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  1. Scripts for TRUMP data analyses. Part II (HLA-related data): statistical analyses specific for hematopoietic stem cell transplantation.

    Science.gov (United States)

    Kanda, Junya

    2016-01-01

    The Transplant Registry Unified Management Program (TRUMP) made it possible for members of the Japan Society for Hematopoietic Cell Transplantation (JSHCT) to analyze large sets of national registry data on autologous and allogeneic hematopoietic stem cell transplantation. However, as the processes used to collect transplantation information are complex and differed over time, the background of these processes should be understood when using TRUMP data. Previously, information on the HLA locus of patients and donors had been collected using a questionnaire-based free-description method, resulting in some input errors. To correct minor but significant errors and provide accurate HLA matching data, the use of a Stata or EZR/R script offered by the JSHCT is strongly recommended when analyzing HLA data in the TRUMP dataset. The HLA mismatch direction, mismatch counting method, and different impacts of HLA mismatches by stem cell source are other important factors in the analysis of HLA data. Additionally, researchers should understand the statistical analyses specific for hematopoietic stem cell transplantation, such as competing risk, landmark analysis, and time-dependent analysis, to correctly analyze transplant data. The data center of the JSHCT can be contacted if statistical assistance is required.

  2. Killing cells by targeting mitosis.

    Science.gov (United States)

    Manchado, E; Guillamot, M; Malumbres, M

    2012-03-01

    Cell cycle deregulation is a common feature of human cancer. Tumor cells accumulate mutations that result in unscheduled proliferation, genomic instability and chromosomal instability. Several therapeutic strategies have been proposed for targeting the cell division cycle in cancer. Whereas inhibiting the initial phases of the cell cycle is likely to generate viable quiescent cells, targeting mitosis offers several possibilities for killing cancer cells. Microtubule poisons have proved efficacy in the clinic against a broad range of malignancies, and novel targeted strategies are now evaluating the inhibition of critical activities, such as cyclin-dependent kinase 1, Aurora or Polo kinases or spindle kinesins. Abrogation of the mitotic checkpoint or targeting the energetic or proteotoxic stress of aneuploid or chromosomally instable cells may also provide further benefits by inducing lethal levels of instability. Although cancer cells may display different responses to these treatments, recent data suggest that targeting mitotic exit by inhibiting the anaphase-promoting complex generates metaphase cells that invariably die in mitosis. As the efficacy of cell-cycle targeting approaches has been limited so far, further understanding of the molecular pathways modulating mitotic cell death will be required to move forward these new proposals to the clinic.

  3. Cell killing by avian leukosis viruses.

    OpenAIRE

    Weller, S K; Temin, H M

    1981-01-01

    Infection of chicken cells with a cytopathic avian leukosis virus resulted in the detachment of killed cells from the culture dish. The detached, dead cells contained more unintegrated viral DNA than the attached cells. These results confirm the hypothesis that cell killing after infection with a cytopathic avian leukosis virus is associated with accumulation of large amounts of unintegrated viral DNA. No accumulation of large amounts of integrated viral DNA was found in cells infected with c...

  4. HIV transcription is induced with some forms of cell killing

    Energy Technology Data Exchange (ETDEWEB)

    Woloschak, G.E. [Argonne National Lab., IL (United States); Schreck, S. [Argonne National Lab., IL (United States)][South Carolina Univ., Columbia, SC (United States). Dept. of Chemistry; Panozzo, J. [Loyola Univ. Medical Center, Maywood, IL (United States); Chang-Liu, C.-M. [Argonne National Lab., IL (United States); Libertin, C.R. [Loyola Univ. Medical Center, Maywood, IL (United States)

    1996-11-01

    Using HeLa cells stably transfected with an HIV-LTR-CAT construct`, we demonstrated a peak in CAT induction that occurs in viable (but not necessarily cell-division-competent) cells 24 h following exposure to some cell-killing agents. {Gamma} rays were the only cell-killing agent which did not induce HIV transcription; this can be attributed to the fact that {gamma}-ray-induced apoptotic death requires function p53, which is missing in HeLa cells. For all other agents, HIV-LTR induction was dose-dependent and correlated with the amount of cell killing that occurred in the culture.

  5. Morphological effect of oscillating magnetic nanoparticles in killing tumor cells

    Science.gov (United States)

    Cheng, Dengfeng; Li, Xiao; Zhang, Guoxin; Shi, Hongcheng

    2014-04-01

    Forced oscillation of spherical and rod-shaped iron oxide magnetic nanoparticles (MNPs) via low-power and low-frequency alternating magnetic field (AMF) was firstly used to kill cancer cells in vitro. After being loaded by human cervical cancer cells line (HeLa) and then exposed to a 35-kHz AMF, MNPs mechanically damaged cell membranes and cytoplasm, decreasing the cell viability. It was found that the concentration and morphology of the MNPs significantly influenced the cell-killing efficiency of oscillating MNPs. In this preliminary study, when HeLa cells were pre-incubated with 100 μg/mL rod-shaped MNPs (rMNP, length of 200 ± 50 nm and diameter of 50 to 120 nm) for 20 h, MTT assay proved that the cell viability decreased by 30.9% after being exposed to AMF for 2 h, while the cell viability decreased by 11.7% if spherical MNPs (sMNP, diameter of 200 ± 50 nm) were used for investigation. Furthermore, the morphological effect of MNPs on cell viability was confirmed by trypan blue assay: 39.5% rMNP-loaded cells and 15.1% sMNP-loaded cells were stained after being exposed to AMF for 2 h. It was also interesting to find that killing tumor cells at either higher (500 μg/mL) or lower (20 μg/mL) concentration of MNPs was less efficient than that achieved at 100 μg/mL concentration. In conclusion, the relatively asymmetric morphological rod-shaped MNPs can kill cancer cells more effectively than spherical MNPs when being exposed to AMF by virtue of their mechanical oscillations.

  6. Newcastle disease virus selectively kills human tumor cells.

    Science.gov (United States)

    Reichard, K W; Lorence, R M; Cascino, C J; Peeples, M E; Walter, R J; Fernando, M B; Reyes, H M; Greager, J A

    1992-05-01

    Newcastle disease virus (NDV), strain 73-T, has previously been shown to be cytolytic to mouse tumor cells. In this study, we have evaluated the ability of NDV to replicate in and kill human tumor cells in culture and in athymic mice. Plaque assays were used to determine the cytolytic activity of NDV on six human tumor cell lines, fibrosarcoma (HT1080), osteosarcoma (KHOS), cervical carcinoma (KB8-5-11), bladder carcinoma (HCV29T), neuroblastoma (IMR32), and Wilm's tumor (G104), and on nine different normal human fibroblast lines. NDV formed plaques on all tumor cells tested as well as on chick embryo cells (CEC), the native host for NDV. Plaques did not form on any of the normal fibroblast lines. To detect NDV replication, virus yield assays were performed which measured virus particles in infected cell culture supernatants. Virus yield increased 10,000-fold within 24 hr in tumor and CEC supernatants. Titers remained near zero in normal fibroblast supernatants. In vivo tumoricidal activity was evaluated in athymic nude Balb-c mice by subcutaneous injection of 9 x 10(6) tumor cells followed by intralesional injection of either live or heat-killed NDV (1.0 x 10(6) plaque forming units [PFU]), or medium. After live NDV treatment, tumor regression occurred in 10 out of 11 mice bearing KB8-5-11 tumors, 8 out of 8 with HT-1080 tumors, and 6 out of 7 with IMR-32 tumors. After treatment with heat-killed NDV no regression occurred (P less than 0.01, Fisher's exact test). Nontumor-bearing mice injected with 1.0 x 10(8) PFU of NDV remained healthy. These results indicate that NDV efficiently and selectively replicates in and kills tumor cells, but not normal cells, and that intralesional NDV causes complete tumor regression in athymic mice with a high therapeutic index.

  7. How Taxol/paclitaxel kills cancer cells.

    Science.gov (United States)

    Weaver, Beth A

    2014-09-15

    Taxol (generic name paclitaxel) is a microtubule-stabilizing drug that is approved by the Food and Drug Administration for the treatment of ovarian, breast, and lung cancer, as well as Kaposi's sarcoma. It is used off-label to treat gastroesophageal, endometrial, cervical, prostate, and head and neck cancers, in addition to sarcoma, lymphoma, and leukemia. Paclitaxel has long been recognized to induce mitotic arrest, which leads to cell death in a subset of the arrested population. However, recent evidence demonstrates that intratumoral concentrations of paclitaxel are too low to cause mitotic arrest and result in multipolar divisions instead. It is hoped that this insight can now be used to develop a biomarker to identify the ∼50% of patients that will benefit from paclitaxel therapy. Here I discuss the history of paclitaxel and our recently evolved understanding of its mechanism of action.

  8. Targeted cytotoxic therapy kills persisting HIV infected cells during ART.

    Science.gov (United States)

    Denton, Paul W; Long, Julie M; Wietgrefe, Stephen W; Sykes, Craig; Spagnuolo, Rae Ann; Snyder, Olivia D; Perkey, Katherine; Archin, Nancie M; Choudhary, Shailesh K; Yang, Kuo; Hudgens, Michael G; Pastan, Ira; Haase, Ashley T; Kashuba, Angela D; Berger, Edward A; Margolis, David M; Garcia, J Victor

    2014-01-01

    Antiretroviral therapy (ART) can reduce HIV levels in plasma to undetectable levels, but rather little is known about the effects of ART outside of the peripheral blood regarding persistent virus production in tissue reservoirs. Understanding the dynamics of ART-induced reductions in viral RNA (vRNA) levels throughout the body is important for the development of strategies to eradicate infectious HIV from patients. Essential to a successful eradication therapy is a component capable of killing persisting HIV infected cells during ART. Therefore, we determined the in vivo efficacy of a targeted cytotoxic therapy to kill infected cells that persist despite long-term ART. For this purpose, we first characterized the impact of ART on HIV RNA levels in multiple organs of bone marrow-liver-thymus (BLT) humanized mice and found that antiretroviral drug penetration and activity was sufficient to reduce, but not eliminate, HIV production in each tissue tested. For targeted cytotoxic killing of these persistent vRNA(+) cells, we treated BLT mice undergoing ART with an HIV-specific immunotoxin. We found that compared to ART alone, this agent profoundly depleted productively infected cells systemically. These results offer proof-of-concept that targeted cytotoxic therapies can be effective components of HIV eradication strategies.

  9. Double suicide genes selectively kill human umbilical vein endothelial cells

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    Liu Lunxu

    2011-02-01

    Full Text Available Abstract Background To construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR promoter on human umbilical vein endothelial cells. Methods Human KDR promoter, Escherichia coli (E. coli cytosine deaminase (CD gene and the herpes simplex virus-thymidine kinase (TK gene were cloned using polymerase chain reaction (PCR. Plasmid pKDR-CDglyTK was constructed with the KDR promoter and CDglyTK genes. A recombinant adenoviral plasmid AdKDR-CDglyTK was then constructed and transfected into 293 packaging cells to grow and harvest adenoviruses. KDR-expressing human umbilical vein endothelial cells (ECV304 and KDR-negative liver cancer cell line (HepG2 were infected with the recombinant adenoviruses at different multiplicity of infection (MOI. The infection rate was measured by green fluorescent protein (GFP expression. The infected cells were cultured in culture media containing different concentrations of prodrugs ganciclovir (GCV and/or 5-fluorocytosine (5-FC. The killing effects were measured using two different methods, i.e. annexin V-FITC staining and terminal transferase-mediated dUTP nick end-labeling (TUNEL staining. Results Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed and they infected ECV304 and HepG2 cells efficiently. The infection rate was dependent on MOI of recombinant adenoviruses. ECV304 cells infected with AdKDR-CDglyTK were highly sensitive to GCV and 5-FC. The cell survival rate was dependent on both the concentration of the prodrugs and the MOI of recombinant adenoviruses. In contrast, there were no killing effects in the HepG2 cells. The combination of two prodrugs was much more effective in killing ECV304 cells than GCV or 5-FC alone. The growth of transgenic ECV304 cells was suppressed in the presence of prodrugs. Conclusion AdKDR-CDglyTK/double prodrog system may be a useful

  10. Bystander Host Cell Killing Effects of Clostridium perfringens Enterotoxin

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    Archana Shrestha

    2016-12-01

    Full Text Available Clostridium perfringens enterotoxin (CPE binds to claudin receptors, e.g., claudin-4, and then forms a pore that triggers cell death. Pure cultures of host cells that do not express claudin receptors, e.g., fibroblasts, are unaffected by pathophysiologically relevant CPE concentrations in vitro. However, both CPE-insensitive and CPE-sensitive host cells are present in vivo. Therefore, this study tested whether CPE treatment might affect fibroblasts when cocultured with CPE-sensitive claudin-4 fibroblast transfectants or Caco-2 cells. Under these conditions, immunofluorescence microscopy detected increased death of fibroblasts. This cytotoxic effect involved release of a toxic factor from the dying CPE-sensitive cells, since it could be reproduced using culture supernatants from CPE-treated sensitive cells. Supernatants from CPE-treated sensitive cells, particularly Caco-2 cells, were found to contain high levels of membrane vesicles, often containing a CPE species. However, most cytotoxic activity remained in those supernatants even after membrane vesicle depletion, and CPE was not detected in fibroblasts treated with supernatants from CPE-treated sensitive cells. Instead, characterization studies suggest that a major cytotoxic factor present in supernatants from CPE-treated sensitive cells may be a 10- to 30-kDa host serine protease or require the action of that host serine protease. Induction of caspase-3-mediated apoptosis was found to be important for triggering release of the cytotoxic factor(s from CPE-treated sensitive host cells. Furthermore, the cytotoxic factor(s in these supernatants was shown to induce a caspase-3-mediated killing of fibroblasts. This bystander killing effect due to release of cytotoxic factors from CPE-treated sensitive cells could contribute to CPE-mediated disease.

  11. Immune Killing Activity of Lymphocytes on Hela Cells Expressing Interleukin-12 In Vitro

    Institute of Scientific and Technical Information of China (English)

    Huiyan WANG; Suhua CHEN

    2008-01-01

    The killing effects of lymphocytes on Hela cells expressing intedeukin-12 (IL-12) in vitro were explored. By using gene transfection technique, full length IL-12 gene was transfected into Hela cells. The expression of IL-12 in Hela cells was detected quantitatively by ELISA; Changes in killing effects of lymphocytes on Hela cells expressing IL-12 were observed by MTT. It was found that Hela cells could express IL-12 between 24h and 72h after transfection. Killing activity of lymphocytes on Hela cells expressing IL-12 was significantly enhanced. It was concluded by cell transfection technique, Hela cells could express IL-12 and were more easily killed by lymphocytes.

  12. IAP antagonists sensitize murine osteosarcoma cells to killing by TNFα

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    Shekhar, Tanmay M.; Miles, Mark A.; Gupte, Ankita; Taylor, Scott; Tascone, Brianna; Walkley, Carl R.; Hawkins, Christine J.

    2016-01-01

    Outcomes for patients diagnosed with the bone cancer osteosarcoma have not improved significantly in the last four decades. Only around 60% of patients and about a quarter of those with metastatic disease survive for more than five years. Although DNA-damaging chemotherapy drugs can be effective, they can provoke serious or fatal adverse effects including cardiotoxicity and therapy-related cancers. Better and safer treatments are therefore needed. We investigated the anti-osteosarcoma activity of IAP antagonists (also known as Smac mimetics) using cells from primary and metastatic osteosarcomas that arose spontaneously in mice engineered to lack p53 and Rb expression in osteoblast-derived cells. The IAP antagonists SM-164, GDC-0152 and LCL161, which efficiently target XIAP and cIAPs, sensitized cells from most osteosarcomas to killing by low levels of TNFα but not TRAIL. RIPK1 expression levels and activity correlated with sensitivity. RIPK3 levels varied considerably between tumors and RIPK3 was not required for IAP antagonism to sensitize osteosarcoma cells to TNFα. IAP antagonists, including SM-164, lacked mutagenic activity. These data suggest that drugs targeting XIAP and cIAP1/2 may be effective for osteosarcoma patients whose tumors express abundant RIPK1 and contain high levels of TNFα, and would be unlikely to provoke therapy-induced cancers in osteosarcoma survivors. PMID:27129149

  13. Killing of glucose-deprived hypoxic cells with moderate hyperthermia. [HeLa cells

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    Kim, J.H.; Kim, S.H.; Hahn, E.W.

    1978-08-01

    Investigations were carried out to determine the importance of glucose as a modifying factor for hyperthermic cellular damage. HeLa S-3 cells were heated under oxic and hypoxic conditions, in the presence and absence of D-glucose. Temperatures as low as 41/sup 0/C selectively enhanced killing of the glucose deprived hypoxic cell. Glucose deprivation (37/sup 0/C, 2 hr) or heat alone (41/sup 0/C for 2 hr in regular medium with glucose) produced minimal cell kill under oxic or hypoxic conditions. No enhancement was seen with hyperthermia under oxic condition in the absence of glucose. The result suggests that the interplay between glucose concentration is important for hyperthermic cell kill.

  14. ABT-737 synergizes with Bortezomib to kill melanoma cells

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    Steven N. Reuland

    2012-02-01

    The BH3 mimetic ABT-737 is a potent inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-XL, and Bcl-w. The Bcl-2 family modulates sensitivity to anticancer drugs in many cancers, including melanomas. In this study, we examined whether ABT-737 is effective in killing melanoma cells either alone or in combination with a proteasome inhibitor already in clinical use (Bortezomib in vitro and in vivo, and further evaluated the mechanisms of action. Results showed that ABT-737 alone induced modest cytotoxicity in melanoma cells, but only at higher doses. Knock-down of the anti-apoptotic proteins Bcl-2, Bcl-XL, or Mcl-1 with siRNAs demonstrated that Mcl-1 is the critical mediator of melanoma's resistance to ABT-737 treatment. However, ABT-737 displayed strong synergistic lethality when combined with Bortezomib. Immunoblot analyses demonstrated that Bortezomib increased expression of Noxa, a pro-apoptotic Bcl-2 member that antagonizes Mcl-1. Additionally, siRNA-mediated inhibition of Noxa expression protected melanoma cells from cytotoxicity induced by the combination treatment. These results demonstrate that Bortezomib synergizes with ABT-737 by neutralizing Mcl-1's function via increased levels of Noxa. In a xenograft mouse model, although drug doses were limited due to toxicity, ABT-737 or Bortezomib slowed melanoma tumor growth compared to the control, and the drug combination significantly decreased growth compared to either drug alone. These data imply that less toxic drugs fulfilling a function similar to Bortezomib to neutralize Mcl-1 are promising candidates for combination with ABT-737 for treating melanomas.

  15. Cell killing and mutation induction on Chinese hamster cells by photoradiations

    Energy Technology Data Exchange (ETDEWEB)

    Lam, C.K.C.

    1982-11-01

    Applying radiation directly on cells, far-uv is more effective than black light, and black light is more effective than white light in inducing proliferative death and in inducing resistance to 6-thioguanine (6-TG), ouabain and diptheria toxin (DT). Gold light has no killing and mutagenic effects on CHO (Chinese hamster ovary) cells. Use of filters showed that a small percentage of shorter wavelengths in the far-uv region is responsible for most of the killing and mutagenic effects in the unfiltered broad spectra of black and white light.

  16. A general functional response of cytotoxic T lymphocyte-mediated killing of target cells.

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    Gadhamsetty, Saikrishna; Marée, Athanasius F M; Beltman, Joost B; de Boer, Rob J

    2014-04-15

    Cytotoxic T lymphocytes (CTLs) kill virus-infected cells and tumor cells, and play a critical role in immune protection. Our knowledge of how the CTL killing efficiency varies with CTL and target cell numbers is limited. Here, we simulate a region of lymphoid tissue using a cellular Potts model to characterize the functional response of CTL killing of target cells, and find that the total killing rate saturates both with the CTL and the target cell densities. The relative saturation in CTL and target cell densities is determined by whether a CTL can kill multiple target cells at the same time, and whether a target cell can be killed by many CTLs together. We find that all the studied regimes can be well described by a double-saturation (DS) function with two different saturation constants. We show that this DS model can be mechanistically derived for the cases where target cells are killed by a single CTL. For the other cases, a biological interpretation of the parameters is still possible. Our results imply that this DS function can be used as a tool to predict the cellular interactions in cytotoxicity data.

  17. Comparative efficiency of HIV-1-infected T cell killing by NK cells, monocytes and neutrophils.

    Science.gov (United States)

    Smalls-Mantey, Adjoa; Connors, Mark; Sattentau, Quentin J

    2013-01-01

    HIV-1 infected cells are eliminated in infected individuals by a variety of cellular mechanisms, the best characterized of which are cytotoxic T cell and NK cell-mediated killing. An additional antiviral mechanism is antibody-dependent cellular cytotoxicity. Here we use primary CD4(+) T cells infected with the BaL clone of HIV-1 as target cells and autologous NK cells, monocytes, and neutrophils as effector cells, to quantify the cytotoxicity mediated by the different effectors. This was carried out in the presence or absence of HIV-1-specific antiserum to assess antibody-dependent cellular cytotoxicity. We show that at the same effector to target ratio, NK cells and monocytes mediate similar levels of both antibody-dependent and antibody-independent killing of HIV-1-infected T cells. Neutrophils mediated significant antibody-dependent killing of targets, but were less effective than monocytes or NK cells. These data have implications for acquisition and control of HIV-1 in natural infection and in the context of vaccination.

  18. Cytotoxic T-lymphocyte-mediated killing of human pancreatic islet cells in vitro.

    Science.gov (United States)

    Campbell, Peter D; Estella, Eugene; Dudek, Nadine L; Jhala, Gaurang; Thomas, Helen E; Kay, Thomas W H; Mannering, Stuart I

    2008-09-01

    Cytotoxic T lymphocytes (CTL) are believed to play an essential role in beta-cell destruction leading to development of type 1 diabetes and allogeneic islet graft failure. We aimed to identify the mechanisms used by CTL to kill human beta cells. CTL clones that recognize epitopes from influenza virus and Epstein-Barr virus restricted by human leukocyte antigen (HLA)-A0201 and -B0801, respectively, were used to investigate the susceptibility of human beta cells to CTL. In a short-term (5-hour) assay, CTL killed human islet cells of the appropriate major histocompatibility complex (MHC) class I type that had been pulsed with viral peptides. Killing was increased by pretreating islets with interferon gamma that increases MHC class I on target cells. Killing was abolished by incubation of CTL with the perforin inhibitor concanamycin A. The Fas pathway did not contribute to killing because blocking with neutralizing anti-Fas ligand antibody did not significantly reduce beta-cell killing. In conclusion, we report a novel way of investigating the interaction between CTL and human islets. Human islets were rapidly killed in vitro by MHC class I-restricted CTL predominantly by the granule exocytosis pathway.

  19. Interactions between neutrophils and macrophages promote macrophage killing of rat muscle cells in vitro

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    Nguyen, Hal X.; Tidball, James G.

    2003-01-01

    Current evidence indicates that the physiological functions of inflammatory cells are highly sensitive to their microenvironment, which is partially determined by the inflammatory cells and their potential targets. In the present investigation, interactions between neutrophils, macrophages and muscle cells that may influence muscle cell death are examined. Findings show that in the absence of macrophages, neutrophils kill muscle cells in vitro by superoxide-dependent mechanisms, and that low concentrations of nitric oxide (NO) protect against neutrophil-mediated killing. In the absence of neutrophils, macrophages kill muscle cells through a NO-dependent mechanism, and the presence of target muscle cells causes a three-fold increase in NO production by macrophages, with no change in the concentration of inducible nitric oxide synthase. Muscle cells that are co-cultured with both neutrophils and macrophages in proportions that are observed in injured muscle show cytotoxicity through a NO-dependent, superoxide-independent mechanism. Furthermore, the concentration of myeloid cells that is necessary for muscle killing is greatly reduced in assays that use mixed myeloid cell populations, rather than uniform populations of neutrophils or macrophages. These findings collectively show that the magnitude and mechanism of muscle cell killing by myeloid cells are modified by interactions between muscle cells and neutrophils, between muscle cells and macrophages and between macrophages and neutrophils.

  20. Retargeting T cells for HER2-positive tumor killing by a bispecific Fv-Fc antibody.

    Directory of Open Access Journals (Sweden)

    Lei Wang

    Full Text Available To exploit the biological and pharmacological properties of immunoglobulin constant domain Fc fragment and increase the killing efficacy of T cells, a single chain variable fragment specific to CD3 was fused with Fcab (Fc antigen binding, a mutant Fc fragment with specificity against Human epidermal growth factor receptor 2 (HER2 developed by F-star. The bispecific fusion named as FcabCD3 was expressed by transient transfection in HEK-293T cells and purified by affinity chromatography. Specific cytolytic activity of retargeted T cells to kill HER2 positive SKBR3 cell line was evaluated in vitro. FcabCD3 was able to retarget T cells to kill both Herceptin insensitive Colo205-luc cell line and HER2 low expression MDA-MB-231-luc cell line. Furthermore, FcabCD3 was effective in eliminating the Colo205 tumor established on BALB/c nu/nu mice.

  1. T cells kill bacteria captured by transinfection from dendritic cells and confer protection in mice.

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    Cruz-Adalia, Aránzazu; Ramirez-Santiago, Guillermo; Calabia-Linares, Carmen; Torres-Torresano, Mónica; Feo, Lidia; Galán-Díez, Marta; Fernández-Ruiz, Elena; Pereiro, Eva; Guttmann, Peter; Chiappi, Michele; Schneider, Gerd; Carrascosa, José López; Chichón, Francisco Javier; Martínez Del Hoyo, Gloria; Sánchez-Madrid, Francisco; Veiga, Esteban

    2014-05-14

    Dendritic cells (DCs) phagocytose, process, and present bacterial antigens to T lymphocytes to trigger adaptive immunity. In vivo, bacteria can also be found inside T lymphocytes. However, T cells are refractory to direct bacterial infection, leaving the mechanisms by which bacteria invade T cells unclear. We show that T cells take up bacteria from infected DCs by the process of transinfection, which requires direct contact between the two cells and is enhanced by antigen recognition. Prior to transfer, bacteria localize to the immunological synapse, an intimate DC/T cell contact structure that activates T cells. Strikingly, T cells efficiently eliminate the transinfecting bacteria within the first hours after infection. Transinfected T cells produced high levels of proinflammatory cytokines and were able to protect mice from bacterial challenge following adoptive transfer. Thus, T lymphocytes can capture and kill bacteria in a manner reminiscent of innate immunity.

  2. Protection of Candida parapsilosis from neutrophil killing through internalization by human endothelial cells.

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    Glass, Kyle A; Longley, Sarah J; Bliss, Joseph M; Shaw, Sunil K

    2015-01-01

    Candida parapsilosis is a fungal pathogen that is associated with hematogenously disseminated disease in premature neonates, acutely ill or immunocompromised patients. In cell culture, C. parapsilosis cells are actively and avidly endocytosed by endothelial cells via actin polymerization mediated by N-WASP. Here we present evidence that C. parapsilosis that were internalized by endothelial cells remained alive, and avoided being acidified or otherwise damaged via the host cell. Internalized fungal cells reproduced intracellularly and eventually burst out of the host endothelial cell. When neutrophils were added to endothelium and C. parapsilosis, they patrolled the endothelial surface and efficiently killed most adherent fungal cells prior to endocytosis. But after endocytosis by endothelial cells, internalized fungal cells evaded neutrophil killing. Silencing endothelial N-WASP blocked endocytosis of C. parapsilosis and left fungal cells stranded on the cell surface, where they were susceptible to neutrophil killing. These observations suggest that for C. parapsilosis to escape from the bloodstream, fungi may adhere to and be internalized by endothelial cells before being confronted and phagocytosed by a patrolling leukocyte. Once internalized by endothelial cells, C. parapsilosis may safely replicate to cause further rounds of infection. Immunosurveillance of the intravascular lumen by leukocytes crawling on the endothelial surface and rapid killing of adherent yeast may play a major role in controlling C. parapsilosis dissemination and infected endothelial cells may be a significant reservoir for fungal persistence.

  3. Recombinant scorpion insectotoxin AaIT kills specifically insect cells but not human cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LCs0 value of 18.4 uM and 0.70 μM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 μM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3μM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.

  4. Characterization of cell lysis in Pseudomonas putida induced upon expression of heterologous killing genes

    DEFF Research Database (Denmark)

    Ronchel, M.C.; Molina, L.; Witte, A.;

    1998-01-01

    Active biological containment systems are based on the controlled expression of killing genes. These systems are of interest for the Pseudomonadaceae because of the potential applications of these microbes as bioremediation agents and biopesticides, The physiological effects that lead to cell death...... upon the induction of expression of two different heterologous killing genes in nonpathogenic Pseudomonas putida KT2440 derivatives have been analyzed, P. putida CMC4 and CMC12 carry in their chromosomes a fusion of the PAl-04/03 promoter to the Escherichia coli gef gene and the phi X174 lysis gene E......, respectively. Expression of the killing genes is controlled by the LacI protein, whose expression is initiated from the XylS-dependent Pm promoter. Under induced conditions, killing of P. putida CMC12 cells mediated by phi X174 lysis protein E was faster than that observed for P. putida CMC4, for which the Gef...

  5. A license to kill : The evolution of NK cell receptors

    NARCIS (Netherlands)

    Carrillo Bustamante, N.P.

    2015-01-01

    Natural killer (NK) cells innate 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 must be tightly regulated. Unlike B and T cells, NK cell do not undergo DNA rearrangeme

  6. Combination of sulindac and dichloroacetate kills cancer cells via oxidative damage.

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    Ayyanathan, Kasirajan; Kesaraju, Shailaja; Dawson-Scully, Ken; Weissbach, Herbert

    2012-01-01

    Sulindac is an FDA-approved non-steroidal anti-inflammatory drug with documented anticancer activities. Our recent studies showed that sulindac selectively enhanced the killing of cancer cells exposed to oxidizing agents via production of reactive oxygen species (ROS) resulting in mitochondrial dysfunction. This effect of sulindac and oxidative stress on cancer cells could be related to the defect in respiration in cancer cells, first described by Warburg 50 years ago, known as the Warburg effect. We postulated that sulindac might enhance the selective killing of cancer cells when combined with any compound that alters mitochondrial respiration. To test this hypothesis we have used dichloroacetate (DCA), which is known to shift pyruvate metabolism away from lactic acid formation to respiration. One might expect that DCA, since it stimulates aerobic metabolism, could stress mitochondrial respiration in cancer cells, which would result in enhanced killing in the presence of sulindac. In this study, we have shown that the combination of sulindac and DCA enhances the selective killing of A549 and SCC25 cancer cells under the conditions used. As predicted, the mechanism of killing involves ROS production, mitochondrial dysfunction, JNK signaling and death by apoptosis. Our results suggest that the sulindac-DCA drug combination may provide an effective cancer therapy.

  7. Combination of sulindac and dichloroacetate kills cancer cells via oxidative damage.

    Directory of Open Access Journals (Sweden)

    Kasirajan Ayyanathan

    Full Text Available Sulindac is an FDA-approved non-steroidal anti-inflammatory drug with documented anticancer activities. Our recent studies showed that sulindac selectively enhanced the killing of cancer cells exposed to oxidizing agents via production of reactive oxygen species (ROS resulting in mitochondrial dysfunction. This effect of sulindac and oxidative stress on cancer cells could be related to the defect in respiration in cancer cells, first described by Warburg 50 years ago, known as the Warburg effect. We postulated that sulindac might enhance the selective killing of cancer cells when combined with any compound that alters mitochondrial respiration. To test this hypothesis we have used dichloroacetate (DCA, which is known to shift pyruvate metabolism away from lactic acid formation to respiration. One might expect that DCA, since it stimulates aerobic metabolism, could stress mitochondrial respiration in cancer cells, which would result in enhanced killing in the presence of sulindac. In this study, we have shown that the combination of sulindac and DCA enhances the selective killing of A549 and SCC25 cancer cells under the conditions used. As predicted, the mechanism of killing involves ROS production, mitochondrial dysfunction, JNK signaling and death by apoptosis. Our results suggest that the sulindac-DCA drug combination may provide an effective cancer therapy.

  8. Kinase domain insert containing receptor promotor controlled suicide gene system kills human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Zong-Hai Huang; Wen-Yu Yang; Qi Cheng; Jing-Long Yu; Zhou Li; Zong-Yan Tong; Hui-Juan Song; Xiao-Yan Che

    2005-01-01

    AIM: To evaluate the killing effect of double suicide gene mediated by adenovirus and regulated under kinase domain insert containing receptor (KDR) promoter on human umbilical vein endothelial cells. METHODS: By PCR technology, human KDR promoter gene, Escherichia coli(E. coli) cytosine deaminase (CD) gene and the herpes simple virus-thymidine kinase (TK) gene were cloned. Plasmid pKDR-CDglyTK was constructed with them. Then, a recombinant adenoviral plasmid pAdKDRCDglyTK was constructed in a "two-step transformation protocol". The newly constructed plasmids were transfected to 293 packaging cells to grow adenoviruses, which were further propagated and purified. Human umbilical vein endothelial cells (HUVEC) were infected with a different multiplicity of infection (MOI) of resultant recombinant adenovirus, the infection rate was measured with the aid of (GFP) expression. Infected cells were cultured in culture media containing different concentrations of (GCV) and/or 5-(FC), and the killing effects were measured.RESULTS: Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed, and they infected HUVEC cells efficiently. Our data indicated that the infection rate was relevant to MOI of recombinant adenoviruses. HUVEC cells infected with AdKDR-CDglyTK were highly sensitive to the prodrugs, their survival rate correlated to both the concentration of the prodrugs and the MOI of recombinant adenoviruses. Our data also indicated that the two prodrugs used in combination were much more effective on killing transgeneic cells than GCV or 5-FC used alone. CONCLUSION: Prodrug/KDR-CDglyTK system is effective on killing HUVEC cells, its killing effect correlates to the concentration of prodrugs and recombinant adenovirus' MOI. Combined use of the two prodrugs confers better killing effects on transgeneic cells.

  9. Galactose/N-acetylgalactosamine lectin: the coordinator of host cell killing

    Indian Academy of Sciences (India)

    Douglas R Boettner; Christopher Huston; William A Petri Jr

    2002-11-01

    Entamoeba histolytica is an enteric parasite that can kill host cells via a contact-dependent mechanism. This killing involves the amoebic surface protein referred to as the Gal/GalNAc lectin. The Gal/GalNAc lectin binds galactose and N-acetylgalactosamine allowing the adherence of amoebas to host cells. Involvement of the lectin in the pathogenesis of E. histolytica infection will be reviewed in this paper. The lectin has been shown to have very specific and substantial effects on adherence, cytotoxicity, and encystation. There is also possible involvement of the lectin in phagocytosis and caspase activation in host cells.

  10. The kinematics of cytotoxic lymphocytes influence their ability to kill target cells.

    Science.gov (United States)

    Bhat, Purnima; Leggatt, Graham; Matthaei, Klaus I; Frazer, Ian H

    2014-01-01

    Cytotoxic lymphocytes (CTL) have been reported to show a range of motility patterns from rapid long-range tracking to complete arrest, but how and whether these kinematics affect their ability to kill target cells is not known. Many in vitro killing assays utilize cell lines and tumour-derived cells as targets, which may be of limited relevance to the kinetics of CTL-mediated killing of somatic cells. Here, live-cell microscopy is used to examine the interactions of CTL and primary murine skin cells presenting antigens. We developed a qualitative and quantitative killing assay using extended-duration fluorescence time-lapse microscopy coupled with large-volume objective software-based data analysis to obtain population data of cell-to-cell interactions, motility and apoptosis. In vivo and ex vivo activated antigen-specific cytotoxic lymphocytes were added to primary keratinocyte targets in culture with fluorometric detection of caspase-3 activation in targets as an objective determinant of apoptosis. We found that activated CTL achieved contact-dependent apoptosis of non-tumour targets after a period of prolonged attachment - on average 21 hours - which was determined by target cell type, amount of antigen, and activation status of CTL. Activation of CTL even without engagement of the T cell receptor was sufficient to mobilise cells significantly above baseline, while the addition of cognate antigen further enhanced their motility. Highly activated CTL showed markedly increased vector displacement, and velocity, and lead to increased antigen-specific target cell death. These data show that the inherent kinematics of CTL correlate directly with their ability to kill non-tumour cells presenting cognate antigen.

  11. The kinematics of cytotoxic lymphocytes influence their ability to kill target cells.

    Directory of Open Access Journals (Sweden)

    Purnima Bhat

    Full Text Available Cytotoxic lymphocytes (CTL have been reported to show a range of motility patterns from rapid long-range tracking to complete arrest, but how and whether these kinematics affect their ability to kill target cells is not known. Many in vitro killing assays utilize cell lines and tumour-derived cells as targets, which may be of limited relevance to the kinetics of CTL-mediated killing of somatic cells. Here, live-cell microscopy is used to examine the interactions of CTL and primary murine skin cells presenting antigens. We developed a qualitative and quantitative killing assay using extended-duration fluorescence time-lapse microscopy coupled with large-volume objective software-based data analysis to obtain population data of cell-to-cell interactions, motility and apoptosis. In vivo and ex vivo activated antigen-specific cytotoxic lymphocytes were added to primary keratinocyte targets in culture with fluorometric detection of caspase-3 activation in targets as an objective determinant of apoptosis. We found that activated CTL achieved contact-dependent apoptosis of non-tumour targets after a period of prolonged attachment - on average 21 hours - which was determined by target cell type, amount of antigen, and activation status of CTL. Activation of CTL even without engagement of the T cell receptor was sufficient to mobilise cells significantly above baseline, while the addition of cognate antigen further enhanced their motility. Highly activated CTL showed markedly increased vector displacement, and velocity, and lead to increased antigen-specific target cell death. These data show that the inherent kinematics of CTL correlate directly with their ability to kill non-tumour cells presenting cognate antigen.

  12. HAMLET kills tumor cells by apoptosis: structure, cellular mechanisms, and therapy.

    Science.gov (United States)

    Gustafsson, Lotta; Hallgren, Oskar; Mossberg, Ann-Kristin; Pettersson, Jenny; Fischer, Walter; Aronsson, Annika; Svanborg, Catharina

    2005-05-01

    New cancer treatments should aim to destroy tumor cells without disturbing normal tissue. HAMLET (human alpha-lactalbumin made lethal to tumor cells) offers a new molecular approach to solving this problem, because it induces apoptosis in tumor cells but leaves normal differentiated cells unaffected. After partial unfolding and binding to oleic acid, alpha-lactalbumin forms the HAMLET complex, which enters tumor cells and freezes their metabolic machinery. The cells proceed to fragment their DNA, and they disintegrate with apoptosis-like characteristics. HAMLET kills a wide range of malignant cells in vitro and maintains this activity in vivo in patients with skin papillomas. In addition, HAMLET has striking effects on human glioblastomas in a rat xenograft model. After convection-enhanced delivery, HAMLET diffuses throughout the brain, selectively killing tumor cells and controlling tumor progression without apparent tissue toxicity. HAMLET thus shows great promise as a new therapeutic with the advantage of selectivity for tumor cells and lack of toxicity.

  13. Optimization of total body irradiation: the match between (maximal) leukemic cell kill and (minimal) late effects

    NARCIS (Netherlands)

    Harteveld, M.L. van

    2007-01-01

    Optimization of total body irradiation: the match between (maximal) leukemic cell kill and (minimal) late effects: In this thesis, cataract formation and renal dysfunction as late effects of high-dose total body irradiation (TBI) as part of the conditioning before hematological stem cell transplanta

  14. Both necrosis and apoptosis contribute to HIV-1-induced killing of CD4 cells

    Science.gov (United States)

    Plymale, D. R.; Tang, D. S.; Comardelle, A. M.; Fermin, C. D.; Lewis, D. E.; Garry, R. F.

    1999-01-01

    BACKGROUND: Data currently available on HIV-1-induced cytopathology is unclear regarding the mechanism of cell killing. OBJECTIVE: To clarify the extent to which apoptosis or necrosis is involved in HIV-1-induced cell death in view of conflicting existing data. METHODS: T lymphoblastoid cells or peripheral blood mononuclear cells were infected by various strains of HIV-1 and the numbers of apoptotic or necrotic cells were quantified at various times after infection using video-image analysis techniques; the results were compared with the amount of fragmented DNA using a quantitative method. Measurement of mitochondrial transmembrane potential (deltapsi(m)) and intracellular calcium concentrations [Ca2+]i was performed with fluorescent probes and fluorescence concentration analysis (FCA). RESULTS: Although lymphoblastoid and monocytoid cells acutely infected by HIV-1 had increased levels of fragmented DNA, a marker of apoptotic cell death, few (killing of CD4 cells.

  15. Doxorubicin loaded 17β-estradiol based SWNT dispersions for target specific killing of cancer cells.

    Science.gov (United States)

    Ghosh, Moumita; Das, Prasanta Kumar

    2016-06-01

    The present work reports the synthesis of a 17β-estradiol based amphiphiles comprising of polyethylene glycol (PEG) moiety linked through succinic acid that non-covalently dispersed (76%) the single walled carbon nanotubes (SWNTs) in water. The superior exfoliation of carbon nanotubes was characterized by microscopic and spectroscopic studies. Significant stability of these SWNT dispersions was observed in the presence of protein in cell culture media and the nanohybrids were highly biocompatible toward mammalian cells. Anticancer drug doxorubicin loaded on these nanohybrids was selectively delivered within estrogen receptor rich cancer cells, MCF7 (breast cancer cell) and A549 (lung cancer cell). Microscopic studies showed the localization of doxorubicin within the cancer cell nucleus whereas no such localization was observed in ER negative cells. Both these ER positive cancer cells were killed by ∼3 fold higher efficiency than that of ER negative MDA-MB-231 (advanced breast cancer cell) and HeLa cells that are deprived of estrogen receptors. Thus, judiciously designed estradiol based nanohybrids proved to be excellent tool for SWNT dispersion and also for selectively killing of ER positive cancer cells. To the best of our knowledge, for the first time non-covalently modified SWNTs by estradiol based amphiphilic dispersing agent have been used for selective killing of ER positive cancer cells by doxorubicin loaded on dispersed SWNTs. It holds immense promise to be exploited as a cancer therapeutic agent.

  16. Cytomegalovirus-Infected Cells Resist T Cell Mediated Killing in an HLA-Recognition Independent Manner.

    Science.gov (United States)

    Proff, Julia; Walterskirchen, Christian; Brey, Charlotte; Geyeregger, Rene; Full, Florian; Ensser, Armin; Lehner, Manfred; Holter, Wolfgang

    2016-01-01

    In order to explore the potential of HLA-independent T cell therapy for human cytomegalovirus (HCMV) infections, we developed a chimeric antigen receptor (CAR) directed against the HCMV encoded glycoprotein B (gB), which is expressed at high levels on the surface of infected cells. T cells engineered with this anti-gB CAR recognized HCMV-infected cells and released cytokines and cytotoxic granules. Unexpectedly, and in contrast to analogous approaches for HIV, Hepatitis B or Hepatitis C virus, we found that HCMV-infected cells were resistant to killing by the CAR-modified T cells. In order to elucidate whether this phenomenon was restricted to the use of CARs, we extended our experiments to T cell receptor (TCR)-mediated recognition of infected cells. To this end we infected fibroblasts with HCMV-strains deficient in viral inhibitors of antigenic peptide presentation and targeted these HLA-class I expressing peptide-loaded infected cells with peptide-specific cytotoxic T cells (CTLs). Despite strong degranulation and cytokine production by the T cells, we again found significant inhibition of lysis of HCMV-infected cells. Impairment of cell lysis became detectable 1 day after HCMV infection and gradually increased during the following 3 days. We thus postulate that viral anti-apoptotic factors, known to inhibit suicide of infected host cells, have evolved additional functions to directly abrogate T cell cytotoxicity. In line with this hypothesis, CAR-T cell cytotoxicity was strongly inhibited in non-infected fibroblasts by expression of the HCMV-protein UL37x1, and even more so by additional expression of UL36. Our data extend the current knowledge on Betaherpesviral evasion from T cell immunity and show for the first time that, beyond impaired antigen presentation, infected cells are efficiently protected by direct blockade of cytotoxic effector functions through viral proteins.

  17. Endocytosis of Cytotoxic Granules Is Essential for Multiple Killing of Target Cells by T Lymphocytes.

    Science.gov (United States)

    Chang, Hsin-Fang; Bzeih, Hawraa; Schirra, Claudia; Chitirala, Praneeth; Halimani, Mahantappa; Cordat, Emmanuelle; Krause, Elmar; Rettig, Jens; Pattu, Varsha

    2016-09-15

    CTLs are serial killers that kill multiple target cells via exocytosis of cytotoxic granules (CGs). CG exocytosis is tightly regulated and has been investigated in great detail; however, whether CG proteins are endocytosed following exocytosis and contribute to serial killing remains unknown. By using primary CTLs derived from a knock-in mouse of the CG membrane protein Synaptobrevin2, we show that CGs are endocytosed in a clathrin- and dynamin-dependent manner. Following acidification, endocytosed CGs are recycled through early and late, but not recycling endosomes. CGs are refilled with granzyme B at the late endosome stage and polarize to subsequent synapses formed between the CTL and new target cells. Importantly, inhibiting CG endocytosis in CTLs results in a significant reduction of their cytotoxic activity. Thus, our data demonstrate that continuous endocytosis of CG membrane proteins is a prerequisite for efficient serial killing of CTLs and identify key events in this process.

  18. Residual chromatin breaks as biodosimetry for cell killing by carbon ions

    Science.gov (United States)

    Suzuki, M.; Kase, Y.; Nakano, T.; Kanai, T.; Ando, K.

    1998-11-01

    We have studied the relationship between cell killing and the induction of residual chromatin breaks on various human cell lines and primary cultured cells obtained by biopsy from patients irradiated with either X-rays or heavy-ion beams to identify potential bio-marker of radiosensitivity for radiation-induced cell killing. The carbon-ion beams were accelerated with the Heavy Ion Medical Accelerator in Chiba (HIMAC). Six primary cultures obtained by biopsy from 6 patients with carcinoma of the cervix were irradiated with two different mono-LET beams (LET = 13 keV/μm, 76 keV/μm) and 200kV X rays. Residual chromatin breaks were measured by counting the number of non-rejoining chromatin fragments detected by the premature chromosome condensation (PCC) technique after a 24 hour post-irradiation incubation period. The induction rate of residual chromatin breaks per cell per Gy was the highest for 76 keV/μm beams on all of the cells. Our results indicated that cell which was more sensitive to the cell killing was similarly more susceptible to induction of residual chromatin breaks. Furthermore there is a good correlation between these two end points in various cell lines and primary cultured cells. This suggests that the detection of residual chromatin breaks by the PCC technique may be useful as a predictive assay of tumor response to cancer radiotherapy.

  19. Poliovirus protease 3C(pro) kills cells by apoptosis.

    Science.gov (United States)

    Barco, A; Feduchi, E; Carrasco, L

    2000-01-20

    The tetracycline-based Tet-Off expression system has been used to analyze the effects of poliovirus protease 3C(pro) on human cells. Stable HeLa cell clones that express this poliovirus protease under the control of an inducible, tightly regulated promoter were obtained. Tetracycline removal induces synthesis of 3C protease, followed by drastic morphological alterations and cellular death. Degradation of cellular DNA in nucleosomes and generation of apoptotic bodies are observed from the second day after 3C(pro) induction. The cleavage of poly(ADP-ribose) polymerase, an enzyme involved in DNA repair, occurs after induction of 3C(pro), indicating caspase activation by this poliovirus protease. The 3C(pro)-induced apoptosis is blocked by the caspase inhibitor z-VAD-fmk. Our findings suggest that the protease 3C is responsible for triggering apoptosis in poliovirus-infected cells by a mechanism that involves caspase activation.

  20. Preferential killing of cancer cells using silicon carbide quantum dots.

    Science.gov (United States)

    Mognetti, Barbara; Barberis, Alessandro; Marino, Silvia; Di Carlo, Francesco; Lysenko, Vladimir; Marty, Olivier; Géloën, Alain

    2010-12-01

    Silicon carbide quantum dots are highly luminescent biocompatible nanoparticles whose properties might be of particular interest for biomedical applications. In this study we investigated Silicon Carbide Quantum Dots (3C-SiC QDs) cellular localisation and influence on viability and proliferation on oral squamous carcinoma (AT-84 and HSC) and immortalized cell lines (S-G). They clearly localize into the nuclei, but the presence of 3C-SiC QDs in culture medium provoke morphological changes in cultured cells. We demonstrate that 3C-SiC QDs display dose- and time-dependent selective cytotoxicity on cancer versus immortalized cells in vitro. Since one of the limitations of classical antineoplastic drugs is their lack of selectivity, these results open a new way in the search for antiproliferative drugs.

  1. Cell kill by megavoltage protons with high LET

    Science.gov (United States)

    Kuperman, Vadim Y.

    2016-07-01

    The aim of the current study is to develop a radiobiological model which describes the effect of linear energy transfer (LET) on cell survival and relative biological effectiveness (RBE) of megavoltage protons. By assuming the existence of critical sites within a cell, analytical expression for cell survival S as a function of LET is derived. The obtained results indicate that in cases where dose per fraction is small, \\ln (S) is a linear-quadratic (LQ) function of dose while both alpha and beta radio-sensitivities are non-linearly dependent on LET. In particular, in the current model alpha increases with increasing LET while beta decreases. Conversely, in the case of large dose per fraction, the LQ dependence of \\ln (S) on dose is invalid. The proposed radiobiological model predicts cell survival probability and RBE which, in general, deviate from the results obtained by using conventional LQ formalism. The differences between the LQ model and that described in the current study are reflected in the calculated RBE of protons.

  2. A novel bispecific antibody, S-Fab, induces potent cancer cell killing.

    Science.gov (United States)

    Li, Li; He, Ping; Zhou, Changhua; Jing, Li; Dong, Bin; Chen, Siqi; Zhang, Ning; Liu, Yawei; Miao, Ji; Wang, Zhong; Li, Qing

    2015-01-01

    Bispecific antibodies that engage immune cells to kill cancer cells have been actively studied in cancer immunotherapy. In this study, we present a novel bispecific format, S-Fab, fabricated by linking a single-domain anti-carcinoembryonic antigen VHH to a conventional anti-CD3 Fab. In contrast to most bispecific antibodies, the S-Fab bispecific antibody can be efficiently expressed and purified from bacteria. The purified S-Fab is stable in serum and is able to recruit T cells to drive potent cancer cell killing. In xenograft models, the S-Fab antibody suppresses tumor growth in the presence of human immune cells. Our study suggested that the bispecific S-Fab format can be applied to a wide range of immunotherapies.

  3. EGFR-targeted magnetic nanoparticle heaters kill cancer cells without a perceptible temperature rise.

    Science.gov (United States)

    Creixell, Mar; Bohórquez, Ana C; Torres-Lugo, Madeline; Rinaldi, Carlos

    2011-09-27

    It is currently believed that magnetic nanoparticle heaters (MNHs) can kill cancer cells only when the temperature is raised above 43 °C due to energy dissipation in an alternating magnetic field. On the other hand, simple heat conduction arguments indicate that in small tumors or single cells the relative rates of energy dissipation and heat conduction result in a negligible temperature rise, thus limiting the potential of MNHs in treating small tumors and metastatic cancer. Here we demonstrate that internalized MNHs conjugated to epidermal growth factor (EGF) and which target the epidermal growth factor receptor (EGFR) do result in a significant (up to 99.9%) reduction in cell viability and clonogenic survival in a thermal heat dose dependent manner, without the need for a perceptible temperature rise. The effect appears to be cell type specific and indicates that magnetic nanoparticles in alternating magnetic fields may effectively kill cancer cells under conditions previously considered as not possible.

  4. Syrosingopine sensitizes cancer cells to killing by metformin

    Science.gov (United States)

    Benjamin, Don; Colombi, Marco; Hindupur, Sravanth K.; Betz, Charles; Lane, Heidi A.; El-Shemerly, Mahmoud Y. M.; Lu, Min; Quagliata, Luca; Terracciano, Luigi; Moes, Suzette; Sharpe, Timothy; Wodnar-Filipowicz, Aleksandra; Moroni, Christoph; Hall, Michael N.

    2016-01-01

    We report that the anticancer activity of the widely used diabetic drug metformin is strongly potentiated by syrosingopine. Synthetic lethality elicited by combining the two drugs is synergistic and specific to transformed cells. This effect is unrelated to syrosingopine’s known role as an inhibitor of the vesicular monoamine transporters. Syrosingopine binds to the glycolytic enzyme α-enolase in vitro, and the expression of the γ-enolase isoform correlates with nonresponsiveness to the drug combination. Syrosingopine sensitized cancer cells to metformin and its more potent derivative phenformin far below the individual toxic threshold of each compound. Thus, combining syrosingopine and codrugs is a promising therapeutic strategy for clinical application for the treatment of cancer. PMID:28028542

  5. NK cell-mediated killing of AML blasts. Role of histamine, monocytes and reactive oxygen metabolites

    Energy Technology Data Exchange (ETDEWEB)

    Brune, M.; Mellqvist, U.H. [Sahlgren`s Univ. Hospital, Dept. of Medicine, Haematology Section, Goeteborg (Sweden); Hansson, M.; Hermodsson, S.; Hellstrand, K. [Sahlgren`s Univ. Hospital, Dept. of Virology, Goeteborg (Sweden)

    1996-10-01

    Blasts recovered from patients with acute myelogenous leukaemia (AML) were lysed by heterologeous natural killer (NK) cells treated with NK cell-activating cytokine-induced killing of AML blasts was inhibited by monocytes, recovered from peripheral blood by counterflow centrifugal elutriation. Histamine, at concentrations exceeding 0.1 {mu}M, abrogated the monocyte-induced inhibition of NK cells; thereby, histamine and IL-2 or histamine and IFN-{alpha} synergistically induced NK cell-mediated destruction of AML blasts. The effect of histamine was completely blocked by the histamine H2-receptor (H2R) antagonist ranitidine but not by its chemical control AH20399AA. Catalase, a scavenger of reactive oxygen metabolites (ROM), reversed the monocyte-induced inhibition of NK cell-mediated killing of blast cells, indicating that the inhibitory signal was mediated by products of the respiratory burst of monocytes. It is concluded that (i) monocytes inhibit anti-leukemic properties of NK cells, (ii) the inhibition is conveyed by monocyte-derived ROM, and (iii) histamine reverses the inhibitory signal and, thereby, synergizes with NK cell-activating cytokines to induce killing of AML blasts. (au) 19 refs.

  6. Cardiomyocytes display low mitochondrial priming and are highly resistant toward cytotoxic T‐cell killing

    Science.gov (United States)

    Zheng, Xiang; Halle, Stephan; Yu, Kai; Mishra, Pooja; Scherr, Michaela; Pietzsch, Stefan; Willenzon, Stefanie; Janssen, Anika; Boelter, Jasmin; Hilfiker‐Kleiner, Denise; Eder, Matthias

    2016-01-01

    Following heart transplantation, alloimmune responses can cause graft rejection by damaging donor vascular and parenchymal cells. However, it remains unclear whether cardiomyocytes are also directly killed by immune cells. Here, we used two‐photon microscopy to investigate how graft‐specific effector CD8+ T cells interact with cardiomyocytes in a mouse heart transplantation model. Surprisingly, we observed that CD8+ T cells are completely impaired in killing cardiomyocytes. Even after virus‐mediated preactivation, antigen‐specific CD8+ T cells largely fail to lyse these cells although both cell types engage in dynamic interactions. Furthermore, we established a two‐photon microscopy‐based assay using intact myocardium to determine the susceptibility of cardiomyocytes to undergo apoptosis. This feature, also known as mitochondrial priming reveals an unexpected weak predisposition of cardiomyocytes to undergo apoptosis in situ. These observations together with the early exhaustion phenotype of graft‐infiltrating specific T cells provide an explanation why cardiomyocytes are largely protected from direct CD8+ T‐cell‐mediated killing. PMID:26970349

  7. Vesicle-associated membrane protein 7 (VAMP-7) is essential for target cell killing in a natural killer cell line.

    Science.gov (United States)

    Marcet-Palacios, Marcelo; Odemuyiwa, Solomon O; Coughlin, Jason J; Garofoli, Daniella; Ewen, Catherine; Davidson, Courtney E; Ghaffari, Mazyar; Kane, Kevin P; Lacy, Paige; Logan, Michael R; Befus, A Dean; Bleackley, R Chris; Moqbel, Redwan

    2008-02-15

    Natural killer cells recognize and induce apoptosis in foreign, transformed or virus-infected cells through the release of perforin and granzymes from secretory lysosomes. Clinically, NK-cell mediated killing is a major limitation to successful allo- and xenotransplantation. The molecular mechanisms that regulate the fusion of granzyme B-containing secretory lysosomes to the plasma membrane in activated NK cells, prior to target cell killing, are not fully understood. Using the NK cell line YT-Indy as a model, we have investigated the expression of SNAP REceptors (SNAREs), both target (t-) and vesicular (v-) SNAREs, and their function in granzyme B-mediated target cell killing. Our data showed that YT-Indy cells express VAMP-7 and SNAP-23, but not VAMP-2. VAMP-7 was associated with granzyme B-containing lysosomal granules. Using VAMP-7 small interfering RNA (siRNA), we successfully knocked down the expression of VAMP-7 protein in YT-Indy to less than 10% of untreated cells in 24h. VAMP7-deficient YT-Indy cells activated via co-culture with Jurkat cells released <1ng/mL of granzyme B, compared to 1.5-2.5 microg/mL from controls. Using Jurkat cells as targets, we showed a 7-fold reduction in NK cell-mediated killing by VAMP-7 deficient YT-Indy cells. Our results show that VAMP-7 is a crucial component of granzyme B release and target cell killing in the NK cell line YT-Indy. Thus, targeting VAMP-7 expression specifically with siRNA, following transplantation, may be a viable strategy for preventing NK cell-mediated transplant rejection, in vivo.

  8. Mechanistic insights into selective killing of OXPHOS-dependent cancer cells by arctigenin.

    Science.gov (United States)

    Brecht, Karin; Riebel, Virginie; Couttet, Philippe; Paech, Franziska; Wolf, Armin; Chibout, Salah-Dine; Pognan, Francois; Krähenbühl, Stephan; Uteng, Marianne

    2017-04-01

    Arctigenin has previously been identified as a potential anti-tumor treatment for advanced pancreatic cancer. However, the mechanism of how arctigenin kills cancer cells is not fully understood. In the present work we studied the mechanism of toxicity by arctigenin in the human pancreatic cell line, Panc-1, with special emphasis on the mitochondria. A comparison of Panc-1 cells cultured in glucose versus galactose medium was applied, allowing assessments of effects in glycolytic versus oxidative phosphorylation (OXPHOS)-dependent Panc-1 cells. For control purposes, the mitochondrial toxic response to treatment with arctigenin was compared to the anti-cancer drug, sorafenib, which is a tyrosine kinase inhibitor known for mitochondrial toxic off-target effects (Will et al., 2008). In both Panc-1 OXPHOS-dependent and glycolytic cells, arctigenin dissipated the mitochondrial membrane potential, which was demonstrated to be due to inhibition of the mitochondrial complexes II and IV. However, arctigenin selectively killed only the OXPHOS-dependent Panc-1 cells. This selective killing of OXPHOS-dependent Panc-1 cells was accompanied by generation of ER stress, mitochondrial membrane permeabilization and caspase activation leading to apoptosis and aponecrosis.

  9. Killing multiple myeloma cells with the small molecule 3-bromopyruvate: implications for therapy.

    Science.gov (United States)

    Majkowska-Skrobek, Grażyna; Augustyniak, Daria; Lis, Paweł; Bartkowiak, Anna; Gonchar, Mykhailo; Ko, Young H; Pedersen, Peter L; Goffeau, Andre; Ułaszewski, Stanisław

    2014-07-01

    The small molecule 3-bromopyruvate (3-BP), which has emerged recently as the first member of a new class of potent anticancer agents, was tested for its capacity to kill multiple myeloma (MM) cancer cells. Human MM cells (RPMI 8226) begin to lose viability significantly within 8 h of incubation in the presence of 3-BP. The Km (0.3 mmol/l) for intracellular accumulation of 3-BP in MM cells is 24 times lower than that in control cells (7.2 mmol/l). Therefore, the uptake of 3-BP by MM cells is significantly higher than that by peripheral blood mononuclear cells. Further, the IC50 values for human MM cells and control peripheral blood mononuclear cells are 24 and 58 µmol/l, respectively. Therefore, specificity and selectivity of 3-BP toward MM cancer cells are evident on the basis of the above. In MM cells the transcription levels of the gene encoding the monocarboxylate transporter MCT1 is significantly amplified compared with control cells. The level of intracellular ATP in MM cells decreases by over 90% within 1 h after addition of 100 µmol/l 3-BP. The cytotoxicity of 3-BP, exemplified by a marked decrease in viability of MM cells, is potentiated by the inhibitor of glutathione synthesis buthionine sulfoximine. In addition, the lack of mutagenicity and its superior capacity relative to Glivec to kill MM cancer cells are presented in this study.

  10. Short Communication: Preferential Killing of HIV Latently Infected CD4+ T Cells by MALT1 Inhibitor

    Science.gov (United States)

    Li, Hongmei; He, Hui; Gong, Leyi; Fu, Mingui

    2016-01-01

    Abstract We report that the addition of an host paracaspase MALT1 inhibitor, MI-2, to HIV latently infected ACH-2, Jurkat E4, and J-LAT cells accelerated cell death in the presence of cell stimuli or the protein kinase C agonist, bryostatin 1. MI-2-mediated cell death correlated with the induction of the cellular RNase MCPIP1 and requires the presence of viral component(s). Altogether, the combination of MI-2 and bryostatin 1 displays selective killing of HIV latently infected CD4+ T cells. PMID:26728103

  11. Short Communication: Preferential Killing of HIV Latently Infected CD4(+) T Cells by MALT1 Inhibitor.

    Science.gov (United States)

    Li, Hongmei; He, Hui; Gong, Leyi; Fu, Mingui; Wang, Tony T

    2016-02-01

    We report that the addition of an host paracaspase MALT1 inhibitor, MI-2, to HIV latently infected ACH-2, Jurkat E4, and J-LAT cells accelerated cell death in the presence of cell stimuli or the protein kinase C agonist, bryostatin 1. MI-2-mediated cell death correlated with the induction of the cellular RNase MCPIP1 and requires the presence of viral component(s). Altogether, the combination of MI-2 and bryostatin 1 displays selective killing of HIV latently infected CD4(+) T cells.

  12. Mechanisms of Enhanced Cell Killing at Low Doses: Implications for Radiation Risk

    Energy Technology Data Exchange (ETDEWEB)

    Dr. Peter J. Johnston; Dr. George D. Wilson

    2003-10-15

    We have shown that cell lethality actually measured after exposure to low-doses of low-LET radiation, is markedly enhanced relative to the cell lethality previously expected by extrapolation of the high-dose cell-killing response. Net cancer risk is a balance between cell transformation and cell kill and such enhanced lethality may more than compensate for transformation at low radiation doses over a least the first 10 cGy of low-LET exposure. This would lead to a non-linear, threshold, dose-risk relationship. Therefore our data imply the possibility that the adverse effects of small radiation doses (<10 cGy) could be overestimated in specific cases. It is now important to research the mechanisms underlying the phenomenon of low-dose hypersensitivity to cell killing, in order to determine whether this can be generalized to safely allow an increase in radiation exposure limits. This would have major cost-reduction implications for the whole EM program.

  13. Pan-Bcl-2 inhibitor AT-101 enhances tumor cell killing by EGFR targeted T cells.

    Directory of Open Access Journals (Sweden)

    Archana Thakur

    Full Text Available Pancreatic cancer is a deadly disease and has the worst prognosis among almost all cancers and is in dire need of new and improved therapeutic strategies. Conditioning of tumor cells with chemotherapeutic drug has been shown to enhance the anti-tumor effects of cancer vaccines and adoptive cell therapy. In this study, we investigated the immunomodulatory effects of pan-Bcl-2 inhibitor AT-101 on pancreatic cancer (PC cell cytotoxicity by activated T cells (ATC. The effects of AT-101 on cytotoxicity, early apoptosis, and Granzyme B (GrzB and IFN-γ signaling pathways were evaluated during EGFR bispecific antibody armed ATC (aATC-mediated killing of L3.6pl and MiaPaCa-2 PC cells pre-sensitized with AT-101. We found that pretreatment of tumor cells with AT-101 enhanced susceptibility of L3.6pl and MiaPaCa-2 tumor cells to ATC and aATC-mediated cytotoxicity, which was in part mediated via enhanced release of cytolytic granule GrzB from ATC and aATC. AT-101-sensitized L3.6pl cells showed up-regulation of IFN-γ-mediated induction in the phosphorylation of Ser(727-Stat1 (pS(727-Stat1, and IFN-γ induced dephosphorylation of phospho-Tyr(705-Stat3 (pY(705-Stat3. Priming (conditioning of PC cells with AT-101 can significantly enhance the anti-tumor activity of EGFRBi armed ATC through increased IFN-γ induced activation of pS(727-Stat1 and inhibition of pY(705-Stat3 phosphorylation, and resulting in increased ratio of pro-apoptotic to anti-apoptotic proteins. Our results verify enhanced cytotoxicity after a novel chemotherapy conditioning strategy against PC that warrants further in vivo and clinical investigations.

  14. Polysaccharide nano-vesicular multidrug carriers for synergistic killing of cancer cells

    Science.gov (United States)

    Pramod, P. S.; Shah, Ruchira; Chaphekar, Sonali; Balasubramanian, Nagaraj; Jayakannan, Manickam

    2014-09-01

    Multi-drug delivery based on polymer nano-scaffolds is an essential protocol to be developed for better administration of anticancer drugs to enhance their therapeutic efficacies against cancer cells. Here, we report dual delivery polysaccharide nano-vesicles that are capable of loading and delivering both water soluble and water insoluble drugs together in a single polymer scaffold. The selective rupture of the nano-vesicular assembly under intracellular enzyme conditions allowed the simultaneous delivery of a hydrophobic drug camptothecin (CPT) and hydrophilic drug doxorubicin (DOX) supporting their synergistic killing of breast and colon cancer cells. The polysaccharide nano-vesicles have allowed us to address a few important questions regarding the need for multiple drug administration in cancer cells including (a) the role of simultaneous drug release, (b) antagonistic versus synergistic effects of drug combinations and (c) how these are affected by the ratio of drugs. Further, evaluation of the role of caveolae in endocytosis of these polymer scaffolds was also made. The vesicular scaffolds were found to preserve and deliver DOX resulting in 50-60% better killing of cancer cells than the free drug. Additionally, dual loaded nano-vesicles when compared to drug cocktails with individual drugs in separate nano-vesicles (at comparable molar ratios) suggest the relative drug concentration following release and mode of delivery to be both important in cancer cell killing. Results from these experiments have revealed newly developed polysaccharide nano-vesicles loaded with DOX and CPT drugs as potential candidates for improved breast cancer cell killing. Thus, these custom-designed polysaccharide nano-vesicles provide a new perspective on multi-anticancer drug delivery systems and their efficacy.Multi-drug delivery based on polymer nano-scaffolds is an essential protocol to be developed for better administration of anticancer drugs to enhance their therapeutic

  15. Glycan elongation beyond the mucin associated Tn antigen protects tumor cells from immune-mediated killing.

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    Caroline B Madsen

    Full Text Available Membrane bound mucins are up-regulated and aberrantly glycosylated during malignant transformation in many cancer cells. This results in a negatively charged glycoprotein coat which may protect cancer cells from immune surveillance. However, only limited data have so far demonstrated the critical steps in glycan elongation that make aberrantly glycosylated mucins affect the interaction between cancer cells and cytotoxic effector cells of the immune system. Tn (GalNAc-Ser/Thr, STn (NeuAcα2-6GalNAc-Ser/Thr, T (Galβ1-3GalNAc-Ser/Thr, and ST (NeuAcα2-6Galβ1-3GalNAc-Ser/Thr antigens are recognized as cancer associated truncated glycans, and are expressed in many adenocarcinomas, e.g. breast- and pancreatic cancer cells. To investigate the role of the cancer associated glycan truncations in immune-mediated killing we created glyco-engineered breast- and pancreatic cancer cells expressing only the shortest possible mucin-like glycans (Tn and STn. Glyco-engineering was performed by zinc finger nuclease (ZFN knockout (KO of the Core 1 enzyme chaperone COSMC, thereby preventing glycan elongation beyond the initial GalNAc residue in O-linked glycans. We find that COSMC KO in the breast and pancreatic cancer cell lines T47D and Capan-1 increases sensitivity to both NK cell mediated antibody-dependent cellular-cytotoxicity (ADCC and cytotoxic T lymphocyte (CTL-mediated killing. In addition, we investigated the association between total cell surface expression of MUC1/MUC16 and NK or CTL mediated killing, and observed an inverse correlation between MUC16/MUC1 expression and the sensitivity to ADCC and CTL-mediated killing. Together, these data suggest that up-regulation of membrane bound mucins protects cells from immune mediated killing, and that particular glycosylation steps, as demonstrated for glycan elongation beyond Tn and STn, can be important for fine tuning of the immune escape mechanisms in cancer cells.

  16. Photocatalytic killing effect of TiO2 nanoparticles on Ls-174-t human colon carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Ai-Ping Zhang; Yan-Ping Sun

    2004-01-01

    AIM: To investigate the photocatalytic killing effect of photoexcited TiO2 nanoparticles on human colon carcinoma cell line (Ls-174-t) and to study the mechanism underlying the action of photoexcited TiO2 nanoparticles on malignant cells.METHODS: Ls-174-t human colon carcinoma cells were cultured in RPMI 1640 medium supplemented with 199 mL/L calf serum in a humidified incubator with an atmosphere of 50 mL/L CO2 at 37 ℃. Viable cells in the samples were measured by using the MlT method. A GGZ-300 W high pressure Hg lamp with a maximum ultraviolet-A (UVA, 320-400 nm) irradiation peak at 365 nm was used as light source in the photocatalytic killing test.RESULTS: The photocatalytic killing of Ls-174-t cells was carried out in vitro with TiO2 nanoparticles. The killing effect was weak by using UVA irradiation without TiO2 nanoparticles.In our studies, the photocatalytic killing effect was correlated with the concentration of TiO2 and illumination time. Once TiO2 was added, Ls-174-t cells were killed at a much higher rate. In the presence of 1 000 μg/mL TiO2, 44% of cells were killed after 10 min of UVA irradiation, and 88% of cells were killed after 30 min of UVA irradiation.CONCLUSION: When the concentration of TiO2 is below 200 μg/mL, the photocatalytic killing effect on human colon carcinoma cells is almost the same as that of UVA irradiation alone. When the concentration of TiO2 is above 200 μg/mL,the remarkable killing effect of photoexcited TiO2 nanoparticles can be found.

  17. Complete Cell Killing by Applying High Hydrostatic Pressure for Acellular Vascular Graft Preparation

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    Atsushi Mahara

    2014-01-01

    Full Text Available Pressure treatment has been developed in tissue engineering application. Although the tissue scaffold prepared by a ultrahydrostatic pressure treatment has been reported, an excessive pressure has a potential to disrupt a structure of extracellular matrix through protein denaturation. It is important to understand the suitable low-pressure condition and mechanisms for cell killing. In this study, cellular morphology, mitochondria activity, and membrane permeability of mammalian cells with various pressure treatments were investigated with in vitro models. When the cells were treated with a pressure of 100 MPa for 10 min, cell morphology and adherence were the same as an untreated cells. Dehydrogenase activity in mitochondria was almost the same as untreated cells. On the other hand, when the cells were treated with the pressure of more than 200 MPa, the cells did not adhere, and the dehydrogenase activity was completely suppressed. However, green fluorescence was observed in the live/dead staining images, and the cells were completely stained as red after above 500 MPa. That is, membrane permeability was disturbed with the pressure treatment of above 500 MPa. These results indicated that the pressure of 200 MPa for 10 min was enough to induce cell killing through inactivation of mitochondria activity.

  18. A Small-Molecule Inhibitor of BCL6 Kills DLBCL Cells In Vitro and In Vivo

    Energy Technology Data Exchange (ETDEWEB)

    Cerchietti, L.C.; Ghetu, A.F.; Zhu, X.; Da Silva, G.F.; Zhong, S.; Matthews, M.; Bunting, K.L.; Polo, J.M.; Fares, C.; Arrowsmith, C.H.; Yang, S.N.; Garcia, M.; Coop, A.; Mackerell, A.D.; Prive, G.G.; Melnick, A. (Cornell)

    2010-09-22

    The BCL6 transcriptional repressor is the most frequently involved oncogene in diffuse large B cell lymphoma (DLBCL). We combined computer-aided drug design with functional assays to identify low-molecular-weight compounds that bind to the corepressor binding groove of the BCL6 BTB domain. One such compound disrupted BCL6/corepressor complexes in vitro and in vivo, and was observed by X-ray crystallography and NMR to bind the critical site within the BTB groove. This compound could induce expression of BCL6 target genes and kill BCL6-positive DLBCL cell lines. In xenotransplantation experiments, the compound was nontoxic and potently suppressed DLBCL tumors in vivo. The compound also killed primary DLBCLs from human patients.

  19. Combination of anti-retroviral drugs and radioimmunotherapy specifically kills infected cells from HIV infected individuals

    Directory of Open Access Journals (Sweden)

    Dina Tsukrov

    2016-09-01

    Full Text Available Eliminating virally infected cells is an essential component of any HIV eradication strategy. Radioimmunotherapy (RIT, a clinically established method for killing cells using radiolabeled antibodies, was recently applied to target HIV-1 gp41 antigen expressed on the surface of infect-ed cells. Since gp41 expression by infected cells is likely down-regulated in patients on an-tiretroviral therapy (ART, we evaluated the ability of RIT to kill ART-treated infected cells us-ing both in vitro models and lymphocytes isolated from HIV-infected subjects. Human peripheral blood mononuclear cells (PBMCs were infected with HIV and cultured in the presence of two clinically relevant ART combinations. Scatchard analysis of the 2556 human monoclonal anti-body to HIV gp41 binding to the infected and ART-treated cells demonstrated sufficient residual expression of gp41 on the cell surface to warrant subsequent RIT. This is the first time the quantification of gp41 post-ART is being reported. Cells were then treated with Bismuth-213-labeled 2556 antibody. conjugated to the human monoclonal antibody 2556, which binds to HIV gp41. Cell survival was quantified by Trypan blue and residual viremia by p24 ELISA. Cell surface gp41 expression was assessed by Scatchard analysis. The experiments were repeated using PBMCs isolated from blood specimens obtained from 15 HIV-infected individuals: ten on ART and five ART-naive. We found that 213Bi-2556 killed ART-treated infected PBMCs and reduced viral production to undetectable levels. ART and RIT co-treatment was more effective at reducing viral load in vitro than either therapy alone, indicating that gp41 expression under ART was sufficient to allow 213Bi-2556 to deliver cytocidal doses of radiation to infected cells. This study provides proof of concept that 213Bi-2556 may represent an innovative and effective targeting method for killing HIV-infected cells treated with ART, and supports continued development of 213Bi

  20. Simultaneous Targeting of FcγRs and FcαRI Enhances Tumor Cell Killing

    NARCIS (Netherlands)

    Brandsma, Arianne M; Ten Broeke, Toine; Nederend, Maaike; Meulenbroek, Laura A P M; van Tetering, Geert; Meyer, Saskia; Jansen, J H Marco; Beltrán Buitrago, M Alejandra; Nagelkerke, Sietse Q; Németh, István; Ubink, Ruud; Rouwendal, Gerard; Lohse, Stefan; Valerius, Thomas; Leusen, Jeanette H W; Boross, Peter

    2015-01-01

    Efficacy of anticancer monoclonal antibodies (mAb) is limited by the exhaustion of effector mechanisms. IgG mAbs mediate cellular effector functions through FcγRs expressed on effector cells. IgA mAbs can also induce efficient tumor killing both in vitro and in vivo. IgA mAbs recruit FcαRI-expressin

  1. IL-1α-induced microvascular endothelial cells promote neutrophil killing by increasing MMP-9 concentration and lysozyme activity.

    Science.gov (United States)

    Liu, Xiaoye; Dong, Hong; Wang, Mingming; Gao, Ying; Zhang, Tao; Hu, Ge; Duan, Huiqing; Mu, Xiang

    2016-02-01

    The recruitment of neutrophils by endothelial cells during infection has been extensively studied, but little is known about the regulation of neutrophils activity by endothelial cells. To examine the role of microvascular endothelial cells in neutrophil killing, we established a transmigration model using rat intestinal microvascular endothelial cells (RIMVECs) and measured the extracellular and intracellular killing of Escherichia coli, Lactobacillus acidophilus, and Staphylococcus aureus by transendothelial neutrophils. We observed that blood neutrophils engulfed bacteria but did not kill them, and lipopolysaccharide- or hemolysin-injured RIMVECs inhibited the extracellular and intracellular bactericidal activity of transendothelial neutrophils. In comparison, interleukin-1α-induced RIMVECs promoted the extracellular and intracellular killing activity of transendothelial neutrophils and significantly increased MMP-9 concentration and lysozyme activity in transendothelial neutrophils (p neutrophils and bacterial toxin damage of endothelial cells led to reduction in bactericidal activity of transendothelial neutrophils. These findings offered new insight into the role of endothelial cells in the bactericidal activity of neutrophils.

  2. Adenanthin targets peroxiredoxin I/II to kill hepatocellular carcinoma cells.

    Science.gov (United States)

    Hou, J-K; Huang, Y; He, W; Yan, Z-W; Fan, L; Liu, M-H; Xiao, W-L; Sun, H-D; Chen, G-Q

    2014-09-04

    Adenanthin, a natural diterpenoid isolated from the leaves of Isodon adenanthus, has recently been reported to induce leukemic cell differentiation by targeting peroxiredoxins (Prx) I and II. On the other hand, increasing lines of evidence propose that these Prx proteins would become potential targets to screen drugs for the prevention and treatment of solid tumors. Therefore, it is of significance to explore the potential activities of adenanthin on solid tumor cells. Here, we demonstrate that Prx I protein is essential for the survival of hepatocellular carcinoma (HCC) cells, and adenanthin can kill these malignant liver cells in vitro and xenografts. We also show that the cell death-inducing activity of adenanthin on HCC cells is mediated by the increased reactive oxygen species (ROS) levels. Furthermore, the silencing of Prx I or Prx II significantly enhances the cytotoxic activity of adenanthin on HCC, whereas the ectopic expression of Prx I and Prx II but not their mutants of adenanthin-bound cysteines can rescue adenanthin-induced cytotoxicity in Prxs-silenced HCC cells. Taken together, our results propose that adenanthin targets Prx I/II to kill HCC cells and its therapeutic significance warrants to be further explored in HCC patients.

  3. Targeted killing of a mammalian cell based upon its specialized metabolic state.

    Science.gov (United States)

    Alexander, Peter B; Wang, Jian; McKnight, Steven L

    2011-09-20

    Mouse ES cells use a mitochondrial threonine dehydrogenase (TDH) enzyme to catabolize threonine into glycine and acetyl-CoA. Measurements of mRNA abundance have given evidence that ES cells express upwards of 1,000-fold higher levels of TDH mRNA than any of seven other mouse tissues tested. When cell culture medium is deprived of threonine, ES cells rapidly discontinue DNA synthesis, arrest cell division, and eventually die. Such studies led to the conclusion that mouse ES cells exist in a threonine-dependent metabolic state. Proceeding with the assumption that the active TDH enzyme should be essential for the growth and viability of mouse ES cells, we performed a drug screen in search of specific inhibitors of the purified TDH enzyme. Such efforts led to the discovery of a class of quinazolinecarboxamide (Qc) compounds that inhibit the ability of the TDH enzyme to catabolize threonine into glycine and acetyl-CoA. Administration of Qc inhibitors of TDH to mouse ES cells impeded cell growth and resulted in the induction of autophagy. By contrast, the same chemicals failed to affect the growth of HeLa cells at concentrations 300-fold higher than that required to kill mouse ES cells. It was likewise observed that the Qc class of TDH inhibitors failed to affect the growth or viability of ES cell-derived embryoid body cells known to have extinguished TDH expression. These studies demonstrate how it is possible to kill a specific mammalian cell type on the basis of its specialized metabolic state.

  4. Protection of hepatocytes from cytotoxic T cell mediated killing by interferon-alpha.

    Directory of Open Access Journals (Sweden)

    Christian B Willberg

    Full Text Available BACKGROUND: Cellular immunity plays a key role in determining the outcome of hepatitis C virus (HCV infection, although the majority of infections become persistent. The mechanisms behind persistence are still not clear; however, the primary site of infection, the liver, may be critical. We investigated the ability of CD8+ T-cells (CTL to recognise and kill hepatocytes under cytokine stimulation. METHODS/PRINCIPLE FINDINGS: Resting hepatocytes cell lines expressed low levels of MHC Class I, but remained susceptible to CTL cytotoxicity. IFN-alpha treatment, in vitro, markedly increased hepatocyte MHC Class I expression, however, reduced sensitivity to CTL cytotoxicity. IFN-alpha stimulated hepatocyte lines were still able to present antigen and induce IFN-gamma expression in interacting CTL. Resistance to killing was not due to the inhibition of the FASL/FAS- pathway, as stimulated hepatocytes were still susceptible to FAS-mediated apoptosis. In vitro stimulation with IFN-alpha, or the introduction of a subgenomic HCV replicon into the HepG2 line, upregulated the expression of the granzyme-B inhibitor-proteinase inhibitor 9 (PI-9. PI-9 expression was also observed in liver tissue biopsies from patients with chronic HCV infection. CONCLUSION/SIGNIFICANCE: IFN-alpha induces resistance in hepatocytes to perforin/granzyme mediate CTL killing pathways. One possible mechanism could be through the expression of the PI-9. Hindrance of CTL cytotoxicity could contribute to the chronicity of hepatic viral infections.

  5. Biodegradable polymeric micelle-encapsulated doxorubicin suppresses tumor metastasis by killing circulating tumor cells

    Science.gov (United States)

    Deng, Senyi; Wu, Qinjie; Zhao, Yuwei; Zheng, Xin; Wu, Ni; Pang, Jing; Li, Xuejing; Bi, Cheng; Liu, Xinyu; Yang, Li; Liu, Lei; Su, Weijun; Wei, Yuquan; Gong, Changyang

    2015-03-01

    Circulating tumor cells (CTCs) play a crucial role in tumor metastasis, but it is rare for any chemotherapy regimen to focus on killing CTCs. Herein, we describe doxorubicin (Dox) micelles that showed anti-metastatic activity by killing CTCs. Dox micelles with a small particle size and high encapsulation efficiency were obtained using a pH-induced self-assembly method. Compared with free Dox, Dox micelles exhibited improved cytotoxicity, apoptosis induction, and cellular uptake. In addition, Dox micelles showed a sustained release behavior in vitro, and in a transgenic zebrafish model, Dox micelles exhibited a longer circulation time and lower extravasation from blood vessels into surrounding tissues. Anti-tumor and anti-metastatic activities of Dox micelles were investigated in transgenic zebrafish and mouse models. In transgenic zebrafish, Dox micelles inhibited tumor growth and prolonged the survival of tumor-bearing zebrafish. Furthermore, Dox micelles suppressed tumor metastasis by killing CTCs. In addition, improved anti-tumor and anti-metastatic activities were also confirmed in mouse tumor models, where immunofluorescent staining of tumors indicated that Dox micelles induced more apoptosis and showed fewer proliferation-positive cells. There were decreased side effects in transgenic zebrafish and mice after administration of Dox micelles. In conclusion, Dox micelles showed stronger anti-tumor and anti-metastatic activities and decreased side effects both in vitro and in vivo, which may have potential applications in cancer therapy.

  6. Dynamic visualization the whole process of cytotoxic T lymphocytes killing the B16 tumor cells in vitro

    Science.gov (United States)

    Qi, Shuhong; Zhang, Zhihong

    2016-03-01

    Cytotoxic T lymphocytes (CTLs) played a key role in the immune system to destroy the tumor cells. Although some mechanisms of CTLs killing the tumor cells are revealed already, the dynamic information of CTLs interaction with tumor cells are still not known very clearly. Here we used confocal microscopy to visualize the whole process of CTLs killing the tumor cells in vitro. The imaging data showed that CTLs destroyed the target tumor cells rapidly and efficiently. Several CTLs surrounded one or some tumor cells and the average time for CTLs destroying one tumor cell is just a few minutes in vitro. The study displayed the temporal events of CTLs interacting with tumor cells at the beginning and finally killing them and directly presented the efficient tumor cell cytotoxicity of the CTLs. The results helped us to deeply understand the mechanism of the CTLs destroying the tumor cells and to develop the cancer immunotherapy.

  7. Repurposing a Prokaryotic Toxin-Antitoxin System for the Selective Killing of Oncogenically Stressed Human Cells.

    Science.gov (United States)

    Preston, Mark A; Pimentel, Belén; Bermejo-Rodríguez, Camino; Dionne, Isabelle; Turnbull, Alice; de la Cueva-Méndez, Guillermo

    2016-07-15

    Prokaryotes express intracellular toxins that pass unnoticed to carrying cells until coexpressed antitoxin partners are degraded in response to stress. Although not evolved to function in eukaryotes, one of these toxins, Kid, induces apoptosis in mammalian cells, an effect that is neutralized by its cognate antitoxin, Kis. Here we engineered this toxin-antitoxin pair to create a synthetic system that becomes active in human cells suffering a specific oncogenic stress. Inspired by the way Kid becomes active in bacterial cells, we produced a Kis variant that is selectively degraded in human cells expressing oncoprotein E6. The resulting toxin-antitoxin system functions autonomously in human cells, distinguishing those that suffer the oncogenic insult, which are killed by Kid, from those that do not, which remain protected by Kis. Our results provide a framework for developing personalized anticancer strategies avoiding off-target effects, a challenge that has been hardly tractable by other means thus far.

  8. A model of radiation-induced cell killing: insights into mechanisms and applications for hadron therapy.

    Science.gov (United States)

    Ballarini, Francesca; Altieri, Saverio; Bortolussi, Silva; Giroletti, Elio; Protti, Nicoletta

    2013-09-01

    A mechanism-based, two-parameter biophysical model of cell killing was developed with the aim of elucidating the mechanisms underlying radiation-induced cell death and predicting cell killing by different radiation types, including protons and carbon ions at energies and doses of interest for cancer therapy. The model assumed that certain chromosome aberrations (dicentrics, rings and large deletions, called "lethal aberrations") lead to clonogenic inactivation, and that aberrations derive from μm-scale misrejoining of chromatin fragments, which in turn are produced by "dirty" double-strand breaks called "cluster lesions" (CLs). The average numbers of CLs per Gy per cell were left as a semi-free parameter and the threshold distance for chromatin-fragment rejoining was defined the second parameter. The model was "translated" into Monte Carlo code and provided simulated survival curves, which were compared with survival data on V79 cells exposed to protons, carbon ions and X rays. The agreement was good between simulations and survival data and supported the assumptions of the model at least for doses up to a few Gy. Dicentrics, rings and large deletions were found to be lethal not only for AG1522 cells exposed to X rays, as already reported by others, but also for V79 cells exposed to protons and carbon ions of different energies. Furthermore, the derived CL yields suggest that the critical DNA lesions leading to clonogenic inactivation are more complex than "clean" DSBs. After initial validation, the model was applied to characterize the particle and LET dependence of proton and carbon cell killing. Consistent with the proton data, the predicted fraction of inactivated cells after 2 Gy protons was 40-50% below 7.7 keV/μm, increased by a factor ∼1.6 between 7.7-30.5 keV/μm, and decreased by a factor ∼1.1 between 30.5-34.6 keV/μm. These LET values correspond to proton energies below a few MeV, which are always present in the distal region of hadron therapy

  9. Killing of targets by effector CD8 T cells in the mouse spleen follows the law of mass action

    Energy Technology Data Exchange (ETDEWEB)

    Ganusov, Vitaly V [Los Alamos National Laboratory

    2009-01-01

    In contrast with antibody-based vaccines, it has been difficult to measure the efficacy of T cell-based vaccines and to correlate the efficacy of CD8 T cell responses with protection again viral infections. In part, this difficulty is due to poor understanding of the in vivo efficacy of CD8 T cells produced by vaccination. Using a: recently developed experimental method of in vivo cytotoxicity we have investigated quantitative aspects of killing of peptide-pulsed targets by effector and memory CD8 T cells, specific to three epitopes of lymphocytic choriomeningitis virus (LCMV), in the mouse spleen. By analyzing data on killing of targets with varying number of epitope-specific effector and memory CD8 T cells, we find that killing of targets by effectors follows the law of mass-action, that is the death rate of peptide-pulsed targets is proportional to the frequency of CTLs in the spleen. In contrast, killing of targets by memory CD8 T cells does not follow the mass action law because the death rate of targets saturates at high frequencies of memory CD8 T cells. For both effector and memory cells, we also find little support for the killing term that includes the decrease of the death rate of targets with target cell density. Interestingly, our analysis suggests that at low CD8 T cell frequencies, memory CD8 T cells on the per capita basis are more efficient at killing peptide-pulsed targets than effectors, but at high frequencies, effectors are more efficient killers than memory T cells. Comparison of the estimated killing efficacy of effector T cells with the value that is predicted from theoretical physics and based on motility of T cells in lymphoid tissues, suggests that limiting step in the killing of peptide-pulsed targets is delivering the lethal hit and not finding the target. Our results thus form a basis for quantitative understanding of the process of killing of virus-infected cells by T cell responses in tissues and can be used to correlate the

  10. Cryptococcus gattii is killed by dendritic cells, but evades adaptive immunity by failing to induce dendritic cell maturation.

    Science.gov (United States)

    Huston, Shaunna M; Li, Shu Shun; Stack, Danuta; Timm-McCann, Martina; Jones, Gareth J; Islam, Anowara; Berenger, Byron M; Xiang, Richard F; Colarusso, Pina; Mody, Christopher H

    2013-07-01

    During adaptive immunity to pathogens, dendritic cells (DCs) capture, kill, process, and present microbial Ags to T cells. Ag presentation is accompanied by DC maturation driven by appropriate costimulatory signals. However, current understanding of the intricate regulation of these processes remains limited. Cryptococcus gattii, an emerging fungal pathogen in the Pacific Northwest of Canada and the United States, fails to stimulate an effective immune response in otherwise healthy hosts leading to morbidity or death. Because immunity to fungal pathogens requires intact cell-mediated immunity initiated by DCs, we asked whether C. gattii causes dysregulation of DC functions. C. gattii was efficiently bound and internalized by human monocyte-derived DCs, trafficked to late phagolysosomes, and killed. Yet, even with this degree of DC activation, the organism evaded pathways leading to DC maturation. Despite the ability to recognize and kill C. gattii, immature DCs failed to mature; there was no increased expression of MHC class II, CD86, CD83, CD80, and CCR7, or decrease of CD11c and CD32, which resulted in suboptimal T cell responses. Remarkably, no increase in TNF-α was observed in the presence of C. gattii. However, addition of recombinant TNF-α or stimulation that led to TNF-α production restored DC maturation and restored T cell responses. Thus, despite early killing, C. gattii evades DC maturation, providing a potential explanation for its ability to infect immunocompetent individuals. We have also established that DCs retain the ability to recognize and kill C. gattii without triggering TNF-α, suggesting independent or divergent activation pathways among essential DC functions.

  11. Sulindac enhances the killing of cancer cells exposed to oxidative stress.

    Directory of Open Access Journals (Sweden)

    Maria Marchetti

    Full Text Available BACKGROUND: Sulindac is an FDA-approved non-steroidal anti-inflammatory drug (NSAID that affects prostaglandin production by inhibiting cyclooxygenases (COX 1 and 2. Sulindac has also been of interest for more than decade as a chemopreventive for adenomatous colorectal polyps and colon cancer. PRINCIPAL FINDINGS: Pretreatment of human colon and lung cancer cells with sulindac enhances killing by an oxidizing agent such as tert-butyl hydroperoxide (TBHP or hydrogen peroxide. This effect does not involve cyclooxygenase (COX inhibition. However, under the conditions used, there is a significant increase in reactive oxygen species (ROS within the cancer cells and a loss of mitochondrial membrane potential, suggesting that cell death is due to apoptosis, which was confirmed by Tunel assay. In contrast, this enhanced killing was not observed with normal lung or colon cells. SIGNIFICANCE: These results indicate that normal and cancer cells handle oxidative stress in different ways and sulindac can enhance this difference. The combination of sulindac and an oxidizing agent could have therapeutic value.

  12. Effective cancer cell killing by hydrophobic nanovoid-enhanced cavitation under safe low-energy ultrasound.

    Science.gov (United States)

    Zhao, Yang; Zhu, Yingchun; Fu, Jingke; Wang, Lianzhou

    2014-03-01

    β-Cyclodextrin (β-CD)-capped mesoporous silica nanoparticles with hydrophobic internal nanovoids were prepared and used for effective cancer cell killing in synergistic combination with low-energy ultrasound (≤1.0 W cm(-2) , 1 MHz). The water-dispersible nanoparticles with hydrophobic internal nanovoids can be taken up by cancer cells and subsequently evoke a remarkable cavitation effect under irradiation with mild low-energy ultrasound (≤1.0 W cm(-2) , 1 MHz). A significant cancer cell killing effect was observed in cancer cells and in a mouse xenograft tumor model treated with the nanoagents together with the low-energy ultrasound, showing a distinct dependence on the concentration of nanoagents and ultrasound intensity. By contrast, an antitumor effect was not observed when either low-energy ultrasound or nanoagents were applied alone. These findings are significant as the technique promises a safe, low-cost, and effective treatment for cancer therapy.

  13. Activated allogeneic NK cells preferentially kill poor prognosis B-cell chronic lymphocytic leukemia cells

    Directory of Open Access Journals (Sweden)

    Diego Sanchez-Martinez

    2016-10-01

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

  14. Activated allogeneic NK cells preferentially kill poor prognosis B-cell chronic lymphocytic leukemia cells

    OpenAIRE

    2016-01-01

    Mutational status of TP53 together with expression of wild type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA mismatched Natural Killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cell...

  15. Decitabine Treatment of Glioma-Initiating Cells Enhances Immune Recognition and Killing

    Science.gov (United States)

    Riccadonna, Cristina; Yacoub Maroun, Céline; Vuillefroy de Silly, Romain; Boehler, Margaux; Calvo Tardón, Marta; Jueliger, Simone; Taverna, Pietro; Barba, Leticia; Marinari, Eliana; Pellegatta, Serena; Bassoy, Esen Yonca; Martinvalet, Denis; Dietrich, Pierre-Yves; Walker, Paul R.

    2016-01-01

    Malignant gliomas are aggressive brain tumours with very poor prognosis. The majority of glioma cells are differentiated (glioma-differentiated cells: GDCs), whereas the smaller population (glioma-initiating cells, GICs) is undifferentiated and resistant to conventional therapies. Therefore, to better target this pool of heterogeneous cells, a combination of diverse therapeutic approaches is envisaged. Here we investigated whether the immunosensitising properties of the hypomethylating agent decitabine can be extended to GICs. Using the murine GL261 cell line, we demonstrate that decitabine augments the expression of the death receptor FAS both on GDCs and GICs. Interestingly, it had a higher impact on GICs and correlated with an enhanced sensitivity to FASL-mediated cell death. Moreover, the expression of other critical molecules involved in cognate recognition by cytotoxic T lymphocytes, MHCI and ICAM-1, was upregulated by decitabine treatment. Consequently, T-cell mediated killing of both GDCs and GICs was enhanced, as was T cell proliferation after reactivation. Overall, although GICs are described to resist classical therapies, our study shows that hypomethylating agents have the potential to enhance glioma cell recognition and subsequent destruction by immune cells, regardless of their differentiation status. These results support the development of combinatorial treatment modalities including epigenetic modulation together with immunotherapy in order to treat heterogenous malignancies such as glioblastoma. PMID:27579489

  16. A CSPG4-specific immunotoxin kills rhabdomyosarcoma cells and binds to primary tumor tissues.

    Science.gov (United States)

    Brehm, Hannes; Niesen, Judith; Mladenov, Radoslav; Stein, Christoph; Pardo, Alessa; Fey, Georg; Helfrich, Wijnand; Fischer, Rainer; Gattenlöhner, Stefan; Barth, Stefan

    2014-10-01

    The treatment of rhabdomyosarcoma (RMS) remains challenging, with metastatic and alveolar RMS offering a particularly poor prognosis. Therefore, the identification and evaluation of novel antigens, which are suitable targets for immunotherapy, is one attractive possibility to improve the treatment of this disease. Here we show that chondroitin sulfate proteoglycan 4 (CSPG4) is expressed on RMS cell lines and RMS patient material. We evaluated the immunotoxin (IT) αMCSP-ETA', which specifically recognizes CSPG4 on the RMS cell lines RD, FL-OH1, TE-671 and Rh30. It is internalized rapidly, induces apoptosis and thus kills RMS cells selectively. We also demonstrate the specific binding of this IT to RMS primary tumor material from three different patients.

  17. Neutrophil killing of human umbilical vein endothelial cells is oxygen radical-mediated and enhanced by TNF-. alpha

    Energy Technology Data Exchange (ETDEWEB)

    Dame, M.K.; Varani, J.; Weinberg, J.M.; Ward, P.A. (Univ. of Michigan, Ann Arbor (United States))

    1991-03-11

    Human umbilical vein endothelial cells are sensitive to killing by activated human neutrophils. Killing is inhibited in the presence of catalase and deferoxamine mesylate but not soybean trypsin inhibitor. Reagent hydrogen peroxide can substitute for activated neutrophils in producing endothelial cell injury. These data suggest that lethal injury is due to the production of oxygen radicals by activated neutrophils. In these respects, the human umbilical vein endothelial cells are similar to rat pulmonary artery endothelial cells in that pretreatment with TNF-{alpha} increases sensitivity to injury by activated neutrophils. In part, the increased endothelial cell sensitivity to killing by neutrophils may be due to up-regulation of surface adhesion molecules. However, it was observed that cells passaged more than two times in culture did not demonstrate increased killing after treatment with TNF-{alpha} while up-regulation of neutrophil adhesion could be detected through several additional passages. Although the human umbilical vein endothelial cells are qualitatively similar to rat pulmonary artery endothelial cells in their sensitivity to killing, they are quantitatively much more resistant. What accounts for the relative resistance of the human umbilical vein endothelial cells is not fully understood. In the rat pulmonary artery endothelial cells, killing is known to be dependent on an intraendothelial source of iron. Pre-treatment of the human umbilical vein endothelial cells with 8-hydroxyquinoline-bound iron increased their sensitivity to oxidant injury. These data suggest that the availability of iron within the human umbilical vein endothelial cells may be a limiting factor in sensitivity to oxygen radical-mediated injury.

  18. Targeted killing of virally infected cells by radiolabeled antibodies to viral proteins.

    Directory of Open Access Journals (Sweden)

    Ekaterina Dadachova

    2006-11-01

    Full Text Available BACKGROUND: The HIV epidemic is a major threat to health in the developing and western worlds. A modality that targets and kills HIV-1-infected cells could have a major impact on the treatment of acute exposure and the elimination of persistent reservoirs of infected cells. The aim of this proof-of-principle study was to demonstrate the efficacy of a therapeutic strategy of targeting and eliminating HIV-1-infected cells with radiolabeled antibodies specific to viral proteins in vitro and in vivo. METHODS AND FINDINGS: Antibodies to HIV-1 envelope glycoproteins gp120 and gp41 labeled with radioisotopes bismuth 213 ((213Bi and rhenium 188 ((188Re selectively killed chronically HIV-1-infected human T cells and acutely HIV-1-infected human peripheral blood mononuclear cells (hPBMCs in vitro. Treatment of severe combined immunodeficiency (SCID mice harboring HIV-1-infected hPBMCs in their spleens with a (213Bi- or (188Re-labeled monoclonal antibody (mAb to gp41 resulted in a 57% injected dose per gram uptake of radiolabeled mAb in the infected spleens and in a greater than 99% elimination of HIV-1-infected cells in a dose-dependent manner. The number of HIV-1-infected thymocytes decreased 2.5-fold in the human thymic implant grafts of SCID mice treated with the (188Re-labeled antibody to gp41 compared with those treated with the (188Re-control mAb. The treatment did not cause acute hematologic toxicity in the treated mice. CONCLUSIONS: The current study demonstrates the effectiveness of HIV-targeted radioimmunotherapy and may provide a novel treatment option in combination with highly active antiretroviral therapy for the eradication of HIV.

  19. Killing of Brain Tumor Cells by Hypoxia-Responsive Element Mediated Expression of BAX

    Directory of Open Access Journals (Sweden)

    Hangjun Ruan

    1999-11-01

    Full Text Available The presence of radioresistant hypoxic cells in human brain tumors limits the overall effectiveness of conventional fractionated radiation therapy. Tumor-specific therapies that target hypoxic cells are clearly needed. We have investigated the expression of suicide genes under hypoxia by a hypoxia-responsive element (HRE, which can be activated through hypoxia-inducible factor-1 (HIF-1. We transfected plasmids containing multiple copies of HIRE into U-87 MG and U-251 MG-NCI human brain tumor cells and tested their ability to induce LacZ gene expression under anoxia. Gene expression under anoxia versus oxia was increased about 12-fold for U-87 MG cells and about fourfold for U-251 MG-NCI cells. At intermediate hypoxic conditions, increased LacZ gene expression in U-87 MG cells was induced by the plasmid that contained three HREs, but not by the plasmid with two HREs. Lastly, when we placed a suicide gene BAX under the control of HREs, cells transfected with the BAX plasmids were preferentially killed through apoptosis under anoxia. Our studies demonstrate that HRE-regulated gene expression is active in brain tumor cells, and that the amount of increased gene expression obtained is dependent on the cell line, the HIRE copy number, and the degree of hypoxia.

  20. TH17 cells promote microbial killing and innate immune sensing of DNA via interleukin 26

    KAUST Repository

    Meller, Stephan

    2015-07-13

    Interleukin 17-producing helper T cells (TH 17 cells) have a major role in protection against infections and in mediating autoimmune diseases, yet the mechanisms involved are incompletely understood. We found that interleukin 26 (IL-26), a human TH17 cell-derived cytokine, is a cationic amphipathic protein that kills extracellular bacteria via membrane-pore formation. Furthermore, TH17 cell-derived IL-26 formed complexes with bacterial DNA and self-DNA released by dying bacteria and host cells. The resulting IL-26-DNA complexes triggered the production of type I interferon by plasmacytoid dendritic cells via activation of Toll-like receptor 9, but independently of the IL-26 receptor. These findings provide insights into the potent antimicrobial and proinflammatory function of TH17 cells by showing that IL-26 is a natural human antimicrobial that promotes immune sensing of bacterial and host cell death. © 2015 Nature America, Inc.

  1. Killing effect of coexpressing cytosine deaminase and thymidine kinase on rat vascular smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    曹慧青; 孟宪敏; 刘冬青; 赵秀文; 丁金凤

    2004-01-01

    Background Vascular smooth muscle cell (VSMC) proliferation following arterial injury plays a critical role in a variety of vascular proliferative disorders, such as atherosclerosis and restenosis after balloon angioplasty. Herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV) and E.coli cytosine deaminase (CD)/5-fluorocytosine (5-Fc) suicide gene systems have been successfully employed in cardiovascular gene therapy, respectively. We reasoned that coexpression of both HSV-TK with CD suicide genes would lead to increased cell killing. To test this imagine, the adenoviral vectors expressing TK and/or CD genes were developed and tested on vascular smooth muscle cells. Methods Adenoviral vectors, including Ad-EF1α-CD-cytomegolovirus (CMV)-TK coexpressing both CD and TK double suicide genes, Ad-EF1α-CD and Ad-CMV-TK expressing CD and TK respectively, and control vector Ad-CMV-LacZ, were constructed and prepared with homologous recombination in RecA+E.coli cells. Integration and expression of CD and/or TK gene were identified by PCR and Western blot. Primary cultured VSMCs were infected at a multiplicity of infection (MOI) of 20 with exposure to their matching prodrugs 5-Fc and GCV. Cell mortality was measured by methyl thiazolyl tetrazolium (MTT) assays. Flow cytometry analysis was used to detect cell death. Apoptotic cells were analyzed using Hoechst 33342 fluorescence dye as a DNA probe. Genomic DNA cleavage of apoptotic VSMCs was tested by agarose gel electrophoresis. Results Recombinant adenovirus expressing CD and/or TK suicide genes were successfully constructed. Both single and double suicide genes could be integrated into adenoviral genome and expressed. Cytotoxic effects of Ad-EF1α-CD-CMV-TK double suicide genes combined with 5-Fc and GCV were higher than those of Ad-CMV-TK and Ad-EF1α-CD single gene groups. The rate of cell survival was only (9±3)% in the Ad-EF1α-CD-CMV-TK group, but (37±3)% in the Ad-CMV-TK and (46±4)% in the Ad-EF1

  2. Induction of Foot-and-Mouth Disease Virus-Specific Cytotoxic T Cell Killing by Vaccination

    DEFF Research Database (Denmark)

    Patch, J.R.; Pedersen, Lasse Eggers; Toka, F.N.;

    2011-01-01

    Foot-and-mouth disease (FMD) continues to be a significant threat to the health and economic value of livestock species. This acute infection is caused by the highly contagious FMD virus (FMDV), which infects cloven-hoofed animals including large and small ruminants and swine. Current vaccine...... strategies are all directed toward the induction of neutralizing antibody responses. However, the role of cytotoxic T lymphocytes (CTLs) has not received a great deal of attention, in part because of the technical difficulties associated with establishing a reliable assay of cell killing for this highly...... cytopathic virus. Here, we have used recombinant human adenovirus vectors as a means of delivering FMDV antigens in a T cell-directed vaccine in pigs. We tested the hypothesis that impaired processing of the FMDV capsid would enhance cytolytic activity, presumably by targeting all proteins for degradation...

  3. A numerical investigation of the electric and thermal cell kill distributions in electroporation-based therapies in tissue.

    Directory of Open Access Journals (Sweden)

    Paulo A Garcia

    Full Text Available Electroporation-based therapies are powerful biotechnological tools for enhancing the delivery of exogeneous agents or killing tissue with pulsed electric fields (PEFs. Electrochemotherapy (ECT and gene therapy based on gene electrotransfer (EGT both use reversible electroporation to deliver chemotherapeutics or plasmid DNA into cells, respectively. In both ECT and EGT, the goal is to permeabilize the cell membrane while maintaining high cell viability in order to facilitate drug or gene transport into the cell cytoplasm and induce a therapeutic response. Irreversible electroporation (IRE results in cell kill due to exposure to PEFs without drugs and is under clinical evaluation for treating otherwise unresectable tumors. These PEF therapies rely mainly on the electric field distributions and do not require changes in tissue temperature for their effectiveness. However, in immediate vicinity of the electrodes the treatment may results in cell kill due to thermal damage because of the inhomogeneous electric field distribution and high current density during the electroporation-based therapies. Therefore, the main objective of this numerical study is to evaluate the influence of pulse number and electrical conductivity in the predicted cell kill zone due to irreversible electroporation and thermal damage. Specifically, we simulated a typical IRE protocol that employs ninety 100-µs PEFs. Our results confirm that it is possible to achieve predominant cell kill due to electroporation if the PEF parameters are chosen carefully. However, if either the pulse number and/or the tissue conductivity are too high, there is also potential to achieve cell kill due to thermal damage in the immediate vicinity of the electrodes. Therefore, it is critical for physicians to be mindful of placement of electrodes with respect to critical tissue structures and treatment parameters in order to maintain the non-thermal benefits of electroporation and prevent

  4. A numerical investigation of the electric and thermal cell kill distributions in electroporation-based therapies in tissue.

    Science.gov (United States)

    Garcia, Paulo A; Davalos, Rafael V; Miklavcic, Damijan

    2014-01-01

    Electroporation-based therapies are powerful biotechnological tools for enhancing the delivery of exogeneous agents or killing tissue with pulsed electric fields (PEFs). Electrochemotherapy (ECT) and gene therapy based on gene electrotransfer (EGT) both use reversible electroporation to deliver chemotherapeutics or plasmid DNA into cells, respectively. In both ECT and EGT, the goal is to permeabilize the cell membrane while maintaining high cell viability in order to facilitate drug or gene transport into the cell cytoplasm and induce a therapeutic response. Irreversible electroporation (IRE) results in cell kill due to exposure to PEFs without drugs and is under clinical evaluation for treating otherwise unresectable tumors. These PEF therapies rely mainly on the electric field distributions and do not require changes in tissue temperature for their effectiveness. However, in immediate vicinity of the electrodes the treatment may results in cell kill due to thermal damage because of the inhomogeneous electric field distribution and high current density during the electroporation-based therapies. Therefore, the main objective of this numerical study is to evaluate the influence of pulse number and electrical conductivity in the predicted cell kill zone due to irreversible electroporation and thermal damage. Specifically, we simulated a typical IRE protocol that employs ninety 100-µs PEFs. Our results confirm that it is possible to achieve predominant cell kill due to electroporation if the PEF parameters are chosen carefully. However, if either the pulse number and/or the tissue conductivity are too high, there is also potential to achieve cell kill due to thermal damage in the immediate vicinity of the electrodes. Therefore, it is critical for physicians to be mindful of placement of electrodes with respect to critical tissue structures and treatment parameters in order to maintain the non-thermal benefits of electroporation and prevent unnecessary damage to

  5. Enhancing effects of gamma interferon on phagocytic cell association with and killing of Trypanosoma cruzi

    Science.gov (United States)

    Wirth, J. J.; Kierszenbaum, F.; Sonnenfeld, G.; Zlotnik, A.

    1985-01-01

    Results are reported from a study of the influence gamma interferon (GIFN) and interleukin 2 (IL2) have on the capability of P388D1 cells and mouse resident peritoneal macrophages (MPM) to attach to the blood-resident parasites Trypanosoma cruzi and kill them. Cultures of trypomastigote forms of the Tulahuen strain of T. cruzi grown in bovine serum were introduced into peritoneal cells of mice, along with P388D1 cells incubated with GIFN, IL2 and both. Control cells were also maintained. Statistical analysis were then performed on data on counts of the number of dead T. Cruzi cells. The GIFN enhanced the interaction of MPM and P388D1 cells with the surface of T. Cruzi, provided the interaction was given over 12 hr to take place. A depression of the cytotoxicity of P388D1 cells was attributed to mediation by H2O2, an effect partially offset by incubation with the lymphokine GIFN.

  6. Strong synergy of heat and modulated electromagnetic field in tumor cell killing

    Energy Technology Data Exchange (ETDEWEB)

    Andocs, Gabor [Frederic Joliot Curie National Research Inst. for Radiobiology and Radiohygiene, Budapest (Hungary)]|[St. Istvan Univ., Budapest (Hungary). Dept. of Pharmacology and Toxicology; Renner, Helmut [Klinikum Nuernberg (Germany). Clinic of Radiooncology; Balogh, Lajos [Frederic Joliot Curie National Research Inst. for Radiobiology and Radiohygiene, Budapest (Hungary); Fonyad, Laszlo [Semmelweis Univ., Budapest (Hungary). 1. Dept. of of Pathology and Experimental Cancer Research; Jakab, Csaba [St. Istvan Univ., Budapest (Hungary). Dept. of Pathology; Szasz, Andras [St. Istvan Univ., Goedoelloe (Hungary). Biotechnics Dept.

    2009-02-15

    Hyperthermia is an emerging complementary method in radiooncology. Despite many positive studies and comprehensive reviews, the method is not widely accepted as a combination to radiotherapy. Modulated electrohyperthermia (mEHT; capacitive, electric field modulated, 13.56 MHz) has been used in clinical practice for almost 2 decades in Germany, Austria and Hungary. This in vivo study in nude mice xenograft tumors compares mEHT with 'classic' radiative hyperthermia (radHT). Nude mice were xenografted with HT29 human colorectal carcinoma cells. 28 mice in four groups with seven animals each and two tumors per animal (totally 56 tumors) were included in the present study: group 1 as untreated control; group 2 treated with radHT at 42 C; group 3 treated with mEHT at identical 42 C; group 4 treated with mEHT at 38 C (by intensively cooling down the tumor). 24 h after treatment, animals were sacrificed and the tumor cross sections studied by precise morphological methods for the respective relative amount of 'dead' tumor cells. The effect of mEHT established a double effect as a synergy between the purely thermal (temperature-dependent) and nonthermal (not directly temperature-dependent) effects. The solely thermal enhancement ratio (TER) of cell killing was shown to be 2.9. The field enhancement ratio (FER) at a constant temperature of 42 C was measured as 3.2. Their complex application significantly increased the therapeutic enhancement to 9.4. mEHT had a remarkable cancer cell-killing effect in a nude mice xenograft model. (orig.)

  7. Merocyanine 540 and Photofrin II as photosensitizers for in vitro killing of duck hepatitis B virus and human hepatoma cells

    Science.gov (United States)

    Lin, Tsung-I.; Shien, Yong-Shau; Kao, Ming-Chien

    1994-03-01

    The feasibility of using merocyanine 540 (MC 540) and Photofrin II (PII) as effective photodynamic therapeutic (PDT) agents for killing hepatoma cells and duck hepatitis B virus (DHBV) in vitro was investigated. Cultured duck hepatocytes infected with DHBV and hepatoma cells, Hep 3B and HCC 36, were used as models. MC 540 and PII effectively inhibits the DHBV growth by 90 - 99% in a dose- and light-dependent manner. Photodynamic killing of MC 540 in the two hepatoma cell lines results in 94 - 99% growth inhibition. However, both photosensitizers exhibit dark cytotoxicity (37 - 56%). The present results suggest that MC 540 and PII could be promising and effective photodynamic agents for killing HBV and hepatoma cells.

  8. A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells

    Directory of Open Access Journals (Sweden)

    Kawamoto Megumi

    2011-08-01

    Full Text Available Abstract Background Transferrin receptor (TfR is a cell membrane-associated glycoprotein involved in the cellular uptake of iron and the regulation of cell growth. Recent studies have shown the elevated expression levels of TfR on cancer cells compared with normal cells. The elevated expression levels of this receptor in malignancies, which is the accessible extracellular protein, can be a fascinating target for the treatment of cancer. We have recently designed novel type of immunotoxin, termed "hybrid peptide", which is chemically synthesized and is composed of target-binding peptide and lytic peptide containing cationic-rich amino acids components that disintegrates the cell membrane for the cancer cell killing. The lytic peptide is newly designed to induce rapid killing of cancer cells due to conformational change. In this study, we designed TfR binding peptide connected with this novel lytic peptide and assessed the cytotoxic activity in vitro and in vivo. Methods In vitro: We assessed the cytotoxicity of TfR-lytic hybrid peptide for 12 cancer and 2 normal cell lines. The specificity for TfR is demonstrated by competitive assay using TfR antibody and siRNA. In addition, we performed analysis of confocal fluorescence microscopy and apoptosis assay by Annexin-V binding, caspase activity, and JC-1 staining to assess the change in mitochondria membrane potential. In vivo: TfR-lytic was administered intravenously in an athymic mice model with MDA-MB-231 cells. After three weeks tumor sections were histologically analyzed. Results The TfR-lytic hybrid peptide showed cytotoxic activity in 12 cancer cell lines, with IC50 values as low as 4.0-9.3 μM. Normal cells were less sensitive to this molecule, with IC50 values > 50 μM. Competition assay using TfR antibody and knockdown of this receptor by siRNA confirmed the specificity of the TfR-lytic hybrid peptide. In addition, it was revealed that this molecule can disintegrate the cell membrane of T47

  9. Silver nanocrystals sensitize magnetic-nanoparticle-mediated thermo-induced killing of cancer cells

    Institute of Scientific and Technical Information of China (English)

    Lianke Liu; Fang Ni; Jianchao Zhang; Xiaoli Jiang; Xiang Lu; Zhirui Guo; Ruizhi Xu

    2011-01-01

    Magnetic nanoparticles (MNPs) can heat up tumor tissues and induce killing of cancer cells under external AC magnetic field. However, magnetic nanoparticles hyperthermia (MNPH) requires high concentration of MNPs that are injected into the tumor in order to obtain clinically needed thermal dose because of the complicated heat transfer in v/vo and the limited heat quality of MNPs. To cut down the dose of MNPs and enhance the effect of this Nanotherapy, we prepared silver nanoparticles (AgNPs) with different sizes and investigated the effects of these AgNPs on cancer cells in MNPH treatment. It was found that AgNPs could enhance thermo-sensitivity of glioma cells and this effect was size dependent. AgNPs could induce cell cycles arrested in G2/M phase and enhanced the apoptosis rate of cancer cells after hyperthermia. In glioma bearing rats model, MNPH combined with AgNPs could enhance Bax expression in cancer cells. Our results suggested that AgNPs could be a potential thermo-sensitizer and could be further developed for the design of Ag nanostructurebased thermal seeds for MNPH therapy.

  10. Study of cell killing and morphology on S180 by ultrasound activating hematoporphyrin derivatives

    Institute of Scientific and Technical Information of China (English)

    刘全宏; 孙世惠; 肖娅萍; 齐浩; 尚志远; 张军平; 张金选; 任耀辉; 李萌; 李青

    2003-01-01

    The inhibition of ascitic S180 and induced sarcoma 180 in vivo was studied with the combination of hematoporphyrin derivatives (HpD) and ultrasound (US) at the frequency of 1.1 MHz and different intensities by light microscopy observation, electronic microscopy observation, cytochemical analysis and fluorescence labeling. The present study indicated that the injury of ascitic S180 increased as time passed and the inhibitory effect was stronger in US plus HpD group than that in other groups. Our results also indicated that the changes in cell structure, cytochrome C oxidase activity, the degradation and missing of DNA were the important factors that inhibited the tumor cell growth and even induced celldeath. The phenomenon of apoptosis of tumor cells indicated that cell death andinduced apoptosis exist in the treatment of sonodynamic therapy (SDT). Our study investigated the mechanism underlying the killing effect of S180 induced by USactivating HpD by the observation of cell morphology and dynamic changes from seminal injury to succeeded injury even to death. It would provide rich referencefor the study of SDT.

  11. Pseudomonas exotoxin antisense RNA selectively kills hepatitis B virus infected cells

    Institute of Scientific and Technical Information of China (English)

    Peter Hafkemeyer; Ulrich Brinkmann; Elizabeth Brinkmann; Ira Pastan; Hubert E Blum; Thomas F Baumert

    2008-01-01

    AIM: To present an approach for selectively killing retrovirus-infected cells that combines the toxicity of Pseudomonas exotoxin (PE) and the presence of reverse transcriptase (RT) in infected cells. METHODS: PE antisense toxin RNA has palindromic stem loops at its 5' and 3' ends enabling self-primed generation of cDNA in the presence of RT. The RT activity expressed in retrovirus-infected cells converts "antisense-toxin-RNA" into a lethal toxin gene exclusively in these cells. RESULTS: Using cotransfection studies with Peexpressing RNAs and β-gal expressing reporter plasmids, we show that, in HepG2 and HepG2. 2. 15 hepatomacells as well as in duck hepatitis B virus (DHBV) infected cells, HBV or DHBV-polymerase reverse transcribe a lethal cDNA copy of an antisense toxin RNA, which is composed of sequences complementary to a PE gene and eukaryotic transcription and translation signals. CONCLUSION: This finding may have important implications as a novel therapeutic strategy aimed at the elimination of HBV infection.

  12. Two-stage model of radon-induced malignant lung tumors in rats: effects of cell killing

    Science.gov (United States)

    Luebeck, E. G.; Curtis, S. B.; Cross, F. T.; Moolgavkar, S. H.

    1996-01-01

    A two-stage stochastic model of carcinogenesis is used to analyze lung tumor incidence in 3750 rats exposed to varying regimens of radon carried on a constant-concentration uranium ore dust aerosol. New to this analysis is the parameterization of the model such that cell killing by the alpha particles could be included. The model contains parameters characterizing the rate of the first mutation, the net proliferation rate of initiated cells, the ratio of the rates of cell loss (cell killing plus differentiation) and cell division, and the lag time between the appearance of the first malignant cell and the tumor. Data analysis was by standard maximum likelihood estimation techniques. Results indicate that the rate of the first mutation is dependent on radon and consistent with in vitro rates measured experimentally, and that the rate of the second mutation is not dependent on radon. An initial sharp rise in the net proliferation rate of initiated cell was found with increasing exposure rate (denoted model I), which leads to an unrealistically high cell-killing coefficient. A second model (model II) was studied, in which the initial rise was attributed to promotion via a step function, implying that it is due not to radon but to the uranium ore dust. This model resulted in values for the cell-killing coefficient consistent with those found for in vitro cells. An "inverse dose-rate" effect is seen, i.e. an increase in the lifetime probability of tumor with a decrease in exposure rate. This is attributed in large part to promotion of intermediate lesions. Since model II is preferable on biological grounds (it yields a plausible cell-killing coefficient), such as uranium ore dust. This analysis presents evidence that a two-stage model describes the data adequately and generates hypotheses regarding the mechanism of radon-induced carcinogenesis.

  13. Anthrax lethal toxin-mediated killing of human and murine dendritic cells impairs the adaptive immune response.

    Directory of Open Access Journals (Sweden)

    Abdelkrim Alileche

    2005-10-01

    Full Text Available Many pathogens have acquired strategies to combat the immune response. Bacillus anthracis interferes with host defenses by releasing anthrax lethal toxin (LT, which inactivates mitogen-activated protein kinase pathways, rendering dendritic cells (DCs and T lymphocytes nonresponsive to immune stimulation. However, these cell types are considered resistant to killing by LT. Here we show that LT kills primary human DCs in vitro, and murine DCs in vitro and in vivo. Kinetics of LT-mediated killing of murine DCs, as well as cell death pathways induced, were dependent upon genetic background: LT triggered rapid necrosis in BALB/c-derived DCs, and slow apoptosis in C57BL/6-derived DCs. This is consistent with rapid and slow killing of LT-injected BALB/c and C57BL/6 mice, respectively. We present evidence that anthrax LT impairs adaptive immunity by specifically targeting DCs. This may represent an immune-evasion strategy of the bacterium, and contribute to anthrax disease progression. We also established that genetic background determines whether apoptosis or necrosis is induced by LT. Finally, killing of C57BL/6-derived DCs by LT mirrors that of human DCs, suggesting that C57BL/6 DCs represent a better model system for human anthrax than the prototypical BALB/c macrophages.

  14. Different Length (DL qPCR for Quantification of Cell Killing by UV-induced DNA Damage

    Directory of Open Access Journals (Sweden)

    Guro Skjefstad

    2010-08-01

    Full Text Available We describe the different length (DL qPCR method for quantification of UV induced DNA damage in cell killing. The principle of DL qPCR is that DNA damage inhibits PCR. Applications with different lengths can therefore be used to detect different levels of UV-induced DNA damage. The assay was evaluated on three strains of Escherichia coli exposed to varying levels of ultraviolet (UV radiation. We show that DL qPCR sensitivity and reproducibility are within the range of practical application to detect the effect of UV cell killing.

  15. The Smac Mimetic BV6 Improves NK Cell-Mediated Killing of Rhabdomyosarcoma Cells by Simultaneously Targeting Tumor and Effector Cells

    Science.gov (United States)

    Fischer, Kyra; Tognarelli, Sara; Roesler, Stefanie; Boedicker, Cathinka; Schubert, Ralf; Steinle, Alexander; Klingebiel, Thomas; Bader, Peter; Fulda, Simone; Ullrich, Evelyn

    2017-01-01

    Rhabdomyosarcoma (RMS), the most common cancer of connective tissues in pediatrics, is often resistant to conventional therapies. One underlying mechanism of this resistance is the overexpression of Inhibitor of Apoptosis (IAP) proteins, leading to a dysfunctional cell death program within tumor cells. Smac mimetics (SM) are small molecules that can reactivate the cell death program by antagonizing IAP proteins and thereby compensating their overexpression. Here, we report that SM sensitize two RMS cell lines (RD and RH30) toward natural killer (NK) cell-mediated killing on the one hand, and increase the cytotoxic potential of NK cells on the other. The SM-induced sensitization of RH30 cells toward NK cell-mediated killing is significantly reduced through blocking tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on NK cells prior to coculture. In addition, the presence of zVAD.fmk, a pancaspase inhibitor, rescues tumor cells from the increase in killing, indicating an apoptosis-dependent cell death. On the NK cell side, the presence of SM in addition to IL-2 during the ex vivo expansion leads to an increase in their cytotoxic activity against RH30 cells. This effect is mainly TNFα-dependent and partially mediated by NK cell activation, which is associated with transcriptional upregulation of NF-κB target genes such as IκBα and RelB. Taken together, our findings implicate that SM represent a novel double-hit strategy, sensitizing tumor and activating NK cells with one single drug.

  16. Synergistically killing activity of aspirin and histone deacetylase inhibitor valproic acid (VPA) on hepatocellular cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiaofei; Zhu, Yanshuang [Department of Infectious Diseases, Yiwu Central Hospita, 519 Nan men Street, Yiwu, Jinhua, Zhejing 322000 (China); He, Huabin [Department of Orthopedics, Yiwu Central Hospita, 519 Nan men Street, Yiwu, Jinhua, Zhejing 322000 (China); Lou, Lianqing; Ye, Weiwei; Chen, Yongxin [Department of Infectious Diseases, Yiwu Central Hospita, 519 Nan men Street, Yiwu, Jinhua, Zhejing 322000 (China); Wang, Jinghe, E-mail: Xiaofeili2000@163.com [Department of Infectious Diseases, Yiwu Central Hospita, 519 Nan men Street, Yiwu, Jinhua, Zhejing 322000 (China)

    2013-06-28

    Highlights: •Novel combination therapy using aspirin and valproic acid (VPA). •Combination of aspirin and VPA elicits synergistic cytotoxic effects. •Combination of aspirin and VPA significantly reduces the drug dosage required alone. •Combination of aspirin and VPA significantly inhibit tumor growth. •Lower dose of aspirin in combination therapy will minimize side effects of aspirin. -- Abstract: Aspirin and valproic acid (VPA) have been extensively studied for inducing various malignancies growth inhibition respectively, despite their severe side effects. Here, we developed a novel combination by aspirin and VPA on hepatocellular cancer cells (HCCs). The viability of HCC lines were analyzed by MTT assay, apoptotic analysis of HepG2 and SMMC-7721 cell was performed. Real time-PCR and Western blotting were performed to determine the expression of apoptosis related genes and proteins such as Survivin, Bcl-2/Bax, Cyclin D1 and p15. Moreover, orthotopic xenograft tumors were challenged in nude mice to establish murine model, and then therapeutic effect was analyzed after drug combination therapy. The viability of HCC lines’ significantly decreased after drug combination treatment, and cancer cell apoptosis in combination group increasingly induced compared with single drug use. Therapeutic effect was significantly enhanced by combination therapy in tumor volume and tumor weight decrease. From the data shown here, aspirin and VPA combination have a synergistic killing effect on hepatocellular cancers cells proliferation and apoptosis.

  17. LuIII parvovirus selectively and efficiently targets, replicates in, and kills human glioma cells.

    Science.gov (United States)

    Paglino, Justin C; Ozduman, Koray; van den Pol, Anthony N

    2012-07-01

    Because productive infection by parvoviruses requires cell division and is enhanced by oncogenic transformation, some parvoviruses may have potential utility in killing cancer cells. To identify the parvovirus(es) with the optimal oncolytic effect against human glioblastomas, we screened 12 parvoviruses at a high multiplicity of infection (MOI). MVMi, MVMc, MVM-G17, tumor virus X (TVX), canine parvovirus (CPV), porcine parvovirus (PPV), rat parvovirus 1A (RPV1A), and H-3 were relatively ineffective. The four viruses with the greatest oncolytic activity, LuIII, H-1, MVMp, and MVM-G52, were tested for the ability, at a low MOI, to progressively infect the culture over time, causing cell death at a rate higher than that of cell proliferation. LuIII alone was effective in all five human glioblastomas tested. H-1 progressively infected only two of five; MVMp and MVM-G52 were ineffective in all five. To investigate the underlying mechanism of LuIII's phenotype, we used recombinant parvoviruses with the LuIII capsid replacing the MVMp capsid or with molecular alteration of the P4 promoter. The LuIII capsid enhanced efficient replication and oncolysis in MO59J gliomas cells; other gliomas tested required the entire LuIII genome to exhibit enhanced infection. LuIII selectively infected glioma cells over normal glial cells in vitro. In mouse models, human glioblastoma xenografts were selectively infected by LuIII when administered intratumorally; LuIII reduced tumor growth by 75%. LuIII also had the capacity to selectively infect subcutaneous or intracranial gliomas after intravenous inoculation. Intravenous or intracranial LuIII caused no adverse effects. Intracranial LuIII caused no infection of mature mouse neurons or glia in vivo but showed a modest infection of developing neurons.

  18. Irradiation Can Selectively Kill Tumor Cells while Preserving Erythrocyte Viability in a Co-Culture System.

    Directory of Open Access Journals (Sweden)

    Ming Gong

    Full Text Available An understanding of how to safely apply intraoperative blood salvage (IBS in cancer surgery has not yet been obtained. Here, we investigated the optimal dose of 137Cs gamma-ray irradiation for killing human hepatocarcinoma (HepG2, gastrocarcinoma (SGC7901, and colonic carcinoma (SW620 tumor cells while preserving co-cultured erythrocytes obtained from 14 healthy adult volunteers. HepG2, SGC7901, or SW620 cells were mixed into the aliquots of erythrocytes. After the mixed cells were treated with 137Cs gamma-ray irradiation (30, 50, and 100 Gy, tumor cells and erythrocytes were separated by density gradient centrifugation in Percoll with a density of 1.063 g/ml. The viability, clonogenicity, DNA synthesis, tumorigenicity, and apoptosis of the tumor cells were determined by MTT assay, plate colony formation, 5-ethynyl-2'-deoxyuridine (EdU incorporation, subcutaneous xenograft implantation into immunocompromised mice, and annexin V/7-AAD staining, respectively. The ATP concentration, 2,3-DPG level, free Hb concentration, osmotic fragility, membrane phosphatidylserine externalization, blood gas variables, reactive oxygen species levels, and superoxide dismutase levels in erythrocytes were analyzed. We found that 137Cs gamma-ray irradiation at 50 Gy effectively inhibited the viability, proliferation, and tumorigenicity of HepG2, SGC7901, and SW620 cells without markedly damaging the oxygen-carrying ability or membrane integrity or increasing the oxidative stress of erythrocytes in vitro. These results demonstrated that 50 Gy irradiation in a standard 137Cs blood irradiator might be a safe and effective method of inactivating HepG2, SGC7901, and SW620 cells mixed with erythrocytes, which might help to safely allow IBS in cancer surgery.

  19. Differential timing of antibody-mediated phagocytosis and cell-free killing of invasive African Salmonella allows immune evasion.

    Science.gov (United States)

    Siggins, Matthew K; O'Shaughnessy, Colette M; Pravin, John; Cunningham, Adam F; Henderson, Ian R; Drayson, Mark T; MacLennan, Calman A

    2014-04-01

    Nontyphoidal Salmonellae commonly cause fatal bacteraemia in African children lacking anti-Salmonella antibodies. These are facultative intracellular bacteria capable of cell-free and intracellular survival within macrophages. To better understand the relationship between extracellular and intracellular infection in blood and general mechanisms of Ab-related protection against Salmonella, we used human blood and sera to measure kinetics of Ab and complement deposition, serum-mediated bactericidal killing and phagocytosis of invasive African Salmonella enterica serovar Typhimurium D23580. Binding of antibodies peaked by 30 s, but C3 deposition lagged behind, peaking after 2-4 min. C5b-9 deposition was undetectable until between 2 and 6 min and peaked after 10 min, after which time an increase in serum-mediated killing occurred. In contrast, intracellular, opsonized Salmonellae were readily detectable within 5 min. By 10 min, around half of monocytes and most neutrophils contained bacteria. The same kinetics of serum-mediated killing and phagocytosis were observed with S. enterica Typhimurium laboratory strain SL1344, and the S. enterica Enteritidis African invasive isolate D24954 and laboratory strain PT4. The differential kinetics between cell-free killing and phagocytosis of invasive nontyphoidal Salmonella allows these bacteria to escape the blood and establish intracellular infection before they are killed by the membrane attack complex.

  20. Antimicrobial design of titanium surface that kill sessile bacteria but support stem cells adhesion

    Science.gov (United States)

    Zhu, Chen; Bao, Ni-Rong; Chen, Shuo; Zhao, Jian-Ning

    2016-12-01

    Implant-related bacterial infection is one of the most severe postoperative complications in orthopedic or dental surgery. In this context, from the perspective of surface modification, increasing efforts have been made to enhance the antibacterial capability of titanium surface. In this work, a hierarchical hybrid surface architecture was firstly constructed on titanium surface by two-step strategy of acid etching and H2O2 aging. Then silver nanoparticles were firmly immobilized on the hierarchical surface by ion implantation, showing no detectable release of silver ions from surface. The designed titanium surface showed good bioactivity. More importantly, this elaborately designed titanium surface can effectively inactivate the adherent S. aureus on surface by virtue of a contact-killing mode. Meanwhile, the designed titanium surface can significantly facilitate the initial adhesion and spreading behaviors of bone marrow mesenchymal stem cells (MSCs) on titanium. The results suggested that, the elaborately designed titanium surface might own a cell-favoring ability that can help mammalian cells win the initial adhesion race against bacteria. We hope the present study can provide a new insight for the better understanding and designing of antimicrobial titanium surface, and pave the way to satisfying clinical requirements.

  1. Surface expression of HSP72 by LPS-stimulated neutrophils facilitates gammadeltaT cell-mediated killing.

    Science.gov (United States)

    Hirsh, Mark I; Hashiguchi, Naoyuki; Chen, Yu; Yip, Linda; Junger, Wolfgang G

    2006-03-01

    During inflammation and sepsis, accumulation of activated neutrophils causes lung tissue damage and organ failure. Effective clearance of neutrophils reduces the risk of organ failure; however, its mechanisms are poorly understood. Because lungs are rich in gammadeltaT cells, we investigated the physiological role of these cells in the protection of lung tissue from infiltrating neutrophils. In a mouse model of sepsis, we found that the lungs of survivors contained significantly higher numbers of gammadeltaT cells than those of mice that died from sepsis. The number of gammadeltaT cells correlated inversely with the number of neutrophils in the lungs and with the degree of lung tissue damage. LPS rapidly elicited the expression of heat shock protein (HSP) 72 on the surface of human neutrophils. Inhibitors of transcription, protein synthesis, and intracellular protein transport blocked HSP72 expression, indicating that de novo synthesis is required. gammadeltaT cells targeted and rapidly killed LPS-treated neutrophils through direct cell-to-cell contact. Pre-treatment with neutralizing antibodies to HSP72 diminished neutrophil killing. Our data indicate that HSP72 expression on the cell surface predisposes inflamed neutrophils to killing by gammadeltaT cells. This intercellular exchange may allow gammadeltaT cells to resolve inflammation and limit host tissue damage during sepsis.

  2. PD-L1 Expression on Retrovirus-Infected Cells Mediates Immune Escape from CD8+ T Cell Killing.

    Directory of Open Access Journals (Sweden)

    Ilseyar Akhmetzyanova

    2015-10-01

    Full Text Available Cytotoxic CD8+ T Lymphocytes (CTL efficiently control acute virus infections but can become exhausted when a chronic infection develops. Signaling of the inhibitory receptor PD-1 is an important mechanism for the development of virus-specific CD8+ T cell dysfunction. However, it has recently been shown that during the initial phase of infection virus-specific CD8+ T cells express high levels of PD-1, but are fully competent in producing cytokines and killing virus-infected target cells. To better understand the role of the PD-1 signaling pathway in CD8+ T cell cytotoxicity during acute viral infections we analyzed the expression of the ligand on retrovirus-infected cells targeted by CTLs. We observed increased levels of PD-L1 expression after infection of cells with the murine Friend retrovirus (FV or with HIV. In FV infected mice, virus-specific CTLs efficiently eliminated infected target cells that expressed low levels of PD-L1 or that were deficient for PD-L1 but the population of PD-L1high cells escaped elimination and formed a reservoir for chronic FV replication. Infected cells with high PD-L1 expression mediated a negative feedback on CD8+ T cells and inhibited their expansion and cytotoxic functions. These findings provide evidence for a novel immune escape mechanism during acute retroviral infection based on PD-L1 expression levels on virus infected target cells.

  3. HER2-Specific T Lymphocytes Kill both Trastuzumab-Resistant and Trastuzumab-Sensitive Breast Cell Lines In Vitro

    Institute of Scientific and Technical Information of China (English)

    Xiao-lin Lin; Xu Liang; Tao Shen; Jun Ren; Xiao-li Wang; Bo Ma; Jun Jia; Ying Yan; Li-jun Di; Yan-hua Yuan; Feng-ling Wan; Yuan-li Lu

    2012-01-01

    Objective:Although the development of trastuzumab has improved the outlook for women with human epidermal growth factor receptor 2 (HER2)-positive breast cancer,the resistance to anti-HER2 therapy is a growing clinical dilemma.We aim to determine whether HER2-specific T cells generated from dendritic cells (DCs) modified with HER2 gene could effectively kill the HER2-positive breast cancer cells,especially the trastuzumab-resistant cells.Methods:The peripheral blood mononuclear cells (PBMCs) from healthy donors,whose HLA haplotypes were compatible with the tumor cell lines,were transfected with reconstructive human adeno-association virus (rhAAV/HER2) to obtain the specific killing activities of T cells,and were evaluated by lactate dehydrogenase (LDH)releasing assay.Results:Trastuzumab produced a significant inhibiting effect on SK-BR-3,the IC50 was 100ng/ml.MDA-MB-453 was resistant to trastuzumab even at a concentration of 10,000 ng/ml in vitro.HER2-specific T lymphocytes killed effectively SK-BR-3 [(69.86±13.41)%] and MDA-MB-453 [(78.36±10.68)%] at 40:1 (effector:target ratio,E:T),but had no significant cytotoxicity against HER2-negative breast cancer cell lines MDA-MB-231 or MCF-7 (less than 10%).Conclusion:The study showed that HER2-specific T lymphocytes generated from DCs modified by rhAAV/HER2 could kill HER2-positive breast cancer cell lines in a HER2-dependent manner,and result in significantly high inhibition rates on the intrinsic trastuzumab-resistant cell line MDA-MB-453 and the tastuzumab-sensitive cell line SK-BR-3.These results imply that this immunotherapy might be a potential treatment to HER2-positive breast cancer.

  4. Relative efficiencies of three ultraviolet radiation wavelengths for cell killing and transformation in mouse cells in vitro.

    Science.gov (United States)

    Papadopoulo, D; Muel, B; Latarjet, R

    1983-09-01

    C3H 10 T 1/2 clone 8 mouse cells were irradiated in vitro with three U.V. wavelengths 280, 254, and 230 nm. Two effects were investigated, survival and malignant transformation, and the relative efficiencies were determined for the three radiations. For transformation, these efficiencies were: 280nm:3.9; 254nm:5.1; 230nm:2.3 (transformations produced by 5 Jm-2 of U.V. for 1000 surviving cells). For cell killing the efficiencies were, in relative units, 34, 100, and 50 respectively. These efficiencies are in agreement with the hypothesis that the main chromophore for both effects is the nucleic acid, and not the protein moiety of the genome. This conclusion agrees with that previously reached by other investigators, but our present results obtained with the short wave-length 230 nm provide an especially strong new argument.

  5. Relative efficiencies of three ultraviolet radiation wavelengths for cell killing and transformation in mouse cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Papadopoulo, D.; Muel, B.; Latarjet, R. (Institut du Radium, 75 - Paris (France). Lab. Curie)

    1983-09-01

    C3H 10 T 1/2 clone 8 mouse cells were irradiated in vitro with three U.V. wavelengths 280, 254, and 230 nm. Two effects were investigated, survival and malignant transformation, and the relative efficiences were determined for the three radiation. For transformation, these efficiences were: 280 nm: 3.9; 254 nm: 5.1; 230 nm: 2.3 (transformations produced by 5 J m/sup -2/ of U.V. for 1000 surviving cells). For cell killing the efficiencies were, in relative units, 34, 100, and 50 respectively. These efficiencies are in agreement with the hypothesis that the main chromophore for both effects is the nucleic acid, and not the protein moiety of the genome. This conclusion agrees with that previously reached by other investigators, but our present results obtained with the short wave-length 230 nm provide an especially strong new argument.

  6. T Cell Receptor Vβ Staining Identifies the Malignant Clone in Adult T cell Leukemia and Reveals Killing of Leukemia Cells by Autologous CD8+ T cells.

    Science.gov (United States)

    Rowan, Aileen G; Witkover, Aviva; Melamed, Anat; Tanaka, Yuetsu; Cook, Lucy B M; Fields, Paul; Taylor, Graham P; Bangham, Charles R M

    2016-11-01

    There is growing evidence that CD8+ cytotoxic T lymphocyte (CTL) responses can contribute to long-term remission of many malignancies. The etiological agent of adult T-cell leukemia/lymphoma (ATL), human T lymphotropic virus type-1 (HTLV-1), contains highly immunogenic CTL epitopes, but ATL patients typically have low frequencies of cytokine-producing HTLV-1-specific CD8+ cells in the circulation. It remains unclear whether patients with ATL possess CTLs that can kill the malignant HTLV-1 infected clone. Here we used flow cytometric staining of TCRVβ and cell adhesion molecule-1 (CADM1) to identify monoclonal populations of HTLV-1-infected T cells in the peripheral blood of patients with ATL. Thus, we quantified the rate of CD8+-mediated killing of the putative malignant clone in ex vivo blood samples. We observed that CD8+ cells from ATL patients were unable to lyse autologous ATL clones when tested directly ex vivo. However, short in vitro culture restored the ability of CD8+ cells to kill ex vivo ATL clones in some donors. The capacity of CD8+ cells to lyse HTLV-1 infected cells which expressed the viral sense strand gene products was significantly enhanced after in vitro culture, and donors with an ATL clone that expressed the HTLV-1 Tax gene were most likely to make a detectable lytic CD8+ response to the ATL cells. We conclude that some patients with ATL possess functional tumour-specific CTLs which could be exploited to contribute to control of the disease.

  7. Impact of prolonged fraction dose-delivery time modeling intensity-modulated radiation therapy on hepatocellular carcinoma cell killing

    Institute of Scientific and Technical Information of China (English)

    Xiao-Kang Zheng; Long-Hua Chen; Xiao Yan; Hong-Mei Wang

    2005-01-01

    AIM: To explore the impact of prolonged fraction dosedelivery time modeling intensity-modulated radiation therapy (IMRT) on cell killing of human hepatocellular carcinoma (HCC) HepG2 and Hep3B cell lines.METHODS: The radiobiological characteristics of human HCC HepG2 and Hep3b cell lines were studied with standard clonogenic assays, using standard linear-quadratic model and incomplete repair model to fit the dose-survival curves. The identical methods were also employed to investigate the biological effectiveness of irradiation protocols modeling clinical conventional fractionated external beam radiotherapy (EBRT, fraction delivery time 3 min) and IMRT with different prolonged fraction delivery time (15, 30, and 45 min). The differences of cell surviving fraction irradiated with different fraction delivery time were tested with paired t-test. Factors determining the impact of prolonged fraction delivery time on cell killing were analyzed.RESULTS: The α/β and repair half-time (T1/2) of HepG2and Hep3b were 3.1 and 7.4 Gy, and 22 and 19 min respectively. The surviving fraction of HepG2 irradiated modeling IMRT with different fraction delivery time was significantly higher than irradiated modeling EBRT and the cell survival increased more pronouncedly with the fraction delivery time prolonged from 15 to 45 min,while no significant differences of cell survival in Hep3b were found between different fraction delivery time protocols.CONCLUSION: The prolonged fraction delivery time modeling IMRT significantly decreased the cell killing in HepG2 but not in Hep3b. The capability of sub-lethal damage repair was the predominant factor determining the cell killing decrease. These effects, if confirmed by clinical studies, should be considered in designing IMRT treatments for HCC.

  8. Ascorbic acid kills Epstein-Barr virus positive Burkitt lymphoma cells and Epstein-Barr virus transformed B-cells in vitro, but not in vivo.

    Science.gov (United States)

    Shatzer, Amber N; Espey, Michael Graham; Chavez, Mayra; Tu, Hongbin; Levine, Mark; Cohen, Jeffrey I

    2013-05-01

    Ascorbic acid has been shown to kill various cancer cell lines at pharmacologic concentrations. We found that Epstein-Barr virus (EBV)-positive Burkitt lymphoma (BL) cells were more susceptible to ascorbic acid-induced cell killing than EBV-negative BL cells or EBV-transformed lymphoblastoid cells (LCLs). Ascorbic acid did not induce apoptosis in any of the tested cells but did induce the production of reactive oxygen species and cell death. Previously, we showed that bortezomib, a proteasome inhibitor, induces cell death in LCLs and EBV-positive BL cells. We found that ascorbic acid is strongly antagonistic for bortezomib-induced cell death in LCLs and EBV-positive BL cells. Finally, ascorbic acid did not prolong survival of severe combined immunodefiency mice inoculated with LCLs either intraperitoneally or subcutaneously. Thus, while ascorbic acid was highly effective at killing EBV-positive BL cells and LCLs in vitro, it antagonized cell killing by bortezomib and was ineffective in an animal model.

  9. Killed but metabolically active Leishmania infantum as a novel whole-cell vaccine for visceral leishmaniasis.

    Science.gov (United States)

    Bruhn, Kevin W; Birnbaum, Ron; Haskell, Jacquelyn; Vanchinathan, Veena; Greger, Stephanie; Narayan, Rupa; Chang, Pei-Lin; Tran, Thu Anh; Hickerson, Suzanne M; Beverley, Stephen M; Wilson, Mary E; Craft, Noah

    2012-04-01

    There are currently no effective vaccines for visceral leishmaniasis, the second most deadly parasitic infection in the world. Here, we describe a novel whole-cell vaccine approach using Leishmania infantum chagasi promastigotes treated with the psoralen compound amotosalen (S-59) and low doses of UV A radiation. This treatment generates permanent, covalent DNA cross-links within parasites and results in Leishmania organisms termed killed but metabolically active (KBMA). In this report, we characterize the in vitro growth characteristics of both KBMA L. major and KBMA L. infantum chagasi. Concentrations of S-59 that generate optimally attenuated parasites were identified. Like live L. infantum chagasi, KBMA L. infantum chagasi parasites were able to initially enter liver cells in vivo after intravenous infection. However, whereas live L. infantum chagasi infection leads to hepatosplenomegaly in mice after 6 months, KBMA L. infantum chagasi parasites were undetectable in the organs of mice at this time point. In vitro, KBMA L. infantum chagasi retained the ability to enter macrophages and induce nitric oxide production. These characteristics of KBMA L. infantum chagasi correlated with the ability to prophylactically protect mice via subcutaneous vaccination at levels similar to vaccination with live, virulent organisms. Splenocytes from mice vaccinated with either live L. infantum chagasi or KBMA L. infantum chagasi displayed similar cytokine patterns in vitro. These results suggest that KBMA technology is a potentially safe and effective novel vaccine strategy against the intracellular protozoan L. infantum chagasi. This approach may represent a new method for whole-cell vaccination against other complex intracellular pathogens.

  10. Integrating Enzymatic Self-Assembly and Mitochondria Targeting for Selectively Killing Cancer Cells without Acquired Drug Resistance.

    Science.gov (United States)

    Wang, Huaimin; Feng, Zhaoqianqi; Wang, Youzhi; Zhou, Rong; Yang, Zhimou; Xu, Bing

    2016-12-14

    Targeting organelles by modulating the redox potential of mitochondria is a promising approach to kill cancer cells that minimizes acquired drug resistance. However, it lacks selectivity because mitochondria perform essential functions for (almost) all cells. We show that enzyme-instructed self-assembly (EISA), a bioinspired molecular process, selectively generates the assemblies of redox modulators (e.g., triphenyl phosphinium (TPP)) in the pericellular space of cancer cells for uptake, which allows selectively targeting the mitochondria of cancer cells. The attachment of TPP to a pair of enantiomeric, phosphorylated tetrapeptides produces the precursors (L-1P or D-1P) that form oligomers. Upon dephosphorylation catalyzed by ectophosphatases (e.g., alkaline phosphatase (ALP)) overexpressed on cancer cells (e.g., Saos2), the oligomers self-assemble to form nanoscale assemblies only on the surface of the cancer cells. The cancer cells thus uptake these assemblies of TPP via endocytosis, mainly via a caveolae/raft-dependent pathway. Inside the cells, the assemblies of TPP-peptide conjugates escape from the lysosome, induce dysfunction of mitochondria to release cytochrome c, and result in cell death, while the controls (i.e., omitting TPP motif, inhibiting ALP, or removing phosphate trigger) hardly kill the Saos2 cells. Most importantly, the repeated stimulation of the cancers by the precursors, unexpectedly, sensitizes the cancer cells to the precursors. As the first example of the integration of subcellular targeting with cell targeting, this study validates the spatial control of the assemblies of nonspecific cytotoxic agents by EISA as a promising molecular process for selectively killing cancer cells without inducing acquired drug resistance.

  11. Structural factors and mechanisms underlying the improved photodynamic cell killing with silicon phthalocyanine photosensitizers directed to lysosomes versus mitochondria.

    Science.gov (United States)

    Rodriguez, Myriam E; Zhang, Ping; Azizuddin, Kashif; Delos Santos, Grace B; Chiu, Song-mao; Xue, Liang-yan; Berlin, Jeffery C; Peng, Xinzhan; Wu, Hongqiao; Lam, Minh; Nieminen, Anna-Liisa; Kenney, Malcolm E; Oleinick, Nancy L

    2009-01-01

    The phthalocyanine photosensitizer Pc 4 has been shown to bind preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, especially Bcl-2, are photodamaged and apoptosis, as indicated by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, is triggered. A series of analogs of Pc 4 were synthesized, and the results demonstrate that Pcs with the aminopropylsiloxy ligand of Pc 4 or a similar one on one side of the Pc ring and a second large axial ligand on the other side of the ring have unexpected properties, including enhanced cell uptake, greater monomerization resulting in greater intracellular fluorescence and three-fold higher affinity constants for liposomes. The hydroxyl-bearing axial ligands tend to reduce aggregation of the Pc and direct it to lysosomes, resulting in four to six times more killing of cells, as defined by loss of clonogenicity, than with Pc 4. Whereas Pc 4-PDT photodamages Bcl-2 and Bcl-xL, Pc 181-PDT causes much less photodamage to Bcl-2 over the same dose-response range relative to cell killing, with earlier cleavage of Bid and slower caspase-3-dependent apoptosis. Therefore, within this series of photosensitizers, these hydroxyl-bearing axial ligands are less aggregated than is Pc 4, tend to localize to lysosomes and are more effective in overall cell killing than is Pc 4, but induce apoptosis more slowly and by a modified pathway.

  12. EGL-1 BH3 mutants reveal the importance of protein levels and target affinity for cell-killing potency.

    Science.gov (United States)

    Lee, E F; Chen, L; Yang, H; Colman, P M; Huang, D C S; Fairlie, W D

    2008-10-01

    Studies of the cell death pathway in the nematode Caenorhabditis elegans provided the first evidence of the evolutionary conservation of apoptosis signalling. Here we show that the worm Bcl-2 homology domain-3 (BH3)-only protein EGL-1 binds mammalian pro-survival proteins very poorly, but can be converted into a high-affinity ligand for Bcl-2 and Bcl-x(L) by subtle mutation of the cysteine residue at position 62 within the BH3 domain. A 100-fold increase in affinity was observed following a single atom change (cysteine to serine substitution), and a further 10-fold increase by replacement with glycine. The low affinity of wild-type EGL-1 for mammalian pro-survival proteins and its poor expression correlates with its weak killing activity in mammalian cells whereas the high-affinity C62G mutant is a very potent killer of cells lacking Mcl-1. Cell killing by the C62S mutant with intermediate affinity only occurs when this EGL-1 BH3 domain is placed in a more stable context, namely that of Bim(S), which allows higher expression, though the kinetics of cell death now vary depending on whether Mcl-1 is neutralized by Noxa or genetically deleted. These results demonstrate how levels of BH3-only proteins, target affinity and the spectrum of neutralization of pro-survival proteins all contribute to killing activity.

  13. The Killing

    DEFF Research Database (Denmark)

    Agger, Gunhild

    2013-01-01

    This article tracks the uncanny locations of The Killing (2007–2012), relating them to place, space and atmosphere, putting bits and pieces from the topographic puzzle together with cues from the symbolic space in order to see how they fit into the overall pattern of Nordic Noir. In The Killing......, the abstract level of space and atmosphere meets the concrete level of place, both influencing the notion of location. This meeting, I suggest, has contributed towards the simultaneous domestic and international appeal of The Killing....

  14. UV-inactivated HSV-1 potently activates NK cell killing of leukemic cells.

    Science.gov (United States)

    Samudio, Ismael; Rezvani, Katayoun; Shaim, Hila; Hofs, Elyse; Ngom, Mor; Bu, Luke; Liu, Guoyu; Lee, Jason T C; Imren, Suzan; Lam, Vivian; Poon, Grace F T; Ghaedi, Maryam; Takei, Fumio; Humphries, Keith; Jia, William; Krystal, Gerald

    2016-05-26

    Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light-inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation-dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell-depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML.

  15. High vancomycin MICs within the susceptible range in Staphylococcus aureus bacteraemia isolates are associated with increased cell wall thickness and reduced intracellular killing by human phagocytes.

    Science.gov (United States)

    Falcón, Rocío; Martínez, Alba; Albert, Eliseo; Madrid, Silvia; Oltra, Rosa; Giménez, Estela; Soriano, Mario; Vinuesa, Víctor; Gozalbo, Daniel; Gil, María Luisa; Navarro, David

    2016-05-01

    Vancomycin minimum inhibitory concentrations (MICs) at the upper end of the susceptible range for Staphylococcus aureus have been associated with poor clinical outcomes of bloodstream infections. We tested the hypothesis that high vancomycin MICs in S. aureus bacteraemia isolates are associated with increased cell wall thickness and suboptimal bacterial internalisation or lysis by human phagocytes. In total, 95 isolates were evaluated. Original vancomycin MICs were determined by Etest. The susceptibility of S. aureus isolates to killing by phagocytes was assessed in a human whole blood assay. Internalisation of bacterial cells by phagocytes was investigated by flow cytometry. Cell wall thickness was evaluated by transmission electron microscopy. Genotypic analysis of S. aureus isolates was performed using a DNA microarray system. Vancomycin MICs were significantly higher (P=0.006) in isolates that were killed suboptimally (killing index 70%) and tended to correlate inversely (P=0.08) with the killing indices. Isolates in both killing groups were internalised by human neutrophils and monocytes with comparable efficiency. The cell wall was significantly thicker (P=0.03) in isolates in the low killing group. No genotypic differences were found between the isolates in both killing groups. In summary, high vancomycin MICs in S. aureus bacteraemia isolates were associated with increased cell wall thickness and reduced intracellular killing by phagocytes.

  16. Mathematical analysis and simulations involving chemotherapy and surgery on large human tumours under a suitable cell-kill functional response.

    Science.gov (United States)

    Rodrigues, Diego Samuel; de Arruda Mancera, Paulo Fernando

    2013-02-01

    Dosage and frequency of treatment schedules are important for successful chemotherapy. However, in this work we argue that cell-kill response and tumoral growth should not be seen as separate and therefore are essential in a mathematical cancer model. This paper presents a mathematical model for sequencing of cancer chemotherapy and surgery. Our purpose is to investigate treatments for large human tumours considering a suitable cell-kill dynamics. We use some biological and pharmacological data in a numerical approach, where drug administration occurs in cycles (periodic infusion) and surgery is performed instantaneously. Moreover, we also present an analysis of stability for a chemotherapeutic model with continuous drug administration. According to Norton and Simon [22], our results indicate that chemotherapy is less efficient in treating tumours that have reached a plateau level of growing and that a combination with surgical treatment can provide better outcomes.

  17. Intracellular growth of Mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells

    Science.gov (United States)

    Mahamed, Deeqa; Boulle, Mikael; Ganga, Yashica; Mc Arthur, Chanelle; Skroch, Steven; Oom, Lance; Catinas, Oana; Pillay, Kelly; Naicker, Myshnee; Rampersad, Sanisha; Mathonsi, Colisile; Hunter, Jessica; Sreejit, Gopalkrishna; Pym, Alexander S; Lustig, Gila; Sigal, Alex

    2017-01-01

    A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states. DOI: http://dx.doi.org/10.7554/eLife.22028.001 PMID:28130921

  18. Kinase domain insert containing receptor promoter controlled suicide gene system selectively kills human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Wen-Yu Yang; Zong-Hai Huang; Li-Jun Lin; Zhou Li; Jing-Long Yu; Hui-Juan Song; Yong Qian; Xiao-Yan Che

    2006-01-01

    AIM: To study the selective killing of human umbilical vein endothelial cells (HUVECs) by a double suicide gene under the regulation of a kinase domain insert containing receptor (KDR) promoter and mediated by an adenoviral gene vector.METHODS: Human KDR promoter was cloned by polymerase chain reaction (PCR), and two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were constructed according to a two-step transformation protocol. These two newly constructed plasmids were then transfected into 293 packaging cells to grow adenovirus, which were further multiplied and purified.HUVECs and LoVo cells were infected with either of the two resultant recombinant adenoviruses (AdKDR-CDglyTK and AdCMV-CDglyTK) respectively, and the infection rates were estimated by detection of green fluorescent protein (GFP) expression. Infected cells were cultured in culture media containing different concentrations of 5-fluorocytosine (5-FC) and ganciclovir (GCV), and the killing effects were measured.RESULTS: The two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were successfully constructed and transfected into 293 cells. The resultant recombinant adenoviruses infected cells caused similar infection rates; and the infected cells exhibited different sensitivity to the prodrugs: HUVECs infected with AdCMV-CDglyTK and LoVo cells infected with AdCMVCDglyTK were highly sensitive to the prodrugs, and HUVECs infected with AdKDR-CDglyTK were similarly sensitive but significantly more sensitive than the LoVo cells infected with AdKDR-CdglyTK (P<0.001).CONCLJSION: Selective killing of HUVECs may be achieved by gene transfer of double suicide gene under the regulation of the KDR promoter. This finding may provide an optional way to target gene therapy of malignant tumors by abrogation of tumor blood vessels.

  19. Assessment of attachment, ingestion, and killing of Escherichia coli by bovine polymorphonuclear cells with combined micromethods.

    Science.gov (United States)

    Rainard, P

    1985-11-01

    A set of microassays separately measuring attachment, ingestion, and overall killing of Escherichia coli by bovine granulocytes was devised and its analytical potential used to test the effect of drugs which block intracellular killing: sodium azide, phenylbutazone, chloroquine phosphate were all inactive, suggesting that O2-dependent systems were not the sole pathway involved in the killing of E.coli by granulocytes. The microtechniques were also used to investigate the opsonic requirements for phagocytosis of two E.coli strains. Absorption of normal bovine serum with the homologous and the heterologous strains showed that specific antibodies were necessary to induce attachment of bacteria to phagocytes. Once bound to granulocytes, the unencapsulated strain P4 was engulfed, whereas for the encapsulated strain B117, complement was required for the internalization step of phagocytosis. With immune serum the need for complement was not absolute.

  20. Combinatorial BTK and MALT1 inhibition augments killing of CD79 mutant diffuse large B cell lymphoma.

    Science.gov (United States)

    Nagel, Daniel; Bognar, Miriam; Eitelhuber, Andrea C; Kutzner, Kerstin; Vincendeau, Michelle; Krappmann, Daniel

    2015-12-08

    Survival of activated B cell-subtype (ABC) of diffuse large B cell lymphoma (DLBCL) is driven by chronic B cell receptor (BCR) signaling that activates the canonical NF-κB pathway. Inhibition of BTK by Ibrutinib has been shown to kill ABC DLBCL cells that carry activating mutations in the BCR adaptor CD79. However, mutations in BTK or in downstream components such as CARMA1/CARD11 can render lymphomas Ibrutinib resistant. Therefore, we assessed here the simultaneous inhibition of BTK and the protease MALT1 that acts downstream of CARMA1 and is essential for ABC DLBCL tumor growth. We show that in CD79 mutant cells BTK is a crucial upstream regulator of MALT1, but dispensable in CARMA1 mutant ABC DLBCL. Combined inhibition of BTK by Ibrutinib and MALT1 by S-Mepazine additively impaired MALT1 cleavage activity and expression of NF-κB pro-survival factors. Thereby, combinatorial Ibrutinib and S-Mepazine treatment enhanced killing of CD79 mutant ABC DLBCL cells. Moreover, while expression of oncogenic CARMA1 in CD79 mutant cells conferred Ibrutinib resistance, double mutant cells were still sensitive to MALT1 inhibition by S-Mepazine. Thus, based on the genetic background combinatorial BTK and MALT1 inhibition may improve effectiveness of therapeutic treatment and reduce the chances for the development of drug resistances.

  1. Cell killing and chromatid damage in primary human bronchial epithelial cells irradiated with accelerated 56Fe ions

    Science.gov (United States)

    Suzuki, M.; Piao, C.; Hall, E. J.; Hei, T. K.

    2001-01-01

    We examined cell killing and chromatid damage in primary human bronchial epithelial cells irradiated with high-energy 56Fe ions. Cells were irradiated with graded doses of 56Fe ions (1 GeV/nucleon) accelerated with the Alternating Gradient Synchrotron at Brookhaven National Laboratory. The survival curves for cells plated 1 h after irradiation (immediate plating) showed little or no shoulder. However, the survival curves for cells plated 24 h after irradiation (delayed plating) had a small initial shoulder. The RBE for 56Fe ions compared to 137Cs gamma rays was 1.99 for immediate plating and 2.73 for delayed plating at the D10. The repair ratio (delayed plating/immediate plating) was 1.67 for 137Cs gamma rays and 1.22 for 56Fe ions. The dose-response curves for initially measured and residual chromatid fragments detected by the Calyculin A-mediated premature chromosome condensation technique showed a linear response. The results indicated that the induction frequency for initially measured fragments was the same for 137Cs gamma rays and 56Fe ions. On the other hand, approximately 85% of the fragments induced by 137Cs gamma rays had rejoined after 24 h of postirradiation incubation; the corresponding amount for 56Fe ions was 37%. Furthermore, the frequency of chromatid exchanges induced by gamma rays measured 24 h after irradiation was higher than that induced by 56Fe ions. No difference in the amount of chromatid damage induced by the two types of radiations was detected when assayed 1 h after irradiation. The results suggest that high-energy 56Fe ions induce a higher frequency of complex, unrepairable damage at both the cellular and chromosomal levels than 137Cs gamma rays in the target cells for radiation-induced lung cancers.

  2. Vitamin D and the human antimicrobial peptide LL-37 enhance group a streptococcus resistance to killing by human cells.

    Science.gov (United States)

    Love, John F; Tran-Winkler, Hien J; Wessels, Michael R

    2012-10-23

    The CsrRS two-component regulatory system of group A Streptococcus (GAS; Streptococcus pyogenes) responds to subinhibitory concentrations of the human antimicrobial peptide LL-37. LL-37 signaling through CsrRS results in upregulation of genes that direct synthesis of virulence factors, including the hyaluronic acid capsule and streptolysin O (SLO). Here, we demonstrate that a consequence of this response is augmented GAS resistance to killing by human oropharyngeal keratinocytes, neutrophils, and macrophages. LL-37-induced upregulation of SLO and hyaluronic acid capsule significantly reduced internalization of GAS by keratinocytes and phagocytic killing by neutrophils and macrophages. Because vitamin D induces LL-37 production by macrophages, we tested its effect on macrophage killing of GAS. In contrast to the reported enhancement of macrophage function in relation to other pathogens, treatment of macrophages with 1α,25-dihydroxy-vitamin D3 paradoxically reduced the ability of macrophages to control GAS infection. These observations demonstrate that LL-37 signals through CsrRS to induce a virulence phenotype in GAS characterized by heightened resistance to ingestion and killing by both epithelial cells and phagocytes. By inducing LL-37 production in macrophages, vitamin D may contribute to this paradoxical exacerbation of GAS infection. IMPORTANCE It remains poorly understood why group A Streptococcus (GAS) causes asymptomatic colonization or localized throat inflammation in most individuals but rarely progresses to invasive infection. The human antimicrobial peptide LL-37, which is produced as part of the innate immune response to GAS infection, signals through the GAS CsrRS two-component regulatory system to upregulate expression of multiple virulence factors. This study reports that two CsrRS-regulated GAS virulence factors-streptolysin O and the hyaluronic acid capsule-are critical in LL-37-induced resistance of GAS to killing by human throat epithelial cells

  3. EFFECTS OF CARBOXYMETHLY DEXTRAN MAGNETIC NANOPARTICLES CARRIER SYSTEM ASSOCIATED WITH EXTERNAL MAGNETIC FIELDS ON KILLING TUMOR CELLS AND GENE TRANSFECTION

    Institute of Scientific and Technical Information of China (English)

    CAO Zheng-guo; ZHOU Si-wei; LIU Ji-hong

    2005-01-01

    Objective: To investigate the preparation of the carboxymethly dextran iron oxide magnetic nanoparticles (CDMN) and the effects of CDMN carrier system associated with external magnetic fields on killing tumor cells and gene transfection in vitro. Methods: Epirubicin-CDMN (EPI-CDMN) and green fluorescent protein (GFP) plasmid-CDMN (GFP-CDMN) were prepared by the oxidation-reduction procedure and their characters were detected, respectively. The effects of EPI-CDMN associated with external pulsed electromagnetic fields (PEMFs) (10 mT) on killing human bladder cancer BIU-87 cells were studied by MTT assay and Annexin-V/PI double-labeled flow cytometry technique, respectively. And the transfection efficiency of GFP when CDMN were used as gene carrier associated with the external magnetic fields was evaluated under fluorescence microscope in vitro. Results: The diameter of EPI-CDMN and GFP-CDMN were about 8~10 nm and 5~9 nm, respectively, and saturation magnetization were 0.22 emu/g and 0.26 emu/g, respectively. EPI-CDMN associated with PEMFs could significantly inhibit the proliferation of BIU-87 cells and induce cells apoptosis, the growth inhibitory rate and apoptosis rate were (21.82(3.18)% and (16.79(3.37)%, respectively. The transfection efficiency of GFP-CDMN combined with PEMFs was significant higher than that of GFP-CDMN without PEMFs [(45.70(4.32)% vs (35.85(2.16)%, P<0.05]. Conclusion: It seemed that EPI-CDMN associated with external magnetic fields could significantly killed human bladder cancer BIU-87 cells and CDMN could effectively transfer GFP gene into tumors cells with the help of external magnetic fields which provided experimental basis for the magnetic targeting therapy of tumor.

  4. Carbon-ion beam irradiation kills X-ray-resistant p53-null cancer cells by inducing mitotic catastrophe.

    Directory of Open Access Journals (Sweden)

    Napapat Amornwichet

    Full Text Available BACKGROUND AND PURPOSE: To understand the mechanisms involved in the strong killing effect of carbon-ion beam irradiation on cancer cells with TP53 tumor suppressor gene deficiencies. MATERIALS AND METHODS: DNA damage responses after carbon-ion beam or X-ray irradiation in isogenic HCT116 colorectal cancer cell lines with and without TP53 (p53+/+ and p53-/-, respectively were analyzed as follows: cell survival by clonogenic assay, cell death modes by morphologic observation of DAPI-stained nuclei, DNA double-strand breaks (DSBs by immunostaining of phosphorylated H2AX (γH2AX, and cell cycle by flow cytometry and immunostaining of Ser10-phosphorylated histone H3. RESULTS: The p53-/- cells were more resistant than the p53+/+ cells to X-ray irradiation, while the sensitivities of the p53+/+ and p53-/- cells to carbon-ion beam irradiation were comparable. X-ray and carbon-ion beam irradiations predominantly induced apoptosis of the p53+/+ cells but not the p53-/- cells. In the p53-/- cells, carbon-ion beam irradiation, but not X-ray irradiation, markedly induced mitotic catastrophe that was associated with premature mitotic entry with harboring long-retained DSBs at 24 h post-irradiation. CONCLUSIONS: Efficient induction of mitotic catastrophe in apoptosis-resistant p53-deficient cells implies a strong cancer cell-killing effect of carbon-ion beam irradiation that is independent of the p53 status, suggesting its biological advantage over X-ray treatment.

  5. Real-time dynamic optical imaging of ACC-M tumor cells killed by HSV-tk/ACV system.

    Science.gov (United States)

    Xiong, Tao; Li, Yongjin; Li, Zhiyang; Xie, Xiangmo; Lu, Lisha

    2013-01-01

    HSV-tk/ACV induced and killed human adenoid cystic carcinoma cell (ACC-M) in vivo and in vitro, which were observed through optical imaging and green fluorescence protein (GFP) tagging technique. ACC-M was transfected with TK-GFP, and the single clone cell ACC-M-TK-GFP was selected by G418. With fluorescent stereomicroscope, whole-body fluorescent imaging system and fluorescent microscope, we could observe ACV treated ACC-M-TK-GFP cells in cell level and nude mice. The therapies of tumor were visualized clearly with optical imaging. This study proves that optical imaging is a very good approach for studying the effect of HSV-tk/ACV on the ACC-M tumor cells and decreasing the amount of vessel about tumors cell. Optical imaging will become a visual groundwork for monitoring tumor growth and evaluating in vivo curative effect of antitumor drugs.

  6. Killing effect of adenoviral mediated cytosine deaminase gene on human pancreatic cancer cell line PaTu 8988

    Institute of Scientific and Technical Information of China (English)

    PAN Xue; LI Zhao-shen; XU Guo-ming; CUI Long; ZHANG Su-zhen; GONG Yan-fang; TU Zhen-xing

    2001-01-01

    Objective: To evaluate the in vitro killing effects of cytosine deaminase gene mediated by adenovirus vector on human pancreatic carcinoma. Methods: Cytosine Deaminase (CD) gene was cloned into pAdTrack-CMV-CD, pAdTrack-CMV-CD and pAdEasy-1 were recombined in bacteria, and the products containing green fluorescent protein (GFP)were propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic carcinoma cell line 8988 were infected with this virus, then 5-FC was added; XTT assay was used to estimate the relative numbers of viable cells. Results: The positive clones were confirmed by using endonuclease digestion, and the titer of the virus containing CD gene was 2 × 1011 pfu/ml. It was found that 5-FC possessed significant cytotoxic activities for CD gene transfected 8988cell line, but had little effects on non-transfected pancreatic carcinoma cells. Conclusion: CD gene mediated by adenovirus has a high infectivity and is efficient for killing cultured pancreatic carcinoma cells, indicating suicide gene may be effective for pancreatic cancer in furure.

  7. Hypothermia postpones DNA damage repair in irradiated cells and protects against cell killing

    Energy Technology Data Exchange (ETDEWEB)

    Baird, Brandon J.; Dickey, Jennifer S.; Nakamura, Asako J.; Redon, Christophe E.; Parekh, Palak [Laboratory of Molecular Pharmacology, CCR, NCI, Bethesda, MD 20892 (United States); Griko, Yuri V. [Radiation and Space Biotechnology Branch, NASA Ames Research Center, Moffett Field, CA 94035 (United States); Aziz, Khaled; Georgakilas, Alexandros G. [Biology Department, East Carolina University, Greenville, NC 27858 (United States); Bonner, William M. [Laboratory of Molecular Pharmacology, CCR, NCI, Bethesda, MD 20892 (United States); Martin, Olga A., E-mail: sedelnio@mail.nih.gov [Laboratory of Molecular Pharmacology, CCR, NCI, Bethesda, MD 20892 (United States)

    2011-06-03

    Hibernation is an established strategy used by some homeothermic organisms to survive cold environments. In true hibernation, the core body temperature of an animal may drop to below 0 {sup o}C and metabolic activity almost cease. The phenomenon of hibernation in humans is receiving renewed interest since several cases of victims exhibiting core body temperatures as low as 13.7 {sup o}C have been revived with minimal lasting deficits. In addition, local cooling during radiotherapy has resulted in normal tissue protection. The experiments described in this paper were prompted by the results of a very limited pilot study, which showed a suppressed DNA repair response of mouse lymphocytes collected from animals subjected to 7-Gy total body irradiation under hypothermic (13 {sup o}C) conditions, compared to normothermic controls. Here we report that human BJ-hTERT cells exhibited a pronounced radioprotective effect on clonogenic survival when cooled to 13 {sup o}C during and 12 h after irradiation. Mild hypothermia at 20 and 30 {sup o}C also resulted in some radioprotection. The neutral comet assay revealed an apparent lack on double strand break (DSB) rejoining at 13 {sup o}C. Extension of the mouse lymphocyte study to ex vivo-irradiated human lymphocytes confirmed lower levels of induced phosphorylated H2AX ({gamma}-H2AX) and persistence of the lesions at hypothermia compared to the normal temperature. Parallel studies of radiation-induced oxidatively clustered DNA lesions (OCDLs) revealed partial repair at 13 {sup o}C compared to the rapid repair at 37 {sup o}C. For both {gamma}-H2AX foci and OCDLs, the return of lymphocytes to 37 {sup o}C resulted in the resumption of normal repair kinetics. These results, as well as observations made by others and reviewed in this study, have implications for understanding the radiobiology and protective mechanisms underlying hypothermia and potential opportunities for exploitation in terms of protecting normal tissues against

  8. Identification and structural analysis of an L-asparaginase enzyme from guinea pig with putative tumor cell killing properties.

    Science.gov (United States)

    Schalk, Amanda M; Nguyen, Hien-Anh; Rigouin, Coraline; Lavie, Arnon

    2014-11-28

    The initial observation that guinea pig serum kills lymphoma cells marks the serendipitous discovery of a new class of anti-cancer agents. The serum cell killing factor was shown to be an enzyme with L-asparaginase (ASNase) activity. As a direct result of this observation, several bacterial L-asparaginases were developed and are currently approved by the Food and Drug Administration for the treatment of the subset of hematological malignancies that are dependent on the extracellular pool of the amino acid asparagine. As drugs, these enzymes act to hydrolyze asparagine to aspartate, thereby starving the cancer cells of this amino acid. Prior to the work presented here, the precise identity of this guinea pig enzyme has not been reported in the peer-reviewed literature. We discovered that the guinea pig enzyme annotated as H0W0T5_CAVPO, which we refer to as gpASNase1, has the required low Km property consistent with that possessed by the cell-killing guinea pig serum enzyme. Elucidation of the ligand-free and aspartate complex gpASNase1 crystal structures allows a direct comparison with the bacterial enzymes and serves to explain the lack of L-glutaminase activity in the guinea pig enzyme. The structures were also used to generate a homology model for the human homolog hASNase1 and to help explain its vastly different kinetic properties compared with gpASNase1, despite a 70% sequence identity. Given that the bacterial enzymes frequently present immunogenic and other toxic side effects, this work suggests that gpASNase1 could be a promising alternative to these bacterial enzymes.

  9. Identification and Structural Analysis of an l-Asparaginase Enzyme from Guinea Pig with Putative Tumor Cell Killing Properties*

    Science.gov (United States)

    Schalk, Amanda M.; Nguyen, Hien-Anh; Rigouin, Coraline; Lavie, Arnon

    2014-01-01

    The initial observation that guinea pig serum kills lymphoma cells marks the serendipitous discovery of a new class of anti-cancer agents. The serum cell killing factor was shown to be an enzyme with l-asparaginase (ASNase) activity. As a direct result of this observation, several bacterial l-asparaginases were developed and are currently approved by the Food and Drug Administration for the treatment of the subset of hematological malignancies that are dependent on the extracellular pool of the amino acid asparagine. As drugs, these enzymes act to hydrolyze asparagine to aspartate, thereby starving the cancer cells of this amino acid. Prior to the work presented here, the precise identity of this guinea pig enzyme has not been reported in the peer-reviewed literature. We discovered that the guinea pig enzyme annotated as H0W0T5_CAVPO, which we refer to as gpASNase1, has the required low Km property consistent with that possessed by the cell-killing guinea pig serum enzyme. Elucidation of the ligand-free and aspartate complex gpASNase1 crystal structures allows a direct comparison with the bacterial enzymes and serves to explain the lack of l-glutaminase activity in the guinea pig enzyme. The structures were also used to generate a homology model for the human homolog hASNase1 and to help explain its vastly different kinetic properties compared with gpASNase1, despite a 70% sequence identity. Given that the bacterial enzymes frequently present immunogenic and other toxic side effects, this work suggests that gpASNase1 could be a promising alternative to these bacterial enzymes. PMID:25320094

  10. Dendritic cells engineered to express defined allo-HLA peptide complexes induce antigen-specific cytotoxic T cells efficiently killing tumour cells

    DEFF Research Database (Denmark)

    Stronen, E; Abrahamsen, I W; Gaudernack, G;

    2009-01-01

    presented by a non-self human leucocyte antigen (HLA) molecule and transferred to cancer patients expressing that HLA molecule. Obtaining allo-restricted CTL of high-avidity and low cross-reactivity has, however, proven difficult. Here, we show that dendritic cells transfected with mRNA encoding HLA-A*0201...... and efficiently killed HLA-A*0201(+) melanoma cells, whilst sparing HLA-A*0201(+) B-cells. Allo-restricted CTL specific for peptides from the leukaemia-associated antigens CD33 and CD19 were obtained with comparable efficiency. Collectively, the results show that dendritic cells engineered to express defined allo......Most tumour-associated antigens (TAA) are non-mutated self-antigens. The peripheral T cell repertoire is devoid of high-avidity TAA-specific cytotoxic T lymphocytes (CTL) due to self-tolerance. As tolerance is major histocompatibility complex-restricted, T cells may be immunized against TAA...

  11. Selective Killing Effects of Cold Atmospheric Pressure Plasma with NO Induced Dysfunction of Epidermal Growth Factor Receptor in Oral Squamous Cell Carcinoma.

    Directory of Open Access Journals (Sweden)

    Jung-Hwan Lee

    Full Text Available The aim of this study is to investigate the effects of cold atmospheric pressure plasma (CAP-induced radicals on the epidermal growth factor receptor (EGFR, which is overexpressed by oral squamous cell carcinoma, to determine the underlying mechanism of selective killing. CAP-induced highly reactive radicals were observed in both plasma plume and cell culture media. The selective killing effect was observed in oral squamous cell carcinoma compared with normal human gingival fibroblast. Degradation and dysfunction of EGFRs were observed only in the EGFR-overexpressing oral squamous cell carcinoma and not in the normal cell. Nitric oxide scavenger pretreatment in cell culture media before CAP treatment rescued above degradation and dysfunction of the EGFR as well as the killing effect in oral squamous cell carcinoma. CAP may be a promising cancer treatment method by inducing EGFR dysfunction in EGFR-overexpressing oral squamous cell carcinoma via nitric oxide radicals.

  12. Selective Killing Effects of Cold Atmospheric Pressure Plasma with NO Induced Dysfunction of Epidermal Growth Factor Receptor in Oral Squamous Cell Carcinoma.

    Science.gov (United States)

    Lee, Jung-Hwan; Om, Ji-Yeon; Kim, Yong-Hee; Kim, Kwang-Mahn; Choi, Eun-Ha; Kim, Kyoung-Nam

    2016-01-01

    The aim of this study is to investigate the effects of cold atmospheric pressure plasma (CAP)-induced radicals on the epidermal growth factor receptor (EGFR), which is overexpressed by oral squamous cell carcinoma, to determine the underlying mechanism of selective killing. CAP-induced highly reactive radicals were observed in both plasma plume and cell culture media. The selective killing effect was observed in oral squamous cell carcinoma compared with normal human gingival fibroblast. Degradation and dysfunction of EGFRs were observed only in the EGFR-overexpressing oral squamous cell carcinoma and not in the normal cell. Nitric oxide scavenger pretreatment in cell culture media before CAP treatment rescued above degradation and dysfunction of the EGFR as well as the killing effect in oral squamous cell carcinoma. CAP may be a promising cancer treatment method by inducing EGFR dysfunction in EGFR-overexpressing oral squamous cell carcinoma via nitric oxide radicals.

  13. Fiber mediated receptor masking in non-infected bystander cells restricts adenovirus cell killing effect but promotes adenovirus host co-existence.

    Directory of Open Access Journals (Sweden)

    Johan Rebetz

    Full Text Available The basic concept of conditionally replicating adenoviruses (CRAD as oncolytic agents is that progenies generated from each round of infection will disperse, infect and kill new cancer cells. However, CRAD has only inhibited, but not eradicated tumor growth in xenograft tumor therapy, and CRAD therapy has had only marginal clinical benefit to cancer patients. Here, we found that CRAD propagation and cancer cell survival co-existed for long periods of time when infection was initiated at low multiplicity of infection (MOI, and cancer cell killing was inefficient and slow compared to the assumed cell killing effect upon infection at high MOI. Excessive production of fiber molecules from initial CRAD infection of only 1 to 2% cancer cells and their release prior to the viral particle itself caused a tropism-specific receptor masking in both infected and non-infected bystander cells. Consequently, the non-infected bystander cells were inefficiently bound and infected by CRAD progenies. Further, fiber overproduction with concomitant restriction of adenovirus spread was observed in xenograft cancer therapy models. Besides the CAR-binding Ad4, Ad5, and Ad37, infection with CD46-binding Ad35 and Ad11 also caused receptor masking. Fiber overproduction and its resulting receptor masking thus play a key role in limiting CRAD functionality, but potentially promote adenovirus and host cell co-existence. These findings also give important clues for understanding mechanisms underlying the natural infection course of various adenoviruses.

  14. Clinical-scale laser-based scanning and processing of live cells: selective photothermal killing of fluorescent tumor targets for autologous stem cell transplantation

    Science.gov (United States)

    Koller, Manfred R.; Hanania, Elie G.; Eisfeld, Timothy; O'Neal, Robert A.; Khovananth, Kevin M.; Palsson, Bernhard O.

    2001-04-01

    High-dose chemotherapy, followed by autologous hematopoietic stem cell (HSC) transplantation, is widely used for the treatment of cancer. However, contaminating tumor cells within HSC harvests continue to be of major concern since re-infused tumor cells have proven to contribute to disease relapse. Many tumor purging methods have been evaluated, but all leave detectable tumor cells in the transplant and result in significant loss of HSCs. These shortcomings cause engraftment delays and compromise the therapeutic value of purging. A novel approach integrating automated scanning cytometry, image analysis, and selective laser-induced killing of labeled cells within a cell mixture is described here. Non-Hodgkin's lymphoma (NHL) cells were spiked into cell mixtures, and fluorochrome-conjugated antibodies were used to label tumor cells within the mixture. Cells were then allowed to settle on a surface, and as the surface was scanned with a fluorescence excitation source, a laser pulse was fired at every detected tumor cell using high-speed beam steering mirrors. Tumor cells were selectively killed with little effect on adjacent non-target cells, demonstrating the feasibility of this automated cell processing approach. This technology has many potential research and clinical applications, one example of which is tumor cell purging for autologous HSC transplantation.

  15. 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.

  16. 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.

  17. Influence of metronidazole and some electron acceptors on the chlorin e6 photosensitized killing of Ehrlich carcinoma cells

    Science.gov (United States)

    Chekulayev, V.; Shevchuk, Igor; Mihkelsoo, Virgo T.; Kallikorm, A. P.

    1992-06-01

    A decrease in the effectiveness of photosensitized killing of neoplasm cells was observed in the presence of chlorin-e6 at a reduced concentration of oxygen. But when metronidazole (MZ) was injected in vitro as well as in vivo, a significant increase in the photosensitized killing of Ehrlich carcinoma cells by chlorin-e6 was observed. Moreover, contrary to the hematoporphyrin derivative (HpD), MZ increases the effectiveness of photodynamic therapy (PDT) by using chlorin-e6 not only in the hypoxic but also in the aerobic conditions. The interaction between MZ and the excited photosensitizer may account for an increased phototoxicity of chlorin-e6. The formation of cytotoxic nitroimidazole radicals as a result of photochemical processes of type 1 is discussed. This property of the photosensitizer may be successfully used in working out a method of potentiating PDT in combination not only with nitroimidazoles, but also with other electron acceptor compounds (EACp), e.g., quinone antitumor antibiotics.

  18. Introducing the RadBioStat Educational Software: Computer-Assisted Teaching of the Random Nature of Cell Killing

    Directory of Open Access Journals (Sweden)

    Safari A

    2014-06-01

    Full Text Available The interaction of radiation with cells and tissues has a random nature. Therefore, understanding the random nature of cell killing that is determined by Poisson distribution statistics is an essential point in education of radiation biology. RadBioStat is a newly developed educational MATLAB-based software designed for computer-assisted learning of the target theory in radiation biology. Although its potential applications is developing rapidly, currently RadBioStat software can be a useful tool in computerassisted education of radiobiological models such as single target single hit, multiple target single hit and multiple target multiple hit. Scholars’ feedback is valuable to the producers of this software and help them continuously improve this product, add new features and increase its desirability and functionality.

  19. GP73-regulated oncolytic adenoviruses possess potent killing effect on human liver cancer stem-like cells

    Science.gov (United States)

    Zhang, Rong; Ma, Buyun; Liu, Tao; Yang, Yu; Xie, Wenjie; Liu, Xianglei; Huang, Fang; Liu, Tao; Zhou, Xiumei; Liu, Xinyuan; Wang, Yigang

    2016-01-01

    Cancer stem cells (CSCs), also known as tumor-initiating cells, are highly metastatic, chemo-resistant and tumorigenic, and are critical for cancer development, maintenance and recurrence. Oncolytic adenovirus could targetedly kill CSCs and has been acted as a promising anticancer agent. Currently, a novel GP73-regulated oncolytic adenovirus GD55 was constructed to specifically treat liver cancer and exhibited obvious cytotoxicity effect. However, there remains to be confirmed that whether GD55 could effectively eliminate liver CSCs. We first utilized the suspension culture to enrich the liver CSCs-like cells, which acquires the properties of liver CSCs in self-renewal, differentiation, quiescence, chemo-resistance and tumorigenicity. The results indicated that GD55 elicited more significant cytotoxicity and stronger oncolytic effect in liver CSC-like cells compared to common oncolytic virus ZD55. Additionally, GD55 possessed the greater efficacy in suppressing the growth of implanted tumors derived from liver CSC-like cells than ZD55. Furthermore, GD55 induced remarkable apoptosis of liver CSC-like cells in vitro and in vivo, and inhibited the propogation of cells and angiogenesis in xenograft tumor tissues. Thus, GD55 may virtually represent an attractive therapeutic agent for targeting liver CSCs to achieve better clinical outcomes for HCC patients. PMID:27121064

  20. In vivo label-free photoacoustic flow cytography and on-the-spot laser killing of single circulating melanoma cells

    Science.gov (United States)

    He, Yun; Wang, Lidai; Shi, Junhui; Yao, Junjie; Li, Lei; Zhang, Ruiying; Huang, Chih-Hsien; Zou, Jun; Wang, Lihong V.

    2016-12-01

    Metastasis causes as many as 90% of cancer-related deaths, especially for the deadliest skin cancer, melanoma. Since hematogenous dissemination of circulating tumor cells is the major route of metastasis, detection and destruction of circulating tumor cells are vital for impeding metastasis and improving patient prognosis. Exploiting the exquisite intrinsic optical absorption contrast of circulating melanoma cells, we developed dual-wavelength photoacoustic flow cytography coupled with a nanosecond-pulsed melanoma-specific laser therapy mechanism. We have successfully achieved in vivo label-free imaging of rare single circulating melanoma cells in both arteries and veins of mice. Further, the photoacoustic signal from a circulating melanoma cell immediately hardware-triggers a lethal pinpoint laser irradiation to kill it on the spot in a thermally confined manner without causing collateral damage. A pseudo-therapy study including both in vivo and in vitro experiments demonstrated the performance and the potential clinical value of our method, which can facilitate early treatment of metastasis by clearing circulating tumor cells from vasculature.

  1. Troxerutin, a natural flavonoid binds to DNA minor groove and enhances cancer cell killing in response to radiation.

    Science.gov (United States)

    Panat, Niranjan A; Singh, Beena G; Maurya, Dharmendra K; Sandur, Santosh K; Ghaskadbi, Saroj S

    2016-05-05

    Troxerutin, a flavonoid best known for its radioprotective and antioxidant properties is of considerable interest of study due to its broad pharmacological activities. The present study on troxerutin highlights its abilities to bind DNA and enhance cancer cell killing in response to radiation. Troxerutin showed strong binding with calf thymus DNA in vitro. Troxerutin-DNA interaction was confirmed by CD spectropolarimetry. The mode of binding of troxerutin to DNA was assessed by competing troxerutin with EtBr or DAPI, known DNA intercalator and a minor groove binder, respectively. DAPI fluorescence was drastically reduced with linear increase in troxerutin concentration suggesting possible binding of troxerutin to DNA minor groove. Further, computational studies of docking of troxerutin molecule on mammalian DNA also indicated possible troxerutin-DNA interaction at minor groove of DNA. Troxerutin was found to mainly localize in the nucleus of prostate cancer cells. It induced cytotoxicity in radioresistant (DU145) and sensitive (PC3) prostate cancer cells. When troxerutin pre-treated DU145 and PC3 cells were exposed to γ-radiation, cytotoxicity as estimated by MTT assay, was found to be further enhanced. In addition, the % subG1 population detected by propidium iodide staining also showed similar response when combined with radiation. A similar trend was observed in terms of ROS generation and DNA damage in DU145 cells when troxerutin and radiation were combined. DNA binding at minor groove by troxerutin may have contributed to strand breaks leading to increased radiation induced cell death.

  2. In vivo label-free photoacoustic flow cytography and on-the-spot laser killing of single circulating melanoma cells

    Science.gov (United States)

    He, Yun; Wang, Lidai; Shi, Junhui; Yao, Junjie; Li, Lei; Zhang, Ruiying; Huang, Chih-Hsien; Zou, Jun; Wang, Lihong V.

    2016-01-01

    Metastasis causes as many as 90% of cancer-related deaths, especially for the deadliest skin cancer, melanoma. Since hematogenous dissemination of circulating tumor cells is the major route of metastasis, detection and destruction of circulating tumor cells are vital for impeding metastasis and improving patient prognosis. Exploiting the exquisite intrinsic optical absorption contrast of circulating melanoma cells, we developed dual-wavelength photoacoustic flow cytography coupled with a nanosecond-pulsed melanoma-specific laser therapy mechanism. We have successfully achieved in vivo label-free imaging of rare single circulating melanoma cells in both arteries and veins of mice. Further, the photoacoustic signal from a circulating melanoma cell immediately hardware-triggers a lethal pinpoint laser irradiation to kill it on the spot in a thermally confined manner without causing collateral damage. A pseudo-therapy study including both in vivo and in vitro experiments demonstrated the performance and the potential clinical value of our method, which can facilitate early treatment of metastasis by clearing circulating tumor cells from vasculature. PMID:28000788

  3. The radioresistance to killing of A1-5 cells derives from activation of the Chk1 pathway

    Science.gov (United States)

    Hu, B.; Zhou, X. Y.; Wang, X.; Zeng, Z. C.; Iliakis, G.; Wang, Y.

    2001-01-01

    Checkpoints respond to DNA damage by arresting the cell cycle to provide time for facilitating repair. In mammalian cells, the G(2) checkpoint prevents the Cdc25C phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. Both Chk1 and Chk2, the checkpoint kinases, can phosphorylate Cdc25C and inactivate its in vitro phosphatase activity. Therefore, both Chk1 and Chk2 are thought to regulate the activation of the G(2) checkpoint. Here we report that A1-5, a transformed rat embryo fibroblast cell line, shows much more radioresistance associated with a much stronger G(2) arrest response when compared with its counterpart, B4, although A1-5 and B4 cells have a similar capacity for nonhomologous end-joining DNA repair. These phenotypes of A1-5 cells are accompanied by a higher Chk1 expression and a higher phosphorylation of Cdc2. On the other hand, Chk2 expression increases slightly following radiation; however, it has no difference between A1-5 and B4 cells. Caffeine or UCN-01 abolishes the extreme radioresistance with the strong G(2) arrest and at the same time reduces the phosphorylation of Cdc2 in A1-5 cells. In addition, Chk1 but not Chk2 antisense oligonucleotide sensitizes A1-5 cells to radiation-induced killing and reduces the G(2) arrest of the cells. Taken together these results suggest that the Chk1/Cdc25C/Cdc2 pathway is the major player for the radioresistance with G(2) arrest in A1-5 cells.

  4. Zika Kills Vital Nervous System Cells in Adult Mice, Study Finds

    Science.gov (United States)

    ... effects of the Zika-related changes in neural stem cell numbers in mice. However, research with animals often doesn't translate to similar findings in humans. SOURCE: Cell Stem Cell , news release, Aug. 18, 2016 HealthDay Copyright ( ...

  5. Killing Cancer Cells with the Help of Infrared Light – Photoimmunotherapy

    Science.gov (United States)

    Near-infrared photoimmunotherapy uses an antibody–photoabsorber conjugate that binds to cancer cells. When near-infrared light is applied, the cells swell and then burst, causing the cancer cell to die. Photoimmunotherapy is in clinical trials in patients with inoperable tumors.

  6. Inductive heating kills cells that contribute to plaque: a proof-of-concept

    Directory of Open Access Journals (Sweden)

    Angelo Gaitas

    2015-04-01

    Full Text Available Inducing cell death by heating targeted particles shows promise in cancer treatment. Here, we aim to demonstrate the feasibility of extending the use of this technique to treat and remove vascular deposits and thrombosis. We used induction heating of macrophages, which are key contributors to atherosclerosis and have demonstrated clear feasibility for heating and destroying these cells using ferromagnetic and pure iron particles. Specifically, iron particles achieved maximum temperatures of 51 ± 0.5 °C and spherical particles achieved a maximum temperature of 43.9 ± 0.2 °C (N = 6 after 30 min of inductive heating. Two days of subsequent observation demonstrated that inductive heating led to a significant reduction in cell number. Prior to induction heating, cell density was 105,000 ± 20,820 cells/ml (N = 3. This number was reduced to 6,666 ± 4,410 cells/ml for the spherical particles and 16,666 ± 9,280 cells/ml for the iron particles 24 h after inductive heating. Though cell density increased on the second day following inductive heating, the growth was minimal. Cells grew to 26,667 ± 6,670 cells/ml and 30,000 ± 15,280 cells/ml respectively. Compared to cell cultures with iron and spherical particles that were not subjected to induction heating, we observed a 97% reduction in cell count for the spherical particles and a 91% reduction for the iron particles after the first 24 h. After 48 h we observed a 95% reduction in cell growth for both spherical and iron particles. Induction heating of microparticles was thus highly effective in reducing the macrophage population and preventing their growth. These results demonstrate the feasibility of targeting cells involved in atherosclerosis and warrant further research into potential clinical applications.

  7. Inductive heating kills cells that contribute to plaque: a proof-of-concept.

    Science.gov (United States)

    Gaitas, Angelo; Kim, Gwangseong

    2015-01-01

    Inducing cell death by heating targeted particles shows promise in cancer treatment. Here, we aim to demonstrate the feasibility of extending the use of this technique to treat and remove vascular deposits and thrombosis. We used induction heating of macrophages, which are key contributors to atherosclerosis and have demonstrated clear feasibility for heating and destroying these cells using ferromagnetic and pure iron particles. Specifically, iron particles achieved maximum temperatures of 51 ± 0.5 °C and spherical particles achieved a maximum temperature of 43.9 ± 0.2 °C (N = 6) after 30 min of inductive heating. Two days of subsequent observation demonstrated that inductive heating led to a significant reduction in cell number. Prior to induction heating, cell density was 105,000 ± 20,820 cells/ml (N = 3). This number was reduced to 6,666 ± 4,410 cells/ml for the spherical particles and 16,666 ± 9,280 cells/ml for the iron particles 24 h after inductive heating. Though cell density increased on the second day following inductive heating, the growth was minimal. Cells grew to 26,667 ± 6,670 cells/ml and 30,000 ± 15,280 cells/ml respectively. Compared to cell cultures with iron and spherical particles that were not subjected to induction heating, we observed a 97% reduction in cell count for the spherical particles and a 91% reduction for the iron particles after the first 24 h. After 48 h we observed a 95% reduction in cell growth for both spherical and iron particles. Induction heating of microparticles was thus highly effective in reducing the macrophage population and preventing their growth. These results demonstrate the feasibility of targeting cells involved in atherosclerosis and warrant further research into potential clinical applications.

  8. Oncolytic adenovirus SG600-IL24 selectively kills hepatocellular carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To investigate the effect of oncolytic adenovirus SG600-IL24 and replication-incompetent adenovirus Ad.IL-24 on hepatocellular carcinoma (HCC) cell lines and normal liver cell line. METHODS: HCC cell lines (HepG2, Hep3B and MHCC97L) and normal liver cell line (L02) with a different p53 status were infected with SG600-IL24 and Ad.IL-24, respectively. Melanoma differentiation-associated (MDA)-7/interleukin (IL)-24 mRNA and protein expressions in infected cells were detected by reverse transcription-polym...

  9. SENSITIZATION OF ACNU KILLING EFFECTS ON HeLa S3 CELLS BY MGMT ANTISENSE RNA TRANSFECTION

    Institute of Scientific and Technical Information of China (English)

    季守平; 由英; 吴英; 陈建敏; 杨军; 章扬培

    1998-01-01

    O’-methylguanlne-DNA-msthybransferase(MGMT)plays a very important role in the ceUular resistsnce to uitrosoureas drugs. Inhibition of MGMT might be a useful approach in tumor chemotherapy. In this study, the depletlon vii MGMT activity hy retroviral-mediated antisense RNA transfectkm were reported. Three retroviral vectors expressing MGMT antisense RNA were constructed and transfected into HeLa S3 cells. The difference of MGMT mRNA, MGMT activity as well as cellular resistance to ACNU before and after transtecfion were ohserved. It was found that antisense RNA targeting 5''region and whole length of MGMT mRNA could partially deplete MGMT activity and enhance killing effects of ACNU.However, 3'' region antisense RNA had no effect on MGMT modulation.

  10. Radiation related basic cancer research : research for radiation induced tumor cell killing

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seung Hoon; Hong, Seok Il; Cho, Kyung Ja; Kim, Byung Gi; Lee, Kee Ho; Nam, Myung Jin

    1999-04-01

    The radioresistant clones was established from human U251 glioblastoma cell line through intermittently exposed to 3 Gy gamma-radiation for six months. Treatment of SNU-16 cells with various doses of radiation, TNF alpha and PMA resulted in a decrease in cell viability. The results prove that cell death of SNU16 is a apoptosis mediated by caspase-3. We have examined the expression of bcl-2 and c-myc in cervical cancer specimens and cervical intraepithelial neoplasia (CIN) to determine the role of coexpression of bcl-3 and c-myc during progression into cervical cancer. The frequent alterations in FHIT expression in many cervical carcinomas and their cell lines suggest that FHIT gene alterations are pla a role in cervical tumorigenesis. According to these correlation between the viability and apoptosis of RD cells, the proper range of the dosage for the investigation of differentiation potency in RD cells was assessed as 1 to 3Gy.

  11. Killing effect of different doses of preoperative iodine 131 therapy on thyroid cancer cells and its effect on salivary gland function

    Institute of Scientific and Technical Information of China (English)

    Xiao-Dong Zheng; Tao Pu; Yi Luo; Xing-An Zhang; Zu-Mao Li

    2015-01-01

    Objective:To study the killing effect of different doses of preoperative iodine 131 therapy on thyroid cancer cells and its effect on salivary gland function.Methods:Patients diagnosed with thyroid cancer in our hospital from May 2013 to June 2014 were enrolled for study, given preoperative iodine 131 therapy and randomly divided into control group, low dose group, middle dose group and high dose group. Then cell apoptotic rate, cell cycle, cancer promoting gene and cancer suppressor gene expression in thyroid carcinoma tissue as well as salivary gland function were detected.Results: (1) cancer cell killing effect: compared with control group, cell apoptotic rates and number of cells in G0/G1 phase of low dose group, middle dose group and high dose group increased, number of cells in S and G2/M phase decreased, BRAF, Livin, MCM7 and CDK2 expression decreased, CCNG2 and PTEN expression increased; cell killing effect of middle dose group and high dose group were better than that of low dose group, and cell killing effect of middle dose group and high dose group had no differences; (2) salivary gland function: compared with control group, UI and SR in bilateral parotid and bilateral submandibular glands of low dose group, middle dose group and high dose group decreased; salivary gland damage effect of low dose group and middle dose group were weaker than that of high dose group, and salivary gland damage effect of low dose group and middle dose group had no differences.Conclusion:Middle dose of iodine 131 can take the killing effect on cancer cells and the protective effect on salivary glands into account; it’s an ideal dosage for preoperative iodine 131 internal radiation therapy of thyroid cancer patients.

  12. Analyses of CD20 Monoclonal Antibody–Mediated Tumor Cell Killing Mechanisms: Rational Design of Dosing Strategies

    Science.gov (United States)

    Lindorfer, Margaret A.

    2014-01-01

    Since approval of rituximab for treatment of B cell non-Hodgkin lymphoma, development of monoclonal antibodies (mAbs) for cancer treatment and elucidation of their cytotoxic mechanisms have been subject to intense investigations. Compelling evidence indicates that rituximab and another CD20 mAb, ofatumumab, must use the body’s cellular and humoral immune effector functions to kill malignant cells. Other U.S. Food and Drug Administration–approved mAbs, including obinutuzumab, cetuximab, and trastuzumab, require, in part, these effector mechanisms to eliminate tumor cells. Although gram quantities of mAbs can be administered to patients, our investigations of CD20 mAb-based therapies for chronic lymphocytic leukemia (CLL), including correlative measurements in clinical trials and studies with primary cells and cell lines, indicate that effector mechanisms necessary for mAb activity can be saturated or exhausted if tumor burdens are high, thus substantially compromising the efficacy of high-dose mAb therapy. Under these conditions, another reaction (trogocytosis) predominates in which bound CD20 mAb and CD20 are removed from targeted cells by effector cells that express Fcγ receptors, thereby allowing malignant cells to escape unharmed and continue to promote disease pathology. To address this problem, we propose that a low-dose strategy, based on administering 30–50 mg of CD20 mAb three times per week, may be far more effective for CLL than standard dosing because it will minimize effector function saturation and reduce trogocytosis. This approach may have general applicability to other mAbs that use immune effector functions, and could be formulated into a subcutaneous treatment strategy that would be more accessible and possibly more efficacious for patients. PMID:24944188

  13. Killing of p53-deficient hepatoma cells by parvovirus H-1 and chemotherapeutics requires promyelocytic leukemia protein

    Institute of Scientific and Technical Information of China (English)

    Maike Sieben; Markus Moehler; Kerstin Herzer; Maja Zeidler; Vera Heinrichs; Barbara Leuchs; Martin Schuler; Jan J Cornelis; Peter R Galle; Jean Rommelaere

    2008-01-01

    AIM: To evaluate the synergistic targeting and killing of human hepatocellular carcinoma (HCC) cells lacking p53 by the oncolytic autonomous parvovirus (PV) H-1 and chemotherapeutic agents and its dependence on functional promyelocytic leukemia protein (PML).METHODS: The role of p53 and PML in regulating cytotoxicity and gene transfer mediated by wild-type (wt)PV H-1 were explored in two pairs of isogenic human hepatoma cell lines with different p53 status.Furthermore,H-1 PV infection was combined with cytostatic drug treatment.RESULTS: While the HCC cells with different p53 status studied were all susceptible to H-1 PV-induced apoptosis,the cytotoxicity of H-1 PV was more pronounced in p53-negative than in p53-positive cells.Apoptosis rates in p53-negative cell lines treated by genotoxic drugs were further enhanced by a treatment with H-1 PV.In flow cytometric analyses,H-1 PV infection resulted in a reduction of the mitochondrial transmembrane potential.In addition,H-1 PV cells showed a significant increase in PML expression.Knocking down PML expression resulted in a striking reduction of the level of H-1 PV infected tumor cell death.CONCLUSION: H-1 PV is a suitable agent to circumvent the resistance of p53-negative HCC cells to genotoxic agents,and it enhances the apoptotic process which is dependent on functional PML.Thus,H-1 PV and its oncolytic vector derivatives may be considered as therapeutic options for HCC,particularly for p53-negative tumors.

  14. Nanostructured Surfaces to Target and Kill Circulating Tumor Cells While Repelling Leukocytes

    Directory of Open Access Journals (Sweden)

    Michael J. Mitchell

    2012-01-01

    Full Text Available Hematogenous metastasis, the process of cancer cell migration from a primary to distal location via the bloodstream, typically leads to a poor patient prognosis. Selectin proteins hold promise in delivering drug-containing nanocarriers to circulating tumor cells (CTCs in the bloodstream, due to their rapid, force-dependent binding kinetics. However, it is challenging to deliver such nanocarriers while avoiding toxic effects on healthy blood cells, as many possess ligands that adhesively interact with selectins. Herein, we describe a nanostructured surface to capture flowing cancer cells, while preventing human neutrophil adhesion. Microtube surfaces with immobilized halloysite nanotubes (HNTs and E-selectin functionalized liposomal doxorubicin (ES-PEG L-DXR significantly increased the number of breast adenocarcinoma MCF7 cells captured from flow, yet also significantly reduced the number of captured neutrophils. Neutrophils firmly adhered and projected pseudopods on surfaces coated only with liposomes, while neutrophils adherent to HNT-liposome surfaces maintained a round morphology. Perfusion of both MCF7 cells and neutrophils resulted in primarily cancer cell adhesion to the HNT-liposome surface, and induced significant cancer cell death. This work demonstrates that nanostructured surfaces consisting of HNTs and ES-PEG L-DXR can increase CTC recruitment for chemotherapeutic delivery, while also preventing healthy cell adhesion and uptake of therapeutic intended for CTCs.

  15. ABT-737, a Bcl-2 Selective Inhibitor, and Chloroquine Synergistically Kill Renal Cancer Cells.

    Science.gov (United States)

    Yin, Pei; Jia, Jinpeng; Li, Jijun; Song, Yan; Zhang, Yiyan; Chen, Fengkun

    2016-01-01

    Renal cell carcinoma (RCC) is the most common malignancy in the kidney in the world, and the 5-year overall survival for patients remains poor due to the lack of effective treatment strategies. Although ABT-737, as a Bcl-2/Bcl-xL inhibitor, has recently emerged as a novel cancer therapeutic reagent, apoptosis induced by ABT-737 is often blocked in several types of cancer cells. This study investigated whether the combination of the small-molecule BH3 mimetic ABT-737 and the lysosome inhibitor chloroquine was an effective strategy for treating renal cancer cells. We found that the combination of ABT-737 and chloroquine synergistically decreased cell viability when compared to treatment with either single reagent. Cell apoptosis induced by a combined treatment was markedly inhibited by the caspase inhibitors z-DEVD-FMK and z-VAD-FMK. It was also inhibited by cathepsin inhibitor E-64 and CTSI (cathepsin inhibitor), which suggested that apoptosis was dependent on the cascade of caspase activation and cathepsins released from lysosomes. Furthermore, we found that ABT-737 could increase the cell level of ROS, which triggers cathepsin-mediated cell death and augments the role of chloroquine in cell death. So the combination of ABT-737 and chloroquine was an effective strategy for the treatment of renal cancer cells, and this combined strategy may widen the therapeutic window of ABT-737 and chloroquine as well as enhance the clinical efficacy of synergistic drug combinations.

  16. Fenugreek, a naturally occurring edible spice, kills MCF-7 human breast cancer cells via an apoptotic pathway.

    Science.gov (United States)

    Khoja, Kholoud K; Shaf, Gowhar; Hasan, Tarique N; Syed, Naveed Ahmed; Al-Khalifa, Abdrohman S; Al-Assaf, Abdullah H; Alshatwi, Ali A

    2011-01-01

    There is growing use of anticancer complementary and alternative medicines worldwide. Trigonella foenum graecum (Fenugreek) is traditionally applied to treat disorders such as diabetes, high cholesterol, wounds, inflammation, and gastrointestinal ailments. Fenugreek is also reported to have anticancer properties due to its active beneficial chemical constituents. The mechanism of action of several anticancer drugs is based on their ability to induce apoptosis. The objective of the study was to characterize the downstream apoptotic genes targeted by FCE in MCF-7 human immortalized breast cells. FCE effectively killed MCF-7 cells through induction of apoptosis,confirmed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and RT-PCR assays. When cells were exposed to 50 μg/mL FCE for 24 hours, 23.2% apoptotic cells resulted, while a 48-hour exposure to 50 μg/mL caused 73.8% apoptosis. This was associated with increased expression of Caspase 3, 8, 9, p53, Fas, FADD, Bax and Bak in a time-and dose-dependent manner, as determined by real- time quantitative PCR. In summary, the induction of apoptosis by FCE is effected by its ability to increase the expression of pro-apoptotic genes and the spice holds promise for consideration in complementary therapy for breast cancer patients.

  17. A partner monoclonal antibody to Moab 730 kills 100% of DU145 and PC3 androgen-independent cancer cells

    Indian Academy of Sciences (India)

    Hemant Kumar Vyas; Rahul Pal; Nirmal K Lohiya; G P Talwar

    2009-12-01

    A number of therapeutic options are available for patients with prostate carcinoma till the time that the tumour is hormone dependent. However, no fully effective therapy is available for the treatment of androgen-independent prostate carcinomas. Antibodies directed at epitopes unique to or overexpressed on the cancer cells could be of therapeutic utility. A monoclonal antibody (Moab) 2C4 has been generated, which binds with cells of two androgenindependent prostate cancers, DU145 and PC3, and does not bind to peripheral blood leukocytes (PBLs) of healthy donors. This antibody, along with the previously developed Moab 730, kills 100% of both DU145 and PC3 cells in the presence of complement and does not have a deleterious effect on PBLs of healthy males. The anti-tumour action of the two antibodies prevents the establishment of DU145 cell tumour in nude mice in vivo. Moab 2C4 in combination with 730 has potential for use as therapy for androgen-independent cancers.

  18. Recombinant human tumor necrosis factor alpha does not potentiate cell killing after photodynamic therapy with a silicon phthalocyanine in A431 human epidermoid carcinoma cells.

    Science.gov (United States)

    Azizuddin, K; Kalka, K; Chiu, S M; Ahmad, N; Mukhtar, H; Separovic, D

    2001-02-01

    Photodynamic therapy (PDT) is a novel cancer treatment utilizing a photosensitizer, visible light and oxygen. PDT with the silicon phthalocyanine Pc 4, a new photosensitizer, is highly effective in cancer cell destruction and tumor ablation. The mechanisms underlying cancer cell killing by PDT are not fully understood. Tumor necrosis factor alpha (TNF) is a multifunctional cytokine that has been implicated in photocytotoxicity. We asked whether recombinant human TNF (rhTNF) affects Pc 4-PDT cytotoxicity in A431 human epidermoid carcinoma cells. Co-treatment of A431 cells with various doses of Pc 4-PDT and a sub-lethal rhTNF dose led to a sub-additive reduction in cell survival. In addition, in the presence of Pc 4-PDT or rhTNF, caspase-3 activity and apoptosis were induced. The combined treatment, however, did not potentiate either caspase-3 activity or apoptosis. Similar to previous findings we observed that Pc 4-PDT initiated a time-dependent extracellular TNF accumulation. The data suggest that: a) PDT and rhTNF induce cancer cell killing through different mechanisms; and b) Pc 4-PDT-induced TNF production is a stress response that may not directly affect photocytotoxicity.

  19. Adenovirus vector expressing mda-7 selectively kills hepatocellular carcinoma cell line Hep3B

    Institute of Scientific and Technical Information of China (English)

    Xin-Bo Xue; Kun Chen; Cong-Jun Wang; Jian-Wei Zheng; Yuan Yu; Zhi-Hai Peng; Zai-De Wu

    2008-01-01

    BACKGROUND: Melanoma differentiation associated gene-7 (mda-7) is a novel tumor suppressor gene, which has suppressor activity in a broad spectrum of human cancer cells both in vitro and in vivo through activation of various intracellular signaling pathways. In this study, we investigated the potential effect of mda-7 on human hepatocellular carcinoma (HCC) in vitro. METHODS: Cells from the human HCC cell line Hep3B and the human liver cell line L-02 were assigned to three groups. One was cultured in Dulbecco's modiifed Eagle's medium without serum (control). The others were transfected with adenovirus expressing the mda-7 gene (Ad.mda-7) or adenovirus vector serving as negative control (Ad.vec). The expression of MDA-7 and Bcl-2 proteins in Hep3B and L-02 cells was conifrmed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. The methyl thiazolyl tetrazolium colorimetric assay and lfow cytometry were used to assess tumor cell proliferation and the cell cycle. Hoechst and Annexin-V/propidium iodide staining were used to study mda-7 gene expression in Hep3B and L-02 cells. The expression of MDA-7, Bcl-2 and Bax proteins were detected by Western blotting. RESULTS: The mda-7 gene was expressed in Hep3B and L-02 cells. The protein concentrations of MDA-7 in supernatants were 790 and 810 pg/ml, respectively. mda-7 induced Hep3B growth suppression and apoptosis, compared with Ad.mda-7 and control (P CONCLUSIONS: mda-7 selectively induces growth inhibi-tion and apoptosis in the HCC cell line Hep3B but not in the normal liver cell line L-02 via downregulating the anti-apoptosis protein Bcl-2. It could be an ideal gene for gene therapy in HCC.

  20. Bispecific Antibodies that Mediate Killing of Cells Infected with Human Immunodeficiency Virus of Any Strain

    Science.gov (United States)

    Berg, Jorg; Lotscher, Erika; Steimer, Kathelyn S.; Capon, Daniel J.; Baenziger, Jurg; Jack, Hans-Martin; Wabl, Matthias

    1991-06-01

    Although AIDS patients lose human immunodeficiency virus (HIV)-specific cytotoxic T cells, their remaining CD8-positive T lymphocytes maintain cytotoxic function. To exploit this fact we have constructed bispecific antibodies that direct cytotoxic T lymphocytes of any specificity to cells that express gp120 of HIV. These bispecific antibodies comprise one heavy/light chain pair from an antibody to CD3, linked to a heavy chain whose variable region has been replaced with sequences from CD4 plus a second light chain. CD3 is part of the antigen receptor on T cells and is responsible for signal transduction. In the presence of these bispecific antibodies, T cells of irrelevant specificity effectively lyse HIV-infected cells in vitro.

  1. ROS accumulation by PEITC selectively kills ovarian cancer cells via UPR-mediated apoptosis

    Directory of Open Access Journals (Sweden)

    Yoon-hee eHong

    2015-07-01

    Full Text Available Unfolded protein response (UPR is crucial for both survival and death of mammalian cells, which is regulated by reactive oxygen species (ROS and nutrient depletion. In this study, we demonstrated the effect of ROS-accumulation, induced by β-phenethyl isothiocyanate (PEITC, on UPR mediated apoptosis in ovarian cancer cells. We used ovarian cancer cell lines, PA-1 and SKOV-3, with different p53 status (wild- and null- type, respectively. PEITC caused increased ROS-accumulation and inhibited proliferation selectively in ovarian cancer cells, and glutathione (GSH depletion in SKOV-3. However, PEITC did not cause any effect in normal ovarian epithelial cells and peripheral blood mononuclear cells. After 48 h of PEITC treatment (5 µM, apoptotic cell death was shown to increase significantly in the ovarian cancer cells and not in the normal cells. The key regulator of UPR-mediated apoptosis, CHOP/GADD153 and ER resident chaperone BiP/GRP78 were parallely up-regulated with activation of two major sensors of the UPR (PERK and ATF-6 in PA-1; PERK, and IRE1α in SKOV-3 in response to ROS accumulation induced by PEITC (5 µM. ROS scavenger, N-acetyl-cysteine (NAC, attenuated the effect of PEITC on UPR signatures (P-PERK, IRE1α, CHOP/GADD153, and BiP/GRP78, suggesting the involvement of ROS in UPR-mediated apoptosis. Altogether, PEITC induces UPR-mediated apoptosis in ovarian cancer cells via accumulation of ROS in a cancer-specific manner.

  2. Human NK cells differ more in their KIR2DL1-dependent thresholds for HLA-Cw6-mediated inhibition than in their maximal killing capacity.

    Directory of Open Access Journals (Sweden)

    Catarina R Almeida

    Full Text Available In this study we have addressed the question of how activation and inhibition of human NK cells is regulated by the expression level of MHC class I protein on target cells. Using target cell transfectants sorted to stably express different levels of the MHC class I protein HLA-Cw6, we show that induction of degranulation and that of IFN-γ secretion are not correlated. In contrast, the inhibition of these two processes by MHC class-I occurs at the same level of class I MHC protein. Primary human NK cell clones were found to differ in the amount of target MHC class I protein required for their inhibition, rather than in their maximum killing capacity. Importantly, we show that KIR2DL1 expression determines the thresholds (in terms of MHC I protein levels required for NK cell inhibition, while the expression of other receptors such as LIR1 is less important. Furthermore, using mathematical models to explore the dynamics of target cell killing, we found that the observed delay in target cell killing is exhibited by a model in which NK cells require some activation or priming, such that each cell can lyse a target cell only after being activated by a first encounter with the same or a different target cell, but not by models which lack this feature.

  3. Targeted alpha therapy in vivo: direct evidence for single cancer cell kill using {sup 149}Tb-rituximab

    Energy Technology Data Exchange (ETDEWEB)

    Beyer, G.J.; Soloviev, D.; Buchegger, F. [Division of Nuclear Medicine, University Hospital of Geneva, 24 Rue Micheli du Crest, 1211, Geneva 14 (Switzerland); Miederer, M. [Department of Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York (United States); Vranjes-Duric, S. [Laboratory of Radioisotopes, Vinca Institute of Nuclear Sciences, Belgrade (Czechoslovakia); Comor, J.J. [Laboratory of Physics, Vinca Institute of Nuclear Sciences, Belgrade (Czechoslovakia); Kuenzi, G.; Hartley, O. [Department of Medical Biochemistry, University Medical Center, Geneva (Switzerland); Senekowitsch-Schmidtke, R. [Clinic of Nuclear Medicine, Technical University of Munich, Munich (Germany)

    2004-04-01

    This study demonstrates high-efficiency sterilisation of single cancer cells in a SCID mouse model of leukaemia using rituximab, a monoclonal antibody that targets CD20, labelled with terbium-149, an alpha-emitting radionuclide. Radio-immunotherapy with 5.5 MBq labelled antibody conjugate (1.11 GBq/mg) 2 days after an intravenous graft of 5.10{sup 6} Daudi cells resulted in tumour-free survival for >120 days in 89% of treated animals. In contrast, all control mice (no treatment or treated with 5 or 300 {mu}g unlabelled rituximab) developed lymphoma disease. At the end of the study period, 28.4%{+-}4% of the long-lived daughter activity remained in the body, of which 91.1% was located in bone tissue and 6.3% in the liver. A relatively high daughter radioactivity concentration was found in the spleen (12%{+-}2%/g), suggesting that the killed cancer cells are mainly eliminated through the spleen. This promising preliminary in vivo study suggests that targeted alpha therapy with {sup 149}Tb is worthy of consideration as a new-generation radio-immunotherapeutic approach. (orig.)

  4. Cytolethal distending toxin B as a cell-killing component of tumor-targeted anthrax toxin fusion proteins.

    Science.gov (United States)

    Bachran, C; Hasikova, R; Leysath, C E; Sastalla, I; Zhang, Y; Fattah, R J; Liu, S; Leppla, S H

    2014-01-16

    Cytolethal distending toxin (Cdt) is produced by Gram-negative bacteria of several species. It is composed of three subunits, CdtA, CdtB, and CdtC, with CdtB being the catalytic subunit. We fused CdtB from Haemophilus ducreyi to the N-terminal 255 amino acids of Bacillus anthracis toxin lethal factor (LFn) to design a novel, potentially potent antitumor drug. As a result of this fusion, CdtB was transported into the cytosol of targeted cells via the efficient delivery mechanism of anthrax toxin. The fusion protein efficiently killed various human tumor cell lines by first inducing a complete cell cycle arrest in the G2/M phase, followed by induction of apoptosis. The fusion protein showed very low toxicity in mouse experiments and impressive antitumor effects in a Lewis Lung carcinoma model, with a 90% cure rate. This study demonstrates that efficient drug delivery by a modified anthrax toxin system combined with the enzymatic activity of CdtB has great potential as anticancer treatment and should be considered for the development of novel anticancer drugs.

  5. Engineered metal nanoparticles in the sub-nanomolar levels kill cancer cells

    Directory of Open Access Journals (Sweden)

    Vodyanoy V

    2016-04-01

    Full Text Available Vitaly Vodyanoy,1 Yasmine Daniels,2 Oleg Pustovyy,1 William A MacCrehan,2 Shin Muramoto,2 Gheorghe Stan21Department of Anatomy, Physiology and Pharmacology, Auburn University College of Veterinary Medicine, Auburn, AL, 2Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MA, USA Background: Small metal nanoparticles obtained from animal blood were observed to be toxic to cultured cancer cells, whereas noncancerous cells were much less affected. In this work, engineered zinc and copper metal nanoparticles were produced from bulk metal rods by an underwater high-voltage discharge method. The metal nanoparticles were characterized by atomic force microscopy and X-ray photoelectron spectroscopy. The metal nanoparticles, with estimated diameters of 1 nm–2 nm, were determined to be more than 85% nonoxidized. A cell viability assay and high-resolution light microscopy showed that exposure of RG2, cultured rat brain glioma cancer cells, to the zinc and copper nanoparticles resulted in cell morphological changes, including decreased cell adherence, shrinking/rounding, nuclear condensation, and budding from cell bodies. The metal-induced cell injuries were similar to the effects of staurosporine, an active apoptotic reagent. The viability experiments conducted for zinc and copper yielded values of dissociation constants of 0.22±0.08 nmol/L (standard error [SE] and 0.12±0.02 nmol/L (SE, respectively. The noncancerous astrocytes were not affected at the same conditions. Because metal nanoparticles were lethal to the cancer cells at sub-nanomolar concentrations, they are potentially important as nanomedicine.Purpose: Lethal concentrations of synthetic metal nanoparticles reported in the literature are a few orders of magnitude higher than the natural, blood-isolated metal nanoparticles; therefore, in this work, engineered metal nanoparticles were examined to mimic the properties of endogenous metal

  6. Stressing the ubiquitin-proteasome system without 20S proteolytic inhibition selectively kills cervical cancer cells.

    Directory of Open Access Journals (Sweden)

    Ravi K Anchoori

    Full Text Available Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate, and for specific signaling pathways, notably HPV E6-targeted degradation of p53 and PDZ proteins. Natural compounds with antioxidant properties including flavonoids and triterpenoids hold promise as anticancer agents by interfering with ubiquitin-dependent protein degradation. An increasing body of evidence indicates that their α-β unsaturated carbonyl system is the molecular determinant for inhibition of ubiquitin-mediated protein degradation up-stream of the catalytic sites of the 20S proteasome. Herein we report the identification and characterization of a new class of chalcone-based, potent and cell permeable chemical inhibitors of ubiquitin-dependent protein degradation, and a lead compound RAMB1. RAMB1 inhibits ubiquitin-dependent protein degradation without compromising the catalytic activities of the 20S proteasome, a mechanism distinct from that of Bortezomib. Treatment of cervical cancer cells with RAMB1 triggers unfolded protein responses, including aggresome formation and Hsp90 stabilization, and increases p53 steady state levels. RAMB1 treatment results in activation of lysosomal-dependent degradation pathways as a mechanism to compensate for increasing levels of poly-ubiquitin enriched toxic aggregates. Importantly, RAMB1 synergistically triggers cell death of cervical cancer cells when combined with the lysosome inhibitor Chloroquine.

  7. Cell killing by simian virus 40: impairment of membrane formation and function.

    Science.gov (United States)

    Norkin, L C

    1977-03-01

    Simian virus 40 infection of the CV-1 line of green monkey kidney cells results in the release of mitochondrial malic dehydrogenase as early as 24 h. Released malic dehydrogenase is detected in the cytoplasm prior to its appearance in the overlay medium. Infected cells lose the ability to consume oxygen between 48 and 56 h, and damage to the elctron transport system is indicated. Nevertheless, cellular ATP levels remain high as late as 72 h. Infection leads to a stimulation of membrane phospholipid synthesis, which reaches a peak at about 32 h. This is followed by a severe decline in new membrane synthesis, which correlates in time with the release of cytoplasmic lactic dehydrogenase into the overlay media. Lactic dehydrogenase release precedes the accumulation of trypan blue-stainable cells by about 6 h. Infection had no effect on the turnover of prelabeled membrane phospholipids. An early simian virus 40 mutant, tsA58, and a late mutant, tsB11, are both less effective than wild-type virus at causing reduced levels of phospholipid synthesis, enzyme release, and the accumulation of trypan blue-stainable cells. Another late mutant, tsB8, is similar to wild-type virus in these respects. At 64 h, there is no detectable cell-associated lactic dehydrogenase and nearly all the cells are trypan blue stainable. Nevertheless, at concentrations of deoxyglucose in the medium below the transport Km, deoxyglucose uptake was similar in infected and control cultures. With higher concentrations of deoxyglucose in the medium, uptake by the infected cultures exceeded that by the control cultures.

  8. Targeting the Human Complement Membrane Attack Complex to Selectively Kill Prostate Cancer Cells

    Science.gov (United States)

    2013-10-01

    tract through the urethra. It must also shield the sperm from immune destruction within the vaginal tract while not eliminating cells within the...first of which plays a role in enhancing B cell immunity. iC3b’s other receptor binding partners, CR3 and CR4, have roles in clearance of pathogens by... Nature 444: 213–216. 25. Wiesmann, C., K. J. Katschke, J. Yin, K. Y. Helmy, M. Steffek, W. J. Fairbrother, S. A. McCallum, L. Embuscado, L. DeForge, P

  9. Killing effect of Chinese hamster V79 cells exposed to accelerated carbon ions and RBE determination

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Survival curves of Chinese hamster V79 cells exposed to accclerated carbon ions with linear energy transfers of 125.5, 200 and 700 keV/μm were measured, respectively. Inactivation cross sections corresponding to the irradiation above were deduced from the V79 cell survival curves. They are 7.86±0.17, 10.44±1.11 and 32.32±3.58 μm2 in turn. With the surviving response of V79 cells to 60Co γ-rays as a reference value, relative biological effectiveness at 10%, 20%, 50% and 80% survival levels were given for the accelerated carbon ions. The results showed that carbon ions with LET of 125.5 keV/μm had a higher value of RBE at all the four survival levels than the carbon ions with other LETs. It was prompted that the maximum value of RBE for the V79 cell surviving as the biological endpoint emerged at the LET below 200 keV/μm for carbon ions.

  10. Killing effect of Chinese hamster V79 cells exposed to accelerated carbon ions and RBE determination

    Institute of Scientific and Technical Information of China (English)

    LIQiang; ZHOUGuang-Ming; 等

    2002-01-01

    Survival curves of Chinese hamster V79 cells exposed to accelerated carbon ions with linear energy transfers of 125.5,200 and 700keV/um were measured,respectively,Inactivation cross sections corresponding to the irradiation above were deduced from the V79 cell survival curves.They are 7.86±0.17,10.44±1.11 and 32.32±3.59um2 in turn.With the surviving response of V79 cells to 60Co γ-rays as a reference value,relative biological effectiveness at 10%,20%,50%and 80% survival levels were given for the accelerated carbon ions,The results showed that carbon ions with LET of 125.5keV/um had a higher value of RBE at all the four survival levels than the carbon ions with other LETs.It was prompted that the maximum value of RBE for the V79 cell surviving as the biological endpoint emerged at the LET below 200keV/um for carbon ions.

  11. Rapid and efficient cancer cell killing mediated by high-affinity death receptor homotrimerizing TRAIL variants.

    Science.gov (United States)

    Reis, C R; van der Sloot, A M; Natoni, A; Szegezdi, E; Setroikromo, R; Meijer, M; Sjollema, K; Stricher, F; Cool, R H; Samali, A; Serrano, L; Quax, W J

    2010-10-21

    The tumour necrosis factor family member TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of cancer cells through the activation of death receptors 4 (DR4) and 5 (DR5) and is considered a promising anticancer therapeutic agent. As apoptosis seems to occur primarily via only one of the two death receptors in many cancer cells, the introduction of DR selectivity is thought to create more potent TRAIL agonists with superior therapeutic properties. By use of a computer-aided structure-based design followed by rational combination of mutations, we obtained variants that signal exclusively via DR4. Besides an enhanced selectivity, these TRAIL-DR4 agonists show superior affinity to DR4, and a high apoptosis-inducing activity against several TRAIL-sensitive and -resistant cancer cell lines in vitro. Intriguingly, combined treatment of the DR4-selective variant and a DR5-selective TRAIL variant in cancer cell lines signalling by both death receptors leads to a significant increase in activity when compared with wild-type rhTRAIL or each single rhTRAIL variant. Our results suggest that TRAIL induced apoptosis via high-affinity and rapid-selective homotrimerization of each DR represent an important step towards an efficient cancer treatment.

  12. Glycan elongation beyond the mucin associated Tn antigen protects tumor cells from immune-mediated killing

    DEFF Research Database (Denmark)

    Mathiesen, Caroline Benedicte Kjærulff; Lavrsen, Kirstine; Wandall, Hans H.;

    2013-01-01

    Membrane bound mucins are up-regulated and aberrantly glycosylated during malignant transformation in many cancer cells. This results in a negatively charged glycoprotein coat which may protect cancer cells from immune surveillance. However, only limited data have so far demonstrated the critical...... steps in glycan elongation that make aberrantly glycosylated mucins affect the interaction between cancer cells and cytotoxic effector cells of the immune system. Tn (GalNAc-Ser/Thr), STn (NeuAcα2-6GalNAc-Ser/Thr), T (Galβ1–3GalNAc-Ser/Thr), and ST (NeuAcα2-6Galβ1–3GalNAc-Ser/Thr) antigens...... only the shortest possible mucin-like glycans (Tn and STn). Glyco-engineering was performed by zinc finger nuclease (ZFN) knockout (KO) of the Core 1 enzyme chaperone COSMC, thereby preventing glycan elongation beyond the initial GalNAc residue in O-linked glycans. We find that COSMC KO in the breast...

  13. Ag nanoparticles generated using bio-reduction and -coating cause microbial killing without cell lysis.

    Science.gov (United States)

    Gade, Aniket; Adams, Joshua; Britt, David W; Shen, Fen-Ann; McLean, Joan E; Jacobson, Astrid; Kim, Young-Cheol; Anderson, Anne J

    2016-04-01

    Cost-effective "green" methods of producing Ag nanoparticles (NPs) are being examined because of the potential of these NPs as antimicrobials. Ag NPs were generated from Ag ions using extracellular metabolites from a soil-borne Pythium species. The NPs were variable in size, but had one dimension less than 50 nm and were biocoated; aggregation and coating changed with acetone precipitation. They had dose-dependent lethal effects on a soil pseudomonad, Pseudomonas chlororaphis O6, and were about 30-fold more effective than Ag(+) ions. A role of reactive oxygen species in cell death was demonstrated by use of fluorescent dyes responsive to superoxide anion and peroxide accumulation. Also mutants of the pseudomonad, defective in enzymes that protect against oxidative stress, were more sensitive than the wild type strain; mutant sensitivity differed between exposure to Ag NPs and Ag(+) ions demonstrating a nano-effect. Imaging of bacterial cells treated with the biocoated Ag NPs revealed no cell lysis, but there were changes in surface properties and cell height. These findings support that biocoating the NPs results in limited Ag release and yet they retained potent antimicrobial activity.

  14. Photodynamic killing of cancer cells by a Platinum(II) complex with cyclometallating ligand

    Science.gov (United States)

    Doherty, Rachel E.; Sazanovich, Igor V.; McKenzie, Luke K.; Stasheuski, Alexander S.; Coyle, Rachel; Baggaley, Elizabeth; Bottomley, Sarah; Weinstein, Julia A.; Bryant, Helen E.

    2016-03-01

    Photodynamic therapy that uses photosensitizers which only become toxic upon light-irradiation provides a strong alternative to conventional cancer treatment due to its ability to selectively target tumour material without affecting healthy tissue. Transition metal complexes are highly promising PDT agents due to intense visible light absorption, yet the majority are toxic even without light. This study introduces a small, photostable, charge-neutral platinum-based compound, Pt(II) 2,6-dipyrido-4-methyl-benzenechloride, complex 1, as a photosensitizer, which works under visible light. Activation of the new photosensitizer at low concentrations (0.1-1 μM) by comparatively low dose of 405 nm light (3.6 J cm-2) causes significant cell death of cervical, colorectal and bladder cancer cell lines, and, importantly, a cisplatin resistant cell line EJ-R. The photo-index of the complex is 8. We demonstrate that complex 1 induces irreversible DNA single strand breaks following irradiation, and that oxygen is essential for the photoinduced action. Neither light, nor compound alone led to cell death. The key advantages of the new drug include a remarkably fast accumulation time (diffusion-controlled, minutes), and photostability. This study demonstrates a highly promising new agent for photodynamic therapy, and attracts attention to photostable metal complexes as viable alternatives to conventional chemotherapeutics, such as cisplatin.

  15. Photodynamic killing of cancer cells by a Platinum(II) complex with cyclometallating ligand.

    Science.gov (United States)

    Doherty, Rachel E; Sazanovich, Igor V; McKenzie, Luke K; Stasheuski, Alexander S; Coyle, Rachel; Baggaley, Elizabeth; Bottomley, Sarah; Weinstein, Julia A; Bryant, Helen E

    2016-03-04

    Photodynamic therapy that uses photosensitizers which only become toxic upon light-irradiation provides a strong alternative to conventional cancer treatment due to its ability to selectively target tumour material without affecting healthy tissue. Transition metal complexes are highly promising PDT agents due to intense visible light absorption, yet the majority are toxic even without light. This study introduces a small, photostable, charge-neutral platinum-based compound, Pt(II) 2,6-dipyrido-4-methyl-benzenechloride, complex 1, as a photosensitizer, which works under visible light. Activation of the new photosensitizer at low concentrations (0.1-1 μM) by comparatively low dose of 405 nm light (3.6 J cm(-2)) causes significant cell death of cervical, colorectal and bladder cancer cell lines, and, importantly, a cisplatin resistant cell line EJ-R. The photo-index of the complex is 8. We demonstrate that complex 1 induces irreversible DNA single strand breaks following irradiation, and that oxygen is essential for the photoinduced action. Neither light, nor compound alone led to cell death. The key advantages of the new drug include a remarkably fast accumulation time (diffusion-controlled, minutes), and photostability. This study demonstrates a highly promising new agent for photodynamic therapy, and attracts attention to photostable metal complexes as viable alternatives to conventional chemotherapeutics, such as cisplatin.

  16. Spontaneous human squamous cell carcinomas are killed by a human cytotoxic T lymphocyte clone recognizing a wild-type p53-derived peptide

    DEFF Research Database (Denmark)

    Röpke, M; Hald, J; Guldberg, Per

    1996-01-01

    A cytotoxic T lymphocyte (CTL) clone generated in vitro from the peripheral blood of a healthy HLA-A2-positive individual against a synthetic p53 protein-derived wild-type peptide (L9V) was shown to kill squamous carcinoma cell lines derived from two head and neck carcinomas, which expressed mutant...

  17. Specific killing of P53 mutated tumor cell lines by a cross-reactive human HLA-A2-restricted P53-specific CTL line

    DEFF Research Database (Denmark)

    Würtzen, P A; Pedersen, L O; Poulsen, H S;

    2001-01-01

    53(65-73) and p53(187-197) peptides was obtained in the T-cell lines. Interestingly, cold target inhibition experiments demonstrated that the simultaneous recognition of the 2 peptides was the result of cross-reactivity, which was confirmed by killing experiments at the clonal CTL level. Furthermore...

  18. HAMLET kills tumor cells by an apoptosis-like mechanism--cellular, molecular, and therapeutic aspects.

    Science.gov (United States)

    Svanborg, Catharina; Agerstam, Helena; Aronson, Annika; Bjerkvig, Rolf; Düringer, Caroline; Fischer, Walter; Gustafsson, Lotta; Hallgren, Oskar; Leijonhuvud, Irene; Linse, Sara; Mossberg, Ann-Kristin; Nilsson, Hanna; Pettersson, Jenny; Svensson, Malin

    2003-01-01

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a protein-lipid complex that induces apoptosis-like death in tumor cells, but leaves fully differentiated cells unaffected. This review summarizes the information on the in vivo effects of HAMLET in patients and tumor models on the tumor cell biology, and on the molecular characteristics of the complex. HAMLET limits the progression of human glioblastomas in a xenograft model and removes skin papillomas in patients. This broad anti-tumor activity includes >40 different lymphomas and carcinomas and apoptosis is independent of p53 or bcl-2. In tumor cells HAMLET enters the cytoplasm, translocates to the perinuclear area, and enters the nuclei where it accumulates. HAMLET binds strongly to histones and disrupts the chromatin organization. In the cytoplasm, HAMLET targets ribosomes and activates caspases. The formation of HAMLET relies on the propensity of alpha-lactalbumin to alter its conformation when the strongly bound Ca2+ ion is released and the protein adopts the apo-conformation that exposes a new fatty acid binding site. Oleic acid (C18:1,9 cis) fits this site with high specificity, and stabilizes the altered protein conformation. The results illustrate how protein folding variants may be beneficial, and how their formation in peripheral tissues may depend on the folding change and the availability of the lipid cofactor. One example is the acid pH in the stomach of the breast-fed child that promotes the formation of HAMLET. This mechanism may contribute to the protective effect of breastfeeding against childhood tumors. We propose that HAMLET should be explored as a novel approach to tumor therapy.

  19. Cocoa Extract Indicated Has Activity on Selectively Killing Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ariza Budi Tunjung Sari

    2015-09-01

    Full Text Available Effect of the cocoa crude extract on mortality of breast cancer cell lines i.e. MCF-7, T47D and normal cell (Vero, was observed. Crude cocoa extract prepared from a freshly dried cocoa bean that was containing 14% catechin and 0.6% caffeine. Catechin and caffeine content were modulated to 2-folds (28% catechin or 1.2% caffeine and 3-folds (42% catechin or 1.8% caffeine by adding pure compounds. Extracts were dissolved in dimethylsulfoxide (DMSO at concentrations ranging from 200 to 1600 μg/ml. The positive control was doxorubicin (0.5-16 μg/ml in DMSO. Cell lines (MCF-7, T47D, and Vero were incubated in test sample for 24h at 37°, prior to 3-(4,4-dimetylthiazole-2-yl-2,5-diphenyltetrazolium bromide (MTT assay. The absorbance of each well was measured at 550 nm, and lethal concentration (LC50 was calculated. The cocoa extract induced mortality of breast cancer cell lines but not in Vero cells. The effect on MCF-7 was greater than on T47D, given the LC50 was 1236 μg/ml (MCF-7 and 1893 μg/ml (T47D. Cytotoxic potential of cocoa extract was much lower than doxorubicin whose LC50 was 0,777 μg/ml (MCF-7 and 0,082 μg/ml (T47D. Increasing catechin content to 2-folds did not significantly affect LC50 value, but 3-folds catechin content reduced LC50 to 1021 μg/ml. Meanwhile increasing caffeine content to 2-folds significantly reduced LC50 to 750 μg/ml, however, 3-fold content resulted in slightly higher LC50 at 780 μg/ml. This indicates that cocoa extract have anti-cancer potential, and purification may improve this property.

  20. Selective killing of cancer cells by iron oxide nanoparticles mediated through reactive oxygen species via p53 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Ahamed, Maqusood, E-mail: maqusood@gmail.com; Alhadlaq, Hisham A.; Khan, M. A. Majeed [King Saud University, King Abdullah Institute for Nanotechnology (Saudi Arabia); Akhtar, Mohd. Javed [University of Lucknow, Department of Zoology (India)

    2013-01-15

    Iron oxide (Fe{sub 3}O{sub 4}) nanoparticles (NPs) are increasingly recognized for their utility in biomedical applications. However, little is known about the anticancer activity of Fe{sub 3}O{sub 4} NPs. This study was designed to investigate whether Fe{sub 3}O{sub 4} NPs induced toxicity in a cell-specific manner and determine the possible mechanisms of toxicity caused by Fe{sub 3}O{sub 4} NPs in cancer cells. Fe{sub 3}O{sub 4} NPs used in this study were synthesized by green method using {alpha}-d-glucose as a reducing agent. Prepared Fe{sub 3}O{sub 4} NPs were spherical in shape with a smooth surface, were fairly distributed, and had an average diameter of 23 nm. Cytotoxicity of Fe{sub 3}O{sub 4} NPs was examined against two types of cancer cells (human hepatocellular carcinoma HepG2 and human lung adenocarcinoma A549) and two normal cells (human lung fibroblast IMR-90 and rat hepatocytes). Fe{sub 3}O{sub 4} NPs exerted distinct effects on cell viability via killing of cancer cells while posing no toxicity on normal cells. Fe{sub 3}O{sub 4} NPs were found to induce depletion of glutathione and induction of reactive oxygen species (ROS) in both types of cancer cells (HepG2 and A549). Further, co-exposure of ascorbic acid significantly attenuated the Fe{sub 3}O{sub 4} NPs-induced oxidative stress. The mRNA levels of tumor suppressor gene p53 and apoptotic genes (caspase-3 and caspase-9) were up-regulated in both types of cancer cells due to Fe{sub 3}O{sub 4} NPs exposure. Protein level of p53, along with the higher activity of caspase-3 and caspase-9 enzymes, was also up-regulated by Fe{sub 3}O{sub 4} NPs. Taken together, our data demonstrated that Fe{sub 3}O{sub 4} NPs selectively induced apoptosis in cancer cells (HepG2 and A549) through up-regulation of p53 that might be mediated by ROS through which most of the anticancer drugs trigger apoptosis. The present study warrants further investigation on anticancer activity of Fe{sub 3}O{sub 4} NPs in relevant

  1. Selective killing of cancer cells by iron oxide nanoparticles mediated through reactive oxygen species via p53 pathway

    Science.gov (United States)

    Ahamed, Maqusood; Alhadlaq, Hisham A.; Khan, M. A. Majeed; Akhtar, Mohd. Javed

    2013-01-01

    Iron oxide (Fe3O4) nanoparticles (NPs) are increasingly recognized for their utility in biomedical applications. However, little is known about the anticancer activity of Fe3O4 NPs. This study was designed to investigate whether Fe3O4 NPs induced toxicity in a cell-specific manner and determine the possible mechanisms of toxicity caused by Fe3O4 NPs in cancer cells. Fe3O4 NPs used in this study were synthesized by green method using α- d-glucose as a reducing agent. Prepared Fe3O4 NPs were spherical in shape with a smooth surface, were fairly distributed, and had an average diameter of 23 nm. Cytotoxicity of Fe3O4 NPs was examined against two types of cancer cells (human hepatocellular carcinoma HepG2 and human lung adenocarcinoma A549) and two normal cells (human lung fibroblast IMR-90 and rat hepatocytes). Fe3O4 NPs exerted distinct effects on cell viability via killing of cancer cells while posing no toxicity on normal cells. Fe3O4 NPs were found to induce depletion of glutathione and induction of reactive oxygen species (ROS) in both types of cancer cells (HepG2 and A549). Further, co-exposure of ascorbic acid significantly attenuated the Fe3O4 NPs-induced oxidative stress. The mRNA levels of tumor suppressor gene p53 and apoptotic genes (caspase-3 and caspase-9) were up-regulated in both types of cancer cells due to Fe3O4 NPs exposure. Protein level of p53, along with the higher activity of caspase-3 and caspase-9 enzymes, was also up-regulated by Fe3O4 NPs. Taken together, our data demonstrated that Fe3O4 NPs selectively induced apoptosis in cancer cells (HepG2 and A549) through up-regulation of p53 that might be mediated by ROS through which most of the anticancer drugs trigger apoptosis. The present study warrants further investigation on anticancer activity of Fe3O4 NPs in relevant animal models.

  2. A designed equine herpes thymidine kinase (EHV4 TK) variant improves ganciclovir-induced cell-killing.

    Science.gov (United States)

    McSorley, Theresa; Ort, Stephan; Monnerjahn, Christian; Konrad, Manfred

    2014-02-01

    The limitations of the ganciclovir (GCV)/herpes simplex virus thymidine kinase (HSV1 TK: EC 2.7.1.21) system as a suicide gene therapy approach have been extensively studied over the years. In our study, we focused on improving the cytotoxic profile of the GCV/equine herpes virus-4 thymidine kinase (EHV4 TK: EC 2.7.1.21) system. Our approach involved the structure-guided mutagenesis of EHV4 TK in order to switch its ability to preferentially phosphorylate the natural substrate deoxythymidine (dT) to that of GCV. We performed steady-state kinetic analysis, genetic complementation in a thymidine kinase-deficient Escherichia coli strain, isothermal titration calorimetry, and analysis of GCV-induced cell killing through generation of HEK 293 stable cell-lines expressing EHV4 TK mutants and wild-type EHV4 TK. We found that the EHV4 TK S144H-GFP mutant preferentially phosphorylates GCV and confers increased GCV-induced cytotoxicity compared to wild-type EHV4 TK.

  3. Identification of protective pneumococcal T(H17 antigens from the soluble fraction of a killed whole cell vaccine.

    Directory of Open Access Journals (Sweden)

    Kristin L Moffitt

    Full Text Available Mucosal or parenteral immunization with a killed unencapsulated pneumococcal whole cell antigen (WCA with an adjuvant protects mice from colonization by a T(H17 CD4+ cell-mediated mechanism. Using preparative SDS gels, we separated the soluble proteins that compose the WCA in order to identify fractions that were immunogenic and protective. We screened these fractions for their ability to stimulate IL-17A secretion from splenocytes obtained from mice immunized with WCA and adjuvant. We identified 12 proteins within the stimulatory fractions by mass spectrometry; these proteins were then cloned, recombinantly expressed and purified using an Escherichia coli expression system. The ability of these proteins to induce IL-17A secretion was then evaluated by stimulation of mouse splenocytes. Of the four most stimulatory proteins, three were protective in a mouse pneumococcal serotype 6B colonization model. This work thus describes a method for identifying immunogenic proteins from the soluble fraction of pneumococcus and shows that several of the proteins identified protect mice from colonization when used as mucosal vaccines. We propose that, by providing protection against pneumococcal colonization, one or more of these proteins may serve as components of a multivalent pneumococcal vaccine.

  4. Enhanced killing activity of regulatory T cells ameliorates inflammation and autoimmunity.

    Science.gov (United States)

    Askenasy, Nadir

    2013-08-01

    Regulatory T cells (Treg) are pivotal suppressor elements in immune homeostasis with potential therapeutic applications in inflammatory and autoimmune disorders. Using Treg as vehicles for targeted immunomodulation, a short-lived Fas-ligand (FasL) chimeric protein (killer Treg) was found efficient in preventing the progression of autoimmune insulitis in NOD mice, and amelioration of chronic colitis and graft versus host disease. The main mechanisms of disease suppression by killer Treg are: a) in the acute phase induction of apoptosis in effector cells at the site of inflammation decreases the pathogenic burden, and b) persistent increase in FoxP3⁺ Treg with variable CD25 co-expression induced by FasL sustains disease suppression over extended periods of time. Reduced sensitivity of Treg to receptor-mediated apoptosis under inflammatory conditions makes them optimal vehicles for targeted immunotherapy using apoptotic agents.

  5. Talking about killing: Cell Phones, Collective Action, and Insurgent Violence in Iraq

    Science.gov (United States)

    2011-05-31

    technology, insurgency, collective action Jacob Shapiro, Nils Weidmann Harvard University Office of Sponsored Research 1350 Massachusetts Ave. Holyoke 727...at the district level changes as a result of the introduction of new cell phone towers. Second, using a novel identification strategy, we examine how... identification strategy, we examine how insurgent operation in the tower’s vicinity is affected by the introduction of coverage. Taken together, our

  6. D-polyglutamine amyloid recruits L-polyglutamine monomers and kills cells

    Science.gov (United States)

    Kar, Karunakar; Arduini, Irene; Drombosky, Kenneth W.; van der Wel, Patrick C. A.; Wetzel, Ronald

    2014-01-01

    Polyglutamine (polyQ) amyloid fibrils are observed in disease tissue and have been implicated as toxic agents responsible for neurodegeneration in expanded CAG repeat diseases like Huntington’s disease (HD). Despite intensive efforts, the mechanism of amyloid toxicity remains unknown. As a novel approach to probing polyQ toxicity, we investigate here how some cellular and physical properties of polyQ amyloid vary with the chirality of the glutamine residues in the polyQ. We challenged PC12 cells with small amyloid fibrils composed of either L- or D-polyQ peptides and found that D-fibrils are as cytotoxic as L-fibrils. We also found using fluorescence microscopy that both aggregates effectively seed the aggregation of cell-produced L-polyQ proteins, suggesting a surprising lack of stereochemical restriction in seeded elongation of polyQ amyloid. To investigate this effect further, we studied chemically synthesized D- and L-polyQ in vitro. We found that, as expected, D-polyQ monomers are not recognized by proteins that recognize L-polyQ monomers. However, amyloid fibrils prepared from D-polyQ peptides can efficiently seed the aggregation of L-polyQ monomers in vitro, and vice versa. This result is consistent with our cell results on polyQ recruitment, but is inconsistent with previous literature reports on the chiral specificity of amyloid seeding. This chiral cross-seeding can be rationalized by a model for seeded elongation featuring a “rippled β-sheet” interface between seed fibril and docked monomers of opposite chirality. The lack of chiral discrimination in polyQ amyloid cytotoxicity is consistent with several toxicity mechanisms, including recruitment of cellular polyQ proteins. PMID:24291210

  7. Chaperone-Targeting Cytotoxin and Endoplasmic Reticulum Stress-Inducing Drug Synergize to Kill Cancer Cells

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    Joseph M. Backer

    2009-11-01

    Full Text Available Diverse physiological and therapeutic insults that increase the amount of unfolded or misfolded proteins in the endoplasmic reticulum (ER induce the unfolded protein response, an evolutionarily conserved protective mechanism that manages ER stress. Glucose-regulated protein 78/immunoglobulin heavy-chain binding protein (GRP78/BiP is an ER-resident protein that plays a central role in the ER stress response and is the only known substrate of the proteolytic A subunit (SubA of a novel bacterial AB5 toxin. Here, we report that an engineered fusion protein, epidermal growth factor (EGF-SubA, combining EGF and SubA, is highly toxic to growing and confluent epidermal growth factor receptor-expressing cancer cells, and its cytotoxicity is mediated by a remarkably rapid cleavage of GRP78/BiP. Systemic delivery of EGF-SubA results in a significant inhibition of human breast and prostate tumor xenografts in mouse models. Furthermore, EGF-SubA dramatically increases the sensitivity of cancer cells to the ER stress-inducing drug thapsigargin, and vice versa, demonstrating the first example of mechanism-based synergism in the action of a cytotoxin and an ER-targeting drug.

  8. Cisplatin in 5% Ethanol Eradicates Cisplatin-Resistant Lung Tumor by Killing Lung Cancer Side Population (SP Cells and Non-SP Cells

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    Qi eNiu

    2013-08-01

    Full Text Available Cancer side population (SP cells with cancer stem cell-like properties are thought to be responsible for lung cancer chemotherapy resistance and currently no drug can efficiently target them. Breast cancer resistance protein (BRCP/ABCG2 is a major drug transporter in protecting lung cancer SP cells from cytotoxic agents. We showed that a low concentration of ethanol, which inhibits many membrane proteins, inhibits ABCG2 in lung cancer SP cells. Furthermore, cytotoxic cisplatin (DDP in 5% (vol/vol ethanol kills SP plus non-SP cancer cells better than either treatment alone in eradicating chemoresistant lung tumors. We found that 5% ethanol did not reduce ABCG2 protein levels, but significantly reduced ABCG2 protein function by a Hoechst 33342 extrusion assay, an ATPase activity assay, and transmission electron microscopy. Further, DDP in 5% ethanol (5% ethanol-DDP induced apoptosis of the SP plus non-SP cancer cells both in vitro and in vivo. In DDP-resistant A549/DDP lung tumor-bearing Balb/C nude mice, intratumoral injection of 5% ethanol-DDP regressed tumors and significantly improved survivals compared with 5% ethanol, DDP alone, or control. Intratumoral injection of 5% ethanol-DDP helped eradicate tumors in 30% (3/10 of the mice after 4 weeks treatment. By killing SP and non-SP cancer cells, 5% ethanol-DDP could eradicate DDP-resistant lung tumor and extend survival, providing a novel way to improve chemoresistant lung cancer survival for clinic.

  9. The Effects of Arolycoricidine and Narciprimine on Tumor Cell Killing and Topoisomerase Activity

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    Buket Bozkurt Sarikaya

    2012-07-01

    Full Text Available In this study, narciprimine and arolycoricidine were isolated from G. rizehensis Stern (Amaryllidaceae. The structures of the alkaloids were elucidated by spectroscopic methods (1D NMR, EI-MS. Due to the previous reports on anti-cancer activity of this group of alkaloids, we investigated their effects on DNA topoisomerase reactions, which are known as the cellular targets of a number of chemotherapeutical drugs. The results revealed that arolycoricidine and narciprimine were effective in both type I and type II DNA topoisomerase reactions in a dose-dependent manner. Topoisomerase-interfering ability of these alkaloids partially correlated with cytostaticity assays, using HeLa (cervix adenocarcinoma, MCF7 (breast adenocarcinoma and A431 (skin epidermoid carcinoma cells. Our results are discussed in relation to the potential significance of these alkaloids in the course of drug-development studies.

  10. Resveratrol and arsenic trioxide act synergistically to kill tumor cells in vitro and in vivo.

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    Xiao-Yan Zhao

    Full Text Available BACKGROUND AND AIMS: Arsenic trioxide (As2O3, which used as an effective agent in the treatment of leukaemia and other solid tumors, is largely limited by its toxicity. QT prolongation, torsades de pointes and sudden heart death have been implicated in the cardiotoxicity of As2O3. The present study was designed to explore whether the combination of As2O3 and resveratrol could generate a more powerful anti-cancer effect both in vitro and in vivo. MATERIALS AND METHODS: MTT assay was performed to assess the proliferation of Hela, MCF-7 and NB4 cells. Isobolographic analysis was used to evaluate combination index values from cell viability data. The apoptosis and the cellular reactive oxygen species (ROS level were assessed by fluorescent microscopy and flow cytometry separately in vitro. The effect of As2O3, alone and in combination with resveratrol on Hela tumor growth in an orthotopic nude mouse model was also investigated. The tumor volume and the immunohistochemical analysis of CD31, CD34 and VEGF were determined. RESULTS: Resveratrol dramatically enhanced the anti-cancer effect induced by As2O3 in vitro. In addition, isobolographic analysis further demonstrated that As2O3 and resveratrol generated a synergistic action. More apoptosis and ROS generation were observed in the combination treatment group. Similar synergistic effects were found in nude mice in vivo. The combination of As2O3 and resveratrol dramatically suppressed both tumor growth and angiogenesis in nude mice. CONCLUSIONS: Combining As2O3 with resveratrol would be a novel strategy to treat cancer in clinical practice.

  11. Bacteriophage-derived CHAP domain protein, P128, kills Staphylococcus cells by cleaving interpeptide cross-bridge of peptidoglycan.

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    Sundarrajan, Sudarson; Raghupatil, Junjappa; Vipra, Aradhana; Narasimhaswamy, Nagalakshmi; Saravanan, Sanjeev; Appaiah, Chemira; Poonacha, Nethravathi; Desai, Srividya; Nair, Sandhya; Bhatt, Rajagopala Narayana; Roy, Panchali; Chikkamadaiah, Ravisha; Durgaiah, Murali; Sriram, Bharathi; Padmanabhan, Sriram; Sharma, Umender

    2014-10-01

    P128 is an anti-staphylococcal protein consisting of the Staphylococcus aureus phage-K-derived tail-associated muralytic enzyme (TAME) catalytic domain (Lys16) fused with the cell-wall-binding SH3b domain of lysostaphin. In order to understand the mechanism of action and emergence of resistance to P128, we isolated mutants of Staphylococcus spp., including meticillin-resistant Staphylococcus aureus (MRSA), resistant to P128. In addition to P128, the mutants also showed resistance to Lys16, the catalytic domain of P128. The mutants showed loss of fitness as shown by reduced rate of growth in vitro. One of the mutants tested was found to show reduced virulence in animal models of S. aureus septicaemia suggesting loss of fitness in vivo as well. Analysis of the antibiotic sensitivity pattern showed that the mutants derived from MRSA strains had become sensitive to meticillin and other β-lactams. Interestingly, the mutant cells were resistant to the lytic action of phage K, although the phage was able to adsorb to these cells. Sequencing of the femA gene of three P128-resistant mutants showed either a truncation or deletion in femA, suggesting that improper cross-bridge formation in S. aureus could be causing resistance to P128. Using glutathione S-transferase (GST) fusion peptides as substrates it was found that both P128 and Lys16 were capable of cleaving a pentaglycine sequence, suggesting that P128 might be killing S. aureus by cleaving the pentaglycine cross-bridge of peptidoglycan. Moreover, peptides corresponding to the reported cross-bridge of Staphylococcus haemolyticus (GGSGG, AGSGG), which were not cleaved by lysostaphin, were cleaved efficiently by P128. This was also reflected in high sensitivity of S. haemolyticus to P128. This showed that in spite of sharing a common mechanism of action with lysostaphin, P128 has unique properties, which allow it to act on certain lysostaphin-resistant Staphylococcus strains.

  12. A multifunctional probe with aggregation-induced emission characteristics for selective fluorescence imaging and photodynamic killing of bacteria over mammalian cells.

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    Gao, Meng; Hu, Qinglian; Feng, Guangxue; Tomczak, Nikodem; Liu, Rongrong; Xing, Bengang; Tang, Ben Zhong; Liu, Bin

    2015-04-02

    A multifunctional probe aggregation-induced emission-Zinc(II)-dipicolylamine (AIE-ZnDPA) is developed for selective targeting, fluorescence imaging, and photodynamic killing of both Gram-positive and Gram-negative bacteria over mammalian cells. The probe has significant advantages in simple probe design, enhanced fluorescence upon bacteria binding, excellent photostability, and broad-spectrum antibacterial activity with almost no harm to mammalian cells.

  13. The engineered thymidylate kinase (TMPK)/AZT enzyme-prodrug axis offers efficient bystander cell killing for suicide gene therapy of cancer.

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    Sato, Takeya; Neschadim, Anton; Lavie, Arnon; Yanagisawa, Teruyuki; Medin, Jeffrey A

    2013-01-01

    We previously described a novel suicide (or 'cell fate control') gene therapy enzyme/prodrug system based on an engineered variant of human thymidylate kinase (TMPK) that potentiates azidothymidine (AZT) activation. Delivery of a suicide gene sequence into tumors by lentiviral transduction embodies a cancer gene therapy that could employ bystander cell killing as a mechanism driving significant tumor regression in vivo. Here we present evidence of a significant bystander cell killing in vitro and in vivo mediated by the TMPK/AZT suicide gene axis that is reliant on the formation of functional gap-junctional intercellular communications (GJICs). Potentiation of AZT activation by the engineered TMPK expressed in the human prostate cancer cell line, PC-3, resulted in effective bystander killing of PC-3 cells lacking TMPK expression--an effect that could be blocked by the GJIC inhibitor, carbenoxolone. Although GJICs are mainly formed by connexins, a new family of GJIC molecules designated pannexins has been recently identified. PC-3 cells expressed both connexin43 (Cx43) and Pannexin1 (Panx1), but Panx1 expression predominated at the plasma membrane, whereas Cx43 expression was primarily localized to the cytosol. The contribution of bystander effects to the reduction of solid tumor xenografts established by the PC-3 cell line was evaluated in an animal model. We demonstrate the contribution of bystander cell killing to tumor regression in a xenograft model relying on the delivery of expression of the TMPK suicide gene into tumors via direct intratumoral injection of recombinant therapeutic lentivirus. Taken together, our data underscore that the TMPK/AZT enzyme-prodrug axis can be effectively utilized in suicide gene therapy of solid tumors, wherein significant tumor regression can be achieved via bystander effects mediated by GJICs.

  14. The engineered thymidylate kinase (TMPK/AZT enzyme-prodrug axis offers efficient bystander cell killing for suicide gene therapy of cancer.

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    Takeya Sato

    Full Text Available We previously described a novel suicide (or 'cell fate control' gene therapy enzyme/prodrug system based on an engineered variant of human thymidylate kinase (TMPK that potentiates azidothymidine (AZT activation. Delivery of a suicide gene sequence into tumors by lentiviral transduction embodies a cancer gene therapy that could employ bystander cell killing as a mechanism driving significant tumor regression in vivo. Here we present evidence of a significant bystander cell killing in vitro and in vivo mediated by the TMPK/AZT suicide gene axis that is reliant on the formation of functional gap-junctional intercellular communications (GJICs. Potentiation of AZT activation by the engineered TMPK expressed in the human prostate cancer cell line, PC-3, resulted in effective bystander killing of PC-3 cells lacking TMPK expression--an effect that could be blocked by the GJIC inhibitor, carbenoxolone. Although GJICs are mainly formed by connexins, a new family of GJIC molecules designated pannexins has been recently identified. PC-3 cells expressed both connexin43 (Cx43 and Pannexin1 (Panx1, but Panx1 expression predominated at the plasma membrane, whereas Cx43 expression was primarily localized to the cytosol. The contribution of bystander effects to the reduction of solid tumor xenografts established by the PC-3 cell line was evaluated in an animal model. We demonstrate the contribution of bystander cell killing to tumor regression in a xenograft model relying on the delivery of expression of the TMPK suicide gene into tumors via direct intratumoral injection of recombinant therapeutic lentivirus. Taken together, our data underscore that the TMPK/AZT enzyme-prodrug axis can be effectively utilized in suicide gene therapy of solid tumors, wherein significant tumor regression can be achieved via bystander effects mediated by GJICs.

  15. "Kill" the messenger: Targeting of cell-derived microparticles in lupus nephritis.

    Science.gov (United States)

    Nielsen, Christoffer T; Rasmussen, Niclas S; Heegaard, Niels H H; Jacobsen, Søren

    2016-07-01

    Immune complex (IC) deposition in the glomerular basement membrane (GBM) is a key early pathogenic event in lupus nephritis (LN). The clarification of the mechanisms behind IC deposition will enable targeted therapy in the future. Circulating cell-derived microparticles (MPs) have been proposed as major sources of extracellular autoantigens and ICs and triggers of autoimmunity in LN. The overabundance of galectin-3-binding protein (G3BP) along with immunoglobulins and a few other proteins specifically distinguish circulating MPs in patients with systemic lupus erythematosus (SLE), and this is most pronounced in patients with active LN. G3BP co-localizes with deposited ICs in renal biopsies from LN patients supporting a significant presence of MPs in the IC deposits. G3BP binds strongly to glomerular basement membrane proteins and integrins. Accordingly, MP surface proteins, especially G3BP, may be essential for the deposition of ICs in kidneys and thus for the ensuing formation of MP-derived electron dense structures in the GBM, and immune activation in LN. This review focuses on the notion of targeting surface molecules on MPs as an entirely novel treatment strategy in LN. By targeting MPs, a double hit may be achieved by attenuating both the autoantigenic fueling of immune complexes and the triggering of the adaptive immune system. Thereby, early pathogenic events may be blocked in contrast to current treatment strategies that primarily target and modulate later events in the cellular and humoral immune response.

  16. Drug embedded PVP coated magnetic nanoparticles for targeted killing of breast cancer cells.

    Science.gov (United States)

    Rose, P Arsula; Praseetha, P K; Bhagat, Madhulika; Alexander, Princy; Abdeen, Sunitha; Chavali, Murthy

    2013-10-01

    Magnetic drug targeting is a drug delivery system that can be used in loco-regional cancer treatment. Coated magnetic particles, called carriers, are very useful for delivering chemotherapeutic drugs. Magnetic carriers were synthesized by co-precipitation of iron oxide followed by coating with polyvinyl pyrrolidone (PVP). Characterization was performed using X-ray diffraction, TEM, TGA, FTIR and UV-Vis Spectroscopy. Magnetite (Fe3O4) remained as the core of the carrier. The amount of PVP bound to the iron oxide nanoparticles was estimated by thermogravimetric analysis (TGA) and the attachment of PVP to the iron oxide nanoparticles confirmed by FTIR analysis. The loading efficiency of Epirubicin hydrochloride onto the PVP coated and uncoated iron oxide nanoparticles was measured at intervals such as 1 hr and 24 hrs by UV-Vis Spectroscopy. The binding of Epirubicin hydrochloride to the PVP coated and uncoated iron oxide nanoparticles were confirmed by FTIR analysis. The present findings showed that Epirubicin hydrochloride loaded PVP coated iron oxide nanoparticles are promising for magnetically targeted drug delivery. The drug displayed increased cell cytotoxicity at lower concentrations when conjugated with the nanoparticles than being administered conventionally as individual drugs.

  17. Enhanced killing of SCC17B human head and neck squamous cell carcinoma cells after photodynamic therapy plus fenretinide via the de novo sphingolipid biosynthesis pathway and apoptosis.

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    Boppana, Nithin B; Stochaj, Ursula; Kodiha, Mohamed; Bielawska, Alicja; Bielawski, Jacek; Pierce, Jason S; Korbelik, Mladen; Separovic, Duska

    2015-05-01

    Because photodynamic therapy (PDT) alone is not always effective as an anticancer treatment, PDT is combined with other anticancer agents for improved efficacy. The clinically-relevant fenretinide [N-(4-hydroxyphenyl) retinamide; 4HPR], was combined with the silicon phthalocyanine photosensitizer Pc4-mediated PDT to test for their potential to enhance killing of SCC17B cells, a clinically-relevant model of human head and neck squamous cell carcinoma. Because each of these treatments induces apoptosis and regulates the de novo sphingolipid (SL) biosynthesis pathway, the role of ceramide synthase, the pathway-associated enzyme, in PDT+4HPR-induced apoptotic cell death was determined using the ceramide synthase inhibitor fumonisin B1 (FB). PDT+4HPR enhanced loss of clonogenicity. zVAD-fmk, a pan-caspase inhibitor, and FB, protected cells from death post-PDT+4HPR. In contrast, the anti-apoptotic protein Bcl2 inhibitor ABT199 enhanced cell killing after PDT+4HPR. Combining PDT with 4HPR led to FB-sensitive, enhanced Bax associated with mitochondria and cytochrome c redistribution. Mass spectrometry data showed that the accumulation of C16-dihydroceramide, a precursor of ceramide in the de novo SL biosynthesis pathway, was enhanced after PDT+4HPR. Using quantitative confocal microscopy, we found that PDT+4HPR enhanced dihydroceramide/ceramide accumulation in the ER, which was inhibited by FB. The results suggest that SCC17B cells are sensitized to PDT by 4HPR via the de novo SL biosynthesis pathway and apoptosis, and imply potential clinical relevance of the combination for cancer treatment.

  18. A whole-genome RNAi screen uncovers a novel role for human potassium channels in cell killing by the parasite Entamoeba histolytica.

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    Marie, Chelsea; Verkerke, Hans P; Theodorescu, Dan; Petri, William A

    2015-09-08

    The parasite Entamoeba histolytica kills human cells resulting in ulceration, inflammation and invasion of the colonic epithelium. We used the cytotoxic properties of ameba to select a genome-wide RNAi library to reveal novel host factors that control susceptibility to amebic killing. We identified 281 candidate susceptibility genes and bioinformatics analyses revealed that ion transporters were significantly enriched among susceptibility genes. Potassium (K(+)) channels were the most common transporter identified. Their importance was further supported by colon biopsy of humans with amebiasis that demonstrated suppressed K(+) channel expression. Inhibition of human K(+) channels by genetic silencing, pharmacologic inhibitors and with excess K(+) protected diverse cell types from E. histolytica-induced death. Contact with E. histolytica parasites triggered K(+) channel activation and K(+) efflux by intestinal epithelial cells, which preceded cell killing. Specific inhibition of Ca(2+)-dependent K(+) channels was highly effective in preventing amebic cytotoxicity in intestinal epithelial cells and macrophages. Blockade of K(+) efflux also inhibited caspase-1 activation, IL-1β secretion and pyroptotic death in THP-1 macrophages. We concluded that K(+) channels are host mediators of amebic cytotoxicity in multiple cells types and of inflammasome activation in macrophages.

  19. Porphyromonas gingivalis Evasion of Autophagy and Intracellular Killing by Human Myeloid Dendritic Cells Involves DC-SIGN-TLR2 Crosstalk

    Science.gov (United States)

    El-Awady, Ahmed R.; Miles, Brodie; Scisci, Elizabeth; Kurago, Zoya B.; Palani, Chithra D.; Arce, Roger M.; Waller, Jennifer L.; Genco, Caroline A.; Slocum, Connie; Manning, Matthew; Schoenlein, Patricia V.; Cutler, Christopher W.

    2015-01-01

    Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs. PMID:25679217

  20. Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk.

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    Ahmed R El-Awady

    2015-02-01

    Full Text Available Signaling via pattern recognition receptors (PRRs expressed on professional antigen presenting cells, such as dendritic cells (DCs, is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs. We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

  1. Cell killing, nuclear damage and apoptosis in Chinese hamster V79 cells after irradiation with heavy-ion beams of (16)O, (12)C and (7)Li.

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    Pathak, Rupak; Dey, Subrata Kumar; Sarma, Asiti; Khuda-Bukhsh, Anisur Rahman

    2007-08-15

    Chinese hamster V79 cells were exposed to high LET (linear energy transfer) (16)O-beam (625keV/mum) radiation in the dose range of 0-9.83Gy. Cell survival, micronuclei (MN), chromosomal aberrations (CA) and induction of apoptosis were studied as a follow up of our earlier study on high LET radiations ((7)Li-beam of 60keV/mum and (12)C-beam of 295keV/mum) as well as (60)Co gamma-rays. Dose dependent decline in surviving fraction was noticed along with the increase of MN frequency, CA frequency as well as percentage of apoptosis as detected by nuclear fragmentation assay. The relative intensity of DNA ladder, which is a useful marker for the determination of the extent of apoptosis induction, was also increased in a dose dependent manner. Additionally, expression of tyrosine kinase lck-1 gene, which plays an important role in response to ionizing radiation induced apoptosis, was increased with the increase of radiation doses and also with incubation time. The present study showed that all the high LET radiations were generally more effective in cell killing and inflicting other cytogenetic damages than that of low LET gamma-rays. The dose response curves revealed that (7)Li-beam was most effective in cell killing as well as inducing other nuclear damages followed by (12)C, (16)O and (60)Co gamma-rays, in that order. The result of this study may have some application in biological dosimetry for assessment of genotoxicity in heavy ion exposed subjects and in determining suitable doses for radiotherapy in cancer patients where various species of heavy ions are now being generally used.

  2. Keyhole limpet hemocyanin augmented the killing activity, cytokine production and proliferation of NK cells, and inhibited the proliferation of Meth A sarcoma cells in vitro

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    Md. Moklesur Rahman Sarker

    2014-01-01

    Full Text Available Objective: Keyhole limpet hemocyanin (KLH is a popular tumor vaccine carrier protein and an immunostimulant. The present study aimed to investigate the immunoregulatory activity of KLH on cytotoxicity, cytokines production, and proliferation of natural killer (NK cells. Moreover, antiproliferative activity of KLH on Meth A sarcoma cells was studied. Materials and Methods: Cytotoxicity was determined with killing ability of NK cells against yeast artificial chromosome (YAC-1 cells. Interferon-gamma (IFN-γ and tumor necrosis factor-alpha (TNF-α productions by NK cells were measured by enzyme-linked immunosorbent assay (ELISA. Proliferations of NK and Meth A cells were determined by [ 3 H]thymidine incorporated proliferation and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT methods, respectively. Results: KLH at 6.25, 12.5, and 25 μg/well augmented cytotoxicity of NK cells against YAC-1 cells by 2.5, three, and five-times, respectively. KLH at 25 μg/well enhanced IFN-γ and TNF-α productions by 17- and 23-folds, respectively. The proliferation of NK cells was three times stimulated by KLH. The proliferation of Meth A cells was markedly inhibited by all the doses; the highest (4-folds higher inhibition was observed at a dose of KLH (25 μg/well. Conclusion: The study demonstrated the anticancer activity of KLH acting through the induction of NK cells and inhibition of cancer cells. KLH, therefore, may be a good candidate for an anticancer agent alone or in combination with other chemotherapeutic agents.

  3. Enhanced Expression of T-Cell Immunoglobulin and Mucin Domain Protein 3 in Endothelial Cells Facilitates Intracellular Killing of Rickettsia heilongjiangensis.

    Science.gov (United States)

    Yang, Xiaomei; Jiao, Jun; Han, Gencheng; Gong, Wenping; Wang, Pengcheng; Xiong, Xiaolu; Wen, Bohai

    2016-01-01

    Rickettsia heilongjiangensis is the pathogen of Far eastern spotted fever, and T-cell immunoglobulin and mucin domain protein 3 (Tim-3) is expressed in human vascular endothelial cells, the major target cells of rickettsiae. In the present study, we investigated the effects of altered Tim-3 expression in vivo in mice and in vitro in human endothelial cells, on day 3 after R. heilongjiangensis infection. Compared with corresponding controls, rickettsial burdens both in vivo and in vitro were significantly higher with blocked Tim-3 signaling or silenced Tim-3 and significantly lower with overexpressed Tim-3. Additionally, the expression of inducible nitric oxide synthase and interferon γ in endothelial cells with blocked Tim-3 signaling or silenced Tim-3 was significantly lower, while the expression of inducible nitric oxide synthase, interferon γ, and tumor necrosis factor α in transgenic mice with Tim-3 overexpression was significantly higher. These results reveal that enhanced Tim-3 expression facilitates intracellular rickettsial killing in a nitric oxide-dependent manner in endothelial cells during the early phase of rickettsial infection.

  4. Nuclear estrogen receptor targeted photodynamic therapy: selective uptake and killing of MCF-7 breast cancer cells by a C17alpha-alkynylestradiol-porphyrin conjugate.

    Science.gov (United States)

    Swamy, Narasimha; Purohit, Ajay; Fernandez-Gacio, Ana; Jones, Graham B; Ray, Rahul

    2006-10-15

    We hypothesized that over-expression of estrogen receptor (ER) in hormone-sensitive breast cancer could be harnessed synergistically with the tumor-migrating effect of porphyrins to selectively deliver estrogen-porphyrin conjugates into breast tumor cells, and preferentially kill the tumor cells upon exposure to red light. In the present work we synthesized four (4) conjugates of C17-alpha-alkynylestradiol and chlorin e6-dimethyl ester with varying tether lengths, and showed that all these conjugates specifically bound to recombinant ER alpha. In a cellular uptake assay with ER-positive MCF-7 and ER-negative MDA-MB 231 human breast cancer cell-lines, we observed that one such conjugate (E17-POR, XIV) was selectively taken up in a dose-dependent and saturable manner by MCF-7 cells, but not by MDA-MB 231 cells. Furthermore, MCF-7 cells, but not MDA-MB 231 cells, were selectively and efficiently killed by exposure to red light after incubation with E17-POR. Therefore, the combination approach, including drug and process modalities has the potential to be applied clinically for hormone-sensitive cancers in organs where ER is significantly expressed. This could potentially be carried out either as monotherapy involving a photo-induced selective destruction of tumor cells and/or adjuvant therapy in post-surgical treatment for the destruction of residual cancer cells in tissues surrounding the tumor.

  5. In vitro killing assays of antisera antibody sheep post-infected with Fasciola gigantica with the presence of macrophages cells against homologous and heterologous liver flukes

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    S.E Estuningsih

    1999-10-01

    Full Text Available The previous artificial infection known that the Indonesian Thin Tail (ITT sheep was resistance against the liver fluke of Fasciola gigantica, the resistances occurred in the early infection. In order to observe the immune resistance, some in vitro studies were undertaken in the laboratory, to assay the ability of the antisera antibody of ITT sheep post-infected with F. gigantica, with the presence of macrophages cells in killing the homologous and heterologous liver flukes. The viability of liver flukes were observed within 24-72 hours of incubation period by observing their motility (motile flukes were designated live and non-motile once were death. The results showed that after 72 hours incubation, the motilities of the Newly Excysted Juvenile (NEJ of F. gigantica incubated with the presence of post-infected sera and macrophages cells solution were significantly lower (P0.05. It seems that the occurrence of homologous antibody to the antigens is very important in the development of killing mechanism. The absence of homologous antibody did not reduce the number of flukes or the ability of macrophages cells in killing F. hepatica was not apparent.

  6. Immunity to Schistosoma mansoni in guinea-pigs vaccinated with radiation-attenuated cercariae. T-cell activation of macrophages for larval killing

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    Gordon, J.R.; McLaren, D.J.

    1988-02-01

    This study addresses macrophage activation in guinea-pigs vaccinated with radiation-attenuated cercariae of Schistosom mansoni. Peritoneal exudate macrophages elicited in vaccinated animals by mineral oil injection were activated to kill larval schistosomes in vitro. Killing efficiency is dependent upon the cell:target ratio employed and is enhanced by, but is not strictly dependent on, the presence of specific antibodies. Macrophages co-cultured with parasites release superoxide radicals and hydrogen peroxide, but the use of inhibitors has shown that neither of these reactive oxygen intermediates are the causal agents of cellular cytotoxicity in this system. Oil-elicited macrophages from naive guinea-pigs do not show comparable activation; they can, however, be activated in vitro by incubation with culture supernatant fluids from schistosome antigen-stimulated spleen, or lymph node cells harvested from vaccinated guinea-pigs. Naive macrophages activated in this way kill schistosomula in vitro and release the activation markers IL-l and superoxide anion. The macrophage-activating factor (MAF) present in spleen cell culture supernatant fluids has a MW of 35,000-55,000, but does not have the chemical characteristics of gamma-interferon.

  7. Comparative analysis of cell killing and autosomal mutation in mouse kidney epithelium exposed to 1 GeV/nucleon iron ions in vitro or in situ.

    Science.gov (United States)

    Kronenberg, Amy; Gauny, Stacey; Kwoh, Ely; Connolly, Lanelle; Dan, Cristian; Lasarev, Michael; Turker, Mitchell S

    2009-11-01

    Astronauts receive exposures to high-energy heavy ions from galactic cosmic radiation. Although high-energy heavy ions are mutagenic and carcinogenic, their mutagenic potency in epithelial cells, where most human cancers develop, is poorly understood. Mutations are a critical component of human cancer, and mutations involving autosomal loci predominate. This study addresses the cytotoxic and mutagenic effects of 1 GeV/nucleon iron ions in mouse kidney epithelium. Mutant fractions were measured for an endogenous autosomal locus (Aprt) that detects all types of mutagenic events contributing to human cancer. Results for kidneys irradiated in situ are compared with results for kidney cells from the same strain exposed in vitro. The results demonstrate dose-dependent cell killing in vitro and for cells explanted 3-4 months postirradiation in situ, but in situ exposures were less likely to result in cell death than in vitro exposures. Prolonged incubation in situ (8-9 months) further attenuated cell killing at lower doses. Iron ions were mutagenic to cells in vitro and for irradiated kidneys. No sparing was seen for mutant frequency with a long incubation period in situ. In addition, the degree of mutation induction (relative increase over background) was similar for cells exposed in vitro or in situ. We speculate that the latent effects of iron-ion exposure contribute to the maintenance of an elevated mutation burden in an epithelial tissue.

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

    Directory of Open Access Journals (Sweden)

    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

  9. Comparative analysis of cell killing and autosomal mutation in mouse kidney epithelium exposed to 1 GeV protons in vitro or in vivo.

    Science.gov (United States)

    Kronenberg, Amy; Gauny, Stacey; Kwoh, Ely; Grossi, Gianfranco; Dan, Cristian; Grygoryev, Dmytro; Lasarev, Michael; Turker, Mitchell S

    2013-05-01

    Human exposure to high-energy protons occurs in space flight scenarios or, where necessary, during radiotherapy for cancer or benign conditions. However, few studies have assessed the mutagenic effectiveness of high-energy protons, which may contribute to cancer risk. Mutations cause cancer and most cancer-associated mutations occur at autosomal loci. This study addresses the cytotoxic and mutagenic effects of 1 GeV protons in mouse kidney epithelium. Mutant fractions were measured for an endogenous autosomal locus (Aprt) that detects all types of mutagenic events. Results for kidneys irradiated in vivo are compared with the results for kidney cells from the same strain exposed in vitro. The results demonstrate dose-dependent cell killing in vitro and for cells explanted 3-4 months postirradiation in vivo. Incubation in vivo for longer periods (8-9 months) further attenuates proton-induced cell killing. Protons are mutagenic to cells in vitro and for in vivo irradiated kidneys. The dose-response for Aprt mutation is curvilinear after in vitro or in vivo exposure, bending upward at the higher doses. While the absolute mutant fractions are higher in vivo, the fold-increase over background is similar for both in vitro and in situ exposures. Results are also presented for a limited study on the effect of dose fractionation on the induction of Aprt mutations in kidney epithelial cells. Dose-fractionation reduces the fraction of proton-induced Aprt mutants in vitro and in vivo and also results in less cell killing. Taken together, the mutation burden in the epithelium is slightly reduced by dose-fractionation. Autosomal mutations accumulated during clinical exposure to high-energy protons may contribute to the risk of treatment-associated neoplasms, thereby highlighting the need for rigorous treatment planning to reduce the dose to normal tissues. For low dose exposures that occur during most space flight scenarios, the mutagenic effects of protons appear to be modest.

  10. Interaction between Salmonella typhimurium and phagocytic cells in pigs - Phagocytosis, oxidative burst and killing in polymorphonuclear leukocytes and monocytes

    DEFF Research Database (Denmark)

    Riber, Ulla; Lind, Peter

    1999-01-01

    Interactions between Salmonella typhimurium and peripheral blood leucocytes from healthy, Salmonella-free pigs were investigated in vitro. Both granulocytes and monocytes phagocytized FITC-labelled heat-killed Salmonella bacteria as shown by flow cytometry. Phagocytosis in whole blood and isolated...

  11. Cryptococcus Neoformans Modulates Extracellular Killing by Neutrophils

    OpenAIRE

    Qureshi, Asfia; Grey, Angus; Rose, Kristie L; Schey, Kevin L.; Del Poeta, Maurizio

    2011-01-01

    We recently established a key role for host sphingomyelin synthase (SMS) in regulating the killing activity of neutrophils against Cryptococcus neoformans. In this paper, we studied the effect of C. neoformans on the killing activity of neutrophils and whether SMS would still be a player against C. neoformans in immunocompromised mice lacking T and natural killer (NK) cells (Tgε26 mice). To this end, we analyzed whether C. neoformans would have any effect on neutrophil survival and killing in...

  12. Killing of Sarcoma Cells by Proapoptotic Bcl-XS: Role of the BH3 Domain and Regulation by Bcl-XL

    Directory of Open Access Journals (Sweden)

    Raj. S. Mitra

    2001-01-01

    Full Text Available Kaposi's sarcoma (KS is the most common tumor affecting AIDS patients with over 20% of these patients afflicted by this disease. Previous studies have demonstrated that KS tumor cells predominantly express the prosurvival protein Bcl-XL compared with Bcl-2. In the current study, we have used an adenoviral vector that expresses Bcl-XS, a functional inhibitor of Bcl-XL, to study the significance of Bcl-XL expression in the KS cell line (SLK or KS primary cultures. The results demonstrate that 75% to 80% of SLK or KS primary cells were killed by the Bcl-XS containing adenovirus whereas KS cells infected with control adenovirus showed no significant cell death or growth inhibition. Overexpression of Bcl-XL, but not Bcl-2, in SILK cells attenuated apoptosis induced by adenovirus Bcl-XS. Immunoprecipitation experiments revealed that adenoviral Bcl-XS associated with Bcl-XL, but not with Bcl-2. Mutational analysis showed that the α2 helical region of Bcl-XS containing the BH3 motif was critical for killing activity and interaction with Bcl-XL. These results suggest that Bcl-XS is a direct killer and Bcl-XL may act by interacting with and sequestering Bcl-XS. These studies also suggest that targeting Bcl-XL may be of therapeutic benefit for the treatment of tumors that are characterized by inappropriate expression of Bcl-XL.

  13. The proteins (12 and 15 kDa) isolated from heat-killed Lactobacillus plantarum L67 induces apoptosis in HT-29 cells.

    Science.gov (United States)

    Song, S; Oh, S; Lim, K T

    2015-03-01

    A number of scientific studies have revealed that Lactobacillus strains have beneficial bioactivities in the gastrointestinal tract. In this study, the production of intracellular reactive oxygen species (ROS) and the amounts of intracellular calcium, protein kinase C activity, cytochrome c, Bid, Bcl-2, Bax and the apoptosis-mediated proteins [caspase-8, caspase-3 and poly ADP ribose polymerase (PARP)] were evaluated to understand the induction of programmed cell death in HT-29 cells by Lactobacillus plantarum L67. The results obtained from this study indicated that the relative intensities of the apoptotic-related factors (intracellular ROS and intracellular calcium) and of apoptotic signals (Bax and t-Bid) increased with increasing concentrations of the membrane proteins isolated from heat-killed L. plantarum L67, whereas the relative intensities of cytochrome c, Bcl-2, caspase-8, caspase-3 and PARP decreased. This study determines whether proteins (12 and 15 kDa) isolated from heat-killed L. plantarum L67 induce programmed cell death in HT-29 cells. Proteins isolated from L. plantarum L67 can stimulate the apoptotic signals and then consequently induce programmed cell death in HT-29 cells. The results in this study suggest that the proteins isolated from L. plantarum L67 could be used as an antitumoural agent in probiotics and as a component of supplements or health foods.

  14. Human papillomavirus E6E7-mediated adenovirus cell killing: selectivity of mutant adenovirus replication in organotypic cultures of human keratinocytes.

    Science.gov (United States)

    Balagué, C; Noya, F; Alemany, R; Chow, L T; Curiel, D T

    2001-08-01

    Replication-competent adenoviruses are being investigated as potential anticancer agents. Exclusive virus replication in cancer cells has been proposed as a safety trait to be considered in the design of oncolytic adenoviruses. From this perspective, we have investigated several adenovirus mutants for their potential to conditionally replicate and promote the killing of cells expressing human papillomavirus (HPV) E6 and E7 oncoproteins, which are present in a high percentage of anogenital cancers. For this purpose, we have employed an organotypic model of human stratified squamous epithelium derived from primary keratinocytes that have been engineered to express HPV-18 oncoproteins stably. We show that, whereas wild-type adenovirus promotes a widespread cytopathic effect in all infected cells, E1A- and E1A/E1B-deleted adenoviruses cause no deleterious effect regardless of the coexpression of HPV18 E6E7. An adenovirus deleted in the CR2 domain of E1A, necessary for binding to the pRB family of pocket proteins, shows no selectivity of replication as it efficiently kills all normal and E6E7-expressing keratinocytes. Finally, an adenovirus mutant deleted in the CR1 and CR2 domains of E1A exhibits preferential replication and cell killing in HPV E6E7-expressing cultures. We conclude that the organotypic keratinocyte culture represents a distinct model to evaluate adenovirus selectivity and that, based on this model, further modifications of the adenovirus genome are required to restrict adenovirus replication to tumor cells.

  15. Combining Heavy Ion Radiation and Artificial MicroRNAs to Target the Homologous Recombination Repair Gene Efficiently Kills Human Tumor Cells

    Energy Technology Data Exchange (ETDEWEB)

    Zheng Zhiming [Department of Neurosurgery, Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan (China); Department of Radiation Oncology, School of Medicine, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States); Wang Ping; Wang Hongyan; Zhang Xiangming [Department of Radiation Oncology, School of Medicine, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States); Wang Minli [Division of Life Sciences, Universities Space Research Association, Houston, Texas (United States); Cucinotta, Francis A. [National Aeronautics and Space Administration, Lyndon B. Johnson Space Center, Houston, Texas (United States); Wang Ya, E-mail: ywang94@emory.edu [Department of Radiation Oncology, School of Medicine, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States)

    2013-02-01

    Purpose: Previously, we demonstrated that heavy ions kill more cells at the same dose than X-rays because DNA-clustered lesions produced by heavy ions affect nonhomologous end-joining (NHEJ) repair but not homologous recombination repair (HRR). We have also shown that our designed artificial microRNAs (amiRs) could efficiently target XRCC4 (an essential factor for NHEJ) or XRCC2 (an essential factor for HRR) and sensitize human tumor cells to X-rays. Based on these data, we were interested in testing the hypothesis that combining heavy ions and amiRs to target HRR but not NHEJ should more efficiently kill human tumor cells. Methods and Materials: Human tumor cell lines (U87MG, a brain tumor cell line, and A549, a lung cancer cell line) and their counterparts, overexpressed with amiR to target XRCC2, XRCC4 or both, were used in this study. Survival sensitivities were examined using a clonogenic assay after these cells were exposed to X-rays or heavy ions. In addition, these cell lines were subcutaneously injected into nude mice to form xenografts and the tumor size was compared after the tumor areas were exposed to X-rays or heavy ions. Results: Although targeting either XRCC4 (NHEJ factor) or XRCC2 (HRR factor) sensitized the human tumor cells to X-rays, in vitro and the xenograft animal model, targeting only XRCC2 but not XRCC4 sensitized the human tumor cells to heavy ions in vitro and in the xenograft animal model. Conclusions: Combining heavy ions with targeting the HRR pathway, but not the NHEJ pathway, could significantly improve the efficiency of tumor cell death.

  16. Afa/Dr diffusely adhering Escherichia coli strain C1845 induces neutrophil extracellular traps that kill bacteria and damage human enterocyte-like cells.

    Science.gov (United States)

    Marin-Esteban, Viviana; Turbica, Isabelle; Dufour, Guillaume; Semiramoth, Nicolas; Gleizes, Aude; Gorges, Roseline; Beau, Isabelle; Servin, Alain L; Lievin-Le Moal, Vanessa; Sandré, Catherine; Chollet-Martin, Sylvie

    2012-05-01

    We recently documented the neutrophil response to enterovirulent diffusely adherent Escherichia coli expressing Afa/Dr fimbriae (Afa/Dr DAEC), using the human myeloid cell line PLB-985 differentiated into fully mature neutrophils. Upon activation, particularly during infections, neutrophils release neutrophil extracellular traps (NETs), composed of a nuclear DNA backbone associated with antimicrobial peptides, histones, and proteases, which entrap and kill pathogens. Here, using fluorescence microscopy and field emission scanning electron microscopy, we observed NET production by PLB-985 cells infected with the Afa/Dr wild-type (WT) E. coli strain C1845. We found that these NETs were able to capture, immobilize, and kill WT C1845 bacteria. We also developed a coculture model of human enterocyte-like Caco-2/TC7 cells and PLB-985 cells previously treated with WT C1845 and found, for the first time, that the F-actin cytoskeleton of enterocyte-like cells is damaged in the presence of bacterium-induced NETs and that this deleterious effect is prevented by inhibition of protease release. These findings provide new insights into the neutrophil response to bacterial infection via the production of bactericidal NETs and suggest that NETs may damage the intestinal epithelium, particularly in situations such as inflammatory bowel diseases.

  17. Comparison of the killing effects between nitrogen-doped and pure TiO2 on HeLa cells with visible light irradiation

    Science.gov (United States)

    Li, Zheng; Pan, Xiaobo; Wang, Tianlong; Wang, Pei-Nan; Chen, Ji-Yao; Mi, Lan

    2013-02-01

    The killing effect of nitrogen-doped titanium dioxide (N-TiO2) nanoparticles on human cervical carcinoma (HeLa) cells by visible light photodynamic therapy (PDT) was higher than that of TiO2 nanoparticles. To study the mechanism of the killing effect, the reactive oxygen species produced by the visible-light-activated N-TiO2 and pure-TiO2 were evaluated and compared. The changes of the cellular parameters, such as the mitochondrial membrane potential (MMP), intracellular Ca2+, and nitrogen monoxide (NO) concentrations after PDT were measured and compared for N-TiO2- and TiO2-treated HeLa cells. The N-TiO2 resulted in more loss of MMP and higher increase of Ca2+ and NO in HeLa cells than pure TiO2. The cell morphology changes with time were also examined by a confocal microscope. The cells incubated with N-TiO2 exhibited serious distortion and membrane breakage at 60 min after the PDT.

  18. Development of a novel in-vivo drug/in-vitro light system to investigate mechanisms of cell killing with photodynamic therapy

    Science.gov (United States)

    Hampton, James A.; Selman, Steven H.

    1991-06-01

    Over the last decade considerable interest has developed in the use of exogenously administered chromophores in combination with visible light for the treatment of human tumors. Whether cells are killed directly, or indirectly as a result of disruption of the tissue, microvasculature is unknown. The authors have developed methods to assess in short term culture the effects of PDT on precision cut tissue slices. The use of these tissue slices provide an important link between in vivo studies and in vitro isolated cultured cells for the following reasons: 1) slices contain all of the normal cells in their proper in vivo architectural arrangement; 2) since slices can be obtained relatively easily and in a very short period of time (a few minutes), animals can be treated with compounds in vivo, the tissue can be removed, sliced and mechanistic studies performed in vitro (without the several hours delay required to produce cultured cells); 3) in vitro comparisons between species, including human, can be readily made; and 4) mechanisms of PDT-induced cell killing can be studied in the absence of a functioning vascular system. Using this in vivo drug/in vitro light system, the results presented will detail the findings using normal rat liver and a transplantable rat tumor model.

  19. Selective killing of human immunodeficiency virus infected cells by non-nucleoside reverse transcriptase inhibitor-induced activation of HIV protease

    Directory of Open Access Journals (Sweden)

    Smeulders Liesbeth

    2010-10-01

    Full Text Available Abstract Background Current antiretroviral therapy against human immunodeficiency virus (HIV-1 reduces viral load and thereby prevents viral spread, but it cannot eradicate proviral genomes from infected cells. Cells in immunological sanctuaries as well as cells producing low levels of virus apparently contribute to a reservoir that maintains HIV persistence in the presence of highly active antiretroviral therapy. Thus, accelerated elimination of virus producing cells may represent a complementary strategy to control HIV infection. Here we sought to exploit HIV protease (PR related cytotoxicity in order to develop a strategy for drug induced killing of HIV producing cells. PR processes the viral Gag and Gag-Pol polyproteins during virus maturation, but is also implicated in killing of virus producing cells through off-target cleavage of host proteins. It has been observed previously that micromolar concentrations of certain non-nucleoside reverse transcriptase inhibitors (NNRTIs can stimulate intracellular PR activity, presumably by enhancing Gag-Pol dimerization. Results Using a newly developed cell-based assay we compared the degree of PR activation displayed by various NNRTIs. We identified inhibitors showing higher potency with respect to PR activation than previously described for NNRTIs, with the most potent compounds resulting in ~2-fold increase of the Gag processing signal at 250 nM. The degree of enhancement of intracellular Gag processing correlated with the compound's ability to enhance RT dimerization in a mammalian two-hybrid assay. Compounds were analyzed for their potential to mediate specific killing of chronically infected MT-4 cells. Levels of cytotoxicity on HIV infected cells determined for the different NNRTIs corresponded to the relative degree of drug induced intracellular PR activation, with CC50 values ranging from ~0.3 μM to above the tested concentration range (10 μM. Specific cytotoxicity was reverted by addition

  20. Design, synthesis, and in vitro and in vivo biological studies of a 3'-deoxythymidine conjugate that potentially kills cancer cells selectively.

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    Qiong Wei

    Full Text Available Thymidine kinases (TKs have been considered one of the potential targets for anticancer therapeutic because of their elevated expressions in cancer cells. However, nucleobase analogs targeting TKs have shown poor selective cytotoxicity in cancer cells despite effective antiviral activity. 3'-Deoxythymidine phenylquinoxaline conjugate (dT-QX was designed as a novel nucleobase analog to target TKs in cancer cells and block cell replication via conjugated DNA intercalating quinoxaline moiety. In vitro cell screening showed that dT-QX selectively kills a variety of cancer cells including liver carcinoma, breast adenocarcinoma and brain glioma cells; whereas it had a low cytotoxicity in normal cells such as normal human liver cells. The anticancer activity of dT-QX was attributed to its selective inhibition of DNA synthesis resulting in extensive mitochondrial superoxide stress in cancer cells. We demonstrate that covalent linkage with 3'-deoxythymidine uniquely directed cytotoxic phenylquinoxaline moiety more toward cancer cells than normal cells. Preliminary mouse study with subcutaneous liver tumor model showed that dT-QX effectively inhibited the growth of tumors. dT-QX is the first molecule of its kind with highly amendable constituents that exhibits this selective cytotoxicity in cancer cells.

  1. Killing effect of TNF-related apoptosis inducing ligand regulated by tetracycline on gastric cancer cell line NCI-N87

    Institute of Scientific and Technical Information of China (English)

    Xiao-Chao Wei; Xin-Juan Wang; Kai-Chen; Lei Zhang; Yu Liang; Xin-Li Lin

    2001-01-01

    AIM To clone the cDNA fragment of human TRAIL (TNFrelated apoptosis inducing ligand) into a tetracyclineregulated gene expression system, the RevTet-On system, transduce expression vectors into a gastric carcinoma cell line-NCl-N87 and examine the effects of controlled expression of TRAIL in vitro on the gastric carcinoma cells. METHODS The full-length cDNA of TRAIL was inserted into a vector under the control of the tetracyclineresponsive element (TRE) to obtain the plasmid pRevTRETRAIL, which was transfected into a packaging cell line PT67. In addition, vector pRev-Tet- On and pRevTRE were also transfected into PT67 separately. After hygromycin and G418 selection, the viral titer was determined. The medium containing retroviral vectors was collected and used to transduce a gastric carcinoma cell line NCI-N87.The resulting cell line NCI-N87-Tet-On-TRE-TRAIL and a control cell line, NCI-N87-Tet-On-TRE, were established.TRAIL expression in the cell line was induced by incubating cells with doxycycline (Dox), which is a tetracycline analogue. The killing effect on gastric carcinoma cells was analyzed after induction. RESULTS The recombinant plasmid pRev-TRE-TRAIL was constructed. After hygromycin or G418 selection, the producer cell lines PT67-TRE, PT67-TRE-TRAIL and PT67TetOn were obtained, with titers of about 108CFU@ L-1 By transducing NCI-N87 cells with retroviral vectors from these cell lines, stable cell lines NCI-N87-Tet-On-TRETRAIL (NN3T) and control cell line NCI-N87-Tet-On-TRE (NN2T) were established. The growth curves of the selected cell lines were the same with the wild type NCIN87. When Dox was added, cell death was obvious in the test groups (29% -77%), whereas no difference was observed in control and wild type cell lines. With the addition of a medium from the test group, human leukemia cell line Jurkat was activated till death (83%), indicating the secretion of active TRAIL proteins from the test cells to the medium. CONCLUSION With the use of the

  2. Effects of the oral administration of viable and heat-killed Streptococcus bovis HC5 cells to pre-sensitized BALB/c mice.

    Directory of Open Access Journals (Sweden)

    Aline D Paiva

    Full Text Available Antimicrobial peptides have been suggested as an alternative to classical antibiotics in livestock production and bacteriocin-producing bacteria could be added to animal feeds to deliver bacteriocins in the gastrointestinal (GI tract of ruminant and monogastric animals. In this study, viable (V and heat-killed (HK Streptococcus bovis HC5 cells were orally administered to pre-sensitized mice in order to assess the effects of a bacteriocin-producing bacteria on histological parameters and the immune response of the GI tract of monogastric animals. The administration of V and HK S. bovis HC5 cells during 58 days to BALB/c mice did not affect weight gain, but an increase in gut permeability was detected in animals receiving the HK cells. Viable and heat killed cells caused similar morphological alterations in the GI tract of the animals, but the most prominent effects were detected in the small intestine. The oral administration of S. bovis HC5 also influenced cytokine production in the small intestine, and the immune-mediated activity differed between V and HK cells. The relative expression of IL-12 and INF-γ was significantly higher in the small intestine of mice treated with V cells, while an increase in IL-5, IL-13 and TNF-α expression was only detected in mice treated with HK cells. Considering that even under a condition of severe challenge (pre-sensitization followed by daily exposure to the same bacterial immunogen the general health of the animals was maintained, it appears that oral administration of S. bovis HC5 cells could be a useful route to deliver bacteriocin in the GI tract of livestock animals.

  3. Defects in the acquisition of tumor-killing capability of CD8+ cytotoxic T cells in streptozotocin-induced diabetic mice.

    Directory of Open Access Journals (Sweden)

    Shu-Ching Chen

    Full Text Available Emerging evidences have shown that diabetes mellitus not only raises risk but also heightens mortality rate of cancer. It is not clear, however, whether antitumor CD8+ cytotoxic T lymphocyte (CTL response is down-modulated in diabetic hosts. We investigated the impact of hyperglycemia on CTLs' acquisition of tumor-killing capability by utilizing streptozotocin-induced diabetic (STZ-diabetic mice. Murine diabetes was induced by intraperitoneal injection of STZ (200 mg/kg in C57BL/6 mice, 2C-T cell receptor (TCR transgenic and P14-TCR transgenic mice. The study found that, despite harboring intact proliferative capacity measured with CFSE labeling and MTT assay, STZ-diabetic CD8+ CTLs displayed impaired effector functions. After stimulation, STZ-diabetic CD8+ CTLs produced less perforin and TNFα assessed by intracellular staining, as well as expressed less CD103 protein. Furthermore, adoptive transfer of STZ-diabetic P14 CD8+ effector cells showed an insufficient recruitment to the B16.gp33 melanoma and inadequate production of perforin, granzyme B and TNFα determined by immunohistochemistry in the tumor milieu. As a result, STZ-diabetic CD8+ effector cells were neither able to eliminate tumor nor to improve survival of tumor-bearing mice. Taken together, our data suggest that CD8+ CTLs are crippled to infiltrate into tumors and thus fail to acquire tumor-killing capability in STZ-diabetic hosts.

  4. Irreducible Killing Tensors from Third Rank Killing-Yano Tensors

    OpenAIRE

    Popa, Florian Catalin; Tintareanu-Mircea, Ovidiu

    2006-01-01

    We investigate higher rank Killing-Yano tensors showing that third rank Killing-Yano tensors are not always trivial objects being possible to construct irreducible Killing tensors from them. We give as an example the Kimura IIC metric were from two rank Killing-Yano tensors we obtain a reducible Killing tensor and from third rank Killing-Yano tensors we obtain three Killing tensors, one reducible and two irreducible.

  5. Central memory Vgamma9Vdelta2 T lymphocytes primed and expanded by bacillus Calmette-Guérin-infected dendritic cells kill mycobacterial-infected monocytes.

    Science.gov (United States)

    Martino, Angelo; Casetti, Rita; Sacchi, Alessandra; Poccia, Fabrizio

    2007-09-01

    In humans, innate immune recognition of mycobacteria, including Mycobacterium tuberculosis and bacillus Calmette-Guérin (BCG), is a feature of cells as dendritic cells (DC) and gammadelta T cells. In this study, we show that BCG infection of human monocyte-derived DC induces a rapid activation of Vgamma9Vdelta2 T cells (the major subset of gammadelta T cell pool in human peripheral blood). Indeed, in the presence of BCG-infected DC, Vgamma9Vdelta2 T cells increase both their expression of CD69 and CD25 and the production of TNF-alpha and IFN-gamma, in contrast to DC treated with Vgamma9Vdelta2 T cell-specific Ags. Without further exogenous stimuli, BCG-infected DC expand a functionally cytotoxic central memory Vgamma9Vdelta2 T cell population. This subset does not display lymph node homing receptors, but express a high amount of perforin. They are highly efficient in the killing of mycobacterial-infected primary monocytes or human monocytic THP-1 cells preserving the viability of cocultured, infected DC. This study provides further evidences about the complex relationship between important players of innate immunity and suggests an immunoregulatory role of Vgamma9Vdelta2 T cells in the control of mycobacterial infection.

  6. N′1,N′3-Dimethyl-N′1,N′3-bis(phenylcarbonothioyl Propanedihydrazide (Elesclomol Selectively Kills Cisplatin Resistant Lung Cancer Cells through Reactive Oxygen Species (ROS

    Directory of Open Access Journals (Sweden)

    Niramol Savaraj

    2009-12-01

    Full Text Available Cisplatin is an important chemotherapeutic agent in lung cancer treatment. The mechanism of drug resistance to cisplatin is complex and historically has been difficult to overcome. We report here that cisplatin resistant lung cancer cell lines possess high basal levels of reactive oxygen species (ROS when compared to normal cells and their parental cell counterparts. These resistant cells also have low thioredoxin (TRX levels which may be one of the contributory factors to high ROS. N′1,N′3-dimethyl-N′1,N'3-bis(phenylcarbonothioyl propanedihydrazide (elesclomol, an agent known to increase ROS is selectively toxic to cisplatin-resistant cells, while sparing normal cells and the parental counterpart. The cytotoxic effect of elesclomol in resistant cells is accompanied by further decreases in TRX and glutathione (GSH antioxidant systems, while opposite results were found in parental cells. The ID50 of elesclomol in cisplatin-resistant cells ranged from 5–10 nM, which is well within clinically achievable ranges. N-Acetylcysteine (NAC, which is known to neutralize ROS, can abolish the cytotoxic effect of elesclomol, suggesting that the cytotoxic effect results from increased ROS. Overall, our data suggest that elesclomol selectively kills cisplatin-resistant tumor cells through increased ROS. This agent may hold potential to overcome cisplatin resistance and should be further explored to treat patients who have failed cisplatin therapy.

  7. Critical high-dimensional state transitions in cell populations or why cancers follow the principle ``What does not kill me makes me stronger''

    Science.gov (United States)

    Huang, Sui

    Transitions between high-dimensional attractor states in the quasi-potential landscape of the gene regulatory network, induced by environmental perturbations and/or facilitated by mutational rewiring of the network, underlie cell phenotype switching in development as well as in cancer progression, including acquisition of drug-resistant phenotypes. Considering heterogeneous cell populations as statistical ensembles of cells, and single-cell resolution gene expression profiling of cell populations undergoing a cell phenotype shift allow us now to map the topography of the landscape and its distortion. From snapshots of single-cell expression patterns of a cell population measured during major transitions we compute a quantity that identifies symmetry-breaking destabilization of attractors (bifurcation) and concomitant dimension-reduction of the state space manifold (landscape distortion) which precede critical transitions to new attractor states. The model predicts, and we show experimentally, the almost inevitable generation of aberrant cells associated with such critical transitions in multi-attractor landscapes: therapeutic perturbations which seek to push cancer cells to the apoptotic state, almost always produce ``rebellious'' cells which move in the ``opposite direction'': instead of dying they become more stem-cell-like and malignant. We show experimentally that the inadvertent generation of more malignant cancer cells by therapy indeed results from transition of surviving (but stressed) cells into unforeseen attractor states and not simply from selection of inherently more resistant cells. Thus, cancer cells follow not so much Darwin, as generally thought (survival of the fittest), but rather Nietzsche (What does not kill me makes me stronger). Supported by NIH (NCI, NIGMS), Alberta Innovates.

  8. Accelerated killing of cancer cells using a multifunctional single-walled carbon nanotube-based system for targeted drug delivery in combination with photothermal therapy

    Directory of Open Access Journals (Sweden)

    Jeyamohan P

    2013-07-01

    Full Text Available Prashanti Jeyamohan, Takashi Hasumura, Yutaka Nagaoka, Yasuhiko Yoshida, Toru Maekawa, D Sakthi Kumar Bio-Nano Electronics Research Centre, Graduate School of Interdisciplinary New Science, Toyo University, Kawagoe, Japan Abstract: The photothermal effect of single-walled carbon nanotubes (SWCNTs in combination with the anticancer drug doxorubicin (DOX for targeting and accelerated destruction of breast cancer cells is demonstrated in this paper. A targeted drug-delivery system was developed for selective killing of breast cancer cells with polyethylene glycol biofunctionalized and DOX-loaded SWCNTs conjugated with folic acid. In our work, in vitro drug-release studies showed that the drug (DOX binds at physiological pH (pH 7.4 and is released only at a lower pH, ie, lysosomal pH (pH 4.0, which is the characteristic pH of the tumor environment. A sustained release of DOX from the SWCNTs was observed for a period of 3 days. SWCNTs have strong optical absorbance in the near-infrared (NIR region. In this special spectral window, biological systems are highly transparent. Our study reports that under laser irradiation at 800 nm, SWCNTs exhibited strong light–heat transfer characteristics. These optical properties of SWCNTs open the way for selective photothermal ablation in cancer therapy. It was also observed that internalization and uptake of folate-conjugated NTs into cancer cells was achieved by a receptor-mediated endocytosis mechanism. Results of the in vitro experiments show that laser was effective in destroying the cancer cells, while sparing the normal cells. When the above laser effect was combined with DOX-conjugated SWCNTs, we found enhanced and accelerated killing of breast cancer cells. Thus, this nanodrug-delivery system, consisting of laser, drug, and SWCNTs, looks to be a promising selective modality with high treatment efficacy and low side effects for cancer therapy. Keywords: cancer, nanotherapy, SWCNTs, targeted drug delivery

  9. Melatonin Protects Human Cells from Clustered DNA Damages, Killing and Acquisition of Soft Agar Growth Induced by X-rays or 970 MeV/n Fe ions

    Energy Technology Data Exchange (ETDEWEB)

    Das, B.; Sutherland, B.; Bennett, P. V.; Cutter, N. C.; Sutherland, J. C.

    2011-06-01

    We tested the ability of melatonin (N-acetyl-5 methoxytryptamine), a highly effective radical scavenger and human hormone, to protect DNA in solution and in human cells against induction of complex DNA clusters and biological damage induced by low or high linear energy transfer radiation (100 kVp X-rays, 970 MeV/nucleon Fe ions). Plasmid DNA in solution was treated with increasing concentrations of melatonin (0.0-3.5 mM) and were irradiated with X-rays. Human cells (28SC monocytes) were also irradiated with X-rays and Fe ions with and without 2 mM melatonin. Agarose plugs containing genomic DNA were subjected to Contour Clamped Homogeneous Electrophoretic Field (CHEF) followed by imaging and clustered DNA damages were measured by using Number Average length analysis. Transformation experiments on human primary fibroblast cells using soft agar colony assay were carried out which were irradiated with Fe ions with or without 2 mM melatonin. In plasmid DNA in solution, melatonin reduced the induction of single- and double-strand breaks. Pretreatment of human 28SC cells for 24 h before irradiation with 2 mM melatonin reduced the level of X-ray induced double-strand breaks by {approx}50%, of abasic clustered damages about 40%, and of Fe ion-induced double-strand breaks (41% reduction) and abasic clusters (34% reduction). It decreased transformation to soft agar growth of human primary cells by a factor of 10, but reduced killing by Fe ions only by 20-40%. Melatonin's effective reduction of radiation-induced critical DNA damages, cell killing, and striking decrease of transformation suggest that it is an excellent candidate as a countermeasure against radiation exposure, including radiation exposure to astronaut crews in space travel.

  10. Antibodies against invasive phenotype-specific antigens increase Mycobacterium avium subspecies paratuberculosis translocation across a polarized epithelial cell model and enhance killing by bovine macrophages

    Science.gov (United States)

    Everman, Jamie L.; Bermudez, Luiz E.

    2015-01-01

    Johne's disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a severe chronic enteritis which affects large populations of ruminants globally. Prevention strategies to combat the spread of Johne's disease among cattle herds involve adhering to strict calving practices to ensure young susceptible animals do not come in contact with MAP-contaminated colostrum, milk, or fecal material. Unfortunately, the current vaccination options available are associated with high cost and suboptimal efficacy. To more successfully combat the spread of Johne's disease to young calves, an efficient method of protection is needed. In this study, we examined passive immunization as a mode of introducing protective antibodies against MAP to prevent the passage of the bacterium to young animals via colostrum and milk. Utilizing the infectious MAP phenotype developed after bacterial exposure to milk, we demonstrate that in vitro opsonization with serum from Johne's-positive cattle results in enhanced translocation across a bovine MDBK polarized epithelial cell monolayer. Furthermore, immune serum opsonization of MAP results in a rapid host cell-mediated killing by bovine macrophages in an oxidative-, nitrosative-, and extracellular DNA trap-independent manner. This study illustrates that antibody opsonization of MAP expressing an infectious phenotype leads to the killing of the bacterium during the initial stage of macrophage infection. PMID:26301206

  11. Water disinfection using silver nanoparticle impregnated activated carbon: Escherichia coli cell-killing in batch and continuous packed column operation over a long duration.

    Science.gov (United States)

    Biswas, Pritam; Bandyopadhyaya, Rajdip

    2016-09-01

    Silver nanoparticles (Ag-NP) were selectively impregnated on the external surface of plasma treated activated carbon (AC) granules (referred to as Ag-AC hybrid, having 0.8 wt% of Ag), for achieving continuous disinfection of water in a single flow-column set-up. First, Ag-NPs (28 nm mean size) were synthesized by UV reduction. Subsequently, Escherichia coli cell-killing experiments were performed in both shake flask (i. e. batch-mode) and flow-column (i. e. continuous-mode) operations, using E. coli K12 (MTCC 1302) as a model organism. Batch results using 8 mg Ag-AC hybrid/ml of cell suspension showed that, 10(4) CFU/ml of cells were killed within 25 min contact time, with cell concentration decaying exponentially in time. Maintaining almost the same contact time as in the batch experiments, three columns packed with Ag-AC (all having a height of 25 cm but increasing diameters of 1, 5 and 8 cm, respectively) were used for monitoring cell-killing performance over a long duration. For all columns, inlet water having 10(4) CFU/ml E. coli could be completely disinfected to produce treated, outlet water having zero cell count. Specifically for the 8 cm diameter column, a maximum throughput of treating 1.62 L of contaminated water per hour could be maintained for at least up to 16 days. Moreover, the Ag concentration in the outlet water was only up to 29.8 μg/L at steady state, which is well within the recommended limit of 100 μg/L for drinking water. Hence, water disinfection for potable quality water (zero E. coli count and <100 μg/L Ag) can be achieved in a continuous manner over a long duration, with our packed Ag-AC column.

  12. Safety of the recombinant cholera toxin B subunit, killed whole-cell (rBS-WC oral cholera vaccine in pregnancy.

    Directory of Open Access Journals (Sweden)

    Ramadhan Hashim

    Full Text Available INTRODUCTION: Mass vaccinations are a main strategy in the deployment of oral cholera vaccines. Campaigns avoid giving vaccine to pregnant women because of the absence of safety data of the killed whole-cell oral cholera (rBS-WC vaccine. Balancing this concern is the known higher risk of cholera and of complications of pregnancy should cholera occur in these women, as well as the lack of expected adverse events from a killed oral bacterial vaccine. METHODOLOGY/PRINCIPAL FINDINGS: From January to February 2009, a mass rBS-WC vaccination campaign of persons over two years of age was conducted in an urban and a rural area (population 51,151 in Zanzibar. Pregnant women were advised not to participate in the campaign. More than nine months after the last dose of the vaccine was administered, we visited all women between 15 and 50 years of age living in the study area. The outcome of pregnancies that were inadvertently exposed to at least one oral cholera vaccine dose and those that were not exposed was evaluated. 13,736 (94% of the target women in the study site were interviewed. 1,151 (79% of the 1,453 deliveries in 2009 occurred during the period when foetal exposure to the vaccine could have occurred. 955 (83% out of these 1,151 mothers had not been vaccinated; the remaining 196 (17% mothers had received at least one dose of the oral cholera vaccine. There were no statistically significant differences in the odds ratios for birth outcomes among the exposed and unexposed pregnancies. CONCLUSIONS/SIGNIFICANCE: We found no statistically significant evidence of a harmful effect of gestational exposure to the rBS-WC vaccine. These findings, along with the absence of a rational basis for expecting a risk from this killed oral bacterial vaccine, are reassuring but the study had insufficient power to detect infrequent events. TRIAL REGISTRATION: ClinicalTrials.gov NCT00709410.

  13. Cryptococcus neoformans modulates extracellular killing by neutrophils

    Directory of Open Access Journals (Sweden)

    Asfia eQureshi

    2011-09-01

    Full Text Available We recently established a key role for host sphingomyelin synthase (SMS in the regulation of the killing activity of neutrophils against Cryptococcus neoformans. In this work, we studied the effect of C. neoformans on the killing activity of neutrophils and whether SMS would still be a player against C. neoformans in immunocompromised mice lacking T and NK cells (Tgε26 mice. To this end, we analyzed whether C. neoformans would have any effect on neutrophil survival and killing in vitro and in vivo. We show that unlike C. albicans, neither the presence nor the capsule size of C. neoformans cells have any effect on neutrophil viability. Interestingly, melanized C. neoformans cells totally abrogated the killing activity of neutrophils. Next, we monitored how exposure of neutrophils to C. neoformans cells would interfere with any further killing activity of the medium and found that pre-incubation with live but not heat-killed fungal cells significantly inhibits further killing activity of the medium. We next studied whether activation of SMS at the site of C. neoformans infection is dependent on T and NK cells. Using matrix-assisted laser desorption-ionization (MALDI tissue imaging in infected lung we found that similarly to previous observations in the isogenic wild type CBA/J mice, SM 16:0 levels are significantly elevated at the site of infection in mice lacking T and NK cells but only at early time points. This study highlights that C. neoformans may negatively regulate the killing activity of neutrophils and that SMS activation in neutrophils appears to be partially independent of T and/or NK cells.

  14. Cryptococcus neoformans modulates extracellular killing by neutrophils.

    Science.gov (United States)

    Qureshi, Asfia; Grey, Angus; Rose, Kristie L; Schey, Kevin L; Del Poeta, Maurizio

    2011-01-01

    We recently established a key role for host sphingomyelin synthase (SMS) in regulating the killing activity of neutrophils against Cryptococcus neoformans. In this paper, we studied the effect of C. neoformans on the killing activity of neutrophils and whether SMS would still be a player against C. neoformans in immunocompromised mice lacking T and natural killer (NK) cells (Tgε26 mice). To this end, we analyzed whether C. neoformans would have any effect on neutrophil survival and killing in vitro and in vivo. We show that unlike Candida albicans, neither the presence nor the capsule size of C. neoformans cells have any effect on neutrophil viability. Interestingly, melanized C. neoformans cells totally abrogated the killing activity of neutrophils. We monitored how exposure of neutrophils to C. neoformans cells would interfere with any further killing activity of the conditioned medium and found that pre-incubation with live but not "heat-killed" fungal cells significantly inhibits further killing activity of the medium. We then studied whether activation of SMS at the site of C. neoformans infection is dependent on T and NK cells. Using matrix-assisted laser desorption-ionization tissue imaging in infected lung we found that similar to previous observations in the isogenic wild-type CBA/J mice, SM 16:0 levels are significantly elevated at the site of infection in mice lacking T and NK cells, but only at early time points. This study highlights that C. neoformans may negatively regulate the killing activity of neutrophils and that SMS activation in neutrophils appears to be partially independent of T and/or NK cells.

  15. Modeling the Effects of Vorinostat In Vivo Reveals both Transient and Delayed HIV Transcriptional Activation and Minimal Killing of Latently Infected Cells.

    Science.gov (United States)

    Ke, Ruian; Lewin, Sharon R; Elliott, Julian H; Perelson, Alan S

    2015-10-01

    Recent efforts to cure human immunodeficiency virus type-1 (HIV-1) infection have focused on developing latency reversing agents as a first step to eradicate the latent reservoir. The histone deacetylase inhibitor, vorinostat, has been shown to activate HIV RNA transcription in CD4+ T-cells and alter host cell gene transcription in HIV-infected individuals on antiretroviral therapy. In order to understand how latently infected cells respond dynamically to vorinostat treatment and determine the impact of vorinostat on reservoir size in vivo, we have constructed viral dynamic models of latency that incorporate vorinostat treatment. We fitted these models to data collected from a recent clinical trial in which vorinostat was administered daily for 14 days to HIV-infected individuals on suppressive ART. The results show that HIV transcription is increased transiently during the first few hours or days of treatment and that there is a delay before a sustained increase of HIV transcription, whose duration varies among study participants and may depend on the long term impact of vorinostat on host gene expression. Parameter estimation suggests that in latently infected cells, HIV transcription induced by vorinostat occurs at lower levels than in productively infected cells. Furthermore, the estimated loss rate of transcriptionally induced cells remains close to baseline in most study participants, suggesting vorinostat treatment does not induce latently infected cell killing and thus reduce the latent reservoir in vivo.

  16. Modeling the Effects of Vorinostat In Vivo Reveals both Transient and Delayed HIV Transcriptional Activation and Minimal Killing of Latently Infected Cells.

    Directory of Open Access Journals (Sweden)

    Ruian Ke

    2015-10-01

    Full Text Available Recent efforts to cure human immunodeficiency virus type-1 (HIV-1 infection have focused on developing latency reversing agents as a first step to eradicate the latent reservoir. The histone deacetylase inhibitor, vorinostat, has been shown to activate HIV RNA transcription in CD4+ T-cells and alter host cell gene transcription in HIV-infected individuals on antiretroviral therapy. In order to understand how latently infected cells respond dynamically to vorinostat treatment and determine the impact of vorinostat on reservoir size in vivo, we have constructed viral dynamic models of latency that incorporate vorinostat treatment. We fitted these models to data collected from a recent clinical trial in which vorinostat was administered daily for 14 days to HIV-infected individuals on suppressive ART. The results show that HIV transcription is increased transiently during the first few hours or days of treatment and that there is a delay before a sustained increase of HIV transcription, whose duration varies among study participants and may depend on the long term impact of vorinostat on host gene expression. Parameter estimation suggests that in latently infected cells, HIV transcription induced by vorinostat occurs at lower levels than in productively infected cells. Furthermore, the estimated loss rate of transcriptionally induced cells remains close to baseline in most study participants, suggesting vorinostat treatment does not induce latently infected cell killing and thus reduce the latent reservoir in vivo.

  17. Enhanced killing of breast cancer cells by a d-amino acid analog of the winter flounder-derived pleurocidin NRC-03.

    Science.gov (United States)

    Hilchie, Ashley L; Haney, Evan F; Pinto, Devanand M; Hancock, Robert E W; Hoskin, David W

    2015-12-01

    Cationic antimicrobial peptides (CAPs) defend against pathogens and, in some cases, exhibit potent anticancer activities. We previously reported that the pleurocidin NRC-03 causes lysis of breast cancer and multiple myeloma cells. NRC-03 also reduces the EC50 of other cytotoxic compounds and prevents tumor growth in vivo. However, the therapeutic utility of NRC-03 may be limited by its susceptibility to degradation by proteases. The goal of this study was to characterize the anticancer activities of a d-amino acid analog of NRC-03 ([D]-NRC-03) that was predicted to be resistant to proteolytic degradation. Unlike NRC-03, [D]-NRC-03 was not degraded by human serum or trypsin and, in comparison to NRC-03, showed increased killing of breast cancer cells, including multidrug-resistant cells; however, [D]-NRC-03 was somewhat more cytotoxic than NRC-03 for several types of normal cells. Importantly, [D]-NRC-03 was more effective than NRC-03 in vivo since 4-fold less peptide was required for an equivalent inhibitory effect on the growth of breast cancer cell xenografts in immune-deficient mice. These findings demonstrate that a d-amino acid analog of NRC-03 overcomes a major limitation to the therapeutic use of NRC-03, namely peptide stability. Further modification of [D]-NRC-03 is required to improve its selectivity for cancer cells.

  18. Treatment with 5-Aza-2'-Deoxycytidine Induces Expression of NY-ESO-1 and Facilitates Cytotoxic T Lymphocyte-Mediated Tumor Cell Killing.

    Directory of Open Access Journals (Sweden)

    Agnes S Klar

    Full Text Available NY-ESO-1 belongs to the cancer/testis antigen (CTA family and represents an attractive target for cancer immunotherapy. Its expression is induced in a variety of solid tumors via DNA demethylation of the promoter of CpG islands. However, NY-ESO-1 expression is usually very low or absent in some tumors such as breast cancer or multiple myeloma. Therefore, we established an optimized in vitro treatment protocol for up-regulation of NY-ESO-1 expression by tumor cells using the hypomethylating agent 5-aza-2'-deoxycytidine (DAC.We demonstrated de novo induction of NY-ESO-1 in MCF7 breast cancer cells and significantly increased expression in U266 multiple myeloma cells. This effect was time- and dose-dependent with the highest expression of NY-ESO-1 mRNA achieved by the incubation of 10 μM DAC for 72 hours. NY-ESO-1 activation was also confirmed at the protein level as shown by Western blot, flow cytometry, and immunofluorescence staining. The detection and quantification of single NY-ESO-1 peptides presented at the tumor cell surface in the context of HLA-A*0201 molecules revealed an increase of 100% and 50% for MCF7 and U266 cells, respectively. Moreover, the enhanced expression of NY-ESO-1 derived peptides at the cell surface was accompanied by an increased specific lysis of MCF7 and U266 cells by HLA-A*0201/NY-ESO-1(157-165 peptide specific chimeric antigen receptor (CAR CD8+ T cells. In addition, the killing activity of CAR T cells correlated with the secretion of higher IFN-gamma levels.These results indicate that NY-ESO-1 directed immunotherapy with specific CAR T cells might benefit from concomitant DAC treatment.

  19. Maximal killing of lymphoma cells by DNA damage-inducing therapy requires not only the p53 targets Puma and Noxa, but also Bim.

    Science.gov (United States)

    Happo, Lina; Cragg, Mark S; Phipson, Belinda; Haga, Jon M; Jansen, Elisa S; Herold, Marco J; Dewson, Grant; Michalak, Ewa M; Vandenberg, Cassandra J; Smyth, Gordon K; Strasser, Andreas; Cory, Suzanne; Scott, Clare L

    2010-12-09

    DNA-damaging chemotherapy is the backbone of cancer treatment, although it is not clear how such treatments kill tumor cells. In nontransformed lymphoid cells, the combined loss of 2 proapoptotic p53 target genes, Puma and Noxa, induces as much resistance to DNA damage as loss of p53 itself. In Eμ-Myc lymphomas, however, lack of both Puma and Noxa resulted in no greater drug resistance than lack of Puma alone. A third B-cell lymphoma-2 homology domain (BH)3-only gene, Bim, although not a direct p53 target, was up-regulated in Eμ-Myc lymphomas incurring DNA damage, and knockdown of Bim levels markedly increased the drug resistance of Eμ-Myc/Puma(-/-)Noxa(-/-) lymphomas both in vitro and in vivo. Remarkably, c-MYC-driven lymphoma cell lines from Noxa(-/-)Puma(-/-)Bim(-/-) mice were as resistant as those lacking p53. Thus, the combinatorial action of Puma, Noxa, and Bim is critical for optimal apoptotic responses of lymphoma cells to 2 commonly used DNA-damaging chemotherapeutic agents, identifying Bim as an additional biomarker for treatment outcome in the clinic.

  20. Anti-tumor activity of heat-killed Lactobacillus plantarum BF-LP284 on Meth-A tumor cells in BALB/c mice.

    Science.gov (United States)

    Shin, Ryoichi; Itoh, Yukie; Kataoka, Motoyuki; Iino-Miura, Shiori; Miura, Ryosuke; Mizutani, Takeo; Fujisawa, Tomohiko

    2016-09-01

    Probiotics exert numerous effects on human well-being. Here, heat-killed Lactobacillus plantarum BF-LP284 (H-Lp) was isolated as a potent immuno-modulator among 15 strains of lactobacilli in terms of TNF-α induction ability in peritoneal macrophages. In vitro TNF-α and IFN-γ induction in Peyer's patch (PP) cells was higher when incubated with H-Lp than with live L. plantarum BF-LP284 (L-Lp). Suppression of syngeneic Meth-A tumors in a murine model by oral administration of H-Lp was also greater than that of L-Lp and of controls. H-Lp stimulated IFN-γ production in spleen cells, which displayed inhibited tumor growth in Winn assays when treated with H-Lp. Moreover, H-Lp increased the ratio of CD3(+ )cells among peripheral blood mononuclear cells in Meth-A tumor-bearing mice, suggesting an H-Lp-mediated anti-tumor mechanism whereby immune cells that are activated by H-Lp in PP and acquire anti-tumor activity in the spleen migrate to tumor sites through lymphocyte homing to inhibit tumor growth.

  1. Isolation and killing of candidate chronic myeloid leukemia stem cells by antibody targeting of IL-1 receptor accessory protein

    DEFF Research Database (Denmark)

    Järås, Marcus; Johnels, Petra; Hansen, Nils Gunder;

    2010-01-01

    will require full eradication of Ph chromosome-positive (Ph(+)) CML stem cells. Here we used gene-expression profiling to identify IL-1 receptor accessory protein (IL1RAP) as up-regulated in CML CD34(+) cells and also in cord blood CD34(+) cells as a consequence of retroviral BCR/ABL1 expression. To test...... their Ph-chromosome status. Interestingly, we found that the CML CD34(+)CD38(-)IL1RAP(+) cells were Ph(+), whereas CML CD34(+)CD38(-)IL1RAP(-) cells were almost exclusively Ph(-). By performing long-term culture-initiating cell assays on the two cell populations, we found that Ph(+) and Ph(-) candidate CML...

  2. Fungal and bacterial killing by neutrophils.

    Science.gov (United States)

    Ermert, David; Zychlinsky, Arturo; Urban, Constantin

    2009-01-01

    Neutrophils are professional phagocytes of the innate immune system that are essential to control bacterial and fungal infections. These cells engulf and kill invading microbes. Additionally, activated neutrophils are able to release neutrophil extracellular traps (NETs). These fibers consist of chromatin decorated with antimicrobial proteins to trap and kill microbes. Appropriate quantitative methods are required to understand the nature of interactions of neutrophils with pathogens. Here we present assays to measure killing mediated by phagocytosis, by NETs, by a combination of both, and by granular extract. As examples, we use Candida albicans for fungal and Shigella flexneri for bacterial pathogens.

  3. A Subpopulation of the K562 Cells Are Killed by Curcumin Treatment after G2/M Arrest and Mitotic Catastrophe

    Science.gov (United States)

    Martinez-Castillo, Macario; Bonilla-Moreno, Raul; Aleman-Lazarini, Leticia; Meraz-Rios, Marco Antonio; Orozco, Lorena; Cedillo-Barron, Leticia; Cordova, Emilio J.

    2016-01-01

    Curcumin is extensively investigated as a good chemo-preventive agent in the development of many cancers and particularly in leukemia, including treatment of chronic myelogenous leukemia and it has been proposed as an adjuvant for leukemia therapies. Human chronic myeloid leukemia cells (K562), were treated with 20 μM of curcumin, and we found that a subpopulation of these cells were arrested and accumulate in the G2/M phase of the cell cycle. Characterization of this cell subpopulation showed that the arrested cells presented nuclear morphology changes resembling those described for mitotic catastrophe. Mitotic cells displayed abnormal chromatin organization, collapse of the mitotic spindle and abnormal chromosome segregation. Then, these cells died in an apoptosis dependent manner and showed diminution in the protein levels of BCL-2 and XIAP. Moreover, our results shown that a transient activation of the nuclear factor κB (NFκB) occurred early in these cells, but decreased after 6 h of the treatment, explaining in part the diminution of the anti-apoptotic proteins. Additionally, P73 was translocated to the cell nuclei, because the expression of the C/EBPα, a cognate repressor of the P73 gene, was decreased, suggesting that apoptosis is trigger by elevation of P73 protein levels acting in concert with the diminution of the two anti-apoptotic molecules. In summary, curcumin treatment might produce a P73-dependent apoptotic cell death in chronic myelogenous leukemia cells (K562), which was triggered by mitotic catastrophe, due to sustained BAX and survivin expression and impairment of the anti-apoptotic proteins BCL-2 and XIAP. PMID:27832139

  4. Innate invariant NKT cells recognize Mycobacterium tuberculosis-infected macrophages, produce interferon-gamma, and kill intracellular bacteria.

    Directory of Open Access Journals (Sweden)

    Isabel Sada-Ovalle

    2008-12-01

    Full Text Available Cellular immunity to Mycobacterium tuberculosis (Mtb requires a coordinated response between the innate and adaptive arms of the immune system, resulting in a type 1 cytokine response, which is associated with control of infection. The contribution of innate lymphocytes to immunity against Mtb remains controversial. We established an in vitro system to study this question. Interferon-gamma is produced when splenocytes from uninfected mice are cultured with Mtb-infected macrophages, and, under these conditions, bacterial replication is suppressed. This innate control of bacterial replication is dependent on CD1d-restricted invariant NKT (iNKT cells, and their activation requires CD1d expression by infected macrophages as well as IL-12 and IL-18. We show that iNKT cells, even in limiting quantities, are sufficient to restrict Mtb replication. To determine whether iNKT cells contribute to host defense against tuberculosis in vivo, we adoptively transferred iNKT cells into mice. Primary splenic iNKT cells obtained from uninfected mice significantly reduce the bacterial burden in the lungs of mice infected with virulent Mtb by the aerosol route. Thus, iNKT cells have a direct bactericidal effect, even in the absence of synthetic ligands such as alpha-galactosylceramide. Our finding that iNKT cells protect mice against aerosol Mtb infection is the first evidence that CD1d-restricted NKT cells mediate protection against Mtb in vivo.

  5. T cells infiltrate the liver and kill hepatocytes in HLA-B(∗)57:01-associated floxacillin-induced liver injury.

    Science.gov (United States)

    Wuillemin, Natascha; Terracciano, Luigi; Beltraminelli, Helmut; Schlapbach, Christoph; Fontana, Stefano; Krähenbühl, Stephan; Pichler, Werner J; Yerly, Daniel

    2014-06-01

    Drug-induced liver injury is a major safety issue. It can cause severe disease and is a common cause of the withdrawal of drugs from the pharmaceutical market. Recent studies have identified the HLA-B(∗)57:01 allele as a risk factor for floxacillin (FLUX)-induced liver injury and have suggested a role for cytotoxic CD8(+) T cells in the pathomechanism of liver injury caused by FLUX. This study aimed to confirm the importance of FLUX-reacting cytotoxic lymphocytes in the pathomechanism of liver injury and to dissect the involved mechanisms of cytotoxicity. IHC staining of a liver biopsy from a patient with FLUX-induced liver injury revealed periportal inflammation and the infiltration of cytotoxic CD3(+) CD8(+) lymphocytes into the liver. The infiltration of cytotoxic lymphocytes into the liver of a patient with FLUX-induced liver injury demonstrates the importance of FLUX-reacting T cells in the underlying pathomechanism. Cytotoxicity of FLUX-reacting T cells from 10 HLA-B(∗)57:01(+) healthy donors toward autologous target cells and HLA-B(∗)57:01-transduced hepatocytes was analyzed in vitro. Cytotoxicity of FLUX-reacting T cells was concentration dependent and required concentrations in the range of peak serum levels after FLUX administration. Killing of target cells was mediated by different cytotoxic mechanisms. Our findings emphasize the role of the adaptive immune system and especially of activated drug-reacting T cells in human leukocyte antigen-associated, drug-induced liver injury.

  6. Cancer cells become susceptible to natural killer cell killing after exposure to histone deacetylase inhibitors due to glycogen synthase kinase-3-dependent expression of MHC class I-related chain A and B

    DEFF Research Database (Denmark)

    Skov, Søren; Pedersen, Marianne Terndrup; Andresen, Lars

    2005-01-01

    by small interfering RNA or by different inhibitors showed that GSK-3 activity is essential for the induced MICA/B expression. We thus present evidence that cancer cells which survive the direct induction of cell death by HDAC inhibitors become targets for NKG2D-expressing cells like NK cells, gammadelta T......We show that histone deacetylase (HDAC) inhibitors lead to functional expression of MHC class I-related chain A and B (MICA/B) on cancer cells, making them potent targets for natural killer (NK) cell-mediated killing through a NK group 2, member D (NKG2D) restricted mechanism. Blocking either...... apoptosis or oxidative stress caused by HDAC inhibitor treatment did not affect MICA/B expression, suggesting involvement of a separate signal pathway not directly coupled to induction of cell death. HDAC inhibitor treatment induced glycogen synthase kinase-3 (GSK-3) activity and down-regulation of GSK-3...

  7. Killing effect of CIK cells and DC-CIK cells on neuroblastoma cells%CIK、DC-CIK细胞对神经母细胞瘤细胞杀伤作用的研究

    Institute of Scientific and Technical Information of China (English)

    邢准; 王秋实; 佟海侠

    2014-01-01

    Objective:To study the killing effects on neuroblastoma( NB)cells of cytokine-induced killer cells ( CIK)co-cultured with dendritic cells( DC). Methods:Peripheral blood mononuelear cells( PBMC)were isolated from healthy subjects and tumor patients. DC and CIK cells were induced by different cytokines. The phenotypes of CIK and DC cells before and after culture were determined by flow cytometry. The killing activity of different CIK groups against NB was determined by MTT. Results:The percentage of CD3+CD56+ cells as well as the cytotoxicity activity of CIK cells originated from PBMC of peripheral blood of healthy people were significantly higher than those of tumor patients(P<0. 05). In addition,compared with CIK cells,the DC-CIK cells presented a significantly higher killing effects on NB cells(P<0. 05). Conclusion:DC-CIK cell is a kind of immune cell which has a higher cyto-toxicity than that in CIK cell. CIK cells generated from PBMC of healthy subjects and tumor patients have significant differences,and provide further experimental basis for improving the clinical curative effect of CIK cells.%目的:研究细胞因子诱导的杀伤细胞( CIK)与树突状细胞( DC)共培养后对神经母细胞瘤( neuro-blastoma,NB)细胞株的杀伤作用。方法:取健康人和肿瘤患者外周血单个核细胞( PBMC),加入不同的细胞因子分别诱导出DC和CIK细胞,用流式细胞术测定诱导培养前后DC和CIK细胞的表型,MTT法测定不同组CIK细胞对NB细胞株的杀伤活性。结果:流式细胞仪检测健康人PBMC培养后CD3+CD56+淋巴细胞百分比以及对NB细胞株的杀伤活性均显著高于肿瘤患者( P<0.05)。此外,与单纯CIK细胞相比,DC-CIK细胞具有更强的杀伤NB细胞株的活性( P<0.05)。结论:DC-CIK细胞是一种细胞毒作用高于单纯CIK细胞的免疫活性细胞。健康人和肿瘤患者的PBMC经诱导培养获得的CIK细胞有显著差别,为临床进

  8. The response of normal and ataxia-telangiectasia cells to bleomycin: relationships between chromosome damage, cell cycle delay and cell killing.

    Science.gov (United States)

    Zampetti-Bosseler, F; Scott, D

    1985-08-01

    In agreement with our earlier observation (Scott and Zampetti-Bosseler, 1982) on X-irradiated normal and ataxia-telangiectasia (A-T) fibroblasts, we now report that after bleomycin or neocarzinostatin treatment also, A-T cells exhibit less G2 delay than normal cells. We confirm that A-T cells sustain more chromosome damage and lethality than normal cells after bleomycin. These observations support the hypothesis (Painter and Young, 1980) that A-T cells are defective in the recognition of certain lesions which normally lead to delays in progression through the cell cycle, during which they are repaired, and which, if unrepaired, lead to cell-lethal chromosome damage. However, we find that after bleomycin, as opposed to X-rays, the contribution of this type of lesion to cell death is minimal. The predominant lesions leading to cell death after bleomycin are not manifested at chromosome aberrations and do not lead to G2 delay or DNA-synthesis inhibition. A-T cells are defective in the recognition and/or repair of both types of lesion.

  9. Autophagy inhibition contributes to the synergistic interaction between EGCG and doxorubicin to kill the hepatoma Hep3B cells.

    Directory of Open Access Journals (Sweden)

    Li Chen

    Full Text Available (--Epigallocatechin-3-O-gallate(EGCG, the highest catechins from green tea, has promisingly been found to sensitize the efficacy of several chemotherapy agents like doxorubicin (DOX in hepatocellular carcinoma (HCC treatment. However, the detailed mechanisms by which EGCG augments the chemotherapeutic efficacy remain unclear. Herein, this study was designed to determine the synergistic impacts of EGCG and DOX on hepatoma cells and particularly to reveal whether the autophagic flux is involved in this combination strategy for the HCC. Electron microscopy and fluorescent microscopy confirmed that DOX significantly increased autophagic vesicles in hepatoma Hep3B cells. Western blot and trypan blue assay showed that the increasing autophagy flux by DOX impaired about 45% of DOX-induced cell death in these cells. Conversely, both qRT-PCR and western blotting showed that EGCG played dose-dependently inhibitory role in autophagy signaling, and that markedly promoted cellular growth inhibition. Amazingly, the combined treatment caused a synergistic effect with 40 to 60% increment on cell death and about 45% augmentation on apoptosis versus monotherapy pattern. The DOX-induced autophagy was abolished by this combination therapy. Rapamycin, an autophagic agonist, substantially impaired the anticancer effect of either DOX or combination with EGCG treatment. On the other hand, using small interference RNA targeting chloroquine autophagy-related gene Atg5 and beclin1 to inhibit autophagy signal, hepatoma cell death was dramatically enhanced. Furthermore, in the established subcutaneous Hep3B cells xenograft tumor model, about 25% reduction in tumor growth as well as 50% increment of apoptotic cells were found in combination therapy compared with DOX alone. In addition, immunohistochemistry analysis indicated that the suppressed tendency of autophagic hallmark microtubule-associated protein light chain 3 (LC3 expressions was consistent with thus combined

  10. Heat Killed Attenuated Leishmania Induces Apoptosis of HepG2 Cells Through ROS Mediated p53 Dependent Mitochondrial Pathway

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    Dipayan Bose

    2016-03-01

    Full Text Available Background/Aims: Cytotoxic effect of attenuated Leishmania on liver cancer cells by inducing ROS generation. Methods: Spectrophotometric study to analyze cell death and levels of different active caspases. Flow cytometric study was done to analyze apoptosis induction and ROS generation and levels of different protein. Western blot analysis was performed to study the levels of protein. Confocal microscopy was done to ascertain the expression of different apoptotic markers. Results: We have now observed that attenuated Leishmania donovani UR6 also has potentiality towards growth inhibition of HepG2 cells and investigated the mechanism of action. The effect is associated with increased DNA fragmentation, rise in number of annexinV positive cells, and cell cycle arrest at G1 phase. The detection of unregulated levels of active PARP, cleaved caspases 3 and 9, cytosolic cytochrome C, Bax, and Bad, along with the observed downregulation of Bcl-2 and loss of mitochondrial membrane potential suggested the involvement of mitochondrial pathway. Enhanced ROS and p53 levels regulate the apoptosis of HepG2 cells. NAC was found to inhibit p53 production but PFT-α has no effect on ROS generation. In conclusion, Leishmania donovani UR6 efficiently induces apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. Conclusion: It has been reported earlier that some parasites show prominent cytotoxic effect and prevent tumor growth. From our study we found that Leishmania donovani UR6 efficiently induced apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. This study has rejuvenated the age old idea of bio-therapy.

  11. Tetrandrine, a Compound Common in Chinese Traditional Medicine, Preferentially Kills Breast Cancer Tumor Initiating Cells (TICs) In Vitro

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    Xu, Wei; Debeb, Bisrat G.; Lacerda, Lara; Li, Jessica; Woodward, Wendy A., E-mail: wwoodward@mdanderson.org [Division of Radiation Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030 (United States)

    2011-05-04

    Tetrandrine is a bisbenzylisoquinoline alkaloid found in Stephania tetrandra, a Chinese medicine commonly used as an anti-inflammatory. It has extensive pharmacological activity, including positive ion channel blockade and inhibition of multiple drug resistance proteins. These activities are very similar to that of salinomycin, a known drug targeting breast cancer initiation cells (TICs). Herein, we tested tetrandrine targeting of breast cancer TICs. SUM-149, an inflammatory breast cancer cell line and SUM-159, a non-inflammatory metaplastic breast cancer cell line were used in these studies. In proliferation assays using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetrazolium (MTS), we found that the IC{sub 50} for inhibition of proliferation is 15.3 ± 4.1 μM for SUM-149 and 24.3 ± 2.1 μM for SUM-159 cells. Tetrandrine also inhibited mammosphere formation, a surrogate for breast cancer TICs growth in vitro with IC{sub 50} around 1 μM for SUM-149 and around 2 μM for SUM-159 cells. Tetrandrine has similar effects on the mammosphere formation from cells isolated from fresh patient sample. Moreover, tetrandrine decreases the aldehyde dehydrogenase (ALDH) positive population in SUM-159 by 45% ± 5.45% P = 0.005. In summary, tetrandrine demonstrates significant efficacy against in vitro surrogates for inflammatory and aggressive breast cancer TICs.

  12. Tetrandrine, a Compound Common in Chinese Traditional Medicine, Preferentially Kills Breast Cancer Tumor Initiating Cells (TICs In Vitro

    Directory of Open Access Journals (Sweden)

    Jessica Li

    2011-05-01

    Full Text Available Tetrandrine is a bisbenzylisoquinoline alkaloid found in Stephania tetrandra, a Chinese medicine commonly used as an anti-inflammatory. It has extensive pharmacological activity, including positive ion channel blockade and inhibition of multiple drug resistance proteins. These activities are very similar to that of salinomycin, a known drug targeting breast cancer initiation cells (TICs. Herein, we tested tetrandrine targeting of breast cancer TICs. SUM-149, an inflammatory breast cancer cell line and SUM-159, a non-inflammatory metaplastic breast cancer cell line were used in these studies. In proliferation assays using 3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium (MTS, we found that the IC50 for inhibition of proliferation is 15.3 ± 4.1 µM for SUM-149 and 24.3 ± 2.1 µM for SUM-159 cells. Tetrandrine also inhibited mammosphere formation, a surrogate for breast cancer TICs growth in vitro with IC50 around 1 µM for SUM-149 and around 2 µM for SUM-159 cells. Tetrandrine has similar effects on the mammosphere formation from cells isolated from fresh patient sample. Moreover, tetrandrine decreases the aldehyde dehydrogenase (ALDH positive population in SUM-159 by 45% ± 5.45% P = 0.005. In summary, tetrandrine demonstrates significant efficacy against in vitro surrogates for inflammatory and aggressive breast cancer TICs.

  13. Specific targeted killing of ErbB2 positive breast cancer by retrovirus-mediated Immunocaspase-3 secreting T cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Lihong; YANG An-gang; JIA Lintao; ZHAO Jing; XU Yanming; WEN Weihong; BAO Wei; CAO Yunxin; SU Chengzhi; WANG Chengji

    2004-01-01

    In this study, Immunocaspase-3 gene was transfected into Jurkat T lymphocytes and the targeted proapoptotic protein Immunocaspase-3 was stably secreted.Its entry to ErbB2 positive SKBr3 breast carcinoma cell line was observed by indirect immunofluorescence staining.Growth of SKBr3 cells was significantly inhibited when they were cultured with medium containing Immunocaspase-3.Next, Immunocaspase-3 gene was cloned into retrovirus vector pLNCX, which was then transfected into PA317 cells to package. Packaged cells producing high titer pseudoviruses were acquired and the pseudoviruses were harvested to infect PBMCs, which had been stimulated to division. The latter were selected and administered to nude mice bearing SKBr3 tumors through tail vein. The results showed that the treatment contributed to an inhibition of tumor growth and prolonged the lifetime of nude mice bearing SKBr3 tumor.The efficiency of inhibition of tumor reached 73.25%, and the average lifetime of treated nude mice was 80.95% longer than that of control group. Immunohistochemical examination revealed the exclusive distribution of Immunocaspase-3proteins only in the tumor tissue samples; and TUNEL assay confirmed the occurrence of apoptosis in tumor cells. The present study suggests that Immunocaspase-3 secreted by T lymphocytes can selectively bind and enter into ErbB2 positive breast cancer cells, where it exhibits a proapoptotic activity and causes tumor suppression in an in vivo tumor model.

  14. Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation

    Science.gov (United States)

    Peng, Yuanlin; Zhang, Qinming; Nagasawa, Hatsumi; Okayasu, Ryuichi; Liber, Howard L.; Bedford, Joel S.

    2002-01-01

    Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

  15. Circulating preproinsulin signal peptide-specific CD8 T cells restricted by the susceptibility molecule HLA-A24 are expanded at onset of type 1 diabetes and kill β-cells.

    Science.gov (United States)

    Kronenberg, Deborah; Knight, Robin R; Estorninho, Megan; Ellis, Richard J; Kester, Michel G; de Ru, Arnoud; Eichmann, Martin; Huang, Guo C; Powrie, Jake; Dayan, Colin M; Skowera, Ania; van Veelen, Peter A; Peakman, Mark

    2012-07-01

    Type 1 diabetes results from T cell-mediated β-cell destruction. The HLA-A*24 class I gene confers significant risk of disease and early onset. We tested the hypothesis that HLA-A24 molecules on islet cells present preproinsulin (PPI) peptide epitopes to CD8 cytotoxic T cells (CTLs). Surrogate β-cell lines secreting proinsulin and expressing HLA-A24 were generated and their peptide ligandome examined by mass spectrometry to discover naturally processed and HLA-A24-presented PPI epitopes. A novel PPI epitope was identified and used to generate HLA-A24 tetramers and examine the frequency of PPI-specific T cells in new-onset HLA-A*24(+) patients and control subjects. We identified a novel naturally processed and HLA-A24-presented PPI signal peptide epitope (PPI(3-11); LWMRLLPLL). HLA-A24 tetramer analysis reveals a significant expansion of PPI(3-11)-specific CD8 T cells in the blood of HLA-A*24(+) recent-onset patients compared with HLA-matched control subjects. Moreover, a patient-derived PPI(3-11)-specific CD8 T-cell clone shows a proinflammatory phenotype and kills surrogate β-cells and human HLA-A*24(+) islet cells in vitro. These results indicate that the type 1 diabetes susceptibility molecule HLA-A24 presents a naturally processed PPI signal peptide epitope. PPI-specific, HLA-A24-restricted CD8 T cells are expanded in patients with recent-onset disease. Human islet cells process and present PPI(3-11), rendering themselves targets for CTL-mediated killing.

  16. Vancomycin-modified mesoporous silica nanoparticles for selective recognition and killing of pathogenic gram-positive bacteria over macrophage-like cells.

    Science.gov (United States)

    Qi, Guobin; Li, Lili; Yu, Faquan; Wang, Hao

    2013-11-13

    Rapid, reliable recognition and detection of bacteria from an authentic specimen have been gained increasing interests in the past decades. Various materials have been designed and prepared for implementation of bacterial recognition and treatment in the artificial systems. However, in the complicated physiological condition, the macrophages always compromise the outcomes of bacterial detection and/or treatment. In this work, we demonstrated the vancomycin-modified mesoporous silica nanoparticles (MSNs is a subset of Van) for efficiently targeting and killing gram-positive bacteria over macrophage-like cells. Owing to the specific hydrogen bonding interactions of vancomycin toward the terminal d-alanyl-d-alanine moieties of gram-positive bacteria, the MSNs is a subset of Van exhibited enhanced recognition for gram-positive bacteria due to the multivalent hydrogen binding effect. Furthermore, the fluorescent molecules (FITC) were covalently decorated inside of mesopores of MSNs for tracking and visualizing the MSNs is a subset of Van during the detection/treatment processes. Upon incubation of FITC decorated MSNs with bacteria (i.e., S. aureus and E. coli as gram-positive and gram-negative bacteria, respectively) or macrophage-like cells (Raw 264.7), the fluorescence signals in S. aureus were 2-4 times higher than that in E. coli and no detectable fluorescence signals were observed in Raw 264.7 cells under the same condition. Finally, the MSNs is a subset of Van showed unambiguous antibacterial efficacy without decrease in cell viability of macrophage-like cells. This new strategy opens a new door for specific detection and treatment of pathogenic bacteria with minimized side effects.

  17. Death ligand TRAIL, secreted by CD1a+ and CD14+ cells in blister fluids, is involved in killing keratinocytes in toxic epidermal necrolysis.

    Science.gov (United States)

    de Araujo, Elisabeth; Dessirier, Valérie; Laprée, Geneviève; Valeyrie-Allanore, Laurence; Ortonne, Nicolas; Stathopoulos, Efstathios N; Bagot, Martine; Bensussan, Armand; Mockenhaupt, Maja; Roujeau, Jean-Claude; Tsapis, Andreas

    2011-02-01

    Toxic epidermal necrolysis (TEN) is characterized by an acute detachment and destruction of keratinocytes, affecting large areas of the skin. It is often related to adverse drug reactions. Previous studies have shown that effector CD8+ T cells, which accumulate in the blister fluid, are functionally cytotoxic and act through a classical perforin/granzyme B pathway. It has recently been shown that these cytotoxic T cells also secrete granulysin peptide, which is lethal to keratinocytes. These cytotoxic T cells exert their killer activity against autologous keratinocytes in the presence of the drug. However, they are unlikely to be the only effectors of TEN. We therefore searched for soluble death factors in the blister fluids that might kill keratinocytes. We found that the amounts of interferon-γ, TRAIL and TNF-α proteins were significantly greater in TEN blister fluids than in all controls (normal sera, TEN sera, burns and Eosinophilic pustular folliculitis blister fluids) and TNF-like weak inducer of apoptosis (TWEAK) amounts are also greater in all controls except burns. We showed that these proteins acted in synergy to induce the death of keratinocytes in vitro. We also found that TRAIL and TWEAK were secreted by CD1a+ and CD14+ cells present in the blister fluids. Thus, in addition to MHC class I-restricted cytotoxic T lymphocytes (CTLs), which lyse keratinocytes, ligands secreted by non-lymphoid cells capable of inducing keratinocyte death in an MHC class I-independent manner, also seem to be present in the blister fluids of patients with TEN.

  18. CD8 T cell-mediated killing of orexinergic neurons induces a narcolepsy-like phenotype in mice.

    Science.gov (United States)

    Bernard-Valnet, Raphaël; Yshii, Lidia; Quériault, Clémence; Nguyen, Xuan-Hung; Arthaud, Sébastien; Rodrigues, Magda; Canivet, Astrid; Morel, Anne-Laure; Matthys, Arthur; Bauer, Jan; Pignolet, Béatrice; Dauvilliers, Yves; Peyron, Christelle; Liblau, Roland S

    2016-09-27

    Narcolepsy with cataplexy is a rare and severe sleep disorder caused by the destruction of orexinergic neurons in the lateral hypothalamus. The genetic and environmental factors associated with narcolepsy, together with serologic data, collectively point to an autoimmune origin. The current animal models of narcolepsy, based on either disruption of the orexinergic neurotransmission or neurons, do not allow study of the potential autoimmune etiology. Here, we sought to generate a mouse model that allows deciphering of the immune mechanisms leading to orexin(+) neuron loss and narcolepsy development. We generated mice expressing the hemagglutinin (HA) as a "neo-self-antigen" specifically in hypothalamic orexin(+) neurons (called Orex-HA), which were transferred with effector neo-self-antigen-specific T cells to assess whether an autoimmune process could be at play in narcolepsy. Given the tight association of narcolepsy with the human leukocyte antigen (HLA) HLA-DQB1*06:02 allele, we first tested the pathogenic contribution of CD4 Th1 cells. Although these T cells readily infiltrated the hypothalamus and triggered local inflammation, they did not elicit the loss of orexin(+) neurons or clinical manifestations of narcolepsy. In contrast, the transfer of cytotoxic CD8 T cells (CTLs) led to both T-cell infiltration and specific destruction of orexin(+) neurons. This phenotype was further aggravated upon repeated injections of CTLs. In situ, CTLs interacted directly with MHC class I-expressing orexin(+) neurons, resulting in cytolytic granule polarization toward neurons. Finally, drastic neuronal loss caused manifestations mimicking human narcolepsy, such as cataplexy and sleep attacks. This work demonstrates the potential role of CTLs as final effectors of the immunopathological process in narcolepsy.

  19. Low pH immobilizes and kills human leukocytes and prevents transmission of cell-associated HIV in a mouse model

    Directory of Open Access Journals (Sweden)

    Markham Richard B

    2005-09-01

    results suggest that physiologic or microbicide-induced acid immobilization and killing of infected white blood cells may be effective in preventing sexual transmission of cell-associated HIV.

  20. Enhanced killing of androgen-independent prostate cancer cells using inositol hexakisphosphate in combination with proteasome inhibitors.

    Science.gov (United States)

    Diallo, J-S; Betton, B; Parent, N; Péant, B; Lessard, L; Le Page, C; Bertrand, R; Mes-Masson, A-M; Saad, F

    2008-11-18

    Effective treatments for androgen-independent prostate cancer (AIPCa) are lacking. To address this, emerging therapeutics such as proteasome inhibitors are currently undergoing clinical trials. Inositol hexakisphosphate (IP6) is an orally non-toxic phytochemical that exhibits antitumour activity against several types of cancer including PCa. We have previously shown that treatment of PC3 cells with IP6 induces the transcription of a subset of nuclear factor-kappaB (NF-kappaB)-responsive and pro-apoptotic BCL-2 family genes. In this study, we report that although NF-kappaB subunits p50/p65 translocate to the nucleus of PC3 cells in response to IP6, inhibition of NF-kappaB-mediated transcription using non-degradable inhibitor of kappaB (IkappaB)-alpha does not modulate IP6 sensitivity. Treatment with IP6 also leads to increased protein levels of PUMA, BIK/NBK and NOXA between 4 and 8 h of treatment and decreased levels of MCL-1 and BCL-2 after 24 h. Although blocking transcription using actinomycin D does not modulate PC3 cell sensitivity to IP6, inhibition of protein translation using cycloheximide has a significant protective effect. In contrast, blocking proteasome-mediated protein degradation using MG-132 significantly enhances the ability of IP6 to reduce cellular metabolic activity in both PC3 and DU145 AIPCa cell lines. This effect of combined treatment on mitochondrial depolarisation is particularly striking and is also reproduced by another proteasome inhibitor (ALLN). The enhanced effect of combined MG132/IP6 treatment is almost completely inhibited by cycloheximide and correlates with changes in BCL-2 family protein levels. Altogether these results suggest a role for BCL-2 family proteins in mediating the combined effect of IP6 and proteasome inhibitors and warrant further pre-clinical studies for the treatment of AIPCa.

  1. Sulforaphane enhances the anticancer activity of taxanes against triple negative breast cancer by killing cancer stem cells.

    Science.gov (United States)

    Burnett, Joseph P; Lim, Gi; Li, Yanyan; Shah, Ronak B; Lim, Rebekah; Paholak, Hayley J; McDermott, Sean P; Sun, Lichao; Tsume, Yasuhiro; Bai, Shuhua; Wicha, Max S; Sun, Duxin; Zhang, Tao

    2017-05-28

    Triple negative breast cancer (TNBC) typically exhibits rapid progression, high mortality and faster relapse rates relative to other breast cancer subtypes. In this report we examine the combination of taxanes (paclitaxel or docetaxel) with a breast cancer stem cell (CSC)-targeting agent sulforaphane for use against TNBC. We demonstrate that paclitaxel or docetaxel treatment induces IL-6 secretion and results in expansion of CSCs in TNBC cell lines. Conversely, sulforaphane is capable of preferentially eliminating CSCs, by inhibiting NF-κB p65 subunit translocation, downregulating p52 and consequent downstream transcriptional activity. Sulforaphane also reverses taxane-induced aldehyde dehydrogenase-positive (ALDH+) cell enrichment, and dramatically reduces the size and number of primary and secondary mammospheres formed. In vivo in an advanced treatment orthotopic mouse xenograft model together with extreme limiting dilution analysis (ELDA), the combination of docetaxel and sulforaphane exhibits a greater reduction in primary tumor volume and significantly reduces secondary tumor formation relative to either treatment alone. These results suggest that treatment of TNBCs with cytotoxic chemotherapy would be greatly benefited by the addition of sulforaphane to prevent expansion of and eliminate breast CSCs.

  2. A compound that inhibits the HOP-Hsp90 complex formation and has unique killing effects in breast cancer cell lines.

    Science.gov (United States)

    Pimienta, Genaro; Herbert, Kristina M; Regan, Lynne

    2011-12-05

    The chaperone Hsp90 is required for the correct folding and maturation of certain "client proteins" within all cells. Hsp90-mediated folding is particularly important in cancer cells, because upregulated or mutant oncogenic proteins are often Hsp90 clients. Hsp90 inhibitors thus represent a route to anticancer agents that have the potential to be active against several different types of cancer. Currently, various Hsp90 inhibitors that bind to Hsp90 at its ATP-binding site are in preclinical and clinical trials. Some of the most promising Hsp90 ATP-binding site inhibitors are the well characterized geldanamycin derivative 17-AAG and the recently described compounds PU-H71 and NVP-AUY922. An undesirable characteristic of these compounds is the transcriptional upregulation of Hsp70 that has prosurvival effects. Here we characterize the activity of a new type of chaperone inhibitor, 1,6-dimethyl-3-propylpyrimido[5,4-e][1,2,4]triazine-5,7-dione (named C9 for simplicity). Using purified protein components in vitro, C9 prevents Hsp90 from interacting with the cochaperone HOP and is thus expected to impair the Hsp90-dependent folding pathway in vivo. We show that this compound is effective in killing various breast cancer cell lines including the highly metastatic MDA-MB-231. An important property of this compound is that it does not induce the transcriptional upregulation of Hsp70. Moreover, when cells are treated with a combination of C9 and either 17-AAG or NVP-AUY922, the overexpression of Hsp70 is counteracted considerably and C9's lethal-IC50 decreases compared to its value when added alone.

  3. Fc-mediated activity of EGFR x c-Met bispecific antibody JNJ-61186372 enhanced killing of lung cancer cells.

    Science.gov (United States)

    Grugan, Katharine D; Dorn, Keri; Jarantow, Stephen W; Bushey, Barbara S; Pardinas, Jose R; Laquerre, Sylvie; Moores, Sheri L; Chiu, Mark L

    2017-01-01

    Epidermal growth factor receptor (EGFR) mutant non-small cell lung cancers acquire resistance to EGFR tyrosine kinase inhibitors through multiple mechanisms including c-Met receptor pathway activation. We generated a bispecific antibody targeting EGFR and c-Met (JNJ-61186372) demonstrating anti-tumor activity in wild-type and mutant EGFR settings with c-Met pathway activation. JNJ-61186372 was engineered with low fucosylation (<10 %), resulting in enhanced antibody-dependent cell-mediated cytotoxicity and FcγRIIIa binding. In vitro and in vivo studies with the single-arm EGFR or c-Met versions of JNJ-61186372 identified that the Fc-activity of JNJ-61186372 is mediated by binding of the anti-EGFR arm and required for inhibition of EGFR-driven tumor cells. In a tumor model driven by both EGFR and c-Met, treatment with Fc-silent JNJ-61186372 or with c-Met single-arm antibody reduced tumor growth inhibition compared to treatment with JNJ-61186372, suggesting that the Fc function of JNJ-61186372 is essential for maximal tumor inhibition. Moreover in this same model, downregulation of both EGFR and c-Met receptors was observed upon treatment with Fc-competent JNJ-61186372, suggesting that the Fc interactions are necessary for down-modulation of the receptors in vivo and for efficacy. These Fc-mediated activities, in combination with inhibition of both the EGFR and c-Met signaling pathways, highlight the multiple mechanisms by which JNJ-61186372 combats therapeutic resistance in EGFR mutant patients.

  4. Inhibition of HSP90 by AUY922 Preferentially Kills Mutant KRAS Colon Cancer Cells by Activating Bim through ER Stress.

    Science.gov (United States)

    Wang, Chun Yan; Guo, Su Tang; Wang, Jia Yu; Liu, Fen; Zhang, Yuan Yuan; Yari, Hamed; Yan, Xu Guang; Jin, Lei; Zhang, Xu Dong; Jiang, Chen Chen

    2016-03-01

    Oncogenic mutations of KRAS pose a great challenge in the treatment of colorectal cancer. Here we report that mutant KRAS colon cancer cells are nevertheless more susceptible to apoptosis induced by the HSP90 inhibitor AUY922 than those carrying wild-type KRAS. Although AUY922 inhibited HSP90 activity with comparable potency in colon cancer cells irrespective of their KRAS mutational statuses, those with mutant KRAS were markedly more sensitive to AUY922-induced apoptosis. This was associated with upregulation of the BH3-only proteins Bim, Bik, and PUMA. However, only Bim appeared essential, in that knockdown of Bim abolished, whereas knockdown of Bik or PUMA only moderately attenuated apoptosis induced by AUY922. Mechanistic investigations revealed that endoplasmic reticulum (ER) stress was responsible for AUY922-induced upregulation of Bim, which was inhibited by a chemical chaperone or overexpression of GRP78. Conversely, siRNA knockdown of GRP78 or XBP-1 enhanced AUY922-induced apoptosis. Remarkably, AUY922 inhibited the growth of mutant KRAS colon cancer xenografts through activation of Bim that was similarly associated with ER stress. Taken together, these results suggest that AUY922 is a promising drug in the treatment of mutant KRAS colon cancers, and the agents that enhance the apoptosis-inducing potential of Bim may be useful to improve the therapeutic efficacy.

  5. Nanoassemblies Based on Supramolecular Complexes of Nonionic Amphiphilic Cyclodextrin and Sorafenib as Effective Weapons to Kill Human HCC Cells.

    Science.gov (United States)

    Bondì, Maria Luisa; Scala, Angela; Sortino, Giuseppe; Amore, Erika; Botto, Chiara; Azzolina, Antonina; Balasus, Daniele; Cervello, Melchiorre; Mazzaglia, Antonino

    2015-12-14

    Sorafenib (Sor), an effective chemiotherapeutic drug utilized against hepatocellular carcinoma (HCC), robustly interacts with nonionic amphiphilic cyclodextrin (aCD, SC6OH), forming, in aqueous solution, supramolecular complexes that behave as building blocks of highly water-dispersible colloidal nanoassemblies. SC6OH/Sor complex has been characterized by complementary spectroscopic techniques, such as UV-vis, steady-state fluorescence and anisotropy, resonance light scattering and (1)H NMR. The spectroscopic evidences and experiments carried out in the presence of an adamantane derivative, which competes with drug for CD cavity, agree with the entrapment of Sor in aCD, pointing out the role of the aCD cavity in the interaction between drug and amphiphile. Nanoassemblies based on SC6OH/Sor display size of ∼200 nm, negative zeta-potential (ζ = -11 mV), and both maximum loading capacity (LC ∼ 17%) and entrapment efficiency (EE ∼ 100%). Kinetic release profiles show a slower release of Sor from nanoassemblies with respect to the free drug. SC6OH/Sor nanoassemblies have very low hemolytic activity and high efficiency in vitro in decreasing cell growth and viability of HCC cell lines, such as HepG2, Hep3B, and PLC/PRF/5, opening promising chances to their in vivo applications.

  6. Investigating CTL mediated killing with a 3D cellular automaton.

    Directory of Open Access Journals (Sweden)

    Frederik Graw

    2009-08-01

    Full Text Available Cytotoxic T lymphocytes (CTLs are important immune effectors against intra-cellular pathogens. These cells search for infected cells and kill them. Recently developed experimental methods in combination with mathematical models allow for the quantification of the efficacy of CTL killing in vivo and, hence, for the estimation of parameters that characterize the effect of CTL killing on the target cell populations. It is not known how these population-level parameters relate to single-cell properties. To address this question, we developed a three-dimensional cellular automaton model of the region of the spleen where CTL killing takes place. The cellular automaton model describes the movement of different cell populations and their interactions. Cell movement patterns in our cellular automaton model agree with observations from two-photon microscopy. We find that, despite the strong spatial nature of the kinetics in our cellular automaton model, the killing of target cells by CTLs can be described by a term which is linear in the target cell frequency and saturates with respect to the CTL levels. Further, we find that the parameters describing CTL killing on the population level are most strongly impacted by the time a CTL needs to kill a target cell. This suggests that the killing of target cells, rather than their localization, is the limiting step in CTL killing dynamics given reasonable frequencies of CTL. Our analysis identifies additional experimental directions which are of particular importance to interpret estimates of killing rates and could advance our quantitative understanding of CTL killing.

  7. Predicting In Silico Which Mixtures of the Natural Products of Plants Might Most Effectively Kill Human Leukemia Cells?

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    Hany A. El-Shemy

    2013-01-01

    Full Text Available The aim of the analysis of just 13 natural products of plants was to predict the most likely effective artificial mixtures of 2-3 most effective natural products on leukemia cells from over 364 possible mixtures. The natural product selected included resveratrol, honokiol, chrysin, limonene, cholecalciferol, cerulenin, aloe emodin, and salicin and had over 600 potential protein targets. Target profiling used the Ontomine set of tools for literature searches of potential binding proteins, binding constant predictions, binding site predictions, and pathway network pattern analysis. The analyses indicated that 6 of the 13 natural products predicted binding proteins which were important targets for established cancer treatments. Improvements in effectiveness were predicted for artificial combinations of 2 or 3 natural products. That effect might be attributed to drug synergism rather than increased numbers of binding proteins bound (dose effects. Among natural products, the combinations of aloe emodin with mevinolin and honokiol were predicted to be the most effective combination for AML-related predicted binding proteins. Therefore, plant extracts may in future provide more effective medicines than the single purified natural products of modern medicine, in some cases.

  8. An actinomycete isolate from solitary wasp mud nest having strong antibacterial activity and kills the Candida cells due the shrinkage and the cytosolic loss

    Directory of Open Access Journals (Sweden)

    Vijay eKumar

    2014-08-01

    Full Text Available An actinomycetes strain designated as MN 2(6 was isolated from the solitary wasp mud nest. The isolate was identified using polyphasic taxonomy. It produced the extensive branched brown substrate and white aerial hyphae that changed into grayish black. The aerial mycelia produced the spiral spore chains with rugose spore surface. The growth was observed between temperature range of 27-37°C, pH 8-10 and below salt concentration of 6% (w/v. The comparative analysis of 16S rRNA gene sequence and phylogenetic relationship showed that strain MN 2(6 lies in clade with Streptomyces hygroscopicus subsp. hygroscopicus NRRL 2387T, Streptomyces sporocinereus NBRC 100766T and Streptomyces demainii NRRL B-1478T with which it shares a 16S rRNA gene sequence similarity of 99.3%. The strain MN 2(6 can be differentiated from type strains based on phenotypic characteristics. The strain MN 2(6 showed most promising activity against Gram-positive, Gram-negative bacteria, acid-fast bacilli and Candida species suggesting broad-spectrum characteristics of the active metabolite. Evaluation of anti-candidal activity of the metabolite of strain MN 2(6 by scanning electron microscopy (SEM revealed changed external morphology of yeast. It kills the Candida cells due to the shrinkage and the cytosolic loss. However, further studies are required to elucidate the structure of the active metabolite produced by the isolate MN 2(6

  9. Theriocide: Naming Animal Killing

    Directory of Open Access Journals (Sweden)

    Piers Beirne

    2014-08-01

    Full Text Available In this essay I recommend ‘theriocide’ as the name for those diverse human actions that cause the deaths of animals. Like the killing of one human by another, theriocide may be socially acceptable or unacceptable, legal or illegal. It may be intentional or unintentional and may involve active maltreatment or passive neglect. Theriocide may occur one-on-one, in small groups or in large-scale social institutions. The numerous and sometimes intersecting sites of theriocide include intensive rearing regimes; hunting and fishing; trafficking; vivisection; militarism; pollution; and human-induced climate change. If the killing of animals by humans is as harmful to them as homicide is to humans, then the proper naming of such deaths offers a remedy, however small, to the extensive privileging of human lives over those of other animals. Inevitably, the essay leads to a shocking question: Is theriocide murder?

  10. 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

  11. Burkholderia pseudomallei Capsule Exacerbates Respiratory Melioidosis but Does Not Afford Protection against Antimicrobial Signaling or Bacterial Killing in Human Olfactory Ensheathing Cells.

    Science.gov (United States)

    Dando, Samantha J; Ipe, Deepak S; Batzloff, Michael; Sullivan, Matthew J; Crossman, David K; Crowley, Michael; Strong, Emily; Kyan, Stephanie; Leclercq, Sophie Y; Ekberg, Jenny A K; St John, James; Beacham, Ifor R; Ulett, Glen C

    2016-07-01

    Melioidosis, caused by the bacterium Burkholderia pseudomallei, is an often severe infection that regularly involves respiratory disease following inhalation exposure. Intranasal (i.n.) inoculation of mice represents an experimental approach used to study the contributions of bacterial capsular polysaccharide I (CPS I) to virulence during acute disease. We used aerosol delivery of B. pseudomallei to establish respiratory infection in mice and studied CPS I in the context of innate immune responses. CPS I improved B. pseudomallei survival in vivo and triggered multiple cytokine responses, neutrophil infiltration, and acute inflammatory histopathology in the spleen, liver, nasal-associated lymphoid tissue, and olfactory mucosa (OM). To further explore the role of the OM response to B. pseudomallei infection, we infected human olfactory ensheathing cells (OECs) in vitro and measured bacterial invasion and the cytokine responses induced following infection. Human OECs killed >90% of the B. pseudomallei in a CPS I-independent manner and exhibited an antibacterial cytokine response comprising granulocyte colony-stimulating factor, tumor necrosis factor alpha, and several regulatory cytokines. In-depth genome-wide transcriptomic profiling of the OEC response by RNA-Seq revealed a network of signaling pathways activated in OECs following infection involving a novel group of 378 genes that encode biological pathways controlling cellular movement, inflammation, immunological disease, and molecular transport. This represents the first antimicrobial program to be described in human OECs and establishes the extensive transcriptional defense network accessible in these cells. Collectively, these findings show a role for CPS I in B. pseudomallei survival in vivo following inhalation infection and the antibacterial signaling network that exists in human OM and OECs.

  12. Melphalan, alone or conjugated to an FSH-β peptide, kills murine testicular cells in vitro and transiently suppresses murine spermatogenesis in vivo.

    Science.gov (United States)

    Amory, John K; Hong, SungWoo; Yu, Xiaozhong; Muller, Charles H; Faustman, Elaine; Goldstein, Alex

    2014-07-01

    New approaches to sterilizing male animals are needed to control captive and wild animal populations. We sought to develop a nonsurgical method of permanent sterilization for male animals by administering the gonadotoxicant melphalan conjugated to peptides derived from the β-chain of FSHβ. We hypothesized that conjugating melphalan to FSHβ peptides would magnify the gonadotoxic effects of melphalan while minimizing systemic toxicity. The ability of conjugates of melphalan and FSHβ peptides to kill murine testicular cells was first tested in vitro in a three-dimensional testicular cell coculture system. In this system, melphalan caused considerable cell death as measured both by increases in lactate dehydrogenase concentrations in the culture supernatant and direct visualization of the cultures. Of the conjugates tested, melphalan conjugated to a 20-amino acid peptide derived from human FSHβ consisting of amino acids 33 to 53 (FSHβ (33-53)-melphalan) was very potent, with cell cytotoxicity and lactate dehydrogenase release roughly one-half that of melphalan. The effects of melphalan and FSHβ (33-53)-melphalan on spermatogenesis were then tested in vivo in mature C56Bl/6 male mice. Four weeks after intraperitoneal injection, all mice treated with either FSHβ (33-53)-melphalan or melphalan had approximately 75% reductions in testicular spermatid counts compared with control animals. Testicular histology revealed significant reduction in mature spermatids and spermatocytes in most tubules. However, 12 weeks after the injection, testicular spermatid counts and histology were similar to controls, except in one animal receiving FSHβ (33-53)-melphalan that had no apparent spermatogenesis. We conclude that melphalan and FSHβ (33-53)-melphalan are potent gonadotoxicants in male mice resulting in marked suppression of spermatogenesis 4 weeks after a single intraperitoneal injection. However, this effect is transient in most mice as spermatogenesis is similar to

  13. Rapid and transient activation of gamma/delta T cells to interferon gamma production, NK cell-like killing and antigen processing during acute virus infection

    Science.gov (United States)

    Gamma/delta T cells are the majority peripheral blood T cells in young cattle. The role of gamma/delta T cells in innate responses against infection with foot-and-mouth disease virus (FMDV) was analyzed on 5 consecutive days following infection. Before infection, bovine gamma/delta T cells expressed...

  14. Vibriocidal antibody responses to a bivalent killed whole-cell oral cholera vaccine in a phase III trial in Kolkata, India.

    Directory of Open Access Journals (Sweden)

    Suman Kanungo

    Full Text Available BACKGROUND: During the development of a vaccine, identification of the correlates of protection is of paramount importance for establishing an objective criterion for the protective performance of the vaccine. However, the ascertainment of correlates of immunity conferred by any vaccine is a difficult task. METHODS: While conducting a phase three double-blind, cluster-randomized, placebo-controlled trial of a bivalent killed whole-cell oral cholera vaccine in Kolkata, we evaluated the immunogenicity of the vaccine in a subset of participants. Randomly chosen participants (recipients of vaccine or placebo were invited to provide blood samples at baseline, 14 days after the second dose and one year after the first dose. At these time points, serum geometric mean titers (GMT of vibriocidal antibodies and seroconversion rates for vaccine and placebo arms were calculated and compared across the age strata (1 to 5 years, 5 to 15 years and more than 15 years as well as for all age groups. RESULTS: Out of 137 subjects included in analysis, 69 were vaccinees and 68 received placebo. There were 5•7 and 5•8 geometric mean fold (GMF rises in titers to Vibrio cholerae Inaba and Ogawa, respectively at 14 days after the second dose, with 57% and 61% of vaccinees showing a four-fold or greater titer rise, respectively. After one year, the titers to Inaba and Ogawa remained 1•7 and 2•8 fold higher, respectively, compared to baseline. Serum vibriocidal antibody response to V. cholerae O139 was much lower than that to Inaba or Ogawa. No significant differences in the GMF-rises were observed among the age groups. CONCLUSIONS: The reformulated oral cholera vaccine induced a statistically significant anti-O1 Inaba and O1 Ogawa vibriocidal antibody response 14 days after vaccination, which although declined after one year remained significantly higher than baseline. Despite this decline, the vaccine remained protective five years after vaccination.

  15. Kill the Killers: terapia con células Natural Killer en pacientes pediátricos con cáncer refractario Kill the Killers: Natural Killer cells therapy against paediatric refractory solid tumours

    Directory of Open Access Journals (Sweden)

    J. Valentín Quiroga

    2012-09-01

    Full Text Available Introducción: En el momento actual, los tumores sólidos refractarios al tratamiento convencional constituyen la principal causa de muerte en la edad pediátrica. Por tanto, es necesario desarrollar y consolidar nuevos tratamientos. Las células Natural Killer (NK constituyen la primera línea de defensa del sistema inmune frente al desarrollo de células tumorales. Planteamos una nueva estrategia de terapia celular antitumoral en niños con cánceres refractarios, inmunoterapia con células NK estimuladas con interleucina 15 (IL-15. Pacientes y Métodos: En 22 pacientes pediátricos con tumores sólidos refractarios y en controles sanos determinamos mediante citometría de flujo multiparamétrica y fluorescencia resuelta en el tiempo, el fenotipo y la actividad citotóxica de las células NK, respectivamente. En ratones inmunodeficientes desarrollamos un modelo de neuroblastoma metastático muy agresivo y terapia de rescate con células Natural Killer estimuladas con IL-15. Resultados: Los pacientes pediátricos con cáncer refractario tienen un mayor porcentaje de células NK bright y una menor actividad citotóxica. La estimulación con IL-15 mejora la citotoxicidad in vitro y disminuye la carga tumoral in vivo. Conclusiones: Las células NK estimuladas con IL-15 constituyen una prometedora estrategia antitumoral.Introduction: Refractory solid tumours lead children deaths. To change this statement, new treatments should be developed. Natural Killer cells constitute the first line of defence against tumour cells. We propose a new strategy for antitumor cell therapy in children with refractory solid malignancies: IL-15 stimulated NK cells. Patients and methods: 22 paediatric patients suffering refractory solid tumours participate in this study. We compare NK cell subsets and K562 cytotoxicity in patients and sex-age pair's healthy controls. We use multiparametric flow and time-resolved fluorescent, respectively. In immunocompromised mice we

  16. Small CD4 Mimetics Prevent HIV-1 Uninfected Bystander CD4+ T Cell Killing Mediated by Antibody-dependent Cell-mediated Cytotoxicity

    Directory of Open Access Journals (Sweden)

    Jonathan Richard

    2016-01-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 infection causes a progressive depletion of CD4+ T cells. Despite its importance for HIV-1 pathogenesis, the precise mechanisms underlying CD4+ T-cell depletion remain incompletely understood. Here we make the surprising observation that antibody-dependent cell-mediated cytotoxicity (ADCC mediates the death of uninfected bystander CD4+ T cells in cultures of HIV-1-infected cells. While HIV-1-infected cells are protected from ADCC by the action of the viral Vpu and Nef proteins, uninfected bystander CD4+T cells bind gp120 shed from productively infected cells and are efficiently recognized by ADCC-mediating antibodies. Thus, gp120 shedding represents a viral mechanism to divert ADCC responses towards uninfected bystander CD4+ T cells. Importantly, CD4-mimetic molecules redirect ADCC responses from uninfected bystander cells to HIV-1-infected cells; therefore, CD4-mimetic compounds might have therapeutic utility in new strategies aimed at specifically eliminating HIV-1-infected cells.

  17. Analysing the Wrongness of Killing

    DEFF Research Database (Denmark)

    Di Nucci, Ezio

    2014-01-01

    This article provides an in-depth analysis of the wrongness of killing by comparing different versions of three influential views: the traditional view that killing is always wrong; the liberal view that killing is wrong if and only if the victim does not want to be killed; and Don Marquis‟ future...... of value account of the wrongness of killing. In particular, I illustrate the advantages that a basic version of the liberal view and a basic version of the future of value account have over competing alternatives. Still, ultimately none of the views analysed here are satisfactory; but the different...... reasons why those competing views fail provide important insights into the ethics of killing....

  18. Killing Spinors -- Beyond Supergravity

    CERN Document Server

    Palomo-Lozano, Alberto

    2012-01-01

    This is a doctoral thesis on the application of techniques originally developed in the programme of characterisation of supersymmetric solutions to Supergravity theories, to finding alternative backgrounds. We start by discussing the concept of a Killing spinor, and how these are paramount to the process of classifying of these aforementioned supersymmetric solutions. Moreover, these geometric objects also have applications when considered in different scenarios (the 'beyond' in the title). In particular, techniques based on a parallelising rule for a spinorial field can be used for obtaining solutions to Einstein-Maxwell-De Sitter theories, as well as a (partial) classification of Lorentzian Einstein-Weyl manifolds, a problem of geometrical interest. The annexe contain an introduction and summary in Spanish language. The appendices discuss the tensorial and spinorial conventions employed, some relevant geometrical information on the scalar manifolds for the matter contents of interest, as well as for the nul...

  19. How to kill creativity.

    Science.gov (United States)

    Amabile, T M

    1998-01-01

    In today's knowledge economy, creativity is more important than ever. But many companies unwittingly employ managerial practices that kill it. How? By crushing their employees' intrinsic motivation--the strong internal desire to do something based on interests and passions. Managers don't kill creativity on purpose. Yet in the pursuit of productivity, efficiency, and control--all worthy business imperatives--they undermine creativity. It doesn't have to be that way, says Teresa Amabile. Business imperatives can comfortably coexist with creativity. But managers will have to change their thinking first. Specifically, managers will need to understand that creativity has three parts: expertise, the ability to think flexibly and imaginatively, and motivation. Managers can influence the first two, but doing so is costly and slow. It would be far more effective to increase employees' intrinsic motivation. To that end, managers have five levers to pull: the amount of challenge they give employees, the degree of freedom they grant around process, the way they design work groups, the level of encouragement they give, and the nature of organizational support. Take challenge as an example. Intrinsic motivation is high when employees feel challenged but not overwhelmed by their work. The task for managers, therefore, becomes matching people to the right assignments. Consider also freedom. Intrinsic motivation--and thus creativity--soars when managers let people decide how to achieve goals, not what goals to achieve. Managers can make a difference when it comes to employee creativity. The result can be truly innovative companies in which creativity doesn't just survive but actually thrives.

  20. Killing Symmetry on Finsler Manifold

    CERN Document Server

    Ootsuka, Takayoshi; Ishida, Muneyuki

    2016-01-01

    Killing vector fields $K$ are defined on Finsler manifold. The Killing symmetry is reformulated simply as $\\delta K^\\flat =0$ by using the Killing non-linear 1-form $K^\\flat$ and the spray operator $\\delta$ with the Finsler non-linear connection. $K^\\flat$ is related to the generalization of Killing tensors on Finsler manifold, and the condition $\\delta K^\\flat =0$ gives an analytical method of finding higher derivative conserved quantities, which may be called hidden conserved quantities. We show two examples: the Carter constant on Kerr spacetime and the Runge-Lentz vectors in Newtonian gravity.

  1. Garlic compounds selectively kill childhood pre-B acute lymphoblastic leukemia cells in vitro without reducing T-cell function: Potential therapeutic use in the treatment of ALL

    Directory of Open Access Journals (Sweden)

    Greg Hodge

    2008-03-01

    Full Text Available Greg Hodge1, Stephen Davis2, Michael Rice1, Heather Tapp1, Ben Saxon1, Tamas Revesz11Haematology/Oncology Department, Women’s and Children’s Hospital, North Adelaide, Australia; 2Department of Mycology, Women’s and Children’s Hospital, North Adelaide, AustraliaAbstract: Drugs used for remission induction therapy for childhood precursor-B acute lymphoblastic leukemia (ALL are nonselective for malignant cells. Several garlic compounds have been shown to induce apoptosis of cancer cells and to alter lymphocyte function. To investigate the effect of garlic on the apoptosis of ALL cells and lymphocyte immune function, cells from newly diagnosed childhood ALL patients were cultured with several commonly used chemotherapeutic agents and several garlic compounds. Apoptosis, lymphocyte proliferation and T-cell cytokine production were determined using multiparameter flow cytometry. At concentrations of garlic compounds that did not result in significant increases in Annexin V and 7-AAD staining of normal lymphocytes, there was a significant increase in apoptosis of ALL cells with no alteration of T-cell proliferation as determined by CD25/CD69 upregulation or interferonγ, interleukin-2 or tumor necrosis factor-α intracellular cytokine production. In contrast, the presence of chemotherapeutic agents resulted in nonselective increases in both lymphocyte and ALL apoptosis and a decrease in T-cell proliferation and cytokine production. In conclusion, we show selective apoptosis of malignant cells by garlic compounds that do not alter T-cell immune function and indicate the potential therapeutic benefit of garlic compounds in the treatment of childhood ALL.Keywords: childhood precursor-B acute lymphoblastic leukemia, garlic, apoptosis, immune function, intracellular cytokines

  2. An impedance-based cytotoxicity assay for real-time and label-free assessment of T-cell-mediated killing of adherent cells.

    Science.gov (United States)

    Peper, Janet Kerstin; Schuster, Heiko; Löffler, Markus W; Schmid-Horch, Barbara; Rammensee, Hans-Georg; Stevanović, Stefan

    2014-03-01

    The in vitro assessment of T-cell-mediated cytotoxicity plays an important and increasingly relevant role both in preclinical target evaluation and during immunomonitoring to accompany clinical trials employing targeted immunotherapies. For a long time, the gold standard for this purpose has been the chromium release assay (CRA). This end point assay, however, shows several disadvantages including the inevitable use of radioactivity. Based on electrical impedance measurements (using the xCELLigence system), we have established a label-free assay, facilitating the real-time monitoring of T-cell-mediated cytotoxicity. The coculture of peptide-specific T-cell lines with peptide-loaded target cells reproducibly led to a decrease in impedance due to induced apoptosis and detachment of target cells. Comparing our results to the standard CRA assay, we could demonstrate that impedance-based measurements show comparable results after short incubation periods (6h) but outperform the CRA both in reproducibility and sensitivity after prolonged incubation (24h), enabling the detection of target cell lysis with an effector to target ratio as low as 0.05:1. The impedance-based assay represents a valuable and highly sensitive tool for label-free real-time high throughput analysis of T-cell-mediated cytotoxicity.

  3. Antiproliferative and proapoptotic effects of viable or heat-killed Lactobacillus paracasei IMPC2.1 and Lactobacillus rhamnosus GG in HGC-27 gastric and DLD-1 colon cell lines.

    Science.gov (United States)

    Orlando, A; Refolo, M G; Messa, C; Amati, L; Lavermicocca, P; Guerra, V; Russo, F

    2012-01-01

    Data from literature suggest the possible use of probiotics as chemopreventive agents against colon cancer, but few investigations are available on their effects on gastric cancer proliferation. In our previous study, a specific Lactobacillus, strain L. paracasei IMPC2.1, was demonstrated to colonize the human gut and positively affect fecal bacteria and biochemical parameters. The aims of the present study were to investigate the effects of L. paracasei IMPC2.1, comparing them with those of Lactobacillus rhamnosus GG (L.GG), either as viable or heat-killed cells, on cell proliferation and apoptosis in a gastric cancer (HGC-27) and a colorectal cancer cell line (DLD-1). Both the gastric and colon cancer cells were sensitive to the growth inhibition and apoptosis induction by both viable or heat-killed cells from L. paracasei IMPC2.1 and L.GG. These findings suggest the possibility for a food supplement, based on dead probiotics, including L. paracasei IMPC2.1 cells, which could represent an effective component of a functional food strategy for cancer growth inhibition, with potential for cancer prevention.

  4. Diphtheria Toxin/Human B-Cell Activating Factor Fusion Protein Kills Human Acute Lymphoblastic Leukemia BALL-1 Cells: An Experimental Study

    Institute of Scientific and Technical Information of China (English)

    Xin-pu Gao; Zheng-min Liu; Yu-lian Jiao; Bin Cui; Yue-ting Zhu; Jie Zhang; Lai-cheng Wang; Yue-ran Zhao

    2012-01-01

    Objective:This study aimed to express a fusion protein of diphtheria toxin and human B ceil-activating factor (DT388sBAFF) in Escherichia coli (E.coli) and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells (BALL-1).Methods:A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde Ⅰ and Xho Ⅰ,and inserted into the expression vector pcold Ⅱ digested with the same enzymes.Recombinants were screened by the colony polymerase chain reaction (PCR) and restriction map.The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG).The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot,and then purified by Ni2+-NTA affinity chromatography.The expression level of B cell-activating factor receptor (BAFF-R) on BALL-1 cells was assessed by real-time PCR.The receptor binding capacity of recombinant protein was determined by cell fluorescent assay.The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay.Results:The expression level of recombinant protein was 50% of total bacterial proteins in E.coli,and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells.Conclusion:The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained,providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia.

  5. Expression of thymidine kinase mediated by a novel non-viral delivery system under the control of vascular endothelial growth factor receptor 2 promoter selectively kills human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Ying Wang; Hui-Xiong Xu; Ming-De Lu; Qing Tang

    2008-01-01

    AIM: To investigate the killing efficiency of a recombinant plasmid containing a thymidine kinase (TK) domain insert driven by the vascular endothelial growth factor receptor 2 (VEGFR2) promoter (KDR) on vascular endothelial cells.METHODS: The KDR-TK fragment was extracted from pBluescript 11 KDR-TK plasmid by enzymatic digestion with XhoI and Sa/I. The enhanced green fluorescence protein (EGFP) carrier was extracted from pEGFP by the same procedure. The KDR-TK was inserted into the pEGFP carrier to construct pEGFP-KDR-TK. Using ultrasound irradiation and microbubble,pEGFP-KDR-TK was transferred into human umbilical vein endothelial cells (HUVECs). The transient infection rate was estimated by green fluorescent protein (GFP)expression. Transfected HUVECs, non-transfected HUVECs, and HepG2 cells were cultured in the presence of different concentrations of ganciclovir (GCV), and the killing efficacy of HSV-TK/GCV was analyzed by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTr) assay.RESULTS: The recombinant pEGFP-KDR-TK was successfully constructed by inserting the KDR-TK fragment into the pEGFP carrier. Transfected HUVECs showed cytoplasmic green fluorescence, and the transient transfection rate was about 20.3%. Pools of G418-resistant cells exhibited a higher sensitivity to the prodrug/GCV compared to non-transfected HUVECs or non-transfected HepG2 cells, respectively.CONCLUSION: KDR promoter and the suicide gene/prodrug system mediated by diagnostic ultrasound combined with microbubble can significantly kill HUVECs.Such therapy may present a novel and attractive approach to target gene therapy on tumor vessels.

  6. TGF-β-induced CD4+Foxp3+ T cells attenuate acute graft-versus-host disease via suppressing expansion and killing of effector CD8+ cells

    OpenAIRE

    Gu, Jian; Lu, Ling; Chen, Maogen; Xu, Lili; Lan, Qin; Li, Qiang; Liu, Zhongmin; Chen, Guihua; Wang, Ping; Wang, Xuehao; Brand, David; Olsen, Nancy; Zheng, Song Guo

    2014-01-01

    TGF-β-induced CD4+Foxp3+ T cells (iTregs) have been identified as important prevention and treatment strategies for cell therapy in autoimmune diseases and other disorders. However, the potential use of iTregs as a treatment modality for acute graft-verse-host disease (GVHD) has not been realized because iTregs may be unstable and less suppressive in this disease. Here we restudied the ability of iTregs to prevent and treat acute GVHD in two different mouse models. Our results showed that so ...

  7. Extracts from Calendula officinalis offer in vitro protection against H2 O2 induced oxidative stress cell killing of human skin cells.

    Science.gov (United States)

    Alnuqaydan, Abdullah M; Lenehan, Claire E; Hughes, Rachel R; Sanderson, Barbara J

    2015-01-01

    The in vitro safety and antioxidant potential of Calendula officinalis flower head extracts was investigated. The effect of different concentrations (0.125, 0.5, 1.0, 2.0 and 5.0% (v/v)) of Calendula extracts on human skin cells HaCaT in vitro was explored. Doses of 1.0% (v/v) (0.88 mg dry weight/mL) or less showed no toxicity. Cells were also exposed to the Calendula extracts for either 4, 24 or 48 h before being exposed to an oxidative insult (hydrogen peroxide H2 O2 ) for 1 h. Using the MTT cytotoxicity assay, it was observed that two independent extracts of C. officinalis gave time-dependent and concentration-dependent H2 O2 protection against induced oxidative stress in vitro using human skin cells. Pre-incubation with the Calendula extracts for 24 and 48 h increased survival relative to the population without extract by 20% and 40% respectively following oxidative challenge. The antioxidant potential of the Calendula extracts was confirmed using a complimentary chemical technique, the DPPH(●) assay. Calendula extracts exhibited free radical scavenging abilities. This study demonstrates that Calendula flower extracts contain bioactive and free radical scavenging compounds that significantly protect against oxidative stress in a human skin cell culture model.

  8. Pathophysiological and immune cell responses in calves prior to and following lung challenge with formalin-killed Pasteurella multocida biotype A:3 and protection studies involving subsequent homologous live challenge.

    Science.gov (United States)

    Dowling, A; Hodgson, J C; Dagleish, M P; Eckersall, P D; Sales, J

    2004-08-01

    Pneumonic pasteurellosis is a common respiratory infection in cattle that has major economic and welfare implications world-wide and the incidence in the UK due to Pasteurella multocida, currently the same as that associated with Mannheimia haemolytica, is increasing. Whereas much is known regarding the pathogenesis of M. haemolytica infections little information is available on the pathogenic process of pasteurellosis initiated by P. multocida. In the present work calf systemic and innate immune responses to intratracheal challenge with formalin-killed P. multocida biotype A:3 and to subsequent experimental lung infection with live P. multocida were investigated. Eight-week-old calves were challenged intratracheally on day 0 with either 10(9) colony forming units (cfu) of formalin-killed P. multocida biotype A:3 in 300 ml saline (n = 10) or 300 ml saline alone (n = 10), followed, at day 21, by challenge with 10(9) cfu live P. multocida. Pathophysiological and lung phagocyte responses were assessed by clinical monitoring, sequential lung lavage and blood sampling. Results for samples obtained before, during and after challenge showed clinical and acute phase protein responses to both bacterial culture and saline control treatments, although higher responses were associated with bacterial challenge. Phagocytosis of P. multocida during 1h incubation periods with lavaged cells in vitro was unaffected by exposure in vivo to killed P. multocida and there was evidence that P. multocida was able to survive intracellularly during this assay. There was no indication that lung exposure to formalin-killed P. multocida conferred protection against subsequent homologous live challenge.

  9. Killing by neutrophil extracellular traps: fact or folklore?

    Science.gov (United States)

    Menegazzi, Renzo; Decleva, Eva; Dri, Pietro

    2012-02-02

    Neutrophil extracellular traps (NETs) are DNA structures released by dying neutrophils and claimed to constitute a new microbicidal mechanism. Killing by NET-forming cells is ascribed to these structures because it is prevented by preincubation with DNase, which has been shown to dismantle NETs, before addition of the target microorganisms. Curiously, the possibility that the microorganisms ensnared in NETs are alive has not been considered. Using Staphylococcus aureus and Candida albicans blastospores, we demonstrate that the microorganisms captured by NETs and thought to be killed are alive because they are released and recovered in cell medium by incubation with DNase. It is concluded that NETs entrap but do not kill microbes.

  10. C16-Ceramide Analog Combined with Pc 4 Photodynamic Therapy Evokes Enhanced Total Ceramide Accumulation, Promotion of DEVDase Activation in the Absence of Apoptosis, and Augmented Overall Cell Killing

    Directory of Open Access Journals (Sweden)

    Duska Separovic

    2011-01-01

    Full Text Available Because of the failure of single modality approaches, combination therapy for cancer treatment is a promising alternative. Sphingolipid analogs, with or without anticancer drugs, can improve tumor response. C16-pyridinium ceramide analog LCL30, was used in combination with photodynamic therapy (PDT, an anticancer treatment modality, to test the hypothesis that the combined treatment will trigger changes in the sphingolipid profile and promote cell death. Using SCCVII mouse squamous carcinoma cells, and the silicone phthalocyanine Pc 4 for PDT, we showed that combining PDT with LCL30 (PDT/LCL30 was more effective than individual treatments in raising global ceramide levels, as well as in reducing dihydrosphingosine levels. Unlike LCL30, PDT, alone or combined, increased total dihydroceramide levels. Sphingosine levels were unaffected by LCL30, but were abolished after PDT or the combination. LCL30-triggered rise in sphingosine-1-phosphate was reversed post-PDT or the combination. DEVDase activation was evoked after PDT or LCL30, and was promoted post- PDT/LCL30. Neither mitochondrial depolarization nor apoptosis were observed after any of the treatments. Notably, treatment with the combination resulted in augmented overall cell killing. Our data demonstrate that treatment with PDT/LCL30 leads to enhanced global ceramide levels and DEVDase activation in the absence of apoptosis, and promotion of total cell killing.

  11. Human alpha-lactalbumin made lethal to tumor cells (HAMLET) kills human glioblastoma cells in brain xenografts by an apoptosis-like mechanism and prolongs survival.

    Science.gov (United States)

    Fischer, Walter; Gustafsson, Lotta; Mossberg, Ann-Kristin; Gronli, Janne; Mork, Sverre; Bjerkvig, Rolf; Svanborg, Catharina

    2004-03-15

    Malignant brain tumors present a major therapeutic challenge because no selective or efficient treatment is available. Here, we demonstrate that intratumoral administration of human alpha-lactalbumin made lethal to tumor cells (HAMLET) prolongs survival in a human glioblastoma (GBM) xenograft model, by selective induction of tumor cell apoptosis. HAMLET is a protein-lipid complex that is formed from alpha-lactalbumin when the protein changes its tertiary conformation and binds oleic acid as a cofactor. HAMLET induces apoptosis in a wide range of tumor cells in vitro, but the therapeutic effect in vivo has not been examined. In this study, invasively growing human GBM tumors were established in nude rats (Han:rnu/rnu Rowett, n = 20) by transplantation of human GBM biopsy spheroids. After 7 days, HAMLET was administered by intracerebral convection-enhanced delivery for 24 h into the tumor area; and alpha-lactalbumin, the native, folded variant of the same protein, was used as a control. HAMLET reduced the intracranial tumor volume and delayed the onset of pressure symptoms in the tumor-bearing rats. After 8 weeks, all alpha-lactalbumin-treated rats had developed pressure symptoms, but the HAMLET-treated rats remained asymptomatic. Magnetic resonance imaging scans revealed large differences in tumor volume (456 versus 63 mm(3)). HAMLET caused apoptosis in vivo in the tumor but not in adjacent intact brain tissue or in nontransformed human astrocytes, and no toxic side effects were observed. The results identify HAMLET as a new candidate in cancer therapy and suggest that HAMLET should be additionally explored as a novel approach to controlling GBM progression.

  12. Modulation of Th1/Th2 Immune Responses by Killed Propionibacterium acnes and Its Soluble Polysaccharide Fraction in a Type I Hypersensitivity Murine Model: Induction of Different Activation Status of Antigen-Presenting Cells

    Directory of Open Access Journals (Sweden)

    Carla Cristina Squaiella-Baptistão

    2015-01-01

    Full Text Available Propionibacterium acnes (P. acnes is a gram-positive anaerobic bacillus present in normal human skin microbiota, which exerts important immunomodulatory effects, when used as heat- or phenol-killed suspensions. We previously demonstrated that heat-killed P. acnes or its soluble polysaccharide (PS, extracted from the bacterium cell wall, suppressed or potentiated the Th2 response to ovalbumin (OVA in an immediate hypersensitivity model, depending on the treatment protocol. Herein, we investigated the mechanisms responsible for these effects, using the same model and focusing on the activation status of antigen-presenting cells (APCs. We verified that higher numbers of APCs expressing costimulatory molecules and higher expression levels of these molecules are probably related to potentiation of the Th2 response to OVA induced by P. acnes or PS, while higher expression of toll-like receptors (TLRs seems to be related to Th2 suppression. In vitro cytokines production in cocultures of dendritic cells and T lymphocytes indicated that P. acnes and PS seem to perform their effects by acting directly on APCs. Our data suggest that P. acnes and PS directly act on APCs, modulating the expression of costimulatory molecules and TLRs, and these differently activated APCs drive distinct T helper patterns to OVA in our model.

  13. Minimal killing unit of the mitochondrial targeting domain of Noxa.

    Science.gov (United States)

    Kim, Ji-Young; Han, Ji Hye; Moon, Ae-Ran; Park, Jung Hee; Chang, Jeong Hwan; Bae, Jeehyeon; Kim, Tae-Hyoung

    2013-08-01

    Noxa is a key player in p53-induced cell death via mitochondrial dysfunction, and the mitochondrial-targeting domain (MTD) of Noxa is responsible for the translocation of Noxa to mitochondria and for the induction of necrotic cell death. The purpose of this study was to define the minimal killing unit of MTD in vitro and in vivo. It was found that the peptides R8:MTD(10), R8:MTD(9), and R8:MTD(8) can kill various human tumor cells (HCT116, HeLa, MCF-7, BJAB), but that R8:MTD(7) abolishes the killing activity of MTD mainly because of the loss of mitochondrial targeting activity. We find it interesting that R8:MTD(8) was found to kill tumor cells but showed a limited killing activity on normal peritoneal macrophages. Furthermore, R8:MTD(10), R8:MTD(9), and R8:MTD(8) limitedly suppressed tumor growth when injected i.v. into BalB/C mice bearing CT26 cell-derived tumors. These results indicate that MTD(8) is the minimal killing unit of MTD.

  14. Phantom metrics with Killing spinors

    Directory of Open Access Journals (Sweden)

    W.A. Sabra

    2015-11-01

    Full Text Available We study metric solutions of Einstein–anti-Maxwell theory admitting Killing spinors. The analogue of the IWP metric which admits a space-like Killing vector is found and is expressed in terms of a complex function satisfying the wave equation in flat (2+1-dimensional space–time. As examples, electric and magnetic Kasner spaces are constructed by allowing the solution to depend only on the time coordinate. Euclidean solutions are also presented.

  15. Human neutrophils dump Candida glabrata after intracellular killing.

    Science.gov (United States)

    Essig, Fabian; Hünniger, Kerstin; Dietrich, Stefanie; Figge, Marc Thilo; Kurzai, Oliver

    2015-11-01

    Interaction between fungal pathogens and human phagocytes can lead to remarkably variable outcomes, ranging from intracellular killing to prolonged survival and replication of the pathogen in the host cell. Using live cell imaging we observed primary human neutrophils that release phagocytosed Candida glabrata yeast cells after intracellular killing. This process, for which we propose the name "dumping", adds a new outcome to phagocyte-fungus interaction which may be of potential immunological importance as it allows professional antigen presenting cells to take up and process neutrophil-inactivated pathogens that in their viable state are able to evade intracellular degradation in these cells.

  16. Enhanced Cancer Cell (HeLa Killing Efficacy of Mixed Αlpha and Gamma Iron Oxide Superparamagnetic Nanoparticles under Combined AC (Alternating Current Magnetic-Field and Photoexcitation

    Directory of Open Access Journals (Sweden)

    Md. Shariful Islam, Yoshihumi Kusumoto, Md. Abdulla Al-Mamun and Yuji Horie

    2011-12-01

    Full Text Available We synthesized mixed α and γ-Fe2O3 nanoparticles and investigated their toxic effects against HeLa cells under induced AC (alternating current magnetic-fields and photoexcited conditions at room temperature. The findings revealed that the cell-killing percentage was increased with increasing dose for all types of treatments. Finally, 99% cancer cells were destructed at 1.2 mL dose when exposed to combined AC magnetic-field and photoexcited conditions (T3 whereas 89 and 83 % of HeLa cells were killed under only AC magnetic-field induced (T1 or only photoexcited (T2 condition at the same dose.ABSTRAK: Campuran α dan zarah γ-Fe2O3 bersaiz nano disintesiskan dan kesan toksidnya terhadap sel HeLa dikaji dibawah aruhan medan magnet arus ulang-alik (alternating current (AC dan keadaan photoexcited (proses ransangan atom atau molekul suatu bahan dengan penyerapan tenaga sinaran pada suhu bilik. Penemuan mendedahkan bahawa peratusan sel yang musnah bertambah dengan pertambahan dos untuk semua jenis rawatan. Akhirnya, 99% sel kanser dimusnahkan pada kadar dos 1.2mL setelah didedahkan terhadap kombinasi medan magnet AC dan keadaan photoexcited (T3 dimana 89% dan 83% sel HeLa dimusnahkan dengan hanya di bawah aruhan medan magnet AC (T1 atau hanya pada keadaan photoexcited (T2 pada kadar dos yang sama.KEY WORDS : Cancer, Hyperthermia, Iron oxide nanoparticles, Heat dissipation,    Cytotoxicity, HeLa cell.

  17. Killing, letting die and euthanasia.

    Science.gov (United States)

    Husak, D N

    1979-12-01

    Medical ethicists debate whether or not the moral assessment of cases of euthanasia should depend on whether the patient is 'killed' or 'allowed to die'. The usual presupposition is that a clear distinction between killing and letting die can be drawn so that this substantive question is not begged. I contend that the categorisation of cases of instances of killing rather than as instances of letting die depends in part on a prior moral assessment of the case. Hence is it trivially rather than substantively true that the distinction has moral significance. But even if a morally neutral (ie non-question begging) distinction could be drawn, its application to the euthanasia controversy is problematic. I illustrate the difficulties of employing this distinction to reach moral conclusions by critically discussing Philippa Foot's recent treatment of euthanasia. I conclude that even if an act of euthanasia is an instance of killing, and there exists a prima facie moral duty not to kill, and no more stringent duty overrides this duty, one still cannot determine such an act to be morally impermissible.

  18. Studies of colicin action on wall-less stable L-forms of Escherichia coli. I. Degree of attachment and of killing effect on rods and stable L-form cells.

    Science.gov (United States)

    Smarda, J; Schuhmann, E

    1979-01-01

    Escherichia coli strains B and K12 W 1655 F+ are able to bind more lethal units of colicins E2, E3, G, H, Ia, and K+ X per one stable L-form cell (of the protoplast type) than per one rod cell; colicin D is bound in a higher amount on E. coli B rods. This pattern remains unchanged, if the same colicins are attached on chloroform-killed cells of both forms. Rods of both E. coli strains are more sensitive to colicins D, E2, E3, K + X (as--in the strain B--to colicin Ia) than cells of the respective L-forms. In the strain W 1655 F+ both cell forms are equally highly sensitive to colicin Ia. The stable L-forms of both strains are much more sensitive to colicins G and H than the rods. Thus the Gram-negative cell wall decreases the probability of a colicin molecule to get attached to its receptor in the cytoplasmic membrane. On the other hand, in E. coli cells the attachment of most colicin molecules to the wall receptors increases the probability of their biological effect. There is no such effect of the wall-attachment on the action of colicins G or H. The strain B is tolerant to colicin E2, while being resistant to E3; thus the cytoplasmic membrane receptor sites for them are not identical.

  19. Extracellular Electron Transfer from Aerobic Bacteria to Au-Loaded TiO2 Semiconductor without Light: A New Bacteria-Killing Mechanism Other than Localized Surface Plasmon Resonance or Microbial Fuel Cells.

    Science.gov (United States)

    Wang, Guomin; Feng, Hongqing; Gao, Ang; Hao, Qi; Jin, Weihong; Peng, Xiang; Li, Wan; Wu, Guosong; Chu, Paul K

    2016-09-21

    Titania loaded with noble metal nanoparticles exhibits enhanced photocatalytic killing of bacteria under light illumination due to the localized surface plasmon resonance (LSPR) property. It has been shown recently that loading with Au or Ag can also endow TiO2 with the antibacterial ability in the absence of light. In this work, the antibacterial mechanism of Au-loaded TiO2 nanotubes (Au@TiO2-NT) in the dark environment is studied, and a novel type of extracellular electron transfer (EET) between the bacteria and the surface of the materials is observed to cause bacteria death. Although the EET-induced bacteria current is similar to the LSPR-related photocurrent, the former takes place without light, and no reactive oxygen species (ROS) are produced during the process. The EET is also different from that commonly attributed to microbial fuel cells (MFC) because it is dominated mainly by the materials' surface, but not the bacteria, and the environment is aerobic. EET on the Au@TiO2-NT surface kills Staphylococcus aureus, but if it is combined with special MFC bacteria, the efficiency of MFC may be improved significantly.

  20. Advanced application of bovine intestinal epithelial cell line for evaluating regulatory effect of lactobacilli against heat-killed enterotoxigenicEscherichia coli-mediated inflammation

    OpenAIRE

    Takanashi, Naoya; Tomosada, Yohsuke; Villena, Julio Cesar; Murata, Kozue; Takahashi, Takuya; Chiba, Eriko; Tohno, Masanori; Tomoyuki Shimazu; Aso, Hisashi; Suda, Yoshihito; Ikegami, Shuji; Itoh, Hiroyuki; Kawai, Yasushi; Tadao Saito; Alvarez, Gladis Susana

    2015-01-01

    Background: Previously, a bovine intestinal epithelial cell line (BIE cells) was successfully established. This work hypothesized that BIE cells are useful in vitro model system for the study of interactions of microbial- or pathogenassociated molecular patterns (MAMPs or PAMPs) with bovine intestinal epithelial cells and for the selection of immunoregulatory lactic acid bacteria (LAB). Results: All toll-like receptor (TLR) genes were expressed in BIE cells, being TLR4 one of the most strong...

  1. HecA, a member of a class of adhesins produced by diverse pathogenic bacteria, contributes to the attachment, aggregation, epidermal cell killing, and virulence phenotypes of Erwinia chrysanthemi EC16 on Nicotiana clevelandii seedlings.

    Science.gov (United States)

    Rojas, Clemencia M; Ham, Jong Hyun; Deng, Wen-Ling; Doyle, Jeff J; Collmer, Alan

    2002-10-01

    Erwinia chrysanthemi is representative of a broad class of bacterial pathogens that are capable of inducing necrosis in plants. The E. chrysanthemi EC16 hecA gene predicts a 3,850-aa member of the Bordetella pertussis filamentous hemagglutinin family of adhesins. A hecATn7 mutant was reduced in virulence on Nicotiana clevelandii seedlings after inoculation without wounding. Epifluorescence and confocal laser-scanning microscopy observations of hecA and wild-type cells expressing the green fluorescent protein revealed that the mutant is reduced in its ability to attach and then form aggregates on leaves and to cause an aggregate-associated killing of epidermal cells. Cell killing also depended on production of the major pectate lyase isozymes and the type II, but not the type III, secretion pathway in E. chrysanthemi. HecA homologs were found in bacterial pathogens of plants and animals and appear to be unique to pathogens and universal in necrogenic plant pathogens. Phylogenetic comparison of the conserved two-partner secretion domains in the proteins and the 16S rRNA sequences in respective bacteria revealed the two datasets to be fundamentally incongruent, suggesting horizontal acquisition of these genes. Furthermore, hecA and its two homologs in Yersinia pestis had a G+C content that was 10% higher than that of their genomes and similar to that of plant pathogenic Ralstonia, Xylella, and Pseudomonas spp. Our data suggest that filamentous hemagglutinin-like adhesins are broadly important virulence factors in both plant and animal pathogens.

  2. 3,4-二苯基香豆素衍生物的合成及其促自然杀伤细胞活性研究%Synthesis of 3,4-Biphenyl Coumarin Derivatives and Their Promoting Effects on the Killing Activity of Natural Killing Cell

    Institute of Scientific and Technical Information of China (English)

    陈华; 周利凯; 李帅; 姚玉超; 谷云景; 李春晓; 李娜; 孟明; 李小六

    2013-01-01

    以水杨酸为起始原料,经Fridel-Crafts酰基化反应、微波促进的Perkin反应和两步亲核取代反应,合成了具有氨基烷氧侧链的3,4-二苯基香豆素衍生物1a~1e.初步生物活性测试表明,化合物1a~1e能显著促进自然杀伤细胞杀伤活性,具有较强的免疫调节活性.%A series of novel 3,4-biphenyl coumarin derivatives containing an amino side chain 1a~1e were synthesized using salicylic acid as starting material via Fridel-Crafts acylation, then the microwave-assisted Perkin reaction and double SN2 substitution reactions. The preliminary biological evaluation showed that compounds 1a~1e had significant promoting effects on the killing activity of natural killer cell, and hold potential as immunomodulating agents.

  3. "The Killing Fields" of Innovation

    DEFF Research Database (Denmark)

    Ingerslev, Karen

    2014-01-01

    This paper points to seemingly contradicted processes of framing innovation, idea generation and killing ideas. It reports from a yearlong innovation project, where health care professionals explored problems and tested ideas for solutions, regarding a future downsizing of the case hospital...

  4. To Kill For An Image

    DEFF Research Database (Denmark)

    Fausing, Bent

    Images can provide both an overview and insight, but also the opposite. This ambivalence has become an even bigger part of the nature of the image, of what is an Image? Today we kill for an image, seen from afar on a screen and captured by a drone. The time also asks: Should it be big data...

  5. Human Neutrophils Kill Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    2005-11-01

    Full Text Available Bacillus anthracis spores cause natural infections and are used as biological weapons. Inhalation infection with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. In this study we infected human neutrophils with either spores or vegetative bacteria of a wild-type strain, or strains, expressing only one of the two major virulence factors. The human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then efficiently killed. Interestingly, neutrophil killing was independent of reactive oxygen species production. We fractionated a human neutrophil granule extract by high-performance liquid chromatography and identified alpha-defensins as the component responsible for B. anthracis killing. These data suggest that the timely recruitment of neutrophils can control cutaneous infections and possibly other forms of B. anthracis infections, and that alpha-defensins play an important role in the potent anti-B. anthracis activity of neutrophils.

  6. Human neutrophils kill Bacillus anthracis.

    Directory of Open Access Journals (Sweden)

    Anne Mayer-Scholl

    2005-11-01

    Full Text Available Bacillus anthracis spores cause natural infections and are used as biological weapons. Inhalation infection with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. In this study we infected human neutrophils with either spores or vegetative bacteria of a wild-type strain, or strains, expressing only one of the two major virulence factors. The human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then efficiently killed. Interestingly, neutrophil killing was independent of reactive oxygen species production. We fractionated a human neutrophil granule extract by high-performance liquid chromatography and identified alpha-defensins as the component responsible for B. anthracis killing. These data suggest that the timely recruitment of neutrophils can control cutaneous infections and possibly other forms of B. anthracis infections, and that alpha-defensins play an important role in the potent anti-B. anthracis activity of neutrophils.

  7. Human neutrophils kill Bacillus anthracis.

    Science.gov (United States)

    Mayer-Scholl, Anne; Hurwitz, Robert; Brinkmann, Volker; Schmid, Monika; Jungblut, Peter; Weinrauch, Yvette; Zychlinsky, Arturo

    2005-11-01

    Bacillus anthracis spores cause natural infections and are used as biological weapons. Inhalation infection with B. anthracis, the etiological agent of anthrax, is almost always lethal, yet cutaneous infections usually remain localized and resolve spontaneously. Neutrophils are typically recruited to cutaneous but seldom to other forms of anthrax infections, raising the possibility that neutrophils kill B. anthracis. In this study we infected human neutrophils with either spores or vegetative bacteria of a wild-type strain, or strains, expressing only one of the two major virulence factors. The human neutrophils engulfed B. anthracis spores, which germinated intracellularly and were then efficiently killed. Interestingly, neutrophil killing was independent of reactive oxygen species production. We fractionated a human neutrophil granule extract by high-performance liquid chromatography and identified alpha-defensins as the component responsible for B. anthracis killing. These data suggest that the timely recruitment of neutrophils can control cutaneous infections and possibly other forms of B. anthracis infections, and that alpha-defensins play an important role in the potent anti-B. anthracis activity of neutrophils.

  8. Introduction of Transplant Registry Unified Management Program 2 (TRUMP2): scripts for TRUMP data analyses, part I (variables other than HLA-related data).

    Science.gov (United States)

    Atsuta, Yoshiko

    2016-01-01

    Collection and analysis of information on diseases and post-transplant courses of allogeneic hematopoietic stem cell transplant recipients have played important roles in improving therapeutic outcomes in hematopoietic stem cell transplantation. Efficient, high-quality data collection systems are essential. The introduction of the Second-Generation Transplant Registry Unified Management Program (TRUMP2) is intended to improve data quality and more efficient data management. The TRUMP2 system will also expand possible uses of data, as it is capable of building a more complex relational database. The construction of an accessible data utilization system for adequate data utilization by researchers would promote greater research activity. Study approval and management processes and authorship guidelines also need to be organized within this context. Quality control of processes for data manipulation and analysis will also affect study outcomes. Shared scripts have been introduced to define variables according to standard definitions for quality control and improving efficiency of registry studies using TRUMP data.

  9. Resveratrol Specifically Kills Cancer Cells by a Devastating Increase in the Ca2+ Coupling Between the Greatly Tethered Endoplasmic Reticulum and Mitochondria

    Science.gov (United States)

    Madreiter-Sokolowski, Corina T.; Gottschalk, Benjamin; Parichatikanond, Warisara; Eroglu, Emrah; Klec, Christiane; Waldeck-Weiermair, Markus; Malli, Roland; Graier, Wolfgang F.

    2017-01-01

    Background/Aims Resveratrol and its derivate piceatannol are known to induce cancer cell-specific cell death. While multiple mechanisms of actions have been described including the inhibition of ATP synthase, changes in mitochondrial membrane potential and ROS levels, the exact mechanisms of cancer specificity of these polyphenols remain unclear. This paper is designed to reveal the molecular basis of the cancer-specific initiation of cell death by resveratrol and piceatannol. Methods The two cancer cell lines EA.hy926 and HeLa, and somatic short-term cultured HUVEC were used. Cell viability and caspase 3/7 activity were tested. Mitochondrial, cytosolic and endoplasmic reticulum Ca2+ as well as cytosolic and mitochondrial ATP levels were measured using single cell fluorescence microscopy and respective genetically-encoded sensors. Mitochondria-ER junctions were analyzed applying super-resolution SIM and ImageJ-based image analysis. Results Resveratrol and piceatannol selectively trigger death in cancer but not somatic cells. Hence, these polyphenols strongly enhanced mitochondrial Ca2+ uptake in cancer exclusively. Resveratrol and piceatannol predominantly affect mitochondrial but not cytosolic ATP content that yields in a reduced SERCA activity. Decreased SERCA activity and the strongly enriched tethering of the ER and mitochondria in cancer cells result in an enhanced MCU/Letm1-dependent mitochondrial Ca2+ uptake upon intracellular Ca2+ release exclusively in cancer cells. Accordingly, resveratrol/piceatannol-induced cancer cell death could be prevented by siRNA-mediated knock-down of MCU and Letm1. Conclusions Because their greatly enriched ER-mitochondria tethering, cancer cells are highly susceptible for resveratrol/piceatannol-induced reduction of SERCA activity to yield mitochondrial Ca2+ overload and subsequent cancer cell death. PMID:27606689

  10. Cytotoxic macrophage-released tumour necrosis factor-alpha (TNF-α) as a killing mechanism for cancer cell death after cold plasma activation

    Science.gov (United States)

    Kaushik, Nagendra Kumar; Kaushik, Neha; Min, Booki; Choi, Ki Hong; Hong, Young June; Miller, Vandana; Fridman, Alexander; Choi, Eun Ha

    2016-03-01

    The present study aims at studying the anticancer role of cold plasma-activated immune cells. The direct anti-cancer activity of plasma-activated immune cells against human solid cancers has not been described so far. Hence, we assessed the effect of plasma-treated RAW264.7 macrophages on cancer cell growth after co-culture. In particular, flow cytometer analysis revealed that plasma did not induce any cell death in RAW264.7 macrophages. Interestingly, immunofluorescence and western blot analysis confirmed that TNF-α released from plasma-activated macrophages acts as a tumour cell death inducer. In support of these findings, activated macrophages down-regulated the cell growth in solid cancer cell lines and induced cell death in vitro. Together our findings suggest plasma-induced reactive species recruit cytotoxic macrophages to release TNF-α, which blocks cancer cell growth and can have the potential to contribute to reducing tumour growth in vivo in the near future.

  11. 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...

  12. WOMEN'S RIGHTS VIOLATION: HONOUR KILLINGS

    Directory of Open Access Journals (Sweden)

    CRISTINA OTOVESCU FRASIE

    2011-04-01

    Full Text Available In this study I have presented the domestic violence concept and the situation regarding the observing of woman’s rights in Syria. We have also evidenced the juridical aspects regarding the honor killing directed against women after the modification of the article 548 from the Penal Code changed by the President al-Asad on July the 1st 2009. The data offered by NGOs have been of great help for the elaboration of the study as also the statistic data presented in Thara E-Magazine regarding the cities where had been done the honor killings and their number, the instrument of the murder, the age of the victim, and the motives for the murders. It must be noticed that, lately, the Government fought for the observing of the woman’s rights and promoted he gender equality by appointing women in leading positions, including the vice-president one.

  13. Photothermal-triggered control of sub-cellular drug accumulation using doxorubicin-loaded single-walled carbon nanotubes for the effective killing of human breast cancer cells

    Science.gov (United States)

    Oh, Yunok; Jin, Jun-O.; Oh, Junghwan

    2017-03-01

    Single-walled carbon nanotubes (SWNTs) are often the subject of investigation as effective photothermal therapy (PTT) agents owing to their unique strong optical absorption. Doxorubicin (DOX)-loaded SWNTs (SWNTs-DOX) can be used as an efficient therapeutic agent for combined near infrared (NIR) cancer photothermal and chemotherapy. However, SWNTs-DOX-mediated induction of cancer cell death has not been fully investigated, particularly the reaction of DOX inside cancer cells by PTT. In this study, we examined how the SWNTs-DOX promoted effective MDA-MB-231 cell death compared to DOX and PTT alone. We successfully synthesized the SWNTs-DOX. The SWNTs-DOX exhibited a slow DOX release, which was accelerated by NIR irradiation. Furthermore, DOX released from the SWNTs-DOX accumulated inside the cells at high concentration and effectively localized into the MDA-MB-231 cell nucleus. A combination of SWNTs-DOX and PTT promoted an effective MDA-MB-231 cell death by mitochondrial disruption and ROS generation. Thus, SWNTs-DOX can be utilized as an excellent anticancer agent for early breast cancer treatment.

  14. Killing efficacy of a new silicon phthalocyanine in human melanoma cells treated with photodynamic therapy by early activation of mitochondrion-mediated apoptosis.

    Science.gov (United States)

    Barge, Jérôme; Decréau, Richard; Julliard, Michel; Hubaud, Jean-Claude; Sabatier, Anne-Sophie; Grob, Jean-Jacques; Verrando, Patrick

    2004-01-01

    Photodynamic therapy (PDT) is a promising therapeutic modality that utilizes a combination of a photosensitizer and visible light for the destruction of diseased tissues. Using human-pigmented melanoma cells, we examined the photokilling efficacy of new silicon-phthalocyanines (SiPc) that bore bulky axial substituents. The bis(cholesteryloxy) derivate (Chol-O-SiPc) displayed the best in vitro photokilling efficacy (LD(50) = 6-8 x 10(-9) M) and was seven to nine times more potent than chloro-aluminium Pc (ClAlPc), a known photosensitizer used as a reference. Although Chol-O-SiPc was half as potent as ClAlPc for promoting photo-oxidative membrane damage in a cell-free assay, early events of mitochondrion-mediated apoptosis upon PDT were triggered much faster, as demonstrated by kinetics studies examining cells with permeabilized mitochondrial membranes, cytochrome c release and caspase-9 activation. Inhibition of caspase-9 activity by a substrate analogue argued for its central role in the proapoptotic events leading to cell death by Chol-O-SiPc PDT. In addition, immunoblots showed that Bcl-2 antiapoptotic oncoprotein was not a primary target of Chol-O-SiPc in M3Dau cells treated with PDT. Conclusively, Chol-O-SiPc is a useful new photosensitizer with the property of triggering cell apoptosis mediated by mitochondria.

  15. Tailored Nanoparticle Codelivery of antimiR-21 and antimiR-10b Augments Glioblastoma Cell Kill by Temozolomide: Toward a "Personalized" Anti-microRNA Therapy.

    Science.gov (United States)

    Ananta, Jeyarama S; Paulmurugan, Ramasamy; Massoud, Tarik F

    2016-09-01

    Glioblastoma remains an aggressive brain malignancy with poor prognosis despite advances in multimodal therapy that include standard use of Temozolomide. MicroRNA-21 (miR-21) and microRNA-10b (miR-10b) are oncomiRs overexpressed in glioblastoma, promoting many aspects of cancer biology. We hypothesized that PLGA nanoparticles carrying antisense miR-21 (antimiR-21) and antisense miR-10b (antimiR-10b) might beneficially knockdown endogenous miR-21 and miR-10b function and reprogram cells prior to Temozolomide treatment. PLGA nanoparticles were effective in intracellular delivery of encapsulated oligonucleotides. Concentrations of delivered antimiR-21 and antimiR-10b were optimized and specifically tailored to copy numbers of intracellular endogenous microRNAs. Coinhibition of miR-21 and miR-10b significantly reduced the number of viable cells (by 24%; p < 0.01) and increased (2.9-fold) cell cycle arrest at G2/M phase upon Temozolomide treatment in U87 MG cells. Cell-tailored nanoparticle-assisted concurrent silencing of miR-21 and miR-10b prior to Temozolomide treatment is an effective molecular therapeutic strategy in cell culture, warranting the need for further studies prior to future in vivo "personalized" medicine applications.

  16. Anti-protozoal and anti-bacterial antibiotics that inhibit protein synthesis kill cancer subtypes enriched for stem cell-like properties.

    Science.gov (United States)

    Cuyàs, Elisabet; Martin-Castillo, Begoña; Corominas-Faja, Bruna; Massaguer, Anna; Bosch-Barrera, Joaquim; Menendez, Javier A

    2015-01-01

    Key players in translational regulation such as ribosomes might represent powerful, but hitherto largely unexplored, targets to eliminate drug-refractory cancer stem cells (CSCs). A recent study by the Lisanti group has documented how puromycin, an old antibiotic derived from Streptomyces alboniger that inhibits ribosomal protein translation, can efficiently suppress CSC states in tumorspheres and monolayer cultures. We have used a closely related approach based on Biolog Phenotype Microarrays (PM), which contain tens of lyophilized antimicrobial drugs, to assess the chemosensitivity profiles of breast cancer cell lines enriched for stem cell-like properties. Antibiotics directly targeting active sites of the ribosome including emetine, puromycin and cycloheximide, inhibitors of ribosome biogenesis such as dactinomycin, ribotoxic stress agents such as daunorubicin, and indirect inhibitors of protein synthesis such as acriflavine, had the largest cytotoxic impact against claudin-low and basal-like breast cancer cells. Thus, biologically aggressive, treatment-resistant breast cancer subtypes enriched for stem cell-like properties exhibit exacerbated chemosensitivities to anti-protozoal and anti-bacterial antibiotics targeting protein synthesis. These results suggest that old/existing microbicides might be repurposed not only as new cancer therapeutics, but also might provide the tools and molecular understanding needed to develop second-generation inhibitors of ribosomal translation to eradicate CSC traits in tumor tissues.

  17. CDK1 Inhibition Targets the p53-NOXA-MCL1 Axis, Selectively Kills Embryonic Stem Cells, and Prevents Teratoma Formation

    Directory of Open Access Journals (Sweden)

    Noelle E. Huskey

    2015-03-01

    Full Text Available Embryonic stem cells (ESCs have adopted an accelerated cell-cycle program with shortened gap phases and precocious expression of cell-cycle regulatory proteins, including cyclins and cyclin-dependent kinases (CDKs. We examined the effect of CDK inhibition on the pathways regulating proliferation and survival of ESCs. We found that inhibiting cyclin-dependent kinase 1 (CDK1 leads to activation of the DNA damage response, nuclear p53 stabilization, activation of a subset of p53 target genes including NOXA, and negative regulation of the anti-apoptotic protein MCL1 in human and mouse ESCs, but not differentiated cells. We demonstrate that MCL1 is highly expressed in ESCs and loss of MCL1 leads to ESC death. Finally, we show that clinically relevant CDK1 inhibitors prevent formation of ESC-derived tumors and induce necrosis in established ESC-derived tumors. Our data demonstrate that ES cells are uniquely sensitive to CDK1 inhibition via a p53/NOXA/MCL1 pathway.

  18. Anti-protozoal and anti-bacterial antibiotics that inhibit protein synthesis kill cancer subtypes enriched for stem cell-like properties

    Science.gov (United States)

    Cuyàs, Elisabet; Martin-Castillo, Begoña; Corominas-Faja, Bruna; Massaguer, Anna; Bosch-Barrera, Joaquim; Menendez, Javier A

    2015-01-01

    Key players in translational regulation such as ribosomes might represent powerful, but hitherto largely unexplored, targets to eliminate drug-refractory cancer stem cells (CSCs). A recent study by the Lisanti group has documented how puromycin, an old antibiotic derived from Streptomyces alboniger that inhibits ribosomal protein translation, can efficiently suppress CSC states in tumorspheres and monolayer cultures. We have used a closely related approach based on Biolog Phenotype Microarrays (PM), which contain tens of lyophilized antimicrobial drugs, to assess the chemosensitivity profiles of breast cancer cell lines enriched for stem cell-like properties. Antibiotics directly targeting active sites of the ribosome including emetine, puromycin and cycloheximide, inhibitors of ribosome biogenesis such as dactinomycin, ribotoxic stress agents such as daunorubicin, and indirect inhibitors of protein synthesis such as acriflavine, had the largest cytotoxic impact against claudin-low and basal-like breast cancer cells. Thus, biologically aggressive, treatment-resistant breast cancer subtypes enriched for stem cell-like properties exhibit exacerbated chemosensitivities to anti-protozoal and anti-bacterial antibiotics targeting protein synthesis. These results suggest that old/existing microbicides might be repurposed not only as new cancer therapeutics, but also might provide the tools and molecular understanding needed to develop second-generation inhibitors of ribosomal translation to eradicate CSC traits in tumor tissues. PMID:25970790

  19. Selenium-platinum coordination compounds as novel anticancer drugs: selectively killing cancer cells via a reactive oxygen species (ROS)-mediated apoptosis route.

    Science.gov (United States)

    Zeng, Lingwu; Li, Yang; Li, Tianyu; Cao, Wei; Yi, Yu; Geng, Weijia; Sun, Zhiwei; Xu, Huaping

    2014-08-01

    We report the preparation of selenium-containing platinum-based anticancer drug EG-Se/Pt. EG-Se/Pt was obtained from the coordination of selenium-containing molecules (EG-Se) with cisplatin (CDDP). The structure of EG-Se/Pt was characterized by (1) H and (77) Se NMR spectroscopy, XPS, ESI-MS, and MALDI-TOF. In aqueous solution, EG-Se/Pt self-assembles to form spherical aggregates. EG-Se/Pt shows enhanced stability against dilution and high salt concentration compared with EG-Se. EG-Se/Pt induces cell apoptosis via reactive oxygen species (ROS), which leads to high selectivity between cancer cells and normal cells in cytotoxicity assays. More importantly, EG-Se/Pt effectively inhibits tumor growth in vivo in tumor-bearing mice. It is anticipated that tuning the ROS level through the assembly of selenium-containing molecules can be a general method to realize anticancer selectivity.

  20. Increased PRAME-specific CTL killing of acute myeloid leukemia cells by either a novel histone deacetylase inhibitor chidamide alone or combined treatment with decitabine.

    Directory of Open Access Journals (Sweden)

    Yushi Yao

    Full Text Available As one of the best known cancer testis antigens, PRAME is overexpressed exclusively in germ line tissues such as the testis as well as in a variety of solid and hematological malignant cells including acute myeloid leukemia. Therefore, PRAME has been recognized as a promising target for both active and adoptive anti-leukemia immunotherapy. However, in most patients with PRAME-expressing acute myeloid leukemia, PRAME antigen-specific CD8(+ CTL response are either undetectable or too weak to exert immune surveillance presumably due to the inadequate PRAME antigen expression and PRAME-specific antigen presentation by leukemia cells. In this study, we observed remarkably increased PRAME mRNA expression in human acute myeloid leukemia cell lines and primary acute myeloid leukemia cells after treatment with a novel subtype-selective histone deacetylase inhibitor chidamide in vitro. PRAME expression was further enhanced in acute myeloid leukemia cell lines after combined treatment with chidamide and DNA demethylating agent decitabine. Pre-treatment of an HLA-A0201(+ acute myeloid leukemia cell line THP-1 with chidamide and/or decitabine increased sensitivity to purified CTLs that recognize PRAME(100-108 or PRAME(300-309 peptide presented by HLA-A0201. Chidamide-induced epigenetic upregulation of CD86 also contributed to increased cytotoxicity of PRAME antigen-specific CTLs. Our data thus provide a new line of evidence that epigenetic upregulation of cancer testis antigens by a subtype-selective HDAC inhibitor or in combination with hypomethylating agent increases CTL cytotoxicity and may represent a new opportunity in future design of treatment strategy targeting specifically PRAME-expressing acute myeloid leukemia.

  1. Synergy of caspofungin with human polymorphonuclear granulocytes for killing Candida albicans

    OpenAIRE

    Tullio, Vivian; Mandras, Narcisa; Scalas, Daniela; Allizond, Valeria; Banche, Giuliana; Roana, Janira; Greco, Deborah; Castagno, Franco; Cuffini, Anna Maria; Carlone, Nicola A.

    2010-01-01

    The influence of caspofungin on polymorphonuclear leukocyte (PMN) phagocytosis and intracellular killing of Candida albicans was investigated. Caspofungin, at all of the concentrations tested (2, 3.2, and 8 μg/ml), significantly increased intracellular killing by PMNs through its direct action on both yeast cells and PMNs, indicating the potential ability of caspofungin to synergize with phagocytes for candidal killing. Caspofungin may therefore constitute an effective therapeutic option for ...

  2. Artesunate Exerts a Direct Effect on Endothelial Cell Activation and NF-κB Translocation in a Mechanism Independent of Plasmodium Killing

    Directory of Open Access Journals (Sweden)

    Mariana C. Souza

    2012-01-01

    Full Text Available Artemisinin and its derivates are an important class of antimalarial drug and are described to possess immunomodulatory activities. Few studies have addressed the effect of artesunate in the murine malaria model or its effect on host immune response during malaria infection. Herein, we study the effect of artesunate treatment and describe an auxiliary mechanism of artesunate in modulating the inflammatory response during experimental malaria infection in mice. Treatment with artesunate did not reduce significantly the parasitemia within 12 h, however, reduced BBB breakdown and TNF-α mRNA expression in the brain tissue of artesunate-treated mice. Conversely, mefloquine treatment was not able to alter clinical features. Notably, artesunate pretreatment failed to modulate the expression of LFA-1 in splenocytes stimulated with parasitized red blood cells (pRBCs in vitro; however, it abrogated the expression of ICAM-1 in pRBC-stimulated endothelial cells. Accordingly, a cytoadherence in vitro assay demonstrated that pRBCs did not adhere to artesunate-treated vascular endothelial cells. In addition, NF-κB nuclear translocation in endothelial cells stimulated with pRBCs was impaired by artesunate treatment. Our results suggest that artesunate is able to exert a protective effect against the P. berghei-induced inflammatory response by inhibiting NF-κB nuclear translocation and the subsequent expression of ICAM-1.

  3. VP22-CD基因工程化载体对胶质瘤细胞体外杀伤作用的研究%Effect of VP22-CD genetically engineered vectors on killing glioma cells in vitro

    Institute of Scientific and Technical Information of China (English)

    米蕊芳; 金贵善; 张俊文; 周益强; 徐恒周; 程森; 刘福生

    2016-01-01

    目的 构建CD以及VP22-CD基因工程化载体,并探讨其在体外对胶质瘤细胞的杀伤作用.方法 利用聚合酶链式反应(PCR)得到CD以及VP22基因片段;利用In-fusion基因克隆方法将得到的基因片段插入到pEGFP-N1载体中,构建CD以及VP22-CD基因工程化载体;利用脂质体转染的方法将构建的基因工程化载体转染到胶质瘤细胞C6及U87细胞中,并通过体外光镜及荧光显微镜观察转染率;利用MTT比色法评价加入前药5-氟胞嘧啶后对转染后胶质瘤细胞的杀伤率.结果 (1)将CD基因或VP22-CD融合基因插入到pEGFP-CD载体中,构建得到pEGFP-CD以及pEGFP-VP22-CD载体.(2)pEGFP-CD及pEGFP-VP22-CD载体可转染入C6或U87胶质瘤细胞,转染率分别为2.5%和3.7%以及2.0%和4.1%.(3)在C6或U87细胞中转染pEGFP-CD或pEGFP-VP22-CD载体并加入前药5-氟胞嘧啶后,细胞的存活率分别为(30.36±0.63)%和(11.75±1.01)%以及(28.78±3.62)%和(18.26±2.27)%,与转染pEGFP-N1组相比差异均有统计学意义(均P<0.05).(4)在C6或U87细胞中加入前药5-氟胞嘧啶后,转染pEGFP-VP22-CD组与转染pEGFP-CD组相比差异均有统计学意义(均P<0.05).结论 CD/5-FC系统在体外对恶性胶质瘤细胞具有显著的杀伤作用和旁观者效应;并且穿梭蛋白VP22可进一步促进系统对细胞的杀伤作用.%Objectives To construct CD and VP22-CD genetically engineered vectors and to investigate their killing effects on glioma cells in vitro.Methods CD and VP22 gene segments were obtain using polymerase chain reaction (PCR).The obtained gene fragments were inserted into pEGFP-N1 vectors using in-fusion gene cloning method.CD and VP22-CD gene engineering vectors were constructed.The constructed gene engineering vectors were transfected into the glioma cells C6 and U87 cells using the liposome transfection method,and the transfection efficiency was observed through in vitro light microscope and fluorescence microscope.The kill

  4. MnTnBuOE-2-PyP protects normal colorectal fibroblasts from radiation damage and simultaneously enhances radio/chemotherapeutic killing of colorectal cancer cells

    Science.gov (United States)

    Kosmacek, Elizabeth A.; Chatterjee, Arpita; Tong, Qiang; Lin, Chi; Oberley, Rebecca E.

    2016-01-01

    Manganese porphyrins have been shown to be potent radioprotectors in a variety of cancer models. However, the mechanism as to how these porphyrins protect normal tissues from radiation damage still remains largely unknown. In the current study, we determine the effects of the manganese porphyrin, MnTnBuOE-2-PyP, on primary colorectal fibroblasts exposed to irradiation. We found that 2 Gy of radiation enhances the fibroblasts' ability to contract a collagen matrix, increases cell size and promotes cellular senesence. Treating fibroblasts with MnTnBuOE-2-PyP significantly inhibited radiation-induced collagen contraction, preserved cell morphology and also inhibited cellular senescence. We further showed that MnTnBuOE-2-PyP enhanced the overall viability of the fibroblasts following exposure to radiation but did not protect colorectal cancer cell viability. Specifically, MnTnBuOE-2-PyP in combination with irradiation, caused a significant decrease in tumor clonogenicity. Since locally advanced rectal cancers are treated with chemoradiation therapy followed by surgery and non-metastatic anal cancers are treated with chemoradiation therapy, we also investigated the effects of MnTnBuOE-2-PyP in combination with radiation, 5-fluorouracil with and without Mitomycin C. We found that MnTnBuOE-2-PyP in combination with Mitomycin C or 5-fluorouracil further enhances those compounds' ability to suppress tumor cell growth. When MnTnBuOE-2-PyP was combined with the two chemotherapeutics and radiation, we observed the greatest reduction in tumor cell growth. Therefore, these studies indicate that MnTnBuOE-2-PyP could be used as a potent radioprotector for normal tissue, while at the same time enhancing radiation and chemotherapy treatment for rectal and anal cancers. PMID:27119354

  5. Inhibition of pre-B cell colony-enhancing factor (PBEF/NAMPT/visfatin) decreases the ability of human neutrophils to generate reactive oxidants but does not impair bacterial killing.

    Science.gov (United States)

    Roberts, Kate J; Cross, Andrew; Vasieva, Olga; Moots, Robert J; Edwards, Steven W

    2013-09-01

    NAMPT, also known as PBEF and visfatin, can act extracellularly as a cytokine-like molecule or intracellularly as a NAMPT, regulating NAD biosynthesis in the NAD salvage pathway. Inhibitors of NAMPT have anti-inflammatory and anticancer activity and are finding use as therapeutic agents. In view of the importance of NAD metabolism in neutrophil function, we determined the effects of NAMPT inhibition on a variety of neutrophil functions associated with their role in host protection against infections. Incubation of human neutrophils with the NAMPT inhibitor APO866 decreased neutrophil NAD(P)/H levels in a dose- and time-dependent manner but without a concomitant change in cell viability. NAMPT inhibition did not affect the expression of a number of cell-surface receptors involved in adhesion and opsono-phagocytosis, but the respiratory burst was decreased significantly. Whereas opsono-phagocytosis of Staphylococcus aureus was unaffected by NAMPT inhibition, intraphagosomal oxidant production was decreased. However, the killing efficiency of neutrophils was unaffected. These data indicate that therapeutic NAMPT inhibition is unlikely to have deleterious effects on host protection against infections, in spite of this ability to down-regulate neutrophil respiratory burst activity significantly.

  6. Development of near-infrared photoactivable phthalocyanine-loaded nanoparticles to kill tumor cells: An improved tool for photodynamic therapy of solid cancers.

    Science.gov (United States)

    Duchi, Serena; Ramos-Romero, Sara; Dozza, Barbara; Guerra-Rebollo, Marta; Cattini, Luca; Ballestri, Marco; Dambruoso, Paolo; Guerrini, Andrea; Sotgiu, Giovanna; Varchi, Greta; Lucarelli, Enrico; Blanco, Jeronimo

    2016-10-01

    Conventional photodynamic therapy has shown to be beneficial in the treatment of a variety of tumors. However, one of its major limitations is the inadequate penetration depth of visible light. In order to overcome this constraint, we developed 80nm poly-methylmethacrylate core-shell fluorescent nanoparticles (FNP) loaded with the photosensitizer tetrasulfonated aluminum phthalocyanine (Ptl). To demonstrate the efficacy of our Ptl@FNP we performed in vitro and in vivo studies using a human prostate tumor model. Our data reveal that Ptl@FNP are internalized by tumor cells, favour Ptl intracellular accumulation, and efficiently trigger cell death through the generation of ROS upon irradiation with 680nm light. When directly injected into tumors intramuscularly induced in SCID mice, Ptl@FNP upon irradiation significantly reduce tumor growth with higher efficiency than the bare Ptl. Collectively, these results demonstrate that the newly developed nanoparticles may be utilized as a delivery system for antitumor phototherapy in solid cancers.

  7. Anti-protozoal and anti-bacterial antibiotics that inhibit protein synthesis kill cancer subtypes enriched for stem cell-like properties

    OpenAIRE

    2015-01-01

    Key players in translational regulation such as ribosomes might represent powerful, but hitherto largely unexplored, targets to eliminate drug-refractory cancer stem cells (CSCs). A recent study by the Lisanti group has documented how puromycin, an old antibiotic derived from Streptomyces alboniger that inhibits ribosomal protein translation, can efficiently suppress CSC states in tumorspheres and monolayer cultures. We have used a closely related approach based on Biolog Phenotype Microarray...

  8. Novel innate cancer killing activity in humans

    Directory of Open Access Journals (Sweden)

    Lovato James

    2011-08-01

    Full Text Available Abstract Background In this study, we pilot tested an in vitro assay of cancer killing activity (CKA in circulating leukocytes of 22 cancer cases and 25 healthy controls. Methods Using a human cervical cancer cell line, HeLa, as target cells, we compared the CKA in circulating leukocytes, as effector cells, of cancer cases and controls. The CKA was normalized as percentages of total target cells during selected periods of incubation time and at selected effector/target cell ratios in comparison to no-effector-cell controls. Results Our results showed that CKA similar to that of our previous study of SR/CR mice was present in human circulating leukocytes but at profoundly different levels in individuals. Overall, males have a significantly higher CKA than females. The CKA levels in cancer cases were lower than that in healthy controls (mean ± SD: 36.97 ± 21.39 vs. 46.28 ± 27.22. Below-median CKA was significantly associated with case status (odds ratio = 4.36; 95% Confidence Interval = 1.06, 17.88 after adjustment of gender and race. Conclusions In freshly isolated human leukocytes, we were able to detect an apparent CKA in a similar manner to that of cancer-resistant SR/CR mice. The finding of CKA at lower levels in cancer patients suggests the possibility that it may be of a consequence of genetic, physiological, or pathological conditions, pending future studies with larger sample size.

  9. Killing(-Yano) Tensors in String Theory

    CERN Document Server

    Chervonyi, Yuri

    2015-01-01

    We construct the Killing(-Yano) tensors for a large class of charged black holes in higher dimensions and study general properties of such tensors, in particular, their behavior under string dualities. Killing(-Yano) tensors encode the symmetries beyond isometries, which lead to insights into dynamics of particles and fields on a given geometry by providing a set of conserved quantities. By analyzing the eigenvalues of the Killing tensor, we provide a prescription for constructing several conserved quantities starting from a single object, and we demonstrate that Killing tensors in higher dimensions are always associated with ellipsoidal coordinates. We also determine the transformations of the Killing(-Yano) tensors under string dualities, and find the unique modification of the Killing-Yano equation consistent with these symmetries. These results are used to construct the explicit form of the Killing(-Yano) tensors for the Myers-Perry black hole in arbitrary number of dimensions and for its charged version.

  10. How To Kill a Penguin

    CERN Document Server

    Haisch, Ulrich

    2007-01-01

    Within constrained minimal-flavor-violation the large destructive flavor-changing Z-penguin managed to survive eradication so far. We give a incisive description of how to kill it using the precision measurements of the Z -> b anti-b pseudo observables. The derived stringent range for the non-standard contribution to the universal Inami-Lim function C leads to tight two-sided limits for the branching ratios of all Z-penguin dominated flavor-changing K- and B-decays.

  11. How Ixtoc 1 was killed

    Energy Technology Data Exchange (ETDEWEB)

    1980-04-01

    More than nine months after it erupted 6/3/79, Petroleos Mexicanos' Ixtoc 1 blowout in Campeche Bay was killed with three cement plugs having a total length of 2885 ft. After drilling of relief wells, 200 sacks of cement were used to form the 685 ft. long bottom plug. After inserting an interval of mud, an additional 200 sacks of cement were pumped down to form a 550 ft. plug. The final up-hole plug was formed by 500 sacks of quick-setting cement, which formed a 1650 ft. long plug.

  12. Pseudomonas Exotoxin A: optimized by evolution for effective killing

    Directory of Open Access Journals (Sweden)

    Marta eMichalska

    2015-09-01

    Full Text Available Pseudomonas Exotoxin A (PE is the most toxic virulence factor of the pathogenic bacterium Pseudomonas aeruginosa. This review describes current knowledge about the intoxication pathways of PE. Moreover, PE represents a remarkable example for pathoadaptive evolution, how bacterial molecules have been structurally and functionally optimized under evolutionary pressure to effectively impair and kill their host cells.

  13. The combination of BH3-mimetic ABT-737 with the alkylating agent temozolomide induces strong synergistic killing of melanoma cells independent of p53.

    Directory of Open Access Journals (Sweden)

    Steven N Reuland

    Full Text Available Metastatic melanoma has poor prognosis and is refractory to most conventional chemotherapies. The alkylating agent temozolomide (TMZ is commonly used in treating melanoma but has a disappointing response rate. Agents that can act cooperatively with TMZ and improve its efficacy are thus highly sought after. The BH3 mimetic ABT-737, which can induce apoptosis by targeting pro-survival Bcl-2 family members, has been found to enhance the efficacy of many conventional chemotherapeutic agents in multiple cancers. We found that combining TMZ and ABT-737 induced strong synergistic apoptosis in multiple human melanoma cell lines. When the drugs were used in combination in a mouse xenograft model, they drastically reduced tumor growth at concentrations where each individual drug had no significant effect. We found that TMZ treatment elevated p53 levels, and that the pro-apoptotic protein Noxa was elevated in TMZ/ABT-737 treated cells. Experiments with shRNA demonstrated that the synergistic effect of TMZ and ABT-737 was largely dependent on Noxa. Experiments with nutlin-3, a p53 inducer, demonstrated that p53 induction was sufficient for synergistic cell death with ABT-737 in a Noxa-dependent fashion. However, p53 was not necessary for TMZ/ABT-737 synergy as demonstrated by a p53-null line, indicating that TMZ and ABT-737 together induce Noxa in a p53-independent fashion. These results demonstrate that targeting anti-apoptotic Bcl-2 members is a promising method for treating metastatic melanoma, and that clinical trials with TMZ and Bcl-2 inhibitors are warranted.

  14. Designing a HER2/neu promoter to drive α1,3galactosyltransferase expression for targeted anti-αGal antibody-mediated tumor cell killing

    Science.gov (United States)

    Lanteri, Marion; Ollier, Laurence; Giordanengo, Valérie; Lefebvre, Jean-Claude

    2005-01-01

    Introduction Our goal was to specifically render tumor cells susceptible to natural cytolytic anti-αGal antibodies by using a murine α1,3galactosyltransferase (mαGalT) transgene driven by a designed form of HER2/neu promoter (pNeu), the transcription of which is frequently observed to be above basal in breast tumors. Indeed, the αGalT activity that promotes Galα1,3Galβ1,4GlcNAc-R (αGal) epitope expression has been mutationally disrupted during the course of evolution, starting from Old World primates, and this has led to the counter-production of large amounts of cytotoxic anti-αGal antibodies in recent primates, including man. Method Expression of the endogenous c-erbB-2 gene was investigated in various cell lines by northern blotting. A mαGalT cDNA was constructed into pcDNA3 vector downstream of the original CMV promoter (pCMV/mαGalT) and various forms of pNeu were prepared by PCR amplification and inserted in the pCMV/mαGalT construct upstream of the mαGalT cDNA, in the place of the CMV promoter. These constructs were transferred into HEK-293 control and breast tumor cell lines. Stably transfected cells were analyzed by northern blotting for their expression of αGalT and c-erbB-2, and by flow cytometry for their binding with fluorescein isothiocyanate-conjugated Griffonia simplicifolia/isolectin B4. Results We show that expression of the mαGalT was up- or down-modulated according to the level of endogenous pNeu activity and the particular form of constructed pNeu. Among several constructs, two particular forms of the promoter, pNeu250 containing the CCAAT box and the PEA3 motif adjacent to the TATAA box, and pNeu664, which has three additional PEA3 motifs upstream of the CCAAT box, were found to promote differential αGalT expression. Conclusion Our results strengthen current concepts about the crucial role played by the proximal PEA3 motif of pNeu, and may represent a novel therapeutic approach for the development of targeted transgene expression

  15. Designing a HER2/neu promoter to drive alpha1,3galactosyltransferase expression for targeted anti-alphaGal antibody-mediated tumor cell killing.

    OpenAIRE

    Lanteri, Marion; Ollier, Laurence; Giordanengo, Valérie; Lefebvre, Jean-Claude

    2005-01-01

    INTRODUCTION: Our goal was to specifically render tumor cells susceptible to natural cytolytic anti-alphaGal antibodies by using a murine alpha1,3galactosyltransferase (malphaGalT) transgene driven by a designed form of HER2/neu promoter (pNeu), the transcription of which is frequently observed to be above basal in breast tumors. Indeed, the alphaGalT activity that promotes Galalpha1,3Galbeta1,4GlcNAc-R (alphaGal) epitope expression has been mutationally disrupted during the course of evoluti...

  16. Synthesis of multivalent glycoconjugates containing the immunoactive LELTE peptide: effect of glycosylation on cellular activation and natural killing by human peripheral blood mononuclear cells.

    Science.gov (United States)

    Renaudet, Olivier; Krenek, Karel; Bossu, Isabelle; Dumy, Pascal; Kádek, Alan; Adámek, David; Vanek, Ondrej; Kavan, Daniel; Gazák, Radek; Sulc, Miroslav; Bezouska, Karel; Kren, Vladimír

    2010-05-19

    Pentapeptide diacidic sequence LELTE, derived from the mycobacterial heat shock protein hsp65, has been recently identified as a "danger" signal of the immune system effective via specific binding to the universal leukocyte triggering receptor CD69. This sequence is not active per se, only after its presentation within the multivalent environment of its parent protein, or after artificial dimerization using a standard bifunctional reagents. Here we describe an entirely new way of presenting of this peptide based on its attachment to a cyclopeptide RAFT scaffold (K-K-K-P-G)(2) through the epsilon-amino group of lysine residues, alone or in combination with the carbohydrate epitope alphaGalNAc. The ability of such RAFT scaffolds to precipitate the target CD69 receptor or to activate CD69-positive cells is enhanced in compounds 2 and 4 possessing combined peptide/carbohydrate expression. Compounds 2 and 4 are highly efficient activators of natural killer lymphocytes, but they are completely inactive from the point of view of activation-induced apoptosis of lymphocytes by the target cells. These unique properties make the combined peptide/carbohydrate RAFTs highly suitable for future evaluation in animal tumor therapies in vivo and predict them to be readily available and efficient immunoactivators.

  17. S. Typhimurium strategies to resist killing by cationic antimicrobial peptides.

    Science.gov (United States)

    Matamouros, Susana; Miller, Samuel I

    2015-11-01

    S. Typhimurium is a broad host range Gram-negative pathogen that must evade killing by host innate immune systems to colonize, replicate, cause disease, and be transmitted to other hosts. A major pathogenic strategy of Salmonellae is entrance, survival, and replication within eukaryotic cell phagocytic vacuoles. These phagocytic vacuoles and gastrointestinal mucosal surfaces contain multiple cationic antimicrobial peptides (CAMPs) which control invading bacteria. S. Typhimurium possesses several key mechanisms to resist killing by CAMPs which involve sensing CAMPs and membrane damage to activate signaling cascades that result in remodeling of the bacterial envelope to reduce its overall negative charge with an increase in hydrophobicity to decrease binding and effectiveness of CAMPs. Moreover Salmonellae have additional mechanisms to resist killing by CAMPs including an outer membrane protease which targets cationic peptides at the surface, and specific efflux pumps which protect the inner membrane from damage. This article is part of a Special Issue entitled: Bacterial Resistance to Antimicrobial Peptides.

  18. Phagocytosis and killing of Streptococcus suis by porcine neutrophils.

    Science.gov (United States)

    Chabot-Roy, Geneviève; Willson, Philip; Segura, Mariela; Lacouture, Sonia; Gottschalk, Marcelo

    2006-07-01

    Streptococcus suis serotype 2 is an important swine pathogen responsible for diverse infections, mainly meningitis. Virulence factors and the pathogenesis of infection are not well understood. Neutrophils may play an important role in the pathogenesis of infection given that infiltration by neutrophils and mononuclear cells are frequently observed in lesions caused by S. suis. The objective of this work was to study the interactions between S. suis serotype 2 and porcine neutrophils. Results showed that suilysin is toxic to neutrophils and this could help S. suis evade innate immunity. Moreover, suilysin appears to affect complement-dependent killing by decreasing the opsonization of S. suis and the bactericidal capacity of neutrophils. Our results confirm that capsule polysaccharide protects S. suis against killing and phagocytosis by neutrophils. We also showed that the presence of specific IgG against S. suis serotype 2 promoted killing by neutrophils, indicating that the induction of a strong humoral response is beneficial for clearance of this pathogen.

  19. Specific killing effect of diphtheria toxin A fragment under control of DF3 promotor on human breast cancer cells%DF3调控下的白喉毒素A片段对人乳腺癌细胞的特异性杀伤作用

    Institute of Scientific and Technical Information of China (English)

    Ming Cai; Wenguang Huang; Wei Luo; Sheng Pan; Tao Yin

    2007-01-01

    Objective:To study the effects of recombinant expression vector containing human breast cancer DF3 promotor and diphtheria toxin A fragment on human breast cancer cells. Methods:Constructing recombinant expression vector PGL3-DF3-DTA and transfecting it into human breast cancer cells of DF3 positive and negative. By means of RT-PCR to measure the expression of DTA in human breast cancer cells. MTT color-imetry was used to examine the effect of PGL3-DF3-DTA on growth of human breast cancer calls. By experiment on nude mice to observe the killing effect of PGL3-DF3-DTA on human breast cancer cells. Results:Recombinant expression vector PGL3-DF3-DTA was highly expressed in human breast cancer cell line of DF3 positive, and it could kill the human breast cancer cells not only in vitro but also in vivo. Conclusion:Recombinant expression vector PGL3-DF3-DTA could produce specific killing effect on human breast cancer cell line of DF3 positive.

  20. 食管癌抗原联合金葡素诱导杀瘤性细胞的实验研究%Study on Tumor-Killing Cells Induced by Esophageal Cancer Antigen and Staphlococcal Enterotoxin

    Institute of Scientific and Technical Information of China (English)

    李树义; 刘洪梅; 张庆波

    2011-01-01

    目的:体外应用超抗原金葡素C型(SEC)联合食管癌可溶性抗原(TSA)刺激外周血淋巴细胞,诱导产生细胞毒T细胞(CTLs),对肿瘤细胞进行杀伤,探讨其治疗肿瘤的可行性.方法:外周血淋巴细胞经TSA、超抗原SEC联合作用,进行体外培养.用酶联免疫吸附测定法(ELISA)法和细胞毒试验测定细胞因子含量和杀伤活性.结果:经TSA与超抗原SEC联合刺激,淋巴细胞增殖活性增强,在刺激后72 h达到峰值;与空白对照组相比,SEC、TSA以及SEC与TSA联合刺激,人肿瘤坏死因子α(TNF-α)活性从第1天开始升高,在3 d 达峰值;并且联合刺激的淋巴细胞组诱导的效应细胞对靶细胞杀伤活性显著高于单纯淋巴细胞组(P<0.05),对TSA来源的食管癌细胞具有选择性杀伤作用.结论:肿瘤可溶性抗原与超抗原SEC联合应用能诱导效应细胞明显增殖、活化、并产生高效特异性的抗肿瘤效果.%Objective:To study the effect of tumor soluble antigen (TSA) and staphlococcal enterotoxin(SEC) on the cyctotoxic T lymphocyte(CTL), and to evaluate the new cell immunotherapy on tumor. Methods: Lymphocyte cells were isolated from peripheral blood and then stimulated in vitro with soluble antigen and superantigen SEC, test levels of cytokine and killing activity by enzyme-linked immunosorbent assay (ELISA) and cytotoxic assay. Results: Proliferation activity of lymphocyte cells group stimulated by TSA and SEC was the strongest, peaked at 72h. Compared with the control group, the levels of tumor necrosis factor-α (TNF-α) in the study groups begun to increase after 1d, and peaked at 3d. The killing activity of lymphocyte cell group stimulated by TSA and SEC against target cells were significantly higher than the control group(P<0.05). Conclusions:The seffector cells stimulated by TSA and SEC induce effective proliferation,activation and high antitumor efficiency and show a potential kind of operative antitumor immunotherapy.

  1. Adenovirus mediated fusion gene system driven by KDR promoter kills selectively pancreatic cancer cells%双自杀基因重组腺病毒对胰腺癌细胞特异性杀伤作用

    Institute of Scientific and Technical Information of China (English)

    闫振宇; 陈旭; 孔恒; 黄宗海; 俞金龙; 厉周

    2008-01-01

    目的 研究腺病毒介导的KDR启动子驱动CD/TK融合基因系统(Ad-KDR-CDTK)对胰腺癌细胞Capan-2特异性的杀伤作用.方法 重组腺病毒体外感染表达KDR的Capaw2细胞株,用不表达KDR的肝癌细胞HepG2做对照.观察其感染效率并以RT-PCR方法 检测转基因细胞CDTK的表达,然后给予不同浓度的前药更昔洛韦(ganciclovir,GCV)和5-氟胞嘧啶(5-fluorocy-tosine,5-FC),MTT法观察该体系对Capan-2和HepG2细胞生长增殖的影响及其旁观者效应;电镜观察细胞的病变;流式细胞仪检测细胞周期的变化和DNA含量的变化.建立Capan-2裸鼠皮下移植瘤模型,瘤内注射Ad-KDR-CD/TK,腹腔注射前药GCV(50 mg·kg-1·d-1)和5-FC(500 mg·kg-1·d-1)14 d,观察肿瘤生长抑制效应.结果 腺病毒对两种细胞株的感染率相似,其感染率随腺病毒滴度的增高而递增.RT-PCR方法 检测发现转染Ad-KDR-CDTK的Capan-2细胞有目的 基因表达.MTT法检测显示前药呈剂量依赖性抑制Capan-2生长,而不表达KDR的肝癌细胞HepG2对前药不敏感,且观察到该体系对Capan-2明显的旁观者效应.电镜下可见Capan-2有凋亡改变.用流式细胞仪测定用药组出现典型的凋亡峰;细胞周期分析显示治疗后细胞G0-G1期比率增多,G2-M及S期细胞减少.在Capan-2裸鼠移植瘤模型中,该双自杀基因系统能够显著抑制肿瘤的生长.结论 KDR启动子可调控双自杀基因体系选择性杀伤胰腺癌细胞Capan-2,诱导胰腺癌细胞凋亡,并可显著抑制人胰腺癌裸鼠移植瘤的生长.%Objective To evaluate the selectively killing effect of adenovirus (Ad) mediated double suicide gene driven by KDR promoter on pancreatic cancer cell Capan-2. Methods KDR-ex-pressing Capan-2 and non-KDR-expressing HepG2 were infected by Ad-KDR-CDTK. The infection rate was observed and the expression of CDTK was detected by RT-PCR. Followed by treatment with 5-FC and GCV,the killing effects were evaluated and bystander effects

  2. 9 CFR 113.207 - Encephalomyelitis Vaccine, Eastern, Western, and Venezuelan, Killed Virus.

    Science.gov (United States)

    2010-01-01

    ... Venezuelan, Killed Virus, shall be prepared from virus-bearing cell culture fluids. Each serial or subserial... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Encephalomyelitis Vaccine, Eastern, Western, and Venezuelan, Killed Virus. 113.207 Section 113.207 Animals and Animal Products ANIMAL...

  3. Mechanisms of Cell Killing Response from Low Linear Energy Transfer (LET Radiation Originating from 177Lu Radioimmunotherapy Targeting Disseminated Intraperitoneal Tumor Xenografts

    Directory of Open Access Journals (Sweden)

    Kwon Joong Yong

    2016-05-01

    Full Text Available Radiolabeled antibodies (mAbs provide efficient tools for cancer therapy. The combination of low energy β−-emissions (500 keVmax; 130 keVave along with a γ-emission for imaging makes 177Lu (T1/2 = 6.7 day a suitable radionuclide for radioimmunotherapy (RIT of tumor burdens possibly too large to treat with α-particle radiation. RIT with 177Lu-trastuzumab has proven to be effective for treatment of disseminated HER2 positive peritoneal disease in a pre-clinical model. To elucidate mechanisms originating from this RIT therapy at the molecular level, tumor bearing mice (LS-174T intraperitoneal xenografts were treated with 177Lu-trastuzumab comparatively to animals treated with a non-specific control, 177Lu-HuIgG, and then to prior published results obtained using 212Pb-trastuzumab, an α-particle RIT agent. 177Lu-trastuzumab induced cell death via DNA double strand breaks (DSB, caspase-3 apoptosis, and interfered with DNA-PK expression, which is associated with the repair of DNA non-homologous end joining damage. This contrasts to prior results, wherein 212Pb-trastuzumab was found to down-regulate RAD51, which is involved with homologous recombination DNA damage repair. 177Lu-trastuzumab therapy was associated with significant chromosomal disruption and up-regulation of genes in the apoptotic process. These results suggest an inhibition of the repair mechanism specific to the type of radiation damage being inflicted by either high or low linear energy transfer radiation. Understanding the mechanisms of action of β−- and α-particle RIT comparatively through an in vivo tumor environment offers real information suitable to enhance combination therapy regimens involving α- and β−-particle RIT for the management of intraperitoneal disease.

  4. Light-Tunable Generation of Singlet Oxygen and Nitric Oxide with a Bichromophoric Molecular Hybrid: a Bimodal Approach to Killing Cancer Cells.

    Science.gov (United States)

    Fraix, Aurore; Blangetti, Marco; Guglielmo, Stefano; Lazzarato, Loretta; Marino, Nino; Cardile, Venera; Graziano, Adriana C E; Manet, Ilse; Fruttero, Roberta; Gasco, Alberto; Sortino, Salvatore

    2016-06-20

    The design, synthesis, photochemical properties, and biological evaluation of a novel photoactivatable bichromophoric conjugate are reported. The compound 1, [4-(4,4-difluoro-2,6-diiodo-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl)-N-(3-((4-nitro-3-(trifluoromethyl)phenyl)amino)propyl)butanamide] combines a 2,6-diiodo-1,3,5,7-tetramethyl BODIPY derivative as singlet oxygen ((1) O2 ) photosensitizer and 4-nitro-3-(trifluoromethyl)aniline (NOPD) as nitric oxide (NO) photodonor, joined by an alkyl spacer. These two chromogenic units absorb in distinct regions of the visible spectrum, and their individual photochemical properties are conserved in the molecular conjugate. Irradiation of the bichromophoric conjugate with green light afforded (1) O2 in high quantum yields, whereas (1) O2 production was negligible with the use of blue light; under this latter condition, NO was released. Photogeneration of NO and cytotoxic (1) O2 can therefore be regulated by appropriately tuning the excitation light wavelength and intensity. Tested on melanoma cancer cells, this resulted in amplified photomortality relative to that of a structurally correlated model compound 2 [4-(4,4-difluoro-2,6-diiodo-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl)-N-(3-(p-tolylamino)propyl)butanamide] deprived of the NO-release capacity. The cellular uptake of 1, evaluated by confocal fluorescence microscopy, showed that the product is localized in the cytoplasm.

  5. Killing tensors in pp-wave spacetimes

    Energy Technology Data Exchange (ETDEWEB)

    Keane, Aidan J [87 Carlton Place, Glasgow G5 9TD, Scotland (United Kingdom); Tupper, Brian O J, E-mail: aidan@countingthoughts.co, E-mail: bt32@rogers.co [Department of Mathematics and Statistics, University of New Brunswick, Fredericton, New Brunswick, E3B 5A3 (Canada)

    2010-12-21

    The formal solution of the second-order Killing tensor equations for the general pp-wave spacetime is given. The Killing tensor equations are integrated fully for some specific pp-wave spacetimes. In particular, the complete solution is given for the conformally flat plane wave spacetimes and we find that irreducible Killing tensors arise for specific classes. The maximum number of independent irreducible Killing tensors admitted by a conformally flat plane wave spacetime is shown to be six. It is shown that every pp-wave spacetime that admits an homothety will admit a Killing tensor of Koutras type and, with the exception of the singular scale-invariant plane wave spacetimes, this Killing tensor is irreducible.

  6. ExoU contributes to late killing of Pseudomonas aeruginosa - infected endothelial cells ExoU contribui para a morte tardia de células endoteliais infectadas por Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Alessandra Mattos Saliba

    2003-11-01

    Full Text Available To ascertain the role of ExoU in late P. aeruginosa cytotoxicity, endothelial cells (EC were exposed to wild type PA103, PA103deltaexoU and PA103::exsA for 1h and to gentamicin in culture medium. After 24h, the viability of PA103-infected cells (33.7 ± 14.3% was significantly lower than the viability of PA103deltaexoU- (77.7 ± 6.3% or PA103::exsA- (79.5 ± 23.3% infected EC. P. aeruginosa cytotoxicity did not depend on the bacterial ability to interact with EC because the percentage of cells with associated PA103 (35.9 ± 15.8% was similar to the percentage in PA103deltaexoU- (34.2 ± 16.0% and lower than the percentage in PA103::exsA-infected cultures (82.9 ± 18.9%. Cell treatment with cytochalasin D reduced the PA103 internalization by EC but did not interfere with its ability to kill host cells.Para determinar o papel de ExoU na citotoxicidade tardia de P. aeruginosa, células endoteliais (CE foram expostas às cepas PA103, PA103deltaxoU e PA103::exsA por 1h e à gentamicina em meio de cultura. Após 24h, a viabilidade das CE infectadas com PA103 (33.7 ± 14.3% foi inferior à de CE infectadas com PA103deltaexoU (77.7 ± 6.3% e PA103::exsA (79.5 ± 23.3%. A citotoxicidade não dependeu da capacidade de interagir com as CE porque o percentual de células com bactérias associadas em culturas expostas a PA103 foi semelhante ao percentual em culturas expostas a PA103deltaexoU e inferior em culturas expostas a PA103::exsA. O tratamento das CE com citocalasina D reduziu a internalização de PA103, mas não interferiu em sua citotoxicidade.

  7. Measurement of bacterial ingestion and killing by macrophages.

    Science.gov (United States)

    Campbell, P A; Canono, B P; Drevets, D A

    2001-05-01

    This unit presents fairly simple assays for measuring the binding of bacteria to macrophages, internalization of bacteria (also called ingestion or phagocytosis), and bacterial killing by macrophages. The first basic protocol describes how to measure the ability of macrophages to ingest bacteria. Because it is critical to remove residual extracellular organisms, the protocol presents two alternative steps to accomplish this: a washing procedure and a more stringent method in which cells are sedimented through sucrose. In addition, it is important to distinguish those bacteria truly ingested by a macrophage from those that are bound to, but not internalized by, the cell. A simple but effective way to do this is described in an alternate protocol. The unit also presents two ways to measure the ability of a macrophage to kill bacteria it has internalized. The first is a straightforward assay in which bacterial colonies are enumerated before and after a killing period; a subsequent colony count will indicate whether the bacteria grew within or were killed by the macrophage. The second protocol describes a way to measure bacterial viability based on bacterial metabolism, in which the ability of bacterial dehydrogenases to mediate the reduction of a tetrazolium salt to purple formazan is monitored by measuring absorbance spectrophotometrically.

  8. Involvement of superoxide and myeloperoxidase in oxygen-dependent killing of Staphylococcus aureus by neutrophils.

    Science.gov (United States)

    Hampton, M B; Kettle, A J; Winterbourn, C C

    1996-09-01

    We have used a quantitative assay that measures independent rate constants for phagocytosis and killing of Staphylococcus aureus to investigate the involvement of superoxide and myeloperoxidase in bacterial killing by human neutrophils. To inhibit superoxide-dependent processes, superoxide dismutase was cross-linked to immunoglobulin G and the conjugate was attached to the surface of S. aureus via protein A in its cell wall. Myeloperoxidase was inhibited with azide, and myeloperoxidase-deficient neutrophils were used. Adding the NADPH oxidase inhibitor diphenyleneiodonium, to prevent superoxide production, decreased the killing rate to 25%, indicating that oxidative killing mechanisms predominate in this system. The rate constant for killing of S. aureus with superoxide dismutase attached was 70% of that for control bacteria linked to inactivated enzyme. Superoxide dismutase had no effect in the presence of diphenyleneiodonium. The rate of killing was decreased to 33% in the presence of azide and to 40% with myeloperoxidase-deficient neutrophils. Superoxide dismutase had no effect in the presence of azide. On the assumption that the oxidative and nonoxidative components of killing can be considered separately, the oxidative rate was decreased by almost half by superoxide dismutase and was about six times lower when myeloperoxidase was inactive. We conclude that myeloperoxidase-dependent processes are strongly favored by human neutrophils as their prime mechanism of oxidative killing of S. aureus and that superoxide makes a direct contribution to killing. Our results also suggest that superoxide acts in conjunction with a myeloperoxidase-dependent pathway.

  9. 突变型胸苷激酶对鼠C6胶质瘤细胞的杀伤效应%Killing effect of mutant herpes simplex virus type 1 thymidine kinase on rat C6 glioma cells

    Institute of Scientific and Technical Information of China (English)

    徐晓燕; 刘伟国; 杨小锋; 郑学胜; 沈罡

    2009-01-01

    Objective To explore the killing effect of mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) and its wild type (HSV1-tk) on rat C6 glioma cells after Ganciclovir (GCV) treatment. Methods Eukaryotic expressing vector pcDNA3.1 containing HSV1-tk or HSV1-sr39tk was transfected into C6 cells and identified with RT-PCR. GCV killing efficiencies after gene transfer of HSV1-tk or HSV1-sr39tk were observed and compared in vitro and in vivo. Results 308bp DNA fragment was amplified through RT-PCR in both cells transfected with HSV1-tk (C6/tk) and those with HSV1-sr39tk (C6/sr39tk). As the prodrug GCV concentrations increasing from 0 μmol/L to 400 μmol/L, the cell survival rates in C6/sr39tk group declined from (99.96 ± 3.54) % to (4. 75 ± 1.79) %, while in C6/tk group from (100.03±2. 95)% to (59. 16±3.48)% and in C6 group from(100. 29±1.20)% to (83.62 ±7.56)%. There was a significant difference in killing effect among C6、C6/tk and C6/sr39tk group after GCV treatment(P < 0. 05). In vivo experiment, the rate of tumor formation was 100%. After 10 day treatment of GCV following tumor formation, tumors in animals implantated with C6/sr39tk、C6/tk or C6 got an average volume equaling (574. 08±107.72) mm3、(928.47±165.61) mm3 and (1287.24±364. 84) mm3 respectively. The most powerful tumor growth inhibition was observed in C6/sr39tk + GCV group, with an inhibition rate of tumor growth about 55.40%. Conclusion C6 cells transfected with HSV1-sr39tk were more sensitive to GCV than those with HSV1-tk both in vitro and in vivo, and thus HSV1-sr39tk can be considered as a mean to improve the overall efficacy of the HSV1-tk/GCV suicide gene system.%目的 观察比较突变型单纯疱疹胸苷激酶(HSV1-sr39tk)/更昔洛韦(GCV)和野生型HSV1-tk/GCV对鼠C6胶质瘤细胞的杀伤效应.方法 利用真核表达载体转染目的 基因到C6细胞,RT-PCR鉴定.通过MTT实验和活体植瘤,比较各组对GCV敏感性.结果

  10. Platelets kill intraerythrocytic malarial parasites and mediate survival to infection.

    Science.gov (United States)

    McMorran, Brendan J; Marshall, Vikki M; de Graaf, Carolyn; Drysdale, Karen E; Shabbar, Meriam; Smyth, Gordon K; Corbin, Jason E; Alexander, Warren S; Foote, Simon J

    2009-02-01

    Platelets play a critical role in the pathogenesis of malarial infections by encouraging the sequestration of infected red blood cells within the cerebral vasculature. But platelets also have well-established roles in innate protection against microbial infections. We found that purified human platelets killed Plasmodium falciparum parasites cultured in red blood cells. Inhibition of platelet function by aspirin and other platelet inhibitors abrogated the lethal effect human platelets exert on P. falciparum parasites. Likewise, platelet-deficient and aspirin-treated mice were more susceptible to death during erythrocytic infection with Plasmodium chabaudi. Both mouse and human platelets bind malarial-infected red cells and kill the parasite within. These results indicate a protective function for platelets in the early stages of erythrocytic infection distinct from their role in cerebral malaria.

  11. Escape of Mycobacterium tuberculosis from oxidative killing by neutrophils.

    Science.gov (United States)

    Corleis, Björn; Korbel, Daniel; Wilson, Robert; Bylund, Johan; Chee, Ronnie; Schaible, Ulrich E

    2012-07-01

    Neutrophils enter sites of infection, where they can eliminate pathogenic bacteria in an oxidative manner. Despite their predominance in active tuberculosis lesions, the function of neutrophils in this important human infection is still highly controversial. We observed that virulent Mycobacterium tuberculosis survived inside human neutrophils despite prompt activation of these defence cells' microbicidal effectors. Survival of M. tuberculosis was accompanied by necrotic cell death of infected neutrophils. Necrotic cell death entirely depended on radical oxygen species production since chronic granulomatous disease neutrophils were protected from M. tuberculosis-triggered necrosis. More, importantly, the M. tuberculosis ΔRD1 mutant failed to induce neutrophil necrosis rendering this strain susceptible to radical oxygen species-mediated killing. We conclude that this virulence function is instrumental for M. tuberculosis to escape killing by neutrophils and contributes to pathogenesis in tuberculosis.

  12. Attachment of Escherichia coli to Listeria monocytogenes for pediocin-mediated killing.

    Science.gov (United States)

    Liu, Shanna; Takala, Timo M; Reunanen, Justus; Saris, Ossian; Saris, Per E J

    2015-02-01

    Listeria phage endolysin cell wall-binding domain (CBD) from the Listeria phage A500 was fused with flagellar subunit FliC in Escherichia coli, aiming at binding of E. coli cells to Listeria cells, followed by enhanced killing of Listeria by pediocin production. FliC::CBD chimeric flagella were expressed and detected by Western blot. However, only few chimeric flagella could be isolated from the recombinant cells compared with sufficient amount of wild-type flagella obtained from the host cells. Interestingly, wild-type flagella extract showed capacity of binding Listeria cells. Pediocin-secreting E. coli cells with Listeria-binding flagella killed approximately 40 % of the Listeria cells, whereas cell-free spent growth medium with the same pediocin concentration only inhibited Listeria growth. These results suggested that binding the Listeria to bacteriocin-secreting cells improves killing.

  13. Dedicated high dose rate 192Ir brachytherapy radiation fields for in vitro cell exposures at variable source-target cell distances: killing of mammalian cells depends on temporal dose rate fluctuation

    Science.gov (United States)

    Veigel, Cornelia; Hartmann, Günther H.; Fritz, Peter; Debus, Jürgen; Weber, Klaus-Josef

    2017-02-01

    Afterloading brachytherapy is conducted by the stepwise movement of a radioactive source through surgically implanted applicator tubes where at predefined dwell positions calculated dwell times optimize spatial dose delivery with respect to a planned dose level. The temporal exposure pattern exhibits drastic fluctuations in dose rate at a given coordinate and within a single treatment session because of the discontinuous and repeated source movement into the target volume. This could potentially affect biological response. Therefore, mammalian cells were exposed as monolayers to a high dose rate 192Ir source by utilizing a dedicated irradiation device where the distance between a planar array of radioactive source positions and the plane of the cell monolayer could be varied from 2.5 mm to 40 mm, thus varying dose rate pattern for any chosen total dose. The Gammamed IIi afterloading system equipped with a nominal 370 GBq (10 Ci) 192-Ir source was used to irradiate V79 Chinese hamster lung fibroblasts from both confluent and from exponential growth phase with dose up to 12 Gy (at room temperature, total exposure not exceeding 1 h). For comparison, V79 cells were also exposed to 6 MV x-rays from a clinical linear accelerator (dose rate of 2.5 Gy min‑1). As biological endpoint, cell survival was determined by standard colony forming assay. Dose measurements were conducted with a diamond detector (sensitive area 7.3 mm2), calibrated by means of 60Co radiation. Additionally, dose delivery was simulated by Monte Carlo calculations using the EGSnrc code system. The calculated secondary electron fluence spectra at the cell location did not indicate a significant change of radiation quality (i.e. higher linear energy transfer) at the lower distances. Clonogenic cell survival curves obtained after brachytherapy exhibited an altered biological response compared to x-rays which was characterized by a significant reduction of the survival curve shoulder when dose rate

  14. The Killing Forms of Lie Triple Systems

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zhi Xue; GAO Rui

    2009-01-01

    For Lie triple systems in the characteristic zero setting, we obtain by means of the Killing forms two criterions for semisimplicity and for solvability respectively, and then investigate the relationship among the Killing forms of a real Lie triple system To, the complexification T of To, and the realification of T.

  15. Antibacterial surface design - Contact kill

    Science.gov (United States)

    Kaur, Rajbir; Liu, Song

    2016-08-01

    Designing antibacterial surfaces has become extremely important to minimize Healthcare Associated Infections which are a major cause of mortality worldwide. A previous biocide-releasing approach is based on leaching of encapsulated biocides such as silver and triclosan which exerts negative impacts on the environment and potentially contributes to the development of bacterial resistance. This drawback of leachable compounds led to the shift of interest towards a more sustainable and environmentally friendly approach: contact-killing surfaces. Biocides that can be bound onto surfaces to give the substrates contact-active antibacterial activity include quaternary ammonium compounds (QACs), quaternary phosphoniums (QPs), carbon nanotubes, antibacterial peptides, and N-chloramines. Among the above, QACs and N-chloramines are the most researched contact-active biocides. We review the engineering of contact-active surfaces using QACs or N-chloramines, the modes of actions as well as the test methods. The charge-density threshold of cationic surfaces for desired antibacterial efficacy and attempts to combine various biocides for the generation of new contact-active surfaces are discussed in detail. Surface positive charge density is identified as a key parameter to define antibacterial efficacy. We expect that this research field will continue to attract more research interest in view of the potential impact of self-disinfective surfaces on healthcare-associated infections, food safety and corrosion/fouling resistance required on industrial surfaces such as oil pipes and ship hulls.

  16. Killing Effect of Lentivirus-mediated Double Suicide Genes on Human Breast Cancer Cells in vivo%慢病毒介导双自杀基因对乳腺癌细胞的体内杀伤作用

    Institute of Scientific and Technical Information of China (English)

    孔恒; 黄宗海; 陈海金; 陶霖玉; 齐柯

    2011-01-01

    Objective: To evaluate the killing effect of lentivirus mediated CD/TK fusion gene controlled by kinase insert domain-containing receptor (KDR) promoter on human breast cancer cells in vivo.Methods: Nude mice were used as hosts for cell line MCF-7 xenografts to establish the animal model of breast cancer.The tumor-bearing mice were randomly divided into 4 groups.Group Ⅰ were blank controls with tumor-bearing mice with no treatment.Group Ⅱ was injected with lentivirus and prodrug ( 5-FC+ GCV ).Group Ⅲ was injected with lentivirus.Group Ⅳ was injected with prodrug.Tumor growth rate, tumor size and weight were observed.The expression of double suicide genes in xenograft tumors was examined by RT-PCR.The tumor tissue was analyzed by hematoxylin-eosin staining and PCNA immunohistochemistry.Flow cytometry (FCM) was used for cell cycle analysis.Results: Tumor growth was obviously inhibited in group Ⅱ.But there was no significant difference in tumor growth among group Ⅰ, group Ⅲ, and group Ⅳ.RT-PCR demonstrated the existence of CD/TK gene in genetically modified xenograft tumor cells.The expression of PCNA was lower in tumor tissue of group Ⅱ than in group Ⅰ.Flow cytometry analysis showed that the proportion of G1 phase cells was increased and the proportion of G2-M phase cells was decreased.Conclusion: Combined with the prodrug, FGW-KDRP-CD/TK can significantly inhibit the growth of implanted breast cancer cells in nude mice and cell cycle arrest, which may be related to its inhibitory effect on PCNA expression.%目的:研究慢病毒介导的KDR启动子驱动的胞嘧啶脱氨酶(CD)/胸苷激酶(TK)融合基因系统(FGW-KDRP-CD/TK)对乳腺癌细胞的体内杀伤作用.方法:培养乳腺癌MCF-7细胞,建立裸鼠荷瘤模型.荷瘤后将裸鼠随机分为4组.Ⅰ组:空白对照,荷瘤但不施加任何处理;Ⅱ组:注射慢病毒与前药(5-FC+GCV);Ⅲ组:仅注射慢病毒;Ⅳ组:仅注射前药.观察肿瘤生长速度,测量瘤体大小及

  17. What do mathematical models tell us about killing rates during HIV-1 infection?

    Science.gov (United States)

    Gadhamsetty, Saikrishna; Beltman, Joost B; de Boer, Rob J

    2015-11-01

    Over the past few decades the extent to which cytotoxic T lymphocytes (CTLs) control human immunodeficiency virus (HIV) replication has been studied extensively, yet their role and mode of action remain controversial. In some studies, CTLs were found to kill a large fraction of the productively infected cells relative to the viral cytopathicity, whereas in others CTLs were suggested to kill only a small fraction of infected cells. In this review, we compile published estimates of CTL-mediated death rates, and examine whether these studies permit determining the rate at which CTLs kill HIV-1 infected cells. We highlight potential misinterpretations of the CTL-killing rates from the escape rates of mutants, and from perturbations of the steady state viral load during chronic infection. Our major conclusion is that CTL-mediated killing rates remain unknown. But contrary to current consensus, we argue that killing rates higher than one per day are perfectly consistent with the experimental data, which would imply that the majority of the productively infected cells could still die from CTL-mediated killing rather than from viral cytopathicity.

  18. Combinatorial stresses kill pathogenic Candida species.

    Science.gov (United States)

    Kaloriti, Despoina; Tillmann, Anna; Cook, Emily; Jacobsen, Mette; You, Tao; Lenardon, Megan; Ames, Lauren; Barahona, Mauricio; Chandrasekaran, Komelapriya; Coghill, George; Goodman, Daniel; Gow, Neil A R; Grebogi, Celso; Ho, Hsueh-Lui; Ingram, Piers; McDonagh, Andrew; de Moura, Alessandro P S; Pang, Wei; Puttnam, Melanie; Radmaneshfar, Elahe; Romano, Maria Carmen; Silk, Daniel; Stark, Jaroslav; Stumpf, Michael; Thiel, Marco; Thorne, Thomas; Usher, Jane; Yin, Zhikang; Haynes, Ken; Brown, Alistair J P

    2012-10-01

    Pathogenic microbes exist in dynamic niches and have evolved robust adaptive responses to promote survival in their hosts. The major fungal pathogens of humans, Candida albicans and Candida glabrata, are exposed to a range of environmental stresses in their hosts including osmotic, oxidative and nitrosative stresses. Significant efforts have been devoted to the characterization of the adaptive responses to each of these stresses. In the wild, cells are frequently exposed simultaneously to combinations of these stresses and yet the effects of such combinatorial stresses have not been explored. We have developed a common experimental platform to facilitate the comparison of combinatorial stress responses in C. glabrata and C. albicans. This platform is based on the growth of cells in buffered rich medium at 30°C, and was used to define relatively low, medium and high doses of osmotic (NaCl), oxidative (H(2)O(2)) and nitrosative stresses (e.g., dipropylenetriamine (DPTA)-NONOate). The effects of combinatorial stresses were compared with the corresponding individual stresses under these growth conditions. We show for the first time that certain combinations of combinatorial stress are especially potent in terms of their ability to kill C. albicans and C. glabrata and/or inhibit their growth. This was the case for combinations of osmotic plus oxidative stress and for oxidative plus nitrosative stress. We predict that combinatorial stresses may be highly significant in host defences against these pathogenic yeasts.

  19. Photoexcited quantum dots for killing multidrug-resistant bacteria

    Science.gov (United States)

    Courtney, Colleen M.; Goodman, Samuel M.; McDaniel, Jessica A.; Madinger, Nancy E.; Chatterjee, Anushree; Nagpal, Prashant

    2016-05-01

    Multidrug-resistant bacterial infections are an ever-growing threat because of the shrinking arsenal of efficacious antibiotics. Metal nanoparticles can induce cell death, yet the toxicity effect is typically nonspecific. Here, we show that photoexcited quantum dots (QDs) can kill a wide range of multidrug-resistant bacterial clinical isolates, including methicillin-resistant Staphylococcus aureus, carbapenem-resistant Escherichia coli, and extended-spectrum β-lactamase-producing Klebsiella pneumoniae and Salmonella typhimurium. The killing effect is independent of material and controlled by the redox potentials of the photogenerated charge carriers, which selectively alter the cellular redox state. We also show that the QDs can be tailored to kill 92% of bacterial cells in a monoculture, and in a co-culture of E. coli and HEK 293T cells, while leaving the mammalian cells intact, or to increase bacterial proliferation. Photoexcited QDs could be used in the study of the effect of redox states on living systems, and lead to clinical phototherapy for the treatment of infections.

  20. Comparison microbial killing efficacy between sonodynamic therapy and photodynamic therapy

    Science.gov (United States)

    Drantantiyas, Nike Dwi Grevika; Astuti, Suryani Dyah; Nasution, Aulia M. T.

    2016-11-01

    Biofilm is a way used by bacteria to survive from their environmental conditions by forming colony of bacteria. Specific characteristic in biofilm formation is the availability of matrix layer, known as extracellular polymer substance. Treatment using antibiotics may lead bacteria to be to resistant. Other treatments to reduce microbial, like biofilm, can be performed by using photodynamic therapy. Successful of this kind of therapy is induced by penetration of light and photosensitizer into target cells. The sonodynamic therapy offers greater penetrating capability into tissues. This research aimed to use sonodynamic therapy in reducing biofilm. Moreover, it compares also the killing efficacy of photodynamic therapy, sonodynamic therapy, and the combination of both therapeutic schemes (known as sono-photodynamic) to achieve higher microbial killing efficacy. Samples used are Staphylococcus aureus biofilm. Treatments were divided into 4 groups, i.e. group under ultrasound treatment with variation of 5 power levels, group of light treatment with exposure of 75s, group of combined ultrasound-light with variation of ultrasound power levels, and group of combined lightultrasound with variation of ultrasound power levels. Results obtained for each treatment, expressed in % efficacy of log CFU/mL, showed that the treatment of photo-sonodynamic provides greater killing efficacy in comparison to either sonodynamic and sono-photodynamic. The photo-sonodynamic shows also greater efficacy to photodynamic. So combination of light-ultrasound (photo-sonodynamic) can effectively kill microbial biofilm. The combined therapy will provide even better efficacy using exogenous photosensitizer.

  1. Yersinia pseudotuberculosis is resistant to killing by human neutrophils.

    Science.gov (United States)

    Laws, Thomas R; Davey, Martin S; Green, Christopher; Cooper, Ian A M; Titball, Richard W; Lukaszewski, Roman A

    2011-06-01

    The interaction between human neutrophils and the Gram negative gastrointestinal pathogen Yersinia pseudotuberculosis was investigated in vitro. Despite the wealth of data describing how Yersinia can affect the function of neutrophils, there are no published studies describing if neutrophil cells can affect the viability of Y. pseudotuberculosis. The wild-type IP32953 strain of Y. pseudotuberculosis was found to be resistant to killing by human neutrophils. Confocal examination and flow-cytometric analysis of this interaction revealed that bacteria were taken up.

  2. Qualitative differences in the early immune response to live and killed Leishmania major: Implications for vaccination strategies against Leishmaniasis.

    Science.gov (United States)

    Okwor, Ifeoma; Liu, Dong; Uzonna, Jude

    2009-04-28

    Recovery from natural or deliberate infection with Leishmania major leads to the development of lifelong immunity against rechallenge infections. In contrast, vaccination with killed parasites or defined leishmanial antigens generally induces only short-term protection. The reasons for this difference are currently not known but may be related to differences in the quality of the early immune responses to live and killed parasites. Here, we report that live and killed L. major parasites elicit comparable early inflammatory response as evidenced by influx and/or proliferation of cells in the draining lymph nodes (dLNs). In contrast, the early cytokine responses were qualitatively different. Cells from mice inoculated with killed parasites produced significantly more antigen-specific IL-4 and less IFN-gamma than those from mice injected with live parasites. Inclusion of CpG ODN into killed parasite preparations changed the early response to killed parasites from IL-4 to a predominantly IFN-gamma response, resulting in better protection following secondary high dose virulent L. major challenge. Interestingly, CpG-mediated enhancement of killed parasites-induced protection was short-lived and waned after 12 weeks. Taken together, these results suggest that the nature of primary immunity induced by killed and live parasites are qualitatively different and that these differences may account for the differential protection seen in mice following vaccination with live and killed parasites. They further suggest that modulating the early response with an appropriate adjuvant could enhance efficacy of killed parasite vaccines.

  3. Killing of Klebsiella pneumoniae by human alveolar macrophages.

    Science.gov (United States)

    Hickman-Davis, Judy M; O'Reilly, Philip; Davis, Ian C; Peti-Peterdi, Janos; Davis, Glenda; Young, K Randall; Devlin, Robert B; Matalon, Sadis

    2002-05-01

    We investigated putative mechanisms by which human surfactant protein A (SP-A) effects killing of Klebsiella pneumoniae by human alveolar macrophages (AMs) isolated from bronchoalveolar lavagates of patients with transplanted lungs. Coincubation of AMs with human SP-A (25 microg/ml) and Klebsiella resulted in a 68% decrease in total colony forming units by 120 min compared with AMs infected with Klebsiella in the absence of SP-A, and this SP-A-mediated effect was abolished by preincubation with N(G)-monomethyl-L-arginine. Incubation of transplant AMs with SP-A increased intracellular Ca(2+) concentration ([Ca(2+)](i)) by 70% and nitrite and nitrate (NO(x)) production by 45% (from 0.24 +/- 0.02 to 1.3 +/- 0.21 nmol small middle dot 10(6) AMs(-1).h(-1)). Preincubation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester inhibited the increase in [Ca(2+)](i) and abrogated the SP-A-mediated Klebsiella phagocytosis and killing. In contrast, incubation of AMs from normal volunteers with SP-A decreased both [Ca(2+)](i) and NO(x) production and did not result in killing of Klebsiella. Significant killing of Klebsiella was also seen in a cell-free system by sustained production of peroxynitrite (>1 microM/min) at pH 5 but not at pH 7.4. These findings indicate that SP-A mediates pathogen killing by AMs from transplant lungs by stimulating phagocytosis and production of reactive oxygen-nitrogen intermediates.

  4. Killing superalgebras for Lorentzian four-manifolds

    CERN Document Server

    de Medeiros, Paul; Santi, Andrea

    2016-01-01

    We determine the Killing superalgebras underpinning field theories with rigid unextended supersymmetry on Lorentzian four-manifolds by re-interpreting them as filtered deformations of $\\mathbb{Z}$-graded subalgebras with maximum odd dimension of the $N{=}1$ Poincar\\'e superalgebra in four dimensions. Part of this calculation involves computing a Spencer cohomology group which, by analogy with a similar result in eleven dimensions, prescribes a notion of Killing spinor, which we identify with the defining condition for bosonic supersymmetric backgrounds of minimal off-shell supergravity in four dimensions. We prove that such Killing spinors always generate a Lie superalgebra, and that this Lie superalgebra is a filtered deformation of a subalgebra of the $N{=}1$ Poincar\\'e superalgebra in four dimensions. Demanding the flatness of the connection defining the Killing spinors, we obtain equations satisfied by the maximally supersymmetric backgrounds. We solve these equations, arriving at the classification of ma...

  5. Homefucking is Killing Prostitution / Taavi Eelmaa

    Index Scriptorium Estoniae

    Eelmaa, Taavi, 1971-

    2008-01-01

    Mis jääb vaatajale teatrietendusest meelde? Ilmus Kris Moori raamat "Homefucking is Killing Prostitution". Raamat sisaldab tekste ja Erki Lauri fotosid Von Krahli Teatri samanimelisest etendusest, mida kordagi ei mängitud

  6. The Geometry of D=11 Killing Spinors

    CERN Document Server

    Gauntlett, J P; Gauntlett, Jerome P.; Pakis, Stathis

    2003-01-01

    We propose a way to classify all supersymmetric configurations of D=11 supergravity using the G-structures defined by the Killing spinors. We show that the most general bosonic geometries admitting a Killing spinor have at least an SU(5) or an (Spin(7)\\ltimes R^8)x R structure, depending on whether the Killing vector constructed from the Killing spinor is timelike or null, respectively. In the former case we determine what kind of SU(5) structure is present and show that almost all of the form of the geometry is determined by the structure. We also deduce what further conditions must be imposed in order that the equations of motion are satisfied. We illustrate the formalism with some known solutions and also present some new solutions including a rotating generalisation of the resolved membrane solutions and generalisations of the recently constructed D=11 Godel solution.

  7. Parricide: Children Who Kill Their Parents

    Science.gov (United States)

    1988-01-01

    felt from his father’s death.3 Lauretta Bender is a psychoanalyst noted for her studies of homicide by children and adolescents. Many of her case...Quarterly 45, no. 1 (1971): 65-69. 3 Freud, 222-42. Lauretta Bender , "Children and Adolescents Who Have Killed," American Journal of Psychiatry 116 (1959...Victims. New York: Charles Scribner’s Sons, 1978. Bender , Lauretta . "Children and Adolescents Who Have Killed." American Journal of Psychiatry 116

  8. Killing vector fields and harmonic superfield theories

    Energy Technology Data Exchange (ETDEWEB)

    Groeger, Josua, E-mail: groegerj@mathematik.hu-berlin.de [Humboldt-Universität zu Berlin, Institut für Mathematik, Rudower Chaussee 25, 12489 Berlin (Germany)

    2014-09-15

    The harmonic action functional allows a natural generalisation to semi-Riemannian supergeometry, also referred to as harmonic, which resembles the supersymmetric sigma models studied in high energy physics. We show that Killing vector fields are infinitesimal supersymmetries of this harmonic action and prove three different Noether theorems in this context. En passant, we provide a homogeneous treatment of five characterisations of Killing vector fields on semi-Riemannian supermanifolds, thus filling a gap in the literature.

  9. Killing Vector Fields and Superharmonic Field Theories

    CERN Document Server

    Groeger, Josua

    2013-01-01

    The harmonic action functional allows a natural generalisation to semi-Riemannian supergeometry, referred to as superharmonic action, which resembles the supersymmetric sigma models studied in high energy physics. We show that Killing vector fields are infinitesimal supersymmetries of the superharmonic action and prove three different Noether theorems in this context. En passant, we provide a homogeneous treatment of five characterisations of Killing vector fields on semi-Riemannian supermanifolds, thus filling a gap in the literature.

  10. Valproic acid enhance the killing effect of cisplatin on HO8910 cells%丙戊酸钠协同顺铂对HO8910细胞杀伤作用机制研究

    Institute of Scientific and Technical Information of China (English)

    张丽娜; 马杰; 曹振平; 李彩荣; 冯定庆; 赵卫东

    2011-01-01

    Objective : To study the mechanism of which Valproic acid enhance the killing effect of cisplatin on human ovanan cancer cell line HO-8910. Methods: Pretreated with lμg/ml DDP and 3mmol/L VPA,the growth inhibition ratio of HO-8910 cells was detected with MTT. Morphological changes of cells in each group were observed with inverted microscope.Flow cytometry analyzed the cell cycle changes. The expression of Ac-H3 , p53 , p21 CyclinD1 gene was deteeed by semiquantitative RT- PCR and Western blot. Results : ( 1 ) VPA can inhibit HO8910 cell growth in a dose and time-dependent. The suppression ratio of treated with VPA combined DDP was higher than that of DDP treatment ( P<0. 05 ) . (2) Cell morphology changes can clearly be seen in the VPA group under light microscope : cell volume reduced , change to long spindle , membrane shrinkage , poor adherence. ( 3 ) VPA arrest ovarian cancer HO8910 cell cycle at G1 phase,but in VPA+DDP group G1/G2 phase cells increased as well as S phase decreased. (4)VPA and VPA combination with DDP can significantly enhance the acetylation of Histone H3 ,increase expression of p53 and p21 and reduce Cyclin Dl expression( P<0. 05 ).Conclusion : VPA can enhance the inhibitory effect of DDP on cell HO89IO;The mechanism of action may include increase in the level of histone acetylation and the expression of p53, p21 gene,decrease in the expression of Cyclin Dl and induction of cell cycle arrest.%目的:探讨丙戊酸钠(VPA)协同顺铂(DDP)对人卵巢癌HO8910细胞的杀伤作用及机制.方法:以1μg/ml DDP联合3mmol/L VPA处理细胞,MTT法检测细胞生长抑制率;光镜下观察药物作用下各组细胞形态学的改变;流式细胞术检测细胞周期阻滞情况;RT-PCR法检测p53、p21 mRNA水平表达的改变;Western blot法检测Ac-H3、p53、p21、Cyclin D1蛋白水平表达的改变.结果:(1)VPA能明显抑制HO8910细胞生长,且呈剂量和时间依赖性,VPA+DDP处理组抑制率高于DDP处理组,组

  11. THE KILLING OF COLON BACILLI BY ULTRAVIOLET LIGHT.

    Science.gov (United States)

    Wyckoff, R W

    1932-01-20

    The survival ratios of colon bacilli subjected to several monochromatic ultraviolet radiations follow semilogarithmic straight lines. For each wave length approximate observations have been made of the energy involved in cell destruction. This energy varies somewhat with frequency in the ultraviolet region; it is furthermore nearly one hundred times as great as the amount of X-ray energy required to bring about the same killing. Preliminary experiments show no measurable difference either in rate of killing or in lethal energy between B. coli and B. aertrycke. Parallel results have already been obtained with X-rays and electrons. The data from colon bacilli are interpreted in terms of the assumptions employed for X-rays. They indicate that though bacterial death should result from a single quantum absorption, millions more such absorptions seemingly are without injurious effect on cell growth and multiplication. The "sensitive volume" within which, according to this picture, the lethal quantum must be stopped proves to be about the same as that of a single protein molecule. If this is the correct description of the phenomena of ultraviolet killing, it seems strange that the millions of non-deadly quanta absorbed per bacillus should not show themselves by altered growth rates or in other ways. That they apparently do not suggests the inapplicability of the statistical picture. The death rate under this kind of radiation then would be primarily an expression of the relative sensitivities of the bacterial population. Additional experiments are required to determine this question.

  12. Increased Lytic Efficiency of Bovine Macrophages Trained with Killed Mycobacteria

    Science.gov (United States)

    Juste, Ramon A.; Alonso-Hearn, Marta; Garrido, Joseba M.; Abendaño, Naiara; Sevilla, Iker A.; Gortazar, Christian; de la Fuente, José; Dominguez, Lucas

    2016-01-01

    Innate immunity is evolutionarily conserved in multicellular organisms and was considered to lack memory until very recently. One of its more characteristic mechanisms is phagocytosis, the ability of cells to engulf, process and eventually destroy any injuring agent. We report the results of an ex vivo experiment in bovine macrophages in which improved clearance of Mycobacterium bovis (M. bovis) was induced by pre-exposure to a heat killed M. bovis preparation. The effects were independent of humoral and cellular adaptive immune responses and lasted up to six months. Specifically, our results demonstrate the existence of a training effect in the lytic phase of phagocytosis that can be activated by killed mycobacteria, thus suggesting a new mechanism of vaccine protection. These findings are compatible with the recently proposed concept of trained immunity, which was developed to explain the observation that innate immune responses provide unspecific protection against pathogens including other than those that originally triggered the immune response. PMID:27820836

  13. Involvement of superoxide and myeloperoxidase in oxygen-dependent killing of Staphylococcus aureus by neutrophils.

    OpenAIRE

    Hampton, M B; A. J. Kettle; Winterbourn, C C

    1996-01-01

    We have used a quantitative assay that measures independent rate constants for phagocytosis and killing of Staphylococcus aureus to investigate the involvement of superoxide and myeloperoxidase in bacterial killing by human neutrophils. To inhibit superoxide-dependent processes, superoxide dismutase was cross-linked to immunoglobulin G and the conjugate was attached to the surface of S. aureus via protein A in its cell wall. Myeloperoxidase was inhibited with azide, and myeloperoxidase-defici...

  14. 嗜酸乳杆菌活菌体和热灭活菌体对 IPEC-J2细胞的黏附和黏附拮抗效应%Adhesion and Adhesion Antagonistic Effect of Live and Heat-Killed Lactobacillus acidophilus on IPEC-J2 Cells

    Institute of Scientific and Technical Information of China (English)

    俞涵丽; 方华; 蔡旋; 徐建雄

    2014-01-01

    The adhesion and adhesion antagonistic effect of live and heat-killed Lactobacillus acidophilus on IPEC-J2 cells were studied in vitro. IPEC-J2 cells were treated with live and heat-killed Lactobacillus acidophi-lus,and then stained the cells and observed the adhesive property. Moreover,the inhibition ratio of live and heat-killed Lactobacillus acidophilus to inhibit the adhesion of Escherichia coli on IPEC-J2 cells through three treatments(competitive inhibition,exclusion inhibition and displacement inhibition)was assessed by eosin-methylene blue agar(EMB)culture medium. The results showed as follows:live and heat-killed Lactobacillus acidophilus all could adhere well to the IPEC-J2 cells. The number of heat-killed bacteria adhering to the per cell[(40±2)CFU]was higher than the number of live bacteria adhering to the per cell[(20±2)CFU]. The two forms of Lactobacillus acidophilus had a good ability to inhibit the Escherichia coli adhere to IPEC-J2 cells. In competitive and displacement inhibition experiments,live bacteria showed a significantly better inhibi-tion ratio than heat-killed bacteria(P<0.05),whereas,in exclusion inhibition experiment,heat-killed bacteria showed a significantly better inhibition ratio than live bacteria( P<0.05). In conclusion,live and heat-killed Lactobacillus acidophilus can adhere well to the IPEC-J2 cells,and are able to reduce the adhesion and inva-sion to IPEC-J2 cells by Escherichia coli. Heat-killed Lactobacillus acidophilus inhibits Escherichia coli adhe-sion mainly through competition for host cell-binding receptors. Apart from competition for host cell-binding re-ceptors,some antibacterial substances from live Lactobacillus acidophilus are able to inhibit the growth and re-production of Escherichia coli.%本试验旨在研究嗜酸乳杆菌活菌体和热灭活菌体在体外模拟条件下对 IPEC-J2细胞的黏附和黏附拮抗效应。用嗜酸乳杆菌活菌体和热灭活菌体处理 IPEC-J2细胞,染色后观察细

  15. Mechanisms of Bacterial (Serratia marcescens) Attachment to, Migration along, and Killing of Fungal Hyphae.

    Science.gov (United States)

    Hover, Tal; Maya, Tal; Ron, Sapir; Sandovsky, Hani; Shadkchan, Yana; Kijner, Nitzan; Mitiagin, Yulia; Fichtman, Boris; Harel, Amnon; Shanks, Robert M Q; Bruna, Roberto E; García-Véscovi, Eleonora; Osherov, Nir

    2016-05-01

    We have found a remarkable capacity for the ubiquitous Gram-negative rod bacterium Serratia marcescens to migrate along and kill the mycelia of zygomycete molds. This migration was restricted to zygomycete molds and several basidiomycete species. No migration was seen on any molds of the phylum Ascomycota. S. marcescens migration did not require fungal viability or surrounding growth medium, as bacteria migrated along aerial hyphae as well.S. marcescens did not exhibit growth tropism toward zygomycete mycelium. Bacterial migration along hyphae proceeded only when the hyphae grew into the bacterial colony. S. marcescens cells initially migrated along the hyphae, forming attached microcolonies that grew and coalesced to generate a biofilm that covered and killed the mycelium. Flagellum-defective strains of S. marcescens were able to migrate along zygomycete hyphae, although they were significantly slower than the wild-type strain and were delayed in fungal killing. Bacterial attachment to the mycelium does not necessitate type 1 fimbrial adhesion, since mutants defective in this adhesin migrated equally well as or faster than the wild-type strain. Killing does not depend on the secretion of S. marcescens chitinases, as mutants in which all three chitinase genes were deleted retained wild-type killing abilities. A better understanding of the mechanisms by which S. marcescens binds to, spreads on, and kills fungal hyphae might serve as an excellent model system for such interactions in general; fungal killing could be employed in agricultural fungal biocontrol.

  16. Killing superalgebras for Lorentzian four-manifolds

    Science.gov (United States)

    de Medeiros, Paul; Figueroa-O'Farrill, José; Santi, Andrea

    2016-06-01

    We determine the Killing superalgebras underpinning field theories with rigid unextended supersymmetry on Lorentzian four-manifolds by re-interpreting them as filtered deformations of mathbb{Z} -graded subalgebras with maximum odd dimension of the N = 1 Poincaré superalgebra in four dimensions. Part of this calculation involves computing a Spencer cohomology group which, by analogy with a similar result in eleven dimensions, prescribes a notion of Killing spinor, which we identify with the defining condition for bosonic supersymmetric backgrounds of minimal off-shell supergravity in four dimensions. We prove that such Killing spinors always generate a Lie superalgebra, and that this Lie superalgebra is a filtered deformation of a subalgebra of the N = 1 Poincaré superalgebra in four dimensions. Demanding the flatness of the connection defining the Killing spinors, we obtain equations satisfied by the maximally supersymmetric backgrounds. We solve these equations, arriving at the classification of maximally supersymmetric backgrounds whose associated Killing superalgebras are precisely the filtered deformations we classify in this paper.

  17. Phg1/TM9 proteins control intracellular killing of bacteria by determining cellular levels of the Kil1 sulfotransferase in Dictyostelium.

    Directory of Open Access Journals (Sweden)

    Marion Le Coadic

    Full Text Available Dictyostelium discoideum has largely been used to study phagocytosis and intracellular killing of bacteria. Previous studies have shown that Phg1A, Kil1 and Kil2 proteins are necessary for efficient intracellular killing of Klebsiella bacteria. Here we show that in phg1a KO cells, cellular levels of lysosomal glycosidases and lysozyme are decreased, and lysosomal pH is increased. Surprisingly, overexpression of Kil1 restores efficient killing in phg1a KO cells without correcting these lysosomal anomalies. Conversely, kil1 KO cells are defective for killing, but their enzymatic content and lysosomal pH are indistinguishable from WT cells. The killing defect of phg1a KO cells can be accounted for by the observation that in these cells the stability and the cellular amount of Kil1 are markedly reduced. Since Kil1 is the only sulfotransferase characterized in Dictyostelium, an (unidentified sulfated factor, defective in both phg1a and kil1 KO cells, may play a key role in intracellular killing of Klebsiella bacteria. In addition, Phg1B plays a redundant role with Phg1A in controlling cellular amounts of Kil1 and intracellular killing. Finally, cellular levels of Kil1 are unaffected in kil2 KO cells, and Kil1 overexpression does not correct the killing defect of kil2 KO cells, suggesting that Kil2 plays a distinct role in intracellular killing.

  18. 75 FR 62469 - Drawbridge Operation Regulations; Newtown Creek, Dutch Kills, English Kills, and Their...

    Science.gov (United States)

    2010-10-12

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF HOMELAND SECURITY Coast Guard 33 CFR Part 117 Drawbridge Operation Regulations; Newtown Creek, Dutch Kills, English Kills, and Their Tributaries, NY, Maintenance AGENCY: Coast Guard, DHS. ACTION: Notice of...

  19. 75 FR 30299 - Drawbridge Operation Regulations; Newtown Creek, Dutch Kills, English Kills, and Their...

    Science.gov (United States)

    2010-06-01

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF HOMELAND SECURITY Coast Guard 33 CFR Part 117 Drawbridge Operation Regulations; Newtown Creek, Dutch Kills, English Kills, and Their Tributaries, NY, Maintenance AGENCY: Coast Guard, DHS. ACTION: Notice of...

  20. 9 CFR 113.206 - Wart Vaccine, Killed Virus.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Wart Vaccine, Killed Virus. 113.206... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.206 Wart Vaccine, Killed Virus. Wart Vaccine, Killed Virus, shall be...

  1. Killing and replacing queen-laid eggs: low cost of worker policing in the honeybee.

    Science.gov (United States)

    Kärcher, Martin H; Ratnieks, Francis L W

    2014-07-01

    Worker honeybees, Apis mellifera, police each other's reproduction by killing worker-laid eggs. Previous experiments demonstrated that worker policing is effective, killing most (∼98%) worker-laid eggs. However, many queen-laid eggs were also killed (∼50%) suggesting that effective policing may have high costs. In these previous experiments, eggs were transferred using forceps into test cells, mostly into unrelated discriminator colonies. We measured both the survival of unmanipulated queen-laid eggs and the proportion of removal errors that were rectified by the queen laying a new egg. Across 2 days of the 3-day egg stage, only 9.6% of the queen-laid eggs in drone cells and 4.1% in worker cells were removed in error. When queen-laid eggs were removed from cells, 85% from drone cells and 61% from worker cells were replaced within 3 days. Worker policing in the honeybee has a high benefit to policing workers because workers are more related to the queen's sons (brothers, r = 0.25) than sister workers' sons (0.15). This study shows that worker policing also has a low cost in terms of the killing of queen-laid eggs, as only a small proportion of queen-laid eggs are killed, most of which are rapidly replaced.

  2. 'Total disability' and the wrongness of killing.

    Science.gov (United States)

    Omelianchuk, Adam

    2015-08-01

    Walter Sinnott-Armstrong and Franklin G Miller recently argued that the wrongness of killing is best explained by the harm that comes to the victim, and that 'total disability' best explains the nature of this harm. Hence, killing patients who are already totally disabled is not wrong. I maintain that their notion of total disability is ambiguous and that they beg the question with respect to whether there are abilities left over that remain relevant for the goods of personhood and human worth. If these goods remain, then something more is lost in death than in 'total disability,' and their explanation of what makes killing wrong comes up short. But if total disability is equivalent with death, then their argument is an interesting one.

  3. Axial symmetry and conformal Killing vectors

    CERN Document Server

    Mars, M; Mars, Marc; Senovilla, Jose M.M.

    1993-01-01

    Axisymmetric spacetimes with a conformal symmetry are studied and it is shown that, if there is no further conformal symmetry, the axial Killing vector and the conformal Killing vector must commute. As a direct consequence, in conformally stationary and axisymmetric spacetimes, no restriction is made by assuming that the axial symmetry and the conformal timelike symmetry commute. Furthermore, we prove that in axisymmetric spacetimes with another symmetry (such as stationary and axisymmetric or cylindrically symmetric spacetimes) and a conformal symmetry, the commutator of the axial Killing vector with the two others mush vanish or else the symmetry is larger than that originally considered. The results are completely general and do not depend on Einstein's equations or any particular matter content.

  4. On the algebraic structure of Killing superalgebras

    CERN Document Server

    Figueroa-O'Farrill, José

    2016-01-01

    We study the algebraic structure of the Killing superalgebra of a supersymmetric $11$-dimensional supergravity background and show that it is isomorphic to a filtered deformation of a $\\mathbb Z$-graded subalgebra of the Poincar\\'e superalgebra. We then re-interpret the classification problem for backgrounds which preserve more than half of the supersymmetry as the classification problem of certain admissible filtered subdeformations of the Poincar\\'e superalgebra. In particular we relate the bosonic field equations of $11$-dimensional supergravity to the Jacobi identity of the Killing superalgebra and show in this way that preserving more than half the supersymmetry implies the bosonic field equations.

  5. Membrane lipid peroxidation in copper alloy-mediated contact killing of Escherichia coli.

    Science.gov (United States)

    Hong, Robert; Kang, Tae Y; Michels, Corinne A; Gadura, Nidhi

    2012-03-01

    Copper alloy surfaces are passive antimicrobial sanitizing agents that kill bacteria, fungi, and some viruses. Studies of the mechanism of contact killing in Escherichia coli implicate the membrane as the target, yet the specific component and underlying biochemistry remain unknown. This study explores the hypothesis that nonenzymatic peroxidation of membrane phospholipids is responsible for copper alloy-mediated surface killing. Lipid peroxidation was monitored with the thiobarbituric acid-reactive substances (TBARS) assay. Survival, TBARS levels, and DNA degradation were followed in cells exposed to copper alloy surfaces containing 60 to 99.90% copper or in medium containing CuSO(4). In all cases, TBARS levels increased with copper exposure levels. Cells exposed to the highest copper content alloys, C11000 and C24000, exhibited novel characteristics. TBARS increased immediately at a very rapid rate but peaked at about 30 min. This peak was associated with the period of most rapid killing, loss in membrane integrity, and DNA degradation. DNA degradation is not the primary cause of copper-mediated surface killing. Cells exposed to the 60% copper alloy for 60 min had fully intact genomic DNA but no viable cells. In a fabR mutant strain with increased levels of unsaturated fatty acids, sensitivity to copper alloy surface-mediated killing increased, TBARS levels peaked earlier, and genomic DNA degradation occurred sooner than in the isogenic parental strain. Taken together, these results suggest that copper alloy surface-mediated killing of E. coli is triggered by nonenzymatic oxidative damage of membrane phospholipids that ultimately results in the loss of membrane integrity and cell death.

  6. Mechanism involved in phagocytosis and killing of Listeria monocytogenes by Acanthamoeba polyphaga.

    Science.gov (United States)

    Akya, Alisha; Pointon, Andrew; Thomas, Connor

    2009-10-01

    Intra-cellular pathogen, Listeria monocytogenes, is capable of invasion and survival within mammalian cells. However, Acanthamoeba polyphaga trophozoites phagocytose and rapidly degrade Listeria cells. In order to provide more information on amoeba phagocytosis and killing mechanisms, this study used several inhibitor agents known to affect the phagocytosis and killing of bacteria by eukaryotes. Amoebae were pre-treated with mannose, cytochalasin D, wortmannin, suramin, ammonium chloride, bafilomycin A and monensin followed by co-culture with bacteria. Phagocytosis and killing of bacterial cells by amoeba trophozoites was assessed using plate counting methods and microscopy. The data presented indicates that actin polymerisation and cytoskeletal rearrangement are involved in phagocytosis of L. monocytogenes cells by A. polyphaga trophozoites. Further, both phagosomal acidification and phagosome-lysosome fusion are involved in killing and degradation of L. monocytogenes cells by A. polyphaga. However, the mannose-binding protein receptor does not play an important role in uptake of bacteria by amoeba trophozoites. In conclusion, this data reveals the similar principles of molecular mechanisms used by different types of eukaryotes in uptake and killing of bacteria.

  7. Tissue Dimensionality Influences the Functional Response of Cytotoxic T Lymphocyte-Mediated Killing of Targets.

    Science.gov (United States)

    Gadhamsetty, Saikrishna; Marée, Athanasius F M; de Boer, Rob J; Beltman, Joost B

    2016-01-01

    Cytotoxic T lymphocyte (CTL)-mediated killing of virus infections and tumors occurs over a wide range of conditions. The spatial environments in which CTLs encounter target cells vary from narrow vessels, to two-dimensional epithelial tissues, to densely populated 3-dimensional (3D) T cell areas within lymphoid tissues. How such spatial environments alter the functional response of CTL-mediated killing, i.e., how the killing efficiency depends on cell densities, is unclear. In this study, we perform cellular Potts model simulations in different spatial configurations to investigate how the dimensionality of the space affects the functional response of CTL-mediated killing. Irrespective of the spatial configuration, the function with separate saturation constants for CTL and for target cell densities that we previously proposed can in all cases describe the response, demonstrating its generality. However, the tissue dimensionality determines at which cell densities the killing rate starts to saturate. We show that saturation in a fully 3D environment is stronger than in a "flat" 3D environment, which is largely due to accompanying differences in the CTL-target encounter rates.

  8. The Real-Time-Based Assessment of the Microbial Killing by the Antimicrobial Compounds of Neutrophils

    Directory of Open Access Journals (Sweden)

    J. T. Atosuo

    2011-01-01

    Full Text Available A recombinant Escherichia coli K-12 strain, transformed with a modified bacterial luciferase gene (luxABCDE from Photorhabdus luminescens, was constructed in order to monitor the activity of various antimicrobial agents on a real-time basis. This E. coli-lux emitted, without any addition of substrate, constitutive bioluminescence (BL, which correlated to the number of viable bacterial cells. The decrease in BL signal correlated to the number of killed bacterial cells. Antimicrobial activity of hydrogen peroxide (H2O2 and myeloperoxidase (MPO was assessed. In high concentrations, H2O2 alone had a bacteriocidic function and MPO enhanced this killing by forming hypochlorous acid (HOCl. Taurine, the known HOCl scavenger, blocked the killing by MPO. When E. coli-lux was incubated with neutrophils, similar killing kinetics was recorded as in H2O2/MPO experiments. The opsonization of bacteria enhanced the killing, and the maximum rate of the MPO release from lysosomes coincided with the onset of the killing.

  9. New combined assay of phagocytosis and intracellular killing of Escherichia coli by polymorphonuclear leukocytes

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, P.J.; Ford, J.M. (Saint Bartholomew' s Hospital, London (UK))

    1982-03-12

    A new combined radiometric assay is described in which adherence, and phagocytosis and killing of Escherichia coli by human polymorphonuclear leucocytes (PMN) are simultaneously measured in the same sample. Pure monolayers of PMN in Petri dishes are allowed to ingest (/sup 14/C)phenylalanine labelled E. coli and excess bacteria are removed by washing. A period of incubation allows intracellular killing to occur while polymyxin-B is added to half the dishes to kill extracellular bacteria. The remaining viable bacteria in all dishes are labelled with (/sup 3/H)thymidine. The number of ingested bacteria and the percentage of intracellular organisms killed is determined from the /sup 14/C and /sup 3/H counts by a simple subtraction technique. By performing protein assays on representative monolayers, the number of PMN adhered in the monolayers and hence the mean bacterial uptake per PMN is estimated. The assay detected killing efficiencies reduced below the normal range, in monolayers treated with sodium azide, phenylbutazone, in polymorphonuclear leukocytes from patients with chronic granulomatous disease, and in immature neutrophils from the promyelocytic leukaemic cell line, HL60. The assay was adapted to measure phagocytosis and killing by cells in suspension.

  10. The real-time-based assessment of the microbial killing by the antimicrobial compounds of neutrophils.

    Science.gov (United States)

    Atosuo, J T; Lilius, E-M

    2011-01-01

    A recombinant Escherichia coli K-12 strain, transformed with a modified bacterial luciferase gene (luxABCDE) from Photorhabdus luminescens, was constructed in order to monitor the activity of various antimicrobial agents on a real-time basis. This E. coli-lux emitted, without any addition of substrate, constitutive bioluminescence (BL), which correlated to the number of viable bacterial cells. The decrease in BL signal correlated to the number of killed bacterial cells. Antimicrobial activity of hydrogen peroxide (H(2)O(2)) and myeloperoxidase (MPO) was assessed. In high concentrations, H(2)O(2) alone had a bacteriocidic function and MPO enhanced this killing by forming hypochlorous acid (HOCl). Taurine, the known HOCl scavenger, blocked the killing by MPO. When E. coli-lux was incubated with neutrophils, similar killing kinetics was recorded as in H(2)O(2)/MPO experiments. The opsonization of bacteria enhanced the killing, and the maximum rate of the MPO release from lysosomes coincided with the onset of the killing.

  11. 白细胞介素-2增强淋巴细胞对乳腺癌细胞的杀伤作用%Killing effect of interlukin-2 on breast cancer cells by promoting expansion of lymphocytes in vitro

    Institute of Scientific and Technical Information of China (English)

    熊一全; 黄韬; 赵向旺; 田元; 张景辉

    2015-01-01

    Objective To discuss the inhibitory effect of lymphocytes and interlukin-2 (IL-2)on hormone receptor positive (ER +) breast cancer cells MCF-7,and to investigate the mechanism by which IL-2 promotes the killing effects of lymphocytes.Methods Lymphocytes and IL-2 with different concentrations were co-cultured with MCF-7 cells respectively.Cell counting kit-8 (CCK-8) was used to analyze the the viability of cancer cells.Results In MCF-7 cells treated with 1 000 U IL-2,the effect of IL-2 promoting expansion of lymphocytes was 30.94%,and the killing effect of IL-2 on MCF-7 cells by lymphocytes was 91.24%.The killing effect of lymphocytes on MCF-7 cells was not enhanced by increasing the concentration of IL-2.Conclusion IL-2 prompted the tumor inhibitory effect on ER + breast cancer cells MCF-7%目的 观察淋巴细胞以及白细胞介素-2(IL-2)联合淋巴细胞对于激素受体阳性(ER+)的乳癌细胞株MCF-7的杀伤作用,探讨IL-2增强淋巴细胞的抑癌效应.方法 分别用不同浓度的淋巴细胞以及IL-2加淋巴细胞与乳腺癌细胞株MCF-7共培养,细胞计数试剂盒(CCK-8)法对比分析淋巴细胞对MCF-7细胞的杀伤效应的差异.结果 每孔1 000 U IL-2促进淋巴细胞增殖效应为30.94%;促进淋巴细胞杀伤MCF-7效应为91.24%,增加IL-2的浓度并未增加淋巴细胞杀伤MCF-7能力.结论 IL-2能促进淋巴细胞对MCF-7的杀伤作用.

  12. Killing Effect of Fusion Suicide Genes on Nasopharyngeal Carcinoma Cells in vivo%融合自杀基因对鼻咽癌细胞的体内杀伤作用

    Institute of Scientific and Technical Information of China (English)

    卿菁; 赵素萍; 蒋卫红; 章华

    2013-01-01

    [目的]研究融合自杀基因对鼻咽癌细胞的体内杀伤作用.[方法]培养鼻咽癌CNE-2细胞,建立裸鼠荷瘤模型.随机分为6组,每组5只,A组:脂质体包裹质粒瘤内注射瘤内组;B组:脂质体包裹质粒瘤内注射+肿瘤局部照射γ射线(总剂量9Gy)组;C组:脂质体包裹质粒瘤内注射+腹腔注射5氟胞嘧啶(5-Fc)组;D组:脂质体包裹质粒瘤内注射+肿瘤局部照射γ射线(总剂量9Gy)+腹腔注射5-Fc组;E组:肿瘤局部照射γ射线(总剂量9Gy)+腹腔注射5-Fc,以及空白对照组.记录肿瘤体积变化;计算各组肿瘤抑制率,绘制肿瘤生长曲线;取肿瘤组织进行病理观察;鉴定肿瘤组织中CD/UPRT.UL49基因.肿瘤体积变化的比较采用单向方差分析,组间多重比较采用LSD法.[结果]成功建立鼻咽癌裸鼠荷瘤模型,生长曲线显示:5组瘤体积差异有统计学意义(F=720.684,P<0.01),测序结果证实,除E组外,其余各处理组均检测到目的基因,且Western blot检测结果示:B和D组肿瘤组织内自杀基因的表达高于其余各组.病理切片证实肿块为低分化鳞癌,其中D组肿瘤组织在干预作用下出现明显坏死.[结论] E6.BARF1p.CD/UPRT.UL49自杀基因载体联合前药5-Fc在放射线的作用下,可明显抑制鼻咽癌移植瘤的生长,转染该融合自杀基因可增强放疗敏感性,为鼻咽癌的基因—放射治疗开辟了新思路.%[Purpose] To explore the killing effect of fusion suicide genes in human nasopharyngeal carcinoma (NPC) in vivo.[Methods] NPC CNE-2 cell line was hosted to establish mice model with tumor which were randomly divided into 6 groups,where there were 5 mice with different interventions.The volumes of tumor were measured and compared,the tumor growth inhibition rates were calculated.The tumor tissue was taken out for analysis by Hematoxylin-Eosin staining,and the existence of CD/UPRT.UL49 was detected in tumor tissue.Analysis of variance was used for the comparison of

  13. Killing Hitler: A Writer's Journey and Angst.

    Science.gov (United States)

    Thaler, Paul

    2002-01-01

    Describes the author's experiences in preparing a talk that "evokes the specter" of Adolf Hitler and in writing an historical account of a British plot to kill Hitler. Address the question of why the British allowed him to live that final year of the war. Muses on why scholars write, and the impact of violence and terrorism. (SG)

  14. Gas Well Blowout Kills 243 People

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    @@ At least 243 people have been killed and scores of others poisoned in a devastating blowout at a natural gas field in Southwest China's Chongqing municipality on December 24. The accident happened at the Chuandongbei gas field in Kaixian county of Chongqing municipality.

  15. Contagion in Mass Killings and School Shootings

    Science.gov (United States)

    Towers, Sherry; Gomez-Lievano, Andres; Khan, Maryam; Mubayi, Anuj; Castillo-Chavez, Carlos

    2015-01-01

    Background Several past studies have found that media reports of suicides and homicides appear to subsequently increase the incidence of similar events in the community, apparently due to the coverage planting the seeds of ideation in at-risk individuals to commit similar acts. Methods Here we explore whether or not contagion is evident in more high-profile incidents, such as school shootings and mass killings (incidents with four or more people killed). We fit a contagion model to recent data sets related to such incidents in the US, with terms that take into account the fact that a school shooting or mass murder may temporarily increase the probability of a similar event in the immediate future, by assuming an exponential decay in contagiousness after an event. Conclusions We find significant evidence that mass killings involving firearms are incented by similar events in the immediate past. On average, this temporary increase in probability lasts 13 days, and each incident incites at least 0.30 new incidents (p = 0.0015). We also find significant evidence of contagion in school shootings, for which an incident is contagious for an average of 13 days, and incites an average of at least 0.22 new incidents (p = 0.0001). All p-values are assessed based on a likelihood ratio test comparing the likelihood of a contagion model to that of a null model with no contagion. On average, mass killings involving firearms occur approximately every two weeks in the US, while school shootings occur on average monthly. We find that state prevalence of firearm ownership is significantly associated with the state incidence of mass killings with firearms, school shootings, and mass shootings. PMID:26135941

  16. Contagion in Mass Killings and School Shootings.

    Directory of Open Access Journals (Sweden)

    Sherry Towers

    Full Text Available Several past studies have found that media reports of suicides and homicides appear to subsequently increase the incidence of similar events in the community, apparently due to the coverage planting the seeds of ideation in at-risk individuals to commit similar acts.Here we explore whether or not contagion is evident in more high-profile incidents, such as school shootings and mass killings (incidents with four or more people killed. We fit a contagion model to recent data sets related to such incidents in the US, with terms that take into account the fact that a school shooting or mass murder may temporarily increase the probability of a similar event in the immediate future, by assuming an exponential decay in contagiousness after an event.We find significant evidence that mass killings involving firearms are incented by similar events in the immediate past. On average, this temporary increase in probability lasts 13 days, and each incident incites at least 0.30 new incidents (p = 0.0015. We also find significant evidence of contagion in school shootings, for which an incident is contagious for an average of 13 days, and incites an average of at least 0.22 new incidents (p = 0.0001. All p-values are assessed based on a likelihood ratio test comparing the likelihood of a contagion model to that of a null model with no contagion. On average, mass killings involving firearms occur approximately every two weeks in the US, while school shootings occur on average monthly. We find that state prevalence of firearm ownership is significantly associated with the state incidence of mass killings with firearms, school shootings, and mass shootings.

  17. 树突状细胞的诱导培养及对胃癌细胞株MKN45的体外杀伤效应的初步研究%Preliminary studying on dendritic cell culture and its killing effect on gastric carcinoma cell line MKN 45

    Institute of Scientific and Technical Information of China (English)

    孙梯业; 颜伟; 孙冬丽; 刘全达; 段伟宏; 周宁新

    2010-01-01

    目的 探讨树突状细胞(DC)体外培养的方法及CpG ODN1826刺激DC对胃癌细胞MKN45的杀伤效应.方法 分离正常人外周血DC,培养至第5天,实验分为4组,A组[粒细胞-巨噬细胞集落刺激因子(GM-CSF)+白细胞介素-4(IL-4)]、B组[GM-CSF+IL-4+肿瘤坏死因子-α(TNF-α)]、C组(nonCpG ODN)和D组(CpG ODN 1826).自外周血单核细胞(PBMC)诱导分离DC,流式细胞仪检测DC表面标志,MTT法测定其刺激对同种异体淋巴细胞增殖的影响及对胃癌细胞的杀伤效应.结果 体外培养至第10天,D组和B组均出现大量典型的DC形态.倒置显微镜下见细胞呈树突状、伪足状突起.流式细胞仪检测,D组显著高表达共刺激分子(CD40、CD1a、CD80、CD86)和MHC-Ⅱ类分子,均分别高于其他各组(P<0.05).在体外能强烈刺激同种异体混合淋巴细胞的增殖,显著增强DC杀伤胃癌细胞MKN45的活性.结论 该培养方法可获得较高纯度典型的DC,同时CpG ODN可显著诱导人外周血DC分化成熟,增强DC对胃癌细胞MKN45的杀伤作用.%Objective To explore the cultivated methods of dendritic cells (DC) and the killing effect of DC stimulated by CpG ODN1826 on gastritic cancer cells MKN45 in vitro. Methods DC was induced from peripheral blood monocytes stimulated by A group ( GM- CSF + IL-4 ), B group ( GM- CSF + IL-4 + TNF- α), C group(nonCpG ODN) and D group( CpG ODN 1826). The surface markers of DC was analyzed via flow cytometry, and the abilities to stimulate proliferation of allogenic lymphocyte by DC and antitumor experiment were detected by MTT assay. Results On day 10, a majority of cells showed typical morphology of DC in D group and B group with visible branching-like and pseudopod-like structures under microscope. The results of flow cytometry showed that there are significantly high expressed co-stimulated molecules such as CD40, CD1a,CD80, CD86 and MHC- Ⅱ in D group compared to other experimental groups ( P < 0.05 ), which

  18. Intracellular killing of bacteria: is Dictyostelium a model macrophage or an alien?

    Science.gov (United States)

    Cosson, Pierre; Lima, Wanessa C

    2014-01-01

    Predation of bacteria by phagocytic cells was first developed during evolution by environmental amoebae. Many of the core mechanisms used by amoebae to sense, ingest and kill bacteria have also been conserved in specialized phagocytic cells in mammalian organisms. Here we focus on recent results revealing how Dictyostelium discoideum senses and kills non-pathogenic bacteria. In this model, genetic analysis of intracellular killing of bacteria has revealed a surprisingly complex array of specialized mechanisms. These results raise new questions on these processes, and challenge current models based largely on studies in mammalian phagocytes. In addition, recent studies suggest one additional level on complexity by revealing how Dictyostelium recognizes specifically various bacterial species and strains, and adapts its metabolism to process them. It remains to be seen to what extent mechanisms uncovered in Dictyostelium are also used in mammalian phagocytic cells. PMID:24628900

  19. Invasive fungal infection and impaired neutrophil killing in human CARD9 deficiency.

    Science.gov (United States)

    Drewniak, Agata; Gazendam, Roel P; Tool, Anton T J; van Houdt, Michel; Jansen, Machiel H; van Hamme, John L; van Leeuwen, Ester M M; Roos, Dirk; Scalais, Emmanuel; de Beaufort, Carine; Janssen, Hans; van den Berg, Timo K; Kuijpers, Taco W

    2013-03-28

    Caspase recruitment domain-containing protein 9 (CARD9) is an adaptor molecule in the cytosol of myeloid cells, required for induction of T-helper cells producing interleukin-17 (Th17 cells) and important in antifungal immunity. In a patient suffering from Candida dubliniensis meningoencephalitis, mutations in the CARD9 gene were found to result in the loss of protein expression. Apart from the reduced numbers of CD4(+) Th17 lymphocytes, we identified a lack of monocyte-derived cytokines in response to Candida strains. Importantly, CARD9-deficient neutrophils showed a selective Candida albicans killing defect with abnormal ultrastructural phagolysosomes and outgrowth of hyphae. The neutrophil killing defect was independent of the generation of reactive oxygen species by the reduced NAD phosphate oxidase system. Taken together, this demonstrates that human CARD9 deficiency results in selective defect in the host defense against invasive fungal infection, caused by an impaired phagocyte killing.

  20. CD8+ lymphocytes that kill allogeneic and xenogeneic major histocompatibility complex class I targets.

    Science.gov (United States)

    Harms, J S; Splitter, G A

    1995-09-01

    CD8+ CTLs generated in a two-way MLR should lyse target cells only if these targets share a class I MHC allele with the original stimulators. Using cattle PBMCs in a two-way MLR, we generated CD8+ CTLs that kill allogeneic and xenogeneic cell lines. We have named these cells MLK cells. PBMCs isolated from two unrelated animals were cultured together. After 14 days microfluorimetry analysis was performed on the MLK cells with results showing > 90% CD8+ cells. RFLP analysis revealed these cells to be predominately of one animal. MLK cells were then used as effector cells in cytotoxicity assays with syngeneic, allogeneic, and xenogeneic target cells. MLK cells were able to kill all targets. Incubating MLK cells with mAbs to CD8 markedly reduced killing, suggesting a TCR-mediated cytolytic pathway. Effective cytolysis of these targets by MLK cells was dependent on class I expression. MHC class I expression-impaired mutants of allogeneic and xenogeneic targets were not susceptible to cytolysis. Comparisons to LAK cells revealed similarities in phenotype and function to the NK1.1-, CD8+ subset.

  1. Anti-Sonic hedgehog blocking antibody enhances killing effect of PBMCs on cervical carcinoma HeLa cells%Sonic hedgehog阻断抗体增强外周血单个核细胞抗宫颈癌HeLa细胞的作用

    Institute of Scientific and Technical Information of China (English)

    孙艳; 黄莉霞; 黄文浩; 黄斌

    2011-01-01

    AIM: To investigate the effect of anti - Sonic hedgehog( Shh ) blocking antibody on the killing effect of peripheral blood mononuclear cells( PBMCs ) on cervical carcinoma HeLa cells. METHODS: The expression levels of Shh and Shh signaling molecules in HeLa cells were detected by immunocytochemistry and RT - PCR. PBMCs from health peoples were isolated by the method of Ficoll density gradient centrifugation, and then co - cultured with HeLa cells in vitro. The expression of CD3, CD69 and CD71 was assayed by flew cytometry. The concentrations of IFN - 7, IL - 10 and IL - 4 in culture supernatants were detected by ELISA. The killing effect of PBMCs on HeLa cells was observed under microscope. RESULTS: Shh and Shh signaling molecules were expressed in HeLa cells. The level of Shh expression didn t change significantly in the 6th passage of HeLa cells. CD3 + cells were increased in the co - culture system. The expression of CD69 and CD 71, and the secretion of IFN - 7 were increased, while the secretion of IL - 10 was decreased in the co - culture system treated with anti - Shh blocking antibody. Anti - Shh blocking antibody has no effect on the secretion of IL -4. The killing effect of PBMCs on HeLa cells was strengthened by anti - Shh blocking antibody. CONCLUSION : Anti - Shh blocking antibody promotes the activation of PBMCs and enhances the killing effect of PBMCs on cervical carcinoma HeLa cells.%目的:探讨Sonic hedgehog(Shh)阻断抗体对外周血单个核细胞(PBMCs)抗宫颈癌HeLa细胞作用的影响.方法:免疫细胞化学技术和RT-PCR法检测Shh及其信号分子在HeLa 细胞中的表达;Ficoll密度梯度离心法分离正常人PBMCs,并与HeLa细胞建立共培养体系;于共培养体系中加入Shh阻断抗体,流式细胞术检测CD3、CD69和CD71分子表达;ELISA法检测细胞因子IFN-γ、IL-10和IL-4分泌;显微镜观察PBMCs 对HeLa细胞的杀伤.结果:HeLa细胞表达Shh及其信号分子,HeLa细胞传代6次对Shh的表达水

  2. Dirac operators and Killing spinors with torsion; Dirac-Operatoren und Killing-Spinoren mit Torsion

    Energy Technology Data Exchange (ETDEWEB)

    Becker-Bender, Julia

    2012-12-17

    On a Riemannian spin manifold with parallel skew torsion, we use the twistor operator to obtain an eigenvalue estimate for the Dirac operator with torsion. We consider the equality case in dimensions four and six. In odd dimensions we describe Sasaki manifolds on which equality in the estimate is realized by Killing spinors with torsion. In dimension five we characterize all Killing spinors with torsion and obtain certain naturally reductive spaces as exceptional cases.

  3. Inefficacy of vancomycin and teicoplanin in eradicating and killing Staphylococcus epidermidis biofilms in vitro.

    Science.gov (United States)

    Claessens, J; Roriz, M; Merckx, R; Baatsen, P; Van Mellaert, L; Van Eldere, J

    2015-04-01

    Biofilm-associated bacteria display a decreased susceptibility towards antibiotics. Routine assessment of antibiotic susceptibility of planktonic bacteria therefore offers an insufficient prediction of the biofilm response. In this study, in vitro biofilms of eight clinical Staphylococcus epidermidis strains were subjected to treatment with vancomycin, teicoplanin, oxacillin, rifampicin and gentamicin. In addition, the biofilms were subjected to combinations of an antibiotic with rifampicin. The effects on the biofilms were assessed by crystal violet staining to determine the total biofilm biomass, staining with XTT to determine bacterial cell viability, and microscopy. Combining these methods showed that treatment of S. epidermidis biofilms with glycopeptides increased the total biofilm biomass and that these antibiotics were not effective in killing bacteria embedded in biofilms. The decreased killing efficacy was more pronounced in biofilms produced by strains that were classified as 'strong' biofilm producers. Rifampicin, oxacillin and gentamicin effectively killed biofilm-associated bacteria of all tested strains. Combining antibiotics with rifampicin increased the killing efficacy without influencing the total biofilm biomass. When vancomycin or teicoplanin were combined with rifampicin, the increase in biofilm biomass was neutralised and also the killing efficacy was influenced in a positive way. We conclude that the combined methodology used in this study showed that glycopeptides were not effective in eradicating S. epidermidis biofilms but that combination with rifampicin improved the killing efficacy in vitro.

  4. Effect of Shark Liver Oil on Peritoneal Murine Macrophages in Responses to Killed-Candida albicans

    Directory of Open Access Journals (Sweden)

    Monire Hajimoradi

    2009-09-01

    Full Text Available Objective(sShark Liver Oil (SLO is an immunomodulator. Macrophages play a key role in host defense against pathogens like fungi. Candida albicans have mechanisms to escape immune system. We determined the effect of killed-Candida on the in vitro viability of macrophages and the effect of SLO on augmentation of this potency.Materials and MethodsPeritoneal macrophages were separated and cultured (3×105/well. At first, the effect of killed-Candida (200 cells/well on macrophage viability was evaluated, using MTT test. Then, MTT was performed on macrophages stimulated with killed-Candida in the presence of SLO. ResultsKilled-Candida suppressed the ability of MTT reduction and hence macrophages viability (P=0.026, but addition of SLO (100 mg/ml significantly enhanced cell viability (P=0.00. So, SLO could neutralize the inhibitory effect of Candida.ConclusionSimultaneous with cytotoxic effect of killed-Candida cells on macrophages viability, SLO augment macrophages viability. So, it can be applied in candidiasis as a complement.

  5. Killing rates for caspofungin against Candida albicans after brief and continuous caspofungin exposure in the presence and absence of serum.

    Science.gov (United States)

    Kovács, Renátó; Gesztelyi, Rudolf; Perlin, David S; Kardos, Gábor; Domán, Marianna; Berényi, Réka; Majoros, László

    2014-10-01

    It was previously demonstrated that brief (≤1 h) exposures to echinocandins are as effective to kill Candida albicans cells as continuous 24-h exposure. However, killing rates after continuous and short (1 h) echinocandin exposures to C. albicans have not yet been evaluated in RPMI-1640 with and without 50 % serum. We evaluated four echinocandin susceptible C. albicans bloodstream isolates, ATCC 10231 type strain and an echinocandin-resistant isolate (DPL20, FKS F645P). Caspofungin MICs, time-kill and postantifungal effect (PAFE) tests were performed in RPMI-1640 with and without 50 % serum. Killing rates (k values) in time-kill and PAFE experiments were determined for each strain and concentration. In time-kill experiments, colony count decreases were isolate- and concentration-dependent at 0.25, 1, 4, 8, 16 and 32 mg/L in RPMI-1640, but concentration-independent at 1, 4, 8, 16 and 32 mg/L in 50 % serum. One-hour caspofungin exposure at 4, 16 and 32 mg/L resulted in CFU decreases comparable with the results obtained in time-kill experiments in RPMI-1640, but 50 % serum at 4, 16 and 32 mg/L allowed growth of all isolates (k values were negative) (P caspofungin exposure. As only a short growth inhibition without killing was observed in 50 % serum, clinical relevance of caspofungin PAFE in vivo is questionable.

  6. Enhanced effect of CD8++ T cells activated by tumor lysate -pulsed DCs on killing autologous tumor cells%通过肿瘤致敏的DCs活化的CD8+T细胞可有效地杀死肿瘤细胞

    Institute of Scientific and Technical Information of China (English)

    唐小龙; 江振友; 蔡淑玉

    2008-01-01

    AIM:To evaluate the ability of dendritic cells (DCs) loaded with tumor lysate to initiate cell mediated immune responses by stimulating naive T cells, and the efficiency of activated T cells to kill autologous tumor cells in vitro. METHODS: The peripheral blood lymphocytes and monocytes were obtained from the advanced renal cell carcinoma patient by eonglutination method. The immature dendritic cells were generated in the presence of interleukin -4(IL-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF) from monocytes of healthy individuals.These cells were pulsed with tumor lysate or not. Induction of tumor-specific cytotoxic T lymphocytes(CTLs) response by mature dendritic cells (mDCs) was evaluated by the CD95(Fas) expression assay through FCM and the cytotoxic assay a gninst autolognns human tumor cells. RESULTS: Human immature dendritic cells and T cells obtained from healthy donors were stimulated with tumor- pulsed dendritic cells. The immature dendritic cells were applied to the cytotoxicity assay a gainst target autologons tumor cells. The CD95 (Fas) expression, IFN-γ, and TNF -α secreted by the CTLs in tumor lysate-plused DC group were higher than those of other groups. The capacity of the CTLs to kill autolognns tumor cells was significantly different(P<0. 05). Antigen-specific DCs vaccine can induce T cells activation and proliferation, thus we can obtain higher proportion of tumor specific cytotoxic T cells(CTLs), and enhance the CTLs to secret IFN-γ and TNF-α. CONCLUSION: Our results indicate that monocyte-derived human dendritic cells pulsed with tumor lysate could in duce the specific antitumor effect against autologons tumors. This in vitro model offers a new and simple approach to the development of DC + CTL - based immunotherapy.%目的:探索肿瘤裂解物负载的DCs诱导活化的初始T细胞介导细胞免疫及活化的T细胞杀死肿瘤细胞的能力.方法:应用黏附法分离外周血中的淋巴细胞

  7. 9 CFR 113.214 - Parvovirus Vaccine, Killed Virus (Canine).

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Parvovirus Vaccine, Killed Virus... REQUIREMENTS Killed Virus Vaccines § 113.214 Parvovirus Vaccine, Killed Virus (Canine). Parvovirus Vaccine... established as follows: (1) Twenty-five parvovirus susceptible dogs (20 vaccinates and 5 controls) shall...

  8. 9 CFR 113.205 - Newcastle Disease Vaccine, Killed Virus.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Newcastle Disease Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.205 Newcastle Disease Vaccine, Killed Virus. Newcastle Disease Vaccine... Newcastle disease virus supplied by or approved by Veterinary Services and the vaccinates observed each...

  9. It's not just conflict that motivates killing of orangutans.

    Directory of Open Access Journals (Sweden)

    Jacqueline T Davis

    Full Text Available We investigated why orangutans are being killed in Kalimantan, Indonesia, and the role of conflict in these killings. Based on an analysis of interview data from over 5,000 respondents in over 450 villages, we also assessed the socio-ecological factors associated with conflict and non-conflict killings. Most respondents never kill orangutans. Those who reported having personally killed an orangutan primarily did so for non-conflict reasons; for example, 56% of these respondents said that the reason they had killed an orangutan was to eat it. Of the conflict-related reasons for killing, the most common reasons orangutans were killed was fear of orangutans or in self-defence. A similar pattern was evident among reports of orangutan killing by other people in the villages. Regression analyses indicated that religion and the percentage of intact forest around villages were the strongest socio-ecological predictors of whether orangutans were killed for conflict or non-conflict related reasons. Our data indicate that between 44,170 and 66,570 orangutans were killed in Kalimantan within the respondents' active hunting lifetimes: between 12,690 and 29,024 for conflict reasons (95%CI and between 26,361 and 41,688 for non-conflict reasons (95% CI. These findings confirm that habitat protection alone will not ensure the survival of orangutans in Indonesian Borneo, and that effective reduction of orangutan killings is urgently needed.

  10. 9 CFR 113.211 - Feline Rhinotracheitis Vaccine, Killed Virus.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Feline Rhinotracheitis Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.211 Feline Rhinotracheitis Vaccine, Killed Virus. Feline... tested for potency as follows: (i) Eight feline rhinotracheitis susceptible cats (five vaccinates...

  11. 9 CFR 113.203 - Feline Panleukopenia Vaccine, Killed Virus.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Feline Panleukopenia Vaccine, Killed... REQUIREMENTS Killed Virus Vaccines § 113.203 Feline Panleukopenia Vaccine, Killed Virus. Feline Panleukopenia... shall be individually tested for neutralizing antibody against feline panleukopenia virus to...

  12. 9 CFR 113.201 - Canine Distemper Vaccine, Killed Virus.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Canine Distemper Vaccine, Killed Virus... REQUIREMENTS Killed Virus Vaccines § 113.201 Canine Distemper Vaccine, Killed Virus. Canine Distemper Vaccine... canine distemper susceptible dogs (20 vaccinates and 5 controls) shall be used as test animals....

  13. 9 CFR 113.204 - Mink Enteritis Vaccine, Killed Virus.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Mink Enteritis Vaccine, Killed Virus... REQUIREMENTS Killed Virus Vaccines § 113.204 Mink Enteritis Vaccine, Killed Virus. Mink Enteritis Vaccine... have developed mink enteritis following inoculation with virulent mink enteritis virus. Each...

  14. 9 CFR 113.212 - Bursal Disease Vaccine, Killed Virus.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Bursal Disease Vaccine, Killed Virus..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.212 Bursal Disease Vaccine, Killed Virus. Bursal Disease...

  15. 9 CFR 113.208 - Avian Encephalomyelitis Vaccine, Killed Virus.

    Science.gov (United States)

    2010-01-01

    ..., Killed Virus. 113.208 Section 113.208 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.208 Avian Encephalomyelitis Vaccine, Killed Virus....

  16. 9 CFR 113.213 - Pseudorabies Vaccine, Killed Virus.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Pseudorabies Vaccine, Killed Virus..., DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.213 Pseudorabies Vaccine, Killed Virus. Pseudorabies Vaccine,...

  17. Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis.

    Science.gov (United States)

    Makarov, Vadim; Manina, Giulia; Mikusova, Katarina; Möllmann, Ute; Ryabova, Olga; Saint-Joanis, Brigitte; Dhar, Neeraj; Pasca, Maria Rosalia; Buroni, Silvia; Lucarelli, Anna Paola; Milano, Anna; De Rossi, Edda; Belanova, Martina; Bobovska, Adela; Dianiskova, Petronela; Kordulakova, Jana; Sala, Claudia; Fullam, Elizabeth; Schneider, Patricia; McKinney, John D; Brodin, Priscille; Christophe, Thierry; Waddell, Simon; Butcher, Philip; Albrethsen, Jakob; Rosenkrands, Ida; Brosch, Roland; Nandi, Vrinda; Bharath, Sowmya; Gaonkar, Sheshagiri; Shandil, Radha K; Balasubramanian, Venkataraman; Balganesh, Tanjore; Tyagi, Sandeep; Grosset, Jacques; Riccardi, Giovanna; Cole, Stewart T

    2009-05-08

    New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphoryl-beta-d-ribose 2'-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB.

  18. Anti-bacterial and anti-toxic immunity induced by a killed whole-cell-cholera toxin B subunit cholera vaccine is essential for protection against lethal bacterial infection in mouse pulmonary cholera model.

    Science.gov (United States)

    Kang, S-S; Yang, J S; Kim, K W; Yun, C-H; Holmgren, J; Czerkinsky, C; Han, S H

    2013-07-01

    The lack of appropriate animal model for studying protective immunity has limited vaccine development against cholera. Here, we demonstrate a pulmonary cholera model conferred by intranasal administration of mice with live Vibrio cholerae. The bacterial components, but not cholera toxin, caused lethal and acute pneumonia by inducing massive inflammation. Intranasal immunization with Dukoral, comprising killed whole bacteria and recombinant cholera toxin B subunit (rCTB), developed both mucosal and systemic antibody responses with protection against the lethal challenge. Either rCTB-free Dukoral or rCTB alone partially protected the mice against the challenge. However, reconstitution of rCTB-free Dukoral with rCTB restored full protection. Parenteral immunization with Dukoral evoked strong systemic immunity without induction of mucosal immunity or protection from the challenge. These results suggest that both anti-bacterial and anti-toxic immunity are required for protection against V. cholerae-induced pneumonia, and this animal model is useful for pre-clinical evaluation of candidate cholera vaccines.

  19. Deprive to kill: Glutamine closes the gate to anticancer monocarboxylic drugs

    OpenAIRE

    Cardaci, Simone; Ciriolo, Maria Rosa

    2012-01-01

    Killing properties of antitumor drugs can be enhanced by strategies targeting biochemical adaptations of cancer cells. Recently, we reported that depriving cancer cells of glutamine is a feasible approach to enhance antitumor effects of the alkylating analog of pyruvic acid, 3-bromopyruvate, which rely on the induction of autophagic cell death by metabolic-oxidative stress. 3-bromopyruvate chemopotentiation is the result of its increased intracellular uptake mediated by the monocarboxylate tr...

  20. 腺病毒介导的KDR-HSV-tk系统对血管内皮细胞的杀伤作用及旁观者效应%The killing and bystander effect of KDR-HSV-tk system mediated by adenovirus on vascular endothelial cells

    Institute of Scientific and Technical Information of China (English)

    朱欠元; 曾友根; 张愈延; 李宝金

    2015-01-01

    目的:研究腺病毒介导的KDR-HSV-tk系统对血管内皮细胞的杀伤作用及旁杀效应。方法体外构建受 KDR启动子和巨细胞病毒( CMV)启动子调控并可表达HSV-tk基因的AdKDR-HSV-tk和AdCMV-HSV-tk,经293细胞包装、扩增后,分别感染表达KDR的人脐静脉血管内皮细胞(HUVEC)和不表达KDR的人鼻咽癌细胞CNE-2,建立HUVEC/tk及CNE-2/tk,观察丙氧鸟苷(GCV)处理后 HUVEC 杀伤率及旁观者效应。结果受感染的HUVEC和CNE-2细胞中均有绿色荧光蛋白的表达。在感染复数( MOI)为100的条件下,GCV浓度由0增至50 mg/L 时含 AdKDR-HSV-tk的HUVEC和CNE-2细胞存活率从100%分别降至(23.1±3.9)%和(69.8±3.1)%(P<0.01);感染AdCMV-HSV-tk的HUVEC和 CNE-2细胞存活率从100%分别降至(13.9±2.9)%和(15.7±5.2)%(P<0.05);HUVEC/tk细胞对GCV的敏感性明显高于亲代HUVEC细胞, HUVEC+HUVEC/tk混合细胞的生存率随HUVEC/tk比例的增加而下降,说明AdKDR-HSV-tk/GCV系统不但能杀伤转染tk基因的HUVEC,也能杀伤未导入转染tk基因的HUVEC细胞,存在明显的旁观者效应。结论腺病毒介导的KDR-HSV-tk/GCV系统对血管内皮细胞具有特异的杀伤作用及旁观者效应,为抗肿瘤血管靶向治疗奠定了基础。%Objective To study the killing and bystander effect of KDR-HSV-tk system study of adenovirus mediated vascular endo-thelial cells.Methods AdKDR-HSV-tk and AdCMV-HSV-tk controlled by KDR promoter and cytomegalovirus (CMV) promoter were con-structed in vitro,and infected human umbilical vein endothelial cells (HUVEC) expressing KDR and human nasopharyngeal carcinoma cell line CNE-2 without KDR to establish HUVEC/tk and CNE-2/tk respectively .The killing rate and bystander effect were observed after ganci-clovir(GCV) treatment.Results The infected HUVEC and CNE-2 cells were

  1. Killing-Yano forms and Killing tensors on a warped space

    CERN Document Server

    Krtous, Pavel; Kolar, Ivan

    2015-01-01

    We formulate several criteria under which the symmetries associated with the Killing and Killing-Yano tensors on the base space can be lifted to the symmetries of the full warped geometry. The procedure is explicitly illustrated on several examples, providing new prototypes of spacetimes admitting such tensors. In particular, we study a warped product of two Kerr-NUT-(A)dS spacetimes and show that it gives rise to a new class of highly symmetric vacuum (with cosmological constant) black hole solutions that inherit many of the properties of the Kerr-NUT-(A)dS geometry.

  2. Micro-sociology of mass rampage killings.

    Science.gov (United States)

    Collins, Randall

    2014-01-01

    Spectacular but very rare violent events such as mass killings by habitual non-criminals cannot be explained by factors which are very widespread, such as possession of firearms, being a victim of bullying, an introvert, or a career failure. A stronger clue is clandestine preparation of attack by one or two individuals, against randomly chosen representatives of a hated collective identity. Mass killers develop a deep back-stage, obsessed with planning their attack, overcoming social inferiority and isolation by an emotion of clandestine excitement.

  3. Two independent killing mechanisms of Candida albicans by human neutrophils: evidence from innate immunity defects

    NARCIS (Netherlands)

    Gazendam, R.P.; Hamme, J.L. van; Tool, A.T.; Houdt, M. van; Verkuijlen, P.J.; Herbst, M.; Liese, J.G.; Veerdonk, F.L. van de; Roos, D.; Berg, T.K. van den; Kuijpers, T.W.

    2014-01-01

    Invasive fungal infections, accompanied by high rates of mortality, represent an increasing problem in medicine. Neutrophils are the major effector immune cells in fungal killing. Based on studies with neutrophils from patients with defined genetic defects, we provide evidence that human neutrophils

  4. 9 CFR 113.202 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus.

    Science.gov (United States)

    2010-01-01

    ..., shall be prepared from virus-bearing cell culture fluids. Only Master Seed Virus which has been... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus. 113.202 Section 113.202 Animals and Animal Products ANIMAL AND...

  5. Two independent killing mechanisms of Candida albicans by human neutrophils: evidence from innate immunity defects.

    Science.gov (United States)

    Gazendam, Roel P; van Hamme, John L; Tool, Anton T J; van Houdt, Michel; Verkuijlen, Paul J J H; Herbst, Martin; Liese, Johannes G; van de Veerdonk, Frank L; Roos, Dirk; van den Berg, Timo K; Kuijpers, Taco W

    2014-07-24

    Invasive fungal infections, accompanied by high rates of mortality, represent an increasing problem in medicine. Neutrophils are the major effector immune cells in fungal killing. Based on studies with neutrophils from patients with defined genetic defects, we provide evidence that human neutrophils use 2 distinct and independent phagolysosomal mechanisms to kill Candida albicans. The first mechanism for the killing of unopsonized C albicans was found to be dependent on complement receptor 3 (CR3) and the signaling proteins phosphatidylinositol-3-kinase and caspase recruitment domain-containing protein 9 (CARD9), but was independent of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The second mechanism for the killing of opsonized C albicans was strictly dependent on Fcγ receptors, protein kinase C (PKC), and reactive oxygen species production by the NADPH oxidase system. Each of the 2 pathways of Candida killing required Syk tyrosine kinase activity, but dectin-1 was dispensable for both of them. These data provide an explanation for the variable clinical presentation of fungal infection in patients suffering from different immune defects, including dectin-1 deficiency, CARD9 deficiency, or chronic granulomatous disease.

  6. 'David and Goliath' of the soil food web - Flagellates that kill nematodes

    DEFF Research Database (Denmark)

    Strandmark, Lisa Bjørnlund; Rønn, Regin

    2008-01-01

    Nematodes and flagellates are important bacterial predators in soil and sediments. Generally, these organisms are considered to be competitors for bacterial food. We studied the interaction among flagellates and nematodes using axenic liquid cultures amended with heat-killed bacteria as food...... and showed for the first time that a small and common soil flagellate (Cercomonas sp.) is able to attack and kill the much larger nematode Caenorhabditis elegans. The killing process is not caused by soluble metabolites but requires direct contact between the flagellate cells and the nematode surface...... and occurs rapidly (within a few hours) at high flagellate density. At lower flagellate density, adult nematodes sometimes avoid attachment of flagellates, feed on them and become the dominant bacterial predator. Considering that bacterial feeders affect bacterial communities differently, and that one...

  7. Nod1 signaling overcomes resistance of S. pneumoniae to opsonophagocytic killing.

    Directory of Open Access Journals (Sweden)

    Elena S Lysenko

    2007-08-01

    Full Text Available Airway infection by the Gram-positive pathogen Streptococcus pneumoniae (Sp leads to recruitment of neutrophils but limited bacterial killing by these cells. Co-colonization by Sp and a Gram-negative species, Haemophilus influenzae (Hi, provides sufficient stimulus to induce neutrophil and complement-mediated clearance of Sp from the mucosal surface in a murine model. Products from Hi, but not Sp, also promote killing of Sp by ex vivo neutrophil-enriched peritoneal exudate cells. Here we identify the stimulus from Hi as its peptidoglycan. Enhancement of opsonophagocytic killing was facilitated by signaling through nucleotide-binding oligomerization domain-1 (Nod1, which is involved in recognition of gamma-D-glutamyl-meso-diaminopimelic acid (meso-DAP contained in cell walls of Hi but not Sp. Neutrophils from mice treated with Hi or compounds containing meso-DAP, including synthetic peptidoglycan fragments, showed increased Sp killing in a Nod1-dependent manner. Moreover, Nod1(-/- mice showed reduced Hi-induced clearance of Sp during co-colonization. These observations offer insight into mechanisms of microbial competition and demonstrate the importance of Nod1 in neutrophil-mediated clearance of bacteria in vivo.

  8. PNP-TK融合自杀基因及PNP单自杀基因对肝癌细胞杀伤效率%Cytotoxic killing effects of a PNP-TK chimeric suicide gene system and a single PNP suicide gene system on hepatoma cells

    Institute of Scientific and Technical Information of China (English)

    周俊立; 蔡晓坤; 易学锋; 王德全; 姚振江; 周卫平

    2009-01-01

    目的 分析PNP-TK融合自杀基因系统及PNP单自杀基因系统对肝癌细胞的杀伤作用及二者对肝癌细胞潜在的杀伤机制. 方法构建融合基因表达载体pcDNA3.0/PNP-TK,经酶切、PCR及测序鉴定重组体,G418筛选获得稳定转染了pcDNA3.0/PNP-TK的HepG2细胞克隆.RT-PCR和Western Blotting检测融合基因在HepG2细胞中的表达.台盼兰排斥法检测细胞的生长曲线,MTT法检测细胞对相应前药的敏感性及分别在一种和两种前药作用下所导致的旁观者效应.结果 融合基因片段PNP-TK正确插入了pcDNA3.0载体中,pcDNA3.0/PNP-TK在肝癌细胞株HepG2中实现了表达.抗性细胞克隆对相应的前药十分敏感.在两种前药的联合作用下,pcDNA3.0/PNP-TK所导致的旁观者效应明显强于只给予MeP-dR一种前药以及pcDNA3.0/PNP单基因系统所致的旁观者效应.结论 具有双自杀基因功能的表达载体pcDNA3.0/PNP-TK对肝癌细胞的杀伤效果优于PNP单自杀基因系统.%Objective To analyze the killing effects of a PNP-TK chimeras gene system on the basis of PNP suicide gene and a single PNP suicide gene system on hepatoma cells and explore the potential mechanism of their killing effects. Methods A vector harboring a chimeric gene,pcDNA3.0/PNP-TK was constructed. The recombinant was identified by recombinant enzyme, PCR and sequencing. Then it was transfected into HepG2 cells by liposome-mediated method. The G-418 resistant cell clone with the stable transfection of pcDNA3.0/PNP-TK was developed. The expression of PNP-TK gene was detected by RT-PCR and Western blotting method. The cellular growth curves were determined by trypan blue exclusion. The sensitivity of cell clones to their respective prodrugs and the bystander effects they resulted in were also assayed by MTT method. Results The PNP-TK fusion gene was inserted into pcDNA3.0 correctly,and the stable expression of PNP-TK gene in HepG2 cells was positive. Two clones stably expressing

  9. Targeted Anticancer Immunotoxins and Cytotoxic Agents with Direct Killing Moieties

    Directory of Open Access Journals (Sweden)

    Koji Kawakami

    2006-01-01

    Full Text Available Despite the progress of the bioinformatics approach to characterize cell-surface antigens and receptors on tumor cells, it remains difficult to generate novel cancer vaccines or neutralizing monoclonal antibody therapeutics. Among targeted cancer therapeutics, biologicals with targetable antibodies or ligands conjugated or fused to toxins or chemicals for direct cell-killing ability have been developed over the last 2 decades. These conjugated or fused chimeric proteins are termed immunotoxins or cytotoxic agents. Two agents, DAB389IL-2 (ONTAKTM targeting the interleukin-2 receptor and CD33-calicheamicin (Mylotarg®, have been approved by the FDA for cutaneous T-cell lymphoma (CTCL and relapsed acute myeloid leukemia (AML, respectively. Such targetable agents, including RFB4(dsFv-PE38 (BL22, IL13-PE38QQR, and Tf-CRM107, are being tested in clinical trials. Several agents using unique technology such as a cleavable adapter or immunoliposomes with antibodies are also in the preclinical stage. This review summarizes the generation, mechanism, and development of these agents. In addition, possible future directions of this therapeutic approach are discussed.

  10. Chlordiazepoxide and diazepam induced mouse killing by rats.

    Science.gov (United States)

    Leaf, R C; Wnek, D J; Gay, P E; Corcia, R M; Lamon, S

    1975-10-14

    Chlordiazepoxide HCl, at dose levels from 2.5 mg/kg to 80 mg/kg, significantly increased the low base rates of mouse killing (3-9%) observed in large samples (N = 100/dose) of Holtzman strain albino male rats. Maximal killing rates were obtained at doses from 7.5 mg/kg to 20 mg/kg. Diazepam was equally effective, and several times more potent than chlordiazepoxide. Pentobarbital did not increase killing. Killing induced by chlordiazepoxide was blocked by d-amphetamine SO4, but not by l-amphetamine, at dose levels similar to those that block undrugged killing in this strain (ED50 = 1.5 mg/kg). Unlike pilocarpine-induced killing, the effects of chlordiazepoxide were not increased or decreased significantly by either peripherally or centrally active anticholinergic drugs, over wide dose ranges of these agents; nor were the effects of chlordiazepoxide increased by repeated daily administration.

  11. 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.

  12. 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.

  13. f(R)-gravity from Killing tensors

    Science.gov (United States)

    Paliathanasis, Andronikos

    2016-04-01

    We consider f(R)-gravity in a Friedmann-Lemaître-Robertson-Walker spacetime with zero spatial curvature. We apply the Killing tensors of the minisuperspace in order to specify the functional form of f(R) and for the field equations to be invariant under Lie-Bäcklund transformations, which are linear in momentum (contact symmetries). Consequently, the field equations to admit quadratic conservation laws given by Noether’s theorem. We find three new integrable f(R)-models, for which, with the application of the conservation laws, we reduce the field equations to a system of two first-order ordinary differential equations. For each model we study the evolution of the cosmological fluid. We find that for each integrable model the cosmological fluid has an equation of state parameter, in which there is linear behavior in terms of the scale factor which describes the Chevallier, Polarski and Linder parametric dark energy model.

  14. Killing Symmetries in $\\mathcal{H}$-Spaces with $\\Lambda$

    CERN Document Server

    Chudecki, Adam

    2013-01-01

    All Killing symmetries in complex $\\mathcal{H}$-spaces with $\\Lambda$ in terms of the Pleba\\'nski - Robinson - Finley coordinate system are found. All $\\mathcal{H}$-metrics with $\\Lambda$ admitting a null Killing vector are explicitly given. It is shown that the problem of non-null Killing vector reduces to looking for solution of the Boyer - Finley - Pleba\\'nski (Toda field) equation

  15. The geometry of D=11 null killing spinors

    Energy Technology Data Exchange (ETDEWEB)

    Gauntlett, Jerome P.; Gutowski, Jan B. E-mail: gutowski@maths.ox.ac.uk; Stathis Pakis

    2003-12-01

    We determine the necessary and sufficient conditions on the metric and the four-form for the most general bosonic supersymmetric configurations of D=11 supergravity which admit a null Killing spinor i.e. a Killing spinor which can be used to construct a null Killing vector. This class covers all supersymmetric time-dependent configurations and completes the classification of the most general supersymmetric configurations initiated in hep-th/0212008. (author)

  16. The Geometry of D=11 Null Killing Spinors

    CERN Document Server

    Gauntlett, J P; Pakis, S

    2003-01-01

    We determine the necessary and sufficient conditions on the metric and the four-form for the most general bosonic supersymmetric configurations of D=11 supergravity which admit a null Killing spinor i.e. a Killing spinor which can be used to construct a null Killing vector. This class covers all supersymmetric time-dependent configurations and completes the classification of the most general supersymmetric configurations initiated in hep-th/0212008.

  17. Killing effect of CIKs on pancreatic cancer cells enhanced by DCs loaded with K-ras mutant peptide%K-ras突变多肽负载的DC细胞增强CIK细胞对胰腺癌细胞的杀伤作用

    Institute of Scientific and Technical Information of China (English)

    窦春鹏; 李奎武; 谭广

    2015-01-01

    Objective:To observe the killing effect of the cytokine induced killer cells (CIKs) atfer co-culture with dendritic cells (DCs) harboring K-ras (12-Val) mutant peptide on pancreatic cancer PANC-1 cells. Methods:DCs and CIKs were induced and enriched from peripheral blood of healthy donors, respectively. DCs were loaded with the K-ras mutant epitope peptide (K-ras-DCs), and CIKs were co-cultured with un-loaded DCs or K-ras-DCs to obtain the DC-CIKs and K-ras-DC-CIKs, respectively. hTe proliferative activities between CIKs and K-ras-DC-CIKs were compared, the difference in immunophenotype between DCs and K-ras-DCs as well as between CIKs and K-ras-DC-CIKs were analyzed, the IFN-γand IL-12 levels in the culture supernatants from CIKs, DC-CIKs and K-ras-DC-CIKs were measured, and the killing abilities of CIKs, DC-CIKs and K-ras-DC-CIKs on PANC-1 cells in vitro were determined. Results:The proliferative ability of K-ras-DC-CIKs was significantly greater than that of the untreated CIKs (P Conclusion:K-ras mutant peptide can promote DCs maturation, and DCs harboring K-ras mutant peptide can increase the proliferation of CIKs and killing effect on pancreatic cancer cells.%目的:观察经K-ras(12-Val)突变多肽负载的树突状细胞(DC)与细胞因子诱导的杀伤细胞(CIK)共培养以后对胰腺癌PANC-1细胞的杀伤作用。  方法:取健康人外周血体外诱导分别扩增出DC和CIK;用K-ras突变体抗原表位肽负载DC(K-ras-DC),将单纯DC或K-ras-DC与CIK共培养,获得DC-CIK或K-ras-DC-CIK。比较CIK与K-ras-DC-CIK的增殖活性;分别分析DC与K-ras-DC以及CIK与K-ras-DC-CIK的免疫表型差异;检测CIK、DC-CIK、K-ras-DC-CIK上清液中IFN-γ、IL-12的水平;检测K-ras-DC-CIK、DC-CIK、CIK对PANC-1细胞的体外杀伤力。  结果:K-ras-DC-CIK的增殖能力明显强于单纯CIK(P  结论:K-ras突变多肽负载后能促进DC的成熟,负载K-ras突变多肽后的DC能增加CIK

  18. The Killing Effect on EJ Bladder Cancer Cells by Combination of Radiation-responsive Promoter Conjoined CD Gene and Ionizing Radiation of 125 I%辐射敏感性启动子调控CD基因表达联合125I照射对膀肤癌EJ细胞的杀伤作用

    Institute of Scientific and Technical Information of China (English)

    李玲; 张春丽; 李囡; 康磊; 卢霞; 张丽; 闫平; 王荣福

    2011-01-01

    以脂质体介导的辐射敏感性基因联合胞嘧啶脱氨酶(cytosine deantinase,CD)基因转染膀胱癌EJ细胞,研究放射性核素125 I照射后5-氟胞嘧啶(5-fluorocytosine,5-FC)对转染膀胧癌EJ细胞的杀伤作用.人工合成辐射敏感性启动子E8,将启动子克隆至质粒pCD2的CD基因上游,构建以E8为启动子、CD基因为目的基因的新质粒,并采用DNA测序法测定E8和CD基因的序列;脂质体Lipofectamine2000介导pE8-CD转染膀胧癌EJ细胞,用[3]I照射(吸收剂量为2舜)后,蛋白质免疫印迹分析(Western blot)测定CD蛋白表达;在转染EJ细胞中分别加人不同剂量125 I勺和5-FC,四哇盐比色法(MTT法)测定各组细胞存活率,并以未经125 I勺照射组、未加5-FC组和5-氟尿啼吮(5-FU)组(阳性对照组)进行对照.DNA测序显示构建的pE8-CD质粒含E8启动子及CD基因序列;Westem blot可检测到CD基因表达;125 I加5-FC组细胞存活率明显低于未经125 I照射组及未加5-FC组,与5-FU组相近.这表明放射性核素与基因治疗联合对肿瘤细胞具有协同杀伤作用.%To investigate the killing effect on EJ human bladder cancer cells by combination of radiation-responsive gene promoter conjoined CD/S-FC system and ionizing radiation with radionuclide 125 I, plasmid vector containing synthetic gene promoter E8 responsive to ionizing radiation(IR)and CD gene in downstream was constructed, analyzed by DNA sequencing, and transiently transfected into EJ bladder cancer cells by liposome-mediated method. Expression of downstream CD gene was detected via ionizing radiation of radionuclide 1311 at 2Gy by Western Blot. The killing effect induced by different doses of 125I and 5-FC in plasmid-transfected EJ cells was investigated by MTT colorimetric assay. DNA sequencing demonstrated that the plasmid vector contained E8 promoter and CD gene sequences , Western Blot detected the protein CD, and MTT colorimetric assay showed that the death rate of plasmid

  19. Human Leukocytes Kill Brugia malayi Microfilariae Independently of DNA-Based Extracellular Trap Release

    Science.gov (United States)

    McCoy, Ciaran J.; Reaves, Barbara J.; Giguère, Steeve; Coates, Ruby; Rada, Balázs

    2017-01-01

    Background Wuchereria bancrofti, Brugia malayi and Brugia timori infect over 100 million people worldwide and are the causative agents of lymphatic filariasis. Some parasite carriers are amicrofilaremic whilst others facilitate mosquito-based disease transmission through blood-circulating microfilariae (Mf). Recent findings, obtained largely from animal model systems, suggest that polymorphonuclear leukocytes (PMNs) contribute to parasitic nematode-directed type 2 immune responses. When exposed to certain pathogens PMNs release extracellular traps (NETs) in the form of chromatin loaded with various antimicrobial molecules and proteases. Principal findings In vitro, PMNs expel large amounts of NETs that capture but do not kill B. malayi Mf. NET morphology was confirmed by fluorescence imaging of worm-NET aggregates labelled with DAPI and antibodies to human neutrophil elastase, myeloperoxidase and citrullinated histone H4. A fluorescent, extracellular DNA release assay was used to quantify and observe Mf induced NETosis over time. Blinded video analyses of PMN-to-worm attachment and worm survival during Mf-leukocyte co-culture demonstrated that DNase treatment eliminates PMN attachment in the absence of serum, autologous serum bolsters both PMN attachment and PMN plus peripheral blood mononuclear cell (PBMC) mediated Mf killing, and serum heat inactivation inhibits both PMN attachment and Mf killing. Despite the effects of heat inactivation, the complement inhibitor compstatin did not impede Mf killing and had little effect on PMN attachment. Both human PMNs and monocytes, but not lymphocytes, are able to kill B. malayi Mf in vitro and NETosis does not significantly contribute to this killing. Leukocytes derived from presumably parasite-naïve U.S. resident donors vary in their ability to kill Mf in vitro, which may reflect the pathological heterogeneity associated with filarial parasitic infections. Conclusions/Significance Human innate immune cells are able to

  20. Antibacterial activity of silver-killed bacteria: the "zombies" effect

    Science.gov (United States)

    Wakshlak, Racheli Ben-Knaz; Pedahzur, Rami; Avnir, David

    2015-04-01

    We report a previously unrecognized mechanism for the prolonged action of biocidal agents, which we denote as the zombies effect: biocidally-killed bacteria are capable of killing living bacteria. The concept is demonstrated by first killing Pseudomonas aeruginosa PAO1 with silver nitrate and then challenging, with the dead bacteria, a viable culture of the same bacterium: Efficient antibacterial activity of the killed bacteria is observed. A mechanism is suggested in terms of the action of the dead bacteria as a reservoir of silver, which, due to Le-Chatelier's principle, is re-targeted to the living bacteria. Langmuirian behavior, as well as deviations from it, support the proposed mechanism.

  1. Adenovirus-mediated and tumor-specific transgene expression of the sodium-iodide symporter from the human telomerase reverse transcriptase promoter enhances killing of lung cancer cell line in vitro

    Institute of Scientific and Technical Information of China (English)

    SHI Yi-zhen; ZHANG Jun; LIU Zeng-li; DU Shou-ying; SHEN Yong-mei

    2010-01-01

    Background The sodium-iodide symporter (NIS) protein can mediate the active radioiodine uptake.The human telomerase reverse transcriptase (hTERT) promoter is known to be selectively reactivated in majority of tumors and hence could be used for tumor targeting.We constructed a recombinant adenovirus containing the human sodium iodide symporter (hNIS) gene directed by the hTERT promoter, characterized the ability of infected cells in uptaking iodide, and explored the therapeutic efficacy of 131I in a lung cancer cell line in vitro.Methods The hTERT promoter was amplified by PCR from DNA isolated from log-phase HepG2 cells, subcloned into lineralized FL*-hNIS/pcDNA3, and then the hTERT-hNIS sequence was subcloned into the shuttle plasmid pAdTrack.The recombinant adenovirus Ad-hTERT-hNIS was constructed by AdEasy system.A positive control adenovirusAd-CMV-hNIS and a negative control adenovirus Ad-CMV were created similarly.A549 cells were transduced with recombinant adenoviruses.125I uptake studies and sodium perchlorate suppression studies were used to confirm hNIS expression and function.Toxic effects of 131I on tumor cells were studied by in vitro clonogenic assay.Results We first successfully constructed an adenovirus mediated transgene expression system of the hNIS under the control of hTERT promoter.When infected with recombinant adenovirus constructs expressing hNIS directed by hTERTand CMV-promoters (Ad-hTERT-hNIS and Ad-CMV-hNIS, respectively), the lung cancer cell line A549 had increased ability to uptake radioiodide up to 23- and 30- fold compared to the control parental cells, respectively.The radioiodide uptake ability of both the Ad-CMV-hNIS and Ad-hTERT-hNIS transduced cell lines were repressed 11-fold by sodium perchlorate (NaCIO4).The subsequent in vitro clonogenic assay of the infected A549 cell line was further repressed to 23% (Ad-CMV-hNIS) and 30% (Ad-hTERT-hNIS) of the control group after receiving radioiodide for 7 hours (P <0.001).Conclusion

  2. Enhancement of the immune response against Salmonella infection of mice by heat-killed multispecies combinations of lactic acid bacteria.

    Science.gov (United States)

    Chen, Chih-Yuan; Tsen, Hau-Yang; Lin, Chun-Li; Lin, Chien-Ku; Chuang, Li-Tsen; Chen, Chin-Shuh; Chiang, Yu-Cheng

    2013-11-01

    Heat-killed lactic acid bacteria (LAB) has advantages over live LAB in that it has a long shelf-life and is therefore easy to store and transport. From four LAB strains selected by immunomodulatory activity and adherent properties, we prepared the heat-killed multispecies combination of LAB (MLAB) and the cell walls from MLAB under two conditions (100 °C for 30 min and 121 °C for 15 min). Different effects on the adherent properties of these four LAB strains were observed, depending on the heating conditions. With mouse macrophage cells, the two heat-killed MLABs (HMLABs) showed significantly higher induction activities on the production of interleukin 12 (IL-12) than their individual strains did. Heat-killed MLABs and cell-wall preparations were able to reduce the Salmonella invasion of Caco-2 and mouse macrophage cells. Feeding mice with HMLAB could inhibit the Salmonella invasion of mice significantly. For these mice, the expression level of pro-inflammatory cytokines, such as TNF-α and IL-6, in mouse serum was reduced while that of the anti-inflammatory cytokine, i.e. IL-10, was enhanced. The HMLABs developed in this study showed higher protective effect against Salmonella invasion either of Caco-2 cells or of mice, relative to the heat-killed lactobacilli, which consisted of Lactobacillus acidophilus strains selected at random. In conclusion, the HMLABs were potentially useful for the protection of mice against Salmonella infection and the induced inflammation.

  3. Methionine oxidation contributes to bacterial killing by the myeloperoxidase system of neutrophils.

    Science.gov (United States)

    Rosen, Henry; Klebanoff, Seymour J; Wang, Yi; Brot, Nathan; Heinecke, Jay W; Fu, Xiaoyun

    2009-11-03

    Reactive oxygen intermediates generated by neutrophils kill bacteria and are implicated in inflammatory tissue injury, but precise molecular targets are undefined. We demonstrate that neutrophils use myeloperoxidase (MPO) to convert methionine residues of ingested Escherichia coli to methionine sulfoxide in high yield. Neutrophils deficient in individual components of the MPO system (MPO, H(2)O(2), chloride) exhibited impaired bactericidal activity and impaired capacity to oxidize methionine. HOCl, the principal physiologic product of the MPO system, is a highly efficient oxidant for methionine, and its microbicidal effects were found to correspond linearly with oxidation of methionine residues in bacterial cytosolic and inner membrane proteins. In contrast, outer envelope proteins were initially oxidized without associated microbicidal effect. Disruption of bacterial methionine sulfoxide repair systems rendered E. coli more susceptible to killing by HOCl, whereas over-expression of a repair enzyme, methionine sulfoxide reductase A, rendered them resistant, suggesting a direct role for methionine oxidation in bactericidal activity. Prominent among oxidized bacterial proteins were those engaged in synthesis and translocation of peptides to the cell envelope, an essential physiological function. Moreover, HOCl impaired protein translocation early in the course of bacterial killing. Together, our findings indicate that MPO-mediated methionine oxidation contributes to bacterial killing by neutrophils. The findings further suggest that protein translocation to the cell envelope is one important pathway targeted for damage.

  4. Interleukin-15 increases Paracoccidioides brasiliensis killing by human neutrophils.

    Science.gov (United States)

    Tavian, Elisandra Garcia; Dias-Melicio, Luciane Alarcão; Acorci, Michele Janegitz; Graciani, Ana Paula Bordon; Peraçoli, Maria Terezinha Serrão; Soares, Angela Maria Victoriano de Campos

    2008-01-01

    Interleukin-15 is a cytokine produced by a wide range of different cell types, including macrophages, in response to lipopolysaccharide or microbial infection. This cytokine may play a crucial role in the activation of phagocytic cells against pathogens, especially during innate immune response. The effects of IL-15 on human polymorphonuclear leukocyte fungicidal activity against a highly virulent Paracoccidioides brasiliensis strain were investigated. Pretreatment of human neutrophils from healthy individuals with IL-15 for 18 hours increased cell fungicidal activity in a dose-dependent manner. In addition, the exposure to IL-15 induced an increase in neutrophil oxidative burst as evaluated by superoxide anion and H(2)O(2) release. Catalase inhibited fungicidal activity supporting a role for H(2)O(2) in fungus killing. In contrast, IL-8 and TNF-alpha levels were not affected by IL-15 suggesting that its effects were not mediated by these cytokines. Together, these results show that IL-15 is a potent stimulant of antifungal activities in human neutrophils, at least in part by a mechanism dependent on oxidative metabolism.

  5. Granzyme B-based cytolytic fusion protein targeting EpCAM specifically kills triple negative breast cancer cells in vitro and inhibits tumor growth in a subcutaneous mouse tumor model

    NARCIS (Netherlands)

    Amoury, Manal; Kolberg, Katharina; Pham, Anh-Tuan; Hristodorov, Dmitrij; Mladenov, Radoslav; Di Fiore, Stefano; Helfrich, Wijnand; Kiessling, Fabian; Fischer, Rainer; Pardo, Alessa; Thepen, Theophilus; Hussain, Ahmad F.; Nachreiner, Thomas; Barth, Stefan

    2016-01-01

    Triple-negative breast cancer (TNBC) is associated with poor prognosis and high prevalence among young premenopausal women. Unlike in other breast cancer subtypes, no targeted therapy is currently available. Overexpression of epithelial cell adhesion molecule (EpCAM) in 60% of TNBC tumors correlates

  6. That which does not kill us makes us stronger--does Nietzsche's quote apply to islets? A re-evaluation of the passenger leukocyte theory, free radicals, and glucose toxicity in islet cell transplantation.

    Science.gov (United States)

    Wright, J R; Xu, B-Y

    2014-07-01

    In clinical islet transplantation, isolated islets are embolized into the liver via the portal vein (PV); however, up to 70% of the islets are lost in the first few days after transplantation (i.e., too quickly to be mediated by the adaptive immune system). Part of early loss is due to instant blood-mediated inflammatory reaction, an immune/thrombotic process caused by islets interacting with complement. We have shown that glucose toxicity (GT) also plays a critical role based upon the observation that islets embolized into the PVs of diabetic athymic mice are rapidly lost but, if recipients are not diabetic, the islet grafts persist. Using donor islets resistant to the β-cell toxin streptozotocin, we have shown that intraportal islets engrafted in non-diabetic athymic mice for as little as 3 days will maintain normoglycemia when streptozotocin is administered destroying the recipient's native pancreas β-cells. What is the mechanism of GT in β-cells? Chronic exposure to hyperglycemia over-exerts β-cells and their electron transport chains leak superoxide radicals during aerobic metabolism. Here we reinterpret old data and present some compelling new data supporting a new model of early intraportal islet graft loss. We hypothesize that diabetes stimulates overproduction of superoxide in both the β-cells of the islet grafts and the endothelial cells lining the intraportal microvasculature adjacent to where the embolized islets become lodged. This double dose of oxidant damage stresses both the islets, which are highly susceptible to free radicals because of inherent low levels of scavenging enzymes, and the adjacent hepatic endothelial cells. This, superimposed upon localized endothelial damage caused by embolization, precipitates inflammation and coagulation which further damages islet grafts. Based upon this model, we predict that pre-exposing islets to sub-lethal hyperoxia should up-regulate islet free radical scavenging enzyme levels and promote initial

  7. Cisplatin and 5-fluorouracil Enhance Expression of NKG2D Ligands and CIK Cells Killing Effect of Human Esophagus Carcinoma Cell EC9706%顺铂和氟尿嘧啶增强食管癌细胞NKG2D配体的表达及CIK细胞的杀伤活性

    Institute of Scientific and Technical Information of China (English)

    李瑞君; 梅家转; 禹萌; 刘桂举; 冯睿婷; 张晓娟

    2012-01-01

    20:1. Results DDP, 5-Fu could decrease the proliferation and survival rate of EC9706 cells,the IC50. was 5 jug/ml and 10 μg/ml, respectively,which increased mRNA expressions of signal pathway molecules involved in DNA damage and repair system. MICA, MICB, ULBP2, ULBP3 on EG9706 cells were over expressed after 72 h cultured with 1/2 IC50 DDP, while expression of MICA, UL.BP2, UI.BP3 were higher after treated by 1/2 IC50 5-Fu. Cytotoxichy of CIK cells against EC9706 cells cultured by 1/2 IC50 DDP, 1/2 IC50 5-Fu were (37. OS ±0.62)%.(59. 33 ± 2. 10)% , (52. 44 ± 0. 97)%,respectively. Cytotoxicity of CIK cells against EC9706 cells cultured by either 1/2 IC50 DDP or 1/2 IC50 5-Fu was significantly enhanced. Conclusion The results indicate that DDP or 5-Fu can enhance the susceptibility of EC9706 cells to CIK cells-mediated killing effect by upregulating the expressions of NKG2D ligands through activating signal pathway molecules involved in DNA damage and repair system.

  8. Control of Influenza and Poliomyelitis with Killed Virus Vaccines

    Science.gov (United States)

    Salk, Jonas; Salk, Darrell

    1977-01-01

    Discusses control of poliomyelitis and influenza by live and killed virus vaccines. Considered are the etiological agents, pathogenic mechanisms and epidemiology of each disease. Reviews recent scientific studies of the diseases. Recommends use of killed virus vaccines in controlling both diseases. (CS)

  9. Killing day-old chicks? Public opinion regarding potential alternatives

    NARCIS (Netherlands)

    Leenstra, F.; Munnichs, G.M.; Beekman, V.; Vromans, E.; Aramyan, L.; Woelders, H.

    2011-01-01

    Throughout the world, male chicks from layer breeds are killed just after hatching, as they are not profitable as regards the production of meat. The Dutch and European parliaments have insisted on research into possible alternatives to the killing of day-old chicks. In the present study we have inv

  10. Defective phagocyte Aspergillus killing associated with recurrent pulmonary Aspergillus infections.

    Science.gov (United States)

    Fietta, A; Sacchi, F; Mangiarotti, P; Manara, G; Gialdroni Grassi, G

    1984-01-01

    An apparently healthy boy was suffering from recurrent Aspergillus infections. No classical conditions of immunodeficiency were found. Studies on the patient's phagocytic system revealed neutrophils and monocytes to function normally except in Aspergillus killing (microbicidal activity for bacteria and Candida was normal). Aspergillus killing mechanisms may be complex and peculiarly selective, possibly involving both oxygen-dependent and independent mechanisms.

  11. Beneath the surface: killing of fish as a moral problem

    NARCIS (Netherlands)

    Bovenkerk, B.; Braithwaite, V.A.

    2016-01-01

    Are we morally justified in killing fish and if so, for what purposes? We do not focus on the suffering that is done during the killing, but on the question whether death itself is harmful for fish. We need to distinguish two questions; first, can death be considered a harm for fish? And second, if

  12. 9 CFR 113.216 - Bovine Rhinotracheitis Vaccine, Killed Virus.

    Science.gov (United States)

    2010-01-01

    ... Virus. 113.216 Section 113.216 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.216 Bovine Rhinotracheitis Vaccine, Killed Virus. Infectious...

  13. f(R)-gravity from Killing Tensors

    CERN Document Server

    Paliathanasis, Andronikos

    2015-01-01

    We consider $f\\left( R\\right) $-gravity in a Friedmann-Lema\\^{\\i}tre-Robertson-Walker spacetime with zero spatial curvature. We apply the Killing tensors of the minisuperspace in order to specify the functional form of $f\\left( R\\right) $ and the field equations to be invariant under Lie-B\\"{a}cklund transformations which are linear in the momentum (contact symmetries). Consequently, the field equations to admit quadratic conservation laws given by Noether's Theorem. We find three new integrable $f\\left( R\\right) $ models, for which with the application of the conservation laws we reduce the field equations to a system of two first-order ordinary differential equations. For each model we study the evolution of the cosmological fluid. Where we find that for the one integrable model the cosmological fluid has an equation of state parameter, in which in the latter there is a linear behavior in terms of the scale factor which describes the CPL parametric dark energy model.

  14. Killing effect of peppermint vapor against pink-slime forming microorganisms.

    Science.gov (United States)

    Ihara, Nozomi; Sakamoto, Jin; Yoshida, Munehiro; Tsuchido, Tetsuaki

    2015-01-01

    The killing effect of peppermint vapor (PMV) against pink-slime forming microorganisms, Methylobacterium mesophilicum as a bacterium and Rhodotorula mucilaginosa as a yeast, was investigated by the agar vapor assay. In this method, microbial cells were spread over the agar surface exposed to PMV in a petri dish, and then transferred into a recovery liquid. When 60μl of the peppermint liquid was added to a paper disc, a marked killing effect of PMV was observed after 48h against M. mesophilicum and after 168h against R. mucilaginosa. M. mesophilicum and R. mucilaginosa were found to be more resistant to PMV than Escherichia coli and Candida albicans, used as reference microorganisms, respectively. With the addition of 0.03% sodium pyruvate as a hydrogen peroxide scavenger in agar, the killing effect of PMV against E. coli and C. albicans was decreased, whereas it was little changed against M. mesophilicum and R. mucilaginosa. In fact, the properties of the killing effect of hydrogen peroxide solution at 0.2-1.0mM was in accord with those of PMV. M. mesophilicum and R. mucilaginosa were more resistant to the oxidant than E. coli and C. albicans, respectively. Results obtained suggested that reactive oxygen species (ROS) may be involved in the killing action of PMV and therefore pink-slime formers are more resistant to PMV than non-pink-slime formers because of the presence of carotenoids as an antioxidant in cells. We also suggest that the use of PMV appeared to be a potential tool for the control of pink-slime forming microorganisms occurring in wet areas of houses such as the bathroom and washing room.

  15. Chronic, low-dose rotenone reproduces Lewy neurites found in early stages of Parkinson's disease, reduces mitochondrial movement and slowly kills differentiated SH-SY5Y neural cells

    Directory of Open Access Journals (Sweden)

    Liu Lei

    2008-12-01

    Full Text Available Abstract Background Parkinson's disease, the most common adult neurodegenerative movement disorder, demonstrates a brain-wide pathology that begins pre-clinically with alpha-synuclein aggregates ("Lewy neurites" in processes of gut enteric and vagal motor neurons. Rostral progression into substantia nigra with death of dopamine neurons produces the motor impairment phenotype that yields a clinical diagnosis. The vast majority of Parkinson's disease occurs sporadically, and current models of sporadic Parkinson's disease (sPD can utilize directly infused or systemic neurotoxins. Results We developed a differentiation protocol for human SH-SY5Y neuroblastoma that yielded non-dividing dopaminergic neural cells with long processes that we then exposed to 50 nM rotenone, a complex I inhibitor used in Parkinson's disease models. After 21 days of rotenone, ~60% of cells died. Their processes retracted and accumulated ASYN-(+ and UB-(+ aggregates that blocked organelle transport. Mitochondrial movement velocities were reduced by 8 days of rotenone and continued to decline over time. No cytoplasmic inclusions resembling Lewy bodies were observed. Gene microarray analyses showed that the majority of genes were under-expressed. qPCR analyses of 11 mtDNA-encoded and 10 nDNA-encoded mitochondrial electron transport chain RNAs' relative expressions revealed small increases in mtDNA-encoded genes and lesser regulation of nDNA-encoded ETC genes. Conclusion Subacute rotenone treatment of differentiated SH-SY5Y neuroblastoma cells causes process retraction and partial death over several weeks, slowed mitochondrial movement in processes and appears to reproduce the Lewy neuritic changes of early Parkinson's disease pathology but does not cause Lewy body inclusions. The overall pattern of transcriptional regulation is gene under-expression with minimal regulation of ETC genes in spite of rotenone's being a complex I toxin. This rotenone-SH-SY5Y model in a

  16. Rapid kill-novel endodontic sealer and Enterococcus faecalis.

    Science.gov (United States)

    Beyth, Nurit; Kesler Shvero, Dana; Zaltsman, Nathan; Houri-Haddad, Yael; Abramovitz, Itzhak; Davidi, Michael Perez; Weiss, Ervin I

    2013-01-01

    With growing concern over bacterial resistance, the identification of new antimicrobial means is paramount. In the oral cavity microorganisms are essential to the development of periradicular diseases and are the major causative factors associated with endodontic treatment failure. As quaternary ammonium compounds have the ability to kill a wide array of bacteria through electrostatic interactions with multiple anionic targets on the bacterial surface, it is likely that they can overcome bacterial resistance. Melding these ideas, we investigated the potency of a novel endodontic sealer in limiting Enterococcus faecalis growth. We used a polyethyleneimine scaffold to synthesize nano-sized particles, optimized for incorporation into an epoxy-based endodontic sealer. The novel endodontic sealer was tested for its antimicrobial efficacy and evaluated for biocompatibility and physical eligibility. Our results show that the novel sealer foundation affixes the nanoparticles, achieving surface bactericidal properties, but at the same time impeding nanoparticle penetration into eukaryotic cells and thereby mitigating a possible toxic effect. Moreover, adequate physical properties are maintained. The nanosized quaternary amine particles interact within minutes with bacteria, triggering cell death across wide pH values. Throughout this study we demonstrate a new antibacterial perspective for endodontic sealers; a novel antibacterial, effective and safe antimicrobial means.

  17. Managing Threat, Cost, and Incentive to Kill: The Short- and Long-Term Effects of Intervention in Mass Killings

    Science.gov (United States)

    Kathman, Jacob D.; Wood, Reed M.

    2011-01-01

    How do third-party interventions affect the severity of mass killings? The authors theorize that episodes of mass killing are the consequence of two factors: (1) the threat perceptions of the perpetrators and (2) the cost of implementing genocidal policies relative to other alternatives. To reduce genocidal hostilities, interveners must address…

  18. Extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval Strongyloides stercoralis.

    Science.gov (United States)

    Bonne-Année, Sandra; Kerepesi, Laura A; Hess, Jessica A; Wesolowski, Jordan; Paumet, Fabienne; Lok, James B; Nolan, Thomas J; Abraham, David

    2014-06-01

    Neutrophils are multifaceted cells that are often the immune system's first line of defense. Human and murine cells release extracellular DNA traps (ETs) in response to several pathogens and diseases. Neutrophil extracellular trap (NET) formation is crucial to trapping and killing extracellular pathogens. Aside from neutrophils, macrophages and eosinophils also release ETs. We hypothesized that ETs serve as a mechanism of ensnaring the large and highly motile helminth parasite Strongyloides stercoralis thereby providing a static target for the immune response. We demonstrated that S. stercoralis larvae trigger the release of ETs by human neutrophils and macrophages. Analysis of NETs revealed that NETs trapped but did not kill larvae. Induction of NETs was essential for larval killing by human but not murine neutrophils and macrophages in vitro. In mice, extracellular traps were induced following infection with S. stercoralis larvae and were present in the microenvironment of worms being killed in vivo. These findings demonstrate that NETs ensnare the parasite facilitating larval killing by cells of the immune system.

  19. Psychological traits underlying different killing methods among Malaysian male murderers.

    Science.gov (United States)

    Kamaluddin, Mohammad Rahim; Shariff, Nadiah Syariani; Nurfarliza, Siti; Othman, Azizah; Ismail, Khaidzir H; Mat Saat, Geshina Ayu

    2014-04-01

    Murder is the most notorious crime that violates religious, social and cultural norms. Examining the types and number of different killing methods that used are pivotal in a murder case. However, the psychological traits underlying specific and multiple killing methods are still understudied. The present study attempts to fill this gap in knowledge by identifying the underlying psychological traits of different killing methods among Malaysian murderers. The study adapted an observational cross-sectional methodology using a guided self-administered questionnaire for data collection. The sampling frame consisted of 71 Malaysian male murderers from 11 Malaysian prisons who were selected using purposive sampling method. The participants were also asked to provide the types and number of different killing methods used to kill their respective victims. An independent sample t-test was performed to establish the mean score difference of psychological traits between the murderers who used single and multiple types of killing methods. Kruskal-Wallis tests were carried out to ascertain the psychological trait differences between specific types of killing methods. The results suggest that specific psychological traits underlie the type and number of different killing methods used during murder. The majority (88.7%) of murderers used a single method of killing. Multiple methods of killing was evident in 'premeditated' murder compared to 'passion' murder, and revenge was a common motive. Examples of multiple methods are combinations of stabbing and strangulation or slashing and physical force. An exception was premeditated murder committed with shooting, when it was usually a single method, attributed to the high lethality of firearms. Shooting was also notable when the motive was financial gain or related to drug dealing. Murderers who used multiple killing methods were more aggressive and sadistic than those who used a single killing method. Those who used multiple methods or

  20. Laser Microbial Killing and Biofilm Disruption

    Science.gov (United States)

    Krespi, Yosef P.; Kizhner, Victor

    2009-06-01

    Objectives: To analyze the ability of NIR lasers to reduce bacterial load and demonstrate the capability of fiber-based Q-switched Nd:YAG laser disrupting biofilm. Study Design: NIR diode laser was tested in vitro and in vivo using pathogenic microorganisms (S. aureus, S. pneumoniae, P. aeruginosa). In addition biofilms were grown from clinical Pseudomonas isolates and placed in culture plates, screws, tympanostomy tubes and PET sutures. Methods: In the animal experiments acute rhinosinusitis model was created by packing the rabbit nose with bacteria soaked solution. The nasal pack was removed in two days and nose was exposed to laser irradiation. A 940 nm diode laser with fiber diffuser was used. Nasal cultures were obtained before and after the laser treatments. Animals were sacrificed fifteen days following laser treatment and bacteriologic/histologic results analyzed. Q-switched Nd:YAG laser generated shockwave pulses were delivered on biofilm using special probes over culture plates, screws, tubes, and PET sutures for the biofilm experiments. Results: Average of two log bacteria reduction was achieved with NIR laser compared to controls. Histologic studies demonstrated preservation of tissue integrity without significant damage to mucosa. Biofilms were imaged before, during and after treatment using a confocal microscope. During laser-generated shockwave application, biofilm was initially seen to oscillate and eventually break off. Large and small pieces of biofilm were totally and instantly removed from the surface to which they were attached in seconds. Conclusions: Significant bacterial reduction was achieved with NIR laser therapy in this experimental in vitro and animal study. In addition we disrupted Pseudomonas aeruginosa biofilms using Q-switched Nd:YAG laser and special probes generating plasma and shockwave. This new and innovative method of bacteria killing and biofilm disruption without injuring host tissue may have clinical application in the

  1. Protein chlorination in neutrophil phagosomes and correlation with bacterial killing.

    Science.gov (United States)

    Green, Jessie N; Kettle, Anthony J; Winterbourn, Christine C

    2014-12-01

    Neutrophils ingest and kill bacteria within phagocytic vacuoles. We investigated where they produce hypochlorous acid (HOCl) following phagocytosis by measuring conversion of protein tyrosine residues to 3-chlorotyrosine. We also examined how varying chloride availability affects the relationship between HOCl formation in the phagosome and bacterial killing. Phagosomal proteins, isolated following ingestion of opsonized magnetic beads, contained 11.4 Cl-Tyr per thousand tyrosine residues. This was 12 times higher than the level in proteins from the rest of the neutrophil and ~6 times higher than previously recorded for protein from ingested bacteria. These results indicate that HOCl production is largely localized to the phagosomes and a substantial proportion reacts with phagosomal protein before reaching the microbe. This will in part detoxify the oxidant but should also form chloramines which could contribute to the killing mechanism. Neutrophils were either suspended in chloride-free gluconate buffer or pretreated with formyl-Met-Leu-Phe, a procedure that has been reported to deplete intracellular chloride. These treatments, alone or in combination, decreased both chlorination in phagosomes and killing of Staphylococcus aureus by up to 50%. There was a strong positive correlation between the two effects. Killing was predominantly oxidant and myeloperoxidase dependent (88% inhibition by diphenylene iodonium and 78% by azide). These results imply that lowering the chloride concentration limits HOCl production and oxidative killing. They support a role for HOCl generation, rather than an alternative myeloperoxidase activity, in the killing process.

  2. Therapeutic relevance of penicillin-induced hypersensitivity of Staphylococcus aureus to killing by polymorphonuclear leukocytes.

    OpenAIRE

    Lam, C; Georgopoulos, A.; Laber, G.; Schütze, E

    1984-01-01

    There is an overwhelming body of evidence that certain Staphylococcus aureus strains become more sensitive to killing by polymorphonuclear leukocytes after their growth in media containing subinhibitory concentrations of penicillin. However, it is not clear to what extent this phenomenon contributes to the curative effect of penicillin in vivo. To explore its therapeutic relevance, we evaluated the interaction of staphylococci pretreated with penicillin in vitro with leukocytes in cell-proof ...

  3. DNase expression allows the pathogen group A streptococcus to escape killing in neutrophil extracellular traps

    OpenAIRE

    Buchanan, John T; Simpson, Amelia J; Aziz, Ramy K.; Liu, George Y.; Kristian, Sascha A.; Kotb, Malak; Feramisco, James; Nizet, Victor

    2006-01-01

    The innate immune response plays a crucial role in satisfactory host resolution of bacterial infection. In response to chemotactic signals, neutrophils are early responding cells that migrate in large numbers to sites of infection. The recent discovery of secreted neutrophil extracellular traps (NETs) composed of DNA and histones opened a novel dimension in our understanding of the microbial killing capacity of these specialized leukocytes. M1 serotype strains of the pathogen Group A Streptoc...

  4. 不同剂量γ射线辐照对小鼠红内期疟原虫杀伤作用的研究%The exposure