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Sample records for activate inkt cells

  1. 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

  2. NF-κB Is Activated in CD4+ iNKT Cells by Sickle Cell Disease and Mediates Rapid Induction of Adenosine A2A Receptors

    PubMed Central

    Yu, Jennifer C.; Ken, Ruey; Neuberg, Donna; Nathan, David G.; Linden, Joel

    2013-01-01

    Reperfusion injury following tissue ischemia occurs as a consequence of vaso-occlusion that is initiated by activation of invariant natural killer T (iNKT) cells. Sickle cell disease (SDC) results in widely disseminated microvascular ischemia and reperfusion injury as a result of vaso-occlusion by rigid and adhesive sickle red blood cells. In mice, iNKT cell activation requires NF-κB signaling and can be inhibited by the activation of anti-inflammatory adenosine A2A receptors (A2ARs). Human iNKT cells are divided into subsets of CD4+ and CD4- cells. In this study we found that human CD4+ iNKT cells, but not CD4- cells undergo rapid NF-κB activation (phosphorylation of NF-κB on p65) and induction of A2ARs (detected with a monoclonal antibody 7F6-G5-A2) during SCD painful vaso-occlusive crises. These findings indicate that SCD primarily activates the CD4+ subset of iNKT cells. Activation of NF-κB and induction of A2ARs is concordant, i.e. only CD4+ iNKT cells with activated NF-κB expressed high levels of A2ARs. iNKT cells that are not activated during pVOC express low levels of A2AR immunoreactivity. These finding suggest that A2AR transcription may be induced in CD4+ iNKT cells as a result of NF-κB activation in SCD. In order to test this hypothesis further we examined cultured human iNKT cells. In cultured cells, blockade of NF-κB with Bay 11–7082 or IKK inhibitor VII prevented rapid induction of A2AR mRNA and protein upon iNKT activation. In conclusion, NF-κB-mediated induction of A2ARs in iNKT cells may serve as a counter-regulatory mechanism to limit the extent and duration of inflammatory immune responses. As activated iNKT cells express high levels of A2ARs following their activation, they may become highly sensitive to inhibition by A2AR agonists. PMID:24124453

  3. The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells

    PubMed Central

    Torreno-Pina, Juan A.; Manzo, Carlo; Salio, Mariolina; Aichinger, Michael C.; Oddone, Anna; Lakadamyali, Melike; Shepherd, Dawn; Besra, Gurdyal S.; Cerundolo, Vincenzo

    2016-01-01

    Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such “tonic” activation of iNKT cells remain unclear. Here, we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen-presenting cells (APCs) modulates activation of iNKT cells. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton. Dual-color single-particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton, preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the Toll-like receptor 7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a previously unidentified mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen-loaded CD1d nanoclusters. PMID:26798067

  4. Α-galactosylceramide analogs with weak agonist activity for human iNKT cells define new candidate anti-inflammatory agents.

    PubMed

    Bricard, Gabriel; Venkataswamy, Manjunatha M; Yu, Karl O A; Im, Jin S; Ndonye, Rachel M; Howell, Amy R; Veerapen, Natacha; Illarionov, Petr A; Besra, Gurdyal S; Li, Qian; Chang, Young-Tae; Porcelli, Steven A

    2010-12-17

    CD1d-restricted natural killer T cells with invariant T cell receptor α chains (iNKT cells) are a unique lymphocyte subset that responds to recognition of specific lipid and glycolipid antigens. They are conserved between mice and humans and exert various immunoregulatory functions through their rapid secretion of a variety of cytokines and secondary activation of dendritic cells, B cells and NK cells. In the current study, we analyzed the range of functional activation states of human iNKT cells using a library of novel analogs of α-galactosylceramide (αGalCer), the prototypical iNKT cell antigen. Measurement of cytokines secreted by human iNKT cell clones over a wide range of glycolipid concentrations revealed that iNKT cell ligands could be classified into functional groups, correlating with weak versus strong agonistic activity. The findings established a hierarchy for induction of different cytokines, with thresholds for secretion being consistently lowest for IL-13, higher for interferon-γ (IFNγ), and even higher for IL-4. These findings suggested that human iNKT cells can be intrinsically polarized to selective production of IL-13 by maintaining a low level of activation using weak agonists, whereas selective polarization to IL-4 production cannot be achieved through modulating the strength of the activating ligand. In addition, using a newly designed in vitro system to assess the ability of human iNKT cells to transactivate NK cells, we found that robust secondary induction of interferon-γ secretion by NK cells was associated with strong but not weak agonist ligands of iNKT cells. These results indicate that polarization of human iNKT cell responses to Th2-like or anti-inflammatory effects may best be achieved through selective induction of IL-13 and suggest potential discrepancies with findings from mouse models that may be important in designing iNKT cell-based therapies in humans.

  5. Transcription factor Bcl11b sustains iNKT1 and iNKT2 cell programs, restricts iNKT17 cell program, and governs iNKT cell survival.

    PubMed

    Uddin, Mohammad Nizam; Sultana, Dil Afroz; Lorentsen, Kyle J; Cho, Jonathan J; Kirst, Mariana E; Brantly, Mark L; Califano, Danielle; Sant'Angelo, Derek B; Avram, Dorina

    2016-07-01

    Invariant natural killer T (iNKT) cells are innate-like T cells that recognize glycolipid antigens and play critical roles in regulation of immune responses. Based on expression of the transcription factors (TFs) Tbet, Plzf, and Rorγt, iNKT cells have been classified in effector subsets that emerge in the thymus, namely, iNKT1, iNKT2, and iNKT17. Deficiency in the TF Bcl11b in double-positive (DP) thymocytes has been shown to cause absence of iNKT cells in the thymus and periphery due to defective self glycolipid processing and presentation by DP thymocytes and undefined intrinsic alterations in iNKT precursors. We used a model of cre-mediated postselection deletion of Bcl11b in iNKT cells to determine its intrinsic role in these cells. We found that Bcl11b is expressed equivalently in all three effector iNKT subsets, and its removal caused a reduction in the numbers of iNKT1 and iNKT2 cells, but not in the numbers of iNKT17 cells. Additionally, we show that Bcl11b sustains subset-specific cytokine production by iNKT1 and iNKT2 cells and restricts expression of iNKT17 genes in iNKT1 and iNKT2 subsets, overall restraining the iNKT17 program in iNKT cells. The total numbers of iNKT cells were reduced in the absence of Bcl11b both in the thymus and periphery, associated with the decrease in iNKT1 and iNKT2 cell numbers and decrease in survival, related to changes in survival/apoptosis genes. Thus, these results extend our understanding of the role of Bcl11b in iNKT cells beyond their selection and demonstrate that Bcl11b is a key regulator of iNKT effector subsets, their function, identity, and survival.

  6. Innate recognition of cell wall β-glucans drives invariant Natural Killer T (iNKT) cell responses against fungi

    PubMed Central

    Cohen, Nadia R.; Tatituri, Raju V.V.; Rivera, Amariliz; Watts, Gerald F.M.; Kim, Edy Y.; Chiba, Asako; Fuchs, Beth B.; Mylonakis, Eleftherios; Besra, Gurdyal S.; Levitz, Stuart M.; Brigl, Manfred; Brenner, Michael B.

    2016-01-01

    SUMMARY iNKT cells are innate T lymphocytes recognizing endogenous and foreign lipid antigens presented in the MHC-like molecule CD1d. The semi-invariant iNKT cell TCR can detect certain bacterial and parasitic lipids, and drive iNKT cell responses. How iNKT cells respond to fungi, however, is unknown. We found that CD1d-deficient mice, which lack iNKT cells, poorly control infection with the fungal pathogen Aspergillus fumigatus. Furthermore, A. fumigatus rapidly activates iNKT cells in vivo and in vitro in the presence of APCs. Surprisingly, despite a requirement for CD1d recognition, the anti-fungal iNKT cell response does not require fungal lipids. Instead, Dectin-1 and MyD88-mediated responses to β-1,3 glucans, major fungal cell-wall polysaccharides, trigger IL-12 production by APCs that drives self-reactive iNKT cells to secrete IFN-γ. Innate recognition of β-1,3 glucans also drives iNKT cell responses against Candida, Histoplasma and Alternaria, suggesting that this mechanism may broadly define the basis for anti-fungal iNKT cell responses. PMID:22100160

  7. Novel immunostimulators with a thiazolidin-4-one ring promote the immunostimulatory effect of human iNKT cells on the stimulation of Th2-like immune responsiveness via GATA3 activation in vitro.

    PubMed

    Meng, Ming; Li, Chunxiao; Yang, Fei; Chen, Hua; Li, Xiaoliu; Yang, Yongbin; Chen, Dongzhi

    2016-10-01

    Invariant natural killer T cells (iNKTs) are important innate immune cells which get involved in various immune responses in both mice and humans. These immune reactions range from self-tolerance to development of autoimmunity and responses to pathogens and tumor development. In this study, we aimed to explore the effects of the novel immunostimulators (CH1b and CH2b) containing thiazolidin-4-one on the functions of human invariant natural killer T cells (iNKTs). First of all, iNKTs in peripheral blood mononuclear cells were expanded with α-Galactosylceramide (α-Galcer) in vitro. Then, the highly purified iNKTs were isolated from PBMCs using magnetic cells sorting (MACS). Next, we investigated the impacts of CH1b and CH2b on proliferation, cytokines production, cytotoxicity, and the associated signaling pathways in iNKT cells. Finally, we found that CH2b could significantly promote the activated iNKTs proliferation, increase the production of Th2 cytokines, and induce Th0 differentiation into Th2 subset via GATA 3 signaling pathway. Besides, CH2b could markedly enhance the cytotoxic ability of the activated iNKTs. Therefore, we concluded that CH2b, a promising candidate immunostimulator, might be used for the treatment of infections, tumors, autoimmune and allergic diseases, and for the correction of Th1/Th2 balance disorders in future. PMID:27543853

  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

    2010-10-01

    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, 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. AIRE deficiency leads to impaired iNKT cell development.

    PubMed

    Lindh, Emma; Rosmaraki, Eleftheria; Berg, Louise; Brauner, Hanna; Karlsson, Mikael C I; Peltonen, Leena; Höglund, Petter; Winqvist, Ola

    2010-02-01

    Autoimmune Polyendocrine Syndrome type I (APS I) is caused by mutations in the Autoimmune Regulator gene (AIRE), and results in the immunological destruction of endocrine organs. Herein we have characterized the CD1d-restricted invariant NKT cells (iNKT) and NK cells in APS I patients and Aire(-/-) mice, two cell populations known to play a role in the regulation of autoimmune disease. We show that the frequency of circulating iNKT cells is reduced in APS I patients compared to healthy controls. In accordance with this, iNKT cells are significantly reduced in the thymus and peripheral organs of Aire(-/-) mice. Bone marrow transfer from wild type donors into lethally irradiated Aire(-/-) recipients led to a decreased iNKT cell population in the liver, suggesting an impaired development of iNKT cells in the absence of Aire expression in radio-resistant cells. In contrast to the iNKT cells, both conventional NK cells and thymus-derived NK cells were unaffected by Aire deficiency and differentiated normally in Aire(-/-) mice. Our results show that expression of Aire in radio-resistant cells is important for the development of iNKT cells, whereas NK cell development and function does not depend on Aire.

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

    PubMed

    Thanabalasuriar, Ajitha; Neupane, Arpan S; Wang, Jing; Krummel, Matthew F; Kubes, Paul

    2016-09-20

    iNKT cells are a subset of innate T cells that recognize glycolipids presented on CD1d molecules and protect against bacterial infections, including S. pneumoniae. Using lung intravital imaging, we examined the behavior and mechanism of pulmonary iNKT cell activation in response to the specific iNKT cell ligand α-galactosylceramide or 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. The neutrophils guided 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 recruitment by blocking CCL17 increased susceptibility to S. pneumoniae infection, suggesting a critical role for the influx of iNKT cells in host defense.

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

    PubMed

    Thanabalasuriar, Ajitha; Neupane, Arpan S; Wang, Jing; Krummel, Matthew F; Kubes, Paul

    2016-09-20

    iNKT cells are a subset of innate T cells that recognize glycolipids presented on CD1d molecules and protect against bacterial infections, including S. pneumoniae. Using lung intravital imaging, we examined the behavior and mechanism of pulmonary iNKT cell activation in response to the specific iNKT cell ligand α-galactosylceramide or 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. The neutrophils guided 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 recruitment by blocking CCL17 increased susceptibility to S. pneumoniae infection, suggesting a critical role for the influx of iNKT cells in host defense. PMID:27653688

  13. iNKT cells require TSC1 for terminal maturation and effector lineage fate decisions

    PubMed Central

    Wu, Jinhong; Yang, Jialong; Yang, Kai; Wang, Hongxia; Gorentla, Balachandra; Shin, Jinwook; Qiu, Yurong; Que, Loretta G.; Foster, W. Michael; Xia, Zhenwei; Chi, Hongbo; Zhong, Xiao-Ping

    2014-01-01

    Terminal maturation of invariant NKT (iNKT) cells from stage 2 (CD44+NK1.1–) to stage 3 (CD44+NK1.1+) is accompanied by a functional acquisition of a predominant IFN-γ–producing (iNKT-1) phenotype; however, some cells develop into IL-17–producing iNKT (iNKT-17) cells. iNKT-17 cells are rare and restricted to a CD44+NK1.1– lineage. It is unclear how iNKT terminal maturation is regulated and what factors mediate the predominance of iNKT-1 compared with iNKT-17. The tumor suppressor tuberous sclerosis 1 (TSC1) is an important negative regulator of mTOR signaling, which regulates T cell differentiation, function, and trafficking. Here, we determined that mice lacking TSC1 exhibit a developmental block of iNKT differentiation at stage 2 and skew from a predominantly iNKT-1 population toward a predominantly iNKT-17 population, leading to enhanced airway hypersensitivity. Evaluation of purified iNKT cells revealed that TSC1 promotes T-bet, which regulates iNKT maturation, but downregulates ICOS expression in iNKT cells by inhibiting mTOR complex 1 (mTORC1). Furthermore, mice lacking T-bet exhibited both a terminal maturation defect of iNKT cells and a predominance of iNKT-17 cells, and increased ICOS expression was required for the predominance of iNKT-17 cells in the population of TSC1-deficient iNKT cells. Our data indicate that TSC1-dependent control of mTORC1 is crucial for terminal iNKT maturation and effector lineage decisions, resulting in the predominance of iNKT-1 cells. PMID:24614103

  14. Reduced iNKT cells numbers in type 1 diabetes patients and their first-degree relatives.

    PubMed

    Beristain-Covarrubias, Nonantzin; Canche-Pool, Elsy; Gomez-Diaz, Rita; Sanchez-Torres, Luvia E; Ortiz-Navarrete, Vianney

    2015-12-01

    Type 1 diabetes (T1D) is an autoimmune disease that is characterized by the specific destruction of insulin-producing pancreatic β cells. Invariant natural killer T (iNKT) cells have been associated with development of T1D. Class I MHC-restricted T cell-associated molecule (CRTAM) is expressed on activated iNKT, CD8(+), and CD4(+) T cells, and it is associated with the pro-inflammatory profiles of these cells. Crtam gene expression in CD3(+) lymphocytes from non-obese diabetic (NOD) mice is associated with T1D onset. However, expression of CRTAM on T cells from patients with T1D has not yet been evaluated. We compared iNKT cell (CD3(+)Vα24(+)Vβ11(+)) numbers and CRTAM expression in a Mexican population with recent-onset T1D and their first-degree relatives with control families. Remarkably, we found lower iNKT cell numbers in T1D families, and we identified two iNKT cell populations in some of the families. One iNKT cell population expressed high iTCR levels (iNKT(hi)), whereas another expressed low levels (iNKT(lo)) and also expressed CRTAM. These findings support a probable genetic determinant of iNKT cell numbers and a possible role for these cells in T1D development. This study also suggests that CRTAM identifies recently activated iNKT lymphocytes.

  15. Mouse Invariant Monoclonal Antibody NKT14: A Novel Tool to Manipulate iNKT Cell Function In Vivo

    PubMed Central

    Scheuplein, Felix; Lamont, Deanna J.; Poynter, Matthew E.; Boyson, Jonathan E.; Serreze, David; Lundblad, Lennart K. A.; Mashal, Robert; Schaub, Robert

    2015-01-01

    Invariant Natural Killer T (iNKT) cells are a T cell subset expressing an invariant T Cell Receptor (TCR) that recognizes glycolipid antigens rather than peptides. The cells have both innate-like rapid cytokine release, and adaptive-like thymic positive selection. iNKT cell activation has been implicated in the pathogenesis of allergic asthma and inflammatory diseases, while reduced iNKT cell activation promotes infectious disease, cancer and certain autoimmune diseases such as Type 1 diabetes (T1D). Therapeutic means to reduce or deplete iNKT cells could treat inflammatory diseases, while approaches to promote their activation may have potential in certain infectious diseases, cancer or autoimmunity. Thus, we developed invariant TCR-specific monoclonal antibodies to better understand the role of iNKT cells in disease. We report here the first monoclonal antibodies specific for the mouse invariant TCR that by modifying the Fc construct can specifically deplete or activate iNKT cells in vivo in otherwise fully immuno-competent animals. We have used both the depleting and activating version of the antibody in the NOD model of T1D. As demonstrated previously using genetically iNKT cell deficient NOD mice, and in studies of glycolipid antigen activated iNKT cells in standard NOD mice, we found that antibody mediated depletion or activation of iNKT cells respectively accelerated and retarded T1D onset. In BALB/c mice, ovalbumin (OVA) mediated airway hyper-reactivity (AHR) was abrogated with iNKT cell depletion prior to OVA sensitization, confirming studies in knockout mice. Depletion of iNKT cells after sensitization had no effect on AHR in the conducting airways but did reduce AHR in the lung periphery. This result raises caution in the interpretation of studies that use animals that are genetically iNKT cell deficient from birth. These activating and depleting antibodies provide a novel tool to assess the therapeutic potential of iNKT cell manipulation. PMID:26474487

  16. 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. PMID:27593966

  17. iNKT cell frequency in peripheral blood of Caucasian children and adolescent: the absolute iNKT cell count is stable from birth to adulthood.

    PubMed

    Bienemann, K; Iouannidou, K; Schoenberg, K; Krux, F; Reuther, S; Feyen, O; Bienemann, K; Schuster, F; Uhrberg, M; Laws, H-J; Borkhardt, A

    2011-10-01

    Human invariant natural killer T cells (iNKT cells) are a unique population of T cells that express a semi-invariantly rearranged T cell receptor (TCR) and are involved in a variety of immunoregulatory processes. We assessed the frequency of peripheral blood iNKT cells in 64 healthy Caucasian children from 7 months to 18 years of age and five cord blood samples by flow cytometry. iNKT cells were measured as CD3(+) cells co-expressing TCRVα24 and TCRVβ11 and using the monoclonal antibody 6B11, which recognizes specifically their invariant TCR rearrangement. The absolute number of iNKT cells ranged from 86 to 10,499 (CD3(+) /TCRVα24(+) / TCRVβ11(+)) and 233 to 11,167 (CD3(+) /6B11(+)) iNKT cells per millilitre of blood. This range is stable from birth to adulthood. The relative iNKT cell count was found to be 0.003-0.71% (CD3(+) /TCRVα24/TCRVβ11) and 0.019-0.776% (CD3/6B11) of peripheral blood T cells and shows only a slight increase with age.

  18. Human iNKT Cells Promote Protective Inflammation by Inducing Oscillating Purinergic Signaling in Monocyte-Derived DCs.

    PubMed

    Xu, Xuequn; Pocock, Ginger M; Sharma, Akshat; Peery, Stephen L; Fites, J Scott; Felley, Laura; Zarnowski, Robert; Stewart, Douglas; Berthier, Erwin; Klein, Bruce S; Sherer, Nathan M; Gumperz, Jenny E

    2016-09-20

    Invariant natural killer T (iNKT) cells are innate T lymphocytes that promote host defense against a variety of microbial pathogens. Whether microbial ligands are required for their protective effects remains unclear. Here, we show that iNKT cells stimulate human-monocyte-derived dendritic cells (DCs) to produce inflammatory mediators in a manner that does not require the presence of microbial compounds. Interleukin 2 (IL-2)-exposed iNKT cells selectively induced repeated cytoplasmic Ca(2+) fluxes in DCs that were dependent on signaling by the P2X7 purinergic receptor and mediated by ATP released during iNKT-DC interactions. Exposure to iNKT cells led to DC cyclooxygenase 2 (PTGS2) gene transcription, and release of PGE2 that was associated with vascular permeabilization in vivo. Additionally, soluble factors were released that induced neutrophil recruitment and activation and enhanced control of Candida albicans. These results suggest that sterile interactions between iNKT cells and monocyte-derived DCs lead to the production of non-redundant inflammatory mediators that promote neutrophil responses. PMID:27653689

  19. Basic techniques for studies of iNKT cells and MAIT cells.

    PubMed

    Chiba, Asako; Miyake, Sachiko

    2014-01-01

    Invariant natural killer T (iNKT) cells and mucosal-associated invariant T cells (MAIT cells) are T cell subsets belonging to innate-like lymphocytes. These innate-like lymphocytes express semi-invariant T cell receptors, but exert diverse functions and thus are involved in various types of immune responses. As iNKT cells and MAIT cells are abundant in human peripheral blood, these cells may hold important physiological roles, and thus it is desired to reveal their functions. Here, we first describe the cell preparation techniques commonly used in studies of innate-like lymphocytes, and then introduce methods for the detection and functional analysis of iNKT cells and MAIT cells.

  20. The Transcriptional Repressor Gfi1 Plays a Critical Role in the Development of NKT1- and NKT2-Type iNKT Cells.

    PubMed

    Yasuoka, Toshiaki; Kuwahara, Makoto; Yamada, Takeshi; Maruyama, Saho; Suzuki, Junpei; Taniguchi, Masaru; Yasukawa, Masaki; Yamashita, Masakatsu

    2016-01-01

    Gfi1 plays an important role in the development and maintenance of many hematopoietic linage cells. However, the impact of Gfi1-deficiency on the iNKT cell differentiation remains unclear. We herein demonstrate a critical role of Gfi1 in regulating the development of iNKT cell subsets. In the thymus of T cell-specific Gfi1-deficient mice, iNKT cells normally developed up to stage 2, while the number of stage 3 NK1.1pos iNKT cells was significantly reduced. Furthermore, CD4pos iNKT cells were selectively reduced in the peripheral organs of T cell-specific Gfi1-deficient mice. The α-GalCer-dependent production of IFN-γand Th2 cytokines, but not IL-17A, was severely reduced in T cell-specific Gfi1-deficient mice. In addition, a reduction of the α-GalCer-induced anti-tumor activity was observed in Gfi1-deficient mice. These findings demonstrate the important role of Gfi1 in regulating the development and function of NKT1- and NKT2-type iNKT cell subsets.

  1. The Transcriptional Repressor Gfi1 Plays a Critical Role in the Development of NKT1- and NKT2-Type iNKT Cells

    PubMed Central

    Yasuoka, Toshiaki; Kuwahara, Makoto; Yamada, Takeshi; Maruyama, Saho; Suzuki, Junpei; Taniguchi, Masaru; Yasukawa, Masaki; Yamashita, Masakatsu

    2016-01-01

    Gfi1 plays an important role in the development and maintenance of many hematopoietic linage cells. However, the impact of Gfi1-deficiency on the iNKT cell differentiation remains unclear. We herein demonstrate a critical role of Gfi1 in regulating the development of iNKT cell subsets. In the thymus of T cell-specific Gfi1-deficient mice, iNKT cells normally developed up to stage 2, while the number of stage 3 NK1.1pos iNKT cells was significantly reduced. Furthermore, CD4pos iNKT cells were selectively reduced in the peripheral organs of T cell-specific Gfi1-deficient mice. The α-GalCer-dependent production of IFN-γand Th2 cytokines, but not IL-17A, was severely reduced in T cell-specific Gfi1-deficient mice. In addition, a reduction of the α-GalCer-induced anti-tumor activity was observed in Gfi1-deficient mice. These findings demonstrate the important role of Gfi1 in regulating the development and function of NKT1- and NKT2-type iNKT cell subsets. PMID:27284976

  2. Jarid2 is induced by TCR signalling and controls iNKT cell maturation.

    PubMed

    Pereira, Renata M; Martinez, Gustavo J; Engel, Isaac; Cruz-Guilloty, Fernando; Barboza, Bianca A; Tsagaratou, Ageliki; Lio, Chan-Wang J; Berg, Leslie J; Lee, Youngsook; Kronenberg, Mitchell; Bandukwala, Hozefa S; Rao, Anjana

    2014-08-08

    Jarid2 is a reported component of three lysine methyltransferase complexes, polycomb repressive complex 2 (PRC2) that methylates histone 3 lysine 27 (H3K27), and GLP-G9a and SETDB1 complexes that methylate H3K9. Here we show that Jarid2 is upregulated upon TCR stimulation and during positive selection in the thymus. Mice lacking Jarid2 in T cells display an increase in the frequency of IL-4-producing promyelocytic leukemia zinc finger (PLZF)(hi) immature invariant natural killer T (iNKT) cells and innate-like CD8(+) cells; Itk-deficient mice, which have a similar increase of innate-like CD8(+) cells, show blunted upregulation of Jarid2 during positive selection. Jarid2 binds to the Zbtb16 locus, which encodes PLZF, and thymocytes lacking Jarid2 show increased PLZF and decreased H3K9me3 levels. Jarid2-deficient iNKT cells perturb Th17 differentiation, leading to reduced Th17-driven autoimmune pathology. Our results establish Jarid2 as a novel player in iNKT cell maturation that regulates PLZF expression by modulating H3K9 methylation.

  3. iNKT Cells Are Responsible for the Apoptotic Reduction of Basophils That Mediate Th2 Immune Responses Elicited by Papain in Mice Following γPGA Stimulation.

    PubMed

    Park, Hyun Jung; Lee, Sung Won; Park, Se-Ho; Hong, Seokmann

    2016-01-01

    Recent studies have demonstrated that Bacillus subtilis-derived poly-gamma glutamic acid (γPGA) treatment suppresses the development of allergic diseases such as atopic dermatitis (AD). Although basophils, an innate immune cell, are known to play critical roles in allergic immune responses and repeated long-term administration of γPGA results in decreased splenic basophils in an AD murine model, the underlying mechanisms by which γPGA regulates basophil frequency remain unclear. To investigate how γPGA modulates basophils, we employed basophil-mediated Th2 induction in vivo model elicited by the allergen papain protease. Repeated injection of γPGA reduced the abundance of basophils and their production of IL4 in mice, consistent with our previous study using NC/Nga AD model mice. The depletion of basophils by a single injection of γPGA was dependent on the TLR4/DC/IL12 axis. CD1d-dependent Vα14 TCR invariant natural killer T (iNKT) cells are known to regulate a variety of immune responses, such as allergy. Because iNKT cell activation is highly sensitive to IL12 produced by DCs, we evaluated whether the effect of γPGA on basophils is mediated by iNKT cell activation. We found that in vivo γPGA treatment did not induce the reduction of basophils in iNKT cell-deficient CD1d KO mice, suggesting the critical role of iNKT cells in γPGA-mediated basophil depletion at the early time points. Furthermore, increased apoptotic basophil reduction triggered by iNKT cells upon γPGA stimulation was mainly attributed to Th1 cytokines such as IFNγ and TNFα, consequently resulting in inhibition of papain-induced Th2 differentiation via diminishing basophil-derived IL4. Taken together, our results clearly demonstrate that γPGA-induced iNKT cell polarization toward the Th1 phenotype induces apoptotic basophil depletion, leading to the suppression of Th2 immune responses. Thus, elucidation of the crosstalk between innate immune cells will contribute to the design and

  4. Role for lysosomal phospholipase A2 in iNKT cell-mediated CD1d recognition

    PubMed Central

    Paduraru, Crina; Bezbradica, Jelena S.; Kunte, Amit; Kelly, Robert; Shayman, James A.; Veerapen, Natacha; Cox, Liam R.; Besra, Gurdyal S.; Cresswell, Peter

    2013-01-01

    Invariant natural killer T (iNKT) cells recognize self lipid antigens presented by CD1d molecules. The nature of the self-antigens involved in the development and maturation of iNKT cells is poorly defined. Lysophospholipids are self-antigens presented by CD1d that are generated through the action of phospholipases A1 and A2. Lysosomal phospholipase A2 (LPLA2, group XV phospholipase A2) resides in the endocytic system, the main site where CD1d antigen acquisition occurs, suggesting that it could be particularly important in CD1d function. We find that Lpla2−/− mice show a decrease in iNKT cell numbers that is neither the result of a general effect on the development of lymphocyte populations nor of effects on CD1d expression. However, endogenous lipid antigen presentation by CD1d is reduced in the absence of LPLA2. Our data suggest that LPLA2 plays a role in the generation of CD1d complexes with thymic lipids required for the normal selection and maturation of iNKT cells. PMID:23493550

  5. Innate and cytokine-driven signals, rather than microbial antigens, dominate in natural killer T cell activation during microbial infection

    PubMed Central

    Tatituri, Raju V.V.; Watts, Gerald F.M.; Bhowruth, Veemal; Leadbetter, Elizabeth A.; Barton, Nathaniel; Cohen, Nadia R.; Hsu, Fong-Fu; Besra, Gurdyal S.

    2011-01-01

    Invariant natural killer T cells (iNKT cells) are critical for host defense against a variety of microbial pathogens. However, the central question of how iNKT cells are activated by microbes has not been fully explained. The example of adaptive MHC-restricted T cells, studies using synthetic pharmacological α-galactosylceramides, and the recent discovery of microbial iNKT cell ligands have all suggested that recognition of foreign lipid antigens is the main driver for iNKT cell activation during infection. However, when we compared the role of microbial antigens versus innate cytokine-driven mechanisms, we found that iNKT cell interferon-γ production after in vitro stimulation or infection with diverse bacteria overwhelmingly depended on toll-like receptor–driven IL-12. Importantly, activation of iNKT cells in vivo during infection with Sphingomonas yanoikuyae or Streptococcus pneumoniae, pathogens which are known to express iNKT cell antigens and which require iNKT cells for effective protection, also predominantly depended on IL-12. Constitutive expression of high levels of IL-12 receptor by iNKT cells enabled instant IL-12–induced STAT4 activation, demonstrating that among T cells, iNKT cells are uniquely equipped for immediate, cytokine-driven activation. These findings reveal that innate and cytokine-driven signals, rather than cognate microbial antigen, dominate in iNKT cell activation during microbial infections. PMID:21555485

  6. 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

  7. Steady-state production of IL-4 modulates immunity in mouse strains and is determined by lineage diversity of iNKT cells.

    PubMed

    Lee, You Jeong; Holzapfel, Keli L; Zhu, Jinfang; Jameson, Stephen C; Hogquist, Kristin A

    2013-11-01

    Invariant natural killer T cells (iNKT cells) can produce copious amounts of interleukin 4 (IL-4) early during infection. However, indirect evidence suggests they may produce this immunomodulatory cytokine in the steady state. Through intracellular staining for transcription factors, we have defined three subsets of iNKT cells (NKT1, NKT2 and NKT17) that produced distinct cytokines; these represented diverse lineages and not developmental stages, as previously thought. These subsets exhibited substantial interstrain variation in numbers. In several mouse strains, including BALB/c, NKT2 cells were abundant and were stimulated by self ligands to produce IL-4. In those strains, steady-state IL-4 conditioned CD8(+) T cells to become 'memory-like', increased serum concentrations of immunoglobulin E (IgE) and caused dendritic cells to produce chemokines. Thus, iNKT cell-derived IL-4 altered immunological properties under normal steady-state conditions.

  8. Steady state production of IL-4 modulates immunity in different strains and is determined by lineage diversity of iNKT cells

    PubMed Central

    Lee, You Jeong; Holzapfel, Keli L.; Zhu, Jinfang; Jameson, Stephen C.; Hogquist, Kristin A.

    2013-01-01

    iNKT cells can produce high levels of IL-4 early during infection. However, indirect evidence suggests they may produce this immunomodulatory cytokine in the steady state. Through intracellular staining for transcription factors, we define 3 subsets of iNKT cells that produce distinct cytokines (NKT1, NKT2, and NKT17), which represent diverse lineages and not developmental stages as previously thought. These subsets exhibit substantial inter-strain variation in numbers. In several strains, including BALB/c, NKT2 cells are abundant and stimulated by self-ligands to produce IL-4. In these strains, steady state IL-4 conditions CD8 T cells to become “memory-like”, increases serum IgE levels, and causes dendritic cells to produce chemokines. Thus iNKT cell derived IL-4 alters immune properties under normal steady state conditions. PMID:24097110

  9. 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.

  10. Preventing and curing citrulline-induced autoimmune arthritis in a humanized mouse model using a Th2-polarizing iNKT cell agonist.

    PubMed

    Walker, Kyle M; Rytelewski, Mateusz; Mazzuca, Delfina M; Meilleur, Shannon A; Mannik, Lisa A; Yue, David; Brintnell, William C; Welch, Ian; Cairns, Ewa; Haeryfar, S M Mansour

    2012-07-01

    Invariant natural killer T (iNKT) cells are innate lymphocytes with unique reactivity to glycolipid antigens bound to non-polymorphic CD1d molecules. They are capable of rapidly releasing pro- and/or anti-inflammatory cytokines and constitute attractive targets for immunotherapy of a wide range of diseases including autoimmune disorders. In this study, we have explored the beneficial effects of OCH, a Th2-polarizing glycolipid agonist of iNKT cells, in a humanized mouse model of rheumatoid arthritis (RA) in which citrullinated human proteins are targeted by autoaggressive immune responses in mice expressing an RA susceptibility human leukocyte antigen (HLA) DR4 molecule. We found for the first time that treatment with OCH both prevents and cures citrulline-induced autoimmune arthritis as evidenced by resolved ankle swelling and reversed histopathological changes associated with arthritis. Also importantly, OCH treatment blocked the arthritogenic capacity of citrullinated antigen-experienced splenocytes without compromising their global responsiveness or altering the proportion of splenic naturally occurring CD4(+)CD25(+)FoxP3(+) regulatory T cells. Interestingly, administering the Th1-promoting iNKT cell glycolipid ligand α-C-galactosylceramide into HLA-DR4 transgenic mice increased the incidence of arthritis in these animals and exacerbated their clinical symptoms, strongly suggesting a role for Th1 responses in the pathogenesis of citrulline-induced arthritis. Therefore, our findings indicate a role for Th1-mediated immunopathology in citrulline-induced arthritis and provide the first evidence that iNKT cell manipulation by Th2-skewing glycolipids may be of therapeutic value in this clinically relevant model, a finding that is potentially translatable to human RA. PMID:21912419

  11. The Role of Hepatic Invariant (I)NKT Cells in Systemic/Local Inflammation and Mortality During Polymicrobial Septic Shock1

    PubMed Central

    Hu, Caroline K.; Venet, Fabienne; Heffernan, David S.; Wang, Yvonne L.; Horner, Brian; Huang, Xin; Chung, Chun-Shiang; Gregory, Stephen H.; Ayala, Alfred

    2009-01-01

    Natural killer T (NKT)4 cells have been described as “innate regulatory cells” because of their rapid response to conserved glycolipids presented on CD1d via their invariant TCR. However, little is known about the contribution of the hepatic NKT cell to the development of a local and/or systemic immune response to acute septic challenge (cecal ligation & puncture; CLP). We found not only that mice deficient in invariant [i] NKT cells (Jα18 -/-) had a marked attenuation in CLP induced mortality, but also exhibited an oblation of the systemic inflammatory response (with little effect on splenic/ peritoneal immune responsiveness). Flow cytometric data indicated that following CLP, there was a marked decline in the % of CD3+αGalCer-CD1d-tetramer+ cells in the mouse C57BL/6J and Balb/c liver non-parenchymal cell population. This was associated with the marked activation of these cells (increased expression of CD69 and CD25) as well as a rise in the frequency of NKT cells positive for both Th1 and Th2 intracellular cytokines. In this respect, when mice were pre-treated in vivo with anti-CD1d blocking antibody we observed not only that this inhibited the systemic rise of IL-6 and IL-10 levels in septic mice and improved overall septic survival, but that the CLP induced changes in liver macrophage IL-6 and IL-10 expressions were inversely effected by this treatment. Together, these findings suggest that the activation of hepatic iNKT cells plays a critical role in regulating the innate immune/ systemic inflammatory response and survival in a model of acute septic shock. PMID:19201902

  12. 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.

  13. 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...

  14. Turned on by danger: activation of CD1d-restricted invariant natural killer T cells.

    PubMed

    Lawson, Victoria

    2012-09-01

    CD1d-restricted invariant natural killer T (iNKT) cells bear characteristics of innate and adaptive lymphocytes, which allow them to bridge the two halves of the immune response and play roles in many disease settings. Recent work has characterized precisely how their activation is initiated and regulated. Novel antigens from important pathogens have been identified, as has an abundant self-antigen, β-glucopyranosylcaramide, capable of mediating an iNKT-cell response. Studies of the iNKT T-cell receptor (TCR)-antigen-CD1d complex show how docking between CD1d-antigen and iNKT TCR is highly conserved, and how small sequence differences in the TCR establish intrinsic variation in iNKT TCR affinity. The sequence of the TCR CDR3β loop determines iNKT TCR affinity for ligand-CD1d, independent of ligand identity. CD1d ligands can promote T helper type 1 (Th1) or Th2 biased cytokine responses, depending on the composition of their lipid tails. Ligands loaded into CD1d on the cell surface promote Th2 responses, whereas ligands with long hydrophobic tails are loaded endosomally and promote Th1 responses. This information is informing the design of synthetic iNKT-cell antigens. The iNKT cells may be activated by exogenous antigen, or by a combination of dendritic cell-derived interleukin-12 and iNKT TCR-self-antigen-CD1d engagement. The iNKT-cell activation is further modulated by recent foreign or self-antigen encounter. Activation of dendritic cells through pattern recognition receptors alters their antigen presentation and cytokine production, strongly influencing iNKT-cell activation. In a range of bacterial infections, dendritic cell-dependent innate activation of iNKT cells through interleukin-12 is the dominant influence on their activity.

  15. Invariant NKT cells: regulation and function during viral infection.

    PubMed

    Juno, Jennifer A; Keynan, Yoav; Fowke, Keith R

    2012-01-01

    Natural killer T cells (NKT cells) represent a subset of T lymphocytes that express natural killer (NK) cell surface markers. A subset of NKT cells, termed invariant NKT cells (iNKT), express a highly restricted T cell receptor (TCR) and respond to CD1d-restricted lipid ligands. iNKT cells are now appreciated to play an important role in linking innate and adaptive immune responses and have been implicated in infectious disease, allergy, asthma, autoimmunity, and tumor surveillance. Advances in iNKT identification and purification have allowed for the detailed study of iNKT activity in both humans and mice during a variety of chronic and acute infections. Comparison of iNKT function between non-pathogenic simian immunodeficiency virus (SIV) infection models and chronic HIV-infected patients implies a role for iNKT activity in controlling immune activation. In vitro studies of influenza infection have revealed novel effector functions of iNKT cells including IL-22 production and modulation of myeloid-derived suppressor cells, but ex vivo characterization of human iNKT cells during influenza infection are lacking. Similarly, as recent evidence suggests iNKT involvement in dengue virus pathogenesis, iNKT cells may modulate responses to a number of emerging pathogens. This Review will summarize current knowledge of iNKT involvement in responses to viral infections in both human and mouse models and will identify critical gaps in knowledge and opportunities for future study. We will also highlight recent efforts to harness iNKT ligands as vaccine adjuvants capable of improving vaccination-induced cellular immune responses.

  16. Invariant NKT cells: regulation and function during viral infection.

    PubMed

    Juno, Jennifer A; Keynan, Yoav; Fowke, Keith R

    2012-01-01

    Natural killer T cells (NKT cells) represent a subset of T lymphocytes that express natural killer (NK) cell surface markers. A subset of NKT cells, termed invariant NKT cells (iNKT), express a highly restricted T cell receptor (TCR) and respond to CD1d-restricted lipid ligands. iNKT cells are now appreciated to play an important role in linking innate and adaptive immune responses and have been implicated in infectious disease, allergy, asthma, autoimmunity, and tumor surveillance. Advances in iNKT identification and purification have allowed for the detailed study of iNKT activity in both humans and mice during a variety of chronic and acute infections. Comparison of iNKT function between non-pathogenic simian immunodeficiency virus (SIV) infection models and chronic HIV-infected patients implies a role for iNKT activity in controlling immune activation. In vitro studies of influenza infection have revealed novel effector functions of iNKT cells including IL-22 production and modulation of myeloid-derived suppressor cells, but ex vivo characterization of human iNKT cells during influenza infection are lacking. Similarly, as recent evidence suggests iNKT involvement in dengue virus pathogenesis, iNKT cells may modulate responses to a number of emerging pathogens. This Review will summarize current knowledge of iNKT involvement in responses to viral infections in both human and mouse models and will identify critical gaps in knowledge and opportunities for future study. We will also highlight recent efforts to harness iNKT ligands as vaccine adjuvants capable of improving vaccination-induced cellular immune responses. PMID:22916008

  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. Critical roles of RasGRP1 for invariant natural killer T cell development

    PubMed Central

    Shen, Shudan; Chen, Yong; Gorentla, Balachandra; Lu, Jianxin; Stone, James C.; Zhong, Xiao-Ping

    2011-01-01

    The invariant NKT (iNKT) cell lineage contains CD4+ and CD4- subsets. The mechanisms that control such subset differentiation and iNKT cell maturation in general have not been fully understood. RasGRP1, a guanine nucleotide exchange factor for T cell receptor-induced activation of the Ras-Erk1/2 pathway, is critical for conventional αβ T cell development but dispensable for generating regulatory T cells. Its role in iNKT cells has been unknown. Here we report severe decreases of iNKT cells in RasGRP1-/- mice through cell intrinsic mechanisms. In the remaining iNKT cells in RasGRP1-/- mice, there is a selective absence of the CD4+ subset. Furthermore, RasGRP1-/- iNKT cells are defective in T cell receptor induced proliferation in vitro. These observations establish that RasGRP1 is not only important for early iNKT cell development, but also for the generation/maintenance of the CD4+ iNKT cells. Our data provides genetic evidence that the CD4+ and CD4- iNKT cells are distinct sub-lineages with differential signaling requirements for their development. PMID:21957144

  19. Bacterial CD1d-restricted glycolipids induce IL-10 production by human regulatory T cells upon cross-talk with invariant NKT cells.

    PubMed

    Venken, Koen; Decruy, Tine; Aspeslagh, Sandrine; Van Calenbergh, Serge; Lambrecht, Bart N; Elewaut, Dirk

    2013-09-01

    Invariant NKT (iNKT) cells and CD4(+)CD25(+)FOXP3(+) regulatory T cells (Tregs) are important immune regulatory T cells with Ag reactivity to glycolipids and peptides, respectively. However, the functional interplay between these cells in humans is poorly understood. We show that Tregs suppress iNKT cell proliferation induced by CD1d-restricted glycolipids, including bacterial-derived diacylglycerols, as well as by innate-like activation. Inhibition was related to the potency of iNKT agonists, making diacylglycerol iNKT responses very prone to suppression. Cytokine production by iNKT cells was differentially modulated by Tregs because IL-4 production was reduced more profoundly compared with IFN-γ. A compelling observation was the significant production of IL-10 by Tregs after cell contact with iNKT cells, in particular in the presence of bacterial diacylglycerols. These iNKT-primed Tregs showed increased FOXP3 expression and superior suppressive function. Suppression of iNKT cell responses, but not conventional T cell responses, was IL-10 dependent, suggesting that there is a clear difference in mechanism between the Treg-mediated inhibition of these cell types. Our data highlight a physiologically relevant interaction between human iNKT and Tregs upon pathogen-derived glycolipid recognition that has a significant impact on the design of iNKT cell-based therapeutics.

  20. CXCL16-positive dendritic cells enhance invariant natural killer T cell-dependent IFNγ production and tumor control

    PubMed Central

    Veinotte, Linnea; Gebremeskel, Simon; Johnston, Brent

    2016-01-01

    ABSTRACT Crosstalk interactions between dendritic cells (DCs) and invariant natural killer T (iNKT) cells are important in regulating antitumor responses elicited by glycolipid antigens. iNKT cells constitutively express the chemokine receptor CXCR6, while cytokine-activated DCs upregulate the transmembrane chemokine ligand, CXCL16. This study examined the co-stimulatory role of CXCR6/CXCL16 interactions in glycolipid-dependent iNKT cell activation and tumor control. Spleen and liver DCs in wild-type mice, but not iNKT cell deficient (Jα18−/−) mice, transiently upregulated surface CXCL16 following in vivo administration of the glycolipid antigen α-galactosylceramide. Recombinant CXCL16 did not directly induce iNKT cell activation in vitro but enhanced interferon (IFN)-γ production when mouse or human iNKT cells were stimulated with plate-bound anti-CD3. Compared with glycolipid-loaded CXCL16neg DCs, CXCL16hi DCs induced higher levels of IFNγ production in iNKT cell cultures and following adoptive transfer in vivo. The number of IFNγ+ iNKT cells and expansion of T-bet+ iNKT cells were reduced in vivo when CXCL16−/− DCs were used to activate iNKT cells. Enhanced IFNγ production in vivo was not dependent on CXCR6 expression on natural killer (NK) cells. Adoptive transfer of glycolipid-loaded CXCL16hi DCs provided superior protection against tumor metastasis compared to CXCL16neg DC transfers. Similarly, wild-type DCs provided superior protection against metastasis compared with CXCL16−/− DCs. These experiments implicate an important role for CXCR6/CXCL16 interactions in regulating iNKT cell IFNγ production and tumor control. The selective use of CXCL16hi DCs in adoptive transfer immunotherapies may prove useful for enhancing T helper (Th) type 1 responses and clinical outcomes in cancer patients. PMID:27471636

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

    PubMed Central

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

    2016-01-01

    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. PMID:27195112

  2. An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen.

    PubMed

    Govindarajan, Srinath; Elewaut, Dirk; Drennan, Michael

    2015-01-01

    The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells. Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5(+)) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5(+) fraction are subsequently stained with αGalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 10(8) iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice. PMID:26555769

  3. 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. PMID:26232432

  4. Homeostatic regulation of marginal zone B cells by invariant natural killer T cells.

    PubMed

    Wen, Xiangshu; Yang, Jun-Qi; Kim, Peter J; Singh, Ram Raj

    2011-01-01

    Marginal zone B cells (MZB) mount a rapid antibody response, potently activate naïve T cells, and are enriched in autoreactive B cells. MZBs express high levels of CD1d, the restriction element for invariant natural killer T cells (iNKT). Here, we examined the effect of iNKT cells on MZB cell activation and numbers in vitro and in vivo in normal and autoimmune mice. Results show that iNKT cells activate MZBs, but restrict their numbers in vitro and in vivo in normal BALB/c and C57/BL6 mice. iNKT cells do so by increasing the activation-induced cell death and curtailing proliferation of MZB cells, whereas they promote the proliferation of follicular B cells. Sorted iNKT cells can directly execute this function, without help from other immune cells. Such MZB regulation by iNKTs is mediated, at least in part, via CD1d on B cells in a contact-dependent manner, whereas iNKT-induced proliferation of follicular B cells occurs in a contact- and CD1d-independent manner. Finally, we show that iNKT cells reduce 'autoreactive' MZB cells in an anti-DNA transgenic model, and limit MZB cell numbers in autoimmune-prone (NZB×NZW)F1 and non-obese diabetic mice, suggesting a potentially new mechanism whereby iNKT cells might regulate pathologic autoimmunity. Differential regulation of follicular B cells versus potentially autoreactive MZBs by iNKT cells has important implications for autoimmune diseases as well as for conditions that require a rapid innate B cell response.

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

    PubMed Central

    Zhou, Yonghua

    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. PMID:27563682

  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. PMID:27563682

  7. 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

  8. 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

  9. 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

  10. KLRG+ invariant natural killer T cells are long-lived effectors.

    PubMed

    Shimizu, Kanako; Sato, Yusuke; Shinga, Jun; Watanabe, Takashi; Endo, Takaho; Asakura, Miki; Yamasaki, Satoru; Kawahara, Kazuyoshi; Kinjo, Yuki; Kitamura, Hiroshi; Watarai, Hiroshi; Ishii, Yasuyuki; Tsuji, Moriya; Taniguchi, Masaru; Ohara, Osamu; Fujii, Shin-ichiro

    2014-08-26

    Immunological memory has been regarded as a unique feature of the adaptive immune response mediated in an antigen-specific manner by T and B lymphocytes. However, natural killer (NK) cells and γδT cells, which traditionally are classified as innate immune cells, have been shown in recent studies to have hallmark features of memory cells. Invariant NKT cell (iNKT cell)-mediated antitumor effects indicate that iNKT cells are activated in vivo by vaccination with iNKT cell ligand-loaded CD1d(+) cells, but not by vaccination with unbound NKT cell ligand. In such models, it previously was thought that the numbers of IFN-γ-producing cells in the spleen returned to the basal level around 1 wk after the vaccination. In the current study, we demonstrate the surprising presence of effector memory-like iNKT cells in the lung. We found long-term antitumor activity in the lungs of mice was enhanced after vaccination with iNKT cell ligand-loaded dendritic cells. Further analyses showed that the KLRG1(+) (Killer cell lectin-like receptor subfamily G, member 1-positive) iNKT cells coexpressing CD49d and granzyme A persisted for several months and displayed a potent secondary response to cognate antigen. Finally, analyses of CDR3β by RNA deep sequencing demonstrated that some particular KLRG1(+) iNKT-cell clones accumulated, suggesting the selection of certain T-cell receptor repertoires by an antigen. The current findings identifying effector memory-like KLRG1(+) iNKT cells in the lung could result in a paradigm shift regarding the basis of newly developed extrathymic iNKT cells and could contribute to the future development of antitumor immunotherapy by uniquely energizing iNKT cells. PMID:25118276

  11. Interleukin-13 Pathway Alterations Impair Invariant Natural Killer T-Cell-Mediated Regulation of Effector T Cells in Type 1 Diabetes.

    PubMed

    Usero, Lorena; Sánchez, Ana; Pizarro, Eduarda; Xufré, Cristina; Martí, Mercè; Jaraquemada, Dolores; Roura-Mir, Carme

    2016-08-01

    Many studies have shown that human natural killer T (NKT) cells can promote immunity to pathogens, but their regulatory function is still being investigated. Invariant NKT (iNKT) cells have been shown to be effective in preventing type 1 diabetes in the NOD mouse model. Activation of plasmacytoid dendritic cells, modulation of B-cell responses, and immune deviation were proposed to be responsible for the suppressive effect of iNKT cells. We studied the regulatory capacity of human iNKT cells from control subjects and patients with type 1 diabetes (T1D) at disease clinical onset. We demonstrate that control iNKT cells suppress the proliferation of effector T cells (Teffs) through a cell contact-independent mechanism. Of note, suppression depended on the secretion of interleukin-13 (IL-13) by iNKT cells because an antibody blocking this cytokine resulted from the abrogation of Teff suppression; however, T1D-derived iNKT cells showed impaired regulation that could be attributed to the decrease in IL-13 secretion. Thus, alteration of the IL-13 pathway at disease onset may lead to the progression of the autoimmune response in T1D. Advances in the study of iNKT cells and the selection of agonists potentiating IL-13 secretion should permit new therapeutic strategies to prevent the development of T1D. PMID:27207542

  12. Tight regulation of diacylglycerol-mediated signaling is critical for proper invariant NKT cell development

    PubMed Central

    Shen, Shudan; Wu, Jinhong; Srivatsan, Sruti; Gorentla, Balachandra; Shin, Jinwook; Xu, Li; Zhong, Xiao-Ping

    2011-01-01

    Type I natural killer T (NKT) cells, or iNKT cells, express a semi-invariant T cell receptor characterized by its unique V α 14-Jα 18 usage (iV α 14TCR). Upon interaction with glycolipid/CD1d complexes, the iV α 14TCRs transduce signals that are essential for iNKT selection and maturation. However, it remains unclear how these signals are regulated and how important such regulations are during iNKT development. Diacylglycerol (DAG) is an essential second messenger downstream of the TCR that activates the PKCθ-IKKα/β-NFκB pathway, known to be crucial for iNKT development, as well as the RasGRP1-Ras-Erk1/2 pathway in T cells. DAG kinases (DGKs) play an important role in controlling intracellular DAG concentration and thereby negatively regulate DAG signaling. Here we report that simultaneous absence of DAG kinase α and ζ causes severe defects in iNKT development, coincident with enhanced IKK-NFκB and Ras-Erk1/2 activation. Moreover, constitutive IKKβ and Ras activities also result in iNKT developmental defects. Thus, DAG-mediated signaling is not only essential but also needs to be tightly regulated for proper iNKT cell development. PMID:21775687

  13. Invariant Natural Killer T cells in lupus patients promote IgG and IgG autoantibody production

    PubMed Central

    Shen, Lei; Zhang, Hong; Caimol, Maria; Benike, Claudia J.; Chakravarty, Eliza F.; Strober, Samuel; Engleman, Edgar G.

    2014-01-01

    IgG autoantibodies, including antibodies to double-stranded DNA (dsDNA), are pathogenic in systemic lupus erythematosus, but the mechanisms controlling their production are not understood. To assess the role of invariant natural killer T (iNKT) cells in this process, we studied 44 lupus patients. We took advantage of the propensity of PBMCs from patients with active disease to spontaneously secrete IgG, in vitro. Despite the rarity of iNKT cells in lupus blood (0.002∼0.05% of CD3-positive T cells), antibody blockade of the conserved iNKT TCR or its ligand, CD1d, or selective depletion of iNKT cells, inhibited spontaneous secretion of total IgG and anti-dsDNA IgG by lupus PBMCs. Addition of anti-iNKT or anti-CD1d antibody to PBMC cultures also reduced the frequency of plasma cells, suggesting that lupus iNKT cells induce B cell maturation. Like fresh iNKT cells, expanded iNKT cell lines from lupus patients, but not healthy subjects, induced autologous B cells to secrete antibodies, including IgG anti-dsDNA. This activity was inhibited by anti-CD40L antibody, as well as anti-CD1d antibody, confirming a role for CD40L-CD40 and TCR-CD1d interactions in lupus iNKT mediated help. These results reveal a critical role for iNKT cells in B cell maturation and autoantibody production in patients with lupus. PMID:25352488

  14. 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

  15. An Essential Role for Medullary Thymic Epithelial Cells during the Intrathymic Development of Invariant NKT Cells

    PubMed Central

    White, Andrea J.; Jenkinson, William E.; Cowan, Jennifer E.; Parnell, Sonia M.; Bacon, Andrea; Jones, Nick D.; Jenkinson, Eric J.

    2014-01-01

    In the thymus, interactions with both cortical and medullary microenvironments regulate the development of self-tolerant conventional CD4+ and CD8+ αβT cells expressing a wide range of αβTCR specificities. Additionally, the cortex is also required for the development of invariant NKT (iNKT) cells, a specialized subset of T cells that expresses a restricted αβTCR repertoire and is linked to the regulation of innate and adaptive immune responses. Although the role of the cortex in this process is to enable recognition of CD1d molecules expressed by CD4+CD8+ thymocyte precursors, the requirements for additional thymus microenvironments during iNKT cell development are unknown. In this study, we reveal a role for medullary thymic epithelial cells (mTECs) during iNKT cell development in the mouse thymus. This requirement for mTECs correlates with their expression of genes required for IL-15 trans-presentation, and we show that soluble IL-15/IL-15Rα complexes restore iNKT cell development in the absence of mTECs. Furthermore, mTEC development is abnormal in iNKT cell–deficient mice, and early stages in iNKT cell development trigger receptor activator for NF-κB ligand–mediated mTEC development. Collectively, our findings demonstrate that intrathymic iNKT cell development requires stepwise interactions with both the cortex and the medulla, emphasizing the importance of thymus compartmentalization in the generation of both diverse and invariant αβT cells. Moreover, the identification of a novel requirement for iNKT cells in thymus medulla development further highlights the role of both innate and adaptive immune cells in thymus medulla formation. PMID:24510964

  16. Third-party CD4+ invariant natural killer T cells protect from murine GVHD lethality.

    PubMed

    Schneidawind, Dominik; Baker, Jeanette; Pierini, Antonio; Buechele, Corina; Luong, Richard H; Meyer, Everett H; Negrin, Robert S

    2015-05-28

    Graft-versus-host disease (GVHD) is driven by extensive activation and proliferation of alloreactive donor T cells causing significant morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Invariant natural killer T (iNKT) cells are a potent immunoregulatory T-cell subset in both humans and mice. Here, we explored the role of adoptively transferred third-party CD4(+) iNKT cells for protection from lethal GVHD in a murine model of allogeneic HCT across major histocompatibility barriers. We found that low numbers of CD4(+) iNKT cells from third-party mice resulted in a significant survival benefit with retained graft-versus-tumor effects. In vivo expansion of alloreactive T cells was diminished while displaying a T helper cell 2-biased phenotype. Notably, CD4(+) iNKT cells from third-party mice were as protective as CD4(+) iNKT cells from donor mice although third-party CD4(+) iNKT cells were rejected early after allogeneic HCT. Adoptive transfer of third-party CD4(+) iNKT cells resulted in a robust expansion of donor CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) that were required for protection from lethal GVHD. However, in vivo depletion of myeloid-derived suppressor cells abrogated both Treg expansion and protection from lethal GVHD. Despite the fact that iNKT cells are a rare cell population, the almost unlimited third-party availability and feasibility of in vitro expansion provide the basis for clinical translation.

  17. 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

  18. 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

  19. Innate Invariant NKT Cell Recognition of HIV-1-Infected Dendritic Cells Is an Early Detection Mechanism Targeted by Viral Immune Evasion.

    PubMed

    Paquin-Proulx, Dominic; Gibbs, Anna; Bächle, Susanna M; Checa, Antonio; Introini, Andrea; Leeansyah, Edwin; Wheelock, Craig E; Nixon, Douglas F; Broliden, Kristina; Tjernlund, Annelie; Moll, Markus; Sandberg, Johan K

    2016-09-01

    Invariant NKT (iNKT) cells are innate-like T cells that respond rapidly with a broad range of effector functions upon recognition of glycolipid Ags presented by CD1d. HIV-1 carries Nef- and Vpu-dependent mechanisms to interfere with CD1d surface expression, indirectly suggesting a role for iNKT cells in control of HIV-1 infection. In this study, we investigated whether iNKT cells can participate in the innate cell-mediated immune response to HIV-1. Infection of dendritic cells (DCs) with Nef- and Vpu-deficient HIV-1 induced upregulation of CD1d in a TLR7-dependent manner. Infection of DCs caused modulation of enzymes in the sphingolipid pathway and enhanced expression of the endogenous glucosylceramide Ag. Importantly, iNKT cells responded specifically to rare DCs productively infected with Nef- and Vpu-defective HIV-1. Transmitted founder viral isolates differed in their CD1d downregulation capacity, suggesting that diverse strains may be differentially successful in inhibiting this pathway. Furthermore, both iNKT cells and DCs expressing CD1d and HIV receptors resided in the female genital mucosa, a site where HIV-1 transmission occurs. Taken together, these findings suggest that innate iNKT cell sensing of HIV-1 infection in DCs is an early immune detection mechanism, which is independent of priming and adaptive recognition of viral Ag, and is actively targeted by Nef- and Vpu-dependent viral immune evasion mechanisms.

  20. Contribution of Invariant Natural Killer T Cells to Skin Wound Healing.

    PubMed

    Tanno, Hiromasa; Kawakami, Kazuyoshi; Ritsu, Masae; Kanno, Emi; Suzuki, Aiko; Kamimatsuno, Rina; Takagi, Naoyuki; Miyasaka, Tomomitsu; Ishii, Keiko; Imai, Yoshimichi; Maruyama, Ryoko; Tachi, Masahiro

    2015-12-01

    In the present study, we determined the contribution of invariant natural killer T (iNKT) cells to the skin wound healing process. In iNKT cell-deficient (Jα18KO) mice lacking iNKT cells, wound closure was significantly delayed compared with wild-type mice. Collagen deposition, expression of α-smooth muscle actin and CD31, and wound breaking strength were significantly attenuated in Jα18KO mice. The adoptive transfer of liver mononuclear cells from wild-type but not from Jα18KO or interferon (IFN)-γ gene-disrupted (IFN-γKO) mice resulted in the reversal of this impaired wound healing in Jα18KO mice. IFN-γ expression was induced in the wounded tissues, which was significantly decreased at 6, 12, and 24 hours, but increased on day 3 after wounding in Jα18KO mice. The main source of the late-phase IFN-γ production in Jα18KO mice were neutrophils rather than NK cells and T cells. Administration of α-galactosylceramide, an activator of iNKT cells, resulted in the acceleration of wound healing on day 3 in wild-type mice. This effect was not observed in IFN-γKO mice. These results indicate that iNKT cells play important roles in wound healing. The iNKT cell-induced IFN-γ production may regulate the wound healing process in the early phase.

  1. Contribution of Invariant Natural Killer T Cells to Skin Wound Healing.

    PubMed

    Tanno, Hiromasa; Kawakami, Kazuyoshi; Ritsu, Masae; Kanno, Emi; Suzuki, Aiko; Kamimatsuno, Rina; Takagi, Naoyuki; Miyasaka, Tomomitsu; Ishii, Keiko; Imai, Yoshimichi; Maruyama, Ryoko; Tachi, Masahiro

    2015-12-01

    In the present study, we determined the contribution of invariant natural killer T (iNKT) cells to the skin wound healing process. In iNKT cell-deficient (Jα18KO) mice lacking iNKT cells, wound closure was significantly delayed compared with wild-type mice. Collagen deposition, expression of α-smooth muscle actin and CD31, and wound breaking strength were significantly attenuated in Jα18KO mice. The adoptive transfer of liver mononuclear cells from wild-type but not from Jα18KO or interferon (IFN)-γ gene-disrupted (IFN-γKO) mice resulted in the reversal of this impaired wound healing in Jα18KO mice. IFN-γ expression was induced in the wounded tissues, which was significantly decreased at 6, 12, and 24 hours, but increased on day 3 after wounding in Jα18KO mice. The main source of the late-phase IFN-γ production in Jα18KO mice were neutrophils rather than NK cells and T cells. Administration of α-galactosylceramide, an activator of iNKT cells, resulted in the acceleration of wound healing on day 3 in wild-type mice. This effect was not observed in IFN-γKO mice. These results indicate that iNKT cells play important roles in wound healing. The iNKT cell-induced IFN-γ production may regulate the wound healing process in the early phase. PMID:26468976

  2. Innate Invariant NKT Cell Recognition of HIV-1–Infected Dendritic Cells Is an Early Detection Mechanism Targeted by Viral Immune Evasion

    PubMed Central

    Paquin-Proulx, Dominic; Gibbs, Anna; Bächle, Susanna M.; Checa, Antonio; Introini, Andrea; Leeansyah, Edwin; Wheelock, Craig E.; Nixon, Douglas F.; Broliden, Kristina; Tjernlund, Annelie; Moll, Markus

    2016-01-01

    Invariant NKT (iNKT) cells are innate-like T cells that respond rapidly with a broad range of effector functions upon recognition of glycolipid Ags presented by CD1d. HIV-1 carries Nef- and Vpu-dependent mechanisms to interfere with CD1d surface expression, indirectly suggesting a role for iNKT cells in control of HIV-1 infection. In this study, we investigated whether iNKT cells can participate in the innate cell–mediated immune response to HIV-1. Infection of dendritic cells (DCs) with Nef- and Vpu-deficient HIV-1 induced upregulation of CD1d in a TLR7-dependent manner. Infection of DCs caused modulation of enzymes in the sphingolipid pathway and enhanced expression of the endogenous glucosylceramide Ag. Importantly, iNKT cells responded specifically to rare DCs productively infected with Nef- and Vpu-defective HIV-1. Transmitted founder viral isolates differed in their CD1d downregulation capacity, suggesting that diverse strains may be differentially successful in inhibiting this pathway. Furthermore, both iNKT cells and DCs expressing CD1d and HIV receptors resided in the female genital mucosa, a site where HIV-1 transmission occurs. Taken together, these findings suggest that innate iNKT cell sensing of HIV-1 infection in DCs is an early immune detection mechanism, which is independent of priming and adaptive recognition of viral Ag, and is actively targeted by Nef- and Vpu-dependent viral immune evasion mechanisms. PMID:27481843

  3. Mutation of the Traj18 gene segment using TALENs to generate Natural Killer T cell deficient mice

    PubMed Central

    Zhang, Jingjing; Bedel, Romain; Krovi, S. Harsha; Tuttle, Kathryn D.; Zhang, Bicheng; Gross, James; Gapin, Laurent; Matsuda, Jennifer L.

    2016-01-01

    Invariant Natural Killer T (iNKT) cells are a unique subset of T lymphocytes that have been implicated in both promoting and suppressing a multitude of immune responses. In mice, iNKT cells express T cell antigen receptors (TCRs) comprising a unique TCRα rearrangement between the Trav11 and Traj18 gene segments. When paired with certain Trbv TCRβ chains, these TCRs recognize lipid antigens presented by the major histocompatibility complex (MHC) class I-like molecule, CD1d. Until recently, the sole model of iNKT deficiency targeted the Jα18, which is absolutely required to form the TCR with the appropriate antigenic specificity. However, these mice were demonstrated to have a large reduction in TCR repertoire diversity, which could confound results arising from studies using these mice. Here, we have created a new NKT-deficient mouse strain using transcription activator-like effector nuclease (TALEN) technology to only disrupt the expression of Jα18, leaving the remaining Jα repertoire unperturbed. We confirm that these mice lack iNKT cells and do not respond to lipid antigen stimulation while the development of conventional T cells, regulatory T cells, and type Ib NKT cells is normal. This new mouse strain will serve as a new model of iNKT cell deficiency to facilitate our understanding of iNKT biology. PMID:27256918

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

    PubMed

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

    2014-01-16

    Coevolution of beneficial microorganisms with the mammalian intestine fundamentally shapes mammalian physiology. Here, 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 (MW = 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.

  5. Recognition of Microbial Glycolipids by Natural Killer T Cells.

    PubMed

    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

  6. 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

  7. Optimizing NKT cell ligands as vaccine adjuvants.

    PubMed

    Carreño, Leandro J; Kharkwal, Shalu Sharma; Porcelli, Steven A

    2014-01-01

    NKT cells are a subpopulation of T lymphocytes with phenotypic properties of both T and NK cells and a wide range of immune effector properties. In particular, one subset of these cells, known as invariant NKT cells (iNKT cells), has attracted substantial attention because of their ability to be specifically activated by glycolipid antigens presented by a cell surface protein called CD1d. The development of synthetic α-galactosylceramides as a family of powerful glycolipid agonists for iNKT cells has led to approaches for augmenting a wide variety of immune responses, including those involved in vaccination against infections and cancers. Here, we review basic, preclinical and clinical observations supporting approaches to improving immune responses through the use of iNKT cell-activating glycolipids. Results from preclinical animal studies and preliminary clinical studies in humans identify many promising applications for this approach in the development of vaccines and novel immunotherapies.

  8. 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.

  9. Human Invariant Natural Killer T cells possess immune-modulating functions during Aspergillus infection.

    PubMed

    Beitzen-Heineke, Antonia; Bouzani, Maria; Schmitt, Anna-Lena; Kurzai, Oliver; Hünniger, Kerstin; Einsele, Hermann; Loeffler, Juergen

    2016-02-01

    Aspergillus fumigatus is the most common cause for invasive fungal infections, a disease associated with high mortality in immune-compromised patients. CD1d-restricted invariant natural killer T (iNKT) cells compose a small subset of T cells known to impact the immune response toward various infectious pathogens. To investigate the role of human iNKT cells during A. fumigatus infection, we studied their activation as determined by CD69 expression and cytokine production in response to distinct fungal morphotypes in the presence of different CD1d(+) antigen presenting cells using flow cytometry and multiplex enzyme-linked immunosorbent assay (ELISA). Among CD1d(+) subpopulations, CD1d(+)CD1c(+) mDCs showed the highest potential to activate iNKT cells on a per cell basis. The presence of A. fumigatus decreased this effect of CD1d(+)CD1c(+) mDCs on iNKT cells and led to reduced secretion of TNF-α, G-CSF and RANTES. Production of other Th1 and Th2 cytokines was not affected by the fungus, suggesting an immune-modulating function for human iNKT cells during A. fumigatus infection.

  10. Evaluation of antibody-dependent cell-mediated cytotoxicity activity and cetuximab response in KRAS wild-type metastatic colorectal cancer patients

    PubMed Central

    Lo Nigro, Cristiana; Ricci, Vincenzo; Vivenza, Daniela; Monteverde, Martino; Strola, Giuliana; Lucio, Francesco; Tonissi, Federica; Miraglio, Emanuela; Granetto, Cristina; Fortunato, Mirella; Merlano, Marco Carlo

    2016-01-01

    AIM: To investigate the prognostic role of invariant natural killer T (iNKT) cells and antibody-dependent cell-mediated cytotoxicity (ADCC) in wild type KRAS metastatic colorectal cancer (mCRC) patients treated with cetuximab. METHODS: Forty-one KRAS wt mCRC patients, treated with cetuximab and irinotecan-based chemotherapy in II and III lines were analyzed. Genotyping of single nucleotide polymorphism (SNP)s in the FCGR2A, FCGR3A and in the 3’ untranslated regions of KRAS and mutational analysis for KRAS, BRAF and NRAS genes was determined either by sequencing or allelic discrimination assays. Enriched NK cells were obtained from lymphoprep-peripheral blood mononuclear cell and iNKT cells were defined by co-expression of CD3, TCRVα24, TCRVβ11. ADCC was evaluated as ex vivo NK-dependent activity, measuring lactate dehydrogenase release. RESULTS: At basal, mCRC patients performing ADCC activity above the median level (71%) showed an improved overall survival (OS) compared to patients with ADCC below (median 16 vs 8 mo; P = 0.026). We did not find any significant correlation of iNKT cells with OS (P = 0.19), albeit we observed a trend to a longer survival after 10 mo in patients with iNKT above median basal level (0.382 cells/microliter). Correlation of OS and progression-free survival (PFS) with interesting SNPs involved in ADCC ability revealed not to be significant. Patients carrying alleles both with A in FCGR2A and TT in FCGR3A presented a trend of longer PFS (median 9 vs 5 mo; P = 0.064). Chemotherapy impacted both iNKT cells and ADCC activity. Their prognostic values get lost when we analysed them after 2 and 4 mo of treatment. CONCLUSION: Our results suggest a link between iNKT cells, basal ADCC activity, genotypes in FCGR2A and FCGR3A, and efficacy of cetuximab in KRAS wt mCRC patients. PMID:26909137

  11. Larger number of invariant natural killer T cells in PBSC allografts correlates with improved GVHD-free and progression-free survival.

    PubMed

    Malard, Florent; Labopin, Myriam; Chevallier, Patrice; Guillaume, Thierry; Duquesne, Alix; Rialland, Fanny; Derenne, Sophie; Peterlin, Pierre; Leauté, Anne-Gaelle; Brissot, Eolia; Gregoire, Marc; Moreau, Philippe; Saas, Philippe; Gaugler, Béatrice; Mohty, Mohamad

    2016-04-01

    We studied the impact of a set of immune cells contained within granulocyte colony-stimulating factor-mobilized peripheral blood stem cell grafts (naïve and memory T-cell subsets, B cells, regulatory T cells, invariant natural killer T cells [iNKTs], NK cells, and dendritic cell subsets) in patients (n = 80) undergoing allogeneic stem cell transplantation (SCT), using the composite end point of graft-versus-host disease (GVHD)-free and progression-free survival (GPFS) as the primary end point. We observed that GPFS incidences in patients receiving iNKT doses above and below the median were 49% vs 22%, respectively (P= .007). In multivariate analysis, the iNKT dose was the only parameter with a significant impact on GPFS (hazard ratio = 0.48; 95% confidence interval, 0.27-0.85;P= .01). The incidences of severe grade III to IV acute GVHD and National Institutes of Health grade 2 to 3 chronic GVHD (12% and 16%, respectively) were low and associated with the use of antithymocyte globulin in 91% of patients. No difference in GVHD incidence was reported according to the iNKT dose. In conclusion, a higher dose of iNKTs within the graft is associated with an improved GPFS. These data may pave the way for prospective and active interventions aiming to manipulate the graft content to improve allo-SCT outcome.

  12. Invariant natural killer T cells and their ligands: focus on multiple sclerosis

    PubMed Central

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

    2015-01-01

    Invariant natural killer T (iNKT) cells are an innate population of T cells identified by the expression of an invariant T-cell receptor and reactivity to lipid-based antigens complexed with CD1d. They account for a small percentage of lymphocytes, but are extremely potent and play central roles in immunity to infection, in some cancers, and in autoimmunity. The list of relevant stimulatory lipids and glycolipid antigens now includes a range of endogenous self-antigens including the myelin-derived acetylated galactosylceramides. Recent progress in studies to identify the nature of lipid recognition for iNKT cells in autoimmune diseases like multiple sclerosis is likely to foster the development of therapeutic strategies aimed at harnessing iNKT cell activity. PMID:25976210

  13. CD252 regulates mast cell mediated, CD1d-restricted NKT-cell activation in mice.

    PubMed

    Gonzalez Roldan, Nestor; Orinska, Zane; Ewers, Hanno; Bulfone-Paus, Silvia

    2016-02-01

    The interaction between tissue-resident mast cells (MCs) and recruited immune cells contributes to tissue immunosurveillance. However, the cells, mechanisms, and receptors involved in this crosstalk remain ill defined. Invariant natural killer T (iNKT) cells are CD1d-restricted innate lymphocytes that recognize glycolipid antigens and have emerged as critical players in immunity. Here, we show that primary mouse peritoneal MCs express surface CD1d, which is upregulated in vivo following administration of alpha-galactosylceramide. In contrast, in BM-derived MCs CD1d was found to be stored intracellularly and to relocate at the cell surface upon IgE-mediated degranulation. Activated BM-derived MCs expressing surface CD1d and loaded with alpha-galactosylceramide were found to induce iNKT-cell proliferation and the release of IFN-γ, IL-13, and IL-4 in a CD1d-restricted manner. Moreover, the costimulatory molecules CD48, CD137L, CD252, CD274, and CD275 affected MC-induced IFN-γ release and iNKT-cell proliferation. Interestingly, among the costimulatory molecules, CD48 and CD252 exhibited a distinctly regulatory activity on iNKT-cell release of both IFN-γ and IL-13. In conclusion, we demonstrate that the crosstalk between MCs and iNKT cells may regulate inflammatory immune responses. PMID:26564814

  14. T-Cell-Specific Deletion of Map3k1 Reveals the Critical Role for Mekk1 and Jnks in Cdkn1b-Dependent Proliferative Expansion

    PubMed Central

    Suddason, Tesha; Anwar, Saba; Charlaftis, Nikolaos; Gallagher, Ewen

    2016-01-01

    Summary MAPK signaling is important for T lymphocyte development, homeostasis, and effector responses. To better understand the role of Mekk1 (encoded by Map3k1) in T cells, we conditionally deleted Map3k1 in LckCre/+Map3k1f/f mice, and these display larger iNKT cell populations within the liver, spleen, and bone marrow. Mekk1 signaling controls splenic and liver iNKT cell expansion in response to glycolipid antigen. LckCre/+Map3k1f/f mice have enhanced liver damage in response to glycolipid antigen. Mekk1 regulates Jnk activation in iNKT cells and binds and transfers Lys63-linked poly-ubiquitin onto Carma1. Map3k1 is critical for the regulation of p27Kip1 (encoded by Cdkn1b). PMID:26774476

  15. Innate immune control of EBV-infected B cells by invariant natural killer T cells.

    PubMed

    Chung, Brian K; Tsai, Kevin; Allan, Lenka L; Zheng, Dong Jun; Nie, Johnny C; Biggs, Catherine M; Hasan, Mohammad R; Kozak, Frederick K; van den Elzen, Peter; Priatel, John J; Tan, Rusung

    2013-10-10

    Individuals with X-linked lymphoproliferative disease lack invariant natural killer T (iNKT) cells and are exquisitely susceptible to Epstein-Barr virus (EBV) infection. To determine whether iNKT cells recognize or regulate EBV, resting B cells were infected with EBV in the presence or absence of iNKT cells. The depletion of iNKT cells increased both viral titers and the frequency of EBV-infected B cells. However, EBV-infected B cells rapidly lost expression of the iNKT cell receptor ligand CD1d, abrogating iNKT cell recognition. To determine whether induced CD1d expression could restore iNKT recognition in EBV-infected cells, lymphoblastoid cell lines (LCL) were treated with AM580, a synthetic retinoic acid receptor-α agonist that upregulates CD1d expression via the nuclear protein, lymphoid enhancer-binding factor 1 (LEF-1). AM580 significantly reduced LEF-1 association at the CD1d promoter region, induced CD1d expression on LCL, and restored iNKT recognition of LCL. CD1d-expressing LCL elicited interferon γ secretion and cytotoxicity by iNKT cells even in the absence of exogenous antigen, suggesting an endogenous iNKT antigen is expressed during EBV infection. These data indicate that iNKT cells may be important for early, innate control of B cell infection by EBV and that downregulation of CD1d may allow EBV to circumvent iNKT cell-mediated immune recognition.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25605948

  17. 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

    2015-02-01

    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.

  18. Adjuvant effects of invariant NKT cell ligand potentiates the innate and adaptive immunity to an inactivated H1N1 swine influenza virus vaccine in pigs.

    PubMed

    Dwivedi, Varun; Manickam, Cordelia; Dhakal, Santosh; Binjawadagi, Basavaraj; Ouyang, Kang; Hiremath, Jagadish; Khatri, Mahesh; Hague, Jacquelyn Gervay; Lee, Chang Won; Renukaradhya, Gourapura J

    2016-04-15

    Pigs are considered as the source of some of the emerging human flu viruses. Inactivated swine influenza virus (SwIV) vaccine has been in use in the US swine herds, but it failed to control the flu outbreaks. The main reason has been attributed to lack of induction of strong local mucosal immunity in the respiratory tract. Invariant natural killer T (iNKT) cell is a unique T cell subset, and activation of iNKT cell using its ligand α-Galactosylceramide (α-GalCer) has been shown to potentiate the cross-protective immunity to inactivated influenza virus vaccine candidates in mice. Recently, we discovered iNKT cell in pig and demonstrated its activation using α-GalCer. In this study, we evaluated the efficacy of an inactivated H1N1 SwIV coadministered with α-GalCer intranasally against a homologous viral challenge. Our results demonstrated the potent adjuvant effects of α-GalCer in potentiating both innate and adaptive immune responses to SwIV Ags in the lungs of pigs, which resulted in reduction in the lung viral load by 3 logs compared to without adjuvant. Immunologically, in the lungs of pigs vaccinated with α-GalCer an increased virus specific IgA response, IFN-α secretion and NK cell-cytotoxicity was observed. In addition, iNKT cell-stimulation enhanced the secretion of Th1 cytokines (IFN-γ and IL-12) and reduced the production of immunosuppressive cytokines (IL-10 and TGF-β) in the lungs of pigs⋅ In conclusion, we demonstrated for the first time iNKT cell adjuvant effects in pigs to SwIV Ags through augmenting the innate and adaptive immune responses in the respiratory tract.

  19. The tumor antigen N-glycolyl-GM3 is a human CD1d ligand capable of mediating B cell and natural killer T cell interaction.

    PubMed

    Gentilini, M Virginia; Pérez, M Eugenia; Fernández, Pablo Mariano; Fainboim, Leonardo; Arana, Eloísa

    2016-05-01

    The expression of N-glycolyl-monosialodihexosyl-ganglioside (NGcGM3) in humans is restricted to cancer cells; therefore, it is a tumor antigen. There are measurable quantities of circulating anti-NGcGM3 antibodies (aNGcGM3 Abs) in human serum. Interestingly, some people have circulating Ag-specific immunoglobulins G (IgGs) that are capable of complement mediated cytotoxicity against NGcGM3 positive cells, which is relevant for tumor surveillance. In light of the chemical nature of Ag, we postulated it as a candidate ligand for CD1d. Furthermore, we hypothesize that the immune mechanism involved in the generation of these Abs entails cross talk between B lymphocytes (Bc) and invariant natural killer T cells (iNKT). Combining cellular techniques, such as flow cytometry and biochemical assays, we demonstrated that CD1d binds to NGcGM3 and that human Bc present NGcGM3 in a CD1d context according to two alternative strategies. We also showed that paraformaldehyde treatment of cells expressing CD1d affects the presentation. Finally, by co-culturing primary human Bc with iNKT and measuring Ki-67 expression, we detected a reproducible increment in the proliferation of the iNKT population when Ag was on the medium. Our findings identify a novel, endogenous, human CD1d ligand, which is sufficiently competent to stimulate iNKT. We postulate that CD1d-restricted Bc presentation of NGcGM3 drives effective iNKT activation, an immunological mechanism that has not been previously described for humans, which may contribute to understanding aNGcGM3 occurrence.

  20. The tumor antigen N-glycolyl-GM3 is a human CD1d ligand capable of mediating B cell and natural killer T cell interaction.

    PubMed

    Gentilini, M Virginia; Pérez, M Eugenia; Fernández, Pablo Mariano; Fainboim, Leonardo; Arana, Eloísa

    2016-05-01

    The expression of N-glycolyl-monosialodihexosyl-ganglioside (NGcGM3) in humans is restricted to cancer cells; therefore, it is a tumor antigen. There are measurable quantities of circulating anti-NGcGM3 antibodies (aNGcGM3 Abs) in human serum. Interestingly, some people have circulating Ag-specific immunoglobulins G (IgGs) that are capable of complement mediated cytotoxicity against NGcGM3 positive cells, which is relevant for tumor surveillance. In light of the chemical nature of Ag, we postulated it as a candidate ligand for CD1d. Furthermore, we hypothesize that the immune mechanism involved in the generation of these Abs entails cross talk between B lymphocytes (Bc) and invariant natural killer T cells (iNKT). Combining cellular techniques, such as flow cytometry and biochemical assays, we demonstrated that CD1d binds to NGcGM3 and that human Bc present NGcGM3 in a CD1d context according to two alternative strategies. We also showed that paraformaldehyde treatment of cells expressing CD1d affects the presentation. Finally, by co-culturing primary human Bc with iNKT and measuring Ki-67 expression, we detected a reproducible increment in the proliferation of the iNKT population when Ag was on the medium. Our findings identify a novel, endogenous, human CD1d ligand, which is sufficiently competent to stimulate iNKT. We postulate that CD1d-restricted Bc presentation of NGcGM3 drives effective iNKT activation, an immunological mechanism that has not been previously described for humans, which may contribute to understanding aNGcGM3 occurrence. PMID:26969612

  1. CDR3β sequence motifs regulate autoreactivity of human invariant NKT cell receptors.

    PubMed

    Chamoto, Kenji; Guo, Tingxi; Imataki, Osamu; Tanaka, Makito; Nakatsugawa, Munehide; Ochi, Toshiki; Yamashita, Yuki; Saito, Akiko M; Saito, Toshiki I; Butler, Marcus O; Hirano, Naoto

    2016-04-01

    Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that recognize lipid ligands presented by monomorphic CD1d. Human iNKT T cell receptor (TCR) is largely composed of invariant Vα24 (Vα24i) TCRα chain and semi-variant Vβ11 TCRβ chain, where complementarity-determining region (CDR)3β is the sole variable region. One of the characteristic features of iNKT cells is that they retain autoreactivity even after the thymic selection. However, the molecular features of human iNKT TCR CDR3β sequences that regulate autoreactivity remain unknown. Since the numbers of iNKT cells with detectable autoreactivity in peripheral blood is limited, we introduced the Vα24i gene into peripheral T cells and generated a de novo human iNKT TCR repertoire. By stimulating the transfected T cells with artificial antigen presenting cells (aAPCs) presenting self-ligands, we enriched strongly autoreactive iNKT TCRs and isolated a large panel of human iNKT TCRs with a broad range autoreactivity. From this panel of unique iNKT TCRs, we deciphered three CDR3β sequence motifs frequently encoded by strongly-autoreactive iNKT TCRs: a VD region with 2 or more acidic amino acids, usage of the Jβ2-5 allele, and a CDR3β region of 13 amino acids in length. iNKT TCRs encoding 2 or 3 sequence motifs also exhibit higher autoreactivity than those encoding 0 or 1 motifs. These data facilitate our understanding of the molecular basis for human iNKT cell autoreactivity involved in immune responses associated with human disease. PMID:26748722

  2. Repeated Activation of Lung Invariant NKT Cells Results in Chronic Obstructive Pulmonary Disease-Like Symptoms.

    PubMed

    Tsao, Cheng-Chiu; Tsao, Po-Nien; Chen, Yi-Guang; Chuang, Ya-Hui

    2016-01-01

    Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation, mucus hypersecretion, and emphysema, which lead to reduced lung function and breathlessness. The pathologies of COPD are due to an abnormal immune response. Invariant natural killer T (iNKT) cells are an important population of innate lymphocytes and have been implicated in the regulation of immune responses associated with a broad range of diseases including COPD. We have here analyzed the role of iNKT cells in a model of COPD induced by repeated intranasal administration of iNKT cell agonist α-galactosylceramide (α-GalCer). Our results demonstrated that mice that received repeated intranasal administration of α-GalCer had molecular and inflammatory features of COPD including airway inflammation with significant increases in infiltration of macrophages and lymphocytes, CD8+ T cells, as well as proinflammatory cytokines IL-6 and TNF-α. In particular, these mice also showed the presence of pulmonary emphysema, mucus production, and pulmonary fibrosis. Furthermore, neutralization of IL-4 reduced α-GalCer induced emphysema. This study indicates the importance of iNKT cells in the pathogenesis of COPD by an IL-4 dependent mechanism.

  3. Repeated Activation of Lung Invariant NKT Cells Results in Chronic Obstructive Pulmonary Disease-Like Symptoms.

    PubMed

    Tsao, Cheng-Chiu; Tsao, Po-Nien; Chen, Yi-Guang; Chuang, Ya-Hui

    2016-01-01

    Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation, mucus hypersecretion, and emphysema, which lead to reduced lung function and breathlessness. The pathologies of COPD are due to an abnormal immune response. Invariant natural killer T (iNKT) cells are an important population of innate lymphocytes and have been implicated in the regulation of immune responses associated with a broad range of diseases including COPD. We have here analyzed the role of iNKT cells in a model of COPD induced by repeated intranasal administration of iNKT cell agonist α-galactosylceramide (α-GalCer). Our results demonstrated that mice that received repeated intranasal administration of α-GalCer had molecular and inflammatory features of COPD including airway inflammation with significant increases in infiltration of macrophages and lymphocytes, CD8+ T cells, as well as proinflammatory cytokines IL-6 and TNF-α. In particular, these mice also showed the presence of pulmonary emphysema, mucus production, and pulmonary fibrosis. Furthermore, neutralization of IL-4 reduced α-GalCer induced emphysema. This study indicates the importance of iNKT cells in the pathogenesis of COPD by an IL-4 dependent mechanism. PMID:26811900

  4. Interleukin-15 enhances the expansion and function of natural killer T cells from adult peripheral and umbilical cord blood.

    PubMed

    Lin, Syh-Jae; Huang, Ying-Cheng; Cheng, Po-Jen; Lee, Pei-Tzu; Hsiao, Hsiu-Shan; Kuo, Ming-Ling

    2015-12-01

    Invariant natural killer T cells (iNKT cells) are innate-like non-conventional T cells restricted by the CD1d molecule that are unique in their ability to play a pivotal role in immune regulation. Deficient iNKT function has been reported in patients receiving umbilical cord blood (UCB) transplantation. We sought to determine the effect of interleukin (IL)-15 on α-galactosylceramide (α-GalCer)-expanded iNKT cell function from UCB and adult peripheral blood (APB) mononuclear cells (MNCs). Fresh APB and UCB MNCs were cultured with IL-15 (50 ng/ml) in the presence or absence of α-GalCer (100 ng/ml) for 10 days. Cells were harvested for examination of cell yield, apoptosis, cytokine production and cytotoxic function of Vα24(+)/Vβ11(+) iNKT cells. We observed that α-GalCer-expanded APB and UCB iNKT cells and such expansion was further enhanced with IL-15. The percentage of CD3(+)CD56(+) NKT-like cells in both APB and UCB MNCs was increased with IL-15 but not with α-GalCer. Apoptosis of UCB iNKT cells was ameliorated by IL-15. Although APB and UCB iNKT cells secreted lower IFN-γ, it could be enhanced with IL-15. The expression of perforin in APB iNKT cells can also be enhanced with IL-15. UCB Vα24(+)Vβ11(+) iNKT cells further augmented K562 cytotoxicity mediated by IL-15. Taken together, these results demonstrated the relative functional deficiencies of α-GalCer induced UCB iNKT cells, which can be ameliorated by IL-15. Our findings suggest a therapeutic benefit of IL-15 immunotherapy during the post-UCB transplant period when iNKT function remains poor.

  5. Invariant NKT Cells Regulate the CD8 T Cell Response during Theiler's Virus Infection

    PubMed Central

    Mars, Lennart T.; Mas, Magali; Beaudoin, Lucie; Bauer, Jan; Leite-de-Moraes, Maria; Lehuen, Agnès; Bureau, Jean-Francois; Liblau, Roland S.

    2014-01-01

    Invariant NKT cells are innate lymphocytes with a broad tissue distribution. Here we demonstrate that iNKT cells reside in the central nervous system (CNS) in the absence of inflammation. Their presence in the CNS dramatically augments following inoculation of C57Bl/6 mice with the neurotropic Theiler's murine encephalomyelitis virus (TMEV). At the peak of inflammation the cellular infiltrate comprises 45 000 iNKT cells for 1 250 CD8 T cells specific for the immunodominant TMEV epitope. To study the interaction between these two T cell subsets, we infected both iNKT cell deficient Jα18-/- mice and iNKT cell enriched Vα14 transgenic mice with TMEV. The CD8 T cell response readily cleared TMEV infection in the iNKT cell deficient mice. However, in the iNKT cell enriched mice TMEV infection persisted and was associated with significant mortality. This was caused by the inhibition of the CD8 T cell response in the cervical lymph nodes and spleen after T cell priming. Taken together we demonstrate that iNKT cells reside in the CNS in the absence of inflammation and that their enrichment is associated with the inhibition of the anti-viral CD8 T cell response and an augmented mortality during acute encephalomyelitis. PMID:24498175

  6. Modulation of invariant natural killer T cell cytokine responses by indoleamine 2,3-dioxygenase

    PubMed Central

    Molano, Alberto; Illarionov, Petr A.; Besra, Gurdyal S.; Putterman, Chaim; Porcelli, Steven A.

    2008-01-01

    1. SUMMARY The intracellular enzyme indoleamine 2,3-dioxygenase (IDO), which degrades the rare and essential aminoacid tryptophan and converts it into a series of biologically active catabolites, has been linked to the regulation of immune tolerance by specific dendritic cell subsets, and to the downmodulation of exacerbated immune responses. Although the immunoregulatory effects of IDO may be in part due to generalized suppression of cell proliferation caused by tryptophan starvation, there is also evidence that tryptophan catabolites could be directly responsible for some of the observed effects. In this report, we investigated the consequences of IDO activity, particularly with regard to the effects of tryptophan-derived catabolites, on the cytokine responses of activated invariant natural killer T (iNKT) cells, a specialized T cell subset known to have immunoregulatory properties. Our results showed that pharmacologic inhibition of IDO skewed cytokine responses of iNKT cells towards a Th1 profile. In contrast, the presence at low micromolar concentrations of the tryptophan catabolites L-kynurenine, 3-hydroxy-kynurenine, or 3-hydroxy-anthranilic acid shifted the cytokine balance towards a Th2 pattern. These findings have implications for our current understanding of immunoregulation, and the mechanisms by which iNKT cells participate in the modulation of immune responses. PMID:18272236

  7. Mouse and Human CD1d-Self-Lipid Complexes Are Recognized Differently by Murine Invariant Natural Killer T Cell Receptors

    PubMed Central

    Guo, Tingxi; Chamoto, Kenji; Nakatsugawa, Munehide; Ochi, Toshiki; Yamashita, Yuki; Anczurowski, Mark; Butler, Marcus O.; Hirano, Naoto

    2016-01-01

    Invariant natural killer T (iNKT) cells recognize self-lipids presented by CD1d through characteristic TCRs, which mainly consist of the invariant Vα14-Jα18 TCRα chain and Vβ8.2, 7 or 2 TCRβ chains with hypervariable CDR3β sequences in mice. The iNKT cell-CD1d axis is conserved between humans and mice, and human CD1d reactivity of murine iNKT cells have been described. However, the detailed differences between the recognition of human and mouse CD1d bound to various self-lipids by mouse iNKT TCRs are largely unknown. In this study, we generated a de novo murine iNKT TCR repertoire with a wider range of autoreactivity compared with that of naturally occurring peripheral iNKT TCRs. Vβ8.2 mouse iNKT TCRs capable of recognizing the human CD1d-self-lipid tetramer were identified, although such clones were not detectable in the Vβ7 or Vβ2 iNKT TCR repertoire. In line with previously reports, clonotypic Vβ8.2 iNKT TCRs with unique CDR3β loops did not discriminate among lipids presented by mouse CD1d. Unexpectedly, however, these iNKT TCRs showed greater ligand selectivity toward human CD1d presenting the same lipids. Our findings demonstrated that the recognition of mouse and human CD1d-self-lipid complexes by murine iNKT TCRs is not conserved, thereby further elucidating the differences between cognate and cross-species reactivity of self-antigens by mouse iNKT TCRs. PMID:27213277

  8. TLR9-induced miR-155 and Ets-1 decrease expression of CD1d on B cells in SLE.

    PubMed

    Liu, Fei; Fan, Hongye; Ren, Deshan; Dong, Guanjun; Hu, Erling; Ji, Jianjian; Hou, Yayi

    2015-07-01

    B cells present lipid antigens to CD1d-restricted invariant natural killer T (iNKT) cells to maintain autoimmune tolerance, and this process is disrupted in systemic lupus erythematosus (SLE). Inflammation may inhibit CD1d expression to exacerbate the pathology of lupus. However, how inflammation regulates CD1d expression on B cells is unclear in SLE. In the present study, we showed that the surface expression of CD1d on B cells from SLE mice was decreased and that stimulation of inflammatory responses through TLR9 decreased the membrane and total CD1d levels of CD1d on B cells. Moreover, inflammation-related microRNA-155 (miR-155) negatively correlated with the expression of CD1d in B cells. miR-155 directly targeted the 3'-untranslated region (3'-UTR) of CD1d upon TLR9 activation in both humans and mice. The inhibitory effects of miR-155 on CD1d expression in B cells impaired their antigen-presenting capacity to iNKT cells. In addition, Ets-1, a susceptibility gene of SLE, also directly regulated the expression of the CD1d gene at the transcriptional level. These findings provide new insight into the mechanism underlying decreased CD1d expression on B cells in SLE, suggesting that inhibition of inflammation may increase CD1d expression in B cells to ameliorate SLE via modulating iNKT cells.

  9. 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

  10. An efferocytosis-induced, IL-4-dependent macrophage-iNKT cell circuit suppresses sterile inflammation and is defective in murine CGD.

    PubMed

    Zeng, Melody Yue; Pham, Duy; Bagaitkar, Juhi; Liu, Jianyun; Otero, Karel; Shan, Ming; Wynn, Thomas A; Brombacher, Frank; Brutkiewicz, Randy R; Kaplan, Mark H; Dinauer, Mary C

    2013-04-25

    Efferocytosis of apoptotic neutrophils by macrophages following tissue injury is fundamental to the resolution of inflammation and initiation of tissue repair. Using a sterile peritonitis model in mice, we identified interleukin (IL)-4-producing efferocytosing macrophages in the peritoneum that activate invariant natural killer T (iNKT) cells to produce cytokines including IL-4, IL-13, and interferon-γ. Importantly, IL-4 from macrophages contributes to alternative activation of peritoneal exudate macrophages and augments type 2 cytokine production from NKT cells to suppress inflammation. The increased peritonitis in mice deficient in IL-4, NKT cells, or IL-4Rα expression on myeloid cells suggested that each is a key component for resolution of sterile inflammation. The reduced NAD phosphate oxidase is also critical for this model, because in mice with X-linked chronic granulomatous disease (X-CGD) that lack oxidase subunits, activation of iNKT cells by X-CGD peritoneal exudate macrophages was impaired during sterile peritonitis, resulting in enhanced and prolonged inflammation in these mice. Therefore, efferocytosis-induced IL-4 production and activation of IL-4-producing iNKT cells by macrophages are immunomodulatory events in an innate immune circuit required to resolve sterile inflammation and promote tissue repair.

  11. Invariant natural killer T cells generated from human adult hematopoietic stem-progenitor cells are poly-functional.

    PubMed

    Sun, Wenji; Wang, Yi; East, James E; Kimball, Amy S; Tkaczuk, Katherine; Kesmodel, Susan; Strome, Scott E; Webb, Tonya J

    2015-03-01

    Invariant natural killer T (iNKT) cells constitute an important subset of T cells that can both directly and indirectly mediate anti-tumor immunity. However, cancer patients have a reduction in both iNKT cell number and function, and these deficits limit the potential clinical application of iNKT cells for cancer therapy. To overcome the problem of limited iNKT cell numbers, we investigated whether iNKT cells can be generated in vitro from bone marrow-derived adult hematopoietic stem-progenitor cells (HSPC). Our data demonstrate that co-culture of HSPC with OP9-DL1 stromal cells, results in a functional CD3(+) T cell population. These T cells can be further differentiated into iNKT cells by secondary culture with CD1d-Ig-based artificial antigen-presenting cells (aAPC). Importantly, these in vitro-generated iNKT cells are functional, as demonstrated by their ability to proliferate and secrete IFN-γ and GM-CSF following stimulation. PMID:25569376

  12. Invariant natural killer T cells generated from human adult hematopoietic stem-progenitor cells are poly-functional

    PubMed Central

    Sun, Wenji; Wang, Yi; East, James E.; Kimball, Amy S.; Tkaczuk, Katherine; Kesmodel, Susan; Strome, Scott E.; Webb, Tonya J.

    2014-01-01

    Invariant natural killer T (iNKT) cells constitute an important subset of T cells that can both directly and indirectly mediate anti-tumor immunity. However, cancer patients have a reduction in both iNKT cell number and function, and these deficits limit the potential clinical application of iNKT cells for cancer therapy. To overcome the problem of limited iNKT cell numbers, we investigated whether iNKT cells can be generated in vitro from bone marrow-derived adult hematopoietic stem-progenitor cells (HSPC). Our data demonstrate that co-culture of HSPC with OP9-DL1 stromal cells, results in a functional CD3+ T cell population. These T cells can be further differentiated into iNKT cells by secondary culture with CD1d-Ig-based artificial antigen-presenting cells (aAPC). Importantly, these in vitro-generated iNKT cells are functional, as demonstrated by their ability to proliferate and secrete IFN-γ and GM-CSF following stimulation. PMID:25569376

  13. Expression of CD11c Is Associated with Unconventional Activated T Cell Subsets with High Migratory Potential.

    PubMed

    Qualai, Jamal; Li, Lin-Xi; Cantero, Jon; Tarrats, Antoni; Fernández, Marco Antonio; Sumoy, Lauro; Rodolosse, Annie; McSorley, Stephen J; Genescà, Meritxell

    2016-01-01

    CD11c is an α integrin classically employed to define myeloid dendritic cells. Although there is little information about CD11c expression on human T cells, mouse models have shown an association of CD11c expression with functionally relevant T cell subsets. In the context of genital tract infection, we have previously observed increased expression of CD11c in circulating T cells from mice and women. Microarray analyses of activated effector T cells expressing CD11c derived from naïve mice demonstrated enrichment for natural killer (NK) associated genes. Here we find that murine CD11c+ T cells analyzed by flow cytometry display markers associated with non-conventional T cell subsets, including γδ T cells and invariant natural killer T (iNKT) cells. However, in women, only γδ T cells and CD8+ T cells were enriched within the CD11c fraction of blood and cervical tissue. These CD11c+ cells were highly activated and had greater interferon (IFN)-γ secretory capacity than CD11c- T cells. Furthermore, circulating CD11c+ T cells were associated with the expression of multiple adhesion molecules in women, suggesting that these cells have high tissue homing potential. These data suggest that CD11c expression distinguishes a population of circulating T cells during bacterial infection with innate capacity and mucosal homing potential. PMID:27119555

  14. Expression of CD11c Is Associated with Unconventional Activated T Cell Subsets with High Migratory Potential

    PubMed Central

    Cantero, Jon; Tarrats, Antoni; Fernández, Marco Antonio; Sumoy, Lauro; Rodolosse, Annie; McSorley, Stephen J.

    2016-01-01

    CD11c is an α integrin classically employed to define myeloid dendritic cells. Although there is little information about CD11c expression on human T cells, mouse models have shown an association of CD11c expression with functionally relevant T cell subsets. In the context of genital tract infection, we have previously observed increased expression of CD11c in circulating T cells from mice and women. Microarray analyses of activated effector T cells expressing CD11c derived from naïve mice demonstrated enrichment for natural killer (NK) associated genes. Here we find that murine CD11c+ T cells analyzed by flow cytometry display markers associated with non-conventional T cell subsets, including γδ T cells and invariant natural killer T (iNKT) cells. However, in women, only γδ T cells and CD8+ T cells were enriched within the CD11c fraction of blood and cervical tissue. These CD11c+ cells were highly activated and had greater interferon (IFN)-γ secretory capacity than CD11c- T cells. Furthermore, circulating CD11c+ T cells were associated with the expression of multiple adhesion molecules in women, suggesting that these cells have high tissue homing potential. These data suggest that CD11c expression distinguishes a population of circulating T cells during bacterial infection with innate capacity and mucosal homing potential. PMID:27119555

  15. 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.

  16. 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

  17. Artificial antigen presenting cell (aAPC) mediated activation and expansion of natural killer T cells.

    PubMed

    East, James E; Sun, Wenji; Webb, Tonya J

    2012-01-01

    Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells(1). Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules(2). NKT cells express an invariant TCRα chain rearrangement: Vα14Jα18 in mice and Vα24Jα18 in humans, which is associated with Vβ chains of limited diversity(3-6), and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d (7). The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with α-Galactosylceramide (α-GalCer) and a variety of cytokines(8). Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines(9,10). In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients(11). However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells(12, 13). Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is

  18. 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.

  19. CD155/CD226-interaction impacts on the generation of innate CD8(+) thymocytes by regulating iNKT-cell differentiation.

    PubMed

    Georgiev, Hristo; Ravens, Inga; Shibuya, Akira; Förster, Reinhold; Bernhardt, Günter

    2016-04-01

    The cell surface receptor CD155 influences a variety of immune processes by binding to its ligands CD226, CD96, or TIGIT. Here, we report that the interaction of CD155 with CD226 in the thymus of BALB/c mice has a dual function. It directly influences the dwell time of memory-like CD8(+) T cells, while it is indirectly involved in generating these cells. It was shown earlier that a massive emergence of memory-like CD8 T cells in thymus crucially depends on abundant IL-4, secreted in steady state by iNKT2 (where iNKT is invariant NKT) cells, a subclass of iNKT cells. Here, we show that absence of either CD155 or CD226 in BALB/c mice causes a profound shift in the iNKT subtype composition in thymus, expanding the frequency and numbers of iNKT1 cells at the expense of iNKT2 cells, as well as iNKT17 cells. This shift results in a drop of available IL-4 and creates a scenario similar to that observed in C57BL/6 mice, where iNKT1 cells predominate and iNKT2 cells are much less frequent when compared with BALB/c mice. Yet also in C57BL/6 mice, lack of CD155 or CD226 provokes a further decline in iNKT2 cells, suggesting that the observed effects are not restricted to a particular inbred strain. PMID:26689152

  20. Innate lymphoid cells and natural killer T cells in the gastrointestinal tract immune system.

    PubMed

    Montalvillo, Enrique; Garrote, José Antonio; Bernardo, David; Arranz, Eduardo

    2014-05-01

    The gastrointestinal tract is equipped with a highly specialized intrinsic immune system. However, the intestine is exposed to a high antigenic burden that requires a fast, nonspecific response -so-called innate immunity- to maintain homeostasis and protect the body from incoming pathogens. In the last decade multiple studies helped to unravel the particular developmental requirements and specific functions of the cells that play a role in innate immunity. In this review we shall focus on innate lymphoid cells, a newly discovered, heterogeneous set of cells that derive from an Id2-dependent lymphoid progenitor cell population. These cells have been categorized on the basis of the pattern of cytokines that they secrete, and the transcription factors that regulate their development and functions. Innate lymphoid cells play a role in the early response to pathogens, the anatomical contention of the commensal flora, and the maintenance of epithelial integrity.Amongst the various innate lymphoid cells we shall lay emphasis on a subpopulation with several peculiarities, namely that of natural killer T cells, a subset of T lymphocytes that express both T-cell and NK-cell receptors. The most numerous fraction of the NKT population are the so-called invariant NKT or iNKT cells. These iNKT cells have an invariant TCR and recognize the glycolipidic structures presented by the CD1d molecule, a homolog of class-I MHC molecules. Following activation they rapidly acquire cytotoxic activity and secrete both Th1 and Th2 cytokines, including IL-17. While their specific role is not yet established, iNKT cells take part in a great variety of intestinal immune responses ranging from oral tolerance to involvement in a number of gastrointestinal conditions.

  1. Natural killer cells and natural killer T cells in Lyme arthritis

    PubMed Central

    2013-01-01

    Introduction Natural killer (NK) and natural killer T (NKT) cells provide a first line of defense against infection. However, these cells have not yet been examined in patients with Lyme arthritis, a late disease manifestation. Lyme arthritis usually resolves with antibiotic treatment. However, some patients have persistent arthritis after spirochetal killing, which may result from excessive inflammation, immune dysregulation and infection-induced autoimmunity. Methods We determined the frequencies and phenotypes of NK cells and invariant NKT (iNKT) cells in paired peripheral blood (PB) and synovial fluid (SF) samples from eight patients with antibiotic-responsive arthritis and fifteen patients with antibiotic-refractory arthritis using flow cytometry and cytokine analyses. Results In antibiotic-responsive patients, who were seen during active infection, high frequencies of CD56bright NK cells were found in SF, the inflammatory site, compared with PB (P <0.001); at both sites, a high percentage of cells expressed the activation receptor NKG2D and the chaperone CD94, a low percentage expressed inhibitory killer immunoglobulin-like receptors (KIR), and a high percentage produced IFN-γ. In antibiotic-refractory patients, who were usually evaluated near the conclusion of antibiotics when few if any live spirochetes remained, the phenotype of CD56bright cells in SF was similar to that in patients with antibiotic-responsive arthritis, but the frequency of these cells was significantly less (P = 0.05), and the frequencies of CD56dim NK cells tended to be higher. However, unlike typical NKdim cells, these cells produced large amounts of IFN-γ, suggesting that they were not serving a cytotoxic function. Lastly, iNKT cell frequencies in the SF of antibiotic-responsive patients were significantly greater compared with that of antibiotic-refractory patients where these cells were often absent (P = 0.003). Conclusions In patients with antibiotic-responsive arthritis

  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. CD4+ invariant natural killer T cells protect from murine GVHD lethality through expansion of donor CD4+CD25+FoxP3+ regulatory T cells.

    PubMed

    Schneidawind, Dominik; Pierini, Antonio; Alvarez, Maite; Pan, Yuqiong; Baker, Jeanette; Buechele, Corina; Luong, Richard H; Meyer, Everett H; Negrin, Robert S

    2014-11-20

    Dysregulated donor T cells lead to destruction of host tissues resulting in graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). We investigated the impact of highly purified (>95%) donor CD4(+) invariant natural killer T (iNKT) cells on GVHD in a murine model of allogeneic HCT. We found that low doses of adoptively transferred donor CD4(+) iNKT cells protect from GVHD morbidity and mortality through an expansion of donor CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). These Tregs express high levels of the Ikaros transcription factor Helios and expand from the Treg pool of the donor graft. Furthermore, CD4(+) iNKT cells preserve T-cell-mediated graft-versus-tumor effects. Our studies reveal new aspects of the cellular interplay between iNKT cells and Tregs in the context of tolerance induction after allogeneic HCT and set the stage for clinical translation. PMID:25293774

  4. Differential dependence on nuclear factor-κB-inducing kinase among natural killer T-cell subsets in their development

    PubMed Central

    Noma, Haruka; Eshima, Koji; Satoh, Masashi; Iwabuchi, Kazuya

    2015-01-01

    Natural killer T cells (NKT cells) are comprised of several subsets. However, the possible differences in their developmental mechanisms have not been fully investigated. To evaluate the dependence of some NKT subpopulations on nuclear factor-κB-inducing kinase (NIK) for their generation, we analysed the differentiation of NKT cells, dividing them into subsets in various tissues of alymphoplasia (aly/aly), a mutant mouse strain that lacks functional NIK. The results indicated that the efficient differentiation of both invariant NKT (iNKT) and non-iNKT cells relied on NIK expression in non-haematopoietic cells; however, the dependence of non-iNKT cells was lower than that of iNKT cells. Especially, the differentiation of CD8+ non-iNKT cells was markedly resistant to the aly mutation. The proportion of two other NKT cell subsets, NK1.1+ γδ T cells and NK1.1− iNKT cells, was also significantly reduced in aly/aly mice, and this defect in their development was reversed in wild-type host mice given aly/aly bone marrow cells. In exerting effector functions, NIK in NKT-αβ cells appeared dispensable, as NIK-deficient NKT-αβ cells could secrete interleukin-4 or interferon-γ and exhibit cytolytic activity at a level comparable to that of aly/+ NKT-αβ cells. Collectively, these results imply that the NIK in thymic stroma may be critically involved in the differentiation of most NKT cell subsets (although the level of NIK dependence may vary among the subsets), and also that NIK in NKT-αβ cells may be dispensable for their effector function. PMID:25988531

  5. Antigen mRNA-transfected, allogeneic fibroblasts loaded with NKT-cell ligand confer antitumor immunity.

    PubMed

    Fujii, Shin-ichiro; Goto, Akira; Shimizu, Kanako

    2009-04-30

    The maturation of dendritic cells (DCs) in situ by danger signals plays a central role in linking innate and adaptive immunity. We previously demonstrated that the activation of invariant natural killer T (iNKT) cells by administration of alpha-galactosylceramide (alpha-GalCer)-loaded tumor cells can act as a cellular adjuvant through the DC maturation. In the current study, we used allogeneic fibroblasts loaded with alpha-GalCer and transfected with antigen-encoding mRNA, thus combining the adjuvant effects of iNKT-cell activation with delivery of antigen to DCs in vivo. We found that these cells produce antigen protein and activate NK and iNKT cells. When injected into major histocompatibility complex (MHC)-mismatched mice, they elicited antigen-specific T-cell responses and provided tumor protection, suggesting that these immune responses depend on host DCs. In addition, antigen-expressing fibroblasts loaded with alpha-GalCer lead to a more potent T-cell response than those expressing NK cell ligands. Thus, glycolipid-loaded, mRNA-transfected allogeneic fibroblasts act as cellular vectors to provide iNKT-cell activation, leading to DC maturation and T-cell immunity. By harnessing the innate immune system and generating an adaptive immune response to a variety of antigens, this unique tool could prove clinically beneficial in the development of immunotherapies against malignant and infectious diseases. PMID:19164596

  6. Human CD1d knock-in mouse model demonstrates potent antitumor potential of human CD1d-restricted invariant natural killer T cells

    PubMed Central

    Wen, Xiangshu; Rao, Ping; Carreño, Leandro J.; Kim, Seil; Lawrenczyk, Agnieszka; Porcelli, Steven A.; Cresswell, Peter; Yuan, Weiming

    2013-01-01

    Despite a high degree of conservation, subtle but important differences exist between the CD1d antigen presentation pathways of humans and mice. These differences may account for the minimal success of natural killer T (NKT) cell-based antitumor therapies in human clinical trials, which contrast strongly with the powerful antitumor effects in conventional mouse models. To develop an accurate model for in vivo human CD1d (hCD1d) antigen presentation, we have generated a hCD1d knock-in (hCD1d-KI) mouse. In these mice, hCD1d is expressed in a native tissue distribution pattern and supports NKT cell development. Reduced numbers of invariant NKT (iNKT) cells were observed, but at an abundance comparable to that in most normal humans. These iNKT cells predominantly expressed mouse Vβ8, the homolog of human Vβ11, and phenotypically resembled human iNKT cells in their reduced expression of CD4. Importantly, iNKT cells in hCD1d knock-in mice exert a potent antitumor function in a melanoma challenge model. Our results show that replacement of mCD1d by hCD1d can select a population of functional iNKT cells closely resembling human iNKT cells. These hCD1d knock-in mice will allow more accurate in vivo modeling of human iNKT cell responses and will facilitate the preclinical assessment of iNKT cell-targeted antitumor therapies. PMID:23382238

  7. Chronic alcohol consumption inhibits melanoma growth but decreases the survival of mice immunized with tumor cell lysate and boosted with α-galactosylceramide

    PubMed Central

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

    2015-01-01

    Alcohol consumption increases the incidence of multiple types of cancer. However, how chronic alcohol consumption affects tumor progression and host survival remains largely unexplored. Using a mouse B16BL6 melanoma model, we studied the effects of chronic alcohol consumption on s.c. tumor growth, iNKT cell antitumor immune response, and host survival. The results indicate that although chronic alcohol consumption inhibits melanoma growth, this does not translate into increased host survival. Immunizing mice with a melanoma cell lysate does not significantly increase the median survival of water-drinking, melanoma-bearing mice, but significantly increases the median survival of alcohol-consuming, melanoma-bearing mice. Even though survival is extended in the alcohol-consuming mice after immunization, the mean survival is not different from the immunized mice in the water-drinking group. Immunization with tumor cell lysate combined with α-galatosylceramide activation of iNKT cells significantly increases host survival of both groups of melanoma-bearing mice compared to their respective non-immunized counterparts; however, the median survival of the alcohol-consuming group is significantly lower than that of the water-drinking group. Alcohol consumption increases NKT cells in the thymus and blood and skews NKT cell cytokine profile from Th1 dominant to Th2 dominant in the tumor-bearing mice. In summary, these results indicate that chronic alcohol consumption activates the immune system, which leads to the inhibition of s.c. melanoma growth and enhances the immune response to immunization with melanoma lysate. With tumor progression, alcohol consumption accelerates iNKT cell dysfunction and compromises antitumor immunity, which leads to decreased survival of melanoma-bearing mice. PMID:26118634

  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. Cell sedimentation with gravity activation.

    PubMed

    Czerlinski, G; Goldman-Leikin, R; Reid, D

    1988-12-01

    Murine monoclonal antibody T101 has been coupled to thinly polymer-coated heavy alloy particles (LaMn2Ge2). These conjugates are coupled to cultured cells of the human T-cell leukemia line RPMI 8402 (T8402). The sedimentation velocities of cells, of particles, and of cells with particles attached are measured. After determining the mean radii of cells, of particles, and of cells with particles attached, one may compute a mean number of 33 particles attached to a cell. Independently one may compute a mean number of 144 particles/cell for surface saturation. The Appendix handles the underlying theory in three parts: number of particles/cell, saturation number of particles/cell, and resolution for gravity activation. Regarding the latter, cell radii from 4 to 10 microns and particle radii from 0.01 to 1 micron are considered.

  11. 504 Participation of Invariant NKT Cells (Vα24Jα18) during Asthma Exacerbation in Children

    PubMed Central

    Carpio-Pedroza, Juan Carlos; del Rio-Navarro, Blanca Estela; del Río-Chivardí, Jaime Mariano; Morales-Flores, Amelia; Jiménez-Zamudio, Luis Antonio; Moreno-Lafont, Martha; Pedraza-Sánchez, Sigifredo; Vaughan-Figueroa, Juan Gilberto; Escobar-Gutiérrez, Alejandro

    2012-01-01

    Background Invariant NKT cells (or type 1 NKT cells) co-express CD3 marker and NK receptors (CD56, CD161) and use a single type of TCRα chain (Vα24Jα18 for humans), comprising CD4-CD8-, CD4+ and CD8+ subsets. Participation of these cells and their cytokines in asthmatic children, in stable conditions and under exacerbation, was studied. Methods Three groups on children (6–12 years old) were selected: 1) asthmatics under exacerbation attack (AE) within the first 24 hours after the attack and before starting any treatment; 2) asthmatics with stable asthma (SA), without symptoms for at least a month before bleeding; and 3) healthy controls (HC) without history of asthma, atopy and with normal lung function were selected in the Allergy and Clinical Immunology Service, Hospital Infantil de Mexico. Invariant NKT cells and subset levels as well as intracellular cytokines were evaluated in whole blood by 4-color flow cytometry (antibodies against CD3, CD4, CD8, CD161, Va24, IL-4 and IFN-g). Results Proportion of iNKT cells among total CD3+ cells in HC group was 0.9%, while in SA patients they were increased up to 2.6%; interestingly, during exacerbation such cells were dimished (1.8%). Concerning iNKT CD4+ cells were 0.6% in HC, 1.8% in SA, and 0.7% in AE, while iNKT CD8+ cells were 0.1% in HC, 0.7% in SA, and 0.4% in AE. Both iNKT cell subsets expressed intracellular IFN-g and IL-4 cytokines in AE, SA and HC but predominantly IFN-g in iNKT CD8+ cells from AE patients. Conclusions iNKT cells participation in asthma pathogenesis was confirmed. Increase of IFN-g production in patients with exacerbations, may provide a regulatory environment to stabilize the condition.

  12. Cernunnos Deficiency Reduces Thymocyte Life Span and Alters the T Cell Repertoire in Mice and Humans

    PubMed Central

    Vera, Gabriella; Rivera-Munoz, Paola; Abramowski, Vincent; Malivert, Laurent; Lim, Annick; Bole-Feysot, Christine; Martin, Christelle; Florkin, Benoit; Latour, Sylvain; Revy, Patrick

    2013-01-01

    Cernunnos is a DNA repair factor of the nonhomologous end-joining machinery. Its deficiency in humans causes radiosensitive severe combined immune deficiency (SCID) with microcephaly, characterized in part by a profound lymphopenia. In contrast to the human condition, the immune system of Cernunnos knockout (KO) mice is not overwhelmingly affected. In particular, Cernunnos is dispensable during V(D)J recombination in lymphoid cells. Nevertheless, the viability of thymocytes is reduced in Cernunnos KO mice, owing to the chronic activation of a P53-dependent DNA damage response. This translates into a qualitative alteration of the T cell repertoire to one in which the most distal Vα and Jα segments are missing. This results in the contraction of discrete T cell populations, such as invariant natural killer T (iNKT) and mucosa-associated invariant T (MAIT) cells, in both humans and mice. PMID:23207905

  13. 3-fluoro- and 3,3-difluoro-3,4-dideoxy-KRN7000 analogues as new potent immunostimulator agents: total synthesis and biological evaluation in human invariant natural killer T cells and mice.

    PubMed

    Hunault, Julie; Diswall, Mette; Frison, Jean-Cédric; Blot, Virginie; Rocher, Jézabel; Marionneau-Lambot, Séverine; Oullier, Thibauld; Douillard, Jean-Yves; Guillarme, Stéphane; Saluzzo, Christine; Dujardin, Gilles; Jacquemin, Denis; Graton, Jérôme; Le Questel, Jean-Yves; Evain, Michel; Lebreton, Jacques; Dubreuil, Didier; Le Pendu, Jacques; Pipelier, Muriel

    2012-02-01

    We propose here the synthesis and biological evaluation of 3,4-dideoxy-GalCer derivatives. The absence of the 3- and 4-hydroxyls on the sphingoid base is combined with the introduction of mono or difluoro substituent at C3 (analogues 8 and 9, respectively) to evaluate their effect on the stability of the ternary CD1d/GalCer/TCR complex which strongly modulate the immune responses. Biological evaluations were performed in vitro on human cells and in vivo in mice and results discussed with support of modeling studies. The fluoro 3,4-dideoxy-GalCer analogues appears as partial agonists compared to KRN7000 for iNKT cell activation, inducing T(H)1 or T(H)2 biases that strongly depend of the mode of antigen presentation, including human vs mouse differences. We evidenced that if a sole fluorine atom is not able to balance the loss of the 3-OH, the presence of a difluorine group at C3 of the sphingosine can significantly restore human iNKT activation.

  14. Acoustofluidic Fluorescence Activated Cell Sorter.

    PubMed

    Nawaz, Ahmad Ahsan; Chen, Yuchao; Nama, Nitesh; Nissly, Ruth Helmus; Ren, Liqiang; Ozcelik, Adem; Wang, Lin; McCoy, J Philip; Levine, Stewart J; Huang, Tony Jun

    2015-12-15

    Selective isolation of cell subpopulations with defined biological characteristics is crucial for many biological studies and clinical applications. In this work, we present the development of an acoustofluidic fluorescence activated cell sorting (FACS) device that simultaneously performs on-demand, high-throughput, high-resolution cell detection and sorting, integrated onto a single chip. Our acoustofluidic FACS device uses the "microfluidic drifting" technique to precisely focus cells/particles three dimensionally and achieves a flow of single-file particles/cells as they pass through a laser interrogation region. We then utilize short bursts (150 μs) of standing surface acoustic waves (SSAW) triggered by an electronic feedback system to sort fluorescently labeled particles/cells with desired biological properties. We have demonstrated continuous isolation of fluorescently labeled HeLa cells from unlabeled cells at a throughput of ∼1200 events/s with a purity reaching 92.3 ± 3.39%. Furthermore, 99.18% postsort cell viability indicates that our acoustofluidic sorting technique maintains a high integrity of cells. Therefore, our integrated acoustofluidic FACS device is demonstrated to achieve two-way cell sorting with high purity, biocompatibility, and biosafety. We believe that our device has significant potential for use as a low-cost, high-performance, portable, and user-friendly FACS instrument.

  15. 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.

  16. 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

  17. Tracking and treating activated T cells

    PubMed Central

    Kim, N.H.; Nadithe, V.; Elsayed, M.; Merkel, O.M.

    2014-01-01

    Upon activation, T cells of various subsets are the most important mediators in cell-mediated immune responses. Activated T cells play an important role in immune system related diseases such as chronic inflammatory diseases, viral infections, autoimmune disease, transplant rejection, Crohn disease, diabetes, and many more. Therefore, efforts have been made to both visualize and treat activated T cells specifically. This review summarizes imaging approaches and selective therapeutics for activated T cells and gives an outlook on how tracking and treating can be combined into theragnositc agents for activated T cells. PMID:24660025

  18. Viral Evasion of Natural Killer Cell Activation

    PubMed Central

    Ma, Yi; Li, Xiaojuan; Kuang, Ersheng

    2016-01-01

    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. PMID:27077876

  19. 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.

  20. Salivary gland NK cells are phenotypically and functionally unique.

    PubMed

    Tessmer, Marlowe S; Reilly, Emma C; Brossay, Laurent

    2011-01-13

    Natural killer (NK) cells and CD8(+) T cells play vital roles in containing and eliminating systemic cytomegalovirus (CMV). However, CMV has a tropism for the salivary gland acinar epithelial cells and persists in this organ for several weeks after primary infection. Here we characterize a distinct NK cell population that resides in the salivary gland, uncommon to any described to date, expressing both mature and immature NK cell markers. Using RORγt reporter mice and nude mice, we also show that the salivary gland NK cells are not lymphoid tissue inducer NK-like cells and are not thymic derived. During the course of murine cytomegalovirus (MCMV) infection, we found that salivary gland NK cells detect the infection and acquire activation markers, but have limited capacity to produce IFN-γ and degranulate. Salivary gland NK cell effector functions are not regulated by iNKT or T(reg) cells, which are mostly absent in the salivary gland. Additionally, we demonstrate that peripheral NK cells are not recruited to this organ even after the systemic infection has been controlled. Altogether, these results indicate that viral persistence and latency in the salivary glands may be due in part to the presence of unfit NK cells and the lack of recruitment of peripheral NK cells.

  1. Dormancy activation mechanism of tracheal stem cells

    PubMed Central

    Li, Xin; Xu, Jing-xian; Jia, Xin-Shan; Li, Wen-ya; Han, Yi-chen; Wang, En-hua; Li, Fang

    2016-01-01

    Accurate markers and molecular mechanisms of stem cell dormancy and activation are poorly understood. In this study, the anti-cancer drug, 5-fluorouracil, was used to selectively kill proliferating cells of human bronchial epithelial (HBE) cell line. This method can enrich and purify stem cell population. The dormant versus active status of stem cells was determined by phosphorylation of RNAp II Ser2. The surviving stem cells were cultured to form stem cell spheres expressing stem cell markers and transplanted into nude mice to form a teratoma. The results demonstrated the properties of stem cells and potential for multi-directional differentiation. Bisulfite sequencing polymerase chain reaction showed that demethylation of the Sox2 promoter by 5-FU resulted in Sox2 expression in the dormant stem cells. This study shows that the dormancy and activation of HBE stem cells is closely related to epigenetic modification. PMID:27009861

  2. 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

  3. Immunoregulation of NKT Cells in Systemic Lupus Erythematosus.

    PubMed

    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

  4. 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

  5. CRISPR-Mediated Triple Knockout of SLAMF1, SLAMF5 and SLAMF6 Supports Positive Signaling Roles in NKT Cell Development

    PubMed Central

    Huang, Bonnie; Gomez-Rodriguez, Julio; Preite, Silvia; Garrett, Lisa J.; Harper, Ursula L.; Schwartzberg, Pamela L.

    2016-01-01

    The SLAM family receptors contribute to diverse aspects of lymphocyte biology and signal via the small adaptor molecule SAP. Mutations affecting SAP lead to X-linked lymphoproliferative syndrome Type 1, a severe immunodysregulation characterized by fulminant mononucleosis, dysgammaglobulinemia, and lymphoproliferation/lymphomas. Patients and mice having mutations affecting SAP also lack germinal centers due to a defect in T:B cell interactions and are devoid of invariant NKT (iNKT) cells. However, which and how SLAM family members contribute to these phenotypes remains uncertain. Three SLAM family members: SLAMF1, SLAMF5 and SLAMF6, are highly expressed on T follicular helper cells and germinal center B cells. SLAMF1 and SLAMF6 are also implicated in iNKT development. Although individual receptor knockout mice have limited iNKT and germinal center phenotypes compared to SAP knockout mice, the generation of multi-receptor knockout mice has been challenging, due to the genomic linkage of the genes encoding SLAM family members. Here, we used Cas9/CRISPR-based mutagenesis to generate mutations simultaneously in Slamf1, Slamf5 and Slamf6. Genetic disruption of all three receptors in triple-knockout mice (TKO) did not grossly affect conventional T or B cell development and led to mild defects in germinal center formation post-immunization. However, the TKO worsened defects in iNKT cells development seen in SLAMF6 single gene-targeted mice, supporting data on positive signaling and potential redundancy between these receptors. PMID:27258160

  6. Antibacterial activity of human natural killer cells

    PubMed Central

    1989-01-01

    The in vitro effects of human NK cells on viability of Gram-negative and Gram-positive bacteria was investigated. PBLs depleted of glass- adherent cells showed a significant antibacterial activity that was increased as the concentration of NK cells became higher. Leu-11- enriched cells exhibited the most efficient bactericidal activity. Stimulation of NK cells with staphylococcal enterotoxin B for 16 h produced a significant increase in the antibacterial activity of all NK cells tested. The antibacterial activity of monocyte-depleted cells and Leu-11-enriched cells was also enhanced after culturing in vitro for 16- 24 h without exogenous cytokines. Dependence of the antibacterial activity on the presence of serum in the culture medium was not found. Ultrastructural studies revealed close contact between NK cell membranes and bacteria, no evidence of phagocytosis, and extracellular bacterial ghosts, after incubation at 37 degrees C. Supernatants from purified NK cells exhibited potent bactericidal activity with kinetics and target specificity similar to that of effector cells. These results document the potent antibacterial activity of purified NK cells and suggest an extracellular mechanism of killing. PMID:2642532

  7. Myosin II Activity Softens Cells in Suspension.

    PubMed

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

    2015-04-21

    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

  8. 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

  9. Myosin II Activity Softens Cells in Suspension.

    PubMed

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

    2015-04-21

    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.

  10. 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.

  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. Cell death sensitization of leukemia cells by opioid receptor activation

    PubMed Central

    Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A.; Debatin, Klaus-Michael; Miltner, Erich

    2013-01-01

    Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies. PMID:23633472

  13. Stem cell tracking with optically active nanoparticles

    PubMed Central

    Gao, Yu; Cui, Yan; Chan, Jerry KY; Xu, Chenjie

    2013-01-01

    Stem-cell-based therapies hold promise and potential to address many unmet clinical needs. Cell tracking with modern imaging modalities offers insight into the underlying biological process of the stem-cell-based therapies, with the goal to reveal cell survival, migration, homing, engraftment, differentiation, and functions. Adaptability, sensitivity, resolution, and non-invasiveness have contributed to the longstanding use of optical imaging for stem cell tracking and analysis. To identify transplanted stem cells from the host tissue, optically active probes are usually used to label stem cells before the administration. In comparison to the traditional fluorescent probes like fluorescent proteins and dyes, nanoparticle-based probes are advantageous in terms of the photo-stabilities and minimal changes to the cell phenotype. The main focus here is to overview the recent development of optically active nanoparticles for stem cells tracking. The related optical imaging modalities include fluorescence imaging, photoacoustic imaging, Raman and surface enhanced Raman spectroscopy imaging. PMID:23638335

  14. 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.

  15. Estrogen Therapy Delays Autoimmune Diabetes and Promotes the Protective Efficiency of Natural Killer T-Cell Activation in Female Nonobese Diabetic Mice.

    PubMed

    Gourdy, Pierre; Bourgeois, Elvire A; Levescot, Anaïs; Pham, Linh; Riant, Elodie; Ahui, Marie-Louise; Damotte, Diane; Gombert, Jean-Marc; Bayard, Francis; Ohlsson, Claes; Arnal, Jean-François; Herbelin, André

    2016-01-01

    Therapeutic strategies focused on restoring immune tolerance remain the main avenue to prevent type 1 diabetes (T1D). Because estrogens potentiate FoxP3+ regulatory T cells (Treg) and invariant natural killer T (iNKT) cells, two regulatory lymphocyte populations that are functionally deficient in nonobese diabetic (NOD) mice, we investigated whether estradiol (E2) therapy influences the course of T1D in this model. To this end, female NOD mice were sc implanted with E2- or placebo-delivering pellets to explore the course of spontaneous and cyclophosphamide-induced diabetes. Treg-depleted and iNKT-cell-deficient (Jα18(-/-)) NOD mice were used to assess the respective involvement of these lymphocyte populations in E2 effects. Early E2 administration (from 4 wk of age) was found to preserve NOD mice from both spontaneous and cyclophosphamide-induced diabetes, and a complete protection was also observed throughout treatment when E2 treatment was initiated after the onset of insulitis (from 12 wk of age). This delayed E2 treatment remained fully effective in Treg-depleted mice but failed to entirely protect Jα18(-/-) mice. Accordingly, E2 administration was shown to restore the cytokine production of iNKT cells in response to in vivo challenge with the cognate ligand α-galactosylceramide. Finally, transient E2 administration potentiated the previously described protective action of α-galactosylceramide treatment in NOD females. This study provides original evidence that E2 therapy strongly protects NOD mice from T1D and reveals the estrogen/iNKT cell axis as a new effective target to counteract diabetes onset at the stage of insulitis. Estrogen-based therapy should thus be considered for T1D prevention. PMID:26485613

  16. Estrogen Therapy Delays Autoimmune Diabetes and Promotes the Protective Efficiency of Natural Killer T-Cell Activation in Female Nonobese Diabetic Mice.

    PubMed

    Gourdy, Pierre; Bourgeois, Elvire A; Levescot, Anaïs; Pham, Linh; Riant, Elodie; Ahui, Marie-Louise; Damotte, Diane; Gombert, Jean-Marc; Bayard, Francis; Ohlsson, Claes; Arnal, Jean-François; Herbelin, André

    2016-01-01

    Therapeutic strategies focused on restoring immune tolerance remain the main avenue to prevent type 1 diabetes (T1D). Because estrogens potentiate FoxP3+ regulatory T cells (Treg) and invariant natural killer T (iNKT) cells, two regulatory lymphocyte populations that are functionally deficient in nonobese diabetic (NOD) mice, we investigated whether estradiol (E2) therapy influences the course of T1D in this model. To this end, female NOD mice were sc implanted with E2- or placebo-delivering pellets to explore the course of spontaneous and cyclophosphamide-induced diabetes. Treg-depleted and iNKT-cell-deficient (Jα18(-/-)) NOD mice were used to assess the respective involvement of these lymphocyte populations in E2 effects. Early E2 administration (from 4 wk of age) was found to preserve NOD mice from both spontaneous and cyclophosphamide-induced diabetes, and a complete protection was also observed throughout treatment when E2 treatment was initiated after the onset of insulitis (from 12 wk of age). This delayed E2 treatment remained fully effective in Treg-depleted mice but failed to entirely protect Jα18(-/-) mice. Accordingly, E2 administration was shown to restore the cytokine production of iNKT cells in response to in vivo challenge with the cognate ligand α-galactosylceramide. Finally, transient E2 administration potentiated the previously described protective action of α-galactosylceramide treatment in NOD females. This study provides original evidence that E2 therapy strongly protects NOD mice from T1D and reveals the estrogen/iNKT cell axis as a new effective target to counteract diabetes onset at the stage of insulitis. Estrogen-based therapy should thus be considered for T1D prevention.

  17. Ex vivo induction and expansion of Natural Killer T cells by CD1d1-Ig coated artificial antigen presenting cells

    PubMed Central

    Webb, Tonya J.; Bieler, Joan G.; Schneck, Jonathan P.; Oelke, Mathias

    2009-01-01

    Natural killer T (NKT) cells play a pivotal role in maintaining immune homostasis. They recognize lipid antigen in the context of CD1d molecules and subsequently produce cytokines that activate cells of both the innate and adaptive immune responses. Many studies examining patients with autoimmune disease or cancer have shown that there is a reduction in both NKT cell number and function. Due to the complexities of manipulating NKT cells in vivo, ex vivo expanded effector NKT cells would be an excellent therapeutic modality. To date, immunotherapy utilizing the NKT/CD1d system has been dependent on the use of autologous DC in the presence or absence of a synthetic glycolipid, α-galactocylceramide. Here we report a novel technique that facilitates the growth and analysis of NKT cells through the use of CD1d-expressing aAPC. CD1d-based aAPC can effectively propagate both canonical (iNKT cells) and noncanonical (Vα14−) NKT cells. Importantly, CD1d-Ig aAPC can expand NKT cells from cancer patients. Thus, CD1d-expressing aAPC will enhance our knowledge of NKT cell biology and could potentially be used as a novel tool in adoptive immunotherapeutic strategies. PMID:19446558

  18. Ex vivo induction and expansion of natural killer T cells by CD1d1-Ig coated artificial antigen presenting cells.

    PubMed

    Webb, Tonya J; Bieler, Joan G; Schneck, Jonathan P; Oelke, Mathias

    2009-07-31

    Natural killer T (NKT) cells play a pivotal role in maintaining immune homostasis. They recognize lipid antigen in the context of CD1d molecules and subsequently produce cytokines that activate cells of both the innate and adaptive immune responses. Many studies examining patients with autoimmune disease or cancer have shown that there is a reduction in both NKT cell number and function. Due to the complexities of manipulating NKT cells in vivo, ex vivo expanded effector NKT cells would be an excellent therapeutic modality. To date, immunotherapy utilizing the NKT/CD1d system has been dependent on the use of autologous DC in the presence or absence of a synthetic glycolipid, alpha-galactocylceramide. Here we report a novel technique that facilitates the growth and analysis of NKT cells through the use of CD1d-expressing aAPC. CD1d-based aAPC can effectively propagate both canonical (iNKT cells) and noncanonical (Valpha14(-)) NKT cells. Importantly, CD1d-Ig aAPC can expand NKT cells from cancer patients. Thus, CD1d-expressing aAPC will enhance our knowledge of NKT cell biology and could potentially be used as a novel tool in adoptive immunotherapeutic strategies. PMID:19446558

  19. Activity-driven fluctuations in living cells

    NASA Astrophysics Data System (ADS)

    Fodor, É.; Guo, M.; Gov, N. S.; Visco, P.; Weitz, D. A.; van Wijland, F.

    2015-05-01

    We propose a model for the dynamics of a probe embedded in a living cell, where both thermal fluctuations and nonequilibrium activity coexist. The model is based on a confining harmonic potential describing the elastic cytoskeletal matrix, which undergoes random active hops as a result of the nonequilibrium rearrangements within the cell. We describe the probe's statistics and we bring forth quantities affected by the nonequilibrium activity. We find an excellent agreement between the predictions of our model and experimental results for tracers inside living cells. Finally, we exploit our model to arrive at quantitative predictions for the parameters characterizing nonequilibrium activity, such as the typical time scale of the activity and the amplitude of the active fluctuations.

  20. Single Cell Analysis of Transcriptional Activation Dynamics

    PubMed Central

    Rafalska-Metcalf, Ilona U.; Powers, Sara Lawrence; Joo, Lucy M.; LeRoy, Gary; Janicki, Susan M.

    2010-01-01

    Background Gene activation is thought to occur through a series of temporally defined regulatory steps. However, this process has not been completely evaluated in single living mammalian cells. Methodology/Principal Findings To investigate the timing and coordination of gene activation events, we tracked the recruitment of GCN5 (histone acetyltransferase), RNA polymerase II, Brd2 and Brd4 (acetyl-lysine binding proteins), in relation to a VP16-transcriptional activator, to a transcription site that can be visualized in single living cells. All accumulated rapidly with the VP16 activator as did the transcribed RNA. RNA was also detected at significantly more transcription sites in cells expressing the VP16-activator compared to a p53-activator. After α-amanitin pre-treatment, the VP16-activator, GCN5, and Brd2 are still recruited to the transcription site but the chromatin does not decondense. Conclusions/Significance This study demonstrates that a strong activator can rapidly overcome the condensed chromatin structure of an inactive transcription site and supercede the expected requirement for regulatory events to proceed in a temporally defined order. Additionally, activator strength determines the number of cells in which transcription is induced as well as the extent of chromatin decondensation. As chromatin decondensation is significantly reduced after α-amanitin pre-treatment, despite the recruitment of transcriptional activation factors, this provides further evidence that transcription drives large-scale chromatin decondensation. PMID:20422051

  1. 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

  2. Kinetic Discrimination in T-Cell Activation

    NASA Astrophysics Data System (ADS)

    Rabinowitz, Joshua D.; Beeson, Craig; Lyons, Daniel S.; Davis, Mark M.; McConnell, Harden M.

    1996-02-01

    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.

  3. Active oxygen and cell death in cereal aleurone cells.

    PubMed

    Fath, Angelika; Bethke, Paul; Beligni, Veronica; Jones, Russell

    2002-05-01

    The cereal aleurone layer is a secretory tissue whose function is regulated by gibberellic acid (GA) and abscisic acid (ABA). Aleurone cells lack functional chloroplasts, thus excluding photosynthesis as a source of active oxygen species (AOS) in cell death. Incubation of barley aleurone layers or protoplasts in GA initiated the cell death programme, but incubation in ABA delays programmed cell death (PCD). Light, especially blue and UV-A light, and H(2)O(2) accelerate PCD of GA-treated aleurone cells, but ABA-treated aleurone cells are refractory to light and H(2)O(2) and are not killed. It was shown that light elevated intracellular H(2)O(2), and that the rise in H(2)O(2) was greater in GA-treated cells compared to cells in ABA. Experiments with antioxidants show that PCD in aleurone is probably regulated by AOS. The sensitivity of GA-treated aleurone to light and H(2)O(2) is a result of lowered amounts of enzymes that metabolize AOS. mRNAs encoding catalase, ascorbate peroxidase and superoxide dismutase are all reduced during 6-18 h of incubation in GA, but these mRNAs were present in higher amounts in cells incubated in ABA. The amounts of protein and enzyme activities encoded by these mRNAs were also dramatically reduced in GA-treated cells. Aleurone cells store and metabolize neutral lipids via the glyoxylate cycle in response to GA, and glyoxysomes are one potential source of AOS in the GA-treated cells. Mitochondria are another potential source of AOS in GA-treated cells. AOS generated by these organelles bring about membrane rupture and cell death.

  4. 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

  5. Active lithium chloride cell for spacecraft power

    NASA Technical Reports Server (NTRS)

    Fleischmann, C. W.; Horning, R. J.

    1988-01-01

    An active thionyl chloride high rate battery is under development for spacecraft operations. It is a 540kC (150 Ah) battery capable of pulses up to 75A. This paper describes the design and initial test data on a 'state-of-the-art' cell that has been selected to be the baseline for the prototype cell for that battery. Initial data indicate that the specification can be met with fresh cells. Data for stored cells and additional environmental test data are in the process of being developed.

  6. Immune Reconstitution After Antithymocyte Globulin-Conditioned Hematopoietic Cell Transplantation

    PubMed Central

    Bosch, Mark; Dhadda, Manveer; Hoegh-Petersen, Mette; Liu, Yiping; Hagel, Laura M; Podgorny, Peter; Ugarte-Torres, Alejandra; Khan, Faisal M.; Luider, Joanne; Auer-Grzesiak, Iwona; Mansoor, Adnan; Russell, James A; Daly, Andrew; Stewart, Douglas A.; Maloney, David; Boeckh, Michael; Storek, Jan

    2013-01-01

    Background Antithymocyte globulin (ATG) has been increasingly used to prevent graft-vs-host disease (GVHD), however, its impact on immune reconstitution is relatively unknown. Here we studied (1) immune reconstitution after ATG-conditioned hematopoietic cell transplantation (HCT), (2) determined factors influencing the reconstitution, and (3) compared it to non-ATG-conditioned HCT. Methods Immune cell subset counts were determined at 1–24 months posttransplant in 125 HCT recipients who received ATG during conditioning. The subset counts were also determined in 46 non-ATG-conditioned patients (similarly treated). Results (1) Reconstitution after ATG-conditioned HCT was fast for innate immune cells, intermediate for B cells and CD8 T cells, and very slow for CD4 T cells and invariant NKT (iNKT) cells. (2) Faster reconstitution after ATG-conditioned HCT was associated with higher number of cells of the same subset transferred with the graft in case of memory B cells, naïve CD4 T cells, naïve CD8 T cells, iNKT cells and myeloid dendritic cells; lower recipient age in case of naïve CD4 T cells and naïve CD8 T cells; cytomegalovirus recipient seropositivity in case of memory/effector T cells; absence of GVHD in case of naïve B cells; lower ATG serum levels in case of most T cell subsets including iNKT cells, and higher ATG levels in case of NK cells and B cells. (3) Compared to non-ATG-conditioned HCT, reconstitution after ATG-conditioned HCT was slower for CD4 T cells, and faster for NK cells and B cells. Conclusions ATG worsens reconstitution of CD4 T cells but improves reconstitution of NK and B cells. PMID:22985195

  7. 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.

  8. 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.

  9. 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.

  10. Hippo Pathway Activity Influences Liver Cell Fate

    PubMed Central

    Yimlamai, Dean; Christodoulou, Constantina; Galli, Giorgio G.; Yanger, Kilangsungla; Pepe-Mooney, Brian; Gurung, Basanta; Shrestha, Kriti; Cahan, Patrick; Stanger, Ben Z.; Camargo, Fernando D.

    2014-01-01

    The Hippo signaling pathway is an important regulator of cellular proliferation and organ size. However, little is known about the role of this cascade in the control of cell fate. Employing a combination of lineage tracing, clonal analysis, and organoid culture approaches, we demonstrate that Hippo-pathway activity is essential for the maintenance of the differentiated hepatocyte state. Remarkably, acute inactivation of Hippo-pathway signaling in vivo is sufficient to de-differentiate, at very high efficiencies, adult hepatocytes into cells bearing progenitor characteristics. These hepatocyte-derived progenitor cells demonstrate self-renewal and engraftment capacity at the single cell level. We also identify the NOTCH signaling pathway as a functional important effector downstream of the Hippo transducer YAP. Our findings uncover a potent role for Hippo/YAP signaling in controlling liver cell fate, and reveal an unprecedented level of phenotypic plasticity in mature hepatocytes, which has implications for the understanding and manipulation of liver regeneration. PMID:24906150

  11. Critical telomerase activity for uncontrolled cell growth.

    PubMed

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

    2016-01-01

    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. PMID:27500377

  12. Activated mast cells promote differentiation of B cells into effector cells

    PubMed Central

    Palm, Anna-Karin E.; Garcia-Faroldi, Gianni; Lundberg, Marcus; Pejler, Gunnar; Kleinau, Sandra

    2016-01-01

    Based on the known accumulation of mast cells (MCs) in B cell-dependent inflammatory diseases, including rheumatoid arthritis, we hypothesized that MCs directly modulate B cells. We show here that degranulated, and to a lesser extent naïve or IgE-sensitized, MCs activate both naïve and B cell receptor-activated B cells. This was shown by increased proliferation, blast formation, and expression of CD19, MHC class II and CD86 in the B cells. Further, MCs stimulated the secretion of IgM and IgG in IgM+ B cells, indicating that MCs can induce class-switch recombination in B cells. We also show that coculture of MCs with B cells promotes surface expression of L-selectin, a homing receptor, on the B cells. The effects of MCs on B cells were partly dependent on cell-cell contact and both follicular and marginal zone B cells could be activated by MCs. Our findings suggest that degranulated MCs support optimal activation of B cells, a finding that is in line with in vivo studies showing that MCs frequently degranulate in the context of B-cell driven pathologies such as arthritis. Together, our findings show that MCs have the capacity to differentiate B cells to effector cells. PMID:26847186

  13. Intracellular mechanisms of lymphoid cell activation.

    PubMed

    Fresa, K; Hameed, M; Cohen, S

    1989-01-01

    Activation of lymphocytes for proliferation is associated with the appearance of an intracellular factor (ADR) that can induce DNA synthesis in isolated quiescent nuclei. ADR plays a role in the sequence of intracellular events leading to activation for IL-2-mediated proliferation. Because of the nature of the defining assay, the locus of ADR action appears to be near the terminal end of the transduction pathway. Interestingly, although lymphocytes from aged individuals respond poorly to proliferative stimuli, they appear to produce normal to above-normal levels of ADR. In contrast, their nuclei are only poorly responsive to stimulation by ADR. Preparations rich in ADR activity have proteolytic activity as well. In addition, aprotinin, as well as a variety of other protease inhibitors, suppresses ADR-induced DNA synthesis in a dose-dependent manner. ADR activity can be removed from active extracts by absorption with aprotinin-conjugated agarose beads, and can be removed from the beads by elution at pH 5.0. This latter suggests that ADR itself is a protease. However, its endogenous substrate is not yet known. We have also detected an inhibitor of ADR activity in the cytoplasm of resting lymphocytes. This is a heat-stable protein of approximately 60,000 Da. In addition to suppressing the interaction of ADR with quiescent nuclei, the inhibitor can suppress DNA synthetic activity of replicative nuclei isolated from mitogen-activated lymphocytes. Interestingly, these preparations had little or no activity on replicative nuclei derived from several neoplastic cell lines. The resistance of tumor cell nuclei to spontaneously occurring cytoplasmic inhibitory factors such as the one described here may provide one explanation for the loss of growth control in neoplastic cells. PMID:2642767

  14. Active mechanics and geometry of adherent cells and cell colonies

    NASA Astrophysics Data System (ADS)

    Banerjee, Shiladitya

    2014-03-01

    Measurements of traction stresses exerted by adherent cells or cell colonies on elastic substrates have yielded new insight on how the mechanical and geometrical properties of the substrate regulate cellular force distribution, mechanical energy, spreading, morphology or stress ber architecture. We have developed a generic mechanical model of adherent cells as an active contractile gel mechanically coupled to an elastic substrate and to neighboring cells in a tissue. The contractile gel model accurately predicts the distribution of cellular and traction stresses as observed in single cell experiments, and captures the dependence of cell shape, traction stresses and stress ber polarization on the substrate's mechanical and geometrical properties. The model further predicts that the total strain energy of an adherent cell is solely regulated by its spread area, in agreement with recent experiments on micropatterned substrates with controlled geometry. When used to describe the behavior of colonies of adherent epithelial cells, the model demonstrates the crucial role of the mechanical cross-talk between intercellular and extracellular adhesion in regulating traction force distribution. Strong intercellular mechanical coupling organizes traction forces to the colony periphery, whereas weaker intercellular coupling leads to the build up of traction stresses at intercellular junctions. Furthermore, in agreement with experiments on large cohesive keratinocyte colonies, the model predicts a linear scaling of traction forces with the colony size. This scaling suggests the emergence of an effective surface tension as a scale-free material property of the adherent tissue, originating from actomyosin contractility.

  15. Place cell activation predicts subsequent memory.

    PubMed

    Robitsek, R Jonathan; White, John A; Eichenbaum, Howard

    2013-10-01

    A major quandary in memory research is how hippocampal place cells, widely recognized as elements of a spatial map, contribute to episodic memory, our capacity to remember unique experiences that depends on hippocampal function. Here we recorded from hippocampal neurons as rats performed a T-maze alternation task in which they were required to remember a preceding experience over a delay in order to make a subsequent spatial choice. As it has been reported previously in other variations of this task, we observed differential firing that predicted correct subsequent choices, even as the animal traversed identical locations prior to the choice. Here we also observed that most place cells also fired differently on correct as compared to error trials. Among these cells, a large majority fired strongly before the delay or during the retrieval phase but were less active or failed to activate when the animal subsequently made an error. These findings join the place cell phenomenon with episodic memory performance dependent on the hippocampus, revealing that memory accuracy can be predicted by the activation of single place cells in the hippocampus.

  16. 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

  17. Mechanically activated artificial cell by using microfluidics.

    PubMed

    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

  18. Mechanically activated artificial cell by using microfluidics.

    PubMed

    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.

  19. Phenotypic models of T cell activation.

    PubMed

    Lever, Melissa; Maini, Philip K; van der Merwe, P Anton; Dushek, Omer

    2014-09-01

    T cell activation is a crucial checkpoint in adaptive immunity, and this activation depends on the binding parameters that govern the interactions between T cell receptors (TCRs) and peptide-MHC complexes (pMHC complexes). Despite extensive experimental studies, the relationship between the TCR-pMHC binding parameters and T cell activation remains controversial. To make sense of conflicting experimental data, a variety of verbal and mathematical models have been proposed. However, it is currently unclear which model or models are consistent or inconsistent with experimental data. A key problem is that a direct comparison between the models has not been carried out, in part because they have been formulated in different frameworks. For this Analysis article, we reformulated published models of T cell activation into phenotypic models, which allowed us to directly compare them. We find that a kinetic proofreading model that is modified to include limited signalling is consistent with the majority of published data. This model makes the intriguing prediction that the stimulation hierarchy of two different pMHC complexes (or two different TCRs that are specific for the same pMHC complex) may reverse at different pMHC concentrations.

  20. 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

  1. Epigenetic Changes during Hepatic Stellate Cell Activation

    PubMed Central

    Götze, Silke; Schumacher, Eva C.; Kordes, Claus; Häussinger, Dieter

    2015-01-01

    Background and Aims Hepatic stellate cells (HSC), which can participate in liver regeneration and fibrogenesis, have recently been identified as liver-resident mesenchymal stem cells. During their activation HSC adopt a myofibroblast-like phenotype accompanied by profound changes in the gene expression profile. DNA methylation changes at single genes have been reported during HSC activation and may participate in the regulation of this process, but comprehensive DNA methylation analyses are still missing. The aim of the present study was to elucidate the role of DNA methylation during in vitro activation of HSC. Methods and Results The analysis of DNA methylation changes by antibody-based assays revealed a strong decrease in the global DNA methylation level during culture-induced activation of HSC. To identify genes which may be regulated by DNA methylation, we performed a genome-wide Methyl-MiniSeq EpiQuest sequencing comparing quiescent and early culture-activated HSC. Approximately 400 differentially methylated regions with a methylation change of at least 20% were identified, showing either hypo- or hypermethylation during activation. Further analysis of selected genes for DNA methylation and expression were performed revealing a good correlation between DNA methylation changes and gene expression. Furthermore, global DNA demethylation during HSC activation was investigated by 5-bromo-2-deoxyuridine assay and L-mimosine treatment showing that demethylation was independent of DNA synthesis and thereby excluding a passive DNA demethylation mechanism. Conclusions In summary, in vitro activation of HSC initiated strong DNA methylation changes, which were associated with gene regulation. These results indicate that epigenetic mechanisms are important for the control of early HSC activation. Furthermore, the data show that global DNA demethylation during activation is based on an active DNA demethylation mechanism. PMID:26065684

  2. Shape memory polymers for active cell culture.

    PubMed

    Davis, Kevin A; Luo, Xiaofan; Mather, Patrick T; Henderson, James H

    2011-07-04

    Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat(1-5). In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, T(trans;) [either the melting temperature (T(m;)) or the glass transition temperature (T(g;))]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its T(trans;) while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through T(trans;) under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape. Cells are capable of surveying the mechanical properties of their surrounding environment(6). The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces(7-14). These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date

  3. Transition metals activate TFEB in overexpressing cells.

    PubMed

    Peña, Karina A; Kiselyov, Kirill

    2015-08-15

    Transition metal toxicity is an important factor in the pathogenesis of numerous human disorders, including neurodegenerative diseases. Lysosomes have emerged as important factors in transition metal toxicity because they handle transition metals via endocytosis, autophagy, absorption from the cytoplasm and exocytosis. Transcription factor EB (TFEB) regulates lysosomal biogenesis and the expression of lysosomal proteins in response to lysosomal and/or metabolic stresses. Since transition metals cause lysosomal dysfunction, we proposed that TFEB may be activated to drive gene expression in response to transition metal exposure and that such activation may influence transition metal toxicity. We found that transition metals copper (Cu) and iron (Fe) activate recombinant TFEB and stimulate the expression of TFEB-dependent genes in TFEB-overexpressing cells. In cells that show robust lysosomal exocytosis, TFEB was cytoprotective at moderate levels of Cu exposure, decreasing oxidative stress as reported by the expression of heme oxygenase-1 (HMOX1) gene. However, at high levels of Cu exposure, particularly in cells with low levels of lysosomal exocytosis, activation of overexpressed TFEB was toxic, increasing oxidative stress and mitochondrial damage. Based on these data, we conclude that TFEB-driven gene network is a component of the cellular response to transition metals. These data suggest limitations and disadvantages of TFEB overexpression as a therapeutic approach. PMID:26251447

  4. Transition metals activate TFEB in overexpressing cells

    PubMed Central

    Peña, Karina A.; Kiselyov, Kirill

    2015-01-01

    Transition metal toxicity is an important factor in the pathogenesis of numerous human disorders, including neurodegenerative diseases. Lysosomes have emerged as important factors in transition metal toxicity because they handle transition metals via endocytosis, autophagy, absorption from the cytoplasm and exocytosis. Transcription factor EB (TFEB) regulates lysosomal biogenesis and the expression of lysosomal proteins in response to lysosomal and/or metabolic stresses. Since transition metals cause lysosomal dysfunction, we proposed that TFEB may be activated to drive gene expression in response to transition metal exposure and that such activation may influence transition metal toxicity. We found that transition metals copper (Cu) and iron (Fe) activate recombinant TFEB and stimulate the expression of TFEB-dependent genes in TFEB-overexpressing cells. In cells that show robust lysosomal exocytosis, TFEB was cytoprotective at moderate levels of Cu exposure, decreasing oxidative stress as reported by the expression of heme oxygenase-1 (HMOX1) gene. However, at high levels of Cu exposure, particularly in cells with low levels of lysosomal exocytosis, activation of overexpressed TFEB was toxic, increasing oxidative stress and mitochondrial damage. Based on these data, we conclude that TFEB-driven gene network is a component of the cellular response to transition metals. These data suggest limitations and disadvantages of TFEB overexpression as a therapeutic approach. PMID:26251447

  5. Fluorescence activated cell sorting of plant protoplasts.

    PubMed

    Bargmann, Bastiaan O R; Birnbaum, Kenneth D

    2010-01-01

    High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis. An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: P(SCR;)::GFP (endodermis and quiescent center) and P(WOX5;)::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution. FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time. This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce

  6. Fluorescence activated cell sorting of plant protoplasts.

    PubMed

    Bargmann, Bastiaan O R; Birnbaum, Kenneth D

    2010-02-18

    High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis. An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: P(SCR;)::GFP (endodermis and quiescent center) and P(WOX5;)::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution. FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time. This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce

  7. Insect cells respiratory activity in bioreactor

    PubMed Central

    Jorge, Soraia Athie Calil; Santos, Mariza Gerdulo; Yokomizo, Adriana Yurie; Pereira, Carlos Augusto; Tonso, Aldo

    2008-01-01

    Specific respiration rate ( \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$ Q_{{{\\text{O}}_{2} }} $$\\end{document}) is a key parameter to understand cell metabolism and physiological state, providing useful information for process supervision and control. In this work, we cultivated different insect cells in a very controlled environment, being able to measure \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$ Q_{{{\\text{O}}_{2} }} $$\\end{document}. Spodoptera frugiperda (Sf9) cells have been used through virus infection as host for foreign protein expression and bioinsecticide production. Transfected Drosophila melanogaster (S2) cells can be used to produce different proteins. The objective of this work is to investigate respiratory activity and oxygen transfer during the growth of different insect cells lines as Spodoptera frugiperda (Sf9), Drosophila melanogaster (S2) wild and transfected for the expression of GPV and EGFP. All experiments were performed in a well-controlled 1-L bioreactor, with SF900II serum free medium. Spodoptera frugiperda (Sf9) cells reached 10.7 × 106 cells/mL and maximum specific respiration rate (\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$ Q_{{{\\text{O}}_{2} \\max }} $$\\end{document}) of 7.3 × 10−17 molO2/cell s. Drosophila melanogaster (S2) cells achieved 51.2 × 106 cells/mL and \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage

  8. Chronic variable stress activates hematopoietic stem cells

    PubMed Central

    Courties, Gabriel; Dutta, Partha; Iwamoto, Yoshiko; Zaltsman, Alex; von zur Muhlen, Constantin; Bode, Christoph; Fricchione, Gregory L.; Denninger, John; Lin, Charles P.; Vinegoni, Claudio; Libby, Peter; Swirski, Filip K.; Weissleder, Ralph; Nahrendorf, Matthias

    2014-01-01

    Exposure to psychosocial stress is a risk factor for many diseases, including atherosclerosis1,2. While incompletely understood, interaction between the psyche and the immune system provides one potential mechanism linking stress and disease inception and progression. Known crosstalk between the brain and immune system includes the hypothalamic–pituitary–adrenal axis, which centrally drives glucocorticoid production in the adrenal cortex, and the sympathetic–adrenal–medullary axis, which controls stress–induced catecholamine release in support of the fight–or–flight reflex3,4. It remains unknown however if chronic stress changes hematopoietic stem cell activity. Here we show that stress increases proliferation of these most primitive progenitors, giving rise to higher levels of disease–promoting inflammatory leukocytes. We found that chronic stress induced monocytosis and neutrophilia in humans. While investigating the source of leukocytosis in mice, we discovered that stress activates upstream hematopoietic stem cells. Sympathetic nerve fibers release surplus noradrenaline, which uses the β3 adrenergic receptor to signal bone marrow niche cells to decrease CXCL12 levels. Consequently, elevated hematopoietic stem cell proliferation increases output of neutrophils and inflammatory monocytes. When atherosclerosis–prone ApoE−/− mice encounter chronic stress, accelerated hematopoiesis promotes plaque features associated with vulnerable lesions that cause myocardial infarction and stroke in humans. PMID:24952646

  9. 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.

  10. Cell Micromanipulation with an Active Handheld Micromanipulator

    PubMed Central

    Tabarés, Jaime Cuevas; MacLachlan, Robert A.; Ettensohn, Charles A.

    2012-01-01

    The paper describes the use of an active handheld micromanipulator, known as Micron, for micromanipulation of cells. The device enables users to manipulate objects on the order of tens of microns in size, with the natural ease of use of a fully handheld tool. Micron senses its own position using a purpose-built microscale optical tracker, estimates the erroneous or undesired component of hand motion, and actively corrects it by deflecting its own tool tip using piezoelectric actuators. Benchtop experiments in tip positioning show that active compensation can reduce positioning error by up to 51% compared to unaided performance. Preliminary experiments in bisection of sea urchin embryos exhibit an increased success rate when performed with the help of Micron. PMID:21096452

  11. 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

  12. Active stochastic stress fluctuations in the cell cytoskeleton stir the cell and activate primary cilia

    NASA Astrophysics Data System (ADS)

    Schmidt, Christoph F.; Fakhri, Nikta; Battle, Christopher; Ott, Carolyn M.; Wessel, Alok D.; Lippincott-Schwartz, Jennifer; Mackintosh, Frederick C.

    2015-03-01

    Cells are active systems with molecular force generation that drives complex dynamics at the supramolecular scale. Much of cellular dynamics is driven by myosin motors interacting with the actin cytoskeleton. We discovered active random ``stirring'' driven by cytoplasmic myosin as an intermediate mode of transport, different from both thermal diffusion and directed motor activity. We found a further manifestation of cytoskeletal dynamics in the active motion patterns of primary cilia generated by epithelial cells. These cilia were thought to be immotile due to the absence of dynein motors, but it turns out that their anchoring deeper inside the cell in combination with the strongly fluctuating cortex results in clearly measurable non-equilibrium fluctuations.

  13. Saposins modulate human invariant Natural Killer T cells self-reactivity and facilitate lipid exchange with CD1d molecules during antigen presentation

    PubMed Central

    Salio, Mariolina; Ghadbane, Hemza; Dushek, Omer; Shepherd, Dawn; Cypen, Jeremy; Gileadi, Uzi; Aichinger, Michael C.; Napolitani, Giorgio; Qi, Xiaoyang; van der Merwe, P. Anton; Wojno, Justyna; Veerapen, Natacha; Cox, Liam R.; Besra, Gurdyal S.; Yuan, Weiming; Cresswell, Peter; Cerundolo, Vincenzo

    2013-01-01

    Lipid transfer proteins, such as molecules of the saposin family, facilitate extraction of lipids from biological membranes for their loading onto CD1d molecules. Although it has been shown that prosaposin-deficient mice fail to positively select invariant natural killer T (iNKT) cells, it remains unclear whether saposins can facilitate loading of endogenous iNKT cell agonists in the periphery during inflammatory responses. In addition, it is unclear whether saposins, in addition to loading, also promote dissociation of lipids bound to CD1d molecules. To address these questions, we used a combination of cellular assays and demonstrated that saposins influence CD1d-restricted presentation to human iNKT cells not only of exogenous lipids but also of endogenous ligands, such as the self-glycosphingolipid β-glucopyranosylceramide, up-regulated by antigen-presenting cells following bacterial infection. Furthermore, we demonstrated that in human myeloid cells CD1d-loading of endogenous lipids after bacterial infection, but not at steady state, requires trafficking of CD1d molecules through an endo-lysosomal compartment. Finally, using BIAcore assays we demonstrated that lipid-loaded saposin B increases the off-rate of lipids bound to CD1d molecules, providing important insights into the mechanisms by which it acts as a “lipid editor,” capable of fine-tuning loading and unloading of CD1d molecules. These results have important implications in understanding how to optimize lipid-loading onto antigen-presenting cells, to better harness iNKT cells central role at the interface between innate and adaptive immunity. PMID:24248359

  14. 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. PMID:26714690

  15. 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.

  16. Application of user-guided automated cytometric data analysis to large-scale immunoprofiling of invariant natural killer T cells.

    PubMed

    Hu, Xinli; Kim, Hyun; Brennan, Patrick J; Han, Buhm; Baecher-Allan, Clare M; De Jager, Philip L; Brenner, Michael B; Raychaudhuri, Soumya

    2013-11-19

    Defining and characterizing pathologies of the immune system requires precise and accurate quantification of abundances and functions of cellular subsets via cytometric studies. At this time, data analysis relies on manual gating, which is a major source of variability in large-scale studies. We devised an automated, user-guided method, X-Cyt, which specializes in rapidly and robustly identifying targeted populations of interest in large data sets. We first applied X-Cyt to quantify CD4(+) effector and central memory T cells in 236 samples, demonstrating high concordance with manual analysis (r = 0.91 and 0.95, respectively) and superior performance to other available methods. We then quantified the rare mucosal associated invariant T cell population in 35 samples, achieving manual concordance of 0.98. Finally we characterized the population dynamics of invariant natural killer T (iNKT) cells, a particularly rare peripheral lymphocyte, in 110 individuals by assaying 19 markers. We demonstrated that although iNKT cell numbers and marker expression are highly variable in the population, iNKT abundance correlates with sex and age, and the expression of phenotypic and functional markers correlates closely with CD4 expression.

  17. 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.

  18. 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

  19. 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…

  20. 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

  1. 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

    2016-01-01

    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. PMID:27337592

  2. Activated Muscle Satellite Cells Chase Ghosts.

    PubMed

    Mourikis, Philippos; Relaix, Frédéric

    2016-02-01

    The in vivo behaviors of skeletal muscle stem cells, i.e., satellite cells, during homeostasis and after injury are poorly understood. In this issue of Cell Stem Cell, Webster et al. (2016) now perform a tour de force intravital microscopic analysis of this population, showing that "ghost fiber" remnants act as scaffolds to guide satellite cell divisions after injury. PMID:26849298

  3. Apoptotic cells actively inhibit the expression of CD69 on Con A activated T lymphocytes.

    PubMed

    Sun, E; Zhang, L; Zeng, Y; Ge, Q; Zhao, M; Gao, W

    2000-03-01

    Although apoptosis is commonly viewed as a silent cell death without damage to adjacent tissues, the effect of apoptosis on immunity has been unclear. We have investigated the influence of apoptotic cells on T-cell activation. The K562 or HL-60 human leukemia cell lines that had been induced apoptosis by FTY720 or cycloheximide (CHX) were added into the culture of mouse spleen cells stimulated with Con A. Six to 20 h later, the expression of CD69, an early T-cell activation antigen, was detected using flowcytometry. Living cells and necrotic cells served as control groups. Apoptotic K562 or HL-60 cells induced by either FTY720 or CHX unanimously inhibited CD69 expression on the CD3+ mouse T cells while living and necrotic cells did not. The inhibition was proportional to the number of apoptotic cells and was different in the T-cell subsets, showing a rapid and transient inhibition on the CD3+CD8+ T-cell activation but with a slow and continuous inhibition on CD3+CD8- T-cell activation. In conclusion, the apoptotic cells actively inhibit a T-cell activation that is independent of the cell lines or the apoptotic inducers, indicating that the apoptotic cells dominantly regulate T-cell immunity. PMID:10736091

  4. Phagocytic cell function in active brucellosis.

    PubMed Central

    Ocon, P; Reguera, J M; Morata, P; Juarez, C; Alonso, A; Colmenero, J D

    1994-01-01

    In this study, we analyzed phagocytic cell function in 51 patients with active brucellosis and its relationship with different clinical, serological, and evolutionary variables. A control group was made up of 30 blood donors of similar geographic extraction, age, and sex, with no previous history of brucellosis or known exposure ot the infection or specific antibodies. The investigations were carried out at the time of diagnosis, at the conclusion of treatment, and after 6 months of follow-up. Polymorphonuclear leukocyte adherence and nitroblue tetrazolium reduction in response to Brucella antigen were significantly increased in the patients at the time of diagnosis with respect to the control group. In contrast, chemotaxis in response to Brucella antigen and phagocytosis were significantly reduced in the patients with respect to the control group. The alterations in phagocytic cell function were greater in patients with bacteremia, with focal forms of the disease, or with a longer diagnostic delay. Most of these initial alterations tended to normalize with treatment, indicating their transient character. PMID:8112863

  5. 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.

  6. Structure and binding kinetics of three different human CD1d-alpha-galactosylceramide-specific T cell receptors.

    PubMed

    Gadola, Stephan D; Koch, Michael; Marles-Wright, Jon; Lissin, Nikolai M; Shepherd, Dawn; Matulis, Gediminas; Harlos, Karl; Villiger, Peter M; Stuart, David I; Jakobsen, Bent K; Cerundolo, Vincenzo; Jones, E Yvonne

    2006-03-20

    Invariant human TCR Valpha24-Jalpha18+/Vbeta11+ NKT cells (iNKT) are restricted by CD1d-alpha-glycosylceramides. We analyzed crystal structures and binding characteristics for an iNKT TCR plus two CD1d-alpha-GalCer-specific Vbeta11+ TCRs that use different TCR Valpha chains. The results were similar to those previously reported for MHC-peptide-specific TCRs, illustrating the versatility of the TCR platform. Docking TCR and CD1d-alpha-GalCer structures provided plausible insights into their interaction. The model supports a diagonal orientation of TCR on CD1d and suggests that complementarity determining region (CDR)3alpha, CDR3beta, and CDR1beta interact with ligands presented by CD1d, whereas CDR2beta binds to the CD1d alpha1 helix. This docking provides an explanation for the dominant usage of Vbeta11 and Vbeta8.2 chains by human and mouse iNKT cells, respectively, for recognition of CD1d-alpha-GalCer.

  7. Elasticity of adherent active cells on a compliant substrate

    NASA Astrophysics Data System (ADS)

    Banerjee, Shiladitya; Mertz, Aaron F.; Dufresne, Eric R.; Marchetti, M. Cristina

    2012-02-01

    We present a continuum mechanical model of rigidity sensing by livings cells adhering to a compliant substrate. The cell or cell colony is modeled as an elastic active gel, adapting recently developed continuum theories of active viscoelastic fluids. The coupling to the substrate enters as a boundary condition that relates the cell's deformation field to local stress gradients. In the presence of activity, the substrate induces spatially inhomogeneous contractile stresses and deformations, with a power law dependence of the total traction forces on cell or colony size. This is in agreement with recent experiments on keratinocyte colonies adhered to fibronectin coated surfaces. In the presence of acto-myosin activity, the substrate also enhances the cell polarization, breaking the cell's front-rear symmetry. Maximal polarization is observed when the substrate stiffness matches that of the cell, in agreement with experiments on stem cells.

  8. MAIT cells are activated during human viral infections

    PubMed Central

    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; Barnes, Eleanor; Ball, Jonathan; Burgess, Gary; Cooke, Graham; Dillon, John; Gore, Charles; Foster, Graham; Guha, Neil; Halford, Rachel; Herath, Cham; Holmes, Chris; Howe, Anita; Hudson, Emma; Irving, William; Khakoo, Salim; Koletzki, Diana; Martin, Natasha; Mbisa, Tamyo; McKeating, Jane; McLauchlan, John; Miners, Alec; Murray, Andrea; Shaw, Peter; Simmonds, Peter; Spencer, Chris; Targett-Adams, Paul; Thomson, Emma; Vickerman, Peter; Zitzmann, Nicole; 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

    2016-01-01

    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. PMID:27337592

  9. Functionally Active Gap Junctions between Connexin 43-Positive Mesenchymal Stem Cells and Glioma Cells.

    PubMed

    Gabashvili, A N; Baklaushev, V P; Grinenko, N F; Levinskii, A B; Mel'nikov, P A; Cherepanov, S A; Chekhonin, V P

    2015-05-01

    The formation of functional gap junctions between mesenchymal stem cells and cells of low-grade rat glioma C6 cells was studied in in vitro experiments. Immunocytochemical analysis with antibodies to connexin 43 extracellular loop 2 showed that mesenchymal stem cells as well as C6 glioma cells express the main astroglial gap junction protein connexin 43. Analysis of migration activity showed that mesenchymal stem cells actively migrate towards C6 glioma cells. During co-culturing, mesenchymal stem cells and glioma C6 form functionally active gap junctions mediating the transport of cytoplasmic dye from glioma cells to mesenchymal stem cells in the opposite direction. Fluorometry showed that the intensity of transport of low-molecular substances through heterologous gap junctions between mesenchymal stem cells and glioma cells is similar to that through homologous gap junctions between glioma cells. This phenomenon can be used for the development of new methods of cell therapy of high-grade gliomas.

  10. Activation of Complement by Cells Infected with Respiratory Syncytial Virus

    PubMed Central

    Smith, Thomas F.; Mcintosh, Kenneth; Fishaut, Mark; Henson, Peter M.

    1981-01-01

    The ability of respiratory syncytial virus (RSV)-infected HEp-2 cells in culture to activate complement was investigated. After incubation of cells with various complement sources and buffer, binding of C3b to surfaces of infected cells was demonstrated by immunofluorescence with a double-staining technique. Nonsyncytial and syncytial (i.e., fused, multinucleated) cells were separately enumerated. Also, lysis of RSV-infected cells was assessed by lactic dehydrogenase release. In this system only RSV-infected cells stained for C3b, and they did so only after incubation with functionally active complement. Blocking of classical pathway activation with ethylenediaminetetraacetic acid diminished the number of infected nonsyncytial cells positively stained for C3b, but had no effect on staining of syncytial cells. Blocking of alternative pathway activation with either zymosan incubation or heat treatment decreased the number of both syncytial and nonsyncytial cells stained for C3b. Decreasing immunoglobulin concentration of the serum used as the complement source also decreased numbers of both cell types stained for C3b. Eliminating specific anti-RSV antibody diminished numbers of both cell types stained for C3b, but staining was not eliminated. Lastly, incubation with functionally active complement markedly increased lactic dehydrogenase release from infected cells. This study demonstrated that RSV-infected nonsyncytial and syncytial cells are able to activate complement by both classical and alternative pathways. Activation of complement by syncytial cells appears to be less dependent on the classical pathway than is activation by nonsyncytial cells, and activation by syncytial cells may require immunoglobulin but not specific antibody. These experiments suggest the possibility of complement activation during respiratory tract infection by RSV. Implications of this are discussed. Images PMID:7263071

  11. Hydrogen peroxide activates activator protein-1 and mitogen-activated protein kinases in pancreatic stellate cells.

    PubMed

    Kikuta, Kazuhiro; Masamune, Atsushi; Satoh, Masahiro; Suzuki, Noriaki; Satoh, Kennichi; Shimosegawa, Tooru

    2006-10-01

    Activated pancreatic stellate cells (PSCs) are implicated in the pathogenesis of pancreatic inflammation and fibrosis, where oxidative stress is thought to play a key role. Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) may act as a second messenger to mediate the actions of growth factors and cytokines. But the role of reactive oxygen species in the activation and regulation of cell functions in PSCs remains largely unknown. We here examined the effects of H(2)O(2) on the activation of signal transduction pathways and cell functions in PSCs. PSCs were isolated from the pancreas of male Wistar rats, and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. Activation of transcription factors was examined by electrophoretic mobility shift assay and luciferase assay. Activation of mitogen-activated protein (MAP) kinases was assessed by Western blotting using anti-phosphospecific antibodies. The effects of H(2)O(2) on proliferation, alpha(1)(I)procollagen gene expression, and monocyte chemoattractant protein-1 production were evaluated. The effect of H(2)O(2) on the transformation of freshly isolated PSCs in culture was also assessed. H(2)O(2) at non-cytotoxic concentrations (up to 100 microM) induced oxidative stress in PSCs. H(2)O(2) activated activator protein-1, but not nuclear factor kappaB. In addition, H(2)O(2) activated three classes of MAP kinases: extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAP kinase. H(2)O(2) induced alpha(1)(I)procollagen gene expression but did not induce proliferation or monocyte chemoattractant protein-1 production. H(2)O(2) did not initiate the transformation of freshly isolated PSCs to myofibroblast-like phenotype. Specific activation of these signal transduction pathways and collagen gene expression by H(2)O(2) may play a role in the pathogenesis of pancreatic fibrosis.

  12. Dendritic cell exosomes directly kill tumor cells and activate natural killer cells via TNF superfamily ligands

    PubMed Central

    Munich, Stephan; Sobo-Vujanovic, Andrea; Buchser, William J.; Beer-Stolz, Donna; Vujanovic, Nikola L.

    2012-01-01

    Autocrine and paracrine cell communication can be conveyed by multiple mediators, including membrane-associate proteins, secreted proteins and exosomes. Exosomes are 30–100 nm endosome-derived vesicles consisting in cytosolic material surrounded by a lipid bilayer containing transmembrane proteins. We have previously shown that dendritic cells (DCs) express on their surface multiple TNF superfamily ligands (TNFSFLs), by which they can induce the apoptotic demise of tumor cells as well as the activation of natural killer (NK) cells. In the present study, we demonstrate that, similar to DCs, DC-derived exosomes (DCex) express on their surface TNF, FasL and TRAIL, by which they can trigger caspase activation and apoptosis in tumor cells. We also show that DCex activate NK cells and stimulate them to secrete interferonγ (IFNγ) upon the interaction of DCex TNF with NK-cell TNF receptors. These data demonstrate that DCex can mediate essential innate immune functions that were previously ascribed to DCs. PMID:23170255

  13. 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)

  14. Targeted disruption of CD1d prevents NKT cell development in pigs

    PubMed Central

    Yang, Guan; Artiaga, Bianca L.; Hackmann, Timothy J.; Samuel, Melissa S.; Walters, Eric M; Salek-Ardakani, Shahram; Driver, John P.

    2016-01-01

    Studies in mice genetically lacking natural killer T (NKT) cells show that these lymphocytes make important contributions to both innate and adaptive immune responses. However, the usefulness of murine models to study human NKT cells is limited by the many differences between mice and humans, including that their NKT cell frequencies, subsets and distribution are dissimilar. A more suitable model may be swine that share many metabolic, physiological and growth characteristics with humans and are also similar for NKT cells. Thus, we analyzed genetically modified pigs made deficient for CD1d that is required for the development of Type I invariant NKT (iNKT) cells that express a semi-invariant T cell receptor (TCR) and Type II NKT cells that use variable TCRs. Peripheral blood analyzed by flow cytometry and interferon-γ (IFNγ) enzyme-linked immuno spot (ELISPOT) assays demonstrated that CD1d-knockout pigs completely lack iNKT cells while other leukocyte populations remain intact. CD1d and NKT cells have been shown to be involved in shaping the composition of the commensal microbiota in mice. Therefore, we also compared the fecal microbiota profile between pigs expressing and lacking NKT cells. However, no differences were found between pigs lacking or expressing CD1d. Our results are the first to show that knocking-out CD1d prevents the development of iNKT cells in a non-rodent species. CD1d-deficient pigs should offer a useful model to more accurately determine the contribution of NKT cells for human immune responses. They also have potential for understanding how NKT cells impact the health of commercial swine. PMID:25930071

  15. 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.

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

    PubMed

    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

  17. 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.

  18. Mycoplasma arthritidis mitogen up-regulates human NK cell activity.

    PubMed Central

    D'Orazio, J A; Cole, B C; Stein-Streilein, J

    1996-01-01

    While the effects of superantigens on T lymphocytes are well characterized, how superantigens interact with other immune cells is less clear. This report examines the effects of Mycoplasma arthritidis mitogen (MAM) on human natural killer (NK) cell activity. Incubation of peripheral blood mononuclear cells (PBMC) with MAM for 16 to 20 h augmented NK cytotoxicity (against K562) in a dose-dependent manner (P < or = 0.05). Superantigen-dependent cellular cytotoxicity, an activity of superantigen-activated cytotoxic T cells, was not involved in lysis of K562 cells because the erythroleukemic tumor target cells expressed no class II major histocompatibility complex by fluorescence-activated cell sorter analysis. Kinetic experiments showed that the largest increase in NK activity induced by MAM occurred within 48 h. Incubation with MAM caused a portion of NK cells to become adherent to tissue culture flasks, a quality associated with activation, and augmented NK activity was found in both adherent and nonadherent subpopulations. Experiments using cytokine-specific neutralizing antibodies showed that interleukin-2 contributed to enhancement of the NK activity observed in superantigen-stimulated PBMC. Interestingly, MAM was able to augment NK lysis of highly purified NK (CD56+) cells in the absence of other immune cells in 9 of 12 blood specimens, with the augmented lytic activity ranging from 110 to 170% of unstimulated NK activity. In summary, data presented in this report show for the first time that MAM affects human NK cells directly by increasing their lytic capacity and indirectly in PBMC as a consequence of cytokines produced by T cells. Results of this work suggest that, in vivo, one consequence of interaction with superantigen-secreting microorganisms may be up-regulation of NK lytic activity. These findings may have clinical application as a means of generating augmented NK effector cells useful in the immunotherapy of parasitic infections or neoplasms. PMID

  19. Dengue Virus Directly Stimulates Polyclonal B Cell Activation

    PubMed Central

    Papa, Michelle Premazzi; de Morais, Ana Theresa Silveira; Peçanha, Ligia Maria Torres; de Arruda, Luciana Barros

    2015-01-01

    Dengue infection is associated to vigorous inflammatory response, to a high frequency of activated B cells, and to increased levels of circulating cross-reactive antibodies. We investigated whether direct infection of B cells would promote activation by culturing primary human B lymphocytes from healthy donors with DENV in vitro. B cells were susceptible, but poorly permissive to infection. Even though, primary B cells cultured with DENV induced substantial IgM secretion, which is a hallmark of polyclonal B cell activation. Notably, DENV induced the activation of B cells obtained from either DENV immune or DENV naïve donors, suggesting that it was not dependent on DENV-specific secondary/memory response. B cell stimulation was dependent on activation of MAPK and CD81. B cells cultured with DENV also secreted IL-6 and presented increased expression of CD86 and HLA-DR, which might contribute to B lymphocyte co-stimulatory function. Indeed, PBMCs, but not isolated B cells, secreted high amounts of IgG upon DENV culture, suggesting that interaction with other cell types in vivo might promote Ig isotype switching and IgG secretion from different B cell clones. These findings suggest that activation signaling pathways triggered by DENV interaction with non-specific receptors on B cells might contribute to the exacerbated response observed in dengue patients. PMID:26656738

  20. 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

  1. The DNA methylation profile of activated human natural killer cells.

    PubMed

    Wiencke, John K; Butler, Rondi; Hsuang, George; Eliot, Melissa; Kim, Stephanie; Sepulveda, Manuel A; Siegel, Derick; Houseman, E Andres; Kelsey, Karl T

    2016-05-01

    Natural killer (NK) cells are now recognized to exhibit characteristics akin to cells of the adaptive immune system. The generation of adaptive memory is linked to epigenetic reprogramming including alterations in DNA methylation. The study herein found reproducible genome wide DNA methylation changes associated with human NK cell activation. Activation led predominately to CpG hypomethylation (81% of significant loci). Bioinformatics analysis confirmed that non-coding and gene-associated differentially methylated sites (DMS) are enriched for immune related functions (i.e., immune cell activation). Known DNA methylation-regulated immune loci were also identified in activated NK cells (e.g., TNFA, LTA, IL13, CSF2). Twenty-one loci were designated high priority and further investigated as potential markers of NK activation. BHLHE40 was identified as a viable candidate for which a droplet digital PCR assay for demethylation was developed. The assay revealed high demethylation in activated NK cells and low demethylation in naïve NK, T- and B-cells. We conclude the NK cell methylome is plastic with potential for remodeling. The differentially methylated region signature of activated NKs revealed similarities with T cell activation, but also provided unique biomarker candidates of NK activation, which could be useful in epigenome-wide association studies to interrogate the role of NK subtypes in global methylation changes associated with exposures and/or disease states. PMID:26967308

  2. Remote Control of T Cell Activation Using Magnetic Janus Particles.

    PubMed

    Lee, Kwahun; Yi, Yi; Yu, Yan

    2016-06-20

    We report a strategy for using magnetic Janus microparticles to control the stimulation of T cell signaling with single-cell precision. To achieve this, we designed Janus particles that are magnetically responsive on one hemisphere and stimulatory to T cells on the other side. By manipulating the rotation and locomotion of Janus particles under an external magnetic field, we could control the orientation of the particle-cell recognition and thereby the initiation of T cell activation. This study demonstrates a step towards employing anisotropic material properties of Janus particles to control single-cell activities without the need of complex magnetic manipulation devices.

  3. Anthocyanidins inhibit activator protein 1 activity and cell transformation: structure-activity relationship and molecular mechanisms.

    PubMed

    Hou, De-Xing; Kai, Keiko; Li, Jian-Jian; Lin, Shigang; Terahara, Norihiko; Wakamatsu, Mika; Fujii, Makoto; Young, Mattew R; Colburn, Nancy

    2004-01-01

    Anthocyanins are the chemical components that give the intense color to many fruits and vegetables, such as blueberries, red cabbages and purple sweet potatoes. Extensive studies have indicated that anthocyanins have strong antioxidant activities. To investigate the mechanism of anthocyanidins as an anticancer food source, six kinds of anthocyanidins representing the aglycons of most anthocyanins, were used to examine their effects on tumor promotion in mouse JB6 cells, a validated model for screening cancer chemopreventive agents and elucidating the molecular mechanisms. Of the six anthocyanins tested, only those with an ortho-dihydroxyphenyl structure on the B-ring suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation and activator protein-1 transactivation, suggesting that the ortho-dihydroxyphenyl may contribute to the inhibitory action. Delphinidin, but not peonidin, blocked the phosphorylation of protein kinases in the extracellular signal-regulated protein kinase (ERK) pathway at early times and the c-Jun N-terminal kinase (JNK) signaling pathway at later times. p38 kinase was not inhibited by delphinidin. Furthermore, two mitogen-activated protein kinase (MAPK) specific inhibitors (SP600125 for JNK and UO126 for ERK) could specifically block the activation of JNK and ERK and cell transformation. Those results demonstrate that anthocyanidins contribute to the inhibition of tumorigenesis by blocking activation of the MAPK pathway. These findings provide the first molecular basis for the anticarcinogenic action of anthocyanidins. PMID:14514663

  4. Anthocyanidins inhibit activator protein 1 activity and cell transformation: structure-activity relationship and molecular mechanisms.

    PubMed

    Hou, De-Xing; Kai, Keiko; Li, Jian-Jian; Lin, Shigang; Terahara, Norihiko; Wakamatsu, Mika; Fujii, Makoto; Young, Mattew R; Colburn, Nancy

    2004-01-01

    Anthocyanins are the chemical components that give the intense color to many fruits and vegetables, such as blueberries, red cabbages and purple sweet potatoes. Extensive studies have indicated that anthocyanins have strong antioxidant activities. To investigate the mechanism of anthocyanidins as an anticancer food source, six kinds of anthocyanidins representing the aglycons of most anthocyanins, were used to examine their effects on tumor promotion in mouse JB6 cells, a validated model for screening cancer chemopreventive agents and elucidating the molecular mechanisms. Of the six anthocyanins tested, only those with an ortho-dihydroxyphenyl structure on the B-ring suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation and activator protein-1 transactivation, suggesting that the ortho-dihydroxyphenyl may contribute to the inhibitory action. Delphinidin, but not peonidin, blocked the phosphorylation of protein kinases in the extracellular signal-regulated protein kinase (ERK) pathway at early times and the c-Jun N-terminal kinase (JNK) signaling pathway at later times. p38 kinase was not inhibited by delphinidin. Furthermore, two mitogen-activated protein kinase (MAPK) specific inhibitors (SP600125 for JNK and UO126 for ERK) could specifically block the activation of JNK and ERK and cell transformation. Those results demonstrate that anthocyanidins contribute to the inhibition of tumorigenesis by blocking activation of the MAPK pathway. These findings provide the first molecular basis for the anticarcinogenic action of anthocyanidins.

  5. Senescence of activated stellate cells limits liver fibrosis

    PubMed Central

    Krizhanovsky, Valery; Yon, Monica; Dickins, Ross A.; Hearn, Stephen; Simon, Janelle; Miething, Cornelius; Yee, Herman; Zender, Lars; Lowe, Scott W.

    2011-01-01

    Summary Cellular senescence acts as a potent mechanism of tumor suppression; however, its functional contribution to non-cancer pathologies has not been examined. Here we show that senescent cells accumulate in murine livers treated to produce fibrosis, a precursor pathology to cirrhosis. The senescent cells are derived primarily from activated hepatic stellate cells, which initially proliferate in response to liver damage and produce the extracellular matrix deposited in the fibrotic scar. In mice lacking key senescence regulators, stellate cells continue to proliferate, leading to excessive liver fibrosis. Furthermore, senescent activated stellate cells exhibit gene expression profile consistent with cell cycle exit, reduced secretion of extracellular matrix components, enhanced secretion of extracellular matrix degrading enzymes, and enhanced immune surveillance. Accordingly natural killer cells preferentially kill senescent activated stellate cells in vitro and in vivo, thereby facilitating the resolution of fibrosis. Therefore, the senescence program limits the fibrogenic response to acute tissue damage. PMID:18724938

  6. Immobilization of Pichia pastoris cells containing alcohol oxidase activity

    PubMed Central

    Maleknia, S; Ahmadi, H; Norouzian, D

    2011-01-01

    Background and Objectives The attempts were made to describe the development of a whole cell immobilization of P. pastoris by entrapping the cells in polyacrylamide gel beads. The alcohol oxidase activity of the whole cell Pichia pastoris was evaluated in comparison with yeast biomass production. Materials and Methods Methylotrophic yeast P. pastoris was obtained from Collection of Standard Microorganisms, Department of Bacterial Vaccines, Pasteur Institute of Iran (CSMPI). Stock culture was maintained on YPD agar plates. Alcohol oxidase was strongly induced by addition of 0.5% methanol as the carbon source. The cells were harvested by centrifugation then permeabilized. Finally the cells were immobilized in polyacrylamide gel beads. The activity of alcohol oxidase was determined by method of Tane et al. Results At the end of the logarithmic phase of cell culture, the alcohol oxidase activity of the whole cell P. Pastoris reached the highest level. In comparison, the alcohol oxidase activity was measured in an immobilized P. pastoris when entrapped in polyacrylamide gel beads. The alcohol oxidase activity of cells was induced by addition of 0.5% methanol as the carbon source. The cells were permeabilized by cetyltrimethylammonium bromide (CTAB) and immobilized. CTAB was also found to increase the gel permeability. Alcohol oxidase activity of immobilized cells was then quantitated by ABTS/POD spectrophotometric method at OD 420. There was a 14% increase in alcohol oxidase activity in immobilized cells as compared with free cells. By addition of 2-butanol as a substrate, the relative activity of alcohol oxidase was significantly higher as compared with other substrates added to the reaction media. Conclusion Immobilization of cells could eliminate lengthy and expensive procedures of enzyme separation and purification, protect and stabilize enzyme activity, and perform easy separation of the enzyme from the reaction media. PMID:22530090

  7. Heteronomous rhythmic activity of neurosecretory cells in the silkmoth.

    PubMed

    Ichikawa, Toshio; Kamimoto, Satoshi

    2003-08-21

    Electrical action potentials of neurosecretory cells producing pheromone biosynthesis-activating neuropeptide (PBAN) and electrocardiograms were recorded from female pupae of Bombyx mori and the correlation between firing activity of the cells and cardiac activity was analyzed. PBAN producing cells localized in the suboesophageal ganglion (SOG) generated clusters of action potentials at an interval of 30-60 min. The firing activity rhythm at a middle pupal period was closely related to heartbeat reversal rhythm: an active phase of the cells was usually apparent during anterograde pulse phases. Electrocardiograms at a late pupal period often revealed brief oscillatory potentials (15-25 Hz in frequency) of unknown origin. The firing activity rhythm of PBAN cells closely correlated with the rhythmic appearance of clustered oscillatory potentials. Transection of connectives between the brain and SOG abolished rhythmic activity of the cells. These results suggest that a rhythmic firing activity of the PBAN cell system is heteronomously generated by a cerebral neuronal mechanism and the cerebral mechanism relates the cell system to other neuronal mechanisms controlling cardiac activity and oscillatory potential rhythms. PMID:12873731

  8. 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

  9. 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.

  10. 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

  11. Borrelia burgdorferi Spirochetes Induce Mast Cell Activation and Cytokine Release

    PubMed Central

    Talkington, Jeffrey; Nickell, Steven P.

    1999-01-01

    The Lyme disease spirochete, Borrelia burgdorferi, is introduced into human hosts via tick bites. Among the cell types present in the skin which may initially contact spirochetes are mast cells. Since spirochetes are known to activate a variety of cell types in vitro, we tested whether B. burgdorferi spirochetes could activate mast cells. We report here that freshly isolated rat peritoneal mast cells or mouse MC/9 mast cells cultured in vitro with live or freeze-thawed B. burgdorferi spirochetes undergo low but detectable degranulation, as measured by [5-3H] hydroxytryptamine release, and they synthesize and secrete the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). In contrast to findings in previous studies, where B. burgdorferi-associated activity was shown to be dependent upon protein lipidation, mast cell TNF-α release was not induced by either lipidated or unlipidated recombinant OspA. This activity was additionally shown to be protease sensitive and surface expressed. Finally, comparisons of TNF-α-inducing activity in known low-, intermediate-, and high-passage B. burgdorferi B31 isolates demonstrated passage-dependent loss of activity, indicating that the activity is probably plasmid encoded. These findings document the presence in low-passage B. burgdorferi spirochetes of a novel lipidation-independent activity capable of inducing cytokine release from host cells. PMID:10024550

  12. 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.

  13. Apoptotic Cells Activate AMP-activated Protein Kinase (AMPK) and Inhibit Epithelial Cell Growth without Change in Intracellular Energy Stores*

    PubMed Central

    Patel, Vimal A.; Massenburg, Donald; Vujicic, Snezana; Feng, Lanfei; Tang, Meiyi; Litbarg, Natalia; Antoni, Angelika; Rauch, Joyce; Lieberthal, Wilfred; Levine, Jerrold S.

    2015-01-01

    Apoptosis plays an indispensable role in the maintenance and development of tissues. We have shown that receptor-mediated recognition of apoptotic target cells by viable kidney proximal tubular epithelial cells (PTECs) inhibits the proliferation and survival of PTECs. Here, we examined the effect of apoptotic targets on PTEC cell growth (cell size during G1 phase of the cell cycle). Using a cell culture model, we show that apoptotic cells potently activate AMP-activated protein kinase (AMPK), a highly sensitive sensor of intracellular energy stores. AMPK activation leads to decreased activity of its downstream target, ribosomal protein p70 S6 kinase (p70S6K), and concomitant inhibition of cell growth. Importantly, these events occur without detectable change in intracellular levels of AMP, ADP, or ATP. Inhibition of AMPK, either pharmacologically by compound C or molecularly by shRNA, diminishes the effects of apoptotic targets and largely restores p70S6K activity and cell size to normal levels. Apoptotic targets also inhibit Akt, a second signaling pathway regulating cell growth. Expression of a constitutively active Akt construct partially relieved cell growth inhibition but was less effective than inhibition of AMPK. Inhibition of cell growth by apoptotic targets is dependent on physical interaction between apoptotic targets and PTECs but independent of phagocytosis. We conclude that receptor-mediated recognition of apoptotic targets mimics the effects of intracellular energy depletion, activating AMPK and inhibiting cell growth. By acting as sentinels of environmental change, apoptotic death may enable nearby viable cells, especially nonmigratory epithelial cells, to monitor and adapt to local stresses. PMID:26183782

  14. Experimental evaluation of decrease in bacterial activity due to cell death and activity decay in activated sludge.

    PubMed

    Hao, Xiaodi; Wang, Qilin; Zhang, Xiangping; Cao, Yali; van Mark Loosdrecht, C M

    2009-08-01

    Decrease in bacterial activity (cell decay) in activated sludge can be attributed to cell death (reduction in the amount of active bacteria) and activity decay (reduction in the specific activity of active bacteria). The aim of this study was to experimentally differentiate between cell death and activity decay as a source of decrease in microbial activity. By means of measuring maximal oxygen uptake rates, verifying membrane integrity by live/dead staining and verifying presence of 16S rRNA with fluorescence in-situ hybridization, the decay rates and the death rates of ammonium oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB) and ordinary heterotrophic organisms (OHOs) were determined respectively in a nitrifying sequencing batch reactor (SBR) and a heterotrophic SBR. The experiments revealed that in the nitrifying system activity decay contributed 47% and 82% to the decreased activities of AOB and NOB and that cell death was responsible for 53% and 18% of decreases in their respective activities. In the heterotrophic system, activity decay took a share of 78% in the decreased activity of OHOs, and cell death was only responsible for 22% of decrease in their activity. The difference between the importance of cell death on the decreased activities of AOB and OHOs might be caused by the mechanisms of substrate storage and/or cryptic growth/death-regeneration of OHOs. The different nutrient sources for AOB and NOB might be the reason for a relatively smaller fraction of cell death in NOB.

  15. GATA3 inhibits GCM1 activity and trophoblast cell invasion

    PubMed Central

    Chiu, Yueh Ho; Chen, Hungwen

    2016-01-01

    Development of human placenta involves the invasion of trophoblast cells from anchoring villi into the maternal decidua. Placental transcription factor GCM1 regulates trophoblast cell invasion via transcriptional activation of HtrA4 gene, which encodes a serine protease enzyme. The GATA3 transcription factor regulates trophoblast cell differentiation and is highly expressed in invasive murine trophoblast giant cells. The regulation of trophoblastic invasion by GCM1 may involve novel cellular factors. Here we show that GATA3 interacts with GCM1 and inhibits its activity to suppress trophoblastic invasion. Immunohistochemistry demonstrates that GATA3 and GCM1 are coexpressed in villous cytotrophoblast cells, syncytiotrophoblast layer, and extravillous trophoblast cells of human placenta. Interestingly, GATA3 interacts with GCM1, but not the GCM2 homologue, through the DNA-binding domain and first transcriptional activation domain in GCM1 and the transcriptional activation domains and zinc finger 1 domain in GATA3. While GATA3 did not affect DNA-binding activity of GCM1, it suppressed transcriptional activity of GCM1 and therefore HtrA4 promoter activity. Correspondingly, GATA3 knockdown elevated HtrA4 expression in BeWo and JEG-3 trophoblast cell lines and enhanced the invasion activities of both lines. This study uncovered a new GATA3 function in placenta as a negative regulator of GCM1 activity and trophoblastic invasion. PMID:26899996

  16. Cystatin F regulates proteinase activity in IL-2-activated natural killer cells.

    PubMed

    Maher, Katarina; Konjar, Spela; Watts, Colin; Turk, Boris; Kopitar-Jerala, Natasa

    2014-01-01

    Cystatin F is a unique member of the cystatin family of cysteine protease inhibitors, which is synthesized as an inactive dimer and it is activated by N-terminal cleavage in the endolysosomes. It is expressed in the cells of the immune system: myeloid cells and the cells involved in target cell killing: natural killer (NK) cells and cytotoxic T cells (CTLs). Upon activation of the NK cells with interleukin 2 (IL-2), cystatin F was found upregulated and co-localized in cytotoxic granules with cathepsin C (CatC) and CatV. However, cystatin F inhibits the CatC in cells only when its N-terminal part is processed. Although cystatin F could inhibit both CatV and CatC, the IL-2 stimulation of the YT cells resulted in an increased CatV activity, while the CatC activity was unchanged. The incubation of IL-2 activated NK cells with a cysteine proteinase inhibitor E-64d increased the cystatin F dimer formation. Our results suggest that cystatin F not only inhibits CatV, but it is processed by the CatV in order to inhibit the CatC activity in cytotoxic granules. The regulation of the CatC activity in the cytotoxic granules of the NK cells by the cystatin F could be important for the processing and activation of granule-associated serine proteases - granzymes.

  17. Preparation of cell lines for single-cell analysis of transcriptional activation dynamics.

    PubMed

    Rafalska-Metcalf, Ilona U; Janicki, Susan M

    2013-01-01

    Imaging molecularly defined regions of chromatin in single living cells during transcriptional activation has the potential to provide new insight into gene regulatory mechanisms. Here, we describe a method for isolating cell lines with multi-copy arrays of reporter transgenes, which can be used for real-time high-resolution imaging of transcriptional activation dynamics in single cells.

  18. 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.

  19. 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.

  20. 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

  1. Neural progenitor cells regulate microglia functions and activity.

    PubMed

    Mosher, Kira I; Andres, Robert H; Fukuhara, Takeshi; Bieri, Gregor; Hasegawa-Moriyama, Maiko; He, Yingbo; Guzman, Raphael; Wyss-Coray, Tony

    2012-11-01

    We found mouse neural progenitor cells (NPCs) to have a secretory protein profile distinct from other brain cells and to modulate microglial activation, proliferation and phagocytosis. NPC-derived vascular endothelial growth factor was necessary and sufficient to exert at least some of these effects in mice. Thus, neural precursor cells may not only be shaped by microglia, but also regulate microglia functions and activity.

  2. Signal transduction pathways in mast cell granule-mediated endothelial cell activation.

    PubMed Central

    Chi, Luqi; Stehno-Bittel, Lisa; Smirnova, Irina; Stechschulte, Daniel J; Dileepan, Kottarappat N

    2003-01-01

    BACKGROUND: We have previously shown that incubation of human endothelial cells with mast cell granules results in potentiation of lipopolysaccharide-induced production of interleukin-6 and interleukin-8. AIMS: The objective of the present study was to identify candidate molecules and signal transduction pathways involved in the synergy between mast cell granules and lipopolysaccharide on endothelial cell activation. METHODS: Human umbilical vein endothelial cells were incubated with rat mast cell granules in the presence and absence of lipopolysaccharide, and IL-6 production was quantified. The status of c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2 activation, nuclear factor-kappaB translocation and intracellular calcium levels were determined to identify the mechanism of synergy between mast cell granules and lipopolysaccaride. RESULTS: Mast cell granules induced low levels of interleukin-6 production by endothelial cells, and this effect was markedly enhanced by lipopolysaccharide. The results revealed that both serine proteases and histamine present in mast cell granules were involved in this activation process. Mast cell granules increased intracellular calcium, and activated c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2. The combination of lipopolysaccharide and mast cell granules prolonged c-Jun amino-terminal kinase activity beyond the duration of induction by either stimulant alone and was entirely due to active proteases. However, both proteases and histamine contributed to calcium mobilization and extracellular signal-regulated kinase 1/2 activation. The nuclear translocation of nuclear factor-kappaB proteins was of greater magnitude in endothelial cells treated with the combination of mast cell granules and lipopolysaccharide. CONCLUSIONS:Mast cell granule serine proteases and histamine can amplify lipopolysaccharide-induced endothelial cell activation, which involves calcium mobilization, mitogen-activated

  3. Light activated cell migration in synthetic extracellular matrices.

    PubMed

    Guo, Qiongyu; Wang, Xiaobo; Tibbitt, Mark W; Anseth, Kristi S; Montell, Denise J; Elisseeff, Jennifer H

    2012-11-01

    Synthetic extracellular matrices provide a framework in which cells can be exposed to defined physical and biological cues. However no method exists to manipulate single cells within these matrices. It is desirable to develop such methods in order to understand fundamental principles of cell migration and define conditions that support or inhibit cell movement within these matrices. Here, we present a strategy for manipulating individual mammalian stem cells in defined synthetic hydrogels through selective optical activation of Rac, which is an intracellular signaling protein that plays a key role in cell migration. Photoactivated cell migration in synthetic hydrogels depended on mechanical and biological cues in the biomaterial. Real-time hydrogel photodegradation was employed to create geometrically defined channels and spaces in which cells could be photoactivated to migrate. Cell migration speed was significantly higher in the photo-etched channels and cells could easily change direction of movement compared to the bulk hydrogels.

  4. Tubular solid oxide fuel cell demonstration activities

    SciTech Connect

    Ray, E.R.; Veyo, S.E.

    1995-12-31

    This reports on a solid oxide fuel cell demonstration program in which utilities are provided fully integrated, automatically controlled, packaged solid oxide fuel cell power generation systems. These field units serve 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.

  5. 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.

  6. ITK tunes IL-4-induced development of innate memory CD8+ T cells in a γδ T and invariant NKT cell-independent manner

    PubMed Central

    Huang, Weishan; Huang, Fei; Kannan, Arun Kumar; Hu, Jianfang; August, Avery

    2014-01-01

    True memory CD8+ T cells develop post antigenic exposure and can provide life-long immune protection. More recently, other types of memory CD8+ T cells have been described, such as the memory-like CD8+ T cells (IMP; CD44hiCD122+) that arise spontaneously in Itk−/− mice, which are suggested to develop as a result of IL-4 secreted by NKT-like γδ T or PLZF+ NKT cells found in Itk−/− mice. However, we report here that whereas IMP CD8+ T cell development in Itk−/− mice is dependent on IL-4/STAT6 signaling, it is not dependent on any γδ T or iNKT cells. Our experiments suggest that the IMP develops as a result of tuning of the CD8+ T cell response to exogenous IL-4 and TCR triggering by ITK and challenge the current model of IMP CD8+ T cell development as a result of NKT-like γδ T or iNKT cells. These findings suggest that some naive CD8+ T cells may be preprogrammed by weak homeostatic TCR signals in the presence of IL-4 to become memory phenotype cells with the ability to elaborate effector function rapidly. The role of ITK in this process suggests a mechanism by which IMP CD8+ T cells can be generated rapidly in response to infection. PMID:24620029

  7. Molecular dissection of AKT activation in lung cancer cell lines

    PubMed Central

    Guo, Yanan; Du, Jinyan; Kwiatkowski, David J

    2013-01-01

    AKT is a critical signaling node downstream of PI3K, which is often activated in cancer. We analyzed the state of activation of AKT in 80 human non-small cell lung cancer cell lines under serum starvation conditions. We identified 13 lines which showed persistent AKT activation in the absence of serum. In 12 of the 13 lines, AKT activation could be attributed to loss of PTEN, activating mutation in EGFR or PIK3CA, or amplification of ERBB2. HCC2429 was the only cell line that had no alterations in those genes, but had high phospho-AKT(Ser473) levels under serum starvation conditions. However, the activation of AKT in HCC2429 was PI3K- and mTORC2-dependent based upon use of specific inhibitors. Kinome tyrosine phosphorylation profiling showed that both Notch and SRC were highly activated in this cell line. Despite the activation of Notch, AKT activation and cell survival were not affected by Notch inhibitors DAPT or Compound E. In contrast, SRC inhibitors PP2 and dasatinib both significantly decreased pAKT(Ser473) levels and reduced cell survival by inducing apoptosis. Further, a combination of SRC and mTOR inhibition synergistically blocked activation of AKT and induced apoptosis. Over-expression of SRC has been identified previously in human lung cancers, and these results suggest that a combination of SRC and mTOR inhibitors may have unique therapeutic benefit for a subset of lung cancers with these molecular features. PMID:23319332

  8. Activation of peripheral blood mononuclear cells in bronchoalveolar lavage fluid from patients with sarcoidosis: visualisation of single cell activation products.

    PubMed Central

    Pantelidis, P.; Southcott, A. M.; Cambrey, A. D.; Laurent, G. J.; du Bois, R. M.

    1994-01-01

    BACKGROUND--Interstitial lung diseases are characterised by the recruitment of mononuclear cells to disease sites where maturation occurs and activation products, including lysozyme (LZM), are released. Analysis of in vitro cell culture supernatants for activation products masks the functional heterogeneity of cell populations. It is therefore necessary to examine the secretion of activation products by single cells to assess whether the activation of newly recruited mononuclear phagocytes at the sites of disease in the lung is uniform and controlled by the local microenvironment. METHODS--The reverse haemolytic plaque assay was used to evaluate, at a single cell level, the ability of bronchoalveolar lavage (BAL) fluid from seven patients with sarcoidosis to activate Ficoll-Hypaque-separated peripheral blood mononuclear cells by comparison with BAL fluid from six normal volunteers and nine patients with systemic sclerosis. Monolayers of peripheral blood mononuclear cells and sheep red blood cells were cultured either alone or in the presence of 20% (v/v) BAL fluid with a polyclonal anti-LZM antibody. LZM/anti-LZM complexes bound to red blood cells surrounding the secreting cells were disclosed following complement lysis of red blood cells and quantification of plaque dimensions using microscopy and image analysis. RESULTS--Bronchoalveolar lavage fluid from all the patients with sarcoidosis increased LZM secretion by peripheral blood mononuclear cells compared with unstimulated mononuclear cells. By contrast, BAL fluid from the other individuals had no effect on LZM secretion. CONCLUSIONS--Single cells activated by BAL fluid can be evaluated by the reverse haemolytic plaque assay. BAL fluid from patients with sarcoidosis, but not from patients with systemic sclerosis or normal individuals, contains components capable of activating mononuclear phagocytes to secrete lysozyme. Images PMID:7831632

  9. Mast cells and dendritic cells form synapses that facilitate antigen transfer for T cell activation

    PubMed Central

    Carroll-Portillo, Amanda; Cannon, Judy L.; te Riet, Joost; Holmes, Anna; Kawakami, Yuko; Kawakami, Toshiaki; Cambi, Alessandra

    2015-01-01

    Mast cells (MCs) produce soluble mediators such as histamine and prostaglandins that are known to influence dendritic cell (DC) function by stimulating maturation and antigen processing. Whether direct cell–cell interactions are important in modulating MC/DC function is unclear. In this paper, we show that direct contact between MCs and DCs occurs and plays an important role in modulating the immune response. Activation of MCs through FcεRI cross-linking triggers the formation of stable cell–cell interactions with immature DCs that are reminiscent of the immunological synapse. Direct cellular contact differentially regulates the secreted cytokine profile, indicating that MC modulation of DC populations is influenced by the nature of their interaction. Synapse formation requires integrin engagement and facilitates the transfer of internalized MC-specific antigen from MCs to DCs. The transferred material is ultimately processed and presented by DCs and can activate T cells. The physiological outcomes of the MC–DC synapse suggest a new role for intercellular crosstalk in defining the immune response. PMID:26304724

  10. Activation-induced necroptosis contributes to B-cell lymphopenia in active systemic lupus erythematosus

    PubMed Central

    Fan, H; Liu, F; Dong, G; Ren, D; Xu, Y; Dou, J; Wang, T; Sun, L; Hou, Y

    2014-01-01

    B-cell abnormality including excessive activation and lymphopenia is a central feature of systemic lupus erythematosus (SLE). Although activation threshold, auto-reaction and death of B cells can be affected by intrinsical and/or external signaling, the underlying mechanisms are unclear. Herein, we demonstrate that co-activation of Toll-like receptor 7 (TLR7) and B-cell receptor (BCR) pathways is a core event for the survival/dead states of B cells in SLE. We found that the mortalities of CD19+CD27- and CD19+IgM+ B-cell subsets were increased in the peripheral blood mononuclear cells (PBMCs) of SLE patients. The gene microarray analysis of CD19+ B cells from active SLE patients showed that the differentially expressed genes were closely correlated to TLR7, BCR, apoptosis, necroptosis and immune pathways. We also found that co-activation of TLR7 and BCR could trigger normal B cells to take on SLE-like B-cell characters including the elevated viability, activation and proliferation in the first 3 days and necroptosis in the later days. Moreover, the necroptotic B cells exhibited mitochondrial dysfunction and hypoxia, along with the elevated expression of necroptosis-related genes, consistent with that in both SLE B-cell microarray and real-time PCR verification. Expectedly, pretreatment with the receptor-interacting protein kinase 1 (RIPK1) inhibitor Necrostatin-1, and not the apoptosis inhibitor zVAD, suppressed B-cell death. Importantly, B cells from additional SLE patients also significantly displayed high expression levels of necroptosis-related genes compared with those from healthy donors. These data indicate that co-activation of TLR7 and BCR pathways can promote B cells to hyperactivation and ultimately necroptosis. Our finding provides a new explanation on B-cell lymphopenia in active SLE patients. These data suggest that extrinsic factors may increase the intrinsical abnormality of B cells in SLE patients. PMID:25210799

  11. 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.

  12. 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

  13. [The presence of an endogenous peroxidase activity in hairy cell leukemia cells].

    PubMed

    Reyes, F; Gourdin, M F; Farcet, J P; Dreyfus, B; Breton-Gorius, J

    1977-02-01

    Mononuclear cells from hairy cell leukemia have been studied in three cases by ultrastructural immunocytochemistry. Cells have fairly detectable surface immunoglobulins, without monoclonal distribution however. In addition these cells have a peroxidatic activity which is revealed in the perinuclear space and strands of endoplasmic reticulum. PMID:404081

  14. Interleukin-7 and Toll-Like Receptor 7 Induce Synergistic B Cell and T Cell Activation

    PubMed Central

    Bikker, Angela; Kruize, Aike A.; van der Wurff-Jacobs, Kim M. G.; Peters, Rogier P.; Kleinjan, Marije; Redegeld, Frank; de Jager, Wilco; Lafeber, Floris P. J. G.; van Roon, Joël A. G.

    2014-01-01

    Objectives To investigate the potential synergy of IL-7-driven T cell-dependent and TLR7-mediated B cell activation and to assess the additive effects of monocyte/macrophages in this respect. Methods Isolated CD19 B cells and CD4 T cells from healthy donors were co-cultured with TLR7 agonist (TLR7A, Gardiquimod), IL-7, or their combination with or without CD14 monocytes/macrophages (T/B/mono; 1 : 1 : 0,1). Proliferation was measured using 3H-thymidine incorporation and Ki67 expression. Activation marker (CD19, HLA-DR, CD25) expression was measured by FACS analysis. Immunoglobulins were measured by ELISA and release of cytokines was measured by Luminex assay. Results TLR7-induced B cell activation was not associated with T cell activation. IL-7-induced T cell activation alone and together with TLR7A synergistically increased numbers of both proliferating (Ki67+) B cells and T cells, which was further increased in the presence of monocytes/macrophages. This was associated by up regulation of activation markers on B cells and T cells. Additive or synergistic induction of production of immunoglobulins by TLR7 and IL-7 was associated by synergistic induction of T cell cytokines (IFNγ, IL-17A, IL-22), which was only evident in the presence of monocytes/macrophages. Conclusions IL-7-induced CD4 T cell activation and TLR7-induced B cell activation synergistically induce T helper cell cytokine and B cell immunoglobulin production, which is critically dependent on monocytes/macrophages. Our results indicate that previously described increased expression of IL-7 and TLR7 together with increased numbers of macrophages at sites of inflammation in autoimmune diseases like RA and pSS significantly contributes to enhanced lymphocyte activation. PMID:24740301

  15. 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.

  16. CD27-CD70 interactions regulate B-cell activation by T cells.

    PubMed Central

    Kobata, T; Jacquot, S; Kozlowski, S; Agematsu, K; Schlossman, S F; Morimoto, C

    1995-01-01

    CD27, a member of the tumor necrosis factor (TNF) receptor family, binds to its ligand CD70, a member of the TNF family, and subsequently induces T-cell costimulation and B-cell activation. CD27 is expressed on resting T and B cells, whereas CD70 is expressed on activated T and B cells. Utilizing transfected murine pre-B-cell lines expressing human CD27 or CD70, we have examined the effect of such transfectant cells on human B-cell IgG production and B-cell proliferation. We show that the addition of CD27-transfected cells to a T-cell-dependent, pokeweed mitogen-driven B-cell IgG synthesis system resulted in marked inhibition of IgG production, whereas the addition of CD70-transfected cells enhanced IgG production. The inhibition and enhancement of pokeweed mitogen-driven IgG production by CD27 and CD70 transfectants were abrogated by pretreatment with anti-CD27 and anti-CD70 monoclonal antibodies, respectively. In contrast, little or no inhibition of IgG production and B-cell proliferation was noted with CD27-transfected cells or either anti-CD27 or CD70 monoclonal antibody in a T-cell-independent Staphylococcus aureus/interleukin 2-driven B-cell activation system. In this same system CD70-transfected cells enhanced B-cell IgG production and B-cell proliferation, and this enhancement could be gradually abrogated by addition of increasing numbers of CD27-transfected cells. These results clearly demonstrate that interactions among subsets of T cells expressing CD27 and CD70 play a key role in regulating B-cell activation and immunoglobulin synthesis. PMID:7479974

  17. Multivalent Antigens for Promoting B and T Cell Activation

    PubMed Central

    Bennett, Nitasha R.; Zwick, Daniel B.; Courtney, Adam H.; Kiessling, Laura L.

    2015-01-01

    Efficacious vaccines require antigens that elicit productive immune system activation. Antigens that afford robust antibody production activate both B and T cells. Elucidating the antigen properties that enhance B–T cell communication is difficult with traditional antigens. We therefore used ring-opening metathesis polymerization to access chemically defined, multivalent antigens containing both B and T cell epitopes to explore how antigen structure impacts B cell and T cell activation and communication. The bifunctional antigens were designed so that the backbone substitution level of each antigenic epitope could be quantified using 19F NMR. The T cell peptide epitope was appended so that it could be liberated in B cells via the action of the endosomal protease cathepsin D, and this design feature was critical for T cell activation. Antigens with high BCR epitope valency induce greater BCR-mediated internalization and T cell activation than did low valency antigens, and these high-valency polymeric antigens were superior to protein antigens. We anticipate that these findings can guide the design of more effective vaccines. PMID:25970017

  18. Bacterial lipopolysaccharide activates CD57-negative human NK cells.

    PubMed

    Kanevskiy, L M; Erokhina, S A; Streltsova, M A; Telford, W G; Sapozhnikov, A M; Kovalenko, E I

    2014-12-01

    NK cells play an important regulatory role in sepsis by induction and augmentation of proinflammatory reactions in early stages of the septic process and by suppression of immune response in later stages of inflammation. The present work was aimed at the effect of bacterial lipopolysaccharide (LPS), the main pathogenic factor of sepsis development, on human NK cells ex vivo. We show that LPS activates immature CD57-negative NK cells, which typically constitute less than half of the normal NK cell population in human peripheral blood. Under conditions of NK cell stimulation with IL-2, addition of LPS provokes an increase in IFN-γ production. However, LPS both increased and inhibited NK cell cytotoxic activity. It is important to note that the activation of NK cells on LPS addition was observed in the absence of TLR4 on the NK cell surface. These results confirm our previous data arguing for a direct interaction of LPS with NK cells and evidence an atypical mechanism of LPS-induced NK cell activation without the involvement of surface TLR4.

  19. 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.

  20. Twin Knudsen Cell Configuration for Activity Measurements by Mass Spectrometry

    NASA Technical Reports Server (NTRS)

    Jacobson, N. S.

    1996-01-01

    A twin Knudsen cell apparatus for alloy activity measurements by mass spectrometry is described. Two Knudsen cells - one containing an alloy and one containing a pure component - are mounted on a single flange and translated into the sampling region via a motorized x-y table. Mixing of the molecular beams from the cells is minimized by a novel system of shutters. Activity measurements were taken on two well-characterized alloys to verify the operation of the system. Silver activity measurements are reported for Ag-Cu alloys and aluminum activity measurements are reported for Fe-Al alloys. The temperature dependence of activity for a 0.474 mol fraction Al-Fe alloy gives a partial molar heat of aluminum. Measurements taken with the twin cell show good agreement with literature values for these alloys.

  1. T cell-activating protein on murine lymphocytes.

    PubMed

    Yeh, E T; Reiser, H; Benacerraf, B; Rock, K L

    1986-12-01

    A functional T cell surface molecule, T cell-activating protein (TAP), has been identified on murine lymphocytes. TAP is a protein with an apparent molecular mass of 10-12 kilodaltons (kDa) nonreduced, 16-17 kDa reduced. Cross-linking of TAP can result in profound activation of T lymphocytes to produce lymphokines and to enter the cell cycle. Furthermore, antibody binding to TAP can modulate antigen-driven T cell stimulation. Current data suggest that TAP is physically distinct from the T cell receptor complex. On unstimulated lymphocytes, TAP is expressed on T cells and defines heterogeneity within the major T cell subsets. Its profile of expression is rapidly altered on cell activation. Ontologically, it is first detected in the thymus, where it is found on both the most immature and the most mature cell subsets, and it is functional on both. Together, these TAP+ cells constitute a small fraction of thymocytes. TAP expression, however, defines the immunocompetent compartment of the thymus, and thus could be involved in functional maturation. Finally, the gene controlling TAP expression has been mapped, and is tightly linked to a locus of hematopoietic antigens (Ly-6). TAP is molecularly distinct from these antigens. Furthermore, all of these proteins show a markedly distinct developmental regulation in their cell surface expression.

  2. 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.

  3. Cytotoxic activity of allogeneic natural killer cells on U251 glioma cells in vitro.

    PubMed

    Guo, Meng; Wu, Tingting; Wan, Lixin

    2016-07-01

    The present study aimed to observe the cytotoxic activity of allogeneic natural killer (NK) cells on U251 glioma cells and to investigate their mechanism of action to establish an effective treatment strategy for neuroglioma. Cell survival curves, colony formation assays and karyotype analysis were performed to investigate the characteristics of U251 glioma cells. The present study demonstrated that natural killer group 2, member D (NKG2D)‑major histocompatibility complex class I‑related chain A/B (MICA/B) interactions contributed to the cytotoxic effect of NK cells on K562 and U251 cells. In antibody‑blocking assays to inhibit NKG2D ligands, the cytotoxic activity was not completely attenuated, which suggested that other signaling pathways contribute to the cytotoxic activity of NK cells on tumor cells in addition to the NKG2D‑mediated activity. The present study identified that the expression levels of NKG2D ligands on the surface of target cells influenced the strength of the NK cell immune response. Furthermore, allogeneic NK cells were observed to kill glioma cells in vitro, and this anticancer activity is associated with the rate of NKG2D expression on the surface of glioma cells.

  4. L1 cell adhesion molecule induces melanoma cell motility by activation of mitogen-activated protein kinase pathways.

    PubMed

    Yi, Young-Su; Baek, Kwang-Soo; Cho, Jae Youl

    2014-06-01

    L1 cell adhesion molecule (L1CAM) is highly expressed in various types of cancer cells and has been implicated in the control of cell proliferation and motility. Recently, L1CAM was reported to induce the motility of melanoma cells, but the mechanism of this induction remains poorly understood. In this study, we investigated the molecular mechanisms by which L1CAM induces the motility of melanoma cells. Unlike other types of cancer cells, B16F10 melanoma cells highly expressed L1CAM at both the RNA and protein levels, and the expression of L1CAM induced AP-1 activity. In accordance to AP-1 activation, MAPK signaling pathways were activated by L1CAM. Inhibition of L1CAM expression by L1CAM-specific siRNA suppressed the activation of MAPKs such as ERK and p38. However, no significant change was observed in JNK activation. As expected, upstream MAP2K, MKK3/6, MAP3K, and TAK1 were also deactivated by the inhibition of L1CAM expression. L1CAM induced the motility of B16F10 cells. Inhibition of L1CAM expression suppressed migration and invasion of B16F10 cells, but no suppressive effect was observed on their proliferation and anti-apoptotic resistance. Treatment of B16F10 cells with U0126, an ERK inhibitor, or SB203580, a p38 inhibitor, suppressed the migration and invasion abilities of B16F10 cells. Taken together, our results suggest that L1CAM induces the motility of B16F10 melanoma cells via the activation of MAPK pathways. This finding provides a more detailed molecular mechanism of L1CAM-mediated induction of melanoma cell motility. PMID:24974583

  5. Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells

    PubMed Central

    Jean, Elise; Laoudj-Chenivesse, Dalila; Notarnicola, Cécile; Rouger, Karl; Serratrice, Nicolas; Bonnieu, Anne; Gay, Stéphanie; Bacou, Francis; Duret, Cédric; Carnac, Gilles

    2011-01-01

    Abstract Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties, we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant-derived cells were incubated with ALDEFLUOR, a fluorescent substrate for ALDH, and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast-CD56+ fraction in those cells, but, we also observed heterogeneity of ALDH activity levels within CD56-purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly, ALDH activity and Aldh1a1 expression levels were very low in mouse, rat, rabbit and non-human primate myoblasts. Using different approaches, from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde, an inhibitor of class I ALDH, to cell fractionation by flow cytometry using the ALDEFLUOR assay, we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H2O2)-induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity, as a purification strategy, could allow non-toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage. PMID:19840193

  6. 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.

  7. 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

  8. 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

  9. Cytolytic activity of Naegleria fowleri cell-free extract.

    PubMed

    Fulford, D E; Marciano-Cabral, F

    1986-11-01

    The cytotoxic activity of a cell-free extract of Naegleria fowleri amebae on B103 rat nerve cells in culture was investigated. The cell-free extract was prepared by subjecting lysed amebae to centrifugation at 100,000 g for 1 h, precipitation of the supernatant fluid with 30-60% saturated ammonium sulfate, and desalting by group exclusion chromatography utilizing Sephadex G-25. The supernatant fluid recovered from this procedure was termed the soluble fraction. The Naegleria cytotoxic activity present in the soluble fraction was assayed by 51Cr released from labeled B103 cells. The Naegleria soluble fraction, when added to nerve cells, elicited blebs on the B103 target cell surface within 5 min after exposure to the fraction. Later, holes were observed in the B103 cell plasma membrane. These alterations were never observed on untreated B103 cells. Phospholipase A, phospholipase C, and protease activities were associated with the desalted ammonium sulfate-precipitable cytotoxic activity of N. fowleri cell-free lysate. The cytotoxic activity was impaired by ethylenediamine-tetraacetate (EDTA), phospholipase A inhibitor (Rosenthal's reagent), heating at 50 degrees C for 15 min, or incubation at pH 10 for 60 min. Repeated freeze-thawing and inhibitors of proteolytic enzymes had no effect on the cytotoxic activity. Small amounts of ethanol (5% v/v) enhanced cytotoxic activity of the fraction. Phospholipases A and C, as well as other as yet unidentified cytolytic factors may be responsible for producing 51Cr release from target cells by the soluble fraction of N. fowleri extracts.

  10. 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. PMID:25184988

  11. Patterns of plasminogen activator production in cultured normal embryonic cells

    PubMed Central

    1977-01-01

    Cultured normal low-passage embryo fibroblasts, from a number of species, and two untransformed clones of a Balb/3T3 line elaborate increasing amounts of plasminogen activator (PA) as they approach confluence; the low-passage cells then lose this PA activity after reaching confluence, while the 3T3 cells retain it indefinitely. Even at their peaks, however, the PA activities of the low-passage cells remain well below those of the corresponding virally or spontaneously transformed cells. The PA increases in normal cells are probably a result of PA production rather than of adsorption of secreted PA to the cell surface, or of changes in cell-associated protease inhibitors. The elaboration of PA by normal cells is dependent upon their metabolic activity, such that the level of serum supplementation and the growth phase of the culture directly influence the level of cell-associated PA observed. In addition, there may be a component of serum which exerts a negative control on PA production and which is not an acid-labile protease inhibitor. PMID:21193

  12. Utilizing Chimeric Antigen Receptors to Direct Natural Killer Cell Activity

    PubMed Central

    Hermanson, David L.; Kaufman, Dan S.

    2015-01-01

    Natural killer (NK) cells represent an attractive lymphocyte population for cancer immunotherapy due to their ability to lyse tumor targets without prior sensitization and without need for human leukocyte antigens-matching. Chimeric antigen receptors (CARs) are able to enhance lymphocyte targeting and activation toward diverse malignancies. CARs consist of an external recognition domain (typically a small chain variable fragment) directed at a specific tumor antigen that is linked with one or more intracellular signaling domains that mediate lymphocyte activation. Most CAR studies have focused on their expression in T cells. However, use of CARs in NK cells is starting to gain traction because they provide a method to redirect these cells more specifically to target refractory cancers. CAR-mediated anti-tumor activity has been demonstrated using NK cell lines, as well as NK cells isolated from peripheral blood, and NK cells produced from human pluripotent stem cells. This review will outline the CAR constructs that have been reported in NK cells with a focus on comparing the use of different signaling domains in combination with other co-activating domains. PMID:25972867

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

    PubMed

    Kim, Neil H; Lee, Gloria; Sherer, Nicholas A; Martini, K Michael; Goldenfeld, Nigel; Kuhlman, Thomas E

    2016-06-28

    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

  14. 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

  15. Autoimmunity, polyclonal B-cell activation and infection.

    PubMed

    Granholm, N A; Cavallo, T

    1992-02-01

    It is widely believed that autoimmunity is an integral part of the immune system, and that genetic, immunologic, hormonal, environmental and other factors contribute to the pathogenesis of autoimmune disease. Thus, autoimmune disease may represent an abnormal expression of immune functions instead of loss of tolerance to self, and it can be organ specific or systemic in its manifestations. We review the various factors that contribute to the development of autoimmune disease; we also review the mechanisms of polyclonal B-cell activation, with emphasis on the role of infectious agents. We consider systemic lupus erythematosus in humans and in experimental animals as prototypic autoimmune disease, and we summarize data to indicate that polyclonal B-cell activation is central to the pathogenesis of systemic autoimmune disease. The effect of polyclonal B-cell activation, brought about by injections of a B-cell activator-lipopolysaccharide from Gram-negative bacteria-is sufficient to cause autoimmune disease in an immunologically normal host. In fact, autoimmune disease can be arrested if excessive polyclonal B-cell activation is suppressed; alternatively, autoimmune disease can be exacerbated if polyclonal B-cell activation is enhanced. We explore the mechanism of tissue injury when autoimmune disease is induced or exacerbated, and we consider the pathogenic roles of autoantibodies, immune complexes, complement, the blood cell carrier system, and the mononuclear phagocyte system. Although polyclonal B-cell activation may be the mechanism whereby various factors can cause or exacerbate systemic autoimmune disease, polyclonal B-cell activation may cause autoimmune disease on its own.

  16. 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.

  17. Cell-based flow cytometry assay to measure cytotoxic activity.

    PubMed

    Noto, Alessandra; Ngauv, Pearline; Trautmann, Lydie

    2013-12-17

    Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU₃₀/10(6) cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

  18. 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

  19. Functional activity of mitochondria in cultured neural precursor cells.

    PubMed

    Plotnikov, E Yu; Marei, M V; Podgornyi, O V; Aleksandrova, M A; Zorov, D B; Sukhikh, G T

    2006-01-01

    We studied mitochondrial transmembrane potential of neural precursor cells forming neurospheres in culture. Uneven energization of mitochondria in neurosphere cells was detected. Heterogeneity of cells by the mitochondrial potential increased with neurosphere enlargement during culturing. Decrease in the mitochondrial potential in the central cells in large spheres, presumably caused by insufficient diffusion of oxygen and nutrients, can provoke their damage and death. Population of cells with high mitochondrial potential responded to addition of the nuclear dye by a decrease in mitochondrial potential, which can indicate functioning of ABCG2 complex in these cells, characteristic of undifferentiated stem cells. These data will help to create optimum conditions for culturing of neural stem cells for the maintenance of their maximum functional and proliferative activity. PMID:16929986

  20. 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.

  1. Cytolytic activity against tumor cells by macrophage cell lines and augmentation by macrophage stimulants.

    PubMed

    Taniyama, T; Holden, H T

    1980-07-15

    Previous studies have shown that macrophage cell lines retained the ability to phagocytize, to secrete lysosomal enzymes, and to function as effector cells in antibody-dependent cellular cytoxicity. In this paper, the cytolytic activity of murine macrophage cell lines against tumor target cells was assessed using an 18-h 51Cr release assay. Of the macrophage cell lines tested, RAW 264, PU5-1.8 and IC-21 had intermediate to high levels of spontaneous cytolytic activity, P388D, and J774 had low to intermediate levels, while /WEHI-3 showed little or no cytolytic activity against RBL-5, MBL-2 and TU-5 target cells. Tumor-cell killing by macrophage cell lines could be augmented by the addition of macrophage stimulants, such as bacterial lipopolysaccharide and poly I:C, indicating that the activation of macrophages by these stimulants does not require the participation of other cell types. Treatment with interferon also augmented the tumor-cell killing by macrophage cell lines. Although the mechanism by which these cell lines exert their spontaneous or boosted cytotoxic activity is not clear, it does not appear to be due to depletion of nutrients since cell lines with high metabolic and proliferative activities, such as WEHI-3 and RBL-5, showed little or no cytotoxicity and supernatants from the macrophage cell lines did not exert any cytotoxic effects in their essay. Thus, it appears that the different macrophage cell lines represent different levels of activation and/or differentiation and may be useful for studying the development of these processes as well as providing a useful tool for analyzing the mechanisms of macrophage-mediated cytolysis. PMID:6165690

  2. Indoleamine 2,3-dioxygenase Activity Contributes to Local Immune Suppression in the Skin Expressing Human Papillomavirus Oncoprotein E7

    PubMed Central

    Mittal, D; Kassianos, AJ; Tran, LS; Bergot, AS; Gosmann, C; Hofmann, J; Blumenthal, A; Leggatt, GR; Frazer, IH

    2013-01-01

    Chronic infection of anogenital epithelium with human papillomavirus (HPV) promotes development of cancer. Many pathogens evoke immunosuppressive mechanisms to enable persistent infection. We have previously shown that grafted skin expressing HPV16 E7 oncoprotein from a keratin-14 promoter (K14E7) is not rejected by a syngeneic, immunocompetent host. In this study we show that indoleamine 2, 3-dioxygenase (IDO) 1, an IFN-γ inducible immunoregulatory molecule, is more highly expressed by langerin−ve dermal dendritic cells from K14E7 skin than nontransgenic control skin. Furthermore, inhibiting IDO activity using 1-D/L-methyl tryptophan promotes K14E7 skin graft rejection. Increased IDO1 expression and activity in K14E7 skin requires IFN-γ and iNKT cells, both of which have been shown to negatively regulate T-cell effector function and suppress K14E7 graft rejection. Further, dendritic cells from K14E7 skin express higher level of IFN-γ receptor (IFN-γR) than dendritic cells from control skin. K14E7 transgenic skin recruits significantly higher number of dendritic cells, independent of IFN-γ and IFN-γR expression. Consistent with these observations in a murine model, we found higher expression of IDO1 and IFN-γ but not IDO2 in the cervical epithelium of patients with HPV-associated cervical intraepithelial neoplasia (CIN) 2/3. Our data support a hypothesis that induction of IDO1 in HPV infected skin contributes to evasion of host immunity. PMID:23652797

  3. 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

    2016-09-01

    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.

  4. 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

    2016-09-01

    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. PMID:27569912

  5. Src activity increases and Yes activity decreases during mitosis of human colon carcinoma cells.

    PubMed Central

    Park, J; Cartwright, C A

    1995-01-01

    Src and Yes protein-tyrosine kinase activities are elevated in malignant and premalignant tumors of the colon. To determine whether Src activity is elevated throughout the human colon carcinoma cell cycle as it is in polyomavirus middle T antigen- or F527 Src-transformed cells, and whether Yes activity, which is lower than that of Src in the carcinoma cells, is regulated differently, we measured their activities in cycling cells. We observed that the activities of both kinases were higher throughout all phases of the HT-29 colon carcinoma cell cycle than in corresponding phases of the fibroblast cycle. In addition, during mitosis of HT-29 cells, Src specific activity increased two- to threefold more, while Yes activity and abundance decreased threefold. The decreased steady-state protein levels of Yes during mitosis appeared to be due to both decreased synthesis and increased degradation of the protein. Inhibition of tyrosine but not serine/threonine phosphatases abolished the mitotic activation of Src. Mitotic Src was phosphorylated at novel serine and threonine sites and dephosphorylated at Tyr-527. Two cellular proteins (p160 and p180) were phosphorylated on tyrosine only during mitosis. Tyrosine phosphorylation of several other proteins decreased during mitosis. Thus, Src in HT-29 colon carcinoma cells, similar to Src complexed to polyomavirus middle T antigen or activated by mutation at Tyr-527, is highly active in all phases of the cell cycle. Moreover, Src activity further increases during mitosis, whereas Yes activity and abundance decrease. Thus, Src and Yes appear to be regulated differently during mitosis of HT-29 colon carcinoma cells. PMID:7739521

  6. Characterization of tissue plasminogen activator binding proteins isolated from endothelial cells and other cell types

    SciTech Connect

    Beebe, D.P.; Wood, L.L.; Moos, M. )

    1990-07-15

    Human tissue plasminogen activator (t-PA) was shown to bind specifically to human osteosarcoma cells (HOS), and human epidermoid carcinoma cells (A-431 cells). Crosslinking studies with DTSSP demonstrated high molecular weight complexes (130,000) between {sup 125}I-t-PA and cell membrane protein on human umbilical vein endothelial cells (HUVEC), HOS, and A-431 cells. A 48-65,000 molecular weight complex was demonstrated after crosslinking t-PA peptide (res. 7-20) to cells. Ligand blotting of cell lysates which had been passed over a t-PA affinity column revealed binding of t-PA to 54,000 and 95,000 molecular weight proteins. Several t-PA binding proteins were identified in immunopurified cell lysates, including tubulin beta chain, plasminogen activator inhibitor type 1 and single chain urokinase.

  7. 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

  8. Role and regulation of CD1d in normal and pathological B cells

    PubMed Central

    Chaudhry, Mohammed S.; Karadimitris, Anastasios

    2015-01-01

    CD1d is a non-polymorphic, MHC class I-like molecule, which presents phosphoand glycosphingo-lipid antigens to a subset of CD1d-restricted T cells called invariant NKT (iNKT) cells. This CD1d-iNKT cell axis regulates nearly all aspects of both the innate and adaptive immune response. Expression of CD1d on B cells is suggestive of the ability of these cells to present antigen to and form cognate interactions with iNKT cells. Herein we summarise key evidence regarding the role and regulation of CD1d in normal B cells and in humoral immunity. We then extend the discussion to B cell disorders, with emphasis on autoimmune disease, viral infection and neoplastic transformation of B lineage cells, where CD1d expression can be altered as a mechanism of immune evasion, and can have both diagnostic and prognostic importance. Finally we highlight current and future therapeutic strategies that aim to target the CD1d-iNKT axis in B cells. PMID:25381357

  9. 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…

  10. Increased Mitochondrial Activity in Anthrax-Induced Cell Death

    PubMed Central

    Li, Chi

    2009-01-01

    Pathogenesis of anthrax lethal toxin (LT) is attributed to its ability to cause death of infected cells. New work has demonstrated that increase of mitochondrial F1F0 ATPase activity and subsequent depletion of cellular ATP level are critical early events during LT-induced cell death. PMID:26124679

  11. Buoyancy-Activated Cell Sorting Using Targeted Biotinylated Albumin Microbubbles

    PubMed Central

    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 florescence-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 2μm 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 10g 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 targeted

  12. Endothelial cell phagocytosis of senescent neutrophils decreases procoagulant activity.

    PubMed

    Gao, Chunyan; Xie, Rui; Li, Wen; Zhou, Jin; Liu, Shuchuan; Cao, Fenglin; Liu, Yue; Ma, Ruishuang; Si, Yu; Liu, Yan; Bi, Yayan; Gilbert, Gary E; Shi, Jialan

    2013-06-01

    Abundant senescent neutrophils traverse the vascular compartment and may contribute to pathologic conditions. For example, they become procoagulant when undergoing apoptosis and may contribute to thrombosis or inflammation. Our previous studies demonstrated a dominant clearance pathway in which the neutrophils can be phagocytosed by liver macrophages. The aim of this study was to explore an alternate pathway of neutrophil clearance by endothelial cells. Phagocytosis of the neutrophils by endothelial cells was performed using various experimental approaches includingflow cytometry, confocal microscopy and electron microscopy assays in vitro and in vivo. Procoagulant activity of cultured neutrophils was evaluated by coagulation time, factor Xase and prothrombinase assays. Lactadherin functioned as a novel probe for the detection of phosphatidylserine on apoptotic cells, an opsonin (bridge) between apoptotic cell and phagocyte for promoting phagocytosis, and an efficient anticoagulant for inhibition of factor Xase and thrombin formation. When cultured, purified human neutrophils spontaneously entered apoptosis and developed procoagulant activity that was directly related to the degree of phosphatidylserine exposure. Co-culture of aged neutrophils and endothelial cells resulted in phagocytosis of the neutrophils and prolonged coagulation time. Lactadherin diminished the procoagulant activity and increased the rate of neutrophil clearance. In vivo, neutrophils were sequestered by endothelial cells after blockade of Kupffer cells, a process that was dependent upon both phosphatidylserine exposure and P-selectin expression. Thus, the ability of endothelial cells to clear senescent neutrophils may limit the procoagulant and/or inflammatory impact of these cells.

  13. 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

  14. Aberrant activation of nuclear factor of activated T cell 2 in lamina propria mononuclear cells in ulcerative colitis

    PubMed Central

    Shih, Tsung-Chieh; Hsieh, Sen-Yung; Hsieh, Yi-Yueh; Chen, Tse-Chin; Yeh, Chien-Yu; Lin, Chun-Jung; Lin, Deng-Yn; Chiu, Cheng-Tang

    2008-01-01

    AIM: To investigate the role of nuclear factor of activated T cell 2 (NFAT2), the major NFAT protein in peripheral T cells, in sustained T cell activation and intractable inflammation in human ulcerative colitis (UC). METHODS: We used two-dimensional gel-electrophoresis, immunohistochemistry, double immunohistochemical staining, and confocal microscopy to inspect the expression of NFAT2 in 107, 15, 48 and 5 cases of UC, Crohn’s disease (CD), non-specific colitis, and 5 healthy individuals, respectively. RESULTS: Up-regulation with profound nucleo-translocation/activation of NFAT2 of lamina propria mononuclear cells (LPMC) of colonic mucosa was found specifically in the affected colonic mucosa from patients with UC, as compared to CD or NC (P < 0.001, Kruskal-Wallis test). Nucleo-translocation/activation of NFAT2 primarily occurred in CD8+T, but was less prominent in CD4+ T cells or CD20+B cells. It was strongly associated with the disease activity, including endoscopic stage (τ = 0.2145, P = 0.0281) and histologic grade (τ = 0.4167, P < 0.001). CONCLUSION: We disclose for the first time the nucleo-translocation/activatin of NFAT2 in lamina propria mononuclear cells in ulcerative colitis. Activation of NFAT2 was specific for ulcerative colitis and highly associated with disease activity. Since activation of NFAT2 is implicated in an auto-regulatory positive feedback loop of sustained T-cell activation and NFAT proteins play key roles in the calcium/calcineurin signaling pathways, our results not only provide new insights into the mechanism for sustained intractable inflammation, but also suggest the calcium-calcineurin/NFAT pathway as a new therapeutic target for ulcerative colitis. PMID:18350607

  15. 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

  16. 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.

  17. The activation of protease-activated receptor 1 mediates proliferation and invasion of nasopharyngeal carcinoma cells.

    PubMed

    Zhu, Qingyao; Luo, Jianchao; Wang, Tao; Ren, Jinghua; Hu, Kai; Wu, Gang

    2012-07-01

    Protease-activated receptor 1 (PAR-1) is a G-coupled membrane protein, which is involved in physiological and malignant invasion processes. It is activated by serine proteases such as thrombin through a unique form or by specific synthetic peptides. In this study, we determined the expression of PAR-1 in five nasopharyngeal carcinoma (NPC) cell lines with different characteristics of invasiveness and metastasis, and found that the levels of PAR-1 expression were higher in invasive or metastatic cell lines than those in low invasive or metastatic ones. Of the five NPC cell lines, CNE1-LMP1 cells had the highest expression levels of PAR-1, which was mainly distributed at the membrane and in the cytoplasm of tumor cells. Further study showed that the thrombin receptor synthetic activating peptide SFLLRN could stimulate the growth of CNE1-LMP1 cells in a dose-dependent manner. However, thrombin itself had a dual effect on the proliferation of NPC cells. Concentrations of thrombin in the range of 0.1-0.5 U/ml promoted cell growth, but concentrations higher than 0.5 U/ml impaired cell growth. Moreover, thrombin and SFLLRN also enhanced the invasive capabilities of CNE1-LMP1 cells in vitro, and this was partly due to enhancing the activities of MMP-2 and MMP-9. Our findings suggest that PAR-1 may contribute to the growth and invasive potential of NPC cells. PMID:22562397

  18. 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.

  19. 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.

  20. Spontaneous Activity of Cochlear Hair Cells Triggered by Fluid Secretion Mechanism in Adjacent Support Cells.

    PubMed

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

    2015-12-01

    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 Ca(2+)-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 supporting 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. 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.

  2. Dengue Virus Infection of Mast Cells Triggers Endothelial Cell Activation

    PubMed Central

    Brown, Michael G.; Hermann, Laura L.; Issekutz, Andrew C.; Marshall, Jean S.; Rowter, Derek; Al-Afif, Ayham; Anderson, Robert

    2011-01-01

    Vascular perturbation is a hallmark of severe forms of dengue disease. We show here that antibody-enhanced dengue virus infection of primary human cord blood-derived mast cells (CBMCs) and the human mast cell-like line HMC-1 results in the release of factor(s) which activate human endothelial cells, as evidenced by increased expression of the adhesion molecules ICAM-1 and VCAM-1. Endothelial cell activation was prevented by pretreatment of mast cell-derived supernatants with a tumor necrosis factor (TNF)-specific blocking antibody, thus identifying TNF as the endothelial cell-activating factor. Our findings suggest that mast cells may represent an important source of TNF, promoting vascular endothelial perturbation following antibody-enhanced dengue virus infection. PMID:21068256

  3. Ghrelin Inhibits Oligodendrocyte Cell Death by Attenuating Microglial Activation

    PubMed Central

    Lee, Jee Youn

    2014-01-01

    Background Recently, we reported the antiapoptotic effect of ghrelin in spinal cord injury-induced apoptotic cell death of oligodendrocytes. However, how ghrelin inhibits oligodendrocytes apoptosis, is still unknown. Therefore, in the present study, we examined whether ghrelin inhibits microglia activation and thereby inhibits oligodendrocyte apoptosis. Methods Using total cell extracts prepared from BV-2 cells activated by lipopolysaccharide (LPS) with or without ghrelin, the levels of p-p38 phosphor-p38 mitogen-activated protein kinase (p-p38MAPK), phospho-c-Jun N-terminal kinase (pJNK), p-c-Jun, and pro-nerve growth factor (proNGF) were examined by Western blot analysis. Reactive oxygen species (ROS) production was investigated by using dichlorodihydrofluorescein diacetate. To examine the effect of ghrelin on oligodendrocyte cell death, oligodendrocytes were cocultured in transwell chambers of 24-well plates with LPS-stimulated BV-2 cells. After 48 hours incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling staining were assessed. Results Ghrelin treatment significantly decreased levels of p-p38MAPK, p-JNK, p-c-Jun, and proNGF in LPS-stimulated BV-2 cells. ROS production increased in LPS-stimulated BV-2 cells was also significantly inhibited by ghrelin treatment. In addition, ghrelin significantly inhibited oligodendrocyte cell death when cocultured with LPS-stimulated BV-2 cells. Conclusion Ghrelin inhibits oligodendrocyte cell death by decreasing proNGF and ROS production as well as p38MAPK and JNK activation in activated microglia as an anti-inflammatory hormone. PMID:25309797

  4. Tryptase Activation of Immortalized Human Urothelial Cell Mitogen-Activated Protein Kinase

    PubMed Central

    Marentette, John O.; Hauser, Paul J.; Hurst, Robert E.; Klumpp, David J.; Rickard, Alice; McHowat, Jane

    2013-01-01

    The pathogenesis of interstitial cystitis/painful bladder syndrome (IC/PBS) is multifactorial, but likely involves urothelial cell dysfunction and mast cell accumulation in the bladder wall. Activated mast cells in the bladder wall release several inflammatory mediators, including histamine and tryptase. We determined whether mitogen-activated protein (MAP) kinases are activated in response to tryptase stimulation of urothelial cells derived from human normal and IC/PBS bladders. Tryptase stimulation of normal urothelial cells resulted in a 2.5-fold increase in extracellular signal regulated kinase 1/2 (ERK 1/2). A 5.5-fold increase in ERK 1/2 activity was observed in urothelial cells isolated from IC/PBS bladders. No significant change in p38 MAP kinase was observed in tryptase-stimulated normal urothelial cells but a 2.5-fold increase was observed in cells isolated from IC/PBS bladders. Inhibition of ERK 1/2 with PD98059 or inhibition of p38 MAP kinase with SB203580 did not block tryptase-stimulated iPLA2 activation. Incubation with the membrane phospholipid-derived PLA2 hydrolysis product lysoplasmenylcholine increased ERK 1/2 activity, suggesting the iPLA2 activation is upstream of ERK 1/2. Real time measurements of impedance to evaluate wound healing of cell cultures indicated increased healing rates in normal and IC/PBS urothelial cells in the presence of tryptase, with inhibition of ERK 1/2 significantly decreasing the wound healing rate of IC/PBS urothelium. We conclude that activation of ERK 1/2 in response to tryptase stimulation may facilitate wound healing or cell motility in areas of inflammation in the bladder associated with IC/PBS. PMID:23922867

  5. 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.

  6. The TACI receptor regulates T-cell-independent marginal zone B cell responses through innate activation-induced cell death.

    PubMed

    Figgett, William A; Fairfax, Kirsten; Vincent, Fabien B; Le Page, Mélanie A; Katik, Indzi; Deliyanti, Devy; Quah, Pin Shie; Verma, Pali; Grumont, Raelene; Gerondakis, Steve; Hertzog, Paul; O'Reilly, Lorraine A; Strasser, Andreas; Mackay, Fabienne

    2013-09-19

    Activation-induced cell death (AICD) plays a critical role in immune homeostasis and tolerance. In T-cell-dependent humoral responses, AICD of B cells is initiated by Fas ligand (FasL) on T cells, stimulating the Fas receptor on B cells. In contrast, T-cell-independent B cell responses involve innate-type B lymphocytes, such as marginal zone (MZ) B cells, and little is known about the mechanisms that control AICD during innate B cell responses to Toll-like receptor (TLR) activation. Here, we show that MZ B cells undergo AICD in response to TLR4 activation in vivo. The transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI) receptor and TLR4 cooperate to upregulate expression of both FasL and Fas on MZ B cells and also to repress inhibitors of Fas-induced apoptosis signaling. These findings demonstrate an unappreciated role for TACI and its ligands in the regulation of AICD during T-cell-independent B cell responses.

  7. Volume Changes During Active Shape Fluctuations in Cells

    NASA Astrophysics Data System (ADS)

    Taloni, Alessandro; Kardash, Elena; Salman, Oguz Umut; Truskinovsky, Lev; Zapperi, Stefano; La Porta, Caterina A. M.

    2015-05-01

    Cells modify their volume in response to changes in osmotic pressure but it is usually assumed that other active shape variations do not involve significant volume fluctuations. Here we report experiments demonstrating that water transport in and out of the cell is needed for the formation of blebs, commonly observed protrusions in the plasma membrane driven by cortex contraction. We develop and simulate a model of fluid-mediated membrane-cortex deformations and show that a permeable membrane is necessary for bleb formation which is otherwise impaired. Taken together, our experimental and theoretical results emphasize the subtle balance between hydrodynamics and elasticity in actively driven cell morphological changes.

  8. Volume Changes During Active Shape Fluctuations in Cells

    NASA Astrophysics Data System (ADS)

    La Porta, Caterina A. M.; Taloni, Alessandro; Kardash, Elena; Salman, Oguz Umut; Truskinovsky, Lev; Zapperi, Stefano

    Cells modify their volume in response to changes in osmotic pressure but it is usually assumed that other active shape variations do not involve significant volume fluctuations. Here we report experiments demonstrating that water transport in and out of the cell is needed for the formation of blebs, commonly observed protrusions in the plasma membrane driven by cortex contraction. We develop and simulate a model of fluid-mediated membrane-cortex deformations and show that a permeable membrane is necessary for bleb formation which is otherwise impaired. Taken together, our experimental and theoretical results emphasize the subtle balance between hydrodynamics and elasticity in actively driven cell morphological changes.

  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. 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

  11. Activation of Human T-Helper/Inducer Cell, T-Cytotoxic Cell, B-Cell, and Natural Killer (NK)-Cells and induction of Natural Killer Cell Activity against K562 Chronic Myeloid Leukemia Cells with Modified Citrus Pectin

    PubMed Central

    2011-01-01

    Background Modified citrus pectin (MCP) is known for its anti-cancer effects and its ability to be absorbed and circulated in the human body. In this report we tested the ability of MCP to induce the activation of human blood lymphocyte subsets like T, B and NK-cells. Methods MCP treated human blood samples were incubated with specific antibody combinations and analyzed in a flow cytometer using a 3-color protocol. To test functionality of the activated NK-cells, isolated normal lymphocytes were treated with increasing concentrations of MCP. Log-phase PKH26-labeled K562 leukemic cells were added to the lymphocytes and incubated for 4 h. The mixture was stained with FITC-labeled active form of caspase 3 antibody and analyzed by a 2-color flow cytometry protocol. The percentage of K562 cells positive for PKH26 and FITC were calculated as the dead cells induced by NK-cells. Monosaccharide analysis of the MCP was performed by high-performance anion-exchange chromatography with pulse amperometric detection (HPAEC-PAD). Results MCP activated T-cytotoxic cells and B-cell in a dose-dependent manner, and induced significant dose-dependent activation of NK-cells. MCP-activated NK-cells demonstrated functionality in inducing cancer cell death. MCP consisted of oligogalacturonic acids with some containing 4,5-unsaturated non-reducing ends. Conclusions MCP has immunostimulatory properties in human blood samples, including the activation of functional NK cells against K562 leukemic cells in culture. Unsaturated oligogalacturonic acids appear to be the immunostimulatory carbohydrates in MCP. PMID:21816083

  12. 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.

  13. Immune suppressor factor confers stromal cell line with enhanced supporting activity for hematopoietic stem cells

    SciTech Connect

    Nakajima, Hideaki . E-mail: hnakajim@ims.u-tokyo.ac.jp; Shibata, Fumi; Fukuchi, Yumi; Goto-Koshino, Yuko; Ito, Miyuki; Urano, Atsushi; Nakahata, Tatsutoshi; Aburatani, Hiroyuki; Kitamura, Toshio

    2006-02-03

    Immune suppressor factor (ISF) is a subunit of the vacuolar ATPase proton pump. We earlier identified a short form of ISF (ShIF) as a stroma-derived factor that supports cytokine-independent growth of mutant Ba/F3 cells. Here, we report that ISF/ShIF supports self-renewal and expansion of primary hematopoietic stem cells (HSCs). Co-culture of murine bone marrow cells with a stromal cell line overexpressing ISF or ShIF (MS10/ISF or MS10/ShIF) not only enhanced their colony-forming activity and the numbers of long-term culture initiating cells, but also maintained the competitive repopulating activity of HSC. This stem cell supporting activity depended on the proton-transfer function of ISF/ShIF. Gene expression analysis of ISF/ShIF-transfected cell lines revealed down-regulation of secreted frizzled-related protein-1 and tissue inhibitor of metalloproteinase-3, and the restoration of their expressions in MS10/ISF cells partially reversed its enhanced LTC-IC supporting activity to a normal level. These results suggest that ISF/ShIF confers stromal cells with enhanced supporting activities for HSCs by modulating Wnt-activity and the extracellular matrix.

  14. Cell proliferation in vitro modulates fibroblast collagenase activity

    SciTech Connect

    Lindblad, W.J.; Flood, L.

    1986-05-01

    Collagenase enzyme activity is regulated by numerous control mechanisms which prevent excessive release and activation of this protease. A primary mechanism for regulating enzyme extracellular activity may be linked to cell division, therefore they have examined the release of collagenase by fibroblasts in vitro in response to cellular proliferation. Studies were performed using fibroblasts derived from adult rat dermis maintained in DMEM containing 10% newborn calf serum, 25 mM tricine buffer, and antibiotics. Cells between subculture 10 and 19 were used with enzyme activity determined with a /sup 14/C-labelled soluble Type I collagen substrate with and without trypsin activation. Fibroblasts, trypsinized and plated at low density secreted 8.5 fold more enzyme than those cells at confluence (975 vs. 115 dpm/..mu..g DNA). This diminution occurred gradually as the cells went from logrithmic growth towards confluence. Confluent fibroblast monolayers were scraped in a grid arrangement, stimulating the remaining cells to divide, without exposure to trypsin. Within 24-48 hr postscraping enzyme levels had increased 260-400%, accompanied by enhanced incorporation of /sup 3/H-thymidine and /sup 3/H-uridine into cell macromolecules. The burst of enzyme release began to subside 12 hr later. These results support a close relationship between fibroblast proliferation and collagenase secretion.

  15. 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.

  16. Activation of Soluble Adenylyl Cyclase Protects against Secretagogue Stimulated Zymogen Activation in Rat Pancreaic Acinar Cells

    PubMed Central

    Kolodecik, Thomas R.; Shugrue, Christine A.; Thrower, Edwin C.; Levin, Lonny R.; Buck, Jochen; Gorelick, Fred S.

    2012-01-01

    An early feature of acute pancreatitis is activation of zymogens, such as trypsinogen, within the pancreatic acinar cell. Supraphysiologic concentrations of the hormone cholecystokinin (CCK; 100 nM), or its orthologue cerulein (CER), induce zymogen activation and elevate levels of cAMP in pancreatic acinar cells. The two classes of adenylyl cyclase, trans-membrane (tmAC) and soluble (sAC), are activated by distinct mechanisms, localize to specific subcellular domains, and can produce locally high concentrations of cAMP. We hypothesized that sAC activity might selectively modulate acinar cell zymogen activation. sAC was identified in acinar cells by PCR and immunoblot. It localized to the apical region of the cell under resting conditions and redistributed intracellularly after treatment with supraphysiologic concentrations of cerulein. In cerulein-treated cells, pre-incubation with a trans-membrane adenylyl cyclase inhibitor did not affect zymogen activation or amylase secretion. However, treatment with a sAC inhibitor (KH7), or inhibition of a downstream target of cAMP, protein kinase A (PKA), significantly enhanced secretagogue-stimulated zymogen activation and amylase secretion. Activation of sAC with bicarbonate significantly inhibited secretagogue-stimulated zymogen activation; this response was decreased by inhibition of sAC or PKA. Bicarbonate also enhanced secretagogue-stimulated cAMP accumulation; this effect was inhibited by KH7. Bicarbonate treatment reduced secretagogue-stimulated acinar cell vacuolization, an early marker of pancreatitis. These data suggest that activation of sAC in the pancreatic acinar cell has a protective effect and reduces the pathologic activation of proteases during pancreatitis. PMID:22844459

  17. 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. PMID:25785438

  18. 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.

  19. Plasmin Activation of Glial Cells through Protease-Activated Receptor 1.

    PubMed

    Greenidge, André R; Hall, Kiana R; Hambleton, Ian R; Thomas, Richelle; Monroe, Dougald M; Landis, R Clive

    2013-01-01

    The objective of this study was to determine whether plasmin could induce morphological changes in human glial cells via PAR1. Human glioblastoma A172 cells were cultured in the presence of plasmin or the PAR1 specific activating hexapeptide, SFLLRN. Cells were monitored by flow cytometry to detect proteolytic activation of PAR1 receptor. Morphological changes were recorded by photomicroscopy and apoptosis was measured by annexinV staining. Plasmin cleaved the PAR1 receptor on glial cells at 5 minutes (P = 0.02). After 30 minutes, cellular processes had begun to retract from the basal substratum and by 4 hours glial cells had become detached. Similar results were obtained by generating plasmin de novo from plasminogen. Morphological transformation was blocked by plasmin inhibitors aprotinin or epsilon-aminocaproic acid (P = 0.03). Cell viability was unimpaired during early morphological changes, but by 24 hours following plasmin treatment 22% of glial cells were apoptotic. PAR1 activating peptide SFLLRN (but not inactive isomer FSLLRN) promoted analogous glial cell detachment (P = 0.03), proving the role for PAR1 in this process. This study has identified a plasmin/PAR1 axis of glial cell activation, linked to changes in glial cell morophology. This adds to our understanding of pathophysiological disease mechanisms of plasmin and the plasminogen system in neuroinjury. PMID:23431500

  20. Angiotensin I converting enzyme activity in rabbit corneal endothelial cells.

    PubMed

    Neels, H M; Vanden Berghe, D A; Neetens, A J; Delgadillo, R A; Scharpe, S L

    1983-01-01

    Angiotensin I converting enzyme (ACE) was studied in Vero cells, rabbit corneal fibroblasts, and rabbit corneal endothelial cells. The enzyme activity was determined by means of an assay employing hippuryl-glycyl-glycine as a substrate. The hippuric acid end product was separated from the substrate by reversed phase liquid chromatography and measured spectrophotometrically at 228 nm. The enzyme was further characterized by a captopril inhibition study. Significant ACE activity was found in rabbit corneal endothelial cells but not in other types of cells tested. This is the first report of the presence of this enzyme in a specific ocular cell type and suggests that angiotensin II may play a role in normal ocular physiology.

  1. Isolation of osteogenic cells from the trauma-activated periosteum

    NASA Astrophysics Data System (ADS)

    Wu, Chang-Hsiao; Bullock, John

    1987-12-01

    Closed, greenstick type fractures were created in adult male white New Zealand rabbits. After a waiting period of 5 days the developing callous and bone approximately 1 cm to each side of the callous was harvested and cell cultures established. Biochemical assays for total protein, alkaline phosphatase activity and glycosamino-glycan content were performed on spent media collected at each change and upon the cells after their termination, in an attempt to more fully characterize the osteoblast population. Since little is known about bone forming cells isolated from this source it is important to establish baseline data so as to be able to relate reactions of these cells to altered environmental conditions.

  2. Capsaicin modulates proliferation, migration, and activation of hepatic stellate cells.

    PubMed

    Bitencourt, Shanna; Mesquita, Fernanda; Basso, Bruno; Schmid, Júlia; Ferreira, Gabriela; Rizzo, Lucas; Bauer, Moises; Bartrons, Ramon; Ventura, Francesc; Rosa, Jose Luis; Mannaerts, Inge; van Grunsven, Leo Adrianus; Oliveira, Jarbas

    2014-03-01

    Capsaicin, the active component of chili pepper, has been reported to have antiproliferative and anti-inflammatory effects on a variety of cell lines. In the current study, we aimed to investigate the effects of capsaicin during HSC activation and maintenance. Activated and freshly isolated HSCs were treated with capsaicin. Proliferation was measured by incorporation of EdU. Cell cycle arrest and apoptosis were investigated using flow cytometry. The migratory response to chemotactic stimuli was evaluated by a modified Boyden chamber assay. Activation markers and inflammatory cytokines were determined by qPCR, immunocytochemistry, and flow cytometry. Our results show that capsaicin reduces HSC proliferation, migration, and expression of profibrogenic markers of activated and primary mouse HSCs. In conclusion, the present study shows that capsaicin modulates proliferation, migration, and activation of HSC in vitro. PMID:23955514

  3. Substrate elasticity affects bovine satellite cell activation kinetics in vitro.

    PubMed

    Lapin, M R; Gonzalez, J M; Johnson, S E

    2013-05-01

    Satellite cells support efficient postnatal skeletal muscle hypertrophy through fusion into the adjacent muscle fiber. Nuclear contribution allows for maintenance of the fiber myonuclear domain and proficient transcription of myogenic genes. Niche growth factors affect satellite cell biology; however, the interplay between fiber elasticity and microenvironment proteins remains largely unknown. The objective of the experiment was to examine the effects of hepatocyte growth factor (HGF) and surface elasticity on bovine satellite cell (BSC) activation kinetics in vitro. Young's elastic modulus was calculated for the semimembranosus (SM) and LM muscles of young bulls (5 d; n = 8) and adult cows (27 mo; n = 4) cattle. Results indicate that LM elasticity decreased (P < 0.05) with age; no difference in Young's modulus for the SM was noted. Bovine satellite cells were seeded atop polyacrylamide bioscaffolds with surface elasticities that mimic young bull and adult cow LM or traditional cultureware. Cells were maintained in low-serum media supplemented with 5 ng/mL HGF or vehicle only for 24 or 48 h. Activation was evaluated by proliferating cell nuclear antigen (PCNA) immunocytochemistry. Results indicate that BSC maintained on rigid surfaces were activated at 24 h and refractive to HGF supplementation. By contrast, fewer (P < 0.05) BSC had exited quiescence after 24 h of culture on surfaces reflective of either young bull (8.1 ± 1.7 kPa) or adult cow (14.6 ± 1.6 kPa) LM. Supplementation with HGF promoted activation of BSC cultured on bioscaffolds as measured by an increase (P < 0.05) in PCNA immunopositive cells. Culture on pliant surfaces affected neither activation kinetics nor numbers of Paired box 7 (Pax7) immunopositive muscle stem cells (P > 0.05). However, with increasing surface elasticity, an increase (P < 0.05) in the numbers of muscle progenitors was observed. These results confirm that biophysical and biochemical signals regulate BSC activation.

  4. Fate of retinoic acid-activated embryonic cell lineages.

    PubMed

    Dollé, Pascal; Fraulob, Valérie; Gallego-Llamas, Jabier; Vermot, Julien; Niederreither, Karen

    2010-12-01

    Retinoic acid (RA), a vitamin A derivative, is synthesized by specific cell populations and acts as a diffusible embryonic signal activating ligand-inducible transcription factors, the RA receptors (RARs). RA-activatable transgenic systems have revealed many discrete, transient sites of RA action during development. However, there has been no attempt to permanently label the RA-activated cell lineages during mouse ontogenesis. We describe the characterization of a RA-activatable Cre transgene, which through crosses with a conditional reporter strain (the ROSA26R lacZ reporter), leads to a stable labeling of the cell populations experiencing RA signaling during embryogenesis. RA response-element (RARE)-driven Cre activity mimics at early stages the known activity of the corresponding RARE-lacZ transgene (Rossant et al.,1991). Stable labeling of the Cre-excised cell populations allows to trace the distribution of the RA-activated cell lineages at later stages. These are described in relationship with current models of RA activity in various developmental systems, including the embryonic caudal region, limb buds, hindbrain, sensory organs, and heart. PMID:21046629

  5. Cell wounding activates phospholipase D in primary mouse keratinocytes

    PubMed Central

    Arun, Senthil N.; Xie, Ding; Howard, Amber C.; Zhong, Quincy; Zhong, Xiaofeng; McNeil, Paul L.; Bollag, Wendy B.

    2013-01-01

    Plasma membrane disruptions occur in mechanically active tissues such as the epidermis and can lead to cell death if the damage remains unrepaired. Repair occurs through fusion of vesicle patches to the damaged membrane region. The enzyme phospholipase D (PLD) is involved in membrane traffickiing; therefore, the role of PLD in membrane repair was investigated. Generation of membrane disruptions by lifting epidermal keratinocytes from the substratum induced PLD activation, whereas removal of cells from the substratum via trypsinization had no effect. Pretreatment with 1,25-dihydroxyvitamin D3, previously shown to increase PLD1 expression and activity, had no effect on, and a PLD2-selective (but not a PLD1-selective) inhibitor decreased, cell lifting-induced PLD activation, suggesting PLD2 as the isoform activated. PLD2 interacts functionally with the glycerol channel aquaporin-3 (AQP3) to produce phosphatidylglycerol (PG); however, wounding resulted in decreased PG production, suggesting a potential PG deficiency in wounded cells. Cell lifting-induced PLD activation was transient, consistent with a possible role in membrane repair, and PLD inhibitors inhibited membrane resealing upon laser injury. In an in vivo full-thickness mouse skin wound model, PG accelerated wound healing. These results suggest that PLD and the PLD2/AQP3 signaling module may be involved in membrane repair and wound healing. PMID:23288946

  6. 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

  7. 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.

  8. Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell-cell fusion

    SciTech Connect

    Schowalter, Rachel M.; Wurth, Mark A.; Aguilar, Hector C.; Lee, Benhur; Moncman, Carole L.; McCann, Richard O.; Dutch, Rebecca Ellis . E-mail: rdutc2@uky.edu

    2006-07-05

    The paramyxovirus fusion protein (F) promotes fusion of the viral envelope with the plasma membrane of target cells as well as cell-cell fusion. The plasma membrane is closely associated with the actin cytoskeleton, but the role of actin dynamics in paramyxovirus F-mediated membrane fusion is unclear. We examined cell-cell fusion promoted by two different paramyxovirus F proteins in three cell types in the presence of constitutively active Rho family GTPases, major cellular coordinators of actin dynamics. Reporter gene and syncytia assays demonstrated that expression of either Rac1{sup V12} or Cdc42{sup V12} could increase cell-cell fusion promoted by the Hendra or SV5 glycoproteins, though the effect was dependent on the cell type expressing the viral glycoproteins. In contrast, RhoA{sup L63} decreased cell-cell fusion promoted by Hendra glycoproteins but had little affect on SV5 F-mediated fusion. Also, data suggested that GTPase activation in the viral glycoprotein-containing cell was primarily responsible for changes in fusion. Additionally, we found that activated Cdc42 promoted nuclear rearrangement in syncytia.

  9. 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.

  10. 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

    2016-10-01

    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.

  11. 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

  12. 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.

  13. Binding of tissue plasminogen activator to cultured human endothelial cells.

    PubMed Central

    Hajjar, K A; Hamel, N M; Harpel, P C; Nachman, R L

    1987-01-01

    Tissue plasminogen activator (t-PA) and urokinase (u-PA), the major activators of plasminogen, are synthesized and released from endothelial cells. We previously demonstrated specific and functional binding of plasminogen to cultured human umbilical vein endothelial cells (HUVEC). In the present study we found that t-PA could bind to HUVEC. Binding of t-PA to HUVEC was specific, saturable, plasminogen-independent, and did not require lysine binding sites. The t-PA bound in a rapid and reversible manner, involving binding sites of both high (Kd, 28.7 +/- 10.8 pM; Bmax, 3,700 +/- 300) and low (Kd, 18.1 +/- 3.8 nM; Bmax 815,000 +/- 146,000) affinity. t-PA binding was 70% inhibited by a 100-fold molar excess of u-PA. When t-PA was bound to HUVEC, its apparent catalytic efficiency increased by three- or fourfold as measured by plasminogen activation. HUVEC-bound t-PA was active site-protected from its rapidly acting inhibitor: plasminogen activator inhibitor. These results demonstrate that t-PA specifically binds to HUVEC and that such binding preserves catalytic efficiency with respect to plasminogen activation. Therefore, endothelial cells can modulate hemostatic and thrombotic events at the cell surface by providing specific binding sites for activation of plasminogen. PMID:3119664

  14. Janus particles as artificial antigen-presenting cells for T cell activation.

    PubMed

    Chen, Bo; Jia, Yilong; Gao, Yuan; Sanchez, Lucero; Anthony, Stephen M; Yu, Yan

    2014-01-01

    Here we show that the multifunctionality of Janus particles can be exploited for in vitro T cell activation. We engineer bifunctional Janus particles on which the spatial distribution of two ligands, anti-CD3 and fibronectin, mimics the "bull's eye" protein pattern formed in the membrane junction between a T cell and an antigen-presenting cell. Different levels of T cell activation can be achieved by simply switching the spatial distribution of the two ligands on the surfaces of the "bull's eye" particles. We find that the ligand pattern also affects clustering of intracellular proteins. This study demonstrates that anisotropic particles, such as Janus particles, can be developed as artificial antigen-presenting cells for modulating T cell activation. PMID:25343426

  15. Active Biochemical Regulation of Cell Volume and a Simple Model of Cell Tension Response.

    PubMed

    Tao, Jiaxiang; Sun, Sean X

    2015-10-20

    Active contractile forces exerted by eukaryotic cells play significant roles during embryonic development, tissue formation, and cell motility. At the molecular level, small GTPases in signaling pathways can regulate active cell contraction. Here, starting with mechanical force balance at the cell cortex, and the recent discovery that tension-sensitive membrane channels can catalyze the conversion of the inactive form of Rho to the active form, we show mathematically that this active regulation of cellular contractility together with osmotic regulation can robustly control the cell size and membrane tension against external mechanical or osmotic shocks. We find that the magnitude of active contraction depends on the rate of mechanical pulling, but the cell tension can recover. The model also predicts that the cell exerts stronger contractile forces against a stiffer external environment, and therefore exhibits features of mechanosensation. These results suggest that a simple system for maintaining homeostatic values of cell volume and membrane tension could explain cell tension response and mechanosensation in different environments. PMID:26488645

  16. Streptococcus induces circulating CLA(+) memory T-cell-dependent epidermal cell activation in psoriasis.

    PubMed

    Ferran, Marta; Galván, Ana B; Rincón, Catalina; Romeu, Ester R; Sacrista, Marc; Barboza, Erika; Giménez-Arnau, Ana; Celada, Antonio; Pujol, Ramon M; Santamaria-Babí, Luis F

    2013-04-01

    Streptococcal throat infection is associated with a specific variant of psoriasis and with HLA-Cw6 expression. In this study, activation of circulating psoriatic cutaneous lymphocyte-associated antigen (CLA)(+) memory T cells cultured together with epidermal cells occurred only when streptococcal throat extracts were added. This triggered the production of Th1, Th17, and Th22 cytokines, as well as epidermal cell mediators (CXCL8, CXCL9, CXCL10, and CXCL11). Streptococcal extracts (SEs) did not induce any activation with either CLA(-) cells or memory T cells cultured together with epidermal cells from healthy subjects. Intradermal injection of activated culture supernatants into mouse skin induced epidermal hyperplasia. SEs also induced activation when we used epidermal cells from nonlesional skin of psoriatic patients with CLA(+) memory T cells. Significant correlations were found between SE induced upregulation of mRNA expression for ifn-γ, il-17, il-22, ip-10, and serum level of antistreptolysin O in psoriatic patients. This study demonstrates the direct involvement of streptococcal infection in pathological mechanisms of psoriasis, such as IL-17 production and epidermal cell activation.

  17. 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.

  18. Plasma-activated medium induced apoptosis on tumor cells

    NASA Astrophysics Data System (ADS)

    Hori, Masaru; Tanaka, Hiromasa; Mizuno, Masaaki; Nakamura, Kae; Kajiyama, Hiroaki; Takeda, Keigo; Ishikawa, Kenji; Kano, Hiroyuki; Kikkawa, Fumitaka

    2013-09-01

    The non-equilibrium atmospheric pressure plasma (NEAPP) has attracted attention in cancer therapy. In this study, the fresh medium was treated with our developed NEAPP, ultra-high electron density (approximately 2 × 1016 cm-3). The medium called the plasma-activated medium (PAM) killed not normal cells but tumor cells through induction of apoptosis. Cell proliferation assays showed that the tumor cells were selectively killed by the PAM. Those cells induced apoptosis using an apoptotic molecular marker, cleaved Caspase3/7. The molecular mechanisms of PAM-mediated apoptosis in the tumor cells were also found that the PAM downregulated the expression of AKT kinase, a marker molecule in a survival signal transduction pathway. These results suggest that PAM may be a promising tool for tumor therapy by downregulating the survival signals in cancers.

  19. Hypoxia promotes drug resistance in osteosarcoma cells via activating AMP-activated protein kinase (AMPK) signaling

    PubMed Central

    Zhao, Changfu; Zhang, Qiao; Yu, Tao; Sun, Shudong; Wang, Wenjun; Liu, Guangyao

    2016-01-01

    Purpose Drug resistance has been recognized to be a major obstacle to the chemotherapy for osteosarcoma. And the potential importance of hypoxia as a target to reverse drug resistance in osteosarcoma has been indicated, though the mechanism underlining such role is not clarified. The present study aims to investigate the role of hypoxia in the drug resistance in osteosarcoma cells via activating AMP-activated protein kinase (AMPK) signaling. Experimental design We investigated the promotion of the resistance to doxorubicin of osteosarcoma MG-63 and U2-os cells in vitro, and then determined the role of hypoxia-inducible factor-1 (HIF-1)α and HIF-1β, the activation and regulatory role of AMPK in the osteosarcoma U2-os cells which were treated with doxorubicin under hypoxia. Results It was demonstrated that hypoxia significantly reduced the sensitivity of MG-63 and U2-os cells to doxorubicin, indicating an inhibited viability reduction and a reduced apoptosis promotion. And such reduced sensitivity was not associated with HIF-1α, though it was promoted by hypoxia in U2-os cells. Interestingly, the AMPK signaling was significantly promoted by hypoxia in the doxorubicin-treated U2-os cells, with a marked upregulation of phosphorylated AMPK (Thr 172) and phosphorylated acetyl-CoA carboxylase (ACC) (Ser 79), which were sensitive to the AMPK activator, AICAR and the AMPK inhibitor, Compound C. Moreover, the promoted AMPK activity by AICAR or the downregulated AMPK activity by Compound C significantly reduced or promoted the sensitivity of U2-os cells to doxorubicin. Conclusion The present study confirmed the AMPK signaling activation in the doxorubicin-treated osteosarcoma cells, in response to hypoxia, and the chemical upregulation or downregulation of AMPK signaling reduced or increased the chemo-sensitivity of osteosarcoma U2-os cells in vitro. Our study implies that AMPK inhibition might be a effective strategy to sensitize osteocarcoma cells to chemotherapy. PMID

  20. 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

  1. Anti-tumor activity of safranal against neuroblastoma cells

    PubMed Central

    Samarghandian, Saeed; Shoshtari, Mohammad Ebrahim; Sargolzaei, Javad; Hossinimoghadam, Hosna; Farahzad, Jabbari Azad

    2014-01-01

    Objective: Safranal (2,6,6-trimethyl-1,3-cyclohexadiene-1-carboxaldehyde, C10H14O) is an active ingredient in the saffron, which is used in traditional medicine, and also, the biological activity of saffron in anti-cancer is in development. It has been reported to have anti-oxidant effects, but its anti-tumor effects remain uncertain. The aim of this study was to evaluate effects of safranal on anti-tumor on neuroblastoma cells. Materials and Methods: Neuroblastoma cells were cultured and exposed to safranal (0, 10, 15, 20, 50 μg/ml). Cell proliferation was examined using the 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Apoptotic cells, cell cycle distribution, and sub-G1 fraction were analyzed using flow cytometric analysis after propidium iodide staining. Results: Safranal inhibited the growth of malignant cells in a dose-and time-dependent manner. The IC (50) values against the neuroblastoma cell line were determined as 11.1 and 23.3 μg/ml after 24 and 48 h, respectively. Safranal induced a sub-G1 peak in the flow cytometry histogram of treated cells compared to control cells indicating that apoptotic cell death is involved in safranal toxicity. Conclusions: Our pre-clinical study demonstrated a neuroblastoma cell line to be highly sensitive to safranal-mediated growth inhibition and apoptotic cell death. Although the molecular mechanisms of safranal action are not yet clearly understood, it appears to have potential as a therapeutic agent. PMID:24991121

  2. Melatonin modulates aromatase activity and expression in endothelial cells.

    PubMed

    Alvarez-García, Virginia; González, Alicia; Martínez-Campa, Carlos; Alonso-González, Carolina; Cos, Samuel

    2013-05-01

    Melatonin is known to suppress the development of endocrine-responsive breast cancers by interacting with the estrogen signaling pathways. Paracrine interactions between malignant epithelial cells and proximal stromal cells are responsible for local estrogen biosynthesis. In human breast cancer cells and peritumoral adipose tissue, melatonin downregulates aromatase, which transforms androgens into estrogens. The presence of aromatase on endothelial cells indicates that endothelial cells may contribute to tumor growth by producing estrogens. Since human umbilical vein endothelial cells (HUVECs) express both aromatase and melatonin receptors, the aim of the present study was to evaluate the ability of melatonin to regulate the activity and expression of aromatase on endothelial cells, thus, modulating local estrogen biosynthesis. In the present study, we demonstrated that melatonin inhibits the growth of HUVECs and reduces the local biosynthesis of estrogens through the downregulation of aromatase. These results are supported by three lines of evidence. Firstly, 1 mM of melatonin counteracted the testosterone-induced cell proliferation of HUVECs, which is dependent on the local biosynthesis of estrogens from testosterone by the aromatase activity of the cells. Secondly, we found that 1 mM of melatonin reduced the aromatase activity of HUVECs. Finally, by real‑time RT-PCR, we demonstrated that melatonin significantly downregulated the expression of aromatase as well as its endothelial-specific aromatase promoter region I.7. We conclude that melatonin inhibits aromatase activity and expression in HUVECs by regulating gene expression of specific aromatase promoter regions, thereby reducing the local production of estrogens. PMID:23450505

  3. LPS induces pulp progenitor cell recruitment via complement activation.

    PubMed

    Chmilewsky, F; Jeanneau, C; Laurent, P; About, I

    2015-01-01

    Complement system, a major component of the natural immunity, has been recently identified as an important mediator of the dentin-pulp regeneration process through STRO-1 pulp cell recruitment by the C5a active fragment. Moreover, it has been shown recently that under stimulation with lipoteichoic acid, a complex component of the Gram-positive bacteria cell wall, human pulp fibroblasts are able to synthesize all proteins required for complement activation. However, Gram-negative bacteria, which are also involved in tooth decay, are known as powerful activators of complement system and inflammation. Here, we investigated the role of Gram-negative bacteria-induced complement activation on the pulp progenitor cell recruitment using lipopolysaccharide (LPS), a major component of all Gram-negative bacteria. Our results show that incubating pulp fibroblasts with LPS induced membrane attack complex formation and C5a release in serum-free fibroblast cultures. The produced C5a binds to the pulp progenitor cells' membrane and induces their migration toward the LPS stimulation chamber, as revealed by the dynamic transwell migration assays. The inhibition of this migration by the C5aR-specific antagonist W54011 indicates that the pulp progenitor migration is mediated by the interaction between C5a and C5aR. Our findings demonstrate, for the first time, a direct interaction between the recruitment of progenitor pulp cells and the activation of complement system generated by pulp fibroblast stimulation with LPS.

  4. 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.

  5. Activated Factor X Induces Endothelial Cell Senescence Through IGFBP-5

    PubMed Central

    Sanada, Fumihiro; Taniyama, Yoshiaki; Muratsu, Jun; Otsu, Rei; Iwabayashi, Masaaki; Carracedo, Miguel; Rakugi, Hiromi; Morishita, Ryuichi

    2016-01-01

    Uncontrolled coagulation contributes to the pathophysiology of several chronic inflammatory diseases. In these conditions, senescent cells are often observed and is involved in the generation of inflammation. The coincidence of hyper-coagulation, cell senescence, and inflammation suggests the existence of a common underlying mechanism. Recent evidence indicates that activated coagulation factor X (FXa) plays a role in the processes beyond blood coagulation. This non-hematologic function entails the mediation of inflammation and tissue remodeling. We therefore tested the hypothesis that FXa induces cell senescence resulting in tissue inflammation and impaired tissue regeneration. Human umbilical vein endothelial cells were stimulated with FXa for 14 days. The proliferation of cells treated with FXa was significantly smaller, and the fraction of senescence-associated β-galactosidase-positive cells was increased as compared to the control group. RT-qPCR array revealed that FXa increased the expression of IGFBP-5, EGR-1, p53, and p16INK4a. Inhibition of FXa by a direct FXa inhibitor, rivaroxaban, or IGFBP-5 by siRNA decreased FXa-induced cell senescence, restoring cell proliferation. Moreover, in an ischemic hind limb mouse model, FXa inhibited neovascularization by endothelial progenitor cell. However, rivaroxaban significantly restored FXa-induced impaired angiogenesis. In summary, FXa induced endothelial cell senescence through IGFBP-5, resulting in impaired angiogenesis. PMID:27752126

  6. Plasminogen activator inhibitor-1 enhances radioresistance and aggressiveness of non-small cell lung cancer cells

    PubMed Central

    Youn, HyeSook; Kim, Joong Sun; Youn, BuHyun

    2016-01-01

    Acquired resistance of tumor cells during treatment limits the clinical efficacy of radiotherapy. Recent studies to investigate acquired resistance under treatment have focused on intercellular communication because it promotes survival and aggressiveness of tumor cells, causing therapy failure and tumor relapse. Accordingly, a better understanding of the functional communication between subpopulations of cells within a tumor is essential to development of effective cancer treatment strategies. Here, we found that conditioned media (CM) from radioresistant non-small cell lung cancer (NSCLC) cells increased survival of radiosensitive cells. Comparative proteomics analysis revealed plasminogen activator inhibitor-1 (PAI-1) as a key molecule in the secretome that acts as an extracellular signaling trigger to strengthen resistance to radiation. Our results revealed that expression and secretion of PAI-1 in radioresistant cells was increased by radiation-induced transcription factors, including p53, HIF-1α, and Smad3. When CM from radioresistant cells was applied to radiosensitive cells, extracellular PAI-1 activated the AKT and ERK1/2 signaling pathway and inhibited caspase-3 activity. Our study also proposed that PAI-1 activates the signaling pathway in radiosensitive cells via extracellular interaction with its binding partners, not clathrin-mediated endocytosis. Furthermore, secreted PAI-1 increased cell migration capacity and expression of EMT markers in vitro and in vivo. Taken together, our findings demonstrate that PAI-1 secreted from radioresistant NSCLC cells reduced radiosensitivity of nearby cells in a paracrine manner, indicating that functional inhibition of PAI-1 signaling has therapeutic potential because it prevents sensitive cells from acquiring radioresistance. PMID:27004408

  7. Differentiation Between Intracellular and Cell Surface Glycosyl Transferases: Galactosyl Transferase Activity in Intact Cells and in Cell Homogenate

    PubMed Central

    Deppert, Wolfgang; Werchau, Hermann; Walter, Gernot

    1974-01-01

    Intact BHK (baby hamster kidney) cells catalyze the hydrolysis of UDP-galactose to free galactose. The generation of galactose from UDP-galactose and its intracellular utilization impede the detection of possible galactosyl transferases on the cell surface of intact cells. Several independent procedures have been used to distinguish between intracellular and cell surface glycosyl transferases. With these procedures, no evidence was obtained for the presence of detectable amounts of galactosyl transferase activity on the surface of BHK cells. The data suggest that galactosyl transferases do not play a general role in the phenomena of cell adhesion and contact inhibition. PMID:4528509

  8. Apoptotic Cells Activate NKT Cells through T Cell Ig-Like Mucin-Like–1 Resulting in Airway Hyperreactivity

    PubMed Central

    Lee, Hyun-Hee; Meyer, Everett H.; Goya, Sho; Pichavant, Muriel; Kim, Hye Young; Bu, Xia; Umetsu, Sarah E.; Jones, Jennifer C.; Savage, Paul B.; Iwakura, Yoichiro; Casasnovas, Jose M.; Kaplan, Gerardo; Freeman, Gordon J.; DeKruyff, Rosemarie H.; Umetsu, Dale T.

    2011-01-01

    T cell Ig-like mucin-like–1 (TIM-1) is an important asthma susceptibility gene, but the immunological mechanisms by which TIM-1 functions remain uncertain. TIM-1 is also a receptor for phosphatidylserine (PtdSer), an important marker of cells undergoing programmed cell death, or apoptosis. We now demonstrate that NKT cells constitutively express TIM-1 and become activated by apoptotic cells expressing PtdSer. TIM-1 recognition of PtdSer induced NKT cell activation, proliferation, and cytokine production. Moreover, the induction of apoptosis in airway epithelial cells activated pulmonary NKT cells and unexpectedly resulted in airway hyperreactivity, a cardinal feature of asthma, in an NKT cell-dependent and TIM-1–dependent fashion. These results suggest that TIM-1 serves as a pattern recognition receptor on NKT cells that senses PtdSer on apoptotic cells as a damage-associated molecular pattern. Furthermore, these results provide evidence for a novel innate pathway that results in airway hyperreactivity and may help to explain how TIM-1 and NKT cells regulate asthma. PMID:20889552

  9. 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

  10. Pevonedistat, a NEDD8-activating enzyme inhibitor, is active in mantle cell lymphoma and enhances rituximab activity in vivo.

    PubMed

    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; Hernandez-Ilizaliturri, Francisco J

    2016-03-01

    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

  11. Unirradiated cells rescue cells exposed to ionizing radiation: Activation of NF-κB pathway in irradiated cells.

    PubMed

    Lam, R K K; Han, Wei; Yu, K N

    2015-12-01

    We studied the involvement of NF-κB pathway activation in the rescue effect in HeLa and NIH/3T3 cells irradiated by α particles. Firstly, upon irradiation by 5 cGy of α particles, for both cell lines, the numbers of 53BP1 foci/cell at 12 h post-irradiation were significantly smaller when only 2.5% of the cell population was irradiated as compared to 100% irradiation, which demonstrated the rescue effect. Secondly, we studied the effect of NF-κB on the rescue effect through the use of the NF-κB activation inhibitor BAY-11-7082. Novel experimental setup and procedures were designed to prepare the medium (CM) which had conditioned the bystander cells previously partnered with irradiated cells, to ensure physical separation between rescue and bystander signals. BAY-11-7082 itself did not inflict DNA damages in the cells or have effects on activation of the NF-κB response pathway in the irradiated cells through direct irradiation. The rescue effect was induced in both cell lines by the CM, which was abrogated if BAY-11-7082 was added to the CM. Thirdly, we studied the effect of NF-κB on the rescue effect through staining for phosphorylated NF-κB (p-NF-κB) expression using the anti-NF-κB p65 (phospho S536) antibody. When the fraction of irradiated cells dropped from 100% to 2.5%, the p-NF-κB expression in the cell nuclei of irradiated NIH/3T3 cells increased significantly, while that in the cell nuclei of irradiated HeLa cells also increased although not significantly. Moreover, the p-NF-κB expression in the cell nuclei of irradiated HeLa cells and NIH/3T3 cells treated with CM also increased significantly. PMID:26524645

  12. Unirradiated cells rescue cells exposed to ionizing radiation: Activation of NF-κB pathway in irradiated cells.

    PubMed

    Lam, R K K; Han, Wei; Yu, K N

    2015-12-01

    We studied the involvement of NF-κB pathway activation in the rescue effect in HeLa and NIH/3T3 cells irradiated by α particles. Firstly, upon irradiation by 5 cGy of α particles, for both cell lines, the numbers of 53BP1 foci/cell at 12 h post-irradiation were significantly smaller when only 2.5% of the cell population was irradiated as compared to 100% irradiation, which demonstrated the rescue effect. Secondly, we studied the effect of NF-κB on the rescue effect through the use of the NF-κB activation inhibitor BAY-11-7082. Novel experimental setup and procedures were designed to prepare the medium (CM) which had conditioned the bystander cells previously partnered with irradiated cells, to ensure physical separation between rescue and bystander signals. BAY-11-7082 itself did not inflict DNA damages in the cells or have effects on activation of the NF-κB response pathway in the irradiated cells through direct irradiation. The rescue effect was induced in both cell lines by the CM, which was abrogated if BAY-11-7082 was added to the CM. Thirdly, we studied the effect of NF-κB on the rescue effect through staining for phosphorylated NF-κB (p-NF-κB) expression using the anti-NF-κB p65 (phospho S536) antibody. When the fraction of irradiated cells dropped from 100% to 2.5%, the p-NF-κB expression in the cell nuclei of irradiated NIH/3T3 cells increased significantly, while that in the cell nuclei of irradiated HeLa cells also increased although not significantly. Moreover, the p-NF-κB expression in the cell nuclei of irradiated HeLa cells and NIH/3T3 cells treated with CM also increased significantly.

  13. 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.

  14. [An electrochemical method for measuring metabolic activity and counting cells].

    PubMed

    Kuznetsov, B a; Khlupova, M e; Shleev, S V; Kaprel'iants, A S; Iaropolov, A I

    2006-01-01

    An express electrochemical method for determining the metabolic activity of live cells based on the possibility of an electron exchange between an electrode and elements of the biological electron transfer chain in the presence of a mediator is proposed. This method is useful for studying any live cells (animal, plant, and microbial), including anaerobic, dormant, and spore cells. The sample preparation and measurement itself does not take more than 30 min. The detection limit in a volume of 15 ml amounts to 10-5 cells/ml. The applicability of the assessment method of the metabolic activity level during the transition of the bacteria Mycobacterium smegmatis into an uncultivable dormant state was demonstrated. This method is of special value for medicine and environmental control, detecting latent forms of pathogens. An optimal combination of the methods for the express analysis of latent pathogens is proposed. PMID:17066962

  15. 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.

  16. The lectin ArtinM binds to mast cells inducing cell activation and mediator release.

    PubMed

    Barbosa-Lorenzi, Valéria Cintra; Buranello, Patrícia Andressa de Almeida; Roque-Barreira, Maria Cristina; Jamur, Maria Célia; Oliver, Constance; Pereira-da-Silva, Gabriela

    2011-12-16

    Mast cells are inflammatory cells that respond to signals of innate and adaptive immunity with immediate and delayed release of mediators. ArtinM, a lectin from Artocarpus integrifolia with immunomodulatory activities, is able to induce mast cell activation, but the mechanisms remain unknown. This study sought to further investigate the effects of the lectin on mast cells. We showed that ArtinM binds to mast cells, possibly to the high affinity receptor for immunoglobulin E (IgE) - FcεRI - and/or to IgE bound to FcεRI. Binding of the lectin resulted in protein tyrosine phosphorylation and release of the pre- and newly-formed mediators, β-hexosaminidase and LTB(4) by mast cells, activities that were potentiated in the presence of IgE. ArtinM also induced the activation of the transcription factors NFκB and NFAT, resulting in expression of some of their target genes such as IL-4 and TNF-α. In view of the established significance of mast cells in many immunological and inflammatory reactions, a better understanding of the mechanisms involved in mast cell activation by ArtinM is crucial to the pharmacological application of the lectin.

  17. Increased Intraepithelial Vα24 Invariant NKT Cells in the Celiac Duodenum.

    PubMed

    Montalvillo, Enrique; Bernardo, David; Martínez-Abad, Beatriz; Allegretti, Yessica; Fernández-Salazar, Luis; Calvo, Carmen; Chirdo, Fernando G; Garrote, José A; Arranz, Eduardo

    2015-11-01

    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

  18. Cytoplasmic myosin-exposed apoptotic cells appear with caspase-3 activation and enhance CLL cell viability.

    PubMed

    Cui, X; Zhang, L; Magli, A R; Catera, R; Yan, X-J; Griffin, D O; Rothstein, T L; Barrientos, J; Kolitz, J E; Allen, S L; Rai, K R; Chiorazzi, N; Chu, C C

    2016-01-01

    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

  19. 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

  20. CCL5 activation of CCR5 regulates cell metabolism to enhance proliferation of breast cancer cells.

    PubMed

    Gao, Darrin; Rahbar, Ramtin; Fish, Eleanor N

    2016-06-01

    In earlier studies, we showed that CCL5 enhances proliferation and survival of MCF-7 breast cancer cells in an mTOR-dependent manner and we provided evidence that, for T cells, CCL5 activation of CCR5 results in increased glycolysis and enhanced ATP production. Increases in metabolic activity of cancer cells, specifically increased glycolytic activity and increased expression of glucose transporters, are associated with tumour progression. In this report, we provide evidence that CCL5 enhances the proliferation of human breast cancer cell lines (MDA-MB-231, MCF-7) and mouse mammary tumour cells (MMTV-PyMT), mediated by CCR5 activation. Concomitant with enhanced proliferation we show that CCL5 increases cell surface expression of the glucose transporter GLUT1, and increases glucose uptake and ATP production by these cells. Blocking CCL5-inducible glucose uptake abrogates the enhanced proliferation induced by CCL5. We provide evidence that increased glucose uptake is associated with enhanced glycolysis, as measured by extracellular acidification. Moreover, CCL5 enhances the invasive capacity of these breast cancer cells. Using metabolomics, we demonstrate that the metabolic signature of CCL5-treated primary mouse mammary tumour cells reflects increased anabolic metabolism. The implications are that CCL5-CCR5 interactions in the tumour microenvironment regulate metabolic events, specifically glycolysis, to promote tumour proliferation and invasion.

  1. CCL5 activation of CCR5 regulates cell metabolism to enhance proliferation of breast cancer cells

    PubMed Central

    Gao, Darrin; Rahbar, Ramtin; Fish, Eleanor N.

    2016-01-01

    In earlier studies, we showed that CCL5 enhances proliferation and survival of MCF-7 breast cancer cells in an mTOR-dependent manner and we provided evidence that, for T cells, CCL5 activation of CCR5 results in increased glycolysis and enhanced ATP production. Increases in metabolic activity of cancer cells, specifically increased glycolytic activity and increased expression of glucose transporters, are associated with tumour progression. In this report, we provide evidence that CCL5 enhances the proliferation of human breast cancer cell lines (MDA-MB-231, MCF-7) and mouse mammary tumour cells (MMTV-PyMT), mediated by CCR5 activation. Concomitant with enhanced proliferation we show that CCL5 increases cell surface expression of the glucose transporter GLUT1, and increases glucose uptake and ATP production by these cells. Blocking CCL5-inducible glucose uptake abrogates the enhanced proliferation induced by CCL5. We provide evidence that increased glucose uptake is associated with enhanced glycolysis, as measured by extracellular acidification. Moreover, CCL5 enhances the invasive capacity of these breast cancer cells. Using metabolomics, we demonstrate that the metabolic signature of CCL5-treated primary mouse mammary tumour cells reflects increased anabolic metabolism. The implications are that CCL5–CCR5 interactions in the tumour microenvironment regulate metabolic events, specifically glycolysis, to promote tumour proliferation and invasion. PMID:27335323

  2. Mucosal Regulatory T Cells and T Helper 17 Cells in HIV-Associated Immune Activation

    PubMed Central

    Pandiyan, Pushpa; Younes, Souheil-Antoine; Ribeiro, Susan Pereira; Talla, Aarthi; McDonald, David; Bhaskaran, Natarajan; Levine, Alan D.; Weinberg, Aaron; Sekaly, Rafick P.

    2016-01-01

    Residual mucosal inflammation along with chronic systemic immune activation is an important feature in individuals infected with human immunodeficiency virus (HIV), and has been linked to a wide range of co-morbidities, including malignancy, opportunistic infections, immunopathology, and cardiovascular complications. Although combined antiretroviral therapy (cART) can reduce plasma viral loads to undetectable levels, reservoirs of virus persist, and increased mortality is associated with immune dysbiosis in mucosal lymphoid tissues. Immune-based therapies are pursued with the goal of improving CD4+ T-cell restoration, as well as reducing chronic immune activation in cART-treated patients. However, the majority of research on immune activation has been derived from analysis of circulating T cells. How immune cell alterations in mucosal tissues contribute to HIV immune dysregulation and the associated risk of non-infectious chronic complications is less studied. Given the significant differences between mucosal T cells and circulating T cells, and the immediate interactions of mucosal T cells with the microbiome, more attention should be devoted to mucosal immune cells and their contribution to systemic immune activation in HIV-infected individuals. Here, we will focus on mucosal immune cells with a specific emphasis on CD4+ T lymphocytes, such as T helper 17 cells and CD4+Foxp3+ regulatory T cells (Tregs), which play crucial roles in maintaining mucosal barrier integrity and preventing inflammation, respectively. We hypothesize that pro-inflammatory milieu in cART-treated patients with immune activation significantly contributes to enhanced loss of Th17 cells and increased frequency of dysregulated Tregs in the mucosa, which in turn may exacerbate immune dysfunction in HIV-infected patients. We also present initial evidence to support this hypothesis. A better comprehension of how pro-inflammatory milieu impacts these two types of cells in the mucosa will shed light

  3. Doxorubicin resistant cancer cells activate myeloid-derived suppressor cells by releasing PGE2

    PubMed Central

    Rong, Yuan; Yuan, Chun-Hui; Qu, Zhen; Zhou, Hu; Guan, Qing; Yang, Na; Leng, Xiao-Hua; Bu, Lang; Wu, Ke; Wang, Fu-Bing

    2016-01-01

    Chemotherapies often induce drug-resistance in cancer cells and simultaneously stimulate proliferation and activation of Myeloid-Derived Suppressor Cells (MDSCs) to inhibit anti-tumor T cells, thus result in poor prognosis of patients with breast cancers. To date, the mechanism underlying the expansion of MDSCs in response to chemotherapies is poorly understood. In the present study, we used in vitro cell culture and in vivo animal studies to demonstrate that doxorubicin-resistant breast cancer cells secret significantly more prostaglandin E2 (PGE2) than their parental doxorubicin-sensitive cells. The secreted PGE2 can stimulate expansion and polymerization of MDSCs by directly target to its receptors, EP2/EP4, on the surface of MDSCs, which consequently triggers production of miR-10a through activating PKA signaling. More importantly, activated MDSCs can inhibit CD4+CD25− T cells as evidenced by reduced proliferation and IFN-γ release. In order to determine the molecular pathway that involves miR-10a mediated activation of MDSCs, biochemical and pharmacological studies were carried out. We found that miR-10a can activate AMPK signaling to promote expansion and activation of MDSCs. Thus, these results reveal, for the first time, a novel role of PGE2/miR-10a/AMPK signaling axis in chemotherapy-induced immune resistance, which might be targeted for treatment of chemotherapy resistant tumors. PMID:27032536

  4. Doxorubicin resistant cancer cells activate myeloid-derived suppressor cells by releasing PGE2.

    PubMed

    Rong, Yuan; Yuan, Chun-Hui; Qu, Zhen; Zhou, Hu; Guan, Qing; Yang, Na; Leng, Xiao-Hua; Bu, Lang; Wu, Ke; Wang, Fu-Bing

    2016-01-01

    Chemotherapies often induce drug-resistance in cancer cells and simultaneously stimulate proliferation and activation of Myeloid-Derived Suppressor Cells (MDSCs) to inhibit anti-tumor T cells, thus result in poor prognosis of patients with breast cancers. To date, the mechanism underlying the expansion of MDSCs in response to chemotherapies is poorly understood. In the present study, we used in vitro cell culture and in vivo animal studies to demonstrate that doxorubicin-resistant breast cancer cells secret significantly more prostaglandin E2 (PGE2) than their parental doxorubicin-sensitive cells. The secreted PGE2 can stimulate expansion and polymerization of MDSCs by directly target to its receptors, EP2/EP4, on the surface of MDSCs, which consequently triggers production of miR-10a through activating PKA signaling. More importantly, activated MDSCs can inhibit CD4(+)CD25(-) T cells as evidenced by reduced proliferation and IFN-γ release. In order to determine the molecular pathway that involves miR-10a mediated activation of MDSCs, biochemical and pharmacological studies were carried out. We found that miR-10a can activate AMPK signaling to promote expansion and activation of MDSCs. Thus, these results reveal, for the first time, a novel role of PGE2/miR-10a/AMPK signaling axis in chemotherapy-induced immune resistance, which might be targeted for treatment of chemotherapy resistant tumors. PMID:27032536

  5. Dectin-1-activated dendritic cells trigger potent antitumour immunity through the induction of Th9 cells

    PubMed Central

    Zhao, Yinghua; Chu, Xiao; Chen, Jintong; Wang, Ying; Gao, Sujun; Jiang, Yuxue; Zhu, Xiaoqing; Tan, Guangyun; Zhao, Wenjie; Yi, Huanfa; Xu, Honglin; Ma, Xingzhe; Lu, Yong; Yi, Qing; Wang, Siqing

    2016-01-01

    Dectin-1 signalling in dendritic cells (DCs) has an important role in triggering protective antifungal Th17 responses. However, whether dectin-1 directs DCs to prime antitumour Th9 cells remains unclear. Here, we show that DCs activated by dectin-1 agonists potently promote naive CD4+ T cells to differentiate into Th9 cells. Abrogation of dectin-1 in DCs completely abolishes their Th9-polarizing capability in response to dectin-1 agonist curdlan. Notably, dectin-1 stimulation of DCs upregulates TNFSF15 and OX40L, which are essential for dectin-1-activated DC-induced Th9 cell priming. Mechanistically, dectin-1 activates Syk, Raf1 and NF-κB signalling pathways, resulting in increased p50 and RelB nuclear translocation and TNFSF15 and OX40L expression. Furthermore, immunization of tumour-bearing mice with dectin-1-activated DCs induces potent antitumour response that depends on Th9 cells and IL-9 induced by dectin-1-activated DCs in vivo. Our results identify dectin-1-activated DCs as a powerful inducer of Th9 cells and antitumour immunity and may have important clinical implications. PMID:27492902

  6. 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.

  7. NKG2D is a Key Receptor for Recognition of Bladder Cancer Cells by IL-2-Activated NK Cells and BCG Promotes NK Cell Activation

    PubMed Central

    García-Cuesta, Eva María; López-Cobo, Sheila; Álvarez-Maestro, Mario; Esteso, Gloria; Romera-Cárdenas, Gema; Rey, Mercedes; Cassady-Cain, Robin L.; Linares, Ana; Valés-Gómez, Alejandro; Reyburn, Hugh Thomson; Martínez-Piñeiro, Luis; Valés-Gómez, Mar

    2015-01-01

    Intravesical instillation of bacillus Calmette–Guérin (BCG) is used to treat superficial bladder cancer, either papillary tumors (after transurethral resection) or high-grade flat carcinomas (carcinoma in situ), reducing recurrence in about 70% of patients. Initially, BCG was proposed to work through an inflammatory response, mediated by phagocytic uptake of mycobacterial antigens and cytokine release. More recently, other immune effectors such as monocytes, natural killer (NK), and NKT cells have been suggested to play a role in this immune response. Here, we provide a comprehensive study of multiple bladder cancer cell lines as putative targets for immune cells and evaluated their recognition by NK cells in the presence and absence of BCG. We describe that different bladder cancer cells can express multiple activating and inhibitory ligands for NK cells. Recognition of bladder cancer cells depended mainly on NKG2D, with a contribution from NKp46. Surprisingly, exposure to BCG did not affect the immune phenotype of bladder cells nor increased NK cell recognition of purified IL-2-activated cell lines. However, NK cells were activated efficiently when BCG was included in mixed lymphocyte cultures, suggesting that NK activation after mycobacteria treatment requires the collaboration of various immune cells. We also analyzed the percentage of NK cells in peripheral blood of a cohort of bladder cancer patients treated with BCG. The total numbers of NK cells did not vary during treatment, indicating that a more detailed study of NK cell activation in the tumor site will be required to evaluate the response in each patient. PMID:26106390

  8. 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.

  9. Structured variability in Purkinje cell activity during locomotion

    PubMed Central

    Sauerbrei, Britton A.; Lubenov, Evgueniy V.; Siapas, Athanassios G.

    2015-01-01

    Summary The cerebellum is a prominent vertebrate brain structure that is critically involved in sensorimotor function. During locomotion, cerebellar Purkinje cells are rhythmically active, shaping descending signals and coordinating commands from higher brain areas with the step cycle. However, the variation in this activity across steps has not been studied, and its statistical structure, afferent mechanisms, and relationship to behavior remain unknown. Here, using multi-electrode recordings in freely moving rats, we show that behavioral variables systematically influence the shape of the step-locked firing rate. This effect depends strongly on the phase of the step cycle and reveals a functional clustering of Purkinje cells. Furthermore, we find a pronounced disassociation between patterns of variability driven by the parallel and climbing fibers. These results suggest that Purkinje cell activity not only represents step phase within each cycle, but is also shaped by behavior across steps, facilitating control of movement under dynamic conditions. PMID:26291165

  10. Optochemical Activation of Kinase Function in Live Cells

    PubMed Central

    Karginov, Andrei V.; Hahn, Klaus M.; Deiters, Alexander

    2015-01-01

    Summary Manipulation of protein kinase activity is widely used to dissect signaling pathways controlling physiological and pathological processes. Common methods often cannot provide the desired spatial and temporal resolution in control of kinase activity. Regulation of kinase activity by photocaged kinase inhibitors has been successfully used to achieve tight temporal and local control, but inhibitors are limited to inactivation of kinases, and often do not provide the desired specificity. Here we report detailed methods for light-mediated activation of kinases in living cells using engineered rapamycin-regulated kinases (RapR-kinases) in conjunction with a photocaged analog of rapamycin. PMID:24718793

  11. 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.

  12. 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.

  13. Fluorometric cell-based assay for β-galactosidase activity in probiotic gram-positive bacterial cells - Lactobacillus helveticus.

    PubMed

    Watson, Amanda L; Chiu, Norman H L

    2016-09-01

    Although methods for measuring β-galactosidase activity in intact gram-negative bacterial cells have been reported, the methods may not be applicable to measuring β-galactosidase activity in gram-positive bacterial cells. This report focuses on the development of a fluorometric cell-based assay for measuring β-galactosidase activity in gram-positive cells.

  14. Endoplasmic reticulum stress activation mediates Ginseng Rg3-induced anti-gallbladder cancer cell activity.

    PubMed

    Wu, Keren; Li, Ning; Sun, Huaqin; Xu, Tao; Jin, Fa; Nie, Jifeng

    2015-10-23

    In the current study, we examined the potential effect of Ginsenoside Rg3 against gallbladder cancer cells, the underlying signaling mechanisms were also studied. We demonstrated that Rg3 exerted potent cytotoxic and pro-apoptotic activity against established and primary human gallbladder cancer cells. Yet it was safe to non-cancerous gallbladder epithelial cells. At the molecular level, we showed that Rg3 induced endoplasmic reticulum (ER) stress activation, the latter was evidenced by C/EBP homologous protein (CHOP) upregulation, inositol-requiring enzyme 1 (IRE1)/PKR-like endoplasmic reticulum kinase (PERK) phosphorylations, and caspase-12 activation in gallbladder cancer cells. Reversely, the ER stress inhibitor salubrinal, the caspase-12 inhibitor z-ATAD-fmk as well as CHOP shRNA knockdown significantly attenuated Rg3-induced cytotoxicity against gallbladder cancer cells. In vivo, we showed that Rg3 oral administration significantly inhibited GBC-SD gallbladder cancer xenograft growth in nude mice, its activity was, however, compromised with co-administration of the ER stress inhibitor salubrinal. Thus, we suggest that ER stress activation mediates Ginseng Rg3-induced anti-gallbladder cancer cell activity in vitro and in vivo. PMID:26361144

  15. Cytotoxic Activity from Curcuma zedoaria Through Mitochondrial Activation on Ovarian Cancer Cells.

    PubMed

    Shin, Yujin; Lee, Yongkyu

    2013-12-31

    α-Curcumene is one of the physiologically active components of Curcuma zedoaria, which is believed to perform anti-tumor activities, the mechanisms of which are poorly understood. In the present study, we investigated the mechanism of the apoptotic effect of α-curcumene on the growth of human overian cancer, SiHa cells. Upon treatment with α-curcumene, cell viability of SiHa cells was inhibited > 73% for 48 h incubation. α-Curcumene treatment showed a characteristic nucleosomal DNA fragmentation pattern and the percentage of sub-diploid cells was increased in a concentration-dependent manner, hallmark features of apoptosis. Mitochondrial cytochrome c activation and an in vitro caspase-3 activity assay demonstrated that the activation of caspases accompanies the apoptotic effect of α-curcumene, which mediates cell death. These results suggest that the apoptotic effect of α-curcumene on SiHa cells may converge caspase-3 activation through the release of mitochondrial cytochrome c.

  16. 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.

  17. 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. PMID:26068799

  18. Schistosoma mansoni larvicidal activity of murine bronchoalveolar lavage cells.

    PubMed

    Lewis, F A; White-Ziegler, C A; Ball, J E; Niemann, G M

    1990-12-01

    We have investigated the ability of cells obtained from both normal and immune mice by bronchoalveolar lavage (BACs) to kill Schistosoma mansoni larvae in vitro. In cultures with mechanically derived schistosomules, high levels of larvicidal activity were displayed by BACs from both normal and irradiated cercaria-immunized C57BL/6 mice. Based on effector-to-target-cell ratios, BAC-mediated killing was two- to threefold more efficient than killing mediated by macrophage-rich cell populations obtained from the peritoneal cavity. BACs from normal A/J mice were essentially as larvicidal as normal C57BL/6 cells. However, BACs from a strain of mouse (P/J) with a known macrophage defect possessed negligible larvicidal activity. Macrophages made up 85 to 95% of BACs from all three strains tested. In contrast to cells of the IC-21 macrophage cell line, B6 BACs did not show enhanced killing activity when preincubated with lymphokine-containing supernatants. Lung schistosomules harvested 10 days after cercarial penetration were refractory to BAC-mediated killing. PMID:2254018

  19. Schistosoma mansoni larvicidal activity of murine bronchoalveolar lavage cells.

    PubMed Central

    Lewis, F A; White-Ziegler, C A; Ball, J E; Niemann, G M

    1990-01-01

    We have investigated the ability of cells obtained from both normal and immune mice by bronchoalveolar lavage (BACs) to kill Schistosoma mansoni larvae in vitro. In cultures with mechanically derived schistosomules, high levels of larvicidal activity were displayed by BACs from both normal and irradiated cercaria-immunized C57BL/6 mice. Based on effector-to-target-cell ratios, BAC-mediated killing was two- to threefold more efficient than killing mediated by macrophage-rich cell populations obtained from the peritoneal cavity. BACs from normal A/J mice were essentially as larvicidal as normal C57BL/6 cells. However, BACs from a strain of mouse (P/J) with a known macrophage defect possessed negligible larvicidal activity. Macrophages made up 85 to 95% of BACs from all three strains tested. In contrast to cells of the IC-21 macrophage cell line, B6 BACs did not show enhanced killing activity when preincubated with lymphokine-containing supernatants. Lung schistosomules harvested 10 days after cercarial penetration were refractory to BAC-mediated killing. PMID:2254018

  20. Biphasic activity of chloroquine in human colorectal cancer cells.

    PubMed

    Park, Deokbae; Lee, Youngki

    2014-12-01

    Autophagy is a homeostatic degradation process that is involved in tumor development and normal development. Autophagy is induced in cancer cells in response to chemotherapeutic agents, and inhibition of autophagy results in enhanced cancer cell death or survival. Chloroquine (CQ), an anti-malarial devrepug, is a lysosomotropic agent and is currently used as a potential anticancer agent as well as an autophagy inhibitor. Here, we evaluate the characteristics of these dual activities of CQ using human colorectal cancer cell line HCT15. The results show that CQ inhibited cell viability in dose-and time-dependent manner in the range between 20 to 80 uM, while CQ did not show any antiproliferative activity at 5 and 10 uM. Cotreatment of CQ with antitumor agent NVP-BEZ235, a dual inhibitor of PI3K/mTOR, rescued the cell viability at low concentrations meaning that CQ acted as an autophagy inhibitor, but CQ induced the lethal effect at high concentrations. Acridine orange staining revealed that CQ at high doses induced lysosomal membrane permeabilization (LMP). High doses of CQ produced cellular reactive oxygen species (ROS) and cotreatment of antioxidants, such as NAC and trolox, with high doses of CQ rescued the cell viability. These results suggest that CQ may exert its dual activities, as autophagy inhibitor or LMP inducer, in concentration-dependent manner. PMID:25949192

  1. Biphasic Activity of Chloroquine in Human Colorectal Cancer Cells

    PubMed Central

    Park, Deokbae; Lee, Youngki

    2014-01-01

    Autophagy is a homeostatic degradation process that is involved in tumor development and normal development. Autophagy is induced in cancer cells in response to chemotherapeutic agents, and inhibition of autophagy results in enhanced cancer cell death or survival. Chloroquine (CQ), an anti-malarial devrepug, is a lysosomotropic agent and is currently used as a potential anticancer agent as well as an autophagy inhibitor. Here, we evaluate the characteristics of these dual activities of CQ using human colorectal cancer cell line HCT15. The results show that CQ inhibited cell viability in dose-and time-dependent manner in the range between 20 to 80 uM, while CQ did not show any antiproliferative activity at 5 and 10 uM. Cotreatment of CQ with antitumor agent NVP-BEZ235, a dual inhibitor of PI3K/mTOR, rescued the cell viability at low concentrations meaning that CQ acted as an autophagy inhibitor, but CQ induced the lethal effect at high concentrations. Acridine orange staining revealed that CQ at high doses induced lysosomal membrane permeabilization (LMP). High doses of CQ produced cellular reactive oxygen species (ROS) and cotreatment of antioxidants, such as NAC and trolox, with high doses of CQ rescued the cell viability. These results suggest that CQ may exert its dual activities, as autophagy inhibitor or LMP inducer, in concentration-dependent manner. PMID:25949192

  2. Regulation of T cell-dendritic cell interactions by IL-7 governs T-cell activation and homeostasis

    PubMed Central

    Saini, Manoj; Pearson, Claire

    2009-01-01

    Interleukin-7 (IL-7) plays a central role in the homeostasis of the T-cell compartment by regulating T-cell survival and proliferation. Whether IL-7 can influence T-cell receptor (TCR) signaling in T cells remains controversial. Here, using IL-7–deficient hosts and TCR-transgenic T cells that conditionally express IL-7R, we examined antigen-specific T-cell responses in vitro and in vivo to viral infection and lymphopenia to determine whether IL-7 signaling influences TCR-triggered cell division events. In vitro, we could find no evidence that IL-7 signaling could costimulate T-cell activation over a broad range of conditions, suggesting that IL-7 does not directly tune TCR signaling. In vivo, however, we found an acute requirement for IL-7 signaling for efficiently triggering T-cell responses to influenza A virus challenge. Furthermore, we found that IL-7 was required for the enhanced homeostatic TCR signaling that drives lymphopenia-induced proliferation by a mechanism involving efficient contacts of T cells with dendritic cells. Consistent with this, saturating antigen-presenting capacity in vivo overcame the triggering defect in response to cognate peptide. Thus, we demonstrate a novel role for IL-7 in regulating T cell–dendritic cell interactions that is essential for both T-cell homeostasis and activation in vivo. PMID:19357399

  3. Antihelminthic niclosamide modulates dendritic cells activation and function.

    PubMed

    Wu, Chieh-Shan; Li, Yi-Rong; Chen, Jeremy J W; Chen, Ying-Che; Chu, Chiang-Liang; Pan, I-Hong; Wu, Yu-Shan; Lin, Chi-Chen

    2014-01-01

    Dendritic cells (DCs) link the sensing of the environment by the innate immune system to the initiation of adaptive immune responses. Accordingly, DCs are considered to be a major target in the development of immunomodulating compounds. In this study, the effect of niclosamide, a Food and Drug Administration-approved antihelminthic drug, on the activation of lipopolysaccharide (LPS)-stimulated murine bone marrow-derived DCs was examined. Our experimental results show that niclosamide reduced the pro-inflammatory cytokine and chemokine expression of LPS-activated DCs. In addition, niclosamide also affected the expression of MHC and costimulatory molecules and influenced the ability of the cells to take up antigens. Therefore, in mixed cell cultures composed of syngeneic OVA-specific T cells and DCs, niclosamide-treated DCs showed a decreased ability to stimulate T cell proliferation and IFN-γ production. Furthermore, intravenous injection of niclosamide also attenuated contact hypersensitivity (CHS) in mice during sensitization with 2,4-dinitro-1-fluorobenzene. Blocking the LPS-induced activation of MAPK-ERK, JNK and NF-κB may contribute to the inhibitory effect of niclosamide on DC activation. Collectively, our findings suggest that niclosamide can manipulate the function of DCs. These results provide new insight into the immunopharmacological role of niclosamide and suggest that it may be useful for the treatment of chronic inflammatory disorders or DC-mediated autoimmune diseases. PMID:24561310

  4. 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.

  5. AEA Cell-Bypass-Switch Activation: An Update

    NASA Technical Reports Server (NTRS)

    Keys, Denney; Rao, Gopalakrishna M.; Wannemacher, Harry

    2002-01-01

    The objectives of this project included the following: (1) verify the performance of AEA cell bypass protection device (CBPD) under simulated EOS-Aqua/Aura flight hardware configuration; (2) assess the safety of the hardware under an inadvertent firing of CBPD switch, as well as the closing of CBPD; and (3) confirm that the mode of operation of CBPD switch is the formation of a continuous low impedance path (a homogeneous low melting point alloy). The nominal performance of AEA CBPD under flight operating conditions (vacuum except zero-G, and high impedance cell) has been demonstrated. There is no evidence of cell rupture or excessive heat production during or after CBPD switch activation under simulated high cell impedance (open-circuit cell failure mode). The formation of a continuous low impedance path (a homogeneous low melting point alloy) has been confirmed.

  6. Rare earth elements activate endocytosis in plant cells

    PubMed Central

    Wang, Lihong; Li, Jigang; Zhou, Qing; Yang, Guangmei; Ding, Xiao Lan; Li, Xiaodong; Cai, Chen Xin; Zhang, Zhao; Wei, Hai Yan; Lu, Tian Hong; Deng, Xing Wang; Huang, Xiao Hua

    2014-01-01

    It has long been observed that rare earth elements (REEs) regulate multiple facets of plant growth and development. However, the underlying mechanisms remain largely unclear. Here, using electron microscopic autoradiography, we show the life cycle of a light REE (lanthanum) and a heavy REE (terbium) in horseradish leaf cells. Our data indicate that REEs were first anchored on the plasma membrane in the form of nanoscale particles, and then entered the cells by endocytosis. Consistently, REEs activated endocytosis in plant cells, which may be the cellular basis of REE actions in plants. Moreover, we discovered that a portion of REEs was successively released into the cytoplasm, self-assembled to form nanoscale clusters, and finally deposited in horseradish leaf cells. Taken together, our data reveal the life cycle of REEs and their cellular behaviors in plant cells, which shed light on the cellular mechanisms of REE actions in living organisms. PMID:25114214

  7. Rare earth elements activate endocytosis in plant cells.

    PubMed

    Wang, Lihong; Li, Jigang; Zhou, Qing; Yang, Guangmei; Ding, Xiao Lan; Li, Xiaodong; Cai, Chen Xin; Zhang, Zhao; Wei, Hai Yan; Lu, Tian Hong; Deng, Xing Wang; Huang, Xiao Hua

    2014-09-01

    It has long been observed that rare earth elements (REEs) regulate multiple facets of plant growth and development. However, the underlying mechanisms remain largely unclear. Here, using electron microscopic autoradiography, we show the life cycle of a light REE (lanthanum) and a heavy REE (terbium) in horseradish leaf cells. Our data indicate that REEs were first anchored on the plasma membrane in the form of nanoscale particles, and then entered the cells by endocytosis. Consistently, REEs activated endocytosis in plant cells, which may be the cellular basis of REE actions in plants. Moreover, we discovered that a portion of REEs was successively released into the cytoplasm, self-assembled to form nanoscale clusters, and finally deposited in horseradish leaf cells. Taken together, our data reveal the life cycle of REEs and their cellular behaviors in plant cells, which shed light on the cellular mechanisms of REE actions in living organisms.

  8. 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.

  9. Endothelial activation by platelets from sickle cell anemia patients.

    PubMed

    Proença-Ferreira, Renata; Brugnerotto, Ana Flávia; Garrido, Vanessa Tonin; Dominical, Venina Marcela; Vital, Daiana Morelli; Ribeiro, Marilene de Fátima Reis; dos Santos, Melissa Ercolin; Traina, Fabíola; Olalla-Saad, Sara T; Costa, Fernando Ferreira; Conran, Nicola

    2014-01-01

    Sickle cell anemia (SCA) is associated with a hypercoagulable state. Increased platelet activation is reported in SCA and SCA platelets may present augmented adhesion to the vascular endothelium, potentially contributing to the vaso-occlusive process. We sought to observe the effects of platelets (PLTs) from healthy control (CON) individuals and SCA individuals on endothelial activation, in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured, in the presence, or not, of washed PLTs from CON or steady-state SCA individuals. Supernatants were reserved for cytokine quantification, and endothelial adhesion molecules (EAM) were analyzed by flow cytometry; gene expressions of ICAM1 and genes of the NF-κB pathway were analyzed by qPCR. SCA PLTs were found to be more inflammatory, displaying increased adhesive properties, an increased production of IL-1β and a tendency towards elevated expressions of P-selectin and activated αIIbβ3. Following culture in the presence of SCA PLTs, HUVEC presented significant augmentations in the expressions of the EAM, ICAM-1 and E-selectin, as well as increased IL-8 production and increased ICAM1 and NFKB1 (encodes p50 subunit of NF-κB) gene expressions. Interestingly, transwell inserts abolished the effects of SCA PLTs on EAM expression. Furthermore, an inhibitor of the NF-κB pathway, BAY 11-7082, also prevented the induction of EAM expression on the HUVEC surface by SCA PLTs. In conclusion, we find further evidence to indicate that platelets circulate in an activated state in sickle cell disease and are capable of stimulating endothelial cell activation. This effect appears to be mediated by direct contact, or even adhesion, between the platelets and endothelial cells and via NFκB-dependent signaling. As such, activated platelets in SCD may contribute to endothelial activation and, therefore, to the vaso-occlusive process. Results provide further evidence to support the use of anti-platelet approaches in association

  10. 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

  11. 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...

  12. KIPase activity is a novel caspase-like activity associated with cell proliferation.

    PubMed

    Medina-Palazon, Cahora; Bernard, Emmanuelle; Frost, Victoria; Morley, Simon; Sinclair, Alison J

    2004-07-01

    A novel caspase-like activity, which is directly regulated with cell proliferation is a candidate to regulate the abundance of the cyclin-dependent kinase inhibitor, p27(KIP1), in human lymphoid cells. This activity, which we term KIPase activity, can also cleave a subset of caspase substrates. Here we demonstrate that KIPase is a novel enzyme distinct from any of the previously characterized human caspases. We show that KIPase is active in a variety of cell lineages, its activity is associated with the proliferation of the human T-cell line, Jurkat, and is not inhibited by the broad spectrum caspase inhibitor z-VAD-fmk. Gel filtration analysis revealed that KIPase has a native molecular mass of approximately 100-200 kDa. Furthermore, the activity of KIPase does not change during apoptosis induced by either ligation of FAS or exposure of cells to etoposide. The uniqueness of KIPase is demonstrated by the fact that none of the human caspases tested (1-10) are able to cleave a specific KIPase substrate (Ac-DPSD-AMC) and that an aldehyde modified derivative of the DPSD tetra peptide is unable to inhibit caspases, but is a good inhibitor of KIPase activity. This supports a hypothesis whereby KIPase is a currently unidentified caspase-like enzyme which regulates the abundance of p27(KIP1) in a proliferation-dependent manner.

  13. Analyzing electrical activities of pancreatic β cells using mathematical models.

    PubMed

    Cha, Chae Young; Powell, Trevor; Noma, Akinori

    2011-11-01

    Bursts of repetitive action potentials are closely related to the regulation of glucose-induced insulin secretion in pancreatic β cells. Mathematical studies with simple β-cell models have established the central principle that the burst-interburst events are generated by the interaction between fast membrane excitation and slow cytosolic components. Recently, a number of detailed models have been developed to simulate more realistic β cell activity based on expanded findings on biophysical characteristics of cellular components. However, their complex structures hinder our intuitive understanding of the underlying mechanisms, and it is becoming more difficult to dissect the role of a specific component out of the complex network. We have recently developed a new detailed model by incorporating most of ion channels and transporters recorded experimentally (the Cha-Noma model), yet the model satisfies the charge conservation law and reversible responses to physiological stimuli. Here, we review the mechanisms underlying bursting activity by applying mathematical analysis tools to representative simple and detailed models. These analyses include time-based simulation, bifurcation analysis and lead potential analysis. In addition, we introduce a new steady-state I-V (ssI-V) curve analysis. We also discuss differences in electrical signals recorded from isolated single cells or from cells maintaining electrical connections within multi-cell preparations. Towards this end, we perform simulations with our detailed pancreatic β-cell model.

  14. Long noncoding RNAs in B-cell development and activation

    PubMed Central

    Brazão, Tiago F.; Johnson, Jethro S.; Müller, Jennifer; Heger, Andreas; Ponting, Chris P.

    2016-01-01

    Long noncoding RNAs (lncRNAs) are potentially important regulators of cell differentiation and development, but little is known about their roles in B lymphocytes. Using RNA-seq and de novo transcript assembly, we identified 4516 lncRNAs expressed in 11 stages of B-cell development and activation. Most of these lncRNAs have not been previously detected, even in the closely related T-cell lineage. Comparison with lncRNAs previously described in human B cells identified 185 mouse lncRNAs that have human orthologs. Using chromatin immunoprecipitation-seq, we classified 20% of the lncRNAs as either enhancer-associated (eRNA) or promoter-associated RNAs. We identified 126 eRNAs whose expression closely correlated with the nearest coding gene, thereby indicating the likely location of numerous enhancers active in the B-cell lineage. Furthermore, using this catalog of newly discovered lncRNAs, we show that PAX5, a transcription factor required to specify the B-cell lineage, bound to and regulated the expression of 109 lncRNAs in pro-B and mature B cells and 184 lncRNAs in acute lymphoblastic leukemia. PMID:27381906

  15. Sorting drops and cells with acoustics: acoustic microfluidic fluorescence-activated cell sorter.

    PubMed

    Schmid, Lothar; Weitz, David A; Franke, Thomas

    2014-10-01

    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.

  16. Tomato waste: Carotenoids content, antioxidant and cell growth activities.

    PubMed

    Stajčić, Sladjana; Ćetković, Gordana; Čanadanović-Brunet, Jasna; Djilas, Sonja; Mandić, Anamarija; Četojević-Simin, Dragana

    2015-04-01

    The carotenoid content, antioxidant and cell growth activities of tomato waste extracts, obtained from five different tomato genotypes, was investigated. High performance liquid chromatography was used to identify and quantify the main carotenoids present in tomato waste extracts. The antioxidant activity of tomato waste extracts was tested using spectrophotometric methods, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity and reducing power assay. The highest DPPH scavenging activity (IC50 = 0.057 mg/ml) was obtained for Bačka extract. The Knjaz extract showed the best reducing power (IC50 = 2.12 mg/ml). Cell growth effects were determined in HeLa, MCF7 and MRC-5 cell lines by sulforhodamine B test. Anti-proliferative effects were observed in all cell lines at higher concentrations (⩾ 0.125 mg/ml). The carotenoid contents exhibited a strong correlation with antioxidant and anti-proliferation activity. The results obtained indicated that tomato waste should be regarded as potential nutraceutic resource and may be used as a functional food ingredient.

  17. Tomato waste: Carotenoids content, antioxidant and cell growth activities.

    PubMed

    Stajčić, Sladjana; Ćetković, Gordana; Čanadanović-Brunet, Jasna; Djilas, Sonja; Mandić, Anamarija; Četojević-Simin, Dragana

    2015-04-01

    The carotenoid content, antioxidant and cell growth activities of tomato waste extracts, obtained from five different tomato genotypes, was investigated. High performance liquid chromatography was used to identify and quantify the main carotenoids present in tomato waste extracts. The antioxidant activity of tomato waste extracts was tested using spectrophotometric methods, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity and reducing power assay. The highest DPPH scavenging activity (IC50 = 0.057 mg/ml) was obtained for Bačka extract. The Knjaz extract showed the best reducing power (IC50 = 2.12 mg/ml). Cell growth effects were determined in HeLa, MCF7 and MRC-5 cell lines by sulforhodamine B test. Anti-proliferative effects were observed in all cell lines at higher concentrations (⩾ 0.125 mg/ml). The carotenoid contents exhibited a strong correlation with antioxidant and anti-proliferation activity. The results obtained indicated that tomato waste should be regarded as potential nutraceutic resource and may be used as a functional food ingredient. PMID:25442547

  18. CTNNB1 Signaling in Sertoli Cells Downregulates Spermatogonial Stem Cell Activity via WNT4

    PubMed Central

    Boyer, Alexandre; Yeh, Jonathan R.; Zhang, Xiangfan; Paquet, Marilène; Gaudin, Aurore; Nagano, Makoto C.; Boerboom, Derek

    2012-01-01

    Constitutive activation of the WNT signaling effector CTNNB1 (β-catenin) in the Sertoli cells of the Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ mouse model results in progressive germ cell loss and sterility. In this study, we sought to determine if this phenotype could be due to a loss of spermatogonial stem cell (SSC) activity. Reciprocal SSC transplants between Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ and wild-type mice showed that SSC activity is lost in Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ testes over time, whereas the mutant testes could not support colonization by wild-type SSCs. Microarray analyses performed on cultured Sertoli cells showed that CTNNB1 induces the expression of genes associated with the female sex determination pathway, which was also found to occur in Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ testes. One CTNNB1 target gene encoded the secreted signaling molecule WNT4. We therefore tested the effects of WNT4 on SSC-enriched germ cell cultures, and found that WNT4 induced cell death and reduced SSC activity without affecting cell cycle. Conversely, conditional inactivation of Wnt4 in the Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ model rescued spermatogenesis and male fertility, indicating that WNT4 is the major effector downstream of CTNNB1 responsible for germ cell loss. Furthermore, WNT4 was found to signal via the CTNNB1 pathway in Sertoli cells, suggesting a self-reinforcing positive feedback loop. Collectively, these data indicate for the first time that ectopic activation of a signaling cascade in the stem cell niche depletes SSC activity through a paracrine factor. These findings may provide insight into the pathogenesis of male infertility, as well as embryonic gonadal development. PMID:22253774

  19. 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.

  20. PARP activation promotes nuclear AID accumulation in lymphoma cells.

    PubMed

    Tepper, Sandra; Jeschke, Julia; Böttcher, Katrin; Schmidt, Angelika; Davari, Kathrin; Müller, Peter; Kremmer, Elisabeth; Hemmerich, Peter; Pfeil, Ines; Jungnickel, Berit

    2016-03-15

    Activation-induced cytidine deaminase (AID) initiates immunoglobulin diversification in germinal center B cells by targeted introduction of DNA damage. As aberrant nuclear AID action contributes to the generation of B cell lymphoma, the protein's activity is tightly regulated, e.g. by nuclear/cytoplasmic shuttling and nuclear degradation. In the present study, we asked whether DNA damage may affect regulation of the AID protein. We show that exogenous DNA damage that mainly activates base excision repair leads to prevention of proteasomal degradation of AID and hence its nuclear accumulation. Inhibitor as well as knockout studies indicate that activation of poly (ADP-ribose) polymerase (PARP) by DNA damaging agents promotes both phenomena. These findings suggest that PARP inhibitors influence DNA damage dependent AID regulation, with interesting implications for the regulation of AID function and chemotherapy of lymphoma.

  1. PARP activation promotes nuclear AID accumulation in lymphoma cells

    PubMed Central

    Böttcher, Katrin; Schmidt, Angelika; Davari, Kathrin; Müller, Peter; Kremmer, Elisabeth; Hemmerich, Peter; Pfeil, Ines; Jungnickel, Berit

    2016-01-01

    Activation-induced cytidine deaminase (AID) initiates immunoglobulin diversification in germinal center B cells by targeted introduction of DNA damage. As aberrant nuclear AID action contributes to the generation of B cell lymphoma, the protein's activity is tightly regulated, e.g. by nuclear/cytoplasmic shuttling and nuclear degradation. In the present study, we asked whether DNA damage may affect regulation of the AID protein. We show that exogenous DNA damage that mainly activates base excision repair leads to prevention of proteasomal degradation of AID and hence its nuclear accumulation. Inhibitor as well as knockout studies indicate that activation of poly (ADP-ribose) polymerase (PARP) by DNA damaging agents promotes both phenomena. These findings suggest that PARP inhibitors influence DNA damage dependent AID regulation, with interesting implications for the regulation of AID function and chemotherapy of lymphoma. PMID:26921193

  2. Necroptosis of Dendritic Cells Promotes Activation of γδ T Cells.

    PubMed

    Collins, Cheryl C; Bashant, Kathleen; Erikson, Cuixia; Thwe, Phyu Myat; Fortner, Karen A; Wang, Hong; Morita, Craig T; Budd, Ralph C

    2016-01-01

    γδ T cells function at the interface between innate and adaptive immunity and have well-demonstrated roles in response to infection, autoimmunity and tumors. A common characteristic of these seemingly disparate conditions may be cellular stress or death. However, the conditions under which ligands for γδ T cells are induced or exposed remain largely undefined. We observed that induction of necroptosis of murine or human dendritic cells (DC) by inhibition of caspase activity paradoxically augments their ability to activate γδ T cells. Furthermore, upregulation of the stabilizer of caspase-8 activity, c-FLIP, by IL-4, not only greatly reduced the susceptibility of DC to necroptosis, but also considerably decreased their ability to activate γδ T cells. Collectively, these findings suggest that the induction of necroptosis in DC upregulates or exposes the expression of γδ T cell ligands, and they support the view that γδ T cells function in the immune surveillance of cell stress. PMID:27431410

  3. 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.

  4. Optical Control of Living Cells Electrical Activity by Conjugated Polymers.

    PubMed

    Martino, Nicola; Bossio, Caterina; Vaquero Morata, Susana; Lanzani, Guglielmo; Antognazza, Maria Rosa

    2016-01-28

    Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity. In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported

  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. Optical Control of Living Cells Electrical Activity by Conjugated Polymers.

    PubMed

    Martino, Nicola; Bossio, Caterina; Vaquero Morata, Susana; Lanzani, Guglielmo; Antognazza, Maria Rosa

    2016-01-01

    Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity. In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported

  7. New thiazolidinediones affect endothelial cell activation and angiogenesis.

    PubMed

    Rudnicki, Martina; Tripodi, Gustavo L; Ferrer, Renila; Boscá, Lisardo; Pitta, Marina G R; Pitta, Ivan R; Abdalla, Dulcineia S P

    2016-07-01

    Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor-γ (PPARγ) agonists used in treating type 2 diabetes that may exhibit beneficial pleiotropic effects on endothelial cells. In this study, we characterized the effects of three new TZDs [GQ-32 (3-biphenyl-4-ylmethyl-5-(4-nitro-benzylidene)-thiazolidine-2,4-dione), GQ-169 (5-(4-chloro-benzylidene)-3-(2,6-dichloro-benzyl)-thiazolidine-2,4-dione), and LYSO-7 (5-(5-bromo-1H-indol-3-ylmethylene)-3-(4-chlorobenzyl)-thiazolidine-2,4-dione)] on endothelial cells. The effects of the new TZDs were evaluated on the production of nitric oxide (NO) and reactive oxygen species (ROS), cell migration, tube formation and the gene expression of adhesion molecules and angiogenic mediators in human umbilical vein endothelial cells (HUVECs). PPARγ activation by new TZDs was addressed with a reporter gene assay. The three new TZDs activated PPARγ and suppressed the tumor necrosis factor α-induced expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. GQ-169 and LYSO-7 also inhibited the glucose-induced ROS production. Although NO production assessed with 4-amino-5-methylamino-2',7'-difluorofluorescein-FM probe indicated that all tested TZDs enhanced intracellular levels of NO, only LYSO-7 treatment significantly increased the release of NO from HUVEC measured by chemiluminescence analysis of culture media. Additionally, GQ-32 and GQ-169 induced endothelial cell migration and tube formation by the up-regulation of angiogenic molecules expression, such as vascular endothelial growth factor A and interleukin 8. GQ-169 also increased the mRNA levels of basic fibroblast growth factor, and GQ-32 enhanced transforming growth factor-β expression. Together, the results of this study reveal that these new TZDs act as partial agonists of PPARγ and modulate endothelial cell activation and endothelial dysfunction besides to stimulate migration and tube formation. PMID:27108791

  8. Intracellular localization of mevalonate-activating enzymes in plant cells

    PubMed Central

    Rogers, L. J.; Shah, S. P. J.; Goodwin, T. W.

    1966-01-01

    Mevalonate-activating enzymes are shown to be present in the chloroplasts of French-bean leaves. The chloroplast membrane is impermeable to mevalonic acid. Mevalonate-activating enzymes also appear to be found outside the chloroplast. These results support the view that terpenoid biosynthesis in the plant cell is controlled by a combination of enzyme segregation and specific membrane permeability. ImagesFig. 1.Fig. 2. PMID:5947149

  9. Interleukin-2 activation of cytotoxic cells in postmastectomy seroma.

    PubMed

    Gercel-Taylor, C; Hoffman, J P; Taylor, D D; Owens, K J; Eisenberg, B L

    1996-02-15

    Lymphocytes were isolated from breast seroma fluids and used to study the mechanism of activation of cytotoxic lymphocytes and possible role of immunological potentiation following surgery in breast cancer patients. Single or serial samples were obtained from patients who had undergone mastectomy or lumpectomy with axillary node dissection. Lymphocytes were activated with rIL-2 (interleukin-2) and their cytotoxic activity was studied against Daudi and K562 cells and against a breast tumor line (SKBr-3). All of the patients (21/21) responded to IL-2 stimulation by significant activation of cytotoxic activity. The unstimulated cytotoxic activity of these cells against NK targets was low with less than 10% specific release in cytotoxicity assays. In simultaneous experiments, autologous seroma fluid was included during activation of lymphocytes to study possible regulatory molecules that may be present. In 17/21 patients, the presence of their seroma fluid, during the activation period, enhanced or did not effect the cytotoxic potential of their lymphocytes; inhibition was observed when seroma fluids from 4/21 patients were included. Analysis of the cytotoxic population derived from combined IL-2 and seroma treatments indicates the presence of cells with increased expression of CD56, and CD2, as well as in some cases CD16 expression. Cytotoxic lymphocytes derived from IL-2 and seroma treatments appeared to be more effective killers. Modulation of CD2 expression with seroma alone appeared to result in the generation of this highly cytotoxic population. This study demonstrates the role of CD2 expression in the effectiveness of LAK cell killing and also potential benefit of an immunotherapeutic approach to the postoperative treatment of carcinoma of the breast.

  10. Oxytocin-stimulated NFAT transcriptional activation in human myometrial cells.

    PubMed

    Pont, Jason N A; McArdle, Craig A; López Bernal, Andrés

    2012-10-01

    Oxytocin (OXT) is a peptide hormone that binds the OXT receptor on myometrial cells, initiating an intracellular signaling cascade, resulting in accumulation of intracellular calcium and smooth muscle contraction. In other systems, an elevation of intracellular Ca(2+) stimulates nuclear translocation of the transcription factor, nuclear factor of activated T cells (NFAT), which is transcriptionally active in arterial and ileal smooth muscle. Here we have investigated the role of NFAT in the mechanism of action of OXT. Human myometrial cells expressed all five NFAT isoforms (NFATC1-C4 and -5). Myometrial cells were transduced with a recombinant adenovirus expressing a NFATC1-EFP reporter, and a semi-automated imaging system was used to monitor effects of OXT on reporter localization in live cells. OXT induced a concentration-dependent nuclear translocation of NFATC1-EFP in a reversible manner, which was inhibited by OXT antagonists and calcineurin inhibitors. Pulsatile stimulation with OXT caused intermittent, pulse-frequency-dependent, nuclear translocation of NFATC1-EFP, which was more efficient than sustained stimulation. OXT induced nuclear translocation of endogenous NFAT that was transcriptionally active, because OXT stimulated activity of a NFAT-response element-luciferase reporter and induced calcineurin-NFAT dependent expression of RGS2, RCAN1, and PTGS2 (COX2) mRNA. Furthermore, OXT-dependent transcription was dependent on protein neosynthesis; cycloheximide abolished RGS2 transcription but augmented RCAN1 and COX2 transcriptional readouts. This study identifies a novel signaling mechanism within the myometrium, whereby calcineurin-NFAT signaling mediates OXT-induced transcriptional activity. Furthermore, we show NFATC1-EFP is responsive to pulses of OXT, a mechanism by which myometrial cells could decode OXT pulse frequency.

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

    SciTech Connect

    Davis, L.; Lipsky, P.E.

    1986-05-15

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

  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. Blockade of mast cell activation reduces cutaneous scar formation.

    PubMed

    Chen, Lin; Schrementi, Megan E; Ranzer, Matthew J; Wilgus, Traci A; DiPietro, Luisa A

    2014-01-01

    Damage to the skin initiates a cascade of well-orchestrated events that ultimately leads to repair of the wound. The inflammatory response is key to wound healing both through preventing infection and stimulating proliferation and remodeling of the skin. Mast cells within the tissue are one of the first immune cells to respond to trauma, and upon activation they release pro-inflammatory molecules to initiate recruitment of leukocytes and promote a vascular response in the tissue. Additionally, mast cells stimulate collagen synthesis by dermal fibroblasts, suggesting they may also influence scar formation. To examine the contribution of mast cells in tissue repair, we determined the effects the mast cell inhibitor, disodium cromoglycate (DSCG), on several parameters of dermal repair including, inflammation, re-epithelialization, collagen fiber organization, collagen ultrastructure, scar width and wound breaking strength. Mice treated with DSCG had significantly reduced levels of the inflammatory cytokines IL-1α, IL-1β, and CXCL1. Although DSCG treatment reduced the production of inflammatory mediators, the rate of re-epithelialization was not affected. Compared to control, inhibition of mast cell activity caused a significant decrease in scar width along with accelerated collagen re-organization. Despite the reduced scar width, DSCG treatment did not affect the breaking strength of the healed tissue. Tryptase β1 exclusively produced by mast cells was found to increase significantly in the course of wound healing. However, DSCG treatment did not change its level in the wounds. These results indicate that blockade of mast cell activation reduces scar formation and inflammation without further weakening the healed wound.

  14. The role of cell surface receptors in the activation of human B cells by phosphorothioate oligonucleotides.

    PubMed

    Liang, H; Reich, C F; Pisetsky, D S; Lipsky, P E

    2000-08-01

    Phosphorothioate oligodeoxynucleotides (sODN) containing the CpG motif or TCG repeats induce T cell-independent polyclonal activation of human B cells. To elucidate the mechanism of this response, the role of cell surface receptors was investigated. Sepharose beads coated with stimulatory but not nonstimulatory sODNs induced B cell proliferation comparably with soluble sODNs. The B cell stimulatory activity of Sepharose-bound sODN did not result from free sODN released from the beads since media incubated with coated beads were inactive. Using FITC-labeled sODNs as probes, binding to human B cells could be detected by flow cytometry. Binding was rapid, saturable, initially temperature independent, but with a rapid off-rate. Competition studies indicated that both stimulatory sODNs and minimally stimulatory sODNs bound to the same receptor. By contrast, phosphodiester oligonucleotides with the same nucleotide sequence as sODNs and bacterial DNA inhibited the binding of sODNs to B cells minimally. Charge appeared to contribute to the binding of sODNs to B cells since binding of sODNs was competitively inhibited by negatively charged molecules, including fucoidan, poly I, and polyvinyl sulfate. These data indicate that human B cells bind sODNs by a receptor-mediated mechanism that is necessary but not sufficient for polyclonal activation.

  15. 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. PMID:7852311

  16. Immunomodulation of phloretin by impairing dendritic cell activation and function.

    PubMed

    Lin, Chi-Chen; Chu, Ching-Liang; Ng, Chin-Sheng; Lin, Ching-Yen; Chen, Der-Yuan; Pan, I-Hong; Huang, Kao-Jean

    2014-05-01

    Dietary compounds in fruits and vegetables have been shown to exert many biological activities. In addition to antioxidant effects, a number of flavonoids are able to modulate inflammatory responses. Here, we demonstrated that phloretin (PT), a natural dihydrochalcone found in many fruits, suppressed the activation and function of mouse dendritic cells (DCs). Phloretin disturbed the multiple intracellular signaling pathways in DCs induced by the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS), including ROS, MAPKs (ERK, JNK, p38 MAPK), and NF-κB, and thereby reducing the production of inflammatory cytokines and chemokines. Phloretin also effectively suppressed the activation of DCs treated with different dosages of LPS or various TLR agonists. The LPS-induced DC maturation was attenuated by phloretin because the expression levels of the MHC class II and the co-stimulatory molecules were down-regulated, which then inhibited the LPS-stimulating DCs and the subsequent naïve T cell activation in a mixed lymphocyte reaction. Moreover, in vivo administration of phloretin suppressed the phenotypic maturation of the LPS-challenged splenic DCs and decreased the IFN-γ production from the activated CD4 T cells. Thus, we suggest that phloretin may potentially be an immunomodulator by impairing the activation and function of DCs and phloretin-contained fruits may be helpful in the improvement of inflammation and autoimmune diseases. PMID:24651121

  17. Cloud Activation Characteristics of Airborne Erwinia carotovora Cells.

    NASA Astrophysics Data System (ADS)

    Franc, Gary D.; Demott, Paul J.

    1998-10-01

    Several strains of plant pathogenic bacteria, Erwinia carotovora carotovora and E. carotovora atroseptica, were observed to be active as cloud condensation nuclei (CCN). The CCN supersaturation spectra of bacterial aerosols were measured in the laboratory and compared to the activity of ammonium sulfate. Approximately 25%-30% of the aerosolized bacterial cells activated droplets at 1% water supersaturation compared to 80% activation of the ammonium sulfate aerosol. Physical and numerical simulations of cloud droplet activation and growth on bacteria were also performed. Both simulations predict that aerosolized bacteria will be incorporated into cloud droplets during cloud formation. Results strongly support the hypothesis that significant numbers of the tested bacterial strains are actively involved in atmospheric cloud formation and precipitation processes following natural aerosolization and vertical transport to cloud levels.

  18. 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.

  19. 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

  20. Active p21-activated kinase 1 rescues MCF10A breast epithelial cells from undergoing anoikis.

    PubMed

    Menard, Raymond E; Jovanovski, Andrew P; Mattingly, Raymond R

    2005-07-01

    The protein kinase, PAK1, is overexpressed in human breast cancer and may contribute to malignancy through induction of proliferation and invasiveness. In this study, we examined the role of PAK1 in the survival of detached MCF10A breast epithelial cells to test whether it may also regulate the early stages of neoplasia. MCF10A cells undergo anoikis, as measured by the cleavage of caspase 3 and poly(ADP-ribose) polymerase (PARP), after more than 8 hours of detachment. Endogenous Akt, PAK1, and BAD are phosphorylated in attached MCF10A cells, but these phosphorylation events are all lost during the first 8 hours of detachment. Expression of constitutively active PAK1 or Akt suppresses the cleavage of caspase 3 and PARP in detached MCF10A cells. Co-overexpression of active PAK1 with dominant-negative Akt, or of active Akt with dominant-negative PAK1, still suppresses anoikis. Thus, Akt and PAK1 enhance survival through pathways that are at least partially independent. PAK1-dependent regulation of anoikis is likely to occur early in the apoptotic cascade as expression of dominant-negative PAK1 increased the cleavage of the upstream caspase 9, while constitutively active PAK1 inhibited caspase 9 activation. These results support a role for activated PAK1 in the suppression of anoikis in MCF10A epithelial cells.

  1. 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.

  2. 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. PMID:26941401

  3. Lysine methylation represses p53 activity in teratocarcinoma cancer cells.

    PubMed

    Zhu, Jiajun; Dou, Zhixun; Sammons, Morgan A; Levine, Arnold J; Berger, Shelley L

    2016-08-30

    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. Lysine methylation represses p53 activity in teratocarcinoma cancer cells.

    PubMed

    Zhu, Jiajun; Dou, Zhixun; Sammons, Morgan A; Levine, Arnold J; Berger, Shelley L

    2016-08-30

    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.

  5. Telomerase activity in non-small cell lung cancer

    PubMed Central

    Dobija-Kubica, Katarzyna; Bruliński, Krzysztof; Rogoziński, Paweł; Wiczkowski, Andrzej; Gawrychowska, Agata; Gawrychowski, Jacek

    2016-01-01

    Introduction High telomerase activity has been detected in the majority of malignant neoplasms including lung cancer. The purpose of the study was to attempt to use telomerase activity as a prognostic factor in patients with non-small cell lung cancer (NSCLC). Material and methods Telomerase activity was analyzed in 47 tissue specimens taken from patients with NSCLC. The control group consisted of 30 specimens of non-cancerous lung parenchyma. Telomerase activity was measured by means of the telomeric repeat amplification protocol (TRAP). Results Telomerase activity in the neoplastic tissue was significantly higher than in the lung parenchyma that was free from neoplastic infiltration. There was no significant association between telomerase activity and age, gender, tobacco smoking, histological type of the tumor, or staging (pTNM). No association was found between the level of telomerase activity in NSCLC specimens and the two-year survival rate of patients (p = 0.326). A higher level of telomerase activity in poorly differentiated tumors (G3) as compared to moderately differentiated tumors (G2) was detected (p = 0.008). A positive association was identified between telomerase activity in pulmonary parenchyma free from tumor infiltration and the presence of leukocyte infiltration (p = 0.0001). Conclusions No association was found between the level of telomerase activity in NSCLC specimens and the two-year survival rate of patients. The study has revealed a positive association between telomerase activity and the grade of differentiation (G) in NSCLC. PMID:27212973

  6. Phosphatidylinositol-3-kinase regulates PKCtheta activity in cytotoxic T cells.

    PubMed

    Puente, Lawrence G; Mireau, Laura R; Lysechko, Tara L; Ostergaard, Hanne L

    2005-06-01

    Protein kinase C (PKC) theta plays a crucial role in T cell activation. We, therefore, examined the regulation of PKCtheta activity in cytotoxic T lymphocytes (CTL). We demonstrated that PMA did not stimulate PKCtheta activation and phospholipase C inhibition did not block anti-CD3-stimulated PKCtheta activation in a CTL clone. This suggests that diacylglycerol is neither sufficient nor required for PKCtheta activation. Furthermore, PKCtheta was only activated in a CTL clone stimulated with plate-bound anti-CD3 but not soluble anti-CD3. However, PMA or cross-linked anti-CD3 stimulated phosphorylation of PKCtheta as measured by a migratory shift, suggesting that phosphorylation was not sufficient for activity. Phosphatidylinositol 3-kinase activity was required for anti-CD3, but not PMA, stimulated phosphorylation and for immobilized anti-CD3-triggered PKCtheta activity. A substantial fraction of PKCtheta was constitutively membrane associated and PMA or CD3 stimulation did not significantly increase membrane association. Our data indicate that phosphorylation of PKCtheta is not a suitable surrogate measurement for PKCtheta activity and that additional, yet to be defined steps, are required for the regulation of PKCtheta enzymatic activity in CTL.

  7. FGF2 activates TRPC and Ca(2+) signaling leading to satellite cell activation.

    PubMed

    Liu, Yewei; Schneider, Martin F

    2014-01-01

    Satellite cells, as stem cells of adult skeletal muscle, are tightly associated with the differentiated muscle fibers and remain quiescent in the absence of muscle damage. In response to an injury, the quiescent satellite cell is activated by soluble factors, including FGFs released from injured myofibers. Using immunostaining, we here first show that TRPC1 channels are highly expressed in satellite cells attached to muscle fibers. Since CD34, a traditional stem cell marker, was recently found to be expressed in skeletal muscle satellite cells we labeled living satellite cells in their physiological niche associated with host FDB fibers using anti-CD34-FITC antibody. We then monitored intra-cellular calcium in anti-CD34-FITC labeled satellite cells attached to muscle fibers using the calcium sensitive dye X rhod-1 which has little fluorescence cross talk with FITC. FGF2 increased intracellular calcium in satellite cells, which was antagonized by the TRPC channel blocker SKF 96365. Immunostaining showed that NFATc3 is highly expressed in satellite cells, but not in host FDB fibers. Elevation of intracellular calcium by FGF2 is accompanied by nuclear translocation of NFATc3 and NFATc2 and by an increase in the number of MyoD positive cells per muscle fiber, both of which were attenuated by TRPC blocker SKF 96365. Our results suggest a novel pathway of satellite cell activation where FGF2 enhances calcium influx through a TRPC channel, and the increased cytosolic calcium leads to both NFATc3 and NFATc2 nuclear translocation and enhanced number of MyoD positive satellite cells per muscle fiber.

  8. Hydrogenated Amorphous Silicon Germanium Active Layer for Top Cell of a Multi Junction Cell Structure.

    PubMed

    Cho, Jaehyun; Iftiquar, S M; Kim, Minbum; Park, Jinjoo; Jung, Junhee; Kim, Jiwoong; Yi, Junsin

    2016-05-01

    Intrinsic hydrogenated amorphous silicon-germanium (a-SiGe:H) alloy is generally used in the bottom cell because of its low band gap. The a-SiGe:H has a higher photo conductivity in comparison to the a-Si:H; thus, it is expected that the a-SiGe:H can show better short circuit current density than that of the a-Si:H based solar cell. Therefore, we optimized a-SiGe:H active layer that can be a suitable choice for the front cell of a multi junction.solar cell. Furthermore, we carried out a comparative study of the solar cells that have a-SiGe:H and a-Si:H as respective active layers. The a-SiGe:H based solar cells show higher short circuit current density, while the a-Si:H based cells show higheropen circuit voltage. The current-voltage characteristics of these cells are as follows: (a) V(oc) = 770 mV, J(sc) = 15.0 mA/cm2, FF = 64.5%, and η = 7.47% for a-SiGe:H based cell; and (b) V(oc) = 826 mV, J(sc) = 13.63 mA/cm2, FF = 72.0%, and η = 8.1% for a-Si:H based cell.

  9. M1 muscarinic receptor activation mediates cell death in M1-HEK293 cells.

    PubMed

    Graham, E Scott; Woo, Kerhan K; Aalderink, Miranda; Fry, Sandie; Greenwood, Jeffrey M; Glass, Michelle; Dragunow, Mike

    2013-01-01

    HEK293 cells have been used extensively to generate stable cell lines to study G protein-coupled receptors, such as muscarinic acetylcholine receptors (mAChRs). The activation of M1 mAChRs in various cell types in vitro has been shown to be protective. To further investigate M1 mAChR-mediated cell survival, we generated stable HEK293 cell-lines expressing the human M1 mAChR. M1 mAChRs were efficiently expressed at the cell surface and efficiently internalised within 1 h by carbachol. Carbachol also induced early signalling cascades similar to previous reports. Thus, ectopically expressed M1 receptors behaved in a similar fashion to the native receptor over short time periods of analysis. However, substantial cell death was observed in HEK293-M1 cells within 24 h after carbachol application. Death was only observed in HEK cells expressing M1 receptors and fully blocked by M1 antagonists. M1 mAChR-stimulation mediated prolonged activation of the MEK-ERK pathway and resulted in prolonged induction of the transcription factor EGR-1 (>24 h). Blockade of ERK signalling with U0126 did not reduce M1 mAChR-mediated cell-death significantly but inhibited the acute induction of EGR-1. We investigated the time-course of cell death using time-lapse microscopy and xCELLigence technology. Both revealed the M1 mAChR cytotoxicity occurs within several hours of M1 activation. The xCELLigence assay also confirmed that the ERK pathway was not involved in cell-death. Interestingly, the MEK blocker did reduce carbachol-mediated cleaved caspase 3 expression in HEK293-M1 cells. The HEK293 cell line is a widely used pharmacological tool for studying G-protein coupled receptors, including mAChRs. Our results highlight the importance of investigating the longer term fate of these cells in short term signalling studies. Identifying how and why activation of the M1 mAChR signals apoptosis in these cells may lead to a better understanding of how mAChRs regulate cell-fate decisions.

  10. Cell-to-cell distances between tumor-infiltrating inflammatory cells have the potential to distinguish functionally active from suppressed inflammatory cells.

    PubMed

    Nagl, S; Haas, M; Lahmer, G; Büttner-Herold, M; Grabenbauer, G G; Fietkau, R; Distel, L V

    2016-05-01

    Beyond their mere presence, the distribution pattern of inflammatory cells is of special interest. Our hypothesis was that random distribution may be a clear indicator of being non-functional as a consequence of lack of interaction. Here, we have assessed the implication of cell-to-cell distances among inflammatory cells in anal squamous cell carcinoma and a possible association with survival data. Thirty-eight patients suffering from anal carcinoma were studied using tissue microarrays, double staining immunohistochemistry, whole slide scanning and image analysis software. Therapy consisted of concurrent radiochemotherapy. Numbers of stromal and intraepithelial tumor-infiltrating inflammatory cells (TIC) and the distances between cells were quantified. Double-staining of FoxP3(+) cells with either CD8(+), CD1a(+) or CD20(+) cells was performed. Measured cell-to-cell distances were compared to computer simulated cell-to-cell distances leading to the assumption of non-randomly distributed and therefore functional immune cells. Intraepithelial CD1a(+) and CD20(+) cells were randomly distributed and therefore regarded as non-functional. In contrary, stromal CD20(+) cells had a non-random distribution pattern. A non-random distance between CD20(+) and FoxP3(+) cells was associated with a clearly unfavorable outcome. Measured distances between FoxP3(+) cells were distinctly shorter than expected and indicate a functional active state of the regulatory T cells (Treg). Analysis of cell-to-cell distances between TIC has the potential to distinguish between suppressed non-functional and functionally active inflammatory cells. We conclude that in this tumor model most of the CD1a(+) cells are non-functional as are the intraepithelial CD20(+) cells, while stromal CD20(+) cells and FoxP3(+) cells are functional cells. PMID:27467940

  11. 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.

  12. 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.

  13. Aurora A kinase activity influences calcium signaling in kidney cells.

    PubMed

    Plotnikova, Olga V; Pugacheva, Elena N; Golemis, Erica A

    2011-06-13

    Most studies of Aurora A (AurA) describe it as a mitotic centrosomal kinase. However, we and others have recently identified AurA functions as diverse as control of ciliary resorption, cell differentiation, and cell polarity control in interphase cells. In these activities, AurA is transiently activated by noncanonical signals, including Ca(2+)-dependent calmodulin binding. These and other observations suggested that AurA might be involved in pathological conditions, such as polycystic kidney disease (PKD). In this paper, we show that AurA is abundant in normal kidney tissue but is also abnormally expressed and activated in cells lining PKD-associated renal cysts. PKD arises from mutations in the PKD1 or PKD2 genes, encoding polycystins 1 and 2 (PC1 and PC2). AurA binds, phosphorylates, and reduces the activity of PC2, a Ca(2+)-permeable nonselective cation channel and, thus, limits the amplitude of Ca(2+) release from the endoplasmic reticulum. These and other findings suggest AurA may be a relevant new biomarker or target in the therapy of PKD.

  14. Dextromethorphan Inhibits Activations and Functions in Dendritic Cells

    PubMed Central

    Chen, Der-Yuan; Song, Pei-Shan; Hong, Jau-Shyong; Chu, Ching-Liang; Pan, I-Horng; Chen, Yi-Ming; Lin, Ching-Hsiung; Lin, Sheng-Hao; Lin, Chi-Chen

    2013-01-01

    Dendritic cells (DCs) play an important role in connecting innate and adaptive immunity. Thus, DCs have been regarded as a major target for the development of immunomodulators. In this study, we examined the effect of dextromethorphan (DXM), a common cough suppressant with a high safety profile, on the activation and function of DCs. In the presence of DXM, the LPS-induced expression of the costimulatory molecules in murine bone marrow-derived dendritic cells (BMDCs) was significantly suppressed. In addition, DXM treatment reduced the production of reactive oxygen species (ROS), proinflammatory cytokines, and chemokines in maturing BMDCs that were activated by LPS. Therefore, DXM abrogated the ability of LPS-stimulated DCs to induce Ag-specific T-cell activation, as determined by their decreased proliferation and IFN-γ secretion in mixed leukocyte cultures. Moreover, the inhibition of LPS-induced MAPK activation and NF-κB translocation may contribute to the suppressive effect of DXM on BMDCs. Remarkably, DXM decreased the LPS-induced surface expression of CD80, CD83, and HLA-DR and the secretion of IL-6 and IL-12 in human monocyte-derived dendritic cells (MDDCs). These findings provide a new insight into the impact of DXM treatment on DCs and suggest that DXM has the potential to be used in treating DC-related acute and chronic diseases. PMID:23781253

  15. Hox proteins: sculpting body parts by activating localized cell death.

    PubMed

    Alonso, Claudio R

    2002-11-19

    Hox proteins shape animal structures by eliciting different developmental programs along the anteroposterior body axis. A recent study reveals that the Drosophila Hox protein Deformed directly activates the cell-death-promoting gene reaper to maintain the boundaries between distinct head segments.

  16. 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

  17. Dextromethorphan inhibits activations and functions in dendritic cells.

    PubMed

    Chen, Der-Yuan; Song, Pei-Shan; Hong, Jau-Shyong; Chu, Ching-Liang; Pan, I-Horng; Chen, Yi-Ming; Lin, Ching-Hsiung; Lin, Sheng-Hao; Lin, Chi-Chen

    2013-01-01

    Dendritic cells (DCs) play an important role in connecting innate and adaptive immunity. Thus, DCs have been regarded as a major target for the development of immunomodulators. In this study, we examined the effect of dextromethorphan (DXM), a common cough suppressant with a high safety profile, on the activation and function of DCs. In the presence of DXM, the LPS-induced expression of the costimulatory molecules in murine bone marrow-derived dendritic cells (BMDCs) was significantly suppressed. In addition, DXM treatment reduced the production of reactive oxygen species (ROS), proinflammatory cytokines, and chemokines in maturing BMDCs that were activated by LPS. Therefore, DXM abrogated the ability of LPS-stimulated DCs to induce Ag-specific T-cell activation, as determined by their decreased proliferation and IFN- γ secretion in mixed leukocyte cultures. Moreover, the inhibition of LPS-induced MAPK activation and NF- κ B translocation may contribute to the suppressive effect of DXM on BMDCs. Remarkably, DXM decreased the LPS-induced surface expression of CD80, CD83, and HLA-DR and the secretion of IL-6 and IL-12 in human monocyte-derived dendritic cells (MDDCs). These findings provide a new insight into the impact of DXM treatment on DCs and suggest that DXM has the potential to be used in treating DC-related acute and chronic diseases. PMID:23781253

  18. Blue light-activated hypocrellin B damages ovarian cancer cells

    NASA Astrophysics Data System (ADS)

    Jiang, Y.; Leung, A. W. N.; Xiang, J. Y.; Xu, C. S.

    2011-10-01

    In the present study, a novel blue light source from LED was used to activate hypocrellin B in ovarian cancer HO-8910 cells. Hyppcrellin B concentration was kept at 2.5 μM and light doses from 0.5-4.0 J/cm2. Photocytotoxicity was investigated using MTT reduction assay and light microscopy after light irradiation. Cellular morphology was observed using transmission electron microscopy (TEM). MTT assay showed that the cytotoxicity of blue light-activated hypocrellin B in HO-8910 cells increased along with light dose. The observations from light microscopy reinforced the above results. TEM showed that microvillin disappearance, vacuole formation, chromatin condensation, and topical apoptotic body were observed in the cells treated by both light and hypocrellin B. The findings demonstrated that blue light from LED source could effectively activate hypocrellin B to cause the destruction of HO-8910 cells, indicating that Blue light-activated hypocrellin B might be potential therapeutic strategy in the management of ovarian cancer.

  19. 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.

  20. Prodigiosin Induces Autolysins in Actively Grown Bacillus subtilis Cells.

    PubMed

    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.

  1. Enrichment of epidermal stem cells of rats by Vario magnetic activated cell sorting system.

    PubMed

    Chen, Wei; Zhang, Wei-wei; Shi, Chunying; Lian, Xiaohua; Yi, Shanghong; Yang, Tian

    2013-09-01

    Epidermal stem cells (ESCs) play an important role in skin homeostasis, wound repair, and tumorigensis which have great potential in scientific research and clinical application. So, the efficient isolation of these infrequent stem cells is very important for researchers to solve the problem of low purity and insufficient quantity of stem cells in vitro. The aim of this study was to investigate a method for the enrichment of ESCs by magnetic activated cell sorting system. The isolation strategy was CD71 depletion followed by α6-integrin positive selection. The percentage of α6(bri)CD71(dim) cells in isolated cells was 94.59%. Transmission electron microscopy results revealed that α6(bri) CD71(dim) cells exhibited some typical characteristics like progenitor cells, such as big nucleus, obvious nucleolus, large nuclear-cytoplasm ratio, and few organelles in cytoplasm. When cultured in vitro, the α6(bri)CD71(dim) cells had greater proliferating potential and higher colony-forming ability, and high levels of epidermal stem cell markers were expressed in our positive cells. ESCs have been successfully isolated from neonatal epidermis using Vario MACS and cultured in vitro. This isolation method is simple, fast, and inexpensive, providing an important tool for tissue engineering and cell transplantation studies.

  2. M2 receptor activation inhibits cell cycle progression and survival in human glioblastoma cells.

    PubMed

    Ferretti, Michela; Fabbiano, Cinzia; Di Bari, Maria; Conte, Claudia; Castigli, Emilia; Sciaccaluga, Miriam; Ponti, Donatella; Ruggieri, Paola; Raco, Antonino; Ricordy, Ruggero; Calogero, Antonella; Tata, Ada Maria

    2013-04-01

    Muscarinic receptors, expressed in several primary and metastatic tumours, appear to be implicated in their growth and propagation. In this work we have demonstrated that M2 muscarinic receptors are expressed in glioblastoma human specimens and in glioblastoma cell lines. Moreover, we have characterized the effects of the M2 agonist arecaidine on cell growth and survival both in two different glioblastoma cell lines (U251MG and U87MG) and in primary cultures obtained from different human biopsies. Cell growth analysis has demonstrated that the M2 agonist arecaidine strongly decreased cell proliferation in both glioma cell lines and primary cultures. This effect was dose and time dependent. FACS analysis has confirmed cell cycle arrest at G1/S and at G2/M phase in U87 cells and U251 respectively. Cell viability analysis has also shown that arecaidine induced severe apoptosis, especially in U251 cells. Chemosensitivity assays have, moreover, shown arecaidine and temozolomide similar effects on glioma cell lines, although IC50 value for arecaidine was significantly lower than temozolomide. In conclusion, we report for the first time that M2 receptor activation has a relevant role in the inhibition of glioma cell growth and survival, suggesting that M2 may be a new interesting therapeutic target to investigate for glioblastoma therapy.

  3. 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 cells towards BEC in human liver; thus they could be manipulated for treatment of biliary disease in the future. PMID:26743076

  4. 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

  5. Communication is key: Reducing DEK1 activity reveals a link between cell-cell contacts and epidermal cell differentiation status.

    PubMed

    Galletti, Roberta; Ingram, Gwyneth C

    2015-01-01

    Plant epidermis development requires not only the initial acquisition of tissue identity, but also the ability to differentiate specific cell types over time and to maintain these differentiated states throughout the plant life. To set-up and maintain differentiation, plants activate specific transcriptional programs. Interfering with these programs can prevent differentiation and/or force differentiated cells to lose their identity and re-enter a proliferative state. We have recently shown that the Arabidopsis Defective Kernel 1 (DEK1) protein is required both for the differentiation of epidermal cells and for the maintenance of their fully differentiated state. Defects in DEK1 activity lead to a deregulation of the expression of epidermis-specific differentiation-promoting HD-ZIP IV transcription factors. Here we propose a working model in which DEK1, by maintaining cell-cell contacts, and thus communication between neighboring cells, influences HD-ZIP IV gene expression and epidermis differentiation. PMID:27064205

  6. Diatom-Derived Polyunsaturated Aldehydes Activate Cell Death in Human Cancer Cell Lines but Not Normal Cells

    PubMed Central

    Sansone, Clementina; Braca, Alessandra; Ercolesi, Elena; Romano, Giovanna; Palumbo, Anna; Casotti, Raffaella; Francone, Maria; Ianora, Adrianna

    2014-01-01

    Diatoms are an important class of unicellular algae that produce bioactive polyunsaturated aldehydes (PUAs) that induce abortions or malformations in the offspring of invertebrates exposed to them during gestation. Here we compare the effects of the PUAs 2-trans,4-trans-decadienal (DD), 2-trans,4-trans-octadienal (OD) and 2-trans,4-trans-heptadienal (HD) on the adenocarcinoma cell lines lung A549 and colon COLO 205, and the normal lung/brunch epithelial BEAS-2B cell line. Using the viability MTT/Trypan blue assays, we show that PUAs have a toxic effect on both A549 and COLO 205 tumor cells but not BEAS-2B normal cells. DD was the strongest of the three PUAs tested, at all time-intervals considered, but HD was as strong as DD after 48 h. OD was the least active of the three PUAs. The effect of the three PUAs was somewhat stronger for A549 cells. We therefore studied the death signaling pathway activated in A549 showing that cells treated with DD activated Tumor Necrosis Factor Receptor 1 (TNFR1) and Fas Associated Death Domain (FADD) leading to necroptosis via caspase-3 without activating the survival pathway Receptor-Interacting Protein (RIP). The TNFR1/FADD/caspase pathway was also observed with OD, but only after 48 h. This was the only PUA that activated RIP, consistent with the finding that OD causes less damage to the cell compared to DD and HD. In contrast, cells treated with HD activated the Fas/FADD/caspase pathway. This is the first report that PUAs activate an extrinsic apoptotic machinery in contrast to other anticancer drugs that promote an intrinsic death pathway, without affecting the viability of normal cells from the same tissue type. These findings have interesting implications also from the ecological viewpoint considering that HD is one of the most common PUAs produced by diatoms. PMID:24992192

  7. Nonclassical MHC-restricted invariant Vα6 T cells are critical for efficient early innate anti-viral immunity in the amphibian X. laevis1

    PubMed Central

    Edholm, Eva-Stina; Grayfer, Leon; De Jesús, Francisco; Robert, Jacques

    2015-01-01

    Nonclassical MHC class Ib (class Ib)-restricted invariant T (iT) cell subsets are attracting interest because of their potential to regulate immune responses against various pathogens. The biological relevance and evolutionary conservation of iT cells has recently been strengthened by the identification of iT cells (iVα6) restricted by the class Ib molecule XNC10 in the amphibian Xenopus laevis. These iVα6 T cells are functionally similar to mammalian CD1d-restricted iNKT cells. Using the amphibian pathogen frog virus 3 (FV3) in combination with XNC10 tetramers and RNAi loss-of-function by transgenesis, we show that XNC10-restricted iVα6 T cells are critical for early antiviral immunity in adult X. laevis. Within hours following intraperitoneal FV3 infection, iVα6 T cells were specifically recruited from the spleen into the peritoneum. XNC10-deficiency and concomitant lack of iVα6 T cells resulted in less effective antiviral and macrophage antimicrobial responses, which lead to impaired viral clearance, increased viral dissemination and more pronounced FV3-induced kidney damage. Together, these findings imply that X. laevis XNC10-restricted iVα6 T cells play important roles in the early anti-FV3 response and that, as has been suggested for mammalian iNKT cells, they may serve as immune regulators polarizing macrophage effector functions towards more effective antiviral states. PMID:26062996

  8. T-Cell Tumor Elimination as a Result of T-Cell Receptor-Mediated Activation

    NASA Astrophysics Data System (ADS)

    Ashwell, Jonathan D.; Longo, Dan L.; Bridges, Sandra H.

    1987-07-01

    It has recently been shown that activation of murine T-cell hybridomas with antigen inhibits their growth in vitro. The ``suicide'' of these neoplastic T cells upon stimulation with antigen suggested the possibility that activation via the antigen-specific receptor could also inhibit the growth of neoplastic T cells in vivo. To test this, mice were subcutaneously inoculated with antigen-specific T-cell hybridomas and then treated intraperitoneally with antigen. Administration of the appropriate antigen immediately after inoculation with the T-cell hybridoma abrogated tumor formation; antigen administered after tumors had become established decreased the tumor burden and, in a substantial fraction of animals, led to long-term survival. The efficacy of antigen therapy was due to both a direct inhibitory effect on tumor growth and the induction of host immunity. These studies demonstrate the utility of cellular activation as a means of inhibiting neoplastic T-cell growth in vivo and provide a rationale for studying the use of less selective reagents that can mimic the activating properties of antigen, such as monoclonal antibodies, in the treatment of T-cell neoplasms of unknown a