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Sample records for activated transfected killer

  1. Cytokine-induced killer cells showing multidrug resistance and remaining cytotoxic activity to tumor cells after transfected with mdr1 cDNA

    Institute of Scientific and Technical Information of China (English)

    李惠芳; 杨永红; 石永进; 王逸群; 朱平

    2004-01-01

    Background Routine treatment of cancer such as surgery, radiation or chemotherapy is sometimes unable to erdiacate metastatic malignant cells. So we tried a new method and increased the adoptive immunotherapy of Cytokine-induced killer (CIK) cells in tumor patients and the multidrug resistance (mdr1) cDNA was transfected into CIK cells. Methods CIK cells were obtained from peripheral blood and induced by IFN-γ, anti-CD3 monoclonal antibody, IL-2 and IL-1. CIK cells were transfected with plasmid PHaMDR containing human mdr1 cDNA by electroporation. RT-PCR was used to detect mdr1 mRNA in transfected CIK cells. P-glycoprotein (P-gp) expressed on surface of CIK cells was assayed by FITC-conjugated anti-P-gp monoclonal antibody and flow cytometry. Multidrug resistance to doxorubicin and colchicine and cytotoxic activity to human breast cancer cell line MCF7 were performed using MTT method.Results mdr1 mRNA was detected in transfected CIK cells. P-gp was expressed on the surface of the transfected CIK cells, and the P-gp positive cells reached 21%-37% of the total CIK cells after transfection. The IC50 to doxorubicin increased to 22.3-45.8 times, and that to colchicines to 6.7-11.35 times, as compared to those of untransfected CIK cells. However, the cytotoxic activity to MCF7 cell line remained unaltered.Conclusions CIK cells were successfully transfected with mdr1 cDNA by using electroporation. The transfected CIK cells had the characteristics of multidrug resistance without change in their cytotoxic activity to tumor cells.

  2. Class I (H-2Kb) gene transfection reduces susceptibility of YAC-1 lymphoma targets to natural killer cells.

    Science.gov (United States)

    Carlow, D A; Payne, U; Hozumi, N; Roder, J C; Czitrom, A A

    1990-04-01

    A "hybrid gene" (MTKb) comprised of the human metallothionein IIA promoter ligated to the genomic sequence of the major histocompatibility complex class I (H-2Kb) gene was subcloned into the expression vector pSV2neo and transfected into the natural killer (NK) cell-sensitive YAC-1 lymphoma. The Kb gene product was readily detectable on the cell surface of G418-resistant transfectants using both Kb-specific monoclonal antibodies and H-2b-specific cytolytic T cells. Unlike control pSV2neo transfectants, MTKb-pSV2neo transfectants were relatively resistant to lysis by NK cells from H-2a, H-2b, H-2k or H-2 (a x b)F1 haplotype mice. These data strongly suggest that the effects of MHC expression on susceptibility to NK cells can be mediated by a single and well-defined class I molecule, Kb.

  3. Viral Evasion of Natural Killer Cell Activation

    OpenAIRE

    Yi Ma; Xiaojuan Li; Ersheng Kuang

    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...

  4. Depressed natural killer cell activity in acute myocardial infarction

    DEFF Research Database (Denmark)

    Klarlund, K; Pedersen, B K; Theander, T G

    1987-01-01

    Natural killer (NK) cell activity against K562 target cells was measured in patients within 24 h of acute myocardial infarction (AMI) and regularly thereafter for 6 weeks. NK cell activity was suppressed on days 1, 3, and 7 (P less than 0.01), day 14 (P less than 0.05) and at 6 weeks (P = 0...

  5. Natural killer cell activity during premedication, anaesthesia and surgery

    DEFF Research Database (Denmark)

    Tønnesen, E; Mickley, H; Grunnet, N

    1983-01-01

    Natural killer (NK) cell activity of peripheral blood mononuclear cells was measured against K-562 target cells in a 51Cr release assay in eight patients undergoing total hip replacement surgery. Eight consecutive blood samples were taken from each patient. A significant increase of NK cell...... activity was observed after premedication with diazepam per os. The activity increased further during a combined anaesthesia (thiopentone + N2O + O2 + buprenorphene + pancuronium) and remained increased during surgery. Postoperatively, NK cell activity fell and remained depressed for a period of at least 5...... days. The findings of this study indicate that premedication, anaesthesia and surgery cause a rapid and transient increase in NK cell activity, followed by a decline in activity postoperatively. The transient increase in activity may be explained by mobilization of natural killer cells from extravasal...

  6. Human Immunodeficiency Syndromes Affecting Human Natural Killer Cell Cytolytic Activity

    OpenAIRE

    Ham, Hyoungjun; Billadeau, Daniel D.

    2014-01-01

    Natural killer (NK) cells are lymphocytes of the innate immune system that secrete cytokines upon activation and mediate the killing of tumor cells and virus-infected cells, especially those that escape the adaptive T cell response caused by the down regulation of MHC-I. The induction of cytotoxicity requires that NK cells contact target cells through adhesion receptors, and initiate activation signaling leading to increased adhesion and accumulation of F-actin at the NK cell cytotoxic synaps...

  7. Neuromodulation of Natural Killer Cell Activity

    Science.gov (United States)

    1989-01-01

    between the pineal gland As we have seen, NK cell function is ex- and the mitotic activity of some tissues. Arch Sci Biol tremely sensitive to many...34 New York: Alan R. Liss. Inc,. pp 151 - plasis. BriJ Med psychol 43:313-331. 160. Das Gupta TIC. Terz J (1967): Infuence of pineal gland Hochman PS...1968; Baron and D Gupta, 1970). responsiveness in patients with cerebral tu- Chemical sympathectomy renders rats highly mors (Brooks et al., 1972

  8. Lactobacilli Differentially Activate Natural Killer Cells

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen; Christensen, Hanne Risager; Frøkiær, Hanne

    Bacteria translocating across the gastrointestinal mucosa are presumed to gain access to NK cell compartments, as consumption of certain lactic acid bacteria has been shown to increase in vivo NK cytotoxicity. On-going research in our lab aims at describing strain-dependent effects of lactic acid...... determined by ELISA. Co-incubation of NK cells and a Lactobacillus acidophilus strain caused increased proliferation of the NK cells and induced IFN-gamma production. The proliferative response was further enhanced in the presence of autologous monocytes, probably because cytokines, secreted by monocytes...... having engulfed bacteria, stimulated the growth of the NK cells. In contrast, a Lactobacillus paracasei strain caused the NK cells to proliferate only in the presence of monocytes. These results demonstrate that various lactobacilli have the capacity to activate NK cells in vitro, in a monocyte dependent...

  9. Activation of Natural Killer cells during microbial infections

    Directory of Open Access Journals (Sweden)

    Amir eHorowitz

    2012-01-01

    Full Text Available Natural killer (NK cells are large granular lymphocytes that express a diverse array of germline encoded inhibitory and activating receptors for MHC Class I and Class I-like molecules, classical co-stimulatory ligands and cytokines. The ability of NK cells to be very rapidly activated by inflammatory cytokines, to secrete effector cytokines and to kill infected or stressed host cells, suggests that they may be among the very early responders during infection. Recent studies have also identified a small number of pathogen-derived ligands that can bind to NK cell surface receptors and directly induce their activation. Here we review recent studies that have begun to elucidate the various pathways by which viral, bacterial and parasite pathogens activate NK cells. We also consider two emerging themes of NK cell-pathogen interactions, namely their contribution to adaptive immune responses and their potential to take on regulatory and immunomodulatory functions.

  10. Lethal effects of /sup 32/P decay on transfecting activity of Bacillus subtillis phage phie DNA

    Energy Technology Data Exchange (ETDEWEB)

    Loveday, K.S.

    1979-07-15

    Disintegration of /sup 32/P present in the DNA of Bacillus subtilis phage phie (a phage containing double-strand DNA) results in the loss of viability of intact phage as well as transfecting activity of isolated DNA. Only 1/12 of the /sup 32/P disintegrations per phage DNA equivalent inactivities the intact phage while nearly every disintegration inactivates the transfecting DNA. This result provides evidence for a single-strand intermediate in the transfection of B. subtilis by phie DNA.

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

    Science.gov (United States)

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

    2009-08-15

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

  12. Killing of naive T cells by CD95L-transfected dendritic cells (DC): in vivo study using killer DC-DC hybrids and CD4(+) T cells from DO11.10 mice.

    Science.gov (United States)

    Kusuhara, Masahiro; Matsue, Keiko; Edelbaum, Dale; Loftus, Julie; Takashima, Akira; Matsue, Hiroyuki

    2002-04-01

    Dendritic cells (DC) play the dual task of initiating cellular immunity against potentially harmful foreign antigens (Ag), while maintaining immunological tolerance to self-Ag and environmental Ag. As an approach to induce Ag-specific suppression, we and others introduced CD95 ligand (L) cDNA into DC. The resulting "killer" DC delivered apoptotic signals, instead of activation signals, to primed CD4(+) T cells in vitro and induced Ag-specific immunosuppression in vivo. To study the impact of killer DC on naive T cells, the fate of Ag-reactive T cells and the extent of their depletion after killer DC treatment, we performed in vitro and in vivo reconstitution experiments using: (a) killer DC-DC hybrids created between CD95L-transduced XS106 DC clone (A/J origin) and splenic DC from BALB/c mice, (b) CD4(+) T cells isolated from DO11.10 transgenic mice (BALB/c background), and (c) OVA(323-339) peptide as relevant Ag. Ovalbumin (OVA)-pulsed killer DC-DC hybrids inhibited DO11.10 T cell activation triggered by conventional DC, instead of inducing their activation. Rapid apoptosis of T cells was observed after co-culture with OVA-pulsed killer DC-DC hybrids, but not with non-pulsed killer DC-DC hybrids or OVA-pulsed control DC-DC hybrids. For in vivo reconstitution, (BALB/cxA/J)F1 mice received subcutaneous administration of killer DC-DC hybrids, followed by intravenous inoculation of DO11.10 T cells. Killer DC-DC hybrids migrated preferentially to draining lymph nodes albeit with relatively low efficiency (0.5-1% recovery) and they induced significant, but incomplete (30-40%) killing of DO11.10 T cells in this location. These results document the abilities of CD95L-transduced DC to trigger apoptosis of naive T cells in an Ag-specific manner, to overrule T cell activation signals delivered by conventional DC, and to reduce local frequencies of Ag-reactive T cells in vivo. Our data also uncover two major limitations (relatively low homing efficiency and incomplete

  13. Emotional stability, anxiety, and natural killer activity under examination stress.

    Science.gov (United States)

    Borella, P; Bargellini, A; Rovesti, S; Pinelli, M; Vivoli, R; Solfrini, V; Vivoli, G

    1999-08-01

    This study was performed to evaluate the relation between a stable personality trait, a mood state and immune response to an examination stress. A self-reported measure of emotional stability (BFQ-ES scale) was obtained in a sample (n = 39) randomly selected from 277 cadets; this personality trait was also investigated by completing a neuroticism scale (Eysenck personality inventory) and a trait-anxiety scale (STAI). Natural killer (NK) cell activity was measured at baseline, long before the examination time and the examination day. The state-anxiety scale evaluated the response to the stressful stimulus. Taking subjects all together, the academic task did not result in significant modification over baseline in NK cell activity. Subjects were then divided into three groups based on emotional stability and state-anxiety scores: high emotional stability/low anxiety, medium, and low emotional stability/high anxiety. Examination stress induced significant increases in NK cell activity in the high emotional stability/low anxiety group, no effect in the medium group, and significant decreases in the low emotional stability/high anxiety group. The repeated-measure ANOVA revealed a significant interaction of group x period (baseline vs. examination) for both lytic units and percent cytolysis. The results did not change after introducing coffee and smoking habits as covariates. Our findings suggest that the state-anxiety acts in concert with a stable personality trait to modulate NK response in healthy subjects exposed to a psychological naturalistic stress. The relation between anxiety and poor immune control has been already described, whereas the ability of emotional stability to associate with an immunoenhancement has not yet reported. The peculiarity of our population, a very homogeneous and healthy group for life style and habits, can have highlighted the role of emotional stability, and may account for the difference with other studies.

  14. Acquisition of enhanced natural killer cell activity under anesthesia.

    Science.gov (United States)

    Hsueh, C M; Lorden, J F; Hiramoto, R N; Ghanta, V K

    1992-01-01

    An increase in natural killer (NK) cell activity can be conditioned with a one trial learning paradigm to demonstrate the interaction between the central nervous system (CNS) and the immune system. In order to demonstrate learning possibilities during 'non-conscious' state, mice were anesthetized with a ketamin/rompun mixture and underwent one trial learning with odor cue as the conditioned stimulus (CS) preceding the unconditioned stimulus (US). The results indicated that mice that were exposed to camphor odor cue under the influence of anesthesia can associate the signal with the poly I:C unconditioned stimulus and were able to recall the conditioned response upon reexposure to the CS. Secondly, the conditioned association made in a conscious state can be recalled by exposure to the same olfactory odor cue in a 'non-conscious' state. The increase in the conditioned change in NK cell activity of both situations was significantly higher than the control group. The results demonstrate that learning can take place and the learned response can be recalled under the reduced awareness caused by anesthesia. The findings we report are unusual and novel in that they demonstrate that the CNS can learn new associations under conditions where the host is apparently unaware of the signals being linked. Anesthesia combined with the long interstimulus interval indicates that certain neuronal pathways in the CNS are receptive to second signals (elicited by the US) even when the second signal is separated by one day. This means the conditioned learning of a physiological response can take place unconsciously at a separate level and under situations where the host is totally unaware of the events which the brain is processing and linking as incoming information.

  15. Design and operation specifications of an active monitoring system for detecting southern resident killer whales

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Zhiqun; Carlson, Thomas J.; Xu, Jinshan; Martinez, Jayson J.; Weiland, Mark A.; Mueller, Robert P.; Myers, Joshua R.; Jones, Mark E.

    2011-09-30

    Before final approval is given to the Snohomish County Public Utility District No. 1 for deploying the first tidal power devices in the United States in an open water environment, a system to manage the potential risk of injury to killer whales due to collision with moving turbine blades must be demonstrated. The Pacific Northwest National Laboratory (PNNL) is tasked with establishing the performance requirements for, constructing, and testing a prototype marine animal alert system for triggering temporary turbine shutdown when there is risk of collision with a killer whale. To develop a system that relies on active sonar two critical areas must be investigated - the target strength of killer whales and the frequency content of commercially available active sonar units. PNNL studied three target strength models: a simple model, the Fourier matching model, and the Kirchoff-ray mode model. Using target strength measurements of bottlenose dolphins obtained by previous researchers and assuming killer whales share similar morphology and structure, PNNL extrapolated the target strength of an adult killer whale 7.5 m in length at a frequency of 67 kHz. To study the frequency content of a commercially available sonar unit, direct measurements of the signal transmitted by the sonar were obtained by using a hydrophone connected to a data acquisition system in both laboratory and field conditions. The measurements revealed that in addition to the primary frequency of 200 kHz, there is a secondary frequency component at 90 kHz, which is within the hearing range of killer whales. The amplitude of the 90-kHz frequency component is above the hearing threshold of killer whales but below the threshold for potential injuries.

  16. Purification of Candida guilliermondii and Pichia ohmeri killer toxin as an active agent against Penicillium expansum.

    Science.gov (United States)

    Coelho, Alexandre Rodrigo; Tachi, Masahico; Pagnocca, Fernando Carlos; Nobrega, Gisele Maria Andrade; Hoffmann, Fernando Leite; Harada, Ken-Ichi; Hirooka, Elisa Yoko

    2009-01-01

    An antifungal assay with cell-free culture supernatant of Pichia ohmeri 158 and Candida guilliermondii P3 was tested against Penicillium expansum strain #2 at 25 degrees C by measuring hyphal length and percentage conidia germination. C. guilliermondii was more effective against P. expansum conidia germination (58.15% inhibition), while P. ohmeri showed higher inhibition of mycelial growth (66.17%), indicating a probable mechanism associated with killer activity. This killer toxin (molecular mass expansum:% inhibition rose from 42.16 to 90.93% (C. guilliermondii) and 39.32 to 91.12% (P. ohmeri) (p Penicillium activity.

  17. Immune functions in beluga whales (Delphinapterus leucas): Evaluation of natural killer cell activity.

    NARCIS (Netherlands)

    S. De Guise (Sylvain); P.S. Ross (Peter); A.D.M.E. Osterhaus (Albert); D. Martineau (Daniel); P. Beland; M. Fournier (Michel)

    1997-01-01

    textabstractNatural killer (NK) activity, an important non-specific defense mechanism against viral infections and tumors, was demonstrated in beluga whales using two different methods: 51Cr release and flow cytometry. Using the 51Cr release assay, NK activity in belugas was shown to be higher again

  18. Killer toxin of Saccharomyces cerevisiae Y500-4L active against Fleischmann and Itaiquara commercial brands of yeast

    Directory of Open Access Journals (Sweden)

    Soares Giselle A.M.

    1999-01-01

    Full Text Available The strain Saccharomyces cerevisiae Y500-4L, previously selected from the must of alcohol producing plants and showing high fermentative and killer capacities, was characterized according to the interactions between the yeasts and examined for curing and detection of dsRNA plasmids, which code for the killer character. The killer yeast S. cerevisiae Y500-4L showed considerable killer activity against the Fleischmann and Itaiquara commercial brands of yeast and also against the standard killer yeasts K2 (S. diastaticus NCYC 713, K4 (Candida glabrata NCYC 388 and K11 (Torulopsis glabrata ATCC 15126. However S. cerevisiae Y500-4L showed sensitivity to the killer toxin produced by the standard killer yeasts K8 (Hansenula anomala NCYC 435, K9 (Hansenula mrakii NCYC 500, K10 (Kluyveromyces drosophilarum NCYC 575 and K11 (Torulopsis glabrata ATCC 15126. No M-dsRNA plasmid was detected in the S. cerevisiae Y500-4L strain and these results suggest that the genetic basis for toxin production is encoded by chromosomal DNA. The strain S. cerevisiae Y500-4L was more resistant to the loss of the phenotype killer with cycloheximide and incubation at elevated temperatures (40oC than the standard killer yeast S. cerevisiae K1.

  19. Killer whale presence in relation to naval sonar activity and prey abundance in northern Norway

    NARCIS (Netherlands)

    Kuningas, S.; Kvadsheim, P.H.; Lam, F.P.A.; Miller, P.J.O.

    2013-01-01

    In this study, retrospective data on naval sonar activity and prey abundance were correlated with killer whale sightings within a fjord basin in northern Norway. In addition, passive acoustic and visual marine mammal surveys were conducted before, during, and after a specific navy exercise in 2006.

  20. Assessment of human natural killer and lymphokine-activated killer cell cytotoxicity against Toxoplasma gondii trophozoites and brain cysts

    Energy Technology Data Exchange (ETDEWEB)

    Dannemann, B.R.; Morris, V.A.; Araujo, F.G.; Remington, J.S. (Palo Alto Medical Foundation, CA (USA))

    1989-10-15

    Because previous work has suggested that NK cells may be important in host resistance against the intracellular parasite Toxoplasma gondii we examined whether human NK cells and lymphokine-activated killer (LAK) cells have activity against trophozoites and cysts of this organism in vitro. A method to radiolabel Toxoplasma trophozoites with 51Cr was developed and direct cytotoxic activity was determined by using modifications of the standard 51Cr release assay. Viability of 51Cr-labeled trophozoites assessed by both methylene blue staining and trypan blue exclusion was greater than 90%. Significantly more 51Cr was released by anti-Toxoplasma antibody and C than by antibody in the absence of C. Incubation of trophozoites with freshly isolated human NK cells or NK cells activated with either rIL-2 or rIFN-alpha did not result in significant release of 51Cr (specific lysis was 0 to 2.3%). In contrast, the average specific lysis of radiolabeled trophozoites by LAK cells was significant. In a series of separate experiments, preincubation of radiolabeled trophozoites with heat-inactivated normal or Toxoplasma antibody-positive human serum increased the cytotoxicity of LAK cells from a mean specific lysis of 15% +/- 4.5 to 39% +/- 8.5, respectively, as assessed by 51Cr release. Because previous work has shown that radioisotope release from parasites may be nonspecific, separate experiments were performed to determine the cytotoxicity of LAK cells against antibody-coated trophozoites by using ethidium bromide-acridine orange staining to assess effector cell damage. LAK cells had a mean specific lysis of 51% against antibody-coated trophozoites by ethidium bromide-acridine orange staining. Preincubation with heat-inactivated Toxoplasma-antibody positive human serum did not increase activity of rIL-2-activated NK cells against 51CR-labeled trophozoites.

  1. Immune functions in beluga whales (Delphinapterus leucas): Evaluation of natural killer cell activity.

    OpenAIRE

    Guise, Sylvain De; Ross, Peter; Osterhaus, Albert; Martineau, Daniel; Beland, P; Fournier, Michel

    1997-01-01

    textabstractNatural killer (NK) activity, an important non-specific defense mechanism against viral infections and tumors, was demonstrated in beluga whales using two different methods: 51Cr release and flow cytometry. Using the 51Cr release assay, NK activity in belugas was shown to be higher against K-562 than against YAC-1 cell lines. Moreover, it was enhanced by the addition of human recombinant interleukin-2 with both cell lines. NK activity evaluated by flow cytometry in the peripheral ...

  2. Induction of lymphokine-activated killer activity in rat splenocyte cultures: The importance of 2-mercaptoethanol and indomethacin

    NARCIS (Netherlands)

    P.J.K. Kuppen (P. J K); A.M.M. Eggermont (Alexander); A.W. Marinelli (Andreas); E. de Heer (Emile); C.J.H. van de Velde (Cornelis); G.J. Fleuren (G.)

    1991-01-01

    textabstractThe role of 2-mercaptoethanol and indomethacin in the induction of lymphokine-activated killer (LAK) activity by interleukin-2 (IL-2) in rat splenocyte cultures was investigated. Spleens from 4-month-old male rats of five different strains were tested. Splenocytes were cultured for 3-5 d

  3. Anger expression and natural killer cell activity in family caregivers participating in a physical activity trial.

    Science.gov (United States)

    Wilcox, S; King, A C; Vitaliano, P P; Brassington, G S

    2000-07-01

    Associations between psychological functioning and natural killer cell activity (NKA) were examined in 23 older (62.2 ± 7.5 years) family caregivers randomized to a moderate intensity four-month exercise program or to a wait-list control condition. At baseline, although NKA was related to anger-control (r = -.42; trend p caregiver burden. After controlling for baseline NKA, changes in anger-control explained 14 percent of the variance in NKA four months later. Decreases in anger-control predicted increases in NKA. Group assignment (exercise vs control) was unrelated to changes in NKA over the four-month period; however, the study was not powered to detect this effect. These results are consistent with reported relationships of anger expression with other physiological measures, and extend the importance of anger expression to immune functioning in older family caregivers.

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

    Science.gov (United States)

    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.

  5. Enhancing magnetic nanoparticle-based DNA transfection: Intracellular-active cassette features

    Science.gov (United States)

    Vernon, Matthew Martin

    Efficient plasmid DNA transfection of embryonic stem cells, mesenchymal stem cells, neural cell lines and the majority of primary cell lines is a current challenge in gene therapy research. Magnetic nanoparticle-based DNA transfection is a gene vectoring technique that is promising because it is capable of outperforming most other non-viral transfection methods in terms of both transfection efficiency and cell viability. The nature of the DNA vector implemented depends on the target cell phenotype, where the particle surface chemistry and DNA binding/unbinding kinetics of the DNA carrier molecule play a critical role in the many steps required for successful gene transfection. Accordingly, Neuromag, an iron oxide/polymer nanoparticle optimized for transfection of neural phenotypes, outperforms many other nanoparticles and lipidbased DNA carriers. Up to now, improvements to nanomagnetic transfection techniques have focused mostly on particle functionalization and transfection parameter optimization (cell confluence, growth media, serum starvation, magnet oscillation parameters, etc.). None of these parameters are capable of assisting the nuclear translocation of delivered plasmid DNA once the particle-DNA complex is released from the endosome and dissociates in the cell's cytoplasm. In this study, incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid DNA confers improved nuclear translocation, demonstrating significant improvement in nanomagnetic transfection efficiency in differentiated SH-SY5Y neuroblastoma cells. Other parameters, such as days in vitro, are also found to play a role and represent potential targets for further optimization.

  6. Effect of ranitidine on postoperative suppression of natural killer cell activity and delayed hypersensitivity

    DEFF Research Database (Denmark)

    Nielsen, Hans Jørgen; Pedersen, B K; Moesgaard, F;

    1989-01-01

    hypersensitivity (DTH) antigens, and blood drawn immediately before and 24 hours after skin incision was analyzed for spontaneous and in vitro stimulated (IL-2, IFN-alpha or indomethacin) natural killer (NK) cell activity and PHA and PPD-stimulated lymphocyte proliferation. Lymphocyte subsets (helper......-cell activity (p less than 0.02). Postoperative decrease in helper/inducer-T cell numbers was not significantly lessened (p = 0.07), and ranitidine did not influence the levels of suppressor-T cells. PHA and PPD responses in peripheral blood mononuclear cells were unaltered. The results may suggest potential...

  7. Serial killers: ordering caspase activation events in apoptosis.

    Science.gov (United States)

    Slee, E A; Adrain, C; Martin, S J

    1999-11-01

    Caspases participate in the molecular control of apoptosis in several guises; as triggers of the death machinery, as regulatory elements within it, and ultimately as a subset of the effector elements of the machinery itself. The mammalian caspase family is steadily growing and currently contains 14 members. At present, it is unclear whether all of these proteases participate in apoptosis. Thus, current research in this area is focused upon establishing the repertoire and order of caspase activation events that occur during the signalling and demolition phases of cell death. Evidence is accumulating to suggest that proximal caspase activation events are typically initiated by molecules that promote caspase aggregation. As expected, distal caspase activation events are likely to be controlled by caspases activated earlier in the cascade. However, recent data has cast doubt upon the functional demarcation of caspases into signalling (upstream) and effector (downstream) roles based upon their prodomain lengths. In particular, caspase-3 may perform an important role in propagating the caspase cascade, in addition to its role as an effector caspase within the death programme. Here, we discuss the apoptosis-associated caspase cascade and the hierarchy of caspase activation events within it.

  8. Lactic Acid Bacteria Differentially Activate Natural Killer Cells

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen; Christensen, Hanne Risager; Frøkiær, Hanne

    . In contrast, a Lactobacillus paracasei strain caused the NK cells to proliferate only in the presence of monocytes. Conclusion: In this study we have demonstrated that various strains of gut flora-derived lactic acid bacteria have the capacity to activate NK cells in vitro, in a monocyte dependent...... antigen presenting cells and T-cells. Bacteria translocating across the gastrointestinal mucosa are presumed to gain access to NK cell compartments, as consumption of certain strains of lactic acid bacteria has been shown to increase in vivo NK cytotoxic activity. On-going research in our lab aims...... at describing strain-dependent effects of lactic acid bacteria on regulatory functions of NK-cells. Here, we have investigated how human gut flora-derived non-pathogenic lactic acid bacteria affect NK cells in vitro, by measuring proliferation and IFN-gamma production of human peripheral blood NK cells upon...

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

    Science.gov (United States)

    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.

  10. Decreased peripheral natural killer cells activity in the immune activated stage of chronic hepatitis B.

    Directory of Open Access Journals (Sweden)

    Yuan Li

    Full Text Available BACKGROUND & AIMS: The natural course of chronic hepatitis B virus (HBV infection is characterized by different immune responses, ranging from immune tolerant (IT to immune activated (IA stages. In our study, we investigated the natural killer (NK cells activity in patients at different immunological stages of chronic HBV infection. METHODS: Blood samples obtained from 57 HBeAg positive patients with chronic hepatitis B (CHB, including 15 patients in the immune tolerant (IT stage, 42 patients in the immune activated (IA stage, and 18 healthy individuals (HI. The analyses included flow cytometry to detect NK cells, the determination of cytokine levels as well as of surface receptor expression and cytotoxicity. RESULTS: NK cells in peripheral blood were significantly lower in patients in the IA stage of CHB compared to HI (p<0.05. Patients in the IA stage of CHB had lower levels of NK cells activating receptor NKp30 and NKG2D expression, cytokine interferon-γ (IFN-γ and tumor necrosis factor-α (TNF-α production, as compared to patients in the IT stage and HI, respectively (p<0.05. Cytotoxicity of NK cells was lower in patients in the IA stage of CHB compared to patients in the IT stage and HI, respectively (p<0.05. The level of IFN-γ but not level of TNF-α and cytotoxicity of NK cells was inversely correlated with serum HBV load in patients with CHB. Peripheral NK cells activity did not correlate with ALT level. CONCLUSION: NK cells activity was lower in CHB patients, especially in those in the IA stage.

  11. Tissue distribution of adoptively transferred adherent lymphokine-activated killer cells assessed by different cell labels

    DEFF Research Database (Denmark)

    Basse, P; Herberman, R B; Hokland, M;

    1992-01-01

    Assessment of the tissue distribution of adoptively transferred adherent lymphokine-activated killer A-LAK) cells by use of 51Cr indicated that these effector cells, after an initial phase in the lungs, distributed in high numbers to liver and spleen (30% and 10% of injected dose, respectively......). However, when this experiment was repeated with 125IdUrd as cell label, fewer than 2% and 0.5% of the injected cells distributed into liver and spleen respectively. To analyse this discrepancy, we compared the tissue distribution of 51Cr- and 125IdUrd-labelled A-LAK cells with that indicated...

  12. Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods

    DEFF Research Database (Denmark)

    Llovera Nadal, Laia; Berthold, Peter; Nielsen, Peter E

    2012-01-01

    peptides as complexing agents and carriers of the nucleic acids. However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters. Considering that cationic transfection complexes bind to and thus may up-concentrate on the cell surface, we...

  13. Activation of killer cells with soluble gastric cancer antigen combined with anti-CD3 McAb

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    @@ INTRODUCTION There have been many reports on cancer therapy with lymphokine-activated killer (LAK) cells and interleukin-2 (IL-2), but the proliferative response and anti-cancer effect of LAK cells are dependent on IL-2 dose. Other methods to improve the anti-tumor activity of cytotoxic T cells by activation with anti-CD3 McAb in conjunction with IL-2 are being investigated in recent years. In this study, we attempted to explore the physiologic and biologic effects of T-killer cells (TAK) co-stimulated with soluble gastric cancer antigen, anti-CD3 McAb and IL-2.

  14. Structure-activity correlation in transfection promoted by pyridinium cationic lipids.

    Science.gov (United States)

    Parvizi-Bahktar, P; Mendez-Campos, J; Raju, L; Khalique, N A; Jubeli, E; Larsen, H; Nicholson, D; Pungente, M D; Fyles, T M

    2016-03-21

    The efficiency of the transfection of a plasmid DNA encoding a galactosidase promoted by a series of pyridinium lipids in mixtures with other cationic lipids and neutral lipids was assessed in CHO-K1 cells. We identify key molecular parameters of the lipids in the mixture - clog P, lipid length, partial molar volume - to predict the morphology of the lipid-DNA lipoplex and then correlate these same parameters with transfection efficiency in an in vitro assay. We define a Transfection Index that provides a linear correlation with normalized transfection efficiency over a series of 90 different lipoplex compositions. We also explore the influence of the same set of molecular parameters on the cytotoxicity of the formulations.

  15. Large-scale isolation and cytotoxicity of extracellular vesicles derived from activated human natural killer cells

    Science.gov (United States)

    Jong, Ambrose Y.; Wu, Chun-Hua; Li, Jingbo; Sun, Jianping; Fabbri, Muller; Wayne, Alan S.; Seeger, Robert C.

    2017-01-01

    ABSTRACT Extracellular vesicles (EVs) have been the focus of great interest, as they appear to be involved in numerous important cellular processes. They deliver bioactive macromolecules such as proteins, lipids, and nucleic acids, allowing intercellular communication in multicellular organisms. EVs are secreted by all cell types, including immune cells such as natural killer cells (NK), and they may play important roles in the immune system. Currently, a large-scale procedure to obtain functional NK EVs is lacking, limiting their use clinically. In this report, we present a simple, robust, and cost-effective method to isolate a large quantity of NK EVs. After propagating and activating NK cells ex vivo and then incubating them in exosome-free medium for 48 h, EVs were isolated using a polymer precipitation method. The isolated vesicles contain the tetraspanin CD63, an EV marker, and associated proteins (fibronectin), but are devoid of cytochrome C, a cytoplasmic marker. Nanoparticle tracking analysis showed a size distribution between 100 and 200 nm while transmission electron microscopy imaging displayed vesicles with an oval shape and comparable sizes, fulfilling the definition of EV. Importantly, isolated EV fractions were cytotoxic against cancer cells. Furthermore, our results demonstrate for the first time that isolated activated NK (aNK) cell EVs contain the cytotoxic proteins perforin, granulysin, and granzymes A and B, incorporated from the aNK cells. Activation of caspase -3, -7 and -9 was detected in cancer cells incubated with aNK EVs, and caspase inhibitors blocked aNK EV-induced cytotoxicity, suggesting that aNK EVs activate caspase pathways in target cells. The ability to isolate functional aNK EVs on a large scale may lead to new clinical applications. Abbreviations: NK: natural killer cells; activated NK (aNK) cells; EVs: extracellular vesicles; ALL: acute lymphoblastic leukaemia; aAPC: artificial antigen-presenting cell; TEM: transmission

  16. Marijuana effects on immunity: suppression of human natural killer cell activity of delta-9-tetrahydrocannabinol.

    Science.gov (United States)

    Specter, S C; Klein, T W; Newton, C; Mondragon, M; Widen, R; Friedman, H

    1986-01-01

    Delta-9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, was tested for its ability to modulate human natural killer (NK) cell function. THC was toxic for peripheral blood lymphocytes at 20 micrograms/ml but not at 10 micrograms/ml or less. This component of marijuana also was inhibitory for NK activity against K562, a human tumor cell line at concentrations down to 5 micrograms/ml when pre-incubated with the effector cells. Suppression of NK function was dependent upon the concentration of THC and the length of time of pre-incubation but was independent of the ratio of effector to target cells. Prostaglandins were not involved in suppression of NK activity.

  17. ERK1/2 contributes negative regulation to STAT3 activity in HSS-transfected HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    Signal transducer and activator of transcription 3 (STAT3) is a recently characterized transcription factor which is essential to liver regeneration. We have previously reported that hepatic stimulator substance (HSS), a novel growthpromoting substance, phosphorylated the epidermal growth factor (EGF) receptors and activated downstream RasMAP kinase (extracellular signal-regulated kinases, ERK1/2) cascade. However, whether HSS signal is related to STAT3pathway remains unclear. The present study is aiming to explore the regulatory effect of activation of ERK1/2 evoked by HSS on STAT3 phosphorylation and STAT3 signaling. Human hepatoma cell line HepG2 was stably transfected with HSS cDNA and HSS expression was measured by Northern blot. The results showed that the transfection of HSS into HepG2 resulted in remarkable increase in cellular proliferation as compared with the non-transfected cells, and it was further proved that the cellular proliferation in the HSS-transfected cells was related to ERK1/2 activation. Treatment of the cells with 50 μM of PD98059, an ERK1/2 specific upstream inhibitor, resulted in ERK1/2 inactivation completely.Inhibition of ERK1/2 allowed the tyrosine of STAT3 to be phosphorylated in a dose-dependent manner to PD98059.Furthermore, transient transfection of STAT3 mutant (STAT3S727A) into HSS-bearing cells could remarkably reverse the inhibitory effect of ERK1/2 on STAT3 phosphorylation. Based upon these results, it is concluded that ERK1/2negatively modulates STAT3 phosphorylation and this function is dependent on residual serine-727 (S727) of STAT3.

  18. Control of immune ligands by members of a cytomegalovirus gene expansion suppresses natural killer cell activation

    Science.gov (United States)

    Fielding, Ceri A; Weekes, Michael P; Nobre, Luis V; Ruckova, Eva; Wilkie, Gavin S; Paulo, Joao A; Chang, Chiwen; Suárez, Nicolás M; Davies, James A; Antrobus, Robin; Stanton, Richard J; Aicheler, Rebecca J; Nichols, Hester; Vojtesek, Borek; Trowsdale, John; Davison, Andrew J; Gygi, Steven P

    2017-01-01

    The human cytomegalovirus (HCMV) US12 family consists of ten sequentially arranged genes (US12-21) with poorly characterized function. We now identify novel natural killer (NK) cell evasion functions for four members: US12, US14, US18 and US20. Using a systematic multiplexed proteomics approach to quantify ~1300 cell surface and ~7200 whole cell proteins, we demonstrate that the US12 family selectively targets plasma membrane proteins and plays key roles in regulating NK ligands, adhesion molecules and cytokine receptors. US18 and US20 work in concert to suppress cell surface expression of the critical NKp30 ligand B7-H6 thus inhibiting NK cell activation. The US12 family is therefore identified as a major new hub of immune regulation. DOI: http://dx.doi.org/10.7554/eLife.22206.001 PMID:28186488

  19. Natural Killer Cell Receptors and Cytotoxic Activity in Phosphomannomutase 2 Deficiency (PMM2-CDG.

    Directory of Open Access Journals (Sweden)

    Roberto García-López

    Full Text Available PMM2-CDG is the most common N-glycosylation defect and shows an increased risk of recurrent and/or severe, sometimes fatal, infections in early life. We hypothesized that natural killer (NK cells, as important mediators of the immune response against microbial pathogens and regulators of adaptive immunity, might be affected in this genetic disorder.To evaluate possible defects on PMM2-CDG NK peripheral blood cell number, killing activity and expression of membrane receptors.We studied fresh and activated NK cells from twelve PMM2-CDG cells. The number and expression of lymphoid surface receptors were studied by flow cytometry. The NK responsiveness (frequency of degranulated NK cells and killing activity against K562 target cells was determined in the NK cytotoxicity assay.We found an increase of blood NK cells in three patients with a severe phenotype. Two of them, who had suffered from moderate/severe viral infections during their first year of life, also had reduced T lymphocyte numbers. Patient activated NK cells showed increased expression of CD54 adhesion molecule and NKG2D and NKp46 activating receptors. NKp46 and 2B4 expression was inversely correlated with the expression of NKG2D in activated PMM2-CDG cells. Maximal NK activity against K562 target cells was similar in control and PMM2-CDG cells. Interestingly, the NK cell responsiveness was higher in patient cells. NKG2D and specially CD54 increased surface expression significantly correlated with the increased NK cell cytolytic activity according to the modulation of the killer activity by expression of triggering receptors and adhesion molecules.Our results indicate that hypoglycosylation in PMM2-CDG altered NK cell reactivity against target cells and the expression of CD54 and NKG2D, NKp46 and 2B4 activating receptors during NK cell activation. This suggests a defective control of NK cell killing activity and the overall anti-viral immune response in PMM2-CDG patients. The present

  20. Natural Killer Cell Receptors and Cytotoxic Activity in Phosphomannomutase 2 Deficiency (PMM2-CDG)

    Science.gov (United States)

    García-López, Roberto; de la Morena-Barrio, María Eugenia; Alsina, Laia; Pérez-Dueñas, Belén; Jaeken, Jaak; Serrano, Mercedes; Casado, Mercedes; Hernández-Caselles, Trinidad

    2016-01-01

    Background PMM2-CDG is the most common N-glycosylation defect and shows an increased risk of recurrent and/or severe, sometimes fatal, infections in early life. We hypothesized that natural killer (NK) cells, as important mediators of the immune response against microbial pathogens and regulators of adaptive immunity, might be affected in this genetic disorder. Objective To evaluate possible defects on PMM2-CDG NK peripheral blood cell number, killing activity and expression of membrane receptors. Methods We studied fresh and activated NK cells from twelve PMM2-CDG cells. The number and expression of lymphoid surface receptors were studied by flow cytometry. The NK responsiveness (frequency of degranulated NK cells) and killing activity against K562 target cells was determined in the NK cytotoxicity assay. Results We found an increase of blood NK cells in three patients with a severe phenotype. Two of them, who had suffered from moderate/severe viral infections during their first year of life, also had reduced T lymphocyte numbers. Patient activated NK cells showed increased expression of CD54 adhesion molecule and NKG2D and NKp46 activating receptors. NKp46 and 2B4 expression was inversely correlated with the expression of NKG2D in activated PMM2-CDG cells. Maximal NK activity against K562 target cells was similar in control and PMM2-CDG cells. Interestingly, the NK cell responsiveness was higher in patient cells. NKG2D and specially CD54 increased surface expression significantly correlated with the increased NK cell cytolytic activity according to the modulation of the killer activity by expression of triggering receptors and adhesion molecules. Conclusions Our results indicate that hypoglycosylation in PMM2-CDG altered NK cell reactivity against target cells and the expression of CD54 and NKG2D, NKp46 and 2B4 activating receptors during NK cell activation. This suggests a defective control of NK cell killing activity and the overall anti-viral immune response

  1. Tumour necrosis factor production and natural killer cell activity in peripheral blood during treatment with recombinant tumour necrosis factor

    OpenAIRE

    Männel, Daniela N.; Kist, A.; Ho, A D; Räth, U.; Reichardt, P; Wiedenmann, B; Schlick, E.; Kirchner, H.

    1989-01-01

    Tumour necrosis factor (TNF) has been found to be an important immunomodulator. Among other functions TNF activates natural killer (NK) cells and stimulates monocytes/macrophages in an autocrine fashion. TNF production and NK activity in peripheral blood mononuclear cells were determined in a clinical phase I study in which recombinant human (rh) TNF was administered as a continuous infusion weekly for a period of 8 weeks. Even though TNF production and NK activity were significantly reduced ...

  2. Estabishment of A Human Liver Cancer Cell Line Transfected with IL-2 cDNA and Its Biologic Activity

    Institute of Scientific and Technical Information of China (English)

    孙跃明; 王学浩; 杜竞辉

    2001-01-01

    Objective To obtain IL-2 gene transfected human liver cancer cells and study IL-2 expression and its biologic activity in vivo. Methods Human liver cancer cells SMMC-7721 were cocultured with recombinant retroviral vector LNC-IL-2,and screening was performed in G418 medium.The exogenous IL-2 cDNA at the DNA,RNA,and protein levels were determined by using dot hybridization,PR-PCR and MTT methods respectively.The tumorigenesis and antitumorigenesis of the screened liver cancer cell with subcutaneous injection in nude mice were observed. Results and Conclusion The IL-2 cDNA was successfully integrated into SMMC-7721 cell genomic DNA and continuously expressed for more than 88 days.Subcutaneous vaccination of the nude mice with transfected cells revealed an obvious suppression of its tumorigenicity,and could induce antitumor activity in vivo.

  3. Cortisol level decreases natural killer cell activity among women with aircraft noise

    Directory of Open Access Journals (Sweden)

    Hartono Hartono

    2016-02-01

    Full Text Available One of the impacts of exposure to noise is stress. Natural killer (NK cells are one of the leukocyte subsets that are responsive to physiological and psychological stress. The objective of the present research was to determine the relationship between cortisol levels and NK cell activity among women with aircraft noise stress in the area of Adi Sumarmo Airport, Solo. This study was an analytical survey with a cross sectional design. The number of subjects was 39, who were divided into 3 groups of 13 subjects each. Groups 1 to 3 were exposed to noise levels of 92.29 dB, 71.79 dB and 52.17 dB, respectively. The sample was taken using simple random sampling. The data were analyzed by Pearson correlation test and Anova followed by post hoc test using LSD test. The Anova test showed that there were significant differences in circulating cortisol levels among all groups (p = 0.018. The Pearson correlation test showed that there was a positive association between circulating cortisol levels and the number of NK cells (r = 0.547; p< 0.05 and a negative association between circulating cortisol levels and NK cell activity (r = - 0.578; p < 0.05. This study indicated that cortisol levels decreased NK cell activity among women with exposure to aircraft noise. Women who experienced aircraft noise stress showed increased cortisol levels and decreased NK cells activity.

  4. Immune functions in beluga whales (Delphinapterus leucas): evaluation of natural killer cell activity.

    Science.gov (United States)

    De Guise, S; Ross, P S; Osterhaus, A D; Martineau, D; Béland, P; Fournier, M

    1997-09-19

    Natural killer (NK) activity, an important non-specific defense mechanism against viral infections and tumors, was demonstrated in beluga whales using two different methods: 51Cr release and flow cytometry. Using the 51Cr release assay, NK activity in belugas was shown to be higher against K-562 than against YAC-1 cell lines. Moreover, it was enhanced by the addition of human recombinant interleukin-2 with both cell lines. NK activity evaluated by flow cytometry in the peripheral blood of eight belugas increased when the effector:target cell (E:T) ratio increased, and averaged 13.9% +/- 3.8% (range 9.9% to 17.8%) at an E:T ratio of 100:1. While NK activity could be readily detected using both methods, the lack of radio-isotopes and related laboratory room make the flow cytometric method a viable and safe alternative. The evaluation of this function in cetaceans could lead to a better understanding of the early events that lead to viral epizootics in populations of marine mammals in different parts of the world, as well as to the high prevalence of neoplasms in St. Lawrence beluga whales.

  5. FHL2 Regulates Natural Killer Cell Development and Activation during Streptococcus pneumoniae Infection

    Science.gov (United States)

    Baranek, Thomas; Morello, Eric; Valayer, Alexandre; Aimar, Rose-France; Bréa, Déborah; Henry, Clemence; Besnard, Anne-Gaelle; Dalloneau, Emilie; Guillon, Antoine; Dequin, Pierre-François; Narni-Mancinelli, Emilie; Vivier, Eric; Laurent, Fabrice; Wei, Yu; Paget, Christophe; Si-Tahar, Mustapha

    2017-01-01

    Recent in silico studies suggested that the transcription cofactor LIM-only protein FHL2 is a major transcriptional regulator of mouse natural killer (NK) cells. However, the expression and role of FHL2 in NK cell biology are unknown. Here, we confirm that FHL2 is expressed in both mouse and human NK cells. Using FHL2−/− mice, we found that FHL2 controls NK cell development in the bone marrow and maturation in peripheral organs. To evaluate the importance of FHL2 in NK cell activation, FHL2−/− mice were infected with Streptococcus pneumoniae. FHL2−/− mice are highly susceptible to this infection. The activation of lung NK cells is altered in FHL2−/− mice, leading to decreased IFNγ production and a loss of control of bacterial burden. Collectively, our data reveal that FHL2 is a new transcription cofactor implicated in NK cell development and activation during pulmonary bacterial infection.

  6. Activation mechanisms of invariant natural killer T cells (iNKTs

    Directory of Open Access Journals (Sweden)

    Baena, Andrés

    2016-01-01

    Full Text Available A great amount of knowledge on natural killer T cells (iNKTs is now available, but a consensus about their activation mechanisms has not been reached. These cells recognize different glycolipid antigens through the CD1d molecule. Such antigens may be endogenous, derived from bacteria (foreign and synthetic, the latter have been developed for clinical applications. There exists much interest in understanding how these different glycolipid compounds induce different types of polarization, but it has been difficult to reach a consensus due to the fact that responses depend on different factors such as: the nature of the molecule, the internalization process and the presentation of the glycolipids. Moreover, activation of iNKT cells is determined by the type and state of the antigen presenting cell, the co-stimulatory molecules, the transactivation mechanisms and the location of the glycolipid-CD1d complexes on the plasma membrane, such as the lipid rafts. This review explores the evidence about the factors that affect activation of iNKT cells in order to understand their immune-modulatory potential.

  7. The relationship between the acoustic behaviour and surface activity of killer whales (Orcinus orca) that feed on herring (Clupea harengus)

    DEFF Research Database (Denmark)

    Simon, Malene Juul; McGregor, Peter K.; Ugarte, Fernando

    2007-01-01

    We describe the acoustic behaviour of piscivorous killer whales in Norwegian and Icelandic waters. Whales were assigned to one of three activities (feeding, travelling or other), and sound recordings were made in their proximity with a single hydrophone and a digital audiotape (DAT) recorder....... A quantitative analysis of the production of pulsed calls, whistles and echolocation clicks in the three activities revealed that there was a significant effect of activity on the production of these sound types. Both killer whales in Icelandic and Norwegian waters produced high rates of clicks and calls during...... behaviour, we suggest that the killer whales in Icelandic and Norwegian waters belong to the same ecotype: Scandinavian herring-eating killer whales. Udgivelsesdato: 18 April 2007...

  8. Alzheimer caregiver stress: basal natural killer cell activity, pituitary-adrenal cortical function, and sympathetic tone.

    Science.gov (United States)

    Irwin, M; Hauger, R; Patterson, T L; Semple, S; Ziegler, M; Grant, I

    1997-01-01

    The association between Alzheimer caregiving and natural killer (NK) cell activity and basal plasma levels of adrenocorticotropic hormone (ACTH), cortisol, beta-endorphin, prolactin, epinephrine, norepinephrine, and neuropeptide Y was determined in 100 spousal Alzheimer caregivers and 33 age- and gender-comparable control volunteers upon intake into a study of the psychological and physiologic impact of caregiving. The relationship between these physiologic measures and individual characteristics such as age, gender, medical status, severity of stress, severity of depressive symptoms, and caregiver burden was tested. In addition, the association between NK activity and alterations of the neuroendocrine measures was investigated. As compared to controls, the Alzheimer caregivers had similar levels of NK activity and of basal plasma neuroendocrine hormones and sympathetic measures. While older age and male gender status were associated with increased levels of ACTH, neither medical caseness, severity of life stress, nor severity of depressive symptoms was associated with alterations in any of the multiple physiologic domains. Classification of Alzheimer caregiver burden identified caregivers who were mismatched in terms of the amount of care they were required to provide and the amount of respite time received. The mismatched caregivers had significantly higher basal plasma ACTH but no change in other physiological measures, as compared to non-mismatched caregivers. NK activity was negatively correlated with plasma levels of neuropeptide Y but not with any of the other neuroendocrine measures. Based on this cross-sectional evaluation of NK activity and neuroendocrine and sympathetic measures, we conclude that most Alzheimer caregivers do not show evidence of altered basal physiology.

  9. [Change in the activity of natural killer cells in normal subjects and in virus diseases on exposure to interferon in vitro].

    Science.gov (United States)

    Petrov, R V; Saidov, M Z; Koval'chuk, L V; Sorokin, A M; Kaganov, B S

    1984-04-01

    The activity of natural killers was examined in peripheral blood of healthy subjects and patients with chronic hepatitis and disseminated sclerosis. An attempt was made to correct natural killer activity by human leukocyte interferon in vitro. To assess the activity of natural killers, use was made of the method of serial dilutions. An optimal effector/target ratio was employed in experiments. The patients with chronic hepatitis and disseminated sclerosis demonstrated a reduction in the activity of natural killers whatever the effector/target ratio. The action of interferon in vitro is specific immunomodulatory in nature. Administration of interferon in a dose of 250 Units/ml raises the magnitude of the cytotoxic index in healthy donors and in patients with chronic hepatitis and disseminated sclerosis, making the shape of the killer activity curve approach that of normal. Such an approach can be used for preliminary assessment of the sensitivity of natural killers to interferon in viral diseases of man. The potentialities and efficacy of interferon in clinical medicine are discussed.

  10. Use of flameless atomic absorption spectroscopy in immune cytolysis for nonradioactive determination of killer cell activity.

    Science.gov (United States)

    Borella, P; Bargellini, A; Salvioli, S; Cossarizza, A

    1996-02-01

    We describe here a novel method to evaluate natural killer (NK) cytolytic activity by use of flameless atomic absorption spectroscopy (GF-AAS). This technique may be adopted for use in laboratories equipped with electrothermal atomic absorption spectrometers. Nonradioactive Cr as Na2CrO4 was used to label target cells (K562), and cell lysis was evaluated by measuring Cr released after 4 h of incubation with the effectors. We selected 520 micrograms/L as the optimal dose for labeling targets, between 12 and 20 h as the optimal incubation time, and 10(4) cells as the optimal target size. Advantages of this method include: (a) exclusion of radioactive tracer, with no risk for workers; (b) limited costs; (c) high sensitivity and reproducibility; (d) possibility to store samples; and (e) better control of Cr used for labeling cells due to well-determined, fixed Cr concentrations in the range of nontoxic and linear cellular uptake. Comparison with data obtained by conventional 51Cr labeling of targets killed by the same effectors was excellent, yielding comparable results and corroborating the method.

  11. Role of killer factors in the inhibitory activity of bio-control yeasts against Penicillium expansum and Aspergillus ochraceus

    Directory of Open Access Journals (Sweden)

    Ciro da Silva Portes

    2013-08-01

    Full Text Available This work evaluated the antagonism of killer positive yeast strains (isolated from 11 samples of different frozen fruit pulps against the strains of Penicillium expansum and Aspergillus ochraceus. Of the total 41 killer yeasts tested in YM agar, 19 showed antibiosis against P. expansum and A. ochraceus, with inhibition zone ranging from 10 to 18 mm and 10 to 19 mm, respectively. In the following step, the extracellular activity of Kluyveromyces sp. FP4(13 was tested performing the assay in YM broth. The antifungal activity of Kluyveromyces sp. FP4(13 cell-free culture supernatant (25ºC/96 h was more effective against the conidia germination, showing inhibition rates of 93.33 and 86.44% for P. expansum and A. ochraceus, respectively. The micelial growth inhibition was 28.45 and 21.0%, respectively. The antagonism showed by the selected yeasts could be used as a promising alternative tool to reduce and control the postharvest fungal spoilage of the fruits. However, further studies should be carried out in order to better elucidate the role of innocuous characters in antagonistic microorganisms, as well as the purification and characterization of new killer toxins.

  12. Identification and functional analysis of ligands for natural killer cell activating receptors in colon carcinoma.

    Science.gov (United States)

    Zhang, Zhang; Su, Tao; He, Liang; Wang, Hongtao; Ji, Gang; Liu, Xiaonan; Zhang, Yun; Dong, Guanglong

    2012-01-01

    Natural killer (NK) cells play important roles in the immune defense against tumor cells. The function of NK cells is determined by a balance between activating and inhibitory signals. DNAX accessory molecule-1 (DNAM-1) and NK group 2 member D (NKG2D) are major NK cell activating receptors, which transduce activating signals after binding their ligands CD155, CD112 and major histocompatibility complex class I-related chains A and B (MICA/B). However, the expression and functions of these ligands in colon carcinoma are still elusive. Here, we show the higher expression of CD155, CD112 and MICA/B in colon carcinoma tissues, although no correlations between the ligands expression and patient clinicopathological parameters were found. The subsequent cytotoxicity assay indicated that NK cells effectively kill colon carcinoma cells. Functional blocking of these ligands and/or receptors with antibodies led to significant inhibition of NK cell cytotoxicity. Importantly, expression of DNAM-1 and NKG2D was reduced in NK cells of colon cancer patients, and this reduction could directly suppress the activation of NK cells. Moreover, colon cancer patients have higher serum concentrations of sCD155 and sMICA/B (soluble ligands, secreted or shed from cells) than those in healthy donors (sCD155, 127.82 ± 44.12 vs. 63.67 ± 22.30 ng/ml; sMICA, 331.51 ± 65.23 vs. 246.74 ± 20.76 pg/ml; and sMICB, 349.42 ± 81.69 vs. 52.61 ± 17.56 pg/ml). The up-regulation of these soluble ligands may down-regulate DNAM-1 and NKG2D on NK cells, ultimately leading to the inhibition of NK cytotoxicity. Colon cancer might be a promising target for NK cell-based adoptive immunotherapy.

  13. The "killer cell story" in recurrent miscarriage: Association between activated peripheral lymphocytes and uterine natural killer cells.

    Science.gov (United States)

    Kuon, R J; Vomstein, K; Weber, M; Müller, F; Seitz, C; Wallwiener, S; Strowitzki, T; Schleussner, E; Markert, U R; Daniel, V; Toth, B

    2017-02-01

    Peripheral and uterine NK cells (pNK, uNK) can be distinguished according to their receptor expression. Recent studies indicate an association of elevated pNK and uNK with recurrent miscarriage (RM). This study aimed to analyze pNK and uNK in patients with RM and healthy controls. Out of n=590 RM patients screened according to a standard diagnostic protocol, n=268 couples with ≥3 consecutive RM were identified. Subgroups consisted of n=151 primary RM (pRM), n=85 secondary RM (sRM), n=32 tertiary RM (tRM) and n=42 healthy controls. Finally, n=147 idiopathic RM (iRM) and n=121 non-iRM patients were identified. Peripheral blood levels of CD45+CD3-CD56+CD16+ NK cells were determined in non-pregnant patients and controls in the mid-luteal phase by FACS. In n=129 RM patients a uterine biopsy was taken to evaluate CD56+ NK cells by immunohistochemistry. PRM showed higher absolute pNK than sRM (median/μl (Q1;Q3): 234 (147;306) vs 176 (128;245), p=0.02). Further a trend towards higher pNK percentages in pRM was detected. UNK numbers did not differ between RM subgroups and did not correlate with pNK. However, the rate of highly elevated uNK was increased in iRM compared to non-iRM patients (p=0.04). Further, higher numbers of CD45+CD3-DR+ (p<0.01) and CD45+CD3+CD8+DR+ (p=0.04) peripheral lymphocytes were associated with higher uNK numbers. In conclusion, elevated pNK were present in pRM patients. Although pNK and uNK numbers did not correlate, the association between high CD45+CD3-DR+ and CD45+CD3+CD8+DR+ peripheral lymphocytes and uNK might indicate that activated NK, B and T cells provide cytokines for the differentiation of uNK.

  14. The anti-canine distemper virus activities of ex vivo-expanded canine natural killer cells.

    Science.gov (United States)

    Park, Ji-Yun; Shin, Dong-Jun; Lee, Soo-Hyeon; Lee, Je-Jung; Suh, Guk-Hyun; Cho, Duck; Kim, Sang-Ki

    2015-04-17

    Natural killer (NK) cells play critical roles in induction of antiviral effects against various viruses of humans and animals. However, few data on NK cell activities during canine distemper virus (CDV) infections are available. Recently, we established a culture system allowing activation and expansion of canine non-B, non-T, large granular NK lymphocytes from PBMCs of normal dogs. In the present study, we explored the ability of such expanded NK cells to inhibit CDV infection in vitro. Cultured CD3-CD5-CD21- NK cells produced large amounts of IFN-γ, exhibited highly upregulated expression of mRNAs encoding NK-cell-associated receptors, and demonstrated strong natural killing activity against canine tumor cells. Although the expanded NK cells were dose-dependently cytotoxic to both normal and CDV-infected Vero cells, CDV infection rendered Vero cells more susceptible to NK cells. Pretreatment with anti-CDV serum from hyperimmunized dogs enhanced the antibody-dependent cellular cytotoxicity (ADCC) of NK cells against CDV-infected Vero cells. The culture supernatants of NK cells, added before or after infection, dose-dependently inhibited both CDV replication and development of CDV-induced cytopathic effects (CPEs) in Vero cells. Anti-IFN-γ antibody neutralized the inhibitory effects of NK cell culture supernatants on CDV replication and CPE induction in Vero cells. Such results emphasize the potential significance of NK cells in controlling CDV infection, and indicate that NK cells may play roles both during CDV infection and in combating such infections, under certain conditions.

  15. Biology Myth-Killers

    Science.gov (United States)

    Lampert, Evan

    2014-01-01

    "Biology Myth-Killers" is an activity designed to identify and correct common misconceptions for high school and college introductory biology courses. Students identify common myths, which double as biology misconceptions, and use appropriate sources to share the "truth" about the myths. This learner-centered activity is a fun…

  16. Renal cell carcinoma-infiltrating natural killer cells express differential repertoires of activating and inhibitory receptors and are inhibited by specific HLA class I allotypes.

    Science.gov (United States)

    Schleypen, Julia S; Von Geldern, Marion; Weiss, Elisabeth H; Kotzias, Nicole; Rohrmann, Karl; Schendel, Dolores J; Falk, Christine S; Pohla, Heike

    2003-10-10

    Among tumor-infiltrating lymphocytes (TILs) directly isolated from renal cell carcinomas (RCCs), we found substantial numbers of natural killer (NK) cells in most tumor tissues. They could be identified reliably in situ with an antibody directed against the activating receptor (AR) NKp46 that is exclusively expressed by all NK cells. NK-enriched TILs (NK-TILs) showed cytotoxicity against major histocompatibility complex (MHC) class I-negative cell lines. The ability to detect lysis of target cells was dependent on the percentage of NK cells within the TILs, and cytotoxicity was only observed after overnight activation with low-dose interleukin-2 (IL-2). Infiltrating NK cells were found to express various inhibitory receptors (IRs); among these the CD94/NKG2A receptor complex was overrepresented compared to the autologous peripheral blood mononuclear cell (PBMC) population. Other IRs were underrepresented, indicating that NK subpopulations vary in their tumor-infiltrating capacity. IRs expressed by NK-TILs are functional since receptor engagement with MHC class I ligands presented by human leukocyte antigen (HLA)-transfected target cell lines was able to inhibit NK-mediated cytotoxicity. NK-TILs were also able to lyse autologous or allogeneic tumor cell lines in vitro. This activity correlated with low HLA class I surface expression since lysis could be inhibited by interferon (IFN)-gamma-expressing RCC transductants that displayed a higher surface density of HLA class I molecules. Therefore, NK cells infiltrating tumor tissues have an inherent ability to recognize transformed cells, but they require cytokine activation and are sensitive to inhibition by IR ligands.

  17. Pollen Killer Gene S35 Function Requires Interaction with an Activator That Maps Close to S24, Another Pollen Killer Gene in Rice

    Directory of Open Access Journals (Sweden)

    Takahiko Kubo

    2016-05-01

    Full Text Available Pollen killer genes disable noncarrier pollens, and are responsible for male sterility and segregation distortion in hybrid populations of distantly related plant species. The genetic networks and the molecular mechanisms underlying the pollen killer system remain largely unknown. Two pollen killer genes, S24 and S35, have been found in an intersubspecific cross of Oryza sativa ssp. indica and japonica. The effect of S24 is counteracted by an unlinked locus EFS. Additionally, S35 has been proposed to interact with S24 to induce pollen sterility. These genetic interactions are suggestive of a single S24-centric genetic pathway (EFS–S24–S35 for the pollen killer system. To examine this hypothetical genetic pathway, the S35 and the S24 regions were further characterized and genetically dissected in this study. Our results indicated that S35 causes pollen sterility independently of both the EFS and S24 genes, but is dependent on a novel gene close to the S24 locus, named incentive for killing pollen (INK. We confirmed the phenotypic effect of the INK gene separately from the S24 gene, and identified the INK locus within an interval of less than 0.6 Mb on rice chromosome 5. This study characterized the genetic effect of the two independent genetic pathways of INK–S35 and EFS–S24 in indica–japonica hybrid progeny. Our results provide clear evidence that hybrid male sterility in rice is caused by several pollen killer networks with multiple factors positively and negatively regulating pollen killer genes.

  18. Activation of human natural killer cells by the soluble form of cellular prion protein

    Energy Technology Data Exchange (ETDEWEB)

    Seong, Yeon-Jae [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Hafis Clinic, Seoul (Korea, Republic of); Sung, Pil Soo; Jang, Young-Soon; Choi, Young Joon [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Park, Bum-Chan [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Park, Su-Hyung [Laboratory of Translational Immunology and Vaccinology, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of); Park, Young Woo [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Shin, Eui-Cheol, E-mail: ecshin@kaist.ac.kr [Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon (Korea, Republic of)

    2015-08-21

    Cellular prion protein (PrP{sup C}) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP{sup C} in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP{sup C} protein on human natural killer (NK) cells. Recombinant soluble PrP{sup C} protein was generated by fusion of human PrP{sup C} with the Fc portion of human IgG{sub 1} (PrP{sup C}-Fc). PrP{sup C}-Fc binds to the surface of human NK cells, particularly to CD56{sup dim} NK cells. PrP{sup C}-Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP{sup C}-Fc facilitated the IL-15-induced proliferation of NK cells. PrP{sup C}-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP{sup C}-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP{sup C} (PrP{sup C}-Fc) was generated by fusion of human PrP{sup C} with IgG1 Fc portion. • PrP{sup C}-Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP{sup C}-Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways.

  19. GnRH receptor activation competes at a low level with growth signaling in stably transfected human breast cell lines

    Directory of Open Access Journals (Sweden)

    Morgan Kevin

    2011-11-01

    Full Text Available Abstract Background Gonadotrophin releasing hormone (GnRH analogs lower estrogen levels in pre-menopausal breast cancer patients. GnRH receptor (GnRH-R activation also directly inhibits the growth of certain cells. The applicability of GnRH anti-proliferation to breast cancer was therefore analyzed. Methods GnRH-R expression in 298 primary breast cancer samples was measured by quantitative immunofluorescence. Levels of functional GnRH-R in breast-derived cell lines were assessed using 125I-ligand binding and stimulation of 3H-inositol phosphate production. Elevated levels of GnRH-R were stably expressed in cells by transfection. Effects of receptor activation on in vitro cell growth were investigated in comparison with IGF-I and EGF receptor inhibition, and correlated with intracellular signaling using western blotting. Results GnRH-R immunoscoring was highest in hormone receptor (triple negative and grade 3 breast tumors. However prior to transfection, functional endogenous GnRH-R were undetectable in four commonly studied breast cancer cell lines (MCF-7, ZR-75-1, T47D and MDA-MB-231. After transfection with GnRH-R, high levels of cell surface GnRH-R were detected in SVCT and MDA-MB-231 clones while low-moderate levels of GnRH-R occurred in MCF-7 clones and ZR-75-1 clones. MCF-7 sub-clones with high levels of GnRH-R were isolated following hygromycin phosphotransferase transfection. High level cell surface GnRH-R enabled induction of high levels of 3H-inositol phosphate and modest growth-inhibition in SVCT cells. In contrast, growth of MCF-7, ZR-75-1 or MDA-MB-231 clones was unaffected by GnRH-R activation. Cell growth was inhibited by IGF-I or EGF receptor inhibitors. IGF-I receptor inhibitor lowered levels of p-ERK1/2 in MCF-7 clones. Washout of IGF-I receptor inhibitor resulted in transient hyper-elevation of p-ERK1/2, but co-addition of GnRH-R agonist did not alter the dynamics of ERK1/2 re-phosphorylation. Conclusions Breast cancers

  20. Analysis of sphingosine kinase activity in single natural killer cells from peripheral blood†

    Science.gov (United States)

    Dickinson, Alexandra J.; Meyer, Megan; Pawlak, Erica A.; Gomez, Shawn; Jaspers, Ilona; Allbritton, Nancy L.

    2015-01-01

    Sphingosine-1-phosphate (S1P), a lipid second messenger formed upon phosphorylation of sphingosine by sphingosine kinase (SK), plays a crucial role in natural killer (NK) cell proliferation, migration, and cytotoxicity. Dysregulation of the S1P pathway has been linked to a number of immune system disorders and therapeutic manipulation of the pathway has been proposed as a method of disease intervention. However, peripheral blood NK cells, as identified by surface markers (CD56+CD45+CD3−CD16) consist of a highly diverse population with distinct phenotypes and functions and it is unknown whether the S1P pathway is similarly diverse across peripheral blood NK cells. In this work, we measured the phosphorylation of sphingosine–fluorescein (SF) and subsequent metabolism of S1P fluorescein (S1PF) to form hexadecanoic acid fluorescein (HAF) in 111 single NK cells obtained from the peripheral blood of four healthy human subjects. The percentage of SF converted to S1PF or HAF was highly variable amongst the cells ranging from 0% to 100% (S1PF) and 0% to 97% (HAF). Subpopulations of cells with varying levels of S1PF formation and metabolism were readily identified. Across all subjects, the average percentage of SF converted to S1PF or HAF was 37 ± 36% and 12 ± 19%, respectively. NK cell metabolism of SF by the different subjects was also distinct with hierarchical clustering suggesting two possible phenotypes: low (50%) producers of S1PF. The heterogeneity of SK and downstream enzyme activity in NK cells may enable NK cells to respond effectively to a diverse array of pathogens as well as incipient tumor cells. NK cells from two subjects were also loaded with S1PF to assess the activity of S1P phosphatase (S1PP), which converts S1P to sphingosine. No NK cells (n = 41) formed sphingosine, suggesting that S1PP was minimally active in peripheral blood NK cells. In contrast to the SK activity, S1PP activity was homogeneous across the peripheral blood NK cells, suggesting a

  1. Analysis of sphingosine kinase activity in single natural killer cells from peripheral blood.

    Science.gov (United States)

    Dickinson, Alexandra J; Meyer, Megan; Pawlak, Erica A; Gomez, Shawn; Jaspers, Ilona; Allbritton, Nancy L

    2015-04-01

    Sphingosine-1-phosphate (S1P), a lipid second messenger formed upon phosphorylation of sphingosine by sphingosine kinase (SK), plays a crucial role in natural killer (NK) cell proliferation, migration, and cytotoxicity. Dysregulation of the S1P pathway has been linked to a number of immune system disorders and therapeutic manipulation of the pathway has been proposed as a method of disease intervention. However, peripheral blood NK cells, as identified by surface markers (CD56(+)CD45(+)CD3(-)CD16) consist of a highly diverse population with distinct phenotypes and functions and it is unknown whether the S1P pathway is similarly diverse across peripheral blood NK cells. In this work, we measured the phosphorylation of sphingosine-fluorescein (SF) and subsequent metabolism of S1P fluorescein (S1PF) to form hexadecanoic acid fluorescein (HAF) in 111 single NK cells obtained from the peripheral blood of four healthy human subjects. The percentage of SF converted to S1PF or HAF was highly variable amongst the cells ranging from 0% to 100% (S1PF) and 0% to 97% (HAF). Subpopulations of cells with varying levels of S1PF formation and metabolism were readily identified. Across all subjects, the average percentage of SF converted to S1PF or HAF was 37 ± 36% and 12 ± 19%, respectively. NK cell metabolism of SF by the different subjects was also distinct with hierarchical clustering suggesting two possible phenotypes: low (50%) producers of S1PF. The heterogeneity of SK and downstream enzyme activity in NK cells may enable NK cells to respond effectively to a diverse array of pathogens as well as incipient tumor cells. NK cells from two subjects were also loaded with S1PF to assess the activity of S1P phosphatase (S1PP), which converts S1P to sphingosine. No NK cells (n = 41) formed sphingosine, suggesting that S1PP was minimally active in peripheral blood NK cells. In contrast to the SK activity, S1PP activity was homogeneous across the peripheral blood NK cells, suggesting

  2. Effects of chloroquine, mefloquine and quinine on natural killer cell activity in vitro. An analysis of the inhibitory mechanism

    DEFF Research Database (Denmark)

    Pedersen, B K; Bygbjerg, I C; Theander, T G;

    1986-01-01

    Natural killer (NK) cell activity against K 562 target cells was inhibited by pharmacological concentrations of chloroquine, mefloquine and quinine. The most potent were mefloquine and quinine. The drug-induced inhibition of the NK cell activity was abolished by addition of alpha-interferon (IF...... NK cell enriched populations in a single cell agarose assay, it was shown that the inhibitory effects of mefloquine, but not of chloroquine and quinine were due to an inhibition of the formation of effector/target cell conjugates....

  3. Augmentation by interleukin-18 of MHC-nonrestricted killer activity of human peripheral blood mononuclear cells in response to interleukin-12.

    Science.gov (United States)

    Singh, S M; Yanagawa, H; Hanibuchi, M; Miki, T; Okamura, H; Sone, S

    2000-01-01

    Interleukin (IL)-18 is a novel cytokine with pleiotropic functions. In the present study, we examined the induction of the killer activity of peripheral blood mononuclear cells (MNC) against lung cancer cell lines upon treatment with IL-18 in combination with IL-12. Cytotoxic activity was measured by standard (51)Cr release assay. IL-18 (100 ng/ml) was found to significantly augment IL-12-induced killer activity in a MHC-nonrestricted manner against allogeneic NK-resistant Daudi cells and lung cancer cell lines: SBC-3, RERF-LC-AI and A549. IL-18 could augment IL-12-induced killer activity both at the optimal as well as suboptimal doses of the latter. However, IL-18 was found to have little effect on the killer activity of MNC induced by optimal or suboptimal dose of IL-2 or IL-15. Treatment of MNC with IL-18 in combination with IL-12 for a period of more than 4 days was observed to optimally induce the killer activity. As for induction of IFN-gamma production by MNC, IL-18 augmented that induced by IL-2 and IL-15, as well as that induced by IL-12. These results show the potential of IL-18 in combination with IL-12 for clinical application in treatment of cancer.

  4. Green Tea Catechin Metabolites Exert Immunoregulatory Effects on CD4(+) T Cell and Natural Killer Cell Activities.

    Science.gov (United States)

    Kim, Yoon Hee; Won, Yeong-Seon; Yang, Xue; Kumazoe, Motofumi; Yamashita, Shuya; Hara, Aya; Takagaki, Akiko; Goto, Keiichi; Nanjo, Fumio; Tachibana, Hirofumi

    2016-05-11

    Tea catechins, such as (-)-epigallocatechin-3-O-gallate (EGCG), have been shown to effectively enhance immune activity and prevent cancer, although the underlying mechanism is unclear. Green tea catechins are instead converted to catechin metabolites in the intestine. Here, we show that these green tea catechin metabolites enhance CD4(+) T cell activity as well as natural killer (NK) cell activity. Our data suggest that the absence of a 4'-hydroxyl on this phenyl group (B ring) is important for the effect on immune activity. In particular, 5-(3',5'-dihydroxyphenyl)-γ-valerolactone (EGC-M5), a major metabolite of EGCG, not only increased the activity of CD4(+) T cells but also enhanced the cytotoxic activity of NK cells in vivo. These data suggest that EGC-M5 might show immunostimulatory activity.

  5. [Expression of activating and inhibitory receptors on peripheral blood natural killer cell subsets of women with reproductive failures].

    Science.gov (United States)

    Baltadzheiva, D; Penkova, K; Stamenov, G; Dimitrova, D; Michailova, A

    2010-01-01

    It is now apparent that immunologic implantation failure and recurrent abortions are more than likely mediated through activation of natural killer (NK) cells. The NK cell activity is mediated by a balance between activating and inhibitory receptors upon recognition of MHC class I molecules. In this study, we investigated by flow cytometry the expression of activating and inhibitory receptors on NK cells of women with reproductive failures- recurrent spontaneous abortion (RSA) and implantation failures (IF). In women with implantation failures CD56+CD16+ NK cell subset was significantly increased (p = 0.017) and CD158a expressing NK cells was significantly decreased (p = 0.027). CD161-activating receptor expressing CD56+ NK cells were significantly decreased in women with RSA (p = 0.033). These data further support an imbalance in NK cell subsets in women with reproductive failures.

  6. Killer behavior within the Candida parapsilosis complex.

    Science.gov (United States)

    Robledo-Leal, Efrén; Elizondo-Zertuche, Mariana; Villarreal-Treviño, Licet; Treviño-Rangel, Rogelio de J; García-Maldonado, Nancy; Adame-Rodríguez, Juan M; González, Gloria M

    2014-11-01

    A group of 29 isolates of Candida parapsilosis sensu stricto, 29 of Candida orthopsilosis, and 4 of Candida metapsilosis were assayed for the presence of killer activity using Saccharomyces cerevisiae ATCC 26609 as a sensitive strain. All C. metapsilosis isolates showed killer activity at 25 °C while strains of C. parapsilosis sensu stricto or C. orthopsilosis did not exhibit this activity. Sensitivity to killer toxins was evaluated using a set of previously reported killer strains of clinical origin. Only 11 isolates of the C. parapsilosis complex were inhibited by at least one killer isolate without resulting in any clear pattern, except for C. parapsilosis sensu stricto ATCC 22019, which was inhibited by every killer strain with the exception of C. parapsilosis and Candida utilis. The lack of sensitivity to killer activity among isolates of the genus Candida suggests that their toxins belong to the same killer type. Differentiation of species within the C. parapsilosis complex using the killer system may be feasible if a more taxonomically diverse panel of killer strains is employed.

  7. Gene-carried hepatoma targeting complex induced high gene transfection efficiency with low toxicity and significant antitumor activity

    Directory of Open Access Journals (Sweden)

    Zhao QQ

    2012-06-01

    Full Text Available Qing-Qing Zhao,1,2 Yu-Lan Hu,1 Yang Zhou,3 Ni Li,1 Min Han,1 Gu-Ping Tang,4 Feng Qiu,2 Yasuhiko Tabata,5 Jian-Qing Gao,11Institute of Pharmaceutics, Zhejiang University, Hangzhou, China; 2Department of Pharmacy, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China; 3Institute of Biochemistry, Iowa State University, Ames, IA, USA; 4Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Hangzhou, China; 5Institute for Frontier Medical Sciences, Kyoto University, Kyoto, JapanBackground: The success of gene transfection is largely dependent on the development of a vehicle or vector that can efficiently deliver a gene to cells with minimal toxicity.Methods: A liver cancer-targeted specific peptide (FQHPSF sequence was successfully synthesized and linked with chitosan-linked polyethylenimine (CP to form a new targeted gene delivery vector called CPT (CP/peptide. The structure of CPT was confirmed by 1H nuclear magnetic resonance spectroscopy and ultraviolet spectrophotometry. The particle size of CPT/DNA complexes was measured using laser diffraction spectrometry and the cytotoxicity of the copolymer was evaluated by methylthiazol tetrazolium method. The transfection efficiency evaluation of the CP copolymer was performed using luciferase activity assay. Cellular internalization of the CP/DNA complex was observed under confocal laser scanning microscopy. The targeting specificity of the polymer coupled to peptide was measured by competitive inhibition transfection study. The liver targeting specificity of the CPT copolymer in vivo was demonstrated by combining the copolymer with a therapeutic gene, interleukin-12, and assessed by its abilities in suppressing the growth of ascites tumor in mouse model.Results: The results showed that the liver cancer-targeted specific peptide was successfully synthesized and linked with CP to form a new targeted gene delivery vector called CPT. The composition of CPT

  8. A brief report of basic science: the effects of preincisional low-dose ketamine on natural killer cell activity in male Fischer 344 rats after intra-abdominal surgery.

    Science.gov (United States)

    Estes, Savannah; Dinh, Tim; Garrett, Normalynn

    2009-01-01

    Although the first line of defense in cancer treatment often is surgery, studies suggest that postoperative pain and anesthetic drugs suppress the activity of cells that lyse metastatic cells, that is, natural killer cells. We assessed the affect of low-dose ketamine on natural killer cell activity. The findings are presented in this brief report.

  9. Activating killer-cell immunoglobulin-like receptors (KIR) and their cognate HLA ligands are significantly increased in autism.

    Science.gov (United States)

    Torres, Anthony R; Westover, Jonna B; Gibbons, Cole; Johnson, Randall C; Ward, David C

    2012-10-01

    Killer-cell immunoglobulin-like receptor (KIR) proteins are expressed on natural killer (NK) cells and appear important in innate and adaptive immunity. There are about 14 KIR genes on chromosome 19q13.4, composed of those that inhibit and those that activate NK cell killing. Haplotypes have different combinations of these genes meaning that not all genes are present in a subject. There are two main classes of cognate human leukocyte antigen (HLA) ligands (HLA-Bw4 and HLA-C1/C2) that bind to the inhibitory/activating receptors. As a general rule, the inhibitory state is maintained except when virally infected or tumor cells are encountered; however, both increased activation and inhibition states have been associated with susceptibility and protection against numerous disease states including cancer, arthritis, and psoriasis. Utilizing DNA from 158 Caucasian subjects with autism and 176 KIR control subjects we show for the first time a highly significant increase in four activating KIR genes (2DS5, 3DS1, 2DS1 and 2DS4) as measured by chi square values and odds ratios. In addition, our data suggests a highly significant increase in the activating KIR gene 2DS1 and its cognate HLA-C2 ligand (2DS1+C2; p = 0.00003 [Odds ratio = 2.87]). This information ties together two major immune gene complexes, the human leukocyte complex and the leukocyte receptor complex, and may partially explain immune abnormalities observed in many subjects with autism.

  10. Effects of 5-azacytidine on natural killer cell activating receptor expression in patients with refractory anemia with excess of blasts

    Directory of Open Access Journals (Sweden)

    Régis T. Costello

    2015-01-01

    Full Text Available Epigenetic drugs modify DNA methylation and are used in refractory anemia with excess of blasts (RAEB. These drugs may reactivate anti-oncogene expression and restore a normal phenotype instead of inducing antitumor toxicity, although they also have immunosuppressive effects on T-lymphocytes [1] In RAEB and acute myeloid leukemia, a defect in natural killer (NK cell cytotoxicity has been shown, which relies on abnormal expression of activating receptors. Previous study has shown that 5-azacytidine impaired mRNA synthesis and induced apoptosis in NK cells [2]. In this study we investigated the effect of the demethylating drug 5-azacytidine (Vidaza® on NK receptors with the hypothesis that demethylation of the promoters of activating NK receptor genes induces gene reactivation and thus may increase their expression.

  11. Conditioned enhancement of natural killer cell activity, but not interferon, with camphor or saccharin-LiCl conditioned stimulus.

    Science.gov (United States)

    Ghanta, V K; Hiramoto, N S; Solvason, H B; Tyring, S K; Spector, N H; Hiramoto, R N

    1987-01-01

    Pavlovian conditioning of the natural killer (NK) cell response has been demonstrated by pairing camphor with polyinosinic:polycytidylic acid (poly I:C) in nine association trials. The NK cell response could be conditioned also by using combined saccharin and lithium chloride (LiCl) as the conditioned stimulus. The camphor and saccharin-LiCl paradigms were tested to determine if the conditioned NK cell activity was the result of conditioning of the interferon response. Interferon levels were measured at 6 hr and NK cell activity at 24 hr after application of the conditioned stimulus. The interferon levels measured in separate experiments were not uniformly elevated in conditioned animals compared with controls.

  12. Homing of radiolabelled recombinant interleukin-2 activated natural killer cells and their efficacy in adoptive immunotherapy against murine fibrosarcoma

    Indian Academy of Sciences (India)

    Anuradha Rai; Ashim K Chakravarty

    2007-12-01

    Natural killer (NK) cells are spontaneously cytotoxic against tumour target cells. Their number was found to be four times more in the spleen of tumour-bearing Swiss albino mice. After activation with recombinant interleukin-2 (rIL-2), NK cells were tested and found to seek out the tumour site when injected intravenously in tumour-bearing mice. Their potential for fighting tumours in vivo was further seen following adoptive transfer of rIL-2 activated NK (A-NK) cells in tumour-bearing mice. After surgical removal of tumour load, adoptive transfer of A-NK cells inhibited tumour recurrence in 92.3% cases, thereby suggesting the use of this protocol for therapeutic purposes to obtain a better outcome.

  13. Skin Cancer Risk Is Modified by KIR/HLA Interactions That Influence the Activation of Natural Killer Immune Cells.

    Science.gov (United States)

    Vineretsky, Karin A; Karagas, Margaret R; Christensen, Brock C; Kuriger-Laber, Jacquelyn K; Perry, Ann E; Storm, Craig A; Nelson, Heather H

    2016-01-15

    Natural killer (NK)-cell phenotype is partially mediated through binding of killer-cell immunoglobulin-like receptors (KIR) with HLA class I ligands. The KIR gene family is highly polymorphic and not well captured by standard genome-wide association study approaches. Here, we tested the hypothesis that variations in KIR gene content combined with HLA class I ligand status is associated with keratinocyte skin cancers using a population-based study of basal cell carcinoma (BCC) and squamous cell carcinomas (SCC). We conducted an interaction analysis of KIR gene content variation and HLA-B (Bw4 vs. Bw6) and HLA-C (C1 vs. C2). KIR centromeric B haplotype was associated with significant risk of multiple BCC tumors (OR, 2.39; 95% confidence interval, 1.10-5.21), and there was a significant interaction between HLA-C and the activating gene KIR2DS3 for BCC (Pinteraction = 0.005). Furthermore, there was significant interaction between HLA-B and telomeric KIR B haplotype (containing the activating genes KIR3DS1 and KIR2DS1) as well as HLA-B and the activating KIR gene KIR2DS5 (Pinteraction 0.001 and 0.012, respectively). Similar but greatly attenuated associations were observed for SCC. Moreover, previous in vitro models demonstrated that p53 is required for upregulation of NK ligands, and accordingly, we observed there was a strong association between the KIR B haplotype and p53 alteration in BCC tumors, with a higher likelihood that KIR B carriers harbor abnormal p53 (P KIR and HLA modify risks of BCC and SCC and that KIR encoded by the B genes provides selective pressure for altered p53 in BCC tumors.

  14. Natural Sphingomonas glycolipids vary greatly in their ability to activate natural killer T cells.

    Science.gov (United States)

    Kinjo, Yuki; Pei, Bo; Bufali, Simone; Raju, Ravinder; Richardson, Stewart K; Imamura, Masakazu; Fujio, Masakazu; Wu, Douglass; Khurana, Archana; Kawahara, Kazuyoshi; Wong, Chi-Huey; Howell, Amy R; Seeberger, Peter H; Kronenberg, Mitchell

    2008-07-21

    Mouse natural killer T (NKT) cells expressing an invariant T cell antigen receptor (TCR) recognize glycosphingolipids (GSLs) from Sphingomonas bacteria. The synthetic antigens previously tested, however, were designed to closely resemble the potent synthetic agonist alpha-galactosyl ceramide (alphaGalCer), which contains a monosaccharide and a C18:0 sphingosine lipid. Some Sphingomonas bacteria, however, also have oligosaccharide-containing GSLs, and they normally synthesize several GSLs with different sphingosine chains including one with a cyclopropyl ring-containing C21:0 (C21cycl) sphingosine. Here we studied the stimulation of NKT cells with synthetic GSL antigens containing natural tetrasaccharide sugars, or the C21cycl sphingosine. Our results indicate that there is a great degree of variability in the antigenic potency of different natural Sphingomonas glycolipids, with the C21cycl sphingosine having intermediate potency and the oligosaccharide-containing antigens exhibiting limited or no stimulatory capacity.

  15. The Development of a Micro Assay for Natural Killer and Lymphokine-Activated Killer Activity and Its Use in Monitoring the Purification of an Interleukin-2 Inhibitor

    Science.gov (United States)

    1989-05-01

    malaise, weight gain, diarrhea, rash, arthralgia, myalgia, fluid retention, anemia , 6 hypotension, dyspnea, and hyperbilirubinemia (21-23). There was...inhibitor levels in humans with these autoimune diseases to determine if there was any correlation between the inhibitor levels and disease activity

  16. Natural killer T cells in adipose tissue are activated in lean mice.

    Science.gov (United States)

    Kondo, Taisuke; Toyoshima, Yujiro; Ishii, Yoshiyuki; Kyuwa, Shigeru

    2013-01-01

    Adipose tissues are closely connected with the immune system. It has been suggested that metabolic syndromes such as type 2 diabetes, arteriosclerosis and liver steatosis can be attributed to adipose tissue inflammation characterized by macrophage infiltration. To understand a physiological and pathological role of natural killer T (NKT) cells on inflammation in adipose tissue, we characterized a subset of NKT cells in abdominal and subcutaneous adipose tissues in C57BL/6J mice fed normal or high-fat diets. NKT cells comprised a larger portion of lymphocytes in adipose tissues compared with the spleen and peripheral blood, with epididymal adipose tissue having the highest number of NKT cells. Furthermore, some NKT cells in adipose tissues expressed higher levels of CD69 and intracellular interferon-γ, whereas the Vβ repertoires of NKT cells in adipose tissues were similar to other cells. In obese mice fed a high-fat diet, adipose tissue inflammation had little effect on the Vβ repertoire of NKT cells in epididymal adipose tissues. We speculate that the NKT cells in adipose tissues may form an equivalent subset in other tissues and that these subsets are likely to participate in adipose tissue inflammation. Additionally, the high expression level of CD69 and intracellular IFN-γ raises the possibility that NKT cells in adipose tissue may be stimulated by some physiological mechanism.

  17. Effects of various Eleutherococcus senticosus cortex on swimming time, natural killer activity and corticosterone level in forced swimming stressed mice.

    Science.gov (United States)

    Kimura, Yoshiyuki; Sumiyoshi, Maho

    2004-12-01

    The cortex of Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. has been used extensively in Russia, China, Korea and Japan as an adaptogen whose properties are the ability to increase as non-specific body resistance to stress and fatigue. Although it has been reported that Eleutherococcus senticosus has anti-fatigue and anti-stress actions, their actions are still unclear on the relationship between immune system, especially natural killer (NK) activity and endocrine system (corticosterone level). We compared the effects of the water extracts (A, B, C, D and E) of five Eleutherococcus senticosus cortex on the swimming time, NK activity and blood corticosterone level using forced swimming stressed mice. Among five kinds, C, D and E extracts significantly prolonged the swimming time. C and D extracts inhibited the reduction of NK activity and the corticosterone elevation induced by forced swimming. The contents of eleutheroside E, isoflaxidin and eleutherosides B plus E were in the order C > D > E > B > A and C > E > D > A > B extracts, respectively. Therefore, it is suggested that eleutheroside E may be contributed to the anti-fatigue action, the recovery of the reduction of NK activity and the inhibition of corticosterone elevation induced by swimming stress.

  18. Improved Activation toward Primary Colorectal Cancer Cells by Antigen-Specific Targeting Autologous Cytokine-Induced Killer Cells

    Directory of Open Access Journals (Sweden)

    Claudia Schlimper

    2012-01-01

    Full Text Available Adoptive therapy of malignant diseases with cytokine-induced killer (CIK cells showed promise in a number of trials; the activation of CIK cells from cancer patients towards their autologous cancer cells still needs to be improved. Here, we generated CIK cells ex vivo from blood lymphocytes of colorectal cancer patients and engineered those cells with a chimeric antigen receptor (CAR with an antibody-defined specificity for carcinoembryonic antigen (CEA. CIK cells thereby gained a new specificity as defined by the CAR and showed increase in activation towards CEA+ colon carcinoma cells, but less in presence of CEA− cells, indicated by increased secretion of proinflammatory cytokines. Redirected CIK activation was superior by CAR-mediated CD28-CD3ζ than CD3ζ signaling only. CAR-engineered CIK cells from colon carcinoma patients showed improved activation against their autologous, primary carcinoma cells from biopsies resulting in more efficient tumour cell lysis. We assume that adoptive therapy with CAR-modified CIK cells shows improved selectivity in targeting autologous tumour lesions.

  19. Spontaneous focal activation of invariant natural killer T (iNKT cells in mouse liver and kidney

    Directory of Open Access Journals (Sweden)

    Zeng Jia

    2010-11-01

    Full Text Available Abstract Background Invariant natural killer T (iNKT cells differ from other T cells by their hyperactive effector T-cell status, in addition to the expression of NK lineage receptors and semi-invariant T-cell receptors. It is generally agreed that the immune phenotype of iNKT cells is maintained by repeated activation in peripheral tissues although no explicit evidence for such iNKT cell activity in vivo has so far been reported. Results We used an interferon (IFN-γ-inducible cytoplasmic protein, Irga6, as a histological marker for local IFN-γ production. Irga6 was intensely expressed in small foci of liver parenchymal cells and kidney tubular epithelium. Focal Irga6 expression was unaffected by germ-free status or loss of TLR signalling and was totally dependent on IFN-γ secreted by T cells in the centres of expression foci. These were shown to be iNKT cells by diagnostic T cell receptor usage and their activity was lost in both CD1 d and Jα-deficient mice. Conclusions This is the first report that supplies direct evidence for explicit activation events of NKT cells in vivo and raises issues about the triggering mechanism and consequences for immune functions in liver and kidney.

  20. Inositol hexaphosphate-induced enhancement of natural killer cell activity correlates with suppression of colon carcinogenesis in rats

    Institute of Scientific and Technical Information of China (English)

    Zheng Zhang; Yang Song; Xiu-Li Wang

    2005-01-01

    AIM: To investigate the anti-neoplastic effect of inositol hexaphosphate (InsP6 or phytic acid) on dimethylhydrazine (DMH)-induced colon tumor in rats and its effect on blood natural killer (NK) cell activity.METHODS: Healthy Wistar rats, 4 wk old, were divided into control group (fed with common food) and TnsP6 group (fed with common food+2% sodium inositol hexaphosphate in the drinking water), 15 rats in each group. Both groups were injected with 1,2-dimethylhydrazine subcutaneously (20 mg/kg body weight) once a week for 20 wk. Rats were killed after 21 wk. The whole large intestine was isolated to determine the general condition of tumors and to test blood NK cell activity by lactate-dehydrogenaserelease assay.RESULTS: Administration of InsP6 significantly increased blood NK cell activity in DMH-induced colorectal tumor in rats. InsP6 group had a smaller tumor size on average and a smaller number of tumors than the control group. Its mortality was also higher than that in control. However, the variables of body weight and tumor incidence were not significantly different between the two groups.CONCLUSION: InsP6 can increase blood NK cell activity in DMH-induced colon tumor in rats and inhibit tumor growth and metastasis in rats.

  1. Expansion and activation of natural killer cells from PBMC for immunotherapy of hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Bao-Gang Peng; Li-Jian Liang; Qiang He; Jie-Fu Huang; Ming-De Lu

    2004-01-01

    AIM: To induce efficient expansion of natural killer (NK) cells from peripheral blood mononuclear cells (PBMCs) using a culture of anchorage-dependent Wilms tumor cell lines, and to provide a reliable supply for adoptive immunotherapy of hepatocellular carcinoma.METHODS: Culture expansion of NK cells was achieved using PBMCs cultured with Wilms tumor cells. Cytotoxicity was measured using a standard 51Cr release assay and crystal violet staining technique. The proportions of CD3+, CD4+, CD8+,CD16+, and CD56+ cells were determined by flow cytometry.RESULTS: After PBMCs from healthy donors and hepatocellular carcinoma (HCC) were cultured with irradiated HFWT cells for 10-21 d, CD56+ CD16+ cells shared more than 50% of the cell population, and more than 80% of fresh HFWT cells were killed at an effector/target ratio of 2 over 24 h. NK-enriched lymphocyte population from HCC patients killed HCC-1 and 2 cells with sensitivities comparable to fresh TKB-17RGB cells. HCC cells proliferated 196-fold with the irradiated HFWT cells at 18 d. Stimulation by HFWT cells required intimate cell-cell interaction with PBMC. However, neither the soluble factors released from HFWT cells nor the fixed HFWT cells were effective for NK expansion. The lymphocytes expanded with IL-2 killed fresh HFWT target cells more effectively than the lymphocytes expanded with the 4-cytokine cocktail (IL-1 β, IL-2, IL-4 and IL-6). IL-2 was the sole cytokine required for NK expansion.CONCLUSION: Wilms tumor is sensitive to human NK cells and is highly efficient for selective expansion of NK cells from PBMCs.

  2. Antitumor activities of human autologous cytokine-induced killer (CIK) cells against hepatocellular carcinoma cells in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    Fu-Sheng Wang; Ming-Xu Liu; Bing Zhang; Ming Shi; Zhou-Yun Lei; Wen-Bing Sun; Qing-You Du; Ju-Mei Chen

    2002-01-01

    AIM: To characterize the anticancer function of cytokine-induced killer cells (CIK) and develop an adoptiveimmunotherapy for the patients with primary hepatocellularcarcinoma (HCC), we evaluated the proliferation rate,phenotype and the antitumor activity of human CIK cellsfrom healthy donors and HCC patients in vitro and in vivo.METHODS: Peripheral blood mononuclear cells (PBMC) fronhealthy donors and patients with primary HCC were incubatedin vitro and induced into ClK cells in the presence of variouscytokines such as interferon-gamma (IFN-γ), interleukin-1(IL-1), IL-2, and monoclonal antibody (mAb) against CD3.The phenotype and characterization of CIK cells wereidentified by flow cytometric analysis. The cytotoxicity of CIKcells was determined by 51 Cr release assay.RESULTS: The CIK cells were shown to be a heterogeneouspopulation with different cellular phenotypes. Thepercentage of CD3+/CD56+ positive cells, the dominanteffector cells, in total CIK cells from healthy donors andHCC patients, significantly increased from 0.1-0.13 % at day0 to 19.0-20.5 % at day 21 incubation, which suggested thatthe CD3+ CD56+ positive cells proliferated faster than othercell populations of CIK cells in the protocol used in thisstudy. After 28 day in vitro incubation, the ClK cells frompatients with HCC and healthy donors increased by morethan 300-fold and 500-fold in proliferation cell number,respectively. CIK cells originated from HCC patientspossessed a higher in vitro antitumor cytotoxic activity onautologous HCC cells than the autologous lymphokine-activated killer (LAK) cells and PBMC cells. In in vivoanimal experiment, CIK cells had stronger effects on theinhibition of tumor growth in Balb/c nude mice bearing BEL-7402-producing tumor than LAK cells (mean inhibitory rate,84.7 % vs 52.8 %, P < 0.05) or PBMC (mean inhibitoryrate, 84.7% vs37.1%, P<0.01).CONCLUSION: Autologous CIK cells are of highly efficientcytotoxic effector cells against primary hepatocellularcarcinoma

  3. Keiko, Killer Whale. [Lesson Plan].

    Science.gov (United States)

    Discovery Communications, Inc., Bethesda, MD.

    This lesson plan presents activities designed to help students understand that Keiko, the killer whale, lived for a long time in an aquarium and had to be taught to live independently; and that computer users can get updates on how Keiko is doing. The main activity of the lesson involves middle school students working in small groups to produce a…

  4. Transfection of malaria parasites.

    Science.gov (United States)

    Waters, A P; Thomas, A W; van Dijk, M R; Janse, C J

    1997-10-01

    The stable genetic transformation of three phylogenetically diverse species of Plasmodium, the parasitic etiological agent of malaria, is now possible. The parasite is haploid throughout the vast majority of its life cycle. Therefore with the single selectable marker activity and protocols currently available, it is possible not only to express introduced transgenes but also to study the effects of site-specific homologous recombination such as gene knockout. Transgene expression will allow the detailed study of many aspects of the cellular biology of malaria parasites, for example, the mechanisms underlying drug resistance and protein trafficking. We describe here the methods for propagation of the two animal models (Plasmodium berghei and Plasmodium knowlesi) and for transfection of these two species and the human parasite, Plasmodium falciparum. Examples of transgene expression are given.

  5. Variation in the emission rate of sounds in a captive group of false killer whales Pseudorca crassidens during feedings: possible food anticipatory vocal activity?

    Science.gov (United States)

    Platto, Sara; Wang, Ding; Wang, Kexiong

    2016-11-01

    This study examines whether a group of captive false killer whales ( Pseudorca crassidens ) showed variations in the vocal rate around feeding times. The high level of motivation to express appetitive behaviors in captive animals may lead them to respond with changes of the behavioral activities during the time prior to food deliveries which are referred to as food anticipatory activity. False killer whales at Qingdao Polar Ocean World (Qingdao, China) showed significant variations of the rates of both the total sounds and sound classes (whistles, clicks, and burst pulses) around feedings. Precisely, from the Transition interval that recorded the lowest vocalization rate (3.40 s/m/d), the whales increased their acoustic emissions upon trainers' arrival (13.08 s/m/d). The high rate was maintained or intensified throughout the food delivery (25.12 s/m/d), and then reduced immediately after the animals were fed (9.91 s/m/d). These changes in the false killer whales sound production rates around feeding times supports the hypothesis of the presence of a food anticipatory vocal activity. Although sound rates may not give detailed information regarding referential aspects of the animal communication it might still shed light about the arousal levels of the individuals during different social or environmental conditions. Further experiments should be performed to assess if variations of the time of feeding routines may affect the vocal activity of cetaceans in captivity as well as their welfare.

  6. Transfection of B7-1 cDNA empowers antigen presentation of blood malignant cells for activation of anti-tumor T cells

    Institute of Scientific and Technical Information of China (English)

    克晓燕; 贾丽萍; 王晶; 王德炳

    2003-01-01

    Objective To define roles of B7-1 co-stimulation factor expressed in human malignant cell lines in mediating anti-tumor T cell immune responses. Methods Examining human leucocyte antigen (HLA) and B7 expressions on 8 human blood malignancies cell lines by flow cytometry. Transfecting B7-1 gene to B7-1 negative (B7*!-) Raji and B7*!- Jurkat cell lines by liposome, and comparing the potencies of blood malignant cell lines in the induction of T cell activation by examination of T cell cytokine mRNAs before and after transfection using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Results High level of HLA Ⅰ and Ⅱ molecules were expressed in most human blood malignant cell lines examined, and the co-stimulatory factor B7-2 was also highly expressed. In contrast, another member of B7 family: B7-1 was either not expressed or very limitedly expressed in most of these hematopoietic malignant cell lines. Most importantly, transfection of B7-1 gene to B7*!-. Raji and B7*!-. Jurkat cell lines made these cell lines better antigen presenting cells for stimulation of anti-tumor T cell activation, which was demonstrated by up regulation of expression of T cell cytokines IL-2, IL-4 and INF-γ mRNAs after incubation of these tumor cells with T cells for 24 h. Conclusions B7 co-stimulation plays an important role in anti-tumor immunity. Transfection of B7-1 gene to the human hematopoietic malignant cell lines that are deficient in the B7-1 expression empowers their antigen presentation potency for activation of anti-tumor T cells. Our results suggested that repairing the deficiency of B7-1 co-stimulatory pathway in tumor cells might be a novel immunotherapeutic approach for human hematopoietic malignancies.

  7. NKG2D functions as an activating receptor on natural killer cells in the common marmoset (Callithrix jacchus).

    Science.gov (United States)

    Watanabe, Masamichi; Kudo, Yohei; Kawano, Mitsuko; Nakayama, Masafumi; Nakamura, Kyohei; Kameda, Mai; Ebara, Masamune; Sato, Takeki; Nakamura, Marina; Omine, Kaito; Kametani, Yoshie; Suzuki, Ryuji; Ogasawara, Kouetsu

    2014-11-01

    The natural killer group 2 membrane D (NKG2D) receptor is an NK-activating receptor that plays an important role in host defense against tumors and viral infections. Although the marmoset is an important and reliable animal model, especially for the study of human-specific viral infections, functional characterization of NKG2D on marmoset NK cells has not previously been conducted. In the present study, we investigated a subpopulation of marmoset NK cells that express NKG2D and exhibit cytolytic potential. On the basis of their CD16 and CD56 expression patterns, marmoset NK cells can be classified into three subpopulations: CD16(+) CD56(-), CD16(-) CD56(+) and CD16(-) CD56(-) cells. NKG2D expression on marmoset CD16(+) CD56(-) and CD16(-) CD56(+) splenocytes was confirmed using an NKG2D ligand composed of an MHC class I chain-related molecule A (MICA)-Fc fusion protein. When marmoset splenocytes were cultured with IL-2 for 4 days, NKG2D expression was retained on CD16(+) CD56(-) and CD16(-) CD56(+). In addition, CD16(+) CD56(+) cells within the marmoset NK population appeared which expressed NKG2D after IL-2 stimulation. IL-2-activated marmoset NK cells showed strong cytolytic activity against K562 target cells and target cells stably expressing MICA. Further, the cytolytic activity of marmoset splenocytes was significantly reduced after addition of MICA-Fc fusion protein. Thus, NKG2D functions as an activating receptor on marmoset NK cells that possesses cytotoxic potential, and phenotypic profiles of marmoset NK cell subpopulations are similar to those seen in humans.

  8. Dendritic cell editing by activated natural killer cells results in a more protective cancer-specific immune response.

    Directory of Open Access Journals (Sweden)

    Barbara Morandi

    Full Text Available Over the last decade, several studies have extensively reported that activated natural killer (NK cells can kill autologous immature dendritic cells (DCs in vitro, whereas they spare fully activated DCs. This led to the proposal that activated NK cells might select a more immunogenic subset of DCs during a protective immune response. However, there is no demonstration that autologous DC killing by NK cells is an event occurring in vivo and, consequently, the functional relevance of this killing remains elusive. Here we report that a significant decrease of CD11c(+ DCs was observed in draining lymph nodes of mice inoculated with MHC-devoid cells as NK cell targets able to induce NK cell activation. This in vivo DC editing by NK cells was perforin-dependent and it was functionally relevant, since residual lymph node DCs displayed an improved capability to induce T cell proliferation. In addition, in a model of anti-cancer vaccination, the administration of MHC-devoid cells together with tumor cells increased the number of tumor-specific CTLs and resulted in a significant increase in survival of mice upon challenge with a lethal dose of tumor cells. Depletion of NK cells or the use of perforin knockout mice strongly decreased the tumor-specific CTL expansion and its protective role against tumor cell challenge. As a whole, our data support the hypothesis that NK cell-mediated DC killing takes place in vivo and is able to promote expansion of cancer-specific CTLs. Our results also indicate that cancer vaccines could be improved by strategies aimed at activating NK cells.

  9. Hiding Lipid Presentation: Viral Interference with CD1d-Restricted Invariant Natural Killer T (iNKT Cell Activation

    Directory of Open Access Journals (Sweden)

    Maaike E. Ressing

    2012-10-01

    Full Text Available The immune system plays a major role in protecting the host against viral infection. Rapid initial protection is conveyed by innate immune cells, while adaptive immunity (including T lymphocytes requires several days to develop, yet provides high specificity and long-lasting memory. Invariant natural killer T (iNKT cells are an unusual subset of T lymphocytes, expressing a semi-invariant T cell receptor together with markers of the innate NK cell lineage. Activated iNKT cells can exert direct cytolysis and can rapidly release a variety of immune-polarizing cytokines, thereby regulating the ensuing adaptive immune response. iNKT cells recognize lipids in the context of the antigen-presenting molecule CD1d. Intriguingly, CD1d-restricted iNKT cells appear to play a critical role in anti-viral defense: increased susceptibility to disseminated viral infections is observed both in patients with iNKT cell deficiency as well as in CD1d- and iNKT cell-deficient mice. Moreover, viruses have recently been found to use sophisticated strategies to withstand iNKT cell-mediated elimination. This review focuses on CD1d-restricted lipid presentation and the strategies viruses deploy to subvert this pathway.

  10. Scorpion venom activates natural killer cells in hepatocellular carcinoma via the NKG2D-MICA pathway.

    Science.gov (United States)

    Chen, Han; Zhidan, Wang; Xia, Ren; Zhaoxia, Wang; Qing, Jia; Qiang, Guo; Haipeng, Yin; Hengxiao, Wang

    2016-06-01

    Previous studies have demonstrated that polypeptides extracted from scorpion venom (PESV) inhibited cell proliferation in several tumors, however, the effect on dysfunctional and exhausted natural killer cells which contribute to tumor escape from immune surveillance remain to be elucidated. In this study, we determined the effect of PESV on NK infiltration into H22 cells orthotopic transplantation tumors and on the expression of MHC class I chain-related proteins A (MICA) in HepG2 cells. We found that tumor growth in mice was significantly inhibited by PESV and the survival time of tumor-bearing mice treated with PESV was significantly prolonged. Moreover, levels of tumor-infiltrating NK cells, NKG2D protein, perforin and granzyme B mRNA were significantly increased in the group treated with PESV compared with the tumor-bearing control group. In addition, In addition, up-regulation of MICA by PESV enhances the susceptibility of HepG2 cells to NK lysis in vitro. These results indicate that the inhibitory effects of PESV on hepatic carcinoma are likely mediated by up-regulation of NK cell activity via the MICA-NKG2D pathway.

  11. Deficient natural killer cell function in preeclampsia

    Energy Technology Data Exchange (ETDEWEB)

    Alanen, A.; Lassila, O.

    1982-11-01

    Natural killer cell activity of peripheral blood lymphocytes was measured against K-562 target cells with a 4-hour /sup 51/Cr release assay in 15 primigravid women with preeclamptic symptoms. Nineteen primigravid women with an uncomplicated pregnancy and 18 nonpregnant women served as controls. The natural killer cell activity of preeclamptic women was observed to be significantly lower than that of both control groups. Natural killer cells in preeclamptic women responded normally to augmentation caused by interferon. These findings give further evidence for the participation of the maternal immune system in this pregnancy disorder.

  12. Spermidine/spermine N1-acetyltransferase (SSAT) activity in human small-cell lung carcinoma cells following transfection with a genomic SSAT construct.

    Science.gov (United States)

    Murray-Stewart, Tracy; Applegren, Nancy B; Devereux, Wendy; Hacker, Amy; Smith, Renee; Wang, Yanlin; Casero, Robert A

    2003-07-15

    Spermidine/spermine N (1)-acetyltransferase (SSAT) activity is typically highly inducible in non-small-cell lung carcinomas in response to treatment with anti-tumour polyamine analogues, and this induction is associated with subsequent cell death. In contrast, cells of the small-cell lung carcinoma (SCLC) phenotype generally do not respond to these compounds with an increase in SSAT activity, and usually are only moderately affected with respect to growth. The goal of the present study was to produce an SSAT-overexpressing SCLC cell line to further investigate the role of SSAT in response to these anti-tumour analogues. To accomplish this, NCI-H82 SCLC cells were stably transfected with plasmids containing either the SSAT genomic sequence or the corresponding cDNA sequence. Individual clones were selected based on their ability to show induced SSAT activity in response to exposure to a polyamine analogue, and an increase in the steady-state SSAT mRNA level. Cells transfected with the genomic sequence exhibited a significant increase in basal SSAT mRNA expression, as well as enhanced SSAT activity, intracellular polyamine pool depletion and growth inhibition following treatment with the analogue N (1), N (11)-bis(ethyl)norspermine. Cells containing the transfected cDNA also exhibited an increase in the basal SSAT mRNA level, but remained phenotypically similar to vector control cells with respect to their response to analogue exposure. These studies indicate that both the genomic SSAT sequence and polyamine analogue exposure play a role in the transcriptional and post-transcriptional regulation and subsequent induction of SSAT activity in these cells. Furthermore, this is the first production of a cell line capable of SSAT protein induction from a generally unresponsive parent line.

  13. In vitro and in vivo activity of a killer peptide against Malassezia pachydermatis causing otitis in dogs.

    Science.gov (United States)

    Cafarchia, Claudia; Immediato, Davide; Paola, Giancarlo Di; Magliani, Walter; Ciociola, Tecla; Conti, Stefania; Otranto, Domenico; Polonelli, Luciano

    2014-05-01

    In order to overcome the limitations inherent in current pharmacological treatments for Malassezia pachydermatis, the cause of otitis externa in dogs, the efficacy of a killer decapeptide (KP) was evaluated in vitro and in vivo. Sixteen dogs with naturally occurring M. pachydermatis otitis externa were enrolled, and the in vitro fungicidal activity of KP was evaluated using yeasts recovered from these animals. The therapeutic activity was evaluated in four groups of four animals each. The dogs were topically treated with KP (150 μl, 2 mg/ml) three times per week (group A) or every day (group B), treated with a scramble peptide every day (group C), or left untreated (group D). Assessment of clinical signs (pruritus, erythema, and lichenification and/or hyperpigmentation), expressed as mean of the total clinical index score (mTCIS), the population size of M. pachydermatis at the cytological examination (mean number of yeast cells at 40× magnification [mYC]), and culture testing (mean number of log10 CFU/swab [mCFU]), were conducted daily from the first day of treatment (T0) until two consecutive negative cultures (mCFU ≤ 2). KP showed an in vitro fungicidal effect against M. pachydermatis isolates, with an MFC90 value of 1 μg/ml. The mTCIS, mYC and mCFU were negative only in animals in group B after T8. Daily administration of KP for 8 days was safe and effective in controlling both clinical signs and the population size of M. pachydermatis causing otitis externa, thus offering an alternative to the currently available therapeutic or prophylactic protocols for recurrent cases of Malassezia otitis in dogs.

  14. Docosahexaenoic acid ingestion inhibits natural killer cell activity and production of inflammatory mediators in young healthy men.

    Science.gov (United States)

    Kelley, D S; Taylor, P C; Nelson, G J; Schmidt, P C; Ferretti, A; Erickson, K L; Yu, R; Chandra, R K; Mackey, B E

    1999-04-01

    The purpose of this study was to examine the effects of feeding docosahexaenoic acid (DHA) as triacylglycerol on the fatty acid composition, eicosanoid production, and select activities of human peripheral blood mononuclear cells (PBMNC). A 120-d study with 11 healthy men was conducted at the Metabolic Research Unit of Western Human Nutrition Reach Center. Four subjects (control group) were fed the stabilization diet throughout the study; the remaining seven subjects were fed the basal diet for the first 30 d, followed by 6 g DHA/d for the next 90 d. DHA replaced an equivalent amount of linoleic acid; the two diets were comparable in their total fat and all other nutrients. Both diets were supplemented with 20 mg D alpha-tocopherol acetate per day. PBMNC fatty acid composition and eicosanoid production were examined on day 30 and 113; immune cell functions were tested on day 22, 30, 78, 85, 106, and 113. DHA feeding increased its concentration from 2.3 to 7.4 wt% in the PBMNC total lipids, and decreased arachidonic acid concentration from 19.8 to 10.7 wt%. It also lowered prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) production, in response to lipopolysaccharide, by 60-75%. Natural killer cell activity and in vitro secretion of interleukin-1beta and tumor necrosis factor alpha were significantly reduced by DHA feeding. These parameters remained unchanged in the subjects fed the control diet. B-cell functions as reported here and T-cell functions that we reported previously were not altered by DHA feeding. Our results show that inhibitory effects of DHA on immune cell functions varied with the cell type, and that the inhibitory effects are not mediated through increased production of PGE2 and LTB4.

  15. Natural killer cell dysfunction is a distinguishing feature of systemic onset juvenile rheumatoid arthritis and macrophage activation syndrome.

    Science.gov (United States)

    Villanueva, Joyce; Lee, Susan; Giannini, Edward H; Graham, Thomas B; Passo, Murray H; Filipovich, Alexandra; Grom, Alexei A

    2005-01-01

    Macrophage activation syndrome (MAS) has been reported in association with many rheumatic diseases, most commonly in systemic juvenile rheumatoid arthritis (sJRA). Clinically, MAS is similar to hemophagocytic lymphohistiocytosis (HLH), a genetic disorder with absent or depressed natural killer (NK) function. We have previously reported that, as in HLH, patients with MAS have profoundly decreased NK activity, suggesting that this abnormality might be relevant to the pathogenesis of the syndrome. Here we examined the extent of NK dysfunction across the spectrum of diseases that comprise juvenile rheumatoid arthritis (JRA). Peripheral blood mononuclear cells (PBMC) were collected from patients with pauciarticular (n = 4), polyarticular (n = 16), and systemic (n = 20) forms of JRA. NK cytolytic activity was measured after co-incubation of PBMC with the NK-sensitive K562 cell line. NK cells (CD56+/T cell receptor [TCR]-alphabeta-), NK T cells (CD56+/TCR-alphabeta+), and CD8+ T cells were also assessed for perforin and granzyme B expression by flow cytometry. Overall, NK cytolytic activity was significantly lower in patients with sJRA than in other JRA patients and controls. In a subgroup of patients with predominantly sJRA, NK cell activity was profoundly decreased: in 10 of 20 patients with sJRA and in only 1 of 20 patients with other JRA, levels of NK activity were below two standard deviations of pediatric controls (P = 0.002). Some decrease in perforin expression in NK cells and cytotoxic T lymphocytes was seen in patients within each of the JRA groups with no statistically significant differences. There was a profound decrease in the proportion of circulating CD56bright NK cells in three sJRA patients, a pattern similar to that previously observed in MAS and HLH. In conclusion, a subgroup of patients with JRA who have not yet had an episode of MAS showed decreased NK function and an absence of circulating CD56bright population, similar to the abnormalities observed

  16. Bovine natural killer cells are present in Escherichia coli infected mammary gland tissue and show antimicrobial activity in vitro

    NARCIS (Netherlands)

    Sipka, Anja; Pomeroy, Brianna; Klaessig, Suzanne; Schukken, Ynte

    2016-01-01

    Natural killer (NK) cells are early responders in bacterial infections but their role in bovine mastitis has not been characterized. For the first time, we show the presence of NK cells (NKp46+/CD3) in bovine mammary gland tissue after an intramammary challenge with Escheri

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

    Science.gov (United States)

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

    2016-09-01

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

  18. Enhanced natural killer cell activation by exopolysaccharides derived from yogurt fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1.

    Science.gov (United States)

    Makino, Seiya; Sato, Asako; Goto, Ayako; Nakamura, Marie; Ogawa, Miho; Chiba, Yoshika; Hemmi, Jun; Kano, Hiroshi; Takeda, Kazuyoshi; Okumura, Ko; Asami, Yukio

    2016-02-01

    Yogurt is generally recognized as a beneficial food for our health, but research into its physiological effects has focused mainly on intestinal dysfunctions such as constipation and diarrhea. We previously found yogurt fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (hereafter OLL1073R-1) could reduce risks of catching the common cold and flu in human trials. It was assumed that immunostimulatory exopolysaccharide (EPS) produced from OLL1073R-1 play an important role in this context. However, few studies have examined the immunostimulatory effects of traditional Bulgarian yogurts fermented with different strains of lactobacilli and their metabolites. Therefore, we screened 139 L. delbrueckii ssp. bulgaricus strains and identified OLL1073R-1 as the most robust producer of EPS. This strain was also the only strain that induced the production of IFN-γ in vitro. Oral administration of the EPS or yogurt fermented with OLL1073R-1 and Streptococcus thermophilus OLS3059 (OLL1073R-1 yogurt) augmented natural killer (NK) cell activity and induced IFN-γ production in spleen cells in mice, whereas 2 other yogurts fermented with other strains had no effect on NK cell activity. Cellular preparations of the OLL1073R-1 strain also slightly augmented NK cell activity, but were less effective than EPS itself. The EPS-dependent stimulation of NK cell activity was abrogated in IFN-γ knockout mice and in myeloid differentiation factor 88 knockout mice. Furthermore, IFN-γ production from spleen cells stimulated with EPS was completely blocked with both anti-IL-12 and anti-IL-18 antibodies in vitro. These findings suggest that NK cell activation by OLL1073R-1 yogurt is EPS-dependent, occurs via IL-12- and IL-18-mediated IFN-γ production, and requires myeloid differentiation factor 88. We showed that traditional Bulgarian yogurt could exert immunostimulatory effects by selecting starter strains and part of the mechanisms depend on IFN-γ inducible EPS produced

  19. Activating killer cell Ig-like receptors in health and disease

    Directory of Open Access Journals (Sweden)

    Martin A Ivarsson

    2014-04-01

    Full Text Available Expression of non-rearranged HLA class I-binding receptors characterizes human and mouse NK cells. The postulation of the missing-self hypothesis some 30 years ago triggered the subsequent search and discovery of inhibitory MHC-receptors, both in humans and mice. These receptors have two functions; i to control the threshold for NK cell activation, a process termed licensing or education, and ii to inhibit NK cell activation during interactions with healthy HLA class I-expressing cells. The discovery of activating forms of KIRs (aKIR challenged the concept of NK cell tolerance in steady state, as well as during immune challenge: what is the biological role of the activating KIR, in particular when NK cells express aKIRs in the absence of inhibitory receptors? Recently it was shown that aKIRs also participate in the education of NK cells. However, instead of lowering the threshold of activation like iKIRs, the expression of aKIRs has the opposite effect, i.e. rendering NK cells hyporesponsive. These findings may have consequences during NK cell response to viral infection, in cancer development, and in the initial stages of pregnancy. Here we review the current knowledge of activating KIRs, including the biological concept of aKIR-dependent NK cell education, and their impact in health and disease.

  20. PENGARUH EKSTRAK JAMU TERHADAP AKTIVITAS SEL NATURAL KILLER DALAM MELISIS ALUR SEL LEUKIMIA (K-562 SECARA IN VITRO [The Effects of Commercial “Jamu” Extracts on Natural Killer Cell Activity in Lysing Leukemic Cell Line (K-562 in vitro

    Directory of Open Access Journals (Sweden)

    Elisa Veronica D.C. 2

    2002-04-01

    Full Text Available Natural killer (NK cell consitutes white blood cells which specifically functions in lysing tumor and virus invected cells. In this research, a commercial “Jamu” was tested to observe its effect on NK cells activity against leukemic cell lines (K562 in vitro. Jamu was extracted with hot water, diluted and added into cell cultures consisted of a mixture of human peripheric limphocyte cells, as the source of the effector NK cells, and K562 cell line i.e., the target cells which were cell line derived from human leukemia and had been labelled with H3-thymidine. The mixture of the cells were made by culturing the two cells at the ratio of 50:1 and 100 : 1, respectively. The results showed that lysing activity of NK cells in the presence of “Jamu” water extract measured as lysing percentage and lysing index increased only slightly, which were not statiscally significant. It should be considered that the test used in this research represents only a part of the lysing mechanism by NK cells against the target cells. An in vivo test for a period of time will be recessary to elucidate ffurther this NK cell activity.

  1. Application of natural killer cell activity assay to the diagnosis and treatment of cancers.

    Institute of Scientific and Technical Information of China (English)

    1995-01-01

    In order to study the mechanism and effect of NK cells in cancer patients a ~3H-TdR incorporation suppressive method was used for dynamic comparison and analysis of NK cell activity in 58 cancer patients and 20 patients with benign lesions before and after operation and immunotherapy. The reults showed that

  2. Natural Killer Cells Are Activated by Lactic Acid Bacteria-Matured Dendritic Cells

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen; Christensen, Hanne Risager; Frøkiær, Hanne

    of certain lactic acid bacteria has been shown to increase in vivo NK cytotoxicity. Here, we investigated how human gut flora-derived lactobacilli affect NK cells in vitro, by measuring proliferation and IFN-gamma production of human NK cells upon bacterial stimulation. Human peripheral blood NK cells were...... with lactic acid bacteria will affect NK cell activation. Such activation of NK cells may potentially skew an on-going or subsequent immune response towards a Th1 response....... incubated with 10 microg/ml UV-inactivated bacteria or 10 microg/ml phytohemagglutinin (PHA) for four days. Proliferation was assessed by incorporation of radioactive thymidine into NK cell DNA. The IFN-gamma concentration was measured by ELISA. Incubation of NK cells with a Lactobacillus acidophilus strain...

  3. ARK, the Apaf-1 related killer in Drosophila, requires diverse domains for its apoptotic activity.

    Science.gov (United States)

    Srivastava, M; Scherr, H; Lackey, M; Xu, D; Chen, Z; Lu, J; Bergmann, A

    2007-01-01

    In mammals and Drosophila, apoptotic caspases are under positive control of the CED-4-like proteins Apaf-1 and ARK, respectively. In an EMS-mutagenesis screen, we isolated 33 ark mutants as recessive suppressors of hid-induced apoptosis. The ark mutants are loss-of-function alleles characterized by reduced developmental apoptosis. Using the phenotypic series of these alleles, we identified helical domain I in the nucleotide oligomerization domain as critical for ARK's apoptotic activity. Interestingly, the WD40 region may also have an unanticipated positive requirement for the apoptotic activity of ARK. Considering structural information, we discuss the roles of these domains for assembly and activity of the ARK apoptosome, and propose that the WD40 region is anti-apoptotic in the absence of apoptotic signals, and pro-apoptotic in the presence of such signals. Furthermore, a defined null allele reveals that ark is required for most, but not all apoptosis suggesting the existence of an ARK-independent apoptotic pathway.

  4. Activating Receptor Signals Drive Receptor Diversity in Developing Natural Killer Cells.

    Science.gov (United States)

    Freund, Jacquelyn; May, Rebecca M; Yang, Enjun; Li, Hongchuan; McCullen, Matthew; Zhang, Bin; Lenvik, Todd; Cichocki, Frank; Anderson, Stephen K; Kambayashi, Taku

    2016-08-01

    It has recently been appreciated that NK cells exhibit many features reminiscent of adaptive immune cells. Considerable heterogeneity exists with respect to the ligand specificity of individual NK cells and as such, a subset of NK cells can respond, expand, and differentiate into memory-like cells in a ligand-specific manner. MHC I-binding inhibitory receptors, including those belonging to the Ly49 and KIR families, are expressed in a variegated manner, which creates ligand-specific diversity within the NK cell pool. However, how NK cells determine which inhibitory receptors to express on their cell surface during a narrow window of development is largely unknown. In this manuscript, we demonstrate that signals from activating receptors are critical for induction of Ly49 and KIR receptors during NK cell development; activating receptor-derived signals increased the probability of the Ly49 bidirectional Pro1 promoter to transcribe in the forward versus the reverse direction, leading to stable expression of Ly49 receptors in mature NK cells. Our data support a model where the balance of activating and inhibitory receptor signaling in NK cells selects for the induction of appropriate inhibitory receptors during development, which NK cells use to create a diverse pool of ligand-specific NK cells.

  5. Increased natural killer activity does not prevent progression of experimental Kala-azar Atividade "natural-killer" aumentada não impede a progressão do Kala-azar experimental

    Directory of Open Access Journals (Sweden)

    Alexandrina SARTORI

    1999-07-01

    Full Text Available Kala-azar is the visceral form of leishmaniasis and it is caused by intracellular parasites from the complex Leishmania donovani. Golden hamster (Mesocricetus auratus infected with Leishmania donovani develop a disease very similar to human Kala-azar. There is conspicuous hipergammaglobulinaemia and their T cells do not respond to stimulation with parasite antigens. We used this experimental model to evaluate the natural killer (NK activity during the initial phase of the disease. Outbred hamsters infected by intravenous route with 5.106 amastigotes of L. donovani 1S showed a concurrent increase in the spleen weight and in the spleen cell number. Using the single cell assay we detected a significant increase in the percentage of NK effector cells on the 4th day of infection. Imprints from spleen and liver showed at days 14 and 28 a significant increase in the parasite burden . These results show that the increased NK activity in the beginning of the infection was not able to restrain the progression of the disease in this experimental model.O Kala-azar é a forma visceral da leishmaniose e é causado pelos parasitas do complexo Leishmania donovani. O hamster dourado (Mesocricetus auratus infectado com L. donovani desenvolve uma doença bastante similar ao Kala-azar humano, apresentando hipergamaglobulinemia e supressão da resposta imune celular específica. Utilizamos este modelo experimental para avaliar a atividade natural killer (NK na fase inicial da infecção. Hamsters não isogênicos infectados por via intravenosa com 5.106 amastigotas de L. donovani 1S apresentaram aumento no peso do baço e no número de células esplênicas. Utilizando o "single cell assay" detectamos um aumento significativo no percentual de células NK efetoras no 4º dia de infecção. "Imprints" de baço e de fígado mostraram aumento significativo na carga parasitária após 14 e 28 dias de infecção. Os resultados mostram que o aumento da atividade NK

  6. Follicular lymphoma: in vitro effects of combining lymphokine-activated killer (LAK) cell-induced cytotoxicity and rituximab- and obinutuzumab-dependent cellular cytotoxicity (ADCC) activity.

    Science.gov (United States)

    García-Muñoz, Ricardo; López-Díaz-de-Cerio, Ascensión; Feliu, Jesus; Panizo, Angel; Giraldo, Pilar; Rodríguez-Calvillo, Mercedes; Grande, Carlos; Pena, Esther; Olave, Mayte; Panizo, Carlos; Inogés, Susana

    2016-04-01

    Follicular lymphoma (FL) is a disease of paradoxes-incurable but with a long natural history. We hypothesized that a combination of lymphokine-activated killer (LAK) cells and monoclonal antibodies might provide a robust synergistic treatment and tested this hypothesis in a phase II clinical trial (NCT01329354). In this trial, in addition to R-CHOP, we alternated the administration of only rituximab with rituximab and autologous LAK cells that were expanded ex vivo. Our objective was to determine the in vitro capability of LAK cells generated from FL patients to produce cytotoxicity against tumor cell lines and to determine rituximab- and obinutuzumab-induced cytotoxicity via antibody-dependent cellular cytotoxicity (ADCC) activity. We analyzed the LAK cell-induced cytotoxicity and rituximab (R)- and obinutuzumab (GA101)-induced ADCC activity. We show that LAK cells generated from FL patients induce cytotoxicity against tumor cell lines. R and GA101 enhance cytolysis through ADCC activity of LAK cells. Impaired LAK cell cytotoxicity and ADCC activity were detected in 50 % of patients. Percentage of NK cells in LAK infusions were correlated with the R- and GA101-induced ADCC. Our results indicate that the combination of R or GA101 and LAK cells should be an option as frontline maintenance therapy in patients with FL.

  7. Suicide in serial killers.

    Science.gov (United States)

    Lester, David; White, John

    2010-02-01

    In a sample of 248 killers of two victims in America from 1900 to 2005, obtained from an encyclopedia of serial killers by Newton (2006), those completing suicide did not differ in sex, race, or the motive for the killing from those who were arrested.

  8. Fucoidan from Sargassum sp. and Fucus vesiculosus reduces cell viability of lung carcinoma and melanoma cells in vitro and activates natural killer cells in mice in vivo.

    Science.gov (United States)

    Ale, Marcel Tutor; Maruyama, Hiroko; Tamauchi, Hidekazu; Mikkelsen, Jørn D; Meyer, Anne S

    2011-10-01

    Fucoidan is known to exhibit crucial biological activities, including anti-tumor activity. In this study, we examined the influence of crude fucoidan extracted from Sargassum sp. (MTA) and Fucus vesiculosus (SIG) on Lewis lung carcinoma cells (LCC) and melanoma B16 cells (MC). In vitro studies were performed using cell viability analysis and showed that SIG and MTA fucoidans significantly decreased the viable number of LCC and MC cells in a dose-response fashion. Histochemical staining showed morphological changes of melanoma B16 cells after exposure to fucoidan. The observed changes were indicative of crude fucoidan induced apoptosis. Male C57BL/6JJCL mice were subjected to daily i.p. injections over 4 days with either SIG or MTA fucoidan (50mg/kg body wt.). The cytolytic activity of natural killer (NK) cells was enhanced by crude fucoidan in a dose-dependent manner as indicated by (51)Cr labeled YAC-1 target cell release. This study provides substantial indications that crude fucoidan exerts bioactive effects on lung and skin cancer model cells in vitro and induces enhanced natural killer cell activity in mice in vivo.

  9. G1-4A, a polysaccharide from Tinospora cordifolia induces peroxynitrite dependent killer dendritic cell (KDC) activity against tumor cells.

    Science.gov (United States)

    Pandey, Vipul K; Amin, Prayag J; Shankar, Bhavani S

    2014-12-01

    Dendritic cells (DC) play a central role in the development of an adaptive immune response against tumor. In addition to its role in antigen presentation, DC also possesses cytotoxic activity against tumor cells. We have earlier shown phenotypic and functional maturation of bone marrow derived dendritic cells (BMDC) by G1-4A, an arabinogalactan derived from Tinospora cordifolia. In this study, we have investigated the killer phenotype of BMDC matured in the presence of G1-4A, [mBMDC (G1-4A)] on tumor cells. We have observed several fold increase in killing of tumor cells by mBMDC (G1-4A). The tumoricidal activity was not specific to syngeneic tumors cells but could kill xenogenic tumors also. Nitric oxide released by mBMDC (G1-4A) generates peroxynitrite in tumor cells and is responsible for killing of target cells. This killing was completely abrogated by inducible nitric oxide synthase (iNOS) inhibitor 1400W and NADPH oxidase inhibitor apocyanin. The killed target cells are phagocytosed by BMDC which further activate syngeneic cytotoxic T cells. These results thus show that G1-4A treated mBMDC acquire killer phenotype along with maturation which plays an important role in activation of cytotoxic T cells.

  10. Killer "Killer Examples" for Design Patterns

    DEFF Research Database (Denmark)

    Caspersen, Michael Edelgaard; Alphonce, Carl; Decker, Adrienne

    2007-01-01

    Giving students an appreciation of the benefits of using design patterns and an ability to use them effectively in developing code presents several interesting pedagogical challenges. This paper discusses pedagogical lessons learned at the "Killer Examples" for Design Patterns and Objects First s...... series of workshops held at the Object Oriented Programming, Systems, Languages and Applications (OOPSLA) conference over the past four years. It also showcases three "killer examples" which can be used to support the teaching of design patterns.......Giving students an appreciation of the benefits of using design patterns and an ability to use them effectively in developing code presents several interesting pedagogical challenges. This paper discusses pedagogical lessons learned at the "Killer Examples" for Design Patterns and Objects First...

  11. Psychoneuroendocrine immunology: perception of stress can alter body temperature and natural killer cell activity.

    Science.gov (United States)

    Hiramoto, R N; Solvason, H B; Hsueh, C M; Rogers, C F; Demissie, S; Hiramoto, N S; Gauthier, D K; Lorden, J F; Ghanta, V K

    1999-01-01

    Psychoimmunology has been credited with using the mind as a way to alter immunity. The problem with this concept is that many of the current psychoimmunology techniques in use are aimed at alleviating stress effects on the immune system rather than at direct augmentation of immunity by the brain. Studies in animals provide a model that permits us to approach the difficulties associated with gaining an understanding of the CNS-immune system connection. A particular advantage of using animals over humans is that psychological and social contributions play a less prominent role for animals than for human subjects, since the animals are all inbred and reared under identical controlled conditions. If the insightful information provided by animal studies is correct, then psychotherapy for the treatment of diseases might be made more effective if some aspect of this knowledge is included in the design of the treatment. We emphasize conditioning as a regimen and an acceptable way to train the brain to remember an output pathway to raise immunity. We propose that a specific drug or perception (mild stress, represented by rotation, total body heating or handling) could substitute and kindle the same output pathway without the need for conditioning. If this view is correct, then instead of using conditioning, it may be possible to use an antigen to activate desired immune cells, and substitute a drug or an external environmental sensory stimulus (perception) to energize the output pathway to these cells. Alternatively, monitoring alterations of body temperature in response to a drug or perception might allow us to follow how effectively the brain is performing in altering immunity. Studies with animals suggest that there are alternative ways to use the mind to raise natural or acquired immunity in man.

  12. 'Killer' character of yeasts isolated from ethanolic fermentations

    Directory of Open Access Journals (Sweden)

    Ceccato-Antonini Sandra Regina

    1999-01-01

    Full Text Available The number of killer, neutral and sensitive yeasts was determined from strains isolated from substrates related to alcoholic fermentations. From 113 isolates, 24 showed killer activity against NCYC 1006 (standard sensitive strain, while 30 were sensitive to NCYC 738 (standard killer strain, and 59 had no reaction in assays at 25-27°C. Two wild yeast strains of Saccharomyces cerevisiae and one of Candida colliculosa were tested against 10 standard killer strains and one standard sensitive strain in a cell x cell and well-test assays at four different pHs. None of the isolates displayed strong killer activity or were sensitive to the standard strains. All belonged to the neutral type. It was concluded that although the number of killer strains was high, this character cannot be used to protect ethanol fermentation processes against yeast contaminants like those which form cell clusters.

  13. Construction of a tissue engineered intervertebral disc with high biological activity using an allogeneic intervertebral disc supplemented with transfected nucleus pulposus cells expressing exogenous dopamine beta-hydroxylase.

    Science.gov (United States)

    Bai, M; Wang, Y H; Yin, H P; Li, S W

    2015-09-09

    This study addressed the in vitro construction and biological activity of tissue engineered intervertebral discs with exogenous human dopamine beta-hydroxylase (DBH) nucleus pulposus cells. pSNAV2.0-DBH expression plasmids were utilized to enhance the survival rates of intervertebral disc tissue cells. Various concentrations of transfected nucleus pulposus cells were injected into the discs, and DBH mRNA expression was determined using polymerase chain reaction amplification. Polysaccharide content and total collagen protein content in the engineered disc nucleus pulposus tissue were determined. The visible fluorescence intensities of the 1 x 10(5) and 1 x 10(6) groups vs the 1 x 10(4) group were significantly increased (P 0.05) at 7 days after injection. DBH mRNA expression could be detected in the all but the EGFP control group at 14 days culture. No significant difference was observed in the protein content between the 1 x 10(4) and the control groups at various times, while the protein content was significantly higher in the 1 x 10(5) vs the control and the 1 x 10(4) groups at 7-, 14-, and 21-day cultures. These results demonstrate that a tissue engineered intervertebral disc with high biological activity can be constructed by utilizing allogeneic intervertebral discs stored in liquid nitrogen and a 1 x 10(5) transfected nucleus pulposus cell complex with in vitro culture for 14 days. This model can be used in animal experiments to study the biological activity of the engineered discs.

  14. The Size of Activating and Inhibitory Killer Ig-like Receptor Nanoclusters Is Controlled by the Transmembrane Sequence and Affects Signaling

    Directory of Open Access Journals (Sweden)

    Anna Oszmiana

    2016-05-01

    Full Text Available Super-resolution microscopy has revealed that immune cell receptors are organized in nanoscale clusters at cell surfaces and immune synapses. However, mechanisms and functions for this nanoscale organization remain unclear. Here, we used super-resolution microscopy to compare the surface organization of paired killer Ig-like receptors (KIR, KIR2DL1 and KIR2DS1, on human primary natural killer cells and cell lines. Activating KIR2DS1 assembled in clusters two-fold larger than its inhibitory counterpart KIR2DL1. Site-directed mutagenesis established that the size of nanoclusters is controlled by transmembrane amino acid 233, a lysine in KIR2DS1. Super-resolution microscopy also revealed two ways in which the nanoscale clustering of KIR affects signaling. First, KIR2DS1 and DAP12 nanoclusters are juxtaposed in the resting cell state but coalesce upon receptor ligation. Second, quantitative super-resolution microscopy revealed that phosphorylation of the kinase ZAP-70 or phosphatase SHP-1 is favored in larger KIR nanoclusters. Thus, the size of KIR nanoclusters depends on the transmembrane sequence and affects downstream signaling.

  15. Fucoidan from Sargassum sp. and Fucus vesiculosus reduces cell viability of lung carcinoma and melanoma cells in vitro and activates natural killer cells in mice in vivo

    DEFF Research Database (Denmark)

    Ale, Marcel Tutor; Maruyama, Hiroko; Tamauchi, Hidekazu

    2011-01-01

    Fucoidan is known to exhibit crucial biological activities, including anti-tumor activity. In this study, we examined the influence of crude fucoidan extracted from Sargassum sp. (MTA) and Fucus vesiculosus (SIG) on Lewis lung carcinoma cells (LCC) and melanoma B16 cells (MC). In vitro studies were...... performed using cell viability analysis and showed that SIG and MTA fucoidans significantly decreased the viable number of LCC and MC cells in a dose–response fashion. Histochemical staining showed morphological changes of melanoma B16 cells after exposure to fucoidan. The observed changes were indicative...... of crude fucoidan induced apoptosis. Male C57BL/6JJCL mice were subjected to daily i.p. injections over 4 days with either SIG or MTA fucoidan (50 mg/kg body wt.). The cytolytic activity of natural killer (NK) cells was enhanced by crude fucoidan in a dose-dependent manner as indicated by 51Cr labeled YAC...

  16. Classifying serial killers.

    Science.gov (United States)

    Promish, D I; Lester, D

    1999-11-01

    We attempted to match the appearance and demeanor of 27 serial killers to the postmortem 'signatures' found on their victims' bodies. Our results suggest that a link may exist between postmortem signatures and two complementary appearance-demeanor types.

  17. A KILLER WITH LOVE

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    Leon is a 1994 French action film. This movie is about a tough girl named Mathilda who wants to become a professional killer when her entire families are killed by a vicious government agent. At the same time, she meets the man-Leon, a professionally paid killer, who she chooses as her teacher. Eventually, she becomes soul mate of Leon. At the end of the film, Leon even dies for saving Mathlida.

  18. CXC chemokine ligand 10 controls viral infection in the central nervous system: evidence for a role in innate immune response through recruitment and activation of natural killer cells.

    Science.gov (United States)

    Trifilo, Matthew J; Montalto-Morrison, Cynthia; Stiles, Linda N; Hurst, Kelley R; Hardison, Jenny L; Manning, Jerry E; Masters, Paul S; Lane, Thomas E

    2004-01-01

    How chemokines shape the immune response to viral infection of the central nervous system (CNS) has largely been considered within the context of recruitment and activation of antigen-specific lymphocytes. However, chemokines are expressed early following viral infection, suggesting an important role in coordinating innate immune responses. Herein, we evaluated the contributions of CXC chemokine ligand 10 (CXCL10) in promoting innate defense mechanisms following coronavirus infection of the CNS. Intracerebral infection of RAG1(-/-) mice with a recombinant CXCL10-expressing murine coronavirus (mouse hepatitis virus) resulted in protection from disease and increased survival that correlated with a significant increase in recruitment and activation of natural killer (NK) cells within the CNS. Accumulation of NK cells resulted in a reduction in viral titers that was dependent on gamma interferon secretion. These results indicate that CXCL10 expression plays a pivotal role in defense following coronavirus infection of the CNS by enhancing innate immune responses.

  19. Stochastic modeling of a serial killer.

    Science.gov (United States)

    Simkin, M V; Roychowdhury, V P

    2014-08-21

    We analyze the time pattern of the activity of a serial killer, who during 12 years had murdered 53 people. The plot of the cumulative number of murders as a function of time is of "Devil's staircase" type. The distribution of the intervals between murders (step length) follows a power law with the exponent of 1.4. We propose a model according to which the serial killer commits murders when neuronal excitation in his brain exceeds certain threshold. We model this neural activity as a branching process, which in turn is approximated by a random walk. As the distribution of the random walk return times is a power law with the exponent 1.5, the distribution of the inter-murder intervals is thus explained. We illustrate analytical results by numerical simulation. Time pattern activity data from two other serial killers further substantiate our analysis.

  20. Bovine natural killer cells are present in Escherichia coli infected mammary gland tissue and show antimicrobial activity in vitro.

    Science.gov (United States)

    Sipka, Anja; Pomeroy, Brianna; Klaessig, Suzanne; Schukken, Ynte

    2016-10-01

    Natural killer (NK) cells are early responders in bacterial infections but their role in bovine mastitis has not been characterized. For the first time, we show the presence of NK cells (NKp46(+)/CD3(-)) in bovine mammary gland tissue after an intramammary challenge with Escherichia (E.) coli. A small number of NK cells was detected in milk from quarters before and during an E. coli challenge. In vitro cultures of primary bovine mammary gland epithelial cells stimulated with UV irradiated E. coli induced significant migration of peripheral blood NK cells (pbNK) within 2h. Furthermore, pbNK cells significantly reduced counts of live E. coli in vitro within 2h of culture. The results show that bovine NK cells have the capacity to migrate to the site of infection and produce antibacterial mediators. These findings introduce NK cells as a leukocyte population in the mammary gland with potential functions in the innate immune response in bovine mastitis.

  1. The Violence of Collection: "Indian Killer"'s Archives

    Science.gov (United States)

    Dean, Janet

    2008-01-01

    At the close of Sherman Alexie's "Indian Killer," in a final chapter titled "Creation Story," a killer carries a backpack containing, among other things, "dozens of owl feathers, a scrapbook, and two bloody scalps in a plastic bag." Readers schooled in the psychopathologies of real and fictional serial killers will be familiar with the detail:…

  2. Activation of the control reporter plasmids pRL-TK and pRL-SV40 by multiple GATA transcription factors can lead to aberrant normalization of transfection efficiency

    Directory of Open Access Journals (Sweden)

    Strauss Jerome F

    2004-04-01

    Full Text Available Abstract Background Members of the GATA transcription factor family have been used in many transfection studies to investigate their roles in the regulation of gene expression. To correct for variations in transfection efficiency, the Renilla luciferase encoding plasmids pRL-TK and pRL-SV40 are commonly used as normalization controls. Results We report here that plasmids expressing GATA-4 or GATA-6 transcription factor increased Renilla luciferase gene expression by 2 to 8 fold when co-transfected with pRL-TK or pRL-SV40. This alteration of the control reporter gene activity was shown to cause erroneous normalization of transfection efficiency and thus misinterpretation of results in a transactivation assay. To circumvent the problem, we generated two mutant control plasmids from which putative GATA response elements were deleted. These deletions rendered pRL-SV40 unresponsive to GATA transcription factor stimulation and reduced the response of pRL-TK. A database search also indicates that consensus GATA binding sequences are present in other commercially available Renilla luciferase encoding plasmids; therefore, the latter can potentially be transactivated by GATA transcription factors. Conclusion Taken together, these findings highlight the importance of the selection of an appropriate control reporter plasmid for the normalization of transfection efficiency.

  3. Grass and weed killer poisoning

    Science.gov (United States)

    ... medlineplus.gov/ency/article/002838.htm Grass and weed killer poisoning To use the sharing features on this page, please enable JavaScript. Many weed killers contain dangerous chemicals that are harmful if ...

  4. Establishment of a reference interval for natural killer cell activity through flow cytometry and its clinical application in the diagnosis of hemophagocytic lymphohistiocytosis.

    Science.gov (United States)

    Chung, H J; Park, C J; Lim, J H; Jang, S; Chi, H S; Im, H J; Seo, J J

    2010-04-01

    Recently, the Histiocyte Society revised the diagnostic criteria for hemophagocytic lymphohistiocytosis (HLH) to include low or absent natural killer (NK) cell activity, according to local laboratory reference. The aim of this study was to establish reference interval for functional NK-cell activity in 63 healthy Korean individuals using a flow-cytometric assay. We used peripheral blood mononuclear cells (PBMCs) as effector cells and Fluorescein isothiocyanate-labeled K562 cells as target cells. NK-cell activity was calculated using the following equation: NK-cell activity (%) = (test lysis - spontaneous lysis) x 100/(maximum lysis - spontaneous lysis). NK-cell activity was analyzed in 13 known HLH patients and 16 suspected non-HLH patients using a flow-cytometric assay. The mean (+/-SD) cytotoxicity of PBMCs from healthy individuals was 20.9 +/- 5.3% and the reference interval was 11.8-31.9%. The mean NK-cell activity of HLH patients (8.3 +/- 8.9%) was significantly lower (P = 0.001) than that of non-HLH patients (20.1 +/- 7.8%). The sequential changes in NK-cell activity in the HLH group corresponded to clinical and laboratory findings following treatment. We successfully developed a functional NK-cell activity test for use in the clinical laboratory and obtained a reference interval of NK-cell activity from healthy donors. This assay, and associated reference interval, was used to analyze 30 clinically relevant specimens and the results were shown to be well correlated.

  5. The evolution of natural killer cell receptors

    NARCIS (Netherlands)

    Carrillo-Bustamante, Paola; Kesmir, C.; de Boer, Rob J

    2016-01-01

    Natural killer (NK) cells are immune cells that play a crucial role against viral infections and tumors. To be tolerant against healthy tissue and simultaneously attack infected cells, the activity of NK cells is tightly regulated by a sophisticated array of germline-encoded activating and inhibitin

  6. Synthetic oligonucleotides with particular base sequences from the cDNA encoding proteins of Mycobacterium bovis BCG induce interferons and activate natural killer cells.

    Science.gov (United States)

    Tokunaga, T; Yano, O; Kuramoto, E; Kimura, Y; Yamamoto, T; Kataoka, T; Yamamoto, S

    1992-01-01

    Thirteen kinds of 45-mer single-stranded oligonucleotide, having sequence randomly selected from the known cDNA encoding BCG proteins, were tested for their capability to augment natural killer (NK) cell activity of mouse spleen cells in vitro. Six out of the 13 oligonucleotides showed the activity, while the others did not. In order to know the minimal and essential sequence(s) responsible for the biological activity, 2 kinds of 30-mer and 5 kinds of 15-mer oligonucleotide fragments of an active 45-mer nucleotide were tested for their activity. One of the 30-mer oligonucleotides, designated BCG-A4a, was active, but the other 30-mer was inactive. All of the 15-mer oligonucleotide fragments were inactive. The BCG-A4a also stimulated the spleen cells to produce interferon (IFN)-alpha and -gamma. An experiment using anti-IFN antisera showed that the NK cell activation by the oligonucleotide was ascribed to the IFN-alpha produced. It was noticed that all of the biologically active oligonucleotides possessed one or more palindrome sequence(s), and the inactive ones did not, with an exception of a 45-mer inactive oligonucleotide containing overlapping palindrome sequences (GGGCCCGGG). These findings strongly suggest that certain palindrome sequences, like GACGTC, GGCGCC and TGCGCA, are essential for 30-mer oligonucleotides, like BCG-A4a, to induce IFNs.

  7. Matched sizes of activating and inhibitory receptor/ligand pairs are required for optimal signal integration by human natural killer cells.

    Directory of Open Access Journals (Sweden)

    Karsten Köhler

    Full Text Available It has been suggested that receptor-ligand complexes segregate or co-localise within immune synapses according to their size, and this is important for receptor signaling. Here, we set out to test the importance of receptor-ligand complex dimensions for immune surveillance of target cells by human Natural Killer (NK cells. NK cell activation is regulated by integrating signals from activating receptors, such as NKG2D, and inhibitory receptors, such as KIR2DL1. Elongating the NKG2D ligand MICA reduced its ability to trigger NK cell activation. Conversely, elongation of KIR2DL1 ligand HLA-C reduced its ability to inhibit NK cells. Whereas normal-sized HLA-C was most effective at inhibiting activation by normal-length MICA, only elongated HLA-C could inhibit activation by elongated MICA. Moreover, HLA-C and MICA that were matched in size co-localised, whereas HLA-C and MICA that were different in size were segregated. These results demonstrate that receptor-ligand dimensions are important in NK cell recognition, and suggest that optimal integration of activating and inhibitory receptor signals requires the receptor-ligand complexes to have similar dimensions.

  8. Cutting edge: activation by innate cytokines or microbial antigens can cause arrest of natural killer T cell patrolling of liver sinusoids.

    Science.gov (United States)

    Velázquez, Peter; Cameron, Thomas O; Kinjo, Yuki; Nagarajan, Niranjana; Kronenberg, Mitchell; Dustin, Michael L

    2008-02-15

    Natural killer T (NKT) cells are innate-like lymphocytes that rapidly secrete large amounts of effector cytokines upon activation. Recognition of alpha-linked glycolipids presented by CD1d leads to the production of IL-4, IFN-gamma, or both, while direct activation by the synergistic action of IL-12 and IL-18 leads to IFN-gamma production only. We previously reported that in vitro cultured dendritic cells can modulate NKT cell activation and, using intravital fluorescence laser scanning microscopy, we reported that the potent stimulation of NKT cells results in arrest within hepatic sinusoids. In this study, we examine the relationship between murine NKT cell patrolling and activation. We report that NKT cell arrest results from activation driven by limiting doses of a bacteria-derived weak agonist, galacturonic acid-containing glycosphingolipid, or a synthetic agonist, alpha-galactosyl ceramide. Interestingly, NKT cell arrest also results from IL-12 and IL-18 synergistic activation. Thus, innate cytokines and natural microbial TCR agonists trigger sinusoidal NKT cell arrest and an effector response.

  9. Transfection microarray and the applications.

    Science.gov (United States)

    Miyake, Masato; Yoshikawa, Tomohiro; Fujita, Satoshi; Miyake, Jun

    2009-05-01

    Microarray transfection has been extensively studied for high-throughput functional analysis of mammalian cells. However, control of efficiency and reproducibility are the critical issues for practical use. By using solid-phase transfection accelerators and nano-scaffold, we provide a highly efficient and reproducible microarray-transfection device, "transfection microarray". The device would be applied to the limited number of available primary cells and stem cells not only for large-scale functional analysis but also reporter-based time-lapse cellular event analysis.

  10. Role of protein kinase C in TBT-induced inhibition of lytic function and MAPK activation in human natural killer cells.

    Science.gov (United States)

    Abraha, Abraham B; Rana, Krupa; Whalen, Margaret M

    2010-11-01

    Human natural killer (NK) cells are lymphocytes that destroy tumor and virally infected cells. Previous studies have shown that exposure of NK cells to tributyltin (TBT) greatly diminishes their ability to destroy tumor cells (lytic function) while activating mitogen-activated protein kinases (MAPK) (p44/42, p38, and JNK) in NK cells. The signaling pathway that regulates NK lytic function appears to include activation of protein kinase C(PKC) as well as MAPK activity. TBT-induced activation of MAPKs would trigger a portion of the NK lytic signaling pathway, which would then leave the NK cell unable to trigger this pathway in response to a subsequent encounter with a target cell. In the present study we evaluated the involvement of PKC in inhibition of NK lysis of tumor cells and activation of MAPKs caused by TBT exposure. TBT caused a 2–3-fold activation of PKC at concentrations ranging from 50 to 300 nM (16–98 ng/ml),indicating that activation of PKC occurs in response to TBT exposure. This would then leave the NK cell unable to respond to targets. Treatment with the PKC inhibitor, bisindolylmaleimide I, caused an 85% decrease in the ability of NK cells to lyse tumor cells, validating the involvement of PKC in the lytic signaling pathway. The role of PKC in the activation of MAPKs by TBT was also investigated using bisindolylmaleimide I. The results indicated that, in NK cells where PKC activation was blocked, there was no activation of the MAPK, p44/42 in response to TBT.However, TBT-induced activation of the MAPKs, p38 and JNK did not require PKC activation. These results indicate the pivotal role of PKC in the TBT-induced loss of NK lytic function including activation of p44/42 by TBT in NK cells.

  11. GMP-compliant, large-scale expanded allogeneic natural killer cells have potent cytolytic activity against cancer cells in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Okjae Lim

    Full Text Available Ex vivo-expanded, allogeneic natural killer (NK cells can be used for the treatment of various types of cancer. In allogeneic NK cell therapy, NK cells from healthy donors must be expanded in order to obtain a sufficient number of highly purified, activated NK cells. In the present study, we established a simplified and efficient method for the large-scale expansion and activation of NK cells from healthy donors under good manufacturing practice (GMP conditions. After a single step of magnetic depletion of CD3(+ T cells, the depleted peripheral blood mononuclear cells (PBMCs were stimulated and expanded with irradiated autologous PBMCs in the presence of OKT3 and IL-2 for 14 days, resulting in a highly pure population of CD3(-CD16(+CD56(+ NK cells which is desired for allogeneic purpose. Compared with freshly isolated NK cells, these expanded NK cells showed robust cytokine production and potent cytolytic activity against various cancer cell lines. Of note, expanded NK cells selectively killed cancer cells without demonstrating cytotoxicity against allogeneic non-tumor cells in coculture assays. The anti-tumor activity of expanded human NK cells was examined in SCID mice injected with human lymphoma cells. In this model, expanded NK cells efficiently controlled lymphoma progression. In conclusion, allogeneic NK cells were efficiently expanded in a GMP-compliant facility and demonstrated potent anti-tumor activity both in vitro and in vivo.

  12. Clemency Pogue: Fairy Killer

    Science.gov (United States)

    Petty, J. T.

    2004-01-01

    This young adult author claims his most enjoyable task as a writer is "intellectual danger, getting into other people's trouble." He asks his readers not to trust him, and then, as evidence, tempts us with a look at a chapter from "Clemency Pogue: Fairy Killer."

  13. Transfection of Platyhelminthes

    Directory of Open Access Journals (Sweden)

    Bárbara Moguel

    2015-01-01

    Full Text Available Flatworms are one of the most diverse groups within Lophotrochozoa with more than 20,000 known species, distributed worldwide in different ecosystems, from the free-living organisms in the seas and lakes to highly specialized parasites living in a variety of hosts, including humans. Several infections caused by flatworms are considered major neglected diseases affecting countries in the Americas, Asia, and Africa. For several decades, a particular interest on free-living flatworms was due to their ability to regenerate considerable portions of the body, implying the presence of germ cells that could be important for medicine. The relevance of reverse genetics for this group is clear; understanding the phenotypic characteristics of specific genes will shed light on developmental traits of free-living and parasite worms. The genetic manipulation of flatworms will allow learning more about the mechanisms for tissue regeneration, designing new and more effective anthelmintic drugs, and explaining the host-parasite molecular crosstalk so far partially inaccessible for experimentation. In this review, availability of transfection techniques is analyzed across flatworms, from the initial transient achievements to the stable manipulations now developed for free-living and parasite species.

  14. Transfection of Platyhelminthes.

    Science.gov (United States)

    Moguel, Bárbara; Bobes, Raúl J; Carrero, Julio C; Laclette, Juan P

    2015-01-01

    Flatworms are one of the most diverse groups within Lophotrochozoa with more than 20,000 known species, distributed worldwide in different ecosystems, from the free-living organisms in the seas and lakes to highly specialized parasites living in a variety of hosts, including humans. Several infections caused by flatworms are considered major neglected diseases affecting countries in the Americas, Asia, and Africa. For several decades, a particular interest on free-living flatworms was due to their ability to regenerate considerable portions of the body, implying the presence of germ cells that could be important for medicine. The relevance of reverse genetics for this group is clear; understanding the phenotypic characteristics of specific genes will shed light on developmental traits of free-living and parasite worms. The genetic manipulation of flatworms will allow learning more about the mechanisms for tissue regeneration, designing new and more effective anthelmintic drugs, and explaining the host-parasite molecular crosstalk so far partially inaccessible for experimentation. In this review, availability of transfection techniques is analyzed across flatworms, from the initial transient achievements to the stable manipulations now developed for free-living and parasite species.

  15. Transfection of Platyhelminthes

    Science.gov (United States)

    Moguel, Bárbara; Bobes, Raúl J.; Carrero, Julio C.; Laclette, Juan P.

    2015-01-01

    Flatworms are one of the most diverse groups within Lophotrochozoa with more than 20,000 known species, distributed worldwide in different ecosystems, from the free-living organisms in the seas and lakes to highly specialized parasites living in a variety of hosts, including humans. Several infections caused by flatworms are considered major neglected diseases affecting countries in the Americas, Asia, and Africa. For several decades, a particular interest on free-living flatworms was due to their ability to regenerate considerable portions of the body, implying the presence of germ cells that could be important for medicine. The relevance of reverse genetics for this group is clear; understanding the phenotypic characteristics of specific genes will shed light on developmental traits of free-living and parasite worms. The genetic manipulation of flatworms will allow learning more about the mechanisms for tissue regeneration, designing new and more effective anthelmintic drugs, and explaining the host-parasite molecular crosstalk so far partially inaccessible for experimentation. In this review, availability of transfection techniques is analyzed across flatworms, from the initial transient achievements to the stable manipulations now developed for free-living and parasite species. PMID:26090388

  16. Targeted delivery of lipid antigen to macrophages via the CD169/sialoadhesin endocytic pathway induces robust invariant natural killer T cell activation

    Science.gov (United States)

    Kawasaki, Norihito; Vela, Jose Luis; Nycholat, Corwin M.; Rademacher, Christoph; Khurana, Archana; van Rooijen, Nico; Crocker, Paul R.; Kronenberg, Mitchell; Paulson, James C.

    2013-01-01

    Invariant natural killer T (iNKT) cells induce a protective immune response triggered by foreign glycolipid antigens bound to CD1d on antigen-presenting cells (APCs). A limitation of using glycolipid antigens to stimulate immune responses in human patients has been the inability to target them to the most effective APCs. Recent studies have implicated phagocytic CD169+ macrophages as major APCs in lymph nodes for priming iNKT cells in mice immunized with glycolipid antigen in particulate form. CD169 is known as sialoadhesin (Sn), a macrophage-specific adhesion and endocytic receptor of the siglec family that recognizes sialic acid containing glycans as ligands. We have recently developed liposomes decorated with glycan ligands for CD169/Sn suitable for targeted delivery to macrophages via CD169/Sn-mediated endocytosis. Here we show that targeted delivery of a lipid antigen to CD169+ macrophages in vivo results in robust iNKT cell activation in liver and spleen using nanogram amounts of antigen. Activation of iNKT cells is abrogated in Cd169−/− mice and is macrophage-dependent, demonstrating that targeting CD169+ macrophages is sufficient for systemic activation of iNKT cells. When pulsed with targeted liposomes, human monocyte–derived dendritic cells expressing CD169/Sn activated human iNKT cells, demonstrating the conservation of the CD169/Sn endocytic pathway capable of presenting lipid antigens to iNKT cells. PMID:23610394

  17. Natural killer cell activity, lymphocyte proliferation, and cytokine profile in tumor-bearing mice treated with MAPA, a magnesium aggregated polymer from Aspergillus oryzae.

    Science.gov (United States)

    Justo, G Z; Durán, N; Queiroz, M L S

    2003-08-01

    The present study examined the effects of MAPA, an antitumor aggregated polymer of protein magnesium ammonium phospholinoleate-palmitoleate anhydride, isolated from Aspergillus oryzae, on concanavalin A (Con A)-induced spleen cell proliferation, cytokine production and on natural killer (NK) cell activity in Ehrlich ascites tumor-bearing mice. The Ehrlich ascites tumor (EAT) growth led to diminished mitogen-induced expansion of spleen cell populations and total NK activity. This was accompanied by striking spleen enlargement, with a marked increase in total cell counts. Moreover, a substantial enhancement in IL-10 levels, paralleled by a significant decrease in IL-2 was observed, while production of IL-4 and interferon-gamma (IFN-gamma) was not altered. Treatment of mice with 5 mg/kg MAPA for 7 days promoted spleen cell proliferation, IL-2 production and NK cell activity regardless of tumor outgrowth. In addition, MAPA treatment markedly enhanced IFN-gamma levels and reduced IL-10 production relative to EAT mice. A 35% reduction in splenomegaly with normal number of nucleated cells was also found. Altogether, our results suggest that MAPA directly and/or indirectly modulates immune cell activity, and probably disengages tumor-induced suppression of these responses. Clearly, MAPA has an impact and may delay tumor outgrowth through immunotherapeutic mechanisms.

  18. No Correlation Exists between Disease Activity and the Expression of Killer-Cell Immunoglobulin-Like Receptors in Patients with Rheumatoid Arthritis

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    Toshiaki Kogure

    2007-01-01

    Full Text Available Objective. The genes for killer-cell immunoglobulin-like receptors (KIRs have been cloned and their functions and expression in patients with rheumatoid arthritis (RA have been partially clarified. However, the correlation between their expression and disease activity has not been analyzed in patients with RA. Thus, we measured KIR expression on lymphocytes in patients with RA, and assessed the correlation between KIR expression and disease activity. Patients and Methods. In the cross-sectional study, 15 patients (9 females and 6 males who fulfilled the diagnostic criteria for RA were assessed. In the longitudinal study, patients who were followed-up for 3 months were assessed. CD158a/b expression on peripheral blood mononuclear cells (PBMC of RA patients was analyzed using flow cytometry. Results. No significant correlation between KIR expression and CRP, ESR, or IgM-RF was observed. There was no remarkable change in the expression of KIRs between the baseline and after 3 months. Additionally, in the 5 patients whose expression of KIRs particularly changed, the time-related changes in the expression of KIRs were independent from those of inflammation parameters and IgM-RF. Conclusion. There was no correlation between KIR expression and disease activity; therefore, the clinical use of KIR expression should be limited, while unnatural KIR expression may be involved in the pathogenesis of RA, but not a recruitment of chronic inflammation to induce joint damage.

  19. Synergistic effects in gene delivery-a structure-activity approach to the optimisation of hybrid dendritic-lipidic transfection agents.

    Science.gov (United States)

    Jones, Simon P; Gabrielson, Nathan P; Pack, Daniel W; Smith, David K

    2008-10-21

    Novel gene delivery agents based on combining cholesterol units with spermine-functionalised dendrons exhibit enhanced transfection ability-we report significant synergistic effects in mixed (hybrid) systems which combine aspects of both main classes of synthetic vectors, i.e., cationic polymers and lipids.

  20. Activation of Natural Killer Cells in Patients with Chronic Bone and Joint Infection due to Staphylococci Expressing or Not the Small Colony Variant Phenotype

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    Sébastien Viel

    2014-01-01

    Full Text Available Chronic bone and joint infections (BJI are devastating diseases. Relapses are frequently observed, as some pathogens, especially staphylococci, can persist intracellularly by expressing a particular phenotype called small colony variant (SCV. As natural killer (NK cells are lymphocytes specialized in the killing of host cells infected by intracellular pathogens, we studied NK cells of patients with chronic BJI due to staphylococci expressing or not SCVs (10 patients in both groups. Controls were patients infected with other bacteria without detectable expression of SCVs, and healthy volunteers. NK cell phenotype was evaluated from PBMCs by flow cytometry. Degranulation capacity was evaluated after stimulation with K562 cells in vitro. We found that NK cells were activated in terms of CD69 expression, loss of CD16 and perforin, in all infected patients in comparison with healthy volunteers, independently of the SCV phenotype. Peripheral NK cells in patients with chronic BJI display signs of recent activation and degranulation in vivo in response to CD16-mediated signals, regardless of the type of bacteria involved. This could involve a universal capacity of isolates responsible for chronic BJI to produce undetectable SCVs in vivo, which might be a target of future intervention.

  1. Low-dose ionizing radiation induces direct activation of natural killer cells and provides a novel approach for adoptive cellular immunotherapy.

    Science.gov (United States)

    Yang, Guozi; Kong, Qingyu; Wang, Guanjun; Jin, Haofan; Zhou, Lei; Yu, Dehai; Niu, Chao; Han, Wei; Li, Wei; Cui, Jiuwei

    2014-12-01

    Recent evidence indicates that limited availability and cytotoxicity have restricted the development of natural killer (NK) cells in adoptive cellular immunotherapy (ACI). While it has been reported that low-dose ionizing radiation (LDIR) could enhance the immune response in animal studies, the influence of LDIR at the cellular level has been less well defined. In this study, the authors aim to investigate the direct effects of LDIR on NK cells and the potential mechanism, and explore the application of activation and expansion of NK cells by LDIR in ACI. The authors found that expansion and cytotoxicity of NK cells were markedly augmented by LDIR. The levels of IFN-γ and TNF-α in the supernatants of cultured NK cells were significantly increased after LDIR. Additionally, the effect of the P38 inhibitor (SB203580) significantly decreased the expanded NK cell cytotoxicity, cytokine levels, and expression levels of FasL and perforin. These findings indicate that LDIR induces a direct expansion and activation of NK cells through possibly the P38-MAPK pathway, which provides a potential mechanism for stimulation of NK cells by LDIR and a novel but simplified approach for ACI.

  2. A novel killer protein from Pichia kluyveri isolated from an Algerian soil: purification and characterization of its in vitro activity against food and beverage spoilage yeasts.

    Science.gov (United States)

    Labbani, Fatima-Zohra Kenza; Turchetti, Benedetta; Bennamoun, Leila; Dakhmouche, Scheherazad; Roberti, Rita; Corazzi, Lanfranco; Meraihi, Zahia; Buzzini, Pietro

    2015-04-01

    A novel killer protein (Pkkp) secreted by a Pichia kluyveri strain isolated from an Algerian soil was active against food and beverage spoilage yeasts of the genera Dekkera, Kluyveromyces, Pichia, Saccharomyces, Torulaspora, Wickerhamomyces and Zygosaccharomyces. After purification by gel filtration chromatography Pkkp revealed an apparent molecular mass of 54 kDa with SDS-PAGE. Minimum inhibitory concentrations (MICs) of purified Pkkp exhibited a high in vitro activity against Dekkera bruxellensis (MICs from 64,000- to 256,000-fold lower than that exhibited by potassium metabisulphite) and Saccharomyces cerevisiae (MICs from 32,000- to 64,000- fold lower than potassium sorbate). No in vitro synergistic interactions (calculated by FIC index - Σ FIC) were observed when Pkkp was used in combination with potassium metabisulphite, potassium sorbate, or ethanol. Pkkp exhibited a dose-response effect against D. bruxellensis and S. cerevisiae in a low-alcoholic drink and fruit juice, respectively. The results of the present study suggest that Pkkp could be proposed as a novel food-grade compound useful for the control of food and beverage spoilage yeasts.

  3. An IL-12/Shh-C domain fusion protein-based IL-12 autocrine loop for sustained natural killer cell activation.

    Science.gov (United States)

    Zhu, Lining; Zhao, Zhihui; Wei, Yanzhang; Marcotte, William; Wagner, Thomas E; Yu, Xianzhong

    2012-08-01

    The dependency of activated natural killer (NK) cells on the continuous support of exogenous interleukin (IL)-2 for their in vivo survival, tumor localization and consequently, their antitumor effect, is a major obstacle for NK cell-mediated tumor therapy. In the present study, a fusion gene between IL-12 and mouse sonic hedgehog C-terminal domain (Shh-C) was constructed. The fusion protein was autocatalytically processed to form cholesterol-modified IL-12 molecules and an autocrine loop of IL-12 was established for the sustained activation of NK cells. The transduced NK cells matured more rapidly in vitro with the enhanced expression of granule-related proteins. NKIL-12/Shh-C cells reached the same proliferation rate as NK cells transduced with enhanced green fluorescent protein (EGFP)/Shh-C (NKEGFP/Shh-C) with Shh-C cells 5 and 7 days after transduction was significantly higher than that in the supernatants of NKIL-12 cells. Immunofluorescent staining of lung tissues from B16-bearing mice which had received an intravenous injection of lentivirus-transduced NK cells without exogenous IL-2 confirmed that donor NK cells successfully infiltrated into the lung tissues. The survival time of the mice which had received NKIL-12/Shh-C cells was significantly prolonged compared to the mice which had received NKEGFP/Shh-C cells.

  4. Delaware's first serial killer.

    Science.gov (United States)

    Inguito, G B; Sekula-Perlman, A; Lynch, M J; Callery, R T

    2000-11-01

    The violent murder of Shirley Ellis on November 29, 1987, marked the beginning of the strange and terrible tale of Steven Bryan Pennell's reign as the state of Delaware's first convicted serial killer. Three more bodies followed the first victim, and all had been brutally beaten and sadistically tortured. The body of a fifth woman has never been found. State and county police collaborated with the FBI to identify and hunt down their suspect, forming a task force of over 100 officers and spending about one million dollars. Through their knowledge and experience with other serial killers, the FBI was able to make an amazingly accurate psychological profile of Delaware's serial killer. After months of around-the-clock surveillance, Steven Pennell was arrested on November 29, 1988, one year to the day after the first victim was found. Pennell was found guilty in the deaths of the first two victims on November 29, 1989, and plead no contest to the murder of two others on October 30, 1991. Still maintaining his innocence, he asked for the death penalty so that he could spare his family further agony. Steven Pennell was executed by lethal injection on March 15, 1992.

  5. Activation of p44/42 MAPK plays a role in the TBT-induced loss of human natural killer (NK) cell function.

    Science.gov (United States)

    Dudimah, Fred D; Griffey, Denisha; Wang, Xiaofei; Whalen, Margaret M

    2010-10-01

    Natural killer (NK) cells destroy (lyse) tumor cells, virally infected cells, and antibody-coated cells. Previous studies indicated that exposure to the environmental contaminant tributyltin (TBT) decreases the lytic function of NK cells and activates mitogen-activated protein kinases (MAPK), including p44/42 (Aluoch and Whalen Toxicology 209:263-277, 2005). If activation of p44/42 is required for TBT-induced decreases of lytic function, then activation of p44/42 to similar extents by pharmacological agents such as phorbol 12-myristate 13-acetate (PMA) should mimic to some extent changes induced in NK cells with TBT exposures. NK cells were exposed to PMA concentrations between 0.25 and 10 nM for 10 min, 1 h, and 6 h before determining the lytic function ((51)Cr release assay) and phosphorylation state of MAPKs (Western blot). A 1-h exposure of NK cells to 5 nM PMA resulted in a loss of lytic function of 47%. Western blot analysis showed that a 1-h exposure to 5 nM PMA caused a sixfold increase in phospho-p44/42 levels. Previous studies showed a fivefold increase in phospho-p44/42 in response to a 1-h exposure to 300 nM TBT. Exposure to 300 nM TBT caused about a 40% decrease in lytic function. This study supports the hypothesis that p44/42 activation (as seen with TBT exposures) can cause a loss of NK-cell lytic function.

  6. Effects of acupuncture treatment on natural killer cell activity, pulse rate, and pain reduction for older adults: an uncontrolled, observational study

    Institute of Scientific and Technical Information of China (English)

    Hidetoshi Mori; Hiroshi Kuge; Tim Hideaki Tanaka; Eiichi Taniwaki; Kazuyo Hanyu; Tateyuki Morisawa

    2013-01-01

    OBJECTIVE:The aim of this study was to examine the changes in natural killer (NK) cell activity,pulse rate,and pain intensity among older adults before and after acupuncture treatment.METHODS:Fifty-six individuals (16 males and 40 females),aged 60 to 82 years (mean age 72.4 ± 5.0),who were experiencing pain in the shoulder,low back,or knee,participated in the study.NK cell activity,leukocyte differentiation (granulocytes and lymphocytes),pulse rate,and blood pressure values were obtained.Pain intensity was evaluated by using the visual analog scale (VAS).The Wilcoxon test was used to analyze NK cell activity,leukocytes (granulocyte counts and granulocyte-to-lymphocyte ratio),and the VAS score in accordance with the location of pain complaints before and after acupuncture treatment.RESULTS:NK cell activity decreased after acupuncture treatment for pain in the shoulder-pain and knee-pain groups.Further,the lymphocyte and granulocyte counts increased after acupuncture treatment for the shoulder-pain group.Pulse rate decreased for the shoulder-pain,low-backpain,and knee-pain groups after acupuncture treatment.The VAS score decreased after acupuncture treatment for the shoulder-pain,low-back-pain,and knee-pain groups.CONCLUSION:This study showed that in older adults,acupuncture treatment decreases pulse rate,relieves pain in the shoulder,low back,and knee,and reduces NK-cell activity.

  7. Human cancer cells with stem cell-like phenotype exhibit enhanced sensitivity to the cytotoxicity of IL-2 and IL-15 activated natural killer cells.

    Science.gov (United States)

    Yin, Tao; Wang, Guoping; He, Sisi; Liu, Qin; Sun, Jianhong; Wang, Yongsheng

    2016-02-01

    Tumors harbor a population of cancer stem cells (CSCs) which can drive tumor progression and therapeutical resistance. Nature killer (NK) cells are best known for their ability to directly recognize and kill malignant cells. However, the susceptibility of cancer stem cells to NK cells is not fully understood. Here we demonstrated that human CD44+CD24- breast CSCs were shown enhanced sensitivity to IL-2 and IL-15 activated NK cells. CD44+CD24- CSCs expressed higher levels of NKG2D ligands ULBP1, ULBP2 and MICA. Blockade assay showed that the sensitivity of CSCs to NK cells-mediated lysis was mainly dependent on NKG2D. Furthermore, redox oxygen species (ROS)-low tumor cells were more sensitive to NK cells. The presence of antioxidant enzymes inhibitor L-S,R-buthionine sulfoximine or H2O2 retarded the cytotoxicity of NK cells to CD44+CD24- CSCs. In addition, NK cells could readily target CD133+ colonal CSCs. Our findings provide novel targets for NK cells-based immunotherapy and are of great importance for translational medicine.

  8. Evaluation on the Clinical Efifcacy of Dendritic Cell-Activated Cytokine-Induced Killer Cells Combined with Conventional Therapy in the Treatment of Malignant Tumors

    Institute of Scientific and Technical Information of China (English)

    WEI Hong; HAN Na-na; CAI Xin-hua

    2016-01-01

    Objective: To evaluate the clinical efficacy of dendritic cell-activated cytokine-induced killer (DC-CIK) cells combined with conventional therapy in the treatment of malignant tumors. Methods: A total of 100 patients with malignant tumors were randomly divided into two groups. Treatment group received conventional therapy combined with DC-CIK while control group received conventional therapy alone. The short-term efficacy, adverse reactions and changes of lymphocyte subpopulation were all compared between two groups after treatment. Results: The overall response rate (ORR) was higher in treatment group (86.00%) than in control group (54.00%), the difference was statistically significant (P0.05). WBC reduced markedly, but the level of alanine aminotransferase (ALT) increased obviously after treatment in control group (P0.05). In treatment group, the levels of CD3+, CD3+CD4+, CD3+CD8+, and CD3+CD56+ increased (P0.05). In control group, the levels of CD3+ and CD3+CD4+ reduced (P0.05). The levels of CD3+, CD3+CD4+, CD3+CD8+and CD3+CD56+ in treatment group were higher than those in control group (P Conclusion:DC-CIK combined with conventional therapy, safe and effective, is capable of promoting the recovery of leukocytes and liver and kidney function, and improving the cellular immune function, which may provide a new therapeutic regimen for patients with malignant tumors.

  9. Ultrasound-targeted transfection of tissue-type plasminogen activator gene carried by albumin nanoparticles to dog myocardium to prevent thrombosis after heart mechanical valve replacement

    Directory of Open Access Journals (Sweden)

    Ji J

    2012-06-01

    Full Text Available Ji Jun, Ji Shang-Yi, Yang Jian-An, He Xia, Yang Xiao-Han, Ling Wen-Ping, Chen Xiao-LingDepartment of Pathology and Cardiovascular Surgery, Shenzhen Sun Yat-Sen Cardiovascular Hospital, Shenzhen, Guangdong, People's Republic of ChinaBackground: There are more than 300,000 prosthetic heart valve replacements each year worldwide. These patients are faced with a higher risk of thromboembolic events after heart valve surgery and long-term or even life-long anticoagulative and antiplatelet therapies are necessary. Some severe complications such as hemorrhaging or rebound thrombosis can occur when the therapy ceases. Tissue-type plasminogen activator (t-PA is a thrombolytic agent. One of the best strategies is gene therapy, which offers a local high expression of t-PA over a prolonged time period to avoid both systemic hemorrhaging and local rebound thrombosis. There are some issues with t-PA that need to be addressed: currently, there is no up-to-date report on how the t-PA gene targets the heart in vivo and the gene vector for t-PA needs to be determined.Aims: To fabricate an albumin nano-t-PA gene ultrasound-targeted agent and investigate its targeting effect on prevention of thrombosis after heart mechanic valve replacement under therapeutic ultrasound.Methods: A dog model of mechanical tricuspid valve replacement was constructed. A highly expressive t-PA gene plasmid was constructed and packaged by nanoparticles prepared with bovine serum albumin. This nanopackaged t-PA gene plasmid was further cross-linked to ultrasonic microbubbles prepared with sucrose and bovine serum albumin to form the ultrasonic-targeted agent for t-PA gene transfection. The agent was given intravenously followed by a therapeutic ultrasound treatment (1 MHz, 1.5 w/cm2, 10 minutes of the heart soon after valve replacement had been performed. The expression of t-PA in myocardium was detected with multiclonal antibodies to t-PA by the indirect immunohistochemical method

  10. The assessment of cytotoxic T cell and natural killer cells activity in residents of high and ordinary background radiation areas of Ramsar-Iran

    Directory of Open Access Journals (Sweden)

    Sajad Borzoueisileh

    2013-01-01

    Full Text Available The effective radiation dose of human from natural sources is about 2.4 mSv/y and the dose limit for radiation workers is 20 mSv/y. Ramsar, a city in Iran, has been the subject of concern in the last forty years for a high level of radiation measured in some spots as high as 260 mSv/y. Carcinogenesis is one of the most studied effects of radiation especially in high doses. Recent studies showed that the high level of natural radiation received by inhabitants of this area, paradoxically don′t have significant health effect. Natural killer (NK cells and cytotoxic T cells are the most important cells in tumor immune surveillance and CD107a is a widely expressed intracellular protein located in the lysosomal/endosomal membrane. CD107a transiently located on the cell membrane can be used as a marker of CD8 + T cell degranulation following stimulation. It is also expressed, to a lower extent, on activated NK cells. In this study, 60 healthy people were selected randomly and their consent obtained and confounding factors such as sex, age, life-styles was matched then the count of activated NK and CD8 + cells was compared in high and normal background radiation areas inhabitants of Ramsar. After filling the questionnaire and measurement of background radiation, blood samples of 30 healthy people from each region were analyzed immediately by means of flowcytometry. The leukocytes and their subsets were not significantly different between two groups and the count of active cells was higher in control group. The result shows that the changes in immune system occur due to radiation and maybe it is as a result of higher radiosensitivity of activated cells.

  11. Orally Administered Salacia reticulata Extract Reduces H1N1 Influenza Clinical Symptoms in Murine Lung Tissues Putatively Due to Enhanced Natural Killer Cell Activity

    Science.gov (United States)

    Romero-Pérez, Gustavo A.; Egashira, Masayo; Harada, Yuri; Tsuruta, Takeshi; Oda, Yuriko; Ueda, Fumitaka; Tsukahara, Takamitsu; Tsukamoto, Yasuhiro; Inoue, Ryo

    2016-01-01

    Influenza is a major cause of respiratory tract infection. Although most cases do not require further hospitalization, influenza periodically causes epidemics in humans that can potentially infect and kill millions of people. To countermeasure this threat, new vaccines need to be developed annually to match emerging influenza viral strains with increased resistance to existing vaccines. Thus, there is a need for finding and developing new anti-influenza viral agents as alternatives to current treatments. Here, we tested the antiviral effects of an extract from the stems and roots of Salacia reticulata (SSRE), a plant rich in phytochemicals, such as salacinol, kotalanol, and catechins, on H1N1 influenza virus-infected mice. Following oral administration of 0.6 mg/day of SSRE, the incidence of coughing decreased in 80% of mice, and only one case of severe pulmonary inflammation was detected. Moreover, when compared with mice given Lactobacillus casei JCM1134, a strain previously shown to help increase in vitro natural killer (NK) cell activity, SSRE-administered mice showed greater and equal NK cell activity in splenocytes and pulmonary cells, respectively, at high effector cell:target cell ratios. Next, to test whether or not SSRE would exert protective effects against influenza in the absence of gut microbiota, mice were given antibiotics before being inoculated influenza virus and subsequently administered SSRE. SSRE administration induced an increase in NK cell activity in splenocytes and pulmonary cells at levels similar to those detected in mice not treated with antibiotics. Based on our results, it can be concluded that phytochemicals in the SSRE exerted protective effects against influenza infection putatively via modulation of the immune response, including enhancement of NK cell activity, although some protective effects were not necessarily through modulation of gut microbiota. Further investigation is necessary to elucidate the molecular mechanisms

  12. Atorvastatin prevents age-related and amyloid-beta-induced microglial activation by blocking interferon-gamma release from natural killer cells in the brain

    LENUS (Irish Health Repository)

    Lyons, Anthony

    2011-03-31

    Abstract Background Microglial function is modulated by several factors reflecting the numerous receptors expressed on the cell surface, however endogenous factors which contribute to the age-related increase in microglial activation remain largely unknown. One possible factor which may contribute is interferon-γ (IFNγ). IFNγ has been shown to increase in the aged brain and potently activates microglia, although its endogenous cell source in the brain remains unidentified. Methods Male Wistar rats were used to assess the effect of age and amyloid-β (Aβ) on NK cell infiltration into the brain. The effect of the anti-inflammatory compound, atorvastatin was also assessed under these conditions. We measured cytokine and chemokine (IFNγ, IL-2, monocyte chemoattractant protein-1 (MCP-1) and IFNγ-induced protein 10 kDa (IP-10)), expression in the brain by appropriate methods. We also looked at NK cell markers, CD161, NKp30 and NKp46 using flow cytometry and western blot. Results Natural killer (NK) cells are a major source of IFNγ in the periphery and here we report the presence of CD161+ NKp30+ cells and expression of CD161 and NKp46 in the brain of aged and Aβ-treated rats. Furthermore, we demonstrate that isolated CD161+ cells respond to interleukin-2 (IL-2) by releasing IFNγ. Atorvastatin, the HMG-CoA reductase inhibitor, attenuates the increase in CD161 and NKp46 observed in hippocampus of aged and Aβ-treated rats. This was paralleled by a decrease in IFNγ, markers of microglial activation and the chemokines, MCP-1 and IP-10 which are chemotactic for NK cells. Conclusions We propose that NK cells contribute to the age-related and Aβ-induced neuroinflammatory changes and demonstrate that these changes can be modulated by atorvastatin treatment.

  13. Consumption of purple sweet potato leaves modulates human immune response: T-lymphocyte functions, lytic activity of natural killer cell and antibody production

    Institute of Scientific and Technical Information of China (English)

    Chiao-Ming Chen; Sing-Chung Li; Ya-Ling Lin; Ching-Yun Hsu; Ming-Jer Shieh; Jen-Fang Liu

    2005-01-01

    AIM: To study the immunological effects of physiological doses of purple sweet potato leaves (PSPL).METHODS: The randomized crossover study (two periods,each lasting for 2 wk) involved 16 healthy non-smoking adults of normal weight. The 6-wk study consisted of a run-in (wk 1) PSPL diet (daily consumption of 200 g PSPL) or a control diet (low polyphenols, with the amount of carotenoids adjusted to the same level as that of PSPL) (wk 2-3), washout diet (wk 4), and switched diet (wk 5-6). Fasting blood was collected weekly in the morning. T-lymphocyte function was assessed via the proliferation and secretion of immunoreactive cytokines.Salivary IgA secretion and the specific cytotoxic activities of cytotoxic T lymphocytes and natural killer (NK) cells were determined.RESULTS: The plasma β-carotene level increased with time in both groups, while the plasma polyphenol level decreased in the control group, and no significant difference was detected between the two groups.Although plasma polyphenol levels did not significantly increase in the PSPL group at the end of the study, they were significantly elevated in urine. PSPL consumption produced a significant increase in proliferation responsiveness of peripheral blood mononuclear cells (PBMC) and their secretion of immunoreactive IL-2 and IL-4. As well, lytic activity in NK cells was elevated in a time-dependent fashion. Salivary TgA secretion significantly decreased in control group after 2 wk, and returned to baseline following dietary switch to PSPL.CONCLUSION: Consumption of PSPL modulates various immune functions including increased proliferation responsiveness of PBMC, secretion of cytokines IL-2 and IL-4, and the lytic activity of NK cells. The responsible determinants of PSPL remain to be elucidated, as does the biological significance of the present observations.

  14. Atorvastatin prevents age-related and amyloid-β-induced microglial activation by blocking interferon-γ release from natural killer cells in the brain

    Directory of Open Access Journals (Sweden)

    Clarke Rachael

    2011-03-01

    Full Text Available Abstract Background Microglial function is modulated by several factors reflecting the numerous receptors expressed on the cell surface, however endogenous factors which contribute to the age-related increase in microglial activation remain largely unknown. One possible factor which may contribute is interferon-γ (IFNγ. IFNγ has been shown to increase in the aged brain and potently activates microglia, although its endogenous cell source in the brain remains unidentified. Methods Male Wistar rats were used to assess the effect of age and amyloid-β (Aβ on NK cell infiltration into the brain. The effect of the anti-inflammatory compound, atorvastatin was also assessed under these conditions. We measured cytokine and chemokine (IFNγ, IL-2, monocyte chemoattractant protein-1 (MCP-1 and IFNγ-induced protein 10 kDa (IP-10, expression in the brain by appropriate methods. We also looked at NK cell markers, CD161, NKp30 and NKp46 using flow cytometry and western blot. Results Natural killer (NK cells are a major source of IFNγ in the periphery and here we report the presence of CD161+ NKp30+ cells and expression of CD161 and NKp46 in the brain of aged and Aβ-treated rats. Furthermore, we demonstrate that isolated CD161+ cells respond to interleukin-2 (IL-2 by releasing IFNγ. Atorvastatin, the HMG-CoA reductase inhibitor, attenuates the increase in CD161 and NKp46 observed in hippocampus of aged and Aβ-treated rats. This was paralleled by a decrease in IFNγ, markers of microglial activation and the chemokines, MCP-1 and IP-10 which are chemotactic for NK cells. Conclusions We propose that NK cells contribute to the age-related and Aβ-induced neuroinflammatory changes and demonstrate that these changes can be modulated by atorvastatin treatment.

  15. Lysis of pig endothelium by IL-2 activated human natural killer cells is inhibited by swine and human major histocompatibility complex (MHC) class I gene products.

    Science.gov (United States)

    Itescu, S; Artrip, J H; Kwiatkowski, P A; Wang, S F; Minanov, O P; Morgenthau, A S; Michler, R E

    1997-01-01

    We have previously described a form of xenograft rejection, mediated by natural killer (NK) cells, occurring in pig-to-primate organ transplants beyond the period of antibody-mediated hyperacute rejection. In this study, two distinct NK activation pathways were identified as mechanisms of pig aortic endotheliual cell (PAEC) lysis by human NK cells. Using an antibody-dependent cellular cytotoxicity (ADCC) assay, a progressive increase in human NK lysis of PAEC was observed following incubation with human IgG at increasing serum titer. In the absence of IgG, a second mechanism of PAEC lysis by human NK cells was observed following activation with IL-2. IL-2 activation of human NK cells increased lysis of PAEC by over 3-fold compared with ADCC. These results indicate that IL-2 activation of human NK cells induces significantly higher levels of lytic activity than does conventional ADCC involving IgG and FcRIII. We next investigated the role of MHC class I molecules in the regulation of NK lysis following IL-2 activation. PAEC expression of SLA class I molecules was increased by up to 75% by treatment with human TNFa. Following treatment with TNFa at 1 u/ml, IL-2 activated human NK lysis of PAEC was inhibited at every effector:target (E:T) ratio tested. Maximal effect occurred at an E:T ratio of 10:1, with TNFa inhibiting specific lysis by 59% (p < 0.01). Incubation with an anti-SLA class I Mab, but not IgG isotype control, abrogated the protective effects of TNFa on NK lysis of PAEC, suggesting direct inhibitory effects of SLA class I molecules on human NK function. To investigate whether human MHC class I molecules might have similar effects on human NK lysis of PAEC, further experiments were performed using a soluble peptide derived from the alpha-helical region of HLA-B7. Incubation with the HLA-B7 derived peptide significantly reduced the IL-2 activated NK lytic activity against PAEC in a dose-dependent fashion. Maximal effect occurred at a concentration of 10 mg

  16. Genome wide transcriptional analysis of resting and IL2 activated human natural killer cells: gene expression signatures indicative of novel molecular signaling pathways

    Directory of Open Access Journals (Sweden)

    Schmitz Alexander

    2007-07-01

    Full Text Available Abstract Background Human natural killer (NK cells are the key contributors of innate immune response and the effector functions of these cells are enhanced by cytokines such as interleukine 2 (IL2. We utilized genome-wide transcriptional profiling to identify gene expression signatures and pathways in resting and IL2 activated NK cell isolated from peripheral blood of healthy donors. Results Gene expression profiling of resting NK cells showed high expression of a number of cytotoxic factors, cytokines, chemokines and inhibitory and activating surface NK receptors. Resting NK cells expressed many genes associated with cellular quiescence and also appeared to have an active TGFβ (TGFB1 signaling pathway. IL2 stimulation induced rapid downregulation of quiescence associated genes and upregulation of genes associated with cell cycle progression and proliferation. Numerous genes that may enhance immune function and responsiveness including activating receptors (DNAM1, KLRC1 and KLRC3, death receptor ligand (TNFSF6 (FASL and TRAIL, chemokine receptors (CX3CR1, CCR5 and CCR7, interleukin receptors (IL2RG, IL18RAB and IL27RA and members of secretory pathways (DEGS1, FKBP11, SSR3, SEC61G and SLC3A2 were upregulated. The expression profile suggested PI3K/AKT activation and NF-κB activation through multiple pathways (TLR/IL1R, TNF receptor induced and TCR-like possibly involving BCL10. Activation of NFAT signaling was supported by increased expression of many pathway members and downstream target genes. The transcription factor GATA3 was expressed in resting cells while T-BET was upregulated on activation concurrent with the change in cytokine expression profile. The importance of NK cells in innate immune response was also reflected by late increased expression of inflammatory chemotactic factors and receptors and molecules involved in adhesion and lymphocyte trafficking or migration. Conclusion This analysis allowed us to identify genes implicated in

  17. Conversion of adipose-derived stem cells into natural killer-like cells with anti-tumor activities in nude mice.

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    Hongxiu Ning

    Full Text Available Efforts to develop peripheral blood-derived nature killer (NK cells into therapeutic products have been hampered by these cells' low abundance and histoincompatibility. On the other hand, derivation of NK-like cells from more abundant cell sources such as embryonic stem cells (ESCs and umbilical cord blood (UCB requires the selection of rare CD34+ cells. Thus, we sought to convert adipose-derived stem cells (ADSCs, which are abundant and natively CD34+, into NK-like cells. When grown in hematopoietic induction medium, ADSCs formed sphere clusters and expressed hematopoietic markers CD34, CD45, and KDR. Further induction in NK cell-specific medium resulted in a population of cells that expressed NK cell marker CD56, and thus termed ADSC-NK. Alternatively, the hematopoietically induced ADSCs were transduced with NK cell-specific transcription factor E4BP4 prior to induction in NK cell-specific medium. This latter population of cells, termed ADSC-NKE, expressed CD56 and additional NK cell markers such as CD16, CD94, CD158, CD314, FasL, and NKp46. ADSC-NKE was as potent as NK leukemia cell NKL in killing breast cancer cell MCF7 and prostate cancer cells DU145, PC3, LnCap, DuPro, C4-2 and CWR22, but exhibited no killing activity toward normal endothelial and smooth muscle cells. In nude mice test ADSC-NKE was able to significantly delay the progression of tumors formed by MCF7 and PC3. When injected into immunocompetent rats, ADSC-NKE was detectable in bone marrow and spleen for at least 5 weeks. Together, these results suggest that ADSCs can be converted into NK-like cells with anti-tumor activities.

  18. Copy number variations of HLA-I and activation of NKp30 pathway determine the sensitivity of gastric cancer cells to the cytotoxicity of natural killer cells.

    Science.gov (United States)

    Xing, R; Li, L; Chen, L; Gao, Z; Wang, H; Li, W; Cui, J; Tian, G; Liang, Q; Yu, J; Sung, J J; Luo, G; Gao, H; Xu, X; Yang, H; Wang, J; Zhang, X; Wang, J M; Huang, J; Yu, Y; Wang, J; Lu, Y

    2016-05-19

    Nude mice are important in vivo model for characterization of cell malignancy behavior; however, many cancer cells fail to form tumors in it. Understanding this defective mechanism may provide novel insights into tumorigenesis and how tumor cells escape innate immunity. Whole-genome sequencing was conducted on two gastric cancer (GC) cells, BGC823 and AGS, which do and do not form tumors in nude mice, to identify their genomic differences relevant to natural killer (NK) cells. We found that the tumorigenic capacity of human GC cell lines was dependent on the recruitment and activation of NK cells in xenograft tumors. We used whole-genome sequence (WGS) on GC cell lines to identify potential genes controlling susceptibility to NK-mediated killing. The tumorigenic cell line BGC823 expressed high levels of HLA-I because of copy gain and was resistant to NK cell killing. In contrast, another cell line AGS expressing low levels of HLA-I with activated NKp30/MAPK/IL-12 (interleukin-12) or IL-2 (interleukin-2) pathway was susceptible to NK lysis. Treatment of tumor bearing mice with systemic administration of IL-12 in combination with intratumor injection of anti-HLA-I antibody significantly increased NK cell recruitment into xenograft tumors, which became sensitive to NK killing, resulting in reduced tumor progression. In human GC specimens, decreased HLA-I expression and increased NK cells surrounding tumor cells were correlated with decreased metastasis potential and better prognosis of patients. Our results provide a mechanistic basis for GC cells to escape NK lysis and a promising prospect of NK immunotherapy for GC cells.

  19. The eyeball killer: serial killings with postmortem globe enucleation.

    Science.gov (United States)

    Coyle, Julie; Ross, Karen F; Barnard, Jeffrey J; Peacock, Elizabeth; Linch, Charles A; Prahlow, Joseph A

    2015-05-01

    Although serial killings are relatively rare, they can be the cause of a great deal of anxiety while the killer remains at-large. Despite the fact that the motivations for serial killings are typically quite complex, the psychological analysis of a serial killer can provide valuable insight into how and why certain individuals become serial killers. Such knowledge may be instrumental in preventing future serial killings or in solving ongoing cases. In certain serial killings, the various incidents have a variety of similar features. Identification of similarities between separate homicidal incidents is necessary to recognize that a serial killer may be actively killing. In this report, the authors present a group of serial killings involving three prostitutes who were shot to death over a 3-month period. Scene and autopsy findings, including the unusual finding of postmortem enucleation of the eyes, led investigators to recognize the serial nature of the homicides.

  20. Irradiation-induced up-regulation of HLA-E on macrovascular endothelial cells confers protection against killing by activated natural killer cells.

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    Isabelle Riederer

    Full Text Available BACKGROUND: Apart from the platelet/endothelial cell adhesion molecule 1 (PECAM-1, CD31, endoglin (CD105 and a positive factor VIII-related antigen staining, human primary and immortalized macro- and microvascular endothelial cells (ECs differ in their cell surface expression of activating and inhibitory ligands for natural killer (NK cells. Here we comparatively study the effects of irradiation on the phenotype of ECs and their interaction with resting and activated NK cells. METHODOLOGY/PRINCIPAL FINDINGS: Primary macrovascular human umbilical vein endothelial cells (HUVECs only express UL16 binding protein 2 (ULBP2 and the major histocompatibility complex (MHC class I chain-related protein MIC-A (MIC-A as activating signals for NK cells, whereas the corresponding immortalized EA.hy926 EC cell line additionally present ULBP3, membrane heat shock protein 70 (Hsp70, intercellular adhesion molecule ICAM-1 (CD54 and HLA-E. Apart from MIC-B, the immortalized human microvascular endothelial cell line HMEC, resembles the phenotype of EA.hy926. Surprisingly, primary HUVECs are more sensitive to Hsp70 peptide (TKD plus IL-2 (TKD/IL-2-activated NK cells than their immortalized EC counterpatrs. This finding is most likely due to the absence of the inhibitory ligand HLA-E, since the activating ligands are shared among the ECs. The co-culture of HUVECs with activated NK cells induces ICAM-1 (CD54 and HLA-E expression on the former which drops to the initial low levels (below 5% when NK cells are removed. Sublethal irradiation of HUVECs induces similar but less pronounced effects on HUVECs. Along with these findings, irradiation also induces HLA-E expression on macrovascular ECs and this correlates with an increased resistance to killing by activated NK cells. Irradiation had no effect on HLA-E expression on microvascular ECs and the sensitivity of these cells to NK cells remained unaffected. CONCLUSION/SIGNIFICANCE: These data emphasize that an irradiation

  1. Stochastic modeling of a serial killer

    CERN Document Server

    Simkin, M V

    2012-01-01

    We analyze the time pattern of the activity of a serial killer, who during twelve years had murdered 53 people. The plot of the cumulative number of murders as a function of time is of "Devil's staircase" type. The distribution of the intervals between murders (step length) follows a power law with the exponent of 1.4. We propose a model according to which the serial killer commits murders when neuronal excitation in his brain exceeds certain threshold. We model this neural activity as a branching process, which in turn is approximated by a random walk. As the distribution of the random walk return times is a power law with the exponent 1.5, the distribution of the inter-murder intervals is thus explained. We confirm analytical results by numerical simulation.

  2. Photodynamic Treatment of Tumor with Bacteria Expressing KillerRed.

    Directory of Open Access Journals (Sweden)

    Libo Yan

    Full Text Available Photodynamic therapy (PDT is a cancer treatment modality in which a photosensitizing dye is administered and exposed to light to kill tumor cells via the production of reactive oxygen species (ROS. A fundamental obstacle for PDT is the low specificity for staining solid tumors with dyes. Recently, a tumor targeting system guided by anaerobic bacteria was proposed for tumor imaging and treatment. Here, we explore the feasibility of the genetically encoded photosensitizer KillerRed, which is expressed in Escherichia coli, to treat tumors. Using nitroblue tetrazolium (NBT, we detected a lengthy ROS diffusion from the bodies of KillerRed-expressing bacteria in vitro, which demonstrated the feasibility of using bacteria to eradicate cells in their surroundings. In nude mice, Escherichia coli (E. coli expressing KillerRed (KR-E. coli were subcutaneously injected into xenografts comprising CNE2 cells, a human nasopharyngeal carcinoma cell line, and HeLa cells, a human cervical carcinoma cell line. KR-E. coli seemed to proliferate rapidly in the tumors as observed under an imaging system. When the intensity of fluorescence increased and the fluorescent area became as large as the tumor one day after KR-E. coli injection, the KR-E. coli-bearing tumor was irradiated with an orange light (λ = 540-580 nm. In all cases, the tumors became necrotic the next day and were completely eliminated in a few days. No necrosis was observed after the irradiation of tumors injected with a vehicle solution or a vehicle carrying the E. coli without KillerRed. In successfully treated mice, no tumor recurrence was observed for more than two months. E. coli genetically engineered for KillerRed expression are highly promising for the diagnosis and treatment of tumors when the use of bacteria in patients is cleared for infection safety.

  3. Activation of Estrogen Receptor Transfected into a Receptor-Negative Brest Cancer Cell Line Decreases the Metastatic and Invasive Potential of the Cells

    Science.gov (United States)

    Garcia, Marcel; Derocq, Danielle; Freiss, Gilles; Rochefort, Henri

    1992-12-01

    Breast cancers containing estrogen receptors are responsive to antiestrogen treatment and have a better prognosis than estrogen receptor-negative tumors. The loss of estrogen and progesterone receptors appears to be associated with a progression to less-differentiated tumors. We transfected the human estrogen receptor into the estrogen receptor-negative metastatic breast cancer cell line MDA-MB-231 in an attempt to restore their sensitivity to antiestrogens. Two stable sublines of MDA-MB-231 cells (HC1 and HE5) expressing functional estrogen receptors were studied for their ability to grow and invade in vitro and to metastasize in athymic nude mice. The number and size of lung metastases developed by these two sublines in ovariectomized nude mice was not markedly altered by tamoxifen but was inhibited 3-fold by estradiol. Estradiol also significantly inhibited in vitro cell proliferation of these sublines and their invasiveness in Matrigel, a reconstituted basement membrane, whereas the antiestrogens 4-hydroxytamoxifen and ICI 164,384 reversed these effects. These results show that estradiol inhibits the metastatic ability of estrogen receptornegative breast cancer cells following transfection with the estrogen receptor, whereas estrogen receptor-positive breast cancers are stimulated by estrogen, indicating that factors other than the estrogen receptor are involved in progression toward hormone independence. Reactivation or transfer of the estrogen receptor gene can therefore be considered as therapeutic approaches to hormone-independent cancers

  4. Pim-1 kinase inhibits the activation of reporter gene expression in Elk-1 and c-Fos reporting systems but not the endogenous gene expression: an artifact of the reporter gene assay by transient co-transfection

    Directory of Open Access Journals (Sweden)

    Yan B.

    2006-01-01

    Full Text Available We have studied the molecular mechanism and signal transduction of pim-1, an oncogene encoding a serine-threonine kinase. This is a true oncogene which prolongs survival and inhibits apoptosis of hematopoietic cells. In order to determine whether the effects of Pim-1 occur by regulation of the mitogen-activated protein kinase pathway, we used a transcriptional reporter assay by transient co-transfection as a screening method. In this study, we found that Pim-1 inhibited the Elk-1 and NFkappaB transcriptional activities induced by activation of the mitogen-activated protein kinase cascade in reporter gene assays. However, Western blots showed that the induction of Elk-1-regulated expression of endogenous c-Fos was not affected by Pim-1. The phosphorylation and activation of neither Erk1/2 nor Elk-1 was influenced by Pim-1. Also, in the gel shift assay, the pattern of endogenous NFkappaB binding to its probe was not changed in any manner by Pim-1. These data indicate that Pim-1 does not regulate the activation of Erk1/2, Elk-1 or NFkappaB. These contrasting results suggest a pitfall of the transient co-transfection reporter assay in analyzing the regulation of transcription factors outside of the chromosome context. It ensures that results from reporter gene expression assay should be verified by study of endogenous gene expression.

  5. The influence of tumor immunity suppressors on the effector stage of human and animal lymphokine-activated killer cells.

    Science.gov (United States)

    Abronina, I F; Indrova, M; Bubenic, J; Figurin, K M; Malakhova, N V; Bykovskaya, S N

    1993-01-01

    Spleen cells of tumor-bearing mice suppressed the cytolytic activity of syngeneic LAK cells when added to the mixture of LAK cells and target cells at the beginning of the cytotoxicity test. Spleen cells of MC 14 tumor-bearing mice acquired the suppressor potential as early as 10 days after tumor transplantation; the suppressor activity in the EL 4 and X63-Ag8.653 tumor-bearing animals was first revealed at the 30th day and manifested itself up to the 120th day. The suppressor activity was expressed in a dose-dependent manner, both by unfractionated spleen cells and nylon wool-passed and plastic-adherent sub-populations. Similar results were obtained during the analysis of anti-tumor immunity suppressors in bladder cancer patients. MNC, nylon wool-passed and plastic-adherent cells of patients with stages I-II disease suppressed the cytotoxicity of autologous LAK cells in 2/6 cases; all patients [4] with III-IV stage possessed such suppressor activity. Presumably, the tumor growth induces the activity of suppressor T cells and monocytes/macrophages. The suppressor activity can interfere with the antitumor effect of autologous (syngeneic) LAK cells at the effector stage.

  6. Natural killer cells in psoriasis.

    LENUS (Irish Health Repository)

    Tobin, A M

    2012-02-01

    Psoriasis is one of the most common immune-mediated disorders. There is evidence that it is mediated by Th1 and, more recently, Th17 cells. The cytokine pattern, particularly the dominance of TNF-alpha, implicates the innate immune system in psoriasis pathogenesis. Of the many components of the innate immune system known to be involved in psoriatic lesions, natural killer and natural killer T cells appear to have a unique role. We review the evidence supporting a role for natural killer cells in psoriasis.

  7. Killer Whale Genetic Data - Southern resident killer whale pedigree analysis

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — In this project, we are using genetic variation to infer mating patterns in the southern killer whale community. In Canada, this population was listed as threatened...

  8. The Internalization of Neurotensin by the Low-Affinity Neurotensin Receptors (NTSR2 and vNTSR2) Activates ERK 1/2 in Glioma Cells and Allows Neurotensin-Polyplex Transfection of tGAS1.

    Science.gov (United States)

    Ayala-Sarmiento, Alberto E; Martinez-Fong, Daniel; Segovia, José

    2015-08-01

    Glioblastoma is the most malignant primary brain tumor and is very resistant to treatment; hence, it has a poor prognosis. Neurotensin receptor type 1 (NTSR1) plays a key role in cancer malignancy and has potential therapeutic applications. However, the presence and function of neurotensin (NTS) receptors in glioblastoma is not clearly established. RT-PCR assays showed that healthy (non-tumor) astroglial cells and C6 glioma cells express NTSR2 and its isoform (vNTSR2) rather than NTSR1. In glioma cells, NTS promotes the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK 1/2), an effect that was completely abolished by blocking the internalization of the NTS/NTSR complex. We demonstrated pharmacologically that the internalization is dependent on the activation of NTSR2 receptors and it was prevented by levocabastine, a NTSR2 receptor antagonist. The internalization of NTSR2 and vNTSR2 was further demonstrated by its ability to mediate gene transfer (transfection) via the NTS-polyplex system. Expression of reporter transgenes and of the pro-apoptotic soluble form of growth arrest specific 1 (tGAS1) was observed in glioma cells. A significant reduction on the viability of C6 cells was determined when tGAS1 was transfected into glioma cells. Conversely, astroglial cells could neither internalize NTS nor activate ERK 1/2 and could not be transfected by the NTS-polyplex. These results demonstrate that the internalization process of NTSR2 receptors is a key regulator necessary to trigger the activation of the ERK 1/2. Our data support a new internalization pathway in glioma C6 cells that involve NTSR2/vNTSR2, which can be used to selectively transfer therapeutic genes using the NTS-polyplex system.

  9. Lysis of endogenously infected CD4+ T cell blasts by rIL-2 activated autologous natural killer cells from HIV-infected viremic individuals.

    Directory of Open Access Journals (Sweden)

    Manuela Fogli

    2008-07-01

    Full Text Available Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However, it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous, endogenously HIV-1-infected CD4+ T cells. Here, we stimulate primary CD4+ T cells, purified ex vivo from HIV-1-infected viremic patients, with PHA and rIL2 (with or without rIL-7. This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that, subsequent to the selective down-modulation of MHC class-I (MHC-I molecules, HIV-1-infected p24(pos blasts become partially susceptible to lysis by rIL-2-activated NK cells, while uninfected p24(neg blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However, the degree of NK cell cytolytic activity against autologous, endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-I(neg cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs and with the high frequency of the anergic CD56(neg/CD16(pos subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively, our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24(pos blasts derived from primary T cells.

  10. Prevention of hepatocellular carcinoma in mice by IL-2 and B7-1genes co-transfected liver cancer cell vaccines

    Institute of Scientific and Technical Information of China (English)

    Ning-Ling Ge; Sheng-Long Ye; Ning Zheng; Rui-Xia Sun; Yin-Kun Liu; Zhao-You Tang

    2003-01-01

    AIM: To study the immunoprotective effect of liver cancer vaccine with co-transfected IL-2 and B7-1 genes on hepatocarcinogenesis in mice.METHODS: The murine liver cancer cell line Hepal-6 was transfected with IL-2 and/or B7-1 gene via recombinant adenoviral vectors and the liver cancer vaccines were prepared. C57BL/6 mice were immunized with these vaccines and challenged with the parental Hepal-6 cells afterwards.The immunoprotection was investigated and the reactive T cell line was assayed.RESULTS: The immunoprotection of the tumor vaccine was demonstrated. The effect of IL-2 and B7-1 genes cotransfected Hepal-6 liver cancer vaccine (Hep6-IL2/B7vaccine) on the onset of tumor formation was the strongest.When attacked with wild Hepal-6 cells, the median survival period of the mice immunized with Hep6-IL2/B7 vaccine was the longest (68 days, χ2=7.70-11.69, P<0.05) and the implanted tumor was the smallest (z =3.20-44.10, P<0.05).The effect of single IL-2 or B7-1 gene-transfected vaccine was next to the IL2/B7 gene co-transfected group, and the mean survival periods were 59 and 54 days, respectively.The mean survival periods of wild or enhanced green fluorescence protein gene modified vaccine immunized group were 51 and 48 days, respectively. The mice in control group all died within 38 days and the implanted tumor was the largest (z=3.20-40.21, P<0.05). The cellular immunofunction test and cytotoxicity study showed that the natural killer (NK) cell, lymphokine activated killer (LAK) cell and cytotoxic T lymphocyte (CTL) activities were significantly increased in mice immunized with the Hep6-IL2/B7 vaccine, (29.5±2.5%,65.0±2.9%, 83.1±1.5% respectively, compared with other groups, P<0.05).CONCLUSION: The Hep6-IL2/B7 liver cancer vaccines can induce the mice to produce activated and specific CTL against the parental tumor cells, and demonstrate stronger effect on the hepatocarcinogenesis than single gene modified or the regular tumor vaccine. Therefore, the

  11. Evaluation on the Clinical Efficacy of Dendritic Cell-Activated Cytokine-Induced Killer Cells Combined with Conventional Therapy in the Treatment of Malignant Tumors

    Directory of Open Access Journals (Sweden)

    Hong WEI

    2016-06-01

    Full Text Available Objective: To evaluate the clinical efficacy of dendritic cell-activated cytokine-induced killer (DC-CIK cells combined with conventional therapy in the treatment of malignant tumors.Methods: A total of 100 patients with malignant tumors were randomly divided into two groups. Treatment group received conventional therapy combined with DC-CIK while control group received conventional therapy alone. The short-term efficacy, adverse reactions and changes of lymphocyte subpopulation were all compared between two groups after treatment.Results: The overall response rate (ORR was higher in treatment group (86.00% than in control group (54.00%, the difference was statistically significant (P<0.05. White blood cell count (WBC reduced after treatment when compared with treatment before (P=0.001, but liver and kidney function had no obvious change in treatment group (P>0.05. WBC reduced markedly, but the level of alanine aminotransferase (ALT increased obviously after treatment in control group (P<0.001. WBC was higher, but the level of ALT was lower in treatment group than in control group (P<0.001. However, there was no difference between two groups regarding serum creatinine (Scr and blood urea nitrogen (BUN (P>0.05. In treatment group, the levels of CD3+, CD3+CD4+, CD3+CD8+, and CD3+CD56+ increased (P<0.05, but the level of CD4+/CD8+ had no significant change (P>0.05. In control group, the levels of CD3+ and CD3+CD4+ reduced (P<0.05, while the levels of CD3+CD8+, CD3+CD56+ and CD4+/CD8+ had no significant change (P>0.05. The levels of CD3+, CD3+CD4+, CD3+CD8+ and CD3+CD56+ in treatment group were higher than those in control group (P<0.01, whereas CD4+/CD8+ was lower than that in control group (P<0.01.Conclusion: DC-CIK combined with conventional therapy, safe and effective, is capable of promoting the recovery of leukocytes and liver and kidney function, and improving the cellular immune function, which may provide a new therapeutic regimen for

  12. Eradication of intractable malignant ascites by abdominocentesis, reinfusion of concentrated ascites, and adoptive immunotherapy with dendritic cells and activated killer cells in a patient with recurrent lung cancer: a case report

    Directory of Open Access Journals (Sweden)

    Kimura Hideki

    2008-12-01

    Full Text Available Abstract Introduction Malignant ascites is often a sign of a terminal stage in several malignant diseases. To control ascites, drainage and intra-abdominal chemotherapy are often used in those patients but eradication of ascites is difficult and prognosis is poor. Case presentation A 55-year-old woman was admitted to our hospital on 26 January 2007 with dyspnea, abdominal distention and oliguria. Abdominocentesis revealed peritoneal carcinomatosis resulting from abdominal recurrence from lung cancer. To alleviate the dyspnea and abdominal distention, we drained the ascites aseptically and infused them intravenously back into the patient after removal of tumor cells by centrifugation, and then concentration by apheresis. After the drainage of ascites, we intraperitoneally infused activated killer cells and dendritic cells from the patient's tumor-draining lymph nodes, together with 4.5 × 105U interleukin-2 in 50 ml saline by 2.1 ml/hour infuser balloon. Drastic decreases in the tumor cell count and in ascite retention were observed after several courses of ascites drainage, intravenous infusion and intraperitoneal immunotherapy. The plasma protein level was maintained during the treatment notwithstanding the repeated drainage of ascites. Cell surface marker analysis, cytotoxic activities against autologous tumor cells and interferon-gamma examination of ascites suggested the possibility that these effects were mediated by immunological responses of activated killer cells and dendritic cells infused intraperitoneally. Conclusion Combination of local administration of immune cells and infusion of concentrated cell free ascites may be applicable for patients afflicted with refractory ascites.

  13. Rapid and Permanent Neuronal Inactivation In Vivo via Subcellular Generation of Reactive Oxygen with the Use of KillerRed

    Directory of Open Access Journals (Sweden)

    Daniel C. Williams

    2013-10-01

    Full Text Available Inactivation of selected neurons in vivo can define their contribution to specific developmental outcomes, circuit functions, and behaviors. Here, we show that the optogenetic tool KillerRed selectively, rapidly, and permanently inactivates different classes of neurons in C. elegans in response to a single light stimulus, through the generation of reactive oxygen species (ROS. Ablation scales from individual neurons in single animals to multiple neurons in populations and can be applied to freely behaving animals. Using spatially restricted illumination, we demonstrate that localized KillerRed activation in either the cell body or the axon triggers neuronal degeneration and death of the targeted cell. Finally, targeting KillerRed to mitochondria results in organelle fragmentation without killing the cell, in contrast to the cell death observed when KillerRed is targeted to the plasma membrane. We expect this genetic tool to have wide-ranging applications in studies of circuit function and subcellular responses to ROS.

  14. Role of inositol phospholipid signaling in natural killer cell biology

    Directory of Open Access Journals (Sweden)

    Matthew eGumbleton

    2013-03-01

    Full Text Available Natural Killer (NK cells are important in the host defense against malignancy and infection. At a cellular level NK cells are activated when signals from activating receptors exceed signaling from inhibitory receptors. At a molecular level NK cells undergo an education process to prevent autoimmunity. Mouse models have shown important roles for inositol phospholipid signaling in lymphocytes. NK cells from mice with deletion in different members of the PI3K signaling pathway have defective development, natural killer cell repertoire expression (NKRR and effector function. Here we review the role of inositol phospholipid signaling in NK cell biology.

  15. Production of functional killer protein in batch cultures upon a shift from aerobic to anaerobic conditions

    Directory of Open Access Journals (Sweden)

    Gildo Almeida da Silva

    2011-06-01

    Full Text Available The aim of this work was to study the production of functional protein in yeast culture. The cells of Saccharomyces cerevisiae Embrapa 1B (K+R+ killed a strain of Saccharomyces cerevisiae Embrapa 26B (K-R-in grape must and YEPD media. The lethal effect of toxin-containing supernatant and the effect of aeration upon functional killer production and the correlation between the products of anaerobic metabolism and the functional toxin formation were evaluated. The results showed that at low sugar concentration, the toxin of the killer strain of Sacch. cerevisiae was only produced under anaerobic conditions . The system of killer protein production showed to be regulated by Pasteur and Crabtree effects. As soon as the ethanol was formed, the functional killer toxin was produced. The synthesis of the active killer toxin seemed to be somewhat associated with the switch to fermentation process and with concomitant alcohol dehydrogenase (ADH activity.

  16. Establishment and application of co-transfection screening method for phytoestrogen active constituents%基于共转染的植物雌激素活性成分筛选方法的建立与应用

    Institute of Scientific and Technical Information of China (English)

    魏华波; 阿布力米提·伊力; 马庆苓; 买迪娜; 王振华; 马海蓉

    2011-01-01

    目的:建立一种以共转染为基础的、高效灵敏的植物雌激素活性成分的细胞筛选方法,应用此方法研究鹰嘴豆提取部位的雌激素效应.方法:RT-PCR方法扩增人雌激素受体α(hERα)cDNA,并构建哺乳细胞表达载体pERα.将此载体与含有雌激素应答序列(3 ×ERE)的Luc报告基因载体(pERE-Luc)以不同比例共转染MCF-7细胞,比较不同比例下Luc的活力,确定最佳共转染比例.用芒柄花素、鹰嘴豆豆芽素A和染料木素等植物雌激素验证了该模型的灵敏性,并进一步测定了鹰嘴豆不同提取部位的Luc活力.结果:将pERα与pERE-Luc共转染MCF-7细胞,与pERE-Luc单转染相比,显著提高Luc的活力,且在10∶1( pERE-Luc∶ pERα)时活力最高,Luc活力提高了5倍.用此转染比例测定芒柄花素、鹰嘴豆豆芽素A和染料木素等植物雌激素Luc活力测定结果表明,共转染能诱导Luc的表达,且ER特异性抑制剂ICI 182,780能抑制其活性.利用此模型发现鹰嘴豆的70%乙醇总提取物、乙酸乙酯部位和石油醚部位均具有大量的雌激素活性物质.ICI 182,780能有效抑制其雌激素效应.结论:成功建立了一种共转染为基础的植物雌激素活性成分的筛选方法,该方法具有较高的特异性和灵敏性,可用于植物雌激素活性成分的筛选.%Objective: To establish a highly sensitive screening method for phytoestrogen active constituents and to primarily screen the phytoestrogenic active constituents from the chickpea extractions by the method. Method: Human Erα cDNA was cloned using MCF-7 total RNA as the template by RT-PCR and then was constructed into a pcDNA3 and named as pERa. The cell line MCF-7 was co-transfected with pERa and the reporter plasmid pERE-Luc which carrying the estrogen response element (ERE) plus the luciferase reporter gene. The luciferase activity was then assayed. The model was optimized by changing the ratio of two plasmids. The feasibility of the

  17. Immunobiology of natural killer cells. Volume II

    Energy Technology Data Exchange (ETDEWEB)

    Lotzova, E.; Herberman, R.B.

    1986-01-01

    This book provides a review of natural killer (NK) cell-mediated immunity in humans and experimental animal system. Topics for the volume include: In vivo activities of NK cells against primary and metastatic tumors in experimental animals; involvement of NK cells in human malignant disease; impaired NK cell profile in leukemia patients; in vivo modulation of NK activity in cancer patients; implications of aberrant NK cell activity in nonmalignant, chronic diseases; NK cell role in regulation of the growth and functions of hemopoietic and lymphoid cells; NK cells active against viral, bacterial, protozoan, and fungal infections; cytokine secretion and noncytotoxic functions of human large granular lymphocytes; augmentation of NK activity; regulation of NK cell activity by suppressor cells; NK cell cloning technology and characteristics of NK cell clones; comparison of antibody-dependent cellular cytotoxicity (ADCC) and NK activity, and index.

  18. Interaction of a dengue virus NS1-derived peptide with the inhibitory receptor KIR3DL1 on natural killer cells.

    Science.gov (United States)

    Townsley, E; O'Connor, G; Cosgrove, C; Woda, M; Co, M; Thomas, S J; Kalayanarooj, S; Yoon, I-K; Nisalak, A; Srikiatkhachorn, A; Green, S; Stephens, H A F; Gostick, E; Price, D A; Carrington, M; Alter, G; McVicar, D W; Rothman, A L; Mathew, A

    2016-03-01

    Killer immunoglobulin-like receptors (KIRs) interact with human leucocyte antigen (HLA) class I ligands and play a key role in the regulation and activation of NK cells. The functional importance of KIR-HLA interactions has been demonstrated for a number of chronic viral infections, but to date only a few studies have been performed in the context of acute self-limited viral infections. During our investigation of CD8(+) T cell responses to a conserved HLA-B57-restricted epitope derived from dengue virus (DENV) non-structural protein-1 (NS1), we observed substantial binding of the tetrameric complex to non-T/non-B lymphocytes in peripheral blood mononuclear cells (PBMC) from a long-standing clinical cohort in Thailand. We confirmed binding of the NS1 tetramer to CD56(dim) NK cells, which are known to express KIRs. Using depletion studies and KIR-transfected cell lines, we demonstrated further that the NS1 tetramer bound the inhibitory receptor KIR3DL1. Phenotypical analysis of PBMC from HLA-B57(+) subjects with acute DENV infection revealed marked activation of NS1 tetramer-binding natural killer (NK) cells around the time of defervescence in subjects with severe dengue disease. Collectively, our findings indicate that subsets of NK cells are activated relatively late in the course of acute DENV illness and reveal a possible role for specific KIR-HLA interactions in the modulation of disease outcomes.

  19. Selective serotonin-reuptake inhibitor and norepinephrine dopamine reuptake inhibitor antidepressants do not affect natural killer cell activity in vitro Antidepressivos inibidores seletivos de recaptação da serotonina e inibidores da recaptação de noradrenalina e dopamina não afetam a atividade celular natural killer in vitro

    Directory of Open Access Journals (Sweden)

    Gabriel J. Chittó Gauer

    2009-01-01

    Full Text Available OBJECTIVE: This study aims to evaluate the citotoxic activity of two commonly used anti-depressants: paroxetine and bupropion. We also evaluated the in vitro natural killer activity (NKA after incubating the blood samples with the antidepressants. METHODS: Peripheral blood samples from 15 healthy volunteers were collected and the mononuclear cells (PBMCs were isolated and incubated for 24h with (or without = control cells paroxetine and bupropion, in concentrations of 30, 100 and 1000 ng/ml. After the incubation period in both groups, the amount of dead cells was calculated using trypam blue technique. NKA was evaluated using the classic51Cr release assay. CONCLUSIONS: PBMCs dead cells occurred in both groups and in proportion to all pharmacological concentrations. Nevertheless, the NKA was not affected, even with the reduction in the number of effective cells.OBJETIVO: Avaliar o efeito citotóxico de dois antidepressivos comumente utilizados na prática, a paroxetina e a bupropriona. Além disso, buscou-se avaliar a atividade natural killer (ANK após a incubação dos linfócitos com esses fármacos. MÉTODOS: Sangue venoso de 15 participantes foi coletado e as células mononucleares (PBMCs foram separadas e incubadas por 24h com (ou sem = grupo-controle concentrações de paroxetina e bupropiona em 30, 100 e 1.000 ng/ml. Após a incubação, a quantidade das células mortas foi contada utilizando-se o método trypan blue. Posteriormente foi avaliada a ANK por meio do ensaio clássico de liberação do Cr51. CONCLUSÕES: Ocorreu morte celular de PBMCs proporcionais às doses dos fármacos, no entanto, a ANK não foi afetada, mesmo com a redução do número de células efetoras.

  20. In Vivo Imaging of Natural Killer Cell Trafficking in Tumors

    NARCIS (Netherlands)

    Galli, Filippo; Rapisarda, Anna Serafina; Stabile, Helena; Malviya, Gaurav; Manni, Isabella; Bonanno, Elena; Piaggio, Giulia; Gismondi, Angela; Santoni, Angela; Signore, Alberto

    2015-01-01

    Natural killer cells (NKs) are important effectors of the innate immune system, with marked antitumor activity. Imaging NK trafficking in vivo may be relevant to following up the efficacy of new therapeutic approaches aiming at increasing tumor-infiltrating NKs (TINKs). The specific aims of present

  1. The transfection of embryonic stem cells with Tet-on system and its responsiveness to doxycycline

    Institute of Scientific and Technical Information of China (English)

    WANG; Yan; CONG; Xiaoqian; LIU; Deli; ZHANG; Wenjie; CUI; L

    2005-01-01

    We transiently transfected pTet-on and pTRE2hyg-luciferase into the mouse embryonic stem cells (ESCs) using lipofectamine, and analyzed its inductive effect by adding serial concentrations of doxycycline (DOX). The results showed that in the transfected group, the luciferase activity of the cells was gradually increased along with the increasing concentration of DOX. While in the non-transfected group, the luciferase activity was not detectable even with DOX treatment. This indicated that the ESCs transfected with Tet-on system could response to DOX very well, and the regulation of target gene expression is dose dependent.

  2. Formation of functional asialoglycoprotein receptor after transfection with cDNAs encoding the receptor proteins.

    OpenAIRE

    McPhaul, M; Berg, P.

    1986-01-01

    The rat asialoglycoprotein receptor (ASGP-R) has been expressed in cultured rat hepatoma cells (HTC cells) after transfection with cloned cDNAs. Fluorescence-activated cell sorting of transfected cells was used to identify the functional cDNA clones and to isolate cells expressing the ASGP-R. Simultaneous or sequential transfections with two cloned cDNAs that encode related but distinctive polypeptide chains were needed to obtain ASGP-R activity; transfection with either cDNA alone failed to ...

  3. Evaluation of in vitro screening system for estrogenicity: comparison of stably transfected human estrogen receptor-α transcriptional activation (OECD TG455) assay and estrogen receptor (ER) binding assay.

    Science.gov (United States)

    Lee, Hae Kyung; Kim, Tae Sung; Kim, Chang Yeong; Kang, Il Hyun; Kim, Mi Gyeong; Jung, Ki Kyung; Kim, Hyung Sik; Han, Soon Young; Yoon, Hae Jung; Rhee, Gyu Seek

    2012-01-01

    The estrogenic activity of industrial chemicals, di(2-ethylhexyl) phthalate (DEHP), di(n-butyl) phthalate (DBP), benzylbutyl phthalate (BBP), diethyl phthalate (DEP), tetrabromobisphenol A (TBBPA), bisphenol A (BPA), and nonylphenol (NP), was compared using OECD test guideline 455(TG455), stably transfected transcriptional activation (STTA) and estrogen receptor (ER) binding assays. The estrogenic activity of BBP, BPA and NP were approximately 180,000-fold (PC(50), 4.32 x 10(-6 )M), 5,000-fold (PC(50), 1.26 x 10(-7) M) and 120,000-fold (PC(50), 2.92 x 10(-6 )M) less than 17β-estradiol (PC(50), 2.43 x 10(-11)M), whereas DEHP, DBP and DEP did not show any estrogenicity activity in the STTA assay. Moreover, binding affinities to human ERα of BBP, BPA, and NP were approximately 200,000-fold (IC(50), 4.91 x 10(-4) M), 8000-fold (IC(50), 1.92 x 10(-5) M) and 1400-fold (IC(50), 3.34 x 10(-6) M) less than 17β-estradiol (IC(50), 2.45 x 10(-9) M) in competitive human ERα binding assay. The relative potencies of STTA assay were very similar to ER binding, E-screen, and Yeast screening assays. Therefore, our results suggested that OECD test guideline TG455 may be useful as a screening test for potential endocrine disruptors.

  4. Serum and glucocorticoid-regulated kinase Sgk1 inhibits insulin-dependent activation of phosphomannomutase 2 in transfected COS-7 cells.

    Science.gov (United States)

    Menniti, Miranda; Iuliano, Rodolfo; Amato, Rosario; Boito, Rosalia; Corea, Monica; Le Pera, Ilaria; Gulletta, Elio; Fuiano, Giorgio; Perrotti, Nicola

    2005-01-01

    Serum- and glucocorticoid-regulated kinase (Sgk1) is considered to be an essential convergence point for peptide and steroid regulation of ENaC-mediated sodium transport. We tried to identify molecular partners of Sgk1 by yeast two-hybrid screening. Yeast two-hybrid screening showed a specific interaction between Sgk1 and phosphomannomutase (PMM)2, the latter of which is an enzyme involved in the regulation of glycoprotein biosynthesis. The interaction was confirmed in intact cells by coimmunoprecipitation and colocalization detected using confocal microscopy. We were then able to demonstrate that Sgk1 phosphorylated PMM2 in an in vitro assay. In addition, we found that the enzymatic activity of PMM2 is upregulated by insulin treatment and that Sgk1 completely inhibits PMM2 activity both in the absence and in the presence of insulin stimulation. These data provide evidence suggesting that Sgk1 may modulate insulin action on the cotranslational glycosylation of glycoproteins.

  5. Activation of the insulin receptor (IR) by insulin and a synthetic peptide has different effects on gene expression in IR-transfected L6 myoblasts

    DEFF Research Database (Denmark)

    Jensen, M.; Palsgaard, J.; Borup, R.;

    2008-01-01

    differentially activates post-receptor signalling, we studied the gene expression profile in response to IR activation by either insulin or S597 using microarray technology. We found striking differences between the patterns induced by these two ligands. Most remarkable was that almost half of the genes...... differentially regulated by insulin and S597 were involved in cell proliferation and growth. Insulin either selectively regulated the expression of these genes or was a more potent regulator. Furthermore, we found that half of the differentially regulated genes interact with the genes involved with the MAPK...... differentially affect gene expression in cells, resulting in a different mitogenicity of the two ligands, a finding which has critical therapeutic implications Udgivelsesdato: 2008/6/15...

  6. killerFLIP: a novel lytic peptide specifically inducing cancer cell death.

    Science.gov (United States)

    Pennarun, B; Gaidos, G; Bucur, O; Tinari, A; Rupasinghe, C; Jin, T; Dewar, R; Song, K; Santos, M T; Malorni, W; Mierke, D; Khosravi-Far, R

    2013-10-31

    One of the objectives in the development of effective cancer therapy is induction of tumor-selective cell death. Toward this end, we have identified a small peptide that, when introduced into cells via a TAT cell-delivery system, shows a remarkably potent cytoxicity in a variety of cancer cell lines and inhibits tumor growth in vivo, whereas sparing normal cells and tissues. This fusion peptide was named killerFLIP as its sequence was derived from the C-terminal domain of c-FLIP, an anti-apoptotic protein. Using structure activity analysis, we determined the minimal bioactive core of killerFLIP, namely killerFLIP-E. Structural analysis of cells using electron microscopy demonstrated that killerFLIP-E triggers cell death accompanied by rapid (within minutes) plasma membrane permeabilization. Studies of the structure of the active core of killerFLIP (-E) indicated that it possesses amphiphilic properties and self-assembles into micellar structures in aqueous solution. The biochemical properties of killerFLIP are comparable to those of cationic lytic peptides, which participate in defense against pathogens and have also demonstrated anticancer properties. We show that the pro-cell death effects of killerFLIP are independent of its sequence similarity with c-FLIPL as killerFLIP-induced cell death was largely apoptosis and necroptosis independent. A killerFLIP-E variant containing a scrambled c-FLIPL motif indeed induced similar cell death, suggesting the importance of the c-FLIPL residues but not of their sequence. Thus, we report the discovery of a promising synthetic peptide with novel anticancer activity in vitro and in vivo.

  7. Killer whale prey - Determining prey selection by southern resident killer whales (SRKW)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Prey selectivity by southern resident killer whales is being determined by analyses of fish scales and tissue from predation events and feces. Information on killer...

  8. An Industrial THz Killer Application?

    CERN Document Server

    van Mechelen, Dook

    2015-01-01

    Terahertz technology is mature enough for large-volume sensing applications. However, Dook van Mechelen says there are a few hurdles preventing its industrial debut. THz spectroscopy has a number of advantages that point to abundant industrial applications, in areas such quality control, security and biomedical imaging. Yet despite those advantages, the search for a THz "killer application"--a novel, innovative use with a business case strong enough to bring the technology into the industrial mainstream--has remained fruitless, and even the hope of finding such an application has begun to falter. Why has a killer app for THz radiation been so elusive? And how can the road to industrial application of this versatile technology be cleared?

  9. Effect of hydrophobic scaffold on the cellular uptake and gene transfection activities of DNA-encapsulating liposomal nanoparticles via intracerebroventricular administration.

    Science.gov (United States)

    Akita, Hidetaka; Nakatani, Taichi; Kuroki, Kimiko; Maenaka, Katsumi; Tange, Kota; Nakai, Yuta; Harashima, Hideyoshi

    2015-07-25

    Efficient DNA carriers are needed as a gene medication for curing brain disorders. In the present study, the function of a neutral lipid envelope-type nanoparticle (LNP) encapsulating pDNA was evaluated after intracerebroventricular administration. The lipid envelope was composed of a series of SS-cleavable and pH-activated lipid like materials (ssPalm) including myristic acid, vitamin A and vitamin E in the hydrophobic scaffold (LNPssPalmM, LNPssPalmA, LNPssPalmE, respectively). The LNPssPalmA and LNPssPalmE were extensively distributed in the corpus callosum, and then gene expression occurred mainly astrocytes in this region, while not in LNPssPalmM. The recombinant human ApoE3-dependent enhancement of the uptake into an astrocyte-derived cell line (KT-5) was observed in LNPssPalmA and LNPssPalmE. Thus, ApoE in the brain plays a key role in the cellular uptake of these particles by astrocytes, and this uptake is dependent on the structure of the hydrophobic scaffold.

  10. Revving up natural killer cells and cytokine-induced killer cells against hematological malignancies

    Directory of Open Access Journals (Sweden)

    Gianfranco ePittari

    2015-05-01

    Full Text Available Natural killer (NK cells belong to innate immunity and exhibit cytolytic activity against infectious pathogens and tumor cells. NK-cell function is finely tuned by receptors that transduce inhibitory or activating signals, such as killer immunoglobulin-like receptors (KIR, NK Group 2 member D (NKG2D, NKG2A/CD94, NKp46 and others, and recognize both foreign and self-antigens expressed by NK-susceptible targets. Recent insights into NK-cell developmental intermediates have translated into a more accurate definition of culture conditions for the in vitro generation and propagation of human NK cells. In this respect, interleukin (IL-15 and IL-21 are instrumental in driving NK-cell differentiation and maturation, and hold great promise for the design of optimal NK-cell culture protocols.Cytokine-induced killer (CIK cells possess phenotypic and functional hallmarks of both T cells and NK cells. Similar to T cells, they express CD3 and are expandable in culture, while not requiring functional priming for in vivo activity, like NK cells. CIK cells may offer some advantages over other cell therapy products, including ease of in vitro propagation and no need for exogenous administration of IL-2 for in vivo priming.NK cells and CIK cells can be expanded using a variety of clinical-grade approaches, before their infusion into patients with cancer. Herein, we discuss GMP-compliant strategies to isolate and expand human NK and CIK cells for immunotherapy purposes, focusing on clinical trials of adoptive transfer to patients with hematological malignancies.

  11. Revving up Natural Killer Cells and Cytokine-Induced Killer Cells Against Hematological Malignancies

    Science.gov (United States)

    Pittari, Gianfranco; Filippini, Perla; Gentilcore, Giusy; Grivel, Jean-Charles; Rutella, Sergio

    2015-01-01

    Natural killer (NK) cells belong to innate immunity and exhibit cytolytic activity against infectious pathogens and tumor cells. NK-cell function is finely tuned by receptors that transduce inhibitory or activating signals, such as killer immunoglobulin-like receptors, NK Group 2 member D (NKG2D), NKG2A/CD94, NKp46, and others, and recognize both foreign and self-antigens expressed by NK-susceptible targets. Recent insights into NK-cell developmental intermediates have translated into a more accurate definition of culture conditions for the in vitro generation and propagation of human NK cells. In this respect, interleukin (IL)-15 and IL-21 are instrumental in driving NK-cell differentiation and maturation, and hold great promise for the design of optimal NK-cell culture protocols. Cytokine-induced killer (CIK) cells possess phenotypic and functional hallmarks of both T cells and NK cells. Similar to T cells, they express CD3 and are expandable in culture, while not requiring functional priming for in vivo activity, like NK cells. CIK cells may offer some advantages over other cell therapy products, including ease of in vitro propagation and no need for exogenous administration of IL-2 for in vivo priming. NK cells and CIK cells can be expanded using a variety of clinical-grade approaches, before their infusion into patients with cancer. Herein, we discuss GMP-compliant strategies to isolate and expand human NK and CIK cells for immunotherapy purposes, focusing on clinical trials of adoptive transfer to patients with hematological malignancies. PMID:26029215

  12. Natural killer cells: In health and disease.

    Science.gov (United States)

    Mandal, Arundhati; Viswanathan, Chandra

    2015-06-01

    Natural killer (NK) cells constitute our bodies' frontline defense system, guarding against tumors and launching attacks against infections. The activities of NK cells are regulated by the interaction of various receptors expressed on their surfaces with cell surface ligands. While the role of NK cells in controlling tumor activity is relatively clear, the fact that they are also linked to various other disease conditions is now being highlighted. Here, we present an overview of the role of NK cells during normal body state as well as under diseased state. We discuss the possible utilization of these powerful cells as immunotherapeutic agents in combating diseases such as asthma, autoimmune diseases, and HIV-AIDS. This review also outlines current challenges in NK cell therapy.

  13. Antiviral Treatment Alters the Frequency of Activating and Inhibitory Receptor-Expressing Natural Killer Cells in Chronic Hepatitis B Virus Infected Patients

    Directory of Open Access Journals (Sweden)

    Juan Lv

    2012-01-01

    Full Text Available Natural killer (NK cells play a critical role in innate antiviral immunity, but little is known about the impact of antiviral therapy on the frequency of NK cell subsets. To this aim, we performed this longitudinal study to examine the dynamic changes of the frequency of different subsets of NK cells in CHB patients after initiation of tenofovir or adefovir therapy. We found that NK cell numbers and subset distribution differ between CHB patients and normal subjects; furthermore, the association was found between ALT level and CD158b+ NK cell in HBV patients. In tenofovir group, the frequency of NK cells increased during the treatment accompanied by downregulated expression of NKG2A and KIR2DL3. In adefovir group, NK cell numbers did not differ during the treatment, but also accompanied by downregulated expression of NKG2A and KIR2DL3. Our results demonstrate that treatment with tenofovir leads to viral load reduction, and correlated with NK cell frequencies in peripheral blood of chronic hepatitis B virus infection. In addition, treatments with both tenofovir and adefovir in chronic HBV infected patients induce a decrease of the frequency of inhibitory receptor+ NK cells, which may account for the partial restoration of the function of NK cells in peripheral blood following treatment.

  14. Asbestos fibres inhibit the in vitro activity of lymphokine-activated killer (LAK) cells from healthy individuals and patients with malignant mesothelioma.

    Science.gov (United States)

    Manning, L S; Davis, M R; Robinson, B W

    1991-01-01

    Asbestos exposure is associated with an increased incidence of several malignancies, including malignant mesothelioma (MM). This study evaluates the relationship between asbestos exposure and the in vitro generation and function of LAK cells, an immune effector cell population with powerful lytic activity against MM cells. Both serpentine (chrysotile) and amphibole (amosite and crocidolite) forms of asbestos fibres suppress LAK cell generation, viability (by 5-11%, P less than 0.02) and cell recovery (by 13-15%, P less than 0.02). However, the LAK cells generated in the presence of the amphiboles were as effective as unexposed cells in lysing both standard tumour cell targets (K562, 56.4% lysis versus 61.5%, respectively, P greater than 0.5; NS; Daudi, 60.5% lysis versus 64.5% P greater than 0.5; NS), and MM tumour cell targets (mean of three MM cell lines 48.3% versus 46.3%, P greater than 0.5; NS), whereas the function of LAK cells generated in the presence of chrysotile was significantly reduced against three out of the five tumour cell targets tested (P less than 0.03). In the presence of asbestos fibres, LAK cell function was reduced against all five tumour cell targets (P less than 0.01), irrespective of whether the cell donors were healthy individuals or patients with MM. NK cell activity was also suppressed (P less than 0.01). The serpentine form of asbestos, chrysotile, was significantly more suppressive of both effector cell functions than either of the amphiboles (P less than 0.01). These findings suggest that asbestos exposure may suppress the function and in some instances the generation of immune effector cell mechanisms, thereby increasing the risk of disease and malignancy. PMID:1846329

  15. Manganese superoxide dismutase: effect of the ala16val polymorphism on protein, activity, and mRNA levels in human breast cancer cell lines and stably transfected mouse embryonic fibroblasts.

    Science.gov (United States)

    McAtee, Britt L; Yager, James D

    2010-02-01

    The manganese superoxide dismutase (MnSOD) ala16val polymorphism has been associated with various diseases including breast cancer. In the present study, we investigated levels of MnSOD protein, enzymatic activity, and mRNA with respect to MnSOD genotype in several human breast carcinoma cell lines and in mouse embryonic fibroblasts (MEF), developed from the MnSOD knockout mouse, stably expressing human MnSOD-ala and MnSOD-val. In human breast cell lines, the MnSOD-ala allele was associated with increased levels of MnSOD protein and MnSOD protein per unit mRNA. In the MEF transformants, MnSOD activity correlated fairly well with MnSOD protein levels. MnSOD mRNA expression was significantly lower in MnSOD-ala versus MnSOD-val lines. MnSOD protein and activity levels were not related to MnSOD genotype in the transformed MEF, although, as observed in the human breast cell lines, the MEF human MnSOD-ala lines produced significantly more human MnSOD protein per unit mRNA than the human MnSOD-val lines. This suggests that there is more efficient production of MnSOD-ala protein compared to MnSOD-val protein. Examination of several indicators of reactive oxygen species levels, including superoxide and hydrogen peroxide, in wild-type MEF and in MEF expressing similar elevated amounts of MnSOD-ala or val activity did not show differences related to the levels of MnSOD protein expression. In conclusion, in both human breast carcinoma cell lines and MEF cell lines stably transfected with human MnSOD, the MnSOD-ala allele was associated with increased production of MnSOD protein per unit mRNA indicating a possible imbalance in MnSOD protein production from the MnSOD-val mRNA.

  16. CL22 - a novel cationic peptide for efficient transfection of mammalian cells.

    Science.gov (United States)

    Haines, A M; Irvine, A S; Mountain, A; Charlesworth, J; Farrow, N A; Husain, R D; Hyde, H; Ketteringham, H; McDermott, R H; Mulcahy, A F; Mustoe, T L; Reid, S C; Rouquette, M; Shaw, J C; Thatcher, D R; Welsh, J H; Williams, D E; Zauner, W; Phillips, R O

    2001-01-01

    Condensing peptide-DNA complexes have great potential as nonviral agents for gene delivery. To date, however, such complexes have given transfection activities greatly inferior to adenovirus and somewhat inferior to cationic lipid-DNA complexes, even for cell lines and primary cells in vitro. We report here the identification of a novel condensing peptide, CL22, which forms DNA complexes that efficiently transfect many cell lines, as well as primary dendritic and endothelial cells. We report studies with sequence and structure variants that define some properties of the peptide that contribute to efficient transfection. We demonstrate that the superior transfection activity of CL22 compared with other DNA condensing peptides is conferred at a step after uptake of the complexes into cells. We show that CL22-DNA complexes have transfection activity that is at least equivalent to the best available nonviral agents.

  17. Phototoxic effects of lysosome-associated genetically encoded photosensitizer KillerRed

    Science.gov (United States)

    Serebrovskaya, Ekaterina O.; Ryumina, Alina P.; Boulina, Maria E.; Shirmanova, Marina V.; Zagaynova, Elena V.; Bogdanova, Ekaterina A.; Lukyanov, Sergey A.; Lukyanov, Konstantin A.

    2014-07-01

    KillerRed is a unique phototoxic red fluorescent protein that can be used to induce local oxidative stress by green-orange light illumination. Here we studied phototoxicity of KillerRed targeted to cytoplasmic surface of lysosomes via fusion with Rab7, a small GTPase that is known to be attached to membranes of late endosomes and lysosomes. It was found that lysosome-associated KillerRed ensures efficient light-induced cell death similar to previously reported mitochondria- and plasma membrane-localized KillerRed. Inhibitory analysis demonstrated that lysosomal cathepsins play an important role in the manifestation of KillerRed-Rab7 phototoxicity. Time-lapse monitoring of cell morphology, membrane integrity, and nuclei shape allowed us to conclude that KillerRed-Rab7-mediated cell death occurs via necrosis at high light intensity or via apoptosis at lower light intensity. Potentially, KillerRed-Rab7 can be used as an optogenetic tool to direct target cell populations to either apoptosis or necrosis.

  18. Modus operandi of female serial killers.

    Science.gov (United States)

    Wilson, W; Hilton, T

    1998-04-01

    The modus operandi of female serial killers was examined from a chronology of 58 cases in America and 47 cases in 17 other countries, compiled over 25-year intervals. Female serial killers in other countries accounted for a disproportionately greater number of victims, but those in America managed a longer killing career when associated with a low profile modus operandi.

  19. Agrobacterium sp.-derived β-1,3-glucan enhances natural killer cell activity in healthy adults: a randomized, double-blind, placebo-controlled, parallel-group study

    Science.gov (United States)

    Lee, Yeon Joo; Paik, Doo-Jin; Kwon, Dae Young; Yang, Hye Jeong

    2017-01-01

    BACKGROUND/OBJECTIVES The present study investigated the hypothesis that a highly pure linear β-1,3-glucan produced by Agrobacterium sp. R259 enhances human natural killer (NK) cell activity and suppresses pro-inflammatory cytokines. SUBJECTS/METHODS In an eight-week, double-blind, randomized, placebo-controlled clinical trial, 83 healthy adults with white blood cell counts of 4,000-8,000 cells/µL were participated and randomly assigned to take two capsules per day containing either 350 mg β-1,3-glucan or placebo. Six participants withdrew their study consent or were excluded due to NK cell activity levels outside the normal range. NK cell activity and serum levels of immunoglobulin G (IgG) and cytokines, such as interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12 and tumor necrosis factor (TNF)-α were measured. RESULTS NK cell activity and the serum levels of IL-10 were significantly higher from baseline to week 8 in the β-glucan group compared with the placebo group (P = 0.048, P = 0.029). Consumption of β-1,3-glucan also significantly increased NK cell activity compared with placebo after adjusting for smoking and stress status (P = 0.009). In particular, the effect of β-1,3-glucan on NK cell activity was greater in participants with severe stress than in those experiencing mild stress. However, the administration β-1,3-glucan did not significantly modulate the levels of IFN-γ, IL-2, IL-4, IL-6, IL-12, TNF-α and IgG compared with the placebo. CONCLUSION The results showed that supplementation with bacterial β-1,3-glucan significantly increased NK cell activity without causing any adverse effects. Additionally, the beneficial effect of β-1,3-glucan on NK cell activity was greater in participants experiencing severe stress.

  20. Improved biolistic transfection of hair cells.

    Directory of Open Access Journals (Sweden)

    Hongyu Zhao

    Full Text Available Transient transfection of hair cells has proven challenging. Here we describe modifications to the Bio-Rad Helios Gene Gun that, along with an optimized protocol, improve transfection of bullfrog, chick, and mouse hair cells. The increased penetrating power afforded by our method allowed us to transfect mouse hair cells from the basal side, through the basilar membrane; this configuration protects hair bundles from damage during the procedure. We characterized the efficiency of transfection of mouse hair cells with fluorescently-tagged actin fusion protein using both the optimized procedure and a published procedure; while the efficiency of the two methods was similar, the morphology of transfected hair cells was improved with the new procedure. In addition, using the improved method, we were able to transfect hair cells in the bullfrog sacculus and chick cochlea for the first time. We used fluorescent-protein fusions of harmonin b (USH1C and PMCA2 (ATP2B2; plasma-membrane Ca(2+-ATPase isoform 2 to examine protein distribution in hair cells. While PMCA2-EGFP localization was similar to endogenous PMCA2 detected with antibodies, high levels of harmonin-EGFP were found at stereocilia tapers in bullfrog and chick, but not mouse; by contrast, harmonin-EGFP was concentrated in stereocilia tips in mouse hair cells.

  1. Cytolysis of oligodendrocytes is mediated by killer (K) cells but not by natural killer (NK) cells.

    Science.gov (United States)

    Satoh, J; Kim, S U; Kastrukoff, L F

    1991-03-01

    The cytotoxic activity of killer (K) cells against enriched cultures of bovine oligodendrocytes (BOL) was investigated in multiple sclerosis (MS) and controls. Human K cells mediated cytotoxicity to primary cultures of BOL in the presence of anti-BOL antiserum in all study groups, while BOL were resistant to human natural killer (NK) cells. Cytotoxic activity was significantly reduced in MS when compared to age-matched normal controls but not when compared to other neurologic disease (OND) patients. K cell-mediated lysis of BOL could also be induced with anti-galactocerebroside antibody but not with other antibodies including those specific for OL antigens (myelin basic protein, proteolipid apoprotein, and 2',3'-cyclic nucleotide 3'-phosphodiesterase). Enrichment of the effector population indicated that antibody-dependent cell-mediated cytotoxicity (ADCC) to BOL was mediated by large granular lymphocytes, and the effector population was further characterized by flow cytometry. The effector cells mediating ADCC could be inhibited by protein A of Staphylococcus aureus, and by K562 cells in cold competition assay. These observations indicate that oligodendrocytes are resistant to NK cells but are susceptible to cytolysis mediated by K cells. This may represent a potentially important immune mechanism in the pathogenesis of MS.

  2. Uterine Natural Killer Cells: Their Choices, Their Missions

    Institute of Scientific and Technical Information of China (English)

    Jianhong Zhang; B Anne Croy; Zhigang Tian

    2005-01-01

    Uterine natural killer (uNK) cells, sharing many characters with peripheral blood natural killer (pNK) cells, are a major uterine lymphocyte population at early gestational stages during normal pregnancy in placental mammals.The functions of uNK cells include cytokine production and cytotoxcity that are regulated by signals through activating and inhibitory receptors. UNK cells differ from pNK cells however and contribute to the structural changes that accompany the differentiation of the maternal-fetal interface. Immunological mechanisms must provide a balanced environment for uNK cell proliferation, differentiation and activation through intricate signaling pathways. An improved knowledge of mechanisms regulating uNK cells development and the cytokine network at the maternal-fetal interface of mice and humans might be useful to harness the power of these cells for maintenance of pregnancy. Cellular & Molecular Immunology. 2005;2(2):123-129.

  3. Uterine Natural Killer Cells: Their Choices, Their Missions

    Institute of Scientific and Technical Information of China (English)

    JianhongZhang; BAnneCroy; ZhigangTian

    2005-01-01

    Uterine natural killer (uNK) cells, sharing many characters with peripheral blood natural killer (pNK) cells, are a major uterine lymphocyte population at early gestational stages during normal pregnancy in placental mammals. The functions of uNK cells include cytokine production and cytotoxcity that are regulated by signals through activating and inhibitory receptors. UNK cells differ from pNK cells however and contribute to the structural changes that accompany the differentiation of the maternal-fetal interface. Immunological mechanisms must provide a balanced environment for uNK cell proliferation, differentiation and activation through intricate signaling pathways. An improved knowledge of mechanisms regulating uNK cells development and the cytokine network at the maternal-fetal interface of mice and humans might be useful to harness the power of these cells for maintenance of pregnancy. Cellular & Molecular Immunology. 2005;2(2):123-129.

  4. Target Strength of Southern Resident Killer Whales (Orcinus orca): Measurement and Modeling

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Jinshan; Deng, Zhiqun; Carlson, Thomas J.; Moore, Brian

    2012-04-04

    A major criterion for tidal power licensing in Washington’s Puget Sound is the management of the risk of injury to killer whales due to collision with moving turbine blades. An active monitoring system is being proposed for killer whale detection, tracking, and alerting that links to and triggers temporary turbine shutdown when there is risk of collision. Target strength (TS) modeling of the killer whale is critical to the design and application of any active monitoring system. A 1996 study performed a high-resolution measurement of acoustic reflectivity as a function of frequency of a female bottlenose dolphin (2.2 m length) at broadside aspect and TS as a function of incident angle at 67 kHz frequency. Assuming that killer whales share similar morphology structure with the bottlenose dolphin, we extrapolated the TS of an adult killer whale 7.5 m in length at 67 kHz frequency with -8 dB at broadside aspect and -28 dB at tail side. The backscattering data from three Southern Resident killer whales were analyzed to obtain the TS measurement. These data were collected at Lime Kiln State Park using a split-beam system deployed from a boat. The TS of the killer whale at higher frequency (200 kHz) was estimated based on a three-layer model for plane wave reflection from the lung of the whale. The TS data of killer whales were in good agreement with our model. In this paper, we also discuss and explain possible causes for measurement estimation error.

  5. Killer Treg restore immune homeostasis and suppress autoimmune diabetes in prediabetic NOD mice.

    Science.gov (United States)

    Kaminitz, Ayelet; Yolcu, Esma S; Stein, Jerry; Yaniv, Isaac; Shirwan, Haval; Askenasy, Nadir

    2011-08-01

    We hypothesized that regulatory T cells (Treg) effectively target diabetogenic cells, and reinforcing their killing capacity will attenuate the course of disease. For proof of concept, Fas-ligand (FasL) protein was conjugated to CD25(+) Treg (killer Treg) to simulate the physiological mechanism of activation-induced cell death. Cytotoxic and suppressive activity of killer Treg was superior to naïve Treg in vitro. Administration of 3-4 × 10(6) Treg prevented hyperglycemia in 65% prediabetic NOD females, however only killer Treg postponed disease onset by 14 weeks. CD25(+) Treg homed to the pancreas and regional lymph nodes of prediabetic NOD females, proliferated and ectopic FasL protein induced apoptosis in CD25(-) T cells in situ. This mechanism of pathogenic cell debulking is specific to killer Treg, as FasL-coated splenocytes have no immunomodulatory effect, and only killer Treg prevent the disease in 80% of NOD.SCID recipients of effector:suppressor T cells (10:1 ratio). All immunomodulated mice displayed increased fractional expression of FoxP3 in the pancreas and draining lymph nodes, which was accompanied by CD25 only in recipients of killer Treg. A therapeutic intervention that uses the affinity of Treg to reduce the pathogenic load has long-term consequences: arrest of destructive insulitis in mice with established disease prior to β-cell extinction.

  6. Notorious Cases of Serial Killers

    Directory of Open Access Journals (Sweden)

    Iosub Elena-Cătălina

    2014-05-01

    Full Text Available The reconstruction of a death scene provides an overall picture of the crime and will indicate the murder as an event or one of a series of events and also the criminal. But when the criminal is declared a serial killer, many questions are raised up. How could a person kill some else without a reason or why people react in such a disorganized way and become so brutal or what made them act like that and so many questions with also so many answers. This project explains the psychology of a murderer, his own way of thinking and acting by presuming that we may accurately discover what is in their minds when they kill. It is about a very complex issue regarding murder investigations, biological factors and psychological profile of a serial killer. Dealing with this problem we will at last reach to the question that could solve finally the puzzle: ―Are serial murderers distorted reflections of society's own values?

  7. Natural killer lytic-associated molecule plays a role in controlling tumor dissemination and metastasis

    Directory of Open Access Journals (Sweden)

    Richard Glenn Hoover

    2012-12-01

    Full Text Available Natural killer lytic-associated molecule (NKLAM is an E3 ubiquitin ligase that plays a major role in the cytolytic activity of NK cells. NKLAM is rapidly synthesized and then targeted to the granule membranes of NK cells upon NK activation. Previous studies have shown an essential role for NKLAM in NK killing activity in vitro. These findings were extended to an in vivo model of NK-mediated tumor killing in which NKLAM-deficient knockout (KO mice injected with B16 melanoma cells were found to have significantly higher numbers of pulmonary tumor nodules than wild type (WT mice. To further investigate the role of NKLAM and NK function in tumor immunity in vivo, we utilized additional tumor models to compare tumor development and progression in NKLAM KO and WT mice. Primary tumor growth, dissemination, and metastasis of RMA-S lymphoma cells and E0771 breast cancer cells were evaluated. Both tumor cell lines were stably transfected with constructs that allow expression of green fluorescent protein (GFP, which serves as a tumor-specific marker. Intravenous injection of NK-sensitive RMA-S lymphoma cells resulted in greater dissemination of lymphoma cells in NKLAM KO mice than in WT mice. Lymphoma cells were found in the lymph nodes and bone marrow of NKLAM KO mice two weeks after injection; few detectable tumor cells remained in WT mice. E0771 syngeneic breast cancer cells were injected into the mammary pads of NKLAM KO and WT mice. Primary tumor growth was greater in NKLAM KO than in WT mice. More significantly, there were four to five fold more tumor cells in the blood and lungs of NKLAM KO than in WT mice two weeks after injection of tumor cells into the mammary pad. These results indicate that NKLAM plays a role in tumor development in vivo, especially in controlling tumor dissemination and metastasis to distant sites.

  8. Natural killer cells and cancer: regulation by the killer cell Ig-like receptors (KIR).

    Science.gov (United States)

    Purdy, Amanda K; Campbell, Kerry S

    2009-12-01

    Natural killer (NK) cells are innate immune effector cells that make up approximately 10-15% of the peripheral blood lymphocytes in humans and are primarily involved in immunosurveillance to eliminate transformed and virally-infected cells. They were originally defined by their ability to spontaneously eliminate rare cells lacking expression of class I major histocompatibility complex (MHC-I) self molecules, which is commonly referred to as "missing self" recognition. The molecular basis for missing self recognition emerges from the expression of MHC-I-specific inhibitory receptors on the NK cell surface that tolerize NK cells toward normal MHC-I-expressing cells. By lacking inhibitory receptor ligands, tumor cells or virus-infected cells that have down-modulated surface MHC-I expression become susceptible to attack by NK cells. Killer cell Ig-like receptors (KIR; CD158) constitute a family of MHC-I binding receptors that plays a major role in regulating the activation thresholds of NK cells and some T cells in humans. Here, we review the multiple levels of KIR diversity that contribute to the generation of a highly varied NK cell repertoire and explain how this diversity can influence susceptibility to a variety of diseases, including cancer. We further describe strategies by which KIR can be manipulated therapeutically to treat cancer, through the exploitation of KIR/MHC-I ligand mismatch to potentiate hematopoietic stem cell transplantation and the use of KIR blockade to enhance tumor cell killing.

  9. Bovine colostrum enhances natural killer cell activity and immune response in a mouse model of influenza infection and mediates intestinal immunity through toll-like receptors 2 and 4.

    Science.gov (United States)

    Wong, Eric B; Mallet, Jean-François; Duarte, Jairo; Matar, Chantal; Ritz, Barry W

    2014-04-01

    Oral administration of bovine colostrum affects intestinal immunity, including an increased percentage of natural killer (NK) cells. However, effects on NK cell cytotoxic activity and resistance to infection as well as a potential mechanism remain unclear. Therefore, we investigated the effects of bovine colostrum (La Belle, Inc, Bellingham, WA) on the NK cytotoxic response to influenza infection and on toll-like receptor (TLR) activity in a primary intestinal epithelial cell culture. We hypothesized that colostrum would increase NK cell activity and that TLR-2 and TLR-4 blocking would reduce interleukin 6 production by epithelial cells in response to contact stimulation with colostrum. Four-month-old female C57BL/6 mice were supplemented with 1 g of colostrum per kilogram of body weight before and after infection with influenza A virus (H1N1). Animals were assessed for weight loss, splenic NK cell activity, and lung virus titers. Colostrum-supplemented mice demonstrated less reduction in body weight after influenza infection, indicating a less severe infection, increased NK cell cytotoxicity, and less virus burden in the lungs compared with controls. Colostrum supplementation enhanced NK cell cytotoxicity and improved the immune response to primary influenza virus infection in mice. To investigate a potential mechanism, a primary culture of small intestine epithelial cells was then stimulated with colostrum. Direct activation of epithelial cells resulted in increased interleukin 6 production, which was inhibited with TLR-2 and TLR-4 blocking antibodies. The interaction between colostrum and immunity may be dependent, in part, on the interaction of colostrum components with innate receptors at the intestinal epithelium, including TLR-2 and TLR-4.

  10. Evolutionary vignettes of natural killer cell receptors.

    Science.gov (United States)

    Sambrook, Jennifer G; Beck, Stephan

    2007-10-01

    The discovery of novel immune receptors has led to a recent renaissance of research into the innate immune system, following decades of intense research of the adaptive immune system. Of particular interest has been the discovery of the natural killer (NK) cell receptors which, depending on type, interact with classical or non-classical MHC class I antigens of the adaptive immune system, thus functioning at the interface of innate and adaptive immunity. Here, we review recent progress with respect to two such families of NK receptors, the killer immunoglobulin-like receptors (KIRs) and the killer cell lectin-like receptors (KLRs), and attempt to trace their evolution across vertebrates.

  11. Isolation, cultivation and transfection of human keratinocytes.

    Science.gov (United States)

    Zare, Sona; Zarei, Mohammad Ali; Ghadimi, Tayyeb; Fathi, Fardin; Jalili, Ali; Hakhamaneshi, Mohammad Saeed

    2014-04-01

    Human keratinocytes could be used in the repair of damaged skin, in tissue engineering applications, gene therapy and recently, the generation of iPS cells. We isolated human keratinocytes from foreskin and subsequently cultured them on fibronectin, collagen type I, gelatin and laminin-coated dishes that contained three different types of serum-free medium (epilife, KSM or CnT). We developed improved conditions for efficient transfection of these human keratinocytes by testing three common transfection methods and a GFP plasmid vector. The isolated cells showed typical keratinocyte morphology and expressed the epithelial cell specific antigen, cytokeratin 14. Collagen type 1, epilife medium and lipofectamin 2000 gave the best results for isolation and transfection of human keratinocytes. Our protocol can be used as a reproducible, simple and efficient method for isolation, cultivation and genetic manipulation of human keratinocytes, which may be useful in cell and gene therapy applications.

  12. [THE USE OF "REAMBERIN" AS BIOLOGICAL RESPONSE MODIFIER TO INCREASE THE NATURAL KILLER CELLS' CYTOTOXICITY IN PATIENTS WITH LUNG TUBERCULOSIS].

    Science.gov (United States)

    Kholamov, A I; Mirzomogomedova, V G; Chernoshey, D A; Lizunov, E S

    2015-01-01

    The effect of the drug "Reamberin" cytotoxic activity of natural killer cells (EC) in an experimental model in the blood samples of patients with pulmonary tuberculosis and healthy donors. Simulation acute systemic inflammation by adding to the culture medium of BCG. After 48 hours, selection was performed mononuclear peripheral blood by gradient centrifugation tests set cytotoxic tumor cell line K-562. Revealed the stimulating effect of the drug "Reamberin" cytotoxic activity of natural killer cells. Metabolic Correction has had a positive impact on the energy metabolism of blood natural killer cells, to increase their survial and cytotoxicity.

  13. The evolution of natural killer cell receptors.

    Science.gov (United States)

    Carrillo-Bustamante, Paola; Keşmir, Can; de Boer, Rob J

    2016-01-01

    Natural killer (NK) cells are immune cells that play a crucial role against viral infections and tumors. To be tolerant against healthy tissue and simultaneously attack infected cells, the activity of NK cells is tightly regulated by a sophisticated array of germline-encoded activating and inhibiting receptors. The best characterized mechanism of NK cell activation is "missing self" detection, i.e., the recognition of virally infected or transformed cells that reduce their MHC expression to evade cytotoxic T cells. To monitor the expression of MHC-I on target cells, NK cells have monomorphic inhibitory receptors which interact with conserved MHC molecules. However, there are other NK cell receptors (NKRs) encoded by gene families showing a remarkable genetic diversity. Thus, NKR haplotypes contain several genes encoding for receptors with activating and inhibiting signaling, and that vary in gene content and allelic polymorphism. But if missing-self detection can be achieved by a monomorphic NKR system why have these polygenic and polymorphic receptors evolved? Here, we review the expansion of NKR receptor families in different mammal species, and we discuss several hypotheses that possibly underlie the diversification of the NK cell receptor complex, including the evolution of viral decoys, peptide sensitivity, and selective MHC-downregulation.

  14. Epstein-Barr virus-negative aggressive natural killer-cell leukaemia with high P-glycoprotein activity and phosphorylated extracellular signal-regulated protein kinases 1 and 2

    Directory of Open Access Journals (Sweden)

    Sanja Perkovic

    2012-09-01

    Full Text Available Aggressive natural killer-cell leukaemia (ANKL is a rare type of disease with fulminant course and poor outcome. The disease is more prevalent among Asians than in other ethnic groups and shows strong association with Epstein-Barr virus (EBV and P-glycoprotein (P-gp expression associated with multidrug resistance. Here we present a case of a 47 year old Caucasian female with a prior medical history of azathioprine treated ulcerative colitis who developed EBV-negative form of ANKL. The patient presented with hepatosplenomegaly, fever and nausea with peripheral blood and bone marrow infiltration with up to 70% of atypical lymphoid cells positive for cCD3, CD2, CD7, CD56, CD38, CD45, TIA1 and granzyme B, and negative for sCD3, CD4, CD5, CD8, CD34 and CD123 indicative of ANKL. Neoplastic CD56+ NK-cells showed high level of P-glycoprotein expression and activity, but also strong expression of phosphorylated extracellular signal-regulated protein kinases 1 and 2 (ERK1/2 MAP kinase. The patient was treated with an intensive polychemotherapy regimen designed for treatment of acute lymphoblastic leukaemia, but one month after admission developed sepsis, coma and died of cardiorespiratory arrest. We present additional evidence that, except for the immunophenotype, leukaemic NK-cells resemble normal NK-cells in terms of P-gp functional capacity and expression of phosphorylated ERK1/2 signalling molecule. In that sense drugs that block P-glycoprotein activity and activated signalling pathways might represent new means for targeted therapy.

  15. Serial killer: il database mondiale

    Directory of Open Access Journals (Sweden)

    Gaetano parente

    2016-07-01

    Full Text Available The complex and multisided study of serial killers is partly made difficult by the current level of progress that has led these deviant people to evolve in relation to the aspects of shrewdness (concerning the staging and mobility. Despite the important work of some scholars who proposed important theories, all this shows that, concerning serial murders, it is still particularly frequent not to pay attention to links among homicides committed by the same person but in different parts of the world. It is therefore crucial to develop a worldwide database that allows all police forces to access information collected on crime scenes of murders which are particularly absurd and committed without any apparent reason. It will then be up to the profiler, through ad hoc and technologically advanced tools, to collect this information on the crime scene that would be made available to all police forces thanks to the worldwide database.

  16. Enhanced apoptotic response to photodynamic therapy after bcl-2 transfection.

    Science.gov (United States)

    Kim, H R; Luo, Y; Li, G; Kessel, D

    1999-07-15

    Apoptosis is a cellular death process involving the sequential activation of a series of caspases, endonucleases, and other enzymes. The initiation of apoptosis can be inhibited by overexpression of bcl-2 and certain other members of a related family of proteins. We examined the effects of bcl-2 overexpression on the apoptotic response to photodynamic therapy (PDT), using aluminum phthalocyanine as the photosensitizing agent. In this study, we compared the immortalized human breast epithelial cell line MCF10A with a subline (MCF10A/bcl-2) transfected with the human bcl-2 gene. The latter was approximately 2-fold more sensitive to the phototoxic effects of PDT. At a 50 mJ/cm2 light dose, photodamage to MCF-10A/bcl-2 resulted in a greater loss of the mitochondrial membrane potential (delta(psi)m), enhanced release of mitochondrial cytochrome c, a more rapid and greater activation of caspase-3, and a greater apoptotic response. Western blot analysis revealed that the transfected cell line showed overexpression of both bcl-2 and bax, and that PDT caused selective destruction of bcl-2, leaving bax unaffected. The greater apoptotic response by the transfected line is, therefore, attributed to the higher bax:bcl-2 ratio after photodamage.

  17. Present and future of allogeneic natural killer cell therapy

    Directory of Open Access Journals (Sweden)

    Okjae eLim

    2015-06-01

    Full Text Available Natural killer (NK cells are innate lymphocytes that are capable of eliminating tumor cells and are therefore used for cancer therapy. Although many early investigators used autologous NK cells, including lymphokine-activated killer cells, the clinical efficacies were not satisfactory. Meanwhile, human leukocyte antigen (HLA-haploidentical hematopoietic stem cell transplantation revealed the anti-tumor effect of allogeneic NK cells, and HLA-haploidentical, killer cell immunoglobulin-like receptor (KIR ligand-mismatched allogeneic NK cells are currently used for many protocols requiring NK cells. Moreover, allogeneic NK cells from non-HLA-related healthy donors have been recently used in cancer therapy. The use of allogeneic NK cells from non-HLA-related healthy donors allows the selection of donor NK cells with higher flexibility and to prepare expanded, cryopreserved NK cells for instant administration without delay for ex vivo expansion. In cancer therapy with allogeneic NK cells, optimal matching of donors and recipients is important to maximize the efficacy of the therapy. In this review, we summarize the present state of allogeneic NK cell therapy and its future directions.

  18. Toxicity of a plant based mosquito repellent/killer.

    Science.gov (United States)

    Singh, Bhoopendra; Singh, Prakash Raj; Mohanty, Manoj Kumar

    2012-12-01

    The mission to make humans less attractive to mosquitoes has fuelled decades of scientific research on mosquito behaviour and control. The search for the perfect topical insect repellent/killer continues. This analysis was conducted to review and explore the scientific information on toxicity produced by the ingredients/contents of a herbal product. In this process of systemic review the following methodology was applied. By doing a MEDLINE search with key words of selected plants, plant based insect repellents/killers pertinent articles published in journals and authentic books were reviewed. The World Wide Web and the Extension Toxicity Network database (IPCS-ITOX) were also searched for toxicology data and other pertinent information. Repellents do not all share a single mode of action and surprisingly little is known about how repellents act on their target insects. Moreover, different mosquito species may react differently to the same repellent. After analysis of available data and information on the ingredient, of the product in relation to medicinal uses, acute and chronic toxicity of the selected medicinal plants, it can be concluded that the ingredients included in the herbal product can be used as active agents against mosquitoes. If the product which contains the powder of the above said plants is applied with care and safety, it is suitable fo use as a mosquito repellent/killer.

  19. Fractalkine expression induces endothelial progenitor cell lysis by natural killer cells.

    Directory of Open Access Journals (Sweden)

    Dilyana Todorova

    Full Text Available BACKGROUND: Circulating CD34(+ cells, a population that includes endothelial progenitors, participate in the maintenance of endothelial integrity. Better understanding of the mechanisms that regulate their survival is crucial to improve their regenerative activity in cardiovascular and renal diseases. Chemokine-receptor cross talk is critical in regulating cell homeostasis. We hypothesized that cell surface expression of the chemokine fractalkine (FKN could target progenitor cell injury by Natural Killer (NK cells, thereby limiting their availability for vascular repair. METHODOLOGY/PRINCIPAL FINDINGS: We show that CD34(+-derived Endothelial Colony Forming Cells (ECFC can express FKN in response to TNF-α and IFN-γ inflammatory cytokines and that FKN expression by ECFC stimulates NK cell adhesion, NK cell-mediated ECFC lysis and microparticles release in vitro. The specific involvement of membrane FKN in these processes was demonstrated using FKN-transfected ECFC and anti-FKN blocking antibody. FKN expression was also evidenced on circulating CD34(+ progenitor cells and was detected at higher frequency in kidney transplant recipients, when compared to healthy controls. The proportion of CD34(+ cells expressing FKN was identified as an independent variable inversely correlated to CD34(+ progenitor cell count. We further showed that treatment of CD34(+ circulating cells isolated from adult blood donors with transplant serum or TNF-α/IFN-γ can induce FKN expression. CONCLUSIONS: Our data highlights a novel mechanism by which FKN expression on CD34(+ progenitor cells may target their NK cell mediated killing and participate to their immune depletion in transplant recipients. Considering the numerous diseased contexts shown to promote FKN expression, our data identify FKN as a hallmark of altered progenitor cell homeostasis with potential implications in better evaluation of vascular repair in patients.

  20. Combined Transfection with EBV-Specific Epitopes and HLA-A2 genes is More Effective than Separate Transfection in Promoting CTL Lysis against Nasopharyngeal Carcinoma

    Institute of Scientific and Technical Information of China (English)

    Weijun Ding; Choylen Fong

    2004-01-01

    To augment specific cytotoxic T lymphocyte (CTL) lysis is a promising strategy for cancer therapy. In this study,we examined the boosting effect of CTLs upon autologous lymphoblastoid B cell lines (LCLs) transfected with diverse plasmids, to explore the possible CTL-based immunotherapy of nasopharyngeal carcinoma (NPC).FCM analysis displayed rather high ratio (>30%) of successfully transfected LCLs by utilizing the DMRIE-C kit. CTL assays demonstrated that substantially higher ratio of CTL specific lysis was observed upon the LCLs transfected with both expression vectors encoding EBV-specific epitopes and their presentation molecule HLA-A2, in contrast with those transfected separately. By transfecting the vector encoding HLA-A2 alone, only the LCLs of HLA-A2+ donors elicited markedly higher CTL lysis. CTL assays also showed that there existed no marked differences upon transfection by either different vectors (pcDNA3, pNGVL3 or pNGVL3-hFlex), or different EBV-derived peptides (LMP2Pep1 or LMP2Pep2), or with or without the doubled DNA sequence encoding peptides. This study indicated a promising immunotherapy strategy on NPC through boosting and eliciting the EBV-specific CTL activation by transferring vectors encoding both EBV-specific epitopes and their presentation molecule HLA-A2 into autologous LCL, the presentation cells of MHC/peptide tetrameric complex.

  1. Combined Transfection with EBV-Specific Epitopes and HLA-A2 genes is More Effective than Separate Transfection in Promoting CTL Lysis against Nasopharyngeal Carcinoma

    Institute of Scientific and Technical Information of China (English)

    WeijunDing; ChoylenFong

    2004-01-01

    To augment specific cytotoxic T lymphocyte (CTL) lysis is a promising strategy for cancer therapy. In this study,we examined the boosting effect of CTLs upon autologous lymphoblastoid B cell lines (LCLs) transfected with diverse plasmids, to explore the possible CTL-based immunotherapy of nasopharyngeal carcinoma (NPC).FCM analysis displayed rather high ratio (>30%) of successfully transfected LCLs by utilizing the DMRIE-C kit. CTL assays demonstrated that substantially higher ratio of CTL specific lysis was observed upon the LCLs transfected with both expression vectors encoding EBV-specific epitopes and their presentation molecule HLA-A2, In contrast with those transfected separately. By transfecting the vector encoding HLA-A2 alone, only the LCLs of HLA-A2+ donors elicited markedly higher CTL lysis. CTL assays also showed that there existed no marked differences upon transfection by either different vectors (pcDNA3, pNGVL3 or pNGVL3-hFIex), or different EBV-derived peptides (LMP2Pep1 or LMP2Pep2), or with or without the doubled DNA sequence encoding peptides. This study indicated a promising immunotherapy strategy on NPC through boosting and eliciting the EBV-specific CTL activation by transferring vectors encoding both EBV-specific epitopes and their presentation molecule HLA-A2 into autologous LCL, the presentation cells of MHC/peptide tetrameric complex.

  2. Killer phenotype of indigenous yeasts isolated from Argentinian wine cellars and their potential starter cultures for winemaking.

    Science.gov (United States)

    de Ullivarri, Miguel Fernández; Mendoza, Lucía M; Raya, Raúl R; Farías, Marta E

    2011-11-01

    Of 31 yeasts, from different surfaces of two cellars from the northwest region of Argentina, 11 expressed killer activity against the sensitive strain Saccharomyces cerevisiae P351. Five of these killer yeasts were identified as S. cerevisiae by phenotypic tests and PCR-RFLP analysis. Two S. cerevisiae killer strains, Cf5 and Cf8, were selected based on their excellent kinetic and enological properties as potential autochthonous mixed starter cultures to be used during wine fermentation. They could dominate the natural microbiota in fermentation vats and keep the typical sensorial characteristics of the wine of this region.

  3. Interleukin-15-activated natural killer cells kill autologous osteoclasts via LFA-1, DNAM-1 and TRAIL, and inhibit osteoclast-mediated bone erosion in vitro

    DEFF Research Database (Denmark)

    Feng, Shan; Madsen, Suzi H; Viller, Natasja N;

    2015-01-01

    -derived osteoclasts from healthy donors in vitro. We show that osteoclasts express numerous ligands for receptors present on activated NK cells. Co-culture experiments revealed that interleukin-15-activated, but not resting, NK cells trigger osteoclast apoptosis in a dose-dependent manner, resulting in drastically...

  4. Activation of natural killer T Cells promotes M2 macrophage polarization in adipose tissue and improves systemic glucose tolerance via interleukin-4 (IL-4)/STAT6 protein signalling axis in obesity

    NARCIS (Netherlands)

    Ji, Y.; Sun, S.; Xu, Aimin; Bhargava, P.; Yang, Liu; Lam, K.S.L.; Gao, Bin; Lee, Chih-Hao; Kersten, A.H.; Qi, L.

    2012-01-01

    Natural killer T (NKT) cells are important therapeutic targets in various disease models and are under clinical trials for cancer patients. However, their function in obesity and type 2 diabetes remains unclear. Our data show that adipose tissues of both mice and humans contain a population of type

  5. Minimally-Invasive Gene Transfection by Chemical and Physical Interaction of Atmospheric Pressure Plasma Flow

    Science.gov (United States)

    Kaneko, Toshiro

    2014-10-01

    Non-equilibrium atmospheric pressure plasma irradiated to the living-cell is investigated for medical applications such as gene transfection, which is expected to play an important role in molecular biology, gene therapy, and creation of induced pluripotent stem (iPS) cells. However, the conventional gene transfection using the plasma has some problems that the cell viability is low and the genes cannot be transferred into some specific lipid cells, which is attributed to the unknown mechanism of the gene transfection using the plasma. Therefore, the time-controlled atmospheric pressure plasma flow is generated and irradiated to the living-cell suspended solution for clarifying the transfection mechanism toward developing highly-efficient and minimally- invasive gene transfection system. In this experiment, fluorescent dye YOYO-1 is used as the simulated gene and LIVE/DEAD Stain is simultaneously used for cell viability assay. By the fluorescence image, the transfection efficiency is calculated as the ratio of the number of transferred and surviving cells to total cell count. It is clarified that the transfection efficiency is significantly increased by the short-time (cell viability (>90%). This result indicates that the physical effects such as the electric field caused by the charged particles arriving at the surface of the cell membrane, and chemical effects associated with plasma-activated products in solution act synergistically to enhance the cell-membrane transport with low-damage. This work was supported by JSPS KAKENHI Grant Number 24108004.

  6. Unique palindromic sequences in synthetic oligonucleotides are required to induce IFN [correction of INF] and augment IFN-mediated [correction of INF] natural killer activity.

    Science.gov (United States)

    Yamamoto, S; Yamamoto, T; Kataoka, T; Kuramoto, E; Yano, O; Tokunaga, T

    1992-06-15

    Thirty-mer single-stranded oligonucleotides, with a sequence chosen from the known cDNA encoding the 64-kDa protein named Ag A or the MPB-70 protein of Mycobacterium bovis BCG and the human cellular proteins such as complement component 1 inhibitor and Ig rearranged lambda-chain, were used to dissect the capability to induce IFN and to augment NK cell activity of mouse spleen cells by coincubation in vitro. Three with the hexamer palindromic sequence as GACGTC were active, whereas two kinds of oligonucleotides with no palindrome were inactive. The oligonucleotides containing at least one of the different palindromic sequences showed no activity. When a portion of the sequence of the inactive oligonucleotides was substituted with either palindromic sequence of GACGTC, AGCGCT, or AACGTT, the oligonucleotide acquired the ability to augment NK activity. In contrast, the oligonucleotides substituted with another palindromic sequence such as ACCGGT was without effect. Furthermore, exchange of two neighboring mononucleotides within, but not outside, the active palindromic sequence destroyed the ability of the oligonucleotides to augment NK cell activity. Stimulation of spleen cells with the substituted oligonucleotide, A4a-AAC, induced production of significant amounts of IFN-alpha/beta and small amounts of IFN-gamma. Augmentation of NK activity of the cells by the oligonucleotide was ascribed to IFN-alpha/beta production. These results strongly suggest that the presence of the unique palindromic sequences, such as GACGTC, AGCGCT, and AACGTT, but not ACCGGT, is essential for the immunostimulatory activity of oligonucleotides.

  7. Impact of "Killer Immunoglobulin-Like Receptor /Ligand" Genotypes on Outcome following Surgery among Patients with Colorectal Cancer: Activating KIRs Are Associated with Long-Term Disease Free Survival.

    Directory of Open Access Journals (Sweden)

    Kemal Beksac

    Full Text Available Approximately 30% of patients with stage II/III colorectal cancer develop recurrence following surgery. How individual regulation of host mediated anti-tumor cytotoxicity is modified by the killer-cell immunoglobulin-like receptor (KIRs genotype is essential for prediction of outcome. We analyzed the frequency of KIR and KIR ligand Human Leukocyte Antigen Class I genotypes, and their effects on recurrence and disease-free survival (DFS. Out of randomly selected 87 colorectal cancer patients who underwent R0 resection operations between 2005 and 2008, 29 patients whose cancers progressed within a median five-year follow-up period were compared with 58 patients with no recurrence within the same time period. Recurrent cases shared similar tumor stages with non-recurrent cases, but had different localizations. We used DNA isolated from pathological archival lymphoid and tumor tissues for KIR and KIR ligand (HLA-C, group C1, group C2, and HLA-A-Bw4 genotyping. Among cases with recurrence, KIR2DL1 (inhibitory KIR and A-Bw4 (ligand for inhibitory KIR3DL1 were observed more frequently (p=0.017 and p=0.024; and KIR2DS2 and KIR2DS3 (both activating KIRs were observed less frequently (p=0.005 and p=0.043. Similarly, in the non-recurrent group, inhibitory KIR-ligand combinations 2DL1-C2 and 2DL3-C1 were less frequent, while the activating combination 2DS2-C1 was more frequent. The lack of KIR2DL1, 2DL1-C2, and 2DL3-C1 improved disease-free survival (DFS (100% vs. 62.3%, p=0.05; 93.8% vs. 60.0%, p=0.035; 73.6% vs. 55.9%, p=0.07. The presence of KIR2DS2, 2DS3, and 2DS2-C1 improved DFS (77.8% vs. 48.5%, p=0.01; 79.4% vs. 58.5%, p=0.003; 76.9% vs. 51.4%, p=0.023. KIR2DS3 reduced the risk of recurrence (HR=0.263, 95% CI = 0.080-0.863, p=0.028. The number of activating KIRs are correlated strongly with DFS, none/ one/ two KIR : 54/77/98 months (p=0.004. In conclusion the inheritance of increasing numbers of activating KIRs and lack of inhibitory KIRs

  8. Impact of "Killer Immunoglobulin-Like Receptor /Ligand" Genotypes on Outcome following Surgery among Patients with Colorectal Cancer: Activating KIRs Are Associated with Long-Term Disease Free Survival.

    Science.gov (United States)

    Beksac, Kemal; Beksac, Meral; Dalva, Klara; Karaagaoglu, Ergun; Tirnaksiz, M Bulent

    2015-01-01

    Approximately 30% of patients with stage II/III colorectal cancer develop recurrence following surgery. How individual regulation of host mediated anti-tumor cytotoxicity is modified by the killer-cell immunoglobulin-like receptor (KIRs) genotype is essential for prediction of outcome. We analyzed the frequency of KIR and KIR ligand Human Leukocyte Antigen Class I genotypes, and their effects on recurrence and disease-free survival (DFS). Out of randomly selected 87 colorectal cancer patients who underwent R0 resection operations between 2005 and 2008, 29 patients whose cancers progressed within a median five-year follow-up period were compared with 58 patients with no recurrence within the same time period. Recurrent cases shared similar tumor stages with non-recurrent cases, but had different localizations. We used DNA isolated from pathological archival lymphoid and tumor tissues for KIR and KIR ligand (HLA-C, group C1, group C2, and HLA-A-Bw4) genotyping. Among cases with recurrence, KIR2DL1 (inhibitory KIR) and A-Bw4 (ligand for inhibitory KIR3DL1) were observed more frequently (p=0.017 and p=0.024); and KIR2DS2 and KIR2DS3 (both activating KIRs) were observed less frequently (p=0.005 and p=0.043). Similarly, in the non-recurrent group, inhibitory KIR-ligand combinations 2DL1-C2 and 2DL3-C1 were less frequent, while the activating combination 2DS2-C1 was more frequent. The lack of KIR2DL1, 2DL1-C2, and 2DL3-C1 improved disease-free survival (DFS) (100% vs. 62.3%, p=0.05; 93.8% vs. 60.0%, p=0.035; 73.6% vs. 55.9%, p=0.07). The presence of KIR2DS2, 2DS3, and 2DS2-C1 improved DFS (77.8% vs. 48.5%, p=0.01; 79.4% vs. 58.5%, p=0.003; 76.9% vs. 51.4%, p=0.023). KIR2DS3 reduced the risk of recurrence (HR=0.263, 95% CI = 0.080-0.863, p=0.028). The number of activating KIRs are correlated strongly with DFS, none/ one/ two KIR : 54/77/98 months (p=0.004). In conclusion the inheritance of increasing numbers of activating KIRs and lack of inhibitory KIRs, independent of

  9. Enhancement of natural killer cell activity in healthy subjects by Immulina®, a Spirulina extract enriched for Braun-type lipoproteins

    DEFF Research Database (Denmark)

    Nielsen, Claus Henrik; Balachandran, Premalatha; Christensen, Ole

    2010-01-01

    Immulina®, a commercial extract of Arthrospira (Spirulina) platensis is a potent activator of THP-1 monocytes and CD4+ T cells IN VITRO and enhances several immunological functions in mice. We further characterized Immulina® by determining that Braun-type lipoproteins are responsible for a major ...

  10. Enhancement of natural killer cell activity in healthy subjects by Immulina®, a Spirulina extract enriched for Braun-type lipoproteins

    DEFF Research Database (Denmark)

    Nielsen, Claus Henrik; Balachandran, Premalatha; Christensen, Ole

    2010-01-01

    Immulina®, a commercial extract of Arthrospira (Spirulina) platensis is a potent activator of THP-1 monocytes and CD4+ T cells IN VITRO and enhances several immunological functions in mice. We further characterized Immulina® by determining that Braun-type lipoproteins are responsible for a major...

  11. Transfection of the protozoan parasite Perkinsus marinus.

    Science.gov (United States)

    Fernández-Robledo, José A; Lin, Zhuoer; Vasta, Gerardo R

    2008-01-01

    Ongoing efforts for sequencing the genome of the protozoan parasite Perkinsus marinus, together with functional genomic initiatives, have continued to provide invaluable information about genes and metabolic pathways that not only will increase our understanding of its biology, but also have the potential to reveal useful targets for intervention. The lack of molecular tools for the functional characterization of genes of interest, however, has hindered progress in this regard. Here we report the development and validation of transfection methodology for this parasite. We first selected from our P. marinus EST collection a highly expressed gene, which we designated "MOE" (PmMOE), to which we fused at the C-terminus the enhanced green fluorescent protein (GFP) as a reporter gene (pPmMOE-GFP). The exogenous DNA was introduced into the trophozoite stage of the parasite by electroporation using the Nucleofector technology. The transfection efficiency was 37.8% with fluorescence detected as early as 14 h after electroporation, with the transfectants still remaining fluorescent after 8 months even in the absence of drug selection. The 5' flanking region was essential for transcription; constructs with 100 and 204 bp flanking the transcription start site also drove transcription effectively. Polymerase chain reaction (PCR) and Southern blot analyses was consistent with integration by non-homologous recombination. This transfection technique, the first one reported for a member of the Perkinsozoa, provides a new tool for studies of gene regulation and expression, protein targeting, and protein-protein interactions, and should significantly contribute to gain further insight into the biology of Perkinsus spp.

  12. Modulation of the Culture Supernatant of Decidual Cells with Exogenous Cytokines on Killing Activity of Natural Killer Cells in Early Pregnancy

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To investigate the important function of cytokines in early pregnancy and to provide basic and experimental evidence for understanding the mechanism of their action. Methods Add interferon-γ (IFN-γ) , interleukin- 2(IL- 2) , interleukin- 6(IL-6) and epidermal growth factor (EGF) to the confluent culturing decidual cells with three different concentrations and harvest the culture supernatant after 12, 24 and 48 h separately. Observe the effect of the supernatant on killing activity of NK cells with radioimmunological assay of 51Cr immersion. Results The culture supernatant of decidual cells can promote the killing activity of NK cells in various degrees, and the effect is independent of the type, concentration and acting time of cytokines. Conclusion In normal pregnancy, decidual cytokine network is in a dynamic equilibri um. Exogenous cytokines would be harm to normal pregnancy by interfering the equi librium state, but the exact mechanism needs further study.

  13. Modulation of the Culture Supernatant of Decidual Cells with Exogenous Cytokines on Killing Activity of Natural Killer Cells in Early Pregnancy

    Institute of Scientific and Technical Information of China (English)

    胡冬梅; 王丽莉; 何援利

    2000-01-01

    Objective To investigate the important function of cytohines in early pregnancy and to provide basic and experimental evidence for understanding the mechanism of their action.Methods Add interferon-γ (IFN-γ) ,interleuhin-2(IL-2) , interleuhin-6(IL-6) andepidermal growth factor(EGF) to the confluent culturing decidual cells with three different concentrations and harvest the culture supernatant after 12, 24 and 48 h separately. Observe the effect of the supernatant on killing activity of NK cells with radioimmunological assay of 51Cr immersion.Results The culture supernatant of decidual cells can promote the killing activity of NK cells in various degrees, and the effect is independent of the type, concentration and acting time of cytokines.Conclusion In normal pregnancy, decidual cytokine network is in a dynamic equilibri-um. Exogenous cytokines would be harm to normal pregnancy by interfering the equi-librium state, but the exact mechanism needs further study.

  14. Review article: Heat shock protein 70 (Hsp70 peptide activated Natural Killer (NK cells for the treatment of patients with non-small cell lung cancer (NSCLC after radiochemotherapy (RCTx – from preclinical studies to a clinical phase II trial

    Directory of Open Access Journals (Sweden)

    Hanno M Specht

    2015-04-01

    Full Text Available Heat shock protein 70 (Hsp70 is frequently overexpressed in tumor cells. An unusual cell surface localization could be demonstrated on a large variety of solid tumors including lung, colorectal, breast, squamous cell carcinomas of the head and neck, prostate and pancreatic carcinomas, glioblastomas, sarcomas and hematological malignancies, but not on corresponding normal tissues. A membrane (mHsp70-positive phenotype can be determined either directly on single cell suspensions of tumor biopsies by flow cytometry using cmHsp70.1 monoclonal antibody or indirectly in the serum of patients using a novel lipHsp70 ELISA. A mHsp70-positive tumor phenotype has been associated with highly aggressive tumors, causing invasion and metastases and resistance to cell death. However, natural killer (NK, but not T cells were found to kill mHsp70-positive tumor cells after activation with a naturally occurring Hsp70 peptide (TKD plus low dose IL-2 (TKD/IL-2. Safety and tolerability of ex vivo TKD/IL-2 stimulated, autologous NK cells has been demonstrated in patients with metastasized colorectal and NSCLC in a phase I clinical trial. Based on promising clinical results of the previous study, a phase II randomized clinical study was initiated in 2015. The primary objective of this multicenter proof-of-concept trial is to examine whether an adjuvant treatment of NSCLC patients after platinum based radiochemotherapy with TKD/IL-2 activated, autologous NK cells is clinically effective. As a mHsp70-positive tumor phenotype is associated with poor clinical outcome only mHsp70-positive tumor patients will be recruited into the trial. The primary endpoint of this study will be the comparison of the progression-free survival of patients treated with ex vivo activated NK cells compared to patients who were treated with radiochemotherapy alone. As secondary endpoints overall survival, toxicity, quality-of-life and biological responses will be determined in both study groups.

  15. Orally administered Salacia reticulata extract reduces H1N1 influenza clinical symptoms in murine lung tissues putatively due to enhanced natural killer cell activity

    Directory of Open Access Journals (Sweden)

    Gustavo Adolfo Romero-Pérez

    2016-03-01

    Full Text Available Influenza is a major cause of respiratory tract infection. Although most cases do not require further hospitalization, influenza periodically causes epidemics in humans that can potentially infect and kill millions of people. To countermeasure this threat, new vaccines need to be developed annually to match emerging influenza viral strains with increased resistance to existing vaccines. Thus, there is a need for finding and developing new anti-influenza viral agents as alternatives to current treatments. Here, we tested the antiviral effects of an extract from the stems and roots of Salacia reticulata (SSRE, a plant rich in phytochemicals such as salacinol, kotalanol and catechins, on H1N1 influenza virus-infected mice. Following oral administration of 0.6 mg/day of SSRE, the incidence of coughing decreased in 80% of mice, and only one case of severe pulmonary inflammation was detected. Moreover, when compared with mice given Lactobacillus casei JCM1134, a strain previously shown to help increase in vitro NK cell activity, SSRE-administered mice showed greater and equal NK cell activity in splenocytes and pulmonary cells, respectively, at high effector cell:target cell ratios. Next, to test whether or not SSRE would exert protective effects against influenza in the absence of gut microbiota, mice were given antibiotics before being inoculated influenza virus and subsequently administered SSRE. SSRE administration induced an increase in NK cell activity in splenocytes and pulmonary cells at levels similar to those detected in mice not treated with antibiotics. Based on our results, it can be concluded that phytochemicals in the SSRE exerted protective effects against influenza infection putatively via modulation of the immune response including enhancement of NK cell activity, although some protective effects were not necessarily through modulation of gut microbiota. Further investigation is necessary to elucidate the molecular mechanisms

  16. CXC Chemokine Ligand 10 Controls Viral Infection in the Central Nervous System: Evidence for a Role in Innate Immune Response through Recruitment and Activation of Natural Killer Cells

    OpenAIRE

    Trifilo, Matthew J.; Montalto-Morrison, Cynthia; Stiles, Linda N.; Hurst, Kelley R.; Hardison, Jenny L.; Manning, Jerry E.; Masters, Paul S.; Lane, Thomas E.

    2004-01-01

    How chemokines shape the immune response to viral infection of the central nervous system (CNS) has largely been considered within the context of recruitment and activation of antigen-specific lymphocytes. However, chemokines are expressed early following viral infection, suggesting an important role in coordinating innate immune responses. Herein, we evaluated the contributions of CXC chemokine ligand 10 (CXCL10) in promoting innate defense mechanisms following coronavirus infection of the C...

  17. Importance of killer immunoglobulin-like receptors in allogeneic hematopoietic stem cell transplantation

    Directory of Open Access Journals (Sweden)

    Danilo Santana Alessio Franceschi

    2011-01-01

    Full Text Available Hematopoietic stem cell transplantation is the treatment of choice for many hematologic diseases, such as multiple myeloma, bone marrow aplasia and leukemia. Human leukocyte antigen (HLA compatibility is an important tool to prevent post-transplant complications such as graft rejection and graft-versus-host disease, but the high rates of relapse limit the survival of transplant patients. Natural Killer cells, a type of lymphocyte that is a key element in the defense against tumor cells, cells infected with viruses and intracellular microbes, have different receptors on their surfaces that regulate their cytotoxicity. Killer immunoglobulin-like receptors are the most important, interacting consistently with human leukocyte antigen class I molecules present in other cells and thus controlling the activation of natural killer cells. Several studies have shown that certain combinations of killer immunoglobulin-like receptors and human leukocyte antigens (in both donors and recipients can affect the chances of survival of transplant patients, particularly in relation to the graft-versusleukemia effect, which may be associated to decreased relapse rates in certain groups. This review aims to shed light on the mechanisms and effects of killer immunoglobulin-like receptors - human leukocyte antigen associations and their implications following hematopoietic stem cell transplantation, and to critically analyze the results obtained by the studies presented herein.

  18. Human NK cells maintain licensing status and are subject to killer immunoglobulin-like receptor (KIR) and KIR-ligand inhibition following ex vivo expansion.

    Science.gov (United States)

    Wang, Wei; Erbe, Amy K; Alderson, Kory A; Phillips, Emily; Gallenberger, Mikayla; Gan, Jacek; Campana, Dario; Hank, Jacquelyn A; Sondel, Paul M

    2016-09-01

    Infusion of allogeneic NK cells is a potential immunotherapy for both hematopoietic malignancies and solid tumors. Interactions between killer immunoglobulin-like receptors (KIR) on human NK cells and KIR-ligands on tumor cells influence the magnitude of NK function. To obtain sufficient numbers of activated NK cells for infusion, one potent method uses cells from the K562 human erythroleukemia line that have been transfected to express activating 41BB ligand (41BBL) and membrane-bound interleukin 15 (mbIL15). The functional importance of KIRs on ex vivo expanded NK cells has not been studied in detail. We found that after a 12-day co-culture with K562-mbIL15-41BBL cells, expanded NK cells maintained inhibition specificity and prior in vivo licensing status determined by KIR/KIR-ligand interactions. Addition of an anti-CD20 antibody (rituximab) induced NK-mediated antibody-dependent cellular cytotoxicity and augmented killing of CD20+ target cells. However, partial inhibition induced by KIR/KIR-ligand interactions persisted. Finally, we found that extended co-cultures of NK cells with stimulatory cells transduced to express various KIR-ligands modified both the inhibitory and activating KIR repertoires of the expanded NK cell product. These studies demonstrate that the licensing interactions known to occur during NK ontogeny also influence NK cell function following NK expansion ex vivo with HLA-null stimulatory cells.

  19. Effect of mutated IκBα transfection on multidrug resistance in hilar cholangiocarcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Ru-Fu Chen; Zhi-Hua Li; Xian-He Kong; Ji-Sheng Chen

    2005-01-01

    AIM: To explore the expression effect of mutated IκBαtransfection on multidrug resistance gene (MDR-1) in hilar cholangiocarcinoma cells by inhibiting the activity of nuclear transcription factor-κB (NF-κB).METHODS: We used the mutated IκBα plasmid to transfect QBC939HCVC+ cells and QBC939 cells, and electrophoretic gel mobility shift assay (EMSA) to detect the binding activity of NF-κB DNA and the effect of the transfrecting mutated IκBα plasmid on multidrug resistance gene (MDR-1) in hilar cholangiocarcinoma cells and its expression protein (P-GP).RFSULTS: Plasmid DNA was digested by restriction enzymes Xbal and Hand Ⅲ, and its product after electrophoresis showed two bands with a big difference in molecular weight,with a size of 4.9 kb and 1.55 kb respectively, which indicated that the carrier was successfully constructed and digested with enzymes. The radioactivity accumulation of QBC939HCVC+and QBC939 cells transfected with mutated IκBα plasmid was significantly lower than that of the control group not transfected with mutated IκBα plasmid. Double densimeter scanning showed that the relative signal density between the tansfection group and non-transfection group was significantly different, which proved that the mutated IκBα plasmid could inhibit the binding activity of NF-κB DNA in hilar cholangiocarcinoma cells. Compared to control group not transfected with m IκBα plasmid, the expression level of MDR-1mRNA in the QBC939 and QBC939HCVC+ cells transfected with mutated IκBα plasmid was lower. The expression intensity of P-GP protein in QBC939 and QBC939HCVC+ cells transfected with mutated IκBα was significantly lower than that of the control group not transfected with mutated IκBα plasmid.CONCLUSION: The mutated IκBα plasmid transfection can markedly reverse the multidrug resistance of hilar cholangiocarcinoma cells. Interruption of NF-κB activity may become a new target in gene therapy for hilar cholangiocarcinogenesic carcinoma.

  20. Development of a semi-automated high throughput transient transfection system.

    Science.gov (United States)

    Bos, Aaron B; Duque, Joseph N; Bhakta, Sunil; Farahi, Farzam; Chirdon, Lindsay A; Junutula, Jagath R; Harms, Peter D; Wong, Athena W

    2014-06-20

    Transient transfection of mammalian cells provides a rapid method of producing protein for research purposes. Combining the transient transfection protein expression system with new automation technologies developed for the biotechnology industry would enable a high throughput protein production platform that could be utilized to generate a variety of different proteins in a short amount of time. These proteins could be used for an assortment of studies including proof of concept, antibody development, and biological structure and function. Here we describe such a platform: a semi-automated process for PEI-mediated transient protein production in tubespins at a throughput of 96 transfections at a time using a Biomek FX(P) liquid handling system. In one batch, 96 different proteins can be produced in milligram amounts by PEI transfection of HEK293 cells cultured in 50 mL tubespins. Methods were developed for the liquid handling system to automate the different processes associated with transient transfections such as initial cell seeding, DNA:PEI complex activation and DNA:PEI complex addition to the cells. Increasing DNA:PEI complex incubation time resulted in lower protein expression. To minimize protein production variability, the methods were further optimized to achieve consistent cell seeding, control the DNA:PEI incubation time and prevent cross-contamination among different tubespins. This semi-automated transfection process was applied to express 520 variants of a human IgG1 (hu IgG1) antibody.

  1. Toward an objective evaluation of cell transfection performance

    Science.gov (United States)

    Orlando, Viviana; Pozzi, Daniela; Caracciolo, Giulio; Augusti-Tocco, Gabriella; Biagioni, Stefano

    2010-10-01

    In this study, we considered the interplay between the efficiency and cytotoxicity of multicomponent cationic liposome/DNA complexes, in cell lines of different origin, as NIH 3T3, HEK 293T, N18TG2, and SK-N-SH. We show that both efficiency and cytotoxicity vary considerably depending on used transfection agents and cells lines. Such variations are largely overcome when transfection is evaluated by a parameter, R, that combines the percentages of transfected, nonviable, and nonadherent cells. These findings provide a strong validation of R as an unbiased indicator for transfection performance across cell lines and transfection agents.

  2. Natural born killers?: the development of the sexually sadistic serial killer.

    Science.gov (United States)

    Johnson, B R; Becker, J V

    1997-01-01

    Today's society seems enthralled with serial killers in the news and the media. Forensic psychiatrists often interview serial killers after they have been caught. There are retrospective studies and case reports of individuals who have committed sexually sadistic serial murders. However, there exists a dearth of case reports on adolescents who have expressed serious fantasies about becoming serial killer prior to actualizing their fantasy. This article presents nine clinical cases of 14- to 18-year-olds who have clinically significant fantasies of becoming a serial killer. Similarities exist in these adolescent cases when compared with retrospective studies and case reports of serial killers on the role of sexually sadistic fantasies and actual killings. Since it has been established that sexual paraphilias may develop at a young age, one can surmise that sadistic paraphilias may also develop in some adolescents. The question is posed, can we predict which of these adolescents may go on to actually become serial killers? This article focuses on how the sexually sadistic fantasy can eventually be acted out and possible motives for the act to be repeated multiple times. Finally, recommendations are made about assessing and treating a youngster who expresses violent sexually sadistic killing fantasies so that attempts can be made to interrupt the progression to actual killing.

  3. Influence of human cytomegalvirus on the expression of natural-killer group 2-members receptors on the natural killer cells

    Institute of Scientific and Technical Information of China (English)

    顾绍庆

    2014-01-01

    Objective To examine the effect of human cytomegalovirus(CMV)on the expressions of natural-killer group2-members(NKG2),including natural-killer group 2-member A(NKG2A),natural-killer group 2-member C(NKG2C)and natural-killer group 2-member D(NKG2D)receptors on the natural killer(NK)cells.Methods NK cells were isolated from the peripheral blood mononuclear cells of 20 healthy individuals using

  4. Killer plasma ready to devour the Earth

    CERN Multimedia

    Uhlig, R; Highfield, R

    2001-01-01

    A chance fluctuation of the 'vacuum universe' could disintegrate all atoms, according to CERN associate, Dr Allanach. Alternatively, so-called killer strangelets could "eat up the universe from the inside out". Should either of these scenarios occur, the most likely starting point is the Relativistic Heavy Ion Collider in Long Island, New York state (1 page).

  5. Positive selection on the killer whale mitogenome

    DEFF Research Database (Denmark)

    Foote, Andrew David; Morin, Phillip A; Durban, John W;

    2011-01-01

    Mitochondria produce up to 95 per cent of the eukaryotic cell's energy. The coding genes of the mitochondrial DNA may therefore evolve under selection owing to metabolic requirements. The killer whale, Orcinus orca, is polymorphic, has a global distribution and occupies a range of ecological niches...

  6. The KP4 killer protein gene family

    Science.gov (United States)

    Killer protein 4 (KP4) is a well studied toxin secreted by the maize smut fungus Ustilago maydis that kills sensitive Ustilago strains as well as inhibits Fusarium and plant root growth. This small, cysteine rich protein is encoded by a virus that depends on host survival for replication. KP4 functi...

  7. Isolation, culture, and transfection of melanocytes.

    Science.gov (United States)

    Godwin, Lauren S; Castle, Joanna T; Kohli, Jaskaren S; Goff, Philip S; Cairney, Claire J; Keith, W Nicol; Sviderskaya, Elena V; Bennett, Dorothy C

    2014-06-03

    Located in the basal epidermis and hair follicles, melanocytes of the integument are responsible for its coloration through production of melanin pigments. Melanin is produced in lysosomal-like organelles called melanosomes. In humans, this skin pigmentation acts as an ultraviolet radiation filter. Abnormalities in the division of melanocytes are quite common, with potentially oncogenic growth usually followed by cell senescence producing benign naevi (moles), or occasionally melanoma. Therefore, melanocytes are a useful model for studying melanoma, as well as pigmentation and organelle transport and the diseases affecting these mechanisms. This chapter focuses on the isolation, culture, and transfection of human and murine melanocytes. The first basic protocol describes the primary culture of melanocytes from human skin and the maintenance of growing cultures. The second basic protocol details the subculture and preparation of mouse keratinocyte feeder cells. The primary culture of melanocytes from mouse skin is described in the third basic protocol, and, lastly, the fourth basic protocol outlines a technique for transfecting melanocytes and melanoma cells.

  8. Natural killer cells facilitate PRAME-specific T-cell reactivity against neuroblastoma

    NARCIS (Netherlands)

    Spel, Lotte; Boelens, Jaap Jan; Van Der Steen, Dirk M.; Blokland, Nina J G; van Noesel, Max M.; Molenaar, Jan J.; Heemskerk, Mirjam H M; Boes, Marianne; Nierkens, Stefan

    2015-01-01

    Neuroblastoma is the most common solid tumor in children with an estimated 5-year progression free survival of 20-40% in stage 4 disease. Neuroblastoma actively avoids recognition by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Although immunotherapy has gained traction for neurobla

  9. TdKT, a new killer toxin produced by Torulaspora delbrueckii effective against wine spoilage yeasts.

    Science.gov (United States)

    Villalba, María Leticia; Susana Sáez, Julieta; Del Monaco, Silvana; Lopes, Christian Ariel; Sangorrín, Marcela Paula

    2016-01-18

    Microbiological spoilage is a major concern throughout the wine industry, and control tools are limited. This paper addresses the identification and partial characterization of a new killer toxin from Torulaspora delbrueckii with potential biocontrol activity of Brettanomyces bruxellensis, Pichia guilliermondii, Pichia manshurica and Pichia membranifaciens wine spoilage. A panel of 18 different wine strains of T. delbrueckii killer yeasts was analysed, and the strain T. delbrueckii NPCC 1033 (TdKT producer) showed a significant inhibitory effect on the growth of all different spoilage yeasts evaluated. The TdKT toxin was then subjected to a partial biochemical characterization. Its estimated molecular weight was N30 kDa and it showed glucanase and chitinase enzymatic activities. The killer activity was stable between pH 4.2 and 4.8 and inactivated at temperature above 40 °C. Pustulan and chitin — but not other cell wall polysaccharides — prevented sensitive yeast cells from being killed by TdKT, suggesting that those may be the first toxin targets in the cell wall. TdKT provoked an increase in necrosis cell death after 3 h treatment and apoptotic cell death after 24 h showing time dependence in its mechanisms of action. Killer toxin extracts were active at oenological conditions, confirming their potential use as a biocontrol tool in winemaking.

  10. Optimization of conditions for transfection with the Sofast gene vector.

    Science.gov (United States)

    Zhou, Lei; Liu, Fan; Qiao, Fang-Fang; Tong, Man-Li; Fu, Zuo-Gen; Dan, Bing; Yang, Tian-Ci; Zhang, Zhong-Ying

    2011-01-01

    We previously reported the synthesis and characterization of a novel cationic polymer gene vector. The present article further explored and optimized the working conditions of the Sofast gene vector both in vitro and in vivo, and improved its performance. The transfection conditions of Sofast, such as cell type, cell density, transfection time, N/P values and analysis time after transfection, were further explored. Moreover, the effects of the fusion peptide diINF-7 on transfection efficiency were examined. Sofast was successfully applied for the transfection of exogenous genes into more than 40 types of cell lines derived from humans, mice, monkeys and other species. When the cells were 50-80% confluent, Sofast possessed a better transfection efficiency. In most cases, Sofast also had a higher transfection efficiency when it was used to transfect cells that were seeded for several hours and had adhered to the substrate. The results from in vitro experiments indicate that the recommended Sofast to DNA mass ratio is 16:1, and the optimum analysis time after transfection is 48 h. The salt concentration in the Sofast working solution markedly affected the transfection efficiency. When conducting in vivo transfection, the working solution should be salt-free, whereas for in vitro transfection, it is more appropriate for the working solution to include certain salt concentrations. Finally, the results confirm that diINF-7 significantly promotes the transfection efficiency of Sofast. In conclusion, the present research not only established the optimal conditions for Sofast in the transfection of commonly used cells, but also built the foundations for in vivo and in vitro applications of Sofast, as well as its use in clinical practice.

  11. Research of morphology and function of different cytokines activated natural killer cells%不同细胞因子活化的NK细胞形态及功能的研究

    Institute of Scientific and Technical Information of China (English)

    余少鸿; 汤荣春; 温小明; 张继红

    2011-01-01

    Objective To investigate shape and function of different cytokines activated NK cells.Methods Natural Killer(NK)cells were cultured and activated with IL-12 10 ng/mL,IL-18 100 ng/mL,and both with IL-2 6 000U/L,3 days later took out NK cells,markers and Forward Scatter(FSC) FACS were detected by FACS,IFN gamma production was detected by FLISA, and B16 melanoma cells as targets for NK cells for cytotoxic assay.Results IFN gamma production from NK cells with IL-2+IL-12,IL-2+IL-18 , and IL-2+ IL-12 +IL-18 were higher and more than with IL-2 respectively , but NK cells with IL-2 + IL-12 , IL-2 +IL-18 , and IL-2 + IL-12+ IL-18 kill less B16 cells than with IL-2.Conclusion NK cells with IL-2 + IL-12 , IL-2 + IL-18 , and IL-2 + IL-12 + IL-18 will produce more IFN gamma, but kill less tumor cells.%目的 研究白细胞介素-2(IL-2),IL-2及白细胞介素-12(IL-12),IL-2及白细胞介素-18(IL-18),IL-2、IL-12及IL-18活化的自然杀伤细胞(NK细胞)的形态及对黑色素瘤细胞的杀伤率.方法 提取NK细胞,IL-2 6 000 IU/mL,IL-2 6 000 IU/mL+IL-12 10 ng/mL,IL-2 6 000 IU/mL+IL-18 100 ng/mL,IL-2 6 000 IU/mL+IL-12 10 ng/mL+IL-18 100 ng/mL活化,3 d后流式细胞仪检测其前向角散射(FSC)值,酶联免疫吸附法(ELISA)检测干扰素γ(IFNγ)含量,以B16黑色素瘤细胞为靶细胞检测NK细胞对肿瘤细胞杀伤率.结果 IL-2+IL-12、IL-2+IL-18、IL-2+IL-12+IL-18活化的NK细胞FSC值明显比仅用IL-2活化的NK细胞大(P<0.05),并且分泌的IFN量高(P<0.05),但对B16黑色素瘤细胞杀伤率低.结论 同时应用IL-12及IL-18与IL-2活化的NK细胞能够分泌较高的IFNγ,但是对肿瘤细胞的杀伤作用较低.

  12. Innate immune suppression enables frequent transfection with RNA encoding reprogramming proteins.

    Directory of Open Access Journals (Sweden)

    Matthew Angel

    Full Text Available BACKGROUND: Generating autologous pluripotent stem cells for therapeutic applications will require the development of efficient DNA-free reprogramming techniques. Transfecting cells with in vitro-transcribed, protein-encoding RNA is a straightforward method of directly expressing high levels of reprogramming proteins without genetic modification. However, long-RNA transfection triggers a potent innate immune response characterized by growth inhibition and the production of inflammatory cytokines. As a result, repeated transfection with protein-encoding RNA causes cell death. METHODOLOGY/PRINCIPAL FINDINGS: RNA viruses have evolved methods of disrupting innate immune signaling by destroying or inhibiting specific proteins to enable persistent infection. Starting from a list of known viral targets, we performed a combinatorial screen to identify siRNA cocktails that could desensitize cells to exogenous RNA. We show that combined knockdown of interferon-beta (Ifnb1, Eif2ak2, and Stat2 rescues cells from the innate immune response triggered by frequent long-RNA transfection. Using this technique, we were able to transfect primary human fibroblasts every 24 hours with RNA encoding the reprogramming proteins Oct4, Sox2, Klf4, and Utf1. We provide evidence that the encoded protein is active, and we show that expression can be maintained for many days, through multiple rounds of cell division. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that suppressing innate immunity enables frequent transfection with protein-encoding RNA. This technique represents a versatile tool for investigating expression dynamics and protein interactions by enabling precise control over levels and timing of protein expression. Our finding also opens the door for the development of reprogramming and directed-differentiation methods based on long-RNA transfection.

  13. Laser-based transfection with conjugated gold nanoparticles

    Institute of Scientific and Technical Information of China (English)

    Cuiping Yao; Xiaochao Qu; Zhenxi Zhang

    2009-01-01

    The irradiation of cells combined with the immunoconjugate of gold nanoparticles by the short pulse laser can make the plasma membrane be transiently permeabilized,which can be used to transfer exogenous molecules into the cells.We explore this technique as a novel gene transfection method for floating cells.Three different floating cells exposed to the laser are selectively transfected with fluorescein isothiocyanatedextran,antibody,and green fluorescent protein (GFP) coding plasmids,and the viability of cells are determined by propidium iodide.For fluorescein isothiocyanate-dextran,the best transfection efficiency of 65% is obtained;for the antibody,it is 74%;whereas for the green fluorescent protein coding plasmids,a very small transfection efficiency is gained.If the transfection efficiency is improved,gold nanoparticles will be very useful as mediator for gene transfection in living cells.

  14. Inhibition of transfected PTEN on human colon cancer

    Institute of Scientific and Technical Information of China (English)

    Shou-Shui Xu; Wen-Lu Shen; Song-Ying Ouyang

    2004-01-01

    AIM: To study the inhibitory effect of transfected PTEN on LoVo cells.METHODS: Human PTEN cDNA was transferred into LoVo cells via lipofectin and PTEN mRNA levels and its expression were analyzed by Western blot and flow cytometry. Before or after transfection, the effects of 5-Fu on inhibiting cell proliferation and inducing apoptosis were measured by flow cytometry, DNA bands and MTT.RESULTS: PTEN transfection significantly up-regulated PTEN expression in LoVo cells. 5-Fu inhibited cell proliferation and induced apoptosis in transfected LoVo cells.CONCLUSION: Transfected PTEN can remark ably up-regulate PTEN expression in LoVo cells and promote the apoptosis.PTEN transfection is associated with 5-Fu treatment effect and has a cooperatively cytotoxic effect.

  15. Regulation of Natural Killer Cell Function by STAT3

    Directory of Open Access Journals (Sweden)

    Nicholas eCacalano

    2016-04-01

    Full Text Available Natural killer (NK cells, key members of a distinct hempatopoietic lineage, innate lymphoid cells (ILCs, are critical effectors that mediate cytotoxicity toward tumor and virally-infected cells but also regulate inflammation, antigen presentation and the adaptive immune response. It has been shown that NK cells can regulate the development and activation of many other components of the immune response such as dendritic cells, which in turn, modulate the function of NK cells in multiple synergistic feed back loops driven by cell-cell contact and the secretion of cytokines and chemokines that control effector function and migration of cells to sites of immune activation. The Signal Transducer and Activator of Transcription (STAT-3 is involved in driving almost all of the pathways that control NK cytolytic activity as well as the reciprocal regulatory interactions between NK cells and other components of the immune system. In the context of tumor immunology, NK cells are a first line of defense that eliminates pre-cancerous and transformed cells early in the process of carcinogenesis, through a mechanism of immune surveillance. Even after tumors become established, NK cells are critical components of anti-cancer immunity: dysfunctional NK cells are often found in the peripheral blood of cancer patients and the lack of NK cells in the tumor microenvironment often correlates with poor prognosis. The pathways and soluble factors activated in tumor-associated NK cells, cancer cells, and regulatory myeloid cells which determine the outcome of cancer immunity are all critically regulated by STAT3. Using the tumor microenvironment as a paradigm, we present here an overview of the research that has revealed fundamental mechanisms through which STAT3 regulates all aspects of natural killer cell biology, including NK development, activation, target cell killing, and fine tuning of the innate and adaptive immune responses.

  16. Osteogenic Potential of Cultured Bone Marrow Stromal CellsTransfected with Transforming Growth Factor β1 Gene in vitro

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    To study the osteogenic potential of cultured bone marrow stromal cells (BMSCs) transfected with transforming growth factor β1 (TGF-β1) gene in vitro, cultured BMSCs were transfected with the complexes of pcDNA3-TGF-β1 and Lipofectamine Reagent in vitro. The cell proliferation was detected by MTT method and the morphological features of transfected BMSCs was observed. ALP stains and PNP method were used to measure ALP activity. In addition, the collagen type Ⅰ propeptides and mineralized matrixes were examined by immunohistochemical staining and tetracycline fluorescence labeling respectively. The morphological and biological characters of the transfected BMSCs were similar to those of osteoblasts and the cell proliferation was promoted. The cell layer displayed strong positive reaction for ALP stains and immunohistochemical staining. ALP activity and collagen type Ⅰ expression increased remarkably after transfection. Mineralized matrixes formed earlier and more in transfected BMSCs as compared with control group. It is concluded that transfecting with TGF-β1 gene could promote the osteogenic potential of cultured BMSCs.

  17. Representation of the serial killer on the Italian Internet.

    Science.gov (United States)

    Villano, P; Bastianoni, P; Melotti, G

    2001-10-01

    The representation of serial killers was examined from the analysis of 317 Web pages in the Italian language to study how the psychological profiles of serial killers are described on the Italian Internet. The correspondence analysis of the content of these Web pages shows that in Italy the serial killer is associated with words such as "monster" and "horror," which suggest and imply psychological perversion and aberrant acts. These traits are peculiar for the Italian scenario.

  18. Immune Monitoring Using mRNA-Transfected Dendritic Cells

    DEFF Research Database (Denmark)

    Borch, Troels Holz; Svane, Inge Marie; Met, Özcan

    2016-01-01

    Dendritic cells are known to be the most potent antigen presenting cell in the immune system and are used as cellular adjuvants in therapeutic anticancer vaccines using various tumor-associated antigens or their derivatives. One way of loading antigen into the dendritic cells is by m......RNA electroporation, ensuring presentation of antigen through major histocompatibility complex I and potentially activating T cells, enabling them to kill the tumor cells. Despite extensive research in the field, only one dendritic cell-based vaccine has been approved. There is therefore a great need to elucidate...... and understand the immunological impact of dendritic cell vaccination in order to improve clinical benefit. In this chapter, we describe a method for performing immune monitoring using peripheral blood mononuclear cells and autologous dendritic cells transfected with tumor-associated antigen-encoding mRNA....

  19. Is killer whale dialect evolution random?

    Science.gov (United States)

    Filatova, Olga A; Burdin, Alexandr M; Hoyt, Erich

    2013-10-01

    The killer whale is among the few species in which cultural change accumulates over many generations, leading to cumulative cultural evolution. Killer whales have group-specific vocal repertoires which are thought to be learned rather than being genetically coded. It is supposed that divergence between vocal repertoires of sister groups increases gradually over time due to random learning mistakes and innovations. In this case, the similarity of calls across groups must be correlated with pod relatedness and, consequently, with each other. In this study we tested this prediction by comparing the patterns of call similarity between matrilines of resident killer whales from Eastern Kamchatka. We calculated the similarity of seven components from three call types across 14 matrilines. In contrast to the theoretical predictions, matrilines formed different clusters on the dendrograms made by different calls and even by different components of the same call. We suggest three possible explanations for this phenomenon. First, the lack of agreement between similarity patterns of different components may be the result of constraints in the call structure. Second, it is possible that call components change in time with different speed and/or in different directions. Third, horizontal cultural transmission of call features may occur between matrilines.

  20. Transcriptional effects of Organochlorine o,p′-DDT and its Metabolite p,p′-DDE in Transfected MDA-MB 231 and MCF-7 Breast Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Ehsan zayerzadeh

    2015-04-01

    Conclusion: In conclusion, our results revealed that o,p’-DDT has not estrogenic activity in a classical mechanism in transfected MDA-MB 231 breast cancer cells while has estrogenic activity in a classical mechanism in transfected MCF-7 human breast cancer cell line.

  1. Clinicopathologic features of intestinal natural killer/T-cell lymphoma

    Institute of Scientific and Technical Information of China (English)

    周军

    2013-01-01

    Objective To study the clinicopathologic features,diagnosis and differential diagnosis of intestinal natural killer(NK)/T-cell lymphoma.Methods The clinical features,histopathology,immunohistochemical

  2. A psychological profile of a serial killer: a case report.

    Science.gov (United States)

    Dogra, T D; Leenaars, Antoon A; Chadha, R K; Manju, Mehta; Lalwani, Sanjeev; Sood, Mamta; Lester, David; Raina, Anupuma; Behera, C

    2012-01-01

    Serial killers have always fascinated society. A serial killer is typically defined as a perpetrator who murders three or more people over a period of time. Most reported cases of serial killers come from the United States and Canada. In India, there are few reported cases. We present, to the best of our knowledge, the first Indian case in the literature. The present case is of a 28-year-old man, Surinder Koli. The Department of Forensic Medicine & Toxicology, All India Institute of Medical Sciences, New Delphi handled the forensic study. We present a most unique psychological investigation into the mind of a serial killer.

  3. Susceptibility of species within the Sporothrix schenckii complex to a panel of killer yeasts.

    Science.gov (United States)

    Stopiglia, Cheila Denise Ottonelli; Heidrich, Daiane; Sorrentino, Julia Medeiros; Vieira, Fabiane Jamono; Landell, Melissa Fontes; Valente, Patrícia; Scroferneker, Maria Lúcia

    2014-06-01

    The Sporothrix schenckii complex is the etiologic agent of sporotrichosis, a subacute or chronic mycosis which can affect humans and animals. Killer yeasts have been used in the medical field for development of novel antimycotics and biotyping of pathogenic fungi. The action of 18 killer yeasts on the growth of 88 characterized S. schenckii, Sporothrix globosa, Sporothrix brasiliensis, and Sporothrix mexicana clinical and environmental isolates was evaluated. Killer studies were performed on Petri dishes containing cheese black starch agar. The yeasts Candida catenulata (QU26, QU31, QU127, LV102); Trichosporon faecale (QU100); Trichosporon japonicum (QU139); Kluyveromyces lactis (QU30, QU99, QU73); Kazachstania unispora (QU49), Trichosporon insectorum (QU89), and Kluyveromyces marxianus (QU103) showed activity against all strains of the S. schenckii complex tested. Observation by optical microscopy of S. brasiliensis 61 within the inhibition haloes around the colonies of the killer yeasts QU100, QU139, and LV102 showed that there was no conidiation, but there was hyphal proliferation. The toxins were fungistatic against S. brasiliensis 61. There was no difference in susceptibility to the toxins among the S. schenckii species complex. Further investigations are necessary to clearly establish the mechanism of action of the toxins.

  4. Paucity of natural killer and cytotoxic T cells in human neuromyelitis optica lesions.

    Science.gov (United States)

    Saadoun, Samira; Bridges, Leslie R; Verkman, A S; Papadopoulos, Marios C

    2012-12-19

    Neuromyelitis optica is a severe inflammatory demyelinating disease of the central nervous system. Most patients with neuromyelitis optica have circulating immunoglobulin G (IgG) antibodies against the astrocytic water channel protein aquaporin-4 (AQP4), which are pathogenic. Anti-AQP4 IgG-mediated complement-dependent astrocyte toxicity is a key mechanism of central nervous system damage in neuromyelitis optica, but the role of natural killer and cytotoxic T cells is unknown. Our objective was to determine whether natural killer and cytotoxic T cells play a role in human neuromyelitis optica lesions. We immunostained four actively demyelinating lesions, obtained from patients with anti-AQP4 IgG positive neuromyelitis optica, for Granzyme B and Perforin. The inflammatory cells were perivascular neutrophils, eosinophils and macrophages, with only occasional Granzyme B+ or Perforin+ cells. Greater than 95% of inflamed vessels in each lesion had no surrounding Granzyme B+ or Perforin+ cells. Granzyme B+ or Perforin+ cells were abundant in human spleen (positive control). Although natural killer cells produce central nervous system damage in mice injected with anti-AQP4 IgG, our findings here indicate that natural killer-mediated and T cell-mediated cytotoxicity are probably not involved in central nervous system damage in human neuromyelitis optica.

  5. Native Killer Yeasts as Biocontrol Agents of Postharvest Fungal Diseases in Lemons

    Science.gov (United States)

    Garnica, Nydia Mercedes; Fernández-Zenoff, María Verónica; Farías, María Eugenia; Sepulveda, Milena; Ramallo, Jacqueline; Dib, Julián Rafael

    2016-01-01

    Economic losses caused by postharvest diseases represent one of the main problems of the citrus industry worldwide. The major diseases affecting citrus are the "green mold" and "blue mold", caused by Penicillium digitatum and P. italicum, respectively. To control them, synthetic fungicides are the most commonly used method. However, often the emergence of resistant strains occurs and their use is becoming more restricted because of toxic effects and environmental pollution they generate, combined with trade barriers to international markets. The aim of this work was to isolate indigenous killer yeasts with antagonistic activity against fungal postharvest diseases in lemons, and to determine their control efficiency in in vitro and in vivo assays. Among 437 yeast isolates, 8.5% show to have a killer phenotype. According to molecular identification, based on the 26S rDNA D1/D2 domain sequences analysis, strains were identified belonging to the genera Saccharomyces, Wickerhamomyces, Kazachstania, Pichia, Candida and Clavispora. Killers were challenged with pathogenic molds and strains that caused the maximum in vitro inhibition of P. digitatum were selected for in vivo assays. Two strains of Pichia and one strain of Wickerhamomyces depicted a significant protection (p <0.05) from decay by P. digitatum in assays using wounded lemons. Thus, the native killer yeasts studied in this work showed to be an effective alternative for the biocontrol of postharvest fungal infections of lemons and could be promising agents for the development of commercial products for the biological control industry. PMID:27792761

  6. The Effects of Bone Morphogenetic Protein 2 Gene Transfection on Fibroblasts

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    目的:探讨骨形成蛋白2(bone morphogenetic protein 2,BMP2)基因转染对成纤维细胞成骨表型的调控作用.方法:利用脂质体将包含BMP2 cDNA全长编码序列的表达载体转染至NIH3T3细胞,原位杂交和免疫组化分别检测BMP2在NIH3T3细胞内的稳定转染和表达,同时观察转染细胞的增殖能力及成骨标志物包括碱性磷酸酶(alkaline phosphatase,ALP)活力和骨钙素(osteocalcin,OC)含量的变化.结果:BMP2只在转染细胞内表达.与未转染细胞相比,BMP2基因转染细胞的增殖能力降低,而ALP活力和OC含量增加.结论:结果表明BMP2基因转染能够衣导成纤维细胞的体外成骨潜能.%Objective: To explore the regulatory effects of Bone Morphogenetic Protein 2 (BMP2) gene transfection on the phenotype of fibroblasts. Methods: A phagemid expression vector containing the full length of human BMP2 cDNA coding sequence was transfected into NIH3T3 cells by using LipofectAMINE. The stable transfection and expression of BMP2 in NIH3T3 cells were determined by in situ hybridization and immunohistochemistry, respectively. The proliferation and the markers for osteogenic features, including alkaline phosphatase (碱性磷酸酶, ALP) activity and osteocalcin (骨钙素, OC) production were also investigated in the transfected cells. Results: The results showed that BMP2 was only expressed in the transfected cells. Compared with the non-transfected cells, the BMP2 gene transfected cells showed decreased proliferative ability, but enhanced both ALP activity and OC production (P < 0.05). Conclusions: The results indicate that BMP2 gene transfection can induce the osteogenic potential of fibroblasts in vitro.

  7. Identification of Anti-tumor Cells Carrying Natural Killer (NK Cell Antigens in Patients With Hematological Cancers

    Directory of Open Access Journals (Sweden)

    Ewelina Krzywinska

    2015-10-01

    Full Text Available Natural killer (NK cells, a cytotoxic lymphocyte lineage, are able to kill tumor cells in vitro and in mouse models. However, whether these cells display an anti-tumor activity in cancer patients has not been demonstrated. Here we have addressed this issue in patients with several hematological cancers. We found a population of highly activated CD56dimCD16+ NK cells that have recently degranulated, evidence of killing activity, and it is absent in healthy donors. A high percentage of these cells expressed natural killer cell p46-related protein (NKp46, natural-killer group 2, member D (NKG2D and killer inhibitory receptors (KIRs and a low percentage expressed NKG2A and CD94. They are also characterized by a high metabolic activity and active proliferation. Notably, we found that activated NK cells from hematological cancer patients have non-NK tumor cell antigens on their surface, evidence of trogocytosis during tumor cell killing. Finally, we found that these activated NK cells are distinguished by their CD45RA+RO+ phenotype, as opposed to non-activated cells in patients or in healthy donors displaying a CD45RA+RO− phenotype similar to naïve T cells. In summary, we show that CD45RA+RO+ cells, which resemble a unique NK population, have recognized tumor cells and degranulate in patients with hematological neoplasias.

  8. Transfection of isolated rainbow trout, Oncorhynchus mykiss, granulosa cells through chemical transfection and electroporation at 12°C.

    Science.gov (United States)

    Marivin, E; Mourot, B; Loyer, P; Rime, H; Bobe, J; Fostier, A

    2015-09-15

    Over-expression or inhibition of gene expression can be efficiently used to analyse the functions and/or regulation of target genes. Modulation of gene expression can be achieved through transfection of exogenous nucleic acids into target cells. Such techniques require the development of specific protocols to transfect cell cultures with nucleic acids. The aim of this study was to develop a method of transfection suitable for rainbow trout granulosa cells in primary culture. After the isolation of rainbow trout granulosa cells, chemical transfection of cells with a fluorescent morpholino oligonucleotide (MO) was tested using FuGENE HD at 12 °C. Electroporation was also employed to transfect these cells with either a plasmid or MO. Transfection was more efficient using electroporation (with the following settings: 1200 V/40 ms/1p) than chemical transfection, but electroporation by itself was deleterious, resulting in a decrease of the steroidogenic capacity of the cells, measured via estradiol production from its androgenic substrate. The disturbance of cell biology induced by the transfection method per se should be taken into account in data interpretation when investigating the effects of under- or over-expression of candidate genes.

  9. Transfection system of amino-functionalized calcium phosphate nanoparticles: in vitro efficacy, biodegradability, and immunogenicity study.

    Science.gov (United States)

    Mostaghaci, Babak; Susewind, Julia; Kickelbick, Guido; Lehr, Claus-Michael; Loretz, Brigitta

    2015-03-11

    Many methods have been developed in order to use calcium phosphate (CaP) for delivering nucleotides into living cells. Surface functionalization of CaP nanoparticles (CaP NPs) with N-(2-aminoethyl)-3-aminopropyltrimethoxysilane was shown recently to achieve dispersed NPs with a positive surface charge, capable of transfection (Chem. Mater. 2013, 25 (18), 3667). In this study, different crystal structures of amino-modified CaP NPs (brushite and hydroxyapatite) were investigated for their interaction in cell culture systems in more detail. Qualitative (confocal laser scanning microscopy) and quantitative (flow cytometry) transfection experiments with two cell lines showed the higher transfection efficacy of brushite versus hydroxyapatite. The transfection also revealed a cell type dependency. HEK293 cells were easier to transfect compared to A549 cells. This result was supported by the cytotoxicity results. A549 cells showed a higher degree of tolerance toward the CaP NPs. Further, the impact of the surface modification on the interaction with macrophages and complement as two important components of the innate immune system were considered. The amine surface functionalization had an effect of decreasing the release of proinflammatory cytokines. The complement interaction investigated by a C3a complement activation assay did show no significant differences between CaP NPs without or with amine modification and overall weak interaction. Finally, the degradation of CaP NPs in biological media was studied with respect to the two crystal structures and at acidic and neutral pH. Both amino-modified CaP NPs disintegrate within days at neutral pH, with a notable faster disintegration of brushite NPs at acidic pH. In summary, the fair transfection capability of this amino functionalized CaP NPs together with the excellent biocompatibility, biodegradability, and low immunogenicity make them interesting candidates for further evaluation.

  10. On The Role of Natural Killer Cells in Neurodegenerative Diseases

    Directory of Open Access Journals (Sweden)

    Azzam A. Maghazachi

    2013-02-01

    Full Text Available Natural killer (NK cells exert important immunoregulatory functions by releasing several inflammatory molecules, such as IFN-γ and members of chemokines, which include CCL3/MIP-1α and CCL4/MIP-1β. These cells also express heptahelical receptors, which are coupled to heterotrimeric G proteins that guide them into inflamed and injured tissues. NK cells have been shown to recognize and destroy transformed cells and virally-infected cells, but their roles in neurodegenerative diseases have not been examined in detail. In this review, I will summarize the effects of NK cells in two neurodegenerative diseases, namely multiple sclerosis and globoid cell leukodystrophy. It is hoped that the knowledge obtained from these diseases may facilitate building rational protocols for treating these and other neurodegenerative or autoimmune diseases using NK cells and drugs that activate them as therapeutic tools.

  11. The secretory synapse: the secrets of a serial killer.

    Science.gov (United States)

    Bossi, Giovanna; Trambas, Christina; Booth, Sarah; Clark, Richard; Stinchcombe, Jane; Griffiths, Gillian M

    2002-11-01

    Cytotoxic T lymphocytes (CTLs) destroy their targets by a process involving secretion of specialized granules. The interactions between CTLs and target can be very brief; nevertheless, adhesion and signaling proteins segregate into an immunological synapse. Secretion occurs in a specialized secretory domain. Use of live and fixed cell microscopy allows this secretory synapse to be visualized both temporally and spatially. The combined use of confocal and electron microscopy has produced some surprising findings, which suggest that the secretory synapse may be important both in delivering the lethal hit and in facilitating membrane transfer from target to CTL. Studies on the secretory synapse in wild-type and mutant CTLs have been used to identify proteins involved in secretion. Further clues as to the signals required for secretion are emerging from comparisons of inhibitory and activating synapses formed by natural killer cells.

  12. Type I Interferons and Natural Killer Cell Regulation in Cancer

    Science.gov (United States)

    Müller, Lena; Aigner, Petra; Stoiber, Dagmar

    2017-01-01

    Type I interferons (IFNs) are known to mediate antitumor effects against several tumor types and have therefore been commonly used in clinical anticancer treatment. However, how IFN signaling exerts its beneficial effects is only partially understood. The clinically relevant activity of type I IFNs has been mainly attributed to their role in tumor immune surveillance. Different mechanisms have been postulated to explain how type I IFNs stimulate the immune system. On the one hand, they modulate innate immune cell subsets such as natural killer (NK) cells. On the other hand, type I IFNs also influence adaptive immune responses. Here, we review evidence for the impact of type I IFNs on immune surveillance against cancer and highlight the role of NK cells therein.

  13. Ustilago maydis killer toxin as a new tool for the biocontrol of the wine spoilage yeast Brettanomyces bruxellensis.

    Science.gov (United States)

    Santos, Antonio; Navascués, Eva; Bravo, Enrique; Marquina, Domingo

    2011-01-31

    Brettanomyces bruxellensis is one of the most damaging species for wine quality, and tools for controlling its growth are limited. In this study, thirty-nine strains belonging to Saccharomyces cerevisiae and B. bruxellensis have been isolated from wineries, identified and then tested against a panel of thirty-nine killer yeasts. Here, for the first time, the killer activity of Ustilago maydis is proven to be effective against B. bruxellensis. Mixed cultures in winemaking conditions show that U. maydis CYC 1410 has the ability to inhibit B. bruxellensis, while S. cerevisiae is fully resistant to its killer activity, indicating that it could be used in wine fermentation to avoid the development of B. bruxellensis without undesirable effects on the fermentative yeast. The characterization of the dsRNAs isolated and purified from U. maydis CYC 1410 indicated that this strain produces a KP6-related toxin. Killer toxin extracts were active against B. bruxellensis at pH values between 3.0 and 4.5 and temperatures comprised between 15 °C and 25 °C, confirming their biocontrol activity in winemaking and wine aging conditions. Furthermore, small amounts (100 AU/ml) of killer toxin extracts from U. maydis significantly reduced the amount of 4-ethylphenol produced by B. bruxellensis, indicating that in addition to the growth inhibition observed for high killer toxin concentrations (ranging from 400 to 2000 AU/ml), small amounts of the toxin are able to reduce the production of volatile phenols responsible for the aroma defects in wines caused by B. bruxellensis.

  14. The virally encoded killer proteins from Ustilago maydis

    Science.gov (United States)

    Several strains of Ustilago maydis, a causal agent of corn smut disease, exhibit a 'killer' phenotype that is due to persistent infection by double-stranded RNA Totiviruses. These viruses produce potent killer proteins that are secreted by the host. This is a rare example of virus/host symbiosis in ...

  15. Natural killer cells: Biology, functions and clinical relevance

    Directory of Open Access Journals (Sweden)

    Vojvodić Svetlana

    2010-01-01

    Full Text Available Introduction. Natural Killer cells (NK cells represent the subset of peripheral lymphocytes that play critical role in the innate immune response to virus-infected and tumor transformed cells. Lysis of NK sensitive target cells could be mediated independently of antigen stimulation and without requirement of peptide presentation by the major histocompatibility complex (MHC molecules. NK cell activity and functions are controlled by a considerable number of cell surface receptors, which exist in both inhibitory and activating isoforms. There are several groups of NK cell surface receptors: 1 killer immunoglobulin like receptors-KIR, 2 C-type lectin receptors,3natural citotoxicity receptors-NCR and 4 Toll-like receptors-TLR. Functions of NK receptors. Defining the biology of NK cell surface receptors has contributed to the concept of the manner how NK cells selectively recognize and lyse tumor and virally infected cells while sparing normal cells. Further, identification of NK receptor ligands and their expression on the normal and transformed cells has led to the development of clinical approaches to manipulating receptor/ligand interactions that showed clinical benefit. NK cells are the first lymphocyte subset that reconstitute the peripheral blood following allogeneic HSCT and multiple roles for alloreactive donor NK cells have been demonstrated, in diminishing Graft vs. Host Disease (GvHD through selective killing recipient dendritic cells, prevention of graft rejection by killing recipient T cells and participation in Graft vs. Leukaemia (GvL effect through destruction of residual host tumor cells. Conclusion. Besides their role in HSCT, NK cell receptors have an important clinical relevance that reflects from the fact that they play a crucial role in the development of some diseases as well as in possibilities of managing all NK receptors through selective expansion and usage of NK cells in cancer immunotherapy.

  16. [Transfection of HL-60 cells by Venus lentiviral vector].

    Science.gov (United States)

    Li, Zheng; Hu, Shao-Yan; Cen, Jian-Nong; Chen, Zi-Xing

    2013-06-01

    In order to study the potential of Venus, lentiviral vector, applied to acute myeloid leukemia, the recombinant vector Venus-C3aR was transfected into 293T packing cells by DNA-calcium phosphate coprecipitation. All virus stocks were collected and transfected into HL-60, the GFP expression in HL-60 cells was measured by flow cytometry. The expression level of C3aR1 in transfected HL-60 cells was identified by RT-PCR and flow cytometry. The lentiviral toxicity on HL-60 was measured by using CCK-8 method and the ability of cell differentiation was observed. The results indicated that the transfection efficacy of lentiviral vector on HL-60 cells was more than 95%, which meets the needs for further study. C3aR1 expression on HL-60 cells increased after being transfected with recombinant lentiviral vector. Before and after transfection, the proliferation and differentiation of cells were not changed much. It is concluded that the lentiviral vector showed a high efficacy to transfect AML cells and can be integrated in genome of HL-60 cells to realize the stable expression of interest gene. Meanwhile, lentiviral vector can not affect HL-60 cell ability to proliferate and differentiate.

  17. East Africa’s Quick Killer

    Institute of Scientific and Technical Information of China (English)

    Maya; Reid

    2011-01-01

    HEALTH and politics don’t always mix.In January,as increasing numbers of returnees from North Sudan entered Southern Sudan to vote on a referendum for independence, the World Health Organization(WHO) reported that a major visceral leishmaniasis epidemic was in danger of breaking out.Incidences of the disease - the world’s second deadliest parasitic killer after malaria - had nearly doubled within the span of a month,according to case records from last November. WHO officials speculated that the surge was due to that fact that returnees typically did not have immunity against the disease.

  18. Recruitment of activation receptors at inhibitory NK cell immune synapses.

    Directory of Open Access Journals (Sweden)

    Nicolas Schleinitz

    Full Text Available Natural killer (NK cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses.

  19. Photobleaching and phototoxicity of KillerRed in tumor spheroids induced by continuous wave and pulsed laser illumination.

    Science.gov (United States)

    Kuznetsova, Daria S; Shirmanova, Marina V; Dudenkova, Varvara V; Subochev, Pavel V; Turchin, Ilya V; Zagaynova, Elena V; Lukyanov, Sergey A; Shakhov, Boris E; Kamensky, Vladislav A

    2015-11-01

    The purpose of this study was to evaluate photobleaching of the genetically encoded photosensitizer KillerRed in tumor spheroids upon pulsed and continuous wave (CW) laser irradiation and to analyze the mechanisms of cancer cell death after the treatment. We observed the light-dose dependent mechanism of KillerRed photobleaching over a wide range of fluence rates. Loss of fluorescence was limited to 80% at light doses of 150 J/cm(2) and more. Based on the bleaching curves, six PDT regimes were applied for irradiation using CW and pulsed regimes at a power density of 160 mW/cm(2) and light doses of 140 J/cm(2) , 170 J/cm(2) and 200 J/cm(2). Irradiation of KillerRed-expressing spheroids in the pulsed mode (pulse duration 15 ns, pulse repetition rate 10 Hz) induced predominantly apoptotic cell death, while in the case of CW mode the cancer cells underwent necrosis. In general, these results improve our understanding of photobleaching mechanisms in GFP-like proteins and show the importance of appropriate selection of treatment mode for PDT with KillerRed. Representative fluorescence image of two KillerRed-expressing spheroids before and immediately after CW irradiation.

  20. [VEGF gene expression in transfected human multipotent stromal cells].

    Science.gov (United States)

    Smirnikhina, S A; Lavrov, A V; Bochkov, N P

    2011-01-01

    Dynamics of VEGF gene expression in transfected multipotent stromal cells from adipose tissue was examined using electroporation and lipofection. Differences in the potency and dynamics of plasmid elimination (up to day 9) between cell cultures were observed. All cultures were divided into fast and slow plasmid-eliminating ones. Interculture differences in VEGF expression were detected. The possibility of a 5-6-fold increase of VEGF expression was shown. There were no differences in transfection potency, plasmid elimination dynamics, and VEGF expression after transfection by both nonviral methods.

  1. Interplay between carbohydrate and lipid in recognition of glycolipid antigens by natural killer T cells.

    Science.gov (United States)

    Pei, Bo; Vela, Jose Luis; Zajonc, Dirk; Kronenberg, Mitchell

    2012-04-01

    Natural killer T (NKT) cells are a T cell subpopulation that were named originally based on coexpression of receptors found on natural killer (NK) cells, cells of the innate immune system, and by T lymphocytes. The maturation and activation of NKT cells requires presentation of glycolipid antigens by CD1d, a cell surface protein distantly related to the major histocompatibility complex (MHC)-encoded antigen presenting molecules. This specificity distinguishes NKT cells from most CD4(+) and CD8(+) T cells that recognize peptides presented by MHC class I and class II molecules. The rapid secretion of a large amount of both Th1 and Th2 cytokines by activated NKT cells endows them with the ability to play a vital role in the host immune defense against various microbial infections. In this review, we summarize progress on identifying the sources of microbe-derived glycolipid antigens recognized by NKT cells and the biochemical basis for their recognition.

  2. Modeling Natural Killer Cell Targeted Immunotherapies

    Science.gov (United States)

    Lopez-Lastra, Silvia; Di Santo, James P.

    2017-01-01

    Animal models have extensively contributed to our understanding of human immunobiology and to uncover the underlying pathological mechanisms occurring in the development of diseases. However, mouse models do not reproduce the genetic and molecular complexity inherent in human disease conditions. Human immune system (HIS) mouse models that are susceptible to human pathogens and can recapitulate human hematopoiesis and tumor immunobiology provide one means to bridge the interspecies gap. Natural killer cells are the founding member of the innate lymphoid cell family. They exert a rapid and strong immune response against tumor and pathogen-infected cells. Their antitumor features have long been exploited for therapeutic purposes in the context of cancer. In this review, we detail the development of highly immunodeficient mouse strains and the models currently used in cancer research. We summarize the latest improvements in adoptive natural killer (NK) cell therapies and the development of novel NK cell sources. Finally, we discuss the advantages of HIS mice to study the interactions between human NK cells and human cancers and to develop new therapeutic strategies.

  3. Natural Killer Cells in Asthma

    Directory of Open Access Journals (Sweden)

    Khalil eKarimi

    2013-06-01

    Full Text Available The worldwide prevalence, morbidity, and mortality of asthma have dramatically increased over the last few decades and there is a clear need to identify new, effective, therapeutic and prophylactic strategies. Despite high numbers of NK cells in the lung and their ability to and generate a variety of immunomodulatory mediators the potential of these cells as therapeutic targets in allergic airway disease has been largely overlooked. The fact that IgE, acting through FcγRIII can activate NK cells resulting in cytokine/chemokine production implies that NK cells may contribute to IgE-mediated allergic responses. Indeed, current evidence suggests that NK cells can promote allergic airway responses during sensitization and ongoing inflammation. In animals models increased NK cells are observed in the lung following antigen challenge, while depletion of NK cells before immunization inhibits allergic airway inflammation. Moreover, in asthmatics NK cell phenotype is altered, and may contribute to promotion of the pro-inflammatory Th2-type environment. Conversely, driving NK cells towards an IFN-γ-secreting phenotype can reduce features of the allergic airway response in animal models. However, we have limited knowledge of the signals that drive the development of distinct subsets and functional phenotypes of NK cells in the lung and thus the role and therapeutic potential of NK cells in the allergic airway remains unclear. Here we review the potentially diverse roles of NK cells in allergic airway disease, identify gaps in current knowledge and discuss the potential of modulating NK cell function as a treatment strategy in asthma

  4. DyNAvectors: dynamic constitutional vectors for adaptive DNA transfection.

    Science.gov (United States)

    Clima, Lilia; Peptanariu, Dragos; Pinteala, Mariana; Salic, Adrian; Barboiu, Mihail

    2015-12-25

    Dynamic constitutional frameworks, based on squalene, PEG and PEI components, reversibly connected to core centers, allow the efficient identification of adaptive vectors for good DNA transfection efficiency and are well tolerated by mammalian cells.

  5. Progesterone and estradiol enhance lipid mediated transfection of Sk-Br-3 mammalian cancer cells.

    Science.gov (United States)

    Köster, Frank; Felberbaum, Ricardo; Finas, Dominique; Wünsch, Kurt; Schulz, Christiane; Diedrich, Klaus; Hauser, Charlotte

    2002-06-01

    Cyclodextrin encapsulated beta-estradiol and progesterone were used for enhancement of gene delivery using the breast cancer cell line Sk-Br-3. A non-toxic concentration of cyclodextrin encapsulated sex steroids (50 microM) added to lipid or liposomal transfection led to a 12-fold increase of reporter gene expression (luciferase) with progesterone and an 8-fold increase with estradiol using Lipofectamine Plus mediated transfection. Using the lipid formulation Fugene-6 the results were a 5.5-fold and a 4.5-fold increase respectively. This enhancement could only be observed if the sex steroids were added to the cells before application of the DNA-Fugene complex supporting the evidence that intracellular processes are responsible for the activity of the steroids. The strong differences between progesterone and estradiol in modifying Lipofectamine Plus transfection in Sk-Br-3 cells may to be explained by differences in the distribution of these receptors in the cellular compartments. These results seem to add evidence on the possibility of using sex steroids to increase the efficiency of non-viral vectors for transfection, and may ultimately prove to be relevant to gene therapy in the treatment of breast cancer as well as other solid tumors.

  6. Intra-arterial adenoviral mediated tumor transfection in a novel model of cancer gene therapy

    Directory of Open Access Journals (Sweden)

    Siemionow Maria

    2006-08-01

    Full Text Available Abstract Background The aim of the present study was to develop and characterize a novel in vivo cancer gene therapy model in which intra-arterial adenoviral gene delivery can be characterized. In this model, the rat cremaster muscle serves as the site for tumor growth and provides convenient and isolated access to the tumor parenchyma with discrete control of arterial and venous access for delivery of agents. Results Utilizing adenovirus encoding the green fluorescent protein we demonstrated broad tumor transfection. We also observed a dose dependant increment in luciferase activity at the tumor site using an adenovirus encoding the luciferase reporter gene. Finally, we tested the intra-arterial adenovirus dwelling time required to achieve optimal tumor transfection and observed a minimum time of 30 minutes. Conclusion We conclude that adenovirus mediated tumor transfection grown in the cremaster muscle of athymic nude rats via an intra-arterial route could be achieved. This model allows definition of the variables that affect intra-arterial tumor transfection. This particular study suggests that allowing a defined intra-tumor dwelling time by controlling the blood flow of the affected organ during vector infusion can optimize intra-arterial adenoviral delivery.

  7. Rapid recombination among transfected plasmids, chimeric episome formation and trans gene expression in Plasmodium falciparum.

    Science.gov (United States)

    Kadekoppala, M; Cheresh, P; Catron, D; Ji, D D; Deitsch, K; Wellems, T E; Seifert, H S; Haldar, K

    2001-02-01

    Although recombination is known to be important to generating diversity in the human malaria parasite P. falciparum, the low efficiencies of transfection and the fact that integration of transfected DNA into chromosomes is observed only after long periods (typically 12 weeks or more) have made it difficult to genetically manipulate the blood stages of this major human pathogen. Here we show that co-transfection of a P. falciparum line with two plasmids, one expressing a green fluorescent protein (gfp) reporter and the other expressing a drug resistance marker (Tgdhfr-ts M23), allowed selection of a population in which about approximately 30% of the parasites produce GFP. In these GFP-producing parasites, the transfected plasmids had recombined into chimeric episomes as large as 20 kb and could be maintained under drug pressure for at least 16 weeks. Our data suggest that chimera formation occurs early (detected by 7--14 days) and that it involves homologous recombination favored by presence of the same P. falciparum 5'hrp3 UTR promoting transcription from each plasmid. This indicates the presence of high levels of homologous recombination activity in blood stage parasites that can be used to drive rapid recombination of newly introduced DNA, study mechanisms of recombination, and introduce genes for trans expression in P. falciparum.

  8. Cationic Liposomes Modified with Polyallylamine as a Gene Carrier: Preparation, Characterization and Transfection Efficiency Evaluation

    Directory of Open Access Journals (Sweden)

    Reza Kazemi Oskuee

    2016-12-01

    Full Text Available Purpose: Cationic polymers and cationic liposomes have shown to be effective non-viral gene delivery vectors. In this study, we tried to improve the transfection efficiency by employing the advantages of both. Methods: For this purpose, modified polyallylamines (PAAs were synthesized. These modifications were done through the reaction of PAA (15 KDa with acrylate and 6-bromoalkanoic acid derivatives. Liposomes comprising of these cationic polymers and cationic lipid were prepared and extruded through polycarbonate filters to obtain desired size. Liposome-DNA nanocomplexes were prepared in three carrier to plasmid (C/P ratios. Size, zeta potential and DNA condensation ability of each complex were characterized separately and finally transfection efficiency and cytotoxicity of prepared vectors were evaluated in Neuro2A cell line. Results: The results showed that mean particle size of all these nanocomplexes was lower than 266 nm with surface charge of 22.0 to 33.9 mV. Almost the same condensation pattern was observed in all vectors and complete condensation was occurred at C/P ratio of 1.5. The lipoplexes containing modified PAA 15 kDa with 10% hexyl acrylate showed the highest transfection efficacy and lowest cytotoxicity in C/P ratio of 0.5. Conclusion: In some cases nanocomplexes consisting of cationic liposome and modified PAA showed better transfection activity and lower cytotoxicity compared to PAA.

  9. Relative rates of homologous and nonhomologous recombination in transfected DNA.

    OpenAIRE

    Roth, D B; Wilson, J H

    1985-01-01

    Both homologous and nonhomologous recombination events occur at high efficiency in DNA molecules transfected into mammalian cells. Both types of recombination occur with similar overall efficiencies, as measured by an endpoint assay, but their relative rates are unknown. In this communication, we measure the relative rates of homologous and nonhomologous recombination in DNA transfected into monkey cells. This measurement is made by using a linear simian virus 40 genome that contains a 131-ba...

  10. Temporal, quantitative, and functional characteristics of single-KIR-positive alloreactive natural killer cell recovery account for impaired graft-versus-leukemia activity after haploidentical hematopoietic stem cell transplantation.

    Science.gov (United States)

    Vago, Luca; Forno, Barbara; Sormani, Maria Pia; Crocchiolo, Roberto; Zino, Elisabetta; Di Terlizzi, Simona; Lupo Stanghellini, Maria Teresa; Mazzi, Benedetta; Perna, Serena K; Bondanza, Attilio; Middleton, Derek; Palini, Alessio; Bernardi, Massimo; Bacchetta, Rosa; Peccatori, Jacopo; Rossini, Silvano; Roncarolo, Maria Grazia; Bordignon, Claudio; Bonini, Chiara; Ciceri, Fabio; Fleischhauer, Katharina

    2008-10-15

    In this study, we have characterized reconstitution of the natural killer (NK) cell repertoire after haploidentical CD34(+) selected hematopoietic stem cell transplantation (HSCT) for high-risk hematologic malignancies. Analysis focused on alloreactive single-KIR(+) NK cells, which reportedly are potent antileukemic effectors. One month after HSCT, CD56(bright)/CD56(dim) NK-cell subsets showed inverted ratio and phenotypic features. CD25 and CD117 down-regulation on CD56(bright), and NKG2A and CD62L up-regulation on CD56(dim), suggest sequential CD56(bright)-to-CD56(dim) NK-cell maturation in vivo. Consistently, the functional potential of these maturation intermediates against leukemic blasts was impaired. Mature receptor repertoire reconstitution took at least 3 months. Importantly, at this time point, supposedly alloreactive, single-KIR(+) NK cells were not yet fully functional. Frequency of these cells was highly variable, independently from predicted NK alloreactivity, and below 1% of NK cells in 3 of 6 alloreactive patients studied. In line with these observations, no clinical benefit of predicted NK alloreactivity was observed in the total cohort of 56 patients. Our findings unravel the kinetics, and limits, of NK-cell differentiation from purified haploidentical hematopoietic stem cells in vivo, and suggest that NK-cell antileukemic potential could be best exploited by infusion of mature single-KIR(+) NK cells selected from an alloreactive donor.

  11. Células natural killer e vigilância imunológica Natural killer cells and immune surveillance

    Directory of Open Access Journals (Sweden)

    Mariana Jobim

    2008-08-01

    Full Text Available OBJETIVOS: Analisar a importância das células natural killer, de seus receptores killer immunoglobulin-like receptors e correspondentes genes (KIR na vigilância imunológica do organismo contra agentes infecciosos, transplantes de células-tronco hematopoiéticas, assim como sua participação na auto-imunidade. As características e o polimorfismo dos genes e receptores KIR na população brasileira serão descritos. FONTES DOS DADOS: Livros, artigos de revisão e artigos científicos recentes são citados e listados na bibliografia. A experiência pessoal é também apresentada. SÍNTESE DOS DADOS: Identificamos o perfil de genes e haplótipos KIR na população caucasóide brasileira, sendo de importância esse conhecimento para a análise da relação desse sistema com doenças. Examinamos 116 indivíduos doadores voluntários de medula óssea, identificando-se 32 genótipos e a presença de 51 e 49% de haplótipos A e B, respectivamente. Foi realizado estudo comparativo entre os nossos genótipos e os de outras populações. CONCLUSÕES: A imunidade inata é uma barreira antiinfecciosa de importância em pediatria. Ela atua de maneira independente da imunidade celular e humoral, sendo mais rápida que as demais fontes de proteção do organismo. Ao mesmo tempo, ela estimula os linfócitos T CD8 a agirem e amplificarem a rede de proteção imunológica. Entretanto, como na maioria das vezes em que a imunidade atua, ela também pode ser prejudicial, agredindo o organismo por mecanismos auto-imunes ou mesmo, na sua ausência, oferecer espaço aos agentes infecciosos para agirem de forma impune.OBJECTIVES: To analyze the importance of natural killer cells, their killer immunoglobulin-like receptors (KIR and genes in autoimmunity and in the immune surveillance against infectious agents and stem cells transplantation. The characteristics and polymorphisms of the KIR genes and receptors in the Brazilian population is described. SOURCES

  12. Natural Killer cell recognition of melanoma: new clues for a more effective immunotherapy

    Directory of Open Access Journals (Sweden)

    Raquel eTarazona

    2016-01-01

    Full Text Available Natural killer cells participate in the early immune response against melanoma and also contribute to the development of an adequate adaptive immune response by their crosstalk with dendritic cells and cytokine secretion. Melanoma resistance to conventional therapies together with its high immunogenicity justifies the development of novel therapies aimed to stimulate effective immune responses against melanoma. However, melanoma cells frequently escape to CD8 T cell recognition by the down-regulation of major histocompatibility complex class I molecules. In this scenario, Natural killer cells emerge as potential candidates for melanoma immunotherapy due to their capacity to recognize and destroy melanoma cells expressing low levels of major histocompatibility complex class I molecules. In addition, the possibility to combine immune checkpoint blockade with other NK cell potentiating strategies (e.g. cytokine induction of activating receptors has opened new perspectives in the potential use of adoptive NK cell-based immunotherapy in melanoma.

  13. Unnatural killer cells to prevent bloodborne metastasis: inspiration from biology and engineering.

    Science.gov (United States)

    Mitchell, Michael J; King, Michael R

    2014-06-01

    Metastasis contributes to over 90% of cancer-related deaths. Many types of cancer metastasize via the bloodstream, where circulating tumor cells (CTCs) originating from the primary tumor can undergo selectin-mediated adhesion with the blood vessel wall and subsequently transmigrate to anatomically distant organs. In an effort to neutralize CTCs with the potential to form metastases, a new therapeutic approach has been developed in which circulating leukocytes are functionalized to target and kill cancer cells in the bloodstream. This approach mimics the cytotoxic activity of natural killer cells and the chemical engineering concept of a fluidized bed reactor, which increases the surface area for surface-catalyzed reactions. The resulting 'unnatural killer cells', proven effective in vitro with human blood and also in the living mouse, holds promise in neutralizing CTCs to interrupt the metastasis process.

  14. Biolistic techniques for transfection of mosquito embryos (Anopheles gambiae).

    Science.gov (United States)

    Mialhe, E; Miller, L H

    1994-05-01

    To compensate for the extremely low rates of transformation by DNA microinjection into mosquito embryos of Anopheles gambiae, biolistic techniques were evaluated for introduction of DNA into large numbers of mosquito embryos. Biolistic experiments were first performed with a commercially available instrument intended for this purpose, according to the recommended procedure. The amount of DNA delivered was measured by the expression of luciferase under the control of the Drosophila heat shock protein (hsp) 70 promoter. Despite attempts to optimize biolistic parameters, the level of luciferase activity was low and highly variable. Two other methods of biolistic delivery of DNA-coated particles in aqueous suspension were then evaluated. One method used the gas explosion of the commercially available instrument (mentioned above) to drive an aqueous suspension of DNA-coated particles at high pressure. This method reproducibly increased the level of expression about 100-fold without greatly reducing embryo viability. Another method, which was recently described for plant transfection, uses lower pressure to deliver the aqueous suspension of DNA-coated particles. The level of expression of luciferase and the survival of embryos were equivalent to that obtained with the instrument modified for aqueous delivery of particles. Thus, both aqueous methods offer the advantages of reproducibly delivering more DNA to the embryos. Moreover, these methods could be suitable for delivering DNA mixed with proteins, such as restriction endonucleases and integrases, that may be destroyed by ethanol precipitation used in the standard PDS-1000/He method.

  15. An alternative in vitro drug screening test using Leishmania amazonensis transfected with red fluorescent protein✩

    Science.gov (United States)

    Rocha, Marcele N.; Corrêa, Célia M.; Melo, Maria N.; Beverley, Stephen M.; Martins-Filho, Olindo Assis; Madureira, Ana Paula; Soares, Rodrigo P.

    2013-01-01

    Fluorescent and colorimetric reporter genes are valuable tools for drug screening models, since microscopy is labor intensive and subject to observer variation. In this work, we propose a fluorimetric method for drug screening using red fluorescent parasites. Fluorescent Leishmania amazonensis were developed after transfection with integration plasmids containing either red (RFP) or green fluorescent protein (GFP) genes. After transfection, wild-type (LaWT) and transfected (LaGFP and LaRFP) parasites were subjected to flow cytometry, macrophage infection, and tests of susceptibility to current antileishmanial agents and propranolol derivatives previously shown to be active against Trypanosoma cruzi. Flow cytometry analysis discriminated LaWT from LaRFP and LaGFP parasites, without affecting cell size or granulosity. With microscopy, transfection with antibiotic resistant genes was not shown to affect macrophage infectivity and susceptibility to amphotericin B and propranolol derivatives. Retention of fluorescence remained in the intracellular amastigotes in both LaGFP and LaRFP transfectants. However, detection of intracellular RFP parasites was only achieved in the fluorimeter. Murine BALB/c macrophages were infected with LaRFP parasites, exposed to standard (meglumine antimoniate, amphotericin B, Miltefosine, and allopurinol) and tested molecules. Although it was possible to determine IC50 values for 4 propranolol derivatives (1, 2b, 3, and 4b), all compounds were considered inactive. This study is the first to develop a fluorimetric drug screening test for L. amazonensis RFP. The fluorimetric test was comparable to microscopy with the advantage of being faster and not requiring manual counting. PMID:23312610

  16. Natural Killer Cells: Biology and Clinical Use in Cancer Therapy

    Institute of Scientific and Technical Information of China (English)

    WilliamH.D.Hallett; WilliamJ.Murphy

    2004-01-01

    Natural killer (NK) cells have the ability to mediate both bone marrow rejection and promote engraftment, as well as the ability to elicit potent anti-tumor effects. However the clinical results for these processes are still elusive. Greater understanding of NK cell biology, from activating and inhibitory receptor functions to the role of NK cells in allogeneic transplantation, needs to be appreciated in order to draw out the clinical potential of NK cells. Mechanisms of bone marrow cell (BMC) rejection are known to be dependant on inhibitory receptors specific for major histocompatibility complex (MHC) molecules and on activating receptors that have many potential ligands. The modulation of activating and inhibitory receptors may hold the key to clinical success involving NK cells. Pre-clinical studies in mice have shown that different combinations of activating and inhibitory receptors on NK cells can reduce graft-versus-host disease (GVHD), promote engraftment, and provide superior graft-versus-tumor (GVT) responses. Recent clinical data have shown that the use of KIR-ligand incompatibility produces tremendous graft-versus-leukemia effect in patients with acute myeloid leukemia at high risk of relapse. This review will attempt to be a synthesis of current knowledge concerning NK cells, their involvement in BMT, and their use as an immunotherapy for cancer and other hematologic malignancies. Cellular & Molecular Immunology. 2004;1(1):12-21.

  17. Natural Killer Cells: Biology and Clinical Use in Cancer Therapy

    Institute of Scientific and Technical Information of China (English)

    William H. D. Hallett; William J. Murphy

    2004-01-01

    Natural killer (NK) cells have the ability to mediate both bone marrow rejection and promote engraftment, as well as the ability to elicit potent anti-tumor effects. However the clinical results for these processes are still elusive. Greater understanding of NK cell biology, from activating and inhibitory receptor functions to the role of NK cells in allogeneic transplantation, needs to be appreciated in order to draw out the clinical potential of NK cells. Mechanisms of bone marrow cell (BMC) rejection are known to be dependant on inhibitory receptors specific for major histocompatibility complex (MHC) molecules and on activating receptors that have many potential ligands. The modulation of activating and inhibitory receptors may hold the key to clinical success involving NK cells. Pre-clinical studies in mice have shown that different combinations of activating and inhibitory receptors on NK cells can reduce graft-versus-host disease (GVHD), promote engraftment, and provide superior graft-versus-tumor (GVT) responses. Recent clinical data have shown that the use of KIR-ligand incompatibility produces tremendous graft-versus-leukemia effect in patients with acute myeloid leukemia at high risk of relapse. This review will attempt to be a synthesis of current knowledge concerning NK cells, their involvement in BMT, and their use as an immunotherapy for cancer and other hematologic malignancies. Cellular & Molecular Immunology. 2004;1(1):12-21.

  18. Activation of natural killer T cells by alpha-galactosylceramide rapidly induces the full maturation of dendritic cells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 T cell immunity to a coadministered protein.

    Science.gov (United States)

    Fujii, Shin-Ichiro; Shimizu, Kanako; Smith, Caroline; Bonifaz, Laura; Steinman, Ralph M

    2003-07-21

    The maturation of dendritic cells (DCs) allows these antigen-presenting cells to initiate immunity. We pursued this concept in situ by studying the adjuvant action of alpha-galactosylceramide (alphaGalCer) in mice. A single i.v. injection of glycolipid induced the full maturation of splenic DCs, beginning within 4 h. Maturation was manifest by marked increases in costimulator and major histocompatibility complex class II expression, interferon (IFN)-gamma production, and stimulation of the mixed leukocyte reaction. These changes were not induced directly by alphaGalCer but required natural killer T (NKT) cells acting independently of the MyD88 adaptor protein. To establish that DC maturation was responsible for the adjuvant role of alphaGalCer, mice were given alphaGalCer together with soluble or cell-associated ovalbumin antigen. Th1 type CD4+ and CD8+ T cell responses developed, and the mice became resistant to challenge with ovalbumin-expressing tumor. DCs from mice given ovalbumin plus adjuvant, but not the non-DCs, stimulated ovalbumin-specific proliferative responses and importantly, induced antigen-specific, IFN-gamma producing, CD4+ and CD8+ T cells upon transfer into naive animals. In the latter instance, immune priming did not require further exposure to ovalbumin, alphaGalCer, NKT, or NK cells. Therefore a single dose of alphaGalCer i.v. rapidly stimulates the full maturation of DCs in situ, and this accounts for the induction of combined Th1 CD4+ and CD8+ T cell immunity to a coadministered protein.

  19. Activation of Natural Killer T Cells by α-Galactosylceramide Rapidly Induces the Full Maturation of Dendritic Cells In Vivo and Thereby Acts as an Adjuvant for Combined CD4 and CD8 T Cell Immunity to a Coadministered Protein

    Science.gov (United States)

    Fujii, Shin-ichiro; Shimizu, Kanako; Smith, Caroline; Bonifaz, Laura; Steinman, Ralph M.

    2003-01-01

    The maturation of dendritic cells (DCs) allows these antigen-presenting cells to initiate immunity. We pursued this concept in situ by studying the adjuvant action of α-galactosylceramide (αGalCer) in mice. A single i.v. injection of glycolipid induced the full maturation of splenic DCs, beginning within 4 h. Maturation was manifest by marked increases in costimulator and major histocompatibility complex class II expression, interferon (IFN)-γ production, and stimulation of the mixed leukocyte reaction. These changes were not induced directly by αGalCer but required natural killer T (NKT) cells acting independently of the MyD88 adaptor protein. To establish that DC maturation was responsible for the adjuvant role of αGalCer, mice were given αGalCer together with soluble or cell-associated ovalbumin antigen. Th1 type CD4+ and CD8+ T cell responses developed, and the mice became resistant to challenge with ovalbumin-expressing tumor. DCs from mice given ovalbumin plus adjuvant, but not the non-DCs, stimulated ovalbumin-specific proliferative responses and importantly, induced antigen-specific, IFN-γ producing, CD4+ and CD8+ T cells upon transfer into naive animals. In the latter instance, immune priming did not require further exposure to ovalbumin, αGalCer, NKT, or NK cells. Therefore a single dose of αGalCer i.v. rapidly stimulates the full maturation of DCs in situ, and this accounts for the induction of combined Th1 CD4+ and CD8+ T cell immunity to a coadministered protein. PMID:12874260

  20. Final Critical Habitat for Southern Resident Killer Whales

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — A geospatial data set depicting the boundaries of marine areas designated as critical habitat under the Endangered Species Act (ESA) for Southern Resident killer...

  1. Gulf of Mexico killer whale photo-ID catalog

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Photo-identification data on killer whales occupying the northern Gulf of Mexico have been collected in association with large vessel surveys since 1991. Photographs...

  2. Antiproton cell experiment: antimatter is a better killer

    CERN Multimedia

    2006-01-01

    "European Organization for Nuclear Research is reporting that results from a three year study of antiprotons for neoplasm irrdiation showed a better cellular killer with a smaller lethal dose." (1,5 page)

  3. Effects of urokinase type plasminogen activator gene transfected bone marrow-derived liver stem cells transplantation on hepatocyte regeneration in liver fibrosis rats%尿激酶型纤溶酶原激活物基因转染骨髓源性肝干细胞移植对肝纤维化大鼠肝细胞再生的影响

    Institute of Scientific and Technical Information of China (English)

    孙超; 李定国; 陈源文; 陈颖伟; 汪保灿

    2011-01-01

    目的 探讨尿激酶型纤溶酶原激活物(uPA)基因修饰骨髓源性肝干细胞(BDLSC)移植对四氯化碳(CCl4)诱导肝纤维化大鼠肝细胞再生的影响.方法 纯系Fisher 344雄性大鼠10只,为BDLSC供体大鼠.体外将AduPA转染雄性大鼠BDLSC.纯系Fisher 344雌性大鼠36只,均分为正常组(皮下注射橄榄油)、模型组(CCl4造模,尾静脉注射0.9%氯化钠)、BDLSC组(CCl4造模,尾静脉输入BDLSC)、转基因组(CCl4造模,尾静脉输入转基因BDLSC).检测大鼠肝功能和肝组织胶原面积.半定量RT-PCR方法检测大鼠肝组织肝细胞生长因子(HGF)及其受体c-met mRNA表达水平.免疫组织化学法检测大鼠肝组织增殖细胞核抗原(PCNA)蛋白表达.结果 正常组、模型组、BDLSC组、转基因组肝组织胶原染色面积分别为0.12%±0.03%、14.49%±1.40%、8.25%±0.82%、5.12%±0.40%,组间差异均有统计学意义(P值均<0.05).与模型组和BDLSC组相比,转基因组大鼠肝功能明显改善,血清透明质酸(HA)、血清Ⅲ型前胶原(PCⅢ)、肝组织羟脯氨酸含量明显降低,肝组织HGF和c-met mRNA水平均显著上调,PCNA蛋白表达显著增加.结论 uPA基因修饰BDLSC移植可能诱导肝细胞增殖,从而改善CCl4诱导肝纤维化大鼠的肝功能.%Objective To explore the effects of urokinase type plasminogen activator (uPA) gene-modified bone marrow-derived liver stem cells ( BDLSC) transplantation on hepatocyte regeneration in CCl4-induced liver fibrosis rats. Methods Ten male Fisher 344 rats were donor rats of BDLSC. The BDLSC of male rat was transfected with AduPA. Thirty-six female Fisher 344 rats were equally divided into normal group (injected subcutaneously with olive oil) , model group (CCl4 induced the model, injected through tail vein with 0. 9% sodium chloride), BDLSC group (CCl4 induced the model, injected through tail vein with BDLSC) and gene transfected group (CCl4 induced the model,injected through tail vein with gene transfected

  4. Recombinant Vaccinia Virus is an Effective and Non-perturbing Vector for Human Dendritic Cells Transfected with Epstein-Barr Virus Latent Membrane Protein 2A

    Institute of Scientific and Technical Information of China (English)

    许继军; 姚堃; 彭光勇; 谢芳艺; 丁传林; 朱建中; 秦健

    2002-01-01

    ObjectiveTo study the effects of dendritic cells (DC) transfected with recombinant vaccinia virus encoding Epstein-Barr virus (EBV) latent membrane protein 2A(LMP2A) gene,and to provide evidence for further investigation on the therapeutic vaccines against EBV-associated malignancies.MethodsMature DC were transfected with EBV-LMP2A recombinant vaccinia virus (rVV-LMP2A).Before and after the transfection,the expression of surface antigens on mature DC including CD1a,CD83,CD40,CD80,HLA-DR was measured by fluorescence activated cell sorter (FACS) and the function of DC to stimulate allogeneic T cells proliferation was measured by mixed leukocyte reactions (MLR).ResultsLMP2A protein was highly expressed (66.1%) in DC after the transfection of rVV-LMP2A.No significant changes in the primary surface antigens expression and in the MLR were detected during the transfection.Transfected DC still had strong potential in stimulating the proliferation of allogeneic T cells.ConclusionRecombinant vaccinia virus was an effective and non-perturbing vector to mediate the transfection of LMP2A into DC.The functions of mature DC were not affected significantly by the transfection of Vac-LMP2A.This study could provide evidence for the further immunotherapy of EBV-associated malignancies,e.g.nasopharyngeal carcinoma (NPC).``

  5. Recombinant Vaccinia Virus is an Effective and Non—perturbing Vector for Human Dendritic Cells Transfected with Epstein—Barr Virus Latent Membrane Protein 2A

    Institute of Scientific and Technical Information of China (English)

    许继军; 姚Kun; 等

    2002-01-01

    Objective To study the effects of dendritic cells(DC) transfected with recombinant vaccinia virus encoding Epstein-Barr virus(EBV) latent membrane protein 2A(LMP2A) gene,and to provide evidence for further investigation on the therapeutic uaccines against EBV-associated malignancies.Methods Mature DC were transfected with EVB-LMP2A recombinant vaccinia virus(rVV-LMP2A).Before and after the transfection,the expression of surface antigens on mature DC including CD1a,CD83,CD40,CD80,HLA-DR was measured by fluorescence activated cell sorter(FACS) and the function of DC to stimulate allogeneic T cells proliferation was measured by mixed leukocyte reactions(MLR).Results LMP2A protein was highly expressed (66.1%) in DC after the transfection of rVV-LMP2A.No significant changes in the primary surface antigens expression and in the MLR were detected during the transfection.Transfected DC still had strong potential in stimulating the proliferation of allogeneic T cells.Conclusion Recombinant vaccinia virus was an effective and non-perturbing vector to mediate the transfection of LMP2A into DC.The functions of mature DC were not affected significantly by the transfection of Vac-LMP2A.This study could provide evidence for the further immunotherapy of EBV-associated malignancies,e.g.nasopharyngeal carcinoma(NPC).

  6. Posttranscriptional control of human gamma interferon gene expression in transfected mouse fibroblasts.

    OpenAIRE

    1986-01-01

    Human gamma interferon genomic DNA was introduced into NIH 3T3 fibroblasts by calcium phosphate precipitation and was not expressed in these cells at the cytoplasmic mRNA or protein level. Treatment of the transfected cells with cycloheximide (1 microgram/ml) induced the accumulation of cytoplasmic gamma interferon mRNA and biologically active human gamma interferon. Analysis of the nuclear enriched RNA from untreated cells indicated that human gamma interferon mRNA was present, suggesting th...

  7. Towards PDT with Genetically Encoded Photosensitizer KillerRed: A Comparison of Continuous and Pulsed Laser Regimens in an Animal Tumor Model.

    Directory of Open Access Journals (Sweden)

    Marina Shirmanova

    Full Text Available The strong phototoxicity of the red fluorescent protein KillerRed allows it to be considered as a potential genetically encoded photosensitizer for the photodynamic therapy (PDT of cancer. The advantages of KillerRed over chemical photosensitizers are its expression in tumor cells transduced with the appropriate gene and direct killing of cells through precise damage to any desired cell compartment. The ability of KillerRed to affect cell division and to induce cell death has already been demonstrated in cancer cell lines in vitro and HeLa tumor xenografts in vivo. However, the further development of this approach for PDT requires optimization of the method of treatment. In this study we tested the continuous wave (593 nm and pulsed laser (584 nm, 10 Hz, 18 ns modes to achieve an antitumor effect. The research was implemented on CT26 subcutaneous mouse tumors expressing KillerRed in fusion with histone H2B. The results showed that the pulsed mode provided a higher rate of photobleaching of KillerRed without any temperature increase on the tumor surface. PDT with the continuous wave laser was ineffective against CT26 tumors in mice, whereas the pulsed laser induced pronounced histopathological changes and inhibition of tumor growth. Therefore, we selected an effective regimen for PDT when using the genetically encoded photosensitizer KillerRed and pulsed laser irradiation.

  8. Illuminating the dynamics of signal integration in Natural Killer cells

    Directory of Open Access Journals (Sweden)

    Sophie Victoria Pageon

    2012-10-01

    Full Text Available Natural Killer (NK cell responses are shaped by the integration of signals transduced from multiple activating and inhibitory receptors at their surface. Biochemical and genetic approaches have identified most of the key proteins involved in signal integration but a major challenge remains in understanding how the spatial and temporal dynamics of their interactions lead to NK cells responding appropriately when encountering ligands on target cells. Well over a decade of research using fluorescence microscopy has revealed much about the architecture of the NK cell immune synapse – the structured interface between NK cells and target cells - and how it varies when inhibition or activation is the outcome of signal integration. However, key questions – such as the proximity of individual activating and inhibitory receptors – have remained unanswered because the resolution of optical microscopy has been insufficient, being limited by diffraction. Recent developments in fluorescence microscopy have broken this limit, seeding new opportunities for studying the nanometre-scale organisation of the NK cell immune synapse. Here, we discuss how these new imaging technologies, including super-resolution imaging and other novel light-based methods, can illuminate our understanding of NK cell biology.

  9. Changes in dive behavior during naval sonar exposure in killer whales, long-finned pilot whales, and sperm whales.

    Science.gov (United States)

    Sivle, L D; Kvadsheim, P H; Fahlman, A; Lam, F P A; Tyack, P L; Miller, P J O

    2012-01-01

    Anthropogenic underwater sound in the environment might potentially affect the behavior of marine mammals enough to have an impact on their reproduction and survival. Diving behavior of four killer whales (Orcinus orca), seven long-finned pilot whales (Globicephala melas), and four sperm whales (Physeter macrocephalus) was studied during controlled exposures to naval sonar [low frequency active sonar (LFAS): 1-2 kHz and mid frequency active sonar (MFAS): 6-7 kHz] during three field seasons (2006-2009). Diving behavior was monitored before, during and after sonar exposure using an archival tag placed on the animal with suction cups. The tag recorded the animal's vertical movement, and additional data on horizontal movement and vocalizations were used to determine behavioral modes. Killer whales that were conducting deep dives at sonar onset changed abruptly to shallow diving (ShD) during LFAS, while killer whales conducting deep dives at the onset of MFAS did not alter dive mode. When in ShD mode at sonar onset, killer whales did not change their diving behavior. Pilot and sperm whales performed normal deep dives (NDD) during MFAS exposure. During LFAS exposures, long-finned pilot whales mostly performed fewer deep dives and some sperm whales performed shallower and shorter dives. Acoustic recording data presented previously indicates that deep diving (DD) is associated with feeding. Therefore, the observed changes in dive behavior of the three species could potentially reduce the foraging efficiency of the affected animals.

  10. Pilot whales attracted to killer whale sounds: acoustically-mediated interspecific interactions in cetaceans.

    Directory of Open Access Journals (Sweden)

    Charlotte Curé

    Full Text Available In cetaceans' communities, interactions between individuals of different species are often observed in the wild. Yet, due to methodological and technical challenges very little is known about the mediation of these interactions and their effect on cetaceans' behavior. Killer whales (Orcinus orca are a highly vocal species and can be both food competitors and potential predators of many other cetaceans. Thus, the interception of their vocalizations by unintended cetacean receivers may be particularly important in mediating interspecific interactions. To address this hypothesis, we conducted playbacks of killer whale vocalizations recorded during herring-feeding activity to free-ranging long-finned pilot whales (Globicephala melas. Using a multi-sensor tag, we were able to track the whales and to monitor changes of their movements and social behavior in response to the playbacks. We demonstrated that the playback of killer whale sounds to pilot whales induced a clear increase in group size and a strong attraction of the animals towards the sound source. These findings provide the first experimental evidence that the interception of heterospecific vocalizations can mediate interactions between different cetacean species in previously unrecognized ways.

  11. Current perspectives on natural killer cell education and tolerance: emerging roles for inhibitory receptors

    Directory of Open Access Journals (Sweden)

    Thomas LM

    2015-03-01

    Full Text Available L Michael Thomas Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA Abstract: Natural killer (NK cells are regulated through the coordinated functions of activating and inhibitory receptors. These receptors can act during the initial engagement of an NK cell with a target cell, or in subsequent NK cell engagements to maintain tolerance. Notably, each individual possesses a sizable minority-population of NK cells that are devoid of inhibitory receptors that recognize the surrounding MHC class I (ie, self-MHC. Since these NK cells cannot perform conventional inhibition, they are rendered less responsive through the process of NK cell education (also known as licensing in order to reduce the likelihood of auto-reactivity. This review will delineate current views on NK cell education, clarify various misconceptions about NK cell education, and, lastly, discuss the relevance of NK cell education in anti-cancer therapies. Keywords: natural killer cell education, natural killer cell inhibitory receptors, immunotherapy, cancer

  12. Rat natural killer cell, T cell and macrophage functions after intracerebroventricular injection of SNC 80.

    Science.gov (United States)

    Nowak, J E; Gomez-Flores, R; Calderon, S N; Rice, K C; Weber, R J

    1998-08-01

    We investigated the effects of (+)-4-[(alpha R)-alpha-((2S, 5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N, N-diethylbenzamide (SNC 80), a nonpeptidic delta-opioid receptor-selective agonist, on rat leukocyte functions. Intracerebroventricular injection of SNC 80 (20 nmol) in Fischer 344N male rats did not affect splenic natural killer cell activity compared with intracerebroventricular saline-injected controls. SNC 80 also had no effect on concanavalin A-, anti-T cell receptor-, interleukin-2- and anti-T cell receptor + interleukin-2-induced splenic and thymic lymphocyte proliferation in most experiments. In some experiments, however, SNC 80 significantly (P SNC 80 did not significantly affect splenic T cell or natural killer cell populations as measured by the expression of T cell receptoralphabeta, and T helper (CD4), T suppressor/cytotoxic (CD8) and natural killer cell surface markers. Finally, SNC 80 did not affect interferon-gamma- or lipopolysaccharide (LPS)-induced splenic nitric oxide, and LPS-induced tumor necrosis factor-alpha production by splenic macrophages. These results suggest that SNC 80 could be useful in the treatment of pain without suppressing immune function. However, the potential immunoenhancing properties of SNC 80 may be also valuable in immunocompromised individuals.

  13. Evolution of male-killer suppression in a natural population.

    Directory of Open Access Journals (Sweden)

    Emily A Hornett

    2006-09-01

    Full Text Available Male-killing bacteria are widespread in arthropods, and can profoundly alter the reproductive biology of their host species. Here we detail the first case of complete suppression of a male killer. The nymphalid butterfly Hypolimnas bolina is infected with a strain of the bacterium Wolbachia, wBol1, which kills male host embryos in Polynesian populations, but does not do so in many areas of Southeast Asia, where both males and female adults are naturally infected, and wBol1-infected females produce a 1:1 sex ratio. We demonstrate that absence of male killing by wBol1 is associated with dominant zygotic suppression of the action of the male killer. Simulations demonstrate host suppressors of male-killer action can spread very rapidly, and historical data indicating the presence of male killing in Southeast Asia in the very recent past suggests suppressor spread has been a very recent occurrence. Thus, male killer/host interactions are much more dynamic than previously recognised, with rapid and dramatic loss of the phenotype. Our results also indicate that suppression can render male killers completely quiescent, leading to the conclusion that some species that do not currently express a male killer may have done so in the past, and thus that more species have had their biology affected by these parasites than previously believed.

  14. Macrophage colony-stimulating factor gene transduction into human lung cancer cells differentially regulates metastasis formations in various organ microenvironments of natural killer cell-depleted SCID mice.

    Science.gov (United States)

    Yano, S; Nishioka, Y; Nokihara, H; Sone, S

    1997-02-15

    We investigated whether local production of macrophage colony-stimulating factor (M-CSF), responsible for migration and activation of monocytes/macrophages at a tumor growth site, affected the metastatic pattern of lung cancer. For this, highly metastatic human squamous (RERF-LC-AI) or small (H69/VP) cell lung carcinoma cells were transduced with the human M-CSF gene inserted into pRc/CMV-MCSF to establish M-CSF-producing clones (MCSF-AI-9-18, MCSF-AI-9-24, and MCSF-VP-5). M-CSF gene transduction had no effect on the expression of surface antigen or on in vitro proliferation. After s.c. injection into SCID mice, the growth rates of M-CSF-producing cells were slower than those of parent or mock-transduced cells. In the metastatic model in SCID mice depleted of natural killer cells, RERF-LC-AI cells formed metastases mainly in the liver and kidneys, whereas H69/VP cells metastasized mainly to the liver and systemic lymph nodes. The numbers of metastatic colonies of MCSF-AI-9-18 and MCSF-AI-9-24 cells in the liver but not the kidneys were significantly reduced. The development of lymph node metastases of MCSF-VP-5 cells was also less than that of parent or mock-transduced cells. Treatment of SCID mice with anti-human M-CSF antibody resulted in a significant increase in liver metastases of their M-CSF gene transfectants. No significant differences were observed in the distributions in mice or in the in vitro invasive potentials of MCSF-AI-9-18 cells and Neo-AI-3 cells. These findings indicate that the antimetastatic effect of M-CSF may be specific to particular organs, suggesting the influence of heterogeneity of organ microenvironments on the metastasis of lung cancer.

  15. Transformation of human liver L-02 cells mediated by stable HBx transfection

    Institute of Scientific and Technical Information of China (English)

    Wei-ying ZHANG; Na CAI; Li-hong YE; Xiao-dong ZHANG

    2009-01-01

    Aim: To explore the mechanism of hepatocarcinogenesis associated with the hepatitis B virus X protein (HBx), we investigated the role of HBx in transformation using human liver L-02 cells stably transfected with HBx as a model.Methods: Plasmids encoding HBx were stably transfected into immortalized human liver L-02 cells and rodent fibroblast NIH/3T3 cells. The expression of alfa-fetoprotein (AFP), c-Myc, HBx, and survivin in the engineered cells was examined by Western blotting. The malignant phenotype of the cells was demonstrated by anchorage-independent colony formation and tumor formation in nude mice. RNA interference assays, Western blotting, luciferase reporter gene assays and flow cytometry analysis were performed. The number of centrosomes in the L-O2-X cells was determined by Y-tubulin immunostaining. The effect of HBx on the transcriptional activity of human telomerase reverse transcriptase (hTERT) and hTERT activity in L-02-X cells and/or 3T3-X cells was detected by the luciferase reporter gene assay and telomerase repeat amplification protocol (TRAP).Results: Stable HBx transfection resulted in a malignant phenotype in the engineered cells in vivo and in vitro. Meanwhile, HBx was able to increase the transcription of the NF-κB, AP-1, and survivin genes and to upregulate the expression levels of c-Myc and survivin.Abnormal centrosome duplication and activated hTERT were responsible for the transformation.Conclusion: Stable HBx transfection leads to genomic instability of host cells, which is responsible for hepatocarcinogenesis; mean-while, transactivation by the HBx protein contributes to the development of hepatocellular carcinoma (HCC). The L-02-X cell line is an ideal model for investigating the mechanism of HBx-mediated transformation.

  16. Transient transfection of mammalian cells using a violet diode laser

    Science.gov (United States)

    Torres-Mapa, Maria Leilani; Angus, Liselotte; Ploschner, Martin; Dholakia, Kishan; Gunn-Moore, Frank J.

    2010-07-01

    We demonstrate the first use of the violet diode laser for transient mammalian cell transfection. In contrast to previous studies, which showed the generation of stable cell lines over a few weeks, we develop a methodology to transiently transfect cells with an efficiency of up to ~40%. Chinese hamster ovary (CHO-K1) and human embryonic kidney (HEK293) cells are exposed to a tightly focused 405-nm laser in the presence of plasmid DNA encoding for a mitochondrial targeted red fluorescent protein. We report transfection efficiencies as a function of laser power and exposure time for our system. We also show, for the first time, that a continuous wave laser source can be successfully applied to selective gene silencing experiments using small interfering RNA. This work is a major step towards an inexpensive and portable phototransfection system.

  17. 96-well electroporation method for transfection of mammalian central neurons.

    Science.gov (United States)

    Buchser, William J; Pardinas, Jose R; Shi, Yan; Bixby, John L; Lemmon, Vance P

    2006-11-01

    Manipulating gene expression in primary neurons has been a goal for many scientists for over 20 years. Vertebrate central nervous system neurons are classically difficult to transfect. Most lipid reagents are inefficient and toxic to the cells, and time-consuming methods such as viral infections are often required to obtain better efficiencies. We have developed an efficient method for the transfection of cerebellar granule neurons and hippocampal neurons with standard plasmid vectors. Using 96-well electroporation plates, square-wave pulses can introduce 96 different plasmids into neurons in a single step. The procedure results in greater than 20% transfection efficiencies and requires only simple solutions of nominal cost. In addition to enabling the rapid optimization of experimental protocols with multiple parameters, this procedure enables the use of high content screening methods to characterize neuronal phenotypes.

  18. Evaluation of the magnetic field requirements for nanomagnetic gene transfection

    Directory of Open Access Journals (Sweden)

    A. Fouriki

    2010-07-01

    Full Text Available The objective of this work was to examine the effects of magnet distance (and by proxy, field strength on nanomagnetic transfection efficiency. Methods: non-viral magnetic nanoparticle-based transfection was evaluated using both static and oscillating magnet arrays. Results: Fluorescence intensity (firefly luciferase of transfected H292 cells showed no increase using a 96-well NdFeB magnet array when the magnets were 5 mm from the cell culture plate or nearer. At 6 mm and higher, fluorescence intensity decreased systematically. Conclusion: In all cases, fluorescence intensity was higher when using an oscillating array compared to a static array. For distances closer than 5 mm, the oscillating system also outperformed Lipofectamine 2000™.

  19. Enhanced antitumor effects of tumor antigen-pulsed dendritic cells by their transfection with GM-CSF gene

    Institute of Scientific and Technical Information of China (English)

    曹雪涛; 章卫平; 马施华; 张明徽; 王建莉; 叶天星

    1997-01-01

    To investigate the biological characterization and antitumor activitites of GM-CSF gene-transfected dendritic cells, the splenic dendritic cells were infected with GM-CSF recombinant replication-deficient adenoviruses in vitro . Their enhanced expression of B7 was demonstrated by FACS analysis, and more potent stimulatory activity was confirmed by allogeneic MLR. Immunization of dendritic cells pulsed with irradiated B16 melanoma cells induced sig-nificant CTL and enabled host to resist the challenge of wild-type B16 cells. When they were transfected with GM-CSF gene subsequently, the induced CTL activity was higher, and the produced protection against B16 cell challenge and therapeutic effect on the mice with preestablished pulmonary melastases more effective. These data suggest that the dendritic cells pulsed with tumor antigen then transfected with GM-CSF gene can be used as an effective vaccine in tumor immunotherapy.

  20. Natural killer cell immunotherapy: from bench to bedside

    Directory of Open Access Journals (Sweden)

    Anna eDomogala

    2015-06-01

    Full Text Available The potential of natural Killer (NK cells to target numerous malignancies in vitro has been well documented, however, only limited success has been seen in the clinic. Although NK cells prove non-toxic and safe regardless of the cell numbers injected, there is often little persistence and expansion observed in a patient which is vital for mounting an effective cellular response. NK cells can be isolated directly from peripheral blood (PB, umbilical cord blood (CB or bone marrow (BM, expanded in vitro using cytokines or differentiated in vitro from hematopoietic stem cells (HSC. Drugs that support NK cell function such as lenalidomide and bortezomib have also been studied in the clinic, however, the optimum combination, which can vary amongst different malignancies, is yet to be identified. NK cell proliferation, persistence and function can further be improved by various activation techniques such as priming and cytokine addition though whether stimulation pre or post injection is more favorable is another obstacle to be tackled. Here we review the various methods of obtaining and activating NK cells for use in the clinic whilst considering the ideal product and drug complement for the most successful cellular therapy.

  1. Antigen specificity of invariant natural killer T-cells

    Directory of Open Access Journals (Sweden)

    Alysia M. Birkholz

    2015-12-01

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

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

    Science.gov (United States)

    Birkholz, Alysia M; Kronenberg, Mitchell

    2015-12-01

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

  3. Stimulation of Natural Killer T Cells by Glycolipids

    Directory of Open Access Journals (Sweden)

    Brian L. Anderson

    2013-12-01

    Full Text Available Natural killer T (NKT cells are a subset of T cells that recognize glycolipid antigens presented by the CD1d protein. The initial discovery of immunostimulatory glycolipids from a marine sponge and the T cells that respond to the compounds has led to extensive research by chemists and immunologists to understand how glycolipids are recognized, possible responses by NKT cells, and the structural features of glycolipids necessary for stimulatory activity. The presence of this cell type in humans and most mammals suggests that it plays critical roles in antigen recognition and the interface between innate and adaptive immunity. Both endogenous and exogenous natural antigens for NKT cells have been identified, and it is likely that glycolipid antigens remain to be discovered. Multiple series of structurally varied glycolipids have been synthesized and tested for stimulatory activity. The structural features of glycolipids necessary for NKT cell stimulation are moderately well understood, and designed compounds have proven to be much more potent antigens than their natural counterparts. Nevertheless, control over NKT cell responses by designed glycolipids has not been optimized, and further research will be required to fully reveal the therapeutic potential of this cell type.

  4. Epizone: Interlaboratory Ring Trial to Compare Dna Transfection Efficiencies

    DEFF Research Database (Denmark)

    Dory, Daniel; Albina, Emmanuel; Kwiatek, Olivier

    Chemical-based transfection of DNA into cultured cells is routinely used to study for example viral or cellular gene functions involved in virus replication, to analyse cellular defence mechanisms or develop specific strategies to interfere with virus replication. Other applications include rescue...... of viruses by reverse genetics and/or generation of mutated viruses. A large number of transfection chemicals like calcium phospate, branched organic compounds, liposomes, cationic polymers etc. are available on the market which are used by different laboratories for different cell lines. To obtain...

  5. “It’s Always the Same, and It’s Always Different” Mythologisation and the Serial Killer in Henry: Portrait of a Serial Killer.

    OpenAIRE

    Smyth, David A.

    2015-01-01

    Serial killers are important in American horror because of their ability to exist between ‘myth’ and ‘reality’. The serial killer is one of the most important American myths, but it is one firmly rooted in real life: unlike Paul Bunyan or Superman, serial killers do exist. This essay examines the relationship between the ‘myth’ and the ‘reality’ of serial killers, and the complex relationship between the American public and the serial killer, using Henry: Portrait of a Serial K...

  6. Octaarginine-modified chitosan as a nonviral gene delivery vector: properties and in vitro transfection efficiency

    Science.gov (United States)

    Zhao, Xiaoli; Li, Zhaoyang; Liu, Wenguang; Lam, Wingmoon; Sun, Peng; Kao, Richard Y. T.; Luk, Keith D. K.; Lu, William W.

    2011-02-01

    Protein transduction domains (PTD) have been identified to have the capacity to facilitate molecular cargo to translocate through cell membrane. This study aims to utilize the cell membrane penetrating ability of octaarginine oligopeptide, a simplified prototype of the PTD, to enhance the transfection efficiency of chitosan. Octaarginine-modified chitosan (R8-CS) was synthesized as a gene transfer carrier by carbodiimide chemistry. The structure and composition of R8-CSs were characterized using FTIR and 1H NMR. Agarose gel electrophoresis assay showed that R8-CS could efficiently condense the DNA. The particle size of R8-CS/DNA complexes were determined to be around 100-200 nm. The nanoparticle complexes exhibited a spherical and compact morphology. R8-CS demonstrated higher transfection activity and lower cytotoxicity as compared to the unmodified chitosan and also showed good serum resistance.

  7. A simple, highly efficient method for heterologous expression in mammalian primary neurons using cationic lipid-mediated mRNA transfection

    Directory of Open Access Journals (Sweden)

    Damian J Williams

    2010-11-01

    Full Text Available Expression of heterologous proteins in adult mammalian neurons is a valuable technique for the study of neuronal function. The postmitotic nature of mature neurons prevents effective DNA transfection using simple, cationic lipid-based methods. Adequate heterologous protein expression is often only achievable using complex techniques that, in many cases, are associated with substantial toxicity. Here, a simple method for high efficiency transfection of mammalian primary neurons using in vitro-transcribed mRNA and the cationic lipid transfection reagent Lipofectamine 2000 is described. Optimal transfection conditions were established in adult mouse dissociated dorsal root ganglion (DRG neurons using a 96-well based luciferase activity assay. Using these conditions, a transfection efficiency of 25% was achieved in DRG neurons transfected with EGFP mRNA. High transfection efficiencies were also obtained in dissociated rat superior cervical ganglion (SCG neurons and mouse cortical and hippocampal cultures. Endogenous Ca2+ currents in EGFP mRNA-transfected SCG neurons were not significantly different from untransfected neurons, which suggested that this technique is well suited for heterologous expression in patch clamp recording experiments. Functional expression of a cannabinoid receptor (CB1R, a G protein inwardly-rectifying K+ channel (GIRK4 and a dominant-negative G protein α-subunit mutant (GoA G203T indicate that the levels of heterologous protein expression attainable using mRNA transfection are suitable for most functional protein studies. This study demonstrates that mRNA transfection is a straightforward and effective method for heterologous expression in neurons and is likely to have many applications in neuroscience research.

  8. Alloreactive natural killer cells for the treatment of acute myeloid leukemia: from stem cell transplantation to adoptive immunotherapy

    Directory of Open Access Journals (Sweden)

    Loredana eRuggeri

    2015-10-01

    Full Text Available Natural killer cells express activating and inhibitory receptors which recognize MHC class I alleles, termed Killer cell Immunoglobulin-like Receptors (KIRs. Preclinical and clinical data from haploidentical T-cell depleted stem cell transplantation have demonstrated that alloreactive KIR-L mismatched natural killer cells play a major role as effectors against acute myeloid leukemia. Outside the transplantation setting, several reports have proven the safety and feasibility of natural killer cell infusion in acute myeloid leukemia patients and, in some cases, provided evidence that transferred NK cells are functionally alloreactive and may have a role in disease control. Aim of the present work is to briefly summarize the most recent advances in the field by moving from the first preclinical and clinical demonstration of donor NK alloreactivity in the transplantation setting to the most recent attempts of exploiting the use of alloreactive NK cell infusion as a means of adoptive immunotherapy against acute myeloid leukemia. Altogether, these data highlight the pivotal role of NK cells for the development of novel immunological approaches in the clinical management of acute myeloid leukemia.

  9. Enhanced photo-transfection efficiency of mammalian cells on graphene coated substrates

    Science.gov (United States)

    Mthunzi, Patience; He, Kuang; Ngcobo, Sandile; Warner, Jamie W.

    2014-03-01

    Literature reports graphene, an atomic-thick sheet of carbon atoms as one of the promising biocompatible scaffolds that promotes cellular proliferation in human mesenchymal stem cells. On the other hand, different mammalian cell lines including the induced pluripotent stem cells exhibited an accelerated proliferation rate when cultured on graphene or graphene oxide coated substrates. These findings provide strong motivation to explore the full capability of graphene in further pluripotent stem cell research activities as there exists an urgent requirement to preserve their therapeutic potential. This therefore calls for non-invasive procedures for handling stem cells in-vitro. For example, resent literature has shown successful laser light driven transfection in both multipotent and pluripotent stem cells. In order to explore the non-invasive nature of optical transfection alongside biocompatible qualities of graphene, in this work we investigated the impact of optically transfecting mouse embryonic stem (mES) cells plated on graphene coated sample chambers. Using Chinese Hamster Ovary cells (CHO-K1), we further studied the influence of graphene on cell viability as well as cell cytotoxicity through assessing changes in levels of mitochondrial adenosine triphosphate (ATP) activity and the release of cytosolic lactate dehydrogenase (LHD) respectively. Our results showed that compared to those treated on plain glass, CHO-K1 cells optically treated while plated on graphene coated substrates exhibited a higher production of ATP and a milder release of LDH. In addition there was enhanced photo-transfection efficiency in both CHO-K1 and mES cells irradiated on graphene sample chambers.

  10. Transtornos de personalidade, psicopatia e serial killers Personality disorders, psychopathy and serial killers

    Directory of Open Access Journals (Sweden)

    Hilda C P Morana

    2006-10-01

    Full Text Available OBJETIVO: Apresentar as características básicas dos diversos transtornos específicos de personalidade, mas centrando-se no transtorno de personalidade anti-social, fazendo sua diferenciação com psicopatia. O estudo ainda se propõe a abordar a figura do serial killer, apontando a presença de aspectos psicopáticos no homicídio seriado. MÉTODO: Uma revisão bibliográfica foi feita no sentido de se abordar convergências e divergências entre diversos autores sobre um assunto tão polêmico, sobretudo quanto à viabilidade de tratamento dessa clientela forense. RESULTADOS: Enquanto o transtorno de personalidade anti-social é um diagnóstico médico, pode-se entender o termo "psicopatia", pertencente à esfera psiquiátrico-forense, como um "diagnóstico legal". Não se pode falar ainda de tratamento eficaz para os chamados "serial killers". CONCLUSÃO: Os transtornos de personalidade, especialmente o tipo anti-social, representam ainda hoje um verdadeiro desafio para a psiquiatria forense. O local mais adequado e justo para seus portadores, bem como recomendação homogênea e padronizada de tratamento são questões ainda não respondidas.OBJECTIVE: To illustrate the basic characteristics of several specific personality disorders, focusing mainly in antisocial personality disorder. The differences between antisocial personality disorder and psychopathy are highlighted. Serial killers and its psychopathic aspects are also discussed. METHOD: A bibliographic review was completed in order to outline convergences and divergences among different authors about this controversial issue, especially those concerning the possibility of treatment. RESULTS: While anti-social personality disorder is a medical diagnosis, the term "psychopathy" (which belongs to the sphere of forensic psychiatry may be understood as a "legal diagnosis". It is not still possible to identify an effective treatment for serial killers. CONCLUSION: Personality disorders

  11. Transfection of Eimeria mitis with yellow fluorescent protein as reporter and the endogenous development of the transgenic parasite.

    Directory of Open Access Journals (Sweden)

    Mei Qin

    Full Text Available BACKGROUND: Advancements have been made in the genetic manipulation of apicomplexan parasites. Both the in vitro transient and in vivo stable transfection of Eimeria tenella have been developed successfully. Herein, we report the transient and stable transfection of Eimeria mitis. METHODS AND FINDINGS: Sporozoites of E. mitis transfected with enhanced yellow fluorescent protein (EYFP expression plasmid were inoculated into chickens via the cloacal route. The recovered fluorescent oocysts were sorted by fluorescence activated cell sorting (FACS and then passaged 6 generations successively in chickens. The resulting population was analyzed by genome walking and Western blot. The endogenous development of the transgenic E. mitis was observed and its reproduction potential was tested. The stable transfection of E. mitis was developed. Genome walking confirmed the random integration of plasmid DNA into the genome; while Western blot analysis demonstrated the expression of foreign proteins. Constitutive expression of EYFP was observed in all stages of merogony, gametogony and sporogony. The peak of the transgenic oocyst output was delayed by 24 h and the total oocyst reproduction was reduced by 7-fold when compared to the parental strain. CONCLUSION: Stable transfection of E. mitis was successfully developed. The expression of foreign antigens in the transgenic parasites will facilitate the development of transgenic E. mitis as a vaccine vector.

  12. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    Science.gov (United States)

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination.

  13. Cell transfection as a tool to study growth hormone action

    DEFF Research Database (Denmark)

    Norstedt, G; Enberg, B; Francis, S;

    1994-01-01

    The isolation of growth hormone receptor (GHR) cDNA clones has made possible the transfection of GHRs into cultured cells. Our aim in this minireview is to show how the application of such approaches have benefited GHR research. GH stimulation of cells expressing GHR cDNAs can cause an alteration...

  14. D-glucosamine promotes transfection efficiency during electroporation.

    Science.gov (United States)

    Igawa, Kazunari; Ohara, Naoko; Kawakubo, Atsushi; Sugimoto, Kouji; Yanagiguchi, Kajiro; Ikeda, Takeshi; Yamada, Shizuka; Hayashi, Yoshihiko

    2014-01-01

    D-Glucosamine is a useful medicament in various fields of medicine and dentistry. With respect to stability of the cell membrane, it has been reported that bradykinin-induced nociceptive responses are significantly suppressed by the direct application of D-glucosamine. Electroporation is usually used to effectively introduce foreign genes into tissue culture cells. Buffers for electroporation with or without D-glucosamine are used in experiments of transfection vectors. This is the first study to indirectly observe the stability and protection of the osteoblast membrane against both electric stress and gene uptake (the proton sponge hypothesis: osmotic rupture during endosomes prior to fusion with lysosomes) in electroporation with D-glucosamine application. The transfection efficiency was evaluated as the fluorescence intensity of the transfected green fluorescent protein (GFP) in the cultured cells (osteoblasts; NOS-1 cells). The transfection efficiency increased over 30% in the electroporation samples treated with D-glucosamine-supplemented buffer after one day. The membrane absorption of D-glucosamine is the primary mechanism of membrane stress induced by electric stress. This new function of D-glucosamine is useful and meaningful for developing more effective transformation procedures.

  15. Small Interfering RNA Transfection Across a Phospholipid Membrane

    Science.gov (United States)

    Ngo, Van; Choubey, Amit; Kalia, Rajiv; Nakano, Aiichiro; Vashishta, Priya

    2012-02-01

    Small interfering RNA (siRNA) molecules play a pivotal role in silencing gene expression via the RNA interference mechanism. We have performed steered MD simulations to study the transfection of a bare siRNA and siRNA/Oleic Acid (OA) complex across the dipalmitoylphosphatidycholine (DPPC) bilayer at T = 323 K. Bare siRNA induces the formation of frustrated lipid gel domains, whereas in the presence of siRNA/OA complex the membrane is found to be in the liquid-ordered phase. In both cases the stress profiles across the membrane indicate that the membrane is under tension near the head groups and highly compressed at the water-hydrophobic interface. During transfection, the membrane is deformed and the lateral stress is significantly lowered for the bare siRNA and siRNA/OA complex. The bare siRNA transfects through a lipid-nanopore of hydrophilic head-groups and hydrophobic carbon chains, whereas the siRNA/OA complex transfects through a lipid-nanopore of hydrophilic head groups.

  16. Cutaneous natural killer/T-cell lymphoma.

    Science.gov (United States)

    Radonich, Michael A; Lazova, Rossitza; Bolognia, Jean

    2002-03-01

    Lymphomas are classified as either Hodgkin's or non-Hodgkin's. The 2 subtypes of non-Hodgkin's lymphoma that can present primarily in the skin are cutaneous T-cell lymphoma and cutaneous B-cell lymphoma, both of which tend to be low-grade malignant neoplasms. Recently another distinct subtype of lymphoma was discovered, the natural killer (NK)/T-cell lymphoma, which can involve the skin in a primary or secondary fashion. The NK/T-cell subtype of lymphoma is characterized by the expression of the NK-cell antigen CD56. These CD56(+) lymphomas are further subdivided into nasal NK/T-cell lymphomas that commonly present as midfacial destructive disease and non-nasal NK/T-cell lymphomas that often arise in extranodal locations, including the skin. We report a case of aggressive NK-cell leukemia/lymphoma with numerous secondary cutaneous lesions and review the clinical and histopathologic spectrum of non-nasal CD56(+) lymphomas, with an emphasis on the dermatologic findings.

  17. Molecular Programming of Immunological Memory in Natural Killer Cells.

    Science.gov (United States)

    Beaulieu, Aimee M; Madera, Sharline; Sun, Joseph C

    2015-01-01

    Immunological memory is a hallmark of the adaptive immune system. Although natural killer (NK) cells have traditionally been classified as a component of the innate immune system, they have recently been shown in mice and humans to exhibit certain features of immunological memory, including an ability to undergo a clonal-like expansion during virus infection, generate long-lived progeny (i.e. memory cells), and mediate recall responses against previously encountered pathogens--all characteristics previously ascribed only to adaptive immune responses by B and T cells in mammals. To date, the molecular events that govern the generation of NK cell memory are not completely understood. Using a mouse model of cytomegalovirus infection, we demonstrate that individual pro-inflammatory IL-12, IL-18, and type I-IFN signaling pathways are indispensible and play non-redundant roles in the generation of virus-specific NK cell memory. Furthermore, we discovered that antigen-specific proliferation and protection by NK cells is mediated by the transcription factor Zbtb32, which is induced by pro-inflammatory cytokines and promotes a cell cycle program in activated NK cells. A greater understanding of the molecular mechanisms controlling NK cell responses will provide novel strategies for tailoring vaccines to target infectious disease.

  18. THE KINETICS OF CYTOPLASMIC GRANULE SECRETION IN NATURAL KILLER CYTOTOXICITY

    Institute of Scientific and Technical Information of China (English)

    龚伊红; R.R.Hcrberman; C.W.Reynolds

    1994-01-01

    Antisexum against purified cytoplasmic granules from rat LGL tumor cells, and protein A-gold inmmnoelec-tron microscopy were used to study the secretory events in lysis of YAC-1 tumor cells by rat LGL tumor cells or by isolated LGL from normal rats. After 30 min incubation of effector and target cells together, gold-labeled cyto-plasmic granules were often seen concentrated in the area of the LGL adjacent to the ~ YAC-1 Within 60min,the grantees were observed to move to the cell border near the conjugazed site. At this point, fine granules were fused with file cell membrane, and subsequently released file gold-labeled contents into the junction between the LGL and the target cell. Gold particles could be seen at the B-T interface, on the surface,or sometimes on the target cell surface.These data provide direct evidence for the hypothesis that under conditions of active cytotoxicity,natural killer cells secrete their cytoplasmic granule contents leading to the deposition of granule material on the target cell surface and the eventual lysis of the cell.

  19. [SEX HORMONE INFLUENCE ON PERIPHERAL NATURAL KILLER CELLS COUNT].

    Science.gov (United States)

    Ivanov, P; Konova, E; Blajeva, Sv; Lukanov, Tsv; Angelova, P; Georgieva, V; Totev, V; Komsa-Penkova, R

    2015-01-01

    Proper evaluation of immunological factors connected with pregnancy establishment increased the possibility for exact treatment in high risk gestation cases. Hormonal changes during an ovarian cycle may affect immune response, which is crucial for the embryonic implantation. Peripheral Natural killer (pNK) cells are key components of immune systems and their activities could be regulated by sex hormones. In the present study we investigated the effects of estrogen fluctuation on the number of NK cells in vivo during the early follicular and middle luteal phase of menstrual cycle. In 63 healthy women with at least one full term pregnancy and regular menstrual cycle with duration between 24 and 32 days, blood samples have been collected twice for investigation of CD3/CD16/CD56 positive lymphocytes. The mean pNK count in follicular phase was 11.6% with 4.7% variation. The median was 10.6%. The mean pNK count in luteal phase was 12.1% with 5.1% variation, respectively median for cell number 11.8%. The two-tailed t-test comparison did not find any statistical difference despite the slight elevation of pNK cells count in luteal phase. The insignificant variation in pNK cells count objected the suggestion to evaluate immunological status in women with adverse pregnancy outcome in specific phase of menstrual cycle.

  20. Processing of pro-CGRP in a rat medullary thyroid carcinoma cell line transfected with protease inhibitors

    DEFF Research Database (Denmark)

    Johansen, Teit Eliot; Schifter, S; Vogel, Charlotte Katrine;

    1991-01-01

    A rat medullary thyroid carcinoma cell line, CA77, was used to study the effect of a series of biosynthesized protease inhibitors on the proteolytic cleavage of the endogenously synthesized pro-CGRP. This cell line efficiently converted the pro-CGRP to mature CGRP as assessed by chromatography...... of cell extracts followed by radioimmunoassay for CGRP. CA77 cells were transfected with expression vectors encoding protease inhibitors: the Arg-serpins, alpha 1-antitrypsin Pittsburgh (358 Met----Arg) and plasminogen activator inhibitor 1, the Kazal type serine protease inhibitor, pancreatic secretory...... trypsin inhibitor, and the general thiol protease inhibitor, cystatin C. Only the chromatography of cell extracts from CA77 cells transfected with a plasmid encoding cystatin C showed an apparent higher content of unprocessed pro-CGRP as compared to non-transfected cells. No effect on pro-CGRP processing...

  1. Chemical transfection of dye-conjugated microRNA precursors for microRNA functional analysis of M2 macrophages.

    Science.gov (United States)

    Ng, Yee Seng; Roca, Hernan; Fuller, David; Sud, Sudha; Pienta, Kenneth J

    2012-05-01

    MicroRNAs (miRNAs) are short noncoding ribonucleic acids known to affect gene expression at the translational level and there is mounting evidence that miRNAs play a role in the function of tumor-associated macrophages (TAMs). To aid the functional analyses of miRNAs in an in-vitro model of TAMs known as M2 macrophages, a transfection method to introduce artificial miRNA constructs or miRNA molecules into primary human monocytes is needed. Unlike differentiated macrophages or dendritic cells, undifferentiated primary human monocytes have been known to show resistance to lentiviral transduction. To circumvent this challenge, other techniques such as electroporation and chemical transfection have been used in other applications to deliver small gene constructs into human monocytes. To date, no studies have compared these two methods objectively to evaluate their suitability in the miRNA functional analysis of M2 macrophages. Of the methods tested, the electroporation of miRNA-construct containing plasmids and the chemical transfection of miRNA precursor molecules are the most efficient approaches. The use of a silencer siRNA labeling kit (Ambion) to conjugate Cy 3 fluorescence dyes to the precursor molecules allowed the isolation of successfully transfected cells with fluorescence-activated cell sorting. The chemical transfection of these dye-conjugated miRNA precursors yield an efficiency of 37.5 ± 0.6% and a cell viability of 74 ± 1%. RNA purified from the isolated cells demonstrated good quality, and was fit for subsequent mRNA expression qPCR analysis. While electroporation of plasmids containing miRNA constructs yield transfection efficiencies comparable to chemical transfection of miRNA precursors, these electroporated primary monocytes seemed to have lost their potential for differentiation. Among the most common methods of transfection, the chemical transfection of dye-conjugated miRNA precursors was determined to be the best-suited approach for the

  2. RAT GDNF GENE TRANSFECTION AND EXPRESSION OF ITS mRNA AND PROTEIN IN SCHWANN CELLS

    Institute of Scientific and Technical Information of China (English)

    平萍; 范志宏; 李青峰; 张涤生

    2004-01-01

    Objective To investigate the possibility of the transfection of glial-cell line derived neurotrophic factor (GDNF) gene into Schwann cells(SCs). Methods SCs cultures from sciatic nerves of neonatal rats were established. A recombinant retrovirus vector containing GDNF gene was constructed and transferred into SCs.Expression levels of GDNF mRNA and protein were respectively identified with reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry. Determination of GDNF synthesis rates from Retro. pLNCX2-GDNF-transduced SCs (GDNF-SCs) in vitro by enzyme-linked immunoassay sensitive assay ( ELISA ). Biololgical activity of conditioned medium from GENF-SCs was analysed by co-culture with rat motoneurons. Results Transfection of GDNF gene into SCs lead to significantly enhanced expression of GDNF mRNA and protein. The rate of GDNF secreted by GDNF-SCs was also enhanced(5. 1-fold), and more motoneurons survived co-cultured with conditioned medium of GNDF-SCs than with that of normal SCs. Conclusion GNDF gene transfection may be a better way to graft SCs promoting regeneration and repairing demyelination in PNS and CNS.

  3. Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells

    Science.gov (United States)

    Zhao, Chun-Peng; Guo, Xiao; Chen, Si-Jia; Li, Chang-Zheng; Yang, Yun; Zhang, Jun-He; Chen, Shao-Nan; Jia, Yan-Long; Wang, Tian-Yun

    2017-01-01

    Matrix attachment regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human β-interferon and β-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian virus (SV) 40 promoter. These were transfected into CHO-K1 cells, which were screened with geneticin; eGFP expression was detected by flow cytometry. The presence of MAR elements increased transfection efficiency and transient and stably expression of eGFP expression under both promoters; the level was higher when the two MARs differed (i.e., iMAR and gMAR) under the CMV but not the SV40 promoter. For the latter, two gMARs showed the highest activity. We also found that MARs increased the ratio of stably transfected positive colonies. These results indicate that combining the CMV promoter with two different MAR elements or the SV40 promoter with two gMARs is effective for inducing high expression level and stability of transgenes. PMID:28216629

  4. Ex vivo generated natural killer cells acquire typical natural killer receptors and display a cytotoxic gene expression profile similar to peripheral blood natural killer cells

    NARCIS (Netherlands)

    Lehmann, D.; Spanholtz, J.; Osl, M.; Tordoir, M.; Lipnik, K.; Bilban, M.; Schlechta, B.; Dolstra, H.; Hofer, E.

    2012-01-01

    Ex vivo differentiation systems of natural killer (NK) cells from CD34+ hematopoietic stem cells are of potential importance for adjuvant immunotherapy of cancer. Here, we analyzed ex vivo differentiation of NK cells from cord blood-derived CD34+ stem cells by gene expression profiling, real-time RT

  5. SRKW seasonal occurence - Patterns of seasonal occurrence of Southern Resident Killer Whales

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Patterns of seasonal occurrence of Southern Resident Killer Whales (SRKW) throughout their range. Southern Resident Killer Whales are listed as a Distinct Population...

  6. Granule-Dependent Natural Killer Cell Cytotoxicity to Fungal Pathogens

    Science.gov (United States)

    Ogbomo, Henry; Mody, Christopher H.

    2017-01-01

    Natural killer (NK) cells kill or inhibit the growth of a number of fungi including Cryptococcus, Candida, Aspergillus, Rhizopus, and Paracoccidioides. Although many fungi are not dangerous, invasive fungal pathogens, such as Cryptococcus neoformans, cause life-threatening disease in individuals with impaired cell-mediated immunity. While there are similarities to cell-mediated killing of tumor cells, there are also important differences. Similar to tumor killing, NK cells directly kill fungi in a receptor-mediated and cytotoxic granule-dependent manner. Unlike tumor cell killing where multiple NK cell-activating receptors cooperate and signal events that mediate cytotoxicity, only the NKp30 receptor has been described to mediate signaling events that trigger the NK cell to mobilize its cytolytic payload to the site of interaction with C. neoformans and Candida albicans, subsequently leading to granule exocytosis and fungal killing. More recently, the NKp46 receptor was reported to bind Candida glabrata adhesins Epa1, 6, and 7 and directly mediate fungal clearance. A number of unanswered questions remain. For example, is only one NK cell-activating receptor sufficient for signaling leading to fungal killing? Are the signaling pathways activated by fungi similar to those activated by tumor cells during NK cell killing? How do the cytolytic granules traffic to the site of interaction with fungi, and how does this process compare with tumor killing? Recent insights into receptor use, intracellular signaling and cytolytic granule trafficking during NK cell-mediated fungal killing will be compared to tumor killing, and the implications for therapeutic approaches will be discussed. PMID:28123389

  7. Phytoestrogens induce differential estrogen receptor alpha- or Beta-mediated responses in transfected breast cancer cells.

    Science.gov (United States)

    Harris, D M; Besselink, E; Henning, S M; Go, V L W; Heber, D

    2005-09-01

    Increased intake of phytoestrogens may be associated with a lower risk of cancer in the breast and several other sites, although there is controversy surrounding this activity. One of the mechanisms proposed to explain the activity of phytoestrogens is their ability to bind and activate human estrogen receptor alpha (ERalpha) and human estrogen receptor beta (ERbeta). Nine phytoestrogens were tested for their ability to transactivate ERalpha or ERbeta at a range of doses. Mammary adenocarcinoma (MCF-7) cells were co-transfected with either ERalpha or ERbeta, and an estrogen-response element was linked to a luciferase reporter gene. Dose-dependent responses were compared with the endogenous ligand 17beta-estradiol. Purified genistein, daidzein, apigenin, and coumestrol showed differential and robust transactivation of ERalpha- and ERbeta-induced transcription, with an up to 100-fold stronger activation of ERbeta. Equol, naringenin, and kaempferol were weaker agonists. When activity was evaluated against a background of 0.5 nM 17beta-estradiol, the addition of genistein, daidzein, and resveratrol superstimulated the system, while kaempferol and quercetin were antagonists at the highest doses. This transfection assay provides an excellent model to evaluate the activation of ERalpha and ERbeta by different phytoestrogens in a breast cancer context and can be used as a screening bioassay tool to evaluate the estrogenic activity of extracts of herbs and foods.

  8. The influence of physicochemical parameters on the efficacy of non-viral DNA transfection complexes : A comparative study

    NARCIS (Netherlands)

    Kneuer, Carsten; Ehrhardt, Carsten; Bakowsky, Heike; Kumar, M. N. V. Ravi; Oberle, Volker; Lehr, Claus M.; Hoekstra, Dick; Bakowsky, Udo

    2006-01-01

    Various polycationic vehicles have been developed to facilitate the transfer of foreign DNA into mammalian cells. Structure-activity studies suggested that biophysical properties, such as size, charge, and morphology of the resulting DNA complexes determine transfection efficiency within one class o

  9. Transfection effect of microbubbles on cells in superposed ultrasound waves and behavior of cavitation bubble.

    Science.gov (United States)

    Kodama, Tetsuya; Tomita, Yukio; Koshiyama, Ken-Ichiro; Blomley, Martin J K

    2006-06-01

    The combination of ultrasound and ultrasound contrast agents (UCAs) is able to induce transient membrane permeability leading to direct delivery of exogenous molecules into cells. Cavitation bubbles are believed to be involved in the membrane permeability; however, the detailed mechanism is still unknown. In the present study, the effects of ultrasound and the UCAs, Optison on transfection in vitro for different medium heights and the related dynamic behaviors of cavitation bubbles were investigated. Cultured CHO-E cells mixed with reporter genes (luciferase or beta-gal plasmid DNA) and UCAs were exposed to 1 MHz ultrasound in 24-well plates. Ultrasound was applied from the bottom of the well and reflected at the free surface of the medium, resulting in the superposition of ultrasound waves within the well. Cells cultured on the bottom of 24-well plates were located near the first node (displacement node) of the incident ultrasound downstream. Transfection activity was a function determined with the height of the medium (wave traveling distance), as well as the concentration of UCAs and the exposure time was also determined with the concentration of UCAs and the exposure duration. Survival fraction was determined by MTT assay, also changes with these values in the reverse pattern compared with luciferase activity. With shallow medium height, high transfection efficacy and high survival fraction were obtained at a low concentration of UCAs. In addition, capillary waves and subsequent atomized particles became significant as the medium height decreased. These phenomena suggested cavitation bubbles were being generated in the medium. To determine the effect of UCAs on bubble generation, we repeated the experiments using crushed heat-treated Optison solution instead of the standard microbubble preparation. The transfection ratio and survival fraction showed no additional benefit when ultrasound was used. These results suggested that cavitation bubbles created by the

  10. 50 CFR 226.206 - Critical habitat for the Southern Resident killer whale (Orcinus orca).

    Science.gov (United States)

    2010-10-01

    ... killer whale (Orcinus orca). 226.206 Section 226.206 Wildlife and Fisheries NATIONAL MARINE FISHERIES... CRITICAL HABITAT § 226.206 Critical habitat for the Southern Resident killer whale (Orcinus orca). Critical habitat is designated for the Southern Resident killer whale as described in this section. The...

  11. File list: DNS.Bld.20.AllAg.Natural_Killer_Cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. File list: Unc.Bld.20.AllAg.Natural_Killer_Cells [Chip-atlas[Archive

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  18. File list: DNS.Bld.10.AllAg.Natural_Killer_Cells [Chip-atlas[Archive

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  13. A Danish killer amendment-when judicial review was banned from the 1849 Constitution

    DEFF Research Database (Denmark)

    Pedersen, M. N.

    2014-01-01

    In real political life "killer amendments" are very rare. William H. Riker was the first political scientist to draw systematic attention to this special "heresthetic" phenomenon, but he was himself only able to identify a handful of successful "killer amendments". Subsequent systematic empirical...... Constitution. The motion was defeated by means of what looks like a nicely orchestrated "killer amendment"....

  14. 腺病毒介导G250基因转染树突状细胞激活免疫效应细胞治疗肾癌的实验研究%Experimental study of transfecting adenovirus carrying G250 antigen gene to dendritic cells and to activate immune effector cells in kidney cancer treatment

    Institute of Scientific and Technical Information of China (English)

    齐桓; 郑少斌

    2010-01-01

    Objective To investigate T cell-mediated effects of dendritic cells(DC)transfected with kidney associated antigen G250 gene using adenovirus(Ad)as vector in the treatment of kidney cancer. Methods Peripheral blood mononuclear cells(PBMC)were isolated by standard FicollPaque density gradient centrifugation of heparinized blood obtained from healthy donors.After removal of the nonadherent ceils,the adherent cells were divided into gene transfer group,protein pulsed group and control group.The gene transfer group was infected with Ad/G250 virus;the protein pulsed group was added with G250 protein; the control group was added with PBS.The DCs were cultured and proliferated in vitro using recombinant human granulocyte macrophage colony stimulating factor and inlerleukin-4.The autologous T cells were added into the 3 DC groups(responders:dendritics,20:1).After 5 d co-culture,3 eytotoxic T lymphocyte(CTL)groups were collected.TheG250 mRNA expression was detected in transduced DCs using RT-PCR.The G250 protein translation products and cell surface marker of DCs were analyzed by flow cytometry.The specific cytolytic activities of CTL to different target cells(renal carcinoma 786-0 cell line and lung cancer A549 cell line)were assessed by MTT method. Results Ad/G250 transfected DC successfully and the G250 expression was confirmed by RT-PCR. The expression levels of CD_(80), CD_(83). HLA-DR and CD_(86) in the Ad/G250 group were higher than those in the other 2 groups. The cytotoxicity to 786-0 of T lymphocytes activated by Ad/G250 transfecting DCs(83. 4±2. 8)% was greater than those of T lymphocytes in the protein pulsed group(79. 6±2. 4)% and control group(77. 3±2. 1)%. There were no significant differences of the cytotyxicities to A549 among the 3 cytotoxic T lymphocyte groups(F=0. 373.P=0.693). Conclusions Ad-loading DCs may be useful for immunotherapeutic protocols against self-antigens for the kidney cancer.%目的 探讨以腺病毒(Ad)载

  15. Tumor priming enhances siRNA delivery and transfection in intraperitoneal tumors.

    Science.gov (United States)

    Wang, Jie; Lu, Ze; Yeung, Bertrand Z; Wientjes, M Guillaume; Cole, David J; Au, Jessie L-S

    2014-03-28

    Cancers originating from the digestive system account for 290,000 or ~20% of all new cancer cases annually in the US. We previously developed paclitaxel-loaded tumor-penetrating microparticles (TPM) for intraperitoneal (IP) treatment of peritoneal tumors (Lu et al., 2008; Tsai et al., 2007; Tsai et al., 2013). TPM is undergoing NIH-supported IND-enabling studies for clinical evaluation. The present study evaluated the hypothesis that TPM, via inducing apoptosis and expanding the interstitial space, promotes the delivery and transfection of lipid vectors containing siRNA. The in vivo model was the metastatic human Hs766T pancreatic tumor that, upon IP injection, produced widely distributed solid tumors and ascites in the peritoneal cavity in 100% of animals. The target gene was survivin, an anti-apoptotic protein induced by chemotherapy and associated with metastases and poor prognosis of patients with gastric and colorectal cancers. The siRNA carrier was pegylated liposomes comprising cationic and neutral lipids plus a fusogenic lipid (PCat). PCat-loaded with survivin siRNA (PCat-siSurvivin) was active in cultured cells (decreased survivin mRNA and protein levels, reduced cell clonogenicity, enhanced paclitaxel activity), but lost its activity in vivo; this difference is consistent with the well-known problem of inadequate delivery and transfection of siRNA in vivo. In comparison, single agent TPM prolonged animal survival and, as expected, induced survivin expression in tumors. Addition of PCat-siSurvivin reversed the TPM-induced survivin expression and enhanced the antitumor activity of TPM. The finding that in vivo survivin knockdown by PCat-siSurvivin was successful only when it was given in combination with TPM provides the proof-of-concept that tumor priming promotes the delivery and transfection of liposomal siRNA. The data further suggest the TPM/PCat-siSurvivin combination as a potentially useful chemo-gene therapy for peritoneal cancer.

  16. Contact-dependent Stimulation and Inhibition of Dendritic Cells by Natural Killer Cells

    OpenAIRE

    Piccioli, Diego; Sbrana, Silverio; Melandri, Emiliano; Valiante, Nicholas M.

    2002-01-01

    Natural killer (NK) cells and dendritic cells (DCs) are two distinct cell types of innate immunity. It is known that the in vitro interaction of human NK cells with autologous DCs results in DC lysis. Here we show that contact-dependent interactions between activated human NK cells and immature DCs (iDCs) provides a “control switch” for the immune system. At low NK/DC ratios, this interaction dramatically amplifies DC responses, whereas at high ratios it completely turns off their responses. ...

  17. Role of Natural Killer T Cells In Immunogenic Chemotherapy for Breast Cancer

    Science.gov (United States)

    2012-09-01

    28–38. 5. Godfrey, D. I., and M. Kronenberg . 2004. Going both ways: immune regulation via CD1d-dependent NKT cells. J. Clin. Invest. 114: 1379–1388. 6...Hong, D. C. Scherer, I. Serizawa, N. Burdin, M. Kronenberg , Y. Koezuka, and L. Van Kaer. 1999. Cutting edge: activation of NK T cells by CD1d and a...Y. Kinjo, and M. Kronenberg . 2007. The unique role of natural killer T cells in the response to microorganisms. Nat. Rev. Microbiol. 5: 405– 417. 45

  18. Progesterone induces cellular differentiation in MDA-MB-231 breast cancer cells transfected with progesterone receptor complementary DNA.

    Science.gov (United States)

    Lin, Valerie Chun-Ling; Jin, Rongxian; Tan, Puay-Hoon; Aw, Swee-Eng; Woon, Chow-Thai; Bay, Boon-Huat

    2003-06-01

    Progesterone is an important regulator of growth and differentiation in breast tissues. In this study, the effect of progesterone on cell differentiation was evaluated in the estrogen receptor-negative and progesterone receptor (PR)-negative MDA-MB-231 cell line which was transfected with PR-complementary DNA. Morphological changes were analyzed at the ultrastructural level by scanning and transmission electron microscopy. Progesterone-treated PR-transfected cells exhibited a more protracted and well spread morphology with an increase in organelles such as mitochondria and rough endoplasmic reticulum as compared to the rounded form of control vehicle (0.1% ethanol)-treated PR-transfected cells. Vehicle and progesterone-treated MDA-MB-231 cells transfected with the pSG5 plasmid (transfection control cells) had similar rounded morphology as control vehicle-treated PR-transfected cells. Immunofluorescence staining revealed that expression of E-cadherin, a differentiation marker, was more prominent in progesterone-treated cells. Expression of keratin and vimentin but not beta-catenin was up-regulated in progesterone treated cells when evaluated by immunoblotting. As signal transducers and activators of transcription (STAT) molecules have been implicated in mammary differentiation, we analyzed the expression of Stat 1, 3, 5a, and 5b proteins and found a significant up-regulation of the Stat 5b protein in progesterone-treated cells. We have provided in vitro evidence of the close association of PR with differentiation in breast cancer. It is likely that the Stat 5b protein may play a major role in progesterone-induced differentiation in breast cancer cells.

  19. Photoporation and cell transfection using a violet diode laser

    Science.gov (United States)

    Paterson, L.; Agate, B.; Comrie, M.; Ferguson, R.; Lake, T. K.; Morris, J. E.; Carruthers, A. E.; Brown, C. T. A.; Sibbett, W.; Bryant, P. E.; Gunn-Moore, F.; Riches, A. C.; Dholakia, Kishan

    2005-01-01

    The introduction and subsequent expression of foreign DNA inside living mammalian cells (transfection) is achieved by photoporation with a violet diode laser. We direct a compact 405 nm laser diode source into an inverted optical microscope configuration and expose cells to 0.3 mW for 40 ms. The localized optical power density of ~1200 MW/m2 is six orders of magnitude lower than that used in femtosecond photoporation (~104 TW/m2). The beam perforates the cell plasma membrane to allow uptake of plasmid DNA containing an antibiotic resistant gene as well as the green fluorescent protein (GFP) gene. Successfully transfected cells then expand into clonal groups which are used to create stable cell lines. The use of the violet diode laser offers a new and simple poration technique compatible with standard microscopes and is the simplest method of laser-assisted cell poration reported to date.

  20. Enhanced transfection of brain tumor suppressor genes by photochemical internalization

    Science.gov (United States)

    Chou, Chih H.; Sun, Chung-Ho; Zhou, Yi-Hong; Madsen, Steen J.; Hirschberg, Henry

    2011-03-01

    One of many limitations for cancer gene therapy is the inability of the therapeutic gene to transfect a sufficient number of tumor cells. Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. The utility of PCI for the delivery of a tumor suppressor gene (PAX-6) was investigated in monolayers and spheroids consisting of F98 rat glioma cells.

  1. Transient transfection and expression of firefly luciferase in Giardia lamblia.

    OpenAIRE

    1995-01-01

    We have developed a gene transfer system for the protozoan parasite Giardia lamblia. This organism is responsible for many cases of diarrhea worldwide and is considered to be one of the most primitive eukaryotes. Expression of a heterologous gene was detected in this parasite after electroporation with appropriate DNA constructs. We constructed a series of transfection plasmids using flanking sequences of the Giardia glutamate dehydrogenase (GDH) gene to drive expression of the firefly lucife...

  2. Graphene and carbon nanotube nanocomposite for gene transfection

    Energy Technology Data Exchange (ETDEWEB)

    Hollanda, L.M. [Laboratory of Biotechnology, Department of Biochemistry, Institute of Biology at UNICAMP, Rua Monteiro Lobato 255, Campinas, SP CEP 13083-862 (Brazil); Lobo, A.O. [Laboratory of Biomedical Nanotechnology, Institute of Research and Development at the UNIVAP, Av. Shishima Hifumi, 2911, CEP: 12244-000 Sao Jose dos Campos, SP (Brazil); Lancellotti, M. [Laboratory of Biotechnology, Department of Biochemistry, Institute of Biology at UNICAMP, Rua Monteiro Lobato 255, Campinas, SP CEP 13083-862 (Brazil); Berni, E. [Biological Chemistry Laboratory, Department of Physical Chemistry, Institute of Chemistry at UNICAMP, R. José de Castro, Campinas, SP CEP 13083-970 (Brazil); Corat, E.J. [Associated Laboratory of Sensors and Materials of the INPE, Av. dos Astronautas 1758, Sao Jose dos Campos CEP: 12227-010 SP (Brazil); Zanin, H., E-mail: hudsonzanin@gmail.com [Associated Laboratory of Sensors and Materials of the INPE, Av. dos Astronautas 1758, Sao Jose dos Campos CEP: 12227-010 SP (Brazil)

    2014-06-01

    Graphene and carbon nanotube nanocomposite (GCN) was synthesised and applied in gene transfection of pIRES plasmid conjugated with green fluorescent protein (GFP) in NIH-3T3 and NG97 cell lines. The tips of the multi-walled carbon nanotubes (MWCNTs) were exfoliated by oxygen plasma etching, which is also known to attach oxygen content groups on the MWCNT surfaces, changing their hydrophobicity. The nanocomposite was characterised by high resolution scanning electron microscopy; energy-dispersive X-ray, Fourier transform infrared and Raman spectroscopies, as well as zeta potential and particle size analyses using dynamic light scattering. BET adsorption isotherms showed the GCN to have an effective surface area of 38.5 m{sup 2}/g. The GCN and pIRES plasmid conjugated with the GFP gene, forming π-stacking when dispersed in water by magnetic stirring, resulting in a helical wrap. The measured zeta potential confirmed that the plasmid was connected to the nanocomposite. The NIH-3T3 and NG97 cell lines could phagocytize this wrap. The gene transfection was characterised by fluorescent protein produced in the cells and pictured by fluorescent microscopy. Before application, we studied GCN cell viability in NIH-3T3 and NG97 line cells using both MTT and Neutral Red uptake assays. Our results suggest that GCN has moderate stability behaviour as colloid solution and has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity and good transfection efficiency. - Highlights: • Facile water dispersible GCN was prepared exfoliating MWCNTs by oxygen plasma etching. • Oxygen content groups were attached on the GCN surfaces changing its hydrophobicity. • Low concentrations of GCN showed low cytotoxicity for NIH-3T3 and NG97 line cells. • The GCN and pIRES plasmid conjugated with the GFP gene. • Gene transfection was studied using this new material.

  3. Stable Transfection of the Diplomonad Parasite Spironucleus salmonicida

    OpenAIRE

    Jerlström-Hultqvist, Jon; Einarsson, Elin; Staffan G Svärd

    2012-01-01

    Eukaryotic microbes are highly diverse, and many lineages remain poorly studied. One such lineage, the diplomonads, a group of binucleate heterotrophic flagellates, has been studied mainly due to the impact of Giardia intestinalis, an intestinal, diarrhea-causing parasite in humans and animals. Here we describe the development of a stable transfection system for use in Spironucleus salmonicida, a diplomonad that causes systemic spironucleosis in salmonid fish. We designed vectors in cassette ...

  4. Stable Transfection of the Diplomonad Parasite Spironucleus salmonicida

    Science.gov (United States)

    Einarsson, Elin; Svärd, Staffan G.

    2012-01-01

    Eukaryotic microbes are highly diverse, and many lineages remain poorly studied. One such lineage, the diplomonads, a group of binucleate heterotrophic flagellates, has been studied mainly due to the impact of Giardia intestinalis, an intestinal, diarrhea-causing parasite in humans and animals. Here we describe the development of a stable transfection system for use in Spironucleus salmonicida, a diplomonad that causes systemic spironucleosis in salmonid fish. We designed vectors in cassette format carrying epitope tags for localization (3×HA [where HA is hemagglutinin], 2× Escherichia coli OmpF linker and mouse langerin fusion sequence [2×OLLAS], 3×MYC) and purification of proteins (2× Strep-Tag II–FLAG tandem-affinity purification tag or streptavidin binding peptide–glutathione S-transferase [SBP-GST]) under the control of native or constitutive promoters. Three selectable gene markers, puromycin acetyltransferase (pac), blasticidin S-deaminase (bsr), and neomycin phosphotransferase (nptII), were successfully applied for the generation of stable transfectants. Site-specific integration on the S. salmonicida chromosome was shown to be possible using the bsr resistance gene. We epitope tagged six proteins and confirmed their expression by Western blotting. Next, we demonstrated the utility of these vectors by recording the subcellular localizations of the six proteins by laser scanning confocal microscopy. Finally, we described the creation of an S. salmonicida double transfectant suitable for colocalization studies. The transfection system described herein and the imminent completion of the S. salmonicida genome will make it possible to use comparative genomics as an investigative tool to explore specific, as well as general, diplomonad traits, benefiting research on both Giardia and Spironucleus. PMID:22983987

  5. Impact of killer immunoglobulin-like receptor-human leukocyte antigens ligand incompatibility among renal transplantation.

    Science.gov (United States)

    Alam, S; Rangaswamy, D; Prakash, S; Sharma, R K; Khan, M I; Sonawane, A; Agrawal, S

    2015-01-01

    Killer immunoglobulin-like receptor (KIR) gene shows a high degree of polymorphism. Natural killer cell receptor gets activated once they bind to self-human leukocyte antigens (HLAs) with specific ligand. KIR gene and HLA ligand incompatibility due to the presence/absence of KIR in the recipient and the corresponding HLA ligand in the allograft may impact graft survival in solid organ transplantation. This study evaluates the effect of matches between KIR genes and known HLA ligands. KIR genotypes were determined using sequence specific primer polymerase chain reaction. Presence of certain KIR in a recipient, where the donor lacked the corresponding HLA ligand was considered a mismatch. The allograft was considered matched when both KIR receptor and HLA alloantigen reveald compatibility among recipient and donor. The data revealed better survival among individuals with matched inhibitory KIR receptors and their corresponding HLA ligands (KIR2DL2/DL3-HLAC2, KIR3DL1-HLABw4). On the contrary, no adverse effect was seen for matched activating KIR receptors and their corresponding HLA ligands. One of the activating gene KIR2DS4 showed risk (P = 0.0413, odds ratio = 1.91, 95% confidence interval = 1.02-3.57) association with renal allograft rejection. We conclude that the presence of inhibitory KIR gene leads to better survival; whereas activating motifs show no significant role in renal allograft survival.

  6. Killer cell immunoglobulin-like receptor gene associations with autoimmune and allergic diseases, recurrent spontaneous abortion, and neoplasms

    Directory of Open Access Journals (Sweden)

    Piotr eKusnierczyk

    2013-01-01

    Full Text Available Killer cell immunoglobulin-like receptors (KIRs are a family of cell surface inhibitory or activating receptors expressed on natural killer cells and some subpopulations of T lymphocytes. KIR genes are clustered in the 19q13.4 region and are characterized by both allelic (high numbers of variants and haplotypic (different numbers of genes for inhibitory and activating receptors on individual chromosomes polymorphism. This contributes to diverse susceptibility to diseases and other clinical situations. Associations of KIR genes, as well as of genes for their ligands, with selected diseases such as psoriasis vulgaris and atopic dermatitis, rheumatoid arthritis, recurrent spontaneous abortion, and non-small cell lung cancer are discussed in the context of NK and T cell functions.

  7. Polyfunctionality of natural killer cell in healthy donors

    Directory of Open Access Journals (Sweden)

    Yupanun WUTTI-IN

    2016-04-01

    Full Text Available Background: Natural killer (NK cells are important guards of the innate immune system, which act by performing as primary effector cells in viral infections. NK cell function is regulated by the engagement of activating and/or inhibitory receptors on individual NK cell surfaces. Subsequent to activation, the release of preformed cytolytic granules or cytokines occurs. Recently, the polyfunctionality of NK cells has been described as a potent NK cell subset that mediates antiviral response in HIV-infected slow progressors. Objectives: To evaluate the polyfunctional NK cells in healthy individuals. Methods: Peripheral blood mononuclear cells (PBMCs were separated from 41 healthy blood donors by Ficoll-Hypaque gradient centrifugation. Multicolor flow cytometry was used to investigate the expression of function markers (degranulation marker (CD107a, IFN-γ, and TNF-a on NK cells following PMA/Ionomycin or K562 stimulation. Results: The percentage of NK cells expressing CD107a, IFN-γ, or TNF-a in response to PMA/Ionomycin were 17.85, 10.56, and 2.66 %, respectively. The NK cells expressing CD107a, IFN-γ, or TNF-a in response to K562 stimulation were 6.43, 2.09, and 0.57 %, respectively. The capability of NK cells to perform polyfunctions was 6.19 % of the total NK cells following PMA/Ionomycin stimulation, while 1.06 % was observed following K562 stimulation. The trifunctional CD107a+ / IFN-γ+ / TNF-a + NK cell subset was found to be 0.95 and 0.04 % following PMA/Ionomycin and K562 stimulation, respectively. Conclusion: A small fraction of NK cells was capable of performing polyfunctions following stimulation, with less than 1 % being able to perform trifunctions in this study setting.

  8. M by Fritz Lang (Germany, 1931) Phenomenology of Evil: a serial killer and his social group.

    Science.gov (United States)

    Secchi, Cesare

    2015-10-01

    In 1931 an unknown murderer of little girls (Peter Lorre) is terrorizing the city of Berlin. We see him entice a new victim, the little Elsie Beckmann, who is coming home from school: whistling a tune by Grieg, he buys her a balloon from a blind beggar. When her corpse is discovered, the police undertake a major mobilization aimed at seeking the serial killer in the criminal underworld; meanwhile, the ever more terrified population starts to see the dangerous murderer in everyone. Since the roundups and incursions into the seediest parts of town disturb the gangsters' activities, the leaders of organized crime, headed by Schränker (Gustav Gründgens), take it upon themselves also to hunt down the solitary child-killer, engaging the community of beggars. Every corner of the city is catalogued and sifted by the dual activity of the police and the gangsters. The 'monster', a former psychiatric patient, mild and harmless in manner, is finally tracked down via two parallel routes: the clue of a cigarette packet enables Inspector Lohmann (Otto Wernicke) to track down the serial killer's address, while the blind beggar recognizes the whistled tune. The murderer is identified by a 'slap' from a young criminal which leaves an M marked in chalk on a shoulder of the man's overcoat. Thus he is caught and undergoes a kind of trial at the hands of the gangsters who tie him up in order to lynch him, but they are interrupted by the arrival of the forces of law and order.

  9. Characterization of cell lines stably transfected with rubella virus replicons

    Energy Technology Data Exchange (ETDEWEB)

    Tzeng, Wen-Pin; Xu, Jie [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States); Frey, Teryl K., E-mail: tfrey@gsu.edu [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States)

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  10. Hormonal induction of transfected genes depends on DNA topology.

    Science.gov (United States)

    Piña, B; Haché, R J; Arnemann, J; Chalepakis, G; Slater, E P; Beato, M

    1990-02-01

    Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thymidine kinase promoter of herpes simplex virus and the reporter gene chloramphenicol acetyltransferase of Escherichia coli respond to glucocorticoids and progestins when transfected into appropriate cells. In the human mammary tumor cell line T47D, the response to progestins, but not to glucocorticoids, is highly dependent on the topology of the transfected DNA. Although negatively supercoiled plasmids respond optimally to the synthetic progestin R5020, their linearized counterparts exhibit markedly reduced progestin inducibility. This is not due to changes in the efficiency of DNA transfection, since the amount of DNA incorporated into the cell nucleus is not significantly dependent on the initial topology of the plasmids. In contrast, cotransfection experiments with glucocorticoid receptor cDNA in the same cell line show no significant influence of DNA topology on induction by dexamethasone. A similar result was obtained with fibroblasts that contain endogenous glucocorticoid receptors. When the distance between receptor-binding sites or between the binding sites and the promoter was increased, the dependence of progestin induction on DNA topology was more pronounced. In contrast to the original plasmid, these constructs also revealed a similar topological dependence for induction by glucocorticoids. The differential influence of DNA topology is not due to differences in the affinity of the two hormone receptors for DNA of various topologies, but probably reflects an influence of DNA topology on the interaction between different DNA-bound receptor molecules and between receptors and other transcription factors.

  11. Laser-based patterning for transfected cell microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Hook, Andrew L; Creasey, Rhiannon; Voelcker, Nicolas H [Flinders University, GPO Box 2100, Bedford Park, SA 5042 (Australia); Hayes, Jason P [MiniFAB, 1 Dalmore Drive, Caribbean Park, Scoresby VIC 3179 (Australia); Thissen, Helmut, E-mail: Nico.Voelcker@flinders.edu.a [CSIRO Molecular and Health Technologies, Bayview Avenue, Clayton VIC 3168 (Australia)

    2009-12-15

    The spatial control over biomolecule- and cell-surface interactions is of great interest to a broad range of biomedical applications, including sensors, implantable devices and cell microarrays. Microarrays in particular require precise spatial control and the formation of patterns with microscale features. Here, we have developed an approach specifically designed for transfected cell microarray (TCM) applications that allows microscale spatial control over the location of both DNA and cells on highly doped p-type silicon substrates. This was achieved by surface modification, involving plasma polymerization of allylamine, grafting of poly(ethylene glycol) and subsequent excimer laser ablation. DNA could be delivered in a spatially defined manner using ink-jet printing. In addition, electroporation was investigated as an approach to transfect attached cells with adsorbed DNA and good transfection efficiencies of approximately 20% were observed. The ability of the microstructured surfaces to spatially direct both DNA adsorption and cell attachment was demonstrated in a functional TCM, making this system an exciting platform for chip-based functional genomics.

  12. Towards gene therapy based on femtosecond optical transfection

    Science.gov (United States)

    Antkowiak, M.; Torres-Mapa, M. L.; McGinty, J.; Chahine, M.; Bugeon, L.; Rose, A.; Finn, A.; Moleirinho, S.; Okuse, K.; Dallman, M.; French, P.; Harding, S. E.; Reynolds, P.; Gunn-Moore, F.; Dholakia, K.

    2012-06-01

    Gene therapy poses a great promise in treatment and prevention of a variety of diseases. However, crucial to studying and the development of this therapeutic approach is a reliable and efficient technique of gene and drug delivery into primary cell types. These cells, freshly derived from an organ or tissue, mimic more closely the in vivo state and present more physiologically relevant information compared to cultured cell lines. However, primary cells are known to be difficult to transfect and are typically transfected using viral methods, which are not only questionable in the context of an in vivo application but rely on time consuming vector construction and may also result in cell de-differentiation and loss of functionality. At the same time, well established non-viral methods do not guarantee satisfactory efficiency and viability. Recently, optical laser mediated poration of cell membrane has received interest as a viable gene and drug delivery technique. It has been shown to deliver a variety of biomolecules and genes into cultured mammalian cells; however, its applicability to primary cells remains to be proven. We demonstrate how optical transfection can be an enabling technique in research areas, such as neuropathic pain, neurodegenerative diseases, heart failure and immune or inflammatory-related diseases. Several primary cell types are used in this study, namely cardiomyocytes, dendritic cells, and neurons. We present our recent progress in optimizing this technique's efficiency and post-treatment cell viability for these types of cells and discuss future directions towards in vivo applications.

  13. Effects of ultraviolet irradiation on natural killer cell function in systemic lupus erythematosus

    Energy Technology Data Exchange (ETDEWEB)

    Nived, O.; Johansson, I.; Sturfelt, G. (University Hospital, Lund (Sweden). Dept. of Rheumatology)

    1992-06-01

    In vitro irradiation with long wavelength ultraviolet light (UV-A), in clinically relevant dosages, of a natural killer cell line containing cell preparations from 17 control subjects reduced natural killer cell cytotoxicity with the cell line K562 as target. The spontaneous function of natural killer cells from 12 patients with systematic lupus erythematosus (SLE) correlated inversely with the one hour erythrocyte sedimentation rate, but not with glucocorticoid doses. After UV-A exposure, natural killer cells from patients with SLE exert either increased or decreased cytotoxicity, and the direction of change is inversely correlated with the spontaneous natural killer cell function. (Author).

  14. Effects of secretive bone morphogenetic protein 2 induced by gene transfection on the biological changes of NIH3T3 cells

    Institute of Scientific and Technical Information of China (English)

    SUN Wei-bin; WANG Juan; LU Chun; TANG Gui-xia

    2005-01-01

    Background Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor beta superfamily, are powerful regulators of cartilage and bone formation. This study investigated the biological changes of NIH3T3 cells incubated with secretive BMP2 that was induced by gene transfection through transwell. Methods Eukaryonic expression vector (pcDNA3.1-B2) was transfered into NIH3T3 cells with SofastTM,a positive compound transfection agent. The positive cell clones were selected with G418. The cytoplasmic and extracellular expressions of BMP2 were determined by immunohistochemical stain and enzyme-linked immunosorbent assay. NIH3T3 cells were co-cultured with hBMP2 gene transfecting cells through transwell, and the ultrastructure, alkaline phosphatase activity and the expression of osteocalcin (the marker of osteogenetic differentiation) changes were observed. Results There were cytoplasmic and extracellular expressions of BMP2 in transfecting NIH3T3 cells. The ultrastructural changes, the high activity of alkaline phosphatase and the positive stain of osteocalcin suggested the osteogenetic differentiation tendency of NIH3T3 cells co-cultured with transfecting NIH3T3 cells. Conclusion Secretive BMP2 that is induced by gene transfection could promote the osteogenetic differentiation of fibroblast cells.

  15. Recognition of microbial glycolipids by Natural Killer T cells

    Directory of Open Access Journals (Sweden)

    Dirk Michael Zajonc

    2015-08-01

    Full Text Available T cells can recognize microbial antigens when presented by dedicated antigen-presenting molecules. While peptides are presented by classical members of the Major Histocompatibility (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

  16. Recognition of Microbial Glycolipids by Natural Killer T Cells

    Science.gov (United States)

    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

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

    NARCIS (Netherlands)

    Schipper, H.S.; Rakhshandehroo, M.; Graaf, van de S.F.J.; Venken, K.; Koppen, A.; Stienstra, R.; Prop, S.; Meerding, J.; Hamers, N.; Besra, G.S.; Boon, den L.; Nieuwenhuis, E.E.S.; Elewaut, D.; Prakken, B.; Kersten, A.H.; Boes, M.; Kalkhoven, E.

    2012-01-01

    Lipid overload and adipocyte dysfunction are key to the development of insulin resistance and can be induced by a high-fat diet. CD1d-restricted invariant natural killer T (iNKT) cells have been proposed as mediators between lipid overload and insulin resistance, but recent studies found decreased i

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

    NARCIS (Netherlands)

    Nieuwenhuis, E.E.S.; Schipper, H.S.; Rakhshandehroo, M.; Graaf, S.F.J. van de; Venken, K.; Koppen, A.; Stienstra, R.; Prop, S.; Meerding, J.M.; Hamers, N.; Besra, G.; Boon, L; Elewaut, D.; Prakken, A.B.J.; Kersten, S.; Boes, M.L.; Kalkhoven, E.

    2012-01-01

    Lipid overload and adipocyte dysfunction are key to the development of insulin resistance and can be induced by a high-fat diet. CD1d-restricted invariant natural killer T (iNKT) cells have been proposed as mediators between lipid overload and insulin resistance, but recent studies found decreased i

  19. Life history evolution: what does a menopausal killer whale do?

    Science.gov (United States)

    Whitehead, Hal

    2015-03-16

    Menopause evolved in humans and whales, presumably because older females can help their kin. But how do they help? New research shows that post-menopausal female killer whales lead foraging groups. This leadership is most significant when food is scarce.

  20. Are natural killer cells protecting the metabolically healthy obese patient?

    LENUS (Irish Health Repository)

    Lynch, Lydia A

    2012-02-01

    With the emerging obesity pandemic, identifying those who appear to be protected from adverse consequences such as type 2 diabetes and certain malignancies will become important. We propose that the circulating immune system plays a role in the development of these comorbidities. Clinical data and blood samples were collected from 52 patients with severe obesity attending a hospital weight-management clinic and 11 lean healthy controls. Patients were classified into metabolically "healthy obese" (n = 26; mean age 42.6 years, mean BMI 46.8 kg\\/m(2)) or "unhealthy obese" (n = 26; mean age 45 years, mean BMI 47.5 kg\\/m(2)) groups, based upon standard cutoff points for blood pressure, lipid profile, and fasting glucose. Circulating lymphoid populations and phenotypes were assessed by flow cytometry. Obese patients had significantly less circulating natural killer (NK) and cytotoxic T lymphocytes (CTL) compared to lean controls. There were significantly higher levels of NK cells and CTLs in the healthy obese group compared to the unhealthy obese group (NK: 11.7% vs. 6.5%, P < 0.0001, CD8 13.4% vs. 9.3%, P = 0.04), independent of age and BMI and these NK cells were also less activated in the healthy compared to the unhealthy group (CD69, 4.1% vs. 11.8%, P = 0.03). This is the first time that quantitative differences in the circulating immune system of obese patients with similar BMI but different metabolic profiles have been described. The significantly higher levels of CTLs and NK cells, which express fewer inhibitory molecules, could protect against malignancy, infection, and metabolic disease seen in obesity.

  1. Increased expression of the major cysteine proteinases by stable episomal transfection underlines the important role of EhCP5 for the pathogenicity of Entamoeba histolytica.

    Science.gov (United States)

    Tillack, Manuela; Nowak, Nicolas; Lotter, Hannelore; Bracha, Rivka; Mirelman, David; Tannich, Egbert; Bruchhaus, Iris

    2006-09-01

    The protozoan Entamoeba histolytica causes intestinal inflammation and liver abscess. Cysteine proteinases (CPs) have been proposed as important virulence factors for amoebiasis. To test the role of the various CPs for amoeba induced pathology, the three major enzymes of the parasite, namely EhCP1, EhCP2 and EhCP5 accounting for about 90% of total proteinase activity, were overexpressed by stable episomal transfection. Total CP activity of recombinant amoebae increased by three- to six-fold depending on the gene transfected. Interestingly, overexpression of the genes for EhCP1 or EhCP2 increased the activity of the corresponding enzyme only, whereas overexpression of the gene for EhCP5 increased the activity of all three enzymes, which is consistent with enzyme-converting activity of EhCP5. Cytopathic activity, measured by in vitro monolayer disruption, was dramatically increased in ehcp5-transfectants (five-fold) but showed only a modest increase in ehcp1- or ehcp2-transfectants (1.5-2-fold). In addition, overexpression of ehcp5 but not of ehcp1 or ehcp2 significantly increased amoebic liver abscess formation in laboratory animals. Moreover, transfection and overexpression of ehcp5 was able to compensate the reduction of in vivo pathogenicity in parasites, which have been silenced for the gene encoding the pore-forming protein amoebapore A. In summary, these results further support the important role of EhCP5 in E. histolytica pathogenicity.

  2. HBV X Gene Transfection Upregulates IL-1β and IL-6 Gene Expression and Induces Rat Glomerular Mesangial Cell Proliferation

    Institute of Scientific and Technical Information of China (English)

    Hongzhu LU; Jianhua ZHOU

    2008-01-01

    The X gene of HBV encodes a 17-KD protein, termed HBx, which has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements. The aim of this study was to investigate the effect of HBx on gene expression of interleukin (IL)-1β and IL-6, and proliferation of rat mesangial cells in vitro. The X gene of HBV was amplified by PCR assay, and inserted into the eukaryotic expression vector pCI-neo. The structure of recombinant pCI-neo-X plasmid was proved by restrict endonuclease digestion and sequencing analysis. pCI-neo-X was transfected into cultured rat mesangial cell line in vitro via liposome. HBx expression in transfected mesangial cells was detected by Western blot. The IL-1β and IL-6 mRNA expression in those cells was assayed by semiquantitative RT-PCR. Mesangial cell proliferation was tested by MTT. The results showed that HBx was obviously expressed in cultured mesangial cell line at 36th and 48th h after transfection. The expression of IL-1β and IL-6 mRNA was simultaneously increased. The cell proliferation was also obvious at the same time. It was concluded that HBx gene transfection could induce IL-1β and IL-6 gene expression and mesangial cell proliferation. HBx may play a critical role in mesangial cell proliferation through upregulation of the IL-1β and IL-6 gene expression.

  3. Association of Killer Cell Immunoglobulin- Like Receptor Genes in Iranian Patients with Rheumatoid Arthritis.

    Directory of Open Access Journals (Sweden)

    Masoumeh Nazari

    Full Text Available Rheumatoid arthritis (RA is a chronic inflammatory disorder characterized by persistent synovitis, ultimately leading to cartilage and bone degeneration. Natural Killer cells and CD28 null T-cells are suspected as role players in RA pathogenesis. These cells are similar in feature and function, as they both exert their cytotoxic effect via Killer Cell Immunoglobulin- Like Receptors (KIR on their surface. KIR genes have either an inhibitory or activating effect depending on their intracytoplasmic structure. Herein we genotyped 16 KIR genes, 3 pseudo genes and 6 HLA class І genes as their corresponding ligands in RA patients and control subjects.In this case-control study, KIR and HLA genes were genotyped in 400 RA patients and 372 matched healthy controls using sequence-specific primers (SSP-PCR. Differences in the frequency of genes and haplotypes were determined by χ² test.KIR2DL2, 2DL5a, 2DL5b and activating KIR: KIR2DS5 and 3DS1 were all protective against RA. KIR2DL5 removal from a full Inhibitory KIR haplotype converted the mild protection (OR = 0.56 to a powerful predisposition to RA (OR = 16.47. Inhibitory haplotype No. 7 comprising KIR2DL5 in the absence of KIR2DL1 and KIR2DL3 confers a 14-fold protective effect against RA.Individuals carrying the inhibitory KIR haplotype No. 6 have a high potential risk for developing RA.

  4. Tissue Engineering Using Transfected Growth-Factor Genes

    Science.gov (United States)

    Madry, Henning; Langer, Robert S.; Freed, Lisa E.; Trippel, Stephen; Vunjak-Novakovic, Gordana

    2005-01-01

    A method of growing bioengineered tissues includes, as a major component, the use of mammalian cells that have been transfected with genes for secretion of regulator and growth-factor substances. In a typical application, one either seeds the cells onto an artificial matrix made of a synthetic or natural biocompatible material, or else one cultures the cells until they secrete a desired amount of an extracellular matrix. If such a bioengineered tissue construct is to be used for surgical replacement of injured tissue, then the cells should preferably be the patient s own cells or, if not, at least cells matched to the patient s cells according to a human-leucocyteantigen (HLA) test. The bioengineered tissue construct is typically implanted in the patient's injured natural tissue, wherein the growth-factor genes enhance metabolic functions that promote the in vitro development of functional tissue constructs and their integration with native tissues. If the matrix is biodegradable, then one of the results of metabolism could be absorption of the matrix and replacement of the matrix with tissue formed at least partly by the transfected cells. The method was developed for articular chondrocytes but can (at least in principle) be extended to a variety of cell types and biocompatible matrix materials, including ones that have been exploited in prior tissue-engineering methods. Examples of cell types include chondrocytes, hepatocytes, islet cells, nerve cells, muscle cells, other organ cells, bone- and cartilage-forming cells, epithelial and endothelial cells, connective- tissue stem cells, mesodermal stem cells, and cells of the liver and the pancreas. Cells can be obtained from cell-line cultures, biopsies, and tissue banks. Genes, molecules, or nucleic acids that secrete factors that influence the growth of cells, the production of extracellular matrix material, and other cell functions can be inserted in cells by any of a variety of standard transfection techniques.

  5. Efficient transfection of chicken cells by lipofection, and introduction of transfected blastodermal cells into the embryo.

    Science.gov (United States)

    Brazolot, C L; Petitte, J N; Etches, R J; Verrinder Gibbins, A M

    1991-12-01

    Chicken blastodermal cells (CBCs) and primary chicken fibroblasts (PCFs) have been lipofected with a variety of lacZ constructs encoding Escherichia coli beta-galactosidase (beta-gal). A reporter construct (phspPTlacZpA) containing a mouse heat-shock protein 68 gene (hsp 68) promoter was used to establish conditions for efficient lipofection. The construct, in circular or linear plasmid form or as reporter sequences alone, was transferred efficiently by incubating the cells for 3.5 h in a mixture of 6.2 micrograms Lipofectin (a cationic liposome preparation from Bethesda Research Laboratories) and 1.55-3.1 micrograms DNA per mL DMEM. These lipofection conditions were used to transfer a reporter construct (pCBcMtlacZ) containing a Zn(2+)-inducible chicken metallothionein (cMt) promoter, and constructs showing constitutive expression due to Rous sarcoma virus plus chicken beta-actin (pmiwZ) or cytomegalovirus (pMaori3) promoters. Endogenous chicken beta-gal and transferred bacterial beta-gal activity could be distinguished clearly by incubating the cells with the substrate, Xgal, at pH 4.3 or 7.4, respectively. Expression of phspPTlacZpA in chicken cells did not appear to require specific induction of the mouse hsp68 promoter, whereas expression of pCBcMtlacZ required treatment of the cells for 6-12 h with 150 microM ZnCl2. Bacterial beta-gal activity was observed following lipofection of CBCs that were cultured in suspension or plated. The efficiency of lipofection was at least 1 in 25 for CBCs, judging by the proportion of cells shown to have beta-gal activity 16-24 h after lipofection treatment began; these events could represent transient or stable incorporation of the construct.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Transient and stable transfection in the protozoan parasite Entamoeba invadens.

    Science.gov (United States)

    Ehrenkaufer, Gretchen M; Singh, Upinder

    2012-07-01

    Entamoeba histolytica is an important human pathogen and a major health problem worldwide. Many aspects of parasite biology can be studied with the exception of stage conversion, which cannot be reproduced adequately in E. histolytica. The reptile parasite Entamoeba invadens is a vital model system for studying stage conversion since it can be induced to undergo both encystation and excystation with high efficiency in vitro. However, functional studies using E. invadens have been limited by the lack of genetic tools in this species. Here, we report a new method for both transient and stable transfection of E. invadens. These new tools will greatly enhance research into Entamoeba development.

  7. Photobiomodulation on KATP Channels of Kir6.2-Transfected HEK-293 Cells

    Directory of Open Access Journals (Sweden)

    Fu-qing Zhong

    2014-01-01

    Full Text Available Background and Objective. ATP-sensitive potassium (KATP channel couples cell metabolism to excitability. To explore role of KATP channels in cellular photobiomodulation, we designed experiment to study effect of low intensity 808 nm laser irradiation on the activity of membrane KATP channel. Study Design/Materials and Methods. Plasmids encoding Kir6.2 was constructed and heterologously expressed in cultured mammalian HEK-293 cells. The patch-clamp and data acquisition systems were used to record KATP channel current before and after irradiation. A laser beam of Ga-As 808 nm at 5 mW/cm2 was used in experiments. A one-way ANOVA test followed by a post hoc Student-Newman-Keuls test was used to assess the statistical differences between data groups. Results. Obvious openings of KATP channels of Kir6.2-transfected HEK-293 cells and excised patches were recorded during and after low intensity 808 nm laser irradiation. Compared with the channels that did not undergo irradiation, open probability, current amplitude, and dwell time of KATP channels after irradiation improved. Conclusions. Low intensity 808 nm laser irradiation may activate membrane KATP channels of Kir6.2-transfected HEK-293 cells and in excised patches.

  8. Enhanced gene transfection performance and biocompatibility of polyethylenimine through pseudopolyrotaxane formation with α-cyclodextrin

    Science.gov (United States)

    Hu, Li-Zhong; Wan, Ning; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

    2017-03-01

    Polyethylenimine (PEI), a commercially available gene transfection reagent, is a promising nonviral vector due to its inherent ability to efficiently condense genetic materials and its successful transfection performance in vitro. However, its low transfection efficiency in vivo, along with its high cytotoxicity, limit any further applications in gene therapy. To enhance the gene transfection performance and reduce the cytotoxicity of linear polyethylenimine, pseudopolyrotaxane PEI25k/CD and the polyrotaxanes PEI25k/CD-PA and PEI25k/CD-PB were prepared and their transfection efficiencies were then evaluated. The pseudopolyrotaxane PEI25k/CD exhibited better transfection efficiency and lower cytotoxicity than the transfection reagent linear PEI25k, even in the presence of serum. It also showed a remarkably higher cell viability, similar DNA protecting capability, and better DNA decondensation and release ability, and could be useful for the development of novel and safe nonviral gene delivery vectors for gene therapy.

  9. Effect of TNF gene-transfected LAK cells on the ascitic liver carcinoma-bearing mice

    Institute of Scientific and Technical Information of China (English)

    Guo Liang Lou; Xue Tao Cao; Bi He Min; Wei Ping Zhang; Pei Lin Meng

    2000-01-01

    AIM To investigate the therapeutic effect of TNF gene transfected LAK cells on ascitic liver carcinoma-bearing mice.METHODS TNF gene was transfected into murine LAK cells by retrovirus. Low dose TNF gene-transfectcdLAK cells and IL-2 were i.p. injected into murine model. Cytotoxicity of gene transfected LAK cells wasstudied in vitro growth and the survival time of murine model was observed.RESULTS TNF gene-transfected LAK cells secreted higher level of TNF than that of normal LAK cells orcontrol gene-transfected LAK ceils. The in vitro growth ability and cytotoxicity of TNF gene-transfectedLAK cells were markedly inhibited by anti-TNF monoclonal antibodies. Significant therapeutic effect onascitic liver carcinoma-bearing mice was achieved.CONCLUSION TNF gene-transfected LAK cells have therapeutic effect on ascitic liver carcinoma-bearingmice.

  10. Lipophosphoramidate-based bipolar amphiphiles: their syntheses and transfection properties.

    Science.gov (United States)

    Berchel, Mathieu; Le Gall, Tony; Lozach, Olivier; Haelters, Jean-Pierre; Montier, Tristan; Jaffrès, Paul-Alain

    2016-03-14

    Six new cationic bolaamphiphiles (also called bipolar amphiphiles, bolaform amphiphiles, or bolalipids) were readily prepared by a thiol-ene click reaction that engaged a mercapto-alcohol (mercapto-ethanol or mercapto-hexanol) and a cationic based lipophosphoramidate. The cationic lipophosphoramidates contain two lipid chains that end in an alkene group and a selected cationic polar head group (trimethylammonium, dimethyl hydroxyethyl ammonium, or methylimidazolium). These compounds were formulated in water (with or without DOPE as a colipid) to produce supramolecular aggregates. These aggregates, before (i.e. bolasomes) and after (i.e. bolaplexes) mixing with plasmid DNA (pDNA) at various charge ratios, were characterized with regard to their sizes and zeta potentials. In the case of bolasomes, the suspensions were unstable since precipitation occurred after only a few hours at room temperature. On the other hand, bolaplex formulations exhibited clearly a better colloidal stability. Then, the gene delivery properties of the cationic bolasomes were investigated using two human-derived epithelial cell lines (A549 and 16HBE). Compared to the commercially available lipofection reagent (Lipofectamine), most of the cationic bolaamphiphiles were able to efficiently transfect these cells when they were formulated with DOPE in a 1 : 1 molar ratio. We report herein that bolaamphiphiles possessing a trimethylammonium or a dimethyl hydroxyethyl ammonium head group were the most efficient in terms of transfection efficiency while exhibiting no significant cytotoxicity.

  11. Relative rates of homologous and nonhomologous recombination in transfected DNA.

    Science.gov (United States)

    Roth, D B; Wilson, J H

    1985-05-01

    Both homologous and nonhomologous recombination events occur at high efficiency in DNA molecules transfected into mammalian cells. Both types of recombination occur with similar overall efficiencies, as measured by an endpoint assay, but their relative rates are unknown. In this communication, we measure the relative rates of homologous and nonhomologous recombination in DNA transfected into monkey cells. This measurement is made by using a linear simian virus 40 genome that contains a 131-base-pair duplication at its termini. Once inside the cell, this molecule must circularize to initiate lytic infection. Circularization can occur either by direct, nonhomologous end-joining or by homologous recombination within the duplicated region. Although the products of the two recombination pathways are different, they are equally infectious. Since homologous and nonhomologous recombination processes are competing for the same substrate, the relative amounts of the products of each pathway should reflect the relative rates of homologous and nonhomologous recombination. Analysis of individual recombinant genomes from 164 plaques indicates that the rate of circularization by nonhomologous recombination is 2- to 3-fold higher than the rate of homologous recombination. The assay system described here may prove to be useful for testing procedures designed to influence the relative rates of homologous and nonhomologous recombination.

  12. Ex vivo transfection of trout pronephros leukocytes, a model for cell culture screening of fish DNA vaccine candidates.

    Science.gov (United States)

    Ortega-Villaizan, M; Martinez-Lopez, A; Garcia-Valtanen, P; Chico, V; Perez, L; Coll, J M; Estepa, A

    2012-09-07

    DNA vaccination opened a new era in controlling and preventing viral diseases since DNA vaccines have shown to be very efficacious where some conventional vaccines have failed, as it occurs in the case of the vaccines against fish novirhabdoviruses. However, there is a big lack of in vitro model assays with immune-related cells for preliminary screening of in vivo DNA vaccine candidates. In an attempt to solve this problem, rainbow trout pronephros cells in early primary culture were transfected with two plasmid DNA constructions, one encoding the green fluorescent protein (GFP) and another encoding the viral haemorrhagic septicaemia virus (VHSV) glycoprotein G (G(VHSV)) - the only viral antigen which has conferred in vivo protection. After assessing the presence of GFP- and G(VHSV)-expressing cells, at transcription and protein levels, the immune response in transfected pronephros cells was evaluated. At 24h post-transfection, G(VHSV) up-regulated migm and tcr transcripts expression, suggesting activation of B and T cells, as well, a high up-regulation of tnfα gene was observed. Seventy-two hours post-transfection, we detected the up-regulation of mx and tnfα genes transcripts and Mx protein which correlated with the induction of an anti-VHSV state. All together we have gathered evidence for successful transfection of pronephros cells with pAE6G, which correlates with in vivo protection results, and is less time-consuming and more rapid than in vivo assays. Therefore, this outcome opens the possibility to use pronephros cells in early primary culture for preliminary screening fish DNA vaccines as well as to further investigate the function that these cells perform in fish immune response orchestration after DNA immunisation.

  13. Dietary methoxychlor exposure modulates splenic natural killer cell activity, antibody-forming cell response and phenotypic marker expression in F0 and F1 generations of Sprague Dawley rats.

    Science.gov (United States)

    White, K L; Germolec, D R; Booker, C D; Hernendez, D M; McCay, J A; Delclos, K B; Newbold, R R; Weis, C; Guo, T L

    2005-02-14

    Methoxychlor, a chlorinated hydrocarbon pesticide, is a persistent environmental contaminant that has been identified in human reproductive tissues. Methoxychlor has been shown to be estrogenic in both in vivo and in vitro studies. As an endocrine disrupter, it may have the potential to adversely affect endocrine, reproductive, and immune systems in animals. The present study evaluated methoxychlor's immunotoxic potential in F0 (dams) and F1 generations of Sprague Dawley rats exposed to an isoflavone-free diet containing methoxychlor at concentrations of 10, 100, and 1000 ppm. In dams, exposure to methoxychlor from gestation day 7 to postpartum day 51 (65 days total exposure) produced a significant increase in the NK activity (1000 ppm) and the percentages of T cells (1000 ppm), helper T cells (1000 ppm) and macrophages (100 and 1000 ppm). In contrast, a decrease in the numbers of splenocytes and B cells was observed at the 100 and 1000 ppm concentrations. In F1 males, exposure to methoxychlor gestationally, lactationally and through feed from postnatal day 22-64 (78 days total exposure) produced an increase in the spleen IgM antibody-forming cell response to sheep red blood cells (100 and 1000 ppm) and the activity of NK cells (1000 ppm). However, there was a decrease in the terminal body weight (1000 ppm), spleen weight (1000 ppm), thymus weight (100 and 1000 ppm), and the numbers of splenocytes (1000 ppm), B cells (100 and 1000 ppm), cytotoxic T cells (1000 ppm) and NK cells (100 and 1000 ppm). In F1 females, exposure to methoxychlor produced a decrease in the terminal body weight (1000 ppm) and the percentages of cytotoxic T cells (10, 100 and 1000 ppm). These results demonstrate that developmental and adult dietary exposure to methoxychlor modulates immune responses in Sprague Dawley rats. Immunological changes were more pronounced in the F1 generation male rats that were exposed during gestation and postpartum, when compared to the F0 and F1 generation

  14. Transfection using hydroxyapatite nanoparticles in the inner ear via an intact round window membrane in chinchilla

    Energy Technology Data Exchange (ETDEWEB)

    Wu Xuewen; Ding Dalian [Central South University, Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital (China); Jiang Haiyan [State University of New York at Buffalo, Center for Hearing and Deafness (United States); XingXiaowei [Central South University, Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital (China); Huang, Suping [Central South University, State Key Laboratory of Powder Metallurgy (China); Liu Hong [Central South University, Department of Otolaryngology Head and Neck Surgery, The Third Xiangya Hospital (China); Chen Zhedong [Central South University, State Key Laboratory of Powder Metallurgy (China); Sun Hong, E-mail: shjhaj@vip.163.com [Central South University, Department of Otolaryngology Head and Neck Surgery, Xiangya Hospital (China)

    2012-01-15

    Hydroxyapatite nanoparticles (nHAT) are known to have excellent biocompatibility, and have attracted increasing attention as new candidates of non-viral vectors for gene therapy. In our previous studies, nHAT carrying a therapeutic gene and a reporter gene were successfully transfected into the spiral ganglion neurons in the inner ear of guinea pigs in vivo as well as in the cultured cell lines, although the transfection efficiencies were never higher than 30%. In this study, the surface modification of nHAT with polyethylenimine (PEI) was made (PEI-nHAT, diameter = 73.09 {+-} 27.32 nm) and a recombinant plasmid carrying enhanced green fluorescent protein (EGFP) gene and neurotrophin-3 (NT-3) gene was constructed as pEGFPC2-NT3. The PEI modified nHAT and the recombinant plasmid was then connected to form the nHAT-based vector-gene complex (PEI-nHAT-pEGFPC2-NT3). This complex was then placed onto the intact round window membranes of the chinchillas for inner ear transfection. Auditory brainstem response (ABR) was tested to evaluate auditory function. Green fluorescence of EGFP was observed using confocal microscopy 48 h after administering vector-gene complexes. There was no significant threshold shift in tone burst-evoked ABR at any tested frequency. Abundant, condensed green fluorescence was found in dark cells on both sides of the crista and around the macula of the utricle. Scattered EGFP signals were also detected in vestibular hair cells, some Schwann cells in the cochlear spiral ganglion region, some outer pillar cells in the organ of Corti, and a few cells in the stria vascularis. The density of green fluorescence-marked cells was obviously higher in the vestibular dark cell area than in other areas of the inner ear, suggesting that vestibular dark cells may have the ability to actively engulf the nHAT-based vector-gene complexes. Considering the high transfection efficiency in the vestibular system, PEI-nHAT may be a potential vector for gene therapy of

  15. Transfection using hydroxyapatite nanoparticles in the inner ear via an intact round window membrane in chinchilla

    Science.gov (United States)

    Wu, Xuewen; Ding, Dalian; Jiang, Haiyan; Xing, Xiaowei; Huang, Suping; Liu, Hong; Chen, Zhedong; Sun, Hong

    2012-01-01

    Hydroxyapatite nanoparticles (nHAT) are known to have excellent biocompatibility, and have attracted increasing attention as new candidates of non-viral vectors for gene therapy. In our previous studies, nHAT carrying a therapeutic gene and a reporter gene were successfully transfected into the spiral ganglion neurons in the inner ear of guinea pigs in vivo as well as in the cultured cell lines, although the transfection efficiencies were never higher than 30%. In this study, the surface modification of nHAT with polyethylenimine (PEI) was made (PEI-nHAT, diameter = 73.09 ± 27.32 nm) and a recombinant plasmid carrying enhanced green fluorescent protein (EGFP) gene and neurotrophin-3 (NT-3) gene was constructed as pEGFPC2-NT3. The PEI modified nHAT and the recombinant plasmid was then connected to form the nHAT-based vector-gene complex (PEI-nHAT-pEGFPC2-NT3). This complex was then placed onto the intact round window membranes of the chinchillas for inner ear transfection. Auditory brainstem response (ABR) was tested to evaluate auditory function. Green fluorescence of EGFP was observed using confocal microscopy 48 h after administering vector-gene complexes. There was no significant threshold shift in tone burst-evoked ABR at any tested frequency. Abundant, condensed green fluorescence was found in dark cells on both sides of the crista and around the macula of the utricle. Scattered EGFP signals were also detected in vestibular hair cells, some Schwann cells in the cochlear spiral ganglion region, some outer pillar cells in the organ of Corti, and a few cells in the stria vascularis. The density of green fluorescence-marked cells was obviously higher in the vestibular dark cell area than in other areas of the inner ear, suggesting that vestibular dark cells may have the ability to actively engulf the nHAT-based vector-gene complexes. Considering the high transfection efficiency in the vestibular system, PEI-nHAT may be a potential vector for gene therapy of inner

  16. Transfected Babesia bovis Expressing a Tick GST as a Live Vector Vaccine

    Science.gov (United States)

    Oldiges, Daiane P.; Laughery, Jacob M.; Tagliari, Nelson Junior; Leite Filho, Ronaldo Viana; Davis, William C.; da Silva Vaz, Itabajara; Termignoni, Carlos; Knowles, Donald P.; Suarez, Carlos E.

    2016-01-01

    The Rhipicephalus microplus tick is a notorious blood-feeding ectoparasite of livestock, especially cattle, responsible for massive losses in animal production. It is the main vector for transmission of pathogenic bacteria and parasites, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine Babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione-S-transferase from Haemaphysalis longicornis (HlGST). The B. bovis S74-T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1α (elongation factor 1 alpha) promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD (green fluorescent protein–blasticidin deaminase), and HlGST fused to the MSA-1 (merozoite surface antigen 1) signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST) in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1α locus. A B. bovis clonal line termed HlGST-Cln expressing intracellular GFP and HlGST in the surface of merozoites was then derived from the mixed parasite line HlGST using a fluorescent activated cell sorter. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a previously described control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1α locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis. Tick egg fertility and fully engorged female tick weight was reduced significantly in R. microplus feeding on HlGST-Cln-immunized calves

  17. Comparison of nanoparticle-mediated transfection methods for DNA expression plasmids: efficiency and cytotoxicity

    Directory of Open Access Journals (Sweden)

    Nolte Ingo

    2011-10-01

    Full Text Available Abstract Background Reproducibly high transfection rates with low methodology-induced cytotoxic side effects are essential to attain the required effect on targeted cells when exogenous DNA is transfected. Different approaches and modifications such as the use of nanoparticles (NPs are being evaluated to increase transfection efficiencies. Several studies have focused on the attained transfection efficiency after NP-mediated approaches. However, data comparing toxicity of these novel approaches with conventional methods is still rare. Transfection efficiency and methodology-induced cytotoxicity were analysed after transfection with different NP-mediated and conventional approaches. Two eukaryotic DNA-expression-plasmids were used to transfect the mammalian cell line MTH53A applying six different transfection protocols: conventional transfection reagent (FuGENE HD, FHD, FHD in combination with two different sizes of stabilizer-free laser-generated AuNPs (PLAL-AuNPs_S1,_S2, FHD and commercially available AuNPs (Plano-AuNP, and two magnetic transfection protocols. 24 h post transfection efficiency of each protocol was analysed using fluorescence microscopy and GFP-based flow cytometry. Toxicity was assessed measuring cell proliferation and percentage of propidium iodide (PI% positive cells. Expression of the respective recombinant proteins was evaluated by immunofluorescence. Results The addition of AuNPs to the transfection protocols significantly increased transfection efficiency in the pIRES-hrGFPII-eIL-12 transfections (FHD: 16%; AuNPs mean: 28%, whereas the magnet-assisted protocols did not increase efficiency. Ligand-free PLAL-AuNPs had no significant cytotoxic effect, while the ligand-stabilized Plano-AuNPs induced a significant increase in the PI% and lower cell proliferation. For pIRES-hrGFPII-rHMGB1 transfections significantly higher transfection efficiency was observed with PLAL-AuNPs (FHD: 31%; PLAL-AuNPs_S1: 46%; PLAL-AuNPs_S2: 50

  18. Role of Killer Immunoglobulin-Like Receptor and Ligand Matching in Donor Selection

    Directory of Open Access Journals (Sweden)

    Meral Beksaç

    2012-01-01

    Full Text Available Despite all efforts to improve HLA typing and immunosuppression, it is still impossible to prevent severe graft versus host disease (GVHD which can be fatal. GVHD is not always associated with graft versus malignancy and can prevent stem cell transplantation from reaching its goals. Overall T-cell alloreactivity is not the sole mechanism modulating the immune defense. Innate immune system has its own antigens, ligands, and mediators. The bridge between HLA and natural killer (NK cell-mediated reactions is becoming better understood in the context of stem cell transplantation. Killer immunoglobulin-like receptors (KIRs constitute a wide range of alleles/antigens segregated independently from the HLA alleles and classified into two major haplotypes which imprints the person's ability to suppress or to amplify T-cell alloreactivity. This paper will summarize the impact of both activating and inhibitory KIRs and their ligands on stem cell transplantation outcome. The ultimate goal is to develop algorithms based on KIR profiles to select donors with maximum antileukemic and minimum antihost effects.

  19. Classification of human natural killer cells based on migration behavior and cytotoxic response.

    Science.gov (United States)

    Vanherberghen, Bruno; Olofsson, Per E; Forslund, Elin; Sternberg-Simon, Michal; Khorshidi, Mohammad Ali; Pacouret, Simon; Guldevall, Karolin; Enqvist, Monika; Malmberg, Karl-Johan; Mehr, Ramit; Önfelt, Björn

    2013-02-21

    Despite intense scrutiny of the molecular interactions between natural killer (NK) and target cells, few studies have been devoted to dissection of the basic functional heterogeneity in individual NK cell behavior. Using a microchip-based, time-lapse imaging approach allowing the entire contact history of each NK cell to be recorded, in the present study, we were able to quantify how the cytotoxic response varied between individual NK cells. Strikingly, approximately half of the NK cells did not kill any target cells at all, whereas a minority of NK cells was responsible for a majority of the target cell deaths. These dynamic cytotoxicity data allowed categorization of NK cells into 5 distinct classes. A small but particularly active subclass of NK cells killed several target cells in a consecutive fashion. These "serial killers" delivered their lytic hits faster and induced faster target cell death than other NK cells. Fast, necrotic target cell death was correlated with the amount of perforin released by the NK cells. Our data are consistent with a model in which a small fraction of NK cells drives tumor elimination and inflammation.

  20. [Music therapy induced alternations in natural killer cell count and function].

    Science.gov (United States)

    Hasegawa, Y; Kubota, N; Inagaki, T; Shinagawa, N

    2001-03-01

    The effects of music therapy on natural killer (NK) cell count and activity (NKCA) were studied in 19 persons. Alzheimer's disease, cerebrovessel disease and Parkinson's disease subjects were assigned to a music therapy. Blood samples were drawn at rest and after completion of music therapy. Music therapy did not change the number of circulating lymphocytes. The percentage of NK cells increased during music therapy, along with an increase in the NK cell activity. The proportion of T cells, CD4 and CD8 did not change significantly during music therapy. One hour after the music therapy session, plasma adrenaline increased but cortisol and noradrenalin did not change. The results indicate that music therapy can significantly increase NK cell count and activity. The change in NK cell and function were independent of neuro-degenerative diseases.

  1. Killer Treg cells ameliorate inflammatory insulitis in non-obese diabetic mice through local and systemic immunomodulation.

    Science.gov (United States)

    Kaminitz, Ayelet; Yolcu, Esma S; Mizrahi, Keren; Shirwan, Haval; Askenasy, Nadir

    2013-08-01

    Treg cells endowed with enhanced killing activity through decoration with Fas-ligand (FasL) protein (killer Treg) have been effective in delay of hyperglycemia in prediabetic non-obese diabetic (NOD) mice. In this study, we assessed the therapeutic efficacy of these cells, harvested from age-matched euglycemic NOD donors, on the course of disease in new-onset diabetics. One dose of 4 × 10(6) killer Treg cells stabilized blood glucose associated with increased insulin levels in 5 of 9 mice and partially reversed the severity of islet inflammation, whereas naive Treg cells did not modulate the course of disease significantly. Killer Treg cells were shown to operate through induction of cell apoptosis within the pancreatic lymph nodes, resulting in reduced efficiency of adoptive disease transfer to NOD/SCID recipients. A second mechanism of action consisted of increased fractions of CD4(+)CD25(-)FoxP3(+) T cells in the pancreas and all lymphoid organs. Immunomodulation with FasL rather than Treg cells enhanced the expression of CD25 and FoxP3 in the thymus, suggesting a possible contribution of thymic output to prolonged stabilization of the glucose levels. Autologous Treg cells evolve as excellent vehicles for targeted delivery of FasL as an immunomodulatory protein, which delete pathogenic cells at the site of inflammation and induce systemic dominance of suppressor subsets.

  2. Phylogenomics of the killer whale indicates ecotype divergence in sympatry.

    Science.gov (United States)

    Moura, A E; Kenny, J G; Chaudhuri, R R; Hughes, M A; Reisinger, R R; de Bruyn, P J N; Dahlheim, M E; Hall, N; Hoelzel, A R

    2015-01-01

    For many highly mobile species, the marine environment presents few obvious barriers to gene flow. Even so, there is considerable diversity within and among species, referred to by some as the 'marine speciation paradox'. The recent and diverse radiation of delphinid cetaceans (dolphins) represents a good example of this. Delphinids are capable of extensive dispersion and yet many show fine-scale genetic differentiation among populations. Proposed mechanisms include the division and isolation of populations based on habitat dependence and resource specializations, and habitat release or changing dispersal corridors during glacial cycles. Here we use a phylogenomic approach to investigate the origin of differentiated sympatric populations of killer whales (Orcinus orca). Killer whales show strong specialization on prey choice in populations of stable matrifocal social groups (ecotypes), associated with genetic and phenotypic differentiation. Our data suggest evolution in sympatry among populations of resource specialists.

  3. Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents.

    Science.gov (United States)

    Stein, C A; Hansen, J Bo; Lai, Johnathan; Wu, SiJian; Voskresenskiy, Anatoliy; Høg, Anja; Worm, Jesper; Hedtjärn, Maj; Souleimanian, Naira; Miller, Paul; Soifer, Harris S; Castanotto, Daniella; Benimetskaya, Luba; Ørum, Henrik; Koch, Troels

    2010-01-01

    For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called 'gymnosis') that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.

  4. Specific transfection of inflamed brain by macrophages: a new therapeutic strategy for neurodegenerative diseases.

    Directory of Open Access Journals (Sweden)

    Matthew J Haney

    Full Text Available The ability to precisely upregulate genes in inflamed brain holds great therapeutic promise. Here we report a novel class of vectors, genetically modified macrophages that carry reporter and therapeutic genes to neural cells. Systemic administration of macrophages transfected ex vivo with a plasmid DNA (pDNA encoding a potent antioxidant enzyme, catalase, produced month-long expression levels of catalase in the brain resulting in three-fold reductions in inflammation and complete neuroprotection in mouse models of Parkinson's disease (PD. This resulted in significant improvements in motor functions in PD mice. Mechanistic studies revealed that transfected macrophages secreted extracellular vesicles, exosomes, packed with catalase genetic material, pDNA and mRNA, active catalase, and NF-κb, a transcription factor involved in the encoded gene expression. Exosomes efficiently transfer their contents to contiguous neurons resulting in de novo protein synthesis in target cells. Thus, genetically modified macrophages serve as a highly efficient system for reproduction, packaging, and targeted gene and drug delivery to treat inflammatory and neurodegenerative disorders.

  5. [A serial killer as exemplified by T. R. Bundy].

    Science.gov (United States)

    Gałeska-Sliwka, Anita

    2008-01-01

    The question of serial homicides and their perpetrators poses a considerable problem of both a definitional and practical nature. T R. Bundy is the first perpetrator who was officially termed a "serial killer". Since that time, this concept has been commonly used and sometimes even overused, e.g. in reference to mass murderers. However, the definitions established for Bundy's case have been preserved and continue to be used in practice.

  6. Public opinion and the politics of the killer robots debate

    OpenAIRE

    Michael C. Horowitz

    2016-01-01

    The possibility that today’s drones could become tomorrow’s killer robots has attracted the attention of people around the world. Scientists and business leaders, from Stephen Hawking to Elon Musk, recently signed a letter urging the world to ban autonomous weapons. Part of the argument against these systems is that they violate the public conscience provision of the Martens Clause due to public opposition, making them illegal under international law. What, however, does the US public think o...

  7. Which serial killers commit suicide? An exploratory study.

    Science.gov (United States)

    Lester, David; White, John

    2012-11-30

    In a sample of 483 serial killers, 6.2% were documented to have committed suicide. Those who committed suicide were found to come from more dysfunctional homes characterized by more psychiatric disturbance in the parents. The sexual acts involved in the murders by the suicides seemed to be more deviant in some aspects, such as committing more bizarre sexual acts or more often taping the murder.

  8. Experimental evidence for action imitation in killer whales (Orcinus orca).

    Science.gov (United States)

    Abramson, José Z; Hernández-Lloreda, Victoria; Call, Josep; Colmenares, Fernando

    2013-01-01

    Comparative experimental studies of imitative learning have focused mainly on primates and birds. However, cetaceans are promising candidates to display imitative learning as they have evolved in socioecological settings that have selected for large brains, complex sociality, and coordinated predatory tactics. Here we tested imitative learning in killer whales, Orcinus orca. We used a 'do-as-other-does' paradigm in which 3 subjects witnessed a conspecific demonstrator's performance that included 15 familiar and 4 novel behaviours. The three subjects (1) learned the copy command signal 'Do that' very quickly, that is, 20 trials on average; (2) copied 100 % of the demonstrator's familiar and novel actions; (3) achieved full matches in the first attempt for 8-13 familiar behaviours (out of 15) and for the 2 novel behaviours (out of 2) in one subject; and (4) took no longer than 8 trials to accurately copy any familiar behaviour, and no longer than 16 trials to copy any novel behaviour. This study provides experimental evidence for body imitation, including production imitation, in killer whales that is comparable to that observed in dolphins tested under similar conditions. These findings suggest that imitative learning may underpin some of the group-specific traditions reported in killer whales in the field.

  9. Destructive hostility: the Jeffrey Dahmer case. A psychiatric and forensic study of a serial killer.

    Science.gov (United States)

    Jentzen, J; Palermo, G; Johnson, L T; Ho, K C; Stormo, K A; Teggatz, J

    1994-12-01

    We were involved as forensic experts in the case of the serial killer Jeffrey Dahmer. We discuss the scene and victim autopsy findings, with a brief consideration of the basic emotion of hostility. These findings support the thesis that at the basis of this serial killer's behavior were primary unconscious feelings of hate that he had channeled into a sadistic programmed destruction of 17 young men. The interview of the serial killer, the photographic scene documentation, and the autopsy findings stress the ambivalent homosexuality of the killer, his sexual sadism, his obsessive fetishism, and his possible cannibalism and necrophilia.

  10. Gold nanoparticle-enhanced electroporation for leukemia cell transfection.

    Science.gov (United States)

    Huang, Shuyan; Zu, Yingbo; Wang, Shengnian

    2014-01-01

    Electroporation serves as an attractive nonviral gene delivery approach for its effectiveness, operational simplicity, and no restrictions of probe or cell type. The commercial electroporation systems have been widely adopted in research and clinics with protocols usually compromising appropriate transfection efficiency and cell viability. By introducing gold nanoparticles (AuNPs), we demonstrated greatly enhanced performance of electroporation from two aspects: the highly conductive, naked AuNPs help reduce the potential drop consumed by the electroporation solution so that the majority of the applied voltage of an electric pulse is truly imposed on cells with enhanced field strength; AuNPs with targeting ligands (e.g., transferrin-AuNPs or Tf-AuNPs) are bound to the cell membrane, working as virtual microelectrodes to create pores on cells with limited opening area while from many different sites. The addition of AuNPs during electroporation therefore benefits not only quicker recovery and better survival of cells but also more efficient uptake of the subjected probes. Such enhancement was successfully confirmed on a chronic myeloid leukemia cell line (i.e., K562 cells) in both a commercial batch electroporation system and a homemade flow system with pWizGFP plasmid DNA probes. The efficiency was found to be dependent on the size, concentration, and mixing ratio of free AuNPs/Tf-AuNPs. An equivalent mixture of free AuNPs and Tf-AuNPs exhibited the best enhancement with the transfection efficiency increase of two to threefold at a minimum sacrifice of the cell viability.

  11. Gold nanoparticles enhanced electroporation for mammalian cell transfection.

    Science.gov (United States)

    Zu, Yingbo; Huang, Shuyan; Liao, Wei-Ching; Lu, Yang; Wang, Shengnian

    2014-06-01

    Electroporation figured prominently as an effective nonviral gene delivery approach for its balance on the transfection efficiency and cell viability, no restrictions of probe or cell type, and operation simplicity. The commercial electroporation systems have been widely adopted in the past two decades while still carry drawbacks associated with the high applied electric voltage, unsatisfied delivery efficiency, and/or low cell viability. By adding highly conductive gold nanoparticles (AuNPs) in electroporation solution, we demonstrated enhanced electroporation performance (i.e., better DNA delivery efficiency and higher cell viability) on mammalian cells from two different aspects: the free, naked AuNPs reduce the resistance of the electroporation solution so that the local pulse strength on cells was enhanced; targeting AuNPs (e.g., Tf-AuNPs) were brought to the cell membrane to work as virtual microelectrodes to porate cells with limited area from many different sites. The enhancement was confirmed with leukemia cells in both a commercial batch electroporation system and a home-made flow-through system using pWizGFP plasmid DNA probes. Such enhancement depends on the size, concentration, and the mixing ratio of free AuNPs/Tf-AuNPs. An equivalent mixture of free AuNPs and Tf-AuNPs exhibited the best enhancement with the transfection efficiency increased 2-3 folds at minimum sacrifice of cell viability. This new delivery concept, the combination of nanoparticles and electroporation technologies, may stimulate various in vitro and in vivo biomedical applications which rely on the efficient delivery of nucleic acids, anticancer drugs, or other therapeutic materials.

  12. Cytokine balance and cytokine-driven natural killer cell dysfunction in systemic juvenile idiopathic arthritis.

    Science.gov (United States)

    Avau, Anneleen; Put, Karen; Wouters, Carine H; Matthys, Patrick

    2015-02-01

    Systemic juvenile idiopathic arthritis (sJIA) is a severe inflammatory childhood disorder, characterized by a specific pattern of systemic features and a typical cytokine profile. Patients are at risk to develop macrophage activation syndrome (MAS), an acute life-threatening condition defined by excessive proliferation and activation of macrophages and T cells. Defects of unknown cause in the natural killer (NK) cell cytotoxic capacity are presumed to underlie the pathogenesis of MAS and have been detected in sJIA patients. Here, we provide an overview of the cytokine profiles in sJIA and related mouse models. We discuss the influence of cytokines on NK cell function, and hypothesize that NK cell dysfunction in sJIA is caused by altered cytokine profiles.

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

    Science.gov (United States)

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

    2013-02-01

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

  14. Spatial and Temporal Control of Cavitation Allows High In Vitro Transfection Efficiency in the Absence of Transfection Reagents or Contrast Agents.

    Directory of Open Access Journals (Sweden)

    Kamel Chettab

    Full Text Available Sonoporation using low-frequency high-pressure ultrasound (US is a non-viral approach for in vitro and in vivo gene delivery. In this study, we developed a new sonoporation device designed for spatial and temporal control of ultrasound cavitation. The regulation system incorporated in the device allowed a real-time control of the cavitation level during sonoporation. This device was evaluated for the in vitro transfection efficiency of a plasmid coding for Green Fluorescent Protein (pEGFP-C1 in adherent and non-adherent cell lines. The transfection efficiency of the device was compared to those observed with lipofection and nucleofection methods. In both adherent and non-adherent cell lines, the sonoporation device allowed high rate of transfection of pEGFP-C1 (40-80%, as determined by flow cytometry analysis of GFP expression, along with a low rate of mortality assessed by propidium iodide staining. The transfection efficiency and toxicity of sonoporation on the non-adherent cell lines Jurkat and K562 were similar to those of nucleofection, while these two cell lines were resistant to transfection by lipofection. Moreover, sonoporation was used to produce three stably transfected human lymphoma and leukemia lines. Significant transfection efficiency was also observed in two fresh samples of human acute myeloid leukemia cells. In conclusion, we developed a user-friendly and cost-effective ultrasound device, well adapted for routine in vitro high-yield transfection experiments and which does not require the use of any transfection reagent or gas micro-bubbles.

  15. Paclitaxel tumor priming promotes delivery and transfection of intravenous lipid-siRNA in pancreatic tumors.

    Science.gov (United States)

    Wang, Jie; Lu, Ze; Wang, Junfeng; Cui, Minjian; Yeung, Bertrand Z; Cole, David J; Wientjes, M Guillaume; Au, Jessie L-S

    2015-10-28

    The major barrier for using small interfering RNA (siRNA) as cancer therapeutics is the inadequate delivery and transfection in solid tumors. We have previously shown that paclitaxel tumor priming, by inducing apoptosis, expands the tumor interstitial space, improves the penetration and dispersion of nanoparticles and siRNA-lipoplexes in 3-dimensional tumor histocultures, and promotes the delivery and transfection efficiency of siRNA-lipoplexes under the locoregional setting in vivo (i.e., intraperitoneal treatment of intraperitoneal tumors). The current study evaluated whether tumor priming is functional for systemically delivered siRNA via intravenous injection, which would subject siRNA to several additional delivery barriers and elimination processes. We used the same pegylated cationic (PCat)-siRNA lipoplexes as in the intraperitoneal study to treat mice bearing subcutaneous human pancreatic Hs766T xenograft tumors. The target gene was survivin, an inducible chemoresistance gene. The results show single agent paclitaxel delayed tumor growth but also significantly induced the survivin protein level in residual tumors, whereas addition of PCat-siSurvivin completely reversed the paclitaxel-induced survivin and enhanced the paclitaxel activity (p<0.05). In comparison, PCat-siSurvivin alone did not yield survivin knockdown or antitumor activity, indicating the in vivo effectiveness of intravenous siRNA-mediated gene silencing requires paclitaxel cotreatment. Additional in vitro studies showed that paclitaxel promoted the cytoplasmic release of siGLO, a 22 nucleotide double-stranded RNA that has no mRNA targets, from its PCat lipoplex and/or endosomes/lysosomes. Taken together, our earlier and current data show paclitaxel tumor priming, by promoting the interstitial transport and cytoplasmic release, is critical to promote the delivery and transfection of siRNA in vivo. In addition, because paclitaxel has broad spectrum activity and is used to treat multiple types

  16. Oscillating Magnet Array−Based Nanomagnetic Gene Transfection: A Valuable Tool for Molecular Neurobiology Studies

    Science.gov (United States)

    Subramanian, Mahendran; Tyler, Aimee-Jayne; Luther, Eva Maria; Daniel, Elena Di; Lim, Jenson; Dobson, Jon

    2017-01-01

    To develop treatments for neurodegenerative disorders, it is critical to understand the biology and function of neurons in both normal and diseased states. Molecular studies of neurons involve the delivery of small biomolecules into cultured neurons via transfection to study genetic variants. However, as cultured primary neurons are sensitive to temperature change, stress, and shifts in pH, these factors make biomolecule delivery difficult, particularly non-viral delivery. Herein we used oscillating nanomagnetic gene transfection to successfully transfect SH-SY5Y cells as well as primary hippocampal and cortical neurons on different days in vitro. This novel technique has been used to effectively deliver genetic material into various cell types, resulting in high transfection efficiency and viability. From these observations and other related studies, we suggest that oscillating nanomagnetic gene transfection is an effective method for gene delivery into hard-to-transfect neuronal cell types. PMID:28336862

  17. A novel human protease similar to the interleukin-1 beta converting enzyme induces apoptosis in transfected cells.

    Science.gov (United States)

    Faucheu, C; Diu, A; Chan, A W; Blanchet, A M; Miossec, C; Hervé, F; Collard-Dutilleul, V; Gu, Y; Aldape, R A; Lippke, J A

    1995-05-01

    We have identified a novel cDNA encoding a protein (named TX) with > 50% overall sequence identity with the interleukin-1 beta converting enzyme (ICE) and approximately 30% sequence identity with the ICE homologs NEDD-2/ICH-1L and CED-3. A computer homology model of TX was constructed based on the X-ray coordinates of the ICE crystal recently published. This model suggests that TX is a cysteine protease, with the P1 aspartic acid substrate specificity retained. Transfection experiments demonstrate that TX is a protease which is able to cleave itself and the p30 ICE precursor, but not to generate mature IL-1 beta from pro-IL-1 beta. In addition, this protein induces apoptosis in transfected COS cells. TX therefore delineates a new member of the growing Ice/ced-3 gene family coding for proteases with cytokine processing activity or involved in programmed cell death.

  18. Suppressive effect of FasL-transfected dendritic cells on experimental autoimmune uveoretinitis%FasL基因转染树突状细胞在抑制实验性自身免疫性葡萄膜炎中的作用

    Institute of Scientific and Technical Information of China (English)

    陈杰; 刘峰; 孙卫民; 宋斗

    2012-01-01

    Objectives To investigate the inhibitory effects of antigen-specific FasL-transfected dendritic cells (DCs) on the development of experimental autoimmune uveoretinitis (EAU). Methods: FasL-transfected DC (FasL-DC) and antigen-loaded FasL-DCs ("killer DCs") were generated from mouse mature DC (mDC) using ade-noviral gene transfer. The expression of FasL gene, survival, the mature phenotype of FasL-DC and T cell proliferation induced by FasL-DC were analyzed. EAU in C57BL/6 mice was induced by immunization with human in-terphotoreceptor retinoid-binding protein (hIRBP) R16 peptide. The effect of proliferation and apoptosis of primary T cells and activated primary T cells in cocultures with antigen-loaded FasL-DC,and the proliferation of activated primary T cells induced antigen-loaded FasL-DC in the presence of a blocking anti-FasL antibody were investigated in vitro. Results: Transduction of DCs resulted in FasL over expression without affecting the survival, maturation of phenotype, and T cell proliferation. The proliferation of primary T cells and activated primary T cells was inhibited in cocultures with antigen-loaded FasL-DC due to induction of apoptosis. The reduction in activated primary T cell proliferation could be blocked by an ami-FasL antibody. Conclusions: The results demonstrate that antigen-loaded FasL-DC eliminates autoreactive T cells in an antigen-specific manner, and manipulation of "killer DCs" might be considered to be a potential therapy for human uveitis with an autoimmune origin.%目的:探讨FasL基因转染的抗原特异性树突状细胞(DC)对实验性自身免疫性葡萄膜炎(EAU)的抑制作用.方法:用携带FasL基因的腺病毒转染DC和负载自体抗原——光感受器间维生素A类结合蛋白(IRBP)R16肽段的DC,制备FasL转染的DC(FasL-DC)和抗原负载FasL-DC(“杀伤性”DC).检测FasL基因在DC中的表达及其对DC存活、表型成熟和T细胞增殖的影响.通过IRBP和完全弗氏佐

  19. Osteo-/odontogenic differentiation of BMP2 and VEGF gene-co-transfected human stem cells from apical papilla.

    Science.gov (United States)

    Zhang, Wen; Zhang, Xiaolei; Ling, Junqi; Wei, Xi; Jian, Yutao

    2016-05-01

    groups following 16 days culture in vitro. In conclusion, lentiviral vector-mediated BMP2 and VEGF gene co-transfection significantly activated the osteo‑/odontogenic differentiation of human SCAP.

  20. Large-Scale mRNA Transfection of Dendritic Cells by Electroporation in Continuous Flow Systems

    DEFF Research Database (Denmark)

    Selmeczi, Dávid; Hansen, Thomas Steen; Met, Özcan

    2016-01-01

    Electroporation is well established for transient mRNA transfection of many mammalian cells, including immune cells such as dendritic cells used in cancer immunotherapy. Therapeutic application requires methods to efficiently electroporate and transfect millions of immune cells in a fast process...... the instrumentation and methods needed for the efficient transfection by electroporation of millions of dendritic cells in one continuous flow process....... with high cell survival. Continuous flow of suspended dendritic cells through a channel incorporating spatially separated microporous meshes with a synchronized electrical pulsing sequence can yield dendritic cell transfection rates of >75 % with survival rates of >90 %. This chapter describes...

  1. Electric pulse current stimulation increases electrophysiological properties of If current reconstructed in mHCN4-transfected canine mesenchymal stem cells.

    Science.gov (United States)

    Feng, Yuanyuan; Luo, Shouming; Yang, Pan; Song, Zhiyuan

    2016-04-01

    The 'funny' current, also known as the If current, play a crucial role in the spontaneous diastolic depolarization of sinoatrial node cells. The If current is primarily induced by the protein encoded by the hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) gene. The functional If channel can be reconstructed in canine mesenchymal stem cells (cMSCs) transfected with mouse HCN4 (mHCN4). Biomimetic studies have shown that electric pulse current stimulation (EPCS) can promote cardiogenesis in cMSCs. However, whether EPCS is able to influence the properties of the If current reconstructed in mHCN4-transfected cMSCs remains unclear. The present study aimed to investigate the effects of EPCS on the If current reconstructed in mHCN4-transfected cMSCs. The cMSCs were transfected with the lentiviral vector pLentis-mHCN4-GFP. Following transfection, these cells were divided into two groups: mHCN4-transfected cMSCs (group A), and mHCN4-transfected cMSCs induced by EPCS (group B). Using a whole cell patch-clamp technique, the If current was recorded, and group A cMSCs showed significant time and voltage dependencies and sensitivity to extracellular Cs+. The half-maximal activation (V1/2) value was -101.2±4.6 mV and the time constant of activation was 324±41 msec under -160 mV. In the group B cells the If current increased obviously and activation curve moved to right. The absolute value of V1/2 increased significantly to -92.4±4.8 mV (P<0.05), and the time constant of activation diminished under the same command voltage (251±44 vs. 324±41, P<0.05). In addition, the mRNA and protein expression levels of HCN4, connexin 43 (Cx43) and Cx45 were upregulated in group B compared with group A, as determined by reverse transcription-quantitative polymerase chain reaction and western blot analyses. Transmission electron micrographs also confirmed the increased gap junctions in group B. Collectively, these results indicated that reconstructed If channels may have a

  2. Biologic effect and immunoisolating behavior of BMP-2 gene-transfected bone marrow-derived mesenchymal stem cells in APA microcapsules.

    Science.gov (United States)

    Ding, H F; Liu, R; Li, B G; Lou, J R; Dai, K R; Tang, T T

    2007-11-03

    We investigated the encapsulation of BMP-2 gene-modified mesenchymal stem cells (MSCs) in alginate-poly-L-lysine (APA) microcapsules for the persistent delivery of bone morphogenic protein-2 (BMP-2) to induce bone formation. An electrostatic droplet generator was employed to produce APA microcapsules containing encapsulated beta-gal or BMP-2 gene-transfected bone marrow-derived MSCs. We found that X-gal staining was still positive 28 days after encapsulation. Encapsulated BMP-2 gene-transfected cells were capable of constitutive delivery of BMP-2 proteins for at least 30 days. The encapsulated BMP-2 gene-transfected MSCs or the encapsulated non-gene transfer MSCs (control group) were cocultured with the undifferentiated MSCs. The gene products from the encapsulated BMP-2 cells could induce the undifferentiated MSCs to become osteoblasts that had higher alkaline phosphatase (ALP) activity than those in the control group (pAPA microcapsules could inhibit the permeation of fluorescein isothiocyanate-conjuncted immunoglobulin G. Mixed lymphocyte reaction also indicates that the APA microcapsules could prevent the encapsulated BMP-2 gene-transfected MSCs from initiating the cellular immune response. These results demonstrated that the nonautologous BMP-2 gene-transfected stem cells are of potential utility for enhancement of bone repair and bone regeneration in vivo.

  3. Alterations in metastatic properties of hepatocellular carcinoma cell following H-ras oncogene transfection

    Institute of Scientific and Technical Information of China (English)

    Qing Wang; Zhi Ying Lin; Xiao Li Feng

    2001-01-01

    AIM To demonstrate the relationship betweenH-ras oncogene and hepatocellular carcinoma(HCC) metastasis.METHODS Activated H-ras oncogene wastransfected into SMMC 7721, a cell line derivedfrom human HCC, by calcium phosphatetransfection method. Some metastasis-relatedparameters were detected in vitro, includingadhesion assay, migration assay, expression ofcollagenase ⅣV (c ⅣV ase) and epidermal growthfactor receptor (EGFR).RESULTS The abilities of H-ras-transfected cellclones in adhesion to laminin (LN) or fibronectin(FN), migration, c Ⅳ ase secretion increasedmarkedly, and the expression of EGFR elevatedmoderately. More importantly, these alterationswere consistent positively with the expressionof p21, the protein product of H-ras oncogene.CONCLUSION H-ras oncogene could inducethe metastatic phenotype of HCC cell in vitro toraise its metastatic potential.

  4. Making memory at birth: understanding the differentiation of natural killer T cells.

    Science.gov (United States)

    Engel, Isaac; Kronenberg, Mitchell

    2012-04-01

    Glycolipid reactive natural killer T cells with an invariant TCR α-chain (iNKT cells) are a conserved population of T lymphocytes with a distinct anatomical distribution and functional properties. The differentiation pathway of iNKT cells branches off from mainstream thymocyte differentiation at the double positive stage, and recent work has revealed how signaling events early in the iNKT cell pathway imprint a memory-like behavior on these cells. Additionally, unique molecular interactions governing iNKT cell development and tissue distribution have been uncovered recently, building up our knowledge of the complex network of interactions that form this population. Novel autologous antigens for these cells have been identified, although it has not yet been resolved if there is single endogenous antigen responsible for both positive selection and/or peripheral activation.

  5. Invariant natural killer T cells recognize glycolipids from pathogenic Gram-positive bacteria.

    Science.gov (United States)

    Kinjo, Yuki; Illarionov, Petr; Vela, José Luis; Pei, Bo; Girardi, Enrico; Li, Xiangming; Li, Yali; Imamura, Masakazu; Kaneko, Yukihiro; Okawara, Akiko; Miyazaki, Yoshitsugu; Gómez-Velasco, Anaximandro; Rogers, Paul; Dahesh, Samira; Uchiyama, Satoshi; Khurana, Archana; Kawahara, Kazuyoshi; Yesilkaya, Hasan; Andrew, Peter W; Wong, Chi-Huey; Kawakami, Kazuyoshi; Nizet, Victor; Besra, Gurdyal S; Tsuji, Moriya; Zajonc, Dirk M; Kronenberg, Mitchell

    2011-09-04

    Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell antigen receptor (TCR), but the forces that drive TCR conservation have remained uncertain. Here we show that NKT cells recognized diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells were required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is present in low concentrations in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR and, most notably, extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.

  6. Altered natural killer cell biology in C57BL/6 mice after leukemogenic split-dose irradiation. [/sup 137/Cs

    Energy Technology Data Exchange (ETDEWEB)

    Parkinson, D.R.; Brightman, R.P.; Waksal, S.D.

    1981-04-01

    Natural killer (NK) cell activity was examined in the spleens of C57BL/6 mice given leukemogenic split-dose irradiation. The radiation protocol resulted in severe depression of spontaneous NK cell activity; this activity was not fully restored after treatment with the interferon inducer poly I:C. In vitro mixing studies provided no evidence for active suppression in vivo as a mechanism for this decrease in activity. In addition, spontaneous activity was restored towards control levels after bone marrow transfusion from nonirradiated mice. The results are most compatible with the radiation-induced loss of a cell with normal NK activity from spleen and bone marrow after the split-dose radiation protocol. In addition, a population of cells able to competitively block normal NK cell lysis of YAC-1 tumor cells is found in the bone marrow, spleen, and thymus of the irradiated mice lacking NK cell activity.

  7. Unraveling the genetic expression of the highly variable immune receptors of a killer

    NARCIS (Netherlands)

    Vendelbosch, S.

    2015-01-01

    The Killer Immunoglobulin-like Receptors (KIRs) are a family of highly variable receptors which regulate cytotoxicity of Natural Killer (NK) cells and a subset of T-cells. The KIR genes, clustered on the genome in the KIR locus, are distributed unequally across the population due to variation in gen

  8. Associations of Killer Cell Immunoglobulin-Like Receptor Genes with Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    S. Ramírez-De los Santos

    2012-01-01

    Full Text Available Objective: Rheumatoid Arthritis (RA is an autoimmune and chronic inflammatory disease of unknown etiology. Killer cell immunoglobulin-like receptors are expressed on the surface of natural killer cells and CD28null T-cells, both present in synovial membrane of RA. Therefore we evaluated the associations of KIR genes with RA.

  9. Suppression of a Natural Killer Cell Response by Simian Immunodeficiency Virus Peptides

    NARCIS (Netherlands)

    Schafer, Jamie L; Ries, Moritz; Guha, Natasha; Connole, Michelle; Colantonio, Arnaud D; Wiertz, EJ; Wilson, Nancy A; Kaur, Amitinder; Evans, David T

    2015-01-01

    Natural killer (NK) cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. We previously reporte

  10. Alteration of some cellular function in amikacin resistant Pseudomonas aeruginosa transfected macrophages:a time dependent approach

    Institute of Scientific and Technical Information of China (English)

    Subhankari Prasad Chakraborty; Santanu KarMahapatra; Sabyasachi Das; Somenath Roy

    2011-01-01

    To evaluate the free radical generation and antioxidant enzymes status in murine peritoneal macrophage during in vitro amikacin resistant Pseudomonas aeruginosa (ARPA) treatment with different time interval. Methods: Peritoneal macrophages were treated with 1×108 CFU/mL ARPA cell suspension in vitro for different time interval (1, 2, 3, 6, 12, and 24 h) and super oxide anion generation, NO generation, reduced glutathione level and antioxidant enzymes status were analyzed. Results: Super oxide anion generation and NO generation got peak at 12 h, indicating maximal free radical generation through activation of NADPH oxidase in murine peritoneal macrophages during ARPA transfection. Reduced glutathione level and antioxidant enzymes status were decreased significantly (P<0.05) with increasing time of ARPA transfection. All the changes in peritoneal macrophages after 12 h in vitro ARPA transfection had significant difference (P<0.05). Conclusions: From this study, it may be summarized that in vitro ARPA infection not only generates excess free radical but also affects the antioxidant system and glutathione cycle in murine peritoneal macrophage.

  11. 76 FR 20870 - Protective Regulations for Killer Whales in the Northwest Region Under the Endangered Species Act...

    Science.gov (United States)

    2011-04-14

    ... Killer Whales in the Northwest Region Under the Endangered Species Act and Marine Mammal Protection Act... from approaching killer whales within 200 yards (182.9 m) and from parking in the path of whales when... of this final rule is to protect killer whales from interference and noise associated with...

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  20. Natural killer cells in highly exposed hepatitis C-seronegative injecting drug users.

    Science.gov (United States)

    Mina, M M; Cameron, B; Luciani, F; Vollmer-Conna, U; Lloyd, A R

    2016-06-01

    Injecting drug use remains the major risk factor for hepatitis C (HCV) transmission. A minority of long-term injecting drug users remain seronegative and aviraemic, despite prolonged exposure to HCV - termed highly exposed seronegative subjects. Natural killer (NK) cells have been implicated in this apparent protection. A longitudinal nested, three group case-control series of subjects was selected from a prospective cohort of seronegative injecting drug users who became incident cases (n = 11), remained seronegative (n = 11) or reported transient high-risk behaviour and remained uninfected (n = 11). The groups were matched by age, sex and initial risk behaviour characteristics. Stored peripheral blood mononuclear cells were assayed in multicolour flow cytometry to enumerate natural killer cell subpopulations and to assess functional activity using Toll-like receptor ligands before measurement of activation, cytokine production and natural cytotoxicity receptor expression. Principal components were derived to describe the detailed phenotypic characteristics of the major NK subpopulations (based on CD56 and CD16 co-expression), before logistic regression analysis to identify associations with exposed, seronegative individuals. The CD56(dim) CD16(+) (P = 0.05, OR 6.92) and CD56(dim) CD16(-) (P = 0.05, OR 6.07) principal components differed between exposed, seronegative individuals and pre-infection samples of the other two groups. These included CD56(dim) CD16(+) and CD56(dim) CD16(-) subsets with CD56(dim) CD16(+) IFN-γ and TNF-α on unstimulated cells, and CD56(dim) CD16(-) CD69(+) , CD107a(+) , IFN-γ and TNF-α following TLR stimulation. The cytotoxic CD56(dim) NK subset thus distinguished highly exposed, seronegative subjects, suggesting NK cytotoxicity may contribute to protection from HCV acquisition. Further investigation of the determinants of this association and prospective assessment of protection against HCV infection are warranted.

  1. IMPROVING P-gp EXPRESSION IN HUMAN MONONUCLEAR CELLS IN VITRO TRANSFECTED BY MULTIDRUG RESISTANCE-1 mRNA

    Institute of Scientific and Technical Information of China (English)

    Yang Xiang; Lei Li; Fang Tian; Xiu-yu Yang

    2005-01-01

    Objective To evaluate the expression and function activity of P-glycoprotein (P-gp) in human mononuclear cells (MNCs) in vitro transfected by multidrug resistance-1 (MDR1) mRNA.Methods Two MDR1 cDNA vectors, pT7TS_MDR1 and pGEM5Zf(+)_MDR1, were constructed and transcripted in vitro. Vector pGEM5Zf(+)_MDR1 only contained the coding region ofmdrl cDNA, and pT7TS_MDR1 also included Xeponus β-globin 5' and 3' untranslated region. MNCs were prepared from peripheral blood of parvicellular lung cancer patient. The two human mdrl mRNAs were then transferred into human MNCs in vitro by DOTAP. And the expression efficiency and pump functon of P-gp were measured with flow cytometry.Results Expression of P-gp significantly elevated in both transferred cells compared with untransferred cells (P< 0.01).And pT7TS_MDR1 showed higher capability in elevating the expression of P-gp than pGEM5Zf(+)_MDR1 (P < 0.01). The P-gp function was elevated in both pT7TS_MDR1 and pGEM5Zf(+)_MDR1 groups. The survival ratio of MNCs in erythrocyte-lysis-solution (ELS, 86.07%) and lymphocyte-isolation-solution (LIS, 83.67%) had no significant difference.The CD34+ cells content of the MNCs used for transfection was 2.65% and 1.01% in ELS and LIS group, respectively (P <0.01).Conclusions It is a feasible approach to improve P-gp expression in human MNCs by transfection of MDR-1 mRNA.And the ELS may be more suitable for purifing MNCs for mRNA transfection than LIS.

  2. A novel cell subset:Interferon-producing killer dendritic cells

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Recent reports introduce a novel cell subset of DCs with antigenic phenotypes shared by both NK cells and B cells, but without surface markers of pDCs and T cells, appearing to be a chimera of NK cells and DCs, namely interferon-producing killer dendritic cells(IKDCs).IKDCs not only secret type I and type II interferons to recognize and kill tumor cells effectively, but also express MHC-II molecules to present antigens.Thus, IKDCs are considered as important immunosurveilance cells for tumors, providing a link between innate and adaptive immunity.

  3. The Need for Killer Examples for Object-Oriented Frameworks

    DEFF Research Database (Denmark)

    Caspersen, Michael Edelgaard; Christensen, Henrik Bærbak

    2003-01-01

    In this paper, we argue in favor of introducing object-oriented frameworks as an important topic in our software engineering teaching. Frameworks provide a basis for students to build interesting and impressive programs even with small programming effort at the introductory level. Frameworks...... of publications and literature about teaching frameworks, it seems that few has realized the potential---it is an under-utilized technology. We therefore suggest that killer examples are highly needed and present some experiences with two frameworks that we have found useful in our teaching....

  4. Google+ is Google's Facebook killer doomed to fail?

    CERN Document Server

    Dino, G

    2012-01-01

    Do you have a Facebook profile? How about a Google+ page? Which do you prefer: Facebook or Google+? You may have both, but not use one as often as the other. You may have just one while waiting for the right time to join the other. The question is: When is the right time? Are you waiting to see which one is the better product? This issue has been the talk of the cybersphere since Google+ was released - will it be the Facebook killer that a number of other social media sites in the past have failed to become?

  5. Natural killer cell cytotoxicity assay with time-resolved fluorimetry

    Institute of Scientific and Technical Information of China (English)

    李建中; 章竹君; 金伯泉; 田方

    1996-01-01

    A new time-resolved fluorimetric method for the measurement of natural killer (NK) cell cytotoxicity has been developed by labelling the target cell K562 with a new synthesized fluorescence marker KLUK. The method has advantages of higher sensitivity, time-saving, good reproducibility and has no radioactivity problems. A satisfactory result is obtained by comparing it with 51Cr release method. It demonstrates that the new marker provides an alternative to currently used radioactive markers for the assessment of in vitro cellular cytotoxicity.

  6. Three faces of mortalin: a housekeeper, guardian and killer.

    Science.gov (United States)

    Kaul, Sunil C; Deocaris, Custer C; Wadhwa, Renu

    2007-04-01

    Mortalin was first cloned as a mortality factor that existed in the cytoplasmic fractions of normal, but not in immortal, mouse fibroblasts. A decade of efforts have expanded its persona from a house keeper protein involved in mitochondrial import, energy generation and chaperoning of misfolded proteins, to a guardian of stress that has multiple binding partners and to a killer protein that contributes to carcinogenesis on one hand and to old age disorders on the other. Being proved to be an attractive target for cancer therapy, it also warrants attention from the perspectives of management of old age diseases and healthy aging.

  7. Natural Killer p46 Controls Hepatitis B Virus Replication and Modulates Liver Inflammation.

    Directory of Open Access Journals (Sweden)

    Wanyu Li

    Full Text Available Natural killer (NK cells play an important role in hepatitis B virus (HBV infection control, and are regulated by a complex network of activating and inhibitory receptors. However, NK cell activity in HBV patients remains poorly understood. The objective of this study was to investigate the phenotypic and functional characteristics of circulating NK cells in patients during different chronic hepatitis B (CHB infection stages. We investigated NK cell phenotypes, receptor expression and function in 86 CHB patients and 20 healthy controls. NK cells were purified and NK cell subsets were characterized by flow cytometry. Cytotoxic activity (CD107a and interferon-gamma (IFN-γ secretion were examined, and Natural Killer p46 (NKP46 blockade and spontaneous NK cell cytolytic activity against K562, HepG2 and HepG2.215 cell lines was studied. Activating NKp46 receptor expression was higher in inactive HBsAg carriers when compared with other groups (p = 0.008. NKp46 expression negatively correlated with HBV DNA (R = -0.253, p = 0.049 and ALT (R = -0.256, p = 0.045 levels. CD107a was higher in immune-activated groups when compared with immune-tolerant groups (p = 0.039. CD107a expression was related to viral load (p = 0.02 and HBeAg status (p = 0.024. In vitro NKp46 blockade reduced NK cell cytolytic activity against HepG2 and HepG2.215 cell lines (p = 0.02; p = 0.039. Furthermore, NK cells from high viral load CHB patients displayed significantly lower specific cytolytic activity against anti-NKp46-loaded K562 targets (p = 0.0321. No significant differences were observed in IFN-γ secretion (p > 0.05. In conclusion, NKp46 expression regulates NK cell cytolytic function. NKp46 may moderate NK cell activity during HBV replication suppression and HBV-associated liver damage and may be critical for NK cell activity during CHB infection.

  8. Transfection of small numbers of human endothelial cells by electroporation and synthetic amphiphiles

    NARCIS (Netherlands)

    van Leeuwen, E B; van der Veen, A Y; Hoekstra, D; Engberts, J B; Halie, M R; van der Meer, J; Ruiters, M H

    1999-01-01

    OBJECTIVES: This study compared the efficiency of electroporation and synthetic amphiphiles. (SAINT-2pp/DOPE) in transfecting small numbers of human endothelial cells. METHODS AND RESULTS: Optimal transfection conditions were tested and appeared to be 400 V and 960 microF for electroporation and a 1

  9. Effect of NCAM-transfection on growth and invasion of a human cancer cell line

    DEFF Research Database (Denmark)

    Edvardsen, K; Bock, E; Jirus, S

    1997-01-01

    A cDNA encoding the human transmembrane 140 kDa isoform of the neural cell adhesion molecule (NCAM) was transfected into the highly invasive MDA-MB-231 human breast cancer cell line. Transfectants with a homogeneous expression of NCAM showed a restricted capacity for penetration of an artificial...... of modulating NCAM expression in vivo. In nude mice, NCAM-transfected cells developed tumors with longer latency periods and slower growth rates than tumors induced by NCAM-negative control cells, implying that NCAM may be involved not only in adhesive and motile behavior of tumor cells but also in their growth...... regulation. There was no indication of differences in cell proliferative characteristics between the different NCAM-transfected and the control transfected cells as determined by flow cytometric DNA analysis, suggesting an increased cell loss as the reason for decreased in vivo growth rate of the NCAM...

  10. Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

    Energy Technology Data Exchange (ETDEWEB)

    Wong-Baeza, Carlos; Bustos, Israel; Serna, Manuel; Tescucano, Alonso; Alcantara-Farfan, Veronica; Ibanez, Miguel [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico); Montanez, Cecilia [Department of Genetics and Molecular Biology, Centre for Research and Advanced Studies (CINVESTAV), IPN, Mexico City 07360 (Mexico); Wong, Carlos [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico); Baeza, Isabel, E-mail: ibaeza@encb.ipn.mx [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico)

    2010-05-28

    Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.

  11. The effects of antisense PTEN gene transfection on the growth and invasion of glioma cells

    Institute of Scientific and Technical Information of China (English)

    CHEN Hong-jie; ZHENG Zhao-cong; WANG Ru-mi; WANG Shou-sen; YANG Wei-zhong

    2006-01-01

    Objective:To study the effects of antisense PTEN gene on the growth and invasion of glioma cells. Methods:A pcDNA3. 1/Hygro (-) recombinant plasmid containing antisense PTEN gene fragment was constructed. Glioma cells of primary culture were transfected with antisense PTEN gene vector and stably transfected clones were selected. Then, the different growth and invasion abilities and the different MMP9 mRNA expressions of three kinds of cells were observed, including the transfected cells, untransfected cells and the cells transfected with empty vector. Results :The abilities of growth and invasion of the transfected cells and the expressions of MMP9 mRNA were obviously enhanced. Conclusion: Antisense PTEN gene could have a negative impact on the growth and invasion of primary culture glioma cells.

  12. Regulation of Murine Natural Killer Cell Commitment

    Directory of Open Access Journals (Sweden)

    Nicholas D Huntington

    2013-01-01

    Full Text Available NK cells can derive from the same precursors as B and T cells, however to achieve lineage specificity, several transcription factors need to be activated or annulled. While a few important transcription factors have identified for NK genesis the mechanisms of how this is achieved is far from resolved. Adding to the complexity of this, NK cells are found and potentially develop in diverse locations in vivo and it remains to be addressed if a common NK cell precursor seeds diverse niches and how transcription factors may differentially regulate NK cell commitment in distinct microenvironments. Here we will summarise some recent findings in NK cell commitment and discuss how a NK cell transcriptional network might be organised, while addressing some misconceptions and anomalies along the way.

  13. Enhancement of transfection efficiency for HeLa cells via incorporating arginine moiety into chitosan

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Arginine-rich peptides have attracted considerable attention due to their distinct internalization mechanism. It was reported that arginine and guanidino moieties were able to translocate through cell membranes and played a critical role in the process of membrane permeation. In this work, arginine was conjugated to the backbone of chitosan to form a novel chitosan derivative, arginine modified chitosan (Arg-CS). Arg-CS/DNA complexes were prepared according to the method of coacervation process. The physicochemical properties of Arg-CS and Arg-CS/DNA complexes were characterized and the transfection activity and efficiency mediated by Arg-CS/DNA complexes were investigated taking HeLa cells as target cells. Arg-CS was characterized by FTIR and 13C NMR. Arg-CS/DNA polyelectrolyte complexes were investigated by agarose gel retardation, dynamic light scattering (DLS) and atomic force microscopy (AFM). The results revealed that the Arg-CS/DNA complexes started to form at N/P ratio of 2:1, and the size of particles varied from 100 to 180 nm. The cytotoxicity of Arg-CS and their complexes with plasmid DNA were determined by MTT assay for HeLa cells, and the results suggested that Arg-CS/DNA complexes were slightly less toxic than Arg-CS. Moreover, the derivative alone and their complexes showed significantly lower toxicity than PEI and PEI/DNA complexes, respectively. Taking HeLa cells as target cells and using pGL3-control as reporter gene, the luciferase expression mediated by Arg-CS was greatly enhanced to about 100 folds compared with the luciferase expression mediated by chitosan at different pH media. These results suggest that Arg-CS is a promising candidate as a safe and efficient vector for gene delivery and transfection.

  14. Development of sociality and emergence of independence in a killer whale (Orcinus orca) calf from birth to 36 months.

    Science.gov (United States)

    Guarino, Sara; Hill, Heather M; Sigman, Julie

    2017-01-01

    Dolphin calves spend most of their time swimming with their mother immediately after birth. As they mature, the calves become increasingly independent, and begin to interact more often with other calves, juveniles, and sub-adults. For bottlenose dolphin calves, sociality is related to maternal behaviors. Unfortunately, much less is known about the development of sociality and emergence of independence for killer whale calves. The purpose of this study was to examine the developmental changes in social behaviors and solitary activities of a killer whale calf across a 36-month period. Focal follow video recordings of a mother-calf pair housed at SeaWorld San Antonio were collected 2-6 times a day for 5-15 min at 6-month intervals. Using a sample of randomly selected video recordings at each month, developmental changes in swims and social interactions with her mother, swims and social interactions with non-maternal partners, and solitary activities (e.g., solitary swims, solitary play) were observed across the months. The calf spent most of her time swimming with the mother across the 36-month period. The time the calf socialized with her mother was greater than the time she socialized with others at each month. Besides her mother, the calf socialized more often with the other adult female compared to adult males. As the calf matured, the increase in the time she spent socializing with adult killer whales other than the mother corresponded with an increase in the rate and time spent in solitary play. The developmental trends of sociality and emerging independence replicate research conducted with calves of other dolphin species. Zoo Biol. 36:11-20, 2017. © 2016 Wiley Periodicals, Inc.

  15. The relationship between vessel traffic and noise levels received by killer whales (Orcinus orca)

    Science.gov (United States)

    Houghton, Juliana; Holt, Marla M.; Giles, Deborah A.; Hanson, M. Bradley; Emmons, Candice K.; Hogan, Jeffrey T.; Branch, Trevor A.; Vanblaricom, Glenn R.

    2015-01-01

    Whale watching has become increasingly popular as an ecotourism activity around the globe and is beneficial for environmental education and local economies. Southern Resident killer whales (Orcinus orca) comprise an endangered population that is frequently observed by a large whale watching fleet in the inland waters of Washington state and British Columbia. One of the factors identified as a risk to recovery for the population is the effect of vessels and associated noise. An examination of the effects of vessels and associated noise on whale behavior utilized novel equipment to address limitations of previous studies. Digital acoustic recording tags (DTAGs) measured the noise levels the tagged whales received while laser positioning systems allowed collection of geo-referenced data for tagged whales and all vessels within 1000 m of the tagged whale. The objective of the current study was to compare vessel data and DTAG recordings to relate vessel traffic to the ambient noise received by tagged whales. Two analyses were conducted, one including all recording intervals, and one that excluded intervals when only the research vessel was present. For all data, significant predictors of noise levels were length (inverse relationship), number of propellers, and vessel speed, but only 15% of the variation in noise was explained by this model. When research-vessel-only intervals were excluded, vessel speed was the only significant predictor of noise levels, and explained 42% of the variation. Simple linear regressions (ignoring covariates) found that average vessel speed and number of propellers were the only significant correlates with noise levels. We conclude that vessel speed is the most important predictor of noise levels received by whales in this study. Thus, measures that reduce vessel speed in the vicinity of killer whales would reduce noise exposure in this population.

  16. Midline 1 directs lytic granule exocytosis and cytotoxicity of mouse killer T cells

    DEFF Research Database (Denmark)

    Boding, Lasse; Hansen, Ann K; Meroni, Germana

    2014-01-01

    upregulated in murine cytotoxic lymphocytes (CTLs), and that it controls TCR signaling, centrosome trafficking and exocytosis of lytic granules. In accordance, we find that the killing capacity of MID1(-/-) CTLs is impaired. Transfection of MID1 into MID1(-/-) CTLs completely rescued lytic granule exocytosis......, and vice versa, knock-down of MID1 inhibited exocytosis of lytic granules in wild-type CTLs, cementing a central role for MID1 in the regulation of granule exocytosis. Thus, MID1 orchestrates multiple events in CTL responses, adding a novel level of regulation to CTL activation and cytotoxicity...

  17. Green fluorescent protein (GFP color reporter gene visualizes parvovirus B19 non-structural segment 1 (NS1 transfected endothelial modification.

    Directory of Open Access Journals (Sweden)

    Thomas Wurster

    Full Text Available BACKGROUND: Human Parvovirus B19 (PVB19 has been associated with myocarditis putative due to endothelial infection. Whether PVB19 infects endothelial cells and causes a modification of endothelial function and inflammation and, thus, disturbance of microcirculation has not been elucidated and could not be visualized so far. METHODS AND FINDINGS: To examine the PVB19-induced endothelial modification, we used green fluorescent protein (GFP color reporter gene in the non-structural segment 1 (NS1 of PVB19. NS1-GFP-PVB19 or GFP plasmid as control were transfected in an endothelial-like cell line (ECV304. The endothelial surface expression of intercellular-adhesion molecule-1 (CD54/ICAM-1 and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147 were evaluated by flow cytometry after NS-1-GFP or control-GFP transfection. To evaluate platelet adhesion on NS-1 transfected ECs, we performed a dynamic adhesion assay (flow chamber. NS-1 transfection causes endothelial activation and enhanced expression of ICAM-1 (CD54: mean ± standard deviation: NS1-GFP vs. control-GFP: 85.3 ± 11.2 vs. 61.6 ± 8.1; P<0.05 and induces endothelial expression of EMMPRIN/CD147 (CD147: mean ± SEM: NS1-GFP vs. control-GFP: 114 ± 15.3 vs. 80 ± 0.91; P<0.05 compared to control-GFP transfected cells. Dynamic adhesion assays showed that adhesion of platelets is significantly enhanced on NS1 transfected ECs when compared to control-GFP (P<0.05. The transfection of ECs was verified simultaneously through flow cytometry, immunofluorescence microscopy and polymerase chain reaction (PCR analysis. CONCLUSIONS: GFP color reporter gene shows transfection of ECs and may help to visualize NS1-PVB19 induced endothelial activation and platelet adhesion as well as an enhanced monocyte adhesion directly, providing in vitro evidence of possible microcirculatory dysfunction in PVB19-induced myocarditis and, thus, myocardial tissue damage.

  18. Occurrence of nodular lymphocyte-predominant hodgkin lymphoma in hermansky-pudlak type 2 syndrome is associated to natural killer and natural killer T cell defects.

    Directory of Open Access Journals (Sweden)

    Luisa Lorenzi

    Full Text Available Hermansky Pudlak type 2 syndrome (HPS2 is a rare autosomal recessive primary immune deficiency caused by mutations on β3A gene (AP3B1 gene. The defect results in the impairment of the adaptor protein 3 (AP-3 complex, responsible for protein sorting to secretory lysosomes leading to oculo-cutaneous albinism, bleeding disorders and immunodeficiency. We have studied peripheral blood and lymph node biopsies from two siblings affected by HPS2. Lymph node histology showed a nodular lymphocyte predominance type Hodgkin lymphoma (NLPHL in both HPS2 siblings. By immunohistochemistry, CD8 T-cells from HPS2 NLPHL contained an increased amount of perforin (Prf + suggesting a defect in the release of this granules-associated protein. By analyzing peripheral blood immune cells we found a significant reduction of circulating NKT cells and of CD56(brightCD16(- Natural Killer (NK cells subset. Functionally, NK cells were defective in their cytotoxic activity against tumor cell lines including Hodgkin Lymphoma as well as in IFN-γ production. This defect was associated with increased baseline level of CD107a and CD63 at the surface level of unstimulated and IL-2-activated NK cells. In summary, these results suggest that a combined and profound defect of innate and adaptive effector cells might explain the susceptibility to infections and lymphoma in these HPS2 patients.

  19. Cord Blood as a Source of Natural Killer Cells

    Directory of Open Access Journals (Sweden)

    Rohtesh S Mehta

    2016-01-01

    Full Text Available Cord blood (CB offers several unique advantages as a graft source for hematopoietic stem cell transplantation (HSCT. The risk of relapse and graft-versus-host disease (GVHD after cord blood transplantation (CBT are lower than what is typically observed after other graft sources with a similar degree of human leukocyte antigen (HLA mismatch. Natural killer (NK cells have a well-defined role in both innate and adaptive immunity and as the first lymphocytes to reconstitute after HSCT and CBT, they play a significant role in protection against early relapse. In this article, we highlight the uses of CB NK cells in transplantation and adoptive immunotherapy. First, we will describe differences in the phenotype and functional characteristics of NK cells in CB as compared with peripheral blood. Then, we will review some of the obstacles we face in using resting CB NK cells for adoptive immunotherapy, and discuss methods to overcome them. We will review the current literature on killer-cell immunoglobulin-like receptors (KIR-ligand mismatch and outcomes after CBT. Finally, we will touch on current strategiesfor the use of CB NK cells in cellular immunotherapy.

  20. Energy Security: From Deal Killers to Game Changers

    Science.gov (United States)

    Cooke, Charlie

    2010-03-01

    Five energy security ``deal killers" are identified: 1) Global warming and CO2 emissions from fossil fuel combustion; 2) Intermittent energy sources (wind, solar) and the presence and stability of the grid; 3) Penetration of plant defenses to produce transportation fuels from biomass; 4) Mimicking nature: artificial photosynthesis for solar energy to fuels; and 5) Spent fuel from nuclear power reactors. Transformational basic research is required to successfully change the ground rules, to transform these ``deal killers" into ``game changers." T hey are: 1) Offsetting carbon capture and storage costs through enhanced oil recovery and methane generation from high temperature geothermal saline aquifers; 2) Electrical energy storage, through batteries and super-capacitors; 3) Genetic modification of plant cell walls, and catalytic methods for transforming plant sugars into fuels; 4) Separation of solar-induced electrons from holes, and catalysis to produce fuels; and 5) Closing the nuclear fuel cycle. Basic research can revolutionize our approach to carbon-free energy by enhancing nature to achieve energy security.

  1. Interrogating the mirror : double-crossings in Hemingway's "The killers"

    Directory of Open Access Journals (Sweden)

    Steven Carter

    1997-12-01

    Full Text Available Among the early critics who commented on the discrepancies between what "is" and what "seems" in "The Killers" was Edward C. Sampson, who pointed out in 1952 that very little in the story is what it appears to be: Henry's, which serves no alcoholic beverages, is a converted tavern run by George; AI and Max order breakfast at a lunch counter whose menu features dinner; the orders are mixed up; the time on the clock is wrong; Mrs. Hirsch runs Mrs. Bell's rooming house; and so on (item 2. Here Sampson let the matter rest, and most subsequent commentators have been content to see these discrepancies as "framing" the portrait of the disillusioned Nick Adams, who by story's end finds that the world of appearances conceals a terrifying reality beyond his imagination. There is, however, more to the narrative poetics of "The Killers" than a handful of discrepancies, which - significant as they are - do not explain the mechanics or, for that matter, the presence of complementarity throughout the story.

  2. Underwater audiogram of a false killer whale (Pseudorca crassidens).

    Science.gov (United States)

    Thomas, J; Chun, N; Au, W; Pugh, K

    1988-09-01

    Underwater audiograms are available for only a few odontocete species. A false killer whale (Pseudorca crassidens) was trained at Sea Life Park in Oahu, Hawaii for an underwater hearing test using a go/no-go response paradigm. Over a 6-month period, auditory thresholds from 2-115 kHz were measured using an up/down staircase psychometric technique. The resulting audiogram showed hearing sensitivities below 64 kHz similar to those of belugas (Delphinapterus leucas) and Atlantic bottlenosed dolphins (Tursiops truncatus). Above 64 kHz, this Pseudorca had a rapid decrease in sensitivity of about 150 dB per octave. A similar decrease in sensitivity occurs at 32 kHz in the killer whale, at 50 kHz in the Amazon River dolphin, at 120 kHz in the beluga, at 140 kHz in the bottlenosed dolphin, and at 140 kHz in the harbor porpoise. The most sensitive range of hearing was from 16-64 kHz (a range of 10 dB from the maximum sensitivity). This range corresponds with the peak frequency of echolocation pulses recorded from captive Pseudorca.

  3. Comparing the immunosuppressive potency of naïve marrow stromal cells and Notch-transfected marrow stromal cells

    Directory of Open Access Journals (Sweden)

    Dao Mo A

    2011-10-01

    Full Text Available Abstract Background SB623 cells are expanded from marrow stromal cells (MSCs transfected with a Notch intracellular domain (NICD-expressing plasmid. In stroke-induced animals, these cells reduce infarct size and promote functional recovery. SB623 cells resemble the parental MSCs with respect to morphology and cell surface markers despite having been in extended culture. MSCs are known to have immunosuppressive properties; whether long-term culture of MSCs impact their immunomodulatory activity has not been addressed. Methods To assess the possible senescent properties of SB623 cells, we performed cell cycle related assays and beta-galactosidase staining. To assess the immunomodulatory activity of these expanded NICD-transfected MSCs, we performed co-cultures of SB623 cells or MSCs with either enriched human T cells or monocytes and assessed cytokine production by flow cytometry. In addition, we monitored the immunosuppressive activity of SB623 cells in both allogenic and xenogenic mixed lymphocyte reaction (MLR. Results Compared to MSCs, we showed that a small number of senescent-like cells appear in each lot of SB623 cells. Nevertheless, we demonstrated that these cells suppress human T cell proliferation in both the allogeneic and xenogeneic mixed lymphocyte reaction (MLR in a manner comparable to MSCs. IL-10 producing T cells were generated and monocyte-dendritic cell differentiation was dampened by co-culture with SB623 cells. Compared to the parental MSCs, SB623 cells appear to exert a greater inhibitory impact on the maturation of dendritic cells as demonstrated by a greater reduction in the surface expression of the co-stimulatory molecule, CD86. Conclusion The results demonstrated that the immunosuppressive activity of the expanded NICD-transfected MSCs is comparable to the parental MSCs, in spite of the appearance of a small number of senescent-like cells.

  4. Hemocompatible pullulan-polyethyleneimine conjugates for liver cell gene delivery: In vitro evaluation of cellular uptake, intracellular trafficking and transfection efficiency.

    Science.gov (United States)

    Rekha, M R; Sharma, Chandra P

    2011-01-01

    Polyethyleneimine (PEI; 25 kDa)-conjugated pullulans (PPE1, PPE2 and PPE3) were developed and investigated for possible use in gene delivery applications. The cytotoxicity, blood component interactions such as red blood cell/white blood cell aggregation, platelet and complement activation, and protein interaction of the pullulan-conjugated PEI was drastically reduced in comparison to PEI-based nanocomplexes. Based on the blood compatibility studies, PPE1 was selected for further study. The buffering capacity of this derivative was similar to that of PEI, which plays an important role in efficient gene transfection. The particle size, zeta potential, stability in the presence of plasma and resistance to nuclease degradation were evaluated. In addition, cellular uptake and localization of plasmid, as well as transgene expression, were evaluated following in vitro transfection of HepG2 cells. Endocytosis inhibitors, confocal laser scanning microscopy and fluorescent labeling techniques were used to visualize the nanoplex uptake mechanism, cellular distribution and nuclear localization. The results from inhibitor experiments in the presence of asialofetuin indicated that the asialoglycoprotein receptor is involved in transfection of hepatocytes with pullulan-PEI complexes. The conjugation of pullulan with PEI did not hinder the plasmid nuclear localization ability of PEI. The transfection efficiency of pullulan conjugate was similar to PEI, with the added advantage of hemocompatibility and non-cytotoxicity. The transfection efficiency of PEI and PPE1 was 1.6- and 2-fold more, respectively, in the presence of serum than in the absence of serum. Therefore, the pullulan-PEI conjugate seems to be a promising gene delivery vector with good hemocompatibility and low toxicity but without compromising the transfection efficacy of PEI.

  5. Factors that affect the efficiency of antisense oligodeoxyribonucleotide transfection by insonated gas-filled lipid microbubbles

    Science.gov (United States)

    Zhao, Ying-Zheng; Lu, Cui-Tao

    2008-03-01

    Objective: To investigate the factors that affect the efficiency of antisense oligodeoxyribonucleotide(AS-ODNs) transfection by insonated gas-filled lipid microbubbles. Methods: Lipid microbubbles filled with two types of gases-air and C3F8, were prepared respectively. An AS-ODNs sequence HA824 and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of insonated microbubbles. Two mixing methods, three levels of mixing speed, different mixing durations and various ultrasound initiation time after mixing were examined respectively. Transfection efficiency was detected by fluorescence microscopy. Results: C3F8 microbubbles gave higher levels of AS-ODNs transfection efficiency than air microbubbles in all test conditions. Transfection efficiency resulted from mixing method A (incubation of HA824 and microbubbles before mixing cells) did not show significant difference with that of mixing method B (without incubation of HA824 and microbubbles before mixing cells). Mixing speed, duration of mixing and ultrasound initiation time after mixing were central to determining HA824 transfection efficiency in vitro. The optimum parameters for SK-BR-3 cells were found at a mixing speed of 40-50 rpm for 30-60 s with less than 60 s delay before ultrasound. Conclusion: Ultrasound-mediated AS-ODNs transfection enhanced by C3F8-filled lipid microbubbles represents an effective avenue for AS-ODNs transfer.

  6. Factors that affect the efficiency of antisense oligodeoxyribonucleotide transfection by insonated gas-filled lipid microbubbles

    Energy Technology Data Exchange (ETDEWEB)

    Zhao Yingzheng [General Hospital of Beijing Military Command of PLA, Department of Clinical Pharmacology (China)], E-mail: lctuua@yahoo.com.cn; Lu Cuitao [Madam Medical Management Group (China)

    2008-03-15

    Objective: To investigate the factors that affect the efficiency of antisense oligodeoxyribonucleotide(AS-ODNs) transfection by insonated gas-filled lipid microbubbles. Methods: Lipid microbubbles filled with two types of gases-air and C{sub 3}F{sub 8}, were prepared respectively. An AS-ODNs sequence HA824 and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of insonated microbubbles. Two mixing methods, three levels of mixing speed, different mixing durations and various ultrasound initiation time after mixing were examined respectively. Transfection efficiency was detected by fluorescence microscopy. Results: C{sub 3}F{sub 8} microbubbles gave higher levels of AS-ODNs transfection efficiency than air microbubbles in all test conditions. Transfection efficiency resulted from mixing method A (incubation of HA824 and microbubbles before mixing cells) did not show significant difference with that of mixing method B (without incubation of HA824 and microbubbles before mixing cells). Mixing speed, duration of mixing and ultrasound initiation time after mixing were central to determining HA824 transfection efficiency in vitro. The optimum parameters for SK-BR-3 cells were found at a mixing speed of 40-50 rpm for 30-60 s with less than 60 s delay before ultrasound. Conclusion: Ultrasound-mediated AS-ODNs transfection enhanced by C{sub 3}F{sub 8}-filled lipid microbubbles represents an effective avenue for AS-ODNs transfer.

  7. Mechanistic investigations and molecular medicine applications of gold nanoparticle mediated (GNOME) laser transfection

    Science.gov (United States)

    Schomaker, M.; Heinemann, D.; Kalies, S.; Willenbrock, S.; Murua Escobar, H.; Buch, A.; Sodeik, B.; Ripken, T.; Meyer, H.

    2014-03-01

    Alternative high throughput transfection methods are required to understand the molecular network of the cell, which is linked to the evaluation of target genes as therapeutic agents. Besides diagnostic purposes, the transfection of primary- and stem cells is of high interest for therapeutic use. Here, the cell release of trans- or exogene proteins is used to develop immune cancer therapies. The basic requirement to accomplish manipulation of cells is an efficient and gentle transfection method. Therefore, we developed an automatized cell manipulation platform providing high throughput by using GNOME laser transfection. Herein, the interaction of moderately focused laser pulses with gold nanoparticles in close vicinity to the cell membrane mediate transient membrane permeabilization. The exact nature of the involved permeabilization effects depends on the applied particles and laser parameters. Hereinafter, we describe investigations considering the parameter regime, the permeabilization mechanism and the safety profile of GNOME laser transfection. The experimental and calculated results imply a combined permeabilization mechanism consisting of both photochemical and photothermal effects. Furthermore, paramount spatial control achieved either by laser illumination with micrometer precision or targeted gold nanoparticle binding to the cells was demonstrated, allowing selective cell manipulation and destruction. Additionally, the possibility to manipulate difficult to transfect primary cells (neurons) is shown. These results give insights in the basic mechanisms involved in GNOME laser transfection and serve as a strong basis to deliver different molecules for therapeutic (e.g. proteins) and diagnostic (siRNA) use.

  8. Effects of molecular size and chemical factor on plasma gene transfection

    Science.gov (United States)

    Ikeda, Yoshihisa; Motomura, Hideki; Kido, Yugo; Satoh, Susumu; Jinno, Masafumi

    2016-07-01

    In order to clarify the mechanism of plasma gene transfection, the relationship between transfection efficiency and transferred molecular size was investigated. Molecules with low molecular mass (less than 50 kDa; dye or dye-labeled oligonucleotide) and high molecular mass (more than 1 MDa; plasmid DNA or fragment of plasmid DNA) were transferred to L-929 cells. It was found that the transfection efficiency decreases with increasing in transferred molecular size and also depends on the tertiary structure of transferred molecules. Moreover, it was suggested the transfection mechanism is different between the molecules with low (less than 50 kDa) and high molecular mass (higher than 1 MDa). For the amount of gene transfection after plasma irradiation, which is comparable to that during plasma irradiation, it is shown that H2O2 molecules are the main contributor. The transfection efficiency decreased to 0.40 ± 0.22 upon scavenging the H2O2 generated by plasma irradiation using the catalase. On the other hand, when the H2O2 solution is dropped into the cell suspension without plasma irradiation, the transfection efficiency is almost 0%. In these results, it is also suggested that there is a synergetic effect of H2O2 with electrical factors or other reactive species generated by plasma irradiation.

  9. mRNA transfection of mouse and human neural stem cell cultures.

    Directory of Open Access Journals (Sweden)

    Samuel McLenachan

    Full Text Available The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

  10. Increases in doxorubicin sensitivity and radioiodide uptake by transfecting shMDR and sodium/iodide symporter gene in cancer cells expressing multidrug resistance

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Sohn Joo; Lee, Yong Jin; Lee, You La; Choi, Chang Ik; Lee, Sang Woo; Yoo, Jeong Soo; Ahn, Byeong Cheol; Lee, In Kyu; Lee, Jae Tae [Kyungpook National University, Daegu (Korea, Republic of)

    2007-06-15

    Multidrug resistance (MDR) of the cancer cells related to mdr1 gene expression can be effectively treated by selective short hairpin RNA for mdr1 gene (shMDR). Sodium/iodide symporter (NIS) gene is well known to have both reporter and therapeutic gene characteristics. We have co-transfected both shMDR and NIS gene into colon cancer cells (HCT15 cell) expressing MDR and Tc-99m sestamibi and I-125 uptake were measured. In addition, cytotoxic effects of doxorubicin and I-131 therapy were also assessed after transfection. At first, shMDR was transfected with liposome reagent into human embryonic kidney cells (HEK293) and HCT cells. shMDR transfection was confirmed by RT-PCR and western blot analysis. Adenovirus expressing NIS (Ad-NIS) gene and shMDR (Ad-shMDR) were co-transfected with Ad-NIS into HCT15 cells. Forty-eight hours after infection, inhibition of P-gycoprotein (Pgp) function by shMDR was analyzed by a change of Tc-99m sestamibi uptake and doxorubicin cytotoxicity, and functional activity of induced NIS gene expression was assessed with I-125 uptake assay. In HEK293 cells transfected with shMDR, mdr1 mRNA and Pgp protein expressions were down regulated. HCT15 cells infected with 20 MOI of Ad-NIS was higher NIS protein expression than control cells. After transfection of 300 MOI of Ad-shMDR either with or without 10 MOI of Ad-NIS, uptake of Tc-99m sestamibi increased up to 1.5-fold than control cells. HCT15 cells infected with 10 MOI of Ad-NIS showed approximately 25-fold higher I-125 uptake than control cells. Cotransfection of Ad-shMDR and Ad-NIS resulted in enhanced cytotoxic by doxorubicin in HCT15 cells. I-131 treatment on HCT15 cells infected with 20 MOI of Ad-NIS revealed increased cytotoxic effect. Suppression of mdr1 gene expression, retention of Tc-99m sestamibi, enhanced doxorubicin cytotoxicity and increases in I-125 uptake were achieved in MDR expressing cancer cell by co-transfection of shMDR and NIS gene. Dual therapy with doxorubicin and

  11. In vitro induction of specific anti-tumoral immunity against laryngeal carcinoma by using human interleukin-12gene-transfected dendritic cells

    Institute of Scientific and Technical Information of China (English)

    LIANG Wen; WANG Xue-feng

    2011-01-01

    Background Objective evaluation of the antitumor effect of interleukin-12 (IL-12) gene-transfected dendritic cell (DC)vaccine on laryngeal carcinoma requires in vivo and in vitro tests. The aim of this study was to investigate the function of IL-12 gene transfected DC at initiating specific immune response to laryngeal carcinoma in vitro.Methods Recombinant adenovirus with IL-12 gene was constructed. DCs were isolated from the peripheral blood of patients with laryngeal carcinoma, pulsed with tumor lysate of laryngeal carcinoma cells (DC+Ag), and transfected with IL-12 (DC-IL-12+Ag). The cells pheotypes including CD83, CD86 and HLA-DR on surface of DCs were assayed by flow cytometry (FCM). The concentration of IL-12 in culture supernatant of DCs and interferon γ (IFN-γ) in culture supernatant of T cells cocultured with DCs were quantified by ELISA. Methyl thiazolys tetrazolium (MTT) was used to evaluate proliferation of autologous T lymphocytes and activation of cytotoxic T lymphocytes (CTL) stimulated by IL-12-transfected DCs pulsed with tumor lysate against laryngeal carcinoma cells.Results The recombinant adenovirus expressing IL-12 gene was constructed successfully. Gene-transfected DC plused with tumor lysate with IL-12 (DC-IL-12+Ag) expressed higher level of CD83, CD86 and produced higher level of IL-12 than untransfected DCs (DC+Ag) (CD83: (60.2±1.8)% vs. (50.7±1.2)%, P <0.05; CD86: (88.9±2.1)% vs.(78.2±3.9)%, P <0.05; IL-12: (262.5±3.0) ng/L vs. (103.8±5.1) ng/L, P <0.05). The proliferation of autologous T lymphocytes and production of IFN-γ stimulated by DC transfected with IL-12 were more obviously than untransfected DCs. Cytotoxicity of CTL stimulated by IL-12-transfected DC pulsed with tumor lysate against laryngeal carcinoma cells were significantly stronger than stimulated by untransfected DC.Conclusion It is a promising approach for IL-12-transfected DC pulsed with tumor lysate to increase the antitumoral effect.

  12. Aspects of nonviral gene therapy: correlation of molecular parameters with lipoplex structure and transfection efficacy in pyridinium-based cationic lipids.

    Science.gov (United States)

    Parvizi, Paria; Jubeli, Emile; Raju, Liji; Khalique, Nada Abdul; Almeer, Ahmed; Allam, Hebatalla; Manaa, Maryem Al; Larsen, Helge; Nicholson, David; Pungente, Michael D; Fyles, Thomas M

    2014-01-30

    This study seeks correlations between the molecular structures of cationic and neutral lipids, the lipid phase behavior of the mixed-lipid lipoplexes they form with plasmid DNA, and the transfection efficacy of the lipoplexes. Synthetic cationic pyridinium lipids were co-formulated (1:1) with the cationic lipid 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and these lipids were co-formulated (3:2) with the neutral lipids 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or cholesterol. All lipoplex formulations exhibited plasmid DNA binding and a level of protection from DNase I degradation. Composition-dependent transfection (beta-galactosidase and GFP) and cytotoxicity was observed in Chinese hamster ovarian-K1 cells. The most active formulations containing the pyridinium lipids were less cytotoxic but of comparable activity to a Lipofectamine 2000™ control. Molecular structure parameters and partition coefficients were calculated for all lipids using fragment additive methods. The derived shape parameter values correctly correlated with observed hexagonal lipid phase behavior of lipoplexes as derived from small-angle X-ray scattering experiments. A transfection index applicable to hexagonal phase lipoplexes derived from calculated parameters of the lipid mixture (partition coefficient, shape parameter, lipoplex packing) produced a direct correlation with transfection efficiency.

  13. GDNF-transfected macrophages produce potent neuroprotective effects in Parkinson's disease mouse model.

    Directory of Open Access Journals (Sweden)

    Yuling Zhao

    Full Text Available The pathobiology of Parkinson's disease (PD is associated with the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc projecting to the striatum. Currently, there are no treatments that can halt or reverse the course of PD; only palliative therapies, such as replacement strategies for missing neurotransmitters, exist. Thus, the successful brain delivery of neurotrophic factors that promote neuronal survival and reverse the disease progression is crucial. We demonstrated earlier systemically administered autologous macrophages can deliver nanoformulated antioxidant, catalase, to the SNpc providing potent anti-inflammatory effects in PD mouse models. Here we evaluated genetically-modified macrophages for active targeted brain delivery of glial cell-line derived neurotropic factor (GDNF. To capitalize on the beneficial properties afforded by alternatively activated macrophages, transfected with GDNF-encoded pDNA cells were further differentiated toward regenerative M2 phenotype. A systemic administration of GDNF-expressing macrophages significantly ameliorated neurodegeneration and neuroinflammation in PD mice. Behavioral studies confirmed neuroprotective effects of the macrophage-based drug delivery system. One of the suggested mechanisms of therapeutic effects is the release of exosomes containing the expressed neurotropic factor followed by the efficient GDNF transfer to target neurons. Such formulations can serve as a new technology based on cell-mediated active delivery of therapeutic proteins that attenuate and reverse progression of PD, and ultimately provide hope for those patients who are already significantly disabled by the disease.

  14. Modulation of natural killer cytotoxicity by muramyl dipeptide and trehalose dimycolate incorporated in squalane droplets.

    Science.gov (United States)

    Masihi, K N; Lange, W; Rohde-Schulz, B

    1987-01-01

    The effect on natural killer (NK) cytotoxicity of splenic cells from BALB/c mice pretreated i.v. with squalane-in-water preparations of muramyl dipeptide (MDP), trehalose dimycolate (TDM), or the combination of MDP-plus-TDM was investigated. MDP or TDM augmented the NK cytotoxicity which peaked 48 h after the pretreatment whereas the combination of MDP and TDM induced an inhibition of the NK activity. Infection with influenza virus, a potent stimulator of NK cells, after the pretreatment with biological response modifiers resulted in a markedly enhanced NK activity on day 2 in MDP and control groups. Mice pretreated with TDM or the combination of MDP and TDM showed only moderate NK activity which peaked on day 3 after influenza infection. The NK activity was susceptible to asialo GM1 and complement treatment. The cytotoxicity of MDP-plus-TDM cells could be significantly enhanced after treatment with anti-macrophage monoclonal antibody and complement. NK activity induced by MDP or TDM was reduced by mixing MDP-plus-TDM cells. Addition of adherent cell-depleted MDP-plus-TDM suspension to MDP or TDM cells had a NK restorative effect. Splenic cells from mice pretreated 2 days earlier with MDP or TDM, but not MDP-plus-TDM, generated enhanced levels of luminol-dependent chemiluminescence.

  15. Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Rocha, C.D.; Trombone, A.P.F.; Lorenzi, J.C.C.; Almeida, L.P.; Gembre, A.F.; Padilha, E. [Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Ramos, S.G. [Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Silva, C.L.; Coelho-Castelo, A.A.M. [Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)

    2012-09-21

    In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

  16. Integrated application of uniform design and least-squares support vector machines to transfection optimization

    Directory of Open Access Journals (Sweden)

    Pan Jin-Shui

    2009-05-01

    Full Text Available Abstract Background Transfection in mammalian cells based on liposome presents great challenge for biological professionals. To protect themselves from exogenous insults, mammalian cells tend to manifest poor transfection efficiency. In order to gain high efficiency, we have to optimize several conditions of transfection, such as amount of liposome, amount of plasmid, and cell density at transfection. However, this process may be time-consuming and energy-consuming. Fortunately, several mathematical methods, developed in the past decades, may facilitate the resolution of this issue. This study investigates the possibility of optimizing transfection efficiency by using a method referred to as least-squares support vector machine, which requires only a few experiments and maintains fairly high accuracy. Results A protocol consists of 15 experiments was performed according to the principle of uniform design. In this protocol, amount of liposome, amount of plasmid, and the number of seeded cells 24 h before transfection were set as independent variables and transfection efficiency was set as dependent variable. A model was deduced from independent variables and their respective dependent variable. Another protocol made up by 10 experiments was performed to test the accuracy of the model. The model manifested a high accuracy. Compared to traditional method, the integrated application of uniform design and least-squares support vector machine greatly reduced the number of required experiments. What's more, higher transfection efficiency was achieved. Conclusion The integrated application of uniform design and least-squares support vector machine is a simple technique for obtaining high transfection efficiency. Using this novel method, the number of required experiments would be greatly cut down while higher efficiency would be gained. Least-squares support vector machine may be applicable to many other problems that need to be optimized.

  17. Repeated Gene Transfection Impairs the Engraftment of Transplanted Porcine Neonatal Pancreatic Cells

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    Min Koo Seo

    2011-02-01

    Full Text Available BackgroundPreviously, we reported that neonatal porcine pancreatic cells transfected with hepatocyte growth factor (HGF gene in an Epstein-Barr virus (EBV-based plasmid (pEBVHGF showed improved proliferation and differentiation compared to those of the control. In this study, we examined if pancreatic cells transfected repeatedly with pEBVHGF can be successfully grafted to control blood glucose in a diabetes mouse model.MethodsNeonatal porcine pancreatic cells were cultured as a monolayer and were transfected with pEBVHGF every other day for a total of three transfections. The transfected pancreatic cells were re-aggregated and transplanted into kidney capsules of diabetic nude mice or normal nude mice. Blood glucose level and body weight were measured every other day after transplantation. The engraftment of the transplanted cells and differentiation into beta cells were assessed using immunohistochemistry.ResultsRe-aggregation of the pancreatic cells before transplantation improved engraftment of the cells and facilitated neovascularization of the graft. Right before transplantation, pancreatic cells that were transfected with pEBVHGF and then re-aggregated showed ductal cell marker expression. However, ductal cells disappeared and the cells underwent fibrosis in a diabetes mouse model two to five weeks after transplantation; these mice also did not show controlled blood glucose levels. Furthermore, pancreatic cells transplanted into nude mice with normal blood glucose showed poor graft survival regardless of the type of transfected plasmid (pCEP4, pHGF, or pEBVHGF.ConclusionFor clinical application of transfected neonatal porcine pancreatic cells, further studies are required to develop methods of overcoming the damage for the cells caused by repeated transfection and to re-aggregate them into islet-like structures.

  18. p38MAPK gene transfection induced the apoptosis of rat glioma cells C6

    Institute of Scientific and Technical Information of China (English)

    ZHANG Bi-cheng; LI Qing; YE Jing; WANG Ying-mei; LIN Sheng-cai

    2001-01-01

    To study the effect ofp38MAPK transfecfion on the biological characteristics of rat glioma cells C6. Methods: p38MAPK was transfected into C6 cells by lipofectin. Expression ofp38MAPK in C6 cells before and after transfection was detected by immunocytochemistry and Western-blot analysis. HE staining,transmission electron microscopy and flow cytometry were used to observe the cell morphology, adhesion and study the cell cycle. Results: p38MAPK expressed in C6 cells after transfection. Cell biological characteristics changed,and apoptotic cells emerged. Conclusion: Exogenous p38MAPK could induce the apoptosis of C6 cells.

  19. Migratory, invasive and metastatic capacity of NCAM transfected rat glioma cells

    DEFF Research Database (Denmark)

    Edvardsen, K; Brünner, N; Spang-Thomsen, M

    1993-01-01

    A cDNA encoding a transmembrane 140 kDa isoform of the neural cell adhesion molecule, NCAM, was transfected into the rat glioma cell line BT4Cn. Transfectants with a homogeneously high expression of NCAM-B showed a decreased capacity for penetration of an artificial basement membrane when compared...... to cells transfected with expression-vector alone or untransfected cells. However, when injected subcutaneously into nude mice, both NCAM expressing cells and control cells produced invasive tumors. Nude mice injected with NCAM positive cells developed tumors with slower growth rates as compared to those...

  20. Epigenetic Regulation of the ERβ Gene on the Estrogen Signal Transfection Pathway in Colon Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    翟荣林; 王国斌; 蔡开琳; 陶凯雄; 许飞; 张万里; 王智勇

    2010-01-01

    We studied the regulatory effects of the estragen receptorβ(ERβ)gene on the downstream estrogen signal transfection pathway in colon cancer cells and the possible mechanisms involved.A human ERβ gene recombinant expression plasmid,pEGFP-C1-ERβ,was constructed and transfected into the Caco-2 colon cancer cell line,a line with low ERβ gene expression.The expression of ERβ mRNA and protein was detected 72h after transfection.RT-PCR was used to examine the expression levels of the progesterone recepror(PR)gene ...