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Sample records for activated inkt cells

  1. Cytokine dependent and independent iNKT cell activation

    PubMed Central

    Reilly, Emma C.; Wands, Jack R.; Brossay, Laurent

    2010-01-01

    Invariant NKT (iNKT) cells have been extensively studied throughout the last decade due to their ability to polarize and amplify the downstream immune response. Only recently however, have the various mechanisms underlying NKT cell activation begun to unfold. iNKT cells have the ability to respond as innate immune cells with minimal TCR involvement as well as through direct TCR recognition of glycolipid antigens. Additionally, the existence of several subsets of iNKT cells creates the potential for other unique pathways, which are not yet clearly defined. Here we provide an overview of the known mechanisms of invariant NKT cell activation, focusing on cytokine driven pathways and the resulting cytokine responses. PMID:20554220

  2. Activation and Function of iNKT and MAIT Cells.

    PubMed

    Chandra, Shilpi; Kronenberg, Mitchell

    2015-01-01

    Over the last two decades, it has been established that peptides are not the only antigens recognized by T lymphocytes. Here, we review information on two T lymphocyte populations that recognize nonpeptide antigens: invariant natural killer T cells (iNKT cells), which respond to glycolipids, and mucosal associated invariant T cells (MAIT cells), which recognize microbial metabolites. These two populations have a number of striking properties that distinguish them from the majority of T cells. First, their cognate antigens are presented by nonclassical class I antigen-presenting molecules; CD1d for iNKT cells and MR1 for MAIT cells. Second, these T lymphocyte populations have a highly restricted diversity of their T cell antigen receptor α chains. Third, these cells respond rapidly to antigen or cytokine stimulation by producing copious amounts of cytokines, such as IFNγ, which normally are only made by highly differentiated effector T lymphocytes. Because of their response characteristics, iNKT and MAIT cells act at the interface of innate and adaptive immunity, participating in both types of responses. In this review, we will compare these two subsets of innate-like T cells, with an emphasis on the various ways that lead to their activation and their participation in antimicrobial responses.

  3. Chronic alcohol consumption enhances iNKT cell maturation and activation

    SciTech Connect

    Zhang, Hui Zhang, Faya; Zhu, Zhaohui; Luong, Dung; Meadows, Gary G.

    2015-01-15

    Alcohol consumption exhibits diverse effects on different types of immune cells. NKT cells are a unique T cell population and play important immunoregulatory roles in different types of immune responses. The effects of chronic alcohol consumption on NKT cells remain to be elucidated. Using a mouse model of chronic alcohol consumption, we found that alcohol increases the percentage of NKT cells, especially iNKT cells in the thymus and liver, but not in the spleen or blood. Alcohol consumption decreases the percentage of NK1.1{sup −} iNKT cells in the total iNKT cell population in all of the tissues and organs examined. In the thymus, alcohol consumption increases the number of NK1.1{sup +}CD44{sup hi} mature iNKT cells but does not alter the number of NK1.1{sup −} immature iNKT cells. A BrdU incorporation assay shows that alcohol consumption increases the proliferation of thymic NK1.1{sup −} iNKT cells, especially the NK1.1{sup −}CD44{sup lo} Stage I iNKT cells. The percentage of NKG2A{sup +} iNKT cells increases in all of the tissues and organs examined; whereas CXCR3{sup +} iNKT cells only increases in the thymus of alcohol-consuming mice. Chronic alcohol consumption increases the percentage of IFN-γ-producing iNKT cells and increases the blood concentration of IFN-γ and IL-12 after in vivo α-galactosylceramide (αGalCer) stimulation. Consistent with the increased cytokine production, the in vivo activation of iNKT cells also enhances the activation of dendritic cells (DC) and NK, B, and T cells in the alcohol-consuming mice. Taken together the data indicate that chronic alcohol consumption enhances iNKT cell maturation and activation, which favors the Th1 immune response. - Highlights: • Chronic alcohol consumption increases iNKT cells in the thymus and liver • Chronic alcohol consumption enhances thymic Stage I iNKT cell proliferation • Chronic alcohol consumption enhances iNKT cell maturation in thymus and periphery • Chronic alcohol

  4. CD169(+) macrophages present lipid antigens to mediate early activation of iNKT cells in lymph nodes.

    PubMed

    Barral, Patricia; Polzella, Paolo; Bruckbauer, Andreas; van Rooijen, Nico; Besra, Gurdyal S; Cerundolo, Vincenzo; Batista, Facundo D

    2010-04-01

    Invariant natural killer T cells (iNKT cells) are involved in the host defense against microbial infection. Although it is known that iNKT cells recognize glycolipids presented by CD1d, how and where they encounter antigen in vivo remains unclear. Here we used multiphoton microscopy to visualize the dynamics and activation of iNKT cells in lymph nodes. After antigen administration, iNKT cells became confined in a CD1d-dependent manner in close proximity to subcapsular sinus CD169(+) macrophages. These macrophages retained, internalized and presented lipid antigen and were required for iNKT cell activation, cytokine production and population expansion. Thus, CD169(+) macrophages can act as true antigen-presenting cells controlling early iNKT cell activation and favoring the fast initiation of immune responses.

  5. The autophagy machinery restrains iNKT cell activation through CD1D1 internalization.

    PubMed

    Keller, Christian W; Loi, Monica; Ewert, Svenja; Quast, Isaak; Theiler, Romina; Gannagé, Monique; Münz, Christian; De Libero, Gennaro; Freigang, Stefan; Lünemann, Jan D

    2017-03-15

    Invariant natural killer T (iNKT) cells are innate T cells with powerful immune regulatory functions that recognize glycolipid antigens presented by the CD1D protein. While iNKT-cell-activating glycolipids are currently being explored for their efficacy to improve immunotherapy against infectious diseases and cancer, little is known about the mechanisms that control CD1D antigen presentation and iNKT cell activation in vivo. CD1D molecules survey endocytic pathways to bind lipid antigens in MHC class II containing compartments (MIICs) before recycling to the plasma membrane. Autophagosomes intersect with MIICs and autophagy-related proteins are known to support antigen loading for increased CD4(+) T cell immunity. Here, we report that mice with dendritic cell (DC)-specific deletion of the essential autophagy gene Atg5 showed better CD1D1-restricted glycolipid presentation in vivo. These effects led to enhanced iNKT cell cytokine production upon antigen recognition and lower bacterial loads during Sphingomonas paucimobilis infection. Enhanced iNKT cell activation was independent of receptor-mediated glycolipid uptake or costimulatory signals. Instead, loss of Atg5 in DCs impaired clathrin-dependent internalization of CD1D1 molecules via the adaptor protein complex 2 (AP2) and, thus, increased surface expression of stimulatory CD1D1-glycolipid complexes. These findings indicate that the autophagic machinery assists in the recruitment of AP2 to CD1D1 molecules resulting in attenuated iNKT cell activation, in contrast to the supporting role of macroautophagy in CD4(+) T cell stimulation.

  6. An intravascular immune response to Borrelia burgdorferi involves Kupffer cells and iNKT cells

    PubMed Central

    Lee, Woo-Yong; Moriarty, Tara J; Wong, Connie H Y; Zhou, Hong; Strieter, Robert M; van Rooijen, Nico; Chaconas, George; Kubes, Paul

    2016-01-01

    Here we investigate the dynamics of the hepatic intravascular immune response to a pathogen relevant to invariant natural killer T cells (iNKT cells). Immobilized Kupffer cells with highly ramified extended processes into multiple sinusoids could effectively capture blood-borne, disseminating Borrelia burgdorferi, creating a highly efficient surveillance and filtering system. After ingesting B. burgdorferi, Kupffer cells induced chemokine receptor CXCR3–dependent clustering of iNKT cells. Kupffer cells and iNKT cells formed stable contacts via the antigen-presenting molecule CD1d, which led to iNKT cell activation. An absence of iNKT cells caused B. burgdorferi to leave the blood and enter the joints more effectively. B. burgdorferi that escaped Kupffer cells entered the liver parenchyma and survived despite Ito cell responses. Kupffer cell–iNKT cell interactions induced a key intravascular immune response that diminished the dissemination of B. burgdorferi. PMID:20228796

  7. Shared and distinct transcriptional programs underlie the hybrid nature of iNKT cells

    PubMed Central

    Cohen, Nadia R.; Brennan, Patrick J.; Shay, Tal; Watts, Gerald F.; Brigl, Manfred; Kang, Joonsoo; Brenner, Michael B.

    2013-01-01

    Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that act as critical regulators of the immune response. To better characterize this population, we profiled iNKT cell gene expression during ontogeny and in peripheral subsets as part of the Immunological Genome Project (ImmGen). High-resolution comparative transcriptional analyses defined developmental and subset-specific iNKT cell gene expression programs. In addition, iNKT cells were found to share an extensive transcriptional program with natural killer (NK) cells, similar in magnitude to that shared with major histocompatibility complex (MHC)-restricted T cells. Strikingly, the NK- iNKT program also operated constitutively in γδT cells and in adaptive T cells following activation. Together, our findings highlight a core effector program regulated distinctly in innate and adaptive lymphocytes. PMID:23202270

  8. Thymic and peripheral microenvironments differentially mediate development and maturation of iNKT cells by IL-15 transpresentation.

    PubMed

    Castillo, Eliseo F; Acero, Luis F; Stonier, Spencer W; Zhou, Dapeng; Schluns, Kimberly S

    2010-10-07

    Invariant NKT (iNKT) cells are an innate type of T cells, which respond rapidly on activation. iNKT cells acquire these innate-like abilities during development; however, the signals driving development and functional maturation remain only partially understood. Because interleukin-15 (IL-15) is crucial for iNKT development and is delivered by transpresentation, we set out to identify the cell types providing IL-15 to developing iNKT cells and determine their role at the various states of development and maturation. We report here that transpresentation of IL-15 by parenchymal cells was crucial for generating normal number of iNKTs in the thymus, whereas both hematopoietic and parenchymal cells regulated iNKT cell numbers in the periphery, particularly in the liver. Specifically, dendritic cells contributed to peripheral iNKT cell numbers by up-regulating Bcl-2 expression and promoting extrathymic iNKT cell ex-pansion and their homeostatic proliferation. Whether IL-15 affects functional maturation of iNKT cells was also examined. In IL-15Rα(-/-) mice, CD44(High)NK1.1(+) iNKT cells displayed decreased T-bet expression and in response to α-galactosylceramide, had deficient interferon-γ expression. Such defects could be reversed by exogenous IL-15 signals. Overall, these studies identify stage-specific functions of IL-15, which are determined by the tissue microenvironment and elucidate the importance of IL-15 in functional maturation.

  9. Thymic and peripheral microenvironments differentially mediate development and maturation of iNKT cells by IL-15 transpresentation

    PubMed Central

    Castillo, Eliseo F.; Acero, Luis F.; Stonier, Spencer W.; Zhou, Dapeng

    2010-01-01

    Invariant NKT (iNKT) cells are an innate type of T cells, which respond rapidly on activation. iNKT cells acquire these innate-like abilities during development; however, the signals driving development and functional maturation remain only partially understood. Because interleukin-15 (IL-15) is crucial for iNKT development and is delivered by transpresentation, we set out to identify the cell types providing IL-15 to developing iNKT cells and determine their role at the various states of development and maturation. We report here that transpresentation of IL-15 by parenchymal cells was crucial for generating normal number of iNKTs in the thymus, whereas both hematopoietic and parenchymal cells regulated iNKT cell numbers in the periphery, particularly in the liver. Specifically, dendritic cells contributed to peripheral iNKT cell numbers by up-regulating Bcl-2 expression and promoting extrathymic iNKT cell ex-pansion and their homeostatic proliferation. Whether IL-15 affects functional maturation of iNKT cells was also examined. In IL-15Rα−/− mice, CD44HighNK1.1+ iNKT cells displayed decreased T-bet expression and in response to α-galactosylceramide, had deficient interferon-γ expression. Such defects could be reversed by exogenous IL-15 signals. Overall, these studies identify stage-specific functions of IL-15, which are determined by the tissue microenvironment and elucidate the importance of IL-15 in functional maturation. PMID:20581314

  10. iNKT Cell Emigration out of the Lung Vasculature Requires Neutrophils and Monocyte-Derived Dendritic Cells in Inflammation

    PubMed Central

    Thanabalasuriar, A; Neupane, A.S; Wang, J; Krummel, M.F; Kubes, P

    2017-01-01

    iNKT cells are a subset of innate T cells that recognize glycolipids presented on CD1d molecules and protect against a variety of bacterial infections including S. pneumoniae. Using lung intravital imaging, we examined the behavior and mechanism of pulmonary iNKT cell activation in response to the potent iNKT cell ligand α-galactosylceramide or during S. pneumoniae infection. In untreated mice the major fraction of iNKT cells resided in the vasculature, but a small critical population resided in the extravascular space in proximity to monocyte-derived DCs. Administration of either α-GalCer or S. pneumoniae, induced CD1d dependent rapid recruitment of neutrophils out of the vasculature. This neutrophil exodus paved the way for extravasation of iNKT cells from the lung vasculature via CCL17. Depletion of monocyte-derived DCs abrogated both the neutrophil and subsequent iNKT cell extravasation. Moreover, impairing iNKT cell migration out of the lung vasculature by blocking CCL17 greatly increased susceptibility to S. pneumoniae infection, suggesting a critical role for the secondary wave of iNKT cells in host defense. PMID:27653688

  11. Direct identification of rat iNKT cells reveals remarkable similarities to human iNKT cells and a profound deficiency in LEW rats.

    PubMed

    Monzon-Casanova, Elisa; Paletta, Daniel; Starick, Lisa; Müller, Ingrid; Sant'Angelo, Derek B; Pyz, Elwira; Herrmann, Thomas

    2013-02-01

    iNKT cells are a particular lymphocyte population with potent immunomodulatory capa-city; by promoting or suppressing immune responses against infections, tumors, and autoimmunity, iNKT cells are a promising target for immunotherapy. The hallmark of iNKT cells is the expression of a semiinvariant TCR (with an invariant α-chain comprising AV14 and AJ18 gene segments), which recognizes glycolipids presented by CD1d. Here, we identified iNKT cells for the first time in the rat using rat CD1d-dimers and PLZF staining. Importantly, in terms of frequencies (1.05% ± 0.52 SD of all intrahepatic αβ T cells), coreceptor expression and in vitro expansion features, iNKT cells from F344 inbred rats more closely resemble human iNKT cells than their mouse counterparts. In contrast, in LEW inbred rats, which are often used as models for organ-specific autoimmune diseases, iNKT cell numbers are near or below the detection limit. Interestingly, the usage of members of the rat AV14 gene family differed between F344 and LEW inbred rats. In conclusion, the similarities between F344 rat and human iNKT cells and the nearly absent iNKT cells in LEW rats make the rat a promising animal model for the study of iNKT cell-based therapies and of iNKT-cell biology.

  12. iNKT Cells Induce FGF21 for Thermogenesis and Are Required for Maximal Weight Loss in GLP1 Therapy.

    PubMed

    Lynch, Lydia; Hogan, Andrew E; Duquette, Danielle; Lester, Chantel; Banks, Alexander; LeClair, Katherine; Cohen, David E; Ghosh, Abhisek; Lu, Bing; Corrigan, Michelle; Stevanovic, Darko; Maratos-Flier, Eleftheria; Drucker, Daniel J; O'Shea, Donal; Brenner, Michael

    2016-09-13

    Adipose-resident invariant natural killer T (iNKT) cells are key players in metabolic regulation. iNKT cells are innate lipid sensors, and their activation, using their prototypic ligand α-galactosylceramide (αGalCer), induces weight loss and restores glycemic control in obesity. Here, iNKT activation induced fibroblast growth factor 21 (FGF21) production and thermogenic browning of white fat. Complete metabolic analysis revealed that iNKT cell activation induced increased body temperature, V02, VC02, and fatty acid oxidation, without affecting food intake or activity. FGF21 induction played a major role in iNKT cell-induced weight loss, as FGF21 null mice lost significantly less weight after αGalCer treatment. The glucagon-like peptide 1 (GLP-1) receptor agonist, liraglutide, also activated iNKT cells in humans and mice. In iNKT-deficient mice, liraglutide promoted satiety but failed to induce FGF21, resulting in less weight loss. These findings reveal an iNKT cell-FGF21 axis that defines a new immune-mediated pathway that could be targeted for glycemic control and weight regulation.

  13. Soluble γc cytokine receptor suppresses IL-15 signaling and impairs iNKT cell development in the thymus

    PubMed Central

    Park, Joo-Young; Jo, Yuna; Ko, Eunhee; Luckey, Megan A.; Park, Yoo Kyoung; Park, Se-Ho; Park, Jung-Hyun; Hong, Changwan

    2016-01-01

    The soluble γc protein (sγc) is a naturally occurring splice isoform of the γc cytokine receptor that is produced by activated T cells and inhibits γc cytokine signaling. Here we show that sγc expression is also highly upregulated in immature CD4+CD8+ thymocytes but then downregulated in mature thymocytes. These results indicate a developmentally controlled mechanism for sγc expression and suggest a potential role for sγc in regulating T cell development in the thymus. Indeed, sγc overexpression resulted in significantly reduced thymocyte numbers and diminished expansion of immature thymocytes, concordant to its role in suppressing signaling by IL-7, a critical γc cytokine in early thymopoiesis. Notably, sγc overexpression also impaired generation of iNKT cells, resulting in reduced iNKT cell percentages and numbers in the thymus. iNKT cell development requires IL-15, and we found that sγc interfered with IL-15 signaling to suppress iNKT cell generation in the thymus. Thus, sγc represents a new mechanism to control cytokine availability during T cell development that constrains mature T cell production and specifically iNKT cell generation in the thymus. PMID:27833166

  14. iNKT and memory B-cell alterations in HHV-8 multicentric Castleman disease.

    PubMed

    Sbihi, Zineb; Dossier, Antoine; Boutboul, David; Galicier, Lionel; Parizot, Christophe; Emarre, Amandine; Hoareau, Bénédicte; Dupin, Nicolas; Marcelin, Anne-Geneviève; Oudin, Anne; Fieschi, Claire; Agbalika, Félix; Autran, Brigitte; Oksenhendler, Eric; Carcelain, Guislaine

    2017-02-16

    Human herpesvirus 8 (HHV-8) is the causative agent of Kaposi sarcoma (KS) and multicentric Castleman disease (MCD), a life-threatening, virally induced B-cell lymphoproliferative disorder. HHV-8 is a B-lymphotropic γ-herpesvirus closely related to the Epstein-Barr virus (EBV). Invariant natural killer T (iNKT) cells are innate-like T cells that play a role in antiviral immunity, specifically in controlling viral replication in EBV-infected B cells. Decline of iNKT cells is associated with age or HIV infection, both situations associated with HHV-8-related diseases. We analyzed iNKT cells in both blood (n = 26) and spleen (n = 9) samples from 32 patients with HHV-8 MCD and compared them with patients with KS (n = 24) and healthy donors (n = 29). We determined that both circulating and splenic iNKT cell frequencies were markedly decreased in patients with HHV-8 MCD and were undetectable in 6 of them. Moreover, iNKT cells from patients with HHV-8 MCD displayed a proliferative defect after stimulation with α-galactosylceramide. These iNKT cell alterations were associated with an imbalance in B-cell subsets, including a significant decrease in memory B cells, particularly of marginal zone (MZ) B cells. Coculture experiments revealed that the decrease in iNKT cells contributed to the alterations in the B-cell subset distribution. These observations contribute to a better understanding of the complex interactions between HHV-8 and immune cells that cause HHV-8-related MCD.

  15. NNKTT120, an anti-iNKT cell monoclonal antibody, produces rapid and sustained iNKT cell depletion in adults with sickle cell disease

    PubMed Central

    Majerus, Elaine; Ataga, Kenneth I.; Vichinsky, Elliot P.; Schaub, Robert; Mashal, Robert; Nathan, David G.

    2017-01-01

    Invariant NKT (iNKT) cells can be activated to stimulate a broad inflammatory response. In murine models of sickle cell disease (SCD), interruption of iNKT cell activity prevents tissue injury from vaso-occlusion. NKTT120 is an anti-iNKT cell monoclonal antibody that has the potential to rapidly and specifically deplete iNKT cells and, potentially, prevent vaso-occlusion. We conducted an open-label, multi-center, single-ascending-dose study of NKTT120 to determine its pharmacokinetics, pharmacodynamics and safety in steady-state patients with SCD. Doses were escalated in a 3+3 study design over a range from 0.001 mg/kg to 1.0 mg/kg. Twenty-one adults with SCD were administered NKTT120 as part of 7 dose cohorts. Plasma levels of NKTT120 predictably increased with higher doses. Median half-life of NKTT120 was 263 hours. All subjects in the higher dose cohorts (0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg) demonstrated decreased iNKT cells below the lower limit of quantification within 6 hours after infusion, the earliest time point at which they were measured. In those subjects who received the two highest doses of NKTT120 (0.3, 1 mg/kg), iNKT cells were not detectable in the peripheral blood for a range of 2 to 5 months. There were no serious adverse events in the study deemed to be related to NKTT120. In adults with SCD, NKTT120 produced rapid, specific and sustained iNKT cell depletion without any infusional toxicity or attributed serious adverse events. The next step is a trial to determine NKTT120’s ability to decrease rate of vaso-occlusive pain episodes. Trial Registration: clinicaltrials.gov NCT01783691. PMID:28152086

  16. Adenosine A2A receptors induced on iNKT and NK cells reduce pulmonary inflammation and injury in mice with sickle cell disease

    PubMed Central

    Wallace, Kori L.

    2010-01-01

    We showed previously that pulmonary function and arterial oxygen saturation in NY1DD mice with sickle cell disease (SCD) are improved by depletion of invariant natural killer T (iNKT) cells or blockade of their activation. Here we demonstrate that SCD causes a 9- and 6-fold induction of adenosine A2A receptor (A2AR) mRNA in mouse pulmonary iNKT and natural killer (NK) cells, respectively. Treating SCD mice with the A2AR agonist ATL146e produced a dose-dependent reversal of pulmonary dysfunction with maximal efficacy at 10 ng/kg/minute that peaked within 3 days and persisted throughout 7 days of continuous infusion. Crossing NY1DD mice with Rag1−/− mice reduced pulmonary injury that was restored by adoptive transfer of 106 purified iNKT cells. Reconstituted injury was reversed by ATL146e unless the adoptively transferred iNKT cells were pretreated with the A2AR alkylating antagonist, FSPTP (5-amino-7-[2-(4-fluorosulfonyl)phenylethyl]-2-(2-furyl)-pryazolo[4,3-ϵ]-1,2,4-triazolo[1,5-c]pyrimidine), which completely prevented pro-tection. In NY1DD mice exposed to hypoxia-reoxygenation, treatment with ATL146e at the start of reoxygenation prevented further lung injury. Together, these data indicate that activation of induced A2ARs on iNKT and NK cells in SCD mice is sufficient to improve baseline pulmonary function and prevent hypoxia-reoxygenation–induced exacerbation of pulmonary injury. A2A agonists have promise for treating diseases associated with iNKT or NK cell activation. PMID:20798237

  17. T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse

    PubMed Central

    Moran, Amy E.; Holzapfel, Keli L.; Xing, Yan; Cunningham, Nicole R.; Maltzman, Jonathan S.; Punt, Jennifer

    2011-01-01

    The ability of antigen receptors to engage self-ligands with varying affinity is crucial for lymphocyte development. To further explore this concept, we generated transgenic mice expressing GFP from the immediate early gene Nr4a1 (Nur77) locus. GFP was up-regulated in lymphocytes by antigen receptor stimulation but not by inflammatory stimuli. In T cells, GFP was induced during positive selection, required major histocompatibility complex for maintenance, and directly correlated with the strength of T cell receptor (TCR) stimulus. Thus, our results define a novel tool for studying antigen receptor activation in vivo. Using this model, we show that regulatory T cells (Treg cells) and invariant NKT cells (iNKT cells) perceived stronger TCR signals than conventional T cells during development. However, although Treg cells continued to perceive strong TCR signals in the periphery, iNKT cells did not. Finally, we show that Treg cell progenitors compete for recognition of rare stimulatory TCR self-ligands. PMID:21606508

  18. Invariant natural killer T (iNKT) cells in asthma: a novel insight into the pathogenesis of asthma and the therapeutic implication of glycolipid ligands for allergic diseases.

    PubMed

    Oki, Shinji; Miyake, Sachiko

    2007-03-01

    Allergic bronchial asthma is a complex inflammatory diseases originated from dysregulated immune responses in the respiratory mucosa. The inflammatory state in asthmatic lung is characterized by massive infiltration with eosinophils, lymphocytes, and mast cells in the airway mucosa leading to airway hyperseisitivity, goblet cell hyperplasia and mucus overproduction. The inflammatory process is thought to be the result of intensive T helper (Th) 2-biased immune response. Over the past several years, there has been enormous progress in understanding the mechanisms for development of Th2-biased responses after inhaled exposure to allergens and the characteristics of CD4+ T cells prominently involved in this process. Recently, a new population of T cells, invariant natural killer T (iNKT) cells has been shown to play an important role in the pathogenesis of mouse model of allergic airway inflammation. iNKT cells are one of the most potent immune modulators through a massive production of a various cytokines including IL-4 and IFN-gamma upon activation, and are involved in a variety of immunoregulations including infection, autoimmunity, and tumor surveillance. The potent pathogenic role of iNKT cells in the development of bronchial asthma is due to their ability to produce predominant Th2 cytokines in a given condition. The involvement of iNKT cells in the pathogenesis of asthma might have been underestimated in the past studies demonstrating the involvement of CD4+ T cells in asthma because of the difficulty in the detection of iNKT cells. Meanwhile, growing evidences have demonstrated that iNKT cells could be a promising target for immune-based therapies for autoimmune diseases, tumor, and infection due to the invariance of their TCR usage, the restriction to the evolutionally-conserved non-polymorphic antigen-presenting molecule CD1d, and their outstanding ability to produce both Th1- and Th2-cytokines. In this review, we will overview current understanding of the

  19. miR-17∼92 family clusters control iNKT cell ontogenesis via modulation of TGF-β signaling.

    PubMed

    Fedeli, Maya; Riba, Michela; Garcia Manteiga, Jose Manuel; Tian, Lei; Viganò, Valentina; Rossetti, Grazisa; Pagani, Massimiliano; Xiao, Changchun; Liston, Adrian; Stupka, Elia; Cittaro, Davide; Abrignani, Sergio; Provero, Paolo; Dellabona, Paolo; Casorati, Giulia

    2016-12-20

    Invariant natural killer T cells (iNKT) cells are T lymphocytes displaying innate effector functions, acquired through a distinct thymic developmental program regulated by microRNAs (miRNAs). Deleting miRNAs by Dicer ablation (Dicer KO) in thymocytes selectively impairs iNKT cell survival and functional differentiation. To unravel this miRNA-dependent program, we systemically identified transcripts that were differentially expressed between WT and Dicer KO iNKT cells at different differentiation stages and predicted to be targeted by the iNKT cell-specific miRNAs. TGF-β receptor II (TGF-βRII), critically implicated in iNKT cell differentiation, was found up-regulated in iNKT Dicer KO cells together with enhanced TGF-β signaling. miRNA members of the miR-17∼92 family clusters were predicted to target Tgfbr2 mRNA upon iNKT cell development. iNKT cells lacking all three miR-17∼92 family clusters (miR-17∼92, miR-106a∼363, miR-106b∼25) phenocopied both increased TGF-βRII expression and signaling, and defective effector differentiation, displayed by iNKT Dicer KO cells. Consistently, genetic ablation of TGF-β signaling in the absence of miRNAs rescued iNKT cell differentiation. These results elucidate the global impact of miRNAs on the iNKT cell developmental program and uncover the targeting of a lineage-specific cytokine signaling by miRNAs as a mechanism regulating innate-like T-cell development and effector differentiation.

  20. Implications of Lymphocyte Anergy to Glycolipids in Multiple Sclerosis (MS): iNKT Cells May Mediate the MS Infectious Trigger

    PubMed Central

    Hogan, Edward L; Podbielska, Maria; O’Keeffe, Joan

    2015-01-01

    Immunogenic lipids may play key roles in host defenses against infection and in generating autoimmune inflammation and organ-specific damage. In multiple sclerosis (MS) there are unequivocal autoimmune features and vulnerability to aggravation or induction by microbial or viral infection. We have found glycolipid-driven anergy of circulating lymphocytes in MS indicating that this immune response is affected in MS and the robust effects of iNKT activation with potent cellular and cytokine activities emphasizes its potential importance. Diverse glycolipids including the endogenous myelin acetylated-galactosylceramides (AcGalCer) can drive activation that could be critical to the inflammatory demyelination in the central nervous system and clinical consequences. The iNKT cells and their invariant or iTCR (Vα24Jα18Vβ11) receptor an innate defense–a discrete immune arm that is separate from peptide-driven acquired immune responses. This offers new possibilities for insight including a likelihood that the pattern recognition of exogenous microbial and myelin immunogens can overlap and cross-react especially in an inflammatory milieu. PMID:26347308

  1. Participation of iNKT cells in the early and late components of Tc1-mediated DNFB contact sensitivity: cooperative role of γδ-T cells.

    PubMed

    Askenase, P W; Majewska-Szczepanik, M; Kerfoot, S; Szczepanik, M

    2011-05-01

    Prior studies of classical 24 h responses in TNP-Cl (picryl chloride) allergic contact sensitivity (CS), showed mediation by Th1 cells in CBA mice, and established that 24 h elicitation of responses requires an early 2 h CS-initiating component dependent on iNKT cells, IL-4 and B-1 B cells. Here, we studied the other form of cytotoxic T cell (Tc1) CS in DNFB sensitized BALB/c mice and determined that similar CS-initiation also is required. We systematically tested each step of the initiation pathway in this model. Thus, DNFB Tc1 CS was significantly impaired in iNKT cell deficient CD1d(-/-) and Jα18(-/-) mice, IL4Rα(-/-) and STAT-6(-/-) mice, and also in pan B-cell deficient JH(-/-) mice. Further, the Tc1 DNFB CS-initiating component, like Th1 response to TNP-Cl, was elicited by only 1-day after immunization, due to B-1 cells. In summary, we show that CS-Initiation also is required in Tc1 CS. Further, we have newly determined regulatory support of both the early and late components of DNFB induced Tc1 CS by iNKT cells and γδ-T cells. In summary, both iNKT cells and assisting γδ-T cells are involved in initiating and effector phases of DNFB induced CS.

  2. Distinct gene expression patterns correlate with developmental and functional traits of iNKT subsets

    PubMed Central

    Georgiev, Hristo; Ravens, Inga; Benarafa, Charaf; Förster, Reinhold; Bernhardt, Günter

    2016-01-01

    Invariant natural killer T (iNKT) cells comprise a subpopulation of innate lymphocytes developing in thymus. A new model proposes subdividing murine iNKT cells into iNKT1, 2 and 17 cells. Here, we use transcriptome analyses of iNKT1, 2 and 17 subsets isolated from BALB/c and C57BL/6 thymi to identify candidate genes that may affect iNKT cell development, migration or function. We show that Fcɛr1γ is involved in generation of iNKT1 cells and that SerpinB1 modulates frequency of iNKT17 cells. Moreover, a considerable proportion of iNKT17 cells express IL-4 and IL-17 simultaneously. The results presented not only validate the usefulness of the iNKT1/2/17-concept but also provide new insights into iNKT cell biology. PMID:27721447

  3. CD169+ MACROPHAGES PRESENT LIPID ANTIGENS TO MEDIATE EARLY ACTIVATION OF INVARIANT NKT CELLS IN LYMPH NODES

    PubMed Central

    Barral, Patricia; Polzella, Paolo; Bruckbauer, Andreas; van Rooijen, Nico; Besra, Gurdyal S.; Cerundolo, Vincenzo; Batista, Facundo D.

    2010-01-01

    Invariant NKT (iNKT) cells are involved in host defence against microbial infections. While it is known that iNKT cells recognize glycolipids presented by CD1d, how and where they encounter antigen in vivo remains unclear. We used multi-photon microscopy to visualize the dynamics and activation of iNKT cells in lymph nodes. Following antigen administration, iNKT cells become confined in a CD1d-dependent manner in close proximity to subcapsular sinus CD169+ macrophages. These macrophages retain, internalize and present lipid antigen, and are required for iNKT cell activation, cytokine production and expansion. Thus, CD169+ macrophages can act as bona fide antigen presenting cells controlling early iNKT cell activation and favouring fast initiation of immune responses. PMID:20228797

  4. Relationships between Th1 or Th2 iNKT Cell Activity and Structures of CD1d-Antigen Complexes: Meta-analysis of CD1d-Glycolipids Dynamics Simulations

    PubMed Central

    Laurent, Xavier; Renault, Nicolas; Farce, Amaury; Chavatte, Philippe; Hénon, Eric

    2014-01-01

    A number of potentially bioactive molecules can be found in nature. In particular, marine organisms are a valuable source of bioactive compounds. The activity of an α-galactosylceramide was first discovered in 1993 via screening of a Japanese marine sponge (Agelas mauritanius). Very rapidly, a synthetic glycololipid analogue of this natural molecule was discovered, called KRN7000. Associated with the CD1d protein, this α-galactosylceramide 1 (KRN7000) interacts with the T-cell antigen receptor to form a ternary complex that yields T helper (Th) 1 and Th2 responses with opposing effects. In our work, we carried out molecular dynamics simulations (11.5 µs in total) involving eight different ligands (conducted in triplicate) in an effort to find out correlation at the molecular level, if any, between chemical modulation of 1 and the orientation of the known biological response, Th1 or Th2. Comparative investigations of human versus mouse and Th1 versus Th2 data have been carried out. A large set of analysis tools was employed including free energy landscapes. One major result is the identification of a specific conformational state of the sugar polar head, which could be correlated, in the present study, to the biological Th2 biased response. These theoretical tools provide a structural basis for predicting the very different dynamical behaviors of α-glycosphingolipids in CD1d and might aid in the future design of new analogues of 1. PMID:25376021

  5. Antibody-dependent cellular cytotoxicity toward neuroblastoma enhanced by activated invariant natural killer T cells.

    PubMed

    Mise, Naoko; Takami, Mariko; Suzuki, Akane; Kamata, Toshiko; Harada, Kazuaki; Hishiki, Tomoro; Saito, Takeshi; Terui, Keita; Mitsunaga, Tetsuya; Nakata, Mitsuyuki; Ikeuchi, Takayuki; Nakayama, Toshinori; Yoshida, Hideo; Motohashi, Shinichiro

    2016-03-01

    Anti-ganglioside GD2 antibodies mainly work through antibody-dependent cellular cytotoxicity (ADCC) and have demonstrated clinical benefit for children with neuroblastoma. However, high-risk neuroblastoma still has a high recurrence rate. For further improvement in patient outcomes, ways to maximize the cytotoxic effects of anti-GD2 therapies with minimal toxicity are required. Activated invariant natural killer T (iNKT) cells enhance both innate and type I acquired anti-tumor immunity by producing several kinds of cytokines. In this report, we investigated the feasibility of combination therapy using iNKT cells and an anti-GD2 antibody. Although some of the expanded iNKT cells expressed natural killer (NK) cell markers, including FcγR, iNKT cells were not directly associated with ADCC. When co-cultured with activated iNKT cells, granzyme A, granzyme B and interferon gamma (IFNγ) production from NK cells were upregulated, and the cytotoxicity of NK cells treated with anti-GD2 antibodies was increased. Not only cytokines produced by activated iNKT cells, but also NK-NKT cell contact or NK cell-dendritic cell contact contributed to the increase in NK cell cytotoxicity and further IFNγ production by iNKT cells and NK cells. In conclusion, iNKT cell-based immunotherapy could be an appropriate candidate for anti-GD2 antibody therapy for neuroblastoma.

  6. Cutting edge: nonglycosidic CD1d lipid ligands activate human and murine invariant NKT cells.

    PubMed

    Silk, Jonathan D; Salio, Mariolina; Reddy, B Gopal; Shepherd, Dawn; Gileadi, Uzi; Brown, James; Masri, S Hajar; Polzella, Paolo; Ritter, Gerd; Besra, Gurdyal S; Jones, E Yvonne; Schmidt, Richard R; Cerundolo, Vincenzo

    2008-05-15

    Invariant NKT cells (iNKT cells) recognize CD1d/glycolipid complexes. We demonstrate that the nonglycosidic compound threitolceramide efficiently activates iNKT cells, resulting in dendritic cell (DC) maturation and the priming of Ag-specific T and B cells. Threitolceramide-pulsed DCs are more resistant to iNKT cell-dependent lysis than alpha-galactosylceramide-pulsed DCs due to the weaker affinity of the human iNKT TCR for CD1d/ threitolceramide than CD1d/alpha-galactosylceramide complexes. iNKT cells stimulated with threitolceramide also recover more quickly from activation-induced anergy. Kinetic and functional experiments showed that shortening or lengthening the threitol moiety by one hydroxymethylene group modulates ligand recognition, as human and murine iNKT cells recognize glycerolceramide and arabinitolceramide differentially. Our data broaden the range of potential iNKT cell agonists. The ability of these compounds to assist the priming of Ag-specific immune responses while minimizing iNKT cell-dependent DC lysis makes them attractive adjuvants for vaccination strategies.

  7. Activation of decidual invariant natural killer T cells promotes lipopolysaccharide-induced preterm birth.

    PubMed

    Li, Liping; Yang, Jing; Jiang, Yao; Tu, Jiaoqin; Schust, Danny J

    2015-04-01

    Invariant natural killer T (iNKT) cells are crucial for host defense against a variety of microbial pathogens, but the underlying mechanisms of iNKT cells activation by microbes are not fully explained. In this study, we investigated the molecular mechanisms of iNKT cell activation in lipopolysaccharide (LPS)-stimulated preterm birth using an adoptive transfer system and diverse neutralizing antibodies (Abs) and inhibitors. We found that adoptive transfer of decidual iNKT cells to LPS-stimulated iNKT cell deficient Jα18(-/-) mice that lack invariant Vα14Jα281T cell receptor (TCR) expression significantly decreased the time to delivery and increased the percentage of decidual iNKT cells. Neutralizing Abs against Toll-like receptor 4 (TLR-4), CD1d, interleukin (IL)-12 and IL-18, and inhibitors blocking the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinase (MAPK) p38 and extracellular signal-regulated kinase (ERK) significantly reduced in vivo percentages of decidual iNKT cells, their intracellular interferon (IFN)-γ production and surface CD69 expression. In vitro, in the presence of the same Abs and inhibitors used as in vivo, decidual iNKT cells co-cultured with LPS-pulsed dendritic cells (DCs) showed significantly decreased extracellular and intracellular IFN-γ secretion and surface CD69 expression. Our data demonstrate that the activation of decidual iNKT cells plays an important role in inflammation-induced preterm birth. Activation of decidual iNKT cells also requires TLR4-mediated NF-κB, MAPK p38 and ERK pathways, the proinflammatory cytokines IL-12 and IL-18, and endogenous glycolipid antigens presented by CD1d.

  8. Development of Spontaneous Anergy in Invariant Natural Killer T Cells in a Mouse Model of Dyslipidemia

    PubMed Central

    Braun, Nicole A.; Mendez-Fernandez, Yanice V.; Covarrubias, Roman; Porcelli, Steven A.; Savage, Paul B.; Yagita, Hideo; Van Kaer, Luc; Major, Amy S.

    2010-01-01

    Objective In this study, we investigated whether dyslipidemia-associated perturbed invariant natural killer T (iNKT) cell function is due to intrinsic changes in iNKT cells or defects in the ability of antigen-presenting cells to activate iNKT cells. Methods and Results We compared iNKT cell expansion and cytokine production in C57BL/6J (B6) and apolipoprotein E-deficient (apoE−/−) mice. In response to in vivo stimulation with α-galactosylceramide, a prototypic iNKT cell glycolipid antigen, apoE−/− mice showed significantly decreased splenic iNKT cell expansion at 3 days after injection, a profile associated with iNKT cell anergy due to chronic stimulation. This decrease in expansion and cytokine production was accompanied by a 2-fold increase in percentage of iNKT cells expressing the inhibitory marker programmed death-1 in apoE−/− mice compared with controls. However, in vivo and in vitro blockade of programmed death-1 using monoclonal antibody was not able to restore functions of iNKT cells from apoE−/− mice to B6 levels. iNKT cells from apoE−/− mice also had increased intracellular T cell receptor and Ly49 expression, a phenotype associated with previous activation. Changes in iNKT cell functions were cell autonomous, because dendritic cells from apoE−/− mice were able to activate B6 iNKT cells, but iNKT cells from apoE−/− mice were not able to respond to B6 dendritic cells. Conclusion These data suggest that chronic dyslipidemia induces an iNKT cell phenotype that is unresponsive to further simulation by exogenous glycolipid and that sustained unresponsiveness is iNKT cell intrinsic. PMID:20539017

  9. Rapid and reliable generation of invariant natural killer T-cell lines in vitro

    PubMed Central

    Chiba, Asako; Cohen, Nadia; Brigl, Manfred; Brennan, Patrick J; Besra, Gurdal S; Brenner, Michael B

    2009-01-01

    Several tools have proved useful in the study of invariant natural killer T (iNKT) cells, including CD1d-deficient mice, Jα281-deficient mice, synthetic lipid antigens and antigen-loaded CD1d tetramers. However, the generation and examination of long-term primary murine iNKT cell lines in vitro has been challenging. Here, we show the rapid generation of iNKT cell lines from splenic iNKT cells of Vα14 T-cell receptor (TCR) transgenic (Tg) mice. These purified iNKT cells were stimulated by bone marrow-derived dendritic cells (BMDCs) loaded with α-galactosylceramide (αGalCer) and cultured with interleukin (IL)-2 and IL-7. iNKT cells proliferated dramatically, and the cell number exhibited a 100-fold increase within 2 weeks and a 105-fold increase in 8 weeks after repeated stimulation with αGalCer. The iNKT cell lines consisted of iNKT cells expressing Vβ chains including Vβ8.1/8.2, Vβ14, Vβ10, Vβ6 and Vβ7, and responded to stimulation with αGalCer presented both by BMDCs and by plate-bound CD1d. In addition, the iNKT cell lines produced interferon (IFN)-γ when activated by lipopolysaccharide (LPS) or CpG oligodeoxynucleotide (ODN)-stimulated BMDCs. Further, we show that iNKT cell lines produced cytokines in response to microbial antigens. In summary, high-yield iNKT cell lines were generated very rapidly and robustly expanded, and these iNKT cells responded to both TCR and cytokine stimulation in vitro. Given the desire to study primary iNKT cells for many purposes, these iNKT cell lines should provide an important tool for the study of iNKT cell subsets, antigen and TCR specificity, activation, inactivation and effector functions. PMID:20067532

  10. Activation strategies for invariant natural killer T cells.

    PubMed

    Kohlgruber, Ayano C; Donado, Carlos A; LaMarche, Nelson M; Brenner, Michael B; Brennan, Patrick J

    2016-08-01

    Invariant natural killer T (iNKT) cells are a specialized T cell subset that plays an important role in host defense, orchestrating both innate and adaptive immune effector responses against a variety of microbes. Specific microbial lipids and mammalian self lipids displayed by the antigen-presenting molecule CD1d can activate iNKT cells through their semi-invariant αβ T cell receptors (TCRs). iNKT cells also constitutively express receptors for inflammatory cytokines typically secreted by antigen-presenting cells (APCs) after recognition of pathogen-associated molecular patterns (PAMPs), and they can be activated through these cytokine receptors either in combination with TCR signals, or in some cases even in the absence of TCR signaling. During infection, experimental evidence suggests that both TCR-driven and cytokine-driven mechanisms contribute to iNKT cell activation. While the relative contributions of these two signaling mechanisms can vary widely depending on the infectious context, both lipid antigens and PAMPs mediate reciprocal activation of iNKT cells and APCs, leading to downstream activation of multiple other immune cell types to promote pathogen clearance. In this review, we discuss the mechanisms involved in iNKT cell activation during infection, focusing on the central contributions of both lipid antigens and PAMP-induced inflammatory cytokines, and highlight in vivo examples of activation during bacterial, viral, and fungal infections.

  11. Significance of para-esophageal lymph nodes in food or aeroallergen-induced iNKT cell-mediated experimental eosinophilic esophagitis.

    PubMed

    Rajavelu, Priya; Rayapudi, Madhavi; Moffitt, Matthew; Mishra, Akanksha; Mishra, Anil

    2012-04-01

    Eosinophilic esophagitis (EoE) is a recently recognized inflammatory disorder driven by food hypersensitivity; however, the specific foods and mechanisms involved are unclear. In patients with EoE, we have found that hypersensitivities to corn and peanuts are the most common. Accordingly, we sensitized and exposed mice either intranasally or intragastrically with corn or peanut extract or saline. Esophageal eosinophilia, the genes of eosinophil-directed cytokines, and allergen-induced antibodies were examined in mice challenged with corn or peanut extract or saline. A high number of esophageal lamina propria eosinophils as well as eosinophilic microabscesses, intraepithelial eosinophils, extracellular eosinophilic granules, thickened and disrupted epithelial mucosa, and mast cell hyperplasia were observed in the esophagus of peanut or corn allergen-challenged mice. Mechanistic analysis indicated that para-esophageal lymph nodes might be critical in the trafficking of eosinophils to the esophagus and in EoE association to airway eosinophilia. Furthermore, experimentation with gene-targeted mice revealed that peanut allergen-induced EoE was dependent on eotaxin and invariant natural killer T (iNKT) cells, as CD1d and eotaxin-1/2 gene-deficient mice were protected from disease induction. Thus we provide evidence that para-esophageal lymph nodes are involved in food- or aeroallergen-induced eosinophilia and patchy EoE pathogenesis, likely a mechanism dependent on eotaxins and iNKT cells.

  12. Maternal low protein diet leads to dysregulation of placental iNKT cells and M1/M2 macrophage ratio, body weight loss in male, neonate Sprague-Dawley rats and increased UCP-1 mediated thermogenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Placental immune cells provide cytokines and growth factors that are necessary for placenta development and function. Invariant natural killer T (iNKT) cells are innate cells specific for glycolipid antigens presented by the CD1d molecule and secrete Th1 cytokines in the placenta, suggesting an imm...

  13. Modulation of human natural killer T cell ligands on TLR-mediated antigen-presenting cell activation.

    PubMed

    Salio, Mariolina; Speak, Anneliese O; Shepherd, Dawn; Polzella, Paolo; Illarionov, Petr A; Veerapen, Natacha; Besra, Gurdyal S; Platt, Frances M; Cerundolo, Vincenzo

    2007-12-18

    Invariant natural killer T (iNKT) cells are a subset of nonconventional T cells recognizing endogenous and/or exogenous glycolipid antigens in the context of CD1d molecules. It remains unclear whether innate stimuli can modify the profile of endogenous lipids recognized by iNKT cells on the surface of antigen-presenting cells (APCs). We report that activation of human APCs by Toll-like receptor ligands (TLR-L) modulates the lipid biosynthetic pathway, resulting in enhanced recognition of CD1d-associated lipids by iNKT cells, as defined by IFN-gamma secretion. APC-derived soluble factors further increase CD1d-restricted iNKT cell activation. Finally, using soluble tetrameric iNKT T cell receptors (TCR) as a staining reagent, we demonstrate specific up-regulation of CD1d-bound ligand(s) on TLR-mediated APC maturation. The ability of innate stimuli to modulate the lipid profile of APCs resulting in iNKT cell activation and APC maturation underscores the role of iNKT cells in assisting priming of antigen-specific immune responses.

  14. Modulation of human natural killer T cell ligands on TLR-mediated antigen-presenting cell activation

    PubMed Central

    Salio, Mariolina; Speak, Anneliese O.; Shepherd, Dawn; Polzella, Paolo; Illarionov, Petr A.; Veerapen, Natacha; Besra, Gurdyal S.; Platt, Frances M.; Cerundolo, Vincenzo

    2007-01-01

    Invariant natural killer T (iNKT) cells are a subset of nonconventional T cells recognizing endogenous and/or exogenous glycolipid antigens in the context of CD1d molecules. It remains unclear whether innate stimuli can modify the profile of endogenous lipids recognized by iNKT cells on the surface of antigen-presenting cells (APCs). We report that activation of human APCs by Toll-like receptor ligands (TLR-L) modulates the lipid biosynthetic pathway, resulting in enhanced recognition of CD1d-associated lipids by iNKT cells, as defined by IFN-γ secretion. APC-derived soluble factors further increase CD1d-restricted iNKT cell activation. Finally, using soluble tetrameric iNKT T cell receptors (TCR) as a staining reagent, we demonstrate specific up-regulation of CD1d-bound ligand(s) on TLR-mediated APC maturation. The ability of innate stimuli to modulate the lipid profile of APCs resulting in iNKT cell activation and APC maturation underscores the role of iNKT cells in assisting priming of antigen-specific immune responses. PMID:18077358

  15. Protection from type 1 diabetes by invariant NK T cells requires the activity of CD4+CD25+ regulatory T cells.

    PubMed

    Ly, Dalam; Mi, Qing-Sheng; Hussain, Shabbir; Delovitch, Terry L

    2006-09-15

    Invariant NK T (iNKT) cells regulate immune responses, express NK cell markers and an invariant TCR, and recognize lipid Ags in a CD1d-restricted manner. Previously, we reported that activation of iNKT cells by alpha-galactosylceramide (alpha-GalCer) protects against type 1 diabetes (T1D) in NOD mice via an IL-4-dependent mechanism. To further investigate how iNKT cells protect from T1D, we analyzed whether iNKT cells require the presence of another subset(s) of regulatory T cells (Treg), such as CD4+ CD25+ Treg, for this protection. We found that CD4+ CD25+ T cells from NOD.CD1d(-/-) mice deficient in iNKT cell function similarly in vitro to CD4+ CD25+ T cells from wild-type NOD mice and suppress the proliferation of NOD T responder cells upon alpha-GalCer stimulation. Cotransfer of NOD diabetogenic T cells with CD4+ CD25+ Tregs from NOD mice pretreated with alpha-GalCer demonstrated that activated iNKT cells do not influence the ability of T(regs) to inhibit the transfer of T1D. In contrast, protection from T1D mediated by transfer of activated iNKT cells requires the activity of CD4+ CD25+ T cells, because splenocytes pretreated with alpha-GalCer and then inactivated by anti-CD25 of CD25+ cells did not protect from T1D. Similarly, mice inactivated of CD4+ CD25+ T cells before alpha-GalCer treatment were also not protected from T1D. Our data suggest that CD4+ CD25+ T cells retain their function during iNKT cell activation, and that the activity of CD4+ CD25+ Tregs is required for iNKT cells to transfer protection from T1D.

  16. Metabolic regulator Fnip1 is crucial for iNKT lymphocyte development.

    PubMed

    Park, Heon; Tsang, Mark; Iritani, Brian M; Bevan, Michael J

    2014-05-13

    Folliculin-interacting protein 1 (Fnip1) is an adaptor protein that physically interacts with AMPK, an energy-sensing kinase that stimulates mitochondrial biogenesis and autophagy in response to low ATP, while turning off energy consumption mediated by mammalian target of rapamycin. Previous studies with Fnip1-null mice revealed that Fnip1 is essential for pre-B-cell development. Here we report a critical role of Fnip1 in invariant natural killer T (iNKT) cell development. Thymic iNKT development in Fnip1(-/-) mice was arrested at stage 2 (NK1.1(-)CD44(+)) but development of CD4, CD8, γδ T-cell, and NK cell lineages proceeded normally. Enforced expression of a Vα14Jα18 iNKT TCR transgene or loss of the proapoptotic protein Bim did not rescue iNKT cell maturation in Fnip1(-/-) mice. Whereas most known essential transcription factors for iNKT cell development were represented normally, Fnip1(-/-) iNKT cells failed to down-regulate Promyelocytic leukemia zinc finger compared with their WT counterparts. Moreover, Fnip1(-/-) iNKT cells contained hyperactive mTOR and reduced mitochondrial number despite lower ATP levels, resulting in increased sensitivity to apoptosis. These results indicate that Fnip1 is vital for iNKT cell development by maintaining metabolic homeostasis in response to metabolic stress.

  17. The adaptor molecule SAP plays essential roles during invariant NKT cell cytotoxicity and lytic synapse formation.

    PubMed

    Das, Rupali; Bassiri, Hamid; Guan, Peng; Wiener, Susan; Banerjee, Pinaki P; Zhong, Ming-Chao; Veillette, André; Orange, Jordan S; Nichols, Kim E

    2013-04-25

    The adaptor molecule signaling lymphocytic activation molecule-associated protein (SAP) plays critical roles during invariant natural killer T (iNKT) cell ontogeny. As a result, SAP-deficient humans and mice lack iNKT cells. The strict developmental requirement for SAP has made it difficult to discern its possible involvement in mature iNKT cell functions. By using temporal Cre recombinase-mediated gene deletion to ablate SAP expression after completion of iNKT cell development, we demonstrate that SAP is essential for T-cell receptor (TCR)-induced iNKT cell cytotoxicity against T-cell and B-cell leukemia targets in vitro and iNKT-cell-mediated control of T-cell leukemia growth in vivo. These findings are not restricted to the murine system: silencing RNA-mediated suppression of SAP expression in human iNKT cells also significantly impairs TCR-induced cytolysis. Mechanistic studies reveal that iNKT cell killing requires the tyrosine kinase Fyn, a known SAP-binding protein. Furthermore, SAP expression is required within iNKT cells to facilitate their interaction with T-cell targets and induce reorientation of the microtubule-organizing center to the immunologic synapse (IS). Collectively, these studies highlight a novel and essential role for SAP during iNKT cell cytotoxicity and formation of a functional IS.

  18. Clinical regressions and broad immune activation following combination therapy targeting human NKT cells in myeloma

    PubMed Central

    Richter, Joshua; Neparidze, Natalia; Zhang, Lin; Nair, Shiny; Monesmith, Tamara; Sundaram, Ranjini; Miesowicz, Fred; Dhodapkar, Kavita M.

    2013-01-01

    Natural killer T (iNKT) cells can help mediate immune surveillance against tumors in mice. Prior studies targeting human iNKT cells were limited to therapy of advanced cancer and led to only modest activation of innate immunity. Clinical myeloma is preceded by an asymptomatic precursor phase. Lenalidomide was shown to mediate antigen-specific costimulation of human iNKT cells. We treated 6 patients with asymptomatic myeloma with 3 cycles of combination of α-galactosylceramide–loaded monocyte-derived dendritic cells and low-dose lenalidomide. Therapy was well tolerated and led to reduction in tumor-associated monoclonal immunoglobulin in 3 of 4 patients with measurable disease. Combination therapy led to activation-induced decline in measurable iNKT cells and activation of NK cells with an increase in NKG2D and CD56 expression. Treatment also led to activation of monocytes with an increase in CD16 expression. Each cycle of therapy was associated with induction of eosinophilia as well as an increase in serum soluble IL2 receptor. Clinical responses correlated with pre-existing or treatment-induced antitumor T-cell immunity. These data demonstrate synergistic activation of several innate immune cells by this combination and the capacity to mediate tumor regression. Combination therapies targeting iNKT cells may be of benefit toward prevention of cancer in humans (trial registered at clinicaltrials.gov: NCT00698776). PMID:23100308

  19. Clinical regressions and broad immune activation following combination therapy targeting human NKT cells in myeloma.

    PubMed

    Richter, Joshua; Neparidze, Natalia; Zhang, Lin; Nair, Shiny; Monesmith, Tamara; Sundaram, Ranjini; Miesowicz, Fred; Dhodapkar, Kavita M; Dhodapkar, Madhav V

    2013-01-17

    Natural killer T (iNKT) cells can help mediate immune surveillance against tumors in mice. Prior studies targeting human iNKT cells were limited to therapy of advanced cancer and led to only modest activation of innate immunity. Clinical myeloma is preceded by an asymptomatic precursor phase. Lenalidomide was shown to mediate antigen-specific costimulation of human iNKT cells. We treated 6 patients with asymptomatic myeloma with 3 cycles of combination of α-galactosylceramide-loaded monocyte-derived dendritic cells and low-dose lenalidomide. Therapy was well tolerated and led to reduction in tumor-associated monoclonal immunoglobulin in 3 of 4 patients with measurable disease. Combination therapy led to activation-induced decline in measurable iNKT cells and activation of NK cells with an increase in NKG2D and CD56 expression. Treatment also led to activation of monocytes with an increase in CD16 expression. Each cycle of therapy was associated with induction of eosinophilia as well as an increase in serum soluble IL2 receptor. Clinical responses correlated with pre-existing or treatment-induced antitumor T-cell immunity. These data demonstrate synergistic activation of several innate immune cells by this combination and the capacity to mediate tumor regression. Combination therapies targeting iNKT cells may be of benefit toward prevention of cancer in humans.

  20. IL-10-producing NKT10 cells are a distinct regulatory invariant NKT cell subset.

    PubMed

    Sag, Duygu; Krause, Petra; Hedrick, Catherine C; Kronenberg, Mitchell; Wingender, Gerhard

    2014-09-01

    Invariant natural killer T (iNKT) cells rapidly produce copious amounts of multiple cytokines after activation, thereby impacting a wide variety of different immune reactions. However, strong activation of iNKT cells with α-galactosylceramide (αGalCer) reportedly induces a hyporeactive state that resembles anergy. In contrast, we determined here that iNKT cells from mice pretreated with αGalCer retain cytotoxic activity and maintain the ability to respond to TCR-dependent as well as TCR-independent cytokine-mediated stimulation. Additionally, αGalCer-pretreated iNKT cells acquired characteristics of regulatory cells, including production and secretion of the immunomodulatory cytokine IL-10. Through the production of IL-10, αGalCer-pretreated iNKT cells impaired antitumor responses and reduced disease in experimental autoimmune encephalomyelitis, a mouse model of autoimmune disease. Furthermore, a subset of iNKT cells with a similar inhibitory phenotype and function were present in mice not exposed to αGalCer and were enriched in mouse adipose tissue and detectable in human PBMCs. These data demonstrate that IL-10-producing iNKT cells with regulatory potential (NKT10 cells) represent a distinct iNKT cell subset.

  1. α-Galactosylceramide-induced airway eosinophilia is mediated through the activation of NKT cells.

    PubMed

    Chuang, Ya-Hui; Wang, Tzu-Chun; Jen, Hsiao-Yu; Yu, Alice L; Chiang, Bor-Luen

    2011-04-15

    Invariant NKT (iNKT) cells bridge innate and adaptive immune responses, resulting in the expansion of Ag-specific B and T cell responses. α-Galactosylceramide (α-GalCer), the most studied glycolipid that activates iNKT cells, has been proposed to be an effective adjuvant against infections and tumors. We found that the activation of iNKT cells by intranasal injection of α-GalCer induced airway eosinophilia in naive mice. Eosinophils, which mediate tissue damage and dysfunction by secreting mediators, play important roles in the pathogenesis of allergic diseases. In this study, we investigated the mechanism of how eosinophils are recruited to the lung by α-GalCer. Our results demonstrated that α-GalCer-induced eosinophil inflammation was mediated through iNKT cells. These cells secreted IL-5 to recruit eosinophils directly to the lung and/or secreted IL-4 and IL-13 to recruit eosinophils indirectly by inducing lung epithelial cells, endothelial cells, and fibroblast to secrete the eosinophil chemoattractant eotaxin. In addition, in the OVA-alum murine model of allergic asthma, α-GalCer administration in OVA-immunized mice also increased airway eosinophilia after challenge. Given our findings, intranasal administration of α-GalCer induced airway eosinophilic inflammation in both naive and allergic mice. Hence, it remains to be determined whether the activation of iNKT cells would be applicable in therapeutics for human diseases.

  2. Synthetic glycolipid activators of natural killer T cells as immunotherapeutic agents.

    PubMed

    Carreño, Leandro J; Saavedra-Ávila, Noemí A; Porcelli, Steven A

    2016-04-01

    Certain types of glycolipids have been found to have remarkable immunomodulatory properties as a result of their ability to activate specific T lymphocyte populations with an extremely wide range of immune effector properties. The most extensively studied glycolipid reactive T cells are known as invariant natural killer T (iNKT) cells. The antigen receptors of these cells specifically recognize certain glycolipids, most notably glycosphingolipids with α-anomeric monosaccharides, presented by the major histocompatibility complex class I-like molecule CD1d. Once activated, iNKT cells can secrete a very diverse array of pro- and anti-inflammatory cytokines to modulate innate and adaptive immune responses. Thus, glycolipid-mediated activation of iNKT cells has been explored for immunotherapy in a variety of disease states, including cancer and a range of infections. In this review, we discuss the design of synthetic glycolipid activators for iNKT cells, their impact on adaptive immune responses and their use to modulate iNKT cell responses to improve immunity against infections and cancer. Current challenges in translating results from preclinical animal studies to humans are also discussed.

  3. Selective Conditions Are Required for the Induction of Invariant NKT Cell Hyporesponsiveness by Antigenic Stimulation.

    PubMed

    Wingender, Gerhard; Birkholz, Alysia M; Sag, Duygu; Farber, Elisa; Chitale, Sampada; Howell, Amy R; Kronenberg, Mitchell

    2015-10-15

    Activation of invariant (i)NKT cells with the model Ag α-galactosylceramide induces rapid production of multiple cytokines, impacting a wide variety of different immune reactions. In contrast, following secondary activation with α-galactosylceramide, the behavior of iNKT cells is altered for months, with the production of most cytokines being strongly reduced. The requirements for the induction of this hyporesponsive state, however, remain poorly defined. In this study, we show that Th1-biasing iNKT cell Ags could induce iNKT cell hyporesponsiveness, as long as a minimum antigenic affinity was reached. In contrast, the Th2-biasing Ag OCH did not induce a hyporesponsive state, nor did cytokine-driven iNKT cell activation by LPS or infections. Furthermore, although dendritic cells and B cells have been reported to be essential for iNKT cell stimulation, neither dendritic cells nor B cells were required to induce iNKT cell hyporesponsiveness. Therefore, our data indicate that whereas some bone marrow-derived cells could induce iNKT cell hyporesponsiveness, selective conditions, dependent on the structure and potency of the Ag, were required to induce hyporesponsiveness.

  4. Role of SHIP1 in Invariant NKT Cell Development and Functions.

    PubMed

    Anderson, Courtney K; Salter, Alexander I; Toussaint, Leon E; Reilly, Emma C; Fugère, Céline; Srivastava, Neetu; Kerr, William G; Brossay, Laurent

    2015-09-01

    SHIP1 is a 5'-inositol phosphatase known to negatively regulate the signaling product of the PI3K pathway, phosphatidylinositol (3-5)-trisphosphate. SHIP1 is recruited to a large number of inhibitory receptors expressed on invariant NK (iNKT) cells. We hypothesized that SHIP1 deletion would have major effects on iNKT cell development by altering the thresholds for positive and negative selection. Germline SHIP1 deletion has been shown to affect T cells as well as other immune cell populations. However, the role of SHIP1 on T cell function has been controversial, and its participation on iNKT cell development and function has not been examined. We evaluated the consequences of SHIP1 deletion on iNKT cells using germline-deficient mice, chimeric mice, and conditionally deficient mice. We found that T cell and iNKT cell development are impaired in germline-deficient animals. However, this phenotype can be rescued by extrinsic expression of SHIP1. In contrast, SHIP1 is required cell autonomously for optimal iNKT cell cytokine secretion. This suggests that SHIP1 calibrates the threshold of iNKT cell reactivity. These data further our understanding of how iNKT cell activation is regulated and provide insights into the biology of this unique cell lineage.

  5. Invariant NKT cells reduce the immunosuppressive activity of influenza A virus–induced myeloid-derived suppressor cells in mice and humans

    PubMed Central

    De Santo, Carmela; Salio, Mariolina; Masri, S. Hajar; Lee, Laurel Yong-Hwa; Dong, Tao; Speak, Anneliese O.; Porubsky, Stefan; Booth, Sarah; Veerapen, Natacha; Besra, Gurdyal S.; Gröne, Hermann-Josef; Platt, Frances M.; Zambon, Maria; Cerundolo, Vincenzo

    2008-01-01

    Infection with influenza A virus (IAV) presents a substantial threat to public health worldwide, with young, elderly, and immunodeficient individuals being particularly susceptible. Inflammatory responses play an important role in the fatal outcome of IAV infection, but the mechanism remains unclear. We demonstrate here that the absence of invariant NKT (iNKT) cells in mice during IAV infection resulted in the expansion of myeloid-derived suppressor cells (MDSCs), which suppressed IAV-specific immune responses through the expression of both arginase and NOS, resulting in high IAV titer and increased mortality. Adoptive transfer of iNKT cells abolished the suppressive activity of MDSCs, restored IAV-specific immune responses, reduced IAV titer, and increased survival rate. The crosstalk between iNKT and MDSCs was CD1d- and CD40-dependent. Furthermore, IAV infection and exposure to TLR agonists relieved the suppressive activity of MDSCs. Finally, we extended these results to humans by demonstrating the presence of myeloid cells with suppressive activity in the PBLs of individuals infected with IAV and showed that their suppressive activity is substantially reduced by iNKT cell activation. These findings identify what we believe to be a novel immunomodulatory role of iNKT cells, which we suggest could be harnessed to abolish the immunosuppressive activity of MDSCs during IAV infection. PMID:19033672

  6. Effects of Invariant NKT Cells on Parasite Infections and Hygiene Hypothesis.

    PubMed

    Yang, Jun-Qi; Zhou, Yonghua; Singh, Ram Raj

    2016-01-01

    Invariant natural killer T (iNKT) cells are unique subset of innate-like T cells recognizing glycolipids. iNKT cells can rapidly produce copious amounts of cytokines upon antigen stimulation and exert potent immunomodulatory activities for a wide variety of immune responses and diseases. We have revealed the regulatory effect of iNKT cells on autoimmunity with a serial of publications. On the other hand, the role of iNKT cells in parasitic infections, especially in recently attractive topic "hygiene hypothesis," has not been clearly defined yet. Bacterial and parasitic cell wall is a cellular structure highly enriched in a variety of glycolipids and lipoproteins, some of which may serve as natural ligands of iNKT cells. In this review, we mainly summarized the recent findings on the roles and underlying mechanisms of iNKT cells in parasite infections and their cross-talk with Th1, Th2, Th17, Treg, and innate lymphoid cells. In most cases, iNKT cells exert regulatory or direct cytotoxic roles to protect hosts against parasite infections. We put particular emphasis as well on the identification of the natural ligands from parasites and the involvement of iNKT cells in the hygiene hypothesis.

  7. Invariant natural killer T cells in adipose tissue: novel regulators of immune-mediated metabolic disease.

    PubMed

    Rakhshandehroo, M; Kalkhoven, E; Boes, M

    2013-12-01

    Adipose tissue (AT) represents a microenvironment where intersection takes place between immune processes and metabolic pathways. A variety of immune cells have been characterized in AT over the past decades, with the most recent addition of invariant natural killer T (iNKT) cells. As members of the T cell family, iNKT cells represent a subset that exhibits both innate and adaptive characteristics and directs ensuing immune responses. In disease conditions, iNKT cells have established roles that include disorders in the autoimmune spectrum in malignancies and infectious diseases. Recent work supports a role for iNKT cells in the maintenance of AT homeostasis through both immune and metabolic pathways. The deficiency of iNKT cells can result in AT metabolic disruptions and insulin resistance. In this review, we summarize recent work on iNKT cells in immune regulation, with an emphasis on AT-resident iNKT cells, and identify the potential mechanisms by which adipocytes can mediate iNKT cell activity.

  8. Invariant natural killer T cells as sensors and managers of inflammation.

    PubMed

    Van Kaer, Luc; Parekh, Vrajesh V; Wu, Lan

    2013-02-01

    Invariant natural killer T (iNKT) cells are a subset of innate-like lymphocytes that recognize glycolipid antigens bound by the major histocompatibility complex (MHC)-class-I-related protein CD1d. iNKT cells are activated early during a variety of infections and inflammatory diseases and contribute to the subsequent development of adaptive immune responses. Consequently, iNKT cells play a critical role in the development and resolution of inflammatory diseases and represent attractive targets for the development of immunotherapies. Recent studies have provided important insight into the mechanisms by which iNKT cells become activated in response to diverse inflammatory stimuli. These new findings should be instrumental to promote the immunomodulatory properties of iNKT cells for treatment of inflammatory diseases.

  9. Toll-Like Receptor 3 Ligand Dampens Liver Inflammation by Stimulating Vα14 Invariant Natural Killer T Cells to Negatively Regulate γδT Cells

    PubMed Central

    Gardner, Tommy R.; Chen, Qingling; Jin, Yijun; Ajuebor, Maureen N.

    2010-01-01

    Vα14 invariant natural killer T (Vα14iNKT) cells are at the interface between the innate and adaptive immune responses and are thus critical for providing full engagement of host defense. We investigated the role of polyriboinosinic:polycytidylic acid (poly I:C), a replication-competent viral double-stranded RNA mimic and a specific agonist that recognizes the cellular sensor Toll-like receptor 3 (TLR3), in regulating Vα14iNKT cell activation. We established for the first time that hepatic Vα14iNKT cells up-regulate TLR3 extracellularly after poly I:C treatment. Notably, activation of TLR3-expressing hepatic Vα14iNKT cells by a TLR3 ligand was suppressed by TLR3 deficiency. Our studies also revealed that Vα14iNKT cell activation in response to poly I:C administration uniquely suppressed the accumulation and activation of intrahepatic γδT cells (but not natural killer cells) by inducing apoptosis. Furthermore, we established that activated hepatic Vα14iNKT cells (via cytokines and possibly reactive oxygen species) influenced the frequency and absolute number of intrahepatic γδT cells, as evidenced by increased hepatic γδT cell accumulation in Vα14iNKT cell-deficient mice after poly I:C treatment relative to wild-type mice. Thus, hepatic Vα14iNKT cells and intrahepatic γδT cells are functionally linked on application of TLR3 agonist. Overall, our results demonstrate a novel and previously unrecognized anti-inflammatory role for activated hepatic Vα14iNKT cells in negatively regulating intrahepatic γδT cell accumulation (probably through TLR3 signaling) and thereby preventing potentially harmful activation of intrahepatic γδT cells. PMID:20167870

  10. Human Dendritic Cells Derived From Embryonic Stem Cells Stably Modified With CD1d Efficiently Stimulate Antitumor Invariant Natural Killer T Cell Response

    PubMed Central

    2014-01-01

    Invariant natural killer T (iNKT) cells are a unique lymphocyte subpopulation that mediates antitumor activities upon activation. A current strategy to harness iNKT cells for cancer treatment is endogenous iNKT cell activation using patient-derived dendritic cells (DCs). However, the limited number and functional defects of patient DCs are still the major challenges for this therapeutic approach. In this study, we investigated whether human embryonic stem cells (hESCs) with an ectopically expressed CD1d gene could be exploited to address this issue. Using a lentivector carrying an optimized expression cassette, we generated stably modified hESC lines that consistently overexpressed CD1d. These modified hESC lines were able to differentiate into DCs as efficiently as the parental line. Most importantly, more than 50% of such derived DCs were CD1d+. These CD1d-overexpressing DCs were more efficient in inducing iNKT cell response than those without modification, and their ability was comparable to that of DCs generated from monocytes of healthy donors. The iNKT cells expanded by the CD1d-overexpressing DCs were functional, as demonstrated by their ability to lyse iNKT cell-sensitive glioma cells. Therefore, hESCs stably modified with the CD1d gene may serve as a convenient, unlimited, and competent DC source for iNKT cell-based cancer immunotherapy. PMID:24292792

  11. Antigen-specific cytotoxicity by invariant NKT cells in vivo is CD95/CD178-dependent and is correlated with antigenic potency.

    PubMed

    Wingender, Gerhard; Krebs, Philippe; Beutler, Bruce; Kronenberg, Mitchell

    2010-09-01

    Invariant NKT (iNKT) cells are a unique subset of T lymphocytes that rapidly carry out effector functions following activation with glycolipid Ags, such as the model Ag alpha-galactosylceramide. Numerous studies have investigated the mechanisms leading to Th1 and Th2 cytokine production by iNKT cells, as well as the effects of the copious amounts of cytokines these cells produce. Less is known, however, about the mechanisms of iNKT cell cytotoxicity. In this study, we investigated the effect of Ag availability and strength, as well as the molecules involved in iNKT cytotoxicity. We demonstrate that the iNKT cell cytotoxicity in vivo correlates directly with the amount of CD1d expressed by the targets as well as the TCR affinity for the target glycolipid Ag. iNKT cells from spleen, liver, and thymus were comparable in their cytotoxicity in vitro. Surprisingly, we show that the Ag-specific cytotoxicity of iNKT cells in vivo depended almost exclusively on the interaction of CD95 (Fas) with CD178 (FasL), and that this mechanism can be efficiently used for tumor protection. Therefore, unlike NK cells, which rely mostly on perforin/granzyme-mediated mechanisms, the Ag-specific cytotoxicity of iNKT cells in vivo is largely restricted to the CD95/CD178 pathway.

  12. Antigen-specific cytotoxicity by invariant NKT cells in vivo is CD95/CD178 dependent and is correlated with antigenic potency

    PubMed Central

    Wingender, Gerhard; Krebs, Philippe; Beutler, Bruce; Kronenberg, Mitchell

    2010-01-01

    Invariant NKT (iNKT) cells are a unique subset of T lymphocytes that rapidly carry out effector functions following activation with glycolipid Ags, such as the model Ag α-galactosylceramide (αGalCer). Numerous studies have investigated the mechanisms leading to Th1- and Th2 cytokine production by iNKT cells, and the effects of the copious amounts of cytokines these cells produce. Less is known, however, about the mechanisms of iNKT cell cytotoxicity. Here we investigated the effect of antigen availability and strength, as well as the molecules involved in iNKT cytotoxicity. We demonstrate that the iNKT cell cytotoxicity in vivo correlates directly with the amount of CD1d expressed by the targets as well as the TCR affinity for the target glycolipid Ag. iNKT cells from spleen, liver and thymus were comparable in their cytotoxicity in vitro. Surprisingly, we show that the antigen-specific cytotoxicity of iNKT cells in vivo depended almost exclusively on the interaction of CD95 (Fas) with CD178 (FasL), and that this mechanism can be efficiently utilized for tumor protection. Therefore unlike NK cells, which rely mostly on perforin/granzyme mediated mechanisms, the antigen-specific cytotoxicity of iNKT cells in vivo is largely restricted to the CD95/CD178 pathway. PMID:20660713

  13. Invariant NKT cells increase drug-induced osteosarcoma cell death

    PubMed Central

    Fallarini, S; Paoletti, T; Orsi Battaglini, N; Lombardi, G

    2012-01-01

    BACKGROUND AND PURPOSE In osteosarcoma (OS) patients, only a limited number of drugs are active and the regimens currently in use include a combination of at least two of these drugs: doxorubicin, cisplatin, methotrexate and ifosfamide. Today, 30–40% of patients still die of OS highlighting the urgent need for new treatments. Invariant NKT (iNKT) cells are a lymphocyte lineage with features of both T and NK cells, playing important roles in tumour suppression. Our aim was to test whether the cytoxicity induced by cisplatin, doxorubicin and methotrexate against OS cells can be enhanced by iNKT cell treatment. EXPERIMENTAL APPROACH iNKT cells were purified from human peripheral blood mononuclear cells by cell sorting (Vα24Vβ11+ cells) and used as effector cells against OS cells (U2-OS, HOS, MG-63). Cell death (calcein-AM method), perforin/granzyme B and Fas/FasL expressions were determined by flow cytometry. CD1d expression was analysed at both the gene and protein level. KEY RESULTS iNKT cells were cytotoxic against OS cells through a CD1d-dependent mechanism. This activity was specific for tumour cells, because human CD1d+ mesenchymal stem cells and CD1d- osteoblasts were not affected. iNKT cell treatment enhanced drug-induced OS cell death in a concentration-dependent manner and this effect was reduced in CD1d-silenced OS cells. CONCLUSION AND IMPLICATIONS iNKT cells kill malignant, but not non-malignant, cells. iNKT cell treatment enhances the cytotoxicity of anti-neoplastic drugs against OS cells in a CD1d-dependent manner. The present data encourage further studies on the use of iNKT cells in OS therapy. PMID:22817659

  14. Donor bone marrow cells are essential for iNKT cell-mediated Foxp3+ Treg cell expansion in a murine model of transplantation tolerance.

    PubMed

    Miyairi, Satoshi; Hirai, Toshihito; Ishii, Rumi; Okumi, Masayoshi; Nunoda, Shinichi; Yamazaki, Kenji; Ishii, Yasuyuki; Tanabe, Kazunari

    2017-01-26

    Mixed chimerism induction is the most reliable method for establishing transplantation tolerance. We previously described a novel treatment using a suboptimal dose of anti-CD40 ligand (anti-CD40L) and liposomal formulation of a ligand for invariant natural killer T cells administered to sub-lethally irradiated recipient mice after donor bone marrow cell (BMC) transfer. Recipient mice treated with this regimen showed expansion of a Foxp3-positive regulatory T(Treg) cell phenotype, and formation of mixed chimera. However, the mechanism of expansion and bioactivity of Treg cells remains unclear. Here, we examine the role of donor BMCs in the expansion of bioactive Treg cells. The mouse model was transplanted with a heart allograft the day after treatment. The results showed that transfer of spleen cells in place of BMCs failed to deplete host interferon (IFN)-γ-producing CD8(+) T cells, expand host Ki67(+) CD4(+) CD25(+) Foxp3(+) Treg cells, and prolong graft survival. Severe combined immunodeficiency mice who received Treg cells obtained from BMC-recipients accepted skin grafts in an allo-specific manner. Myeloid-derived suppressor cells, which were a copious cell subset in BMCs, enhanced the Ki67 expression of Treg cells. This suggests that donor BMCs are indispensable for the expansion of host bioactive Treg cells in our novel treatment for transplant tolerance induction.

  15. Antigen specificity of invariant natural killer T-cells.

    PubMed

    Birkholz, Alysia M; Kronenberg, Mitchell

    2015-12-01

    Natural killer T-cells, with an invariant T-cell antigen receptor α-chain (iNKT cells), are unique and conserved subset of lymphocytes capable of altering the immune system through their rapid and potent cytokine responses. They are reactive to lipid antigens presented by the CD1d molecule, an antigen-presenting molecule that is not highly polymorphic. iNKT cell responses frequently involve mixtures of cytokines that work against each other, and therefore attempts are underway to develop synthetic antigens that elicit only strong interferon-gamma (IFNγ) or only strong interleukin-4 responses but not both. Strong IFNγ responses may correlate with tighter binding to CD1d and prolonged stimulation of iNKT cells, and this may be useful for vaccine adjuvants and for stimulating anti-tumor responses. iNKT cells are self-reactive although the structure of the endogenous antigen is controversial. By contrast, bacterial and fungal lipids that engage the T-cell receptor and activate IFNγ from iNKT cells have been identified from both pathogenic and commensal organisms and the responses are in some cases highly protective from pathogens in mice. It is possible that the expanding knowledge of iNKT cell antigens and iNKT cell activation will provide the basis for therapies for patients suffering from infectious and immune diseases and cancer.

  16. Essential role for autophagy during invariant NKT cell development

    PubMed Central

    Salio, Mariolina; Puleston, Daniel J.; Mathan, Till S. M.; Shepherd, Dawn; Stranks, Amanda J.; Adamopoulou, Eleni; Veerapen, Natacha; Besra, Gurdyal S.; Hollander, Georg A.; Simon, Anna Katharina; Cerundolo, Vincenzo

    2014-01-01

    Autophagy is an evolutionarily conserved cellular homeostatic pathway essential for development, immunity, and cell death. Although autophagy modulates MHC antigen presentation, it remains unclear whether autophagy defects impact on CD1d lipid loading and presentation to invariant natural killer T (iNKT) cells and on iNKT cell differentiation in the thymus. Furthermore, it remains unclear whether iNKT and conventional T cells have similar autophagy requirements for differentiation, survival, and/or activation. We report that, in mice with a conditional deletion of the essential autophagy gene Atg7 in the T-cell compartment (CD4 Cre-Atg7−/−), thymic iNKT cell development—unlike conventional T-cell development—is blocked at an early stage and mature iNKT cells are absent in peripheral lymphoid organs. The defect is not due to altered loading of intracellular iNKT cell agonists; rather, it is T-cell–intrinsic, resulting in enhanced susceptibility of iNKT cells to apoptosis. We show that autophagy increases during iNKT cell thymic differentiation and that it developmentally regulates mitochondrial content through mitophagy in the thymus of mice and humans. Autophagy defects result in the intracellular accumulation of mitochondrial superoxide species and subsequent apoptotic cell death. Although autophagy-deficient conventional T cells develop normally, they show impaired peripheral survival, particularly memory CD8+ T cells. Because iNKT cells, unlike conventional T cells, differentiate into memory cells while in the thymus, our results highlight a unique autophagy-dependent metabolic regulation of adaptive and innate T cells, which is required for transition to a quiescent state after population expansion. PMID:25512546

  17. Influence of a non-NK complex region of chromosome 6 on CD4+ invariant NK T cell homeostasis.

    PubMed

    Vallois, David; Gagnerault, Marie-Claude; Avner, Philip; Rogner, Ute C; Boitard, Christian; Benlagha, Kamel; Herbelin, André; Lepault, Françoise

    2008-08-01

    The number and function of immunoregulatory invariant NKT (iNKT) cells are genetically controlled. A defect of iNKT cell ontogeny and function has been implicated as one causal factor of NOD mouse susceptibility to type 1 diabetes. Other factors of diabetes susceptibility, such as a decrease of regulatory T cell function or an increase in TLR1 expression, are corrected in diabetes-resistant Idd6 NOD.C3H 6.VIII congenic mice. Thus, we surmised that the iNKT cell defects found in NOD mice may also be rescued in congenic mice. Unexpectedly, we found, in both the thymus and the periphery, a 50% reduction in iNKT cell number in NOD.C3H 6.VIII mice as compared with NOD mice. This reduction only affected CD4(+) iNKT cells, and left the double negative iNKT cells unchanged. In parallel, the production of IL-4 and IFN-gamma following alpha-GalCer stimulation was proportionally reduced. Using three subcongenic strains, we have narrowed down the region controlling iNKT development within Idd6 (5.8 Mb) to Idd6.2 region (2.5 Mb). Idd6 region had no effect on NK cell number and in vivo cytotoxic activity. These results indicate that the role of iNKT cells in diabetes development is equivocal and more complex than initially considered. In addition, they bring strong evidence that the regulation of CD4(+) iNKT cell production is independent from that of DN iNKT cells, and involves genes of the Idd6 locus.

  18. Invariant natural killer T cells: front line fighters in the war against pathogenic microbes.

    PubMed

    Crosby, Catherine M; Kronenberg, Mitchell

    2016-08-01

    Invariant natural killer T (iNKT) cells constitute a unique subset of innate-like T cells that have been shown to have crucial roles in a variety of immune responses. iNKT cells are characterized by their expression of both NK cell markers and an invariant T cell receptor (TCR) α chain, which recognizes glycolipids presented by the MHC class I-like molecule CD1d. Despite having a limited antigen repertoire, the iNKT cell response can be very complex, and participate in both protective and harmful immune responses. The protective role of these cells against a variety of pathogens has been particularly well documented. Through the use of these pathogen models, our knowledge of the breadth of the iNKT cell response has been expanded. Specific iNKT cell antigens have been isolated from several different bacteria, from which iNKT cells are critical for protection in mouse models. These responses can be generated by direct, CD1d-mediated activation, or indirect, cytokine-mediated activation, or a combination of the two. This can lead to secretion of a variety of different Th1, Th2, or Th17 cytokines, which differentially impact the downstream immune response against these pathogens. This critical role is emphasized by the conservation of these cells between mice and humans, warranting further investigation into how iNKT cells participate in protective immune responses, with the ultimate goal of harnessing their potential for treatment.

  19. Invariant natural killer T cell-based immunotherapy for cancer.

    PubMed

    Motohashi, Shinichiro; Nakayama, Toshinori

    2009-01-01

    Human Valpha24 invariant natural killer T (iNKT) cells are a distinct lymphocyte population, characterized by an invariant T-cell receptor Valpha24 chain paired mainly with Valpha11. Valpha24 iNKT cells are activated by a glycolipid ligand - alpha-galactosylceramide - and produce a large amount of Th1 and Th2 cytokines, thereby modulating the function of other cells. iNKT cells have the capability to control a wide variety of immune responses, including antitumor immunity. Abnormalities in the number and function of Valpha24 iNKT cells have been observed in patients with malignant diseases accompanied with a poor clinical outcome. Therefore, therapeutic strategies that focused on the restoration of Valpha24 iNKT cell population and function would be a reasonable rationale for the treatment of cancer. In this article, the progress to date in the clinical studies of iNKT cell-based immunotherapy is briefly reviewed and the role of Valpha24 iNKT cells in cancer immunotherapy is highlighted.

  20. A natural protective function of invariant NKT cells in a mouse model of innate-cell-driven lung inflammation.

    PubMed

    Bourgeois, Elvire A; Levescot, Anaïs; Diem, Séverine; Chauvineau, Angélique; Bergès, Hortense; Milpied, Pierre; Lehuen, Agnès; Damotte, Diane; Gombert, Jean-Marc; Schneider, Elke; Girard, Jean-Philippe; Gourdy, Pierre; Herbelin, André

    2011-02-01

    Activation of invariant natural killer T (iNKT) cells by treatment with their α-galactosyl ceramide ligand provides therapeutic benefits in several immune inflammatory settings. Given the artificial nature of this stimulation, the natural regulatory functions of iNKT remain uncertain. Addressing this issue in a mouse model of innate-cell-driven lung inflammation induced by the cytokine/alarmin IL-33 that targets iNKT cells, we found that eosinophil and neutrophil recruitment was markedly increased in treated iNKT cell-deficient (Jα18 KO) mice, as was the local production of eotaxin and keratinocyte chemoattractant chemokines. By contrast, lung inflammation decreased after adoptive transfer of iNKT cells, which restored the WT inflammatory response in Jα18 KO mice. Finally, we established that this natural anti-inflammatory function of iNKT cells depends on their IFN-γ production and on endogenous IL-12. Our study provides the first evidence of a protective role of iNKT cells during lung inflammation that does not require pharmacological TCR engagement.

  1. Blockade of programmed death-1/programmed death ligand pathway enhances the antitumor immunity of human invariant natural killer T cells.

    PubMed

    Kamata, Toshiko; Suzuki, Akane; Mise, Naoko; Ihara, Fumie; Takami, Mariko; Makita, Yuji; Horinaka, Atsushi; Harada, Kazuaki; Kunii, Naoki; Yoshida, Shigetoshi; Yoshino, Ichiro; Nakayama, Toshinori; Motohashi, Shinichiro

    2016-12-01

    The role of invariant natural killer T (iNKT) cells in antitumor immunity has been studied extensively, and clinical trials in patients with advanced cancer have revealed a prolonged survival in some cases. In recent years, humanized blocking antibodies against co-stimulatory molecules such as PD-1 have been developed. The enhancement of T cell function is reported to improve antitumor immunity, leading to positive clinical effects. However, there are limited data on the role of PD-1/programmed death ligand (PDL) molecules in human iNKT cells. In this study, we investigated the interaction between PD-1 on iNKT cells and PDL on antigen-presenting cells (APCs) in the context of iNKT cell stimulation. The blockade of PDL1 at the time of stimulation resulted in increased release of helper T cell (Th) 1 cytokines from iNKT cells, leading to the activation of NK cells. The direct antitumor function of iNKT cells was also enhanced after stimulation with anti-PDL1 antibody-treated APCs. According to these results, we conclude that the co-administration of anti-PDL1 antibody and alpha-galactosylceramide (αGalCer)-pulsed APCs enhances iNKT cell-mediated antitumor immunity.

  2. Temporal regulation of Wnt/β-catenin signaling is important for invariant NKT cell development and terminal maturation.

    PubMed

    Pyaram, Kalyani; Sen, Jyoti Misra; Chang, Cheong-Hee

    2017-02-13

    The Wnt/β-catenin signaling pathway plays important roles during various cellular functions including survival and proliferation of immune cells. The critical role of this pathway in conventional T cell development is established but little is known about its contributions to innate T cell development. In this study, we found that β-catenin level, an indication of the strength of Wnt/β-catenin signaling, is regulated during invariant NKT (iNKT) cell development. β-catenin levels were greatly increased during iNKT cell selection from double positive thymocytes to Stage 0 of iNKT cell development and during subsequent development to Stage 1. Thereafter, β-catenin levels decrease from Stage 2, which is essential for the terminal maturation of iNKT cells. Failure to dampen Wnt/β-catenin signaling as in mice expressing a stabilized active form of β-catenin (CATtg) resulted in increased Stage 2 and decreased Stage 3 iNKT cells. Inefficient transition from Stage 2 to 3 in CATtg iNKT cells seems to be contributed by poor expression of IL-15R (CD122) and transcription factor T-bet, both of which are necessary for terminal maturation of iNKT cells in the thymus. Consequently, IFN-γ+ iNKT cells were greatly reduced in CATtg mice. Together, our findings reveal that proper regulation of β-catenin and in turn Wnt signaling plays an important role in the terminal maturation and function of iNKT cells.

  3. Invariant NKT Cell Response to Dengue Virus Infection in Human

    PubMed Central

    Matangkasombut, Ponpan; Chan-in, Wilawan; Opasawaschai, Anunya; Pongchaikul, Pisut; Tangthawornchaikul, Nattaya; Vasanawathana, Sirijitt; Limpitikul, Wannee; Malasit, Prida; Duangchinda, Thaneeya; Screaton, Gavin; Mongkolsapaya, Juthathip

    2014-01-01

    Background Dengue viral infection is a global health threat without vaccine or specific treatment. The clinical outcome varies from asymptomatic, mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF). While adaptive immune responses were found to be detrimental in the dengue pathogenesis, the roles of earlier innate events remain largely uninvestigated. Invariant natural killer T (iNKT) cells represent innate-like T cells that could dictate subsequent adaptive response but their role in human dengue virus infection is not known. We hypothesized that iNKT cells play a role in human dengue infection. Methods Blood samples from a well-characterized cohort of children with DF, DHF, in comparison to non-dengue febrile illness (OFI) and healthy controls at various time points were studied. iNKT cells activation were analyzed by the expression of CD69 by flow cytometry. Their cytokine production was then analyzed after α-GalCer stimulation. Further, the CD1d expression on monocytes, and CD69 expression on conventional T cells were measured. Results iNKT cells were activated during acute dengue infection. The level of iNKT cell activation associates with the disease severity. Furthermore, these iNKT cells had altered functional response to subsequent ex vivo stimulation with α-GalCer. Moreover, during acute dengue infection, monocytic CD1d expression was also upregulated and conventional T cells also became activated. Conclusion iNKT cells might play an early and critical role in the pathogenesis of severe dengue viral infection in human. Targeting iNKT cells and CD1d serve as a potential therapeutic strategy for severe dengue infection in the future. PMID:24945350

  4. Exogenous Activation of Invariant Natural Killer T Cells by α-Galactosylceramide Reduces Pneumococcal Outgrowth and Dissemination Postinfluenza

    PubMed Central

    Barthelemy, Adeline; Ivanov, Stoyan; Hassane, Maya; Fontaine, Josette; Heurtault, Béatrice; Frisch, Benoit; Faveeuw, Christelle; Paget, Christophe

    2016-01-01

    ABSTRACT Influenza A virus infection can predispose to potentially devastating secondary bacterial infections. Invariant natural killer T (iNKT) cells are unconventional, lipid-reactive T lymphocytes that exert potent immunostimulatory functions. Using a mouse model of postinfluenza invasive secondary pneumococcal infection, we sought to establish whether α-galactosylceramide (α-GalCer [a potent iNKT cell agonist that is currently in clinical development]) could limit bacterial superinfection. Our results highlighted the presence of a critical time window during which α-GalCer treatment can trigger iNKT cell activation and influence resistance to postinfluenza secondary pneumococcal infection. Intranasal treatment with α-GalCer during the acute phase (on day 7) of influenza virus H3N2 and H1N1 infection failed to activate (gamma interferon [IFN-γ] and interleukin-17A [IL-17A]) iNKT cells; this effect was associated with a strongly reduced number of conventional CD103+ dendritic cells in the respiratory tract. In contrast, α-GalCer treatment during the early phase (on day 4) or during the resolution phase (day 14) of influenza was associated with lower pneumococcal outgrowth and dissemination. Less intense viral-bacterial pneumonia and a lower morbidity rate were observed in superinfected mice treated with both α-GalCer (day 14) and the corticosteroid dexamethasone. Our results open the way to alternative (nonantiviral/nonantibiotic) iNKT-cell-based approaches for limiting postinfluenza secondary bacterial infections. PMID:27803187

  5. Cellular Adjuvant Properties, Direct Cytotoxicity of Re-differentiated Vα24 Invariant NKT-like Cells from Human Induced Pluripotent Stem Cells

    PubMed Central

    Kitayama, Shuichi; Zhang, Rong; Liu, Tian-Yi; Ueda, Norihiro; Iriguchi, Shoichi; Yasui, Yutaka; Kawai, Yohei; Tatsumi, Minako; Hirai, Norihito; Mizoro, Yasutaka; Iwama, Tatsuaki; Watanabe, Akira; Nakanishi, Mahito; Kuzushima, Kiyotaka; Uemura, Yasushi; Kaneko, Shin

    2016-01-01

    Summary Vα24 invariant natural killer T (iNKT) cells are a subset of T lymphocytes implicated in the regulation of broad immune responses. They recognize lipid antigens presented by CD1d on antigen-presenting cells and induce both innate and adaptive immune responses, which enhance effective immunity against cancer. Conversely, reduced iNKT cell numbers and function have been observed in many patients with cancer. To recover these numbers, we reprogrammed human iNKT cells to pluripotency and then re-differentiated them into regenerated iNKT cells in vitro through an IL-7/IL-15-based optimized cytokine combination. The re-differentiated iNKT cells showed proliferation and IFN-γ production in response to α-galactosylceramide, induced dendritic cell maturation and downstream activation of both cytotoxic T lymphocytes and NK cells, and exhibited NKG2D- and DNAM-1-mediated NK cell-like cytotoxicity against cancer cell lines. The immunological features of re-differentiated iNKT cells and their unlimited availability from induced pluripotent stem cells offer a potentially effective immunotherapy against cancer. PMID:26862702

  6. Innate-like effector differentiation of human invariant NKT cells driven by IL-7.

    PubMed

    de Lalla, Claudia; Festuccia, Nicola; Albrecht, Inka; Chang, Hyun-Dong; Andolfi, Grazia; Benninghoff, Ulrike; Bombelli, Ferdinando; Borsellino, Giovanna; Aiuti, Alessandro; Radbruch, Andreas; Dellabona, Paolo; Casorati, Giulia

    2008-04-01

    Conventional MHC-restricted T lymphocytes leave thymus with a naive phenotype and require Ag-dependent stimulation coupled to proliferation to acquire effector functions. Invariant (i)NKT cells are a subset of T lymphocytes considered innate because they display an effector memory phenotype independent of TCR stimulation by foreign Ags. We investigated the effector differentiation program followed by human iNKT cells by studying cells from a relevant set of fetal thymi and umbilical cord blood samples. We find that human fetal iNKT cells have already started a differentiation program that activates the epigenetic and transcriptional control of ifng and il4 genes, leading at birth to cells that express these cytokines upon TCR signaling but independently of proliferation in vitro. Both ex vivo and in vitro analysis of fetal and neonatal iNKT cells delineate an effector differentiation program linked to cell division in vivo, and they identify IL-7 as one of the crucial signals driving this program in the apparent absence of Ag stimulation. Consistent with these data, human fetal and neonatal iNKT cells are hyperresponsive in vitro to IL-7 in comparison to conventional T cells, owing to an increased expression and signaling function of the IL-7 receptor alpha-chain. The innate nature of human iNKT cells could thus derive from lineage-specific developmental cues that selectively make these cells efficient IL-7 responders following thymic selection.

  7. CD1d Expression and Invariant NKT Cell Responses in Herpesvirus Infections

    PubMed Central

    Chung, Brian K.; Priatel, John J.; Tan, Rusung

    2015-01-01

    Invariant natural killer T (iNKT) cells are a highly conserved subset of unconventional T lymphocytes that express a canonical, semi-invariant T cell receptor and surface markers shared with the natural killer cell lineage. iNKT cells recognize exogenous and endogenous glycolipid antigens restricted by non-polymorphic CD1d molecules, and are highly responsive to the prototypical agonist, α-galactosylceramide. Upon activation, iNKT cells rapidly coordinate signaling between innate and adaptive immune cells through the secretion of proinflammatory cytokines, leading to the maturation of antigen-presenting cells, and expansion of antigen-specific CD4+ and CD8+ T cells. Because of their potent immunoregulatory properties, iNKT cells have been extensively studied and are known to play a pivotal role in mediating immune responses against microbial pathogens including viruses. Here, we review evidence that herpesviruses manipulate CD1d expression to escape iNKT cell surveillance and establish lifelong latency in humans. Collectively, published findings suggest that iNKT cells play critical roles in anti-herpesvirus immune responses and could be harnessed therapeutically to limit viral infection and viral-associated disease. PMID:26161082

  8. Invariant natural killer T cells contribute to chronic-plus-binge ethanol-mediated liver injury by promoting hepatic neutrophil infiltration.

    PubMed

    Mathews, Stephanie; Feng, Dechun; Maricic, Igor; Ju, Cynthia; Kumar, Vipin; Gao, Bin

    2016-03-01

    Neutrophil infiltration is a hallmark of alcoholic steatohepatitis; however, the underlying mechanisms remain unclear. We previously reported that chronic-plus-binge ethanol feeding synergistically induces hepatic recruitment of neutrophils, which contributes to liver injury. In this paper, we investigated the roles of invariant natural killer T (iNKT) cells in chronic-plus-binge ethanol feeding-induced hepatic neutrophil infiltration and liver injury. Wild-type and two strains of iNKT cell-deficient mice (CD1d- and Jα18-deficient mice) were subjected to chronic-plus-binge ethanol feeding. Liver injury and inflammation were examined. Chronic-plus-binge ethanol feeding synergistically increased the number of hepatic iNKT cells and induced their activation, compared with chronic feeding or binge alone. iNKT cell-deficient mice were protected from chronic-plus-binge ethanol-induced hepatic neutrophil infiltration and liver injury. Moreover, chronic-plus-binge ethanol feeding markedly upregulated the hepatic expression of several genes associated with inflammation and neutrophil recruitment in wild-type mice, but induction of these genes was abrogated in iNKT cell-deficient mice. Importantly, several cytokines and chemokines (e.g., MIP-2, MIP-1, IL-4, IL-6 and osteopontin) involved in neutrophil infiltration were upregulated in hepatic NKT cells isolated from chronic-plus-binge ethanol-fed mice compared to pair-fed mice. Finally, treatment with CD1d blocking antibody, which blocks iNKT cell activation, partially prevented chronic-plus-binge ethanol-induced liver injury and inflammation. Chronic-plus-binge ethanol feeding activates hepatic iNKT cells, which play a critical role in the development of early alcoholic liver injury, in part by releasing mediators that recruit neutrophils to the liver, and thus, iNKT cells represent a potential therapeutic target for the treatment of alcoholic liver disease.

  9. Functional invariant NKT cells in pig lungs regulate the airway hyperreactivity: a potential animal model.

    PubMed

    Renukaradhya, Gourapura J; Manickam, Cordelia; Khatri, Mahesh; Rauf, Abdul; Li, Xiangming; Tsuji, Moriya; Rajashekara, Gireesh; Dwivedi, Varun

    2011-04-01

    Important roles played by invariant natural killer T (iNKT) cells in asthma pathogenesis have been demonstrated. We identified functional iNKT cells and CD1d molecules in pig lungs. Pig iNKT cells cultured in the presence of α-GalCer proliferated and secreted Th1 and Th2 cytokines. Like in other animal models, direct activation of pig lung iNKT cells using α-GalCer resulted in acute airway hyperreactivity (AHR). Clinically, acute AHR-induced pigs had increased respiratory rate, enhanced mucus secretion in the airways, fever, etc. In addition, we observed petechial hemorrhages, infiltration of CD4(+) cells, and increased Th2 cytokines in AHR-induced pig lungs. Ex vivo proliferated iNKT cells of asthma induced pigs in the presence of C-glycoside analogs of α-GalCer had predominant Th2 phenotype and secreted more of Th2 cytokine, IL-4. Thus, baby pigs may serve as a useful animal model to study iNKT cell-mediated AHR caused by various environmental and microbial CD1d-specific glycolipid antigens.

  10. Sphingolipids from a symbiotic microbe regulate homeostasis of host intestinal natural killer T cells

    PubMed Central

    An, Dingding; Oh, Sungwhan F.; Olszak, Torsten; Neves, Joana F.; Avci, Fikri; Erturk-Hasdemir, Deniz; Lu, Xi; Zeissig, Sebastian; Blumberg, Richard S.; Kasper, Dennis L.

    2014-01-01

    Summary Co-evolution of beneficial microorganisms with the mammalian intestine fundamentally shapes mammalian physiology. Herein we report that the intestinal microbe Bacteroides fragilis modifies the homeostasis of host invariant natural killer T (iNKT) cells by supplementing the host’s endogenous lipid antigen milieu with unique inhibitory sphingolipids. The process occurs early in life and effectively impedes iNKT cell proliferation during neonatal development. Consequently, total colonic iNKT cell numbers are restricted into adulthood and hosts are protected against experimental iNKT cell–mediated, oxazolone-induced colitis. In studies with neonatal mice lacking access to bacterial sphingolipids, we found that treatment with B. fragilis glycosphingolipids—exemplified by an isolated peak (M.W.=717.6) called GSL-Bf717—reduces colonic iNKT cell numbers and confers protection against oxazolone-induced colitis in adulthood. Our results suggest that the distinctive inhibitory capacity of GSL-Bf717 and similar molecules may prove useful in the treatment of autoimmune and allergic disorders in which iNKT cell activation is destructive. PMID:24439373

  11. Diverse Endogenous Antigens for Mouse Natural Killer T Cells: Self-Antigens That Are Not Glycosphingolipids

    PubMed Central

    Pei, Bo; Speak, Anneliese O; Shepherd, Dawn; Butters, Terry; Cerundolo, Vincenzo; Platt, Frances M; Kronenberg, Mitchell

    2011-01-01

    Natural killer T cells with an invariant antigen receptor (iNKT cells) represent a highly conserved and unique subset of T lymphocytes having properties of innate and adaptive immune cells. They have been reported to regulate a variety of immune responses, including the response to cancers and the development of autoimmunity. The development and activation of iNKT cells is dependent on self-antigens presented by the CD1d antigen-presenting molecule. It is widely believed that these self-antigens are glycosphingolipids (GSLs), molecules that contain ceramide as the lipid backbone. Here we used a variety of methods to show that mammalian antigens for mouse iNKT cells need not be GSLs, including the use of cell lines deficient in GSL biosynthesis and an inhibitor of GSL biosynthesis. Presentation of these antigens required the expression of CD1d molecules that could traffic to late endosomes, the site where self-antigen is acquired. Extracts of antigen-presenting cells (APCs) contain a self-antigen that could stimulate iNKT cells when added to plates coated with soluble, recombinant CD1d molecules. The antigen(s) in these extracts are resistant to sphingolipid-specific hydrolase digestion, consistent with the results using live APCs. Lyosphosphatidylcholine, a potential self-antigen that activated human iNKT cell lines, did not activate mouse iNKT cell hybridomas. Our data indicate that there may be more than one type of self-antigen for iNKT cells, that the self-antigens comparing mouse and human may not be conserved, and that the search to identify these molecules should not be confined to GSLs. PMID:21191069

  12. Diverse endogenous antigens for mouse NKT cells: self-antigens that are not glycosphingolipids.

    PubMed

    Pei, Bo; Speak, Anneliese O; Shepherd, Dawn; Butters, Terry; Cerundolo, Vincenzo; Platt, Frances M; Kronenberg, Mitchell

    2011-02-01

    NKT cells with an invariant Ag receptor (iNKT cells) represent a highly conserved and unique subset of T lymphocytes having properties of innate and adaptive immune cells. They have been reported to regulate a variety of immune responses, including the response to cancers and the development of autoimmunity. The development and activation of iNKT cells is dependent on self-Ags presented by the CD1d Ag-presenting molecule. It is widely believed that these self-Ags are glycosphingolipids (GSLs), molecules that contain ceramide as the lipid backbone. In this study, we used a variety of methods to show that mammalian Ags for mouse iNKT cells need not be GSLs, including the use of cell lines deficient in GSL biosynthesis and an inhibitor of GSL biosynthesis. Presentation of these Ags required the expression of CD1d molecules that could traffic to late endosomes, the site where self-Ag is acquired. Extracts of APCs contain a self-Ag that could stimulate iNKT cells when added to plates coated with soluble, rCD1d molecules. The Ag(s) in these extracts are resistant to sphingolipid-specific hydrolase digestion, consistent with the results using live APCs. Lyosphosphatidylcholine, a potential self-Ag that activated human iNKT cell lines, did not activate mouse iNKT cell hybridomas. Our data indicate that there may be more than one type of self-Ag for iNKT cells, that the self-Ags comparing mouse and human may not be conserved, and that the search to identify these molecules should not be confined to GSLs.

  13. Recognition of Microbial Glycolipids by Natural Killer T Cells

    PubMed Central

    Zajonc, Dirk M.; Girardi, Enrico

    2015-01-01

    T cells can recognize microbial antigens when presented by dedicated antigen-presenting molecules. While peptides are presented by classical members of the major histocompatibility complex (MHC) family (MHC I and II), lipids, glycolipids, and lipopeptides can be presented by the non-classical MHC member, CD1. The best studied subset of lipid-reactive T cells are type I natural killer T (iNKT) cells that recognize a variety of different antigens when presented by the non-classical MHCI homolog CD1d. iNKT cells have been shown to be important for the protection against various microbial pathogens, including B. burgdorferi, the causative agents of Lyme disease, and S. pneumoniae, which causes pneumococcal meningitis and community-acquired pneumonia. Both pathogens carry microbial glycolipids that can trigger the T cell antigen receptor (TCR), leading to iNKT cell activation. iNKT cells have an evolutionary conserved TCR alpha chain, yet retain the ability to recognize structurally diverse glycolipids. They do so using a conserved recognition mode, in which the TCR enforces a conserved binding orientation on CD1d. TCR binding is accompanied by structural changes within the TCR binding site of CD1d, as well as the glycolipid antigen itself. In addition to direct recognition of microbial antigens, iNKT cells can also be activated by a combination of cytokines (IL-12/IL-18) and TCR stimulation. Many microbes carry TLR antigens, and microbial infections can lead to TLR activation. The subsequent cytokine response in turn lower the threshold of TCR-mediated iNKT cell activation, especially when weak microbial or even self-antigens are presented during the cause of the infection. In summary, iNKT cells can be directly activated through TCR triggering of strong antigens, while cytokines produced by the innate immune response may be necessary for TCR triggering and iNKT cell activation in the presence of weak antigens. Here, we will review the molecular basis of iNKT cell

  14. Specific deletion of LDL receptor-related protein on macrophages has skewed in vivo effects on cytokine production by invariant natural killer T cells.

    PubMed

    Covarrubias, Roman; Wilhelm, Ashley J; Major, Amy S

    2014-01-01

    Expression of molecules involved in lipid homeostasis such as the low density lipoprotein receptor (LDLr) on antigen presenting cells (APCs) has been shown to enhance invariant natural killer T (iNKT) cell function. However, the contribution to iNKT cell activation by other lipoprotein receptors with shared structural and ligand binding properties to the LDLr has not been described. In this study, we investigated whether a structurally related receptor to the LDLr, known as LDL receptor-related protein (LRP), plays a role in iNKT cell activation. We found that, unlike the LDLr which is highly expressed on all immune cells, the LRP was preferentially expressed at high levels on F4/80+ macrophages (MΦ). We also show that CD169+ MΦs, known to present antigen to iNKT cells, exhibited increased expression of LRP compared to CD169- MΦs. To test the contribution of MΦ LRP to iNKT cell activation we used a mouse model of MΦ LRP conditional knockout (LRP-cKO). LRP-cKO MΦs pulsed with glycolipid alpha-galactosylceramide (αGC) elicited normal IL-2 secretion by iNKT hybridoma and in vivo challenge of LRP-cKO mice led to normal IFN-γ, but blunted IL-4 response in both serum and intracellular expression by iNKT cells. Flow cytometric analyses show similar levels of MHC class-I like molecule CD1d on LRP-cKO MΦs and normal glycolipid uptake. Survey of the iNKT cell compartment in LRP-cKO mice revealed intact numbers and percentages and no homeostatic disruption as evidenced by the absence of programmed death-1 and Ly-49 surface receptors. Mixed bone marrow chimeras showed that the inability iNKT cells to make IL-4 is cell extrinsic and can be rescued in the presence of wild type APCs. Collectively, these data demonstrate that, although MΦ LRP may not be necessary for IFN-γ responses, it can contribute to iNKT cell activation by enhancing early IL-4 secretion.

  15. Invariant NKT cells modulate the suppressive activity of Serum Amyloid A-differentiated IL-10-secreting neutrophils

    PubMed Central

    De Santo, Carmela; Arscott, Ramon; Booth, Sarah; Karydis, Ioannis; Jones, Margaret; Asher, Ruth; Salio, Mariolina; Middleton, Mark; Cerundolo, Vincenzo

    2010-01-01

    Neutrophils are the primary effector cells during inflammation, but can also control excessive inflammatory responses by secreting anti-inflammatory cytokines. However, the mechanisms modulating their plasticity remain unclear. We now show that systemic serum amyloid A-1 (SAA-1) controls the plasticity of neutrophil differentiation. SAA-1 not only induced anti-inflammatory IL-10-secreting neutrophils but also promoted invariant NKT (iNKT) cell interaction with these neutrophils, a process that limits their suppressive activity by reducing IL-10 and enhancing IL-12 production. Because SAA-1-producing melanomas promote differentiation of IL-10-secreting neutrophils, harnessing iNKT cells could be useful therapeutically by reducing the frequency of immunosuppressive neutrophils and restoring tumor specific immune responses. PMID:20890286

  16. Innate-like recognition of microbes by invariant natural killer T cells.

    PubMed

    Kronenberg, Mitchell; Kinjo, Yuki

    2009-08-01

    Invariant natural killer T cells (iNKT cells) express a restricted T cell antigen receptor (TCR) repertoire and they respond rapidly to glycolipid antigens presented by CD1d. These glycolipid antigens have hexose sugars in alpha-linkage to two types of lipids that can bind to CD1d. Recent work has shown that the responses of iNKT cells to antigen-bearing microbes can have a profound impact on the development of inflammatory diseases. iNKT cells overcome the limitation of their limited TCR diversity by also responding in a foreign antigen-independent fashion to some infectious agents, similar to NK cells. Recent results demonstrate several mechanisms for the indirect activation of iNKT cells by viruses or TLR ligands, dependent on self-antigen recognition and/or different cytokines produced by antigen presenting cells. The means by which iNKT cells influence other cell types and overall host defense are likewise diverse, illustrating the flexibility and functional diversity of this T lymphocyte sublineage.

  17. The regulatory role of invariant NKT cells in tumor immunity

    PubMed Central

    McEwen-Smith, Rosanna M; Salio, Mariolina; Cerundolo, Vincenzo

    2015-01-01

    Invariant natural killer T (iNKT) cells are a unique population of T lymphocytes, which lie at the interface between the innate and adaptive immune systems, and are important mediators of immune responses and tumor-surveillance. iNKT cells recognize lipid antigens in a CD1d-dependent manner; their subsequent activation results in a rapid and specific downstream response, which enhances both innate and adaptive immunity. The capacity of iNKT cells to modify the immune-microenvironment influences the ability of the host to control tumor growth, making them an important population to be harnessed in the clinic for the development of anti-cancer therapeutics. Indeed, the identification of strong iNKT cell agonists, such as α-galactosylceramide (α-GalCer) and its analogues, has led to the development of synthetic lipids which have shown potential in vaccination and treatment against cancers. In this Masters of Immunology article we discuss these latest findings, and summarise the major discoveries in iNKT cell biology, which have enabled the design of potent strategies for immune-mediated tumor destruction. PMID:25941354

  18. Exploiting the CD1d-iNKT cell axis for potentiation of DC-based cancer vaccines.

    PubMed

    Lameris, Roeland; Schneiders, Famke L; de Gruijl, Tanja D; van der Vliet, Hans J

    2014-01-01

    Invariant natural killer T cells (iNKT) and dendritic cells (DC) play a central role in tumor immunity through downstream activation of immune effector cells by pro-inflammatory cytokines. Evidence is accumulating that the CD1d-iNKT cell axis can be effectively used to potentiate DC-based cancer vaccines. Here, we provide a detailed methodology for the generation of (CD1d-expressing) monocyte-derived DC (moDC) and their subsequent loading with the iNKT cell agonist α-galactosylceramide (α-GalCer) or their direct ligation by agonistic anti-CD1d monoclonal antibodies.

  19. Cutting edge: the mechanism of invariant NKT cell responses to viral danger signals.

    PubMed

    Tyznik, Aaron J; Tupin, Emmanuel; Nagarajan, Niranjana A; Her, Min J; Benedict, Chris A; Kronenberg, Mitchell

    2008-10-01

    Invariant NK T (iNKT) cells influence the response to viral infections, although the mechanisms are poorly defined. In this study we show that these innate-like lymphocytes secrete IFN-gamma upon culture with CpG oligodeoxynucleotide-stimulated dendritic cells (DCs) from mouse bone marrow. This requires TLR9 signaling and IL-12 secretion by the activated DCs, but it does not require CD1d expression. iNKT cells also produce IFN-gamma in response to mouse CMV infection. Their mechanism of mouse CMV detection is quite similar to that of CpG, requiring both TLR9 signaling and IL-12 secretion, while the need for CD1d expression is relatively minor. Consequently, iNKT cells have the ability to respond to a variety of microbes, including viruses, in an Ag-independent manner, suggesting they may play a broad role in antipathogen defenses despite their limited TCR repertoire.

  20. α‐Galactosylceramide‐activated murine NK1.1+ invariant‐NKT cells in the myometrium induce miscarriages in mice

    PubMed Central

    Ichikawa, Tomoko; Negishi, Yasuyuki; Shimizu, Masumi; Takeshita, Toshiyuki

    2016-01-01

    Innate immunity, which is unable to discriminate self from allo‐antigens, is thought to be important players in the induction of miscarriages. Here, we show that the administration of IL‐12 to syngeneic‐mated C57BL/6 mice on gestation day 7.5 (Gd 7.5), drives significant miscarriages in pregnant females. Furthermore, the administration on Gd 7.5 of α‐galactosylceramide (α‐GalCer), which is known to activate invariant natural killer T (iNKT) cells, induced miscarriages in both syngeneic‐mated C57BL/6 mice and allogeneic‐mated mice (C57BL/6 (♀) × BALB/c (♂)). Surprisingly, the percentages of both DEC‐205+ DCs and CD1d‐restricted NK1.1+ iNKT cells were higher in the myometrium of pregnant mice treated i.p. with α‐GalCer than in the decidua. IL‐12 secreted from α‐GalCer‐activated DEC‐205+ DCs stimulated the secretion of cytokines, including IL‐2, IL‐4, IFN‐γ, TNF‐α, perforin, and granzyme B, from the NK1.1+ iNKT cells in the myometrium, leading to fetal loss in pregnant mice. Finally, the i.p. administration of IL‐12 and/or α‐GalCer in iNKT‐deficient Jα18(‐/‐) (Jα18 KO) mice did not induce miscarriages. This study provides a new perspective on the importance of the myometrium, rather than the decidua, in regulating pregnancy and a mechanism of miscarriage mediated by activated DEC‐205+ DCs and NK1.1+ iNKT cells in the myometrium of pregnant mice. PMID:27198610

  1. Essential functions for ID proteins at multiple checkpoints in invariant NKT cell development.

    PubMed

    Verykokakis, Mihalis; Krishnamoorthy, Veena; Iavarone, Antonio; Lasorella, Anna; Sigvardsson, Mikael; Kee, Barbara L

    2013-12-15

    Invariant NKT (iNKT) cells display characteristics of both adaptive and innate lymphoid cells (ILCs). Like other ILCs, iNKT cells constitutively express ID proteins, which antagonize the E protein transcription factors that are essential for adaptive lymphocyte development. However, unlike ILCs, ID2 is not essential for thymic iNKT cell development. In this study, we demonstrated that ID2 and ID3 redundantly promoted iNKT cell lineage specification involving the induction of the signature transcription factor PLZF and that ID3 was critical for development of TBET-dependent NKT1 cells. In contrast, both ID2 and ID3 limited iNKT cell numbers by enforcing the postselection checkpoint in conventional thymocytes. Therefore, iNKT cells show both adaptive and innate-like requirements for ID proteins at distinct checkpoints during iNKT cell development.

  2. Invariant NKT cells are resistant to circulating CD15+ myeloid-derived suppressor cells in patients with head and neck cancer.

    PubMed

    Horinaka, Atsushi; Sakurai, Daiju; Ihara, Fumie; Makita, Yuji; Kunii, Naoki; Motohashi, Shinichiro; Nakayama, Toshinori; Okamoto, Yoshitaka

    2016-03-01

    Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature and progenitor myeloid cells with an immunosuppressive role in various types of cancer, including head and neck squamous cell carcinoma (HNSCC). However, the effect on the host immune system, especially on invariant NKT (iNKT) cells with potent anti-tumor activity, remains unclear. In this study, we investigated the effects of circulating MDSC subsets on the peripheral lymphocytes of patients with head and neck tumors. A significant accumulation of CD15+ granulocytic MDSC (G-MDSC) and CD14+ monocytic MDSC (M-MDSC) was demonstrated in HNSCC patients. The percentage of G-MDSC showed an inverse correlation with the percentage of T cells in the peripheral blood. The increased G-MDSC was significantly associated with advanced clinical stage and poor prognosis of HNSCC patients. The proliferation and viability of T cells were suppressed by CD15+ cells, and the suppression was reversed by adding the hydrogen peroxide scavenger catalase. However, iNKT cell activation upon α-galactosylceramide (αGalCer) stimulation was not affected by the presence or absence of CD15+ G-MDSC. These results indicate that increased G-MDSC negatively affects peripheral T cell immunity, but not iNKT cells, in HNSCC patients, and that T cells are more sensitive to hydrogen peroxide produced by G-MDSC than iNKT cells. Cancer immunotherapy designed to enhance the antitumor activity of iNKT cells by stimulation with αGalCer may remain effective in the presence of G-MDSC.

  3. Invariant and Noninvariant Natural Killer T Cells Exert Opposite Regulatory Functions on the Immune Response during Murine Schistosomiasis▿

    PubMed Central

    Mallevaey, Thierry; Fontaine, Josette; Breuilh, Laetitia; Paget, Christophe; Castro-Keller, Alexandre; Vendeville, Catherine; Capron, Monique; Leite-de-Moraes, Maria; Trottein, François; Faveeuw, Christelle

    2007-01-01

    CD1d-restricted natural killer T (NKT) cells represent a heterogeneous population of innate memory immune cells expressing both NK and T-cell markers distributed into two major subsets, i.e., invariant NKT (iNKT) cells, which express exclusively an invariant T-cell receptor (TCR) α chain (Vα14Jα18 in mice), and non-iNKT cells, which express more diverse TCRs. NKT cells quickly produce Th1- and/or Th2-type cytokines following stimulation with glycolipid antigen (Ag) and, through this property, play potent immunoregulatory roles in autoimmune diseases, cancer, and infection. No study has addressed the role of NKT cells in metazoan parasite infections so far. We show that during murine schistosomiasis, the apparent frequency of both iNKT cells and non-iNKT cells decreased in the spleen as early as 3 weeks postinfection (p.i.) and that both populations expressed a greater amount of the activation marker CD69 at 6 weeks p.i., suggesting an activated phenotype. Two different NKT-cell-deficient mouse models, namely, TCR Jα18−/− (exclusively deficient in iNKT cells) and CD1d−/− (deficient in both iNKT and non-iNKT cells) mice, were used to explore the implication of these subsets in infection. We show that whereas both iNKT and non-iNKT cells do not have a major impact on the immune response during the early phase (1 and 4 weeks) of infection, they exert important, although opposite, effects on the immune response during the acute phase of the disease (7 and 12 weeks), after schistosome egg production. Indeed, iNKT cells contribute to Th1 cell differentiation whereas non-iNKT cells might be mostly implicated in Th2 cell differentiation in response to parasite Ag. Our findings suggest, for the first time, that helminths activate both iNKT and non-iNKT cells in vivo, enabling them to differentially influence the Th1/Th2 balance of the immune response. PMID:17353286

  4. NKT cells mediate pulmonary inflammation and dysfunction in murine sickle cell disease through production of IFN-γ and CXCR3 chemokines

    PubMed Central

    Wallace, Kori L.; Marshall, Melissa A.; Ramos, Susan I.; Lannigan, Joanne A.; Field, Joshua J.; Strieter, Robert M.

    2009-01-01

    Ischemia-reperfusion injury (IRI) triggers an inflammatory cascade that is initiated by the activation of CD1d-restricted iNKT cells. In sickle cell disease (SCD), misshapen erythrocytes evoke repeated transient bouts of microvascular IRI. Compared with C57BL/6 controls, NY1DD mice have more numerous and activated (CD69+, interferon-γ+ [IFN-γ+]) lung, liver, and spleen iNKT cells that are hyperresponsive to hypoxia/reoxygenation. NY1DD mice have increased pulmonary levels of IFN-γ, IFN-γ–inducible chemokines (CXCL9, CXCL10), and elevated numbers of lymphocytes expressing the chemokine receptor CXCR3. Treating NY1DD mice with anti-CD1d antibody to inhibit iNKT cell activation reverses baseline pulmonary dysfunction manifested as elevated vascular permeability, decreased arterial oxygen saturation, and increased numbers of activated leukocytes. Anti-CD1d antibodies decrease pulmonary levels of IFN-γ and CXCR3 chemokines. Neutralization of CXCR3 receptors ameliorates pulmonary dysfunction. Crossing NY1DD to lymphocyte-deficient Rag1−/− mice decreases pulmonary dysfunction. This is counteracted by the adoptive transfer of 1 million NKT cells. Like mice, people with SCD have increased numbers of activated circulating iNKT cells expressing CXCR3. Together, these data indicate that iNKT cells play a pivotal role in sustaining inflammation in SCD mice by a pathway involving IFN-γ and production of chemotactic CXCR3 chemokines and that this mechanism may translate to human disease. PMID:19433855

  5. CD38 is required for the peripheral survival of immunotolerogenic CD4+ invariant NK T cells in nonobese diabetic mice.

    PubMed

    Chen, Yi-Guang; Chen, Jing; Osborne, Melissa A; Chapman, Harold D; Besra, Gurdyal S; Porcelli, Steven A; Leiter, Edward H; Wilson, S Brian; Serreze, David V

    2006-09-01

    T cell-mediated autoimmune type-1 diabetes (T1D) in NOD mice partly results from this strain's numerical and functional defects in invariant NK T (iNKT) cells. T1D is inhibited in NOD mice treated with the iNKT cell superagonist alpha-galactosylceramide through a process involving enhanced accumulation of immunotolerogenic dendritic cells in pancreatic lymph nodes. Conversely, T1D is accelerated in NOD mice lacking CD38 molecules that play a role in dendritic cell migration to inflamed tissues. Unlike in standard NOD mice, alpha-galactosylceramide pretreatment did not protect the CD38-deficient stock from T1D induced by an adoptively transferred pancreatic beta cell-autoreactive CD8 T cell clone (AI4). We found that in the absence of CD38, ADP-ribosyltransferase 2 preferentially activates apoptotic deletion of peripheral iNKT cells, especially the CD4+ subset. Therefore, this study documents a previously unrecognized role for CD38 in maintaining survival of an iNKT cell subset that preferentially contributes to the maintenance of immunological tolerance.

  6. Genetic engineering of hematopoietic stem cells to generate invariant natural killer T cells.

    PubMed

    Smith, Drake J; Liu, Siyuan; Ji, Sunjong; Li, Bo; McLaughlin, Jami; Cheng, Donghui; Witte, Owen N; Yang, Lili

    2015-02-03

    Invariant natural killer T (iNKT) cells comprise a small population of αβ T lymphocytes. They bridge the innate and adaptive immune systems and mediate strong and rapid responses to many diseases, including cancer, infections, allergies, and autoimmunity. However, the study of iNKT cell biology and the therapeutic applications of these cells are greatly limited by their small numbers in vivo (∼0.01-1% in mouse and human blood). Here, we report a new method to generate large numbers of iNKT cells in mice through T-cell receptor (TCR) gene engineering of hematopoietic stem cells (HSCs). We showed that iNKT TCR-engineered HSCs could generate a clonal population of iNKT cells. These HSC-engineered iNKT cells displayed the typical iNKT cell phenotype and functionality. They followed a two-stage developmental path, first in thymus and then in the periphery, resembling that of endogenous iNKT cells. When tested in a mouse melanoma lung metastasis model, the HSC-engineered iNKT cells effectively protected mice from tumor metastasis. This method provides a powerful and high-throughput tool to investigate the in vivo development and functionality of clonal iNKT cells in mice. More importantly, this method takes advantage of the self-renewal and longevity of HSCs to generate a long-term supply of engineered iNKT cells, thus opening up a new avenue for iNKT cell-based immunotherapy.

  7. Natural killer T cells in adipose tissue prevent insulin resistance.

    PubMed

    Schipper, Henk S; Rakhshandehroo, Maryam; van de Graaf, Stan F J; Venken, Koen; Koppen, Arjen; Stienstra, Rinke; Prop, Serge; Meerding, Jenny; Hamers, Nicole; Besra, Gurdyal; Boon, Louis; Nieuwenhuis, Edward E S; Elewaut, Dirk; Prakken, Berent; Kersten, Sander; Boes, Marianne; Kalkhoven, Eric

    2012-09-01

    Lipid overload and adipocyte dysfunction are key to the development of insulin resistance and can be induced by a high-fat diet. CD1d-restricted invariant natural killer T (iNKT) cells have been proposed as mediators between lipid overload and insulin resistance, but recent studies found decreased iNKT cell numbers and marginal effects of iNKT cell depletion on insulin resistance under high-fat diet conditions. Here, we focused on the role of iNKT cells under normal conditions. We showed that iNKT cell-deficient mice on a low-fat diet, considered a normal diet for mice, displayed a distinctive insulin resistance phenotype without overt adipose tissue inflammation. Insulin resistance was characterized by adipocyte dysfunction, including adipocyte hypertrophy, increased leptin, and decreased adiponectin levels. The lack of liver abnormalities in CD1d-null mice together with the enrichment of CD1d-restricted iNKT cells in both mouse and human adipose tissue indicated a specific role for adipose tissue-resident iNKT cells in the development of insulin resistance. Strikingly, iNKT cell function was directly modulated by adipocytes, which acted as lipid antigen-presenting cells in a CD1d-mediated fashion. Based on these findings, we propose that, especially under low-fat diet conditions, adipose tissue-resident iNKT cells maintain healthy adipose tissue through direct interplay with adipocytes and prevent insulin resistance.

  8. Pivotal Advance: Invariant NKT cells reduce accumulation of inflammatory monocytes in the lungs and decrease immune-pathology during severe influenza A virus infection.

    PubMed

    Kok, Wai Ling; Denney, Laura; Benam, Kambez; Cole, Suzanne; Clelland, Colin; McMichael, Andrew J; Ho, Ling-Pei

    2012-03-01

    Little is known of how a strong immune response in the lungs is regulated to minimize tissue injury during severe influenza A virus (IAV) infection. Here, using a model of lethal, high-pathogenicity IAV infection, we first show that Ly6C(hi)Ly6G(-) inflammatory monocytes, and not neutrophils, are the main infiltrate in lungs of WT mice. Mice devoid of iNKT cells (Jα18(-/-) mice) have increased levels of inflammatory monocytes, which correlated with increased lung injury and mortality (but not viral load). Activation of iNKT cells correlated with reduction of MCP-1 levels and improved outcome. iNKT cells were able to selectively lyse infected, MCP-1-producing monocytes in vitro, in a CD1d-dependent process. Our study provides a detailed profile and kinetics of innate immune cells in the lungs during severe IAV infection, highlighting inflammatory monocytes as the major infiltrate and identifying a role for iNKT cells in control of these cells and lung immune-pathology.

  9. Natural killer T cells in adipose tissue prevent insulin resistance

    PubMed Central

    Schipper, Henk S.; Rakhshandehroo, Maryam; van de Graaf, Stan F.J.; Venken, Koen; Koppen, Arjen; Stienstra, Rinke; Prop, Serge; Meerding, Jenny; Hamers, Nicole; Besra, Gurdyal; Boon, Louis; Nieuwenhuis, Edward E.S.; Elewaut, Dirk; Prakken, Berent; Kersten, Sander; Boes, Marianne; Kalkhoven, Eric

    2012-01-01

    Lipid overload and adipocyte dysfunction are key to the development of insulin resistance and can be induced by a high-fat diet. CD1d-restricted invariant natural killer T (iNKT) cells have been proposed as mediators between lipid overload and insulin resistance, but recent studies found decreased iNKT cell numbers and marginal effects of iNKT cell depletion on insulin resistance under high-fat diet conditions. Here, we focused on the role of iNKT cells under normal conditions. We showed that iNKT cell–deficient mice on a low-fat diet, considered a normal diet for mice, displayed a distinctive insulin resistance phenotype without overt adipose tissue inflammation. Insulin resistance was characterized by adipocyte dysfunction, including adipocyte hypertrophy, increased leptin, and decreased adiponectin levels. The lack of liver abnormalities in CD1d-null mice together with the enrichment of CD1d-restricted iNKT cells in both mouse and human adipose tissue indicated a specific role for adipose tissue–resident iNKT cells in the development of insulin resistance. Strikingly, iNKT cell function was directly modulated by adipocytes, which acted as lipid antigen-presenting cells in a CD1d-mediated fashion. Based on these findings, we propose that, especially under low-fat diet conditions, adipose tissue–resident iNKT cells maintain healthy adipose tissue through direct interplay with adipocytes and prevent insulin resistance. PMID:22863618

  10. Expression of activation-induced cytidine deaminase enhances the clearance of pneumococcal pneumonia: evidence of a subpopulation of protective anti-pneumococcal B1a cells.

    PubMed

    Yamamoto, Natsuo; Kerfoot, Steven M; Hutchinson, Andrew T; Dela Cruz, Charles S; Nakazawa, Naomi; Szczepanik, Marian; Majewska-Szczepanik, Monika; Nazimek, Katarzyna; Ohana, Noboru; Bryniarski, Krzysztof; Mori, Tsutomu; Muramatsu, Masamichi; Kanemitsu, Keiji; Askenase, Philip W

    2016-01-01

    We describe a protective early acquired immune response to pneumococcal pneumonia that is mediated by a subset of B1a cells. Mice deficient in B1 cells (xid), or activation-induced cytidine deaminase (AID(-/-) ), or invariant natural killer T (iNKT) cells (Jα18(-/-) ), or interleukin-13 (IL-13(-/-) ) had impaired early clearance of pneumococci in the lung, compared with wild-type mice. In contrast, AID(-/-) mice adoptively transferred with AID(+/+) B1a cells, significantly cleared bacteria from the lungs as early as 3 days post infection. We show that this early bacterial clearance corresponds to an allergic contact sensitivity-like cutaneous response, probably due to a subpopulation of initiating B1a cells. In the pneumonia model, these B1a cells were found to secrete higher affinity antigen-specific IgM. In addition, as in contact sensitivity, iNKT cells were required for the anti-pneumococcal B1a cell initiating response, probably through early production of IL-13, given that IL-13(-/-) mice also failed to clear infection. Our study is the first to demonstrate the importance of AID in generating an appropriate B1a cell response to pathogenic bacteria. Given the antibody affinity and pneumonia resistance data, natural IgM produced by conventional B1a cells are not responsible for pneumonia clearance compared with the AID-dependent subset.

  11. Discrete TCR Binding Kinetics Control Invariant NKT Cell Selection and Central Priming.

    PubMed

    Cruz Tleugabulova, Mayra; Escalante, Nichole K; Deng, Shenglou; Fieve, Stephanie; Ereño-Orbea, June; Savage, Paul B; Julien, Jean-Philippe; Mallevaey, Thierry

    2016-11-15

    Invariant NKT (iNKT) cells develop and differentiate in the thymus, segregating into iNKT1/2/17 subsets akin to Th1/2/17 classical CD4(+) T cells; however, iNKT TCRs recognize Ags in a fundamentally different way. How the biophysical parameters of iNKT TCRs influence signal strength in vivo and how such signals affect the development and differentiation of these cells are unknown. In this study, we manipulated TCRs in vivo to generate clonotypic iNKT cells using TCR retrogenic chimeras. We report that the biophysical properties of CD1d-lipid-TCR interactions differentially impacted the development and effector differentiation of iNKT cells. Whereas selection efficiency strongly correlated with TCR avidity, TCR signaling, cell-cell conjugate formation, and iNKT effector differentiation correlated with the half-life of CD1d-lipid-TCR interactions. TCR binding properties, however, did not modulate Ag-induced iNKT cytokine production. Our work establishes that discrete TCR interaction kinetics influence iNKT cell development and central priming.

  12. Pre-transplant donor CD4(-) invariant NKT cell expansion capacity predicts the occurrence of acute graft-versus-host disease.

    PubMed

    Rubio, M-T; Bouillié, M; Bouazza, N; Coman, T; Trebeden-Nègre, H; Gomez, A; Suarez, F; Sibon, D; Brignier, A; Paubelle, E; Nguyen-Khoc, S; Cavazzana, M; Lantz, O; Mohty, M; Urien, S; Hermine, O

    2017-04-01

    Clinically useful pre-transplant predictive factors of acute graft-versus-host-disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-SCT) are lacking. We prospectively analyzed HSC graft content in CD34(+), NK, conventional T, regulatory T and invariant natural killer T (iNKT) cells in 117 adult patients before allo-SCT. Results were correlated with occurrence of aGVHD and relapse. In univariate analysis, iNKT cells were the only graft cell populations associated with occurrence of aGVHD. In multivariate analysis, CD4(-) iNKT/T cell frequency could predict grade II-IV aGVHD in bone marrow and peripheral blood stem cell (PBSC) grafts, while CD4(-) iNKT expansion capacity was predictive in PBSC grafts. Receiver operating characteristic analyses determined the CD4(-) iNKT expansion factor as the best predictive factor of aGVHD. Incidence of grade II-IV aGVHD was reduced in patients receiving a graft with an expansion factor above versus below 6.83 (9.7 vs 80%, P<0.0001), while relapse incidence at two years was similar (P=0.5).The test reached 94% sensitivity and 100% specificity in the subgroup of patients transplanted with human leukocyte antigen 10/10 PBSCs without active disease. Analysis of this CD4(-) iNKT expansion capacity test may represent the first diagnostic tool allowing selection of the best donor to avoid severe aGVHD with preserved graft-versus-leukemia effect after peripheral blood allo-SCT.

  13. The Hypothesis of the Human iNKT/Innate CD8(+) T-Cell Axis Applied to Cancer: Evidence for a Deficiency in Chronic Myeloid Leukemia

    PubMed Central

    Jacomet, Florence; Cayssials, Emilie; Barbarin, Alice; Desmier, Deborah; Basbous, Sara; Lefèvre, Lucie; Levescot, Anaïs; Robin, Aurélie; Piccirilli, Nathalie; Giraud, Christine; Guilhot, François; Roy, Lydia; Herbelin, André; Gombert, Jean-Marc

    2017-01-01

    We recently identified a new human subset of NK-like [KIR/NKG2A(+)] CD8(+) T cells with a marked/memory phenotype, high Eomesodermin expression, potent antigen-independent cytotoxic activity, and the capacity to generate IFN-γ rapidly after exposure to pro-inflammatory cytokines. These features support the hypothesis that this new member of the innate T cell family in humans, hereafter referred to as innate CD8(+) T cells, has a role in cancer immune surveillance analogous to invariant natural killer T (iNKT) cells. Here, we report the first quantitative and functional analysis of innate CD8(+) T cells in a physiopathological context in humans, namely chronic myeloid leukemia (CML), a well-characterized myeloproliferative disorder. We have chosen CML based on our previous report that IL-4 production by iNKT cells was deficient in CML patients at diagnosis and considering the recent evidence in mice that IL-4 promotes the generation/differentiation of innate CD8(+) T cells. We found that the pool of innate CD8(+) T cells was severely reduced in the blood of CML patients at diagnosis. Moreover, like iNKT and NK cells, innate CD8(+) T cells were functionally impaired, as attested by their loss of antigen-independent cytotoxic activity and IFN-γ production in response to innate-like stimulation with IL-12 + IL-18. Remarkably, as previously reported for IL-4 production by iNKT cells, both quantitative and functional deficiencies of innate CD8(+) T cells were at least partially corrected in patients having achieved complete cytogenetic remission following tyrosine kinase inhibitor therapy. Finally, direct correlation between the functional potential of innate CD8(+) T and iNKT cells was found when considering all healthy donors and CML patients in diagnosis and remission, in accordance with the iNKT cell-dependent generation of innate CD8(+) T cells reported in mice. All in all, our data demonstrate that CML is associated with deficiencies of innate CD8(+) T cells

  14. TIM-4 is expressed on invariant NKT cells but dispensable for their development and function.

    PubMed

    Zhang, Xilin; Gu, Jun; Zhou, Li; Mi, Qing-Sheng

    2016-11-01

    T cell immunoglobulin and mucin-4 (TIM-4), mainly expressed on antigen presenting cells, plays a versatile role in immunoregulation. CD1d-restricted invariant natural killer T (iNKT) cells are potent cells involved in the diverse immune responses. It was recently reported that recombinant TIM-4 (rTIM-4) alone enhanced cytokine production in NKT hybridoma, DN32.D3 cells. Hence, we hypothesized that TIM-4 might regulate iNKT cell biology, especially their function of cytokine secretion. For the first time, we identified that TIM-4 was expressed in thymus iNKT cells, and its expression increased upon iNKT cell migration to the secondary lymphoid organs, especially in lymph nodes. Using TIM-4-deficient mice, we found that lack of TIM-4 did not disturb iNKT cell development, maturation, peripheral homeostasis and cytokine secretion. Moreover, TIM-4 deficiency did not alter the polarization of iNKT sublineages, including NKT1, NKT2 and NKT17. Finally, the mixed bone marrow transfer experiments further confirmed normal iNKT cell development and function from TIM-4-deficient bone marrow. In conclusion, our data suggest that TIM-4 is expressed on iNKT cells but dispensable for their development and function.

  15. TIM-4 is expressed on invariant NKT cells but dispensable for their development and function

    PubMed Central

    Zhang, Xilin; Gu, Jun; Zhou, Li; Mi, Qing-Sheng

    2016-01-01

    T cell immunoglobulin and mucin-4 (TIM-4), mainly expressed on antigen presenting cells, plays a versatile role in immunoregulation. CD1d-restricted invariant natural killer T (iNKT) cells are potent cells involved in the diverse immune responses. It was recently reported that recombinant TIM-4 (rTIM-4) alone enhanced cytokine production in NKT hybridoma, DN32.D3 cells. Hence, we hypothesized that TIM-4 might regulate iNKT cell biology, especially their function of cytokine secretion. For the first time, we identified that TIM-4 was expressed in thymus iNKT cells, and its expression increased upon iNKT cell migration to the secondary lymphoid organs, especially in lymph nodes. Using TIM-4-deficient mice, we found that lack of TIM-4 did not disturb iNKT cell development, maturation, peripheral homeostasis and cytokine secretion. Moreover, TIM-4 deficiency did not alter the polarization of iNKT sublineages, including NKT1, NKT2 and NKT17. Finally, the mixed bone marrow transfer experiments further confirmed normal iNKT cell development and function from TIM-4-deficient bone marrow. In conclusion, our data suggest that TIM-4 is expressed on iNKT cells but dispensable for their development and function. PMID:27662666

  16. A Novel Glycolipid Antigen for NKT Cells That Preferentially Induces IFN-γ Production.

    PubMed

    Birkholz, Alysia M; Girardi, Enrico; Wingender, Gerhard; Khurana, Archana; Wang, Jing; Zhao, Meng; Zahner, Sonja; Illarionov, Petr A; Wen, Xiangshu; Li, Michelle; Yuan, Weiming; Porcelli, Steven A; Besra, Gurdyal S; Zajonc, Dirk M; Kronenberg, Mitchell

    2015-08-01

    In this article, we characterize a novel Ag for invariant NKT (iNKT) cells capable of producing an especially robust Th1 response. This glycosphingolipid, DB06-1, is similar in chemical structure to the well-studied α-galactosylceramide (αGalCer), with the only change being a single atom: the substitution of a carbonyl oxygen with a sulfur atom. Although DB06-1 is not a more effective Ag in vitro, the small chemical change has a marked impact on the ability of this lipid Ag to stimulate iNKT cells in vivo, with increased IFN-γ production at 24 h compared with αGalCer, increased IL-12, and increased activation of NK cells to produce IFN-γ. These changes are correlated with an enhanced ability of DB06-1 to load in the CD1d molecules expressed by dendritic cells in vivo. Moreover, structural studies suggest a tighter fit into the CD1d binding groove by DB06-1 compared with αGalCer. Surprisingly, when iNKT cells previously exposed to DB06-1 are restimulated weeks later, they have greatly increased IL-10 production. Therefore, our data are consistent with a model whereby augmented and or prolonged presentation of a glycolipid Ag leads to increased activation of NK cells and a Th1-skewed immune response, which may result, in part, from enhanced loading into CD1d. Furthermore, our data suggest that strong antigenic stimulation in vivo may lead to the expansion of IL-10-producing iNKT cells, which could counteract the benefits of increased early IFN-γ production.

  17. A novel glycolipid antigen for NKT cells that preferentially induces IFN-γ production

    PubMed Central

    Birkholz, Alysia M.; Girardi, Enrico; Wingender, Gerhard; Khurana, Archana; Wang, Jing; Zhao, Meng; Zahner, Sonja; Illarionov, Petr A.; Wen, Xiangshu; Li, Michelle; Yuan, Weiming; Porcelli, Steven A.; Besra, Gurdyal S.; Zajonc, Dirk M.; Kronenberg, Mitchell

    2015-01-01

    Here we characterize a novel Ag for invariant natural killer T-cells (iNKT cells) capable of producing an especially robust Th1 response. This glycosphingolipid (GSL), DB06-1, is similar in chemical structure to the well-studied α-galactosylceramide (αGalCer), the only change being in a single atom, the substitution of a carbonyl oxygen with a sulfur atom. Although DB06-1 is not a more effective Ag in vitro, the small chemical change has a marked impact on the ability of this lipid Ag to stimulate iNKT cells in vivo, with increased IFN-γ production at 24 h compared to αGalCer, increased IL-12, and increased activation of NK cells to produce IFN-γ. These changes are correlated with an enhanced ability of DB06-1 to load in the CD1d molecules expressed by DCs in vivo. Moreover, structural studies suggest a tighter fit into the CD1d binding groove by DB061 compared to αGalCer. Surprisingly, when iNKT cells previously exposed to DB06-1 are restimulated weeks later, they have greatly increased IL-10 production. Our data are therefore consistent with a model whereby augmented and or prolonged presentation of a glycolipid Ag leads to increased activation of NK cells and a Th1-skewed immune response, which may result in part from enhanced loading into CD1d. Furthermore, our data suggest that strong antigenic stimulation in vivo may lead to the expansion of IL-10 producing iNKT cells, which could counteract the benefits of increased, early IFN-γ production. PMID:26078271

  18. Negative Checkpoint Regulatory Molecule 2B4 (CD244) Upregulation Is Associated with Invariant Natural Killer T Cell Alterations and Human Immunodeficiency Virus Disease Progression

    PubMed Central

    Ahmad, Fareed; Shankar, Esaki M.; Yong, Yean K.; Tan, Hong Y.; Ahrenstorf, Gerrit; Jacobs, Roland; Larsson, Marie; Schmidt, Reinhold E.; Kamarulzaman, Adeeba; Ansari, Abdul W.

    2017-01-01

    The CD1d-restricted invariant natural killer T (iNKT) cells are implicated in innate immune responses against human immunodeficiency virus (HIV). However, the determinants of cellular dysfunction across the iNKT cells subsets are seldom defined in HIV disease. Herein, we provide evidence for the involvement of the negative checkpoint regulator (NCR) 2B4 in iNKT cell alteration in a well-defined cohort of HIV-seropositive anti-retroviral therapy (ART) naïve, ART-treated, and elite controllers (ECs). We report on exaggerated 2B4 expression on iNKT cells of HIV-infected treatment-naïve individuals. In sharp contrast to CD4−iNKT cells, 2B4 expression was significantly higher on CD4+ iNKT cell subset. Notably, an increased level of 2B4 on iNKT cells was strongly correlated with parameters associated with HIV disease progression. Further, iNKT cells from ART-naïve individuals were defective in their ability to produce intracellular IFN-γ. Together, our results suggest that the levels of 2B4 expression and the downstream co-inhibitory signaling events may contribute to impaired iNKT cell responses.

  19. Invariant natural killer T cells act as an extravascular cytotoxic barrier for joint-invading Lyme Borrelia

    PubMed Central

    Lee, Woo-Yong; Sanz, Maria-Jesus; Wong, Connie H. Y.; Hardy, Pierre-Olivier; Salman-Dilgimen, Aydan; Moriarty, Tara J.; Chaconas, George; Marques, Adriana; Krawetz, Roman; Mody, Christopher H.; Kubes, Paul

    2014-01-01

    CXCR6-GFP+ cells, which encompass 70% invariant natural killer T cells (iNKT cells), have been found primarily patrolling inside blood vessels in the liver. Although the iNKT cells fail to interact with live pathogens, they do respond to bacterial glycolipids presented by CD1d on liver macrophage that have caught the microbe. In contrast, in this study using dual laser multichannel spinning-disk intravital microscopy of joints, the CXCR6-GFP, which also made up 60–70% iNKT cells, were not found in the vasculature but rather closely apposed to and surrounding the outside of blood vessels, and to a lesser extent throughout the extravascular space. These iNKT cells also differed in behavior, responding rapidly and directly to joint-homing pathogens like Borrelia burgdorferi, which causes Lyme disease. These iNKT cells interacted with B. burgdorferi at the vessel wall and disrupted dissemination attempts by these microbes into joints. Successful penetrance of B. burgdorferi out of the vasculature and into the joint tissue was met by a lethal attack by extravascular iNKT cells through a granzyme-dependent pathway, an observation also made in vitro for iNKT cells from joint but not liver or spleen. These results suggest a novel, critical extravascular iNKT cell immune surveillance in joints that functions as a cytotoxic barrier and explains a large increase in pathogen burden of B. burgdorferi in the joint of iNKT cell-deficient mice, and perhaps the greater susceptibility of humans to this pathogen because of fewer iNKT cells in human joints. PMID:25205813

  20. Invariant natural killer T cells act as an extravascular cytotoxic barrier for joint-invading Lyme Borrelia.

    PubMed

    Lee, Woo-Yong; Sanz, Maria-Jesus; Wong, Connie H Y; Hardy, Pierre-Olivier; Salman-Dilgimen, Aydan; Moriarty, Tara J; Chaconas, George; Marques, Adriana; Krawetz, Roman; Mody, Christopher H; Kubes, Paul

    2014-09-23

    CXCR6-GFP(+) cells, which encompass 70% invariant natural killer T cells (iNKT cells), have been found primarily patrolling inside blood vessels in the liver. Although the iNKT cells fail to interact with live pathogens, they do respond to bacterial glycolipids presented by CD1d on liver macrophage that have caught the microbe. In contrast, in this study using dual laser multichannel spinning-disk intravital microscopy of joints, the CXCR6-GFP, which also made up 60-70% iNKT cells, were not found in the vasculature but rather closely apposed to and surrounding the outside of blood vessels, and to a lesser extent throughout the extravascular space. These iNKT cells also differed in behavior, responding rapidly and directly to joint-homing pathogens like Borrelia burgdorferi, which causes Lyme disease. These iNKT cells interacted with B. burgdorferi at the vessel wall and disrupted dissemination attempts by these microbes into joints. Successful penetrance of B. burgdorferi out of the vasculature and into the joint tissue was met by a lethal attack by extravascular iNKT cells through a granzyme-dependent pathway, an observation also made in vitro for iNKT cells from joint but not liver or spleen. These results suggest a novel, critical extravascular iNKT cell immune surveillance in joints that functions as a cytotoxic barrier and explains a large increase in pathogen burden of B. burgdorferi in the joint of iNKT cell-deficient mice, and perhaps the greater susceptibility of humans to this pathogen because of fewer iNKT cells in human joints.

  1. Invariant NKT cells require autophagy to coordinate proliferation and survival signals during differentiation.

    PubMed

    Pei, Bo; Zhao, Meng; Miller, Brian C; Véla, Jose Luis; Bruinsma, Monique W; Virgin, Herbert W; Kronenberg, Mitchell

    2015-06-15

    Autophagy regulates cell differentiation, proliferation, and survival in multiple cell types, including cells of the immune system. In this study, we examined the effects of a disruption of autophagy on the differentiation of invariant NKT (iNKT) cells. Using mice with a T lymphocyte-specific deletion of Atg5 or Atg7, two members of the macroautophagic pathway, we observed a profound decrease in the iNKT cell population. The deficit is cell-autonomous, and it acts predominantly to reduce the number of mature cells, as well as the function of peripheral iNKT cells. In the absence of autophagy, there is reduced progression of iNKT cells in the thymus through the cell cycle, as well as increased apoptosis of these cells. Importantly, the reduction in Th1-biased iNKT cells is most pronounced, leading to a selective reduction in iNKT cell-derived IFN-γ. Our findings highlight the unique metabolic and genetic requirements for the differentiation of iNKT cells.

  2. Inflammatory mechanisms in sepsis: elevated invariant natural killer T-cell numbers in mouse and their modulatory effect on macrophage function.

    PubMed

    Heffernan, Daithi S; Monaghan, Sean F; Thakkar, Rajan K; Tran, Mai L; Chung, Chun-Shiang; Gregory, Stephen H; Cioffi, William G; Ayala, Alfred

    2013-08-01

    Invariant natural killer T cells (iNKT) cells are emerging as key mediators of innate immune cellular and inflammatory responses to sepsis and peritonitis. Invariant natural killer T cells mediate survival following murine septic shock. Macrophages are pivotal to survival following sepsis. Invariant natural killer T cells have been shown to modulate various mediators of the innate immune system, including macrophages. We demonstrate sepsis-inducing iNKT-cell exodus from the liver appearing in the peritoneal cavity, the source of the sepsis. This migration was affected by programmed death receptor 1. Programmed death receptor 1 is an inhibitory immune receptor, reported as ubiquitously expressed at low levels on iNKT cells. Programmed death receptor 1 has been associated with markers of human critical illness. Programmed death receptor 1-deficient iNKT cells failed to demonstrate similar migration. To the extent that iNKT cells affected peritoneal macrophage function, we assessed peritoneal macrophages' ability to phagocytose bacteria. Invariant natural killer T(-/-) mice displayed dysfunctional macrophage phagocytosis and altered peritoneal bacterial load. This dysfunction was reversed when peritoneal macrophages from iNKT(-/-) mice were cocultured with wild-type iNKT cells. Together, our results indicate that sepsis induces liver iNKT-cell exodus into the peritoneal cavity mediated by programmed death receptor 1, and these peritoneal iNKT cells appear critical to regulation of peritoneal macrophage phagocytic function. Invariant natural killer T cells offer therapeutic targets for modulating immune responses and detrimental effects of sepsis.

  3. Coupling of T cell receptor specificity to natural killer T cell development by bivalent histone H3 methylation.

    PubMed

    Dobenecker, Marc-Werner; Kim, Jong Kyong; Marcello, Jonas; Fang, Terry C; Prinjha, Rab; Bosselut, Remy; Tarakhovsky, Alexander

    2015-03-09

    The fidelity of T cell immunity depends greatly on coupling T cell receptor signaling with specific T cell effector functions. Here, we describe a chromatin-based mechanism that enables integration of TCR specificity into definite T cell lineage commitment. Using natural killer T cells (iNKT cell) as a model of a T cell subset that differentiates in response to specific TCR signaling, we identified a key role of histone H3 lysine 27 trimethylation (H3K27me3) in coupling iNKT cell TCR specificity with the generation of iNKT cells. We found that the Zbtb16/PLZF gene promoter that drives iNKT cell differentiation possesses a bivalent chromatin state characterized by the simultaneous presence of negative and positive H3K27me3 and H3K4me3 modifications. Depletion of H3K27me3 at the Zbtb16/PLZF promoter leads to uncoupling of iNKT cell development from TCR specificity and is associated with accumulation of iNKT-like CD4(+) cells that express a non-iNKT cell specific T cell repertoire. In turn, stabilization of H3K27me3 leads to a drastic reduction of the iNKT cell population. Our data suggest that H3K27me3 levels at the bivalent Zbtb16/PLZF gene define a threshold enabling precise coupling of TCR specificity to lineage commitment.

  4. NKT cells prevent chronic joint inflammation after infection with Borrelia burgdorferi.

    PubMed

    Tupin, Emmanuel; Benhnia, Mohammed Rafii-El-Idrissi; Kinjo, Yuki; Patsey, Rebeca; Lena, Christopher J; Haller, Matthew C; Caimano, Melissa J; Imamura, Masakazu; Wong, Chi-Huey; Crotty, Shane; Radolf, Justin D; Sellati, Timothy J; Kronenberg, Mitchell

    2008-12-16

    Borrelia burgdorferi is the etiologic agent of Lyme disease, a multisystem inflammatory disorder that principally targets the skin, joints, heart, and nervous system. The role of T lymphocytes in the development of chronic inflammation resulting from B. burgdorferi infection has been controversial. We previously showed that natural killer T (NKT) cells with an invariant (i) TCR alpha chain (iNKT cells) recognize glycolipids from B. burgdorferi, but did not establish an in vivo role for iNKT cells in Lyme disease pathogenesis. Here, we evaluate the importance of iNKT cells for host defense against these pathogenic spirochetes by using Valpha14i NKT cell-deficient (Jalpha18(-/-)) BALB/c mice. On tick inoculation with B. burgdorferi, Jalpha18(-/-) mice exhibited more severe and prolonged arthritis as well as a reduced ability to clear spirochetes from infected tissues. Valpha14i NKT cell deficiency also resulted in increased production of antibodies directed against both B. burgdorferi protein antigens and borrelial diacylglycerols; the latter finding demonstrates that anti-glycolipid antibody production does not require cognate help from Valpha14i NKT cells. Valpha14i NKT cells in infected wild-type mice expressed surface activation markers and produced IFNgamma in vivo after infection, suggesting a participatory role for this unique population in cellular immunity. Our data are consistent with the hypothesis that the antigen-specific activation of Valpha14i NKT cells is important for the prevention of persistent joint inflammation and spirochete clearance, and they counter the long-standing notion that humoral rather than cellular immunity is sufficient to facilitate Lyme disease resolution.

  5. T cell activation.

    PubMed

    Smith-Garvin, Jennifer E; Koretzky, Gary A; Jordan, Martha S

    2009-01-01

    This year marks the 25th anniversary of the first Annual Review of Immunology article to describe features of the T cell antigen receptor (TCR). In celebration of this anniversary, we begin with a brief introduction outlining the chronology of the earliest studies that established the basic paradigm for how the engaged TCR transduces its signals. This review continues with a description of the current state of our understanding of TCR signaling, as well as a summary of recent findings examining other key aspects of T cell activation, including cross talk between the TCR and integrins, the role of costimulatory molecules, and how signals may negatively regulate T cell function.Acronyms and DefinitionsAdapter protein: cellular protein that functions to bridge molecular interactions via characteristic domains able to mediate protein/protein or protein/lipid interactions Costimulation: signals delivered to T cells by cell surface receptors other than the TCR itself that potentiate T cell activation cSMAC: central supramolecular activation cluster Immunoreceptor tyrosine-based activation motif (ITAM): a short peptide sequence in the cytoplasmic tails of key surface receptors on hematopoietic cells that is characterized by tyrosine residues that are phosphorylated by Src family PTKs, enabling the ITAM to recruit activated Syk family kinases Inside-out signaling: signals initiated by engagement of immunoreceptors that lead to conformational changes and clustering of integrins, thereby increasing the affinity and avidity of the integrins for their ligands NFAT: nuclear factor of activated T cells PI3K: phosphoinositide 3-kinase PKC: protein kinase C PLC: phospholipase C pMHC: peptide major histocompatibility complex (MHC) complex pSMAC: peripheral supramolecular activation cluster PTK: protein tyrosine kinase Signal transduction: biochemical events linking surface receptor engagement to cellular responses TCR: T cell antigen receptor

  6. Invariant natural killer T cells are not affected by lysosomal storage in patients with Niemann-Pick disease type C.

    PubMed

    Speak, Anneliese O; Platt, Nicholas; Salio, Mariolina; te Vruchte, Danielle; Smith, David A; Shepherd, Dawn; Veerapen, Natacha; Besra, Gurdyal S; Yanjanin, Nicole M; Simmons, Louise; Imrie, Jackie; Wraith, James E; Lachmann, Robin H; Hartung, Ralf; Runz, Heiko; Mengel, Eugen; Beck, Michael; Hendriksz, Christian J; Porter, Forbes D; Cerundolo, Vincenzo; Platt, Frances M

    2012-07-01

    Invariant natural killer T (iNKT) cells are a specialised subset of T cells that are restricted to the MHC class I like molecule, CD1d. The ligands for iNKT cells are lipids, with the canonical superagonist being α-galactosylceramide, a non-mammalian glycosphingolipid. Trafficking of CD1d through the lysosome is required for the development of murine iNKT cells. Niemann-Pick type C (NPC) disease is a lysosomal storage disorder caused by dysfunction in either of two lysosomal proteins, NPC1 or NPC2, resulting in the storage of multiple lipids, including glycosphingolipids. In the NPC1 mouse model, iNKT cells are virtually undetectable, which is likely due to the inability of CD1d to be loaded with the selecting ligand due to defective lysosomal function and/or CD1d trafficking. However, in this study we have found that in NPC1 patients iNKT cells are present at normal frequencies, with no phenotypic or functional differences. In addi-tion, antigen-presenting cells derived from NPC1 patients are functionally competent to present several different CD1d/iNKT-cell ligands. This further supports the hypothesis that there are different trafficking requirements for the development of murine and human iNKT cells, and a functional lysosomal/late-endosomal compartment is not required for human iNKT-cell development.

  7. Raman activated cell sorting.

    PubMed

    Song, Yizhi; Yin, Huabing; Huang, Wei E

    2016-08-01

    Single cell Raman spectra (SCRS) are intrinsic biochemical profiles and 'chemical images' of single cells which can be used to characterise phenotypic changes, physiological states and functions of cells. On the base of SCRS, Raman activated cell sorting (RACS) provides a label-free cell sorting approach, which can link single cells to their chemical or phenotypic profiles. Overcoming naturally weak Raman signals, establishing Raman biomarker as sorting criteria to RACS and improving specific sorting technology are three challenges of developing RACS. Advances on Raman spectroscopy such as stimulated Raman scattering (SRS) and pre-screening helped to increase RACS sorting speed. Entire SCRS can be characterised using pattern recognition methods, and specific Raman bands can be extracted as biomarkers for RACS. Recent advances on cell sorting technologies based on microfluidic device and surface-ejection enable accurate and reliable single cell sorting from complex samples. A high throughput RACS will be achievable in near future by integrating fast Raman detection system such as SRS with microfluidic RACS and Raman activated cell ejection (RACE).

  8. Lysophospholipid presentation by CD1d and recognition by a human Natural Killer T-cell receptor

    PubMed Central

    López-Sagaseta, Jacinto; Sibener, Leah V; Kung, Jennifer E; Gumperz, Jenny; Adams, Erin J

    2012-01-01

    Invariant Natural Killer T (iNKT) cells use highly restricted αβ T cell receptors (TCRs) to probe the repertoire of lipids presented by CD1d molecules. Here, we describe our studies of lysophosphatidylcholine (LPC) presentation by human CD1d and its recognition by a native, LPC-specific iNKT TCR. Human CD1d presenting LPC adopts an altered conformation from that of CD1d presenting glycolipid antigens, with a shifted α1 helix resulting in an open A' pocket. Binding of the iNKT TCR requires a 7-Å displacement of the LPC headgroup but stabilizes the CD1d–LPC complex in a closed conformation. The iNKT TCR CDR loop footprint on CD1d–LPC is anchored by the conserved positioning of the CDR3α loop, whereas the remaining CDR loops are shifted, due in part to amino-acid differences in the CDR3β and Jβ segment used by this iNKT TCR. These findings provide insight into how lysophospholipids are presented by human CD1d molecules and how this complex is recognized by some, but not all, human iNKT cells. PMID:22395072

  9. Lysophospholipid presentation by CD1d and recognition by a human Natural Killer T-cell receptor

    SciTech Connect

    López-Sagaseta, Jacinto; Sibener, Leah V.; Kung, Jennifer E.; Gumperz, Jenny; Adams, Erin J.

    2014-10-02

    Invariant Natural Killer T (iNKT) cells use highly restricted {alpha}{beta} T cell receptors (TCRs) to probe the repertoire of lipids presented by CD1d molecules. Here, we describe our studies of lysophosphatidylcholine (LPC) presentation by human CD1d and its recognition by a native, LPC-specific iNKT TCR. Human CD1d presenting LPC adopts an altered conformation from that of CD1d presenting glycolipid antigens, with a shifted {alpha}1 helix resulting in an open A pocket. Binding of the iNKT TCR requires a 7-{angstrom} displacement of the LPC headgroup but stabilizes the CD1d-LPC complex in a closed conformation. The iNKT TCR CDR loop footprint on CD1d-LPC is anchored by the conserved positioning of the CDR3{alpha} loop, whereas the remaining CDR loops are shifted, due in part to amino-acid differences in the CDR3{beta} and J{beta} segment used by this iNKT TCR. These findings provide insight into how lysophospholipids are presented by human CD1d molecules and how this complex is recognized by some, but not all, human iNKT cells.

  10. Id2 regulates hyporesponsive invariant natural killer T cells

    PubMed Central

    Stradner, Martin H; Cheung, Kitty P; Lasorella, Anna; Goldrath, Ananda W; D’Cruz, Louise M

    2016-01-01

    While the invariant natural killer T (iNKT)-cell response to primary stimulation with the glycolipid, α-galactosylceramide (αGalCer), is robust, the secondary response to this stimulus is muted resulting in a hyporesponsive state characterized by anti-inflammatory interleukin-10 (IL-10) production and high expression of programmed cell death 1 (PD1) and neuropilin 1 (NRP1). The E protein transcription factors and their negative regulators, the Id proteins, have previously been shown to regulate iNKT cell thymic development, subset differentiation and peripheral survival. Here, we provide evidence that the expression of the transcriptional regulator Id2 is downregulated upon stimulation of iNKT cells with their cognate antigen. Moreover, loss of Id2 expression by iNKT cells resulted in a hyporesponsive state, with splenic Id2-deficient iNKT cells expressing low levels of TBET, high levels of PD1 and NRP1 and production of IL-10 upon stimulation. We propose that downregulation of Id2 expression is an essential component of induction of the anti-inflammatory, hyporesponsive state in iNKT cells. PMID:26880074

  11. Active Cells for Multifunctional Structures

    DTIC Science & Technology

    2014-09-24

    techniques to explore a variety of cell designs.  Designed a simplified active cell using Nitinol as the actuation method and relying on Joule heating...for contraction of the cell.  Developed manufacturing techniques for reliably creating Nitinol spring coils in a variety of diameters and gauges...design of the active cells to maximum the stroked length of the active cells by tuning the stiffness of a passive spring in parallel with the Nitinol

  12. Effect of PD-1: PD-L1 in Invariant Natural Killer T-Cell Emigration and Chemotaxis Following Sepsis.

    PubMed

    Young, John S; Heffernan, Daithi S; Chung, Chun-Shiang; Kettenmann, Maude L; Young, Whitney A; Guillen, Valeria Sanabria; Cioffi, William G; Ayala, Alfred

    2016-05-01

    Invariant natural killer T-cells (iNKT) are a subset of T-cells that play a regulatory role in sepsis. Following cecal ligation and puncture (CLP), iNKT cells emigrate from the liver and into the circulation and peritoneum in a manner dependent upon coinhibitory molecule Programmed Cell Death Receptor 1 (PD-1). We hypothesized that the effect of PD-1 on iNKT-cell emigration was dependent upon the direct PD-1:PD-L1 interaction, and that PD-1 and PD-L1 would play a role in chemotaxis and chemokine receptor expression. Adoptive transfer of Vybrant-labeled wild-type (WT) cells showed the donor iNKT cells migrated from the liver to the peritoneum following CLP, but PD-L1 deficient donor iNKT cells did not. In a chemotaxis assay, WT-iNKT cells chemotaxed to CXCL12, but PD-1 and PD-L1 deficient iNKT cells did not. Using flow cytometry to evaluate chemokine receptor expression, peritoneal iNKT expression of CXCR4 increased following CLP in the WT, PD-1, and PD-L1 deficient animals, and CXCR6 increased in the WT and PD-1 deficient animals. In conclusion here we document that the hepatic emigration of iNKT cells following CLP to the peritoneum appears dependent upon the direct PD-1:PD-L1 interaction; however, although PD-1 and PD-L1 appear to play a role in chemotaxis, this is unlikely a reflection of iNKT-cell chemokine receptor expression changes.

  13. Binding Strength and Dynamics of Invariant Natural Killer Cell T Cell Receptor/CD1d-Glycosphingolipid Interaction on Living Cells by Single Molecule Force Spectroscopy*

    PubMed Central

    Bozna, Bianca L.; Polzella, Paolo; Rankl, Christian; Zhu, Rong; Salio, Mariolina; Shepherd, Dawn; Duman, Memed; Cerundolo, Vincenzo; Hinterdorfer, Peter

    2011-01-01

    Invariant natural killer T (iNKT) cells are a population of T lymphocytes that play an important role in regulating immunity to infection and tumors by recognizing endogenous and exogenous CD1d-bound lipid molecules. Using soluble iNKT T cell receptor (TCR) molecules, we applied single molecule force spectroscopy for the investigation of the iNKT TCR affinity for human CD1d molecules loaded with glycolipids differing in the length of the phytosphingosine chain using either recombinant CD1d molecules or lipid-pulsed THP1 cells. In both settings, the dissociation of the iNKT TCR from human CD1d molecules loaded with the lipid containing the longer phytosphingosine chain required higher unbinding forces compared with the shorter phytosphingosine lipid. Our findings are discussed in the context of previous results obtained by surface plasmon resonance measurements. We present new insights into the energy landscape and the kinetic rate constants of the iNKT TCR/human CD1d-glycosphingolipid interaction and emphasize the unique potential of single molecule force spectroscopy on living cells. PMID:21454514

  14. Binding strength and dynamics of invariant natural killer cell T cell receptor/CD1d-glycosphingolipid interaction on living cells by single molecule force spectroscopy.

    PubMed

    Bozna, Bianca L; Polzella, Paolo; Rankl, Christian; Zhu, Rong; Salio, Mariolina; Shepherd, Dawn; Duman, Memed; Cerundolo, Vincenzo; Hinterdorfer, Peter

    2011-05-06

    Invariant natural killer T (iNKT) cells are a population of T lymphocytes that play an important role in regulating immunity to infection and tumors by recognizing endogenous and exogenous CD1d-bound lipid molecules. Using soluble iNKT T cell receptor (TCR) molecules, we applied single molecule force spectroscopy for the investigation of the iNKT TCR affinity for human CD1d molecules loaded with glycolipids differing in the length of the phytosphingosine chain using either recombinant CD1d molecules or lipid-pulsed THP1 cells. In both settings, the dissociation of the iNKT TCR from human CD1d molecules loaded with the lipid containing the longer phytosphingosine chain required higher unbinding forces compared with the shorter phytosphingosine lipid. Our findings are discussed in the context of previous results obtained by surface plasmon resonance measurements. We present new insights into the energy landscape and the kinetic rate constants of the iNKT TCR/human CD1d-glycosphingolipid interaction and emphasize the unique potential of single molecule force spectroscopy on living cells.

  15. Fluorescence activated cell sorting.

    NASA Technical Reports Server (NTRS)

    Bonner, W. A.; Hulett, H. R.; Sweet, R. G.; Herzenberg, L. A.

    1972-01-01

    An instrument has been developed for sorting biological cells. The cells are rendered differentially fluorescent and incorporated into a small liquid stream illuminated by a laser beam. The cells pass sequentially through the beam, and fluorescent light from the cells gives rise to electrical signals. The stream is broken into a series of uniform size drops downstream of the laser. The cell signals are used to give appropriate electrostatic charges to drops containing the cells. The drops then pass between two charged plates and are deflected to appropriate containers. The system has proved capable of providing fractions containing large numbers of viable cells highly enriched in a particular functional type.

  16. Bacterial activation of mast cells.

    PubMed

    Chi, David S; Walker, Elaine S; Hossler, Fred E; Krishnaswamy, Guha

    2006-01-01

    Mast cells often are found in a perivascular location but especially in mucosae, where they may response to various stimuli. They typically associate with immediate hypersensitive responses and are likely to play a critical role in host defense. In this chapter, a common airway pathogen, Moraxella catarrhalis, and a commensal bacterium, Neiserria cinerea, are used to illustrate activation of human mast cells. A human mast cell line (HMC-1) derived from a patient with mast cell leukemia was activated with varying concentrations of heat-killed bacteria. Active aggregation of bacteria over mast cell surfaces was detected by scanning electron microscopy. The activation of mast cells was analyzed by nuclear factor-kappaB (NF-kappaB) activation and cytokine production in culture supernatants. Both M. catarrhalis and N. cinerea induce mast cell activation and the secretion of two key inflammatory cytokines, interleukin-6 and MCP-1. This is accompanied by NF-kappaB activation. Direct bacterial contact with mast cells appears to be essential for this activation because neither cell-free bacterial supernatants nor bacterial lipopolysaccharide induce cytokine secretion.

  17. Protective Role of Interleukin-17 in Murine NKT Cell-Driven Acute Experimental Hepatitis

    PubMed Central

    Wondimu, Zenebech; Santodomingo-Garzon, Tania; Le, Tai; Swain, Mark G.

    2010-01-01

    NKT cells are highly enriched within the liver. On activation NKT cells rapidly release large quantities of different cytokines which subsequently activate, recruit, or modulate cells important for the development of hepatic inflammation. Recently, it has been demonstrated that NKT cells can also produce interleukin-17 (IL-17), a proinflammatory cytokine that is also known to have diverse immunoregulatory effects. The role played by IL-17 in hepatic inflammation is unclear. Here we show that during α-galactosylceramide (αGalCer)-induced hepatitis in mice, a model of hepatitis driven by specific activation of the innate immune system via NKT cells within the liver, NK1.1+ and CD4+ iNKT cells rapidly produce IL-17 and are the main IL-17-producing cells within the liver. Administration of IL-17 neutralizing monoclonal antibodies before αGalCer injection significantly exacerbated hepatitis, in association with a significant increase in hepatic neutrophil and proinflammatory monocyte (ie, producing IL-12, tumor necrosis factor-α) recruitment, and increased hepatic mRNA and protein expression for the relevant neutrophil and monocyte chemokines CXCL5/LIX and CCL2/MCP-1, respectively. In contrast, administration of exogenous recombinant murine IL-17 before α-GalCer injection ameliorated hepatitis and inhibited the recruitment of inflammatory monocytes into the liver. Our results demonstrate that hepatic iNKT cells specifically activated with α-GalCer rapidly produce IL-17, and IL-17 produced after α-GalCer administration inhibits the development of hepatitis. PMID:20847291

  18. Centriole polarisation to the immunological synapse directs secretion from cytolytic cells of both the innate and adaptive immune systems

    PubMed Central

    2011-01-01

    Background Cytolytic cells of the immune system destroy pathogen-infected cells by polarised exocytosis of secretory lysosomes containing the pore-forming protein perforin. Precise delivery of this lethal hit is essential to ensuring that only the target cell is destroyed. In cytotoxic T lymphocytes (CTLs), this is accomplished by an unusual movement of the centrosome to contact the plasma membrane at the centre of the immunological synapse formed between killer and target cells. Secretory lysosomes are directed towards the centrosome along microtubules and delivered precisely to the point of target cell recognition within the immunological synapse, identified by the centrosome. We asked whether this mechanism of directing secretory lysosome release is unique to CTL or whether natural killer (NK) and invariant NKT (iNKT) cytolytic cells of the innate immune system use a similar mechanism to focus perforin-bearing lysosome release. Results NK cells were conjugated with B-cell targets lacking major histocompatibility complex class I 721.221 cells, and iNKT cells were conjugated with glycolipid-pulsed CD1-bearing targets, then prepared for thin-section electron microscopy. High-resolution electron micrographs of the immunological synapse formed between NK and iNKT cytolytic cells with their targets revealed that in both NK and iNKT cells, the centrioles could be found associated (or 'docked') with the plasma membrane within the immunological synapse. Secretory clefts were visible within the synapses formed by both NK and iNKT cells, and secretory lysosomes were polarised along microtubules leading towards the docked centrosome. The Golgi apparatus and recycling endosomes were also polarised towards the centrosome at the plasma membrane within the synapse. Conclusions These results reveal that, like CTLs of the adaptive immune system, the centrosomes of NK and iNKT cells (cytolytic cells of the innate immune system) direct secretory lysosomes to the immunological

  19. Immunotherapy and mast cell activation.

    PubMed

    Carlos, A G; Carlos, M L; Santos, M A; Pedro, E; Santos, S; Lopes-Pregal, A

    1998-10-01

    Tryptase is the more specific markers for mast cell activation and mediators release and can be used as an index of mast cell activation after challenge. Nasal provocation tests have been done in patients allergic to the pollen of Parietaria (pellitory wall) before and after specific systemic immunotherapy and tryptase release evaluated in nasal lavage fluid. After specific immunotherapy the concentration of tryptase in nasal lavage was significantly decreased to all the concentrations used in challenge and the peack of tryptase release was delayed. These results confirm that assays of tryptase in nasal fluid after nasal provocation are a reliable markers of mast cell activation. Immunotherapy with specific allergen decreases mast cell reactivity to the same allergen.

  20. ATP Binding Cassette Transporter ABCA7 Regulates NKT Cell Development and Function by Controlling CD1d Expression and Lipid Raft Content

    PubMed Central

    Nowyhed, Heba N.; Chandra, Shilpi; Kiosses, William; Marcovecchio, Paola; Andary, Farah; Zhao, Meng; Fitzgerald, Michael L.; Kronenberg, Mitchell; Hedrick, Catherine C.

    2017-01-01

    ABCA7 is an ABC transporter expressed on the plasma membrane, and actively exports phospholipid complexes from the cytoplasmic to the exocytoplasmic leaflet of membranes. Invariant NKT (iNKT) cells are a subpopulation of T lymphocytes that recognize glycolipid antigens in the context of CD1d-mediated antigen presentation. In this study, we demonstrate that ABCA7 regulates the development of NKT cells in a cell-extrinsic manner. We found that in Abca7−/− mice there is reduced expression of CD1d accompanied by an alteration in lipid raft content on the plasma membrane of thymocytes and antigen presenting cells. Together, these alterations caused by absence of ABCA7 negatively affect NKT cell development and function. PMID:28091533

  1. Helicobacter pylori Cholesteryl α-Glucosides Contribute to Its Pathogenicity and Immune Response by Natural Killer T Cells

    PubMed Central

    Ito, Yuki; Vela, Jose Luis; Matsumura, Fumiko; Hoshino, Hitomi; Tyznik, Aaron; Lee, Heeseob; Girardi, Enrico; Zajonc, Dirk M.; Liddington, Robert; Kobayashi, Motohiro; Bao, Xingfeng; Bugaytsova, Jeanna; Borén, Thomas; Jin, Rongsheng; Zong, Yinong; Seeberger, Peter H.; Nakayama, Jun; Kronenberg, Mitchell; Fukuda, Minoru

    2013-01-01

    Approximately 10–15% of individuals infected with Helicobacter pylori will develop ulcer disease (gastric or duodenal ulcer), while most people infected with H. pylori will be asymptomatic. The majority of infected individuals remain asymptomatic partly due to the inhibition of synthesis of cholesteryl α-glucosides in H. pylori cell wall by α1,4-GlcNAc-capped mucin O-glycans, which are expressed in the deeper portion of gastric mucosa. However, it has not been determined how cholesteryl α-glucosyltransferase (αCgT), which forms cholesteryl α-glucosides, functions in the pathogenesis of H. pylori infection. Here, we show that the activity of αCgT from H. pylori clinical isolates is highly correlated with the degree of gastric atrophy. We investigated the role of cholesteryl α-glucosides in various aspects of the immune response. Phagocytosis and activation of dendritic cells were observed at similar degrees in the presence of wild-type H. pylori or variants harboring mutant forms of αCgT showing a range of enzymatic activity. However, cholesteryl α-glucosides were recognized by invariant natural killer T (iNKT) cells, eliciting an immune response in vitro and in vivo. Following inoculation of H. pylori harboring highly active αCgT into iNKT cell-deficient (Jα18−/−) or wild-type mice, bacterial recovery significantly increased in Jα18−/− compared to wild-type mice. Moreover, cytokine production characteristic of Th1 and Th2 cells dramatically decreased in Jα18−/− compared to wild-type mice. These findings demonstrate that cholesteryl α-glucosides play critical roles in H. pylori-mediated gastric inflammation and precancerous atrophic gastritis. PMID:24312443

  2. Circulating CD56dim natural killer cells and CD56+ T cells that produce interferon-γ or interleukin-10 are expanded in asymptomatic, E antigen-negative patients with persistent hepatitis B virus infection.

    PubMed

    Conroy, M J; Mac Nicholas, R; Grealy, R; Taylor, M; Otegbayo, J A; O'Dea, S; Mulcahy, F; Ryan, T; Norris, S; Doherty, D G

    2015-03-01

    Infection with hepatitis B virus (HBV) can result in spontaneous resolution or chronic infection, which can remain asymptomatic or can progress to cirrhosis and/or hepatocellular carcinoma. The host immune response is thought to be a major determinant of the outcome of HBV infection and virus-specific cytotoxic T lymphocytes (CTL) can mediate immunity against the virus and cause liver pathology. Antigen-nonspecific innate lymphocytes may also contribute to HBV infection and liver disease, therefore, we examined the frequencies, phenotypes, cytolytic activities and cytokine profiles of circulating natural killer (NK) cells, CD1d-restricted invariant natural killer T (iNKT) cells and CD56(+) T cells in 102 asymptomatic HBV-infected patients and compared them with those in 66 uninfected control subjects. NK cells expressing low levels of CD56 (CD56(dim)) and CD56(+) T cells were significantly expanded in the circulation of HBV-infected patients compared with control subjects. CD1d expression and iNKT cell frequencies were similar in both groups. Despite these expansions, we did not detect augmented natural or cytokine-induced cytotoxicity in the HBV-infected subjects. All lymphocyte populations studied produced interferon-γ (IFN-γ) significantly more frequently when taken from HBV-infected patients compared with when taken from healthy controls. Additionally, NK cells from the patients more frequently produced interleukin-10. As our HBV-infected cohort consisted of asymptomatic patients with low viral loads, we propose that CD56(dim) NK cells and CD56(+) T cells control HBV infection by noncytolytic mechanisms.

  3. The Epstein-Barr Virus Glycoprotein gp150 Forms an Immune-Evasive Glycan Shield at the Surface of Infected Cells

    PubMed Central

    Gram, Anna M.; Oosenbrug, Timo; Lindenbergh, Marthe F. S.; Büll, Christian; Comvalius, Anouskha; Dickson, Kathryn J. I.; Wiegant, Joop; Vrolijk, Hans; Lebbink, Robert Jan; Wolterbeek, Ron; Adema, Gosse J.; Griffioen, Marieke; Heemskerk, Mirjam H. M.; Tscharke, David C.; Hutt-Fletcher, Lindsey M.; Ressing, Maaike E.

    2016-01-01

    Cell-mediated immunity plays a key role in host control of viral infection. This is exemplified by life-threatening reactivations of e.g. herpesviruses in individuals with impaired T-cell and/or iNKT cell responses. To allow lifelong persistence and virus production in the face of primed immunity, herpesviruses exploit immune evasion strategies. These include a reduction in viral antigen expression during latency and a number of escape mechanisms that target antigen presentation pathways. Given the plethora of foreign antigens expressed in virus-producing cells, herpesviruses are conceivably most vulnerable to elimination by cell-mediated immunity during the replicative phase of infection. Here, we show that a prototypic herpesvirus, Epstein-Barr virus (EBV), encodes a novel, broadly acting immunoevasin, gp150, that is expressed during the late phase of viral replication. In particular, EBV gp150 inhibits antigen presentation by HLA class I, HLA class II, and the non-classical, lipid-presenting CD1d molecules. The mechanism of gp150-mediated T-cell escape does not depend on degradation of the antigen-presenting molecules nor does it require gp150’s cytoplasmic tail. Through its abundant glycosylation, gp150 creates a shield that impedes surface presentation of antigen. This is an unprecedented immune evasion mechanism for herpesviruses. In view of its likely broader target range, gp150 could additionally have an impact beyond escape of T cell activation. Importantly, B cells infected with a gp150-null mutant EBV displayed rescued levels of surface antigen presentation by HLA class I, HLA class II, and CD1d, supporting an important role for iNKT cells next to classical T cells in fighting EBV infection. At the same time, our results indicate that EBV gp150 prolongs the timespan for producing viral offspring at the most vulnerable stage of the viral life cycle. PMID:27077376

  4. Viral Evasion of Natural Killer Cell Activation.

    PubMed

    Ma, Yi; Li, Xiaojuan; Kuang, Ersheng

    2016-04-12

    Natural killer (NK) cells play a key role in antiviral innate defenses because of their abilities to kill infected cells and secrete regulatory cytokines. Additionally, NK cells exhibit adaptive memory-like antigen-specific responses, which represent a novel antiviral NK cell defense mechanism. Viruses have evolved various strategies to evade the recognition and destruction by NK cells through the downregulation of the NK cell activating receptors. Here, we review the recent findings on viral evasion of NK cells via the impairment of NK cell-activating receptors and ligands, which provide new insights on the relationship between NK cells and viral actions during persistent viral infections.

  5. Roles of 3′ and 4′ Hydroxyls in α-Galactosylceramide Stimulation of Invariant Natural Killer T Cells

    PubMed Central

    Zhang, Wenpeng; Zhang, Yalong; Chen, Wenlan; Nadas, Janos; Severin, Ryan; Woodward, Robert; Wang, Bin; Wang, Xin; Kronenberg, Mitchell

    2012-01-01

    The marine-derived α-galactosylceramide is an exogenous ligand for natural killer T cells and leads to the secretion of both T help 1 (Th1) and Th2 cytokines. The relationship between the sugar moiety structure and invariant natural killer T (iNKT) cell stimulation ability has not been fully understood. With the series α-galactosylceramide analogues varied on C3′ and C4′ position, subjected to a murine system, we discovered that the 3′ hydroxyl is very crucial in maintaining the molecule’s immunogenicity. Any modification on this position will lead to the losing of activity. We also found that the C4′ position is not so sensitive and can tolerate some small modifications on it. Moreover, the C4′ substituted analogues induced biased Th2 cytokines release was observed. PMID:19780098

  6. Mast cells enhance T cell activation: Importance of mast cell-derived TNF

    NASA Astrophysics Data System (ADS)

    Nakae, Susumu; Suto, Hajime; Kakurai, Maki; Sedgwick, Jonathon D.; Tsai, Mindy; Galli, Stephen J.

    2005-05-01

    Mast cells are not only important effector cells in immediate hypersensitivity reactions and immune responses to pathogens but also can contribute to T cell-mediated disorders. However, the mechanisms by which mast cells might influence T cells in such settings are not fully understood. We find that mast cells can enhance proliferation and cytokine production in multiple T cell subsets. Mast cell-dependent enhancement of T cell activation can be promoted by FcRI-dependent mast cell activation, TNF production by both mast cells and T cells, and mast cell-T cell contact. However, at high concentrations of cells, mast cells can promote T cell activation independent of IgE or TNF. Finally, mast cells also can promote T cell activation by means of soluble factors. These findings identify multiple mechanisms by which mast cells can influence T cell proliferation and cytokine production. allergy | asthma | autoimmunity | cytokines | immune response

  7. Evolution of nonclassical MHC-dependent invariant T cells.

    PubMed

    Edholm, Eva-Stina; Grayfer, Leon; Robert, Jacques

    2014-12-01

    TCR-mediated specific recognition of antigenic peptides in the context of classical MHC molecules is a cornerstone of adaptive immunity of jawed vertebrate. Ancillary to these interactions, the T cell repertoire also includes unconventional T cells that recognize endogenous and/or exogenous antigens in a classical MHC-unrestricted manner. Among these, the mammalian nonclassical MHC class I-restricted invariant T cell (iT) subsets, such as iNKT and MAIT cells, are now believed to be integral to immune response initiation as well as in orchestrating subsequent adaptive immunity. Until recently the evolutionary origins of these cells were unknown. Here we review our current understanding of a nonclassical MHC class I-restricted iT cell population in the amphibian Xenopus laevis. Parallels with the mammalian iNKT and MAIT cells underline the crucial biological roles of these evolutionarily ancient immune subsets.

  8. Evolution of nonclassical MHC-dependent invariant T cells

    PubMed Central

    Edholm, Eva-Stina; Grayfer, Leon; Robert, Jacques

    2014-01-01

    TCR-mediated specific recognition of antigenic peptides in the context of classical MHC molecules is a cornerstone of adaptive immunity of jawed vertebrate. Ancillary to these interactions, the T cell repertoire also includes unconventional T cells that recognize endogenous and/or exogenous antigens in a classical MHC-unrestricted manner. Among these, the mammalian nonclassical MHC class I-restricted invariant T cell (iT) subsets, such as iNKT and MAIT cells, are now believed to be integral to immune response initiation as well as in orchestrating subsequent adaptive immunity. Until recently the evolutionary origins of these cells were unknown. Here we review our current understanding of a nonclassical MHC class I-restricted iT cell population in the amphibian Xenopus laevis. Parallels with the mammalian iNKT and MAIT cells underline the crucial biological roles of these evolutionarily ancient immune subsets. PMID:25117267

  9. Immunoregulation of NKT Cells in Systemic Lupus Erythematosus

    PubMed Central

    Chen, Junwei; Wu, Meng; Wang, Jing; Li, Xiaofeng

    2015-01-01

    Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease with different variety of clinical manifestations. Natural killer T (NKT) cells are innate lymphocytes that play a regulatory role during broad range of immune responses. A number of studies demonstrated that the quantity and quality of invariant NKT (iNKT) cells showed marked defects in SLE patients in comparison to healthy controls. This finding suggests that iNKT cells may play a regulatory role in the occurrence and development of this disease. In this review, we mainly summarized the most recent findings about the behavior of NKT cells in SLE patients and mouse models, as well as how NKT cells affect the proportion of T helper cells and the production of autoreactive antibodies in the progress of SLE. This will help people better understand the role of NKT cells in the development of SLE and improve the therapy strategy. PMID:26819956

  10. Myosin II Activity Softens Cells in Suspension

    PubMed Central

    Chan, Chii J.; Ekpenyong, Andrew E.; Golfier, Stefan; Li, Wenhong; Chalut, Kevin J.; Otto, Oliver; Elgeti, Jens; Guck, Jochen; Lautenschläger, Franziska

    2015-01-01

    The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells. PMID:25902426

  11. Active cell mechanics: Measurement and theory.

    PubMed

    Ahmed, Wylie W; Fodor, Étienne; Betz, Timo

    2015-11-01

    Living cells are active mechanical systems that are able to generate forces. Their structure and shape are primarily determined by biopolymer filaments and molecular motors that form the cytoskeleton. Active force generation requires constant consumption of energy to maintain the nonequilibrium activity to drive organization and transport processes necessary for their function. To understand this activity it is necessary to develop new approaches to probe the underlying physical processes. Active cell mechanics incorporates active molecular-scale force generation into the traditional framework of mechanics of materials. This review highlights recent experimental and theoretical developments towards understanding active cell mechanics. We focus primarily on intracellular mechanical measurements and theoretical advances utilizing the Langevin framework. These developing approaches allow a quantitative understanding of nonequilibrium mechanical activity in living cells. This article is part of a Special Issue entitled: Mechanobiology.

  12. Choreography of MAGUKs during T cell activation.

    PubMed

    Rincón, Mercedes; Davis, Roger J

    2007-02-01

    T cell receptor activation requires the membrane-associated guanylate kinase CARMA1. A new study finds that a second such kinase, Dlgh1, is also required specifically for activation of the alternative p38 kinase pathway.

  13. Measurement of myeloid cell immune suppressive activity.

    PubMed

    Dolcetti, Luigi; Peranzoni, Elisa; Bronte, Vincenzo

    2010-11-01

    This unit presents simple methods to assess the immunosuppressive properties of immunoregulatory cells of myeloid origin, such as myeloid-derived suppressor cells (MDSCs), both in vitro and in vivo. These methods are general and could be adapted to test the impact of different suppressive populations on T cell activation, proliferation, and cytotoxic activity; moreover they could be useful to assess the influence exerted on immune suppressive pathways by genetic modifications, chemical inhibitors, and drugs.

  14. Function and expression of CD1d and invariant natural killer T-cell receptor in the cotton rat (Sigmodon hispidus).

    PubMed

    Fichtner, Alina Suzann; Paletta, Daniel; Starick, Lisa; Schumann, Richard F; Niewiesk, Stefan; Herrmann, Thomas

    2015-12-01

    The cotton rat (Sigmodon hispidus) belongs to the rodent family of Cricetidae and provides a powerful model to study the pathogenesis of human respiratory viruses and measles virus. Recent studies in other rodent models have suggested a role for invariant natural killer T (iNKT) cells in antiviral immunity and vaccination against respiratory virus infections. Using new experimental tools, we provide the first evidence for a functional CD1d cell molecule (crCD1d) and iNKT T-cell receptor in cotton rats. The crCD1d cDNA sequence was identified and crCD1d transductants showed that monoclonal antibody WTH-2 stains crCD1d as efficiently as mouse or rat CD1d. The expression of crCD1d was clearly weaker for thymocytes and B cells, and higher for T cells, which is different to what is found in murine species. The antigen-presenting capacity of crCD1d was demonstrated with crCD1d-immunoglobulin dimers loaded with the glycolipid PBS57, which bound iNKT T-cell receptors. Evidence for functional cotton rat iNKT cells was provided by detection of interferon-γ and interleukin-4 in cultures of splenocytes stimulated with PBS57 and α-galactosylceramide and by specific staining of about 0·2% of splenocytes with PBS57-loaded crCD1d dimers. Canonical AV14/AJ18 rearrangements were identified and found to contain multiple members of the AV14 (AV11) family. One of them was expressed and found to bind CD1d dimers. In summary, these data provide the first evidence for functional CD1d molecules and iNKT T-cell receptors in cotton rats and provide the tools to analyse them both in the cotton rat model of infectious diseases.

  15. Active gel model of amoeboid cell motility

    NASA Astrophysics Data System (ADS)

    Callan-Jones, A. C.; Voituriez, R.

    2013-02-01

    We develop a model of amoeboid cell motility based on active gel theory. Modeling the motile apparatus of a eukaryotic cell as a confined layer of finite length of poroelastic active gel permeated by a solvent, we first show that, due to active stress and gel turnover, an initially static and homogeneous layer can undergo a contractile-type instability to a polarized moving state in which the rear is enriched in gel polymer. This agrees qualitatively with motile cells containing an actomyosin-rich uropod at their rear. We find that the gel layer settles into a steadily moving, inhomogeneous state at long times, sustained by a balance between contractility and filament turnover. In addition, our model predicts an optimal value of the gel-substrate adhesion leading to maximum layer speed, in agreement with cell motility assays. The model may be relevant to motility of cells translocating in complex, confining environments that can be mimicked experimentally by cell migration through microchannels.

  16. Receptor Dissociation and B-Cell Activation.

    PubMed

    Yang, Jianying; Reth, Michael

    2016-01-01

    The B-cell antigen receptor (BCR) is one of the most abundant receptors on the surface of B cells with roughly 100,000-200,000 copies per cell. Signaling through the BCR is crucial for the activation and differentiation of B cells. Unlike other receptors, the BCR can be activated by a large set of structurally different ligands, but the molecular mechanism of BCR activation is still a matter of controversy. Although dominant for a long time, the cross-link model (CLM) of BCR activation is not supported by recent studies of the nanoscale organization of the BCR on the surface of resting B cells. In contrast to the prediction of CLM, the numerous BCR complexes on these cells are not randomly distributed monomers but rather form oligomers which reside within membrane confinements. This finding is more in line with the dissociation activation model (DAM), wherein B-cell activation is accompanied by an opening of the auto-inhibited BCR oligomers instead of a cross-linking of the BCR monomers. In this review, we discuss in detail the new findings and their implications for BCR signaling.

  17. T cell activation requires force generation

    PubMed Central

    Hu, Kenneth H.

    2016-01-01

    Triggering of the T cell receptor (TCR) integrates both binding kinetics and mechanical forces. To understand the contribution of the T cell cytoskeleton to these forces, we triggered T cells using a novel application of atomic force microscopy (AFM). We presented antigenic stimulation using the AFM cantilever while simultaneously imaging with optical microscopy and measuring forces on the cantilever. T cells respond forcefully to antigen after calcium flux. All forces and calcium responses were abrogated upon treatment with an F-actin inhibitor. When we emulated the forces of the T cell using the AFM cantilever, even these actin-inhibited T cells became activated. Purely mechanical stimulation was not sufficient; the exogenous forces had to couple through the TCR. These studies suggest a mechanical–chemical feedback loop in which TCR-triggered T cells generate forceful contacts with antigen-presenting cells to improve access to antigen. PMID:27241914

  18. [Hydrogen ion activity in the cell].

    PubMed

    Sorokin, Z A

    1976-07-01

    Literature data and results of our experiments evidence for a heterogenous hydrogen distribution in cells. Intracellular pH should be regarded as a mean activity of hydrogen ions which is the sum of activities in different phases of a cell. Intracellular pH value does not depend on the transmembrane action potential difference, and is resistant to respiratory and metabolic disorders of acid-base equilibrium in the body. It also slightly changes with changing the electrolyte composition and pH of the medium and is not influenced by metabolic inhibitors. A low hydrogen activity in the cell has a certain functional significance. The pH stability is ensured by a number of regulatory mechanism: the buffer properties of the protoplasm itself, and the active hydrogen transport into the medium. Hydrogen released from cells is supposed to be connected with functioning of a specific respiratory chain of superficial protoplasmic membranes.

  19. Kinetic discrimination in T-cell activation.

    PubMed Central

    Rabinowitz, J D; Beeson, C; Lyons, D S; Davis, M M; McConnell, H M

    1996-01-01

    We propose a quantitative model for T-cell activation in which the rate of dissociation of ligand from T-cell receptors determines the agonist and antagonist properties of the ligand. The ligands are molecular complexes between antigenic peptides and proteins of the major histocompatibility complex on the surfaces of antigen-presenting cells. Binding of ligand to receptor triggers a series of biochemical reactions in the T cell. If the ligand dissociates after these reactions are complete, the T cell receives a positive activation signal. However, dissociation of ligand after completion of the first reaction but prior to generation of the final products results in partial T-cell activation, which acts to suppress a positive response. Such a negative signal is brought about by T-cell ligands containing the variants of antigenic peptides referred to as T-cell receptor antagonists. Results of recent experiments with altered peptide ligands compare favorably with T-cell responses predicted by this model. PMID:8643643

  20. Activated Membrane Patches Guide Chemotactic Cell Motility

    PubMed Central

    Hecht, Inbal; Skoge, Monica L.; Charest, Pascale G.; Ben-Jacob, Eshel; Firtel, Richard A.; Loomis, William F.; Levine, Herbert; Rappel, Wouter-Jan

    2011-01-01

    Many eukaryotic cells are able to crawl on surfaces and guide their motility based on environmental cues. These cues are interpreted by signaling systems which couple to cell mechanics; indeed membrane protrusions in crawling cells are often accompanied by activated membrane patches, which are localized areas of increased concentration of one or more signaling components. To determine how these patches are related to cell motion, we examine the spatial localization of RasGTP in chemotaxing Dictyostelium discoideum cells under conditions where the vertical extent of the cell was restricted. Quantitative analyses of the data reveal a high degree of spatial correlation between patches of activated Ras and membrane protrusions. Based on these findings, we formulate a model for amoeboid cell motion that consists of two coupled modules. The first module utilizes a recently developed two-component reaction diffusion model that generates transient and localized areas of elevated concentration of one of the components along the membrane. The activated patches determine the location of membrane protrusions (and overall cell motion) that are computed in the second module, which also takes into account the cortical tension and the availability of protrusion resources. We show that our model is able to produce realistic amoeboid-like motion and that our numerical results are consistent with experimentally observed pseudopod dynamics. Specifically, we show that the commonly observed splitting of pseudopods can result directly from the dynamics of the signaling patches. PMID:21738453

  1. Bursts of activity in collective cell migration

    PubMed Central

    Chepizhko, Oleksandr; Giampietro, Costanza; Mastrapasqua, Eleonora; Nourazar, Mehdi; Ascagni, Miriam; Sugni, Michela; Fascio, Umberto; Leggio, Livio; Malinverno, Chiara; Scita, Giorgio; Santucci, Stéphane; Alava, Mikko J.; Zapperi, Stefano; La Porta, Caterina A. M.

    2016-01-01

    Dense monolayers of living cells display intriguing relaxation dynamics, reminiscent of soft and glassy materials close to the jamming transition, and migrate collectively when space is available, as in wound healing or in cancer invasion. Here we show that collective cell migration occurs in bursts that are similar to those recorded in the propagation of cracks, fluid fronts in porous media, and ferromagnetic domain walls. In analogy with these systems, the distribution of activity bursts displays scaling laws that are universal in different cell types and for cells moving on different substrates. The main features of the invasion dynamics are quantitatively captured by a model of interacting active particles moving in a disordered landscape. Our results illustrate that collective motion of living cells is analogous to the corresponding dynamics in driven, but inanimate, systems. PMID:27681632

  2. Mechanisms of Invariant Natural Killer T Cell-Mediated Immunoregulation in Cancer

    DTIC Science & Technology

    2011-05-01

    immunoregulatory phenotype. Specifically, three non-mutually exclusive hypothesis will be tested –1) that a lipid antigen derived from 4T1 tumor cells can be...made significant progress in demonstrating that lipid antigen/s derived from 4T1 tumors can differentially modulate the maturational markers in...4 INTRODUCTION Invariant natural killer T (iNKT) cells comprise a unique group of immune cells that specifically recognize lipid antigens

  3. Metabolic activity is necessary for activation of T suppressor cells by B cells

    SciTech Connect

    Elkins, K.L.; Stashak, P.W.; Baker, P.J. )

    1990-04-15

    Ag-primed B cells must express cell-surface IgM, but not IgD or Ia Ag, and must remain metabolically active, in order to activate suppressor T cells (Ts) specific for type III pneumococcal polysaccharide. Ag-primed B cells that were gamma-irradiated with 1000r, or less, retained the ability to activate Ts; however, Ag-primed B cells exposed to UV light were not able to do so. gamma-Irradiated and UV-treated Ag-primed B cells both expressed comparable levels of cell-surface IgM, and both localized to the spleen after in vivo transfer; neither could proliferate in vitro in response to mitogens. By contrast, gamma-irradiated primed B cells were still able to synthesize proteins, whereas UV-treated primed B cells could not. These findings suggest that in order for Ag-primed B cells to activate Ts, they must (a) express cell-associated IgM (sIgM) antibody bearing the idiotypic determinants of antibody specific for type III pneumococcal polysaccharide, and (b) be able to synthesize protein for either the continued expression of sIgM after cell transfer, or for the elaboration of another protein molecule that is also required for the activation of Ts; this molecule does not appear to be Ia Ag.

  4. Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells

    SciTech Connect

    Parekkadan, Biju; Poll, Daan van; Megeed, Zaki; Kobayashi, Naoya; Tilles, Arno W.; Berthiaume, Francois; Yarmush, Martin L.

    2007-11-16

    Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to prevent the development of liver fibrosis in a number of pre-clinical studies. Marked changes in liver histopathology and serological markers of liver function have been observed without a clear understanding of the therapeutic mechanism by which stem cells act. We sought to determine if MSCs could modulate the activity of resident liver cells, specifically hepatic stellate cells (SCs) by paracrine mechanisms using indirect cocultures. Indirect coculture of MSCs and activated SCs led to a significant decrease in collagen deposition and proliferation, while inducing apoptosis of activated SCs. The molecular mechanisms underlying the modulation of SC activity by MSCs were examined. IL-6 secretion from activated SCs induced IL-10 secretion from MSCs, suggesting a dynamic response of MSCs to the SCs in the microenvironment. Blockade of MSC-derived IL-10 and TNF-{alpha} abolished the inhibitory effects of MSCs on SC proliferation and collagen synthesis. In addition, release of HGF by MSCs was responsible for the marked induction of apoptosis in SCs as determined by antibody-neutralization studies. These findings demonstrate that MSCs can modulate the function of activated SCs via paracrine mechanisms provide a plausible explanation for the protective role of MSCs in liver inflammation and fibrosis, which may also be relevant to other models of tissue fibrosis.

  5. (+)-Catechin attenuates activation of hepatic stellate cells.

    PubMed

    Bragança de Moraes, Cristina Machado; Bitencourt, Shanna; de Mesquita, Fernanda Cristina; Mello, Denizar; de Oliveira, Leticia Paranhos; da Silva, Gabriela Viegas; Lorini, Vinicius; Caberlon, Eduardo; de Souza Basso, Bruno; Schmid, Julia; Ferreira, Gabriela Acevedo; de Oliveira, Jarbas Rodrigues

    2014-04-01

    (+)-Catechin is a type of catechin present in large amounts in açaí fruits and cocoa seeds. Besides its antioxidant and anti-inflammatory activities, little is known about its effects in the liver, especially during hepatic fibrosis. We report here the effects of (+)-catechin on hepatic stellate cells. (+)-Catechin induced quiescent phenotype in GRX cells, along with an increase in lipid droplets. Proliferator-activated receptor γ mRNA expression was upregulated, whereas type I collagen mRNA expression was downregulated. Pro-inflammatory cytokines were not influenced by (+)-catechin, whereas the levels of interleukin 10 were significantly increased. The data provide evidence that (+)-catechin can reduce hepatic stellate cell activation.

  6. Entangled active matter: From cells to ants

    NASA Astrophysics Data System (ADS)

    Hu, D. L.; Phonekeo, S.; Altshuler, E.; Brochard-Wyart, F.

    2016-07-01

    Both cells and ants belong to the broad field of active matter, a novel class of non-equilibrium materials composed of many interacting units that individually consume energy and collectively generate motion or mechanical stresses. However cells and ants differ from fish and birds in that they can support static loads. This is because cells and ants can be entangled, so that individual units are bound by transient links. Entanglement gives cells and ants a set of remarkable properties usually not found together, such as the ability to flow like a fluid, spring back like an elastic solid, and self-heal. In this review, we present the biology, mechanics and dynamics of both entangled cells and ants. We apply concepts from soft matter physics and wetting to characterize these systems as well as to point out their differences, which arise from their differences in size. We hope that our viewpoints will spur further investigations into cells and ants as active materials, and inspire the fabrication of synthetic active matter.

  7. Critical telomerase activity for uncontrolled cell growth

    NASA Astrophysics Data System (ADS)

    Wesch, Neil L.; Burlock, Laura J.; Gooding, Robert J.

    2016-08-01

    The lengths of the telomere regions of chromosomes in a population of cells are modelled using a chemical master equation formalism, from which the evolution of the average number of cells of each telomere length is extracted. In particular, the role of the telomere-elongating enzyme telomerase on these dynamics is investigated. We show that for biologically relevant rates of cell birth and death, one finds a critical rate, R crit, of telomerase activity such that the total number of cells diverges. Further, R crit is similar in magnitude to the rates of mitosis and cell death. The possible relationship of this result to replicative immortality and its associated hallmark of cancer is discussed.

  8. CD8+NKT-like cells regulate the immune response by killing antigen-bearing DCs

    PubMed Central

    Wang, Chao; Liu, Xi; Li, Zhengyuan; Chai, Yijie; Jiang, Yunfeng; Wang, Qian; Ji, Yewei; Zhu, Zhongli; Wan, Ying; Yuan, Zhenglong; Chang, Zhijie; Zhang, Minghui

    2015-01-01

    CD1d-dependent NKT cells have been extensively studied; however, the function of CD8+NKT-like cells, which are CD1d-independent T cells with NK markers, remains unknown. Here, we report that CD1d-independent CD8+NKT-like cells, which express both T cell markers (TCRβ and CD3) and NK cell receptors (NK1.1, CD49b and NKG2D), are activated and significantly expanded in mice immunized with GFP-expressing dendritic cells. Distinct from CD1d-dependent NKT cells, CD8+NKT-like cells possess a diverse repertoire of TCRs and secrete high levels of IFN-gamma but not IL-4. CD8+NKT-like cell development is normal in CD1d−/− mice, which suggests that CD8+NKT-like cells undergo a unique development pathway that differs from iNKT cells. Further functional analyses show that CD8+NKT-like cells suppress T-cell responses through elimination of dendritic cells in an antigen-specific manner. Adoptive transfer of antigen-specific CD8+NKT-like cells into RIP-OVA mice prevented subsequent development of diabetes in the animals induced by activated OT-I CD8 T cells. Our study suggests that CD8+NKT-like cells can function as antigen-specific suppressive cells to regulate the immune response through killing antigen-bearing DCs. Antigen-specific down regulation may provide an active and precise method for constraining an excessive immune response and avoiding bypass suppression of necessary immune responses to other antigens. PMID:26369936

  9. Aging and defective lymphoid cell activation.

    PubMed

    Coffman, F D; Cohen, S

    1989-01-01

    Activation of lymphocytes for proliferation is a crucial process in the immune response. Age-related deficiencies in this cellular response strongly correlate with deficiencies in the immune system response, with concomitant increase in disease severity and mortality. Defects associated with the transmission of the initial activation signal and with IL-2 production contribute to the depressed response, but defects in the IL-2 response mechanism also play important roles. A major factor in this area is the inability of the nuclei of these cells to respond to the intracellular factor ADR, which plays a crucial role in the initiation of DNA replication. These cells produce normal levels of ADR; thus, either the nuclei cannot bind ADR in a productive manner or the defect lies beyond the point of ADR binding, perhaps in one of the other proteins of the initiation complex. An interesting contrast to the age-related failure of nuclei to respond to ADR is the failure of neoplastic nuclei to respond to the ADR inhibitor. This inhibitor, found in the cytoplasm of quiescent cells, suppresses both the activation of quiescent nuclei by ADR and the ongoing DNA synthesis in isolated nuclei from activated cells. Nuclei from spontaneous proliferating cell lines were not affected by this inhibitor, which may be an important factor in the uncontrolled growth seen in neoplastic cells. The investigation of ADR has given hints that perhaps two of the fundamental questions in biology, namely why some cells don't proliferate and why some others won't stop proliferating, may be two sides of the same coin.

  10. Mechanically activated artificial cell by using microfluidics

    PubMed Central

    Ho, Kenneth K. Y.; Lee, Lap Man; Liu, Allen P.

    2016-01-01

    All living organisms sense mechanical forces. Engineering mechanosensitive artificial cell through bottom-up in vitro reconstitution offers a way to understand how mixtures of macromolecules assemble and organize into a complex system that responds to forces. We use stable double emulsion droplets (aqueous/oil/aqueous) to prototype mechanosensitive artificial cells. In order to demonstrate mechanosensation in artificial cells, we develop a novel microfluidic device that is capable of trapping double emulsions into designated chambers, followed by compression and aspiration in a parallel manner. The microfluidic device is fabricated using multilayer soft lithography technology, and consists of a control layer and a deformable flow channel. Deflections of the PDMS membrane above the main microfluidic flow channels and trapping chamber array are independently regulated pneumatically by two sets of integrated microfluidic valves. We successfully compress and aspirate the double emulsions, which result in transient increase and permanent decrease in oil thickness, respectively. Finally, we demonstrate the influx of calcium ions as a response of our mechanically activated artificial cell through thinning of oil. The development of a microfluidic device to mechanically activate artificial cells creates new opportunities in force-activated synthetic biology. PMID:27610921

  11. Mechanically activated artificial cell by using microfluidics

    NASA Astrophysics Data System (ADS)

    Ho, Kenneth K. Y.; Lee, Lap Man; Liu, Allen P.

    2016-09-01

    All living organisms sense mechanical forces. Engineering mechanosensitive artificial cell through bottom-up in vitro reconstitution offers a way to understand how mixtures of macromolecules assemble and organize into a complex system that responds to forces. We use stable double emulsion droplets (aqueous/oil/aqueous) to prototype mechanosensitive artificial cells. In order to demonstrate mechanosensation in artificial cells, we develop a novel microfluidic device that is capable of trapping double emulsions into designated chambers, followed by compression and aspiration in a parallel manner. The microfluidic device is fabricated using multilayer soft lithography technology, and consists of a control layer and a deformable flow channel. Deflections of the PDMS membrane above the main microfluidic flow channels and trapping chamber array are independently regulated pneumatically by two sets of integrated microfluidic valves. We successfully compress and aspirate the double emulsions, which result in transient increase and permanent decrease in oil thickness, respectively. Finally, we demonstrate the influx of calcium ions as a response of our mechanically activated artificial cell through thinning of oil. The development of a microfluidic device to mechanically activate artificial cells creates new opportunities in force-activated synthetic biology.

  12. Parthenolide enhances dacarbazine activity against melanoma cells.

    PubMed

    Koprowska, Kamila; Hartman, Mariusz L; Sztiller-Sikorska, Malgorzata; Czyz, Malgorzata E

    2013-09-01

    Dacarbazine induces a clinical response only in 15% of melanoma patients. New treatment strategies may involve combinations of drugs with different modes of action to target the tumor heterogeneity. We aimed to determine whether the combined treatment of heterogeneous melanoma cell populations in vitro with the alkylating agent dacarbazine and the nuclear factor-κB inhibitor parthenolide could be more effective than either drug alone. A panel of melanoma cell lines, including highly heterogeneous populations derived from surgical specimens, was treated with dacarbazine and parthenolide. The effect of drugs on the viable cell number was examined using an acid phosphatase activity assay, and the combination effect was determined by median-effect analysis. Cell death and cell-cycle arrest were assessed by flow cytometry. Gene expression was measured by real-time PCR and changes in the protein levels were evaluated by western blotting. Secretion of vascular endothelial growth factor and interleukin-8 was determined using an enzyme-linked immunosorbent assay. The self-renewing capacity was assessed using a clonogenic assay. Dacarbazine was less effective in heterogeneous melanoma populations than in the A375 cell line. Parthenolide and dacarbazine synergistically reduced the viable cell numbers. Both drugs induced cell-cycle arrest and apoptotic cell death. Importantly, parthenolide abrogated the baseline and dacarbazine-induced vascular endothelial growth factor secretion from melanoma cells in heterogeneous populations, whereas interleukin-8 secretion was not significantly affected by either drug. Parthenolide eradicated melanoma cells with self-renewing capacity also in cultures simultaneously treated with dacarbazine. The combination of parthenolide and dacarbazine might be considered as a new therapeutic modality against metastatic melanoma.

  13. Glycolate kinase activity in human red cells.

    PubMed

    Fujii, S; Beutler, E

    1985-02-01

    Human red cells manifest glycolate kinase activity. This activity copurifies with pyruvate kinase and is decreased in the red cells of subjects with hereditary pyruvate kinase deficiency. Glycolate kinase activity was detected in the presence of FDP or glucose-1,6-P2. In the presence of 1 mmol/L FDP, the Km for adenosine triphosphate (ATP) was 0.28 mmol/L and a half maximum velocity for glycolate was obtained at 40 mmol/L. The pH optimum of the reaction was over 10.5 With 10 mumol/L FDP, 500 mumol/L glucose-1,6-P2, 2 mmol/L ATP, 5 mmol/L MgCl2, and 50 mmol/L glycolate at pH 7.5, glycolate kinase activity was calculated to be approximately 0.0013 U/mL RBC. In view of this low activity even in the presence of massive amounts of glycolate, the glycolate kinase reaction cannot account for the maintenance of the reported phosphoglycolate level in human red cells.

  14. Cell Cholesterol Homeostasis: Mediation by Active Cholesterol

    PubMed Central

    Steck, Theodore L.; Lange, Yvonne

    2010-01-01

    Recent evidence suggests that the major pathways mediating cell cholesterol homeostasis respond to a common signal: active membrane cholesterol. Active cholesterol is that fraction which exceeds the complexing capacity of the polar bilayer lipids. Increments in plasma membrane cholesterol exceeding this threshold have an elevated chemical activity (escape tendency) and redistribute via diverse transport proteins to both circulating plasma lipoproteins and intracellular organelles. Active cholesterol prompts several feedback responses thereby. It is the substrate for its own esterification and for the synthesis of regulatory side-chain oxysterols. It also stimulates manifold pathways that down-regulate the biosynthesis, curtail the ingestion and increase the export of cholesterol. Thus, the abundance of cholesterol is tightly coupled to that of its polar lipid partners through active cholesterol. PMID:20843692

  15. T cell immunoregulation in active ocular toxoplasmosis.

    PubMed

    Cordeiro, Cynthia A; Vieira, Erica L M; Castro, Vinicius M; Dutra, Walderez O; Costa, Rogerio A; Orefice, Juliana L; Campos, Wesley R; Orefice, Fernando; Young, Lucy H; Teixeira, Antonio Lucio

    2017-04-01

    Toxoplasma gondii infection is an important cause of infectious ocular disease. The physiopathology of retinochoroidal lesions associated with this infection is not completely understood. The present study was undertaken to investigate cytokine production by T cells from individuals with active toxoplasmic retinochoroiditis (TR) comparing with controls. Eighteen patients with active TR and 15 healthy controls (6 controls IgG(+) to Toxoplasma and 9 negative controls) were included in the study. Peripheral blood mononuclear cells were incubated in the presence or absence of T. gondii antigen (STAg), and stained against CD4, CD8, TNF, IL-10 and IFN-γ. Baseline expression of cytokines was higher in TR/IgG(+) patients in comparison with controls. Cytokine expression was not increased by STAg in vitro stimulation in controls. After stimulation, TR/IgG(+) patients' lymphocytes increased cytokine as compared to cultures from both controls. While T cells were the main source of IL-10, but also IFN-γ and TNF, other lymphocyte populations were relevant source of inflammatory cytokines. Interestingly, it was observed a negative correlation between ocular lesion size and IL-10 expression by CD4(+) lymphocytes. This study showed that T cells are the main lymphocyte populations expressing IL-10 in patients with TR. Moreover, expression of IL-10 plays a protective role in active TR.

  16. Design, synthesis, and functional activity of labeled CD1d glycolipid agonists.

    PubMed

    Jervis, Peter J; Polzella, Paolo; Wojno, Justyna; Jukes, John-Paul; Ghadbane, Hemza; Garcia Diaz, Yoel R; Besra, Gurdyal S; Cerundolo, Vincenzo; Cox, Liam R

    2013-04-17

    Invariant natural killer T cells (iNKT cells) are restricted by CD1d molecules and activated upon CD1d-mediated presentation of glycolipids to T cell receptors (TCRs) located on the surface of the cell. Because the cytokine response profile is governed by the structure of the glycolipid, we sought a method for labeling various glycolipids to study their in vivo behavior. The prototypical CD1d agonist, α-galactosyl ceramide (α-GalCer) 1, instigates a powerful immune response and the generation of a wide range of cytokines when it is presented to iNKT cell TCRs by CD1d molecules. Analysis of crystal structures of the TCR-α-GalCer-CD1d ternary complex identified the α-methylene unit in the fatty acid side chain, and more specifically the pro-S hydrogen at this position, as a site for incorporating a label. We postulated that modifying the glycolipid in this way would exert a minimal impact on the TCR-glycolipid-CD1d ternary complex, allowing the labeled molecule to function as a good mimic for the CD1d agonist under investigation. To test this hypothesis, the synthesis of a biotinylated version of the CD1d agonist threitol ceramide (ThrCer) was targeted. Both diastereoisomers, epimeric at the label tethering site, were prepared, and functional experiments confirmed the importance of substituting the pro-S, and not the pro-R, hydrogen with the label for optimal activity. Significantly, functional experiments revealed that biotinylated ThrCer (S)-10 displayed behavior comparable to that of ThrCer 5 itself and also confirmed that the biotin residue is available for streptavidin and antibiotin antibody recognition. A second CD1d agonist, namely α-GalCer C20:2 4, was modified in a similar way, this time with a fluorescent label. The labeled α-GalCer C20:2 analogue (11) again displayed functional behavior comparable to that of its unlabeled substrate, supporting the notion that the α-methylene unit in the fatty acid amide chain should be a suitable site for attaching

  17. Design, Synthesis, and Functional Activity of Labeled CD1d Glycolipid Agonists

    PubMed Central

    2013-01-01

    Invariant natural killer T cells (iNKT cells) are restricted by CD1d molecules and activated upon CD1d-mediated presentation of glycolipids to T cell receptors (TCRs) located on the surface of the cell. Because the cytokine response profile is governed by the structure of the glycolipid, we sought a method for labeling various glycolipids to study their in vivo behavior. The prototypical CD1d agonist, α-galactosyl ceramide (α-GalCer) 1, instigates a powerful immune response and the generation of a wide range of cytokines when it is presented to iNKT cell TCRs by CD1d molecules. Analysis of crystal structures of the TCR−α-GalCer–CD1d ternary complex identified the α-methylene unit in the fatty acid side chain, and more specifically the pro-S hydrogen at this position, as a site for incorporating a label. We postulated that modifying the glycolipid in this way would exert a minimal impact on the TCR–glycolipid–CD1d ternary complex, allowing the labeled molecule to function as a good mimic for the CD1d agonist under investigation. To test this hypothesis, the synthesis of a biotinylated version of the CD1d agonist threitol ceramide (ThrCer) was targeted. Both diastereoisomers, epimeric at the label tethering site, were prepared, and functional experiments confirmed the importance of substituting the pro-S, and not the pro-R, hydrogen with the label for optimal activity. Significantly, functional experiments revealed that biotinylated ThrCer (S)-10 displayed behavior comparable to that of ThrCer 5 itself and also confirmed that the biotin residue is available for streptavidin and antibiotin antibody recognition. A second CD1d agonist, namely α-GalCer C20:2 4, was modified in a similar way, this time with a fluorescent label. The labeled α-GalCer C20:2 analogue (11) again displayed functional behavior comparable to that of its unlabeled substrate, supporting the notion that the α-methylene unit in the fatty acid amide chain should be a suitable site for

  18. Inhibitors of aminoglycoside resistance activated in cells.

    PubMed

    Vong, Kenward; Tam, Ingrid S; Yan, Xuxu; Auclair, Karine

    2012-03-16

    The most common mechanism of resistance to aminoglycoside antibiotics entails bacterial expression of drug-metabolizing enzymes, such as the clinically widespread aminoglycoside N-6'-acetyltransferase (AAC(6')). Aminoglycoside-CoA bisubstrates are highly potent AAC(6') inhibitors; however, their inability to penetrate cells precludes in vivo studies. Some truncated bisubstrates are known to cross cell membranes, yet their activities against AAC(6') are in the micromolar range at best. We report here the synthesis and biological activity of aminoglycoside-pantetheine derivatives that, although devoid of AAC(6') inhibitory activity, can potentiate the antibacterial activity of kanamycin A against an aminoglycoside-resistant strain of Enterococcus faecium. Biological studies demonstrate that these molecules are potentially extended to their corresponding full-length bisubstrates by enzymes of the coenzyme A biosynthetic pathway. This work provides a proof-of-concept for the utility of prodrug compounds activated by enzymes of the coenzyme A biosynthetic pathway, to resensitize resistant strains of bacteria to aminoglycoside antibiotics.

  19. Activated Allogeneic NK Cells Preferentially Kill Poor Prognosis B-Cell Chronic Lymphocytic Leukemia Cells.

    PubMed

    Sánchez-Martínez, Diego; Lanuza, Pilar M; Gómez, Natalia; Muntasell, Aura; Cisneros, Elisa; Moraru, Manuela; Azaceta, Gemma; Anel, Alberto; Martínez-Lostao, Luis; Villalba, Martin; Palomera, Luis; Vilches, Carlos; García Marco, José A; Pardo, Julián

    2016-01-01

    Mutational status of TP53 together with expression of wild-type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA-mismatched Natural killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs) the most effective stimulus to activate NK cells. Here, we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell-activating receptors (NKG2D and NCRs) and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV) are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells, and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments.

  20. Activated Allogeneic NK Cells Preferentially Kill Poor Prognosis B-Cell Chronic Lymphocytic Leukemia Cells

    PubMed Central

    Sánchez-Martínez, Diego; Lanuza, Pilar M.; Gómez, Natalia; Muntasell, Aura; Cisneros, Elisa; Moraru, Manuela; Azaceta, Gemma; Anel, Alberto; Martínez-Lostao, Luis; Villalba, Martin; Palomera, Luis; Vilches, Carlos; García Marco, José A.; Pardo, Julián

    2016-01-01

    Mutational status of TP53 together with expression of wild-type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA-mismatched Natural killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs) the most effective stimulus to activate NK cells. Here, we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell-activating receptors (NKG2D and NCRs) and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV) are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells, and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments. PMID:27833611

  1. Complement activation by a B cell superantigen.

    PubMed

    Kozlowski, L M; Soulika, A M; Silverman, G J; Lambris, J D; Levinson, A I

    1996-08-01

    Staphylococcal protein A (SpA), acting as a B cell superantigen, binds to the Fab region of human VH3+ Igs. Using SpA abrogated of its IgG Fc binding activity (Mod SpA) as a model B cell superantigen, we determined whether such an interaction causes complement activation. Addition of Mod SpA to human serum led to complement consumption and the generation of C3a. To determine whether this complement activation 1) was due to an interaction between VH3+ Igs and the Fab binding site of SpA and 2) proceeded via the classical complement pathway, we tested a panel of monoclonal IgM proteins for the ability to hind C1q following interaction with SpA. C1q binding was restricted to SpA-reactive, VH3+ IgM proteins. To formally determine whether the binding of SpA to the reactive VH3+ IgM proteins led to complement activation, we reconstituted the serum from a hypogammaglobulinemic patient with monoclonal IgM proteins and measured complement consumption and C3a generation following the addition of Mod SpA. We observed complement consumption and C3a production only in Mod SpA-treated serum reconstituted with a VH3+, SpA-binding, IgM protein. Taken together, these results provide compelling evidence that the interaction of the Fab binding site of SpA and VH3+ Igs can lead to complement activation via the classical pathway. This novel interaction may have significant implications for the in vivo properties of a B cell superantigen.

  2. Persistent neural activity in head direction cells

    NASA Technical Reports Server (NTRS)

    Taube, Jeffrey S.; Bassett, Joshua P.; Oman, C. M. (Principal Investigator)

    2003-01-01

    Many neurons throughout the rat limbic system discharge in relation to the animal's directional heading with respect to its environment. These so-called head direction (HD) cells exhibit characteristics of persistent neural activity. This article summarizes where HD cells are found, their major properties, and some of the important experiments that have been conducted to elucidate how this signal is generated. The number of HD and angular head velocity cells was estimated for several brain areas involved in the generation of the HD signal, including the postsubiculum, anterior dorsal thalamus, lateral mammillary nuclei and dorsal tegmental nucleus. The HD cell signal has many features in common with what is known about how neural integration is accomplished in the oculomotor system. The nature of the HD cell signal makes it an attractive candidate for using neural network models to elucidate the signal's underlying mechanisms. The conditions that any network model must satisfy in order to accurately represent how the nervous system generates this signal are highlighted and areas where key information is missing are discussed.

  3. Innate-like and conventional T cell populations from hemodialyzed and kidney transplanted patients are equally compromised.

    PubMed

    Baron, Marine; Belo, Renata; Cathelin, Dominique; Moreira-Teixeira, Lucia; Cartery, Claire; Rondeau, Eric; Mesnard, Laurent; Leite-de-Moraes, Maria

    2014-01-01

    Clinicians are well aware of existing pharmacologically-induced immune deficient status in kidney-transplanted patients that will favor their susceptibility to bacterial or viral infections. Previous studies indicated that advanced Stage 4-5 Chronic Kidney Disease might also be regarded as an immune deficiency-like status as well, even though the mechanisms are not fully understood. Here, we analyzed the ex vivo frequency and the functional properties of both conventional and innate-like T (ILT) lymphocyte subsets in the peripheral blood of 35 patients on hemodialysis, 29 kidney transplanted patients and 38 healthy donors. We found that peripheral blood cell count of ILT cells, as iNKT (invariant Natural Killer T) and MAIT (mucosal-associated invariant T), were significantly decreased in hemodialyzed patients compared to healthy controls. This deficiency was also observed regarding conventional T cells, including the IL-17-producing CD4(+) Th17 cells. Pertaining to regulatory T cells, we also noticed major modifications in the global frequency of CD4(+)CD25(+)Foxp3(+) T lymphocytes, including the resting suppressive CD45RA(+)Foxp3lo and activated suppressive CD45RA-Foxp3hi T cell subpopulations. We found no significant differences between the immune status of hemodialyzed and kidney-transplanted subjects. In conclusion, we demonstrated that both ILT and conventional T cell numbers are equally impaired in hemodialyzed and kidney-transplanted patients.

  4. Transgelin-2 in B-Cells Controls T-Cell Activation by Stabilizing T Cell - B Cell Conjugates

    PubMed Central

    Chae, Myoung-Won; Kim, Hye-Ran; Kim, Chang-Hyun; Jun, Chang-Duk; Park, Zee-Yong

    2016-01-01

    The immunological synapse (IS), a dynamic and organized junction between T-cells and antigen presenting cells (APCs), is critical for initiating adaptive immunity. The actin cytoskeleton plays a major role in T-cell reorganization during IS formation, and we previously reported that transgelin-2, an actin-binding protein expressed in T-cells, stabilizes cortical F-actin, promoting T-cell activation in response to antigen stimulation. Transgelin-2 is also highly expressed in B-cells, although no specific function has been reported. In this study, we found that deficiency in transgelin-2 (TAGLN2-/-) in B-cells had little effect on B-cell development and activation, as measured by the expression of CD69, MHC class II molecules, and CD80/86. Nevertheless, in B-cells, transgelin-2 accumulated in the IS during the interaction with T-cells. These results led us to hypothesize that transgelin-2 may also be involved in IS stability in B-cells, thereby influencing T-cell function. Notably, we found that transgelin-2 deficiency in B-cells reduced T-cell activation, as determined by the release of IL-2 and interferon-γ and the expression of CD69. Furthermore, the reduced T-cell activation was correlated with reduced B-cell–T-cell conjugate formation. Collectively, these results suggest that actin stability in B-cells during IS formation is critical for the initiation of adaptive T-cell immunity. PMID:27232882

  5. Human lymphokine-activated killer cells are cytotoxic against cells infected with Toxoplasma gondii

    PubMed Central

    1992-01-01

    Experiments were conducted to determine whether human lymphokine- activated killer (LAK) cells are cytotoxic against cells infected with Toxoplasma gondii. Nylon wool nonadherent (NWNA) peripheral blood lymphocytes, as well as purified natural killer cell (NK) (CD3- CD16+ CD56+) and T (CD3+ CD16- CD56-) cells obtained from five healthy T. gondii seronegative volunteers exhibited minimal cytotoxic activity against T. gondii-infected cells. When standard LAK (S-LAK) cell preparations were induced by incubation of NWNA cells with recombinant interleukin 2, induction of remarkable cytotoxic activity against T. gondii-infected cells. When standard in LAK cell preparations from each of the volunteers. The phenotype of the LAK precursor and effector cells varied depending on the target cell used. Whereas the precursor and the effector cells of most of the LAK activity against K562 and Daudi cells were cells with NK phenotype, when T. gondii-infected cells were used as targets, both cells with NK and T cell phenotypes were precursors and effectors of the lysis. When cytotoxic activity of S-LAK cells was compared with the activity of adherent LAK (A-LAK) cells, A- LAK cells displayed higher cytotoxic activity against T. gondii- infected cells, as well as against K562 and Daudi cells. Cold target inhibition experiments suggested that there is a subset of LAK effector cells capable of lysing both T. gondii-infected cells and Daudi cells, whereas other subsets preferentially or exclusively lyse one of these target cells. PMID:1460415

  6. Probing cell activity in random access modality

    NASA Astrophysics Data System (ADS)

    Sacconi, L.; Crocini, C.; Lotti, J.; Coppini, R.; Ferrantini, C.; Tesi, C.; Yan, P.; Loew, L. M.; Cerbai, E.; Poggesi, C.; Pavone, F. S.

    2013-06-01

    We combined the advantage of an ultrafast random access microscope with novel labelling technologies to study the intra- and inter-cellular action potential propagation in neurons and cardiac myocytes with sub-millisecond time resolution. The random accesses microscopy was used in combination with a new fluorinated voltage sensitive dye with improved photostability to record membrane potential from multiple Purkinje cells with near simultaneous sampling. The RAMP system rapidly scanned between lines drawn in the membranes of neurons to perform multiplex measurements of the TPF signal. This recording was achieved by rapidly positioning the laser excitation with the AOD to sample a patch of membrane from each cell in <100 μs for recording from five cells, multiplexing permits a temporal resolution of 400 μs sufficient to capture every spike. The system is capable to record spontaneous activity over 800 ms from five neighbouring cells simultaneously, showing that spiking is not temporally correlated. The system was also used to investigate the electrical properties of tubular system (TATS) in isolated rat ventricular myocytes.

  7. Hymenoptera Allergy and Mast Cell Activation Syndromes.

    PubMed

    Bonadonna, Patrizia; Bonifacio, Massimiliano; Lombardo, Carla; Zanotti, Roberta

    2016-01-01

    Mast cell activation syndrome (MCAS) can be diagnosed in patients with recurrent, severe symptoms from mast cell (MC)-derived mediators, which are transiently increased in serum and are attenuated by mediator-targeting drugs. When KIT-mutated, clonal MC are detected in these patients, a diagnosis of primary MCAS can be made. Severe systemic reactions to hymenoptera venom (HV) represent the most common form of anaphylaxis in patients with mastocytosis. Patients with primary MCAS and HV anaphylaxis are predominantly males and do not have skin lesions in the majority of cases, and anaphylaxis is characterized by hypotension and syncope in the absence of urticaria and angioedema. A normal value of tryptase (≤11.4 ng/ml) in these patients does not exclude a diagnosis of mastocytosis. Patients with primary MCAS and HV anaphylaxis have to undergo lifelong venom immunotherapy, in order to prevent further potentially fatal severe reactions.

  8. Notch reporter activity in breast cancer cell lines identifies a subset of cells with stem cell activity.

    PubMed

    D'Angelo, Rosemarie C; Ouzounova, Maria; Davis, April; Choi, Daejin; Tchuenkam, Stevie M; Kim, Gwangil; Luther, Tahra; Quraishi, Ahmed A; Senbabaoglu, Yasin; Conley, Sarah J; Clouthier, Shawn G; Hassan, Khaled A; Wicha, Max S; Korkaya, Hasan

    2015-03-01

    Developmental pathways such as Notch play a pivotal role in tissue-specific stem cell self-renewal as well as in tumor development. However, the role of Notch signaling in breast cancer stem cells (CSC) remains to be determined. We utilized a lentiviral Notch reporter system to identify a subset of cells with a higher Notch activity (Notch(+)) or reduced activity (Notch(-)) in multiple breast cancer cell lines. Using in vitro and mouse xenotransplantation assays, we investigated the role of the Notch pathway in breast CSC regulation. Breast cancer cells with increased Notch activity displayed increased sphere formation as well as expression of breast CSC markers. Interestingly Notch(+) cells displayed higher Notch4 expression in both basal and luminal breast cancer cell lines. Moreover, Notch(+) cells demonstrated tumor initiation capacity at serial dilutions in mouse xenografts, whereas Notch(-) cells failed to generate tumors. γ-Secretase inhibitor (GSI), a Notch blocker but not a chemotherapeutic agent, effectively targets these Notch(+) cells in vitro and in mouse xenografts. Furthermore, elevated Notch4 and Hey1 expression in primary patient samples correlated with poor patient survival. Our study revealed a molecular mechanism for the role of Notch-mediated regulation of breast CSCs and provided a compelling rationale for CSC-targeted therapeutics.

  9. Notch reporter activity in breast cancer cell lines identifies a subset of cells with stem cell activity

    PubMed Central

    Davis, April; Choi, Daejin; Tchuenkam, Stevie M.; Kim, Gwangil; Luther, Tahra; Quraishi, Ahmed A.; Senbabaoglu, Yasin; Conley, Sarah J.; Clouthier, Shawn G.; Hassan, Khaled A.; Wicha, Max S.; Korkaya, Hasan

    2015-01-01

    Developmental pathways such as Notch play a pivotal role in tissue specific stem cell self-renewal as well as in tumor development. However, the role of Notch signaling in breast cancer stem cells (CSC) remains to be determined. We utilized a lentiviral Notch reporter system to identify a subset of cells with a higher Notch activity (Notch+) or reduced activity (Notch-) in multiple breast cancer cell lines. Using in vitro and mouse xenotransplantation assays we investigated the role of Notch pathway in breast CSC regulation. Breast cancer cells with increased Notch activity displayed increased sphere formation as well as expression of breast CSC markers. Interestingly Notch+ cells displayed higher Notch4 expression in both basal and luminal breast cancer cell lines. Moreover, Notch+ cells demonstrated tumor initiation capacity at serial dilutions in mouse xenografts while Notch- cells failed to generate tumors. Gamma-secretase inhibitor (GSI), a Notch blocker but not a chemotherapeutic agent effectively targets these Notch+ cells in vitro and in mouse xenografts. Furthermore, elevated Notch4 and Hey1 expression in primary patient samples correlated with poor patient survival. Our studies reveal molecular mechanism for the role of Notch mediated regulation of breast CSCs and provide a compelling rationale for CSC targeted therapeutics. PMID:25673823

  10. Sertoli Cells Maintain Leydig Cell Number and Peritubular Myoid Cell Activity in the Adult Mouse Testis

    PubMed Central

    Monteiro, Ana; Milne, Laura; Cruickshanks, Lyndsey; Jeffrey, Nathan; Guillou, Florian; Freeman, Tom C.; Mitchell, Rod T.; Smith, Lee B.

    2014-01-01

    The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR) specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health. PMID:25144714

  11. Bursts of Active Transport in Living Cells

    NASA Astrophysics Data System (ADS)

    Wang, Bo; Kuo, James; Granick, Steve

    2013-11-01

    We show, using a large new data set, that the temporally resolved speed of active cargo transport in living cells follows a scaling law over several decades of time and length. The statistical regularities display a time-averaged shape that we interpret to reflect stress buildup, followed by rapid release. The scaling power law agrees quantitatively with those reported in inanimate systems (jammed colloids and granular media, and magnetic Barkhausen noise), suggesting a common origin in pushing through a crowded environment in a weak force regime. The implied regulation of the speed of active cellular transport due to environmental obstruction results in bursts of speed and acceleration. These findings extend the classical notion of molecular crowding.

  12. Bursts of active transport in living cells.

    PubMed

    Wang, Bo; Kuo, James; Granick, Steve

    2013-11-15

    We show, using a large new data set, that the temporally resolved speed of active cargo transport in living cells follows a scaling law over several decades of time and length. The statistical regularities display a time-averaged shape that we interpret to reflect stress buildup, followed by rapid release. The scaling power law agrees quantitatively with those reported in inanimate systems (jammed colloids and granular media, and magnetic Barkhausen noise), suggesting a common origin in pushing through a crowded environment in a weak force regime. The implied regulation of the speed of active cellular transport due to environmental obstruction results in bursts of speed and acceleration. These findings extend the classical notion of molecular crowding.

  13. T helper cell activation and human retroviral pathogenesis.

    PubMed Central

    Copeland, K F; Heeney, J L

    1996-01-01

    T helper (Th) cells are of central importance in regulating many critical immune effector mechanisms. The profile of cytokines produced by Th cells correlates with the type of effector cells induced during the immune response to foreign antigen. Th1 cells induce the cell-mediated immune response, while Th2 cells drive antibody production. Th cells are the preferential targets of human retroviruses. Infections with human T-cell leukemia virus (HTLV) or human immunodeficiency virus (HIV) result in the expansion of Th cells by the action of HTLV (adult T-cell leukemia) or the progressive loss of T cells by the action of HIV (AIDS). Both retrovirus infections impart a high-level activation state in the host immune cells as well as systemically. However, diverging responses to this activation state have contrasting effects on the Th-cell population. In HIV infection, Th-cell loss has been attributed to several mechanisms, including a selective elimination of cells by apoptosis. The induction of apoptosis in HIV infection is complex, with many different pathways able to induce cell death. In contrast, infection of Th cells with HTLV-1 affords the cell a protective advantage against apoptosis. This advantage may allow the cell to escape immune surveillance, providing the opportunity for the development of Th-cell cancer. In this review, we will discuss the impact of Th-cell activation and general immune activation on human retrovirus expression with a focus upon Th-cell function and the progression to disease. PMID:8987361

  14. Kinase Activity Studied in Living Cells Using an Immunoassay

    ERIC Educational Resources Information Center

    Bavec, Aljos?a

    2014-01-01

    This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more…

  15. Regulation of polymorphonuclear cell activation by thrombopoietin.

    PubMed Central

    Brizzi, M F; Battaglia, E; Rosso, A; Strippoli, P; Montrucchio, G; Camussi, G; Pegoraro, L

    1997-01-01

    Thrombopoietin (TPO) regulates early and late stages of platelet formation as well as platelet activation. TPO exerts its effects by binding to the receptor, encoded by the protooncogene c-mpl, that is expressed in a large number of cells of hematopoietic origin. In this study, we evaluated the expression of c-Mpl and the effects of TPO on human polymorphonuclear cells (PMN). We demonstrate that PMN express the TPO receptor c-Mpl and that TPO induces STAT1 tyrosine phosphorylation and the formation of a serum inducible element complex containing STAT1. The analysis of biological effects of TPO on PMN demonstrated that TPO, at concentrations of 1-10 ng/ml, primes the response of PMN to n-formyl-met-leu-phe (FMLP) by inducing an early oxidative burst. TPO-induced priming on FMLP-stimulated PMN was also detected on the tyrosine phosphorylation of a protein with a molecular mass of approximately 28 kD. Moreover, we demonstrated that TPO by itself was able to stimulate, at doses ranging from 0.05 to 10 ng/ml, early release and delayed synthesis of interleukin 8 (IL-8). Thus, our data indicate that, in addition to sustaining megakaryocytopoiesis, TPO may have an important role in regulating PMN activation. PMID:9120001

  16. Epigenomics of T cell activation, differentiation and memory

    PubMed Central

    Cuddapah, Suresh; Barski, Artem; Zhao, Keji

    2010-01-01

    Activation of T cells is an essential step in the immunological response to infection. While activation of naïve T cells results in proliferation and slow differentiation into cytokine-producing effector cells, antigen engagement with memory cells leads to cytokine production immediately. Even though the cell surface signaling events are similar in both the cases, the outcome is different, suggesting that distinct regulatory mechanisms may exist downstream of the activation signals. Recent advances in the understanding of global epigenetic patterns in T cells have resulted in the appreciation of the role of epigenetic mechanisms in processes such as activation and differentiation. In this review we discuss recent data suggesting that naïve T cell activation, differentiation and lineage commitment results in epigenetic changes and a fine balance between different histone modifications is required. On the other hand, memory T cells are poised and do not require epigenetic changes for short-term activation. PMID:20226645

  17. MAIT cells are activated during human viral infections.

    PubMed

    van Wilgenburg, Bonnie; Scherwitzl, Iris; Hutchinson, Edward C; Leng, Tianqi; Kurioka, Ayako; Kulicke, Corinna; de Lara, Catherine; Cole, Suzanne; Vasanawathana, Sirijitt; Limpitikul, Wannee; Malasit, Prida; Young, Duncan; Denney, Laura; Moore, Michael D; Fabris, Paolo; Giordani, Maria Teresa; Oo, Ye Htun; Laidlaw, Stephen M; Dustin, Lynn B; Ho, Ling-Pei; Thompson, Fiona M; Ramamurthy, Narayan; Mongkolsapaya, Juthathip; Willberg, Christian B; Screaton, Gavin R; Klenerman, Paul

    2016-06-23

    Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation-driving cytokine release and Granzyme B upregulation-is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/β. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology.

  18. Nylon wool purification alters the activation of T cells.

    PubMed

    Wohler, Jillian E; Barnum, Scott R

    2009-02-01

    Purification of lymphocytes, particularly T cells, is commonly performed using nylon wool. This enrichment method selectively retains B cells and some myeloid cells allowing a significantly more pure T cell population to flow through a nylon wool column. T cells purified in this fashion are assumed to be unaltered and functionally naïve, however some studies have suggested aberrant in vitro T cell responses after nylon wool treatment. We found that nylon wool purification significantly altered T cell proliferation, expression of activation markers and production of cytokines. Our results suggest that nylon wool treatment modifies T cell activation responses and that caution should be used when choosing this purification method.

  19. The Vα14 invariant natural killer T cell TCR forces microbial glycolipids and CD1d into a conserved binding mode

    PubMed Central

    Li, Yali; Girardi, Enrico; Wang, Jing; Yu, Esther Dawen; Painter, Gavin F.; Kronenberg, Mitchell

    2010-01-01

    Invariant natural killer T cells (iNKT cells) rapidly produce effector cytokines. In this study, we report the first crystal structures of the iNKT cell T cell receptor (TCR) bound to two natural, microbial glycolipids presented by CD1d. Binding of the TCR induced CDR3-α–dependent structural changes in the F′ roof of CD1d; these changes resemble those occurring in the absence of TCR engagement when the highly potent synthetic antigen α-galactosylceramide (α-GalCer) binds CD1d. Furthermore, in the Borrelia burgdorferi α–galactosyl diacylglycerol–CD1d complex, TCR binding caused a marked repositioning of the galactose sugar into an orientation that closely resembles α-GalCer. The TCR-dependent reorientation of the sugar, together with the induced CD1d fit, may explain the weaker potency of the microbial antigens compared with α-GalCer. We propose that the TCR of iNKT cells binds with a conserved footprint onto CD1d, regardless of the bound glycolipid antigen, and that for microbial antigens this unique binding mode requires TCR-initiated conformational changes. PMID:20921281

  20. Isomaltulose is actively metabolized in plant cells.

    PubMed

    Wu, Luguang; Birch, Robert G

    2011-12-01

    Isomaltulose is a structural isomer of sucrose (Suc). It has been widely used as a nonmetabolized sugar in physiological studies aimed at better understanding the regulatory roles and transport of sugars in plants. It is increasingly used as a nutritional human food, with some beneficial properties including low glycemic index and acariogenicity. Cloning of genes for Suc isomerases opened the way for direct commercial production in plants. The understanding that plants lack catabolic capabilities for isomaltulose indicated a possibility of enhanced yields relative to Suc. However, this understanding was based primarily on the treatment of intact cells with exogenous isomaltulose. Here, we show that sugarcane (Saccharum interspecific hybrids), like other tested plants, does not readily import or catabolize extracellular isomaltulose. However, among intracellular enzymes, cytosolic Suc synthase (in the breakage direction) and vacuolar soluble acid invertase (SAI) substantially catabolize isomaltulose. From kinetic studies, the specificity constant of SAI for isomaltulose is about 10% of that for Suc. Activity varied against other Suc isomers, with V(max) for leucrose about 6-fold that for Suc. SAI activities from other plant species varied substantially in substrate specificity against Suc and its isomers. Therefore, in physiological studies, the blanket notion of Suc isomers including isomaltulose as nonmetabolized sugars must be discarded. For example, lysis of a few cells may result in the substantial hydrolysis of exogenous isomaltulose, with profound downstream signal effects. In plant biotechnology, different V(max) and V(max)/K(m) ratios for Suc isomers may yet be exploited, in combination with appropriate developmental expression and compartmentation, for enhanced sugar yields.

  1. Hyperoxia Inhibits T Cell Activation in Mice

    NASA Astrophysics Data System (ADS)

    Hughes-Fulford, M.; Meissler, J.; Aguayo, E. T.; Globus, R.; Aguado, J.; Candelario, T.

    2013-02-01

    , spleens were removed and the splenocytes were isolated and kept as individual biological samples. We have also examined transcription factors (JASPAR) and pathways of the immune system to help us understand the mechanism of regulation. Results: Our recent mouse immunology experiment aboard STS-131 suggests that the early T cell immune response was inhibited in animals that have been exposed to spaceflight, even 24 hours after return to earth. Moreover, recent experiments in hyperoxic mice show that many of the same genes involved in early T cell activation were altered. Specifically, expression of IL-2Rα, Cxcl2, TNFα, FGF2, LTA and BCL2 genes are dysregulated in mice exposed to hyperoxia. Conclusions: If these hyperoxia-induced changes of gene expression in early T cell activation are additive to the changes seen in the microgravity of spaceflight, there could be an increased infection risk to EVA astronauts, which should be addressed prior to conducting a Mars or other long-term mission.

  2. Lysis of primary hepatic tumours by lymphokine activated killer cells.

    PubMed Central

    Hsieh, K H; Shu, S Y; Lee, C S; Chu, C T; Yang, C S; Chang, K J

    1987-01-01

    Lymphokine activated killer cell is a newly described lytic system against a variety of solid tumours and is distinct in several respects from the classic cytolytic T cell and the natural killer systems. This study was conducted to evaluate the lytic activity of lymphokine activated killer cells against fresh autologous and allogeneic, as well as cultured hepatocellular carcinoma cells. Lymphokine activated killer cell was generated by incubating peripheral blood mononuclear cells with various concentrations of recombinant IL-2 (rIL-2, Cetus, USA) for various periods of time. A four hour 51Cr release assay was used to measure cytotoxicity. The results show that fresh and cultured hepatocellular carcinoma cells were only slightly susceptible to natural killer cells. Normal hepatocytes were resistant to lymphokine activated killer-mediated lysis. Lymphokine activated killer cells could be generated from mononuclear cells of hepatocellular carcinoma patients and normal subjects with lytic activity against fresh autologous and allogeneic and cultured hepatocellular carcinoma cells, but lymphokine activated killer cells from the former was less efficient than that from the latter. It is concluded that the adoptive immunotherapy with combined rIL-2 and lymphokine activated killer may be worth trying in early cases of primary hepatocellular carcinoma. PMID:3030899

  3. A Simple Laboratory Exercise Illustrating Active Transport in Yeast Cells.

    ERIC Educational Resources Information Center

    Stambuk, Boris U.

    2000-01-01

    Describes a simple laboratory activity illustrating the chemiosmotic principles of active transport in yeast cells. Demonstrates the energy coupling mechanism of active a-glucoside uptake by Saccaromyces cerevisiae cells with a colorimetric transport assay using very simple equipment. (Contains 22 references.) (Author/YDS)

  4. Mechanism of human natural killer cell activation by Haemophilus ducreyi.

    PubMed

    Li, Wei; Janowicz, Diane M; Fortney, Kate R; Katz, Barry P; Spinola, Stanley M

    2009-08-15

    The role of natural killer (NK) cells in the host response to Haemophilus ducreyi infection is unclear. In pustules obtained from infected human volunteers, there was an enrichment of CD56bright NK cells bearing the activation markers CD69 and HLA-DR, compared with peripheral blood. To study the mechanism by which H. ducreyi activated NK cells, we used peripheral blood mononuclear cells from uninfected volunteers. H. ducreyi activated NK cells only in the presence of antigen-presenting cells. H. ducreyi-infected monocytes and monocyte-derived macrophages activated NK cells in a contact- and interleukin-18 (IL-18)-dependent manner, whereas monocyte-derived dendritic cells induced NK activation through soluble IL-12. More lesional NK cells than peripheral blood NK cells produced IFN-gamma in response to IL-12 and IL-18. We conclude that NK cells are recruited to experimental lesions and likely are activated by infected macrophages and dendritic cells. IFN-gamma produced by lesional NK cells may facilitate phagocytosis of H. ducreyi.

  5. Active unjamming of confluent cell layers

    NASA Astrophysics Data System (ADS)

    Marchetti, M. Cristina

    Cell motion inside dense tissues governs many biological processes, including embryonic development and cancer metastasis, and recent experiments suggest that these tissues exhibit collective glassy behavior. Motivated by these observations, we have studied a model of dense tissues that combines self-propelled particle models and vertex models of confluent cell layers. In this model, referred to as self-propelled Voronoi (SPV), cells are described as polygons in a Voronoi tessellation with directed noisy cell motility and interactions governed by a shape energy that incorporates the effects of cell volume incompressibility, contractility and cell-cell adhesion. Using this model, we have demonstrated a new density-independent solid-liquid transition in confluent tissues controlled by cell motility and a cell-shape parameter measuring the interplay of cortical tension and cell-cell adhesion. An important insight of this work is that the rigidity and dynamics of cell layers depends sensitively on cell shape. We have also used the SPV model to test a new method developed by our group to determine cellular forces and tissue stresses from experimentally accessible cell shapes and traction forces, hence providing the spatio-temporal distribution of stresses in motile dense tissues. This work was done with Dapeng Bi, Lisa Manning and Xingbo Yang. MCM was supported by NSF-DMR-1305184 and by the Simons Foundation.

  6. Mycoplasma pneumoniae induces cytotoxic activity in guinea pig bronchoalveolar cells

    SciTech Connect

    Kist, M.; Koester, H.; Bredt, W.

    1985-06-01

    Precultured guinea pig alveolar macrophages (AM) and freshly harvested alveolar cells (FHAC) activated by interaction with Mycoplasma pneumoniae were cytotoxic for xenogeneic /sup 75/selenomethionine-labeled tumor target cells. Phagocytosis of whole opsonized or nonopsonized M. pneumoniae cells was more effective in eliciting cytotoxicity than uptake of sonicated microorganisms. The addition of living mycoplasma cells to the assay system enhanced the cytotoxic effect considerably. Target cells were significantly more susceptible to the cytotoxic action of phagocytes if they were coated with mycoplasma antigen or cocultured together with M. pneumoniae. The activation of the phagocytes could be inhibited by 2-deoxy-D-glucose but not by antimicrobial substances suppressing mycoplasma protein synthesis. It was accompanied by /sup 51/Cr release without detectable signs of cell damage. The supernatants of activated cells were cytotoxic for approximately 24 h. Inhibition, release, and cytotoxic activity indicate the necessity of an intact metabolism of the effector cells and suggest a secretion of cytotoxic substances.

  7. Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation

    PubMed Central

    Bruno, Stefania; Grange, Cristina; Tapparo, Marta; Pasquino, Chiara; Romagnoli, Renato; Dametto, Ennia; Amoroso, Antonio; Tetta, Ciro; Camussi, Giovanni

    2016-01-01

    Human liver stem cells (HLSCs) are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs), and dendritic cells (DCs) in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2) and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs), HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response. PMID:27127520

  8. Organizer activity of the polar cells during Drosophila oogenesis.

    PubMed

    Grammont, Muriel; Irvine, Kenneth D

    2002-11-01

    Patterning of the Drosophila egg requires the establishment of several distinct types of somatic follicle cells, as well as interactions between these follicle cells and the oocyte. The polar cells occupy the termini of the follicle and are specified by the activation of Notch. We have investigated their role in follicle patterning by creating clones of cells mutant for the Notch modulator fringe. This genetic ablation of polar cells results in cell fate defects within surrounding follicle cells. At the anterior, the border cells, the immediately adjacent follicle cell fate, are absent, as are the more distant stretched and centripetal follicle cells. Conversely, increasing the number of polar cells by expressing an activated form of the Notch receptor increases the number of border cells. At the posterior, elimination of polar cells results in abnormal oocyte localization. Moreover, when polar cells are mislocalized laterally, the surrounding follicle cells adopt a posterior fate, the oocyte is located adjacent to them, and the anteroposterior axis of the oocyte is re-oriented with respect to the ectopic polar cells. Our observations demonstrate that the polar cells act as an organizer that patterns surrounding follicle cells and establishes the anteroposterior axis of the oocyte. The origin of asymmetry during Drosophila development can thus be traced back to the specification of the polar cells during early oogenesis.

  9. Activating Cell Death Ligand Signaling Through Proteasome Inhibition

    DTIC Science & Technology

    2009-05-01

    Activating Cell Death Ligand Signaling Through Proteasome Inhibition PRINCIPAL INVESTIGATOR: Steven R Schwarze...SUBTITLE Activating Cell Death Ligand Signaling Through 5a. CONTRACT NUMBER Proteasome Inhibition 5b. GRANT NUMBER W81XWH-08-1-0392 5c...proteasome inhibition can act as an anti-neoplastic agent in vivo by sensitizing cancer cells to cell death ligands in the tumor microenvironment

  10. Immune activation: death, danger and dendritic cells.

    PubMed

    Pulendran, Bali

    2004-01-06

    Dendritic cells are critical for host immunity, and sense microbes with pathogen recognition receptors. New evidence indicates that these cells also sense uric acid crystals in dead cells, suggesting that the immune system is conscious not only of pathogens, but also of death and danger.

  11. Shape control and compartmentalization in active colloidal cells

    PubMed Central

    Spellings, Matthew; Engel, Michael; Klotsa, Daphne; Sabrina, Syeda; Drews, Aaron M.; Nguyen, Nguyen H. P.; Bishop, Kyle J. M.; Glotzer, Sharon C.

    2015-01-01

    Small autonomous machines like biological cells or soft robots can convert energy input into control of function and form. It is desired that this behavior emerges spontaneously and can be easily switched over time. For this purpose we introduce an active matter system that is loosely inspired by biology and which we term an active colloidal cell. The active colloidal cell consists of a boundary and a fluid interior, both of which are built from identical rotating spinners whose activity creates convective flows. Similarly to biological cell motility, which is driven by cytoskeletal components spread throughout the entire volume of the cell, active colloidal cells are characterized by highly distributed energy conversion. We demonstrate that we can control the shape of the active colloidal cell and drive compartmentalization by varying the details of the boundary (hard vs. flexible) and the character of the spinners (passive vs. active). We report buckling of the boundary controlled by the pattern of boundary activity, as well as formation of core–shell and inverted Janus phase-separated configurations within the active cell interior. As the cell size is increased, the inverted Janus configuration spontaneously breaks its mirror symmetry. The result is a bubble–crescent configuration, which alternates between two degenerate states over time and exhibits collective migration of the fluid along the boundary. Our results are obtained using microscopic, non–momentum-conserving Langevin dynamics simulations and verified via a phase-field continuum model coupled to a Navier–Stokes equation. PMID:26253763

  12. Shape control and compartmentalization in active colloidal cells.

    PubMed

    Spellings, Matthew; Engel, Michael; Klotsa, Daphne; Sabrina, Syeda; Drews, Aaron M; Nguyen, Nguyen H P; Bishop, Kyle J M; Glotzer, Sharon C

    2015-08-25

    Small autonomous machines like biological cells or soft robots can convert energy input into control of function and form. It is desired that this behavior emerges spontaneously and can be easily switched over time. For this purpose we introduce an active matter system that is loosely inspired by biology and which we term an active colloidal cell. The active colloidal cell consists of a boundary and a fluid interior, both of which are built from identical rotating spinners whose activity creates convective flows. Similarly to biological cell motility, which is driven by cytoskeletal components spread throughout the entire volume of the cell, active colloidal cells are characterized by highly distributed energy conversion. We demonstrate that we can control the shape of the active colloidal cell and drive compartmentalization by varying the details of the boundary (hard vs. flexible) and the character of the spinners (passive vs. active). We report buckling of the boundary controlled by the pattern of boundary activity, as well as formation of core-shell and inverted Janus phase-separated configurations within the active cell interior. As the cell size is increased, the inverted Janus configuration spontaneously breaks its mirror symmetry. The result is a bubble-crescent configuration, which alternates between two degenerate states over time and exhibits collective migration of the fluid along the boundary. Our results are obtained using microscopic, non-momentum-conserving Langevin dynamics simulations and verified via a phase-field continuum model coupled to a Navier-Stokes equation.

  13. Control of Experimental Autoimmune Encephalomyelitis by T Cells Responding to Activated T Cells

    NASA Astrophysics Data System (ADS)

    Lohse, Ansgar W.; Mor, Felix; Karin, Nathan; Cohen, Irun R.

    1989-05-01

    T cell vaccination against experimental autoimmune disease is herein shown to be mediated in part by anti-ergotypic T cells, T cells that recognize and respond to the state of activation of other T cells. The anti-ergotypic response thus combines with the previously shown anti-idiotypic T cell response to regulate autoimmunity.

  14. Recruitment and Activation of Natural Killer (Nk) Cells in Vivo Determined by the Target Cell Phenotype

    PubMed Central

    Glas, Rickard; Franksson, Lars; Une, Clas; Eloranta, Maija-Leena; Öhlén, Claes; Örn, Anders; Kärre, Klas

    2000-01-01

    Natural killer (NK) cells can spontaneously lyse certain virally infected and transformed cells. However, early in immune responses NK cells are further activated and recruited to tissue sites where they perform effector functions. This process is dependent on cytokines, but it is unclear if it is regulated by NK cell recognition of susceptible target cells. We show here that infiltration of activated NK cells into the peritoneal cavity in response to tumor cells is controlled by the tumor major histocompatibility complex (MHC) class I phenotype. Tumor cells lacking appropriate MHC class I expression induced NK cell infiltration, cytotoxic activation, and induction of transcription of interferon γ in NK cells. The induction of these responses was inhibited by restoration of tumor cell MHC class I expression. The NK cells responding to MHC class I–deficient tumor cells were ∼10 times as active as endogenous NK cells on a per cell basis. Although these effector cells showed a typical NK specificity in that they preferentially killed MHC class I–deficient cells, this specificity was even more distinct during induction of the intraperitoneal response. Observations are discussed in relation to a possible adaptive component of the NK response, i.e., recruitment/activation in response to challenges that only NK cells are able to neutralize. PMID:10620611

  15. Invariant natural killer T cells and mucosal-associated invariant T cells in multiple sclerosis.

    PubMed

    Bianchini, Elena; De Biasi, Sara; Simone, Anna Maria; Ferraro, Diana; Sola, Patrizia; Cossarizza, Andrea; Pinti, Marcello

    2017-03-01

    Multiple sclerosis (MS) is a chronic progressive inflammatory demyelinating disorder of the central nervous system, and in several countries is a leading cause of permanent neurological disability in young adults, particularly women. MS is considered an autoimmune disease, caused by an aberrant immune response to environmental triggers in genetically susceptible subjects. However, the contribution of the innate or of the adaptive immune system to the development and progression of the disease has not yet been fully elucidated. Innate-like T lymphocytes are unconventional T cells that bridge the innate and adaptive arms of the immune system, because they use a T cell receptor to sense external ligands, but behave like innate cells when they rapidly respond to stimuli. These cells could play an important role in the pathogenesis of MS. Here, we focus on invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells, and we review the current knowledge on their biology and possible involvement in MS. Although several studies have evaluated the frequency and functions of iNKT and MAIT cells both in MS patients and in experimental mouse models, contradictory observations have been reported, and it is not clear whether they exert a protective or a pro-inflammatory and harmful role. A better understanding of how immune cells are involved in MS, and of their interactions could be of great interest for the development of new therapeutic strategies.

  16. Cutting edge: inhibition of T cell activation by TIM-2.

    PubMed

    Knickelbein, Jared E; de Souza, Anjali J; Tosti, Richard; Narayan, Preeti; Kane, Lawrence P

    2006-10-15

    T cell Ig and mucin domain protein 2 (TIM-2) has been shown to regulate T cell activation in vitro and T cell-mediated disease in vivo. However, it is still not clear whether TIM-2 acts mainly to augment T cell function or to inhibit it. We have directly examined the function of TIM-2 in murine and human T cell lines. Our results indicate that expression of TIM-2 significantly impairs the induction of NFAT and AP-1 transcriptional reporters by not only TCR ligation but also by the pharmacological stimuli PMA and ionomycin. This does not appear to be due to a general effect on cell viability, and the block in NFAT activation can be bypassed by expression of activated alleles of Ras or calcineurin, or MEK kinase, in the case of AP-1. Thus, our data are consistent with a model whereby TIM-2 inhibits T cell activation.

  17. Alloantigen presentation by B cells: analysis of the requirement for B-cell activation.

    PubMed Central

    Wilson, J L; Cunningham, A C; Kirby, J A

    1995-01-01

    This paper describes a model for investigation of the functional implications of B-cell activation for antigen presentation. Mixed lymphocyte cultures were used to assess the ability of freshly isolated B cells, mitogen-activated B cells and Epstein-Barr virus (EBV)-transformed B-cell lines to stimulate the activation and proliferation of allogeneic T cells under a variety of experimental conditions. It was found that resting B cells presented antigen poorly, while activated cells were highly immunogenic. Paraformaldehyde fixation completely eliminated antigen presentation by resting B cells, despite constitutive expression of class II MHC antigens. However, fixation had little effect on antigen presentation by activated B cells that expressed B7-1 and B7-2 in addition to class II major histocompatibility complex (MHC) molecules. Arrest of B-cell activation by serial fixation after treatment with F(ab')2 fragments of goat anti-human IgM produced cells with variable antigen-presenting capacity. Optimal antigen presentation was observed for cells fixed 72 hr after the initiation of B-cell activation. Although both B7-1 and B7-2 antigen expression increased after B-cell activation, it was found that the rate of T-cell proliferation correlated most closely with B7-2 expression. Stimulation of T cells by fixed activated B lymphocytes could be blocked by antibodies directed at class II MHC molecules, indicating involvement of the T-cell antigen receptor. In addition, T-cell proliferation was inhibited by antibodies specific for B7-1 and B7-2 and by the fusion protein CTLA4-Ig, demonstrating a requirement for CD28 signal transduction. The sole requirement of B7 family expression for antigen presentation by B lymphocytes was shown by demonstration of T-cell stimulation by fixed resting B cells in the presence of CD28 antibody as a source of artificial costimulation. PMID:8550066

  18. Visualizing how T cells collect activation signals in vivo.

    PubMed

    Moreau, Hélène D; Bousso, Philippe

    2014-02-01

    A decade ago the first movies depicting T cell behavior in vivo with the help of two-photon microscopy were generated. These initial experiments revealed that T cells migrate rapidly and randomly in secondary lymphoid organs at steady state and profoundly alter their behavior during antigen recognition, establishing both transient and stable contacts with antigen-presenting cells (APCs). Since then, in vivo imaging has continuously improved our understanding of T cell activation. In particular, recent studies uncovered how T cells may be guided in their search for the best APCs. Additionally, the development of more sophisticated fluorescent tools has permitted not only to visualize T cell-APC contacts but also to probe their functional impact on T cell activation. These recent progresses are providing new insights into how T cells sense antigen, collect activation signals during distinct types of interaction and integrate information over successive encounters.

  19. Activated leukocyte cell adhesion molecule regulates the interaction between pancreatic cancer cells and stellate cells

    PubMed Central

    Zhang, Wei-Wei; Zhan, Shu-Hui; Geng, Chang-Xin; Sun, Xin; Erkan, Mert; Kleeff, Jörg; Xie, Xiang-Jun

    2016-01-01

    Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a transmembrane glycoprotein that is involved in tumor progression and metastasis. In the present study, the expression and functional role of ALCAM in pancreatic cancer cells and pancreatic stellate cells (PSCs) was investigated. Tissue specimens were obtained from patients with pancreatic ductal adenocarcinoma (n=56) or chronic pancreatitis (CP; n=10), who underwent pancreatic resection, and from normal pancreatic tissue samples (n=10). Immunohistochemistry was used to analyze the localization and expression of ALCAM in pancreatic tissues. Subsequently, reverse transcription-quantitative polymerase chain reaction and immunoblotting were applied to assess the expression of ALCAM in pancreatic cancer Panc-1 and T3M4 cells, as well as in PSCs. An enzyme-linked immunosorbent assay was used to measure ALCAM levels in cell culture medium stimulated by hypoxia, tumor necrosis factor (TNF)-α and transforming growth factor-β. Silencing of ALCAM was performed using ALCAM small interfering (si)RNA and immunocytochemistry was used to analyze the inhibition efficiency. An invasion assay and a cell interaction assay were performed to assess the invasive ability and co-cultured adhesive potential of Panc-1 and T3M4 cells, as well as PSCs. Histologically, ALCAM expression was generally weak or absent in pancreatic cancer cells, but was markedly upregulated in PSCs in pancreatic cancer tissues. ALCAM was highly expressed in PSCs from CP tissues and PSCs surrounding pancreatic intraepithelial neoplasias, as well as in pancreatic cancer cells. ALCAM mRNA was highly expressed in PSCs, with a low to moderate expression in T3M4 and Panc-1 cells. Similar to the mRNA expression, immunoblotting demonstrated that ALCAM protein levels were high in PSCs and T3M4 cells, but low in Panc-1 cells. The expression of TNF-α increased, while hypoxia decreased the secretion of ALCAM in pancreatic cancer Panc-1 and T3M4 cells, and also in

  20. Regulation of tissue factor coagulant activity on cell surfaces

    PubMed Central

    RAO, L.V.M.; PENDURTHI, U.R.

    2012-01-01

    Summary Tissue factor (TF) is a transmembrane glycoprotein and an essential component of factor VIIa-TF enzymatic complex that triggers activation of the coagulation cascade. Formation of TF-FVIIa complexes on cell surfaces not only trigger the coagulation cascade but also transduce cell signaling via activation of protease-activated receptors. Tissue factor is expressed constitutively on cell surfaces of a variety of extravascular cell types, including fibroblasts and pericytes in and surrounding blood vessel walls and epithelial cells but generally absent on cells that come in contact with blood directly. However, TF expression could be induced in some blood cells, such as monocytes and endothelial cells, following an injury or pathological stimuli. Tissue factor is essential for hemostasis, but aberrant expression of TF leads to thrombosis. Therefore, a proper regulation of TF activity is critical for the maintenance of hemostatic balance and health in general. TF-FVIIa coagulant activity at the cell surface is influenced not only by TF protein expression levels but also independently by a variety of mechanisms, including alterations in membrane phospholipid composition and cholesterol content, thiol-dependent modifications of TF allosteric disulfide bond, and other post-translational modifications of TF. In this article, we critically review key literature on mechanisms by which TF coagulant activity is regulated at the cell surface in the absence of changes in TF protein levels with specific emphasis on recently published data and provide the authors’ perspective on the subject. PMID:23006890

  1. Activated AKT regulates NF-kappaB activation, p53 inhibition and cell survival in HTLV-1-transformed cells.

    PubMed

    Jeong, Soo-Jin; Pise-Masison, Cynthia A; Radonovich, Michael F; Park, Hyeon Ung; Brady, John N

    2005-10-06

    AKT activation enhances resistance to apoptosis and induces cell survival signaling through multiple downstream pathways. We now present evidence that AKT is activated in HTLV-1-transformed cells and that Tax activation of AKT is linked to NF-kappaB activation, p53 inhibition and cell survival. Overexpression of AKT wild type (WT), but not a kinase dead (KD) mutant, resulted in increased Tax-mediated NF-kappaB activation. Blocking AKT with the PI3K/AKT inhibitor LY294002 or AKT SiRNA prevented NF-kappaB activation and inhibition of p53. Treatment of C81 cells with LY294002 resulted in an increase in the p53-responsive gene MDM2, suggesting a role for AKT in the Tax-mediated regulation of p53 transcriptional activity. Further, we show that LY294002 treatment of C81 cells abrogates in vitro IKKbeta phosphorylation of p65 and causes a reduction of p65 Ser-536 phosphorylation in vivo, steps critical to p53 inhibition. Interestingly, blockage of AKT function did not affect IKKbeta phosphorylation of IkappaBalpha in vitro suggesting selective activity of AKT on the IKKbeta complex. Finally, AKT prosurvival function in HTLV-1-transformed cells is linked to expression of Bcl-xL. We suggest that AKT plays a role in the activation of prosurvival pathways in HTLV-1-transformed cells, possibly through NF-kappaB activation and inhibition of p53 transcription activity.

  2. Cytotoxic activity of CD4+ T cells against autologous tumor cells.

    PubMed

    Konomi, Y; Sekine, T; Takayama, T; Fuji, M; Tanaka, T

    1995-09-01

    The 51Cr-release assay is mostly applied to detecting the cytotoxic activity of CD8+ T cells, and little is known about the activity of CD4+ T cells. Therefore, the correlation between the cytotoxic activity of CD4+ or CD8+ T cells and the incubation period with autologous tumor cells was analyzed by two methods. The incubation periods were 4 and 20 h (4 h and 20 h assay) for the 51Cr-release assay. Eight pairs of tumor cells and T cells were assayed. T cells were fractionated into CD4+ and CD8+ T cells by using magnetic beads and panning methods, and those cells were activated by culture with recombinant interleukin-2 and immobilized anti-CD3 monoclonal antibody. In 6 out of 8 cases, no cytotoxic activity of CD4+ T cells was detected by the 4 h assay, whereas cytotoxic activity was detected in all cases in the 20 h assay. The cytotoxic activities in 20 h assay of CD4+ T cells were increased 67-fold in comparison with the activities in 4 h assay (range: 5-197). In the case of CD8+ T cells, cytotoxic activities were detected in 6 out of 8 cases in the 4 h assay. The lytic unit ratio of CD4+ and CD8+ T cells was calculated as 1.5 in the 20 h assay (range: 0.2- > 7.2) versus 0.4 in the 4 h assay (range: < 0.1-1.3). Cytotoxic activities in colorimetric assay using Crystal Violet with a 24 h incubation were similar to those in the 20 h 51Cr-release assay in all eight cases. These results indicate that CD4+ T cells have cytotoxic activity as strong as that of CD8+ T cells towards autologous tumor cells.

  3. Automatically activated, 300 ampere-hour silver-zinc cell

    NASA Technical Reports Server (NTRS)

    Hennigan, T. J.

    1972-01-01

    A prototype silver zinc cell is reported for which the electrolyte is being stored in a separate tank; the cell is being activated when additional power is required by collapsing the neoprene bellows container and thus forcing the electrolyte into cell through a plastic connection. A solar array is proposed as main power source for the flow actuator.

  4. Reduced DNA topoisomerase II activity in ataxia-telangiectasia cells.

    PubMed Central

    Singh, S P; Mohamed, R; Salmond, C; Lavin, M F

    1988-01-01

    Considerable evidence supports a defect at the level of chromatin structure or recognition of that structure in cells from patients with the human genetic disorder ataxia-telangiectasia. Accordingly, we have investigated the activities of enzymes that alter the topology of DNA in Epstein Barr Virus-transformed lymphoblastoid cells from patients with this syndrome. Reduced activity of DNA topoisomerase II, determined by unknotting of P4 phage DNA, was observed in partially purified extracts from 5 ataxia-telangiectasia cell lines. The levels of enzyme activity was reduced substantially in 4 of these cell lines and to a lesser extent in the other cell line compared to controls. DNA topoisomerase I, assayed by relaxation of supercoiled DNA, was found to be present at comparable levels in both cell types. Reduced activity of topoisomerase II in ataxia-telangiectasia is compatible with the molecular, cellular and clinical changes described in this syndrome. Images PMID:2836804

  5. Effects of Neuroendocrine CB1 Activity on Adult Leydig Cells

    PubMed Central

    Cobellis, Gilda; Meccariello, Rosaria; Chianese, Rosanna; Chioccarelli, Teresa; Fasano, Silvia; Pierantoni, Riccardo

    2016-01-01

    Endocannabinoids control male reproduction acting at central and local level via cannabinoid receptors. The cannabinoid receptor CB1 has been characterized in the testis, in somatic and germ cells of mammalian and non-mammalian animal models, and its activity related to Leydig cell differentiation, steroidogenesis, spermiogenesis, sperm quality, and maturation. In this short review, we provide a summary of the insights concerning neuroendocrine CB1 activity in male reproduction focusing on adult Leydig cell ontogenesis and steroid biosynthesis. PMID:27375550

  6. Calcium alloy as active material in secondary electrochemical cell

    DOEpatents

    Roche, Michael F.; Preto, Sandra K.; Martin, Allan E.

    1976-01-01

    Calcium alloys such as calcium-aluminum and calcium-silicon, are employed as active material within a rechargeable negative electrode of an electrochemical cell. Such cells can use a molten salt electrolyte including calcium ions and a positive electrode having sulfur, sulfides, or oxides as active material. The calcium alloy is selected to prevent formation of molten calcium alloys resulting from reaction with the selected molten electrolytic salt at the cell operating temperatures.

  7. Microwave-induced thermogenetic activation of single cells

    SciTech Connect

    Safronov, N. A.; Fedotov, I. V.; Ermakova, Yu. G.; Matlashov, M. E.; Belousov, V. V.; Sidorov-Biryukov, D. A.; Fedotov, A. B.; Zheltikov, A. M.

    2015-04-20

    Exposure to a microwave field is shown to enable thermogenetic activation of individual cells in a culture of cell expressing thermosensitive ion channels. Integration of a microwave transmission line with an optical fiber and a diamond quantum thermometer has been shown to allow thermogenetic single-cell activation to be combined with accurate local online temperature measurements based on an optical detection of electron spin resonance in nitrogen–vacancy centers in diamond.

  8. Melanoma cells inhibit natural killer cell function by modulating the expression of activating receptors and cytolytic activity.

    PubMed

    Pietra, Gabriella; Manzini, Claudia; Rivara, Silvia; Vitale, Massimo; Cantoni, Claudia; Petretto, Andrea; Balsamo, Mirna; Conte, Romana; Benelli, Roberto; Minghelli, Simona; Solari, Nicola; Gualco, Marina; Queirolo, Paola; Moretta, Lorenzo; Mingari, Maria Cristina

    2012-03-15

    Natural killer (NK) cells play a key role in tumor immune surveillance. However, adoptive immunotherapy protocols using NK cells have shown limited clinical efficacy to date, possibly due to tumor escape mechanisms that inhibit NK cell function. In this study, we analyzed the effect of coculturing melanoma cells and NK cells on their phenotype and function. We found that melanoma cells inhibited the expression of major NK receptors that trigger their immune function, including NKp30, NKp44, and NKG2D, with consequent impairment of NK cell-mediated cytolytic activity against various melanoma cell lines. This inhibitory effect was primarily mediated by indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2). Together, our findings suggest that immunosuppressive barriers erected by tumors greatly hamper the antitumor activity of human NK cells, thereby favoring tumor outgrowth and progression.

  9. Effects of dexamethasone on palate mesenchymal cell phospholipase activity

    SciTech Connect

    Bulleit, R.F.; Zimmerman, E.F.

    1984-09-15

    Corticosteroids will induce cleft palate in mice. One suggested mechanism for this effect is through inhibition of phospholipase activity. This hypothesis was tested by measuring the effects of dexamethasone, a synthetic corticosteroid, on phospholipase activity in cultures of palate mesenchymal cells. Palate mesenchymal cells were prelabeled with (3H)arachidonic acid. The cells were subsequently treated with various concentrations of dexamethasone. Concurrently, cultures of M-MSV-transformed 3T3 cells were prepared identically. After treatment, phospholipase activity was stimulated by the addition of serum or epidermal growth factor (EGF), and radioactivity released into the medium was taken as a measure of phospholipase activity. Dexamethasone (1 X 10(-5) or 1 X 10(-4) M) could inhibit serum-stimulated phospholipase activity in transformed 3T3 cells after 1 to 24 hr of treatment. However, no inhibition of activity was measured in palate mesenchymal cells following this period of treatment. Not until 120 hr of treatment with dexamethasone (1 X 10(-4) M) was any significant inhibition of serum-stimulated phospholipase activity observed in palate mesenchymal cells. When EGF was used to stimulate phospholipase activity, dexamethasone (1 X 10(-5) M) caused an increase in phospholipase activity in palate mesenchymal cells. These observations suggested that phospholipase in transformed 3T3 cells was sensitive to inhibition by dexamethasone. However, palate mesenchymal cell phospholipase is only minimally sensitive to dexamethasone, and in certain instances can be enhanced. These results cannot support the hypothesis that corticosteroids mediate their teratogenic effect via inhibition of phospholipase activity.

  10. Dormancy activation mechanism of oral cavity cancer stem cells.

    PubMed

    Chen, Xiang; Li, Xin; Zhao, Baohong; Shang, Dehao; Zhong, Ming; Deng, Chunfu; Jia, Xinshan

    2015-07-01

    Radiotherapy and chemotherapy are targeted primarily at rapidly proliferating cancer cells and are unable to eliminate cancer stem cells in the G0 phase. Thus, these treatments cannot prevent the recurrence and metastasis of cancer. Understanding the mechanisms by which cancer stem cells are maintained in the dormant G0 phase, and how they become active is key to developing new cancer therapies. The current study found that the anti-cancer drug 5-fluorouracil, acting on the oral squamous cell carcinoma KB cell line, selectively killed proliferating cells while sparing cells in the G0 phase. Bisulfite sequencing PCR showed that demethylation of the Sox2 promoter led to the expression of Sox2. This then resulted in the transformation of cancer stem cells from the G0 phase to the division stage and suggested that the transformation of cancer stem cells from the G0 phase to the division stage is closely related to an epigenetic modification of the cell.

  11. Nylon Wool Purification Alters the Activation of T Cells

    PubMed Central

    Wohler, Jillian E.; Barnum, Scott R.

    2009-01-01

    Purification of lymphocytes, particularly T cells, is commonly performed using nylon wool. This enrichment method selectively retains B cells and some myeloid cells allowing a significantly more pure T cell population to flow through a nylon wool column. T cells purified in this fashion are assumed to be unaltered and functionally naïve, however some studies have suggested aberrant in vitro T cell responses after nylon wool treatment. We found that nylon wool purification significantly altered T cell proliferation, expression of activation markers and production of cytokines. Our results suggest that nylon wool treatment modifies T cell activation responses and that caution should be used when choosing this purification method. PMID:18952296

  12. Cooperativity of peptidoglycan synthases active in bacterial cell elongation.

    PubMed

    Banzhaf, Manuel; van den Berg van Saparoea, Bart; Terrak, Mohammed; Fraipont, Claudine; Egan, Alexander; Philippe, Jules; Zapun, André; Breukink, Eefjan; Nguyen-Distèche, Martine; den Blaauwen, Tanneke; Vollmer, Waldemar

    2012-07-01

    Growth of the bacterial cell wall peptidoglycan sacculus requires the co-ordinated activities of peptidoglycan synthases, hydrolases and cell morphogenesis proteins, but the details of these interactions are largely unknown. We now show that the Escherichia coli peptidoglycan glycosyltrasferase-transpeptidase PBP1A interacts with the cell elongation-specific transpeptidase PBP2 in vitro and in the cell. Cells lacking PBP1A are thinner and initiate cell division later in the cell cycle. PBP1A localizes mainly to the cylindrical wall of the cell, supporting its role in cell elongation. Our in vitro peptidoglycan synthesis assays provide novel insights into the cooperativity of peptidoglycan synthases with different activities. PBP2 stimulates the glycosyltransferase activity of PBP1A, and PBP1A and PBP2 cooperate to attach newly synthesized peptidoglycan to sacculi. PBP2 has peptidoglycan transpeptidase activity in the presence of active PBP1A. Our data also provide a possible explanation for the depletion of lipid II precursors in penicillin-treated cells.

  13. BMP2 Transfer to Neighboring Cells and Activation of Signaling.

    PubMed

    Alborzinia, Hamed; Shaikhkarami, Marjan; Hortschansky, Peter; Wölfl, Stefan

    2016-09-01

    Morphogen gradients and concentration are critical features during early embryonic development and cellular differentiation. Previously we reported the preparation of biologically active, fluorescently labeled BMP2 and quantitatively analyzed their binding to the cell surface and followed BMP2 endocytosis over time on the level of single endosomes. Here we show that this internalized BMP2 can be transferred to neighboring cells and, moreover, also activates downstream BMP signaling in adjacent cells, indicated by Smad1/5/8 phosphorylation and activation of the downstream target gene id1. Using a 3D matrix to modulate cell-cell contacts in culture we could show that direct cell-cell contact significantly increased BMP2 transfer. Using inhibitors of vesicular transport, transfer was strongly inhibited. Interestingly, cotreatment with the physiological BMP inhibitor Noggin increased BMP2 uptake and transfer, albeit activation of Smad signaling in neighboring cells was completely suppressed. Our findings present a novel and interesting mechanism by which morphogens such as BMP2 can be transferred between cells and how this is modulated by BMP antagonists such as Noggin, and how this influences activation of Smad signaling by BMP2 in neighboring cells.

  14. Phenotypic Approaches to Identify Inhibitors of B Cell Activation

    PubMed Central

    Kim, Suzie; Wiener, Jake; Rao, Navin L.; Milla, Marcos E.; DiSepio, Daniel

    2015-01-01

    An EPIC label-free phenotypic platform was developed to explore B cell receptor (BCR) and CD40R-mediated B cell activation. The phenotypic assay measured the association of RL non-Hodgkin’s lymphoma B cells expressing lymphocyte function-associated antigen 1 (LFA-1) to intercellular adhesion molecule 1 (ICAM-1)-coated EPIC plates. Anti-IgM (immunoglobulin M) mediated BCR activation elicited a response that was blocked by LFA-1/ICAM-1 specific inhibitors and a panel of Bruton’s tyrosine kinase (BTK) inhibitors. LFA-1/ICAM-1 association was further increased on coapplication of anti-IgM and mega CD40L when compared to individual application of either. Anti-IgM, mega CD40L, or the combination of both displayed distinct kinetic profiles that were inhibited by treatment with a BTK inhibitor. We also established a FLIPR-based assay to measure B cell activation in Ramos Burkitt’s lymphoma B cells and an RL cell line. Anti-IgM-mediated BCR activation elicited a robust calcium response that was inhibited by a panel of BTK inhibitors. Conversely, CD40R activation did not elicit a calcium response in the FLIPR assay. Compared to the FLIPR, the EPIC assay has the propensity to identify inhibitors of both BCR and CD40R-mediated B cell activation and may provide more pharmacological depth or novel mechanisms of action for inhibition of B cell activation. PMID:25948491

  15. Mitogen-activated Tasmanian devil blood mononuclear cells kill devil facial tumour disease cells.

    PubMed

    Brown, Gabriella K; Tovar, Cesar; Cooray, Anne A; Kreiss, Alexandre; Darby, Jocelyn; Murphy, James M; Corcoran, Lynn M; Bettiol, Silvana S; Lyons, A Bruce; Woods, Gregory M

    2016-08-01

    Devil facial tumour disease (DFTD) is a transmissible cancer that has brought the host species, the Tasmanian devil, to the brink of extinction. The cancer cells avoid allogeneic immune recognition by downregulating cell surface major histocompatibility complex (MHC) I expression. This should prevent CD8(+) T cell, but not natural killer (NK) cell, cytotoxicity. The reason why NK cells, normally reactive to MHC-negative cells, are not activated to kill DFTD cells has not been determined. The immune response of wild devils to DFTD, if it occurs, is uncharacterised. To investigate this, we tested 12 wild devils with DFTD, and found suggestive evidence of low levels of antibodies against DFTD cells in one devil. Eight of these devils were also analysed for cytotoxicity, however, none showed evidence for cytotoxicity against cultured DFTD cells. To establish whether mimicking activation of antitumour responses could induce cytotoxic activity against DFTD, Tasmanian devil peripheral blood mononuclear cells (PBMCs) were treated with either the mitogen Concanavalin A, the Toll-like receptor agonist polyinosinic:polycytidylic acid or recombinant Tasmanian devil IL-2. All induced the PBMC cells to kill cultured DFTD cells, suggesting that activation does not occur after encounter with DFTD cells in vivo, but can be induced. The identification of agents that activate cytotoxicity against DFTD target cells is critical for developing strategies to protect against DFTD. Such agents could function as adjuvants to induce functional immune responses capable of targeting DFTD cells and tumours in vivo.

  16. Magnetic Field-Induced T Cell Receptor Clustering by Nanoparticles Enhances T Cell Activation and Stimulates Antitumor Activity

    PubMed Central

    2015-01-01

    Iron–dextran nanoparticles functionalized with T cell activating proteins have been used to study T cell receptor (TCR) signaling. However, nanoparticle triggering of membrane receptors is poorly understood and may be sensitive to physiologically regulated changes in TCR clustering that occur after T cell activation. Nano-aAPC bound 2-fold more TCR on activated T cells, which have clustered TCR, than on naive T cells, resulting in a lower threshold for activation. To enhance T cell activation, a magnetic field was used to drive aggregation of paramagnetic nano-aAPC, resulting in a doubling of TCR cluster size and increased T cell expansion in vitro and after adoptive transfer in vivo. T cells activated by nano-aAPC in a magnetic field inhibited growth of B16 melanoma, showing that this novel approach, using magnetic field-enhanced nano-aAPC stimulation, can generate large numbers of activated antigen-specific T cells and has clinically relevant applications for adoptive immunotherapy. PMID:24564881

  17. Tubular solid oxide fuel cell demonstration activities

    SciTech Connect

    Veyo, S.E.

    1995-08-01

    The development of a viable fuel cell driven electrical power generation system involves not only the development of cell and stack technology, but also the development of the overall system concept, the strategy for control, and the ancillary subsystems. The design requirements used to guide system development must reflect a customer focus in order to evolve a commercial product. In order to obtain useful customer feedback, Westinghouse has practiced the deployment with customers of fully integrated, automatically controlled, packaged solid oxide fuel cell power generation systems. These field units have served to demonstrate to customers first hand the beneficial attributes of the SOFC, to expose deficiencies through experience in order to guide continued development, and to garner real world feedback and data concerning not only cell and stack parameters, but also transportation, installation, permitting and licensing, start-up and shutdown, system alarming, fault detection, fault response, and operator interaction.

  18. Activation of B cells by non-canonical helper signals.

    PubMed

    Cerutti, Andrea; Cols, Montserrat; Puga, Irene

    2012-09-01

    Cognate interaction between T and B lymphocytes of the adaptive immune system is essential for the production of high-affinity antibodies against microbes, and for the establishment of long-term immunological memory. Growing evidence shows that--in addition to presenting antigens to T and B cells--macrophages, dendritic cells and other cells of the innate immune system provide activating signals to B cells, as well as survival signals to antibody-secreting plasma cells. Here, we discuss how these innate immune cells contribute to the induction of highly diversified and temporally sustained antibody responses, both systemically and at mucosal sites of antigen entry.

  19. Substrate Stiffness Regulates Filopodial Activities in Lung Cancer Cells

    PubMed Central

    Liou, Yu-Ren; Torng, Wen; Kao, Yu-Chiu; Sung, Kung-Bin; Lee, Chau-Hwang; Kuo, Po-Ling

    2014-01-01

    Microenvironment stiffening plays a crucial role in tumorigenesis. While filopodia are generally thought to be one of the cellular mechanosensors for probing environmental stiffness, the effects of environmental stiffness on filopodial activities of cancer cells remain unclear. In this work, we investigated the filopodial activities of human lung adenocarcinoma cells CL1-5 cultured on substrates of tunable stiffness using a novel platform. The platform consists of an optical system called structured illumination nano-profilometry, which allows time-lapsed visualization of filopodial activities without fluorescence labeling. The culturing substrates were composed of polyvinyl chloride mixed with an environmentally friendly plasticizer to yield Young's modulus ranging from 20 to 60 kPa. Cell viability studies showed that the viability of cells cultured on the substrates was similar to those cultured on commonly used elastomers such as polydimethylsiloxane. Time-lapsed live cell images were acquired and the filopodial activities in response to substrates with varying degrees of stiffness were analyzed. Statistical analyses revealed that lung cancer cells cultured on softer substrates appeared to have longer filopodia, higher filopodial densities with respect to the cellular perimeter, and slower filopodial retraction rates. Nonetheless, the temporal analysis of filopodial activities revealed that whether a filopodium decides to extend or retract is purely a stochastic process without dependency on substrate stiffness. The discrepancy of the filopodial activities between lung cancer cells cultured on substrates with different degrees of stiffness vanished when the myosin II activities were inhibited by treating the cells with blebbistatin, which suggests that the filopodial activities are closely modulated by the adhesion strength of the cells. Our data quantitatively relate filopodial activities of lung cancer cells with environmental stiffness and should shed light

  20. A Single Subset of Dendritic Cells Controls the Cytokine Bias of Natural Killer T Cell Responses to Diverse Glycolipid Antigens

    PubMed Central

    Arora, Pooja; Baena, Andres; Yu, Karl O.A.; Saini, Neeraj K.; Kharkwal, Shalu S.; Goldberg, Michael F.; Kunnath-Velayudhan, Shajo; Carreño, Leandro J.; Venkataswamy, Manjunatha M.; Kim, John; Lazar-Molnar, Eszter; Lauvau, Gregoire; Chang, Young-tae; Liu, Zheng; Bittman, Robert; Al-Shamkhani, Aymen; Cox, Liam R.; Jervis, Peter J.; Veerapen, Natacha; Besra, Gurdyal S.; Porcelli, Steven A.

    2014-01-01

    Summary Many hematopoietic cell types express CD1d and are capable of presenting glycolipid antigens to invariant natural killer T cells (iNKT cells). However, the question of which cells are the principal presenters of glycolipid antigens in vivo remains controversial, and it has been suggested that this might vary depending on the structure of a particular glycolipid antigen. Here we have shown that a single type of cell, the CD8α+ DEC-205+ dendritic cell, was mainly responsible for capturing and presenting a variety of different glycolipid antigens, including multiple forms of α-galactosylceramide that stimulate widely divergent cytokine responses. After glycolipid presentation, these dendritic cells rapidly altered their expression of various costimulatory and coinhibitory molecules in a manner that was dependent on the structure of the antigen. These findings show flexibility in the outcome of two-way communication between CD8α+ dendritic cells and iNKT cells, providing a mechanism for biasing toward either proinflammatory or anti-inflammatory responses. PMID:24412610

  1. A single subset of dendritic cells controls the cytokine bias of natural killer T cell responses to diverse glycolipid antigens.

    PubMed

    Arora, Pooja; Baena, Andres; Yu, Karl O A; Saini, Neeraj K; Kharkwal, Shalu S; Goldberg, Michael F; Kunnath-Velayudhan, Shajo; Carreño, Leandro J; Venkataswamy, Manjunatha M; Kim, John; Lazar-Molnar, Eszter; Lauvau, Gregoire; Chang, Young-tae; Liu, Zheng; Bittman, Robert; Al-Shamkhani, Aymen; Cox, Liam R; Jervis, Peter J; Veerapen, Natacha; Besra, Gurdyal S; Porcelli, Steven A

    2014-01-16

    Many hematopoietic cell types express CD1d and are capable of presenting glycolipid antigens to invariant natural killer T cells (iNKT cells). However, the question of which cells are the principal presenters of glycolipid antigens in vivo remains controversial, and it has been suggested that this might vary depending on the structure of a particular glycolipid antigen. Here we have shown that a single type of cell, the CD8α(+) DEC-205(+) dendritic cell, was mainly responsible for capturing and presenting a variety of different glycolipid antigens, including multiple forms of α-galactosylceramide that stimulate widely divergent cytokine responses. After glycolipid presentation, these dendritic cells rapidly altered their expression of various costimulatory and coinhibitory molecules in a manner that was dependent on the structure of the antigen. These findings show flexibility in the outcome of two-way communication between CD8α(+) dendritic cells and iNKT cells, providing a mechanism for biasing toward either proinflammatory or anti-inflammatory responses.

  2. T Cell Activation Thresholds are Affected by Gravitational

    NASA Technical Reports Server (NTRS)

    Adams, Charley; Gonzalez, M.; Nelman-Gonzalez, M.

    1999-01-01

    T cells stimulated in space flight by various mitogenic signals show a dramatic reduction in proliferation and expression of early activation markers. Similar results are also obtained in a ground based model of microgravity, clinorotation, which provides a vector-averaged reduction of the apparent gravity on cells without significant shear force. Here we demonstrate that T cell inhibition is due to an increase in the required threshold for activation. Dose response curves indicate that cells activated during clinorotation require higher stimulation to achieve the same level of activation, as measured by CD69 expression. Interleukin 2 receptor expression, and DNA synthesis. The amount of stimulation necessary for 50% activation is 5 fold in the clinostat relative to static. Correlation of TCR internalization with activation also exhibit a dramatic right shift in clinorotation, demonstrating unequivocally that signal transduction mechanism independent of TCR triggering account for the increased activation threshold. Previous results from space flight experiments are consistent with the dose response curves obtained for clinorotation. Activation thresholds are important aspects of T cell memory, autoimmunity and tolerance Clinorotation is a useful, noninvasive tool for the study of cellular and biochemical event regulating T cell activation threshold and the effects of gravitation forces on these systems.

  3. Detection of activity of single microalgae cells in a new microfluidic cell capturing chip

    NASA Astrophysics Data System (ADS)

    Wang, Junsheng; Meng, Xiongfei; Song, Yongxin; Pan, Xinxiang; Li, Dongqing

    2016-12-01

    The analysis of single microalgae cell plays a very important role in understanding the activity of microalgae cell. In this paper, a new method of capturing and monitoring a microalgae cell is presented. This method uses the surface of an air bubble formed in an aqueous solution in a microchannel to capture a microalgae cell. The aliveness of the captured microalgae cell can be monitored quantitatively by measuring chlorophyll fluorescence intensity of the microalgae cell. To demonstrate the performance of this method, two species of microalgae cells, Dunaliella salina and Tetraselmis Chui, were taken as samples. The effect of pH on the cell capture was studied experimentally. The cells were treated by NaClO or Formaldehyde solutions of different concentrations. The kinetics of the photosynthesis activity of the captured single microalgae cells was investigated under different treatment conditions. The results show that the viability of single microalgae cells can be studied individually and accurately by this method.

  4. Aldehyde dehydrogenase activity in cancer stem cells from canine mammary carcinoma cell lines.

    PubMed

    Michishita, M; Akiyoshi, R; Suemizu, H; Nakagawa, T; Sasaki, N; Takemitsu, H; Arai, T; Takahashi, K

    2012-08-01

    Increasing evidence suggests that diverse solid tumours arise from a small population of cells known as cancer stem cells or tumour-initiating cells. Cancer stem cells in several solid tumours are enriched for aldehyde dehydrogenase (ALDH) activity. High levels of ALDH activity (ALDH(high)) were detected in four cell lines derived from canine mammary carcinomas. ALDH(high) cells were enriched in a CD44(+)CD24(-) population having self-renewal capacity. Xenotransplantation into immunodeficient mice demonstrated that 1×10(4) ALDH(high) cells were sufficient for tumour formation in all injected mice, whereas 1×10(4) ALDH(low) cells failed to initiate any tumours. ALDH(high)-derived tumours contained both ALDH(+) and ALDH(-) cells, indicating that these cells had cancer stem cell-like properties.

  5. Retinal Pigment Epithelial Cell Line Suppression of Phagolysosome Activation.

    PubMed

    Taylor, A W; Dixit, S; Yu, J

    2015-01-29

    The eye is an immune privileged tissue with multiple mechanisms of immunosuppression to protect the light gathering tissues from the damage of inflammation. One of theses mechanisms involves retinal pigment epithelial cell suppression of phagosome activation in macrophages. The objective of this work is to determine if the human RPE cell line ARPE-19 is capable of suppressing the activation of the phagolysosome in macrophages in a manner similar to primary RPE. The conditioned media of RPE eyecups, sub-confluent, just confluent cultures, or established confluent cultures of human ARPE-19 cells were generated. These condition media were used to treat macrophages phagocytizing pHrodo bioparticles. After 24 hours incubation the macrophages were imaged by fluorescent microscopy, and fluorescence was measured. The fluorescent intensity is proportional to the amount of bioparticles phagocytized and are in an activated phagolysosome. The conditioned media of in situ mouse RPE eyecups significantly suppressed the activation of phagolysosome. The conditioned media from cultures of human ARPE-19 cells, grown to sub-confluence (50%) or grown to confluence had no effect on phagolysosome activation. In contrast, the conditioned media from established confluent cultures significantly suppressed phagolysosome activation. The neuropeptides alpha-MSH and NPY were depleted from the conditioned media of established confluent ARPE-19 cell cultures. This depleted conditioned media had diminished suppression of phagolysosome activation while promoting macrophage cell death. In addition, the condition media from cultures of ARPE-19 monolayers wounded with a bisecting scrape was diminished in suppressing phagolysosome activation. This technical report suggests that like primary RPE monolayers, established confluent cultures of ARPE-19 cells produce soluble factors that suppress the activation of macrophages, and can be used to study the molecular mechanisms of retinal immunobiology. In

  6. Determination of telomerase activity in stem cells and non-stem cells of breast cancer.

    PubMed

    Li, Zhi; He, Yanli; Zhang, Jiahua; Zhang, Jinghui; Huang, Tao

    2007-07-01

    Although all normal tissue cells, including stem cells, are genetically homologous, variation in gene expression patterns has already determined the distinct roles for individual cells in the physiological process due to the occurrence of epigenetic modification. This is of special importance for the existence of tissue stem cells because they are exclusively immortal within the body, capable of self-replicating and differentiating by which tissues renew and repair itself and the total tissue cell population maintains a steady-state. Impairment of tissue stem cells is usually accompanied by a reduction in cell number, slows down the repair process and causes hypofunction. For instance, chemotherapy usually leads to depression of bone marrow and hair loss. Cellular aging is closely associated with the continuous erosion of the telomere while activation of telomerase repairs and maintains telomeres, thus slowing the aging process and prolonging cell life. In normal adults, telomerase activation mainly presents in tissue stem cells and progenitor cells giving them unlimited growth potential. Despite the extensive demonstration of telomerase activation in malignancy (> 80%), scientists found that heterogeneity also exists among the tumor cells and only minorities of cells, designated as cancer stem cells, undergo processes analogous to the self-renewal and differentiation of normal stem cells while the rest have limited lifespans. In this study, telomerase activity was measured and compared in breast cancer stem cells and non-stem cells that were phenotypically sorted by examining surface marker expression. The results indicated that cancer stem cells show a higher level of enzyme activity than non-stem cells. In addition, associated with the repair of cancer tissue (or relapse) after chemotherapy, telomerase activity in stem cells was markedly increased.

  7. An alpha-galactosylceramide C20:2 N-acyl variant enhances anti-inflammatory and regulatory T cell-independent responses that prevent type 1 diabetes.

    PubMed

    Ly, D; Tohn, R; Rubin, B; Blumenfeld, H; Besra, G S; Veerapen, N; Porcelli, S A; Delovitch, T L

    2010-05-01

    Protection from type 1 diabetes (T1D), a T helper type 1 (Th1)-mediated disease, is achievable in non-obese diabetic (NOD) mice by treatment with alpha-galactosylceramide (alpha-GalCer) glycolipids that stimulate CD1d-restricted invariant natural killer T (iNK T) cells. While we have reported previously that the C20:2 N-acyl variant of alpha-GalCer elicits a Th2-biased cytokine response and protects NOD mice from T1D more effectively than a form of alpha-GalCer that induces mixed Th1 and Th2 responses, it remained to determine whether this protection is accompanied by heightened anti-inflammatory responses. We show that treatment of NOD mice with C20:2 diminished the activation of 'inflammatory' interleukin (IL)-12 producing CD11c(high)CD8+ myeloid dendritic cells (mDCs) and augmented the function of 'tolerogenic' DCs more effectively than treatment with the prototypical iNKT cell activator KRN7000 (alpha-GalCer C26:0) that induces Th1- and Th2-type responses. These findings correlate with a reduced capacity of C20:2 to sustain the early transactivation of T, B and NK cells. They may also explain our observation that C20:2 activated iNK T cells depend less than KRN7000 activated iNK T cells upon regulation by regulatory T cells for cytokine secretion and protection from T1D. The enhanced anti-inflammatory properties of C20:2 relative to KRN7000 suggest that C20:2 should be evaluated further as a drug to induce iNK T cell-mediated protection from T1D in humans.

  8. MERTK as negative regulator of human T cell activation

    PubMed Central

    Cabezón, Raquel; Carrera-Silva, E. Antonio; Flórez-Grau, Georgina; Errasti, Andrea E.; Calderón-Gómez, Elisabeth; Lozano, Juan José; España, Carolina; Ricart, Elena; Panés, Julián; Rothlin, Carla Vanina; Benítez-Ribas, Daniel

    2015-01-01

    The aim of this study was to test the hypothesis whether MERTK, which is up-regulated in human DCs treated with immunosuppressive agents, is directly involved in modulating T cell activation. MERTK is a member of the TAM family and contributes to regulating innate immune response to ACs by inhibiting DC activation in animal models. However, whether MERTK interacts directly with T cells has not been addressed. Here, we show that MERTK is highly expressed on dex-induced human tol-DCs and participates in their tolerogenic effect. Neutralization of MERTK in allogenic MLR, as well as autologous DC–T cell cultures, leads to increased T cell proliferation and IFN-γ production. Additionally, we identify a previously unrecognized noncell-autonomous regulatory function of MERTK expressed on DCs. Mer-Fc protein, used to mimic MERTK on DCs, suppresses naïve and antigen-specific memory T cell activation. This mechanism is mediated by the neutralization of the MERTK ligand PROS1. We find that MERTK and PROS1 are expressed in human T cells upon TCR activation and drive an autocrine proproliferative mechanism. Collectively, these results suggest that MERTK on DCs controls T cell activation and expansion through the competition for PROS1 interaction with MERTK in the T cells. In conclusion, this report identified MERTK as a potent suppressor of T cell response. PMID:25624460

  9. Protein kinase C activators inhibit capillary endothelial cell growth

    SciTech Connect

    Doctrow, S.R.

    1986-05-01

    Phorbol 12,13-dibutyrate (PDBu) binds specifically to bovine capillary endothelial (BCE) cells (K/sub d/ = 8nM) and inhibits the proliferation (K/sub 50/ = 6 +/- 4 nM). Under similar conditions, PDBu does not inhibit the growth of bovine aortic endothelial or smooth muscle cells. PDBu markedly attenuates the response of BCE cells to purified human hepatoma-derived growth factor which, in the absence of PDBu, stimulates BCE cell growth by about 3-fold. Several observations suggest that the inhibition of BCE cell growth by PDBu is mediated by protein kinase C: (1) different phorbol compounds inhibit BCE cell growth according to the relative potencies as protein kinase C activators (12-tetradecanoylphorbol 13-acetate > PDBu >> phorbol 12,13-diacetate >>>..beta..-phorbol; ..cap alpha..-phorbol 12,13-didecanoate). (2) Specific binding of PDBu to BCE cells is displaced by sn-1,2-dioctanoylglycerol (diC/sub 8/), a protein kinase C activator and an analog of the putative second messenger activating this kinase in vivo. The weak protein kinase C activator, sn-1,2-dibutyrylglycerol, does not affect PDBu binding. (3) A cytosolic extract from BCE cells contains a Ca/sup 2 +//phosphatidylserine-dependent kinase that is activated by diC/sub 8/ and PDBu, but not by ..beta..-phorbol. These results support a role for protein kinase C in suppressing capillary endothelial cell growth and may therefore have implications in the intracellular regulation of angiogenesis.

  10. Growth Factor Midkine Promotes T-Cell Activation through Nuclear Factor of Activated T Cells Signaling and Th1 Cell Differentiation in Lupus Nephritis.

    PubMed

    Masuda, Tomohiro; Maeda, Kayaho; Sato, Waichi; Kosugi, Tomoki; Sato, Yuka; Kojima, Hiroshi; Kato, Noritoshi; Ishimoto, Takuji; Tsuboi, Naotake; Uchimura, Kenji; Yuzawa, Yukio; Maruyama, Shoichi; Kadomatsu, Kenji

    2017-04-01

    Activated T cells play crucial roles in the pathogenesis of autoimmune diseases, including lupus nephritis (LN). The activation of calcineurin/nuclear factor of activated T cells (NFAT) and STAT4 signaling is essential for T cells to perform various effector functions. Here, we identified the growth factor midkine (MK; gene name, Mdk) as a novel regulator in the pathogenesis of 2,6,10,14-tetramethylpentadecane-induced LN via activation of NFAT and IL-12/STAT4 signaling. Wild-type (Mdk(+/+)) mice showed more severe glomerular injury than MK-deficient (Mdk(-/-)) mice, as demonstrated by mesangial hypercellularity and matrix expansion, and glomerular capillary loops with immune-complex deposition. Compared with Mdk(-/-) mice, the frequency of splenic CD69(+) T cells and T helper (Th) 1 cells, but not of regulatory T cells, was augmented in Mdk(+/+) mice in proportion to LN disease activity, and was accompanied by skewed cytokine production. MK expression was also enhanced in activated CD4(+) T cells in vivo and in vitro. MK induced activated CD4(+) T cells expressing CD69 through nuclear activation of NFAT transcription and selectively increased in vitro differentiation of naive CD4(+) T cells into Th1 cells by promoting IL-12/STAT4 signaling. These results suggest that MK serves an indispensable role in the NFAT-regulated activation of CD4(+) T cells and Th1 cell differentiation, eventually leading to the exacerbation of LN.

  11. Cytotoxic activity of natural killer cells in vitro under microgravity

    NASA Astrophysics Data System (ADS)

    Grigorieva, O. V.; Buravkova, L. B.; Rykova, M. P.

    2005-08-01

    Changes in the immune response during space flight are close relation to functions of NK lymphocytes and their ability to interact with target cells. The aim of this research was to study NK cells cytotoxic activity and their ability to produce cytokines under microgravity in vitro. The modification of the method to study NK cells cytotoxic activity with the use of human peripheral blood mononuclear cells and myeloblasts K-562 (as target cells) proved highly effective (Buravkova et al., 2004). The flight experiment "Cell-to-cell interaction" with the use of the special device "Fibroblast-1" was carried out by Russian cosmonauts within the first two days after the docking when a new crew was taking over on International Space Station (ISS 8 - 10). The data collected on board ISS revealed that NK lymphocytes cytotoxic activity in vitro can increase under microgravity. The ground-based simulation experiments showed that long-term changes in gravity vector direction clinorotation resulted in a smaller increase of NK cells cytotoxic activity than it did in microgravity. As lymphocytes produce cytokines while interacting with target cells, the levels of TNF-α, IL-1α, IL- 2, IL-6 in cell-conditioned medium were assessed. The data showed that microgravity has varied effects on cytokines production level.

  12. Activation of ion transport systems during cell volume regulation

    SciTech Connect

    Eveloff, J.L.; Warnock, D.G.

    1987-01-01

    This review discusses the activation of transport pathways during volume regulation, including their characteristics, the possible biochemical pathways that may mediate the activation of transport pathways, and the relations between volume regulation and transepithelial transport in renal cells. Many cells regulate their volume when exposed to an anisotonic medium. The changes in cell volume are caused by activation of ion transport pathways, plus the accompanying osmotically driven water movement such that cell volume returns toward normal levels. The swelling of hypertonically shrunken cells is termed regulatory volume increase (RVI) and involves an influx of NaCl into the cell via either activation of Na-Cl, Na-K-2Cl cotransport systems, or Na/sup +/-H/sup +/ and Cl/sup -/-HCO/sub 3//sup -/ exchangers. The reshrinking of hypotonically swollen cells is termed regulatory volume decrease (RVD) and involves an efflux of KCl and water from the cell by activation of either separate K/sup +/ and Cl/sup -/ conductances, a K-Cl cotransport system, or parallel K/sup +/-H/sup +/ and Cl/sup -/-HCO/sub 3//sup -/ exchangers. The biochemical mechanisms involved in the activation of transport systems are largely unknown, however, the phosphoinositide pathway may be implicated in RVI; phorbol esters, cGMP, and Ca/sup 2 +/ affect the process of volume regulation. Renal tubular cells, as well as the blood cells that transverse the medulla, are subjected to increasing osmotic gradients from the corticomedullary junction to the papillary tip, as well as changing interstitial and tubule fluid osmolarity, depending on the diuretic state of the animal. Medullary cells from the loop of Henle and the papilla can volume regulate by activating Na-K-2Cl cotransport or Na/sup +/-H/sup +/ and Cl/sup -/-HCO/sub 3//sup -/ exchange systems.

  13. M-cadherin-mediated intercellular interactions activate satellite cell division.

    PubMed

    Marti, Merce; Montserrat, Núria; Pardo, Cristina; Mulero, Lola; Miquel-Serra, Laia; Rodrigues, Alexandre Miguel Cavaco; Andrés Vaquero, José; Kuebler, Bernd; Morera, Cristina; Barrero, María José; Izpisua Belmonte, Juan Carlos

    2013-11-15

    Adult muscle stem cells and their committed myogenic precursors, commonly referred to as the satellite cell population, are involved in both muscle growth after birth and regeneration after damage. It has been previously proposed that, under these circumstances, satellite cells first become activated, divide and differentiate, and only later fuse to the existing myofiber through M-cadherin-mediated intercellular interactions. Our data show that satellite cells fuse with the myofiber concomitantly to cell division, and only when the nuclei of the daughter cells are inside the myofiber, do they complete the process of differentiation. Here we demonstrate that M-cadherin plays an important role in cell-to-cell recognition and fusion, and is crucial for cell division activation. Treatment of satellite cells with M-cadherin in vitro stimulates cell division, whereas addition of anti-M-cadherin antibodies reduces the cell division rate. Our results suggest an alternative model for the contribution of satellite cells to muscle development, which might be useful in understanding muscle regeneration, as well as muscle-related dystrophies.

  14. Innate response activator B cells: origins and functions

    PubMed Central

    Swirski, Filip K.

    2015-01-01

    Innate response activator (IRA) B cells are a subset of B-1a derived B cells that produce the growth factors granulocyte macrophage colony stimulating factor and IL-3. In mouse models of sepsis and pneumonia, B-1a B cells residing in serosal sites recognize bacteria, migrate to the spleen or lung, and differentiate to IRA B cells that then contribute to the host response by amplifying inflammation and producing polyreactive IgM. In atherosclerosis, IRA B cells accumulate in the spleen, where they promote extramedullary hematopoiesis and activate classical dendritic cells. In this review, we focus on the ontogeny and function of IRA B cells in acute and chronic inflammation. PMID:25957266

  15. Active Cellular Mechanics and Information Processing in the Living Cell

    NASA Astrophysics Data System (ADS)

    Rao, M.

    2014-07-01

    I will present our recent work on the organization of signaling molecules on the surface of living cells. Using novel experimental and theoretical approaches we have found that many cell surface receptors are organized as dynamic clusters driven by active currents and stresses generated by the cortical cytoskeleton adjoining the cell surface. We have shown that this organization is optimal for both information processing and computation. In connecting active mechanics in the cell with information processing and computation, we bring together two of the seminal works of Alan Turing.

  16. Protease-activated receptor 2 modulates proliferation and invasion of oral squamous cell carcinoma cells.

    PubMed

    Al-Eryani, Kamal; Cheng, Jun; Abé, Tatsuya; Maruyama, Satoshi; Yamazaki, Manabu; Babkair, Hamzah; Essa, Ahmed; Saku, Takashi

    2015-07-01

    Based on our previous finding that protease-activated receptor 2 (PAR-2) regulates hemophagocytosis of oral squamous cell carcinoma (SCC) cells, which induces their heme oxygenase 1-dependent keratinization, we have formulated a hypothesis that PAR-2 functions in wider activities of SCC cells. To confirm this hypothesis, we investigated immunohistochemical profiles of PAR-2 in oral SCC tissues and its functional roles in cell proliferation and invasion in SCC cells in culture. The PAR-2 expression modes were determined in 48 surgical tissue specimens of oral SCC. Using oral SCC-derived cell systems, we determined both gene and protein expression levels of PAR-2. SCC cell proliferation and invasive properties were also examined in conditions in which PAR-2 was activated by the synthetic peptide SLIGRL. PAR-2 was immunolocalized in oral SCC and carcinoma in situ cells, especially in those on the periphery of carcinoma cell foci (100% of cases), but not in normal oral epithelia. Its expression at both gene and protein levels was confirmed in 3 oral SCC cell lines including ZK-1. Activation of PAR-2 induced ZK-1 cell proliferation in a dose-dependent manner. PAR-2-activated ZK-1 cells invaded faster than nonactivated ones. The expression of PAR-2 is specific to oral malignancies, and PAR-2 regulates the growth and invasion of oral SCC cells.

  17. Cell associated urokinase activity and colonic epithelial cells in health and disease.

    PubMed Central

    Gibson, P R; van de Pol, E; Doe, W F

    1991-01-01

    It is not known if urokinase-type plasminogen activator (uPA) is associated with normal colonic epithelial cells. The aims of this study were to determine if normal colonic epithelial cells have uPA activity and whether this is concentrated at the cell membrane. In addition, the contribution of colonic epithelial cell associated uPA activity to disease related pertubations of mucosal uPA activity were examined. A highly enriched population of colonic epithelial cells was isolated from resected colon or biopsy specimens by an enzymatic technique. uPA activity was measured in cell homogenates by a specific and sensitive colorimetric method and expressed relative to cellular DNA. In two experiments subcellular fractionation of colonic epithelial cells was performed by nitrogen cavitation followed by ultracentrifugation over a linear sucrose gradient. The fractions collected were analysed for uPA and organelle-specific enzyme activities. Normal colonic epithelial cells have cell associated uPA activity (mean (SEM) 5.6 (1.1) IU/mg, n = 18). This colocalised with fractions enriched for leucine-beta-naphthylamidase and 5'-nucleotidase, markers of plasma membrane. uPA activities in epithelial cells from cancerous colons (9.8 (3.1) n = 7) or from mucosa affected by inflammatory bowel disease (3.8 (0.7) n = 15) were not significantly different from normal (paired t test), while that in epithelial cells from greatly inflamed mucosa was similar to that from autologous normal or mildly inflamed areas (4.4 (1.2) v 5.9 (3.6), n = 9). Thus normal colonic epithelial cells have cell associated uPA activity which is concentrated on the plasma membranes, suggesting the presence of uPA receptors. Increased mucosal levels of uPA previously reported in patients with inflammatory bowel disease are not due to increased colonic epithelial cell associated uPA. PMID:1650741

  18. Activated microglia cause reversible apoptosis of pheochromocytoma cells, inducing their cell death by phagocytosis

    PubMed Central

    Hornik, Tamara C.; Vilalta, Anna; Brown, Guy C.

    2016-01-01

    ABSTRACT Some apoptotic processes, such as phosphatidylserine exposure, are potentially reversible and do not necessarily lead to cell death. However, phosphatidylserine exposure can induce phagocytosis of a cell, resulting in cell death by phagocytosis: phagoptosis. Phagoptosis of neurons by microglia might contribute to neuropathology, whereas phagoptosis of tumour cells by macrophages might limit cancer. Here, we examined the mechanisms by which BV-2 microglia killed co-cultured pheochromocytoma (PC12) cells that were either undifferentiated or differentiated into neuronal cells. We found that microglia activated by lipopolysaccharide rapidly phagocytosed PC12 cells. Activated microglia caused reversible phosphatidylserine exposure on and reversible caspase activation in PC12 cells, and caspase inhibition prevented phosphatidylserine exposur and decreased subsequent phagocytosis. Nitric oxide was necessary and sufficient to induce the reversible phosphatidylserine exposure and phagocytosis. The PC12 cells were not dead at the time they were phagocytised, and inhibition of their phagocytosis left viable cells. Cell loss was inhibited by blocking phagocytosis mediated by phosphatidylserine, MFG-E8, vitronectin receptors or P2Y6 receptors. Thus, activated microglia can induce reversible apoptosis of target cells, which is insufficient to cause apoptotic cell death, but sufficient to induce their phagocytosis and therefore cell death by phagoptosis. PMID:26567213

  19. Activated microglia cause reversible apoptosis of pheochromocytoma cells, inducing their cell death by phagocytosis.

    PubMed

    Hornik, Tamara C; Vilalta, Anna; Brown, Guy C

    2016-01-01

    Some apoptotic processes, such as phosphatidylserine exposure, are potentially reversible and do not necessarily lead to cell death. However, phosphatidylserine exposure can induce phagocytosis of a cell, resulting in cell death by phagocytosis: phagoptosis. Phagoptosis of neurons by microglia might contribute to neuropathology, whereas phagoptosis of tumour cells by macrophages might limit cancer. Here, we examined the mechanisms by which BV-2 microglia killed co-cultured pheochromocytoma (PC12) cells that were either undifferentiated or differentiated into neuronal cells. We found that microglia activated by lipopolysaccharide rapidly phagocytosed PC12 cells. Activated microglia caused reversible phosphatidylserine exposure on and reversible caspase activation in PC12 cells, and caspase inhibition prevented phosphatidylserine exposur and decreased subsequent phagocytosis. Nitric oxide was necessary and sufficient to induce the reversible phosphatidylserine exposure and phagocytosis. The PC12 cells were not dead at the time they were phagocytised, and inhibition of their phagocytosis left viable cells. Cell loss was inhibited by blocking phagocytosis mediated by phosphatidylserine, MFG-E8, vitronectin receptors or P2Y6 receptors. Thus, activated microglia can induce reversible apoptosis of target cells, which is insufficient to cause apoptotic cell death, but sufficient to induce their phagocytosis and therefore cell death by phagoptosis.

  20. Monocytic Cells Become Less Compressible but More Deformable upon Activation

    PubMed Central

    Ravetto, Agnese; Wyss, Hans M.; Anderson, Patrick D.; den Toonder, Jaap M. J.; Bouten, Carlijn V. C.

    2014-01-01

    Aims Monocytes play a significant role in the development of atherosclerosis. During the process of inflammation, circulating monocytes become activated in the blood stream. The consequent interactions of the activated monocytes with the blood flow and endothelial cells result in reorganization of cytoskeletal proteins, in particular of the microfilament structure, and concomitant changes in cell shape and mechanical behavior. Here we investigate the full elastic behavior of activated monocytes in relation to their cytoskeletal structure to obtain a better understanding of cell behavior during the progression of inflammatory diseases such as atherosclerosis. Methods and Results The recently developed Capillary Micromechanics technique, based on exposing a cell to a pressure difference in a tapered glass microcapillary, was used to measure the deformation of activated and non-activated monocytic cells. Monitoring the elastic response of individual cells up to large deformations allowed us to obtain both the compressive and the shear modulus of a cell from a single experiment. Activation by inflammatory chemokines affected the cytoskeletal organization and increased the elastic compressive modulus of monocytes with 73–340%, while their resistance to shape deformation decreased, as indicated by a 25–88% drop in the cell’s shear modulus. This decrease in deformability is particularly pronounced at high strains, such as those that occur during diapedesis through the vascular wall. Conclusion Overall, monocytic cells become less compressible but more deformable upon activation. This change in mechanical response under different modes of deformation could be important in understanding the interplay between the mechanics and function of these cells. In addition, our data are of direct relevance for computational modeling and analysis of the distinct monocytic behavior in the circulation and the extravascular space. Lastly, an understanding of the changes of monocyte

  1. Ionizing Radiation Impairs T Cell Activation by Affecting Metabolic Reprogramming.

    PubMed

    Li, Heng-Hong; Wang, Yi-Wen; Chen, Renxiang; Zhou, Bin; Ashwell, Jonathan D; Fornace, Albert J

    2015-01-01

    Ionizing radiation has a variety of acute and long-lasting adverse effects on the immune system. Whereas measureable effects of radiation on immune cell cytotoxicity and population change have been well studied in human and animal models, little is known about the functional alterations of the surviving immune cells after ionizing radiation. The objective of this study was to delineate the effects of radiation on T cell function by studying the alterations of T cell receptor activation and metabolic changes in activated T cells isolated from previously irradiated animals. Using a global metabolomics profiling approach, for the first time we demonstrate that ionizing radiation impairs metabolic reprogramming of T cell activation, which leads to substantial decreases in the efficiency of key metabolic processes required for activation, such as glucose uptake, glycolysis, and energy metabolism. In-depth understanding of how radiation impacts T cell function highlighting modulation of metabolism during activation is not only a novel approach to investigate the pivotal processes in the shift of T cell homeostasis after radiation, it also may lead to new targets for therapeutic manipulation in the combination of radiotherapy and immune therapy. Given that appreciable effects were observed with as low as 10 cGy, our results also have implications for low dose environmental exposures.

  2. CD4 T cell activation by B cells in human Leishmania (Viannia) infection

    PubMed Central

    2014-01-01

    Background An effective adaptive immune response requires activation of specific CD4 T cells. The capacity of B cells to activate CD4 T cells in human cutaneous leishmaniasis caused by Leishmania (Viannia) has not been evaluated. Methods CD4 T cell activation by B cells of cutaneous leishmaniasis patients was evaluated by culture of PBMCs or purified B cells and CD4 T cells with Leishmania panamensis antigens. CD4 T cell and B cell activation markers were evaluated by flow cytometry and 13 cytokines were measured in supernatants with a bead-based capture assay. The effect of Leishmania antigens on BCR-mediated endocytosis of ovalbumin was evaluated in the Ramos human B cell line by targeting the antigen with anti-IgM-biotin and anti-biotin-ovalbumin-FITC. Results Culture of PBMCs from cutaneous leishmaniasis patients with Leishmania antigens resulted in upregulation of the activation markers CD25 and CD69 as well as increased frequency of CD25hiCD127- cells among CD4 T cells. Concomitantly, B cells upregulated the costimulatory molecule CD86. These changes were not observed in PBMCs from healthy subjects, indicating participation of Leishmania-specific lymphocytes expanded in vivo. Purified B cells from these patients, when interacting with purified CD4 T cells and Leishmania antigens, were capable of inducing significant increases in CD25 and CD69 expression and CD25hiCD127- frequency in CD4 T cells. These changes were associated with upregulation of CD86 in B cells. Comparison of changes in CD4 T cell activation parameters between PBMC and B cell/CD4 T cell cultures showed no statistically significant differences; further, significant secretion of IFN-γ, TNF-α, IL-6 and IL-13 was induced in both types of cultures. Additionally, culture with Leishmania antigens enhanced BCR-mediated endocytosis of ovalbumin in Ramos human B cells. Conclusions The capacity of B cells specific for Leishmania antigens in peripheral blood of cutaneous leishmaniasis patients to

  3. Sclerostin Antibody Administration Converts Bone Lining Cells Into Active Osteoblasts.

    PubMed

    Kim, Sang Wan; Lu, Yanhui; Williams, Elizabeth A; Lai, Forest; Lee, Ji Yeon; Enishi, Tetsuya; Balani, Deepak H; Ominsky, Michael S; Ke, Hua Zhu; Kronenberg, Henry M; Wein, Marc N

    2016-11-14

    Sclerostin antibody (Scl-Ab) increases osteoblast activity, in part through increasing modeling-based bone formation on previously quiescent surfaces. Histomorphometric studies have suggested that this might occur through conversion of bone lining cells into active osteoblasts. However, direct data demonstrating Scl-Ab-induced conversion of lining cells into active osteoblasts are lacking. Here, we used in vivo lineage tracing to determine if Scl-Ab promotes the conversion of lining cells into osteoblasts on periosteal and endocortical bone surfaces in mice. Two independent, tamoxifen-inducible lineage-tracing strategies were used to label mature osteoblasts and their progeny using the DMP1 and osteocalcin promoters. After a prolonged "chase" period, the majority of labeled cells on bone surfaces assumed a thin, quiescent morphology. Then, mice were treated with either vehicle or Scl-Ab (25 mg/kg) twice over the course of the subsequent week. After euthanization, marked cells were enumerated, their thickness quantified, and proliferation and apoptosis examined. Scl-Ab led to a significant increase in the average thickness of labeled cells on periosteal and endocortical bone surfaces, consistent with osteoblast activation. Scl-Ab did not induce proliferation of labeled cells, and Scl-Ab did not regulate apoptosis of labeled cells. Therefore, direct reactivation of quiescent bone lining cells contributes to the acute increase in osteoblast numbers after Scl-Ab treatment in mice. © 2017 American Society for Bone and Mineral Research.

  4. Increased proteasome activity determines human embryonic stem cell identity

    PubMed Central

    Vilchez, David; Boyer, Leah; Morantte, Ianessa; Lutz, Margaret; Merkwirth, Carsten; Joyce, Derek; Spencer, Brian; Page, Lesley; Masliah, Eliezer; Berggren, W. Travis; Gage, Fred H.; Dillin, Andrew

    2016-01-01

    Embryonic stem cells are able to replicate continuously in the absence of senescence and, therefore, are immortal in culture1,2. While genome stability is central for survival of stem cells; proteome stability may play an equally important role in stem cell identity and function. Additionally, with the asymmetric divisions invoked by stem cells, the passage of damaged proteins to daughter cells could potentially destroy the resulting lineage of cells. We hypothesized that stem cells have an increased proteostasis ability compared to their differentiated counterparts and asked whether proteasome activity differed among human embryonic stem cells (hESCs). Notably, hESC populations exhibit a high proteasome activity that is correlated with increased levels of the 19S proteasome subunit PSMD11/RPN-63–5 and a corresponding increased assembly of the 26S/30S proteasome. Ectopic expression of PSMD11 is sufficient to increase proteasome assembly and activity. Proteasome inhibition affects pluripotency of hESCs inducing differentiation towards specific cell lineages. FOXO4, an insulin/IGF-1 responsive transcription factor associated with long lifespan in invertebrates6,7, regulates proteasome activity by modulating the expression of PSMD11 in hESCs. Our results establish a novel regulation of proteostasis in hESCs that links longevity and stress resistance in invertebrates with hESC function and identity. PMID:22972301

  5. Real-time transposable element activity in individual live cells

    PubMed Central

    Lee, Gloria; Martini, K. Michael

    2016-01-01

    The excision and reintegration of transposable elements (TEs) restructure their host genomes, generating cellular diversity involved in evolution, development, and the etiology of human diseases. Our current knowledge of TE behavior primarily results from bulk techniques that generate time and cell ensemble averages, but cannot capture cell-to-cell variation or local environmental and temporal variability. We have developed an experimental system based on the bacterial TE IS608 that uses fluorescent reporters to directly observe single TE excision events in individual cells in real time. We find that TE activity depends upon the TE’s orientation in the genome and the amount of transposase protein in the cell. We also find that TE activity is highly variable throughout the lifetime of the cell. Upon entering stationary phase, TE activity increases in cells hereditarily predisposed to TE activity. These direct observations demonstrate that real-time live-cell imaging of evolution at the molecular and individual event level is a powerful tool for the exploration of genome plasticity in stressed cells. PMID:27298350

  6. Regulation of Cell Surface CB2 Receptor during Human B Cell Activation and Differentiation.

    PubMed

    Castaneda, Julie T; Harui, Airi; Roth, Michael D

    2017-03-31

    Cannabinoid receptor type 2 (CB2) is the primary receptor pathway mediating the immunologic consequences of cannabinoids. We recently reported that human peripheral blood B cells express CB2 on both the extracellular membrane and at intracellular sites, where-as monocytes and T cells only express intracellular CB2. To better understand the pattern of CB2 expression by human B cells, we examined CD20(+) B cells from three tissue sources. Both surface and intracellular expression were present and uniform in cord blood B cells, where all cells exhibited a naïve mature phenotype (IgD(+)/CD38(Dim)). While naïve mature and quiescent memory B cells (IgD(-)/CD38(-)) from tonsils and peripheral blood exhibited a similar pattern, tonsillar activated B cells (IgD(-)/CD38(+)) expressed little to no surface CB2. We hypothesized that regulation of the surface CB2 receptor may occur during B cell activation. Consistent with this, a B cell lymphoma cell line known to exhibit an activated phenotype (SUDHL-4) was found to lack cell surface CB2 but express intracellular CB2. Furthermore, in vitro activation of human cord blood resulted in a down-regulation of surface CB2 on those B cells acquiring the activated phenotype but not on those retaining IgD expression. Using a CB2 expressing cell line (293 T/CB2-GFP), confocal microscopy confirmed the presence of both cell surface expression and multifocal intracellular expression, the latter of which co-localized with endoplasmic reticulum but not with mitochondria, lysosomes, or nucleus. Our findings suggest a dynamic multi-compartment expression pattern for CB2 in B cells that is specifically modulated during the course of B cell activation.

  7. Surface free energy activated high-throughput cell sorting.

    PubMed

    Zhang, Xinru; Zhang, Qian; Yan, Tao; Jiang, Zeyi; Zhang, Xinxin; Zuo, Yi Y

    2014-09-16

    Cell sorting is an important screening process in microbiology, biotechnology, and clinical research. Existing methods are mainly based on single-cell analysis as in flow cytometric and microfluidic cell sorters. Here we report a label-free bulk method for sorting cells by differentiating their characteristic surface free energies (SFEs). We demonstrated the feasibility of this method by sorting model binary cell mixtures of various bacterial species, including Pseudomonas putida KT2440, Enterococcus faecalis ATCC 29212, Salmonella Typhimurium ATCC 14028, and Escherichia coli DH5α. This method can effectively separate 10(10) bacterial cells within 30 min. Individual bacterial species can be sorted with up to 96% efficiency, and the cell viability ratio can be as high as 99%. In addition to its capacity of sorting evenly mixed bacterial cells, we demonstrated the feasibility of this method in selecting and enriching cells of minor populations in the mixture (presenting at only 1% in quantity) to a purity as high as 99%. This SFE-activated method may be used as a stand-alone method for quickly sorting a large quantity of bacterial cells or as a prescreening tool for microbial discrimination. Given its advantages of label-free, high-throughput, low cost, and simplicity, this SFE-activated cell sorting method has potential in various applications of sorting cells and abiotic particles.

  8. T Cell Receptor-induced Activation and Apoptosis In Cycling Human T Cells Occur throughout the Cell Cycle

    PubMed Central

    Karas, Michael; Zaks, Tal Z.; JL, Liu; LeRoith, Derek

    1999-01-01

    Previous studies have found conflicting associations between susceptibility to activation-induced cell death and the cell cycle in T cells. However, most of the studies used potentially toxic pharmacological agents for cell cycle synchronization. A panel of human melanoma tumor-reactive T cell lines, a CD8+ HER-2/neu-reactive T cell clone, and the leukemic T cell line Jurkat were separated by centrifugal elutriation. Fractions enriched for the G0–G1, S, and G2–M phases of the cell cycle were assayed for T cell receptor-mediated activation as measured by intracellular Ca2+ flux, cytolytic recognition of tumor targets, and induction of Fas ligand mRNA. Susceptibility to apoptosis induced by recombinant Fas ligand and activation-induced cell death were also studied. None of the parameters studied was specific to a certain phase of the cell cycle, leading us to conclude that in nontransformed human T cells, both activation and apoptosis through T cell receptor activation can occur in all phases of the cell cycle. PMID:10588669

  9. Acetaminophen Induces Human Neuroblastoma Cell Death through NFKB Activation

    PubMed Central

    Posadas, Inmaculada; Santos, Pablo; Ceña, Valentín

    2012-01-01

    Neuroblastoma resistance to apoptosis may contribute to the aggressive behavior of this tumor. Therefore, it would be relevant to activate endogenous cellular death mechanisms as a way to improve neuroblastoma therapy. We used the neuroblastoma SH-SY5Y cell line as a model to study the mechanisms involved in acetaminophen (AAP)-mediated toxicity by measuring CYP2E1 enzymatic activity, NFkB p65 subunit activation and translocation to the nucleus, Bax accumulation into the mitochondria, cytochrome c release and caspase activation. AAP activates the intrinsic death pathway in the SH-SY5Y human neuroblastoma cell line. AAP metabolism is partially responsible for this activation, because blockade of the cytochrome CYP2E1 significantly reduced but did not totally prevent, AAP-induced SH-SY5Y cell death. AAP also induced NFkB p65 activation by phosphorylation and its translocation to the nucleus, where NFkB p65 increased IL-1β production. This increase contributed to neuroblastoma cell death through a mechanism involving Bax accumulation into the mitochondria, cytochrome c release and caspase3 activation. Blockade of NFkB translocation to the nucleus by the peptide SN50 prevented AAP-mediated cell death and IL-1β production. Moreover, overexpression of the antiapoptotic protein Bcl-xL did not decrease AAP-mediated IL-1β production, but prevented both AAP and IL-1β-mediated cell death. We also confirmed the AAP toxic actions on SK-N-MC neuroepithelioma and U87MG glioblastoma cell lines. The results presented here suggest that AAP activates the intrinsic death pathway in neuroblastoma cells through a mechanism involving NFkB and IL-1β. PMID:23166834

  10. Novel APC-like properties of human NK cells directly regulate T cell activation

    PubMed Central

    Hanna, Jacob; Gonen-Gross, Tsufit; Fitchett, Jonathan; Rowe, Tony; Daniels, Mark; Arnon, Tal I.; Gazit, Roi; Joseph, Aviva; Schjetne, Karoline W.; Steinle, Alexander; Porgador, Angel; Mevorach, Dror; Goldman-Wohl, Debra; Yagel, Simcha; LaBarre, Michael J.; Buckner, Jane H.; Mandelboim, Ofer

    2004-01-01

    Initiation of the adaptive immune response is dependent on the priming of naive T cells by APCs. Proteomic analysis of unactivated and activated human NK cell membrane–enriched fractions demonstrated that activated NK cells can efficiently stimulate T cells, since they upregulate MHC class II molecules and multiple ligands for TCR costimulatory molecules. Furthermore, by manipulating antigen administration, we show that NK cells possess multiple independent unique pathways for antigen uptake. These results highlight NK cell–mediated cytotoxicity and specific ligand recognition by cell surface–activating receptors on NK cells as unique mechanisms for antigen capturing and presentation. In addition, we analyzed the T cell–activating potential of human NK cells derived from different clinical conditions, such as inflamed tonsils and noninfected and CMV-infected uterine decidual samples, and from transporter-associated processing antigen 2–deficient patients. This in vivo analysis revealed that proinflammatory, but not immune-suppressive, microenvironmental requirements can selectively dictate upregulation of T cell–activating molecules on NK cells. Taken together, these observations offer new and unexpected insights into the direct interactions between NK and T cells and suggest novel APC-like activating functions for human NK cells. PMID:15578093

  11. Laser activated nanothermolysis of leukemia cells monitored by photothermal microscopy

    NASA Astrophysics Data System (ADS)

    Lapotko, Dmitri; Lukianova, Ekaterina; Shnip, Alexander; Zheltov, George; Potapnev, Michail; Savitsky, Valeriy; Klimovich, Olga; Oraevsky, Alexander

    2005-04-01

    We are developing new diagnostic and therapeutic technologies for leukemia based on selective targeting of leukemia cells with gold nanoparticles and thermomechanical destruction of the tumor cells with laser-induced microbubbles. Clusters of spherical gold nanoparticles that have strong optical absorption of laser pulses at 532 nm served as nucleation sites of vapor microbubbles. The nanoparticles were targeted selectively to leukemia cells using leukemia-specific surface receptors and a set of two monoclonal antibodies. Application of a primary myeloid-specific antibody to tumor cells followed by targeting the cells with 30-nm nanoparticles conjugated with a secondary antibody (IgG) resulted in formation of nanoparticulate clusters due to aggregation of IgGs. Formation of clusters resulted in substantial decrease of the damage threshold for target cells. The results encourage development of Laser Activated Nanothermolysis as a Cell Elimination Therapy (LANCET) for leukemia. The proposed technology can be applied separately or in combination with chemotherapy for killing leukemia cells without damage to other blood cells. Potential applications include initial reduction of concentration of leukemia cells in blood prior to chemotherapy and treatment of residual tumor cells after the chemotherapy. Laser-induced bubbles in individual cells and cell damage were monitored by analyzing profile of photothermal response signals over the entire cell after irradiation with a single 10-ns long laser pulse. Photothermal microscopy was utilized for imaging formation of microbubbles around nanoparticulate clusters.

  12. Edwardsiella tarda invasion of fish cell lines and the activation of divergent cell death pathways.

    PubMed

    Wang, Bin; Yu, Tong; Dong, Xue; Zhang, Zenghu; Song, Lin; Xu, Ying; Zhang, Xiao-Hua

    2013-05-03

    Edwardsiella tarda is an important gram-negative intracellular pathogen of fish. However, the invasive features of E. tarda to fish cells and the pathogenesis of host cell death have not been thoroughly investigated. In this study, two fish cell models were used to investigate the interactions between E. tarda and its cellular hosts. E. tarda invaded and replicated in both cell lines. Epithelioma papulosum cyprini (EPC) cells were more sensitive to E. tarda infection than the flounder gill cell line FG-9307, with higher levels of intracellular bacteria in the former. The invasion and intracellular replication of E. tarda in FG-9307 cells were studied at the ultrastructural level, and infected cells with large amounts of replicated bacteria and destroyed organelles were observed. Apoptosis was observed in EPC cells upon infection, characterized by the occurrence of apoptotic bodies, DNA ladder, increased Annexin V binding and the activation of caspase-3, whereas E. tarda infected FG-9307 cells were negative for all of those features. E. tarda infection in FG-9307 cells failed to protect the staurosporine-induced apoptosis. Moreover, both intrinsic and extrinsic pathways were activated in EPC cells upon E. tarda infection. The present study revealed that E. tarda interacts with fish cells in different manners, and divergent pathways were activated in these cellular hosts to mediate cell death. These results provided new information on the interactions between E. tarda and fish cells.

  13. Xyloglucan Endotransglycosylase Activity in Carrot Cell Suspensions during cell Elongation and Somatic Embryogenesis.

    PubMed

    Hetherington, P. R.; Fry, S. C.

    1993-11-01

    Xyloglucan endotransglycosylase (XET) has been proposed to contribute to cell elongation through wall loosening. To explore this relationship further, we assayed this enzyme activity in suspensions of carrot (Daucus carota L.) cells exhibiting various rates of cell elongation. In one cell line, elongation was induced by dilution into dichlorophenoxyacetic acid (2,4-D)-free medium. During this elongation, 93% of the XET activity was found in the culture medium; in nonelongating controls, by contrast, 68% was found in the cell extracts even though the specific activity of these extracts was lower than in the elongating cells. By far the highest rates of XET secretion per cell were in the elongating cells. A second cell line was induced to undergo somatic embryogenesis by dilution into 2,4-D-free medium. During the first 6 d, numerous globular embryoids composed of small, isodiametric cells were formed in the absence of cell elongation; extracellular XET activity was almost undetectable, and intracellular specific activity markedly declined. After 6 d, heart, torpedo, and cotyledonary embryoids began to appear (i.e. cell elongation resumed); the intracellular specific activity of XET rose rapidly and >80% of the XET activity accumulated in the medium. Thus, nonexpanding cell suspensions (whether or not they were rapidly dividing) produced and secreted less XET activity than did expanding cells. We propose that a XET molecule has an ephemeral wall-loosening role while it passes through the load-bearing layer of the wall on its way from the protoplast into the culture medium.

  14. Xyloglucan Endotransglycosylase Activity in Carrot Cell Suspensions during cell Elongation and Somatic Embryogenesis.

    PubMed Central

    Hetherington, P. R.; Fry, S. C.

    1993-01-01

    Xyloglucan endotransglycosylase (XET) has been proposed to contribute to cell elongation through wall loosening. To explore this relationship further, we assayed this enzyme activity in suspensions of carrot (Daucus carota L.) cells exhibiting various rates of cell elongation. In one cell line, elongation was induced by dilution into dichlorophenoxyacetic acid (2,4-D)-free medium. During this elongation, 93% of the XET activity was found in the culture medium; in nonelongating controls, by contrast, 68% was found in the cell extracts even though the specific activity of these extracts was lower than in the elongating cells. By far the highest rates of XET secretion per cell were in the elongating cells. A second cell line was induced to undergo somatic embryogenesis by dilution into 2,4-D-free medium. During the first 6 d, numerous globular embryoids composed of small, isodiametric cells were formed in the absence of cell elongation; extracellular XET activity was almost undetectable, and intracellular specific activity markedly declined. After 6 d, heart, torpedo, and cotyledonary embryoids began to appear (i.e. cell elongation resumed); the intracellular specific activity of XET rose rapidly and >80% of the XET activity accumulated in the medium. Thus, nonexpanding cell suspensions (whether or not they were rapidly dividing) produced and secreted less XET activity than did expanding cells. We propose that a XET molecule has an ephemeral wall-loosening role while it passes through the load-bearing layer of the wall on its way from the protoplast into the culture medium. PMID:12231995

  15. NITRIC OXIDE, MITOCHONDRIAL HYPERPOLARIZATION AND T-CELL ACTIVATION

    PubMed Central

    Nagy, Gyorgy; Koncz, Agnes; Fernandez, David; Perl, Andras

    2007-01-01

    T lymphocyte activation is associated with nitric oxide (NO) production that plays an essential role in multiple T cell functions. NO acts as a messenger, activating soluble guanyl cyclase and participating in the transduction signaling pathways involving cyclic GMP. NO modulates mitochondrial events that are involved in apoptosis and regulates mitochondrial membrane potential and mitochondrial biogenesis in many cell types, including lymphocytes. Mitochondrial hyperpolarization (MHP), an early and reversible event during both T lymphocyte activation and apoptosis, is regulated by NO. Here, we discuss recent evidence that NO-induced MHP represents a molecular switch in multiple T cell signaling pathways. Overproduction of NO in systemic lupus erythematosus (SLE) induces mitochondrial biogenesis and alters Ca2+ signaling. Thus, while NO plays a physiological role in lymphocyte cell signaling, its overproduction may disturb normal T cell function, contributing to the pathogenesis of autoimmunity. PMID:17462531

  16. The regulation and activation of lupus-associated B cells.

    PubMed

    Fields, Michele L; Hondowicz, Brian D; Wharton, Gina N; Adair, Brigette S; Metzgar, Michele H; Alexander, Shawn T; Caton, Andrew J; Erikson, Jan

    2005-04-01

    Anti-double-stranded DNA (anti-dsDNA) B cells are regulated in non-autoimmune mice. While some are deleted or undergo receptor editing, a population of anti-dsDNA (VH3H9/V lambda 1) B cells that emigrate into the periphery has also been identified. These cells have an altered phenotype relative to normal B cells in that they have a reduced lifespan, appear developmentally arrested, and localize primarily to the T/B-cell interface in the spleen. This phenotype may be the consequence of immature B cells encountering antigen in the absence of T-cell help. When provided with T-cell help, the anti-dsDNA B cells differentiate into antibody-forming cells. In the context of the autoimmune-prone lpr/lpr or gld/gld mutations, the VH3H9/V lambda 1 anti-dsDNA B cells populate the B-cell follicle and by 12 weeks of age produce serum autoantibodies. The early event of anti-dsDNA B-cell follicular entry, in the absence of autoantibody production, is dependent upon CD4(+) T cells. We hypothesize that control of autoantibody production in young autoimmune-prone mice may be regulated by the counterbalancing effect of T-regulatory (T(reg)) cells. Consistent with this model, we have demonstrated that T(reg) cells are able to prevent autoantibody production induced by T-cell help. Additional studies are aimed at investigating the mechanisms of this suppression as well as probing the impact of distinct forms of T-cell-dependent and -independent activation on anti-dsDNA B cells.

  17. Dog mastocytoma cells secrete a 92-kD gelatinase activated extracellularly by mast cell chymase.

    PubMed Central

    Fang, K C; Raymond, W W; Lazarus, S C; Caughey, G H

    1996-01-01

    Gelatinolytic metalloproteinases implicated in connective tissue remodeling and tumor invasion are secreted from several types of cells in the form of inactive zymogens. In this report, characterization of gelatinase activity secreted by the BR line of dog mastocytoma cells reveals a phorbol-inducible, approximately 92-kD, Ca2+ - and Zn2+ -dependent proenzyme cleaved over time to smaller, active forms. Incubation of cells with the general serine protease inhibitor, PMSF, prevented proenzyme cleavage and permitted its purification free of activation products. The NH2-terminal 13 amino acids of the purified mastocytoma progelatinase are 50-67% identical to those of human, mouse, and rabbit 92-kD progelatinase (gelatinase B; matrix metalloproteinase-9). Degranulation of mastocytoma cells using ionophore A23187 greatly accelerated proenzyme cleavage, suggesting that a serine protease present in secretory granules hydrolyzed the progelatinase to active fragments. To identify the activating protease, cells were coincubated with ionophore and a panel of selective serine protease inhibitors. Soybean trypsin inhibitor and succinyl-L-Ala-Ala-Pro-Phe-chloromethylketone, which inhibit mast cell chymase, prevented progelatinase activation. Inhibitors of tryptase and dog mast cell protease (dMCP)-3, i.e., aprotinin or bis(5-amidino-2-benzimidazolyl) methane (BABIM), did not. In further experiments using highly purified enzymes, mastocytoma cell chymase activated 92-kD progelatinase in the absence of other enzymes or cofactors; tryptase and dMCP-3, however, had no effect. These data demonstrate that dog mastocytoma cells secrete a metalloproteinase related to progelatinase B that is directly activated outside of the cell by exocytosed chymase, and provide the first demonstration of a cell that activates a matrix metalloproteinase it secretes by cosecreting an activating enzyme. In mastocytomas, this pathway may facilitate tumor invasion of surrounding tissues, and in normal mast

  18. trans-Activation of a globin promoter in nonerythroid cells.

    PubMed Central

    Evans, T; Felsenfeld, G

    1991-01-01

    We show that expression in fibroblasts of a single cDNA, encoding the erythroid DNA-binding protein Eryf1 (GF-1, NF-E1), very efficiently activates transcription of a chicken alpha-globin promoter, trans-Activation in these cells occurred when Eryf1 bound to a single site within a minimal globin promoter. In contrast, efficient activation in erythroid cells required multiple Eryf1 binding sites. Our results indicate that mechanisms exist that are capable of modulating the trans-acting capabilities of Eryf1 in a cell-specific manner, without affecting DNA binding. The response of the minimal globin promoter to Eryf1 in fibroblasts was at least as great as for optimal constructions in erythroid cells. Therefore, the assay provides a very simple and sensitive system with which to study gene activation by a tissue-specific factor. Images PMID:1990287

  19. Asbestos exposure increases human bronchial epithelial cell fibrinolytic activity.

    PubMed

    Gross, T J; Cobb, S M; Gruenert, D C; Peterson, M W

    1993-03-01

    Chronic exposure to asbestos fibers results in fibrotic lung disease. The distal pulmonary epithelium is an early target of asbestos-mediated injury. Local plasmin activity may be important in modulating endoluminal inflammatory responses in the lung. We studied the effects of asbestos exposure on cell-mediated plasma clot lysis as a marker of pericellular plasminogen activation. Exposing human bronchial epithelial (HBE) cells to 100 micrograms/ml of asbestos fibers for 24 h resulted in increased plasma clot lysis. Fibrinolytic activity was augmented in a dose-dependent fashion, was not due to secreted protease, and occurred only when there was direct contact between the plasma clot and the epithelial monolayer. Further analysis showed that asbestos exposure increased HBE cell-associated urokinase-type plasminogen activator (uPA) activity in a time-dependent manner. The increased cell-associated PA activity could be removed by acid washing. The increase in PA activity following asbestos exposure required new protein synthesis because it was abrogated by treatment with either cycloheximide or actinomycin D. Therefore, asbestos exposure increases epithelial-mediated fibrinolysis by augmenting expression of uPA activity at the cell surface by mechanisms that require new RNA and protein synthesis. These observations suggest a novel mechanism whereby exposure of the distal epithelium to inhaled particulates may result in a chronic inflammatory response that culminates in the development of fibrotic lung disease.

  20. Isosmotic modulation of cell volume and intracellular ion activities during stimulation of single exocrine cells.

    PubMed

    Foskett, J K; Wong, M M; Sue-A-Quan, G; Robertson, M A

    1994-02-01

    Stimulation of salivary secretion is associated with a rise of [Ca2+]i in acinar cells. We examined the osmotic and ionic consequences of activation of Ca(2+)-dependent K+ and Cl- channels, by simultaneous optical determinations of cell volume and [Ca2+]i, [Cl-]i or [Na+]i during muscarinic stimulation of single salivary acinar cells, using a differential interference contrast (DIC)-fluorescence microscope. Carbachol caused a rapid rise of [Ca2+]i, as well as a substantial cell shrinkage. Despite variability in the level and kinetics of the subsequent sustained phase of the [Ca2+]i response, cell volume was correlated with [Ca2+]i in all cases. Elevated [Ca2+]i was both necessary and sufficient to cause these changes in cell volume. The proposition that changes in cell volume reflected changes in cell solute content was confirmed by simultaneously measuring [Cl-]i and cell volume. Simultaneous determinations of cell volume and [Na+]i indicated that the initial cell shrinkage was due entirely to K+ and Cl- efflux. Subsequent to the initial shrinkage, [Na+]i rose to high levels, primarily due to activation of Na+/H+ exchange. Thus, modulation of ion transport activities under isosmotic conditions results in substantial changes in cell solute content and cell volume. Subsequent to the early Ca(2+)-induced changes in these parameters, other transporters become active, but it is unclear what signals their activation. Cell swelling by osmotic dilution of the bath resulted in compensatory cell shrinkage (RVD) which was sensitive to K+ and Cl- gradients. Nevertheless, a rise of [Ca2+]i was not necessary for RVD. Osmotic shrinkage and/or cell acidification were insufficient to activate Na+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Paclitaxel inhibits the hyper-activation of spleen cells by lipopolysaccharide and induces cell death

    PubMed Central

    Kim, Hyun-Ji

    2016-01-01

    Paclitaxel was isolated from the bark of the Pacific yew, Taxus brevifolia, and used as an anticancer agent. Paclitaxel prevents cancer cell division by inhibiting spindle fiber function, inducing cell death. A recent study demonstrated that paclitaxel binds to myeloid differentiation protein-2 of Toll-like receptor 4 and prevents the signal transduction of lipopolysaccharide (LPS). Paclitaxel converts immune cells hypo-responsive to LPS. In this study, we investigated whether paclitaxel can inhibit the phenotype and function of immune cells. To accomplish this, we used spleen cells, a major type of immune cell, LPS, a representative inflammatory agent and a mitogen for B lymphocytes. LPS profoundly increased the activation and cytokine production of spleen cells. However, paclitaxel significantly inhibited LPS-induced hyper-activation of spleen cells. Furthermore, we found that paclitaxel induced cell death of LPS-treated spleen cells. These results suggest that paclitaxel can inhibit the hyper-immune response of LPS in spleen cells via a variety of mechanisms. These findings suggest that paclitaxel can be used as a modulating agent for diseases induced by hyper-activation of B lymphocytes. Taken together, these results demonstrate that paclitaxel inhibits the function of spleen cells activated by LPS, and further induces cell death. PMID:27030196

  2. γδ T Cells Support Pancreatic Oncogenesis by Restraining αβ T Cell Activation.

    PubMed

    Daley, Donnele; Zambirinis, Constantinos Pantelis; Seifert, Lena; Akkad, Neha; Mohan, Navyatha; Werba, Gregor; Barilla, Rocky; Torres-Hernandez, Alejandro; Hundeyin, Mautin; Mani, Vishnu Raj Kumar; Avanzi, Antonina; Tippens, Daniel; Narayanan, Rajkishen; Jang, Jung-Eun; Newman, Elliot; Pillarisetty, Venu Gopal; Dustin, Michael Loran; Bar-Sagi, Dafna; Hajdu, Cristina; Miller, George

    2016-09-08

    Inflammation is paramount in pancreatic oncogenesis. We identified a uniquely activated γδT cell population, which constituted ∼40% of tumor-infiltrating T cells in human pancreatic ductal adenocarcinoma (PDA). Recruitment and activation of γδT cells was contingent on diverse chemokine signals. Deletion, depletion, or blockade of γδT cell recruitment was protective against PDA and resulted in increased infiltration, activation, and Th1 polarization of αβT cells. Although αβT cells were dispensable to outcome in PDA, they became indispensable mediators of tumor protection upon γδT cell ablation. PDA-infiltrating γδT cells expressed high levels of exhaustion ligands and thereby negated adaptive anti-tumor immunity. Blockade of PD-L1 in γδT cells enhanced CD4(+) and CD8(+) T cell infiltration and immunogenicity and induced tumor protection suggesting that γδT cells are critical sources of immune-suppressive checkpoint ligands in PDA. We describe γδT cells as central regulators of effector T cell activation in cancer via novel cross-talk.

  3. Macroautophagy regulates energy metabolism during effector T cell activation.

    PubMed

    Hubbard, Vanessa M; Valdor, Rut; Patel, Bindi; Singh, Rajat; Cuervo, Ana Maria; Macian, Fernando

    2010-12-15

    Macroautophagy is a highly conserved mechanism of lysosomal-mediated protein degradation that plays a key role in maintaining cellular homeostasis by recycling amino acids, reducing the amount of damaged proteins, and regulating protein levels in response to extracellular signals. We have found that macroautophagy is induced after effector T cell activation. Engagement of the TCR and CD28 results in enhanced microtubule-associated protein 1 light chain 3 (LC3) processing, increased numbers of LC3-containing vesicles, and increased LC3 flux, indicating active autophagosome formation and clearance. The autophagosomes formed in stimulated T cells actively fuse with lysosomes to degrade their cargo. Using a conditional KO mouse model where Atg7, a critical gene for macroautophagy, is specifically deleted in T cells, we have found that macroautophagy-deficient effector Th cells have defective IL-2 and IFN-γ production and reduced proliferation after stimulation, with no significant increase in apoptosis. We have found that ATP generation is decreased when autophagy is blocked, and defects in activation-induced cytokine production are restored when an exogenous energy source is added to macroautophagy-deficient T cells. Furthermore, we present evidence showing that the nature of the cargo inside autophagic vesicles found in resting T cells differs from the cargo of autophagosomes in activated T cells, where mitochondria and other organelles are selectively excluded. These results suggest that macroautophagy is an actively regulated process in T cells that can be induced in response to TCR engagement to accommodate the bioenergetic requirements of activated T cells.

  4. Cell membrane stretch activates intermediate-conductance Ca2+-activated K+ channels in arterial smooth muscle cells.

    PubMed

    Hayabuchi, Yasunobu; Nakaya, Yutaka; Mawatari, Kazuaki; Inoue, Miki; Sakata, Miho; Kagami, Shoji

    2011-01-01

    The aim of this study is to determine the signal transduction of membrane stretch on intermediate-conductance Ca(2+)-activated K(+) (IKca) channels in rat aorta smooth muscle cells using the patch-clamp technique. To stretch the cell membrane, both suction to the rear end of patch pipette and hypotonic shock were used. In cell-attached and inside-out patch configurations, the open probability of IKca channels increased when 20- to 45-mmHg suction was applied. Hyposmotic swelling efficiently increased IKca channel current. When the Ca(2+)-free solution was superfused, the activation of IKca current by the hyposmotic swelling was reduced. Furthermore, gadolinium (Gd(3+)) attenuated the activation of IKca channels induced by hyposmotic swelling, whereas nicardipine did not. In the experiments with Ca(2+)-free bath solution, pretreatment with GF109203X, a protein kinase C (PKC) inhibitor, completely abolished the stretch-induced activation of IKca currents. The stretch-induced activation of IKca channels was strongly inhibited by cytochalasin D, indicating a role for the F-actin in modulation of IKca channels by changes in cell stretching. These data suggest that cell membrane stretch activates IKca channels. In addition, the activation is associated with extracellular Ca(2+) influx through stretch-activated nonselective cation channels, and is also modulated by the F-actin cytoskeleton and the activation of PKC.

  5. The Nrf2 activator tBHQ inhibits T cell activation of primary human CD4 T cells.

    PubMed

    Turley, Alexandra E; Zagorski, Joseph W; Rockwell, Cheryl E

    2015-02-01

    The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates a battery of antioxidant, detoxification, and cell stress genes. It is activated by oxidative stress and a number of exogenous compounds, one of which is tert-butylhydroquinone (tBHQ), a widely used food preservative. Nrf2 modulates immune responses in numerous rodent models of inflammation, but its effects on human immune cells are not well characterized. The purpose of these studies was to evaluate the effects of the Nrf2 activator tBHQ on early events of T cell activation in primary human cells. Treatment with tBHQ induced mRNA expression of the Nrf2 target genes HMOX-1, GCLC, and NQO1, and also increased NRF2 mRNA expression, albeit to a lesser extent than the other target genes. tBHQ decreased production of the cytokines IL-2 and IFN-γ at both the protein and mRNA levels after stimulation with anti-CD3/anti-CD28 in human peripheral blood mononuclear cells and to an even greater extent in isolated CD4 T cells. Likewise, tBHQ decreased induction of CD25 and CD69 in peripheral blood mononuclear cells (PBMCs) and this decrease was even more marked in isolated CD4 T cells. In addition, tBHQ inhibited induction of NFκB DNA binding in anti-CD3/anti-CD28-activated PBMCs. Collectively, these data suggest that tBHQ inhibits activation of primary human CD4 T cells, which correlates with activation of Nrf2 and inhibition of NFκB DNA binding. Although these studies suggest the food additive tBHQ negatively impacts T cell activation, further studies will be needed to fully elucidate the effect of tBHQ on human immune responses.

  6. Thrombomodulin inhibits the activation of eosinophils and mast cells.

    PubMed

    Roeen, Ziaurahman; Toda, Masaaki; D'Alessandro-Gabazza, Corina N; Onishi, Masahiro; Kobayashi, Tetsu; Yasuma, Taro; Urawa, Masahito; Taguchi, Osamu; Gabazza, Esteban C

    2015-01-01

    Eosinophils and mast cells play critical roles in the pathogenesis of bronchial asthma. Activation of both cells leads to the release of pro-inflammatory mediators in the airway of asthmatic patients. Recently, we have shown that inhaled thrombomodulin inhibits allergic bronchial asthma in a mouse model. In the present study, we hypothesize that thrombomodulin can inhibit the activation of eosinophils and mast cells. The effect of thrombomodulin on the activation and release of inflammatory mediators from eosinophils and mast cells was evaluated. Thrombomodulin inhibited the eotaxin-induced chemotaxis, upregulation of CD11b and degranulation of eosinophils. Treatment with thrombomodulin also significantly suppressed the degranulation and synthesis of inflammatory cytokines and chemokines in eosinophils and mast cells. Mice treated with a low-dose of inhaled thrombomodulin have decreased number of eosinophils and activated mast cells and Th2 cytokines in the lungs compared to untreated mice. The results of this study suggest that thrombomodulin may modulate allergic responses by inhibiting the activation of both eosinophils and mast cells.

  7. Activation of antitumor cytotoxic T lymphocytes by fusions of human dendritic cells and breast carcinoma cells

    PubMed Central

    Gong, Jianlin; Avigan, David; Chen, Dongshu; Wu, Zekui; Koido, Shigeo; Kashiwaba, Masahiro; Kufe, Donald

    2000-01-01

    We have reported that fusions of murine dendritic cells (DCs) and murine carcinoma cells reverse unresponsiveness to tumor-associated antigens and induce the rejection of established metastases. In the present study, fusions were generated with primary human breast carcinoma cells and autologous DCs. Fusion cells coexpressed tumor-associated antigens and DC-derived costimulatory molecules. The fusion cells also retained the functional potency of DCs and stimulated autologous T cell proliferation. Significantly, the results show that autologous T cells are primed by the fusion cells to induce MHC class I-dependent lysis of autologous breast tumor cells. These findings demonstrate that fusions of human breast cancer cells and DCs activate T cell responses against autologous tumors. PMID:10688917

  8. A Model Approach to the Electrochemical Cell: An Inquiry Activity

    ERIC Educational Resources Information Center

    Cullen, Deanna M.; Pentecost, Thomas C.

    2011-01-01

    In an attempt to address some student misconceptions in electrochemistry, this guided-inquiry laboratory was devised to give students an opportunity to use a manipulative that simulates the particulate-level activity within an electrochemical cell, in addition to using an actual electrochemical cell. Students are led through a review of expected…

  9. GSK621 Targets Glioma Cells via Activating AMP-Activated Protein Kinase Signalings

    PubMed Central

    Jiang, Hong; Liu, Wei; Zhan, Shi-Kun; Pan, Yi-Xin; Bian, Liu-Guan; Sun, Bomin; Sun, Qing-Fang; Pan, Si-Jian

    2016-01-01

    Here, we studied the anti-glioma cell activity by a novel AMP-activated protein kinase (AMPK) activator GSK621. We showed that GSK621 was cytotoxic to human glioma cells (U87MG and U251MG lines), possibly via provoking caspase-dependent apoptotic cell death. Its cytotoxicity was alleviated by caspase inhibitors. GSK621 activated AMPK to inhibit mammalian target of rapamycin (mTOR) and downregulate Tetraspanin 8 (Tspan8) in glioma cells. AMPK inhibition, through shRNA knockdown of AMPKα or introduction of a dominant negative (T172A) AMPKα, almost reversed GSK621-induced AMPK activation, mTOR inhibition and Tspan8 degradation. Consequently, GSK621’s cytotoxicity in glioma cells was also significantly attenuated by AMPKα knockdown or mutation. Further studies showed that GSK621, at a relatively low concentration, significantly potentiated temozolomide (TMZ)’s sensitivity and lethality against glioma cells. We summarized that GSK621 inhibits human glioma cells possibly via activating AMPK signaling. This novel AMPK activator could be a novel and promising anti-glioma cell agent. PMID:27532105

  10. TLR3-induced activation of mast cells modulates CD8+ T-cell recruitment.

    PubMed

    Orinska, Zane; Bulanova, Elena; Budagian, Vadim; Metz, Martin; Maurer, Marcus; Bulfone-Paus, Silvia

    2005-08-01

    Mast cells play an important role in host defense against various pathogens, but their role in viral infection has not been clarified in detail. dsRNA, synthesized by various types of viruses and mimicked by polyinosinic-polycytidylic acid (poly(I:C)) is recognized by Toll-like receptor 3 (TLR3). In this study, we demonstrate that poly(I:C) injection in vivo potently stimulates peritoneal mast cells to up-regulate a number of different costimulatory molecules. Therefore, we examined the expression and the functional significance of TLR3 activation in mast cells. Mast cells express TLR3 on the cell surface and intracellularly. After stimulation of mast cells with poly(I:C) and Newcastle disease virus (NDV), TLR3 is phosphorylated and the expression of key antiviral response cytokines (interferon beta, ISG15) and chemokines (IP10, RANTES) is upregulated. Interestingly, mast cells activated via TLR3-poly(I:C) potently stimulate CD8+ T-cell recruitment. Indeed, mast-cell-deficient mice (KitW/KitW-v) given an intraperitoneal injection of poly(I:C) show a decreased CD8+ T-cell recruitment, whereas granulocytes normally migrate to the peritoneal cavity. Mast-cell reconstitution of KitW/KitW-v mice normalizes the CD8+ T-cell influx. Thus, mast cells stimulated through engagement of TLR3 are potent regulators of CD8+ T-cell activities in vitro and in vivo.

  11. mTOR activation is critical for betulin treatment in renal cell carcinoma cells.

    PubMed

    Cheng, Wenlong; Ji, Shiqi; Zhang, Haijian; Han, Zhixing; Liu, Qingjun; Wang, Jianwen; Ping, Hao

    2017-01-22

    Betulin, a natural product isolated from the bark of the birch trees, exhibits multiple anticancer effects. Activation of mTOR signaling pathway has been found in numerous cancers, including renal cell carcinoma (RCC). Here, we attempted to study whether mTOR signaling was essential for betulin to treat RCC. Based on cell survival and colony formation assays, we found that mTOR hyperactive RCC cell line 786-O cells were more sensitive to betulin treatment compared with mTOR-inactive Caki-2 cells. Knockdown of TSC2 in Caki-2 cells had similar results to 786-O cells, and mTOR silencing in 786-O cells rescued the inhibitory effect of betulin, indicating that betulin inhibited RCC cell proliferation in an mTOR-dependent manner. Furthermore, betulin treatment decreases the levels of glucose consumption and lactate production in 786-O cells, while minimal effects were observed in Caki-2 cells. In addition, betulin significantly inhibited the expression of PKM2 and HK2 in 786-O cells. Finally, knockdown of PKM2 or HK2 in 786-O reversed the anti-proliferative effects of betulin, and overexpression of PKM2 or HK2 in Caki-2 cells enhanced the sensitivity to betulin treatment. Taken together, these findings demonstrated the critical role of mTOR activation in RCC cells to betulin treatment, suggesting that betulin might be valuable for targeted therapies in RCC patients with mTOR activation.

  12. Buoyancy-activated cell sorting using targeted biotinylated albumin microbubbles.

    PubMed

    Liou, Yu-Ren; Wang, Yu-Hsin; Lee, Chia-Ying; Li, Pai-Chi

    2015-01-01

    Cell analysis often requires the isolation of certain cell types. Various isolation methods have been applied to cell sorting, including fluorescence-activated cell sorting and magnetic-activated cell sorting. However, these conventional approaches involve exerting mechanical forces on the cells, thus risking cell damage. In this study we applied a novel isolation method called buoyancy-activated cell sorting, which involves using biotinylated albumin microbubbles (biotin-MBs) conjugated with antibodies (i.e., targeted biotin-MBs). Albumin MBs are widely used as contrast agents in ultrasound imaging due to their good biocompatibility and stability. For conjugating antibodies, biotin is conjugated onto the albumin MB shell via covalent bonds and the biotinylated antibodies are conjugated using an avidin-biotin system. The albumin microbubbles had a mean diameter of 2 μm with a polydispersity index of 0.16. For cell separation, the MDA-MB-231 cells are incubated with the targeted biotin-MBs conjugated with anti-CD44 for 10 min, centrifuged at 10 g for 1 min, and then allowed 1 hour at 4 °C for separation. The results indicate that targeted biotin-MBs conjugated with anti-CD44 antibodies can be used to separate MDA-MB-231 breast cancer cells; more than 90% of the cells were collected in the MB layer when the ratio of the MBs to cells was higher than 70:1. Furthermore, we found that the separating efficiency was higher for targeted biotin-MBs than for targeted avidin-incorporated albumin MBs (avidin-MBs), which is the most common way to make targeted albumin MBs. We also demonstrated that the recovery rate of targeted biotin-MBs was up to 88% and the sorting purity was higher than 84% for a a heterogenous cell population containing MDA-MB-231 cells (CD44(+)) and MDA-MB-453 cells (CD44-), which are classified as basal-like breast cancer cells and luminal breast cancer cells, respectively. Knowing that the CD44(+) is a commonly used cancer-stem-cell biomarker, our

  13. Activated Rac1 requires gp130 for Stat3 activation, cell proliferation and migration

    SciTech Connect

    Arulanandam, Rozanne; Geletu, Mulu; Feracci, Helene; Raptis, Leda

    2010-03-10

    Rac1 (Rac) is a member of the Rho family of small GTPases which controls cell migration by regulating the organization of actin filaments. Previous results suggested that mutationally activated forms of the Rho GTPases can activate the Signal Transducer and Activator of Transcription-3 (Stat3), but the exact mechanism is a matter of controversy. We recently demonstrated that Stat3 activity of cultured cells increases dramatically following E-cadherin engagement. To better understand this pathway, we now compared Stat3 activity levels in mouse HC11 cells before and after expression of the mutationally activated Rac1 (Rac{sup V12}), at different cell densities. The results revealed for the first time a dramatic increase in protein levels and activity of both the endogenous Rac and Rac{sup V12} with cell density, which was due to inhibition of proteasomal degradation. In addition, Rac{sup V12}-expressing cells had higher Stat3, tyrosine-705 phosphorylation and activity levels at all densities, indicating that Rac{sup V12} is able to activate Stat3. Further examination of the mechanism of Stat3 activation showed that Rac{sup V12} expression caused a surge in mRNA of Interleukin-6 (IL6) family cytokines, known potent Stat3 activators. Knockdown of gp130, the common subunit of this family reduced Stat3 activity, indicating that these cytokines may be responsible for the Stat3 activation by Rac{sup V12}. The upregulation of IL6 family cytokines was required for cell migration and proliferation induced by Rac{sup V12}, as shown by gp130 knockdown experiments, thus demonstrating that the gp130/Stat3 axis represents an essential effector of activated Rac for the regulation of key cellular functions.

  14. Upregulation of swelling-activated Cl− channel sensitivity to cell volume by activation of EGF receptors in murine mammary cells

    PubMed Central

    Abdullaev, Iskandar F; Sabirov, Ravshan Z; Okada, Yasunobu

    2003-01-01

    Whole-cell recordings showed that, in mouse mammary C127 cells transfected with the full genome of the bovine papilloma virus (BPV), a hypotonic challenge induced the activation of outwardly rectifying Cl− currents with a peak amplitude 2.7 times greater than that in control C127 cells. Cell-attached single-channel recordings showed that BPV-induced augmentation of the peak amplitude of the whole-cell current could not chiefly be explained by a small increase (1.2 times) in unitary conductance. There was no difference between control and BPV-transfected cells in the osmotic cell swelling rate, and hence, osmotic water permeability. However, a plot of the whole-cell current density as a function of cell volume, which was measured simultaneously, showed that the BPV-transfected cells had a strikingly greater volume sensitivity than control cells. Since the E5 protein of BPV has been reported to induce constitutive activation of the epidermal growth factor (EGF) receptor and platelet-derived growth factor (PDGF) receptor in a variety of cell lines including C127 cells, effects of the growth factors on volume-sensitive outwardly rectifying (VSOR) Cl− currents were examined in C127 cells. Application of PDGF peptides failed to affect the Cl− currents in control and BPV-transfected cells, although C127 cells are known to endogenously express PDGF receptors. In contrast, EGF peptides significantly increased the VSOR Cl− current in control cells. However, they failed to induce further augmentation of the current in BPV-transfected cells. VSOR Cl− currents were inhibited by tyrphostin B46, an inhibitor of the EGF receptor tyrosine kinase, in both control and BPV-transfected cells. The IC50 value in BPV-transfected cells (12 μm) was lower than that in control cells (31 μm). However, the VSOR Cl− currents in both cell types were insensitive to tyrphostin AG1296, an inhibitor of the PDGF receptor tyrosine kinase. The rate of regulatory volume decrease (RVD) was

  15. Substrate rigidity regulates human T cell activation and proliferation.

    PubMed

    O'Connor, Roddy S; Hao, Xueli; Shen, Keyue; Bashour, Keenan; Akimova, Tatiana; Hancock, Wayne W; Kam, Lance C; Milone, Michael C

    2012-08-01

    Adoptive immunotherapy using cultured T cells holds promise for the treatment of cancer and infectious disease. Ligands immobilized on surfaces fabricated from hard materials such as polystyrene plastic are commonly employed for T cell culture. The mechanical properties of a culture surface can influence the adhesion, proliferation, and differentiation of stem cells and fibroblasts. We therefore explored the impact of culture substrate stiffness on the ex vivo activation and expansion of human T cells. We describe a simple system for the stimulation of the TCR/CD3 complex and the CD28 receptor using substrates with variable rigidity manufactured from poly(dimethylsiloxane), a biocompatible silicone elastomer. We show that softer (Young's Modulus [E] < 100 kPa) substrates stimulate an average 4-fold greater IL-2 production and ex vivo proliferation of human CD4(+) and CD8(+) T cells compared with stiffer substrates (E > 2 MPa). Mixed peripheral blood T cells cultured on the stiffer substrates also demonstrate a trend (nonsignificant) toward a greater proportion of CD62L(neg), effector-differentiated CD4(+) and CD8(+) T cells. Naive CD4(+) T cells expanded on softer substrates yield an average 3-fold greater proportion of IFN-γ-producing Th1-like cells. These results reveal that the rigidity of the substrate used to immobilize T cell stimulatory ligands is an important and previously unrecognized parameter influencing T cell activation, proliferation, and Th differentiation. Substrate rigidity should therefore be a consideration in the development of T cell culture systems as well as when interpreting results of T cell activation based upon solid-phase immobilization of TCR/CD3 and CD28 ligands.

  16. Substrate rigidity regulates human T cell activation and proliferation1

    PubMed Central

    O’Connor, Roddy S.; Hao, Xueli; Shen, Keyue; Bashour, Keenan; Akimova, Tatiana; Hancock, Wayne W.; Kam, Lance; Milone, Michael C.

    2012-01-01

    Adoptive immunotherapy using cultured T cells holds promise for the treatment of cancer and infectious disease. Ligands immobilized on surfaces fabricated from hard materials such as polystyrene plastic are commonly employed for T cell culture. The mechanical properties of a culture surface can influence the adhesion, proliferation, and differentiation of stem cells and fibroblasts. We therefore explored the impact of culture substrate stiffness on the ex vivo activation and expansion of human T cells. We describe a simple system for the stimulation of the TCR/CD3 complex and the CD28 receptor using substrates with variable rigidity manufactured from poly(dimethylsiloxane) (PDMS), a biocompatible silicone elastomer. We show that softer (Young’s Modulus [E] < 100 kPa) substrates stimulate an average 4-fold greater IL-2 production and ex vivo proliferation of human CD4+ and CD8+ T cells compared with stiffer substrates (E >2 MPa). Mixed peripheral blood T cells cultured on the stiffer substrates also demonstrate a trend (non-significant) towards a greater proportion of CD62Lneg, effector-differentiated CD4+ and CD8+ T cells. Naïve CD4+ T cells expanded on softer substrates yield an average 3-fold greater proportion of IFN-γ producing TH1-like cells. These results reveal that the rigidity of the substrate used to immobilize T cell stimulatory ligands is an important and previously unrecognized parameter influencing T cell activation, proliferation and TH differentiation. Substrate rigidity should therefore be a consideration in the development of T cell culture systems as well as when interpreting results of T cell activation based upon solid-phase immobilization of TCR/CD3 and CD28 ligands. PMID:22732590

  17. Activated T cells sustain myeloid-derived suppressor cell-mediated immune suppression

    PubMed Central

    Damuzzo, Vera; Francescato, Samuela; Pozzuoli, Assunta; Berizzi, Antonio; Mocellin, Simone; Rossi, Carlo Riccardo; Bronte, Vincenzo; Mandruzzato, Susanna

    2016-01-01

    The expansion of myeloid derived suppressor cells (MDSCs), a suppressive population able to hamper the immune response against cancer, correlates with tumor progression and overall survival in several cancer types. We have previously shown that MDSCs can be induced in vitro from precursors present in the bone marrow and observed that these cells are able to actively proliferate in the presence of activated T cells, whose activation level is critical to drive the suppressive activity of MDSCs. Here we investigated at molecular level the mechanisms involved in the interplay between MDSCs and activated T cells. We found that activated T cells secrete IL-10 following interaction with MDSCs which, in turn, activates STAT3 phosphorylation on MDSCs then leading to B7-H1 expression. We also demonstrated that B7-H1+ MDSCs are responsible for immune suppression through a mechanism involving ARG-1 and IDO expression. Finally, we show that the expression of ligands B7-H1 and MHC class II both on in vitro-induced MDSCs and on MDSCs in the tumor microenvironment of cancer patients is paralleled by an increased expression of their respective receptors PD-1 and LAG-3 on T cells, two inhibitory molecules associated with T cell dysfunction. These findings highlight key molecules and interactions responsible for the extensive cross-talk between MDSCs and activated T cells that are at the basis of immune suppression. PMID:26700461

  18. Activated T cells sustain myeloid-derived suppressor cell-mediated immune suppression.

    PubMed

    Pinton, Laura; Solito, Samantha; Damuzzo, Vera; Francescato, Samuela; Pozzuoli, Assunta; Berizzi, Antonio; Mocellin, Simone; Rossi, Carlo Riccardo; Bronte, Vincenzo; Mandruzzato, Susanna

    2016-01-12

    The expansion of myeloid derived suppressor cells (MDSCs), a suppressive population able to hamper the immune response against cancer, correlates with tumor progression and overall survival in several cancer types. We have previously shown that MDSCs can be induced in vitro from precursors present in the bone marrow and observed that these cells are able to actively proliferate in the presence of activated T cells, whose activation level is critical to drive the suppressive activity of MDSCs. Here we investigated at molecular level the mechanisms involved in the interplay between MDSCs and activated T cells. We found that activated T cells secrete IL-10 following interaction with MDSCs which, in turn, activates STAT3 phosphorylation on MDSCs then leading to B7-H1 expression. We also demonstrated that B7-H1+ MDSCs are responsible for immune suppression through a mechanism involving ARG-1 and IDO expression. Finally, we show that the expression of ligands B7-H1 and MHC class II both on in vitro-induced MDSCs and on MDSCs in the tumor microenvironment of cancer patients is paralleled by an increased expression of their respective receptors PD-1 and LAG-3 on T cells, two inhibitory molecules associated with T cell dysfunction. These findings highlight key molecules and interactions responsible for the extensive cross-talk between MDSCs and activated T cells that are at the basis of immune suppression.

  19. Fluorescence-Activated Cell Sorting of Live Versus Dead Bacterial Cells and Spores

    NASA Technical Reports Server (NTRS)

    Bernardini, James N.; LaDuc, Myron T.; Diamond, Rochelle; Verceles, Josh

    2012-01-01

    This innovation is a coupled fluorescence-activated cell sorting (FACS) and fluorescent staining technology for purifying (removing cells from sampling matrices), separating (based on size, density, morphology, and live versus dead), and concentrating cells (spores, prokaryotic, eukaryotic) from an environmental sample.

  20. Spontaneous activity of cochlear hair cells triggered by fluid secretion mechanism in adjacent support cells

    PubMed Central

    Wang, Han Chin; Lin, Chun-Chieh; Cheung, Rocky; Zhang-Hooks, YingXin; Agarwal, Amit; Ellis-Davies, Graham; Rock, Jason; Bergles, Dwight E.

    2015-01-01

    Summary Spontaneous electrical activity of neurons in developing sensory systems promotes their maturation and proper connectivity. In the auditory system, spontaneous activity of cochlear inner hair cells (IHCs) is initiated by the release of ATP from glia-like inner supporting cells (ISCs), facilitating maturation of central pathways before hearing onset. Here, we find that ATP stimulates purinergic autoreceptors in ISCs, triggering Cl− efflux and osmotic cell shrinkage by opening TMEM16A Ca2+-activated Cl− channels. Release of Cl− from ISCs also forces K+ efflux, causing transient depolarization of IHCs near ATP release sites. Genetic deletion of TMEM16A markedly reduces the spontaneous activity of IHCs and spiral ganglion neurons in the developing cochlea, and prevents ATP-dependent shrinkage of supporting cells. These results indicate that support cells in the developing cochlea have adapted a pathway used for fluid secretion in other organs to induce periodic excitation of hair cells. PMID:26627734

  1. Antiproliferative activities of Garcinia bracteata extract and its active ingredient, isobractatin, against human tumor cell lines.

    PubMed

    Shen, Tao; Li, Wei; Wang, Yan-Yan; Zhong, Qing-Qing; Wang, Shu-Qi; Wang, Xiao-Ning; Ren, Dong-Mei; Lou, Hong-Xiang

    2014-03-01

    In our cell based screening of antitumor ingredients from plants, the EtOH extract of Garcinia bracteata displayed antiproliferative effect against human lung adenocarcinoma A549 cells, human breast cancer MCF-7 cells, and human prostate cancer PC3 cells. Phytochemical investigation of this active extract produced nine ingredients, and their structures were established by analysis of MS and NMR spectra. Antiproliferative evaluation of isolated ingredients on A549, MCF-7 and PC3 cells indicated that a xanthone named isobractatin (1) exhibited potent antiproliferative activity against the above three human cancer cell lines with IC50 values ranging from 2.90 to 4.15 μM. Treatment of PC3 cells with 1 led to an enhancement of the cell apoptosis, and arrested cell cycle in the G0/G1 phase. The G0/G1 phase cycle-related proteins analysis showed that the expressions of cyclins D1 and E were reduced by 1, whereas the protein level of cyclin dependent kinase (CDK) inhibitor P21 was induced. Additionally, 1 enhanced PC3 cell apoptosis by activations of Bax, caspases 3 and 9, and by inhibition of Bcl-2. Our combined data illustrated that isobractatin (1) was the antiproliferative ingredient of G. bracteata against three human cancer cell lines, which exerted its antiproliferatrive effect via cell cycle arrest and induction of apoptosis.

  2. An integrated optofluidic platform for Raman-activated cell sorting.

    PubMed

    Lau, Adrian Y; Lee, Luke P; Chan, James W

    2008-07-01

    We report on integrated optofluidic Raman-activated cell sorting (RACS) platforms that combine multichannel microfluidic devices and laser tweezers Raman spectroscopy (LTRS) for delivery, identification, and simultaneous sorting of individual cells. The system allows label-free cell identification based on Raman spectroscopy and automated continuous cell sorting. Two optofluidic designs using hydrodynamic focusing and pinch-flow fractionation are evaluated based on their sorting design and flow velocity effect on the laser trapping efficiency at different laser power levels. A proof-of-principle demonstration of the integrated optofluidic LTRS system for the identification and sorting of two leukemia cell lines is presented. This functional prototype lays the foundation for the development of a label-free cell sorting platform based on intrinsic Raman markers for automated sampling and sorting of a large number of individual cells in solution.

  3. Enhancement of endothelial cell migration by constitutively active LPA{sub 1}-expressing tumor cells

    SciTech Connect

    Kitayoshi, Misaho; Kato, Kohei; Tanabe, Eriko; Yoshikawa, Kyohei; Fukui, Rie; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer Mutated LPA{sub 1} stimulates cell migration of endothelial cells. Black-Right-Pointing-Pointer VEGF expressions are increased by mutated LPA{sub 1}. Black-Right-Pointing-Pointer LPA signaling via mutated LPA{sub 1} is involved in angiogenesis. Black-Right-Pointing-Pointer Mutated LPA{sub 1} promotes cancer cell progression. -- Abstract: Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors (LPA receptors; LPA{sub 1} to LPA{sub 6}). They indicate a variety of cellular response by the interaction with LPA, including cell proliferation, migration and differentiation. Recently, we have reported that constitutive active mutated LPA{sub 1} induced the strong biological effects of rat neuroblastoma B103 cells. In the present study, we examined the effects of mutated LPA{sub 1} on the interaction between B103 cells and endothelial F-2 cells. Each LPA receptor expressing B103 cells were maintained in serum-free DMEM and cell motility assay was performed with a Cell Culture Insert. When F-2 cells were cultured with conditioned medium from Lpar1 and Lpar3-expressing cells, the cell motility of F-2 cells was significantly higher than control cells. Interestingly, the motile activity of F-2 cells was strongly induced by mutated LPA{sub 1} than other cells, correlating with the expression levels of vascular endothelial growth factor (Vegf)-A and Vegf-C. Pretreatment of LPA signaling inhibitors inhibited F-2 cell motility stimulated by mutated LPA{sub 1}. These results suggest that activation of LPA signaling via mutated LPA{sub 1} may play an important role in the promotion of angiogenesis in rat neuroblastoma cells.

  4. A role for CD9 molecules in T cell activation

    PubMed Central

    1996-01-01

    Costimulation mediated by the CD28 molecule plays an important role in optimal activation of T cells. However, CD28-deficient mice can mount effective T cell-dependent immune responses, suggesting the existence of other costimulatory systems. In a search for other costimulatory molecules on T cells, we have developed a monoclonal antibody (mAb) that can costimulate T cells in the absence of antigen-presenting cells (APC). The molecule recognized by this mAb, 9D3, was found to be expressed on almost all mature T cells and to be a protein of approximately 24 kD molecular mass. By expression cloning, this molecule was identified as CD9, 9D3 (anti-CD9) synergized with suboptimal doses of anti-CD3 mAb in inducing proliferation by virgin T cells. Costimulation was induced by independent ligation of CD3 and CD9, suggesting that colocalization of these two molecules is not required for T cell activation. The costimulation by anti-CD9 was as potent as that by anti-CD28. Moreover, anti-CD9 costimulated in a CD28- independent way because anti-CD9 equally costimulated T cells from the CD28-deficient as well as wild-type mice. Thus, these results indicate that CD9 serves as a molecule on T cells that can deliver a potent CD28- independent costimulatory signal. PMID:8760830

  5. Caerulomycin A Suppresses Immunity by Inhibiting T Cell Activity

    PubMed Central

    Chauhan, Arun; Khatri, Neeraj; Vohra, Rakesh M.; Jolly, Ravinder S.; Agrewala, Javed N.

    2014-01-01

    Background Caerulomycin A (CaeA) is a known antifungal and antibiotic agent. Further, CaeA is reported to induce the expansion of regulatory T cell and prolongs the survival of skin allografts in mouse model of transplantation. In the current study, CaeA was purified and characterized from a novel species of actinomycetes, Actinoalloteichus spitiensis. The CaeA was identified for its novel immunosuppressive property by inhibiting in vitro and in vivo function of T cells. Methods Isolation, purification and characterization of CaeA were performed using High Performance Flash Chromatography (HPFC), NMR and mass spectrometry techniques. In vitro and in vivo T cell studies were conducted in mice using flowcytometry, ELISA and thymidine-[methyl-3H] incorporation. Results CaeA significantly suppressed T cell activation and IFN-γ secretion. Further, it inhibited the T cells function at G1 phase of cell cycle. No apoptosis was noticed by CaeA at a concentration responsible for inducing T cell retardation. Furthermore, the change in the function of B cells but not macrophages was observed. The CaeA as well exhibited substantial inhibitory activity in vivo. Conclusion This study describes for the first time novel in vitro and in vivo immunosuppressive function of CaeA on T cells and B cells. CaeA has enough potential to act as a future immunosuppressive drug. PMID:25286329

  6. Gamma Delta T-Cells Regulate Wound Myeloid Cell Activity After Burn

    DTIC Science & Technology

    2014-03-01

    GAMMA DELTA T CELLS REGULATE WOUND MYELOID CELL ACTIVITY AFTER BURN Meenakshi Rani ,* Qiong Zhang,* and Martin G. Schwacha*† *Department of Surgery...Cell Activity After Burn 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Rani M., Zhang Q., Schwacha M. G., 5d...WT mice. 138 SHOCK VOL. 42, NO. 2 RANI ET AL. Copyright © 2014 by the Shock Society. Unauthorized reproduction of this article is prohibited. systemic

  7. Minimal model for spontaneous cell polarization and edge activity in oscillating, rotating and migrating cells

    NASA Astrophysics Data System (ADS)

    Raynaud, Franck; Ambühl, Mark E.; Gabella, Chiara; Bornert, Alicia; Sbalzarini, Ivo F.; Meister, Jean-Jacques; Verkhovsky, Alexander B.

    2016-04-01

    How cells break symmetry and organize activity at their edges to move directionally is a fundamental question in cell biology. Physical models of cell motility commonly incorporate gradients of regulatory proteins and/or feedback from the motion itself to describe the polarization of this edge activity. These approaches, however, fail to explain cell behaviour before the onset of polarization. We use polarizing and moving fish epidermal cells as a model system to bridge the gap between cell behaviours before and after polarization. Our analysis suggests a novel and simple principle of self-organizing cell activity, in which local cell-edge dynamics depends on the distance from the cell centre, but not on the orientation with respect to the front-back axis. We validate this principle with a stochastic model that faithfully reproduces a range of cell-migration behaviours. Our findings indicate that spontaneous polarization, persistent motion and cell shape are emergent properties of the local cell-edge dynamics controlled by the distance from the cell centre.

  8. The potential of a dielectrophoresis activated cell sorter (DACS) as a next generation cell sorter

    NASA Astrophysics Data System (ADS)

    Lee, Dongkyu; Hwang, Bohyun; Kim, Byungkyu

    2016-12-01

    Originally introduced by H. A. Pohl in 1951, dielectrophoretic (DEP) force has been used as a striking tool for biological particle manipulation (or separation) for the last few decades. In particular, dielectrophoresis activated cell sorters (DACSes) have been developed for applications in various biomedical fields. These applications include cell replacement therapy, drug screening and medical diagnostics. Since a DACS does not require a specific bio-marker, it is able to function as a biological particle sorting tool with numerous configurations for various cells [e.g. red blood cells (RBCs), white blood cells (WBCs), circulating tumor cells, leukemia cells, breast cancer cells, bacterial cells, yeast cells and sperm cells]. This article explores current DACS capabilities worldwide, and it also looks at recent developments intended to overcome particular limitations. First, the basic theories are reviewed. Then, representative DACSes based on DEP trapping, traveling wave DEP systems, DEP field-flow fractionation and DEP barriers are introduced, and the strong and weak points of each DACS are discussed. Finally, for the purposes of commercialization, prerequisites regarding throughput, efficiency and recovery rates are discussed in detail through comparisons with commercial cell sorters (e.g. fluorescent activated and magnetic activated cell sorters).

  9. Synergistic activation of cells by Epstein-Barr virus and B-cell growth factor.

    PubMed Central

    Hutt-Fletcher, L M

    1987-01-01

    Infection with Epstein-Barr virus (EBV) is initiated by virus binding to the C3dg-C3d receptor CR2. Several workers have implicated this receptor in the control of B-cell activation by examining the effects of antibodies to CR2 and isolated C3d on B-cell proliferation and differentiation. We report here on the activating effects of irradiated EBV, which retains its capacity to bind to CR2 but loses its ability to function as a T-independent B-cell activator. EBV synergized with B-cell growth factor in the induction of uptake of tritiated thymidine by T cell-depleted leukocytes from seronegative donors but did not induce secretion of immunoglobulin. Synergism could be inhibited with an anti-viral antibody that inhibited binding of EBV to CR2. No similar synergism was found between EBV and recombinant interleukin 2, interleukin 1 alpha, or gamma interferon or with the lipid A fraction of bacterial lipopolysaccharide. EBV may thus initiate B-cell activation as it binds to CR2. Infectious virus may, under normal circumstances, induce the cell to make those growth factors necessary to support B-cell proliferation; the difficulty of transforming cells with transfected EBV DNA may in part reflect the absence of an activation event provided by intact virus as it attaches to CR2. The synergism of EBV and B-cell growth factor more clearly distinguishes the effects of B-cell growth factor from those of interleukin 1 and interleukin 2 in other models of B-cell activation. Thus, this may be a useful model for further delineation of unique effects of B-cell growth factor on B-cell function. PMID:3027404

  10. Zinc modulates PPARgamma signaling and activation of porcine endothelial cells.

    PubMed

    Meerarani, Purushothaman; Reiterer, Gudrun; Toborek, Michal; Hennig, Bernhard

    2003-10-01

    Dietary zinc has potent antioxidant and anti-inflammatory properties and is a critical component of peroxisome proliferator-activated receptor (PPAR) gene expression and regulation. To assess the protective mechanisms of PPARgamma in endothelial cell dysfunction and the role of zinc in the modulation of PPARgamma signaling, cultured porcine pulmonary artery endothelial cells were exposed to the membrane-permeable zinc chelator N,N,N'N'-tetrakis (2-pyridylmethyl)-ethylene diamine (TPEN), thiazolidinedione (TZD; PPARgamma agonist) or bisphenol A diglycidyl ether (BADGE; PPARgamma antagonist). Subsequently, endothelial cells were activated by treatment with linoleic acid (90 micro mol/L) for 6 h. Zinc chelation by TPEN increased the DNA binding activity of nuclear factor (NF)-kappaB and activator protein (AP)-1, decreased PPARgamma expression and activation as well as up-regulated interleukin (IL)-6 expression and production. These effects were fully reversed by zinc supplementation. In addition, exposure to TZD down-regulated linoleic acid-induced DNA binding activity of NF-kappaB and AP-1, whereas BADGE further induced activation of these oxidative stress-sensitive transcription factors. Most importantly, the TZD-mediated down-regulation of NF-kappaB and AP-1 and reduced inflammatory response were impaired during zinc chelation. These data suggest that zinc plays a critical role in PPARgamma signaling in linoleic acid-induced endothelial cell activation and indicate that PPARgamma signaling is impaired during zinc deficiency.

  11. Decrease in T Cell Activation and Calcium Flux during Clinorotation

    NASA Technical Reports Server (NTRS)

    Sams, Clarence; Holtzclaw, J. David

    2006-01-01

    We investigated the effect of altered gravitational environments on T cell activation. We isolated human, naive T cells (CD3+CD14-CD19-CD16-CD56-CD25-CD69-CD45RA-) following IRB approved protocols. These purified T cells were then incubated with 6 mm polystyrene beads coated with OKT3 (Ortho Biotech, Raritan, NJ) and antiCD28 (Becton Dickinson (BD), San Jose, CA) at 37 C for 24 hours. Antibodies were at a 1:1 ratio and the bead-to-cell ratio was 2:1. Four incubation conditions existed: 1) static or "1g"; 2) centrifugation at 10 relative centrifugal force (RCF) or "10g"; 3) clinorotation at 25 RPM (functional weightlessness or "0g"); and 4) clinorotation at 80 RPM ("1g" plus net shear force approx.30 dynes/sq cm). Following incubation, T cells were stained for CD25 expression (BD) and intracellular calcium (ratio of Fluo4 to Fura Red, Molecular Probes, Eugene, OR) and analyzed by flow cytometry (Coulter EPICS XL, Miami, FL). Results: Static or "1g" T cells had the highest level of CD25 expression and intracellular calcium. T cells centrifuged at 10 RCF ("10g") had lower CD25 expression and calcium levels compared to the static control. However, cells centrifuged at 10 RCF had higher CD25 expression and calcium levels than those exposed to 24 RPM clinorotation ("0g"). T cells exposed to 24 RPM clinorotation had lower CD25 expression, but the approximately the same calcium levels than T cells exposed to 80 RPM clinorotation. These data suggest that stress-activated calcium channel exist in T cells and may play a role during T cell activation.

  12. Direct determination of phosphatase activity from physiological substrates in cells.

    PubMed

    Ren, Zhongyuan; Do, Le Duy; Bechkoff, Géraldine; Mebarek, Saida; Keloglu, Nermin; Ahamada, Saandia; Meena, Saurabh; Magne, David; Pikula, Slawomir; Wu, Yuqing; Buchet, René

    2015-01-01

    A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min(-1) mg(-1) for PPi, to 56 ± 11 nmol min(-1) mg(-1) for AMP, to 79 ± 23 nmol min(-1) mg(-1) for beta-glycerophosphate and to 73 ± 15 nmol min(-1) mg(-1) for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole--a TNAP inhibitor--served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.

  13. Nattokinase-promoted tissue plasminogen activator release from human cells.

    PubMed

    Yatagai, Chieko; Maruyama, Masugi; Kawahara, Tomoko; Sumi, Hiroyuki

    2008-01-01

    When heated to a temperature of 70 degrees C or higher, the strong fibrinolytic activity of nattokinase in a solution was deactivated. Similar results were observed in the case of using Suc-Ala-Ala-Pro-Phe-pNA and H-D-Val-Leu-Lys-pNA, which are synthetic substrates of nattokinase. In the current study, tests were conducted on the indirect fibrinolytic effects of the substances containing nattokinase that had been deactivated through heating at 121 degrees C for 15 min. Bacillus subtilis natto culture solutions made from three types of bacteria strain were heat-treated and deactivated, and it was found that these culture solutions had the ability to generate tissue plasminogen activators (tPA) from vascular endothelial cells and HeLa cells at certain concentration levels. For example, it was found that the addition of heat-treated culture solution of the Naruse strain (undiluted solution) raises the tPA activity of HeLa cells to about 20 times that of the control. Under the same conditions, tPA activity was raised to a level about 5 times higher for human vascular endothelial cells (HUVEC), and to a level about 24 times higher for nattokinase sold on the market. No change in cell count was observed for HeLa cells and HUVEC in the culture solution at these concentrations, and the level of activity was found to vary with concentration.

  14. Idelalisib and caffeine reduce suppression of T cell responses mediated by activated chronic lymphocytic leukemia cells

    PubMed Central

    Hock, Barry D.; MacPherson, Sean A.; McKenzie, Judith L.

    2017-01-01

    Chronic lymphocytic leukemia (CLL) is associated with T cell dysfunction. Activated CLL cells are found within the lymphoid tumor micro-environment and overcoming immuno-suppression induced by these cells may improve anti-CLL immune responses. However, the mechanisms by which activated CLL cells inhibit T cell responses, and reagents targeting such mechanisms have not been identified. Here we demonstrate that the ability of in vitro activated CLL cells to suppress T cell proliferation is not reversed by the presence of ecto-nuclease inhibitors or blockade of IL-10, PD-1 and CTLA-4 pathways. Caffeine is both an adenosine receptor antagonist and a phosphatidylinositol-3-kinase, p110δ (PI3Kδ) inhibitor and, at physiologically relevant levels, significantly reversed suppression. Significant reversal of suppression was also observed with the PI3Kδ specific inhibitor Idelalisib but not with adenosine receptor specific antagonists. Furthermore, addition of caffeine or Idelalisib to activated CLL cells significantly inhibited phosphorylation of AKT, a downstream kinase of PI3K, but did not affect CLL viability. These results suggest that caffeine, in common with Idelalisib, reduces the immuno-suppressive activity of activated CLL cells by inhibiting PI3Kδ. These findings raise the possibility that these compounds may provide a useful therapeutic adjunct by reducing immuno-suppression within the tumor micro-environment of CLL. PMID:28257435

  15. Calcium Activation Profile In Electrically Stimulated Intact Rat Heart Cells

    NASA Astrophysics Data System (ADS)

    Geerts, Hugo; Nuydens, Rony; Ver Donck, Luc; Nuyens, Roger; De Brabander, Marc; Borgers, Marcel

    1988-06-01

    Recent advances in fluorescent probe technology and image processing equipment have made available the measurement of calcium in living systems on a real-time basis. We present the use of the calcium indicator Fura-2 in intact normally stimulated rat heart cells for the spatial and dynamic measurement of the calcium excitation profile. After electric stimulation (1 Hz), the activation proceeds from the center of the myocyte toward the periphery. Within two frame times (80 ms), the whole cell is activated. The activation is slightly faster in the center of the cell than in the periphery. The mean recovery time is 200-400 ms. There is no difference along the cell's long axis. The effect of a beta-agonist and of a calcium antagonist is described.

  16. Activity-based probes for detection of active MALT1 paracaspase in immune cells and lymphomas.

    PubMed

    Eitelhuber, Andrea C; Vosyka, Oliver; Nagel, Daniel; Bognar, Miriam; Lenze, Dido; Lammens, Katja; Schlauderer, Florian; Hlahla, Daniela; Hopfner, Karl-Peter; Lenz, Georg; Hummel, Michael; Verhelst, Steven H L; Krappmann, Daniel

    2015-01-22

    MALT1 paracaspase is activated upon antigen receptor stimulation to promote lymphocyte activation. In addition, deregulated MALT1 protease activity drives survival of distinct lymphomas such as the activated B cell type of diffuse large B cell lymphoma (ABC-DLBCL). Here, we designed fluorophore or biotin-coupled activity based-probes (ABP) that covalently modify the active center of MALT1. MALT1-ABPs are exclusively labeling an active modified full length form of MALT1 upon T cell stimulation. Further, despite the CARMA1 requirement for initial MALT1 activation, the MALT1-ABPs show that protease activity is not confined to the high-molecular CARMA1-BCL10-MALT1 (CBM) complex. Using biotin-coupled ABPs, we developed a robust assay for sensitive and selective detection of active MALT1 in cell lines, primary lymphocytes, and DLBCL tumor biopsies. Taken together, MALT1-ABPs represent powerful chemical tools to measure cellular MALT1 activation, determine efficacy of small molecule inhibitors, and classify lymphomas based on MALT1 activity status.

  17. Evidence for B cell activation in patients with active rheumatoid arthritis.

    PubMed Central

    Youinou, P Y; Irving, W L; Shipley, M; Hayes, J; Lydyard, P M

    1984-01-01

    Peripheral blood lymphocytes and in some cases synovial eluate cells from 51 patients with rheumatoid arthritis (RA), were analysed for the percentages of cells bearing surface light chains (total B cells), IgM and IgD. In addition, their capacity to form rosettes with mouse erythrocytes (mRFC)--a property of a B cell subpopulation--was determined. Activity of the disease was assessed by clinical and laboratory criteria and classified as very active, moderately active and inactive. Normal, age and sex matched individuals and a group of patients with a variety of other rheumatological disorders, were used as control populations. Although there was no significant difference in percentages of total B cells in any of the groups compared with normal controls, there was a small but significant increase in the ratio of cells bearing IgM to those bearing IgD in patients with very active disease. This was paralleled by a significant decrease in the mRFC in this disease activity group. Patients with inactive disease showed no change in their proportions of IgM:IgD, but did show a significant increase in mRFC. These results are discussed in terms of the presence of activated B cells in patients with very active RA. PMID:6607144

  18. Cinnamon effectively inhibits the activity of leukemia stem cells.

    PubMed

    Guan, X; Su, M C; Zhao, R B; Ouyang, H M; Dong, X D; Hu, P; Pei, Q; Lu, J; Li, Z F; Zhang, C R; Yang, T-H

    2016-08-19

    Cinnamon is the main component of Sanyangxuedai, which is one of the effective traditional Chinese medicines for treating malignancies. Leukemia is a prevalent malignant disease that Sanyangxuedai has been used to treat. Although successful in several studies, there is a lack of solid evidence as to why Sanyangxuedai has an effect on leukemia, and little is known about the underlying mechanisms. In this study, the active ingredients of cinnamon were isolated, purified, and identified. The transwell transport pool formed with the Caco-2 cell model was used to filter the active ingredients of cinnamon by simulating the gastrointestinal barrier in vitro. Moreover, the cell morphology, cell cycle status, apoptosis status, and antigenic variation of the cell surface antigens were observed and measured in K562 cells after treatment with the active ingredients of cinnamon. Our results showed that 50-75 μM was a safe concentration of cinnamon extract for treatment of K562 cells for 72 h. The cinnamon extract caused growth inhibition of K562 cells. Cinnamon extract seemed to arrest the cells at the G1 stage and increased the apoptosis rate significantly. Interestingly, cinnamon extract treatment upregulated the expression of erythroid and myeloid differentiation antigens and downregulated that of the megakaryocytic differentiation antigens in a dose-dependent manner. Our findings indicate that cinnamon extract from Sanyangxuedai may be effective for treating leukemia.

  19. Synaptic background activity influences spatiotemporal integration in single pyramidal cells.

    PubMed Central

    Bernander, O; Douglas, R J; Martin, K A; Koch, C

    1991-01-01

    The standard one-dimensional Rall cable model assumes that the electrotonic structure of neurons does not change in response to synaptic input. This model is used in a great number of both theoretical and anatomical-physiological structure-function studies. In particular, the membrane time constant, tau m, the somatic input resistance, Rin, and the electrotonic length are used to characterize single cells. However, these studies do not take into account that neurons are embedded in a network of spontaneously active cells. Synapses from these cells will contribute significantly to the membrane conductance, especially if recent evidence of very high specific membrane resistance, Rm = 100 k omega.cm2, is taken into account. We numerically simulated the electrical behavior of an anatomically reconstructed layer V cortical pyramidal cell receiving input from 4000 excitatory and 1000 inhibitory cells firing spontaneously at 0-7 Hz. We found that, over this range of synaptic background activity, tau m and Rin change by a factor of 10 (80-7 msec, 110-14 M omega) and the electrotonic length of the cell changes by a factor of 3. We show that this significantly changes the response of the cell to temporal desynchronized versus temporal synchronized synaptic input distributed throughout the neuron. Thus, the global activity of the network can control how individual cells perform spatial and temporal integration. PMID:1763072

  20. Phagocytosis by macrophages and endothelial cells inhibits procoagulant and fibrinolytic activity of acute promyelocytic leukemia cells.

    PubMed

    Xie, Rui; Gao, Chunyan; Li, Wen; Zhu, Jiuxin; Novakovic, Valerie; Wang, Jing; Ma, Ruishuang; Zhou, Jin; Gilbert, Gary E; Shi, Jialan

    2012-03-08

    The coagulopathy of acute promyelocytic leukemia (APL) is mainly related to procoagulant substances and fibrinolytic activators of APL blasts, but the fate of these leukemic cells is unknown. The aim of this study was to investigate the removal of APL blasts by macrophages and endothelial cells in vitro and consequent procoagulant and fibrinolytic activity of APL cells. We found that human umbilical vein endothelial cells as well as THP-1 and monocyte-derived macrophages bound, engulfed, and subsequently degraded immortalized APL cell line NB4 and primary APL cells. Lactadherin promoted phagocytosis of APL cells in a time-dependent fashion. Furthermore, factor Xa and prothrombinase activity of phosphatidylserine-exposed target APL cells was time-dependently decreased after incubation with phagocytes (THP-1-derived macrophages or HUVECs). Thrombin production on target APL cells was reduced by 40%-45% after 2 hours of coincubation with phagocytes and 80% by a combination of lactadherin and phagocytes. Moreover, plasmin generation of target APL cells was inhibited 30% by 2 hours of phagocytosis and ∼ 50% by lactadherin-mediated engulfment. These results suggest that engulfment by macrophages and endothelial cells reduce procoagulant and fibrinolytic activity of APL blasts. Lactadherin and phagocytosis could cooperatively ameliorate the clotting disorders in APL.

  1. Xenobiotic induction of quinone oxidoreductase activity in lens epithelial cells.

    PubMed

    Tumminia, S J; Rao, P V; Zigler, J S; Russell, P

    1993-12-08

    Xenobiotic regulatory elements have been identified for enzymes which ameliorate oxidative damage in cells. Zeta (zeta)-crystallin, a taxon-specific enzyme/crystallin shown to be a novel NADPH-dependent quinone reductase, is found in a number of tissues and cell types. This study shows that zeta-crystallin is present in mouse lens epithelium, as well as in the alpha TN4 mouse lens epithelial cell line. To determine whether zeta-crystallin is an inducible quinone reductase, cell cultures were exposed to the xenobiotics, 1,2-naphthoquinone and beta-naphthoflavone. Assays of cellular homogenates showed that quinone reductase activity was stimulated greater than 70% and 90%, respectively, over the control cells. This observed activity was sensitive to dicumarol, a potent inhibitor of quinone reductase activity. 1,2-Naphthoquinone- and beta-naphthoflavone-exposed cells were found to exhibit 1.47- and 1.68-fold increases, respectively, in zeta-crystallin protein concentration. A comparable increase in zeta-crystallin mRNA was indicative of an induction in zeta-crystallin expression in response to naphthalene challenge. Lens epithelial cells were also checked for DT-diaphorase, a well-known cellular protective enzyme which can catalyze the two-electron reduction of quinones. Slot blot analyses indicated that alpha TN4 cells exposed to 1,2-naphthoquinone and beta-naphthoflavone exhibited 2.71- and 6.81-fold increases in DT-diaphorase concentration when compared to the control cells. The data suggest that while DT-diaphorase is most likely responsible for the majority of the observed increase in quinone reductase activity, the zeta-crystallin gene also undergoes activation which is apparently mediated by a xenobiotic-responsive element.

  2. Effect of Fibroblast-Like Cells of Mesenchymal Origin of Cytotoxic Activity of Lymphocytes against NK-Sensitive Target Cells.

    PubMed

    Lupatov, A Yu; Kim, Ya S; Bystrykh, O A; Vakhrushev, I V; Pavlovich, S V; Yarygin, K N; Sukhikh, G T

    2017-02-01

    We studied immunosuppressive properties of skin fibroblasts and mesenchymal stromal cells against NK cells. In vitro experiments showed that mesenchymal stromal cells isolated from human umbilical cord and human skin fibroblasts can considerably attenuate cytotoxic activity of NK cells against Jurkat cells sensitive to NK-mediated lysis. NK cells cultured in lymphocyte population exhibited higher cytotoxic activity than isolated NK cells. Mesenchymal stromal cells or fibroblasts added 1:1 to lymphocyte culture almost completely suppressed NK cell cytotoxicity. This suggests that fibroblast-like cells can suppress not only isolated NK cells, but also NK cells in natural cell microenvironment.

  3. CD1d- and MR1-Restricted T Cells in Sepsis

    PubMed Central

    Szabo, Peter A.; Anantha, Ram V.; Shaler, Christopher R.; McCormick, John K.; Haeryfar, S.M. Mansour

    2015-01-01

    Dysregulated immune responses to infection, such as those encountered in sepsis, can be catastrophic. Sepsis is typically triggered by an overwhelming systemic response to an infectious agent(s) and is associated with high morbidity and mortality even under optimal critical care. Recent studies have implicated unconventional, innate-like T lymphocytes, including CD1d- and MR1-restricted T cells as effectors and/or regulators of inflammatory responses during sepsis. These cell types are typified by invariant natural killer T (iNKT) cells, variant NKT (vNKT) cells, and mucosa-associated invariant T (MAIT) cells. iNKT and vNKT cells are CD1d-restricted, lipid-reactive cells with remarkable immunoregulatory properties. MAIT cells participate in antimicrobial defense, and are restricted by major histocompatibility complex-related protein 1 (MR1), which displays microbe-derived vitamin B metabolites. Importantly, NKT and MAIT cells are rapid and potent producers of immunomodulatory cytokines. Therefore, they may be considered attractive targets during the early hyperinflammatory phase of sepsis when immediate interventions are urgently needed, and also in later phases when adjuvant immunotherapies could potentially reverse the dangerous state of immunosuppression. We will highlight recent findings that point to the significance or the therapeutic potentials of NKT and MAIT cells in sepsis and will also discuss what lies ahead in research in this area. PMID:26322041

  4. Blockade of T-cell activation by dithiocarbamates involves novel mechanisms of inhibition of nuclear factor of activated T cells.

    PubMed Central

    Martínez-Martínez, S; Gómez del Arco, P; Armesilla, A L; Aramburu, J; Luo, C; Rao, A; Redondo, J M

    1997-01-01

    Dithiocarbamates (DTCs) have recently been reported as powerful inhibitors of NF-kappaB activation in a number of cell types. Given the role of this transcription factor in the regulation of gene expression in the inflammatory response, NF-kappaB inhibitors have been suggested as potential therapeutic drugs for inflammatory diseases. We show here that DTCs inhibited both interleukin 2 (IL-2) synthesis and membrane expression of antigens which are induced during T-cell activation. This inhibition, which occurred with a parallel activation of c-Jun transactivating functions and expression, was reflected by transfection experiments at the IL-2 promoter level, and involved not only the inhibition of NF-kappaB-driven reporter activation but also that of nuclear factor of activated T cells (NFAT). Accordingly, electrophoretic mobility shift assays (EMSAs) indicated that pyrrolidine DTC (PDTC) prevented NF-kappaB, and NFAT DNA-binding activity in T cells stimulated with either phorbol myristate acetate plus ionophore or antibodies against the CD3-T-cell receptor complex and simultaneously activated the binding of AP-1. Furthermore, PDTC differentially targeted both NFATp and NFATc family members, inhibiting the transactivation functions of NFATp and mRNA induction of NFATc. Strikingly, Western blotting and immunocytochemical experiments indicated that PDTC promoted a transient and rapid shuttling of NFATp and NFATc, leading to their accelerated export from the nucleus of activated T cells. We propose that the activation of an NFAT kinase by PDTC could be responsible for the rapid shuttling of the NFAT, therefore transiently converting the sustained transactivation of this transcription factor that occurs during lymphocyte activation, and show that c-Jun NH2-terminal kinase (JNK) can act by directly phosphorylating NFATp. In addition, the combined inhibitory effects on NFAT and NF-KB support a potential use of DTCs as immunosuppressants. PMID:9343406

  5. Passive versus active local microrheology in mammalian cells and amoebae

    NASA Astrophysics Data System (ADS)

    Riviere, C.; Gazeau, F.; Marion, S.; Bacri, J.-C.; Wilhelm, C.

    2004-12-01

    We compare in this paper the rotational magnetic microrheology detailed by Marion et al [18] and Wilhelm et al [19] to the passive tracking microrheology. The rotational microrheology has been designed to explore, using magnetic rotating probes, the local intracellular microenvironment of living cells in terms of viscoelasticity. Passive microrheology techniques is based on the analysis of spontaneous diffusive motions of Brownian probes. The dependence of mean square displacement (MSD) with the time then directly reflects the type of movement (sub-, hyper- or diffusive motions). Using the same intracellular probes, we performed two types of measurements (active and passive). Based on the fluctuation-dissipation theorem, one should obtain the same information from the both techniques in a thermally equilibrium system. Interestingly, our measurements differ, and the discordances directly inform on active biological processes, which add to thermally activated fluctuations in our out-of equilibrium systems. In both cell models used, mammalian Hela cells and amoebae Entamoeba Histolytica, a hyper-diffusive regime at a short time is observed, which highlights the presence of an active non-thermal driving force, acting on the probe. However, the nature of this active force in mammalian cells and amoebae is different, according to their different phenotypes. In mammalian cells active processes are governed by the transport, via molecular motors, on the microtubule network. In amoebae, which are highly motile cells free of microtubule network, the active processes are dominated by strong fluxes of cytoplasm driven by extension of pseudopodia, in random directions, leading to an amplitude of motion one order of magnitude higher than for mammalian cells. Figs 7, Refs 32.

  6. High efficiency cell-specific targeting of cytokine activity

    NASA Astrophysics Data System (ADS)

    Garcin, Geneviève; Paul, Franciane; Staufenbiel, Markus; Bordat, Yann; van der Heyden, José; Wilmes, Stephan; Cartron, Guillaume; Apparailly, Florence; de Koker, Stefaan; Piehler, Jacob; Tavernier, Jan; Uzé, Gilles

    2014-01-01

    Systemic toxicity currently prevents exploiting the huge potential of many cytokines for medical applications. Here we present a novel strategy to engineer immunocytokines with very high targeting efficacies. The method lies in the use of mutants of toxic cytokines that markedly reduce their receptor-binding affinities, and that are thus rendered essentially inactive. Upon fusion to nanobodies specifically binding to marker proteins, activity of these cytokines is selectively restored for cell populations expressing this marker. This ‘activity-by-targeting’ concept was validated for type I interferons and leptin. In the case of interferon, activity can be directed to target cells in vitro and to selected cell populations in mice, with up to 1,000-fold increased specific activity. This targeting strategy holds promise to revitalize the clinical potential of many cytokines.

  7. Focal Adhesion Kinase Modulates Cell Adhesion Strengthening via Integrin Activation

    PubMed Central

    Michael, Kristin E.; Dumbauld, David W.; Burns, Kellie L.; Hanks, Steven K.

    2009-01-01

    Focal adhesion kinase (FAK) is an essential nonreceptor tyrosine kinase regulating cell migration, adhesive signaling, and mechanosensing. Using FAK-null cells expressing FAK under an inducible promoter, we demonstrate that FAK regulates the time-dependent generation of adhesive forces. During the early stages of adhesion, FAK expression in FAK-null cells enhances integrin activation to promote integrin binding and, hence, the adhesion strengthening rate. Importantly, FAK expression regulated integrin activation, and talin was required for the FAK-dependent effects. A role for FAK in integrin activation was confirmed in human fibroblasts with knocked-down FAK expression. The FAK autophosphorylation Y397 site was required for the enhancements in adhesion strengthening and integrin-binding responses. This work demonstrates a novel role for FAK in integrin activation and the time-dependent generation of cell–ECM forces. PMID:19297531

  8. Efficient Killing of High Risk Neuroblastoma Using Natural Killer Cells Activated by Plasmacytoid Dendritic Cells

    PubMed Central

    Cordeau, Martine; Belounis, Assila; Lelaidier, Martin; Cordeiro, Paulo; Sartelet, Hervé; Duval, Michel

    2016-01-01

    High-risk neuroblastoma (NB) remains a major therapeutic challenge despite the recent advent of disialoganglioside (GD2)-antibody treatment combined with interleukin (IL)-2 and granulocyte monocyte-colony stimulating factor (GM-CSF). Indeed, more than one third of the patients still die from this disease. Here, we developed a novel approach to improve the current anti-GD2 immunotherapy based on NK cell stimulation using toll-like receptor (TLR)-activated plasmacytoid dendritic cells (pDCs). We demonstrated that this strategy led to the efficient killing of NB cells. When the expression of GD2 was heterogeneous on NB cells, the combination of pDC-mediated NK-cell activation and anti-GD2 treatment significantly increased the cytotoxicity of NK cells against NB cells. Activation by pDCs led to a unique NK-cell phenotype characterized by increased surface expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), with increased expression of CD69 on CD56dim cytotoxic cells, and strong interferon-γ production. Additionally, NB-cell killing was mediated by the TRAIL death-receptor pathway, as well as by the release of cytolytic granules via the DNAX accessory molecule 1 pathway. NK-cell activation and lytic activity against NB was independent of cell contact, depended upon type I IFN produced by TLR-9-activated pDCs, but was not reproduced by IFN-α stimulation alone. Collectively, these results highlighted the therapeutic potential of activated pDCs for patients with high-risk NB. PMID:27716850

  9. Activated NKT cells imprint NK-cell differentiation, functionality and education.

    PubMed

    Riese, Peggy; Trittel, Stephanie; May, Tobias; Cicin-Sain, Luka; Chambers, Benedict J; Guzmán, Carlos A

    2015-06-01

    NK cells represent a vital component of the innate immune system. The recent discoveries demonstrating that the functionality of NK cells depends on their differentiation and education status underscore their potential as targets for immune intervention. However, to exploit their full potential, a detailed understanding of the cellular interactions involved in these processes is required. In this regard, the cross-talk between NKT cells and NK cells needs to be better understood. Our results provide strong evidence for NKT cell-induced effects on key biological features of NK cells. NKT-cell activation results in the generation of highly active CD27(high) NK cells with improved functionality. In this context, degranulation activity and IFNγ production were mainly detected in the educated subset. In a mCMV infection model, we also demonstrated that NKT-cell stimulation induced the generation of highly functional educated and uneducated NK cells, crucial players in viral control. Thus, our findings reveal new fundamental aspects of the NKT-NK cell axis that provide important hints for the manipulation of NK cells in clinical settings.

  10. A PTEN inhibitor displays preclinical activity against hepatocarcinoma cells

    PubMed Central

    Augello, Giuseppa; Puleio, Roberto; Emma, Maria Rita; Cusimano, Antonella; Loria, Guido R.; McCubrey, James A.; Montalto, Giuseppe; Cervello, Melchiorre

    2016-01-01

    ABSTRACT Phosphatase and tensin homolog (PTEN) gene is considered a tumor suppressor gene. However, PTEN mutations rarely occur in hepatocellular carcinoma (HCC), whereas heterozygosity of PTEN, resulting in reduced PTEN expression, has been observed in 32–44% of HCC patients. In the present study, we investigated the effects of the small molecule PTEN inhibitor VO-OHpic in HCC cells. VO-OHpic inhibited cell viability, cell proliferation and colony formation, and induced senescence-associated β-galactosidase activity in Hep3B (low PTEN expression) and to a lesser extent in PLC/PRF/5 (high PTEN expression) cells, but not in PTEN-negative SNU475 cells. VO-OHpic synergistically inhibited cell viability when combined with PI3K/mTOR and RAF/MEK/ERK pathway inhibitors, but only in Hep3B cells, and significantly inhibited tumor growth in nude mice bearing xenografts of Hep3B cells. Therefore, we demonstrated for the first time that VO-OHpic inhibited cell growth and induced senescence in HCC cells with low PTEN expression, and that the combination of VO-OHpic with PI3K/mTOR and RAF/MEK/ERK inhibitors resulted in a more effective tumor cell kill. Our findings, hence, provide proof-of-principle evidence that pharmacological inhibition of PTEN may represent a promising approach for HCC therapy in a subclass of patients with a low PTEN expression. PMID:26794644

  11. Inhibitory Activity of (+)-Usnic Acid against Non-Small Cell Lung Cancer Cell Motility.

    PubMed

    Yang, Yi; Nguyen, Thanh Thi; Jeong, Min-Hye; Crişan, Florin; Yu, Young Hyun; Ha, Hyung-Ho; Choi, Kyung Hee; Jeong, Hye Gwang; Jeong, Tae Cheon; Lee, Kwang Youl; Kim, Kyung Keun; Hur, Jae-Seoun; Kim, Hangun

    2016-01-01

    Lichens are symbiotic organisms that produce various unique chemicals that can be used for pharmaceutical purposes. With the aim of screening new anti-cancer agents that inhibit cancer cell motility, we tested the inhibitory activity of seven lichen species collected from the Romanian Carpathian Mountains against migration and invasion of human lung cancer cells and further investigated the molecular mechanisms underlying their anti-metastatic activity. Among them, Alectoria samentosa, Flavocetraria nivalis, Alectoria ochroleuca, and Usnea florida showed significant inhibitory activity against motility of human lung cancer cells. HPLC results showed that usnic acid is the main compound in these lichens, and (+)-usnic acid showed similar inhibitory activity that crude extract have. Mechanistically, β-catenin-mediated TOPFLASH activity and KITENIN-mediated AP-1 activity were decreased by (+)-usnic acid treatment in a dose-dependent manner. The quantitative real-time PCR data showed that (+)-usnic acid decreased the mRNA level of CD44, Cyclin D1 and c-myc, which are the downstream target genes of both β-catenin/LEF and c-jun/AP-1. Also, Rac1 and RhoA activities were decreased by treatment with (+)-usnic acid. Interestingly, higher inhibitory activity for cell invasion was observed when cells were treated with (+)-usnic acid and cetuximab. These results implied that (+)-usnic acid might have potential activity in inhibition of cancer cell metastasis, and (+)-usnic acid could be used for anti-cancer therapy with a distinct mechanisms of action.

  12. Inhibitory Activity of (+)-Usnic Acid against Non-Small Cell Lung Cancer Cell Motility

    PubMed Central

    Yang, Yi; Nguyen, Thanh Thi; Jeong, Min-Hye; Crişan, Florin; Yu, Young Hyun; Ha, Hyung-Ho; Choi, Kyung Hee; Jeong, Hye Gwang; Jeong, Tae Cheon; Lee, Kwang Youl; Kim, Kyung Keun; Hur, Jae-Seoun; Kim, Hangun

    2016-01-01

    Lichens are symbiotic organisms that produce various unique chemicals that can be used for pharmaceutical purposes. With the aim of screening new anti-cancer agents that inhibit cancer cell motility, we tested the inhibitory activity of seven lichen species collected from the Romanian Carpathian Mountains against migration and invasion of human lung cancer cells and further investigated the molecular mechanisms underlying their anti-metastatic activity. Among them, Alectoria samentosa, Flavocetraria nivalis, Alectoria ochroleuca, and Usnea florida showed significant inhibitory activity against motility of human lung cancer cells. HPLC results showed that usnic acid is the main compound in these lichens, and (+)-usnic acid showed similar inhibitory activity that crude extract have. Mechanistically, β-catenin-mediated TOPFLASH activity and KITENIN-mediated AP-1 activity were decreased by (+)-usnic acid treatment in a dose-dependent manner. The quantitative real-time PCR data showed that (+)-usnic acid decreased the mRNA level of CD44, Cyclin D1 and c-myc, which are the downstream target genes of both β-catenin/LEF and c-jun/AP-1. Also, Rac1 and RhoA activities were decreased by treatment with (+)-usnic acid. Interestingly, higher inhibitory activity for cell invasion was observed when cells were treated with (+)-usnic acid and cetuximab. These results implied that (+)-usnic acid might have potential activity in inhibition of cancer cell metastasis, and (+)-usnic acid could be used for anti-cancer therapy with a distinct mechanisms of action. PMID:26751081

  13. Raloxifene induces autophagy-dependent cell death in breast cancer cells via the activation of AMP-activated protein kinase.

    PubMed

    Kim, Dong Eun; Kim, Yunha; Cho, Dong-Hyung; Jeong, Seong-Yun; Kim, Sung-Bae; Suh, Nayoung; Lee, Jung Shin; Choi, Eun Kyung; Koh, Jae-Young; Hwang, Jung Jin; Kim, Choung-Soo

    2015-01-01

    Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells. Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death. Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.

  14. Simvastatin requires activation in accessory cells to modulate T-cell responses in asthma and COPD.

    PubMed

    Knobloch, Jürgen; Yakin, Yakup; Körber, Sandra; Grensemann, Barbara; Bendella, Zeynep; Boyaci, Niyazi; Gallert, Willem-Jakob; Yanik, Sarah Derya; Jungck, David; Koch, Andrea

    2016-10-05

    T-cell-dependent airway and systemic inflammation triggers the progression of chronic obstructive pulmonary disease (COPD) and asthma. Retrospective studies suggest that simvastatin has anti-inflammatory effects in both diseases but it is unclear, which cell types are targeted. We hypothesized that simvastatin modulates T-cell activity. Circulating CD4+ and CD8+ T-cells, either pure, co-cultured with monocytes or alveolar macrophages (AM) or in peripheral blood mononuclear cells (PBMCs), were ex vivo activated towards Th1/Tc1 or Th2/Tc2 and incubated with simvastatin. Markers for Th1/Tc1 (IFNγ) and Th2/Tc2 (IL-5, IL-13) were measured by ELISA; with PBMCs this was done comparative between 11 healthy never-smokers, 11 current smokers without airflow limitation, 14 smokers with COPD and 11 never-smokers with atopic asthma. T-cell activation induced IFNγ, IL-5 and IL-13 in the presence and absence of accessory cells. Simvastatin did not modulate cytokine expression in pure T-cell fractions. β-hydroxy-simvastatin acid (activated simvastatin) suppressed IL-5 and IL-13 in pure Th2- and Tc2-cells. Simvastatin suppressed IL-5 and IL-13 in Th2-cells co-cultivated with monocytes or AM, which was partially reversed by the carboxylesterase inhibitor benzil. Simvastatin suppressed IL-5 production of Th2/Tc2-cells in PBMCs without differences between cohorts and IL-13 stronger in never-smokers and asthma compared to COPD. Simvastatin induced IFNγ in Th1/Tc1-cells in PBMCs of all cohorts except asthmatics. Simvastatin requires activation in accessory cells likely by carboxylesterase to suppress IL-5 and IL-13 in Th2/Tc2-cells. The effects on Il-13 are partially reduced in COPD. Asthma pathogenesis prevents simvastatin-induced IFNγ up-regulation. Simvastatin has anti-inflammatory effects that could be of interest for asthma therapy.

  15. Ca2+-activated K channels in parotid acinar cells

    PubMed Central

    Romanenko, Victor G; Thompson, Jill

    2010-01-01

    Fluid secretion relies on a close interplay between Ca2+-activated Cl and K channels. Salivary acinar cells contain both large conductance, BK, and intermediate conductance, IK1, K channels. Physiological fluid secretion occurs with only modest (<500 nM) increases in intracellular Ca2+ levels but BK channels in many cell types and in heterologous expression systems require very high concentrations for significant activation. We report here our efforts to understand this apparent contradiction. We determined the Ca2+ dependence of IK1 and BK channels in mouse parotid acinar cells. IK1 channels activated with an apparent Ca2+ affinity of about 350 nM and a hill coefficient near 3. Native parotid BK channels activated at similar Ca2+ levels unlike the BK channels in other cell types. Since the parotid BK channel is encoded by an uncommon splice variant, we examined this clone in a heterologous expression system. In contrast to the native parotid channel, activation of this expressed “parslo” channel required very high levels of Ca2+. In order to understand the functional basis for the special properties of the native channels, we analyzed the parotid BK channel in the context of the horrigan-Aldrich model of BK channel gating. We found that the shifted activation of parotid BK channels resulted from a hyperpolarizing shift of the voltage dependence of voltage sensor activation and channel opening and included a large change in the coupling of these two processes. PMID:20519930

  16. Behavioral inspiratory inhibition: inactivated and activated respiratory cells.

    PubMed

    Orem, J

    1989-11-01

    1. Eleven adult cats were trained to stop inspiration in response to a conditioning stimulus. The conditioning stimuli were presented at the onset of inspiration at intervals of approximately 20-30 s. Intratracheal pressures, diaphragmatic activity, and the extracellular activity of single medullary respiratory neurons were recorded while the animals performed this response. 2. Inactivation of the diaphragm to the conditioning stimuli occurred at latencies that varied from 40 to 110 ms and averaged 74 +/- 32 (SD) ms. 3. The subjects of this report are 38 inspiratory neurons that were inactivated and 19 cells that were activated when inspiration was stopped behaviorally. These cells were located in the region of n. ambiguus and the ventrolateral n. of tractus solitarius. 4. The inspiratory cells that were inactivated behaviorally had the following characteristics: 1) Most had an augmenting inspiratory profile with (n = 14) or without (n = 9) postinspiratory activity. Other types were inspiratory throughout (n = 5), decrementing inspiratory (n = 3), tonic inspiratory (n = 4), early inspiratory (n = 2), and expiratory-inspiratory (n = 1). 2) Their mean discharge rate was 39 +/- 2.7 (SE) Hz. 3) The latency of their inactivation in response to the task averaged 81 +/- 4.9 (SE) ms, and 4) Their activity corresponded closely to breathing not only during the behavioral response but also during eupnea (eta 2 = 0.62 +/- 0.04, mean +/- SE) and respiratory acts such as sneezing, sniffing, meowing, and purring. 5. The cells that were activated when inspiration was stopped behaviorally had the following characteristics. 1) As a group, they had discharge profiles related to every phase of the respiratory cycle. 2) They were recorded in the same region as, and often simultaneously with, respiratory cells that were inactivated. 3) Their activity patterns were highly variable such that the signal strength and consistency of the respiratory component of that activity were weak (eta 2

  17. Acute psychological stress increases peripheral blood CD3+CD56+ natural killer T cells in healthy men: possible implications for the development and treatment of allergic and autoimmune disorders.

    PubMed

    Atanackovic, Djordje; Nowottne, Ulrike; Freier, Eva; Weber, Cora Stefanie; Meyer, Sabrina; Bartels, Katrin; Hildebrandt, York; Cao, Yanran; Kröger, Nicolaus; Brunner-Weinzierl, Monika Christine; Bokemeyer, Carsten; Deter, Hans-Christian

    2013-07-01

    Acute psychological stress has primarily been investigated regarding its effects on conventional lymphocytes such as natural killer (NK) cells and CD4(+) and CD8(+) T cells. However, it might be important to focus on more "specialized" lymphocyte subsets, playing a role, for instance, in allergic conditions and autoimmunity, to identify links between stress, the immune system and somatic diseases. Using flow cytometry we determined frequencies of circulating T helper (Th)1-type (CD226(+)) and Th2-type (CRTH2(+)) T cells, γδ T cells, conventional CD56(+) natural killer T (NKT) cells and invariant NKT cells (iNKT) in healthy young males (N = 31; median age 26 years) undergoing a laboratory computer-based stressor lasting 12 min. We found that acute psychological stress induced a prolonged increase in CD4(+) and CD8(+) T cells expressing a Th2 phenotype. We also detected an acute increase in CD4(-) and CD8(-) double negative γδ T cells. Finally, we found that the well-known increase in NK cells under stressful conditions was paralleled by a significant increase in numbers of conventional CD56(+) NKT cells. In contrast, numbers of iNKT was not altered by stress. This study adds further evidence to a psychoneuroimmunological model proposing that under stressful conditions certain lymphocyte subsets, including iNKT and less mature T cells, are retained in lymphoid tissues while antigen-experienced effector-type T cells with a Th2 phenotype, γδ T cells and conventional CD56(+) NKT cells are mobilized into the peripheral blood. We suggest that in the case of frequent stress exposure, this might result in the promotion of, for example, allergic conditions.

  18. Biofuel cell operating on activated THP-1 cells: A fuel and substrate study.

    PubMed

    Javor, Kristina; Tisserant, Jean-Nicolas; Stemmer, Andreas

    2017-01-15

    It is known that electrochemical energy can be harvested from mammalian cells, more specifically from white blood cells (WBC). This study focuses on an improved biofuel cell operating on phorbol myristate acetate (PMA) activated THP-1 human monocytic cells. Electrochemical investigation showed strong evidence pointing towards hydrogen peroxide being the primary current source, confirming that the current originates from NADPH oxidase activity. Moreover, an adequate substrate for differentiation and activation of THP-1 cells was examined. ITO, gold, platinum and glass were tested and the amount of superoxide anion produced by NADPH oxidase was measured by spectrophotometry through WST-1 reduction at 450nm and used as an indicator of cellular activity and viability. These substrates were subsequently used in a conventional two-compartment biofuel cell where the power density output was recorded. The material showing the highest cell activity compared to the reference cell culture plate and the highest power output was ITO. Under our experimental conditions, a power density of 4.5μW/cm(2) was reached. To the best of our knowledge, this is a threefold higher power output than other leukocyte biofuel cells.

  19. Radiosensitivity of human natural killer cells: Binding and cytotoxic activities of natural killer cell subsets

    SciTech Connect

    Rana, R.; Vitale, M.; Mazzotti, G.; Manzoli, L.; Papa, S. )

    1990-10-01

    The sensitivity of human natural killer (NK) cell activities (both binding and killing) after exposure of peripheral blood mononuclear cells to different doses of gamma radiation was studied. A panel of monoclonal antibodies was used to identify the NK and T-lymphocyte subsets and to evaluate their radiosensitivity. Peripheral blood mononuclear cells were irradiated with low (2-6 Gy) and high (10-30 Gy) doses and NK cell binding and cytotoxic activity against K562 target cells were studied after 3 h and 48 h in culture. The primary damage to NK cell activity was identified at the postbinding level and affected mainly the lytic machinery. After 48 h culture postirradiation, an overall depression of cytotoxic activity was observed, but ionizing radiation produced either a selection of the more cytotoxic NK cell subsets, which therefore might be considered more resistant to radiation damage than the less cytotoxic NK cells, or a long-term stimulation of cytotoxic activity in surviving cells.

  20. Controlling T-Cell Activation with Synthetic Dendritic Cells Using the Multivalency Effect

    PubMed Central

    2017-01-01

    Artificial antigen-presenting cells (aAPCs) have recently gained a lot of attention. They efficiently activate T cells and serve as powerful replacements for dendritic cells in cancer immunotherapy. Focusing on a specific class of polymer-based aAPCs, so-called synthetic dendritic cells (sDCs), we have investigated the importance of multivalent binding on T-cell activation. Using antibody-functionalized sDCs, we have tested the influence of polymer length and antibody density. Increasing the multivalent character of the antibody-functionalized polymer lowered the effective concentration required for T-cell activation. This was evidenced for both early and late stages of activation. The most important effect observed was the significantly prolonged activation of the stimulated T cells, indicating that multivalent sDCs sustain T-cell signaling. Our results highlight the importance of multivalency for the design of aAPCs and will ultimately allow for better mimics of natural dendritic cells that can be used as vaccines in cancer treatment. PMID:28393131

  1. Distinct FAK activities determine progenitor and mammary stem cell characteristics

    PubMed Central

    Luo, Ming; Zhao, Xiaofeng; Chen, Song; Liu, Suling; Wicha, Max S.; Guan, Jun-Lin

    2013-01-01

    Mammary stem (MaSCs) and progenitor cells are important for mammary gland development and maintenance and may give rise to mammary cancer stem cells (MaCSCs). Yet there remains limited understanding of how these cells contribute to tumorigenesis. Here we show that conditional deletion of focal adhesion kinase (FAK) in embryonic mammary epithelial cells (MaECs) decreases luminal progenitors (LPs) and basal MaSCs, reducing their colony-forming and regenerative potentials in a cell autonomous manner. Loss of FAK kinase activity in MaECs specifically impaired LP proliferation and alveologenesis, whereas a kinase-independent activity of FAK supported ductal invasion and basal MaSC activity. Deficiency in LPs suppressed tumorigenesis and MaCSC formation in a mouse model of breast cancer. In contrast to the general inhibitory effect of FAK attenuation, inhibitors of FAK kinase preferentially inhibited proliferation and tumorsphere formation of LP-like, but not MaSC-like, human breast cancer cells. Our findings establish distinct kinase dependent and independent activities of FAK that differentially regulate LPs and basal MaSCs. We suggest that targeting these distinct functions may tailor therapeutic strategies to address breast cancer heterogeneity more effectively. PMID:23832665

  2. Shed syndecan-2 enhances tumorigenic activities of colon cancer cells

    PubMed Central

    Choi, Sojoong; Choi, Youngsil; Jun, Eunsung; Kim, In-San; Kim, Seong-Eun; Jung, Sung-Ae; Oh, Eok-Soo

    2015-01-01

    Because earlier studies showed the cell surface heparan sulfate proteoglycan, syndecan-2, sheds from colon cancer cells in culture, the functional roles of shed syndecan-2 were assessed. A non-cleavable mutant of syndecan-2 in which the Asn148-Leu149 residues were replaced with Asn148-Ile149, had decreased shedding, less cancer-associated activities of syndecan-2 in vitro, and less syndecan-2-mediated metastasis of mouse melanoma cells in vivo, suggesting the importance of shedding on syndecan-2-mediated pro-tumorigenic functions. Indeed, shed syndecan-2 from cancer-conditioned media and recombinant shed syndecan-2 enhanced cancer-associated activities, and depletion of shed syndecan-2 abolished these effects. Similarly, shed syndecan-2 was detected from sera of patients from advanced carcinoma (625.9 ng/ml) and promoted cancer-associated activities. Furthermore, a series of syndecan-2 deletion mutants showed that the tumorigenic activity of shed syndecan-2 resided in the C-terminus of the extracellular domain and a shed syndecan-2 synthetic peptide (16 residues) was sufficient to establish subcutaneous primary growth of HT29 colon cancer cells, pulmonary metastases (B16F10 cells), and primary intrasplenic tumor growth and liver metastases (4T1 cells). Taken together, these results demonstrate that shed syndecan-2 directly enhances colon cancer progression and may be a promising therapeutic target for controlling colon cancer development. PMID:25686828

  3. Dopamine Modulates the Activity of Sensory Hair Cells

    PubMed Central

    Toro, Cecilia; Trapani, Josef G.; Pacentine, Itallia; Maeda, Reo; Sheets, Lavinia; Mo, Weike

    2015-01-01

    The senses of hearing and balance are subject to modulation by efferent signaling, including the release of dopamine (DA). How DA influences the activity of the auditory and vestibular systems and its site of action are not well understood. Here we show that dopaminergic efferent fibers innervate the acousticolateralis epithelium of the zebrafish during development but do not directly form synapses with hair cells. However, a member of the D1-like receptor family, D1b, tightly localizes to ribbon synapses in inner ear and lateral-line hair cells. To assess modulation of hair-cell activity, we reversibly activated or inhibited D1-like receptors (D1Rs) in lateral-line hair cells. In extracellular recordings from hair cells, we observed that D1R agonist SKF-38393 increased microphonic potentials, whereas D1R antagonist SCH-23390 decreased microphonic potentials. Using ratiometric calcium imaging, we found that increased D1R activity resulted in larger calcium transients in hair cells. The increase of intracellular calcium requires Cav1.3a channels, as a Cav1 calcium channel antagonist, isradipine, blocked the increase in calcium transients elicited by the agonist SKF-38393. Collectively, our results suggest that DA is released in a paracrine fashion and acts at ribbon synapses, likely enhancing the activity of presynaptic Cav1.3a channels and thereby increasing neurotransmission. SIGNIFICANCE STATEMENT The neurotransmitter dopamine acts in a paracrine fashion (diffusion over a short distance) in several tissues and bodily organs, influencing and regulating their activity. The cellular target and mechanism of the action of dopamine in mechanosensory organs, such as the inner ear and lateral-line organ, is not clearly understood. Here we demonstrate that dopamine receptors are present in sensory hair cells at synaptic sites that are required for signaling to the brain. When nearby neurons release dopamine, activation of the dopamine receptors increases the activity of

  4. Brucella and Osteoarticular Cell Activation: Partners in Crime.

    PubMed

    Giambartolomei, Guillermo H; Arriola Benitez, Paula C; Delpino, M Victoria

    2017-01-01

    Osteoarticular brucellosis is the most common presentation of human active disease although its prevalence varies widely. The three most common forms of osteoarticular involvement are sacroiliitis, spondylitis, and peripheral arthritis. The molecular mechanisms implicated in bone damage have been recently elucidated. B. abortus induces bone damage through diverse mechanisms in which TNF-α and the receptor activator of nuclear factor kappa-B ligand (RANKL)-the natural modulator of bone homeostasis are involved. These processes are driven by inflammatory cells, like monocytes/macrophages, neutrophils, Th17 CD4(+) T, and B cells. In addition, Brucella abortus has a direct effect on osteoarticular cells and tilts homeostatic bone remodeling. These bacteria inhibit bone matrix deposition by osteoblasts (the only bone cells involved in bone deposition), and modify the phenotype of these cells to produce matrix metalloproteinases (MMPs) and cytokine secretion, contributing to bone matrix degradation. B. abortus also affects osteoclasts (cells naturally involved in bone resorption) by inducing an increase in osteoclastogenesis and osteoclast activation; thus, increasing mineral and organic bone matrix resorption, contributing to bone damage. Given that the pathology induced by Brucella species involved joint tissue, experiments conducted on synoviocytes revealed that besides inducing the activation of these cells to secrete chemokines, proinflammatory cytokines and MMPS, the infection also inhibits synoviocyte apoptosis. Brucella is an intracellular bacterium that replicates preferentially in the endoplasmic reticulum of macrophages. The analysis of B. abortus-infected synoviocytes indicated that bacteria also replicate in their reticulum suggesting that they could use this cell type for intracellular replication during the osteoarticular localization of the disease. Finally, the molecular mechanisms of osteoarticular brucellosis discovered recently shed light on how the

  5. Brucella and Osteoarticular Cell Activation: Partners in Crime

    PubMed Central

    Giambartolomei, Guillermo H.; Arriola Benitez, Paula C.; Delpino, M. Victoria

    2017-01-01

    Osteoarticular brucellosis is the most common presentation of human active disease although its prevalence varies widely. The three most common forms of osteoarticular involvement are sacroiliitis, spondylitis, and peripheral arthritis. The molecular mechanisms implicated in bone damage have been recently elucidated. B. abortus induces bone damage through diverse mechanisms in which TNF-α and the receptor activator of nuclear factor kappa-B ligand (RANKL)-the natural modulator of bone homeostasis are involved. These processes are driven by inflammatory cells, like monocytes/macrophages, neutrophils, Th17 CD4+ T, and B cells. In addition, Brucella abortus has a direct effect on osteoarticular cells and tilts homeostatic bone remodeling. These bacteria inhibit bone matrix deposition by osteoblasts (the only bone cells involved in bone deposition), and modify the phenotype of these cells to produce matrix metalloproteinases (MMPs) and cytokine secretion, contributing to bone matrix degradation. B. abortus also affects osteoclasts (cells naturally involved in bone resorption) by inducing an increase in osteoclastogenesis and osteoclast activation; thus, increasing mineral and organic bone matrix resorption, contributing to bone damage. Given that the pathology induced by Brucella species involved joint tissue, experiments conducted on synoviocytes revealed that besides inducing the activation of these cells to secrete chemokines, proinflammatory cytokines and MMPS, the infection also inhibits synoviocyte apoptosis. Brucella is an intracellular bacterium that replicates preferentially in the endoplasmic reticulum of macrophages. The analysis of B. abortus-infected synoviocytes indicated that bacteria also replicate in their reticulum suggesting that they could use this cell type for intracellular replication during the osteoarticular localization of the disease. Finally, the molecular mechanisms of osteoarticular brucellosis discovered recently shed light on how the

  6. Anticancer activities of epigallocatechin-3-gallate against cholangiocarcinoma cells

    PubMed Central

    Kwak, Tae Won; Park, Su Bum; Kim, Hyun-Jung; Jeong, Young-IL; Kang, Dae Hwan

    2017-01-01

    Purpose Epigallocatechin-3-gallate (EGCG) is an antioxidant agent derived from green tea. Because it has chemopreventive and anti-invasive effect against various cancer cells, EGCG can be used to inhibit proliferation and invasion of cholangiocarcinoma (CCA) cells. Methods The anticancer effects of EGCG were studied using human CCA cells (HuCC-T1). Apoptosis was analyzed by Western blotting. Invasion and migration of cancer cells were assessed with Matrigel® and wound healing assays. An animal tumor xenograft model of HuCC-T1 was used to study the in vivo antitumor activities of EGCG. Results EGCG effectively inhibited the growth of HuCC-T1 cells with no adverse effects on the viability of 293T cells. EGCG induced apoptotic cell death at 5 µg/mL concentration. It inhibited the expression of mutant p53 and induced apoptotic molecular signals such as Bax/Bcl-2, Caspase, and cytochrome C. Furthermore, EGCG dose-dependently inhibited the activity of matrix metalloproteinase (MMP)-2/9, invasion, and migration. In the animal tumor xenograft model of HuCC-T1 cells, EGCG was subcutaneously administered beside the tumor for local treatment. EGCG efficiently inhibited growth of the tumor and suppressed carcinogenic molecular signals such as Notch1, MMP-2/9, and proliferating cell nuclear antigen. Conclusion EGCG induced apoptosis of cancer cells without adverse effects on normal cells. EGCG inhibited growth, invasion, and migration of HuCC-T1 cells. We suggest EGCG as a promising candidate for local treatment of CCA. PMID:28053547

  7. Massively augmented hippocampal dentate granule cell activation accompanies epilepsy development.

    PubMed

    Dengler, Christopher G; Yue, Cuiyong; Takano, Hajime; Coulter, Douglas A

    2017-02-20

    In a mouse model of temporal lobe epilepsy, multicellular calcium imaging revealed that disease emergence was accompanied by massive amplification in the normally sparse, afferent stimulation-induced activation of hippocampal dentate granule cells. Patch recordings demonstrated reductions in local inhibitory function within the dentate gyrus at time points where sparse activation was compromised. Mimicking changes in inhibitory synaptic function and transmembrane chloride regulation was sufficient to elicit the dentate gyrus circuit collapse evident during epilepsy development. Pharmacological blockade of outward chloride transport had no effect during epilepsy development, and significantly increased granule cell activation in both control and chronically epileptic animals. This apparent occlusion effect implicates reduction in chloride extrusion as a mechanism contributing to granule cell hyperactivation specifically during early epilepsy development. Glutamine plays a significant role in local synthesis of GABA in synapses. In epileptic mice, sparse granule cell activation could be restored by glutamine application, implicating compromised GABA synthesis. Glutamine had no effect on granule cell activation earlier, during epilepsy development. We conclude that compromised feedforward inhibition within the local circuit generates the massive dentate gyrus circuit hyperactivation evident in animals during and following epilepsy development. However, the mechanisms underlying this disinhibition diverge significantly as epilepsy progresses.

  8. Massively augmented hippocampal dentate granule cell activation accompanies epilepsy development

    PubMed Central

    Dengler, Christopher G.; Yue, Cuiyong; Takano, Hajime; Coulter, Douglas A.

    2017-01-01

    In a mouse model of temporal lobe epilepsy, multicellular calcium imaging revealed that disease emergence was accompanied by massive amplification in the normally sparse, afferent stimulation-induced activation of hippocampal dentate granule cells. Patch recordings demonstrated reductions in local inhibitory function within the dentate gyrus at time points where sparse activation was compromised. Mimicking changes in inhibitory synaptic function and transmembrane chloride regulation was sufficient to elicit the dentate gyrus circuit collapse evident during epilepsy development. Pharmacological blockade of outward chloride transport had no effect during epilepsy development, and significantly increased granule cell activation in both control and chronically epileptic animals. This apparent occlusion effect implicates reduction in chloride extrusion as a mechanism contributing to granule cell hyperactivation specifically during early epilepsy development. Glutamine plays a significant role in local synthesis of GABA in synapses. In epileptic mice, sparse granule cell activation could be restored by glutamine application, implicating compromised GABA synthesis. Glutamine had no effect on granule cell activation earlier, during epilepsy development. We conclude that compromised feedforward inhibition within the local circuit generates the massive dentate gyrus circuit hyperactivation evident in animals during and following epilepsy development. However, the mechanisms underlying this disinhibition diverge significantly as epilepsy progresses. PMID:28218241

  9. Inner ear cell therapy targeting hereditary deafness by activation of stem cell homing factors.

    PubMed

    Kamiya, Kazusaku

    2015-01-01

    Congenital deafness affects about 1 in 1000 children and more than half of them have a genetic background such as Connexin26 (CX26) gene mutation. Inner ear cell therapy for sensorineural hearing loss has been expected to be an effective therapy for hereditary deafness. Previously, we developed a novel strategy for inner ear cell therapy using bone marrow mesenchymal stem cells as a supplement for cochlear fibrocytes functioning for cochlear ion transport. For cell therapy targeting hereditary deafness, a more effective cell delivery system to induce the stem cells into cochlear tissue is required, because gene mutations affect all cochlear cells cochlear cells expressing genes such as GJB2 encoding CX26. Stem cell homing is one of the crucial mechanisms to be activated for efficient cell delivery to the cochlear tissue. In our study, monocyte chemotactic protein-1, stromal cell-derived factor-1 and their receptors were found to be a key regulator for stem cell recruitment to the cochlear tissue. Thus, the activation of stem cell homing may be an efficient strategy for hearing recovery in hereditary deafness.

  10. Cancer cell-binding peptide fused Fc domain activates immune effector cells and blocks tumor growth

    PubMed Central

    Mobergslien, Anne; Peng, Qian; Vasovic, Vlada; Sioud, Mouldy

    2016-01-01

    Therapeutic strategies aiming at mobilizing immune effector cells to kill tumor cells independent of tumor mutational load and MHC expression status are expected to benefit cancer patients. Recently, we engineered various peptide-Fc fusion proteins for directing Fcg receptor-bearing immune cells toward tumor cells. Here, we investigated the immunostimulatory and anti-tumor effects of one of the engineered Fc fusion proteins (WN-Fc). In contrast to the Fc control, soluble WN-Fc-1 fusion protein activated innate immune cells (e.g. monocytes, macrophages, dendritic cells, NK cells), resulting in cytokine production and surface display of the lytic granule marker CD107a on NK cells. An engineered Fc-fusion variant carrying two peptide sequences (WN-Fc-2) also activated immune cells and bound to various cancer cell types with high affinity, including the murine 4T1 breast carcinoma cells. When injected into 4T1 tumor-bearing BALB/c mice, both peptide-Fc fusions accumulated in tumor tissues as compared to other organs such as the lungs. Moreover, treatment of 4T1 tumor-bearing BALB/c mice by means of two intravenous injections of the WN-Fc fusion proteins inhibited tumor growth with WN-Fc-2 being more effective than WN-Fc-1. Treatment resulted in tumor infiltration by T cells and NK cells. These new engineered WN-Fc fusion proteins may be a promising alternative to existing immunotherapies for cancer. PMID:27713158

  11. Identification of a potent microbial lipid antigen for diverse Natural Killer T cells1

    PubMed Central

    Wolf, Benjamin J.; Tatituri, Raju V. V.; Almeida, Catarina F.; Le Nours, Jérôme; Bhowruth, Veemal; Johnson, Darryl; Uldrich, Adam P.; Hsu, Fong-Fu; Brigl, Manfred; Besra, Gurdyal S.; Rossjohn, Jamie; Godfrey, Dale I.; Brenner, Michael B.

    2016-01-01

    Invariant Natural Killer T (iNKT) cells are a well-characterized CD1d-restricted T cell subset. The availability of potent antigens and tetramers for iNKT cells has allowed this population to be extensively studied and has revealed their central roles in infection, autoimmunity, and tumor immunity. In contrast, diverse Natural Killer T (dNKT) cells are poorly understood because the lipid antigens they recognize are largely unknown. We sought to identify dNKT cell lipid antigen(s) by interrogating a panel of dNKT mouse cell hybridomas with lipid extracts from the pathogen Listeria monocytogenes. We identified Listeria phosphatidylglycerol (PG) as a microbial antigen that was significantly more potent than a previously characterized dNKT cell antigen, mammalian PG. Further, while mammalian PG loaded CD1d tetramers did not stain dNKT cells, the Listeria-derived PG loaded tetramers did. The structure of Listeria PG was distinct from mammalian PG since it contained shorter, fully-saturated anteiso fatty acid lipid tails. CD1d binding lipid displacement studies revealed that the microbial PG antigen binds significantly better to CD1d than counterparts with the same headgroup. These data reveal a highly-potent microbial lipid antigen for a subset of dNKT cells and provide an explanation for its increased antigen potency compared to the mammalian counterpart. PMID:26254340

  12. Unbalanced acetylcholinesterase activity in larynx squamous cell carcinoma.

    PubMed

    Castillo-González, Ana Cristina; Pelegrín-Hernández, Juan Pablo; Nieto-Cerón, Susana; Madrona, Antonio Piñero; Noguera, José Antonio; López-Moreno, María Fuensanta; Rodríguez-López, José Neptuno; Vidal, Cecilio J; Hellín-Meseguer, Diego; Cabezas-Herrera, Juan

    2015-11-01

    Previous reports have demonstrated that a non-neuronal cholinergic system is expressed aberrantly in airways. A proliferative effect is exerted directly by cholinergic agonists through the activation of nicotinic and muscarinic receptors. In cancer, particularly those related with smoking, the mechanism through which tumour cells respond to aberrantly activated cholinergic signalling is a key question. Fifty paired pieces of larynx squamous cell carcinoma and adjacent non-cancerous tissue were compared in terms of their acetylcholinesterase activity (AChE). The AChE activity in non-cancerous tissues (0.248 ± 0.030 milliunits per milligram of wet tissue; mU/mg) demonstrates that upper respiratory tissues express sufficient AChE activity for controlling the level of acetylcholine (ACh). In larynx carcinomas, the AChE activity decreased to 0.157 ± 0.024 mU/mg (p=0.009). Larynx cancer patients exhibiting low ACh-degrading enzymatic activity had a significantly shorter overall survival (p=0.031). Differences in the mRNA levels of alternatively spliced AChE isoforms and molecular compositions were noted between glottic and supraglottic cancers. Our results suggest that the low AChE activity observed in larynx squamous cell carcinoma may be useful for predicting the outcome of patients.

  13. Hili inhibits HIV replication in activated T cells.

    PubMed

    Peterlin, B Matija; Liu, Pingyang; Wang, Xiaoyun; Cary, Daniele; Shao, Wei; Leoz, Marie; Hong, Tian; Pan, Tao; Fujinaga, Koh

    2017-03-22

    Piwil proteins restrict the replication of mobile genetic elements in the germline. They are also expressed in many transformed cell lines. In this report, we discovered that the human piwil 2 (hili) can also inhibit HIV replication, especially in activated CD4+ T cells that are the preferred target cells for this virus in the infected host. Although resting cells did not express hili, it was rapidly induced following T cell activation. In these cells and transformed cell lines, depletion of hili increased levels of viral proteins and new viral particles. Further studies revealed that hili binds to tRNA. Some of them represent rare tRNA species, whose codons are over-represented in the viral genome. Targeting tRNA(Arg)(UCU) with an antisense oligonucleotide replicated effects of hili and also inhibited HIV replication. Finally, hili also inhibited the retrotransposition of the endogenous intracysternal A particle (IAP) by a similar mechanism. Thus, hili joins a list of host proteins that inhibit the replication of HIV and other mobile genetic elements.IMPORTANCE Piwil proteins inhibit the movement of mobile genetic elements in the germline. In their absence, sperm does not form and male mice are sterile. This inhibition is thought to occur via small piRNAs. However, in some species and in human somatic cells, piwil proteins bind primarily to tRNA. In this report, we demonstrate that human piwil proteins, especially hili, not only bind to select tRNA species that include rare tRNAs, but also inhibit HIV replication. Importantly, T cell activation induces the expression of hili in CD4+ T cells. Since hili also inhibited the movement of an endogenous retrovirus (IAP), our finding shed new light on this intracellular resistance to exogenous and endogenous retroviruses as well as other mobile genetic elements.

  14. Vinculin contributes to Cell Invasion by Regulating Contractile Activation

    NASA Astrophysics Data System (ADS)

    Mierke, Claudia Tanja

    2008-07-01

    Vinculin is a component of the focal adhesion complex and is described as a mechano-coupling protein connecting the integrin receptor and the actin cytoskeleton. Vinculin knock-out (k.o.) cells (vin-/-) displayed increased migration on a 2-D collagen- or fibronectin-coated substrate compared to wildtype cells, but the role of vinculin in cell migration through a 3-D connective tissue is unknown. We determined the invasiveness of established tumor cell lines using a 3-D collagen invasion assay. Gene expression analysis of 4 invasive and 4 non-invasive tumor cell lines revealed that vinculin expression was significantly increased in invasive tumor cell lines. To analyze the mechanisms by which vinculin increased cell invasion in a 3-D gel, we studied mouse embryonic fibroblasts wildtype and vin-/- cells. Wildtype cells were 3-fold more invasive compared vin-/- cells. We hypothesized that the ability to generate sufficient traction forces is a prerequisite for tumor cell migration in a 3-D connective tissue matrix. Using traction microscopy, we found that wildtype exerted 3-fold higher tractions on fibronectin-coated polyacrylamide gels compared to vin-/- cells. These results show that vinculin controls two fundamental functions that lead to opposite effects on cell migration in a 2-D vs. a 3-D environment: On the one hand, vinculin stabilizes the focal adhesions (mechano-coupling function) and thereby reduces motility in 2-D. On the other hand, vinculin is also a potent activator of traction generation (mechano-regulating function) that is important for cell invasion in a 3-D environment.

  15. Antitumor activity of dobutamine on human osteosarcoma cells

    PubMed Central

    YIN, JUN; DONG, QIRONG; ZHENG, MINQIAN; XU, XIAOZU; ZOU, GUOYOU; MA, GUOLIN; LI, KEFENG

    2016-01-01

    Dobutamine has been widely used for the treatment of heart failure and cardiogenic shock since the 1970s. Osteosarcoma is the most commonly observed malignant bone tumor in children. Currently, there are no effective drugs for the treatment of osteosarcoma. In the present study, the potential anticancer activity of dobutamine on human osteosarcoma cells was examined. Human osteosarcoma MG-63 cells were treated with dobutamine at various concentrations and for various incubation times. The inhibition of cell growth by dobutamine was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometry was utilized to evaluate the effect of dobutamine on cell apoptosis and the cell cycle. Furthermore, the expression levels of caspase-3 and caspase-9 were assessed by western blot analysis. The influence of dobutamine on cancer cell migration and invasion was additionally evaluated using wound-healing assay and the Boyden Chamber migration method. Dobutamine significantly inhibited the growth of MG-63 cells at a concentration of 10 µM or higher when incubated for 12 h or longer (P=0.023). Dobutamine augmented cell apoptosis and arrested the cell cycle in the G2/M phase. Western blot analysis revealed that dobutamine induces expression of caspase-3 and caspase-9. In addition, the invasiveness and migration of MG-63 cells was inhibited by dobutamine in a concentration-dependent manner. The results of the present study may lead to novel applications for dobutamine in the treatment of osteosarcoma. PMID:27284371

  16. Pevonedistat, a NEDD8-activating enzyme inhibitor, is active in mantle cell lymphoma and enhances rituximab activity in vivo

    PubMed Central

    Czuczman, Natalie M.; Barth, Matthew J.; Gu, Juan; Neppalli, Vishala; Mavis, Cory; Frys, Sarah E.; Hu, Qiang; Liu, Song; Klener, Pavel; Vockova, Petra; Czuczman, Myron S.

    2016-01-01

    Mantle cell lymphoma (MCL) is characterized by an aggressive clinical course and inevitable development of refractory disease, stressing the need to develop alternative therapeutic strategies. To this end, we evaluated pevonedistat (MLN4924), a novel potent and selective NEDD8-activating enzyme inhibitor in a panel of MCL cell lines, primary MCL tumor cells, and 2 distinct murine models of human MCL. Pevonedistat exposure resulted in a dose-, time-, and caspase-dependent cell death in the majority of the MCL cell lines and primary tumor cells tested. Of interest, in the MCL cell lines with lower half-maximal inhibitory concentration (0.1-0.5 μM), pevonedistat induced G1-phase cell cycle arrest, downregulation of Bcl-xL levels, decreased nuclear factor (NF)-κB activity, and apoptosis. In addition, pevonedistat exhibited additive/synergistic effects when combined with cytarabine, bendamustine, or rituximab. In vivo, as a single agent, pevonedistat prolonged the survival of 2 MCL-bearing mouse models when compared with controls. Pevonedistat in combination with rituximab led to improved survival compared with rituximab or pevonedistat monotherapy. Our data suggest that pevonedistat has significant activity in MCL preclinical models, possibly related to effects on NF-κB activity, Bcl-xL downregulation, and G1 cell cycle arrest. Our findings support further investigation of pevonedistat with or without rituximab in the treatment of MCL. PMID:26675347

  17. Ethanol Metabolism Activates Cell Cycle Checkpoint Kinase, Chk2

    PubMed Central

    Clemens, Dahn L.; Mahan Schneider, Katrina J.; Nuss, Robert F.

    2011-01-01

    Chronic ethanol abuse results in hepatocyte injury and impairs hepatocyte replication. We have previously shown that ethanol metabolism results in cell cycle arrest at the G2/M transition, which is partially mediated by inhibitory phosphorylation of the cyclin-dependent kinase, Cdc2. To further delineate the mechanisms by which ethanol metabolism mediates this G2/M arrest, we investigated the involvement of upstream regulators of Cdc2 activity. Cdc2 is activated by the phosphatase Cdc25C. The activity of Cdc25C can, in turn, be regulated by the checkpoint kinase, Chk2, which is regulated by the kinase ataxia telangiectasia mutated (ATM). To investigate the involvement of these regulators of Cdc2 activity, VA-13 cells, which are Hep G2 cells modified to efficiently express alcohol dehydrogenase, were cultured in the presence or absence of 25 mM ethanol. Immunoblots were performed to determine the effects of ethanol metabolism on the activation of Cdc25C, Chk2, and ATM. Ethanol metabolism increased the active forms of ATM, and Chk2, as well as the phosphorylated form of Cdc25C. Additionally, inhibition of ATM resulted in approximately 50% of the cells being rescued from the G2/M cell cycle arrest, and ameliorated the inhibitory phosphorylation of Cdc2. Our findings demonstrate that ethanol metabolism activates ATM. ATM can activate the checkpoint kinase Chk2, resulting in phosphorylation of Cdc25C, and ultimately in the accumulation of inactive Cdc2. This may, in part, explain the ethanol metabolism-mediated impairment in hepatocyte replication, which may be important in the initiation and progression of alcoholic liver injury. PMID:21924579

  18. Ionizing Radiation Activates AMP-Activated Kinase (AMPK): A Target for Radiosensitization of Human Cancer Cells

    SciTech Connect

    Sanli, Toran; Rashid, Ayesha; Liu Caiqiong

    2010-09-01

    Purpose: Adenosine monophosphate (AMP)-activated kinase (AMPK) is a molecular energy sensor regulated by the tumor suppressor LKB1. Starvation and growth factors activate AMPK through the DNA damage sensor ataxia-telangiectasia mutated (ATM). We explored the regulation of AMPK by ionizing radiation (IR) and its role as a target for radiosensitization of human cancer cells. Methods and Materials: Lung, prostate, and breast cancer cells were treated with IR (2-8 Gy) after incubation with either ATM or AMPK inhibitors or the AMPK activator metformin. Then, cells were subjected to either lysis and immunoblotting, immunofluorescence microscopy, clonogenic survival assays, or cell cycle analysis. Results: IR induced a robust phosphorylation and activation of AMPK in all tumor cells, independent of LKB1. IR activated AMPK first in the nucleus, and this extended later into cytoplasm. The ATM inhibitor KU-55933 blocked IR activation of AMPK. AMPK inhibition with Compound C or anti-AMPK {alpha} subunit small interfering RNA (siRNA) blocked IR induction of the cell cycle regulators p53 and p21{sup waf/cip} as well as the IR-induced G2/M arrest. Compound C caused resistance to IR, increasing the surviving fraction after 2 Gy, but the anti-diabetic drug metformin enhanced IR activation of AMPK and lowered the surviving fraction after 2 Gy further. Conclusions: We provide evidence that IR activates AMPK in human cancer cells in an LKB1-independent manner, leading to induction of p21{sup waf/cip} and regulation of the cell cycle and survival. AMPK appears to (1) participate in an ATM-AMPK-p21{sup waf/cip} pathway, (2) be involved in regulation of the IR-induced G2/M checkpoint, and (3) may be targeted by metformin to enhance IR responses.

  19. Active and passive calcium transport systems in plant cells

    SciTech Connect

    Sze, H.

    1990-01-01

    The ability to change cytoplasmic Ca{sup 2+} levels ((Ca{sup 2+})) by cells has made this cation a key regulator of many biological processes. Cytoplasmic (Ca{sup 2+}) is determined by the coordination of passive Ca{sup 2+} fluxes which increase cytosolic (Ca{sup 2+}) and active Ca{sup 2+} transport systems that lower cytosolic (Ca{sup 2+}). The mechanisms by which plant cells achieve this is poorly understood. We have initially used isolated vesicles from the plasma membrane or organellar membranes to study Ca{sup 2+} transport systems in oat roots (a monocot) and carrot suspension cells (a dicot). The objectives of the proposal were to identify and characterize active (energy-dependent) and passive calcium transport systems that work together to regulate calcium levels in the cytoplasm of plant cells. 10 figs., 2 tabs.

  20. Active and passive calcium transport systems in plant cells

    SciTech Connect

    Sze, H.

    1991-01-01

    The ability to change cytoplasmic Ca{sup 2+} levels ((Ca{sup 2+})) by cells has made this cation a key regulator of many biological processes. Cytoplasmic (Ca{sup 2+}) is determined by the coordination of passive Ca{sup 2+} fluxes which increase cytosolic (Ca{sup 2+}) and active Ca{sup 2+} transport systems that lower cytosolic (Ca{sup 2+}). The mechanisms by which plant cells achieve this is poorly understood. We have initially used isolated vesicles from the plasma membrane or organellar membranes to study Ca{sup 2+} transport systems in oat roots (a monocot) and carrot suspension cells (a dicot). The objectives of the proposal were to identify and characterize active (energy-dependent) and passive calcium transport systems that work together to regulate calcium levels in the cytoplasm of plant cells.

  1. Cell Activation-Induced Phosphoinositide 3-Kinase Alpha/Beta Dimerization Regulates PTEN Activity

    PubMed Central

    Pérez-García, Vicente; Redondo-Muñoz, Javier; Kumar, Amit

    2014-01-01

    The phosphoinositide 3-kinase (PI3K)/PTEN (phosphatase and tensin homolog) pathway is one of the central routes that enhances cell survival, division, and migration, and it is frequently deregulated in cancer. PI3K catalyzes formation of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] after cell activation; PTEN subsequently reduces these lipids to basal levels. Activation of the ubiquitous p110α isoform precedes that of p110β at several points during the cell cycle. We studied the potential connections between p110α and p110β activation, and we show that cell stimulation promotes p110α and p110β association, demonstrating oligomerization of PI3K catalytic subunits within cells. Cell stimulation also promoted PTEN incorporation into this complex, which was necessary for PTEN activation. Our results show that PI3Ks dimerize in vivo and that PI3K and PTEN activities modulate each other in a complex that controls cell PI(3,4,5)P3 levels. PMID:24958106

  2. Spectral perspective on the electromagnetic activity of cells.

    PubMed

    Kučera, Ondrej; Červinková, Kateřina; Nerudová, Michaela; Cifra, Michal

    2015-01-01

    In this mini-review, we summarize the current hypotheses, theories and experimental evidence concerning the electromagnetic activity of living cells. We systematically classify the bio-electromagnetic phenomena in terms of frequency and we assess their general acceptance in scientific community. We show that the electromagnetic activity of cells is well established in the low frequency range below 1 kHz and on optical wavelengths, while there is only limited evidence for bio-electromagnetic processes in radio- frequency and millimeter-wave ranges. This lack of generally accepted theory or trustful experimental results is the cause for controversy which accompanies this topic. We conclude our review with the discussion of the relevance of the electromagnetic activity of cells to human medicine.

  3. RNase L Suppresses Androgen Receptor Signaling, Cell Migration and Matrix Metalloproteinase Activity in Prostate Cancer Cells.

    PubMed

    Dayal, Shubham; Zhou, Jun; Manivannan, Praveen; Siddiqui, Mohammad Adnan; Ahmad, Omaima Farid; Clark, Matthew; Awadia, Sahezeel; Garcia-Mata, Rafael; Shemshedini, Lirim; Malathi, Krishnamurthy

    2017-03-01

    The interferon antiviral pathways and prostate cancer genetics converge on a regulated endoribonuclease, RNase L. Positional cloning and linkage studies mapped Hereditary Prostate Cancer 1 (HPC1) to RNASEL. To date, there is no correlation of viral infections with prostate cancer, suggesting that RNase L may play additional roles in tumor suppression. Here, we demonstrate a role of RNase L as a suppressor of androgen receptor (AR) signaling, cell migration and matrix metalloproteinase activity. Using RNase L mutants, we show that its nucleolytic activity is dispensable for both AR signaling and migration. The most prevalent HPC1-associated mutations in RNase L, R462Q and E265X, enhance AR signaling and cell migration. RNase L negatively regulates cell migration and attachment on various extracellular matrices. We demonstrate that RNase L knockdown cells promote increased cell surface expression of integrin β1 which activates Focal Adhesion Kinase-Sarcoma (FAK-Src) pathway and Ras-related C3 botulinum toxin substrate 1-guanosine triphosphatase (Rac1-GTPase) activity to increase cell migration. Activity of matrix metalloproteinase (MMP)-2 and -9 is significantly increased in cells where RNase L levels are ablated. We show that mutations in RNase L found in HPC patients may promote prostate cancer by increasing expression of AR-responsive genes and cell motility and identify novel roles of RNase L as a prostate cancer susceptibility gene.

  4. RNase L Suppresses Androgen Receptor Signaling, Cell Migration and Matrix Metalloproteinase Activity in Prostate Cancer Cells

    PubMed Central

    Dayal, Shubham; Zhou, Jun; Manivannan, Praveen; Siddiqui, Mohammad Adnan; Ahmad, Omaima Farid; Clark, Matthew; Awadia, Sahezeel; Garcia-Mata, Rafael; Shemshedini, Lirim; Malathi, Krishnamurthy

    2017-01-01

    The interferon antiviral pathways and prostate cancer genetics converge on a regulated endoribonuclease, RNase L. Positional cloning and linkage studies mapped Hereditary Prostate Cancer 1 (HPC1) to RNASEL. To date, there is no correlation of viral infections with prostate cancer, suggesting that RNase L may play additional roles in tumor suppression. Here, we demonstrate a role of RNase L as a suppressor of androgen receptor (AR) signaling, cell migration and matrix metalloproteinase activity. Using RNase L mutants, we show that its nucleolytic activity is dispensable for both AR signaling and migration. The most prevalent HPC1-associated mutations in RNase L, R462Q and E265X, enhance AR signaling and cell migration. RNase L negatively regulates cell migration and attachment on various extracellular matrices. We demonstrate that RNase L knockdown cells promote increased cell surface expression of integrin β1 which activates Focal Adhesion Kinase-Sarcoma (FAK-Src) pathway and Ras-related C3 botulinum toxin substrate 1-guanosine triphosphatase (Rac1-GTPase) activity to increase cell migration. Activity of matrix metalloproteinase (MMP)-2 and -9 is significantly increased in cells where RNase L levels are ablated. We show that mutations in RNase L found in HPC patients may promote prostate cancer by increasing expression of AR-responsive genes and cell motility and identify novel roles of RNase L as a prostate cancer susceptibility gene. PMID:28257035

  5. SNX17 Affects T Cell Activation by Regulating T Cell Receptor and Integrin Recycling

    PubMed Central

    Osborne, Douglas G.; Piotrowski, Joshua T.; Dick, Christopher J.; Zhang, Jin-San; Billadeau, Daniel D.

    2015-01-01

    A key component in T cell activation is the endosomal recycling of receptors to the cell surface, thereby allowing continual integration of signaling and antigen recognition. One protein potentially involved in T cell receptor transport is sorting nexin 17 (SNX17). SNX proteins have been found to bind proteins involved in T cell activation, but specifically the role of SNX17 in receptor recycling and T cell activation is unknown. Using immunofluorescence, we find that SNX17 co-localizes with TCR and localizes to the immune synapse in T-APC conjugates. Significantly, knockdown of the SNX17 resulted in fewer T-APC conjugates, lower CD69, TCR, and LFA-1 surface expression, as well as lower overall TCR recycling compared to control T cells. Lastly, we identified the FERM-domain of SNX17 as being responsible in the binding and trafficking of TCR and LFA-1 to the cell surface. These data suggest that SNX17 plays a role in the maintenance of normal surface levels of activating receptors and integrins to permit optimum T cell activation at the immune synapse. PMID:25825439

  6. Effect Of Simulated Microgravity On Activated T Cell Gene Transcription

    NASA Technical Reports Server (NTRS)

    Morrow, Maureen A.

    2003-01-01

    Studies of T lymphocytes under the shear stress environment of clinorotation have demonstrated an inhibition of activation in response to TCR mediated signaling. These results mimic those observed during space flight. This work investigates the molecular signaling events of T lymphocyte activation with clinorotation. Purified human T lymphocytes and the T cell clone Jurkat exhibit an uncoupling of signaling as mediated through the TCR. Activation of the transcription factor AP-1 is inhibited while activation of NFAT occurs. NFAT dephosphorylation and activation is dependent on sustained Ca(++) influx. Alternatively, AP-1, which consists of two transcription factors, jun and fos, is activated by PKC and Ras mediated pathways. TCR signaling is known to be dependent on cytoskeletal rearrangements, in particular, raft aggregation is critical. Raft aggregation, as mediated through GM, crosslinking, overcomes the inhibition of T lymphocyte activation with clinorotation, indicating that the block is occurring upstream of raft aggregation. Clinorotation is shown to have an effect similar to a weak TCR signal.

  7. Nuclear cathepsin L activity is required for cell cycle progression of colorectal carcinoma cells.

    PubMed

    Tamhane, Tripti; Lllukkumbura, Rukshala; Lu, Shiying; Maelandsmo, Gunhild M; Haugen, Mads H; Brix, Klaudia

    2016-03-01

    Prominent tasks of cysteine cathepsins involve endo-lysosomal proteolysis and turnover of extracellular matrix constituents or plasma membrane proteins for maintenance of intestinal homeostasis. Here we report on enhanced levels and altered subcellular localization of distinct cysteine cathepsins in adenocarcinoma tissue in comparison to adjacent normal colon. Immunofluorescence and immunoblotting investigations revealed the presence of cathepsin L in the nuclear compartment in addition to its expected endo-lysosomal localization in colorectal carcinoma cells. Cathepsin L was represented as the full-length protein in the nuclei of HCT116 cells from which stefin B, a potent cathepsin L inhibitor, was absent. Fluorescence activated cell sorting analyses with synchronized cell cultures revealed deceleration of cell cycle progression of HCT116 cells upon inhibition of cathepsin L activity, while expression of cathepsin L-enhanced green fluorescent protein chimeras accelerated S-phase entry. We conclude that the activity of cathepsin L is high in the nucleus of colorectal carcinoma cells because of lacking stefin B inhibitory activity. Furthermore, we hypothesize that nuclear cathepsin L accelerates cell cycle progression of HCT116 cells thereby supporting the notion that cysteine cathepsins may play significant roles in carcinogenesis due to deregulated trafficking.

  8. IFNγ Regulates Activated Vδ2+ T Cells through a Feedback Mechanism Mediated by Mesenchymal Stem Cells

    PubMed Central

    Fechter, Karoline; Dorronsoro, Akaitz; Jakobsson, Emma; Ferrin, Izaskun; Lang, Valérie; Sepulveda, Pilar; Pennington, Daniel J.; Trigueros, César

    2017-01-01

    γδ T cells play a role in a wide range of diseases such as autoimmunity and cancer. The majority of circulating human γδ T lymphocytes express a Vγ9Vδ2+ (Vδ2+) T cell receptor (TCR) and following activation release pro-inflammatory cytokines. In this study, we show that IFNγ, produced by Vδ2+ cells, activates mesenchymal stem cell (MSC)-mediated immunosupression, which in turn exerts a negative feedback mechanism on γδ T cell function ranging from cytokine production to proliferation. Importantly, this modulatory effect is limited to a short period of time (<24 hours) post-T cell activation, after which MSCs can no longer exert their immunoregulatory capacity. Using genetically modified MSCs with the IFNγ receptor 1 constitutively silenced, we demonstrate that IFNγ is essential to this process. Activated γδ T cells induce expression of several factors by MSCs that participate in the depletion of amino acids. In particular, we show that indolamine 2,3-dioxygenase (IDO), an enzyme involved in L-tryptophan degradation, is responsible for MSC-mediated immunosuppression of Vδ2+ T cells. Thus, our data demonstrate that γδ T cell responses can be immuno-modulated by different signals derived from MSC. PMID:28076364

  9. PPARγ negatively regulates T cell activation to prevent follicular helper T cells and germinal center formation.

    PubMed

    Park, Hong-Jai; Kim, Do-Hyun; Choi, Jin-Young; Kim, Won-Ju; Kim, Ji Yun; Senejani, Alireza G; Hwang, Soo Seok; Kim, Lark Kyun; Tobiasova, Zuzana; Lee, Gap Ryol; Craft, Joseph; Bothwell, Alfred L M; Choi, Je-Min

    2014-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor that regulates lipid and glucose metabolism. Although studies of PPARγ ligands have demonstrated its regulatory functions in inflammation and adaptive immunity, its intrinsic role in T cells and autoimmunity has yet to be fully elucidated. Here we used CD4-PPARγKO mice to investigate PPARγ-deficient T cells, which were hyper-reactive to produce higher levels of cytokines and exhibited greater proliferation than wild type T cells with increased ERK and AKT phosphorylation. Diminished expression of IκBα, Sirt1, and Foxo1, which are inhibitors of NF-κB, was observed in PPARγ-deficient T cells that were prone to produce all the signature cytokines under Th1, Th2, Th17, and Th9 skewing condition. Interestingly, 1-year-old CD4-PPARγKO mice spontaneously developed moderate autoimmune phenotype by increased activated T cells, follicular helper T cells (TFH cells) and germinal center B cells with glomerular inflammation and enhanced autoantibody production. Sheep red blood cell immunization more induced TFH cells and germinal centers in CD4-PPARγKO mice and the T cells showed increased of Bcl-6 and IL-21 expression suggesting its regulatory role in germinal center reaction. Collectively, these results suggest that PPARγ has a regulatory role for TFH cells and germinal center reaction to prevent autoimmunity.

  10. Premalignant Oral Lesion Cells Elicit Increased Cytokine Production and Activation of T-cells

    PubMed Central

    JOHNSON, SARA D.; LEVINGSTON, CORINNE; YOUNG, M. RITA I.

    2016-01-01

    Background Head and neck squamous cell carcinomas (HNSCC) are known to evade the host immune response. How premalignant oral lesions modulate the immune response, however, has yet to be elucidated. Materials and Methods A mouse model of oral carcinogenesis was used to determine how mediators from premalignant oral lesion cells vs. HNSCC cells impact on immune cytokine production and activation. Results Media conditioned by premalignant lesion cells elicited an increased production of T cell-associated cytokines and proinflammatory mediators from cervical lymph node cells compared to media conditioned by HNSCC cells or media alone. In the presence of premalignant lesion cell-conditioned media, CD4+ T cell expression of the IL-2 receptor CD25 and CD8+ T cell expression of the activation marker CD69 was greater, compared to what was induced in HNSCC cell-conditioned media or media alone. Conclusion Premalignant lesion cells promote a proinflammatory environment and induce immune changes before HNSCC tumors are established. PMID:27354582

  11. Cytoplasmic myosin exposed apoptotic cells appear with caspase-3 activation and enhance CLL cell viability

    PubMed Central

    Cui, Xiaoxuan; Zhang, Lu; Magli, Amanda R.; Catera, Rosa; Yan, Xiao-Jie; Griffin, Daniel O.; Rothstein, Thomas L.; Barrientos, Jacqueline; Kolitz, Jonathan E.; Allen, Steven L.; Rai, Kanti R.; Chiorazzi, Nicholas; Chu, Charles C.

    2015-01-01

    The degree of chronic lymphocytic leukemia (CLL) B-cell antigen receptor (BCR) binding to myosin exposed apoptotic cells (MEACs) correlates with worse patient outcomes, suggesting a link to disease activity. Therefore, we studied MEAC formation and the effects of MEAC binding on CLL cells. In cell line studies, both intrinsic (spontaneous or camptothecin-induced) and extrinsic (FasL- or anti-Fas-induced) apoptosis created a high percent of MEACs over time in a process associated with caspase-3 activation, leading to cytoplasmic myosin cleavage and trafficking to cell membranes. The involvement of common apoptosis pathways suggests that most cells can produce MEACs and indeed CLL cells themselves form MEACs. Consistent with the idea that MEAC formation may be a signal to remove dying cells, we found that natural IgM antibodies bind to MEACs. Functionally, co-culture of MEACs with CLL cells, regardless of immunoglobulin heavy chain variable region gene mutation status, improved leukemic cell viability. Based on inhibitor studies, this improved viability involved BCR signaling molecules. These results support the hypothesis that stimulation of CLL cells with antigen, such as those on MEACs, promotes CLL cell viability, which in turn could lead to progression to worse disease. PMID:26220042

  12. Stochasticity and spatial heterogeneity in T-cell activation.

    PubMed

    Burroughs, Nigel J; van der Merwe, P Anton

    2007-04-01

    Stochastic and spatial aspects are becoming increasingly recognized as an important factor in T-cell activation. Activation occurs in an intrinsically noisy environment, requiring only a handful of agonist peptide-major histocompatibility complex molecules, thus making consideration of signal to noise of prime importance in understanding sensitivity and specificity. Furthermore, it is widely established that surface-bound ligands are more effective at activation than soluble forms, while surface patternation has highlighted the role of spatial relocation in activation. Here we consider the results of a number of models of T-cell activation, from a realistic model of kinetic segregation-induced T-cell receptor (TCR) triggering through to simple queuing theory models. These studies highlight the constraints on cell activation by a surface receptor that recruits kinases. Our analysis shows that TCR triggering based on trapping of bound TCRs in regions of close proximity that exclude large ectodomain-containing molecules, such as the phosphatases CD45 and CD148, can effectively reproduce known signaling characteristics and is a viable 'signal transduction' mechanism distinct from oligomerization and conformation-based mechanisms. A queuing theory analysis shows the interrelation between sensitivity and specificity, emphasizing that these are properties of individual cell functions and need not be, nor are likely to be, uniform across different functions. In fact, threshold-based mechanisms of detection are shown to be poor at ligand discrimination because, although they can be highly specific, that specificity is limited to a small range of peptide densities. Time integration mechanisms however are able to control noise effectively, while kinetic proofreading mechanisms endow them with good specificity properties. Thus, threshold mechanisms are likely to be important for rapidly detecting minimal signaling requirements, thus achieving efficient scanning of antigen

  13. T-Cell Immunophenotyping Distinguishes Active From Latent Tuberculosis

    PubMed Central

    Pollock, Katrina M.; Whitworth, Hilary S.; Montamat-Sicotte, Damien J.; Grass, Lisa; Cooke, Graham S.; Kapembwa, Moses S.; Kon, Onn M.; Sampson, Robert D.; Taylor, Graham P.; Lalvani, Ajit

    2013-01-01

    Background. Changes in the phenotype and function of Mycobacterium tuberculosis (M. tuberculosis)-specific CD4+ and CD8+ T-cell subsets in response to stage of infection may allow discrimination between active tuberculosis and latent tuberculosis infection. Methods. A prospective comparison of M. tuberculosis-specific cellular immunity in subjects with active tuberculosis and latent tuberculosis infection, with and without human immunodeficiency virus (HIV) coinfection. Polychromatic flow cytometry was used to measure CD4+ and CD8+ T-cell subset phenotype and secretion of interferon γ (IFN-γ), interleukin 2 (IL-2), and tumor necrosis factor α (TNF-α). Results. Frequencies of CD4+ and CD8+ cells secreting IFN-γ-only, TNF-α-only and dual IFN-γ/TNF-α were greater in active tuberculosis vs latent tuberculosis infection. All M. tuberculosis-specific CD4+ subsets, with the exception of IL-2-only cells, switched from central to effector memory phenotype in active tuberculosis vs latent tuberculosis infection, accompanied by a reduction in IL-7 receptor α (CD127) expression. The frequency of PPD-specific CD4+ TNF-α-only-secreting T cells with an effector phenotype accurately distinguished active tuberculosis from latent tuberculosis infection with an area under the curve of 0.99, substantially more discriminatory than measurement of function alone. Conclusions. Combined measurement of T-cell phenotype and function defines a highly discriminatory biomarker of tuberculosis disease activity. Unlocking the diagnostic and monitoring potential of this combined approach now requires validation in large-scale prospective studies. PMID:23966657

  14. Effect of millimeter waves on natural killer cell activation.

    PubMed

    Makar, V R; Logani, M K; Bhanushali, A; Kataoka, M; Ziskin, M C

    2005-01-01

    Millimeter wave therapy (MMWT) is being widely used for the treatment of many diseases in Russia and other East European countries. MMWT has been reported to reduce the toxic effects of chemotherapy on the immune system. The present study was undertaken to investigate whether millimeter waves (MMWs) can modulate the effect of cyclophosphamide (CPA), an anticancer drug, on natural killer (NK) cell activity. NK cells play an important role in the antitumor response. MMWs were produced with a Russian-made YAV-1 generator. The device produced modulated 42.2 +/- 0.2 GHz radiation through a 10 x 20 mm rectangular output horn. Mice, restrained in plastic tubes, were irradiated on the nasal area. Peak SAR at the skin surface and peak incident power density were measured as 622 +/- 100 W/kg and 31 +/- 5 mW/cm2, respectively. The maximum temperature elevation, measured at the end of 30 min, was 1 degrees C. The animals, restrained in plastic tubes, were irradiated on the nasal area. CPA injection (100 mg/kg) was given intraperitoneally on the second day of 3-days exposure to MMWs. All the irradiation procedures were performed in a blinded manner. NK cell activation and cytotoxicity were measured after 2, 5, and 7 days following CPA injection. Flow cytometry of NK cells showed that CPA treatment caused a marked enhancement in NK cell activation. The level of CD69 expression, which represents a functional triggering molecule on activated NK cells, was increased in the CPA group at all the time points tested as compared to untreated mice. However, the most enhancement in CD69 expression was observed on day 7. A significant increase in TNF-alpha level was also observed on day 7 following CPA administration. On the other hand, CPA caused a suppression of the cytolytic activity of NK cells. MMW irradiation of the CPA treated groups resulted in further enhancement of CD69 expression on NK cells, as well as in production of TNF-alpha. Furthermore, MMW irradiation restored CPA

  15. Investigation of MEK activity in COS7 cells entering mitosis.

    PubMed

    Shi, Huaiping; Zhang, Tianying; Yi, Yongqing; Luo, Jun

    2014-12-01

    Although the mitogen-activated protein kinase (MAPK) pathway has been extensively investigated, numerous events remain unclear. In the present study, we examined mitogen-activated protein kinase kinase (MEK) expression from interphase to mitosis. Following nocodazole treatment, COS7 cells gradually became round as early as 4 h after treatment. Cyclin B1 expression gradually increased from 4 to 24 h in the presence of nocodazole. When cells were treated with nocodazole for 4 h, the level of epidermal growth factor (EGF)-mediated MEK phosphorylation did not significantly change between nocodazole-untreated and -treated (4 h) cells (P>0.05). However, EGF-mediated MEK phosphorylation was significantly inhibited upon treatment with nocodazole for 8 and 24 h compared to nocodazole-untreated cells (P<0.05). MEK phosphorylation levels were comparable between 1, 5, 10 and 50 ng/ml EGF treatments. Phorbol 12-myristic 13-acetate (PMA) did not activate MEK in mitotic cells. Following treatment of COS7 cells at the interphase with AG1478 or U0126, MEK phosphorylation was blocked. In addition, the investigation of the expression of proteins downstream of MEK demonstrated that EGF does not significantly affect the phosphorylation level of extracellular-signal-regulated kinase (ERK), ribosomal protein S6 kinase (RSK) and Elk in mitotic cells (P>0.05). The results showed that MEK expression is gradually inhibited from cell interphase to mitosis, and that MEK downstream signaling is affected by this inhibition, which probably reflects the requirements of cell physiology during mitosis.

  16. Paeonol Suppresses Neuroinflammatory Responses in LPS-Activated Microglia Cells.

    PubMed

    He, Li Xia; Tong, Xiaoyun; Zeng, Jing; Tu, Yuanqing; Wu, Saicun; Li, Manping; Deng, Huaming; Zhu, Miaomiao; Li, Xiucun; Nie, Hong; Yang, Li; Huang, Feng

    2016-12-01

    In this work, we assessed the anti-inflammatory effects of paeonol (PAE) in LPS-activated N9 microglia cells, as well as its underlying molecular mechanisms. PAE had no adverse effect on the viability of murine microglia N9 cell line within a broad range (0.12∼75 μM). When N9 cell line was activated by LPS, PAE (0.6, 3, 15 μM) significantly suppressed the release of proinflammatory products, such as nitric oxide (NO), interleukin-1β (IL-1β), and prostaglandin E2 (PGE2), demonstrated by the ELISA assay. Moreover, the levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were significantly reduced in PAE-treated N9 microglia cells. We also examined some proteins involved in immune signaling pathways and found that PAE treatment significantly decreased the expression of TLR4, MyD88, IRAK4, TNFR-associated factor 6 (TRAF6), p-IkB-α, and NF-kB p65, as well as the mitogen-activated protein kinase (MAPK) pathway molecules p-P38, p-JNK, and p-ERK, indicating that PAE might act on these signaling pathways to inhibit inflammatory responses. Overall, we found that PAE had anti-inflammatory effect on LPS-activated N9 microglia cells, possibly via inhibiting the TLR4 signaling pathway, and it could be a potential drug therapy for inflammation-associated neurodegenerative diseases.

  17. Femtosecond laser fabricated microfluorescence-activated cell sorter for single cell recovery

    NASA Astrophysics Data System (ADS)

    Bragheri, F.; Paiè, P.; Nava, G.; Yang, T.; Minzioni, P.; Martinez Vazquez, R.; Bellini, N.; Ramponi, R.; Cristiani, I.; Osellame, R.

    2014-03-01

    Manipulation, sorting and recovering of specific live cells from samples containing less than a few thousand cells is becoming a major hurdle in rare cell exploration such as stem cell research or cell based diagnostics. Moreover the possibility of recovering single specific cells for culturing and further analysis would be of great impact in many biological fields ranging from regenerative medicine to cancer therapy. In recent years considerable effort has been devoted to the development of integrated and low-cost optofluidic devices able to handle single cells, which usually rely on microfluidic circuits that guarantee a controlled flow of the cells. Among the different microfabrication technologies, femtosecond laser micromachining (FLM) is ideally suited for this purpose as it provides the integration of both microfluidic and optical functions on the same glass chip leading to monolithic, robust and portable devices. Here a new optofluidic device is presented, which is capable of sorting and recovering of single cells, through optical forces, on the basis of their fluorescence and. Both fluorescence detection and single cell sorting functions are integrated in the microfluidic chip by FLM. The device, which is specifically designed to operate with a limited amount of cells but with a very high selectivity, is fabricated by a two-step process that includes femtosecond laser irradiation followed by chemical etching. The capability of the device to act as a micro fluorescence-activated cell sorter has been tested on polystyrene beads and on tumor cells and the results on the single live cell recovery are reported.

  18. T cell-mediated activation and regulation of anti-chromatin B cells.

    PubMed

    Pagán, Antonio J; Ramón, Hilda E; Hondowicz, Brian D; Erikson, Jan

    2006-07-01

    We have taken an immunoglobulin transgenic approach to study how self-reactive B cells are held in check in healthy mice and what parameters contribute to their activation in autoimmunity. Using this strategy, we have documented that a population of anti-chromatin B cells migrate to the periphery. In a healthy background, these cells have a reduced lifespan, appear developmentally arrested, and localize primarily to the T/B cell interface in the spleen. Importantly, they are capable of differentiating into antibody-forming cells when provided with T cell help. T(H)1 and T(H)2 cells induce IgG2a and IgG1 autoantibodies, respectively. In the context of the autoimmune-prone lpr/lpr or gld/gld mutations, these autoreactive B cells populate the B cell follicle, and this is dependent upon CD4 T cells. However, after 10 weeks of age serum autoantibodies are produced. We hypothesize that control of autoantibody production in young autoimmune-prone mice is regulated by the counterbalancing influence of regulatory T cells. We show that while autoantibody production is blocked in the context of regulatory T cells, early events characterizing a productive T cell-B cell interaction are not disturbed, with the notable exceptions of T(H) ICOS levels and IFN-gamma and IL-10 production.

  19. Calcium-dependent activation of mitochondrial metabolism in mammalian cells

    PubMed Central

    Gaspers, Lawrence D.; Thomas, Andrew P.

    2008-01-01

    Endogenous fluorophores provide a simple, but elegant means to investigate the relationship between agonist-evoked Ca2+ signals and the activation of mitochondrial metabolism. In this article, we discuss the methods and strategies to measure cellular pyridine nucleotide and flavoprotein fluorescence alone or in combination with Ca2+-sensitive indicators. These methods were developed using primary cultured hepatocytes and neurons, which contain relatively high levels of endogenous fluorophores and robust metabolic responses. Nevertheless, these methods are amendable to a wide variety of primary cell types and cell lines that maintain active mitochondrial metabolism. PMID:18854213

  20. Muscarinic activation of mitogen-activated protein kinase in PC12 cells.

    PubMed

    Berkeley, J L; Levey, A I

    2000-08-01

    Muscarinic acetylcholine receptors (mAChRs) activate many downstream signaling pathways, some of which can lead to mitogen-activated protein kinase (MAPK) phosphorylation and activation. MAPKs play roles in regulating cell growth, differentiation, and synaptic plasticity. Here, the activation of MAPK was examined in PC12 cells endogenously expressing mAChRs. Western blot analysis using a phosphospecific MAPK antibody revealed a dose-dependent and atropine-sensitive increase in MAPK phosphorylation in cells stimulated with carbachol (CCh). The maximal response occurred after 5 min and was rapidly reduced to baseline. To investigate the receptors responsible for CCh activation of MAPK in PC12 cells, the mAChR subtypes present were determined using RT-PCR and immunoprecipitation. RT-PCR was used to amplify fragments of the appropriate sizes for m1, m4, and m5, and the identities of the bands were confirmed with restriction digests. Immunoprecipitation using subtype-specific antibodies showed that approximately 95% of the expressed receptors were m4, whereas the remaining approximately 5% were m1 and m5. A highly specific m1 toxin completely blocked MAPK phosphorylation in response to CCh stimulation. The mAChR-induced MAPK activation was abolished by protein kinase C down-regulation and partially inhibited by pertussis toxin. Although m1 represents a small proportion of the total mAChR population, pharmacological evidence suggests that m1 is responsible for MAPK activation in PC12 cells.

  1. Benfotiamine upregulates antioxidative system in activated BV-2 microglia cells

    PubMed Central

    Bozic, Iva; Savic, Danijela; Stevanovic, Ivana; Pekovic, Sanja; Nedeljkovic, Nadezda; Lavrnja, Irena

    2015-01-01

    Chronic microglial activation and resulting sustained neuroinflammatory reaction are generally associated with neurodegeneration. Activated microglia acquires proinflammatory cellular profile that generates oxidative burst. Their persistent activation exacerbates inflammation, which damages healthy neurons via cytotoxic mediators, such as superoxide radical anion and nitric oxide. In our recent study, we have shown that benfotiamine (S-benzoylthiamine O-monophosphate) possesses anti-inflammatory effects. Here, the effects of benfotiamine on the pro-oxidative component of activity of LPS-stimulated BV-2 cells were investigated. The activation of microglia was accompanied by upregulation of intracellular antioxidative defense, which was further promoted in the presence of benfotiamine. Namely, activated microglia exposed to non-cytotoxic doses of benfotiamine showed increased levels and activities of hydrogen peroxide- and superoxide-removing enzymes—catalase and glutathione system, and superoxide dismutase. In addition, benfotiamine showed the capacity to directly scavenge superoxide radical anion. As a consequence, benfotiamine suppressed the activation of microglia and provoked a decrease in NO and ·O−2 production and lipid peroxidation. In conclusion, benfotiamine might silence pro-oxidative activity of microglia to alleviate/prevent oxidative damage of neighboring CNS cells. PMID:26388737

  2. Activation of AMPK Stimulates Neurotensin Secretion in Neuroendocrine Cells

    PubMed Central

    Li, Jing; Song, Jun; Weiss, Heidi L.; Weiss, Todd; Townsend, Courtney M.

    2016-01-01

    AMP-activated protein kinase (AMPK), a critical fuel-sensing enzyme, regulates the metabolic effects of various hormones. Neurotensin (NT) is a 13-amino acid peptide predominantly localized in enteroendocrine cells of the small bowel and released by fat ingestion. Increased fasting plasma levels of pro-NT (a stable NT precursor fragment produced in equimolar amounts relative to NT) are associated with an increased risk of diabetes, cardiovascular disease, and mortality; however, the mechanisms regulating NT release are not fully defined. We previously reported that inhibition of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) increases NT secretion and gene expression through activation of the MEK/ERK pathway. Here, we show that activation of AMPK increases NT secretion from endocrine cell lines (BON and QGP-1) and isolated mouse crypt cells enriched for NT-positive cells. In addition, plasma levels of NT increase in mice treated with 5-aminoimidazole-4-carboxamide riboside, a pharmacologic AMPK activator. Small interfering RNA-mediated knockdown of AMPKα decrease, whereas overexpression of the subunit significantly enhances, NT secretion from BON cells treated with AMPK activators or oleic acid. Similarly, small interfering RNA knockdown of the upstream AMPK kinases, liver kinase B1 and Ca2+ calmodulin-dependent protein kinase kinase 2, also attenuate NT release and AMPK phosphorylation. Moreover, AMPK activation increases NT secretion through inhibition of mTORC1 signaling. Together, our findings show that AMPK activation enhances NT release through inhibition of mTORC1 signaling, thus demonstrating an important cross talk regulation for NT secretion. PMID:26528831

  3. Activation of AMPK Stimulates Neurotensin Secretion in Neuroendocrine Cells.

    PubMed

    Li, Jing; Song, Jun; Weiss, Heidi L; Weiss, Todd; Townsend, Courtney M; Evers, B Mark

    2016-01-01

    AMP-activated protein kinase (AMPK), a critical fuel-sensing enzyme, regulates the metabolic effects of various hormones. Neurotensin (NT) is a 13-amino acid peptide predominantly localized in enteroendocrine cells of the small bowel and released by fat ingestion. Increased fasting plasma levels of pro-NT (a stable NT precursor fragment produced in equimolar amounts relative to NT) are associated with an increased risk of diabetes, cardiovascular disease, and mortality; however, the mechanisms regulating NT release are not fully defined. We previously reported that inhibition of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) increases NT secretion and gene expression through activation of the MEK/ERK pathway. Here, we show that activation of AMPK increases NT secretion from endocrine cell lines (BON and QGP-1) and isolated mouse crypt cells enriched for NT-positive cells. In addition, plasma levels of NT increase in mice treated with 5-aminoimidazole-4-carboxamide riboside, a pharmacologic AMPK activator. Small interfering RNA-mediated knockdown of AMPKα decrease, whereas overexpression of the subunit significantly enhances, NT secretion from BON cells treated with AMPK activators or oleic acid. Similarly, small interfering RNA knockdown of the upstream AMPK kinases, liver kinase B1 and Ca(2+) calmodulin-dependent protein kinase kinase 2, also attenuate NT release and AMPK phosphorylation. Moreover, AMPK activation increases NT secretion through inhibition of mTORC1 signaling. Together, our findings show that AMPK activation enhances NT release through inhibition of mTORC1 signaling, thus demonstrating an important cross talk regulation for NT secretion.

  4. The influence of tetracyclines on T cell activation.

    PubMed Central

    Kloppenburg, M; Verweij, C L; Miltenburg, A M; Verhoeven, A J; Daha, M R; Dijkmans, B A; Breedveld, F C

    1995-01-01

    Minocycline has been shown to have an anti-inflammatory effect in patients with rheumatoid arthritis (RA). Since there is evidence that RA is a T cell-mediated disease, we investigated the effect of minocycline on human T cell clones derived from the synovium of an RA patient. The T cells, when activated via the T cell receptor (TCR)/CD3 complex, were suppressed functionally by minocycline, resulting in a dose-dependent inhibition of T cell proliferation and reduction in production of IL-2, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha). Besides an inhibition of IL-2 production, minocycline exerted its effect on T cell proliferation by induction of a decreased IL-2 responsiveness. We showed that the chelating capacity of minocycline plays a crucial role in the inhibitory effect on T cell function, since the inhibitory effect on T cell proliferation could be annulled by addition of exogenous Ca2+. However, minocycline did not markedly influence the typical TCR/CD3-induced intracellular Ca2+ mobilization. Taken together, the results clearly indicate that minocycline has immunomodulating effects on human T cells. PMID:8536384

  5. Activation of cells using femtosecond laser beam (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Batabyal, Subrata; Satpathy, Sarmishtha; Kim, Young-tae; Mohanty, Samarendra K.

    2016-03-01

    Study of communication in cellular systems requires precise activation of targeted cell(s) in the network. In contrast to chemical, electrical, thermal, mechanical stimulation, optical stimulation is non-invasive and is better suited for stimulation of targeted cells. As compared to visible lasers, the near infrared (NIR) microsecond/nanosecond pulsed laser beams are being used as preferred stimulation tool as they provide higher penetration depth in tissues. Femotosecond (FS) laser beams in NIR are also being used for direct and indirect (i.e. via two-photon optogenetics) stimulation of cells. Here, we present a comparative evaluation of efficacy of NIR FS laser beam for direct (no optogenetic sensitization) and 2ph optogenetic stimulation of cells. Further, for the first time, we demonstrate the use of blue (~450 nm, obtained by second harmonic generation) FS laser beam for stimulation of cells with and without Channelrhodopisn-2 (ChR2) expression. Comparative analysis of photocurrent generated by blue FS laser beam and continuous wave blue light for optogenetics stimulation of ChR2 transfected HEK cells will be presented. The use of ultrafast laser micro-beam for focal, non-contact, and repeated stimulation of single cells in a cellular circuitry allowed us to study the communication between different cell types.

  6. Calcineurin functions in Ca(2+)-activated cell death in mammalian cells

    PubMed Central

    1995-01-01

    Calcineurin is a calcium-dependent protein phosphatase that functions in T cell activation. We present evidence that calcineurin functions more generally in calcium-triggered apoptosis in mammalian cells deprived of growth factors. Specifically, expression of epitope-tagged calcineurin A induces rapid cell death upon calcium signaling in the absence of growth factors. We show that this apoptosis does not require new protein synthesis and therefore calcineurin must operate through existing substrates. Co-expression of the Bcl-2 protooncogene efficiently blocks calcineurin-induced cell death. Significantly, we demonstrate that a calcium-independent calcineurin mutant induces apoptosis in the absence of calcium, and that this apoptotic response is a direct consequence of calcineurin's phosphatase activity. These data suggest that calcineurin plays an important role in mediating the upstream events in calcium-activated cell death. PMID:7593193

  7. Pattern matching based active optical sorting of colloids/cells

    NASA Astrophysics Data System (ADS)

    Verma, R. S.; Dasgupta, R.; Ahlawat, S.; Kumar, N.; Uppal, A.; Gupta, P. K.

    2013-08-01

    We report active optical sorting of colloids/cells by employing a cross correlation based pattern matching technique for selection of the desired objects and thereafter sorting using dynamically controllable holographic optical traps. The problem of possible collision between the different sets of objects during sorting was avoided by raising one set of particles to a different plane. We also present the results obtained on using this approach for some representative applications such as sorting of silica particles of two different sizes, of closely packed colloids and of white blood cells and red blood cells from a mixture of the two.

  8. Boundaries in gravitational and magnetic activation of cells for sorting.

    PubMed

    Czerlinski, G H

    1991-06-01

    Standard deviations in the distribution of radii of cells and particles are considered to arrive at realistic limits in the use of gravitational and magnetic activation of cells for sorting. Using a specific fractionation design, it is shown that the radius of particles (or cells) may be fractionated down to a precision of +/- 0.76%. Although higher precisions could be obtained with other designs, the number of particles available per fraction is inversely proportional to the precision desired. Thus, one would prefer to keep the precision as moderate as permissible by the experiments.

  9. Responses of cells in plasma-activated medium

    NASA Astrophysics Data System (ADS)

    Tanaka, Hiromasa; Mizuno, Masaaki; Ishikawa, Kenji; Takeda, Keigo; Hashizume, Hiroshi; Nakamura, Kae; Kajiyama, Hiroaki; Kano, Hiroyuki; Okazaki, Yasumasa; Toyokuni, Shinya; Maruyama, Shoichi; Kodera, Yasuhiro; Terasaki, Hiroko; Adachi, Tetsuo; Kato, Masashi; Kikkawa, Fumitaka; Hori, Masaru

    2015-09-01

    Plasma consists of electrons, ions, radicals, and lights, and produces various reactive species in gas and liquid phase. Cells receive various inputs from their circumstances, and induce several physiological outputs. Our goal is to clarify the relationships between plasma inputs and physiological outputs. Plasma-activated medium (PAM) is a circumstance that plasma provides cells and our previous studies suggest that PAM is a promising tool for cancer therapy. However, the mode of actions remains to be elucidated. We propose survival and proliferation signaling networks as well as redox signaling networks are key factors to understand cellular responses of PAM-treated glioblastoma cells.

  10. Endothelial juxtaposition of distinct adult stem cells activates angiogenesis signaling molecules in endothelial cells.

    PubMed

    Mohammadi, Elham; Nassiri, Seyed Mahdi; Rahbarghazi, Reza; Siavashi, Vahid; Araghi, Atefeh

    2015-12-01

    Efficacy of therapeutic angiogenesis needs a comprehensive understanding of endothelial cell (EC) function and biological factors and cells that interplay with ECs. Stem cells are considered the key components of pro- and anti-angiogenic milieu in a wide variety of physiopathological states, and interactions of EC-stem cells have been the subject of controversy in recent years. In this study, the potential effects of three tissue-specific adult stem cells, namely rat marrow-derived mesenchymal stem cells (rBMSCs), rat adipose-derived stem cells (rADSCs) and rat muscle-derived satellite cells (rSCs), on the endothelial activation of key angiogenic signaling molecules, including VEGF, Ang-2, VEGFR-2, Tie-2, and Tie2-pho, were investigated. Human umbilical vein endothelial cells (HUVECs) and rat lung microvascular endothelial cells (RLMECs) were cocultured with the stem cells or incubated with the stem cell-derived conditioned media on Matrigel. Following HUVEC-stem cell coculture, CD31-positive ECs were flow sorted and subjected to western blotting to analyze potential changes in the expression of the pro-angiogenic signaling molecules. Elongation and co-alignment of the stem cells were seen along the EC tubes in the EC-stem cell cocultures on Matrigel, with cell-to-cell dye communication in the EC-rBMSC cocultures. Moreover, rBMSCs and rADSCs significantly improved endothelial tubulogenesis in both juxtacrine and paracrine manners. These two latter stem cells dynamically up-regulated VEGF, Ang-2, VREGR-2, and Tie-2 but down-regulated Tie2-pho and the Tie2-pho/Tie-2 ratio in HUVECs. Induction of pro-angiogenic signaling in ECs by marrow- and adipose-derived MSCs further indicates the significance of stem cell milieu in angiogenesis dynamics.

  11. Use of a human plaque-forming cell assay to study peripheral blood bursa-equivalent cell activation and excessive suppressor cell activity in humoral immunodeficiency.

    PubMed Central

    Herrod, H G; Buckley, R H

    1979-01-01

    A plaque assay that detects human mononuclear blood cells producing immunoglobulin (Ig)M antibody to sheep erythrocytes was investigated for its usefulness in studying B-cell activation and regulation in 24 patients with humoral immunodeficiency. Cells from 3 of 15 patients with common variable agammaglobulinemia produced some plaques (range 40--160/10(6) cells; normal range 80--1240/10(6)), but those from the other 12, from all 7 with x-linked agammaglobulinemia and from the 2 with x-linked immunodeficiency with hyper-IgM failed to produce any detectable plaques. In co-cultures of patient and normal cells a very good correlation was seen between results of the plaque assay and an IgM biosynthesis assay in detecting excessive suppressor cell activity. Cells from 7 of 15 common variable agammaglobulinemics, from 3 of 7 x-linked agammaglobulinemics, and from both patients with hyper-IgM caused significant suppression of IgM biosynthesis and(or) plaque formation by normal cells. The observations in the last two groups and discordance for excess suppressor activity in identical twins with common variable agammaglobulinemia suggest that the activity develops secondarily to whatever their primary defects may be. Culturing non-T cells from common variable agammaglobulinemics exhibiting excessive suppressor cell activity with normal T cells resulted in plaque formation in four of five patients so studied; in all five the suppressor activity was found in the T-cell population. The availability of a plaque assay for the study of blood cells from immunodeficient patients provides a new probe to examine the cellular nature of such defects. PMID:376549

  12. Functional Anatomy of T Cell Activation and Synapse Formation

    PubMed Central

    Fooksman, David R.; Vardhana, Santosh; Vasiliver-Shamis, Gaia; Liese, Jan; Blair, David; Waite, Janelle; Sacristán, Catarina; Victora, Gabriel; Zanin-Zhorov, Alexandra; Dustin, Michael L.

    2010-01-01

    T cell activation and function require a structured engagement of antigen-presenting cells. These cell contacts are characterized by two distinct dynamics in vivo: transient contacts resulting from promigratory junctions called immunological kinapses or prolonged contacts from stable junctions called immunological synapses. Kinapses operate in the steady state to allow referencing to self-peptide-MHC (pMHC) and searching for pathogen-derived pMHC. Synapses are induced by T cell receptor (TCR) interactions with agonist pMHC under specific conditions and correlate with robust immune responses that generate effector and memory T cells. High-resolution imaging has revealed that the synapse is highly coordinated, integrating cell adhesion, TCR recognition of pMHC complexes, and an array of activating and inhibitory ligands to promote or prevent T cell signaling. In this review, we examine the molecular components, geometry, and timing underlying kinapses and synapses. We integrate recent molecular and physiological data to provide a synthesis and suggest ways forward. PMID:19968559

  13. Expression of activated Ras during Dictyostelium development alters cell localization and changes cell fate.

    PubMed

    Jaffer, Z M; Khosla, M; Spiegelman, G B; Weeks, G

    2001-03-01

    There is now a body of evidence to indicate that Ras proteins play important roles in development. Dictyostelium expresses several ras genes and each appears to perform a distinct function. Previous data had indicated that the overexpression of an activated form of the major developmentally regulated gene, rasD, caused a major aberration in morphogenesis and cell type determination. We now show that the developmental expression of an activated rasG gene under the control of the rasD promoter causes a similar defect. Our results indicate that the expression of activated rasG in prespore cells results in their transdifferentiation into prestalk cells, whereas activated rasG expression in prestalk causes gross mislocalization of the prestalk cell populations.

  14. ASBESTOS-INDUCED ACTIVATION OF CELL SIGNALING PATHWAYS IN HUMAN BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Using respiratory epithelial cells transfected with either superoxide dismutase (SOD) or catalase, the authors tested the hypothesis that the activation of the epidermal growth factor (EGF) receptor signal pathway after asbestos exposure involves an oxidative stress. Western blot...

  15. Berberine-induced anticancer activities in FaDu head and neck squamous cell carcinoma cells.

    PubMed

    Seo, Yo-Seob; Yim, Min-Ji; Kim, Bok-Hee; Kang, Kyung-Rok; Lee, Sook-Young; Oh, Ji-Su; You, Jae-Seek; Kim, Su-Gwan; Yu, Sang-Joun; Lee, Gyeong-Je; Kim, Do Kyung; Kim, Chun Sung; Kim, Jin-Soo; Kim, Jae-Sung

    2015-12-01

    In the present study, we investigated berberine‑induced apoptosis and the signaling pathways underlying its activity in FaDu head and neck squamous cell carcinoma cells. Berberine did not affect the viability of primary human normal oral keratinocytes. In contrast, the cytotoxicity of berberine was significantly increased in FaDu cells stimulated with berberine for 24 h. Furthermore, berberine increased nuclear condensation and apoptosis rates in FaDu cells than those in untreated control cells. Berberine also induced the upregulation of apoptotic ligands, such as FasL and TNF-related apoptosis-inducing ligand, and triggered the activation of caspase-8, -7 and -3, and poly(ADP ribose) polymerase, characteristic of death receptor-dependent extrinsic apoptosis. Moreover, berberine activated the mitochondria‑dependent apoptotic signaling pathway by upregulating pro-apoptotic factors, such as Bax, Bad, Apaf-1, and the active form of caspase-9, and downregulating anti-apoptotic factors, such as Bcl-2 and Bcl-xL. In addition, berberine increased the expression of the tumor suppressor p53 in FaDu cells. The pan-caspase inhibitor Z-VAD-fmk suppressed the activation of caspase-3 and prevented cytotoxicity in FaDu cells treated with berberine. Interestingly, berberine suppressed cell migration through downregulation of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, and MMP-9. Moreover, the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and p38, components of the mitogen-activated protein kinase pathway that are associated with the expression of MMP and VEGF, was suppressed in FaDu cells treated with berberine for 24 h. Therefore, these data suggested that berberine exerted anticancer effects in FaDu cells through induction of apoptosis and suppression of migration. Berberine may have potential applications as a chemotherapeutic agent for the management of head and neck squamous carcinoma.

  16. Detection of programmed cell death in cells exposed to genotoxic agents using a caspase activation assay.

    PubMed

    Gupta, Madhu; Santra, Madhumita; Koty, Patrick P

    2014-01-01

    Many toxins that individuals are exposed to cause DNA damage. Cells that have sustained DNA damage may attempt to repair the damage prior to replication. However, if a cell has sustained serious damage it cannot repair, it will commit suicide through a genetically regulated programmed cell death (PCD) pathway. Crucial to the ultimate execution of PCD is a family of cysteine proteases called caspases. Activation of these enzymes occurs late enough in the PCD pathway that a cell can no longer avoid cell death, but still earlier than PCD-associated morphological changes or DNA fragmentation. This protocol details a method for using fluorochrome-conjugated caspase inhibitors for the detection of activated caspases in intact cells. The analysis and documentation is performed using fluorescence microscopy.

  17. Detection of programmed cell death in cells exposed to genotoxic agents using a caspase activation assay.

    PubMed

    Gehring, Michael E; Koty, Patrick P

    2005-01-01

    Many environmental toxins cause DNA damage. Cells that have sustained significant DNA damage must attempt to repair the damage prior to replication, in which aberrant base incorporation can result in an irreversible mutation. If a cell cannot repair the damage, however, it may commit suicide through a genetically regulated programmed cell death (PCD) pathway. Crucial to the ultimate execution of PCD is a family of cysteine proteases called caspases. Activation of these enzymes occurs late in the PCD pathway, when a cell can no longer avoid cell death, but earlier than other PCD markers, such as morphological changes or DNA fragmentation. This protocol details a method for using fluorochrome-conjugated caspase inhibitors for the detection of activated caspases in intact cells using fluorescent microscopy.

  18. Single-cell transcriptome analyses reveal signals to activate dormant neural stem cells.

    PubMed

    Luo, Yuping; Coskun, Volkan; Liang, Aibing; Yu, Juehua; Cheng, Liming; Ge, Weihong; Shi, Zhanping; Zhang, Kunshan; Li, Chun; Cui, Yaru; Lin, Haijun; Luo, Dandan; Wang, Junbang; Lin, Connie; Dai, Zachary; Zhu, Hongwen; Zhang, Jun; Liu, Jie; Liu, Hailiang; deVellis, Jean; Horvath, Steve; Sun, Yi Eve; Li, Siguang

    2015-05-21

    The scarcity of tissue-specific stem cells and the complexity of their surrounding environment have made molecular characterization of these cells particularly challenging. Through single-cell transcriptome and weighted gene co-expression network analysis (WGCNA), we uncovered molecular properties of CD133(+)/GFAP(-) ependymal (E) cells in the adult mouse forebrain neurogenic zone. Surprisingly, prominent hub genes of the gene network unique to ependymal CD133(+)/GFAP(-) quiescent cells were enriched for immune-responsive genes, as well as genes encoding receptors for angiogenic factors. Administration of vascular endothelial growth factor (VEGF) activated CD133(+) ependymal neural stem cells (NSCs), lining not only the lateral but also the fourth ventricles and, together with basic fibroblast growth factor (bFGF), elicited subsequent neural lineage differentiation and migration. This study revealed the existence of dormant ependymal NSCs throughout the ventricular surface of the CNS, as well as signals abundant after injury for their activation.

  19. Sorting drops and cells with acoustics: acoustic microfluidic fluorescence-activated cell sorter.

    PubMed

    Schmid, Lothar; Weitz, David A; Franke, Thomas

    2014-10-07

    We describe a versatile microfluidic fluorescence-activated cell sorter that uses acoustic actuation to sort cells or drops at ultra-high rates. Our acoustic sorter combines the advantages of traditional fluorescence-activated cell (FACS) and droplet sorting (FADS) and is applicable for a multitude of objects. We sort aqueous droplets, at rates as high as several kHz, into two or even more outlet channels. We can also sort cells directly from the medium without prior encapsulation into drops; we demonstrate this by sorting fluorescently labeled mouse melanoma cells in a single phase fluid. Our acoustic microfluidic FACS is compatible with standard cell sorting cytometers, yet, at the same time, enables a rich variety of more sophisticated applications.

  20. Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting.

    PubMed

    Liu, Ling; Cheung, Tom H; Charville, Gregory W; Rando, Thomas A

    2015-10-01

    The prospective isolation of purified stem cell populations has dramatically altered the field of stem cell biology, and it has been a major focus of research across tissues in different organisms. Muscle stem cells (MuSCs) are now among the most intensely studied stem cell populations in mammalian systems, and the prospective isolation of these cells has allowed cellular and molecular characterizations that were not dreamed of a decade ago. In this protocol, we describe how to isolate MuSCs from limb muscles of adult mice by fluorescence-activated cell sorting (FACS). We provide a detailed description of the physical and enzymatic dissociation of mononucleated cells from limb muscles, a procedure that is essential in order to maximize cell yield. We also describe a FACS-based method that is used subsequently to obtain highly pure populations of either quiescent or activated MuSCs (VCAM(+)CD31(-)CD45(-)Sca1(-)). The isolation process takes ∼5-6 h to complete. The protocol also allows for the isolation of endothelial cells, hematopoietic cells and mesenchymal stem cells from muscle tissue.

  1. Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma.

    PubMed

    Roy, Ishan; Boyle, Kathleen A; Vonderhaar, Emily P; Zimmerman, Noah P; Gorse, Egal; Mackinnon, A Craig; Hwang, Rosa F; Franco-Barraza, Janusz; Cukierman, Edna; Tsai, Susan; Evans, Douglas B; Dwinell, Michael B

    2017-03-01

    The mechanisms by which the extreme desmoplasia observed in pancreatic tumors develops remain unknown and its role in pancreatic cancer progression is unsettled. Chemokines have a key role in the recruitment of a wide variety of cell types in health and disease. Transcript and protein profile analyses of human and murine cell lines and human tissue specimens revealed a consistent elevation in the receptors CCR10 and CXCR6, as well as their respective ligands CCL28 and CXCL16. Elevated ligand expression was restricted to tumor cells, whereas receptors were in both epithelial and stromal cells. Consistent with its regulation by inflammatory cytokines, CCL28 and CCR10, but not CXCL16 or CXCR6, were upregulated in human pancreatitis tissues. Cytokine stimulation of pancreatic cancer cells increased CCL28 secretion in epithelial tumor cells but not an immortalized activated human pancreatic stellate cell line (HPSC). Stellate cells exhibited dose- and receptor-dependent chemotaxis in response to CCL28. This functional response was not linked to changes in activation status as CCL28 had little impact on alpha smooth muscle actin levels or extracellular matrix deposition or alignment. Co-culture assays revealed CCL28-dependent chemotaxis of HPSC toward cancer but not normal pancreatic epithelial cells, consistent with stromal cells being a functional target for the epithelial-derived chemokine. These data together implicate the chemokine CCL28 in the inflammation-mediated recruitment of cancer-associated stellate cells into the pancreatic cancer parenchyma.

  2. Regulation of B cell fate by chronic activity of the IgE B cell receptor

    PubMed Central

    Yang, Zhiyong; Robinson, Marcus J; Chen, Xiangjun; Smith, Geoffrey A; Taunton, Jack; Liu, Wanli; Allen, Christopher D C

    2016-01-01

    IgE can trigger potent allergic responses, yet the mechanisms regulating IgE production are poorly understood. Here we reveal that IgE+ B cells are constrained by chronic activity of the IgE B cell receptor (BCR). In the absence of cognate antigen, the IgE BCR promoted terminal differentiation of B cells into plasma cells (PCs) under cell culture conditions mimicking T cell help. This antigen-independent PC differentiation involved multiple IgE domains and Syk, CD19, BLNK, Btk, and IRF4. Disruption of BCR signaling in mice led to consistently exaggerated IgE+ germinal center (GC) B cell but variably increased PC responses. We were unable to confirm reports that the IgE BCR directly promoted intrinsic apoptosis. Instead, IgE+ GC B cells exhibited poor antigen presentation and prolonged cell cycles, suggesting reduced competition for T cell help. We propose that chronic BCR activity and access to T cell help play critical roles in regulating IgE responses. DOI: http://dx.doi.org/10.7554/eLife.21238.001 PMID:27935477

  3. Regulation of B cell fate by chronic activity of the IgE B cell receptor.

    PubMed

    Yang, Zhiyong; Robinson, Marcus J; Chen, Xiangjun; Smith, Geoffrey A; Taunton, Jack; Liu, Wanli; Allen, Christopher D C

    2016-12-09

    IgE can trigger potent allergic responses, yet the mechanisms regulating IgE production are poorly understood. Here we reveal that IgE(+) B cells are constrained by chronic activity of the IgE B cell receptor (BCR). In the absence of cognate antigen, the IgE BCR promoted terminal differentiation of B cells into plasma cells (PCs) under cell culture conditions mimicking T cell help. This antigen-independent PC differentiation involved multiple IgE domains and Syk, CD19, BLNK, Btk, and IRF4. Disruption of BCR signaling in mice led to consistently exaggerated IgE(+) germinal center (GC) B cell but variably increased PC responses. We were unable to confirm reports that the IgE BCR directly promoted intrinsic apoptosis. Instead, IgE(+) GC B cells exhibited poor antigen presentation and prolonged cell cycles, suggesting reduced competition for T cell help. We propose that chronic BCR activity and access to T cell help play critical roles in regulating IgE responses.

  4. Regulation of myeloid cells by activated T cells determines the efficacy of PD-1 blockade.

    PubMed

    Eissler, Nina; Mao, Yumeng; Brodin, David; Reuterswärd, Philippa; Andersson Svahn, Helene; Johnsen, John Inge; Kiessling, Rolf; Kogner, Per

    2016-01-01

    Removal of immuno-suppression has been reported to enhance antitumor immunity primed by checkpoint inhibitors. Although PD-1 blockade failed to control tumor growth in a transgenic murine neuroblastoma model, concurrent inhibition of colony stimulating factor 1 receptor (CSF-1R) by BLZ945 reprogrammed suppressive myeloid cells and significantly enhanced therapeutic effects. Microarray analysis of tumor tissues identified a significant increase of T-cell infiltration guided by myeloid cell-derived chemokines CXCL9, 10, and 11. Blocking the responsible chemokine receptor CXCR3 hampered T-cell infiltration and reduced antitumor efficacy of the combination therapy. Multivariate analysis of 59 immune-cell parameters in tumors and spleens detected the correlation between PD-L1-expressing myeloid cells and tumor burden. In vitro, anti-PD-1 antibody Nivolumab in combination with BLZ945 increased the activation of primary human T and NK cells. Importantly, we revealed a previously uncharacterized pathway, in which T cells secreted M-CSF upon PD-1 blockade, leading to enhanced suppressive capacity of monocytes by upregulation of PD-L1 and purinergic enzymes. In multiple datasets of neuroblastoma patients, gene expression of CD73 correlated strongly with myeloid cell markers CD163 and CSF-1R in neuroblastoma tumors, and associated with worse survival in high-risk patients. Altogether, our data reveal the dual role of activated T cells on myeloid cell functions and provide a rationale for the combination therapy of anti-PD-1 antibody with CSF-1R inhibitor.

  5. XIAP reverses various functional activities of FRNK in endothelial cells

    SciTech Connect

    Ahn, Sunyoung; Kim, Hyun Jeong; Chi, Sung-Gil; Park, Heonyong

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer FRNK domain is recruited into focal adhesion (FA), controlling endothelial cell adhesion. Black-Right-Pointing-Pointer XIAP binds the FRNK domain of FAK. Black-Right-Pointing-Pointer XIAP inhibits recruitment of FRNK into Fas and FRNK-promoted cell adhesion. Black-Right-Pointing-Pointer XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK. -- Abstract: In endothelial cells, focal adhesion kinase (FAK) regulates cell proliferation, migration, adhesion, and shear-stimulated activation of MAPK. We recently found that FAK is recruited into focal adhesion (FA) sites through interactions with XIAP (X-chromosome linked inhibitor of apoptosis protein) and activated by Src kinase in response to shear stress. In this study, we examined which domain(s) of FAK is(are) important for various vascular functions such as FA recruiting, XIAP-binding and shear stress-stimulated ERK activation. Through a series of experiments, we determined that the FRNK domain is recruited into FA sites and promotes endothelial cell adhesion. Interestingly, XIAP knockdown was shown to reduce FA recruitment of FRNK and the cell adhesive effect of FRNK. In addition, we found that XIAP interacts with FRNK, suggesting cross-talk between XIAP and FRNK. We also demonstrated that FRNK inhibits endothelial cell migration and shear-stimulated ERK activation. These inhibitory effects of FRNK were reversed by XIAP knockdown. Taken together, we can conclude that XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK.

  6. Murraya koenigii leaf extract inhibits proteasome activity and induces cell death in breast cancer cells

    PubMed Central

    2013-01-01

    Background Inhibition of the proteolytic activity of 26S proteasome, the protein-degrading machine, is now considered a novel and promising approach for cancer therapy. Interestingly, proteasome inhibitors have been demonstrated to selectively kill cancer cells and also enhance the sensitivity of tumor cells to chemotherapeutic agents. Recently, polyphenols/flavonoids have been reported to inhibit proteasome activity. Murraya koenigii Spreng, a medicinally important herb of Indian origin, has been used for centuries in the Ayurvedic system of medicine. Here we show that Murraya koenigii leaves (curry leaves), a rich source of polyphenols, inhibit the proteolytic activity of the cancer cell proteasome, and cause cell death. Methods Hydro-methanolic extract of curry leaves (CLE) was prepared and its total phenolic content [TPC] determined by, the Folin-Ciocalteau’s method. Two human breast carcinoma cell lines: MCF-7 and MDA-MB-231 and a normal human lung fibroblast cell line, WI-38 were used for the studies. Cytotoxicity of the CLE was assessed by the MTT assay. We studied the effect of CLE on growth kinetics using colony formation assay. Growth arrest was assessed by cell cycle analysis and apoptosis by Annexin-V binding using flow cytometry. Inhibition of the endogenous 26S proteasome was studied in intact cells and cell extracts using substrates specific to 20S proteasomal enzymes. Results CLE decreased cell viability and altered the growth kinetics in both the breast cancer cell lines in a dose-dependent manner. It showed a significant arrest of cells in the S phase albeit in cancer cells only. Annexin V binding data suggests that cell death was via the apoptotic pathway in both the cancer cell lines. CLE treatment significantly decreased the activity of the 26S proteasome in the cancer but not normal cells. Conclusions Our study suggests M. koenigii leaves to be a potent source of proteasome inhibitors that lead to cancer cell death. Therefore, identification

  7. Isolation of cell type-specific apoptotic bodies by fluorescence-activated cell sorting

    PubMed Central

    Atkin-Smith, Georgia K.; Paone, Stephanie; Zanker, Damien J.; Duan, Mubing; Phan, Than K.; Chen, Weisan; Hulett, Mark D.; Poon, Ivan K. H.

    2017-01-01

    Apoptotic bodies (ApoBDs) are membrane-bound extracellular vesicles that can mediate intercellular communication in physiological and pathological settings. By combining recently developed analytical strategies with fluorescence-activated cell sorting (FACS), we have developed a method that enables the isolation of ApoBDs from cultured cells to 99% purity. In addition, this approach also enables the identification and isolation of cell type-specific ApoBDs from tissue, bodily fluid and blood-derived samples. PMID:28057919

  8. Photodynamic activation as a molecular switch to promote osteoblast cell differentiation via AP-1 activation

    PubMed Central

    Kushibiki, Toshihiro; Tu, Yupeng; Abu-Yousif, Adnan O.; Hasan, Tayyaba

    2015-01-01

    In photodynamic therapy (PDT), cells are impregnated with a photosensitizing agent that is activated by light irradiation, thereby photochemically generating reactive oxygen species (ROS). The amounts of ROS produced depends on the PDT dose and the nature of the photosensitizer. Although high levels of ROS are cytotoxic, at physiological levels they play a key role as second messengers in cellular signaling pathways, pluripotency, and differentiation of stem cells. To investigate further the use of photochemically triggered manipulation of such pathways, we exposed mouse osteoblast precursor cells and rat primary mesenchymal stromal cells to low-dose PDT. Our results demonstrate that low-dose PDT can promote osteoblast differentiation via the activation of activator protein-1 (AP-1). Although PDT has been used primarily as an anti-cancer therapy, the use of light as a photochemical “molecular switch” to promote differentiation should expand the utility of this method in basic research and clinical applications. PMID:26279470

  9. HIV-1 latency in actively dividing human T cell lines

    PubMed Central

    Jeeninga, Rienk E; Westerhout, Ellen M; van Gerven, Marja L; Berkhout, Ben

    2008-01-01

    Background Eradication of HIV-1 from an infected individual cannot be achieved by current drug regimens. Viral reservoirs established early during the infection remain unaffected by anti-retroviral therapy and are able to replenish systemic infection upon interruption of the treatment. Therapeutic targeting of viral latency will require a better understanding of the basic mechanisms underlying the establishment and long-term maintenance of HIV-1 in resting memory CD4 T cells, the most prominent reservoir of transcriptional silent provirus. However, the molecular mechanisms that permit long-term transcriptional control of proviral gene expression in these cells are still not well understood. Exploring the molecular details of viral latency will provide new insights for eventual future therapeutics that aim at viral eradication. Results We set out to develop a new in vitro HIV-1 latency model system using the doxycycline (dox)-inducible HIV-rtTA variant. Stable cell clones were generated with a silent HIV-1 provirus, which can subsequently be activated by dox-addition. Surprisingly, only a minority of the cells was able to induce viral gene expression and a spreading infection, eventhough these experiments were performed with the actively dividing SupT1 T cell line. These latent proviruses are responsive to TNFα treatment and alteration of the DNA methylation status with 5-Azacytidine or genistein, but not responsive to the regular T cell activators PMA and IL2. Follow-up experiments in several T cell lines and with wild-type HIV-1 support these findings. Conclusion We describe the development of a new in vitro model for HIV-1 latency and discuss the advantages of this system. The data suggest that HIV-1 proviral latency is not restricted to resting T cells, but rather an intrinsic property of the virus. PMID:18439275

  10. DOCK2 regulates cell proliferation through Rac and ERK activation in B cell lymphoma

    SciTech Connect

    Wang, Lei; Nishihara, Hiroshi; Kimura, Taichi; Kato, Yasutaka; Tanino, Mishie; Nishio, Mitsufumi; Obara, Masato; Endo, Tomoyuki; Koike, Takao; Tanaka, Shinya

    2010-04-23

    DOCK2; a member of the CDM protein family, regulates cell motility and cytokine production through the activation of Rac in mammalian hematopoietic cells and plays a pivotal role in the modulation of the immune system. Here we demonstrated the alternative function of DOCK2 in hematopoietic tumor cells, especially in terms of its association with the tumor progression. Immunostaining for DOCK2 in 20 cases of human B cell lymphoma tissue specimens including diffuse large B cell lymphoma and follicular lymphoma revealed the prominent expression of DOCK2 in all of the lymphoma cells. DOCK2-knockdown (KD) of the B cell lymphoma cell lines, Ramos and Raji, using the lentiviral shRNA system presented decreased cell proliferation compared to the control cells. Furthermore, the tumor formation of DOCK2-KD Ramos cell in nude mice was significantly abrogated. Western blotting analysis and pull-down assay using GST-PAK-RBD kimeric protein suggested the presence of DOCK2-Rac-ERK pathway regulating the cell proliferation of these lymphoma cells. This is the first report to clarify the prominent role of DOCK2 in hematopoietic malignancy.

  11. Role of the Bp35 cell surface polypeptide in human B-cell activation.

    PubMed Central

    Clark, E A; Shu, G; Ledbetter, J A

    1985-01-01

    A 35-kDa polypeptide, Bp35, expressed on the surface of all B cells, plays a role in B-cell activation. Monoclonal antibodies to Bp35 stimulate human tonsillar B cells to proliferate. The activation induced by anti-Bp35 is similar to anti-Ig-mediated in several ways: the activation does not require T cells but is augmented by T-cell-derived allogeneic factors; monovalent Fab fragments to Bp35 do not trigger proliferation but instead block activation by whole antibody, indicating that cross-linking is required; and induction by anti-Bp35, like the induction by anti-Ig, is inhibited by monoclonal anti-IgM via an Fc domain-dependent mechanism. However, several features of anti-Bp35-mediated proliferation are clearly different from activation by anti-Ig: anti-Bp35 monoclonal antibodies do not require attachment to beads to function, the proliferation induced by anti-Bp35 and anti-Ig is additive, and Fab fragments of anti-Bp35 augment proliferation induced by anti-Ig. Models for the possible function of the Bp35 polypeptide as either a "bridge" or a "second signal" with surface Ig in B-cell activation are discussed. PMID:3872456

  12. Identification of a novel gene expressed in activated natural killer cells and T cells

    SciTech Connect

    Dahl, C.A.; Schall, R.P.; He, H.; Cairns, J.S. )

    1992-01-15

    The authors have isolated a cDNA clone from a human activated NK cell-derived cDNA library that identifies a transcript [NK4] that is selectively expressed in lymphocytes. The expression of this transcript is increased after activation of T cells by mitogens or activation of NK cells by IL-2 (lymphokine-activated killer cells). The transcript levels demonstrated by Northern blot analysis increase by 12 h after activation, remain high for at least 48 h, and require protein synthesis for expression. Southern blot analysis of B lymphoblastoid lines derived from 18 unrelated individuals reveal variable banding patterns suggestive of polymorphism within the NK4 gene. No homology was found between the sequence of the coding region of this transcript and any sequences in the GenBank data base. Sequence homology to the U1 small nuclear RNA was found within the 3[prime] untranslated region immediately upstream of the site of polyadenylation, suggesting a possible role for U1 in the polyadenylation process. Sequence analysis indicates the transcript would encode a protein having a mass of 27 kDa. The presence of a signal sequence and lack of a transmembrane region suggests that the protein is secreted. In addition, the protein contains an RGD sequence that may be involved in cellular adhesion. This transcript appears to encode a novel product common to the activation pathways of both NK cells and T cells. 50 refs., 8 figs.

  13. Dehydroascorbate uptake activity correlates with cell growth and cell division of tobacco bright yellow-2 cell cultures.

    PubMed

    Horemans, Nele; Potters, Geert; De Wilde, Leen; Caubergs, Roland J

    2003-09-01

    Recently, ascorbate (ASC) concentration and the activity of a number of enzymes from the ASC metabolism have been proven to correlate with differences in growth or cell cycle progression. Here, a possible correlation between growth and the activity of a plasma membrane dehydroascorbate (DHA) transporter was investigated. Protoplasts were isolated from a tobacco (Nicotiana tabacum) Bright Yellow-2 cell culture at different intervals after inoculation and the activity of DHA transport was tested with (14)C-labeled ASC. Ferricyanide (1 mM) or dithiothreitol (1 mM) was included in the test to keep the external (14)C-ASC in its oxidized respectively reduced form. Differential uptake activity was observed, correlating with growth phases of the cell culture. Uptake of DHA in cells showed a peak in exponential growth phase, whereas uptake in the presence of dithiothreitol did not. The enhanced DHA uptake was not due to higher endogenous ASC levels that are normally present in exponential phase because preloading of protoplasts of different ages did not affect DHA uptake. Preloading was achieved by incubating cells before protoplastation for 4 h in a medium supplemented with 1 mM DHA. In addition to testing cells at different growth phases, uptake of DHA into the cells was also followed during the cell cycle. An increase in uptake activity was observed during M phase and the M/G1 transition. These experiments are the first to show that DHA transport activity into plant cells differs with cell growth. The relevance of the data to the action of DHA and ASC in cell growth will be discussed.

  14. Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

    PubMed

    Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

    1995-02-10

    Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain.

  15. Phosphatidylinositol turnover is associated with human natural killer cell activation by tumor target cells

    SciTech Connect

    Steele, T.A.; Brahmi, Z.

    1986-03-01

    Natural Killer (NK) cell activity has been shown to be a binding-dependent event leading to the destruction of various targets. This suggests a possible role for plasma membrane phospholipid turnover in coupling a receptor-mediated binding event with transduction of a intracellular signal to result in the activation of the effector cell. Currently, phosphatidylinositol (PI) turnover is implicated in several immune cell systems. Therefore, in this study, the authors examined phospholipid turnover in human NK cells upon exposure to a sensitive (K562) and a resistant (YAC-1) target cell (TC). NK cell membrane phospholipids were labelled with Phosphorus-32 (/sup 32/P) and, following stimulation, were extracted and run on silica gel thin-layer chromatography. Labelled phospholipids were visualized by autoradiography then scraped and counted in a liquid scintillation counter. A 2.5 fold increase in label incorporation into PI relative to controls was shown to occur when NK cells were stimulated by K562 for 2 hours. In contrast, no increased labelling of PI relative to controls was noted when NK cells were stimulated by YAC-1 for the same period of time. No change in incorporation of /sup 32/P into phosphatidylcholine or phosphatidylethanolamine occurred in either set of conditions. These results suggest that PI turnover may be an early activation event in NK cells following binding of K562.

  16. A bioluminescent caspase-1 activity assay rapidly monitors inflammasome activation in cells.

    PubMed

    O'Brien, Martha; Moehring, Danielle; Muñoz-Planillo, Raúl; Núñez, Gabriel; Callaway, Justin; Ting, Jenny; Scurria, Mike; Ugo, Tim; Bernad, Laurent; Cali, James; Lazar, Dan

    2017-03-04

    Inflammasomes are protein complexes induced by diverse inflammatory stimuli that activate caspase-1, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent, plate-based assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized lytic reagent added directly to cultured cells. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor in the lytic reagent and by use of a caspase-1 inhibitor to confirm activity. This approach enables a specific and rapid determination of caspase-1 activation. Caspase-1 activity is stable in the reagent thereby providing assay convenience and flexibility. Using this assay system, caspase-1 activation has been determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, gramicidin, MSU, R848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages (BMDMs) from mice, as well as in human primary monocytes. Caspase-1 activity was not detected in treated BMDMs derived from Casp1(-/-) mice, further confirming the specificity of the assay. Caspase-1 activity can be measured directly in cultured cells using the lytic reagent, or caspase-1 activity released into medium can be monitored by assay of transferred supernatant. The caspase-1 assay can be multiplexed with other assays to monitor additional parameters from the same cells, such as IL-1β release or cell death. The caspase-1 assay in combination with a sensitive real-time monitor of cell death allows one to accurately establish pyroptosis. This assay system provides a rapid, convenient, and flexible method to specifically and quantitatively monitor caspase-1 activation in cells in a plate-based format. This will allow a more efficient and effective

  17. Nanomapping of CD1d-glycolipid complexes on THP1 cells by using simultaneous topography and recognition imaging.

    PubMed

    Duman, Memed; Chtcheglova, Lilia A; Zhu, Rong; Bozna, Bianca L; Polzella, Paolo; Cerundolo, Vicenzo; Hinterdorfer, Peter

    2013-09-01

    CD1d molecule, a monomorphic major histocompatibility complex class I-like molecule, presents different types of glycolipids to invariant natural killer T (iNKT) cells that play an important role in immunity to infection and tumors, as well as in regulating autoimmunity. Here, we present simultaneous topography and recognition imaging (TREC) analysis to detect density, distribution and localization of single CD1d molecules on THP1 cells that were loaded with different glycolipids. TREC was conducted using magnetically coated atomic force microscopy tips functionalized with a biotinylated iNKT cell receptor (TCR). The recognition map revealed binding sites visible as dark spots, resulting from oscillation amplitude reduction during specific binding between iNKT TCR and the CD1d-glycolipid complex. THP1 cells were pulsed with three different glycolipids (α-GalCer, C20 and OCH12) for 4 and 16 hr. Whereas CD1d-α-GalCer and CD1d-C20:2 complexes on cellular membrane formed smaller microdomains up to ~10 000 nm(2) (dimension area), OCH12 loaded CD1d complexes presented larger clusters with a dimension up to ~30 000 nm(2). Moreover, the smallest size of recognition spots was about 25 nm, corresponding to a single CD1d binding site. TREC successfully revealed the distribution and localization of CD1d-glycolipid complexes on THP1 cell with single molecule resolution under physiological conditions.

  18. Increased Intraepithelial Vα24 Invariant NKT Cells in the Celiac Duodenum

    PubMed Central

    Montalvillo, Enrique; Bernardo, David; Martínez-Abad, Beatriz; Allegretti, Yessica; Fernández-Salazar, Luis; Calvo, Carmen; Chirdo, Fernando G.; Garrote, José A.; Arranz, Eduardo

    2015-01-01

    Celiac Disease (CD) is an interferon (IFN)γ-mediated duodenal hypersensitivity to wheat gluten occurring in genetically predisposed individuals. Gluten-free diet (GFD) leads to a complete remission of the disease. Vα24-restricted invariant NKT (iNKT) cells are important to maintain immune homeostasis in the gut mucosa because of their unique capacity to rapidly produce large quantities of both T-helper (Th)1 and Th2 cytokines upon stimulation. We studied the presence of these cells in the CD duodenum. Duodenal biopsies were obtained from 45 untreated-CD patients (uCD), 15 Gluten Free Diet-CD patients (GFD-CD), 44 non-inflamed non-CD controls (C-controls) and 15 inflamed non-CD controls (I-controls). Two populations from Spain and Argentina were recruited. Messenger RNA (mRNA) expression of Vα24-Jα18 (invariant TCRα chain of human iNKT cells), IFNγ and intracellular transcription factor Forkhead Box P3 (Foxp3), and flow cytometry intraepithelial lymphocyte (IEL) profile were determined. Both uCD and GFD-CD patients had higher Vα24-Jα18 mRNA levels than non-CD controls (I and C-controls). The expression of Vα24-Jα18 correlated with Marsh score for the severity of mucosal lesion and also with increased mRNA IFNγ levels. uCD and GFD-CD patients had decreased mRNA expression of FoxP3 but increased expression of Vα24-Jα18, which revealed a CD-like molecular profile. Increased numbers of iNKT cells were confirmed by flow cytometry within the intraepithelial lymphocyte compartment of uCD and GFD-CD patients and correlated with Vα24-Jα18 mRNA expression. In conclusion, we have found an increased number of iNKT cells in the duodenum from both uCD and GFD-CD patients, irrespective of the mucosal status. A CD-like molecular profile, defined by an increased mRNA expression of Vα24-Jα18 together with a decreased expression of FoxP3, may represent a pro-inflammatory signature of the CD duodenum. PMID:26529008

  19. A Micro Fluorescent Activated Cell Sorter for Astrobiology Applications

    NASA Technical Reports Server (NTRS)

    Platt, Donald W.; Hoover, Richard B.

    2009-01-01

    A micro-scale Fluorescent Activated Cell Sorter (microFACS) for astrobiology applications is under development. This device is designed to have a footprint of 7 cm x 7 cm x 4 cm and allow live-dead counts and sorting of cells that have fluorescent characteristics from staining. The FACS system takes advantage of microfluidics to create a cell sorter that can fit in the palm of the hand. A micron-scale channel allows cells to pass by a blue diode which causes emission of marker-expressed cells which are detected by a filtered photodetector. A small microcontroller then counts cells and operates high speed valves to select which chamber the cell is collected in (a collection chamber or a waste chamber). Cells with the expressed characteristic will be collected in the collection chamber. This system has been built and is currently being tested. We are also designing a system with integrated MEMS-based pumps and valves for a small and compact unit to fly on small satellite-based biology experiments.

  20. CNR-TAE`s activity on fuel cells

    SciTech Connect

    Staiti, P.; Freni, S.; Passalacqua, E.; Antonucci, V.

    1997-07-01

    The recognition of the advantages and efficiencies implicit in an economy based upon the electrochemical conversion of fuels has led to intensive efforts toward the development of the fuel cells technology. On phosphoric acid fuel cells (PAFC), the CNR-TAE owns a full capability in PAFC technology and has built and tested 1 kW power plant in an ENEA supported program. On molten carbonate fuel cells (MCFC), the CNR-TAE has matured a sound experience on the modeling of energy balances of MCFC with external or internal reforming, screening design and testing of reforming catalysts, on mechanisms of components aging, catalysts formulation and innovative methods to control catalyst poisoning by means of porous ceramic membranes. In solid oxide fuel cells (SOFC) research, a comparison between steam internal and external reforming, exhaust gas recycling reforming and use of partial oxidation has been performed. Basic researches on the mechanism of conduction in solids and search for novel electrolytes are on course. In the field of fuel cells operating at low temperatures, the activity is addressed to the development of low Pt loading electrodes for polymer electrolyte fuel cells (PEFC) and development of ternary catalysts supported on carbon black for electrochemical oxidation of methanol in direct methanol fuel cells (DMFC). Further research is on the fuel cell utilizing a new type of electrolyte; in this field, an heteropolyacid lab-scale monocell has been realized and successful tested.

  1. Contact dependent suppression of CD4 T cell activation and proliferation by B cells activated through IgD cross-linking.

    PubMed

    Preciado-Llanes, Lorena; Wing, James B; Foster, Rachel A; Carlring, Jennifer; Lees, Andrew; Read, Robert C; Heath, Andrew W

    2014-09-20

    Although the co-stimulatory interaction between B and T cells is well defined, recent evidence suggests that B cells also have a regulatory role. Here, we show that B cells activated using anti-IgD conjugated to dextran (α-δ-dex) directly inhibit TCR-induced CD4 T cell activation, proliferation and cytokine production. This effect was observed in CD4 T cells activated both with and without CD28 co-stimulation. T cell viability was unaffected, and the T cell suppressive effect was mediated by contact with IgD activated purified B cells and not by IL-10 or other soluble factors. This is the first evidence of IgD activated B cells mediating inhibition of activation and proliferation of CD4 T cells in humans. This article is protected by copyright. All rights reserved.

  2. Activation of resting human B cells by helper T-cell clone supernatant: characterization of a human B-cell-activating factor.

    PubMed Central

    Diu, A; Gougeon, M L; Moreau, J L; Reinherz, E L; Thèze, J

    1987-01-01

    The effects of helper T-cell clone supernatants on resting human B cells were investigated. Four different helper T-cell clones (two T4+ and two T8+) were stimulated by anti-T3 monoclonal antibodies on Sepharose beads or anti-T11(2) plus anti-T11(3) monoclonal antibodies. The supernatants from these activated clones induced the proliferation of highly purified resting B lymphocytes from the peripheral blood. The B cells exhibited a cell size and a surface-antigen pattern (4F2 antigen and transferrin receptor) of phase G0 B cells, and they were functionally resting. In response to T-cell supernatants a large fraction of the B cells enlarged and expressed 4F2 antigens and transferrin receptors. In gel filtration, the corresponding activity migrated with an apparent Mr of 12,000-15,000. Our findings strongly support the existence of a human B-cell-activating factor acting on resting B cells and causing them to enter phase G1 of the cell cycle. PMID:2962196

  3. Lysine methylation represses p53 activity in teratocarcinoma cancer cells

    PubMed Central

    Zhu, Jiajun; Dou, Zhixun; Sammons, Morgan A.; Levine, Arnold J.; Berger, Shelley L.

    2016-01-01

    TP53 (which encodes the p53 protein) is the most frequently mutated gene among all human cancers, whereas tumors that retain the wild-type TP53 gene often use alternative mechanisms to repress the p53 tumor-suppressive function. Testicular teratocarcinoma cells rarely contain mutations in TP53, yet the transcriptional activity of wild-type p53 is compromised, despite its high expression level. Here we report that in the teratocarcinoma cell line NTera2, p53 is subject to lysine methylation at its carboxyl terminus, which has been shown to repress p53’s transcriptional activity. We show that reduction of the cognate methyltransferases reactivates p53 and promotes differentiation of the NTera2 cells. Furthermore, reconstitution of methylation-deficient p53 mutants into p53-depleted NTera2 cells results in elevated expression of p53 downstream targets and precocious loss of pluripotent gene expression compared with re-expression of wild-type p53. Our results provide evidence that lysine methylation of endogenous wild-type p53 represses its activity in cancer cells and suggest new therapeutic possibilities of targeting testicular teratocarcinoma. PMID:27535933

  4. UV-inactivated HSV-1 potently activates NK cell killing of leukemic cells.

    PubMed

    Samudio, Ismael; Rezvani, Katayoun; Shaim, Hila; Hofs, Elyse; Ngom, Mor; Bu, Luke; Liu, Guoyu; Lee, Jason T C; Imren, Suzan; Lam, Vivian; Poon, Grace F T; Ghaedi, Maryam; Takei, Fumio; Humphries, Keith; Jia, William; Krystal, Gerald

    2016-05-26

    Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light-inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation-dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell-depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML.

  5. Leptin deficiency in vivo enhances the ability of splenic dendritic cells to activate T cells

    PubMed Central

    Ramirez, Oscar

    2014-01-01

    Leptin is a pleiotropic adipokine that is critical for regulating food intake and energy expenditure and also participates in functions of the immune system, including those of antigen-presenting cells. Here, we assess the effect of leptin deficiency on the function splenic dendritic cells (sDC). sDC from leptin-deficient mice (Lepob) were evaluated ex vivo for phenotype, ability to respond to inflammatory stimuli, to acquire and process antigens and to activate T cells. The data show that Lepob sDC express activation markers similar to controls and respond similarly to LPS activation or anti-CD40 cross-linking. In addition, antigen acquisition and processing by Lepob sDC was similar to controls. However, Lepob sDC elicited higher production of IFN-γ in mixed lymphocyte reactions and increased production of IL-2 by antigen-specific T-cell hybridoma relative to controls. To assess Lepob sDC activation of T cells in vivo, Lepob and control mice were infected systemically with Mycobacterium avium. Lepob mice were significantly better at neutralizing the infection as measured by splenic bacterial load over time. This was mirrored with an increased percentage of activated T cells in M. avium-infected Lepob mice. Thus, although no changes were detected in sDC phenotype, activation, antigen processing or presentation, these DC surprisingly presented an enhanced ability to activate T cells ex vivo and in vivo. These data demonstrate that leptin can modulate DC function and suggest that leptin may dampen T-cell responsiveness in the physiological setting. PMID:24966213

  6. CD39 modulates endothelial cell activation and apoptosis.

    PubMed Central

    Goepfert, C.; Imai, M.; Brouard, S.; Csizmadia, E.; Kaczmarek, E.; Robson, S. C.

    2000-01-01

    BACKGROUND: CD39 is the dominant vascular nucleoside triphosphate diphosphohydrolase (NTPDase) that exerts major effects on platelet reactivity by the regulated hydrolysis of extracellular adenine nucleotides. The effects of NTPDases on endothelial cell (EC) activation and apoptosis remain unexplored. MATERIAL AND METHODS: Recombinant replication-deficient adenoviruses were constructed with human CD39 cDNA (rAdCD39) or the bacterial beta-galactosidase (rAdbetagal). RESULTS: Intact human umbilical vein EC cultures infected with rAdCD39 had substantial and stable increases in NTPDase biochemical activity (14.50 +/- 3.50 Pi nmole/well/min), when contrasted with noninfected cells (0.95 +/- 0.002) and rAdbetagal infected cells (1.01 +/- 0.02; p<0.005). Increased NTPDase activity efficiently inhibited immediate type 2Y purinergic receptor (P2Y)-mediated EC activation responses viz. von Willebrand factor secretion in response to extracellular ATP. In addition, CD39 up-regulation blocked ATP-induced translocation of the transcription nuclear factor (NF)-kappaB to the cell nucleus, and abrogated transcription of mRNA encoding E-selectin, and consequent protein synthesis. CD39 also decreased the extent of apoptosis triggered by putative type-2X purinergic (P2X7) receptors in response to high concentrations of extracellular ATP in vitro. CONCLUSION: These properties of CD39 indicate primary vascular protective effects with potential therapeutic applications. PMID:10997340

  7. Prodigiosin Induces Autolysins in Actively Grown Bacillus subtilis Cells

    PubMed Central

    Danevčič, Tjaša; Borić Vezjak, Maja; Tabor, Maja; Zorec, Maša; Stopar, David

    2016-01-01

    Prodigiosin produced by marine bacterium Vibrio ruber DSM 14379 exhibits a potent antimicrobial activity against a broad range of Gram positive and Gram negative bacteria. The mechanism of prodigiosin antimicrobial action, however, is not known. In this work, the effect of prodigiosin on Bacillus subtilis growth, cell membrane leakage, and induction of autolysins was studied. Treating B. subtilis with prodigiosin resulted in rapid decline of optical density and increased cell membrane leakage measured by β-galactosidase activity. Cell lysis was initiated immediately after treatment with prodigiosin in the middle exponential phase and was completed within 2 h. Lytic activity of prodigiosin in mutant strains with impaired autolysin genes lytABCD decreased for 80% compared to the wild type strain, while in lytABCDEF mutant strain prodigiosin had no bacteriolytic but only bacteriostatic effect. Fast prodigiosin lytic activity on individual B. subtilis cells was confirmed by a modified comet assay. The results indicate that prodigiosin autolysin induction in B. subtilis is growth phase dependent. PMID:26858704

  8. ACTIVATION OF AP-1 IN UROTSA CELLS BY METHYLATED ARSENICALS

    EPA Science Inventory

    ACTIVATION OF AP-1 IN UROTSA CELLS BY METHYLATED TRIVALENT ARSENICALS. Z Drobna1, I Jaspers2, D J Thomas3 and M Styblo1. 1Department of Pediatrics; 2Center for Environmental Medicine and Lung Biology, University of North Carolina at Chapel Hill, NC, USA; 3US EPA, RTP, NC, USA.

  9. Stra13 regulates satellite cell activation by antagonizing Notch signaling

    PubMed Central

    Sun, Hong; Li, Li; Vercherat, Cécile; Gulbagci, Neriman Tuba; Acharjee, Sujata; Li, Jiali; Chung, Teng-Kai; Thin, Tin Htwe; Taneja, Reshma

    2007-01-01

    Satellite cells play a critical role in skeletal muscle regeneration in response to injury. Notch signaling is vital for satellite cell activation and myogenic precursor cell expansion but inhibits myogenic differentiation. Thus, precise spatial and temporal regulation of Notch activity is necessary for efficient muscle regeneration. We report that the basic helix-loop-helix transcription factor Stra13 modulates Notch signaling in regenerating muscle. Upon injury, Stra13−/− mice exhibit increased cellular proliferation, elevated Notch signaling, a striking regeneration defect characterized by degenerated myotubes, increased mononuclear cells, and fibrosis. Stra13−/− primary myoblasts also exhibit enhanced Notch activity, increased proliferation, and defective differentiation. Inhibition of Notch signaling ex vivo and in vivo ameliorates the phenotype of Stra13−/− mutants. We demonstrate in vitro that Stra13 antagonizes Notch activity and reverses the Notch-imposed inhibition of myogenesis. Thus, Stra13 plays an important role in postnatal myogenesis by attenuating Notch signaling to reduce myoblast proliferation and promote myogenic differentiation. PMID:17502421

  10. Decorin binds myostatin and modulates its activity to muscle cells

    SciTech Connect

    Miura, Takayuki; Kishioka, Yasuhiro; Wakamatsu, Jun-ichi; Hattori, Akihito; Hennebry, Alex; Berry, Carole J.; Sharma, Mridula; Kambadur, Ravi; Nishimura, Takanori . E-mail: nishi@anim.agr.hokudai.ac.jp

    2006-02-10

    Myostatin, a member of TGF-{beta} superfamily of growth factors, acts as a negative regulator of skeletal muscle mass. The mechanism whereby myostatin controls the proliferation and differentiation of myogenic cells is mostly clarified. However, the regulation of myostatin activity to myogenic cells after its secretion in the extracellular matrix (ECM) is still unknown. Decorin, a small leucine-rich proteoglycan, binds TGF-{beta} and regulates its activity in the ECM. Thus, we hypothesized that decorin could also bind to myostatin and participate in modulation of its activity to myogenic cells. In order to test the hypothesis, we investigated the interaction between myostatin and decorin by surface plasmon assay. Decorin interacted with mature myostatin in the presence of concentrations of Zn{sup 2+} greater than 10 {mu}M, but not in the absence of Zn{sup 2+}. Kinetic analysis with a 1:1 binding model resulted in dissociation constants (K {sub D}) of 2.02 x 10{sup -8} M and 9.36 x 10{sup -9} M for decorin and the core protein of decorin, respectively. Removal of the glycosaminoglycan chain by chondroitinase ABC digestion did not affect binding, suggesting that decorin could bind to myostatin with its core protein. Furthermore, we demonstrated that immobilized decorin could rescue the inhibitory effect of myostatin on myoblast proliferation in vitro. These results suggest that decorin could trap myostatin and modulate its activity to myogenic cells in the ECM.

  11. Clonal mast cell activation syndrome with anaphylaxis to sulfites.

    PubMed

    Cifuentes, Liliana; Ring, Johannes; Brockow, Knut

    2013-01-01

    Sulfites are rarely suspected as causative agents of immediate-type hypersensitivity. We report on a 49-year-old male patient who developed recurrent severe hypotension after food ingestion. A diagnosis of monoclonal mast cell activation syndrome was established. In the double-blind, placebo-controlled food challenge, the patient reacted to potassium metabisulfite with anaphylaxis.

  12. Active cells for redundant and configurable articulated structures

    NASA Astrophysics Data System (ADS)

    Swensen, John P.; Nawroj, Ahsan I.; Pounds, Paul E. I.; Dollar, Aaron M.

    2014-10-01

    The proposed research effort explores the development of active cells—simple contractile electro-mechanical units that can be used as the material basis for larger articulable structures. Each cell, which might be considered a ‘muscle unit,’ consists of a contractile Nitinol Shape Memory Alloy (SMA) core with conductive terminals. Large numbers of these cells might be combined and externally powered to change phase, contracting to either articulate with a large strain or increase the stiffness of the ensemble, depending on the cell design. Unlike traditional work in modular robotics, the approach presented here focuses on cells that have a simplistic design and function, are inexpensive to fabricate, and are eventually scalable to sub-millimeter sizes, working toward our vision of articulated and robotic structures that can be custom-fabricated from large numbers of general cell units, similar to biological structures. In this paper, we present the design of the active cells and demonstrate their usage with three articulated structures built with them.

  13. Suppressor cell activity in a proliferative disorder of T lymphocytes.

    PubMed

    Kupa, A; Thomas, M E; Moore, H; Bradley, J; Zola, H; Hooper, M; Harding, P

    1981-06-01

    We report details of the immunological profile of a patient with the candidiasis endocrinopathy syndrome who has developed T-type chronic lymphocytic leukaemia. The patient is anergic to a panel of delayed hypersensitivity skin tests, and has poor in vitro mitogenic responses, but B cell function in vivo is not impaired. Subsequent functional studies have revealed that cells from the patient have a significant suppressive effect in coculture (P less than 0.05) on the responses of healthy donor lymphocytes (NR) to the mitogen phytohaemagglutinin (PHA). A degree of selectivity for the suppressive effect is suggested by the lack of similar effects on coculture responses to the mitogens concanavalin A (Con A) and pokeweed mitogen (PWM). Mitomycin C treatment of the patient's cells reduced their suppressive activity but significant suppression was still observed in the majority of PHA cocultures. The suppressor activity required the presence of the patient's cells in cocultures, as no suppression was observed when the patient's serum or cell culture supernatant were included instead of the patient's cells in NR cultures.

  14. Multi-neuronal activity and functional connectivity in cell assemblies.

    PubMed

    Roudi, Yasser; Dunn, Benjamin; Hertz, John

    2015-06-01

    Our ability to collect large amounts of data from many cells has been paralleled by the development of powerful statistical models for extracting information from this data. Here we discuss how the activity of cell assemblies can be analyzed using these models, focusing on the generalized linear models and the maximum entropy models and describing a number of recent studies that employ these tools for analyzing multi-neuronal activity. We show results from simulations comparing inferred functional connectivity, pairwise correlations and the real synaptic connections in simulated networks demonstrating the power of statistical models in inferring functional connectivity. Further development of network reconstruction techniques based on statistical models should lead to more powerful methods of understanding functional anatomy of cell assemblies.

  15. Active scaffolds for on-demand drug and cell delivery

    PubMed Central

    Zhao, Xuanhe; Kim, Jaeyun; Cezar, Christine A.; Huebsch, Nathaniel; Lee, Kangwon; Bouhadir, Kamal; Mooney, David J.

    2011-01-01

    Porous biomaterials have been widely used as scaffolds in tissue engineering and cell-based therapies. The release of biological agents from conventional porous scaffolds is typically governed by molecular diffusion, material degradation, and cell migration, which do not allow for dynamic external regulation. We present a new active porous scaffold that can be remotely controlled by a magnetic field to deliver various biological agents on demand. The active porous scaffold, in the form of a macroporous ferrogel, gives a large deformation and volume change of over 70% under a moderate magnetic field. The deformation and volume variation allows a new mechanism to trigger and enhance the release of various drugs including mitoxantrone, plasmid DNA, and a chemokine from the scaffold. The porous scaffold can also act as a depot of various cells, whose release can be controlled by external magnetic fields. PMID:21149682

  16. Activity of quinone alkylating agents in quinone-resistant cells.

    PubMed

    Begleiter, A; Leith, M K

    1990-05-15

    The role of the quinone group in the antitumor activity of quinone alkylating agents, such as mitomycin C and 2,5-diaziridinyl-3,5-bis(carboethoxyamino)-1,4-benzoquinone, is still uncertain. The quinone group may contribute to antitumor activity by inducing DNA strand breaks through the formation of free radicals and/or by influencing the alkylating activity of the quinone alkylators. The cytotoxic activity and DNA damage produced by the model quinone alkylating agents, benzoquinone mustard and benzoquinone dimustard, were compared in L5178Y murine lymphoblasts sensitive and resistant to the model quinone antitumor agent, hydrolyzed benzoquinone mustard. The resistant cell lines, L5178Y/HBM2 and L5178Y/HBM10, have increased concentrations of glutathione and elevated catalase, superoxide dismutase, glutathione S-transferase, and DT-diaphorase activity. L5178Y/HBM2 and L5178Y/HBM10 cells were 7.4- and 8.5-fold less sensitive to benzoquinone mustard and 1.7- and 4.3-fold less sensitive to benzoquinone dimustard, respectively, compared with sensitive cells, but showed no resistance to the non-quinone alkylating agent, aniline mustard. The formation of DNA double strand breaks by benzoquinone mustard was reduced by 2- and 8-fold in L5178Y/HBM2 and L5178Y/HBM10 cells, respectively, while double strand break formation by benzoquinone dimustard was reduced only in the L5178Y/HBM10 cells. The number of DNA-DNA cross-links produced by benzoquinone mustard was 3- and 6-fold lower, and the number produced by benzoquinone dimustard was 35% and 2-fold lower in L5178Y/HBM2 and L5178Y/HBM10 cells, respectively, compared with L5178Y parental cells. In contrast, cross-linking by aniline mustard was unchanged in sensitive and resistant cells. Dicoumarol, an inhibitor of DT-diaphorase, increased the cytotoxic activity of both benzoquinone mustard and benzoquinone dimustard in L5178Y/HBM10 cells. This study provides evidence that elevated DT-diaphorase activity in the resistant cells

  17. Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MR1

    PubMed Central

    Jeffery, Hannah C.; van Wilgenburg, Bonnie; Kurioka, Ayako; Parekh, Krishan; Stirling, Kathryn; Roberts, Sheree; Dutton, Emma E.; Hunter, Stuart; Geh, Daniel; Braitch, Manjit K.; Rajanayagam, Jeremy; Iqbal, Tariq; Pinkney, Thomas; Brown, Rachel; Withers, David R.; Adams, David H.; Klenerman, Paul; Oo, Ye H.

    2016-01-01

    Background & Aims Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells characterised by the invariant TCR-chain, Vα7.2-Jα33, and are restricted by MR1, which presents bacterial vitamin B metabolites. They are important for antibacterial immunity at mucosal sites; however, detailed characteristics of liver-infiltrating MAIT (LI-MAIT) and their role in biliary immune surveillance remain unexplored. Methods The phenotype and intrahepatic localisation of human LI-MAIT cells was examined in diseased and normal livers. MAIT cell activation in response to E. coli-exposed macrophages, biliary epithelial cells (BEC) and liver B cells was assessed with/without anti-MR1. Results Intrahepatic MAIT cells predominantly localised to bile ducts in the portal tracts. Consistent with this distribution, they expressed biliary tropic chemokine receptors CCR6, CXCR6, and integrin αEβ7. LI-MAIT cells were also present in the hepatic sinusoids and possessed tissue-homing chemokine receptor CXCR3 and integrins LFA-1 and VLA-4, suggesting their recruitment via hepatic sinusoids. LI-MAIT cells were enriched in the parenchyma of acute liver failure livers compared to chronic diseased livers. LI-MAIT cells had an activated, effector memory phenotype, expressed α4β7 and receptors for IL-12, IL-18, and IL-23. Importantly, in response to E. coli-exposed macrophages, liver B cells and BEC, MAIT cells upregulated IFN-γ and CD40 Ligand and degranulated in an MR1-dependent, cytokine-independent manner. In addition, diseased liver MAIT cells expressed T-bet and RORγt and the cytokines IFN-γ, TNF-α, and IL-17. Conclusions Our findings provide the first evidence of an immune surveillance effector response for MAIT