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Sample records for cell specific cytotoxicity

  1. Concanavalin A-induced activation of lymphocytic choriomeningitis virus memory lymphocytes into specifically cytotoxic T cells

    DEFF Research Database (Denmark)

    Marker, O; Thomsen, Allan Randrup; Andersen, G T

    1977-01-01

    When spleen cells, which have been primed to Lymphocytic Choriomeningitis (LCM) virus during a primary infection several months previously, are stimulated in vitro with Con A. highly specific secondary cytotoxic effector cells are generated. The degree of cytotoxicity revealed by such Con A......-stimulated cells is higher than that of non-incubated spleen cells harvested nine days following the primary infection, and the effect is totally inhibited by anti-theta serum plus complement treatment of the effector cells immediately before the cytotoxic test....

  2. In vitro cell-mediated cytotoxicity to the male specific (H-Y) antigen in man.

    Science.gov (United States)

    Singal, D P; Wadia, Y J; Naipaul, N

    1981-02-01

    We have studied the role of HLA antigens in restricting specificity of the cytotoxic lymphocytes (CTL). CTL's developed between female responder/male stimulator combinations, were tested for H-Y antigen killing in cell-mediated lympholysis. Two CTL's demonstrated HLA-restricted H-Y cytotoxicity. In both instances, the responders are married parous females and both are positive for HLA-A2. These CTL's lysed target cells from donors who are either positive for the sensitizing HLA antigen or who are HLA-A2-positive males. On the other hand, one CTL where the HLA-A2-positive responder is not a parous female did not show HLA-restricted H-Y cytotoxicity. Also, CTL's where responders do not carry HLA-A2 showed no H-Y cytotoxicity. The data suggest that pregnancy(ies) is sufficient in itself to induce HLA-restricted H-Y cytotoxicity and that it can be recalled by in vitro stimulation with lymphocytes from an unrelated male donor. Also, in these studies HLA-restricted H-Y cytotoxicity was obtained only with targets that shared HLA-A2 with the effectors.

  3. Indoleamine 2,3-dioxygenase specific, cytotoxic T cells as immune regulators

    DEFF Research Database (Denmark)

    Sørensen, Rikke Bæk; Hadrup, Sine Reker; Svane, Inge Marie

    2011-01-01

    Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme that is implicated in suppressing T-cell immunity in normal and pathologic settings. Here, we describe that spontaneous cytotoxic T-cell reactivity against IDO exists not only in patients with cancer but also in healthy persons. We...... show that the presence of such IDO-specific CD8(+) T cells boosted T-cell immunity against viral or tumor-associated antigens by eliminating IDO+ suppressive cells. This had profound effects on the balance between interleukin-17 (IL-17)-producing CD4(+) T cells and regulatory T cells. Furthermore......, this caused an increase in the production of the proinflammatory cytokines IL-6 and tumor necrosis factor-alpha while decreasing the IL-10 production. Finally, the addition of IDO-inducing agents (ie, the TLR9 ligand cytosine-phosphate- guanosine, soluble cytotoxic T lymphocyte-associated antigen 4...

  4. Mechanism of arctigenin-mediated specific cytotoxicity against human lung adenocarcinoma cell lines.

    Science.gov (United States)

    Susanti, Siti; Iwasaki, Hironori; Inafuku, Masashi; Taira, Naoyuki; Oku, Hirosuke

    2013-12-15

    The lignan arctigenin (ARG) from the herb Arctium lappa L. possesses anti-cancer activity, however the mechanism of action of ARG has been found to vary among tissues and types of cancer cells. The current study aims to gain insight into the ARG mediated mechanism of action involved in inhibiting proliferation and inducing apoptosis in lung adenocarcinoma cells. This study also delineates the cancer cell specificity of ARG by comparison with its effects on various normal cell lines. ARG selectively arrested the proliferation of cancer cells at the G0/G1 phase through the down-regulation of NPAT protein expression. This down-regulation occurred via the suppression of either cyclin E/CDK2 or cyclin H/CDK7, while apoptosis was induced through the modulation of the Akt-1-related signaling pathway. Furthermore, a GSH synthase inhibitor specifically enhanced the cytotoxicity of ARG against cancer cells, suggesting that the intracellular GSH content was another factor influencing the susceptibility of cancer cells to ARG. These findings suggest that specific cytotoxicity of ARG against lung cancer cells was explained by its selective modulation of the expression of NPAT, which is involved in histone biosynthesis. The cytotoxicity of ARG appeared to be dependent on the intracellular GSH level.

  5. Allorestricted cytotoxic T cells specific for human CD45 show potent antileukemic activity.

    Science.gov (United States)

    Amrolia, Persis J; Reid, Steven D; Gao, Liquan; Schultheis, Beate; Dotti, Gianpietro; Brenner, Malcolm K; Melo, Junia V; Goldman, John M; Stauss, Hans J

    2003-02-01

    Recent advances have made haploidentical transplantation for leukemia feasible, but the rigorous T-cell depletion used contributes to the high relapse rates observed. We have attempted to improve the graft-versus-leukemia (GVL) effect by generating allorestricted cytotoxic T lymphocytes (CTLs) directed against human CD45. Such CTLs should recognize patient hematopoietic cells including leukemia, enhancing donor cell engraftment and improving the GVL effect, but they should not recognize host nonhematopoietic tissues or donor cells from the graft. Using the T2 binding assay, 4 CD45-derived peptides were found to bind HLA-A2 molecules. These peptides were used to generate cytotoxic T-cell lines from HLA-A2(-) donors by sequential stimulation with peptide-pulsed HLA-A2(+) stimulators, and the lines obtained were screened for peptide-specific cytotoxicity. Using one of these peptides (P1218), it was possible to generate peptide-specific, allorestricted CTLs in 3 of 7 responders. P1218-specific CTL lines show potent cytotoxicity against hematopoietic cell lines coexpressing HLA-A2 and CD45 but not CD45 loss variants. Studies with stable transfectants of 293 cells demonstrated recognition by P1218-specific CTLs of endogenously expressed CD45. Likewise P1218-specific CTLs recognized peripheral blood mononuclear cells (PBMCs) from HLA-A2(+) patients with chronic myeloid leukemia (CML) and leukemic blasts in HLA-A2(+) patients with acute myeloid leukemia (AML), but they were unable to lyse HLA-A2(+) fibroblasts or HLA-A2(-) normal PBMCs. Coculture of CD34(+) PBMCs and bone marrow mononuclear cells (BMMCs) with P1218-specific CTL significantly inhibited colony-forming unit-granulocyte macrophage (CFU-GM) formation in HLA-A2(+) healthy controls and CML patients but resulted in no significant inhibition in HLA-A2(-) healthy controls. These studies demonstrate that P1218-specific cytotoxic T lymphocytes (CTLs) have potent activity against leukemic progenitors and suggest that

  6. Indoleamine 2,3-dioxygenase specific, cytotoxic T cells as immune regulators

    DEFF Research Database (Denmark)

    Sørensen, Rikke Bæk; Hadrup, Sine Reker; Svane, Inge Marie;

    2011-01-01

    , this caused an increase in the production of the proinflammatory cytokines IL-6 and tumor necrosis factor-alpha while decreasing the IL-10 production. Finally, the addition of IDO-inducing agents (ie, the TLR9 ligand cytosine-phosphate- guanosine, soluble cytotoxic T lymphocyte-associated antigen 4...... show that the presence of such IDO-specific CD8(+) T cells boosted T-cell immunity against viral or tumor-associated antigens by eliminating IDO+ suppressive cells. This had profound effects on the balance between interleukin-17 (IL-17)-producing CD4(+) T cells and regulatory T cells. Furthermore...

  7. Cytotoxicity of tumor antigen specific human T cells is unimpaired by arginine depletion.

    Directory of Open Access Journals (Sweden)

    Markus Munder

    Full Text Available Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8(+ T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i CD8(+ T cells with specificity against the MART-1aa26-35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii clonal CMV pp65aa495-503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495-503 specific T cell receptor were analyzed. Our data demonstrate that human CD8(+ T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency.

  8. HBV-specific CD4+ cytotoxic T cells in hepatocellular carcinoma are less cytolytic toward tumor cells and suppress CD8+ T cell-mediated antitumor immunity.

    Science.gov (United States)

    Meng, Fanzhi; Zhen, Shoumei; Song, Bin

    2017-08-01

    In East Asia and sub-Saharan Africa, chronic infection is the main cause of the development of hepatocellular carcinoma, an aggressive cancer with low survival rate. Cytotoxic T cell-based immunotherapy is a promising treatment strategy. Here, we investigated the possibility of using HBV-specific CD4(+) cytotoxic T cells to eliminate tumor cells. The naturally occurring HBV-specific cytotoxic CD4(+) and CD8(+) T cells were identified by HBV peptide pool stimulation. We found that in HBV-induced hepatocellular carcinoma patients, the HBV-specific cytotoxic CD4(+) T cells and cytotoxic CD8(+) T cells were present at similar numbers. But compared to the CD8(+) cytotoxic T cells, the CD4(+) cytotoxic T cells secreted less cytolytic factors granzyme A (GzmA) and granzyme B (GzmB), and were less effective at eliminating tumor cells. In addition, despite being able to secrete cytolytic factors, CD4(+) T cells suppressed the cytotoxicity mediated by CD8(+) T cells, even when CD4(+) CD25(+) regulator T cells were absent. Interestingly, we found that interleukin 10 (IL-10)-secreting Tr1 cells were enriched in the cytotoxic CD4(+) T cells. Neutralization of IL-10 abrogated the suppression of CD8(+) T cells by CD4(+) CD25(-) T cells. Neither the frequency nor the absolute number of HBV-specific CD4(+) cytotoxic T cells were correlated with the clinical outcome of advanced stage hepatocellular carcinoma patients. Together, this study demonstrated that in HBV-related hepatocellular carcinoma, CD4(+) T cell-mediated cytotoxicity was present naturally in the host and had the potential to exert antitumor immunity, but its capacity was limited and was associated with immunoregulatory properties. © 2017 APMIS. Published by John Wiley & Sons Ltd.

  9. Antigen-specific B cells reactivate an effective cytotoxic T cell response against phagocytosed Salmonella through cross-presentation.

    Directory of Open Access Journals (Sweden)

    Jelle de Wit

    Full Text Available BACKGROUND: The eradication of facultative intracellular bacterial pathogens, like Salmonella typhi, requires the concerted action of both the humoral immune response and the cytotoxic CD8(+ T cell response. Dendritic cells (DCs are considered to orchestrate the cytotoxic CD8(+ T cell response via cross-presentation of bacterial antigens onto MHC class I molecules. Cross-presentation of Salmonella by DCs however, is accompanied by the induction of apoptosis in the DCs. Besides antibody production, B cells are required to clear Salmonella infection for other unknown reasons. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that Salmonella-specific B cells that phagocytose Salmonella upon BCR-ligation reactivate human memory CD8(+ T cells via cross-presentation yielding a Salmonella-specific cytotoxic T cell response. The reactivation of CD8(+ T cells is dependent on CD4(+ T cell help. Unlike the DCs, B cell-mediated cross-presentation of Salmonella does not coincide with apoptosis. CONCLUSIONS/SIGNIFICANCE: B cells form a new player in the activation of the cytotoxic effector arm of the immune response and the generation of effective adaptive immunity in Salmonella infection.

  10. GENETICALLY MODIFIED DENDRITIC CELLS INDUCED SPECIFIC CYTOTOXITY AGAINST HUMAN HCC CELLS IN VITRO

    Institute of Scientific and Technical Information of China (English)

    刘彬彬; 叶胜龙; 贺平; 郑宁; 赵燕; 孙瑞霞; 刘银坤; 汤钊猷

    2004-01-01

    Objective: to transduce the tumor associated antigen gene MAGE-1 and/or IL-12 gene into dendritic cells (DC) and to observe the in vitro cytotoxic effect induced by the genetically modified DC against the human hepatocellular carcinoma (HCC) cell line SMMC7721. Methods: the MAGE-1 gene was inserted into the retrovirus vector LXSN to construct the recombinant retrovirus LMSN. The monocyte-derived DCs were transfected at appropriate differentiation stage by LMSN and/or a recombinant adenovirus AdmiL-12, which containing murine IL-12 gene. The control groups included retrovirus LXSN transfected, adenovirus AdBGFP transfected and non-transfected DCs. The MAGE-1 gene expression was identified by western blot and the mIL-12 p70 secretion was detected by ELISA assay. The in vitro cytotoxicities against SMMC7721 induced by genetically modified and control groups of DC were tested by MTT assay. Results: The MAGE-1 expression was detected by a monoclonal antibody in DCs tranfected with LMSN but not in control groups. At 16 h, 24 h and 48 h after transfection with AdmIL-12, the concentration of the mIL-12 p70 in the culture medium was 580pg/106 cells, 960pg/106 cells and 1100pg/106 cells respectively. The mIL-12 p70 secretions were not detected in other groups. The lytic activity (as judged by % lysis) induced by each groups of DC was 94.2(5.2% (LMSN and AdmIL-12 cotransfected group), 78.9(3.6% (LMSN transfected groups), 52.6(9.7% (AdmIL-12 transfected group), 34.7(4.3% (LXSN transfected group), 36.3(3.8% (AdBGFP transfected group) and 3.9(2.0% (non-transfected group) respectively. Except for LXSN transfected and AdBGFP transfected group, the difference of the lytic activities between other groups were statistically significant (P<0.05). Conclusion: The MAGE-1 gene modified DCs can induce relatively specific cytotoxicty against SMMC7721 in vitro and thus suggested that those genetically engineered DCs have the potential to serve as novel vaccine for HCC. Transduction of

  11. Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

    Energy Technology Data Exchange (ETDEWEB)

    Kurayoshi, Kenta [Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan); Ozono, Eiko [Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary, University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ (United Kingdom); Iwanaga, Ritsuko; Bradford, Andrew P. [Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Anschutz Medical Campus, 12800 East 19th Avenue, Aurora, CO 80045 (United States); Komori, Hideyuki [Center for Stem Cell Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109 (United States); Ohtani, Kiyoshi, E-mail: btm88939@kwansei.ac.jp [Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan)

    2014-07-18

    Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter

  12. Specific induction of anti-leukemia effects by umbilical cord cell-derived CD8~+ T cytotoxic lymphocytes

    Institute of Scientific and Technical Information of China (English)

    刘芯

    2006-01-01

    Objective To explore the specific anti-leukemia immune response of CD8+ cytotoxic T lymphocyte (CTL) derived from cord blood (CB) ex vivo and evaluate the feasibilities and values of the CTL for specific immunotherapy. Methods Dendritic cells (DC) were induced from mononuclear cells (MNC) by combination cytokines in 10 CB samples. Loading U937 cell lysate antigen on

  13. Enhanced Eradication of Lymphoma by Tumor-Specific Cytotoxic T-Cells Secreting an Engineered Tumor-Specific Immunotoxin

    Science.gov (United States)

    2010-06-01

    5% CO2. The level of proliferation was then assessed using an ATP assay (CellTiter-Glo G7570; Promega, Madison, WI). Phenotyping. Cell-surface...trypan blue or an ATP assay (Promega). Cytotoxicity assay. T cell cytotoxic activity was evaluated in a flow cytometry based assay. Target cells...ml PMA and 1µM ionomycin. Cells were collected and lysed, and the lysate analyzed for bioluminescent signal. This revealed that the promoter induced

  14. A Tetrameric Peptide Derived from Bovine Lactoferricin Exhibits Specific Cytotoxic Effects against Oral Squamous-Cell Carcinoma Cell Lines

    Science.gov (United States)

    Solarte, Víctor A.; Rosas, Jaiver E.; Rivera, Zuly J.; Arango-Rodríguez, Martha L.; García, Javier E.; Vernot, Jean-Paul

    2015-01-01

    Several short linear peptides derived from cyclic bovine lactoferricin were synthesized and tested for their cytotoxic effect against the oral cavity squamous-cell carcinoma (OSCC) cell lines CAL27 and SCC15. As a control, an immortalized and nontumorigenic cell line, Het-1A, was used. Linear peptides based on the RRWQWR core sequence showed a moderate cytotoxic effect and specificity towards tumorigenic cells. A tetrameric peptide, LfcinB(20–25)4, containing the RRWQWR motif, exhibited greater cytotoxic activity (>90%) in both OSCC cell lines compared to the linear lactoferricin peptide or the lactoferrin protein. Additionally, this tetrameric peptide showed the highest specificity towards tumorigenic cells among the tested peptides. Interestingly, this effect was very fast, with cell shrinkage, severe damage to cell membrane permeability, and lysis within one hour of treatment. Our results are consistent with a necrotic effect rather than an apoptotic one and suggest that this tetrameric peptide could be considered as a new candidate for the therapeutic treatment of OSCC. PMID:26609531

  15. A Tetrameric Peptide Derived from Bovine Lactoferricin Exhibits Specific Cytotoxic Effects against Oral Squamous-Cell Carcinoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Víctor A. Solarte

    2015-01-01

    Full Text Available Several short linear peptides derived from cyclic bovine lactoferricin were synthesized and tested for their cytotoxic effect against the oral cavity squamous-cell carcinoma (OSCC cell lines CAL27 and SCC15. As a control, an immortalized and nontumorigenic cell line, Het-1A, was used. Linear peptides based on the RRWQWR core sequence showed a moderate cytotoxic effect and specificity towards tumorigenic cells. A tetrameric peptide, LfcinB(20–254, containing the RRWQWR motif, exhibited greater cytotoxic activity (>90% in both OSCC cell lines compared to the linear lactoferricin peptide or the lactoferrin protein. Additionally, this tetrameric peptide showed the highest specificity towards tumorigenic cells among the tested peptides. Interestingly, this effect was very fast, with cell shrinkage, severe damage to cell membrane permeability, and lysis within one hour of treatment. Our results are consistent with a necrotic effect rather than an apoptotic one and suggest that this tetrameric peptide could be considered as a new candidate for the therapeutic treatment of OSCC.

  16. Tumor specific cytotoxicity of glucosylceramide.

    Science.gov (United States)

    Oku, Hirosuke; Wongtangtintharn, Sawitree; Iwasaki, Hironori; Inafuku, Masashi; Shimatani, Masayuki; Toda, Takayoshi

    2007-11-01

    To develop a new taxon of anti-cancer agent with lower side effect, this study described a tumor selective cytotoxicity of glucosylceramide extracted from malt feed of beer brewing waste. Interpretation of (13)C- and (1)H-NMR spectra identified the chemical structure of major component of glucosylceramide as 1-O-beta-D: -glucopyranosyl-2(2'-hydroxyeicosanoylamino)-4,11-octadecadiene-1,3-diol. Selective cytotoxicity was studied with three pairs of normal and cancer cells: liver, skin and lung. The glucosylceramide selectively lowered the relative viability of cancer cells. Of the pairs, the selectivity was most pronounced with the liver cells, and, for this reason, further experiment was conducted with this pair of normal (CS-HC) and cancer cells (HepG2) to get more insight into the selective toxicity. The glucosylceramide significantly increased the cell population at G(2)/M phase in HepG2 cells, and also increased the numbers of apoptotic (sub-G(0)/G(1)) cells, but to much lesser extent compared with the increase in G(2)/M phase. Treatment of HepG2 cells with this agent selectively disrupted the mitochondrial membrane integrity without activation of caspase pathway to induce apoptosis. These findings suggested that the glucosylceramide specifically suppressed the growth of cancer cells by inhibiting cell renewal capacity rather than induction of apoptosis. The underlying mechanism for the selectivity remains to be answered in the forthcoming study.

  17. Monitoring of WT1-specific cytotoxic T lymphocytes after allogeneic hematopoietic stem cell transplantation.

    Science.gov (United States)

    Morita, Yuriko; Heike, Yuji; Kawakami, Mami; Miura, Osamu; Nakatsuka, Shin-Ichi; Ebisawa, Michiko; Mori, Shin-Ichiro; Tanosaki, Ryuji; Fukuda, Takahiro; Kim, Sung-Won; Tobinai, Kensei; Takaue, Yoichi

    2006-09-15

    Donor-derived cytotoxic T lymphocytes (CTL) that respond to tumor antigens emerge after hematopoietic stem cell transplantation (HSCT), particularly in association with the status of immune recovery. To analyze the frequency of CTL against PR1, PRAME and WT1 after HSCT, a tetramer-based analysis was performed in 97 samples taken from 35 patients (9 AML, 11 MDS, 2 CML, 4 ALL, 7 lymphoma and 2 renal cell carcinoma [RCC]) with the HLA-A02 phenotype. Regarding PR1, only 1 sample showed the presence of tetramer-positive cells (0.04%/lymphocyte). Similarly, in PRAME, only 10 of 97 samples were sporadically positive with low titers. For WT1, positive results were detected in 39 of 97 samples and 7 (2 CML, 1 ALL, 2 lymphoma and 2 RCC) patients clearly showed positive results more than once. On the basis of these results, we performed serial analyses of WT1-specific CTL during the clinical course in 2 patients with RCC, who underwent HSCT with a reduced-intensity regimen, to examine the precise correlation between the kinetics of CTL, the occurrence of GVHD and the observed clinical response. A higher positive rate for WT1-specific CTL and a correlation with the clinical response suggest that WT1 may be a useful antigen for a wider monitoring application.

  18. Antigen-Specific B Cells Reactivate an Effective Cytotoxic T Cell Response against Phagocytosed Salmonella through Cross-Presentation

    NARCIS (Netherlands)

    de Wit, J.; Souwer, Y.; Jorritsma, T.; Bos, H.; ten Brinke, A.; Neefjes, J.; Ham, S.M.

    2010-01-01

    Background: The eradication of facultative intracellular bacterial pathogens, like Salmonella typhi, requires the concerted action of both the humoral immune response and the cytotoxic CD8(+) T cell response. Dendritic cells (DCs) are considered to orchestrate the cytotoxic CD8(+) T cell response vi

  19. Measuring melanoma-specific cytotoxic T lymphocytes elicited by dendritic cell vaccines with a tumor inhibition assay in vitro.

    Science.gov (United States)

    Paczesny, Sophie; Shi, Honhgzhen; Saito, Hiroaki; Mannoni, Patrice; Fay, Joseph; Banchereau, Jacques; Palucka, A Karolina

    2005-01-01

    Improving cancer vaccines depends on assays measuring elicited tumor-specific T-cell immunity. Cytotoxic effector cells are essential for tumor clearance and are commonly evaluated using 51Cr release from labeled target cells after a short (4 hours) incubation with T cells. The authors used a tumor inhibition assay (TIA) that assesses the capacity of cytotoxic T lymphocytes (CTLs) to control the survival/growth of EGFP-labeled tumor cell lines. TIA was validated using CD8+ T cells primed in vitro against melanoma and breast cancer cells. TIA was then used to assess the CTL function of cultured CD8+ T cells isolated from patients with metastatic melanoma who underwent vaccination with peptide-pulsed CD34+ HPCs-derived DCs. After the DC vaccination, T cells from six of eight patients yielded CTLs that could inhibit the survival/growth of melanoma cells. The results of TIA correlated with killing of tumor cells in a standard 4-hour 51Cr release assay, yet TIA allowed detection of CTL activities that appeared marginal in the 51Cr release assay. Thus, TIA might prove valuable for measuring spontaneous and induced antigen-specific cytotoxic T cells.

  20. Myeloid leukemic progenitor cells can be specifically targeted by minor histocompatibility antigen LRH-1-reactive cytotoxic T cells.

    Science.gov (United States)

    Norde, Wieger J; Overes, Ingrid M; Maas, Frans; Fredrix, Hanny; Vos, Johanna C M; Kester, Michel G D; van der Voort, Robbert; Jedema, Inge; Falkenburg, J H Frederik; Schattenberg, Anton V; de Witte, Theo M; Dolstra, Harry

    2009-03-05

    CD8(+) T cells recognizing minor histocompatibility antigens (MiHAs) on leukemic stem and progenitor cells play a pivotal role in effective graft-versus-leukemia reactivity after allogeneic stem cell transplantation (SCT). Previously, we identified a hematopoiesis-restricted MiHA, designated LRH-1, which is presented by HLA-B7 and encoded by the P2X5 purinergic receptor gene. We found that P2X5 is significantly expressed in CD34(+) leukemic subpopulations from chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) patients. Here, we demonstrate that LRH-1-specific CD8(+) T-cell responses are frequently induced in myeloid leukemia patients following donor lymphocyte infusions. Patients with high percentages of circulating LRH-1-specific CD8(+) T cells had no or only mild graft-versus-host disease. Functional analysis showed that LRH-1-specific cytotoxic T lymphocytes (CTLs) isolated from 2 different patients efficiently target LRH-1-positive leukemic CD34(+) progenitor cells from both CML and AML patients, whereas mature CML cells are only marginally lysed due to down-regulation of P2X5. Furthermore, we observed that relative resistance to LRH-1 CTL-mediated cell death due to elevated levels of antiapoptotic XIAP could be overcome by IFN-gamma prestimulation and increased CTL-target ratios. These findings provide a rationale for use of LRH-1 as immunotherapeutic target antigen to treat residual or persisting myeloid malignancies after allogeneic SCT.

  1. Cytotoxic murine monoclonal antibody LAM8 with specificity for human small cell carcinoma of the lung.

    Science.gov (United States)

    Stahel, R A; O'Hara, C J; Mabry, M; Waibel, R; Sabbath, K; Speak, J A; Bernal, S D

    1986-04-01

    The reactivity of the murine immunoglobulin monoclonal antibody LAM8 directed against a membrane antigen of human small cell carcinoma (SCC) of the lung was investigated on human cell lines and tissues. Indirect immunofluorescence staining, radioimmunoassays, and cytotoxicity assays showed LAM8 antibody to selectively react with SCC but not with non-SCC lung cancer cell lines and extrapulmonary tumor cell lines. Unlike other SCC antibodies, including those we have previously described, highly preferential reactivity with SCC tissues was also demonstrated by immunoperoxidase staining of deparaffinized formalin-fixed tissue sections. Membrane and cytoplasmic staining was seen in of 9 of 12 SCC tissues. No significant staining was seen in non-SCC lung cancer and a wide range of other tumors, including mesothelioma and bronchial carcinoids. Significant LAM8 reactivity was also absent in normal tissues of all major organs. Few tumors and epithelial tissues, including bronchial epithelium had rare LAM8 positive cells which were always less than 2% of the entire cell population. In vitro treatment with antibody and human complement was highly cytotoxic to SCC cells, but had not effect on bone marrow progenitor cells. Immunoblotting of membrane extracts separated on sodium dodecyl sulfate-polyacrylamide gels showed the LAM8 antigen to have a band of an approximate molecular weight of 135,000 and a cluster of bands with approximate molecular weights of 90,000. This reactivity was lost after incubation of the extracts with periodate. LAM8 antibody shows a highly preferential reactivity with SCC cell lines and formalin-fixed paraffin-embedded SCC tissues and is selectively cytotoxic to cells expressing LAM8 antigen.

  2. Induction of cancer-specific cytotoxicity towards human prostate and skin cells using quercetin and ultrasound

    OpenAIRE

    Paliwal, S.; Sundaram, J; Mitragotri, S

    2005-01-01

    Bioflavonoids, such as quercetin, have recently emerged as a new class of chemotherapeutic drugs for the treatment of various cancer types, but are marred by their low potency and poor selectivity. We report that a short application of low-frequency ultrasound selectively sensitises prostate and skin cancer cells against quercetin. Pretreatment of cells with ultrasound (20 kHz, 2 W cm−2, 60 s) selectively induced cytotoxicity in skin and prostate cancer cells, while having minimal effect on c...

  3. Antigen-Specific B Cells Reactivate an Effective Cytotoxic T Cell Response against Phagocytosed Salmonella through Cross-Presentation

    OpenAIRE

    Jelle de Wit; Yuri Souwer; Tineke Jorritsma; Hanny Klaasse Bos; Anja ten Brinke; Jacques Neefjes; S Marieke van Ham

    2010-01-01

    BACKGROUND: The eradication of facultative intracellular bacterial pathogens, like Salmonella typhi, requires the concerted action of both the humoral immune response and the cytotoxic CD8(+) T cell response. Dendritic cells (DCs) are considered to orchestrate the cytotoxic CD8(+) T cell response via cross-presentation of bacterial antigens onto MHC class I molecules. Cross-presentation of Salmonella by DCs however, is accompanied by the induction of apoptosis in the DCs. Besides antibody pro...

  4. Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus.

    Science.gov (United States)

    He, Wenqian; Tan, Gene S; Mullarkey, Caitlin E; Lee, Amanda J; Lam, Mannie Man Wai; Krammer, Florian; Henry, Carole; Wilson, Patrick C; Ashkar, Ali A; Palese, Peter; Miller, Matthew S

    2016-10-18

    The generation of strain-specific neutralizing antibodies against influenza A virus is known to confer potent protection against homologous infections. The majority of these antibodies bind to the hemagglutinin (HA) head domain and function by blocking the receptor binding site, preventing infection of host cells. Recently, elicitation of broadly neutralizing antibodies which target the conserved HA stalk domain has become a promising "universal" influenza virus vaccine strategy. The ability of these antibodies to elicit Fc-dependent effector functions has emerged as an important mechanism through which protection is achieved in vivo. However, the way in which Fc-dependent effector functions are regulated by polyclonal influenza virus-binding antibody mixtures in vivo has never been defined. Here, we demonstrate that interactions among viral glycoprotein-binding antibodies of varying specificities regulate the magnitude of antibody-dependent cell-mediated cytotoxicity induction. We show that the mechanism responsible for this phenotype relies upon competition for binding to HA on the surface of infected cells and virus particles. Nonneutralizing antibodies were poor inducers and did not inhibit antibody-dependent cell-mediated cytotoxicity. Interestingly, anti-neuraminidase antibodies weakly induced antibody-dependent cell-mediated cytotoxicity and enhanced induction in the presence of HA stalk-binding antibodies in an additive manner. Our data demonstrate that antibody specificity plays an important role in the regulation of ADCC, and that cross-talk among antibodies of varying specificities determines the magnitude of Fc receptor-mediated effector functions.

  5. HIV-specific cytotoxic T-cell activity in an HIV-exposed but uninfected infant.

    Science.gov (United States)

    Rowland-Jones, S L; Nixon, D F; Aldhous, M C; Gotch, F; Ariyoshi, K; Hallam, N; Kroll, J S; Froebel, K; McMichael, A

    1993-04-03

    The factors necessary for protective immunity against HIV-1 are unknown. Important information about these factors should come from study of people at high risk of HIV infection who have not apparently become infected. Among these are the estimated 60-85% of children who may be exposed in utero or perinatally to HIV-1 but do not become infected. We observed the transient appearance of HIV-specific cytotoxic T-lymphocyte (CTL) activity in a baby born to HIV-1-infected parents, in whom all standard markers of infection remained negative. These findings suggest that HIV-specific CTLs may be a marker for recently exposed, but uninfected, individuals.

  6. Selection of restriction specificities of virus-specific cytotoxic T cells in the thymus: no evidence for a crucial role of antigen-presenting cells

    Energy Technology Data Exchange (ETDEWEB)

    Zinkernagel, R.M.

    1982-12-01

    The proposal was tested that (P1 X P2) F1 leads to P1 irradiation bone marrow chimeras expressed predominantly P1-restricted T cells because donor derived stem cells were exposed to recipient derived antigen-presenting cells in the thymus. Because P1 recipient-derived antigen-presenting cells are replaced only slowly after 6-8 wk by (P1 X P2) donor-derived antigen-presenting cells in the thymus and because replenished pools of mature T cells may by then prevent substantial numbers of P2-restricted T cells to be generated, a large portion of thymus cells and mature T cells were eliminated using the following treatments of 12-20-wk-old (P1 X P2) F1 leads to P1 irradiation bone marrow chimeras: (a) cortisone plus antilymphocyte serum, (b) Cytoxan, (c) three doses of sublethal irradiation (300 rad) 2d apart, and (d) lethal irradiation (850 rad) and reconstitution with T cell-depleted (P1 X P2) F1 stem cells. 12-20 wk after this second treatment, (P1 X P2) leads to P1 chimeras were infected with vaccinia-virus. Virus-specific cytotoxic T cell reactivity was expressed by chimeric T cells of (P1 X P(2) F1 origin and was restricted predominantly to P1. Virus-specific cytotoxic T cells, therefore, do not seem to be selected to measurable extent by the immigrating donor-derived antigen-presenting cells in the thymus; their selection depends apparently from the recipient-derived radioresistant thymus cells.

  7. Effective Delivery of Antigen-Encapsulin Nanoparticle Fusions to Dendritic Cells Leads to Antigen-Specific Cytotoxic T Cell Activation and Tumor Rejection.

    Science.gov (United States)

    Choi, Bongseo; Moon, Hyojin; Hong, Sung Joon; Shin, Changsik; Do, Yoonkyung; Ryu, Seongho; Kang, Sebyung

    2016-08-23

    In cancer immunotherapy, robust and efficient activation of cytotoxic CD8(+) T cell immune responses is a promising, but challenging task. Dendritic cells (DCs) are well-known professional antigen presenting cells that initiate and regulate antigen-specific cytotoxic CD8(+) T cells that kill their target cells directly as well as secrete IFN-γ, a cytokine critical in tumor rejection. Here, we employed recently established protein cage nanoparticles, encapsulin (Encap), as antigenic peptide nanocarriers by genetically incorporating the OT-1 peptide of ovalbumin (OVA) protein to the three different positions of the Encap subunit. With them, we evaluated their efficacy in activating DC-mediated antigen-specific T cell cytotoxicity and consequent melanoma tumor rejection in vivo. DCs efficiently engulfed Encap and its variants (OT-1-Encaps), which carry antigenic peptides at different positions, and properly processed them within phagosomes. Delivered OT-1 peptides were effectively presented by DCs to naïve CD8(+) T cells successfully, resulting in the proliferation of antigen-specific cytotoxic CD8(+) T cells. OT-1-Encap vaccinations in B16-OVA melanoma tumor bearing mice effectively activated OT-1 peptide specific cytotoxic CD8(+) T cells before or even after tumor generation, resulting in significant suppression of tumor growth in prophylactic as well as therapeutic treatments. A large number of cytotoxic CD8(+) T cells that actively produce both intracellular and secretory IFN-γ were observed in tumor-infiltrating lymphocytes collected from B16-OVA tumor masses originally vaccinated with OT-1-Encap-C upon tumor challenges. The approaches we describe herein may provide opportunities to develop epitope-dependent vaccination systems that stimulate and/or modulate efficient and epitope-specific cytotoxic T cell immune responses in nonpathogenic diseases.

  8. Production of Nfa1-specific monoclonal antibodies that influences the in vitro cytotoxicity of Naegleria fowleri trophozoites on microglial cells.

    Science.gov (United States)

    Lee, Yang-Jin; Kim, Jong-Hyun; Jeong, Seok-Ryoul; Song, Kyoung-Ju; Kim, Kyongmin; Park, Sun; Park, Moon-Sung; Shin, Ho-Joon

    2007-10-01

    Naegleria fowleri, agent of fatal primary amoebic meningoencephalitis, appears to induce cytotoxicity mechanically through its contact with the cell. The nfa1 gene cloned from a cDNA library of pathogenic N. fowleri by immunoscreening consists of 360 bp and expresses a 13.1-kDa recombinant protein (rNfa1) that demonstrated localization in the pseudopodia when examined using immunocytochemistry. To study the mechanisms involved in N. fowleri cytotoxicity, we developed a large volume of rNfa1-specific monoclonal antibody (McAb) against a 17-kDa His-tag fusion rNfa1 protein using a cell fusion technique. We established eight McAb-producing hybridoma cells. The antibodies were all immunoglobulin G2b and reacted strongly with a 17-kDa band representing the rNfa1 fusion protein in Western blotting, demonstrating immunoreactivity to the Nfa1 protein in pseudopodia (especially in the food cups) of N. fowleri trophozoites. A 51Cr-release assay indicated N. fowleri cytotoxicity by demonstrating that it eliminated 37.8, 60.6, and 98.8% of the target (microglial) cells 6, 12, and 24 h after co-incubation, respectively. When an anti-Nfa1 McAb was added to the coculture system, N. fowleri cytotoxicity decreased to 29.8, 44.1, and 66.3%, respectively.

  9. Dengue virus-specific, human CD4+ CD8- cytotoxic T-cell clones: multiple patterns of virus cross-reactivity recognized by NS3-specific T-cell clones.

    OpenAIRE

    Kurane, I; Brinton, M.A.; Samson, A L; Ennis, F A

    1991-01-01

    Thirteen dengue virus-specific, cytotoxic CD4+ CD8- T-cell clones were established from a donor who was infected with dengue virus type 3. These clones were examined for virus specificity and human leukocyte antigen (HLA) restriction in cytotoxic assays. Six patterns of virus specificities were determined. Two serotype-specific clones recognized only dengue virus type 3. Two dengue virus subcomplex-specific clones recognized dengue virus types 2, 3, and 4, and one subcomplex-specific clone re...

  10. 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...

  11. Antigen-specific cytotoxic T lymphocytes target airway CD103+ and CD11b+ dendritic cells to suppress allergic inflammation.

    Science.gov (United States)

    Daniels, N J; Hyde, E; Ghosh, S; Seo, K; Price, K M; Hoshino, K; Kaisho, T; Okada, T; Ronchese, F

    2016-01-01

    Allergic airway inflammation is driven by the recognition of inhaled allergen by T helper type 2 (Th2) cells in the airway and lung. Allergen-specific cytotoxic T lymphocytes (CTLs) can strongly reduce airway inflammation, however, the mechanism of their inhibitory activity is not fully defined. We used mouse models to show that allergen-specific CTLs reduced early cytokine production by Th2 cells in lung, and their subsequent accumulation and production of interleukin (IL)-4 and IL-13. In addition, treatment with specific CTLs also increased the proportion of caspase(+) dendritic cells (DCs) in mediastinal lymph node (MLN), and decreased the numbers of CD103(+) and CD11b(+) DCs in the lung. This decrease required expression of the cytotoxic mediator perforin in CTLs and of the appropriate MHC-antigen ligand on DCs, suggesting that direct CTL-DC contact was necessary. Lastly, lung imaging experiments revealed that in airway-challenged mice XCR1-GFP(+) DCs, corresponding to the CD103(+) DC subset, and XCR1-GFP(-) CD11c(+) cells, which include CD11b(+) DCs and alveolar macrophages, both clustered in the areas surrounding the small airways and were closely associated with allergen-specific CTLs. Thus, allergen-specific CTLs reduce allergic airway inflammation by depleting CD103(+) and CD11b(+) DC populations in the lung, and may constitute a mechanism through which allergic immune responses are regulated.

  12. Cytotoxic Drug Dispersal, Cytotoxic Safety, and Cytotoxic Waste Management: Practices and Proposed India-specific Guidelines.

    Science.gov (United States)

    Capoor, Malini R; Bhowmik, Kumar Tapas

    2017-01-01

    This article deals with practices related to cytotoxic drug dispersal, cytotoxic safety, and cytotoxic waste management and attempts at India-specific guidelines for their dispersal and disposal. The articles related to cytotoxic drug dispersal, cytotoxic safety, and cytotoxic waste management were reviewed from PubMed and their applicability in Indian health-care facilities (HCFs) was also reviewed. All HCFs dealing with cytotoxic drugs should consider cytotoxic policy, patient safety and health-care worker safety, and environmental monitoring program as per the available international guidelines customized as per Indian conditions. Utmost care in handling cytotoxic waste is quintessential. The formation of India-specific cytotoxic guidelines requires the inputs from all stakeholders. Cytotoxic waste, cytotoxic safety, and cytotoxic waste management should be the subject of a national strategy with an infrastructure, cradle-to-grave legislation, competent regulatory authority, and trained personnel.

  13. Dendritic cells transduced with Rsf-1/HBXAP gene generate specific cytotoxic T lymphocytes against ovarian cancer in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Li [Department of Gynecology Oncology, Shan Dong Tumor Hospital, Jinan, Shandong (China); Department of Obstetrics and Gynecology, Qilu Hospital, Shandong University, Jinan, Shandong (China); Kong, Beihua, E-mail: kongbeihua@sdu.edu.cn [Department of Obstetrics and Gynecology, Qilu Hospital, Shandong University, Jinan, Shandong (China); Sheng, Xiugui [Department of Gynecology Oncology, Shan Dong Tumor Hospital, Jinan, Shandong (China); Sheu, Jim Jinn-Chyuan [Human Genetic Center, China Medical University Hospital and Graduate Institute of Chinese Medical Science, China Medical University, Taichung City, Taiwan (China); Shih, Ie-Ming [Departments of Pathology, Oncology, and Gynecology and Obstetrics, Johns Hopkins Medical Institutions, Baltimore, MD 21231 (United States)

    2010-04-09

    Recently, some studies have indicated that Rsf-1/HBXAP plays a role in chromatin remodeling and transcriptional regulation that may contribute to tumorigenesis in ovarian cancer. The present study demonstrates that using dendritic cells (DCs) from human cord blood CD34{sup +} cells transduced with Rsf-1/HBXAP DNA plasmids by nucleofection generate specific cytotoxic T lymphocytes (CTL) against ovarian cancer in vitro. After transfection, DCs were analyzed for Rsf-1/HBXAP mRNA expression by RT-PCR and protein expression by Western blot. Then the DC phenotypes, T-cell stimulatory capacity, endocytic activity and migration capacity were explored by flow cytometry analysis, allogeneic mixed lymphocyte reaction, endocytosis and transwell chemotaxis assay, respectively. After transfection, Rsf-1/HBXAP expression was detected at mRNA and protein levels. Allogeneic T-cell proliferation induced by transfected DCs was obviously higher than non-transfected DCs, but the endocytosis capacity and migratory ability were not different. Rsf-1/HBXAP gene-transduced DCs could induce antigen-specific CTL and generate a very potent cytotoxicity to OVCAR3 cells. These data suggest that Rsf-1/HBXAP gene-transduced DCs may be a potential adjuvant immunotherapy for ovarian cancer in clinical applications.

  14. Application of magnetic field hyperthermia and superparamagnetic iron oxide nanoparticles to HIV-1-specific T-cell cytotoxicity

    Directory of Open Access Journals (Sweden)

    Williams JP

    2013-07-01

    Full Text Available James P Williams,1 Paul Southern,2 Anya Lissina,3 Helen C Christian,4 Andrew K Sewell,3 Rodney Phillips,1,5,6 Quentin Pankhurst,2 John Frater1,5,6 1Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford, UK; 2Davy-Faraday Research Laboratory, Royal Institution of Great Britain, London, UK; 3Institute of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK; 4Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; 5James Martin 21st Century School, John Radcliffe Hospital, Oxford, UK; 6Oxford NIHR Biomedical Research Centre, Oxford, UK Abstract: The latent HIV-1 reservoir remains the major barrier to HIV-1 eradication. Although successful at limiting HIV replication, highly active antiretroviral therapy is unable to cure HIV infection, thus novel therapeutic strategies are needed to eliminate the virus. Magnetic field hyperthermia (MFH generates thermoablative cytotoxic temperatures in target-cell populations, and has delivered promising outcomes in animal models, as well as in several cancer clinical trials. MFH has been proposed as a strategy to improve the killing of HIV-infected cells and for targeting the HIV latent reservoirs. We wished to determine whether MFH could be used to enhance cytotoxic T-lymphocyte (CTL targeting of HIV-infected cells in a proof-of-concept study. Here, for the first time, we apply MFH to an infectious disease (HIV-1 using the superparamagnetic iron oxide nanoparticle FeraSpin R. We attempt to improve the cytotoxic potential of T-cell receptor-transfected HIV-specific CTLs using thermotherapy, and assess superparamagnetic iron oxide nanoparticle toxicity, uptake, and effect on cell function using more sensitive methods than previously described. FeraSpin R exhibited only limited toxicity, demonstrated efficient uptake and cell-surface attachment, and only modestly impacted T-cell function. In contrast to the cancer models, insufficient MFH was

  15. 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...

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

    Science.gov (United States)

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

    2010-09-01

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

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

    Science.gov (United States)

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

    2010-01-01

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

  18. Paraflagellar rod protein-specific CD8+ cytotoxic T lymphocytes target Trypanosoma cruzi-infected host cells.

    Science.gov (United States)

    Wrightsman, Ruth A; Luhrs, Keith A; Fouts, David; Manning, Jerry E

    2002-08-01

    Our previous studies show that in mice immunized with the paraflagellar rod (PFR) proteins of Trypanosoma cruzi protective immunity against this protozoan parasite requires MHC class I-restricted T cell function. To determine whether PFR-specific CD8+ T cell subsets are generated during T. cruzi infection, potential CTL targets in the PFR proteins were identified by scanning the amino acid sequences of the four PFR proteins for regions of 8-10 amino acids that conform to predicted MHC class I H-2b binding motifs. A subset of the peptide sequences identified were synthesized and tested as target antigen in 51Cr-release assays with effector cells from chronically infected T. cruzi mice. Short-term cytotoxic T lymphocyte (CTL) lines specific for two of the peptides, PFR-1(164-171) and PFR-3(123-130), showed high levels of lytic activity against peptide-pulsed target cells, secreted interferon (IFN)-gamma in response to parasite-infected target cells, and were found to be CD8+, CD4-, CD3+, TCRalphabeta+ cells of the Tc1 subset. Challenge of PFR immunized CD8-/- and perforin-deficient (PKO) mice confirmed that while CD8+ cells are required for survival of T. cruzi challenge infection, perforin activity is not required. Furthermore, while lytic activity of PFR-specific CD8+ T cell lines derived from PKO mice was severely impaired, the IFN-gamma levels secreted by CTLs from PKO mice were equivalent to that of normal mice, suggesting that the critical role played by CD8+ T cells in immunity to the parasite may be secretion of type 1 cytokines rather than lysis of parasite infected host cells.

  19. The cell transmembrane pH gradient in tumors enhances cytotoxicity of specific weak acid chemotherapeutics.

    Science.gov (United States)

    Kozin, S V; Shkarin, P; Gerweck, L E

    2001-06-15

    The extracellular pH is lower in tumor than in normal tissue, whereas their intracellular pH is similar. In this study, we show that the tumor-specific pH gradient may be exploited for the treatment of cancer by weak acid chemotherapeutics. i.v.-injected glucose substantially decreased the electrode estimated extracellular pH in a xenografted human tumor while its intracellular pH, evaluated by (31)P magnetic resonance spectroscopy, remained virtually unchanged. The resulting increase in the average cell pH gradient caused a parallel increase in tumor growth delay by the weak acid chlorambucil (CHL). Regardless of glucose administration, the effect of CHL was significantly greater in tumors preirradiated with a large dose of ionizing radiation. This suggests that CHL was especially pronounced in radioresistant hypoxic cells possessing a larger transmembrane pH gradient. These results indicate that the naturally occurring cell pH gradient difference between tumor and normal tissue is a major and exploitable determinant of the uptake of weak acids in the complex tumor microenvironment. The use of such drugs may be especially effective in combination with radiation.

  20. Development of a recombinant hCG-specific single chain immunotoxin cytotoxic to hCG expressing cancer cells.

    Science.gov (United States)

    Nand, Kripa N; Gupta, Jagdish C; Panda, A K; Jain, S K

    2015-02-01

    A large number of cancers express human chorionic gonadotropin (hCG) or its subunits ectopically. Patients harboring such cancers have poor prognosis and adverse survival. PiPP is a monoclonal antibody of high affinity and specificity for hCGβ/hCG. Work was carried out to develop a PiPP based recombinant immunotoxin for the immunotherapy of hCG expressing cancers. Recombinant PiPP antibody was constructed in scFv format in which gene encoding the VH and VL domains were joined through a linker. This scFv gene was fused to the gene expressing Pseudomonas exotoxin (PE38), and cloned in a Escherichia coli based expression vector under the control of strong bacteriophage T7 promoter. Immunotoxin conjugating scFv(PiPP) and PE38, was expressed in E. coli as recombinant protein. Recombinant PiPP immunotoxin was purified from the bacterial cell lysate and tested for binding and killing of hCGβ expressing lymphoma, T-lymphoblastic leukemia and lung carcinoma cells in vitro. Immunotoxin showed nearly 90% killing on the cells. This is the first ever report on recombinant immunotoxin for binding and cytotoxicity to hCG expressing cancer cells, and thus can be a potential candidate for the immunotherapy of hCG expressing cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Frankincense oil derived from Boswellia carteri induces tumor cell specific cytotoxicity

    Directory of Open Access Journals (Sweden)

    Ashley Richard A

    2009-03-01

    Full Text Available Abstract Background Originating from Africa, India, and the Middle East, frankincense oil has been important both socially and economically as an ingredient in incense and perfumes for thousands of years. Frankincense oil is prepared from aromatic hardened gum resins obtained by tapping Boswellia trees. One of the main components of frankincense oil is boswellic acid, a component known to have anti-neoplastic properties. The goal of this study was to evaluate frankincense oil for its anti-tumor activity and signaling pathways in bladder cancer cells. Methods Frankincense oil-induced cell viability was investigated in human bladder cancer J82 cells and immortalized normal bladder urothelial UROtsa cells. Temporal regulation of frankincense oil-activated gene expression in bladder cancer cells was identified by microarray and bioinformatics analysis. Results Within a range of concentration, frankincense oil suppressed cell viability in bladder transitional carcinoma J82 cells but not in UROtsa cells. Comprehensive gene expression analysis confirmed that frankincense oil activates genes that are responsible for cell cycle arrest, cell growth suppression, and apoptosis in J82 cells. However, frankincense oil-induced cell death in J82 cells did not result in DNA fragmentation, a hallmark of apoptosis. Conclusion Frankincense oil appears to distinguish cancerous from normal bladder cells and suppress cancer cell viability. Microarray and bioinformatics analysis proposed multiple pathways that can be activated by frankincense oil to induce bladder cancer cell death. Frankincense oil might represent an alternative intravesical agent for bladder cancer treatment.

  2. Artificial antigen-presenting cells expressing AFP(158-166) peptide and interleukin-15 activate AFP-specific cytotoxic T lymphocytes.

    Science.gov (United States)

    Sun, Longhao; Guo, Hao; Jiang, Ruoyu; Lu, Li; Liu, Tong; Zhang, Zhixiang; He, Xianghui

    2016-04-05

    Professional antigen-presenting cells (APCs) are potent generators of tumor antigen-specific cytotoxic T lymphocytes (CTLs) for adoptive immunotherapy; however, generation of APCs is cumbersome, expensive, and subject to the tumor microenvironment. Artificial APCs (aAPCs) have been developed as a cost-effective alternative to APCs. We developed a cellular aAPC that efficiently generated alpha-fetoprotein (AFP)-specific CTLs. We genetically modified the human B cell lymphoma cell line BJAB with a lentiviral vector to establish an aAPC called BA15. The expression of AFP(158-166)-HLA-A*02:01 complex, CD80, CD86, and interleukin (IL)-15 in BA15 cells was assessed. The efficiency of BA15 at generating AFP-specific CTLs and the specific cytotoxicity of CTLs against AFP+ cells were also determined. BA15 cells expressed high levels of AFP(158-166) peptide, HLA-A2, CD80, CD86, and IL-15. BA15 cells also exhibited higher efficiency in generating AFP-specific CTLs than did dendritic cells. These CTLs had greater cytotoxicity against AFP+ hepatocellular carcinoma cells than did CTLs obtained from dendritic cells in vitro and in vivo. Our novel aAPC system could provide a robust platform for the generation of functional AFP-specific CTLs for adoptive immunotherapy of hepatocellular carcinoma.

  3. Low dose decitabine treatment induces CD80 expression in cancer cells and stimulates tumor specific cytotoxic T lymphocyte responses.

    Science.gov (United States)

    Wang, Li-Xin; Mei, Zhen-Yang; Zhou, Ji-Hao; Yao, Yu-Shi; Li, Yong-Hui; Xu, Yi-Han; Li, Jing-Xin; Gao, Xiao-Ning; Zhou, Min-Hang; Jiang, Meng-Meng; Gao, Li; Ding, Yi; Lu, Xue-Chun; Shi, Jin-Long; Luo, Xu-Feng; Wang, Jia; Wang, Li-Li; Qu, Chunfeng; Bai, Xue-Feng; Yu, Li

    2013-01-01

    Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC), a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL) response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight) once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8(+), but not CD4(+) T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy.

  4. Low dose decitabine treatment induces CD80 expression in cancer cells and stimulates tumor specific cytotoxic T lymphocyte responses.

    Directory of Open Access Journals (Sweden)

    Li-Xin Wang

    Full Text Available Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC, a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS, acute myeloid leukemia (AML and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8(+, but not CD4(+ T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy.

  5. Induction of leukemia-specific cytotoxic response by cross-presentation of late-apoptotic leukemic blasts by autologous dendritic cells of nonleukemic origin.

    Science.gov (United States)

    Spisek, Radek; Chevallier, Patrice; Morineau, Nadine; Milpied, Noël; Avet-Loiseau, Herve; Harousseau, Jean-Luc; Meflah, Khaled; Gregoire, Marc

    2002-05-15

    Acute myeloid leukemias (AMLs) are monoclonal proliferations of undifferentiated myeloid progenitors in blood and bone marrow. Long-term remissions are achieved in AML leukemic blasts can acquire the morphology and phenotype of dendritic cells (DCs), i.e., differentiate into leukemic DCs. However, this method has limitations as a potential immunotherapy. The alternative approach for the induction of leukemia-specific cytotoxicity we explored in this study consisted of using DCs of nonleukemic origin, pulsed with autologous apoptotic leukemic blasts. We show that mature pulsed nonleukemic DCs were successfully generated from remission samples of all tested patients with minimal interindividual differences. Mature pulsed DCs were used as antigen-presenting cells for leukemia-specific CTL induction. Specific cytotoxic activity against autologous AML blasts was demonstrated. Tumor lysis was autologous blast specific, with no killing activity against allogeneic leukemic cells or autologous mature unpulsed DCs and was MHC class I and class II restricted. In one patient, autologous CTLs stimulated by leukemic DCs or pulsed nonleukemic DCs showed similar significant cytotoxic activity against autologous AML cells. These findings demonstrate the induction of leukemia-specific cytotoxic response by nonleukemic mature DCs cross-presenting apoptotic leukemic blasts and offer a complementary approach to the use of leukemic DCs. We believe that this strategy permits the generation of DC vaccines for the majority of patients with hematological malignancies.

  6. Induction of leukemia-specific CD8+ cytotoxic T cells with autologous myeloid leukemic cells maturated with a fiber-modified adenovirus encoding TNF-alpha.

    Science.gov (United States)

    Saudemont, Aurore; Corm, Selim; Wickham, Thomas; Hetuin, Dominique; Quesnel, Bruno

    2005-06-01

    Acute myeloid leukemia (AML) cells can be differentiated into dendritic cells (DCs) using appropriate combinations of cytokines but generation of autologous antileukemic cytotoxic T cells using leukemic DCs remains difficult. Transduction by adenoviral vectors has been reported to induce efficient maturation of monocyte-derived DCs but AML cells are generally resistant to adenoviral gene transfer. In this study we tested the effects of adenoviral TNF-alpha gene transfer on maturation of AML cells using the fiber-modified AdTNF.F(pK7) adenovirus. All samples expressed high and sustained levels of TNF-alpha following transduction. AdTNF.F(pK7) induced significantly greater maturation of AML cells into antigen-presenting cells (APC) than did recombinant TNF-alpha or control adenoviral vector. Maturation of leukemic cells into APCs was mediated at least partially via a PI3K/mTOR pathway, as the inhibitors LY294002, wortmannin, and rapamycin inhibited the maturation effect induced by the AdTNF.F(pK7) adenovirus. In addition, CD8+ T cells expanded with AdTNF.F(pK7)-transduced AML cells showed greater expansion and specific CD8+ CTL activity against autologous AML cells than T cells expanded by other means. Thus, fiber-modified adenoviral vectors encoding TNF-alpha are able to maturate AML cells into APCs with high efficacy and reproducibility, providing a useful tool to generate efficiently specific CD8+ CTLs against leukemic disease.

  7. Recombinant mammaglobin A adenovirus-infected dendritic cells induce mammaglobin A-specific CD8+ cytotoxic T lymphocytes against breast cancer cells in vitro.

    Directory of Open Access Journals (Sweden)

    Huixia Cui

    Full Text Available Mammaglobin A (MGBA is a novel breast cancer-associated antigen almost exclusively over-expressed in primary and metastatic human breast cancers, making it a potential therapeutic target for breast cancer. The development of dendritic cell (DC-induced tumor antigen specific CD8(+ cytotoxic T lymphocytes (CTLs may hold promise in cancer immunotherapy. In this study we constructed recombinant replication-defective adenoviral (Ad vectors encoding MGBA and evaluated their ability to trigger anti-tumor immunity in vitro. DCs were isolated from the human peripheral blood monocyte cells (PBMCs of two HLA-A33(+ healthy female volunteers, and infected with adenovirus carrying MGBA cDNA (Ad-MGBA. After that, the Ad-MGBA-infected DCs were used to stimulate CD8(+ CTLs in vitro and the latter was used for co-culture with breast cancer cell lines. The data revealed that infection with Ad-MGBA improved DC maturation and up-regulated the expression of co-stimulatory molecules and the secretion of interleukin-12 (IL-12, but down-regulated interleukin-10 (IL-10 secretion from DCs. Ad-MGBA-infected DC-stimulated CD8(+CTLs displayed the highest cytotoxicity towards HLA-A33(+/MGBA(+ breast cancer MDA-MB-415 cells compared with other CD8(+CTL populations, and compared with the cytotoxicity towards HLA-A33(-/MGBA(+ breast cancer HBL-100 cells and HLA-A33(-/MGBA(- breast cancer MDA-MB 231 cells. In addition, Ad-MGBA-infected DC-stimulated CD8(+ CTLs showed a high level of IFNγ secretion when stimulated with HLA-A33(+/MGBA(+ breast cancer MDA-MB-415 cells, but not when stimulated with HLA-A33(-/MGBA(+ HBL-100 and HLA-A33(-/MGBA(-MDA-MB-231 cells. In addition, killing of CD8(+CTLs against breast cancer was in a major histocompability complex (MHC-limited pattern. Finally, the data also determined the importance of TNF-α in activating DCs and T cells. These data together suggest that MGBA recombinant adenovirus-infected DCs could induce specific anti-tumor immunity

  8. Artificial antigen-presenting cells plus IL-15 and IL-21 efficiently induce melanoma-specific cytotoxic CD8+CD28+ T lymphocyte responses

    Institute of Scientific and Technical Information of China (English)

    Xia Yu; Yuan Fang; Xi Li; Nuo Zhou; Yong-Xiang Zhao; Xiao-Ling Lu; Jian He; Sodaly Mongkhoune; Yi Peng; Yuan Xie; Jing Su; Su-Fang Zhou; Xiao-Xun Xie; Guo-Rong Luo

    2013-01-01

    To develop a novel artificial antigen-presenting system for efficiently inducing melanoma-specific CD8+CD28+ cytotoxic T lymphocyte (CTL) responses. Methods: Cell-sized Dynabeads® M-450 Epoxy beads coated with H-2Kb:Ig-TRP2180-188 and anti-CD28 antibody were used as artificial antigen-presenting cells (aAPCs) to induce melanoma-specific CD8+CD28+CTL responses with the help of IL-21 and IL-15. Dimer staining, proliferation, ELISPOT, and cytotoxicity experiments were conducted to evaluate the frequency and activity of induced CTLs. Results: Dimer staining demonstrated that the new artificial antigen-presenting system efficiently induced melanoma TRP2-specific CD8+CD28+ CTLs. Proliferation and ELISPOT assays indicated that the induced CTLs rapidly proliferate and produce increased IFN-γ under the stimulation of H-2Kb:Ig-TRP2-aAPCs, IL-15, and IL-21. In addition, cytotoxicity experiments showed that induced CTLs have specific killing activity of target cells. Conclusions: The new artificial antigen-presenting system including aAPCs plus IL-21 and IL-15 can induce a large number of antigen-specific CD8+CD28+ CTLs against the melanoma. Our study provides evidence for a novel adoptive immunotherapy against tumors.

  9. DNAM-1 mediates epithelial cell-specific cytotoxicity of aberrant intraepithelial lymphocyte lines from refractory celiac disease type II patients.

    Science.gov (United States)

    Tjon, Jennifer M-L; Kooy-Winkelaar, Yvonne M C; Tack, Greetje J; Mommaas, A Mieke; Schreurs, Marco W J; Schilham, Marco W; Mulder, Chris J; van Bergen, Jeroen; Koning, Frits

    2011-06-01

    In refractory celiac disease (RCD), intestinal epithelial damage persists despite a gluten-free diet. Characteristic for RCD type II (RCD II) is the presence of aberrant surface TCR-CD3(-) intraepithelial lymphocytes (IELs) that can progressively replace normal IELs and eventually give rise to overt lymphoma. Therefore, RCD II is considered a malignant condition that forms an intermediate stage between celiac disease (CD) and overt lymphoma. We demonstrate in this study that surface TCR-CD3(-) IEL lines isolated from three RCD II patients preferentially lyse epithelial cell lines. FACS analysis revealed that DNAM-1 was strongly expressed on the three RCD cell lines, whereas other activating NK cell receptors were not expressed on all three RCD cell lines. Consistent with this finding, cytotoxicity of the RCD cell lines was mediated mainly by DNAM-1 with only a minor role for other activating NK cell receptors. Furthermore, enterocytes isolated from duodenal biopsies expressed DNAM-1 ligands and were lysed by the RCD cell lines ex vivo. Although DNAM-1 on CD8(+) T cells and NK cells is known to mediate lysis of tumor cells, this study provides, to our knowledge, the first evidence that (pre)malignant cells themselves can acquire the ability to lyse epithelial cells via DNAM-1. This study confirms previous work on epithelial lysis by RCD cell lines and identifies a novel mechanism that potentially contributes to the gluten-independent tissue damage in RCD II and RCD-associated lymphoma.

  10. Stimulation by means of dendritic cells followed by Epstein-Barr virus-transformed B cells as antigen-presenting cells is more efficient than dendritic cells alone in inducing Aspergillus f16-specific cytotoxic T cell responses.

    Science.gov (United States)

    Zhu, F; Ramadan, G; Davies, B; Margolis, D A; Keever-Taylor, C A

    2008-02-01

    Adoptive immunotherapy with in vitro expanded antigen-specific cytotoxic T lymphocytes (CTLs) may be an effective approach to prevent, or even treat, Aspergillus (Asp) infections. Such lines can be generated using monocyte-derived dendritic cells (DC) as antigen-presenting cells (APC) but requires a relatively high volume of starting blood. Here we describe a method that generates Asp-specific CTL responses more efficiently using a protocol of antigen presented on DC followed by Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (BLCL) as APC. Peripheral blood mononuclear cells were stimulated weekly (2-5x) with a complete pool of pentadecapeptides (PPC) spanning the coding region of Asp f16 pulsed onto autologous mature DC. Cultures were split and stimulated subsequently with either PPC-DC or autologous PPC-pulsed BLCL (PPC-BLCL). Lines from the DC/BLCL arm demonstrated Asp f16-specific cytotoxicity earlier and to a higher degree than lines generated with PPC-DC alone. The DC/BLCL-primed lines showed a higher frequency of Asp f16-specific interferon (IFN)-gamma producing cells but an identical effector cell phenotype and peptide specificity compared to PPC-DC-only-primed lines. Tumour necrosis factor (TNF)-alpha, but not IL-10, appeared to play a role in the effectiveness of BLCL as APC. These results demonstrate that BLCL serve as highly effective APC for the stimulation of Asp f16-specific T cell responses and that a culture approach using initial priming with PPC-DC followed by PPC-BLCL may be a more effective method to generate Asp f16-specific T cell lines and requires less starting blood than priming with PPC-DC alone.

  11. Myeloid leukemic progenitor cells can be specifically targeted by minor histocompatibility antigen LRH-1-reactive cytotoxic T cells.

    NARCIS (Netherlands)

    Norde, W.J.; Overes, I.M.; Maas, F.M.H.M.; Fredrix, J.M.; Vos, A.; Kester, M.G.; Voort, R. van der; Jedema, I.; Falkenburg, J.H.F.; Schattenberg, A.V.M.B.; Witte, T.J.M. de; Dolstra, H.

    2009-01-01

    CD8(+) T cells recognizing minor histocompatibility antigens (MiHAs) on leukemic stem and progenitor cells play a pivotal role in effective graft-versus-leukemia reactivity after allogeneic stem cell transplantation (SCT). Previously, we identified a hematopoiesis-restricted MiHA, designated LRH-1,

  12. Epstein-Barr virus-transformed B-cells as efficient antigen presenting cells to propagate Aspergillus-specific cytotoxic T-lymphocytes.

    Science.gov (United States)

    Ramadan, Gamal

    2008-01-01

    To overcome the cytotoxic T-lymphocytes (CTL) expansion limitations imposed by the lack of sufficient dendritic cells (DC) alternative sources of autologous antigen presenting cells (APC) such as Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (BLCL), which are easy to establish in vitro, have been considered and studied in the present work. Non-adherent peripheral blood mononuclear cells of three healthy donors were repeatedly primed with autologous Aspergillus fumigatus commercial culture-filtrate antigen-pulsed fast monocyte-derived DC (Aspf-CFA-DC) alone, Aspf-CFA-pulsed BLCL (Aspf-CFA-BLCL) alone or Aspf-CFA-BLCL after one, two, or three primings with Aspf-CFA-DC (1DC/BLCL, 2DC/BLCL or 3DCIBLCL; respectively). After 5th priming, lines generated by Aspf-CFA-BLCL only showed strong/weak lytic activity for EBV/Aspf; respectively. Aspf-specific lytic activity in all donors was increased by increasing the number of primings with Aspf-CFA-DC before switching to Aspf-CFA-BLCL (18.20 +/- 1.65% versus 35.67 +/- 1.02% and 40.03 +/- 1.41% in bulk cultures generated by 1DC/BLCL versus 2DC/BLCL and 3DC/BLCL, respectively). Bulk cultures generated by Aspf-CFA-BLCL after at least two primings with Aspf-CFA-DC showed approximately the same Aspf-specific lytic activity, effector cell phenotype, expansion level and percentage expression of IFN-gamma, CD69 and CD107a without any significant differences (p > 0.05) as standard bulk cultures generated by only Aspf-CFA-DC. Thus, this study explored the use of a combined DC/BLCL protocol to establish/propagate Aspf-specific CTL for adoptive immunotherapy to prevent or treat invasive pulmonary aspergillosis.

  13. Generation of cytotoxic T lymphocytes specific for B-cell acute lymphoblastic leukemia family-shared peptides derived from immunoglobulin heavy chain framework region

    Institute of Scientific and Technical Information of China (English)

    LIU Ying; ZHU Ping; HU Ya-mei

    2007-01-01

    Background Immunoglobulin heavy chain variable region (IgHV) is a well-characterized tumor antigen for B-cell malignancies. It can function as a target for T cell-mediated immune response. Clinical trials of IgHV protein vaccines against lymphoma have demonstrated induction of tumor-specific cytotoxic T lymphocyte (CTL) responses. However,complementary determining regions-based individual vaccines have disadvantages for wide clinical application. Although a recent study demonstrated that immunogenic peptides are derived from framework regions (FR) shared among patients with B-cell lymphoma, how to choose the appropriate peptides for each patient is still unsolved. The aim of this study was to investigate whether immunoglobulin heavy chain FR-derived peptides shared in each IgHV family are potential CTL epitopes presented by B-cell acute lymphoblastic leukemia (B-ALL). Such CTL epitopes might be beneficial to shifting vaccination strategies against B-ALL from individual specificity to family specificity.Methods Seven IgHV gene families were amplified respectively by PCR and sequenced directly from 71 childhood B-ALL cases. Bioinformatics was applied in analyzing characteristics of sequences available and predicting HLA-A*0201-restricted CTL epitopes for each IgHV family. An antigen-specific T cell expansion system was used to generate peptide-specific CTLs. The cytotoxicity of CTLs against B-ALL cells was assessed in the lactate dehydrogenase release assay.Results Complete IgHV rearrangements were identified in all of the 71 B-ALL cases. All of 40 sequences available showed ≥98% homology with the nearest germline IgHV genes, indicating IgHV genes in B-ALL of germline nature.Twelve nonapeptides of high HLA-A*0201-binding scores were obtained from 26 productive IgHV protein sequences. Ten (83%) of the peptides were located in FR1 and FR3 shared among the corresponding IgHV family. CTLs specific for the peptide QLVQSGAEV located in FR1 (3-11) shared among the IgHV1

  14. Persistent hepatitis C virus (HCV) infection impairs HCV-specific cytotoxic T cell reactivity through Mcl-1/Bim imbalance due to CD127 down-regulation.

    Science.gov (United States)

    Larrubia, J R; Lokhande, M U; García-Garzón, S; Miquel, J; González-Praetorius, A; Parra-Cid, T; Sanz-de-Villalobos, E

    2013-02-01

    In persistent hepatitis C virus (HCV) infection, HCV-specific cytotoxic T lymphocyte (CTL) reactivity is impaired and this affects HCV control. Interleukin-7 receptor (CD127) expression on these cells could regulate CTL reactivity through Mcl-1/Bim balance modulation. Bim is a pro-apoptotic molecule blocked by the action of Mcl-1. Mcl-1/Bim expression and T cell reactivity on HCV-specific CTLs were compared according to CD127 phenotype. Peripheral blood lymphocytes (PBL) from HLA-A2(+) HCV(+) patients were obtained. HCV-specific CTLs were visualized by staining PBL with anti-CD8 and HLA-A2/peptide pentameric complexes (pentamer). Mcl-1/Bim/CD127 phenotype of HCV-specific CTLs was tested by staining detectable CD8(+)/pentamer(+) cells with anti-Mcl-1/Bim/CD127 antibodies. HCV-specific CTL proliferation ability after specific in vitro challenge was tested in the presence and absence of pancaspase inhibitor z-VAD-fmk. All stained cells were analysed by flow cytometry. CD127(low)-expressing HCV-specific CTLs associated with high HCV viraemia, while CD127(high) correlated with undetectable viral loads (P Bim was up-regulated after antigen encounter (P Bim expression on pentamer(+) cells correlated positively with CD127 expression level (P Bim up-regulation after antigen encounter are involved in CD127(low) HCV-specific CTL hyporeactivity during chronic infection, but it can be overcome by apoptosis blockade.

  15. In vitro priming of tumor-specific cytotoxic T lymphocytes using allogeneic dendritic cells derived from the human MUTZ-3 cell line.

    Science.gov (United States)

    Santegoets, Saskia J A M; Schreurs, Marco W J; Masterson, Allan J; Liu, Ying Poi; Goletz, Steffen; Baumeister, Hans; Kueter, Esther W M; Lougheed, Sinéad M; van den Eertwegh, Alfons J M; Scheper, Rik J; Hooijberg, Erik; de Gruijl, Tanja D

    2006-12-01

    The adoptive transfer of in vitro-induced and expanded tumor-specific cytotoxic T lymphocytes (CTL) presents a promising immunotherapeutic approach for the treatment of cancer. The in vitro induction of tumor-reactive CTL requires repeated stimulation of CTL precursors with dendritic cells (DC). To circumvent problems like scarcity of blood DC precursors and donor variability, it would be attractive to use DC from a non-autologous, unlimited source. DCs derived from the human acute myeloid leukemia (AML) cell line MUTZ-3 are attractive candidates since these DCs closely resemble monocyte-derived DC (MoDC) in terms of phenotype and T cell stimulatory capacity. Here we demonstrate that functional CTL clones could be generated against multiple tumor-associated antigens, i.e., human telomerase reverse transcriptase (hTERT), ErbB3-binding protein-1 (Ebp1), carcinoembryonic antigen (CEA) and Her-2/neu, by stimulating CD8beta(+) CTL precursors with peptide-loaded allogeneic, HLA-A2-matched MUTZ-3-derived DC. A consistent induction capacity, as determined by MHC tetramer-binding, was found in multiple donors and comparable to autologous peptide-loaded MoDC. Functional characterization at the clonal level revealed the priming of CTL that recognized endogenously processed epitopes on tumor cell lines in an HLA-A2-restricted fashion. Our data indicate that MUTZ-3-derived DC can be used as stimulator cells for in vitro priming and expansion of functional TAA-specific effector CTL. MUTZ-3-derived DCs thus represent a ready and standardized source of allogeneic DC to generate CTL for therapeutic adoptive transfer strategies.

  16. Specific Colon Cancer Cell Cytotoxicity Induced by Bacteriophage E Gene Expression under Transcriptional Control of Carcinoembryonic Antigen Promoter

    Directory of Open Access Journals (Sweden)

    Ana R. Rama

    2015-06-01

    Full Text Available Colorectal cancer is one of the most prevalent cancers in the world. Patients in advanced stages often develop metastases that require chemotherapy and usually show a poor response, have a low survival rate and develop considerable toxicity with adverse symptoms. Gene therapy may act as an adjuvant therapy in attempts to destroy the tumor without affecting normal host tissue. The bacteriophage E gene has demonstrated significant antitumor activity in several cancers, but without any tumor-specific activity. The use of tumor-specific promoters may help to direct the expression of therapeutic genes so they act against specific cancer cells. We used the carcinoembryonic antigen promoter (CEA to direct E gene expression (pCEA-E towards colon cancer cells. pCEA-E induced a high cell growth inhibition of human HTC-116 colon adenocarcinoma and mouse MC-38 colon cancer cells in comparison to normal human CCD18co colon cells, which have practically undetectable levels of CEA. In addition, in vivo analyses of mice bearing tumors induced using MC-38 cells showed a significant decrease in tumor volume after pCEA-E treatment and a low level of Ki-67 in relation to untreated tumors. These results suggest that the CEA promoter is an excellent candidate for directing E gene expression specifically toward colon cancer cells.

  17. HIV-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC) -Mediating Antibodies Decline while NK Cell Function Increases during Antiretroviral Therapy (ART).

    Science.gov (United States)

    Jensen, Sanne Skov; Fomsgaard, Anders; Borggren, Marie; Tingstedt, Jeanette Linnea; Gerstoft, Jan; Kronborg, Gitte; Rasmussen, Line Dahlerup; Pedersen, Court; Karlsson, Ingrid

    2015-01-01

    Understanding alterations in HIV-specific immune responses during antiretroviral therapy (ART), such as antibody-dependent cellular cytotoxicity (ADCC), is important in the development of novel strategies to control HIV-1 infection. This study included 53 HIV-1 positive individuals. We evaluated the ability of effector cells and antibodies to mediate ADCC separately and in combination using the ADCC-PanToxiLux assay. The ability of the peripheral blood mononuclear cells (PBMCs) to mediate ADCC was significantly higher in individuals who had been treated with ART before seroconversion, compared to the individuals initiating ART at a low CD4+ T cell count (ART-naïve individuals. The frequency of CD16 expressing natural killer (NK) cells correlated with both the duration of ART and Granzyme B (GzB) activity. In contrast, the plasma titer of antibodies mediating ADCC declined during ART. These findings suggest improved cytotoxic function of the NK cells if initiating ART early during infection, while the levels of ADCC mediating antibodies declined during ART.

  18. ssDNA Aptamer Specifically Targets and Selectively Delivers Cytotoxic Drug Doxorubicin to HepG2 Cells.

    Directory of Open Access Journals (Sweden)

    Ge Yu

    Full Text Available Hepatocellular carcinoma (HCC is the third leading cause of death due to cancer worldwide with over 500,000 people affected annually. Although chemotherapy has been widely used to treat patients with HCC, alternate modalities to specifically deliver therapeutic cargos to cancer cells have been sought in recent years due to the severe side effects of chemotherapy. In this respect, aptamer-based tumor targeted drug delivery has emerged as a promising approach to increase the efficacy of chemotherapy and reduce or eliminate drug toxicity. In this study, we developed a new HepG2-specific aptamer (HCA#3 by a procedure known as systematic evolution of ligands by exponential enrichment (SELEX and exploited its role as a targeting ligand to deliver doxorubicin (Dox to HepG2 cells in vitro. The selected 76-base nucleotide aptamer preferentially bound to HepG2 hepatocellular carcinoma cells but not to control cells. The aptamer HCA#3 was modified with paired CG repeats at the 5'-end to carry and deliver a high payload of intercalated Dox molecules at the CG sites. Four Dox molecules (mol/mol were fully intercalated in each conjugate aptamer-Dox (ApDC molecule. Biostability analysis showed that the ApDC molecules are stable in serum. Functional analysis showed that ApDC specifically targeted and released Dox within HepG2 cells but not in control cells, and treatment with HCA#3 ApDC induced HepG2 cell apoptosis but had minimal effect on control cells. Our study demonstrated that HCA#3 ApDC is a promising aptamer-targeted therapeutic that can specifically deliver and release a high doxorubicin payload in HCC cells.

  19. CERT depletion predicts chemotherapy benefit and mediates cytotoxic and polyploid‐specific cancer cell death through autophagy induction

    DEFF Research Database (Denmark)

    Lee, Alvin J. X.; Roylance, Rebecca; Sander, Jil

    2012-01-01

    Chromosomal instability (CIN) has been implicated in multidrug resistance and the silencing of the ceramide transporter, CERT, promotes sensitization to diverse cytotoxics. An improved understanding of mechanisms governing multidrug sensitization might provide insight into pathways contributing...

  20. Identification of swine influenza virus epitopes and analysis of multiple specificities expressed by cytotoxic T cell subsets

    DEFF Research Database (Denmark)

    Pedersen, Lasse Eggers; Breum, Solvej Østergaard; Riber, Ulla

    2014-01-01

    to elimination of viruses such as swine influenza virus (SwIV). This study describes the identification of SLA-presented peptide epitopes that are targets for a swine CTL response, and further analyses multiple specificities expressed by SwIV activated CTL subsets. Findings: Four SwIV derived peptides were...... identified as T cell epitopes using fluorescent influenza: SLA tetramers. In addition, multiple CTL specificities were analyzed using peptide sequence substitutions in two of the four epitope candidates analyzed. Interestingly both conserved and substituted peptides were found to stain the CD4-CD8+ T cell...

  1. Induction/Engineering, Detection, Selection, and Expansion of Clinical-Grade Human Antigen-Specific CD8+ Cytotoxic T Cell Clones for Adoptive Immunotherapy

    Directory of Open Access Journals (Sweden)

    Matjaž Jeras

    2010-01-01

    Full Text Available Adoptive transfer of effector antigen-specific immune cells is becoming a promising treatment option in allogeneic transplantation, infectious diseases, cancer, and autoimmune disorders. Within this context, the important role of CD8+ cytotoxic T cells (CTLs is objective of intensive studies directed to their in vivo and ex vivo induction, detection, selection, expansion, and therapeutic effectiveness. Additional questions that are being addressed by the scientific community are related to the establishment and maintenance of their longevity and memory state as well as to defining critical conditions underlying their transitions between discrete, but functionally different subtypes. In this article we review and comment latest approaches and techniques used for preparing large amounts of antigen-specific CTLs, suitable for clinical use.

  2. Adeno-associated virus type 2 (AAV2) capsid-specific cytotoxic T lymphocytes eliminate only vector-transduced cells coexpressing the AAV2 capsid in vivo.

    Science.gov (United States)

    Li, Chengwen; Hirsch, Matthew; Asokan, Aravind; Zeithaml, Brian; Ma, Hong; Kafri, Tal; Samulski, R Jude

    2007-07-01

    A recent clinical trial has suggested that recombinant adeno-associated virus (rAAV) vector transduction in humans induces a cytotoxic T-lymphocyte (CTL) response against the AAV2 capsid. To directly address the ability of AAV capsid-specific CTLs to eliminate rAAV-transduced cells in vitro and in vivo in mice, we first demonstrated that AAV2 capsid-specific CTLs could be induced by dendritic cells with endogenous AAV2 capsid expression or pulsed with AAV2 vectors. These CTLs were able to kill a cell line stable for capsid expression in vitro and also in a mouse tumor xenograft model in vivo. Parent colon carcinoma (CT26) cells transduced with a large amount of AAV2 vectors in vitro were also destroyed by these CTLs. To determine the effect of CTLs on the elimination of target cells transduced by AAV2 vectors in vivo, we carried out adoptive transfer experiments. CTLs eliminated liver cells with endogenous AAV2 capsid expression but not liver cells transduced by AAV2 vectors, regardless of the reporter genes. Similar results were obtained for rAAV2 transduction in muscle. Our data strongly suggest that AAV vector-transduced cells are rarely eliminated by AAV2 capsid-specific CTLs in vivo, even though the AAV capsid can induce a CTL response. In conclusion, AAV capsid-specific CTLs do not appear to play a role in elimination of rAAV-transduced cells in a mouse model. In addition, our data suggest that the mouse model may not mimic the immune response noted in humans and additional modification to AAV vectors may be required for further study in order to elicit a similar cellular immune response.

  3. Adeno-Associated Virus Type 2 (AAV2) Capsid-Specific Cytotoxic T Lymphocytes Eliminate Only Vector-Transduced Cells Coexpressing the AAV2 Capsid In Vivo▿

    Science.gov (United States)

    Li, Chengwen; Hirsch, Matthew; Asokan, Aravind; Zeithaml, Brian; Ma, Hong; Kafri, Tal; Samulski, R. Jude

    2007-01-01

    A recent clinical trial has suggested that recombinant adeno-associated virus (rAAV) vector transduction in humans induces a cytotoxic T-lymphocyte (CTL) response against the AAV2 capsid. To directly address the ability of AAV capsid-specific CTLs to eliminate rAAV-transduced cells in vitro and in vivo in mice, we first demonstrated that AAV2 capsid-specific CTLs could be induced by dendritic cells with endogenous AAV2 capsid expression or pulsed with AAV2 vectors. These CTLs were able to kill a cell line stable for capsid expression in vitro and also in a mouse tumor xenograft model in vivo. Parent colon carcinoma (CT26) cells transduced with a large amount of AAV2 vectors in vitro were also destroyed by these CTLs. To determine the effect of CTLs on the elimination of target cells transduced by AAV2 vectors in vivo, we carried out adoptive transfer experiments. CTLs eliminated liver cells with endogenous AAV2 capsid expression but not liver cells transduced by AAV2 vectors, regardless of the reporter genes. Similar results were obtained for rAAV2 transduction in muscle. Our data strongly suggest that AAV vector-transduced cells are rarely eliminated by AAV2 capsid-specific CTLs in vivo, even though the AAV capsid can induce a CTL response. In conclusion, AAV capsid-specific CTLs do not appear to play a role in elimination of rAAV-transduced cells in a mouse model. In addition, our data suggest that the mouse model may not mimic the immune response noted in humans and additional modification to AAV vectors may be required for further study in order to elicit a similar cellular immune response. PMID:17475652

  4. In vitro cytotoxicity of {sup 211}At-labeled trastuzumab in human breast cancer cell lines: effect of specific activity and HER2 receptor heterogeneity on survival fraction

    Energy Technology Data Exchange (ETDEWEB)

    Akabani, Gamal [Department of Radiology, Duke University Medical Center, P.O. Box 3808, Durham, NC 27710 (United States); Carlin, Sean [Department of Radiology, Duke University Medical Center, P.O. Box 3808, Durham, NC 27710 (United States); Welsh, Phil [Department of Radiology, Duke University Medical Center, P.O. Box 3808, Durham, NC 27710 (United States); Zalutsky, Michael R. [Department of Radiology, Duke University Medical Center, P.O. Box 3808, Durham, NC 27710 (United States)]. E-mail: zalut001@mc.duke.edu

    2006-04-15

    Introduction: Radioimmunotherapy with anti-HER2 monoclonal antibodies (mAbs) such as trastuzumab is a promising strategy for treating HER2-positive breast and ovarian carcinoma patients. The objective of this study was to determine the cytotoxic effectiveness of trastuzumab labeled with the 7.2-h half-life {alpha}-particle emitter {sup 211}At. Methods: Experiments were performed on SKBr-3, BT-474 and the transfected MCF7/HER2-18 human breast carcinoma cell lines. Intrinsic radiosensitivity was determined after exposure to external beam irradiation. The cytotoxicity of {sup 211}At-labeled trastuzumab was measured by clonogenic assays. The distribution of HER2 receptor expression on the cell lines was measured using fluorescence-activated cell sorting. A pharmacokinetic (PK)/microdosimetric model was established to assess the effects of specific activity (SA), HER2 receptor expression and absorbed dose on survival fraction (SF). Results: With external beam irradiation, the 2-Gy SF for BT-474, SKBr-3 and MCF7/HER2-18 cells was 0.78, 0.53 and 0.64 Gy, respectively. Heterogeneous HER2 expression was observed, with a subpopulation of cells lacking measurable receptor (14.5%, SKBr-3; 0.34%, MCF-7/HER2; 1.73%, BT-474). When plotted as a function of activity concentration, SF curves were biphasic and inversely proportional to SA; however, when the model was applied and absorbed doses calculated, the SF curve was monoexponential independent of SA. Thus, the PK model was able to demonstrate the effects of competition between cold and labeled mAb. These studies showed that the relative biological effectiveness of {sup 211}At-labeled trastuzaumab was about 10 times higher than that of external beam therapy. Conclusion: These in vitro studies showed that {sup 211}At-labeled trastuzumab mAb is an effective cytotoxic agent for the treatment of HER2-positive tumor cells. The SA of the labeled mAb and the homogeneity of HER2 receptor expression are important variables influencing

  5. FastDC derived from human monocytes within 48 h effectively prime tumor antigen-specific cytotoxic T cells.

    Science.gov (United States)

    Dauer, Marc; Schad, Katharina; Herten, Jan; Junkmann, Jana; Bauer, Christian; Kiefl, Rosemarie; Endres, Stefan; Eigler, Andreas

    2005-07-01

    Previously, we have shown that dendritic cells (DCs) with full T-cell stimulatory capacity can be derived from human monocytes after 48 h of in vitro culture (FastDC). Compared to a standard 7-day protocol, this new strategy not only reduces the time span and the amount of recombinant cytokines required, but may also resemble DC development in vivo more closely. Using a melanoma antigen model, we show here that FastDC prime CTL responses against tumor antigens as effectively as standard monocyte-derived DCs (moDCs). FastDC and moDCs derived from monocytes of HLA-A2(+) donors were loaded with the melanoma-associated, HLA-A(*)0201-restricted peptide Melan-A and cocultured with autologous CD3(+) T cells. After two weekly restimulations with freshly prepared, peptide-loaded FastDC or moDCs, binding of CD8(+) T cells to fluorescently labeled MHC-I/Melan-A-peptide complexes and intracellular cytokine staining revealed that the two DC preparations had an equal capacity to prime Melan-A-specific, IFN-gamma producing CD8(+) T cells. CTLs derived from cocultures with FastDC lysed Melan-A-loaded T2 cells even more effectively than CTLs primed by moDCs. Comparative analysis also revealed that FastDC possess an equal capacity to migrate in response to the chemokine receptor CCR-7 ligand 6Ckine. Importantly, DCs can be generated with higher yield and purity using the FastDC-protocol. The reliability and efficacy of this new strategy for DC development from monocytes may facilitate clinical investigation of DC-based tumor immunotherapy.

  6. Cell-type-specific and differentiation-status-dependent variations in cytotoxicity of tributyltin in cultured rat cerebral neurons and astrocytes.

    Science.gov (United States)

    Oyanagi, Koshi; Tashiro, Tomoko; Negishi, Takayuki

    2015-08-01

    Tributyltin (TBT) is an organotin used as an anti-fouling agent for fishing nets and ships and it is a widespread environmental contaminant at present. There is an increasing concern about imperceptible but serious adverse effect(s) of exposure to chemicals existing in the environment on various organs and their physiological functions, e.g. brain and mental function. Here, so as to contribute to improvement of and/or advances in in vitro cell-based assay systems for evaluating brain-targeted adverse effect of chemicals, we tried to evaluate cell-type-specific and differentiation-status-dependent variations in the cytotoxicity of TBT towards neurons and astrocytes using the four culture systems differing in the relative abundance of these two types of cells; primary neuron culture (> 95% neurons), primary neuron-astrocyte (2 : 1) mix culture, primary astrocyte culture (> 95% astrocytes), and passaged astrocyte culture (100% proliferative astrocytes). Cell viability was measured at 48 hr after exposure to TBT in serum-free medium. IC50's of TBT were 198 nM in primary neuron culture, 288 nM in primary neuron-astrocyte mix culture, 2001 nM in primary astrocyte culture, and 1989 nM in passaged astrocyte culture. Furthermore, in primary neuron-astrocyte mix culture, vulnerability of neurons cultured along with astrocytes to TBT toxicity was lower than that of neurons cultured purely in primary neuron culture. On the other hand, astrocytes in primary neuron-astrocyte mix culture were considered to be more vulnerable to TBT than those in primary or passaged astrocyte culture. The present study demonstrated variable cytotoxicity of TBT in neural cells depending on the culture condition.

  7. Cytotoxic capacity of SIV-specific CD8(+ T cells against primary autologous targets correlates with immune control in SIV-infected rhesus macaques.

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    Daniel Mendoza

    2013-02-01

    Full Text Available Although the study of non-human primates has resulted in important advances for understanding HIV-specific immunity, a clear correlate of immune control over simian immunodeficiency virus (SIV replication has not been found to date. In this study, CD8(+ T-cell cytotoxic capacity was examined to determine whether this function is a correlate of immune control in the rhesus macaque (RM SIV infection model as has been suggested in chronic HIV infection. SIVmac251-infected human reverse transcriptase (hTERT-transduced CD4(+ T-cell clone targets were co-incubated with autologous macaque effector cells to measure infected CD4(+ T-cell elimination (ICE. Twenty-three SIV-infected rhesus macaques with widely varying plasma viral RNA levels were evaluated in a blinded fashion. Nineteen of 23 subjects (83% were correctly classified as long-term nonprogressor/elite controller (LTNP/EC, slow progressor, progressor or SIV-negative rhesus macaques based on measurements of ICE (weighted Kappa 0.75. LTNP/EC had higher median ICE than progressors (67.3% [22.0-91.7%] vs. 23.7% [0.0-58.0%], p = 0.002. In addition, significant correlations between ICE and viral load (r = -0.57, p = 0.01, and between granzyme B delivery and ICE (r = 0.89, p<0.001 were observed. Furthermore, the CD8(+ T cells of LTNP/EC exhibited higher per-cell cytotoxic capacity than those of progressors (p = 0.004. These findings support that greater lytic granule loading of virus-specific CD8(+ T cells and efficient delivery of active granzyme B to SIV-infected targets are associated with superior control of SIV infection in rhesus macaques, consistent with observations of HIV infection in humans. Therefore, such measurements appear to represent a correlate of control of viral replication in chronic SIV infection and their role as predictors of immunologic control in the vaccine setting should be evaluated.

  8. HPV16E7-HSP70 Hybrid DNA Vaccine Induces E7-Specific Cytotoxic T Cells and Antitumor Immunity

    Institute of Scientific and Technical Information of China (English)

    ZHU Liqin; LI Hui; XIONG Jinhu; WANG Tongxiang; OU Xuan; WEI Yun; WU Xinxing

    2006-01-01

    Using human papillomavirus type 16 (HPV16) E7 as an antigen and Heat Shock Protein 70 as adjuvant, we constructed a DNA vaccine by linking HSP70 gene to E7C91G gene. Mice, after being immunized with E7C91G-HSP70, E7C91G/HSP70, E7C91G, and wild E7 DNA vaccines respectively, produced E7 specific CD8+ T-cell precursor frequencies oF280. 33±2.52, 144.34±4. 04, 164.34±5.13 and 82.33± 3.51 respectively within every 1 × 105 mouse splenocytes. This proves that E7C91G-HSP70 fusion vaccine can significantly enhance the E7 specific cellular immunity within the mice body(p<0.01). After being immunized with E7C91G-HSP70 fusion vaccine, tumor-bearing mice of the group being treated have significantly longer latency and survival periods, comparing with other three categories of E7 vaccines. Experiment shows that this vaccine has a significant effect on enhancing E7 positive tumor-treatment within mice body. After being immunized with E7C91G-HSP70 vaccine, there were no pathological changes found in livers, kidneys and spleens of the mice, which proves that the vaccine is quite safe. After all,E7C91G-HSP70 fusion vaccine has a much stronger tumor- treatment effect than that of wild type E7 DNA vaccine.

  9. Identification of an MSI-H Tumor-Specific Cytotoxic T Cell Epitope Generated by the (−1 Frame of U79260(FTO

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    Michael Linnebacher

    2010-01-01

    Full Text Available Microsatellite instability (MSI-H induced by defects of the DNA mismatch repair system results in insertion or deletion of single nucleotides at short repetitive DNA sequences. About 15% of sporadic and approximately 90% of hereditary nonpolyposis colorectal cancers display MSI-H. When affecting coding regions, MSI-H results in frameshift mutations and expression of corresponding frameshift peptides (FSPs. Functional tumor promoting relevance has been demonstrated for a growing number of genes frequently hit by MSI-H. Contrary, immune reactions against FSPs are involved in the immune surveillance of MSI-H cancers. Here, we provide conclusive data that the (−1 frame of U79260(FTO encodes an HLA-A0201-restricted cytotoxic T cell epitope (FSP11; TLSPGWSAV. T cells specific for FSP11 efficiently recognized HLA-A0201(pos tumor cells harboring the mutated reading frame. Considering the exceptionally high mutation rate of U79260(FTO in MSI-H colorectal carcinoma (81.8%, this recommends that FSP11 be a component of future vaccines.

  10. NJK14013, a novel synthetic estrogen receptor-α agonist, exhibits estrogen receptor-independent, tumor cell-specific cytotoxicity.

    Science.gov (United States)

    Kim, Hye-In; Kim, Taelim; Kim, Ji-Eun; Lee, Jun; Heo, Jinyuk; Lee, Na-Rae; Kim, Nam-Jung; Inn, Kyung-Soo

    2015-07-01

    Estrogens act through interactions with estrogen receptors (ERs) to play diverse roles in various pathophysiological conditions. A number of synthetic selective estrogen receptor modulators (SERMs), such as tamoxifen and raloxifene, have been developed and used to treat ER-related diseases, including breast cancer and osteoporosis. Here, we identified a novel compound, bis(4-hydroxyphenyl)methanone-O-isopentyl oxime, designated NJK14013, as an ER agonist. NJK14013 activated ER-dependent transcription in a concentration-dependent manner, while suppressing androgen receptor-dependent transcriptional activity. It induced the activation-related phosphorylation of ER and enhanced the transcription of growth regulation by estrogen in breast cancer 1 (GREB1), further supporting its ER-stimulating activity. NJK14013 exerted anti-proliferative effects on various cancer cell lines, including an ER-negative breast cancer cell line, suggesting that it is capable of suppressing the growth of cancer cells independent of its ER-modulating activity. In addition, NJK14013 treatment resulted in significant apoptotic death of MCF7 and Ishikawa cancer cells, but did not induce apoptosis in non-cancer human umbilical vein endothelial cells. Collectively, our findings demonstrate that NJK14013 is a novel SERM that can activate ER-mediated transcription in MCF7 cells and suppress the proliferation of various cancer cells, including breast cancer cells and endometrial cancer cells. These results suggest that NJK14013 has potential as a novel SERM for anticancer or hormone-replacement therapy with reduced risk of carcinogenesis.

  11. Polysaccharopeptides derived from Coriolus versicolor potentiate the S-phase specific cytotoxicity of Camptothecin (CPT on human leukemia HL-60 cells

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    Jiang Pingping

    2010-04-01

    Full Text Available Abstract Background Polysaccharopeptide (PSP from Coriolus versicolor (Yunzhi is used as a supplementary cancer treatment in Asia. The present study aims to investigate whether PSP pre-treatment can increase the response of the human leukemia HL-60 cells to apoptosis induction by Camptothecin (CPT. Methods We used bivariate bromodeoxyuridine/propidium iodide (BrdUrd/PI flow cytometry analysis to measure the relative movement (RM of the BrdUrd positively labeled cells and DNA synthesis time (Ts on the HL-60 cell line. We used annexin V/PI flow cytometry analysis to quantify the viable, necrotic and apoptotic cells. The expression of cyclin E and cyclin B1 was determined with annexin V/PI flow cytometry and western blotting. Human peripheral blood mononuclear cells were used to test the cytotoxicity of PSP and CPT. Results PSP reduced cellular proliferation; inhibited cells progression through both S and G2 phase, reduced 3H-thymidine uptake and prolonged DNA synthesis time (Ts in HL-60 cells. PSP-pretreated cells enhanced the cytotoxicity of CPT. The sensitivity of cells to the cytotoxic effects of CPT was seen to be the highest in the S-phase and to a small extent of the G2 phase of the cell cycle. On the other hand, no cell death (measured by annexin V/PI was evident with the normal human peripheral blood mononuclear cells with treatment of either PSP or CPT. Conclusion The present study shows that PSP increases the sensitization of the HL-60 cells to undergo effective apoptotic cell death induced by CPT. The pattern of sensitivity of cancer cells is similar to that of HL-60 cells. PSP rapidly arrests and/or kills cells in S-phase and did not interfere with the anticancer action of CPT. PSP is a potential adjuvant to treat human leukemia as rapidly proliferating tumors is characterized by a high proportion of S-phase cells.

  12. Mechanism of Arctigenin-Induced Specific Cytotoxicity against Human Hepatocellular Carcinoma Cell Lines: Hep G2 and SMMC7721.

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    Zheng Lu

    Full Text Available Arctigenin (ARG has been previously reported to exert high biological activities including anti-inflammatory, antiviral and anticancer. In this study, the anti-tumor mechanism of ARG towards human hepatocellular carcinoma (HCC was firstly investigated. We demonstrated that ARG could induce apoptosis in Hep G2 and SMMC7721 cells but not in normal hepatic cells, and its apoptotic effect on Hep G2 was stronger than that on SMMC7721. Furthermore, the following study showed that ARG treatment led to a loss in the mitochondrial out membrane potential, up-regulation of Bax, down-regulation of Bcl-2, a release of cytochrome c, caspase-9 and caspase-3 activation and a cleavage of poly (ADP-ribose polymerase in both Hep G2 and SMMC7721 cells, suggesting ARG-induced apoptosis was associated with the mitochondria mediated pathway. Moreover, the activation of caspase-8 and the increased expression levels of Fas/FasL and TNF-α revealed that the Fas/FasL-related pathway was also involved in this process. Additionally, ARG induced apoptosis was accompanied by a deactivation of PI3K/p-Akt pathway, an accumulation of p53 protein and an inhibition of NF-κB nuclear translocation especially in Hep G2 cells, which might be the reason that Hep G2 was more sensitive than SMMC7721 cells to ARG treatment.

  13. Mechanism of Arctigenin-Induced Specific Cytotoxicity against Human Hepatocellular Carcinoma Cell Lines: Hep G2 and SMMC7721.

    Science.gov (United States)

    Lu, Zheng; Cao, Shengbo; Zhou, Hongbo; Hua, Ling; Zhang, Shishuo; Cao, Jiyue

    2015-01-01

    Arctigenin (ARG) has been previously reported to exert high biological activities including anti-inflammatory, antiviral and anticancer. In this study, the anti-tumor mechanism of ARG towards human hepatocellular carcinoma (HCC) was firstly investigated. We demonstrated that ARG could induce apoptosis in Hep G2 and SMMC7721 cells but not in normal hepatic cells, and its apoptotic effect on Hep G2 was stronger than that on SMMC7721. Furthermore, the following study showed that ARG treatment led to a loss in the mitochondrial out membrane potential, up-regulation of Bax, down-regulation of Bcl-2, a release of cytochrome c, caspase-9 and caspase-3 activation and a cleavage of poly (ADP-ribose) polymerase in both Hep G2 and SMMC7721 cells, suggesting ARG-induced apoptosis was associated with the mitochondria mediated pathway. Moreover, the activation of caspase-8 and the increased expression levels of Fas/FasL and TNF-α revealed that the Fas/FasL-related pathway was also involved in this process. Additionally, ARG induced apoptosis was accompanied by a deactivation of PI3K/p-Akt pathway, an accumulation of p53 protein and an inhibition of NF-κB nuclear translocation especially in Hep G2 cells, which might be the reason that Hep G2 was more sensitive than SMMC7721 cells to ARG treatment.

  14. Trastuzumab Labeled to High Specific Activity with (111)In by Site-Specific Conjugation to a Metal-Chelating Polymer Exhibits Amplified Auger Electron-Mediated Cytotoxicity on HER2-Positive Breast Cancer Cells.

    Science.gov (United States)

    Ngo Ndjock Mbong, Ghislaine; Lu, Yijie; Chan, Conrad; Cai, Zhongli; Liu, Peng; Boyle, Amanda J; Winnik, Mitchell A; Reilly, Raymond M

    2015-06-01

    Our objective was to evaluate the cytotoxicity toward HER2-positive human breast cancer (BC) cells of trastuzumab modified site-specifically with a metal-chelating polymer (MCP) that presents multiple DTPA chelators for complexing (111)In. (111)In emits subcellular range Auger electrons that induce multiple lethal DNA double-strand breaks (DSBs) in cells. MCPs were synthesized with a polyglutamide backbone with 24 or 29 pendant DTPA groups, with or without nuclear translocation sequence (NLS) peptide modification and a terminal hydrazide group for reaction with aldehydes generated by sodium periodate (NaIO4)-oxidation of glycans on the Fc-domain of trastuzumab. Trastuzumab was site-specifically modified with two DTPA and labeled with (111)In for comparison (trastuzumab-NH-Bn-DTPA-(111)In). The maximum specific activity (SA) for labeling trastuzumab-Hy-MCP with (111)In was 90-fold greater than for trastuzumab-NH-Bn-DTPA-(111)In [8.9 MBq/μg (1.5 × 10(6) MBq/μmol) vs 0.1 MBq/μg (1.2 × 10(4) MBq/μmol)]. Trastuzumab-Hy-MCP-(111)In was bound, internalized, and imported into the nucleus of SK-BR-3 cells. NLS peptide modification of MCPs did not increase nuclear importation. A greater density of DNA DSBs was found for BC cells exposed to high SA (5.5 MBq/μg) than low SA (0.37 MBq/μg) radioimmunoconjugates. At 20 nmol/L, high SA trastuzumab-Hy-MCP-(111)In was 6-fold more effective at reducing the clonogenic survival (CS) of HER2 overexpressed and HER2 gene-amplified SK-BR-3 cells (1.3 × 10(6) receptors/cell) than low SA MCP-radioimmunoconjugates (CS = 1.8 ± 1.3 vs 10.9 ± 0.7%; P = 0.001). Low SA trastuzumab-NH-Bn-DTPA-(111)In (20 nmol/L) reduced the CS of SK-BR-3 cells to 15.8 ± 2.1%. The CS of ZR-75-1 cells with intermediate HER2 density (4 × 10(5) receptors/cell) but without HER2 gene amplification was reduced to 20.5 ± 4.3% by high SA trastuzumab-Hy-MCP-(111)In (20 nmol/L). The CS of HER2-overexpressed (5 × 10(5) HER2/cell) but trastuzumab-resistant TrR1

  15. Cell Death Mechanisms Induced by Cytotoxic Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    Ch(a)vez-Gal(a)n L; Arenas-Del Angel MC; Zenteno E; Ch(a)vez R; Lascurain R

    2009-01-01

    One of the functions of the immune system is to recognize and destroy abnormal or infected cells to maintain homeostasis. This is accomplished by cytotoxic lymphocytes. Cytotoxicity is a highly organized multifactor process. Here, we reviewed the apoptosis pathways induced by the two main cytotoxic lymphocyte subsets, natural killer (NK) cells and CD8+T cells. In base to recent experimental evidence, we reviewed NK receptors involved in recognition of target-cell, as well as lytic molecules such as perforin, granzymes-A and -B, and granulysin. In addition, we reviewed the Fas-FasL intercellular linkage mediated pathway, and briefly the cross-linking of tumor necrosis factor (TNF) and TNF receptor pathway. We discussed three models of possible molecular interaction between lyric molecules from effector cytotoxic cells and target-cell membrane to induction of apoptosis.

  16. Induction of MTG8-specific cytotoxic T-cell lines: MTG8 is probably a tumour antigen that is recognized by cytotoxic T cells in AML1-MTG8-fused gene-positive acute myelogenous leukaemia.

    Science.gov (United States)

    Maeda, M; Otsuka, T; Kimura, N; Kozu, T; Fukuyama, T; Uchida, N; Sugio, Y; Itoh, Y; Iino, T; Inaba, S; Niho, Y

    2000-11-01

    Several reports have demonstrated the persistent detection of AML1-MTG8 fusion products, representing minimal residual disease (MRD), in patients with t(8;21) acute myelogenous leukaemia (AML) who are in long-term remission. It is probable that immune-mediated mechanisms that are able to suppress the expansion of MRD may result in the continuance of remission. It was previously shown that some t(8;21) AML patients had high anti-MTG8 antibody titres. MTG8 expression in normal adult tissues is limited to the brain or heart in which human leucocyte antigen (HLA) class I cell-surface antigens are either not or are only faintly detectable. We hypothesized that the overexpression of the MTG8 gene in t(8;21) AML cells could act as a possible tumour antigen, which might be able to induce the immune-mediated suppression of the expansion of MRD. We were able to induce HLA-A0201-restricted cytotoxic T-lymphocyte (CTL) lines against an MTG8 peptide (MTG8b amino acids 182-191) using monocyte-derived dendritic cells from a healthy donor. T-cell receptor (TCR)Valpha17, TCRVbeta14 and 15, and TCRJbeta2.1 and 2.3 are predominantly used in these CTL lines. Our data, which suggest that the MTG8 protein could be one of the tumour antigens recognized by CTLs, may be helpful in further investigations of TCR analysis in t(8;21) AML patients with HLA-A0201 who are in long-term remission.

  17. Dequalinium induces cytotoxicity in human leukemia NB4 cells by downregulation of Raf/MEK/ERK and PI3K/Akt signaling pathways and potentiation of specific inhibitors of these pathways.

    Science.gov (United States)

    García-Pérez, Ana I; Galeano, Eva; Nieto, Elena; Estañ, M Cristina; Sancho, Pilar

    2014-07-01

    Delocalized lipophilic cation dequalinium (DQA) selectively accumulates in mitochondria and displays anticancer activity in different malignancies. Our previous studies indicate a DQA-induced cytotoxicity in human acute promyelocytic leukemia NB4 cells by early disturbance in mitochondrial function and oxidative stress. This study shows the ability of DQA to downregulate Raf/MEK/ERK1/2 and PI3K/Akt signaling pathways in NB4 cells which leads to cell death by apoptosis and/or necrosis. Moreover, DQA potentiates the action of specific inhibitors of these pathways. These DQA effects could be mediated by redox regulation of Akt. Our results contribute to a better understanding of the cytotoxic DQA mechanism on leukemia cells and encourage the performance of further studies in combination with other agents such as kinase inhibitors for improving the efficacy of therapies against acute promyelocytic leukemia.

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

    Science.gov (United States)

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

    1996-02-15

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

  19. Altered effector function of peripheral cytotoxic cells in COPD

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    Corne Jonathan M

    2009-06-01

    Full Text Available Abstract Background There is mounting evidence that perforin and granzymes are important mediators in the lung destruction seen in COPD. We investigated the characteristics of the three main perforin and granzyme containing peripheral cells, namely CD8+ T lymphocytes, natural killer (NK; CD56+CD3- cells and NKT-like (CD56+CD3+ cells. Methods Peripheral blood mononuclear cells (PBMCs were isolated and cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD8+ T lymphocytes, NK (CD56+CD3- and NKT-like (CD56+CD3+ cells were used in an LDH release assay to determine cytotoxicity and cytotoxic mechanisms were investigated by blocking perforin and granzyme B with relevant antibodies. Results The proportion of peripheral blood NKT-like (CD56+CD3+ cells in smokers with COPD (COPD subjects was significantly lower (0.6% than in healthy smokers (smokers (2.8%, p +CD3- cells from COPD subjects were significantly less cytotoxic than in smokers (16.8% vs 51.9% specific lysis, p +CD3+ cells (16.7% vs 52.4% specific lysis, p +CD3- and NKT-like (CD56+CD3+ cells from smokers and HNS. Conclusion In this study, we show that the relative numbers of peripheral blood NK (CD56+CD3- and NKT-like (CD56+CD3+ cells in COPD subjects are reduced and that their cytotoxic effector function is defective.

  20. Activation of nonspecific cytotoxic cells (NCC) with synthetic oligodeoxynucleotides and bacterial genomic DNA: binding, specificity and identification of unique immunostimulatory motifs.

    Science.gov (United States)

    Oumouna, M; Jaso-Friedmann, L; Evans, D L

    2002-04-01

    We have analyzed the effects of synthetic oligodeoxynucleotides (sODNs) and bacterial DNA (bDNA) on the in vitro activation of NCC. Teleost NCC recognition of DNA appeared to differ from that which occurs in higher vertebrates. NCC contain at least two different receptor specificities for DNA. Both oligodeoxyguanosine 20-mers (dG20) and 5'-TGCTGCTTGTGCTTGTGCTT-3' (4GC-2T) bound specifically to NCC. The existence of different receptor specificities was indicated by reciprocal cold target inhibition experiments. dG20 competed with 4GC-2T binding but sODNs composed of GpC or CpG nests did not compete with recognition by NCC of the dG20. ODN binding by NCC primarily depended on the presence of GpC or CpG nests with a preference for -G- serving as the anchor nucleotide. Secondarily, and similar to models of ODN activation in mammals, palindrome sequences of pu-pu-CpG-py-py activated NCC cytotoxicity. Additional analysis of the requirements for ODN activation indicated that guanosine could not substitute for adenosine as a purine spacer and that CpG motifs containing flanking thymidine (i.e.-GTCpGTT-) augmented the activity of the sODN containing this flanking base. Other evidence for the participation of both G and C in the recognition of specific nucleotides by NCC was that poly-dC20, dA20 or dT20 had no activating properties. Methylation of all cytosine nucleotides within an ODN abrogated activation. A canonical ODN motif of 5'-C/AT/AGCTT-3' can now be suggested for teleosts. Additional studies were done to examine the effects of in vitro treatment of NCC with bDNA. bDNA from three different disease isolates of Streptococcus iniae activated NCC cytotoxicity. Treatment of the bDNA with DNase abrogated the enhancement of cytotoxicity. Also, treatment of NCC with eukaryotic DNA had no effects on cytotoxicity. These studies suggested that NCC recognize bacterial nonmethylated DNA. The consequences of these interactions may be increased innate and acquired anti

  1. Clonal Expansion and Cytotoxicity of TCRVβ Subfamily T Cells Induced by CML and K562 Cells

    Institute of Scientific and Technical Information of China (English)

    YupingZHang; YangqiuLi; ShaohuaChen; LijianYang; GengxinLuo; XueliZhang

    2004-01-01

    OBJECTIVE To investigate the anti-leukemia effect, the distribution and clonal expansion of TCRVβ subfamily T cells in T cells from cord blood and adult peripheral blood induced by CML cells and K562 cells in vitro. METHODS Peripheral blood T cells from one adult donor and 3 cases of cord blood were stimulated with CML cells and K562 cells and further amplified by a suspended T cell-bulk culture,in order to induce CML specific cytotoxic T lymphocytes. The induced T cells were further analyzed for the specific cytotoxicity in CML by LDH assay, the phenotype identification by indirect immunofiuorescence technique and the distribution and clonal expansion of TCRVβ subfamily by using reverse transcriptase-polymerase chain reaction (RT-PCR) and genescan analysis, respectively. RESULTS Oligoclonal and oligoclonal tendency T cells with higher specific cytotoxicity from cord blood and adult peripheral blood could be induced by stimulation with CML cells and K562 cells. CONCLUSIONS Specific cytotoxic T cells for an anti-CML effect could be induced by CML cells and K562 cells .The induced T cells which have the characteristic of specific cytotoxicity against CML cells may come from the clonal expansion of TCRVβ subfamily T cells.

  2. Microchip screening platform for single cell assessment of NK cell cytotoxicity

    Directory of Open Access Journals (Sweden)

    Karolin eGuldevall

    2016-04-01

    Full Text Available Here we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK cells within larger populations. Human primary NK cells were distributed across a silicon-glass microchip containing 32 400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75% were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3 target cells within the 12 hours long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g. in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.

  3. Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity

    Science.gov (United States)

    Guldevall, Karolin; Brandt, Ludwig; Forslund, Elin; Olofsson, Karl; Frisk, Thomas W.; Olofsson, Per E.; Gustafsson, Karin; Manneberg, Otto; Vanherberghen, Bruno; Brismar, Hjalmar; Kärre, Klas; Uhlin, Michael; Önfelt, Björn

    2016-01-01

    Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon–glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy. PMID:27092139

  4. Tumor specific cytotoxicity of arctigenin isolated from herbal plant Arctium lappa L.

    Science.gov (United States)

    Susanti, Siti; Iwasaki, Hironori; Itokazu, Yukiyoshi; Nago, Mariko; Taira, Naoyuki; Saitoh, Seikoh; Oku, Hirosuke

    2012-10-01

    The effectiveness of cancer chemotherapy is often limited by the toxicity to other tissues in the body. Therefore, the identification of non-toxic chemotherapeutics from herbal medicines remains to be an attractive goal to advance cancer treatments. This study evaluated the cytotoxicity profiles of 364 herbal plant extracts, using various cancer and normal cell lines. The screening found occurrence of A549 (human lung adenocarcinoma) specific cytotoxicity in nine species of herbal plants, especially in the extract of Arctium lappa L. Moreover, purification of the selective cytotoxicity in the extract of Arctium lappa L. resulted in the identification of arctigenin as tumor specific agent that showed cytotoxicity to lung cancer (A549), liver cancer (HepG2) and stomach cancer (KATO III) cells, while no cytotoxicity to several normal cell lines. Arctigenin specifically inhibited the proliferation of cancer cells, which might consequently lead to the induction of apoptosis. In conclusion, this study found that arctigenin was one of cancer specific phytochemicals, and in part responsible for the tumor selective cytotoxicity of the herbal medicine.

  5. Induction of the Epstein-Barr Virus Latent Membrane Protein 2 Antigen-specific Cytotoxic T Lymphocytes Using Human Leukocyte Antigen Tetramer-based Artificial Antigen-presenting Cells

    Institute of Scientific and Technical Information of China (English)

    Xiao-Ling LU; Zhi-Hui LIANG; Cai-E ZHANG; Sheng-Jun LU; Xiu-Fang WENG; Xiong-Wen WU

    2006-01-01

    Cytotoxic T lymphocytes (CTLs) specific for the Epstein-Barr virus (EBV) latent membrane protein 2 (LMP2) antigen are important reagents for the treatment of some EBV-associated malignancies,such as EBV-positive Hodgkin's disease and nasopharyngeal carcinoma. However, the therapeutic amount of CTLs is often hampered by the limited supply of antigen-presenting cells. To address this issue, an artificial antigen-presenting cell (aAPC) was made by coating a human leukocyte antigen (HLA)-pLMP2 tetrameric complex, anti-CD28 antibody and CD54 molecule to a cell-sized latex bead, which provided the dual signals required for T cell activation. By co-culture of the HLA-A2-LMP2 bearing aAPC and peripheral blood mononuclear cells from HLA-A2 positive healthy donors, LMP2 antigen-specific CTLs were induced and expanded in vitro. The specificity of the aAPC-induced CTLs was demonstrated by both HLA-A2-LMP2tetramer staining and cytotoxicity against HLA-A2-LMP2 bearing T2 cell, the cytotoxicity was inhibited by the anti-HLA class I antibody (W6/32). These results showed that LMP2 antigen-specific CTLs could be induced and expanded in vitro by the HLA-A2-LMP2-bearing aAPC. Thus, aAPCs coated with an HLApLMP2 complex, anti-CD28 and CD54 might be promising tools for the enrichment of LMP2-specific CTLs for adoptive immunotherapy.

  6. Generation in vivo of peptide-specific cytotoxic T cells and presence of regulatory T cells during vaccination with hTERT (class I and II peptide-pulsed DCs

    Directory of Open Access Journals (Sweden)

    Satthaporn Sukchai

    2009-03-01

    Full Text Available Abstract Background Optimal techniques for DC generation for immunotherapy in cancer are yet to be established. Study aims were to evaluate: (i DC activation/maturation milieu (TNF-α +/- IFN-α and its effects on CD8+ hTERT-specific T cell responses to class I epitopes (p540 or p865, (ii CD8+ hTERT-specific T cell responses elicited by vaccination with class I alone or both class I and II epitope (p766 and p672-pulsed DCs, prepared without IFN-α, (iii association between circulating T regulatory cells (Tregs and clinical responses. Methods Autologous DCs were generated from 10 patients (HLA-0201 with advanced cancer by culturing CD14+ blood monocytes in the presence of GM-CSF and IL-4 supplemented with TNF-α [DCT] or TNF-α and IFN-α [DCTI]. The capacity of the DCs to induce functional CD8+ T cell responses to hTERT HLA-0201 restricted nonapeptides was assessed by MHC tetramer binding and peptide-specific cytotoxicity. Each DC preparation (DCT or DCTI was pulsed with only one type of hTERT peptide (p540 or p865 and both preparations were injected into separate lymph node draining regions every 2–3 weeks. This vaccination design enabled comparison of efficacy between DCT and DCTI in generating hTERT peptide specific CD8+ T cells and comparison of class I hTERT peptide (p540 or p865-loaded DCT with or without class II cognate help (p766 and p672 in 6 patients. T regulatory cells were evaluated in 8 patients. Results (i DCTIs and DCTs, pulsed with hTERT peptides, were comparable (p = 0.45, t-test in inducing peptide-specific CD8+ T cell responses. (ii Class II cognate help, significantly enhanced (p (iii Clinical responders had significantly lower (p Conclusion Addition of IFN-α to ex vivo monocyte-derived DCs, did not significantly enhance peptide-specific T cell responses in vivo, compared with TNF-α alone. Class II cognate help significantly augments peptide-specific T cell responses. Clinically favourable responses were seen in patients

  7. TCR down-regulation boosts T-cell-mediated cytotoxicity and protection against poxvirus infections

    DEFF Research Database (Denmark)

    Hansen, Ann Kathrine; Regner, Matthias; Bonefeld, Charlotte Menne

    2011-01-01

    Cytotoxic T (Tc) cells play a key role in the defense against virus infections. Tc cells recognize infected cells via the T-cell receptor (TCR) and subsequently kill the target cells by one or more cytotoxic mechanisms. Induction of the cytotoxic mechanisms is finely tuned by the activation signals...... from the TCR. To determine whether TCR down-regulation affects the cytotoxicity of Tc cells, we studied TCR down-regulation-deficient CD3¿LLAA mice. We found that Tc cells from CD3¿LLAA mice have reduced cytotoxicity due to a specific deficiency in exocytosis of lytic granules. To determine whether......-regulation critically increases Tc cell cytotoxicity and protection against poxvirus infection....

  8. A novel mechanism of methylglyoxal cytotoxicity in prostate cancer cells.

    Science.gov (United States)

    Antognelli, Cinzia; Mezzasoma, Letizia; Fettucciari, Katia; Talesa, Vincenzo Nicola

    2013-04-01

    Methylglyoxal is one of the most powerful glycating agents of proteins and other important cellular components and has been shown to be toxic to cultured cells. Methylglyoxal cytotoxicity appears to occur through cell-cycle arrest but, more often, through induction of apoptosis. In this study we examined whether, and through which molecular mechanism, methylglyoxal affects the growth of poorly aggressive LNCaP and invasive PC3 human prostate cancer cells, where its role has not been exhaustively investigated yet. We demonstrated that methylglyoxal is cytotoxic on LNCaP and PC3 and that such cytotoxicity occurs not via cell proliferation but apoptosis control. Moreover, we demonstrated that methylglyoxal cytotoxicity, potentiated by the silencing of its major scavenging enzyme Glyoxalase I, occurred via different apoptotic responses in LNCaP and PC3 cells that also showed a different susceptibility to this metabolite. Finally, we showed that the observed methylglyoxal apoptogenic role involved different molecular pathways, specifically mediated by methylglyoxal or methylglyoxal-derived argpyrimidine intracellular accumulation and NF-kB signaling-pathway. In particular, in LNCaP cells, methylglyoxal, through the accumulation of argpyrimidine, desensitized the key cell survival NF-kB signaling pathway, which was consistent with the modulation of NF-kB-regulated genes, triggering a mitochondrial apoptotic pathway. The results suggest that this physiological compound merits investigation as a potential chemo-preventive/-therapeutic agent, in differently aggressive prostate cancers.

  9. Engineered cytotoxic T lymphocytes with AFP-specific TCR gene for adoptive immunotherapy in hepatocellular carcinoma.

    Science.gov (United States)

    Sun, Longhao; Guo, Hao; Jiang, Ruoyu; Lu, Li; Liu, Tong; He, Xianghui

    2016-01-01

    Alpha-fetoprotein (AFP) is overexpressed in hepatocellular carcinoma (HCC) and could serve as a tumor-associated antigen (TAA) and potential target for adoptive immunotherapy. However, low frequency and severe functional impairment of AFP-specific T cells in vivo hamper adoptive infusion. TAA-specific T cell receptor (TCR) gene transfer could be an efficient and reliable alternation to generate AFP-specific cytotoxic T lymphocytes (CTLs). Autologous dendritic cells (DC) pulsed with AFP158-166 peptides were used to stimulate AFP-specific CTLs. TCR α/β chain genes of AFP-specific CTLs were cloned and linked by 2A peptide to form full-length TCR coding sequence synthesized into a lentiviral vector. Nonspecific activated T cells were engineered by lentivirus infection. Transgenetic CTLs were evaluated for transfection efficiency, expression of AFP158-166-specific TCR, interferon (IFN)-γ secretion, and specific cytotoxicity toward AFP+ HCC cells in vitro and in vivo. Flow cytometry revealed the AFP158-166-MHC-Pentamer positive transgenetic CTLs was 9.86 %. The number of IFN-γ secretion T cells and the specific cytotoxicity toward HpeG2 in vitro and in tumor-bearing NOD/SCID mice were significantly raised in transgenetic CTLs than that of AFP158-166-specific CTLs obtained by peptide-pulsed DCs or control group. TCR gene transfer is a promising strategy to generate AFP158-166-specific CTLs for the treatment of HCC.

  10. T cell retargeting with MHC class I-restricted antibodies: the CD28 costimulatory domain enhances antigen-specific cytotoxicity and cytokine production

    NARCIS (Netherlands)

    R.A. Willemsen (Ralph); C. Ronteltap; P. Chames; J.E.M.A. Debets (Reno); R.L.H. Bolhuis (Reinder)

    2005-01-01

    textabstractT cells require both primary and costimulatory signals for optimal activation. The primary Ag-specific signal is delivered by engagement of the TCR. The second Ag-independent costimulatory signal is mediated by engagement of the T cell surface costimulatory molecule CD2

  11. Arecoline is cytotoxic for human endothelial cells.

    Science.gov (United States)

    Ullah, Mafaz; Cox, Stephen; Kelly, Elizabeth; Boadle, Ross; Zoellner, Hans

    2014-11-01

    Oral submucous fibrosis is a pre-malignant fibrotic condition caused by areca nut use and involves reduced mucosal vascularity. Arecoline is the principal areca nut alkaloid and is cytotoxic for epithelium and fibroblasts. Endothelial cell cycle arrest is reported on exposure to arecoline, as is cytotoxicity for endothelial-lung carcinoma hybrid cells. We here describe cytotoxicity for primary human endothelial cultures from seven separate donors. Human umbilical vein endothelial cells were exposed to increasing concentrations of arecoline and examined by: phase-contrast microscopy, haemocytometer counts, transmission electron microscopy, lactate dehydrogenase release and the methyl-thiazol-tetrazolium assay. Vacuolation and detachment of endothelium were observed at and above arecoline concentrations of 333 μg/ml or more. Ultrastructural features of cellular stress were seen after 24-h treatment with 111 μg/ml arecoline and included reduced ribosomal studding of endoplasmic reticulum, increased autophagolysosomal structures, increased vacuolation and reduced mitochondrial cristae with slight swelling. Similar changes were seen at 4 h with arecoline at 333 μg/ml or above, but with more severe mitochondrial changes including increased electron density of mitochondrial matrix and greater cristal swelling, while by 24 h, these cells were frankly necrotic. Haemocytometer counts were paralleled by both lactate dehydrogenase release and the methyl-thiazol-tetrazolium assays. Arecoline is cytotoxic via necrosis for endothelium, while biochemical assays indicate no appreciable cellular leakage before death and detachment, as well as no clear effect on mitochondrial function in viable cells. Arecoline toxicity may thus contribute to reduced vascularity in oral submucous fibrosis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Trastuzumab labeled to high specific activity with ¹¹¹In by conjugation to G4 PAMAM dendrimers derivatized with multiple DTPA chelators exhibits increased cytotoxic potency on HER2-positive breast cancer cells.

    Science.gov (United States)

    Chan, Conrad; Cai, Zhongli; Reilly, Raymond M

    2013-08-01

    To conjugate trastuzumab with/without NLS peptides to G4 PAMAM dendrimers derivatized with DTPA and determine the specific radioactivity (SA) for (111)In labeling, HER2 immunoreactivity and cytotoxicity on breast cancer (BC) cells. G4 dendrimers were reacted with DTPA then conjugated through a thiol to maleimide-derivatized trastuzumab. The SA achievable was determined by incubating 2 to 20 μg with 60 MBq of (111)In. HER2 immunoreactivity, internalization and nuclear importation were measured. The effect of (111)In-DTPA-G4-trastuzumab (5.9 MBq/μg) on the clonogenic survival (CS) of SK-Br-3 or MDA-MB-231 cells with high or low HER2 density, respectively was compared to (111)In-DTPA-NLS-trastuzumab (0.5 MBq/μg). DNA double-strand breaks (DSBs) were measured. DTPA-G4-trastuzumab was labeled with (111)In to a SA (23.6 MBq/μg) which was 100-fold higher than (111)In-DTPA-NLS-trastuzumab. (111)In-DTPA-G4-trastuzumab and (111)In-DTPA-G4-NLS-trastuzumab retained HER2 immunoreactivity and were internalized and imported into the nucleus of BC cells. G4-radioimmunoconjugates were 2-4 fold and 9-fold more cytotoxic to SK-Br-3 and MDA-MB-231 cells, respectively than (111)In-DTPA-NLS-trastuzumab which was associated with an increase in DNA DSBs. Conjugation of trastuzumab to G4 PAMAM dendrimers modified with 30 DTPA permitted high SA (111)In labeling which increased their cytotoxic potency for BC cells with high or low HER2 density.

  13. Nanomaterial cytotoxicity is composition, size, and cell type dependent.

    Science.gov (United States)

    Sohaebuddin, Syed K; Thevenot, Paul T; Baker, David; Eaton, John W; Tang, Liping

    2010-08-21

    Despite intensive research efforts, reports of cellular responses to nanomaterials are often inconsistent and even contradictory. Additionally, relationships between the responding cell type and nanomaterial properties are not well understood. Using three model cell lines representing different physiological compartments and nanomaterials of different compositions and sizes, we have systematically investigated the influence of nanomaterial properties on the degrees and pathways of cytotoxicity. In this study, we selected nanomaterials of different compositions (TiO2 and SiO2 nanoparticles, and multi-wall carbon nanotubes [MWCNTs]) with differing size (MWCNTs of different diameters 50 nm; but same length 0.5-2 microm) to analyze the effects of composition and size on toxicity to 3T3 fibroblasts, RAW 264.7 macrophages, and telomerase-immortalized (hT) bronchiolar epithelial cells. Following characterization of nanomaterial properties in PBS and serum containing solutions, cells were exposed to nanomaterials of differing compositions and sizes, with cytotoxicity monitored through reduction in mitochondrial activity. In addition to cytotoxicity, the cellular response to nanomaterials was characterized by quantifying generation of reactive oxygen species, lysosomal membrane destabilization and mitochondrial permeability. The effect of these responses on cellular fate - apoptosis or necrosis - was then analyzed. Nanomaterial toxicity was variable based on exposed cell type and dependent on nanomaterial composition and size. In addition, nanomaterial exposure led to cell type dependent intracellular responses resulting in unique breakdown of cellular functions for each nanomaterial: cell combination. Nanomaterials induce cell specific responses resulting in variable toxicity and subsequent cell fate based on the type of exposed cell. Our results indicate that the composition and size of nanomaterials as well as the target cell type are critical determinants of

  14. Nanomaterial cytotoxicity is composition, size, and cell type dependent

    Directory of Open Access Journals (Sweden)

    Sohaebuddin Syed K

    2010-08-01

    Full Text Available Abstract Background Despite intensive research efforts, reports of cellular responses to nanomaterials are often inconsistent and even contradictory. Additionally, relationships between the responding cell type and nanomaterial properties are not well understood. Using three model cell lines representing different physiological compartments and nanomaterials of different compositions and sizes, we have systematically investigated the influence of nanomaterial properties on the degrees and pathways of cytotoxicity. In this study, we selected nanomaterials of different compositions (TiO2 and SiO2 nanoparticles, and multi-wall carbon nanotubes [MWCNTs] with differing size (MWCNTs of different diameters 50 nm; but same length 0.5-2 μm to analyze the effects of composition and size on toxicity to 3T3 fibroblasts, RAW 264.7 macrophages, and telomerase-immortalized (hT bronchiolar epithelial cells. Results Following characterization of nanomaterial properties in PBS and serum containing solutions, cells were exposed to nanomaterials of differing compositions and sizes, with cytotoxicity monitored through reduction in mitochondrial activity. In addition to cytotoxicity, the cellular response to nanomaterials was characterized by quantifying generation of reactive oxygen species, lysosomal membrane destabilization and mitochondrial permeability. The effect of these responses on cellular fate - apoptosis or necrosis - was then analyzed. Nanomaterial toxicity was variable based on exposed cell type and dependent on nanomaterial composition and size. In addition, nanomaterial exposure led to cell type dependent intracellular responses resulting in unique breakdown of cellular functions for each nanomaterial: cell combination. Conclusions Nanomaterials induce cell specific responses resulting in variable toxicity and subsequent cell fate based on the type of exposed cell. Our results indicate that the composition and size of nanomaterials as well as the

  15. Differential phenotypic and functional profiles of TcCA-2 -specific cytotoxic CD8+ T cells in the asymptomatic versus cardiac phase in Chagasic patients.

    Science.gov (United States)

    Egui, Adriana; Thomas, M Carmen; Carrilero, Bartolomé; Segovia, Manuel; Alonso, Carlos; Marañón, Concepción; López, Manuel Carlos

    2015-01-01

    It has been reported that the immune response mediated by T CD8+ lymphocytes plays a critical role in the control of Trypanosoma cruzi infection and that the clinical symptoms of Chagas disease appear to be related to the competence of the CD8+ T immune response against the parasite. Herewith, in silico prediction and binding assays on TAP-deficient T2 cells were used to identify potential HLA-A*02:01 ligands in the T. cruzi TcCA-2 protein. The TcCA-2-specific CD8+ T cells were functionality evaluated by Granzyme B and cytokine production in peripheral blood mononuclear cells (PBMC) from Chagas disease patients stimulated with the identified HLA-A*02:01 peptides. The specific cells were phenotypically characterized by flow cytometry using several surface markers and HLA-A*02:01 APC-labeled dextramer loaded with the peptides. In the T. cruzi TcCA-2 protein four T CD8+ epitopes were identified which are processed and presented during Chagas disease. Interestingly, a differential cellular phenotypic profile could be correlated with the severity of the disease. The TcCA-2-specific T CD8+ cells from patients with cardiac symptoms are mainly effector memory cells (TEM and TEMRA) while, those present in the asymptomatic phase are predominantly naive cells (TNAIVE). Moreover, in patients with cardiac symptoms the percentage of cells with senescence features is significantly higher than in patients at the asymptomatic phase of the disease. We consider that the identification of these new class I-restricted epitopes are helpful for designing biomarkers of sickness pathology as well as the development of immunotherapies against T. cruzi infection.

  16. Differential phenotypic and functional profiles of TcCA-2 -specific cytotoxic CD8+ T cells in the asymptomatic versus cardiac phase in Chagasic patients.

    Directory of Open Access Journals (Sweden)

    Adriana Egui

    Full Text Available It has been reported that the immune response mediated by T CD8+ lymphocytes plays a critical role in the control of Trypanosoma cruzi infection and that the clinical symptoms of Chagas disease appear to be related to the competence of the CD8+ T immune response against the parasite. Herewith, in silico prediction and binding assays on TAP-deficient T2 cells were used to identify potential HLA-A*02:01 ligands in the T. cruzi TcCA-2 protein. The TcCA-2-specific CD8+ T cells were functionality evaluated by Granzyme B and cytokine production in peripheral blood mononuclear cells (PBMC from Chagas disease patients stimulated with the identified HLA-A*02:01 peptides. The specific cells were phenotypically characterized by flow cytometry using several surface markers and HLA-A*02:01 APC-labeled dextramer loaded with the peptides. In the T. cruzi TcCA-2 protein four T CD8+ epitopes were identified which are processed and presented during Chagas disease. Interestingly, a differential cellular phenotypic profile could be correlated with the severity of the disease. The TcCA-2-specific T CD8+ cells from patients with cardiac symptoms are mainly effector memory cells (TEM and TEMRA while, those present in the asymptomatic phase are predominantly naive cells (TNAIVE. Moreover, in patients with cardiac symptoms the percentage of cells with senescence features is significantly higher than in patients at the asymptomatic phase of the disease. We consider that the identification of these new class I-restricted epitopes are helpful for designing biomarkers of sickness pathology as well as the development of immunotherapies against T. cruzi infection.

  17. Dengue virus-specific cross-reactive CD8+ human cytotoxic T lymphocytes.

    OpenAIRE

    Bukowski, J F; Kurane, I; Lai, C J; Bray, M.; Falgout, B.; Ennis, F A

    1989-01-01

    Stimulation with live dengue virus of peripheral blood mononuclear cells from a dengue virus type 4-immune donor generated virus-specific, serotype-cross-reactive, CD8+, class I-restricted cytotoxic T lymphocytes (CTL) capable of lysing dengue virus-infected cells and cells pulsed with dengue virus antigens of all four serotypes. These CTL lysed autologous fibroblasts infected with vaccinia virus-dengue virus recombinant viruses containing the E gene or several nonstructural dengue virus type...

  18. Ultrastructural studies of time-course and cellular specificity of interleukin-1 mediated islet cytotoxicity

    DEFF Research Database (Denmark)

    Mandrup-Poulsen, T; Egeberg, J; Nerup, J

    1987-01-01

    of incubation and examined by electron microscopy in a blinded fashion. Already after 30 min, accumulation of opaque intracytoplasmic bodies without apparent surrounding membranes, and autophagic vacuoles were seen in about 20% of the beta cells examined in rat islets exposed to interleukin-1. After 16 h......Previous electron-microscopic studies of isolated islets of Langerhans exposed to the monokine interleukin-1 for 7 days have indicated that interleukin-1 is cytotoxic to all islet cells. To study the time-course and possible cellular specificity of interleukin-1 cytotoxicity to islets exposed...... of incubation with interleukin-1, more than 80% of rat beta cells showed signs of degeneration. Beta cell specific changes similar to those observed in rat islets exposed to IL-1 for 30 min were seen in human islets exposed to IL-1 for 24 h. The described changes were not observed in alpha cells in interleukin...

  19. Differential Phenotypic and Functional Profiles of TcCA-2 -Specific Cytotoxic CD8+ T Cells in the Asymptomatic versus Cardiac Phase in Chagasic Patients

    OpenAIRE

    2015-01-01

    It has been reported that the immune response mediated by T CD8+ lymphocytes plays a critical role in the control of Trypanosoma cruzi infection and that the clinical symptoms of Chagas disease appear to be related to the competence of the CD8+ T immune response against the parasite. Herewith, in silico prediction and binding assays on TAP-deficient T2 cells were used to identify potential HLA-A*02:01 ligands in the T. cruzi TcCA-2 protein. The TcCA-2-specific CD8+ T cells were functionality ...

  20. Cytotoxicity and Effects on Cell Viability of Nickel Nanowires

    KAUST Repository

    Rodriguez, Jose E.

    2013-05-01

    Recently, magnetic nanoparticles are finding an increased use in biomedical applications and research. Nanobeads are widely used for cell separation, biosensing and cancer therapy, among others. Due to their properties, nanowires (NWs) are gaining ground for similar applications and, as with all biomaterials, their cytotoxicity is an important factor to be considered before conducting biological studies with them. In this work, the cytotoxic effects of nickel NWs (Ni NWs) were investigated in terms of cell viability and damage to the cellular membrane. Ni NWs with an average diameter of 30-34 nm were prepared by electrodeposition in nanoporous alumina templates. The templates were obtained by a two-step anodization process with oxalic acid on an aluminum substrate. Characterization of NWs was done using X-Ray diffraction (XRD) and energy dispersive X-Ray analysis (EDAX), whereas their morphology was observed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Cell viability studies were carried out on human colorectal carcinoma cells HCT 116 by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell proliferation colorimetric assay, whereas the lactate dehydrogenase (LDH) homogenous membrane fluorimetric assay was used to measure the degree of cell membrane rupture. The density of cell seeding was calculated to obtain a specific cell number and confluency before treatment with NWs. Optical readings of the cell-reduced MTT products were measured at 570 nm, whereas fluorescent LDH membrane leakage was recorded with an excitation wavelength of 525 nm and an emission wavelength of 580 - 640 nm. The effects of NW length, cell exposure time, as well as NW:cell ratio, were evaluated through both cytotoxic assays. The results show that cell viability due to Ni NWs is affected depending on both exposure time and NW number. On the other hand, membrane rupture and leakage was only significant at later exposure times. Both

  1. Differential cytotoxicity but augmented IFN-γ secretion by NK cells after interaction with monocytes from humans, and those from wild type and myeloid specific COX-2 knockout mice

    Directory of Open Access Journals (Sweden)

    Han-Ching eTseng

    2015-06-01

    Full Text Available The list of genes which augment NK cell function when knocked out in neighboring cells is increasing, and may point to the fundamental function of NK cells targeting cells with diminished capability to differentiate optimally since NK cells are able to target less differentiated cells, and aid in their differentiation. In this paper we aimed at understanding the effect of monocytes from targeted knockout of COX-2 in myeloid cells (Cox-2flox/flox;LysMCre/+ and from control littermates (Cox-2flox/flox;LysM+/+ on ex vivo function of NK cells. Furthermore, we compared the effect of monocytes treated with and without lipopolysaccharide (LPS on NK cells from mice and humans. NK cells purified from Cox-2flox/flox;LysMCre/+ mice had heightened cytotoxic activity when compared to those obtained from control littermates. In addition, NK cells cultured with autologous Cox-2flox/flox;LysMCre/+ monocytes and DCs, mouse embryonic fibroblasts (MEFs from global knock out COX-2, but not with knock out of COX-2 in T cells, had increased cytotoxic function as well as augmented IFN-γ secretion when compared to NK cells from control littermates cultured with monocytes. LPS inhibited NK cell cytotoxicity while increasing IFN-γ secretion when cultured in the presence of monocytes from either Cox-2flox/flox;LysMCre/+ or control littermates. In contrast to mice, NK cells from humans when cultured with monocytes lost cytotoxic function and gained ability to secrete large amounts of IFN-γ, a process which we had previously coined as split anergy. Similar to mice, LPS potentiated the loss of human NK cell cytotoxicity while increasing IFN-γ secretion in the presence of monocytes. Greater loss of cytotoxicity and larger secretion of IFN-γ in NK cells induced by gene knock out cells may be important for the greater need of these cells for differentiation.

  2. Bulk culture levels of specific cytotoxic T-cell activity against HIV-1 proteins are not associated with risk of death

    DEFF Research Database (Denmark)

    Aladdin, H; Ullum, H; Lepri, A Cozzi

    1999-01-01

    The ability of cytotoxic T lymphocytes (CTL) to control and influence the outcome of human immunodeficiency virus (HIV) infection is not fully understood. The association between HIV-CTL activity and disease progression was evaluated prospectively in 36 HIV-1-infected individuals with a median fo...

  3. Heat Shock Proteins 60 and 70 Specific Proinflammatory and Cytotoxic Response of CD4+CD28null Cells in Chronic Kidney Disease

    Directory of Open Access Journals (Sweden)

    Ashok K. Yadav

    2013-01-01

    Full Text Available Background. CD4+CD28null T cells are expanded in peripheral blood of patients with chronic kidney disease and associated with subclinical atherosclerosis. However, triggers for the oligoclonal expansion and activation of these cells are not clear. Methods. We investigated twenty-five stage V-IV chronic kidney disease (CKD patients and eight healthy subjects (HC. Peripheral mononuclear cells were isolated and incubated with heat shock protein- (HSP 60 and 70. CD4+CD28null and CD4+CD28+ cells were sorted by flowcytometry and antigen specific response was assessed by the mRNA and protein expression of interferon (IFN-γ, perforin, and granzyme B using qRT-PCR and Elispot. Results. The basal mRNA expression of IFN-γ, perforin, and granzyme B in CD4+CD28null cells was higher in subjects with CKD compared to that in HC (P<0.0001. Subjects with CKD also showed expression of IFN-γ, perforin, and granzyme B in the CD4+CD28+ subset, but this was much weaker than that seen in the CD4+CD28null population (P<0.0001. We did not note the expression of these molecules at mRNA or protein level in either subset of CD4 cells in HC. After incubation with HSP60 and HSP70, CD4+CD28null cells showed increased expression at mRNA (P<0.001 and protein level (P<0.001. CD4+CD28+ cells also showed a weak increase in expression. No antigen-specific response was noted in HC. Conclusion. These data show that CD4+CD28null cells in subjects with CKD react with HSP60 and HSP70 by upregulating the expression of IFN-γ, perforin and granzyme B. Increased circulating level of HSP60 and HSP70 might play a role in initiation and/or progression of atherosclerosis in CKD subjects through perturbation of CD4+CD28null cells.

  4. Establishment and operation of a Good Manufacturing Practice-compliant allogeneic Epstein-Barr virus (EBV)-specific cytotoxic cell bank for the treatment of EBV-associated lymphoproliferative disease.

    Science.gov (United States)

    Vickers, Mark A; Wilkie, Gwen M; Robinson, Nicolas; Rivera, Nadja; Haque, Tanzina; Crawford, Dorothy H; Barry, Jacqueline; Fraser, Neil; Turner, David M; Robertson, Victoria; Dyer, Phil; Flanagan, Peter; Newlands, Helen R; Campbell, John; Turner, Marc L

    2014-11-01

    Epstein-Barr virus (EBV) is associated with several malignancies, including post-transplant lymphoproliferative disorder (PTLD). Conventional treatments for PTLD are often successful, but risk organ rejection and cause significant side effects. EBV-specific cytotoxic T lymphocytes (CTLs) generated in vitro from peripheral blood lymphocytes provide an alternative treatment modality with few side effects, but autologous CTLs are difficult to use in clinical practice. Here we report the establishment and operation of a bank of EBV-specific CTLs derived from 25 blood donors with human leucocyte antigen (HLA) types found at high frequency in European populations. Since licensure, there have been enquiries about 37 patients, who shared a median of three class I and two class II HLA types with these donors. Cells have been infused into ten patients with lymphoproliferative disease, eight of whom achieved complete remission. Neither patient with refractory disease was matched for HLA class II. Both cases of EBV-associated non-haematopoietic sarcoma receiving cells failed to achieve complete remission. Thirteen patients died before any cells could be issued, emphasizing that the bank should be contacted before patients become pre-terminal. Thus, this third party donor-derived EBV-specific CTL cell bank can supply most patients with appropriately matched cells and most recipients have good outcomes.

  5. CYTOTOXICITY TESTING OF WOUND DRESSINGS USING METHYLCELLULOSE CELL-CULTURE

    NARCIS (Netherlands)

    VANLUYN, MJA; VANWACHEM, PB; NIEUWENHUIS, P; JONKMAN, MF

    1992-01-01

    Wound dressings may induce cytotoxic effects. In this study, we check several, mostly commercially available, wound dressings for cytotoxicity. We used our previously described, newly developed and highly sensitive 7 d methylcellulose cell culture with fibroblasts as the test system. Cytotoxicity is

  6. Proteolytically modified human beta 2-microglobulin augments the specific cytotoxic activity in murine mixed lymphocyte culture

    DEFF Research Database (Denmark)

    Nissen, Mogens Holst; Claësson, M H

    1987-01-01

    (M-beta 2-m) bind to murine lymphocytes expressing H-2 class I antigens; M-beta 2-m, when added at day 0 and 1 of culture in nanomolar concentrations to a one-way murine allogeneic mixed lymphocyte culture (MLC) augments the generation of specific cytotoxic T lymphocytes; M-beta 2-m increases...... the endogenous production of interleukin 2 in the MLC culture; monoclonal antibody which reacts with both the native beta 2-m and M-beta 2-m molecule blocks the augmentation of cytotoxic T lymphocyte production induced by M-beta 2-m; murine as well as human MLC responder cells can proteolytically modify native...

  7. Targeted cytosine deaminase-uracil phosphoribosyl transferase suicide gene therapy induces small cell lung cancer-specific cytotoxicity and tumor growth delay

    DEFF Research Database (Denmark)

    Christensen, Camilla L; Gjetting, Torben; Poulsen, Thomas Tuxen

    2010-01-01

    Small cell lung cancer (SCLC) is a highly malignant cancer for which there is no curable treatment. Novel therapies are therefore in great demand. In the present study we investigated the therapeutic effect of transcriptionally targeted suicide gene therapy for SCLC based on the yeast cytosine...... deaminase (YCD) gene alone or fused with the yeast uracil phosphoribosyl transferase (YUPRT) gene followed by administration of 5-fluorocytosine (5-FC) prodrug. Experimental design: The YCD gene or the YCD-YUPRT gene was placed under regulation of the SCLC-specific promoter insulinoma-associated 1 (INSM1...

  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. PMID:24415939

  9. 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.

  10. Natural killer cell mediated cytotoxic responses in the Tasmanian devil.

    Directory of Open Access Journals (Sweden)

    Gabriella K Brown

    Full Text Available The Tasmanian devil (Sarcophilus harrisii, the world's largest marsupial carnivore, is under threat of extinction following the emergence of an infectious cancer. Devil facial tumour disease (DFTD is spread between Tasmanian devils during biting. The disease is consistently fatal and devils succumb without developing a protective immune response. The aim of this study was to determine if Tasmanian devils were capable of forming cytotoxic antitumour responses and develop antibodies against DFTD cells and foreign tumour cells. The two Tasmanian devils immunised with irradiated DFTD cells did not form cytotoxic or humoral responses against DFTD cells, even after multiple immunisations. However, following immunisation with xenogenic K562 cells, devils did produce cytotoxic responses and antibodies against this foreign tumour cell line. The cytotoxicity appeared to occur through the activity of natural killer (NK cells in an antibody dependent manner. Classical NK cell responses, such as innate killing of DFTD and foreign cancer cells, were not observed. Cells with an NK-like phenotype comprised approximately 4 percent of peripheral blood mononuclear cells. The results of this study suggest that Tasmanian devils have NK cells with functional cytotoxic pathways. Although devil NK cells do not directly recognise DFTD cancer cells, the development of antibody dependent cell-mediated cytotoxicity presents a potential pathway to induce cytotoxic responses against the disease. These findings have positive implications for future DFTD vaccine research.

  11. Correlation between luminescence intensity and cytotoxicity in cell-based cytotoxicity assay using luciferase.

    Science.gov (United States)

    Wakuri, S; Yamakage, K; Kazuki, Y; Kazuki, K; Oshimura, M; Aburatani, S; Yasunaga, M; Nakajima, Y

    2017-04-01

    The luciferase reporter assay has become one of the conventional methods for cytotoxicity evaluation. Typically, the decrease of luminescence expressed by a constitutive promoter is used as an index of cytotoxicity. However, to our knowledge, there have been no reports of the correlation between cytotoxicity and luminescence intensity. In this study, to accurately verify the correlation between them, beetle luciferase was stably expressed in human hepatoma HepG2 cells harboring the multi-integrase mouse artificial chromosome vector. We showed that the cytotoxicity assay using luciferase does not depend on the stability of luciferase protein and the kind of constitutive promoter. Next, HepG2 cells in which green-emitting beetle luciferase was expressed under the control of CAG promoter were exposed to 58 compounds. The luminescence intensity and cytotoxicity curves of cells exposed to 48 compounds showed similar tendencies, whereas those of cells exposed to 10 compounds did not do so, although the curves gradually approached each other with increasing exposure time. Finally, we demonstrated that luciferase expressed under the control of a constitutive promoter can be utilized both as an internal control reporter for normalizing a test reporter and for monitoring cytotoxicity when two kinds of luciferases are simultaneously used in the cytotoxicity assay. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Extracellular thiol-assisted selenium uptake dependent on the x(c)(-) cystine transporter explains the cancer-specific cytotoxicity of selenite

    DEFF Research Database (Denmark)

    Olm, E.; Fernandes, A. P.; Hebert, C.

    2009-01-01

    The selenium salt selenite (SeO32-) is cytotoxic in low to moderate concentrations, with a remarkable specificity for cancer cells resistant to conventional chemotherapy. Our data show that selenium uptake and accumulation, rather than intracellular events, are crucial to the specific selenite...... cytotoxicity observed in resistant cancer cells. We show that selenium uptake depends on extracellular reduction, and that the extracellular environment is a key factor specific to selenite cytotoxicity. The extracellular reduction is mediated by cysteine, and the efficacy is determined by the uptake...... and sensitivity to selenite cytotoxicity, and highlights the great therapeutic potential in treating multidrug-resistant cancer....

  13. Recognition of clonogenic leukemic cells, remission bone marrow and HLA-identical donor bone marrow by CD8+ or CD4+ minor histocompatibility antigen-specific cytotoxic T lymphocytes.

    Science.gov (United States)

    Faber, L M; van der Hoeven, J; Goulmy, E; Hooftman-den Otter, A L; van Luxemburg-Heijs, S A; Willemze, R; Falkenburg, J H

    1995-08-01

    We investigated whether minor histocompatibility (mH) antigen-specific cytotoxic T lymphocytes (CTL) can discriminate between leukemic hematopoietic progenitor cells (leukemic-HPC) from AML or CML patients, the HPC from their remission bone marrow (remission-HPC), and normal HPC from their HLA-identical sibling bone marrow donor (donor-HPC). Specific lysis by CD8+ CTL clones was observed not only of the leukemic-HPC but also of the donor-HPC in 3/4 patient/donor combinations expressing mH antigen HA-1, 3/5 combinations expressing mH antigen HA-2, 2/3 combinations expressing mH antigen HA-3, and 2/2 combinations expressing mH antigen HY-A1. In four patient/donor combinations the recognition of the donor-HPC was clearly less than of the leukemic-HPC, indicating differential susceptibility to lysis by these mH CTL clones. In addition, differential recognition of leukemic-HPC and remission-HPC within seven patients was analyzed. In one patient expressing the HA-2 antigen on the leukemic cells the recognition of the remission-HPC was clearly less than of the leukemic-HPC. One CD4+ CTL clone showed specific lysis of the leukemic-HPC from an AML patient and a CML patient as well as of normal remission-HPC and donor-HPC. These results illustrate that in general CD8+ and CD4+ mH antigen specific CTL clones do not differentially recognize leukemic-HPC and normal-HPC. However, differences in susceptibility to lysis of malignant versus normal cells may contribute to a differential GVL effect.

  14. CD56 marks human dendritic cell subsets with cytotoxic potential

    NARCIS (Netherlands)

    Roothans, D.; Smits, E.; Lion, E.; Tel, J.; Anguille, S.

    2013-01-01

    Human plasmacytoid and myeloid dendritic cells (DCs), when appropriately stimulated, can express the archetypal natural killer (NK)-cell surface marker CD56. In addition to classical DC functions, CD56+ DCs are endowed with an unconventional cytotoxic capacity.

  15. Analysis of the Effects of Cell Stress and Cytotoxicity on In ...

    Science.gov (United States)

    Chemical toxicity can arise from disruption of specific biomolecular functions or through more generalized cell stress and cytotoxicity-mediated processes. Here, concentration-dependent responses of 1063 chemicals including pharmaceuticals, natural products, pesticidals, consumer, and industrial chemicals across a diverse battery of 821 in vitro assay endpoints from 7 high-throughput assay technology platforms were analyzed in order to better distinguish between these types of activities. Both cell-based and cell-free assays showed a rapid increase in the frequency of responses at concentrations where cell stress / cytotoxicity responses were observed in cell-based assays. Chemicals that were positive on at least two viability/cytotoxicity assays within the concentration range tested (typically up to 100 M) activated a median of 12% of assay endpoints while those that were not cytotoxic in this concentration range activated 1.3% of the assays endpoints. The results suggest that activity can be broadly divided into: (1) specific biomolecular interactions against one or more targets (e.g., receptors or enzymes) at concentrations below which overt cytotoxicity-associated activity is observed; and (2) activity associated with cell stress or cytotoxicity, which may result from triggering of specific cell stress pathways, chemical reactivity, physico-chemical disruption of proteins or membranes, or broad low-affinity non-covalent interactions. Chemicals showing a g

  16. A new class of pluripotent stem cell cytotoxic small molecules.

    Directory of Open Access Journals (Sweden)

    Mark Richards

    Full Text Available A major concern in Pluripotent Stem Cell (PSC-derived cell replacement therapy is the risk of teratoma formation from contaminating undifferentiated cells. Removal of undifferentiated cells from differentiated cultures is an essential step before PSC-based cell therapies can be safely deployed in a clinical setting. We report a group of novel small molecules that are cytotoxic to PSCs. Our data indicates that these molecules are specific and potent in their activity allowing rapid eradication of undifferentiated cells. Experiments utilizing mixed PSC and primary human neuronal and cardiomyocyte cultures demonstrate that up to a 6-fold enrichment for specialized cells can be obtained without adversely affecting cell viability and function. Several structural variants were synthesized to identify key functional groups and to improve specificity and efficacy. Comparative microarray analysis and ensuing RNA knockdown studies revealed involvement of the PERK/ATF4/DDIT3 ER stress pathway. Surprisingly, cell death following ER stress induction was associated with a concomitant decrease in endogenous ROS levels in PSCs. Undifferentiated cells treated with these molecules preceding transplantation fail to form teratomas in SCID mice. Furthermore, these molecules remain non-toxic and non-teratogenic to zebrafish embryos suggesting that they may be safely used in vivo.

  17. Serglycin determines secretory granule repertoire and regulates natural killer cell and cytotoxic T lymphocyte cytotoxicity.

    Science.gov (United States)

    Sutton, Vivien R; Brennan, Amelia J; Ellis, Sarah; Danne, Jill; Thia, Kevin; Jenkins, Misty R; Voskoboinik, Ilia; Pejler, Gunnar; Johnstone, Ricky W; Andrews, Daniel M; Trapani, Joseph A

    2016-03-01

    The anionic proteoglycan serglycin is a major constituent of secretory granules in cytotoxic T lymphocyte (CTL)/natural killer (NK) cells, and is proposed to promote the safe storage of the mostly cationic granule toxins, granzymes and perforin. Despite the extensive defects of mast cell function reported in serglycin gene-disrupted mice, no comprehensive study of physiologically relevant CTL/NK cell populations has been reported. We show that the cytotoxicity of serglycin-deficient CTL and NK cells is severely compromised but can be partly compensated in both cell types when they become activated. Reduced intracellular granzyme B levels were noted, particularly in CD27(+) CD11b(+) mature NK cells, whereas serglycin(-/-) TCR-transgenic (OTI) CD8 T cells also had reduced perforin stores. Culture supernatants from serglycin(-/-) OTI T cells and interleukin-2-activated NK contained increased granzyme B, linking reduced storage with heightened export. By contrast, granzyme A was not significantly reduced in cells lacking serglycin, indicating differentially regulated trafficking and/or storage for the two granzymes. A quantitative analysis of different granule classes by transmission electronmicroscopy showed a selective loss of dense-core granules in serglycin(-/-) CD8(+) CTLs, although other granule types were maintained quantitatively. The findings of the present study show that serglycin plays a critical role in the maturation of dense-core cytotoxic granules in cytotoxic lymphocytes and the trafficking and storage of perforin and granzyme B, whereas granzyme A is unaffected. The skewed retention of cytotoxic effector molecules markedly reduces CTL/NK cell cytotoxicity, although this is partly compensated for as a result of activating the cells by physiological means.

  18. HIV-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC) - Mediating Antibodies Decline while NK Cell Function Increases during Antiretroviral Therapy (ART)

    DEFF Research Database (Denmark)

    Jensen, Sanne Skov; Fomsgaard, Anders; Borggren, Marie

    2015-01-01

    the ability of effector cells and antibodies to mediate ADCC separately and in combination using the ADCC-PanToxiLux assay. The ability of the peripheral blood mononuclear cells (PBMCs) to mediate ADCC was significantly higher in individuals who had been treated with ART before seroconversion, compared...

  19. Expression of Glycosylphosphatidylinositol-Anchored CD59 on Target Cells Enhances Human NK Cell-Mediated Cytotoxicity1

    OpenAIRE

    2006-01-01

    NK cell-mediated cytotoxicity of target cells is the result of a balance between the activating and inhibitory signals provided by their respective ligand-receptor interactions. In our current study, we have investigated the significance of CD59 on human target cells in modulating this process. A range of CD59 site-specific Abs were used in NK cytotoxicity blocking studies against the CD59-expressing K562 target cell line. Significantly reduced cytotoxicity was observed in the presence of Abs...

  20. Ultrastructural studies of time-course and cellular specificity of interleukin-1 mediated islet cytotoxicity

    DEFF Research Database (Denmark)

    Mandrup-Poulsen, T; Egeberg, J; Nerup, J

    1987-01-01

    Previous electron-microscopic studies of isolated islets of Langerhans exposed to the monokine interleukin-1 for 7 days have indicated that interleukin-1 is cytotoxic to all islet cells. To study the time-course and possible cellular specificity of interleukin-1 cytotoxicity to islets exposed...... to interleukin-1 for short time periods, isolated rat or human islets were incubated with or without 25 U/ml highly purified human interleukin-1 for 24 h. Samples of rat islets were taken after 5 min, 30 min, 1, 2, 4, 6, 8, 10, 12, 16, 20 and 24 h and samples of human islets after 5 min, 30 min and 24 h...... of incubation and examined by electron microscopy in a blinded fashion. Already after 30 min, accumulation of opaque intracytoplasmic bodies without apparent surrounding membranes, and autophagic vacuoles were seen in about 20% of the beta cells examined in rat islets exposed to interleukin-1. After 16 h...

  1. Cytotoxic T lymphocytes specific for the simian immunodeficiency virus.

    Science.gov (United States)

    Letvin, N L; Schmitz, J E; Jordan, H L; Seth, A; Hirsch, V M; Reimann, K A; Kuroda, M J

    1999-08-01

    A non-human primate model for acquired immunodeficiency syndrome (AIDS), the simian immunodeficiency virus (SIV)-infected rhesus monkey, was used to explore the role of the AIDS virus-specific cytotoxic T-lymphocyte (CTL) response in disease pathogenesis. This CTL response was measured using the major histocompatibility complex (MHC) class I/peptide tetramer technology. Large numbers of tetramer-binding CD8+ T lymphocytes were demonstrable not only in the peripheral blood, but in lymph nodes and even in semen of chronically SIV-infected monkeys. The central role of these effector T lymphocytes in containing SIV spread during primary infection was demonstrated by showing that early SIV clearance during primary infection correlated with the emergence of the tetramer binding CD8+ T lymphocytes and that in vivo depletion of CD8+ lymphocytes eliminated the ability of the infected monkeys to contain SIV replication. These observations suggest that an effective AIDS vaccine should elicit a potent virus-specific CTL response. In fact, a live, recombinant SIV vaccine constructed using the attenuated pox virus vector modified vaccinia Ankara (MVA) elicited a high-frequency CTL response, comparable in magnitude to that elicited by SIV infection itself. This suggests that vaccine modalities such as MVA may prove useful in creating an effective human immunodeficiency virus (HIV) vaccine. These studies also indicate the power of both the SIV/macaque model and MHC class I/peptide tetramers for assessing AIDS vaccine strategies.

  2. Fas involvement in Ca(2+)-independent T cell-mediated cytotoxicity.

    Science.gov (United States)

    Rouvier, E; Luciani, M F; Golstein, P

    1993-01-01

    Mechanisms of T cell-mediated cytotoxicity remain poorly defined at the molecular level. To investigate some of these mechanisms, we used as target cells, on the one hand, thymocytes from lpr and gld mouse mutants, and on the other hand, L1210 cells transfected or not with the apoptosis-inducing Fas molecule. These independent mutant or transfectant-based approaches both led to the conclusion that Fas was involved in the Ca(2+)-independent component of cytotoxicity mediated by at least two sources of T cells, namely nonantigen-specific in vitro activated hybridoma cells, and antigen-specific in vivo raised peritoneal exudate lymphocytes. Thus, in these cases, T cell-mediated cytotoxicity involved transduction via Fas of the target cell death signal.

  3. Targeted cytosine deaminase-uracil phosphoribosyl transferase suicide gene therapy induces small cell lung cancer-specific cytotoxicity and tumor growth delay

    DEFF Research Database (Denmark)

    Christensen, Camilla L; Gjetting, Torben; Poulsen, Thomas Tuxen

    2010-01-01

    Small cell lung cancer (SCLC) is a highly malignant cancer for which there is no curable treatment. Novel therapies are therefore in great demand. In the present study we investigated the therapeutic effect of transcriptionally targeted suicide gene therapy for SCLC based on the yeast cytosine de...

  4. Tracking the elusive cytotoxic T cell response in pigs

    DEFF Research Database (Denmark)

    Jungersen, Gregers; Nielsen, Morten; Overgaard, Nana Haahr;

    Quantitative and qualitative assessment of antigen-specific cytotoxic T cell (CTL) responses in pigs is not a straightforward process. Through the years we have developed a series of reagents, tools and protocols to characterize peptide-specific CTL responses in pigs. The most common recombinant ...... SLA heavy chains were produced and peptide binding motifs were determined by assays measuring the affinity and stability of the peptide-SLA complex (pSLA) interaction. These results have been used to train neural networks to predict the binding of any pSLA (http......://www.cbs.dtu.dk/services/). Recombinant SLA molecules complexed with verified binding peptides can be assembled to SLA multimers for staining of peptide-specific CTLs, and measured by flow cytometry, as we have shown with FMDV and influenza. This, however, requires SLA-matched pigs for which we have developed two methods: a sequence...

  5. Deep immune profiling by mass cytometry links human T and NK cell differentiation and cytotoxic molecule expression patterns.

    Science.gov (United States)

    Bengsch, Bertram; Ohtani, Takuya; Herati, Ramin Sedaghat; Bovenschen, Niels; Chang, Kyong-Mi; Wherry, E John

    2017-03-19

    The elimination of infected or tumor cells by direct lysis is a key T and NK cell effector function. T and NK cells can kill target cells by coordinated secretion of cytotoxic granules containing one or both pore-forming proteins, perforin and granulysin and combinations of granzyme (Gzm) family effector proteases (in humans: Gzm A, B, K, M and H). Understanding the pattern of expression of cytotoxic molecules and the relationship to different states of T and NK cells may have direct relevance for immune responses in autoimmunity, infectious disease and cancer. Approaches capable of simultaneously evaluating expression of multiple cytotoxic molecules with detailed information on T and NK differentiation state, however, remain limited. Here, we established a high dimensional mass cytometry approach to comprehensively interrogate single cell proteomic expression of cytotoxic programs and lymphocyte differentiation. This assay identified a coordinated expression pattern of cytotoxic molecules linked to CD8 T cell differentiation stages. Coordinated high expression of perforin, granulysin, Gzm A, Gzm B and Gzm M was associated with markers of late effector memory differentiation and expression of chemokine receptor CX3CR1. However, classical gating and dimensionality reduction approaches also identified other discordant patterns of cytotoxic molecule expression in CD8 T cells, including reduced perforin, but high Gzm A, Gzm K and Gzm M expression. When applied to non-CD8 T cells, this assay identified different patterns of cytotoxic molecule co-expression by CD56(hi) versus CD56(dim) defined NK cell developmental stages; in CD4 T cells, low expression of cytotoxic molecules was found mainly in TH1 phenotype cells, but not in Tregs or T follicular helper cells (TFH). Thus, this comprehensive, single cell, proteomic assessment of cytotoxic protein co-expression patterns demonstrates specialized cytotoxic programs in T cells and NK cells linked to their differentiation

  6. Flow cytometry evaluation of cell-mediated cytotoxicity.

    Science.gov (United States)

    Zarcone, D; Tilden, A B; Cloud, G; Friedman, H M; Landay, A; Grossi, C E

    1986-11-20

    A novel flow cytometry method for the evaluation of cell-mediated cytotoxicity is described. This method uses flow cytometry analysis to distinguish target cells from effector cells by differences in volume and light scatter characteristics. Non-viable target cells, following their interaction with effector cells, are determined via propidium iodide (PI) dye exclusion and then expressed as a percentage of the total target cell population. This assay is suitable both for analysis of systems which allow recycling of cytotoxic effector cells (total cell cytotoxicity assays, TCCA), and of systems in which recycling does not occur (single cell cytotoxicity assays, SCCA). Natural killer (NK) cell-mediated cytotoxicity evaluated by flow cytometry is significantly correlated with the standard 51Cr release assay. Flow cytometry can also be used to evaluate the competitive inhibition that certain cell types exert on the cell-mediated killing of NK-sensitive targets. A prerequisite for this assay is that competitor cells and target cells are distinguishable through their volume and light scatter characteristics. Advantages and pitfalls of the flow cytometry method are discussed, in comparison with the 51Cr-release assay.

  7. Propranolol sensitizes thyroid cancer cells to cytotoxic effect of vemurafenib.

    Science.gov (United States)

    Wei, Wei-Jun; Shen, Chen-Tian; Song, Hong-Jun; Qiu, Zhong-Ling; Luo, Quan-Yong

    2016-09-01

    Treatment options for advanced metastatic or progressive thyroid cancers are limited. Although targeted therapy specifically inhibiting intracellular kinase signaling pathways has markedly changed the therapeutic landscape, side-effects and resistance of single agent targeted therapy often leads to termination of the treatment. The objective of the present study was to identify the antitumor property of the non-selective β-adrenergic receptor antagonist propranolol for thyroid cancers. Human thyroid cancer cell lines 8505C, K1, BCPAP and BHP27 were used in the present study. Broad β-blocker propranolol and β2-specific antagonist ICI118551, but not β1-specific antagonist atenolol, inhibited the growth of 8505C and K1 cells. Propranolol treatment inhibited growth and induced apoptosis of 8505C cells in vitro and in vivo, which are closely associated with decreased expressions of cyclin D1 and anti-apoptotic Bcl-2. Expression of hexokinase 2 (HK2) and glucose transporter 1 (GLUT1) also decreased following propranolol intervention. 18F-FDG PET/CT imaging of the 8505C xenografts validated shrinkage of the tumors in the propranolol-treated group when compared to the phosphate‑buffered saline treated group. Finally, we found that propranolol can amplify the cytotoxicity of vemurafenib and sensitize thyroid cancer cells to cytotoxic effect of vemurafenib. Our present results suggest that propranolol has potential activity against thyroid cancers and investigation of the combination with targeted molecular therapy for progressive thyroid cancers could be beneficial.

  8. Enhanced cytotoxicity and decreased CD8 dependence of human cancer-specific cytotoxic T lymphocytes after vaccination with low peptide dose.

    Science.gov (United States)

    Lövgren, Tanja; Baumgaertner, Petra; Wieckowski, Sébastien; Devêvre, Estelle; Guillaume, Philippe; Luescher, Immanuel; Rufer, Nathalie; Speiser, Daniel E

    2012-06-01

    In mice, vaccination with high peptide doses generates higher frequencies of specific CD8+ T cells, but with lower avidity compared to vaccination with lower peptide doses. To investigate the impact of peptide dose on CD8+ T cell responses in humans, melanoma patients were vaccinated with 0.1 or 0.5 mg Melan-A/MART-1 peptide, mixed with CpG 7909 and Incomplete Freund's adjuvant. Neither the kinetics nor the amplitude of the Melan-A-specific CD8+ T cell responses differed between the two vaccination groups. Also, CD8+ T cell differentiation and cytokine production ex vivo were similar in the two groups. Interestingly, after low peptide dose vaccination, Melan-A-specific CD8+ T cells showed enhanced degranulation upon peptide stimulation, as assessed by CD107a upregulation and perforin release ex vivo. In accordance, CD8+ T cell clones derived from low peptide dose-vaccinated patients showed significantly increased degranulation and stronger cytotoxicity. In parallel, Melan-A-specific CD8+ T cells and clones from low peptide dose-vaccinated patients expressed lower CD8 levels, despite similar or even stronger binding to tetramers. Furthermore, CD8+ T cell clones from low peptide dose-vaccinated patients bound CD8 binding-deficient tetramers more efficiently, suggesting that they may express higher affinity TCRs. We conclude that low peptide dose vaccination generated CD8+ T cell responses with stronger cytotoxicity and lower CD8 dependence.

  9. Reduced LAK cytotoxicity of peripheral blood mononuclear cells in patients with bladder cancer

    DEFF Research Database (Denmark)

    Hermann, G G; Petersen, K R; Steven, K

    1990-01-01

    were analyzed using monoclonal antibodies against T cells, natural killer (NK) -cells, monocytes, and activation markers. The cytotoxicities of US-PBMC, PS-PBMC, and LAK cells were all significantly lower in the cancer patients than in the controls (P less than 0.05). The percentages of PBMC positive......The cytotoxicity of unstimulated peripheral blood mononuclear cells (US-PBMC), phytohemagglutinin (PHA)-stimulated PBMC (PS-PBMC) and interleukin-2 (IL-2)-activated PBMC (LAK cells) was assessed in patients with noninvasive and invasive transitional-cell bladder cancer and compared with those...... determined in healthy controls. The differences in the cytotoxicities were correlated with specific changes in the subsets of peripheral blood mononuclear cells (PBMC). PBMC from 37 patients and 13 healthy controls were tested against the bladder cancer cell line T24 in 51Cr-release assays. The PBMC subsets...

  10. Cytotoxic T-lymphocyte clones, established by stimulation with the HLA-A2 binding p5365-73 wild type peptide loaded on dendritic cells In vitro, specifically recognize and lyse HLA-A2 tumour cells overexpressing the p53 protein

    DEFF Research Database (Denmark)

    Barfoed, Annette Malene; Petersen, T R; Kirkin, A F;

    2000-01-01

    to carry identical T-cell receptors. The CTL clone, 2D9, was shown to specifically lyse the HLA-A*0201+ squamous carcinoma cell line SCC9 and the breast cancer cell line MDA-MB-468. Our data demonstrate that human peripheral blood lymphocytes from normal healthy individuals comprise T cells capable...... of recognizing p53 derived wild type (self) peptides. Furthermore, the capacity of R9V specific T cell clones to exert HLA restricted cytotoxicity, argues that the R9V peptide is naturally presented on certain cancer cells. This supports the view that p53 derived wild type peptides might serve as candidate...... target antigens for the immunotherapeutic treatment of cancer....

  11. Flow cytometric assay detecting cytotoxicity against human endogenous retrovirus antigens expressed on cultured multiple sclerosis cells

    DEFF Research Database (Denmark)

    Møller-Larsen, A; Brudek, T; Petersen, T

    2013-01-01

    as control antibody. Without antibodies this system is suitable for analyses of natural killer cell activity. In optimization of the assay we have used effector lymphocytes from healthy donors. The most effective effector cells are CD56(+) cells. CD8(+) T cells also express CD107a in ADCC. Using the adapted......Damage of target cells by cytotoxicity, either mediated by specific lymphocytes or via antibody-dependent reactions, may play a decisive role in causing the central nervous system (CNS) lesions seen in multiple sclerosis (MS). Relevant epitopes, antibodies towards these epitopes and a reliable...... assay are all mandatory parts in detection and evaluation of the pertinence of such cytotoxicity reactions. We have adapted a flow cytometry assay detecting CD107a expression on the surface of cytotoxic effector cells to be applicable for analyses of the effect on target cells from MS patients...

  12. Demethylating agent decitabine induces autologous cancer testis antigen specific cytotoxic T lymphocytes in vivo

    Institute of Scientific and Technical Information of China (English)

    ZHOU Ji-hao; YAO Yu-shi; WANG Li-xin; WANG Jia; LI Yong-hui; JIANG Meng-meng; ZHOU Min-hang

    2013-01-01

    Background Cancer testis antigens (CTAs) are a novel group of tumor associated antigens.Demethylating agent decitabine was reported to be able to up-regulate CTAs through its hypomethylation mechanism,thus enhance the immunogenicity of leukemia cells.However,few researches have ever focused on the questions that whether this immunostimulatory effect of decitabine could induce autologous CTA specific cytotoxic T lymphocytes (CTLs) in vivo,and if so,whether this effect contributes to disease control.In this study,we aimed to show that decitabine could induce specific autologous CTLs against some mouse CTAs in leukemia cells in vitro and in vivo.Methods Several mouse CTAs were screened by RT-PCR.CTL specific to one of the CTAs named P1A was detected and sorted by P1A specific dimer by flow cytometry.The activity of specific CTLs was measured by real time RT-PCR.Results We firstly screened expression of some CTAs in mouse leukemia cells before and after decitabine treatment and found that decitabine treatment did up-regulate expression of many CTAs.Then we measured the CTLs' activity specific to a mouse CTA P1A in vivo and showed that this activity increased after decitabine treatment.Finally,we sorted these in vivo induced P1A specific CTLs by flow cytometry and demonstrated their cytotoxicity against decitabine treated leukemia cells.Conclusions Our study showed the autologous immune response induced by decitabine in vivo.And more importantly,we firstly proved that this response may contribute to disease control.We believe that this immunostimulatory effect is another anti-cancer mechanism of decitabine,and this special effect would inspire new applications of decitabine in the field of leukemia treatment in the future.

  13. Rhamnolipids elicit the same cytotoxic sensitivity between cancer cell and normal cell by reducing surface tension of culture medium.

    Science.gov (United States)

    Jiang, Lifang; Shen, Chong; Long, Xuwei; Zhang, Guoliang; Meng, Qin

    2014-12-01

    Biosurfactant rhamnolipids have been claimed to show biological activities of inhibiting the proliferation of cancer cells. In this study, the cytotoxicity of rhamnolipids was examined on four cancer cells (HepG2, Caco-2, Hela, MCF-7 cells) and two normal cells (HK-2 cell, primary hepatocyte). Interestingly, both cancer cells and normal cells exhibited similar sensitivities to the addition of rhamnolipids in culture medium, and the cytotoxicity was largely attenuated by the presence of fetal bovine serum (FBS) in culture medium. In correlation of the mono-/di-rhamnolipid cytotoxicity with the surface tension of culture medium, it was found that rhamnolipids triggered cytotoxicity whenever the surface tension of culture medium decreased below 41 mN/m irrespective of the FBS content in culture medium, cell line, or rhamnolipid congener. Similarly, each chemical surfactant (Tween-80, sodium dodecyl sulfate, and sodium dodecyl benzene sulfonate) could cause cytotoxicity on HepG2 cells whenever its addition made the surface tension under 41 mN/m in culture medium with or without the presence of FBS. It seems that rhamnolipids, like chemical surfactants, exhibited cytotoxicity by reducing the surface tension of culture medium rather than by changing its specific molecular structure, which had no selection on tumor cells. This study could offer helps to correct the misleading biological activity of rhamnolipids and to avoid the possible large wastes of time and expenses on developing the applications in antitumor drugs.

  14. Human heat shock protein-specific cytotoxic T lymphocytes display potent antitumour immunity in multiple myeloma

    Science.gov (United States)

    Li, Rong; Qian, Jianfei; Zhang, Wenhao; Fu, Weijun; Du, Juan; Jiang, Hua; Zhang, Hui; Zhang, Chunyang; Xi, Hao; Yi, Qing; Hou, Jian

    2014-01-01

    Tumour cell–derived heat shock proteins (HSPs) are used as vaccines for immunotherapy of cancer patients. However, it is proposed that the peptide chaperoned on HSPs, not HSPs themselves, elicited a potent immune response. Given that HSPs are highly expressed by most myeloma cells and vital to myeloma cell survival, we reasoned that HSPs themselves might be an ideal myeloma antigen. In the present study, we explored the feasibility of targeting HSPs themselves for treating multiple myeloma. We identified and chose HLA-A*0201-binding peptides from human HSPB1 (HSP27) and HSP90AA1 (HSP90), and confirmed their immunogenicity in HLA-A*0201 transgenic mice. Dendritic cells pulsed with HSPB1 and HSP90AA1 peptides were used to stimulate peripheral blood mononuclear cells from healthy volunteers and myeloma patients to generate HSP peptide-specific cytotoxic T lymphocytes (CTLs). HSP peptide-specific CTLs efficiently lysed HLA-A*0201+ myeloma cells (established cell lines and primary plasma cells) but not HLA-A*0201− myeloma cells in vitro, indicating that myeloma cells naturally express HSP peptides in the context of major histocompatibility complex class I molecules. More importantly, HSP peptide-specific CTLs effectively reduced tumour burden in the xenograft mouse model of myeloma. Our study clearly demonstrated that HSPs might be novel tumour antigens for immunotherapy of myeloma. PMID:24824351

  15. Antigen processing influences HIV-specific cytotoxic T lymphocyte immunodominance

    DEFF Research Database (Denmark)

    Tenzer, Stefan; Wee, Edmund; Burgevin, Anne

    2009-01-01

    Although cytotoxic T lymphocytes (CTLs) in people infected with human immunodeficiency virus type 1 can potentially target multiple virus epitopes, the same few are recognized repeatedly. We show here that CTL immunodominance in regions of the human immunodeficiency virus type 1 group-associated ......Although cytotoxic T lymphocytes (CTLs) in people infected with human immunodeficiency virus type 1 can potentially target multiple virus epitopes, the same few are recognized repeatedly. We show here that CTL immunodominance in regions of the human immunodeficiency virus type 1 group...

  16. Virus elimination in acute lymphocytic choriomeningitis virus infection. Correlation with virus-specific delayed-type hypersensitivity rather than cytotoxicity

    DEFF Research Database (Denmark)

    Thomsen, Allan Randrup; Volkert, M; Bro-Jørgensen, K

    1983-01-01

    The immunological effector mechanism responsible for the elimination of virus in murine acute non-fatal extracranial lymphocytic choriomeningitis virus infection was studied. In this infection virus clearance is generally regarded as the result of a direct action of virus-specific cytotoxic T cells...

  17. MODERATE CYTOTOXICITY OF PROANTHOCYANIDINS TO HUMAN TUMOR-CELL LINES

    NARCIS (Netherlands)

    KOLODZIEJ, H; HABERLAND, C; WOERDENBAG, HJ; KONINGS, AWT

    1995-01-01

    In the present study the cytotoxicity of 16 proanthocyanidins was evaluated in GLC(4), a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values ranging from 18 to >200 mu m following continuous

  18. Cytotoxic effects of octenidine mouth rinse on human fibroblasts and epithelial cells - an in vitro study.

    Science.gov (United States)

    Schmidt, J; Zyba, V; Jung, K; Rinke, S; Haak, R; Mausberg, R F; Ziebolz, D

    2016-01-01

    This study compared the cytotoxicity of a new octenidine mouth rinse (MR) against gingival fibroblasts and epithelial cells with different established MRs. The following MRs were used: Octenidol (OCT), Chlorhexidine 0.2% (CHX), Listerine (LIS), Meridol (MER), Betaisodona (BET); and control (medium only). Human primary gingiva fibroblasts and human primary nasal epithelial cells were cultivated in cell-specific media (2 × 10(5) cells/ml) and treated with MR for 1, 5, and 15 min. Each test was performed 12 times. Metabolism activity was measured using a cytotoxicity assay. A cellometer analyzed cell viability, cell number, and cell diameter. The data were analyzed by two-way analysis of variance with subsequent Dunnett's test and additional t-tests. The cytotoxic effects of all MRs on fibroblasts and epithelial cells compared to the control depended on the contact time (p 0.005). Cell numbers of both cell types at all contact times revealed that OCT showed a less negative effect (p > 0.005), especially for epithelial cells compared to CHX after 15 min (p 0.005), but MER showed less influence than OCT in epithelial cells (p < 0.005). OCT is a potential alternative to CHX regarding cytotoxicity because of its lower cell-toxic effect against fibroblasts and epithelial cells.

  19. Real time assays for quantifying cytotoxicity with single cell resolution.

    Directory of Open Access Journals (Sweden)

    Sonny C Hsiao

    Full Text Available A new live cell-based assay platform has been developed for the determination of complement dependent cytotoxicity (CDC, antibody dependent cellular cytotoxicity (ADCC, and overall cytotoxicity in human whole blood. In these assays, the targeted tumor cell populations are first labeled with fluorescent Cell Tracker dyes and immobilized using a DNA-based adhesion technique. This allows the facile generation of live cell arrays that are arranged arbitrarily or in ordered rectilinear patterns. Following the addition of antibodies in combination with serum, PBMCs, or whole blood, cell death within the targeted population can be assessed by the addition of propidium iodide (PI as a viability probe. The array is then analyzed with an automated microscopic imager. The extent of cytotoxicity can be quantified accurately by comparing the number of surviving target cells to the number of dead cells labeled with both Cell Tracker and PI. Excellent batch-to-batch reproducibility has been achieved using this method. In addition to allowing cytotoxicity analysis to be conducted in real time on a single cell basis, this new assay overcomes the need for hazardous radiochemicals. Fluorescently-labeled antibodies can be used to identify individual cells that bear the targeted receptors, but yet resist the CDC and ADCC mechanisms. This new approach also allows the use of whole blood in cytotoxicity assays, providing an assessment of antibody efficacy in a highly relevant biological mixture. Given the rapid development of new antibody-based therapeutic agents, this convenient assay platform is well-poised to streamline the drug discovery process significantly.

  20. Establishment of Vero cells/MTT method for determination of specific cytotoxicity of diphtheria toxin%检测白喉毒素特异性细胞毒性的Vero细胞/MTT法的建立

    Institute of Scientific and Technical Information of China (English)

    邓杰; 张珂; 袁涛; 张雪梅

    2013-01-01

    目的 建立检测白喉毒素(diphtheria toxin,DT)特异性细胞毒性的Vero细胞/MTT法.方法 用MEM培养液对DT进行不同倍数的预稀释,用目测法确定DT的最适预稀释倍数.用建立的方法检测以最适预稀释倍数稀释的DT,以确定该法的灵敏度和细胞病变半数抑制浓度(IC50),同时绘制剂量-反应曲线以验证该法的重复性.用建立的方法于不同pH、盐浓度或蔗糖浓度条件下检测DT,计算DT回收率以验证该法的耐用性.结果 目测法确定的DT最适预稀释倍数为106倍.建立的方法的平均检测灵敏度为(6.01±1.44)×10-5 lf/ml DT,细胞病变半数抑制率对数值(log IC50)范围为5.02 ~ 5.82,变异系数为5.82%,该法具有较好的重复性.用建立的方法检测不同条件下的DT显示,DT的平均回收率为93.7% ~ 110.4%,该法具有较好的耐用性.结论 Vero细胞/MTr法可用于检测DT特异性细胞毒性.%Objective To establish Vero cells / MTT method for determination of specific cytotoxicity of diphtheria toxin (DT).Methods DT was diluted multiply with MEM medium.The optimal pre-dilution multiple of DT was detected by microscopic observation.DT diluted in definitive time was detected by the established method to determine the sensitivity and half cytopathic inhibition concentration (IC50) of the method.The dosage-effect curves were drawn to validate repeatability of the method.DTs under different pH,salt concentration or sucrose concentration conditions were detected by the established method,and recovery rates of DT were calculated to validate durability of the method.Results The optimal pre-dilution multiple of DT detected by microscopic observation was 106 time.The sensitivity of the established method was (6.01 ± 1.44) × 10-5 lf/ml DT,logIC50 ranged from 5.02 to 5.82,and its variable coefficient was 5.82%.The average recovery rates of DT under different conditions were 93.7%-110.4%.Conclusion Vero cells/MTT method has good

  1. Pathogenic Trichomonas vaginalis cytotoxicity to cell culture monolayers.

    Science.gov (United States)

    Alderete, J F; Pearlman, E

    1984-04-01

    Exposure of monolayer cultures of human urogenital and vaginal (HeLa), human epithelial (HEp-2), normal baboon testicular (NBT), and monkey kidney (Vero) cells to live pathogenic Trichomonas vaginalis resulted in extensive disruption of monolayers. Trypan blue was taken up by all host cells released from cell monolayers, which indicated irreversible damage of these cell types by trichomonads. Time and dose related data on cytotoxicity kinetics were obtained using increasing ratios of parasites to cells. All cell types were most sensitive to trichomonads at a multiplicity of infection of one. Release of tritiated thymidine (3H-thymidine) of the deoxyribonucleic acid (DNA) of prelabelled host cells after incubation with T vaginalis corroborated that extensive cytotoxicity was caused by pathogenic trichomonads in man. Only living parasites were cytotoxic, and no trichomonal toxic products were implicated in disruption of the cell monolayer cultures. A pathogenic bovine trichomonad, Tritrichomonas foetus KV-1, produced half as much cell damage as did T vaginalis. Trichomonas tenax, a non-pathogenic member of the normal flora of the oral cavity in man, produced no measurable cytotoxicity to HeLa cells when compared with the pathogenic human trichomonads.

  2. Surface charge-specific cytotoxicity and cellular uptake of tri-block copolymer nanoparticles

    NARCIS (Netherlands)

    Bhattacharjee, S.; Ershov, D.S.; Gucht, van der J.; Alink, G.M.; Rietjens, I.; Zuilhof, H.; Marcelis, A.T.M.

    2013-01-01

    A series of monodisperse (45 ± 5 nm) fluorescent nanoparticles from tri-block copolymers (polymeric nanoparticles (PNPs)) bearing different surface charges were synthesised and investigated for cytotoxicity in NR8383 and Caco-2 cells. The positive PNPs were more cytotoxic and induced a higher intrac

  3. HLA class I-restricted cytotoxic T-cell epitopes of the respiratory syncytial virus fusion protein

    NARCIS (Netherlands)

    A.H. Brandenburg (Afke); L. de Waal (Leon); H.H. Timmerman (Helga); P. Hoogerhout (Peter); R.L. de Swart (Rik); A.D.M.E. Osterhaus (Albert)

    2000-01-01

    textabstractVirus-specific cytotoxic T lymphocytes (CTL) play a major role in the clearance of respiratory syncytial virus (RSV) infection. We have generated cytotoxic T-cell clones (TCC) from two infants who had just recovered from severe RSV infection. These TCC were

  4. Melatonin Cytotoxicity Is Associated to Warburg Effect Inhibition in Ewing Sarcoma Cells.

    Directory of Open Access Journals (Sweden)

    Ana M Sanchez-Sanchez

    Full Text Available Melatonin kills or inhibits the proliferation of different cancer cell types, and this is associated with an increase or a decrease in reactive oxygen species, respectively. Intracellular oxidants originate mainly from oxidative metabolism, and cancer cells frequently show alterations in this metabolic pathway, such as the Warburg effect (aerobic glycolysis. Thus, we hypothesized that melatonin could also regulate differentially oxidative metabolism in cells where it is cytotoxic (Ewing sarcoma cells and in cells where it inhibits proliferation (chondrosarcoma cells. Ewing sarcoma cells but not chondrosarcoma cells showed a metabolic profile consistent with aerobic glycolysis, i.e. increased glucose uptake, LDH activity, lactate production and HIF-1α activation. Melatonin reversed Ewing sarcoma metabolic profile and this effect was associated with its cytotoxicity. The differential regulation of metabolism by melatonin could explain why the hormone is harmless for a wide spectrum of normal and only a few tumoral cells, while it kills specific tumor cell types.

  5. Concanavalin A-mediated in vitro activation of a secondary cytotoxic T-cell response in virus-primed splenocytes

    DEFF Research Database (Denmark)

    Thomsen, Allan Randrup; Jensen, B L

    1980-01-01

    In a recent report it was shown that what appeared to be secondary cytotoxic T cells could be obtained from lymphocytic choriomeningitis virus (LCMV)-primed splenocytes after stimulation in vitro with the non-specific T cell mitogen concanavalin A (Con A). The present experiments attempt to chara......In a recent report it was shown that what appeared to be secondary cytotoxic T cells could be obtained from lymphocytic choriomeningitis virus (LCMV)-primed splenocytes after stimulation in vitro with the non-specific T cell mitogen concanavalin A (Con A). The present experiments attempt...... to characterize further these effector cells and, in particular, to establish whether the Con A-activated cytotoxic effectors are qualitatively different from the secondary cytotoxic T cells induced by restimulation with the homologous antigen. It was found that: (1) in vitro activation with Con A could......, since no evidence was found to indicate a role for other cell types or soluble (cytotoxic or arming) factors; (4) cytotoxicity was specific with regard to both virus and 'self'. By comparison with previous data on LCMV-induced cytotoxic T cells, it is concluded that Con A induces the generation...

  6. UVA radiation augments cytotoxic activity of psoralens in melanoma cells.

    Science.gov (United States)

    Wrześniok, Dorota; Beberok, Artur; Rok, Jakub; Delijewski, Marcin; Hechmann, Anna; Oprzondek, Martyna; Rzepka, Zuzanna; Bacler-Żbikowska, Barbara; Buszman, Ewa

    2017-07-01

    Melanoma is an aggressive form of skin cancer. The aim of the study was to evaluate the influence of UVA radiation and psoralens: 5-methoxypsoralen (5-MOP) or 8-methoxypsoralen (8-MOP) on melanoma cells viability. The amelanotic C32 and melanotic COLO829 human melanoma cell lines were exposed to increasing concentrations of psoralens (0.1-100 μM) in the presence or absence of UVA radiation. Cell viability was evaluated by the WST-1 assay. We demonstrated that 8-MOP, in contrast to 5-MOP, has no cytotoxic effect on both melanoma cell lines. Simultaneous exposure of cells to 8-MOP and UVA radiation caused significant cytotoxic response in C32 cells where the EC50 value was estimated to be 131.0 μM (UVA dose: 1.3 J/cm(2)) and 105.3 μM (UVA dose: 2.6 J/cm(2)). The cytotoxicity of 5-MOP on both C32 and COLO829 cells was significantly augmented by UVA radiation - the EC50 was estimated to be 22.7 or 7.9 μM (UVA dose: 1.3 J/cm(2)) and 24.2 or 7.0 μM (UVA dose: 2.6 J/cm(2)), respectively. The demonstrated high cytotoxic response after simultaneous exposure of melanoma cells to psoralens and UVA radiation in vitro suggests the usefulness of PUVA therapy to treat melanoma in vivo.

  7. Human immunodeficiency virus type 1 infection of antigen-specific CD4 cytotoxic T lymphocytes.

    Science.gov (United States)

    Robbins, P A; Roderiquez, G L; Peden, K W; Norcross, M A

    1998-11-01

    The effect of macrophage (M)-tropic and T cell line (T)-tropic human immunodeficiency virus type 1 (HIV-1) infection on antigen-specific CD4 cytotoxic T lymphocytes (CTLs) has been studied using a CD4 CTL line specific for a peptide from influenza B virus hemagglutinin. In the absence of antigen presentation, the production of CC chemokines was low. Both the M-tropic HIV-1 strain (HIV-1AD) and the T-tropic HIV-1 strain (HIV-1LAI) established productive infections in the CD4 CTLs, decreasing antigen-specific cytotoxicity. Peptide presented to the CD4 CTLs increased their secretion of RANTES and MIP-1beta, suppressed M-tropic HIV-1 replication, downmodulated CCR5 expression, and preserved CTL recognition. The suppression of M-tropic HIV-1 replication and downmodulation of the CCR5 receptor likely resulted from CC chemokine secretion since antibodies to CC chemokines restored M-tropic HIV-1 replication. Antigen presentation did not protect CD4 CTLs from T-tropic HIV-1 infection or preserve their CTL recognition. Thus, these CD4 CTLs do not make suppressor factors that inhibit the T-tropic HIV-1LAI isolate. The results indicate that these CD4 CTLs can either harbor or suppress M-tropic HIV-1 infection, depending on whether antigen is present. CD4 CTLs might therefore provide some protection in the early stages of HIV-1 infection when M-tropic isolates are present.

  8. A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts

    Directory of Open Access Journals (Sweden)

    Bendinelli Mauro

    2009-03-01

    Full Text Available Abstract Background Cytotoxic T lymphocytes play a crucial role in the immunological control of microbial infections and in the design of vaccines and immunotherapies. Measurement of cytotoxic T lymphocyte activity requires that the test antigen is presented by target cells having the same or compatible class I major hystocompatibility complex antigens as the effector cells. Conventional assays use target cells labeled with 51chromium and infer cytotoxic T lymphocyte activity by measuring the isotope released by the target cells lysed following incubation with antigen-specific cytotoxic T lymphocytes. This assay is sensitive but needs manipulation and disposal of hazardous radioactive reagents and provides a bulk estimate of the reporter released, which may be influenced by spontaneous release of the label and other poorly controllable variables. Here we describe a novel method for producing target in outbred hosts and assessing cytotoxic T lymphocyte activity by flow cytometry. Results The method consists of culturing skin fibroblasts, immortalizing them with a replication defective clone of simian virus 40, and finally transducing them with a bicistronic vector encoding the target antigen and the reporter green fluorescent protein. When used in a flow cytometry-based assay, the target cells obtained with this method proved valuable for assessing the viral envelope protein specific cytotoxic T lymphocyte activity in domestic cats acutely or chronically infected with feline immunodeficiency virus, a lentivirus similar to human immunodeficiency virus and used as animal model for AIDS studies. Conclusion Given the versatility of the bicistronic vector used, its ability to deliver multiple and large transgenes in target cells, and its extremely wide cell specificity when pseudotyped with the vesicular stomatitis virus envelope protein, the method is potentially exploitable in many animal species.

  9. Effects of different transferrin forms on transferrin receptor expression, iron uptake, and cellular proliferation of human leukemic HL60 cells. Mechanisms responsible for the specific cytotoxicity of transferrin-gallium

    Energy Technology Data Exchange (ETDEWEB)

    Chitambar, C.R.; Seligman, P.A.

    1986-12-01

    We have previously shown that human leukemic cells proliferate normally in serum-free media containing various transferrin forms, but the addition of transferrin-gallium leads to inhibition of cellular proliferation. Because gallium has therapeutic potential, the effects of transferrin-gallium on leukemic cell proliferation, transferrin receptor expression, and cellular iron utilization were studied. The cytotoxicity of gallium is considerably enhanced by its binding to transferrin and cytotoxicity can be reversed by transferrin-iron but not by other transferrin forms. Exposure to transferrin-gallium leads to a marked increase in cell surface transferrin binding sites, but despite this, cellular /sup 59/Fe incorporation is inappropriately low. Although shunting of transferrin-gallium to another cellular compartment has not been ruled out, other studies suggest that transferrin-gallium impairs intracellular release of /sup 59/Fe from transferrin by interfering with processes responsible for intracellular acidification. These studies, taken together, demonstrate that inhibition of cellular iron incorporation by transferrin-gallium is a prerequisite for inhibition of cellular proliferation.

  10. Natural killer cell cytotoxicity assay with time-resolved fluorimetry

    Institute of Scientific and Technical Information of China (English)

    李建中; 章竹君; 金伯泉; 田方

    1996-01-01

    A new time-resolved fluorimetric method for the measurement of natural killer (NK) cell cytotoxicity has been developed by labelling the target cell K562 with a new synthesized fluorescence marker KLUK. The method has advantages of higher sensitivity, time-saving, good reproducibility and has no radioactivity problems. A satisfactory result is obtained by comparing it with 51Cr release method. It demonstrates that the new marker provides an alternative to currently used radioactive markers for the assessment of in vitro cellular cytotoxicity.

  11. Differential cytotoxicity of copper ferrite nanoparticles in different human cells.

    Science.gov (United States)

    Ahmad, Javed; Alhadlaq, Hisham A; Alshamsan, Aws; Siddiqui, Maqsood A; Saquib, Quaiser; Khan, Shams T; Wahab, Rizwan; Al-Khedhairy, Abdulaziz A; Musarrat, Javed; Akhtar, Mohd Javed; Ahamed, Maqusood

    2016-10-01

    Copper ferrite nanoparticles (NPs) have the potential to be applied in biomedical fields such as cell labeling and hyperthermia. However, there is a lack of information concerning the toxicity of copper ferrite NPs. We explored the cytotoxic potential of copper ferrite NPs in human lung (A549) and liver (HepG2) cells. Copper ferrite NPs were crystalline and almost spherically shaped with an average diameter of 35 nm. Copper ferrite NPs induced dose-dependent cytotoxicity in both types of cells, evident by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide and neutral red uptake assays. However, we observed a quite different susceptibility in the two kinds of cells regarding toxicity of copper ferrite NPs. Particularly, A549 cells showed higher susceptibility against copper ferrite NP exposure than those of HepG2 cells. Loss of mitochondrial membrane potential due to copper ferrite NP exposure was observed. The mRNA level as well as activity of caspase-3 enzyme was higher in cells exposed to copper ferrite NPs. Cellular redox status was disturbed as indicated by induction of reactive oxygen species (oxidant) generation and depletion of the glutathione (antioxidant) level. Moreover, cytotoxicity induced by copper ferrite NPs was efficiently prevented by N-acetylcysteine treatment, which suggests that reactive oxygen species generation might be one of the possible mechanisms of cytotoxicity caused by copper ferrite NPs. To the best of our knowledge, this is the first report showing the cytotoxic potential of copper ferrite NPs in human cells. This study warrants further investigation to explore the mechanisms of differential toxicity of copper ferrite NPs in different types of cells. Copyright © 2016 John Wiley & Sons, Ltd.

  12. Cytotoxicity and genotoxicity of phenazine in two human cell lines.

    Science.gov (United States)

    McGuigan, Claire F; Li, Xing-Fang

    2014-06-01

    Phenazine was recently identified as a drinking water disinfection byproduct (DBP), but little is known of its toxic effects. We examined in vitro cytotoxicity and genotoxicity of phenazine (1.9-123 μM) in HepG2 and T24 cell lines. Cytotoxicity was determined by an impedance-based real-time cell analysis instrument. The BrdU (5-bromo-2'-deoxyuridine) proliferation and MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assays were used to examine mechanisms of cytotoxicity. Genotoxicity was determined using the alkaline comet assay. Concentration-dependent cytotoxicity was observed in HepG2 cells, primarily due to an antiproliferative effect (BrdU 24 h IC50: 11 μM; 48 h IC50: 7.8 μM) observed as low as 1.9 μM. T24 cells experienced a minor antiproliferative effect (BrdU 24 h IC50: 47 μM; 48 h IC50: 17 μM). IC50 values for HepG2 proliferation and viability were 54-77% lower compared to T24 cells. In both cell lines, IC50 values for proliferation were 66-90% lower than those for viability. At phenazine concentrations producing equivalent cytotoxicity, HepG2 cells (1.9-30.8 μM) experienced no significant genotoxic effects, while T24 cells (7.7-123 μM) experienced significant genotoxicity at ⩾61.5 μM. While these effects were seen at phenazine concentrations above those found in disinfected water, the persistence of the antiproliferative effect and the differential toxicity in each cell line deserves further study.

  13. Transfection of NY-ESO-1 specific TCR improves the specific cytotoxicity of human PBMC to NY-ESO-1 positive tumor cells%转染NY-ESO-1特异性TCR增强人PBMC对NY-ESO-1阳性肿瘤细胞的特异性细胞毒性

    Institute of Scientific and Technical Information of China (English)

    郭佳; 田洲; 顾娜; 徐珩; 闾军

    2013-01-01

    Objective To study whether NY-ESO-1-specific TCR gene transduced into human PBMC (Peripheral Blood Mononuclear Cell, PBMC) can improve its abilities of specifically recognizing and killing tumor cells. Methods pCDNA3. 1-ESO-TCR plasmid was electroporately transferred into PBMC separated from healthy people in vitro and confirmed by RT-PCR. The phenotype analysis after eletroporation was measured by flow cytometry method. The NY-ESO-1 specific peptide (pl57-165) was added into the culture of electroporated PBMC, the IFN-γ level secreted by electroporated PBMC was detected by ELISPOT assay. The specific cytotoxicity in vitro was detected by real time cell analysis (ACEA biosciences). Results The NY-ESO-1-specific TCR fragments in electroporated PBMC were detected by RT-PCR. The specific expression of NY-ESO-TCR in transferred PBMC was significantly higher than that in PBMC transfered with empty vector (P < 0. 05). The positive dots of IFN-γ secretion in transferred PBMC stimulated by peptide pl57-165 was significantly more than that in PBMC transfered with empty vector (P <0. 05); The specific cytotoxicity of transferred PBMC was obviously enhanced than PBMC transduced with empty vector. Conclusions The specific cytotoxicity of PBMC against NY-ESO-1 positive HepG2 cells is elevated by transduced PBMC with NY-ESO-1 specific TCR, which may stimulate more researches about adoptive immunotherapy of hepatocellular carcinoma.%目的 探讨体外转导NY-ESO-1特异性T细胞受体至人外周血淋巴细胞中,是否可增强转导后细胞体外特异性识别并杀伤肿瘤细胞的能力.方法 将NY-ESO-1特异性TCR质粒(pCDNA3.1-ESO-TCR)体外电转入新分离的正常人PBMC中,RT-PCR法鉴定转导是否成功;采用流式细胞仪分析转导后PBMC表型;用特异性NY-ESO-1b抗原肽(p157-165)刺激转导PBMC后,用ELISPOT法检测PBMC分泌IFN-γ的能力,用实时无标记动态细胞分析仪检测PBMC特异性细胞毒性作用.结果 电转后PBMC

  14. Cytotoxicity of lymphocytes from melanoma patients against autologous tumor cells and its potentiation in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Bykovskaya, S.N.; Iobadze, M.S.; Kupriyanova, T.A.; Demidov, L.V.

    1987-06-01

    The specific and natural cytotoxicity of peripheral blood lymphocytes from patients with melanomas was compared and stimulation with autologous tumor cells or a pool of allogeneic lymphocytes from five healthy blood donors also was used to potentiate the specific antitumor activity of the patients' lymphocytes. To assess cytolytic ability, cells of an autologous tumor, cells of the K-562 line, autologous peripheral blood lymphocytes, and blast cells obtained from these lymphocytes after stimulation by phytohemagglutinin were used as the target cells. The target cells were incubated in a medium containing sodium chromate and were labelled with the chromium 51 isotope.

  15. Cytotoxicity Effects of Amoora rohituka and chittagonga on Breast and Pancreatic Cancer Cells

    Directory of Open Access Journals (Sweden)

    Leo L. Chan

    2011-01-01

    Full Text Available Chemotherapeutic agents for cancer are highly toxic to healthy tissues and hence alternative medicine avenues are widely researched. Majority of the recent studies on alternative medicine suggested that Amoora rohituka possesses considerable antitumor and antibacterial properties. In this work, rohituka and chittagonga, fractionated with petroleum ether, dichloromethane, and ethanol, were explored for their anticancer potential against two breast cancer (MCF-7 and HTB-126 and three pancreatic cancer (Panc-1, Mia-Paca2, and Capan1. The human foreskin fibroblast, Hs68, was also included. Cytotoxicity of each extract was analyzed using the MTT assay and label-free photonic crystal biosensor assay. A concentration series of each extract was performed on the six cell lines. For MCF-7 cancer cells, the chittagonga (Pet-Ether and CH2Cl2 and rohituka (Pet-Ether extracts induced cytotoxicity; the chittagonga (EtoAC and rohituka (MeOH extracts did not induce cytotoxicity. For HTB126, Panc-1, Mia-Paca2, and Capan-1 cancer cells, only the chittagonga CH2Cl2 extract showed a significant cytotoxic effect. The extracts were not cytotoxic to normal fibroblast Hs68 cells, which may be correlated to the specificity of Amoora extracts in targeting cancerous cells. Based on these results, further examination of the potential anticancer properties Amoora species and the identification of the active ingredients of these extracts is warranted.

  16. Fluorophore-tagged pharmacophores for antitumor cytotoxicity: Modified chiral lipidic dialkynylcarbinols for cell imaging.

    Science.gov (United States)

    Listunov, Dymytrii; Mazères, Serge; Volovenko, Yulian; Joly, Etienne; Génisson, Yves; Maraval, Valérie; Chauvin, Remi

    2015-10-15

    Chiral lipidic dialkynylcarbinols (DACs), recently highlighted as antitumoral pharmacophores, have been conjugated to difluoroboron-dipyrromethene (bodipy), 7-hydroxy-coumarine, and 7-nitro-benzoxadiazole (NBD) fluorophore motifs through triazole clips. The labeled lipids preserve cytotoxic activity against HCT116 cells, and fluorescence microscopy of the stained cells showed clear signals in the intra-cellular membrane system. While the bodipy conjugate also labels lipid droplets very brightly, as expected, the coumarine and NBD probes appear as promising specific tools for the identification of the intra-cellular targets of DACs' cytotoxicity.

  17. Spontaneous cytotoxic T-Cell reactivity against indoleamine 2,3-dioxygenase-2

    DEFF Research Database (Denmark)

    Sørensen, Rikke Bæk; Køllgaard, Tania; Andersen, Rikke Sick;

    2011-01-01

    by the D stereoisomer of the IDO blocker 1-methyl-tryptophan (1MT), which tends to be more active than the L-isomer in a variety of biological assays for IDO function, suggests that IDO2 may be important to sustain immune escape and growth of tumors. Especially, D-1MT heightens chemotherapeutic efficacy...... in mouse models of cancer in a nontoxic fashion. Here, we describe the immunogenicity of IDO2 by showing the presence of spontaneous cytotoxic T-cell reactivity against IDO2 in peripheral blood of both healthy donors and cancer patients. Furthermore, we show that these IDO2-specific T cells are cytotoxic...

  18. Peptide microarrays for the profiling of cytotoxic T-lymphocyte activity using minimum numbers of cells.

    NARCIS (Netherlands)

    Hoff, A.; Bagu, A.C.; Andre, T.; Roth, G.; Wiesmuller, K.H.; Guckel, B.; Brock, R.E.

    2010-01-01

    The identification of epitopes that elicit cytotoxic T-lymphocyte activity is a prerequisite for the development of cancer-specific immunotherapies. However, especially the parallel characterization of several epitopes is limited by the availability of T cells. Microarrays have enabled an unpreceden

  19. Evaluation of cell cytotoxic effect on herbal extracts mixtures

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yong Soo; Gwon, Hui Jeong; Choi, Bo Ram; Lim, Youn Mook; Nho, Young Chang [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

    2009-12-15

    Herbal extracts (HE) such as Houttuynia cordata Thunb., Eucommia ulimoides, Plantago asiatica var., Morus alba L., and Ulmus davidiana var., are known to suppress an atopic dermatitis like skin lesions. In this study, to evaluate the cell cytotoxicity effect on L929, HaCaT and HMC-1 cell by the HE, the herbs were extracted with distilled water (at 75 .deg. C) and then the HE mixtures were freeze-dried for 5 days and sterilized with {gamma}-rays. The cytotoxicity was measured by Cell Counting Kit-8 (CCK-8) assay. The result showed that the HE mixtures did not significantly affect cell viability and had no toxicity on the cells. These findings indicate that the HE mixtures can be used as a potential therapeutic agent.

  20. Production of good manufacturing practice-grade cytotoxic T lymphocytes specific for Epstein-Barr virus, cytomegalovirus and adenovirus to prevent or treat viral infections post-allogeneic hematopoietic stem cell transplant.

    Science.gov (United States)

    Sili, Uluhan; Leen, Ann M; Vera, Juan F; Gee, Adrian P; Huls, Helen; Heslop, Helen E; Bollard, Catherine M; Rooney, Cliona M

    2012-01-01

    Infections with a range of common community viruses remain a major cause of mortality and morbidity after allogeneic hematopoietic stem cell transplantation. T cells specific for cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenoviruses can safely prevent and infections with these three most common culprits, but the manufacture of individual T cell lines for each virus would be prohibitive in terms of time and cost. We have demonstrated that T cells specific for all three viruses can be manufactured in a single culture using monocytes and EBV-transformed B lymphoblastoid cell lines (LCLs), both transduced with an adenovirus vector expressing pp65 of CMV, as antigen-presenting cells. Trivirus-specific T cell lines produced from healthy stem cell donors could prevent and treat infections with all three viruses, not only in the designated recipient, but in unrelated, partially-HLA-matched third party recipients. We now provide the details and logistics of T cell manufacture.

  1. Profiling flavonoid cytotoxicity in human breast cancer cell lines: determination of structure-function relationships.

    Science.gov (United States)

    Yadegarynia, Sina; Pham, Anh; Ng, Alex; Nguyen, Duong; Lialiutska, Tetiana; Bortolazzo, Anthony; Sivryuk, Valentin; Bremer, Martina; White, J Brandon

    2014-05-01

    Flavonoids have been shown to be cytotoxic to cancer cells. However, the mechanism of cytotoxicity has not been clearly defined. It has previously been reported that HER2/ERBB2, the estrogen receptor, progesterone receptor, and p53 were required for flavonoid induced cytotoxicity in breast cancer cell lines. We have used a panel of breast cancer cell lines, known to contain as well as be deficient in these signaling pathways, to screen fourteen different flavonoids. Comparing the cytotoxicity for all flavonoids allows us to determine if a structure-functional relationship exists between cytotoxicity and flavonoid, and if a particular signaling pathway is required for cytotoxicity. We show that several flavonoids are cytotoxic to all cell lines including primary mammary epithelial cells tested. The cytotoxic flavonoids are also able to inhibit Mitochondrial Outer Membrane Permeability while at the same time stimulate ATP levels whereas the non-cytotoxic flavonoids are not able to do this. We also show that both cytotoxic and non-cytotoxic flavonoids can transverse the cell membrane to enter MDA-MB-231 cells at different levels. Finally, all flavonoids regardless of their cytotoxicity were able to induce some form of cell cycle arrest. We conclude that for flavonoids to be strongly cytotoxic, they must possess the 2,3-double bond in the C-ring and we believe the cytotoxicity occurs through mitochondrial poisoning in both cancer and normal cells.

  2. Human infection with Trypanosoma cruzi induces parasite antigen-specific cytotoxic T lymphocyte responses.

    Science.gov (United States)

    Wizel, B; Palmieri, M; Mendoza, C; Arana, B; Sidney, J; Sette, A; Tarleton, R

    1998-09-01

    Experimental models of Chagas' disease, an infection caused by the intracellular protozoan Trypanosoma cruzi, have demonstrated the crucial immunoprotective role played by CD8(+) T lymphocytes. These cells dominate inflammatory foci in parasitized tissues and their elimination from mice leads to uncontrolled parasite replication and subsequent death of the infected host. A trypomastigote surface antigen, TSA-1, and two amastigote surface molecules, ASP-1 and ASP-2, were recently identified as targets of CD8(+) cytotoxic T lymphocytes (CTL) in T. cruzi-infected mice. Until now, however, there was no evidence for the development of parasite-specific CTL in T. cruzi-infected humans. In this study, human CTL specific for TSA-1-, ASP-1-, and ASP-2-derived peptides were detected in the peripheral blood mononuclear cells from 21 of 24 HLA-A2(+) T. cruzi-infected patients. CTL recognition was antigen specific, A2-restricted, and CD8(+) T cell-dependent. Demonstration of human CTL against T. cruzi and against target molecules identified using the murine model provides important information for the optimal design and evaluation of vaccines to prevent or ameliorate Chagas' disease.

  3. Poly (D,L-lactide-co-glycolide nanoparticles: Uptake by epithelial cells and cytotoxicity

    Directory of Open Access Journals (Sweden)

    J. H. Hamman

    2014-03-01

    Full Text Available Nanoparticles as drug delivery systems offer benefits such as protection of the encapsulated drug against degradation, site-specific targeting and prolonged blood circulation times. The aim of this study was to investigate nanoparticle uptake into Caco-2 cell monolayers, their co-localization within the lysosomal compartment and their cytotoxicity in different cell lines. Rhodamine-6G labelled poly(D,L-lactide-co-glycolide (PLGA nanoparticles were prepared by a double emulsion solvent evaporation freeze-drying method. Uptake and co-localisation of PLGA nanoparticles in lysosomes were visualized by confocal laser scanning microscopy. The cytotoxicity of the nanoparticles was evaluated on different mammalian cells lines by means of Trypan blue exclusion and the MTS assay. The PLGA nanoparticles accumulated in the intercellular spaces of Caco-2 cell monolayers, but were also taken up transcellularly into the Caco-2 cells and partially co-localized within the lysosomal compartment indicating involvement of endocytosis during uptake. PLGA nanoparticles did not show cytotoxic effects in all three cell lines. Intact PLGA nanoparticles are therefore capable of moving across epithelial cell membranes partly by means of endocytosis without causing cytotoxic effects.

  4. Klebsiella pneumoniae triggers a cytotoxic effect on airway epithelial cells

    Directory of Open Access Journals (Sweden)

    Llobet-Brossa Enrique

    2009-08-01

    Full Text Available Abstract Background Klebsiella pneumoniae is a capsulated Gram negative bacterial pathogen and a frequent cause of nosocomial infections. Despite its clinical relevance, little is known about the features of the interaction between K. pneumoniae and lung epithelial cells on a cellular level, neither about the role of capsule polysaccharide, one of its best characterised virulence factors, in this interaction. Results The interaction between Klebsiella pneumoniae and cultured airway epithelial cells was analysed. K. pneumoniae infection triggered cytotoxicity, evident by cell rounding and detachment from the substrate. This effect required the presence of live bacteria and of capsule polysaccharide, since it was observed with isolates expressing different amounts of capsule and/or different serotypes but not with non-capsulated bacteria. Cytotoxicity was analysed by lactate dehydrogenase and formazan measurements, ethidium bromide uptake and analysis of DNA integrity, obtaining consistent and complementary results. Moreover, cytotoxicity of non-capsulated strains was restored by addition of purified capsule during infection. While a non-capsulated strain was avirulent in a mouse infection model, capsulated K. pneumoniae isolates displayed different degrees of virulence. Conclusion Our observations allocate a novel role to K. pneumoniae capsule in promotion of cytotoxicity. Although this effect is likely to be associated with virulence, strains expressing different capsule levels were not equally virulent. This fact suggests the existence of other bacterial requirements for virulence, together with capsule polysaccharide.

  5. Construction of HA-1-DC Nucleic-acid Vaccine and Induction of Specific Cytotoxic T Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    WANG Yaya; ZHANG Donghua; HU Jinmei; LIU Wenli; ZHOU hongsheng; ZHANG Lu; LIU Dan; HUANG Zhenqian; TAN Huo

    2007-01-01

    An HA-1-DC nucleic-acid vaccine was constructed to induce anti-leukemia effect after hematopoietic stem cell transplantation (HSCT). DCs were generated from HSCT donors in vitro, and its immunologic activity was assayed by using flow cytometry and mixed lymphocytes reaction.HA-1 gene was electroporated into the cultured DCs to construct a DC nucleic-acid vaccine. After transfection for 48 h, the expression of HA-1 protein could be detected by using Western blot. The DCs were cultured with syngenic lymphocytes to induce specific cytotoxic T lymphocytes (CTLs).The cytoxicity of the CTLs was detected by LDH assay. The results showed that The DCs derived from peripheral blood monocytes (PBMCs) expressed the phenotype of DCs, and were effective in stimulating proliferation of the allogenic lymphocytes. After electroporating for 48-h, HA-1 protein was detected by using Western blot. The cytotoxity of inducing CTLs was higher than the control group. It was suggested that minor histocompatibility antigen HA-1 could be considered as a target of immunotherapy against leukemia after HSCT.

  6. Universal cytotoxic activity of a HTLV-1 Tax-specific T cell clone from an HLA-A*24:02⁺ patient with adult T-cell leukemia against a variety of HTLV-I-infected T-cells.

    Science.gov (United States)

    Tanaka, Yukie; Yamazaki, Rie; Terasako-Saito, Kiriko; Nakasone, Hideki; Akahoshi, Yu; Nakano, Hirofumi; Ugai, Tomotaka; Wada, Hidenori; Yamasaki, Ryoko; Ishihara, Yuko; Kawamura, Koji; Sakamoto, Kana; Ashizawa, Masahiro; Sato, Miki; Kimura, Shun-ichi; Kikuchi, Misato; Kako, Shinichi; Kanda, Junya; Tanihara, Aki; Nishida, Junji; Kanda, Yoshinobu

    2014-01-01

    Adult T cell leukemia/lymphoma (ATL) is an aggressive mature T cell malignancy that is causally associated with human T cell lymphotropic virus type 1 (HTLV-1) infection. The HTLV-1 regulatory protein Tax aggressively accelerates the proliferation of host cells and is also an important target antigen for CD8(+) cytotoxic T cells (CTLs). We previously reported that several predominant HLA-A*24:02-restricted HTLV-1 Tax301-309-specific CTL clones commonly expressed a particular amino acid sequence motif (P-D-R) in complementarity-determining region 3 of T-cell receptor (TCR)-β chain among unrelated ATL patients who underwent allogeneic stem cell transplantation (allo-HSCT). Furthermore, a PDR-motif(+) CTL clone persistently existed in a long-term survivor as a central CTL clone with strong CTL activities after HSCT. Although a larger analysis of the relationship between PDR-motif(+) CTLs and the clinical course is required, the expression of PDR-motif(+) TCR on CD8(+) T cells may play a critical role in the management of anti-HTLV-1 activities for HLA-A24:02(+) ATL patients. Therefore, in this study, we prepared an HTLV-1 Tax301-309 peptide-specific CTL clone (HT-9) expressing PDR-motif(+) TCR isolated from a long-term survivor after HSCT, and evaluated its CTL activity against a variety of HTLV-1-infected T-cells from HLA-A*24:02(+) ATL patients. Before the assay of CTL function, we confirmed that HT-9 expressed less-differentiated effector-memory phenotypes (CD45RA(-)CCR7(-)CD27(+)CD28(+/-)CD57(+/-)) and T-cell exhaustion marker PD-1(+). In assays of CTL function, HT-9 recognized HTLV-1 Tax in an HLA-restricted fashion and demonstrated strong CTL activities against a variety of HTLV-1-infected T-cells from HLA-A*24:02(+) ATL patients regardless of whether the sources were autologous or allogeneic, but not normal cells. These data indicate that PDR-motif(+) TCR could be an important TCR candidate for TCR-gene immunotherapy for HLA-A24:02(+) ATL patients, provided

  7. Control of mucosal virus infection by influenza nucleoprotein-specific CD8+ cytotoxic T lymphocytes

    Directory of Open Access Journals (Sweden)

    Couch Robert B

    2007-06-01

    Full Text Available Abstract Background MHC class I-restricted CD8+ cytotoxic T lymphocytes (CTL are thought to play a major role in clearing virus and promoting recovery from influenza infection and disease. This has been demonstrated for clearance of influenza virus from the lungs of infected mice. However, human influenza infection is primarily a respiratory mucosal infection involving the nasopharynx and tracheobronchial tree. The role of CD8+ CTL directed toward the influenza nucleoprotein (NP in defense against influenza virus infection at the respiratory mucosa was evaluated in two separate adoptive transfer experiments. Methods Influenza nucleoprotein (NP-specific CD8+ CTL were generated from splenocytes obtained from Balb/c mice previously primed with influenza A/Taiwan/1/86 (H1N1 infection or with influenza A/PR/8/34 (H1N1-derived NP plasmid DNA vaccine followed by infection with A/Hong Kong/68 (H3N2 virus. After in vitro expansion by exposure to an influenza NP-vaccinia recombinant, highly purified CD8+ T cells exhibited significant lysis in vitro of P815 target cells infected with A/Hong Kong/68 (H3N2 virus while the CD8- fraction (CD4+ T cells, B cells and macrophages had no CTL activity. Purified CD8+ and CD8- T cells (1 × 107 were injected intravenously or interperitoneally into naive mice four hours prior to intranasal challenge with A/HK/68 (H3N2 virus. Results The adoptively transferred NP-vaccinia-induced CD8+ T cells caused significant reduction of virus titers in both the lungs and nasal passages when compared to CD8- cells. Neither CD8+ nor CD8- T cells from cultures stimulated with HIV gp120-vaccinia recombinant reduced virus titers. Conclusion The present data demonstrate that influenza NP-specific CD8+ CTL can play a direct role in clearance of influenza virus from the upper respiratory mucosal surfaces.

  8. TRANSDENTINAL CYTOTOXICITY OF GLUTARALDEHYDE ON ODONTOBLAST-LIKE CELLS

    Science.gov (United States)

    Scheffel, Débora Lopes Salles; Soares, Diana Gabriela; Basso, Fernanda Gonçalves; de Souza Costa, Carlos Alberto; Pashley, David Henry; Hebling, Josimeri

    2015-01-01

    Objectives This study investigated the transdentinal cytotoxicity of glutahaldehyde-containing solutions/materials on odontoblast-like cells. Methods Dentin discs were adapted to artificial pulp chambers. MDPC-23 cells were seeded on the pulpal side of the discs and the occlusal surface was treated with the following solutions: water, 2%glutaraldehyde (GA), 5%GA, 10%GA, Gluma Comfort Bond+Desensitizer (GCB+De) or Gluma Desensitizer (GDe). Cell viability and morphology were assessed by the Alamar Blue assay and SEM. The eluates were collected and applied on cells seeded in 24-well plates. After 7 or 14 days the total protein (TP) production, alkaline phosphatase activity (ALP) and deposition of mineralized nodules (MN) were evaluated. Results Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (p<0.05). GA solutions were not cytotoxic against MDPC-23. GCB+De (85.1%) and GDe (77.2%) reduced cell viability as well as TP production and ALP activity at both periods. After 14 days, GCB+De and GDe groups produced less MN. Affected MDPC-23 presented deformation of the cytoskeleton and reduction of cellular projections. Conclusions The treatment with 2.5%, 5% and 10%GA was not harmful to odontoblast-like cells. Conversely, when GA was combined with other components like HEMA, the final material became cytotoxic. Clinical significance Glutaraldehyde has been used to decrease dentin hypersensitivity. This substance is also capable of preventing resin-dentin bond degradation by cross-linking collagen and MMPs. This study showed that GA might be safe when applied on acid etched dentin. However, when combined with HEMA the product becomes cytotoxic. PMID:25985981

  9. Differential Cytotoxic Potential of Silver Nanoparticles in Human Ovarian Cancer Cells and Ovarian Cancer Stem Cells

    Directory of Open Access Journals (Sweden)

    Yun-Jung Choi

    2016-12-01

    Full Text Available The cancer stem cell (CSC hypothesis postulates that cancer cells are composed of hierarchically-organized subpopulations of cells with distinct phenotypes and tumorigenic capacities. As a result, CSCs have been suggested as a source of disease recurrence. Recently, silver nanoparticles (AgNPs have been used as antimicrobial, disinfectant, and antitumor agents. However, there is no study reporting the effects of AgNPs on ovarian cancer stem cells (OvCSCs. In this study, we investigated the cytotoxic effects of AgNPs and their mechanism of causing cell death in A2780 (human ovarian cancer cells and OvCSCs derived from A2780. In order to examine these effects, OvCSCs were isolated and characterized using positive CSC markers including aldehyde dehydrogenase (ALDH and CD133 by fluorescence-activated cell sorting (FACS. The anticancer properties of the AgNPs were evaluated by assessing cell viability, leakage of lactate dehydrogenase (LDH, reactive oxygen species (ROS, and mitochondrial membrane potential (mt-MP. The inhibitory effect of AgNPs on the growth of ovarian cancer cells and OvCSCs was evaluated using a clonogenic assay. Following 1–2 weeks of incubation with the AgNPs, the numbers of A2780 (bulk cells and ALDH+/CD133+ colonies were significantly reduced. The expression of apoptotic and anti-apoptotic genes was measured by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR. Our observations showed that treatment with AgNPs resulted in severe cytotoxicity in both ovarian cancer cells and OvCSCs. In particular, AgNPs showed significant cytotoxic potential in ALDH+/CD133+ subpopulations of cells compared with other subpopulation of cells and also human ovarian cancer cells (bulk cells. These findings suggest that AgNPs can be utilized in the development of novel nanotherapeutic molecules for the treatment of ovarian cancers by specific targeting of the ALDH+/CD133+ subpopulation of cells.

  10. Withania somnifera Root Extract Has Potent Cytotoxic Effect against Human Malignant Melanoma Cells.

    Science.gov (United States)

    Halder, Babli; Singh, Shruti; Thakur, Suman S

    2015-01-01

    In Ayurveda, Withania somnifera is commonly known as Ashwagandha, its roots are specifically used in medicinal and clinical applications. It possesses numerous therapeutic actions which include anti-inflammatory, sedative, hypnotic and narcotic. Extracts from this plant have been reported for its anticancer properties. In this study we evaluated for the first time, the cytotoxic effect of Withania root extract on human malignant melanoma A375 cells. The crude extract of Withania was tested for cytotoxicity against A375 cells by MTT assay. Cell morphology of treated A375 cells was visualized through phase contrast as well as fluorescence microscopy. Agarose gel electrophoresis was used to check DNA fragmentation of the crude extract treated cells. Crude extract of Withania root has the potency to reduce viable cell count in dose as well as time dependent manner. Morphological change of the A375 cells was also observed in treated groups in comparison to untreated or vehicle treated control. Apoptotic body and nuclear blebbing were observed in DAPI stained treated cells under fluorescence microscope. A ladder of fragmented DNA was noticed in treated cells. Thus it might be said that the crude water extract of Withania somnifera has potent cytotoxic effect on human malignant melanoma A375 cells.

  11. Evaluation of cytotoxicity and gelatinases activity in 3T3 fibroblast cell by root repair materials

    Directory of Open Access Journals (Sweden)

    Varol Basak

    2016-09-01

    Full Text Available The aim of this study was to investigate the effects of calcium silicate-based products on cytotoxicity in the 3T3 fibroblast and gelatinolytic activity of matrix metalloproteinases (MMPs. 3T3 fibroblasts were incubated directly with Ortho Mineral trioxide aggregate (MTA, BioAggregate, Biodentine, MTA Plus, MTA Angelus and MTA Cerkamed for 24 hours and seven days. The cytotoxicity was determined using an MTT assay. Supernatants were collected to determine MMP-2 and MMP-9. Data were analysed using IBM SPSS 22. Seventh day extracts of Ortho MTA and Biodentine showed reduced cell viability. Specific characterization of MMPs in cell culture demonstrated that MMP-2 (62 kPa in the cell culture supernatants by gelatin zymography showed induced expression in four out of seven groups by 3T3 cells. No MMP-9 expression was observed. The cytotoxicity of materials revealed a significant difference in cell viability between the groups on the first and seventh days. The results of this study revealed minor cytotoxic effects for Ortho MTA and Biodentine. This study suggests that endodontic sealers induced production of MMP-2. MMP-9 might be expressed in small amounts when compared with MMP-2.

  12. PDMS/glass microfluidic cell culture system for cytotoxicity tests and cells passage

    DEFF Research Database (Denmark)

    Ziolkowska, K.; Jedrych, E.; Kwapiszewski, R.

    2010-01-01

    microdevice was developed and successfully tested. The MCCS microdevice is fully reusable, i.e. it can be used several times for various cell culture and cytotoxic experiments. The suitability of designed MCCS for cell-based cytotoxicity assay application was verified using 1,4-dioxane as a model toxic agent....... The series of cytotoxicity tests in the microdevice as well as in classic way using 96-well cell culture plates were performed to compare results obtained in micro- and macroscale. Fluorescein dibutyrate (FDB) and iodide propidine (PI) were used as viable and dead cells' markers, respectively. Fabricated...... MCCS microdevices were reproducible and apart from cell culture for long period of time, including cell passaging, it allowed cell-based cytotoxicity assays performance. The MCCS can be applied in high-throughput cell-based assays providing important informations on potential drug targets, substances...

  13. Evaluation of alginate hydrogel cytotoxicity on three-dimensional culture of type A spermatogonial stem cells.

    Science.gov (United States)

    Jalayeri, Maryam; Pirnia, Afshin; Najafabad, Elaheh Poorazizi; Varzi, Ali Mohammad; Gholami, Mohammadreza

    2017-02-01

    The culture of spermatogonial cells for future transplantation, based on the specific biology of these cells is important and necessary. Recently, the use of scaffolds especially alginate for culturing stem cells has been the focus of many researchers. The aim of this study was to evaluate the cytotoxicity of alginate hydrogels to cultures of type A spermatogonial stem cells. Spermatogonial stem cells of 6day-old immature mice were isolated by surgery; thereafter, the cells were purified by MACS using antibodies against thy-1 and C-kit and cultured on a layer of laminin. After purification, spermatogonial stem cells were encapsulated in alginate hydrogels. After one month of encapsulation and culture in DMEM culture medium containing 10ng/ml GDNF, cells were removed from hydrogel and were examined for viability, cell morphology and structure, cytotoxicity and expression of apoptosis genes Fas, P53, Bax, Bcl2, Caspase3 by staining with trypan blue, scanning electron microscopy, LDH test, and Real time PCR, respectively. The encapsulation did not change the morphology and viability of spermatogonial stem cells. Investigations showed that spermatogonial stem cells preserve by the high viability (74.08%) and cytotoxicity of alginate hydrogel was estimated to be 5%. Expression of Fas gene increased in main group compared with the control group, and expression of Bax and P53 was reduced in main group compared with the control group. Expression of Bcl2 and Caspase3 genes did not show any significant difference between the main group and the control group. Considering the lack of cytotoxicity and antioxidant properties of alginate hydrogel scaffold and high viability of cells, this three-dimensional scaffold is applicable for culturing and encapsulation of spermatogonial stem cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Myrtus comunis and Eucalyptus camaldulensis cytotoxicity on breast cancer cells

    Directory of Open Access Journals (Sweden)

    Hrubik Jelena D.

    2012-01-01

    Full Text Available In vitro cytotoxicity of methanol, ethyl acetate, n-buthanol, and water extracts of Myrtus communis L. and Eucalyptus camaldulensis Dehnh. was examined against two human breast cancer cell lines (MCF 7 and MDA-MB-231 using MTT and SRB assays. The results showed significant cytotoxic potential of examined extracts, with IC50 values ranging from 7 to 138 μg/ml for M. communis and 3-250 μg/ml for E. camaldulensis. The two plants generally expressed similar activity, and no significant difference in cell line’s sensitivity towards extracts was observed. The results indicate to M. communis and E. camaldulensis as candidates for thorough chemical analyses for identification of active compounds, and eventually for attention in the process of discovery of new natural products in the control of cancer. [Projekat Ministarstva nauke Republike Srbije, br. 173037 i br. 172058

  15. Cytotoxic Effect of Lipophilic Bismuth Dimercaptopropanol Nanoparticles on Epithelial Cells.

    Science.gov (United States)

    Rene, Hernandez-Delgadillo; Badireddy, Appala Raju; José, Martínez-Sanmiguel Juan; Francisco, Contreras-Cordero Juan; Israel, Martinez-Gonzalez Gustavo; Isela, Sánchez-Nájera Rosa; Chellam, Shankararaman; Claudio, Cabral-Romero

    2016-01-01

    Bismuth nanoparticles have many interesting properties to be applied in biomedical and medicinal sectors, however their safety in humans have not been comprehensively investigated. The objective of this research was to determine the cytotoxic effect of bismuth dimercaptopropanol nanoparticles (BisBAL NPs) on epithelial cells. The nanoparticles are composed of 18.7 nm crystallites on average and have a rhombohedral structure, agglomerating into chains-like or clusters of small nanoparticles. Based on MTT viability assay and fluorescence microscopy, cytotoxicity was not observed on monkey kidney cells after growing with 5 µM of BisBAL NPs for 24 h. Employing same techniques, identical results were obtained with human epithelial cells (HeLa), showing a not strain-dependent phenomenon. The absence of toxic effects on epithelial cells growing with BisBAL NPs was corroborated with long-time experiments (24-72 hrs.), showing no difference in comparison with growing control (cells without nanoparticles). Further, genotoxicity assays, comet assay and fluorescent microscopy and electrophoresis in bromide-stained agarose gel revealed no damage to genomic DNA of MA104 cells after 24 h. of exposition to BisBAL NPs. Finally, the effect of bismuth nanoparticles on protein synthesis was studied in cells growing with BisBAL NPs for 24 h. SDS-PAGE assays showed no difference between treated and untreated cells, suggesting that BisBAL NPs did not interfere with protein synthesis. Hence BisBAL NPs do not appear to exert cytotoxic effects suggesting their biological compatibility with epithelial cells.

  16. Emergence of cytotoxic resistance in cancer cell populations*

    Directory of Open Access Journals (Sweden)

    Lorenzi Tommaso

    2015-01-01

    Full Text Available We formulate an individual-based model and an integro-differential model of phenotypic evolution, under cytotoxic drugs, in a cancer cell population structured by the expression levels of survival-potential and proliferation-potential. We apply these models to a recently studied experimental system. Our results suggest that mechanisms based on fundamental laws of biology can reversibly push an actively-proliferating, and drug-sensitive, cell population to transition into a weakly-proliferative and drug-tolerant state, which will eventually facilitate the emergence of more potent, proliferating and drug-tolerant cells.

  17. Early spatiotemporal-specific changes in intermediate signals are predictive of cytotoxic sensitivity to TNFα and co-treatments

    Science.gov (United States)

    Loo, Lit-Hsin; Bougen-Zhukov, Nicola Michelle; Tan, Wei-Ling Cecilia

    2017-03-01

    Signaling pathways can generate different cellular responses to the same cytotoxic agents. Current quantitative models for predicting these differential responses are usually based on large numbers of intracellular gene products or signals at different levels of signaling cascades. Here, we report a study to predict cellular sensitivity to tumor necrosis factor alpha (TNFα) using high-throughput cellular imaging and machine-learning methods. We measured and compared 1170 protein phosphorylation events in a panel of human lung cancer cell lines based on different signals, subcellular regions, and time points within one hour of TNFα treatment. We found that two spatiotemporal-specific changes in an intermediate signaling protein, p90 ribosomal S6 kinase (RSK), are sufficient to predict the TNFα sensitivity of these cell lines. Our models could also predict the combined effects of TNFα and other kinase inhibitors, many of which are not known to target RSK directly. Therefore, early spatiotemporal-specific changes in intermediate signals are sufficient to represent the complex cellular responses to these perturbations. Our study provides a general framework for the development of rapid, signaling-based cytotoxicity screens that may be used to predict cellular sensitivity to a cytotoxic agent, or identify co-treatments that may sensitize or desensitize cells to the agent.

  18. α-Mangostin Enhances Betulinic Acid Cytotoxicity and Inhibits Cisplatin Cytotoxicity on HCT 116 Colorectal Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Amin Malik Shah Abdul Majid

    2012-03-01

    Full Text Available Despite the progress in colon cancer treatment, relapse is still a major obstacle. Hence, new drugs or drug combinations are required in the battle against colon cancer. α-Mangostin and betulinic acid (BA are cytotoxic compounds that work by inducing the mitochondrial apoptosis pathway, and cisplatin is one of the most potent broad spectrum anti-tumor agents. This study aims to investigate the enhancement of BA cytotoxicity by α-mangostin, and the cytoprotection effect of α-mangostin and BA on cisplatin-induced cytotoxicity on HCT 116 human colorectal carcinoma cells. Cytotoxicity was investigated by the XTT cell proliferation test, and the apoptotic effects were investigated on early and late markers including caspases-3/7, mitochondrial membrane potential, cytoplasmic shrinkage, and chromatin condensation. The effect of α-mangostin on four signalling pathways was also investigated by the luciferase assay. α-Mangostin and BA were more cytotoxic to the colon cancer cells than to the normal colonic cells, and both compounds showed a cytoprotective effect against cisplatin-induced cytotoxicity. On the other hand, α-mangostin enhanced the cytotoxic and apoptotic effects of BA. Combination therapy hits multiple targets, which may improve the overall response to the treatment, and may reduce the likelihood of developing drug resistance by the tumor cells. Therefore, α-mangostin and BA may provide a novel combination for the treatment of colorectal carcinoma. The cytoprotective effect of the compounds against cisplatin-induced cytotoxicity may find applications as chemopreventive agents against carcinogens, irradiation and oxidative stress, or to neutralize cisplatin side effects.

  19. A novel L-fucose-binding lectin from Fenneropenaeus indicus induced cytotoxicity in breast cancer cells.

    Science.gov (United States)

    Chatterjee, Biji; Ghosh, Krishna; Yadav, Nitin; Kanade, Santosh R

    2017-01-01

    Lectins are omnipresent in almost all life forms, being the proteins which specifically bind to carbohydrate moieties on the cell surface; they have been explored for their anti-tumour activities. In this study, we purified a fucose specific-lectin (IFL) from Fenneropenaeus indicus haemolymph using fucose-affinity column and characterized for its haemagglutination activity, carbohydrate specificity, dependency on cations and cytotoxicity against cancer cells. The lectin showed non-specificity against human erythrocytes. It was a Ca(2+)-dependent lectin which remained stable over wide pH and temperature ranges. The lectin showed effective dose dependent cytotoxicity against different human cancer cell lines and induced apoptosis in MCF-7 cells as evidenced by DNA ladder assay and PARP cleavage in a dose dependent manner. Moreover, an increased p21 level corresponding to cyclin D downregulation in response to IFL treatment was observed which might work as probable factors to inhibit cell growth and induce apoptosis of MCF-7 cells. Therefore, we report a novel lectin from the prawn haemolymph with high specificity for L-fucose and antiproliferative towards human cancer cells. However, further establishment of the modus operandi of this lectin is required to enable its biotechnological applications.

  20. C22:0- and C24:0-dihydroceramides confer mixed cytotoxicity in T-cell acute lymphoblastic leukemia cell lines.

    Directory of Open Access Journals (Sweden)

    Michael W Holliday

    Full Text Available We previously reported that fenretinide (4-HPR was cytotoxic to acute lymphoblastic leukemia (ALL cell lines in vitro in association with increased levels of de novo synthesized dihydroceramides, the immediate precursors of ceramides. However, the cytotoxic potentials of native dihydroceramides have not been defined. Therefore, we determined the cytotoxic effects of increasing dihydroceramide levels via de novo synthesis in T-cell ALL cell lines and whether such cytotoxicity was dependent on an absolute increase in total dihydroceramide mass versus an increase of certain specific dihydroceramides. A novel method employing supplementation of individual fatty acids, sphinganine, and the dihydroceramide desaturase-1 (DES inhibitor, GT-11, was used to increase de novo dihydroceramide synthesis and absolute levels of specific dihydroceramides and ceramides. Sphingolipidomic analyses of four T-cell ALL cell lines revealed strong positive correlations between cytotoxicity and levels of C22:0-dihydroceramide (ρ = 0.74-0.81, P ≤ 0.04 and C24:0-dihydroceramide (ρ = 0.84-0.90, P ≤ 0.004, but not between total or other individual dihydroceramides, ceramides, or sphingoid bases or phosphorylated derivatives. Selective increase of C22:0- and C24:0-dihydroceramide increased level and flux of autophagy marker, LC3B-II, and increased DNA fragmentation (TUNEL assay in the absence of an increase of reactive oxygen species; pan-caspase inhibition blocked DNA fragmentation but not cell death. C22:0-fatty acid supplemented to 4-HPR treated cells further increased C22:0-dihydroceramide levels (P ≤ 0.001 and cytotoxicity (P ≤ 0.001. These data demonstrate that increases of specific dihydroceramides are cytotoxic to T-cell ALL cells by a caspase-independent, mixed cell death mechanism associated with increased autophagy and suggest that dihydroceramides may contribute to 4-HPR-induced cytotoxicity. The targeted increase of specific acyl chain dihydroceramides

  1. Granule-Dependent Natural Killer Cell Cytotoxicity to Fungal Pathogens

    Science.gov (United States)

    Ogbomo, Henry; Mody, Christopher H.

    2017-01-01

    Natural killer (NK) cells kill or inhibit the growth of a number of fungi including Cryptococcus, Candida, Aspergillus, Rhizopus, and Paracoccidioides. Although many fungi are not dangerous, invasive fungal pathogens, such as Cryptococcus neoformans, cause life-threatening disease in individuals with impaired cell-mediated immunity. While there are similarities to cell-mediated killing of tumor cells, there are also important differences. Similar to tumor killing, NK cells directly kill fungi in a receptor-mediated and cytotoxic granule-dependent manner. Unlike tumor cell killing where multiple NK cell-activating receptors cooperate and signal events that mediate cytotoxicity, only the NKp30 receptor has been described to mediate signaling events that trigger the NK cell to mobilize its cytolytic payload to the site of interaction with C. neoformans and Candida albicans, subsequently leading to granule exocytosis and fungal killing. More recently, the NKp46 receptor was reported to bind Candida glabrata adhesins Epa1, 6, and 7 and directly mediate fungal clearance. A number of unanswered questions remain. For example, is only one NK cell-activating receptor sufficient for signaling leading to fungal killing? Are the signaling pathways activated by fungi similar to those activated by tumor cells during NK cell killing? How do the cytolytic granules traffic to the site of interaction with fungi, and how does this process compare with tumor killing? Recent insights into receptor use, intracellular signaling and cytolytic granule trafficking during NK cell-mediated fungal killing will be compared to tumor killing, and the implications for therapeutic approaches will be discussed. PMID:28123389

  2. Cytotoxicity of bovine and porcine collagen membranes in mononuclear cells.

    Science.gov (United States)

    Moura, Camilla Christian Gomes; Soares, Priscilla Barbosa Ferreira; Carneiro, Karine Fernandes; Souza, Maria Aparecida de; Magalhães, Denildo

    2012-01-01

    This study compared the cytotoxicity and the release of nitric oxide induced by collagen membranes in human mononuclear cells. Peripheral blood was collected from each patient and the separation of mononuclear cells was performed by Ficoll. Then, 2x10(5) cells were plated in 48-well culture plates under the membranes in triplicate. The polystyrene surface was used as negative control. Cell viability was assessed by measuring mitochondrial activity (MTT) at 4, 12 and 24 h, with dosage levels of nitrite by the Griess method for the same periods. Data had non-normal distribution and were analyzed by the Kruskal-Wallis test (pporcine membrane induced a higher release of nitrite compared with the control and bovine membrane, respectively (pporcine collagen membrane induces an increased production of proinflammatory mediators by mononuclear cells in the first hours of contact, decreasing with time.

  3. Cytotoxicity of citral against melanoma cells: The involvement of oxidative stress generation and cell growth protein reduction.

    Science.gov (United States)

    Sanches, Larissa Juliani; Marinello, Poliana Camila; Panis, Carolina; Fagundes, Tatiane Renata; Morgado-Díaz, José Andrés; de-Freitas-Junior, Julio Cesar Madureira; Cecchini, Rubens; Cecchini, Alessandra Lourenço; Luiz, Rodrigo Cabral

    2017-03-01

    Citral is a natural compound that has shown cytotoxic and antiproliferative effects on breast and hematopoietic cancer cells; however, there are few studies on melanoma cells. Oxidative stress is known to be involved in all stages of melanoma development and is able to modulate intracellular pathways related to cellular proliferation and death. In this study, we hypothesize that citral exerts its cytotoxic effect on melanoma cells by the modulation of cellular oxidative status and/or intracellular signaling. To test this hypothesis, we investigated the antiproliferative and cytotoxic effects of citral on B16F10 murine melanoma cells evaluating its effects on cellular oxidative stress, DNA damage, cell death, and important signaling pathways, as these pathways, namely, extracellular signal-regulated kinases 1/2 (ERK1/2), AKT, and phosphatidylinositol-3 kinase, are involved in cell proliferation and differentiation. The p53 and nuclear factor kappa B were also investigated due to their ability to respond to intracellular stress. We observed that citral exerted antiproliferative and cytotoxic effects in B16F10; induced oxidative stress, DNA lesions, and p53 nuclear translocation; and reduced nitric oxide levels and nuclear factor kappa B, ERK1/2, and AKT. To investigate citral specificity, we used non-neoplastic human and murine cells, HaCaT (human skin keratinocytes) and NIH-3T3 cells (murine fibroblasts), and observed that although citral effects were not specific for cancer cells, non-neoplastic cells were more resistant to citral than B16F10. These findings highlight the potential clinical utility of citral in melanoma, with a mechanism of action involving the oxidative stress generation, nitric oxide depletion, and interference in signaling pathways related to cell proliferation.

  4. Low direct cytotoxicity of nabumetone on gastric mucosal cells.

    Science.gov (United States)

    Arai, Yasuhiro; Tanaka, Ken-Ichiro; Ushijima, Hironori; Tomisato, Wataru; Tsutsumi, Shinji; Aburaya, Mayuko; Hoshino, Tatsuya; Yokomizo, Kazumi; Suzuki, Keitarou; Katsu, Takashi; Tsuchiya, Tomofusa; Mizushima, Tohru

    2005-09-01

    Prodrugs of non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for clinical purposes because they are not harmful to the gastrointestinal mucosa. We recently showed that NSAIDs have direct cytotoxicity in NSAID-induced gastric lesions. We show here that under conditions where the NSAIDs indomethacin and celecoxib clearly induce cell death, an NSAID prodrug, nabumetone, and its active metabolite 6-methoxy-2-naphthylacetic acid (6MNA), did not have such effects. Moreover, nabumetone and 6MNA exhibited much lower membrane permeabilizing activities than did indomethacin and celecoxib. We recently reported that when an orally administered NSAID was used in combination with a low dose of intravenously administered indomethacin, the severity of gastric lesions produced in rats depended on the cytotoxicity of the orally administered NSAID. Using a similar protocol, we show here that gastric lesions were produced when the orally administered NSAID was celecoxib, but not when nabumetone was used. We thus propose that the low direct cytotoxicity of nabumetone observed in vitro is maintained in vivo, and that the use of nabumetone does not harm the gastric mucosa.

  5. Analysis of HBV-specific cytotoxic T lymphocytes in patients with hepatitis B

    Directory of Open Access Journals (Sweden)

    Ning DING

    2011-04-01

    Full Text Available Objective To investigate the expression feature of hepatitis B virus(HBV-specific cytotoxic T lymphocytes(CTL,and analyze the role of CTL in immune response.Methods Peripheral venous blood was collected from 40 acute hepatitis B(AHB patients hospitalized from Oct.2008 to Jun.2010 in 302 Hospital of PLA for the detection of human leucocyte antigen-A2(HLA-A2.Out of the 40 patients,18 were HLA-A2-positive,and their peripheral blood mononuclear cells were isolated at acute phase and convalescent phase if the patients could be contacted.Six HLA-A2-negative AHB patients,18 HLA-A2-positive chronic HB patients and 6 HLA-A2-positive normal persons served as controls.The CTL was stained by fluorescence-labeled anti-CD3 monoclonal antibody,anti-CD8 monoclonal antibody and HBV epitopic pentamers(S183-191,S335-343,and examined with flow cytometry,then analyzed by software Cell-quest.The peak value of alanine aminotransferase(ALT and HBV load was detected at acute phase of patients,and the correlation was analyzed.Results The average frequencies were 0.23%±0.18% for HBsAg183-191-specific CTL and 0.49%±0.31% for HBsAg335-343-specific CTL at the acute phase of the 18 AHB patients,and they were significantly higher than those in patients with chronic HB,in whom CTL frequencies were 0.07%±0.03% for HBsAg183-191-specific CTL and 0.08%±0.04% for HBsAg335-343-specific CTL(P < 0.01.There was no correlation between the frequency of HBsAg183-191-specific CTL and the peak value of ALT(r=0.4506,P=0.0528,while the frequency of HBsAg335-343-specific CTL was correlated with ALT level(r=0.5642,P=0.0119.Neither the two CTL frequencies showed correlation with virus load.Compared with the acute phase,the CTL frequencies obviously decreased at convalescent phase(P=0.011.Both kinds of CTL were not detectable in HLA-A2-negative AHB patients and HLA-A2-positive healthy individuals.Conclusion HBV-specific CTL may play an important role in both virus clearance and liver

  6. [Cytotoxicity studies on T-3262 in cultured Chinese hamster cells].

    Science.gov (United States)

    Yoneda, T; Nakamura, S; Nojima, Y; Nishio, Y

    1989-04-01

    T-3262 is an antibacterial drug which belongs to the group of pyridonecarboxylic acids. In this study, we investigated cytotoxicity of T-3262 for inhibition of cell growth and effects on viability of, and morphological changes in cultured Chinese hamster cells (V79 cells). The following results were obtained. 1. The 50% inhibition dose of T-3262 for cell growth (ID50, cultured for 48 hours) was 12 micrograms/ml, showing that the inhibitory effect of T-3262 on the cell growth was stronger than that of enoxacin (ENX: ID50 44 micrograms/ml), norfloxacin (NFLX: ID50 105 micrograms/ml) or ofloxacin (OFLX: ID50 145 micrograms/ml). 2. The number of cells increased and dead cells were scarcely seen at the highest concentration tested in culture medium (40 micrograms/ml of T-3262 for 48 hours). At this concentration, degeneration of cytoplasm (atrophy and round shape) and decrease of mitotic cells were observed. These morphological changes were similar to those of the cells treated 400 micrograms/ml of NFLX or OFLX for 48 hours. 3. After the removal of T-3262 from culture medium, the cells began to grow actively and recovered from the morphological changes. The similar phenomenon was observed with ENX treated cells but not with fluorouracil or mitomycin C treated cells.

  7. Soluble fibrin inhibits monocyte adherence and cytotoxicity against tumor cells: implications for cancer metastasis

    Directory of Open Access Journals (Sweden)

    Patel Shonak

    2006-08-01

    Full Text Available Abstract Background Soluble fibrin (sFn is a marker for disseminated intravascular coagulation and may have prognostic significance, especially in metastasis. However, a role for sFn in the etiology of metastatic cancer growth has not been extensively studied. We have reported that sFn cross-linked platelet binding to tumor cells via the major platelet fibrin receptor αIIbβ3, and tumor cell CD54 (ICAM-1, which is the receptor for two of the leukocyte β2 integrins (αLβ2 and aMβ2. We hypothesized that sFn may also affect leukocyte adherence, recognition, and killing of tumor cells. Furthermore, in a rat experimental metastasis model sFn pre-treatment of tumor cells enhanced metastasis by over 60% compared to untreated cells. Other studies have shown that fibrin(ogen binds to the monocyte integrin αMβ2. This study therefore sought to investigate the effect of sFn on β2 integrin mediated monocyte adherence and killing of tumor cells. Methods The role of sFn in monocyte adherence and cytotoxicity against tumor cells was initially studied using static microplate adherence and cytotoxicity assays, and under physiologically relevant flow conditions in a microscope perfusion incubator system. Blocking studies were performed using monoclonal antibodies specific for β2 integrins and CD54, and specific peptides which inhibit sFn binding to these receptors. Results Enhancement of monocyte/tumor cell adherence was observed when only one cell type was bound to sFn, but profound inhibition was observed when sFn was bound to both monocytes and tumor cells. This effect was also reflected in the pattern of monocyte cytotoxicity. Studies using monoclonal blocking antibodies and specific blocking peptides (which did not affect normal coagulation showed that the predominant mechanism of fibrin inhibition is via its binding to αMβ2 on monocytes, and to CD54 on both leukocytes and tumor cells. Conclusion sFn inhibits monocyte adherence and cytotoxicity of

  8. Cytotoxicity of yellow sand in lung epithelial cells

    Indian Academy of Sciences (India)

    Y H Kim; K S Kim; N J Kwak; K H Lee; S A Kweon; Y Lim

    2003-02-01

    The present study was carried out to observe the cytotoxicity of yellow sand in comparison with silica and titanium dioxide in a rat alveolar type II cell line (RLE-6TN). Yellow sand (China Loess) was obtained from the loess layer in the Gunsu Province of China. The mean particle diameter of yellow sand was about 0.003 ± 0.001 mm. Major elements of yellow sand were Si(27.7 ± 0.6%), Al(6.01 ± 0.17%), and Ca(5.83 ± 0.23%) in that order. Silica and yellow sand significantly decreased cell viability and increased [Ca2+]i. All three particles increased the generation of H2O2. TiO2 did not change Fenton activity, while silica induced a slight increase of Fenton activity. In contrast, yellow sand induced a significant increase of Fenton activity. Silica, yellow sand and TiO2 induced significant nitrite formations in RLE-6TN cells. Silica showed the highest increase in nitrite formation, while yellow sand induced the least formation of nitrite. Silica and yellow sand increased the release of TNF-. Based on these results, we suggest that yellow sand can induce cytotoxicity in RLE-6TN cells and reactive oxygen species, Fenton activity and reactive nitrogen species might be involved in this toxicity.

  9. In vitro cytotoxicity following specific activation of amygdalin by beta-glucosidase conjugated to a bladder cancer-associated monoclonal antibody.

    Science.gov (United States)

    Syrigos, K N; Rowlinson-Busza, G; Epenetos, A A

    1998-12-09

    We describe a novel version of antibody-directed enzyme prodrug therapy (ADEPT), with the use of amygdalin as prodrug. Amygdalin is a naturally occurring cyanogenic glycoside, which can be cleaved by sweet almond beta-glucosidase to yield free cyanide. If amygdalin could be activated specifically at the tumour site, then malignant cells would be killed without the systemic toxicity usually associated with chemotherapy. To this end, we have conjugated beta-glucosidase to a tumour-associated monoclonal antibody (MAb) (HMFG1) and the conjugate has been tested in vitro for specificity and cytotoxicity subsequent to activation of amygdalin. Amygdalin was cytotoxic to HT1376 bladder cancer cells only at high concentrations, whereas the combination of amygdalin with HMFG1-beta-glucosidase enhanced the cytotoxic effect of amygdalin by 36-fold. When 2 concentrations of HMFG1-beta-glucosidase were compared, the toxic effect was dose dependent. The cytotoxicity of amygdalin was also enhanced by the MAb-enzyme conjugate even when the unbound conjugate was removed from the medium prior to exposure to amygdalin and the cells were washed. In addition to the cytotoxic effect, we also demonstrated specificity, using a MAb-enzyme conjugate that does not recognise the HT1376 bladder cancer cells. Finally, we studied the cytotoxic effect of the conjugate in co-culture of HMFG1-positive and-negative cell lines (HT 1376 and U87MG cells). We demonstrated that the rate of surviving cells corresponds well to the percentage of U87MG (HMFG1-negative) cells in the flask. Our findings indicate that ADEPT is more effective than non-directed enzyme activation of a prodrug and can result in a non-toxic cancer therapy.

  10. Cytotoxic effects of catechols to glial and neuronal cells

    Directory of Open Access Journals (Sweden)

    Ramon Santos El-Bachá

    2015-04-01

    Full Text Available Catechols are compounds that autoxidises under physiological conditions leading to the formation of reactive oxygen species (ROS, semiquinones, and quinones. These molecules can be formed in organisms because of the metabolism of exogenous aromatic substances, such as benzene. However, there are several important endogenous catechols, which have physiological functions, such as catecholamines. Furthermore, several pharmacological agents are catechols, such as apomorphine, or can be metabolised to generate these compounds. In this presentation we will show that apomorphine can unspecifically bind to proteins during its autoxidation, a phenomenon that is inhibited by thiols. Brain endothelial cells and glial cells express xenobiotic-metabolising enzymes as components of the metabolic blood-brain barrier in an attempt to protect the central nervous system against drugs. Since UDP-glucuronosyltransferases (EC 2.4.1.17 are among these enzymes, we investigated the ability of brain microsomes to conjugate catechols with glucuronate. Despite the fact that 1-naphtol could be glucuronidated in the presence of brain cortex microsomes, the same was not observed for most of catechols that were tested. Therefore, this is not the main mechanism used to protect the brain against them. Indeed, catechols may inhibit other xenobiotic-metabolising enzymes. We showed that apomorphine inhibited the cytochrome P450-dependent dealkylation activity. The production of ROS and reactive quinones, as well as their effects on protein functions, seems to be involved in the cytotoxicity of catechols. Glial cells are more resistant than neuronal cells. Apomorphine was more toxic to rat neurons than to rat C6 glioma cells. 1,2-Dihydroxybenzene (catechol killed human GL-15 cells with an EC50 of 230 uM after 72 h, a effect that was significantly inhibited by superoxide dismutase (EC 1.15.1.1. Another mechanism that we found to be involved in catechol cytotoxicity is the inhibition

  11. A chimeric fusion of the hASH1 and EZH2 promoters mediates high and specific reporter and suicide gene expression and cytotoxicity in small cell lung cancer cells

    DEFF Research Database (Denmark)

    Poulsen, T.T.; Pedersen, N.; Juel, H.

    2008-01-01

    Transcriptionally targeted gene therapy is a promising experimental modality for treatment of systemic malignancies such as small cell lung cancer (SCLC). We have identified the human achaete-scute homolog 1 (hASH1) and enhancer of zeste homolog 2 (EZH2) genes as highly upregulated in SCLC compar...

  12. Cytotoxicity of monodispersed chitosan nanoparticles against the Caco-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Loh, Jing Wen [Laboratory for Drug Delivery, Pharmacy, Characterisation and Analysis, University of Western Australia (Australia); Saunders, Martin [Centre for Microscopy, Characterisation and Analysis, University of Western Australia (Australia); Lim, Lee-Yong, E-mail: lee.lim@uwa.edu.au [Laboratory for Drug Delivery, Pharmacy, Characterisation and Analysis, University of Western Australia (Australia); School of Biomedical, Biomolecular and Chemical Sciences, 35 Stirling Hwy, Crawley 6009 (Australia)

    2012-08-01

    Published toxicology data on chitosan nanoparticles (NP) often lack direct correlation to the in situ size and surface characteristics of the nanoparticles, and the repeated NP assaults as experienced in chronic use. The aim of this paper was to breach these gaps. Chitosan nanoparticles synthesized by spinning disc processing were characterised for size and zeta potential in HBSS and EMEM at pHs 6.0 and 7.4. Cytotoxicity against the Caco-2 cells was evaluated by measuring the changes in intracellular mitochondrial dehydrogenase activity, TEER and sodium fluorescein transport data and cell morphology. Cellular uptake of NP was observed under the confocal microscope. Contrary to established norms, the collective data suggest that the in vitro cytotoxicity of NP against the Caco-2 cells was less influenced by positive surface charges than by the particle size. Particle size was in turn determined by the pH of the medium in which the NP was dispersed, with the mean size ranging from 25 to 333 nm. At exposure concentration of 0.1%, NP of 25 ± 7 nm (zeta potential 5.3 ± 2.8 mV) was internalised by the Caco-2 cells, and the particles were observed to inflict extensive damage to the intracellular organelles. Concurrently, the transport of materials along the paracellular pathway was significantly facilitated. The Caco-2 cells were, however, capable of recovering from such assaults 5 days following NP removal, although a repeat NP exposure was observed to produce similar effects to the 1st exposure, with the cells exhibiting comparable resiliency to the 2nd assault. -- Highlights: ► Chitosan nanoparticles reduced mitochondrial dehydrogenase activity. ► Cellular uptake of chitosan nanoparticles was observed. ► Chitosan nanoparticles inflicted extensive damage to the cell morphology. ► The transport of materials along the paracellular pathway was facilitated.

  13. Cytotoxic effect of root extract of Tiliacora racemosa and oil of Semecarpus anacardium nut in human tumour cells.

    Science.gov (United States)

    Chakraborty, Sutapa; Roy, Madhumita; Taraphdar, Amit K; Bhattacharya, R K

    2004-08-01

    Tiliacora racemosa and Semecarpus anacardium, the two plants frequently used in Ayurvedic medicine for the treatment of cancerous diseases, have been selected to examine their action in four human tumour cell lines: acute myeloblastic leukaemia (HL-60), chronic myelogenic leukaemia (K-562), breast adenocarcinoma (MCF-7) and cervical epithelial carcinoma (HeLa). In cells grown in appropriate media the ethanol extract of T. racemosa root, the total alkaloids isolated from this organ and S. anacardium nut oil prepared according to the Ayurvedic principle were found to have cytotoxic activity. The alkaloid fraction from T. racemosa had maximum cytotoxicity and was effective against all four cell lines. S. anacardium oil was cytotoxic only in leukaemic cells. These herbal preparations were not cytotoxic towards normal human lymphocytes, suggesting their action is specific for tumour cells. On microscopic examination the cells treated with these agents exhibited characteristic morphological features of apoptosis, such as cell shrinkage, and the formation of apoptotic bodies. Fluorescent staining with propidium iodide revealed distinct chromatin condensation and nuclear fragmentation. The apoptotic index paralleled the cytotoxic parameters, and fragmented DNA extracted free of genomic DNA from treated cells displayed a typical ladder pattern on gel electrophoresis. Apoptosis induced by alkaloids and phenolics, the active principles present in T. racemosa and S. anacardium, respectively, was found to be mediated by the activation of caspases. Copyright (c) 2004 John Wiley & Sons, Ltd.

  14. Non-specific SIRT inhibition as a mechanism for the cytotoxicity of ginkgolic acids and urushiols.

    Science.gov (United States)

    Ryckewaert, Lucie; Sacconnay, Lionel; Carrupt, Pierre-Alain; Nurisso, Alessandra; Simões-Pires, Claudia

    2014-09-02

    Ginkgolic acids and urushiols are natural alkylphenols known for their mutagenic, carcinogenic and genotoxic potential. However, the mechanism of toxicity of these compounds has not been thoroughly elucidated so far. Considering that the SIRT inhibitory potential of anacardic acids has been hypothesized by in silico techniques, we herein demonstrated through both in vitro and computational methods that structurally related compounds such as ginkgolic acids and urushiols are able to modulate SIRT activity. Moreover, their SIRT inhibitory profile and cytotoxicity were comparable to sirtinol, a non-specific SIRT inhibitor (SIRT1 and SIRT2), and different from EX-527, a SIRT1 specific inhibitor. This is the first report on the SIRT inhibition of ginkgolic acids and urushiols. The results reported here are in line with previously observed effects on the induction of apoptosis by this class of compounds, and the non-specific SIRT inhibition is suggested as a new mechanism for their in vitro cytotoxicity.

  15. Cell-mediated immunity to Plasmodium falciparum infection: evidence against the involvement of cytotoxic lymphocytes

    DEFF Research Database (Denmark)

    Theander, T G; Andersen, B J; Pedersen, B K;

    1988-01-01

    by either SPag or PPD in the presence of immune serum. Studies on subpopulations of PBMC indicated that the inhibitory cells resided among the adherent cell fraction. Furthermore we tested PBMC for cytotoxic activity against P. falciparum-infected autologous or heterologous erythrocytes. Experiments were...... done both in the absence and the presence of immune serum. Neither fresh PBMC nor PBMC activated by SPag or PPD for 7 days prior to assay were cytotoxic, indicating that cytotoxic T cells, natural killer (NK) cells, and K cells did not possess cytotoxic activity directed against parasitized...

  16. Enhanced Growth Inhibition of Osteosarcoma by Cytotoxic Polymerized Liposomal Nanoparticles Targeting the Alcam Cell Surface Receptor

    Directory of Open Access Journals (Sweden)

    Noah Federman

    2012-01-01

    Full Text Available Osteosarcoma is the most common primary malignancy of bone in children, adolescents, and adults. Despite extensive surgery and adjuvant aggressive high-dose systemic chemotherapy with potentially severe bystander side effects, cure is attainable in about 70% of patients with localized disease and only 20%–30% of those patients with metastatic disease. Targeted therapies clearly are warranted in improving our treatment of this adolescent killer. However, a lack of osteosarcoma-associated/specific markers has hindered development of targeted therapeutics. We describe a novel osteosarcoma-associated cell surface antigen, ALCAM. We, then, create an engineered anti-ALCAM-hybrid polymerized liposomal nanoparticle immunoconjugate (α-AL-HPLN to specifically target osteosarcoma cells and deliver a cytotoxic chemotherapeutic agent, doxorubicin. We have demonstrated that α-AL-HPLNs have significantly enhanced cytotoxicity over untargeted HPLNs and over a conventional liposomal doxorubicin formulation. In this way, α-AL-HPLNs are a promising new strategy to specifically deliver cytotoxic agents in osteosarcoma.

  17. Parasporal Proteins from Bacillus thuringiensis and Their Cytotoxicity on Human Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    WANG Lei; LV Yuan; YI Yin-sha; YI Shang-hui; LI Lin

    2015-01-01

    Parasporins(PSs) represent a novel functional category of crystal proteins (Cry) produced by non-insecticidal Bacillus thuringiensisA distinct feature for PSs is their specific cytotoxicity against human cancer cells from diverse origins, other than hemolytic or insecticidal activityAs structurally/functionally Cry proteins, parasporins are expressed as protoxins that require protease cleavage for activationCurrently, identified PSs is classified into 6 groups:PS1, PS2, PS3, PS4, PS5 and PS6, which are heterogeneous in cytotoxic spectrum and activity levelSome PSs have been explored for their mode of anticancer activities, reports mainly include pore formation induced by binding to putative receptors on cell membrane and apoptosis by intracellular Ca 2+concentrationFurther work should focus on the identification of new PS or PS homologs and better understanding of their anticancer mechanism before possible application in cancer therapy.

  18. Dietary gluten increases natural killer cell cytotoxicity and cytokine secretion.

    Science.gov (United States)

    Larsen, Jesper; Dall, Morten; Antvorskov, Julie Christine; Weile, Christian; Engkilde, Kåre; Josefsen, Knud; Buschard, Karsten

    2014-10-01

    Dietary gluten influences the development of type 1 diabetes in nonobese diabetic (NOD) mice and biobreeding rats, and has been shown to influence a wide range of immunological factors in the pancreas and gut. In the present study, the effects of gluten on NK cells were studied in vitro and in vivo. We demonstrated that gliadin increased direct cytotoxicity and IFN-γ secretion from murine splenocytes and NK cells toward the pancreatic beta-cell line MIN6 cells. Additionally, stimulation of MIN6 cells led to a significantly increased proportion of degranulating C57BL/6 CD107a(+) NK cells. Stimulation of C57BL/6 pancreatic islets with gliadin significantly increased secretion of IL-6 more than ninefold. In vivo, the gluten-containing diet led to a higher expression of NKG2D and CD71 on NKp46(+) cells in all lymphoid organs in BALB/c and NOD mice compared with the gluten-free diet. Collectively, our data suggest that dietary gluten increases murine NK-cell activity against pancreatic beta cells. This mechanism may contribute to development of type 1 diabetes and explain the higher disease incidence associated with gluten intake in NOD mice.

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

    Science.gov (United States)

    Guo, Meng; Wu, Tingting; Wan, Lixin

    2016-07-01

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

  20. Most influenza A virus-specific memory cytotoxic T lymphocytes react with antigenic epitopes associated with internal virus determinants

    OpenAIRE

    1984-01-01

    This paper shows that most murine (C57BL/6) influenza A virus-specific memory cytotoxic T lymphocyte (CTL) clones tested in limiting dilution did not react with the influenza A virus surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). This lysis of syngeneic target cells infected with the influenza A virus strains, Aichi (H3N2), PR8 (H1N1), or recombinant strain X31 (H3N2) indicates that most antigenic epitopes recognized are associated with internal virus determinants. X31 and ...

  1. In Vitro Generation of Antigen-Specific T Cells from Induced Pluripotent Stem Cells of Antigen-Specific T Cell Origin.

    Science.gov (United States)

    Kaneko, Shin

    2016-01-01

    Induced pluripotent stem (iPS) cells derived from T lymphocyte (T-iPS cells) preserve the T cell receptor (TCR) α and β gene rearrangements identical to the original T cell clone. Re-differentiated CD8 single positive αβ T cells from the T-iPS cells exhibited antigen-specific cytotoxicity, improved proliferative response, and elongation of telomere indicating rejuvenation of antigen specific T cell immunity in vitro. To regenerate antigen specific cytotoxic T lymphocytes (CTL), first, we have optimized a method for reprogramming-resistant CD8 T cell clones into T-iPS cells by using sendaiviral vectors. Second, we have optimized stepwise differentiation methods for inducing hematopoietic progenitor cells, T cell progenitors, and functionally matured CD8 single positive CTL. These protocols provide useful in vitro tools and models both for research of antigen-specific T cell immunotherapy and for research of normal and pathological thymopoiesis.

  2. Cytotoxicity of Portuguese Propolis: The Proximity of the In Vitro Doses for Tumor and Normal Cell Lines

    Directory of Open Access Journals (Sweden)

    Ricardo C. Calhelha

    2014-01-01

    Full Text Available With a complex chemical composition rich in phenolic compounds, propolis (resinous substance collected by Apis mellifera from various tree buds exhibits a broad spectrum of biological activities. Recently, in vitro and in vivo data suggest that propolis has anticancer properties, but is the cytoxicity of propolis specific for tumor cells? To answer this question, the cytotoxicity of phenolic extracts from Portuguese propolis of different origins was evaluated using human tumor cell lines (MCF7—breast adenocarcinoma, NCI-H460—non-small cell lung carcinoma, HCT15—colon carcinoma, HeLa—cervical carcinoma, and HepG2—hepatocellular carcinoma, and non-tumor primary cells (PLP2. The studied propolis presented high cytotoxic potential for human tumor cell lines, mostly for HCT15. Nevertheless, excluding HCT15 cell line, the extracts at the GI50 obtained for tumor cell lines showed, in general, cytotoxicity for normal cells (PLP2. Propolis phenolic extracts comprise phytochemicals that should be further studied for their bioactive properties against human colon carcinoma. In the other cases, the proximity of the in vitro cytotoxic doses for tumor and normal cell lines should be confirmed by in vivo tests and may highlight the need for selection of specific compounds within the propolis extract.

  3. Identification of cytotoxic drugs that selectively target tumor cells with MYC overexpression.

    Directory of Open Access Journals (Sweden)

    Anna Frenzel

    Full Text Available Expression of MYC is deregulated in a wide range of human cancers, and is often associated with aggressive disease and poorly differentiated tumor cells. Identification of compounds with selectivity for cells overexpressing MYC would hence be beneficial for the treatment of these tumors. For this purpose we used cell lines with conditional MYCN or c-MYC expression, to screen a library of 80 conventional cytotoxic compounds for their ability to reduce tumor cell viability and/or growth in a MYC dependent way. We found that 25% of the studied compounds induced apoptosis and/or inhibited proliferation in a MYC-specific manner. The activities of the majority of these were enhanced both by c-MYC or MYCN over-expression. Interestingly, these compounds were acting on distinct cellular targets, including microtubules (paclitaxel, podophyllotoxin, vinblastine and topoisomerases (10-hydroxycamptothecin, camptothecin, daunorubicin, doxorubicin, etoposide as well as DNA, RNA and protein synthesis and turnover (anisomycin, aphidicholin, gliotoxin, MG132, methotrexate, mitomycin C. Our data indicate that MYC overexpression sensitizes cells to disruption of specific pathways and that in most cases c-MYC and MYCN overexpression have similar effects on the responses to cytotoxic compounds. Treatment of the cells with topoisomerase I inhibitors led to down-regulation of MYC protein levels, while doxorubicin and the small molecule MYRA-A was found to disrupt MYC-Max interaction. We conclude that the MYC pathway is only targeted by a subset of conventional cytotoxic drugs currently used in the clinic. Elucidating the mechanisms underlying their specificity towards MYC may be of importance for optimizing treatment of tumors with MYC deregulation. Our data also underscores that MYC is an attractive target for novel therapies and that cellular screenings of chemical libraries can be a powerful tool for identifying compounds with a desired biological activity.

  4. Identification of cytotoxic drugs that selectively target tumor cells with MYC overexpression.

    Science.gov (United States)

    Frenzel, Anna; Zirath, Hanna; Vita, Marina; Albihn, Ami; Henriksson, Marie Arsenian

    2011-01-01

    Expression of MYC is deregulated in a wide range of human cancers, and is often associated with aggressive disease and poorly differentiated tumor cells. Identification of compounds with selectivity for cells overexpressing MYC would hence be beneficial for the treatment of these tumors. For this purpose we used cell lines with conditional MYCN or c-MYC expression, to screen a library of 80 conventional cytotoxic compounds for their ability to reduce tumor cell viability and/or growth in a MYC dependent way. We found that 25% of the studied compounds induced apoptosis and/or inhibited proliferation in a MYC-specific manner. The activities of the majority of these were enhanced both by c-MYC or MYCN over-expression. Interestingly, these compounds were acting on distinct cellular targets, including microtubules (paclitaxel, podophyllotoxin, vinblastine) and topoisomerases (10-hydroxycamptothecin, camptothecin, daunorubicin, doxorubicin, etoposide) as well as DNA, RNA and protein synthesis and turnover (anisomycin, aphidicholin, gliotoxin, MG132, methotrexate, mitomycin C). Our data indicate that MYC overexpression sensitizes cells to disruption of specific pathways and that in most cases c-MYC and MYCN overexpression have similar effects on the responses to cytotoxic compounds. Treatment of the cells with topoisomerase I inhibitors led to down-regulation of MYC protein levels, while doxorubicin and the small molecule MYRA-A was found to disrupt MYC-Max interaction. We conclude that the MYC pathway is only targeted by a subset of conventional cytotoxic drugs currently used in the clinic. Elucidating the mechanisms underlying their specificity towards MYC may be of importance for optimizing treatment of tumors with MYC deregulation. Our data also underscores that MYC is an attractive target for novel therapies and that cellular screenings of chemical libraries can be a powerful tool for identifying compounds with a desired biological activity.

  5. Mycobacterium avium and purified protein derivative-specific cytotoxicity mediated by CD4+ lymphocytes from healthy HIV-seropositive and-seronegative individuals

    DEFF Research Database (Denmark)

    Ravn, P; Pedersen, B K

    1996-01-01

    to respond to mycobacterial antigens. The immunological dysfunction leading to reduced mycobacterial-specific cytotoxic activity in healthy HIV-seropositive subjects is caused either by a reduction in the number of antigen-responsive CD4+ T cells (memory) or by an impairment of their ability to respond...

  6. Cytotoxicity of PEGylated graphene oxide on lymphoma cells.

    Science.gov (United States)

    Du, Li; Wu, Shaoling; Li, Yanhui; Zhao, Xindong; Ju, Xiaoyan; Wang, Yuzhen

    2014-01-01

    Graphene oxide (GO) is a hotspot, especially in the field of biomedical. However, the clinical application of GO is still faces a lot of challenges. In order to improve the solubility and biocompatibility of GO, polyethylene glycol (PEG) was grafted on the surface of graphene oxide by amide reaction. PEGylated graphene oxide (PEG-GO) was characterized using Fourier transform infrared spectroscopy (FTIR). The stability of PEG-GO detected in different solutions. Raji cell was selected as a lymphoma cell model to study the cytotoxicity of PEG-GO. Cell viability was detected using the Cell Counting Kit-8 assay. Cells were treated with different concentrations (10-100 μg/mL) of PEG-GO at different time points (6, 12, and 24 h). The FTIR spectrum of PEG-GO indicated that polyethylene glycol was successfully grafted onto GO. PEG-GO had excellent stability in all solutions. Cells treated with PEG-GO (10-100 μg/mL) for 24 hours had survival rates were over 80%. These results demonstrate that PEG-GO had an excellent dispersion in biological solutions and the toxicity of PEG-GO to lymphoma cells was low. The paper may provide cytological evidence for the application of PEG-GO in medicine.

  7. Analysis of the Effects of Cell Stress and Cytotoxicity on In Vitro Assay Activity Across a Diverse Chemical and Assay Space

    Data.gov (United States)

    U.S. Environmental Protection Agency — Chemical toxicity can arise from disruption of specific biomolecular functions or through more generalized cell stress and cytotoxicity-mediated processes. Here,...

  8. Analysis of the Effects of Cell Stress and Cytotoxicity on In Vitro Assay Activity Across a Diverse Chemical and Assay Space

    Science.gov (United States)

    Chemical toxicity can arise from disruption of specific biomolecular functions or through more generalized cell stress and cytotoxicity-mediated processes. Here, concentration-dependent responses of 1063 chemicals including pharmaceuticals, natural products, pesticidals, consumer...

  9. Sulfasalazine intensifies temozolomide cytotoxicity in human glioblastoma cells.

    Science.gov (United States)

    Ignarro, Raffaela Silvestre; Facchini, Gustavo; Vieira, André Schwambach; De Melo, Daniela Rodrigues; Lopes-Cendes, Iscia; Castilho, Roger Frigério; Rogerio, Fabio

    2016-07-01

    Temozolomide (TMZ) is an alkylating agent used to treat glioblastoma. This tumor type synthesizes the antioxidant glutathione through system X c (-) , which is inhibited by sulfasalazine (SAS). We exposed A172 and T98G human glioblastoma cells to a presumably clinically relevant concentration of TMZ (25 µM) and/or 0.5 mM SAS for 1, 3, or 5 days and assessed cell viability. For both cell lines, TMZ alone did not alter viability at any time point, while the coadministration of TMZ and SAS significantly reduced cell viability after 5 days. The drug combination exerted a synergistic effect on A172 cells after 3 and 5 days. Therefore, this particular lineage was subjected to complementary analyses on the genetic (transcriptome) and functional (glutathione and proliferating cell nuclear antigen (PCNA) protein) levels. Cellular pathways containing differentially expressed genes related to the cell cycle were modified by TMZ alone. On the other hand, SAS regulated pathways associated with glutathione metabolism and synthesis, irrespective of TMZ. Moreover, SAS, but not TMZ, depleted the total glutathione level. Compared with the vehicle-treated cells, the level of PCNA protein was lower in cells treated with TMZ alone or in combination with SAS. In conclusion, our data showed that the association of TMZ and SAS is cytotoxic to T98G and A172 cells, thus providing useful insights for improving TMZ clinical efficacy through testing this novel drug combination. Moreover, the present study not only reports original information on differential gene expression in glioblastoma cells exposed to TMZ and/or SAS but also describes an antiproliferative effect of TMZ, which has not yet been observed in A172 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. CCL22-specific T Cells

    DEFF Research Database (Denmark)

    Martinenaite, Evelina; Munir Ahmad, Shamaila; Hansen, Morten

    2016-01-01

    Tumor cells and tumor-infiltrating macrophages produce the chemokine CCL22, which attracts regulatory T cells (Tregs) into the tumor microenvironment, decreasing anticancer immunity. Here, we investigated the possibility of targeting CCL22-expressing cells by activating specific T cells. We...... analyzed the CCL22 protein signal sequence, identifying a human leukocyte antigen A2- (HLA-A2-) restricted peptide epitope, which we then used to stimulate peripheral blood mononuclear cells (PMBCs) to expand populations of CCL22-specific T cells in vitro. T cells recognizing an epitope derived from...... the signal-peptide of CCL22 will recognize CCL22-expressing cells even though CCL22 is secreted out of the cell. CCL22-specific T cells recognized and killed CCL22-expressing cancer cells. Furthermore, CCL22-specific T cells lysed acute monocytic leukemia cells in a CCL22 expression-dependent manner. Using...

  13. Cytotoxic activity of marine algae against cancerous cells

    Directory of Open Access Journals (Sweden)

    Élica A. C. Guedes

    2013-08-01

    Full Text Available This paper presents an investigation on the cytotoxic activity in human tumor cell from dichloromethane, chloroform, methanol, ethanol, water extracts, and hexane and chloroform fractions from green, brown and red algae collected at Riacho Doce Beach, north coast of Alagoas, Brazil, against the cancer cells K562 (chronic myelocytic leukemia, HEp-2 (laryngeal epidermoid carcinoma and NCI-H292 (human lung mucoepidermoid carcinoma through the MTT colorimetric method. The dichloromethane extract and chloroform fraction of Hypnea musciformis showed the best cytotoxic activity against K562 (3.8±0.2 µg.mL-1 and 6.4±0.4 µg.mL-1, respectively. Dichloromethane extracts of Dictyota dichotoma (16.3±0.3 µg.mL-1 and the chloroform fraction of H. musciformis (6.0±0.03 µg.mL-1 and chloroform fraction of P. gymnospora (8.2±0.4 were more active against HEp-2 as well as ethanol extracts of P. gymnospora (15.9±2.8 µg.mL-1 and chloroform fraction of H. musciformis (15.0±1.3 µg.mL-1 against the cell NCI-H292. The constituents with higher anticancer action are present in the extracts of dichloromethane and chloroform and in the chloroform fraction of H. musciformis, Digenea simplex, P. gymnospora, and D.dichotoma. In the case of the seaweed S. vulgare, the anticancer constituents are present in the aqueous extract.

  14. Cytotoxicity of methanol extracts of Elaeis guineensis on MCF-7 and Vero cell lines

    Institute of Scientific and Technical Information of China (English)

    Soundararajan Vijayarathna; Sreenivasan Sasidharan

    2012-01-01

    To investigate the cytotoxic effect of Elaeis guineensis methanol extract on MCF-7 and Vero cell. Methods: In vitro cytotoxicity was evaluated in by MTT assay. Cell morphological changes were observed by using light microscope. Results: The MTT assay indicated that methanol extract of the plant exhibited significant cytotoxic effects on MCF-7. Morphological alteration of the cell lines after exposure with Elaeis guineensis extract were observed under phase contrast microscope in the dose dependent manner. Conclusions: The results suggest the probable use of the Elaeis guineensis methanol extract in preparing recipes for cancer-related ailments. Further studies on isolation of metabolites and their in vivo cytotoxicity are under investigation.

  15. Cytotoxicity of methanol extracts of Elaeis guineensis on MCF-7 and Vero cell lines.

    Science.gov (United States)

    Vijayarathna, Soundararajan; Sasidharan, Sreenivasan

    2012-10-01

    To investigate the cytotoxic effect of Elaeis guineensis methanol extract on MCF-7 and Vero cell. In vitro cytotoxicity was evaluated in by MTT assay. Cell morphological changes were observed by using light microscope. The MTT assay indicated that methanol extract of the plant exhibited significant cytotoxic effects on MCF-7. Morphological alteration of the cell lines after exposure with Elaeis guineensis extract were observed under phase contrast microscope in the dose dependent manner. The results suggest the probable use of the Elaeis guineensis methanol extract in preparing recipes for cancer-related ailments. Further studies on isolation of metabolites and their in vivo cytotoxicity are under investigation.

  16. Serine protease inhibitor 6 protects cytotoxic T cells from self-inflicted injury by ensuring the integrity of cytotoxic granules.

    Science.gov (United States)

    Zhang, Manling; Park, Sun-Mi; Wang, Yue; Shah, Ramila; Liu, Ni; Murmann, Andrea E; Wang, Chyung-Ru; Peter, Marcus E; Ashton-Rickardt, Philip G

    2006-04-01

    How cytotoxic T lymphocytes (CTLs) kill intracellular pathogens without killing themselves has been a recurring question ever since their discovery. By using mice deficient in Serine Protease Inhibitor 6 (Spi6), we show that by inhibiting granzyme B (GrB), Spi6 protects CTLs from self-inflicted injury. Infection with either Lymphocytic Choriomeningitis virus (LCMV) or Listeria monocytogenes (LM) revealed increased apoptosis and diminished survival of Spi6 knockout (KO) CTLs, which was cell autonomous and could be corrected by GrB deficiency. Spi6 KO mice in turn were impaired in their ability to clear LCMV infection. Spi6 KO CTLs revealed a breakdown in the integrity of cytotoxic granules, increased cytoplasmic GrB, and ensuing apoptosis. We conclude that Spi6 protects CTLs from suicide caused by GrB-mediated breakdown of cytotoxic granules.

  17. In vitro testing of basal cytotoxicity: establishment of an adverse outcome pathway from chemical insult to cell death

    Science.gov (United States)

    Vinken, Mathieu; Blaauboer, Bas J.

    2017-01-01

    In this paper, an in vitro basal cytotoxicity testing strategy is described for new chemical entities that lack any pre-existing information on potential toxicity. Special attention is paid to the selection of the cellular system, cytotoxicity assay and exposure conditions. This approach is based on a newly proposed generic adverse outcome pathway from chemical insult to cell death that consists of 3 steps, including initial cell injury, mitochondrial dysfunction and cell demise. The suggested strategy to consider in vitro basal cytotoxicity as a first step in evaluating the toxicity of new chemical entities can be placed in a tiered strategy that could be continued by evaluating more specific types of toxicity. PMID:27939612

  18. In vitro testing of basal cytotoxicity: Establishment of an adverse outcome pathway from chemical insult to cell death.

    Science.gov (United States)

    Vinken, Mathieu; Blaauboer, Bas J

    2017-03-01

    In this paper, an in vitro basal cytotoxicity testing strategy is described for new chemical entities that lack any pre-existing information on potential toxicity. Special attention is paid to the selection of the cellular system, cytotoxicity assay and exposure conditions. This approach is based on a newly proposed generic adverse outcome pathway from chemical insult to cell death that consists of 3 steps, including initial cell injury, mitochondrial dysfunction and cell demise. The suggested strategy to consider in vitro basal cytotoxicity as a first step in evaluating the toxicity of new chemical entities can be placed in a tiered strategy that could be continued by evaluating more specific types of toxicity. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Tumor acidosis enhances cytotoxic effects and autophagy inhibition by salinomycin on cancer cell lines and cancer stem cells

    Science.gov (United States)

    Pellegrini, Paola; Dyczynski, Matheus; Sbrana, Francesca Vittoria; Karlgren, Maria; Buoncervello, Maria; Hägg-Olofsson, Maria; Ma, Ran; Hartman, Johan; Bajalica-Lagercrantz, Svetlana; Grander, Dan; Kharaziha, Pedram; De Milito, Angelo

    2016-01-01

    Sustained autophagy contributes to the metabolic adaptation of cancer cells to hypoxic and acidic microenvironments. Since cells in such environments are resistant to conventional cytotoxic drugs, inhibition of autophagy represents a promising therapeutic strategy in clinical oncology. We previously reported that the efficacy of hydroxychloroquine (HCQ), an autophagy inhibitor under clinical investigation is strongly impaired in acidic tumor environments, due to poor uptake of the drug, a phenomenon widely associated with drug resistance towards many weak bases. In this study we identified salinomycin (SAL) as a potent inhibitor of autophagy and cytotoxic agent effective on several cancer cell lines under conditions of transient and chronic acidosis. Since SAL has been reported to specifically target cancer-stem cells (CSC), we used an established model of breast CSC and CSC derived from breast cancer patients to examine whether this specificity may be associated with autophagy inhibition. We indeed found that CSC-like cells are more sensitive to autophagy inhibition compared to cells not expressing CSC markers. We also report that the ability of SAL to inhibit mammosphere formation from CSC-like cells was dramatically enhanced in acidic conditions. We propose that the development and use of clinically suitable SAL derivatives may result in improved autophagy inhibition in cancer cells and CSC in the acidic tumor microenvironment and lead to clinical benefits. PMID:27248168

  20. Dendritic cells decreased the concomitant expanded Tregs and Tregs related IL-35 in cytokine-induced killer cells and increased their cytotoxicity against leukemia cells.

    Science.gov (United States)

    Pan, Ying; Tao, Qianshan; Wang, Huiping; Xiong, Shudao; Zhang, Rui; Chen, Tianping; Tao, Lili; Zhai, Zhimin

    2014-01-01

    Regulatory T cells (Tregs) are potent immunosuppressive cells and essential for inducing immune tolerance. Recent studies have reported that Tregs and Tregs related cytokines can inhibit the antitumor activity of cytokine-induced killer (CIK) cells, but dendritic cells co-cultured CIK (DC-CIK) cells can be used for induction of a specific immune response by blocking of Tregs and TGF-β, IL-10. As a novel identified cytokine, IL-35 is specially produced by Tregs and plays an essential role in immune regulation. However, it remains unknown whether IL-35 roles in tumor immunotherapy mediated by CIK and DC-CIK cells. In this study, we cultured CIK and DC-CIK cells from the same healthy adult samples, and investigated their phenotype, proliferation, cytotoxic activity against leukemia cell lines K562 and NB4 by FCM and CCK-8, measured IL-35, TGF-β and IL-10 protein by ELISA, detected Foxp3, IL-35 and IL-35 receptor mRNA by Real-time PCR, respectively. We found Tregs and IL-35 concomitantly expanded by a time-dependent way during the generation of CIK cells, but DC significantly down-regulated the expression of them and simultaneously up-regulated the proliferation ability as well as cytotoxic activity of CIK cells against leukemia cell lines. Therefore, our data suggested that DC decreased concomitant expanded Tregs and Tregs related IL-35 in CIK cells and might contribute to improve their cytotoxicity against leukemia cells in vitro.

  1. Mechanisms of Dihydroartemisinin and Dihydroartemisinin/Holotransferrin Cytotoxicity in T-Cell Lymphoma Cells.

    Directory of Open Access Journals (Sweden)

    Qiuyan Wang

    Full Text Available The validated therapeutic effects of dihydroartemisinin (DHA in solid tumors have encouraged us to explore its potential in treating T-cell lymphoma. We found that Jurkat cells (a T-cell lyphoma cell line were sensitive to DHA treatment with a IC50 of dihydroartemisinin. The cytotoxic effect of DHA in Jurkat cells showed a dose- and time- dependent manner. Interestingly, the cytotoxic effect of DHA was further enhanced by holotransferrin (HTF due to the high expression of transferrin receptors in T-cell lymphoma. Mechanistically, DHA significantly increased the production of intracellular reactive oxygen species, which led to cell cycle arrest and apoptosis. The DHA treatment also inhibited the expression of protumorgenic factors including VEGF and telomerase catalytic subunit. Our results have proved the therapeutic effect of DHA in T-cell lymphoma. Especially in combination with HTF, DHA may provide a novel efficient approach in combating the deadly disease.

  2. PHA-induced cytotoxicity of human lymphocytes against adherent hela-cells

    NARCIS (Netherlands)

    Huges-Law, G.; de Gast, G. C.; The, T. Hauw

    1978-01-01

    The conditions for a phytohaemagglutinin(PHA)-induced cytotoxicity test of human peripheral blood lymphocytes were investigated. [3H]thymidine prelabelled HeLa cells were used as target cells. Stimulation with 10 μl PHA/ml during 24 h gave the best measure of lymphocyte cytotoxic capacity. Supernata

  3. Reduced cytotoxicity in PCB-exposed Chinese Hamster Ovary (CHO) cells pretreated with vitamin E.

    Science.gov (United States)

    Murati, Teuta; Šimić, Branimir; Pleadin, Jelka; Vukmirović, Maja; Miletić, Marina; Durgo, Ksenija; Kniewald, Jasna; Kmetič, Ivana

    2017-01-01

    The aim of this study was to evaluate protective effects of vitamin E (50 -150 μM) in ovary cells upon cytotoxic effects induced by two structurally distinct PCB congeners - planar "dioxin-like" PCB 77 and non-planar di-ortho-substituted PCB 153 with an emphasis on identifying differences in the mechanism of vitamin E action depending on the structure of congeners. Application of three bioassays confirmed that PCBs decrease ovarian cell proliferation with slightly profound effects of PCB 77. PCB - induced ROS production and lipid peroxidation were significant for both congeners with also more noticeable effect for PCB 77. Vitamin E pre-incubation has improved viability of cells, reduced ROS formation and lipid peroxidation induced by PCBs' treatment. Preincubation with vitamin E was more effective when cells where treated with non-planar PCB 153. Altogether, vitamin E action was protective, congener specific and more effective when ovary cells were exposed to ortho-substituted PCB congener.

  4. Cytotoxic effects of curcumin in human retinal pigment epithelial cells.

    Directory of Open Access Journals (Sweden)

    Margrit Hollborn

    Full Text Available BACKGROUND: Curcumin from turmeric is an ingredient in curry powders. Due to its antiinflammatory, antioxidant and anticarcinogenic effects, curcumin is a promising drug for the treatment of cancer and retinal diseases. We investigated whether curcumin alters the viability and physiological properties of human retinal pigment epithelial (RPE cells in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Cellular proliferation was investigated with a bromodeoxy-uridine immunoassay, and chemotaxis was investigated with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation ELISA. Gene expression was determined by real-time PCR, and secretion of VEGF and bFGF was examined with ELISA. The phosphorylation level of proteins was revealed by Western blotting. The proliferation of RPE cells was slightly increased by curcumin at 10 µM and strongly reduced by curcumin above 50 µM. Curcumin at 50 µM increased slightly the chemotaxis of the cells. Curcumin reduced the expression and secretion of VEGF under control conditions and abolished the VEGF secretion induced by PDGF and chemical hypoxia. Whereas low concentrations of curcumin stimulated the expression of bFGF and HGF, high concentrations caused downregulation of both factors. Curcumin decreased dose-dependently the viability of RPE cells via induction of early necrosis (above 10 µM and delayed apoptosis (above 1 µM. The cytotoxic effect of curcumin involved activation of caspase-3 and calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress, increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt protein. CONCLUSION: It is concluded that curcumin at concentrations described to be effective in the treatment of tumor cells and in inhibiting death of retinal neurons (∼10 µM has adverse effects on RPE cells. It is suggested that, during the intake of curcumin as

  5. Cytotoxic Effects of Curcumin in Human Retinal Pigment Epithelial Cells

    Science.gov (United States)

    Hollborn, Margrit; Chen, Rui; Wiedemann, Peter; Reichenbach, Andreas; Bringmann, Andreas; Kohen, Leon

    2013-01-01

    Backround Curcumin from turmeric is an ingredient in curry powders. Due to its antiinflammatory, antioxidant and anticarcinogenic effects, curcumin is a promising drug for the treatment of cancer and retinal diseases. We investigated whether curcumin alters the viability and physiological properties of human retinal pigment epithelial (RPE) cells in vitro. Methodology/Principal Findings Cellular proliferation was investigated with a bromodeoxy-uridine immunoassay, and chemotaxis was investigated with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation ELISA. Gene expression was determined by real-time PCR, and secretion of VEGF and bFGF was examined with ELISA. The phosphorylation level of proteins was revealed by Western blotting. The proliferation of RPE cells was slightly increased by curcumin at 10 µM and strongly reduced by curcumin above 50 µM. Curcumin at 50 µM increased slightly the chemotaxis of the cells. Curcumin reduced the expression and secretion of VEGF under control conditions and abolished the VEGF secretion induced by PDGF and chemical hypoxia. Whereas low concentrations of curcumin stimulated the expression of bFGF and HGF, high concentrations caused downregulation of both factors. Curcumin decreased dose-dependently the viability of RPE cells via induction of early necrosis (above 10 µM) and delayed apoptosis (above 1 µM). The cytotoxic effect of curcumin involved activation of caspase-3 and calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress, increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt protein. Conclusion It is concluded that curcumin at concentrations described to be effective in the treatment of tumor cells and in inhibiting death of retinal neurons (∼10 µM) has adverse effects on RPE cells. It is suggested that, during the intake of curcumin as concomitant therapy of

  6. Increasing a Robust Antigen-Specific Cytotoxic T Lymphocyte Response by FMDV DNA Vaccination with IL-9 Expressing Construct

    Directory of Open Access Journals (Sweden)

    Qiang Zou

    2010-01-01

    Full Text Available Various chemokines and cytokines as adjuvants can be used to improve efficacy of DNA vaccination. In this study, we sought to investigate if a DNA construct expressing IL-9 (designed as proV-IL9 as a molecular adjuvant enhance antigen specific immune responses elicited by the pcD-VP1 DNA vaccination. Mice immunized with pcD-VP1 combined with proV-IL9 developed a strong humoral response. In addition, the coinoculation induced significant higher level of antigen-specific cell proliferation and cytotoxic response. This agreed well with higher expression level of IFN-γ and perforin in CD8+ T cells, but not with IL-17 in these T cells. The results indicate that IL-9 induces the development of IFN-γ-producing CD8+ T cells (Tc1, but not the IL-17-producing CD8+ T cells (Tc17. Up-regulated expressions of BCL-2 and BCL-XL were exhibited in these Tc1 cells, suggesting that IL-9 may trigger antiapoptosis mechanism in these cells. Together, these results demonstrated that IL-9 used as molecular adjuvant could enhance the immunogenicity of DNA vaccination, in augmenting humoral and cellular responses and particularly promoting Tc1 activations. Thus, the IL-9 may be utilized as a potent Tc1 adjuvant for DNA vaccines.

  7. Primary WWOX phosphorylation and JNK activation during etoposide induces cytotoxicity in HEK293 cells

    Directory of Open Access Journals (Sweden)

    M Jamshidiha

    2010-06-01

    Full Text Available "n  "nBackground and the purpose of the study: Etoposide is an antineoplastic agent used in multiple cancers. It is known that etoposide induce cell death via interaction with topoisomerase II; however, the etopoisde cellular response is poorly understood. Upon etoposide induced DNA damage, many stress signaling pathways including JNK are activated. In response to DNA damage, it has been shown that WWOX, a recently introduced tumor suppressor, can be activated. In this study the activation of WWOX and JNK and their interaction following etoposide treatment were evaluated. "nMaterials and Methods:HEK293 cells treated with etoposide were lysed in a time course manner. The whole cell lysates were used to evaluate JNK and WWOX activation pattern using Phospho specific antibodies on western blots. The viability of cells treated with etoposide, JNK specific inhibitor and their combination was examined using MTT assay. "nResults:Findings of this study indicate that WWOX and JNK are activated in a simultaneous way in response to DNA damage. Moreover, JNK inhibition enhances etoposide induced cytotoxicity in HEK293. "nConclusion:Taken together, our results indicate that etoposide induces cytotoxicity and WWOX phosphorylation and the cytotoxicty is augmented by blocking JNK pathway.

  8. Online Measurement of Real-Time Cytotoxic Responses Induced by Multi-Component Matrices, such as Natural Products, through Electric Cell-Substrate Impedance Sensing (ECIS

    Directory of Open Access Journals (Sweden)

    Adyary Fallarero

    2015-11-01

    Full Text Available Natural products are complex matrices of compounds that are prone to interfere with the label-dependent methods that are typically used for cytotoxicity screenings. Here, we developed a label-free Electric Cell-substrate Impedance Sensing (ECIS-based cytotoxicity assay that can be applied in the assessment of the cytotoxicity of natural extracts. The conditions to measure the impedance using ECIS were first optimized in mice immortalized hypothalamic neurons GT1-7 cells. The performance of four natural extracts when tested using three conventional cytotoxicity assays in GT1-7 cells, was studied. Betula pendula (silver birch tree was found to interfere with all of the cytotoxicity assays in which labels were applied. The silver birch extract was also proven to be cytotoxic and, thus, served as a proof-of-concept for the use of ECIS. The extract was fractionated and the ECIS method permitted the distinction of specific kinetic patterns of cytotoxicity on the fractions as well as the extract’s pure constituents. This study offers evidence that ECIS is an excellent tool for real-time monitoring of the cytotoxicity of complex extracts that are difficult to work with using conventional (label-based assays. Altogether, it offers a very suitable cytotoxicity-screening assay making the work with natural products less challenging within the drug discovery workflow.

  9. Online Measurement of Real-Time Cytotoxic Responses Induced by Multi-Component Matrices, such as Natural Products, through Electric Cell-Substrate Impedance Sensing (ECIS).

    Science.gov (United States)

    Fallarero, Adyary; Batista-González, Ana E; Hiltunen, Anna K; Liimatainen, Jaana; Karonen, Maarit; Vuorela, Pia M

    2015-11-12

    Natural products are complex matrices of compounds that are prone to interfere with the label-dependent methods that are typically used for cytotoxicity screenings. Here, we developed a label-free Electric Cell-substrate Impedance Sensing (ECIS)-based cytotoxicity assay that can be applied in the assessment of the cytotoxicity of natural extracts. The conditions to measure the impedance using ECIS were first optimized in mice immortalized hypothalamic neurons GT1-7 cells. The performance of four natural extracts when tested using three conventional cytotoxicity assays in GT1-7 cells, was studied. Betula pendula (silver birch tree) was found to interfere with all of the cytotoxicity assays in which labels were applied. The silver birch extract was also proven to be cytotoxic and, thus, served as a proof-of-concept for the use of ECIS. The extract was fractionated and the ECIS method permitted the distinction of specific kinetic patterns of cytotoxicity on the fractions as well as the extract's pure constituents. This study offers evidence that ECIS is an excellent tool for real-time monitoring of the cytotoxicity of complex extracts that are difficult to work with using conventional (label-based) assays. Altogether, it offers a very suitable cytotoxicity-screening assay making the work with natural products less challenging within the drug discovery workflow.

  10. Silver nanoparticles-induced cytotoxicity requires ERK activation in human bladder carcinoma cells.

    Science.gov (United States)

    Castiglioni, Sara; Cazzaniga, Alessandra; Perrotta, Cristiana; Maier, Jeanette A M

    2015-09-17

    Silver nanoparticles are toxic both in vitro and in vivo. We have investigated the possibility to exploit the cytotoxic potential of silver nanoparticles in T24 bladder carcinoma cells using both bare and PolyVinylPyrrolidone-coated silver nanoparticles. We show that the two types of silver nanoparticles promote morphological changes and cytoskeletal disorganization, are cytotoxic and induce cell death. These effects are due to the increased production of reactive oxygen species which are responsible, at least in part, for the sustained activation of ERK1/2. Indeed, both cytotoxicity and ERK1/2 activation are prevented by exposing the cells to the anti-oxidant N-acetylcysteine. Also blocking the ERK1/2 pathway with the MEK inhibitor PD98059 protects the cells from nanoparticles' cytotoxicity. Our findings suggest that ERK activation plays a role in silver nanoparticle-mediated cytotoxicity in T24 cells. Copyright © 2015. Published by Elsevier Ireland Ltd.

  11. Continuously expanding CAR NK-92 cells display selective cytotoxicity against B-cell leukemia and lymphoma.

    Science.gov (United States)

    Oelsner, Sarah; Friede, Miriam E; Zhang, Congcong; Wagner, Juliane; Badura, Susanne; Bader, Peter; Ullrich, Evelyn; Ottmann, Oliver G; Klingemann, Hans; Tonn, Torsten; Wels, Winfried S

    2017-02-01

    Natural killer (NK) cells can rapidly respond to transformed and stressed cells and represent an important effector cell type for adoptive immunotherapy. In addition to donor-derived primary NK cells, continuously expanding cytotoxic cell lines such as NK-92 are being developed for clinical applications. To enhance their therapeutic utility for the treatment of B-cell malignancies, we engineered NK-92 cells by lentiviral gene transfer to express chimeric antigen receptors (CARs) that target CD19 and contain human CD3ζ (CAR 63.z), composite CD28-CD3ζ or CD137-CD3ζ signaling domains (CARs 63.28.z and 63.137.z). Exposure of CD19-positive targets to CAR NK-92 cells resulted in formation of conjugates between NK and cancer cells, NK-cell degranulation and selective cytotoxicity toward established B-cell leukemia and lymphoma cells. Likewise, the CAR NK cells displayed targeted cell killing of primary pre-B-ALL blasts that were resistant to parental NK-92. Although all three CAR NK-92 cell variants were functionally active, NK-92/63.137.z cells were less effective than NK-92/63.z and NK-92/63.28.z in cell killing and cytokine production, pointing to differential effects of the costimulatory CD28 and CD137 domains. In a Raji B-cell lymphoma model in NOD-SCID IL2R γ(null) mice, treatment with NK-92/63.z cells, but not parental NK-92 cells, inhibited disease progression, indicating that selective cytotoxicity was retained in vivo. Our data demonstrate that it is feasible to generate CAR-engineered NK-92 cells with potent and selective antitumor activity. These cells may become clinically useful as a continuously expandable off-the-shelf cell therapeutic agent. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  12. Cytotoxic Effect Of Verapamil On Human Embryonic Kidney Cell Line

    Directory of Open Access Journals (Sweden)

    Jamil L Ahmad

    2015-08-01

    Full Text Available Introduction The link between long term use of verapamil and cancer development has been suggested in literature many years back. However there are numerous controversies surrounding this association with several epidemiological studies in the positive negative and non-association between verapamil and cancer development. Aim To investigate in mechanistic terms the link between chronic use of a calcium channel blocker verapamil and cancer development using human embryonic kidney HEK293 cell line. Method Trypan blue dye exclusion cell counting and 3-amp615314 5-Dimethylthiazol-2-ylamp61533-2 5-diphenyl-tetrazolium bromide MTT assays were used to determine the proliferative as well as cytotoxic effects of verapamil. Results Verapamil had a growth inhibitory rather than proliferative effect on HEK293 cells and the growth inhibition was found to be significant p0.05. Conclusion The long term use of verapamil is associated with cellular growth inhibition and this possibly explained the rationale behind its use as part of combination chemotherapy for some human cancers.

  13. Surface-modified gold nanorods for specific cell targeting

    Science.gov (United States)

    Wang, Chan-Ung; Arai, Yoshie; Kim, Insun; Jang, Wonhee; Lee, Seonghyun; Hafner, Jason H.; Jeoung, Eunhee; Jung, Deokho; Kwon, Youngeun

    2012-05-01

    Gold nanoparticles (GNPs) have unique properties that make them highly attractive materials for developing functional reagents for various biomedical applications including photothermal therapy, targeted drug delivery, and molecular imaging. For in vivo applications, GNPs need to be prepared with very little or negligible cytotoxicitiy. Most GNPs are, however, prepared using growth-directing surfactants such as cetyl trimethylammonium bromide (CTAB), which are known to have considerable cytotoxicity. In this paper, we describe an approach to remove CTAB to a non-toxic concentration. We optimized the conditions for surface modification with methoxypolyethylene glycol thiol (mPEG), which replaced CTAB and formed a protective layer on the surface of gold nanorods (GNRs). The cytotoxicities of pristine and surface-modified GNRs were measured in primary human umbilical vein endothelial cells and human cell lines derived from hepatic carcinoma cells, embryonic kidney cells, and thyroid papillary carcinoma cells. Cytotoxicity assays revealed that treating cells with GNRs did not significantly affect cell viability except for thyroid papillary carcinoma cells. Thyroid cancer cells were more susceptible to residual CTAB, so CTAB had to be further removed by dialysis in order to use GNRs for thyroid cell targeting. PEGylated GNRs are further modified to present monoclonal antibodies that recognize a specific surface marker, Na-I symporter, for thyroid cells. Antibody-conjugated GNRs specifically targeted human thyroid cells in vitro.

  14. Bacillus cereus enterotoxins act as major virulence factors and exhibit distinct cytotoxicity to different human cell lines.

    Science.gov (United States)

    Jeßberger, Nadja; Dietrich, Richard; Bock, Stefanie; Didier, Andrea; Märtlbauer, Erwin

    2014-01-01

    A comparative analysis on the relevance of the Bacillus cereus enterotoxins Nhe (nonhemolytic enterotoxin), HBL (haemolysin BL) and CytK (cytotoxin K) was accomplished, concerning their toxic activity towards different target cell lines. Overall, among the components secreted by the reference strains for Nhe and HBL, the enterotoxin complexes accounted for over 90% of the total toxicity. Vero and primary endothelial cells (HUVEC) were highly susceptible to Nhe, whereas Hep-G2, Vero and A549 reacted most sensitive to Nhe plus HBL. For CytK the highest toxicity was observed on CaCo-2 cells. As HBL positive strains always produce Nhe in parallel, the specific contribution of both enterotoxin complexes to the overall observed cytotoxic effects was determined by consecutively removing their single components. While in most cell lines Nhe and HBL contributed more or less equally (40-60%) to cytotoxicity, the relative activity of Nhe was approximately 90% in HUVEC, and that of HBL 75% in A549 cells. With U937, a nearly Nhe resistant cell line was identified for the first time. This distinct susceptibility of cell lines was confirmed by investigating a set of 37 B. cereus strains. Interestingly, whereas Nhe is the enterotoxin mainly responsible for cell death as determined by WST-1 bioassays, more rapid pore formation was observed when HBL was present, pointing to a different mode of action of the two enterotoxin complexes. Furthermore, correlation was observed between cytotoxicity of solely Nhe producing strains and NheB. Cytotoxicity of Nhe/HBL producing isolates correlated with the expression of HBL L1, NheB and HBL B. In conclusion, the observed susceptibilities of target cell lines of different histological origin underline that B. cereus enterotoxins represent major virulence factors and that toxicity is not restricted to gastrointestinal infections. The varying contribution of Nhe and HBL to total cytotoxicity strongly indicates that Nhe as well as HBL specific B

  15. Cytotoxic drug sensitivity of Epstein-Barr virus transformed lymphoblastoid B-cells

    Directory of Open Access Journals (Sweden)

    Olah Eva

    2006-11-01

    Full Text Available Abstract Background Epstein-Barr virus (EBV is the causative agent of immunosuppression associated lymphoproliferations such as post-transplant lymphoproliferative disorder (PTLD, AIDS related immunoblastic lymphomas (ARL and immunoblastic lymphomas in X-linked lymphoproliferative syndrome (XLP. The reported overall mortality for PTLD often exceeds 50%. Reducing the immunosuppression in recipients of solid organ transplants (SOT or using highly active antiretroviral therapy in AIDS patients leads to complete remission in 23–50% of the PTLD/ARL cases but will not suffice for recipients of bone marrow grafts. An additional therapeutic alternative is the treatment with anti-CD20 antibodies (Rituximab or EBV-specific cytotoxic T-cells. Chemotherapy is used for the non-responding cases only as the second or third line of treatment. The most frequently used chemotherapy regimens originate from the non-Hodgkin lymphoma protocols and there are no cytotoxic drugs that have been specifically selected against EBV induced lymphoproliferative disorders. Methods As lymphoblastoid cell lines (LCLs are well established in vitro models for PTLD, we have assessed 17 LCLs for cytotoxic drug sensitivity. After three days of incubation, live and dead cells were differentially stained using fluorescent dyes. The precise numbers of live and dead cells were determined using a custom designed automated laser confocal fluorescent microscope. Results Independently of their origin, LCLs showed very similar drug sensitivity patterns against 29 frequently used cytostatic drugs. LCLs were highly sensitive for vincristine, methotrexate, epirubicin and paclitaxel. Conclusion Our data shows that the inclusion of epirubicin and paclitaxel into chemotherapy protocols against PTLD may be justified.

  16. Mogoltacin enhances vincristine cytotoxicity in human transitional cell carcinoma (TCC) cell line.

    Science.gov (United States)

    Behnam Rassouli, F; Matin, M M; Iranshahi, M; Bahrami, A R; Neshati, V; Mollazadeh, S; Neshati, Z

    2009-03-01

    Bladder cancer is the second common cancer of the genitourinary system throughout the world and intravesical chemotherapy is usually used to reduce tumour recurrence and progression. Human transitional cell carcinoma (TCC) is an epithelial-like adherent cell line originally established from primary bladder carcinoma. Here we report the effect of mogoltacin, a sesquiterpene coumarin from Ferula badrakema on TCC cells. Mogoltacin was isolated from the fruits of F. badrakema, using silica gel column chromatography and preparative thin layer chromatography. Mogoltacin did not have any significant cytotoxicity effect on neoplastic TCC cells at 16, 32, 64, 128, 200 and 600 microg ml(-1) concentrations. In order to analyse its combination effect, TCC cells were cultured in the presence of various combining concentrations of mogoltacin and vincristine. Cells were then observed for morphological changes (by light microscopy) and cytotoxicity using MTT assay. The effect of mogoltacin on vincristine toxicity was studied after 24, 48 and 72 h of drug administration. The results of MTT assay showed that mogoltacin can significantly enhance the cytotoxicity of vincristine and confirmed the morphological observations. Results revealed that combination of 40 microg ml(-1) vincristine with 16 microg ml(-1) mogoltacin increased the cytotoxicity of vincristine after 48 h by 32.8%.

  17. Screening a hybridoma producing a specific monoclonal antibody to HLA-A24+Bw4 antigen by cytotoxicity inhibition assay.

    Science.gov (United States)

    Hiroishi, S; Kaneko, T; Arita, J

    1987-02-01

    A hybridoma secreting a monoclonal antibody (Tsa-1, IgG3) reacting specifically to HLA-A24+Bw4 was screened by cytotoxicity inhibition assay and micrototoxicity test. The R value of the antibody was 0.843.

  18. Specific cytotoxic T lymphocyte responses in Chinese HIV/AIDS patients

    Institute of Scientific and Technical Information of China (English)

    尚红; 韩晓旭; 王亚男; 周立平; 张子宁; 姜拥军; 张旻; 于旭

    2004-01-01

    Human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T lymphocytes (CTL) are considered to play a central role in the immune response against HIV-1. During untreated acute HIV-1 infection, virus-specific CTL activity is associated with the initial decrease in viremia.1 After HIV infection, those individuals with a slow progressive course develop stronger CTL responses than those with typical disease progression.2,3 Restoring HIV-1-specific CTL responses have been considered a key factor in immune reconstitution and vaccine development. Here we present the analysis of HIV-1 Gag-specific CTL responses in 23 Chinese HIV/AIDS cases in order to take the initial steps at identifying the epitopes that dominate the CTL response in Chinese patients.

  19. Cytotoxic compounds against cancer cells from Bombyx mori inoculated with Cordyceps militaris.

    Science.gov (United States)

    Qiu, Weitao; Wu, Jing; Choi, Jae-Hoon; Hirai, Hirofumi; Nishida, Hiroshi; Kawagishi, Hirokazu

    2017-06-01

    Two compounds, 3'-deoxyinosine and cordycepin, were isolated from Bombyx mori inoculated with Cordyceps militaris. In the bioassay examining cytotoxicity against cancer cells, both compounds showed toxicity against A549, PANC-1, and MCF-7 cancer cells.

  20. [Effect of astragalus polysaccharide on sensitivity of leukemic cell line HL-60 to NK cell cytotoxicity and its mechanism].

    Science.gov (United States)

    Zeng, Peng-Yun; Deng, Li-Li; Yue, Ling-Ling; Zhang, Lian-Sheng

    2012-08-01

    The objective of this study was to explore the effect of astragalus polysaccharide (APS) on sensitivity of leukemic cell line HL-60 to NK cell cytotoxicity and its mechanism. The cytotoxicities of NK cells against HL-60 cells were analyzed by LDH releasing assay at different effect-to-target cell ratios (E:T) before and after treated with APS. The gene expression of MHC class I chain-related (MICA) in HL-60 cells before and after APS treatment was assayed with RT-PCR. Protein expression of MICA in HL-60 cells was assayed by flow cytometry before and after treated by APS. The results showed that after treated with APS 15 mg/ml for 48 h, the cytotoxicities of NK cells against HL-60 cells enhanced at different effect-to-target (P HL-60 cells were up-regulated (P HL-60 cells, thus enhance sensitivity of HL-60 cells to cytotoxicity of NK cells.

  1. Enhanced cytotoxic T-cell function and inhibition of tumor progression by Mst1 deficiency.

    Science.gov (United States)

    Yasuda, Kaneki; Ueda, Yoshihiro; Ozawa, Madoka; Matsuda, Tadashi; Kinashi, Tatsuo

    2016-01-01

    Mammalian ste-20 like kinase Mst1 plays important roles during apoptosis, proliferation, cell polarity, and migration. Here, we report a novel role of Mst1 for cytotoxic T-cell responses and tumor suppression. The defect of Mst1 caused decreased levels of FoxO, and promoted cytotoxicity in vitro. Mst1(-/-) cytotoxic T cells also exhibited enhanced T-bet expression that was associated with elevated expression levels of IFNγ and granzyme B. Moreover, Mst1(-/-) cytotoxic T cells suppressed tumor growth in vivo. The data suggest that Mst1 inhibits cytotoxicity via T-bet suppression by FoxO1 and FoxO3a. Thus, Mst1 is a potential therapeutic target for tumor immunotherapy.

  2. Cytotoxicity against KB and NCI-H187 cell lines of modified flavonoids from Kaempferia parviflora.

    Science.gov (United States)

    Yenjai, Chavi; Wanich, Suchana

    2010-05-01

    Flavones 1-4 isolated from Kaempferia parviflora were used for structural modification. Sixteen flavonoid derivatives, including four new derivatives, were synthesized and evaluated for cytotoxicity against KB and NCI-H187 cell lines. Flavanones 2a-4a demonstrated higher cytotoxic activity than the parent compounds. Cytotoxicity against KB cell line of oxime 1c was about 7 times higher than the ellipticine standard. Interestingly, oximes 1c and 2c exhibited highly potent cytotoxicity against NCI-H187 cell line with IC(50) values of 0.014 and 0.23 microM, respectively. Oximes 4c and 5c showed strong cytotoxicity against NCI-H187 cell line with IC(50) values of 4.04 and 2.32 microM, respectively. 2010 Elsevier Ltd. All rights reserved.

  3. NK Cell Maturation and Cytotoxicity are Controlled by the Intramembrane Aspartyl Protease SPPL31

    Science.gov (United States)

    Hamblet, Corinne E.; Makowski, Stefanie L.; Tritapoe, Julia M.; Pomerantz, Joel L.

    2016-01-01

    NK cell maturation is critical for normal effector function and the innate immune response to tumors and pathogens. However, the molecular pathways that control NK cell maturation remain largely undefined. Here, we investigate the role of SPPL3, an intramembrane aspartyl protease, in murine NK cell biology. We find that deletion of SPPL3 in the hematopoietic system reduces numbers of peripheral NK cells, clearance of MHC Class I-deficient tumors in vivo, and cytotoxicity against tumor cells in vitro. This phenotype is concomitant with reduced numbers of CD27+CD11b+ and CD27−CD11b+ NK cells, indicating a requirement for SPPL3 in efficient NK cell maturation. NK cell-specific deletion of SPPL3 results in the same deficiencies, revealing a cell-autonomous role for SPPL3 in these processes. CRISPR/Cas9 genomic editing in murine zygotes was used to generate knock-in mice with a catalytically compromised SPPL3 D271A allele. Mice engineered to express only SPPL3 D271A in NK cells phenocopy mice deleted for SPPL3, indicating a requirement for SPPL3 protease activity in NK cell biology. Our results identify SPPL3 as a cell-autonomous molecular determinant of NK cell maturation and expand the role of intramembrane aspartyl proteases in innate immunity. PMID:26851218

  4. NK Cell Maturation and Cytotoxicity Are Controlled by the Intramembrane Aspartyl Protease SPPL3.

    Science.gov (United States)

    Hamblet, Corinne E; Makowski, Stefanie L; Tritapoe, Julia M; Pomerantz, Joel L

    2016-03-15

    NK cell maturation is critical for normal effector function and the innate immune response to tumors and pathogens. However, the molecular pathways that control NK cell maturation remain largely undefined. In this article, we investigate the role of SPPL3, an intramembrane aspartyl protease, in murine NK cell biology. We find that deletion of SPPL3 in the hematopoietic system reduces numbers of peripheral NK cells, clearance of MHC class I-deficient tumors in vivo, and cytotoxicity against tumor cells in vitro. This phenotype is concomitant with reduced numbers of CD27(+)CD11b(+) and CD27(-)CD11b(+) NK cells, indicating a requirement for SPPL3 in efficient NK cell maturation. NK cell-specific deletion of SPPL3 results in the same deficiencies, revealing a cell-autonomous role for SPPL3 in these processes. CRISPR/Cas9 genomic editing in murine zygotes was used to generate knockin mice with a catalytically compromised SPPL3 D271A allele. Mice engineered to express only SPPL3 D271A in NK cells phenocopy mice deleted for SPPL3, indicating a requirement for SPPL3 protease activity in NK cell biology. Our results identify SPPL3 as a cell-autonomous molecular determinant of NK cell maturation and expand the role of intramembrane aspartyl proteases in innate immunity. Copyright © 2016 by The American Association of Immunologists, Inc.

  5. Endothelial cell effects of cytotoxics: balance between desired and unwanted effects.

    Science.gov (United States)

    de Vos, F Y F L; Willemse, P H B; de Vries, E G E; Gietema, J A

    2004-10-01

    Since Folkman defined angiogenesis more than 25 years ago as the most important process in tumour growth and metastasis, specific anti-angiogenic agents have been developed. One obvious route to block this process was until recently overlooked, however. Tumour endothelial cells are different from normal endothelial cells and may respond differently to conventional cytotoxics. Chemotherapeutic-induced vascular toxicity has been observed in various clinical studies and seems to be based on endothelial cell damage as seen in vitro in human umbilical vein endothelial cells (HUVEC) models with protracted low-dose cytostatic exposure. Translated into the clinical setting, such "metronomically" administered chemotherapy could lead to anti-angiogenesis enhancing anti-tumour efficacy of cytostatic drugs. This paper reviews the desired anti-tumour endothelial activity versus the unwanted general vascular toxicity of cytostatic drugs. Several ways to enhance the anti-tumour activity and to circumvent the unwanted vascular toxicity of these "accidental" anti-angiogenic drugs will be discussed.

  6. A Model of Cytotoxic T Antitumor Activation Stimulated by Pulsed Dendritic Cells

    Science.gov (United States)

    Pennisi, Marzio; Pappalardo, Francesco; Chiacchio, Ferdinando; Motta, Santo

    2011-09-01

    We present a preliminary ODE model to sketch the immune response of cytotoxic T cells against cancer through the use of pulsed autologous dendritic cells. The model is partially based on data coming from experiments that are presently in progress in the wet lab of our collaborators, but it can be applied in principle to different tumors. To this end, we show the immune response of cytotoxic T cells stimulated by autologous dendritic cells for different cancers.

  7. Garcinielliptone FC: antiparasitic activity without cytotoxicity to mammalian cells.

    Science.gov (United States)

    Silva, Ana P; Silva, Marcos P; Oliveira, Cristiano G; Monteiro, Daniela C; Pinto, Pedro L; Mendonça, Ronaldo Z; Costa Júnior, Joaquim S; Freitas, Rivelilson M; de Moraes, Josué

    2015-06-01

    Garcinielliptone FC (GFC) is a natural prenylated benzophenone found in the seeds of Platonia insignis Mart. (Clusiaceae), a native Brazilian plant. It has been chemically characterized and it is known that GFC has several biological activities such as antioxidant and vasorelaxant properties. In this study, we report the in vitro effect of GFC against the blood fluke Schistosoma mansoni, the parasite responsible for schistosomiasis mansoni. The anti-S. mansoni activity and cytotoxicity toward mammalian cells were determined for the compound. GFC⩾6.25 μM showed antischistosomal activity and confocal laser scanning microscopy analysis demonstrated several morphological alterations on the tegument of worms, and a correlation between viability and tegumental damage was observed. In addition, at sub-lethal concentrations of GFC (⩽3.125 μM), the number of S. mansoni eggs was reduced. More importantly, GFC exhibited no activity toward mammalian cells and, therefore, there is an appreciable selectivity of this compound against the helminths. In conclusion, these findings indicate the potential of GFC as an antiparasitic agent. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Potentiated cytotoxic effects of statins and ajoene in murine melanoma cells.

    Science.gov (United States)

    Ledezma, Eliades; Wittig, Olga; Alonso, Jose; Cardier, Jose E

    2009-04-01

    Because statins and ajoene inhibit the 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, we evaluated the hypothesis that the cytotoxic effect of these compounds may be potentiated when both are used in combination on tumor cells. We showed that cotreatment of the murine melanoma B16F10 cell with statins (atorvastatin and pravastatin) and ajoene, all at nontoxic doses, dramatically increased their cytotoxicity. B16F10 cell death induced by statins, but not by ajoene, was prevented by mevalonate and geranylgeranylpyrophosphate. To our knowledge, this is the first report that the combination of statins and ajoene, which alters the mevalonate pathway, might potentiate their cytotoxic effects on tumor cells.

  9. Cytotoxicity and mutagenicity of cola and grape flavored soft drinks in bone marrow cells of rodents

    Directory of Open Access Journals (Sweden)

    Elisângela Düsman

    2013-03-01

    Full Text Available Due to the large consumption of soft drinks in Brazil and worldwide in recent years and considering that some of the components present in their composition pose potential risks to human health, the aim of this study was to evaluate the cytotoxic and mutagenic potential of specific cola and grape-flavored soft drink brands. Bone marrow cells of Wistar rats were initially treated by gavage with one single dose of Cola or Grape soft drink, which was next offered ad libitum (instead of water for 24 hours. A negative control treatment was performed by administering one single dose of water and a positive control administering cyclophosphamide intraperitoneally. Statistical analysis showed that the Cola and Grape soft drinks studied were not cytotoxic. However, the Cola soft drink proved mutagenic in this experiment treatment time. Therefore, this study serves as a warning about the consumption of Cola-flavored soft drink and for the need for further subchronic and chronic studies on soft drinks in order to evaluate the long term mutagenic and cytotoxic effects of these substances.

  10. The effect of different anesthetics on tumor cytotoxicity by natural killer cells.

    Science.gov (United States)

    Tazawa, Kazumasa; Koutsogiannaki, Sophia; Chamberlain, Matthew; Yuki, Koichi

    2017-01-15

    A number of retrospective studies have suggested that choice of anesthetic drugs during surgical tumor resection might affect tumor recurrence/metastasis, or outcome of patients. The recent study showed that volatile anesthetics-based general anesthesia was associated with the worse outcomes than intravenous anesthetics-based general anesthesia. However, the underlying mechanism is yet to be determined. Because natural killer (NK) cells are implicated as important immune cells for tumor recurrence/metastasis in the perioperative period, we examined the effect of different anesthetics on NK cell-mediated tumor cytotoxicity. Because adhesion molecule leukocyte function-associated antigen-1 (LFA-1) is functionally important in NK cells and is inhibited by commonly used volatile anesthetics isoflurane and sevoflurane, we hypothesized that these anesthetics would attenuate NK cell-mediated cytotoxicity. Using human NK cell line NK92-MI cells and tumor cell line K562 cells as a model system, we performed cytotoxicity, proliferation, conjugation and degranulation assays. Lytic granule polarization was also assessed. We showed that isoflurane, sevoflurane and LFA-1 inhibitor BIRT377 attenuated cytotoxicity, and reduced conjugation and polarization, but not degranulation of NK cells. Our data suggest that isoflurane and sevoflurane attenuated NK cell-mediated cytotoxicity at least partly by their LFA-1 inhibition in vitro. Whether or not isoflurane and sevoflurane attenuate NK cell-mediated tumor cytotoxicity in patients needs to be determined in the future. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  11. Mortalin inhibitors sensitize K562 leukemia cells to complement-dependent cytotoxicity.

    Science.gov (United States)

    Pilzer, David; Saar, Moran; Koya, Keizo; Fishelson, Zvi

    2010-03-15

    Mortalin, the mitochondrial hsp70, is a vital constitutively expressed heat shock protein. Its elevated expression has been correlated with malignant transformation and poor cancer prognosis. Cancer cells exhibit increased resistance to complement-dependent cytotoxicity, partly due to their capacity to eliminate the complement membrane attack complex (MAC) from their cell surface. As we have previously reported, mortalin and the complement membrane attack complexes are released in membrane vesicles from complement attacked cells. As shown here, knock down of mortalin with specific siRNA reduces MAC elimination and enhances cell sensitivity to MAC-induced cell death. Similar results were obtained with MKT-077, a cationic rhodacyanine dye that inhibits mortalin. Treatment of human erythroleukemia K562 and colorectal carcinoma HCT116 cells with MKT-077 sensitizes them to cell death mediated by MAC but not by streptolysin O. Pre-treatment of cells with MKT-077 also reduces the extent of MAC-mortalin vesiculation following a sublytic complement attack. In the presence of MKT-077, the direct binding of mortalin to complement C9, the major MAC component, is inhibited. The tumor suppressor protein p53 is a known mortalin client protein. The effect of MKT-077 on complement-mediated lysis of HCT116 p53(+/+) and p53(-/-) cells was found to be independent on the presence of p53. Our results also demonstrate that recombinant human mortain inhibits complement-mediated hemolysis of rabbit erythrocytes as well as zinc-induced C9 polymerization. We conclude that mortalin supports cancer cell resistance to complement-dependent cytotoxicity and propose consideration of mortalin as a novel target for cancer adjuvant immunotherapy.

  12. Genetic vaccination against the melanocyte lineage-specific antigen gp100 induces cytotoxic T lymphocyte-mediated tumor protection.

    Science.gov (United States)

    Schreurs, M W; de Boer, A J; Figdor, C G; Adema, G J

    1998-06-15

    Melanocyte lineage-specific antigens, such as gp100, have been shown to induce both cellular and humoral immune responses against melanoma. Therefore, these antigens are potential targets for specific antimelanoma immunotherapy. A novel approach to induce both cellular and humoral immunity is genetic vaccination, the injection of antigen-encoding naked plasmid DNA. In a mouse model, we investigated whether genetic vaccination against the human gp100 antigen results in specific antitumor immunity. The results demonstrate that vaccinated mice were protected against a lethal challenge with syngeneic B16 melanoma-expressing human gp100, but not control-transfected B16. Both cytotoxic T cells and IgG specific for human gp100 could be detected in human gp100-vaccinated mice. However, only adoptive transfer of spleen-derived lymphocytes, not of the serum, isolated from protected mice was able to transfer antitumor immunity to nonvaccinated recipients, indicating that CTLs are the predominant effector cells. CTI, lines generated from human gp100-vaccinated mice specifically recognized human gp100. Interestingly, one of the CTL lines cross-reacted between human and mouse gp100, indicating the recognition of a conserved epitope. However, these CTLs did not appear to be involved in the observed tumor protection. Collectively, our results indicate that genetic vaccination can result in a potent antitumor response in vivo and constitutes a potential immunotherapeutic strategy to fight cancer.

  13. Compound A398, a novel podophyllotoxin analogue: cytotoxicity and induction of apoptosis in human leukemia cells.

    Directory of Open Access Journals (Sweden)

    Alethéia L Silveira

    Full Text Available Despite advances in oncology research, cancer is one of the leading causes of death worldwide. Thus, there is a demand for the development of more selective and effective antitumor agents. This study showed that A398, a novel podophyllotoxin analogue, was cytotoxic to the HT-29, MCF-7, MOLT-4 and HL-60 tumor cell lines, being less active in human peripheral blood mononuclear cells and normal cell lines FGH and IEC-6. Tests using the HepG2 lineage indicated that its metabolites do not contribute to its cytotoxicity. In the HL-60 cells, A398 induced apoptosis in a time and concentration-dependent manner, promoting mitochondrial depolarization, inhibition of Bcl-2, phosphatidylserine exposure, activation of caspases -8, -9 and -3, and DNA fragmentation. The production of reactive oxygen species does not seem to be a crucial event for the apoptotic process. Pretreatment with specific inhibitors of kinases ERK1/2, JNK and p38 resulted in an increased percentage of death induced by A398. These results indicate that the compound induced apoptosis through activation of intrinsic and extrinsic death pathways with the mechanism involving the inhibition of the MAPKs and Bcl-2. Taken together, our findings suggest that A398 has an anticancer potential, proving itself to be a candidate for preclinical studies.

  14. A three-dimensional organotypic assay to measure target cell killing by cytotoxic T lymphocytes.

    NARCIS (Netherlands)

    Weigelin, B.; Friedl, P.H.A.

    2010-01-01

    Cytotoxic T lymphocytes (CTL) mediate antigen- and cell-cell contact dependent killing of target cells, such as cancer cells and virus-infected cells. In vivo, this process requires the active migration of CTL towards and away from target cells. We here describe an organotypic 3D collagen matrix

  15. Hsp70 confines tumor progression of rat histiocytoma and impedes the cytotoxicity induced by natural killer cells and peritoneal macrophages

    Institute of Scientific and Technical Information of China (English)

    Amere Subbarao Sreedhar

    2010-01-01

    Objective:To study the role of inducible form of heat shock protein 70 (Hsp70) in the host tumor regression of rat tumor model.Methods: We examined the role of Hsp70 in host tumorigenicity andin vitro cellular cytotoxicity using a rat histocytoma. The differential tumor growth and regression kinetics were studied and correlated with the expression of Hsp70, activation of macrophages and natural killer (NK) cells, and circulating or tumor infiltrating immune molecules in the host system.Results: The sub cuteaneous (s.c.) tumor regression was correlated with increased serum cytokines such as IL-12, TNFα,IFNγ and Hsp70. Despite of similar increase of Hsp70 in intraperitoneal (i.p.) tumor implanted animals, animals succumb to tumor growth, further, evidently, no immune molecule activation was observed. The viral promoter driven Hsp70 over expression in these tumor cells restrained solid tumor growth, however, failed to inhibit ascites growth. The NK cells from s.c. immunized animals induces cytotoxicity in the presence of anti-tumor antibody, which necessitated CD40-L expression, conversely, NK cells from i.p. immunized animals failed to induce cytotoxicity. The NK cells from s.c. or i.p. implanted animals with Hsp70 positive tumor cells failed to induce such cytotoxicity. The peritoneal macrophages isolated from s.c. tumor implanted animals when co-cultured with parental BC-8 cells lyses tumor cells, nevertheless entail macrophage specific TNFα expression. On the contrary, Hsp70 expressing BC-8 tumor cells were resistant to peritoneal macrophage induced cytolysis.Conclusions:This study brings out that Hsp70 possibly involved in regulating the host tumor response and cellular cytotoxicity.

  16. METHYLCELLULOSE CELL-CULTURE AS A NEW CYTOTOXICITY TEST SYSTEM FOR BIOMATERIALS

    NARCIS (Netherlands)

    VANLUYN, MJA; VANWACHEM, PB; NIEUWENHUIS, P; DAMINK, LO; TENHOOPEN, H; FEIJEN, J

    1991-01-01

    The cytotoxicity of biomaterials can be tested in vitro using various culture systems. Liquid culture systems may detect cytotoxicity of a material either by culture of cells with extracts or with the material itself. In the latter instance, renewing the medium will remove possible released cytotoxi

  17. Cytotoxic Activity and G1 Cell Cycle Arrest of a Dienynone from Echinacea pallida

    DEFF Research Database (Denmark)

    Chicca, Andrea; Adinolfi, Barbara; Pellati, Federica;

    2009-01-01

    In the present study, a further investigation of the cytotoxic activity of an acetylenic constituent of ECHINACEA PALLIDA roots, namely, pentadeca-(8 Z,13 Z)-dien-11-yn-2-one, was performed, revealing a concentration-dependent cytotoxicity on several human cancer cell lines, including leukemia...

  18. NK cell mediated antibody-dependent cellular cytotoxicity in cancer immunotherapy

    Directory of Open Access Journals (Sweden)

    Wei eWang

    2015-07-01

    Full Text Available Natural killer (NK cells play a major role in cancer immunotherapies that involve tumor-antigen targeting by monoclonal antibodies (mAbs. NK cells express a variety of activating and inhibitory receptors that serve to regulate the function and activity of the cells. In the context of targeting cells, NK cells can be specifically activated through certain Fc receptors that are expressed on their cell surface. NK cells can express FcγRIIIA and/or FcγRIIC, which can bind to the Fc portion of immunoglobulins, transmitting activating signals within NK cells. Once activated through Fc receptors by antibodies bound to target cells, NK cells are able to lyse target cells without priming, and secrete cytokines like interferon gamma to recruit adaptive immune cells. This antibody-dependent cell-mediated cytotoxicity (ADCC of tumor cells is utilized in the treatment of various cancers overexpressing unique antigens, such as neuroblastoma, breast cancer, B cell lymphoma, and others. NK cells also express a family of receptors called Killer Immunoglobulin-like Receptors (KIRs, which regulate the function and response of NK cells towards target cells through their interaction with their cognate ligands that are expressed on tumor cells. Genetic polymorphisms in KIR and KIR ligands, as well as FcγRs may influence NK cell responsiveness in conjunction with mAb immunotherapies. This review focuses on current therapeutic mAbs, different strategies to augment the anti-tumor efficacy of ADCC, and genotypic factors that may influence patient responses to antibody-dependent immunotherapies.

  19. Comparison of cytotoxicity, genotoxicity and immunological inflammatory biomarker activity of several endodontic sealers against immortalized human pulp cells.

    Science.gov (United States)

    Martinho, F C; Camargo, S E A; Fernandes, A M M; Campos, M S; Prado, R F; Camargo, C H R; Valera, M C

    2017-04-25

    To establish an SV40 T-Ag-transfected cell line of human pulp-derived cells in order to compare the cytotoxicity, genotoxicity and to investigate the activities of immunological biomarkers of several endodontic sealers. Primary human pulp cells and transfected cells were cultured. Cell morphology and proliferation were analysed, and the expression of cell-specific gene transcripts and proteins was detected by RT-PCR and immunohistochemistry. Transfection of human pulp-derived cells resulted in an immortalized cell line retaining phenotypic characteristics from the primarily cells tested. The SV40 T-Ag-transfected cells were cultured and stimulated by sealers (Apexit Plus, Real Seal, AH Plus, and EndoREZ) to evaluate the cytotoxicity and genotoxicity by MTT and MTN assays, respectively. Immunological inflammatory biomarkers (IL6, IL8 and TNF-α) were determined by ELISA assay. The differences between median values were statistically analysed using Kruskal-Wallis and Dunn's tests at 5% significance level. The cytotoxicity assay revealed that multimethacrylate (Real Seal) was the most cytotoxic sealer (P root canal sealers tested were able to stimulate the immortalized pulp cells to produce IL-6, IL-8 and TNF-α, with differences in relation to the control group (P sealers tested (P epoxy resin-based sealer (AHPlus), single-methacrylate sealer (EndoREZ) and calcium hydroxide-based sealer (Apexit Plus), regardless of the cytokine investigated (all P > 0.05). A SV40 T-Ag-transfected cell line of human pulp-derived cells was established. The methacrylate resin-based sealer (Real Seal) exhibited the greatest cytoxicity and inflammatory potential against immortalized pulp cells compared to an epoxy resin-based sealer (AH Plus), a methacrylate-based sealer (EndoRez) and a calcium hydroxide-based sealer (Apexit). © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  20. The cytotoxic and proapoptotic activities of hypnophilin are associated with calcium signaling in UACC-62 cells.

    Science.gov (United States)

    Pinto, Mauro C X; Cota, Betania B; Rodrigues, Michele A; Leite, Maria F; de Souza-Fagundes, Elaine M

    2013-11-01

    Hypnophilin (HNP) is a sesquiterpene that is isolated from Lentinus cf. strigosus and has cytotoxic activities. Here, we studied the calcium signaling and cytotoxic effects of HNP in UACC-62 cells, a human skin melanoma cell line. HNP was able to increase the intracellular calcium concentration in UACC-62 cells, which was blocked in cells stimulated in Ca(2+) -free media. HNP treatment with BAPTA-AM, an intracellular Ca(2+) chelator, caused an increase in calcium signals. HNP showed cytotoxicity against UACC-62 cells in which it induced DNA fragmentation and morphological alterations, including changes in the nuclear chromatin profile and increased cytoplasmatic vacuolization, but it had no effect on the plasma membrane integrity. These data suggest that cytotoxicity in UACC-62 cells, after treatment with HNP, is associated with Ca(2+) influx. Together, these findings suggest that HNP is a relevant tool for the further investigation of new anticancer approaches.

  1. Cytotoxic activity of interferon alpha induced dendritic cells as a biomarker of glioblastoma

    Science.gov (United States)

    Mishinov, S. V.; Stupak, V. V.; Tyrinova, T. V.; Leplina, O. Yu.; Ostanin, A. A.; Chernykh, E. R.

    2016-08-01

    Dendritic cells (DCs) are the most potent antigen presenting cells that can play direct role in anti-tumor immune response as killer cells. DC tumoricidal activity can be stimulated greatly by type I IFN (IFNα and IFNβ). In the present study, we examined cytostatic and cytotoxic activity of monocyte-derived IFNα-induced DCs generated from patients with brain glioma and evaluated the potential use of these parameters in diagnostics of high-grade gliomas. Herein, we demonstrated that patient DCs do not possess the ability to inhibit the growth of tumor HEp-2 cell line but low-grade and high-grade glioma patients do not differ significantly in DC cytostatic activity. However, glioma patient DCs are characterized by reduced cytotoxic activity against HEp-2 cells. The impairment of DC cytotoxic function is observed mainly in glioblastoma patients. The cytotoxic activity of DCs against HEp-2 cells below 9% is an informative marker for glioblastomas.

  2. Effector cell mediated cytotoxicity measured by intracellular Granzyme B release in HIV infected subjects

    Directory of Open Access Journals (Sweden)

    Mahajan Supriya D.

    2003-01-01

    Full Text Available CD8+ cytotoxic T lymphocyte (CTL activity is currently believed to be one of the key immunologic mechanisms responsible for the prevention or attenuation of HIV-1 infection. The induction of CD8+ T cell activation may also result in the production of soluble or non-classical lytic factors that are associated with protection from infection or slower disease progression. Traditionally, CD8+ CTL responses have been measured by the classic chromium release assay, monitoring the ability of T cells (Effector cells to lyse radiolabelled HLA – matched “target cells” that express the appropriate antigen-MHC complex. This method is not only labor intensive, semi quantitative assay at best, but also needs fresh, non-cryopreserved cells. Recently, cytokine specific ELISPOT assays or tetrameric MHC-I/ peptide complexes have utilized to directly quantitate circulating CD8+ effector cells, and these assays are more sensitive, quantitative and reproducible than the traditional CTL lysis assay and can also be performed on cryopreserved cells. Although these are reproducible assays for the assessment of soluble antiviral activity secreted by activated T cell populations they can be extremely expensive to perform. We have used FACS Analysis to measure Granzyme B release as a function of cell mediated cytotoxicity. This method helps quantitate the CTL activity and also identifies the phenotype of the cells elucidating this immune response. The method described not only monitors immunological response but also is also simple to perform, precise and extremely time efficient and is ideal for screening a large number of samples.

  3. Isolation and characterization of cytotoxic effector cells and antibody producing cells from human intestine.

    Science.gov (United States)

    MacDermott, R P

    1985-01-01

    We have examined the ability of intestinal and peripheral blood mononuclear cells isolated from patients with inflammatory bowel disease to mediate killing against cell line targets in spontaneous, antibody-dependent, lectin-induced, and interferon-induced cell-mediated cytotoxicity assays, as well as responsiveness in the allogeneic mixed leukocyte reaction, and effector capabilities in cell-mediated lympholysis. IMC were poor mediators of spontaneous or antibody-dependent cellular cytotoxicity with cell line cells as targets (in comparison to normal PBMC, but were capable of killing antibody coated chicken red blood cells. Although IMC were capable of responding to allogeneic cell surface antigens in the mixed leukocyte reaction, they did not exhibit effector function in cell-mediated lympholysis. Mitogenic lectins induced cell-mediated cytotoxicity by isolated intestinal mononuclear cells from controls and patients. HFIF induces cytotoxicity by control but not inflammatory bowel disease intestinal cells. Pokeweed mitogen was the lectin which induced the greatest amount of killing against human cell line targets. We therefore speculate that exogenous agents, or endogenous factors released during viral infection, could play a role in inducing cell mediated cytotoxic damage to the intestine in inflammatory bowel disease patients. In addition, the functional differences between IMC and PBMC indicate that intestinal MNC may have unique cell capabilities which must be better understood prior to the delineation of immunopathologic events in solid organ tissues. We have also examined the secretion of IgA, IgM, and IgG by isolated human IMC, human bone marrow MNC from rib specimens, and PBMC from patients with CD, UC, SLE, or Henoch-Schoenlein purpura (HSP). Control IMC exhibited high spontaneous secretion of IgA, while intestinal MNC from UC and CD patients exhibited only modest increases in IgA secretion. PBMC from patients with CD, UC, SLE, or HSP exhibited markedly

  4. Cytotoxic T lymphocytes and natural killer cells display impaired cytotoxic functions and reduced activation in patients with alcoholic hepatitis.

    Science.gov (United States)

    Støy, Sidsel; Dige, Anders; Sandahl, Thomas Damgaard; Laursen, Tea Lund; Buus, Christian; Hokland, Marianne; Vilstrup, Hendrik

    2015-02-15

    The dynamics and role of cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, and NKT cells in the life-threatening inflammatory disease alcoholic hepatitis is largely unknown. These cells directly kill infected and damaged cells through, e.g., degranulation and interferon-γ (IFNγ) production, but cause tissue damage if overactivated. They also assist tissue repair via IL-22 production. We, therefore, aimed to investigate the frequency, functionality, and activation state of such cells in alcoholic hepatitis. We analyzed blood samples from 24 severe alcoholic hepatitis patients followed for 30 days after diagnosis. Ten healthy abstinent volunteers and 10 stable abstinent alcoholic cirrhosis patients were controls. Using flow cytometry we assessed cell frequencies, NK cell degranulation capacity following K562 cell stimulation, activation by natural killer group 2 D (NKG2D) expression, and IL-22 and IFNγ production. In alcoholic hepatitis we found a decreased frequency of CTLs compared with healthy controls (P cells (P = 0.089). The NK cell degranulation capacity was reduced by 25% compared with healthy controls (P = 0.02) and by 50% compared with cirrhosis patients (P = 0.04). Accordingly, the NKG2D receptor expression was markedly decreased on NK cells, CTLs, and NKT cells (P cells were doubled compared with healthy controls (P < 0.05, all) but not different from cirrhosis patients. This exploratory study for the first time showed impaired cellular cytotoxicity and activation in alcoholic hepatitis. This is unlikely to cause hepatocyte death but may contribute toward the severe immune incompetence. The results warrant detailed and mechanistic studies. Copyright © 2015 the American Physiological Society.

  5. Evaluation of the cytotoxicity of geosmin and 2-methylisoborneol using cultured human, monkey, and dog cells.

    Science.gov (United States)

    Mochida, Kyo

    2009-03-01

    The cytotoxicity of musty odor-emitting substances, geosmin (GM) and 2-methylisoborneol, at a concentration of 10 ng/L - 300 mg/L was investigated using cultured mammalian cells. These two compounds exhibited no cytotoxicity in either the colony-formation of human KB cells or WST-1 assays of human-, monkey-, and dog-derived cells. These results suggest that the maximum concentration (700 ng/L) of GM found in the water of Lake Shinji is not toxic.

  6. T cytotoxic-1 CD8+ T cells are effector cells against pneumocystis in mice.

    Science.gov (United States)

    McAllister, Florencia; Mc Allister, Florencia; Steele, Chad; Zheng, Mingquan; Young, Erana; Shellito, Judd E; Marrero, Luis; Kolls, Jay K

    2004-01-15

    Host defenses are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes. A hallmark of HIV infection is Pneumocystis carinii (PC) pneumonia. Recently, CD8+ T cells, which are recruited to the lung in large numbers in response to PC infection, have been associated with some level of host defense as well as contributing to lung injury in BALB/c mice. In this study, we show that CD8+ T cells that have a T cytotoxic-1 response to PC in BALB/c mice, as determined by secretion of IFN-gamma, have in vitro killing activity against PC and effect clearance of the organism in adoptive transfer studies. Moreover, non-T cytotoxic-1 CD8+ T cells lacked in vitro effector activity and contributed to lung injury upon adoptive transfer. This dichotomous response in CD8+ T cell response may in part explain the clinical heterogeneity in the severity of PC pneumonia.

  7. Neolignans from Nectandra megapotamica (Lauraceae Display in vitro Cytotoxic Activity and Induce Apoptosis in Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Vitor Ponci

    2015-07-01

    Full Text Available Nectandra megapotamica (Spreng. Mez. (Lauraceae is a well-known Brazilian medicinal plant that has been used in folk medicine to treat several diseases. In continuation of our ongoing efforts to discover new bioactive natural products from the Brazilian flora, this study describes the identification of cytotoxic compounds from the MeOH extract of N. megapotamica (Lauraceae leaves using bioactivity-guided fractionation. This approach resulted in the isolation and characterization of eight tetrahydrofuran neolignans: calopeptin (1, machilin-G (2, machilin-I (3, aristolignin (4, nectandrin A (5, veraguensin (6, ganschisandrin (7, and galgravin (8. Different assays were conducted to evaluate their cytotoxic activities and to determine the possible mechanism(s related to the activity displayed against human leukemia cells. The most active compounds 4, 5 and 8 gave IC50 values of 14.2 ± 0.7, 16.9 ± 0.8 and 16.5 ± 0.8 µg/mL, respectively, against human leukemia (HL-60 tumor cells. Moreover, these compounds induced specific apoptotic hallmarks, such as plasma membrane bleb formation, nuclear DNA condensation, specific chromatin fragmentation, phosphatidyl-serine exposure on the external leaflet of the plasma membrane, cleavage of PARP as well as mitochondrial damage, which as a whole could be related to the intrinsic apoptotic pathway.

  8. Limited transplantation of antigen-expressing hematopoietic stem cells induces long-lasting cytotoxic T cell responses.

    Directory of Open Access Journals (Sweden)

    Warren L Denning

    Full Text Available Harnessing the ability of cytotoxic T lymphocytes (CTLs to recognize and eradicate tumor or pathogen-infected cells is a critical goal of modern immune-based therapies. Although multiple immunization strategies efficiently induce high levels of antigen-specific CTLs, the initial increase is typically followed by a rapid contraction phase resulting in a sharp decline in the frequency of functional CTLs. We describe a novel approach to immunotherapy based on a transplantation of low numbers of antigen-expressing hematopoietic stem cells (HSCs following nonmyeloablative or partially myeloablative conditioning. Continuous antigen presentation by a limited number of differentiated transgenic hematopoietic cells results in an induction and prolonged maintenance of fully functional effector T cell responses in a mouse model. Recipient animals display high levels of antigen-specific CTLs four months following transplantation in contrast to dendritic cell-immunized animals in which the response typically declines at 4-6 weeks post-immunization. Majority of HSC-induced antigen-specific CD8+ T cells display central memory phenotype, efficiently kill target cells in vivo, and protect recipients against tumor growth in a preventive setting. Furthermore, we confirm previously published observation that high level engraftment of antigen-expressing HSCs following myeloablative conditioning results in tolerance and an absence of specific cytotoxic activity in vivo. In conclusion, the data presented here supports potential application of immunization by limited transplantation of antigen-expressing HSCs for the prevention and treatment of cancer and therapeutic immunization of chronic infectious diseases such as HIV-1/AIDS.

  9. Treatment of solid organ transplant recipients with autologous Epstein Barr virus–specific cytotoxic T lymphocytes (CTLs)

    Science.gov (United States)

    Savoldo, Barbara; Goss, John A.; Hammer, Markus M.; Zhang, Lan; Lopez, Teresita; Gee, Adrian P.; Lin, Yu-Feng; Quiros-Tejeira, Ruben E.; Reinke, Petra; Schubert, Stephan; Gottschalk, Stephen; Finegold, Milton J.; Brenner, Malcolm K.; Rooney, Cliona M.; Heslop, Helen E.

    2006-01-01

    We have investigated the in vivo safety, efficacy, and persistence of autologous Epstein Barr virus (EBV)–specific cytotoxic T lymphocytes (CTLs) for the treatment of solid organ transplant (SOT) recipients at high risk for EBV-associated posttransplantation lymphoproliferative disease (PTLD). EBV-CTLs generated from 35 patients expanded with normal kinetics contained both CD8 and CD4 lymphocytes and produced significant specific killing of autologous EBV-transformed B lymphoblastoid cell lines (LCLs). Twelve SOT recipients at high risk for PTLD, or with active disease, received autologous CTL infusions without toxicity. Real-time polymerase chain reaction (PCR) monitoring of EBV-DNA showed a transient increase in plasma EBV-DNA suggestive of lysis of EBV-infected cells, although there was no consistent decrease in virus load in peripheral-blood mononuclear cells. Interferon-γ enzyme-linked immunospot (ELISPOT) assay and tetramer analysis showed an increase in the frequency of EBV-responsive T cells, which returned to preinfusion levels after 2 to 6 months. None of the treated patients developed PTLD. One patient with liver PTLD showed a complete response, and one with ocular disease has had a partial response stable for over one year. These data are consistent with an expansion and persistence of adoptively transferred EBV-CTLs that is limited in the presence of continued immunosuppression but that nonetheless produces clinically useful antiviral activity. PMID:16835376

  10. Cell cycle analysis and cytotoxic potential of Ruta graveolens against human tumor cell lines.

    Science.gov (United States)

    Varamini, P; Soltani, M; Ghaderi, A

    2009-01-01

    There are reports on the presence of various compounds exerting different biological activities in Ruta graveolens, a plant of Rutaceae family. The aim of the present study was to evaluate in vitro cytotoxicity of the total extract of R. graveolens against tumor cell lines of different origin. Aerial parts of the plant was extracted with 70% ethanol by sonication method and cytotoxic activity was examined on RAJI, RAMOS, RPMI8866, U937, Jurkat, MDA-MB-453, MCF-7, LNCap-FGC-10, 5637, HeLa, SK-OV-3, A549, Mehr-80 and also peripheral blood mononuclear cells (PBMC) by the use of WST-1 assay. Results were expressed as IC(50) values. R. graveolens extract showed high cytotoxic activity against RAJI and RAMOS, two Burkitt's lymphoma cell lines, with an IC(50) equal to 24.3 microg/ml and 35.2 microg/ml respectively and LNCap-FGC-10, a prostate adenocarcinoma cell line with an IC(50) equal to 27.6 microg/ml as well as Mehr-80, a newly established Large Cell Lung Carcinoma (IC(50)=46.2 microg/ml). No significant anti-proliferative activity was observed on other cell lines including MCF-7, MDA-MB-453, SK-OV-3, HeLa, 5637, JURKAT and RPMI8866. Adverse cytotoxic effect of R. graveolens was investigated against PBMCs and a significantly lower effect of this extract (IC(50)=104 microg/ml) was seen on normal cells compared with RAJI and RAMOS, two haematopoietic cell lines.

  11. Effects of Ginkgo biloba extract on cell proliferation and cytotoxicity in human hepatocellular carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Jane CJ Chao; Chia Chou Chu

    2004-01-01

    AIM: To study the effect of Ginkgo biloba extract (EGb 761)containing 22-27% fiavonoids (ginkgo-flavone glycosides)and 5-7% terpenoids (ginkgolides and bilobalides) on cell proliferation and cytotoxicity in human hepatocellular carcinoma (HCC) cells.METHODS: Human HCC cell lines (HepG2 and Hep3B) were incubated with various concentrations (0-1 000 mg/L) of EGb 761 solution. After 24 h incubation, cell proliferation and cytotoxicity were determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and lactate dehydrogenase (LDH)release, respectively. After 48 h incubation, the expression of proliferating cell nuclear antigen (PCNA) and p53 protein was measured by Western blotting.RESULTS: The results showed that EGb 761 (50-1 000 mg/L)significantly suppressed cell proliferation and increased LDH release (P<0.05) in HepG2 and Hep3B cells compared with the control group. The cell proliferation of HepG2 and Hep3B cells treated with EGb 761 (1 000 mg/L) was 45% and 39% of the control group (P<0.05), respectively. LDH release of HepG2 cells without and with EGb 761 (1 000 mg/L) treatment was 6.7% and 37.7%, respectively, and that of Hep3B cells without and with EGb 761 (1 000 mg/L) treatment was 7.2% and 40.3%, respectively. The expression of PCNA and p53 protein in HepG2 cells treated with EGb 761 (1 000 mg/L)was 85% and 174% of the control group, respectively.CONCLUSION: Ginkgobilobaextract significantly can suppress proliferation and increase cytotoxicity in HepG2 and Hep3B cells. Additionally, Ginkgo biloba extract can decrease PCNA and increase p53 expression in HepG2 cells.

  12. Cytotoxic effects of the lipid peroxidation product 2,4-decadienal in vascular smooth muscle cells.

    Science.gov (United States)

    Cabré, Anna; Girona, Josefa; Vallvé, Joan-C; Heras, Mercedes; Masana, Lluís

    2003-08-01

    It is well known that oxidized LDL can be cytotoxic to smooth muscle cells (SMC) and then contribute to the progression of atherosclerosis. Nevertheless, which oxidized compound and which mechanism are involved in cell death is still under study. In this work we have studied the role of two representative apolar aldehydes (hexanal and 2,4-decadienal (2,4-DDE)), derived from polyunsaturated fatty acids oxidation, on human SMC cytotoxicity. Cell cytotoxicity was assessed by means of lactate deshydrogenase (LDH) release, cell morphology and DNA fragmentation. Results showed that hexanal up to 50 microM for 24 h was not cytotoxic to cells. However, 2,4-DDE at 50 microM for 24 h induced a 48% LDH leakage. The observed cytotoxic effect of 2,4-DDE was not due to a programmed cell death as no DNA ladder was detected. After aldehydes exposition a decreased expression of p53 and c-myc mRNA, genes involved in cell death regulation, was also demonstrated by RT-PCR. These observations suggest that 2,4-DDE may be the molecular cause of lipid oxidation cytotoxicity to human vascular SMC. By inducing cell necrosis in advanced stages, lipid oxidation may contribute to the cell debris core which is growing in the atherosclerotic lesion leading to a weakened and unstable plaque.

  13. Chemosensitizing and cytotoxic effects of 2-deoxy-D-glucose on breast cancer cells

    Directory of Open Access Journals (Sweden)

    Zhang Fanjie

    2009-09-01

    Full Text Available Background: Accelerated glucose uptake for anerobic glycolysis is one of the major metabolic changes found in malignant cells. This property has been exploited for imaging malignancies and as a possible anticancer therapy. The nonmetabolizable glucose analog 2-deoxyglucose (2 DG interferes with glucose metabolism leading to breast cancer cell death. Aims: To determine whether 2DG can synergize with chemotherapeutic agents commonly used in breast cancer treatment and identify cellular characteristics associated with sensitivity to 2DG. Materials and Methods: SkBr3 breast cancer cells were incubated with varying concentrations of 5-fluorouracil (5FU, doxorubicin, cisplatin, cyclophosphamide, or herceptin with or without 2DG. Cell viability was measured using the MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay. Results: Combining 2DG with doxorubicin, 5 FU, cyclophosphamide, and herceptin resulted in enhanced cell death compared with each agent alone, while in combination with cisplatin, the amount of cell death was additive. Mouse embryo fibroblasts (MEF mutated for p53 (-/- were 30% more sensitive to the cytotoxic effects of 2DG than the parental cell lines. Cells mutated for Bax/Bac, genes involved in protection from apoptosis, are slightly more sensitive than the parental cell lines. Conclusions: These results indicate that 2DG acts synergistically with specific chemotherapeutic agents in causing cell death and the class of chemicals most sensitive appear to be those which cause DNA damage.

  14. Probing of Actinobacillus pleuropneumoniae ApxIIIA toxin-dependent cytotoxicity towards mammalian peripheral blood mononucleated cells

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    Fett Thomas

    2008-12-01

    Full Text Available Abstract Background Actinobacillus pleuropneumoniae, the causative bacterial agent of porcine pleuropneumonia, produces Apx toxins which belong to RTX toxin family and are recognized as the major virulence factors. So far, their target receptor(s has not been identified and the disease cytopathogenesis remains poorly understood. Production of an active Apx toxin and characterization of its toxic activity constitute the premises necessary to the description of its interaction with a potential receptor. From this point of view, we produced an active recombinant ApxIIIA toxin in order to characterize its toxicity on peripheral blood mononucleated cells (PBMCs isolated from several species. Findings Toxin preparation exercises a strong cytotoxic action on porcine PBMCs which is directly related to recombinant ApxIIIA since preincubation with polymyxin B does not modify the cytotoxicity rate while preincubation with a monospecific polyclonal antiserum directed against ApxIIIA does. The cell death process triggered by ApxIIIA is extremely fast, the maximum rate of toxicity being already reached after 20 minutes of incubation. Moreover, ApxIIIA cytotoxicity is species-specific because llama, human, dog, rat and mouse PBMCs are resistant. Interestingly, bovine and caprine PBMCs are slightly sensitive to ApxIIIA toxin too. Finally, ApxIIIA cytotoxicity is cell type-specific as porcine epithelial cells are resistant. Conclusion We have produced an active recombinant ApxIIIA toxin and characterized its specific cytotoxicity on porcine PBMCs which will allow us to get new insights on porcine pleuropneumonia pathogenesis in the future.

  15. Regulatory effect of Ganoderma lucidum polysaccharides on cytotoxic T-lymphocytes induced by dendritic cells in vitro

    Institute of Scientific and Technical Information of China (English)

    CAOLi-Zhen; LINZhi-Bin

    2003-01-01

    AIM:To study the regulatory effects of Ganoderma lucidum polysaccharides(Gl-PS)on cytotoxicity and mechanism of specific cytotoxic T-lymphocytes(CTL)induced by dendritic cells(DC)in vitro during the stage of antigen presentation.METHODS:Cultured murine bone marrow-derived DC were pulsed with P815 tumor cell lysates and co-incubated with or without various concentrations of Gl-PS(0.8,3.2,or 12.8mg/L)at the same time.P815 specific CTL were induced by spleen lymphocytes stimulated with mature DC.Non-adherent cells and culture supernatants were harvested on d 5 for analysis of specific cytotoxicity with lactate dehydrogenase(LDH)activity assay,mRNA expression of IFNγ,granzyme B with RT-PCR assay,and protein expression of IFNγ,granzyme B with ELISA or Western blot assay,respectively,RESULTS:Three concentrations of Gl-PS promoted LDH activities released into culture supernatants(P<0.01).It also increased mRNA expression of IFNγin CTL(Gl-PS 12.8mg/L vs RPMI medium 1640,P<0.05)and granzyme B in CTL(P<0.01).Protein production of IFNγin culture supernatants(P<0.05)and protein expression of granzyme B in CTL(Gl-PS 12.8mg/L vs RPMI medium 1640,P<0.05)were also augmented by Gl-PS,CONCLUSION:Gl-PS is shown to promote the cytotoxicity of specific CTL induced by DC which were pulsed with P815 tumor antigen during the stage of antigen presentation,and the mechanism of cytotoxicity is believed to be going through IFNγ and granzyme B pathways.

  16. Cytotoxicity of Different Excipients on RPMI 2650 Human Nasal Epithelial Cells

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    Tamás Horváth

    2016-05-01

    Full Text Available The nasal route receives a great deal of attention as a non-invasive method for the systemic administration of drugs. For nasal delivery, specific formulations containing excipients are used. Because of the sensitive respiratory mucosa, not only the active ingredients, but also additives need to be tested in appropriate models for toxicity. The aim of the study was to measure the cytotoxicity of six pharmaceutical excipients, which could help to reach larger residence time, better permeability, and increased solubility dissolution rate. The following excipients were investigated on RPMI 2650 human nasal septum tumor epithelial cells: β-d-mannitol, sodium hyaluronate, α and β-cyclodextrin, polyvinyl alcohol and methylcellulose. 3-(4,5-dimethyltiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT dye conversion assay and real-time impedance analysis were used to investigate cytotoxicity. No excipient showed toxicity at 0.3% (w/v concentration or below while 1% concentration a significantly reduced metabolic activity was measured by MTT assay for methylcellulose and cyclodextrins. Using impedance measurements, only β-cyclodextrin (1% was toxic to cells. Mannitol at 1% concentration had a barrier opening effect on epithelial cells, but caused no cellular damage. Based on the results, all additives at 0.3%, sodium hyaluronate and polyvinyl alcohol at 1% concentrations can be safely used for nasal formulations.

  17. Cytotoxicity and genotoxicity induced by coal and coal fly ash particles samples in V79 cells.

    Science.gov (United States)

    León-Mejía, Grethel; Silva, Luis F O; Civeira, Matheus S; Oliveira, Marcos L S; Machado, Miriana; Villela, Izabel Vianna; Hartmann, Andreas; Premoli, Suziane; Corrêa, Dione Silva; Da Silva, Juliana; Henriques, João Antônio Pêgas

    2016-12-01

    Exposure to coal and coal ashes can cause harmful effects in in vitro and in vivo systems, mainly by the induction of oxidative damage. The aim of this work was to assess cytotoxic and genotoxic effects using the V79 cell line treated with coal and coal fly ash particles derived from a coal power plant located in Santa Catarina, Brazil. Two coal samples (COAL11 and COAL16) and two coal fly ash samples (CFA11 and CFA16) were included in this study. COAL16 was co-firing with a mixture of fuel oil and diesel oil. The comet assay data showed that exposure of V79 cells to coal and coal fly ash particles induced primary DNA lesions. Application of lesion-specific endonucleases (FPG and ENDO III) demonstrated increased DNA effects indicating the presence of high amounts of oxidative DNA lesions. The cytokinesis-block micronucleus cytome assay analysis showed that exposure of V79 cells to high concentrations of coal and coal fly ash particles induced cytotoxic effects (apoptosis and necrosis) and chromosomal instability (nucleoplasmic bridges, nuclear buds, and micronucleus (MN) formation). These results may be associated with compounds contained in the surface of the particles as hazardous elements, ultrafine/nanoparticles, and polycyclic aromatic hydrocarbons (PAHs) which were detected in the samples. Graphical abstract ᅟ.

  18. Withania somnifera Induces Cytotoxic and Cytostatic Effects on Human T Leukemia Cells.

    Science.gov (United States)

    Turrini, Eleonora; Calcabrini, Cinzia; Sestili, Piero; Catanzaro, Elena; de Gianni, Elena; Diaz, Anna Rita; Hrelia, Patrizia; Tacchini, Massimo; Guerrini, Alessandra; Canonico, Barbara; Papa, Stefano; Valdrè, Giovanni; Fimognari, Carmela

    2016-01-01

    Cancer chemotherapy is characterized by an elevated intrinsic toxicity and the development of drug resistance. Thus, there is a compelling need for new intervention strategies with an improved therapeutic profile. Immunogenic cell death (ICD) represents an innovative anticancer strategy where dying cancer cells release damage-associated molecular patterns promoting tumor-specific immune responses. The roots of Withania somnifera (W. somnifera) are used in the Indian traditional medicine for their anti-inflammatory, immunomodulating, neuroprotective, and anticancer activities. The present study is designed to explore the antileukemic activity of the dimethyl sulfoxide extract obtained from the roots of W. somnifera (WE). We studied its cytostatic and cytotoxic activity, its ability to induce ICD, and its genotoxic potential on a human T-lymphoblastoid cell line by using different flow cytometric assays. Our results show that WE has a significant cytotoxic and cytostatic potential, and induces ICD. Its proapoptotic mechanism involves intracellular Ca(2+) accumulation and the generation of reactive oxygen species. In our experimental conditions, the extract possesses a genotoxic potential. Since the use of Withania is suggested in different contexts including anti-infertility and osteoarthritis care, its genotoxicity should be carefully considered for an accurate assessment of its risk-benefit profile.

  19. Induction of cytotoxic CD8+CD56+ T cells from human thymocytes by interleukin-15

    DEFF Research Database (Denmark)

    Thulesen, S; Nissen, Mogens Holst; Ødum, N;

    2001-01-01

    CD8(+) CD56(+) cells isolated from human peripheral blood lymphocytes have been shown recently to represent a population of cytotoxic active T cells. However, it is not known if these cells are intrathymically or extrathymically developed or how these cells are influenced by growth factors...... of thymocytes. The majority of the IL-15-grown CD8(+) CD56(+) cells were CD45R0(+), representing a memory phenotype, and showed high expression of the IL-15R-complex and high numbers of CD69(+) cells. Moreover, cytotoxic activity was confined to this cell population....

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

    Science.gov (United States)

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

    2016-04-01

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

  1. Lactobacillus delbrueckii ssp. bulgaricus B-30892 can inhibit cytotoxic effects and adhesion of pathogenic Clostridium difficile to Caco-2 cells

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    Banerjee Pratik

    2009-04-01

    Full Text Available Abstract Background Probiotic microorganisms are receiving increasing interest for use in the prevention, treatment, or dietary management of certain diseases, including antibiotic-associated diarrhea (AAD. Clostridium difficile is the most common cause of AAD and the resulting C. difficile – mediated infection (CDI, is potentially deadly. C. difficile associated diarrhea (CDAD is manifested by severe inflammation and colitis, mostly due to the release of two exotoxins by C. difficile causing destruction of epithelial cells in the intestine. The aim of this study was to determine the effect of probiotic bacteria Lactobacillus delbrueckii ssp. bulgaricus B-30892 (LDB B-30892 on C. difficile-mediated cytotoxicity using Caco-2 cells as a model. Methods Experiments were carried out to test if the cytotoxicity induced by C. difficile-conditioned-medium on Caco-2 cells can be altered by cell-free supernatant (CFS from LDB B-30892 in different dilutions (1:2 to 1:2048. In a similar experimental setup, comparative evaluations of other probiotic strains were made by contrasting the results from these strains with the results from LDB B-30892, specifically the ability to affect C. difficile induced cytotoxicity on Caco-2 monolayers. Adhesion assays followed by quantitative analysis by Giemsa staining were conducted to test if the CFSs from LDB B-30892 and other probiotic test strains have the capability to alter the adhesion of C. difficile to the Caco-2 monolayer. Experiments were also performed to evaluate if LDB B-30892 or its released components have any bactericidal effect on C. difficile. Results and discussion Co-culturing of LDB B-30892 with C. difficile inhibited the C. difficile-mediated cytotoxicity on Caco-2 cells. When CFS from LDB B-30892-C. difficile co-culture was administered (up to a dilution of 1:16 on Caco-2 monolayer, there were no signs of cytotoxicity. When CFS from separately grown LDB B-30892 was mixed with the cell-free toxin

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1989-10-15

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

  3. Amphotericin B, an Anti-Fungal Medication, Directly Increases the Cytotoxicity of NK Cells

    Science.gov (United States)

    Kim, Nayoung; Choi, Ji-Wan; Park, Hye-Ran; Kim, Inki; Kim, Hun Sik

    2017-01-01

    Immunomodulatory drugs (IMiDs) present one example of immunomodulatory agents that improve cancer immunotherapy. Based on the cytotoxic activity of natural killer (NK) cells against cancer cells, a high throughput screening method for the identification of novel immunomodulatory molecules with the potential to stimulate NK cell cytotoxicity against cancer cells was designed and tested using an approved drug library. Among the primary hit compounds, the anti-fungal drug amphotericin B (AMP-B) increased the cytotoxicity of NK cell line and human primary NK cells in a direct manner. The increase in NK cell activity was related to increased formation of NK-target cell conjugates and the subsequent granule polarization toward target cells. The results of the present study indicate that AMP-B could serve a dual function as an anti-fungal and immunomodulatory drug. PMID:28608807

  4. Identification of Melanoma-specific Peptide Epitopes by HLA-A2.1-restricted Cytotoxic T Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    Hai-Liang GE; Ying WANG; Shu-Jun WANG; Yong ZHANG

    2006-01-01

    HLA-A2.1-associated peptides, extracted from human melanoma cells, were used to study epitopes for melanoma-specific HLA-A2.1-restricted cytotoxic T lymphocytes (CTLs) by epitope reconstitution, active peptide sequence characterization and synthetic peptide verification. CTL were generated from tumor-involved nodes by in vitro stimulation, initially with autologous melanoma cells and subsequently with allogeneic HLA-A2.1 positive melanoma cells. The CTLs could lyse autologous and allogeneic HLA-A2.1 positive melanomas, but not HLA-A2.1 negative melanomas or HLA-A2.1 positive non-melanomas. The lysis of melanomas could be inhibited by anti-CD3, anti-HLA class Ⅰ and anti-HLA-A2.1 monoclonal antibodies.HLA-A2.1 molecules were purified from detergent-solubilized human melanoma cells by immunoaffinity column chromatography and further fractionated by reversed phase high performance liquid chromatography.The fractions were assessed for their ability to reconstitute melanoma-specific epitopes with HLA-A2.1 positive antigen-processing mutant T2 cells. Three reconstitution peaks were observed in lactate dehydrogenase release assay. Mass spectrometry and ion-exchange high performance liquid chromatography analysis were used to identify peptide epitopes. Peptides with a mass-to-charge ratio of 948 usually consist of nine amino acid residues. The data from reconstitution experiments confirmed that the synthetic peptides contained epitopes and that the peptides associated with HLA-A2.1 and recognized by melanoma-specific CTL were present in these different melanoma cells. These peptides could be potentially exploited in novel peptide-based antitumor vaccines in immunotherapy for CTL.

  5. Analysis of Epstein-Barr viral DNA load, EBV-LMP2 specific cytotoxic T-lymphocytes and levels of CD4+CD25+T cells in patients with nasopharyngeal carcinomas positive for IgA antibody to EBV viral capsid antigen

    Institute of Scientific and Technical Information of China (English)

    MO Wu-ning; TANG An-zhou; ZHOU Ling; HUANG Guang-wu; WANG Zhan; ZENG Yi

    2009-01-01

    Background Epstein-Barr virus (EBV) is a herpesvirus commonly associated with several malignant diseases including nasopharyngeal carcinoma (NPC), which is a common cancer in Southeastem Asia. Previous studies showed that plasma levels of EBV-DNA might be a sensitive and reliable biomarker for the diagnosis, staging and evaluating of therapy for NPC. There are a few analyses of the levels of EBV-latent membrane protein 2 (LMP2)-specific cytotoxic T-lymphocytes (CTLs) in patients with NPC. This study was conducted to investigate the levels of EBV-LMP2-specific CTLs, EBV-DNA load and the level of CD4+CD25+T cells in such patients.Methods From February 2006 to April 2006, 62 patients with NPC, 40 healthy virus carders positive for EBV viral capsid antigen (EBV-IgA-VCA) and 40 controls were enrolled in the study. We used a highly sensitive ELISPOT assay,real-time polymerase chain reaction (PCR) and flow cytometry to measure the EBV-LMP2-specific CTL response, the EBV DNA load and the level of CD4+CD25+T cells, respectively.Results The EBV-LMP2-specific CTL responses of the samples from the control, healthy virus carders and patients with NPC were significantly different from the LMP2 epitopes, with the control and healthy virus carder samples displaying a stronger response in three cases. There were significant differences in EBV DNA load in serum between NPC and the healthy groups; patients with NPC at stages Ⅲ or Ⅳ had significantly higher viral loads compared with those at stages Ⅰ or Ⅱ. A significantly higher percentage of CD4+CD25+ T lymphocytes were detected in the patients, compared with healthy virus carriers and healthy controls. Moreover, patients with advanced stages of NPC (Ⅲ and Ⅲ) had significantly higher percentages than the patients with early stages (Ⅰ and Ⅱ).Conclusions Patients with NPC are frequently unable to establish or maintain sufficient immunosurveillance to control proliferating B cells harboring EBV and to destroy the tumor

  6. Study on the Cytotoxic T Lymphocytes Clone Specific for the Nucleocapsid Protein of Hantaan Virus from Peripheral Blood in Patients with Hemorrhagic Fever with Renal Syndrome

    Institute of Scientific and Technical Information of China (English)

    潘蕾; 白雪帆; 黄长形; 李光玉

    2003-01-01

    In order to elucidate the molecular and immunological mechanisms as well as the pathogenesis of hemorrhagic fever with renal syndrome (HFRS), the CD8+ cytotoxic T lymphecytes (CTL) clone was established directly from peripheral blood mononuclear cells (PBMC) of patients with HFRS. The activities of CTL were detected as usual with EBV-transformed lymphoblastoid cell line (BLCL) as target cells. The results showed that the CTL clone could recognized and killed the targetcells with specificity of nucleocapsid protein of Hantaan virus (HTNVNP) with the cytotoxicity percentages of 50.2%,25.4% and 39.0% respectively. These results demonstrated that the antigenic epitopes of HTNVNP mainly located on the C-temainal of the viral nucleocapsid protein.

  7. Doxorubicin has a synergistic cytotoxicity with cucurbitacin B in anaplastic thyroid carcinoma cells.

    Science.gov (United States)

    Kim, Si Hyoung; Kang, Jun Goo; Kim, Chul Sik; Ihm, Sung-Hee; Choi, Moon Gi; Yoo, Hyung Joon; Lee, Seong Jin

    2017-02-01

    In this study, the combined effect of doxorubicin with cucurbitacin B on survival of anaplastic thyroid carcinoma cells was evaluated. For experiments, 8505C and CAL62 human anaplastic thyroid carcinoma cells were used. Cell viability, the percentage of viable cells, and cytotoxic activity were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, multiplexed cytotoxicity assay, and cytotoxicity assay, respectively. Reactive oxygen species production was measured. In experiments, doxorubicin and cucurbitacin B reduced cell viability in a dose- and time-dependent manner. Cotreatment of doxorubicin and cucurbitacin B, compared with treatment of doxorubicin alone, decreased the percentage of viable cells and increased cytotoxic activity. All of the combination index values were lower than 1.0, suggesting the synergism between doxorubicin and cucurbitacin B in induction of cytotoxicity. In cells treated with both doxorubicin and cucurbitacin B, compared with doxorubicin alone, the protein levels of cleaved poly(adenosine diphosphate-ribose) polymerase and cyclooxygenase 2 and reactive oxygen species production were enhanced. In contrast, the protein levels of B-cell chronic lymphocytic leukemia/lymphoma 2 and survivin and B-cell chronic lymphocytic leukemia/lymphoma 2/B-cell chronic lymphocytic leukemia/lymphoma 2-associated x protein ratio were diminished. The protein levels of Janus kinase 2 and signal transducer and activator of transcription 3 were reduced, while phospho-extracellular signal-regulated kinase 1/2 protein levels were elevated without change in total extracellular signal-regulated kinase 1/2 protein levels. These results suggest that doxorubicin synergizes with cucurbitacin B in induction of cytotoxicity in anaplastic thyroid carcinoma cells. Moreover, synergistic cytotoxicity of doxorubicin with cucurbitacin B is mediated by B-cell chronic lymphocytic leukemia/lymphoma 2 family proteins, survivin, and reactive oxygen

  8. Identification of a human cyclin D1-derived peptide that induces human cytotoxic CD4 T cells.

    Directory of Open Access Journals (Sweden)

    Tao Dao

    Full Text Available Cyclin D1 is over-expressed in various human tumors and therefore can be a potential oncogenic target antigen. However, only a limited number of T cell epitopes has been characterized. We aimed at identifying human cyclin D1-derived peptides that include both CD4 and CD8 T cell epitopes and to test if such multi-epitope peptides could yield improved cytotoxic CD8 T cell responses as well as cytotoxic CD4 T cells. Five HLA-DR.B1-binding peptides containing multiple overlapping CD4 epitopes and HLA-A0201-restricted CD8 T cell epitopes were predicted by computer algorithms. Immunogenicity of the synthetic peptides was assessed by stimulating T cells from healthy donors in vitro and the epitope recognition was measured by IFN-gamma ELISPOT and (51Chromium release assays. A HLA-DR.B1 peptide, designed "DR-1", in which a HLA-A0201-binding epitopes (D1-1 was imbedded, induced CD3 T cell responses against both DR-1 and D1-1 peptides in IFN-gamma ELISPOT assay. This suggested processing of the shorter D1-1 epitope from the DR-1 sequence. However, only DR-1-stimulated CD4 or CD3 T cells possessed cytotoxicity against peptide-pulsed autologous DCs and a cancer cell line, that expresses a high level of cyclin D1. Monoclonal antibody to HLA-DR abrogated the epitope-specific responses of both CD3 and CD4 T cells, demonstrating class II-mediated killing. Our studies suggest a possible role of CD4 T cells in anti-tumor immunity as cytotoxic effectors against HLA-DR expressing cancers and provide a rationale for designing peptide vaccines that include CD4 epitopes.

  9. DAP10 contributes to CD8(+) T cell-mediated cytotoxic effector mechanisms during Mycobacterium tuberculosis infection.

    Science.gov (United States)

    Hessmann, Manuela; Rausch, Alexandra; Rückerl, Dominik; Adams, Pamela Scott; Simon, Markus; Gilfillan, Susan; Colonna, Marco; Ehlers, Stefan; Hölscher, Christoph

    2011-05-01

    The activating C-type lectin-like receptor NKG2D, which is expressed by mouse NK cells and activated CD8 T cells, was previously demonstrated to be involved in tumor rejection and as a defense mechanism against viral and bacterial infections. Because CD8 T cells are important for protective immune responses during chronic Mycobacterium tuberculosis (Mtb) infection and represent a promising target for new vaccine strategies to prevent human pulmonary tuberculosis (TB), we studied the immune response in mice deficient for the NKG2D adapter molecule DAP10 during experimental TB. After aerosol infection, DAP10-defcient mice displayed an unimpaired recruitment, activation and development of antigen-specific CD8 T cells. Whereas the frequency of interferon-gamma-producing CD8 T cells from Mtb-infected DAP10-defcient mice was not affected, CD8 T cell-mediated cytotoxicity was significantly reduced in the absence of DAP10. The loss of cytotoxic activity in DAP10-deficient CD8 T cells was associated with an impaired release of cytotoxic granules. Together, our results suggest that during Mtb infection DAP10 is required for maximal cytolytic activity of CD8 T cells.

  10. The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung epithelial cells.

    Science.gov (United States)

    Xie, Hong; Smith, Leah J; Holmes, Amie L; Zheng, Tongzhang; Pierce Wise, John

    2016-05-01

    Cobalt is a toxic metal used in various industrial applications leading to adverse lung effects by inhalation. Cobalt is considered a possible human carcinogen with the lung being a primary target. However, few studies have considered cobalt-induced toxicity in human lung cells, especially normal lung epithelial cells. Therefore, in this study, we sought to determine the cytotoxicity and genotoxicity of particulate and soluble cobalt in normal primary human lung epithelial cells. Cobalt oxide and cobalt chloride were used as representative particulate and soluble cobalt compounds, respectively. Exposure to both particulate and soluble cobalt induced a concentration-dependent increase in cytotoxicity, genotoxicity, and intracellular cobalt ion levels. Based on intracellular cobalt ion levels, we found that soluble and particulate cobalt induced similar cytotoxicity while soluble cobalt was more genotoxic than particulate cobalt. These data indicate that cobalt compounds are cytotoxic and genotoxic to human lung epithelial cells.

  11. Electrochemical monitoring of phytochelatin accumulation in Nicotiana tabacum cells exposed to sub-cytotoxic and cytotoxic levels of cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Fojta, Miroslav [Laboratory of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic)]. E-mail: fojta@ibp.cz; Fojtova, Miloslava [Laboratory of Molecular Epigenetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic); Havran, Ludek [Laboratory of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic); Pivonkova, Hana [Laboratory of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic); Dorcak, Vlastimil [Laboratory of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno (Czech Republic); Sestakova, Ivana [J. Heyrovsky Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Dolejskova 3, 182 23 Prague 8 (Czech Republic)

    2006-02-03

    Cadmium belongs to the most dangerous environmental pollutants among the toxic heavy metals seriously affecting vital functions in both animal and plant cells. It has been previously shown that cadmium ions at 50-100 {mu}M concentrations caused tobacco BY-2 (TBY-2) cells to enter apoptosis within several days of exposure. Phytochelatins (PCs), the 'plant metallothioneins', are cysteine-rich peptides involved in detoxification of heavy metals in plants. The PCs are synthesized in response to the heavy metal exposure. In this paper, we utilized electrochemical analysis to monitor accumulation of PCs in the TBY-2 cells exposed to cadmium ions. Measurements of a characteristic PC signal at mercury electrode in the presence of cobalt ions made it possible to detect changes in the cellular PC levels during the time of cultivation, starting from 30 min after exposure. Upon TBY-2 cultivation in the presence of cytotoxic cadmium concentrations, the PC levels remarkably increased during the pre-apoptotic phase and reached a limiting value at cultivation times coinciding with apoptosis trigger. The PC level observed for a sub-cytotoxic cadmium concentration (10 {mu}M) was about three-times lower than that observed for the 50 or 100 {mu}M cadmium ions after 5 days of exposure. We show that using a simple electrochemical analysis, synthesis of PCs in plant cells can be easily followed in parallel with other tests of the cellular response to the toxic heavy metal stress.

  12. The Mechanism of Melanocytes-Specific Cytotoxicity Induced by Phenol Compounds Having a Prooxidant Effect, relating to the Appearance of Leukoderma

    Directory of Open Access Journals (Sweden)

    Takeshi Nagata

    2015-01-01

    Full Text Available Specific phenol compounds including rhododendrol (RD, a skin-brightening ingredient in cosmetics, are reported to induce leukoderma, inducing a social problem, and the elucidation of mechanism of leukoderma is strongly demanded. This study investigated the relationship among the cytotoxicities of six phenol compounds on B16F10 melanoma cells and HaCaT keratinocytes and generated reactive oxygen species (ROS. As a result, the cytotoxicity of RD on B16F10 cells was higher than that on HaCaT cells, and RD significantly increased intracellular ROS and hydrogen peroxide (H2O2 levels in B16F10 cells. Furthermore, although raspberry ketone (RK, RD derivative, also increased intracellular ROS in B16F10 cells, increase in ROS was suppressed by disodium dihydrogen ethylenediaminetetraacetate dehydrate (EDTA. The amounts of increased ROS with RK in HaCaT cells without melanocyte were further increased by tyrosinase. Therefore, tyrosinase, a metalloprotein having copper, was speculated to be one of causative agents allowing phenol compounds to work as a prooxidant. Hydroxyl radical was generated by adding a mixture of tyrosinase and H2O2 to RD, and the amount of the radical was further increased by UVB, indicating that RD cytotoxicity was caused by intracellularly increased ROS, which possibly related to phenol induced prooxidants.

  13. Streptozotocin-Induced Cytotoxicity, Oxidative Stress and Mitochondrial Dysfunction in Human Hepatoma HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Haider Raza

    2012-05-01

    Full Text Available Streptozotocin (STZ is an antibiotic often used in the treatment of different types of cancers. It is also highly cytotoxic to the pancreatic beta-cells and therefore is commonly used to induce experimental type 1 diabetes in rodents. Resistance towards STZ-induced cytotoxicity in cancer cells has also been reported. Our previous studies have reported organ-specific toxicity and metabolic alterations in STZ-induced diabetic rats. STZ induces oxidative stress and metabolic complications. The precise molecular mechanism of STZ-induced toxicity in different tissues and carcinomas is, however, unclear. We have, therefore, investigated the mechanism of cytotoxicity of STZ in HepG2 hepatoma cells in culture. Cells were treated with different doses of STZ for various time intervals and the cytotoxicity was studied by observing the alterations in oxidative stress, mitochondrial redox and metabolic functions. STZ induced ROS and RNS formation and oxidative stress as measured by an increase in the lipid peroxidation as well as alterations in the GSH-dependent antioxidant metabolism. The mitochondria appear to be a highly sensitive target for STZ toxicity. The mitochondrial membrane potential and enzyme activities were altered in STZ treated cells resulting in the inhibition of ATP synthesis. ROS-sensitive mitochondrial aconitase activity was markedly inhibited suggesting increased oxidative stress in STZ-induced mitochondrial toxicity. These results suggest that STZ-induced cytotoxicity in HepG2 cells is mediated, at least in part, by the increase in ROS/RNS production, oxidative stress and mitochondrial dysfunction. Our study may be significant for better understanding the mechanisms of STZ action in chemotherapy and drug induced toxicity.

  14. The Granzyme B ELISPOT assay: an alternative to the 51Cr-release assay for monitoring cell-mediated cytotoxicity

    Directory of Open Access Journals (Sweden)

    Baseler Michael

    2003-12-01

    Full Text Available Abstract Background The interferon-γ (IFN-γ ELISPOT assay is one of the most useful techniques for immunological monitoring of cancer vaccine trials and has gained increased application as a measure of specific T cell activation. However, it does not assess cell-mediated cytotoxicity directly as IFN-γ secretion is not limited to only cytolytic cells. Granzyme B (GrB is a key mediator of target cell death via the granule-mediated pathway. Therefore, the release of GrB by cytolytic lymphocytes upon effector-target interaction may be a more specific indicator of CTL and NK cytotoxic ability than IFN-γ secretion. Methods We assessed whether the GrB ELISPOT assay is a viable alternative to the 51Cr-release and IFN-γ ELISPOT assays for measuring antigen-specific CTL cytotoxicity. Direct comparisons between the three assays were made using human CTL cell lines (αEN-EBV and αJY and an in vitro stimulated anti-Flu matrix peptide (FMP-specific CTL. Results When the GrB ELISPOT was directly compared to the IFN-γ ELISPOT and 51Cr-release assays, excellent cross-correlation between all three assays was shown. However, measurable IFN-γ secretion in the ELISPOT assay was observed only after 1 hour of incubation and cytotoxicity assessed via the 51Cr-release assay after 4 hours, whereas GrB secretion was detectable within 10 min of effector-target contact with significant secretion observed after 1 h. Titration studies demonstrated a strong correlation between the number of effector cells and GrB spots per well. Irrelevant targets or antigens did not induce significant GrB secretion. Additionally, GrB secretion was abrogated when CTL cultures were depleted of CD8+ cells. Conclusion Our findings demonstrate that the GrB ELISPOT assay is a superior alternative to the 51Cr-release assay since it is significantly more sensitive and provides an estimation of cytotoxic effector cell frequency. Additionally, unlike the IFN-γ ELISPOT assay, the GrB ELISPOT

  15. The cytotoxic effect of 2-acylated-1,4-naphthohydroquinones on leukemia/lymphoma cells

    Science.gov (United States)

    Pedroza, Diego A.; De Leon, Fernando; Varela-Ramirez, Armando; Lema, Carolina; Aguilera, Renato J.; Mito, Shizue

    2014-01-01

    Here, we tested seven 2-acylated-1,4-hydronaphthoquinones for their cytotoxic effects on a panel of cancer lymphoma/leukemia cells and compared to a non-cancer origin cell line. Several naphthohydroquinones exhibited selective cytotoxic effects on lymphoma/leukemia cells with lowest activity on non-cancer cells. The mode of cell death induced by an acylated naphthohydroquinone, which has a long alkyl chain, was found to be via apoptosis. Furthermore, the naphthohydroquinone provoked mitochondria depolarization and activation of its downstream effector, caspase-3, thus implicating the intrinsic apoptotic pathway as its mechanism to exert cell death. PMID:24368029

  16. IFN-γ is required for cytotoxic T cell-dependent cancer genome immunoediting

    Science.gov (United States)

    Takeda, Kazuyoshi; Nakayama, Masafumi; Hayakawa, Yoshihiro; Kojima, Yuko; Ikeda, Hiroaki; Imai, Naoko; Ogasawara, Kouetsu; Okumura, Ko; Thomas, David M.; Smyth, Mark J.

    2017-01-01

    Genetic evolution that occurs during cancer progression enables tumour heterogeneity, thereby fostering tumour adaptation, therapeutic resistance and metastatic potential. Immune responses are known to select (immunoedit) tumour cells displaying immunoevasive properties. Here we address the role of IFN-γ in mediating the immunoediting process. We observe that, in several mouse tumour models such as HA-expressing 4T1 mammary carcinoma cells, OVA-expressing EG7 lymphoma cells and CMS5 MCA-induced fibrosarcoma cells naturally expressing mutated extracellular signal-regulated kinase (ERK) antigen, the action of antigen-specific cytotoxic T cell (CTL) in vivo results in the emergence of resistant cancer cell clones only in the presence of IFN-γ within the tumour microenvironment. Moreover, we show that exposure of tumours to IFN-γ-producing antigen-specific CTLs in vivo results in copy-number alterations (CNAs) associated with DNA damage response and modulation of DNA editing/repair gene expression. These results suggest that enhanced genetic instability might be one of the mechanisms by which CTLs and IFN-γ immunoedits tumours, altering their immune resistance as a result of genetic evolution. PMID:28233863

  17. Novel mathematical models for cell-mediated cytotoxicity assays without applying enzyme kinetics but with combinations and probability: bystanders in bulk effector cells influence results of cell-mediated cytotoxicity assays.

    Science.gov (United States)

    Takayanagi, Toshiaki

    2011-07-01

    Cell-mediated cytotoxicity assays are widely implemented to evaluate cell-mediated cytotoxic activity, and some assays are analyzed using the analogy of enzyme kinetics. In the analogy, the effector cell is regarded as the enzyme, the target cell as the substrate, the effector cell-target cell conjugate as the enzyme-substrate complex and the dead target cell as the product. However, the assumptions analogous to those of enzyme kinetics are not always true in cell-mediated cytotoxicity assays, and the parameter analogous to the Michaelis-Menten constant is not constant but is dependent on the number of effector cells. Therefore I present novel mathematical models for cell-mediated cytotoxicity assays without applying enzyme kinetics. I instead use combinations and probability, because analysis of cell-mediated cytotoxicity assays by applying enzyme kinetics seems controversial. With my original models, I demonstrate simulations of the data in previously published papers. The results are exhibited in the same forms as the corresponding data. Comparing the simulation results with the published data, the results seem to agree well with the data. From simulations of cytotoxic assays with bulk effector cells, it appears that bystanders in bulk effector cells increase both the cytotoxic activity and the motility of effector cells.

  18. Inhibiting Heat Shock Proteins Can Potentiate the Cytotoxic Effect of Cannabidiol in Human Glioma Cells.

    Science.gov (United States)

    Scott, Katherine A; Dennis, Jayne L; Dalgleish, Angus G; Liu, Wai M

    2015-11-01

    Cannabinoids possess a number of characteristics that make them putative anticancer drugs, and their value as such is currently being explored in a number of clinical studies. To further understand the roles that cannabinoids may have, we performed gene expression profiling in glioma cell lines cultured with cannabidiol (CBD) and/or Δ9-tetrahydrocannabinol (THC), and pursued targets identified by this screening. Results showed that a large number of genes belonging to the heat shock protein (HSP) super-family were up-regulated following treatment, specifically with CBD. Increases were observed both at the gene and protein levels and arose as a consequence of increased generation of ROS by CBD, and correlated with an increase in a number of HSP client proteins. Furthermore, increases impeded the cytotoxic effect of CBD; an effect that was improved by co-culture with pharmacalogical inhibitors of HSPs. Similarly, culturing glioma cells with CBD and HSP inhibitors increased radiosensitivity when compared to CBD-alone. Taken together, these data indicate that the cytotoxic effects of CBD can be diminished by HSPs that indirectly rise as a result of CBD use, and that the inclusion of HSP inhibitors in CBD treatment regimens can enhance the overall effect.

  19. High folic acid intake reduces natural killer cell cytotoxicity in aged mice.

    Science.gov (United States)

    Sawaengsri, Hathairat; Wang, Junpeng; Reginaldo, Christina; Steluti, Josiane; Wu, Dayong; Meydani, Simin Nikbin; Selhub, Jacob; Paul, Ligi

    2016-04-01

    Presence of unmetabolized folic acid in plasma, which is indicative of folic acid intake beyond the metabolic capacity of the body, is associated with reduced natural killer (NK) cell cytotoxicity in postmenopausal women ≥50years. NK cells are cytotoxic lymphocytes that are part of the innate immune system critical for surveillance and defense against virus-infected and cancer cells. We determined if a high folic acid diet can result in reduced NK cell cytotoxicity in an aged mouse model. Female C57BL/6 mice (16-month-old) were fed an AIN-93M diet with the recommended daily allowance (1× RDA, control) or 20× RDA (high) folic acid for 3months. NK cytotoxicity was lower in splenocytes from mice fed a high folic acid diet when compared to mice on control diet (Pfolic acid fed mice could be due to their lower mature cytotoxic/naïve NK cell ratio (P=.03) when compared to the control mice. Splenocytes from mice on high folic acid diet produced less interleukin (IL)-10 when stimulated with lipopolysaccharide (Pfolic acid group was at least partially due to reduced IL-10 production. This study demonstrates a causal relationship between high folic acid intake and reduced NK cell cytotoxicity and provides some insights into the potential mechanisms behind this relationship.

  20. Rhinovirus infection induces cytotoxicity and delays wound healing in bronchial epithelial cells

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    Constantopoulos Andreas G

    2005-10-01

    Full Text Available Abstract Background Human rhinoviruses (RV, the most common triggers of acute asthma exacerbations, are considered not cytotoxic to the bronchial epithelium. Recent observations, however, have questioned this knowledge. The aim of this study was to evaluate the ability of RV to induce epithelial cytotoxicity and affect epithelial repair in-vitro. Methods Monolayers of BEAS-2B bronchial epithelial cells, seeded at different densities were exposed to RV serotypes 1b, 5, 7, 9, 14, 16. Cytotoxicity was assessed chromatometrically. Epithelial monolayers were mechanically wounded, exposed or not to RV and the repopulation of the damaged area was assessed by image analysis. Finally epithelial cell proliferation was assessed by quantitation of proliferating cell nuclear antigen (PCNA by flow cytometry. Results RV1b, RV5, RV7, RV14 and RV16 were able to induce considerable epithelial cytotoxicity, more pronounced in less dense cultures, in a cell-density and dose-dependent manner. RV9 was not cytotoxic. Furthermore, RV infection diminished the self-repair capacity of bronchial epithelial cells and reduced cell proliferation. Conclusion RV-induced epithelial cytotoxicity may become considerable in already compromised epithelium, such as in the case of asthma. The RV-induced impairment on epithelial proliferation and self-repair capacity may contribute to the development of airway remodeling.

  1. Participation of CD45, NKR-P1A and ANK61 antigen in rat hepatic NK cell (pit cell)-mediated target cell cytotoxicity

    Institute of Scientific and Technical Information of China (English)

    Dian Zhong Luo; David Vermijlen; B lent Ahishali; Vasilis Triantis; Eddie Wisse; Karin Vanderkerken; Peter J.K. Kuppen

    2000-01-01

    AIM Several triggering receptors have been described to be involved in natural killer (NK) cellmediated target cytotoxicity. In these studies, NK cells derived from blood or spleen were used. Pit cells are liver-specific NK cells that possess a higher level of natural cytotoxicity and a different morphology when compared to blood NK cells. The aim of this study was to characterize the role of the NK-triggering molecules NKR-P1A, ANK61 antigen, and CD45 in pit cell-mediated killing of target cells. METHODS 51 Cr-release and DNA fragmentation were used to quantify target cell lysis and apoptosis, respectively. RESULTS Flow cytometric analysis showed that pit cells expressed CD45, NKR-P1A, and ANK61 antigen. Treatment of pit cells with monoclonal antibody ( mAb ) to CD45 ( ANK74 ) not only inhibited CC531s or YAC-1 target lysis but also apoptosis induced by pit cells. The mAbs to NKRP1A (3.2.3) and ANK61 antigen (ANK61) had no effect on pit cell-mediated CC531s or YAC-1 target cytolysis or apoptosis, while they did increase the Fcγ receptor positive (FcγR+) P815 cytolysis and apoptosis. This enhanced cytotoxicity could he inhibited by 3,4-dichloroisocoumarin, an inhibitor of granzymes. CONCLUSION These results indicate that CD45 participates in pit cell-mediated CC531s and YAC-1 target cytolysis and apoptosis. NKR-P1A and ANK61 antigen on pit cells function as activation structures against FcγR+ P815 cells, which was mediated by the perforin/granzyme pathway.

  2. Clinical-Grade Human Multipotent Adult Progenitor Cells Block CD8+ Cytotoxic T Lymphocytes.

    Science.gov (United States)

    Plessers, Jeroen; Dekimpe, Emily; Van Woensel, Matthias; Roobrouck, Valerie D; Bullens, Dominique M; Pinxteren, Jef; Verfaillie, Catherine M; Van Gool, Stefaan W

    2016-12-01

    : MultiStem cells are clinical-grade multipotent adult bone marrow-derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T-cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8(+) cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL-mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was-even after major histocompatibility complex class I upregulation-insensitive to alloantigen-specific CTL-mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T-cell activation phase. As a consequence, MultiStem dose-dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact-dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T-cell activation markers CD25, CD69, and human leukocyte antigen-DR. The MultiStem-induced CD8(-)CD69(+) T-cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed-lymphocyte culture. Finally, the killer activity of activated antigen-specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical-grade MAPCs are an immune-modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs. Because multipotent adult progenitor cells (MAPCs) are among the noteworthy adult mesenchymal stem cell populations for immune

  3. Synthesis of Chromonylthiazolidines and Their Cytotoxicity to Human Cancer Cell Lines

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    Hoang Le Tuan Anh

    2015-01-01

    Full Text Available Nine new chromonylthiazolidine derivatives were successfully semi-synthesized from paeonol. All of the compounds, including starting materials, the intermediate compound and products, were evaluated for their cytotoxic effects toward eight human cancer cell lines. The synthesized chromonylthiazolidines displayed weak cytotoxic effects against the tested cancer cell lines, but selective cytotoxic effects were observed. Compounds 3a and 3b showed the most selective cytotoxic effects against human epidermoid carcinoma (IC50 44.1 ± 3.6 μg/mL and breast cancer (IC50 32.8 ± 1.4 μg/mL cell lines, respectively. The results suggest that chromoylthiazolidines are potential low-cost, and selective anticancer agents.

  4. Dose-dependent cytotoxic and mutagenic effects of antineoplastic alkylating agents on human lymphoblastoid cells

    Energy Technology Data Exchange (ETDEWEB)

    Sanderson, B.J.S.; Johnson, K.J.; Henner, W.D. (Oregon Health Sciences Univ., Portland (United States))

    1991-01-01

    The alkylating agents in clinical use as antineoplastics are strongly implicated as human carcinogens on the basis of animal studies and human epidemiologic studies. However, there is little quantitative information on the extent to which exposure to these drugs is mutagenic for normal (non-malignant) cells and the extent to which such mutagenicity correlates with cytotoxicity of these agents. Human lymphoblastoid cells (WIL2-NS) were exposed to graded doses of eight antineoplastic alkylating agents. Dose-dependent decreases in survival were used to calculate IC{sub 50}s for each of the drugs tested. The mutagenicity of these agents is correlated strongly with cytotoxicity. These results quantitate the dose-dependent cytotoxic and mutagenic effects of these bifunctional alkylating agents on human cells. All are cytotoxic and mutagenic, although their mutagenic efficiency varies.

  5. Cytotoxicity of methanol extracts of Elaeis guineensis on MCF-7 and Vero cell lines

    Institute of Scientific and Technical Information of China (English)

    Soundararajan; Vijayarathna; Sreenivasan; Sasidharan

    2012-01-01

    Objective:To investigate the cytotoxic effect of Elaeis guineensis methanol extract on MCF-7and Vero cell.Methods:In vitro cytotoxicity was evaluated in by MTT assay.Cell morphological changes were observed by using light microscope.Results:The MTT assay indicated that methanol extract of the plant exhibited significant cytotoxic effects on MCF-7.Morphological alteration of the cell lines after exposure with lilaeis guineensis extract were observed under phase contrast microscope in the dose dependent manner.Conclusions:The results suggest the probable use of the Elaeis guineensis methanol extract in preparing recipes for cancer-related ailments.Further studies on isolation of metabolites and their in vivo cytotoxicity are under investigation.

  6. Cytotoxic Effects of Strawberry, Korean Raspberry, and Mulberry Extracts on Human Ovarian Cancer A2780 Cells

    Science.gov (United States)

    Lee, Dahae; Kang, Ki Sung; Lee, Sanghyun; Cho, Eun Ju; Kim, Hyun Young

    2016-01-01

    Reactive oxygen species are tumorigenic by their ability to increase cell proliferation, survival, and cellular migration. The purpose of the present study was to compare the antioxidant activity and cytotoxic effects of 3 berry extracts (strawberry, Korean raspberry, and mulberry) in A2780 human ovarian carcinoma cells. Except for raspberry, the ethyl acetate or methylene chloride fractions of berries containing phenolic compounds exerted dose dependent free radical scavenging activities. In the raspberry fractions, the hexane fraction also exhibited potent antioxidant activity. The cytotoxic effects of berries extracts in A2780 human ovarian carcinoma cells were measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Surprisingly, co-treatment with n-butanol (BuOH) fractions of berries showed stronger cytotoxic effects compared to the other fractions. These findings suggest that potent anticancer molecules are found in the BuOH fractions of berries that have stronger cytotoxic activity than antioxidants. PMID:28078263

  7. Cytotoxic and apoptotic effects of six herbal plants against the human hepatocarcinoma (HepG2 cell line

    Directory of Open Access Journals (Sweden)

    Nonpunya Apiyada

    2011-10-01

    Full Text Available Abstract Background Six plants from Thailand were evaluated for their cytotoxicity and apoptosis induction in human hepatocarcinoma (HepG2 as compared to normal African green monkey kidney epithelial cell lines. Methods Ethanol-water crude extracts of the six plants were tested with neutral red assay for their cytotoxicity after 24 hours of exposure to the cells. Apoptotic induction was tested in the HepG2 cells with diamidino-2-phenylindole staining. DNA fragmentation, indicative of apoptosis, was analyzed with agarose gel electrophoresis. Alkylation, indicative of DNA damage, was also evaluated in vitro by 4-(4'-nitrobenzyl pyridine assay. Results The extract of Pinus kesiya showed the highest selectivity (selectivity index = 9.6 and potent cytotoxicity in the HepG2 cell line, with an IC50 value of 52.0 ± 5.8 μg/ml (mean ± standard deviation. Extract of Catimbium speciosum exerted cytotoxicity with an IC50 value of 55.7 ± 8.1 μg/ml. Crude extracts from Glochidion daltonii, Cladogynos orientalis, Acorus tatarinowii and Amomum villosum exhibited cytotoxicity with IC50 values ranging 100-500 μg/ml. All crude extracts showed different alkylating abilities in vitro. Extracts of P. kesiya, C. speciosum and C. orientalis caused nuclei morphological changes and DNA laddering. Conclusion The extracts of C. speciosum, C. orientalis and P. kesiya induced apoptosis. Among the three plants, P. kesiya possessed the most robust anticancer activity, with specific selectivity against HepG2 cells.

  8. Endothelial cell cytotoxicity of cotton bracts tannin and aqueous cotton bracts extract

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    Johnson, C.M.; Hanson, M.N.; Rohrbach, M.S.

    1986-04-01

    Using an in vitro cytotoxicity assay based on the release of /sup 51/Cr from cultured porcine thoracic aortic and pulmonary arterial endothelial cells, we have demonstrated that cotton bracts tannin is a potent endothelial cell cytotoxin. It produces dose-dependent lethal injury to both types of endothelial cells with the aortic cells, being somewhat more sensitive to tannin-mediated injury than the pulmonary arterial cells. Cytotoxic injury to the cells was biphasic. During the first 3 hr of exposure to tannin, no lethal injury was detected. However, during this period, profound changes in morphology were observed suggesting sublethal injury to the cells preceded the ultimate toxic damage. Comparison of the cytotoxicity dose curves for aqueous bracts extracts with those for tannin demonstrated that tannin was major cytotoxin present in bracts.

  9. Cytotoxicity of arctigenin and matairesinol against the T-cell lymphoma cell line CCRF-CEM.

    Science.gov (United States)

    Su, Shan; Cheng, Xinlai; Wink, Michael

    2015-09-01

    Arctigenin and matairesinol possess a diversity of bioactivities. Here we investigated the cytotoxicity of arctigenin and matairesinol against a T-cell lymphoma cell line CCRF-CEM and the underlying mechanisms that have not been explored before. The cytotoxic activity was investigated using MTT assay. The cell cycle arrest and reactive oxygen species (ROS) accumulation were determined by flow cytometric analysis. The apoptosis induction was assessed using Annexin V/Propidium Iodide assay. The gene quantification analysis was measured through real-time polymerase chain reaction. Arctigenin and matairesinol exhibited significant antiproliferative activity against CCRF-CEM cells after 72 h treatment with IC50 values of 1.21 ± 0.15 μm and 4.27 ± 0.41 μm, respectively. In addition, both lignans arrest CCRF-CEM cells in the S phase. Furthermore, they could induce apoptosis in CCRF-CEM cells in a concentration- and time-dependent manner. Interestingly, the lignans differentially regulated the expression of several key genes involved in apoptosis pathways, including Bax, Bad and caspase-9. Moreover, both lignans could increase ROS levels in CCRF-CEM cells. Our study provides an insight into the potential of arctigenin and matairesinol as good candidates for the development of novel agents against T-cell lymphoma. © 2015 Royal Pharmaceutical Society.

  10. Relation between parthenolideinduced cytotoxicity and COX—2 expression in nasopharyngeal carcinoma cell

    Institute of Scientific and Technical Information of China (English)

    LinYL; OngCN

    2002-01-01

    To investigate the potential anti-tumor effects and its relative its relative mechanism of active component in chrysanthemum,on human nasopharyngeal carcinoma(NPC).Parthenolide(PN) was used as tested substance and exposed to NPC cell lines,CNE1 and CNE2.Cell toxicity indexes and cyclooxygenase-2(COX-2) expression were determined.Results showed that in CNE1 cells,with high level COX-2,there were significant cytotoxicities after PN-treated.However,there were not inducible effects on CNE2 cells with low level COX-2.It was indicated that PN could induce cytotoxicity in NPC cell,which correlated with its intracellular COX-2 expression.

  11. Cytotoxicity Induced by a Redox-silent Analog of Tocotrienol in Human Mesothelioma H2452 Cell Line via Suppression of Cap-dependent Protein Translation.

    Science.gov (United States)

    Sato, Ayami; Ueno, Haruka; Takase, Akari; Ando, Akira; Sekine, Yuko; Yano, Tomohiro

    2016-04-01

    De novo synthesis of proteins is regulated by cap-dependent protein translation. Aberrant activation of the translation is a hallmark of many cancer types including malignant mesothelioma (MM). We previously reported that a redox-silent analog of α-tocotrienol, 6-O-carboxypropyl-α-tocotrienol (T3E) induces potent cytotoxicity against human MM cells. However, the detailed mechanism of cytotoxicity of T3E remains unclear. In this study, we investigated if T3E induced potent cytotoxicity aganist MM cells. T3E reduced the formation of the cap-dependent translation complex and induced inactivation of oncogene from rat sarcoma virus (RAS). These events were associated with T3E cytotoxicity in MM cells. Furthermore, atorvastatin, an inhibitor of RAS function, had similar effects on MM cells. Moreover, 4EGI-1, a specific inhibitor of the cap-dependent translation complex, induced severe cytotoxicity in MM cells. Overall, T3E had a cytotoxic effect on MM cells via disruption of the activated cap-dependent translation complex through inactivation of RAS.

  12. Cytotoxicity and inhibitory effects of low-concentration triclosan on adipogenic differentiation of human mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Li-Wu [Division of Personalized Nutrition and Medicine, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079 (United States); Wu, Qiangen [Division of Biochemical Toxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079 (United States); Green, Bridgett; Nolen, Greg [Division of Personalized Nutrition and Medicine, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079 (United States); Shi, Leming [Division of Systems Biology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079 (United States); LoSurdo, Jessica [Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892 (United States); Deng, Helen [Arkansas Department of Health, Little Rock, AR 72205 (United States); Bauer, Steven [Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892 (United States); Fang, Jia-Long, E-mail: jia-long.fang@fda.hhs.gov [Division of Biochemical Toxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079 (United States); Ning, Baitang, E-mail: baitang.ning@fda.hhs.gov [Division of Personalized Nutrition and Medicine, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079 (United States)

    2012-07-15

    Humans at all ages are continually exposed to triclosan (TCS), a widely used antimicrobial agent that can be found in many daily hygiene products, such as toothpastes and shampoos; however, the toxicological and biological effects of TCS in the human body after long-term and low-concentration exposure are far from being well understood. In the current study, we investigated the effects of TCS on the differentiation of human mesenchymal stem cells (hMSCs) by measuring the cytotoxicity, morphological changes, lipid accumulation, and the expression of adipocyte differentiation biomarkers during 21-day adipogenesis. Significant cytotoxicity was observed in un-induced hMSCs treated with high-concentration TCS (≥ 5.0 μM TCS), but not with low-concentration treatments (≤ 2.5 μM TCS). TCS inhibited adipocyte differentiation of hMSCs in a concentration-dependent manner in the 0.156 to 2.5 μM range as indicated by morphological changes with Oil Red O staining, which is an index of lipid accumulation. The inhibitory effect was confirmed by a decrease in gene expression of specific adipocyte differentiation biomarkers including adipocyte protein 2, lipoprotein lipase, and adiponectin. Our study demonstrates that TCS inhibits adipocyte differentiation of hMSCs under concentrations that are not cytotoxic and in the range observed in human blood. -- Highlights: ► TCS is cytotoxic to un-induced hMSCs at concentrations ≥ 5.0 μM. ► TCS at concentrations ≤ 2.5 μM is not cytotoxic to induced hMSCs. ► TCS at non-cytotoxic concentrations inhibits lipid formation in induced hMSCs. ► TCS decreases the expression of specific biomarkers of adipocyte differentiation. ► TCS at concentrations observed in human blood inhibits adipogenesis of hMSCs.

  13. Natural mineral particles are cytotoxic to rainbow trout gill epithelial cells in vitro.

    Science.gov (United States)

    Michel, Christian; Herzog, Simon; de Capitani, Christian; Burkhardt-Holm, Patricia; Pietsch, Constanze

    2014-01-01

    Worldwide increases in fluvial fine sediment are a threat to aquatic animal health. Fluvial fine sediment is always a mixture of particles whose mineralogical composition differs depending on the sediment source and catchment area geology. Nonetheless, whether particle impact in aquatic organisms differs between mineral species remains to be investigated. This study applied an in vitro approach to evaluate cytotoxicity and uptake of four common fluvial mineral particles (quartz, feldspar, mica, and kaolin; concentrations: 10, 50, 250 mg L(-1)) in the rainbow trout epithelial gill cell line RTgill-W1. Cells were exposed for 24, 48, 72, and 96 h. Cytotoxicity assays for cell membrane integrity (propidium iodide assay), oxidative stress (H2DCF-DA assay), and metabolic activity (MTT assay) were applied. These assays were complemented with cell counts and transmission electron microscopy. Regardless of mineral species, particles ≤ 2 µm in diameter were taken up by the cells, suggesting that particles of all mineral species came into contact and interacted with the cells. Not all particles, however, caused strong cytotoxicity: Among all assays the tectosilicates quartz and feldspar caused sporadic maximum changes of 0.8-1.2-fold compared to controls. In contrast, cytotoxicity of the clay particles was distinctly stronger and even differed between the two particle types: mica induced concentration-dependent increases in free radicals, with consistent 1.6-1.8-fold-changes at the 250 mg L(-1) concentration, and a dilated endoplasmic reticulum. Kaolin caused concentration-dependent increases in cell membrane damage, with consistent 1.3-1.6-fold increases at the 250 mg L(-1) concentration. All effects occurred in the presence or absence of 10% fetal bovine serum. Cell numbers per se were marginally affected. Results indicate that (i.) natural mineral particles can be cytotoxic to gill epithelial cells, (ii.) their cytotoxic potential differs between mineral species, with

  14. Natural mineral particles are cytotoxic to rainbow trout gill epithelial cells in vitro.

    Directory of Open Access Journals (Sweden)

    Christian Michel

    Full Text Available Worldwide increases in fluvial fine sediment are a threat to aquatic animal health. Fluvial fine sediment is always a mixture of particles whose mineralogical composition differs depending on the sediment source and catchment area geology. Nonetheless, whether particle impact in aquatic organisms differs between mineral species remains to be investigated. This study applied an in vitro approach to evaluate cytotoxicity and uptake of four common fluvial mineral particles (quartz, feldspar, mica, and kaolin; concentrations: 10, 50, 250 mg L(-1 in the rainbow trout epithelial gill cell line RTgill-W1. Cells were exposed for 24, 48, 72, and 96 h. Cytotoxicity assays for cell membrane integrity (propidium iodide assay, oxidative stress (H2DCF-DA assay, and metabolic activity (MTT assay were applied. These assays were complemented with cell counts and transmission electron microscopy. Regardless of mineral species, particles ≤ 2 µm in diameter were taken up by the cells, suggesting that particles of all mineral species came into contact and interacted with the cells. Not all particles, however, caused strong cytotoxicity: Among all assays the tectosilicates quartz and feldspar caused sporadic maximum changes of 0.8-1.2-fold compared to controls. In contrast, cytotoxicity of the clay particles was distinctly stronger and even differed between the two particle types: mica induced concentration-dependent increases in free radicals, with consistent 1.6-1.8-fold-changes at the 250 mg L(-1 concentration, and a dilated endoplasmic reticulum. Kaolin caused concentration-dependent increases in cell membrane damage, with consistent 1.3-1.6-fold increases at the 250 mg L(-1 concentration. All effects occurred in the presence or absence of 10% fetal bovine serum. Cell numbers per se were marginally affected. Results indicate that (i. natural mineral particles can be cytotoxic to gill epithelial cells, (ii. their cytotoxic potential differs between mineral

  15. 重组融合蛋白C-CPE-ETA′对卵巢癌细胞的细胞毒作用%Target-specific cytotoxic activity of recombinant fusion toxin C-CPE-ETA′ against CLDN-3, 4-overexpressing ovarian cancer cells

    Institute of Scientific and Technical Information of China (English)

    姚勤; 郑庆梅; 温君凤; 吕腾; 魏明谦; 戴淑真

    2010-01-01

    Objective The aim of this study was to explore the possibility of creating a toxin,C-CPE-ETA′, by fusing C-terminal high affinity binding domain of CPE(C-CPE)with a truncated form of Pseudomonas aeruginosa exotoxin A(ETA')and to examine whether C-CPE-ETA' could specifically target CLDN-3,4 molecule and the targeted toxin was cytotoxic against CLDN-3,4-overexpressing ovarian cancer.Methods CLDN-3 and CLDN-4 expressions were analyzed at the mRNA level in three ovarian cancer cell liues and epithelial ovarian cancer tissues from 20 patients. After transforming an expression plasmid of C-CPE-ETA′ into E. coli BL21(DE3)plysS strain, the recombinant protein was purified using His-Bind resin chromatography column and analyzed by Western blot and Coomassie blue staining. The specific binding, proapoptotic and cytolytic activities were evaluated by flow cytometry, fluorescence microscopy with the JC-1 probe and MTT assay in CLDN-3,4-overexpressing ovarian cancer cells. Results Quantitive RT-PCR results showed there existed high levels of CLDN-3 and CLDN-4 in ovarian cancer cells, CAOV3,OVCAR3 and SKOV3. Moreover, high expressions of CLDN-3 and CLDN-4 were observed in 90.0%(18/20)and 60.0%(12/20)of ovarian cancer tissues, with an expression level 10-fold higher than that in the normal ovarian tissue. A 58 000 recombinant protein C-CPE-ETA′ was demonstrated by Western blot and Coomassie blue staining. Purified and recombinant C-CPE-ETA' was bound with high affinity to CLDN-3,4-overexpressing ovarian cancer cells, CAOV3, OVCAR3 and SKOV3 cells. C-CPE-ETA′ was strongly proapoptotic and cytotoxic towards the CLDN-3,4-overexpressing ovarian cancer cells. The concentration of IC50 was 7. 364 ng/ml for CAOV3 cells, 8.110 ng/ml for OVCAR3 cells and 22. 340 ng/ml for SKOV3cells, respectively. However, control CLDN-3,4-deficient cell line HUVEC was not susceptible to the recombinant C-CPE-ETA' at a concentration up to 10 μg/ml. Conclusions The C-CPE-ETA' protein exhibits

  16. Phytochemicals and Cytotoxicity of Launaea procumbens on Human Cancer Cell Lines

    Science.gov (United States)

    Rawat, Preeti; Saroj, Lokesh M.; Kumar, Anil; Singh, Tryambak D.; Tewari, SK.; Pal, Mahesh

    2016-01-01

    Background: The plant Launaea procumbens belongs to the family Asteraceae and traditionally used in the treatment rheumatism, kidney, liver dysfunctions and eye diseases. In the present study Phytochemical analysis and fractions of methanolic extract of L. procumbens leaves were tested in vitro for their cytotoxicity. Objectives: Phytochemical analysis and cytotoxic activity of methanolic extract and fractions of Launaea procumbens against four cancer cell lines K562, HeLa, MIA-Pa-Ca-2 and MCF-2 by SRB assay. Materials and Methods: Powdered leaves of Launaea procumbens were extracted sequentially with hexane, ethyl acetate, butanol and water by cold extraction. Phytochemical analysis and cytotoxicity assay were carried out for these fractions using SRB assay against four human cancer cell lines, namely leukemia (K562), cervix (HeLa), pancreatic (MIA-Pa-Ca-2) and breast (MCF-7). Results: Ethyl acetate extract exerts potent cytotoxicity against human leukemia (K562), cervix (HeLa) and breast (MCF-7) cell lines IC50 value of 25.30±0.50, 19.80±0.10 and 36.90±4.90 μg/ml respectively. Moderately cytotoxic effect found in hexane extract IC50 value of 41±8 and 48.20±0.50 μg/ml against leukemia (K562), and breast (MCF-7) cancer cell line respectively. The Chemical composition analyzed by GC-MS showed considerable differences in solvent fractions of Launaea procumbens. Conclusion: This study revealed the cytotoxic potential of ethyl acetate and hexane fractions of L. procumbens leaves on different cancer cell lines. SUMMARY Ethyl acetate and Hexane fractions of Launaea procumbens plant exhibit cytotoxicity. Among the different fractions Ethyl acetate showed relatively higher cytotoxicity.Ethyl acetate found more cytotoxic against leukemia (K 562), cervix (HeLa) and breast (MCF-7) cancer cell lines. Moderete cytotoxicity found in hexane fraction against leukemia (K 562) and breast (MCF-7) cancer cell line.GC-MS results showed L. procumbens is a rich source of 1-H

  17. Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity

    Directory of Open Access Journals (Sweden)

    Tana A. Omokoko

    2016-01-01

    Full Text Available Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard 51Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the 51Cr release and further confirmed the assay’s ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay’s combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies.

  18. Cytotoxic and apoptotic effects of chalcone derivatives of 2-acetyl thiophene on human colon adenocarcinoma cells.

    Science.gov (United States)

    de Vasconcelos, Alana; Campos, Vinicius Farias; Nedel, Fernanda; Seixas, Fabiana Kömmling; Dellagostin, Odir A; Smith, Kevin R; de Pereira, Cláudio Martin Pereira; Stefanello, Francieli Moro; Collares, Tiago; Barschak, Alethéa Gatto

    2013-06-01

    Recent studies report that chalcones exhibit cytotoxicity to human cancer cell lines. Typically, the form of cell death induced by these compounds is apoptosis. In the context of the discovery of new anticancer agents and in light of the antitumour potential of several chalcone derivatives, in the present study, we synthesized and tested the cytotoxicity of six chalcone derivatives on human colon adenocarcinoma cells. Six derivatives of 3-phenyl-1-(thiophen-2-yl) prop-2-en-1-one were prepared and characterized on the basis of their (1) H and (13) C NMR spectra. HT-29 cells were treated with synthesized chalcones on two concentrations by three different incubation times. Cells were evaluated by cell morphology, Tetrazolium dye (MTT) colorimetric assay, live/dead, flow cytometry (annexin V) and gene expression analyses to determine the cytotoxic way. Chalcones 3-(4-bromophenyl)-1-(thiophen-2-yl)prop-2-en-1-one (C06) and 3-(2-nitrophenyl)-1-(thiophen-2-yl)prop-2-en-1-one (C09) demonstrated higher cytotoxicity than other chalcones as shown by cell morphology, live/dead and MTT assays. In addition, C06 induced apoptosis on flow cytometry annexin V assay. These data were confirmed by a decreased expression of anti-apoptotic genes and increased pro-apoptotic genes. Our findings indicate in summary that the cytotoxic activity of chalcone C06 on colorectal carcinoma cells occurs by apoptosis.

  19. Cytotoxicity of Thymus vulgaris essential oil towards human oral cavity squamous cell carcinoma.

    Science.gov (United States)

    Sertel, Serkan; Eichhorn, Tolga; Plinkert, Peter K; Efferth, Thomas

    2011-01-01

    Oral cavity squamous cell carcinoma (OCSCC) accounts for 2% to 3% of all malignancies and has a high mortality rate. The majority of anticancer drugs are of natural origin. However, it is unknown whether the medicinal plant Thymus vulgaris L. (thyme) is cytotoxic towards head and neck squamous cell carcinoma (HNSCC). Cytotoxicity of thyme essential oil was investigated on the HNSCC cell line, UMSCC1. The IC₅₀ of thyme essential oil extract was 369 μg/ml. Moreover, we performed pharmacogenomics analyses. Genes involved in the cell cycle, cell death and cancer were involved in the cytotoxic activity of thyme essential oil at the transcriptional level. The three most significantly regulated pathways by thyme essential oil were interferon signaling, N-glycan biosynthesis and extracellular signal-regulated kinase 5 (ERK5) signaling. Thyme essential oil inhibits human HNSCC cell growth. Based on pharmacogenomic approaches, novel insights into the molecular mode of anticancer activity of thyme are presented.

  20. Cytotoxic effect of Cousinia verbascifolia Bunge against OVCAR-3 and HT-29 cancer cells

    Directory of Open Access Journals (Sweden)

    Sajjadi Seyed Ebrahim

    2015-01-01

    Full Text Available Introduction: Little information is available about phytochemical and biological properties of Cousinia genus. In a primary study, seven Cousinia species including C. verbascifolia showed cytotoxic activity ranged between 18.4 ± 0.59 to 87.9 ± 0.58 μg/mL. To the best of our knowledge, no other biological studies have been conducted on this plant. Therefore, in this study the cytotoxic effect of Cousinia verbascifolia Bunge against OVCAR-3 and HT-29 cancer cells was evaluated. Methods: Filtration and in vacuo concentration of methanol extract resulted in a green gum which was subjected on reverse column chromatography. Semi polar fraction (41.3 g eluted with water: methanol (20:80, was then subjected on a silica gel column chromatography using hexane/acetone and resulted in 11 fractions. Finally, cytotoxic activities against ovarian and colon cancer cells were determined at a wavelength of 570 nm by Matrix metalloproteinase protein (MTT standard method. Results: None of the fractions showed highly cytotoxic activity. Based on NCI, fractions Fr. 1, Fr. 2, Fr. 4, Fr. 5, Fr. 6, Fr. 8 and Fr. 10 showed moderately cytotoxicity with IC50 values ranged between 119 to 190 μg/mL against OVCAR-3 cells. Fractions Fr. 1, Fr. 2, Fr. 6, Fr. 7 and Fr. 8 showed moderately cytotoxic activity ranged between 118 to 194 μg/mL against HT-29 cells. Fr. 10 and Fr. 11 showed no cytotoxic activity. Conclusion: Due to the inhibitory properties of extract and its fractions on cancer cells, identification of responsible compounds possessing cytotoxic effects for generating possible new approach in medicinal chemistry are recommended.

  1. Differential cytotoxic effects of gold nanoparticles in different mammalian cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Chueh, Pin Ju [Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan (China); Graduate Institute of Basic Medicine, China Medical University, Taichung 40402, Taiwan (China); Department of Medical Research, China Medical University Hospital, Taichung 40402, Taiwan (China); Department of Biotechnology, Asia University, Taichung 41354, Taiwan (China); Liang, Ruei-Yue; Lee, Yi-Hui; Zeng, Zih-Ming [Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan (China); Chuang, Show-Mei, E-mail: smchuang@dragon.nchu.edu.tw [Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan (China)

    2014-01-15

    Highlights: • AuNPs induce apoptosis in Vero cells. • AuNPs-induced attenuation of cell growth in NIH3T3 cells through autophagy. • Cell-cycle delay was associated with the resistance to AuNPs in MRC-5 cells. • Cell growth was continuously monitored using the measurement of cell impedance. -- Abstract: Gold nanoparticles (AuNPs) possess unique properties that have been exploited in several medical applications. However, a more comprehensive understanding of the environmental safety of AuNPs is imperative for use of these nanomaterials. Here, we describe the impacts of AuNPs in various mammalian cell models using an automatic and dye-free method for continuous monitoring of cell growth based on the measurement of cell impedance. Several well-established cytotoxicity assays were also used for comparison. AuNPs induced a concentration-dependent decrease in cell growth. This inhibitory effect was associated with apoptosis induction in Vero cells but not in MRC-5 or NIH3T3 cells. Interestingly, cDNA microarray analyses in MRC-5 cells supported the involvement of DNA damage and repair responses, cell-cycle regulation, and oxidative stress in AuNP-induced cytotoxicity and genotoxicity. Moreover, autophagy appeared to play a role in AuNPs-induced attenuation of cell growth in NIH3T3 cells. In this study, we present a comprehensive overview of AuNP-induced cytotoxicity in a variety of mammalian cell lines, comparing several cytotoxicity assays. Collectively, these assays offer convincing evidence of the cytotoxicity of AuNPs and support the value of a systematic approach for analyzing the toxicology of nanoparticles.

  2. Cytotoxicity of Selected Medicinal and Nonmedicinal Plant Extracts to Microbial and Cervical Cancer Cells

    Directory of Open Access Journals (Sweden)

    Gary M. Booth

    2012-01-01

    Full Text Available This study investigated the cytotoxicity of 55 species of plants. Each plant was rated as medicinal, or nonmedicinal based on the existing literature. About 79% of the medicinal plants showed some cytotoxicity, while 75% of the nonmedicinal plants showed bioactivity. It appears that Asteraceae, Labiatae, Pinaceae, and Chenopodiaceae were particularly active against human cervical cancer cells. Based on the literature, only three of the 55 plants have been significantly investigated for cytotoxicity. It is clear that there is much toxicological work yet to be done with both medicinal and nonmedicinal plants.

  3. Ex vivo induction of anti-leukemia cytotoxic T cell effect by dendritic cells from human umbilical cord blood cell origin.

    Science.gov (United States)

    Tan, Huo; Zeng, Lin-Juan; Ye, Xu

    2005-06-01

    To explore the possibility of in vitro induction of cord blood cell-derived lymphocytes into cytotoxic T lymphocytes (CTL) with anti-leukemia specificity, umbilical cord blood (UCB)-derived mononuclear cells were cultured with multiple cytokines to generate dendritic cells (DC) in vitro. Leukemia cells were irradiated with (137)Cs and activated by premature cytokines. The characteristics of maturation of DC were evaluated through morphology examination and flow cytometry. DC pulsed with leukemic antigens were co-cultured with lymphocytes. Cytotoxicity of the CTL to corresponding leukemic cells was measured with lactate dehydrogenase-release assay. The results showed that UCB-derived monocytes could be induced into typical DC in all of the 12 samples. Expression of immunological markers such as CD1a(+), HLA-DR(+), CD86(+), CD83(+) on DC were significantly up-regulated (P CTL with a killing rate of (44.76 +/- 17.42)% at the E:T ratio of 50:1 against AML cells and a killing rate of (8.50 +/- 4.25)% at the E:T ratio of 50:1 against ALL cells. Whereas, these CTL present almost no killing effect on the mononuclear cells collected from the same patients in complete remission phase. It is concluded that (1) it is possible to induce UCB-derived monocytes into mature DC with typical morphology. (2) Cord blood derived mature DC presenting leukemia antigen can generate leukemia-specific CTL with vigorous cytotoxic activity against the same leukemia blasts and low killing activity against bone marrow cells of the same patients in complete remission phase.

  4. Mechanisms for high methoxymorpholino doxorubicin cytotoxicity in doxorubicin-resistant tumor cell lines

    NARCIS (Netherlands)

    Bakker, M; Renes, J; Groenhuijzen, A; Visser, P; TimmerBosscha, H; Muller, M; Groen, HJM; Smit, EF; deVries, EGE

    1997-01-01

    Methoxymorpholino doxorubicin (MMRDX) is an anthracycline analogue that is able to overcome tumor cell resistance to classical anthracyclines. Mechanisms for increased MMRDX cytotoxicity were analyzed in a small cell lung carcinoma cell line (GLC(4)), its 300-fold doxorubicin-resistant and multidrug

  5. HLA-G inhibits xenogenetic cytotoxicity mediated by human NK cells and T lymphocytes against PECs

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    In order to investigate whether the non-classi-cal HLA-G classⅠmolecule protects the porcine endothelial cells (PECs) from the lysis mediated by human immune cells in pig to human discordant xenotransplantation, we have cloned HLA-G cDNA from a human placenta by RT-PCR. Mammalian expression vector, pEFG-neo, was constructed by insertion of HLA-G cDNA in pEF-neo. We obtained efficiently expressed PECs by stable transfection. Cytotoxicity assay showed that overexpression of HLA-G on PECs was sufficient to inhibit human NK-92 cell lysis. The level of lysis was equal to or less than that of the lysis of human umbilical vein endothelial cells mediated by human NK-92 cells. It also indicated that HLA-G inhibited the lysis of PECs mediated by xeno-antigen specific T lymphocytes. The reduction of lysis ranged between 59.1% and 88.9%. These findings suggest that the transgenic approach to overexpress HLA-G is believed to be a new immunotherapy in overcoming the immune rejections in xenotransplantion, including delayed xenograft rejection and cell-mediated rejection.

  6. Analysis of the cytotoxicity of carbon-based nanoparticles, diamond and graphite, in human glioblastoma and hepatoma cell lines

    DEFF Research Database (Denmark)

    Zakrzewska, Karolina Ewa; Samluk, Anna; Wierzbicki, Mateusz

    2015-01-01

    Nanoparticles have attracted a great deal of attention as carriers for drug delivery to cancer cells. However, reports on their potential cytotoxicity raise questions of their safety and this matter needs attentive consideration. In this paper, for the first time, the cytotoxic effects of two...... are suitable for drug cytotoxicity tests....

  7. Editor's Highlight: Analysis of the Effects of Cell Stress and Cytotoxicity on In Vitro Assay Activity Across a Diverse Chemical and Assay Space.

    Science.gov (United States)

    Judson, Richard; Houck, Keith; Martin, Matt; Richard, Ann M; Knudsen, Thomas B; Shah, Imran; Little, Stephen; Wambaugh, John; Woodrow Setzer, R; Kothya, Parth; Phuong, Jimmy; Filer, Dayne; Smith, Doris; Reif, David; Rotroff, Daniel; Kleinstreuer, Nicole; Sipes, Nisha; Xia, Menghang; Huang, Ruili; Crofton, Kevin; Thomas, Russell S

    2016-08-01

    Chemical toxicity can arise from disruption of specific biomolecular functions or through more generalized cell stress and cytotoxicity-mediated processes. Here, responses of 1060 chemicals including pharmaceuticals, natural products, pesticidals, consumer, and industrial chemicals across a battery of 815 in vitro assay endpoints from 7 high-throughput assay technology platforms were analyzed in order to distinguish between these types of activities. Both cell-based and cell-free assays showed a rapid increase in the frequency of responses at concentrations where cell stress/cytotoxicity responses were observed in cell-based assays. Chemicals that were positive on at least 2 viability/cytotoxicity assays within the concentration range tested (typically up to 100 μM) activated a median of 12% of assay endpoints whereas those that were not cytotoxic in this concentration range activated 1.3% of the assays endpoints. The results suggest that activity can be broadly divided into: (1) specific biomolecular interactions against one or more targets (eg, receptors or enzymes) at concentrations below which overt cytotoxicity-associated activity is observed; and (2) activity associated with cell stress or cytotoxicity, which may result from triggering specific cell stress pathways, chemical reactivity, physico-chemical disruption of proteins or membranes, or broad low-affinity non-covalent interactions. Chemicals showing a greater number of specific biomolecular interactions are generally designed to be bioactive (pharmaceuticals or pesticidal active ingredients), whereas intentional food-use chemicals tended to show the fewest specific interactions. The analyses presented here provide context for use of these data in ongoing studies to predict in vivo toxicity from chemicals lacking extensive hazard assessment.

  8. Immunoglobulin fragments, F(ab')2, that are cytotoxic to enzyme-treated cells.

    Science.gov (United States)

    Holtgrewe, E M; Killion, J J

    1984-07-01

    Bivalent immunoglobulin fragments of IgG, F(ab')2, prepared from normal murine sera were found to be cytotoxic to neuraminidase-treated cells. The fragments were cytotoxic to both allogenic and syngeneic targets (with respect to the source of the sera), suggesting that the antigen bound by the F(ab')2 is not related to the major histocompatibility locus of mice (H-2).

  9. Cellular uptake and cytotoxicity of positively charged chitosan gold nanoparticles in human lung adenocarcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Seon Young; Jang, Soo Hwa [Seoul National University, Laboratory of Veterinary Pharmacology, College of Veterinary Medicine and Institute for Veterinary Science (Korea, Republic of); Park, Jin; Jeong, Saeromi; Park, Jin Ho; Ock, Kwang Su [Soongsil University, Department of Chemistry (Korea, Republic of); Lee, Kangtaek [Yonsei University, Department of Chemical and Biomolecular Engineering (Korea, Republic of); Yang, Sung Ik [Kyung Hee University, College of Environment and Applied Chemistry (Korea, Republic of); Joo, Sang-Woo, E-mail: sjoo@ssu.ac.kr [Soongsil University, Department of Chemistry (Korea, Republic of); Ryu, Pan Dong; Lee, So Yeong, E-mail: leeso@snu.ac.kr [Seoul National University, Laboratory of Veterinary Pharmacology, College of Veterinary Medicine and Institute for Veterinary Science (Korea, Republic of)

    2012-12-15

    Cellular uptake, cytotoxicity, and mechanisms of cytotoxicity of the positively charged Au nanoparticles (NPs) were examined in A549 cells, which are one of the most characterized pulmonary cellular systems. Positively charged Au NPs were prepared by chemical reduction using chitosan. The dimension and surface charge of Au NPs were examined by transmission electron microscopy (TEM), dynamic light scattering, and zeta potential measurements. The uptake of Au NPs into A549 cells was also monitored using TEM and dark-field microscopy (DFM) and z-stack confocal microRaman spectroscopy. DFM live cell imaging was also performed to monitor the entry of chitosan Au NPs in real time. The cytotoxic assay, using both methylthiazol tetrazolium and lactate dehydrogenase assays revealed that positively charged Au NPs decreased cell viability. Flow cytometry, DNA fragmentation, real-time PCR, and western blot analysis suggest that positively charged chitosan Au NPs provoke cell damage through both apoptotic and necrotic pathways.

  10. Cyclamen exerts cytotoxicity in solid tumor cell lines: a step toward new anticancer agents?

    Science.gov (United States)

    Yildiz, Mustafa; Bozcu, Hakan; Tokgun, Onur; Karagur, Ege Riza; Akyurt, Oktay; Akca, Hakan

    2013-01-01

    Cyclamen coum is a traditional medicinal plant in the Turkey. Its anticancer properties and whether cyclamen extract induces any cytotoxicity in solid cancer cell lines have not been thoroughly investigated previously. Therefore we examined cytotoxic effects on cervical cancer, HeLa, and non small cell lung cancer cell, H1299, lines. Cyclamen extract induced cellular death of both HeLa and H1299 cells in a dose dependent manner. We also analyzed the capacity of cyclamen extract to induce apoptosis by the TUNEL method. Here, we for the first time report that the extract of Cyclamen coum, an endemic plant for Turkey, can induce cytotoxicity via apoptosis in HeLa and H1299 cells. These results imply that cyclamen extract can be further analyzed to potentially find novel anticancer compounds.

  11. Paclitaxel augments cytotoxic effect of photodynamic therapy using verteporfin in gastric and bile duct cancer cells.

    Science.gov (United States)

    Park, Seungwoo; Hong, Sung Pil; Oh, Tae Yoon; Bang, Seungmin; Chung, Jae Bock; Song, Si Young

    2008-07-01

    Photodynamic therapy (PDT) shows a limited antitumor effect in treating gastrointestinal tumors because of improper light penetration or insufficient photosensitizer uptake. The aim of this study was to evaluate the cytotoxic effect of PDT combined with paclitaxel on in vitro cancer cells. In vitro photodynamic therapy was performed in gastric cancer cells (NCI-N87) and bile duct cancer cells (YGIC-6B) using verteporfin (2 ug mL(-1)) and a PTH light source (1 000 W, Oriel Co.) with 665-675 nm narrow band pass filter. Cytotoxicity was compared using the MTT assay between cancer cells treated with PDT alone or pretreated with paclitaxel (IC(25)). Apoptotic changes were evaluated using DAPI staining, DNA fragmentation analysis, Annexin V-FITC apoptosis assay, cell cycle analysis, and western blots for cytochrome c, Bax, and Bid. The PDT-induced cytotoxicity was potentiated by pretreating with low dose paclitaxel (P cancer therapy.

  12. Cytotoxicity of various chemicals and mycotoxins in fresh primary duck embryonic fibroblasts: a comparison to HepG2 cells.

    Science.gov (United States)

    Chen, Xi; Murdoch, Rhonda; Shafer, Daniel J; Ajuwon, Kolapo M; Applegate, Todd J

    2016-11-01

    To screen cost-effectively the overall toxicity of a sample, particularly in the case of food and feed ingredient quality control, a sensitive bioassay is necessary. With the wide variety of cytotoxicity assays, performance comparison between assays using different cells has become of interest. Fresh primary duck embryonic fibroblasts (DEF) were hypothesized to be a sensitive tool for in vitro cytotoxicity screening; cell viability of DEF in response to various cytotoxins was determined and compared with response of HepG2 cells. The IC50 values by the alamar blue assay in the DEF cells had a high correlation (R(2)  = 0.96) with those obtained in HepG2 cells. Within the same toxin, primary DEF yielded significantly lower IC50 values than that obtained from HepG2 cells using the MTT and alamar blue assay. Additionally, primary DEF responded to all mycotoxins tested using the alamar blue assay, while HepG2 was less sensitive, particularly at short exposure times. The estimated IC50 for aflatoxin B1 , fumonisins B1 and deoxynivalenol in DEF after 72 h incubation were 3.69, 4.19 and 1.26 μg ml(-1) , respectively. Results from the current study suggest that primary DEF are more sensitive to cytotoxins and mycotoxins compared to HepG2, and thus may have great potential as an effective tool for cytotoxicity assessment. The question remains whether in vitro IC50 values can accurately predict in vivo toxicity; however, the current study accentuates the need for further attention to identify sensitive cell models for in vitro cytotoxicity screening and subsequent exploration of species-specific prediction models for in vivo toxicity. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  13. Spontaneous presence of FOXO3-specific T cells in cancer patients

    DEFF Research Database (Denmark)

    Larsen, Stine Kiaer; Ahmad, Shamaila Munir; Idorn, Manja

    2014-01-01

    In the present study, we describe forkhead box O3 (FOXO3)-specific, cytotoxic CD8(+) T cells existent among peripheral-blood mononuclear cells (PBMCs) of cancer patients. FOXO3 immunogenicity appears specific, as we did not detect reactivity toward FOXO3 among T cells in healthy individuals. FOXO3...... may naturally serve as a target antigen for tumor-reactive T cells as it is frequently over-expressed in cancer cells. In addition, expression of FOXO3 plays a critical role in immunosuppression mediated by tumor-associated dendritic cells (TADCs). Indeed, FOXO3-specific cytotoxic T lymphocytes (CTLs......) were able to specifically recognize and kill both FOXO3-expressing cancer cells as well as dendritic cells. Thus, FOXO3 was processed and presented by HLA-A2 on the cell surface of both immune cells and cancer cells. As FOXO3 programs TADCs to become tolerogenic, FOXO3 signaling thereby comprises...

  14. Specific nature of Trichomonas vaginalis parasitism of host cell surfaces.

    Science.gov (United States)

    Alderete, J F; Garza, G E

    1985-01-01

    The adherence of Trichomonas vaginalis NYH 286 to host cells was evaluated by using monolayer cultures of HeLa and HEp-2 epithelial cells and human fibroblast cell lines. Saturation of sites on HeLa cells was achieved, yielding a maximal T. vaginalis NYH 286-to-cell ratio of two. The ability of radiolabeled NYH 286 to compete with unlabeled trichomonads for attachment and the time, temperature, and pH-dependent nature of host cell parasitism reinforced the idea of specific parasite-cell associations. Other trichomonal isolates (JH31A, RU375, and JHHR) were also found to adhere to cell monolayers, albeit to different degrees, and all isolates produced maximal contact-dependent HeLa cell cytotoxicity. The avirulent trichomonad, Trichomonas tenax, did not adhere to cell monolayers and did not cause host cell damage. Interestingly, parasite cytadherence was greater with HeLa and HEp-2 epithelial cells than with fibroblast cells. In addition, cytotoxicity with fibroblast cells never exceeded 20% of the level of cell killing observed for epithelial cells. Elucidation of properties of the pathogenic human trichomonads that allowed for host cell surface parasitism was also attempted. Treatment of motile T. vaginalis NYH 286 with trypsin diminished cell parasitism. Incubation of trypsinized organisms in growth medium allowed for regeneration of trichomonal adherence, and cycloheximide inhibited the regeneration of attachment. Organisms poisoned with metronidazole or iodoacetate failed to attach to host cells, and adherent trichomonads exposed to metronidazole or iodoacetate were readily released from parasitized cells. Coincubation experiments with polycationic proteins and sugars and pretreatment of parasites or cells with neuraminidase or periodate had no effect on host cell parasitism. Colchicine and cytochalasin B, however, did produce some inhibition of adherence to HeLa cells. The data suggest that metabolizing T. vaginalis adheres to host cells via parasite surface

  15. Cytotoxic and apoptotic effects of bortezomib and gefitinib compared to alkylating agents on human glioblastoma cells.

    Science.gov (United States)

    Pédeboscq, Stéphane; L'Azou, Béatrice; Passagne, Isabelle; De Giorgi, Francesca; Ichas, François; Pometan, Jean-Paul; Cambar, Jean

    2008-01-01

    Glioblastoma is a malignant astrocytic tumor with a median survival of about 12 months for which new therapeutic strategies are required. We therefore examined the cytotoxicity of anticancer drugs with different mechanisms of action on two human glioblastoma cell lines expressing various levels of EGFR (epidermal growth factor receptor). Apoptosis induced by these anticancer agents was evaluated by flow cytometry. The cytotoxicity of alkylating drugs followed a dose-effect curve and cytotoxicity index values were lower with carboplatin than with BCNU and temozolomide. Anti-EGFR gefitinib (10 microM) cytotoxicity on DBTRG.05-MG expressing high levels of EGFR was significantly higher than on U87-MG expressing low levels of EGFR. Carboplatin and temozolomide cytotoxicity was potentiated with the addition of gefitinib on DBTRG.05-MG. Among the anticancer agents tested, the proteasome inhibitor bortezomib was the most cytotoxic with very low IC50 on the two cell lines. Moreover, all anticancer drugs tested induced apoptosis in a concentration-dependent manner. Bortezomib proved to be a more potent inductor of apoptosis than gefitinib and alkylating agents. These results show the efficacy of bortezomib and of the association between conventional chemotherapy and gefitinib on glioblastoma cells and therefore suggest the interest of these molecules in the treatment of glioblastoma.

  16. Uremic Toxins Enhance Statin-Induced Cytotoxicity in Differentiated Human Rhabdomyosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Hitoshi Uchiyama

    2014-09-01

    Full Text Available The risk of myopathy and rhabdomyolysis is considerably increased in statin users with end-stage renal failure (ESRF. Uremic toxins, which accumulate in patients with ESRF, exert cytotoxic effects that are mediated by various mechanisms. Therefore, accumulation of uremic toxins might increase statin-induced cytotoxicity. The purpose of this study was to determine the effect of four uremic toxins—hippuric acid, 3-carboxy-4-methyl-5-propyl-2-furanpropionate, indole-3-acetic acid, and 3-indoxyl sulfate—on statin-induced myopathy. Differentiated rhabdomyosarcoma cells were pre-treated with the uremic toxins for seven days, and then the cells were treated with pravastatin or simvastatin. Cell viability and apoptosis were assessed by viability assays and flow cytometry. Pre-treatment with uremic toxins increased statin- but not cisplatin-induced cytotoxicity (p < 0.05 vs. untreated. In addition, the pre-treatment increased statin-induced apoptosis, which is one of the cytotoxic factors (p < 0.05 vs. untreated. However, mevalonate, farnesol, and geranylgeraniol reversed the effects of uremic toxins and lowered statin-induced cytotoxicity (p < 0.05 vs. untreated. These results demonstrate that uremic toxins enhance statin-induced apoptosis and cytotoxicity. The mechanism underlying this effect might be associated with small G-protein geranylgeranylation. In conclusion, the increased severity of statin-induced rhabdomyolysis in patients with ESRF is likely due to the accumulation of uremic toxins.

  17. Reduced antibody-dependent cellular cytotoxicity to herpes simplex virus-infected cells of salivary polymorphonuclear leukocytes and inhibition of peripheral blood polymorphonuclear leukocyte cytotoxicity by saliva.

    Science.gov (United States)

    Ashkenazi, M; Kohl, S

    1990-06-15

    Blood polymorphonuclear leukocytes (BPMN) have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) against HSV-infected cells. Although HSV infections are frequently found in the oral cavity, the ADCC capacity of salivary PMN (SPMN) has not been studied, mainly because methods to isolate SPMN were not available. We have recently developed a method to isolate SPMN, and in this study have evaluated their ADCC activity against HSV-infected cells. SPMN were obtained by repeated washings of the oral cavity, and separated from epithelial cells by nylon mesh filtration. ADCC was quantitatively determined by 51Cr release from HSV-infected Chang liver cells. SPMN in the presence of antibody were able to destroy HSV-infected cells, but SPMN were much less effective in mediating ADCC than BPMN (3.4% vs 40.7%, p less than 0.0001). In the presence of antiviral antibody, SPMN were able to adhere to HSV-infected cells, but less so than BPMN (34% vs 67%), and specific antibody-induced adherence was significantly lower in SPMN (p less than 0.04). The spontaneous adherence to HSV-infected cells was higher for SPMN than BPMN. SPMN demonstrated up-regulation of the adhesion glycoprotein CD18, but down-regulation of the FcRIII receptor. Incubation with saliva decreased ADCC capacity of BPMN, up-regulated CD18 expression, and down-regulated FcRIII expression.

  18. Cytotoxicity of protein corona-graphene oxide nanoribbons on human epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Mbeh, Doris A. [Laboratory for Innovation and Analysis of Bio-Performance, École Polytechnique, C.P. 6079, Succursale Centre-ville, Montréal, Québec H3C 3A7 (Canada); Akhavan, Omid, E-mail: oakhavan@sharif.edu [Department of Physics, Sharif University of Technology, P.O. Box 11155-9161, Tehran (Iran, Islamic Republic of); Institute for Nanoscience and Nanotechnology, Sharif University of Technology, P.O. Box 14588-89694, Tehran (Iran, Islamic Republic of); Javanbakht, Taraneh [Laboratory for Innovation and Analysis of Bio-Performance, École Polytechnique, C.P. 6079, Succursale Centre-ville, Montréal, Québec H3C 3A7 (Canada); Mahmoudi, Morteza [Department of Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences (Iran, Islamic Republic of); Yahia, L’Hocine [Laboratory for Innovation and Analysis of Bio-Performance, École Polytechnique, C.P. 6079, Succursale Centre-ville, Montréal, Québec H3C 3A7 (Canada)

    2014-11-30

    Highlights: • Graphene oxide nanoribons (GONRs) were synthesized by unzipping of multi-walled carbon nanotubes. • GONRs were functionalized by the albumin originated from the two different protein sources. • Concentration-dependent cytotoxicity of the functionalized GONRs was investigated on human epithelial cells. - Abstract: Graphene oxide nanoribbons (GONRs) were synthesized using an oxidative unzipping of multi-walled carbon nanotubes. The interactions of the GONRs with various concentrations of fetal bovine serum or human plasma serum indicated that the GONRs were functionalized substantially by the albumin originated from the two different protein sources. Then, concentration-dependent cytotoxicity of the protein-functionalized GONRs on human epithelial cells was studied. Although the GONRs with concentrations ≤50 μg/mL did not exhibit significant cytotoxicity on the cells (with the cell viability >85%), the concentration of 100 μg/mL exhibited significant cytotoxicity including prevention of cell proliferation and induction of cell apoptosis. These results can provide more in-depth understanding about cytotoxic effects of graphene nanostructures which can be functionalized by the proteins of media.

  19. Arecoline induced cell cycle arrest, apoptosis, and cytotoxicity to human endothelial cells.

    Science.gov (United States)

    Tseng, Shuei-Kuen; Chang, Mei-Chi; Su, Cheng-Yao; Chi, Lin-Yang; Chang, Jenny Zwei-Ching; Tseng, Wan-Yu; Yeung, Sin-Yuet; Hsu, Ming-Lun; Jeng, Jiiang-Huei

    2012-08-01

    Betel quid (BQ) chewing is a common oral habit in South Asia and Taiwan. BQ consumption may increase the risk of oral squamous cell carcinoma (OSCC), oral submucous fibrosis (OSF), and periodontitis as well as systemic diseases (atherosclerosis, hypertension, etc.). However, little is known about the toxic effect of BQ components on endothelial cells that play important roles for angiogenesis, carcinogenesis, tissue fibrosis, and cardiovascular diseases. EAhy 926 (EAHY) endothelial cells were exposed to arecoline, a major BQ alkaloid, for various time periods. Cytotoxicity was estimated by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. The cell cycle distribution of EAHY cells residing in sub-G0/G1, G0/G1, S-, and G2/M phases was analyzed by propidium iodide staining of cellular DNA content and flow cytometry. Some EAHY cells retracted, became round-shaped in appearance, and even detached from the culture plate after exposure to higher concentrations of arecoline (> 0.4 mM). At concentrations of 0.4 and 0.8 mM, arecoline induced significant cytotoxicity to EAHY cells. At similar concentrations, arecoline induced G2/M cell cycle arrest and increased sub-G0/G1 population, a hallmark of apoptosis. Interestingly, prolonged exposure to arecoline (0.1 mM) for 12 and 21 days significantly suppressed the proliferation of EAHY cells, whereas EAHY cells showed adaptation and survived when exposed to 0.05 mM arecoline. These results suggest that BQ components may contribute to the pathogenesis of OSF and BQ chewing-related cardiovascular diseases via toxicity to oral or systemic endothelial cells, leading to impairment of vascular function. During BQ chewing, endothelial damage may be induced by areca nut components and associate with the pathogenesis of OSF, periodontitis, and cardiovascular diseases.

  20. The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung fibroblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Leah J.; Holmes, Amie L. [Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300 (United States); Maine Center for Environmental Toxicology and Health, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300 (United States); Department of Applied Medical Science, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300 (United States); Kandpal, Sanjeev Kumar; Mason, Michael D. [Department of Chemical and Biological Engineering, University of Maine, Orono, ME (United States); Zheng, Tongzhang [Department of Environmental Health Sciences, Yale School of Public Health, New Haven, CT (United States); Wise, John Pierce, E-mail: John.Wise@usm.maine.edu [Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300 (United States); Maine Center for Environmental Toxicology and Health, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300 (United States); Department of Applied Medical Science, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300 (United States)

    2014-08-01

    Cobalt exposure is increasing as cobalt demand rises worldwide due to its use in enhancing rechargeable battery efficiency, super-alloys, and magnetic products. Cobalt is considered a possible human carcinogen with the lung being a primary target. However, few studies have considered cobalt-induced toxicity in human lung cells. Therefore, in this study, we sought to determine the cytotoxicity and genotoxicity of particulate and soluble cobalt in human lung cells. Cobalt oxide and cobalt chloride were used as representative particulate and soluble cobalt compounds, respectively. Exposure to both particulate and soluble cobalt induced a concentration-dependent increase in cytotoxicity, genotoxicity, and intracellular cobalt ion levels. Based on intracellular cobalt ion levels, we found that soluble cobalt was more cytotoxic than particulate cobalt while particulate and soluble cobalt induced similar levels of genotoxicity. However, soluble cobalt induced cell cycle arrest indicated by the lack of metaphases at much lower intracellular cobalt concentrations compared to cobalt oxide. Accordingly, we investigated the role of particle internalization in cobalt oxide-induced toxicity and found that particle-cell contact was necessary to induce cytotoxicity and genotoxicity after cobalt exposure. These data indicate that cobalt compounds are cytotoxic and genotoxic to human lung fibroblasts, and solubility plays a key role in cobalt-induced lung toxicity. - Highlights: • Particulate and soluble cobalt are cytotoxic and genotoxic to human lung cells. • Soluble cobalt induces more cytotoxicity compared to particulate cobalt. • Soluble and particulate cobalt induce similar levels of genotoxicity. • Particle-cell contact is required for particulate cobalt-induced toxicity.

  1. Enhanced cytotoxicity of natural killer cells following the acquisition of chimeric antigen receptors through trogocytosis.

    Directory of Open Access Journals (Sweden)

    Fu-Nan Cho

    Full Text Available Natural killer (NK cells have the capacity to target tumors and are ideal candidates for immunotherapy. Viral vectors have been used to genetically modify in vitro expanded NK cells to express chimeric antigen receptors (CARs, which confer cytotoxicity against tumors. However, use of viral transduction methods raises the safety concern of viral integration into the NK cell genome. In this study, we used trogocytosis as a non-viral method to modify NK cells for immunotherapy. A K562 cell line expressing high levels of anti-CD19 CARs was generated as a donor cell to transfer the anti-CD19 CARs onto NK cells via trogocytosis. Anti-CD19 CAR expression was observed in expanded NK cells after these cells were co-cultured for one hour with freeze/thaw-treated donor cells expressing anti-CD19 CARs. Immunofluorescence analysis confirmed the localization of the anti-CD19 CARs on the NK cell surface. Acquisition of anti-CD19 CARs via trogocytosis enhanced NK cell-mediated cytotoxicity against the B-cell acute lymphoblastic leukemia (B-ALL cell lines and primary B-ALL cells derived from patients. To our knowledge, this is the first report that describes the increased cytotoxicity of NK cells following the acquisition of CARs via trogocytosis. This novel strategy could be a potential valuable therapeutic approach for the treatment of B-cell tumors.

  2. Evaluation of mercury mediated in vitro cytotoxicity among cell lines established from green sea turtles.

    Science.gov (United States)

    Wang, Hong; Tong, Jing; Bi, Yongyi; Wang, Chunhong; Guo, Liqiao; Lu, Yuanan

    2013-04-01

    In vitro cell cultures are currently tested for their application as a biological tool for enhanced monitoring and field evaluation of environmental toxic chemical pollution. Here cell lines established from green sea turtles (GSTs) were comparatively tested for their cytotoxic responses to mercury chloride (HgCl2) exposure and also their potential use as a biological tool for effective monitoring and screening of mercury contamination in environmental waters. Following a 24-h exposure to different concentrations of mercury solution, marine turtle cells were evaluated for their cytotoxic responses using three different endpoint bioassays: tetrazolium salt reduction (MTT), neutral red uptake (NR), and Coomassie blue (CB) methods. Cytotoxic sensitivities of GST cell lines to HgCl2 were determined and compared basing on their 50% inhibition concentration (IC50) values calculated from these tests. These marine turtle cells share a very different pattern of cytotoxic sensitivities and reactions to inorganic Mercury. Among these nine turtle cell lines, turtle liver cells (GST-LV) appear to be the most tolerant one to mercury exposure while turtle lung cells (GST-LG) exhibit to be the most sensitive one. Results from this in vitro study correlate well with in vivo examination of mercury concentration in the tissues of marine turtles and are also validated and ascertained by calculated regression equations showing a significant correlation (Pturtle cells and also shows GST-LG to be a cell line with potential application in field monitoring and assessing mercury contamination as a bioindicator.

  3. A comparative assessment of cytotoxicity of commonly used agricultural insecticides to human and insect cells.

    Science.gov (United States)

    Yun, Xinming; Huang, Qingchun; Rao, Wenbing; Xiao, Ciying; Zhang, Tao; Mao, Zhifan; Wan, Ziyi

    2017-03-01

    The cytotoxic potential of 13 commonly used agricultural insecticides was examined using cell-based systems with three human HepG2, Hek293, HeLa cells and three insect Tn5B1-4, Sf-21, and Drosophila S2 cells. Data showed that (1) an enhancement of some insecticides (e.g. pyrethroids) on cells proliferation; (2) an inhibition of some insecticides on cells viability; (3) various levels of susceptibility of different cells to the same insecticide; and (4) the cell type dependent sensitivity to different insecticides. The degree of cytotoxicity of insecticides on human cells was significantly lower than that on insect cells (Pinsecticides against intact cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Selective Cytotoxicity of Goniothalamin against Hepatoblastoma HepG2 Cells

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    Noorjahan B. Alitheen

    2011-04-01

    Full Text Available Liver cancer has become one of the major types of cancer with high mortality and liver cancer is not responsive to the current cytotoxic agents used in chemotherapy. The purpose of this study was to examine the in vitro cytotoxicity of goniothalamin on human hepatoblastoma HepG2 cells and normal liver Chang cells. The cytotoxicity of goniothalamin against HepG2 and liver Chang cell was tested using MTT cell viability assay, LDH leakage assay, cell cycle flow cytometry PI analysis, BrdU proliferation ELISA assay and trypan blue dye exclusion assay. Goniothalamin selectively inhibited HepG2 cells [IC50 = 4.6 (±0.23 µM in the MTT assay; IC50 = 5.20 (±0.01 µM for LDH assay at 72 hours], with less sensitivity in Chang cells [IC50 = 35.0 (±0.09 µM for MTT assay; IC50 = 32.5 (±0.04 µM for LDH assay at 72 hours]. In the trypan blue dye exclusion assay, the Viability Indexes were 52 ± 1.73% for HepG2 cells and 62 ± 4.36% for Chang cells at IC50 after 72 hours. Cytotoxicity of goniothalamin was related to inhibition of DNA synthesis, as revealed by the reduction of BrdU incorporation. At 72 hours, the lowest concentration of goniothalamin (2.3 µL retained 97.6% of normal liver Chang cells proliferation while it reduced HepG2 cell proliferation to 19.8% as compared to control. Besides, goniothalamin caused accumulation of hypodiploid apoptosis and different degree of G2/M arrested as shown in cell cycle analysis by flow cytometry. Goniothalamin selectively killed liver cancer cell through suppression of proliferation and induction of apoptosis. These results suggest that goniothalamin shows potential cytotoxicity against hepatoblastoma HepG2 cells.

  5. Preliminary Study on Cytotoxic Effect of Biodegradation of Magnesium on Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    Yu Zhang; Ling Ren; Mei Li; Xiao Lin; Huafu Zhao; Ke Yang

    2012-01-01

    Biodegradation of magnesium (Mg) based metals in body fluid can lead to a strong alkalinity as well as an increase of Mg2+ concentration in its surrounding environment. In vitro cytotoxic effects of the extracts of pure Mg with and without micro arc oxidation (MAO) coating on osteosarcoma U2-OS cells, a kind of bone cancer cells, were preliminarily studied, independently considering the increase of either alkalinity or Mg2+ concentration. The results indicated that the high alkalinity, i.e., a great increase of pH value, caused by the degradations of Mg with and without MAO coating in the culture medium all showed strong cytotoxic effects on U2-OS cells. However, the increase of Mg2+ concentration had no such cytotoxic effect. This finding may provide an alternative way to cure bone cancers through creating a high alkalinity surrounding the cancer cells.

  6. NK Cell Maturation and Cytotoxicity are Controlled by the Intramembrane Aspartyl Protease SPPL31

    OpenAIRE

    Hamblet, Corinne E.; Makowski, Stefanie L.; Tritapoe, Julia M.; Pomerantz, Joel L.

    2016-01-01

    NK cell maturation is critical for normal effector function and the innate immune response to tumors and pathogens. However, the molecular pathways that control NK cell maturation remain largely undefined. Here, we investigate the role of SPPL3, an intramembrane aspartyl protease, in murine NK cell biology. We find that deletion of SPPL3 in the hematopoietic system reduces numbers of peripheral NK cells, clearance of MHC Class I-deficient tumors in vivo, and cytotoxicity against tumor cells i...

  7. Oxidative stress and cytotoxicity generated by dental composites in human pulp cells.

    Science.gov (United States)

    Krifka, Stephanie; Seidenader, Claudia; Hiller, Karl-Anton; Schmalz, Gottfried; Schweikl, Helmut

    2012-02-01

    Dental composites are a source of residual monomers that are released into the oral environment. Since monomers act on cultured cells through reactive oxygen species (ROS), we hypothesized that composites generate ROS associated with cytotoxicity. Human pulp-derived cells were exposed to extracts of methacrylate-based materials including triethylene glycol dimethacrylate and 2-hydroxyethyl methacrylate-free composites (Tetric Ceram, Tetric EvoCeram, els, els flow, Solitaire 2) and a silorane-based composite (Hermes III). The materials were polymerized in the presence and absence of a polyester film and then extracted in culture medium. The generation of ROS was measured by flow cytometry, and cytotoxicity was determined as well. Methacrylate-based composites reduced cell survival but varied in efficiency. Undiluted extracts of Solitaire 2 specimens prepared in the absence of a polyester film reduced cell survival to 26% compared with untreated cultures. Cytotoxicity was reduced when specimens were covered with a polyester film during preparation. Cytotoxicity of the composites was ranked as follows: Solitaire 2 > els flow > Tetric Ceram = Tetric EvoCeram = els > Hermes III. The generation of ROS followed the same pattern as detected with cytotoxic effects. A positive correlation was found between ROS production and cell survival caused by extracts made from materials not covered with a polyester film. These findings suggest that components released from composites affect cellular signaling networks through ROS formation. Regenerative and reparative capacities of the dentine-pulp complex may be impaired by biologically active resin monomers released from composite restorations.

  8. Cytotoxicity effect of Zataria multiflora Boiss. on two human colon carcinoma cell lines

    Directory of Open Access Journals (Sweden)

    F. Sharififar

    2017-10-01

    Full Text Available Background and objectives: Natural products are one of the major sources for investigations of novel medicines. Zataria multiflora Boiss (ZM has shown pharmacological activities especially in gastrointestinal tract; however, there are limited studies about its cytotoxicity effects. In this study, the effect of Zataria multiflora was examined on two colon cancer cell lines (SW-48 and HT-29. Methods: Hydro-alcoholic extract of ZM and its fractions including chloroform, petroleum ether and methanol extract were prepared by warm maceration method. Different concentrations were prepared and examined on SW-48 and HT-29 cell lines using 2-(4, 5-dimethylthiazol-2-yl 2, 5-diphenyltetrazolium bromide (MTT assay. Results: The results of the present study have shown the cytotoxic effect of some fractions of ZM. The most considerable cytotoxic effect was shown against HT-29 cell line. Also, total ZM extract and the petroleum ether fraction demonstrated cytotoxic effects with IC50 values of 44.22 and 33.42 µg/ml on SW-48 and HT-29 cell lines, respectively. Conclusion: Zataria multiflora was cytotoxic to against colon cancer cell lines HT-29 and SW-48.

  9. Accurate non-invasive image-based cytotoxicity assays for cultured cells

    Directory of Open Access Journals (Sweden)

    Brouwer Jaap

    2010-06-01

    Full Text Available Abstract Background The CloneSelect™ Imager system is an image-based visualisation system for cell growth assessment. Traditionally cell proliferation is measured with the colorimetric MTT assay. Results Here we show that both the CloneSelect Imager and the MTT approach result in comparable EC50 values when assaying the cytotoxicity of cisplatin and oxaliplatin on various cell lines. However, the image-based technique was found non-invasive, considerably quicker and more accurate than the MTT assay. Conclusions This new image-based technique has the potential to replace the cumbersome MTT assay when fast, unbiased and high-throughput cytotoxicity assays are requested.

  10. Investigation of the cytotoxic effects of titanate nanotubes on Caco-2 cells.

    Science.gov (United States)

    Fenyvesi, Ferenc; Kónya, Zoltán; Rázga, Zsolt; Vecsernyés, Miklós; Kása, Péter; Pintye-Hódi, Klára; Bácskay, Ildikó

    2014-08-01

    Titanate nanotubes can be used as drug delivery systems, but limited information is available on their interactions with intestinal cells. In this study, we investigated the cytotoxicity and cellular uptake of titanate nanotubes on Caco-2 monolayers and found that up to 5 mg/ml concentration, these nanotubes are not cytotoxic and not able to permeate through the intestinal cell layer. Transmission electron microscopic experiments showed that titanate nanotubes are not taken up by cells, only caused a high-density granulation on the surface of the endoplasmic reticulum. According to these results, titanate nanotubes are suitable systems for intestinal drug delivery.

  11. Cytotoxic activity of kaempferol glycosides against human leukaemic cell lines in vitro.

    Science.gov (United States)

    Dimas, K; Demetzos, C; Mitaku, S; Marselos, M; Tzavaras, T; Kokkinopoulos, D

    2000-01-01

    Two kaempferol coumaroyl glycosides (i.e. platanoside and tiliroside) isolated from the methanolic extract of Platanus orientalis L. buds, were examined for their in vitro cytotoxic activity against a panel of human leukaemic cell lines. Platanoside (1) exhibited cytotoxic activity against most of the cell lines tested, while tiliroside (2) was active against two of the nine tested cell lines. Compound 1, was examined for its effect on the uptake of [(3)H]thymidine as a marker of DNA synthesis. Kaempferol was used as a control.

  12. Phenolics-saponins rich fraction of defatted kenaf seed meal exhibits cytotoxicity towards cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    Latifah Saiful Yazan; Napsiah Abd Rahman; Kim Wei Chan; Wan Nor Hafiza Wan Abd Ghani; Yin Sim Tor; Jhi Biau Foo

    2016-01-01

    Objectives: To determine the cytotoxicity of crude ethanolic extract, n-butanol fraction and aqueous fraction on selected cancer cell lines, and to observe the morphological changes of the cancer cells treated with n-butanol fraction.Methods: The cytotoxic effect of n-butanol fraction, crude ethanolic extract and aqueous fraction on breast cancer(MCF-7 and MDA-MB-231), colon cancer(HT29), lung cancer(A549), cervical cancer(He La) and normal mouse fibroblast(3T3) cell lines was determined using MTT assay. The morphological changes of the treated cells were observed under an inverted light microscope.Results: n-Butanol fraction was the most cytotoxic towards HT29 and MCF-7 cells in a dose-dependent manner compared to crude ethanolic extract and aqueous fraction(P < 0.05). The IC50 of n-butanol fraction for HT29 and MCF-7 was(780.00 ± 28.28)and(895.00 ± 7.07) mg/m L, respectively. Cell shrinkage, membrane blebbing and formation of apoptotic bodies were noted following treatment of HT29 cells with n-butanol fraction.Conclusions: In conclusion, n-butanol fraction was more cytotoxic than crude ethanolic extract and aqueous fraction towards the selected cancerous cell lines and induced apoptosis in HT29 cells.

  13. NK cells, displaying early activation, cytotoxicity and adhesion molecules, are associated with mild dengue disease

    Science.gov (United States)

    Azeredo, E L; De Oliveira-Pinto, L M; Zagne, S M; Cerqueira, D I S; Nogueira, R M R; Kubelka, C F

    2006-01-01

    During the innate immune response against infections, Natural Killer (NK) cells are as important effector cells as are Cytotoxic T lymphocytes (CTL) generated after antigenic stimulation in the adaptative response. NK cells increase in numbers, after viral infection or vaccination. We investigated the NK cell and CD8 T lymphocyte status in 55 dengue infected patients. The NK (CD56+CD3−) and CD56+ T cell (CD56+CD3+) rates rise during the acute phase of disease. The majority of NK cells from dengue patients display early markers for activation (CD69, HLA-DR, and CD38) and cell adhesion molecules (CD44, CD11a) during the acute phase of disease. The intracellular cytotoxic granule, TIA-1, is also up-regulated early in NK cells. Most of these markers appear also on CD8+ T lymphocytes but during the late acute phase. Circulating IL-15 is elevated in a significant number of patients during early acute infection and its values were statistically correlated with NK frequencies and cytotoxic markers on NKs. We have therefore shown that dengue virus infection is very likely stimulating a cytotoxic response that may be efficient in controlling the virus in synergism with CD8+ T lymphocytes. Interestingly, the heightened CD56+CD3−, CD56+CD3+, CD56+TIA-1+ and CD56+CD11a+ cell rates are associated with mild dengue clinical manifestations and might indicate a good prognosis of the disease. PMID:16412060

  14. Discrepancy between direct and antibody-dependent cytotoxic activities of human LAK cells.

    Science.gov (United States)

    Potapnev, M P; Garbuzenco, T S; Goncharova, N V; Zobnin, V D; Shadrin, O V; Bykovskaya, S N

    1994-06-01

    Human lymphokine-activated killer (LAK) cells display cytotoxic activity against natural killer (NK)-resistant tumor cells in an antibody-independent and -dependent manner. We compared LAK cell-mediated antibody-independent cytotoxicity (LAK activity) and antibody-dependent cellular cytotoxicity (ADCC) against untreated and antibody-coated Raji cells, respectively. Human lymphocytes showed drastically increased LAK activity after stimulation with interleukin-2 (IL-2) for 3 or 7 days when compared to non-activated cells. The level of ADCC was reduced for 3-day-generated LAK cells and augmented for 7-day-generated LAK cells as compared to non-activated cultured lymphocytes. Phenotypical analysis revealed IL-2-induced up-regulation of the proportion of CD11b+ (but not CD16+) lymphocyte subpopulation in 7-day-generated LAK cells. The data imply that human LAK cells exhibit antibody-dependent and -independent cytotoxic activities via distinct effector pathways at different stages of generation. These stages may be associated with changes in adhesion molecule (CD11b/CD18) expression on the surface of IL-2-activated lymphocytes.

  15. NK cells, displaying early activation, cytotoxicity and adhesion molecules, are associated with mild dengue disease.

    Science.gov (United States)

    Azeredo, E L; De Oliveira-Pinto, L M; Zagne, S M; Cerqueira, D I S; Nogueira, R M R; Kubelka, C F

    2006-02-01

    During the innate immune response against infections, Natural Killer (NK) cells are as important effector cells as are Cytotoxic T lymphocytes (CTL) generated after antigenic stimulation in the adaptative response. NK cells increase in numbers, after viral infection or vaccination. We investigated the NK cell and CD8 T lymphocyte status in 55 dengue infected patients. The NK (CD56+CD3-) and CD56+ T cell (CD56+CD3+) rates rise during the acute phase of disease. The majority of NK cells from dengue patients display early markers for activation (CD69, HLA-DR, and CD38) and cell adhesion molecules (CD44, CD11a) during the acute phase of disease. The intracellular cytotoxic granule, TIA-1, is also up-regulated early in NK cells. Most of these markers appear also on CD8+ T lymphocytes but during the late acute phase. Circulating IL-15 is elevated in a significant number of patients during early acute infection and its values were statistically correlated with NK frequencies and cytotoxic markers on NKs. We have therefore shown that dengue virus infection is very likely stimulating a cytotoxic response that may be efficient in controlling the virus in synergism with CD8+ T lymphocytes. Interestingly, the heightened CD56+CD3-, CD56+CD3+, CD56+TIA-1+ and CD56+CD11a+ cell rates are associated with mild dengue clinical manifestations and might indicate a good prognosis of the disease.

  16. Cytotoxic effects of new MTA-based cement formulations on fibroblast-like MDPL-20 cells.

    Science.gov (United States)

    Garcia, Lucas da Fonseca Roberti; Santos, Alailson Domingos dos; Moraes, João Carlos Silos; Costa, Carlos Alberto de Souza

    2016-01-01

    The present study aimed at evaluating the cytotoxic effects of a novel cement called CER on periodontal fibroblast-like cells of mice (MDPL-20), in comparison with different formulations of Mineral Trioxide Aggregate (MTA), by means of the cell viability test (MTT) and cell morphology analysis. Thirty-two round-shaped samples were fabricated with the following cements: white MTA, white and gray CER and experimental white MTA. The samples were immersed in serum-free culture medium for 24 hours or 7 days (n = 16). The extracts (culture medium + components released from the cements) were applied for 24 hours to previously cultured cells (40.000 cells/cm2) in the wells of 24-well plates. Cells seeded in complete culture medium were used as a negative control. Cell viability was assessed using the MTT assay. Two samples of each cement were used for cell morphology analysis by Scanning Electron Microscopy (SEM). The extracts obtained at the 7-day period presented higher cytotoxicity compared with the 24-hour period (p MTA presented the lowest, similar to the control (p > 0.05). However, at the 7-day period, the experimental white MTA presented no significant difference in comparison with the other cements (p > 0.05). At the 7-day period, CER cement presented cytotoxic effects on fibroblast-like cells, similar to different MTA formulations. However, the immersion period in the culture medium influenced the cytotoxicity of the cements, which was greater for CER cement at 24 hours.

  17. Induction of NKG2D ligands and increased sensitivity of tumor cells to NK cell-mediated cytotoxicity by hematoporphyrin-based photodynamic therapy.

    Science.gov (United States)

    Park, Min-Ju; Bae, Jae-Ho; Chung, Joo Seop; Kim, Sun-Hee; Kang, Chi-Dug

    2011-01-01

    Natural killer (NK) cells are important innate effector cells which can irradicate tumor cells through specific interactions between activating receptors on NK cells and their cognate ligands on cancer cells. Recently, it has been known that induction of activating NKG2D ligands including MHC class I chain-related (MIC) and UL16-binding protein (ULBP) families on tumor cells by various stresses makes them more susceptible to NK cell-mediated cytotoxicity. Therefore, it was investigated whether sublethal dose of hematoporphyrin-based photodynamic therapy (PDT) could up-regulate NKG2D ligands on tumor cells and increase the susceptibility of cancer cells against NK cells. Treatment with sublethal dose of hematoporphyrin-based PDT increased mRNA transcription and surface expression of ULBP1 and ULBP2 genes in SNU-1 human gastric tumor cell line and MICA/B, ULBP1, ULBP2 and ULBP3 genes in SW-900 human lung cancer cell line. These results were followed by increased susceptibility of cancer cells to NK cell-mediated cytotoxicity after sublethal PDT, which was abolished by addition of a blocking NKG2D mAb. Therefore, it could be suggested that the effect of hematoporphyrin-based PDT might be mediated in part by the increased susceptibility to NK cells via induction of NKG2D ligands on tumor cells, which survived after treatment with PDT.

  18. In vitro cytotoxicity of Indonesian stingless bee products against human cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    Paula M Kustiawan; Songchan Puthong; Enos T Arung; Chanpen Chanchao

    2014-01-01

    Objective: To screen crude extracts of propolis, bee pollen and honey from four stingless bee species [Trigona incisa (T. incisa)], Timia apicalis, Trigona fusco-balteata and Trigona fuscibasis) native to East Kalimantan, Indonesia for cytotoxic activity against five human cancer cell lines (HepG2, SW620, ChaGo-I, KATO-III and BT474).Methods:All samples were extracted with methanol, and then subpartitioned with n-hexane and ethyl acetate. Each crude extract was screened at 20 µg/mL for in vitro cytotoxicity against the cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, four previously shown bioactive components from propolis (apigenin, caffeic acid phenyl ester, kaempferol and naringenin) and two chemotherapeutic drugs (doxorubicin and 5-fluorouracil) were used to evaluate the sensitivity of the cell lines.Results:Overall, crude extracts from propolis and honey had higher cytotoxic activities than bee pollen, but the activity was dependent upon the extraction solvent, bee species and cell line. Propolis extracts from T. incisa and Timia apicalis showed the highest and lowest cytotoxic activity, respectively. Only the HepG2 cell line was broadly sensitive to the honey extracts. For pure compounds, doxorubicin was the most cytotoxic, the four propolis compounds the least, but the ChaGo-I cell line was sensitive to kaempferol at 10 µg/mL and KATO-III was sensitive to kaempferol and apigenin at 10 µg/mL. All pure compounds were effective against the BT474 cell line.Conclusions:Propolis from T. incisa and Trigona fusco-balteata contain an in vitro cytotoxic activity against human cancer cell lines. Further study is required, including the isolation and characterization of the active antiproliferative agent(s).

  19. Cytotoxicity and genotoxicity assessment of Euphorbia hirta in MCF-7 cell line model using comet assay

    Institute of Scientific and Technical Information of China (English)

    Kwan Yuet Ping; Ibrahim Darah; Yeng Chen; Sreenivasan Sasidharan

    2013-01-01

    Objective:To evaluate the cytotoxicity and genotoxicity activity of Euphorbia hirta (E. hirta) in MCF-7 cell line model using comet assay. Methods: The cytotoxicity of E. hirta extract was investigated by employing brine shrimp lethality assay and the genotoxicity of E. hirta was assessed by using Comet assay. Results: Both toxicity tests exhibited significant toxicity result. In the comet assay, the E. hirta extract exhibited genotoxicity effects against MCF-7 DNA in a time-dependent manner by increasing mean percentage of DNA damage. The extract of E. hirta showed significant toxicity against brine shrimp with an LC50 value of 620.382 μg/mL (24 h). Comparison with positive control potassium dichromate signifies that cytotoxicity exhibited by the methanol extract might have moderate activity. Conclusion:The present work confirmed the cytotoxicity and genotoxicity of E. hirta. However, the observed toxicity of E. hirta extracts needs to be confirmed in additional studies.

  20. Myricitrin Inhibits Acrylamide-Mediated Cytotoxicity in Human Caco-2 Cells by Preventing Oxidative Stress

    Science.gov (United States)

    Chen, Wei; Feng, Lina; Shen, Yang; Su, Hongming; Li, Ya; Zhuang, Jingjing; Zhang, Lingxia; Zheng, Xiaodong

    2013-01-01

    Oxidative stress was thought to be associated with acrylamide cytotoxicity, but the link between oxidative stress and acrylamide cytotoxicity in the gastrointestinal tract, the primary organ in contact with dietary acrylamide, is still unclear. This study was conducted to evaluate the antioxidant activity of natural dietary compound myricitrin and its protective role against acrylamide cytotoxicity. We found that myricitrin can effectively scavenge multiple free radicals (including DPPH free radical, hydroxyl radical, and ABTS free radical) in a concentration-dependent manner. Our results further indicated that the presence of myricitrin (2.5–10 μg/mL) was found to significantly inhibit acrylamide-induced cytotoxicity in human gastrointestinal Caco-2 cells. Moreover, acrylamide-induced cytotoxicity is closely related to oxidative stress in Caco-2 cells. Interestingly, myricitrin was able to suppress acrylamide toxicity by inhibiting ROS generation. Taken together, these results demonstrate that myricitrin had a profound antioxidant effect and can protect against acrylamide-mediated cytotoxicity. PMID:24224177

  1. HER2 as a promising target for cytotoxicity T cells in human melanoma therapy.

    Directory of Open Access Journals (Sweden)

    Juan Ma

    Full Text Available Anti-HER2/neu antibody therapy has been reported to mediate tumor regression of HER2/ neu(+ tumors. Here we demonstrated the expression of HER2 in a wide range of human melanoma cells including a primary culture and seven cell lines, and we further investigated whether HER2 could be served as a target for T cell mediated immunotherapy of human melanoma. Specific cytolytic activity of activated T cells (ATC armed with anti-CD3 x anti-HER2 bispecific antibody (HER2Bi-Ab against Malme-3M-luc cells was evaluated by bioluminescent signal generated by luciferase reporter which did not alter HER2 expression or proliferation ability of Malme-3M cells. Contrast with unarmed ATC, increased cytotoxic activity of HER2Bi-armed ATC against Malme-3M-luc cells was observed at effector/target (E/T ratios of 1:1, 5:1, and 20:1. Moreover, HER2Bi-armed ATC expressed higher level of activation marker CD69 and secreted significantly higher level of IFN-γ than unarmed ATC counterpart at the E/T ratio of 20:1. In addition, compared with anti-HER2 mAb (Herceptin® or unarmed ATC, HER2Bi-armed ATC showed remarkable suppression effect on Malme-3M-luc tumor cells. Furthermore, in melanoma tumor cell xenograft mice, infusion of HER2Bi-armed ATC successfully inhibited the growth of melanoma tumors. The anti-tumor effect of HER2Bi-armed ATC may provide a promising immunotherapy for melanoma in the future.

  2. [Cytotoxic effects of local anesthesia through lidocaine/ropivacaine on human melanoma cell lines].

    Science.gov (United States)

    Kang, Ding-Kun; Zhao, Li-Yan; Wang, Hong-Li

    Local anesthetics (LAs) are generally considered as safe, but cytotoxicity has been reported for several local anesthetics used in humans, which is not well investigated. In the present study, the cytotoxicity of lidocaine, ropivacaine and the combination of lidocaine and ropivacaine were evaluated on human melanoma cell lines. Melphalan, a nitrogen mustard alkylating agent, was used as a control agent for comparison of cytotoxic activity. Melanoma cell lines, A375 and Hs294T, were exposed to 1h to different concentrations of above agents. Cell-viability after exposure was determined by flow cytometry. Investigated LAs showed detrimental cytotoxicity on studied melanoma cell lines in time- (p<0.001), concentration- (p<0.001), and agent dependant. In both A375 and Hs294T cell lines, minimum cell viability rates were found after 72h of exposure to these agents. Lidocaine 2% caused a reduction of vital cells to 10%±2% and 14%±2% in A375 and Hs294T, respectively after 72h of exposure. Ropivacaine 0.75% after 72h reduced viable cells to 15%±3% and 25%±3% in A375 and Hs294T, respectively. Minimum cell viability after 72h exposure to the combination was 10%±2% and 18%±2% in A375 and Hs294T, respectively. Minimum cell viability after 72h exposure to melphalan was 8%±1% and 12%±2%, in A375 and Hs294T, respectively. LAs have cytotoxic activity on human melanoma cell lines in a time-, concentration- and agent-dependant manner. Apoptosis in the cell lines was mediated through activity of caspases-3 and caspases-8. Copyright © 2016 Sociedade Brasileira de Anestesiologia. Publicado por Elsevier Editora Ltda. All rights reserved.

  3. Cytotoxic effects of local anesthesia through lidocaine/ropivacaine on human melanoma cell lines.

    Science.gov (United States)

    Kang, Ding-Kun; Zhao, Li-Yan; Wang, Hong-Li

    Local anesthetics (LAs) are generally considered as safe, but cytotoxicity has been reported for several local anesthetics used in humans, which is not well investigated. In the present study, the cytotoxicity of lidocaine, ropivacaine and the combination of lidocaine and ropivacaine were evaluated on human melanoma cell lines. Melphalan, a nitrogen mustard alkylating agent, was used as a control agent for comparison of cytotoxic activity. Melanoma cell lines, A375 and Hs294T, were exposed to 1h to different concentrations of above agents. Cell-viability after exposure was determined by flow cytometry. Investigated LAs showed detrimental cytotoxicity on studied melanoma cell lines in time- (p<0.001), concentration- (p<0.001), and agent dependant. In both A375 and Hs294T cell lines, minimum cell viability rates were found after 72h of exposure to these agents. Lidocaine 2% caused a reduction of vital cells to 10%±2% and 14%±2% in A375 and Hs294T, respectively after 72h of exposure. Ropivacaine 0.75% after 72h reduced viable cells to 15%±3% and 25%±3% in A375 and Hs294T, respectively. Minimum cell viability after 72h exposure to the combination was 10%±2% and 18%±2% in A375 and Hs294T, respectively. Minimum cell viability after 72h exposure to melphalan was 8%±1% and 12%±2%, in A375 and Hs294T, respectively. LAs have cytotoxic activity on human melanoma cell lines in a time-, concentration- and agent-dependant manner. Apoptosis in the cell lines was mediated through activity of caspases-3 and caspases-8. Copyright © 2016 Sociedade Brasileira de Anestesiologia. Published by Elsevier Editora Ltda. All rights reserved.

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

    Science.gov (United States)

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

    2015-01-01

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

  5. Fast intracellular dissolution and persistent cellular uptake of silver nanoparticles in CHO-K1 cells: implication for cytotoxicity

    DEFF Research Database (Denmark)

    Jiang, Xiumei; Miclaus, Teodora; Wang, Liming

    2015-01-01

    species, and cytotoxicity of Ag NPs (15.9 ± 7.6 nm) in Chinese hamster ovary cell subclone K1 cells, a cell line recommended by the OECD for genotoxicity studies. Quantification of intracellular nanoparticle uptake and ion release was performed through inductively coupled plasma mass spectrometry. X....... Subsequent cytotoxicity studies show that Ag NPs decrease cell viability and increase ROS production. Pre-incubation with N-acetyl-l-cysteine, an efficient antioxidant and Ag+ chelator, diminished the cytotoxicity caused by Ag NPs or Ag+ exposure. Our study suggests that the cytotoxicity mechanism of Ag NPs...

  6. Melanoma cells treated with GGTI and IFN-gamma allow murine vaccination and enhance cytotoxic response against human melanoma cells.

    Directory of Open Access Journals (Sweden)

    Guillaume Sarrabayrouse

    Full Text Available BACKGROUND: Suboptimal activation of T lymphocytes by melanoma cells is often due to the defective expression of class I major histocompatibility antigens (MHC-I and costimulatory molecules. We have previously shown that geranylgeranyl transferase inhibition (done with GGTI-298 stimulates anti-melanoma immune response through MHC-I and costimulatory molecule expression in the B16F10 murine model [1]. METHODOLOGY/PRINCIPAL FINDINGS: In this study, it is shown that vaccination with mIFN-gand GGTI-298 pretreated B16F10 cells induces a protection against untreated tumor growth and pulmonary metastases implantation. Furthermore, using a human melanoma model (LB1319-MEL, we demonstrated that in vitro treatment with hIFN-gamma and GGTI-298 led to the up regulation of MHC-I and a costimulatory molecule CD86 and down regulation of an inhibitory molecule PD-1L. Co-culture experiments with peripheral blood mononuclear cells (PBMC revealed that modifications induced by hIFN-gamma and GGTI-298 on the selected melanoma cells, enables the stimulation of lymphocytes from HLA compatible healthy donors. Indeed, as compared with untreated melanoma cells, pretreatment with hIFN-gamma and GGTI-298 together rendered the melanoma cells more efficient at inducing the: i activation of CD8 T lymphocytes (CD8+/CD69+; ii proliferation of tumor-specific CD8 T cells (MelanA-MART1/TCR+; iii secretion of hIFN-gamma; and iv anti-melanoma specific cytotoxic cells. CONCLUSIONS/SIGNIFICANCE: These data indicate that pharmacological treatment of melanoma cell lines with IFN-gamma and GGTI-298 stimulates their immunogenicity and could be a novel approach to produce tumor cells suitable for vaccination and for stimulation of anti-melanoma effector cells.

  7. Suppressor T cells, distinct from "veto cells," are induced by alloantigen priming and mediate transferable suppression of cytotoxic T lymphocyte responses in vivo

    DEFF Research Database (Denmark)

    Owens, T; Crispe, I N

    1985-01-01

    Primary and secondary cytotoxic T lymphocyte responses to minor alloantigens can be suppressed by priming host mice with a high dose (10(8) cells) of alloantigenic donor spleen cells (SC). Such suppression is antigen specific and transferable into secondary hosts with T cells. One interpretation...... of this is that antigen-specific host suppressor T cells (Ts) are activated. Alternatively, donor Lyt-2+ T cells, introduced in the priming inoculum, may inactivate host CTL precursors (CTLp) that recognize the priming (donor) alloantigens. Donor cells that act in this way are termed veto T cells. The experiments...... for the transfer of suppression of a secondary CTL response to B10 minors was of the host Thy-1 allotype, and so originated in the host spleen and was not introduced in the priming inoculum. Secondly, antigen-specific Ts generated in CBA female mice against B10 minors could act on CTL responses to an unequivocally...

  8. Cytotoxic activity to acute myeloid leukemia cells by Antp-TPR hybrid peptide targeting Hsp90.

    Science.gov (United States)

    Horibe, Tomohisa; Kawamoto, Megumi; Kohno, Masayuki; Kawakami, Koji

    2012-07-01

    We previously reported that Antp-TPR hybrid peptide inhibited the interaction of Hsp90 with TPR2A and had selective cytotoxic activity discriminating between normal and cancer cells to induce cancer cell death. In this study, we investigated the cytotoxic activity of Antp-TPR peptide toward acute myeloid leukemia (AML) cells. It was demonstrated that Antp-TPR peptide induced AML cell death in cell lines such as U937, K562, THP-1, and HL-60 via activation of caspases 3 and 7, and disruption of mitochondrial membrane potential. Conversely, Antp-TPR peptide did not reduce the viability of normal cells including peripheral blood mononuclear cells (PBMCs), although both geldanamycin and 17-AAG, small-molecule inhibitors of Hsp90, mediated cytotoxicity to these normal cells at low concentrations. In addition, mutation analysis of TPR peptide demonstrated that the highly conserved amino acids Lys and Arg were critical to the cytotoxic activity. These results indicated that Antp-TPR hybrid peptide would provide potent and selective therapeutic options in the treatment of AML.

  9. [Cytotoxicity of polyphenolic/flavonoid compounds in a leukaemia cell culture].

    Science.gov (United States)

    Josipović, Pavle; Orsolić, Nada

    2008-12-01

    Flavonoid components of propolis are biologically active substances with antioxidative, immunostimulative, immunomodulative, and anti-inflamatory properties. The aim of the study was to investigate their cytotoxic effect on different leukaemia cell lines. For this purpose we used five different flavonoids (quercetin, caffeic acid, chrysin, naringenin, and naringin) and five types of leukemia cell lines (MOLT, JURKAT, HL-60, RAJI and U937). Cells were cultured at 37 degrees C in the RPMI-1640 medium supplemented with 10% FCS in humified atmosphere with 5% of CO2. Flavonoids were added in the following concentrations: 100 microg mL(-1), 50 microg mL(-1), 25 microg mL(-1), or 12.5 microg mL(-1). The results show different dose- and cell-type-dependent cytotoxicity. Among the flavonoids, quercetin showed the strongest cytotoxic effect in all cell lines. Caffeic acid and chrisyn also expressed a high level of cytotoxicty. Treatment of U937 and HL-60 cell lines with low concentrations of chrisyn or naringenin stimulated cell proliferation. These results suggest a biphase effect of the tested compounds on monocyte cell lines. Cytotoxicity and growth stimulation mechanisms caused directly by flavonoids should further be investigated on the molecular level.

  10. Melanoma inhibitor of apoptosis protein (ML-IAP) specific cytotoxic T lymphocytes cross-react with an epitope from the auto-antigen SS56

    DEFF Research Database (Denmark)

    Baek Sørensen, Rikke; Faurschou, Mikkel; Troelsen, Lone;

    2009-01-01

    A large proportion of melanoma patients host a spontaneous T-cell response specifically against ML-IAP-derived peptides. In this study, we describe that some ML-IAP-specific cytotoxic T cells isolated from melanoma patients cross react with an epitope from the auto-antigen SS56. SS56 is a recently...... described target of autoantibody responses in Sjögren's syndrome (SS) as well as systemic lupus erythematosus (SLE). Here, we describe that SS56 is also an auto-antigen for T cells in SS and SLE. Hence, SS56-specific T cells could readily be detected in circulation and among the infiltrating cells of SLE...... skin lesions. SS56-specific T cells were able to lyse target cells presenting the peptide epitope on the surface. Notably, SS56-specific CD8 T cells isolated from an SS patient cross reacted with the ML-IAP epitope. This early evidence of a target for auto-reactive CTL in SS and SLE patients...

  11. The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung fibroblast cells.

    Science.gov (United States)

    Smith, Leah J; Holmes, Amie L; Kandpal, Sanjeev Kumar; Mason, Michael D; Zheng, Tongzhang; Wise, John Pierce

    2014-08-01

    Cobalt exposure is increasing as cobalt demand rises worldwide due to its use in enhancing rechargeable battery efficiency, super-alloys, and magnetic products. Cobalt is considered a possible human carcinogen with the lung being a primary target. However, few studies have considered cobalt-induced toxicity in human lung cells. Therefore, in this study, we sought to determine the cytotoxicity and genotoxicity of particulate and soluble cobalt in human lung cells. Cobalt oxide and cobalt chloride were used as representative particulate and soluble cobalt compounds, respectively. Exposure to both particulate and soluble cobalt induced a concentration-dependent increase in cytotoxicity, genotoxicity, and intracellular cobalt ion levels. Based on intracellular cobalt ion levels, we found that soluble cobalt was more cytotoxic than particulate cobalt while particulate and soluble cobalt induced similar levels of genotoxicity. However, soluble cobalt induced cell cycle arrest indicated by the lack of metaphases at much lower intracellular cobalt concentrations compared to cobalt oxide. Accordingly, we investigated the role of particle internalization in cobalt oxide-induced toxicity and found that particle-cell contact was necessary to induce cytotoxicity and genotoxicity after cobalt exposure. These data indicate that cobalt compounds are cytotoxic and genotoxic to human lung fibroblasts, and solubility plays a key role in cobalt-induced lung toxicity.

  12. Ptaquiloside-induced cytotoxicity in Crandall feline kidney and HGC-27 cells.

    Science.gov (United States)

    Yurdakok, Begum; Kismali, Gorkem; Ozen, Dogukan

    2014-10-01

    Ptaquiloside (PTA) is a potent genotoxic carcinogenic compound, which is found in bracken ferns and predominantly causes gastric tumors in humans, as well as bladder tumors and chronic enzootic hematuria in cattle. The underlying molecular mechanisms of PTA remain a topic for interdisciplinary investigation. The aim of the present study was to determine the possible cytotoxic effect of 24 h of PTA exposure in Crandall feline kidney (CrFK) and human gastric cells (the HGC-27 cell line) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactose dehydrogenase (LDH) analysis. The cytotoxic effects of PTA (0.0005-500 μg/ml) were found to increase in a dose-dependent manner, whereby the half maximal inhibitory concentration values were 11.17 and 11.86 μg/ml in the CrFK cells, and 2.03 and 2.56 μg/ml in the HGC-27 cells, by LDH and MTT assay, respectively. The results of the present study are consistent with those of previous studies associated with the cytotoxicity of PTA; however, cytotoxicity was identified to occur at significantly lower doses. This cytotoxic effect in vitro at particularly high doses may be linked to the initiation of carcinogenesis as a result of oxidative stress.

  13. Cytotoxicity Induced by Engineered Silver Nanocrystallites is Dependent on Surface Coatings and Cell Types

    Energy Technology Data Exchange (ETDEWEB)

    Suresh, Anil K [ORNL; Pelletier, Dale A [ORNL; Wang, Wei [ORNL; Morrell-Falvey, Jennifer L [ORNL; Doktycz, Mitchel John [ORNL

    2012-01-01

    Due to their unique antimicrobial properties silver nanocrystallites have garnered substantial recognition and are used extensively in biomedical applications such as wound dressing, surgical instruments and as bone substitute material. They are also released into unintended locations such as the environment or biosphere. Therefore it is imperative to understand the potential interactions, fate and transport of nanoparticles with environmental biotic systems. Although numerous factors including the composition, size, shape, surface charge and capping molecule of nanoparticles are known to influence the cell cytotoxicity, our results demonstrate for the first time that surface coatings are a major determinant in eliciting the potential cytotoxicity and cell interactions of silver nanoparticles. In the present investigation, silver nanocrystallites with nearly uniform size and shape distribution but with different surface coatings, imparting overall high negativity to high positivity, were synthesized. These nanoparticles were poly (diallyldimethylammonium) chloride-Ag, biogenic-Ag, colloidal-Ag (uncoated) and oleate-Ag with zeta potentials +45 5 mV, -12 2 mV, -42 5 mV and -45 5 mV respectively; the particles were thoroughly purified so as to avoid false cytotoxicity interpretations. A systematic investigation on the cytotoxic effects, cellular response and membrane damage caused by these four different silver nanoparticles were evaluated using multiple toxicity measurements on mouse macrophage (RAW-264.7) and lung epithelial (C-10) cell lines. From a toxicity perspective, our results clearly indicated that the cytotoxicity was depend on various factors such as synthesis procedure, surface coat or surface charge and the cell-type for the different silver nanoparticles that were investigated. Poly (diallyldimethylammonium) chloride -Ag was found to be the most toxic, followed by biogenic-Ag and oleate-Ag, whereas uncoated-Ag was found to be least toxic to both

  14. In vitro Acute Cytotoxicity of Abamectin to the Gill Cell Line of Flounder Paralichthy olivaceus

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The cytotoxicity of abamectin to the Gill Cell Line of Flounder (FG cell line) was examined in this study. It was found that the exposure of FG cells to abamectin caused the decreases of both cell growth rate and antioxidant enzyme activities, and the increase of intracellular O2- content. It was proposed that the reduction of antioxidant enzyme activities in FG cells caused the accumulation of O2- content in FG cells, leading to the change of cell morphology and even the death of cells. The results showed that FG cell line is suitable for the evaluation of the acute toxicity of abamectin.

  15. Antiproliferative, Cytotoxic, and Apoptotic Activity of Steroidal Oximes in Cervicouterine Cell Lines

    Directory of Open Access Journals (Sweden)

    Luis Sánchez-Sánchez

    2016-11-01

    Full Text Available Steroidal sapogenins have shown antiproliferative effects against several tumor cell lines; and their effects on human cancer cells are currently under study. Changes in the functionality on the steroidal structure make it possible to modify the biological activity of compounds. Herein, we report the synthesis and in vitro antitumor activity of two steroidal oxime compounds on cervical cancer cells. These derivatives were synthesized from the steroidal sapogenin diosgenin in good yields. The in vitro assays show that the steroidal oximes show significant antiproliferative activity compared to the one observed for diosgenin. Cell proliferation, cell death, and the cytotoxic effects were determined in both cervical cancer cells and human lymphocytes. The cancer cells showed apoptotic morphology and an increased presence of active caspase-3, providing the notion of a death pathway in the cell. Significantly, the steroidal oximes did not exert a cytotoxic effect on lymphocytes.

  16. Chloroquine enhances gefitinib cytotoxicity in gefitinib-resistant nonsmall cell lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Mei-Chuan Tang

    Full Text Available Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs, including gefitinib, are effective for non-small cell lung cancer (NSCLC patients with EGFR mutations. However, these patients eventually develop resistance to EGFR-TKI. The goal of the present study was to investigate the involvement of autophagy in gefitinib resistance. We developed gefitinib-resistant cells (PC-9/gef from PC-9 cells (containing exon 19 deletion EGFR after long-term exposure in gefitinib. PC-9/gef cells (B4 and E3 were 200-fold more resistant to gefitinib than PC-9/wt cells. Compared with PC-9/wt cells, both PC-9/gefB4 and PC-9/gefE3 cells demonstrated higher basal LC3-II levels which were inhibited by 3-methyladenine (3-MA, an autophagy inhibitor and potentiated by chloroquine (CQ, an inhibitor of autophagolysosomes formation, indicating elevated autophagy in PC-9/gef cells. 3-MA and CQ concentration-dependently inhibited cell survival of both PC-9wt and PC-9/gef cells, suggesting that autophagy may be pro-survival. Furthermore, gefitinib increased LC3-II levels and autolysosome formation in both PC-9/wt cells and PC-9/gef cells. In PC-9/wt cells, CQ potentiated the cytotoxicity by low gefitinib (3 nM. Moreover, CQ overcame the acquired gefitinib resistance in PC-9/gef cells by enhancing gefitinib-induced cytotoxicity, activation of caspase 3 and poly (ADP-ribose polymerase cleavage. Using an in vivo model xenografting with PC-9/wt and PC-9/gefB4 cells, oral administration of gefitinib (50 mg/kg completely inhibited the tumor growth of PC-9/wt but not PC-9/gefB4cells. Combination of CQ (75 mg/kg, i.p. and gefitinib was more effective than gefitinib alone in reducing the tumor growth of PC-9/gefB4. Our data suggest that inhibition of autophagy may be a therapeutic strategy to overcome acquired resistance of gefitinib in EGFR mutation NSCLC patients.

  17. Cytotoxic CD4 T Cells: Differentiation, Function, and Application to Dengue Virus Infection

    Directory of Open Access Journals (Sweden)

    Yuan Tian

    2016-12-01

    Full Text Available Dengue virus (DENV has spread through most tropical and subtropical areas of the world and represents a serious public health problem. The control of DENV infection has not yet been fully successful due to lack of effective therapeutics or vaccines. Nevertheless, a better understanding of the immune responses against DENV infection may reveal new strategies for eliciting and improving antiviral immunity. T cells provide protective immunity against various viral infections by generating effector cells that cooperate to eliminate antigens and memory cells that can survive for long periods with enhanced abilities to control recurring pathogens. Following activation, CD8 T cells can migrate to sites of infection and kill infected cells, whereas CD4 T cells contribute to the elimination of pathogens by trafficking to infected tissues and providing help to innate immune responses, B cells, as well as CD8 T cells. However, it is now evident that CD4 T cells can also perform cytotoxic functions and induce the apoptosis of target cells. Importantly, accumulating studies demonstrate that cytotoxic CD4 T cells develop following DENV infections and may play a crucial role in protecting the host from severe dengue disease. We review our current understanding of the differentiation and function of cytotoxic CD4 T cells, with a focus on DENV infection, and discuss the potential of harnessing these cells for the prevention and treatment of DENV infection and disease.

  18. Cytotoxic CD4 T Cells: Differentiation, Function, and Application to Dengue Virus Infection.

    Science.gov (United States)

    Tian, Yuan; Sette, Alessandro; Weiskopf, Daniela

    2016-01-01

    Dengue virus (DENV) has spread through most tropical and subtropical areas of the world and represents a serious public health problem. The control of DENV infection has not yet been fully successful due to lack of effective therapeutics or vaccines. Nevertheless, a better understanding of the immune responses against DENV infection may reveal new strategies for eliciting and improving antiviral immunity. T cells provide protective immunity against various viral infections by generating effector cells that cooperate to eliminate antigens and memory cells that can survive for long periods with enhanced abilities to control recurring pathogens. Following activation, CD8 T cells can migrate to sites of infection and kill infected cells, whereas CD4 T cells contribute to the elimination of pathogens by trafficking to infected tissues and providing help to innate immune responses, B cells, as well as CD8 T cells. However, it is now evident that CD4 T cells can also perform cytotoxic functions and induce the apoptosis of target cells. Importantly, accumulating studies demonstrate that cytotoxic CD4 T cells develop following DENV infections and may play a crucial role in protecting the host from severe dengue disease. We review our current understanding of the differentiation and function of cytotoxic CD4 T cells, with a focus on DENV infection, and discuss the potential of harnessing these cells for the prevention and treatment of DENV infection and disease.

  19. Effects of spider Macrothele raven venom on cell proliferation and cytotoxicity in HeLa cells

    Institute of Scientific and Technical Information of China (English)

    Li GAO; Bao-en SHAN; Jing CHEN; Jiang-hui LIU; Da-xiang SONG; Bao-cheng ZHU

    2005-01-01

    Aim: To examine the effect of venom from the spider Macrothele raven on cell proliferation and cytotoxicity in human cervical carcinoma, HeLa cells. Methods:Morphological and biochemical signs of apoptosis appeared using acridine orange-ethidium bromide (AO/EB) staining. Marked morphological changes in HeLa cells after treatment with spider venom were observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Cell proliferation and cytotoxicity were determined by [methyl-3H] thymidine assay ([3H]TdR) and lactate dehydrogenase (LDH) release, respectively. DNA fragmentation and cell cycle distribution were monitored using flow cytometry. In addition, Western blot analysis was used to evaluate the level of caspase-3 expression. In vivo examination of the inhibition of the size of tumors in nude mice treated with spider venom was measured. Results: Marked morphological changes were observed using AO/EB staining, SEM and TEM assay. Spider venom at concentrations of 10-40 mg/L caused dose- and time-dependent inhibition of HeLa cell proliferation.The ratio of apoptosis and necrosis increased. The activity of caspase-3 was upregulated after spider venom treatment. In vivo study of tumor size revealed that tumors significantly decreased in size from controls to tumors treated for 3 weeks with spider venom (P<0.05). Conclusion: The inhibition of HeLa cells by the venom of the spider Macrothele raveni was carried out in three ways: induction of apoptosis, necrosis of toxicity damage and direct lysis. Spider venom is a novel anti-tumor material both in vitro and in vivo.

  20. Suppression of NK cell-mediated cytotoxicity against PRRSV-infected porcine alveolar macrophages in vitro.

    Science.gov (United States)

    Cao, Jun; Grauwet, Korneel; Vermeulen, Ben; Devriendt, Bert; Jiang, Ping; Favoreel, Herman; Nauwynck, Hans

    2013-06-28

    The adaptive immunity against PRRSV has already been studied in depth, but only limited data are available on the innate immune responses against this pathogen. In the present study, we analyzed the interaction between porcine natural killer (NK) cells and PRRSV-infected primary porcine alveolar macrophages (PAMs), since NK cells are one of the most important components of innate immunity and PAMs are primary target cells of PRRSV infection. NK cytotoxicity assays were performed using enriched NK cells as effector cells and virus-infected or mock-inoculated PAMs as target cells. The NK cytotoxicity against PRRSV-infected PAMs was decreased starting from 6h post inoculation (hpi) till the end of the experiment (12 hpi) and was significantly lower than that against pseudorabies virus (PrV)-infected PAMs. UV-inactivated PRRSV also suppressed NK activity, but much less than infectious PRRSV. Furthermore, co-incubation with PRRSV-infected PAMs inhibited degranulation of NK cells. Finally, using the supernatant of PRRSV-infected PAMs collected at 12 hpi showed that the suppressive effect of PRRSV on NK cytotoxicity was not mediated by soluble factors. In conclusion, PRRSV-infected PAMs showed a reduced susceptibility toward NK cytotoxicity, which may represent one of the multiple evasion strategies of PRRSV.

  1. Cytotoxicity testing of silver-containing burn treatments using primary and immortal skin cells.

    Science.gov (United States)

    Boonkaew, Benjawan; Kempf, Margit; Kimble, Roy; Cuttle, Leila

    2014-12-01

    A novel burn wound hydrogel dressing has been previously developed which is composed of 2-acrylamido-2-methylpropane sulfonic acid sodium salt with silver nanoparticles (silver AMPS). This study compared the cytotoxicity of this dressing to the commercially available silver products; Acticoat™, PolyMem Silver(®) and Flamazine™ cream. Human keratinocytes (HaCaT and primary HEK) and normal human fibroblasts (NHF) were exposed to dressings incubated on Nunc™ polycarbonate inserts for 24, 48 and 72h. Four different cytotoxicity assays were performed including; Trypan Blue cell count, MTT, Celltiter-Blue™ and Toluidine Blue surface area assays. The results were expressed as relative cell viability compared to an untreated control. The cytotoxic effects of Acticoat™ and Flamazine™ cream were dependent on exposure time and cell type. After 24h exposure, Acticoat™ and Flamazine™ cream were toxic to all tested cell lines. Surprisingly, HaCaTs treated with Acticoat™ and Flamazine™ had an improved ability to survive at 48 and 72h while HEKs and NHFs had no improvement in survival with any treatment. The novel silver hydrogel and PolyMem Silver(®) showed low cytotoxicity to all tested cell lines at every time interval and these results support the possibility of using the novel silver hydrogel as a burn wound dressing. Researchers who rely on HaCaT cells as an accurate keratinocyte model should be aware that they can respond differently to primary skin cells.

  2. Phenolics-saponins rich fraction of defatted kenaf seed meal exhibits cytotoxicity towards cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    Latifah Saiful Yazan; Napsiah Abd Rahman; Kim Wei Chan; Wan Nor Hafiza Wan Abd Ghani; Yin Sim Tor; Jhi Biau Foo

    2016-01-01

    Objectives: To determine the cytotoxicity of crude ethanolic extract, n-butanol fraction and aqueous fraction on selected cancer cell lines, and to observe the morphological changes of the cancer cells treated with n-butanol fraction. Methods: The cytotoxic effect of n-butanol fraction, crude ethanolic extract and aqueous fraction on breast cancer (MCF-7 and MDA-MB-231), colon cancer (HT29), lung cancer (A549), cervical cancer (HeLa) and normal mouse fibroblast (3T3) cell lines was deter-mined using MTT assay. The morphological changes of the treated cells were observed under an inverted light microscope. Results: n-Butanol fraction was the most cytotoxic towards HT29 and MCF-7 cells in a dose-dependent manner compared to crude ethanolic extract and aqueous fraction (P Conclusions: In conclusion, n-butanol fraction was more cytotoxic than crude ethanolic extract and aqueous fraction towards the selected cancerous cell lines and induced apoptosis in HT29 cells.

  3. Dengue virus infection elicits highly polarized CX3CR1+ cytotoxic CD4+ T cells associated with protective immunity.

    Science.gov (United States)

    Weiskopf, Daniela; Bangs, Derek J; Sidney, John; Kolla, Ravi V; De Silva, Aruna D; de Silva, Aravinda M; Crotty, Shane; Peters, Bjoern; Sette, Alessandro

    2015-08-04

    Dengue virus (DENV) is a rapidly spreading pathogen with unusual pathogenesis, and correlates of protection from severe dengue disease and vaccine efficacy have not yet been established. Although DENV-specific CD8(+) T-cell responses have been extensively studied, the breadth and specificity of CD4(+) T-cell responses remains to be defined. Here we define HLA-restricted CD4(+) T-cell epitopes resulting from natural infection with dengue virus in a hyperepidemic setting. Ex vivo flow-cytometric analysis of DENV-specific CD4(+) T cells revealed that the virus-specific cells were highly polarized, with a strong bias toward a CX3CR1(+) Eomesodermin(+) perforin(+) granzyme B(+) CD45RA(+) CD4 CTL phenotype. Importantly, these cells correlated with a protective HLA DR allele, and we demonstrate that these cells have direct ex vivo DENV-specific cytolytic activity. We speculate that cytotoxic dengue-specific CD4(+) T cells may play a role in the control of dengue infection in vivo, and this immune correlate may be a key target for dengue virus vaccine development.

  4. The anti-tumor immune response of specific cytotoxic T lymphocytes induced by breast cancer stem cells RNA-DCs vaccine%乳腺癌干细胞RNA-DCs疫苗诱导特异性细胞毒性T淋巴细胞抗肿瘤免疫应答

    Institute of Scientific and Technical Information of China (English)

    钟志宏; 鄢俊; 施华球

    2013-01-01

    目的 制备负载乳腺癌干细胞RNA的树突状细胞(DCs)疫苗,研究其诱导的特异性细胞毒性T淋巴细胞(CTLs)的抗肿瘤免疫反应.方法 采用细胞毒性试剂盒检测2种乳腺癌干细胞疫苗[(A组,CD44+ CD24-+MCF-7-CTLs)、(B组,MCF-7-CTLs)]和DC-CTLs(C组)的体外细胞杀伤能力.取24只小鼠均分为四组:A1组皮下接种活化的A组疫苗+MCF-7乳腺癌细胞;B1组接种活化的B组疫苗+MCF-7乳腺癌细胞;C1组皮下注射活化的DCs-CTLs+ MCF-7乳腺癌细胞;D1组单用MCF-7乳腺癌细胞作为对照.分析裸鼠接种后肿瘤在体内的生长情况.结果 体外对CD44+CD24-+ MCF-7乳腺癌细胞杀伤能力强度:A组>B组>C组(P<0.05).体外对MCF-7乳腺癌细胞杀伤能力强度:B组>A组>C组(P<0.05).在体成瘤实验显示,B1组和A1组的成瘤时间分别为第7周和第6周,明显长于D1组(第1周)和C1组(第2周)(P<0.05).结论 CD44+CD24乳腺癌干细胞RNA-DCs疫苗诱导的CTLs能够产生特异性免疫应答,从而抑制乳腺癌细胞成瘤.%Objective To prepare dendritic cells(DCs) vaccine loaded breast cancer stem cells RNA and explore the anti-tumor immune response of specific cytotoxic T lymphocytes (CTLs).Methods Killing capacity of breast cancer stem cells vaccines in vitro was detected by cytotoxicity kit in groups of A(treated with CD44+ CD24-+MCF-7-CTLs),B(treated with MCF-7-CTLs) and C (treated with DC-CTLs).Twenty-four mice were equally assigned into 4 groups of A1 (subcutaneous injection of activated CD44+ CD24-+ MCF-7-CTLs + MCF-7 breast cancer cells),B1 (activated MCF-7-CTLs+ MCF-7 breast cancer cells),C1 (activated DC-CTLs+ MCF-7 breast cancer cells) and D1(MCF-7 breast cancer cells alone).The growth of xenografts was observed.Results In vitro,the killing capacity on CD44+ CD24-+MCF-7 breast cancer cells was in an order of group A >group B >group C(P<0.05) and that on MCF-7 breast cancer cells was in an order of group B >group A > grop C(P<0.05).The

  5. Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity

    DEFF Research Database (Denmark)

    Lavrsen, Kirstine; Madsen, Caroline B; Rasch, Morten G

    2013-01-01

    not covered by immunological tolerance in MUC1 humanized mice and man. The objective of this study was to determine if mouse antibodies to this Tn-MUC1 epitope induce antibody-dependent cellular cytotoxicity (ADCC) pivotal for their potential use in cancer immunotherapy. Binding affinity of mAb 5E5 directed...... is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity suggesting that antibodies targeting glycopeptide epitopes on mucins are strong candidates for cancer-specific immunotherapies.......Protein glycosylation often changes during cancer development, resulting in the expression of cancer-associated carbohydrate antigens. In particular mucins such as MUC1 are subject to these changes. We previously identified an immunodominant Tn-MUC1 (GalNAc-α-MUC1) cancer-specific epitope...

  6. 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.

  7. Cytotoxic Activity and Antiproliferative Effects of Crude Skin Secretion from Physalaemus nattereri (Anura: Leptodactylidae on in vitro Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Andréa Cruz e Carvalho

    2015-10-01

    Full Text Available Anuran secretions are rich sources of bioactive molecules, including antimicrobial and antitumoral compounds. The aims of this study were to investigate the therapeutic potential of Physalaemus nattereri skin secretion against skin cancer cells, and to assess its cytotoxic action mechanisms on the murine melanoma cell line B16F10. Our results demonstrated that the crude secretion reduced the viability of B16F10 cells, causing changes in cell morphology (e.g., round shape and structure shrinkage, reduction in mitochondrial membrane potential, increase in phosphatidylserine exposure, and cell cycle arrest in S-phase. Together, these changes suggest that tumor cells die by apoptosis. This skin secretion was also subjected to chromatographic fractioning using RP-HPLC, and eluted fractions were assayed for antiproliferative and antibacterial activities. Three active fractions showed molecular mass components in a range compatible with peptides. Although the specific mechanisms causing the reduced cell viability and cytotoxicity after the treatment with crude secretion are still unknown, it may be considered that molecules, such as the peptides found in the secretion, are effective against B16F10 tumor cells. Considering the growing need for new anticancer drugs, data presented in this study strongly reinforce the validity of P. nattereri crude secretion as a rich source of new anticancer molecules.

  8. Selenium cytotoxicity in cancer.

    Science.gov (United States)

    Wallenberg, Marita; Misra, Sougat; Björnstedt, Mikael

    2014-05-01

    Selenium is an essential trace element with growth-modulating properties. Decades of research clearly demonstrate that selenium compounds inhibit the growth of malignant cells in diverse experimental model systems. However, the growth-modulating and cytotoxic mechanisms are diverse and far from clear. Lately, a remarkable tumour selective cytotoxicity of selenium compounds has been shown, indicating the potential of selenium in the treatment of cancer. Of particular interest are the redox-active selenium compounds exhibiting cytotoxic potential to tumour cells. These selenium compounds elicit complex patterns of pharmacodynamics and pharmacokinetics, leading to cell death pathways that differ among compounds. Modern oncology often focuses on targeted ligand-based therapeutic strategies that are specific to their molecular targets. These drugs are initially efficient, but the tumour cells often rapidly develop resistance against these drugs. In contrast, certain redox-active selenium compounds induce complex cascades of pro-death signalling at pharmacological concentrations with superior tumour specificity. The target molecules are often the ones that are important for the survival of cancer cells and often implicated in drug resistance. Therefore, the chemotherapeutic applications of selenium offer great possibilities of multi-target attacks on tumour cells. This MiniReview focuses on the tumour-specific cytotoxic effects of selenium, with special emphasis on cascades of cellular events induced by the major groups of pharmacologically active selenium compounds. Furthermore, the great pharmacological potential of selenium in the treatment of resistant cancers is discussed.

  9. HIV-TB coinfection impairs CD8(+) T-cell differentiation and function while dehydroepiandrosterone improves cytotoxic antitubercular immune responses.

    Science.gov (United States)

    Suarez, Guadalupe V; Angerami, Matías T; Vecchione, María B; Laufer, Natalia; Turk, Gabriela; Ruiz, Maria J; Mesch, Viviana; Fabre, Bibiana; Maidana, Patricia; Ameri, Diego; Cahn, Pedro; Sued, Omar; Salomón, Horacio; Bottasso, Oscar A; Quiroga, María F

    2015-09-01

    Tuberculosis (TB) is the leading cause of death among HIV-positive patients. The decreasing frequencies of terminal effector (TTE ) CD8(+) T cells may increase reactivation risk in persons latently infected with Mycobacterium tuberculosis (Mtb). We have previously shown that dehydroepiandrosterone (DHEA) increases the protective antitubercular immune responses in HIV-TB patients. Here, we aimed to study Mtb-specific cytotoxicity, IFN-γ secretion, memory status of CD8(+) T cells, and their modulation by DHEA during HIV-TB coinfection. CD8(+) T cells from HIV-TB patients showed a more differentiated phenotype with diminished naïve and higher effector memory and TTE T-cell frequencies compared to healthy donors both in total and Mtb-specific CD8(+) T cells. Notably, CD8(+) T cells from HIV-TB patients displayed higher Terminal Effector (TTE ) CD45RA(dim) proportions with lower CD45RA expression levels, suggesting a not fully differentiated phenotype. Also, PD-1 expression levels on CD8(+) T cells from HIV-TB patients increased although restricted to the CD27(+) population. Interestingly, DHEA plasma levels positively correlated with TTE in CD8(+) T cells and in vitro DHEA treatment enhanced Mtb-specific cytotoxic responses and terminal differentiation in CD8(+) T cells from HIV-TB patients. Our data suggest that HIV-TB coinfection promotes a deficient CD8(+) T-cell differentiation, whereas DHEA may contribute to improving antitubercular immunity by enhancing CD8(+) T-cell functions during HIV-TB coinfection. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Detection of cytotoxic activity on Vero cells in clinical isolates of Serratia marcescens

    Directory of Open Access Journals (Sweden)

    G.V. Carbonell

    1997-11-01

    Full Text Available Cytotoxin production was studied in 60 Serratia marcescens strains isolated from hospitalized patients. Association of cytotoxic activity with serotype, source of isolation and presence of plasmids was also evaluated. Thirteen of the 60 S. marcescens strains produced a cytotoxic effect on Vero cells. These strains were isolated from distinct clinical sources and classified into seven different serotypes (O1:H7; O4:NM; O10:NT; O19:NM; O6,14:H4; O6,14:NM and O6,14:H1. No relationship was observed between cytotoxic activity and clinical source or serotypes of the strains. Plasmids from five cytotoxin-producing S. marcescens strains were transferred to E. coli K12/711. The transconjugants did not exhibit cytotoxicity, indicating that the cytotoxic effect is not plasmid-mediated among these strains. Although a cytotoxic activity was demonstrated in filtrates of some S. marcescens strains, further studies should be performed to assess the role of this toxin in pathogenesis

  11. Neutral red uptake assay for the estimation of cell viability/cytotoxicity.

    Science.gov (United States)

    Repetto, Guillermo; del Peso, Ana; Zurita, Jorge L

    2008-01-01

    The neutral red uptake assay provides a quantitative estimation of the number of viable cells in a culture. It is one of the most used cytotoxicity tests with many biomedical and environmental applications. It is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in the lysosomes. Most primary cells and cell lines from diverse origin may be successfully used. Cells are seeded in 96-well tissue culture plates and are treated for the appropriate period. The plates are then incubated for 2 h with a medium containing neutral red. The cells are subsequently washed, the dye is extracted in each well and the absorbance is read using a spectrophotometer. The procedure is cheaper and more sensitive than other cytotoxicity tests (tetrazolium salts, enzyme leakage or protein content). Once the cells have been treated, the assay can be completed in <3 h.

  12. Screening ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay

    Science.gov (United States)

    An Adherent Cell Differentiation and Cytotoxicity (ACDC) in vitro assay with mouse embryonic stem cells was used to screen the ToxCast Phase I chemical library for effects on cellular differentiation and cell number. The U.S. Environmental Protection Agency (EPA) established the ...

  13. A sialic acid-specific lectin from the mushroom Paecilomyces Japonica that exhibits hemagglutination activity and cytotoxicity.

    Science.gov (United States)

    Park, Jee Hun; Ryu, Chang Soo; Kim, Ha Na; Na, Young Jun; Park, Hyun Joo; Kim, Hahyung

    2004-12-01

    The mushroom Paecilomyces japonica, grown on the silkworm larvae, has been used in Asia as a nutraceutical, tea, and Chinese medicine. In the present study, a sialic acid-specific lectin has been purified from the mushroom P. japonica using affinity chromatography on a fetuin-agarose column. Electrophoretical analyses indicated that this lectin, designated P. japonica agglutinin (PJA), is an acidic protein with a molecular mass of 16 kDa, and has no intermolecular disulfide bonds. PJA induced hemagglutination activity in human ABO, mouse, rat, and rabbit erythrocytes. This activity was inhibited by sialic acid and sialoglycoproteins, but not by any other carbohydrates. PJA was stable at pH 4.0-8.0, and at temperatures below 55 degrees C. The activity of PJA was independent of EDTA and divalent cations. In addition, PJA exerts cytotoxic effects on the following cancer cell lines: human stomach cancer SNU-1, human pancreas cancer AsPc-1, and human breast cancer MDA-MB-231.

  14. Synthesis and cytotoxicity evaluation of 4-amino-4-dehydroxylarctigenin derivatives in glucose-starved A549 tumor cells.

    Science.gov (United States)

    Lei, Min; Gan, Xianwen; Zhao, Kun; Yu, Qiang; Hu, Lihong

    2015-02-01

    The natural product arctigenin (ATG) demonstrated preferential cytotoxicity to cancer cells under glucose starvation. A series of 4-amino-4-dehydroxylarctigenin derivatives based on lead compound ATG were designed and synthesized by bioisosteric modifications. Their cytotoxicities were evaluated in glucose-starved A549 tumor cells and the results indicated that the 4-amino-4-dehydroxylarctigenin showed more potent cytotoxicity than arctigenin, and the further substituent group on 4-amino would result in the cytotoxicities decreased significantly. 4-Substituted-arctigenin could selectively target on glucose-starved A549 tumor cells which provide an alternative strategy for anticancer drug development with minimal normal tissue toxicity.

  15. Bystander cells enhance NK cytotoxic efficiency by reducing search time.

    Science.gov (United States)

    Zhou, Xiao; Zhao, Renping; Schwarz, Karsten; Mangeat, Matthieu; Schwarz, Eva C; Hamed, Mohamed; Bogeski, Ivan; Helms, Volkhard; Rieger, Heiko; Qu, Bin

    2017-03-13

    Natural killer (NK) cells play a central role during innate immune responses by eliminating pathogen-infected or tumorigenic cells. In the microenvironment, NK cells encounter not only target cells but also other cell types including non-target bystander cells. The impact of bystander cells on NK killing efficiency is, however, still elusive. In this study we show that the presence of bystander cells, such as P815, monocytes or HUVEC, enhances NK killing efficiency. With bystander cells present, the velocity and persistence of NK cells were increased, whereas the degranulation of lytic granules remained unchanged. Bystander cell-derived H2O2 was found to mediate the acceleration of NK cell migration. Using mathematical diffusion models, we confirm that local acceleration of NK cells in the vicinity of bystander cells reduces their search time to locate target cells. In addition, we found that integrin β chains (β1, β2 and β7) on NK cells are required for bystander-enhanced NK migration persistence. In conclusion, we show that acceleration of NK cell migration in the vicinity of H2O2-producing bystander cells reduces target cell search time and enhances NK killing efficiency.

  16. Evaluation of cytotoxicity of Moringa oleifera Lam. callus and leaf extracts on Hela cells

    Directory of Open Access Journals (Sweden)

    Abbas Jafarain

    2014-01-01

    Full Text Available Background: There are considerable attempts worldwide on herbal and traditional compounds to validate their use as anti-cancer drugs. Plants from Moringaceae family including Moringa oleifera possess several activities such as antitumor effect on tumor cell lines. In this study we sought to determine if callus and leaf extracts of M. oleifera possess any cytotoxicity. Materials and Methods: Ethanol-water (70-30 extracts of callus and leaf of M. oleifera were prepared by maceration method. The amount of phenolic compounds of the extracts was determined by Folin Ciocalteu method. The cytotoxicity of the extracts against Hela tumor cells was carried out using MTT assay. Briefly, cells were seeded in microplates and different concentrations of the extract were added. Cells were incubated for 48 h and their viability was evaluated by addition of tetrazolium salt solution. After 3 h medium was aspirated, dimethyl sulfoxide was added and absorbance was determined at 540 nm with an ELISA plate reader. Cytotoxicity was considered when more than 50% reduction on cell survival was observed. Results: Callus and leaf extracts of M. oleifera significantly decreased the viability of Hela cells in a concentration-dependent manner. However, leaf extract of M. oleifera were more potent than that of callus extract. Conclusion: As the content of phenolic compounds of leaf extract was higher than that of callus extract, it can be concluded that phenolic compounds are involved in the cytotoxicity of M. oleifera.

  17. Size-dependent cytotoxicity of europium doped NaYF{sub 4} nanoparticles in endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Shizhu; Zhang, Cuimiao; Jia, Guang; Duan, Jianlei; Wang, Shuxiang, E-mail: wsx@hbu.edu.cn; Zhang, Jinchao, E-mail: jczhang6970@163.com

    2014-10-01

    Lanthanide-doped sodium yttrium fluoride (NaYF{sub 4}) nanoparticles exhibit novel optical properties which make them be widely used in various fields. The extensive applications increase the chance of human exposure to these nanoparticles and thus raise deep concerns regarding their riskiness. In the present study, we have synthesized europium doped NaYF{sub 4} (NaYF{sub 4}:Eu{sup 3+}) nanoparticles with three diameters and used endothelial cells (ECs) as a cell model to explore the potential toxic effect. The cell viability, cytomembrane integrity, cellular uptake, intracellular localization, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), apoptosis detection, caspase-3 activity and expression of inflammatory gene were studied. The results indicated that these nanoparticles could be uptaken into ECs and decrease the cell viability, induce the intracellular lactate dehydrogenase (LDH) release, increase the ROS level, and decrease the cell MMP in a size-dependent manner. Besides that, the cells were suffered to apoptosis with the caspase-3 activation, and the inflammation specific gene expressions (ICAM1 and VCAM1) were also increased. Our results suggest that the damage pathway may be related to the ROS generation and mitochondrial damage. The results provide novel evidence to elucidate their toxicity mechanisms and may be helpful for more rational applications of these compounds in the future. - Highlights: • NaYF{sub 4}:Eu{sup 3+} nanoparticles with three diameters have been synthesized. • NaYF{sub 4}:Eu{sup 3+} nanoparticles could be uptaken by endothelial cells (ECs). • NaYF{sub 4}:Eu{sup 3+} nanoparticles show a significant cytotoxicity on ECs. • The size of NaYF{sub 4}:Eu{sup 3+} nanoparticles may be important to their toxicology effect.

  18. A cationic amphiphilic peptide ABP-CM4 exhibits selective cytotoxicity against leukemia cells.

    Science.gov (United States)

    Chen, Yu Qing; Min, Cui; Sang, Ming; Han, Yang Yang; Ma, Xiao; Xue, Xiao Qing; Zhang, Shuang Quan

    2010-08-01

    Some cationic antibacterial peptides exhibit a broad spectrum of cytotoxic activity against cancer cells, which could provide a new class of anticancer drugs. In the present study, the anticancer activity of ABP-CM4, an antibacterial peptide from Bombyx mori, against leukemic cell lines THP-1, K562 and U937 was evaluated, and the cytotoxicity compared with the effects on non-cancerous mammalian cells, including peripheral blood mononuclear cells (PBMCs), HEK-293 and erythrocytes. ABP-CM4 reduced the number of viable cells of the leukemic cell lines after exposure for 24h. The reduction was concentration dependent, and the IC50 values ranged from 14 to 18 microM. Conversely, ABP-CM4, even at 120 microM, exhibited no cytotoxicity toward HEK-293 or PBMCs, indicating that there was no significant effect on these two types of non-cancer cells. ABP-CM4 at a concentration of 200 microM had no hemolytic activity on mammalian erythrocytes. Together, these results suggested a selective cytotoxicity in leukemia cells. Flow cytometry demonstrated that the binding activity of ABP-CM4 to leukemia cells was much higher than that to HEK-293 or PBMCs, and there was almost no binding to erythrocytes. FITC-labeled ABP-CM4 molecules were examined under a confocal microscope and found to be concentrated at the surface of leukemia cells and changes of the cell membrane were determined by a cell permeability assay, which led us to the conclusion that ABP-CM4 could act at the cell membrane for its anticancer activity on leukemia cells. Collectively, our results indicated that ABP-CM4 has the potential for development as a novel antileukemic agent.

  19. Oxaliplatin regulates expression of stress ligands in ovarian cancer cells and modulates their susceptibility to natural killer cell-mediated cytotoxicity.

    Science.gov (United States)

    Siew, Yin-Yin; Neo, Soek-Ying; Yew, Hui-Chuing; Lim, Shun-Wei; Ng, Yi-Cheng; Lew, Si-Min; Seetoh, Wei-Guang; Seow, See-Voon; Koh, Hwee-Ling

    2015-12-01

    Selected cytotoxic chemicals can provoke the immune system to recognize and destroy malignant tumors. Most of the studies on immunogenic cell death are focused on the signals that operate on a series of receptors expressed by dendritic cells to induce tumor antigen-specific T-cell responses. Here, we explored the effects of oxaliplatin, an immunogenic cell death inducer, on the induction of stress ligands and promotion of natural killer (NK) cell-mediated cytotoxicity in human ovarian cancer cells. The results indicated that treatment of tumor cells with oxaliplatin induced the production of type I interferons and chemokines and enhanced the expression of major histocompatibility complex class I-related chains (MIC) A/B, UL16-binding protein (ULBP)-3, CD155 and TNF-related apoptosis-inducing ligand (TRAIL)-R1/R2. Furthermore, oxaliplatin but not cisplatin treatment enhanced susceptibility of ovarian cancer cells to NK cell-mediated cytolysis. In addition, activated NK cells completely abrogated the growth of cancer cells that were pretreated with oxaliplatin. However, cancer cells pretreated with the same concentration of oxaliplatin alone were capable of potentiating regrowth over a period of time. These results suggest an advantage in combining oxaliplatin and NK cell-based therapy in the treatment of ovarian cancer. Further investigation on such potential combination therapy is warranted.

  20. Carbon dots of different composition and surface functionalization: cytotoxicity issues relevant to fluorescence cell imaging.

    Science.gov (United States)

    Wang, Yanli; Anilkumar, Parambath; Cao, Li; Liu, Jia-Hui; Luo, Pengju G; Tackett, Kenneth N; Sahu, Sushant; Wang, Ping; Wang, Xin; Sun, Ya-Ping

    2011-11-01

    Nanoscale carbon particles have emerged as versatile precursors for a new class of highly fluorescent nanomaterials that resemble semiconductor quantum dots. The surface-passivated fluorescent carbon nanoparticles, dubbed 'carbon dots', were already demonstrated for their potential optical bioimaging applications in vitro and in vivo. In this study, we conducted a systematic cytotoxicity evaluation on the carbon dots prepared by various combinations of precursor carbon nanoparticles and molecules for the particle surface functionalization. The results suggested that the cytotoxicity of carbon dots was dependent on the selection of surface passivation molecules. Those dots showing more significant cytotoxicity at higher concentrations were also evaluated for their effects on the fluorescence imaging of live cells. The implications of the results on the eventual use of carbon dots as cell imaging agents are discussed.

  1. Resazurin Live Cell Assay: Setup and Fine-Tuning for Reliable Cytotoxicity Results.

    Science.gov (United States)

    Rodríguez-Corrales, José Á; Josan, Jatinder S

    2017-01-01

    In vitro cytotoxicity tests allow for fast and inexpensive screening of drug efficacy prior to in vivo studies. The resazurin assay (commercialized as Alamar Blue(®)) has been extensively utilized for this purpose in 2D and 3D cell cultures, and high-throughput screening. However, improper or lack of assay validation can generate unreliable results and limit reproducibility. Herein, we report a detailed protocol for the optimization of the resazurin assay to determine relevant analytical (limits of detection, quantification, and linear range) and biological (growth kinetics) parameters, and, thus, provide accurate cytotoxicity results. Fine-tuning of the resazurin assay will allow accurate and fast quantification of cytotoxicity for drug discovery. Unlike more complicated methods (e.g., mass spectrometry), this assay utilizes fluorescence spectroscopy and, thus, provides a less costly alternative to observe changes in the reductase proteome of the cells.

  2. Cytotoxicity assessment of graphene-based nanomaterials on human dental follicle stem cells.

    Science.gov (United States)

    Olteanu, Diana; Filip, Adriana; Socaci, Crina; Biris, Alexandru Radu; Filip, Xenia; Coros, Maria; Rosu, Marcela Corina; Pogacean, Florina; Alb, Camelia; Baldea, Ioana; Bolfa, Pompei; Pruneanu, Stela

    2015-12-01

    Graphene-oxide (GO) and its most encountered derivatives, thermally reduced graphene oxide (TRGO) and nitrogen-doped graphene (N-Gr), were synthesized and structurally characterized by spectroscopic techniques, like Raman and (13)C MAS solid state NMR. Several biological effects (cytotoxicity, oxidative stress induction, and cellular and mithocondrial membrane alterations) induced by such graphene-based materials on human dental follicle stem cells were investigated. Graphene oxide shows the lowest cytotoxic effect, followed by the nitrogen-doped graphene, while thermally reduced graphene oxide exhibits high cytotoxic effects. Graphene oxide induces oxidative stress without causing cell membrane damage. Nitrogen-doped graphene shows a slight antioxidant activity; however, at high doses (20 and 40 μg/ml) it causes membrane damage. Both graphene oxide and nitrogen-doped graphene seem to be valuable candidates for usage in dental nanocomposites.

  3. Cytotoxic and apoptogenic effects of Strobilanthes crispa Blume extracts on nasopharyngeal cancer cells.

    Science.gov (United States)

    Koh, Rhun Yian; Sim, Yi Chi; Toh, Hwee Jin; Liam, Liang Kuan; Ong, Rachael Sze Lynn; Yew, Mei Yeng; Tiong, Yee Lian; Ling, Anna Pick Kiong; Chye, Soi Moi; Ng, Khuen Yen

    2015-10-01

    The chemotherapeutic agents used to treat nasopharyngeal cancer (NPC) exhibit low efficacy. Strobilanthes crispa Blume is widely used for its anticancer, diuretic and anti‑diabetic properties. The present study aimed to determine the cytotoxic and apoptogenic effects of S. crispa on CNE‑1 NPC cells. A 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5 diphenyl tetrazolium bromide assay was used to evaluate the cytotoxic effects of S. crispa against CNE‑1 cells. The rate of apoptosis was determined using propidium iodide staining and caspase assays. Ethyl acetate, hexane and chloroform extracts of S. crispa leaves all exhibited cytotoxic effects on CNE‑1 cells, at a half maximal inhibitory concentration (IC50) of 119, 123.5 and 161.7 µg/ml, respectively. In addition, hexane, chloroform and ethyl acetate extracts of S. crispa stems inhibited CNE‑1 cell proliferation, at a IC50 of 49.4, 148.3 and 163.5 µg/ml, respectively. Flow cytometric analysis revealed an increased proportion of cells in the sub G1 phase and a decreased proportion of cells in the G2/M phase, following treatment with the extracts. However, the extracts did not alter the activities of caspase ‑3/7, ‑8 and ‑9. No cytotoxic effect was observed when the cells were treated with the methanol and water extracts of S. crispa stems and leaves. In conclusion, the S. crispa extracts were cytotoxic against CNE‑1 cells and these extracts were able to induce apoptosis, independent of caspase activation.

  4. Evaluation of Cytotoxicity of Sagebrush Plain Extract on Human Breast Cancer MCF7 Cells

    Directory of Open Access Journals (Sweden)

    B Gordanian

    2013-07-01

    Full Text Available Abstract Background & aim: Several studies have reported anti-cancer properties of sagebrush plain. The aim of this study was to evaluate the cytotoxicity of the methanol extract of sagebrush plain on human breast cancer MCF7 cells. Methods: In the present experimental study, the toxic effects of methanol extracts of flowers, leaves, stems and roots of sagebrush plain from of Khorassan and Esfahan province were tested on human breast cancer cells MCF-7 and normal cells HEK293 . Plant samples were extracted by methanol and their toxic effects on normal and breast cancer cells at concentrations of 5.62, 125, 250 and 500 µg/ml was determined by MTT. Both breast cancer cells MCF-7 and normal HEK293 cells were cultured in RPMI-1640 medium and DMEM containing 10% fetal calf serums were cultured. Data were analyzed by one-way ANOVA. Results: The methanol extract of sagebrush showed toxicity on MCF7 cells. The extract of Khorasan showed higher toxicity than Esfahan province. IC50 of sagebrush plant for all parts of the plant were obtained more than 500 µg/ml, but the IC50 of sagebrush plant of Khorasan region in leaf and flower were 205 ± 1.3 and 213 ± 5.3µg respectively. The leaves and flowers in both cases had the highest cytotoxicity. Plant extracts in both regions did not show significant cytotoxicity on normal HEK293 cells. Conclusion: The extract of the sagebrush plain region of Khorasan region showed greater cytotoxicity than Esfahan. It seems that different environmental conditionshas considerable cytotoxicity. Keywords: Sagebrush Plain, MTT, Breast Cancer

  5. The best time of cytotoxicity for extracted cell wall from Lactobacillus casei and paracasei in K562 cell line

    Directory of Open Access Journals (Sweden)

    Riki M

    2013-02-01

    Full Text Available Background: The aim of this study was to evaluate the effect of extracted cell walls from Lactobacillus casei and Lactobacillus paracasei as probiotic bacteria (isolated from common carp intestine on K562 and the role of cell concentration on the results of MTT [3-(4,5-Dimethylthiazol-2-yl2,5- Diphenyl tetrazolium Bromide] test.Methods: For this purpose, bacteria were cultured in specific medium (MRS broth at anaerobic condition for 24-48 hour. After incubation period culture medium was centri-fuged, then the cells were washed twice with PBS buffer to remove additional medium. Finally, collected bacterial cell disrupted by Sonication and cell walls were separated from other components by centrifugation. After that, different concentrations of cell walls (500, 1000, 2000 and 4000 µg/ml were prepared in RPMI medium for each bacteria, separately. Then anticancer properties of the cell walls were determined in vitro at 12, 24, 48 and 72 h, also the effect of K562 concentration was assayed with MTT technique.Results: The results showed extracted cell wall from both probiotic statistically (P=0.098 have anti turmeric properties in K562 and their properties will arise in relation with concentration. As well as, we found that the number of cell had not any affect on the result of MTT assay.Conclusion: We conclude that the cytotoxicity property of extracted cell wall is related in the type of bacteria, but this anticancer property would warrant further study on the clinical application of extracted cell wall.

  6. Real-time cell toxicity profiling of Tox21 10K compounds reveals cytotoxicity dependent toxicity pathway linkage.

    Science.gov (United States)

    Hsieh, Jui-Hua; Huang, Ruili; Lin, Ja-An; Sedykh, Alexander; Zhao, Jinghua; Tice, Raymond R; Paules, Richard S; Xia, Menghang; Auerbach, Scott S

    2017-01-01

    Cytotoxicity is a commonly used in vitro endpoint for evaluating chemical toxicity. In support of the U.S. Tox21 screening program, the cytotoxicity of ~10K chemicals was interrogated at 0, 8, 16, 24, 32, & 40 hours of exposure in a concentration dependent fashion in two cell lines (HEK293, HepG2) using two multiplexed, real-time assay technologies. One technology measures the metabolic activity of cells (i.e., cell viability, glo) while the other evaluates cell membrane integrity (i.e., cell death, flor). Using glo technology, more actives and greater temporal variations were seen in HEK293 cells, while results for the flor technology were more similar across the two cell types. Chemicals were grouped into classes based on their cytotoxicity kinetics profiles and these classes were evaluated for their associations with activity in the Tox21 nuclear receptor and stress response pathway assays. Some pathways, such as the activation of H2AX, were associated with the fast-responding cytotoxicity classes, while others, such as activation of TP53, were associated with the slow-responding cytotoxicity classes. By clustering pathways based on their degree of association to the different cytotoxicity kinetics labels, we identified clusters of pathways where active chemicals presented similar kinetics of cytotoxicity. Such linkages could be due to shared underlying biological processes between pathways, for example, activation of H2AX and heat shock factor. Others involving nuclear receptor activity are likely due to shared chemical structures rather than pathway level interactions. Based on the linkage between androgen receptor antagonism and Nrf2 activity, we surmise that a subclass of androgen receptor antagonists cause cytotoxicity via oxidative stress that is associated with Nrf2 activation. In summary, the real-time cytotoxicity screen provides informative chemical cytotoxicity kinetics data related to their cytotoxicity mechanisms, and with our analysis, it is

  7. Cell-mediated cytotoxicity in rainbow trout, Oncorhynchus mykiss, infected with viral haemorrhagic septicaemia virus

    DEFF Research Database (Denmark)

    Utke, K.; Bergmann, S.; Lorenzen, Niels

    2007-01-01

    Mammalian cytotoxic T cells as part of the adaptive immune system recognize virus-infected target cells by binding of their T-cell receptors (TCR) to classical MHC class I molecules loaded with viral peptides. Our previous studies have shown that the allele of the single dominant polymorphic...... level of the CD8 alpha gene which is a typical marker for mammalian cytotoxic T cells. Concurrently, the expression of the natural killer cell enhancement factor (NKEF)-like gene was enhanced as measured by real-time RT-PCR. Taken together, these results suggest that both innate and adaptive cell....... This is contradictory to the generally accepted rule that innate immune mechanisms represent the first line of defence after viral infections....

  8. Effect of Inhalational Anesthetics on Cytotoxicity and Intracellular Calcium Differently in Rat Pheochromocytoma Cells (PC12)

    Institute of Scientific and Technical Information of China (English)

    Qiujun WANG; Kezhong LI; Shanglong YAO

    2008-01-01

    Isoflurane, a commonly used inhaled anesthetic, induces apoptosis in rat pheochromo-cytoma cells (PC12) in a concentration- and time-dependent manner with unknown mechanism. We hypothesized that isoflurane induced apoptosis by causing abnormal calcium release from the endo-plasmic reticulum (ER) via activation of inositol 1,4,5-trisphosphate (IP3) receptors. Alzheimer's pre-senilin-1 (PS1) mutation increased activity of IP3 receptors and therefore rendered cells vulnerable to isoflurane-induced cytotoxicity. Sevoflurane and desflurane had less ability to disrupt intraceUular calcium homeostasis and thus being less potent to cause cytotoxicity. This study examined and com- pared the cytotoxic effects of various inhaled anesthetics on PC12 cells transfected with the Alz- heimer's mutated Psi (L286V) and the disruption of intracellular calcium homeostasis. PC12 cells transfected with wild type (WT) and mutated PS1 (L286V) were treated with equivalent of 1 MAC of isoflurane, sevoflurane and desflurane for 12 h. MTT reduction and LDH release assays were per- formed to evaluate cell viability. Changes of calcium concentration in cytosolic space ([Ca2+]c) were determined after exposing different types of cells to various inhalational anesthetics. The effects of IP3 receptor antagonist xestospongin C on isoflurane-induced cytotoxicity and calcium release from the ER in L286V PC12 cells were also determined. The results showed that isoflurane at 1 MAC for 12 h induced cytoxicity in L286V but not WT PC12 cells, which was also associated with greater and faster elevation of peak [Ca2+]c in L286V than in the WT cells. Xestospongin C significantly amelio- rated isoflurane cytotoxicity in L286V cells, as well as inhibited the calcium release from the ER in L286V cells. Sevoflurane and desflurane at equivalent exposure to isoflurane did not induce similar cytotoxicity or elevation of peak [Ca2+]c in L286V PC12 cells. These results suggested that isoflurane induced cytoxicity by

  9. Synergistic cytotoxic effect of sulindac and pyrrolidine dithiocarbamate against ovarian cancer cells.

    Science.gov (United States)

    Jakubowska-Mućka, Anna; Sieńko, Jacek; Zapała, Łukasz; Wolny, Rafał; Lasek, Witold

    2012-04-01

    Sulindac, a non-steroidal anti-inflammatory drug, suppresses carcinogenesis and inhibits growth of tumor cells. Pyrrolidine dithiocarbamate (PDTC), a potent NF-κB inhibitor, has been also identified as a potential anti-neoplastic agent. We hypothesized that combination of sulindac and PDTC could result in augmentation of cytotoxicity against ovarian cancer cells. The effect of sulindac and PDTC was examined on several ovarian cancer lines. Tumor cell viability was assessed using the MTT assay. Annexin-V/PI staining was used to detect apoptosis, cell cycle distribution was analyzed in FACS, and expression of cellular proteins was detected by western blotting. Incubation of OVA-14, OVP-10 and CAOV-1 ovarian cancer cells with sulindac and PDTC resulted in significantly greater inhibition of cell viability compared to either compound alone. In a model of OVA-14 cells it was evident that this effect was not related to the expression of COX enzymes since both active (sulindac sulfide) and inactive (sulindac) in vitro compounds affected the growth of tumor cells to a similar extent and synergized in cytotoxicity with PDTC. Combination of sulindac and PDTC lead to G0 arrest and massive apoptosis in co-treated cultures. Western blotting analysis argued for induction of the mitochondrial apoptotic pathway. These data demonstrate the synergistic cytotoxic effect of sulindac and PDTC on ovarian cancer cells through apoptosis and cell cycle arrest and prompt to test the efficacy of this combination in animal models.

  10. Annona muricata leaves have strongest cytotoxic activity against breast cancer cells

    Directory of Open Access Journals (Sweden)

    Susi Endrini

    2015-12-01

    Full Text Available BACKGROUND Plant-derived herbal compounds have a long history of clinical use, better patient tolerance and acceptance. They are freely available natural compounds that can be safely used to prevent various ailments. Plants have been the basis of traditional medicine throughout the world for thousands of years and are providing mankind with new remedies. The objective of this study was to determine the cytotoxicity of soursop (Anona muricata Linn leaves and pearl grass (Hedyotis corymbosa (L. Lam. on the hormone-dependent human breast carcinoma Michigan Cancer Foundation-7 (MCF-7 cell line. METHODS This study used two types of solvents (water and ethanol in the extraction process and two incubation times (24 hours and 48 hours in the MTT assays to analyze the cytotoxic effects of both plants. RESULTS Preliminary results showed that the ethanolic extract of soursop leaves (SE displayed cytotoxic effects against MCF-7 on 24- and 48-hour incubation times with IC50 values of 88.788 μg/ml and 14.678 μg/ml, respectively. Ethanolic pearl grass extract (PE showed similar results, with IC50 values of 65.011 μg/ ml on 24-hour incubation time and 52.329 μg/ml on 48-hour incubation time against MCF-7 cell line. However, the water extract of both plants displayed lower cytotoxic effect against MCF-7 cell line. CONCLUSION The ethanolic extract of both plants displayed cytotoxic effect against MCF-7. Soursop (Anona muricata Linn leaves have the strongest cytotoxic activity against MCF-7 breast cancer cell line.

  11. Cytotoxicity of fucosterol containing fraction of marine algae against breast and colon carcinoma cell line.

    Science.gov (United States)

    Khanavi, Mahnaz; Gheidarloo, Razieh; Sadati, Nargess; Ardekani, Mohammad Reza Shams; Nabavi, Seyed Mohammad Bagher; Tavajohi, Shohreh; Ostad, Seyed Nasser

    2012-01-01

    Marine algae produce different secondary metabolites with a wide range of biological activities. Many studies have been achieved on the screening of biological effects of marine organisms and a lot of active compounds were isolated and characterized. In an attempt to find cytotoxic compound of hexane fraction, isolation, identification, and cytotoxicity of active compound of this fraction were performed. In this study, total methanolic (70%) extract and partition fractions of hexane, chloroform (CHCl(3)), ethyl acetate (EtOAc), and MeOH-H(2)O of Sargassum angustifolium, Chondria dasyphylla, and Ulva flexuosa, collected from coastlines of the Persian Gulf in south of Iran, were studied against colon carcinoma (HT-29), colorectal adenocarcinoma (Caco-2), breast ductal carcinoma (T47D), and Swiss mouse embryo fibroblast (NIH 3T3) cell lines by MTT assay. IC(50) (median growth inhibitory concentration) values were calculated by Sigmaplot (10) software. Hexane fraction of Chondria dasyphylla (IC(50) 82.26 ± 4.09 μg/ml) and MeOH-H(2)O fraction of Ulva flexuosa (IC(50) 116.92 ± 8.58 μg/ml) showed cytotoxic activity against proliferation of T47D cells. Hexane fraction of Sargassum angustifolium was also observed for cytotoxicity against T47D and HT-29 cell lines (IC(50) 166.42 ± 26.7 and 190.24 ± 52.8 μg/ml), respectively. An investigation of a component from the hexane fraction of Sargassum angustifolium yielded a steroidal metabolite, fucosterol, with cytotoxicity in T47D and HT29 (IC(50) 27.94 ± 9.3 and 70.41 ± 7.5 μg/ml). These results indicated that fucosterol, the most abundant phytosterol in brown algae, is responsible for cytotoxic effect of this extract against breast and colon carcinoma cell lines.

  12. Corchorus olitorius (jute) extract induced cytotoxicity and genotoxicity on human multiple myeloma cells (ARH-77).

    Science.gov (United States)

    İşeri, Özlem Darcansoy; Yurtcu, Erkan; Sahin, Feride Iffet; Haberal, Mehmet

    2013-06-01

    Corchorus olitorius L. (Malvaceae) has industrial importance in world jute production and is a widely cultivated and consumed crop in Cyprus and in some Arabic countries. The present study investigated cytotoxic and genotoxic effects of leaf extracts (LE) and seed extracts (SE) of the C. olitorius on the multiple myeloma-derived ARH-77 cells. The extracts were also evaluated for their total phenol content (TPC) and free radical scavenging activity (FRSA). C. olitorius was collected from Nicosia, Cyprus. TPC and FRSA were measured by Folin-Ciocalteu and DPPH free radical methods, respectively. Cytotoxicity was evaluated by the MTT assay (4-2048 µg/mL range), and DNA damage (at IC50 and ½IC50) was measured by the comet assay. The LE had significantly higher total phenol (78 mg GAE/g extract) than the SE (2 mg GAE/g extract) with significantly higher FRSA (IC50 LE: 23 µg/mL and IC50 SE: 10 401 µg/mL). Both LE and SE exerted cytotoxic effects on cells after 48 h. The IC50 of SE (17 µg/mL) was lower than LE (151 µg/mL), which demonstrates its higher cytotoxicity on cells. The extracts were applied at 150 and 75 µg/mL for LE and at 17 and 8.5 µg/mL for SE, and the results of the comet assay revealed that the extracts induced genotoxic damage on ARH-77 cells. In both 48 h leaf and seed extract treatments, genotoxic damage significantly increased with increasing concentrations at relevant cytotoxic concentrations. To our knowledge, this is the first report demonstrating the high cytotoxic potential of C. olitorius SE and the genotoxic potential of LE and SE.

  13. Cytotoxicity of fucosterol containing fraction of marine algae against breast and colon carcinoma cell line

    Science.gov (United States)

    Khanavi, Mahnaz; Gheidarloo, Razieh; Sadati, Nargess; Ardekani, Mohammad Reza Shams; Nabavi, Seyed Mohammad Bagher; Tavajohi, Shohreh; Ostad, Seyed Nasser

    2012-01-01

    Context: Marine algae produce different secondary metabolites with a wide range of biological activities. Many studies have been achieved on the screening of biological effects of marine organisms and a lot of active compounds were isolated and characterized. Aims: In an attempt to find cytotoxic compound of hexane fraction, isolation, identification, and cytotoxicity of active compound of this fraction were performed. Materials and Methods: In this study, total methanolic (70%) extract and partition fractions of hexane, chloroform (CHCl3), ethyl acetate (EtOAc), and MeOH–H2O of Sargassum angustifolium, Chondria dasyphylla, and Ulva flexuosa, collected from coastlines of the Persian Gulf in south of Iran, were studied against colon carcinoma (HT-29), colorectal adenocarcinoma (Caco-2), breast ductal carcinoma (T47D), and Swiss mouse embryo fibroblast (NIH 3T3) cell lines by MTT assay. Statistical Analysis Used: IC50 (median growth inhibitory concentration) values were calculated by Sigmaplot (10) software. Results: Hexane fraction of Chondria dasyphylla (IC50 82.26 ± 4.09 μg/ml) and MeOH-H2O fraction of Ulva flexuosa (IC50 116.92 ± 8.58 μg/ml) showed cytotoxic activity against proliferation of T47D cells. Hexane fraction of Sargassum angustifolium was also observed for cytotoxicity against T47D and HT-29 cell lines (IC50 166.42 ± 26.7 and 190.24 ± 52.8 μg/ml), respectively. An investigation of a component from the hexane fraction of Sargassum angustifolium yielded a steroidal metabolite, fucosterol, with cytotoxicity in T47D and HT29 (IC50 27.94 ± 9.3 and 70.41 ± 7.5 μg/ml). Conclusions: These results indicated that fucosterol, the most abundant phytosterol in brown algae, is responsible for cytotoxic effect of this extract against breast and colon carcinoma cell lines. PMID:22438665

  14. Cytotoxicity of fucosterol containing fraction of marine algae against breast and colon carcinoma cell line

    Directory of Open Access Journals (Sweden)

    Mahnaz Khanavi

    2012-01-01

    Full Text Available Context: Marine algae produce different secondary metabolites with a wide range of biological activities. Many studies have been achieved on the screening of biological effects of marine organisms and a lot of active compounds were isolated and characterized. Aims: In an attempt to find cytotoxic compound of hexane fraction, isolation, identification, and cytotoxicity of active compound of this fraction were performed. Materials and Methods: In this study, total methanolic (70% extract and partition fractions of hexane, chloroform (CHCl 3 , ethyl acetate (EtOAc, and MeOH-H 2 O of Sargassum angustifolium, Chondria dasyphylla, and Ulva flexuosa, collected from coastlines of the Persian Gulf in south of Iran, were studied against colon carcinoma (HT-29, colorectal adenocarcinoma (Caco-2, breast ductal carcinoma (T47D, and Swiss mouse embryo fibroblast (NIH 3T3 cell lines by MTT assay. Statistical Analysis Used: IC 50 (median growth inhibitory concentration values were calculated by Sigmaplot (10 software. Results: Hexane fraction of Chondria dasyphylla (IC 50 82.26 ± 4.09 μg/ml and MeOH-H 2 O fraction of Ulva flexuosa (IC 50 116.92 ± 8.58 μg/ml showed cytotoxic activity against proliferation of T47D cells. Hexane fraction of Sargassum angustifolium was also observed for cytotoxicity against T47D and HT-29 cell lines (IC 50 166.42 ± 26.7 and 190.24 ± 52.8 μg/ml, respectively. An investigation of a component from the hexane fraction of Sargassum angustifolium yielded a steroidal metabolite, fucosterol, with cytotoxicity in T47D and HT29 (IC 50 27.94 ± 9.3 and 70.41 ± 7.5 μg/ml. Conclusions: These results indicated that fucosterol, the most abundant phytosterol in brown algae, is responsible for cytotoxic effect of this extract against breast and colon carcinoma cell lines.

  15. Influence of uranium speciation on normal rat kidney (NRK-52E) proximal cell cytotoxicity.

    Science.gov (United States)

    Carrière, M; Avoscan, L; Collins, R; Carrot, F; Khodja, H; Ansoborlo, E; Gouget, B

    2004-03-01

    Uranium is a naturally occurring heavy metal. Its extensive use in the nuclear cycle and for military applications has focused attention on its potential health effects. Acute exposures to uranium are toxic to the kidneys where they mainly cause damage to proximal tubular epithelium. The purpose of this study was to investigate the biological consequences of acute in vitro uranyl exposure and the influence of uranyl speciation on its cytotoxicity. NRK-52E cells, representative of rat kidney proximal epithelium, were exposed to uranyl-carbonate and -citrate complexes, which are the major complexes transiting through renal tubules after acute in vivo contamination. Before NRK-52E cell exposure, these complexes were diluted in classical or modified cell culture media, which can possibly modify uranyl speciation. In these conditions, uranium cytotoxicity appears after 16 h of exposure. The CI50 cytotoxicity index, the uranium concentration leading to 50% dead cells after 24 h of exposure, is 500 microM (+/-100 microM) and strongly depends on uranyl counterion and cell culture medium composition. Computer modeling of uranyl speciation is reported, enabling one to draw a parallel between uranyl speciation and its cytotoxicity.

  16. Cytotoxicity and genotoxicity of intravitreal adalimumab administration in rabbit retinal cells

    Directory of Open Access Journals (Sweden)

    Álcio Coutinho de Paula

    2015-04-01

    Full Text Available Purpose: To assess the cytotoxicity and genotoxicity of intravitreal adalimumab treatment in an animal experimental model using cytological and molecular techniques. Methods: Eighteen rabbits were randomly assigned to three groups: control, adalimumab treatment, and placebo. Cytotoxicity on retinal cells was evaluated using flow cytometry assays to determine the level of apoptosis and necrosis. Genotoxicity was evaluated by comet assays to assess DNA damage, and quantitative real-time polymerase chain reaction (qPCR was used to evaluate expression of apoptosis-inducing caspases (8 and 3. Results: No cytotoxicity or genotoxicity was observed in any of the two treatment groups (adalimumab and placebo following intravitreal administration compared with the control group. Flow cytometry analysis revealed that more than 90% of the cells were viable, and only a low proportion of retinal cells presented apoptotic (~10% or necrotic (<1% activity across all groups. Molecular damage was also low with a maximum of 6.4% DNA degradation observed in the comet assays. In addition, no increase in gene expression of apoptosis-inducing caspases was observed on retinal cells by qPCR in both the adalimumab and placebo groups compared with the control group. Conclusion: The use of adalimumab resulted in no detectable cytotoxicity or genotoxicity on retinal cells for up to 60 days upon administration. These results therefore indicate that adalimumab may be a safe option for intravitreal application to treat ocular inflammatory diseases in which TNF-α is involved.

  17. Chemical composition of Schinus molle essential oil and its cytotoxic activity on tumour cell lines.

    Science.gov (United States)

    Díaz, Cecilia; Quesada, Silvia; Brenes, Oscar; Aguilar, Gilda; Cicció, José F

    2008-01-01

    The leaf essential oil hydrodistilled from Schinus molle grown in Costa Rica was characterised in terms of its chemical composition, antioxidant activity, ability to induce cytotoxicity and the mechanism of cell death involved in the process. As a result, 42 constituents, accounting for 97.2% of the total oil, were identified. The major constituents of the oil were beta-pinene and alpha-pinene. The antioxidant activity showed an IC(50) of 36.3 microg mL(-1). The essential oil was cytotoxic in several cell lines, showing that it is more effective on breast carcinoma and leukemic cell lines. The LD(50) for cytotoxicity at 48 h in K562 corresponded to 78.7 microg mL(-1), which was very similar to the LD(50) obtained when apoptosis was measured. The essential oil did not induce significant necrosis up to 200 microg mL(-1), which together with the former results indicate that apoptosis is the main mechanism of toxicity induced by S. molle essential oil in this cell line. In conclusion, the essential oil tested was weak antioxidant and induced cytotoxicity in different cell types by a mechanism related to apoptosis. It would be interesting to elucidate the role that different components of the oil play in the effect observed here, since some of them could have potential anti-tumoural effects, either alone or in combination.

  18. Synthesis and cytotoxic activity of certain benzothiazole derivatives against human MCF-7 cancer cell line.

    Science.gov (United States)

    Mohamed, Lamia W; Taher, Azza T; Rady, Ghada S; Ali, Mamdouh M; Mahmoud, Abeer E

    2016-10-04

    A new series of benzothiazole has been synthesized as cytotoxic agents. The new derivatives were tested for their cytotoxic activity toward the human breast cancer MCF-7 cell line against cisplatin as the reference drug. Many derivatives revealed good cytotoxic effect, whereas four of them, 4, 5c, 5d, and 6b, were more potent than cisplatin, with IC50 values being 8.64, 7.39, 7.56, and 5.15 μm compared to 13.33 μm of cisplatin. The four derivatives' cytotoxic activity was accompanied by regulating free radicals production, by increasing the activity of superoxide dismutase and depletion of intracellular reduced glutathione, catalase, and glutathione peroxidase activities, accordingly, the high production of hydrogen peroxide, nitric oxide, and other free radicals causing tumor cell death as monitored by reduction in the synthesis of protein and nucleic acids. Most of the tested compounds showed potent to moderate growth inhibitory activity; in particular, compound 6b exhibited the highest activity suggesting it is a lead compound in cytotoxic activity.

  19. A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity.

    Science.gov (United States)

    Wu, Yuling; Li, Jia J; Kim, Hyun Jun; Liu, Xu; Liu, Weiyi; Akhgar, Ahmad; Bowen, Michael A; Spitz, Susan; Jiang, Xu-Rong; Roskos, Lorin K; White, Wendy I

    2015-11-01

    Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35 ng/mL [80% maximal effective concentration (EC80)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10 μg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5 μg/mL) in the presence of 0.78 μg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function.

  20. Fullerene C60 Penetration into Leukemic Cells and Its Photoinduced Cytotoxic Effects

    Science.gov (United States)

    Franskevych, D.; Palyvoda, K.; Petukhov, D.; Prylutska, S.; Grynyuk, I.; Schuetze, C.; Drobot, L.; Matyshevska, O.; Ritter, U.

    2017-01-01

    Fullerene C60 as a representative of carbon nanocompounds is suggested to be promising agent for application in photodynamic therapy due to its unique physicochemical properties. The goal of this study was to estimate the accumulation of fullerene C60 in leukemic cells and to investigate its phototoxic effect on parental and resistant to cisplatin leukemic cells. Stable homogeneous water colloid solution of pristine C60 with average 50-nm diameter of nanoparticles was used in experiments. Fluorescent labeled C60 was synthesized by covalent conjugation of C60 with rhodamine B isothiocyanate. The results of confocal microscopy showed that leukemic Jurkat cells could effectively uptake fullerene C60 from the medium. Light-emitting diode lamp (100 mW cm-2, λ = 420-700 nm) was used for excitation of accumulated C60. A time-dependent decrease of viability was detected when leukemic Jurkat cells were exposed to combined treatment with C60 and visible light. The cytotoxic effect of photoexcited C60 was comparable with that induced by H2O2, as both agents caused 50% decrease of cell viability at 24 h at concentrations about 50 μM. Using immunoblot analysis, protein phosphotyrosine levels in cells were estimated. Combined action of C60 and visible light was followed by decrease of cellular proteins phosphorylation on tyrosine residues though less intensive as compared with that induced by H2O2 or protein tyrosine kinase inhibitor staurosporine. All tested agents reduced phosphorylation of 55, 70, and 90 kDa proteins while total suppression of 26 kDa protein phosphorylation was specific only for photoexcited C60.

  1. Genipin-induced inhibition of uncoupling protein-2 sensitizes drug-resistant cancer cells to cytotoxic agents.

    Directory of Open Access Journals (Sweden)

    Ryan J Mailloux

    Full Text Available Uncoupling protein-2 (UCP2 is known to suppress mitochondrial reactive oxygen species (ROS production and is employed by drug-resistant cancer cells to mitigate oxidative stress. Using the drug-sensitive HL-60 cells and the drug-resistant MX2 subline as model systems, we show that genipin, a UCP2 inhibitor, sensitizes drug-resistant cells to cytotoxic agents. Increased MX2 cell death was observed upon co-treatment with genipin and different doses of menadione, doxorubicin, and epirubicin. DCFH-DA fluorimetry revealed that the increase in MX2 cell death was accompanied by enhanced cellular ROS levels. The drug-induced increase in ROS was linked to genipin-mediated inhibition of mitochondrial proton leak. State 4 and resting cellular respiratory rates were higher in the MX2 cells in comparison to the HL-60 cells, and the increased respiration was readily suppressed by genipin in the MX2 cells. UCP2 accounted for a remarkable 37% of the resting cellular oxygen consumption indicating that the MX2 cells are functionally reliant on this protein. Higher amounts of UCP2 protein were detected in the MX2 versus the HL-60 mitochondria. The observed effects of genipin were absent in the HL-60 cells pointing to the selectivity of this natural product for drug-resistant cells. The specificity of genipin for UCP2 was confirmed using CHO cells stably expressing UCP2 in which genipin induced an ∼22% decrease in state 4 respiration. These effects were absent in empty vector CHO cells expressing no UCP2. Thus, the chemical inhibition of UCP2 with genipin sensitizes multidrug-resistant cancer cells to cytotoxic agents.

  2. Artemisinin-derived sesquiterpene lactones as potential antitumour compounds : Cytotoxic action against bone marrow and tumour cells

    NARCIS (Netherlands)

    Beekman, AC; Wierenga, PK; Woerdenbag, HJ; Van Uden, W; Pras, N; Konings, AWT; El-Feraly, FS; Galal, AM; Wikstrom, HV

    1998-01-01

    We determined the in vitro cytotoxic activity of the sesquiterpene lactone endoperoxide artemisinin (1) and some chemically prepared derivatives, which have been found to display cytotoxicity to cloned murine Ehrlich ascites tumour (EAT) cells and human HeLa cells and against murine bone marrow usin

  3. Enhancement of cisplatin cytotoxicity by benzyl isothiocyanate in HL-60 cells.

    Science.gov (United States)

    Lee, Younghyun; Kim, Yang Jee; Choi, Young Joo; Lee, Joong Won; Lee, Sunyeong; Chung, Hai Won

    2012-07-01

    Cis-diamminedichloroplatinum (II) (cisplatin) is one of the most widely used chemotherapeutic drugs, but its effectiveness is limited by tumor cell resistance and the severe side effects it causes. One strategy for overcoming this problem is the concomitant use of natural dietary compounds as therapeutic agents. Benzyl isothiocyanate (BITC) is a promising chemopreventive agent found in cruciferous vegetables and papaya fruits. The aim of this study was to investigate the effects of BITC on cisplatin-induced cytotoxicity in human promyelocytic leukemia cells and normal human lymphocytes. The combined treatment of HL-60 cells with BITC followed by cisplatin (BITC/cisplatin) caused a significant decrease in cell viability. BITC also increased apoptotic cell death compared to cisplatin treatment alone. In normal human lymphocytes, BITC did not enhance the cytotoxic effects of cisplatin. Cellular exposure to BITC/cisplatin increased reactive oxygen species (ROS) generation but decreased the total glutathione (GSH) level in HL-60 cells. Pretreatment of HL-60 cells with N-acetylcysteine or glutathione monoethyl ester effectively decreased BITC/cisplatin-induced cell death. The addition of the extracellular signal-regulated kinase (ERK) inhibitor PD98059 abolished BITC/cisplatin-induced apoptosis. Taken together, our results suggest that BITC enhances cisplatin-induced cytotoxicity through the generation of ROS, depletion of GSH, and ERK signaling in HL-60 cells.

  4. Modulation of doxorubicin cytotoxicity by resveratrol in a human breast cancer cell line

    Directory of Open Access Journals (Sweden)

    Osman Abdel-Moneim M

    2012-11-01

    Full Text Available Abstract Background Breast cancer is the most common cancer in the Arab world and it ranked first among Saudi females. Doxorubicin (DOX, an anthracycline antibiotic is one of the most effective anticancer agents used to treat breast cancer. chronic cardiotoxicity is a major limiting factor of the use of doxorubicin. Therefore, our study was designed to assess the role of a natural product resveratrol (RSVL on sensitization of human breast cancer cells (MCF-7 to the action of DOX in an attempt to minimize doxorubicin effective dose and thereby its side effects. Methods Human breast cancer cell line MCF-7, was used in this study. Cytotoxic activity of DOX was determined using (sulforhodamine SRB method. Apoptotic cells were quantified after treatment by annexin V-FITC- propidium iodide (PI double staining using flow-cytometer. Cell cycle disturbance and doxorubicin uptake were determined after RSVL or DOX treatment. Results Treatment of MCF-7 cells with 15 μg/ml RSVL either simultaneously or 24 h before DOX increased the cytotoxicity of DOX, with IC50 were 0.056 and 0.035 μg/ml, respectively compared to DOX alone IC50 (0.417 μg/ml. Moreover, flow cytometric analysis of the MCF-7 cells treated simultaneously with DOX (0.5 μg/ml and RSVL showed enhanced arrest of the cells in G0 (80%. On the other hand, when RSVL is given 24 h before DOX although there was more increased in the cytotoxic effect of DOX against the growth of the cells, however, there was decreased in percentage arrest of cells in G0, less inhibition of DOX-induced apoptosis and reduced DOX cellular uptake into the cells. Conclusion RSVL treatment increased the cytotoxic activity of DOX against the growth of human breast cancer cells when given either simultaneously or 24 h before DOX.

  5. The Adaptor Protein SAP Regulates Type II NKT Cell Development, Cytokine Production and Cytotoxicity Against Lymphoma1

    Science.gov (United States)

    Weng, Xiufang; Liao, Chia-Min; Bagchi, Sreya; Cardell, Susanna L.; Stein, Paul L.; Wang, Chyung-Ru

    2014-01-01

    CD1d-restricted NKT cells represent a unique lineage of immunoregulatory T cells that are divided into two groups, type I and type II, based on their TCR usage. Because there are no specific tools to identify type II NKT cells, little is known about their developmental requirements and functional regulation. In our previous study, we showed that signaling lymphocytic activation molecule-associated protein (SAP) is essential for the development of type II NKT cells. Here, using a type II NKT cell TCR transgenic mouse model (24αβTg), we demonstrated that CD1d-expressing hematopoietic cells but not thymic epithelial cells meditate efficient selection of type II NKT cells. Further, we showed that SAP regulates type II NKT cell development by controlling Egr2 and PLZF expression. SAP-deficient 24αβ transgenic T cells (24αβ T cells) exhibited an immature phenotype with reduced Th2 cytokine-producing capacity and diminished cytotoxicity to CD1d-expressing lymphoma cells. The impaired IL-4 production by SAP-deficient 24αβ T cells was associated with reduced IRF4 and GATA-3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X-linked lymphoproliferative disease caused by mutations in SAP. PMID:25236978

  6. Canonical Wnt pathway inhibitor ICG-001 induces cytotoxicity of multiple myeloma cells in Wnt-independent manner.

    Directory of Open Access Journals (Sweden)

    Eileen R Grigson

    Full Text Available Canonical Wnt signaling has been implicated in the regulation of multiple myeloma (MM growth. Here, we investigated whether the targeting of this pathway with a novel pharmacological inhibitor ICG-001 would result in an anti-tumor effect and improvement of chemosensitivity in MM. As expected, ICG-001 specifically down-regulated β-catenin/TCF-mediated transcription in MM cells. Treatment with ICG-001 resulted in growth arrest and apoptosis in MM cell lines and primary MM cells. Moreover, ICG-001 enhanced the cytotoxic effects of doxorubicin and melphalan and abrogated chemoresistance of MM cells to these chemotherapeutics induced by bone marrow stroma. The cytotoxic effect of ICG-001 was caspase-dependent and mediated through transcriptional up-regulation of BH3-only pro-apoptotic members of the Bcl-2 family Noxa and Puma but not through inhibition of canonical Wnt signaling. ICG-001 selectively induced apoptosis in primary MM cells but did not affect non-MM cells of the bone marrow microenvironment. Experiments using a xenograft model of MM showed substantial anti-tumor effects of this compound in vivo. Thus, our study demonstrated that the small molecule inhibitor ICG-001 has strong anti-MM effects and could be developed further for therapeutic intervention in this disease.

  7. The Cytotoxicity and Genotoxicity of Particulate and Soluble Cobalt in Human Urothelial Cells.

    Science.gov (United States)

    Speer, Rachel M; The, Therry; Xie, Hong; Liou, Louis; Adam, Rosalyn M; Wise, John Pierce

    2017-03-21

    Cobalt use is increasing particularly due to its use as one of the primary metals in cobalt-chromium-molybdenum (CoCrMo) metal-on-metal prosthetics. CoCrMo is a high-strength, wear-resistant alloy with reduced risk for prosthetic loosening and device fracture. More than 500,000 people receive hip implants each year in the USA which puts them at potential risk for exposure to metal ions and particles released by the prosthetic implants. Data show cobalt ions released from prosthetics reach the bloodstream and accumulate in the bladder. As patients with failed hip implants show increased urinary and blood cobalt levels, no studies have considered the effects of cobalt on human urothelial cells. Accordingly, we investigated the cytotoxic and genotoxic effects of particulate and soluble cobalt in urothelial cells. Exposure to both particulate and soluble cobalt resulted in a concentration-dependent increase in cytotoxicity, genotoxicity, and intracellular cobalt ions. Based on intracellular cobalt ion levels, we found, when compared to particulate cobalt, soluble cobalt was more cytotoxic, but induced similar levels of genotoxicity. Interestingly, at similar intracellular cobalt ion concentrations, soluble cobalt induced cell cycle arrest indicated by a lack of metaphases not observed after particulate cobalt treatment. These data indicate that cobalt compounds are cytotoxic and genotoxic to human urothelial cells and solubility may play a key role in cobalt-induced toxicity.

  8. CYTOTOXICITY OF ARTEMISININ-RELATED ENDOPEROXIDES TO EHRLICH ASCITES TUMOR-CELLS

    NARCIS (Netherlands)

    WOERDENBAG, HJ; MOSKAL, TA; PRAS, N; MALINGRE, TM; ELFERALY, FS; KAMPINGA, HH; KONINGS, AWT

    1993-01-01

    A series of artemisinin-related endoperoxides was tested for cytotoxicity to Ehrlich ascites tumor (EAT) cells using the microculture tetrazolium (MTT) assay. Artemisinin [1] had an IC50 value of 29.8 muM. Derivatives of dihydroartemisinin [2], being developed as antimalarial drugs (artemether [3],

  9. Digitoxin-induced cytotoxicity in cancer cells is mediated through distinct kinase and interferon signaling networks.

    Science.gov (United States)

    Prassas, Ioannis; Karagiannis, George S; Batruch, Ihor; Dimitromanolakis, Apostolos; Datti, Alessandro; Diamandis, Eleftherios P

    2011-11-01

    Cardiac glycosides (e.g., digoxin, digitoxin) constitute a diverse family of plant-derived sodium pump inhibitors that have been in clinical use for the treatment of heart-related diseases (congestive heart failure, atrial arrhythmia) for many years. Recently though, accumulating in vitro and in vivo evidence highlight potential anticancer properties of these compounds. Despite the fact that members of this family have advanced to clinical trial testing in cancer therapeutics, their cytotoxic mechanism is not yet elucidated. In this study, we investigated the cytotoxic properties of cardiac glycosides against a panel of pancreatic cancer cell lines, explored their apoptotic mechanism, and characterized the kinetics of cell death induced by these drugs. Furthermore, we deployed a high-throughput kinome screening approach and identified several kinases of the Na-K-ATPase-mediated signal transduction circuitry (epidermal growth factor receptor, Src, pkC, and mitogen-activated protein kinases) as important mediators downstream of cardiac glycoside cytotoxic action. To further extend our knowledge on their mode of action, we used mass-spectrometry-based quantitative proteomics (stable isotope labeling of amino acids in cell culture) coupled with bioinformatics to capture large-scale protein perturbations induced by a physiological dose of digitoxin in BxPC-3 pancreatic cancer cells and identified members of the interferon family as key regulators of the main protein/protein interactions downstream of digitoxin action. Hence, our findings provide more in-depth information regarding the molecular mechanisms underlying cardiac glycoside-induced cytotoxicity.

  10. Suitability of Cell-Based Label-Free Detection for Cytotoxicity Screening of Carbon Nanotubes

    Directory of Open Access Journals (Sweden)

    Claudia Meindl

    2013-01-01

    Full Text Available Cytotoxicity testing of nanoparticles (NPs by conventional screening assays is often complicated by interference. Carbon nanotubes (CNTs are particularly difficult to assess. To test the suitability of cell-based label-free techniques for this application, a panel of CNTs with different diameters and surface functionalizations was assessed by impedance-based technique (xCELLigence RTCA and automated microscopy (Cell-IQ compared to formazan bioreduction (MTS assay. For validation of the label-free systems different concentrations of ethanol and of amine (AMI polystyrene NPs were used. CNTs were evaluated in various cell lines, but only endothelial EAhy926 cells and L929 and V79 fibroblasts could be evaluated in all systems. Polystyrene particles obtained similar results in all assays. All systems identified thin (20 nm CNTs, but detection by xCELLigence system was less sensitive to CNT-induced cytotoxicity. Despite advantages, such as continuous monitoring and more detailed analysis of cytotoxic effects, label-free techniques cannot be generally recommended for cytotoxicity screening of NPs.

  11. Fasting-Mimicking Diet Reduces HO-1 to Promote T Cell-Mediated Tumor Cytotoxicity.

    Science.gov (United States)

    Di Biase, Stefano; Lee, Changhan; Brandhorst, Sebastian; Manes, Brianna; Buono, Roberta; Cheng, Chia-Wei; Cacciottolo, Mafalda; Martin-Montalvo, Alejandro; de Cabo, Rafael; Wei, Min; Morgan, Todd E; Longo, Valter D

    2016-07-11

    Immune-based interventions are promising strategies to achieve long-term cancer-free survival. Fasting was previously shown to differentially sensitize tumors to chemotherapy while protecting normal cells, including hematopoietic stem and immune cells, from its toxic side effects. Here, we show that the combination of chemotherapy and a fasting-mimicking diet (FMD) increases the levels of bone marrow common lymphoid progenitor cells and cytotoxic CD8(+) tumor-infiltrating lymphocytes (TILs), leading to a major delay in breast cancer and melanoma progression. In breast tumors, this effect is partially mediated by the downregulation of the stress-responsive enzyme heme oxygenase-1 (HO-1). These data indicate that FMD cycles combined with chemotherapy can enhance T cell-dependent targeted killing of cancer cells both by stimulating the hematopoietic system and by enhancing CD8(+)-dependent tumor cytotoxicity.

  12. Protein-binding, cytotoxicity in vitro and cell cycle arrest of ruthenium(II) polypyridyl complexes

    Science.gov (United States)

    Liu, Si-Hong; Zhu, Jian-Wei; Xu, Hui-Hua; Wang, Yan; Liu, Ya-Min; Liang, Jun-Bo; Zhang, Gui-Qiang; Cao, Di-Hua; Lin, Yang-Yang; Wu, Yong; Guo, Qi-Feng

    2016-05-01

    The cytotoxic activity of two Ru(II) complexes against A549, BEL-7402, HeLa, PC-12, SGC-7901 and SiHa cell lines was investigated by MTT method. Complexes 1 and 2 show moderate cytotoxicity toward BEL-7402 cells with an IC50 value of 53.9 ± 3.4 and 39.3 ± 2.1 μM. The effects of t