Sample records for antibody-dependent cell-mediated cytotoxicity

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

    Directory of Open Access Journals (Sweden)

    Wei eWang


    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.

  2. Enhancement of antibody-dependent cell mediated cytotoxicity: a new era in cancer treatment

    Directory of Open Access Journals (Sweden)

    Rajasekaran N


    Full Text Available Narendiran Rajasekaran,1,* Cariad Chester,1,* Atsushi Yonezawa,1,2 Xing Zhao,1,3 Holbrook E Kohrt1 1Division of Oncology, Stanford School of Medicine, Stanford University, Stanford, CA, USA; 2Department of Clinical Pharmacology and Therapeutics, Kyoto University Hospital, Kyoto, Japan; 3Tissue Engineering and Stem Cells Research Center, Department of Immunology, Guiyang Medical University, Guiyang, Guizhou Province, People's Republic of China *These authors contributed equally to this work Abstract: The therapeutic efficacy of some anti-tumor monoclonal antibodies (mAbs depends on the capacity of the mAb to recognize the tumor-associated antigen and induce cytotoxicity via a network of immune effector cells. This process of antibody-dependent cell-mediated cytotoxicity (ADCC against tumor cells is triggered by the interaction of the fragment crystallizable (Fc portion of the mAb with the Fc receptors on effector cells like natural killer cells, macrophages, γδ T cells, and dendritic cells. By augmenting ADCC, the antitumor activity of mAbs can be significantly increased. Currently, identifying and developing therapeutic agents that enhance ADCC is a growing area of research. Combining existing tumor-targeting mAbs and ADCC-promoting agents that stimulate effector cells will translate to greater clinical responses. In this review, we discuss strategies for enhancing ADCC and emphasize the potential of combination treatments that include US Food and Drug Administration-approved mAbs and immunostimulatory therapeutics. Keywords: ADCC, NK cell, reovirus, TLR, CD137

  3. A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity. (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


    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.

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


    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. Characterization of in vitro antibody-dependent cell-mediated cytotoxicity activity of therapeutic antibodies - impact of effector cells. (United States)

    Chung, Shan; Lin, Yuwen L; Reed, Chae; Ng, Carl; Cheng, Zhijie Jey; Malavasi, Fabio; Yang, Jihong; Quarmby, Valerie; Song, An


    Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action implicated in the clinical efficacy of several therapeutic antibodies. In vitro ADCC assays employing effector cells capable of inducing lysis of target cells bound by antibodies are routinely performed to support the research and development of therapeutic antibodies. ADCC assays are commonly performed using peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells or engineered cell lines as effector cells. In this study we evaluated the impact of different effector cell types including primary PBMCs, primary NK cells, engineered NK cell lines, and an engineered reporter cell line, on the in vitro ADCC activity of two glycoforms of a humanized IgG1 antibody. The results of this study show the differential effects on both the efficacy and potency of the antibodies by different effector cells and the finding that both the allotype and the expression level of CD16a affect the potency of effector cells in ADCC assays. Our results also show that engineered NK or reporter cell lines provide reduced variability compared to primary effector cells for in vitro ADCC assays.

  6. 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


    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...

  7. Reactive oxygen species induced by therapeutic CD20 antibodies inhibit natural killer cell-mediated antibody-dependent cellular cytotoxicity against primary CLL cells (United States)

    Werlenius, Olle; Aurelius, Johan; Hallner, Alexander; Akhiani, Ali A.; Simpanen, Maria; Martner, Anna; Andersson, Per-Ola; Hellstrand, Kristoffer; Thorén, Fredrik B.


    The antibody-dependent cellular cytotoxicity (ADCC) of natural killer (NK) cells is assumed to contribute to the clinical efficacy of monoclonal antibodies (mAbs) in chronic lymphocytic leukemia (CLL) and other hematopoietic malignancies of B cell origin. We sought to determine whether reactive oxygen species (ROS)-producing monocytes regulate the ADCC of NK cells against primary CLL cells using anti-CD20 as the linking antibody. The monoclonal CD20 antibodies rituximab and ofatumumab were found to trigger substantial release of ROS from monocytes. Antibody-exposed monocytes induced NK cell apoptosis and restricted NK cell-mediated ADCC against autologous CLL cells. The presence of inhibitors of ROS formation and scavengers of ROS preserved NK cell viability and restored NK cell-mediated ADCC against primary CLL cells. We propose that limiting the antibody-induced induction of immunosuppressive ROS may improve the anti-leukemic efficacy of anti-CD20 therapy in CLL. PMID:27097113

  8. Potential for Natural Killer Cell-Mediated Antibody-Dependent Cellular Cytotoxicity for Control of Human Cytomegalovirus

    Directory of Open Access Journals (Sweden)

    Rebecca J. Aicheler


    Full Text Available Human cytomegalovirus (HCMV is an important pathogen that infects the majority of the population worldwide, yet, currently, there is no licensed vaccine. Despite HCMV encoding at least seven Natural Killer (NK cell evasion genes, NK cells remain critical for the control of infection in vivo. Classically Antibody-Dependent Cellular Cytotoxicity (ADCC is mediated by CD16, which is found on the surface of the NK cell in a complex with FcεRI-γ chains and/or CD3ζ chains. Ninety percent of NK cells express the Fc receptor CD16; thus, they have the potential to initiate ADCC. HCMV has a profound effect on the NK cell repertoire, such that up to 10-fold expansions of NKG2C+ cells can be seen in HCMV seropositive individuals. These NKG2C+ cells are reported to be FcεRI-γ deficient and possess variable levels of CD16+, yet have striking ADCC functions. A subset of HCMV cell surface proteins will induce robust antibody responses that could render cells susceptible to ADCC. We will consider how the strong anti-HCMV function of NKG2C+ FcεRI-γ-deficient NK cells could potentially be harnessed in the clinic to treat patients suffering from HCMV disease and in the development of an efficacious HCMV vaccine.

  9. Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcγ receptor binding and antibody-dependent cell-mediated cytotoxicity activities. (United States)

    Chung, Shan; Quarmby, Valerie; Gao, Xiaoying; Ying, Yong; Lin, Linda; Reed, Chae; Fong, Chris; Lau, Wendy; Qiu, Zhihua J; Shen, Amy; Vanderlaan, Martin; Song, An


    The presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward FcγRIIIa as well as their antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, the quantitative nature of this structure-function relationship remains unclear. In this study, the in vitro biological activity of an afucosylated anti-CD20 antibody was fully characterized. Further, the effect of fucose reduction on Fc effector functions was quantitatively evaluated using the afucosylated antibody, its "regular" fucosylated counterpart and a series of mixtures containing varying proportions of "regular" and afucosylated materials. Compared with the "regular" fucosylated antibody, the afucosylated antibody demonstrated similar binding interactions with the target antigen (CD20), C1q and FcγRIa, moderate increases in binding to FcγRIIa and IIb, and substantially increased binding to FcγRIIIa. The afucosylated antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity. Based on EC 50 values derived from dose-response curves, our results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcγRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the extent of ADCC enhancement due to fucose depletion was not affected by the FcγRIIIa genotype of the effector cells.

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

    Directory of Open Access Journals (Sweden)

    Jonathan Richard


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

  11. Antibody-Dependent Cell-Mediated Cytotoxicity Effector-Enhanced EphA2 Agonist Monoclonal Antibody Demonstrates Potent Activity against Human Tumors

    Directory of Open Access Journals (Sweden)

    Elizabeth M. Bruckheimer


    Full Text Available EphA2 is a receptor tyrosine kinase that has been shown to be overexpressed in a variety of human tumor types. Previous studies demonstrated that agonist monoclonal antibodies targeting EphA2 induced the internalization and degradation of the receptor, thereby abolishing its oncogenic effects. In this study, the in vitro and in vivo antibody-dependent cell-mediated cytotoxicity (ADCC activity of EphA2 effector-enhanced agonist monoclonal antibodies was evaluated. With tumor cell lines and healthy human peripheral blood monocytes, the EphA2 antibodies demonstrated ∼80% tumor cell killing. In a dose-dependent manner, natural killer (NK cells were required for the in vitro ADCC activity and became activated as demonstrated by the induction of cell surface expression of CD107a. To assess the role of NK cells on antitumor efficacy in vivo, the EphA2 antibodies were evaluated in xenograft models in severe compromised immunodeficient (SCID mice (which have functional NK cells and monocytes and SCID nonobese diabetic (NOD mice (which largely lack functional NK cells and monocytes. Dosing of EphA2 antibody in the SCID murine tumor model resulted in a 6.2-fold reduction in tumor volume, whereas the SCID/nonobese diabetic model showed a 1.6-fold reduction over the isotype controls. Together, these results demonstrate that the anti-EphA2 monoclonal antibodies may function through at least two mechanisms of action: EphA2 receptor activation and ADCC-mediated activity. These novel EphA2 monoclonal antibodies provide additional means by which host effector mechanisms can be activated for selective destruction of EphA2-expressing tumor cells.

  12. The combination of trastuzumab and pertuzumab administered at approved doses may delay development of trastuzumab resistance by additively enhancing antibody-dependent cell-mediated cytotoxicity. (United States)

    Tóth, Gábor; Szöőr, Árpád; Simon, László; Yarden, Yosef; Szöllősi, János; Vereb, György


    Although the recently concluded CLEOPATRA trial showed clinical benefits of combining trastuzumab and pertuzumab for treating HER2-positive metastatic breast cancer, trastuzumab monotherapy is still the mainstay in adjuvant settings. Since trastuzumab resistance occurs in over half of these cancers, we examined the mechanisms by which treatment of intrinsically trastuzumab-resistant and -sensitive tumors can benefit from the combination of these antibodies. F(ab')2 of both trastuzumab and pertuzumab were generated and validated in order to separately analyze antibody-dependent cell-mediated cytotoxicity (ADCC)-based and direct biological effects of the antibodies. Compared to monotherapy, combination of the two antibodies at clinically permitted doses enhanced the recruitment of natural killer cells responsible for ADCC, and significantly delayed the outgrowth of xenografts from intrinsically trastuzumab-resistant JIMT-1 cells. Antibody dose-response curves of in vitro ADCC showed that antibody-mediated killing can be saturated, and the two antibodies exert an additive effect at sub-saturation doses. Thus, the additive effect in vivo indicates that therapeutic tissue levels likely do not saturate ADCC. Additionally, isobole studies with the in vitro trastuzumab-sensitive BT-474 cells showed that the direct biological effect of combined treatment is additive, and surpasses the maximum effect of either monotherapy. Our results suggest the combined therapy is expected to give results that are superior to monotherapy, whatever the type of HER2-positive tumor may be. The combination of both antibodies at maximum clinically approved doses should thus be administered to patients to recruit maximum ADCC and cause maximum direct biological growth inhibition.

  13. A Human Anti-M2 Antibody Mediates Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC and Cytokine Secretion by Resting and Cytokine-Preactivated Natural Killer (NK Cells.

    Directory of Open Access Journals (Sweden)

    Venkateswara R Simhadri

    Full Text Available The highly conserved matrix protein 2 (M2 is a good candidate for the development of a broadly protective influenza vaccine that induces long-lasting immunity. In animal models, natural killer (NK cells have been proposed to play an important role in the protection provided by M2-based vaccines through a mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC. We investigated the ability of the human anti-M2 Ab1-10 monoclonal antibody (mAb to activate human NK cells. They mediated ADCC against M2-expressing cells in the presence of Ab1-10 mAb. Furthermore, NK cell pro-inflammatory cytokine and chemokine secretion is also enhanced when Ab1-10 mAb is present. We also generated cytokine-preactivated NK cells and showed that they still displayed increased effector functions in the presence of Ab1-10 mAb. Thus, our study has demonstrated that human resting and cytokine-preactivated NK cells may have a very important role in the protection provided by anti-M2 Abs.

  14. Discrepancy between direct and antibody-dependent cytotoxic activities of human LAK cells. (United States)

    Potapnev, M P; Garbuzenco, T S; Goncharova, N V; Zobnin, V D; Shadrin, O V; Bykovskaya, S N


    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. Antibody-dependent cellular cytotoxicity and skin disease

    Energy Technology Data Exchange (ETDEWEB)

    Norris, D.A.; Lee, L.A.


    Antibody dependent cellular cytotoxicity (ADCC) is a recently described mechanism of immunologic lysis in which cellular targets sensitized by specific antibodies are efficiently and selectively lysed by Fc receptor (FcR) bearing nonspecific effectors. Immunoglobulins of various classes (IgG, IgM, IgA, IgE) and various cellular effectors (large granular lymphocytes, monocyte/macrophages, T lymphocytes, neutrophils, and eosinophils) can induce ADCC in vitro, and the importance of ADCC in vivo is being tested experimentally in resistance to viral, bacterial, and parasitic infection, in tumor surveillance, in allograft rejection, and in inflammatory diseases. There is much indirect evidence that ADCC may be the mechanism of damage of different cellular targets in skin diseases, but the best direct evidence concerns immunologic keratinocyte damage, especially in cutaneous lupus erythematosus (LE). The authors have shown that keratinocytes of several species are highly susceptible to lymphocyte and monocyte-mediated ADCC, but not to neutrophil or eosinophil ADCC in vitro using two different cytotoxicity assays. In contrast, complement was a relatively ineffective mediator of lysis of metabolically intact keratinocyte targets. Patients with certain cutaneous lupus syndromes have serum antibodies capable of inducing monocyte and lymphocyte ADCC of targets coated with extractable nuclear antigens. The authors have shown that these antigens apparently move to the cell membrane of keratinocytes in vitro following ultraviolet irradiation. In an animal model, they have shown that antibodies to SSA/Ro bind to human keratinocytes in vivo, especially after ultraviolet irradiation.

  16. Flow cytometry evaluation of cell-mediated cytotoxicity. (United States)

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


    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.

  17. Malignant monoblasts can function as effector cells in natural killer cell and antibody-dependent cellular cytotoxicity assays

    DEFF Research Database (Denmark)

    Hokland, P; Hokland, M; Ellegaard, J


    This is the first report describing natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) of malignant monoblasts. Pure acute monoblastic leukemia was diagnosed in bone marrow aspirations from two patients by use of conventional cytochemical methods as well as multiple immunologic...

  18. CD16 is indispensable for antibody-dependent cellular cytotoxicity by human monocytes (United States)

    Yeap, Wei Hseun; Wong, Kok Loon; Shimasaki, Noriko; Teo, Esmeralda Chi Yuan; Quek, Jeffrey Kim Siang; Yong, Hao Xiang; Diong, Colin Phipps; Bertoletti, Antonio; Linn, Yeh Ching; Wong, Siew Cheng


    Antibody-dependent cellular cytotoxicity (ADCC) is exerted by immune cells expressing surface Fcγ receptors (FcγRs) against cells coated with antibody, such as virus-infected or transformed cells. CD16, the FcγRIIIA, is essential for ADCC by NK cells, and is also expressed by a subset of human blood monocytes. We found that human CD16− expressing monocytes have a broad spectrum of ADCC capacities and can kill cancer cell lines, primary leukemic cells and hepatitis B virus-infected cells in the presence of specific antibodies. Engagement of CD16 on monocytes by antibody bound to target cells activated β2-integrins and induced TNFα secretion. In turn, this induced TNFR expression on the target cells, making them susceptible to TNFα-mediated cell death. Treatment with TLR agonists, DAMPs or cytokines, such as IFNγ, further enhanced ADCC. Monocytes lacking CD16 did not exert ADCC but acquired this property after CD16 expression was induced by either cytokine stimulation or transient transfection. Notably, CD16+ monocytes from patients with leukemia also exerted potent ADCC. Hence, CD16+ monocytes are important effectors of ADCC, suggesting further developments of this property in the context of cellular therapies for cancer and infectious diseases. PMID:27670158

  19. Clinical Cancer Therapy by NK Cells via Antibody-Dependent Cell-Mediated Cytotoxicity

    Directory of Open Access Journals (Sweden)

    Kory L. Alderson


    Full Text Available Natural killer (NK cells are powerful effector cells that can be directed to eliminate tumor cells through tumor-targeted monoclonal antibodies (mAbs. Some tumor-targeted mAbs have been successfully applied in the clinic and are included in the standard of care for certain malignancies. Strategies to augment the antitumor response by NK cells have led to an increased understanding of how to improve their effector responses. Next-generation reagents, such as molecularly modified mAbs and mAb-cytokine fusion proteins (immunocytokines, ICs designed to augment NK-mediated killing, are showing promise in preclinical and some clinical settings. Continued research into the antitumor effects induced by NK cells and tumor-targeted mAbs suggests that additional intrinsic and extrinsic factors may influence the antitumor response. Therefore more research is needed that focuses on evaluating which NK cell and tumor criteria are best predictive of a clinical response and which combination immunotherapy regimens to pursue for distinct clinical settings.

  20. Affinity-purified respiratory syncytial virus antibodies from intravenous immunoglobulin exert potent antibody-dependent cellular cytotoxicity.

    Directory of Open Access Journals (Sweden)

    Nimesh Gupta

    Full Text Available Mixed infections are one of the major therapeutic challenges, as the current strategies have had limited success. One of the most common and widespread conditions of mixed infection is respiratory syncytial virus-mediated pathology of the respiratory tract in children. There is a dire need for the development of novel therapeutic approaches during mixed infections. Therapeutic intravenous immunoglobulin preparations, obtained from plasma pools of healthy donors have been used in immune deficiencies. This study was thus designed to characterize the functional efficacy of RSV-specific antibodies in IVIg. To explore the functional ability of these affinity-purified RSV-specific antibodies, the antibody-dependent and complement dependent cytotoxicity was determined using peripheral cells of healthy donors. This study demonstrates the existence of highly potent RSV-specific antibodies in IVIg preparations and provides the basis for the use of IVIg as broad-spectrum protective shield to RSV-infected children during mixed infections.

  1. Augmentation of natural killer cell and antibody-dependent cellular cytotoxicity in BALB/c mice by sulforaphane, a naturally occurring isothiocyanate from broccoli through enhanced production of cytokines IL-2 and IFN-gamma. (United States)

    Thejass, P; Kuttan, G


    Effect of sulforaphane on cell-mediated immune (CMI) response was studied in normal as well as Ehrlich ascites tumor-bearing BALB/c mice. Administration of sulforaphane significantly enhanced natural killer (NK) cell activity in both normal as well as tumor-bearing animals, and the activity was observed earlier than in tumor-bearing control animals. Antibody-dependent cellular cytotoxicity (ADCC) also was enhanced significantly in both normal as well as tumor-bearing animals after sulforaphane administration compared with untreated control tumor-bearing animals. An early antibody-dependent complement-mediated cytotoxicity (ACC) also was observed in sulforaphane-treated normal and tumor-bearing animals. Administration of sulforaphane significantly enhanced the production of Interleukin-2 and Interferon-gamma in normal as well as tumor-bearing animals. In addition, sulforaphane significantly enhanced the proliferation of splenocytes, bone marrow cells, and thymocytes by stimulating the mitogenic potential of various mitogens such as concanavalin A, phytohaemagglutinin, poke weed mitogen, and lipopolysaccharide.

  2. 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


    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...... 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....

  3. Antibody-dependent-cellular-cytotoxicity-inducing antibodies significantly affect the post-exposure treatment of Ebola virus infection (United States)

    Liu, Qiang; Fan, Changfa; Li, Qianqian; Zhou, Shuya; Huang, Weijin; Wang, Lan; Sun, Chunyun; Wang, Meng; Wu, Xi; Ma, Jian; Li, Baowen; Xie, Liangzhi; Wang, Youchun


    Passive immunotherapy with monoclonal antibodies (mAbs) is an efficacious treatment for Ebola virus (EBOV) infections in animal models and humans. Understanding what constitutes a protective response is critical for the development of novel therapeutic strategies. We generated an EBOV-glycoprotein-pseudotyped Human immunodeficiency virus to develop sensitive neutralizing and antibody-dependent cellular cytotoxicity (ADCC) assays as well as a bioluminescent-imaging-based mouse infection model that does not require biosafety level 4 containment. The in vivo treatment efficiencies of three novel anti-EBOV mAbs at 12 h post-infection correlated with their in vitro anti-EBOV ADCC activities, without neutralizing activity. When they were treated with these mAbs, natural killer cell (NK)-deficient mice had lower viral clearance than WT mice, indicating that the anti-EBOV mechanism of the ADCC activity of these mAbs is predominantly mediated by NK cells. One potent anti-EBOV mAb (M318) displayed unprecedented neutralizing and ADCC activities (neutralization IC50, 0.018 μg/ml; ADCC EC50, 0.095 μg/ml). These results have important implications for the efficacy of antiviral drugs and vaccines as well as for pathogenicity studies of EBOV. PMID:28358050

  4. Tumour antigen targeted monoclonal antibodies incorporating a novel multimerisation domain significantly enhance antibody dependent cellular cytotoxicity against colon cancer. (United States)

    Jain, Ajay; Poonia, Bhawna; So, Edward C; Vyzasatya, Ravi; Burch, Erin E; Olsen, Henrik S; Mérigeon, Emmanuel Y; Block, David S; Zhang, Xiaoyu; Schulze, Dan H; Hanna, Nader N; Twadell, William S; Yfantis, Harris G; Chan, Siaw L; Cai, Ling; Strome, Scott E


    Tumour antigen targeted antibodies (mAbs) can induce natural killer (NK) cells to kill tumours through antibody dependent cellular cytotoxicity (ADCC) upon engagement of NK cell expressed FcγRIIIa. FcγRIIIa polymorphisms partially dictate the potency of the ADCC response. The high affinity FcγRIIIa-158-valine (V) polymorphism is associated with more potent ADCC response than the low affinity FcγRIIIa-158-phenylalanine (F) polymorphism. Because approximately 45% of patients are homozygous for the FcγRIIIa-158-F polymorphism (FF genotype), their ability to mount ADCC is impaired. We investigated whether a novel mAb capable of binding multiple antigen specific targets and engaging multiple low affinity FcγRIIIa receptors could further enhance ADCC against colon cancer in vitro. Specifically, we generated a novel anti-epidermal growth factor receptor (EGFR) antibody (termed a stradobody) consisting of an unmodified Fab sequence and two Immunoglobulin G, subclass 1 (IgG1) Fc domains separated by an isoleucine zipper domain and the 12 amino-acid IgG2 hinge. The stradobody framework induced multimerisation and was associated with increased binding to the EGFR and FcγRIIIa. From a functional perspective, when compared to an unmodified anti-EGFR mAb with a sequence identical to cetuximab (a commercially available anti-EGFR mAb), stradobodies significantly enhanced ADCC. These effects were observed using both KRAS wild type HT29 and KRAS mutant SW480 colon cancer cells as targets, and by NK cells obtained from healthy donors and a cohort of patients with colon cancer. These data suggest that high avidity cross-linking of multiple tumour surface antigens and multiple NK cell associated FcγRIIIa molecules can enhance ADCC and partially overcome impaired ADCC by FF genotype individuals in vitro.

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

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


    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.

  6. 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. (United States)

    Ashkenazi, M; Kohl, S


    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.

  7. A novel antibody-dependent cellular cytotoxicity mechanism involved in defense against malaria requires costimulation of monocytes FcgammaRII and FcgammaRIII

    DEFF Research Database (Denmark)

    Jafarshad, Ali; Dziegiel, Morten Hanefeld; Lundquist, Rasmus


    Clinical experiments have shown that the Ab-dependent cell-mediated inhibition of Plasmodium falciparum is a major mechanism controlling malaria parasitemia and thereby symptoms. In this study, we demonstrate that a single merozoite per monocyte (MN) is sufficient to trigger optimal antiparasitic......-dependent cellular cytotoxicity and implies that all MN are not equally effective. These findings have both fundamental and practical implications, particularly for vaccine discovery....

  8. 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. (United States)

    Takayanagi, Toshiaki


    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.

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

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


    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.

  10. Neutralizing Antibody Response and Antibody-Dependent Cellular Cytotoxicity in HIV-1-Infected Individuals from Guinea-Bissau and Denmark

    DEFF Research Database (Denmark)

    Borggren, Marie; Jensen, Sanne Skov; Heyndrickx, Leo


    cytotoxicity (ADCC) against local and nonlocal circulating HIV-1 strains. The neutralizing activity did not demonstrate higher potential against local circulating strains according to geography and subtype determination, but the plasma from Danish individuals demonstrated significantly higher inhibitory...

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

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


    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.

  12. KIR/HLA interactions negatively affect rituximab- but not GA101 (obinutuzumab)-induced antibody-dependent cellular cytotoxicity. (United States)

    Terszowski, Grzegorz; Klein, Christian; Stern, Martin


    Ab-dependent cellular cytotoxicity (ADCC) mediated by NK cells is regulated by inhibitory killer cell Ig-like receptors (KIRs), which interact with target cell HLA class I. We analyzed how KIR/HLA interactions influence ADCC induced by rituximab and by GA101, a novel type II CD20 Ab glycoengineered for increased FcgRIII binding and ADCC capacity. We found that KIR/HLA interactions strongly and selectively inhibit rituximab-induced in vitro ADCC toward target cells expressing cognate HLA KIR ligands. NK cells of donors carrying all three ligands to inhibitory KIR showed weak activation and target cell depletion capacity when incubated with rituximab and KIR-ligand matched target B cells. In contrast, NK cells from individuals missing one or more KIR ligands activated more strongly and depleted KIR ligand-matched target B cells more efficiently in the presence of rituximab. NK cells expressing a KIR for which the ligand was absent were the main effectors of ADCC in these donors. Notably, the influence of KIR/HLA interactions on NK cell activation was synergistic with the effect of the V158F FCGR3A single nucleotide polymorphism. In contrast, GA101 induced activation of NK cells irrespective of inhibitory KIR expression, and efficiency of target cell depletion was not negatively affected by KIR/HLA interactions. These data show that modification of the Fc fragment to enhance ADCC can be an effective strategy to augment the efficacy of therapeutic mAbs by recruiting NK cells irrespective of their inhibitory KIR expression.

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



    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...

  14. Narcolepsy-Associated HLA Class I Alleles Implicate Cell-Mediated Cytotoxicity (United States)

    Tafti, Mehdi; Lammers, Gert J.; Dauvilliers, Yves; Overeem, Sebastiaan; Mayer, Geert; Nowak, Jacek; Pfister, Corinne; Dubois, Valérie; Eliaou, Jean-François; Eberhard, Hans-Peter; Liblau, Roland; Wierzbicka, Aleksandra; Geisler, Peter; Bassetti, Claudio L.; Mathis, Johannes; Lecendreux, Michel; Khatami, Ramin; Heinzer, Raphaël; Haba-Rubio, José; Feketeova, Eva; Baumann, Christian R.; Kutalik, Zoltán; Tiercy, Jean-Marie


    Study Objectives: Narcolepsy with cataplexy is tightly associated with the HLA class II allele DQB1*06:02. Evidence indicates a complex contribution of HLA class II genes to narcolepsy susceptibility with a recent independent association with HLA-DPB1. The cause of narcolepsy is supposed be an autoimmune attack against hypocretin-producing neurons. Despite the strong association with HLA class II, there is no evidence for CD4+ T-cell-mediated mechanism in narcolepsy. Since neurons express class I and not class II molecules, the final effector immune cells involved might include class I-restricted CD8+ T-cells. Methods: HLA class I (A, B, and C) and II (DQB1) genotypes were analyzed in 944 European narcolepsy with cataplexy patients and in 4,043 control subjects matched by country of origin. All patients and controls were DQB1*06:02 positive and class I associations were conditioned on DQB1 alleles. Results: HLA-A*11:01 (OR = 1.49 [1.18–1.87] P = 7.0*10−4), C*04:01 (OR = 1.34 [1.10–1.63] P = 3.23*10−3), and B*35:01 (OR = 1.46 [1.13–1.89] P = 3.64*10−3) were associated with susceptibility to narcolepsy. Analysis of polymorphic class I amino-acids revealed even stronger associations with key antigen-binding residues HLA-A-Tyr9 (OR = 1.32 [1.15–1.52] P = 6.95*10−5) and HLA-C-Ser11 (OR = 1.34 [1.15–1.57] P = 2.43*10−4). Conclusions: Our findings provide a genetic basis for increased susceptibility to infectious factors or an immune cytotoxic mechanism in narcolepsy, potentially targeting hypocretin neurons. Citation: Tafti M, Lammers GJ, Dauvilliers Y, Overeem S, Mayer G, Nowak J, Pfister C, Dubois V, Eliaou JF, Eberhard HP, Liblau R, Wierzbicka A, Geisler P, Bassetti CL, Mathis J, Lecendreux M, Khatami R, Heinzer R, Haba-Rubio J, Feketeova E, Baumann CR, Kutalik Z, Tiercy JM. Narcolepsy-associated HLA class I alleles implicate cell-mediated cytotoxicity. SLEEP 2016;39(3):581–587. PMID:26518595

  15. Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

    Directory of Open Access Journals (Sweden)

    Cavazzoni Andrea


    Full Text Available Abstract Background The epidermal growth factor receptor (EGFR is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. Results In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. Conclusion Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib.

  16. A Phase I Trial to Evaluate Antibody-Dependent Cellular Cytotoxicity of Cetuximab and Lenalidomide in Advanced Colorectal and Head and Neck Cancer. (United States)

    Bertino, Erin M; McMichael, Elizabeth L; Mo, Xiaokui; Trikha, Prashant; Davis, Melanie; Paul, Bonnie; Grever, Michael; Carson, William E; Otterson, Gregory A


    mAbs can induce antibody-dependent cellular cytotoxicity (ADCC) via the innate immune system's ability to recognize mAb-coated cancer cells and activate immune effector cells. Lenalidomide is an immunomodulatory agent with the capacity to stimulate immune cell cytokine production and ADCC activity. This phase I trial evaluated the combination of cetuximab with lenalidomide for the treatment of advanced colorectal and head and neck squamous cell cancers (HNSCC). This trial included patients with advanced colorectal cancer or HNSCC. Treatment consisted of cetuximab 500 mg/m(2) i.v. every two weeks with lenalidomide given orally days 1-21 on a 28-day cycle. Three dose levels of lenalidomide were evaluated (15, 20, 25 mg). Correlative studies included measurement of ADCC, FcγRIIIA polymorphism genotyping, measurement of serum cytokine levels, and flow cytometric analysis of immune cell subtypes. Twenty-two patients were enrolled (19 colorectal cancer, 3 HNSCC). Fatigue was the only dose-limiting toxicity. One partial response was observed and 8 patients had stable disease at least 12 weeks. The recommended phase II dose is cetuximab 500 mg/m(2) with lenalidomide 25 mg daily, days 1-21. Correlative studies demonstrated a dose-dependent increase in natural killer cytotoxic activity with increasing doses of lenalidomide. Cetuximab and lenalidomide were well tolerated. There was a lenalidomide dose-dependent increase in ADCC with higher activity in patients enrolled in cohort 3 than those enrolled in cohorts 1/2. Although response was not a primary endpoint, there was evidence of antitumor activity for the combination therapy. Further investigation of lenalidomide as an immunomodulator in solid tumors is warranted. Mol Cancer Ther; 15(9); 2244-50. ©2016 AACR.

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

    Directory of Open Access Journals (Sweden)

    Mahajan Supriya D.


    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.

  18. Hyperimmune antisera against synthetic peptides representing the glycoprotein of human immunodeficiency virus type 2 can mediate neutralization and antibody-dependent cytotoxic activity. (United States)

    Björling, E; Broliden, K; Bernardi, D; Utter, G; Thorstensson, R; Chiodi, F; Norrby, E


    Twenty-five 13- to 35-amino-acid-long peptides representing regions of human immunodeficiency virus type 2 (HIV-2), strain SBL6669, envelope proteins were evaluated for their immunogenic activity in guinea pigs. The peptides were selected to provide homologous representation of sites in the HIV-1 envelope proteins that were previously documented to have a particular immunogenic importance. A number of the HIV-2 peptides were found to be capable of inducing strain SBL6669 neutralizing and antibody-dependent cellular cytotoxicity (ADCC) antibodies. Two overlapping peptides covering amino acids 311-337 representing the central and C-terminal part of the variable third (V3) region, terminology according to Modrow et al. [Modrow, S., Hahn, B., Shaw, G. M., Gallo, R. C., Wong-Staal, F. & Wolf, H. (1987) J. Virol. 61, 570-578], showed the most pronounced capacity to induce neutralizing antibodies. One of the peptides (amino acids 318-337) also induced antibodies mediating ADCC. Two additional regions in the large glycoprotein, gp125, containing linear sites reacting with neutralizing antibodies were identified (amino acids, 119-137 and 472-509). The transmembrane protein, gp36, of HIV-2 harbored two regions of importance for induction of neutralizing antibodies (amino acids 595-614 and 714-729). ADCC activity was induced by two additional gp125-specific peptides (amino acids 291-311 and 446-461). Thus, except for the single V3-specific site there was no correlation between linear immunogenic sites stimulating neutralizing antibody and ADCC activity. These findings pave the way for development of synthetic vaccines against HIV-2 and possibly also simian immunodeficiency virus infections. The capacity of such a product to induce protective immunity can be evaluated in macaque monkeys. Images PMID:2068087

  19. Influenza virus A(H1N1)2009 antibody-dependent cellular cytotoxicity in young children prior to the H1N1 pandemic. (United States)

    Mesman, Annelies W; Westerhuis, Brenda M; Ten Hulscher, Hinke I; Jacobi, Ronald H; de Bruin, Erwin; van Beek, Josine; Buisman, Annemarie M; Koopmans, Marion P; van Binnendijk, Robert S


    Pre-existing immunity played a significant role in protection during the latest influenza A virus H1N1 pandemic, especially in older age groups. Structural similarities were found between A(H1N1)2009 and older H1N1 virus strains to which humans had already been exposed. Broadly cross-reactive antibodies capable of neutralizing the A(H1N1)2009 virus have been implicated in this immune protection in adults. We investigated the serological profile of a group of young children aged 9 years (n=55), from whom paired blood samples were available, just prior to the pandemic wave (March 2009) and shortly thereafter (March 2010). On the basis of A(H1N1)2009 seroconversion, 27 of the 55 children (49 %) were confirmed to be infected between these two time points. Within the non-infected group of 28 children (51 %), high levels of seasonal antibodies to H1 and H3 HA1 antigens were detected prior to pandemic exposure, reflecting past infection with H1N1 and H3N2, both of which had circulated in The Netherlands prior to the pandemic. In some children, this reactivity coincided with specific antibody reactivity against A(H1N1)2009. While these antibodies were not able to neutralize the A(H1N1)2009 virus, they were able to mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro upon interaction with the A(H1N1)2009 virus. This finding suggests that cross-reactive antibodies could contribute to immune protection in children via ADCC.

  20. Mechanisms of pancreatic islet cell destruction. Dose-dependent cytotoxic effect of soluble blood mononuclear cell mediators on isolated islets of Langerhans

    DEFF Research Database (Denmark)

    Mandrup-Poulsen, T; Bendtzen, K; Nerup, J;


    reconstituted with tuberculin or phytohaemagglutinin did not impair islet function. Electron microscopy demonstrated that supernatants were cytotoxic to islet cells. The cytotoxic mononuclear cell mediator(s) was non-dialysable, sensitive to heating to 56 degrees C, labile even when stored at -70 degrees C...

  1. 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


    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....

  2. 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;


    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...

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

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


    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.

  4. Intracellular protease activation in apoptosis and cell-mediated cytotoxicity characterized by cell-permeable fluorogenic protease substrates

    Institute of Scientific and Technical Information of China (English)

    Beverly Z Packard; Akira Komoriya


    Over the past decade the importance of signaling from reporter molecules inside live cells and tissues has been clearly established. Biochemical events related to inflammation, tumor metastasis and proliferation, and viral infectivity and replication are examples of processes being further defined as more molecular tools for live cell measurements become available. Moreover, in addition to quantitating parameters related to physiologic processes, real-time imaging of molecular interactions that compose basic cellular activities are providing insights into understanding disease mechanisms as well as extending clinical efficacy of therapeutic regimens. In this review the use of highly cell-permeable fluorogenic substrates that report protease activities inside live cells is described; applications to defining the molecular events of two cellular processes, i.e., apoptosis and cell-mediated cytotoxicity, are then illustrated.

  5. Cell-mediated cytotoxicity in rainbow trout, Oncorhynchus mykiss, infected with viral haemorrhagic septicaemia virus

    DEFF Research Database (Denmark)

    Utke, K.; Bergmann, S.; Lorenzen, Niels


    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...... classical MHC class I locus Onmy-UBA is identical in the rainbow trout clone C25 and in the permanent rainbow trout cell line RTG-2. This enabled us to develop an assay to measure antiviral cytotoxicity in rainbow trout using a system of MHC class I-matched effector and target cells. Peripheral blood...... 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...

  6. Suppression of NK cell-mediated cytotoxicity against PRRSV-infected porcine alveolar macrophages in vitro. (United States)

    Cao, Jun; Grauwet, Korneel; Vermeulen, Ben; Devriendt, Bert; Jiang, Ping; Favoreel, Herman; Nauwynck, Hans


    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.

  7. Fasting-Mimicking Diet Reduces HO-1 to Promote T Cell-Mediated Tumor Cytotoxicity. (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


    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.

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

    Directory of Open Access Journals (Sweden)

    Christian B Willberg

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

  9. 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


    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

  10. Down-regulation of LAK cell-mediated cytotoxicity: cancer and ageing. (United States)

    Bykovskaya, S N; Abronina, I F; Kupriyanova, T A; Bubenik, J


    A comparative study was made of the generation of lymphokine-activated killer (LAK) cells in patients with melanoma and healthy donors of different age groups. Significant reduction of effector cell cytotoxicity in patients following 72 h culture with 1,000 U/ml or recombinant IL-2 (rIL-2) as well as a decreased ability to generate LAK cells in elderly individuals were shown to be correlated with suppressor cell activation in rIL-2 stimulated cell population. Suppressor effect depends on monocytes and T-lymphocytes: partial abolition of suppression in LAK cells was observed following removal of adherent cells or treatment with OKT8 monoclonal antibodies and complement.

  11. Combination of two anti-CD5 monoclonal antibodies synergistically induces complement-dependent cytotoxicity of chronic lymphocytic leukaemia cells

    DEFF Research Database (Denmark)

    Klitgaard, Josephine L; Koefoed, Klaus; Geisler, Christian


    secondary effector functions represent an attractive opportunity for CLL treatment. Here, a repertoire of mAbs against human CD5 was generated and tested for ability to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) both as single m...

  12. Induction of Wnt-inducible signaling protein-1 correlates with invasive breast cancer oncogenesis and reduced type 1 cell-mediated cytotoxic immunity: a retrospective study. (United States)

    Klinke, David J


    Innate and type 1 cell-mediated cytotoxic immunity function as important extracellular control mechanisms that maintain cellular homeostasis. Interleukin-12 (IL12) is an important cytokine that links innate immunity with type 1 cell-mediated cytotoxic immunity. We recently observed in vitro that tumor-derived Wnt-inducible signaling protein-1 (WISP1) exerts paracrine action to suppress IL12 signaling. The objective of this retrospective study was three fold: 1) to determine whether a gene signature associated with type 1 cell-mediated cytotoxic immunity was correlated with overall survival, 2) to determine whether WISP1 expression is increased in invasive breast cancer, and 3) to determine whether a gene signature consistent with inhibition of IL12 signaling correlates with WISP1 expression. Clinical information and mRNA expression for genes associated with anti-tumor immunity were obtained from the invasive breast cancer arm of the Cancer Genome Atlas study. Patient cohorts were identified using hierarchical clustering. The immune signatures associated with the patient cohorts were interpreted using model-based inference of immune polarization. Reverse phase protein array, tissue microarray, and quantitative flow cytometry in breast cancer cell lines were used to validate observed differences in gene expression. We found that type 1 cell-mediated cytotoxic immunity was correlated with increased survival in patients with invasive breast cancer, especially in patients with invasive triple negative breast cancer. Oncogenic transformation in invasive breast cancer was associated with an increase in WISP1. The gene expression signature in invasive breast cancer was consistent with WISP1 as a paracrine inhibitor of type 1 cell-mediated immunity through inhibiting IL12 signaling and promoting type 2 immunity. Moreover, model-based inference helped identify appropriate immune signatures that can be used as design constraints in genetically engineering better pre

  13. Immunomodulatory activity of xanthohumol: inhibition of T cell proliferation, cell-mediated cytotoxicity and Th1 cytokine production through suppression of NF-κB


    Gao, Xiaohua; Deeb, Dorrah; Liu, Yongbo; DULCHAVSKY, SCOTT A.; Gautam, Subhash C.


    Xanthohumol (XN), a prenylated chalcone present in hops (Humulus lupus L.) and beer, exhibits anti-inflammatory, antioxidant and antiproliferative activity, but has not been studied for effects on T cell-mediated immune responses. Here we demonstrate that XN has profound immunosuppressive effects on T cell proliferation, development of IL-2 activated killer (LAK) cells, cytotoxic T lymphocytes (CTLs), and production of Th1 cytokines (IL-2, IFN-γ and TNF-α). The suppression of these cell-media...

  14. An impedance-based cytotoxicity assay for real-time and label-free assessment of T-cell-mediated killing of adherent cells. (United States)

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


    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.

  15. Peritoneal lavage cells of Indonesian thin-tail sheep mediate antibody-dependent superoxide radical cytotoxicity in vitro against newly excysted juvenile Fasciola gigantica but not juvenile Fasciola hepatica. (United States)

    Piedrafita, David; Estuningsih, Endah; Pleasance, Jill; Prowse, Rhoda; Raadsma, Herman W; Meeusen, Els N T; Spithill, Terry W


    Indonesian thin-tail (ITT) sheep resist infection by Fasciola gigantica by an immunological mechanism within 2 to 4 weeks of infection yet are susceptible to F. hepatica infection. Studies of ITT sheep show that little liver damage occurs following F. gigantica infection, suggesting that the invading parasites are killed within the peritoneum or shortly after reaching the liver. We investigated whether cells isolated from the peritoneums of ITT sheep could kill newly excysted juvenile F. gigantica in vitro and act as a potential mechanism of resistance against F. gigantica infection. Peritoneal cells from F. gigantica-infected sheep, rich in macrophages and eosinophils, mediated antibody-dependent cytotoxicity against juvenile F. gigantica in vitro. Cytotoxicity was dependent on contact between the parasite and effector cells. Isolated mammary gland eosinophils of F. gigantica-infected sheep, or resident peritoneal monocytes/macrophages from uninfected sheep, also killed the juvenile parasites in vitro. By using inhibitors, we show that the molecular mechanism of killing in these assays was dependent on the production of superoxide radicals by macrophages and eosinophils. In contrast, this cytotoxic mechanism was ineffective against juvenile F. hepatica parasites in vitro. Analysis of superoxide dismutase activity and mRNA levels showed that activity and gene expression were higher in F. hepatica than in F. gigantica, suggesting a possible role for this enzyme in the resistance of F. hepatica to superoxide-mediated killing. We suggest that ovine macrophages and eosinophils, acting in concert with a specific antibody, may be important effector cells involved in the resistance of ITT sheep to F. gigantica.

  16. 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


    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.

  17. Anti-Glycoprotein G Antibodies of Herpes Simplex Virus 2 Contribute to Complete Protection after Vaccination in Mice and Induce Antibody-Dependent Cellular Cytotoxicity and Complement-Mediated Cytolysis

    Directory of Open Access Journals (Sweden)

    Staffan Görander


    Full Text Available We investigated the role of antibodies against the mature portion of glycoprotein G (mgG-2 of herpes simplex virus 2 (HSV-2 in protective immunity after vaccination. Mice were immunized intramuscularly with mgG-2 and oligodeoxynucleotides containing two CpG motifs plus alum as adjuvant. All C57BL/6 mice survived and presented no genital or systemic disease. High levels of immunoglobulin G subclass 1 (IgG1 and IgG2 antibodies were detected and re-stimulated splenic CD4+ T cells proliferated and produced IFN-γ. None of the sera from immunized mice exhibited neutralization, while all sera exerted antibody-dependent cellular cytotoxicity (ADCC and complement-mediated cytolysis (ACMC activity. Passive transfer of anti-mgG-2 monoclonal antibodies, or immune serum, to naive C57BL/6 mice did not limit disease progression. Immunized B‑cell KO mice presented lower survival rate and higher vaginal viral titers, as compared with vaccinated B-cell KO mice after passive transfer of immune serum and vaccinated C57BL/6 mice. Sera from mice that were vaccinated subcutaneously and intranasally with mgG-2 presented significantly lower titers of IgG antibodies and lower ADCC and ACMC activity. We conclude that anti-mgG-2 antibodies were of importance to limit genital HSV‑2 infection. ADCC and ACMC activity are potentially important mechanisms in protective immunity, and could tentatively be evaluated in future animal vaccine studies and in clinical trials.

  18. Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2 Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

    Directory of Open Access Journals (Sweden)

    Masaki Kurogochi

    Full Text Available Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain, and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC and complement dependent cytotoxicity (CDC. To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases, one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2, high-mannose type (Man4-9GlcNAc2, and complex type (Man3GlcNAc3-4 N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL, the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1 were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q, and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2 was performed using SKBR-3 and BT-474 as target

  19. Induction of NKG2D ligands and increased sensitivity of tumor cells to NK cell-mediated cytotoxicity by hematoporphyrin-based photodynamic therapy. (United States)

    Park, Min-Ju; Bae, Jae-Ho; Chung, Joo Seop; Kim, Sun-Hee; Kang, Chi-Dug


    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.

  20. Oxaliplatin regulates expression of stress ligands in ovarian cancer cells and modulates their susceptibility to natural killer cell-mediated cytotoxicity. (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


    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.

  1. The Role of Antibody Isotypes Against Fasciola Gigantica In Immune Response With An Emphasis On Antibody Dependent Cell Mediated Cytoxicity%抗巨片形吸虫同型抗体在动物巨片形吸虫感染所致的抗体依赖性细胞介导的细胞毒反应( ADCC )免疫应答作用的研究

    Institute of Scientific and Technical Information of China (English)

    陈思礼; 陈强; 周雨丝; 李玲; 陈思义; 吴风娇; 陈思祗; 丁书茂; SPITHILL Terry


    The study was carried out in two breeds of sheep, Indonesia Thin Tail (ITT) and Merino, both infected with F. gigantica. Kinetic analysis of Fasciola gigantica specific antibody responses revealed that resistant ITT do not produce a parasite specific IgG2 response; whereas susceptible Merino sheep produce a high parasite specific IgG2 response. The IgG2 produced in Merino sheep might act as a blocking antibody for antibody dependant cell mediated cytotoxicity by macrophages. Whereas, ITT appeared to down regulate IgG2 response, which might enable these sheep to possess an enhanced capacity of killing F. gigantica. The purified IgG1 and IgG2 were used in in vitro killing assays. IgG1 promotes the highest amount of killing compared to immune sera and IgG2 using purified Merino sera antibodies for in vitro killing assays. F. gigantica cultured in increasing amount of IgG1 had a trend of increasing the death rate. Parasites cultured in 10 ug/ml of IgG1 showed the highest amount of death. F. gigantica cultured in eosinophils with immune ITT sera (Day35) was 55% death after 24 hours. It should be that using purified eosinophils with immune ITT serum resulted in a strong trend of killing occurring in those wells which contained a combination of immune sera complement, IL-5 and eosinophils.%以印尼短尾羊(ITT)和美利奴细毛羊( Merino )两种品系的绵羊为实验动物,研究动物感染巨片形吸虫后所产生的抗体依赖性细胞介导的细胞毒反应的免疫应答.结果显示:对巨片形吸虫感染具有抗性的印尼短尾羊不产生特异性的IgG2, 而易感品系的美利奴细毛羊感染巨片形吸虫后体内产生高滴度特异性IgG2抗体.特异性IgG2抗体在免疫应答中起着封闭抗体的作用,抑制巨噬细胞抗体依赖性细胞介导的细胞毒反应.由于印尼短尾羊不产生特异性IgG2抗体,对巨噬细胞抗体依赖性细胞介导的细胞毒反应不产生抑制作用,因此印尼短尾羊对巨片形吸

  2. T cell-mediated cytotoxicity against p53-protein derived peptides in bulk and limiting dilution cultures of healthy donors

    DEFF Research Database (Denmark)

    Röpke, M; Regner, M; Claesson, M H


    -I restricted epitopes for T cell recognition and p53-derived peptides have been suggested as targets for tumour-specific cytotoxic T lymphocytes (CTL). Our primary aim was to estimate the frequencies of p53-peptide reactive CTL precursors (CTLp) in peripheral blood from healthy young individuals. We selected...

  3. An oral chitosan DNA vaccine against nodavirus improves transcription of cell-mediated cytotoxicity and interferon genes in the European sea bass juveniles gut and survival upon infection. (United States)

    Valero, Yulema; Awad, Elham; Buonocore, Francesco; Arizcun, Marta; Esteban, M Ángeles; Meseguer, José; Chaves-Pozo, Elena; Cuesta, Alberto


    Vaccines for fish need to be improved for the aquaculture sector, with DNA vaccines and the oral administration route providing the most promising improvements. In this study, we have created an oral chitosan-encapsulated DNA vaccine (CP-pNNV) for the nodavirus (NNV) in order to protect the very susceptible European sea bass (Dicentrarchus labrax). Our data show that the oral CP-pNNV vaccine failed to induce serum circulating or neutralizing specific antibodies (immunoglobulin M) or to up-regulate their gene expression in the posterior gut. However, the vaccine up-regulated the expression of genes related to the cell-mediated cytotoxicity (CMC; tcrb and cd8a) and the interferon pathway (IFN; ifn, mx and ifng). In addition, 3 months after vaccination, challenged fish showed a retarded onset of fish death and lower cumulative mortality with a relative survival of 45%. Thus, we created a chitosan-encapsulated DNA vaccine against NNV that is partly protective to European sea bass juveniles and up-regulates the transcription of genes related to CMC and IFN. However, further studies are needed to improve the anti-NNV vaccine and to understand its mechanisms.

  4. 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


    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...... on their surface. Polyclonal antibodies against defined peptides in the Env- and Gag-regions of the HERVs were raised in rabbits and used in antibody-dependent cell-mediated cytotoxicity (ADCC) -assays. Rituximab® (Roche), a chimeric monoclonal antibody against CD20 expressed primarily on B cells, was used...

  5. Enhancement of antibody-dependent mechanisms of tumor cell lysis by a targeted activator of complement. (United States)

    Imai, Masaki; Ohta, Rieko; Varela, Juan C; Song, Hongbin; Tomlinson, Stephen


    Complement inhibitors expressed on tumor cells provide a hindrance to the therapeutic efficacy of some monoclonal antibodies (mAb). We investigated a novel strategy to overwhelm complement inhibitor activity and amplify complement activation on tumor cells. The C3-binding domain of human complement receptor 2 (CR2; CD21) was linked to the complement-activating Fc region of human IgG1 (CR2-Fc), and the ability of the construct to target and amplify complement deposition on tumor cells was investigated. CR2 binds C3 activation fragments, and CR2-Fc targeted tumor cells by binding to C3 initially deposited by a tumor-specific antibody. Complement deposition on Du145 cells (human prostate cancer cell line) and anti-MUC1 mAb-mediated complement-dependent lysis of Du145 cells were significantly enhanced by CR2-Fc. Anti-MUC1 antibody-dependent cell-mediated cytotoxicity of Du145 by human peripheral blood mononuclear cells was also significantly enhanced by CR2-Fc in both the presence and the absence of complement. Radiolabeled CR2-Fc targeted to s.c. Du145 tumors in nude mice treated with anti-MUC1 mAb, validating the targeting strategy in vivo. A metastatic model was used to investigate the effect of CR2-Fc in a therapeutic paradigm. Administration of CR2-Fc together with mAb therapy significantly improved long-term survival of nude mice challenged with an i.v. injection of EL4 cells. The data show that CR2-Fc enhances the therapeutic efficacy of antibody therapy, and the construct may provide particular benefits under conditions of limiting antibody concentration or low tumor antigen density.

  6. Cytotoxic activity against human neuroblastoma and melanoma cells mediated by IgM antibodies derived from peripheral blood of healthy donors. (United States)

    Devarapu, Satish Kumar; Mamidi, Srinivas; Plöger, Frank; Dill, Othmar; Blixt, Ola; Kirschfink, Michael; Schwartz-Albiez, Reinhard


    A small percentage of healthy donors identified in the Western population carry antibodies in their peripheral blood which convey cytotoxic activity against certain human melanoma and neuroblastoma cell lines. We measured the cytotoxic activity of sera and plasmas from healthy donors on the human neuroblastoma cell line Kelly and various melanoma cell lines. Antibodies of IgM isotype, presumably belonging to the class of naturally occurring antibodies, exerted cytotoxic activity in a complement-dependent fashion. Apart from complement-dependent tumor cell lysis, we observed C3 opsonization in all tumor cell lines upon treatment with cytotoxic plasmas. Cell lines tested primarily expressed membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59 to various extents. Blocking of mCRPs by monoclonal antibodies enhanced cell lysis and opsonization, though some melanoma cells remained resistant to complement attack. Epitopes recognized by cytotoxic antibodies were represented by gangliosides such as GD2 and GD3, as evidenced by cellular sialidase pretreatment and enhanced expression of distinct gangliosides. It remains to be clarified why only a small fraction of healthy persons carry these antitumor cytotoxic antibodies.

  7. A Lectin from Chinese Mistletoe Increases γδ T Cell-mediated Cytotoxicity through Induction of Caspase-dependent Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Fang GONG; Dongqing ZHANG; Yanhui MA; Anlun MA; Qiwen YU; Jiying ZHANG; Hong NIE; Xuehua CHEN; Baihua SHEN; Ningli LI


    In this study, a mistletoe lectin (ML) was purified from Chinese mistletoe and the effect of this 60 kDa Chinese ML on human γδ T cell cytotoxicity, apoptosis and modulation of the cytokine network was studied. The cytotoxic properties of δ T cells was evaluated by using a 51Cr release test and employed fluorescence-activated cell sorting and enzyme-linked immunosorbent assay analysis to quantify translocation of the cell membrane phospholipid, phosphatidylserine and nuclear DNA fragmentation during apoptosis.It was found that: (i) ML effectively stimulated γδ T cell proliferation in a dose- and time-dependent manner;(ii) ML increased γδ T cell cytotoxicity; (iii) ML could modulate lipopolysaccharide-induced cytokine release in a pro-inflammatory manner by increasing tumor necrosis factor (TNF)-α release and inhibiting the release of anti-inflammatory interleukin (IL)-10; (iv) ML induced apoptosis in caspase-dependent and CD95-independent manner. The results indicated that ML is a potent immunomodulator to human γδ T cell cytotoxicity, apoptosis and cytokine production.

  8. Cell-mediated immune response

    DEFF Research Database (Denmark)

    Meyer, Sonja Izquierdo; Fuglsang, Katrine; Blaakaer, Jan


    OBJECTIVE: This clinical review aims to assess the efficacy of human papillomavirus 16/18 (HPV16/18) vaccination on the cell-mediated immune response in women with existing cervical intraepithelial neoplasia or cervical cancer induced by HPV16 or HPV18. DATA SOURCES AND STUDY SELECTION: A focused...... and thorough literature search conducted in five different databases found 996 publications. Six relevant articles were chosen for further review. In total, 154 patients (>18 years of age) were enrolled in prospective study trials with 3-15 months of follow up. The vaccine applications were administered two...... triggered a detectable cell-mediated immune response, some of which were statistically significant. Correlations between immunological response and clinical outcome (histopathology) were not significant, so neoplasms may not be susceptible to vaccine-generated cytotoxic T cells (CD8(+)). CONCLUSIONS...

  9. Obinutuzumab (GA101) compared to rituximab significantly enhances cell death and antibody-dependent cytotoxicity and improves overall survival against CD20(+) rituximab-sensitive/-resistant Burkitt lymphoma (BL) and precursor B-acute lymphoblastic leukaemia (pre-B-ALL): potential targeted therapy in patients with poor risk CD20(+) BL and pre-B-ALL. (United States)

    Awasthi, Aradhana; Ayello, Janet; Van de Ven, Carmella; Elmacken, Mona; Sabulski, Anthony; Barth, Matthew J; Czuczman, Myron S; Islam, Humayun; Klein, Christian; Cairo, Mitchell S


    Obinutuzumab is a novel glycoengineered Type-II CD20 monoclonal antibody. CD20 is expressed in approximately 100% of children and adolescents with Burkitt lymphoma (BL) and 40% with precursor B-cell acute lymphoblastic leukaemia (pre-B-ALL). We evaluated the anti-tumour activity of obinutuzumab versus rituximab against rituximab-resistant (Raji 4RH) and -sensitive (Raji) BL and pre-B-ALL (U698-M) cells in vitro and in human BL or Pre-B-ALL xenografted mice. We demonstrated that obinutuzumab compared to rituximab significantly enhanced cell death against Raji 35·6 ± 3·1% vs. 25·1 ± 2·0%, (P = 0·001), Raji4RH 19·7 ± 2·2% vs. 7·9 ± 1·5% (P = 0·001) and U-698-M 47·3 ± 4·9% vs. 23·2 ± 0·5% (P = 0·001), respectively. Obinutuzumab versus rituximab also induced a significant increase in antibody-dependent cellular cytotoxicity (ADCC) with K562-IL15-41BBL expanded NK cells against Raji 73·8 ± 8·1% vs. 56·81 ± 4·6% (P = 0·001), Raji-4RH 40·0 ± 1·6% vs. 0·5 ± 1·1% (P = 0·001) and U-698-M 70·0 ± 1·6% vs. 45·5 ± 0·1% (P = 0·001), respectively. Overall survival in tumour xenografted mice receiving 30 mg/kg of obinutuzumab was significantly increased when compared to those receiving 30 mg/kg of rituximab in BL; Raji (P = 0·05), Raji4RH (P = 0·02) and U698-M (P = 0·03), respectively. These preclinical data suggest obinutuzumab is significantly superior to rituximab in inducing cell death, ADCC and against rituximab-sensitive/-resistant BL and pre-B-ALL xenografted mice. Taken together, these preclinical results provide evidence to suggest that future investigation of obinutuzumab is warranted in patients with relapsed/refractory CD20(+) BL and/or pre-B-ALL.

  10. Isolation and characterization of cytotoxic effector cells and antibody producing cells from human intestine. (United States)

    MacDermott, R P


    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

  11. Antibody dependent enhancement of frog virus 3 infection

    Directory of Open Access Journals (Sweden)

    Penny Emily


    Full Text Available Abstract Background Viruses included in the family Iridoviridae are large, icosahedral, dsDNA viruses that are subdivided into 5 genera. Frog virus 3 (FV3 is the type species of the genus Ranavirus and the best studied iridovirus at the molecular level. Typically, antibodies directed against a virus act to neutralize the virus and limit infection. Antibody dependent enhancement occurs when viral antibodies enhance infectivity of the virus rather than neutralize it. Results Here we show that anti-FV3 serum present at the time of FV3 infection enhances infectivity of the virus in two non-immune teleost cell lines. We found that antibody dependent enhancement of FV3 was dependent on the Fc portion of anti-FV3 antibodies but not related to complement. Furthermore, the presence of anti-FV3 serum during an FV3 infection in a non-immune mammalian cell line resulted in neutralization of the virus. Our results suggest that a cell surface receptor specific to teleost cell lines is responsible for the enhancement. Conclusions This report represents the first evidence of antibody dependent enhancement in iridoviruses. The data suggests that anti-FV3 serum can either neutralize or enhance viral infection and that enhancement is related to a novel antibody dependent enhancement pathway found in teleosts that is Fc dependent.

  12. Studies of cytotoxic antibodies against eye muscle antigens in patients with thyroid-associated ophthalmopathy

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Z.-G.; Hiromatsu, Y.; Salvi, M.; Triller, H.; Bernard, N.; Wall, J.R. (Thyroid Research Unit, The Montreal General Hospital Research Institute, Montreal, Quebec (Canada)); Medeiros-Neto, G.; Iacona, A.; Lima, N. (Thyroid Clinic, Hospital das Clinicas, Sao Paulo (Brazil))


    We have studied the prevalence and significance of cytotoxic antibodies against human eye muscle cells, as detected in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated antibody-dependent cytotoxicity (CMAC) in {sup 51}Cr release assays, in patients with Graves' ophthalmopathy or Hashimoto's thyroiditis. A high prevalence of positive ADCC tests was found in all groups of patients with ophthalmopathy tested. Tests were positive in 64% of patients with Graves' ophthalmopathy from an area of severe iodine deficiency (Sao Paulo) and in 64% of such patients from an iodine replete area (Montreal). In patients with so-called ''euthyroid ophthalmopathy'', i.e. eye disease associated with thyroiditis, ADCC tests were positive in 75 and 38% of patients from the two areas, respectively, while tests were positive in 40 and 22%, respectively, of patients with Graves' hyperthyroidism without evident eye disease. In normal subjects, levels of {sup 51}Cr release was always at background levels. In a group of patients from the high-iodine area, levels of antibodies in ADCC correlated positively with the intraocular pressure (mmHg) in primary position as a parameter of eye muscle dysfunction. In patients with ophthalmopathy, positive ADCC tests were assciated with antibodies to eye muscle membrane antigens of 55,65 and 95 kD as detected by immunoblotting, although the correlation was not close for any antigen. in contrast, CMAC tests were negative in all patients with ophthalmopathy. We also tested 9 mouse and 10 human monoclonal antibodies, reactive with orbital antigens in an enzyme-linked immunosorbent assay, for cytotoxic activity, in ADCC and CMAC, against eye muscle and thyroid cells. All monoclonal antibodies were of the IgM class and negative in ADCC assays. (Abstract Truncated)

  13. Proteasome Inhibition Suppresses Dengue Virus Egress in Antibody Dependent Infection.

    Directory of Open Access Journals (Sweden)

    Milly M Choy


    Full Text Available The mosquito-borne dengue virus (DENV is a cause of significant global health burden, with an estimated 390 million infections occurring annually. However, no licensed vaccine or specific antiviral treatment for dengue is available. DENV interacts with host cell factors to complete its life cycle although this virus-host interplay remains to be fully elucidated. Many studies have identified the ubiquitin proteasome pathway (UPP to be important for successful DENV production, but how the UPP contributes to DENV life cycle as host factors remains ill defined. We show here that proteasome inhibition decouples infectious virus production from viral RNA replication in antibody-dependent infection of THP-1 cells. Molecular and imaging analyses in β-lactone treated THP-1 cells suggest that proteasome function does not prevent virus assembly but rather DENV egress. Intriguingly, the licensed proteasome inhibitor, bortezomib, is able to inhibit DENV titers at low nanomolar drug concentrations for different strains of all four serotypes of DENV in primary monocytes. Furthermore, bortezomib treatment of DENV-infected mice inhibited the spread of DENV in the spleen as well as the overall pathological changes. Our findings suggest that preventing DENV egress through proteasome inhibition could be a suitable therapeutic strategy against dengue.

  14. 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


    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.

  15. Antibody-dependent NK cell activation is associated with late kidney allograft dysfunction and the complement-independent alloreactive potential of donor-specific antibodies

    Directory of Open Access Journals (Sweden)

    Tristan Legris


    Full Text Available Although kidney transplantation remains the best treatment for end-stage renal failure, it is limited by chronic humoral aggression of the graft vasculature by donor-specific antibodies (DSAs. The complement-independent mechanisms that lead to the antibody-mediated rejection (ABMR of kidney allografts remain poorly understood. Increasing lines of evidence have revealed the relevance of natural killer (NK cells as innate immune effectors of antibody-dependent cellular cytotoxicity, but few studies have investigated their alloreactive potential in the context of solid organ transplantation. Our study aimed to investigate the potential contribution of the antibody-dependent alloreactive function of NK cells to kidney graft dysfunction. We first conducted an observational study to investigate whether the cytotoxic function of NK cells is associated with chronic allograft dysfunction. The NK-Cellular Humoral Activation Test (NK-CHAT was designed to evaluate the recipient and antibody-dependent reactivity of NK cells against allogeneic target cells. The release of CD107a/Lamp1+ cytotoxic granules, resulting from the recognition of rituximab-coated B cells by NK cells, was analyzed in 148 kidney transplant recipients (KTRs, mean graft duration: 6.2 years. Enhanced ADCC responsiveness was associated with reduced graft function and identified as an independent risk factor predicting a decline in the estimated glomerular filtration rate (eGFR over a 1-year period (hazard ratio: 2.83. In a second approach, we used the NK-CHAT to reveal the cytotoxic potential of circulating alloantibodies in vitro. The level of CD16 engagement resulting from the in vitro recognition of serum-coated allogeneic B cells or splenic cells was further identified as a specific marker of DSA-induced ADCC. The NK-CHAT scoring of sera obtained from 40 patients at the time of transplant biopsy was associated with ABMR diagnosis. Our findings indicate that despite the administration

  16. Antibody-dependent enhancement of dengue virus infection is inhibited by SA-17, a doxorubicin derivative

    NARCIS (Netherlands)

    Ayala Nunez, Vanesa; Jarupathirun, Patsaporn; Kaptein, Suzanne; Neyts, Johan; Smit, Jolanda


    Antibody-dependent enhancement (ADE) is thought to play a critical role in the exacerbation of dengue virus (DENV)-induced disease during a heterologous re-infection. Despite ADE's clinical impact, only a few antiviral compounds have been assessed for their anti-ADE activity. We reported earlier tha

  17. Cell-mediated mutagenesis by chemical carcinogens

    Energy Technology Data Exchange (ETDEWEB)

    Huberman, E.; Langenbach, R.


    The cell-mediated mutation system, with the proper choice of metabolizing cells, can be used to detect the mutagenic activities of different classes of chemical carcinogens. When fibroblastic cells were used as the metabolizing cells, a correlation between the in vivo carcinogenic activity and the in vitro mutagenic activity of 11 aromatic polycyclic hydrocarbons was observed. When primary liver cells were used as the metabolizing cells, three known liver carcinogens were demonstrated to be mutagenic by the cell-mediated assay, while two non-carcinogenic analogues were not mutagenic. These results from the cell-mediated system suggest that the reactive intermediates of the carcinogens are stable enough to be transferred from the metabolizing cells to the V79 cells. The cell-mediated mutagenesis system is a simple in vitro assay which may simulate the in vivo situation. It was concluded that this approach could be extended to the co-cultivation of cells from other organs or tissues with mutable mammalian cells.

  18. Ageing and cell-mediated immunity. (United States)

    Fixa, B; Komárková, O; Chmelar, V


    The lymphocyte transformation test with phytohemagglutinin as mitogen estimated according to the incorporation of 2-(14)C-thymidine in DNA was used as an indicator of cell-mediated reactivity in 53 healthy subjects. Three age groups were examined: up to 20 years (21 subjects), 21-40 years (10 subjects) and over 70 years (22 subjects). The responsiveness of lymphocytes decreased significantly with age. In the highest age group 12 pathologically low values were found.

  19. Impact of the NK Cell Receptor LIR-1 (ILT-2/CD85j/LILRB1) on Cytotoxicity against Multiple Myeloma

    NARCIS (Netherlands)

    Heidenreich, Silke; Eulenburg, Christine Zu; Hildebrandt, York; Stuebig, Thomas; Sierich, Heidi; Badbaran, Anita; Eiermann, Thomas H.; Binder, Thomas M. C.; Kroeger, Nicolaus


    The role of different receptors in natural-killer- (NK-) cell-mediated cytotoxicity against multiple myeloma (MM) cells is unknown. We investigated if an enhancement of NK-cell-mediated cytotoxicity against MM could be reached by blocking of the inhibitory leukocyte immunoglobulin-like receptor 1 (L

  20. Mast Cell-Mediated Mechanisms of Nociception. (United States)

    Aich, Anupam; Afrin, Lawrence B; Gupta, Kalpna


    Mast cells are tissue-resident immune cells that release immuno-modulators, chemo-attractants, vasoactive compounds, neuropeptides and growth factors in response to allergens and pathogens constituting a first line of host defense. The neuroimmune interface of immune cells modulating synaptic responses has been of increasing interest, and mast cells have been proposed as key players in orchestrating inflammation-associated pain pathobiology due to their proximity to both vasculature and nerve fibers. Molecular underpinnings of mast cell-mediated pain can be disease-specific. Understanding such mechanisms is critical for developing disease-specific targeted therapeutics to improve analgesic outcomes. We review molecular mechanisms that may contribute to nociception in a disease-specific manner.

  1. In vivo Cytotoxicity of Type I CD20 Antibodies Critically Depends on Fc Receptor ITAM Signaling

    NARCIS (Netherlands)

    de Haij, Simone; Jansen, J. H. Marco; Boross, Peter; Beurskens, Frank J.; Bakema, Jantine E.; Bos, Desiree L.; Martens, Anton; Verbeek, J. Sjef; Parren, Paul W. H. I.; van de Winkel, Jan G. J.; Leusen, Jeanette H. W.


    Antibody-Fc receptor (FcR) interactions play an important role in the mechanism of action of most therapeutic antibodies against cancer. Effector cell activation through FcR triggering may induce tumor cell killing via antibody-dependent cellular cytotoxicity (ADCC). Reciprocally, FcR cross-linking

  2. CD8+ T cells prevent antigen-induced antibody-dependent enhancement of dengue disease in mice


    Zellweger, Raphaël M.; Eddy, William E.; Tang, William W.; Miller, Robyn; Shresta, Sujan


    Dengue virus (DENV) causes pathologies ranging from the febrile illness dengue fever to the potentially lethal severe dengue disease. A major risk factor for developing severe dengue disease is the presence of sub-protective DENV-reactive antibodies from a previous infection (or from an immune mother), which can induce antibody-dependent enhancement of infection (ADE). However, infection in the presence of sub-protective anti-DENV antibodies does not always result in severe disease, suggestin...

  3. Cell-mediated immune responses in rainbow trout after DNA immunization against the viral hemorrhagic septicemia virus

    DEFF Research Database (Denmark)

    Utke, Katrin; Kock, Holger; Schuetze, Heike


    To identify viral proteins that induce cell-mediated cytotoxicity (CMC) against viral hemorrhagic septicemia virus (VHSV)-infected cells, rainbow trout were immunized with DNA vectors encoding the glycoprotein G or the nucleocapsid protein N of VHSV. The G protein was a more potent trigger...... injection site rather than to injection sites of heterologous vaccines, suggesting the antigen specificity of homing. By demonstrating CMC responses to distinct viral proteins and homing in rainbow trout, these results substantially contribute to the understanding of the teleost immune system....... of cytotoxic cells than the N protein. Peripheral blood leukocytes (PBL) isolated from trout immunized against the G protein killed both VHSV-infected MHC class I matched (RTG-2) and VHSV-infected xenogeneic (EPC) target cells, suggesting the involvement of both cytotoxic T lymphocytes (CTL) and NK cells...

  4. A cytotoxic anti-IL-3Rα antibody targets key cells and cytokines implicated in systemic lupus erythematosus (United States)

    Oon, Shereen; Huynh, Huy; Tai, Tsin Yee; Ng, Milica; Monaghan, Katherine; Biondo, Mark; Maraskovsky, Eugene; Nash, Andrew D.; Wicks, Ian P.; Wilson, Nicholas J.


    To date, the major target of biologic therapeutics in systemic lupus erythematosus (SLE) has been the B cell, which produces pathogenic autoantibodies. Recently, targeting type I IFN, which is elaborated by plasmacytoid dendritic cells (pDCs) in response to endosomal TLR7 and TLR9 stimulation by SLE immune complexes, has shown promising results. pDCs express high levels of the IL-3Rα chain (CD123), suggesting an alternative potential targeting strategy. We have developed an anti-CD123 monoclonal antibody, CSL362, and show here that it affects key cell types and cytokines that contribute to SLE. CSL362 potently depletes pDCs via antibody-dependent cell-mediated cytotoxicity, markedly reducing TLR7, TLR9, and SLE serum-induced IFN-α production and IFN-α-upregulated gene expression. The antibody also inhibits TLR7- and TLR9-induced plasmablast expansion by reducing IFN-α and IL-6 production. These effects are more pronounced than with IFN-α blockade alone, possibly because pDC depletion reduces production of other IFN subtypes, such as type III, as well as non-IFN proinflammatory cytokines, such as IL-6. In addition, CSL362 depletes basophils and inhibits IL-3 signaling. These effects were confirmed in cells derived from a heterogeneous population of SLE donors, various IFN-dependent autoimmune diseases, and healthy controls. We also demonstrate in vivo activity of CSL362 following its s.c. administration to cynomolgus monkeys. This spectrum of effects provides a preclinical rationale for the therapeutic evaluation of CSL362 in SLE. PMID:27699260

  5. Different NK cell-activating receptors preferentially recruit Rab27a or Munc13-4 to perforin-containing granules for cytotoxicity

    DEFF Research Database (Denmark)

    Wood, Stephanie M; Meeths, Marie; Chiang, Samuel C C


    of perforin-containing lytic granules induced by signals for natural and antibody-dependent cellular cytotoxicity. We demonstrate here that these signals fail to induce degranulation in resting NK cells from Rab27a-deficient patients. In resting NK cells from healthy subjects, endogenous Rab27a and Munc13...... functional antigen-1, NKG2D, or 2B4 induced colocalization of Rab27a, but not Munc13-4, with perforin. Conversely, engagement of antibody-dependent cellular cytotoxicity receptor CD16 induced colocalization of Munc13-4, but not Rab27a, with perforin. Furthermore, colocalization of Munc13-4 with perforin...

  6. Mesenchymal stem cell-mediated functional tooth regeneration in swine.

    Directory of Open Access Journals (Sweden)

    Wataru Sonoyama

    Full Text Available Mesenchymal stem cell-mediated tissue regeneration is a promising approach for regenerative medicine for a wide range of applications. Here we report a new population of stem cells isolated from the root apical papilla of human teeth (SCAP, stem cells from apical papilla. Using a minipig model, we transplanted both human SCAP and periodontal ligament stem cells (PDLSCs to generate a root/periodontal complex capable of supporting a porcelain crown, resulting in normal tooth function. This work integrates a stem cell-mediated tissue regeneration strategy, engineered materials for structure, and current dental crown technologies. This hybridized tissue engineering approach led to recovery of tooth strength and appearance.

  7. Mesenchymal stem cell-mediated functional tooth regeneration in swine. (United States)

    Sonoyama, Wataru; Liu, Yi; Fang, Dianji; Yamaza, Takayoshi; Seo, Byoung-Moo; Zhang, Chunmei; Liu, He; Gronthos, Stan; Wang, Cun-Yu; Wang, Songlin; Shi, Songtao


    Mesenchymal stem cell-mediated tissue regeneration is a promising approach for regenerative medicine for a wide range of applications. Here we report a new population of stem cells isolated from the root apical papilla of human teeth (SCAP, stem cells from apical papilla). Using a minipig model, we transplanted both human SCAP and periodontal ligament stem cells (PDLSCs) to generate a root/periodontal complex capable of supporting a porcelain crown, resulting in normal tooth function. This work integrates a stem cell-mediated tissue regeneration strategy, engineered materials for structure, and current dental crown technologies. This hybridized tissue engineering approach led to recovery of tooth strength and appearance.

  8. Narcolepsy-Associated HLA Class I Alleles Implicate Cell-Mediated Cytotoxicity

    NARCIS (Netherlands)

    Tafti, M.; Lammers, G.J.; Dauvilliers, Y.; Overeem, S.; Mayer, G.; Nowak, J.; Pfister, C.; Dubois, V.; Eliaou, J.F.; Eberhard, H.P.; Liblau, R.; Wierzbicka, A.; Geisler, P.; Bassetti, C.L.; Mathis, J.; Lecendreux, M.; Khatami, R.; Heinzer, R.; Haba-Rubio, J.; Feketeova, E.; Baumann, C.R.; Kutalik, Z.; Tiercy, J.M.


    STUDY OBJECTIVES: Narcolepsy with cataplexy is tightly associated with the HLA class II allele DQB1*06:02. Evidence indicates a complex contribution of HLA class II genes to narcolepsy susceptibility with a recent independent association with HLA-DPB1. The cause of narcolepsy is supposed be an autoi

  9. Cord blood T cells mediate enhanced antitumor effects compared with adult peripheral blood T cells. (United States)

    Hiwarkar, Prashant; Qasim, Waseem; Ricciardelli, Ida; Gilmour, Kimberly; Quezada, Sergio; Saudemont, Aurore; Amrolia, Persis; Veys, Paul


    Unrelated cord blood transplantation (CBT) without in vivo T-cell depletion is increasingly used to treat high-risk hematologic malignancies. Following T-replete CBT, naïve CB T cells undergo rapid peripheral expansion with memory-effector differentiation. Emerging data suggest that unrelated CBT, particularly in the context of HLA mismatch and a T-replete graft, may reduce leukemic relapse. To study the role of CB T cells in mediating graft-versus-tumor responses and dissect the underlying immune mechanisms for this, we compared the ability of HLA-mismatched CB and adult peripheral blood (PB) T cells to eliminate Epstein-Barr virus (EBV)-driven human B-cell lymphoma in a xenogeneic NOD/SCID/IL2rg(null) mouse model. CB T cells mediated enhanced tumor rejection compared with equal numbers of PB T cells, leading to improved survival in the CB group (P cells that were autologous vs allogeneic to the lymphoma demonstrated that this antitumor effect was mediated by alloreactive rather than EBV-specific T cells. Analysis of tumor-infiltrating lymphocytes demonstrated that CB T cells mediated this enhanced antitumor effect by rapid infiltration of the tumor with CCR7(+)CD8(+) T cells and prompt induction of cytotoxic CD8(+) and CD4(+) T-helper (Th1) T cells in the tumor microenvironment. In contrast, in the PB group, this antilymphoma effect is impaired because of delayed tumoral infiltration of PB T cells and a relative bias toward suppressive Th2 and T-regulatory cells. Our data suggest that, despite being naturally programmed toward tolerance, reconstituting T cells after unrelated T-replete CBT may provide superior Tc1-Th1 antitumor effects against high-risk hematologic malignancies.

  10. Susceptibility to T cell-mediated liver injury is enhanced in asialoglycoprotein receptor-deficient mice. (United States)

    McVicker, Benita L; Thiele, Geoffrey M; Casey, Carol A; Osna, Natalia A; Tuma, Dean J


    T cell activation and associated pro-inflammatory cytokine production is a pathological feature of inflammatory liver disease. It is also known that liver injury is associated with marked impairments in the function of many hepatic proteins including a hepatocyte-specific binding protein, the asialoglycoprotein receptor (ASGPR). Recently, it has been suggested that hepatic ASGPRs may play an important role in the physiological regulation of T lymphocytes, leading to our hypothesis that ASGPR defects correlate with inflammatory-mediated events in liver diseases. Therefore, in this study we investigated whether changes in hepatocellular ASGPR expression were related to the dysregulation of intrahepatic T lymphocytes and correlate with the development of T-cell mediated hepatitis. Mice lacking functional ASGPRs (receptor-deficient, RD), and wild-type (WT) controls were intravenously injected with T-cell mitogens, Concanavalin A (Con A) or anti-CD3 antibody. As a result of T cell mitogen treatment, RD mice lacking hepatic ASGPRs displayed enhancements in liver pathology, transaminase activities, proinflammatory cytokine expression, and caspase activation compared to that observed in normal WT mice. Furthermore, FACS analysis demonstrated that T-cell mitogen administration resulted in a significant rise in the percentage of CD8+ lymphocytes present in the livers of RD animals versus WT mice. Since these two mouse strains differ only in whether they express the hepatic ASGPR, it can be concluded that proper ASGPR function exerts a protective effect against T cell mediated hepatitis and that impairments to this hepatic receptor could be related to the accumulation of cytotoxic T cells that are observed in inflammatory liver diseases.

  11. The role of cell-mediated immunity in typhoid. (United States)

    Mabel, T J; Paniker, C K


    The cell-mediated immunity in typhoid was assessed by the leukocyte migration inhibition test and delayed hypersensitivity skin test in 60 clinical typhoid patients. The property of leukocyte migration inhibition appeared first and was positive in 28 of 60 (46.7%) patients on admission and 45 of 60 (75%) at the time of discharge. This difference was definitely more in blood culture positive patients. The delayed hypersensitivity appeared later and was positive in 18 of 60 (30%) on admission and 31 of 60 (51.7%) at the time of discharge. Patients with positive cellular-immune response against typhoid antigen did not develop relapse. On the whole cell-mediated immunity seems to play an important role in typoid. The control groups--the medical and surgical patients, doctors, clinical students and preclinical students--showed positive cellular immune response of 43.3 81.3, 40.7 and 25% respectively. The significance of these results is discussed.

  12. Suppression of cell-mediated immunity by misonidazole

    Energy Technology Data Exchange (ETDEWEB)

    Rockwell, S.; Neaderland, M.H. (Yale Univ., New Haven, CT (USA). School of Medicine)


    The data presented in this report demonstrate that single treatments with large doses of misonidazole (l mg/g) produce significant inhibition of delayed hypersensitivity to DNFB. Contact sensitivity to DNFB is generally considered to be a cell-mediated immune response (Asherson and Ptak 1968, Moorhead 1978, Phanuphak et al. 1974, Zembala and Asherson 1973). The authors' histological observations and the lack of ear swelling in the nude mice support this interpretation.

  13. Enhancement of NK cell-mediated lysis of non-small lung cancer cells by nPKC activator, ingenol 3,20 dibenzoate. (United States)

    Gong, Chenyuan; Yao, Chao; Xu, Zihang; Ni, Zhongya; Zhu, Xiaowen; Wang, Lixin; Yan, Xuewei; Zhou, Wuxiong; Zhu, Shiguo


    The IFN-γ production is crucial for NK cell-mediated lysis of cancer cells. Thus increasing the IFN-γ production by NK cells may be an ideal strategy to improve their tumoricidal effect. Since the focus on new drug development has shifted towards natural products, limited information is out there about natural products that enhance the IFN-γ production by NK cells. In this study, through a high-throughput screening, we have identified a natural product ingenol 3,20 dibenzoate (IDB), an activator of tumor suppressor protein kinase C (PKC) isozymes, could increase the IFN-γ production and degranulation by NK cells, especially when NK cells were stimulated by non-small lung cancer (NSCLC) cells. IDB also significantly enhanced the NK cell-mediated lysis of NSCLC cells. Furthermore, PKC inhibitor, sotrastaurin abrogated IDB-induced IFN-γ production, degranulation and cytotoxicity, but did not affect IFN-γ production by NK cells without IDB treatment and NSCLC cell stimulation. The IFN-γ neutralization reversed the IDB-induced enhancement of NK cell mediated killing. In conclusion, our study indicated that IDB enhanced NK cell-mediated lysis of NSCLC cells is dependent on specific PKC mediated IFN-γ production and degranulation. Thus, IDB may have a promising application in clinic for NK cell-based cancer immunotherapy.

  14. Neonatal Fc receptor expression in dendritic cells mediates protective immunity against colorectal cancer. (United States)

    Baker, Kristi; Rath, Timo; Flak, Magdalena B; Arthur, Janelle C; Chen, Zhangguo; Glickman, Jonathan N; Zlobec, Inti; Karamitopoulou, Eva; Stachler, Matthew D; Odze, Robert D; Lencer, Wayne I; Jobin, Christian; Blumberg, Richard S


    Cancers arising in mucosal tissues account for a disproportionately large fraction of malignancies. Immunoglobulin G (IgG) and the neonatal Fc receptor for IgG (FcRn) have an important function in the mucosal immune system that we have now shown extends to the induction of CD8(+) T cell-mediated antitumor immunity. We demonstrate that FcRn within dendritic cells (DCs) was critical for homeostatic activation of mucosal CD8(+) T cells that drove protection against the development of colorectal cancers and lung metastases. FcRn-mediated tumor protection was driven by DCs activation of endogenous tumor-reactive CD8(+) T cells via the cross-presentation of IgG complexed antigens (IgG IC), as well as the induction of cytotoxicity-promoting cytokine secretion, particularly interleukin-12, both of which were independently triggered by the FcRn-IgG IC interaction in murine and human DCs. FcRn thus has a primary role within mucosal tissues in activating local immune responses that are critical for priming efficient anti-tumor immunosurveillance.

  15. Antibody-dependent enhancement infection facilitates dengue virus-regulated signaling of IL-10 production in monocytes.

    Directory of Open Access Journals (Sweden)

    Tsung-Ting Tsai


    Full Text Available Interleukin (IL-10 levels are increased in dengue virus (DENV-infected patients with severe disorders. A hypothetical intrinsic pathway has been proposed for the IL-10 response during antibody-dependent enhancement (ADE of DENV infection; however, the mechanisms of IL-10 regulation remain unclear.We found that DENV infection and/or attachment was sufficient to induce increased expression of IL-10 and its downstream regulator suppressor of cytokine signaling 3 in human monocytic THP-1 cells and human peripheral blood monocytes. IL-10 production was controlled by activation of cyclic adenosine monophosphate response element-binding (CREB, primarily through protein kinase A (PKA- and phosphoinositide 3-kinase (PI3K/PKB-regulated pathways, with PKA activation acting upstream of PI3K/PKB. DENV infection also caused glycogen synthase kinase (GSK-3β inactivation in a PKA/PI3K/PKB-regulated manner, and inhibition of GSK-3β significantly increased DENV-induced IL-10 production following CREB activation. Pharmacological inhibition of spleen tyrosine kinase (Syk activity significantly decreased DENV-induced IL-10 production, whereas silencing Syk-associated C-type lectin domain family 5 member A caused a partial inhibition. ADE of DENV infection greatly increased IL-10 expression by enhancing Syk-regulated PI3K/PKB/GSK-3β/CREB signaling. We also found that viral load, but not serotype, affected the IL-10 response. Finally, modulation of IL-10 expression could affect DENV replication.These results demonstrate that, in monocytes, IL-10 production is regulated by ADE through both an extrinsic and an intrinsic pathway, all involving a Syk-regulated PI3K/PKB/GSK-3β/CREB pathway, and both of which impact viral replication.

  16. Molecular imaging of cell-mediated cancer immunotherapy. (United States)

    Lucignani, Giovanni; Ottobrini, Luisa; Martelli, Cristina; Rescigno, Maria; Clerici, Mario


    New strategies based on the activation of a patient's immune response are being sought to complement present conventional exogenous cancer therapies. Elucidating the trafficking pathways of immune cells in vivo, together with their migratory properties in relation to their differentiation and activation status, is useful for understanding how the immune system interacts with cancer. Methods based on tissue sampling to monitor immune responses are inadequate for repeatedly characterizing the responses of the immune system in different organs. A solution to this problem might come from molecular and cellular imaging - a branch of biomedical sciences that combines biotechnology and imaging methods to characterize, in vivo, the molecular and cellular processes involved in normal and pathologic states. The general concepts of noninvasive imaging of targeted cells as well as the technology and probes applied to cell-mediated cancer immunotherapy imaging are outlined in this review.

  17. Comparison of stem-cell-mediated osteogenesis and dentinogenesis. (United States)

    Batouli, S; Miura, M; Brahim, J; Tsutsui, T W; Fisher, L W; Gronthos, S; Robey, P Gehron; Shi, S


    The difference between stem-cell-mediated bone and dentin regeneration is not yet well-understood. Here we use an in vivo stem cell transplantation system to investigate differential regulation mechanisms of bone marrow stromal stem cells (BMSSCs) and dental pulp stem cells (DPSCs). Elevated expression of basic fibroblast growth factor (bFGF) and matrix metalloproteinase 9 (MMP-9, gelatinase B) was found to be associated with the formation of hematopoietic marrow in BMSSC transplants, but not in the connective tissue of DPSC transplants. The expression of dentin sialoprotein (DSP) specifically marked dentin synthesis in DPSC transplants. Moreover, DPSCs were found to be able to generate reparative dentin-like tissue on the surface of human dentin in vivo. This study provided direct evidence to suggest that osteogenesis and dentinogenesis mediated by BMSSCs and DPSCs, respectively, may be regulated by distinct mechanisms, leading to the different organization of the mineralized and non-mineralized tissues.

  18. Cell mediated immune response in human antirabies revaccination

    Directory of Open Access Journals (Sweden)

    Débora Regina Veiga


    Full Text Available The occurrence of secondary cell mediated immune response (CMI in human antirabies immunization was studied. The Puenzalida & Palácios vaccine was used because it is routinely used in Brazil. CMI was evaluated by lymphoblastic transformation indices obtained in whole blood culture in the presence of rabies and control (nervous tissue antigens. Eleven volunteers submitted to revaccination constituted the group under study, while three other volunteers submitted primo vaccination were utilized as control group. A clear secondary CMI to rabies antigen was detected in all the revaccinated volunteers who showed earlier and more intense response than the control group. Response to the control antigen, however, present in all the components of the first group was not detectable in two out of the three primovaccinated and very low in the third one.

  19. Fatal meningitis following lymphocytic choriomeningitis virus infection reflects delayed-type hypersensitivity rather than cytotoxicity

    DEFF Research Database (Denmark)

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


    Fatal meningitis following intracerebral inoculation of lymphocytic choriomeningitis virus (LCMV) reflects an immunopathological lesion believed to be mediated by cytotoxic T cells. The results presented here demonstrate that pretreatment with cyclophosphamide (Cy; 150 mg/kg body weight) 2 days...... before intracerebral infection significantly reduced the lethality of the infection. However, this treatment did not impair the antiviral cytotoxic response as measured in the spleen. On the other hand, virus-specific delayed-type hypersensitivity (DTH) was significantly reduced. This reduction seems...... not solely represent T-cell-mediated cytotoxicity in vivo but that a fatal outcome of the infection critically involves not only effector T cells but also ancillary cells....

  20. Studies of cell-mediated immune responses to influenza vaccination in systemic lupus erythematosus

    NARCIS (Netherlands)

    Holvast, Albert; Van Assen, Sander; De Haan, Aalzen; Huckriede, Anke; Benne, Cornelis A.; Westra, Johanna; Palache, Abraham; Wilschut, Jan; Kallenberg, Cornelis; Bijl, Marc


    Objective. Both antibody and cell-mediated responses are involved in the defense against influenza. In patients with systemic lupus erythematosus (SLE), a decreased antibody response to subunit influenza vaccine has been demonstrated, but cell-mediated responses have not yet been assessed. This stud

  1. The Smac Mimetic BV6 Improves NK Cell-Mediated Killing of Rhabdomyosarcoma Cells by Simultaneously Targeting Tumor and Effector Cells (United States)

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


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

  2. Cell-mediated immunity to insulin: a new criterion for differentiation of diabetes mellitus? (United States)

    Asfandiyarova, Nailya S


    Any classification is a step forward and it should help to determine the reason, the course, the prognosis, the treatment of a disease. The current classification of diabetes mellitus (DM) is really very convenient for work, but it has some drawbacks, and the absence of differentiation of type 2 diabetes is the main. The problem is the absence of an adequate criterion, based on pathogenesis for differentiation. We suppose that cell mediated immunity (CMI) to insulin plays the central role in the diabetes genesis. Autoimmune process may be triggered by viruses family Paramyxoviridae, in 10-20% of type 1 diabetes patients the disease is a consequence of direct cytotoxic effect of other viruses to the islet cells of pancreas. In acute phase of viral infection (measles, mumps, parainfluenza) CMI against viruses is developed, in some patients CMI to insulin appeared. We suppose that autoimmune reactions in these cases are the result of cross reaction between viral antigens and insulin. The majorities of patients suppress these reactions and recover from acute infection diseases with the antiviral immunity development and without any complications. Other patients are not able to suppress autoimmune reactions to insulin and pathological process is triggered. Type 1A diabetes is a result of direct CMI to insulin, and this process is responsible for beta-cells destruction; may be type 1B DM is due to the direct cytotoxic effect of other viruses or toxins to them. Some patients with acute viral infection cannot destroy the aggressive clone and they suppress autoimmune reaction to insulin by prostaglandin synthesizing cells (PGSC) or сells with histamine receptors (CHR). As a result of this process the insulin resistance is developed, because these cells or their cytokines form a block to the insulin receptors not only on immunocompetent cells, but in insulin sensitive tissues too. Patients with different reactions to insulin have different courses and outcomes of DM. We

  3. Mast cell mediators and peritoneal adhesion formation in the rat. (United States)

    Langer, J C; Liebman, S M; Monk, P K; Pelletier, G J


    We have previously shown that mast cell stabilization attenuates peritoneal adhesion formation in the rat. The present study investigated the mechanism of this protection. Adhesions were created in weanling rats using cecal scraping and application of 95% ethanol. Rats received specific blockers for the mast cell products histamine, serotonin (5HT), leukotriene D4, and platelet activating factor intraperitoneally 30 min before laparotomy and at the time of abdominal closure. Control animals received saline. Adhesions were assessed blindly 1 week later using a standardized scale. Adhesion formation was not affected by histamine blockade using combined mepyramine and ranitidine, 5-HT1 blockade using methysergide, 5-HT3 blockade using ondansetron, leukotriene D4 blockade using MK-571, or platelet activating factor blockade using WEB-2086. However, blockade of the 5-HT2 receptor using ketanserin resulted in significant dose-dependent attenuation of adhesions compared to saline. These data suggest that mast cells mediate peritoneal adhesion formation in the rat through release of serotonin acting on 5HT2 receptors. Further understanding of this process may lead to new strategies for the prevention of postoperative adhesions.

  4. Lung Regeneration: Endogenous and Exogenous Stem Cell Mediated Therapeutic Approaches. (United States)

    Akram, Khondoker M; Patel, Neil; Spiteri, Monica A; Forsyth, Nicholas R


    The tissue turnover of unperturbed adult lung is remarkably slow. However, after injury or insult, a specialised group of facultative lung progenitors become activated to replenish damaged tissue through a reparative process called regeneration. Disruption in this process results in healing by fibrosis causing aberrant lung remodelling and organ dysfunction. Post-insult failure of regeneration leads to various incurable lung diseases including chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis. Therefore, identification of true endogenous lung progenitors/stem cells, and their regenerative pathway are crucial for next-generation therapeutic development. Recent studies provide exciting and novel insights into postnatal lung development and post-injury lung regeneration by native lung progenitors. Furthermore, exogenous application of bone marrow stem cells, embryonic stem cells and inducible pluripotent stem cells (iPSC) show evidences of their regenerative capacity in the repair of injured and diseased lungs. With the advent of modern tissue engineering techniques, whole lung regeneration in the lab using de-cellularised tissue scaffold and stem cells is now becoming reality. In this review, we will highlight the advancement of our understanding in lung regeneration and development of stem cell mediated therapeutic strategies in combating incurable lung diseases.

  5. Lung Regeneration: Endogenous and Exogenous Stem Cell Mediated Therapeutic Approaches

    Directory of Open Access Journals (Sweden)

    Khondoker M. Akram


    Full Text Available The tissue turnover of unperturbed adult lung is remarkably slow. However, after injury or insult, a specialised group of facultative lung progenitors become activated to replenish damaged tissue through a reparative process called regeneration. Disruption in this process results in healing by fibrosis causing aberrant lung remodelling and organ dysfunction. Post-insult failure of regeneration leads to various incurable lung diseases including chronic obstructive pulmonary disease (COPD and idiopathic pulmonary fibrosis. Therefore, identification of true endogenous lung progenitors/stem cells, and their regenerative pathway are crucial for next-generation therapeutic development. Recent studies provide exciting and novel insights into postnatal lung development and post-injury lung regeneration by native lung progenitors. Furthermore, exogenous application of bone marrow stem cells, embryonic stem cells and inducible pluripotent stem cells (iPSC show evidences of their regenerative capacity in the repair of injured and diseased lungs. With the advent of modern tissue engineering techniques, whole lung regeneration in the lab using de-cellularised tissue scaffold and stem cells is now becoming reality. In this review, we will highlight the advancement of our understanding in lung regeneration and development of stem cell mediated therapeutic strategies in combating incurable lung diseases.

  6. Effects of chrysotherapy on cell mediated immune response. (United States)

    Lorber, A; Jackson, W H; Simon, T M


    Auranofin (AF) differs significantly from gold sodium thiomalate (GSTM) in formulation, i.e., aurous gold is stabilized by dual sulfur and phosphorus ligands, hydrophobic rather than hydrophilic characteristics, and lack of ionic charge. These attributes facilitate: oral absorption of AF, plasma membrane penetration, increase in intracellular lymphocyte gold concentration; and perhaps thereby influence lymphocyte function. AF treated subjects recorded prompt and sharp declines in mitogen-induced lymphoproliferative response (LMR) greater than 80%; suppressed response to skin testing with dinitrochlorobenezene (DNCB) in 11 of 14 subjects; and blebbing of lymphocyte membranes by scanning electron microscopy. In contrast, lymphocytes from a matched group of GSTM treated subjects recorded later onset and less suppression of LMR; normal response to DNCB skin testing; and did not manifest membrane blebbing. Accordingly, the therapeutic action of AF on immune response was observed in the 16 subjects receiving 6 mg/d of an average of 45 weeks to effect primarily cell mediated rather than humoral immune response when compared with a matched group of GSTM treated patients.

  7. CXCR5+ T helper cells mediate protective immunity against tuberculosis (United States)

    Slight, Samantha R.; Rangel-Moreno, Javier; Gopal, Radha; Lin, Yinyao; Fallert Junecko, Beth A.; Mehra, Smriti; Selman, Moises; Becerril-Villanueva, Enrique; Baquera-Heredia, Javier; Pavon, Lenin; Kaushal, Deepak; Reinhart, Todd A.; Randall, Troy D.; Khader, Shabaana A.


    One third of the world’s population is infected with Mycobacterium tuberculosis (Mtb). Although most infected people remain asymptomatic, they have a 10% lifetime risk of developing active tuberculosis (TB). Thus, the current challenge is to identify immune parameters that distinguish individuals with latent TB from those with active TB. Using human and experimental models of Mtb infection, we demonstrated that organized ectopic lymphoid structures containing CXCR5+ T cells were present in Mtb-infected lungs. In addition, we found that in experimental Mtb infection models, the presence of CXCR5+ T cells within ectopic lymphoid structures was associated with immune control. Furthermore, in a mouse model of Mtb infection, we showed that activated CD4+CXCR5+ T cells accumulated in Mtb-infected lungs and produced proinflammatory cytokines. Mice deficient in Cxcr5 had increased susceptibility to TB due to defective T cell localization within the lung parenchyma. We demonstrated that CXCR5 expression in T cells mediated correct T cell localization within TB granulomas, promoted efficient macrophage activation, protected against Mtb infection, and facilitated lymphoid follicle formation. These data demonstrate that CD4+CXCR5+ T cells play a protective role in the immune response against TB and highlight their potential use for future TB vaccine design and therapy. PMID:23281399

  8. Cell-mediated mutagenesis and cell transformation by chemical carcinogens

    Energy Technology Data Exchange (ETDEWEB)

    Huberman, E.; Langenbach, R.


    Results are reported from studies that showed that mutagenesis of mammalian cells can be achieved by carcinogenic polycyclic hydrocarbons, nitrosamines, and aflatoxins when tested in the presence of fibroblasts and hepatocytes which are able to metabolize these carcinogens. Further, we have found that there is a relationship between the degree of mutant induction and the degree of carcinogenicity of the different chemicals tested. By simultaneously measuring the frequency of cell transformation and the frequency of mutation at one locus (ouabain resistance) in the same cell system, it was possible to estimate the genetic target site for cell transformation. The results indicated that the target site for transformation is approximately 20 times larger than that determined for ouabain resistance. The results suggest that cell transformation may be due to a mutational event and the mutation can occur in one out of a small number of the same or different genes, and that the cell-mediated mutagenesis approach may be a valuable means of detecting tissue-specific carcinogens.

  9. ELISPOT Assay for Monitoring Cytotoxic T Lymphocytes (CTL Activity in Cancer Vaccine Clinical Trials

    Directory of Open Access Journals (Sweden)

    Thomas J. Sayers


    Full Text Available The profiling and monitoring of immune responses are key elements in the evaluation of the efficacy and development of new biotherapies, and a number of assays have been introduced for analyzing various immune parameters before, during, and after immunotherapy. The choice of immune assays for a given clinical trial depends on the known or suggested immunomodulating mechanisms associated with the tested therapeutic modality. Cell-mediated cytotoxicity represents a key mechanism in the immune response to various pathogens and tumors. Therefore, the selection of monitoring methods for the appropriate assessment of cell-mediated cytotoxicity is thought to be crucial. Assays that can detect both cytotoxic T lymphocytes (CTL frequency and function, such as the IFN-γ enzyme-linked immunospot assay (ELISPOT have gained increasing popularity for monitoring clinical trials and in basic research. Results from various clinical trials, including peptide and whole tumor cell vaccination and cytokine treatment, have shown the suitability of the IFN-γ ELISPOT assay for monitoring T cell responses. However, the Granzyme B ELISPOT assay and Perforin ELISPOT assay may represent a more direct analysis of cell-mediated cytotoxicity as compared to the IFN-γ ELISPOT, since Granzyme B and perforin are the key mediators of target cell death via the granule-mediated pathway. In this review we analyze our own data and the data reported by others with regard to the application of various modifications of ELISPOT assays for monitoring CTL activity in clinical vaccine trials.

  10. 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.

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


    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.

  12. Evaluation of antibody-dependent cell-mediatedcytotoxicity activity and cetuximab response in KRAS wildtypemetastatic colorectal cancer patients

    Institute of Scientific and Technical Information of China (English)


    AIM To investigate the prognostic role of invariantnatural killer T (iNKT) cells and antibody-dependentcell-mediated cytotoxicity (ADCC) in wild type KRASmetastatic colorectal cancer (mCRC) patients treated withcetuximab.METHODS: Forty-one KRAS wt mCRC patients, treatedwith cetuximab and irinotecan-based chemotherapyin Ⅱ and Ⅲ lines were analyzed. Genotyping ofsingle nucleotide polymorphism (SNP)s in the FCGR2A ,FCGR3A and in the 3' untranslated regions of KRAS andmutational analysis for KRAS , BRAF and NRAS genes wasdetermined either by sequencing or allelic discriminationassays. Enriched NK cells were obtained from lymphoprepperipheralblood mononuclear cell and iNKT cells weredefined by co-expression of CD3, TCRVα24, TCRVβ11.ADCC was evaluated as ex vivo NK-dependent activity,measuring lactate dehydrogenase release.RESULTS: At basal, mCRC patients performing ADCCactivity above the median level (71%) showed an improvedoverall survival (OS) compared to patients with ADCC below (median 16 vs 8 mo; P = 0.026). We did not findany significant correlation of iNKT cells with OS (P = 0.19),albeit we observed a trend to a longer survival after 10mo in patients with iNKT above median basal level (0.382cells/microliter). Correlation of OS and progression-freesurvival (PFS) with interesting SNPs involved in ADCC abilityrevealed not to be significant. Patients carrying alleles bothwith A in FCGR2A and TT in FCGR3A presented a trend oflonger PFS (median 9 vs 5 mo; P = 0.064). Chemotherapyimpacted both iNKT cells and ADCC activity. Their prognosticvalues get lost when we analysed them after 2 and 4 mo oftreatment.CONCLUSION: Our results suggest a link between iNKTcells, basal ADCC activity, genotypes in FCGR2A andFCGR3A, and efficacy of cetuximab in KRAS wt mCRCpatients.

  13. Effects of feed supplementation with glycine chelate and iron sulfate on selected parameters of cell-mediated immune response in broiler chickens. (United States)

    Jarosz, Łukasz; Kwiecień, Małgorzata; Marek, Agnieszka; Grądzki, Zbigniew; Winiarska-Mieczan, Anna; Kalinowski, Marcin; Laskowska, Ewa


    Because little is known about the impact of chelated (Fe-Gly, Fe-Gly+F) and inorganic (FeSO4, FeSO4+F) iron products on immune response parameters in broiler chickens, the objective of the study was to determine the effects of inorganic and organic forms of iron on selected parameters of the cell-mediated immune response in broiler chickens by assessing the percentage of CD3(+)CD4(+), CD3(+)CD8(+), CD25(+), and MHC Class II lymphocytes, as well as the CD4(+)/CD8(+) ratio and IL-2 concentration in the peripheral blood. The experiments were conducted using 50day-old Ross 308 roosters. The test material was peripheral blood. Flow cytometry was used to determine selected cell-mediated immune response parameters. The results obtained indicate that the use of iron chelates in the diet of broiler chickens may stimulate cellular defense mechanisms. As a result of the experiment an increase was observed in the percentage of Th1, mainly T CD4(+) and T CD8(+). It was also noted that application of chelated iron can increase production of T CD8(+) cytotoxic cells and IL-2, which promotes the body's natural response to developing inflammation. There were no changes in T CD4(+), T CD8(+), T CD25(+) or MHC II lymphocyte subpopulations in the chickens following application of the inorganic form of iron.

  14. Granule-Dependent Natural Killer Cell Cytotoxicity to Fungal Pathogens (United States)

    Ogbomo, Henry; Mody, Christopher H.


    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

  15. Assessing humoral and cell-mediated immune response in Hawaiian green turtles, Chelonia mydas (United States)

    Work, T.M.; Balazs, G.H.; Rameyer, R.A.; Chang, S.P.; Berestecky, J.


    Seven immature green turtles, Chelonia mydas, captured from Kaneohe Bay on the island of Oahu were used to evaluate methods for assessing their immune response. Two turtles each were immunized intramuscularly with egg white lysozyme (EWL) in Freunda??s complete adjuvant, Gerbu, or ISA-70; a seventh turtle was immunized with saline only and served as a control. Humoral immune response was measured with an indirect enzyme linked immunosorbent assay (ELISA). Cell-mediated immune response was measured using in vitro cell proliferation assays (CPA) using whole blood or peripheral blood mononuclear cells (PBM) cultured with concanavalin A (ConA), phytohaemagglutinin (PHA), or soluble egg EWL antigen. All turtles, except for one immunized with Gerbu and the control, produced a detectable humoral immune response by 6 weeks which persisted for at least 14 weeks after a single immunization. All turtles produced an anamnestic humoral immune response after secondary immunization. Antigen specific cell-mediated immune response in PBM was seen in all turtles either after primary or secondary immunization, but it was not as consistent as humoral immune response; antigen specific cell-mediated immune response in whole blood was rarely seen. Mononuclear cells had significantly higher stimulation indices than whole blood regardless of adjuvant, however, results with whole blood had lower variability. Both Gerbu and ISA-70 appeared to potentiate the cell-mediated immune response when PBM or whole blood were cultured with PHA. This is the first time cell proliferation assays have been compared between whole blood and PBM for reptiles. This is also the first demonstration of antigen specific cell-mediated response in reptiles. Cell proliferation assays allowed us to evaluate the cell-mediated immune response of green turtles. However, CPA may be less reliable than ELISA for detecting antigen specific immune response. Either of the three adjuvants appears suitable to safely elicit a

  16. Role of IL-33 and Its Receptor in T Cell-Mediated Autoimmune Diseases

    Directory of Open Access Journals (Sweden)

    Qing Zhao


    Full Text Available Interleukin-33 (IL-33 is a new cytokine of interleukin-1 family, whose specific receptor is ST2. IL-33 exerts its functions via its target cells and plays different roles in diseases. ST2 deletion and exclusion of IL-33/ST2 axis are accompanied by enhanced susceptibility to dominantly T cell-mediated organ-specific autoimmune diseases. It has been reported that IL-33/ST2 pathway plays a key role in host defense and immune regulation in inflammatory and infectious diseases. This review focuses on new findings in the roles of IL-33 and ST2 in several kinds of T cell-mediated autoimmune diseases.

  17. Analysis of cell-mediated immune responses in support of dengue vaccine development efforts. (United States)

    Rothman, Alan L; Currier, Jeffrey R; Friberg, Heather L; Mathew, Anuja


    Dengue vaccine development has made significant strides, but a better understanding of how vaccine-induced immune responses correlate with vaccine efficacy can greatly accelerate development, testing, and deployment as well as ameliorate potential risks and safety concerns. Advances in basic immunology knowledge and techniques have already improved our understanding of cell-mediated immunity of natural dengue virus infection and vaccination. We conclude that the evidence base is adequate to argue for inclusion of assessments of cell-mediated immunity as part of clinical trials of dengue vaccines, although further research to identify useful correlates of protective immunity is needed.

  18. Intravital imaging reveals improved Kupffer cell-mediated phagocytosis as a mode of action of glycoengineered anti-CD20 antibodies (United States)

    Grandjean, Capucine L.; Montalvao, Fabricio; Celli, Susanna; Michonneau, David; Breart, Beatrice; Garcia, Zacarias; Perro, Mario; Freytag, Olivier; Gerdes, Christian A.; Bousso, Philippe


    Anti-CD20 monoclonal antibodies (mAbs) represent an effective treatment for a number of B cell malignancies and autoimmune disorders. Glycoengineering of anti-CD20mAb may contribute to increased anti-tumor efficacy through enhanced antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP) as reported by in vitro studies. However, where and how glycoengineered Ab may potentiate therapeutic responses in vivo is yet to be elucidated. Here, we have performed mouse liver transplants to demonstrate that the liver is sufficient to mediate systemic B cells depletion after anti-CD20 treatment. Relying on intravital two-photon imaging of human CD20-expressing mice, we provide evidence that ADP by Kupffer cells (KC) is a major mechanism for rituximab-mediated B cell depletion. Notably, a glycoengineered anti-mouse CD20 Ab but not its wild-type counterpart triggered potent KC-mediated B cell depletion at low doses. Finally, distinct thresholds for KC phagocytosis were also observed for GA101 (obinutuzumab), a humanized glycoengineered type II anti-CD20 Ab and rituximab. Thus, we propose that enhanced phagocytosis of circulating B cells by KC represents an important in vivo mechanism underlying the improved activity of glycoengineered anti-CD20 mAbs. PMID:27698437

  19. Substitution of the precursor peptide prevents anti-prM antibody-mediated antibody-dependent enhancement of dengue virus infection. (United States)

    Wang, Ying; Si, Lu-Lu; Guo, Xiao-Lan; Cui, Guo-Hui; Fang, Dan-Yun; Zhou, Jun-Mei; Yan, Hui-Jun; Jiang, Li-Fang


    Antibody-dependent enhancement (ADE) is currently considered as the mechanism underlying the pathogenesis of severe dengue disease. Many studies have shown that precursor (pr) peptide-specific antibodies do not efficiently neutralize infection but potently promote ADE of dengue virus (DENV) infection. To explore the effect of pr peptide substitution on neutralization and ADE of DENV infection, the rabbit anti-prM polyclonal antibodies (pAbs) and anti-JEVpr/DENV-M pAbs were prepared, and the neutralization and ADE of these two pAbs were further compared. Here, we report that both anti-JEVpr/DENV-M and anti-prM pAbs exhibited broad cross-reactivity and only partial neutralization with four DENV serotypes and immature DENV. Rabbit anti-prM pAbs showed a significant enhancement in a broad range of serum dilutions. However, there was no statistically significant difference in the enhancing activity of rabbit anti-JEVpr/DENV-M pAbs at various levels of dilution. These results demonstrate that anti-prM antibody-mediated ADE can be prevented by JEV pr peptide replacement. The present study contribute further to research on the pathogenesis of DENV infection.

  20. Fcγ-receptor IIa-mediated Src Signaling Pathway Is Essential for the Antibody-Dependent Enhancement of Ebola Virus Infection (United States)

    Furuyama, Wakako; Marzi, Andrea; Maruyama, Junki; Kuroda, Makoto; Miyamoto, Hiroko; Manzoor, Rashid; Yoshida, Reiko; Igarashi, Manabu; Feldmann, Heinz; Takada, Ayato


    Antibody-dependent enhancement (ADE) of Ebola virus (EBOV) infection has been demonstrated in vitro, raising concerns about the detrimental potential of some anti-EBOV antibodies. ADE has been described for many viruses and mostly depends on the cross-linking of virus-antibody complexes to cell surface Fc receptors, leading to enhanced infection. However, little is known about the molecular mechanisms underlying this phenomenon. Here we show that Fcγ-receptor IIa (FcγRIIa)-mediated intracellular signaling through Src family protein tyrosine kinases (PTKs) is required for ADE of EBOV infection. We found that deletion of the FcγRIIa cytoplasmic tail abolished EBOV ADE due to decreased virus uptake into cellular endosomes. Furthermore, EBOV ADE, but not non-ADE infection, was significantly reduced by inhibition of the Src family protein PTK pathway, which was also found to be important to promote phagocytosis/macropinocytosis for viral uptake into endosomes. We further confirmed a significant increase of the Src phosphorylation mediated by ADE. These data suggest that antibody-EBOV complexes bound to the cell surface FcγRIIa activate the Src signaling pathway that leads to enhanced viral entry into cells, providing a novel perspective for the general understanding of ADE of virus infection. PMID:28036370

  1. A longitudinal study of cell-mediated immunity in pigs infected with porcine parvovirus

    DEFF Research Database (Denmark)

    Ladekjaer-Mikkelsen, A.S.; Nielsen, Jens


    Porcine parvovirus (PPV) is an ubiquitous pathogen causing reproductive failure in swine. Protection against reproductive failure caused by acute PPV infection has commonly been related to the presence of specific antibodies in the dam. However, the role of cell-mediated immunity during chronic PPV...

  2. Signaling factors in stem cell-mediated repair of infarcted myocardium

    NARCIS (Netherlands)

    Vandervelde, S; van Luyn, MJA; Tio, RA; Harmsen, MC


    Myocardial infarction leads to scar formation and subsequent reduced cardiac performance. The ultimate therapy after myocardial infarction would pursue stem cell-based regeneration. The aim of stem cell-mediated cardiac repair embodies restoration of cardiac function by regeneration of healthy myoca


    Directory of Open Access Journals (Sweden)

    Michel Leclerc


    Full Text Available Cell-mediated immune responses occur in sea star system. In Asterias rubens it is said that B sea star lymphocytes and T sea star lymphocytes exist in the axial organ which can be considered as an ancestral lymphoid organ. In the same manner the origin of lymphocytes can be found in Invertebrates such as Echinodermal.

  4. Role of macrophage inflammatory protein-1alpha in T-cell-mediated immunity to viral infection

    DEFF Research Database (Denmark)

    Madsen, Andreas N; Nansen, Anneline; Christensen, Jan P


    The immune response to lymphocytic choriomeningitis virus in mice lacking macrophage inflammatory protein-1alpha (MIP-1alpha) was evaluated. Generation of virus-specific effector T cells is unimpaired in MIP-1alpha-deficient mice. Furthermore, MIP-1alpha is not required for T-cell-mediated virus...

  5. Recombinant nucleocapsid-like particles from dengue-2 induce functional serotype-specific cell-mediated immunity in mice. (United States)

    Gil, Lázaro; Bernardo, Lídice; Pavón, Alequis; Izquierdo, Alienys; Valdés, Iris; Lazo, Laura; Marcos, Ernesto; Romero, Yaremis; Guzmán, María G; Guillén, Gerardo; Hermida, Lisset


    The interplay of different inflammatory cytokines induced during dengue virus infection plays a role in either protection or increased disease severity. In this sense, vaccine strategies incorporating whole virus are able to elicit both functional and pathological responses. Therefore, an ideal tetravalent vaccine candidate against dengue should be focused on serotype-specific sequences. In the present work, a new formulation of nucleocapsid-like particles (NLPs) obtained from the recombinant dengue-2 capsid protein was evaluated in mice to determine the level of protection against homologous and heterologous viral challenge and to measure the cytotoxicity and cytokine-secretion profiles induced upon heterologous viral stimulation. As a result, a significant protection rate was achieved after challenge with lethal dengue-2 virus, which was dependent on CD4(+) and CD8(+) cells. In turn, no protection was observed after heterologous challenge. In accordance, in vitro-stimulated spleen cells from mice immunized with NLPs from the four dengue serotypes showed a serotype-specific response of gamma interferon- and tumour necrosis factor alpha-secreting cells. A similar pattern was detected when spleen cells from dengue-immunized animals were stimulated with the capsid protein. Taking these data together, we can assert that NLPs constitute an attractive vaccine candidate against dengue. They induce a functional immune response mediated by CD4(+) and CD8(+) cells in mice, which is protective against viral challenge. In turn, they are potentially safe due to two important facts: induction of serotype specific cell-mediated immunity and lack of induction of antiviral antibodies. Further studies in non-human primates or humanized mice should be carried out to elucidate the usefulness of the NLPs as a potential vaccine candidate against dengue disease.

  6. Cytotoxicity of carbon nanotubes

    Institute of Scientific and Technical Information of China (English)

    ZHU Ying; LI WenXin


    With large-scale production and application at large scale, carbon nanotubes (CNTs) may cause ad-verse response to the environment and human health. Thus, study on bio-effects and safety of CNTs has attracted great attention from scientists and governments worldwide. This report briefly summa-rizes the main results from the in vitro toxicity study of CNTs. The emphasis is placed on the descrip-tion of a variety of factors affecting CNTs cytotoxicity, including species of CNTs, impurities contained,lengths of CNTs, aspect ratios, chemical modification, and assaying methods of cytotoxicity. However,experimental information obtained thus far on CNTs' cytotoxicity is lacking in comparability, and some-times there is controversy about it. In order to assess more accurately the potential risks of CNTs to human health, we suggest that care should be taken for issues such as chemical modification and quantitative characterization of CNTa in cytotoxicity assessment. More importantly, studies on physical and chemical mechanisms of CNTs' cytotoxicity should be strengthened; assaying methods and evaluating criteria characterized by nanotoxicology should be gradually established.

  7. Cytotoxicity of carbon nanotubes

    Institute of Scientific and Technical Information of China (English)


    With large-scale production and application at large scale, carbon nanotubes (CNTs) may cause ad-verse response to the environment and human health. Thus, study on bio-effects and safety of CNTs has attracted great attention from scientists and governments worldwide. This report briefly summa-rizes the main results from the in vitro toxicity study of CNTs. The emphasis is placed on the descrip-tion of a variety of factors affecting CNTs cytotoxicity, including species of CNTs, impurities contained, lengths of CNTs, aspect ratios, chemical modification, and assaying methods of cytotoxicity. However, experimental information obtained thus far on CNTs’ cytotoxicity is lacking in comparability, and some-times there is controversy about it. In order to assess more accurately the potential risks of CNTs to human health, we suggest that care should be taken for issues such as chemical modification and quantitative characterization of CNTs in cytotoxicity assessment. More importantly, studies on physical and chemical mechanisms of CNTs’ cytotoxicity should be strengthened; assaying methods and evaluating criteria characterized by nanotoxicology should be gradually established.

  8. The combination of IL-21 and IFN-alpha boosts STAT3 activation, cytotoxicity and experimental tumor therapy

    DEFF Research Database (Denmark)

    Eriksen, Karsten W; Søndergaard, Henrik; Woetmann, Anders


    a selective increase in MHC class I expression and NK- and CD8(+) T-cell-mediated cytotoxicity. In an experimental in vivo model of renal carcinoma, the combined treatment of IFN-alpha and IL-21 also produces a significant anti-cancer effect as judged by an inhibition of tumor growth and an increased survival....... Taken together our data show that the combined use of IFN-alpha and IL-21 boosts STAT3 signaling, cytotoxicity, and anti-tumor efficacy, suggesting that a combinatorial therapeutic use of these cytokines may benefit cancer patients....

  9. Molecular determinants of dengue virus 2 envelope protein important for virus entry in FcγRIIA-mediated antibody-dependent enhancement of infection

    Energy Technology Data Exchange (ETDEWEB)

    Chotiwan, Nunya; Roehrig, John T. [Arboviral Diseases Branch, Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Schlesinger, Jacob J. [Department of Medicine, University of Rochester, Rochester, NY 14642 (United States); Blair, Carol D. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Huang, Claire Y.-H., E-mail: [Arboviral Diseases Branch, Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States)


    Antibody-dependent enhancement (ADE) of infection may cause severe illness in patients suffering a secondary infection by a heterologous dengue virus (DENV) serotype. During ADE of infection, cross-reactive non- or poorly-neutralizing antibodies form infectious virus-Ab complexes with the newly infecting serotype and enhance virus infection by binding to the Fcγ receptors (FcγR) on FcγR-bearing cells. In this study, we determined that molecular determinants of DENV2 envelope protein critical for virus entry during non-ADE infection are also required for ADE infection mediated by FcγRIIA, and binding of virus-Ab complexes with FcγRIIA alone is not sufficient for ADE of infection. The FcγRIIA mainly plays an auxiliary role in concentrating the virus–Ab complex to the cell surface, and other primary cellular receptors are required for virus entry. Understanding the viral entry pathway in ADE of DENV infection will greatly facilitate rational designs of anti-viral therapeutics against severe dengue disease associated with ADE. - Highlights: • KKK305/307/310 in DENV2 E-DIII is critical for virus attachment in ADE and non-ADE infection. • Binding of DENV2–Ab complex with FcγRII alone is not sufficient for virus entry in ADE infection. • Other primary receptors were required for DENV2 internalization during FcγRII–mediated ADE. • G104 and L135 of DENV2 E are critical for virus-mediated membrane fusion. • DENV2 virus-mediated membrane fusion is required for both ADE and non-ADE infection.

  10. Sulfated polysaccharide, curdlan sulfate, efficiently prevents entry/fusion and restricts antibody-dependent enhancement of dengue virus infection in vitro: a possible candidate for clinical application. (United States)

    Ichiyama, Koji; Gopala Reddy, Sindhoora Bhargavi; Zhang, Li Feng; Chin, Wei Xin; Muschin, Tegshi; Heinig, Lars; Suzuki, Youichi; Nanjundappa, Haraprasad; Yoshinaka, Yoshiyuki; Ryo, Akihide; Nomura, Nobuo; Ooi, Eng Eong; Vasudevan, Subhash G; Yoshida, Takashi; Yamamoto, Naoki


    Curdlan sulfate (CRDS), a sulfated 1→3-β-D glucan, previously shown to be a potent HIV entry inhibitor, is characterized in this study as a potent inhibitor of the Dengue virus (DENV). CRDS was identified by in silico blind docking studies to exhibit binding potential to the envelope (E) protein of the DENV. CRDS was shown to inhibit the DENV replication very efficiently in different cells in vitro. Minimal effective concentration of CRDS was as low as 0.1 µg/mL in LLC-MK2 cells, and toxicity was observed only at concentrations over 10 mg/mL. CRDS can also inhibit DENV-1, 3, and 4 efficiently. CRDS did not inhibit the replication of DENV subgenomic replicon. Time of addition experiments demonstrated that the compound not only inhibited viral infection at the host cell binding step, but also at an early post-attachment step of entry (membrane fusion). The direct binding of CRDS to DENV was suggested by an evident reduction in the viral titers after interaction of the virus with CRDS following an ultrafiltration device separation, as well as after virus adsorption to an alkyl CRDS-coated membrane filter. The electron microscopic features also showed that CRDS interacted directly with the viral envelope, and caused changes to the viral surface. CRDS also potently inhibited DENV infection in DC-SIGN expressing cells as well as the antibody-dependent enhancement of DENV-2 infection. Based on these data, a probable binding model of CRDS to DENV E protein was constructed by a flexible receptor and ligand docking study. The binding site of CRDS was predicted to be at the interface between domains II and III of E protein dimer, which is unique to this compound, and is apparently different from the β-OG binding site. Since CRDS has already been tested in humans without serious side effects, its clinical application can be considered.

  11. The role of IgG subclass of mouse monoclonal antibodies in antibody-dependent enhancement of feline infectious peritonitis virus infection of feline macrophages. (United States)

    Hohdatsu, T; Tokunaga, J; Koyama, H


    Antibody-dependent enhancement (ADE) of feline infectious peritonitis virus (FIPV) infection was studied in feline alveolar macrophages and human monocyte cell line U937 using mouse neutralizing monoclonal antibodies (MAbs) directed to the spike protein of FIPV. Even among the MAbs that have been shown to recognize the same antigenic site, IgG 2a MAbs enhanced FIPV infection strongly, whereas IgG 1 MAbs did not. These IgG 2a MAbs enhanced the infection even when macrophages pretreated with the MAb were washed and then inoculated with the virus. Immunofluorescence flow cytometric analysis of the macrophages treated with each of the MAbs showed that the IgG 2a MAbs but not the IgG 1 MAbs bound to feline alveolar macrophages. Treatment of the IgG 2a MAb with protein A decreased the binding to the macrophages and, in parallel, diminished the ADE activity. Although no infection was observed by inoculation of FIPV to human monocyte cell line U937 cells, FIPV complexed with either the IgG 2a MAb or the IgG 1 MAb caused infection in U937 cells which are shown to express Fc gamma receptor (Fc gamma R) I and II that can bind mouse IgG 2a and IgG 1, respectively. These results suggest that the enhancing activity of MAb is closely correlated with IgG subclass and that the correlation is involved in binding of MAb to Fc gamma R on feline macrophage.

  12. Sulfated polysaccharide, curdlan sulfate, efficiently prevents entry/fusion and restricts antibody-dependent enhancement of dengue virus infection in vitro: a possible candidate for clinical application.

    Directory of Open Access Journals (Sweden)

    Koji Ichiyama

    Full Text Available Curdlan sulfate (CRDS, a sulfated 1→3-β-D glucan, previously shown to be a potent HIV entry inhibitor, is characterized in this study as a potent inhibitor of the Dengue virus (DENV. CRDS was identified by in silico blind docking studies to exhibit binding potential to the envelope (E protein of the DENV. CRDS was shown to inhibit the DENV replication very efficiently in different cells in vitro. Minimal effective concentration of CRDS was as low as 0.1 µg/mL in LLC-MK2 cells, and toxicity was observed only at concentrations over 10 mg/mL. CRDS can also inhibit DENV-1, 3, and 4 efficiently. CRDS did not inhibit the replication of DENV subgenomic replicon. Time of addition experiments demonstrated that the compound not only inhibited viral infection at the host cell binding step, but also at an early post-attachment step of entry (membrane fusion. The direct binding of CRDS to DENV was suggested by an evident reduction in the viral titers after interaction of the virus with CRDS following an ultrafiltration device separation, as well as after virus adsorption to an alkyl CRDS-coated membrane filter. The electron microscopic features also showed that CRDS interacted directly with the viral envelope, and caused changes to the viral surface. CRDS also potently inhibited DENV infection in DC-SIGN expressing cells as well as the antibody-dependent enhancement of DENV-2 infection. Based on these data, a probable binding model of CRDS to DENV E protein was constructed by a flexible receptor and ligand docking study. The binding site of CRDS was predicted to be at the interface between domains II and III of E protein dimer, which is unique to this compound, and is apparently different from the β-OG binding site. Since CRDS has already been tested in humans without serious side effects, its clinical application can be considered.

  13. Intranasal administration of HIV-DNA vaccine formulated with a polymer, carboxymethylcellulose, augments mucosal antibody production and cell-mediated immune response. (United States)

    Hamajima, K; Sasaki, S; Fukushima, J; Kaneko, T; Xin, K Q; Kudoh, I; Okuda, K


    We previously reported that intramuscular (i.m.) immunization of DNA vaccine encoding human immunodeficiency virus type 1 (HIV-1)IIIB env and rev genes alone or in combination with appropriate adjuvant induces substantial and enhanced immune response against HIV-1. In the present study, we examined whether a polymer, low-viscosity carboxymethylcellulose sodium salt (CMCS-L), has an adjuvant effect on immune response induced by DNA vaccination. BALB/c mice were immunized with HIV-DNA vaccine formulated with CMCS-L via the intranasal (i.n.) and i.m. routes. The combination with the polymer elicited higher levels of antigen-specific serum IgG and fecal IgA antibodies than DNA vaccine alone. For cell-mediated immunity, HIV-specific delayed-type hypersensitivity response and cytotoxic T lymphocyte activity were measured by the footpad-swelling test and the 51Cr-release assay, respectively. Both were enhanced by the combination with CMCS-L via i.n. and i.m. inoculation. Cytokine analysis in culture media of bulk splenocytes harvested from immunized animals showed higher levels of IL-4 production in i.n. -immunized mice compared with i.m.-immunized mice. Nevertheless, the increased IFN-gamma production resulting from the combination with CMCS-L was observed only in i.n.-immunized mice. These data indicate that i.n. immunization of HIV-DNA vaccine formulated with CMCS-L enhances HIV-specific mucosal antibody (Ab) and systemic Ab and cell-mediated immune response.

  14. Selenium cytotoxicity in cancer. (United States)

    Wallenberg, Marita; Misra, Sougat; Björnstedt, Mikael


    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.

  15. Role of very late antigen-1 in T-cell-mediated immunity to systemic viral infection

    DEFF Research Database (Denmark)

    Ørding Kauffmann, Susanne; Thomsen, Allan Randrup; Christensen, Jan Pravsgaard


    The T-cell response to lymphocytic choriomeningitis virus was studied in mice lacking very late antigen-1 (VLA-1). The generation of virus-specific effector T cells was unimpaired in VLA-1(-/-) mice. In the memory phase, VLA-1 deficiency did not influence the number of memory CD8(+) T cells or th......, the current findings indicate that the expression of VLA-1 is not pivotal for T-cell-mediated antiviral immunity to a systemic infection.......-cell-mediated inflammation, no significant influence of VLA-1 was found either in the primary response or in the memory phase. However, alpha-VLA-4 antibody reduced the DTH-like reaction in VLA-1(-/-) mice to a higher degree than in wt mice, suggesting a synergistic effect of blocking both integrins. Taken together...

  16. Effect of disodium cromoglycate on mast cell-mediated immediate-type allergic reactions. (United States)

    Shin, Hye-Young; Kim, Jung-Sook; An, Nyeon-Hyoung; Park, Rae-Kil; Kim, Hyung-Min


    We investigated the effect of disodium cromoglycate (DSCG) on mast cell-mediated immediate-type hypersensitivity. DSCG inhibited systemic allergic reaction induced by compound 48/80 dose-dependently. Passive cutaneous anaphylaxis was inhibited by 71.6% by oral administration of DSCG (1 g/kg). When DSCG was pretreated at concentration rang from 0.01-1000 g/kg, the serum histamine levels were reduced in a dose dependent manner. DSCG also significantly inhibited histamine release from rat peritoneal mast cell (RPMC) by compound 48/80. We confirmed that DSCG inhibited compound 48/80-induced degranulation of RPMC by alcian blue/nuclear fast red staining. In addition, DSCG showed a significant inhibitory effect on anti-dinitrophenyl IgE-mediated tumor necrosis factor-alpha production. These results indicate that DSCG inhibits mast cell-mediated immediate-type allergic reaction.

  17. Impaired cell mediated immunity in haemophilia in the absence of infection with human immunodeficiency virus. (United States)

    Madhok, R; Gracie, A; Lowe, G D; Burnett, A; Froebel, K; Follett, E; Forbes, C D


    The cell mediated immune response was evaluated in vivo in 29 patients with clinically severe haemophilia by means of the dinitrochlorobenzene skin test. All patients had a response below the median normal value, and in 19 the response was on or below the lower limit of the normal range. There was no difference in skin response between patients positive and negative for the human immunodeficiency virus (HIV; formerly known as human T cell lymphotropic virus III or lymphadenopathy associated virus). In the whole group, and in seronegative patients (n = 17), there was an inverse relation between exposure to clotting factor and skin response. In seropositive patients (n = 12) no such association was apparent. This study shows that clotting factor concentrate impairs the cell mediated immune response to a new antigen in the absence of infection with HIV.

  18. Saos-2 cell-mediated mineralization on collagen gels: Effect of densification and bioglass incorporation. (United States)

    Liu, Gengbo; Pastakia, Meet; Fenn, Michael B; Kishore, Vipuil


    Plastic compression is a collagen densification process that has been widely used for the development of mechanically robust collagen-based materials. Incorporation of bioglass within plastically compressed collagen gels has been shown to mimic the microstructural properties of native bone and enhance in vitro cell-mediated mineralization. The current study seeks to decouple the effects of collagen densification and bioglass incorporation to understand the interplay between collagen packing density and presence of bioglass on cell-mediated mineralization. Saos-2 cell-mediated mineralization was assessed as a measure of the osteoconductivity of four different collagen gels: (1) uncompressed collagen gel (UC), (2) bioglass incorporated uncompressed collagen gel (UC + BG), (3) plastically compressed collagen gel (PC), and (4) bioglass incorporated plastically compressed collagen gel (PC + BG). The results indicated that collagen densification enhanced mineralization as shown by SEM, increased alkaline phosphatase activity and produced significantly higher amounts of mineralized nodules on PC gels compared to UC gels. Further, the amount of nodule formation on PC gels was significantly higher compared to UC + BG gels indicating that increase in matrix stiffness due to collagen densification had a greater effect on cell-mediated mineralization compared to bioglass incorporation into loosely packed UC gels. Incorporation of bioglass into PC gels further enhanced mineralization as evidenced by significantly larger nodule size and higher amount of mineralization on PC + BG gels compared to PC gels. In conclusion, collagen densification via plastic compression improves the osteoconductivity of collagen gels. Further, incorporation of bioglass within PC gels has an additive effect and further enhances the osteoconductivity of collagen gels.

  19. Activation of cell-mediated immunity by Morinda citrifolia fruit extract and its constituents. (United States)

    Murata, Kazuya; Abe, Yumi; Futamura-Masudaa, Megumi; Uwaya, Akemi; Isami, Fumiyuki; Matsuda, Hideaki


    Morinda citrifolia, commonly known as noni, is a traditional natural medicine in French Polynesia and Hawaii. Functional foods derived from M. citrifolia fruit have been marketed to help prevent diseases and promote good health. The objective of this study was to assess the effects of M. citrifolia fruit on cell-mediated immunity. In the picryl chloride-induced contact dermatitis test, M. citrifolia fruit extract (Noni-ext) inhibited the suppression of cell-mediated immunity by immunosuppressive substances isolated from freeze-dried ascites of Ehrlich carcinoma-bearing mice (EC-sup). In addition, Noni-ext inhibited reduction of IL-2 production in EC-sup-treated mice and activated natural killer cells in normal mice. These results suggest that Noni-ext has multiple effects on the recovery of cell-mediated immunity. Furthermore, we investigated the active principles of Noni-ext and identified an iridoid glycoside, deacetylasperulosidic acid. Oral administration of deacetylasperulosidic acid inhibited the reduction of ear swelling, and also cancelled the suppression of IL-2 production along with the activation of natural killer cells in the same manner as that of Noni-ext.

  20. Flow cytometric readout based on Mitotracker Red CMXRos staining of live asexual blood stage malarial parasites reliably assesses antibody dependent cellular inhibition

    Directory of Open Access Journals (Sweden)

    Jogdand Prajakta S


    Full Text Available Abstract Background Functional in vitro assays could provide insights into the efficacy of malaria vaccine candidates. For estimating the anti-parasite effect induced by a vaccine candidate, an accurate determination of live parasite count is an essential component of most in vitro bioassays. Although traditionally parasites are counted microscopically, a faster, more accurate and less subjective method for counting parasites is desirable. In this study mitochondrial dye (Mitotracker Red CMXRos was used for obtaining reliable live parasite counts through flow cytometry. Methods Both asynchronous and tightly synchronized asexual blood stage cultures of Plasmodium falciparum were stained with CMXRos and subjected to detection by flow cytometry and fluorescence microscopy. The parasite counts obtained by flow cytometry were compared to standard microscopic counts obtained through examination of Giemsa-stained thin smears. A comparison of the ability of CMXRos to stain live and compromised parasites (induced by either medium starvation or by anti-malarial drug treatment was carried out. Finally, parasite counts obtained by CMXRos staining through flow cytometry were used to determine specific growth inhibition index (SGI in an antibody-dependent cellular inhibition (ADCI assay. Results Mitotracker Red CMXRos can reliably detect live intra-erythrocytic stages of P. falciparum. Comparison between staining of live with compromised parasites shows that CMXRos predominantly stains live parasites with functional mitochondria. Parasite counts obtained by CMXRos staining and flow cytometry were highly reproducible and can reliably determine the ability of IgG from hyper-immune individuals to inhibit parasite growth in presence of monocytes in ADCI assay. Further, a dose-dependent parasite growth inhibitory effect could be detected for both total IgG purified from hyper-immune sera and affinity purified IgGs against the N-terminal non-repeat region of GLURP

  1. [Tumor/cytotoxic effector cross-talk in the control of tumor susceptibility to lysis]. (United States)

    Gati, Asma; Dorothée, Guillaume; Thiéry, Jérôme; Guerra, Nadia; Richon, Catherine; Gaudin, Catherine; Mami-Chouaib, Fathia; Caignard, Anne; Diarra-Mehrpour, Maryam; Chouaib, Salem


    During the two least decades, the field of tumor immunology has met an expansion of knowledge about the molecular and cellular bases of immune regulation. The identification of cancer antigens has been of critical importance and cancer vaccine is at present a very fast moving field. However, the immunotherapy approaches in cancer are of modest success. This is mainly due to the capacity of tumor cells to escape from immunological detection and to resist to cell mediated cytotoxicity. We will discuss some mechanisms associated with the acquisition of this tumor resistance and the alteration of T cell function and how cancer profiling through genomics approaches may help to reconceptualize immunotherapy strategies.

  2. Cytotoxicity of organophosphate anticholinesterases. (United States)

    Cao, C J; Mioduszewski, R J; Menking, D E; Valdes, J J; Katz, E J; Eldefrawi, M E; Eldefrawi, A T


    Organophosphate (OP) anticholinesterases were found to modulate metabolic activities of human neuroblastoma cells and hepatocytes, which was detectable by the Cytosensor microphysiometer. The nerve gas ethyl-S-2-diisopropylaminoethyl methylphosphorothiolate (VX), at 10 microM, produced significant reduction in cell metabolism within 2 min, as measured by changes in the acidification rate of the medium. The reduction was dose- and time-dependent and irreversible after 4 h of exposure. Two alkaline degradation products of VX produced no cytotoxicity. Exposure for 24 h to 3 microM VX caused 36% and 94% irreversible loss of metabolism in hepatocytes and neuroblastoma cells, respectively. The insecticides parathion and chlorpyrifos stimulated hepatocyte metabolism but inhibited neuroblastoma cells. Their oxons were more active. Exposure of neuroblastoma cells for 4 h to VX, parathion, paraoxon, diisopropylfluorophosphate or chlorpyrifos gave an LC50 of 65, 775, 640, 340, or 672 microM, respectively, whereas 24 h gave an LC50 of 0.7, 3.7, 2.5, 29, and 31 microM, respectively. Preincubation of hepatocytes with phenobarbital enhanced their response to parathion and VX due to metabolic bioactivation. Atropine partially blocked the effects of VX and paraoxon on both cell types, which suggests the involvement of a muscarinic receptor as the target for cytotoxicity. There was no correlation between OP in vivo neurotoxicity and in vitro cytotoxicity. It is suggested that the former results from their cholinesterase inhibition, while the latter results from action on different targets and requires much higher concentrations.

  3. The impact of long-term haemofiltration (continuous veno-venous haemofiltration) on cell-mediated immunity during endotoxaemia

    DEFF Research Database (Denmark)

    Toft, P; Nilsen, B U; Bollen, P;


    BACKGROUND: Increased survival with high-volume continuous veno-venous haemofiltration (CVVH) has been demonstrated in critically ill patients. This may be the result of intensified blood purification or an effect on the immune system. We hypothesized that CVVH modifies the cell-mediated immunity....... However, in the long term, CVVH was unable to modify the endotoxin-induced changes in cell-mediated immunity....

  4. The PHA test reflects acquired T-cell mediated immunocompetence in birds.

    Directory of Open Access Journals (Sweden)

    José L Tella

    Full Text Available BACKGROUND: cological immunology requires techniques to reliably measure immunocompetence in wild vertebrates. The PHA-skin test, involving subcutaneous injection of a mitogen (phytohemagglutinin, PHA and measurement of subsequent swelling as a surrogate of T-cell mediated immunocompetence, has been the test of choice due to its practicality and ease of use in the field. However, mechanisms involved in local immunological and inflammatory processes provoked by PHA are poorly known, and its use and interpretation as an acquired immune response is currently debated. METHODOLOGY: Here, we present experimental work using a variety of parrot species, to ascertain whether PHA exposure produces larger secondary than primary responses as expected if the test reflects acquired immunocompetence. Moreover, we simultaneously quantified T-lymphocyte subsets (CD4(+, CD5(+ and CD8(+ and plasma proteins circulating in the bloodstream, potentially involved in the immunological and inflammatory processes, through flow cytometry and electrophoresis. PRINCIPAL FINDINGS: Our results showed stronger responses after a second PHA injection, independent of species, time elapsed and changes in body mass of birds between first and second injections, thus supporting the adaptive nature of this immune response. Furthermore, the concomitant changes in the plasma concentrations of T-lymphocyte subsets and globulins indicate a causal link between the activation of the T-cell mediated immune system and local tissue swelling. CONCLUSIONS/SIGNIFICANCE: These findings justify the widespread use of the PHA-skin test as a reliable evaluator of acquired T-cell mediated immunocompetence in diverse biological disciplines. Further experimental research should be aimed at evaluating the relative role of innate immunocompetence in wild conditions, where the access to dietary proteins varies more than in captivity, and to ascertain how PHA responses relate to particular host

  5. Dehydroeffusol effectively inhibits human gastric cancer cell-mediated vasculogenic mimicry with low toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Wenming; Meng, Mei; Zhang, Bin; Du, Longsheng; Pan, Yanyan; Yang, Ping; Gu, Zhenlun; Zhou, Quansheng, E-mail:; Cao, Zhifei, E-mail:


    Accumulated data has shown that various vasculogenic tumor cells, including gastric cancer cells, are able to directly form tumor blood vessels via vasculogenic mimicry, supplying oxygen and nutrients to tumors, and facilitating progression and metastasis of malignant tumors. Therefore, tumor vasculogenic mimicry is a rational target for developing novel anticancer therapeutics. However, effective antitumor vasculogenic mimicry-targeting drugs are not clinically available. In this study, we purified 2,7-dihydroxyl-1-methyl-5-vinyl-phenanthrene, termed dehydroeffusol, from the traditional Chinese medicinal herb Juncus effusus L., and found that dehydroeffusol effectively inhibited gastric cancer cell-mediated vasculogenic mimicry in vitro and in vivo with very low toxicity. Dehydroeffusol significantly suppressed gastric cancer cell adhesion, migration, and invasion. Molecular mechanistic studies revealed that dehydroeffusol markedly inhibited the expression of a vasculogenic mimicry master gene VE-cadherin and reduced adherent protein exposure on the cell surface by inhibiting gene promoter activity. In addition, dehydroeffusol significantly decreased the expression of a key vasculogenic gene matrix metalloproteinase 2 (MMP2) in gastric cancer cells, and diminished MMP2 protease activity. Together, our results showed that dehydroeffusol effectively inhibited gastric cancer cell-mediated vasculogenic mimicry with very low toxicity, suggesting that dehydroeffusol is a potential drug candidate for anti-gastric cancer neovascularization and anti-gastric cancer therapy. - Highlights: • Dehydroeffusol markedly inhibits gastric cancer cell-mediated vasculogenic mimicry. • Dehydroeffusol suppresses the expression of vasculogenic mimicry key gene VE-cadherin. • Dehydroeffusol decreases the MMP2 expression and activity in gastric cancer cells. • Dehydroeffusol is a potential anti-cancer drug candidate with very low toxicity.


    Directory of Open Access Journals (Sweden)

    Anis Ahmed


    Full Text Available Varicella zoster virus causes primary infection as chickenpox, at which time latencyis established in the neurons of the dorsal root ganglia or ganglia of the cranial nerves.Reactivation produces herpes zoster infection (HZI, commonly called shingles. Anunderstanding of the mechanisms of latency is crucial in developing effective therapies forVZV infections of the nervous system. This article describes the pathogenesis of VZVwhich includes immune response to the virus, immune evasion by the virus, mechanism ofits latency and cell-mediated immunity.

  7. Antigenic role of stress-induced catalase of Salmonella typhimurium in cell-mediated immunity.


    Kagaya, K; Miyakawa, Y; Watanabe, K; Fukazawa, Y.


    The ability of the H2O2-induced catalase of Salmonella typhimurium to induce cell-mediated immunity against S. typhimurium infection in mice was examined. When exponentially growing cells of S. typhimurium were treated with 20 microM H2O2, the cells resisted killing by 1 mM H2O2 and showed the induction of a new species of catalase in addition to the constitutively produced one. Two molecules of catalases in S. typhimurium were isolated from mutant strains: H2O2-induced catalase (catalase II,...

  8. Reactivating effect of levamisole on cell-mediated immunity in gastrointestinal cancer patients

    Directory of Open Access Journals (Sweden)



    Full Text Available Cell-mediated immunity was studied in 23 cases of advanced gastrointestinal cancer. The patients received levamisole at 150 mg/day for three consecutive days each week for four weeks. In cases at the terminal stage of gastrointestinal cancer, the blastformation rate of peripheral blood lymphocytes against phytohemagglutinin (PHA after the administration of levamisole showed a slight increase, but cases with blastformation rates over 40% increased markedly three or four weeks after the initial administration of levamisole. The peripheral blood lymphocyte count showed little change in these cases.

  9. Induction of ATM/ATR pathway combined with Vγ2Vδ2 T cells enhances cytotoxicity of ovarian cancer cells. (United States)

    Lu, Jingwei; Das, Manjusri; Kanji, Suman; Aggarwal, Reeva; Joseph, Matthew; Ray, Alo; Shapiro, Charles L; Pompili, Vincent J; Das, Hiranmoy


    Many ovarian cancer cells express stress-related molecule MICA/B on their surface that is recognized by Vγ2Vδ2 T cells through their NKG2D receptor, which is transmitted to downstream stress-signaling pathway. However, it is yet to be established how Vγ2Vδ2 T cell-mediated recognition of MICA/B signal is transmitted to downstream stress-related molecules. Identifying targeted molecules would be critical to develop a better therapy for ovarian cancer cells. It is well established that ATM/ATR signal transduction pathways, which is modulated by DNA damage, replication stress, and oxidative stress play central role in stress signaling pathway regulating cell cycle checkpoint and apoptosis. We investigated whether ATM/ATR and its down stream molecules affect Vγ2Vδ2 T cell-mediated cytotoxicity. Herein, we show that ATM/ATR pathway is modulated in ovarian cancer cells in the presence of Vγ2Vδ2 T cells. Furthermore, downregulation of ATM pathway resulted downregulation of MICA, and reduced Vγ2Vδ2 T cell-mediated cytotoxicity. Alternately, stimulating ATM pathway enhanced expression of MICA, and sensitized ovarian cancer cells for cytotoxic lysis by Vγ2Vδ2 T cells. We further show that combining currently approved chemotherapeutic drugs, which induced ATM signal transduction, along with Vγ2Vδ2 T cells enhanced cytotoxicity of resistant ovarian cancer cells. These findings indicate that ATM/ATR pathway plays an important role in tumor recognition, and drugs promoting ATM signaling pathway might be considered as a combination therapy together with Vγ2Vδ2 T cells for effectively treating resistant ovarian cancer cells.

  10. Autoxidation and cytotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Borg, D C; Schaich, K M; Elmore, Jr, J J


    A comprehensive synthesis, or reaction schema, to relate autoxidations of non-lipid compounds to lipid chain peroxidation in vivo is presented. This is done in the context of cytotoxic autoxidation reactions, and it is concluded that hydroxyl radicals produced by iron-dependent Fenton reactions serve as both primary toxicants and as sources of secondary toxicants. The latter stem from lipid chain peroxidation initiated by the Fenton-derived hydroxyl radicals, which are visualized as the obligate coupling step linking enzyme-dependent and non-enzymic autoxidations to potentially toxic outcomes.

  11. Cytotoxic glycosides from Albizia julibrissin. (United States)

    Ikeda, T; Fujiwara, S; Araki, K; Kinjo, J; Nohara, T; Miyoshi, T


    During the course of a study of leguminous plants, cytotoxicity was demonstrated by the crude saponin fraction of Albizia julibrissin. Following chromatographic purification, the structures of three novel saponins, julibrosides I-III (1-3), inclusive of a cytotoxic principle, were elucidated. A comparison of the cytotoxicity of julibrosides (1-3) and their prosapogenins (4-15) prepared by alkaline hydrolysis clearly indicated that both an alpha-L-arabinofuranosyl-(1-->4)-[beta-D-glucopyranosyl-(1-->3)]-alpha- L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl ester unit and a monoterpene-quinovopyranosyl moiety are crucial substituents for cytotoxicity among this class of compounds. The hydroxy group at C-16 of aglycon may play an important role in mediating cytotoxicity, and the N-acetyl-glucosamine moiety at C-3 seems to enhance activity because 3 showed the strongest cytotoxicity.

  12. Cytotoxic T lymphocyte activity in children infected with HIV. (United States)

    Froebel, K S; Aldhous, M C; Mok, J Y; Hayley, J; Arnott, M; Peutherer, J F


    Of the Edinburgh cohort of approximately 130 children born to HIV-infected women, 9 are infected and alive. This article describes results from the first 18 months of a natural history study of seven of these, and two adopted children, studying the CD8 T cell-mediated cytotoxicity against HIV proteins (Gag, Tat, Pol, and Env), over time, and relating it to clinical progression and viral activity. Autologous EBV cell lines infected with vaccinia-HIV constructs were used as target cells, and bulk-cultured peripheral blood mononuclear cells as effector cells. The children ranged in age from 0 to 93 months, with six of the nine showing CTL activity to one or more HIV proteins. The specificity of the response was directed against Tat in the younger children, switching to Pol, then Gag or Env. Preliminary analysis of virological data showed no association between CTL and virus activity. The children with CTLs tended to be well clinically, but the cohort needs to be studied longer before conclusions can be made about CTL activity and HIV disease progression. Cytotoxic T lymphocyte activity has also been observed in two children diagnosed as HIV uninfected. These results show the importance of looking at CTL specificity, and may have implications in vaccine design.

  13. The susceptibility to cytotoxic T lymphocyte mediated lysis of chemically induced sarcomas from immunodeficient and normal mice

    DEFF Research Database (Denmark)

    Svane, I M; Engel, A M; Thomsen, Allan Randrup;


    tested for susceptibility to cytolysis by virus specific cytotoxic T cells. Tumour cells originating from tumours induced in immunocompetent C.B.-17 mice presented virus antigen more efficiently than tumour cells from immunodeficient SCID mice. No significant difference in virus antigen presentation...... was found between tumours from nude and nu/+ BALB/c mice. The sensitivity of target cells from the individual tumours to cytotoxic T lymphocyte (CTL) mediated lysis correlated negatively with their sensitivity to natural killer (NK) cell mediated lysis. There was a positive correlation between...... the sensitivity to CTL mediated lysis and surface expression of the MHC class I molecule Ld of the tumour cells. Tumour cells incapable of in vitro presentation of viral antigen to specific cytotoxic T cells originated from tumours known from previous experiments to be readily accepted after transplantation...

  14. C1-esterase inhibitor blocks T lymphocyte proliferation and cytotoxic T lymphocyte generation in vitro

    DEFF Research Database (Denmark)

    Nissen, Mogens Holst; Bregenholt, S; Nording, J A


    We have previously shown that activated C1s complement and activated T cells cleave beta2-microglobulin (beta2m) in vitro leading to the formation of desLys58 beta2m. This process can specifically be inhibited by C1-esterase inhibitor (C1-inh). Furthermore we showed that exogenously added desLys58...... beta2m in nanomolar amounts to a one-way allogenic mixed lymphocyte culture (MLC) increased the endogenous production of IL-2 and the generation of allo-specific cytotoxic T lymphocytes. C1-inh was purified from fresh human plasma and added to human or murine MLC and mitogen-stimulated lymphocyte...... cultures grown in the presence of complement-inactivated serum. Read-outs were cell proliferation, lymphokine production and development of T cell-mediated cytotoxicity. We found that addition of C1-inh to MLC and mitogen-exposed murine and human lymphocyte cultures inhibited proliferation, the development...

  15. Stephanthraniline A suppressed CD4(+) T cell-mediated immunological hepatitis through impairing PKCθ function. (United States)

    Chen, Feng-Yang; Zhou, Li-Fei; Li, Xiao-Yu; Zhao, Jia-Wen; Xu, Shi-Fang; Huang, Wen-Hai; Gao, Li-Juan; Hao, Shu-Juan; Ye, Yi-Ping; Sun, Hong-Xiang


    Stephanthraniline A (STA), a C21 steroid isolated from Stephanotis mucronata (Blanco) Merr., was previously shown to inhibit T cells activation and proliferation in vitro and in vivo. The purpose of this study was to further evaluate the in vivo immunosuppressive activity of STA and to elucidate its potential mechanisms. The results showed that pretreatment with STA significantly attenuated concanavalin A (Con A)-induced hepatitis and reduced CD4(+) T cells activation and aggregation in hepatic tissue in mice. STA directly suppressed the activation and proliferation of Con A-induced CD4(+) T cells, and inhibited NFAT, NFκB and MAPK signaling cascades in activated CD4(+) T cells in vitro. Moreover, it was proved that STA inhibited T cells activation and proliferation through proximal T cell-receptor (TCR) signaling- and Ca(2+) signaling-independent way. The molecular docking studies predicted that STA could tight bind to PKCθ via five hydrogen. The further findings indicated STA directly inhibited PKCθ kinase activity, and its phosphorylation in activated CD4(+) T cells in vitro. Collectively, the present study indicated that STA could protect against CD4(+) T cell-mediated immunological hepatitis in mice through PKCθ and its downstream NFAT, NFκB and MAPK signaling cascades. These results highlight the potential of STA as an effective leading compound for use in the treatment of CD4(+) T cell-mediated inflammatory and autoimmune diseases.

  16. Cordyceps militaris Enhances Cell-Mediated Immunity in Healthy Korean Men. (United States)

    Kang, Ho Joon; Baik, Hyun Wook; Kim, Sang Jung; Lee, Seong Gyu; Ahn, Hong Yup; Park, Ju Sang; Park, Sang Jong; Jang, Eun Jeong; Park, Sang Woon; Choi, Jin Young; Sung, Ji Hee; Lee, Seung Min


    Cordyceps militaris is a mushroom traditionally used for diverse pharmaceutical purposes in East Asia, including China, and has been found to be effective for enhancing immunity through various types of animal testing. The aim of this study is to determine the efficacy of C. militaris for enhancing cell-mediated immunity and its safety in healthy male adults. Healthy male adults were divided into the experimental group (n = 39), given 1.5 g/day of ethanol treated C. militaris in capsules, and the control group (n = 40), given the same number of identical placebo capsules filled with microcrystalline cellulose and lactose for 4 weeks from February 13 to March 14, 2012; the natural killer (NK) cell activity, lymphocyte proliferation index (PI), and T-helper cell 1 (Th1) cytokine cluster (interferon [IFN]-γ, interleukin [IL]-12, IL-2, and tumor necrosis factor [TNF]-α) were measured, along with stability test, at weeks 0, 2, and 4. The C. militaris group showed a statistically significant greater increase in NK200 (P = .0010), lymphocyte PI (P ≤ .0001), IL-2 (P = .0096), and IFN-γ (P = .0126), compared with the basal level, than the placebo group. There was no statistically significant adverse reaction. C. militaris enhanced the NK cell activity and lymphocyte proliferation and partially increased Th1 cytokine secretion. Therefore, C. militaris is safe and effective for enhancing cell-mediated immunity of healthy male adults.

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

  18. Cell-mediated BMP-2 liberation promotes bone formation in a mechanically unstable implant environment. (United States)

    Hägi, Tobias T; Wu, Gang; Liu, Yuelian; Hunziker, Ernst B


    The flexible alloplastic materials that are used in bone-reconstruction surgery lack the mechanical stability that is necessary for sustained bone formation, even if this process is promoted by the application of an osteogenic agent, such as BMP-2. We hypothesize that if BMP-2 is delivered gradually, in a cell-mediated manner, to the surgical site, then the scaffolding material's lack of mechanical stability becomes a matter of indifference. Flexible discs of Ethisorb were functionalized with BMP-2, which was either adsorbed directly onto the material (rapid release kinetics) or incorporated into a calcium-phosphate coating (slow release kinetics). Unstabilized and titanium-plate-stabilized samples were implanted subcutaneously in rats and retrieved up to 14 days later for a histomorphometric analysis of bone and cartilage volumes. On day 14, the bone volume associated with titanium-plate-stabilized discs bearing an adsorbed depot of BMP-2 was 10-fold higher than that associated with their mechanically unstabilized counterparts. The bone volume associated with discs bearing a coating-incorporated depot of BMP-2 was similar in the mechanically unstabilized and titanium-plate-stabilized groups, and comparable to that associated with the titanium-plate-stabilized discs bearing an adsorbed depot of BMP-2. Hence, if an osteogenic agent is delivered in a cell-mediated manner (via coating degradation), ossification can be promoted even within a mechanically unstable environment.

  19. T cell mediated cerebral hemorrhages and microhemorrhages during passive Aβ immunization in APPPS1 transgenic mice

    Directory of Open Access Journals (Sweden)

    de Calignon Alix


    Full Text Available Abstract Background Immunization against amyloid-β (Aβ, the peptide that accumulates in the form of senile plaques and in the cerebrovasculature in Alzheimer's disease (AD, causes a dramatic immune response that prevents plaque formation and clears accumulated Aβ in transgenic mice. In a clinical trial of Aβ immunization, some patients developed meningoencephalitis and hemorrhages. Neuropathological investigations of patients who died after the trial showed clearance of amyloid pathology, but also a powerful immune response involving activated T cells probably underlying the negative effects of the immunization. Results To define the impact of T cells on this inflammatory response we used passive immunization and adoptive transfer to separate the effect of IgG and T cell mediated effects on microhemorrhage in APPPS1 transgenic mice. Neither anti Aβ IgG nor adoptively transferred T cells, alone, led to increased cerebrovascular damage. However, the combination of adoptively transferred T cells and passive immunization led to massive cerebrovascular bleeding that ranged from multiple microhemorrhages in the parenchyma to large hematomas. Conclusions Our results indicate that vaccination can lead to Aβ and T cell induced cerebral micro-hemorrhages and acute hematomas, which are greatly exacerbated by T cell mediated activity.

  20. Cytotoxicity of halogenated graphenes (United States)

    Teo, Wei Zhe; Khim Chng, Elaine Lay; Sofer, Zdeněk; Pumera, Martin


    Graphene and its family of derivatives possess unique and remarkable physicochemical properties which make them valuable materials for applications in many areas like electronics, energy storage and biomedicine. In response to the possibility of its large-scale manufacturing as commercial products in the future, an investigation was conducted to determine the cytotoxicity of one particular family of graphene derivatives, the halogenated graphenes, for the first time. Halogenated graphenes were prepared through thermal exfoliation of graphite oxide in gaseous chlorine, bromine or iodine atmospheres to yield chlorine- (TRGO-Cl), bromine- (TRGO-Br) and iodine-doped graphene (TRGO-I) respectively. 24 h exposure of human lung carcinoma epithelial cells (A549) to the three halogenated graphenes and subsequent cell viability assessments using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and water-soluble tetrazolium salt (WST-8) assays revealed that all the halogenated graphenes examined are rather cytotoxic at the concentrations tested (3.125 μg mL-1 to 200 μg mL-1) and the effects are dose-dependent, with TRGO-Cl reducing the cell viability to as low as 25.7% at the maximum concentration of 200 μg mL-1. Their levels of cytotoxicity can be arranged in the order of TRGO-Cl > TRGO-Br > TRGO-I, and it is suggested that the amount of halogen present in the graphene material is the determining factor for the observed trend. Control experiments were carried out to test for possible nanomaterial-induced interference as a consequence of reaction between the halogenated graphenes and the viability markers (MTT/WST-8 reagent) or binding of the formazan products under cell-free conditions. The data obtained eliminate the probability of significant influence by these interferents as the change in the normalized percentage of formazan formed is relatively small and thorough washings were performed prior to the viability assessments to reduce the amount of halogenated

  1. A non-surgical approach for male germ cell mediated gene transmission through transgenesis. (United States)

    Usmani, Abul; Ganguli, Nirmalya; Sarkar, Hironmoy; Dhup, Suveera; Batta, Suryaprakash R; Vimal, Manoj; Ganguli, Nilanjana; Basu, Sayon; Nagarajan, P; Majumdar, Subeer S


    Microinjection of foreign DNA in male pronucleus by in-vitro embryo manipulation is difficult but remains the method of choice for generating transgenic animals. Other procedures, including retroviral and embryonic stem cell mediated transgenesis are equally complicated and have limitations. Although our previously reported technique of testicular transgenesis circumvented several limitations, it involved many steps, including surgery and hemicastration, which carried risk of infection and impotency. We improved this technique further, into a two step non-surgical electroporation procedure, for making transgenic mice. In this approach, transgene was delivered inside both testes by injection and modified parameters of electroporation were used for in-vivo gene integration in germ cells. Using variety of constructs, germ cell integration of the gene and its transmission in progeny was confirmed by PCR, slot blot and immunohistochemical analysis. This improved technique is efficient, requires substantially less time and can be easily adopted by various biomedical researchers.

  2. Immunotherapy: Shifting the Balance of Cell-Mediated Immunity and Suppression in Human Prostate Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Tucker, Jo A.; Jochems, Caroline [Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Gulley, James L. [Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Medical Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Schlom, Jeffrey, E-mail:; Tsang, Kwong Y. [Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)


    Active immunotherapy is dependent on the ability of the immune system to recognize and respond to tumors. Despite overwhelming evidence to support a cell-mediated immune response to prostate cancer, it is insufficient to eradicate the disease. This is likely due to a high level of suppression at the tumor site from a variety of sources, including immunosuppressive cells. Immune cells entering the tumor microenvironment may be inhibited directly by the tumor, stromal cells or other immune cells that have been induced to adopt a suppressive phenotype. The resurgence of interest in immunotherapy following the approval of sipuleucel-T and ipilimumab by the Food and Drug Administration has brought about new strategies for overcoming tumor-mediated suppression and bolstering anti-tumor responses. Improved understanding of the immune response to prostate cancer can lead to new combination therapies, such as the use of vaccine with small molecule and checkpoint inhibitors or other immunotherapies.

  3. Immunotherapy: Shifting the Balance of Cell-Mediated Immunity and Suppression in Human Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Jeffrey Schlom


    Full Text Available Active immunotherapy is dependent on the ability of the immune system to recognize and respond to tumors. Despite overwhelming evidence to support a cell-mediated immune response to prostate cancer, it is insufficient to eradicate the disease. This is likely due to a high level of suppression at the tumor site from a variety of sources, including immunosuppressive cells. Immune cells entering the tumor microenvironment may be inhibited directly by the tumor, stromal cells or other immune cells that have been induced to adopt a suppressive phenotype. The resurgence of interest in immunotherapy following the approval of sipuleucel-T and ipilimumab by the Food and Drug Administration has brought about new strategies for overcoming tumor-mediated suppression and bolstering anti-tumor responses. Improved understanding of the immune response to prostate cancer can lead to new combination therapies, such as the use of vaccine with small molecule and checkpoint inhibitors or other immunotherapies.

  4. Modulations in cell-mediated immunity of Mytilus edulis following the `Sea Empress` oil spill

    Energy Technology Data Exchange (ETDEWEB)

    Dyrynda, E.A.; Dyrynda, P.E.J.; Ratcliffe, N.A. [University of Wales Swansea (United Kingdom). School of Biological Sciences; Law, R.J.; Kelly, C.A.; Graham, K.L. [MAFF Fisheries Lab., Burnham-on-Crouch (United Kingdom); Pipe, R.K. [Plymouth Marine Lab. (United Kingdom)


    The `Sea Empress` oil tanker grounded outside Milford Haven (Wales, UK) in February 1996, spilling {approx} 70,000 tonnes of crude oil and contaminating over 100 km of coastline, causing mass mortalities and strandings of at least 11 mollusc species. Intensive field monitoring commenced after the spill, examining immunity and hydrocarbon levels in the mussel, Mytilus edulis (Mollusca: Bivalvia), a commercially-harvested species which can accumulate contaminants. Comparisons of mussels from oiled and reference sites revealed significant modulations in cell-mediated immunity. Elevations in blood cell (haemocyte) numbers and decreases in superoxide generation and phagocytosis were identified in contaminated animals. The immune response of contaminated mussels gradually improved and generally showed no significant differences compared with clean mussels after 11 weeks. By then, total hydrocarbon content in contaminated mussels had declined by 70-90%, while polycyclic aromatic hydrocarbon content had decreased by over 90%. (author)

  5. IL-10 polymorphism and cell-mediated immune response to Chlamydia trachomatis

    DEFF Research Database (Denmark)

    Öhman, H.; Tiitinen, A; Halttunen, M.


    Chlamydia trachomatis infection induces an inflammatory response that is crucial in resolving acute infection but may also play a key role in the pathogenesis of C trachomatis associated infertility. The immune response is linked to cytokine secretion pattern which is influenced by the host genetic...... background. To study a relationship between interleukin-10 (IL-10) promoter -1082 polymorphism and cell-mediated immune response during C trachomatis infection in vitro, lymphocyte proliferation and cytokine (IL-10, IFN-gamma, TNF-alpha, IL-2, IL-4 and IL-5) secretion were analysed in subjects with different...... IL-10 genotypes. Enhanced IL-10 secretion and reduced antigen-specific lymphocyte proliferative and IFN-gamma responses were found in subjects with IL-10 -1082 GG genotype when compared to those with -1082 AA genotype. CD14+ monocytes were main source of IL-10 indicating that these cells...

  6. Cell-mediated mutagenesis and cell transformation of mammalian cells by chemical carcinogens. [Rats, hamsters

    Energy Technology Data Exchange (ETDEWEB)

    Huberman, E.; Langenbach, R.


    We have developed a cell-mediated mutagenesis assay in which cells with the appropriate markers for mutagenesis are co-cultivated with either lethally irradiated rodent embryonic cells that can metabolize carcinogenic hydrocarbons or with primary rat liver cells that can metabolize chemicals carcinogenic to the liver. During co-cultivation, the reactive metabolites of the procarcinogen appear to be transmitted to the mutable cells and induce mutations in them. Assays of this type make it possible to demonstrate a relationship between carcinogenic potency of the chemicals and their ability to induce mutations in mammalian cells. In addition, by simultaneously comparing the frequencies of transformation and mutation induced in normal diploid hamster cells by benzo(a)pyrene (BP) and one of its metabolites, it is possible to estimate the genetic target size for cell transformation in vitro.

  7. Cell-mediated infection of cervix derived epithelial cells with primary isolates of human immunodeficiency virus. (United States)

    Tan, X; Phillips, D M


    We have previously demonstrated that HIV-infected transformed T-cells or monocytes adhere to monolayers of CD4-negative epithelial cells. Adhesion is soon followed by budding of HIV from infected mononuclear cells onto the surface of epithelial cells. Epithelial cells subsequently take up virus and become productively infected. Based on these findings, we proposed that sexual transmission of HIV may involve cell-mediated infection of intact mucosal epithelia of the urogenital tract. However, it has become increasingly clear that primary cells and HIV strains isolated from patients are more appropriate models for HIV infection than established cell lines and lab strains of virus. In the studies described here, we infected cervix-derived epithelial monolayers with primary monocytes infected with patient isolates of non-syncytial inducing (NSI) macrophage-tropic strains of HIV. Under the culture conditions employed, HIV-infected primary monocytes do not remain adherent to the apical surface of the epithelium, as did HIV-infected transformed cells. Instead, following adherence, the primary cells migrate between epithelial cells. Virus is secreted from a pseudopod as HIV-infected primary monocytes pass between cells of the epithelium. Productive infection of the epithelium was detected by p24 ELISA and PCR Southern blot analysis. Infection can be blocked by sera from HIV-seropositive individuals or by certain sulfated polysaccharides. These findings support the supposition that transmission of HIV may occur via cell-mediated infection of intact epithelia. The observations also hint at the possibility that-HIV-infected monocyte/macrophages in semen or cervical-vaginal secretions could cross intact epithelia by passing between epithelial cells. Blocking studies suggest that it may be possible to inhibit sexual transmission of HIV either by antibodies in genital tract secretions or by a topical formulation containing certain sulfated polysaccharides.

  8. Inhibitory effects of atractylone on mast cell-mediated allergic reactions. (United States)

    Han, Na-Ra; Moon, Phil-Dong; Nam, Sun-Young; Ryu, Ka-Jung; Yoou, Myoung-Schook; Choi, Jung-Hye; Hwang, Sung-Yeoun; Kim, Hyung-Min; Jeong, Hyun-Ja


    This study investigated a salutary effect of atractylone (Atr) which is an active constituent of Pyeongwee-San (KMP6) on mast cell-mediated allergic reactions. Our previous report indicated that KMP6 regulated allergic reactions. Thus, this study sought to determine the potential of Atr in vitro models, compound 48/80-stimulated rat peritoneal mast cells (RPMCs), phorbol 12-myristate 13-acetate (PMA) plus A23187-stimulated human mast cell line (HMC-1) cells, and stem cell factor (SCF)-stimulated RPMCs as well as in vivo models, IgE-mediated passive cutaneous anaphylaxis (PCA), compound 48/80-induced systemic anaphylaxis, and compound 48/80-induced ear swelling. The results showed that Atr inhibited compound 48/80-induced RPMCs degranulation, intracellular calcium level, tryptase release, and histamine release. Atr inhibited the up-regulation of p56(lck) tyrosine kinase activity by compound 48/80. And Atr reduced tryptase and histamine releases from PMA plus A23187-stimulated HMC-1 cells. In addition, Atr decreased histidine decarboxylase activity and expression in the activated HMC-1 cells. Atr inhibited SCF-induced morphological alteration and filamentous actin formation in RPMCs. Atr improved IgE-induced PCA reaction by decreasing the levels of histamine, IgE, interleukin (IL)-4, IL-5, IL-6, vascular endothelial growth factor, and IL-13 in the serum of PCA-induced mice. Furthermore, Atr mitigated compound 48/80-induced systemic anaphylaxis and ear swelling. Taken together, these results of this study indicate that Atr regulates the degranulation of mast cell, proving its potential in the treatment of mast cell-mediated allergic reactions.

  9. STIM1 controls T cell-mediated immune regulation and inflammation in chronic infection. (United States)

    Desvignes, Ludovic; Weidinger, Carl; Shaw, Patrick; Vaeth, Martin; Ribierre, Theo; Liu, Menghan; Fergus, Tawania; Kozhaya, Lina; McVoy, Lauren; Unutmaz, Derya; Ernst, Joel D; Feske, Stefan


    Chronic infections induce a complex immune response that controls pathogen replication, but also causes pathology due to sustained inflammation. Ca2+ influx mediates T cell function and immunity to infection, and patients with inherited mutations in the gene encoding the Ca2+ channel ORAI1 or its activator stromal interaction molecule 1 (STIM1) are immunodeficient and prone to chronic infection by various pathogens, including Mycobacterium tuberculosis (Mtb). Here, we demonstrate that STIM1 is required for T cell-mediated immune regulation during chronic Mtb infection. Compared with WT animals, mice with T cell-specific Stim1 deletion died prematurely during the chronic phase of infection and had increased bacterial burdens and severe pulmonary inflammation, with increased myeloid and lymphoid cell infiltration. Although STIM1-deficient T cells exhibited markedly reduced IFN-γ production during the early phase of Mtb infection, bacterial growth was not immediately exacerbated. During the chronic phase, however, STIM1-deficient T cells displayed enhanced IFN-γ production in response to elevated levels of IL-12 and IL-18. The lack of STIM1 in T cells was associated with impaired activation-induced cell death upon repeated TCR engagement and pulmonary lymphocytosis and hyperinflammation in Mtb-infected mice. Chronically Mtb-infected, STIM1-deficient mice had reduced levels of inducible regulatory T cells (iTregs) due to a T cell-intrinsic requirement for STIM1 in iTreg differentiation and excessive production of IFN-γ and IL-12, which suppress iTreg differentiation and maintenance. Thus, STIM1 controls multiple aspects of T cell-mediated immune regulation to limit injurious inflammation during chronic infection.

  10. Effect of vitamin E levels on the cell-mediated immunity of broilers vaccinated against coccidiosis

    Directory of Open Access Journals (Sweden)

    ICM da Silva


    Full Text Available Studies on the relationships between animal nutrition and immunity have sought reliable methodologies to measure responses. Cell-mediated immune response is similarly studied in humans. The cutaneous basophil hypersensitivity test (CBH is one of the methods to measure that response and consists in the infiltration of inflammatory cells, particularly of lymphocytes and basophils, as result of the application of substances capable of inducing cell proliferation in determined sites, such as wings, wattle, and interdigital space in birds. CBH is considered a simple and fast method and can be applied in birds of different ages. In immunocompetence studies with poultry, phytohemagglutinin-P (PHA-P is a commonly used substance, despite the variability of the response related to the method of application (intradermal injection and the antigens used. In the present experiment, PHA-P was used to observe the cell-mediated immune response of 216 chicks fed three dietary levels of vitamin E from 1 to 36 days of age. All birds were immunologically challenged by vaccination against coccidiosis at three days of age and against Newcastle Disease (NCD at 14 and 30 days of age. At 36 days of age, birds were submitted to the CBH test according to the methodology of Corrier & DeLoach (1990. Birds fed 65mg/kg of vitamin E presented lasting cell reaction (p<0.08, which indicates that this vitamin E level improved cell immune response of birds due to its antioxidant and immunomodulating properties. The use of this vitamin E level can be considered by nutritionists under practical conditions, aiming to improve broiler immunity.

  11. A convergent and essential interneuron pathway for Mauthner-cell-mediated escapes. (United States)

    Lacoste, Alix M B; Schoppik, David; Robson, Drew N; Haesemeyer, Martin; Portugues, Ruben; Li, Jennifer M; Randlett, Owen; Wee, Caroline L; Engert, Florian; Schier, Alexander F


    The Mauthner cell (M-cell) is a command-like neuron in teleost fish whose firing in response to aversive stimuli is correlated with short-latency escapes [1-3]. M-cells have been proposed as evolutionary ancestors of startle response neurons of the mammalian reticular formation [4], and studies of this circuit have uncovered important principles in neurobiology that generalize to more complex vertebrate models [3]. The main excitatory input was thought to originate from multisensory afferents synapsing directly onto the M-cell dendrites [3]. Here, we describe an additional, convergent pathway that is essential for the M-cell-mediated startle behavior in larval zebrafish. It is composed of excitatory interneurons called spiral fiber neurons, which project to the M-cell axon hillock. By in vivo calcium imaging, we found that spiral fiber neurons are active in response to aversive stimuli capable of eliciting escapes. Like M-cell ablations, bilateral ablations of spiral fiber neurons largely eliminate short-latency escapes. Unilateral spiral fiber neuron ablations shift the directionality of escapes and indicate that spiral fiber neurons excite the M-cell in a lateralized manner. Their optogenetic activation increases the probability of short-latency escapes, supporting the notion that spiral fiber neurons help activate M-cell-mediated startle behavior. These results reveal that spiral fiber neurons are essential for the function of the M-cell in response to sensory cues and suggest that convergent excitatory inputs that differ in their input location and timing ensure reliable activation of the M-cell, a feedforward excitatory motif that may extend to other neural circuits.

  12. Are diamond nanoparticles cytotoxic? (United States)

    Schrand, Amanda M; Huang, Houjin; Carlson, Cataleya; Schlager, John J; Omacr Sawa, Eiji; Hussain, Saber M; Dai, Liming


    Finely divided carbon particles, including charcoal, lampblack, and diamond particles, have been used for ornamental and official tattoos since ancient times. With the recent development in nanoscience and nanotechnology, carbon-based nanomaterials (e.g., fullerenes, nanotubes, nanodiamonds) attract a great deal of interest. Owing to their low chemical reactivity and unique physical properties, nanodiamonds could be useful in a variety of biological applications such as carriers for drugs, genes, or proteins; novel imaging techniques; coatings for implantable materials; and biosensors and biomedical nanorobots. Therefore, it is essential to ascertain the possible hazards of nanodiamonds to humans and other biological systems. We have, for the first time, assessed the cytotoxicity of nanodiamonds ranging in size from 2 to 10 nm. Assays of cell viability such as mitochondrial function (MTT) and luminescent ATP production showed that nanodiamonds were not toxic to a variety of cell types. Furthermore, nanodiamonds did not produce significant reactive oxygen species. Cells can grow on nanodiamond-coated substrates without morphological changes compared to controls. These results suggest that nanodiamonds could be ideal for many biological applications in a diverse range of cell types.

  13. Cytomegalovirus-Infected Cells Resist T Cell Mediated Killing in an HLA-Recognition Independent Manner. (United States)

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


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

  14. Cell-mediated immune responses differentiate infections with Brucella suis from Yersinia enterocolitica serotype O : 9 in pigs

    DEFF Research Database (Denmark)

    Riber, Ulla; Jungersen, Gregers


    serological Brucellosis reactions. While 36 of the 39 FPSR pigs were also FPSR in a second test, none of the pigs were test positive in whole blood IFN-gamma assay or Brucellergene OCB skin test. In conclusion, use of IFN-gamma assay and skin test as measurements of cell-mediated immune responses to non...

  15. Novel antigens for detection of cell mediated immune responses to Mycobacterium avium subsp. paratuberculosis infection in cattle

    DEFF Research Database (Denmark)

    Mikkelsen, Heidi; Aagaard, Claus; Nielsen, Søren Saxmose;


    Paratuberculosis is a chronic infection of the intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Early stage MAP infection can be detected by measuring specific cell mediated immune responses, using the whole blood interferon-γ (IFN-γ) assay. Available IFN-γ assa...

  16. Flow cytometric assessment of chicken T cell-mediated immune responses after Newcastle disease virus vaccination and challenge

    DEFF Research Database (Denmark)

    Dalgaard, T. S.; Norup, L. R.; Pedersen, A.R.;


    The objective of this study was to use flow cytometry to assess chicken T cell-mediated immune responses. In this study two inbred genetic chicken lines (L130 and L133) were subjected to two times vaccination against Newcastle disease (ND) and a subsequent challenge by ND virus (NDV) infection...

  17. Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy

    DEFF Research Database (Denmark)

    Da Roit, F.; Engelberts, P. J.; Taylor, R. P.


    -treatment with ibrutinib did not inhibit complement activation or complement-mediated lysis. In contrast, ibrutinib strongly inhibited all cell-mediated mechanisms induced by anti-CD20 antibodies rituximab, ofatumumab or obinutuzumab, either in purified systems or whole blood assays. Activation of natural killer cells...

  18. A 17 year experience in perioperative anaphylaxis 1998-2015: harmonising optimal detection of mast cell mediator release.


    Egner, W.; Sargur, R.; Shrimpton, A.; York, M.; Green, K


    BACKGROUND: Sheffield NARCOS (National Adverse Reactions Advisory Service) investigates suspected perioperative anaesthetic reactions using serial tryptase, urinary methylhistamine and clinical information. Further recommendations for additional allergy clinic assessment are provided. OBJECTIVE: To establish a robustly measurable protocol for identifying mast cell mediator (MMR) release in this cohort. To compare these thresholds with previous suggested thresholds and algorithms. METHOD: A re...

  19. Chlamydia trachomatis and chlamydial heat shock protein 60-specific antibody and cell-mediated responses predict tubal factor infertility

    DEFF Research Database (Denmark)

    Tiitinen, A.; Surcel, H.-M.; Halttunen, M.


    BACKGROUND: To evaluate the role of Chlamydia trachomatis-induced humoral and cell-mediated immune (CMI) responses in predicting tubal factor infertility (TFI). METHODS: Blood samples were taken from 88 women with TFI and 163 control women. C. trachomatis and chlamydial heat shock protein 60 (CHSP...

  20. Sialylation of IgG Fc domain impairs complement-dependent cytotoxicity. (United States)

    Quast, Isaak; Keller, Christian W; Maurer, Michael A; Giddens, John P; Tackenberg, Björn; Wang, Lai-Xi; Münz, Christian; Nimmerjahn, Falk; Dalakas, Marinos C; Lünemann, Jan D


    IgG molecules exert both pro- and antiinflammatory effector functions based on the composition of the fragment crystallizable (Fc) domain glycan. Sialylated IgG Fc domains have antiinflammatory properties that are attributed to their ability to increase the activation threshold of innate effector cells to immune complexes by stimulating the upregulation of the inhibitory Fcγ receptor IIB (FcγRIIB). Here, we report that IgG Fc sialylation of human monoclonal IgG1 molecules impairs their efficacy to induce complement-mediated cytotoxicity (CDC). Fc sialylation of a CD20-targeting antibody had no impact on antibody-dependent cellular cytotoxicity and did not change the affinity of the antibody for activating Fcγ receptors. In contrast, the presence of sialic acid abrogated the increased binding of C1q to Fc-galactosylated IgG1 and resulted in decreased levels of C3b deposition on the cell surface. Similar to monoclonal antibodies, sialic acid inhibited the increased C1q binding to galactosylated Fc fragments in human polyclonal IgG. In sera derived from patients with chronic inflammatory demyelinating polyneuropathy, an autoimmune disease of the peripheral nervous system in which humoral immune responses mediate tissue damage, induction of IgG Fc sialylation was associated with clinical disease remission. Thus, impairment of CDC represents an FcγR-independent mechanism by which Fc-sialylated glycovariants might limit proinflammatory IgG effector functions.

  1. 5-Azacytidine treatment sensitizes tumor cells to T-cell mediated cytotoxicity and modulates NK cells in patients with myeloid malignancies

    DEFF Research Database (Denmark)

    Gang, A O; Frøsig, T M; Brimnes, M K


    Treatment with the demethylating agent 5-Azacytidine leads to prolonged survival for patients with myelodysplastic syndrome, and the demethylation induces upregulation of cancer-testis antigens. Cancer-testis antigens are well-known targets for immune recognition in cancer, and the immune system ...

  2. Cell-mediated immune suppression effect of rocket kerosene through dermal exposure in mice

    Directory of Open Access Journals (Sweden)

    Bing-xin XU


    Full Text Available Objective To study the effect of cell-mediated immune suppression effect of rocket kerosene (RK through dermal application in mice. Methods Skin delayed type hypersensitivity (DTH was used to observe the relation of the RK amount the skin exposed and the cellular immune inhibitory function. Different amount of the undiluted fuel was smeared directly onto the dorsal skin of mice. Mice in negative and positive control groups were treated with acetone. After the last exposure, all the mice except those in negative control group were allergized by evenly smearing with 1% dinitrofluorobenzene (DNFB solution on their dorsum. Five days after allergy, 1% DNFB solution was smeared onto right ear of all mice to stimulate the allergic reaction. Twenty-four hours after attack, the auricle swelling, spleen index and thymus index in corresponding mice were determined. In the first series of experiments, different dosages of RK were applied once, and the ICR mice were randomly divided into negative control group, positive control group and experimental group (0.5ml/kg.BW×1, 1ml/kg.BW×1 and 2ml/kg.BW×1 group. In the second series of experiments, the certain and same dosage of RK was applied for different times, and the ICR mice were randomly divided into negative control group, positive control group and experimental group (0.5ml/kg.BW×1, 0.5mL/kg.BW×2, 0.5ml/kg.BW×3, 0.5ml/kg.BW×4 and 0.5mL/kg.BW×5 group. In the third series of experiments, the different dosages of RK were applied more than once, and the ICR mice were randomly divided into negative control group, positive control group and experimental group (0.5ml/kg.BW×5, 1ml/kg.BW×5 and 2ml/kg.BW×5 group. Lymphocyte proliferation experiment in vitrowas conducted to observe the persistent time of the cell-mediated immune suppression in mice by RK dermal exposure. The lymphocyte proliferation induced by concanavalin A (Con A was analyzed by MTT assay, and T lymphocyte subsets (CD3+, CD4+ and CD

  3. Stimulatory effect of Eucalyptus essential oil on innate cell-mediated immune response

    Directory of Open Access Journals (Sweden)

    Rasi Guido


    Full Text Available Abstract Background Besides few data concerning the antiseptic properties against a range of microbial agents and the anti-inflammatory potential both in vitro and in vivo, little is known about the influence of Eucalyptus oil (EO extract on the monocytic/macrophagic system, one of the primary cellular effectors of the immune response against pathogen attacks. The activities of this natural extract have mainly been recognized through clinical experience, but there have been relatively little scientific studies on its biological actions. Here we investigated whether EO extract is able to affect the phagocytic ability of human monocyte derived macrophages (MDMs in vitro and of rat peripheral blood monocytes/granulocytes in vivo in absence or in presence of immuno-suppression induced by the chemotherapeutic agent 5-fluorouracil (5-FU. Methods Morphological activation of human MDMs was analysed by scanning electron microscopy. Phagocytic activity was tested: i in vitro in EO treated and untreated MDMs, by confocal microscopy after fluorescent beads administration; ii in vivo in monocytes/granulocytes from peripheral blood of immuno-competent or 5-FU immuno-suppressed rats, after EO oral administration, by flow cytometry using fluorescein-labelled E. coli. Cytokine release by MDMs was determined using the BD Cytometric Bead Array human Th1/Th2 cytokine kit. Results EO is able to induce activation of MDMs, dramatically stimulating their phagocytic response. EO-stimulated internalization is coupled to low release of pro-inflammatory cytokines and requires integrity of the microtubule network, suggesting that EO may act by means of complement receptor-mediated phagocytosis. Implementation of innate cell-mediated immune response was also observed in vivo after EO administration, mainly involving the peripheral blood monocytes/granulocytes. The 5-FU/EO combined treatment inhibited the 5-FU induced myelotoxicity and raised the phagocytic activity of the

  4. The Ebola Interferon Inhibiting Domains Attenuate and Dysregulate Cell-Mediated Immune Responses (United States)

    Meyer, Michelle; Koup, Richard A.; Bukreyev, Alexander


    Ebola virus (EBOV) infections are characterized by deficient T-lymphocyte responses, T-lymphocyte apoptosis and lymphopenia. We previously showed that disabling of interferon-inhibiting domains (IIDs) in the VP24 and VP35 proteins effectively unblocks maturation of dendritic cells (DCs) and increases the secretion of cytokines and chemokines. Here, we investigated the role of IIDs in adaptive and innate cell-mediated responses using recombinant viruses carrying point mutations, which disabled IIDs in VP24 (EBOV/VP24m), VP35 (EBOV/VP35m) or both (EBOV/VP35m/VP24m). Peripheral blood mononuclear cells (PBMCs) from cytomegalovirus (CMV)-seropositive donors were inoculated with the panel of viruses and stimulated with CMV pp65 peptides. Disabling of the VP35 IID resulted in increased proliferation and higher percentages of CD4+ T cells secreting IFNγ and/or TNFα. To address the role of aberrant DC maturation in the IID-mediated suppression of T cell responses, CMV-stimulated DCs were infected with the panel of viruses and co-cultured with autologous T-lymphocytes. Infection with EBOV/VP35m infection resulted in a significant increase, as compared to wt EBOV, in proliferating CD4+ cells secreting IFNγ, TNFα and IL-2. Experiments with expanded CMV-specific T cells demonstrated their increased activation following co-cultivation with CMV-pulsed DCs pre-infected with EBOV/VP24m, EBOV/VP35m and EBOV/VP35m/VP24m, as compared to wt EBOV. Both IIDs were found to block phosphorylation of TCR complex-associated adaptors and downstream signaling molecules. Next, we examined the effects of IIDs on the function of B cells in infected PBMC. Infection with EBOV/VP35m and EBOV/VP35m/VP24m resulted in significant increases in the percentages of phenotypically distinct B-cell subsets and plasma cells, as compared to wt EBOV, suggesting inhibition of B cell function and differentiation by VP35 IID. Finally, infection with EBOV/VP35m increased activation of NK cells, as compared to wt

  5. Mutation of neutralizing/antibody-dependent enhancing epitope on spike protein and 7b gene of feline infectious peritonitis virus: influences of viral replication in monocytes/macrophages and virulence in cats. (United States)

    Takano, Tomomi; Tomiyama, Yoshika; Katoh, Yasuichiroh; Nakamura, Michiyo; Satoh, Ryoichi; Hohdatsu, Tsutomu


    We previously prepared neutralizing monoclonal antibody (MAb)-resistant (mar) mutant viruses using a laboratory strain feline infectious peritonitis virus (FIPV) 79-1146 (Kida et al., 1999). Mar mutant viruses are mutated several amino acids of the neutralizing epitope of Spike protein, compared with the parent strain, FIPV 79-1146. We clarified that MAb used to prepare mar mutant viruses also lost its activity to enhance homologous mar mutant viruses, strongly suggesting that neutralizing and antibody-dependent enhancing epitopes are present in the same region in the strain FIPV 79-1146. We also discovered that amino acid mutation in the neutralizing epitope reduced viral replication in monocytes/macrophages. We also demonstrated that the mutation or deletion of two nucleotides in 7b gene abrogate the virulence of strain FIPV 79-1146.

  6. Analysis of the mechanism of antibody-dependent enhancement of feline infectious peritonitis virus infection: aminopeptidase N is not important and a process of acidification of the endosome is necessary. (United States)

    Takano, Tomomi; Katada, Yukari; Moritoh, Saiko; Ogasawara, Mika; Satoh, Kumi; Satoh, Ryoichi; Tanabe, Maki; Hohdatsu, Tsutomu


    Infection of the monocyte/macrophage lineage with feline infectious peritonitis virus (FIPV) is enhanced in the presence of anti-FIPV antibodies (antibody-dependent enhancement or ADE). We investigated the following unclear points concerning ADE of FIPV infection: (i) involvement of the virus receptor, feline aminopeptidase N (fAPN), in ADE activity in FIPV infection; (ii) necessity of acidification of the endosome in cellular invasion of FIPV. Virus receptor-blocking experiments using anti-fAPN antibodies at 4 or 37 degrees C and experiments using fAPN-negative U937 cells revealed that fAPN is not involved in ADE of FIPV infection. Experiments using lysosomotropic agents clarified that acidification of the endosome is necessary for cellular invasion by FIPV, regardless of the presence or absence of antibodies. These findings may be very important for understanding the mechanism of ADE of FIPV infection.

  7. Advances in antibody-dependent enhancement for dengue virus%登革病毒抗体依赖性感染增强作用的研究进展

    Institute of Scientific and Technical Information of China (English)

    宋克玉; 李晓峰; 江振友; 秦成峰


    Antibody-dependent enhancement ( ADE ) present in the infection of dengue virus ( DENV ) has huge effect on the effect of treatment. ADE of virus replication is believed to occur when heterotypic, nonneutralizing antibody present in the host from a previous DENV infection binds to the virus via binding to the Fey receptors ( Fc-yR ) during a subsequent heterotypic infection but is unable to neutralize the virus. The overall result is an increase in virus replication and the level of viremia, which is associated with an increase in disease severity. Studies on the immune response of ADE can contribute to the derelopment of effective vaccines and drugs. In this review, the resent advances of ADE in dengue virus are discussed.%抗体依赖性感染增强作用(antibody-dependent enhancement,ADE)在登革病毒的致病机制中发挥重要作用.在存在亚中和浓度抗体的条件下,登革病毒可通过Fc受体或补体受体等途径进入靶细胞,进而导致ADE的发生.ADE的发生受病毒和宿主一系列复杂因素的调节,直接影响患者的临床表现和病情进展,对其作用机制的研究有助于加深对登革出血热发病机制的认识,对登革热疫苗以及治疗性抗体的研发具有重要意义.本文综述了登革病毒ADE的研究进展.

  8. Influence of histone deacetylase inhibitors and DNA-methyltransferase inhibitors on the NK cell-mediated lysis of pediatric B-lineage leukemia.

    Directory of Open Access Journals (Sweden)

    Matthias Manuel Pfeiffer


    Full Text Available Epigenetic drugs like histone deacetylase inhibitors and DNA methyltransferase inhibitors have been shown to be effective against a variety of tumor entities. Among different molecular anticancer activities of epigenetic active substances, up-regulation of NK cell ligands was described to contribute to an enhanced NK cell-mediated killing of tumor cell lines. So far, no data is available on this effect in childhood acute lymphoblastic leukemia. We investigated the effect of two HDACi (vorinostat, VPA and two DNMTi (azacytidine, decitabine on the viability, expression of NK ligands and NK-susceptibility of the pre-B-cell-ALL cell line MHH-CALL-4. Whereas vorinostat, azacytidine and decitabine directly reduced viability of the cell line, VPA had no direct cytotoxic effect. NKG2D ligands were expressed only at very low levels and not affected by epigenetic treatment. Higher expression was found for the DNAM1 ligands with significant up regulation of CD112 after treatment with VPA (p=0.02. No significant increase in lysis mediated by resting NK cells could be observed, whereas incubation of target cells with decitabine resulted in a significant increase in lysis mediated by IL-2 activated NK cells (p=0.0051, p=0.06 for azacytidine. Vorinostat and VPA could increase the lysis by expanded NK cells which was statistically not significant due to high inter-individual variability. Furthermore, HDACi but not DNMTi reduced the NK mediated lysis of MHH-CALL-4 after incubation of effector cells. In conclusion, there is a synergistic effect between epigenetic drugs and NK cells against MHH-CALL-4 which is not as strong as in other tumor entities. In situations where NK mediated control of leukemia is assumed or wanted, a sophisticated combination of single epigenetic drugs and ex vivo expanded NK cells is needed to maximize the synergistic effect of both treatment strategies and DNMTIs may be preferred based on the direct inhibitory effect of HDACi on NK cell

  9. Hepatic non-parenchymal cells and extracellular matrix participate in oval cell-mediated liver regeneration

    Institute of Scientific and Technical Information of China (English)

    Wei Zhang; Xiao-Ping Chen; Wan-Guang Zhang; Feng Zhang; Shuai Xiang; Han-Hua Dong; Lei Zhang


    AIM: To elucidate the interaction between nonparenchymal cells, extracellular matrix and oval cells during the restituting process of liver injury induced by partial hepatectomy (PH). METHODS: We examined the localization of oval cells, non-parenchymal cells, and the extracellular matrix components using immunohistochemical and double immunofluorescent analysis during the proliferation and differentiation of oval cells in N-2-acetylaminofluorene (2-AAF)/PH rat model. RESULTS: By day 2 after PH, small oval cells began to proliferate around the portal area. Most of stellate cells and laminin were present along the hepatic sinusoids in the periportal area. Kupffer cells and fibronectin markedly increased in the whole hepatic lobule. From day 4 to 9, oval cells spread further into hepatic parenchyma, closely associated with stellate cells, fibronectin and laminin. Kupffer cells admixed with oval cells by day 6 and then decreased in the periportal zone. From day 12 to 15, most of hepatic stellate cells (HSCs), laminin and fibronectin located around the small hepatocyte nodus, and minority of them appeared in the nodus. Kupffer cells were mainly limited in the pericentral sinusoids. After day 18, the normal liver lobule structures began to recover.CONCLUSION: Local hepatic microenvironment may participate in the oval cell-mediated liver regeneration through the cell-cell and cell-matrix interactions.

  10. Neo-lymphoid aggregates in the adult liver can initiate potent cell-mediated immunity.

    Directory of Open Access Journals (Sweden)

    Melanie Greter


    Full Text Available Subcutaneous immunization delivers antigen (Ag to local Ag-presenting cells that subsequently migrate into draining lymph nodes (LNs. There, they initiate the activation and expansion of lymphocytes specific for their cognate Ag. In mammals, the structural environment of secondary lymphoid tissues (SLTs is considered essential for the initiation of adaptive immunity. Nevertheless, cold-blooded vertebrates can initiate potent systemic immune responses even though they lack conventional SLTs. The emergence of lymph nodes provided mammals with drastically improved affinity maturation of B cells. Here, we combine the use of different strains of alymphoplastic mice and T cell migration mutants with an experimental paradigm in which the site of Ag delivery is distant from the site of priming and inflammation. We demonstrate that in mammals, SLTs serve primarily B cell priming and affinity maturation, whereas the induction of T cell-driven immune responses can occur outside of SLTs. We found that mice lacking conventional SLTs generate productive systemic CD4- as well as CD8-mediated responses, even under conditions in which draining LNs are considered compulsory for the initiation of adaptive immunity. We describe an alternative pathway for the induction of cell-mediated immunity (CMI, in which Ag-presenting cells sample Ag and migrate into the liver where they induce neo-lymphoid aggregates. These structures are insufficient to support antibody affinity maturation and class switching, but provide a novel surrogate environment for the initiation of CMI.

  11. TRESK channel as a potential target to treat T-cell mediated immune dysfunction

    Energy Technology Data Exchange (ETDEWEB)

    Han, Jaehee [Medical Research Center for Neural Dysfunction, Department of Physiology, Institute of Health Sciences, Gyeongsang National University, School of Medicine, Jinju 660-751 (Korea, Republic of); Kang, Dawon, E-mail: [Medical Research Center for Neural Dysfunction, Department of Physiology, Institute of Health Sciences, Gyeongsang National University, School of Medicine, Jinju 660-751 (Korea, Republic of)


    In this review, we propose that TRESK background K{sup +} channel could serve as a potential therapeutic target for T-cell mediated immune dysfunction. TRESK has many immune function-related properties. TRESK is abundantly expressed in the thymus, the spleen, and human leukemic T-lymphocytes. TRESK is highly activated by Ca{sup 2+}, calcineurin, acetylcholine, and histamine which induce hypertrophy, whereas TRESK is inhibited by immunosuppressants, such as cyclosporin A and FK506. Cyclosporine A and FK506 target the binding site of nuclear factor of activated T-cells (NFAT) to inhibit calcineurin. Interestingly, TRESK possesses an NFAT-like docking site that is present at its intracellular loop. Calcineurin has been found to interact with TRESK via specific NFAT-like docking site. When the T-cell is activated, calcineurin can bind to the NFAT-docking site of TRESK. The activation of both TRESK and NFAT via Ca{sup 2+}-calcineurin-NFAT/TRESK pathway could modulate the transcription of new genes in addition to regulating several aspects of T-cell function.

  12. Epistasis between microRNAs 155 and 146a during T cell-mediated antitumor immunity (United States)

    Huffaker, Thomas B.; Hu, Ruozhen; Runtsch, Marah C.; Bake, Erin; Chen, Xinjian; Zhao, Jimmy; Round, June L.; Baltimore, David; O’Connell, Ryan M.


    SUMMARY An increased understanding of antitumor immunity is necessary to improve cell-based immunotherapies against human cancers. Here, we investigated the roles of two immune system-expressed microRNAs (miRNAs), miR-155 and miR-146a, in the regulation of antitumor immune responses. Our results indicate that miR-155 promotes and miR-146a inhibits IFNγ responses by T cells and reduced solid tumor growth in vivo. Using a novel double knockout (DKO) mouse strain deficient in both miR-155 and miR-146a, we have also identified an epistatic relationship between these two miRNAs. DKO mice had defective T cell responses and tumor growth phenotypes similar to miR-155−/− mice. Further analysis of the T cell compartment revealed that miR-155 modulates IFNγ expression through a mechanism involving repression of Ship1. Our work reveals critical roles for miRNAs in the reciprocal regulation of CD4+ and CD8+ T cell-mediated antitumor immunity, and demonstrates the dominant nature of miR-155 during its promotion of immune responses. PMID:23200854

  13. Epistasis between MicroRNAs 155 and 146a during T Cell-Mediated Antitumor Immunity

    Directory of Open Access Journals (Sweden)

    Thomas B. Huffaker


    Full Text Available An increased understanding of antitumor immunity is necessary for improving cell-based immunotherapies against human cancers. Here, we investigated the roles of two immune system-expressed microRNAs (miRNAs, miR-155 and miR-146a, in the regulation of antitumor immune responses. Our results indicate that miR-155 promotes and miR-146a inhibits interferon γ (IFNγ responses by T cells and reduces solid tumor growth in vivo. Using a double-knockout (DKO mouse strain deficient in both miR-155 and miR-146a, we have also identified an epistatic relationship between these two miRNAs. DKO mice had defective T cell responses and tumor growth phenotypes similar to miR-155−/− mice. Further analysis of the T cell compartment revealed that miR-155 modulates IFNγ expression through a mechanism involving repression of Ship1. Our work reveals critical roles for miRNAs in the reciprocal regulation of CD4+ and CD8+ T cell-mediated antitumor immunity and demonstrates the dominant nature of miR-155 during its promotion of immune responses.

  14. Loss of STAT3 in Lymphoma Relaxes NK Cell-Mediated Tumor Surveillance

    Energy Technology Data Exchange (ETDEWEB)

    Putz, Eva Maria [Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Veterinaerplatz 1, Vienna 1210 (Austria); Hoelzl, Maria Agnes [Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna (MUV), Waehringer Strasse 13A, Vienna 1090 (Austria); Baeck, Julia [Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Veterinaerplatz 1, Vienna 1210 (Austria); Bago-Horvath, Zsuzsanna [Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Veterinaerplatz 1, Vienna 1210 (Austria); Clinical Institute of Pathology, Medical University of Vienna (MUV), Waehringer Gürtel 18-20, Vienna 1090 (Austria); Schuster, Christian [Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna (MUV), Waehringer Strasse 13A, Vienna 1090 (Austria); Reichholf, Brian [Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Veterinaerplatz 1, Vienna 1210 (Austria); Kern, Daniela; Aberger, Fritz [Department of Molecular Biology, University of Salzburg, Hellbrunnerstrasse 34, Salzburg 5020 (Austria); Sexl, Veronika; Hoelbl-Kovacic, Andrea, E-mail: [Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Veterinaerplatz 1, Vienna 1210 (Austria)


    The transcription factors and proto-oncogenes STAT3 and STAT5 are highly activated in hematological malignancies and represent promising therapeutic targets. Whereas the importance of STAT5 as tumor promoter is beyond doubt, the role of STAT3 in hematological cancers is less well understood. Both, enforced as well as attenuated expression of STAT3 were reported in hematopoietic malignancies. Recent evidence implicates STAT3 as key player for tumor immune surveillance as it both mediates the production of and response to inflammatory cytokines. Here we investigated the effects of STAT3 deletion in a BCR/ABL-induced lymphoma model, which is tightly controlled by natural killer (NK) cells in vivo. Upon STAT3 deletion tumor growth is significantly enhanced when compared to STAT3-expressing controls. The increased tumor size upon loss of STAT3 was accompanied by reduced NK cell infiltration and decreased levels of the cytokine IFN-γ and the chemokine RANTES. Upon transplantation into NK cell-deficient mice differences in lymphoma size were abolished indicating that STAT3 expression in the tumor cells controls NK cell-dependent tumor surveillance. Our findings indicate that STAT3 inhibition in lymphoma patients will impair NK cell-mediated tumor surveillance, which needs to be taken into account when testing STAT3 inhibitors in preclinical or clinical trials.

  15. Epstein-Barr virus reactivation associated with diminished cell-mediated immunity in antarctic expeditioners (United States)

    Mehta, S. K.; Pierson, D. L.; Cooley, H.; Dubow, R.; Lugg, D.


    Epstein-Barr virus (EBV) reactivation and cell-mediated immune (CMI) responses were followed in 16 Antarctic expeditioners during winter-over isolation at 2 Australian National Antarctic Research Expedition stations. Delayed-type hypersensitivity (DTH) skin testing was used as an indicator of the CMI response, that was evaluated 2 times before winter isolation and 3 times during isolation. At all 5 evaluation times, 8 or more of the 16 subjects had a diminished CMI response. Diminished DTH was observed on every test occasion in 4/16 subjects; only 2/16 subjects exhibited normal DTH responses for all 5 tests. A polymerase chain reaction (PCR) assay was used to detect EBV DNA in saliva specimens collected before, during, and after the winter isolation. EBV DNA was present in 17% (111/642) of the saliva specimens; all 16 subjects shed EBV in their saliva on at least 1 occasion. The probability of EBV shedding increased (P = 0.013) from 6% before or after winter isolation to 13% during the winter period. EBV appeared in saliva during the winter isolation more frequently (P viruses.

  16. Micro-RNA dysregulation in multiple sclerosis favours pro-inflammatory T-cell-mediated autoimmunity. (United States)

    Guerau-de-Arellano, Mireia; Smith, Kristen M; Godlewski, Jakub; Liu, Yue; Winger, Ryan; Lawler, Sean E; Whitacre, Caroline C; Racke, Michael K; Lovett-Racke, Amy E


    Pro-inflammatory T cells mediate autoimmune demyelination in multiple sclerosis. However, the factors driving their development and multiple sclerosis susceptibility are incompletely understood. We investigated how micro-RNAs, newly described as post-transcriptional regulators of gene expression, contribute to pathogenic T-cell differentiation in multiple sclerosis. miR-128 and miR-27b were increased in naïve and miR-340 in memory CD4(+) T cells from patients with multiple sclerosis, inhibiting Th2 cell development and favouring pro-inflammatory Th1 responses. These effects were mediated by direct suppression of B lymphoma Mo-MLV insertion region 1 homolog (BMI1) and interleukin-4 (IL4) expression, resulting in decreased GATA3 levels, and a Th2 to Th1 cytokine shift. Gain-of-function experiments with these micro-RNAs enhanced the encephalitogenic potential of myelin-specific T cells in experimental autoimmune encephalomyelitis. In addition, treatment of multiple sclerosis patient T cells with oligonucleotide micro-RNA inhibitors led to the restoration of Th2 responses. These data illustrate the biological significance and therapeutic potential of these micro-RNAs in regulating T-cell phenotypes in multiple sclerosis.

  17. Activation by SLAM Family Receptors Contributes to NK Cell Mediated "Missing-Self" Recognition. (United States)

    Alari-Pahissa, Elisenda; Grandclément, Camille; Jeevan-Raj, Beena; Leclercq, Georges; Veillette, André; Held, Werner


    Natural Killer (NK) cells attack normal hematopoietic cells that do not express inhibitory MHC class I (MHC-I) molecules, but the ligands that activate NK cells remain incompletely defined. Here we show that the expression of the Signaling Lymphocyte Activation Molecule (SLAM) family members CD48 and Ly9 (CD229) by MHC-I-deficient tumor cells significantly contributes to NK cell activation. When NK cells develop in the presence of T cells or B cells that lack inhibitory MHC-I but express activating CD48 and Ly9 ligands, the NK cells' ability to respond to MHC-I-deficient tumor cells is severely compromised. In this situation, NK cells express normal levels of the corresponding activation receptors 2B4 (CD244) and Ly9 but these receptors are non-functional. This provides a partial explanation for the tolerance of NK cells to MHC-I-deficient cells in vivo. Activating signaling via 2B4 is restored when MHC-I-deficient T cells are removed, indicating that interactions with MHC-I-deficient T cells dominantly, but not permanently, impair the function of the 2B4 NK cell activation receptor. These data identify an important role of SLAM family receptors for NK cell mediated "missing-self" reactivity and suggest that NK cell tolerance in MHC-I mosaic mice is in part explained by an acquired dysfunction of SLAM family receptors.

  18. Cell-mediated immune response of synovial fluid lymphocytes to ureaplasma antigen in Reiter's syndrome

    Directory of Open Access Journals (Sweden)

    Pavlica Ljiljana


    Full Text Available INTRODUCTION Reiter's syndrome (RS is an seronegative arthritis that occurs after urogenital or enteric infection which in addition with occular and/or mucocutaneous manifestations presents complete form of disease. According to previous understanding arthritis in the RS is the reactive one, which means that it is impossible to isolate its causative agent. However, there are the more and more authors suggesting that arthritis in the urogenital form of disease is caused by the infective agent in the affected joint. This suggestion is based on numerous studies on the presence of Chlmaydia trachomatis and Ureaplasma urealyticum in the inflamed joint by using new diagnostic methods in molecular biology published in the recent literature [1-3]. Besides, numerous studies of the humoral and cell-mediated immune response to "triggering" bacteria in the affected joint have supported previous suggestions [4-7]. Aim of the study was to determine whether synovial fluid T-cells specifically recognize the "triggering" bacteria presumably responsible for the Reiter's syndrome. METHOD The 3H-thymidine uptake procedure for measuring lymphocyte responses was applied to lymphocytes derived concurrently from synovial fluid (SF and from peripheral blood (PB [8]. Ureaplasma antigen and mitogen PHA stimulated lymphocytes in 24 RS patients (24 PB samples, 9 SF samples and the results were compared with those found in 10 patients with rheumatoid arthritis (RA (10 PB samples, 5 SF samples. Preparation of ureaplasma antigen. Ureaplasma was cultured on cell-free liquid medium [9]. Sample of 8 ml was heat-inactivated for 15 minutes at 601C and permanently stirred with magnetic mixer. The sample was centrifuged at 2000 x g for 40 minutes and than deposits carefully carried to other sterile glass tubes (Corex and recentrifuged at 9000 x g for 30 minutes. The deposit was washed 3 times in sterile 0.9% NaCl, and final sediment was resuspended in 1.2 ml sterile 0.9% Na

  19. Human mast cell mediator cocktail excites neurons in human and guinea-pig enteric nervous system. (United States)

    Schemann, M; Michel, K; Ceregrzyn, M; Zeller, F; Seidl, S; Bischoff, S C


    Neuroimmune interactions are an integral part of gut physiology and involved in the pathogenesis of inflammatory and functional bowel disorders. Mast cells and their mediators are important conveyors in the communication from the innate enteric immune system to the enteric nervous system (ENS). However, it is not known whether a mediator cocktail released from activated human mast cells affects neural activity in the ENS. We used the Multi-Site Optical Recording Technique to image single cell activity in guinea-pig and human ENS after application of a mast cell mediator cocktail (MCMC) that was released from isolated human intestinal mucosa mast cells stimulated by IgE-receptor cross-linking. Local application of MCMC onto individual ganglia evoked an excitatory response consisting of action potential discharge. This excitatory response occurred in 31%, 38% or 11% neurons of guinea-pig submucous plexus, human submucous plexus, or guinea-pig myenteric plexus, respectively. Compound action potentials from nerve fibres or fast excitatory synaptic inputs were not affected by MCMC. This study demonstrates immunoneural signalling in the human gut and revealed for the first time that an MCMC released from stimulated human intestinal mast cells induces excitatory actions in the human and guinea-pig ENS.

  20. Paper-based bioactive scaffolds for stem cell-mediated bone tissue engineering. (United States)

    Park, Hyun-Ji; Yu, Seung Jung; Yang, Kisuk; Jin, Yoonhee; Cho, Ann-Na; Kim, Jin; Lee, Bora; Yang, Hee Seok; Im, Sung Gap; Cho, Seung-Woo


    Bioactive, functional scaffolds are required to improve the regenerative potential of stem cells for tissue reconstruction and functional recovery of damaged tissues. Here, we report a paper-based bioactive scaffold platform for stem cell culture and transplantation for bone reconstruction. The paper scaffolds are surface-engineered by an initiated chemical vapor deposition process for serial coating of a water-repellent and cell-adhesive polymer film, which ensures the long-term stability in cell culture medium and induces efficient cell attachment. The prepared paper scaffolds are compatible with general stem cell culture and manipulation techniques. An optimal paper type is found to provide structural, physical, and mechanical cues to enhance the osteogenic differentiation of human adipose-derived stem cells (hADSCs). A bioactive paper scaffold significantly enhances in vivo bone regeneration of hADSCs in a critical-sized calvarial bone defect. Stacking the paper scaffolds with osteogenically differentiated hADSCs and human endothelial cells resulted in vascularized bone formation in vivo. Our study suggests that paper possesses great potential as a bioactive, functional, and cost-effective scaffold platform for stem cell-mediated bone tissue engineering. To the best of our knowledge, this is the first study reporting the feasibility of a paper material for stem cell application to repair tissue defects.

  1. Induction of cell-mediated immunity during early stages of infection with intracellular protozoa

    Directory of Open Access Journals (Sweden)

    Gazzinelli R.T.


    Full Text Available Toxoplasma gondii and Trypanosoma cruzi are intracellular parasites which, as part of their life cycle, induce a potent cell-mediated immunity (CMI maintained by Th1 lymphocytes and IFN-g. In both cases, induction of a strong CMI is thought to protect the host against rapid parasite multiplication and consequent pathology and lethality during the acute phase of infection. However, the parasitic infection is not eliminated by the immune system and the vertebrate host serves as a parasite reservoir. In contrast, Leishmania sp, which is a slow growing parasite, appears to evade induction of CMI during early stages of infection as a strategy for surviving in a hostile environment (i.e., inside the macrophages which are their obligatory niche in the vertebrate host. Recent reports show that the initiation of IL-12 synthesis by macrophages during these parasitic infections is a key event in regulating CMI and disease outcome. The studies reviewed here indicate that activation/inhibition of distinct signaling pathways and certain macrophage functions by intracellular protozoa are important events in inducing/modulating the immune response of their vertebrate hosts, allowing parasite and host survival and therefore maintaining parasite life cycles.

  2. Melatonin treatment prevents modulation of cell-mediated immune response induced by propoxur in rats. (United States)

    Suke, Sanvidhan G; Pathak, Rahul; Ahmed, Rafat S; Tripathi, A K; Banerjee, B D


    The effect of melatonin, a major secretory product of the pineal gland, in attenuation of propoxur (2-isopropoxy phenyl N-methyl carbamate)-induced modulation of cell-mediated immune (CMI) response was studied in rats. Male Wistar albino rats were exposed to propoxur (a widely used pesticide) orally (10 mg/kg) and/or melatonin (10 mg/kg) orally for 4 weeks. CMI was measured by delayed-type hypersensitivity (DTH), leucocyte and macrophage migration inhibition (LMI and MMI) responses and estimation of cytokines TNF-alpha and IFN-gamma levels. Rats exposed to propoxur for 4 weeks showed significant decrease in DTH, LMI and MMI responses. Propoxur also suppressed TNF-alpha and IFN-gamma production significantly. Administration of melatonin alone caused a significant increase in DTH response. Although there were no changes in the LMI and MMI response, the cytokine levels were significantly increased, as compared to control. Co-administration of melatonin along with propoxur significantly nullified the effect of the pesticide on the CMI response, except DTH and reversed levels of cytokines to near control/normal values. Thus, melatonin treatment considerably attenuated immunomodulation caused by sub-chronic treatment of propoxur in experimental animals.

  3. Changes in cell-mediated immune response after lung resection surgery for MDR-TB patients. (United States)

    Park, Seung-Kyu; Hong, Sunghee; Eum, Seok-Yong; Lee, In Hee; Shin, Donk Ok; Cho, Jang Eun; Cho, Sungae; Cho, Sang-Nae


    The immune responses of multidrug-resistant tuberculosis (MDR-TB) patients undergoing lung resection surgery were investigated in order to understand the mechanism of strong immune suppression in MDR-TB. We examined changes in cell-mediated immune response (CMI) of a total of sixteen MDR-TB patients, three of them extensively drug-resistant tuberculosis (XDR-TB) patients, after the removal of the heavily diseased lung section. The IFN-γ response to Mycobacterium tuberculosis culture filtrate proteins (Mtb-CFP), one of the most important CMI to defend TB, showed a statistically significant elevation in 2-4 months after operation when compared to the preoperative CMI in patients who were converted into AFB negative and cured in two years' follow-up, suggesting that the recovery of CMI may be one of the key factors in the successful treatment of MDR-TB. Interestingly, IL-10 response to Mtb-CFP was also elevated in 2-4 months after surgery in cured patients although both proliferative response and PBMC composition were not significantly changed. Infection with first- or second-line drugs resistant Mtb reduces the efficiency of chemotherapeutic treatment of MDR-TB to about 50%. Thus, this study suggests that chemotherapeutic treatment of MDR-TB may be more effective when combined with accompanying therapy that increases CMI, includes lung resection surgery.

  4. Induction of cell-mediated immunity to Mycobacterium leprae in mice

    Energy Technology Data Exchange (ETDEWEB)

    Patel, P.J.; Lefford, M.J.


    The immune response of mice to armadillo-derived, irradiation-killed Mycobacterium leprae (I-ML) was investigated. Following injection of 100 microgram of I-ML into the left hind footpads of mice, a state of cell-mediated immunity (CMI) was engendered to antigens of M. leprae. The evidence for CMI was as follows: (1) development of delayed-type hypersensitivity to both human tuberculin purified protein derivative and soluble M. leprae antigens; (2) T-lymphocyte-dependent macrophage activation at the inoculation site; (3) specific systemaic resistance to the cross-reactive species M. tuberculosis; and (4) immunopotentiation of the delayed-type hypersensitivity response to an unrelated antigen. The CMI induced by I-ML in aqueous suspension was greater than that obtained with the same antigen in water-in-oil emulsion, even though the latter generated a more severe reaction at the site of immunization. I-ML also induced a stronger CMI response than the corresponding dose of heat-killed BCG.

  5. Xanthohumol attenuates tumour cell-mediated breaching of the lymphendothelial barrier and prevents intravasation and metastasis. (United States)

    Viola, Katharina; Kopf, Sabine; Rarova, Lucie; Jarukamjorn, Kanokwan; Kretschy, Nicole; Teichmann, Mathias; Vonach, Caroline; Atanasov, Atanas G; Giessrigl, Benedikt; Huttary, Nicole; Raab, Ingrid; Krieger, Sigurd; Strnad, Miroslav; de Martin, Rainer; Saiko, Philipp; Szekeres, Thomas; Knasmüller, Siegfried; Dirsch, Verena M; Jäger, Walter; Grusch, Michael; Dolznig, Helmut; Mikulits, Wolfgang; Krupitza, Georg


    Health beneficial effects of xanthohumol have been reported, and basic research provided evidence for anti-cancer effects. Furthermore, xanthohumol was shown to inhibit the migration of endothelial cells. Therefore, this study investigated the anti-metastatic potential of xanthohumol. MCF-7 breast cancer spheroids which are placed on lymphendothelial cells (LECs) induce "circular chemorepellent-induced defects" (CCIDs) in the LEC monolayer resembling gates for intravasating tumour bulks at an early step of lymph node colonisation. NF-κB reporter-, EROD-, SELE-, 12(S)-HETE- and adhesion assays were performed to investigate the anti-metastatic properties of xanthohumol. Western blot analyses were used to elucidate the mechanisms inhibiting CCID formation. Xanthohumol inhibited the activity of CYP, SELE and NF-kB and consequently, the formation of CCIDs at low micromolar concentrations. More specifically, xanthohumol affected ICAM-1 expression and adherence of MCF-7 cells to LECs, which is a prerequisite for CCID formation. Furthermore, markers of epithelial-to-mesenchymal transition (EMT) and of cell mobility such as paxillin, MCL2 and S100A4 were suppressed by xanthohumol. Xanthohumol attenuated tumour cell-mediated defects at the lymphendothelial barrier and inhibited EMT-like effects thereby providing a mechanistic explanation for the anti-intravasative/anti-metastatic properties of xanthohumol.

  6. Fibrocyte-like cells mediate acquired resistance to anti-angiogenic therapy with bevacizumab. (United States)

    Mitsuhashi, Atsushi; Goto, Hisatsugu; Saijo, Atsuro; Trung, Van The; Aono, Yoshinori; Ogino, Hirokazu; Kuramoto, Takuya; Tabata, Sho; Uehara, Hisanori; Izumi, Keisuke; Yoshida, Mitsuteru; Kobayashi, Hiroaki; Takahashi, Hidefusa; Gotoh, Masashi; Kakiuchi, Soji; Hanibuchi, Masaki; Yano, Seiji; Yokomise, Hiroyasu; Sakiyama, Shoji; Nishioka, Yasuhiko


    Bevacizumab exerts anti-angiogenic effects in cancer patients by inhibiting vascular endothelial growth factor (VEGF). However, its use is still limited due to the development of resistance to the treatment. Such resistance can be regulated by various factors, although the underlying mechanisms remain incompletely understood. Here we show that bone marrow-derived fibrocyte-like cells, defined as alpha-1 type I collagen-positive and CXCR4-positive cells, contribute to the acquired resistance to bevacizumab. In mouse models of malignant pleural mesothelioma and lung cancer, fibrocyte-like cells mediate the resistance to bevacizumab as the main producer of fibroblast growth factor 2. In clinical specimens of lung cancer, the number of fibrocyte-like cells is significantly increased in bevacizumab-treated tumours, and correlates with the number of treatment cycles, as well as CD31-positive vessels. Our results identify fibrocyte-like cells as a promising cell biomarker and a potential therapeutic target to overcome resistance to anti-VEGF therapy.

  7. Loss of STAT3 in Lymphoma Relaxes NK Cell-Mediated Tumor Surveillance

    Directory of Open Access Journals (Sweden)

    Eva Maria Putz


    Full Text Available The transcription factors and proto-oncogenes STAT3 and STAT5 are highly activated in hematological malignancies and represent promising therapeutic targets. Whereas the importance of STAT5 as tumor promoter is beyond doubt, the role of STAT3 in hematological cancers is less well understood. Both, enforced as well as attenuated expression of STAT3 were reported in hematopoietic malignancies. Recent evidence implicates STAT3 as key player for tumor immune surveillance as it both mediates the production of and response to inflammatory cytokines. Here we investigated the effects of STAT3 deletion in a BCR/ABL-induced lymphoma model, which is tightly controlled by natural killer (NK cells in vivo. Upon STAT3 deletion tumor growth is significantly enhanced when compared to STAT3-expressing controls. The increased tumor size upon loss of STAT3 was accompanied by reduced NK cell infiltration and decreased levels of the cytokine IFN-γ and the chemokine RANTES. Upon transplantation into NK cell-deficient mice differences in lymphoma size were abolished indicating that STAT3 expression in the tumor cells controls NK cell-dependent tumor surveillance. Our findings indicate that STAT3 inhibition in lymphoma patients will impair NK cell-mediated tumor surveillance, which needs to be taken into account when testing STAT3 inhibitors in preclinical or clinical trials.

  8. Acquired pMHC I Complexes Greatly Enhance CD4+ Th Cell's Stimulatory Effect on CD8+ T Cell-Mediated Diabetes in Transgenic RIP-mOVA Mice

    Institute of Scientific and Technical Information of China (English)

    Khawaja Ashfaque Ahmed; Yufeng Xie; Xueshu Zhang; Jim Xiang


    CD4+ helper T (Th) cells play pivotal roles in induction of CD8+ CTL immunity. However, the mechanism of CD4+ T cell help delivery to CD8+ T cells in vivo is still elusive. In this study, we used ovalbumin (OVA)-pulsed dendritic cells (DCovA) to activate OT-Ⅱ mouse CD4+ T cells, and then studied the help effect of these CD4+ T cells on CD8+ cytotoxic T lymphocyte (CTL) responses. We also examined CTL mediated islet β cell destruction which leaded to diabetes in wild-type C57BL/6 mice and transgenic rat insulin promoter (RIP)-mOVA mice expressing β cell antigen OVA with self OVA-specific tolerance, respectively. In adoptive transfer experiments, we demonstrated that help, in the form of peptide/major histocompatibility complex (pMHC) I acquired from DCovA by DCovA activation, was required for induction of OVA-specific CTL responses in C57BL/6 mice. However, in combination with TCR transgenic OT-I mouse CD8+ T cells, the tolerogenic dosage of CD4+ Th cells with acquired pMHC I, but not CD4+ (Kb-/-) Th cells without acquired pMHC I were able to cause diabetes in 8/10 (80%) RIP-mOVA mice.This study thus expands the current knowledge in T cell-mediated autoimmunity and provides insight into the nature of CD4+ T cell-mediated help in CD8+ CTL induction. Cellular & Molecular Immunology. 2008;5(6):407-415.

  9. Paucity of natural killer and cytotoxic T cells in human neuromyelitis optica lesions. (United States)

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


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

  10. 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.

  11. Two sides of one coin: massive hepatic necrosis and progenitor cell-mediated regeneration in acute liver failure. (United States)

    Weng, Hong-Lei; Cai, Xiaobo; Yuan, Xiaodong; Liebe, Roman; Dooley, Steven; Li, Hai; Wang, Tai-Ling


    Massive hepatic necrosis is a key event underlying acute liver failure, a serious clinical syndrome with high mortality. Massive hepatic necrosis in acute liver failure has unique pathophysiological characteristics including extremely rapid parenchymal cell death and removal. On the other hand, massive necrosis rapidly induces the activation of liver progenitor cells, the so-called "second pathway of liver regeneration." The final clinical outcome of acute liver failure depends on whether liver progenitor cell-mediated regeneration can efficiently restore parenchymal mass and function within a short time. This review summarizes the current knowledge regarding massive hepatic necrosis and liver progenitor cell-mediated regeneration in patients with acute liver failure, the two sides of one coin.

  12. Endogenous n-3 polyunsaturated fatty acids attenuate T cell-mediated hepatitis via autophagy activation

    Directory of Open Access Journals (Sweden)

    Yanli Li


    Full Text Available Omega-3 polyunsaturated fatty acids (n-3 PUFAs exert anti-inflammatory effects in several liver disorders, including cirrhosis, acute liver failure, and fatty liver disease. To date, little is known about their role in immune-mediated liver diseases. In this study, we used fat-1 transgenic mice rich in endogenous n-3 PUFAs to examine the role of n-3 PUFAs in immune-mediated liver injury. Concanavalin A (Con A was administered intravenously to wild-type (WT and fat-1 transgenic mice to induce T cell-mediated hepatitis. Reduced liver damage was shown in Con A-administrated fat-1 transgenic mice, as evidenced by decreased mortality, attenuated hepatic necrosis, lessened serum alanine aminotransferase (ALT activity, and inhibited production of pro-inflammatory cytokines (e.g. TNF-α, IL-6, IL-17A and IFN-γ. In vivo and in vitro studies demonstrated that n-3 PUFAs significantly inhibited the activation of hepatic T cells and the differentiation of Th1 cells after Con A challenge. Further studies showed that n-3 PUFAs markedly increased autophagy level in Con A-treated fat-1 T cells compared with the WT counterparts. Blocking hepatic autophagy activity with chloroquine diminished the differences in T cell activation and liver injury between Con A-injected WT and fat-1 transgenic mice. We conclude that n-3 PUFAs limit Con A-induced hepatitis via an autophagy-dependent mechanism, and could be exploited as a new therapeutic approach for autoimmune hepatitis.

  13. Endogenous n-3 Polyunsaturated Fatty Acids Attenuate T Cell-Mediated Hepatitis via Autophagy Activation (United States)

    Li, Yanli; Tang, Yuan; Wang, Shoujie; Zhou, Jing; Zhou, Jia; Lu, Xiao; Bai, Xiaochun; Wang, Xiang-Yang; Chen, Zhengliang; Zuo, Daming


    Omega-3 polyunsaturated fatty acids (n-3 PUFAs) exert anti-inflammatory effects in several liver disorders, including cirrhosis, acute liver failure, and fatty liver disease. To date, little is known about their role in immune-mediated liver diseases. In this study, we used fat-1 transgenic mice rich in endogenous n-3 PUFAs to examine the role of n-3 PUFAs in immune-mediated liver injury. Concanavalin A (Con A) was administered intravenously to wild-type (WT) and fat-1 transgenic mice to induce T cell-mediated hepatitis. Reduced liver damage was shown in Con A-administrated fat-1 transgenic mice, as evidenced by decreased mortality, attenuated hepatic necrosis, lessened serum alanine aminotransferase activity, and inhibited production of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-17A, and IFN-γ). In vivo and in vitro studies demonstrated that n-3 PUFAs significantly inhibited the activation of hepatic T cells and the differentiation of Th1 cells after Con A challenge. Further studies showed that n-3 PUFAs markedly increased autophagy level in Con A-treated fat-1 T cells compared with the WT counterparts. Blocking hepatic autophagy activity with chloroquine diminished the differences in T cell activation and liver injury between Con A-injected WT and fat-1 transgenic mice. We conclude that n-3 PUFAs limit Con A-induced hepatitis via an autophagy-dependent mechanism and could be exploited as a new therapeutic approach for autoimmune hepatitis. PMID:27679638

  14. MYSM1-dependent checkpoints in B cell lineage differentiation and B cell-mediated immune response. (United States)

    Förster, Michael; Farrington, Kyo; Petrov, Jessica C; Belle, Jad I; Mindt, Barbara C; Witalis, Mariko; Duerr, Claudia U; Fritz, Jörg H; Nijnik, Anastasia


    MYSM1 is a chromatin-binding histone deubiquitinase. MYSM1 mutations in humans result in lymphopenia whereas loss of Mysm1 in mice causes severe hematopoietic abnormalities, including an early arrest in B cell development. However, it remains unknown whether MYSM1 is required at later checkpoints in B cell development or for B cell-mediated immune responses. We analyzed conditional mouse models Mysm1(fl/fl)Tg.mb1-cre, Mysm1(fl/fl)Tg.CD19-cre, and Mysm1(fl/fl)Tg.CD21-cre with inactivation of Mysm1 at prepro-B, pre-B, and follicular B cell stages of development. We show that loss of Mysm1 at the prepro-B cell stage in Mysm1(fl/fl)Tg.mb1-cre mice results in impaired B cell differentiation, with an ∼2-fold reduction in B cell numbers in the lymphoid organs. Mysm1(fl/fl)Tg.mb1-cre B cells also showed increased expression of activation markers and impaired survival and proliferation. In contrast, Mysm1 was largely dispensable from the pre-B cell stage onward, with Mysm1(fl/fl)Tg.CD19-cre and Mysm1(fl/fl)Tg.CD21-cre mice showing no alterations in B cell numbers and largely normal responses to stimulation. MYSM1, therefore, has an essential role in B cell lineage specification but is dispensable at later stages of development. Importantly, MYSM1 activity at the prepro-B cell stage of development is important for the normal programming of B cell responses to stimulation once they complete their maturation process.

  15. Curcuma oil reduces endothelial cell-mediated inflammation in postmyocardial ischemia/reperfusion in rats. (United States)

    Manhas, Amit; Khanna, Vivek; Prakash, Prem; Goyal, Dipika; Malasoni, Richa; Naqvi, Arshi; Dwivedi, Anil K; Dikshit, Madhu; Jagavelu, Kumaravelu


    Endothelial cells initiated inflammation persisting in postmyocardial infarction needs to be controlled and moderated for avoiding fatal complications. Curcuma oil (C.oil, Herbal Medicament), a standardized hexane soluble fraction of Curcuma longa has possessed neuroprotective effect. However, its effect on myocardial ischemia/reperfusion (MI/RP) and endothelial cells remains incompletely defined. Here, using in vivo rat MI/RP injury model and in vitro cellular approaches using EA.hy926 endothelial cells, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and myograph, we provide evidence that with effective regimen and preconditioning of rats with C.oil (250 mg/kg, PO), before and after MI/RP surgery protects rats from MI/RP-induced injury. C.oil treatment reduces left ventricular ischemic area and endothelial cell-induced inflammation, specifically in the ischemic region (*P < 0.0001) and improved endothelial function by reducing the expression of proinflammatory genes and adhesion factors on endothelial cells both in vitro and in vivo. Furthermore, mechanistic studies have revealed that C.oil reduced the expression of adhesion factors like E-selectin (#P = 0.0016) and ICAM-1 ($P = 0.0069) in initiating endothelial cells-induced inflammation. In line to the real-time polymerase chain reaction expression data, C.oil reduced the adhesion of inflammatory cells to endothelial cells as assessed by the interaction of THP-1 monocytes with the endothelial cells using flow-based adhesion and under inflammatory conditions. These studies provide evidence that salutary effect of C.oil on MI/RP could be achieved with pretreatment and posttreatment of rats, C.oil reduced MI/RP-induced injury by reducing the endothelial cell-mediated inflammation, specifically in the ischemic zone of MI/RP rat heart.

  16. Precision Subtypes of T Cell-Mediated Rejection Identified by Molecular Profiles (United States)

    Kadota, Paul Ostrom; Hajjiri, Zahraa; Finn, Patricia W.; Perkins, David L.


    Among kidney transplant recipients, the treatment of choice for acute T cell-mediated rejection (TCMR) with pulse steroids or antibody protocols has variable outcomes. Some rejection episodes are resistant to an initial steroid pulse, but respond to subsequent antibody protocols. The biological mechanisms causing the different therapeutic responses are not currently understood. Histological examination of the renal allograft is considered the gold standard in the diagnosis of acute rejection. The Banff Classification System was established to standardize the histopathological diagnosis and to direct therapy. Although widely used, it shows variability among pathologists and lacks criteria to guide precision individualized therapy. The analysis of the transcriptome in allograft biopsies, which we analyzed in this study, provides a strategy to develop molecular diagnoses that would have increased diagnostic precision and assist the development of individualized treatment. Our hypothesis is that the histological classification of TCMR contains multiple subtypes of rejection. Using R language algorithms to determine statistical significance, multidimensional scaling, and hierarchical, we analyzed differential gene expression based on microarray data from biopsies classified as TCMR. Next, we identified KEGG functions, protein–protein interaction networks, gene regulatory networks, and predicted therapeutic targets using the integrated database ConsesnsusPathDB (CPDB). Based on our analysis, two distinct clusters of biopsies termed TCMR01 and TCMR02 were identified. Despite having the same Banff classification, we identified 1933 differentially expressed genes between the two clusters. These genes were further divided into three major groups: a core group contained within both the TCMR01 and TCMR02 subtypes, as well as genes unique to TCMR01 or TCMR02. The subtypes of TCMR utilized different biological pathways, different regulatory networks and were predicted to

  17. HLA-G inhibits xenogenetic cytotoxicity mediated by human NK cells and T lymphocytes against PECs

    Institute of Scientific and Technical Information of China (English)


    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.

  18. CDK8-Mediated STAT1-S727 Phosphorylation Restrains NK Cell Cytotoxicity and Tumor Surveillance

    Directory of Open Access Journals (Sweden)

    Eva Maria Putz


    Full Text Available The transcription factor STAT1 is important in natural killer (NK cells, which provide immediate defense against tumor and virally infected cells. We show that mutation of a single phosphorylation site (Stat1-S727A enhances NK cell cytotoxicity against a range of tumor cells, accompanied by increased expression of perforin and granzyme B. Stat1-S727A mice display significantly delayed disease onset in NK cell-surveilled tumor models including melanoma, leukemia, and metastasizing breast cancer. Constitutive phosphorylation of S727 depends on cyclin-dependent kinase 8 (CDK8. Inhibition of CDK8-mediated STAT1-S727 phosphorylation may thus represent a therapeutic strategy for stimulating NK cell-mediated tumor surveillance.

  19. 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.

  20. Upregulated Expression of Cytotoxicity-Related Genes in IFN-γ Knockout Mice with Schistosoma japonicum Infection

    Directory of Open Access Journals (Sweden)

    Xiaotang Du


    Full Text Available It is well accepted that IFN-γ is important to the development of acquired resistance against murine schistosomiasis. However, the in vivo role of this immunoregulatory cytokine in helminth infection needs to be further investigated. In this study, parasite burden and host immune response were observed in IFN-γ knockout mice (IFNg KO infected with Schistosoma japonicum for 6 weeks. The results suggested that deficiency in IFN-γ led to decreased egg burden in mice, with low schistosome-specific IgG antibody response and enhanced activation of T cells during acute infection. Microarray and qRT-PCR data analyses showed significant upregulation of some cytotoxicity-related genes, including those from the granzyme family, tumor necrosis factor, Fas Ligand, and chemokines, in the spleen cells of IFNg KO mice. Furthermore, CD8+ cells instead of NK cells of IFNg KO mice exhibited increased transcription of cytotoxic genes compared with WT mice. Additionally, Schistosoma japonicum-specific egg antigen immunization also could activate CD8+ T cells to upregulate the expression of cytotoxic genes in IFNg KO mice. Our data suggest that IFN-γ is not always a positive regulator of immune responses. In certain situations, the disruption of IFN-γ signaling may up-regulate the cytotoxic T-cell-mediated immune responses to the parasite.

  1. Cloning and expression analysis of nonspecific cytotoxic cell receptor 1 (Ls-NCCRP1) from red snapper (Lutjanus sanguineus). (United States)

    Cai, Jia; Wei, Shina; Wang, Bei; Huang, Yucong; Tang, Jufen; Lu, Yishan; Wu, Zaohe; Jian, Jichang


    It is well known that nonspecific cytotoxic cells (NCCs) are kinds of natural killer cell mediated innate immune responses in teleosts. The nonspecific cytotoxic cell receptor protein 1 (NCCRP-1) is an important cell surface protein on NCC, which serves crucial functions in target cell recognition and cytotoxicity activation. In the present study, a nonspecific cytotoxic cell receptor protein NCCRP-1 (Ls-NCCRP1) was cloned from red snapper, Lutjanus sanguineus. The Ls-NCCRP1 cDNA is composed of 986bp with a 43bp of 5'-UTR, 702bp open reading frame (ORF) and 241bp 3'-UTR, encoding a polypeptide of 233 amino acids (GenBank accession no: ADK32635). Phylogenetic analysis revealed that Ls-NCCRP1 showed highest similarity to sea bream NCCRP-1. Quantitative real-time PCR (qRT-PCR) analysis showed that Ls-NCCRP1 had relatively high expression level in the head kidney, spleen and liver. After Vibrio alginolyticus infection, transcripts of Ls-NCCRP1 increased and reached its peak at 4h p.i. These results indicated that Ls-NCCRP1 may play an important role in innate immune response to bacteria.

  2. Cell mediated therapeutics for cancer treatment: Tumor homing cells as therapeutic delivery vehicles (United States)

    Balivada, Sivasai

    Many cell types were known to have migratory properties towards tumors and different research groups have shown reliable results regarding cells as delivery vehicles of therapeutics for targeted cancer treatment. Present report discusses proof of concept for 1. Cell mediated delivery of Magnetic nanoparticles (MNPs) and targeted Magnetic hyperthermia (MHT) as a cancer treatment by using in vivo mouse cancer models, 2. Cells surface engineering with chimeric proteins for targeted cancer treatment by using in vitro models. 1. Tumor homing cells can carry MNPs specifically to the tumor site and tumor burden will decrease after alternating magnetic field (AMF) exposure. To test this hypothesis, first we loaded Fe/Fe3O4 bi-magnetic NPs into neural progenitor cells (NPCs), which were previously shown to migrate towards melanoma tumors. We observed that NPCs loaded with MNPs travel to subcutaneous melanoma tumors. After alternating magnetic field (AMF) exposure, the targeted delivery of MNPs by the NPCs resulted in a mild decrease in tumor size (Chapter-2). Monocytes/macrophages (Mo/Ma) are known to infiltrate tumor sites, and also have phagocytic activity which can increase their uptake of MNPs. To test Mo/Ma-mediated MHT we transplanted Mo/Ma loaded with MNPs into a mouse model of pancreatic peritoneal carcinomatosis. We observed that MNP-loaded Mo/Ma infiltrated pancreatic tumors and, after AMF treatment, significantly prolonged the lives of mice bearing disseminated intraperitoneal pancreatic tumors (Chapter-3). 2. Targeted cancer treatment could be achieved by engineering tumor homing cell surfaces with tumor proteases cleavable, cancer cell specific recombinant therapeutic proteins. To test this, Urokinase and Calpain (tumor specific proteases) cleavable; prostate cancer cell (CaP) specific (CaP1 targeting peptide); apoptosis inducible (Caspase3 V266ED3)- rCasp3V266ED3 chimeric protein was designed in silico. Hypothesized membrane anchored chimeric protein (rCasp3V

  3. Cell mediated immune responses in the placenta following challenge of vaccinated pregnant heifers with Neospora caninum. (United States)

    Hecker, Y P; Cantón, G; Regidor-Cerrillo, J; Chianini, F; Morrell, E; Lischinsky, L; Ortega-Mora, L M; Innes, E A; Odeón, A; Campero, C M; Moore, D P


    The aim of the present study was to investigate and correlate the cell-mediated immune response and pathological changes at the maternal-fetal interface of Neospora-challenged pregnant cattle previously immunized with live and inactivated experimental vaccines. Pregnant heifers naïve to Neospora caninum were divided in 5 groups of 4 animals, each one immunized before mating: Group A heifers were intravenously (iv) immunized with 6.25 × 10(7) live tachyzoites of the NC-6 strain; group B heifers were immunized twice subcutaneously (sc) 3 weeks apart with native antigen extract of the NC-6 strain formulated with ISCOMs; group C heifers were sc immunized twice 3 weeks apart with three recombinant proteins (rNcSAG1, rNcHSP20, rNcGRA7) of the NC-1 strain formulated with ISCOMs; group D heifers were sc injected with sterile phosphate-buffered saline (PBS) and group E heifers received sc ISCOM-matrix (ISCOMs without antigen). All groups were iv-challenged with 4.7 × 10(7) NC-1 tachyzoites at 70 days of gestation. Heifers were culled at day 104 of gestation and placentomes were examined to evaluate lesions and local cellular immune responses using histopathology, immunohistochemistry and real time-PCR. Immunohistochemistry was performed using bovine leucocyte specific antibodies. Cytokine expression and levels (IFN-γ, IL-4, IL-10, IL-12 and TNF-α) were measured using real-time reverse transcription-PCR and ELISA, respectively. Minimal inflammation was observed in group A placentomes; while placentomes from group B, C, D and E had moderate to severe infiltration with CD3(+), CD4(+), γδ-T cells, CD8(+) cells and macrophages being more numerous in groups B and E placentomes, when compared with groups C and D (P<0.001). Cytokine levels were significantly increased in the caruncles of animals of groups B and C in comparison with the other animal groups (P < 0.001). The results from this study showed that the strongest cellular immune responses were observed in the

  4. Cytotoxicity of zinc in vitro. (United States)

    Borovanský, J; Riley, P A


    The effect of zinc ions on B16 mouse melanoma lines, HeLa cells and I-221 epithelial cells was investigated in vitro in order to ascertain whether sensitivity to Zn2+ is a general feature of cells in vitro and in an attempt to elucidate the mechanism(s) of zinc cytotoxicity. The proliferation of B16, HeLa and I-221 cell lines was inhibited by 1.25 x 10(-4), 1.50 x 10(-4) and 1.50 x 10(-4) mol/l Zn2+, respectively. The free radical scavengers, methimazole and ethanol, did not suppress the toxicity of Zn2+, neither did superoxide dismutase or catalase. The addition of the chelating agent EDTA reduced the zinc cytotoxicity. It was possible to suppress the cytotoxicity of zinc by increasing the concentration of either Fe2+ or Ca2+ but not Mg2+, which suggests that a prerequisite for the toxic action of zinc is entry into cells using channels that are shared with iron or calcium. This view was supported by experiments in which transferrin intensified the cytotoxic action of zinc in serum-free medium. Another agent facilitating zinc transport, prostaglandin E2, inhibited the proliferation of the B16 melanoma cell line. There were no conspicuous differences in zinc toxicity to pigmented and unpigmented cells. The toxic effect of zinc in the cell systems studied exceeded that of iron, copper, manganese and cobalt in the same concentration range. In vitro, Zn2+ should be regarded as a dangerous cation.

  5. Cytotoxic Compounds from Brucea mollis


    Tung, Mai Hung Thanh; Đuc, Ho Viet; Huong, Tran Thu; Nguyen Thanh DUONG; Do Thi PHUONG; Thao, Do Thi; Tai, Bui Huu; Kim, Young Ho; Bach, Tran The; Cuong, Nguyen Manh


    Ten compounds, including soulameanone (1), isobruceine B (2), 9-methoxy-canthin-6-one (3), bruceolline F (4), niloticine (5), octatriacontan-1-ol (6), bombiprenone (7), α-tocopherol (8), inosine (9), and apigenin 7-O-β-D-glucopyranoside (10), were isolated from the leaves, stems, and roots of Brucea mollis Wall. ex Kurz. Their structures were determined using one- and two-dimensional NMR spectroscopy and mass spectrometry. All compounds were evaluated for their cytotoxic activity against KB (...

  6. Structure-cytotoxicity relationships for dietary flavonoids

    DEFF Research Database (Denmark)

    Breinholt, V.; Dragsted, L.O.


    The cytotoxicity of a large series of dietary flavonoids was tested in a non-tumorigenic mouse and two human cancer cell lines, using the neutral red dye exclusion assay. All compounds tested exhibited a concentration-dependent cytotoxic action in the employed cell lines. The relative cytotoxicity...... of the flavonoids, however, Tvas found to vary greatly among the different cell Lines. With a few exceptions, the investigated flavonoids were more cytotoxic to the human cancer cell lines, than the mouse cell line. The differences in cytotoxicity were accounted for in part by differences in cellular uptake...... and metabolic capacity among the different cell types. In 3T3 cells fairly consistent structure-cytotoxicity relationships were found. The most cytotoxic structures tested in 3T3 cells were flavonoids with adjacent 3',4' hydroxy groups on the B-ring, such as luteolin, quercetin, myricetin, fisetin, eriodictyol...

  7. Comparative cytotoxicity of periodontal bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Stevens, R.H.; Hammond, B.F.


    The direct cytotoxicity of sonic extracts (SE) from nine periodontal bacteria for human gingival fibroblasts (HGF) was compared. Equivalent dosages (in terms of protein concentration) of SE were used to challenge HGF cultures. The cytotoxic potential of each SE was assessed by its ability to (1) inhibit HGF proliferation, as measured by direct cell counts; (2) inhibit 3H-thymidine incorporation in HGF cultures; or (3) cause morphological alterations of the cells in challenged cultures. The highest concentration (500 micrograms SE protein/ml) of any of the SEs used to challenge the cells was found to be markedly inhibitory to the HGFs by all three of the criteria of cytotoxicity. At the lowest dosage tested (50 micrograms SE protein/ml); only SE from Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, and Fusobacterium nucleatum caused a significant effect (greater than 90% inhibition or overt morphological abnormalities) in the HGFs as determined by any of the criteria employed. SE from Capnocytophaga sputigena, Eikenella corrodens, or Wolinella recta also inhibited cell proliferation and thymidine incorporation at this dosage; however, the degree of inhibition (5-50%) was consistently, clearly less than that of the first group of three organisms named above. The SE of the three other organisms tested (Actinomyces odontolyticus, Bacteroides intermedius, and Streptococcus sanguis) had little or no effect (0-10% inhibition) at this concentration. The data suggest that the outcome of the interaction between bacterial components and normal resident cells of the periodontium is, at least in part, a function of the bacterial species.

  8. Cytotoxic Compounds from Brucea mollis

    Directory of Open Access Journals (Sweden)

    Mai Hung Thanh TUNG


    Full Text Available Ten compounds, including soulameanone (1, isobruceine B (2, 9-methoxy-canthin-6-one (3, bruceolline F (4, niloticine (5, octatriacontan-1-ol (6, bombiprenone (7, α-tocopherol (8, inosine (9, and apigenin 7-O-β-D-glucopyranoside (10, were isolated from the leaves, stems, and roots of Brucea mollis Wall. ex Kurz. Their structures were determined using one- and two-dimensional NMR spectroscopy and mass spectrometry. All compounds were evaluated for their cytotoxic activity against KB (human carcinoma of the mouth, LU-1 (human lung adenocarcinoma, LNCaP (human prostate adeno-carcinoma, and HL-60 (human promyelocytic leukemia cancer cell lines. Compound 2 showed significant cytotoxic activity against KB, LU-1, LNCaP, and HL-60 cancer cells with IC50 values of 0.39, 0.40, 0.34, and 0.23 μg/mL, respectively. In addition, compounds 3 and 5 showed significant cytotoxic activity against KB, LU-1, LNCaP, and HL-60 cancer cells with IC50 values around 1–4 μg/mL. Compounds 9-methoxycanthin-6-one (3 and niloticine (5 have been discovered for the first time from the Brucea genus.

  9. Novel antigens used to detect cell-mediated immune responses over time in Mycobacterium avium subsp. paratuberculosis infected cattle

    DEFF Research Database (Denmark)

    Mikkelsen, Heidi; Aagaard, Claus; Nielsen, Søren Saxmose;

    Early stage Mycobacterium avium subsp. paratuberculosis (MAP) infection of cattle can be detected by measuring specific cell mediated immune responses, using the interferon gamma (IFN-γ) test. Available IFN-γ tests are using purified protein derivatives of MAP (PPDj) which are crude products...... on the same 30 heifers from a known MAP infected herd. Determination of cut-off for each antigen was based on samples from a non-infected herd, including 60 heifers. Based on PPDj stimulations, more than 50% of the heifers tested MAP positive at the first two samplings, whereas only 20% tested positive...

  10. Effects of depression on parameters of cell-mediated immunity in patients with digestive tract cancers

    Institute of Scientific and Technical Information of China (English)

    Ke-Jun Nan; Yong-Chang Wei; Fu-Ling Zhou; Chun-Li Li; Chen-Guang Sui; Ling-Yun Hui; Cheng-Ge Gao


    AIM: To evaluate the effects of depression on parameters of cell-mediated immunity in patients with cancers of the digestive tract.METHODS: One hundred and eight adult patients of both sexes with cancers of the digestive tract admitted between March 2001 and February 2002 in the Department of Medical Oncology, First Affiliated Hospital of Xi'an Jiaotong University were randomly enrolled in the study. The Zung self-rating depression scale (SDS), Zung self-rating anxiety scale(SAS), numeric rating scale (NRS) and social support rating scale (SSRS) were employed to evaluate the degree of depression and their contributing factors. In terms of their SDS index scores, the patients were categorized into depression group (SDS≥50) and non-depression group(SDS<50). Immunological parameters such as T-lymphocyte subsets and natural killer (NK) cell activities in peripheral blood were determined and compared between the two groups of patients.RESULTS: The SDS index was from 33.8 to 66.2 in the 108 cases, 50% of these patients had a SDS index more than 50. Similarly, the SAS index of all the patients ranged from 35.0 to 62.0 and 46.3% of the cases had a SAS index above 50. Cubic curve estimation showed that the depression was positively correlated with anxiety and negatively with social support. Furthermore, the depression correlated with the tumor type, which manifested in a descending order as stomach, gallbladder, pancreas, intestine, esophagus,duodenum and rectum, according to their correlativity. Step-wise regression analysis suggested that hyposexuality,dispidtment, agitation, palpitation, low CD56 and anxiety were the significant factors contributing to depression. More severe anxiety (49.7±7.5 vs 45.3±6.9, P<0.05), pain (6.5±2.8 vs4.6±3.2, P<0.05), poor social support (6.8±2.0 vs 7.6±2.1,P<0.05), as well as decline of lymphocyte count (0.33±0.09vs0.39±0.87, P<0.05) and CD56 (0.26±0.11 vs0.29±0.11,P<0.05) were noted in the depression group compared

  11. Liposomal formulations of cytotoxic drugs. (United States)

    Janknegt, R


    Liposomes are microscopic particles of lipid bilayer membrane that enclose aqueous internal compartments. These drug-delivery systems offer a very interesting opportunity for delivering cytotoxic drugs with equal or improved clinical efficacy and reduced toxicity. The most important clinical application of liposomes until now has been the inclusion of amphotericin B. At the same dose level, liposomal amphotericin B is as effective or slightly less effective than the conventional formulation, but much higher dosages, up to 5-7 mg kg-1day-1, can be given with acceptable toxicity. There are three preparations of cytotoxic drugs in an advanced stage of commercial development. Two of these (Doxil and TLD D99) contain doxorubicin and the other (DaunoXome) contains daunorubicin. The cardiac toxicity of the three preparations under clinical evaluation appears to be low in comparison with conventional doxorubicin or daunorubicin. No direct comparisons between the new formulations are available, so it is not yet possible to make any statements concerning their relative efficacy and toxicity. DaunoXome is the only drug that is approved in any country, and is also the best documented. It is too early to make recommendations concerning the place of these drugs in therapy. The marked increase in concentrations at the site of the tumour has yet to lead to increased therapeutic efficacy. These findings need further investigation. The efficacy of liposomal preparations in Kaposi's sarcoma appears to be similar to that of standard therapy and the clinical tolerance is good. Perhaps combination therapy with other cytotoxic agents could result in improved clinical efficacy. Their cost will probably be high in comparison with standard therapies.

  12. T Cell-Mediated Modulation of Mast Cell Function: Heterotypic Adhesion-Induced Stimulatory or Inhibitory Effects

    Directory of Open Access Journals (Sweden)

    Yoseph A. Mekori


    Full Text Available Close physical proximity between mast cells and T cells has been demonstrated in several T cell mediated inflammatory processes such as rheumatoid arthritis and sarcoidosis. However, the way by which mast cells are activated in these T cell-mediated immune responses has not been fully elucidated. We have identified and characterized a novel mast cell activation pathway initiated by physical contact with activated T cells, and showed that this pathway is associated with degranulation and cytokine release. The signaling events associated with this pathway of mast cell activation have also been elucidated confirming the activation of the Ras MAPK systems. More recently, we hypothesized and demonstrated that mast cells may also be activated by microparticles released from activated T cells that are considered as miniature version of a cell. By extension, microparticles might affect the activity of mast cells, which are usually not in direct contact with T cells at the inflammatory site. Recent works have also focused on the effects of regulatory T cells on mast cells. These reports highlighted the importance of the cytokines IL-2 and IL-9, produced by mast cells and T cells, respectively, in obtaining optimal immune suppression. Finally, physical contact, associated by OX40-OX40L engagement has been found to underlie the down-regulatory effects exerted by regulatory T cells on mast cell function.

  13. Type I NKT-cell-mediated TNF-α is a positive regulator of NLRP3 inflammasome priming. (United States)

    Chow, Melvyn T; Duret, Helene; Andrews, Daniel M; Faveeuw, Christelle; Möller, Andreas; Smyth, Mark J; Paget, Christophe


    The NLRP3 inflammasome plays a crucial role in the innate immune response to pathogens and exogenous or endogenous danger signals. Its activity must be precisely and tightly regulated to generate tailored immune responses. However, the immune cell subsets and cytokines controlling NLRP3 inflammasome activity are still poorly understood. Here, we have shown a link between NKT-cell-mediated TNF-α and NLRP3 inflammasome activity. The NLRP3 inflammasome in APCs was critical to potentiate NKT-cell-mediated immune responses, since C57BL/6 NLRP3 inflammasome-deficient mice exhibited reduced responsiveness to α-galactosylceramide. Importantly, NKT cells were found to act as regulators of NLRP3 inflammasome signaling, as NKT-cell-derived TNF-α was required for optimal IL-1β and IL-18 production by myeloid cells in response to α-galactosylceramide, by acting on the NLRP3 inflammasome priming step. Thus, NKT cells play a role in the positive regulation of NLRP3 inflammasome priming by mediating the production of TNF-α, thus demonstrating another means by which NKT cells control early inflammation.

  14. Antagonizing arachidonic acid-derived eicosanoids reduces inflammatory Th17 and Th1 cell-mediated inflammation and colitis severity. (United States)

    Monk, Jennifer M; Turk, Harmony F; Fan, Yang-Yi; Callaway, Evelyn; Weeks, Brad; Yang, Peiying; McMurray, David N; Chapkin, Robert S


    During colitis, activation of two inflammatory T cell subsets, Th17 and Th1 cells, promotes ongoing intestinal inflammatory responses. n-6 polyunsaturated fatty acid- (PUFA-) derived eicosanoids, such as prostaglandin E2 (PGE2), promote Th17 cell-mediated inflammation, while n-3 PUFA antagonize both Th17 and Th1 cells and suppress PGE2 levels. We utilized two genetic mouse models, which differentially antagonize PGE2 levels, to examine the effect on Th17 cells and disease outcomes in trinitrobenzene sulfonic acid- (TNBS-) induced colitis. Fat-1 mice contain the ω3 desaturase gene from C. elegans and synthesize n-3 PUFA de novo, thereby reducing the biosynthesis of n-6 PUFA-derived eicosanoids. In contrast, Fads1 Null mice contain a disrupted Δ5 desaturase gene and produce lower levels of n-6 PUFA-derived eicosanoids. Compared to Wt littermates, Fat-1 and Fads1 Null mice exhibited a similar colitic phenotype characterized by reduced colonic mucosal inflammatory eicosanoid levels and mRNA expression of Th17 cell markers (IL-17A, RORγτ, and IL-23), decreased percentages of Th17 cells and, improved colon injury scores (P ≤ 0.05). Thus, during colitis, similar outcomes were obtained in two genetically distinct models, both of which antagonize PGE2 levels via different mechanisms. Our data highlight the critical impact of n-6 PUFA-derived eicosanoids in the promotion of Th17 cell-mediated colonic inflammation.

  15. Antagonizing Arachidonic Acid-Derived Eicosanoids Reduces Inflammatory Th17 and Th1 Cell-Mediated Inflammation and Colitis Severity

    Directory of Open Access Journals (Sweden)

    Jennifer M. Monk


    Full Text Available During colitis, activation of two inflammatory T cell subsets, Th17 and Th1 cells, promotes ongoing intestinal inflammatory responses. n-6 polyunsaturated fatty acid- (PUFA- derived eicosanoids, such as prostaglandin E2 (PGE2, promote Th17 cell-mediated inflammation, while n-3 PUFA antagonize both Th17 and Th1 cells and suppress PGE2 levels. We utilized two genetic mouse models, which differentially antagonize PGE2 levels, to examine the effect on Th17 cells and disease outcomes in trinitrobenzene sulfonic acid- (TNBS- induced colitis. Fat-1 mice contain the ω3 desaturase gene from C. elegans and synthesize n-3 PUFA de novo, thereby reducing the biosynthesis of n-6 PUFA-derived eicosanoids. In contrast, Fads1 Null mice contain a disrupted Δ5 desaturase gene and produce lower levels of n-6 PUFA-derived eicosanoids. Compared to Wt littermates, Fat-1 and Fads1 Null mice exhibited a similar colitic phenotype characterized by reduced colonic mucosal inflammatory eicosanoid levels and mRNA expression of Th17 cell markers (IL-17A, RORγτ, and IL-23, decreased percentages of Th17 cells and, improved colon injury scores (P≤0.05. Thus, during colitis, similar outcomes were obtained in two genetically distinct models, both of which antagonize PGE2 levels via different mechanisms. Our data highlight the critical impact of n-6 PUFA-derived eicosanoids in the promotion of Th17 cell-mediated colonic inflammation.

  16. Human CD8+ T cells mediate protective immunity induced by a human malaria vaccine in human immune system mice. (United States)

    Li, Xiangming; Huang, Jing; Zhang, Min; Funakoshi, Ryota; Sheetij, Dutta; Spaccapelo, Roberta; Crisanti, Andrea; Nussenzweig, Victor; Nussenzweig, Ruth S; Tsuji, Moriya


    A number of studies have shown that CD8+ T cells mediate protective anti-malaria immunity in a mouse model. However, whether human CD8+ T cells play a role in protection against malaria remains unknown. We recently established human immune system (HIS) mice harboring functional human CD8+ T cells (HIS-CD8 mice) by transduction with HLA-A∗0201 and certain human cytokines using recombinant adeno-associated virus-based gene transfer technologies. These HIS-CD8 mice mount a potent, antigen-specific HLA-A∗0201-restricted human CD8+ T-cell response upon immunization with a recombinant adenovirus expressing a human malaria antigen, the Plasmodium falciparum circumsporozoite protein (PfCSP), termed AdPfCSP. In the present study, we challenged AdPfCSP-immunized HIS-CD8 mice with transgenic Plasmodium berghei sporozoites expressing full-length PfCSP and found that AdPfCSP-immunized (but not naïve) mice were protected against subsequent malaria challenge. The level of the HLA-A∗0201-restricted, PfCSP-specific human CD8+ T-cell response was closely correlated with the level of malaria protection. Furthermore, depletion of human CD8+ T cells from AdPfCSP-immunized HIS-CD8 mice almost completely abolished the anti-malaria immune response. Taken together, our data show that human CD8+ T cells mediate protective anti-malaria immunity in vivo.

  17. Cytotoxic quassinoids from Simaba cedron. (United States)

    Ozeki, A; Hitotsuyanagi, Y; Hashimoto, E; Itokawa, H; Takeya, K; de Mello Alves, S


    Four new quassinoids, cedronolactones A-D (1-4), together with nine known compounds, simalikalactone D (5), chaparrinone (6), chaparrin (7), glaucarubolone (8), glaucarubol (9), samaderine Z (10), guanepolide (11), ailanquassin A (12), and polyandrol (13), were isolated from the wood of Simaba cedron. The chemical structures of 1-4 were elucidated on the basis of their chemical and spectral properties. Cedronolactone A (1) was shown to exhibit a significant in vitro cytotoxicity (IC50 0.0074 microg/mL) against P-388 cells.

  18. γδ T Cell-Mediated Immunity to Cytomegalovirus Infection (United States)

    Khairallah, Camille; Déchanet-Merville, Julie; Capone, Myriam


    γδ T lymphocytes are unconventional immune cells, which have both innate- and adaptive-like features allowing them to respond to a wide spectrum of pathogens. For many years, we and others have reported on the role of these cells in the immune response to human cytomegalovirus in transplant patients, pregnant women, neonates, immunodeficient children, and healthy people. Indeed, and as described for CD8+ T cells, CMV infection leaves a specific imprint on the γδ T cell compartment: (i) driving a long-lasting expansion of oligoclonal γδ T cells in the blood of seropositive individuals, (ii) inducing their differentiation into effector/memory cells expressing a TEMRA phenotype, and (iii) enhancing their antiviral effector functions (i.e., cytotoxicity and IFNγ production). Recently, two studies using murine CMV (MCMV) have corroborated and extended these observations. In particular, they have illustrated the ability of adoptively transferred MCMV-induced γδ T cells to protect immune-deficient mice against virus-induced death. In vivo, expansion of γδ T cells is associated with the clearance of CMV infection as well as with reduced cancer occurrence or leukemia relapse risk in kidney transplant patients and allogeneic stem cell recipients, respectively. Taken together, all these studies show that γδ T cells are important immune effectors against CMV and cancer, which are life-threatening diseases affecting transplant recipients. The ability of CMV-induced γδ T cells to act independently of other immune cells opens the door to the development of novel cellular immunotherapies that could be particularly beneficial for immunocompromised transplant recipients.

  19. Hitchhiking nanoparticles: Reversible coupling of lipid-based nanoparticles to cytotoxic T lymphocytes. (United States)

    Wayteck, Laura; Dewitte, Heleen; De Backer, Lynn; Breckpot, Karine; Demeester, Jo; De Smedt, Stefaan C; Raemdonck, Koen


    Following intravenous injection of anti-cancer nanomedicines, many barriers need to be overcome en route to the tumor. Cell-mediated delivery of nanoparticles (NPs) is promising in terms of overcoming several of these barriers based on the tumoritropic migratory properties of particular cell types. This guided transport aims to enhance the NP accumulation in the tumor and moreover enhance the infiltration of regions that are typically inaccessible for free NPs. Within this study, cytotoxic CD8(+) T cells were selected as carriers based on both their ability to migrate to the tumor and their intrinsic cytolytic activity against tumor cells. Many anti-cancer nanomedicines require tumor cell internalization to mediate cytosolic drug delivery and enhance the anti-cancer effect. This proof-of-concept therefore reports on the reversible attachment of liposomes to the surface of cytotoxic T lymphocytes via a reduction sensitive coupling. The activation status of the T cells and the liposome composition are shown to strongly influence the loading efficiency. Loading the cells with liposomes does not compromise T cell functionalities like proliferation and cytolytic function. Additionally, the triggered liposome release is demonstrated upon the addition of glutathione. Based on this optimization using liposomes as model NPs, a small interfering RNA (siRNA)-loaded NP was developed that can be coupled to the surface of CD8(+) T cells.

  20. The adaptor molecule SAP plays essential roles during invariant NKT cell cytotoxicity and lytic synapse formation. (United States)

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


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

  1. Ex vivo-expanded cynomolgus macaque regulatory T cells are resistant to alemtuzumab-mediated cytotoxicity. (United States)

    Dons, E M; Raimondi, G; Zhang, H; Zahorchak, A F; Bhama, J K; Lu, L; Ezzelarab, M; Ijzermans, J N M; Cooper, D K C; Thomson, A W


    Alemtuzumab (Campath-1H) is a humanized monoclonal antibody (Ab) directed against CD52 that depletes lymphocytes and other leukocytes, mainly by complement-dependent mechanisms. We investigated the influence of alemtuzumab (i) on ex vivo-expanded cynomolgus monkey regulatory T cells (Treg) generated for prospective use in adoptive cell therapy and (ii) on naturally occurring Treg following alemtuzumab infusion. Treg were isolated from PBMC and lymph nodes and expanded for two rounds. CD52 expression, binding of alemtuzumab and both complement-mediated killing and Ab-dependent cell-mediated cytotoxicity (ADCC) were compared between freshly isolated and expanded Treg and effector T cells. Monkeys undergoing allogeneic heart transplantation given alemtuzumab were monitored for Treg and serum alemtuzumab activity. Ex vivo-expanded Treg showed progressive downregulation of CD52 expression, absence of alemtuzumab binding, minimal change in complement inhibitory protein (CD46) expression and no complement-dependent killing or ADCC. Infusion of alemtuzumab caused potent depletion of all lymphocytes, but a transient increase in the incidence of circulating Treg. After infusion of alemtuzumab, monkey serum killed fresh PBMC, but not expanded Treg. Thus, expanded cynomolgus monkey Treg are resistant to alemtuzumab-mediated, complement-dependent cytotoxicity. Furthermore, our data suggest that these expanded monkey Treg can be infused into graft recipients given alemtuzumab without risk of complement-mediated killing.

  2. The cytotoxic effect of denture base polymers. (United States)

    Hensten-Pettersen, A; Wictorin, L


    The cytotoxic potential of autopolymerized pour and dough type resins and heat cured resins was studied by in vitro cell culture techniques. Human epithelial cells (NCTC 2544) were grown in Eagle's minimal essential medium on the surface of the polymer disks. The cell multiplication on the surface of the specimens was measured. One heat cured resin and one pour type resin demonstrated a slight cytotoxic effect. The other polymers gave a moderate cytotoxic effect. The study did not indicate any difference in the cytotoxicity of the polymers when manufactured by alternate processing methods.

  3. Cytotoxicity of Ustilago maydis isolated from maize

    Directory of Open Access Journals (Sweden)

    Magdalena Twarużek


    Full Text Available The main pathogen of maize are fungi of the genus Fusarium. Besides phytopathogenic Fusarium, Ustilago maydis is another fungal genus affecting maize yields, causing lesions, known as smut. The objective of the study was evaluation of the cytotoxicity of Ustilago maydis isolated from maize. Nine Ustilago maydis strains were selected to a detailed evaluation of their cytotoxicity using a 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT test. Ustilago maydis strains showed medium and high cytotoxicity compared to control. High levels of cytotoxicity of Ustilago maydis may be indicative of their toxigenic potential.

  4. Tumor specific cytotoxicity of glucosylceramide. (United States)

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


    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.

  5. Cytotoxicity of Mycoplasma pneumoniae Membranes (United States)

    Gabridge, Michael G.; Johnson, Cynthia K.; Cameron, Alexander M.


    Organ cultures of adult hamster trachea were used to evaluate the cytotoxic potential of cell fractions of Mycoplasma pneumoniae. Cytoplasm was essentially devoid of activity, whereas viable cells and membrane preparations, at a level of 25 μg of protein per ml, induced necrosis. Damage, as revealed by light and electron microscopy, included ciliostasis, vacuolization, loss of ciliated respiratory epithelial cells, disorganization, and a loss of polarity. Dose response data indicated that the speed and degree of cytotoxicity was directly related to the concentration of membranes. Doses of 30 to 60 μg of protein per ml could reduce relative ciliary activity to 20% of the control level within 4 days. Membranes prepared after freeze-thaw lysis of cells were almost twice as active as those isolated after a combination of osmotic and sonic shock. Membranes of M. fermentans were inactive, though both the FH and M129 strains of M. pneumoniae were toxic. These data indicate that the toxic factor responsible for M. pneumoniae may be located in the cell membrane. Images PMID:16558100

  6. Induction of potent NK cell-dependent anti-myeloma cytotoxic T cells in response to combined mapatumumab and bortezomib. (United States)

    Neeson, Paul J; Hsu, Andy K; Chen, Yin R; Halse, Heloise M; Loh, Joanna; Cordy, Reece; Fielding, Kate; Davis, Joanne; Noske, Josh; Davenport, Alex J; Lindqvist-Gigg, Camilla A; Humphreys, Robin; Tai, Tsin; Prince, H Miles; Trapani, Joseph A; Smyth, Mark J; Ritchie, David S


    There is increasing evidence that some cancer therapies can promote tumor immunogenicity to boost the endogenous antitumor immune response. In this study, we used the novel combination of agonistic anti-TRAIL-R1 antibody (mapatumumab, Mapa) with low dose bortezomib (LDB) for this purpose. The combination induced profound myeloma cell apoptosis, greatly enhanced the uptake of myeloma cell apoptotic bodies by dendritic cell (DC) and induced anti-myeloma cytotoxicity by both CD8(+) T cells and NK cells. Cytotoxic lymphocyte expansion was detected within 24 h of commencing therapy and was maximized when myeloma-pulsed DC were co-treated with low dose bortezomib and mapatumumab (LDB+Mapa) in the presence of NK cells. This study shows that Mapa has two distinct but connected modes of action against multiple myeloma (MM). First, when combined with LDB, Mapa produced powerful myeloma cell apoptosis; secondly, it promoted DC priming and an NK cell-mediated expansion of anti-myeloma cytotoxic lymphocyte (CTL). Overall, this study indicates that Mapa can be used to drive potent anti-MM immune responses.

  7. A cytotoxic substance from Sangre de Grado. (United States)

    Itokawa, H; Ichihara, Y; Mochizuki, M; Enomori, T; Morita, H; Shirota, O; Inamatsu, M; Takeya, K


    Taspine has been isolated as a cytotoxic substance from Sangre de Grado, sap of Croton palanostigma (Euphorbiaceae), by bioassay guided fractionation. The cytotoxicity (IC50) of taspine was found to be 0.39 microgram/ml against KB cells and 0.17 microgram/ml against V-79 cells.

  8. Differential cell-mediated immune response to S. mutans in children with low and high dental caries. (United States)

    Parkash, H; Sharma, A; Banerjee, U; Sidhu, S S; Sundaram, K R


    Role of cell-mediated immune response (CMI) in dental caries was studied in 171 subjects, comprising of 86 children with low caries (LC), 31 with high caries (HC), and 54 age matched controls. [3H]thymidine mediated lymphoblast transformation test (LTT) using mutans streptococci antigen as stimulant was used to study the stimulation index (SI) of in vitro cultured lymphocytes from these children. The analysis revealed low stimulation index in high caries children whereas low caries children exhibited high stimulation index normally ranging between 2 to 6. The findings indicated that low caries children had strong CMI response as compared to high caries children. Although, the findings are based on limited number of samples, it certainly lays emphasis on protective or regulatory role of CMI in different phases of dental caries.

  9. Effect of renal and non-renal ischemia/reperfusion on cell-mediated immunity in organs and plasma

    DEFF Research Database (Denmark)

    Brøchner, Anne Craveiro; Dagnæs-Hansen, Frederik; Toft, Palle


    study, 80 mice were divided into four groups. The following surgeries were performed on the groups compared: bilateral renal I/R by clamping, unilateral renal ischemia, anesthesia only, and unilateral hind leg I/R. Half of the animals were killed after 2 h and the other half after 24 h. To assess...... following renal I/R. All kinds of I/R induced an upregulation of the adhesion molecule CD 11b and a downregulation of MHC II. Renal and non-renal I/R induced neutrophil infiltration in distant organs. Renal I/R does not induce a larger cell-mediated inflammatory response in blood and organs than non-renal I/R....

  10. The role of cytokines and chemokines in the T-cell-mediated autoimmune process in alopecia areata. (United States)

    Ito, Taisuke; Tokura, Yoshiki


    The aetiology of alopecia areata (AA) is still not fully understood. However, recent clinical and experimental studies have provided insights into the pathomechanisms of AA and revealed that it is an organ-specific and cell-mediated autoimmune disease. Some triggers, such as viral infections, trauma, hormones and emotional/physical stressors, may cause activation of autoreactive T cells that target hair follicle (HF) autoantigens. In these immunological responses, cytokines and chemokines are regarded as key players that mediate the autoimmune inflammation. This results in the collapse of HF immune privilege, which is central to the pathogenesis of AA. This essay will focus on how cytokines and chemokines contribute to the immunological aspects of AA. The management of AA often remains difficult in a number of cases. Our review suggests that novel therapies for AA may involve targeting cytokines and chemokines.

  11. Memory T-Cell-Mediated Immune Responses Specific to an Alternative Core Protein in Hepatitis C Virus Infection (United States)

    Bain, Christine; Parroche, Peggy; Lavergne, Jean Pierre; Duverger, Blandine; Vieux, Claude; Dubois, Valérie; Komurian-Pradel, Florence; Trépo, Christian; Gebuhrer, Lucette; Paranhos-Baccala, Glaucia; Penin, François; Inchauspé, Geneviève


    In vitro studies have described the synthesis of an alternative reading frame form of the hepatitis C virus (HCV) core protein that was named F protein or ARFP (alternative reading frame protein) and includes a domain coded by the +1 open reading frame of the RNA core coding region. The expression of this protein in HCV-infected patients remains controversial. We have analyzed peripheral blood from 47 chronically or previously HCV-infected patients for the presence of T lymphocytes and antibodies specific to the ARFP. Anti-ARFP antibodies were detected in 41.6% of the patients infected with various HCV genotypes. Using a specific ARFP 99-amino-acid polypeptide as well as four ARFP predicted class I-restricted 9-mer peptides, we show that 20% of the patients display specific lymphocytes capable of producing gamma interferon, interleukin-10, or both cytokines. Patients harboring three different viral genotypes (1a, 1b, and 3) carried T lymphocytes reactive to genotype 1b-derived peptides. In longitudinal analysis of patients receiving therapy, both core and ARFP-specific T-cell- and B-cell-mediated responses were documented. The magnitude and kinetics of the HCV antigen-specific responses differed and were not linked with viremia or therapy outcome. These observations provide strong and new arguments in favor of the synthesis, during natural HCV infection, of an ARFP derived from the core sequence. Moreover, the present data provide the first demonstration of the presence of T-cell-mediated immune responses directed to this novel HCV antigen. PMID:15367612

  12. Effect of rosemary (Rosmarinus officinalis extract on weight, hematology and cell-mediated immune response of newborn goat kids

    Directory of Open Access Journals (Sweden)

    Borhan Shokrollahi


    Full Text Available This study aimed at evaluating the effects of different levels of rosemary (Rosmarinus officinalis extract on growth rate, hematology and cell-mediated immune response in Markhoz newborn goat kids. Twenty four goat kids (aged 7±3 days were randomly allotted to four groups with six replicates. The groups included: control, T1, T2 and T3 groups which received supplemented-milk with 0, 100, 200 and 400mg aqueous rosemary extract per kg of live body weight per day for 42 days. Body weights of kids were measured weekly until the end of the experiment. On day 42, 10 ml blood samples were collected from each kid through the jugular vein. Cell-mediated immune response was assessed through the double skin thickness after intradermal injection of phyto-hematoglutinin (PHA at day 21 and 42. No significant differences were seen in initial body weight, average daily gain (ADG and total gain. However, significant differences in globulin (P<0.05, and white blood cells (WBC (P<0.001 were observed. There were no significant differences in haemoglobin (Hb, packed cell volume (PCV, red blood cells (RBC, lymphocytes and neutrophils between the treatments. Skin thickness in response to intra dermal injection of PHA significantly increased in the treated groups as compared to the control group at day 42 (P<0.01 with the T3 group showing the highest response to PHA injection. In conclusion, the results indicated that aqueous rosemary extract supplemented-milk had a positive effect on immunity and skin thickness of newborn goat kids.

  13. Cytotoxic Effects of Bangladeshi Medicinal Plant Extracts

    Directory of Open Access Journals (Sweden)

    Shaikh J. Uddin


    Full Text Available To investigate the cytotoxic effect of some Bangladeshi medicinal plant extracts, 16 Bangladeshi medicinal plants were successively extracted with n-hexane, dichloromethane, methanol and water. The methanolic and aqueous extracts were screened for cytotoxic activity against healthy mouse fibroblasts (NIH3T3 and three human cancer-cell lines (gastric: AGS; colon: HT-29; and breast: MDA-MB-435S using the MTT assay. Two methanolic extracts (Hygrophila auriculata and Hibiscus tiliaceous and one aqueous extract (Limnophila indica showed no toxicity against healthy mouse fibroblasts, but selective cytotoxicity against breast cancer cells (IC50 1.1–1.6 mg mL−1. Seven methanolic extracts from L. indica, Clerodendron inerme, Cynometra ramiflora, Xylocarpus moluccensis, Argemone mexicana, Ammannia baccifera and Acrostichum aureum and four aqueous extracts from Hygrophila auriculata, Bruguiera gymnorrhiza, X. moluccensis and Aegiceras corniculatum showed low toxicity (IC50 > 2.5 mg mL−1 against mouse fibroblasts but selective cytotoxicity (IC50 0.2–2.3 mg mL−1 against different cancer cell lines. The methanolic extract of Blumea lacera showed the highest cytotoxicity (IC50 0.01–0.08 mg mL−1 against all tested cell lines among all extracts tested in this study. For some of the plants their traditional use as anticancer treatments correlates with the cytotoxic results, whereas for others so far unknown cytotoxic activities were identified.

  14. Cytotoxicity of Southeast Asian snake venoms

    Directory of Open Access Journals (Sweden)

    A Jamunaa


    Full Text Available Cytotoxicity of venoms from eleven medically important snakes found in Southeast Asia (Naja kaouthia, Naja siamensis, Naja sumatrana, Ophiophagus hannah, Bungarus candidus, Bungarus fasciatus, Enhydrina schistosa, Calloselasma rhodostoma, Trimeresurus purpureomaculatus and Tropidolaemus sumatranus was determined, based on the MTS cytotoxicity assay, which determines the survival of viable cells in monolayer MDCK and Vero cell cultures upon exposure to the snake venoms. Snake venom toxicity was expressed as the venom dose that killed 50% of the cells (CTC50 under the assay conditions. Venoms of C. rhodostoma (2.6 µg/mL, 1.4 µg/mL and O. hannah were the most cytotoxic (3.8 µg/mL, 1.7 µg/mL whereas N. siamensis venom showed the least cytotoxicity (51.9 µg/mL, 45.7 µg/mL against Vero and MDCK cells, respectively. All the viper venoms showed higher cytotoxic potency towards both Vero and MDCK cell lines, in comparison to krait and cobra venoms. E. schistosa did not cause cytotoxicity towards MDCK or Vero cells at the tested concentrations. The cytotoxicity correlates well with the known differences in the composition of venoms from cobras, kraits, vipers and sea snakes.

  15. Serial dilution microchip for cytotoxicity test (United States)

    Bang, Hyunwoo; Lim, Sun Hee; Lee, Young Kyung; Chung, Seok; Chung, Chanil; Han, Dong-Chul; Chang, Jun Keun


    Today's pharmaceutical industry is facing challenges resulting from the vast increases in sample numbers produced by high-throughput screening (HTS). In addition, the bottlenecks created by increased demand for cytotoxicity testing (required to assess compound safety) are becoming a serious problem. We have developed a polymer PDMS (polydimethylsiloxane) based microfluidic device that can perform a cytotoxicity test in a rapid and reproducible manner. The concept that the device includes is well adjustable to automated robots in huge HTS systems, so we can think of it as a potential dilution and delivery module. Cytotoxicity testing is all about the dilution and dispensing of a drug sample. Previously, we made a PDMS based microfluidic device which automatically and precisely diluted drugs with a buffer solution with serially increasing concentrations. This time, the serially diluted drug solution was directly delivered to 96 well plates for cytotoxicity testing. Cytotoxic paclitaxel solution with 2% RPMI 1640 has been used while carrying out cancerous cell based cytotoxicity tests. We believe that this rapid and robust use of the PDMS microchip will overcome the growing problem in cytotoxicity testing for HTS.

  16. Characteristics of Cell-mediated, Anti-listerial Immunity Induced by A Naturally Avirulent Listeria monocytogenes Serotype 4a Strain HCC23 (United States)

    The characteristics of cell-mediated, anti-listerial immune response initiated by an avirulent Listeria monocytogenes serotype 4a strain HCC23 was assessed. Similar to virulent strain EGD, avirulent strain HCC23 grew readily within macrophage-like J774 cells, but nonhemolytic strain ATCC 15313 did n...

  17. Isolation of Mallory bodies and an attempt to demonstrate cell mediated immunity to Mallory body isolate in patients with alcoholic liver disease

    DEFF Research Database (Denmark)

    Gluud, C; Hardt, F; Aldershvile, J;


    in haematoxylin-eosin stained smears. Electron microscopy confirmed the presence of Mallory bodies in the isolates. The Mallory body isolate was used as antigen in the agarose leucocyte migration inhibition test in order to test the cell-mediated immunity. No significant difference in leucocyte migration...

  18. Caloric Restriction reduces inflammation and improves T cell-mediated immune response in obese mice but concomitant consumption of curcumin/piperine adds no further benefit (United States)

    Obesity is associated with low-grade inflammation and impaired immune response. Caloric restriction (CR) has been shown to inhibit inflammatory response and enhance cell-mediated immune function. Curcumin, the bioactive phenolic component of turmeric spice, is proposed to have anti-obesity and anti-...

  19. CD4+ type II NKT cells mediate ICOS and programmed death-1-dependent regulation of type 1 diabetes

    DEFF Research Database (Denmark)

    Kadri, Nadir; Korpos, Eva; Gupta, Shashank


    Type 1 diabetes (T1D) is a chronic autoimmune disease that results from T cell-mediated destruction of pancreatic ß cells. CD1d-restricted NKT lymphocytes have the ability to regulate immunity, including autoimmunity. We previously demonstrated that CD1d-restricted type II NKT cells, which carry ...

  20. Comparative efficiency of HIV-1-infected T cell killing by NK cells, monocytes and neutrophils. (United States)

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


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

  1. The cytotoxic activity of ursolic acid derivatives. (United States)

    Ma, Chao-Mei; Cai, Shao-Qing; Cui, Jing-Rong; Wang, Rui-Qing; Tu, Peng-Fei; Hattori, Masao; Daneshtalab, Mohsen


    Ursolic acid and 2alpha-hydroxyursolic acid isolated from apple peels were found to show growth inhibitory activity against four tumor cell lines, HL-60, BGC, Bel-7402 and Hela. Structural modifications were performed on the C-3, C-28 and C-11 positions of ursolic acid and the cytotoxicity of the derivatives was evaluated. The SAR revealed that the triterpenes possessing two hydrogen-bond forming groups (an H-donor and a carbonyl group) at positions 3 and 28 exhibit cytotoxic activity. The configuration at C-3 was found to be important for the activity. Introduction of an amino group increased the cytotoxicity greatly. A 3beta-amino derivative was 20 times more potent than the parent ursolic acid. The 28-aminoalkyl dimer compounds showed selective cytotoxicity.

  2. 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


    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.

  3. Cell-mediated lympholysis of trinitrophenyl-modified autologous lymphocytes. Effector cell specificity to modified cell surface components controlled by H-2K and H-2D serological regions of the murine major histocompatibility complex. (United States)

    Shearer, G M; Rehn, T G; Garbarino, C A


    Splenic lymphocytes from four C57BL/10 congenic resistant mouse strains were sensitized in vitro with trinitrophenyl (TNP)-modified autologous spleen cellsmthe effector cells generated were incubated with 51-Cr-labeled unmodified or TNP-modified spleen or tumor target cells, and the percentage of specific lympholysis determined. The results obtained using syngeneic-, congenic-, recombinante, and allogeneic-modified target cells indicated that TNP modification of the target cells was a necessary but insufficient requirement for lympholysis. Intra-H-2 homology either between modified stimulating cells and modified target cells or between responding lymphocytes and modified target cells was also important in the specificity for lysis. Homology at the K serological region or at K plus I-A in the B10.A and B10BR strains, and at either the D serological region or at some other region (possibly K) in the B10.D2 and C57BL/10 strains were shown to be necessary in order to detect lympholysis. Experiments using (B10itimes C57BL/10)F1 responding lymphocytes sensitized and assayed with TNP-modified parental cells indicated that the homology required for lympholysis was between modified stimulating and modified target cellsmthe possibility is raised that histocompatibility antigens may serve in the autologous system as cell surface components which are modified by viruses or autoimmune complexes to form cell-bound modified-self antigens, which are particularly suited for cell-mediated immune reactions. Evidence is presented suggesting that H-2-linked Ir genes are expressed in the TNP-modified autologous cytotoxic system. These findings imply that the major histocompatibility complex can be functionally involved both in the response potential to and in the formation of new antigenic determinants involving modified-self components.

  4. Upregulation of CD54 and downregulation of HLA‑ABC contribute to the novel enhancement of the susceptibility of HL-60 cells to NK cell-mediated cytolysis induced by ATRA plus VPA. (United States)

    Zou, Huijuan; Li, Lianlian; Han, Yang; Ma, Ruiping; Liao, Qiong; Tian, Jing; Zhang, Xiaoyu; Ren, Xia; Song, Guanhua; Guo, Qiang; Li, Xia; Ding, Huifang; Jiang, Guosheng


    Enhancement of the susceptibility of HL-60 cells to NK cell-mediated cytolysis induced by all-trans-retinoic acid (ATRA) plus valproate (VPA) was evaluated. In addition to the synergistic effect of ATRA plus VPA on HL-60 cells, the optimal concentration of 1 mM VPA plus 0.5 µM ATRA increased the cytotoxic sensitivity of HL-60 cells to NK cells. The expression of the activated receptors NKp30 and NKG2D on NK-92 cells was higher compared with the levels noted for the other receptors, and the expression of NKG2D ligands MICA/B on HL-60 cells was not significantly upregulated in the ATRA plus VPA goup compared with the control. Moreover, it was observed that the ligands of NKp30 on HL-60 cells presented the same variation trend. As to the co-stimulatory and adhesion molecules on NK-92 and their ligands on HL-60 cells post exposure to ATRA and VPA alone or their combination, there was no obvious change in the expression of CD112, CD48 and CD70 on the HL-60 cells. However, the expression of CD54 on HL-60 cells was significantly upregulated. In contrast, the expression of NKG2A ligands HLA-ABC on HL-60 cells was obviously downregulated. In addition, the expression of HLA-E on the HL-60 cells in the group treated with ATRA plus VPA was not significantly increased. In conclusion, the combination of VPA and ATRA not only induced the differentiation of HL-60 cells, but also induced enhancement of the sensitivity of HL-60 cells to NK cells by downregulating the expression of HLA-ABC and upregulating the expression of CD54, but not MICA/MICB. The results provide experimental and theoretical basis for the clinical combination of a low-dose of ATRA plus VPA for the treatment of leukemia.

  5. Host genetic background influences the response to the opportunistic Pseudomonas aeruginosa infection altering cell-mediated immunity and bacterial replication.

    Directory of Open Access Journals (Sweden)

    Maura De Simone

    Full Text Available Pseudomonas aeruginosa is a common cause of healthcare-associated infections including pneumonia, bloodstream, urinary tract, and surgical site infections. The clinical outcome of P. aeruginosa infections may be extremely variable among individuals at risk and patients affected by cystic fibrosis. However, risk factors for P. aeruginosa infection remain largely unknown. To identify and track the host factors influencing P. aeruginosa lung infections, inbred immunocompetent mouse strains were screened in a pneumonia model system. A/J, BALB/cJ, BALB/cAnNCrl, BALB/cByJ, C3H/HeOuJ, C57BL/6J, C57BL/6NCrl, DBA/2J, and 129S2/SvPasCRL mice were infected with P. aeruginosa clinical strain and monitored for body weight and mortality up to seven days. The most deviant survival phenotypes were observed for A/J, 129S2/SvPasCRL and DBA/2J showing high susceptibility while BALB/cAnNCrl and C3H/HeOuJ showing more resistance to P. aeruginosa infection. Next, one of the most susceptible and resistant mouse strains were characterized for their deviant clinical and immunological phenotype by scoring bacterial count, cell-mediated immunity, cytokines and chemokines profile and lung pathology in an early time course. Susceptible A/J mice showed significantly higher bacterial burden, higher cytokines and chemokines levels but lower leukocyte recruitment, particularly neutrophils, when compared to C3H/HeOuJ resistant mice. Pathologic scores showed lower inflammatory severity, reduced intraluminal and interstitial inflammation extent, bronchial and parenchymal involvement and diminished alveolar damage in the lungs of A/J when compared to C3H/HeOuJ. Our findings indicate that during an early phase of infection a prompt inflammatory response in the airways set the conditions for a non-permissive environment to P. aeruginosa replication and lock the spread to other organs. Host gene(s may have a role in the reduction of cell-mediated immunity playing a critical role in

  6. Host genetic background influences the response to the opportunistic Pseudomonas aeruginosa infection altering cell-mediated immunity and bacterial replication. (United States)

    De Simone, Maura; Spagnuolo, Lorenza; Lorè, Nicola Ivan; Rossi, Giacomo; Cigana, Cristina; De Fino, Ida; Iraqi, Fuad A; Bragonzi, Alessandra


    Pseudomonas aeruginosa is a common cause of healthcare-associated infections including pneumonia, bloodstream, urinary tract, and surgical site infections. The clinical outcome of P. aeruginosa infections may be extremely variable among individuals at risk and patients affected by cystic fibrosis. However, risk factors for P. aeruginosa infection remain largely unknown. To identify and track the host factors influencing P. aeruginosa lung infections, inbred immunocompetent mouse strains were screened in a pneumonia model system. A/J, BALB/cJ, BALB/cAnNCrl, BALB/cByJ, C3H/HeOuJ, C57BL/6J, C57BL/6NCrl, DBA/2J, and 129S2/SvPasCRL mice were infected with P. aeruginosa clinical strain and monitored for body weight and mortality up to seven days. The most deviant survival phenotypes were observed for A/J, 129S2/SvPasCRL and DBA/2J showing high susceptibility while BALB/cAnNCrl and C3H/HeOuJ showing more resistance to P. aeruginosa infection. Next, one of the most susceptible and resistant mouse strains were characterized for their deviant clinical and immunological phenotype by scoring bacterial count, cell-mediated immunity, cytokines and chemokines profile and lung pathology in an early time course. Susceptible A/J mice showed significantly higher bacterial burden, higher cytokines and chemokines levels but lower leukocyte recruitment, particularly neutrophils, when compared to C3H/HeOuJ resistant mice. Pathologic scores showed lower inflammatory severity, reduced intraluminal and interstitial inflammation extent, bronchial and parenchymal involvement and diminished alveolar damage in the lungs of A/J when compared to C3H/HeOuJ. Our findings indicate that during an early phase of infection a prompt inflammatory response in the airways set the conditions for a non-permissive environment to P. aeruginosa replication and lock the spread to other organs. Host gene(s) may have a role in the reduction of cell-mediated immunity playing a critical role in the control of P

  7. Cytotoxic Activity of Selected Nigerian Plants


    Sowemimo, A; Venter, M.; Baatjies, L; Koekemoer, T


    Cancer is one of the most prominent human diseases which has stimulated scientific and commercial interest in the discovery of new anticancer agents from natural sources. The current study investigates the cytotoxic activity of ethanolic extracts of sixteen Nigerian plants used locally for the treatment of cancer using the MTT assay on the HeLa cell line. Sapium ellipticum leaves showed activity comparable to the reference compound Cisplatin and greater cytotoxic activity than Combretum panic...

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

    Directory of Open Access Journals (Sweden)

    Ilseyar Akhmetzyanova


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


    DEFF Research Database (Denmark)

    Rasmussen, Josephine Skovgaard; Riber, Ulla; Lauritsen, Klara Tølbøll;

    -antigen and recombinant IL-18, significantly higher level of IFN-γ was produced in the vaccine group compared to the placebo group one day before challenge with Mhs. In contrast, significantly higher level of IFN-γ was measured in the placebo group compared to the vaccine group at day 6 after challenge. The latter could...... (Germany) September 10-13, 2009), testing a new vaccine (formalin inactivated Mhs and EMULSIGEN-BCL® (MVP Laboratories)), cell-mediated immune response was measured in the vaccine group (n=13) and not in the placebo group (n=13). In an IFN-γ assay, where whole-blood was co-cultured with Mhs...... to be CD4 and especially CD4CD8 double positive T-cells simultaneously expressing CD25. Interestingly, the proportion of CD4CD8 double positive T-cells within the total population of CD4 positive cells increased in the vaccine group after challenge, indicating that generation of specific T-cell memory had...

  10. Empirical evidence of cold stress induced cell mediated and humoral immune response in common myna ( Sturnus tristis) (United States)

    Sandhu, Mansur A.; Zaib, Anila; Anjum, Muhammad S.; Qayyum, Mazhar


    Common myna ( Sturnus tristis) is a bird indigenous to the Indian subcontinent that has invaded many parts of the world. At the onset of our investigation, we hypothesized that the immunological profile of myna makes it resistant to harsh/new environmental conditions. In order to test this hypothesis, a number of 40 mynas were caught and divided into two groups, i.e., 7 and 25 °C for 14 days. To determine the effect of cold stress, cell mediated and humoral immune responses were assessed. The macrophage engulfment percentage was significantly ( P blood cells (SRBC). Macrophage engulfment/cell and nitric oxide production behaved in a similar manner. However, splenic cells plaque formation, heterophil to lymphocyte (H/L) ratio, and serum IgM or IgG production remained non-significant. There was a significant increase of IgG antibody production after a second immunization by SRBC. To the best of our knowledge, these findings have never been reported in the progression of this bird's invasion in frosty areas of the world. The results revealed a strengthened humoral immune response of myna and made this bird suitable for invasion in the areas of harsh conditions.

  11. Inter- and intra-individual variation in tests of cell-mediated immunity in young and old women. (United States)

    Molls, Roshni R; Ahluwalia, Namanjeet; Fick, Tara; Mastro, Andrea M; Wagstaff, David; Handte, Gordon; Ball, Rick


    Exploring means to maintain or improve immunity in older persons has been receiving attention. To establish relationships between immune function and variables of interest, it is important to determine these variables accurately and precisely. Precision relates to the degree of variation in the laboratory test. The nature and magnitude of variability in tests of immune function has not been described extensively. We examined inter- and intra-individual variation in tests of cell-mediated immunity (CMI) in generally healthy and well-nourished young (20-40 years; n=15) and old (60-80 years; n=15) women. Subjects provided blood samples on 2 days within a week to determine leukocyte subsets, T-cell proliferation response to phytohemagglutinin A and concanavalin A, and interleukin (IL)-1beta, IL-2 and IL-6 production by stimulated mononuclear cells. Intra-individual variation was partitioned into day-to-day biological and analytical variation. Inter-individual variation was greater than intra-individual variability for most tests of CMI for both age groups. Furthermore, all CMI tests exhibited large day-to-day intra-individual variation (CV approximately 15% or greater) which was primarily due to biological rather than analytical sources, for both age groups. In conclusion, both age groups showed large between-person and considerable within-person variation in CMI tests. Therefore, assessment of CMI based on a single blood draw may not provide a reliable estimate of immune function.

  12. The Ameliorative Effect of Sophoricoside on Mast Cell-Mediated Allergic Inflammation in Vivo and in Vitro

    Directory of Open Access Journals (Sweden)

    Jae-Young Um


    Full Text Available Sophoricoside exhibits numerous pharmacological effects, including anti- inflammatory and anti-cancer actions, yet the exact mechanism that accounts for the anti-allergic effects of sophoricoside is not completely understood. The aim of the present study was to elucidate whether and how sophoricoside modulates the mast cell-mediated allergic inflammation in vitro and in vivo. We investigated the pharmacological effects of sophoricoside on both compound 48/80 or histamine-induced scratching behaviors and 2,4-dinitrochlorobenzene (DNCB-induced atopic dermatitis in mice. Additionally, to find a possible explanation for the anti-inflammatory effects of sophoricoside, we evaluated the effects of sophoricoside on the production of histamine and inflammatory cytokines and activation of nuclear factor-κB (NF-κB and caspase-1 in phorbol 12-myristate 13-acetate plus calcium ionophore A23187 (PMACI-stimulated human mast cells (HMC-1. The finding of this study demonstrated that sophoricoside reduced compound 48/80 or histamine-induced scratching behaviors and DNCB-induced atopic dermatitis in mice. Additionally, sophoricoside inhibited the production of inflammatory cytokines as well as the activation of NF-κB and caspase-1 in stimulated HMC-1. Collectively, the findings of this study provide us with novel insights into the pharmacological actions of sophoricoside as a potential molecule for use in the treatment of allergic inflammation diseases.

  13. IL-7 inhibits tumor growth by promoting T cell-mediated antitumor immunity in Meth A model. (United States)

    Tang, Jian-Cai; Shen, Guo-Bo; Wang, Shi-Min; Wan, Yong-Sheng; Wei, Yu-Quan


    Immune suppression is well documented during tumor progression, which includes loss of effect of T cells and expansion of T regulatory (Treg) cells. IL-7 plays a key role in the proliferation, survival and homeostasis of T cells and displays a potent antitumor activity in vivo. In the present study, we investigated the antitumor effect of IL-7 in Meth A model. IL-7 inhibited tumor growth and prolonged the survival of tumor-bearing mice with corresponding increases in the frequency of CD4 and CD8 T cells, Th1 (CD4(+)IFN-γ(+)), Tc1 (CD8(+)IFN-γ(+)) and T cells cytolytic activity against Meth A cells. Neutralization of CD4 or CD8 T cells reversed the antitumor benefit of IL-7. Furthermore, IL-7 decreased regulatory T Foxp3 as well as cells suppressive activity with a reciprocal increase in SMAD7. In addition, we observed an increase of the serum concentrations of IL-6 and IFN-γ, and a significant decrease of TGF-β and IL-10 after IL-7 treatment. Taken together, these results indicate that IL-7 augments T cell-mediated antitumor immunity and improves the effect of antitumor in Meth A model.

  14. A Possible Role for CD8+ T Lymphocytes in the Cell-Mediated Pathogenesis of Pemphigus Vulgaris

    Directory of Open Access Journals (Sweden)

    Federica Giurdanella


    Full Text Available Pemphigus vulgaris (PV is an autoimmune blistering disease whose pathogenesis involves both humoral and cell-mediated immune response. Though the pathogenetic role of autoantibodies directed against desmoglein 3 is certain, a number of other factors have been suggested to determine acantholysis in PV. In this study we examined the possible role of CD8+ T cells in the development of acantholysis by a passive transfer of PV autoantibodies using CD8 deficient mice, and we also studied the inflammatory infiltrate of PV skin lesions by immunohistochemical staining. The results of the immunohistochemical staining to study the expression of CD3, CD4, and CD8 in PV skin lesions showed that CD4+ are more expressed than CD8+ in the inflammatory infiltrate of PV lesions, confirming the data of the previous literature. The passive transfer study showed a lower incidence of pemphigus in the group of CD8 deficient mice compared to the control one of wild-type mice. These results suggest that CD8+ T cells may play a role in the pathogenesis of PV, perhaps through the Fas/FasL pathway.

  15. The cytotoxicity study of praziquantel enantiomers

    Directory of Open Access Journals (Sweden)

    Sun Q


    Full Text Available Qian Sun, Ruifeng Mao, Dongling Wang, Changyan Hu, Yang Zheng, Dequn Sun Department of Pharmacy, Marine College, Shandong University, Weihai, People’s Republic of China Abstract: Praziquantel (PZQ is prescribed as a racemic mixture (racemic-PZQ, rac-PZQ, which is composed of (R-PZQ and (S-PZQ. In this work, the cytotoxicity of rac-PZQ and its two enantiomers (R-PZQ and (S-PZQ on eight cell lines (L-02, HepG2, prf-plc-5, SH-SY5Y, HUVEC, A549, HCT-15, Raw264.7 was evaluated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays. The morphology of apoptotic cells was studied by fluorescence microscope using Hoechst 33342 staining, and the cytotoxicity of the compounds was also tested by lactate dehydrogenase assay. Results revealed that (R-PZQ had negligible cytotoxicity against L-02, SH-SY5Y, HUVEC, A549, HCT-15, and Raw264.7 cells but selectively inhibited tumor cell lines (prf-plc-5 and HepG2. However, in contrast to (R-PZQ, the (S-isomer showed higher cytotoxicity against L-02 cells and lower inhibition on prf-plc-5 and HepG2 cells. Besides, (R-PZQ showed lower cytotoxicity on SH-SY5Y cells than (S-PZQ. Meanwhile, (R-PZQ at <80 µM concentration could promote proliferation of macrophage cells (Raw264.7. Our research revealed that (R-PZQ has lower cytotoxicity than (S-PZQ and has similar cytotoxicity with rac-PZQ. (S-PZQ is the principal enantiomer to cause side effects on human definitive hosts. These findings gave the reasonable reasons for World Health Organization to produce (R-PZQ as a replacement for rac-PZQ for the treatment of schistosomiasis. Keywords: isomer, MTT, selectivity, (R-PZQ

  16. Cytotoxicity and intracellular dissolution of nickel nanowires

    KAUST Repository

    Perez, Jose E.


    The assessment of cytotoxicity of nanostructures is a fundamental step for their development as biomedical tools. As widely used nanostructures, nickel nanowires (Ni NWs) seem promising candidates for such applications. In this work, Ni NWs were synthesized and then characterized using vibrating sample magnetometry, energy dispersive X-Ray analysis and electron microscopy. After exposure to the NWs, cytotoxicity was evaluated in terms of cell viability, cell membrane damage and induced apoptosis/necrosis on the model human cell line HCT 116. The influence of NW to cell ratio (10:1 to 1000:1) and exposure times up to 72 hours was analyzed for Ni NWs of 5.4 µm in length, as well as for Ni ions. The results show that cytotoxicity markedly increases past 24 hours of incubation. Cellular uptake of NWs takes place through the phagocytosis pathway, with a fraction of the dose of NWs dissolved inside the cells. Cell death results from a combination of apoptosis and necrosis, where the latter is the outcome of the secondary necrosis pathway. The cytotoxicity of Ni ions and Ni NWs dissolution studies suggest a synergistic toxicity between NW aspect ratio and dissolved Ni, with the cytotoxic effects markedly increasing after 24 hours of incubation.

  17. Isoflavanones from Desmodium oxyphyllum and their cytotoxicity. (United States)

    Li, Yan-Ping; Li, Yin-Ke; Du, Gang; Yang, Hai-Yin; Gao, Xue-Mei; Hu, Qiu-Fen


    Two new isoflavanones, (3R)-7-hydroxy-4'-methoxy-5-methoxycarbonyl-isoflavanone (1) and (3R)-8-hydroxy-4'-methoxy-7-methoxycarbonyl-isoflavanone (2), together with seven known isoflavanones (3-9) were isolated from Desmodium oxyphyllum of the Leguminosae family. Their structures were elucidated by spectroscopic methods, including extensive 1D and 2D NMR techniques. Compound 1 showed good cytotoxicity against NB4 and SHSY5Y cell lines with IC50 values of 3.1 and 2.5 μM; compound 2 exhibited cytotoxicity against PC3 cell lines with a IC50 value of 3.6 μM; compound 4 showed cytotoxicity against A549 and SHSY5Y cell lines with IC50 values of 3.6 and 2.8 μM; and compound 5 displayed cytotoxicity against NB4, SHSY5Y, and MCF7 cell lines with IC50 values of 2.6, 3.8, and 2.8 μM, respectively. Other compounds also showed moderate cytotoxicity for some tested cell lines with IC50 values between 5.4 and 8.8 μM.

  18. Alterations of Cell-Mediated Immunity in Patients with Type 2 Diabetes Mellitus%2型糖尿病细胞免疫功能的变化

    Institute of Scientific and Technical Information of China (English)

    姚远; 郑佳; 杨敏


    Objective To investigate the alterations of cell- mediated immunity in patients with type 2 diabetes mellitus. Methods The level of CD3 CD4 CD8 in 30 normal subjects (NC group) and47 type 2 diabetes mellitus was measured by flow cytometry. Result Type - 2 diabetics had lower levels of CD4 and higher levels of CD8 than the non - diabetic control,especially during the 5 ~ 15 years of diabetes courses. The ratios of CD4 to CD8 was decreased. The correlation analysis showed that the level of CD3 CD4 CD8 and CD4/CD8 was not positively correlated with c - peptide. Conclusion Type - 2 diabeties have alterations of cell- mediated immunity.

  19. Opposing effects of CXCR3 and CCR5 deficiency on CD8+ T cell-mediated inflammation in the central nervous system of virus-infected mice

    DEFF Research Database (Denmark)

    de Lemos, Carina; Christensen, Jeanette Erbo; Nansen, Anneline;


    T cells play a key role in the control of viral infection in the CNS but may also contribute to immune-mediated cell damage. To study the redundancy of the chemokine receptors CXCR3 and CCR5 in regulating virus-induced CD8+ T cell-mediated inflammation in the brain, CXCR3/CCR5 double-deficient mice...... and therefore protect mice against the otherwise fatal CD8+ T cell-mediated immune attack. Contrary to expectations, the accumulation of mononuclear cells in cerebrospinal fluid was only slightly delayed compared with mice with normal expression of both receptors. Even more surprising, CXCR3/CCR5 double...... an important role in controlling CNS inflammation, other receptors but not CCR5 also contribute significantly. Additionally, our results suggest that CCR5 primarily functions as a negative regulator of the antiviral CD8+ T cell response....

  20. Inducible nitric-oxide synthase plays a minimal role in lymphocytic choriomeningitis virus-induced, T cell-mediated protective immunity and immunopathology

    DEFF Research Database (Denmark)

    Bartholdy, C; Nansen, A; Christensen, Jeanette Erbo;


    -mediated immune response was found to be unaltered in iNOS-deficient mice compared with wild-type C57BL/6 mice, and LCMV- induced general immunosuppression was equally pronounced in both strains. In vivo analysis revealed identical kinetics of virus clearance, as well as unaltered clinical severity of systemic......By using mice with a targetted disruption in the gene encoding inducible nitric-oxide synthase (iNOS), we have studied the role of nitric oxide (NO) in lymphocytic choriomeningitis virus (LCMV)-induced, T cell-mediated protective immunity and immunopathology. The afferent phase of the T cell....... This might suggest a role of NO in regulating vascular reactivity in the context of T cell-mediated inflammation. In conclusion, these findings indicate a minimal role for iNOS/NO in the host response to LCMV. Except for a reduced local oedema in the knockout mice, iNOS/NO seems to be redundant...

  1. Clinical study of non-specific cell mediated immunity in the patients with esophageal cancer. Influence of preoperative irradiation and surgical intervention

    Energy Technology Data Exchange (ETDEWEB)

    Nakata, Yoshitaka


    Few data are available to elucidate the influence of combined preoperative irradiation and surgery on the non-specific cell mediated immunity of patients with esophageal cancer. In vitro and in vivo examinations of the non-specific cell mediated immunity were made before and after irradiation and surgery in 108 patients with esophageal cancer. Decreased immune competence was noticeable one month after surgery in the irradiated group, as compared with the non-irradiated group. Simultaneously, the ratio of concanavalin A to phytohemagglutinin was significantly higher in the irradiated group than the non-irradiated group (p < 0.01). Two months later, both findings in the two groups were similar. There was no consistent tendency toward altered immune competence between the group with curative surgery and the group with non-curative surgery. (Namekawa, K.).

  2. Schwann Cell-Mediated Preservation of Vision in Retinal Degenerative Diseases via the Reduction of Oxidative Stress: A Possible Mechanism (United States)



    After injury to the central nervous system (CNS), regeneration is often inadequate, except in the case of remyelination. This remyelination capacity of the CNS is a good example of a stem/precursor cell-mediated renewal process. Schwann cells have been found to act as remyelinating agents in the peripheral nervous system (PNS), but several studies have highlighted their potential role in remyelination in the CNS too. Schwann cells are able to protect and support retinal cells by secreting growth factors such as brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and basic fibroblast growth factor. Retinal degenerative diseases can be highly debilitating, and they are a major concern in countries with an ageing populations. One of the leading causes of permanent loss of vision in the West is a retinal degenerative disease known as age-related macular degeneration (AMD). In the United States, nearly 1.75 million people over the age of 40 have advanced AMD, and it is estimated that this number will increase to approximately 3 million people by 2020. One of the most common pathways involved in the initiation and development of retinal diseases is the oxidative stress pathway. In patients with diabetes, Schwann cells have been shown to be able to secrete large amounts of antioxidant enzymes that protect the PNS from the oxidative stress that results from fluctuations in blood glucose levels. This antioxidant ability may be involved in the mechanism by which Schwann cells are able to promote reconstruction in the CNS, especially in individuals with retinal injuries and degenerative diseases. PMID:28293647

  3. Immunotoxicity of aflatoxin B1: impairment of the cell-mediated response to vaccine antigen and modulation of cytokine expression. (United States)

    Meissonnier, Guylaine M; Pinton, Philippe; Laffitte, Joëlle; Cossalter, Anne-Marie; Gong, Yun Yun; Wild, Christopher P; Bertin, Gérard; Galtier, Pierre; Oswald, Isabelle P


    Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus or A. parasiticus, is a frequent contaminant of food and feed. This toxin is hepatotoxic and immunotoxic. The present study analyzed in pigs the influence of AFB1 on humoral and cellular responses, and investigated whether the immunomodulation observed is produced through interference with cytokine expression. For 28 days, pigs were fed a control diet or a diet contaminated with 385, 867 or 1807 microg pure AFB1/kg feed. At days 4 and 15, pigs were vaccinated with ovalbumin. AFB1 exposure, confirmed by an observed dose-response in blood aflatoxin-albumin adduct, had no major effect on humoral immunity as measured by plasma concentrations of total IgA, IgG and IgM and of anti-ovalbumin IgG. Toxin exposure did not impair the mitogenic response of lymphocytes but delayed and decreased their specific proliferation in response to the vaccine antigen, suggesting impaired lymphocyte activation in pigs exposed to AFB1. The expression level of pro-inflammatory (TNF-alpha, IL-1beta, IL-6, IFN-gamma) and regulatory (IL-10) cytokines was assessed by real-time PCR in spleen. A significant up-regulation of all 5 cytokines was observed in spleen from pigs exposed to the highest dose of AFB1. In pigs exposed to the medium dose, IL-6 expression was increased and a trend towards increased IFN-gamma and IL-10 was observed. In addition we demonstrate that IL-6 impaired in vitro the antigenic- but not the mitogenic-induced proliferation of lymphocytes from control pigs vaccinated with ovalbumin. These results indicate that AFB1 dietary exposure decreases cell-mediated immunity while inducing an inflammatory response. These impairments in the immune response could participate in failure of vaccination protocols and increased susceptibility to infections described in pigs exposed to AFB1.

  4. Trypanosoma cruzi adjuvants potentiate T cell-mediated immunity induced by a NY-ESO-1 based antitumor vaccine.

    Directory of Open Access Journals (Sweden)

    Caroline Junqueira

    Full Text Available Immunological adjuvants that induce T cell-mediate immunity (TCMI with the least side effects are needed for the development of human vaccines. Glycoinositolphospholipids (GIPL and CpGs oligodeoxynucleotides (CpG ODNs derived from the protozoa parasite Trypanosoma cruzi induce potent pro-inflammatory reaction through activation of Toll-Like Receptor (TLR4 and TLR9, respectively. Here, using mouse models, we tested the T. cruzi derived TLR agonists as immunological adjuvants in an antitumor vaccine. For comparison, we used well-established TLR agonists, such as the bacterial derived monophosphoryl lipid A (MPL, lipopeptide (Pam3Cys, and CpG ODN. All tested TLR agonists were comparable to induce antibody responses, whereas significant differences were noticed in their ability to elicit CD4(+ T and CD8(+ T cell responses. In particular, both GIPLs (GTH, and GY and CpG ODNs (B344, B297 and B128 derived from T. cruzi elicited interferon-gamma (IFN-γ production by CD4(+ T cells. On the other hand, the parasite derived CpG ODNs, but not GIPLs, elicited a potent IFN-γ response by CD8(+ T lymphocytes. The side effects were also evaluated by local pain (hypernociception. The intensity of hypernociception induced by vaccination was alleviated by administration of an analgesic drug without affecting protective immunity. Finally, the level of protective immunity against the NY-ESO-1 expressing melanoma was associated with the magnitude of both CD4(+ T and CD8(+ T cell responses elicited by a specific immunological adjuvant.

  5. Multivalent TB vaccines targeting the esx gene family generate potent and broad cell-mediated immune responses superior to BCG. (United States)

    Villarreal, Daniel O; Walters, Jewell; Laddy, Dominick J; Yan, Jian; Weiner, David B


    Development of a broad-spectrum synthetic vaccine against TB would represent an important advance to the limited vaccine armamentarium against TB. It is believed that the esx family of TB antigens may represent important vaccine candidates. However, only 4 esx antigens have been studied as potential vaccine antigens. The challenge remains to develop a vaccine that simultaneously targets all 23 members of the esx family to induce enhanced broad-spectrum cell-mediated immunity. We sought to investigate if broader cellular immune responses could be induced using a multivalent DNA vaccine representing the esx family protein members delivered via electroporation. In this study, 15 designed esx antigens were created to cross target all members of the esx family. They were distributed into groups of 3 self-processing antigens each, resulting in 5 trivalent highly optimized DNA plasmids. Vaccination with all 5 constructs elicited robust antigen-specific IFN-γ responses to all encoded esx antigens and induced multifunctional CD4 Th1 and CD8 T cell responses. Importantly, we show that when all constructs are combined into a cocktail, the RSQ-15 vaccine, elicited substantial broad Ag-specific T cell responses to all esx antigens as compared with vaccination with BCG. Moreover, these vaccine-induced responses were highly cross-reactive with BCG encoded esx family members and were highly immune effective in a BCG DNA prime-boost format. Furthermore, we demonstrate the vaccine potential and immunopotent profile of several novel esx antigens never previously studied. These data highlight the likely importance of these novel immunogens for study as preventative or therapeutic synthetic TB vaccines in combination or as stand alone antigens.

  6. Trypanosoma cruzi adjuvants potentiate T cell-mediated immunity induced by a NY-ESO-1 based antitumor vaccine. (United States)

    Junqueira, Caroline; Guerrero, Ana Tereza; Galvão-Filho, Bruno; Andrade, Warrison A; Salgado, Ana Paula C; Cunha, Thiago M; Ropert, Catherine; Campos, Marco Antônio; Penido, Marcus L O; Mendonça-Previato, Lúcia; Previato, José Oswaldo; Ritter, Gerd; Cunha, Fernando Q; Gazzinelli, Ricardo T


    Immunological adjuvants that induce T cell-mediate immunity (TCMI) with the least side effects are needed for the development of human vaccines. Glycoinositolphospholipids (GIPL) and CpGs oligodeoxynucleotides (CpG ODNs) derived from the protozoa parasite Trypanosoma cruzi induce potent pro-inflammatory reaction through activation of Toll-Like Receptor (TLR)4 and TLR9, respectively. Here, using mouse models, we tested the T. cruzi derived TLR agonists as immunological adjuvants in an antitumor vaccine. For comparison, we used well-established TLR agonists, such as the bacterial derived monophosphoryl lipid A (MPL), lipopeptide (Pam3Cys), and CpG ODN. All tested TLR agonists were comparable to induce antibody responses, whereas significant differences were noticed in their ability to elicit CD4(+) T and CD8(+) T cell responses. In particular, both GIPLs (GTH, and GY) and CpG ODNs (B344, B297 and B128) derived from T. cruzi elicited interferon-gamma (IFN-γ) production by CD4(+) T cells. On the other hand, the parasite derived CpG ODNs, but not GIPLs, elicited a potent IFN-γ response by CD8(+) T lymphocytes. The side effects were also evaluated by local pain (hypernociception). The intensity of hypernociception induced by vaccination was alleviated by administration of an analgesic drug without affecting protective immunity. Finally, the level of protective immunity against the NY-ESO-1 expressing melanoma was associated with the magnitude of both CD4(+) T and CD8(+) T cell responses elicited by a specific immunological adjuvant.

  7. Cytotoxic falcarinol oxylipins from Dendropanax arboreus. (United States)

    Bernart, M W; Cardellina, J H; Balaschak, M S; Alexander, M R; Shoemaker, R H; Boyd, M R


    The crude organic extract of Dendropanax arboreus was selected as a candidate for bioassayguided fractionation on the basis of its relatively selective cytotoxicity to a subset of cell lines within the National Cancer Institute's disease-oriented in vitro tumor-screening panel. The major compound responsible for the in vitro cytotoxicity was falcarinol (1). Several other known compounds were isolated and found to be cytotoxic, including dehydrofalcarinol (2), a diyenne (3), falcarindiol (4), and dehydrofalcarindiol (5). In addition, two novel polyacetylenes, dendroarboreols A (6) and B (7), were isolated and characterized by standard and inverse-detected NMR methods. Compounds were selected from this series for absolute stereochemical determination using the modified Mosher method and preliminary in vivo evaluation using a LOX melanoma mouse xenograft model.

  8. Influence of Ganoderma lucidum (Curt.: Fr.) P. Karst. on T-cell-mediated immunity in normal and immunosuppressed mice line CBA/Ca. (United States)

    Nizhenkovska, Iryna V; Pidchenko, Vitalii T; Bychkova, Nina G; Bisko, Nina A; Rodnichenko, Angela Y; Kozyko, Natalya O


    The article presents the results of the investigation of the effect of biomass powder of the fungus Ganoderma lucidum on T-cell-mediated immunity in normal and immunosuppressed mice CBA/Ca. Delayed-type hypersensitivity assay was used. Experimental immunodeficiency was established with intraperitoneal injection of the immunosuppressant cyclophosphamide at a single dose of 150 mg/kg on the first day of the experiment. Results of the study show that the administration of biomass powder of Ganoderma lucidum in a dose of 0.5 mg/kg orally for 10 days increases the delayed-type hypersensitivity response in normal mice CBA/Ca. Administration of 0.5 mg/kg of biomass powder of the fungus Ganoderma lucidum for 10 days blocked the development of the T-cell-mediated immunosuppression, induced by administration of cyclophosphamide and restored the delayed-type hypersensitivity response in immunosuppressed mice. Key words: fungus Ganoderma lucidum cyclophosphamide immunodeficiency T-cell-mediated immunity delayed-type hypersensitivity.

  9. Cytotoxicity associated with electrospun polyvinyl alcohol. (United States)

    Pathan, Saif G; Fitzgerald, Lisa M; Ali, Syed M; Damrauer, Scott M; Bide, Martin J; Nelson, David W; Ferran, Christiane; Phaneuf, Tina M; Phaneuf, Matthew D


    Polyvinyl alcohol (PVA) is a synthetic, water-soluble polymer, with applications in industries ranging from textiles to biomedical devices. Research on electrospinning of PVA has been targeted toward optimizing or finding novel applications in the biomedical field. However, the effects of electrospinning on PVA biocompatibility have not been thoroughly evaluated. In this study, the cytotoxicity of electrospun PVA (nPVA) which was not crosslinked after electrospinning was assessed. PVA polymers of several molecular weights were dissolved in distilled water and electrospun using the same parameters. Electrospun PVA materials with varying molecular weights were then dissolved in tissue culture medium and directly compared against solutions of nonelectrospun PVA polymer in human coronary artery smooth muscle cells and human coronary artery endothelial cells cultures. All nPVA solutions were cytotoxic at a threshold molar concentration that correlated with the molecular weight of the starting PVA polymer. In contrast, none of the nonelectrospun PVA solutions caused any cytotoxicity, regardless of their concentration in the cell culture. Evaluation of the nPVA material by differential scanning calorimetry confirmed that polymer degradation had occurred after electrospinning. To elucidate the identity of the nPVA component that caused cytotoxicity, nPVA materials were dissolved, fractionated using size exclusion columns, and the different fractions were added to HCASMC and human coronary artery endothelial cells cultures. These studies indicated that the cytotoxic component of the different nPVA solutions were present in the low-molecular-weight fraction. Additionally, the amount of PVA present in the 3-10 kg/mol fraction was approximately sixfold greater than that in the nonelectrospun samples. In conclusion, electrospinning of PVA resulted in small-molecular-weight fractions that were cytotoxic to cells. This result demonstrates that biocompatibility of electrospun

  10. Cytotoxic Aaptamines from Malaysian Aaptos aaptos

    Directory of Open Access Journals (Sweden)

    Kee Cheng Ling


    Full Text Available In a preliminary screen, Aaptos aaptos showed significant cytotoxic activity towards a panel of cell lines and was thus subjected to bioassay-guided isolation of the bioactive constituents. In addition to the known aaptamine, two new derivatives of the alkaloid were isolated from the bioactive chloroform fraction of the crude methanolic extract. Detailed analysis by NMR and mass spectroscopy enabled their identification to be 3-(phenethylaminodemethyl(oxyaaptamine and 3-(isopentylaminodemethyl(oxy aaptamine. The cytotoxic activities of the three alkaloids were further evaluated against CEM-SS cells.

  11. Incipient cytotoxicity: A time-independent measure of cytotoxic potency in vitro. (United States)

    Gülden, Michael; Kähler, Daria; Seibert, Hasso


    Time is an important determinant of toxicity but largely ignored in in vitro toxicity assays where exposure times chosen are rather arbitrary. To investigate the impact of time on the cytotoxic potency of chemicals in vitro, the concentration dependent cytotoxic action of selected chemicals (surfactants, metals, oxidative stressors, a mitochondrial poison) was determined after various exposure times (1-72 h) in cultures of Balb/c 3T3 cells. Time affected the cytotoxic potency as well as the cytotoxic efficacy. The median cytotoxic concentrations, EC50, decreased and in most cases approached an "incipient" value, EC50,∞, within 72 h. Cytotoxicity due to mitochondrial insult occurred after a threshold time which was dependent on the medium glucose concentration. Within the chemicals studied the extent of potency change with time ranged from 3- to >1000-fold and the "time to incipient cytotoxicity", tic, from 4 to >72 h. Hence, also the relative cytotoxic potencies depend on exposure time. Ignoring this may lead to severe bias in toxicological hazard and risk assessment. Therefore it is recommended to determine the incipient cytotoxic potency of chemical compounds, represented by, e.g., the incipient median effect (EC50,∞), no effect (NEC∞) or lowest effect concentrations (LEC∞) instead of measures obtained after arbitrary exposure times. If this is not possible, the 72 h-potency measurements appear to be useful surrogates. These time-independent incipient potency values can be reasonably compared between substances, endpoints, cells and biological test systems and may serve to define points of departure for quantitative in vitro-in vivo extrapolations.

  12. Expression and cytotoxic effect of transmembrane form of human blood group A antigen mimotope vaccine in a malignant melanoma cell line B16%跨膜型血型A抗原模拟肽疫苗在黑素瘤细胞B16中表达及细胞毒效应检测

    Institute of Scientific and Technical Information of China (English)

    岑东芝; 孟辉; 张积仁


    Objective To establish a stable cell line expressing transmembrane form of human blood group A antigen mimotope vaccine by transfecting malignant melanoma cell line B16, and to detect the cytotoxicity of the vaccine against melanoma cells. Methods Cultured B16 cells were classified into 4 groups, i.e.,P/F-M-pIRES group [transfected with the recombinant plasmid mimotope peptide/Fas-macrophage inflammatory protein (Mip)-pIRES], P/F-pIRES group (transfected with the recombinant plasmid mimotope peptide/FaspIRES), M-pIRES group (transfected with the recombinant plasmid Mip-pIRES), and pIRES group (transfected with the empty plasmid pIRES). B 16 cells were transfected through Lipofectamine 2000. Subsequently, RT-PCR and Western blotting were performed to detect the mRNA and protein expressions of the mimotope peptide/Fas fusion gene and Mip3β in transfected B16 cells. Cell counting kit-8 (CCK-8) was used to evaluate the vaccinemediated complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)against B16 cells. Results RT-PCR yielded specific DNA fragments with expected size. Western blotting revealed the anti-A antibody-binding activity of the recombinant mimotope peptide/Fas fusion protein. Factor analysis indicated significant differences in CDC (F = 244.522, P < 0.01 ) and ADCC (F = 71.593, P < 0.01 )against B16 cells between the 4 groups. Group comparisons demonstrated more intense CDC and ADCC in P/FM-pIRES and P/F-pIRES groups compared with M-plRES and pIRES groups, stronger ADCC in P/F-M-pIRES group in comparison with P/F-pIRES group (F = 15.42, P < 0.05), but no significant difference in CDC was observed between M-pIRES and pIRES group. Conclusions The transmembrane form of human blood group A antigen mimotope vaccine could be stably expressed in B16 cells, and mediate ADCC and CDC against B16 cells in vitro.%目的 建立稳定表达跨膜型血型A抗原模拟肽疫苗的恶性黑素瘤细胞株B16,并检

  13. Measuring mucosal damage induced by cytotoxic therapy.

    NARCIS (Netherlands)

    Blijlevens, N.M.A.; Land, B. van 't; Donnelly, J.P.; Rabet, L. M'; Pauw, B.E. de


    We scored oral mucositis and gut toxicity and measured sugar permeability testing among 56 recipients of a haematopoietic stem cell transplant (HSCT) given myeloablative conditioning with idarubicin, cyclophosphamide and TBI, and a group of 18 patients given cytotoxic chemotherapy for newly diagnose

  14. Cytotoxicity Potentials of Eleven Bangladeshi Medicinal Plants

    Directory of Open Access Journals (Sweden)

    Amina Khatun


    Full Text Available Various forms of cancer are rising all over the world, requiring newer therapy. The quest of anticancer drugs both from natural and synthetic sources is the demand of time. In this study, fourteen extracts of different parts of eleven Bangladeshi medicinal plants which have been traditionally used for the treatment of different types of carcinoma, tumor, leprosy, and diseases associated with cancer were evaluated for their cytotoxicity for the first time. Extraction was conceded using methanol. Phytochemical groups like reducing sugars, tannins, saponins, steroids, gums, flavonoids, and alkaloids were tested using standard chromogenic reagents. Plants were evaluated for cytotoxicity by brine shrimp lethality bioassay using Artemia salina comparing with standard anticancer drug vincristine sulphate. All the extracts showed potent to moderate cytotoxicity ranging from LC50 2 to 115 µg/mL. The highest toxicity was shown by Hygrophila spinosa seeds (LC50=2.93 µg/mL and the lowest by Litsea glutinosa leaves (LC50=114.71 µg/mL in comparison with standard vincristine sulphate (LC50=2.04 µg/mL. Among the plants, the plants traditionally used in different cancer and microbial treatments showed highest cytotoxicity. The results support their ethnomedicinal uses and require advanced investigation to elucidate responsible compounds as well as their mode of action.

  15. Cytotoxic activity of four Mexican medicinal plants. (United States)

    Vega-Avila, Elisa; Espejo-Serna, Adolfo; Alarcón-Aguilar, Francisco; Velasco-Lezama, Rodolfo


    Ibervillea sonorae Greene, Cucurbita ficifolia Bouché, Tagetes lucida Cav and Justicia spicigera Scheltdd are Mexican native plants used in the treatment of different illnesses. The ethanolic extract of J. spicigera and T. lucida as well as aqueous extracts from I. sonorae, C. ficifolia, T. lucida and J. spicigera were investigated using sulforhodamine B assay. These extracts were assessed using two cell line: T47D (Human Breast cancer) and HeLa (Human cervix cancer). Colchicine was used as the positive control. Data are presented as the dose that inhibited 50% control growth (ED50). All of the assessed extracts were cytotoxic (ED50 < 20 microg/ml) against T47D cell line, meanwhile only the aqueous extract from T. lucida and the ethanolic extract from J. spicigera were cytotoxic to HeLa cell line. Ethanolic extract from J. spicigera presented the best cytotoxic effect. The cytotoxic activity of J. spicigera correlated with one of the popular uses, the treatment of cancer.

  16. Targeting cytotoxic T lymphocytes for cancer immunotherapy


    Maher, J; Davies, E. T.


    In light of their preeminent role in cellular immunity, there is considerable interest in targeting of cytotoxic T-lymphocytes to cancer. This review summarises the active and passive immunotherapeutic approaches under development to achieve this goal, emphasising how recent advances in tumour immunology and gene transfer have impacted upon this field.

  17. Cytotoxicity of the rhizome of medicinal plants

    Institute of Scientific and Technical Information of China (English)

    Shakhawoat Hossain; Golam Kader; Farjana Nikkon; Tanzima Yeasmin


    Objective:To investigate the cytotoxicity of the crude ethanol extract of the rhizome of Zingiber zerumbet (Z. zerumbet) (L) Smith. and Curcuma zedoaria (C. zedoaria) Rosc. against Artemia salina Leach. Methods:Fresh rhizomes of Z. zerumbet (L) Smith. and C. zedoaria Rosc. were extracted separately in cold with ethanol (2.5 L) and after concentration a brownish syrupy suspension of ethanol extracts of Z. zerumbet (L) Smith. and C. zedoaria Rosc. was obtained. The cytotoxic effect of the crude ethanol extracts of both plants was determined by brine shrimp lethality bioassay. Results: Crude ethanol extracts of the rhizome of Z. zerumbet (L) Smith. showed the highest cytotoxicity (LC50 was 1.24μg/mL) against brine shrimp nauplii as compared with C. zedoaria Rosc. (LC50 was 33.593μg/mL) after 24 h of exposure. Conclusions:It can be concluded that the rhizome of Z. zerumbet (L) Smith. and C. zedoaria Rosc. can be used as a source of cytotoxic agent.

  18. Cytotoxic oxoisoaporphine alkaloids from Menispermum dauricum. (United States)

    Yu, B W; Meng, L H; Chen, J Y; Zhou, T X; Cheng, K F; Ding, J; Qin, G W


    Four new oxoisoaporphine alkaloids, daurioxoisoporphines A-D (1-4), were isolated from the rhizomes of Menispermum dauricum. The structures of these alkaloids were established by spectroscopic methods. The cytotoxic evaluation of 1 and 2 is reported against four cancer cell lines.

  19. Evidence of T-cell mediated neuronal injury in stiff-person syndrome with anti-amphiphysin antibodies. (United States)

    Poh, Mervyn Q W; Simon, Neil G; Buckland, Michael E; Salisbury, Elizabeth; Watson, Shaun


    Paraneoplastic stiff-person syndrome (SPS) has been associated with antibodies against amphiphysin. Current evidence supports a pathogenic role for anti-amphiphysin antibodies. A 74-year-old female was diagnosed with amphiphysin-associated paraneoplastic stiff-person syndrome and associated encephalomyelitis. She had initial response to IVIG, however her symptoms worsened after two months and were resistant to further treatment. Subsequently the patient died and a post-mortem was performed. Neuropathology revealed perivascular and parenchymal lymphocytic infiltrates, with neuronophagia mediated by CD8+ T cells and microglia in brainstem, spinal cord, and mesial temporal lobe structures. These findings suggest a pathogenic role of cytotoxic CD8+ T-cells, with potential implication for therapy of future patients.


    NARCIS (Netherlands)



    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

  1. Gold Nanoparticles Inhibit Matrix Metalloproteases without Cytotoxicity. (United States)

    Hashimoto, M; Sasaki, J I; Yamaguchi, S; Kawai, K; Kawakami, H; Iwasaki, Y; Imazato, S


    Nanoparticles (NPs) are currently the focus of considerable attention for dental applications; however, their biological effects have not been fully elucidated. The long-term, slow release of matrix metalloproteases (MMPs) digests collagen fibrils within resin-dentin bonds. Therefore, MMP inhibitors can prolong the durability of resin-dentin bonds. However, there have been few reports evaluating the combined effect of MMP inhibition and the cytotoxic effects of NPs for dentin bonding. The aim of this study was to evaluate MMP inhibition and cytotoxic responses to gold (AuNPs) and platinum nanoparticles (PtNPs) stabilized by polyvinylpyrrolidone (PVP) in cultured murine macrophages (RAW264) by using MMP inhibition assays, measuring cell viability and inflammatory responses (quantitative reverse transcription polymerase chain reaction [RT-qPCR]), and conducting a micromorphological analysis by fluorescence and transmission electron microscopy. Cultured RAW264 cells were exposed to metal NPs at various concentrations (1, 10, 100, and 400 µg/mL). AuNPs and PtNPs markedly inhibited MMP-8 and MMP-9 activity. Although PtNPs were cytotoxic at high concentrations (100 and 400 µg/mL), no cytotoxic effects were observed for AuNPs at any concentration. Transmission electron microscopy images showed a significant nonrandom intercellular distribution for AuNPs and PtNPs, which were mostly observed to be localized in lysosomes but not in the nucleus. RT-qPCR analysis demonstrated inflammatory responses were not induced in RAW264 cells by AuNPs or PtNPs. The cytotoxicity of nanoparticles might depend on the core metal composition and arise from a "Trojan horse" effect; thus, MMP inhibition could be attributed to the surface charge of PVP, which forms the outer coating of NPs. The negative charge of the surface coating of PVP binds to Zn(2+) from the active center of MMPs by chelate binding and results in MMP inhibition. In summary, AuNPs are attractive NPs that effectively

  2. Identification of iGb3 and iGb4 in melanoma B16F10-Nex2 cells and the iNKT cell-mediated antitumor effect of dendritic cells primed with iGb3

    Directory of Open Access Journals (Sweden)

    Almeida Igor C


    , modulated negatively by GM3 which is also produced in these cells. iGb3-primed BMDC exerted a significant iNKT cell-mediated anti-tumor activity in mice challenged with melanoma cells.

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

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


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

  4. Cell-mediated immune responses to Malassezia furfur serovars A, B and C in patients with pityriasis versicolor, seborrheic dermatitis and controls. (United States)

    Ashbee, H R; Ingham, E; Holland, K T; Cunliffe, W J


    It has been postulated that patients with Malassezia furfur-associated dermatoses have a deficient cell-mediated immune response to M. furfur. This study examined the cell-mediated immune responses to M. furfur serovars A, B and C of 10 patients with pityriasis versicolor and 10 age- and sex-matched controls; and 10 patients with seborrheic dermatitis and 10 age- and sex-matched controls. The responses to each serovar of M. furfur were assessed using the lymphocyte transformation assay and the leukocyte migration inhibition assay. The lymphocyte transformation responses of the patients with pityriasis versicolor to M. furfur serovars A, B and C (0/10, 6/10 and 5/10 respectively) were not significantly different from those of controls (0/10, 2/10 and 1/10). However, for patients with seborrheic dermatitis, significantly more patients' lymphocytes responded to serovars B and C (6/10 and 6/10 respectively) than those of controls (1/10 and 1/10). No patient or control responded to serovar A. In the leukocyte migration inhibition assay, the leukocytes from a greater proportion of patients with pityriasis versicolor (5/7) responded to serovar B than controls (2/10); and the leukocytes from a greater proportion of patients with seborrheic dermatitis (4/10) responded to serovar C than controls (0/9). Thus, this data did not indicate the presence of any cell-mediated immune deficiency to M. furfur in patients with pityriasis versicolor or seborrheic dermatitis, as measured by the lymphocyte transformation assay or the leukocyte migration inhibition assay. The greater responsiveness of T lymphocytes from patients may indicate that T lymphocytes might be involved in the pathogenesis of these diseases.

  5. Cytotoxic constituents of ethyl acetate fraction from Dianthus superbus. (United States)

    Ding, Chengli; Zhang, Wu; Li, Jie; Lei, Jiachuan; Yu, Jianqing


    The ethyl acetate fraction (EE-DS) from Dianthus superbus was found to possess the cytotoxic activity against cancer cells in previous study. To investigate cytotoxic constituents, the bioassay-guided isolation of compounds from EE-DS was performed. Two dianthramides (1 and 2), three flavonoids (3-5), two coumarins (6 and 7) and three other compounds (8-10) were obtained. Structures of isolated compounds were identified by spectroscopic analysis. Cytotoxicity of the compounds against HepG2 cells was evaluated. Compound 1 showed the strongest cytotoxicity, compounds 10, 4, 3 and 5 had moderate cytotoxicity.

  6. Cytotoxic compounds from endemic Arnebia purpurea. (United States)

    Yuzbasioglu, Merve; Kuruuzum-Uz, Ayse; Guvenalp, Zuhal; Simon, András; Tóth, Gabór; Harput, U Sebnem; Kazaz, Cavit; Bilgili, Bilgehan; Duman, Hayri; Saracoglu, Iclal; Demirezer, L Omur


    Phytochemical studies of the roots and aerial parts of endemic Arnebia purpurea S. Erik & H. Sumbul resulted in the isolation and characterization of four naphthoquinones [isovalerylalkannin (1), α-methyl-n-butanoyl alkannin (2), acetylalkannin (3), and alkannin (4)], a triterpene derivative [3-O-acetyl-oleanolic acid (5)], a steroid [β-sitosterol (6)], three flavonoid glycosides [isorhamnetin-3-O-rutinoside (7), kaempferol-3-O-rutinoside (8), kaempferol 3-O-(5"-acetyl) apiofuranoside 7-O-rhamnopyranoside (9)] and a phenolic acid [rosmarinic acid (10)]. 3-O-Acetyl-oleanolic acid, isorhamnetin-3-O-rutinoside, kaempferol-3-O-mrutinoside, and kaempferol 3-O-(5"-acetyl) apiofuranoside 7-O-rhamnopyranoside are reported from an Arnebia species for the first time. Cytotoxic activities on L929 murine fibrosarcoma cell line of the isolated compounds were investigated using MTT assay. Naphthoquinones (1-4) showed intermediate cytotoxic activity in comparison with the standard, doxorubicin.

  7. Molecular Mechanisms of Lymphocyte-Mediated Cytotoxicity

    Institute of Scientific and Technical Information of China (English)

    Zusen Fan; Qixiang Zhang


    Granule-mediated cytotoxicity is the major mechanism for lymphocytes to kill viruses, intracellular bacteria and tumors. The cytotoxic granules move to the immunological synapse by exocytosis after recognition of a killer cell.The contents of the granules are delivered into target cells with the help of perforin by endocytosis. A group of serine protease granzymes cleave their critical substrates to initiate DNA damage and cell death. The most abundant granzymes are granzyme A and B. They induce cell death through alternate and nonoverlapping pathways. The substrates and functions of the majority of the orphan granzymes have not yet been identified. It is possible that the diversity of granzymes provides fail-safe mechanisms for killing viruses and tumor cells.

  8. Novel cytotoxic annonaceous acetogenins from Annona muricata. (United States)

    Chang, F R; Wu, Y C


    Seven new annonaceous acetogenins, muricins A-G (1-7), as well as five known compounds, a mixture of muricatetrocin A (8) and muricatetrocin B (9), longifolicin (10), corossolin (11), and corossolone (12), were isolated from the seeds of Annona muricata. The structures of all isolates were elucidated and characterized by spectral and chemical methods. These acetogenins showed significantly selective in vitro cytotoxicities toward the human hepatoma cell lines Hep G(2) and 2,2,15.

  9. A cytotoxic diacetylene from Dendropanax arboreus. (United States)

    Setzer, W N; Green, T J; Whitaker, K W; Moriarity, D M; Yancey, C A; Lawton, R O; Bates, R B


    The crude ethanol extract from the leaves of Dendropanax arboreus (Araliaceae) from Monteverde, Costa Rica, exhibits cytotoxic activity against Hep-G2, A-431, H-4IIE, and L-1210 tumor cell lines, but is not toxic against normal hepatocytes. The active component has been isolated by activity-directed separation and identified by 1H- and 13C-NMR spectroscopy as the acetylenic compound cis-1,9,16-heptadecatriene-4,6-diyne-3,8-diol.

  10. Cytotoxic activity of extracts from Hypochaeris radicata. (United States)

    MacKay, R J; Wyer, S; Gilmour, A; Kongara, K; Harding, D R; Clark, S; Mayhew, I G; Thomson, C E


    Pasture-associated stringhalt is an acquired equine disease characterized by peripheral neuropathy and hyperflexion of the pelvic limbs. The disease occurs most commonly during periods of drought in horses grazing pastures heavily contaminated by Hypochaeris radicata. We hypothesized that stringhalt is caused by neurotoxins elaborated by H. radicata in response to the stress of drought conditions. Supernates were collected from H. radicata that were stressed (or not) by immersion in copper chloride solution, then extracted with ethyl acetate and dried. Dilutions of extracts from stressed (SE) and control, unstressed (UE) plants were incubated with myelinating spinal cord cultures (MSCC) established from fetal Swiss mice, and with spinal ganglion cultures (SGC) and dermal fibroblast cultures derived from neonatal mouse tissues. Cytotoxicity in culture monolayers was evaluated both morphologically by microscopy and by release of lactate dehydrogenase activity into culture supernates. Three different SGC preparations were exposed to a single H. radicata extract and single preparations of fibroblasts and MSCC were exposed to three different extracts. Repin, a plant-derived sesquiterpene lactone neurotoxin, was included as a positive control. Significant dose-dependent cytotoxicity was seen within 24 h in all three culture types when incubated with SE or repin. Complete morphologic destruction of culture monolayers was induced by the highest concentrations tested of SE (100 μg/mL) and repin (30 μg/mL). Cytotoxic effect of SE was significantly greater than that of UE for all three cell types and was not due to copper contamination of the extract. This study has identified a cytotoxic activity in leaf exudates of H. radicata that was upregulated by the model stressor, copper chloride.

  11. Cytotoxic Effects of Fucoidan Nanoparticles against Osteosarcoma


    Ryuichiro Kimura; Takayoshi Rokkaku; Shinji Takeda; Masachika Senba; Naoki Mori


    In this study, we analyzed the size-dependent bioactivities of fucoidan by comparing the cytotoxic effects of native fucoidan and fucoidan lipid nanoparticles on osteosarcoma in vitro and in vivo. In vitro experiments indicated that nanoparticle fucoidan induced apoptosis of an osteosarcoma cell line more efficiently than native fucoidan. The more potent effects of nanoparticle fucoidan, relative to native fucoidan, were confirmed in vivo using a xenograft osteosarcoma model. Caco-2 cell tran...

  12. Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 antibody with enhanced direct and immune effector cell-mediated B-cell cytotoxicity

    NARCIS (Netherlands)

    Moessner, Ekkehard; Bruenker, Peter; Moser, Samuel; Puentener, Ursula; Schmidt, Carla; Herter, Sylvia; Grau, Roger; Gerdes, Christian; Nopora, Adam; van Puijenbroek, Erwin; Ferrara, Claudia; Sondermann, Peter; Jaeger, Christiane; Strein, Pamela; Fertig, Georg; Friess, Thomas; Schuell, Christine; Bauer, Sabine; Dal Porto, Joseph; Del Nagro, Christopher; Dabbagh, Karim; Dyer, Martin J. S.; Poppema, Sibrand; Klein, Christian; Umana, Pablo


    CD20 is an important target for the treatment of B-cell malignancies, including non-Hodgkin lymphoma as well as autoimmune disorders. B-cell depletion therapy using monoclonal antibodies against CD20, such as rituximab, has revolutionized the treatment of these disorders, greatly improving overall s

  13. Mutagenic and cytotoxic activities of benfuracarb insecticide. (United States)

    Eren, Yasin; Erdoğmuş, Sevim Feyza; Akyıl, Dilek; Özkara, Arzu


    Benfuracarb is a carbamate insecticide used to control insect pests in vegetables and it has anti-acetylcholinesterase activity lower than other carbamates. Cytotoxic effects of benfuracarb were evaluated by using root growth inhibition (EC50), mitotic index (MI), and mitotic phase determinations on the root meristem cells of Allium cepa and mutagenic effects were determined in Salmonella typhymurium Ames test by TA98 and TA100 strains with and without metabolic activation. In Allium test, 1 % DMSO was used as negative control group and 10 ppm MMS was used as positive control group. 75 ppm concentration of benfuracarb was found as EC50. In MI and mitotic phases determination study, 37.5, 75 and 150 ppm doses of benfuracarb were used. Dose-dependent cytotoxic activity was found by root growth inhibition and MI studies. It was identified that mitotic inhibition activity of benfuracarb was higher than 10 ppm MMS. In Ames test, mutagenic activity was not observed and over 200 µg/plate of benfuracarb was determined as cytotoxic to S. typhymurium strains. Benfuracarb can be called as "mitotic inhibitor" but not called as mutagen.

  14. Initial cytotoxicity of novel titanium alloys. (United States)

    Koike, M; Lockwood, P E; Wataha, J C; Okabe, T


    We assessed the biological response to several novel titanium alloys that have promising physical properties for biomedical applications. Four commercial titanium alloys [Super-TIX(R) 800, Super-TIX(R) 51AF, TIMETAL(R) 21SRx, and Ti-6Al-4V (ASTM grade 5)] and three experimental titanium alloys [Ti-13Cr-3Cu, Ti-1.5Si and Ti-1.5Si-5Cu] were tested. Specimens (n = 6; 5.0 x 5.0 x 3.0 mm(3)) were cast in a centrifugal casting machine using a MgO-based investment and polished to 600 grit, removing 250 mum from each surface. Commercially pure titanium (CP Ti: ASTM grade 2) and Teflon (polytetrafluoroethylene) were used as positive controls. The specimens were cleaned and disinfected, and then each cleaned specimen was placed in direct contact with Balb/c 3T3 fibroblasts for 72 h. The cytotoxicity [succinic dehydrogenase (SDH) activity] of the extracts was assessed using the MTT method. Cytotoxicity of the metals tested was not statistically different compared to the CP Ti and Teflon controls (p > 0.05). These novel titanium alloys pose cytotoxic risks no greater than many other commonly used alloys, including commercially pure titanium. The promising short-term biocompatibility of these Ti alloys is probably due to their excellent corrosion resistance under static conditions, even in biological environments.

  15. The Escape of Cancer from T Cell-Mediated Immune Surveillance: HLA Class I Loss and Tumor Tissue Architecture (United States)

    Garrido, Federico; Perea, Francisco; Bernal, Mónica; Sánchez-Palencia, Abel; Aptsiauri, Natalia; Ruiz-Cabello, Francisco


    Tumor immune escape is associated with the loss of tumor HLA class I (HLA-I) expression commonly found in malignant cells. Accumulating evidence suggests that the efficacy of immunotherapy depends on the expression levels of HLA class I molecules on tumors cells. It also depends on the molecular mechanism underlying the loss of HLA expression, which could be reversible/“soft” or irreversible/“hard” due to genetic alterations in HLA, β2-microglobulin or IFN genes. Immune selection of HLA-I negative tumor cells harboring structural/irreversible alterations has been demonstrated after immunotherapy in cancer patients and in experimental cancer models. Here, we summarize recent findings indicating that tumor HLA-I loss also correlates with a reduced intra-tumor T cell infiltration and with a specific reorganization of tumor tissue. T cell immune selection of HLA-I negative tumors results in a clear separation between the stroma and the tumor parenchyma with leucocytes, macrophages and other mononuclear cells restrained outside the tumor mass. Better understanding of the structural and functional changes taking place in the tumor microenvironment may help to overcome cancer immune escape and improve the efficacy of different immunotherapeutic strategies. We also underline the urgent need for designing strategies to enhance tumor HLA class I expression that could improve tumor rejection by cytotoxic T-lymphocytes (CTL). PMID:28264447

  16. HPV-E7 Delivered by Engineered Exosomes Elicits a Protective CD8+ T Cell-Mediated Immune Response

    Directory of Open Access Journals (Sweden)

    Paola Di Bonito


    Full Text Available We developed an innovative strategy to induce a cytotoxic T cell (CTL immune response against protein antigens of choice. It relies on the production of exosomes, i.e., nanovesicles spontaneously released by all cell types. We engineered the upload of huge amounts of protein antigens upon fusion with an anchoring protein (i.e., HIV-1 Nefmut, which is an inactive protein incorporating in exosomes at high levels also when fused with foreign proteins. We compared the immunogenicity of engineered exosomes uploading human papillomavirus (HPV-E7 with that of lentiviral virus-like particles (VLPs incorporating equivalent amounts of the same antigen. These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs. It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G. Most important, we provide evidence that the inoculation in mouse of exosomes uploading HPV-E7 induces production of anti-HPV E7 CTLs, blocks the growth of syngeneic tumor cells inoculated after immunization, and controls the development of tumor cells inoculated before the exosome challenge. These results represent the proof-of-concept about both feasibility and efficacy of the Nefmut-based exosome platform for the induction of CD8+ T cell immunity.

  17. Human dendritic cells mediate cellular apoptosis via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). (United States)

    Fanger, N A; Maliszewski, C R; Schooley, K; Griffith, T S


    TRAIL (TNF-related apoptosis-inducing ligand) is a member of the TNF family that induces apoptosis in a variety of cancer cells. In this study, we demonstrate that human CD11c(+) blood dendritic cells (DCs) express TRAIL after stimulation with either interferon (IFN)-gamma or -alpha and acquire the ability to kill TRAIL-sensitive tumor cell targets but not TRAIL-resistant tumor cells or normal cell types. The DC-mediated apoptosis was TRAIL specific, as soluble TRAIL receptor blocked target cell death. Moreover, IFN-stimulated interleukin (IL)-3 receptor (R)alpha(+) blood precursor (pre-)DCs displayed minimal cytotoxicity toward the same target cells, demonstrating a clear functional difference between the CD11c(+) DC and IL-3Ralpha(+) pre-DC subsets. These results indicate that TRAIL may serve as an innate effector molecule on CD11c(+) DCs for the elimination of spontaneously arising tumor cells and suggest a means by which TRAIL-expressing DCs may regulate or eliminate T cells responding to antigen presented by the DCs.

  18. The Escape of Cancer from T Cell-Mediated Immune Surveillance: HLA Class I Loss and Tumor Tissue Architecture

    Directory of Open Access Journals (Sweden)

    Federico Garrido


    Full Text Available Tumor immune escape is associated with the loss of tumor HLA class I (HLA-I expression commonly found in malignant cells. Accumulating evidence suggests that the efficacy of immunotherapy depends on the expression levels of HLA class I molecules on tumors cells. It also depends on the molecular mechanism underlying the loss of HLA expression, which could be reversible/“soft” or irreversible/“hard” due to genetic alterations in HLA, β2-microglobulin or IFN genes. Immune selection of HLA-I negative tumor cells harboring structural/irreversible alterations has been demonstrated after immunotherapy in cancer patients and in experimental cancer models. Here, we summarize recent findings indicating that tumor HLA-I loss also correlates with a reduced intra-tumor T cell infiltration and with a specific reorganization of tumor tissue. T cell immune selection of HLA-I negative tumors results in a clear separation between the stroma and the tumor parenchyma with leucocytes, macrophages and other mononuclear cells restrained outside the tumor mass. Better understanding of the structural and functional changes taking place in the tumor microenvironment may help to overcome cancer immune escape and improve the efficacy of different immunotherapeutic strategies. We also underline the urgent need for designing strategies to enhance tumor HLA class I expression that could improve tumor rejection by cytotoxic T-lymphocytes (CTL.

  19. The ROS-induced cytotoxicity of ascorbate is attenuated by hypoxia and HIF-1alpha in the NCI60 cancer cell lines. (United States)

    Sinnberg, Tobias; Noor, Seema; Venturelli, Sascha; Berger, Alexander; Schuler, Paul; Garbe, Claus; Busch, Christian


    Intravenous application of high-dose ascorbate is used in complementary palliative medicine to treat cancer patients. Pharmacological doses of ascorbate in the mM range induce cytotoxicity in cancer cells mediated by reactive oxygen species (ROS), namely hydrogen peroxide and ascorbyl radicals. However, little is known about intrinsic or extrinsic factors modulating this ascorbate-mediated cytotoxicity. Under normoxia and hypoxia, ascorbate IC50 values were determined on the NCI60 cancer cells. The cell cycle, the influence of cobalt chloride-induced hypoxia-inducible factor-1α (HIF-1α) and the glucose transporter 1 (GLUT-1) expression (a pro-survival HIF-1α-downstream-target) were analysed after ascorbate exposure under normoxic and hypoxic conditions. The amount of ascorbyl radicals increased with rising serum concentrations. Hypoxia (0.1% O2 ) globally increased the IC50 of ascorbate in the 60 cancer cell lines from 4.5 ± 3.6 mM to 10.1 ± 5.9 mM (2.2-fold increase, P ascorbate. This ascorbate resistance depended on HIF-1α-signalling, but did not correlate with cell line-specific expression of the ascorbate transporter GLUT-1. However, under normoxic and hypoxic conditions, ascorbate treatment at the individual IC50 reduced the expression of GLUT-1 in the cancer cells. Our data show a ROS-induced, HIF-1α- and O2 -dependent cytotoxicity of ascorbate on 60 different cancer cells. This suggests that for clinical application, cancer patients should additionally be oxygenized to increase the cytotoxic efficacy of ascorbate.

  20. Development of a lipopolysaccharide (LPS)-supplemented adjuvant and its effects on cell-mediated and humoral immune responses in male rats immunized against sperm (United States)

    NOGUCHI, Junko; WATANABE, Shinya; NGUYEN, Thanh Q. Dang; KIKUCHI, Kazuhiro; KANEKO, Hiroyuki


    Supplementation with lipopolysaccharide (LPS) from non-pathogenic Escherichia coli was found to enhance the adjuvant effects of a veterinary vaccine adjuvant (ISA 71VG®). Sperm immunization using 71VG as an adjuvant in the immature period induced infertility in 25% of male rats, whereas this increased to 62.5% after immunization with 71VG + LPS or Freund′s complete adjuvant (FCA). Mean testicular weight of non-sterile males in the 71VG + LPS group was significantly lower than that in the 71VG or FCA group. Histological examination of testicular tissue from sterile males demonstrated severe impairment of spermatogenesis due to experimental autoimmune orchitis, a cell-mediated autoimmune condition. The serum anti-sperm titer was elevated in the three sperm-immunized groups relative to male rats treated with adjuvant alone, but the titer was higher in the 71VG + LPS and FCA groups than in the 71VG group. We consider that this LPS-supplemented adjuvant stimulates both humoral and cell-mediated immune responses to an extent comparable to FCA. PMID:27890874

  1. A suicidal DNA vaccine expressing the fusion protein of peste des petits ruminants virus induces both humoral and cell-mediated immune responses in mice. (United States)

    Wang, Yong; Yue, Xiaolin; Jin, Hongyan; Liu, Guangqing; Pan, Ling; Wang, Guijun; Guo, Hao; Li, Gang; Li, Yongdong


    Peste des petits ruminants (PPR), a highly contagious disease induced by PPR virus (PPRV), affects sheep and goats. PPRV fusion (F) protein is important for the induction of immune responses against PPRV. We constructed a Semliki Forest virus (SFV) replicon-vectored DNA vaccine ("suicidal DNA vaccine") and evaluated its immunogenicity in BALB/c mice. The F gene of PPRV was cloned and inserted into the SFV replicon-based vector pSCA1. The antigenicity of the resultant plasmid pSCA1/F was identified by indirect immunofluorescence and western blotting. BALB/c mice were then intramuscularly injected with pSCA1/F three times at 14-d intervals. Specific antibodies and virus-neutralizing antibodies against PPRV were quantified by indirect ELISA and microneutralization tests, respectively. Cell-mediated immune responses were examined by cytokine and lymphocyte proliferation assays. The pSCA1/F expressed F protein in vitro and induced specific and neutralizing antibody production, and lymphocyte proliferation in mice. Mice vaccinated with pSCA1/F had increased IL-2 and IL-10 levels after 24-h post first immunization. IFN-γ and TNF-α levels increased from that time point and gradually decreased thereafter. Thus, the Semliki Forest virus replicon-vectored DNA vaccine expressing the F protein of PPRV induced both humoral and cell-mediated immune responses in mice. This could be considered as a novel strategy for vaccine development against PPR.

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


    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.

  3. Dual Pressure from Antiretroviral Therapy and Cell-Mediated Immune Response on the Human Immunodeficiency Virus Type 1 Protease Gene (United States)

    Karlsson, Annika C.; Deeks, Steven G.; Barbour, Jason D.; Heiken, Brandon D.; Younger, Sophie R.; Hoh, Rebecca; Lane, Meghan; Sällberg, Matti; Ortiz, Gabriel M.; Demarest, James F.; Liegler, Teri; Grant, Robert M.; Martin, Jeffrey N.; Nixon, Douglas F.


    Human immunodeficiency virus (HIV)-specific CD8+ T-lymphocyte pressure can lead to the development of viral escape mutants, with consequent loss of immune control. Antiretroviral drugs also exert selection pressures on HIV, leading to the emergence of drug resistance mutations and increased levels of viral replication. We have determined a minimal epitope of HIV protease, amino acids 76 to 84, towards which a CD8+ T-lymphocyte response is directed. This epitope, which is HLA-A2 restricted, includes two amino acids that commonly mutate (V82A and I84V) in the face of protease inhibitor therapy. Among 29 HIV-infected patients who were treated with protease inhibitors and who had developed resistance to these drugs, we show that the wild-type PR82V76-84 epitope is commonly recognized by cytotoxic T lymphocytes (CTL) in HLA-A2-positive patients and that the CTL directed to this epitope are of high avidity. In contrast, the mutant PR82A76-84 epitope is generally not recognized by wild-type-specific CTL, or when recognized it is of low to moderate avidity, suggesting that the protease inhibitor-selected V82A mutation acts both as a CTL and protease inhibitor escape mutant. Paradoxically, the absence of a mutation at position 82 was associated with the presence of a high-avidity CD8+ T-cell response to the wild-type virus sequence. Our results indicate that both HIV type 1-specific CD8+ T cells and antiretroviral drugs provide complex pressures on the same amino acid sequence of the HIV protease gene and, thus, can influence viral sequence evolution. PMID:12767994

  4. Identification of MHC class II restricted T-cell-mediated reactivity against MHC class I binding Mycobacterium tuberculosis peptides. (United States)

    Wang, Mingjun; Tang, Sheila T; Stryhn, Anette; Justesen, Sune; Larsen, Mette V; Dziegiel, Morten H; Lewinsohn, David M; Buus, Søren; Lund, Ole; Claesson, Mogens H


    Major histocompatibility complex (MHC) class I restricted cytotoxic T lymphocytes (CTL) are known to play an important role in the control of Mycobacterium tuberculosis infection so identification of CTL epitopes from M. tuberculosis is of importance for the development of effective peptide-based vaccines. In the present work, bioinformatics technology was employed to predict binding motifs of 9mer peptides derived from M. tuberculosis for the 12 HLA-I supertypes. Subsequently, the predicted peptides were synthesized and assayed for binding to HLA-I molecules in a biochemically based system. The antigenicity of a total of 157 peptides with measured affinity for HLA-I molecules of K(D) ≤ 500 nM were evaluated using peripheral blood T cells from strongly purified protein derivative reactive healthy donors. Of the 157 peptides, eight peptides (5%) were found to induce T-cell responses. As judged from blocking with HLA class I and II subtype antibodies in the ELISPOT assay culture, none of the eight antigenic peptides induced HLA class I restricted CD8(+) T-cell responses. Instead all responses were blocked by pan-HLA class II and anti-HLA-DR antibodies. In addition, CD4(+) T-cell depletion before the 10 days of expansion, resulted in total loss of reactivity in the ELISPOT culture for most peptide specificities. FACS analyses with intracellular interferon-γ staining of T cells expanded in the presence of M. tuberculosis peptides confirmed that the responsive cells were indeed CD4(+). In conclusion, T-cell immunity against HLA-I binding 9mer M. tuberculosis-derived peptides might in many cases turn out to be mediated by CD4(+) T cells and restricted by HLA-II molecules. The use of 9mer peptides recognized by both CD8(+) and CD4(+) T cells might be of importance for the development of future M. tuberculosis peptide-based vaccines.

  5. Nature of the suppressor cells mediating prolonged graft survival after administration of extracted histocompatibility antigen and cyclosporine

    Energy Technology Data Exchange (ETDEWEB)

    Yoshimura, N.; Kahan, B.D.


    Antigen-specific suppressor T cells are induced by donor histocompatibility antigen extracted from spleen cells with 3M KCl combined with cyclosporine (Ag-CsA). A single i.v. injection of 5 mg 3M-KCl-extracted donor Buffalo (Buf, RT1b) antigen (Ag) combined with a three day course of CsA prolonged renal allograft survival in Wistar-Furth (WFu, RT1u) hosts to a greater extent (MST 26.5 days) than CsA alone (MST 11.8 days). Peripheral blood lymphocytes (PBL) or spleen cells harvested from Ag-CsA-treated recipients ten days after transplantation inhibited the mixed lymphocyte reaction (MLR) between normal responder WFu cells and irradiated Buf cells (55.6% and 64.4% suppression, respectively, P less than 0.025), but not third-party Brown-Norway (BN, RT1n) stimulator cells (13.6% and -18.3% suppression, respectively, NS). The suppressor effect was not mediated by cytolytic cells; there was neither primary nor secondary cytolytic activity against /sup 51/Cr-labeled Con-A blastoid Buf cells. The suppressor cells were neither adherent to plastic dishes nor to nylon-wool columns. PBL irradiated with 800 rads, but not 1500 rads, suppressed the MLR. A single injection of cyclophosphamide (CY, 25 mg/kg) seven days after transplantation abrogated the suppression induced by Ag-CsA treatment. Moreover, PBL from Ag-CsA recipients failed to suppress the MLR, if depleted either of all T cells by treatment with monoclonal antibody (Mab) W3/13 HLK (pan T cells; % suppression -15.8), or of cytotoxic/suppressor cells with Mab OX-8 (-19.3% suppression) together with rabbit antimouse immunoglobulin and complement.

  6. CD8 T cell-mediated killing of orexinergic neurons induces a narcolepsy-like phenotype in mice. (United States)

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


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

  7. 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.

  8. Cytotoxic phorbol esters of Croton tiglium. (United States)

    Zhang, Xiao-Long; Wang, Lun; Li, Fu; Yu, Kai; Wang, Ming-Kui


    Chemical investigation of the seeds of Croton tiglium afforded eight new phorbol diesters (three phorbol diesters, 1-3, and five 4-deoxy-4α-phorbol diesters, 4-8), together with 11 known phorbol diesters (nine phorbol diesters, 9-17, and two 4-deoxy-4α-phorbol diesters, 18 and 19). The structures of compounds 1-8 were determined by spectroscopic data information and chemical degradation experiments. The cytotoxic activities of the phorbol diesters were evaluated against the SNU387 hepatic tumor cell line, and compound 3 exhibited the most potent activity (IC50 1.2 μM).

  9. New Cytotoxic Tigliane Diterpenoids from Croton caudatus. (United States)

    Chen, Ying-Ying; Yang, Kun-Xian; Yang, Xing-Wei; Khan, Afsar; Liu, Lu; Wang, Bei; Zhao, Yun-Li; Liu, Ya-Ping; Li, Yan; Luo, Xiao-Dong


    Three new tigliane-type diterpenoids were isolated from the methanolic extract of the twigs and leaves of Croton caudatus, trivially named crotusins A-C (1-3). The structures of compounds 1-3 were elucidated on the basis of extensive spectral methods. These new compounds were highly oxygenated and heavily substituted. Cytotoxic activity against five human tumor cell lines was assessed for compounds 1-3 of which compound 3 showed significant inhibitory activity with IC50 values ranging from 0.49 to 4.19 µM against these cells, while crotusins A and B exhibited moderate activity.

  10. Cytotoxic Effects of Fucoidan Nanoparticles against Osteosarcoma

    Directory of Open Access Journals (Sweden)

    Ryuichiro Kimura


    Full Text Available In this study, we analyzed the size-dependent bioactivities of fucoidan by comparing the cytotoxic effects of native fucoidan and fucoidan lipid nanoparticles on osteosarcoma in vitro and in vivo. In vitro experiments indicated that nanoparticle fucoidan induced apoptosis of an osteosarcoma cell line more efficiently than native fucoidan. The more potent effects of nanoparticle fucoidan, relative to native fucoidan, were confirmed in vivo using a xenograft osteosarcoma model. Caco-2 cell transport studies showed that permeation of nanoparticle fucoidan was higher than native fucoidan. The higher bioactivity and superior bioavailability of nanoparticle fucoidan could potentially be utilized to develop novel therapies for osteosarcoma.

  11. Mitigation of quantum dot cytotoxicity by microencapsulation.

    Directory of Open Access Journals (Sweden)

    Amelia Romoser

    Full Text Available When CdSe/ZnS-polyethyleneimine (PEI quantum dots (QDs are microencapsulated in polymeric microcapsules, human fibroblasts are protected from acute cytotoxic effects. Differences in cellular morphology, uptake, and viability were assessed after treatment with either microencapsulated or unencapsulated dots. Specifically, QDs contained in microcapsules terminated with polyethylene glycol (PEG mitigate contact with and uptake by cells, thus providing a tool to retain particle luminescence for applications such as extracellular sensing and imaging. The microcapsule serves as the "first line of defense" for containing the QDs. This enables the individual QD coating to be designed primarily to enhance the function of the biosensor.

  12. Orthodontic rare earth magnets--in vitro assessment of cytotoxicity. (United States)

    Bondemark, L; Kurol, J; Wennberg, A


    The aim of this study was to assess and compare in vitro the cytotoxic effects of uncoated and parylene-coated rare earth magnets, used in orthodontics. Cytotoxicity of samarium-cobalt magnets (SmCo5 and Sm2Co17) and neodymium-iron-boron magnets (Nd2Fe14B) was assessed by two in vitro methods, the millipore filter method and an extraction method. Orthodontic stainless steel brackets served as controls. Uncoated SmCo5-magnets showed high cytotoxicity while uncoated Sm2Co17-magnets demonstrated moderate cytotoxicity. Uncoated neodymium-iron-boron magnets, as well as parylene coated Sm2Co17-magnets and parylene-coated neodymium-iron-boron magnets, showed negligible cytotoxicity. Short-term exposure to a static magnetic field did not cause any cytotoxic effect on the cells.

  13. Novel CD8(+) cytotoxic T cell epitopes in bovine leukemia virus with cattle. (United States)

    Bai, Lanlan; Takeshima, Shin-Nosuke; Isogai, Emiko; Kohara, Junko; Aida, Yoko


    Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis and is closely related to human T cell leukemia virus (HTLV). The cytotoxic T lymphocyte (CTL) plays a key role in suppressing the progression of disease caused by BLV. T and B cell epitopes in BLV have been studied, but CD8(+) CTL epitopes remain poorly understood. We used a library of 115 synthetic peptides covering the entirety of the Env proteins (gp51 and gp30), the Gag proteins (p15, p24, and p12), and the Tax protein of BLV to identify 11 novel CD8(+) T cell epitopes (gp51N5, gp51N11, gp51N12, gp30N5, gp30N6, gp30N8, gp30N16, tax16, tax18, tax19, and tax20) in four calves experimentally infected with BLV. The number of CD8(+) T cell epitopes that could be identified in each calf correlated with the BLV proviral load. Interestingly, among the 11 epitopes identified, only gp51N11 was capable of inducing CD8(+) T cell-mediated cytotoxicity in all four calves, but it is not a suitable vaccine target because it shows a high degree of polymorphism according to the Wu-Kabat variability index. By contrast, no CTL epitopes were identified from the Gag structural protein. In addition, several epitopes were obtained from gp30 and Tax, indicating that cellular immunity against BLV is strongly targeted to these proteins. CD8(+) CTL epitopes from gp30 and Tax were less polymorphic than epitopes from. Indeed, peptides tax16, tax18, tax19, and tax20 include a leucine-rich activation domain that encompasses a transcriptional activation domain, and the gp30N16 peptide contains a proline-rich region that interacts with a protein tyrosine phosphatase SHP1 to regulate B cell activation. Moreover, at least one CD8(+) CTL epitope derived from gp30 was identified in each of the four calves. These results indicate that BLV gp30 may be the best candidate for the development of a BLV vaccine.




    An alcoholic extract of Asclepias curassavica L., a plant widely used in folk medicine for treating cancer and warts, shows cytotoxic activity when tested in vitro against cells derived from human carcinoma of the nasopharynx. Systematic fractionation of the extract has led to isolation and characterization of calotropin as a cytotoxic principle. Calotropin is similar in structure to two cardiac glycosides recently shown to be responsible for the cytotoxicity of Apocynum cannabinum L.

  15. Intracellular haemolytic agents of Heterocapsa circularisquama exhibit toxic effects on H. circularisquama cells themselves and suppress both cell-mediated haemolytic activity and toxicity to rotifers (Brachionus plicatilis). (United States)

    Nishiguchi, Tomoki; Cho, Kichul; Yasutomi, Masumi; Ueno, Mikinori; Yamaguchi, Kenichi; Basti, Leila; Yamasaki, Yasuhiro; Takeshita, Satoshi; Kim, Daekyung; Oda, Tatsuya


    A harmful dinoflagellate, Heterocapsa circularisquama, is highly toxic to shellfish and the zooplankton rotifer Brachionus plicatilis. A previous study found that H. circularisquama has both light-dependent and -independent haemolytic agents, which might be responsible for its toxicity. Detailed analysis of the haemolytic activity of H. circularisquama suggested that light-independent haemolytic activity was mediated mainly through intact cells, whereas light-dependent haemolytic activity was mediated by intracellular agents which can be discharged from ruptured cells. Because H. circularisquama showed similar toxicity to rotifers regardless of the light conditions, and because ultrasonic ruptured H. circularisquama cells showed no significant toxicity to rotifers, it was suggested that live cell-mediated light-independent haemolytic activity is a major factor responsible for the observed toxicity to rotifers. Interestingly, the ultrasonic-ruptured cells of H. circularisquama suppressed their own lethal effect on the rotifers. Analysis of samples of the cell contents (supernatant) and cell fragments (precipitate) prepared from the ruptured H. circularisquama cells indicated that the cell contents contain inhibitors for the light-independent cell-mediated haemolytic activity, toxins affecting H. circularisquama cells themselves, as well as light-dependent haemolytic agents. Ethanol extract prepared from H. circularisquama, which is supposed to contain a porphyrin derivative that displays photosensitising haemolytic activity, showed potent toxicity to Chattonella marina, Chattonella antiqua, and Karenia mikimotoi, as well as to H. circularisquama at the concentration range at which no significant toxicity to rotifers was observed. Analysis on a column of Sephadex LH-20 revealed that light-dependent haemolytic activity and inhibitory activity on cell-mediated light-independent haemolytic activity existed in two separate fractions (f-2 and f-3), suggesting that both

  16. Vitamin B12 and Folic Acid Imbalance Modifies NK Cytotoxicity, Lymphocytes B and Lymphoprolipheration in Aged Rats

    Directory of Open Access Journals (Sweden)

    Teresa Partearroyo


    Full Text Available Different vitamin B12 and folic acid concentrations could exacerbate the immune response. The aim was to evaluate different dietary folic acid and vitamin B12 levels on the immune response in aged rats. Male Sprague Dawley aged rats were assigned to three folic acid groups (deficient, control, supplemented each in absence of vitamin B12 for 30 days. Several parameters of innate and acquired immune responses were measured. Serum and hepatic folate levels increased according to folic acid dietary level, while vitamin B12 levels decreased. There was a significant decrease in natural killer cell-mediated cytotoxicity in the spleen for the vitamin B12 deficient diet and folic acid control diet groups. Significant changes in CD45 lymphocyte subsets were also observed according to dietary imbalance. Lymphoproliferative response to concanavalin A and phytohemagglutinin did not differ significantly between groups. The spleen response to lipopolysaccharide increased significantly, but was unmodified for the other organs. An imbalance between dietary vitamin B12 and folic acid concentrations alters some immunological parameters in aged rats. Therefore, the ratio between folate and vitamin B12 could be as important as their absolute dietary concentrations.

  17. Cytotoxicity and genotoxicity of biogenic silver nanoparticles (United States)

    Lima, R.; Feitosa, L. O.; Ballottin, D.; Marcato, P. D.; Tasic, L.; Durán, N.


    Biogenic silver nanoparticles with 40.3 ± 3.5 nm size and negative surface charge (- 40 mV) were prepared with Fusarium oxysporum. The cytotoxicity of 3T3 cell and human lymphocyte were studied by a TaliTM image-based cytometer and the genotoxicity through Allium cepa and comet assay. The results of BioAg-w (washed) and BioAg-nw (unwashed) biogenic silver nanoparticles showed cytotoxicity exceeding 50 μg/mL with no significant differences of response in 5 and 10 μg/mL regarding viability. Results of genotoxicity at concentrations 5.0 and 10.0 ug/mL show some response, but at concentrations 0.5 and 1.0 μg/mL the washed and unwashed silver nanoparticles did not present any effect. This in an important result since in tests with different bacteria species and strains, including resistant, MIC (minimal inhibitory concentration) had good answers at concentrations less than 1.9 μg/mL. This work concludes that biogenic silver nanoparticles may be a promising option for antimicrobial use in the range where no cyto or genotoxic effect were observed. Furthermore, human cells were found to have a greater resistance to the toxic effects of silver nanoparticles in comparison with other cells.

  18. Cytotoxic activity of lignans from Justicia procumbens. (United States)

    Jin, Hong; Yin, Hai-Long; Liu, Shi-Jun; Chen, Li; Tian, Ying; Li, Bin; Wang, Qiong; Dong, Jun-Xing


    Three new lignans, Pronaphthalide A (1), Procumbiene (2), and Procumbenoside J (3), along with a novel natural product Juspurpudin (4), and twelve other known lignans were isolated from Justicia procumbens. The structures of the new compounds were elucidated by extensive spectroscopic analyses and the data of 3 provided insight into the conformational equilibria existing in it. All compounds were evaluated for their in vitro cytotoxic activity against Human LoVo and BGC-823 cell lines except for compound 2, and eight of them were found to possess potent cytotoxicity. The structure-activity relationship (SAR) analysis revealed that (i) the parent structure of 2-carbonyl arylnaphthalide lactone attached with 6 and 7-OMe was the essential element; (ii) the polarity of substituents on C-4 might significantly affect the activity; (iii) a proper cyclic lipophilic group at the C-3″ and C-5″ of apiofuranose on C-4 might enhance the activity, which could optimize the application of 3 similar to VP-16.

  19. Cytotoxic effects of curcumin in osteosarcoma cells

    Directory of Open Access Journals (Sweden)

    Moran JM


    Full Text Available JM Moran, FJ Rodriguez-Velasco, R Roncero-Martin, V Vera, JD Pedrera-Zamorano Metabolic Bone Diseases Research Group, University of Extremadura, Cáceres, SpainWe read with interest the results of the study from Chang et al1 in the International Journal of Nanomedicine. This article shows that curcumin (diferuloymethane, a phenolic compound extracted from the natural plant Curcuma longa L., exerts higher cytotoxicity in osteosarcoma MG-63 cells than in healthy human osteoblasts. Based on the dosages provided by the authors it is hypothesized that with the appropriate drug carriers, curcumin could be delivered to bone tumors and selectively kill osteosarcoma cells without harming healthy osteoblasts. This hypothesis is based in the data provided that claims the dose needed to exert a significant cytotoxicity on osteosarcoma cells was much lower than that needed to exert the same effect on healthy human osteoblasts. The topic of this study is of importance for both cancer and metabolic bone disease fields, we appreciate the methodology of the study, nevertheless we believe that the hypothesis formulated by the authors and their conclusion deserves further comment.View original paper by Chang and colleagues.

  20. Cytotoxicity study of plant Aloe vera (Linn

    Directory of Open Access Journals (Sweden)

    Atul N Chandu


    Full Text Available Background: The objective of this study has been to evaluate the in-vitro antitumor activity of Aloe vera extract of in cultured B16F10 melanoma cell line by measuring cell viability using "Trypan blue exclusion assay" method. Aim: To find out such kind of anticancer drug which is a cheap, safe, less toxic, and more potent drug compared to chemotherapy drug. Materials and Methods: In-vitro antitumor activity cell culture1, drug treatment (standard and test extract and Trypan blue exclusion assay growth and viability test 1 were used. Treatment of Aloe vera extract against B16F10 melanoma cell line, in all concentration range, showed decrease in percent cell viability, as compared to that of negative when examined by "Trypan blue exclusion assay". Results: In overall variation of test samples, Aloe vera extract showed its best activity in the concentration of 300 μg/ml, which was approximately equal to the activity of standard drug doxorubicin. Evaluation of in-vitro antitumor activity revealed that Aloe vera extract exhibits good cytotoxic activity. The best cytotoxic activity by Aloe vera was shown at 200 μg/ml concentration. Conclusion: The study of cytoprotection against normal cells by micronucleus assay has shown that the herbal extracts have less toxic effects to the normal blood lymphocytes, as compared to that of standard anticancer drug.

  1. Cytotoxicity and genotoxicity of butyl cyclohexyl phthalate. (United States)

    Köksal, Çinel; Nalbantsoy, Ayse; Karabay Yavaşoğlu, N Ülkü


    Butyl cyclohexyl phthalate (BCP) is frequently used in personal care products, medical and household applications. The aim of this study is therefore to evaluate possible cytotoxicity and genotoxicity of BCP using in vitro and in vivo assays. The in vitro cytotoxic effect of BCP was investigated on mouse fibroblastic cell line (L929 cells) by MTT assay. The result showed that BCP inhibits cell proliferation in a concentration-dependent manner (IC50 value = 0.29 µg/mL). For genotoxicity assessment, tested concentrations of BCP demonstrated mutagenic activity in the presence of S9 mix with the Salmonella strain TA100 in the Ames test. Results showed that BCP is a secondary mutagenic substance even in low concentrations. The data obtained from 28-days repeated toxicity tests on mice revealed that BCP caused abnormalities of chromosome number, in a dose-dependent manner. Additionally, DNA damage, particularly DNA strand breaks, was assessed by Comet assay. The test result shows that BCP seemed to have genotoxic potential at a high level of exposure.

  2. Pyruvate anions neutralize peritoneal dialysate cytotoxicity. (United States)

    Mahiout, A; Brunkhorst, R


    A new peritoneal dialysate containing pyruvate anions was developed in order to avoid cytotoxic effect of conventional lactate-based dialysate. The dialysate has a final pH of 5.4 to 5.6 and is composed of 1.36-3.86% glucose-monohydrate; 132 mmol/l sodium; 1.75 mmol/l calcium; 0.75 mmol/l magnesium; 102 mmol/l chloride and 35 mmol/l pyruvate. For cytotoxicity testing peritoneal macrophages, and mesothelial cells (MC) were exposed to conventional lactate dialysate, and pyruvate dialysate. We investigated the O2- generation and cytokine synthesis after endotoxin stimulation in peritoneal macrophages and the proliferation of mesothelial cells of cultured human MC. After exposure to lactate dialysate O2- generation and cytokine synthesis in peritoneal macrophages and proliferation of mesothelial cells were inhibited when compared to solution containing pyruvate and the control solution. After preincubation with 3.86% glucose containing solutions, all negative effects became even more pronounced in the lactate group whereas after pre-exposure to pyruvate containing solution the toxic effects were absent. These results suggest that the acute toxic effects of commercially available peritoneal dialysates can be avoided by the use of sodium pyruvate instead of sodium lactate.

  3. Daratumumab-mediated lysis of primary multiple myeloma cells is enhanced in combination with the human anti-KIR antibody IPH2102 and lenalidomide

    DEFF Research Database (Denmark)

    Nijhof, I. S.; Lammerts van Bueren, J. J.; van Kessel, B.;


    killer cell inhibitory receptors with the human monoclonal anti-KIR antibody IPH2102, next to activation of natural killer cells with the immune modulatory drug lenalidomide. In 4-hour antibody-dependent cell-mediated cytotoxicity assays, IPH2102 did not induce lysis of multiple myeloma cell lines......RIIa-131R allele, who bind IgG1 with lower affinity than patients carrying the FcgammaRIIIa-158V allele or the FcgammaRIIa-131H allele. Finally, a further synergistically improved myeloma cell lysis with the daratumumab-IPH2102 combination was observed by adding lenalidomide, which suggests that more...

  4. Developing a Successful HIV Vaccine. (United States)

    Gallo, Robert C


    Human immunodeficiency virus (HIV) genome integration indicates that persistent sterilizing immunity will be needed for a successful vaccine candidate. This suggests a need for broad antibodies targeting the Env protein. Immunogens targeting gp120 have been developed that block infection in monkeys and mimic the modest success of the RV144 clinical trial in that protection is short-lived following a decline in antibody-depending cell-mediated cytotoxicity-like antibodies. Attempts to induce antibody persistence have been complicated by a loss of efficacy, presumably by increasing the number of HIV-target cells. The key seems to be achieving an immune balance.

  5. Co-incubation with IL-18 potentiates antigen-specific IFN-γ response in a whole-blood stimulation assay for measurement of cell-mediated immune responses in pigs experimentally infected with Lawsonia intracellularis

    DEFF Research Database (Denmark)

    Riber, Ulla; Boesen, Henriette Toft; Jakobsen, Jeanne Toft


    The whole-blood interferon-gamma (IFN-γ) assay is a quantitative in-vitro assay for a direct read out of Ag-specific cell-mediated immune (CMI) responses to infectious diseases. The IFN-γ assay is robust in severe intracellular infections like Brucella or mycobacteria, but more difficult to evalu......The whole-blood interferon-gamma (IFN-γ) assay is a quantitative in-vitro assay for a direct read out of Ag-specific cell-mediated immune (CMI) responses to infectious diseases. The IFN-γ assay is robust in severe intracellular infections like Brucella or mycobacteria, but more difficult...

  6. IL-18 potentiated whole blood IFN-γ assay can identify cell-mediated immune responses towards Lawsonia intracellularis in experimentally infected pigs

    DEFF Research Database (Denmark)

    Riber, Ulla; Jakobsen, Jeanne Toft; Hvass, Henriette Cordes;

    Lawsonia intracellularis is an obligate intracellular bacteria causing proliferative enteropathy (PE) in pigs. The infection causes diarrhoea, retarded growth and sudden death in pigs and is one of the most economically important diseases in the swine industry worldwide. The infection is one...... indications that cell-mediated immune responses (CMI) are important for the protection against infections with L. intracellularis and in mice models IFN-γ has been shown to play a key role in the host defence against experimental infections . In L. intracellularis infected pigs, IFN-γ is only sparsely...... exhibited a much lower level of IFN-γ response. Thus, age seems to be an important parameter in measurement of IFN-γ in response to L. intracellularis infection. In the young pigs antibiotic treatment (from 3 weeks. p.i.) cleared the L. intracellularis infection. In contrast to the low response observed...

  7. Angiogenesis in patients with psoriasis and psoriatic arthritis: cell-mediated and humoral mechanisms, its role in pathogenesis, and searching for promising therapeutic targets

    Directory of Open Access Journals (Sweden)

    T.V. Korotaeva


    Full Text Available Modern concepts of the role of angiogenesis in pathogenesis of psoriasis and psoriatic arthritis (PsA are analyzed. The features of cell-mediated and humoral immune responses resulting in proliferation of synovial membrane and vessel overgrowth in patients with this pathology are discussed. A number of angiogenesis mediators, pro-angiogenic cytokines, growth factors, matrix metalloproteinases, etc. are shown to be involved in progression of neovascularization. The role of tumor necrosis factor α as a key therapeutic target for treatment of psoriasis and PsA is emphasized. Drugs inhibiting some stages of angiogenesis, which are either used in clinical practice or are being developed, are discussed. 


    Directory of Open Access Journals (Sweden)

    H. Khorami


    Full Text Available Neopterin, apyrazinopyrimidine compound, is produced by macrophages after induction by interferon gamma (IFN-y and serves as a marker of cellular immune system activation followed by oxidative stress. The aim of this study was to determine urinary neopterin to creatinine ratio (UNCR as a surrogate marker of cell-mediated immune activation in multiple sclerosis (MS. Three weekly early morning urine samples were collected from 27 patients with MS and 31 age- and sex-matched apparently healthy subjects. Urinary neopterin and creatinine were determined using reversed phase high-performance liquid chromatography and Jaffe reaction, respectively. UNCR was significantly higher in patients than in healthy controls indicating IFN-y-induced cellular immunity activation and oxidative stress in multiple sclerosis. As a non-invasive method, UNCR determination may be helpful in monitoring disease progression and the effects of therapies, as well.

  9. Aspects of T Cell-Mediated Immunity Induced in Mice by a DNA Vaccine Based on the Dengue-NS1 Antigen after Challenge by the Intracerebral Route (United States)

    Oliveira, Edson R. A.; Gonçalves, Antônio J. S.; Costa, Simone M.; Azevedo, Adriana S.; Mantuano-Barradas, Marcio; Nogueira, Ana Cristina M. A.


    Dengue disease has emerged as a major public health issue across tropical and subtropical countries. Infections caused by dengue virus (DENV) can evolve to life-threatening forms, resulting in about 20,000 deaths every year worldwide. Several animal models have been described concerning pre-clinical stages in vaccine development against dengue, each of them presenting limitations and advantages. Among these models, a traditional approach is the inoculation of a mouse-brain adapted DENV variant in immunocompetent animals by the intracerebral (i.c.) route. Despite the historical usage and relevance of this model for vaccine testing, little is known about the mechanisms by which the protection is developed upon vaccination. To cover this topic, a DNA vaccine based on the DENV non-structural protein 1 (pcTPANS1) was considered and investigations were focused on the induced T cell-mediated immunity against i.c.-DENV infection. Immunophenotyping assays by flow cytometry revealed that immunization with pcTPANS1 promotes a sustained T cell activation in spleen of i.c.-infected mice. Moreover, we found that the downregulation of CD45RB on T cells, as an indicator of cell activation, correlated with absence of morbidity upon virus challenge. Adoptive transfer procedures supported by CFSE-labeled cell tracking showed that NS1-specific T cells induced by vaccination, proliferate and migrate to peripheral organs of infected mice, such as the liver. Additionally, in late stages of infection (from the 7th day onwards), vaccinated mice also presented reduced levels of circulating IFN-γ and IL-12p70 in comparison to non-vaccinated animals. In conclusion, this work presented new aspects about the T cell-mediated immunity concerning DNA vaccination with pcTPANS1 and the i.c. infection model. These insights can be explored in further studies of anti-dengue vaccine efficacy. PMID:27631083

  10. Sex-Specific Effects of High Yolk Androgen Levels on Constitutive and Cell-Mediated Immune Responses in Nestlings of an Altricial Passerine. (United States)

    Muriel, Jaime; Pérez-Rodríguez, Lorenzo; Ortiz-Santaliestra, Manuel E; Puerta, Marisa; Gil, Diego

    Avian embryos are exposed to yolk androgens that are incorporated into the egg by the ovulating female. These steroids can affect several aspects of embryo development, often resulting in increases in overall size or the speed of growth of different traits. However, several studies suggest that they also entail immune costs to the offspring. In this study, we explored whether variation in yolk androgen concentration affected several measures of the constitutive and cell-mediated immune axes in the spotless starling (Sturnus unicolor). Using a within-brood design, we injected different doses of androgens (testosterone and androstenedione) into the eggs. Our study showed that experimentally increased yolk androgens led to sex-specific immunosuppression in both the innate and adaptive axes of the immune system. Both cell-mediated immune response (CMI) and lysozyme activity decreased with increasing androgen levels injected into the egg in the case of male nestlings, whereas there were no effects on females. The effects that we found were always linear: no quadratic or threshold patterns were detected. We found no effects of the experimental treatment in hemolysis or agglutination capacity, but these measures were negatively correlated with CMI, suggesting negative correlation among different branches of the immune system. Blood (trypanosomes and hemosporidians) and intestinal (coccidia) parasites were not affected by the experimental increase of yolk androgen levels. Our results show that in our study species yolk androgens induce immunosuppression in some axes of the male nestling immune system. Further studies should analyze the proximate causes for these contrasting effects in different axes of the immune system and the reason for the differential impact on males and females.

  11. Detection of herpes simplex virus type 2 (HSV-2) -specific cell-mediated immune responses in guinea pigs during latent HSV-2 genital infection. (United States)

    Perry, Clarice L; Banasik, Brianne N; Gorder, Summer R; Xia, Jingya; Auclair, Sarah; Bourne, Nigel; Milligan, Gregg N


    Genital infections with herpes simplex virus type 2 (HSV-2) are a source of considerable morbidity and are a health concern for newborns exposed to virus during vaginal delivery. Additionally, HSV-2 infection diminishes the integrity of the vaginal epithelium resulting in increased susceptibility of individuals to infection with other sexually transmitted pathogens. Understanding immune protection against HSV-2 primary infection and immune modulation of virus shedding events following reactivation of the virus from latency is important for the development of effective prophylactic and therapeutic vaccines. Although the murine model of HSV-2 infection is useful for understanding immunity following immunization, it is limited by the lack of spontaneous reactivation of HSV-2 from latency. Genital infection of guinea pigs with HSV-2 accurately models the disease of humans including the spontaneous reactivation of HSV-2 from latency and provides a unique opportunity to examine virus-host interactions during latency. Although the guinea pig represents an accurate model of many human infections, relatively few reagents are available to study the immunological response to infection. To analyze the cell-mediated immune response of guinea pigs at extended periods of time after establishment of HSV-2 latency, we have modified flow-cytometry based proliferation assays and IFN-γ ELISPOT assays to detect and quantify HSV-specific cell-mediated responses during latent infection of guinea pigs. Here we demonstrate that a combination of proliferation and ELISPOT assays can be used to quantify and characterize effecter function of virus-specific immune memory responses during HSV-latency.

  12. Chronic active hepatitis induced by Helicobacter hepaticus in the A/JCr mouse is associated with a Th1 cell-mediated immune response. (United States)

    Whary, M T; Morgan, T J; Dangler, C A; Gaudes, K J; Taylor, N S; Fox, J G


    Helicobacter hepaticus infection in A/JCr mice results in chronic active hepatitis characterized by perivascular, periportal, and parenchymal infiltrates of mononuclear and polymorphonuclear cells. This study examined the development of hepatitis and the immune response of A/JCr mice to H. hepaticus infection. The humoral and cell-mediated T helper immune response was profiled by measuring the postinfection (p.i.) antibody response in serum, feces, and bile and by the production of cytokines and proliferative responses by splenic mononuclear cells to H. hepaticus antigens. Secretory immunoglobulin A (IgA) and systemic IgG2a antibody developed by 4 weeks p.i. and persisted through 12 months. Splenocytes from infected mice proliferated and produced more gamma interferon (IFN-gamma) than interleukin-4 (IL-4) or IL-5 when cultured with H. hepaticus outer membrane proteins. The predominantly IgG2a antibody response in serum and the in vitro production of IFN-gamma in excess of IL-4 or IL-5 are consistent with a Th1 immune response reported in humans and mice infected with Helicobacter pylori and Helicobacter felis, respectively. Mice infected with H. hepaticus developed progressively severe perivascular, periportal, and hepatic parenchymal lesions consisting of lymphohistiocytic and plasmacytic cellular infiltrates. In addition, transmural typhlitis was observed at 12 months p.i. The characterization of a cell-mediated Th1 immune response to H. hepaticus infection in the A/JCr mouse should prove valuable as a model for experimental regimens which manipulate the host response to Helicobacter.

  13. Inhibitors of histone deacetylase 1 reverse the immune evasion phenotype to enhance T-cell mediated lysis of prostate and breast carcinoma cells. (United States)

    Gameiro, Sofia R; Malamas, Anthony S; Tsang, Kwong Y; Ferrone, Soldano; Hodge, James W


    The clinical promise of cancer immunotherapy relies on the premise that the immune system can recognize and eliminate tumor cells identified as non-self. However, tumors can evade host immune surveillance through multiple mechanisms, including epigenetic silencing of genes involved in antigen processing and immune recognition. Hence, there is an unmet clinical need to develop effective therapeutic strategies that can restore tumor immune recognition when combined with immunotherapy, such as immune checkpoint blockade and therapeutic cancer vaccines. We sought to examine the potential of clinically relevant exposure of prostate and breast human carcinoma cells to histone deacetylase (HDAC) inhibitors to reverse tumor immune escape to T-cell mediated lysis. Here we demonstrate that prostate (LNCAP) and breast (MDA-MB-231) carcinoma cells are more sensitive to T-cell mediated lysis in vitro after clinically relevant exposure to epigenetic therapy with either the pan-HDAC inhibitor vorinostat or the class I HDAC inhibitor entinostat. This pattern of immunogenic modulation was observed against a broad range of tumor-associated antigens, such as CEA, MUC1, PSA, and brachyury, and associated with augmented expression of multiple proteins involved in antigen processing and tumor immune recognition. Genetic and pharmacological inhibition studies identified HDAC1 as a key determinant in the reversal of carcinoma immune escape. Further, our findings suggest that the observed reversal of tumor immune evasion is driven by a response to cellular stress through activation of the unfolded protein response. This offers the rationale for combining HDAC inhibitors with immunotherapy, including therapeutic cancer vaccines.

  14. In vitro cytotoxicity of metallic ions released from dental alloys

    NARCIS (Netherlands)

    Milheiro, A.; Nozaki, K.; Kleverlaan, C.J.; Muris, J.; Miura, H.; Feilzer, A.J.


    The cytotoxicity of a dental alloy depends on, but is not limited to, the extent of its corrosion behavior. Individual ions may have effects on cell viability that are different from metals interacting within the alloy structure. We aimed to investigate the cytotoxicity of individual metal ions in c

  15. Evaluation Of Potential Cytotoxic Effects Of Herbal Extracts

    Directory of Open Access Journals (Sweden)

    Radovanovic Ana


    Full Text Available Herbal medicines have played an important role in treating different diseases since ancient times. Bioactive components of medicinal plants are a good starting point for discovering new drugs such as chemotherapeutics. Currently, there are four classes of plant-derived chemotherapeutic drugs used in clinical practice. However, to discover new potential cytotoxic molecules, the research effort on herbal extracts has not diminished. The aim of this review was to evaluate the chemical constituents of plants that possess cytotoxicity, the signalling pathways responsible for this effect, and the influence of solvent polarity on potential cytotoxic effect and to present the cytotoxic activity of selected herbal extracts. The polyphenolic, anthraquinon, diterpneoid, triterpenoid, flavonoid, betulinic acid and berberine content contributes to cytotoxicity of herbal extracts. The inhibitory effect on cancer cells viability could be a consequence of the non-apoptotic processes, such as cell cycle arrestment, and the apoptotic process in tumour cells through different signalling pathways. The influence of solvent polarity on potential cytotoxic effect of herbal extracts should not be ignored. In general, the best cytotoxic activity was found in nonpolar and moderately polar herbal extracts. The herbal extract with IC50 below 30 μg/ml could be considered a very strong cytotoxic agent. Considering that many antitumor drugs have been discovered from natural products, further research on plants and plant-derived chemicals may result in the discovery of potent anticancer agents.

  16. Antibody-Dependent Enhancement of Marburg Virus Infection


    Nakayama, Eri; Tomabechi, Daisuke; Matsuno, Keita; Kishida, Noriko; Yoshida, Reiko; Feldmann, Heinz; Takada, Ayato


    Background. Marburg virus (MARV) and Ebola virus (EBOV) cause severe hemorrhagic fever in primates. Earlier studies demonstrated that antibodies to particular epitopes on the glycoprotein (GP) of EBOV enhanced virus infectivity in vitro.

  17. Cytotoxic withanolide constituents of Physalis longifolia. (United States)

    Zhang, Huaping; Samadi, Abbas K; Gallagher, Robert J; Araya, Juan J; Tong, Xiaoqin; Day, Victor W; Cohen, Mark S; Kindscher, Kelly; Gollapudi, Rao; Timmermann, Barbara N


    Fourteen new withanolides, 1-14, named withalongolides A-N, respectively, were isolated from the aerial parts of Physalis longifolia together with eight known compounds (15-22). The structures of compounds 1-14 were elucidated through spectroscopic techniques and chemical methods. In addition, the structures of withanolides 1, 2, 3, and 6 were confirmed by X-ray crystallographic analysis. Using a MTS viability assay, eight withanolides (1, 2, 3, 7, 8, 15, 16, and 19) and four acetylated derivatives (1a, 1b, 2a, and 2b) showed potent cytotoxicity against human head and neck squamous cell carcinoma (JMAR and MDA-1986), melanoma (B16F10 and SKMEL-28), and normal fetal fibroblast (MRC-5) cells with IC₅₀ values in the range between 0.067 and 9.3 μM.

  18. New Cytotoxic Saponins from Lysimachia davurica Ledeb.

    Institute of Scientific and Technical Information of China (English)


    To investigate the saponins from whole plants of Lysimachia davurica Ledeb., two new saponins named davuricoside I (compound 1) and E (compound 2) were isolated. Their chemical structures were elucidated as 3β,16α, 28, 29-tetrihydroxy-olean-12-en-3-O-β-D-glucopyranosyl-(1-→2)-β-D-glucuronopyranoside (compound 1)and 3β,16α, 29-trihydroxy-13, 28-epoxy-oleanane-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranoside (compound 2) on the basis of their one- and two-dimensional nuclear magnetic resonance and mass spectrometry data, and chemical methods. Compound 1 showed significant cytotoxic activity against human A2780 cells.

  19. Antitumor Activity of Cytotoxic Cyclooxygenase-2 Inhibitors (United States)

    Uddin, Md. Jashim; Crews, Brenda C.; Xu, Shu; Ghebreselasie, Kebreab; Daniel, Cristina K.; Kingsley, Philip J.; Banerjee, Surajit; Marnett, Lawrence J.


    Targeted delivery of chemotherapeutic agents to tumors has been explored as a means to increase the selectivity and potency of cytotoxicity. Most efforts in this area have exploited the molecular recognition of proteins highly expressed on the surface of cancer cells followed by internalization. A related approach that has received less attention is the targeting of intracellular proteins by ligands conjugated to anti-cancer drugs. An attractive target for this approach is the enzyme cyclooxygenase-2 (COX-2), which is highly expressed in a range of malignant tumors. Herein, we describe the synthesis and evaluation of a series of chemotherapeutic agents targeted to COX-2 by conjugation to indomethacin. Detailed characterization of compound 12, a conjugate of indomethacin with podophyllotoxin, revealed highly potent and selective COX-2 inhibition in vitro and in intact cells. Kinetics and X-ray crystallographic studies demonstrated that compound 12 is a slow, tight-binding inhibitor that likely binds to COX-2’s allosteric site with its indomethacin moiety in a conformation similar to that of indomethacin. Compound 12 exhibited cytotoxicity in cell culture similar to that of podophyllotoxin with no evidence of COX-2-dependent selectivity. However, in vivo, compound 12 accumulated selectively in and more effectively inhibited the growth of a COX-2-expressing xenograft compared to a xenograft that did not express COX-2. Compound 12, which we have named chemocoxib A, provides proof-of-concept for the in vivo targeting of chemotherapeutic agents to COX-2, but suggests that COX-2-dependent selectivity may not be evident in cell culture-based assays. PMID:27588346

  20. Synthesis and cytotoxic activities of semisynthetic zearalenone analogues. (United States)

    Tadpetch, Kwanruthai; Kaewmee, Benyapa; Chantakaew, Kittisak; Kantee, Kawalee; Rukachaisirikul, Vatcharin; Phongpaichit, Souwalak


    Zearalenone is a β-resorcylic acid macrolide with various biological activities. Herein we report the synthesis and cytotoxic activities of 34 zearalenone analogues against human oral epidermoid carcinoma (KB) and human breast adenocarcinoma (MCF-7) cells as well as noncancerous Vero cells. Some zearalenone analogues showed moderately enhanced cytotoxic activities against the two cancer cell lines. We have discovered the potential lead compounds with diminished or no cytotoxicity to Vero cells. Preliminary structure-activity relationship studies revealed that the double bond at the 1' and 2' positions of zearalenone core was crucial for cytotoxic activities on both cell lines. In addition, for zearalenol analogues, the unprotected hydroxyl group at C-2 and an alkoxy substituent at C-4 played key roles on cytotoxic effects of both cell lines.

  1. Serum-free culture of H pylori intensifies cytotoxicity

    Institute of Scientific and Technical Information of China (English)

    Hiroyuki Ohno; Akiyuki Murano


    AIM: To perform a long culture passage of H pylori without serum, taking into account its cytotoxicity and the presence of the probable new cytotoxic factor.METHODS: One sample of H pylori 60190 (ATCC49503) was grown on Brain Heart Infusion (BHI) agar containing 0.5% 2,6-di-O-methyl-β-cyclodextrin without any serum, being passaged 70-100 times every 3-4 d for approximately 2 h, while another sample of H pylori contained 70 mL/L fetal calf serum without 2,6-di-Omethyl-β-cyclodextrin. Their supernatant and extract after 16 h in culture were evaluated for changes in cell morphology and for cell viability using HeLa cells. Furthermore, the characteristics of the probable cytotoxic factor in the extract were examined on partial purification studies and its cytotoxicity was evaluated in various human cells.RESULTS: The supernatant and the extract of the bacterium grown on serum-free medium had strong cytotoxicity compared with those grown on serumcontaining medium. They irreversibly damaged HeLa cells without vacuolation that was altogether different from that of the bacterium when grown with serum.Their cytotoxicity was easily measured by cell viability assay. The probable cytotoxic factor partially purified and detected by chromatography had characteristics difference from that of vacuolating toxin and a broad cytotoxicity toward various cell lines.CONCLUSION: Serum-free long culture method of H pylori makes its supematant and its extract cytotoxic enough to be easily measured by cell viability assay. The probable cytotoxic factor has a unique characteristic and might be a new cytotoxin.

  2. Sublethal exposure to alpha radiation (223Ra dichloride) enhances various carcinomas' sensitivity to lysis by antigen-specific cytotoxic T lymphocytes through calreticulin-mediated immunogenic modulation. (United States)

    Malamas, Anthony S; Gameiro, Sofia R; Knudson, Karin M; Hodge, James W


    Radium-223 dichloride (Xofigo®; 223Ra) is an alpha-emitting radiopharmaceutical FDA-approved for the treatment of bone metastases in patients with advanced castration-resistant prostate cancer. It is also being examined clinically in patients with breast and lung carcinoma and patients with multiple myeloma. As with other forms of radiation, the aim of 223Ra is to reduce tumor burden by directly killing tumor cells. External beam (photon) and proton radiation have been shown to augment tumor sensitivity to antigen-specific CD8+ cytotoxic T lymphocytes (CTLs). However, little is known about whether treatment with 223Ra can also induce such immunogenic modulation in tumor cells that survive irradiation. We examined these effects in vitro by exposing human prostate, breast, and lung carcinoma cells to sublethal doses of 223Ra. 223Ra significantly enhanced T cell-mediated lysis of each tumor type by CD8+ CTLs specific for MUC-1, brachyury, and CEA tumor antigens. Immunofluorescence analysis revealed that the increase in CTL killing was accompanied by augmented protein expression of MHC-I and calreticulin in each tumor type, molecules that are essential for efficient antigen presentation. Enhanced tumor-cell lysis was facilitated by calreticulin surface translocation following 223Ra exposure. The phenotypic changes observed after treatment appear to be mediated by induction of the endoplasmic reticulum stress response pathway. By rendering tumor cells more susceptible to T cell-mediated lysis, 223Ra may potentially be effective in combination with various immunotherapies, particularly cancer vaccines that are designed to generate and expand patients' endogenous antigen-specific T-cell populations against specific tumor antigens.

  3. Cytotoxicity of alginate for orthodontic use

    Directory of Open Access Journals (Sweden)

    Matheus Melo Pithon


    Full Text Available OBJECTIVE: To evaluate the cytotoxicity of three different alginate impression materials for orthodontic use. METHODS: Three different brands of alginate were divided into three groups, namely, Group JCO (Jeltrate Chromatic Ortho, OP (Orthoprint and CO (Cavex Orthotrace. Three control groups were also included: Group C+ (positive control, consisting of detergent Tween 80; Group C- (negative control, consisting of PBS, and Group CC (cell control, consisting of cells not exposed to any material. After manipulating the materials according to the respective manufacturer instructions, samples were made with the use of silicon rings. Then the samples were immersed in Eagle's minimum essential medium (MEM for 2 minutes. The supernatants were then removed and brought into direct contact with L929 fibroblasts. After exposure to the medium, the cells were incubated for 24 hours. Then 100 µl of 0.01% neutral red dye were added. The cells were incubated again for 3 hours so that the dye could be absorbed. After this 3-hour period, the cells were fixed to perform the viable cell count, using a spectrophotometer (BioTek, Winooski, Vermont, USA at a wavelength of 492 nm. RESULTS: Statistical differences were found when Groups CC and C- were compared with the other experimental groups. Group JCO had the highest cytotoxicity, followed by Groups OP and CO. CONCLUSION: Based on the results obtained in this work, it was concluded that all alginate impression materials are potentially cytotoxic.OBJETIVO: avaliar a citotoxicidade de três diferentes alginatos de uso ortodôntico. MÉTODOS: foram avaliados três diferentes alginatos divididos em três grupos, denominados grupo JCO (Jeltrate Chromatic Ortho, OP (Orthoprint e CO (Carrex Orthotrace. Três grupos controle também participaram: controle + (C+, constituído pelo detergente celular Tween 80; controle - (C- PBS; e controle de célula (CC onde as células não foram expostas a nenhum material. Após manipula

  4. Interleukin-2 activation of cytotoxic cells in postmastectomy seroma. (United States)

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


    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.

  5. Stability and cytotoxicity of crystallin amyloid nanofibrils (United States)

    Kaur, Manmeet; Healy, Jackie; Vasudevamurthy, Madhusudan; Lassé, Moritz; Puskar, Ljiljana; Tobin, Mark J.; Valery, Celine; Gerrard, Juliet A.; Sasso, Luigi


    Previous work has identified crystallin proteins extracted from fish eye lenses as a cheap and readily available source for the self-assembly of amyloid nanofibrils. However, before exploring potential applications, the biophysical aspects and safety of this bionanomaterial need to be assessed so as to ensure that it can be effectively and safely used. In this study, crude crystallin amyloid fibrils are shown to be stable across a wide pH range, in a number of industrially relevant solvents, at both low and high temperatures, and in the presence of proteases. Crystallin nanofibrils were compared to well characterised insulin and whey protein fibrils using Thioflavin T assays and TEM imaging. Cell cytotoxicity assays suggest no adverse impact of both mature and fragmented crystallin fibrils on cell viability of Hec-1a endometrial cells. An IR microspectroscopy study supports long-term structural integrity of crystallin nanofibrils.Previous work has identified crystallin proteins extracted from fish eye lenses as a cheap and readily available source for the self-assembly of amyloid nanofibrils. However, before exploring potential applications, the biophysical aspects and safety of this bionanomaterial need to be assessed so as to ensure that it can be effectively and safely used. In this study, crude crystallin amyloid fibrils are shown to be stable across a wide pH range, in a number of industrially relevant solvents, at both low and high temperatures, and in the presence of proteases. Crystallin nanofibrils were compared to well characterised insulin and whey protein fibrils using Thioflavin T assays and TEM imaging. Cell cytotoxicity assays suggest no adverse impact of both mature and fragmented crystallin fibrils on cell viability of Hec-1a endometrial cells. An IR microspectroscopy study supports long-term structural integrity of crystallin nanofibrils. Electronic supplementary information (ESI) available: ThT fluorescence graphs of buffers and solvents used for

  6. DNA prime/Adenovirus boost malaria vaccine encoding P. falciparum CSP and AMA1 induces sterile protection associated with cell-mediated immunity.

    Directory of Open Access Journals (Sweden)

    Ilin Chuang

    Full Text Available BACKGROUND: Gene-based vaccination using prime/boost regimens protects animals and humans against malaria, inducing cell-mediated responses that in animal models target liver stage malaria parasites. We tested a DNA prime/adenovirus boost malaria vaccine in a Phase 1 clinical trial with controlled human malaria infection. METHODOLOGY/PRINCIPAL FINDINGS: The vaccine regimen was three monthly doses of two DNA plasmids (DNA followed four months later by a single boost with two non-replicating human serotype 5 adenovirus vectors (Ad. The constructs encoded genes expressing P. falciparum circumsporozoite protein (CSP and apical membrane antigen-1 (AMA1. The regimen was safe and well-tolerated, with mostly mild adverse events that occurred at the site of injection. Only one AE (diarrhea, possibly related to immunization, was severe (Grade 3, preventing daily activities. Four weeks after the Ad boost, 15 study subjects were challenged with P. falciparum sporozoites by mosquito bite, and four (27% were sterilely protected. Antibody responses by ELISA rose after Ad boost but were low (CSP geometric mean titer 210, range 44-817; AMA1 geometric mean micrograms/milliliter 11.9, range 1.5-102 and were not associated with protection. Ex vivo IFN-γ ELISpot responses after Ad boost were modest (CSP geometric mean spot forming cells/million peripheral blood mononuclear cells 86, range 13-408; AMA1 348, range 88-1270 and were highest in three protected subjects. ELISpot responses to AMA1 were significantly associated with protection (p = 0.019. Flow cytometry identified predominant IFN-γ mono-secreting CD8+ T cell responses in three protected subjects. No subjects with high pre-existing anti-Ad5 neutralizing antibodies were protected but the association was not statistically significant. SIGNIFICANCE: The DNA/Ad regimen provided the highest sterile immunity achieved against malaria following immunization with a gene-based subunit vaccine (27%. Protection

  7. Cytotoxic activities of some benzothiazole-piperazine derivatives. (United States)

    Gurdal, Enise Ece; Durmaz, Irem; Cetin-Atalay, Rengul; Yarim, Mine


    Synthesis, characterization and cytotoxic activities of ten benzothiazole-piperazine derivatives were reported. In vitro cytotoxic activities of compounds were screened against hepatocellular (HUH-7), breast (MCF-7) and colorectal (HCT-116) cancer cell lines by sulphorhodamine B assay. Based on the GI50 values of the compounds, most of the benzothiazole-piperazine derivatives are active against HUH-7, MCF-7 and HCT-116 cancer cell lines. Compound 1d is highly cytotoxic against all tested cancer cell lines. Further investigation of compound 1d by Hoechst Staining and Fluorescence-Activated Cell Sorting Analysis (FACS) revealed that this compound causes apoptosis by cell cycle arrest at subG1 phase.

  8. Cytotoxic alkaloids from stems, leaves and twigs of Dasymaschalon blumei. (United States)

    Chanakul, Waraporn; Tuchinda, Patoomratana; Anantachoke, Natthinee; Pohmakotr, Manat; Piyachaturawat, Pawinee; Jariyawat, Surawat; Suksen, Kanoknetr; Jaipetch, Tharworn; Nuntasaen, Narong; Reutrakul, Vichai


    Bioassay-guided fractionation of the cytotoxic ethyl acetate extract from the stems of Dasymaschalon blumei (Annonaceae) led to the isolation of four aristololactam alkaloids, including the hitherto unknown 3,5-dihydroxy-2,4-dimethoxyaristolactam (1), as well as the three known compounds, aristolactam BI, goniopedaline, and griffithinam. Additionally, the cytotoxic extract from the combined leaves and twigs of the same plant yielded three known oxoaporphine alkaloids, oxodiscoguattine, dicentrinone, and duguevalline. The structures of aristolactams and oxoaporphine alkaloids were elucidated on the basis of spectroscopic methods. All isolates were evaluated for cytotoxicity against a panel of mammalian cancer cell lines and a noncancerous human embryonic kidney cell Hek 293.

  9. Design and synthesis of novel cytotoxic podophyllotoxin derivatives

    Institute of Scientific and Technical Information of China (English)

    Wen Li Xi; Qian Cai; Yan Bo Tang; Hua Sun; Zhi Yan Xiao


    In order to investigate the effect of different C4 linkage moieties on the cytotoxicity of podophyllotoxin derivatives, novel 4-N-and 4-C-substituted 4'-O-demethylepipodophyllotoxin derivatives were designed and synthesized. All the compounds were tested against A549 and MCF-7 tumor cells in vitro, and six compounds showed significant cytotoxicity. The most active compound 9f was superior to GL-331, and exhibited potent cytotoxicity with IC50 value at 10-7 mol/L level.

  10. Stability, accumulation and cytotoxicity of an albumin-cisplatin adduct

    DEFF Research Database (Denmark)

    Møller, Charlotte; Tastesen, Hanne Sørup; Gammelgaard, Bente


    The accumulation and cytotoxicity of a 10 µmol L¿¹ equimolar human serum albumin-cisplatin adduct (HSA-Pt) was investigated in suspension Ehrlich Ascites Tumor Cells (EATC) and adherent Ehrlich Lettré Ascites Cells (Lettré). HSA-Pt did not induce apoptosis nor was it taken up by the cells to any...... significant amount within 24 h incubation. The accumulation and cytotoxicity of HSA-Pt was compared to 10 µmol L¿¹ cisplatin for which a larger accumulation and cytotoxicity were observed in EATC compared to Lettré. The experiment was performed with cell medium exchange every fourth hour as HSA...

  11. Nanomaterial Induced Immune Responses and Cytotoxicity. (United States)

    Ali, Ashraf; Suhail, Mohd; Mathew, Shilu; Shah, Muhammad Ali; Harakeh, Steve M; Ahmad, Sultan; Kazmi, Zulqarnain; Alhamdan, Mohammed Abdul Rahman; Chaudhary, Adeel; Damanhouri, Ghazi Abdullah; Qadri, Ishtiaq


    Nanomaterials are utilized in a wide array of end user products such as pharmaceuticals, electronics, clothes and cosmetic products. Due to its size (< 100 nm), nanoparticles have the propensity to enter through the airway and skin, making its path perilous with the potential to cause damages of varying severity. Once within the body, these particles have unconstrained access to different tissues and organs including the brain, liver, and kidney. As a result, nanomaterials may cause the perturbation of the immune system eliciting an inflammatory response and cytotoxicity. This potential role is dependent on many factors such as the characteristics of the nanomaterials, presence or absence of diseases, and genetic predisposition. Cobalt and nickel nanoparticles, for example, were shown to have inflammogenic properties, while silver nanoparticles were shown to reduce allergic inflammation. Just as asbestos fibers, carbon nanotubes were shown to cause lungs damage. Some nanomaterials were shown, based on animal studies, to result in cell damage, leading to the formation of pre-cancerous lesions. This review highlights the impact of nanomaterials on immune system and its effect on human health with toxicity consideration. It recommends the development of suitable animal models to study the toxicity and bio-clearance of nanomaterials and propose safety guidelines.

  12. Copper Nanoparticle Induced Cytotoxicity to Nitrifying Bacteria ... (United States)

    With the inclusion of engineered nanomaterials in industrial processes and consumer products, wastewater treatments plants (WWTPs) will serve as a major sink for these emerging contaminants. Previous research has demonstrated that nanomaterials are potentially toxic to microbial communities utilized in biological wastewater treatment (BWT). Copper-based nanoparticles (CuNPs) are of particular interest based on their increasing use in wood treatment, paints, household products, coatings, and byproducts of semiconductor manufacturing. A critical step in BWT is nutrient removal via denitrification. This study examined the potential toxicity of bare and polyvinylpyrrolidone (PVP) coated CuO, and Cu2O nanoparticles, as well as Cu ions to microbial communities responsible for nitrogen removal in BWT. Inhibition was inferred from changes to the specific oxygen uptake rate (sOUR) in the absence and presence of Cu ions and CuNPs. X-ray absorption fine structure spectroscopy, with Linear Combination Fitting (LCF), was utilized to track changes to Cu speciation throughout exposure. Results indicate that the dissolution of Cu ions from CuNPs drive microbial inhibition. The presence of a PVP coating on CuNPs has little effect on inhibition. LCF fitting of the biomass combined with metal partitioning analysis supports the current hypothesis that Cu-induced cytotoxicity is primarily caused by reactive oxygen species formed from ionic Cu in solution via catalytic reaction inter

  13. Earlier low-dose TBI or DST overcomes CD8+ T-cell-mediated alloresistance to allogeneic marrow in recipients of anti-CD40L. (United States)

    Takeuchi, Yasuo; Ito, Hiroshi; Kurtz, Josef; Wekerle, Thomas; Ho, Leon; Sykes, Megan


    Treatment with a single injection of anti-CD40L (CD154) monoclonal antibody (mAb) and fully mismatched allogeneic bone marrow transplant (BMT) allows rapid tolerization of CD4+ T cells to the donor. The addition of in vivo CD8 T-cell depletion leads to permanent mixed hematopoietic chimerism and tolerance. We now describe two approaches that obviate the requirement for CD8 T-cell depletion by rapidly tolerizing recipient CD8 T cells in addition to CD4 cells. Administration of donor-specific transfusion (DST) to mice receiving 3 Gy total body irradiation (TBI), BMT and anti-CD40L mAb on day 0 uniformly led to permanent mixed chimerism and tolerance, compared with only 40% of mice receiving similar treatment without DST. In the absence of DST, moving the timing of 3 Gy TBI to day -1 or day -2 instead of day 0 led to rapid (by 2 weeks) induction of CD8+ cell tolerance, and also permitted uniform achievement of permanent mixed chimerism and donor-specific tolerance in recipients of anti-CD40L and BMT on day 0. These nontoxic regimens overcome CD8+ and CD4+ T-cell-mediated alloresistance without requiring host T-cell depletion, permitting the induction of permanent mixed chimerism and tolerance.

  14. Effects of iridovirus-like agent on the cell-mediated immunity in sheatfish (Silurus glanis)--an in vitro study. (United States)

    Siwicki, A K; Pozet, F; Morand, M; Volatier, C; Terech-Majewska, E


    An iridovirus-like agent was tested for the first time in vitro on cell-mediated immunity in sheatfish (Silurus glanis). The influence of the iridovirus-like agent on pronephric macrophage metabolism was examined at two temperatures, 20 and 30 degrees C, by studying the respiratory burst activity stimulated by phorbol myristate acetate as well as the proliferative ability of lymphocytes stimulated by concanavalin A (ConA) and lipopolysaccharide (LPS) measured by MTT assay. The results showed that the iridovirus-like agent decreased the macrophage activity at incubation temperatures of 20 and 30 degrees C. The highest inhibitory effect was observed at 30 degrees C. The proliferative ability of pronephric lymphocytes had a similar pattern. The results showed that applying a virus at the same time or after the mitogen at 20 and 30 degrees C decreased the lymphocyte proliferation that was stimulated by either ConA or LPS. The highest suppressive effect was observed when virus was applied 14 h after the mitogen. This preliminary in vitro study demonstrated a strong suppressive influence of the iridovirus-like agent on pronephric macrophage and lymphocyte activity in sheatfish.

  15. CD4+ T cell-mediated presentation of non-infectious HIV-1virion antigens to HIV-specific CD8+ T cells

    Institute of Scientific and Technical Information of China (English)

    XU Jian-qing; Franco Lori; Julianna Lisziewicz


    Background The mechanism of chronic immune activation and impairment of HIV-specific immune responses during chronic infection is not fully understood. However, it is known that high immune activation leads to more rapid progression to AIDS. We hypothesize that CD4+ T cell-mediated viral antigen presentation contributes to this pathologic immune activation in HIV-infected individuals.Methods HIV-specific T cells, responding to noninfectious HIV-1 virions as antigen, were measured by flow cytometric assays. These experimental conditions reflect the in vivo condition where noninfectious HIV-1 represents more than 99% of the antigens.Results CD4+ T cells purified from HIV-infected individuals were capable of cross presenting exogenous noninfectious HIV-1 virions to HIV-1-specific CD8+ T cells. Cross presentation required the entry of HIV-1 to CD4+ T cells and antigen translocation from endoplasmic reticulum to the Golgi complex. Blocking CD4+mediated activation of HIV-specific CD8+ T cells and redirecting the viral antigens to antigen presenting cells improved HIV-specific T cell responses.Conclusions One possible cause of chronic immune activation and impairment of HIV-1 specific T cell responses is represented by HIV-1 harboring CD4+ T cells cross presenting HIV-1 antigen to activate CD8+ T cells. This new mechanism provides the first evidence that cross presentation of noninfectious HIV-1. Virions play a role in the immunopathogenesis of HIV-1 infection.

  16. In vitro effect of p21WAF-1/CIP1 gene on growth of human glioma cells mediated by EGFR targeted non-viral vector GE7 system

    Institute of Scientific and Technical Information of China (English)

    陈永新; 许秀兰; 张光霁; 王韦; 金海英; 卢亦成; 朱诚; 顾健人


    Objective: To construct the EGFR targeted non-viral vector GE7 system and explore the in vitro effect of p21WAF-1/CIP1 gene on growth of human glioma cells mediated by the GE7 system. Methods: The EGFR targeted non-viral vector GE7 gene delivery system was constructed. The malignant human glioma cell line U251MG was transfected in vitro with β-galactosidase gene(reporter gene) and p21WAF-1/CIP1 gene (therapeutic gene) using the GE7 system. By means of X-gal staining, MTS and FACS, the transfection efficiency of exogenous gene and apoptosis rate of tumor cells were examined. The expression of p21WAF-1/CIP1 gene in transfected U251MG cell was examined by immunohistochemistry staining. Results: The highest transfer rate of exogenous gene was 70%. After transfection with p21WAF-1/CIP1 gene, the expression of WAF-1 increased remarkably and steadily; the growth of U251MG cells were inhibited evidently. FACS examination showed G1 arrest. The average apoptosis rate was 25.2%. Conclusion: GE7 system has the ability to transfer exogenous gene to targeted cells efficiently, and expression of p21WAF-1/CIP1 gene can induce apoptosis of glioma cell and inhibit its growth.

  17. Immunization of Mice with a Live Transconjugant Shigella Hybrid Strain Induced Th1 and Th17 Cell-Mediated Immune Responses and Confirmed Passive Protection Against Heterologous Shigellae. (United States)

    Nag, D; Koley, H; Sinha, R; Mukherjee, P; Sarkar, C; Withey, J H; Gachhui, R


    An avirulent, live transconjugant Shigella hybrid (LTSHΔstx) strain was constructed in our earlier study by introducing a plasmid vector, pPR1347, into a Shiga toxin gene deleted Shigella dysenteriae 1. Three successive oral administrations of LTSHΔstx to female adult mice produced comprehensive passive heterologous protection in their offspring against challenge with wild-type shigellae. Production of NO and different cytokines such asIL-12p70, IL-1β and IL-23 in peritoneal mice macrophages indicated that LTSHΔstx induced innate and adaptive immunity in mice. Furthermore, production of IFN-γ, IL-10 and IL-17 in LTSH-primed splenic CD4+ T cell suggested that LTSHΔstx may induce Th1 and Th17 cell-mediated immune responses. Exponential increase of the serum IgG and IgA titre against whole shigellae was observed in immunized adult mice during and after the immunization with the highest peak on day 35. Antigen-specific sIgA was also determined from intestinal lavage of immunized mice. The stomach extracts of neonates from immunized mice, mainly containing mother's milk, contained significant levels of anti-LTSHΔstx immunoglobulin. These studies suggest that the LTSHΔstx could be a new live oral vaccine candidate against shigellosis in the near future.

  18. Humoral and Cell-mediated Autoimmune Reactions to Human Acidic Ribosomal P2 Protein in Individuals Sensitized to Aspergillus fumigatus P2 Protein (United States)

    Mayer, Christina; Appenzeller, Ulrich; Seelbach, Heike; Achatz, Gernot; Oberkofler, Hannes; Breitenbach, Michael; Blaser, Kurt; Crameri, Reto


    A panel of cDNAs encoding allergenic proteins was isolated from an Aspergillus fumigatus cDNA library displayed on the surface of filamentous phage. Solid phase–immobilized serum immunoglobulin E (IgE) from A. fumigatus–allergic individuals was used to enrich phage displaying IgE-binding molecules. One of the cDNAs encoded a 11.1-kD protein that was identified as acidic ribosomal phosphoprotein type 2 (P2 protein). The allergen, formally termed rAsp f 8, shares >62% sequence identity and >84% sequence homology to corresponding eukaryotic P2 proteins, including human P2 protein. The sequences encoding human and fungal P2 protein were subcloned, expressed in Escherichia coli as His6-tagged fusion proteins, and purified by Ni2+–chelate affinity chromatography. Both recombinant P2 proteins were recognized by IgE antibodies from allergic individuals sensitized to the A. fumigatus P2 protein and elicited strong type 1–specific skin reactions in these individuals. Moreover, human and fungal P2 proteins induced proliferative responses in peripheral blood mononuclear cells of A. fumigatus– allergic subjects sensitized to the fungal P2 protein. These data provide strong evidence for in vitro and in vivo humoral and cell-mediated autoreactivity to human P2 protein in patients suffering from chronic A. fumigatus allergy. PMID:10224291

  19. Dim light at night interferes with the development of the short-day phenotype and impairs cell-mediated immunity in Siberian hamsters (Phodopus sungorus). (United States)

    Aubrecht, Taryn G; Weil, Zachary M; Nelson, Randy J


    Winter is a challenging time to survive and breed outside of the tropics. Animals use day length (photoperiod) to regulate seasonally appropriate adaptations in anticipation of challenging winter conditions. The net result of these photoperiod-mediated adjustments is enhanced immune function and increased survival. Thus, the ability to discriminate day length information is critical for survival and reproduction in small animals. However, during the past century, urban and suburban development has rapidly expanded and filled the night sky with light from various sources, obscuring crucial light-dark signals, which alters physiological interpretation of day lengths. Furthermore, reduced space, increased proximity to people, and the presence of light at night may act as stressors for small animals. Whereas acute stressors typically enhance immune responses, chronic exposure to stressors often impairs immune responses. Therefore, we hypothesized that the combination of dim light at night and chronic stress interferes with enhanced cell-mediated immunity observed during short days. Siberian hamsters (Phodopus sungorus) were assigned to short or long days with dark nights (0 lux) or dim (5 lux) light at night for 10 weeks. Following 2 weeks of chronic restraint (6 hr/day), a model of chronic stress, delayed type hypersensitivity (DTH) responses were assessed. Both dim light at night and restraint reduced the DTH response. Dim light at night during long nights produced an intermediate short day phenotype. These results suggest the constant presence of light at night could negatively affect survival of photoperiodic rodents by disrupting the timing of breeding and immune responses.

  20. Designing the method for optical in vitro monitoring of the cell-mediated scaffold technology for bone regeneration based on laser-induced fluorescence spectroscopy (United States)

    Larionov, P. M.; Maslov, N. A.; Papaeva, E. O.; Tereshchenko, V. P.; Khlestkin, V. K.; Bogachev, S. S.; Proskurina, A. S.; Titov, A. T.; Filipenko, M. L.; Pavlov, V. V.; Kudrov, G. A.; Orishich, A. M.


    One of the main unsolved problems in traumatology and orthopedics is reconstruction of critical-sized segmental bone defects. We believe that implementation of noninvasive monitoring of the bioengineering stages for cell-mediated bone scaffold by laser-induced fluorescence (LIF) can become a positive aspect in mastering this technique. An electrospun scaffold model (parameters: 10 wt. % polycaprolactone; 5% wt type A gelatin; mean fiber diameter 877.1 ± 169.1, and contact angle 45.3°) seeded with BHK IR cell culture (182 ± 38 cells/mm2) was used to show the principal possibility of differentiating between the scaffold seeded and unseeded with cells. First of all, the fluorescence spectra of the cell-seeded scaffold contain a peak at 305 nm for the excitation range of 230-290 nm, which can be used to differentiate between the samples. An increase in fluorescence intensity of the cell-seeded scaffold in the range of 400- 580 nm upon excitation at 230-340 nm is also noticeable. The wavelength of 250 nm is characterized by high signal intensity and is most suitable for differentiation between the samples.

  1. CD56 marks human dendritic cell subsets with cytotoxic potential

    NARCIS (Netherlands)

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


    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.

  2. Evaluation of cytotoxic activity of Schisandra chinensis lignans. (United States)

    Smejkal, Karel; Slapetová, Tereza; Krmenčík, Pavel; Babula, Petr; Dall'Acqua, Stefano; Innocenti, Gabbriella; Vančo, Ján; Casarin, Elisabetta; Carrara, Maria; Kalvarová, Karolína; Dvorská, Margita; Slanina, Jiří; Kramářová, Eva; Julínek, Ondřej; Urbanová, Marie


    Using exhaustive chromatographic separation we have isolated (-)-tigloyl-deangeloyl-gomisin F as a novel dibenzocyclooctadiene lignan from schisandra chinensis. With the help of HPLC, we further isolated (+)-schisandrin, (+)-deoxyschisandrin, (+)-γ-schisandrin, (-)-gomisin J, (+)-gomisin A, (-)-gomisin N, (-)-tigloyl-gomisin P, (-)-wuweizisu C, (-)-gomisin D, rubrisandrin A, (-)-gomisin G, (+)-gomisin K (3) and (-)-schisantherin C. A full NMR description of (-)-schisantherin C was carried out with the aim to confirm previous reports of its structure. Compounds isolated were identified on the basis of UV, IR, (1)H- and (13)C-NMR and MS. The cytotoxicity of lignans was tested for the BY-2 cell line alone and as a synergistic effect with the cytotoxic agent camptothecin. Lignans showed various toxicity and synergistic and antagonistic effects on camptothecin-induced cytotoxicity. Cytotoxicity against colon cancer cell line LoVo was also tested.

  3. Antioxidant and cytotoxic activity of Acanthus ilicifolius flower

    Institute of Scientific and Technical Information of China (English)

    Muhamad Firdaus; Asep Awaludin Prihanto; Rahmi Nurdiani


    Objective: To investigate the antioxidant and cytotoxic activity of the flower of Acanthus ilicifolius (A. ilicifolius). Methods: Antioxidant activity was determined as antiradical efficiency with diphenyl picrylhydrazil (DPPH) method and cytotoxic assay was undertaken using brine shrimp lethal toxicity test. Results: A. ilicifolius flower contained terpenoid, phenolic compounds, and alkaloid. The methanol extract of A. ilicifolius flower showed the highest antiradical efficiency (AE=1.41í10-3) against DPPH radicals and the highest cytotoxicity (LC50=22 μg/mL) against brine shrimp nauplii. Conclusions: It is suggested that active compounds of A. ilicifolius flower solved in methanol play a role to inhibit free radical activity and kill Artemia salina nauplii. The substances can be considered as potential antioxidant and cytotoxic agents as well as imminent candidate for cancer therapy.

  4. Antioxidant and cytotoxic activity of Acanthus ilicifolius flower

    Institute of Scientific and Technical Information of China (English)

    Muhamad; Firdaus; Asep; Awaludin; Prihanto; Rahmi; Nurdiani


    Objective:To investigate the antioxidant and cytotoxic activity of the flower of Acanthus ilicifolius(A.ilicifolius).Methods:Antioxidant activity was determined as antiradical efficiency with diphenyl picrylhydrazil(DPPH)method and cytotoxic assay was undertaken using brine shrimp lethal toxicity test.Results:A.ilicifolius flower contained terpenoid,phenolic compounds,and alkaloid.The methanol extract of A.ilicifolius flower showed the highest antiradical efficiency(AE=1.41×10-3)against DPPH radicals and the highest cytotoxicity(LC50=22μg/mL)against brine shrimp nauplii.Conclusions:It is suggested that active compounds of A.ilicifolius flower solved in methanol play a role to inhibit free radical activity and kill Artemia salina nauplii.The substances can be considered as potential antioxidant and cytotoxic agents as well as imminent candidate for cancer therapy.

  5. Antimicrobial and cytotoxic activities of Abroma augusta Lnn. leaves extract

    Institute of Scientific and Technical Information of China (English)

    FK Saikot; Alam Khan; MF Hasan


    Objective: To evaluate the antimicrobial and cytotoxic activity of acetone extract of leaves ofAbroma augusta. Methods: Disc diffusion method was used to demonstrate antibacterial and antifungal activities. Cytotoxicity was determined against brine shrimp nauplii. In addition, minimum inhibitory concentration (MIC) was determined using serial dilution technique to determine antibacterial potency. Results: The extract showed significant antibacterial activities against three gram-positive (Bacillus subtilis, Bacillus megaterium and Staphylococcus aureus) and four gram-negative (Escherichia coli, Shigella dysenteriae, Shigella sonnei and Salmonella typhi) bacteria. The antifungal activity was found strong against five fungi (Aspergillus flavus, Aspergillus niger, Candida albicans, Rhizopus oryzae and Aspergillus fumigatus). In cytotoxicity determination, LC50 of the extract against brine shrimp nauplii was 7.06μg/ml. Conclusions: The Abroma leaves extract may be consider as a potent antimicrobial and cytotoxic agent for further advance research.

  6. Icogenin, a new cytotoxic steroidal saponin isolated from Dracaena draco. (United States)

    Hernández, Juan C; León, Francisco; Quintana, José; Estévez, Francisco; Bermejo, Jaime


    This paper reports on the cytotoxic effect induced by a new natural steroidal saponin, icogenin, on the myeloid leukemia cell line HL-60. Icogenin was found to be a cytotoxic compound IC(50) 2.6+/-0.9microM at 72h, with growth inhibition caused by the induction of apoptosis, as determined by microscopy of nuclear changes and the fragmentation of poly(ADP-ribose) polymerase-1.

  7. Phytochemical, antioxidant, antiviral and cytotoxic evaluation of Opuntia dillenii flowers


    Arthanari Saravana Kumar; Mani Ganesh; Mei Mei Peng; Jang Hyun Tae


    Opuntia dillenii used in Asian traditional medicine especially in China. We here report on the investigation of the phytochemical content, antioxidant, cytotoxicity and antiviral activity of methanolic extract of O. dillenii flowers. The antioxidant activity was measured with the DPPH, hydrogen peroxide and hydroxyl radicals scavenging method. In the antiviral and cytotoxic assay we used different viruses in different cell lines. In antioxidant assay, the DPPH assay exhibited potent antioxid...

  8. Two New Cytotoxic Sesquiterpenoids from Eupatorium lindleyanum DC.

    Institute of Scientific and Technical Information of China (English)


    Two new sesquiterpenoid lactones, namely eupalinilides K and L, were isolated from Eupatorium lindleyanum DC. Their structures were determined by spectral methods, including 1D- and 2D-NMR spectra. Cytotoxic evaluation showed that eupalinilide L exhibited potent cytotoxicity against P-388 and A-549 tumor cell lines with IC50values of 0.17 and 2.60 μ mol/L, respectively.

  9. Molecular cytotoxic mechanisms of chlorpromazine in isolated rat hepatocytes. (United States)

    MacAllister, Stephanie L; Young, Cheryl; Guzdek, Anna; Zhidkov, Nickholas; O'Brien, Peter J


    Chlorpromazine (CPZ), a member of the largest class of first-generation antipsychotic agents, is known to cause hepatotoxicity in the form of cholestasis and hepatocellular necrosis in some patients. The mechanism of CPZ hepatotoxicity is unclear, but is thought to result from reactive metabolite formation. The goal of this research was to assess potential cytotoxic mechanisms of CPZ using the accelerated cytotoxicity mechanism screening (ACMS) technique with freshly isolated rat hepatocytes. This study identified CPZ cytotoxicity and inhibition of mitochondrial membrane potential (MMP) to be concentration-dependent. Furthermore, inhibition of cytochrome P450s (CYPs), including CYP2D1 and 1A2, delayed CPZ cytotoxicity, suggesting a role for CYP activation of CPZ to a toxic metabolite(s) in this model. Metabolism studies also demonstrated glucuronide and glutathione (GSH) requirement for CPZ detoxification in hepatocytes. Inactivating the 2-electron reduction pathway, NAD(P)H quinone oxidoreductase (NQO1), caused a significant increase in hepatocyte susceptibility to CPZ, indicating quinoneimine contribution to CPZ cytotoxicity. Nontoxic concentrations of peroxidase/H(2)O(2) (inflammatory model) increased cytotoxicity in CPZ-treated hepatocytes and caused additional mitochondrial toxicity. Inflammation further depleted GSH and increased oxidized glutathione (GSSG) levels. Results suggest activation of CPZ to reactive metabolites by 2 pathways in hepatocytes: (i) a CYP-catalyzed quinoneimine pathway, and (ii) a peroxidase-catalyzed oxidation of CPZ to CPZ radicals.

  10. Phytochemical and Cytotoxic Investigations of Curcuma mangga Rhizomes

    Directory of Open Access Journals (Sweden)

    Sri Nurestri A. Malek


    Full Text Available Investigations on the cytotoxic effects of the crude methanol and fractionated extracts (hexane, ethyl acetate C. mangga against six human cancer cell lines, namely the hormone-dependent breast cell line (MCF-7, nasopharyngeal epidermoid cell line (KB, lung cell line (A549, cervical cell line (Ca Ski, colon cell lines (HCT 116 and HT-29, and one non-cancer human fibroblast cell line (MRC-5 were conducted using an in-vitro neutral red cytotoxicity assay. The crude methanol and fractionated extracts (hexane and ethyl acetate displayed good cytotoxic effects against MCF-7, KB, A549, Ca Ski and HT-29 cell lines, but exerted no damage on the MRC-5 line. Chemical investigation from the hexane and ethyl acetate fractions resulted in the isolation of seven pure compounds, namely (E-labda-8(17,12-dien-15,16-dial (1, (E-15,16-bisnor-labda-8(17,11-dien-13-on (2, zerumin A (3, β-sitosterol, curcumin, demethoxycurcumin and bis-demethoxycurcumin. Compounds 1 and 3 exhibited high cytotoxic effects against all six selected cancer cell lines, while compounds 2 showed no anti-proliferative activity on the tested cell lines. Compound 1 also demonstrated strong cytotoxicity against the normal cell line MRC-5. This paper reports for the first time the cytotoxic activities of C. mangga extracts on KB, A549, Ca Ski, HT-29 and MRC-5, and the occurrence of compound 2 and 3 in C. mangga.

  11. Cytotoxicity of commonly used luting cements -An in vitro study. (United States)

    Trumpaite-Vanagiene, Rita; Bukelskiene, Virginija; Aleksejuniene, Jolanta; Puriene, Alina; Baltriukiene, Daiva; Rutkunas, Vygandas


    The study aimed to 1) evaluate the cytotoxicity of luting cements: Hoffmann's Zinc Phosphate (Hoffmann's ZP), GC Fuji Plus Resin Modified Glass Ionomer (Fuji Plus RMGI) and 3M ESPE RelyX Unicem Resin Cement (RelyX Unicem RC) and 2) test if pre-washing reduces the cements' cytotoxicity. In vitro human gingival fibroblast (HGF) culture model was chosen. The cytotoxicity was evaluated by MTT test, the cell viability -by staining the cells with AO/EB dye mixture. The means±SD of Cell Survival Ratio (CSR%) were compared among different cement types under two testing conditions, with or without cement pre-washing. The CSR%s were compared by ANOVA and linear multiple regression (LMR). Hoffmann's ZPC was less cytotoxic, while Fuji Plus RMGIC and RelyX Unicem RC were more cytotoxic (ANOVA, ptype of cement and cement pre-washing jointly explained 90% of cell survival (LMR, p<0.001, adjusted squared R=0.889). The commonly used luting cements such as Hoffmann's ZP, Fuji Plus RMGI and RelyX Unicem RC may have a cytotoxic potential.

  12. Antioxidants protect keratinocytes against M. ulcerans mycolactone cytotoxicity.

    Directory of Open Access Journals (Sweden)

    Alvar Grönberg

    Full Text Available BACKGROUND: Mycobacterium ulcerans is the causative agent of necrotizing skin ulcerations in distinctive geographical areas. M. ulcerans produces a macrolide toxin, mycolactone, which has been identified as an important virulence factor in ulcer formation. Mycolactone is cytotoxic to fibroblasts and adipocytes in vitro and has modulating activity on immune cell functions. The effect of mycolactone on keratinocytes has not been reported previously and the mechanism of mycolactone toxicity is presently unknown. Many other macrolide substances have cytotoxic and immunosuppressive activities and mediate some of their effects via production of reactive oxygen species (ROS. We have studied the effect of mycolactone in vitro on human keratinocytes--key cells in wound healing--and tested the hypothesis that the cytotoxic effect of mycolactone is mediated by ROS. METHODOLOGY/PRINCIPAL FINDINGS: The effect of mycolactone on primary skin keratinocyte growth and cell numbers was investigated in serum free growth medium in the presence of different antioxidants. A concentration and time dependent reduction in keratinocyte cell numbers was observed after exposure to mycolactone. Several different antioxidants inhibited this effect partly. The ROS inhibiting substance deferoxamine, which acts via chelation of Fe(2+, completely prevented mycolactone mediated cytotoxicity. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that mycolactone mediated cytotoxicity can be inhibited by deferoxamine, suggesting a role of iron and ROS in mycolactone induced cytotoxicity of keratinocytes. The data provide a basis for the understanding of Buruli ulcer pathology and the development of improved therapies for this disease.

  13. Long-term cytotoxic effects of contemporary root canal sealers

    Directory of Open Access Journals (Sweden)

    Emmanuel Joao Nogueira Leal da SILVA


    Full Text Available Objectives The aim of the present study was to investigate the effects of root canal sealers on the cytotoxicity of 3T3 fibroblasts during a period of 5 weeks. Material and Methods Fibroblasts (3T3, 1×105 cells per well were incubated with elutes of fresh specimens from eight root canal sealers (AH Plus, Epiphany, Endomethasone N, EndoREZ, MTA Fillapex, Pulp Canal Sealer EWT, RoekoSeal and Sealapex and with elutes of the same specimens for 5 succeeding weeks after immersing in simulated body fluid. The cytotoxicity of all root canal sealers was determined using the MTT assay. Data were analyzed using ANOVA and Tukey's test. Results RoekoSeal was the only sealer that did not show any cytotoxic effects (p<0.05. All the other tested sealers exhibited severe toxicity initially (week 0. MTA Fillapex remained moderately cytotoxic after the end of experimental period. Toxicity of the other tested sealers decreased gradually over time. The evaluated root canal sealers presented varying degrees of cytotoxicity, mainly in fresh mode. Conclusions RoekoSeal had no cytotoxic effect both freshly mixed and in the other tested time points. MTA Fillapex was associated with significantly less cell viability when compared to the other tested root canal sealers.

  14. Intravitelline injection of cultured rat embryos: An improved method for the identification of cytotoxic and non-cytotoxic teratogens. (United States)

    Cumberland, P F; Richold, M; Parsons, J F; Pratten, M K


    A preliminary study of a novel developmental toxicity screen has been carried out. The technique involves the direct injection into the vitelline circulation of the 11.5-day rat conceptus, by-passing the metabolically active visceral yolk sac. The evaluation was performed blind using four coded model compounds: sulphanilamide (non-cytotoxic, non-teratogen), retinoic acid (teratogen) and methotrexate and cyclophosphamide (both cytotoxic teratogens). Seven parameters of teratogenicity and cytotoxicity were measured (yolk sac diameter, crown-rump length, somite number, yolk sac protein, yolk sac DNA, embryo protein, embryo DNA) and morphological abnormalities were also noted. The results showed that this technique successfully identified the developmental toxins and, moreover, differentiated between teratogens and cytotoxic teratogens. Additionally, the results show that methotrexate and cyclophosphamide produced an effect without prior exogenous activation as is necessary in other in vitro tests.

  15. 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


    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.

  16. Cytotoxic activity of Thai medicinal plants for cancer treatment

    Directory of Open Access Journals (Sweden)

    Chawaboon Dechsukum


    Full Text Available Twelve Thai medicinal plants as the ingredients of a Southern Thai traditional formula for cancer treatment were selected to test cytotoxicity activity against two types of human cancer cell lines ; large cell lung carcinoma (CORL-23 and prostate cancer cell lines (PC3 and one type of normal human cell line, fibroblast cells (10FS. SRB assay was used to test cytotoxic activity against all the cell types. Two of the extracts (water and ethanolic extracts procedures used were similar to those practised by Thai traditional doctors. One concentration (50 μg/ml of two different extracts was tested first against cell lines and the active plant extracts were diluted and tested for calculating IC50. The ethanolic extracts of six plants (Bridelia ovata, Curcuma zedoaria, Derris scandens, Dioscorea membranacea, Nardostachys jatamansi and Rhinacanthus nasutus showed cytotoxic activity (IC50< 30 μg/ml against lung and prostate cancer cell lines. Dioscorea membranacea roots showed the highest cytotoxic activity against lung cancer cell lines ( IC50= 4.6 μg/ml but it exhibited low cytotoxic activity against prostate cancer cell lines (IC50= 17.55 μg/ml and less cytotoxic activity against normal cell lines (IC50= 66.05 μg/ml. Curcuma zedoaria showed cytotoxic activity against COR L-23 and PC3 but less cytotoxic activity against 10FS (IC50 = 6.05, 17.84 and 55.50 μg/ml respectively Rhinacanthus nasutus root extract showed the highest cytotoxic activity against PC3 ( IC50 = 2.01 μg/ml and this extract also showed high activity against COR L-23 and 10FS (IC50=5.05 and 10.95 μg/ml respectively. The water extract of all plants exhibited no activity against all types of human cells. Two ethanolic plant extracts (Dioscorea membranacea and Curcuma zedoaria which showed specific activity against lung cancer cell lines and less cytotoxic activity against normal cells should be further investigated for active compounds against lung cancer cell.

  17. Protective effects of rilmenidine and AGN 192403 on oxidative cytotoxicity and mitochondrial inhibitor-induced cytotoxicity in astrocytes. (United States)

    Choi, Dong-Hee; Kim, Dong-Hoon; Park, Yun-Gyu; Chun, Boe-Gwun; Choi, Sang-Hyun


    Oxidative stress and mitochondrial dysfunction are important aspects of pathogenesis, particularly in the brain, which is highly dependent on oxygen, and the protection of astrocytes is essential for neuroprotection. In this context, imidazoline drugs have been reported to be neuroprotective. Our recent study showed that imidazoline drugs, including guanabenz, inhibit the naphthazarin-induced oxidative cytotoxicity associated with lysosomal destabilization. We now report on a study into the protective effects of rilmenidine and AGN 192403, which have affinity for imidazoline-1 receptors, on the cytotoxicity induced by naphthazarin and inhibitors of mitochondrial respiration in astrocytes. Cytotoxicity was measured grossly by LDH release and by measuring changes in lysosomal membrane stability and features of mitochondrial membrane permeabilization. Naphthazarin-induced cytotoxicity was evidenced by the ordered development of lysosomal acridine orange relocation, decrease in mitochondrial potential, cytochrome c release, and caspase-9 activation, and was inhibited by guanabenz, rilmenidine, and AGN 192403. Antimycin A and rotenone induced mitochondrial dysfunction primarily, and their cytotoxicities were inhibited only by AGN 192403. Rilmenidine and guanabenz may have a lysosomal stabilizing effect, which underlies their protective effects. AGN 192403 might affect the mitochondrial cell death cascades, and had a novel protective effect on the cytotoxicity associated with mitochondrial dysfunction.

  18. High pro-inflammatory cytokine secretion and loss of high avidity cross-reactive cytotoxic T-cells during the course of secondary dengue virus infection.

    Directory of Open Access Journals (Sweden)

    Tao Dong

    Full Text Available BACKGROUND: Dengue is one of the most important human diseases transmitted by an arthropod vector and the incidence of dengue virus infection has been increasing - over half the world's population now live in areas at risk of infection. Most infections are asymptomatic, but a subset of patients experience a potentially fatal shock syndrome characterised by plasma leakage. Severe forms of dengue are epidemiologically associated with repeated infection by more than one of the four dengue virus serotypes. Generally attributed to the phenomenon of antibody-dependent enhancement, recent observations indicate that T-cells may also influence disease phenotype. METHODS AND FINDINGS: Virus-specific cytotoxic T lymphocytes (CTL showing high level cross reactivity between dengue serotypes could be expanded from blood samples taken during the acute phase of secondary dengue infection. These could not be detected in convalescence when only CTL populations demonstrating significant serotype specificity were identified. Dengue cross-reactive CTL clones derived from these patients were of higher avidity than serotype-specific clones and produced much higher levels of both type 1 and certain type 2 cytokines, many previously implicated in dengue pathogenesis. CONCLUSION: Dengue serotype cross-reactive CTL clones showing high avidity for antigen produce higher levels of inflammatory cytokines than serotype-specific clones. That such cells cannot be expanded from convalescent samples suggests that they may be depleted, perhaps as a consequence of activation-induced cell death. Such high avidity cross-reactive memory CTL may produce inflammatory cytokines during the course of secondary infection, contributing to the pathogenesis of vascular leak. These cells appear to be subsequently deleted leaving a more serotype-specific memory CTL pool. Further studies are needed to relate these cellular observations to disease phenotype in a large group of patients. If

  19. The role of a novel protein ligand hMSH 2 in Vγ9δ2 T cell-mediated anti-cervix cancer immunity

    Institute of Scientific and Technical Information of China (English)



    Objective To explore the role of hMSH2,a novel endogenous tumor-associated protein ligand recognized by Vγ9δ2 T cells,in innate anti-cervix cancer immunity.Methods hMSH2 that expressed on the surface of cervical cancer cell line HeLa cells was blocked by specific antibody.Then the differences in their effects on Vγ9δ2T cells before and after antibody blockage were evaluated by cytotoxicity of Vγ9δ2 T cells and cytokines secretion.

  20. Intracellular concentrations determine the cytotoxicity of adefovir, cidofovir and tenofovir. (United States)

    Zhang, Xun; Wang, Ruduan; Piotrowski, Mary; Zhang, Hui; Leach, Karen L


    Lack of in vitro to in vivo translation is a major challenge in safety prediction during early drug discovery.One of the most common in vitro assays to evaluate the probability of a compound to cause adverse effects is a cytotoxicity assay. Cytotoxicity of a compound is often measured by dose–response curves assuming the administered doses and intracellular exposures are equal at the time of measurement.However, this may not be true for compounds with low membrane permeability or those which are substrates for drug transporters as intracellular concentrations are determined both by passive permeability and active uptake through drug transporters. We show here that three antiviral drugs, adefovir, cidofovir and tenofovir exhibit significantly increased cytotoxicity in HEK293 cells transfected with organic anion transporter (OAT) 1 and 3 compared to a lack of cytotoxicity in HEK293 wildtype cells. A further look at the media and intracellular drug concentrations showed that 24 h after dosing, all three drugs had higher intracellular drug concentrations than that of media in the HEK-OAT1 cells whereas the intracellular drug concentrations in the wildtype cells were much lower than the administered doses. Comparing cytotoxicity IC(50) values of adefovir, cidofovir and tenofovir based on administered doses and measured intracellular concentrations in HEK-OAT1 cells revealed that intracellular drug concentrations have significant impact on calculated IC(50) values. Tenofovir showed much less intrinsic cytotoxicity than adefovir and cidofovir using intracellular concentrations rather than media concentration. Our data suggest that for low permeable drugs or drugs that are substrates for drug transporters, the choice of cellular model is critical for providing an accurate determination of cytotoxicity.

  1. Novel plant virus-based vaccine induces protective cytotoxic T-lymphocyte-mediated antiviral immunity through dendritic cell maturation. (United States)

    Lacasse, Patrick; Denis, Jérôme; Lapointe, Réjean; Leclerc, Denis; Lamarre, Alain


    Currently used vaccines protect mainly through the production of neutralizing antibodies. However, antibodies confer little or no protection for a majority of chronic viral infections that require active involvement of cytotoxic T lymphocytes (CTLs). Virus-like particles (VLPs) have been shown to be efficient inducers of cell-mediated immune responses, but administration of an adjuvant is generally required. We recently reported the generation of a novel VLP system exploiting the self-assembly property of the papaya mosaic virus (PapMV) coat protein. We show here that uptake of PapMV-like particles by murine splenic dendritic cells (DCs) in vivo leads to their maturation, suggesting that they possess intrinsic adjuvant-like properties. DCs pulsed with PapMV-like particles displaying the lymphocytic choriomeningitis virus (LCMV) p33 immunodominant CTL epitope (PapMV-p33) efficiently process and cross-present the viral epitope to p33-specific transgenic T cells. Importantly, the CTL epitope is also properly processed and presented in vivo, since immunization of p33-specific T-cell receptor transgenic mice with PapMV-p33 induces the activation of large numbers of specific CTLs. C57BL/6 mice immunized with PapMV-p33 VLPs in the absence of adjuvant develop p33-specific effector CTLs that rapidly expand following LCMV challenge and protect vaccinated mice against LCMV infection in a dose-dependent manner. These results demonstrate the efficiency of this novel plant virus-based vaccination platform in inducing DC maturation leading to protective CTL responses.

  2. Effects of in ovo exposure to PCBs (coplanar congener, kanechlor mixture, hydroxylated metabolite) on the developing cell-mediated immunity in chickens

    Energy Technology Data Exchange (ETDEWEB)

    Hasegawa, J.; Matsuda, M.; Kawano, M.; Wakimoto, T. [Faculty of Agriculture, Ehime Univ., Matsuyama, Ehime (Japan); Kashima, Y. [Dept. of Hygiene, Yokohama City Univ. School of Medicine, Yokohama (Japan)


    Polychlorinated biphenyls (PCBs) are wide spread environmental contaminants and known to cause various adverse effects on health of human and wildlife. Immune system is one of the several targets for toxic effects of PCBs and its normal balance is often disrupted by the exposure of the compounds. For example, PCBs may induce immune suppression and result in increased susceptibility to bacterial and viral infections, or conversely, excessive immune enhancement may cause adverse outcomes including as autoimmune disease and anergy. Therefore immune function is regarded as one of an important endpoint in toxicological risk assessment. There are a number of studies shown that neonatal organisms perinatally exposed to polyhalogenated aromatic hydrocarbons (PHAHs) such as PCBs have severer effects on their immune system than adult. Dioxins and coplanar PCB congeners, structurally planar PHAHs are known to have high affinity for aryl hydrocarbon receptor (AhR). 2,3,7,8-tetrachlorinated dibenzo-p-dioxin (TCDD) have the strongest affinity among such compounds and these are considered to act on immune system through AhR. On the other hand, such as non-planar PCB congeners with low affinity for AhR, which are abundantly contained in commercial PCB preparations have non-additive (antagonistic) effects on immune function. Prenatal exposure of TCDD to rodent induced abnormal lymphoid development in the thymus and thymus-dependent immune functions were remarkably disturbed. Although several experimental studies in mammals have been carried out on the developmental immunotoxicity of PCBs, there are still limited information available on avian species. Thus in this study, prenatal exposure to low level of PCBs and the effects on the developing immune system were investigated with chicken as a model animal of avian species, especially it is focused on the cell-mediated immune function.

  3. Thiol dependent NF-κB suppression and inhibition of T-cell mediated adaptive immune responses by a naturally occurring steroidal lactone Withaferin A. (United States)

    Gambhir, Lokesh; Checker, Rahul; Sharma, Deepak; Thoh, M; Patil, Anand; Degani, M; Gota, Vikram; Sandur, Santosh K


    Withaferin A (WA), a steroidal lactone isolated from ayurvedic medicinal plant Withania somnifera, was shown to inhibit tumor growth by inducing oxidative stress and suppressing NF-κB pathway. However, its effect on T-cell mediated adaptive immune responses and the underlying mechanism has not been investigated. Since both T-cell responses and NF-κB pathway are known to be redox sensitive, the present study was undertaken to elucidate the effect of WA on adaptive immune responses in vitro and in vivo. WA inhibited mitogen induced T-cell and B-cell proliferation in vitro without inducing any cell death. It inhibited upregulation of T-cell (CD25, CD69, CD71 and CD54) and B-cell (CD80, CD86 and MHC-II) activation markers and secretion of Th1 and Th2 cytokines. WA induced oxidative stress by increasing the basal ROS levels and the immunosuppressive effects of WA were abrogated only by thiol anti-oxidants. The redox modulatory effects of WA in T-cells were attributed to its ability to directly interact with free thiols. WA inhibited NF-κB nuclear translocation in lymphocytes and prevented the direct binding of nuclear NF-κB to its consensus sequence. MALDI-TOF analysis using a synthetic NF-κB-p50 peptide containing Cys-62 residue suggested that WA can modify the cysteine residue of NF-κB. The pharmacokinetic studies for WA were also carried out and in vivo efficacy of WA was studied using mouse model of Graft-versus-host disease. In conclusion, WA is a potent inhibitor of T-cell responses and acts via a novel thiol dependent mechanism and inhibition of NF-κB pathway.

  4. Tetherin does not significantly restrict dendritic cell-mediated HIV-1 transmission and its expression is upregulated by newly synthesized HIV-1 Nef

    Directory of Open Access Journals (Sweden)

    Wu Li


    Full Text Available Abstract Background Dendritic cells (DCs are among the first cells to encounter HIV-1 and play important roles in viral transmission and pathogenesis. Immature DCs allow productive HIV-1 replication and long-term viral dissemination. The pro-inflammatory factor lipopolysaccharide (LPS induces DC maturation and enhances the efficiency of DC-mediated HIV-1 transmission. Type I interferon (IFN partially inhibits HIV-1 replication and cell-cell transmission in CD4+ T cells and macrophages. Tetherin is a type I IFN-inducible restriction factor that blocks HIV-1 release and modulates CD4+ T cell-mediated cell-to-cell transmission of HIV-1. However, the role of type I IFN and tetherin in HIV-1 infection of DCs and DC-mediated viral transmission remains unknown. Results We demonstrated that IFN-alpha (IFNα-induced mature DCs restricted HIV-1 replication and trans-infection of CD4+ T cells. Tetherin expression in monocyte-derived immature DCs was undetectable or very low. High levels of tetherin were transiently expressed in LPS- and IFNα-induced mature DCs, while HIV-1 localized into distinct patches in these DCs. Knockdown of induced tetherin in LPS- or IFNα-matured DCs modestly enhanced HIV-1 transmission to CD4+ T cells, but had no significant effect on wild-type HIV-1 replication in mature DCs. Intriguingly, we found that HIV-1 replication in immature DCs induced significant tetherin expression in a Nef-dependent manner. Conclusions The restriction of HIV-1 replication and transmission in IFNα-induced mature DCs indicates a potent anti-HIV-1 response; however, high levels of tetherin induced in mature DCs cannot significantly restrict wild-type HIV-1 release and DC-mediated HIV-1 transmission. Nef-dependent tetherin induction in HIV-1-infected immature DCs suggests an innate immune response of DCs to HIV-1 infection.

  5. Hemagglutinin-based polyanhydride nanovaccines against H5N1 influenza elicit protective virus neutralizing titers and cell-mediated immunity

    Directory of Open Access Journals (Sweden)

    Ross KA


    Full Text Available Kathleen A Ross,1 Hyelee Loyd,2 Wuwei Wu,2 Lucas Huntimer,3 Shaheen Ahmed,4 Anthony Sambol,5 Scott Broderick,6 Zachary Flickinger,2 Krishna Rajan,6 Tatiana Bronich,4 Surya Mallapragada,1 Michael J Wannemuehler,3 Susan Carpenter,2 Balaji Narasimhan1 1Chemical and Biological Engineering, Iowa State University, Ames, IA, USA; 2Animal Science, Iowa State University, Ames, IA, USA; 3Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA, USA; 4Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA; 5Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA; 6Materials Science and Engineering, Iowa State University, Ames, IA, USA Abstract: H5N1 avian influenza is a significant global concern with the potential to become the next pandemic threat. Recombinant subunit vaccines are an attractive alternative for pandemic vaccines compared to traditional vaccine technologies. In particular, polyanhydride nanoparticles encapsulating subunit proteins have been shown to enhance humoral and cell-mediated immunity and provide protection upon lethal challenge. In this work, a recombinant H5 hemagglutinin trimer (H53 was produced and encapsulated into polyanhydride nanoparticles. The studies performed indicated that the recombinant H53 antigen was a robust immunogen. Immunizing mice with H53 encapsulated into polyanhydride nanoparticles induced high neutralizing antibody titers and enhanced CD4+ T cell recall responses in mice. Finally, the H53-based polyanhydride nanovaccine induced protective immunity against a low-pathogenic H5N1 viral challenge. Informatics analyses indicated that mice receiving the nanovaccine formulations and subsequently challenged with virus were similar to naïve mice that were not challenged. The current studies provide a basis to further exploit the advantages of polyanhydride nanovaccines in pandemic scenarios. Keywords: polymer, nanoparticle, vaccine, subunit

  6. Anandamide attenuates Th-17 cell-mediated delayed-type hypersensitivity response by triggering IL-10 production and consequent microRNA induction.

    Directory of Open Access Journals (Sweden)

    Austin R Jackson

    Full Text Available Endogenous cannabinoids [endocannabinoids] are lipid signaling molecules that have been shown to modulate immune functions. However, their role in the regulation of Th17 cells has not been studied previously. In the current study, we used methylated Bovine Serum Albumin [mBSA]-induced delayed type hypersensitivity [DTH] response in C57BL/6 mice, mediated by Th17 cells, as a model to test the anti-inflammatory effects of endocannabinoids. Administration of anandamide [AEA], a member of the endocannabinoid family, into mice resulted in significant mitigation of mBSA-induced inflammation, including foot pad swelling, cell infiltration, and cell proliferation in the draining lymph nodes [LN]. AEA treatment significantly reduced IL-17 and IFN-γ production, as well as decreased RORγt expression while causing significant induction of IL-10 in the draining LNs. IL-10 was critical for the AEA-induced mitigation of DTH response inasmuch as neutralization of IL-10 reversed the effects of AEA. We next analyzed miRNA from the LN cells and found that 100 out of 609 miRNA species were differentially regulated in AEA-treated mice when compared to controls. Several of these miRNAs targeted proinflammatory mediators. Interestingly, many of these miRNA were also upregulated upon in vitro treatment of LN cells with IL-10. Together, the current study demonstrates that AEA may suppress Th-17 cell-mediated DTH response by inducing IL-10 which in turn triggers miRNA that target proinflammatory pathways.

  7. 21 Days head-down bed rest induces weakening of cell-mediated immunity - Some spaceflight findings confirmed in a ground-based analog. (United States)

    Kelsen, Jens; Bartels, Lars Erik; Dige, Anders; Hvas, Christian Lodberg; Frings-Meuthen, Petra; Boehme, Gisela; Thomsen, Marianne Kragh; Fenger-Grøn, Morten; Dahlerup, Jens Frederik


    Several studies indicate a weakening of cell-mediated immunity (CMI) and reactivation of latent herpes viruses during spaceflight. We tested the hypothesis that head-down bed rest (HDBR), a ground-based analog of spaceflight, mimics the impact of microgravity on human immunity. Seven healthy young males underwent two periods of 3 weeks HDBR in the test facility of the German Aerospace Center. As a nutritional countermeasure aimed against bone demineralisation, 90 mmol potassium bicarbonate (KHCO(3)) was administered daily in a crossover design. Blood samples were drawn on five occasions. Whole blood was stimulated with antigen i.e. Candida albicans, purified protein derivative (PPD) tuberculin, tetanus toxoid and Cytomegalovirus (CMV) (CMV-QuantiFERON). Flow cytometric analysis included CD4(+)CD25(+)CD127(-)FOXP3(+) regulatory T cells (Tregs), γδ T cells, B cells, NK cells and dendritic cells. In one of the two bed rest periods, we observed a significant decrease in production of interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) following phytohemagglutinin (PHA) stimulation, with a rapid normalization being observed after HDBR. The cytokine levels showed a V-shaped pattern that led to a relativeTh2-shift in cytokine balance. Only three individuals responded to the specific T cell antigens without showing signs of an altered response during HDBR, nor did we observe reactivation of CMV or Epstein-Barr virus (EBV). Of unknown significance, dietary supplementation with KHCO(3) counteracted the decrease in IL-2 levels during HDBR, while there was no impact on other immunological parameters. We conclude that discrete alterations in CMI may be induced by HDBR in selected individuals.

  8. Study of biomaterial-induced macrophage activation, cell-mediated immune response and molecular oxidative damage in patients with dermal bioimplants. (United States)

    Sánchez, Olga; Rodríguez-Sureda, Víctor; Domínguez, Carmen; Fernández-Figueras, Teresa; Vilches, Angel; Llurba, Elisa; Alijotas-Reig, Jaume


    Several soft-tissue dermal fillers have been reported to provoke immunogenicity and may cause adverse reactions despite claims regarding their safety. This study aimed to assess biomaterial-induced macrophage activation, cell-mediated immune response and oxidative stress in 169 patients with dermal bioimplants. To this end, we analysed plasma concentrations of myeloperoxidase (MPO), the chitinase-like proteins chitotriosidase and YKL-40 and molecular oxidative damage. The present study shows, for the first time, that the components of innate immunity: chitotriosidase and YKL-40, are significantly higher in patients with certain bioimplants and these markers of monocyte/macrophage activation rose progressively as adverse reactions (AR) evolved. Plasma MPO levels increased 4-fold in filler users with AR and 3-fold in those without. Analysis by filler type showed subjects injected with calcium hydroxylapatite, methacrylate, acrylamides and silicone to have values significantly above those of non-filler subjects for at least two plasma biomarkers, probably because the afore-mentioned biomaterials are permanent and prone to trigger AR in the long term. By contrast, hyaluronic acid alone elicited little immune response. Plasma concentrations of markers of oxidative damage to lipids and proteins were found to be significantly higher in users of four of the nine dermal fillers studied. These diffusible products of molecular peroxidation would stem from the reaction catalysed by MPO that generates potent oxidants, leading to cell oxidative damage which, in turn, may exert deleterious effects on the organism. Overall, the results of this study on the effects of a range of dermal fillers point to chronic activation of the immune response mediated by macrophages and PMNs. The increases in plasma of MPO, chitotriosidase and YKL-40 proteins and products of macromolecular peroxidation suggests that these molecules could serve as blood-based biochemical markers and alert to the

  9. An antibody against the surfactant protein A (SP-A)-binding domain of the SP-A receptor inhibits T cell-mediated immune responses to Mycobacterium tuberculosis. (United States)

    Samten, Buka; Townsend, James C; Sever-Chroneos, Zvjezdana; Pasquinelli, Virginia; Barnes, Peter F; Chroneos, Zissis C


    Surfactant protein A (SP-A) suppresses lymphocyte proliferation and IL-2 secretion, in part, by binding to its receptor, SP-R210. However, the mechanisms underlying this effect are not well understood. Here, we studied the effect of antibodies against the SP-A-binding (neck) domain (alpha-SP-R210n) or nonbinding C-terminal domain (alpha-SP-R210ct) of SP-R210 on human peripheral blood T cell immune responses against Mycobacterium tuberculosis. We demonstrated that both antibodies bind to more than 90% of monocytes and 5-10% of CD3+ T cells in freshly isolated PBMC. Stimulation of PBMC from healthy tuberculin reactors [purified protein derivative-positive (PPD+)] with heat-killed M. tuberculosis induced increased antibody binding to CD3+ cells. Increased antibody binding suggested enhanced expression of SP-R210, and this was confirmed by Western blotting. The antibodies (alpha-SP-R210n) cross-linking the SP-R210 through the SP-A-binding domain markedly inhibited cell proliferation and IFN-gamma secretion by PBMC from PPD+ donors in response to heat-killed M. tuberculosis, whereas preimmune IgG and antibodies (alpha-SP-R210ct) cross-linking SP-R210 through the non-SP-A-binding, C-terminal domain had no effect. Anti-SP-R210n also decreased M. tuberculosis-induced production of TNF-alpha but increased production of IL-10. Inhibition of IFN-gamma production by alpha-SP-R210n was abrogated by the combination of neutralizing antibodies to IL-10 and TGF-beta1. Together, these findings support the hypothesis that SP-A, via SP-R210, suppresses cell-mediated immunity against M. tuberculosis via a mechanism that up-regulates secretion of IL-10 and TGF-beta1.

  10. Primordial germ cell-mediated chimera technology produces viable pure-line Houbara bustard offspring: potential for repopulating an endangered species.

    Directory of Open Access Journals (Sweden)

    Ulrich Wernery

    Full Text Available BACKGROUND: The Houbara bustard (Chlamydotis undulata is a wild seasonal breeding bird populating arid sandy semi-desert habitats in North Africa and the Middle East. Its population has declined drastically during the last two decades and it is classified as vulnerable. Captive breeding programmes have, hitherto, been unsuccessful in reviving population numbers and thus radical technological solutions are essential for the long term survival of this species. The purpose of this study was to investigate the use of primordial germ cell-mediated chimera technology to produce viable Houbara bustard offspring. METHODOLOGY/PRINCIPAL FINDINGS: Embryonic gonadal tissue was dissected from Houbara bustard embryos at eight days post-incubation. Subsequently, Houbara tissue containing gonadal primordial germ cells (gPGCs was injected into White Leghorn chicken (Gallus gallus domesticus embryos, producing 83/138 surviving male chimeric embryos, of which 35 chimeric roosters reached sexual maturity after 5 months. The incorporation and differentiation of Houbara gPGCs in chimeric chicken testis were assessed by PCR with Houbara-specific primers and 31.3% (5/16 gonads collected from the injected chicken embryos showed the presence of donor Houbara cells. A total of 302 semen samples from 34 chimeric roosters were analyzed and eight were confirmed as germline chimeras. Semen samples from these eight roosters were used to artificially inseminate three female Houbara bustards. Subsequently, 45 Houbara eggs were obtained and incubated, two of which were fertile. One egg hatched as a male live born Houbara; the other was female but died before hatching. Genotyping confirmed that the male chick was a pure-line Houbara derived from a chimeric rooster. CONCLUSION: This study demonstrates for the first time that Houbara gPGCs can migrate, differentiate and eventually give rise to functional sperm in the chimeric chicken testis. This approach may provide a promising

  11. The influence of ascorbic acid, TGF-beta1, and cell-mediated remodeling on the bulk mechanical properties of 3-D PEG-fibrinogen constructs. (United States)

    Kim, Peter D; Peyton, Shelly R; VanStrien, Amy J; Putnam, Andrew J


    Two-dimensional cell culture studies have shown that matrix rigidity can influence cell function, but little is known about how matrix physical properties, and their changes with time, influence cell function in 3-D. Biosynthetic hydrogels based on PEGylated fibrinogen permit the initial decoupling of matrix chemical and mechanical properties, and are thus potentially attractive for addressing this question. However, the mechanical stability of these gels due to passive hydrolysis and cell-mediated remodeling has not previously been addressed. Here, we show that the bulk mechanical properties of acellular PEG-fibrinogen hydrogels significantly decrease over time in PBS regardless of matrix cross-linking density in 7 days. To compensate, smooth muscle cells (SMCs) were encapsulated and stimulated to produce their own matrix using ascorbic acid or TGF-beta1. Ascorbic acid treatment improved the mechanical properties of the constructs after 14 days in less cross-linked matrices, but TGF-beta1 did not. The increase in matrix modulus of the constructs was not due to an increase in type I collagen deposition, which remained low and pericellular regardless of cross-link density or the soluble factor applied. Instead, ascorbic acid, but not TGF-beta1, preferentially enhanced the contractile SMC phenotype in the less cross-linked gels. Inhibition of contractility reduced cell spreading and the expression of contractile markers, and eliminated any beneficial increase in matrix modulus induced by cell-generated contraction of the gels. Together, these data show that PEG-fibrinogen hydrogels are susceptible to both hydrolysis and proteolysis, and suggest that some soluble factors may stimulate matrix remodeling by modulating SMC phenotype instead of inducing ECM synthesis in a 3-D matrix.

  12. 2-Hydroxy-3-methoxybenzoic acid attenuates mast cell-mediated allergic reaction in mice via modulation of the FcεRI signaling pathway. (United States)

    Kim, Yeon-Yong; Je, In-Gyu; Kim, Min Jong; Kang, Byeong-Cheol; Choi, Young-Ae; Baek, Moon-Chang; Lee, Byungheon; Choi, Jin Kyeong; Park, Hae Ran; Shin, Tae-Yong; Lee, Soyoung; Yoon, Seung-Bin; Lee, Sang-Rae; Khang, Dongwoo; Kim, Sang-Hyun


    Mast cells are important effector cells in immunoglobulin (Ig) E-mediated allergic reactions such as asthma, atopic dermatitis and rhinitis. Vanillic acid, a natural product, has shown anti-oxidant and anti-inflammatory activities. In the present study, we investigated the anti-allergic inflammatory effects of ortho-vanillic acid (2-hydroxy-3-methoxybenzoic acid, o-VA) that was a derivative of vanillic acid isolated from Amomum xanthioides. In mouse anaphylaxis models, oral administration of o-VA (2, 10, 50 mg/kg) dose-dependently attenuated ovalbumin-induced active systemic anaphylaxis and IgE-mediated cutaneous allergic reactions such as hypothermia, histamine release, IgE production and vasodilation; administration of o-VA also suppressed the mast cell degranulator compound 48/80-induced anaphylaxis. In cultured mast cell line RBL-2H3 and isolated rat peritoneal mast cells in vitro, pretreatment with o-VA (1-100 μmol/L) dose-dependently inhibited DNP-HSA-induced degranulation of mast cells by decreasing the intracellular free calcium level, and suppressed the expression of pro-inflammatory cytokines TNF-α and IL-4. Pretreatment of RBL-2H3 cells with o-VA suppressed DNP-HSA-induced phosphorylation of Lyn, Syk, Akt, and the nuclear translocation of nuclear factor-κB. In conclusion, o-VA suppresses the mast cell-mediated allergic inflammatory response by blocking the signaling pathways downstream of high affinity IgE receptor (FcεRI) on the surface of mast cells.

  13. Assay of mast cell mediators

    DEFF Research Database (Denmark)

    Rådinger, Madeleine; Jensen, Bettina M; Swindle, Emily


    Mediator release from activated mast cells is a major initiator of the symptomology associated with allergic disorders such as anaphylaxis and asthma. Thus, methods to monitor the generation and release of such mediators have widespread applicability in studies designed to understand the processes...... regulating mast cell activation and for the identification of therapeutic approaches to block mast cell-driven disease. In this chapter, we discuss approaches used for the determination of mast cell degranulation, lipid-derived inflammatory mediator production, and cytokine/chemokine gene expression as well...

  14. Data mining the NCI60 to predict generalized cytotoxicity. (United States)

    Lee, Adam C; Shedden, Kerby; Rosania, Gustavo R; Crippen, Gordon M


    Elimination of cytotoxic compounds in the early and later stages of drug discovery can help reduce the costs of research and development. Through the application of principal components analysis (PCA), we were able to data mine and prove that approximately 89% of the total log GI 50 variance is due to the nonspecific cytotoxic nature of substances. Furthermore, PCA led to the identification of groups of structurally unrelated substances showing very specific toxicity profiles, such as a set of 45 substances toxic only to the Leukemia_SR cancer cell line. In an effort to predict nonspecific cytotoxicity on the basis of the mean log GI 50, we created a decision tree using MACCS keys that can correctly classify over 83% of the substances as cytotoxic/noncytotoxic in silico, on the basis of the cutoff of mean log GI 50 = -5.0. Finally, we have established a linear model using least-squares in which nine of the 59 available NCI60 cancer cell lines can be used to predict the mean log GI 50. The model has R (2) = 0.99 and a root-mean-square deviation between the observed and calculated mean log GI 50 (RMSE) = 0.09. Our predictive models can be applied to flag generally cytotoxic molecules in virtual and real chemical libraries, thus saving time and effort.

  15. Cytotoxic activity and phytochemical analysis of Arum palaestinum Boiss

    Institute of Scientific and Technical Information of China (English)

    Mai Mohammed Farid; Sameh Reda Hussein; Lamiaa Fawzy Ibrahim; Mohammed Ali El Desouky; Amr Mohammed Elsayed; Ahmad Ali El Oqlah; Mahmoud Mohammed Saker


    Objective:To evaluate the in vitro cytotoxic activity of the fractionated extract as well as isolated compounds of Arum palaestinum Boiss. (A. palaestinum) (black calla lily), and to identify the volatile components which may be responsible for the potential antitumor activity. Methods: A. palaestinum was collected from its natural habitats and subjected to phytochemical analysis for separation of pure compounds. In vitro cytotoxic activity was investigated against four human carcinoma cell lines Hep2, HeLa, HepG2 and MCF7 for the fractionated extract and isolated compounds. While, the diethyl ether fraction was subjected to GC–MS analysis as it exhibited the most potent cytotoxic effect to evaluate the active constituents responsible for the cytotoxic activities. Results:Four flavonoid compounds were isolated (luteolin, chrysoeriol, isoorientin, isovitexin) from the diethyl ether and ethyl acetate. The extracts and the pure isolated compounds showed a significant high antiproliferative activity against all investigated cell lines. The GC–MS analysis revealed the separation and identification of 15 compounds representing 95.01%of the extract and belonging to different groups of chemical compounds. Conclusions:The present study is considered to be the first report on the cytotoxic activities carried out on different selected fractions and pure compounds of A. palaestinum to provide evidences for its strong antitumor activities. In addition, chrysoeriol and isovitexin compounds were isolated for the first time from the studied taxa.

  16. CRTAM determines the CD4+ cytotoxic T lymphocyte lineage. (United States)

    Takeuchi, Arata; Badr, Mohamed El Sherif Gadelhaq; Miyauchi, Kosuke; Ishihara, Chitose; Onishi, Reiko; Guo, Zijin; Sasaki, Yoshiteru; Ike, Hiroshi; Takumi, Akiko; Tsuji, Noriko M; Murakami, Yoshinori; Katakai, Tomoya; Kubo, Masato; Saito, Takashi


    Naive T cells differentiate into various effector T cells, including CD4(+) helper T cell subsets and CD8(+) cytotoxic T cells (CTL). Although cytotoxic CD4(+) T cells (CD4 +: CTL) also develop from naive T cells, the mechanism of development is elusive. We found that a small fraction of CD4(+) T cells that express class I-restricted T cell-associated molecule (CRTAM) upon activation possesses the characteristics of both CD4(+) and CD8(+) T cells. CRTAM(+) CD4(+) T cells secrete IFN-γ, express CTL-related genes, such as eomesodermin (Eomes), Granzyme B, and perforin, after cultivation, and exhibit cytotoxic function, suggesting that CRTAM(+) T cells are the precursor of CD4(+)CTL. Indeed, ectopic expression of CRTAM in T cells induced the production of IFN-γ, expression of CTL-related genes, and cytotoxic activity. The induction of CD4(+)CTL and IFN-γ production requires CRTAM-mediated intracellular signaling. CRTAM(+) T cells traffic to mucosal tissues and inflammatory sites and developed into CD4(+)CTL, which are involved in mediating protection against infection as well as inducing inflammatory response, depending on the circumstances, through IFN-γ secretion and cytotoxic activity. These results reveal that CRTAM is critical to instruct the differentiation of CD4(+)CTL through the induction of Eomes and CTL-related gene.

  17. Arsenic Induction of Metallothionein and Metallothionein Induction Against Arsenic Cytotoxicity. (United States)

    Rahman, Mohammad Tariqur; De Ley, Marc

    Human exposure to arsenic (As) can lead to oxidative stress that can become evident in organs such as the skin, liver, kidneys and lungs. Several intracellular antioxidant defense mechanisms including glutathione (GSH) and metallothionein (MT) have been shown to minimize As cytotoxicity. The current review summarizes the involvement of MT as an intracellular defense mechanism against As cytotoxicity, mostly in blood. Zinc (Zn) and selenium (Se) supplements are also proposed as a possible remediation of As cytotoxicity. In vivo and in vitro studies on As toxicity were reviewed to summarize cytotoxic mechanisms of As. Intracellular antioxidant defense mechanisms of MT are linked in relation to As cytotoxicity. Arsenic uses a different route, compared to major metal MT inducers such as Zn, to enter/exit blood cells. A number of in vivo and in vitro studies showed that upregulated MT biosynthesis in blood components are related to toxic levels of As. Despite the cysteine residues in MT that aid to bind As, MT is not the preferred binding protein for As. Nonetheless, intracellular oxidative stress due to As toxicity can be minimized, if not eliminated, by MT. Thus MT induction by essential metals such as Zn and Se supplementation could be beneficial to fight against As toxicity.

  18. Carbon nanotubes enhance cytotoxicity mediated by human lymphocytes in vitro.

    Directory of Open Access Journals (Sweden)

    Zhao Sun

    Full Text Available With the expansion of the potential applications of carbon nanotubes (CNT in biomedical fields, the toxicity and biocompatibility of CNT have become issues of growing concern. Since the immune system often mediates tissue damage during pathogenesis, it is important to explore whether CNT can trigger cytotoxicity through affecting the immune functions. In the current study, we evaluated the influence of CNT on the cytotoxicity mediated by human lymphocytes in vitro. The results showed that while CNT at low concentrations (0.001 to 0.1 µg/ml did not cause obvious cell death or apoptosis directly, it enhanced lymphocyte-mediated cytotoxicity against multiple human cell lines. In addition, CNT increased the secretion of IFN-γ and TNF-α by the lymphocytes. CNT also upregulated the NF-κB expression in lymphocytes, and the blockage of the NF-κB pathway reduced the lymphocyte-mediated cytotoxicity triggered by CNT. These results suggest that CNT at lower concentrations may prospectively initiate an indirect cytotoxicity through affecting the function of lymphocytes.

  19. The Use of Isolated Human Lymphocytes in Mycotoxin Cytotoxicity Testing

    Directory of Open Access Journals (Sweden)

    Mike F. Dutton


    Full Text Available The cytotoxicity of selected mycotoxins against isolated human lymphocytes was investigated, as a means of detecting mycotoxins in extracts derived from cereal samples. The methodology was based on the ability of viable cells to reduce methyl tetrazolium bromide to a purple formazan dye that could be quantitated by spectrophometric means and hence give a measure of the cytotoxicity of added substances. The results showed that there was good correlation with the occurrence of identified mycotoxins with only a minimum of false positives. For example, of the 13 samples of barley or barley derivatives that were positive for the mycotoxins, fumonisin B1 (FB1 deoxynivalenol (DON and ochratoxin A (OTA, all gave positive cytotoxicity responses. Two samples negative for mycotoxins gave no cytotoxicity responses. There was little variation between the results for lymphocytes drawn from the same healthy volunteer on three different occasions. Furthermore, for two of the mycotoxins tested (FB1 and DON it was possible to correlate general levels of mycotoxins present to the cytotoxic response of the lymphocytes but not for OTA, where it was concluded that interfering substances prevented direct correlation. It was concluded that this method was suited for general application as it could handle relatively high number of samples in a short period of time.

  20. Evaluation of Northern Iran Mentha pulegium L. cytotoxicity

    Directory of Open Access Journals (Sweden)

    Farshad H. Shirazi


    Full Text Available In vitro tests could be a valuable tool for the evaluation of medicinal plants’ cytotoxicity. One of the most frequently used Iranian traditional plants is Mentha Pulegium from Labiatae family. In the present study, essential oil and the methanolic extract of Mentha pulegium, were analyzed for cytotoxicity on human ovary adenocarcinoma SK-OV-3, human malignant cervix carcinoma Hela, and human lung carcinoma A549 cell lines. Two different assays of clonogenic and neutral red (NR were used for evaluation of cytotoxicity. Although the methanolic extract of Mentha pulegium did not show any cytotoxic effects, the essential oil of this plant proved to be a potent cytotoxic agent on the above three cell lines. According to the clonogenic assay, LD50s of the essential oil on SK-OV-3, Hela and A549 cell lines are 14.10, 59.10 and 18.76 µg/ml, respectively. Our findings suggest that Mentha pulegium essential oil might be considered as a potentially toxic agent on human cancer cell lines, and a possible candidate for human cancer chemotherapy. However, further biological tests on the efficacy and side effects of this plant are necessary before its use in human

  1. Cytotoxic activity and phytochemical analysis of Arum palaestinum Boiss.

    Institute of Scientific and Technical Information of China (English)

    Mai; Mohammed; Farid; Sameh; Reda; Hussein; Lamiaa; Fawzy; Ibrahim; Mohammed; Ali; El; Desouky; Amr; Mohammed; Elsayed; Ahmad; Ali; El; Oqlah; Mahmoud; Mohammed; Saker


    Objective: To evaluate the in vitro cytotoxic activity of the fractionated extract as well as isolated compounds of Arum palaestinum Boiss.(A. palaestinum)(black calla lily), and to identify the volatile components which may be responsible for the potential antitumor activity.Methods: A. palaestinum was collected from its natural habitats and subjected to phytochemical analysis for separation of pure compounds. In vitro cytotoxic activity was investigated against four human carcinoma cell lines Hep2, He La, Hep G2 and MCF7 for the fractionated extract and isolated compounds. While, the diethyl ether fraction was subjected to GC–MS analysis as it exhibited the most potent cytotoxic effect to evaluate the active constituents responsible for the cytotoxic activities.Results: Four flavonoid compounds were isolated(luteolin, chrysoeriol, isoorientin,isovitexin) from the diethyl ether and ethyl acetate. The extracts and the pure isolated compounds showed a significant high antiproliferative activity against all investigated cell lines. The GC–MS analysis revealed the separation and identification of 15 compounds representing 95.01% of the extract and belonging to different groups of chemical compounds.Conclusions: The present study is considered to be the first report on the cytotoxic activities carried out on different selected fractions and pure compounds of A. palaestinum to provide evidences for its strong antitumor activities. In addition,chrysoeriol and isovitexin compounds were isolated for the first time from the studied taxa.

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

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


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

  3. Profiling flavonoid cytotoxicity in human breast cancer cell lines: determination of structure-function relationships. (United States)

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


    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.

  4. Cytotoxic Constituents from the Stems of Clausena lansium (Lour. Skeels

    Directory of Open Access Journals (Sweden)

    Shu Shan Du


    Full Text Available Six compounds were isolated from the stems of Clausena lansium (Lour. Skeels by repeated sillica gel column chromatography. Their chemical structures were elucidated on the basic of physicochemical and spectroscopic data. Among them, 8-geranyloxypsolaren (3 and 2-methoxy-1-(3-methyl-buten-1-yl-9H-carbazole-3-carbaldehyde (6 were isolated for the first time from this plant. These compounds were screened for cytotoxicity in human cervical cancer (Hela, leukemia (K562, lung cancer (A549, non-small lung carcinoma (H1299 and liver cancer (SMMC-7721. Within the series of cytotoxic tests, compounds 4–6 displayed potent cytotoxic activity against H1299 and SMMC-7721, with the IC50 values of 6.19 to 26.84 μg/mL.

  5. Evaluation of in vitro cytotoxic effect of Trichosanthes dioica root

    Directory of Open Access Journals (Sweden)

    Sanjib Bhattacharya


    Full Text Available Background: Trichosanthes dioica Roxb. (Cucurbitaceae, called pointed gourd in English is a dioecious climber grown in India and used traditionally for various medicinal purposes. Methods: Present study was aimed to evaluate in vitro cytotoxic effect of dichloromethane (DCTD, methanol (METD, and aqueous (AQTD extracts of T. dioica root using Allium cepa root meristems by keeping them in different concentrations of each test extract under specific experimental conditions followed by determination of root growth inhibition (root length and number and mitotic index. Results: All the extracts significantly demonstrated concentration-dependent inhibition of root length and number and reduction in mitotic index, indicating antimitotic activity demonstrating cytotoxicity and genotoxicity. DCTD was found to be the most potent (EC 50 : 2.8 mg/ml, followed by METD and AQTD. Conclusion: The present study therefore, establishes promising in vitro cytotoxic and genotoxic property of T. dioica root against the test system.

  6. Cytotoxic components of Pereskia bleo (Kunth) DC. (Cactaceae) leaves. (United States)

    Malek, Sri Nurestri Abdul; Shin, Sim Kae; Wahab, Norhanom Abdul; Yaacob, Hashim


    Dihydroactinidiolide (1) and a mixture of sterols [campesterol (2), stigmasterol (3) and beta-sitosterol (4)], together with the previously isolated individual compounds beta-sitosterol (4), 2,4-di-tert-butylphenol (5), alpha-tocopherol (6), phytol (7) were isolated from the active ethyl acetate fraction of Pereskia bleo (Kunth) DC. (Cactaceae) leaves. Cytotoxic activities of the above mentioned compounds against five human carcinoma cell lines, namely the human nasopharyngeal epidermoid carcinoma cell line (KB), human cervical carcinoma cell line (CasKi), human colon carcinoma cell line (HCT 116), human hormone-dependent breast carcinoma cell line (MCF7) and human lung carcinoma cell line (A549); and non-cancer human fibroblast cell line (MRC-5) were investigated. Compound 5 possessed very remarkable cytotoxic activity against KB cells, with an IC(50 )value of 0.81microg/mL. This is the first report on the cytotoxic activities of the compounds isolated from Pereskia bleo.

  7. Synthesis and cytotoxic activity of novel curcumin analogues

    Institute of Scientific and Technical Information of China (English)

    Qin Zhang; Yao Fu; Hao Wei Wang; Tao Gong; Yong Qin; Zhi Rong Zhang


    Five novel curcumin analogues bearing different substituents at 4-position of phenyl group were synthesized. Their structures were confirmed by NMR and HRMS spectrum. Their cytotoxic activities against six tumor cell lines were tested by the standard MTT assay in vitro. The results indicated that four analogues (1A-1C, 1E) with solubilizing moieties showed selective potent cytotoxicity against HepG2, HeLa and CT26 cell lines, and analogue 1A and 1C exhibited more potent cytotoxicity than curcumin against CT26 cell line. It was suggested that introduction of appropriate substituents to 4-position of phenyl group might be a potential option for structural modification of curcumin.

  8. Antimicrobial and cytotoxic effects of Mexican medicinal plants. (United States)

    Jacobo-Salcedo, Maria del Rosario; Alonso-Castro, Angel Josabad; Salazar-Olivo, Luis A; Carranza-Alvarez, Candy; González-Espíndola, Luis Angel; Domínguez, Fabiola; Maciel-Torres, Sandra Patricia; García-Lujan, Concepción; González-Martínez, Marisela del Rocio; Gómez-Sánchez, Maricela; Estrada-Castillón, Eduardo; Zapata-Bustos, Rocio; Medellin-Milán, Pedro; García-Carrancá, Alejandro


    The antimicrobial effects of the Mexican medicinal plants Guazuma ulmifolia, Justicia spicigera, Opuntia joconostle, O. leucotricha, Parkinsonia aculeata, Phoradendron longifolium, P. serotinum, Psittacanthus calyculatus, Tecoma stans and Teucrium cubense were tested against several human multi-drug resistant pathogens, including three Gram (+) and five Gram (-) bacterial species and three fungal species using the disk-diffusion assay. The cytotoxicity of plant extracts on human cancer cell lines and human normal non-cancerous cells was also evaluated using the MTT assay. Phoradendron longifolium, Teucrium cubense, Opuntia joconostle, Tecoma stans and Guazuma ulmifolia showed potent antimicrobial effects against at least one multidrug-resistant microorganism (inhibition zone > 15 mm). Only Justicia spicigera and Phoradendron serotinum extracts exerted active cytotoxic effects on human breast cancer cells (IC50 plant species may be important sources of antimicrobial and cytotoxic agents.

  9. Antioxidant Activities and Cytotoxicity of Zingiber zerumbet (L. Smith Rhizome

    Directory of Open Access Journals (Sweden)

    Anish Nag


    Full Text Available Zingiber zerumbet (L. Smith, popularly known as shampoo ginger, is one of the most commonly used ingredients in Indo-Malaysian traditional medicines. The antioxidant and cytotoxic activities of the ethanolic extract of Z. zerumbet rhizome (ZZ was investigated. 2,2-di(4-tert-octylphenyl-1- picryl-hydrazyl (DPPH and hydroxyl radical scavenging assays showed significant radical scavenging activities of ZZ. The extract was rich in polyphenol and flavonoids. Cytotoxicity was assessed in vitro by trypan blue exclusion test. Human peripheral blood lymphocyte cells were incubated in different concentrations of ZZ (0, 15, 30, 60, 120, 300 and 600 µg/ml for 3 h at 37 °C. The rhizome extract was found to be cytotoxic at concentrations higher for human consumption. In addition, HPLC analysis revealed ZZ as a rich source of kaempferol. Based on the results of the present investigation the rhizome may be used safely as a therapeutic antioxidant.

  10. Phytochemistry, cytotoxicity and antiviral activity of Eleusine indica (sambau) (United States)

    Iberahim, Rashidah; Yaacob, Wan Ahmad; Ibrahim, Nazlina


    Goose grass also known as Eleusine indica (EI) is a local medicinal plant that displays antioxidant, antimicrobial and anticancer activities. The present study is to determine the phytochemical constituents, cytotoxicity and antiviral activities for both crude extract and fraction obtained from the plant. The crude extract contained more secondary metabolites compared to the hexane fraction as gauged using standard phytochemical tests. Cytotoxicity screening against Vero cells using MTT assay showed that the CC50 values for crude extract and hexane fraction were 2.07 and 5.62 mg/ml respectively. The antiviral activity towards Herpes Simplex Virus type 1 (HSV-1) was determined using plaque reduction assay. The selective indices (SI = CC50 / EC50) for both methanol extract and hexane fraction were 12.2 and 6.2 respectively. These results demonstrate that the extract prepared from E. indica possesses phytochemical compound that was non cytotoxic to the cell with potential antiviral activity.

  11. Internalization of Ineffective Platinum Complex in Nanocapsules Renders It Cytotoxic. (United States)

    Vrana, Oldrich; Novohradsky, Vojtech; Medrikova, Zdenka; Burdikova, Jana; Stuchlikova, Olga; Kasparkova, Jana; Brabec, Viktor


    Anticancer therapy by platinum complexes, based on nanocarrier-based delivery, may offer a new approach to improve the efficacy and tolerability of the platinum family of anticancer drugs. The original rules for the design of new anticancer platinum drugs were affected by the fact that, although cisplatin (cis-[PtCl2 (NH3)2) was an anticancer drug, its isomer transplatin was not cytotoxic. For the first time, it is demonstrated that simple encapsulation of an inactive platinum compound in phospholipid bilayers transforms it into an efficient cytotoxic agent. Notably, the encapsulation of transplatin makes it possible to overcome the resistance mechanisms operating in cancer cells treated with cisplatin and prevents inactivation of transplatin in the extracellular environment. It is also shown that transplatin delivered to the cells in nanocapsules, in contrast to free (nonencapsulated) complex, forms cytotoxic cross-links on DNA.

  12. Cytotoxic immunotherapy strategies for cancer: mechanisms and clinical development. (United States)

    Aguilar, Laura K; Guzik, Brian W; Aguilar-Cordova, Estuardo


    Traditional therapies for cancer include surgery, chemotherapy, and radiation. Chemotherapy has widespread systemic cytotoxic effects against tumor cells but also affects normal cells. Radiation has more targeted local cytotoxicity but is limited to killing cells in the radiation field. Immunotherapy has the potential for systemic, specific killing of tumor cells. However, if the immune response is specific to a single antigen, tumor evasion can occur by down-regulation of that antigen. An immunotherapy approach that induces polyvalent immunity to autologous tumor antigens can provide a personalized vaccine with less potential for immunologic escape. A cytotoxic immunotherapy strategy creates such a tumor vaccine in situ. Immunogenic tumor cell death provides tumor antigen targets for the adaptive immune response and stimulates innate immunity. Attraction and activation of antigen presenting cells such as dendritic cells is important to process and present tumor antigens to T cells. These include cytotoxic T cells that kill tumor cells and T cells which positively and negatively regulate immunity. Tipping the balance in favor of anti-tumor immunity is an important aspect of an effective strategy. Clinically, immunotherapies may be most effective when combined with standard therapies in a complimentary way. An example is gene-mediated cytotoxic immunotherapy (GMCI) which uses an adenoviral vector, AdV-tk, to deliver a cytotoxic and immunostimulatory gene to tumor cells in vivo in combination with standard therapies creating an immunostimulatory milieu. This approach, studied extensively in animal models and early stage clinical trials, is now entering a definitive Phase 3 trial for prostate cancer.

  13. Cytotoxicity of selenium nanoparticles in rat dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    Ramos JF


    Full Text Available Joseph F Ramos,1 Thomas J Webster21School of Engineering, Center of Biomedical Engineering, 2School of Engineering and Department of Orthopedics, Brown University, Providence, RI, USABackground: Ventilator-associated pneumonia is a deadly nosocomial infection caused by contaminated endotracheal tubes. It has been shown that polyvinyl chloride (PVC, the endotracheal tube substrate coated with elemental selenium nanoparticles reduces bacterial adherence and proliferation on PVC by over 99%. However, it is not known if selenium nanoparticles elicit a cytotoxic effect in vitro. The purpose of this study was to investigate the cytotoxic effects of PVC coated with selenium nanoparticles on fibroblasts, which are mammalian cells central to endotracheal tube intubation.Methods: Different concentrations of selenium nanoparticles were precipitated onto the PVC surface by reduction of selenium salts using glutathione. Characterization of PVC coated with selenium nanoparticles was done by scanning electron microscopy, energy dispersive x-ray, and contact angle measurements. For the cytotoxicity experiments, fibroblasts were seeded at a density of 5000 cm2 onto PVC coated with three different concentrations of selenium nanoparticles (high, medium, low and incubated for 4 hours (adhesion as well as for 24 hours and 72 hours (proliferation. The half-maximal inhibitory concentration (IC50 value was determined after 72 hours using an ultrahigh concentration. MTT assays were used to assess cell viability at the indicated time points.Results: The three concentrations of selenium nanoparticles did not elicit a cytotoxic effect after 72 hours (P < 0.01, n = 3. It was found that the IC50 value was at the ultrahigh concentration of selenium nanoparticles. The nanoparticulate elemental selenium concentration previously shown to decrease the function of bacteria was shown not to cause a cytotoxic effect on fibroblasts in vitro.Conclusion: These findings demonstrate

  14. Synthesis and cytotoxic activity of some derivatives of alkyl piperidine. (United States)

    Jahan, Sarwat; Akhtar, Shamim; Saify, Zafar Saied; Mushtaq, Nousheen; Sial, Ali Akbar; Kamil, Arfa; Arif, Muhammed


    Synthesis of novel phenacyl derivatives of alkyl piperidine as cytotoxic agents via simple and single step reaction procedure is going to be reported here. Twelve new compounds were successfully synthesized in moderate yield and in solid form. Their synthesis was confirmed by TLC, melting point, CHN analysis and through different spectral studies such as UV, IR, Mass and proton NMR. The advantages of this synthetic route are simple operation, mild reaction conditions and good yields. These newly synthesized derivatives were extensively explored for their cytotoxicity by brine shrimp lethality assay.

  15. Sesquiterpene lactones from Ferula oopoda and their cytotoxic properties. (United States)

    Kasaian, Jamal; Iranshahy, Milad; Masullo, Milena; Piacente, Sonia; Ebrahimi, Fatemeh; Iranshahi, Mehrdad


    Two new sesquiterpene lactones, namely feruhodin A and feruhodin B, together with six known compounds, daucoeudesmanolactone, dehydrooopodin, oopodin, badkhysin, 7-demethylplastochromenol, and scoparone, were isolated from the roots of Ferula oopoda. The structures of these compounds were elucidated by 1D and 2D NMR techniques as well as high-resolution mass spectrometry. Cytotoxic effects of these compounds were evaluated against two human cancer cell lines including MCF7and K562 using the Alamar blue assay. The results showed that dehydrooopodin possessed significant cytotoxic effects with IC₅₀ values of 15 and 5 μM against MCF7 and K562, respectively.

  16. Chemical constituents of Lecythis pisonis and cytotoxic activity

    Directory of Open Access Journals (Sweden)

    Jocélia P. C. Oliveira


    Full Text Available The phytochemical investigation of the ethanol extract from leaves of Lecythis pisonis Cambess., Lecythidaceae, resulted in the isolation of seven triterpenes: α- and β-amyrin, uvaol and erythrodiol, ursolic and oleanolic acids and 3β-friedelinol, as well as a mixture of sitosterol and stigmasterol steroids and a diterpene (E-phytol. The structures of these compounds were identified by¹H and 13C NMR spectral analysis and compared with literature data. The mixture of triterpenes ursolic and oleanolic acids isolated from the active ethereal fraction showed moderate cytotoxic activity. This paper describes for the first time the phytochemical and cytotoxic study of Lecythis pisonis' leaves.

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

    DEFF Research Database (Denmark)

    Tenzer, Stefan; Wee, Edmund; Burgevin, Anne


    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...

  18. Synthesis and in vitro cytotoxicity of haloderivatives of noscapine. (United States)

    Verma, Akhilesh Kumar; Bansal, Sandhya; Singh, Jaspal; Tiwari, Rakesh Kumar; Kasi Sankar, V; Tandon, Vibha; Chandra, Ramesh


    Three haloderivatives of noscapine 2-4 were synthesized chemoselectively and their in vitro cytotoxicity was assessed by MTT assay on U-87 human glioblastoma cell lines. At 50 microM concentration after 72 h, 9-chloronoscapine 2, 9-bromonoscapine 3 (EM011), and 9-iodonoscapine 4 killed 87.8%, 51.2%, and 56.8% cells, respectively, however noscapine kills only 40% of the cells; revealing 9-chloronoscapine as a potential cytotoxic agent than noscapine and 9-bromonoscapine (EM011). At low concentration (1 microM) 9-bromonoscapine (46.7%) and 9-chloronoscapine (45.7%) did not show any significant difference.

  19. New Cytotoxic 24-Homoscalarane Sesterterpenoids from the Sponge Ircinia felix. (United States)

    Lai, Ya-Yuan; Chen, Li-Chai; Wu, Chug-Fung; Lu, Mei-Chin; Wen, Zhi-Hong; Wu, Tung-Ying; Fang, Lee-Shing; Wang, Li-Hsueh; Wu, Yang-Chang; Sung, Ping-Jyun


    Two new 24-homoscalarane sesterterpenoids, felixins F (1) and G (2), were isolated from the sponge Ircinia felix. The structures of new homoscalaranes 1 and 2 were elucidated by extensive spectroscopic methods, particularly with one-dimensional (1D) and two-dimensional (2D) NMR, and, by comparison, the spectral data with those of known analogues. The cytotoxicity of 1 and 2 against the proliferation of a limited panel of tumor cell lines was evaluated and 1 was found to show cytotoxicity toward the leukemia K562, MOLT-4, and SUP-T1 cells (IC50 ≤ 5.0 μM).

  20. New Cytotoxic 24-Homoscalarane Sesterterpenoids from the Sponge Ircinia felix

    Directory of Open Access Journals (Sweden)

    Ya-Yuan Lai


    Full Text Available Two new 24-homoscalarane sesterterpenoids, felixins F (1 and G (2, were isolated from the sponge Ircinia felix. The structures of new homoscalaranes 1 and 2 were elucidated by extensive spectroscopic methods, particularly with one-dimensional (1D and two-dimensional (2D NMR, and, by comparison, the spectral data with those of known analogues. The cytotoxicity of 1 and 2 against the proliferation of a limited panel of tumor cell lines was evaluated and 1 was found to show cytotoxicity toward the leukemia K562, MOLT-4, and SUP-T1 cells (IC50 ≤ 5.0 μM.

  1. 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


    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.

  2. Lactate dehydrogenase assay for assessment of polycation cytotoxicity

    DEFF Research Database (Denmark)

    Parhamifar, Ladan; Andersen, Helene; Moghimi, Seyed Moien


    Cellular toxicity and/or cell death entail complex mechanisms that require detailed evaluation for proper characterization. A detailed mechanistic assessment of cytotoxicity is essential for design and construction of more effective polycations for nucleic acid delivery. A single toxicity assay...... cannot stand alone in determining the type and extent of damage or cell death mechanism. In this chapter we describe a lactate dehydrogenase (LDH) assay for high-throughput screening that can be used as a starting point for further detailed cytotoxicity determination. LDH release is considered an early...

  3. Gynecomastia Following Cytotoxic Therapy in a Patient with Testicular Cancer

    Directory of Open Access Journals (Sweden)

    Barış Akıncı


    Full Text Available Gynecomastia is the development of abnormal breast tissue in men. Relatively increased estrogen action on tissue level is believed to play a main role in the pathogenesis of the entity. Here, we describe a patient with painless gynecomastia presenting after cytotoxic chemotherapy for testicular cancer. Further investigations showed no evidence of disease progression, recurrence, or metastasis. We suggest that the clinicians should be aware that gynecomastia may follow cytotoxic chemotherapy for testicular cancer and does not reflect the return of malignancy. Turk Jem 2008; 12: 86-7

  4. Levamisole Enhances Cell-Mediated Immune Responses and Reduces Shedding of H9N2 Avian Influenza Virus in Japanese Quails (Coturnix coturnix japonica

    Directory of Open Access Journals (Sweden)

    Tahoora Shomali


    Full Text Available Problem statement: Regarding the role of Japanese quails (Coturnix coturnix japonica in reassortment and spreading of avian influenza (AI viruses and inadequate protection of vaccination in this species, the present study aimed to evaluate the effect of levamisole as an immunomodulatory agent on cell-mediated immunity (CMI, antibody responses and shedding of H9N2 AI virus in experimentally infected quails. Approach: On day 20 of age, 100 quails randomly allocated into 4 equal groups. Birds in groups 2, 3 and 4 were inoculated with virus where group 1 kept as control. Groups 3 and 4 orally received 15 mg kg-1 levamisole for three consecutive days just before virus inoculation which was repeated 10 days post inoculation (PI only in group 4. Antibody titers and CMI of all birds were assayed by HI and delayed type hypersensitivity (DTH test respectively and virus detection in fecal and tracheal samples performed by RT-PCR method. Data analyzed by one-way ANOVA and Tukey’s test. Results: Levamisole in both regimens had no appreciable effect on antibody titers (p>0.05 while repeated regimen resulted in higher CMI response than group 2 at 48 and 72 h post DTH test (p = 0.011 and p = 0.031 respectively. Total fecal samples positive for virus from birds in group 3 and 4 were 34.4 and 40% lower than group 2 respectively. For trachea, the positive samples were 33.3% (group 3 and 46.7% (group 4 lower than group 2. Moreover; fecal and tracheal samples from levamisole treated birds (especially from group 4 became void of virus earlier than group 2. Conclusion/Recommendations: Levamisole administration in a repeated regimen enhances CMI response against H9N2 AI virus and reduces virus shedding in quails. This may pave the road for further investigations on potential positive effects of this agent on prevention and management of H9N2 AI infections in quail industry.

  5. Strategy for eliciting antigen-specific CD8+ T cell-mediated immune response against a cryptic CTL epitope of merkel cell polyomavirus large T antigen

    Directory of Open Access Journals (Sweden)

    Gomez Bianca P


    Full Text Available Abstract Background Merkel cell carcinoma (MCC is a relatively new addition to the expanding category of oncovirus-induced cancers. Although still comparably rare, the number of cases has risen dramatically in recent years. Further complicating this trend is that MCC is an extremely aggressive neoplasm with poor patient prognosis and limited treatment options for advanced disease. The causative agent of MCC has been identified as the merkel cell polyomavirus (MCPyV. The MCPyV-encoded large T (LT antigen is an oncoprotein that is theorized to be essential for virus-mediated tumorigenesis and is therefore, an excellent MCC antigen for the generation of antitumor immune responses. As a foreign antigen, the LT oncoprotein avoids the obstacle of immune tolerance, which normally impedes the development of antitumor immunity. Ergo, it is an excellent target for anti-MCC immunotherapy. Since tumor-specific CD8+ T cells lead to better prognosis for MCC and numerous other cancers, we have generated a DNA vaccine that is capable of eliciting LT-specific CD8+ T cells. The DNA vaccine (pcDNA3-CRT/LT encodes the LT antigen linked to a damage-associated molecular pattern, calreticulin (CRT, as it has been demonstrated that the linkage of CRT to antigens promotes the induction of antigen-specific CD8+ T cells. Results The present study shows that DNA vaccine-induced generation of LT-specific CD8+ T cells is augmented by linking CRT to the LT antigen. This is relevant since the therapeutic effects of the pcDNA3-CRT/LT DNA vaccine is mediated by LT-specific CD8+ T cells. Mice vaccinated with the DNA vaccine produced demonstrably more LT-specific CD8+ T cells. The DNA vaccine was also able to confer LT-specific CD8+ T cell-mediated protective and therapeutic effects to prolong the survival of mice with LT-expressing tumors. In the interest of determining the LT epitope which most MCC-specific CD8+ T cells recognize, we identified the amino acid sequence of the

  6. Expression of human IAP-like protein in skeletal muscle: a possible explanation for the rare incidence of muscle fiber apoptosis in T-cell mediated inflammatory myopathies. (United States)

    Li, M; Dalakas, M C


    In Polymyositis (PM) and sporadic Inclusion Body Myositis (s-IBM), the CD8(+) cytotoxic T cells invade the muscle membrane and release perforin and granzyme B to induce cell death. Although granzyme B is a direct activator of executioner caspases, there is no convincing evidence of apoptosis in the muscle fibers of these patients. To search for an explanation, we examined the muscle expression of the human IAP-Like Protein (hILP), an evolutionarily conserved cell death suppressor, that exerts major anti-apoptotic effects by inhibiting the executioner caspases. Muscle biopsy specimens from patients with inflammatory myopathies and controls were studied with: (a) immunocytochemistry using antibodies against hILP and caspase-3 in single and double-labeled confocal laser microscopy; (b) immunoblotting of muscle extracts immunoreacted with anti-hILP antibodies; and (c) subcellular fractionation of muscle lysates immunoreacted with antibodies against hILP. We found that hILP is expressed on the sarcolemmal region and co-localizes with dystrophin. Caspase-3 is undetectable. Subcellular fractionation of the muscle specimens confirmed that hILP is a membrane-associated protein. By immunoblotting, the 57 kD hILP was abundantly expressed in the normal as well as the diseased muscles. We conclude that in s-IBM and PM the expression of hILP, a major cell death suppressor, on the muscle membrane may prevent the induction of apoptosis by the autoinvasive cytotoxic T cells on the cell surface, by inhibiting the caspase activation.

  7. CD107a Expression and IFN-γ Production as Markers for Evaluation of Cytotoxic CD3+ CD8+ T Cell Response to CMV Antigen in Women with Recurrent Spontaneous Abortion

    Directory of Open Access Journals (Sweden)

    Batoul Tarokhian


    Full Text Available Background: Some evidence has shown a relationship between primary human cytomegalovirus (CMV infection and pregnancy loss. The impact of CMV infection reactivation during pregnancy on adverse pregnancy outcomes is not completely understood. It is proposed that altered immune response, and therefore, recurrence or reactivation of latent CMV infection may relate to recurrent spontaneous abortion (RSA; however, few data are available in this regard. To find out about any cell mediated defect and reactivation of latent CMV infection in women with RPL, cellular immunity to the virus has been evaluated by specific cytotoxic T lymphocyte (CTL response to CMV. Materials and Methods: In a case control study, CTL CD107a expression and intercellular IFN-γ production in response to CMV pp65 antigen and staphylococcus enterotoxin B (SEB in women with RSA were assessed by flow cytometric analysis. Forty-four cases with history of recurrent pregnancy and forty-four controls with history of successful pregnancies were included. The FACSCaliber flow cytometer were used for analysis. Results: No significant difference was observed between CD107a expression and IFN-γ production in response to CMV PP65 antigen in RPL patients and control group. However, the cytotoxic response to SEB antigen in patients with RPL was significantly lower than control group (p=0.042. Conclusion: The results of this study show that impaired CD107a expression and IFN-γ production as CTL response to CMV does not appear to be a major contributing and immune incompetence factor in patients with RPL, but cytotoxic T cell response defect to other antigens requires to be assessed further in these patients.

  8. Efficiency of immunotoxin cytotoxicity is modulated by the intracellular itinerary.

    Directory of Open Access Journals (Sweden)

    Lori L Tortorella

    Full Text Available Pseudomonas exotoxin-based immunotoxins, including LMB-2 (antiTac(F(v-PE38, are proposed to traffic to the trans-Golgi network (TGN and move by a retrograde pathway to the endoplasmic reticulum, where they undergo translocation to the cytoplasm, a step that is essential for cytotoxicity. The retrograde transport pathways used by LMB-2 are not completely understood, so it is unclear if transit through specific organelles is critical for maximal cytotoxic activity. In this study, we used Chinese hamster ovary (CHO cell lines that express chimeric constructs of CD25, the Tac antigen, attached to the cytoplasmic domain of the TGN-targeted transmembrane proteins, TGN38 and furin. These chimeras are both targeted to the TGN, but the itineraries they follow are quite different. LMB-2 was incubated with the two cell lines, and the efficiency of cell killing was determined using cell viability and cytotoxicity assays. LMB-2 that is targeted through the endocytic recycling compartment to the TGN via Tac-TGN38 kills the cells more efficiently than immunotoxins delivered through the late endosomes by Tac-furin. Although the processing to the 37 kDa active fragment was more efficient in Tac-furin cells than in Tac-TGN38 cells, this was not associated with enhanced cytotoxicity - presumably because the toxin was also degraded more rapidly in these cells. These data indicate that trafficking through specific organelles is an important factor modulating toxicity by LMB-2.

  9. Cytotoxicity evaluation of methacrylate- and silorane-based composite resins

    Directory of Open Access Journals (Sweden)

    Gulsah Goktolga Akin


    Full Text Available

    Objectives: The objective of this study was to investigate and compare the cytotoxic effects of four composite resin materials with different content.

    Material and Methods: Two traditional methacrylate-based (Clearfil AP-X, RefleXions, as well as a self-adhering methacrylate-based (Vertise Flow and a silorane-based (Filtek Silorane composite resin were tested in the experiment. Ten cylindrical specimens were made of each material, using a mould (2mm. thick and 8 mm. in diameter. An agar diffusion method was employed, and cytotoxicity rankings were determined using lysis index scores. For statistical analysis, Kruskal-Wallis and Mann-Whitney U-tests were used.

    Results: Amongst the composite resins, the silorane-based composite was found to be less cytotoxic than the methacrylate-based composite resins, which all had the same cytotoxicity ranking.

    Conclusions: The silorane-based composite resin was considered more biocompatible than the methacrylate-based composite resins.

  10. Cytotoxic activity and phytochemical analysis of Arum palaestinum Boiss.

    Directory of Open Access Journals (Sweden)

    Mai Mohammed Farid


    Conclusions: The present study is considered to be the first report on the cytotoxic activities carried out on different selected fractions and pure compounds of A. palaestinum to provide evidences for its strong antitumor activities. In addition, chrysoeriol and isovitexin compounds were isolated for the first time from the studied taxa.

  11. Three New Cytotoxic ent-Kaurane Diterpenes from Isodon excisoides. (United States)

    Dai, Li-Ping; Li, Chun; Yang, Han-Ze; Lu, Yan-Qing; Yu, Hong-Yan; Gao, Hui-Min; Wang, Zhi-Min


    Three types of ent-kaurane diterpenoids were isolated from the aerial parts of Isodon excisoides, including three new diterpenoids, 1α,7α,14β-trihydroxy-20-acetoxy-ent-kaur-15-one (1); 1α,7α,14β,18-tetrahydroxy-20-acetoxy-ent-kaur-15-one (2); and 1α-acetoxy-14β-hydroxy-7α,20-epoxy-ent-kaur-16-en-15-one (3); together with six known diterpenes henryin (4); kamebanin (5); reniformin C (6); kamebacetal A (7); kamebacetal B (8); and oridonin (9). The structures of the isolated compounds were elucidated by means of nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry in conjunction with published data for their analogs, as well as their fragmentation patterns. Compounds 5 and 9 were isolated from Isodon excisoides for the first time. To explore the structure-activity relationships of the isolated compounds, they were tested for their cytotoxic effects against five human cancer cell lines: HCT-116, HepG2, A2780, NCI-H1650, and BGC-823. Most of the isolated compounds showed certain cytotoxic activity against the five cancer cell lines with IC50 values ranging from 1.09-8.53 µM. Among the tested compounds, compound 4 exhibited the strongest cytotoxic activity in the tested cell lines, with IC50 values ranging from 1.31-2.07 µM. Compounds 1, 6, and 7 exhibited selective cytotoxic activity.

  12. Altered effector function of peripheral cytotoxic cells in COPD

    Directory of Open Access Journals (Sweden)

    Corne Jonathan M


    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.

  13. Immunoadsorption using protein A columns complicates interpretation of cytotoxic crossmatch

    DEFF Research Database (Denmark)

    Koefoed-Nielsen, Pernille Bundgaard; Bistrup, Claus; Christiansen, Mette


    . The immunological pretransplant monitoring consists of luminex-based monitoring of DSA strength combined with flowcytometric and cytotoxic complement dependent crossmatching (CDC). The goal of pretreatment is to obtain a negative CDC T-cell crossmatch and a significant reduction in DSA strength. From 2012-2013 we...

  14. Terpenoids from Ainsliaea latifolia and their cytotoxic activities. (United States)

    Dong, Xue-Yun; Wang, Guo-Wei; Zhuo, Zhi-Guo; Lv, Chao; Fang, Xin; Shi, Zhi-Ran; Zeng, Ren-Tao; Shen, Yun-Heng; Zhang, Wei-Dong


    Two new compounds including one new sesquiterpenoid and one new monoterpenoid, together with 10 known compounds were isolated from the whole plants of Ainsliaea latifolia. The structures of these compounds were elucidated by analysis of spectroscopic data. All compounds were evaluated for their cytotoxic activities.

  15. Cytotoxicity of plants used in traditional medicine in Yemen. (United States)

    Al-Fatimi, M; Friedrich, U; Jenett-Siems, K


    Twenty-five extracts obtained from 14 plant species used in the traditional medicine in Yemen have been screened for cytotoxic activity against human ECV-304 cells. Extracts of Dracaena cinnabari, Eucalyptus camaldulensis, Euclea divinorum, Euphorbia cactus, Pulicaria crispa, and Withania somnifera displayed a remarkable activity.

  16. Synthesis of Obyanamide, a Marine Cytotoxic Cyclic Depsipeptide

    Institute of Scientific and Technical Information of China (English)


    The synthesis of a marine cytotoxic cyclic depsipeptide obyanamide has been accomplished. The key steps include assembling liner pentapeptide via Yamaguchi esterification and HATU-promoted ring closing. The structure of the synthetic sample was identified by 1H and 13C NMR, H-H COSY, HMQC, HMBC, and HRESIMS, but appears to be different from that of the marine natural product.

  17. Scyphozoan jellyfish venom metalloproteinases and their role in the cytotoxicity. (United States)

    Lee, Hyunkyoung; Jung, Eun-sun; Kang, Changkeun; Yoon, Won Duk; Kim, Jong-Shu; Kim, Euikyung


    The present study, for the first time, comparatively investigated the enzymatic activities (proteases and hyaluronidases) in the venoms of four Scyphozoan jellyfish species, including Nemopilema nomurai, Rhopilema esculenta, Cyanea nozakii, and Aurelia aurita. For this, various zymographic analyses were performed using assay specific substrates. Interestingly, all the four jellyfish venoms showed gelatinolytic, caseinolytic, and fibrinolytic activities, each of which contains a multitude of enzyme components with molecular weights between 17 and 130 kDa. These four jellyfish venoms demonstrated a huge variation in their proteolytic activities in quantitative and qualitative manner depending on the species. Most of these enzymatic activities were disappeared by the treatment of 1,10-phenanthroline, suggesting they might be belonged to metalloproteinases. Toxicological significance of these venom proteases was examined by comparing their proteolytic activity and the cytotoxicity in NIH 3T3 cells. The relative cytotoxic potency was C. nozakii > N. nomurai > A. aurita > R. esculenta. The cytotoxicity of jellyfish venom shows a positive correlation with its overall proteolytic activity. The metalloproteinases appear to play an important role in the induction of jellyfish venom toxicities. In conclusion, the present report proposes a novel finding of Scyphozoan jellyfish venom metalloproteinases and their potential role in the cytotoxicity.

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

    Directory of Open Access Journals (Sweden)

    Llobet-Brossa Enrique


    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.

  19. Evaluation of Ciprofloxacin Cytotoxic Effect in Rat Testis

    Directory of Open Access Journals (Sweden)

    A. Khaki


    Full Text Available Introduction & Objective: Ciprofloxacin is a synthetic antibacterial agent belonging to the family of fluoroquinolones with a very broad spectrum against microbial pathogens, especially Gram-negative infectious diseases, that has been approved in more than 100 countries world-wide. The aim of this study was to see histopathological and cytotoxic effects of ciprofloxacin after inducement in rat testis. Materials & Methods: The twenty male wistar rat were selected and randomly divided into two groups; control (n=10 and test (n=10. The test group was received 12.5mg/kg (PO ciprofloxacin daily for sixty day; however the control group just received plate. On sixtieth day the testis tissue of rat in both groups were removed and were prepared for light microscopy and cytotoxic studies. Results: Study about cytotoxic effects was indicated that absorption of radiation rate after five day in control group was increased when as compared with experimental group, (Control: 92.8±1.5 & Test: 65±6, P<0.05 and the studies of testis tissue slices of test group showed many changes such as necrosis in spermatocyt I cells plus diameter of nuclei in spermatocyt I, was increased, (P<0.01. Conclusion: Since in our study ciprofloxacin had cytotoxic side effect on spermatocyt I cells, and rate of cell death may be increased in this cells then consequently ciprofloxacin inducement is harmful for sperm health ability parameters and due decrease fertility rates in human.

  20. Phytochemical, antioxidant, antiviral and cytotoxic evaluation of Opuntia dillenii flowers

    Directory of Open Access Journals (Sweden)

    Arthanari Saravana Kumar


    Full Text Available Opuntia dillenii used in Asian traditional medicine especially in China. We here report on the investigation of the phytochemical content, antioxidant, cytotoxicity and antiviral activity of methanolic extract of O. dillenii flowers. The antioxidant activity was measured with the DPPH, hydrogen peroxide and hydroxyl radicals scavenging method. In the antiviral and cytotoxic assay we used different viruses in different cell lines. In antioxidant assay, the DPPH assay exhibited potent antioxidant abilities with IC50 of 58.7 µg/mL. In antiviral assay, the extract possess strongest antiviral activity against herpes simplex 1(EC50= 25 µg/mL and 2 (EC50= 20 µg/mL, vaccinia (EC50= 100 µg/mL and moderate activity for remaining viruses (EC50= >100 µg/mL. The cytotoxicity effect was evaluated using MTT assay and the results revealed that the extracts exhibited cytotoxicity above the range of 100 µg/mL. Our present reports confirmed that the O. dillenii could be a potential antioxidant and antimicrobial agent in near future.

  1. An efficient analysis of nanomaterial cytotoxicity based on bioimpedance (United States)

    Kandasamy, Karthikeyan; Choi, Cheol Soo; Kim, Sanghyo


    In the emerging nanotechnology field, there is an urgent need for the development of a significant and sensitive method that can be used to analyse and compare the cytotoxicities of nanomaterials such as carbon nanotubes (CNTs) and gold nanoparticles (AuNPs), since such materials can be applied as contrast agents or drug delivery carriers. The bioimpedance system possesses great potential in many medical research fields including nanotechnology. Electric cell-substrate impedance sensing (ECIS) is a particular bioimpedance system that offers a real-time, non-invasive, and quantitative measurement method for the cytotoxicity of various materials. The present work compared the cytotoxicity of AuNPs to that of purchased single-walled carbon nanotubes (SWCNTs). The size-controlled and monodispersed AuNPs were synthesized under autoclaved conditions and reduced by ascorbic acid (AA) whereas the purchased SWCNTs were used without any surface modifications. Bioimpedance results were validated by conventional WST-1 and trypan blue assays, and transmission electron microscopy (TEM) and field emission scanning electron microscopy (FE-SEM) were performed to examine nanomaterials inside the VERO cells. This research evaluates the ability of the ECIS system compared to those of conventional methods in analyzing the cytotoxicity of AuNPs and SWCNTs with higher sensitivity under real-time conditions.

  2. An efficient analysis of nanomaterial cytotoxicity based on bioimpedance

    Energy Technology Data Exchange (ETDEWEB)

    Kandasamy, Karthikeyan; Kim, Sanghyo [College of Bionanotechnology, Kyungwon University, Gyeonggi-do (Korea, Republic of); Choi, Cheol Soo, E-mail: [Lee Gil Ya Diabetes and Cancer Institute, Gachon University of Medicine and Science, Incheon (Korea, Republic of)


    In the emerging nanotechnology field, there is an urgent need for the development of a significant and sensitive method that can be used to analyse and compare the cytotoxicities of nanomaterials such as carbon nanotubes (CNTs) and gold nanoparticles (AuNPs), since such materials can be applied as contrast agents or drug delivery carriers. The bioimpedance system possesses great potential in many medical research fields including nanotechnology. Electric cell-substrate impedance sensing (ECIS) is a particular bioimpedance system that offers a real-time, non-invasive, and quantitative measurement method for the cytotoxicity of various materials. The present work compared the cytotoxicity of AuNPs to that of purchased single-walled carbon nanotubes (SWCNTs). The size-controlled and monodispersed AuNPs were synthesized under autoclaved conditions and reduced by ascorbic acid (AA) whereas the purchased SWCNTs were used without any surface modifications. Bioimpedance results were validated by conventional WST-1 and trypan blue assays, and transmission electron microscopy (TEM) and field emission scanning electron microscopy (FE-SEM) were performed to examine nanomaterials inside the VERO cells. This research evaluates the ability of the ECIS system compared to those of conventional methods in analyzing the cytotoxicity of AuNPs and SWCNTs with higher sensitivity under real-time conditions.

  3. Cytotoxic sesquiterpene lactones from the aerial parts of Inula aucheriana.

    Directory of Open Access Journals (Sweden)

    Ahmad Reza Gohari


    Full Text Available Inula aucheriana DC is a member of the family Asteraceae which is known to produce cytotoxic secondary metabolites noted as sesquiterpene lactones. In the present study, sesquiterpene lactones inuchinenolide B, 6-deoxychamissonolide (stevin and 14-acetoxy-1β,5α,7αH-4β-hydroxy-guai-9(10,11(13-dien-12,8α-olide were isolated from I. aucheriana. Inuchinenolide B and 14-acetoxy-1β,5α,7αH-4β-hydroxy-guai-9(10,11(13-dien-12,8α-olide were further evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide assay to demonstrate cytotoxic activity with IC50 values of (56.6, 19.0, (39.0, 11.8, and (55.7, 15.3 μg/mL against HepG-2, MCF-7 and A-549 cells, respectively. The cytotoxic activity of the two evaluated sesquiterpene lactones partly explains the cytotoxic activity that was previously observed for the extracts of Inula aucheriana. The isolated compounds could be further investigated in cancer research studies.

  4. New cytotoxic steroids from the leaves of Clerodendrum trichotomum. (United States)

    Xu, Rui-Lan; Wang, Rui; Ding, Lan; Shi, Yan-Ping


    A phytochemical investigation of the leaves of Clerodendrum trichotomum led to the isolation of five new (2-6) and two known (1 and 7) steroids, whose structures and relative configurations were elucidated by comprehensive spectroscopic analysis and by comparison of their NMR data with those of related compounds. Steroids 2 and 5 exhibited moderate cytotoxicity in vitro against HeLa cell line.

  5. Antioxidant and cytotoxic activity of Acanthus ilicifolius flower

    Directory of Open Access Journals (Sweden)

    Muhamad Firdaus


    Conclusions: It is suggested that active compounds of A. ilicifolius flower solved in methanol play a role to inhibit free radical activity and kill Artemia salina nauplii. The substances can be considered as potential antioxidant and cytotoxic agents as well as imminent candidate for cancer therapy.

  6. A novel mechanism of methylglyoxal cytotoxicity in prostate cancer cells. (United States)

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


    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.


    NARCIS (Netherlands)



    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

  8. Cytotoxicity of natural ginseng glycosides and semisynthetic analogues

    NARCIS (Netherlands)

    Atopkina, LN; Malinovskaya, GV; Elyakov, GB; Uvarova, NI; Woerdenbag, HJ; Koulman, A; Potier, P


    The cytotoxicity of natural glycosides from Ginseng, semisynthetic analogues and related triterpenes of the dammarane series, isolated from the leaves of the Far-East species of the genus Betula was studied in order to elucidate structure-activity relationships. Some of the compounds studied were ac

  9. Three New Cytotoxic ent-Kaurane Diterpenes from Isodon excisoides

    Directory of Open Access Journals (Sweden)

    Li-Ping Dai


    Full Text Available Three types of ent-kaurane diterpenoids were isolated from the aerial parts of Isodon excisoides, including three new diterpenoids, 1α,7α,14β-trihydroxy-20-acetoxy-ent-kaur-15-one (1; 1α,7α,14β,18-tetrahydroxy-20-acetoxy-ent-kaur-15-one (2; and 1α-acetoxy-14β-hydroxy-7α,20-epoxy-ent-kaur-16-en-15-one (3; together with six known diterpenes henryin (4; kamebanin (5; reniformin C (6; kamebacetal A (7; kamebacetal B (8; and oridonin (9. The structures of the isolated compounds were elucidated by means of nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry in conjunction with published data for their analogs, as well as their fragmentation patterns. Compounds 5 and 9 were isolated from Isodon excisoides for the first time. To explore the structure-activity relationships of the isolated compounds, they were tested for their cytotoxic effects against five human cancer cell lines: HCT-116, HepG2, A2780, NCI-H1650, and BGC-823. Most of the isolated compounds showed certain cytotoxic activity against the five cancer cell lines with IC50 values ranging from 1.09–8.53 µM. Among the tested compounds, compound 4 exhibited the strongest cytotoxic activity in the tested cell lines, with IC50 values ranging from 1.31–2.07 µM. Compounds 1, 6, and 7 exhibited selective cytotoxic activity.

  10. 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


    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

  11. Cytotoxic Constituents from the Leaves of Zanthoxylum schinifolium

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Zhe; Min, Byung Sun; Kim, Ae Kyong; Woo, Mi Hee [Catholic Univ. of Daegu, Gyeongsan (Korea, Republic of); Jun, Do Youn; Kim, Young Ho [Kyungpook National Univ., Daegu (Korea, Republic of)


    The roots, stems, pericarps, and seeds of Z. schinifolium were each extracted with MeOH, and the leaves were extracted with 80% MeOH and concentrated. These extracts were examined on MTT for cytotoxicity against Jurkat T cell clone E6.1. The results showed that the leaves extract had the strongest MTT cytotoxicity. The MeOH extract of Z. schinifolium leaves was subsequently fractionated into four parts: methylene chloride, ethyl acetate, n-butanol and water. These fractions were examined on MTT for cytotoxicity. The results showed that the methylene chloride fraction exhibited the strongest MTT cytotoxicity. Chromatographic separation of the methylene chloride and butanol fractions had yielded a quinolin (1), three phenylpropanoids (2, 3, 12), four coumarins (4 ∼ 7), three triterpenoids (8 ∼ 10), an alkaloid (11), an alcohol glucoside (13) and three monoterpene glucosides (14, 15, 16). One of these compounds were identified as new threo-6-amino-5-hydroxy-5-methyl-1,3-oxazinan-4-one (11) together with fifteen known, 3-heptyl-2-methylisoquinolin-1(2H)-one (1), integrifoliodiol (2), cuspidiol (3), bergapten (4), aurapten (5), 8-hydroxy-7-methoxy-chromen-2-one (6), 6,7-dimethoxy-2H-naphthalen-1-one (7), lupeol (8), lupeone (9), β-sitosterol (10), syringin (12), 2-propyl alchol β-D-glucopyranoside (13), vomifoliol-9-O-β-D-glucopyranoside (14), betulalbuside A (15) and cnidioside C (16) on the basis of spectroscopic and chemical evidences. All of the compounds were isolated for the first time from this plant except 5 and 7. In the MTT cytotoxicity assay against Jurkat T cell clone E6.1, IC{sub 50} values of cuspidiol (3) and auraptene (5) were obtained at 7.3 μg/mL and 16.5 μg/mL, respectively.

  12. Cytotoxicity and genotoxicity of phenazine in two human cell lines. (United States)

    McGuigan, Claire F; Li, Xing-Fang


    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. Mutagenic and cytotoxic activities of Limonium globuliferum methanol extracts. (United States)

    Eren, Yasin


    Unmonitored use of plant extractions alone or in combination with drugs may cause important health problems and toxic effects. Limonium (Plumbaginaceae) plants are known as antibacterial, anticancer and antivirus agent. But it is possible that this genus may have toxic effects. This study evaluated the mutagenic and cytotoxic effects of Limonium globuliferum (Boiss. et Heldr.) O. Kuntze (Plumbaginaceae) acetone/methanol (2:1), and methanol extracts of root, stem, and leaf. Different parts of this species were used in order to compare the mutagenic and cytotoxic effects of these parts. Ames test was carried out with S. typhimurium TA98, and TA100 strains. Strains were incubated at 37 °C for 72 h. MDBK cell line was used in MTT test. 10,000, 1000, 100, 10, 1 and 0.1 µg/plate concentrations of plant extracts were used in Ames test. 50, 25, 12.5, 6.25 and 3.125 µg/ml concentrations of root, stem and leaf acetone/methanol (2:1) and methanol extracts were used in MTT test. Ames test results indicated that only methanol leaf extract (10,000 µg/plate) had mutagenic activity. L. globuliferum root methanol extracts (3.125 and 6.25 µg/ml) increased the proliferation rates. Root acetone/methanol (2:1) extracts were found highly cytotoxic in all treatments. The results indicated that leaf extracts had lower cytotoxic effects than root and stem extracts. High concentrations of L. globuliferum stem and leaf methanol extracts showed cytotoxic activity in all treatment periods while low concentrations of the stem methanol extracts increased the proliferation rates.

  14. Contribution of physicochemical characteristics of nano-oxides to cytotoxicity. (United States)

    Xu, Mingsheng; Fujita, Daisuke; Kajiwara, Shoko; Minowa, Takashi; Li, Xianglan; Takemura, Taro; Iwai, Hideo; Hanagata, Nobutaka


    To identify the key physicochemical properties of nano-oxides governing cytotoxicity, we investigate the contribution of the size, shape, morphology, and electronic properties of ten types of insulator (SiO(2), CeO(2) and Al(2)O(3)) and semiconductor (ZnO and CuO) nano-oxides to cytotoxicity using the NIH3T3 and A549 cell lines as models. We find that the shape of the Al(2)O(3) (nanoparticle versus nanowhisker) and the morphology of the SiO(2) (porous versus non-porous nanoparticles) did not have obvious effect on the observed cytotoxicity, and the size of the nano-oxides cannot be regarded as an indicator of cytotoxicity. By contrast, we find that the cell viability exposed to the semiconductor nano-oxides was much lower than that exposed to the insulator nano-oxides. Moreover, the Al-doped ZnO nanoparticle (NP) was more toxic than the non-doped ZnO NP, whereas the Al-doped CuO NP was less toxic than the non-doped CuO NP but more toxic than the Al(2)O(3) NP. Correspondingly, the valence band X-ray photoelectron spectra of the nano-oxides show the density of states of the Al-doped ZnO NP (the Al-doped CuO NP) is higher (lower) than that of the non-doped ZnO NP (the non-doped CuO NP). These results suggest that the electronic properties of nano-oxides may play an important role in the observed cytotoxicity. The results have implications for selectively tailoring the toxic effect and establishing predictive models for the design of various types of nanomaterials with unique properties and for the understanding of interactions between nanomaterials with biological system.

  15. Nanoparticle incorporation of melittin reduces sperm and vaginal epithelium cytotoxicity.

    Directory of Open Access Journals (Sweden)

    Andrew P Jallouk

    Full Text Available Melittin is a cytolytic peptide component of bee venom which rapidly integrates into lipid bilayers and forms pores resulting in osmotic lysis. While the therapeutic utility of free melittin is limited by its cytotoxicity, incorporation of melittin into the lipid shell of a perfluorocarbon nanoparticle has been shown to reduce its toxicity in vivo. Our group has previously demonstrated that perfluorocarbon nanoparticles containing melittin at concentrations <10 µM inhibit HIV infectivity in vitro. In the current study, we assessed the impact of blank and melittin-containing perfluorocarbon nanoparticles on sperm motility and the viability of both sperm and vaginal epithelial cells. We found that free melittin was toxic to sperm and vaginal epithelium at concentrations greater than 2 µM (p<0.001. However, melittin nanoparticles were not cytotoxic to sperm (p = 0.42 or vaginal epithelium (p = 0.48 at an equivalent melittin concentration of 10 µM. Thus, nanoparticle formulation of melittin reduced melittin cytotoxicity fivefold and prevented melittin toxicity at concentrations previously shown to inhibit HIV infectivity. Melittin nanoparticles were toxic to vaginal epithelium at equivalent melittin concentrations ≥20 µM (p<0.001 and were toxic to sperm at equivalent melittin concentrations ≥40 µM (p<0.001. Sperm cytotoxicity was enhanced by targeting of the nanoparticles to the sperm surface antigen sperm adhesion molecule 1. While further testing is needed to determine the extent of cytotoxicity in a more physiologically relevant model system, these results suggest that melittin-containing nanoparticles could form the basis of a virucide that is not toxic to sperm and vaginal epithelium. This virucide would be beneficial for HIV serodiscordant couples seeking to achieve natural pregnancy.

  16. Differential cytotoxicity of copper ferrite nanoparticles in different human cells. (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


    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.

  17. Electrical potential difference across the stomach wall and gastric morphology in anaesthetized pigs after intravenous administration of cytotoxic drugs

    DEFF Research Database (Denmark)

    Fabrin, B.; Højgaard, L.; Olesen, H.P.;


    Oncologi, cytotoxic drugs, electrical potential difference, medicin, cander, gastric, side effects, chemotherapy......Oncologi, cytotoxic drugs, electrical potential difference, medicin, cander, gastric, side effects, chemotherapy...

  18. Gender-specific effects of genetic variants within Th1 and Th17 cell-mediated immune response genes on the risk of developing rheumatoid arthritis.

    Directory of Open Access Journals (Sweden)

    Rafael Cáliz

    Full Text Available The present study was conducted to explore whether single nucleotide polymorphisms (SNPs in Th1 and Th17 cell-mediated immune response genes differentially influence the risk of rheumatoid arthritis (RA in women and men. In phase one, 27 functional/tagging polymorphisms in C-type lectins and MCP-1/CCR2 axis were genotyped in 458 RA patients and 512 controls. Carriers of Dectin-2 rs4264222T allele had an increased risk of RA (OR = 1.47, 95%CI 1.10-1.96 whereas patients harboring the DC-SIGN rs4804803G, MCP-1 rs1024611G, MCP-1 rs13900T and MCP-1 rs4586C alleles had a decreased risk of developing the disease (OR = 0.66, 95%CI 0.49-0.88; OR = 0.66, 95%CI 0.50-0.89; OR = 0.73, 95%CI 0.55-0.97 and OR = 0.68, 95%CI 0.51-0.91. Interestingly, significant gender-specific differences were observed for Dectin-2 rs4264222 and Dectin-2 rs7134303: women carrying the Dectin-2 rs4264222T and Dectin-2 rs7134303G alleles had an increased risk of RA (OR = 1.93, 95%CI 1.34-2.79 and OR = 1.90, 95%CI 1.29-2.80. Also five other SNPs showed significant associations only with one gender: women carrying the MCP-1 rs1024611G, MCP-1 rs13900T and MCP-1 rs4586C alleles had a decreased risk of RA (OR = 0.61, 95%CI 0.43-0.87; OR = 0.67, 95%CI 0.47-0.95 and OR = 0.60, 95%CI 0.42-0.86. In men, carriers of the DC-SIGN rs2287886A allele had an increased risk of RA (OR = 1.70, 95%CI 1.03-2.78, whereas carriers of the DC-SIGN rs4804803G had a decreased risk of developing the disease (OR = 0.53, 95%CI 0.32-0.89. In phase 2, we genotyped these SNPs in 754 RA patients and 519 controls, leading to consistent gender-specific associations for Dectin-2 rs4264222, MCP-1 rs1024611, MCP-1 rs13900 and DC-SIGN rs4804803 polymorphisms in the pooled sample (OR = 1.38, 95%CI 1.08-1.77; OR = 0.74, 95%CI 0.58-0.94; OR = 0.76, 95%CI 0.59-0.97 and OR = 0.56, 95%CI 0.34-0.93. SNP-SNP interaction analysis of significant SNPs also showed a

  19. Physico-chemical properties based differential toxicity of graphene oxide/reduced graphene oxide in human lung cells mediated through oxidative stress (United States)

    Mittal, Sandeep; Kumar, Veeresh; Dhiman, Nitesh; Chauhan, Lalit Kumar Singh; Pasricha, Renu; Pandey, Alok Kumar


    Goraphene derivatives (GD) are currently being evaluated for technological and biomedical applications owing to their unique physico-chemical properties over other carbon allotrope such as carbon nanotubes (CNTs). But, the possible association of their properties with underlying in vitro effects have not fully examined. Here, we assessed the comparative interaction of three GD - graphene oxide (GO), thermally reduced GO (TRGO) and chemically reduced GO (CRGO), which significantly differ in their lateral size and functional groups density, with phenotypically different human lung cells; bronchial epithelial cells (BEAS-2B) and alveolar epithelial cells (A549). The cellular studies demonstrate that GD significantly ineternalize and induce oxidative stress mediated cytotoxicity in both cells. The toxicity intensity was in line with the reduced lateral size and increased functional groups revealed more toxicity potential of TRGO and GO respectively. Further, A549 cells showed more susceptibility than BEAS-2B which reflected cell type dependent differential cellular response. Molecular studies revealed that GD induced differential cell death mechanism which was efficiently prevented by their respective inhibitors. This is prior study to the best of our knowledge involving TRGO for its safety evaluation which provided invaluable information and new opportunities for GD based biomedical applications.

  20. Demonstration of NK cell-mediated lysis of varicella-zoster virus (VZV)-infected cells: characterization of the effector cells

    Energy Technology Data Exchange (ETDEWEB)

    Tilden, A.B.; Cauda, R.; Grossi, C.E.; Balch, C.M.; Lakeman, A.D.; Whitley, R.J.


    Infection with varicella-zoster virus (VZV) rendered RAJI cells more susceptible to lysis by non-adherent blood lymphocytes. At an effector to target ratio of 80:1 the mean percentage of /sup 51/Cr release of VZV-infected RAJI cells was 41 +/- 12%, whereas that of uninfected RAJI cells was 15 +/- 6%. The increased susceptibility to lysis was associated with increased effector to target conjugate formation in immunofluorescence binding assays. The effector cells cytotoxic for VZV-infected RAJI cells were predominantly Leu-11a/sup +/ Leu-4/sup -/ granular lymphocytes as demonstrated by fluorescence-activated cell sorting. The effector cell active against VZV-infected RAJI cells appeared similar to those active against herpes simplex virus (HSV)-infected cells, because in cold target competition experiments the lysis of /sup 51/Cr-labeled VZV-infected RAJI cells was efficiently inhibited by either unlabeled VZV-infected RAJI cells (mean 71% inhibition, 2:1 ratio unlabeled to labeled target) or HSV-infected RAJI cells (mean 69% inhibition) but not by uninfected RAJI cells (mean 10% inhibition). In contrast, competition experiments revealed donor heterogeneity in the overlap between effector cells for VZV- or HSV-infected RAJI vs K-562 cells.

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

    Directory of Open Access Journals (Sweden)

    Hrubik Jelena D.


    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

  2. Preparation of Conjugates of Cytotoxic Lupane Triterpenes with Biotin. (United States)

    Soural, Miroslav; Hodon, Jiri; Dickinson, Niall J; Sidova, Veronika; Gurska, Sona; Dzubak, Petr; Hajduch, Marian; Sarek, Jan; Urban, Milan


    To better understand the mechanism of action of antitumor triterpenes, we are developing methods to identify their molecular targets. A promising method is based on combination of quantitative proteomics with SILAC and uses active compounds anchored to magnetic beads via biotin-streptavidin interaction. We developed a simple and fast solid-phase synthetic technique to connect terpenes to biotin through a linker. Betulinic acid was biotinylated from three different conjugation sites for use as a standard validation tool since many molecular targets of this triterpene are already known. Then, a set of four other cytotoxic triterpenoids was biotinylated. Biotinylated terpenes were similarly cytotoxic to their nonbiotinylated parents, which suggests that the target identification should not be influenced by linker or biotin. The developed solid-phase synthetic approach is the first attempt to use solid-phase synthesis to connect active triterpenes to biotin and is applicable as a general procedure for routine conjugation of triterpenes with other molecules of choice.

  3. Cytotoxic diterpenoid pseudodimers from the Korean sponge Phorbas gukhulensis. (United States)

    Jeon, Ju-eun; Liao, Lijuan; Kim, Heegyu; Sim, Chung J; Oh, Dong-Chan; Oh, Ki-Bong; Shin, Jongheon


    Four new cytotoxic diterpenoid pseudodimers (2-5), along with a previously reported one, gukulenin A (1), were isolated from the marine sponge Phorbas gukhulensis collected off the coast of Gagu-do, Korea. These novel compounds, designated gukulenins C-F (2-5), were determined by extensive spectroscopic analyses to be pseudodimers of the gagunins, like gukulenin A. The termini of the tropolone-containing side chains in gukulenins C-E (2-4) were found to have diverse modifications involving acetamides or taurine, whereas gukulenin F (5) was formed from 1 by the ring-opening of a cyclic hemiketal. The relative and absolute configurations were assigned by Murata's and modified Snatzke's methods using a HETLOC experiment and a CD measurement of a dimolybdenum complex, respectively. All of these compounds exhibited significant cytotoxicity against the K562 and A549 cell lines.

  4. Cytotoxic effects of selective species of Caryophyllaceae in Iran

    Directory of Open Access Journals (Sweden)

    F. Naghibi


    Full Text Available Cancer is a major cause of death worldwide and causes serious problems in human life. It is developed by uncontrolled growth of a cell or a group of cells. There are many difficulties in treatment of cancer and many researchers are involved in investigating for effective drugs to treat the disease. Caryophyllaceae is a large family of about 86 genera and 2200 herbaceous or subshrub species. The family is known for its ornamental plants and saponin compounds. In the present study, the potential cytotoxic activity of 17 selected species from Caryophyllaceae has been investigated against MCF-7, HepG-2, A-549, HT-29 and MDBK cells using MTT assay. Five species exhibited cytotoxic effects with IC50 values < 100 μg/mL. Silene ampullata and Acanthophyllum bracteatum extracts were toxic only against MCF-7 cell line suggesting them as suitable candidates for more investigations of breast cancer studies.

  5. Sesquiterpenoids from Pulicaria canariensis and their cytotoxic activities. (United States)

    Triana, Jorge; López, Mariana; Pérez, Francisco J; González-Platas, Javier; Quintana, José; Estévez, Francisco; León, Francisco; Bermejo, Jaime


    Thirteen new sesquiterpenes, pulicanadiene A (1), B (2), and C (3), pulicanone (4), pulicanol (5), pulicanarals A (6), B (7), and C (8), pulicanadienals A (9) and B (10), pulicanadienol (11), and pulioplopanones A (12) and B (13), and seven known compounds, stigmasterol, ergosterol peroxide, calenduladiol, 7,4'-di-O-methyldihydrokaempferol, 5,7-dihydroxy-3,3',4'-trimethoxyflavone, dihydroquercetin 7,3'-dimethyl ether, and 6,15alpha-epoxy-1beta,4beta-dihydroxyeudesmane, were isolated from Pulicaria canariensis. Compound 4a showed cytotoxicity on the human myeloid leukemia cell line HL-60. The cytotoxicity was caused by induction of apoptosis as determined by microscopy of nuclear changes, activation of caspases, and the cleavage of poly(ADP-ribose) polymerase-1.

  6. In vitro cytotoxic activity of Brazilian Middle West plant extracts

    Directory of Open Access Journals (Sweden)

    Talal Suleiman Mahmoud


    Full Text Available Cytotoxic activity of eight plant extracts, native from the Mid-West of Brazil comprising Cerrado, Pantanal and semideciduous forest, was evaluated for MDA-MB-435, SF-295, and HCT-8 cancer cell strains. A single 100 µg.mL-1 dose of each extract was employed with 72 h of incubation for all tests. Doxorubicin (1 µg.mL-1 was used as the positive control and the MTT method was used to detect the activity. Cytotoxicity of distinct polarities was observed in thirty extracts (46%, from different parts of the following species: Tabebuia heptaphylla (Vell. Toledo, Bignoniaceae, Tapirira guianensis Aubl., Anacardiaceae, Myracrodruon urundeuva Allemão, Anacardiaceae, Schinus terebinthifolius Raddi, Anacardiaceae, Gomphrena elegans Mart., Amaranthaceae, Attalea phalerata Mart. ex Spreng., Arecaceae, Eugenia uniflora L., Myrtaceae, and Annona dioica A. St.-Hil., Annonaceae. Extracts of at least two tested cell strains were considered to be highly active since their inhibition rate was over 75%.

  7. Mechanisms of the statins cytotoxicity in freshly isolated rat hepatocytes. (United States)

    Abdoli, Narges; Heidari, Reza; Azarmi, Yadollah; Eghbal, Mohammad Ali


    Statins are potent drugs, used as lipid-lowering agents in cardiovascular diseases. Hepatotoxicity is one of the serious adverse effects of statins, and the exact mechanism of hepatotoxicity is not yet clear. In this study, the cytotoxic effects of the most commonly used statins, that is, atorvastatin, lovastatin, and simvastatin toward isolated rat hepatocytes, were evaluated. Markers, such as cell death, reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential, and the amount of reduced and oxidized glutathione in the statin-treated hepatocytes, were investigated. It was found that the statins caused cytotoxicity toward rat hepatocytes dose dependently. An elevation in ROS formation, accompanied by a significant amount of lipid peroxidation and mitochondrial depolarization, was observed. Cellular glutathione reservoirs were decreased, and a significant amount of oxidized glutathione was formed. This study suggests that the adverse effect of statins toward hepatocytes is mediated through oxidative stress and the hepatocytes mitochondria play an important role in the statin-induced toxicity.

  8. Synthesis and Cytotoxicity of Chalcones and 5-Deoxyflavonoids

    Directory of Open Access Journals (Sweden)

    Jing Zhang


    Full Text Available Chalcones 1~8 and 5-deoxyflavonoids 9~22 were synthesized in good yields by aldol condensation, Algar-Flynn-Oyamada reaction, glycosidation, and deacetylation reaction, respectively, starting from 2-acetyl phenols substituted by methoxy or methoxymethoxy group and appropriately benzaldehydes substituted by methoxy, methoxymethoxy group, or chlorine. Among them, 13 and 17~22 are new compounds. The cytotoxicity bioassays of these chalcones and 5-deoxyflavonoids were screened using the sulforhodamine B (SRB protein staining method, and the results showed that compounds 2, 4, 5, 6, 10, 15, and 19 exhibited moderate cytotoxicity against the cancer cell line of MDA-MB-231, U251, BGC-823, and B16 in comparison with control drugs (HCPT, Vincristine, and Taxol.

  9. Antimycobacterial and cytotoxicity activity of synthetic and natural compounds

    Energy Technology Data Exchange (ETDEWEB)

    Souza, Ana O. de [Instituto Butantan, Sao Paulo, SP (Brazil). Lab. de Bioquimica e Biofisica]. E-mail:; Galetti, Fabio C.S.; Silva, Celio L. [Universidade de Sao Paulo (USP), Ribeirao Preto, SP (Brazil). Faculdade de Medicina. Dept. de Bioquimica e Imunologia] (and others)


    Antimycobacterial and cytotoxicity activity of synthetic and natural compounds. Secondary metabolites from Curvularia eragrostidis and Drechslera dematioidea, Clusia sp. floral resin, alkaloids from Pilocarpus alatus, salicylideneanilines, piperidine amides, the amine 1-cinnamylpiperazine and chiral pyridinium salts were assayed on Mycobacterium tuberculosis H37Rv. N-(salicylidene)-2-hydroxyaniline was the most effective compound with a minimal inhibitory concentration (MIC) of 8 {mu}mol/L. Dihydrocurvularin was moderately effective with a MIC of 40 {mu}mol/L. Clusia sp. floral resin and a gallocatechin-epigallocatechin mixture showed MIC of 0.02 g/L and 38 {mu}mol/L, respectively. The cytotoxicity was evaluated for N-(salicylidene)-2-hydroxyaniline, curvularin, dihydrocurvularin and Clusia sp. floral resin, and the selectivity indexes were > 125, 0.47, 0.75 and 5, respectively. (author)

  10. Gymnasterkoreaynes A-F, cytotoxic polyacetylenes from Gymnaster koraiensis. (United States)

    Jung, Hyun-Ju; Min, Byung-Sun; Park, Jin-Young; Kim, Young-Ho; Lee, Hyeong-Kyu; Bae, Ki-Hwan


    Six new polyacetylenes, gymnasterkoreaynes A-F (1-6), were isolated from the roots of Gymnaster koraiensis, together with 2,9,16-heptadecatrien-4,6-diyn-8-ol (7) and 1,9,16-heptadecatriene-4,6-diyn-3,8-diol (8), by bioassay-guided fractionation using the L1210 tumor cell line as a model for cytotoxicity. The structures of compounds 1-6 were established spectroscopically, which included 2D NMR experiments. Gymnasterkoreaynes A-F (1-6) are linear diacetylenes and are structurally related to falcarinol, panaxynol, panaxydiol, and panaxytriol. Of the compounds isolated, gymnasterkoreaynes B (2), C (3), F (6), and 1,9,16-heptadecatrien-4,6-diyn-3,8-diol (8) exhibited significant cytotoxicity against L1210 tumor cells with ED(50) values of 0.12-3.3 microg/mL.

  11. A Cytotoxic Neolignan from Schisandra propinqua (Wall.) Baill.

    Institute of Scientific and Technical Information of China (English)

    Li-Jia Xu; Feng Huang; Si-Bao Chen; Lian-Niang Li; Shi-Lin Chen; Pei-Gen Xiao


    In the course of our study of bioactive natural products from Schisandra plants, we isolated a neolignan from an EtOAc extract of the stems of Schisandra propinqua (Wall.) Baill. The structure of the new compound was determined to be 4, 4-di (4-hydroxy-3-methoxyphenly)-2, 3-dimethylbutanol (compound 1) on the basis of 1H- and 13C-NMR spectra and 2D NMR methods. Eight known compounds, compounds 2-9, were also isolated and identified, of which compounds 3, 4, 6 and 9 were isolated for the first time from this plant. In addition, compounds 1-4 were evaluated for cytotoxicity by an 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Compound 1 showed significant potential cytotoxic ability in the bioassay.

  12. In vitro bioactivity and cytotoxicity of chemically treated glass fibers

    Directory of Open Access Journals (Sweden)

    Ângela Leão Andrade


    Full Text Available Samples of a commercial glass fiber FM® (Fiber Max were used to test the efficacy of a chemical sol-gel surface treatment to enhance their bioactivity. After treatment with tetraethoxysilane (TEOS, individual fiber samples were soaked into a simulated body fluid (SBF solution, from which they were removed at intervals of 5 and 10 days. Micrographs obtained by scanning electron microscopy (SEM analysis of samples chemically treated with TEOS revealed the formation of a hydroxyapatite (HA coating layer after 5 days into SBF solution. Fourier transform infrared spectroscopic (FTIR analyses confirmed that the coating layer has P-O vibration bands characteristic of HA. The in vitro cytotoxicity was evaluated using a direct contact test, minimum essential medium elution test (ISO 10993-5 and MTT assay. Fibers immersed in SBF and their extracts exhibited lower cytotoxicity than the controls not subjected to immersion, suggesting that SBF treatment improves the biocompatibility of the fiber.

  13. Cytotoxity of two new coumarin derivatives isolated from Launaea mucronata. (United States)

    El-Sharkawy, Eman R; Mahmoud, Khaled


    The chloroform fraction of methanol (MeOH) extract of the aerial parts of Launaea mucronata was in vitro investigated for cytotoxicity against HCT116, HepG2 and MCF-7 cell lines, and resulted with IC50 = 20.0, 18.6 and 14.30 μg/mL, respectively. The chloroform fraction of the MeOH extract was subjected to further fractionation, which led to the isolation of two new coumarin compounds (6-isobutyl coumarin and 6-isobutyl-7-methyl- coumarin). The structures of the new compounds were elucidated by high field 1D and 2D NMR and ESI-MS spectroscopies. Low polar fractions revealed the isolation of other known triterpene compounds which were identified according to its spectral data and comparison with the literature. New coumarin compounds show high cytotoxicity against MCF-7, HCT116 and HepG2 cell lines.

  14. Cytotoxic Activity of Some Novel Dicoumarin Derivatives in vitro

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hui; YU Tian-zhi; ZHAO Yu-ling; FAN Duo-wang; DING Lan; ZHANG Shi-dong


    Some novel dicoumarin derivatives, triethylene-glycol dibenzo[5,6] coumarin-3-carboxylate(1a). PEG (600) dibenzo[5,6]coumarin-3-carboxylate(1b), triethylene-glycol di[7-(N,N'-diethylamino)]-coumarin-3-carboxy-late(2a), were synthesized. The cytotoxic effect of these compounds, along with benzo[5,6]coumarin-3-carboxylic acid(1) and 7-(N,N'-diethylamino)-coumarin-3-carboxylic acid(2), against the SGC-7901 cell lines were determined by Sulforhodamine B(SRB) assay. The preliminary cytotoxicity screening process revealed that the investigated dicoumarin derivatives induced 50% inhibition of the cell viability of SGC-7901 cells at micromolar concentrations. Compound 2a was proved superior to compound la according to the IC_(50) values obtained and the agent with PEG moiety has more contribution to cell-killing ability of the molecules than the remaining agents.

  15. Acetaminophen metabolism, cytotoxicity, and genotoxicity in rat primary hepatocyte cultures

    Energy Technology Data Exchange (ETDEWEB)

    Milam, K.M.; Byard, J.L.


    Acetaminophen (APAP) metabolism, cytotoxicity, and genotoxicity were measured in primary cultures of rat hepatocytes. Although 3 mM APAP caused a slight increase in cellular release of lactate dehydrogenase into the culture medium, cellular glutathione concentration (an index of APAP metabolism) was reduced by 50%. APAP at 7 mM was significantly more toxic to these hepatocytes and had a similar but more marked effect on glutathione concentrations. In spite of its cytotoxicity, neither dose of APAP stimulated DNA repair synthesis when monitored by the rate of incorporation of (/sup 3/H)thymidine into DNA following exposure to APAP. Thus, although APAP has been shown to be both hepato- and nephrotoxic in several in vivo and in vitro systems, the reactive toxic metabolite of APAP is not genotoxic in rat primary hepatocyte cultures.

  16. Lipid Rafts Disruption Increases Ochratoxin A Cytotoxicity to Hepatocytes. (United States)

    Zhang, Yu; Qi, Xiaozhe; Zheng, Juanjuan; Luo, Yunbo; Zhao, Changhui; Hao, Junran; Li, Xiaohong; Huang, Kunlun; Xu, Wentao


    Lipid rafts are microdomains in plasma membrane and can mediate cytotoxicity. In this study, the role of lipid rafts in ochratoxin A-induced toxicity was investigated using Hepatoblastoma Cell Line HepG-2 cells. Disruption of cholesterol-containing lipid rafts enhanced Ochratoxin A (OTA) toxicity, as shown by increased lactate dehydrogenase leakage, increased reactive oxygen species level and reduction of superoxide dismutase activity in a time-dependent manner. Isobaric tags for relative and absolute quantitation-based proteomics of the cell membranes showed that nearly 85.5% proteins were downregulated by OTA, indicating that OTA inhibited the membrane protein synthesis. Most of altered proteins were involved in Gene Ontology "transport", "cell adhesion" and "vesicle-mediated transport". In conclusion, lipid rafts play a key role in OTA-induced cytotoxicity. This study provides insight into how OTA toxicity is regulated by the plasma membrane, especially the lipid rafts.

  17. Triterpene Glycosides from Sea Cucumber Holothuria scabra with Cytotoxic Activity

    Institute of Scientific and Technical Information of China (English)

    HAN Hua; LI Ling; YI Yang-hua; WANG Xiao-hua; PAN Min-xiang


    Objective To study the new triterpene glycosides from sea cucumber Holothuria scabra with cytotoxic activity.Methods Triterpene glycosides from H.scabra were separated and purified by chromatography on DA-101,silica gel,and reversed-phase silica gel column,as well as RP-HPLC.Their structures were elucidated on the basis of spectral data and chemical evidence.Results Three triterpene glycosides were identified as scabraside D (1),fuscocineroside C (2),and 24-dehydroechinoside A (3).Their inhibition on P-388,A549,MKN-28,HCT116,and MCF-7 cells were significant.Conclusion Scabraside D (1) is a new triterpene glycoside,and compounds 2 and 3 are isolated from H.scabra for the first time.The glycosides 1-3 show the in vitro cytotoxicity against five human tumor cell lines in comparison to 10-hydroxycamptothecin.

  18. A New Natural Compound with Cytotoxic Activity from Tripterygium hypoglaucum

    Institute of Scientific and Technical Information of China (English)

    LI Wei; WANG Fang; ZHAO Yu-qing


    Objective To find antitumor cOnstituents from Tripterygium hypoglaucum.Methods The chloroform extract of T.hypoglaucum was separated by silica gel column chromatography and preparative HPLC.The structures of compounds isolated were identified by spectral analysis and chemical evidence.Results Seven compounds were isolated and identified as rhein ethyl ester(1),chrysophenol(2),physcion(3),emodin(4),wilfordine(5),wilforgine(6),and wilforine(7).The cytotoxic activities of the compounds against cancer cell lines were assayed.Conclusion Compound 1 is a new natural compound with strong activities against human cancer cell lines (A2780 and OVCAR-3).Compounds 2-4 are isolated from this genus plants for the first time.The possible structure-activity relationship among compounds 1-4 shows that the methoxy group or oxyethyl moiety might be responsible for the cytotoxity.

  19. Cytotoxic Oxygenated Steroids from the Soft Coral Nephthea erecta. (United States)

    Tsai, Tsung-Chang; Huang, Yu-Ting; Chou, Shih-Kai; Shih, Ming-Cheng; Chiang, Ching-Ying; Su, Jui-Hsin


    A new 10-demethylated steroid, nephtheasteroid A (1), a new 19-oxygenated steroid, nephtheasteroid B (2) as well as five known steroids 3-7 were isolated from the organic extract of a Taiwanese soft coral Nephthea erecta. The structure was determined by means of IR, MS, and NMR techniques. Among these metabolites, 1 is rarely found in steroids possessing a 19-norergostane skeleton. In vitro cytotoxicity study using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that compounds 3 and 4 exhibited cytotoxicity against human chronic myelogenous leukemia (K562), human acute lymphoblastic leukemia (Molt-4), human T lymphoblastoid (Sup-T1), and human leukemic monocyte lymphoma (U937), with IC50 of 6.5-14.0 µM.

  20. New Cytotoxic Oxygenated Sterols from the Marine Bryozoan Cryptosula pallasiana

    Directory of Open Access Journals (Sweden)

    Ping-Hu Zhang


    Full Text Available Six new sterols (1–6, together with seven known sterols (7–13, were isolated from the CCl4 extract of the marine bryozoan Cryptosula pallasiana, four (3–6 of which have already been reported as synthetic sterols. This is the first time that these compounds (3–6 are reported as natural sterols. The structures of the new compounds were determined on the basis of the extensive spectroscopic analysis, including two-dimensional (2D NMR and HR-ESI-MS data. Compounds 1–4, 7 and 10–13 were evaluated for their cytotoxicity against HL-60 human myeloid leukemia cell line, and all of the evaluated compounds exhibited moderate cytotoxicity to HL-60 cells with a range of IC50 values from 14.73 to 22.11 µg/mL except for compounds 12 and 13.