binding of distal enhancers and that transitions in POU partner factors may control tissue-specific transcriptional programs. Our findings have important implications for understanding lineage specification and somatic cell reprogramming, where SOX2, OCT4, and BRN2 have been shown to be key factors.

  19. Distinct and atypical intrinsic and extrinsic cell death pathways between photoreceptor cell types upon specific ablation of Ranbp2 in cone photoreceptors.

    Directory of Open Access Journals (Sweden)

    Kyoung-In Cho

    2013-06-01

    Full Text Available Non-autonomous cell-death is a cardinal feature of the disintegration of neural networks in neurodegenerative diseases, but the molecular bases of this process are poorly understood. The neural retina comprises a mosaic of rod and cone photoreceptors. Cone and rod photoreceptors degenerate upon rod-specific expression of heterogeneous mutations in functionally distinct genes, whereas cone-specific mutations are thought to cause only cone demise. Here we show that conditional ablation in cone photoreceptors of Ran-binding protein-2 (Ranbp2, a cell context-dependent pleiotropic protein linked to neuroprotection, familial necrotic encephalopathies, acute transverse myelitis and tumor-suppression, promotes early electrophysiological deficits, subcellular erosive destruction and non-apoptotic death of cones, whereas rod photoreceptors undergo cone-dependent non-autonomous apoptosis. Cone-specific Ranbp2 ablation causes the temporal activation of a cone-intrinsic molecular cascade highlighted by the early activation of metalloproteinase 11/stromelysin-3 and up-regulation of Crx and CoREST, followed by the down-modulation of cone-specific phototransduction genes, transient up-regulation of regulatory/survival genes and activation of caspase-7 without apoptosis. Conversely, PARP1+ -apoptotic rods develop upon sequential activation of caspase-9 and caspase-3 and loss of membrane permeability. Rod photoreceptor demise ceases upon cone degeneration. These findings reveal novel roles of Ranbp2 in the modulation of intrinsic and extrinsic cell death mechanisms and pathways. They also unveil a novel spatiotemporal paradigm of progression of neurodegeneration upon cell-specific genetic damage whereby a cone to rod non-autonomous death pathway with intrinsically distinct cell-type death manifestations is triggered by cell-specific loss of Ranbp2. Finally, this study casts new light onto cell-death mechanisms that may be shared by human dystrophies with distinct

  20. Resolution of Chlamydia trachomatis Infection Is Associated with a Distinct T Cell Response Profile.

    Science.gov (United States)

    Picard, Michele D; Bodmer, Jean-Luc; Gierahn, Todd M; Lee, Alexander; Price, Jessica; Cohane, Kenya; Clemens, Veronica; DeVault, Victoria L; Gurok, Galina; Kohberger, Robert; Higgins, Darren E; Siber, George R; Flechtner, Jessica Baker; Geisler, William M

    2015-11-01

    Chlamydia trachomatis is the causative agent of the most frequently reported bacterial sexually transmitted infection, the total burden of which is underestimated due to the asymptomatic nature of the infection. Untreated C. trachomatis infections can cause significant morbidities, including pelvic inflammatory disease and tubal factor infertility (TFI). The human immune response against C. trachomatis, an obligate intracellular bacterium, is poorly characterized but is thought to rely on cell-mediated immunity, with CD4(+) and CD8(+) T cells implicated in protection. In this report, we present immune profiling data of subjects enrolled in a multicenter study of C. trachomatis genital infection. CD4(+) and CD8(+) T cells from subjects grouped into disease-specific cohorts were screened using a C. trachomatis proteomic library to identify the antigen specificities of recall T cell responses after natural exposure by measuring interferon gamma (IFN-γ) levels. We identified specific T cell responses associated with the resolution of infection, including unique antigens identified in subjects who spontaneously cleared infection and different antigens associated with C. trachomatis-related sequelae, such as TFI. These data suggest that novel and unique C. trachomatis T cell antigens identified in individuals with effective immune responses can be considered as targets for vaccine development, and by excluding antigens associated with deleterious sequelae, immune-mediated pathologies may be circumvented. PMID:26446421

  1. Blue Light Induces a Distinct Starch Degradation Pathway in Guard Cells for Stomatal Opening.

    Science.gov (United States)

    Horrer, Daniel; Flütsch, Sabrina; Pazmino, Diana; Matthews, Jack S A; Thalmann, Matthias; Nigro, Arianna; Leonhardt, Nathalie; Lawson, Tracy; Santelia, Diana

    2016-02-01

    Stomatal pores form a crucial interface between the leaf mesophyll and the atmosphere, controlling water and carbon balance in plants [1]. Major advances have been made in understanding the regulatory networks and ion fluxes in the guard cells surrounding the stomatal pore [2]. However, our knowledge on the role of carbon metabolism in these cells is still fragmentary [3-5]. In particular, the contribution of starch in stomatal opening remains elusive [6]. Here, we used Arabidopsis thaliana as a model plant to provide the first quantitative analysis of starch turnover in guard cells of intact leaves during the diurnal cycle. Starch is present in guard cells at the end of night, unlike in the rest of the leaf, but is rapidly degraded within 30 min of light. This process is critical for the rapidity of stomatal opening and biomass production. We exploited Arabidopsis molecular genetics to define the mechanism and regulation of guard cell starch metabolism, showing it to be mediated by a previously uncharacterized pathway. This involves the synergistic action of β-amylase 1 (BAM1) and α-amylase 3 (AMY3)-enzymes that are normally not required for nighttime starch degradation in other leaf tissues. This pathway is under the control of the phototropin-dependent blue-light signaling cascade and correlated with the activity of the plasma membrane H(+)-ATPase. Our results show that guard cell starch degradation has an important role in plant growth by driving stomatal responses to light. PMID:26774787

  2. Effects of Corroded and Non-Corroded Biodegradable Mg and Mg Alloys on Viability, Morphology and Differentiation of MC3T3-E1 Cells Elicited by Direct Cell/Material Interaction

    Science.gov (United States)

    Mostofi, Sepideh; Bonyadi Rad, Ehsan; Wiltsche, Helmar; Fasching, Ulrike; Szakacs, Gabor; Ramskogler, Claudia; Srinivasaiah, Sriveena; Ueçal, Muammer; Willumeit, Regine; Weinberg, Annelie-Martina; Schaefer, Ute

    2016-01-01

    This study investigated the effect of biodegradable Mg and Mg alloys on selected properties of MC3T3-E1 cells elicited by direct cell/material interaction. The chemical composition and morphology of the surface of Mg and Mg based alloys (Mg2Ag and Mg10Gd) were analysed by scanning electron microscopy (SEM) and EDX, following corrosion in cell culture medium for 1, 2, 3 and 8 days. The most pronounced difference in surface morphology, namely crystal formation, was observed when Pure Mg and Mg2Ag were immersed in cell medium for 8 days, and was associated with an increase in atomic % of oxygen and a decrease of surface calcium and phosphorous. Crystal formation on the surface of Mg10Gd was, in contrast, negligible at all time points. Time-dependent changes in oxygen, calcium and phosphorous surface content were furthermore not observed for Mg10Gd. MC3T3-E1 cell viability was reduced by culture on the surfaces of corroded Mg, Mg2Ag and Mg10Gd in a corrosion time-independent manner. Cells did not survive when cultured on 3 day pre-corroded Pure Mg and Mg2Ag, indicating crystal formation to be particular detrimental in this regard. Cell viability was not affected when cells were cultured on non-corroded Mg and Mg alloys for up to 12 days. These results suggest that corrosion associated changes in surface morphology and chemical composition significantly hamper cell viability and, thus, that non-corroded surfaces are more conducive to cell survival. An analysis of the differentiation potential of MC3T3-E1 cells cultured on non-corroded samples based on measurement of Collagen I and Runx2 expression, revealed a down-regulation of these markers within the first 6 days following cell seeding on all samples, despite persistent survival and proliferation. Cells cultured on Mg10Gd, however, exhibited a pronounced upregulation of collagen I and Runx2 between days 8 and 12, indicating an enhancement of osteointegration by this alloy that could be valuable for in vivo orthopedic

  3. Identification of distinct human invariant natural killer T-cell response phenotypes to alpha-galactosylceramide

    Directory of Open Access Journals (Sweden)

    Besra Gurdyal S

    2008-12-01

    Full Text Available Abstract Background Human CD1d-restricted, invariant natural killer T cells (iNKT are a unique class of T lymphocytes that recognise glycolipid antigens such as α-galactosylceramide (αGalCer and upon T cell receptor (TCR activation produce both Th1 and Th2 cytokines. iNKT cells expand when cultured in-vitro with αGalCer and interleukin 2 (IL-2 in a CD1d-restricted manner. However, the expansion ratio of human iNKT cells varies between individuals and this has implications for attempts to manipulate this pathway therapeutically. We have studied a panel of twenty five healthy human donors to assess the variability in their in-vitro iNKT cell expansion responses to stimulation with CD1d ligands and investigated some of the factors that may influence this phenomenon. Results Although all donors had comparable numbers of circulating iNKT cells their growth rates in-vitro over 14 days in response to a range of CD1d ligands and IL-2 were highly donor-dependent. Two reproducible donor response patterns of iNKT expansion were seen which we have called 'strong' or 'poor' iNKT responders. Donor response phenotype did not correlate with age, gender, frequency of circulating iNKT, or with the CD1d ligand utilised. Addition of exogenous recombinant human interleukin 4 (IL-4 to 'poor' responder donor cultures significantly increased their iNKT proliferative capacity, but not to levels equivalent to that of 'strong' responder donors. However in 'strong' responder donors, addition of IL-4 to their cultures did not significantly alter the frequency of iNKT cells in the expanded CD3+ population. Conclusion (i in-vitro expansion of human iNKT cells in response to CD1d ligand activation is highly donor variable, (ii two reproducible patterns of donor iNKT expansion were observed, which could be classified into 'strong' and 'poor' responder phenotypes, (iii donor iNKT response phenotypes did not correlate with age, gender, frequency of circulating iNKT cells, or

  4. Distinct Dasatinib-Induced Mechanisms of Apoptotic Response and Exosome Release in Imatinib-Resistant Human Chronic Myeloid Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Juan Liu

    2016-04-01

    Full Text Available Although dasatinib is effective in most imatinib mesylate (IMT-resistant chronic myeloid leukemia (CML patients, the underlying mechanism of its effectiveness in eliminating imatinib-resistant cells is only partially understood. This study investigated the effects of dasatinib on signaling mechanisms driving-resistance in imatinib-resistant CML cell line K562 (K562RIMT. Compared with K562 control cells, exsomal release, the phosphoinositide 3-kinase (PI3K/protein kinase B (Akt/ mammalian target of rapamycin (mTOR signaling and autophagic activity were increased significantly in K562RIMT cells and mTOR-independent beclin-1/Vps34 signaling was shown to be involved in exosomal release in these cells. We found that Notch1 activation-mediated reduction of phosphatase and tensin homolog (PTEN was responsible for the increased Akt/mTOR activities in K562RIMT cells and treatment with Notch1 γ-secretase inhibitor prevented activation of Akt/mTOR. In addition, suppression of mTOR activity by rapamycin decreased the level of activity of p70S6K, induced upregulation of p53 and caspase 3, and led to increase of apoptosis in K562RIMT cells. Inhibition of autophagy by spautin-1 or beclin-1 knockdown decreased exosomal release, but did not affect apoptosis in K562RIMT cells. In summary, in K562RIMT cells dasatinib promoted apoptosis through downregulation of Akt/mTOR activities, while preventing exosomal release and inhibiting autophagy by downregulating expression of beclin-1 and Vps34. Our findings reveal distinct dasatinib-induced mechanisms of apoptotic response and exosomal release in imatinib-resistant CML cells.

  5. Electrophysiological, morphological, and topological properties of two histochemically distinct subpopulations of cerebellar unipolar brush cells.

    Science.gov (United States)

    Kim, Jin-Ah; Sekerková, Gabriella; Mugnaini, Enrico; Martina, Marco

    2012-12-01

    Unipolar brush cells (UBCs) are excitatory cerebellar granular layer interneurons whose brush-like dendrites receive one-to-one mossy fiber inputs. Subclasses of UBCs differ primarily by expressing metabotropic glutamate receptor (mGluR) 1α or calretinin. We used GENSAT Tg(Grp-EGFP) BAC transgenic mice, which selectively express enhanced green fluorescent protein (EGFP) in mGluR1α-positive UBCs to compare the functional properties of the two subclasses. Compared to EGFP-negative UBCs, which include the calretinin-positive cells, EGFP-positive UBCs had smaller somata (area 48 vs 63 μm(2)), lower specific membrane resistance (6.4 vs. 13.7 KΩ cm(2)), were less prone to intrinsic firing, and showed more irregular firing (in cell-attached ~49 % were firing vs. ~88 %, and the CV was 0.53 vs. 0.32 for EGFP-negative cells). Some of these differences are attributable to higher density of background K(+) currents in EGFP-positive cells (at -120 mV, the barium-sensitive current was 94 vs. 37 pA in EGFP-negative cells); Ih, on the contrary, was more abundantly expressed in EGFP-negative cells (at -140 mV, it was -122 vs. -54 pA in EGFP-positive neurons); furthermore, while group II mGluR modulation of the background potassium current in EGFP-negative UBCs was maintained after intracellular dialysis, mGluR modulation in EGFP-positive UBCs was lost in whole-cell recordings. Finally, cell-attached firing was reversibly abolished by the GABA(B) activation in EGFP-positive, but not in EGFP-negative UBCs. Immunohistochemistry showed that EGFP-negative UBCs express GIRK2 at high density, while mGluR1α UBCs are GIRK2 negative, suggesting that GIRK2 mediates the mGluR-sensitive current in EGFP-negative UBCs. These data suggest that the two subclasses perform different functions in the cerebellar microcircuits. PMID:22528965

  6. Lipid Droplets Form from Distinct Regions of the Cell in the Fission Yeast Schizosaccharomyces pombe.

    Science.gov (United States)

    Meyers, Alex; Del Rio, Zuania P; Beaver, Rachael A; Morris, Ryan M; Weiskittel, Taylor M; Alshibli, Amany K; Mannik, Jaana; Morrell-Falvey, Jennifer; Dalhaimer, Paul

    2016-06-01

    Eukaryotic cells store cholesterol/sterol esters (SEs) and triacylglycerols (TAGs) in lipid droplets, which form from the contiguous endoplasmic reticulum (ER) network. However, it is not known if droplets preferentially form from certain regions of the ER over others. Here, we used fission yeast Schizosaccharomyces pombe cells where the nuclear and cortical/peripheral ER domains are distinguishable by light microscopy to show that SE-enriched lipid droplets form away from the nucleus at the cell tips, whereas TAG-enriched lipid droplets form around the nucleus. Sterols localize to the regions of the cells where droplets enriched in SEs are observed. TAG droplet formation around the nucleus appears to be a strong function of diacylglycerol (DAG) homeostasis with Cpt1p, which coverts DAG into phosphatidylcholine and phosphatidylethanolamine localized exclusively to the nuclear ER. Also, Dgk1p, which converts DAG into phosphatidic acid localized strongly to the nuclear ER over the cortical/peripheral ER. We also show that TAG more readily translocates from the ER to lipid droplets than do SEs. The results augment the standard lipid droplet formation model, which has SEs and TAGs flowing into the same nascent lipid droplet regardless of its biogenesis point in the cell. PMID:26990381

  7. Unambiguous distinction between diffusion length and surface recombination velocity of solar cells at different excitation levels

    Science.gov (United States)

    Wawer, P.; Rochel, M.; Wagemann, H.-G.

    1999-06-01

    In this work we present a conclusive separation of bulk and surface recombination properties of solar cells. For this purpose, bifacial silicon solar cells were fabricated. The backside differential spectral response of the cells has been measured in the presence of bias light, both with and without backside passivation by means of corona charging on top of a thermal oxide. Employing the common one-dimensional Shockley model, the measurement curves have been simulated. This enables the base diffusion length to be distinguished from the backside surface recombination velocity. As such, their values have been determined individually. Repeating this procedure for different intensities of bias light has yielded the nonlinear behavior of the recombination mechanisms. By applying the Schockley-Read-Hall recombination theory, it was deduced that Fe interstitials presumably are the predominant bulk recombination centers.

  8. Distinct nonrandom patterns of chromosomal deletions in giant-cell lesions of bone

    Directory of Open Access Journals (Sweden)

    Baruffi Marcelo Razera

    2002-01-01

    Full Text Available Cytogenetic analyses were performed on a bone giant cell reparative granuloma (GCRG and on three bone giant cell tumors (GCT. The present GCRG case is the second to be described cytogenetically. A modal chromosome number of 46 was observed in all samples. Clonal chromosome abnormalities were detected in all cases. The numerical alterations most frequently observed involved the loss of chromosomes 17 and 18. Among the structural anomalies observed, there was preferential involvement of chromosomes 6 and 10. Three GCT cases presented del(10(p13 and two cases presented del(6(q25 (1 GCRG and 1 GCT. These breakpoints mapped on 10p and 6q may harbour genes of importance in the development of bone giant cell tumors.

  9. Syndecans promote integrin-mediated adhesion of mesenchymal cells in two distinct pathways

    DEFF Research Database (Denmark)

    Whiteford, James; Behrends, Volker; Kirby, Hishani; Kusche-Gullberg, Marion; Muramatsu, Takashi; Couchman, John R

    2007-01-01

    Syndecans are transmembrane proteoglycans that support integrin-mediated adhesion. Well documented is the contribution of syndecan-4 that interacts through its heparan sulphate chains to promote focal adhesion formation in response to fibronectin domains. This process has requirements for integrin...... and signaling through the cytoplasmic domain of syndecan-4. Here an alternate pathway mediated by the extracellular domains of syndecans-2 and -4 is characterized that is independent of both heparan sulphate and syndecan signaling. This pathway is restricted to mesenchymal cells and was not seen in...... any epithelial cell line tested, apart from vascular endothelia. The syndecan ectodomains coated as substrates promoted integrin-dependent attachment, spreading and focal adhesion formation. Syndecan-4 null cells were competent, as were fibroblasts compromised in heparan sulphate synthesis that were...

  10. The expression of CD8α discriminates distinct T cell subsets in teleost fish.

    Science.gov (United States)

    Takizawa, Fumio; Dijkstra, Johannes Martinus; Kotterba, Paul; Korytář, Tomáš; Kock, Holger; Köllner, Bernd; Jaureguiberry, Beltran; Nakanishi, Teruyuki; Fischer, Uwe

    2011-07-01

    CD8, belonging to the TCR complex, is the main marker molecule of CTLs. Although CD8 genes have been detected in many fish species, the analysis of teleost CD8+ cells has been limited because of the lack of antibodies. Using newly established mAbs against rainbow trout CD8α, we found high ratios of CD8α+ cells in trout thymus, gill and intestine, but relatively low abundance in pronephros, spleen and blood. Accordingly, tissue sections revealed many CD8α+ cells in thymus, numerous intra- and subepithelial CD8α+ cells in intestine and gill and few scattered CD8α+ cells in spleen and pronephros. In secondary lymphoid tissues, CD8α+ lymphocytes, which did not react with anti-thrombocyte or anti-IgM mAbs, expressed CD8α, CD8β and TCRα, while Ig and CD4 transcripts were found in CD8α⁻ lymphocytes. In contrast, considerable CD4 expression in CD8α+ thymocytes suggests the presence of double-positive early T cells. Highly expressed TCRγ, LAG3 and CTLA4 in CD8α+ lymphocytes imply that they constitute a heterogeneous population different from found in non-mucosal tissues. PHA stimulation resulted in an up-regulation of CTL effector genes (perforin, granulysin and IFN-γ) in CD8α+ pronephrocytes, while both Th1 (IFN-γ) and Th2 (IL-4/13A) cytokines were up-regulated in CD8α⁻ pronephrocytes. Although the basic characteristics of CD8α+ lymphocytes seem similar in teleost and mammals, features such as the low proportion of teleost CD8α+ lymphocytes in blood and their high abundance in respiratory tissue reveal a unique dynamics and distribution. PMID:21352850

  11. Human decidual macrophages and NK cells differentially express Toll-like receptors and display distinct cytokine profiles upon TLR stimulation.

    Directory of Open Access Journals (Sweden)

    ElisabethMenu

    2014-07-01

    We report for the first time that both human dMs and dNK cells express mRNAs encoding TLRs 1-9, albeit with a higher expression level in dMs. TLR2, TLR3 and TLR4 protein expression checked by flow cytometry was positive for both dMs and dNK cells. In vitro treatment of primary dMs and dNK cells with specific TLR2, TLR3, TLR4, TLR7/8 and TLR9 agonists enhanced their secretion of pro- and anti-inflammatory cytokines, as well as cytokines and chemokines involved in immune cell crosstalk. Only dNK cells released IFN-γ, whereas only dMs released IL-1β, IL-10 and IL-12. TLR9 activation of dMs resulted in a distinct pattern of cytokine expression compared to the other TLRs. The cytokine profiles expressed by dMs and dNK cells upon TLR activation are compatible with maintenance of the fetotolerant immune environment during initiation of immune responses to pathogens at the maternofetal interface.

  12. In situ expansion of T cells that recognize distinct self-antigens sustains autoimmunity in the CNS.

    Science.gov (United States)

    Ramadan, Abdulraouf; Lucca, Liliana E; Carrié, Nadège; Desbois, Sabine; Axisa, Pierre-Paul; Hayder, Myriam; Bauer, Jan; Liblau, Roland S; Mars, Lennart T

    2016-05-01

    Polyspecific T cells recognizing multiple distinct self-antigens have been identified in multiple sclerosis and other organ-specific autoimmune diseases, but their pathophysiological relevance remains undetermined. Using a mouse model of multiple sclerosis, we show that autoimmune encephalomyelitis induction is strictly dependent on reactivation of pathogenic T cells by a peptide (35-55) derived from myelin oligodendrocyte glycoprotein (MOG). This disease-inducing response wanes after onset. Strikingly, the progression of disease is driven by the in situ activation and expansion of a minority of MOG35-55-specific T cells that also recognize neurofilament-medium (NF-M)15-35, an intermediate filament protein expressed in neurons. This mobilization of bispecific T cells is critical for disease progression as adoptive transfer of NF-M15-35/MOG35-55 bispecific T cell lines caused full-blown disease in wild-type but not NF-M-deficient recipients. Moreover, specific tolerance through injection of NF-M15-35 peptide at the peak of disease halted experimental autoimmune encephalomyelitis progression. Our findings highlight the importance of polyspecific autoreactive T cells in the aggravation and perpetuation of central nervous system autoimmunity. PMID:27000832

  13. Electrophysiological, morphological and topological properties of two histochemically distinct subpopulations of cerebellar unipolar brush cells

    OpenAIRE

    Kim, Jin-Ah; Sekerková, Gabriella; Mugnaini, Enrico; Martina, Marco

    2012-01-01

    Unipolar brush cells (UBCs) are excitatory cerebellar granular layer interneurons whose brush-like dendrites receive one-to-one mossy fiber inputs. Subclasses of UBCs differ primarily by expressing metabotropic glutamate receptor (mGluR) 1α or calretinin. We used GENSAT Tg(Grp-EGFP) BAC-transgenic mice, which selectively express EGFP in mGluR1α-positive UBCs to compare the functional properties of the two subclasses. Compared to EGFP-negative UBCs, which include the calretinin-positive cells,...

  14. Distinct mathematical behavior of apoptotic vs. non-apoptotic tumor cell death

    International Nuclear Information System (INIS)

    Purpose: A quantitative description of cancer cell death behavior is of potential importance in identifying prognostically meaningful treatment responses and mechanisms underlying those responses. Here we examine clonogenic survival curves for two genetically related tumor cell lines differing in the presence or absence of p53, for whom solid tumor radiosensitivity patterns have been previously described. Materials and Methods: Oncogene-transformed fibroblasts derived from E1A+Ras tranfections of p53 wildtype or p53 null mouse embryonic fibroblasts were plated in single cell suspension. Cells were irradiated at increasing radiation doses from 1.5 Gy to 11 Gy. Dishes were scored for colonies at day 11. Survival curves were generated by least-squares regression over dose of log (survival) onto the quadratic (αD + βD2), each point being weighted inversely to the variance among replicates. Apoptosis was assessed morphologically by staining with flourescent nuclear dye DAPI, by DNA fragmentation with the APOPTAG Apoptosis Detection Kit (Oncor), and by measurement of apoptotic cysteine protease cleavage activity in cytosolic extracts. Results: Whereas radiation triggers massive apoptosis in the presence of p53, in this system it produces no measurable DNA fragmentation, apoptotic cysteine protease cleavage activity, or morphologic changes of apoptosis in the cells lacking p53. These contrasting mechanisms of death display dramatically different quantitative behavior: log-survival of apoptotic cells is linearly proportional to dose (S=e-nD) whereas non-apoptotic survival is quadratically related to dose (S=e-nD2). Radioresistance at clinical doses (about 2 Gy) were largely mirrored by the initial slopes. For quadratic killing, survival was nearly flat at 77%, whereas it was only 12% for apoptosis owing to its steep linear slope. Conclusions: Apoptosis exhibits single hit kinetics and is seen to produce a high α/β ratio and no significant shoulder, whereas non

  15. The special case of hepatocytes : unique tissue architecture calls for a distinct mode of cell division

    NARCIS (Netherlands)

    Slim, Christiaan L; van IJzendoorn, Sven C D; Lázaro-Diéguez, Francisco; Müsch, Anne

    2014-01-01

    Columnar epithelia (e.g., kidney, intestine) and hepatocytes embody the two major organizational phenotypes of non-stratified epithelial cells. Columnar epithelia establish their apical and basal domains at opposing poles and organize in monolayered cysts and tubules, in which their apical surfaces

  16. Angiogenesis interactome and time course microarray data reveal the distinct activation patterns in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Liang-Hui Chu

    Full Text Available Angiogenesis involves stimulation of endothelial cells (EC by various cytokines and growth factors, but the signaling mechanisms are not completely understood. Combining dynamic gene expression time-course data for stimulated EC with protein-protein interactions associated with angiogenesis (the "angiome" could reveal how different stimuli result in different patterns of network activation and could implicate signaling intermediates as points for control or intervention. We constructed the protein-protein interaction networks of positive and negative regulation of angiogenesis comprising 367 and 245 proteins, respectively. We used five published gene expression datasets derived from in vitro assays using different types of blood endothelial cells stimulated by VEGFA (vascular endothelial growth factor A. We used the Short Time-series Expression Miner (STEM to identify significant temporal gene expression profiles. The statistically significant patterns between 2D fibronectin and 3D type I collagen substrates for telomerase-immortalized EC (TIME show that different substrates could influence the temporal gene activation patterns in the same cell line. We investigated the different activation patterns among 18 transmembrane tyrosine kinase receptors, and experimentally measured the protein level of the tyrosine-kinase receptors VEGFR1, VEGFR2 and VEGFR3 in human umbilical vein EC (HUVEC and human microvascular EC (MEC. The results show that VEGFR1-VEGFR2 levels are more closely coupled than VEGFR1-VEGFR3 or VEGFR2-VEGFR3 in HUVEC and MEC. This computational methodology can be extended to investigate other molecules or biological processes such as cell cycle.

  17. Patient-derived acute myeloid leukemia (AML) bone marrow cells display distinct intracellular kinase phosphorylation patterns

    International Nuclear Information System (INIS)

    Multiparametric analyses of phospho-protein activation in patients with acute myeloid leukemia (AML) offers a quantitative measure to monitor the activity of novel intracellular kinase (IK) inhibitors. As recent clinical investigation with FMS-like tyrosine-3 inhibitors demonstrated, targeting IK with selective inhibitors can have a modest clinical benefit. Because multiple IKs are active in patients with AML, multikinase inhibitors may provide the necessary inhibition profile to achieve a more sustained clinical benefit. We here describe a method of assessing the activation of several IKs by flow cytometry. In 40 different samples of patients with AML we observed hyper-activated phospho-proteins at baseline, which is modestly increased by adding stem cell factor to AML cells. Finally, AML cells had a significantly different phospho-protein profile compared with cells of the lymphocyte gate. In conclusion, our method offers a way to determine the activation status of multiple kinases in AML and hence is a reliable assay to evaluate the pharmacodynamic activity of novel multikinase inhibitors

  18. Human decidual macrophages and NK cells differentially express Toll-like receptors and display distinct cytokine profiles upon TLR stimulation.

    Science.gov (United States)

    Duriez, Marion; Quillay, Héloïse; Madec, Yoann; El Costa, Hicham; Cannou, Claude; Marlin, Romain; de Truchis, Claire; Rahmati, Mona; Barré-Sinoussi, Françoise; Nugeyre, Marie-Thérèse; Menu, Elisabeth

    2014-01-01

    Maternofetal pathogen transmission is partially controlled at the level of the maternal uterine mucosa at the fetal implantation site (the decidua basalis), where maternal and fetal cells are in close contact. Toll-like receptors (TLRs) may play an important role in initiating rapid immune responses against pathogens in the decidua basalis, however the tolerant microenvironment should be preserved in order to allow fetal development. Here we investigated the expression and functionality of TLRs expressed by decidual macrophages (dMs) and NK cells (dNKs), the major decidual immune cell populations. We report for the first time that both human dMs and dNK cells express mRNAs encoding TLRs 1-9, albeit with a higher expression level in dMs. TLR2, TLR3, and TLR4 protein expression checked by flow cytometry was positive for both dMs and dNK cells. In vitro treatment of primary dMs and dNK cells with specific TLR2, TLR3, TLR4, TLR7/8, and TLR9 agonists enhanced their secretion of pro- and anti-inflammatory cytokines, as well as cytokines and chemokines involved in immune cell crosstalk. Only dNK cells released IFN-γ, whereas only dMs released IL-1β, IL-10, and IL-12. TLR9 activation of dMs resulted in a distinct pattern of cytokine expression compared to the other TLRs. The cytokine profiles expressed by dMs and dNK cells upon TLR activation are compatible with maintenance of the fetotolerant immune environment during initiation of immune responses to pathogens at the maternofetal interface. PMID:25071732

  19. Distinct populations of dendritic cells are present in the subepithelial dome and T cell regions of the murine Peyer's patch

    Science.gov (United States)

    1996-01-01

    Despite the fact that the Peyer's patch (PP) is the primary site for antigen uptake in the intestine, the cellular basis of antigen handling after transport into the PP is poorly understood. We performed immunohistology of murine PPs using the dendritic cell (DC)-reactive monoclonal antibodies N418, NLDC-145, M342, and 2A1, as well as antibodies to other T cell, B cell, and macrophage markers. N418+, 2A1+, NLDC-145-, M342- cells form a dense layer of cells in the subepithelial dome (SED), just beneath the follicle epithelium, and are scattered throughout the follicle, sparing the germinal center. In contrast, N418+, 2A1+, NLDC-145+, and M342+ DCs are present in the interfollicular T cell regions (IFR). CD3+ and CD4+, but no CD8+ T cells were present in the SED and the follicle, including the germinal center, while CD3+, CD4+, and CD8+ T cells were present in the IFR. B cells and macrophages were poorly represented in the SED as no B220+ cells, only few Mac-1lo cells, and no F4/80+ cells were present at this site. In contrast, Mac-1hi cells were found in the IFR and lamina propria of intestinal villi, while F4/80+ cells were found only in the latter. In further phenotypic studies, we analyzed surface molecules of PP and spleen DCs by flow cytometry and found that these cells had similar fluorescence profiles when stained with N418, NLDC-145, and 33D1 DC-reactive antibodies, and antibodies to the costimulatory molecules B7-1 (1G10) and B7-2 (GL1). In contrast, PP DCs expressed 5- 10-fold higher levels of major histocompatibility complex class II antigens (IEk) than spleen DCs. Finally, in functional studies, we demonstrated that both PP and spleen DCs process soluble protein antigens during overnight culture and induce similar levels of proliferation in CD3+ T cells, and CD4+/Mel 14hi T cells from T cell receptor transgenic mice. The in vivo relevance of such presentation was shown by the fact that PP DCs isolated from Balb/c mice after being fed ovalbumin stimulated

  20. Distinct activation of primary human BDCA1(+) dendritic cells upon interaction with stressed or infected β cells.

    Science.gov (United States)

    Schulte, B M; Kers-Rebel, E D; Bottino, R; Piganelli, J D; Galama, J M D; Engelse, M A; de Koning, E J P; Adema, G J

    2016-06-01

    Derailment of immune responses can lead to autoimmune type 1 diabetes, and this can be accelerated or even induced by local stress caused by inflammation or infection. Dendritic cells (DCs) shape both innate and adaptive immune responses. Here, we report on the responses of naturally occurring human myeloid BDCA1(+) DCs towards differentially stressed pancreatic β cells. Our data show that BDCA1(+) DCs in human pancreas-draining lymph node (pdLN) suspensions and blood-derived BDCA1(+) DCs both effectively engulf β cells, thus mimicking physiological conditions. Upon uptake of enterovirus-infected, but not mock-infected cells, BDCA1(+) DCs induced interferon (IFN)-α/β responses, co-stimulatory molecules and proinflammatory cytokines and chemokines. Notably, induction of stress in β cells by ultraviolet irradiation, culture in serum-free medium or cytokine-induced stress did not provoke strong DC activation, despite efficient phagocytosis. DC activation correlated with the amount of virus used to infect β cells and required RNA within virally infected cells. DCs encountering enterovirus-infected β cells, but not those incubated with mock-infected or stressed β cells, suppressed T helper type 2 (Th2) cytokines and variably induced IFN-γ in allogeneic mixed lymphocyte reaction (MLR). Thus, stressed β cells have little effect on human BDCA1(+) DC activation and function, while enterovirus-infected β cells impact these cells significantly, which could help to explain their role in development of autoimmune diabetes in individuals at risk. PMID:26888163

  1. Distinct functional properties of highly purified hematopoietic stem cells from mouse strains differing in stem cell numbers.

    Science.gov (United States)

    de Haan, G; Szilvassy, S J; Meyerrose, T E; Dontje, B; Grimes, B; Van Zant, G

    2000-08-15

    We have previously demonstrated that young adult DBA/2 (DBA) mice have more stem cells than C57BL/6 (B6) mice, as measured in a cobblestone area-forming cell (CAFC) assay using unfractionated marrow. To study the nature of this difference, we have now compared the proliferative fate of single, highly enriched Sca-1(+)c-kit(+)Lin(-) stem cells from these strains. Although equal in frequency, functional comparison revealed that Sca-1(+)c-kit(+)Lin(-) cells from DBA mice contained twice as many cells with CAFC activity. DBA clones persisted much longer in vitro, and developed later in time. To assess whether these differences were of any functional relevance in vivo, we compared engraftment of lethally irradiated mice transplanted with 1000 B6 or DBA Sca-1(+)c-kit(+)Lin(-) cells. Recipients of enriched DBA cells recovered much faster than animals transplanted with B6 cells. We also studied endogenous hematopoietic recovery after 5-fluorouracil (5-FU) treatment in vivo. Progenitors and peripheral blood cells recovered twice as fast in DBA mice. Thus, DBA stem cells have superior proliferative potential compared with phenotypically identical stem cells obtained from B6 mice. Such genetically determined quantitative and qualitative differences in stem cell behavior likely contribute to the dramatically different hematopoietic recovery rates observed in human transplant patients. (Blood. 2000;96:1374-1379) PMID:10942380

  2. Distinct linkage between post-translational processing and differential secretion of progastrin derivatives in endocrine cells

    DEFF Research Database (Denmark)

    Bundgaard, J.R.; Rehfeld, Jens Frederik

    2008-01-01

    Prohormones often undergo extensive cellular processing prior to secretion. These post-translational processing events occur in organelles of the constitutive or regulated secretory pathway. The aim of this study was to examine the relationship between post-translational modifications and the...... secretory pathways taken by peptides derived from progastrin, the prohormone of gastrin, which in vivo is secreted by cells of the pyloric glands and stimulates the release of gastric acid. Targeting progastrin to compartments of the early secretory pathway shows that endoproteolytic processing is initiated...... in a pre-trans-Golgi network compartment of endocrine but not non-endocrine cells. The resulting N-terminal fragments of progastrin are secreted via the constitutive pathway, whereas endoproteolytically processed C-terminal fragments are secreted via the regulated or constitutive-like pathways. C...

  3. The differential loading of two barley CENH3 variants into distinct centromeric substructures is cell type- and development-specific.

    Science.gov (United States)

    Ishii, Takayoshi; Karimi-Ashtiyani, Raheleh; Banaei-Moghaddam, Ali Mohammad; Schubert, Veit; Fuchs, Jörg; Houben, Andreas

    2015-06-01

    The organization of centromeric chromatin of diploid barley (Hordeum vulgare) encoding two (α and β) CENH3 variants was analysed by super-resolution microscopy. Antibody staining revealed that both CENH3 variants are organized in distinct but intermingled subdomains in interphase, mitotic and meiotic centromeres. Artificially extended chromatin fibres illustrate that these subdomains are formed by polynucleosome clusters. Thus, a CENH3 variant-specific loading followed by the arrangement into specific intermingling subdomains forming the centromere region appears. The CENH3 composition and transcription vary among different tissues. In young embryos, most interphase centromeres are composed of both CENH3 variants, while in meristematic root cells, a high number of nuclei contain βCENH3 mainly dispersed within the nucleoplasm. A similar distribution and no preferential arrangement of the two CENH3 variants in relationship to the spindle poles suggest that both homologs meet the same function in metaphase cells. PMID:25688006

  4. A nuclear circularity-based classifier for diagnostic distinction of desmoplastic from spindle cell melanoma in digitized histological images

    Directory of Open Access Journals (Sweden)

    Manuel Schöchlin

    2014-01-01

    Full Text Available Context: Distinction of spindle cell melanoma (SM and desmoplastic melanoma (DM is clinically important due to differences in metastatic rate and prognosis; however, histological distinction is not always straightforward. During a routine review of cases, we noted differences in nuclear circularity between SM and DM. Aim: The primary aim in our study was to determine whether these differences in nuclear circularity, when assessed using a basic ImageJ-based threshold extraction, can serve as a diagnostic classifier to distinguish DM from SM. Settings and Design: Our retrospective analysis of an established patient cohort (SM n = 9, DM n = 9 was employed to determine discriminatory power. Subjects and Methods: Regions of interest (total n = 108; 6 images per case were selected from scanned H and E-stained histological sections, and nuclear circularity was extracted and quantified by computational image analysis using open source tools (plugins for ImageJ. Statistical Analysis: Using analysis of variance, t-tests, and Fisher′s exact tests, we compared extracted quantitative shape measures; statistical significance was defined as P < 0.05. Results: Classifying circularity values into four shape categories (spindled, elongated, oval, round demonstrated significant differences in the spindled and round categories. Paradoxically, DM contained more spindled nuclei than SM (P = 0.011 and SM contained more round nuclei than DM (P = 0.026. Performance assessment using a combined shape-classification of the round and spindled fractions showed 88.9% accuracy and a Youden index of 0.77. Conclusions: Spindle cell melanoma and DM differ significantly in their nuclear morphology with respect to fractions of round and spindled nuclei. Our study demonstrates that quantifying nuclear circularity can be used as an adjunct diagnostic tool for distinction of DM and SM.

  5. Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes

    Science.gov (United States)

    Sutton, F.; Paul, S. S.; Wang, X. Q.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

    2000-01-01

    Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K(+) current (I(Kin)) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K(+) current (I(Kout)). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, I(Kout) was generated that displayed similar properties to I(Kout) observed from direct analysis of mesophyll cell protoplasts. I(Kout) expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K(+) channel expressed in guard cells, resulted in I(Kin) that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced I(Kin) that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K(+) channels.

  6. Three Distinct Modes of Exocytosis Revealed by Amperometry in Neuroendocrine Cells

    OpenAIRE

    van Kempen, G. Th. H.; vanderLeest, H.T.; Van den Berg, R J; Eilers, P.; Westerink, R.H.S.

    2011-01-01

    Neurotransmission requires Ca2+-dependent release of secretory products through fusion pores that open and reclose (partial membrane distention) or open irreversibly (complete membrane distention). It has been challenging to distinguish between these release modes; however, in the work presented here, we were able to deduce different modes of depolarization-evoked exocytosis in neuroendocrine chromaffin and PC12 cells solely by analyzing amperometric recordings. After we determined the quanta...

  7. Disruption of E-cadherin-mediated adhesion induces a functionally distinct pathway of dendritic cell maturation

    OpenAIRE

    Jiang, Aimin; Bloom, Ona; Ono, Satoru; Cui, Weiguo; Unternaehrer, Juli; Jiang, Shan; Whitney, J. Andrew; Connolly, John; Banchereau, Jacques; Mellman, Ira

    2007-01-01

    The maturation of dendritic cells (DCs) following exposure to microbial products or inflammatory mediators plays a critical role in initiating the immune response. We now find that maturation can also occur under steady state conditions, triggered by alterations in E-cadherin-mediated DC-DC adhesion. Selective disruption of these interactions induces the typical features of DC maturation including the upregulation of costimulatory molecules, MHC class II, and chemokine receptors. These events...

  8. Prominent Lymphatic Vessel Hyperplasia with Progressive Dysfunction and Distinct Immune Cell Infiltration in Lymphedema.

    Science.gov (United States)

    Gousopoulos, Epameinondas; Proulx, Steven T; Scholl, Jeannette; Uecker, Maja; Detmar, Michael

    2016-08-01

    Lymphedema is a common complication that occurs after breast cancer treatment in up to 30% of the patients undergoing surgical lymph node excision. It is associated with tissue swelling, fibrosis, increased risk of infection, and impaired wound healing. Despite the pronounced clinical manifestations of the disease, little is known about the morphological and functional characteristics of the lymphatic vasculature during the course of lymphedema progression. We used an experimental murine tail lymphedema model where sustained fluid stasis was generated on disruption of lymphatic flow, resulting in chronic edema formation with fibrosis and adipose tissue deposition. Morphological analysis of the lymphatic vessels revealed a dramatic expansion during the course of the disease, with active proliferation of lymphatic endothelial cells at the early stages of lymphedema. The lymphatic capillaries exhibited progressively impaired tracer filling and retrograde flow near the surgery site, whereas the collecting lymphatic vessels showed a gradually decreasing contraction amplitude with unchanged contraction frequency, leading to lymphatic contraction arrest at the later stages of the disease. Lymphedema onset was associated with pronounced infiltration by immune cells, predominantly Ly6G(+) and CD4(+) cells, which have been linked to impaired lymphatic vessel function. PMID:27315777

  9. Distinct Neurodegenerative Changes in an Induced Pluripotent Stem Cell Model of Frontotemporal Dementia Linked to Mutant TAU Protein

    Directory of Open Access Journals (Sweden)

    Marc Ehrlich

    2015-07-01

    Full Text Available Frontotemporal dementia (FTD is a frequent form of early-onset dementia and can be caused by mutations in MAPT encoding the microtubule-associated protein TAU. Because of limited availability of neural cells from patients’ brains, the underlying mechanisms of neurodegeneration in FTD are poorly understood. Here, we derived induced pluripotent stem cells (iPSCs from individuals with FTD-associated MAPT mutations and differentiated them into mature neurons. Patient iPSC-derived neurons demonstrated pronounced TAU pathology with increased fragmentation and phospho-TAU immunoreactivity, decreased neurite extension, and increased but reversible oxidative stress response to inhibition of mitochondrial respiration. Furthermore, FTD neurons showed an activation of the unfolded protein response, and a transcriptome analysis demonstrated distinct, disease-associated gene expression profiles. These findings indicate distinct neurodegenerative changes in FTD caused by mutant TAU and highlight the unique opportunity to use neurons differentiated from patient-specific iPSCs to identify potential targets for drug screening purposes and therapeutic intervention.

  10. Single-cell RNA-seq reveals distinct injury responses in different types of DRG sensory neurons

    Science.gov (United States)

    Hu, Ganlu; Huang, Kevin; Hu, Youjin; Du, Guizhen; Xue, Zhigang; Zhu, Xianmin; Fan, Guoping

    2016-01-01

    Peripheral nerve injury leads to various injury-induced responses in sensory neurons including physiological pain, neuronal cell death, and nerve regeneration. In this study, we performed single-cell RNA-sequencing (scRNA-seq) analysis of mouse nonpeptidergic nociceptors (NP), peptidergic nociceptors (PEP), and large myelinated sensory neurons (LM) under both control and injury conditions at 3 days after sciatic nerve transection (SNT). After performing principle component and weighted gene co-expression network analysis, we categorized dorsal root ganglion (DRG) neurons into different subtypes and discovered co-regulated injury-response genes including novel regeneration associated genes (RAGs) in association with neuronal development, protein translation and cytoplasm transportation. In addition, we found significant up-regulation of the genes associated with cell death such as Pdcd2 in a subset of NP neurons after axotomy, implicating their actions in neuronal cell death upon nerve injury. Our study revealed the distinctive and sustained heterogeneity of transcriptomic responses to injury at single neuron level, implicating the involvement of different gene regulatory networks in nerve regeneration, neuronal cell death and neuropathy in different population of DRG neurons. PMID:27558660

  11. Dynamic Recruitment of Functionally Distinct Swi/Snf Chromatin Remodeling Complexes Modulates Pdx1 Activity in Islet β Cells

    Directory of Open Access Journals (Sweden)

    Brian McKenna

    2015-03-01

    Full Text Available Pdx1 is a transcription factor of fundamental importance to pancreas formation and adult islet β cell function. However, little is known about the positive- and negative-acting coregulators recruited to mediate transcriptional control. Here, we isolated numerous Pdx1-interacting factors possessing a wide range of cellular functions linked with this protein, including, but not limited to, coregulators associated with transcriptional activation and repression, DNA damage response, and DNA replication. Because chromatin remodeling activities are essential to developmental lineage decisions and adult cell function, our analysis focused on investigating the influence of the Swi/Snf chromatin remodeler on Pdx1 action. The two mutually exclusive and indispensable Swi/Snf core ATPase subunits, Brg1 and Brm, distinctly affected target gene expression in β cells. Furthermore, physiological and pathophysiological conditions dynamically regulated Pdx1 binding to these Swi/Snf complexes in vivo. We discuss how context-dependent recruitment of coregulatory complexes by Pdx1 could impact pancreas cell development and adult islet β cell activity.

  12. T cell clones from Schistosoma haematobium infected and exposed individuals lacking distinct cytokine profiles for Th1/Th2 polarisation

    Directory of Open Access Journals (Sweden)

    Mduluza T

    2001-01-01

    Full Text Available T cell clones were derived from peripheral blood mononuclear cells of Schistosoma haematobium infected and uninfected individuals living in an endemic area. The clones were stimulated with S. haematobium worm and egg antigens and purified protein derivative. Attempts were made to classify the T cell clones according to production of the cytokines IL-4, IL-5 and IFN-gamma. All the T cell clones derived were observed to produce cytokines used as markers for the classification of Th1/Th2 subsets. However, the 'signature' cytokines marking each subset were produced at different levels. The classification depended on the dominating cytokine type, which was having either Th0/1 or Th0/2 subsets. The results indicated that no distinct cytokine profiles for polarisation of Th1/Th2 subsets were detected in these S. haematobium infected humans. The balance in the profiles of cytokines marking each subset were related to infection and re-infection status after treatment with praziquantel. In the present study, as judged by the changes in infection status with time, the T cell responses appeared to be less stable and more dynamic, suggesting that small quantitative changes in the balance of the cytokines response could result in either susceptibility or resistant to S. haematobium infection.

  13. Immunogold electron microscopy and confocal analyses reveal distinctive patterns of histone H3 phosphorylation during mitosis in MCF-7 cells.

    Science.gov (United States)

    Yan, Yitang; Cummings, Connie A; Sutton, Deloris; Yu, Linda; Castro, Lysandra; Moore, Alicia B; Gao, Xiaohua; Dixon, Darlene

    2016-04-01

    Histone phosphorylation has a profound impact on epigenetic regulation of gene expression, chromosome condensation and segregation, and maintenance of genome integrity. Histone H3 Serine 10 is evolutionally conserved and heavily phosphorylated during mitosis. To examine Histone H3 Serine 10 phosphorylation (H3S10ph) dynamics in mitosis, we applied immunogold labeling and confocal microscopy to visualize H3S10ph expression in MCF-7 cells. Confocal observations showed that MCF-7 cells had abundant H3S10ph expression in prophase and metaphase. In anaphase, the H3S10ph expression was significantly decreased and displayed only sparsely localized staining that mainly associated with the chromatid tips. We showed that immunogold bead density distribution followed the H3S10ph expression patterns observed in confocal analysis. At a higher magnification in metaphase, the immunogold beads were readily visible and the bead distribution along the condensed chromosomes was distinctive, indicating the specificity and reliability of the immunogold staining procedure. In anaphase, the beads were found to distribute focally in specific regions of chromatids, reinforcing the confocal observations of differential H3 phosphorylation. To our knowledge, this is the first report to show the specific H3S10ph expression with an immunogold technique and transmission electron microscopy. Additionally, with confocal microscopy, we analyzed H3S10ph expression in an immortalized cell line derived from benign uterine smooth muscle tumor cells. H3S10ph epitope was expressed more abundantly during anaphase in the benign tumor cells, and there was no dramatic differential expression within the condensed chromatid clusters as observed in MCF-7 cells. The differences in H3S10ph expression pattern and dynamics may contribute to the differential proliferative potential between benign tumor cells and MCF-7 cells. Published 2016. This article is a U.S. Government work and is in the public domain in the

  14. The new vaccines: building viruses that elicit antitumor immunity

    OpenAIRE

    Restifo, Nicholas P

    1996-01-01

    Whereas cancer cells are poor immunogens, some viruses are capable of eliciting powerful and lifelong immunity. Recombinant viruses and plasmid DNA encoding tumor-associated antigens can elicit powerful and specific immune responses that can be enhanced by the use of cytokines and costimulatory molecules. These immune responses have destroyed growing tumor cells in experimental animal models. For the first time, immunotherapeutic strategies that employ recombinant viruses are being tested in ...

  15. Intraepithelial p63-dependent expression of distinct components of cell adhesion complexes in normal esophageal mucosa and squamous cell carcinoma.

    Science.gov (United States)

    Thépot, Amélie; Hautefeuille, Agnès; Cros, Marie-Pierre; Abedi-Ardekani, Behnoush; Pétré, Aurélia; Damour, Odile; Krutovskikh, Vladimir; Hainaut, Pierre

    2010-11-01

    TP63 gene is a member of TP53 tumor suppressor gene family that encodes several protein isoforms involved in the process of epithelial stratification and in epithelial-mesenchyme interactions. TP63 is amplified in a significant proportion of squamous cell carcinoma of the esophagus (ESCC), resulting in the hyper-expression of DeltaNp63 as the major p63 isoform. To better understand the contribution of this high expression to tumorigenesis, we have analyzed the impact of intraepithelial p63 expression on the expression of cell adhesion complexes in normal esophagus and in ESCC cell lines. Cells expressing p63 showed an adhesion pattern characterized by lack of tight junctions and presence of adherens junctions. Cell differentiation was accompanied by a decrease in p63 and by a shift to adhesion patterns involving tight junctions. Silencing of p63 mRNA in ESCC cell lines resulted in a similar shift, characterized by increased expression of component of tight junctions, decreased cell-to-cell communication and downregulation of cell proliferation. These results indicate that DeltaNp63 may contribute to esophageal squamous carcinogenesis by maintaining cell adhesion patterns compatible with cell proliferation. PMID:20127860

  16. Trypanosoma cruzi-elicited CD8+ T cell-mediated myocarditis: chemokine receptors and adhesion molecules as potential therapeutic targets to control chronic inflammation?

    Directory of Open Access Journals (Sweden)

    Joseli Lannes-Vieira

    2003-04-01

    Full Text Available In Chagas disease, during the acute phase, the establishment of inflammatory processes is crucial for Trypanosoma cruzi control in target tissues and for the establishment of host/parasite equilibrium. However, in about 30% of the patients, inflammation becomes progressive, resulting in chronic disease, mainly characterized by myocarditis. Although several hypothesis have been raised to explain the pathogenesis of chagasic myocardiopathy, including the persistence of the parasite and/or participation of autoimmune processes, the molecular mechanisms underlying the establishment of the inflammatory process leading to parasitism control but also contributing to the maintenance of T. cruzi-elicited chronic myocarditis remain unsolved. Trying to shed light on these questions, we have for several years been working with murine models for Chagas disease that reproduce the acute self-resolving meningoencephalitis, the encephalitis resulting of reactivation described in immunodeficient individuals, and several aspects of the acute and chronic myocarditis. In the present review, our results are summarized and discussed under the light of the current literature. Furthermore, rational therapeutic intervention strategies based on integrin-mediated adhesion and chemokine receptor-driven recruitment of leukocytes are proposed to control T. cruzi-elicited unbalanced inflammation.

  17. Disruption of Escherichia coli Nissle 1917 K5 capsule biosynthesis, through loss of distinct kfi genes, modulates interaction with intestinal epithelial cells and impact on cell health.

    Directory of Open Access Journals (Sweden)

    Jonathan Nzakizwanayo

    Full Text Available Escherichia coli Nissle 1917 (EcN is among the best characterised probiotics, with a proven clinical impact in a range of conditions. Despite this, the mechanisms underlying these "probiotic effects" are not clearly defined. Here we applied random transposon mutagenesis to identify genes relevant to the interaction of EcN with intestinal epithelial cells. This demonstrated mutants disrupted in the kfiB gene, of the K5 capsule biosynthesis cluster, to be significantly enhanced in attachment to Caco-2 cells. However, this phenotype was distinct from that previously reported for EcN K5 deficient mutants (kfiC null mutants, prompting us to explore further the role of kfiB in EcN:Caco-2 interaction. Isogenic mutants with deletions in kfiB (EcNΔkfiB, or the more extensively characterised K5 capsule biosynthesis gene kfiC (EcNΔkfiC, were both shown to be capsule deficient, but displayed divergent phenotypes with regard to impact on Caco-2 cells. Compared with EcNΔkfiC and the EcN wild-type, EcNΔkfiB exhibited significantly greater attachment to Caco-2 cells, as well as apoptotic and cytotoxic effects. In contrast, EcNΔkfiC was comparable to the wild-type in these assays, but was shown to induce significantly greater COX-2 expression in Caco-2 cells. Distinct differences were also apparent in the pervading cell morphology and cellular aggregation between mutants. Overall, these observations reinforce the importance of the EcN K5 capsule in host-EcN interactions, but demonstrate that loss of distinct genes in the K5 pathway can modulate the impact of EcN on epithelial cell health.

  18. Disruption of Escherichia coli Nissle 1917 K5 capsule biosynthesis, through loss of distinct kfi genes, modulates interaction with intestinal epithelial cells and impact on cell health.

    Science.gov (United States)

    Nzakizwanayo, Jonathan; Kumar, Sandeep; Ogilvie, Lesley A; Patel, Bhavik A; Dedi, Cinzia; Macfarlane, Wendy M; Jones, Brian V

    2015-01-01

    Escherichia coli Nissle 1917 (EcN) is among the best characterised probiotics, with a proven clinical impact in a range of conditions. Despite this, the mechanisms underlying these "probiotic effects" are not clearly defined. Here we applied random transposon mutagenesis to identify genes relevant to the interaction of EcN with intestinal epithelial cells. This demonstrated mutants disrupted in the kfiB gene, of the K5 capsule biosynthesis cluster, to be significantly enhanced in attachment to Caco-2 cells. However, this phenotype was distinct from that previously reported for EcN K5 deficient mutants (kfiC null mutants), prompting us to explore further the role of kfiB in EcN:Caco-2 interaction. Isogenic mutants with deletions in kfiB (EcNΔkfiB), or the more extensively characterised K5 capsule biosynthesis gene kfiC (EcNΔkfiC), were both shown to be capsule deficient, but displayed divergent phenotypes with regard to impact on Caco-2 cells. Compared with EcNΔkfiC and the EcN wild-type, EcNΔkfiB exhibited significantly greater attachment to Caco-2 cells, as well as apoptotic and cytotoxic effects. In contrast, EcNΔkfiC was comparable to the wild-type in these assays, but was shown to induce significantly greater COX-2 expression in Caco-2 cells. Distinct differences were also apparent in the pervading cell morphology and cellular aggregation between mutants. Overall, these observations reinforce the importance of the EcN K5 capsule in host-EcN interactions, but demonstrate that loss of distinct genes in the K5 pathway can modulate the impact of EcN on epithelial cell health. PMID:25790373

  19. Two distinct sites in sonic Hedgehog combine for heparan sulfate interactions and cell signaling functions

    DEFF Research Database (Denmark)

    Chang, Shu-Chun; Mulloy, Barbara; Magee, Anthony I;

    2011-01-01

    Hedgehog (Hh) proteins are morphogens that mediate many developmental processes. Hh signaling is significant for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancer. Hh proteins require heparan sulfate proteoglycans (HSPGs) for...... their normal distribution and signaling activity. Here, we have used molecular modeling to examine the heparin-binding domain of sonic hedgehog (Shh). In biochemical and cell biological assays, the importance of specific residues of the putative heparin-binding domain for signaling was assessed. It was...

  20. Distinct Glycoprotein O Complexes Arise in a Post-Golgi Compartment of Cytomegalovirus-Infected Cells

    OpenAIRE

    Theiler, Regan N.; Compton, Teresa

    2002-01-01

    Human cytomegalovirus (CMV) glycoproteins H, L, and O (gH, gL, and gO, respectively) form a heterotrimeric disulfide-bonded complex that participates in the fusion of the viral envelope with the host cell membrane. During virus maturation, this complex undergoes a series of intracellular assembly and processing events which are not entirely defined (M. T. Huber and T. Compton, J. Virol. 73:3886-3892, 1999). Here, we demonstrate that gO does not undergo the same posttranslational processing in...

  1. Identification of Distinct Breast Cancer Stem Cell Populations Based on Single-Cell Analyses of Functionally Enriched Stem and Progenitor Pools

    Directory of Open Access Journals (Sweden)

    Nina Akrap

    2016-01-01

    Full Text Available The identification of breast cancer cell subpopulations featuring truly malignant stem cell qualities is a challenge due to the complexity of the disease and lack of general markers. By combining extensive single-cell gene expression profiling with three functional strategies for cancer stem cell enrichment including anchorage-independent culture, hypoxia, and analyses of low-proliferative, label-retaining cells derived from mammospheres, we identified distinct stem cell clusters in breast cancer. Estrogen receptor (ERα+ tumors featured a clear hierarchical organization with switch-like and gradual transitions between different clusters, illustrating how breast cancer cells transfer between discrete differentiation states in a sequential manner. ERα− breast cancer showed less prominent clustering but shared a quiescent cancer stem cell pool with ERα+ cancer. The cellular organization model was supported by single-cell data from primary tumors. The findings allow us to understand the organization of breast cancers at the single-cell level, thereby permitting better identification and targeting of cancer stem cells.

  2. Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines

    International Nuclear Information System (INIS)

    The inability of cancer cells to present antigen on the cell surface via MHC class I molecules is one of the mechanisms by which tumor cells evade anti-tumor immunity. Alterations of Jak-STAT components of interferon (IFN)-mediated signaling can contribute to the mechanism of cell resistance to IFN, leading to lack of MHC class I inducibility. Hence, the identification of IFN-γ-resistant tumors may have prognostic and/or therapeutic relevance. In the present study, we investigated a mechanism of MHC class I inducibility in response to IFN-γ treatment in human melanoma cell lines. Basal and IFN-induced expression of HLA class I antigens was analyzed by means of indirect immunofluorescence flow cytometry, Western Blot, RT-PCR, and quantitative real-time RT-PCR (TaqMan® Gene Expression Assays). In demethylation studies cells were cultured with 5-aza-2'-deoxycytidine. Electrophoretic Mobility Shift Assay (EMSA) was used to assay whether IRF-1 promoter binding activity is induced in IFN-γ-treated cells. Altered IFN-γ mediated HLA-class I induction was observed in two melanoma cells lines (ESTDAB-004 and ESTDAB-159) out of 57 studied, while treatment of these two cell lines with IFN-α led to normal induction of HLA class I antigen expression. Examination of STAT-1 in ESTDAB-004 after IFN-γ treatment demonstrated that the STAT-1 protein was expressed but not phosphorylated. Interestingly, IFN-α treatment induced normal STAT-1 phosphorylation and HLA class I expression. In contrast, the absence of response to IFN-γ in ESTDAB-159 was found to be associated with alterations in downstream components of the IFN-γ signaling pathway. We observed two distinct mechanisms of loss of IFN-γ inducibility of HLA class I antigens in two melanoma cell lines. Our findings suggest that loss of HLA class I induction in ESTDAB-004 cells results from a defect in the earliest steps of the IFN-γ signaling pathway due to absence of STAT-1 tyrosine-phosphorylation, while absence

  3. Different BAG-1 isoforms have distinct functions in modulating chemotherapeutic-induced apoptosis in breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    Hongyu LIU; Zhuomin WANG; Yun BAI; Min WANG; Ying LI; Sen WEI; Qinghua ZHOU; Jun CHEN

    2009-01-01

    Aim:BAG-1 is a multifunctional anti-apoptotic gene with four isoforms,and different BAG-1 isoforms have different anti-apoptotic functions.In this study,we transfected BAG-1 isoforms into the human breast cancer cell lines Hs578T (ER nega-tive) and MCF-7 (ER positive) to study their effect on apoptosis with or without estrogens.Methods: The constructed recombinant expression vectors carrying individual BAG-1 isoforms was used to transfect human breast cancer cell lines Hs578T (ER negative) and MCF-7 (ER positive).After stable cell lines were made,a variety of apoptosis-inducing agents,including doxorubicin,docetaxel,and 5-FU,was used to treat these cell lines with or without estrogen to test the role of BAG-1.The mechanism by which BAG-1 affected the function of Bcl-2 was exploredby using the cycloheximide chase assay.Results: The BAG-1 p50 and p46 isoforms significantly enhanced the resistance to apoptosis in both cell lines according to flow cytometry analysis.BAG-1 p33 and p29 failed to protect the transfected cells from apoptosis.The cell viability assay showed that only BAG-1 p50,but not p46,p33,or p29,increased estrogen-dependent function in ER-positive cell line MCF-7.Only BAG-1 p50 dramatically increased its anti-apoptotic ability in the presence of estrogen,while estrogen has very little effect on the anti-apoptotic ability of other BAG-1 isoforms.In the detection of the expression of K-ras,Hsp70,cytochrome c,Raf-1,ER-α,and Bcl-2 in MCF-7 cells by Western blot,only Bcl-2 protein expression was significantly increased in MCF-7 cells transfected with BAG-1 p50 and p46,respectively.Furthermore,the cycloheximide chase assay indicated that the degradation of Bcl-2 protein was extended in the BAG-1 p50 and p46 transfected MCF-7 cells.Conclusion: Distinct isoforrns of BAG-1 have different anti-apoptotic functions in breast cancer cells,and that the BAG-1 p50 isoform can potentiate the role of estrogen in ER-positive breast cancer.

  4. Two Distinct Pathways to Development of Squamous Cell Carcinoma of the Vulva

    Directory of Open Access Journals (Sweden)

    Yutaka Ueda

    2011-01-01

    Full Text Available Squamous cell carcinoma (SCC accounts for approximately 95% of the malignant tumors of the vaginal vulva and is mostly found in elderly women. The future numbers of patients with vulvar SCC is expected to rise, mainly because of the proportional increase in the average age of the general population. Two different pathways for vulvar SCC have been put forth. The first pathway is triggered by infection with a high-risk-type Human Papillomavirus (HPV. Integration of the HPV DNA into the host genome leads to the development of a typical vulvar intraepithelial neoplasia (VIN, accompanied with overexpression of p14ARF and p16INK4A. This lesion subsequently forms a warty- or basaloid-type SCC. The HPV vaccine is a promising new tool for prevention of this HPV related SCC of the vulva. The second pathway is HPV-independent. Keratinizing SCC develops within a background of lichen sclerosus (LS through a differentiated VIN. It has a different set of genetic alterations than those in the first pathway, including p53 mutations, allelic imbalances (AI, and microsatellite instability (MSI. Further clinical and basic research is still required to understand and prevent vulvar SCC. Capsule. Two pathway for pathogenesis of squamous cell carcinoma of the value are reviewed.

  5. P. falciparum isolate-specific distinct patterns of induced apoptosis in pulmonary and brain endothelial cells.

    Directory of Open Access Journals (Sweden)

    Nadine N'Dilimabaka

    Full Text Available The factors implicated in the transition from uncomplicated to severe clinical malaria such as pulmonary oedema and cerebral malaria remain unclear. It is known that alterations in vascular integrity due to endothelial cell (EC activation and death occur during severe malaria. In this study, we assessed the ability of different P. falciparum clinical isolates to induce apoptosis in ECs derived from human lung and brain. We observed that induction of EC apoptosis was sensitive to the environmental pH and required direct contact between the parasite and the cell, though it was not correlated to the ability of the parasite to cytoadhere. Moreover, the extent of induced apoptosis in the two EC types varied with the isolate. Analysis of parasite genes transcript led us to propose that the activation of different pathways, such as Plasmodium apoptosis-linked pathogenicity factors (PALPF, PALPF-2, PALPF-5 and PF11_0521, could be implied in EC death. These observations provide an experimental framework to decipher the molecular mechanism implicated in the genesis of severe malaria.

  6. Coexistence of a colon carcinoma with two distinct renal cell carcinomas: a case report

    Directory of Open Access Journals (Sweden)

    Giannopoulos Lambros A

    2011-04-01

    Full Text Available Abstract Introduction We present the case of a patient with two tumors in his left kidney and a synchronous colon cancer. While coexisting tumors have been previously described in the same kidney or the kidney and other organs, or the colon and other organs, to the best of our knowledge no such concurrency of three primary tumors has been reported in the literature to date. Case presentation A 72-year-old man of Greek nationality presenting with pain in the right hypochondrium underwent a series of examinations that revealed gallstones, a tumor in the hepatic flexure of the colon and an additional tumor in the upper pole of the left kidney. He was subjected to a right hemicolectomy, left nephrectomy and cholecystectomy, and his postoperative course was uneventful. Histopathology examinations showed a mucinous colon adenocarcinoma, plus two tumors in the left kidney, a papillary renal cell carcinoma and a chromophobe renal cell carcinoma. Conclusion This case underlines the need to routinely scan patients pre-operatively in order to exclude coexisting tumors, especially asymptomatic renal tumors in patients with colorectal cancer, and additionally to screen concurrent tumors genetically in order to detect putative common genetic alterations.

  7. A Distinct Subpopulation of Bone Marrow Mesenchymal Stem Cells, Muse Cells, Directly Commit to the Replacement of Liver Components.

    Science.gov (United States)

    Katagiri, H; Kushida, Y; Nojima, M; Kuroda, Y; Wakao, S; Ishida, K; Endo, F; Kume, K; Takahara, T; Nitta, H; Tsuda, H; Dezawa, M; Nishizuka, S S

    2016-02-01

    Genotyping graft livers by short tandem repeats after human living-donor liver transplantation (n = 20) revealed the presence of recipient or chimeric genotype cases in hepatocytes (6 of 17, 35.3%), sinusoidal cells (18 of 18, 100%), cholangiocytes (15 of 17, 88.2%) and cells in the periportal areas (7 of 8, 87.5%), suggesting extrahepatic cell involvement in liver regeneration. Regarding extrahepatic origin, bone marrow mesenchymal stem cells (BM-MSCs) have been suggested to contribute to liver regeneration but compose a heterogeneous population. We focused on a more specific subpopulation (1-2% of BM-MSCs), called multilineage-differentiating stress-enduring (Muse) cells, for their ability to differentiate into liver-lineage cells and repair tissue. We generated a physical partial hepatectomy model in immunodeficient mice and injected green fluorescent protein (GFP)-labeled human BM-MSC Muse cells intravenously (n = 20). Immunohistochemistry, fluorescence in situ hybridization and species-specific polymerase chain reaction revealed that they integrated into regenerating areas and expressed liver progenitor markers during the early phase and then differentiated spontaneously into major liver components, including hepatocytes (≈74.3% of GFP-positive integrated Muse cells), cholangiocytes (≈17.7%), sinusoidal endothelial cells (≈2.0%), and Kupffer cells (≈6.0%). In contrast, the remaining cells in the BM-MSCs were not detected in the liver for up to 4 weeks. These results suggest that Muse cells are the predominant population of BM-MSCs that are capable of replacing major liver components during liver regeneration. PMID:26663569

  8. Distinct nuclear gene expression profiles in cells with mtDNA depletion and homoplasmic A3243G mutation

    Energy Technology Data Exchange (ETDEWEB)

    Jahangir Tafrechi, Roshan S. [Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden (Netherlands); Svensson, Peter J. [Department of Toxicogenetics, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden (Netherlands); Department of Oncology, Radiology and Clinical Immunology, University Hospital, 75185 Uppsala (Sweden); Janssen, George M.C. [Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden (Netherlands); Szuhai, Karoly [Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden (Netherlands); Maassen, J. Antonie [Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden (Netherlands); Raap, Anton K. [Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden (Netherlands)]. E-mail: A.K.Raap@lumc.nl

    2005-10-15

    The pathobiochemical pathways determining the wide variability in phenotypic expression of mitochondrial DNA (mtDNA) mutations are not well understood. Most pathogenic mtDNA mutations induce a general defect in mitochondrial respiration and thereby ATP synthesis. Yet phenotypic expression of the different mtDNA mutations shows large variations that are difficult to reconcile with ATP depletion as sole pathogenic factor, implying that additional mechanisms contribute to the phenotype. Here, we use DNA microarrays to identify changes in nuclear gene expression resulting from the presence of the A3243G diabetogenic mutation and from a depletion of mtDNA ({rho}{sup 0} cells). We find that cells respond mildly to these mitochondrial states with both general and specific changes in nuclear gene expression. This observation indicates that cells can sense the status of mtDNA. A number of genes show divergence in expression in {rho}{sup 0} cells compared to cells with the A3243G mutation, such as genes involved in oxidative phosphorylation. As a common response in A3243G and {rho}{sup 0} cells, mRNA levels for extracellular matrix genes are up-regulated, while the mRNA levels of genes involved in ubiquitin-mediated protein degradation and in ribosomal protein synthesis is down-regulated. This reduced expression is reflected at the level of cytosolic protein synthesis in both A3243G and {rho}{sup 0} cells. Our finding that mitochondrial dysfunction caused by different mutations affects nuclear gene expression in partially distinct ways suggests that multiple pathways link mitochondrial function to nuclear gene expression and contribute to the development of the different phenotypes in mitochondrial disease.

  9. Live and inactivated Salmonella enterica serovar Typhimurium stimulate similar but distinct transcriptome profiles in bovine macrophages and dendritic cells.

    Science.gov (United States)

    Jensen, Kirsty; Gallagher, Iain J; Kaliszewska, Anna; Zhang, Chen; Abejide, Oluyinka; Gallagher, Maurice P; Werling, Dirk; Glass, Elizabeth J

    2016-01-01

    Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of gastroenteritis in cattle and humans. Dendritic cells (DC) and macrophages (Mø) are major players in early immunity to Salmonella, and their response could influence the course of infection. Therefore, the global transcriptional response of bovine monocyte-derived DC and Mø to stimulation with live and inactivated S. Typhimurium was compared. Both cell types mount a major response 2 h post infection, with a core common response conserved across cell-type and stimuli. However, three of the most affected pathways; inflammatory response, regulation of transcription and regulation of programmed cell death, exhibited cell-type and stimuli-specific differences. The expression of a subset of genes associated with these pathways was investigated further. The inflammatory response was greater in Mø than DC, in the number of genes and the enhanced expression of common genes, e.g., interleukin (IL) 1B and IL6, while the opposite pattern was observed with interferon gamma. Furthermore, a large proportion of the investigated genes exhibited stimuli-specific differential expression, e.g., Mediterranean fever. Two-thirds of the investigated transcription factors were significantly differentially expressed in response to live and inactivated Salmonella. Therefore the transcriptional responses of bovine DC and Mø during early S. Typhimurium infection are similar but distinct, potentially due to the overall function of these cell-types. The differences in response of the host cell will influence down-stream events, thus impacting on the subsequent immune response generated during the course of the infection. PMID:27000047

  10. Modes of correlated angular motion in live cells across three distinct time scales

    International Nuclear Information System (INIS)

    Particle tracking experiments with high speed digital microscopy yield the positions and trajectories of lipid droplets inside living cells. Angular correlation analysis shows that the lipid droplets have uncorrelated motion at short time scales (τ 10 ms, becomes persistent, indicating directed movement. The motion at all time scales is associated with the lipid droplets being tethered to and driven along the microtubule network. The point at which the angular correlation changes from anti-persistent to persistent motion corresponds to the cross over between sub-diffusive and super diffusive motion, as observed by mean square displacement analysis. Correct analysis of the angular correlations of the detector noise is found to be crucial in modelling the observed phenomena. (paper)

  11. Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle signatures in joint diseases.

    Directory of Open Access Journals (Sweden)

    Bence György

    Full Text Available INTRODUCTION: Microvesicles (MVs, earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. METHODS: In this study, we analyzed synovial fluid (SF samples of patients with osteoarthritis (OA, rheumatoid arthritis (RA and juvenile idiopathic arthritis (JIA. To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM, Nanoparticle Tracking Analysis (NTA and mass spectrometry (MS. For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. RESULTS: EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3(+ and CD8(+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p=0.027 and p=0.009, respectively, after Bonferroni corrections. In JIA, we identified reduced numbers of B cell-derived MVs (p=0.009, after Bonferroni correction. CONCLUSIONS: Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.

  12. Distinctive Patterns of CTNNB1 (β-Catenin) Alterations in Salivary Gland Basal Cell Adenoma and Basal Cell Adenocarcinoma.

    Science.gov (United States)

    Jo, Vickie Y; Sholl, Lynette M; Krane, Jeffrey F

    2016-08-01

    Salivary gland basaloid neoplasms are diagnostically challenging. Limited publications report that some basal cell adenomas harbor CTNNB1 mutations, and nuclear β-catenin expression is prevalent. We evaluated β-catenin expression in basal cell adenomas and adenocarcinomas in comparison with salivary tumors in the differential diagnosis and performed targeted genetic analysis on a subset of cases. β-catenin immunohistochemistry was performed on formalin-fixed, paraffin-embedded whole sections from 73 tumors. Nuclear staining was scored semiquantitatively by extent and intensity. DNA was extracted from 6 formalin-fixed, paraffin-embedded samples (5 basal cell adenomas, 1 basal cell adenocarcinoma) for next-generation sequencing. Nuclear β-catenin staining was present in 18/22 (82%) basal cell adenomas; most were diffuse and strong and predominant in the basal component. Two of 3 basal cell adenocarcinomas were positive (1 moderate focal; 1 moderate multifocal). All adenoid cystic carcinomas (0/20) and pleomorphic adenomas (0/20) were negative; 2/8 epithelial-myoepithelial carcinomas showed focal nuclear staining. Most β-catenin-negative tumors showed diffuse membranous staining in the absence of nuclear staining. Four of 5 basal cell adenomas had exon 3 CTNNB1 mutations, all c.104T>C (p.I35T). Basal cell adenocarcinoma showed a more complex genomic profile, with activating mutations in PIK3CA, biallelic inactivation of NFKBIA, focal CYLD deletion, and without CTNNB1 mutation despite focal β-catenin expression. Nuclear β-catenin expression has moderate sensitivity (82%) for basal cell adenoma but high specificity (96%) in comparison with its morphologic mimics. CTNNB1 mutation was confirmed in most basal cell adenomas tested, and findings in basal cell adenocarcinoma suggest possible tumorigenic mechanisms, including alterations in PI3K and NF-κB pathways and transcriptional regulation. PMID:27259009

  13. The target cell of transformation is distinct from the leukemia stem cell in murine CALM/AF10 leukemia models.

    Science.gov (United States)

    Dutta, S; Krause, A; Vosberg, S; Herold, T; Ksienzyk, B; Quintanilla-Martinez, L; Tizazu, B; Chopra, M; Graf, A; Krebs, S; Blum, H; Greif, P A; Vetter, A; Metzeler, K; Rothenberg-Thurley, M; Schneider, M R; Dahlhoff, M; Spiekermann, K; Zimber-Strobl, U; Wolf, E; Bohlander, S K

    2016-05-01

    The CALM/AF10 fusion gene is found in various hematological malignancies including acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia and malignant lymphoma. We have previously identified the leukemia stem cell (LSC) in a CALM/AF10-driven murine bone marrow transplant AML model as B220+ lymphoid cells with B-cell characteristics. To identify the target cell for leukemic transformation or 'cell of origin of leukemia' (COL) in non-disturbed steady-state hematopoiesis, we inserted the CALM/AF10 fusion gene preceded by a loxP-flanked transcriptional stop cassette into the Rosa26 locus. Vav-Cre-induced panhematopoietic expression of the CALM/AF10 fusion gene led to acute leukemia with a median latency of 12 months. Mice expressing CALM/AF10 in the B-lymphoid compartment using Mb1-Cre or CD19-Cre inducer lines did not develop leukemia. Leukemias had a predominantly myeloid phenotype but showed coexpression of the B-cell marker B220, and had clonal B-cell receptor rearrangements. Using whole-exome sequencing, we identified an average of two to three additional mutations per leukemia, including activating mutations in known oncogenes such as FLT3 and PTPN11. Our results show that the COL for CALM/AF10 leukemia is a stem or early progenitor cell and not a cell of B-cell lineage with a phenotype similar to that of the LSC in CALM/AF10+ leukemia. PMID:26686248

  14. A Distinct Slow-Cycling Cancer Stem-like Subpopulation of Pancreatic Adenocarcinoma Cells is maintained in Vivo

    Energy Technology Data Exchange (ETDEWEB)

    Dembinski, Jennifer L., E-mail: jennifer.dembinski@rr-research.no; Krauss, Stefan [Cellular and Genetic Therapy, Department of Microbiology, Cancer Stem Cell Innovation Center (CAST), Oslo University Hospital, Rikshospitalet, Oslo (Norway)

    2010-11-29

    Pancreatic adenocarcinoma has the worst prognosis of any major malignancy, with <5% of patients surviving five years. This can be contributed to the often late diagnosis, lack of sufficient treatment and metastatic spread. Heterogeneity within tumors is increasingly becoming a focus in cancer research, as novel therapies are required to target the most aggressive subpopulations of cells that are frequently termed cancer stem cells (CSCs). In the current study, we describe the identification of a slow-cycling cancer stem-like population of cells in vivo in BxPC-3 and Panc03.27 xenografts. A distinct slow-cycling label-retaining population of cells (DiI+/SCC) was found both at the edge of tumors, and in small circumscribed areas within the tumors. DiI+/SCC in these areas display an epithelial-to-mesenchymal transition (EMT) fingerprint, including an upregulation of the mesenchymal markers vimentin and N-cadherin and a loss of the epithelial marker E-cadherin. DiI+/SCC also displayed a critical re-localization of beta-catenin from the membrane to the nucleus. Additionally, the DiI+/SCC population was found to express the developmental signaling molecule sonic hedgehog. This study represents a novel step in defining the biological activities of a tumorigenic subpopulation within the heterogeneous tumor microenvironment in vivo. Understanding the interactions and functions of a CSC population within the context of the tumor microenvironment is critical to design targeted therapeutics.

  15. A Distinct Slow-Cycling Cancer Stem-like Subpopulation of Pancreatic Adenocarcinoma Cells is maintained in Vivo

    International Nuclear Information System (INIS)

    Pancreatic adenocarcinoma has the worst prognosis of any major malignancy, with <5% of patients surviving five years. This can be contributed to the often late diagnosis, lack of sufficient treatment and metastatic spread. Heterogeneity within tumors is increasingly becoming a focus in cancer research, as novel therapies are required to target the most aggressive subpopulations of cells that are frequently termed cancer stem cells (CSCs). In the current study, we describe the identification of a slow-cycling cancer stem-like population of cells in vivo in BxPC-3 and Panc03.27 xenografts. A distinct slow-cycling label-retaining population of cells (DiI+/SCC) was found both at the edge of tumors, and in small circumscribed areas within the tumors. DiI+/SCC in these areas display an epithelial-to-mesenchymal transition (EMT) fingerprint, including an upregulation of the mesenchymal markers vimentin and N-cadherin and a loss of the epithelial marker E-cadherin. DiI+/SCC also displayed a critical re-localization of beta-catenin from the membrane to the nucleus. Additionally, the DiI+/SCC population was found to express the developmental signaling molecule sonic hedgehog. This study represents a novel step in defining the biological activities of a tumorigenic subpopulation within the heterogeneous tumor microenvironment in vivo. Understanding the interactions and functions of a CSC population within the context of the tumor microenvironment is critical to design targeted therapeutics

  16. Simultaneous Observation of an Intraband Transition and Distinct Transient Species in the Infrared Region for Perovskite Solar Cells.

    Science.gov (United States)

    Narra, Sudhakar; Chung, Chih-Chun; Diau, Eric Wei-Guang; Shigeto, Shinsuke

    2016-07-01

    Solar cells based on organometal-halide perovskites such as CH3NH3PbI3 have emerged as a promising next-generation photovoltaic system, but the underlying photophysics and photochemistry remain to be established because of the limited availability of methods to implement the simultaneous and direct measurement of various charge carriers and ions that play a crucial role in the operating device. We used nanosecond time-resolved infrared (IR) spectroscopy to investigate, with high molecular specificity, distinct transient species that are formed in perovskite solar cells after photoexcitation. In CH3NH3PbI3 planar-heterojuction solar cells, we simultaneously observed infrared spectral signatures that are associated with an intraband transition of conduction-band electrons, Fano resonance, and the spiro-OMeTAD cation having an exceptionally short lifetime of 1.0 μs (at ∼1485 cm(-1)). The present results show that the time-resolved IR method offers a unique capability to elucidate these important transients in perovskite solar cells and their dynamic interplay in a comprehensive manner. PMID:27302315

  17. Differential progression of structural and functional alterations in distinct retinal ganglion cell types in a mouse model of glaucoma.

    Science.gov (United States)

    Della Santina, Luca; Inman, Denise M; Lupien, Caroline B; Horner, Philip J; Wong, Rachel O L

    2013-10-30

    Intraocular pressure (IOP) elevation is a principal risk factor for glaucoma. Using a microbead injection technique to chronically raise IOP for 15 or 30 d in mice, we identified the early changes in visual response properties of different types of retinal ganglion cells (RGCs) and correlated these changes with neuronal morphology before cell death. Microbead-injected eyes showed reduced optokinetic tracking as well as cell death. In such eyes, multielectrode array recordings revealed that four RGC types show diverse alterations in their light responses upon IOP elevation. OFF-transient RGCs exhibited a more rapid decline in both structural and functional organizations compared with other RGCs. In contrast, although the light-evoked responses of OFF-sustained RGCs were perturbed, the dendritic arbor of this cell type remained intact. ON-transient and ON-sustained RGCs had normal functional receptive field sizes but their spontaneous and light-evoked firing rates were reduced. ON- and OFF-sustained RGCs lost excitatory synapses across an otherwise structurally normal dendritic arbor. Together, our observations indicate that there are changes in spontaneous activity and light-evoked responses in RGCs before detectable dendritic loss. However, when dendrites retract, we found corresponding changes in receptive field center size. Importantly, the effects of IOP elevation are not uniformly manifested in the structure and function of diverse RGC populations, nor are distinct RGC types perturbed within the same time-frame by such a challenge. PMID:24174678

  18. MUCOSAL IMMUNOLOGY. Individual intestinal symbionts induce a distinct population of RORγ⁺ regulatory T cells.

    Science.gov (United States)

    Sefik, Esen; Geva-Zatorsky, Naama; Oh, Sungwhan; Konnikova, Liza; Zemmour, David; McGuire, Abigail Manson; Burzyn, Dalia; Ortiz-Lopez, Adriana; Lobera, Mercedes; Yang, Jianfei; Ghosh, Shomir; Earl, Ashlee; Snapper, Scott B; Jupp, Ray; Kasper, Dennis; Mathis, Diane; Benoist, Christophe

    2015-08-28

    T regulatory cells that express the transcription factor Foxp3 (Foxp3(+) T(regs)) promote tissue homeostasis in several settings. We now report that symbiotic members of the human gut microbiota induce a distinct T(reg) population in the mouse colon, which constrains immuno-inflammatory responses. This induction—which we find to map to a broad, but specific, array of individual bacterial species—requires the transcription factor Rorγ, paradoxically, in that Rorγ is thought to antagonize FoxP3 and to promote T helper 17 (T(H)17) cell differentiation. Rorγ's transcriptional footprint differs in colonic T(regs) and T(H)17 cells and controls important effector molecules. Rorγ, and the T(regs) that express it, contribute substantially to regulating colonic T(H)1/T(H)17 inflammation. Thus, the marked context-specificity of Rorγ results in very different outcomes even in closely related cell types. PMID:26272906

  19. Quinuclidine compounds differently act as agonists of Kenyon cell nicotinic acetylcholine receptors and induced distinct effect on insect ganglionic depolarizations.

    Science.gov (United States)

    Mathé-Allainmat, Monique; Swale, Daniel; Leray, Xavier; Benzidane, Yassine; Lebreton, Jacques; Bloomquist, Jeffrey R; Thany, Steeve H

    2013-12-01

    We have recently demonstrated that a new quinuclidine benzamide compound named LMA10203 acted as an agonist of insect nicotinic acetylcholine receptors. Its specific pharmacological profile on cockroach dorsal unpaired median neurons (DUM) helped to identify alpha-bungarotoxin-insensitive nAChR2 receptors. In the present study, we tested its effect on cockroach Kenyon cells. We found that it induced an inward current demonstrating that it bounds to nicotinic acetylcholine receptors expressed on Kenyon cells. Interestingly, LMA10203-induced currents were completely blocked by the nicotinic antagonist α-bungarotoxin. We suggested that LMA10203 effect occurred through the activation of α-bungarotoxin-sensitive receptors and did not involve α-bungarotoxin-insensitive nAChR2, previously identified in DUM neurons. In addition, we have synthesized two new compounds, LMA10210 and LMA10211, and compared their effects on Kenyon cells. These compounds were members of the 3-quinuclidinyl benzamide or benzoate families. Interestingly, 1 mM LMA10210 was not able to induce an inward current on Kenyon cells compared to LMA10211. Similarly, we did not find any significant effect of LMA10210 on cockroach ganglionic depolarization, whereas these three compounds were able to induce an effect on the central nervous system of the third instar M. domestica larvae. Our data suggested that these three compounds could bind to distinct cockroach nicotinic acetylcholine receptors. PMID:23884575

  20. Distinct Sources of Hematopoietic Progenitors Emerge before HSCs and Provide Functional Blood Cells in the Mammalian Embryo

    Directory of Open Access Journals (Sweden)

    Kathleen E. McGrath

    2015-06-01

    Full Text Available Hematopoietic potential arises in mammalian embryos before adult-repopulating hematopoietic stem cells (HSCs. At embryonic day 9.5 (E9.5, we show the first murine definitive erythro-myeloid progenitors (EMPs have an immunophenotype distinct from primitive hematopoietic progenitors, maturing megakaryocytes and macrophages, and rare B cell potential. EMPs emerge in the yolk sac with erythroid and broad myeloid, but not lymphoid, potential. EMPs migrate to the fetal liver and rapidly differentiate, including production of circulating neutrophils by E11.5. Although the surface markers, transcription factors, and lineage potential associated with EMPs overlap with those found in adult definitive hematopoiesis, they are present in unique combinations or proportions that result in a specialized definitive embryonic progenitor. Furthermore, we find that embryonic stem cell (ESC-derived hematopoiesis recapitulates early yolk sac hematopoiesis, including primitive, EMP, and rare B cell potential. EMPs do not have long-term potential when transplanted in immunocompromised adults, but they can provide transient adult-like RBC reconstitution.

  1. Distinctive effects of eicosapentaenoic and docosahexaenoic acids in regulating neural stem cell fate are mediated via endocannabinoid signalling pathways.

    Science.gov (United States)

    Dyall, S C; Mandhair, H K; Fincham, R E A; Kerr, D M; Roche, M; Molina-Holgado, F

    2016-08-01

    Emerging evidence suggests a complex interplay between the endocannabinoid system, omega-3 fatty acids and the immune system in the promotion of brain self-repair. However, it is unknown if all omega-3 fatty acids elicit similar effects on adult neurogenesis and if such effects are mediated or regulated by interactions with the endocannabinoid system. This study investigated the effects of DHA and EPA on neural stem cell (NSC) fate and the role of the endocannabinoid signalling pathways in these effects. EPA, but not DHA, significantly increased proliferation of NSCs compared to controls, an effect associated with enhanced levels of the endocannabinoid 2-arachidonylglycerol (2-AG) and p-p38 MAPK, effects attenuated by pre-treatment with CB1 (AM251) or CB2 (AM630) receptor antagonists. Furthermore, in NSCs derived from IL-1β deficient mice, EPA significantly decreased proliferation and p-p38 MAPK levels compared to controls, suggesting a key role for IL-1β signalling in the effects observed. Although DHA similarly increased 2-AG levels in wild-type NSCs, there was no concomitant increase in proliferation or p-p38 MAPK activity. In addition, in NSCs from IL-1β deficient mice, DHA significantly increased proliferation without effects on p-P38 MAPK, suggesting effects of DHA are mediated via alternative signalling pathways. These results provide crucial new insights into the divergent effects of EPA and DHA in regulating NSC proliferation and the pathways involved, and highlight the therapeutic potential of their interplay with endocannabinoid signalling in brain repair. PMID:27044662

  2. Distinct roles of Shh and Fgf signaling in regulating cell proliferation during zebrafish pectoral fin development

    Directory of Open Access Journals (Sweden)

    Neumann Carl J

    2008-09-01

    Full Text Available Abstract Background Cell proliferation in multicellular organisms must be coordinated with pattern formation. The major signaling pathways directing pattern formation in the vertebrate limb are well characterized, and we have therefore chosen this organ to examine the interaction between proliferation and patterning. Two important signals for limb development are members of the Hedgehog (Hh and Fibroblast Growth Factor (Fgf families of secreted signaling proteins. Sonic hedgehog (Shh directs pattern formation along the anterior/posterior axis of the limb, whereas several Fgfs in combination direct pattern formation along the proximal/distal axis of the limb. Results We used the genetic and pharmacological amenability of the zebrafish model system to dissect the relative importance of Shh and Fgf signaling in regulating proliferation during development of the pectoral fin buds. In zebrafish mutants disrupting the shh gene, proliferation in the pectoral fin buds is initially normal, but later is strongly reduced. Correlating with this reduction, Fgf signaling is normal at early stages, but is later lost in shh mutants. Furthermore, pharmacological inhibition of Hh signaling for short periods has little effect on either Fgf signaling, or on expression of G1- and S-phase cell-cycle genes, whereas long periods of inhibition lead to the downregulation of both. In contrast, even short periods of pharmacological inhibition of Fgf signaling lead to strong disruption of proliferation in the fin buds, without affecting Shh signaling. To directly test the ability of Fgf signaling to regulate proliferation in the absence of Shh signaling, we implanted beads soaked with Fgf protein into shh mutant fin buds. We find that Fgf-soaked beads rescue proliferation in the pectoral find buds of shh mutants, indicating that Fgf signaling is sufficient to direct proliferation in zebrafish fin buds in the absence of Shh. Conclusion Previous studies have shown that both

  3. Dendritic Cells and Monocytes with Distinct Inflammatory Responses Reside in Lung Mucosa of Healthy Humans.

    Science.gov (United States)

    Baharom, Faezzah; Thomas, Saskia; Rankin, Gregory; Lepzien, Rico; Pourazar, Jamshid; Behndig, Annelie F; Ahlm, Clas; Blomberg, Anders; Smed-Sörensen, Anna

    2016-06-01

    Every breath we take contains potentially harmful pathogens or allergens. Dendritic cells (DCs), monocytes, and macrophages are essential in maintaining a delicate balance of initiating immunity without causing collateral damage to the lungs because of an exaggerated inflammatory response. To document the diversity of lung mononuclear phagocytes at steady-state, we performed bronchoscopies on 20 healthy subjects, sampling the proximal and distal airways (bronchial wash and bronchoalveolar lavage, respectively), as well as mucosal tissue (endobronchial biopsies). In addition to a substantial population of alveolar macrophages, we identified subpopulations of monocytes, myeloid DCs (MDCs), and plasmacytoid DCs in the lung mucosa. Intermediate monocytes and MDCs were highly frequent in the airways compared with peripheral blood. Strikingly, the density of mononuclear phagocytes increased upon descending the airways. Monocytes from blood and airways produced 10-fold more proinflammatory cytokines than MDCs upon ex vivo stimulation. However, airway monocytes were less inflammatory than blood monocytes, suggesting a more tolerant nature. The findings of this study establish how to identify human lung mononuclear phagocytes and how they function in normal conditions, so that dysregulations in patients with respiratory diseases can be detected to elucidate their contribution to immunity or pathogenesis. PMID:27183618

  4. Aldehydes with high and low toxicities inactivate cells by damaging distinct cellular targets.

    Science.gov (United States)

    Xie, Ming-Zhang; Shoulkamy, Mahmoud I; Salem, Amir M H; Oba, Shunya; Goda, Mizuki; Nakano, Toshiaki; Ide, Hiroshi

    2016-04-01

    Aldehydes are genotoxic and cytotoxic molecules and have received considerable attention for their associations with the pathogenesis of various human diseases. In addition, exposure to anthropogenic aldehydes increases human health risks. The general mechanism of aldehyde toxicity involves adduct formation with biomolecules such as DNA and proteins. Although the genotoxic effects of aldehydes such as mutations and chromosomal aberrations are directly related to DNA damage, the role of DNA damage in the cytotoxic effects of aldehydes is poorly understood because concurrent protein damage by aldehydes has similar effects. In this study, we have analysed how saturated and α,β-unsaturated aldehydes exert cytotoxic effects through DNA and protein damage. Interestingly, DNA repair is essential for alleviating the cytotoxic effect of weakly toxic aldehydes such as saturated aldehydes but not highly toxic aldehydes such as long α,β-unsaturated aldehydes. Thus, highly toxic aldehydes inactivate cells exclusively by protein damage. Our data suggest that DNA interstrand crosslinks, but not DNA-protein crosslinks and DNA double-strand breaks, are the critical cytotoxic DNA damage induced by aldehydes. Further, we show that the depletion of intracellular glutathione and the oxidation of thioredoxin 1 partially account for the DNA damage-independent cytotoxicity of aldehydes. On the basis of these findings, we have proposed a mechanistic model of aldehyde cytotoxicity mediated by DNA and protein damage. PMID:26917342

  5. Metformin and rapamycin have distinct effects on the AKT pathway and proliferation in breast cancer cells.

    Science.gov (United States)

    Zakikhani, Mahvash; Blouin, Marie-José; Piura, Esther; Pollak, Michael N

    2010-08-01

    Rapamycin and its analogues inhibit mTOR, which leads to decreased protein synthesis and decreased cancer cell proliferation in many experimental systems. Adenosine 5'- monophosphate-activated protein kinase (AMPK) activators such as metformin have similar actions, in keeping with the TSC2/1 pathway linking activation of AMPK to inhibition of mTOR. As mTOR inhibition by rapamycin is associated with attenuation of negative feedback to IRS-1, rapamycin is known to increase activation of AKT, which may reduce its anti-neoplastic activity. We observed that metformin exposure decreases AKT activation, an action opposite to that of rapamycin. We show that metformin (but not rapamycin) exposure leads to increased phosphorylation of IRS-1 at Ser(789), a site previously reported to inhibit downstream signaling and to be an AMPK substrate phosphorylated under conditions of cellular energy depletion. siRNA methods confirmed that reduction of AMPK levels attenuates both the IRS-1 Ser(789) phosphorylation and the inhibition of AKT activation associated with metformin exposure. Although both rapamycin and metformin inhibit mTOR (the former directly and the latter through AMPK signaling), our results demonstrate previously unrecognized differences between these agents. The data are consistent with the observation that maximal induction of apoptosis and inhibition of proliferation are greater for metformin than rapamycin. PMID:20135346

  6. Distinct functional responses to stressors of bone marrow derived dendritic cells from diverse inbred chicken lines.

    Science.gov (United States)

    Van Goor, Angelica; Slawinska, Anna; Schmidt, Carl J; Lamont, Susan J

    2016-10-01

    Differences in responses of chicken bone marrow derived dendritic cells (BMDC) to in vitro treatment with lipopolysaccharide (LPS), heat, and LPS + heat were identified. The Fayoumi is more disease resistant and heat tolerant than the Leghorn line. Nitric Oxide (NO) production, phagocytic ability, MHC II surface expression and mRNA expression were measured. NO was induced in BMDC from both lines in response to LPS and LPS + heat stimulation; Fayoumi produced more NO with LPS treatment. Fayoumi had higher phagocytic ability and MHC II surface expression. Gene expression for the heat-related genes BAG3, HSP25, HSPA2, and HSPH1 was strongly induced with heat and few differences existed between lines. Expression for the immune-related genes CCL4, CCL5, CD40, GM-CSF, IFN-γ, IL-10, IL-12β, IL-1β, IL-6, IL-8, and iNOS was highly induced in response to LPS and different between lines. This research contributes to the sparse knowledge of genetic differences in chicken BMDC biology and function. PMID:27238770

  7. Bovine mammary dendritic cells: a heterogeneous population, distinct from macrophages and similar in phenotype to afferent lymph veiled cells.

    Science.gov (United States)

    Maxymiv, Nicolas G; Bharathan, Mini; Mullarky, Isis K

    2012-01-01

    Dendritic cells (DC) are a heterogeneous population of professional antigen presenting cells and are potent stimulators of naïve T-cells. However, there is little previous research describing DC in bovine mammary tissue, primarily because of the difficulty distinguishing these cells from macrophages, which possess a similar phenotype. Using immunohistofluorescence and a combination of markers (MHC-II, CD205, CD11c), DC were localized in the bovine mammary gland and supramammary lymph node. In mammary tissue DC were found within the alveolar epithelium and within the intralobular connective tissue. In the lymph node DC were found on the periphery of B-cell areas, in the cortex, and among T-cells in the paracortex and medulla. DC in mammary parenchyma and supramammary lymph nodes were quantified and further characterized using flow cytometry. DC were CD11c(hi), CD14(lo) cells that expressed MHC-II and CD205. DC could be distinguished from macrophages based on their low CD14 expression. This research provides a better understanding of mammary gland immunology, while potentially aiding in the targeting of antigens to mucosal DC for vaccine development. PMID:22019401

  8. A prospective study on contrast-enhanced magnetic resonance imaging of testicular lesions: distinctive features of Leydig cell tumours

    Energy Technology Data Exchange (ETDEWEB)

    Manganaro, Lucia; Vinci, Valeria; Saldari, Matteo; Bernardo, Silvia; Cantisani, Vito; Catalano, Carlo [Sapienza University of Rome, Department of Radiology, Rome (Italy); Pozza, Carlotta; Gianfrilli, Daniele; Pofi, Riccardo; Lenzi, Andrea; Isidori, Andrea M. [Sapienza University of Rome, Department of Experimental Medicine, Rome (Italy); Scialpi, Michele [Perugia University, S. Maria della Misericordia Hospital, Department of Surgical and Biomedical Sciences, Division of Radiology 2, Perugia (Italy)

    2015-12-15

    Up to 20 % of incidentally found testicular lesions are benign Leydig cell tumours (LCTs). This study evaluates the role of contrast-enhanced magnetic resonance imaging (MRI) in the identification of LCTs in a large prospective cohort study. We enrolled 44 consecutive patients with at least one solid non-palpable testicular lesion who underwent scrotal MRI. Margins of the lesions, signal intensity and pattern of wash-in and wash-out were analysed by two radiologists. The frequency distribution of malignant and benign MRI features in the different groups was compared by using the chi-squared or Fisher's exact test. Sensitivity, specificity, positive and negative predictive value, and diagnostic accuracy were calculated. The sensitivity of scrotal MRI to diagnose LCTs was 89.47 % with 95.65 % specificity; sensitivity for malignant lesions was 95.65 % with 80.95 % specificity. A markedly hypointense signal on T2-WI, rapid and marked wash-in followed by a prolonged washout were distinctive features significantly associated with LCTs. Malignant lesions were significantly associated with blurred margins, weak hypointense signal on T2-WI,and weak and progressive wash-in. The overall diagnostic accuracy was 93 %. LCTs have distinctive contrast-enhanced MRI features that allow the differential diagnosis of incidental testicular lesions. (orig.)

  9. Differentiated neuroprogenitor cells incubated with human or canine adenovirus, or lentiviral vectors have distinct transcriptome profiles.

    Directory of Open Access Journals (Sweden)

    Stefania Piersanti

    Full Text Available Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD human (HAd and canine (CAV-2 adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS. With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways--but in opposite directions--suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer.

  10. Distinct mechanisms account for acquired von Willebrand syndrome in plasma cell dyscrasias.

    Science.gov (United States)

    Dicke, Christina; Schneppenheim, Sonja; Holstein, Katharina; Spath, Brigitte; Bokemeyer, Carsten; Dittmer, Rita; Budde, Ulrich; Langer, Florian

    2016-05-01

    Acquired von Willebrand syndrome (AVWS) is a rare bleeding disorder that may cause life-threatening hemorrhages in patients with plasma cell dyscrasias (PCDs). Early diagnosis and treatment require a thorough understanding of its underlying pathophysiology. Two patients with IgG MGUS presented with dramatically decreased plasma von Willebrand factor (VWF) and a severe type-1 pattern on multimer analysis. A prompt response to intravenous immunoglobulins (IVIG), but not to VWF/FVIII, was consistent with accelerated immunologic clearance of plasma VWF. Another IgG MGUS patient showed a type-2 pattern and a less pronounced response to IVIG, suggesting that additional mechanism(s) contributed to AVWS evolution. In a patient with Waldenström's macroglobulinemia and severe depletion of plasma VWF, multimer analysis indicated association of the IgM paraprotein with VWF before, but not after plasmapheresis, resulting in destruction of the agarose gel and a characteristically distorted band structure of VWF multimers. A type-2 pattern with highly abnormal VWF triplets and laboratory evidence of excessive fibrinolytic activity suggested that plasmin-mediated VWF degradation contributed to AVWS in a patient with multiple myeloma (MM) and AL amyloidosis. Finally, in a patient with IgG MM, maximally prolonged PFA-100® closure times and a specific defect in ristocetin-induced platelet agglutination, both of which resolved after remission induction, indicated interference of the paraprotein with VWF binding to platelet GPIb. Importantly, in none of the six patients, circulating autoantibodies to VWF were detected by a specific in-house ELISA. In summary, when evaluating PCD patients with severe bleeding symptoms, AVWS due to various pathogenic mechanisms should be considered. PMID:27040683

  11. A 3D in vitro model reveals differences in the astrocyte response elicited by potential stem cell therapies for CNS injury

    OpenAIRE

    East, Emma; Johns, Noemie; Georgiou, Melanie; Golding, Jon; Loughlin, Jane; Kingham, Paul; Phillips, James

    2013-01-01

    Aim: This study aimed to develop a 3D culture model to test the extent to which transplanted stem cells modulate astrocyte reactivity, where exacerbated glial cell activation could be detrimental to CNS repair success. Materials & methods: The reactivity of rat astrocytes to bone marrow mesenchymal stem cells, neural crest stem cells (NCSCs) and differentiated adipose-derived stem cells was assessed after 5 days. Schwann cells were used as a positive control. Results: NCSCs and differ...

  12. Molecular Stratification of Clear Cell Renal Cell Carcinoma by Consensus Clustering Reveals Distinct Subtypes and Survival Patterns

    OpenAIRE

    Brannon, A. Rose; Reddy, Anupama; Seiler, Michael; Arreola, Alexandra; Moore, Dominic T.; Pruthi, Raj S.; Wallen, Eric M.; Nielsen, Matthew E; Liu, Huiqing; Nathanson, Katherine L.; Ljungberg, Börje; Zhao, Hongjuan; BROOKS, JAMES D.; Ganesan, Shridar; Bhanot, Gyan

    2010-01-01

    Clear cell renal cell carcinoma (ccRCC) is the predominant RCC subtype, but even within this classification, the natural history is heterogeneous and difficult to predict. A sophisticated understanding of the molecular features most discriminatory for the underlying tumor heterogeneity should be predicated on identifiable and biologically meaningful patterns of gene expression. Gene expression microarray data were analyzed using software that implements iterative unsupervised consensus cluste...

  13. Different concentrations of berberine result in distinct cellular localization patterns and cell cycle effects in a melanoma cell line

    OpenAIRE

    Serafim, Teresa; Oliveira, Paulo; Sardao, Vilma; Perkins, Ed; Parke, Donna; Holy, Jon

    2008-01-01

    Abstract Purpose Natural products represent a rich reservoir of potential small molecule inhibitors exhibiting antiproliferative and tumoricidal properties. An example is the isoquinoline alkaloid berberine, which is found in plants such as goldenseal (Hydrastis canadensis). Studies have shown that berberine is able to trigger apoptosis in different malignant cell lines, and can also lead to cell cycle arrest at sub-apoptotic doses. A particularly interesting feature of berberine is the fact...

  14. Giant-cell arteritis without cranial manifestations: Working diagnosis of a distinct disease pattern.

    Science.gov (United States)

    de Boysson, Hubert; Lambert, Marc; Liozon, Eric; Boutemy, Jonathan; Maigné, Gwénola; Ollivier, Yann; Ly, Kim; Manrique, Alain; Bienvenu, Boris; Aouba, Achille

    2016-06-01

    Diagnosis of giant-cell arteritis (GCA) is challenging in the absence of cardinal cranial symptoms/signs. We aimed to describe the clinical presentation, diagnostic process, and disease course of GCA patients without cranial symptoms, and to compare them to those of patients with typical cranial presentation. In this retrospective multicenter study, we enrolled patients with GCA who satisfied at least 3 of the 5 American College of Rheumatology criteria for GCA, or 2 criteria associated with contributory vascular biopsy other than temporal artery biopsy or with demonstration of large-vessel involvement; underwent iconographic evaluation of large arterial vessels (aortic CT scan or a positron emission tomography with F-fluorodeoxyglucose combined with computed tomography (FDG-PET/CT) scan or cardiac echography combined with a large-vessel Doppler) at diagnosis. We divided the cohort into 2 groups, distinguishing between patients without cranial symptoms/signs (i.e., headaches, clinical temporal artery anomaly, jaw claudication, ophthalmologic symptoms) and those with cranial symptoms/signs. In the entire cohort of 143 patients, all of whom underwent vascular biopsy and vascular imaging, we detected 31 (22%) patients with no cranial symptoms/signs. In the latter, diagnosis was biopsy proven in an arterial sample in 23 cases (74% of patients, on a temporal site in 20 cases and on an extratemporal site in 3). One-third of these 31 patients displayed extracranial symptoms/signs whereas the remaining two-thirds presented only with constitutional symptoms and/or inflammatory laboratory test results. Compared to the 112 patients with cardinal cranial clinical symptoms/signs, patients without cranial manifestations displayed lower levels of inflammatory laboratory parameters (C-reactive level: 68 [9-250] mg/L vs 120 [3-120] mg/L; P < 0.01), highest rate of aorta and aortic branch involvement identified (19/31 (61%) vs 42/112 (38%); P = 0.02) and also a lower rate of

  15. Distinct cellular properties of oncogenic KIT receptor tyrosine kinase mutants enable alternative courses of cancer cell inhibition.

    Science.gov (United States)

    Shi, Xiarong; Sousa, Leiliane P; Mandel-Bausch, Elizabeth M; Tome, Francisco; Reshetnyak, Andrey V; Hadari, Yaron; Schlessinger, Joseph; Lax, Irit

    2016-08-16

    Large genomic sequencing analysis as part of precision medicine efforts revealed numerous activating mutations in receptor tyrosine kinases, including KIT. Unfortunately, a single approach is not effective for inhibiting cancer cells or treating cancers driven by all known oncogenic KIT mutants. Here, we show that each of the six major KIT oncogenic mutants exhibits different enzymatic, cellular, and dynamic properties and responds distinctly to different KIT inhibitors. One class of KIT mutants responded well to anti-KIT antibody treatment alone or in combination with a low dose of tyrosine kinase inhibitors (TKIs). A second class of KIT mutants, including a mutant resistant to imatinib treatment, responded well to a combination of TKI with anti-KIT antibodies or to anti-KIT toxin conjugates, respectively. We conclude that the preferred choice of precision medicine treatments for cancers driven by activated KIT and other RTKs may rely on clear understanding of the dynamic properties of oncogenic mutants. PMID:27482095

  16. Integrative genome-wide expression profiling identifies three distinct molecular subgroups of renal cell carcinoma with different patient outcome

    International Nuclear Information System (INIS)

    Renal cell carcinoma (RCC) is characterized by a number of diverse molecular aberrations that differ among individuals. Recent approaches to molecularly classify RCC were based on clinical, pathological as well as on single molecular parameters. As a consequence, gene expression patterns reflecting the sum of genetic aberrations in individual tumors may not have been recognized. In an attempt to uncover such molecular features in RCC, we used a novel, unbiased and integrative approach. We integrated gene expression data from 97 primary RCC of different pathologic parameters, 15 RCC metastases as well as 34 cancer cell lines for two-way nonsupervised hierarchical clustering using gene groups suggested by the PANTHER Classification System. We depicted the genomic landscape of the resulted tumor groups by means of Single Nuclear Polymorphism (SNP) technology. Finally, the achieved results were immunohistochemically analyzed using a tissue microarray (TMA) composed of 254 RCC. We found robust, genome wide expression signatures, which split RCC into three distinct molecular subgroups. These groups remained stable even if randomly selected gene sets were clustered. Notably, the pattern obtained from RCC cell lines was clearly distinguishable from that of primary tumors. SNP array analysis demonstrated differing frequencies of chromosomal copy number alterations among RCC subgroups. TMA analysis with group-specific markers showed a prognostic significance of the different groups. We propose the existence of characteristic and histologically independent genome-wide expression outputs in RCC with potential biological and clinical relevance

  17. Distinct Signaling Pathways After Higher or Lower Doses of Radiation in Three Closely Related Human Lymphoblast Cell Lines

    International Nuclear Information System (INIS)

    Purpose: The tumor suppressor p53 plays an essential role in cellular responses to DNA damage caused by ionizing radiation; therefore, this study aims to further explore the role that p53 plays at different doses of radiation. Materials and Methods: The global cellular responses to higher-dose (10 Gy) and lower dose (iso-survival dose, i.e., the respective D0 levels) radiation were analyzed using microarrays in three human lymphoblast cell lines with different p53 status: TK6 (wild-type p53), NH32 (p53-null), and WTK1 (mutant p53). Total RNAs were extracted from cells harvested at 0, 1, 3, 6, 9, and 24 h after higher and lower dose radiation exposures. Template-based clustering, hierarchical clustering, and principle component analysis were applied to examine the transcriptional profiles. Results: Differential expression profiles between 10 Gy and iso-survival radiation in cells with different p53 status were observed. Moreover, distinct gene expression patterns were exhibited among these three cells after 10 Gy radiation treatment, but similar transcriptional responses were observed in TK6 and NH32 cells treated with iso-survival radiation. Conclusions: After 10 Gy radiation exposure, the p53 signaling pathway played an important role in TK6, whereas the NFkB signaling pathway appeared to replace the role of p53 in WTK1. In contrast, after iso-survival radiation treatment, E2F4 seemed to play a dominant role independent of p53 status. This study dissected the impacts of p53, NFkB and E2F4 in response to higher or lower doses of γ-irradiation.

  18. Nifedipine, a calcium channel blocker, inhibits advanced glycation end product (AGE)-elicited mesangial cell damage by suppressing AGE receptor (RAGE) expression via peroxisome proliferator-activated receptor-gamma activation

    International Nuclear Information System (INIS)

    The interaction between advanced glycation end products (AGE) and their receptor RAGE mediates the progressive alteration in renal architecture and loss of renal function in diabetic nephropathy. Oxidative stress generation and inflammation also play a central role in diabetic nephropathy. This study investigated whether and how nifedipine, a calcium channel blocker (CCB), blocked the AGE-elicited mesangial cell damage in vitro. Nifedipine, but not amlodipine, a control CCB, down-regulated RAGE mRNA levels and subsequently reduced reactive oxygen species (ROS) generation in AGE-exposed mesangial cells. AGE increased mRNA levels of vascular cell adhesion molecule-1 (VCAM-1) and induced monocyte chemoattractant protein-1 (MCP-1) production in mesangial cells, both of which were prevented by the treatment with nifedipine, but not amlodipine. The beneficial effects of nifedipine on AGE-exposed mesangial cells were blocked by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-γ (PPAR-γ). Although nifedipine did not affect expression levels of PPAR-γ, it increased the PPAR-γ transcriptional activity in mesangial cells. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-inflammatory agent against AGE by suppressing RAGE expression in cultured mesangial cells via PPAR-γ activation.

  19. Nifedipine, a calcium channel blocker, inhibits advanced glycation end product (AGE)-elicited mesangial cell damage by suppressing AGE receptor (RAGE) expression via peroxisome proliferator-activated receptor-gamma activation

    Energy Technology Data Exchange (ETDEWEB)

    Matsui, Takanori [Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011 (Japan); Yamagishi, Sho-ichi, E-mail: shoichi@med.kurume-u.ac.jp [Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011 (Japan); Takeuchi, Masayoshi [Department of Pathophysiological Science, Faculty of Pharmaceutical Science, Hokuriku University, Kanazawa (Japan); Ueda, Seiji; Fukami, Kei; Okuda, Seiya [Department of Medicine, Kurume University School of Medicine, Kurume (Japan)

    2009-07-24

    The interaction between advanced glycation end products (AGE) and their receptor RAGE mediates the progressive alteration in renal architecture and loss of renal function in diabetic nephropathy. Oxidative stress generation and inflammation also play a central role in diabetic nephropathy. This study investigated whether and how nifedipine, a calcium channel blocker (CCB), blocked the AGE-elicited mesangial cell damage in vitro. Nifedipine, but not amlodipine, a control CCB, down-regulated RAGE mRNA levels and subsequently reduced reactive oxygen species (ROS) generation in AGE-exposed mesangial cells. AGE increased mRNA levels of vascular cell adhesion molecule-1 (VCAM-1) and induced monocyte chemoattractant protein-1 (MCP-1) production in mesangial cells, both of which were prevented by the treatment with nifedipine, but not amlodipine. The beneficial effects of nifedipine on AGE-exposed mesangial cells were blocked by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}). Although nifedipine did not affect expression levels of PPAR-{gamma}, it increased the PPAR-{gamma} transcriptional activity in mesangial cells. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-inflammatory agent against AGE by suppressing RAGE expression in cultured mesangial cells via PPAR-{gamma} activation.

  20. Recursive Distinctioning

    CERN Document Server

    Isaacson, Joel

    2016-01-01

    Recursive distinctioning (RD) is a name coined by Joel Isaacson in his original patent document describing how fundamental patterns of process arise from the systematic application of operations of distinction and description upon themselves. Recursive distinctioning means just what it says. A pattern of distinctions is given in a space based on a graphical structure (such as a line of print or a planar lattice or given graph). Each node of the graph is occupied by a letter from some arbitrary alphabet. A specialized alphabet is given that can indicate distinctions about neighbors of a given node. The neighbors of a node are all nodes that are connected to the given node by edges in the graph. The letters in the specialized alphabet (call it SA) are used to describe the states of the letters in the given graph and at each stage in the recursion, letters in SA are written at all nodes in the graph, describing its previous state. The recursive structure that results from the iteration of descriptions is called ...

  1. Distinct accessory cell requirements define two types of rat T cell hybridomas specific for unique determinants in the encephalitogenic 68-86 region of myelin basic protein

    International Nuclear Information System (INIS)

    Six clonotypically unique T cell hybridomas from Lewis rats were used to study accessory cell activities required for class II MHC restricted T cell responses to the 68-86 encephalitogenic sequence of myelin basic protein (MBP). T cell hybrids which were cultured with GP68-86 68-86 sequence of guinea pig MBP (GPMBP) and naive splenocytes (SPL) were induced to produce IL-2 as measured by the CTLL indicator cell line. The hybrids were categorized into two subsets (designated THYB-1 and THYB-2), because two distinct subset-specific pathways of communication between accessory cells and T cells were involved in GPMBP-induced IL-2 production. These pathways were distinguished by the following six observations. First, when the duration of a pulse of SPL with GPMBP was lengthened from 1 to 4 h, these SPL lost their ability to induce IL-2 production by THYB-2 hybrids yet nevertheless retained full stimulatory activity for THYB-1 hybrids. Second, paraformaldehyde fixation of GPMBP-pulsed SPL abrogated an activity necessary for Ag-induced IL-2 production by THYB-2 hybrids. These fixed SPL were nevertheless able to stimulate THYB-1 hybrids, albeit to a lesser extent than viable unfixed SPL. Third, the addition of either cycloheximide, cytochalasin B, or 2-deoxyglucose to an Ag pulse of SPL with GPMBP dramatically inhibited the subsequent responses of THYB-2 hybrids yet had little or no effect upon the reactivity of THYB-1 hybrids. Fourth, thymocytes lacked necessary activities for GPMBP evoked IL-2 production by THYB-2 hybrids yet strongly promoted THYB-1 hybrid responses. Fifth, exposure of SPL to as little as 500 rad of gamma-irradiation markedly attenuated THYB-2 hybrid response to GPMBP but did not affect THYB-1 responses. Sixth, anti-GPMBP responses by THYB-2 hybrids were observed only in the presence of both radioresistant adherent SPL and a distinct population of radiosensitive nonadherent SPL

  2. Primary cutaneous peripheral T-cell lymphoma, unspecified with an indolent clinical course: a distinct peripheral T-cell lymphoma?

    LENUS (Irish Health Repository)

    Ryan, A J A

    2012-02-01

    Primary cutaneous peripheral T-cell lymphomas (PTL), unspecified, are rare lymphomas, with a poor prognosis. They grow and disseminate rapidly, leading to widespread disease. We report a case of PTL, unspecified occurring on the nose. Despite its aggressive histology, this tumour behaved indolently. It is remarkably similar, clinically and histologically, to four recently described cases that occurred on the ear.

  3. Two distinct pathways responsible for the loading of CENP-A to centromeres in the fission yeast cell cycle.

    Science.gov (United States)

    Takahashi, Kohta; Takayama, Yuko; Masuda, Fumie; Kobayashi, Yasuyo; Saitoh, Shigeaki

    2005-03-29

    CENP-A is a centromere-specific histone H3 variant that is- essential for faithful chromosome segregation in all eukaryotes thus far investigated. We genetically identified two factors, Ams2 and Mis6, each of which is required for the correct centromere localization of SpCENP-A (Cnp1), the fission yeast homologue of CENP-A. Ams2 is a cell-cycle-regulated GATA factor that localizes on the nuclear chromatin, including on centromeres, during the S phase. Ams2 may be responsible for the replication-coupled loading of SpCENP-A by facilitating nucleosomal formation during the S phase. Consistently, overproduction of histone H4, but not that of H3, suppressed the defect of SpCENP-A localization in Ams2-deficient cells. We demonstrated the existence of at least two distinct phases for SpCENP-A loading during the cell cycle: the S phase and the late-G2 phase. Ectopically induced SpCENP-A was efficiently loaded onto the centromeres in G2-arrested cells, indicating that SpCENP-A probably undergoes replication-uncoupled loading after the completion of S phase. This G2 loading pathway of SpCENP-A may require Mis6, a constitutive centromere-binding protein that is also implicated in the Mad2-dependent spindle attachment checkpoint response. Here, we discuss the functional relationship between the flexible loading mechanism of CENP-A and the plasticity of centromere chromatin formation in fission yeast. PMID:15897182

  4. A synthetic lymph node containing inactivated Treponema pallidum cells elicits strong, antigen-specific humoral and cellular immune responses in mice.

    Science.gov (United States)

    Stamm, Lola V; Drapp, Rebecca L

    2014-02-01

    The goal of this study was to investigate the use of a synthetic lymph node (SLN) for delivery of Treponema pallidum (Tp) antigens. Immune responses of C57BL/6 mice were analyzed at 4, 8, and 12 weeks after SLN implantation. Group 1 mice received SLN with no antigen; Group 2, SLN with formalin-inactivated Tp (f-Tp); and Group 3, SLN with f-Tp plus a CpG oligodeoxynucleotide. When tested by ELISA, sera from Group 2 and Group 3 mice showed stronger IgG antibody reactivity than sera from Group 1 mice to sonicates of f-Tp or untreated Tp, but not to sonicate of normal rabbit testicular extract at all times. The IgG1 level was higher than IgG2c level for Group 2 mice at all times and for Group 3 mice at 4 and 8 weeks. IgG1 and IgG2c levels were nearly equivalent for Group 3 mice at 12 weeks. Immunoblotting showed that IgG from Group 2 and Group 3 mice recognized several Tp proteins at all times. Supernatants of splenocytes from Group 2 and Group 3 mice contained significantly more IFNγ than those from Group 1 mice after stimulation with f-Tp at all times. A significant level of IL-4 was not detected in any supernatants. These data show that strong humoral and cellular immune responses to Tp can be elicited via a SLN. PMID:24106125

  5. Distinct effects of nuclear membrane localization on gene transcription silencing in Drosophila S2 cells and germ cells

    Institute of Scientific and Technical Information of China (English)

    Lu Sui; Yanhong Yang

    2011-01-01

    Nuclear envelope proteins have important roles in chromatin organization and signal-dependent transcriptional regulation. A previous study reported that the inner nuclear membrane protein, Otefin (Ote), was essential for germline stem cell (GSC) maintenance via interaction with Smad complex. The interaction of Otc with the Smad complex recruits the bam locus to the nuclear periphery and subsequently results in bam transcriptional silencing, revealing that nuclear peripheral localization is essential for bam gene regulation. However, it remains unknown whether the nuclear peripheral localization is sufficient for bam silencing. To address this issue, we have established a tethering system, in which the Gal4 DNA binding domain (DBD) of the Flag:Gal4 DBD:Ote △ LEM fusion protein physically interacts with the Gal4 binding sites upstream of bamP-gfp to artificially recruit the reporter gene gfp to the nuclear membrane. Our data demonstrated that the nuclear peripheral localization seemed to affect the expression of the target naked gene in S2 cells. By contrast, in Drosophila germ cells, the nuclear membrane localization was not sufficient for gene silencing.

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

    Science.gov (United States)

    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

  7. Novel Human Embryonic Stem Cell Regulators Identified by Conserved and Distinct CpG Island Methylation State.

    Directory of Open Access Journals (Sweden)

    Steve Pells

    Full Text Available Human embryonic stem cells (hESCs undergo epigenetic changes in vitro which may compromise function, so an epigenetic pluripotency "signature" would be invaluable for line validation. We assessed Cytosine-phosphate-Guanine Island (CGI methylation in hESCs by genomic DNA hybridisation to a CGI array, and saw substantial variation in CGI methylation between lines. Comparison of hESC CGI methylation profiles to corresponding somatic tissue data and hESC mRNA expression profiles identified a conserved hESC-specific methylation pattern associated with expressed genes. Transcriptional repressors and activators were over-represented amongst genes whose associated CGIs were methylated or unmethylated specifically in hESCs, respectively. Knockdown of candidate transcriptional regulators (HMGA1, GLIS2, PFDN5 induced differentiation in hESCs, whereas ectopic expression in fibroblasts modulated iPSC colony formation. Chromatin immunoprecipitation confirmed interaction between the candidates and the core pluripotency transcription factor network. We thus identify novel pluripotency genes on the basis of a conserved and distinct epigenetic configuration in human stem cells.

  8. Distinctive Citizenship

    DEFF Research Database (Denmark)

    Kaur, Ravinder

    2009-01-01

    The refugee, in India's Partition history, appears as an enigmatic construct - part pitiful, part heroic, though mostly shorn of agency - representing the surface of the human tragedy of Partition. Yet this archetype masks the undercurrent of social distinctions that produced hierarchies of post...

  9. A human immune data-informed vaccine concept elicits strong and broad T-cell specificities associated with HIV-1 control in mice and macaques

    OpenAIRE

    Mothe, B; Hu, Xintao; Llano, Anuska; Rosati, Margherita; Olvera, Alex; Kulkarni, Viraj; Valentin, Antonio; Alicea, Candido; Pilkington, Guy R.; Sardesai, Niranjan J.; Rocafort, Muntsa; Crespo, Manel; Carrillo, Jorge; Marco, Andrés; Mullins, James I.

    2015-01-01

    Background None of the HIV T-cell vaccine candidates that have reached advanced clinical testing have been able to induce protective T cell immunity. A major reason for these failures may have been suboptimal T cell immunogen designs. Methods To overcome this problem, we used a novel immunogen design approach that is based on functional T cell response data from more than 1,000 HIV-1 clade B and C infected individuals and which aims to direct the T cell response to the most vulnerable sites o...

  10. Odontogenic ameloblast-associated protein (ODAM) inhibits growth and migration of human melanoma cells and elicits PTEN elevation and inactivation of PI3K/AKT signaling

    International Nuclear Information System (INIS)

    The Odontogenic Ameloblast-associated Protein (ODAM) is expressed in a wide range of normal epithelial, and neoplastic tissues, and we have posited that ODAM serves as a novel prognostic biomarker for breast cancer and melanoma. Transfection of ODAM into breast cancer cells yields suppression of cellular growth, motility, and in vivo tumorigenicity. Herein we have extended these studies to the effects of ODAM on cultured melanoma cell lines. The A375 and C8161 melanoma cell lines were stably transfected with ODAM and assayed for properties associated with tumorigenicity including cell growth, motility, and extracellular matrix adhesion. In addition, ODAM–transfected cells were assayed for signal transduction via AKT which promotes cell proliferation and survival in many neoplasms. ODAM expression in A375 and C8161 cells strongly inhibited cell growth and motility in vitro, increased cell adhesion to extracellular matrix, and yielded significant cytoskeletal/morphologic rearrangement. Furthermore, AKT activity was downregulated by ODAM expression while an increase was noted in expression of the PTEN (phosphatase and tensin homolog on chromosome 10) tumor suppressor gene, an antagonist of AKT activation. Increased PTEN in ODAM-expressing cells was associated with increases in PTEN mRNA levels and de novo protein synthesis. Silencing of PTEN expression yielded recovery of AKT activity in ODAM-expressing melanoma cells. Similar PTEN elevation and inhibition of AKT by ODAM was observed in MDA-MB-231 breast cancer cells while ODAM expression had no effect in PTEN-deficient BT-549 breast cancer cells. The apparent anti-neoplastic effects of ODAM in cultured melanoma and breast cancer cells are associated with increased PTEN expression, and suppression of AKT activity. This association should serve to clarify the clinical import of ODAM expression and any role it may serve as an indicator of tumor behavior

  11. Distinct populations of hepatic stellate cells in the mouse liver have different capacities for retinoid and lipid storage.

    Directory of Open Access Journals (Sweden)

    Diana N D'Ambrosio

    Full Text Available Hepatic stellate cell (HSC lipid droplets are specialized organelles for the storage of retinoid, accounting for 50-60% of all retinoid present in the body. When HSCs activate, retinyl ester levels progressively decrease and the lipid droplets are lost. The objective of this study was to determine if the HSC population in a healthy, uninjured liver demonstrates heterogeneity in its capacity for retinoid and lipid storage in lipid droplets. To this end, we utilized two methods of HSC isolation, which leverage distinct properties of these cells, including their vitamin A content and collagen expression. HSCs were isolated either from wild type (WT mice in the C57BL/6 genetic background by flotation in a Nycodenz density gradient, followed by fluorescence activated cell sorting (FACS based on vitamin A autofluorescence, or from collagen-green fluorescent protein (GFP mice by FACS based on GFP expression from a GFP transgene driven by the collagen I promoter. We show that GFP-HSCs have: (i increased expression of typical markers of HSC activation; (ii decreased retinyl ester levels, accompanied by reduced expression of the enzyme needed for hepatic retinyl ester synthesis (LRAT; (iii decreased triglyceride levels; (iv increased expression of genes associated with lipid catabolism; and (v an increase in expression of the retinoid-catabolizing cytochrome, CYP2S1.Our observations suggest that the HSC population in a healthy, uninjured liver is heterogeneous. One subset of the total HSC population, which expresses early markers of HSC activation, may be "primed" and ready for rapid response to acute liver injury.

  12. Distinct Cytoplasmic Expression of KL-6 Mucin in Chromophobe Renal Cell Carcinoma: A Comparative Immunohistochemical Study with Other Renal Epithelial Cell Tumors

    International Nuclear Information System (INIS)

    The presence of cytoplasmic sialyl glycoproteins is a conspicuous feature in chromophobe renal cell carcinoma (RCC). We compared the immunohistochemical expression of sialyl glycoproteins in chromophobe RCC with that in other types of renal tumors. Formalin-fixed, paraffin-embedded tissues of surgically resected renal tumors (chromophobe RCC, 14 cases [10 cases of classic type and 4 cases of eosinophilic variant]; oncocytoma, 7 cases; and clear cell RCC, 9 cases) and kidneys from immature infants (4 cases) were immunostained with antibodies against sialyl glycoproteins (anti-KL-6 and anti-sialyl MUC1 antibodies). Cytoplasmic expression of KL-6 and sialyl MUC1 was distinctive in the chromophobe RCC and renal oncocytoma cells, and in the intercalated cells in collecting duct epithelia. Apical-surface staining of these sialyl glycoproteins was predominantly observed in clear RCC, in the epithelia of the distal tubule and collecting duct, and in the neonatal renal proximal tubule, but not in those of the adult renal proximal tubule. The above-mentioned observations provide additional evidence for similar phenotypic profiles of chromophobe RCC and renal oncocytoma, and the intercalated cells in collecting ducts and the oncofetal expression of sialyl glycoproteins in clear cell RCC. KL-6 is a potential tumor marker for renal tumors

  13. Cathepsin K induces platelet dysfunction and affects cell signaling in breast cancer - molecularly distinct behavior of cathepsin K in breast cancer

    OpenAIRE

    Andrade, Sheila Siqueira; Gouvea, Iuri Estrada; Silva, Mariana Cristina C.; Castro, Eloísa Dognani; de Paula, Cláudia A. A.; Okamoto, Debora; Oliveira, Lilian; Peres, Giovani Bravin; Ottaiano, Tatiana; Facina, Gil; Nazário, Afonso Celso Pinto; Campos, Antonio Hugo J. F. M.; Paredes-Gamero, Edgar Julian; Juliano, Maria; da Silva, Ismael D. C. G.

    2016-01-01

    Background Breast cancer comprises clinically and molecularly distinct tumor subgroups that differ in cell histology and biology and show divergent clinical phenotypes that impede phase III trials, such as those utilizing cathepsin K inhibitors. Here we correlate the epithelial-mesenchymal-like transition breast cancer cells and cathepsin K secretion with activation and aggregation of platelets. Cathepsin K is up-regulated in cancer cells that proteolyze extracellular matrix and contributes t...

  14. Deleted in malignant brain tumors 1 (DMBT1) elicits increased VEGF and decreased IL-6 production in type II lung epithelial cells

    DEFF Research Database (Denmark)

    Müller, Hanna; Nagel, Christian; Weiss, Christel;

    2015-01-01

    between VEGF and IL-6 levels to DMBT1 expression in the lungs of preterm and term infants and in lung epithelial cells in vitro. METHODS: We examined by ELISA VEGF levels in 120 tracheal aspirates of 57 preterm and term infants and tested for correlation with different perinatal factors as well as with...... DMBT1 levels. To examine the effect of DMBT1 on VEGF and IL-6 expression we compared type II lung epithelial A549 cells stably transfected with a DMBT1 expression plasmid (DMBT1+ cells) to A549 cells stably transfected with an empty expression plasmid (DMBT1- cells). The concentrations of VEGF and IL-6...... that DMBT1 promotes VEGF and suppresses IL-6 production in alveolar tissues, which could point to DMBT1 having a possible role in the transition from inflammation to regeneration and being a potentially useful clinical marker....

  15. Silencing of ribosomal protein S9 elicits a multitude of cellular responses inhibiting the growth of cancer cells subsequent to p53 activation.

    Directory of Open Access Journals (Sweden)

    Mikael S Lindström

    Full Text Available BACKGROUND: Disruption of the nucleolus often leads to activation of the p53 tumor suppressor pathway through inhibition of MDM2 that is mediated by a limited set of ribosomal proteins including RPL11 and RPL5. The effects of ribosomal protein loss in cultured mammalian cells have not been thoroughly investigated. Here we characterize the cellular stress response caused by depletion of ribosomal protein S9 (RPS9. METHODOLOGY/PRINCIPAL FINDINGS: Depletion of RPS9 impaired production of 18S ribosomal RNA and induced p53 activity. It promoted p53-dependent morphological differentiation of U343MGa Cl2:6 glioma cells as evidenced by intensified expression of glial fibrillary acidic protein and profound changes in cell shape. U2OS osteosarcoma cells displayed a limited senescence response with increased expression of DNA damage response markers, whereas HeLa cervical carcinoma cells underwent cell death by apoptosis. Knockdown of RPL11 impaired p53-dependent phenotypes in the different RPS9 depleted cell cultures. Importantly, knockdown of RPS9 or RPL11 also markedly inhibited cell proliferation through p53-independent mechanisms. RPL11 binding to MDM2 was retained despite decreased levels of RPL11 protein following nucleolar stress. In these settings, RPL11 was critical for maintaining p53 protein stability but was not strictly required for p53 protein synthesis. CONCLUSIONS: p53 plays an important role in the initial restriction of cell proliferation that occurs in response to decreased level of RPS9. Our results do not exclude the possibility that other nucleolar stress sensing molecules act upstream or in parallel to RPL11 to activate p53. Inhibiting the expression of certain ribosomal proteins, such as RPS9, could be one efficient way to reinitiate differentiation processes or to induce senescence or apoptosis in rapidly proliferating tumor cells.

  16. Immunization of volunteers with Salmonella enterica serovar Typhi strain Ty21a elicits the oligoclonal expansion of CD8+ T cells with predominant Vbeta repertoires.

    Science.gov (United States)

    Salerno-Gonçalves, Rosângela; Wahid, Rezwanul; Sztein, Marcelo B

    2005-06-01

    CD8(+) T cells are likely to play an important role in host defense against Salmonella enterica serovar Typhi by several effector mechanisms, including lysis of infected cells (cytotoxicity) and gamma interferon (IFN-gamma) secretion. In an effort to better understand these responses, we studied the T-cell receptor (TCR) repertoire of serovar Typhi-specific CD8(+) T cells in humans. To this end, we determined the TCR beta chain (Vbeta) usage of CD8(+) T cells from three volunteers orally immunized with Ty21a typhoid vaccine by flow cytometry using a panel of monoclonal antibodies. Although TCR Vbeta usage varied among volunteers, we identified oligoclonal Vbeta subset expansions in individual volunteers (Vbeta 2, 5.1, 8, 17, and 22 in volunteer 1; Vbeta 1, 2, 5.1, 14, 17, and 22 in volunteer 2; and Vbeta 3, 8, 14, and 16 in volunteer 3). These subsets were antigen specific, as shown by cytotoxicity and IFN-gamma secretion assays on Vbeta sorted cells and on T-cell clones derived from these volunteers. Moreover, eight-color flow cytometric analysis showed that these clones exhibited a T effector memory phenotype (i.e., CCR7(-) CD27(-) CD45RO(+) CD62L(-)) and coexpressed gut homing molecules (e.g., high levels of integrin alpha4beta7, intermediate levels of CCR9, and low levels of CD103). In conclusion, our results show that long-term T-cell responses to serovar Typhi in Ty21a vaccinees are oligoclonal, involving multiple TCR Vbeta families. Moreover, these serovar Typhi-specific CD8(+) T cells bearing defined Vbeta specificities are phenotypically and functionally consistent with T effector memory cells with preferential gut homing potential. PMID:15908381

  17. Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression

    Energy Technology Data Exchange (ETDEWEB)

    Changchien, Jung-Jung; Chen, Ying-Jung; Huang, Chia-Hui [Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Cheng, Tian-Lu [Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Lin, Shinne-Ren [Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Chang, Long-Sen, E-mail: lschang@mail.nsysu.edu.tw [Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Department of Biotechnology, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China)

    2015-04-01

    Although previous studies have revealed the anti-cancer activity of quinacrine, its effect on leukemia is not clearly resolved. We sought to explore the cytotoxic effect and mechanism of quinacrine action in human leukemia K562 cells. Quinacrine induced K562 cell apoptosis accompanied with ROS generation, mitochondrial depolarization, and down-regulation of BCL2L1 and BCL2. Upon exposure to quinacrine, ROS-mediated p38 MAPK activation and ERK inactivation were observed in K562 cells. Quinacrine-induced cell death and mitochondrial depolarization were suppressed by the p38MAPK inhibitor SB202190 and constitutively active MEK1 over-expression. Activation of p38 MAPK was shown to promote BCL2 degradation. Further, ERK inactivation suppressed c-Jun-mediated transcriptional expression of BCL2L1. Over-expression of BCL2L1 and BCL2 attenuated quinacrine-evoked mitochondrial depolarization and rescued the viability of quinacrine-treated cells. Taken together, our data indicate that quinacrine-induced K562 cell apoptosis is mediated through mitochondrial alterations triggered by p38 MAPK-mediated BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression. - Highlights: • Quinacrine induces K562 cell apoptosis via down-regulation of BCL2 and BCL2L1. • Quinacrine induces p38 MAPK activation and ERK inactivation in K562 cells. • Quinacrine elicits p38 MAPK-mediated BCL2 down-regulation. • Quinacrine suppresses ERK/c-Jun-mediated BCL2L1 expression.

  18. Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression

    International Nuclear Information System (INIS)

    Although previous studies have revealed the anti-cancer activity of quinacrine, its effect on leukemia is not clearly resolved. We sought to explore the cytotoxic effect and mechanism of quinacrine action in human leukemia K562 cells. Quinacrine induced K562 cell apoptosis accompanied with ROS generation, mitochondrial depolarization, and down-regulation of BCL2L1 and BCL2. Upon exposure to quinacrine, ROS-mediated p38 MAPK activation and ERK inactivation were observed in K562 cells. Quinacrine-induced cell death and mitochondrial depolarization were suppressed by the p38MAPK inhibitor SB202190 and constitutively active MEK1 over-expression. Activation of p38 MAPK was shown to promote BCL2 degradation. Further, ERK inactivation suppressed c-Jun-mediated transcriptional expression of BCL2L1. Over-expression of BCL2L1 and BCL2 attenuated quinacrine-evoked mitochondrial depolarization and rescued the viability of quinacrine-treated cells. Taken together, our data indicate that quinacrine-induced K562 cell apoptosis is mediated through mitochondrial alterations triggered by p38 MAPK-mediated BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression. - Highlights: • Quinacrine induces K562 cell apoptosis via down-regulation of BCL2 and BCL2L1. • Quinacrine induces p38 MAPK activation and ERK inactivation in K562 cells. • Quinacrine elicits p38 MAPK-mediated BCL2 down-regulation. • Quinacrine suppresses ERK/c-Jun-mediated BCL2L1 expression

  19. HIF-1alpha and HIF-2alpha are differentially activated in distinct cell populations in retinal ischaemia.

    Directory of Open Access Journals (Sweden)

    Freya M Mowat

    Full Text Available BACKGROUND: Hypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF and erythropoietin (Epo by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO. CONCLUSIONS/SIGNIFICANCE: Both HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation

  20. Andrographis paniculata elicits anti-invasion activities by suppressing TM4SF3 gene expression and by anoikis-sensitization in esophageal cancer cells.

    Science.gov (United States)

    Yue, Grace Gar-Lee; Lee, Julia Kin-Ming; Li, Lin; Chan, Kar-Man; Wong, Eric Chun-Wai; Chan, Judy Yuet-Wah; Fung, Kwok-Pui; Lui, Vivian Wai Yan; Chiu, Philip Wai-Yan; Lau, Clara Bik-San

    2015-01-01

    Esophageal cancer is the sixth most common cancer in male causing death worldwide. It is usually diagnosed at advanced stage with high postoperative recurrence and systemic metastasis, which leads to poor prognosis. The potential inhibitory effect of herbal medicines on metastasis of esophageal cancer has drawn researchers' great attention. In the present study, the anti-invasion activities of Andrographis paniculata (AP) have been evaluated in two esophageal cancer cell lines, EC-109 and KYSE-520, as well as human microvascular endothelial cells (HMEC-1). The anti-tumor and anti-metastatic activities of AP were also evaluated in human esophageal xenograft-bearing mouse models. Our results demonstrated for the first time that aqueous extract of AP inhibited the motility and invasion of esophageal cancer cells, which is the initial step of metastasis, without cytotoxicity. Anoikis resistance has also been reversed in AP-treated cancer cells. Besides, the expression of metastasis-related gene TM4SF3 in EC-109 cells was significantly decreased in AP extract-treated cells in a concentration-dependent manner. Furthermore, the anti-tumor and anti-metastatic efficacies in subcutaneous and intraperitoneal esophageal xenograft-bearing mice were demonstrated after oral administration of AP aqueous extract for 3 weeks. Last but not least, the active component, isoandrographolide, responsible for the anti-migratory activity was firstly revealed here. In conclusion, the AP aqueous extract exerted inhibitory activities on the migration and anoikis resistance of esophageal cancer cells EC-109 and KYSE-520, as well as suppressed the proliferation and motility of endothelial cells. Combining the mentioned effects may account for the anti-tumor and anti-metastasis effects of AP aqueous extract in xenograft-bearing mice. The findings in the present study further enhance the understanding of the therapeutic mechanisms of the herb AP, which may lead to clinical applications. PMID

  1. Recombinant parvovirus-like particles as an antigen carrier: A novel nonreplicative exogenous antigen to elicit protective antiviral cytotoxic T cells

    OpenAIRE

    Sedlik, C.; Saron, M.-F.; Sarraseca, J.; Casal, I; Leclerc, C.

    1997-01-01

    To develop a strategy that promotes efficient antiviral immunity, hybrid virus-like particles (VLP) were prepared by self-assembly of the modified porcine parvovirus VP2 capsid protein carrying a CD8+ T cell epitope from the lymphocytic choriomeningitis virus nucleoprotein. Immunization of mice with these hybrid pseudoparticles, without adjuvant, induced strong cytotoxic T lymphocyte (CTL) responses against both peptide-coated- or virus-infected-target cells. This CD8+ class I-restricted cyto...

  2. Effect of Botrytis cinerea infection and elicitation on ß-1,3-glucanase and chitinase activity in bean leaves and cell cultures

    OpenAIRE

    Elżbieta Kuźniak; Henryk Urbanek; Aneta Michalak; Katarzyna Herka

    2013-01-01

    The activity of ß-1,3-glucanase and chitinase in bean plants treated with B. cinerea products or/and infected and in cell cultures after application of fungal products has been studied. Botrytis cinerea infection and culture filtrates, ethanol precipitates, glucan and conidial extract treatment markedly enhanced the activity of both hydrolases. Cell cultures treated with B.cinerea products reacted similarly to intact plants. In plants pretreated with 2-day culture filtrate and conidial extrac...

  3. Elicitation of ostomy pouch preferences

    DEFF Research Database (Denmark)

    Bonnichsen, Ole

    2011-01-01

    stoma care would affect patients and, to the author's knowledge, the present study is the first to elicit preferences for potential improvements in ostomy pouches in the form of monetary values. Objective: This article examines and measures Swedish patients' preferences for potential improvements in...... the utility that patients would obtain from a potential improvement in their ostomy pouch. This provides information as to how treatment options in terms of stoma management can be structured so as to maximize the benefits for patients....

  4. Survey Based Reviewof Elicitation Problems

    OpenAIRE

    Sidra Arshad

    2016-01-01

    Any software development process is the combination of multiple development activities and each activity has a vital role in the software development cycle. Requirement Engineering is the main and basic branch of Software Engineering, it has many phases but the most initial phase is Requirement Elicitation. In this phase requirements are gathered for system development. This paper provides a literature review of the requirements engineering processes performed in traditional and ...

  5. RSK is a principal effector of the RAS-ERK pathway for eliciting a coordinate promotile/invasive gene program and phenotype in epithelial cells

    DEFF Research Database (Denmark)

    Doehn, Ulrik; Hauge, Camilla; Frank, Scott R;

    2009-01-01

    The RAS-stimulated RAF-MEK-ERK pathway confers epithelial cells with critical motile and invasive capacities during development, tissue regeneration, and carcinoma progression, often via promoting the epithelial-mesenchymal transition (EMT). Many mechanisms by which ERK exerts this control remain...... elusive. We demonstrate that the ERK-activated kinase RSK is necessary to induce mesenchymal motility and invasive capacities in nontransformed epithelial and carcinoma cells. RSK is sufficient to induce certain motile responses. Expression profiling analysis revealed that a primary role of RSK is to...... stimulate motility and invasion. These findings uncover a mechanism whereby the RAS-ERK pathway controls epithelial cell motility by identifying RSK as a key effector, from which emanate multiple highly coordinate transcription-dependent mechanisms for stimulation of motility and invasive properties....

  6. Effect of Botrytis cinerea infection and elicitation on ß-1,3-glucanase and chitinase activity in bean leaves and cell cultures

    Directory of Open Access Journals (Sweden)

    Elżbieta Kuźniak

    2013-12-01

    Full Text Available The activity of ß-1,3-glucanase and chitinase in bean plants treated with B. cinerea products or/and infected and in cell cultures after application of fungal products has been studied. Botrytis cinerea infection and culture filtrates, ethanol precipitates, glucan and conidial extract treatment markedly enhanced the activity of both hydrolases. Cell cultures treated with B.cinerea products reacted similarly to intact plants. In plants pretreated with 2-day culture filtrate and conidial extract and then infected, ß-1,3-glucanase and chitinase were induced stronger than after infection without pretreatment.

  7. Activation of Plasmacytoid Dendritic Cells in Colon-Draining Lymph Nodes during Citrobacter rodentium Infection Involves Pathogen-Sensing and Inflammatory Pathways Distinct from Conventional Dendritic Cells.

    Science.gov (United States)

    Toivonen, Raine; Kong, Lingjia; Rasool, Omid; Lund, Riikka J; Lahesmaa, Riitta; Hänninen, Arno

    2016-06-01

    Dendritic cells (DCs) bear the main responsibility for initiation of adaptive immune responses necessary for antimicrobial immunity. In the small intestine, afferent lymphatics convey Ags and microbial signals to mesenteric lymph nodes (LNs) to induce adaptive immune responses against microbes and food Ags derived from the small intestine. Whether the large intestine is covered by the same lymphatic system or represents its own lymphoid compartment has not been studied until very recently. We identified three small mesenteric LNs, distinct from small intestinal LNs, which drain lymph specifically from the colon, and studied DC responses to the attaching and effacing pathogen Citrobacter rodentium in these. Transcriptional profiling of conventional (CD11c(high)CD103(high)) DC and plasmacytoid (plasmacytoid DC Ag-1(high)B220(+)CD11c(int)) DC (pDC) populations during steady-state conditions revealed activity of distinct sets of genes in these two DC subsets, both in small intestinal and colon-draining LNs. C. rodentium activated DC especially in colon-draining LNs, and gene expression changed in pDC more profoundly than in conventional DC. Among the genes most upregulated in pDC were C-type lectin receptor CLEC4E, IL-1Rs (IL-1R1 and -2), proinflammatory cytokines (IL-1a and IL-6), and TLR6. Our results indicate that colon immune surveillance is distinct from that of the small intestine in terms of draining LNs, and identify pDC as active sentinels of colonic inflammation and/or microbial dysbiosis. PMID:27183629

  8. [Two recombinant adenovirus vaccine candidates containing neuraminidase Gene of H5N1 influenza virus (A/Anhui/1/2005) elicited effective cell-mediated immunity in mice].

    Science.gov (United States)

    Ma, Jing; Zhang, Xiao-Guang; Chen, Hong; Li, Kui-Biao; Zhang, Xiao-Mei; Zhang, Ke; Yang, Liang; Xu, Hong; Shu, Yue-Long; Tan, Wen-Jie; Zeng, Yi

    2009-09-01

    The aim of this study is to develop the recombinant adenovirus vaccine (rAdV) candidates containing neuraminidase (NA) gene of H5N1 influenza virus and test in BALB/c mice the effect of cell-mediated immunity. In this study, two kind of NA gene (WtNA gene, the wild type; Mod. NA gene, the codon-modified type) derived from H5N1 influenza virus (A/Anhui/1/2005) were cloned and inserted respectively into plasmid of adenovirus vector, then the rAdV vaccines candidates (rAdV-WtNA and rAdV-Mod. NA) were developed and purified, followed by immunization intramuscularly (10(9) TCID50 per dose, double injection at 0 and 4th week) in BALB/c mice, the effect of cell-mediated immunity were analysed at 5th week. Results indicated that: (i) NA protein expression was detected in two rAdV vaccines candidates by Western blotting; (ii) the rAdV-Mod. NA vaccine could elicit more robust NA specific cell-mediated immunity in mice than that of rAdV-WtNA vaccine (P = 0. 016) by IFN-gamma ELIspot assay. These findings suggested rAdV-Mod. NA vaccine was a potential vaccine candidate against H5N1 influenza and worthy of further investigation. PMID:19954107

  9. Bone marrow stromal cells elicit tissue sparing after acute but not delayed transplantation into the contused adult rat thoracic spinal cord.

    NARCIS (Netherlands)

    Tewarie, R.D.; Hurtado, A.; Ritfeld, G.J.; Rahiem, S.T.; Wendell, D.F.; Barroso, M.M.; Grotenhuis, J.A.; Oudega, M.

    2009-01-01

    Bone marrow stromal cells (BMSC) transplanted into the contused spinal cord may support repair by improving tissue sparing. We injected allogeneic BMSC into the moderately contused adult rat thoracic spinal cord at 15 min (acute) and at 3, 7, and 21 days (delayed) post-injury and quantified tissue s

  10. Cutaneous sensitivity induced by immunization with irradiated Schistosoma mansoni cercariae. I. Induction, elicitation, and adoptive transfer analysis of cell-mediated cutaneous sensitivity

    International Nuclear Information System (INIS)

    Exposure of C57BL/6 mice to highly irradiated (50 kR) cercariae of Schistosoma mansoni leads to the development of partial resistance against subsequent challenge with unattenuated cercariae. We have analyzed the cellular immune responses that occur during the afferent and efferent phases of this protective sensitization. Mice were immunized by exposure to irradiated S. mansoni cercariae. After challenge with irradiated cercariae, delayed-type (18-72 hr) cutaneous sensitivity reaction sites were rich in mononuclear cells and eosinophils. This reactivity was established by 4 days after sensitization, reached its maximum between 7 and 14 days after sensitization, and was maintained for over 20 weeks. These challenge reactions could be abrogated by treatment with either 200 mg/kg cyclophosphamide or 5 mg of hydrocortisone. Syngeneic adoptive transfer of cutaneous sensitivity was accomplished with lymphoid cells from the draining lymph nodes or spleens of mice sensitized 7-14 days previously. Negative selection studies of nylon-wool non-adherent cells from sensitized donors demonstrated that the cells responsible for transferring this eosinophil-rich, delayed-type cutaneous sensitivity to S. mansoni irradiated cercariae were Thy-1+, Lyt1+, Lyt2-, surface Ig- lymphocytes

  11. Cutaneous sensitivity induced by immunization with irradiated Schistosoma mansoni cercariae. I. Induction, elicitation, and adoptive transfer analysis of cell-mediated cutaneous sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Ch' ang, L.Y.; Colley, D.G.

    1986-06-01

    Exposure of C57BL/6 mice to highly irradiated (50 kR) cercariae of Schistosoma mansoni leads to the development of partial resistance against subsequent challenge with unattenuated cercariae. We have analyzed the cellular immune responses that occur during the afferent and efferent phases of this protective sensitization. Mice were immunized by exposure to irradiated S. mansoni cercariae. After challenge with irradiated cercariae, delayed-type (18-72 hr) cutaneous sensitivity reaction sites were rich in mononuclear cells and eosinophils. This reactivity was established by 4 days after sensitization, reached its maximum between 7 and 14 days after sensitization, and was maintained for over 20 weeks. These challenge reactions could be abrogated by treatment with either 200 mg/kg cyclophosphamide or 5 mg of hydrocortisone. Syngeneic adoptive transfer of cutaneous sensitivity was accomplished with lymphoid cells from the draining lymph nodes or spleens of mice sensitized 7-14 days previously. Negative selection studies of nylon-wool non-adherent cells from sensitized donors demonstrated that the cells responsible for transferring this eosinophil-rich, delayed-type cutaneous sensitivity to S. mansoni irradiated cercariae were Thy/sup -1 +/, Lyt/sup 1 +/, Lyt/sup 2 -/, surface Ig/sup -/ lymphocytes.

  12. Influence of FcγRIIa-Expressing Cells on the Assessment of Neutralizing and Enhancing Serum Antibodies Elicited by a Live-Attenuated Tetravalent Dengue Vaccine.

    Science.gov (United States)

    Byers, Anthony M; Broder, Ryan; Haupfear, Kelly; Timiryasova, Tatyana M; Hu, Branda T; Boaz, Mark; Warren, William L; Jackson, Nicholas; Moser, Janice M; Guy, Bruno

    2015-12-01

    Background.  Recent trials of recombinant, live-attenuated chimeric yellow fever-dengue tetravalent dengue vaccine (CYD-TDV) demonstrated efficacy against symptomatic, virologically confirmed dengue disease with higher point estimates of efficacy toward dengue virus (DENV)3 and DENV4 and moderate levels toward DENV1 and DENV2. It is interesting to note that serotype-specific efficacy did not correlate with absolute neutralizing antibody (nAb) geometric mean titer (GMT) values measured in a Vero-based plaque reduction neutralization test assay. The absence of Fcγ receptors on Vero cells may explain this observation. Methods.  We performed parallel seroneutralization assays in Vero cells and CV-1 cells that express FcγRIIa (CV-1-Fc) to determine the neutralizing and enhancing capacity of serotype-specific DENV Abs present in CYD-TDV clinical trial sera. Results.  Enhancement of DENV infection was observed in CV-1-Fc cells in naturally exposed nonvaccine sera, mostly for DENV3 and DENV4, at high dilutions. The CYD-TDV-vaccinated sera showed similar enhancement patterns. The CV-1-Fc nAb GMT values were 2- to 9-fold lower than Vero for all serotypes in both naturally infected individuals and CYD-TDV-vaccinated subjects with and without previous dengue immunity. The relative (CV-1-Fc/Vero) GMT decrease for anti-DENV1 and anti-DENV2 responses was not greater than for the other serotypes. Conclusions.  In vitro neutralization assays utilizing FcγRIIa-expressing cells provide evidence that serotype-specific Ab enhancement may not be a primary factor in the serotype-specific efficacy differences exhibited in the CYD-TDV trials. PMID:26719844

  13. Methotrexate-Loaded Four-Arm Star Amphiphilic Block Copolymer Elicits CD8+ T Cell Response against a Highly Aggressive and Metastatic Experimental Lymphoma.

    Science.gov (United States)

    Hira, Sumit Kumar; Ramesh, Kalyan; Gupta, Uttam; Mitra, Kheyanath; Misra, Nira; Ray, Biswajit; Manna, Partha Pratim

    2015-09-16

    We have synthesized a well-defined four-arm star amphiphilic block copolymer [poly(DLLA)-b-poly(NVP)]4 [star-(PDLLA-b-PNVP)4] that consists of D,L-lactide (DLLA) and N-vinylpyrrolidone (NVP) via the combination of ring-opening polymerization (ROP) and xanthate-mediated reversible addition-fragmentation chain transfer (RAFT) polymerization. Synthesis of the polymer was verified by 1H NMR spectroscopy and gel permeation chromatography (GPC). The amphiphilic four-arm star block copolymer forms spherical micelles in water as demonstrated by transmission electron microscopy (TEM) and 1H NMR spectroscopy. Pyrene acts as a probe to ascertain the critical micellar concentration (cmc) by using fluorescence spectroscopy. Methotrexate (MTX)-loaded polymeric micelles of star-(PDLLA15-b-PNVP10)4 amphiphilic block copolymer were prepared and characterized by fluorescence and TEM studies. Star-(PDLLA15-b-PNVP10)4 copolymer was found to be significantly effective with respect to inhibition of proliferation and lysis of human and murine lymphoma cells. The amphiphilic block copolymer causes cell death in parental and MTX-resistant Dalton lymphoma (DL) and Raji cells. The formulation does not cause hemolysis in red blood cells and is tolerant to lymphocytes compared to free MTX. Therapy with MTX-loaded star-(PDLLA15-b-PNVP10)4 amphiphilic block copolymer micelles prolongs the life span of animals with neoplasia by reducing the tumor load, preventing metastasis and augmenting CD8+ T cell-mediated adaptive immune responses. PMID:26323031

  14. Transgenerational Inheritance of Increased Fat Depot Size, Stem Cell Reprogramming, and Hepatic Steatosis Elicited by Prenatal Exposure to the Obesogen Tributyltin in Mice

    OpenAIRE

    Chamorro-García, Raquel; Sahu, Margaret; Abbey, Rachelle J; Laude, Jhyme; Pham, Nhieu; Blumberg, Bruce

    2013-01-01

    Background: We have previously shown that exposure to tributyltin (TBT) modulates critical steps of adipogenesis through RXR/PPARγ and that prenatal TBT exposure predisposes multipotent mesenchymal stem cells (MSCs) to become adipocytes by epigenetic imprinting into the memory of the MSC compartment. Objective: We tested whether the effects of prenatal TBT exposure were heritable in F2 and F3 generations. Methods: We exposed C57BL/6J female mice (F0) to DMSO vehicle, the pharmaceutical obesog...

  15. Rabies virus-based vaccines elicit neutralizing antibodies, poly-functional CD8+ T cell, and protect rhesus macaques from AIDS-like disease after SIVmac251 challenge

    Science.gov (United States)

    Faul, Elizabeth J.; Aye, Pyone P.; Papaneri, Amy B.; Pahar, Bapi; McGettigan, James P.; Schiro, Faith; Chervoneva, Inna; Montefiori, David C.; Lackner, Andrew A.; Schnell, Matthias J.

    2010-01-01

    Highly attenuated rabies virus (RV) vaccine vectors were evaluated for their ability to protect against highly pathogenic SIVmac251 challenge. Mamu-A*01 negative rhesus macaques were immunized in groups of four with either: RV expressing SIVmac239-GagPol, a combination of RV expressing SIVmac239-Env and RV expressing SIVmac239-GagPol, or with empty RV vectors. Eight weeks later animals received a booster immunization with a heterologous RV expressing the same antigens. At twelve weeks post-boost, all animals were challenged intravenously with 100 TCID50 of pathogenic SIVmac251-CX. Immunized macaques in both vaccine groups had 1.3–1.6-log fold decrease in viral set point compared to control animals. The GagPol/Env immunized animals also had a significantly lower peak viral load. When compared to control animals following challenge, vaccinated macaques had a more rapid induction of SIVmac251 neutralizing antibodies and of CD8+ T cell responses to various SIV epitopes. Moreover, vaccinated macaques better-maintained peripheral memory CD4+ T cells and were able to mount a poly-functional CD8+ T cell response in the mucosa. These findings indicate promise for RV-based vectors and have important implications for the development of an efficacious HIV vaccine. PMID:19879223

  16. Tricho- and atrichoblast cell files show distinct PIN2 auxin efflux carrier exploitations and are jointly required for defined auxin-dependent root organ growth.

    Science.gov (United States)

    Löfke, Christian; Scheuring, David; Dünser, Kai; Schöller, Maria; Luschnig, Christian; Kleine-Vehn, Jürgen

    2015-08-01

    The phytohormone auxin is a vital growth regulator in plants. In the root epidermis auxin steers root organ growth. However, the mechanisms that allow adjacent tissues to integrate growth are largely unknown. Here, the focus is on neighbouring epidermal root tissues to assess the integration of auxin-related growth responses. The pharmacologic, genetic, and live-cell imaging approaches reveal that PIN2 auxin efflux carriers are differentially controlled in tricho- and atrichoblast cells. PIN2 proteins show lower abundance at the plasma membrane of trichoblast cells, despite showing higher rates of intracellular trafficking in these cells. The data suggest that PIN2 proteins display distinct cell-type-dependent trafficking rates to the lytic vacuole for degradation. Based on this insight, it is hypothesized that auxin-dependent processes are distinct in tricho- and atrichoblast cells. Moreover, genetic interference with epidermal patterning supports this assumption and suggests that tricho- and atrichoblasts have distinct importance for auxin-sensitive root growth and gravitropic responses. PMID:26041320

  17. Glial Cell-Elicited Activation of Brain Microvasculature in Response to Brucella abortus Infection Requires ASC Inflammasome-Dependent IL-1β Production.

    Science.gov (United States)

    Miraglia, M Cruz; Costa Franco, Miriam M; Rodriguez, Ana M; Bellozi, Paula M Q; Ferrari, Carina C; Farias, Maria I; Dennis, Vida A; Barrionuevo, Paula; de Oliveira, Antonio C P; Pitossi, Fernando; Kim, Kwang Sik; Delpino, M Victoria; Oliveira, Sergio Costa; Giambartolomei, Guillermo H

    2016-05-01

    Blood-brain barrier activation and/or dysfunction are a common feature of human neurobrucellosis, but the underlying pathogenic mechanisms are largely unknown. In this article, we describe an immune mechanism for inflammatory activation of human brain microvascular endothelial cells (HBMEC) in response to infection with Brucella abortus Infection of HBMEC with B. abortus induced the secretion of IL-6, IL-8, and MCP-1, and the upregulation of CD54 (ICAM-1), consistent with a state of activation. Culture supernatants (CS) from glial cells (astrocytes and microglia) infected with B. abortus also induced activation of HBMEC, but to a greater extent. Although B. abortus-infected glial cells secreted IL-1β and TNF-α, activation of HBMEC was dependent on IL-1β because CS from B. abortus-infected astrocytes and microglia deficient in caspase-1 and apoptosis-associated speck-like protein containing a CARD failed to induce HBMEC activation. Consistently, treatment of CS with neutralizing anti-IL-1β inhibited HBMEC activation. Both absent in melanoma 2 and Nod-like receptor containing a pyrin domain 3 are partially required for caspase-1 activation and IL-1β secretion, suggesting that multiple apoptosis-associated speck-like protein containing CARD-dependent inflammasomes contribute to IL-1β-induced activation of the brain microvasculature. Inflammasome-mediated IL-1β secretion in glial cells depends on TLR2 and MyD88 adapter-like/TIRAP. Finally, neutrophil and monocyte migration across HBMEC monolayers was increased by CS from Brucella-infected glial cells in an IL-1β-dependent fashion, and the infiltration of neutrophils into the brain parenchyma upon intracranial injection of B. abortus was diminished in the absence of Nod-like receptor containing a pyrin domain 3 and absent in melanoma 2. Our results indicate that innate immunity of the CNS set in motion by B. abortus contributes to the activation of the blood-brain barrier in neurobrucellosis and IL-1β mediates

  18. Eliciting expert knowledge in conservation science.

    Science.gov (United States)

    Martin, Tara G; Burgman, Mark A; Fidler, Fiona; Kuhnert, Petra M; Low-Choy, Samantha; McBride, Marissa; Mengersen, Kerrie

    2012-02-01

    Expert knowledge is used widely in the science and practice of conservation because of the complexity of problems, relative lack of data, and the imminent nature of many conservation decisions. Expert knowledge is substantive information on a particular topic that is not widely known by others. An expert is someone who holds this knowledge and who is often deferred to in its interpretation. We refer to predictions by experts of what may happen in a particular context as expert judgments. In general, an expert-elicitation approach consists of five steps: deciding how information will be used, determining what to elicit, designing the elicitation process, performing the elicitation, and translating the elicited information into quantitative statements that can be used in a model or directly to make decisions. This last step is known as encoding. Some of the considerations in eliciting expert knowledge include determining how to work with multiple experts and how to combine multiple judgments, minimizing bias in the elicited information, and verifying the accuracy of expert information. We highlight structured elicitation techniques that, if adopted, will improve the accuracy and information content of expert judgment and ensure uncertainty is captured accurately. We suggest four aspects of an expert elicitation exercise be examined to determine its comprehensiveness and effectiveness: study design and context, elicitation design, elicitation method, and elicitation output. Just as the reliability of empirical data depends on the rigor with which it was acquired so too does that of expert knowledge. PMID:22280323

  19. Stem/Progenitor Cells Derived from the Cochlear Sensory Epithelium Give Rise to Spheres with Distinct Morphologies and Features

    OpenAIRE

    Diensthuber, Marc; Oshima, Kazuo; Heller, Stefan

    2009-01-01

    Nonmammalian vertebrates regenerate lost sensory hair cells by means of asymmetric division of supporting cells. Inner ear or lateral line supporting cells in birds, amphibians, and fish consequently serve as bona fide stem cells resulting in high regenerative capacity of hair cell-bearing organs. Hair cell regeneration does not happen in the mammalian cochlea, but cells with proliferative capacity can be isolated from the neonatal cochlea. These cells have the ability to form clonal floating...

  20. Protein phosphorylation profiling using an in situ proximity ligation assay: phosphorylation of AURKA-elicited EGFR-Thr654 and EGFR-Ser1046 in lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Tzu-Chi Chen

    Full Text Available The epidermal growth factor receptor (EGFR, which is up-regulated in lung cancer, involves the activation of mitogenic signals and triggers multiple signaling cascades. To dissect these EGFR cascades, we used 14 different phospho-EGFR antibodies to quantify protein phosphorylation using an in situ proximity ligation assay (in situ PLA. Phosphorylation at EGFR-Thr654 and -Ser1046 was EGF-dependent in the wild-type (WT receptor but EGF-independent in a cell line carrying the EGFR-L858R mutation. Using a ProtoAarray™ containing ∼5000 recombinant proteins on the protein chip, we found that AURKA interacted with the EGFR-L861Q mutant. Moreover, overexpression of EGFR could form a complex with AURKA, and the inhibitors of AURKA and EGFR decreased EGFR-Thr654 and -Ser1046 phosphorylation. Immunohistochemical staining of stage I lung adenocarcinoma tissues demonstrated a positive correlation between AURKA expression and phosphorylation of EGFR at Thr654 and Ser1046 in EGFR-mutant specimens, but not in EGFR-WT specimens. The interplay between EGFR and AURKA provides an explanation for the difference in EGF dependency between EGFR-WT and EGFR-mutant cells and may provide a new therapeutic strategy for lung cancer patients carrying EGFR mutations.

  1. Hemagglutinin-based polyanhydride nanovaccines against H5N1 influenza elicit protective virus neutralizing titers and cell-mediated immunity

    Directory of Open Access Journals (Sweden)

    Ross KA

    2014-12-01

    Full Text Available Kathleen A Ross,1 Hyelee Loyd,2 Wuwei Wu,2 Lucas Huntimer,3 Shaheen Ahmed,4 Anthony Sambol,5 Scott Broderick,6 Zachary Flickinger,2 Krishna Rajan,6 Tatiana Bronich,4 Surya Mallapragada,1 Michael J Wannemuehler,3 Susan Carpenter,2 Balaji Narasimhan1 1Chemical and Biological Engineering, Iowa State University, Ames, IA, USA; 2Animal Science, Iowa State University, Ames, IA, USA; 3Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA, USA; 4Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA; 5Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA; 6Materials Science and Engineering, Iowa State University, Ames, IA, USA Abstract: H5N1 avian influenza is a significant global concern with the potential to become the next pandemic threat. Recombinant subunit vaccines are an attractive alternative for pandemic vaccines compared to traditional vaccine technologies. In particular, polyanhydride nanoparticles encapsulating subunit proteins have been shown to enhance humoral and cell-mediated immunity and provide protection upon lethal challenge. In this work, a recombinant H5 hemagglutinin trimer (H53 was produced and encapsulated into polyanhydride nanoparticles. The studies performed indicated that the recombinant H53 antigen was a robust immunogen. Immunizing mice with H53 encapsulated into polyanhydride nanoparticles induced high neutralizing antibody titers and enhanced CD4+ T cell recall responses in mice. Finally, the H53-based polyanhydride nanovaccine induced protective immunity against a low-pathogenic H5N1 viral challenge. Informatics analyses indicated that mice receiving the nanovaccine formulations and subsequently challenged with virus were similar to naïve mice that were not challenged. The current studies provide a basis to further exploit the advantages of polyanhydride nanovaccines in pandemic scenarios. Keywords: polymer, nanoparticle, vaccine, subunit

  2. Eliciting Spill: A methodological note

    Directory of Open Access Journals (Sweden)

    Alvita Nathaniel, Ph.D.

    2008-03-01

    Full Text Available Classic grounded theory is an inductive process that focuses on the experiences and perceptions of research participants (Glaser, 1978, 1998. Although grounded theorists may utilize other types of data, most are likely to gather information through qualitative interviews. The theorist seeks to understand what is going on as people resolve their main concern in a given substantive area. People know what is important to them and most want to tell their stories. They feel encouraged to talk when they recognize that their stories are valued. Once the informant realizes that he or she is being heard, the story flows. This is what Glaser refers to as “spill.” When this occurs, the theorist becomes a vessel to receive the story. As Glaser describes it, “The researcher will become a ‘big ear’ to pour into incessantly” (1998, p. 124. But, as easy as this seems, the researcher must overcome certain positivist tendencies to allow this to happen. Rather than asking a list of pre-planned questions, the grounded theorist will try to develop one question that will trigger the telling of a story. Eliciting spill requires a deliberate process that employs a deep understanding of the fundamentals of classic grounded theory. Derived from Glaser’s writings, the following are suggestions intended to help the novice grounded theorist to elicit spill.

  3. PAF-receptor is preferentially expressed in a distinct synthetic phenotype of smooth muscle cells cloned from human internal thoracic artery: Functional implications in cell migration

    International Nuclear Information System (INIS)

    Platelet-activating-Factor (PAF) and its structural analogues formed upon low density lipoprotein oxidation are involved in atherosclerotic plaque formation and may signal through PAF-receptor (PAF-R) expressed in human macrophages and in certain smooth muscle cells (SMCs) in the media, but rarely in the intima of human plaques. Our aim was to determine which SMC phenotype expresses PAF-R and whether this receptor is functional in cell migration. Circulating SMC progenitors and two phenotypically distinct clones of proliferative, epithelioid phenotype vs contractile, spindle-shaped SMCs from the media of adult internal thoracic artery were studied for the presence of PAF-receptor (PAF-R). The levels of specific mRNA were obtained by reverse transcription/real-time PCR, the protein expression was deduced from immunohistochemistry staining, and the functional transmigration assay was performed by Boyden chamber-type chemotaxis assay. Only SMCs of spindle-shape and synthetic phenotype expressed both mRNA and PAF-R protein and in the functional test migrated at low concentrations of PAF. Two unrelated, specific PAF-R antagonists inhibited PAF-induced migration, but did not modify the migration initiated by PDGF. The presence of functional PAF-R in arterial spindle-shaped SMCs of synthetic phenotype may be important for their migration from the media into the intima and atherosclerotic plaques formation

  4. Targeting surface nucleolin with multivalent HB-19 and related Nucant pseudopeptides results in distinct inhibitory mechanisms depending on the malignant tumor cell type

    International Nuclear Information System (INIS)

    Nucleolin expressed at the cell surface is a binding protein for a variety of ligands implicated in tumorigenesis and angiogenesis. By using a specific antagonist that binds the C-terminal RGG domain of nucleolin, the HB-19 pseudopeptide, we recently reported that targeting surface nucleolin with HB-19 suppresses progression of established human breast tumor cells in the athymic nude mice, and delays development of spontaneous melanoma in the RET transgenic mice. By the capacity of HB-19 to bind stably surface nucleolin, we purified and identified nucleolin partners at the cell surface. HB-19 and related multivalent Nucant pseudopeptides, that present pentavalently or hexavalently the tripeptide Lysψ(CH2N)-Pro-Arg, were then used to show that targeting surface nucleolin results in distinct inhibitory mechanisms on breast, prostate, colon carcinoma and leukemia cells. Surface nucleolin exists in a 500-kDa protein complex including several other proteins, which we identified by microsequencing as two Wnt related proteins, Ku86 autoantigen, signal recognition particle subunits SRP68/72, the receptor for complement component gC1q-R, and ribosomal proteins S4/S6. Interestingly, some of the surface-nucleolin associated proteins are implicated in cell signaling, tumor cell adhesion, migration, invasion, cell death, autoimmunity, and bacterial infections. Surface nucleolin in the 500-kDa complex is highly stable. Surface nucleolin antagonists, HB-19 and related multivalent Nucant pseudopeptides, exert distinct inhibitory mechanisms depending on the malignant tumor cell type. For example, in epithelial tumor cells they inhibit cell adhesion or spreading and induce reversion of the malignant phenotype (BMC cancer 2010, 10:325) while in leukemia cells they trigger a rapid cell death associated with DNA fragmentation. The fact that these pseudopeptides do not cause cell death in epithelial tumor cells indicates that cell death in leukemia cells is triggered by a specific

  5. Targeting surface nucleolin with multivalent HB-19 and related Nucant pseudopeptides results in distinct inhibitory mechanisms depending on the malignant tumor cell type

    Directory of Open Access Journals (Sweden)

    Hovanessian Ara G

    2011-08-01

    Full Text Available Abstract Background Nucleolin expressed at the cell surface is a binding protein for a variety of ligands implicated in tumorigenesis and angiogenesis. By using a specific antagonist that binds the C-terminal RGG domain of nucleolin, the HB-19 pseudopeptide, we recently reported that targeting surface nucleolin with HB-19 suppresses progression of established human breast tumor cells in the athymic nude mice, and delays development of spontaneous melanoma in the RET transgenic mice. Methods By the capacity of HB-19 to bind stably surface nucleolin, we purified and identified nucleolin partners at the cell surface. HB-19 and related multivalent Nucant pseudopeptides, that present pentavalently or hexavalently the tripeptide Lysψ(CH2N-Pro-Arg, were then used to show that targeting surface nucleolin results in distinct inhibitory mechanisms on breast, prostate, colon carcinoma and leukemia cells. Results Surface nucleolin exists in a 500-kDa protein complex including several other proteins, which we identified by microsequencing as two Wnt related proteins, Ku86 autoantigen, signal recognition particle subunits SRP68/72, the receptor for complement component gC1q-R, and ribosomal proteins S4/S6. Interestingly, some of the surface-nucleolin associated proteins are implicated in cell signaling, tumor cell adhesion, migration, invasion, cell death, autoimmunity, and bacterial infections. Surface nucleolin in the 500-kDa complex is highly stable. Surface nucleolin antagonists, HB-19 and related multivalent Nucant pseudopeptides, exert distinct inhibitory mechanisms depending on the malignant tumor cell type. For example, in epithelial tumor cells they inhibit cell adhesion or spreading and induce reversion of the malignant phenotype (BMC cancer 2010, 10:325 while in leukemia cells they trigger a rapid cell death associated with DNA fragmentation. The fact that these pseudopeptides do not cause cell death in epithelial tumor cells indicates that cell

  6. Δ9-Tetrahydrocannabinol-mediated epigenetic modifications elicit myeloid-derived suppressor cell activation via STAT3/S100A8.

    Science.gov (United States)

    Sido, Jessica Margaret; Yang, Xiaoming; Nagarkatti, Prakash S; Nagarkatti, Mitzi

    2015-04-01

    MDSCs are potent immunosuppressive cells that are induced during inflammatory responses, as well as by cancers, to evade the anti-tumor immunity. We recently demonstrated that marijuana cannabinoids are potent inducers of MDSCs. In the current study, we investigated the epigenetic mechanisms through which THC, an exogenous cannabinoid, induces MDSCs and compared such MDSCs with the naïve MDSCs found in BM of BL6 (WT) mice. Administration of THC into WT mice caused increased methylation at the promoter region of DNMT3a and DNMT3b in THC-induced MDSCs, which correlated with reduced expression of DNMT3a and DNMT3b. Furthermore, promoter region methylation was decreased at Arg1 and STAT3 in THC-induced MDSCs, and consequently, such MDSCs expressed higher levels of Arg1 and STAT3. In addition, THC-induced MDSCs secreted elevated levels of S100A8, a calcium-binding protein associated with accumulation of MDSCs in cancer models. Neutralization of S100A8 by use of anti-S100A8 (8H150) in vivo reduced the ability of THC to trigger MDSCs. Interestingly, the elevated S100A8 expression also promoted the suppressive function of MDSCs. Together, the current study demonstrates that THC mediates epigenetic changes to promote MDSC differentiation and function and that S100A8 plays a critical role in this process. PMID:25713087

  7. Transient and sustained afterdepolarizations in accessory olfactory bulb mitral cells are mediated by distinct mechanisms that are differentially regulated by neuromodulators

    Directory of Open Access Journals (Sweden)

    Guy eShpak

    2015-01-01

    Full Text Available Social interactions between mammalian conspecifics rely heavily on molecular communication via the main and accessory olfactory systems. These two chemosensory systems show high similarity in the organization of information flow along their early stages: social chemical cues are detected by the sensory neurons of the main olfactory epithelium and the vomeronasal organ. These neurons then convey sensory information to the main (MOB and accessory (AOB olfactory bulbs, respectively, where they synapse upon mitral cells that project to higher brain areas. Yet, the functional difference between these two chemosensory systems remains unclear. We have previously shown that MOB and AOB mitral cells exhibit very distinct intrinsic biophysical properties leading to different types of information processing. Specifically, we found that unlike MOB mitral cells, AOB neurons display persistent firing responses to strong stimuli. These prolonged responses are mediated by long-lasting calcium-activated non-selective cationic current (Ican. In the current study we further examined the firing characteristics of these cells and their modulation by several neuromodulators. We found that AOB mitral cells display transient depolarizing afterpotentials (DAPs following moderate firing. These DAPs are not found in MOB mitral cells that show instead robust hyperpolarizing afterpotentials. Unlike Ican, the DAPs of AOB mitral cells are activated by low levels of intracellular calcium and are relatively insensitive to flufenamic acid. Moreover, the cholinergic agonist carbachol exerts opposite effects on the persistent firing and DAPs of AOB mitral cells. We conclude that these phenomena are mediated by distinct biophysical mechanisms that may serve to mediate different types of information processing in the AOB at distinct brain states.

  8. Transient and sustained afterdepolarizations in accessory olfactory bulb mitral cells are mediated by distinct mechanisms that are differentially regulated by neuromodulators.

    Science.gov (United States)

    Shpak, Guy; Zylbertal, Asaph; Wagner, Shlomo

    2014-01-01

    Social interactions between mammalian conspecifics rely heavily on molecular communication via the main and accessory olfactory systems. These two chemosensory systems show high similarity in the organization of information flow along their early stages: social chemical cues are detected by the sensory neurons of the main olfactory epithelium and the vomeronasal organ. These neurons then convey sensory information to the main (MOB) and accessory (AOB) olfactory bulbs, respectively, where they synapse upon mitral cells that project to higher brain areas. Yet, the functional difference between these two chemosensory systems remains unclear. We have previously shown that MOB and AOB mitral cells exhibit very distinct intrinsic biophysical properties leading to different types of information processing. Specifically, we found that unlike MOB mitral cells, AOB neurons display persistent firing responses to strong stimuli. These prolonged responses are mediated by long-lasting calcium-activated non-selective cationic current (Ican). In the current study we further examined the firing characteristics of these cells and their modulation by several neuromodulators. We found that AOB mitral cells display transient depolarizing afterpotentials (DAPs) following moderate firing. These DAPs are not found in MOB mitral cells that show instead robust hyperpolarizing afterpotentials. Unlike Ican, the DAPs of AOB mitral cells are activated by low levels of intracellular calcium and are relatively insensitive to flufenamic acid. Moreover, the cholinergic agonist carbachol exerts opposite effects on the persistent firing and DAPs of AOB mitral cells. We conclude that these phenomena are mediated by distinct biophysical mechanisms that may serve to mediate different types of information processing in the AOB at distinct brain states. PMID:25642164

  9. Intra-articular nuclear factor-κB blockade ameliorates collagen-induced arthritis in mice by eliciting regulatory T cells and macrophages.

    Science.gov (United States)

    Min, S-Y; Yan, M; Du, Y; Wu, T; Khobahy, E; Kwon, S-R; Taneja, V; Bashmakov, A; Nukala, S; Ye, Y; Orme, J; Sajitharan, D; Kim, H-Y; Mohan, C

    2013-05-01

    Nuclear factor (NF)-κB is a transcription factor implicated in the pathogenesis of autoimmune disorders such as rheumatoid arthritis (RA). Here we have examined the effect of intra-articular administration of the IKK inhibitor, NEMO-binding domain peptide (NBD), on the severity of collagen-induced arthritis (CIA). NBD peptides were injected intra-articularly into the knee joints of DBA/1J mice after the onset of disease. Collagen-injected mice given a scrambled peptide served as controls. Arthritis severity was determined by visual examination of paws. Intra-articular NBD injection reduced the arthritis score and ameliorated morphological signs of bone destruction compared to the controls. Serum levels of type-II collagen-specific immunoglobulin (Ig)G2a antibodies were lower in NBD-treated mice versus the control mice, whereas the levels of type-II collagen-specific IgG1 antibodies were increased by NBD treatment. NBD treatment diminished the proinflammatory cytokines interleukin (IL)-17 and interferon (IFN)-γ in serum, but increased the regulatory cytokine IL-10. NBD-treated CIA mice exhibited significantly higher percentages and numbers of forkhead box protein 3 (FoxP3(+)) CD4(+) CD25(+) regulatory T cells than controls. Immunofluorescence analysis of NBD-treated mice revealed that FoxP3 and Ym1, a marker of alternatively activated macrophages, were juxtaposed to each other within draining inguinal lymph nodes. Intra-articular administration of NBD peptide is effective as an experimental therapy in a murine model of RA. Nevertheless, the intra-articular treatment modality is still associated with systemic effects on the immune system. PMID:23574318

  10. Identification of a distinct small cell population from human bone marrow reveals its multipotency in vivo and in vitro.

    Directory of Open Access Journals (Sweden)

    James Wang

    Full Text Available Small stem cells, such as spore-like cells, blastomere-like stem cells (BLSCs, and very-small embryonic-like stem cells (VSELs have been described in recent studies, although their multipotency in human tissues has not yet been confirmed. Here, we report the discovery of adult multipotent stem cells derived from human bone marrow, which we call StemBios (SB cells. These isolated SB cells are smaller than 6 ìm and are DAPI+ and Lgr5+ (Leucine-Rich Repeat Containing G Protein-Coupled Receptor 5. Because Lgr5 has been characterized as a stem cell marker in the intestine, we hypothesized that SB cells may have a similar function. In vivo cell tracking assays confirmed that SB cells give rise to three types of cells, and in vitro studies demonstrated that SB cells cultured in proprietary media are able to grow to 6-25 ìm in size. Once the SB cells have attached to the wells, they differentiate into different cell lineages upon exposure to specific differentiation media. We are the first to demonstrate that stem cells smaller than 6 ìm can differentiate both in vivo and in vitro. In the future, we hope that SB cells will be used therapeutically to cure degenerative diseases.

  11. Rat bone marrow-derived dendritic cells generated with GM-CSF/IL-4 or FLT3L exhibit distinct phenotypical and functional characteristics.

    Science.gov (United States)

    N'diaye, Marie; Warnecke, Andreas; Flytzani, Sevasti; Abdelmagid, Nada; Ruhrmann, Sabrina; Olsson, Tomas; Jagodic, Maja; Harris, Robert A; Guerreiro-Cacais, Andre Ortlieb

    2016-03-01

    Dendritic cells are professional APCs that play a central role in the initiation of immune responses. The limited ex vivo availability of dendritic cells inspires the widespread use of bone marrow-derived dendritic cells as an alternative in research. However, the functional characteristics of bone marrow-derived dendritic cells are incompletely understood. Therefore, we compared functional and phenotypic characteristics of rat bone marrow-derived dendritic cells generated with GM-CSF/IL-4 or FLT3 ligand bone marrow-derived dendritic cells. A comparison of surface markers revealed that FLT3 ligand-bone marrow-derived dendritic cells expressed signal regulatory protein α, CD103, and CD4 and baseline levels of MHC class II, CD40, and CD86, which were highly up-regulated upon stimulation. Conversely, GM-CSF/IL-4-bone marrow-derived dendritic cells constitutively expressed signal regulatory protein α, CD11c, and CD11b but only mildly up-regulated MHC class II, CD40, or CD86 following stimulation. Expression of dendritic cell-associated core transcripts was restricted to FLT3 ligand-bone marrow-derived dendritic cells . GM-CSF/IL-4-bone marrow-derived dendritic cells were superior at phagocytosis but were outperformed by FLT3 ligand-bone marrow-derived dendritic cells at antigen presentation and T cell stimulation in vitro. Stimulated GM-CSF/IL-4-bone marrow-derived dendritic cells secreted more TNF, CCL5, CCL20, and NO, whereas FLT3 ligand-bone marrow-derived dendritic cells secreted more IL-6 and IL-12. Finally, whereas GM-CSF/IL-4-bone marrow-derived dendritic cell culture supernatants added to resting T cell cultures promoted forkhead box p3(+) regulatory T cell populations, FLT3 ligand-bone marrow-derived dendritic cell culture supernatants drove Th17 differentiation. We conclude that rat GM-CSF/IL-4-bone marrow-derived dendritic cells and FLT3 ligand-bone marrow-derived dendritic cells are functionally distinct. Our data support the current rationale that FLT3

  12. Probenecid-resistant J774 cell expression of enhanced organic anion transport by a mechanism distinct from multidrug resistance.

    Science.gov (United States)

    Cao, C; Steinberg, T H; Neu, H C; Cohen, D; Horwitz, S B; Hickman, S; Silverstein, S C

    1993-08-01

    Macrophages possess organic anion transporters that carry membrane-impermeant fluorescent dyes, such as lucifer yellow (LY) and carboxy-fluorescein, from the cytoplasm into endosomes and out of the cells. Probenecid, an organic anion transport inhibitor, blocks these processes. Prolonged incubation of J774 cells in medium containing 2.5 mM probenecid eventually kills most of these cells. To identify J774 variants that express increased organic anion transport activity, we selected probenecid-resistant (PBR) J774 cells by growing them in medium containing increasing concentrations of probenecid. When PBR and unselected J774 cells were loaded with LY by ATP4- permeabilization, the amount of LY accumulated by the PBR cells was about half that in the unselected cells. This difference was abolished by adding 10 mM probenecid to the medium in which the cells were loaded, suggesting that the diminished LY accumulation in PBR cells was due to enhanced LY secretion and that the PBR cells expressed increased organic anion transport activity. Direct comparison of LY efflux from J774 and PBR J774 cells showed a faster initial rate of secretion of LY from PBR J774 cells than from unselected J774 cells. To determine whether LY efflux is mediated by P-glycoprotein, we compared LY efflux in unselected J774 cells, PBR J774 cells, and multidrug-resistant J774 cells (J7.C1). LY efflux from J7.C1 cells was not sensitive to verapamil, which inhibits multidrug-resistance transporters, and reverses the multidrug-resistant phenotype of J7.C1 cells. The rates of LY efflux from unselected J774 and J7.C1 cells were virtually identical.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7909709

  13. Downregulation of the autophagy protein ATG-7 correlates with distinct sphingolipid profile in MCF-7 cells sensitized to photodamage

    Science.gov (United States)

    Separovic, Duska; Kelekar, Ameeta; Tarca, Adi L.; Bielawski, Jacek; Kessel, David

    2009-06-01

    The objective of this study was to determine the sphingolipid (SL) profile in autophagy-defective cells and overall cell death after PDT with Pc 4 (PDT). Human breast cancer MCF-7 cells with downregulated autophagy protein ATG-7 and their scrambled controls (Scr) were used. Exposure of ATG-7 knockdown cells to PDT led to defective processing of the autophagy marker LC3, and increased overall cell killing. In both cell types PDT evoked an early (2 h) increase in ceramides and dihydroceramides (DHceramides). When the two cell types were compared regarding time (2 and 24 h) and treatment conditions (with and without PDT), the levels of several ceramides and DHceramides were reduced, whereas the concentrations of C14-ceramide, C16-ceramide and C12-DHceramide were higher in ATG-7 knockdown cells. The data imply that the SL profile might be a marker of autophagy-deficiency in cells sensitized to PDT.

  14. Characterization of distinct mesenchymal-like cell populations from human skeletal muscle in situ and in vitro

    International Nuclear Information System (INIS)

    Human skeletal muscle is an essential source of various cellular progenitors with potential therapeutic perspectives. We first used extracellular markers to identify in situ the main cell types located in a satellite position or in the endomysium of the skeletal muscle. Immunohistology revealed labeling of cells by markers of mesenchymal (CD13, CD29, CD44, CD47, CD49, CD62, CD73, CD90, CD105, CD146, and CD15 in this study), myogenic (CD56), angiogenic (CD31, CD34, CD106, CD146), hematopoietic (CD10, CD15, CD34) lineages. We then analysed cell phenotypes and fates in short- and long-term cultures of dissociated muscle biopsies in a proliferation medium favouring the expansion of myogenic cells. While CD56+ cells grew rapidly, a population of CD15+ cells emerged, partly from CD56+ cells, and became individualized. Both populations expressed mesenchymal markers similar to that harboured by human bone marrow-derived mesenchymal stem cells. In differentiation media, both CD56+ and CD15+ cells shared osteogenic and chondrogenic abilities, while CD56+ cells presented a myogenic capacity and CD15+ cells presented an adipogenic capacity. An important proportion of cells expressed the CD34 antigen in situ and immediately after muscle dissociation. However, CD34 antigen did not persist in culture and this initial population gave rise to adipogenic cells. These results underline the diversity of human muscle cells, and the shared or restricted commitment abilities of the main lineages under defined conditions.

  15. Genetic and Epigenetic Tumor Suppressor Gene Silencing Are Distinct Molecular Phenotypes Driven by Growth Promoting Mutations in Nonsmall Cell Lung Cancer

    OpenAIRE

    MARSIT, CARMEN J.; E. Andres Houseman; Nelson, Heather H; Karl T Kelsey

    2008-01-01

    Both genetic and epigenetic alterations characterize human nonsmall cell lung cancer (NSCLC), but the biological processes that create or select these alterations remain incompletely investigated. Our hypothesis posits that a roughly reciprocal relationship between the propensity for promoter hypermethylation and a propensity for genetic deletion leads to distinct molecular phenotypes of lung cancer. To test this hypothesis, we examined promoter hypermethylation of 17 tumor suppressor genes, ...

  16. Distinct Mechanisms of Receptor and Nonreceptor Tyrosine Kinase Activation by Reactive Oxygen Species in Vascular Smooth Muscle Cells: Role of Metalloprotease and Protein Kinase C-δ

    OpenAIRE

    Frank, Gerald D.; Mifune, Mizuo; Inagami, Tadashi; Ohba, Motoi; Sasaki, Terukatsu; Higashiyama, Shigeki; Dempsey, Peter J; Eguchi, Satoru

    2003-01-01

    Reactive oxygen species (ROS) are implicated in cardiovascular diseases. ROS, such as H2O2, act as second messengers to activate diverse signaling pathways. Although H2O2 activates several tyrosine kinases, including the epidermal growth factor (EGF) receptor, JAK2, and PYK2, in vascular smooth muscle cells (VSMCs), the intracellular mechanism by which ROS activate these tyrosine kinases remains unclear. Here, we identified two distinct signaling pathways required for receptor and nonreceptor...

  17. Distinct N-Glycan Glycosylation of P-glycoprotein Isolated from the Human Uterine Sarcoma Cell Line MES-SA/D×5

    OpenAIRE

    Greer, D.A.; Ivey, S.

    2007-01-01

    The uterine sarcoma human cell line MES-SA/D×5 overexpresses the MDR1 gene product, P-glycoprotein (Pgp). Pgp is a heavily glycosylated, ATP-dependent drug efflux pump expressed in many human cancers. There are more than 50 known isoforms of Pgp, which complicates the characterization of Pgp glycans because each isoform could present a different glycome. The contribution of these oligosaccharides to the structure and function of Pgp remains unclear. We identified distinct Pgp glycans recogniz...

  18. Distinct early molecular responses to mutations causing vLINCL and JNCL presage ATP synthase subunit C accumulation in cerebellar cells.

    Directory of Open Access Journals (Sweden)

    Yi Cao

    Full Text Available Variant late-infantile neuronal ceroid lipofuscinosis (vLINCL, caused by CLN6 mutation, and juvenile neuronal ceroid lipofuscinosis (JNCL, caused by CLN3 mutation, share clinical and pathological features, including lysosomal accumulation of mitochondrial ATP synthase subunit c, but the unrelated CLN6 and CLN3 genes may initiate disease via similar or distinct cellular processes. To gain insight into the NCL pathways, we established murine wild-type and CbCln6(nclf/nclf cerebellar cells and compared them to wild-type and CbCln3(Δex7/8/Δex7/8 cerebellar cells. CbCln6(nclf/nclf cells and CbCln3(Δex7/8/Δex7/8 cells both displayed abnormally elongated mitochondria and reduced cellular ATP levels and, as cells aged to confluence, exhibited accumulation of subunit c protein in Lamp 1-positive organelles. However, at sub-confluence, endoplasmic reticulum PDI immunostain was decreased only in CbCln6(nclf/nclf cells, while fluid-phase endocytosis and LysoTracker® labeled vesicles were decreased in both CbCln6(nclf/nclf and CbCln3(Δex7/8/Δex7/8 cells, though only the latter cells exhibited abnormal vesicle subcellular distribution. Furthermore, unbiased gene expression analyses revealed only partial overlap in the cerebellar cell genes and pathways that were altered by the Cln3(Δex7/8 and Cln6(nclf mutations. Thus, these data support the hypothesis that CLN6 and CLN3 mutations trigger distinct processes that converge on a shared pathway, which is responsible for proper subunit c protein turnover and neuronal cell survival.

  19. Investigating Oxidative Stress and Inflammatory Responses Elicited by Silver Nanoparticles Using High-Throughput Reporter Genes in HepG2 Cells: Effect of Size, Surface Coating, and Intracellular Uptake

    Science.gov (United States)

    Abstract Silver nanoparticles (Ag NP) have been shown to generate reactive oxygen species; however, the association between physicochemical characteristics of nanoparticles and cellular stress responses elicited by exposure has not been elucidated. Here, we examined three key...

  20. Differential regulation of vitamin D receptor expression in distinct leukemic cell lines upon phorbol ester-induced growth arrest

    Directory of Open Access Journals (Sweden)

    Folgueira M.A.A.K.

    2000-01-01

    Full Text Available A close correlation between vitamin D receptor (VDR abundance and cell proliferation rate has been shown in NIH-3T3 fibroblasts, MCF-7 breast cancer and in HL-60 myeloblastic cells. We have now determined if this association occurs in other leukemic cell lines, U937 and K562, and if VDR content is related to c-myc expression, which is also linked to cell growth state. Upon phorbol myristate acetate (PMA treatment, cells from the three lineages (HL-60, U937 and K562 differentiated and expressed specific surface antigens. All cell lines analyzed were growth inhibited by PMA and the doubling time was increased, mainly due to an increased fraction of cells in the G0/G1 phase, as determined by flow cytometry measurements of incorporated bromodeoxyuridine and cell DNA content. C-myc mRNA expression was down-regulated and closely correlated to cell growth arrest. However, VDR expression in leukemic cell lines, as determined by immunofluorescence and Northern blot assays, was not consistently changed upon inhibition of cell proliferation since VDR levels were down-regulated only in HL-60 cells. Our data suggest that VDR expression cannot be explained simply as a reflection of the leukemic cell growth state.

  1. Conditional IFNAR1 ablation reveals distinct requirements of Type I IFN signaling for NK cell maturation and tumor surveillance

    Science.gov (United States)

    Mizutani, Tatsuaki; Neugebauer, Nina; Putz, Eva M.; Moritz, Nadine; Simma, Olivia; Zebedin-Brandl, Eva; Gotthardt, Dagmar; Warsch, Wolfgang; Eckelhart, Eva; Kantner, Hans-Peter; Kalinke, Ulrich; Lienenklaus, Stefan; Weiss, Siegfried; Strobl, Birgit; Müller, Mathias; Sexl, Veronika; Stoiber, Dagmar

    2012-01-01

    Mice with an impaired Type I interferon (IFN) signaling (IFNAR1- and IFNβ-deficient mice) display an increased susceptibility toward v-ABL-induced B-cell leukemia/lymphoma. The enhanced leukemogenesis in the absence of an intact Type I IFN signaling is caused by alterations within the tumor environment. Deletion of Ifnar1 in tumor cells (as obtained in Ifnar1f/f CD19-Cre mice) failed to impact on disease latency or type. In line with this observation, the initial transformation and proliferative capacity of tumor cells were unaltered irrespective of whether the cells expressed IFNAR1 or not. v-ABL-induced leukemogenesis is mainly subjected to natural killer (NK) cell-mediated tumor surveillance. Thus, we concentrated on NK cell functions in IFNAR1 deficient animals. Ifnar1-/- NK cells displayed maturation defects as well as an impaired cytolytic activity. When we deleted Ifnar1 selectively in mature NK cells (by crossing Ncr1-iCre mice to Ifnar1f/f animals), maturation was not altered. However, NK cells derived from Ifnar1f/f Ncr1-iCre mice showed a significant cytolytic defect in vitro against the hematopoietic cell lines YAC-1 and RMA-S, but not against the melanoma cell line B16F10. Interestingly, this defect was not related to an in vivo phenotype as v-ABL-induced leukemogenesis was unaltered in Ifnar1f/f Ncr1-iCre compared with Ifnar1f/f control mice. Moreover, the ability of Ifnar1f/f Ncr1-iCre NK cells to kill B16F10 melanoma cells was unaltered, both in vitro and in vivo. Our data reveal that despite the necessity for Type I IFN in NK cell maturation the expression of IFNAR1 on mature murine NK cells is not required for efficient tumor surveillance. PMID:23170251

  2. Distinct microRNA expression profile and targeted biological pathways in functional myeloid-derived suppressor cells induced by Δ9-tetrahydrocannabinol in vivo: regulation of CCAAT/enhancer-binding protein α by microRNA-690.

    Science.gov (United States)

    Hegde, Venkatesh L; Tomar, Sunil; Jackson, Austin; Rao, Roshni; Yang, Xiaoming; Singh, Udai P; Singh, Narendra P; Nagarkatti, Prakash S; Nagarkatti, Mitzi

    2013-12-27

    Δ(9)-Tetrahydrocannabinol (THC), the major bioactive component of marijuana, has been shown to induce functional myeloid-derived suppressor cells (MDSCs) in vivo. Here, we studied the involvement of microRNA (miRNA) in this process. CD11b(+)Gr-1(+) MDSCs were purified from peritoneal exudates of mice administered with THC and used for genome-wide miRNA profiling. Expression of CD31 and Ki-67 confirmed that the THC-MDSCs were immature and proliferating. THC-induced MDSCs exhibited distinct miRNA expression signature relative to various myeloid cells and BM precursors. We identified 13 differentially expressed (>2-fold) miRNA in THC-MDSCs relative to control BM precursors. In silico target prediction for these miRNA and pathway analysis using multiple bioinformatics tools revealed significant overrepresentation of Gene Ontology clusters within hematopoiesis, myeloid cell differentiation, and regulation categories. Insulin-like growth factor 1 signaling involved in cell growth and proliferation, and myeloid differentiation pathways were among the most significantly enriched canonical pathways. Among the differentially expressed, miRNA-690 was highly overexpressed in THC-MDSCs (∼16-fold). Transcription factor CCAAT/enhancer-binding protein α (C/EBPα) was identified as a potential functional target of miR-690. Supporting this, C/EBPα expression was attenuated in THC-MDSCs as compared with BM precursors and exhibited an inverse relation with miR-690. miR-690 knockdown using peptide nucleic acid-antagomiR was able to unblock and significantly increase C/EBPα expression establishing the functional link. Further, CD11b(+)Ly6G(+)Ly6C(+) and CD11b(+)Ly6G(-)Ly6C(+) purified subtypes showed high levels of miR-690 with attenuated C/EBPα expression. Moreover, EL-4 tumor-elicited MDSCs showed increased miR-690 expression. In conclusion, miRNA are significantly altered during the generation of functional MDSC from BM. Select miRNA such as miR-690 targeting genes involved in

  3. Genome-wide analyses of transcription factor GATA3-mediated gene regulation in distinct T cell types

    OpenAIRE

    Wei, Gang; Abraham, Brian J.; Yagi, Ryoji; Jothi, Raja; Cui, Kairong; Sharma, Suveena; Narlikar, Leelavati; Northrup, Daniel L.; Tang, Qingsong; Paul, William E.; Zhu, Jinfang; Zhao, Keji

    2011-01-01

    The transcription factor GATA3 plays an essential role during T cell development and T helper 2 (Th2) cell differentiation. To understand GATA3-mediated gene regulation, we identified genome-wide GATA3 binding sites in ten well-defined developmental and effector T lymphocyte lineages. In the thymus, GATA3 directly regulated many critical factors, including Th-POK, Notch1 and T cell receptor subunits. In the periphery, GATA3 induced a large number of Th2 cell-specific as well as Th2 cell non-s...

  4. Characterization of distinct mesenchymal-like cell populations from human skeletal muscle in situ and in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Lecourt, Severine, E-mail: severine.lecourt@sls.aphp.fr [UPMC/AIM UMR S 974, Groupe Hospitalier Pitie-Salpetriere, Paris (France); INSERM U974, Groupe Hospitalier Pitie-Salpetriere, Paris (France); CNRS UMR 7215, Groupe Hospitalier Pitie-Salpetriere, Paris (France); Laboratoire de Therapie Cellulaire, Hopital Saint Louis, Paris (France); Marolleau, Jean-Pierre, E-mail: Marolleau.Jean-Pierre@chu-amiens.fr [Laboratoire de Therapie Cellulaire, Hopital Saint Louis, Paris (France); CHU Amiens Hopital Sud, Service d' Hematologie Clinique, UPJV, Amiens (France); Fromigue, Olivia, E-mail: olivia.fromigue@larib.inserm.fr [INSERM U606, Universite Paris 07, Hopital Lariboisiere, Paris (France); Vauchez, Karine, E-mail: k.vauchez@institut-myologie.org [UPMC/AIM UMR S 974, Groupe Hospitalier Pitie-Salpetriere, Paris (France); INSERM U974, Groupe Hospitalier Pitie-Salpetriere, Paris (France); CNRS UMR 7215, Groupe Hospitalier Pitie-Salpetriere, Paris (France); Genzyme S.A.S., Saint-Germain en Laye (France); Andriamanalijaona, Rina, E-mail: rinandria@yahoo.fr [Laboratoire de Biochimie des Tissus Conjonctifs, Faculte de Medecine, Caen (France); Ternaux, Brigitte, E-mail: brigitte.ternaux@orange.fr [Laboratoire de Therapie Cellulaire, Hopital Saint Louis, Paris (France); Lacassagne, Marie-Noelle, E-mail: mnlacassagne@free.fr [Laboratoire de Therapie Cellulaire, Hopital Saint Louis, Paris (France); Robert, Isabelle, E-mail: isa-robert@hotmail.fr [Laboratoire de Therapie Cellulaire, Hopital Saint Louis, Paris (France); Boumediene, Karim, E-mail: karim.boumediene@unicaen.fr [Laboratoire de Biochimie des Tissus Conjonctifs, Faculte de Medecine, Caen (France); Chereau, Frederic, E-mail: fchereau@pervasistx.com [Myosix S.A., Saint-Germain en Laye (France); Marie, Pierre, E-mail: pierre.marie@larib.inserm.fr [INSERM U606, Universite Paris 07, Hopital Lariboisiere, Paris (France); and others

    2010-09-10

    Human skeletal muscle is an essential source of various cellular progenitors with potential therapeutic perspectives. We first used extracellular markers to identify in situ the main cell types located in a satellite position or in the endomysium of the skeletal muscle. Immunohistology revealed labeling of cells by markers of mesenchymal (CD13, CD29, CD44, CD47, CD49, CD62, CD73, CD90, CD105, CD146, and CD15 in this study), myogenic (CD56), angiogenic (CD31, CD34, CD106, CD146), hematopoietic (CD10, CD15, CD34) lineages. We then analysed cell phenotypes and fates in short- and long-term cultures of dissociated muscle biopsies in a proliferation medium favouring the expansion of myogenic cells. While CD56{sup +} cells grew rapidly, a population of CD15{sup +} cells emerged, partly from CD56{sup +} cells, and became individualized. Both populations expressed mesenchymal markers similar to that harboured by human bone marrow-derived mesenchymal stem cells. In differentiation media, both CD56{sup +} and CD15{sup +} cells shared osteogenic and chondrogenic abilities, while CD56{sup +} cells presented a myogenic capacity and CD15{sup +} cells presented an adipogenic capacity. An important proportion of cells expressed the CD34 antigen in situ and immediately after muscle dissociation. However, CD34 antigen did not persist in culture and this initial population gave rise to adipogenic cells. These results underline the diversity of human muscle cells, and the shared or restricted commitment abilities of the main lineages under defined conditions.

  5. Transcriptome profiling of primary murine monocytes, lung macrophages and lung dendritic cells reveals a distinct expression of genes involved in cell trafficking

    Directory of Open Access Journals (Sweden)

    Lohmeyer Jürgen

    2009-01-01

    Full Text Available Abstract Background Peripheral blood monocytes (PBMo originate from the bone marrow, circulate in the blood and emigrate into various organs where they differentiate into tissue resident cellular phenotypes of the mononuclear phagocyte system, including macrophages (Mϕ and dendritic cells (DC. Like in other organs, this emigration and differentiation process is essential to replenish the mononuclear phagocyte pool in the lung under both inflammatory and non-inflammatory steady-state conditions. While many studies have addressed inflammation-driven monocyte trafficking to the lung, the emigration and pulmonary differentiation of PBMo under non-inflammatory conditions is much less understood. Methods In order to assess the transcriptional profile of circulating and lung resident mononuclear phagocyte phenotypes, PBMo, lung Mϕ and lung DC from naïve mice were flow-sorted to high purity, and their gene expression was compared by DNA microarrays on a genome-wide scale. Differential regulation of selected genes was validated by quantitative PCR and on protein level by flow cytometry. Results Differentially-expressed genes related to cell traffic were selected and grouped into the clusters (i matrix metallopeptidases, (ii chemokines/chemokine receptors, and (iii integrins. Expression profiles of clustered genes were further assessed at the mRNA and protein levels in subsets of circulating PBMo (GR1- vs GR1+ and lung resident macrophages (alveolar vs interstitial Mϕ. Our data identify differentially activated genetic programs in circulating monocytes and their lung descendents. Lung DC activate an extremely diverse set of gene families but largely preserve a mobile cell profile with high expression levels of integrin and chemokine/chemokine receptors. In contrast, interstitial and even more pronounced alveolar Mϕ, stepwise downregulate gene expression of these traffic relevant communication molecules, but strongly upregulate a distinct set of

  6. Combination therapy with vemurafenib (PLX4032/RG7204 and metformin in melanoma cell lines with distinct driver mutations

    Directory of Open Access Journals (Sweden)

    Recio Juan A

    2011-05-01

    Full Text Available Abstract Background A molecular linkage between the MAPK and the LKB1-AMPK energy sensor pathways suggests that combined MAPK oncogene inhibition and metabolic modulation of AMPK would be more effective than either manipulation alone in melanoma cell lines. Materials and methods The combination of the BRAF inhibitor vemurafenib (formerly PLX4032 and metformin were tested against a panel of human melanoma cell lines with defined BRAF and NRAS mutations for effects on viability, cell cycle and apoptosis. Signaling molecules in the MAPK, PI3K-AKT and LKB1-AMPK pathways were studied by Western blot. Results Single agent metformin inhibited proliferation in 12 out of 19 cell lines irrespective of the BRAF mutation status, but in one NRASQ61K mutant cell line it powerfully stimulated cell growth. Synergistic anti-proliferative effects of the combination of metformin with vemurafenib were observed in 6 out of 11 BRAFV600E mutants, including highly synergistic effects in two BRAFV600E mutant melanoma cell lines. Antagonistic effects were noted in some cell lines, in particular in BRAFV600E mutant cell lines resistant to single agent vemurafenib. Seven out of 8 BRAF wild type cell lines showed marginally synergistic anti-proliferative effects with the combination, and one cell line had highly antagonistic effects with the combination. The differential effects were not dependent on the sensitivity to each drug alone, effects on cell cycle or signaling pathways. Conclusions The combination of vemurafenib and metformin tended to have stronger anti-proliferative effects on BRAFV600E mutant cell lines. However, determinants of vemurafenib and metformin synergism or antagonism need to be understood with greater detail before any potential clinical utility of this combination.

  7. The TNF Family Molecules LIGHT and Lymphotoxin αβ Induce a Distinct Steroid-Resistant Inflammatory Phenotype in Human Lung Epithelial Cells.

    Science.gov (United States)

    da Silva Antunes, Ricardo; Madge, Lisa; Soroosh, Pejman; Tocker, Joel; Croft, Michael

    2015-09-01

    Lung epithelial cells are considered important sources of inflammatory molecules and extracellular matrix proteins that contribute to diseases such as asthma. Understanding the factors that stimulate epithelial cells may lead to new insights into controlling lung inflammation. This study sought to investigate the responsiveness of human lung epithelial cells to the TNF family molecules LIGHT and lymphotoxin αβ (LTαβ). Bronchial and alveolar epithelial cell lines, and primary human bronchial epithelial cells, were stimulated with LIGHT and LTαβ, and expression of inflammatory cytokines and chemokines and markers of epithelial-mesenchymal transition and fibrosis/remodeling was measured. LTβ receptor, the receptor shared by LIGHT and LTαβ, was constitutively expressed on all epithelial cells. Correspondingly, LIGHT and LTαβ strongly induced a limited but highly distinct set of inflammatory genes in all epithelial cells tested, namely the adhesion molecules ICAM-1 and VCAM-1; the chemokines CCL5, CCL20, CXCL1, CXCL3, CXCL5, and CXCL11; the cytokines IL-6, activin A and GM-CSF; and metalloproteinases matrix metalloproteinase-9 and a disintegrin and metalloproteinase domain-8. Importantly, induction of the majority of these inflammatory molecules was insensitive to the suppressive effects of the corticosteroid budesonide. LIGHT and LTαβ also moderately downregulated E-cadherin, a protein associated with maintaining epithelial integrity, but did not significantly drive production of extracellular matrix proteins or α-smooth muscle actin. Thus, LIGHT and LTαβ induce a distinct steroid-resistant inflammatory signature in airway epithelial cells via constitutively expressed LTβ receptor. These findings support our prior murine studies that suggested the receptors for LIGHT and LTαβ contribute to development of lung inflammation characteristic of asthma and idiopathic pulmonary fibrosis. PMID:26209626

  8. Distinct effect of CacyBP/SIP on the ERK1/2-CREB-BDNF pathway in undifferentiated and differentiated neuroblastoma NB2a cells.

    Science.gov (United States)

    Rosińska, Sara; Leśniak, Wiesława; Filipek, Anna

    2016-07-01

    CacyBP/SIP, a protein expressed to high extent in the brain, has been shown to act as ERK1/2 phosphatase in vitro and in cultured cells. It has been demonstrated recently that CacyBP/SIP can modulate the activity of some transcription factors in neurons and glioma cells. In the present work we have examined the effect of CacyBP/SIP overexpression and silencing on the phosphorylation/activity of ERK1/2 (pERK1/2) and CREB (pCREB) and on the level of BDNF mRNA in differentiated and undifferentiated neuroblastoma NB2a cells. We have shown that in undifferentiated cells the amount of pERK1/2 decreased upon CacyBP/SIP overexpression. Further studies have shown that the activity of CREB and the level of BDNF mRNA, downstream effectors of the ERK1/2 signaling pathway, also depended on the CacyBP/SIP level and strictly matched the level of pERK1/2. Interestingly, in differentiated NB2a cells, overexpression of CacyBP/SIP appeared to have a distinct effect on the pERK1/2 level from that observed in undifferentiated cells. Subsequent studies have revealed that distinct function of CacyBP/SIP in undifferentiated and differentiated NB2a cells might be due to changes in its posttranslational modifications and protein ligands. Altogether, our studies suggest that CacyBP/SIP is involved in the ERK1/2-CREB-BDNF pathway and that it might regulate this pathway depending on the stage of NB2a cell differentiation. PMID:27180052

  9. Fourier transform infrared spectroscopy for the distinction of MCF-7 cells treated with different concentrations of 5-fluorouracil

    OpenAIRE

    WU, BI-BO; Gong, Yi-Ping; Wu, Xin-Hong; Chen, Yuan-Yuan; Chen, Fang-Fang; Jin, Li-Ting; Cheng, Bo-Ran; Hu, Fen; Xiong, Bin

    2015-01-01

    Background In order to provide personalized treatment to patients with breast cancer, an accurate, reliable and cost-efficient analytical technique is needed for drug screening and evaluation of tumor response to chemotherapy. Methods Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was used as a tool to assess cancer cell response to chemotherapy. MCF-7 cells (human breast adenocarcinoma cell line) were treated with different concentrations of 5-fluorouracil (5...

  10. Analysis of Thalassiosira pseudonana Silicon Transporters Indicates Distinct Regulatory Levels and Transport Activity through the Cell Cycle▿

    OpenAIRE

    Thamatrakoln, Kimberlee; Hildebrand, Mark

    2006-01-01

    An analysis of the expression and activity of silicon transporters (SITs) was done on synchronously growing cultures of the diatom Thalassiosira pseudonana to provide insight into the role these proteins play in cellular silicon metabolism during the cell cycle. The first SIT-specific polyclonal peptide antibody was generated and used in the immunoblot analysis of whole-cell protein lysates to monitor SIT protein levels during synchronized progression through the cell cycle. Peaks in SIT prot...

  11. Mode of Bioenergetic Metabolism during B Cell Differentiation in the Intestine Determines the Distinct Requirement for Vitamin B1

    Directory of Open Access Journals (Sweden)

    Jun Kunisawa

    2015-10-01

    Full Text Available Bioenergetic metabolism varies during cell differentiation, but details of B cell metabolism remain unclear. Here, we show the metabolic changes during B cell differentiation in the intestine, where B cells differentiate into IgA+ plasma cells (PCs. Naive B cells in the Peyer’s patches (PPs and IgA+ PCs in the intestinal lamina propria (iLP both used the tricarboxylic acid (TCA cycle, but only IgA+ PCs underwent glycolysis. These metabolic differences reflected their dependencies on vitamin B1, an essential cofactor for the TCA cycle. Indeed, the diminished activity of the TCA cycle after dietary vitamin B1 depletion decreased the number of naive B cells in PPs without affecting IgA+ PCs in the iLP. The maintenance of naive B cells by dietary vitamin B1 was required to induce—but not maintain—intestinal IgA responses against oral antigens. These findings reveal the diet-mediated maintenance of B cell immunometabolism in organized and diffuse intestinal tissues.

  12. Distinct CPT-induced deaths in lung cancer cells caused by clathrin-mediated internalization of CP micelles

    Science.gov (United States)

    Liu, Yu-Sheng; Cheng, Ru-You; Lo, Yu-Lun; Hsu, Chin; Chen, Su-Hwei; Chiu, Chien-Chih; Wang, Li-Fang

    2016-02-01

    We previously synthesized a chondroitin sulfate-graft-poly(ε-caprolactone) copolymer (H-CP) with a high content of poly(ε-caprolactone) (18.7 mol%), which self-assembled in water into a rod-like micelle to encapsulate hydrophobic camptothecin (CPT) in the core (micelle/CPT) for tumor-targeted drug delivery. As a result of the recognition of the micelle by CD44, the micelle/CPT entered CRL-5802 cells efficiently and released CPT efficaciously, resulting in higher tumor suppression than commercial CPT-11. In this study, H1299 cells were found to have a higher CD44 expression than CRL-5802 cells. However, the lower CD44-expressing CRL-5802 cells had a higher percentage of cell death and higher cellular uptake of the micelle/CPT than the higher CD44-expressing H1299 cells. Examination of the internalization pathway of the micelle/CPT in the presence of different endocytic chemical inhibitors showed that the CRL-5802 cells involved clathrin-mediated endocytosis, which was not found in the H1299 cells. Analysis of the cell cycle of the two cell lines exposed to the micelle/CPT revealed that the CRL-5802 cells arrested mainly in the S phase and the H1299 cells arrested mainly in the G2-M phase. A consistent result was also found in the evaluation of γ-H2AX expression, which was about three-fold higher in the CRL-5802 cells than in the H1299 cells. A near-infrared dye, IR780, was encapsulated into the micelle to observe the in vivo biodistribution of the micelle/IR780 in tumor-bearing mice. The CRL-5802 tumor showed a higher fluorescence intensity than the H1299 tumor at any tracing time after 1 h. Thus we tentatively concluded that CRL-5802 cells utilized the clathrin-mediated internalization pathway and arrested in the S phase on exposure to the micelle/CPT; all are possible reasons for the better therapeutic outcome in CRL-5802 cells than in H1299 cells.We previously synthesized a chondroitin sulfate-graft-poly(ε-caprolactone) copolymer (H-CP) with a high content of

  13. Impact of HIV on CD8+ T cell CD57 expression is distinct from that of CMV and aging.

    Directory of Open Access Journals (Sweden)

    Sulggi A Lee

    Full Text Available Chronic antigenic stimulation by cytomegalovirus (CMV is thought to increase "immunosenesence" of aging, characterized by accumulation of terminally differentiated CD28- CD8+ T cells and increased CD57, a marker of proliferative history. Whether chronic HIV infection causes similar effects is currently unclear.We compared markers of CD8+ T cell differentiation (e.g., CD28, CD27, CCR7, CD45RA and CD57 expression on CD28- CD8+ T cells in healthy HIV-uninfected adults with and without CMV infection and in both untreated and antiretroviral therapy (ART-suppressed HIV-infected adults with asymptomatic CMV infection.Compared to HIV-uninfected adults without CMV (n=12, those with asymptomatic CMV infection (n=31 had a higher proportion of CD28-CD8+ T cells expressing CD57 (P=0.005. Older age was also associated with greater proportions of CD28-CD8+ T cells expressing CD57 (rho: 0.47, P=0.007. In contrast, untreated HIV-infected CMV+ participants (n=55 had much lower proportions of CD28- CD8+ cells expressing CD57 than HIV-uninfected CMV+ participants (P<0.0001 and were enriched for less well-differentiated CD28- transitional memory (TTR CD8+ T cells (P<0.0001. Chronically HIV-infected adults maintaining ART-mediated viral suppression (n=96 had higher proportions of CD28-CD8+ T cells expressing CD57 than untreated patients (P<0.0001, but continued to have significantly lower levels than HIV-uninfected controls (P=0.001. Among 45 HIV-infected individuals initiating their first ART regimen, the proportion of CD28-CD8+ T cells expressing CD57 declined (P<0.0001, which correlated with a decline in percent of transitional memory CD8+ T cells, and appeared to be largely explained by a decline in CD28-CD57- CD8+ T cell counts rather than an expansion of CD28-CD57+ CD8+ T cell counts.Unlike CMV and aging, which are associated with terminal differentiation and proliferation of effector memory CD8+ T cells, HIV inhibits this process, expanding less well

  14. Morphogenesis of respiratory syncytial virus in human primary nasal ciliated epithelial cells occurs at surface membrane microdomains that are distinct from cilia

    International Nuclear Information System (INIS)

    The distribution of cilia and the respiratory syncytial virus (RSV) nucleocapsid (N) protein, fusion (F) protein, attachment (G) protein, and M2-1 protein in human ciliated nasal epithelial cells was examined at between 1 and 5 days post-infection (dpi). All virus structural proteins were localized at cell surface projections that were distinct from cilia. The F protein was also trafficked into the cilia, and while its presence increased as the infection proceeded, the N protein was not detected in the cilia at any time of infection. The presence of the F protein in the cilia correlated with cellular changes in the cilia and reduced cilia function. At 5 dpi extensive cilia loss and further reduced cilia function was noted. These data suggested that although RSV morphogenesis occurs at non-cilia locations on ciliated nasal epithelial cells, RSV infection induces changes in the cilia body that leads to extensive cilia loss. - Highlights: • Respiratory syncytial virus (RSV) infects nasal ciliated epithelial cells. • Virus morphogenesis occurs within filamentous projections distinct from cilia. • The RSV N protein was not detected in the cilia at any time during infection. • Trafficking of the F protein into the cilia occurred early in infection. • Presence of the F protein in cilia correlated with impaired cilia function

  15. Morphogenesis of respiratory syncytial virus in human primary nasal ciliated epithelial cells occurs at surface membrane microdomains that are distinct from cilia

    Energy Technology Data Exchange (ETDEWEB)

    Jumat, Muhammad Raihan [School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Yan, Yan [Department of Otolaryngology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore 119228 (Singapore); Ravi, Laxmi Iyer [School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Wong, Puisan [Detection and Diagnostics Laboratory, DSO National Laboratories, 27 Medical Drive, Singapore 117510 (Singapore); Huong, Tra Nguyen [School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Li, Chunwei [Department of Otolaryngology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore 119228 (Singapore); Tan, Boon Huan [Detection and Diagnostics Laboratory, DSO National Laboratories, 27 Medical Drive, Singapore 117510 (Singapore); Wang, De Yun [Department of Otolaryngology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore 119228 (Singapore); Sugrue, Richard J., E-mail: rjsugrue@ntu.edu.sg [School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore)

    2015-10-15

    The distribution of cilia and the respiratory syncytial virus (RSV) nucleocapsid (N) protein, fusion (F) protein, attachment (G) protein, and M2-1 protein in human ciliated nasal epithelial cells was examined at between 1 and 5 days post-infection (dpi). All virus structural proteins were localized at cell surface projections that were distinct from cilia. The F protein was also trafficked into the cilia, and while its presence increased as the infection proceeded, the N protein was not detected in the cilia at any time of infection. The presence of the F protein in the cilia correlated with cellular changes in the cilia and reduced cilia function. At 5 dpi extensive cilia loss and further reduced cilia function was noted. These data suggested that although RSV morphogenesis occurs at non-cilia locations on ciliated nasal epithelial cells, RSV infection induces changes in the cilia body that leads to extensive cilia loss. - Highlights: • Respiratory syncytial virus (RSV) infects nasal ciliated epithelial cells. • Virus morphogenesis occurs within filamentous projections distinct from cilia. • The RSV N protein was not detected in the cilia at any time during infection. • Trafficking of the F protein into the cilia occurred early in infection. • Presence of the F protein in cilia correlated with impaired cilia function.

  16. Par1b links lumen polarity with LGN-NuMA positioning for distinct epithelial cell division phenotypes

    NARCIS (Netherlands)

    Lazaro-Dieguez, Francisco; Cohen, David; Fernandez, Dawn; Hodgson, Louis; van IJzendoorn, Sven C. D.; Muesch, Anne

    2013-01-01

    Columnar epithelia establish their luminal domains and their mitotic spindles parallel to the basal surface and undergo symmetric cell divisions in which the cleavage furrow bisects the apical domain. Hepatocyte lumina interrupt the lateral domain of neighboring cells perpendicular to two basal doma

  17. Unique and shared signaling pathways cooperate to regulate the differentiation of human CD4+ T cells into distinct effector subsets.

    Science.gov (United States)

    Ma, Cindy S; Wong, Natalie; Rao, Geetha; Nguyen, Akira; Avery, Danielle T; Payne, Kathryn; Torpy, James; O'Young, Patrick; Deenick, Elissa; Bustamante, Jacinta; Puel, Anne; Okada, Satoshi; Kobayashi, Masao; Martinez-Barricarte, Ruben; Elliott, Michael; Sebnem Kilic, Sara; El Baghdadi, Jamila; Minegishi, Yoshiyuki; Bousfiha, Aziz; Robertson, Nic; Hambleton, Sophie; Arkwright, Peter D; French, Martyn; Blincoe, Annaliesse K; Hsu, Peter; Campbell, Dianne E; Stormon, Michael O; Wong, Melanie; Adelstein, Stephen; Fulcher, David A; Cook, Matthew C; Stepensky, Polina; Boztug, Kaan; Beier, Rita; Ikincioğullari, Aydan; Ziegler, John B; Gray, Paul; Picard, Capucine; Boisson-Dupuis, Stéphanie; Phan, Tri Giang; Grimbacher, Bodo; Warnatz, Klaus; Holland, Steven M; Uzel, Gulbu; Casanova, Jean-Laurent; Tangye, Stuart G

    2016-07-25

    Naive CD4(+) T cells differentiate into specific effector subsets-Th1, Th2, Th17, and T follicular helper (Tfh)-that provide immunity against pathogen infection. The signaling pathways involved in generating these effector cells are partially known. However, the effects of mutations underlying human primary immunodeficiencies on these processes, and how they compromise specific immune responses, remain unresolved. By studying individuals with mutations in key signaling pathways, we identified nonredundant pathways regulating human CD4(+) T cell differentiation in vitro. IL12Rβ1/TYK2 and IFN-γR/STAT1 function in a feed-forward loop to induce Th1 cells, whereas IL-21/IL-21R/STAT3 signaling is required for Th17, Tfh, and IL-10-secreting cells. IL12Rβ1/TYK2 and NEMO are also required for Th17 induction. Strikingly, gain-of-function STAT1 mutations recapitulated the impact of dominant-negative STAT3 mutations on Tfh and Th17 cells, revealing a putative inhibitory effect of hypermorphic STAT1 over STAT3. These findings provide mechanistic insight into the requirements for human T cell effector function, and explain clinical manifestations of these immunodeficient conditions. Furthermore, they identify molecules that could be targeted to modulate CD4(+) T cell effector function in the settings of infection, vaccination, or immune dysregulation. PMID:27401342

  18. Genetic cell targeting uncovers specific neuronal types and distinct subregions in the bed nucleus of the stria terminalis.

    Science.gov (United States)

    Nguyen, Amanda Q; Dela Cruz, Julie A D; Sun, Yanjun; Holmes, Todd C; Xu, Xiangmin

    2016-08-15

    The bed nucleus of the stria terminalis (BNST) plays an important role in fear, stress, and anxiety. It contains a collection of subnuclei delineated by gross cytoarchitecture features; however, there has yet to be a systematic examination of specific BNST neuronal types and their associated neurochemical makeup. The present study focuses on improved characterization of the anterior BNST based on differing molecular and chemical expression aided by mouse genetics. Specific Cre driver lines crossed with a fluorescent reporter line were used for genetic cell targeting and immunochemical staining. Using this new approach, we were able to robustly identify specific excitatory and inhibitory cell types in the BNST. The presence and distribution of excitatory neurons were firmly established; glutamatergic neurons in the anterior BNST accounted for about 14% and 31% of dorsal and ventral BNST cells, respectively. GABAergic neurons expressing different isoforms of glutamic acid decarboxylase were found to have differential subregional distributions. Almost no parvalbumin-expressing cells were found in the BNST, while somatostatin-expressing cells and calretinin-expressing cells account for modest proportions of BNST cells. In addition, vasoactive intestinal peptide-expressing axonal plexuses were prominent in the oval and juxtacapsular subregions. In addition, we discovered that corticotropin-releasing hormone-expressing cells contain GABAergic and glutamatergic subpopulations. Together, this study reveals new information on excitatory and inhibitory neurons in the BNST, which will facilitate genetic dissection and functional studies of BNST subregions. J. Comp. Neurol. 524:2379-2399, 2016. © 2016 Wiley Periodicals, Inc. PMID:26718312

  19. Partial restoration of mutant enzyme homeostasis in three distinct lysosomal storage disease cell lines by altering calcium homeostasis.

    Directory of Open Access Journals (Sweden)

    Ting-Wei Mu

    2008-02-01

    Full Text Available A lysosomal storage disease (LSD results from deficient lysosomal enzyme activity, thus the substrate of the mutant enzyme accumulates in the lysosome, leading to pathology. In many but not all LSDs, the clinically most important mutations compromise the cellular folding of the enzyme, subjecting it to endoplasmic reticulum-associated degradation instead of proper folding and lysosomal trafficking. A small molecule that restores partial mutant enzyme folding, trafficking, and activity would be highly desirable, particularly if one molecule could ameliorate multiple distinct LSDs by virtue of its mechanism of action. Inhibition of L-type Ca2+ channels, using either diltiazem or verapamil-both US Food and Drug Administration-approved hypertension drugs-partially restores N370S and L444P glucocerebrosidase homeostasis in Gaucher patient-derived fibroblasts; the latter mutation is associated with refractory neuropathic disease. Diltiazem structure-activity studies suggest that it is its Ca2+ channel blocker activity that enhances the capacity of the endoplasmic reticulum to fold misfolding-prone proteins, likely by modest up-regulation of a subset of molecular chaperones, including BiP and Hsp40. Importantly, diltiazem and verapamil also partially restore mutant enzyme homeostasis in two other distinct LSDs involving enzymes essential for glycoprotein and heparan sulfate degradation, namely alpha-mannosidosis and type IIIA mucopolysaccharidosis, respectively. Manipulation of calcium homeostasis may represent a general strategy to restore protein homeostasis in multiple LSDs. However, further efforts are required to demonstrate clinical utility and safety.

  20. Self-enforcing Feedback Activation between BCL6 and Pre-B Cell Receptor Signaling Defines a Distinct Subtype of Acute Lymphoblastic Leukemia

    Science.gov (United States)

    Geng, Huimin; Hurtz, Christian; Lenz, Kyle B.; Chen, Zhengshan; Baumjohann, Dirk; Thompson, Sarah; Goloviznina, Natalya; Chen, Wei-Yi; Huan, Jianya; LaTocha, Dorian; Ballabio, Erica; Xiao, Gang; Lee, Jae-Woong; Deucher, Anne; Qi, Zhongxia; Park, Eugene; Huang, Chuanxin; Nahar, Rahul; Kweon, Soo-Mi; Shojaee, Seyedmehdi; Chan, Lai N.; Yu, Jingwei; Kornblau, Steven M.; Bijl, Janetta J.; Ye, B. Hilda; Ansel, Mark; Paietta, Elisabeth; Melnick, Ari; Hunger, Stephen P.; Kurre, Peter; Tyner, Jeffrey W.; Loh, Mignon L.; Roeder, Robert G.; Druker, Brian J.; Burger, Jan. A.; Milne, Thomas A.; Chang, Bill H.; Müschen, Markus

    2015-01-01

    SUMMARY Studying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR+ ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR+ ALL. PMID:25759025

  1. BRCA1-deficient breast cancer cell lines are resistant to MEK inhibitors and show distinct sensitivities to 6-thioguanine.

    Science.gov (United States)

    Gu, Yuexi; Helenius, Mikko; Väänänen, Kristiina; Bulanova, Daria; Saarela, Jani; Sokolenko, Anna; Martens, John; Imyanitov, Evgeny; Kuznetsov, Sergey

    2016-01-01

    Germ-line or somatic inactivation of BRCA1 is a defining feature for a portion of human breast cancers. Here we evaluated the anti-proliferative activity of 198 FDA-approved and experimental drugs against four BRCA1-mutant (HCC1937, MDA-MB-436, SUM1315MO2, and SUM149PT) and four BRCA1-wild-type (MDA-MB-231, SUM229PE, MCF10A, and MCF7) breast cancer cell lines. We found that all BRCA1-mutant cell lines were insensitive to inhibitors of mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) Selumetinib and Pimasertib in contrast to BRCA1-wildtype control cell lines. However, unexpectedly, only two BRCA1-mutant cell lines, HCC1937 and MDA-MB-436, were hypersensitive to a nucleotide analogue 6-thioguanine (6-TG). SUM149PT cells readily formed radiation-induced RAD51-positive nuclear foci indicating a functional homologous recombination, which may explain their resistance to 6-TG. However, the reason underlying 6-TG resistance of SUM1315MO2 cells remains unclear. Our data reveal a remarkable heterogeneity among BRCA1-mutant cell lines and provide a reference for future studies. PMID:27313062

  2. Snai2 and Snai3 transcriptionally regulate cellular fitness and functionality of T cell lineages through distinct gene programs.

    Science.gov (United States)

    Pioli, Peter D; Whiteside, Sarah K; Weis, Janis J; Weis, John H

    2016-05-01

    T lymphocytes are essential contributors to the adaptive immune system and consist of multiple lineages that serve various effector and regulatory roles. As such, precise control of gene expression is essential to the proper development and function of these cells. Previously, we identified Snai2 and Snai3 as being essential regulators of immune tolerance partly due to the impaired function of CD4(+) regulatory T cells in Snai2/3 conditional double knockout mice. Here we extend those previous findings using a bone marrow transplantation model to provide an environmentally unbiased view of the molecular changes imparted onto various T lymphocyte populations once Snai2 and Snai3 are deleted. The data presented here demonstrate that Snai2 and Snai3 transcriptionally regulate the cellular fitness and functionality of not only CD4(+) regulatory T cells but effector CD8(α+) and CD4(+) conventional T cells as well. This is achieved through the modulation of gene sets unique to each cell type and includes transcriptional targets relevant to the survival and function of each T cell lineage. As such, Snai2 and Snai3 are essential regulators of T cell immunobiology. PMID:26831822

  3. Discrete levels of Twist activity are required to direct distinct cell functions during gastrulation and somatic myogenesis.

    Directory of Open Access Journals (Sweden)

    Ming-Ching Wong

    Full Text Available Twist (Twi, a conserved basic helix-loop-helix transcriptional regulator, directs the epithelial-to-mesenchymal transition (EMT, and regulates changes in cell fate, cell polarity, cell division and cell migration in organisms from flies to humans. Analogous to its role in EMT, Twist has been implicated in metastasis in numerous cancer types, including breast, pancreatic and prostate. In the Drosophila embryo, Twist is essential for discrete events in gastrulation and mesodermal patterning. In this study, we derive a twi allelic series by examining the various cellular events required for gastrulation in Drosophila. By genetically manipulating the levels of Twi activity during gastrulation, we find that coordination of cell division is the most sensitive cellular event, whereas changes in cell shape are the least sensitive. Strikingly, we show that by increasing levels of Snail expression in a severe twi hypomorphic allelic background, but not a twi null background, we can reconstitute gastrulation and produce viable adult flies. Our results demonstrate that the level of Twi activity determines whether the cellular events of ventral furrow formation, EMT, cell division and mesodermal migration occur.

  4. The role of endogenous proteins in the protein-free maintenance of 3 distinct tumor-cell lines invitro.

    Science.gov (United States)

    Watanabe, H; Chigira, M

    1992-09-01

    We established new two protein-free culture subclones from murine well-characterized Ehrlich ascites carcinoma and P815 mastocytoma using intermittent protein-free culture performed previously for a protein-free subclone of fibrosarcoma (Gc-4 PF). The Ehrlich protein-free subclone (Ehrlich PF) grew much more slowly than the original cell line and showed a proliferative response to FCS. On the other hand, like Gc-4 PF, the P815 protein-free subclone (P815 PF) showed a similar growth rate to that of the original counterpart. Interestingly the original P815 mastocytoma cells also grew exponentially in protein-free medium. Although the protein-free culture exhibited cells that were more spheroid and spread less in each of these three cell lines, the major structure protein bands demonstrated on SDS-PAGE were virtually identical between the original and protein-free culture cells. In contrast to the structural peptides, the distribution of the secretory peptide differed among the three protein-free culture cell lines, which may reflect their state of differentiation. Growth-inhibiting activities were detected from the supernatant of all three protein-free culture cells, while no protein-free culture cells secreted predominantly growth-stimulating activity into their cultured media. These results suggest that autonomy in tumor cell proliferation may result from the acquisition of the ability to escape from negative control in multicellular organisms, as shown in monads, rather than an acquisition of further response to growth-stimulating control. PMID:21584570

  5. Requirements Elicitation Problems: A Literature Analysis

    Directory of Open Access Journals (Sweden)

    Bill Davey

    2015-06-01

    Full Text Available Requirements elicitation is the process through which analysts determine the software requirements of stakeholders. Requirements elicitation is seldom well done, and an inaccurate or incomplete understanding of user requirements has led to the downfall of many software projects. This paper proposes a classification of problem types that occur in requirements elicitation. The classification has been derived from a literature analysis. Papers reporting on techniques for improving requirements elicitation practice were examined for the problem the technique was designed to address. In each classification the most recent or prominent techniques for ameliorating the problems are presented. The classification allows the requirements engineer to be sensitive to problems as they arise and the educator to structure delivery of requirements elicitation training.

  6. Immortalization of distinct human mammary epithelial cell types by human papilloma virus 16 E6 or E7.

    OpenAIRE

    Wazer, D E; X. L. Liu; Chu, Q.; Gao, Q.; Band, V

    1995-01-01

    Multiple mammary epithelial cell (MEC) types are observed both in mammary ducts in vivo and in primary cultures in vitro; however, the oncogenic potential of different cell types remains unknown. Here, we used human papilloma virus 16 E6 and E7 oncogenes, which target p53 and Rb tumor suppressor proteins, respectively, to immortalize MECs present in early or late passages of human mammary tissue-derived cultures or in milk. One MEC subtype was exclusively immortalized by E6; such cells predom...

  7. Characterization of a distinct population of circulating human non-adherent endothelial forming cells and their recruitment via intercellular adhesion molecule-3.

    Directory of Open Access Journals (Sweden)

    Sarah L Appleby

    Full Text Available Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133(+ population of non-adherent endothelial forming cells (naEFCs which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38 together with mature endothelial cell markers (VEGFR2, CD144 and CD31. These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8 or myeloid markers (CD11b and CD14 which distinguishes them from 'early' endothelial progenitor cells (EPCs. Functional studies demonstrated that these naEFCs (i bound Ulex europaeus lectin, (ii demonstrated acetylated-low density lipoprotein uptake, (iii increased vascular cell adhesion molecule (VCAM-1 surface expression in response to tumor necrosis factor and (iv in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs. Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis.

  8. Reduced Wall Acetylation Proteins Play Vital and Distinct Roles in Cell Wall O-Acetylation in Arabidopsis

    DEFF Research Database (Denmark)

    Manabe, Yuzuki; Verhertbruggen, Yves; Gille, Sascha;

    2013-01-01

    The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwa2, which has about 20% lower level of O-acetylation in leaf cell walls and no obvious growth or developmental phenotype. In this study, we generated double...... quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco) mannan, and xyloglucan as well as overall cell wall acetylation is affected differently...... in different combinations of triple mutants, suggesting their diversity in substrate preference. The overall degree of wall acetylation in the rwa quadruple mutant was reduced by 63% compared with the wild type, and histochemical analysis of the rwa quadruple mutant stem indicates defects in cell...

  9. Distinct regions of loss of heterozygosity on 22q in different sites of head and neck squamous cell carcinomas

    DEFF Research Database (Denmark)

    dos Reis, Patricia Pintor; Poli-Frederico, Regina Célia; dos Santos, Rodrigo Mattos;

    2002-01-01

    laryngeal, and 31 pharyngeal carcinomas. RESULTS: Two separate regions of LOH were identified in the laryngeal (22q11.2-12.1) and oral cavity (22q13.1-13.31) tumors. When the different anatomical sites were compared, a statistically significant difference was found between the presence of LOH at D22S421 (p......<0.001), D22S315 (p=0.014) and D22S929 (p=0.026) in the laryngeal tumors. CONCLUSIONS: These data suggest that distinct regions on 22q are involved in LOH in oral cavity and laryngeal tumorigenesis, but do not support a similar association between the development of pharyngeal tumors and genes...

  10. Distinct Mechanisms of Ferritin Delivery to Lysosomes in Iron-Depleted and Iron-Replete Cells

    OpenAIRE

    Asano, Takeshi; Komatsu, Masaaki; Yamaguchi-Iwai, Yuko; Ishikawa, Fuyuki; Mizushima, Noboru; Iwai, Kazuhiro

    2011-01-01

    Ferritin is a cytosolic protein that stores excess iron, thereby protecting cells from iron toxicity. Ferritin-stored iron is believed to be utilized when cells become iron deficient; however, the mechanisms underlying the extraction of iron from ferritin have yet to be fully elucidated. Here, we demonstrate that ferritin is degraded in the lysosome under iron-depleted conditions and that the acidic environment of the lysosome is crucial for iron extraction from ferritin and utilization by ce...

  11. Type I interferon drives a distinctive dendritic cell maturation phenotype that allows continued class II MHC synthesis and antigen processing

    OpenAIRE

    Simmons, Daimon P.; Wearsch, Pamela A.; Canaday, David H; Meyerson, Howard J.; Liu, Yi C.; Wang, Ying; Boom, W Henry; Harding, Clifford V.

    2012-01-01

    Microbial molecules or cytokines can stimulate dendritic cell (DC) maturation, which involves DC migration to lymph nodes and enhanced presentation of Ag to launch T cell responses. Microbial Toll-like receptor (TLR) agonists are the most studied inducers of DC maturation, but type I interferon (IFN-I) also promotes DC maturation. In response to TLR stimulation, DC maturation involves a burst of Ag processing with enhanced expression of peptide-MHC-II complexes and co-stimulator molecules. Su...

  12. An HPV 16 L1-based chimeric human papilloma virus-like particles containing a string of epitopes produced in plants is able to elicit humoral and cytotoxic T-cell activity in mice

    Directory of Open Access Journals (Sweden)

    Weiss-Steider Benny

    2009-01-01

    Full Text Available Abstract Background Even though two prophylactic vaccines against HPV are currently licensed, infections by the virus continue to be a major health problem mainly in developing countries. The cost of the vaccines limits wide-scale application in poor countries. A promising strategy for producing affordable and efficient vaccines involves the expression of recombinant immunogens in plants. Several HPV genes have been expressed in plants, including L1, which can self-assemble into virus-like particles. A plant-based, dual prophylactic/therapeutic vaccine remains an attractive possibility. Results We sought to express in tomato plants chimeric HPV 16 VLPs containing L1 fused to a string of epitopes from HPV 16 E6 and E7 proteins. The L1 employed had been modified to eliminate a strong inhibitory region at the 5' end of the molecule to increase expression levels. Several tomato lines were obtained expressing either L1 alone or L1-E6/E7 from 0.05% to 0.1% of total soluble protein. Stable integration of the transgenes was verified by Southern blot. Northern and western blot revealed successful expression of the transgenes at the mRNA and protein level. The chimeric VLPs were able to assemble adequately in tomato cells. Intraperitoneal administration in mice was able to elicit both neutralizing antibodies against the viral particle and cytotoxic T-lymphocytes activity against the epitopes. Conclusion In this work, we report for the first time the expression in plants of a chimeric particle containing the HPV 16 L1 sequence and a string of T-cell epitopes from HPV 16 E6 and E7 fused to the C-terminus. The particles were able to induce a significant antibody and cytotoxic T-lymphocytes response. Experiments in vivo are in progress to determine whether the chimeric particles are able to induce regression of disease and resolution of viral infection in mice. Chimeric particles of the type described in this work may potentially be the basis for developing

  13. Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231.

    Science.gov (United States)

    Tenorio, María J; Ross, Breyan H; Luchsinger, Charlotte; Rivera-Dictter, Andrés; Arriagada, Cecilia; Acuña, Diego; Aguilar, Marcelo; Cavieres, Viviana; Burgos, Patricia V; Ehrenfeld, Pamela; Mardones, Gonzalo A

    2016-01-01

    Golgi phosphoprotein 3 (GOLPH3) has been implicated in the development of carcinomas in many human tissues, and is currently considered a bona fide oncoprotein. Importantly, several tumor types show overexpression of GOLPH3, which is associated with tumor progress and poor prognosis. However, the underlying molecular mechanisms that connect GOLPH3 function with tumorigenicity are poorly understood. Experimental evidence shows that depletion of GOLPH3 abolishes transformation and proliferation of tumor cells in GOLPH3-overexpressing cell lines. Conversely, GOLPH3 overexpression drives transformation of primary cell lines and enhances mouse xenograft tumor growth in vivo. This evidence suggests that overexpression of GOLPH3 could result in distinct features of GOLPH3 in tumor cells compared to that of non-tumorigenic cells. GOLPH3 is a peripheral membrane protein mostly localized at the trans-Golgi network, and its association with Golgi membranes depends on binding to phosphatidylinositol-4-phosphate. GOLPH3 is also contained in a large cytosolic pool that rapidly exchanges with Golgi-associated pools. GOLPH3 has also been observed associated with vesicles and tubules arising from the Golgi, as well as other cellular compartments, and hence it has been implicated in several membrane trafficking events. Whether these and other features are typical to all different types of cells is unknown. Moreover, it remains undetermined how GOLPH3 acts as an oncoprotein at the Golgi. Therefore, to better understand the roles of GOLPH3 in cancer cells, we sought to compare some of its biochemical and cellular properties in the human breast cancer cell lines MCF7 and MDA-MB-231 with that of the non-tumorigenic breast human cell line MCF 10A. We found unexpected differences that support the notion that in different cancer cells, overexpression of GOLPH3 functions in diverse fashions, which may influence specific tumorigenic phenotypes. PMID:27123979

  14. Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

    Science.gov (United States)

    Luchsinger, Charlotte; Rivera-Dictter, Andrés; Arriagada, Cecilia; Acuña, Diego; Aguilar, Marcelo; Cavieres, Viviana; Burgos, Patricia V.; Ehrenfeld, Pamela; Mardones, Gonzalo A.

    2016-01-01

    Golgi phosphoprotein 3 (GOLPH3) has been implicated in the development of carcinomas in many human tissues, and is currently considered a bona fide oncoprotein. Importantly, several tumor types show overexpression of GOLPH3, which is associated with tumor progress and poor prognosis. However, the underlying molecular mechanisms that connect GOLPH3 function with tumorigenicity are poorly understood. Experimental evidence shows that depletion of GOLPH3 abolishes transformation and proliferation of tumor cells in GOLPH3-overexpressing cell lines. Conversely, GOLPH3 overexpression drives transformation of primary cell lines and enhances mouse xenograft tumor growth in vivo. This evidence suggests that overexpression of GOLPH3 could result in distinct features of GOLPH3 in tumor cells compared to that of non-tumorigenic cells. GOLPH3 is a peripheral membrane protein mostly localized at the trans-Golgi network, and its association with Golgi membranes depends on binding to phosphatidylinositol-4-phosphate. GOLPH3 is also contained in a large cytosolic pool that rapidly exchanges with Golgi-associated pools. GOLPH3 has also been observed associated with vesicles and tubules arising from the Golgi, as well as other cellular compartments, and hence it has been implicated in several membrane trafficking events. Whether these and other features are typical to all different types of cells is unknown. Moreover, it remains undetermined how GOLPH3 acts as an oncoprotein at the Golgi. Therefore, to better understand the roles of GOLPH3 in cancer cells, we sought to compare some of its biochemical and cellular properties in the human breast cancer cell lines MCF7 and MDA-MB-231 with that of the non-tumorigenic breast human cell line MCF 10A. We found unexpected differences that support the notion that in different cancer cells, overexpression of GOLPH3 functions in diverse fashions, which may influence specific tumorigenic phenotypes. PMID:27123979

  15. Natural killer cell cytotoxicity of breast cancer targets is enhanced by two distinct mechanisms of antibody-dependent cellular cytotoxicity against LFA-3 and HER2/neu.

    Science.gov (United States)

    Cooley, S; Burns, L J; Repka, T; Miller, J S

    1999-10-01

    Treatment of advanced breast cancer with autologous stem cell transplantation is limited by a high probability of disease relapse. In clinical trials, interleukin 2 (IL-2) alone can expand natural killer (NK) cells in vivo and increase their cytotoxic activity against breast cancer cell lines, but this increase is modest. Understanding the mechanisms that mediate NK cell lysis of breast cancer targets may lead to improvements of current immunotherapy strategies. NK cells from normal donors or patients receiving subcutaneous IL-2 were tested in cytotoxicity assays against five breast cancer cell lines. The role of adhesion molecules and antibodies that interact through Fc receptors on NK cells was explored. NK cell lysis of breast cancer targets is variable and is partially dependent on recognition through ICAM-1 and CD18. While blocking CD2 slightly decreased cytotoxicity, contrary to expectations, an antibody against CD58 (the ligand for CD2), failed to block killing and instead mediated an increased cytotoxicity that correlated with target density of CD58. The CD58 antibody-enhanced killing was dependent not only on FcRgammaIII but also on CD2 and ICAM-1/CD18. To further elucidate the mechanism of this CD58 antibody-dependent cellular cytotoxicity (ADCC), another antibody was tested. Trastuzumab (Herceptin), a humanized antibody against HER2/neu, mediated potent ADCC against all the HER2/neu positive breast cancer targets. Unlike CD58 antibody-mediated ADCC, Herceptin ADCC was minimally affected by blocking antibodies to CD2 or ICAM-1/CD18, which suggests a different mechanism of action. This study shows that multiple mechanisms are involved in NK cell lysis of breast cancer targets, that none of the targets are inherently resistant to killing, and that two distinct mechanisms of ADCC can target immunotherapy to breast cancer cells. PMID:10517495

  16. A functional gammadeltaTCR/CD3 complex distinct from gammadeltaT cells is expressed by human eosinophils.

    Directory of Open Access Journals (Sweden)

    Fanny Legrand

    Full Text Available BACKGROUND: Eosinophils are effector cells during parasitic infections and allergic responses. However, their contribution to innate immunity has been only recently unravelled. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that human eosinophils express CD3 and gammadelta T Cell Receptor (TCR but not alphabeta TCR. Surface expression of gammadeltaTCR/CD3 is heterogeneous between eosinophil donors and inducible by mycobacterial ligands. Surface immunoprecipitation revealed expression of the full gammadeltaTCR/CD3 complex. Real-time PCR amplification for CD3, gamma and delta TCR constant regions transcripts showed a significantly lower expression in eosinophils than in gammadeltaT cells. Limited TCR rearrangements occur in eosinophils as shown by spectratyping analysis of CDR3 length profiles and in situ hybridization. Release by eosinophils of Reactive Oxygen Species, granule proteins, Eosinophil Peroxidase and Eosinophil-Derived Neurotoxin and cytokines (IFN-gamma and TNF-alpha was observed following activation by gammadeltaTCR-specific agonists or by mycobacteria. These effects were inhibited by anti-gammadeltaTCR blocking antibodies and antagonists. Moreover, gammadeltaTCR/CD3 was involved in eosinophil cytotoxicity against tumor cells. CONCLUSIONS/SIGNIFICANCE: Our results provide evidence that human eosinophils express a functional gammadeltaTCR/CD3 with similar, but not identical, characteristics to gammadeltaTCR from gammadeltaT cells. We propose that this receptor contributes to eosinophil innate responses against mycobacteria and tumors and may represent an additional link between lymphoid and myeloid lineages.

  17. Cytokeratin and protein expression patterns in squamous cell carcinoma of the oral cavity provide evidence for two distinct pathogenetic pathways

    Science.gov (United States)

    FROHWITTER, GESCHE; BUERGER, HORST; VAN DIEST, PAUL J.; KORSCHING, EBERHARD; KLEINHEINZ, JOHANNES; FILLIES, THOMAS

    2016-01-01

    Squamous cell carcinoma (SCC) of the oral cavity is a morphological heterogeneous disease. Various cytokeratin (CK) expression patterns with different prognostic values have been described, but little is known concerning the underlying biological cell mechanisms. Therefore, the present study investigated 193 cases of oral SCCs using immunohistochemistry for α/β/γ-catenin, glucose transporter 1, caspase-3, X-linked inhibitor of apoptosis protein, hypoxia inducible factor-1α, carbonic anhydrase 9, heat shock protein (hsp) 70, mast/stem cell growth factor receptor, p21, p27, p16, p53, B-cell lymphoma 6, epidermal growth factor receptor, cyclin D1 and CK1, 5/6, 8/18, 10, 14 and 19. Expression patterns were analyzed with biomathematical permutation analysis. The present results revealed a significant association between the expression of low-molecular weight CK8/18 and 19 and a high-tumor grade, β and γ-catenin expression, deregulated cell cycle proteins and a predominant localization of the tumor on the floor of the mouth. By contrast, expression of high-molecular weight CK1, 5/6, 10 and 14 was significantly associated with the expression of p21 and hsp70. In conclusion, the current study presents evidence for the existence of two parallel pathogenetic pathways in oral SCCs, characterized by the expression of low- and high-molecular weight CKs. Additional studies are required to demonstrate the extent that these results may be used to improve therapeutic regimens. PMID:27347109

  18. Distinct CDR3 Conformations in T Cell Receptors Determine the Level of Cross-Reactivity for Diverse Antigens, but Not the Docking Orientation

    OpenAIRE

    Jones, Lindsay L.; Colf, Leremy A.; Stone, Jennifer D.; Garcia, K. Christopher; Kranz, David M.

    2008-01-01

    T cells are known to cross-react with diverse peptide MHC antigens through their αβ TCRs. To explore the basis of such cross-reactivity, we examined the 2C TCR that recognizes two structurally distinct ligands, SIY-Kb and alloantigen QL9-Ld. Here we characterized the crossreactivity of several high affinity 2C TCR variants that contained mutations only in the CDR3α loop. Two of the TCRs lost their ability to cross-react with the reciprocal ligand (SIY-Kb), while another TCR (m67) maintained r...

  19. CD4+/CD25+ regulatory cells inhibit activation of tumor-primed CD4+ T cells with IFN-gamma-dependent antiangiogenic activity, as well as long-lasting tumor immunity elicited by peptide vaccination

    OpenAIRE

    Casares, N. (Noelia); L. Arribillaga; Sarobe, P.; Dotor, J. (Javier); Lopez-Diaz-de-Cerio, A. (Ascensión); Melero, I; Lasarte, J.J. (Juan José)

    2003-01-01

    CD25(+) regulatory T (T reg) cells suppress the activation/proliferation of other CD4(+) or CD8(+) T cells in vitro. Also, down-regulation of CD25(+) T reg cells enhance antitumor immune responses. In this study, we show that depletion of CD25(+) T reg cells allows the host to induce both CD4(+) and CD8(+) antitumoral responses following tumor challenge. Simultaneous depletion of CD25(+) and CD8(+) cells, as well as adoptive transfer experiments, revealed that tumor-specific CD4(+) T cells, w...

  20. DNA damage activates a spatially distinct late cytoplasmic cell-cycle checkpoint network controlled by MK2-mediated RNA stabilization

    DEFF Research Database (Denmark)

    Reinhardt, H Christian; Hasskamp, Pia; Schmedding, Ingolf;

    2010-01-01

    Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1...... pathway. We used functional genetics to dissect the contributions of Chk1 and MK2 to checkpoint control. We show that nuclear Chk1 activity is essential to establish a G(2)/M checkpoint, while cytoplasmic MK2 activity is critical for prolonged checkpoint maintenance through a process of...... posttranscriptional mRNA stabilization. Following DNA damage, the p38/MK2 complex relocalizes from nucleus to cytoplasm where MK2 phosphorylates hnRNPA0, to stabilize Gadd45α mRNA, while p38 phosphorylates and releases the translational inhibitor TIAR. In addition, MK2 phosphorylates PARN, blocking Gadd45α m...

  1. Interleukin-3, GM-CSF, and TPA induce distinct phosphorylation events in an interleukin 3-dependent multipotential cell line

    International Nuclear Information System (INIS)

    The mechanism of action of the hemopoietic growth factor, murine interleukin-3 (mIL-3), was investigated using an mIL-3-dependent multipotential hematopoietic cell line, B6SUtA1. Murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) was as potent as mIL-3 in stimulating these cells. In addition, sodium orthovanadate, an inhibitor of phosphotyrosine phosphatase, and 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a known activator of protein kinase C, also stimulated DNA synthesis in these cells, suggesting that protein phosphorylation might be involved in the mechanism of action of mIL-3 and mGM-CSF. To assess this possibility, intact B6SUtA1 cells exposed for brief periods to mIL-3, mGM-CSF, and TPA were analyzed for changes in phosphorylation patterns using metabolic 32P-labeling and antibodies to phosphotyrosine. Both mIL-3 and mGM-CSF induced the serine-specific phosphorylation of a 68-Kd cytosolic protein, whereas all three agents stimulated the serine-specific phosphorylation of a 68-Kd membrane protein. Furthermore, mIL-3 stimulated tyrosine phosphorylation of the 68-Kd membrane protein, as well as of 140-, 90-, 55, and 40-Kd proteins. The 90-Kd protein was also tyrosine phosphorylated in response to mGM-CSF. These phosphotyrosine containing proteins were not detected in TPA-treated cells. These results indicate that protein phosphorylations on tyrosine and serine residues occur in B6SUtA1 cells following short-term incubation with mIL-3 or mGM-CSF and that most of these phosphorylation events are mediated by kinases other than protein kinase C

  2. Evolutionary divergence of β-expansin structure and function in grasses parallels emergence of distinctive primary cell wall traits.

    Science.gov (United States)

    Sampedro, Javier; Guttman, Mara; Li, Lian-Chao; Cosgrove, Daniel J

    2015-01-01

    Expansins are wall-loosening proteins that promote the extension of primary cell walls without the hydrolysis of major structural components. Previously, proteins from the EXPA (α-expansin) family were found to loosen eudicot cell walls but to be less effective on grass cell walls, whereas the reverse pattern was found for EXPB (β-expansin) proteins obtained from grass pollen. To understand the evolutionary and structural bases for the selectivity of EXPB action, we assessed the extension (creep) response of cell walls from diverse monocot families to EXPA and EXPB treatments. Cell walls from Cyperaceae and Juncaceae (families closely related to grasses) displayed a typical grass response ('β-response'). Walls from more distant monocots, including some species that share with grasses high levels of arabinoxylan, responded preferentially to α-expansins ('α-response'), behaving in this regard like eudicots. An expansin with selective activity for grass cell walls was detected in Cyperaceae pollen, coinciding with the expression of genes from the divergent EXPB-I branch that includes grass pollen β-expansins. The evolutionary origin of this branch was located within Poales on the basis of phylogenetic analyses and its association with the 'sigma' whole-genome duplication. Accelerated evolution in this branch has remodeled the protein surface in contact with the substrate, potentially for binding highly substituted arabinoxylan. We propose that the evolution of the divergent EXPB-I group made a fundamental change in the target and mechanism of wall loosening in the grass lineage possible, involving a new structural role for xylans and the expansins that target them. PMID:25353668

  3. Escherichia coli Nissle 1917 Distinctively Modulates T-Cell Cycling and Expansion via Toll-Like Receptor 2 Signaling

    OpenAIRE

    Sturm, Andreas; Rilling, Klaus; Baumgart, Daniel C.; Gargas, Konstantinos; Abou-Ghazalé, Tay; Raupach, Bärbel; Eckert, Jana; Schumann, Ralf R.; Enders, Corinne; Sonnenborn, Ulrich; Wiedenmann, Bertram; Dignass, Axel U

    2005-01-01

    Although the probiotic Escherichia coli strain Nissle 1917 has been proven to be efficacious for the treatment of inflammatory bowel diseases, the underlying mechanisms of action still remain elusive. The aim of the present study was to analyze the effects of E. coli Nissle 1917 on cell cycling and apoptosis of peripheral blood and lamina propria T cells (PBT and LPT, respectively). Anti-CD3-stimulated PBT and LPT were treated with E. coli Nissle 1917-conditioned medium (E. coli Nissle 1917-C...

  4. Monocyte-derived macrophages exhibit distinct and more restricted HIV-1 integration site repertoire than CD4+ T cells

    OpenAIRE

    Yik Lim Kok; Valentina Vongrad; Mohaned Shilaih; Francesca Di Giallonardo; Herbert Kuster; Roger Kouyos; Günthard, Huldrych F.; Metzner, Karin J.

    2016-01-01

    The host genetic landscape surrounding integrated HIV-1 has an impact on the fate of the provirus. Studies analysing HIV-1 integration sites in macrophages are scarce. We studied HIV-1 integration site patterns in monocyte-derived macrophages (MDMs) and activated CD4+ T cells derived from seven antiretroviral therapy (ART)-treated HIV-1-infected individuals whose cells were infected ex vivo with autologous HIV-1 isolated during the acute phase of infection. A total of 1,484 unique HIV-1 integ...

  5. Recombinant interleukin 2 and gamma-interferon act synergistically on distinct steps of in vitro terminal human B cell maturation.

    OpenAIRE

    Lê thi Bich-Thuy; Fauci, A S

    1986-01-01

    The effects of recombinant interleukin 2 (IL-2) on the in vitro differentiation of human tonsillar B cells which were not preincubated with Staphylococcus aureus Cowan I or with anti-human IgM were investigated. IL-2 was shown to induce the generation of Ig-containing cells in a dose-dependent fashion from 2.5 to 2,500 U IL-2/ml. Conversely, the quantities of Ig secreted in the culture supernatant were found in the majority of experiments to peak at 25 U/ml. The possible presence, in cultures...

  6. Distinct functions of the transcription factors Gata3 and ThPOK during intrathymic CD4 T cell differentiation*

    OpenAIRE

    Wang, Lie; Wildt, Kathryn F.; Zhu, Jinfang; Zhang, Xianyu; Feigenbaum, Lionel; Tessarollo, Lino; Paul, William E.; Fowlkes, B.J.; Bosselut, Rémy

    2008-01-01

    The transcription factors Gata3 and Zbtb7b are required for intrathymic CD4 T cell differentiation, but their precise roles in this process remain unclear. Here we show that, contrary to previous findings, Gata3 disruption blocked CD4 T cell lineage differentiation before CD4 lineage commitment, and in some contexts permitted ‘redirection’ of MHC class II-restricted thymocytes into the CD8 lineage. We found that Gata3 promotes Zbtb7b expression, and binds within a region of the Zbtb7b locus e...

  7. EdnrB Governs Regenerative Response of Melanocyte Stem Cells by Crosstalk with Wnt Signaling

    OpenAIRE

    Makoto Takeo; Wendy Lee; Piul Rabbani; Qi Sun; Hai Hu; Chae Ho Lim; Prashiela Manga; Mayumi Ito

    2016-01-01

    Delineating the crosstalk between distinct signaling pathways is key to understanding the diverse and dynamic responses of adult stem cells during tissue regeneration. Here, we demonstrate that the Edn/EdnrB signaling pathway can interact with other signaling pathways to elicit distinct stem cell functions during tissue regeneration. EdnrB signaling promotes proliferation and differentiation of melanocyte stem cells (McSCs), dramatically enhancing the regeneration of hair and epidermal melano...

  8. The binucleate cell of okapi and giraffe placenta shows distinctive glycosylation compared with other ruminants: a lectin histochemical study.

    Science.gov (United States)

    Jones, Carolyn J P; Wilsher, Sandra A; Wooding, F B P; Benirschke, K; Allen, W R

    2015-02-01

    The placenta of ruminants contains characteristic binucleate cells (BNC) with a highly conserved glycan structure which evolved early in Ruminant phylogenesis. Giraffe and Okapi placentae also contain these cells and it is not known whether they have a similar glycan array. We have used lectin histochemistry to examine the glycosylation of these cells in these species and compare them with bovine BNC which have a typical ruminant glycan composition. Two placentae, mid and near term, from Giraffe (Giraffa camelopardalis) and two term placenta of Okapi (Okapia johnstoni) were embedded in resin and stained with a panel of 23 lectins and compared with near-term bovine (Bos taurus) placenta. Significant differences were found in the glycans of Giraffe and Okapi BNC compared with those from the bovine, with little or no expression of terminal αN-acetylgalactosamine bound by Dolichos biflorus and Vicia villosa agglutinins which instead bound to placental blood vessels. Higher levels of N-acetylglucosamine bound by Lycopersicon esculentum and Phytolacca americana agglutinins were also apparent. Some differences between Okapi and Giraffe were evident. Most N-linked glycans were similarly expressed in all three species as were fucosyl residues. Interplacentomal areas in Giraffe and Bovine showed differences from the placentomal cells though no intercotyledonary BNC were apparent in Okapi. In conclusion, Giraffidae BNC developed different glycan biosynthetic pathways following their split from the Bovidae with further differences evolving as Okapi and Giraffe diverged from each other, affecting both inter and placentomal BNC which may have different functions during development. PMID:25527317

  9. Distinct target cell-dependent forms of short-term plasticity of the central visceral afferent synapses of the rat

    Directory of Open Access Journals (Sweden)

    Watabe Ayako M

    2010-10-01

    Full Text Available Abstract Background The visceral afferents from various cervico-abdominal sensory receptors project to the dorsal vagal complex (DVC, which is composed of the nucleus of the solitary tract (NTS, the area postrema and the dorsal motor nucleus of the vagus nerve (DMX, via the vagus and glossopharyngeal nerves and then the solitary tract (TS in the brainstem. While the excitatory transmission at the TS-NTS synapses shows strong frequency-dependent suppression in response to repeated stimulation of the afferents, the frequency dependence and short-term plasticity at the TS-DMX synapses, which also transmit monosynaptic information from the visceral afferents to the DVC neurons, remain largely unknown. Results Recording of the EPSCs activated by paired or repeated TS stimulation in the brainstem slices of rats revealed that, unlike NTS neurons whose paired-pulse ratio (PPR is consistently below 0.6, the distribution of the PPR of DMX neurons shows bimodal peaks that are composed of type I (PPR, 0.6-1.5; 53% of 120 neurons recorded and type II (PPR, Conclusions These two general types of short-term plasticity might contribute to the differential activation of distinct vago-vagal reflex circuits, depending on the firing frequency and type of visceral afferents.

  10. Interaction between Epstein-Barr virus and a T cell line (HSB-2) via a receptor phenotypically distinct from complement receptor type 2.

    Science.gov (United States)

    Hedrick, J A; Watry, D; Speiser, C; O'Donnell, P; Lambris, J D; Tsoukas, C D

    1992-05-01

    Epstein-Barr virus (EBV), the causative agent of mononucleosis and several human cancers, infects cells via complement receptor type 2 (CR2, CD21) which also serves as the receptor for the third complement component, C3. Expression of this receptor is restricted to B lymphocytes, immature thymocytes, and certain epithelial cells. In the present investigation; we describe the presence of a seemingly novel EBV receptor which is phenotypically distinct from CR2. Among various leukemic T cells studied, one, HSB-2, demonstrates no reactivity to several anti-CR2 antibodies, yet it reacts strongly with EBV as detected by incubation with biotin-conjugated virus and streptavidin-phycoerythrin. The virus binding is specific as demonstrated by blocking with anti-EBV antibodies and with non-conjugated virus. Aggregated C3 also binds HSB-2 and is capable of partially inhibiting EBV binding. The absence of CR2 on HSB-2 is further supported by the lack of expression of specific mRNA, assessed by Northern blotting analysis and polymerase chain reaction. Viral internalization and infection is demonstrated with electron microscopy, with detection of EBV-DNA by Southern blotting, and with detection of EBNA-1 transcripts by the polymerase chain reaction. Even though HSB-2 does not express CR2, it nevertheless displays transcripts which have some homology to a CR2 cDNA probe under low stringency hybridization conditions. This probe encompasses approximately the N-terminal half of CR2 which includes the EBV-binding epitope(s). The HSB-2 message is 5.2 kb, a size distinct from the 4.7-kb message of B cell CR2s. In contrast, the 5.2-kb message in not seen, under similar hybridization conditions, with a probe comprising the C-terminal half of CR2. Collectively, the data indicate that a receptor molecule having distinct phenotypic characteristics from the known CR2 protein on B cells is utilized by EBV to target human T lymphocytes. PMID:1315687

  11. Human Immunodeficiency Virus Protein Tat Induces Synapse Loss via a Reversible Process that is Distinct from Cell Death

    OpenAIRE

    Kim, Hee Jung; Martemyanov, Kirill A.; Thayer, Stanley A.

    2008-01-01

    Human immunodeficiency virus (HIV)-1 infection of the CNS produces changes in dendritic morphology that correlate with cognitive decline in patients with HIV-1 associated dementia (HAD). Here we investigated the effects of HIV-1 transactivator of transcription (Tat), a protein released by virus-infected cells, on synapses between hippocampal neurons using an imaging-based assay that quantified clusters of the scaffolding protein postsynaptic density 95 fused to green fluorescent protein (PSD9...

  12. Definition of Genetically Distinct Attenuation Mechanisms in Naturally Virulence-Attenuated Listeria monocytogenes by Comparative Cell Culture and Molecular Characterization

    OpenAIRE

    Roberts, Angela; Chan, Yvonne; Wiedmann, Martin

    2005-01-01

    Listeria monocytogenes is a foodborne pathogen able to cause serious disease in humans and animals. Not all isolates are equally pathogenic, however, and several isolates have been characterized as naturally virulence attenuated. We sought to identify the genetic basis of natural virulence attenuation using cell culture assays and molecular techniques. By comparing the phenotypes of naturally virulence-attenuated isolates to those of defined virulence gene mutants in plaque, cytotoxicity, and...

  13. Human decidual macrophages and NK cells differentially express Toll-like receptors and display distinct cytokine profiles upon TLR stimulation.

    OpenAIRE

    ElisabethMenu; MarionDuriez; HéloïseQuillay; YoannMadec; RomainMarlin; Clairede Truchis; MonaRahmati

    2014-01-01

    Maternofetal pathogen transmission is partially controlled at the level of the maternal uterine mucosa at the fetal implantation site (the decidua basalis), where maternal and fetal cells are in close contact. Toll-like receptors (TLRs) may play an important role in initiating rapid immune responses against pathogens in the decidua basalis, however the tolerant microenvironment should be preserved in order to allow fetal development. Here we investigated the expression and functionality of TL...

  14. Human decidual macrophages and NK cells differentially express Toll-like receptors and display distinct cytokine profiles upon TLR stimulation

    OpenAIRE

    Duriez, Marion; Quillay, Héloïse; Madec, Yoann; El Costa, Hicham; Cannou, Claude; Marlin, Romain; De Truchis, Claire; Rahmati, Mona; Barré-Sinoussi, Françoise; Nugeyre, Marie-Thérèse; Menu, Elisabeth

    2014-01-01

    Maternofetal pathogen transmission is partially controlled at the level of the maternal uterine mucosa at the fetal implantation site (the decidua basalis), where maternal and fetal cells are in close contact. Toll-like receptors (TLRs) may play an important role in initiating rapid immune responses against pathogens in the decidua basalis, however the tolerant microenvironment should be preserved in order to allow fetal development. Here we investigated the expression and functionality of TL...

  15. Contribution of distinct homeodomain DNA binding specificities to Drosophila embryonic mesodermal cell-specific gene expression programs.

    Directory of Open Access Journals (Sweden)

    Brian W Busser

    Full Text Available Homeodomain (HD proteins are a large family of evolutionarily conserved transcription factors (TFs having diverse developmental functions, often acting within the same cell types, yet many members of this family paradoxically recognize similar DNA sequences. Thus, with multiple family members having the potential to recognize the same DNA sequences in cis-regulatory elements, it is difficult to ascertain the role of an individual HD or a subclass of HDs in mediating a particular developmental function. To investigate this problem, we focused our studies on the Drosophila embryonic mesoderm where HD TFs are required to establish not only segmental identities (such as the Hox TFs, but also tissue and cell fate specification and differentiation (such as the NK-2 HDs, Six HDs and identity HDs (I-HDs. Here we utilized the complete spectrum of DNA binding specificities determined by protein binding microarrays (PBMs for a diverse collection of HDs to modify the nucleotide sequences of numerous mesodermal enhancers to be recognized by either no or a single subclass of HDs, and subsequently assayed the consequences of these changes on enhancer function in transgenic reporter assays. These studies show that individual mesodermal enhancers receive separate transcriptional input from both I-HD and Hox subclasses of HDs. In addition, we demonstrate that enhancers regulating upstream components of the mesodermal regulatory network are targeted by the Six class of HDs. Finally, we establish the necessity of NK-2 HD binding sequences to activate gene expression in multiple mesodermal tissues, supporting a potential role for the NK-2 HD TF Tinman (Tin as a pioneer factor that cooperates with other factors to regulate cell-specific gene expression programs. Collectively, these results underscore the critical role played by HDs of multiple subclasses in inducing the unique genetic programs of individual mesodermal cells, and in coordinating the gene regulatory

  16. Two distinct signalling pathways are involved in FGF2-stimulated proliferation of choriocapillary endothelial cells: a comparative study with VEGF.

    Science.gov (United States)

    Zubilewicz, A; Hecquet, C; Jeanny, J C; Soubrane, G; Courtois, Y; Mascarelli, F

    2001-03-22

    In the retina, angiogenesis is an important component of normal physiological events such as embryonic vascular development. It is also involved in pathological processes including diabetic retinopathies and age-related macular degeneration, and tumour growth such as choroidal melanoma. Fibroblast growth factor (FGF) 2 and vascular endothelial cell growth factor (VEGF) are the two major angiogenic factors in the retina. We investigated the mechanism of proliferation and the regulation of the mitogenic properties of FGF2 and VEGF in cultures of chorocapillary endothelial cells (CEC). FGF2 is a strong mitogen for CEC and induced a 2.5-fold increase in cell proliferation after 4 days in culture in the absence of serum. In contrast, VEGF is a poor mitogen for CEC. FGF2, but not VEGF induces a large activation of MEK1, ERK1/2 and P90(RSK) during CEC proliferation. Pharmacological inhibition of Ras processing, and of MEK1 and ERK1/2 activation reduced only by 50% FGF2-induced cell proliferation, suggesting that there is another signalling pathway for CEC proliferation. Pharmacological inhibition of the PI 3-Kinase also inhibits by half FGF2-induced CEC proliferation. FGF2 stimulates the activation of the PI 3-K, P70(S6K) and Akt. Inhibition of both ERK1/2 and PI 3-K activities suppressed FGF2-induced CEC proliferation, demonstrating that CEC proliferation requires both ERKs and PI 3-K pathways. These data on the molecular mechanism and signalling may have important implications for providing more selective methods for anti-angiogenic and anti-tumoural therapy. PMID:11313884

  17. Distinct signalling pathways of murine histamine H1- and H4-receptors expressed at comparable levels in HEK293 cells.

    Directory of Open Access Journals (Sweden)

    Silke Beermann

    Full Text Available Histamine (HA is recognized by its target cells via four G-protein-coupled receptors, referred to as histamine H1-receptor (H1R, H2R, H3R, and H4R. Both H1R and H4R exert pro-inflammatory functions. However, their signal transduction pathways have never been analyzed in a directly comparable manner side by side. Moreover, the analysis of pharmacological properties of the murine orthologs, representing the main targets of pre-clinical research, is very important. Therefore, we engineered recombinant HEK293 cells expressing either mouse (mH1R or mH4R at similar levels and analyzed HA-induced signalling in these cells. HA induced intracellular calcium mobilization via both mH1R and mH4R, with the mH1R being much more effective. Whereas cAMP accumulation was potentiated via the mH1R, it was reduced via the mH4R. The regulation of both second messengers via the H4R, but not the H1R, was sensitive to pertussis toxin (PTX. The mitogen-activated protein kinases (MAPKs ERK 1/2 were massively activated downstream of both receptors and demonstrated a functional involvement in HA-induced EGR-1 gene expression. The p38 MAPK was moderately activated via both receptors as well, but was functionally involved in HA-induced EGR-1 gene expression only in H4R-expressing cells. Surprisingly, in this system p38 MAPK activity reduced the HA-induced gene expression. In summary, using this system which allows a direct comparison of mH1R- and mH4R-induced signalling, qualitative and quantitative differences on the levels of second messenger generation and also in terms of p38 MAPK function became evident.

  18. Structure-function analysis of Leishmania lipophosphoglycan. Distinct domains that mediate binding and inhibition of endothelial cell function.

    Science.gov (United States)

    Ho, J L; Kim, H K; Sass, P M; He, S; Geng, J; Xu, H; Zhu, B; Turco, S J; Lo, S K

    1996-10-01

    We have shown that Leishmania lipophosphoglycan (LPG) inhibits IL-1 beta gene expression in human monocytes. Here, we show that LPG can bind in a time-dependent manner and suppress endothelial cell activation, possibly via specific LPG domains. Endotoxin (10 ng/ml, 4 h) consistently caused endothelium to increase monocyte adhesion (approximately 20-fold). LPG pretreatment (2 microM, 2 h) completely blocked endotoxin-mediated monocyte adhesion. LPG did not grossly suppress endothelial functions because TNF-alpha- and IL-1 beta-mediated adhesion toward monocytes were not affected. Using four highly purified LPG fragments (namely, repeating phosphodisaccharide (PGM), phosphoglycan, phosphosaccharide core-lyso-alkyl-phosphatidylinositol (core-PI), and lyso-alkyl-phosphatidylinositol (lyso-PI)), we examined whether these fragments can independently inhibit endothelial adhesion. In contrast to that of intact LPG, neither the four LPG fragments (2 microM, 2 h) independently nor the co-addition of phosphoglycan and core-P1 fragments blocked the endotoxin-mediated adhesion to monocytes. To determine whether the fragments can reverse the effect of intact LPG, endothelial cells were first pretreated with the LPG fragments (10 microM, 15 min), followed by the addition of LPG (2 microM). All four LPG fragments fully reversed the effect of LPG. Simultaneous addition of LPG fragments and intact LPG caused only partial suppression (approximately 45%), while the addition of LPG fragments 14 min later had no reversal effect. Flow cytometry revealed that only core-P1 and lyso-P1 competitively inhibited (approximately 30%) LPG binding. Conversely, LPG competed with the binding of [3H]lyso-P1 (approximately 30%). Furthermore, mAb against the PGM reversed (approximately 70%) the effect of LPG. Thus, the lyso-P1 domain on LPG mediates binding to endothelial cells, whereas the PGM domain mediates the cell inhibitory effect. PMID:8816410

  19. The membrane composition of coated pits, microvilli, endosomes, and lysosomes is distinctive in the rat kidney proximal tubule cell

    OpenAIRE

    1986-01-01

    The distribution of a number of membrane proteins on plasmalemmal microdomains (microvilli, coated pits) and in endosomes and lysosomes of the proximal tubule epithelial cell was determined in normal rat kidneys by immunofluorescence and immunoelectron microscopy. Two major brush border proteins, 130 and 94 kD, and gamma-glutamyl transpeptidase were detected on the membranes of the microvilli but were not found on membranes of coated pits. Gp330, the Heymann nephritis antigen, and clathrin we...

  20. Distinct Roles for Intestinal Epithelial Cell-Specific Hdac1 and Hdac2 in the Regulation of Murine Intestinal Homeostasis.

    Science.gov (United States)

    Gonneaud, Alexis; Turgeon, Naomie; Boudreau, François; Perreault, Nathalie; Rivard, Nathalie; Asselin, Claude

    2016-02-01

    The intestinal epithelium responds to and transmits signals from the microbiota and the mucosal immune system to insure intestinal homeostasis. These interactions are in part conveyed by epigenetic modifications, which respond to environmental changes. Protein acetylation is an epigenetic signal regulated by histone deacetylases, including Hdac1 and Hdac2. We have previously shown that villin-Cre-inducible intestinal epithelial cell (IEC)-specific Hdac1 and Hdac2 deletions disturb intestinal homeostasis. To determine the role of Hdac1 and Hdac2 in the regulation of IEC function and the establishment of the dual knockout phenotype, we have generated villin-Cre murine models expressing one Hdac1 allele without Hdac2, or one Hdac2 allele without Hdac1. We have also investigated the effect of short-term deletion of both genes in naphtoflavone-inducible Ah-Cre and tamoxifen-inducible villin-Cre(ER) mice. Mice with one Hdac1 allele displayed normal tissue architecture, but increased sensitivity to DSS-induced colitis. In contrast, mice with one Hdac2 allele displayed intestinal architecture defects, increased proliferation, decreased goblet cell numbers as opposed to Paneth cells, increased immune cell infiltration associated with fibrosis, and increased sensitivity to DSS-induced colitis. In comparison to dual knockout mice, intermediary activation of Notch, mTOR, and Stat3 signaling pathways was observed. While villin-Cre(ER) Hdac1 and Hdac2 deletions led to an impaired epithelium and differentiation defects, Ah-Cre-mediated deletion resulted in blunted proliferation associated with the induction of a DNA damage response. Our results suggest that IEC determination and intestinal homeostasis are highly dependent on Hdac1 and Hdac2 activity levels, and that changes in the IEC acetylome may alter the mucosal environment. PMID:26174178

  1. Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease

    OpenAIRE

    Butler, Timothy M.; Johnson-Camacho, Katherine; Peto, Myron; Wang, Nicholas J.; Macey, Tara A.; Korkola, James E.; Koppie, Theresa M.; Corless, Christopher L.; Joe W. Gray; Spellman, Paul T

    2015-01-01

    The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA) sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, thro...

  2. Distinct single cell signal transduction signatures in leukocyte subsets stimulated with khat extract, amphetamine-like cathinone, cathine or norephedrine

    OpenAIRE

    Bredholt, Therese; Ersvær, Elisabeth; Erikstein, Bjarte Skoe; Sulen, André; Reikvam, Håkon; Aarstad, Hans Jørgen; Johannessen, Anne Christine; Vintermyr, Olav Karsten; Bruserud, Øystein; Gjertsen, Bjørn Tore

    2013-01-01

    Background: Amphetamine and amphetamine derivatives are suggested to induce an immunosuppressive effect. However, knowledge of how amphetamines modulate intracellular signaling pathways in cells of the immune system is limited. We have studied phosphorylation of signal transduction proteins (Akt, CREB, ERK1/2, NF-κB, c-Cbl, STAT1/3/5/6) and stress sensors (p38 MAPK, p53) in human leukocyte subsets following in vitro treatment with the natural amphetamine cathinone, the cathinone d...

  3. Distinctive features of single nucleotide alterations in induced pluripotent stem cells with different types of DNA repair deficiency disorders.

    Science.gov (United States)

    Okamura, Kohji; Sakaguchi, Hironari; Sakamoto-Abutani, Rie; Nakanishi, Mahito; Nishimura, Ken; Yamazaki-Inoue, Mayu; Ohtaka, Manami; Periasamy, Vaiyapuri Subbarayan; Alshatwi, Ali Abdullah; Higuchi, Akon; Hanaoka, Kazunori; Nakabayashi, Kazuhiko; Takada, Shuji; Hata, Kenichiro; Toyoda, Masashi; Umezawa, Akihiro

    2016-01-01

    Disease-specific induced pluripotent stem cells (iPSCs) have been used as a model to analyze pathogenesis of disease. In this study, we generated iPSCs derived from a fibroblastic cell line of xeroderma pigmentosum (XP) group A (XPA-iPSCs), a rare autosomal recessive hereditary disease in which patients develop skin cancer in the areas of skin exposed to sunlight. XPA-iPSCs exhibited hypersensitivity to ultraviolet exposure and accumulation of single-nucleotide substitutions when compared with ataxia telangiectasia-derived iPSCs that were established in a previous study. However, XPA-iPSCs did not show any chromosomal instability in vitro, i.e. intact chromosomes were maintained. The results were mutually compensating for examining two major sources of mutations, nucleotide excision repair deficiency and double-strand break repair deficiency. Like XP patients, XPA-iPSCs accumulated single-nucleotide substitutions that are associated with malignant melanoma, a manifestation of XP. These results indicate that XPA-iPSCs may serve a monitoring tool (analogous to the Ames test but using mammalian cells) to measure single-nucleotide alterations, and may be a good model to clarify pathogenesis of XP. In addition, XPA-iPSCs may allow us to facilitate development of drugs that delay genetic alteration and decrease hypersensitivity to ultraviolet for therapeutic applications. PMID:27197874

  4. Distinctive features of single nucleotide alterations in induced pluripotent stem cells with different types of DNA repair deficiency disorders

    Science.gov (United States)

    Okamura, Kohji; Sakaguchi, Hironari; Sakamoto-Abutani, Rie; Nakanishi, Mahito; Nishimura, Ken; Yamazaki-Inoue, Mayu; Ohtaka, Manami; Periasamy, Vaiyapuri Subbarayan; Alshatwi, Ali Abdullah; Higuchi, Akon; Hanaoka, Kazunori; Nakabayashi, Kazuhiko; Takada, Shuji; Hata, Kenichiro; Toyoda, Masashi; Umezawa, Akihiro

    2016-01-01

    Disease-specific induced pluripotent stem cells (iPSCs) have been used as a model to analyze pathogenesis of disease. In this study, we generated iPSCs derived from a fibroblastic cell line of xeroderma pigmentosum (XP) group A (XPA-iPSCs), a rare autosomal recessive hereditary disease in which patients develop skin cancer in the areas of skin exposed to sunlight. XPA-iPSCs exhibited hypersensitivity to ultraviolet exposure and accumulation of single-nucleotide substitutions when compared with ataxia telangiectasia-derived iPSCs that were established in a previous study. However, XPA-iPSCs did not show any chromosomal instability in vitro, i.e. intact chromosomes were maintained. The results were mutually compensating for examining two major sources of mutations, nucleotide excision repair deficiency and double-strand break repair deficiency. Like XP patients, XPA-iPSCs accumulated single-nucleotide substitutions that are associated with malignant melanoma, a manifestation of XP. These results indicate that XPA-iPSCs may serve a monitoring tool (analogous to the Ames test but using mammalian cells) to measure single-nucleotide alterations, and may be a good model to clarify pathogenesis of XP. In addition, XPA-iPSCs may allow us to facilitate development of drugs that delay genetic alteration and decrease hypersensitivity to ultraviolet for therapeutic applications. PMID:27197874

  5. Distinctive toxicity of TiO2 rutile/anatase mixed phase nanoparticles on Caco-2 cells.

    Science.gov (United States)

    Gerloff, Kirsten; Fenoglio, Ivana; Carella, Emanuele; Kolling, Julia; Albrecht, Catrin; Boots, Agnes W; Förster, Irmgard; Schins, Roel P F

    2012-03-19

    Titanium dioxide has a long-standing use as a food additive. Micrometric powders are, e.g., applied as whiteners in confectionary or dairy products. Possible hazards of ingested nanometric TiO(2) particles for humans and the potential influence of varying specific surface area (SSA) are currently under discussion. Five TiO(2)-samples were analyzed for purity, crystallinity, primary particle size, SSA, ζ potential, and aggregation/agglomeration. Their potential to induce cytotoxicity, oxidative stress, and DNA damage was evaluated in human intestinal Caco-2 cells. Only anatase-rutile containing samples, in contrast to the pure anatase samples, induced significant LDH leakage or mild DNA damage (Fpg-comet assay). Evaluation of the metabolic competence of the cells (WST-1 assay) revealed a highly significant correlation between the SSA of the anatase samples and cytotoxicity. The anatase/rutile samples showed higher toxicity per unit surface area than the pure anatase powders. However, none of the samples affected cellular markers of oxidative stress. Our findings suggest that both SSA and crystallinity are critical determinants of TiO(2)-toxicity toward intestinal cells. PMID:22263745

  6. Definition of genetic events directing the development of distinct types of brain tumors from postnatal neural stem/progenitor cells.

    Science.gov (United States)

    Hertwig, Falk; Meyer, Katharina; Braun, Sebastian; Ek, Sara; Spang, Rainer; Pfenninger, Cosima V; Artner, Isabella; Prost, Gaëlle; Chen, Xinbin; Biegel, Jaclyn A; Judkins, Alexander R; Englund, Elisabet; Nuber, Ulrike A

    2012-07-01

    Although brain tumors are classified and treated based upon their histology, the molecular factors involved in the development of various tumor types remain unknown. In this study, we show that the type and order of genetic events directs the development of gliomas, central nervous system primitive neuroectodermal tumors, and atypical teratoid/rhabdoid-like tumors from postnatal mouse neural stem/progenitor cells (NSC/NPC). We found that the overexpression of specific genes led to the development of these three different brain tumors from NSC/NPCs, and manipulation of the order of genetic events was able to convert one established tumor type into another. In addition, loss of the nuclear chromatin-remodeling factor SMARCB1 in rhabdoid tumors led to increased phosphorylation of eIF2α, a central cytoplasmic unfolded protein response (UPR) component, suggesting a role for the UPR in these tumors. Consistent with this, application of the proteasome inhibitor bortezomib led to an increase in apoptosis of human cells with reduced SMARCB1 levels. Taken together, our findings indicate that the order of genetic events determines the phenotypes of brain tumors derived from a common precursor cell pool, and suggest that the UPR may represent a therapeutic target in atypical teratoid/rhabdoid tumors. PMID:22719073

  7. Stress-Induced In Vivo Recruitment of Human Cytotoxic Natural Killer Cells Favors Subsets with Distinct Receptor Profiles and Associates with Increased Epinephrine Levels.

    Directory of Open Access Journals (Sweden)

    Marc B Bigler

    Full Text Available Acute stress drives a 'high-alert' response in the immune system. Psychoactive drugs induce distinct stress hormone profiles, offering a sought-after opportunity to dissect the in vivo immunological effects of acute stress in humans.3,4-methylenedioxymethamphetamine (MDMA, methylphenidate (MPH, or both, were administered to healthy volunteers in a randomized, double-blind, placebo-controlled crossover-study. Lymphocyte subset frequencies, natural killer (NK cell immune-phenotypes, and changes in effector function were assessed, and linked to stress hormone levels and expression of CD62L, CX3CR1, CD18, and stress hormone receptors on NK cells.MDMA/MPH > MDMA > MPH robustly induced an epinephrine-dominant stress response. Immunologically, rapid redistribution of peripheral blood lymphocyte-subsets towards phenotypically mature NK cells occurred. NK cytotoxicity was unaltered, but they expressed slightly reduced levels of the activating receptor NKG2D. Preferential circulation of mature NK cells was associated with high epinephrine receptor expression among this subset, as well as expression of integrin ligands previously linked to epinephrine-induced endothelial detachment.The acute epinephrine-induced stress response was characterized by rapid accumulation of mature and functional NK cells in the peripheral circulation. This is in line with studies using other acute stressors and supports the role of the acute stress response in rapidly mobilizing the innate immune system to counteract incoming threats.

  8. Autophagosome Proteins LC3A, LC3B and LC3C Have Distinct Subcellular Distribution Kinetics and Expression in Cancer Cell Lines

    Science.gov (United States)

    Koukourakis, Michael I.; Kalamida, Dimitra; Giatromanolaki, Alexandra; Zois, Christos E.; Sivridis, Efthimios; Pouliliou, Stamatia; Mitrakas, Achilleas; Gatter, Kevin C.; Harris, Adrian L.

    2015-01-01

    LC3s (MAP1-LC3A, B and C) are structural proteins of autophagosomal membranes, widely used as biomarkers of autophagy. Whether these three LC3 proteins have a similar biological role in autophagy remains obscure. We examine in parallel the subcellular expression patterns of the three LC3 proteins in a panel of human cancer cell lines, as well as in normal MRC5 fibroblasts and HUVEC, using confocal microscopy and western blot analysis of cell fractions. In the cytoplasm, there was a minimal co-localization between LC3A, B and C staining, suggesting that the relevant autophagosomes are formed by only one out of the three LC3 proteins. LC3A showed a perinuclear and nuclear localization, while LC3B was equally distributed throughout the cytoplasm and localized in the nucleolar regions. LC3C was located in the cytoplasm and strongly in the nuclei (excluding nucleoli), where it extensively co-localized with the LC3A and the Beclin-1 autophagy initiating protein. Beclin 1 is known to contain a nuclear trafficking signal. Blocking nuclear export function by Leptomycin B resulted in nuclear accumulation of all LC3 and Beclin-1 proteins, while Ivermectin that blocks nuclear import showed reduction of accumulation, but not in all cell lines. Since endogenous LC3 proteins are used as major markers of autophagy in clinical studies and cell lines, it is essential to check the specificity of the antibodies used, as the kinetics of these molecules are not identical and may have distinct biological roles. The distinct subcellular expression patterns of LC3s provide a basis for further studies. PMID:26378792

  9. Oscillatory fluid flow elicits changes in morphology, cytoskeleton and integrin-associated molecules in MLO-Y4 cells, but not in MC3T3-E1 cells.

    Science.gov (United States)

    Xu, Huiyun; Zhang, Jian; Wu, Jiawei; Guan, Ying; Weng, Yuanyuan; Shang, Peng

    2012-01-01

    Interstitial fluid flow stress is one of the most important mechanical stimulations of bone cells under physiological conditions. Osteocytes and osteoblasts act as primary mechanosensors within bones, and in vitro are able to respond to fluid shear stress, both morphologically and functionally. However, there is little information about the response of integrin-associated molecules using both osteoblasts and osteocytes. In this study, we investigated the changes in response to 2 hours of oscillatory fluid flow stress in the MLO-Y4 osteocyte-like cell line and the MC3T3-E1 osteoblast-like cell line. MLO-Y4 cells exhibited a significant increase in the expression of integrin-associated molecules, including OPN, CD44, vinculin and integrin αvβ3. However, there was no or limited increase observed in MC3T3-E1 osteoblast-like cells. Cell area and fiber stress formation were also markedly promoted by fluid flow only in MLO-Y4 cells. But the numbers of processes per cell remain unaffected in both cell lines. PMID:23096360

  10. Keratoderma-like T cell dyscrasia: A report of 13 cases and its distinction from mycosis fungoides palmaris et plantaris

    Directory of Open Access Journals (Sweden)

    Cynthia M Magro

    2016-01-01

    Full Text Available Background: Atypical epitheliotropic T cell lymphocytic infiltrates are commonly encountered in routine and consultative dermatopathology practices and typically do not represent mycosis fungoides. Other conditions can mimic certain light microscopic and phenotypic findings encountered in mycosis fungoides, comprising a diverse spectrum of conditions including the lymphomatoid drug reaction, collagen vascular disease, viral hypersensitivity reactions and cutaneous T cell dyscrasia. Aims: To examine biopsies obtained from cutaneous T cell dyscrasia localized to the palms and soles and to evaluate whether it exhibits a morphologic and pathogenetic continuum with mycosis fungoides plantaris et palmaris. Methods: We examined 13 biopsies showing an epidermotropic superficial lymphocytic infiltrate from thirteen patients who presented with a palmar and/or plantar keratoderma without other sites of cutaneous involvement. Conventional light microscopy, immunophenotyping and clonality studies were carried out. The clinical features were recorded. Results: Biopsies showed a variably dense, superficial, angiocentric CD4 or CD8 dominant lymphocytic infiltrate accompanied by a non-destructive pattern of epidermotropism. Low-grade cerebriform atypia along with variable diminution in the expression of CD7 and CD62L was noted. In three cases, statins were suspected to be the cause. Due to lack of familiarity with the entity, treatment interventions were inconsistent and not aggressively pursued. There was no evidence of disease progression to mycosis fungoides in any case. Limitations: The limitations of this study include the lack of long-term follow up and information on the nature of the therapeutic interventions and responses to treatment. Conclusion: The spectrum of cutaneous lymphoid dyscrasias should be expanded to include cases manifesting as palmo-plantar keratoderma. These cases are to be distinguished from mycosis fungoides palmaris et plantaris. As

  11. Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease.

    Directory of Open Access Journals (Sweden)

    Timothy M Butler

    Full Text Available The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome. In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient's resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor.

  12. Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease

    Science.gov (United States)

    Butler, Timothy M.; Johnson-Camacho, Katherine; Peto, Myron; Wang, Nicholas J.; Macey, Tara A.; Korkola, James E.; Koppie, Theresa M.; Corless, Christopher L.; Gray, Joe W.; Spellman, Paul T.

    2015-01-01

    The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA) sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome. In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R) which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient’s resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor. PMID:26317216

  13. CD105 (endoglin-negative murine mesenchymal stromal cells define a new multipotent subpopulation with distinct differentiation and immunomodulatory capacities.

    Directory of Open Access Journals (Sweden)

    Per Anderson

    Full Text Available Administration of in vitro expanded mesenchymal stromal cells (MSCs represents a promising therapy for regenerative medicine and autoimmunity. Both mouse and human MSCs ameliorate autoimmune disease in syn-, allo- and xenogeneic settings. However, MSC preparations are heterogeneous which impairs their therapeutic efficacy and endorses variability between experiments. This heterogeneity has also been a main hurdle in translating experimental MSC data from mouse models to human patients. The objective of the present manuscript has been to further characterize murine MSCs (mMSCs with the aim of designing more efficient and specific MSC-based therapies. We have found that mMSCs are heterogeneous for endoglin (CD105 expression and that this heterogeneity is not due to different stages of MSC differentiation. CD105 is induced on a subpopulation of mMSCs early upon in vitro culture giving rise to CD105(+ and CD105(- MSCs. CD105(+ and CD105(- mMSCs represent independent subpopulations that maintain their properties upon several passages. CD105 expression on CD105(+ mMSCs was affected by passage number and cell confluency while CD105(- mMSCs remained negative. The CD105(+ and CD105(- mMSC subpopulations had similar growth potential and expressed almost identical mMSC markers (CD29(+CD44(+Sca1 (+ MHC-I(+ and CD45(-CD11b(-CD31(- but varied in their differentiation and immunoregulatory properties. Interestingly, CD105(- mMSCs were more prone to differentiate into adipocytes and osteocytes and suppressed the proliferation of CD4(+ T cells more efficiently compared to CD105(+ mMSCs. Based on these studies we propose to redefine the phenotype of mMSCs based on CD105 expression.

  14. Maitotoxin activates cation channels distinct from the receptor-activated non-selective cation channels of HL-60 cells

    OpenAIRE

    Musgrave, I. F.; Seifert, Roland; Schultz, Günter

    1994-01-01

    We investigated whether maitotoxin activates non-selective cation channels, as was recently proposed [Soergel, Yasumoto, Daly and Gusovsky (1992) Mol. Pharmacol. 41, 487-493]. Stimulation of dibutyryl cyclic AMP-differentiated HL-60 cells with the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; 0.1 microM), the Ca(2+)-ATPase inhibitor thapsigargin (0.1 microM) or maitotoxin (25 ng/ml) resulted in an increase in cytoplasmic free calcium concentration ([Ca2+]i). Unlike ...

  15. Met-ase: Cloning and distinct chromosomal location of a serine protease preferentially expressed in human natural killer cells

    Energy Technology Data Exchange (ETDEWEB)

    Smyth, M.J.; Trapani, J.A. (Austin Research Institute, Austin Hospital, Victoria (Australia)); Sayers, T.J.; Wiltrout, T. (NCI-Frederick Cancer Research and Development Center, Frederick, MD (United States)); Powers, J.C. (Georgia Institute of Technology, Atlanta, GA (United States))

    1993-12-01

    A cDNA clone encoding a human NK serine protease was obtained by screening a [lambda]-gt10 library