Protoplast preparation from monokaryotic mycelium of Pleurotus sajor-caju using lysing enzyme

International Nuclear Information System (INIS)

Hassan Hamdani Mutaat; Mat Rasol Awang

2004-01-01

The objective of this study was to determine the optimum parameters of the factors influencing protoplast isolation from monokaryotic mycelium of Pleurotus sajor-caju using lysing enzyme from Trichoderma harzianurm. The study was conducted by manipulating the variables of the factors affecting protoplast isolation, such as age of mycelium culture, period for lysing of mycelium, concentration of lysing enzyme and concentration of osmotic stabilizer. The highest protoplast yield of 8.3 x 104 protoplast/ml was achieved when a 3-day P. sajor-caju mycelium, cultured statically, was incubated for 3 hours in a lytic mixture containing 7.5 mg/ml lysing enzyme and 1.2 M ammonium sulfate as osmotic stabilizer. This protoplast yield, however, is insufficient for regeneration and protoplast fusion works. (Author)

Human CD4+ T cells lyse target cells via granzyme/perforin upon circumvention of MHC class II restriction by an antibody-like immunoreceptor.

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Hombach, Andreas; Köhler, Heike; Rappl, Gunter; Abken, Hinrich

2006-10-15

Immune elimination of tumor cells requires the close cooperation between CD8+ CTL and CD4+ Th cells. We circumvent MHC class II-restriction of CD4+ T cells by expression of a recombinant immunoreceptor with an Ab-derived binding domain redirecting specificity. Human CD4+ T cells grafted with an immunoreceptor specific for carcinoembryonic Ag (CEA) are activated to proliferate and secrete cytokines upon binding to CEA+ target cells. Notably, redirected CD4+ T cells mediate cytolysis of CEA+ tumor cells with high efficiencies. Lysis by redirected CD4+ T cells is independent of death receptor signaling via TNF-alpha or Fas, but mediated by perforin and granzyme because cytolysis is inhibited by blocking the release of cytotoxic granules, but not by blocking of Fas ligand or TNF-alpha. CD4+ T cells redirected by Ab-derived immunoreceptors in a MHC class II-independent fashion substantially extend the power of an adoptive, Ag-triggered immunotherapy not only by CD4+ T cell help, but also by cytolytic effector functions. Because cytolysis is predominantly mediated via granzyme/perforin, target cells that are resistant to death receptor signaling become sensitive to a cytolytic attack by engineered CD4+ T cells.

A novel murine T-cell receptor targeting NY-ESO-1.

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Rosati, Shannon F; Parkhurst, Maria R; Hong, Young; Zheng, Zhili; Feldman, Steven A; Rao, Mahadev; Abate-Daga, Daniel; Beard, Rachel E; Xu, Hui; Black, Mary A; Robbins, Paul F; Schrump, David A; Rosenberg, Steven A; Morgan, Richard A

2014-04-01

Cancer testis antigens, such as NY-ESO-1, are expressed in a variety of prevalent tumors and represent potential targets for T-cell receptor (TCR) gene therapy. DNA encoding a murine anti-NY-ESO-1 TCR gene (mTCR) was isolated from immunized HLA-A*0201 transgenic mice and inserted into a γ-retroviral vector. Two mTCR vectors were produced and used to transduce human PBL. Transduced cells were cocultured with tumor target cell lines and T2 cells pulsed with the NY-ESO-1 peptide, and assayed for cytokine release and cell lysis activity. The most active TCR construct was selected for production of a master cell bank for clinical use. mTCR-transduced PBL maintained TCR expression in short-term and long-term culture, ranging from 50% to 90% efficiency 7-11 days after stimulation and 46%-82% 10-20 days after restimulation. High levels of interferon-γ secretion were observed (1000-12000 pg/mL), in tumor coculture assays and recognition of peptide-pulsed cells was observed at 0.1 ng/mL, suggesting that the new mTCR had high avidity for antigen recognition. mTCR-transduced T cells also specifically lysed human tumor targets. In all assays, the mTCR was equivalent or better than the comparable human TCR. As the functional activity of TCR-transduced cells may be affected by the formation of mixed dimers, mTCRs, which are less likely to form mixed dimers with endogenous hTCRs, may be more effective in vivo. This new mTCR targeted to NY-ESO-1 represents a novel potential therapeutic option for adoptive cell-transfer therapy for a variety of malignancies.

Immunologic targeting of FOXP3 in inflammatory breast cancer cells.

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Smita Nair

Full Text Available The forkhead transcription factor FOXP3 is necessary for induction of regulatory T lymphocytes (Tregs and their immunosuppressive function. We have previously demonstrated that targeting Tregs by vaccination of mice with murine FOXP3 mRNA-transfected dendritic cells (DCs elicits FOXP3-specific T cell responses and enhances tumor immunity. It is clear that FOXP3 expression is not restricted to T-cell lineage and herein, using RT-PCR, flow cytometry, and western immunoblot we demonstrate for the first time that FOXP3 is expressed in inflammatory breast cancer (IBC cells, SUM149 (triple negative, ErbB1-activated and SUM190 (ErbB2-overexpressing. Importantly, FOXP3-specific T cells generated in vitro using human FOXP3 RNA-transfected DCs as stimulators efficiently lyse SUM149 cells. Interestingly, an isogenic model (rSUM149 derived from SUM149 with an enhanced anti-apoptotic phenotype was resistant to FOXP3-specific T cell mediated lysis. The MHC class I cellular processing mechanism was intact in both cell lines at the protein and transcription levels suggesting that the resistance to cytolysis by rSUM149 cells was not related to MHC class I expression or to the MHC class I antigen processing machinery in these cells. Our data suggest that FOXP3 may be an effective tumor target in IBC cells however increased anti-apoptotic signaling can lead to immune evasion.

Concerning the role of cell lysis-cryptic growth in anaerobic side-stream reactors: the single-cell analysis of viable, dead and lysed bacteria.

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Foladori, P; Velho, V F; Costa, R H R; Bruni, L; Quaranta, A; Andreottola, G

2015-05-01

In the Anaerobic Side-Stream Reactor (ASSR), part of the return sludge undergoes alternating aerobic and anaerobic conditions with the aim of reducing sludge production. In this paper, viability, enzymatic activity, death and lysis of bacterial cells exposed to aerobic and anaerobic conditions for 16 d were investigated at single-cell level by flow cytometry, with the objective of contributing to the understanding of the mechanisms of sludge reduction in the ASSR systems. Results indicated that total and viable bacteria did not decrease during the anaerobic phase, indicating that anaerobiosis at ambient temperature does not produce a significant cell lysis. Bacteria decay and lysis occurred principally under aerobic conditions. The aerobic decay rate of total bacteria (bTB) was considered as the rate of generation of lysed bacteria. Values of bTB of 0.07-0.11 d(-1) were measured in anaerobic + aerobic sequence. The enzymatic activity was not particularly affected by the transition from anaerobiosis to aerobiosis. Large solubilisation of COD and NH4(+) was observed only under anaerobic conditions, as a consequence of hydrolysis of organic matter, but not due to cell lysis. The observations supported the proposal of two independent mechanisms contributing equally to sludge reduction: (1) under anaerobic conditions: sludge hydrolysis of non-bacterial material, (2) under aerobic conditions: bacterial cell lysis and oxidation of released biodegradable compounds. Copyright © 2015 Elsevier Ltd. All rights reserved.

Genetic Engineering of T Cells to Target HERV-K, an Ancient Retrovirus on Melanoma.

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Krishnamurthy, Janani; Rabinovich, Brian A; Mi, Tiejuan; Switzer, Kirsten C; Olivares, Simon; Maiti, Sourindra N; Plummer, Joshua B; Singh, Harjeet; Kumaresan, Pappanaicken R; Huls, Helen M; Wang-Johanning, Feng; Cooper, Laurence J N

2015-07-15

The human endogenous retrovirus (HERV-K) envelope (env) protein is a tumor-associated antigen (TAA) expressed on melanoma but not normal cells. This study was designed to engineer a chimeric antigen receptor (CAR) on T-cell surface, such that they target tumors in advanced stages of melanoma. Expression of HERV-K protein was analyzed in 220 melanoma samples (with various stages of disease) and 139 normal organ donor tissues using immunohistochemical (IHC) analysis. HERV-K env-specific CAR derived from mouse monoclonal antibody was introduced into T cells using the transposon-based Sleeping Beauty (SB) system. HERV-K env-specific CAR(+) T cells were expanded ex vivo on activating and propagating cells (AaPC) and characterized for CAR expression and specificity. This includes evaluating the HERV-K-specific CAR(+) T cells for their ability to kill A375-SM metastasized tumors in a mouse xenograft model. We detected HERV-K env protein on melanoma but not in normal tissues. After electroporation of T cells and selection on HERV-K(+) AaPC, more than 95% of genetically modified T cells expressed the CAR with an effector memory phenotype and lysed HERV-K env(+) tumor targets in an antigen-specific manner. Even though there is apparent shedding of this TAA from tumor cells that can be recognized by HERV-K env-specific CAR(+) T cells, we observed a significant antitumor effect. Adoptive cellular immunotherapy with HERV-K env-specific CAR(+) T cells represents a clinically appealing treatment strategy for advanced-stage melanoma and provides an approach for targeting this TAA on other solid tumors. ©2015 American Association for Cancer Research.

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

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Bendinelli Mauro

2009-03-01

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

Defining the Role of BTLA in Breast Cancer Immunosurveillance and Selective Targeting of the BTLA-HVEM-LIGHT Costimulatory System

Science.gov (United States)

2010-05-01

assay was conducted as reported by Jedema et al. (41), with alterations noted below. Briefly, allogeneic P815 (H-2Kd) or syngeneic EL4 (H-2Kb) target...cells lysed (CFSE7- AAD) was determined by FACS. CTL activity was not observed toward syngeneic (H-2Kb) EL4 cell targets (data not shown). The mean...could induce BTLA phosphor- ylation (Fig. 4c,d). We analyzed BTLA phosphorylation in EL4 cells using immunoprecipitation immunoblot analysis as

Lysis of typhus-group rickettsia-infected targets by lymphokine activated killers

International Nuclear Information System (INIS)

Carl, M.; Dasch, G.A.

1986-01-01

The authors recently described a subset of OKT8, OKT3-positive lymphocytes from typhus-group rickettsia immune individuals which were capable of lysing autologous PHA-blasts or Epstein-Barr virus transformed B cells (LCL) infected with typhus-group rickettsiae. In order to determine if killing by these effectors was HLA-restricted, they stimulated peripheral blood mononuclear cells (PBMC) from typhus-group rickettsia immune individuals in vitro with typhus-group rickettsia-derived antigen for one week and then measured lysis of autologous LCL or HLA-mismatched LCL in a 4-6 hour Cr 51 -release assay. There was significant lysis of both the autologous and the HLA-mismatched infected targets as compared to the corresponding uninfected targets. Since this suggested that the effectors were lymphokine activated killers (LAK) rather than cytotoxic T lymphocytes, they then tested this hypothesis by stimulating PBMC from both immune and non-immune individuals in vitro for one week with purified interleukin 2 and measuring lysis of infected, autologous LCL. PBMC thus treated, from both immune and non-immune individuals, were capable of significantly lysing autologous, infected LCL as compared to the non-infected control. They therefore conclude that targets infected with typhus-group rickettsiae are susceptible to lysis to LAK

In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

International Nuclear Information System (INIS)

Rowe, Alexander M.; Brundage, Kathleen M.; Barnett, John B.

2007-01-01

The herbicide atrazine is a known immunotoxicant and an inhibitor of human natural killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell-mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24-h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesized that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell-target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4-h incubation with K562 target cells. The relative level of intracellular lytic protein was 25-50% higher, and the amount of lytic protein released was 55-65% less in atrazine-treated cells than vehicle-treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells

CAR-T cells targeting CLL-1 as an approach to treat acute myeloid leukemia.

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Wang, Jinghua; Chen, Siyu; Xiao, Wei; Li, Wende; Wang, Liang; Yang, Shuo; Wang, Weida; Xu, Liping; Liao, Shuangye; Liu, Wenjian; Wang, Yang; Liu, Nawei; Zhang, Jianeng; Xia, Xiaojun; Kang, Tiebang; Chen, Gong; Cai, Xiuyu; Yang, Han; Zhang, Xing; Lu, Yue; Zhou, Penghui

2018-01-10

Acute myeloid leukemia (AML) is one of the most common types of adult acute leukemia. Standard chemotherapies can induce complete remission in selected patients; however, a majority of patients eventually relapse and succumb to the disease. Thus, the development of novel therapeutics for AML is urgently needed. Human C-type lectin-like molecule-1 (CLL-1) is a type II transmembrane glycoprotein, and its expression is restricted to myeloid cells and the majority of AML blasts. Moreover, CLL-1 is expressed in leukemia stem cells (LSCs), but absent in hematopoietic stem cells (HSCs), which may provide a potential therapeutic target for AML treatment. We tested the expression of CLL-1 antigen on peripheral blood cells and bone marrow cells in healthy donor and AML patients. Then, we developed a chimeric antigen receptor (CAR) containing a CLL1-specific single-chain variable fragment, in combination with CD28, 4-1BB costimulatory domains, and CD3-ζ signaling domain. We further investigate the function of CLL-1 CAR-T cells. The CLL-1 CAR-T cells specifically lysed CLL-1 + cell lines as well as primary AML patient samples in vitro. Strong anti-leukemic activity was observed in vivo by using a xenograft model of disseminated AML. Importantly, CLL-1 + myeloid progenitor cells and mature myeloid cells were specifically eliminated by CLL-1 CAR-T cells, while normal HSCs were not targeted due to the lack of CLL-1 expression. CLL-1 CAR-T represents a promising immunotherapy for the treatment of AML.

A cell-penetrating peptide analogue, P7, exerts antimicrobial activity against Escherichia coli ATCC25922 via penetrating cell membrane and targeting intracellular DNA.

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Li, Lirong; Shi, Yonghui; Cheng, Xiangrong; Xia, Shufang; Cheserek, Maureen Jepkorir; Le, Guowei

2015-01-01

The antibacterial activities and mechanism of a new P7 were investigated in this study. P7 showed antimicrobial activities against five harmful microorganisms which contaminate and spoil food (MIC=4-32 μM). Flow cytometry and scanning electron microscopy analyses demonstrated that P7 induced pore-formation on the cell surface and led to morphological changes but did not lyse cell. Confocal fluorescence microscopic observations and flow cytometry analysis expressed that P7 could penetrate the Escherichia coli cell membrane and accumulate in the cytoplasm. Moreover, P7 possessed a strong DNA binding affinity. Further cell cycle analysis and change in gene expression analysis suggested that P7 induced a decreased expression in the genes involved in DNA replication. Up-regulated expression genes encoding DNA damage repair. This study suggests that P7 could be applied as a candidate for the development of new food preservatives as it exerts its antibacterial activities by penetrating cell membranes and targets intracellular DNA. Copyright © 2014 Elsevier Ltd. All rights reserved.

Aliphatic long chain quaternary ammonium compounds analysis by ion-pair chromatography coupled with suppressed conductivity and UV detection in lysing reagents for blood cell analysers.

Science.gov (United States)

Giovannelli, D; Abballe, F

2005-08-26

A method has been developed which allows simultaneous determination of three linear alkyl trimethylammonium salts. Dodecyltrimethylammonium chloride, tetradecyltrimethylammonium bromide and hexadecyltrimethylammonium chloride are widely used as main active ingredients of lysing reagents for blood cell analyzers which perform white blood cells differential determination into two or more sub-populations by impedance analysis. The ion-pair on styrene-divinyl benzene chromatographic phase looks like a suitable, reliable and long term stable tool for separation of such quaternary compounds. The detection based on suppressed conductivity was chosen because of the lack of significance chromophores. A micromembrane suppressor device compatible with high solvent concentration (up to 80%) was used in order to minimize the conductivity background before the detection. In the present work we show how the chemical post column derivatization makes the alkyl chain detectable also by UV direct detection at 210 nm.

Development of Augmented Leukemia/Lymphoma-Specific T-Cell Immunotherapy for Deployment with Haploidentical, Hematompoietic Progenitor-Cell Transplant

Science.gov (United States)

2011-05-01

cells exhibited redirected specific lysis of a genetically modified EL4 target expressing 92% human CD19 (Figure 6a). T cells not genetically modified...but propagated by crosslinking CD3 using g-irradiated aAPC loaded with OKT3 did not appreciably lyse CD19 EL4 cells (Figure 6b). DISCUSSION We and...by genetically modified T cells. Lysis by chromium release assay of CD19+ EL4 tumor cells compared with CD19 parental EL4 cells by (a) T cells

NYESO-1/LAGE-1s and PRAME are targets for antigen specific T cells in chondrosarcoma following treatment with 5-Aza-2-deoxycitabine.

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Seth M Pollack

Full Text Available Chondrosarcoma has no proven systemic option in the metastatic setting. The development of a non-cross-resistant strategy, such as cellular immunotherapy using antigen-specific T cells would be highly desirable. NY-ESO-1 and PRAME are members of the Cancer Testis Antigen (CTA family that have been identified as promising targets for T cell therapy. LAGE-1 is a cancer testis antigen 90% homologous to NY-ESO-1, sharing the 157-165 A*0201 NY-ESO-1 epitope with its transcript variant, LAGE-1s. A number of CTA's have been induced using 5-Aza-2-Deoxycitabine (5-Aza-dC in other cancers. We sought to evaluate the feasibility of targeting chondrosarcoma tumors using NY-ESO-1/LAGE-1s and PRAME specific T cells using 5-Aza-dC to induce antigen expression.We used 11 flash frozen tumors from the University of Washington tumor bank to test for the expression of NY-ESO-1, PRAME, LAGE-1s and LAGE-1L in chondrosarcoma tumors. Using four chondrosarcoma cell lines we tested the expression of these CTA's with and without 5-Aza-dC treatments. Finally, using NY-ESO-1/LAGE-1s and PRAME specific effectors that we generated from sarcoma patients, we evaluated the ability of these T cells to lyse A*0201 expressing chondrosarcoma cell lines in vitro both with and without 5-Aza-dC treatment.A minority (36% of chondrosarcoma tumors expressed either NY-ESO-1 or LAGE-1s at >10% of our reference value and none expressed PRAME at that level. However, in all four of the chondrosarcoma cell lines tested, NY-ESO-1 and PRAME expression could be induced following treatment with 5-Aza-dC including in cell lines where expression was absent or barely detectable. Furthermore, NY-ESO-1/LAGE-1s and PRAME specific CD8+ effector T cells were able to specifically recognize and lyse A*0201 expressing chondrosarcoma cell lines following 5-Aza-dC treatment.These data suggest that adoptive immunotherapy in combination with 5-Aza-dC may be a potential strategy to treat unresectable or metastatic

Targeting Alpha-Fetoprotein (AFP)-MHC Complex with CAR T-Cell Therapy for Liver Cancer.

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Liu, Hong; Xu, Yiyang; Xiang, Jingyi; Long, Li; Green, Shon; Yang, Zhiyuan; Zimdahl, Bryan; Lu, Jingwei; Cheng, Neal; Horan, Lucas H; Liu, Bin; Yan, Su; Wang, Pei; Diaz, Juan; Jin, Lu; Nakano, Yoko; Morales, Javier F; Zhang, Pengbo; Liu, Lian-Xing; Staley, Binnaz K; Priceman, Saul J; Brown, Christine E; Forman, Stephen J; Chan, Vivien W; Liu, Cheng

2017-01-15

The majority of tumor-specific antigens are intracellular and/or secreted and therefore inaccessible by conventional chimeric antigen receptor (CAR) T-cell therapy. Given that all intracellular/secreted proteins are processed into peptides and presented by class I MHC on the surface of tumor cells, we used alpha-fetoprotein (AFP), a specific liver cancer marker, as an example to determine whether peptide-MHC complexes can be targets for CAR T-cell therapy against solid tumors. We generated a fully human chimeric antigen receptor, ET1402L1-CAR (AFP-CAR), with exquisite selectivity and specificity for the AFP 158-166 peptide complexed with human leukocyte antigen (HLA)-A*02:01. We report that T cells expressing AFP-CAR selectively degranulated, released cytokines, and lysed liver cancer cells that were HLA-A*02:01 + /AFP + while sparing cells from multiple tissue types that were negative for either expressed proteins. In vivo, intratumoral injection of AFP-CAR T cells significantly regressed both Hep G2 and AFP 158 -expressing SK-HEP-1 tumors in SCID-Beige mice (n = 8 for each). Moreover, intravenous administration of AFP-CAR T cells in Hep G2 tumor-bearing NSG mice lead to rapid and profound tumor growth inhibition (n = 6). Finally, in an established intraperitoneal liver cancer xenograft model, AFP-CAR T cells showed robust antitumor activity (n = 6). This study demonstrates that CAR T-cell immunotherapy targeting intracellular/secreted solid tumor antigens can elicit a potent antitumor response. Our approach expands the spectrum of antigens available for redirected T-cell therapy against solid malignancies and offers a promising new avenue for liver cancer immunotherapy. Clin Cancer Res; 23(2); 478-88. ©2016 AACR. ©2016 American Association for Cancer Research.

Wind characterization for design and comparison with standards, an example from Lyse at the Swedish west coast

Energy Technology Data Exchange (ETDEWEB)

Ganander, H. [Teknikgruppen AB, Sollentuna (Sweden); Carlen, I. [Chalmers Univ. of Technology, Goeteborg (Sweden). Div. of Building Technology; Bergstroem, H. [Uppsala Univ. (Sweden). Dept. of Meteorology

1996-12-01

The Lyse site at the Swedish west coast is an area with an archipelago of rocky islands to the west and an equally rocky mainland to the east. In between there are some open sea areas. As being the responsible project manager for the erection and the operation of a turbine at a site like Lyse, the question arises about characterization of the wind for design or purchase of a wind turbine. Or in other words what wind turbine class has to be used for the design, according to existing standards like for example IEC-1400 ? 3 refs, 10 figs

Bacteriophage enzymes for the prevention and treatment of bacterial infections: Stability and stabilization of the enzyme lysing Streptococcus pyogenes cells

Energy Technology Data Exchange (ETDEWEB)

Klyachko, N. L.; Dmitrieva, N. F.; Eshchina, A. S.; Ignatenko, O. V.; Filatova, L. Y.; Rainina, Evguenia I.; Kazarov, A. K.; Levashov, A. V.

2008-06-01

Recombinant, phage associated lytic enzyme Ply C capable to lyse streptococci of groups A and C was stabilized in the variety of the micelles containing compositions to improve the stability of the enzyme for further application in medicine. It was shown that, in the micellar polyelectrolyte composition M16, the enzyme retained its activity for 2 months; while in a buffer solution under the same conditions ((pH 6.3, room temperature), it completely lost its activity in 2 days

Protection efficacy of the Brucella abortus ghost vaccine candidate lysed by the N-terminal 24-amino acid fragment (GI24) of the 36-amino acid peptide PMAP-36 (porcine myeloid antimicrobial peptide 36) in murine models.

Science.gov (United States)

Kwon, Ae Jeong; Moon, Ja Young; Kim, Won Kyong; Kim, Suk; Hur, Jin

2016-11-01

Brucella abortus cells were lysed by the N-terminal 24-amino acid fragment (GI24) of the 36-amino acid peptide PMAP-36 (porcine myeloid antimicrobial peptide 36). Next, the protection efficacy of the lysed fragment as a vaccine candidate was evaluated. Group A mice were immunized with sterile PBS, group B mice were intraperitoneally (ip) immunized with 3 × 10 8 colony-forming units (CFUs) of B. abortus strain RB51, group C mice were immunized ip with 3 × 10 8 cells of the B. abortus vaccine candidate, and group D mice were orally immunized with 3 × 10 9 cells of the B. abortus vaccine candidate. Brucella lipopolysaccharide (LPS)-specific serum IgG titers were considerably higher in groups C and D than in group A. The levels of interleukin (IL)-4, IL-10, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) were significantly higher in groups B-D than in group A. After an ip challenge with B. abortus 544, only group C mice showed a significant level of protection as compared to group A. Overall, these results show that ip immunization with a vaccine candidate lysed by GI24 can effectively protect mice from systemic infection with virulent B. abortus.

Rigid Polyurethane Foam from Glyco lysed Polyethylene Terephthalate Dissolved in Palm-based Polyol

International Nuclear Information System (INIS)

Khairiah Badri; Lily Iliyana Mohd Dawi; Nur Ashikin Abdul Aziz

2013-01-01

An investigation on the thermal and mechanical properties of rigid polyurethane (PU) foam from polyethylene terephthalate (PET) waste (of plastic drinking bottles) was conducted. The PET waste was glyco lysed with ethylene glycol prior to blending with palm based-polyol (PKO-p). This blend was then reacted with 2, 4-methylene diphenyl diisocyanate (MDI) at a ratio of 1:1 to form the PU foam. The incorporation of the glyco lysed PET (g-PET) into the PKO-p was studied at 50, 70 and 100 % w/ w loading. PU foam prepared from 100 % w/ w g-PET (without PKO-p) resulted in PU with high glass transition temperature and mechanical strength. This water-blown foam has molded and core densities of 182 kg m -3 and 179 kg m -3 , respectively, with maximum compressive stress and modulus at 396 kPa and 1920 kPa, respectively. An initial enthalpy value of 3164.8 cal g -1 and a glass transition temperature of 65 degree Celsius were observed. (author)

Chemovirotherapy for head and neck squamous cell carcinoma with EGFR-targeted and CD/UPRT-armed oncolytic measles virus.

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Zaoui, K; Bossow, S; Grossardt, C; Leber, M F; Springfeld, C; Plinkert, P K; Kalle, C von; Ungerechts, G

2012-03-01

First-line treatment of recurrent and/or refractory head and neck squamous cell carcinoma (HNSCC) is based on platinum, 5-fluorouracil (5-FU) and the monoclonal antiEGFR antibody cetuximab. However, in most cases this chemoimmunotherapy does not cure the disease, and more than 50% of HNSCC patients are dying because of local recurrence of the tumors. In the majority of cases, HNSCC overexpress the epidermal growth factor receptor (EGFR), and its presence is associated with a poor outcome. In this study, we engineered an EGFR-targeted oncolytic measles virus (MV), armed with the bifunctional enzyme cytosine deaminase/uracil phosphoribosyltransferase (CD/UPRT). CD/UPRT converts 5-fluorocytosine (5-FC) into the chemotherapeutic 5-FU, a mainstay of HNSCC chemotherapy. This virus efficiently replicates in and lyses primary HNSCC cells in vitro. Arming with CD/UPRT mediates efficient prodrug activation with high bystander killing of non-infected tumor cells. In mice bearing primary HNSCC xenografts, intratumoral administration of MV-antiEGFR resulted in statistically significant tumor growth delay and prolongation of survival. Importantly, combination with 5-FC is superior to virus-only treatment leading to significant tumor growth inhibition. Thus, chemovirotherapy with EGFR-targeted and CD/UPRT-armed MV is highly efficacious in preclinical settings with direct translational implications for a planned Phase I clinical trial of MV for locoregional treatment of HNSCC.

Upregulation of adhesion molecules on leukemia targets improves the efficacy of cytotoxic T cells transduced with chimeric anti-CD19 receptor.

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Laurin, David; Marin, Virna; Biagi, Ettore; Pizzitola, Irene; Agostoni, Valentina; Gallot, Géraldine; Vié, Henri; Jacob, Marie Christine; Chaperot, Laurence; Aspord, Caroline; Plumas, Joël

2013-04-01

T lymphocytes engineered to express chimeric antigen receptors (CARs) interact directly with cell surface molecules, bypassing MHC antigen presentation dependence. We generated human anti-CD19ζ CAR cytotoxic T lymphocytes and cytokine-induced killer cells and studied their sensitivity to the expression of adhesion molecules for the killing of primary B-lineage acute lymphoblastic leukemia (B-ALL) targets. Despite a very low basal expression of surface adhesion molecules, B-ALL blasts were lysed by the anti-CD19ζ-CAR transduced effectors as expected. We next investigated the regulatory role of adhesion molecules during CAR-mediated cytolysis. The blocking of these accessory molecules strongly limited the chimeric effector's cytotoxicity. Thereafter, B-ALL cells surface adhesion molecule level expression was induced by IFN-γ or by the combined use of CD40L and IL-4 and the cells were submitted to anti-CD19ζ-CAR transduced effectors lysis. Upregulation of adhesion molecules expression by blasts potentiated their killing. The improved cytotoxicity observed was dependent on target surface expression of adhesion molecules, particularly CD54. Taken together, these results indicate that adhesion molecules, and principally CD54, are involved in the efficiency of recognition by effector chimeric ζ. These observations have potential implications for the design of immunotherapy treatment approaches for hematological malignancies and tumors based on the adoption of CAR effector cells.

Gamma-H2AX biodosimetry for use in large scale radiation incidents: comparison of a rapid ‘96 well lyse/fix’ protocol with a routine method

Directory of Open Access Journals (Sweden)

Jayne Moquet

2014-03-01

Full Text Available Following a radiation incident, preliminary dose estimates made by γ-H2AX foci analysis can supplement the early triage of casualties based on clinical symptoms. Sample processing time is important when many individuals need to be rapidly assessed. A protocol was therefore developed for high sample throughput that requires less than 0.1 ml blood, thus potentially enabling finger prick sampling. The technique combines red blood cell lysis and leukocyte fixation in one step on a 96 well plate, in contrast to the routine protocol, where lymphocytes in larger blood volumes are typically separated by Ficoll density gradient centrifugation with subsequent washing and fixation steps. The rapid ‘96 well lyse/fix’ method reduced the estimated sample processing time for 96 samples to about 4 h compared to 15 h using the routine protocol. However, scoring 20 cells in 96 samples prepared by the rapid protocol took longer than for the routine method (3.1 versus 1.5 h at zero dose; 7.0 versus 6.1 h for irradiated samples. Similar foci yields were scored for both protocols and consistent dose estimates were obtained for samples exposed to 0, 0.2, 0.6, 1.1, 1.2, 2.1 and 4.3 Gy of 250 kVp X-rays at 0.5 Gy/min and incubated for 2 h. Linear regression coefficients were 0.87 ± 0.06 (R2 = 97.6% and 0.85 ± 0.05 (R2 = 98.3% for estimated versus actual doses for the routine and lyse/fix method, respectively. The lyse/fix protocol can therefore facilitate high throughput processing for γ-H2AX biodosimetry for use in large scale radiation incidents, at the cost of somewhat longer foci scoring times.

Suppression of in vitro cell-mediated lympholysis generation by alloactivated lymphocytes. Examination of radioresistant suppressive activity

International Nuclear Information System (INIS)

Orosz, C.G.; Ferguson, R.M.

1986-01-01

We investigated the radioresistant (1000 rads) suppression of CML generation mediated by alloactivated murine splenocytes. Suppressive cells were generated in MLCs by stimulation of (A X 6R)F1 splenocytes with irradiated C57BL/10 splenocytes. Suppressive cells could lyse targets bearing H-2b alloantigens, but would not lyse parental B10.T(6R) or B10.A targets. Suppressive activity was detected by including the alloactivated (A X 6R)F1 cells in B10.T(6R) anti-B10.A(1R) MLCs. Relative to the suppressive (A X 6R)F1 cells, the B10.A(1R) lymphocytes display both parental and suppressor-inducing alloantigens. In the absence of a suppressive population, B10.A(1R) stimulators cause B10.T(6R) splenocytes to generate cytolytic activity specific for both H-2Db (suppressor-inducing) and H-2Kk (suppressor-borne) target determinants. The irradiated, alloactivated (A X 6R)F1 cells decrease the H-2Db-specific CML generated in this system, thus mediating apparent antigen-specific suppression. However, cytolytic activity concomitantly generated in the same culture against the unrelated H-2Kk target determinants is similarly reduced by the (A X 6R)F1 cells. Thus, radioresistant suppression by alloactivated splenocytes is not necessarily antigen-specific. The irradiated (A X 6R)F1 cells would not suppress the generation of H-2Kk-specific CTL in B10.T(6R) anti-B10.A MLCs. Hence, the irradiated (A X 6R)F1 cells can impede CML generation against third-party alloantigens if, and only if, those alloantigens are coexpressed with suppressor-inducing alloantigens on the stimulator cells in suppressed MLCs. Similar results were also obtained using a different histoincompatible lymphocyte combination

CCL22-specific T Cells

DEFF Research Database (Denmark)

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

2016-01-01

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

Microfluidic device for acoustic cell lysis

Science.gov (United States)

Branch, Darren W.; Cooley, Erika Jane; Smith, Gennifer Tanabe; James, Conrad D.; McClain, Jaime L.

2015-08-04

A microfluidic acoustic-based cell lysing device that can be integrated with on-chip nucleic acid extraction. Using a bulk acoustic wave (BAW) transducer array, acoustic waves can be coupled into microfluidic cartridges resulting in the lysis of cells contained therein by localized acoustic pressure. Cellular materials can then be extracted from the lysed cells. For example, nucleic acids can be extracted from the lysate using silica-based sol-gel filled microchannels, nucleic acid binding magnetic beads, or Nafion-coated electrodes. Integration of cell lysis and nucleic acid extraction on-chip enables a small, portable system that allows for rapid analysis in the field.

Target cells in internal dosimetry

Energy Technology Data Exchange (ETDEWEB)

Goessner, W

2003-07-01

Data related to radium induced bone sarcomas in humans are used as a model for defining target cells on bone surfaces and in the bone marrow. The differential distribution of radiation induced bone sarcoma types with a high ratio of non-bone producing, mainly fibroblastic tumours, challenges the ICRP concept that the bone lining cells are target cells. Multipotential mesenchymal stem cells are located within the range of alpha particles, and are the most likely target cells for the fibroblastic type of bone sarcoma. The histogenesis of bone sarcomas after irradiation with alpha emitters shows that their final histopathology is not dependent on a single target cell. Each target cell has a microenvironment, which has to be regarded as a synergistic morpho-functional tissue unit. For this the concept of 'histion', a term used in general pathology, is proposed. Interactions between target cells that have been hit by alpha-particles, leading to lethal, mutational or transformation events with all components of a 'histion', will prove critical to understanding the pathogenesis of both deterministic and stochastic late effects. (author)

Target cells in internal dosimetry

International Nuclear Information System (INIS)

Goessner, W.

2003-01-01

Data related to radium induced bone sarcomas in humans are used as a model for defining target cells on bone surfaces and in the bone marrow. The differential distribution of radiation induced bone sarcoma types with a high ratio of non-bone producing, mainly fibroblastic tumours, challenges the ICRP concept that the bone lining cells are target cells. Multipotential mesenchymal stem cells are located within the range of alpha particles, and are the most likely target cells for the fibroblastic type of bone sarcoma. The histogenesis of bone sarcomas after irradiation with alpha emitters shows that their final histopathology is not dependent on a single target cell. Each target cell has a microenvironment, which has to be regarded as a synergistic morpho-functional tissue unit. For this the concept of 'histion', a term used in general pathology, is proposed. Interactions between target cells that have been hit by alpha-particles, leading to lethal, mutational or transformation events with all components of a 'histion', will prove critical to understanding the pathogenesis of both deterministic and stochastic late effects. (author)

Regulation of Ribulose-1,5-Bisphosphate Carboxylase Activity by the Activase System in Lysed Spinach Chloroplasts

Science.gov (United States)

Parry, Martin A. J.; Keys, Alfred J.; Foyer, Christine H.; Furbank, Robert T.; Walker, David A.

1988-01-01

Ribulose-1,5-bisphosphate (RuBP) carboxylase in lysed spinach (Spinacia oleracea L. cv virtuosa) chloroplasts that had been partly inactivated at low CO2 and Mg2+ by incubating in darkness with 4 millimolar partially purified RuBP was reactivated by light. If purified RuBP was used to inhibit dark activation of the enzyme, reactivation by light was not observed unless fructose-1,6-bisphosphate, ATP, or ADP plus inorganic phosphate were also added. Presumably, ADP plus inorganic phosphate acted as an ATP-generating system with a requirement for the generation of ΔpH across the thylakoid membrane. When the RuBP obtained from Sigma Chemical Co. was used, light did not reactivate the enzyme. There was no direct correlation between ΔpH and activation. Therefore, thylakoids are required in the ribulose-1,5-bisphosphate carboxylase activase system largely to synthesize ATP. Inactivation of RuBP carboxylase in isolated chloroplasts or in the lysed chloroplast system was not promoted simply by a transition from light to dark conditions but was caused by low CO2 and Mg2+. PMID:16666184

UV-C irradiation of HSV-1 infected fibroblasts (HSV-FS) enhances human natural killer (NK) cell activity against these targets

International Nuclear Information System (INIS)

Pettera, L.; Fitzgerald-Bocarsly, P.

1991-01-01

Expression of Herpes Simplex Virus Type 1 (HSV-1) immediate early gene products has been bound to be sufficient for NK cell mediated lysis of HSV-1 infected FS. To block the targets at various stages in the infectious cycle, HSV-FS were irradiated with UV light for 1 min at 2, 6, and 20 hr post-infection. NK mediated lysis of 2 hr and 5 hr UV treated HSV-FS was 2-fold higher than non-UV treated HSV-FS despite a >99% inhibition in virus yield. In contrast, 20 hr infected targets were lysed less well than 2 and 6 hr targets despite strong glycoprotein expression and induction of high levels of interferon-alpha (IFN-α) production by effector PBMC's; this lysis was not enhanced by UV treatment. Since NK lysis of HSV-FS has been found to be dependent on an HLA-DR + accessory cell (AC), lysis of irradiated HSV-FS by PBMC's depleted of AC was measured. Such depletion eradicated NK lysis of the UV treated HSV-FS indicating that irradiation does not overcome the AC requirement for NK lysis. UV irradiation of another HLA-DR + dependent target, Vesicular Stomatitis Virus (VSV) infected FS led to a dramatic reduction in both NK lysis and IFN-α induction. HSV-1 is a DNA virus whose genes are expressed in a cascade fashion whereas VSV is an RNA virus. The authors hypothesize that the enhancement in AC dependent NK activity observed for UV irradiated HSV-FS, but not VSV-FS, targets is due to overproduction of either a cellular or viral gene product which specifically occurs early in the HSV-1 infectious cycle and is downregulated by 20 hr post-infection

Soft sensor for monitoring biomass subpopulations in mammalian cell culture processes.

Science.gov (United States)

Kroll, Paul; Stelzer, Ines V; Herwig, Christoph

2017-11-01

Biomass subpopulations in mammalian cell culture processes cause impurities and influence productivity, which requires this critical process parameter to be monitored in real-time. For this reason, a novel soft sensor concept for estimating viable, dead and lysed cell concentration was developed, based on the robust and cheap in situ measurements of permittivity and turbidity in combination with a simple model. It could be shown that the turbidity measurements contain information about all investigated biomass subpopulations. The novelty of the developed soft sensor is the real-time estimation of lysed cell concentration, which is directly correlated to process-related impurities such as DNA and host cell protein in the supernatant. Based on data generated by two fed-batch processes the developed soft sensor is described and discussed. The presented soft sensor concept provides a tool for viable, dead and lysed cell concentration estimation in real-time with adequate accuracy and enables further applications with respect to process optimization and control.

Whole-cell hybridization of Methanosarcina cells with two new oligonucleotide probes

DEFF Research Database (Denmark)

Sørensen, A.H.; Torsvik, V.L.; Torsvik, T.

1997-01-01

Two new oligonucleotide probes targeting the 16S rRNA of the methanogenic genus Methanosarcina were developed. The probes have the following sequences (Escherichia coli numbering): probe SARCI551, 5'-GAC CCAATAATCACGATCAC-3', and probe SARCI645, 5'-TCCCGGTTCCAAGTCTGGC-3'. In situ hybridization...... with the fluorescently labelled probes required several modifications of standard procedures. Cells of Methanosarcina mazeii S-6 were found to lyse during the hybridization step if fixed in 3% formaldehyde and stored in 50% ethanol. Lysis was, however, not observed with cells fixed and stored in 1.6% formaldehyde-0.......85% NaCl. Extensive autofluorescence of the cells was found upon hybridization in the presence of 5 mM EDTA, but successful hybridization could be obtained without addition of this compound. The mounting agent Citifluor AF1, often used in conjugation with the fluorochrome fluorescein, was found to wash...

The use of Listeria monocytogenes as a DNA delivery vector for cancer gene therapy.

LENUS (Irish Health Repository)

Tangney, Mark

2012-01-31

Listeria monocytogenes is an intracellular pathogen that lyses the phagosomal vacuole of infected cells, proliferates in the host cell cytoplasm and can actively enter adjacent cells. The pathogen is therefore well suited to exploitation as a vector for the delivery of DNA to target cells as the lifecycle favors cellular targeting with vector amplification and the potential for cell-to-cell spread. We have recently demonstrated DNA transfer by L. monocytogenes in growing tumors in murine models. Our approach exploited an ampicillin sensitive stain of L. monocytogenes which can be lysed through systemic administration of ampicillin to facilitate release of plasmid DNA for expression by infected mammalian cells. Here, we discuss the implications of this technology and the potential for future improvements of the system.

Preparing nuclei from cells in monolayer cultures suitable for counting and for following synchronized cells through the cell cycle.

Science.gov (United States)

Butler, W B

1984-08-15

A procedure is described for preparing nuclei from cells in monolayer culture so that they may be counted using an electronic particle counter. It takes only 10 to 15 min, and consists of swelling the cells in hypotonic buffer and then lysing them with the quaternary ammonium salt, ethylhexadecyldimethylammonium bromide. The cells are completely lysed, yielding a suspension of clean single nuclei which is stable, free of debris, and easily counted. The method was developed for a cell line of epithelial origin (MCF-7), which is often difficult to trypsinize to single cells. It works equally well at all cell densities up to and beyond confluence, and has been used with a variety of cells in culture, including 3T3 cells, bovine macrophages, rat mammary epithelial cells, mouse mammary tumor cell lines, and human fibroblasts. The size of the nuclei produced by this procedure is related to their DNA content, and the method is thus suitable for following cultures of synchronized cells through the cell cycle, and for performing differential counts of cells with substantial differences in DNA content.

Cooperative tumour cell membrane targeted phototherapy

Science.gov (United States)

Kim, Heegon; Lee, Junsung; Oh, Chanhee; Park, Ji-Ho

2017-06-01

The targeted delivery of therapeutics using antibodies or nanomaterials has improved the precision and safety of cancer therapy. However, the paucity and heterogeneity of identified molecular targets within tumours have resulted in poor and uneven distribution of targeted agents, thus compromising treatment outcomes. Here, we construct a cooperative targeting system in which synthetic and biological nanocomponents participate together in the tumour cell membrane-selective localization of synthetic receptor-lipid conjugates (SR-lipids) to amplify the subsequent targeting of therapeutics. The SR-lipids are first delivered selectively to tumour cell membranes in the perivascular region using fusogenic liposomes. By hitchhiking with extracellular vesicles secreted by the cells, the SR-lipids are transferred to neighbouring cells and further spread throughout the tumour tissues where the molecular targets are limited. We show that this tumour cell membrane-targeted delivery of SR-lipids leads to uniform distribution and enhanced phototherapeutic efficacy of the targeted photosensitizer.

Targeting of Mesenchymal Stromal Cells by Cre-Recombinase Transgenes Commonly Used to Target Osteoblast Lineage Cells.

Science.gov (United States)

Zhang, Jingzhu; Link, Daniel C

2016-11-01

The targeting specificity of tissue-specific Cre-recombinase transgenes is a key to interpreting phenotypes associated with their use. The Ocn-Cre and Dmp1-Cre transgenes are widely used to target osteoblasts and osteocytes, respectively. Here, we used high-resolution microscopy of bone sections and flow cytometry to carefully define the targeting specificity of these transgenes. These transgenes were crossed with Cxcl12 gfp mice to identify Cxcl12-abundant reticular (CAR) cells, which are a perivascular mesenchymal stromal population implicated in hematopoietic stem/progenitor cell maintenance. We show that in addition to osteoblasts, Ocn-Cre targets a majority of CAR cells and arteriolar pericytes. Surprisingly, Dmp1-Cre also targets a subset of CAR cells, in which expression of osteoblast-lineage genes is enriched. Finally, we introduce a new tissue-specific Cre-recombinase, Tagln-Cre, which efficiently targets osteoblasts, a majority of CAR cells, and both venous sinusoidal and arteriolar pericytes. These data show that Ocn-Cre and Dmp1-Cre target broader stromal cell populations than previously appreciated and may aid in the design of future studies. Moreover, these data highlight the heterogeneity of mesenchymal stromal cells in the bone marrow and provide tools to interrogate this heterogeneity. © 2016 American Society for Bone and Mineral Research. © 2016 American Society for Bone and Mineral Research.

Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

Science.gov (United States)

Alvarez-Vallina, L; Yañez, R; Blanco, B; Gil, M; Russell, S J

2000-04-01

Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.

Lung Squamous Cell Carcinoma Stem Cells as Immunotherapy Targets

Science.gov (United States)

2017-08-01

AWARD NUMBER: W81XWH-16-1-0260 TITLE: Lung Squamous Cell Carcinoma Stem Cells as Immunotherapy Targets PRINCIPAL INVESTIGATOR: Carla Kim... Cell Carcinoma Stem Cells as Immunotherapy Targets 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-16-1-0260 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...SUPPLEMENTARY NOTES 14. ABSTRACT Lung squamous cell carcinoma (SCC) is the second most common type of lung cancer, and immunotherapy is a promising new

Human lactoferricin derived di-peptides deploying loop structures induce apoptosis specifically in cancer cells through targeting membranous phosphatidylserine.

Science.gov (United States)

Riedl, Sabrina; Leber, Regina; Rinner, Beate; Schaider, Helmut; Lohner, Karl; Zweytick, Dagmar

2015-11-01

Host defense-derived peptides have emerged as a novel strategy for the development of alternative anticancer therapies. In this study we report on characteristic features of human lactoferricin (hLFcin) derivatives which facilitate specific killing of cancer cells of melanoma, glioblastoma and rhabdomyosarcoma compared with non-specific derivatives and the synthetic peptide RW-AH. Changes in amino acid sequence of hLFcin providing 9-11 amino acids stretched derivatives LF11-316, -318 and -322 only yielded low antitumor activity. However, the addition of the repeat (di-peptide) and the retro-repeat (di-retro-peptide) sequences highly improved cancer cell toxicity up to 100% at 20 μM peptide concentration. Compared to the complete parent sequence hLFcin the derivatives showed toxicity on the melanoma cell line A375 increased by 10-fold and on the glioblastoma cell line U-87mg by 2-3-fold. Reduced killing velocity, apoptotic blebbing, activation of caspase 3/7 and formation of apoptotic DNA fragments proved that the active and cancer selective peptides, e.g. R-DIM-P-LF11-322, trigger apoptosis, whereas highly active, though non-selective peptides, such as DIM-LF11-318 and RW-AH seem to kill rapidly via necrosis inducing membrane lyses. Structural studies revealed specific toxicity on cancer cells by peptide derivatives with loop structures, whereas non-specific peptides comprised α-helical structures without loop. Model studies with the cancer membrane mimic phosphatidylserine (PS) gave strong evidence that PS only exposed by cancer cells is an important target for specific hLFcin derivatives. Other negatively charged membrane exposed molecules as sialic acid, heparan and chondroitin sulfate were shown to have minor impact on peptide activity. Copyright © 2015. Published by Elsevier B.V.

Human immune cell targeting of protein nanoparticles - caveospheres

Science.gov (United States)

Glass, Joshua J.; Yuen, Daniel; Rae, James; Johnston, Angus P. R.; Parton, Robert G.; Kent, Stephen J.; de Rose, Robert

2016-04-01

Nanotechnology has the power to transform vaccine and drug delivery through protection of payloads from both metabolism and off-target effects, while facilitating specific delivery of cargo to immune cells. However, evaluation of immune cell nanoparticle targeting is conventionally restricted to monocultured cell line models. We generated human caveolin-1 nanoparticles, termed caveospheres, which were efficiently functionalized with monoclonal antibodies. Using this platform, we investigated CD4+ T cell and CD20+ B cell targeting within physiological mixtures of primary human blood immune cells using flow cytometry, imaging flow cytometry and confocal microscopy. Antibody-functionalization enhanced caveosphere binding to targeted immune cells (6.6 to 43.9-fold) within mixed populations and in the presence of protein-containing fluids. Moreover, targeting caveospheres to CCR5 enabled caveosphere internalization by non-phagocytic CD4+ T cells--an important therapeutic target for HIV treatment. This efficient and flexible system of immune cell-targeted caveosphere nanoparticles holds promise for the development of advanced immunotherapeutics and vaccines.

The biology of NK cells and their receptors affects clinical outcomes after hematopoietic cell transplantation (HCT).

Science.gov (United States)

Foley, Bree; Felices, Martin; Cichocki, Frank; Cooley, Sarah; Verneris, Michael R; Miller, Jeffrey S

2014-03-01

Natural killer (NK) cells were first identified for their capacity to reject bone marrow allografts in lethally irradiated mice without prior sensitization. Subsequently, human NK cells were detected and defined by their non-major histocompatibility complex (MHC)-restricted cytotoxicity toward transformed or virally infected target cells. Karre et al. later proposed 'the missing self hypothesis' to explain the mechanism by which self-tolerant cells could kill targets that had lost self MHC class I. Subsequently, the receptors that recognize MHC class I to mediate tolerance in the host were identified on NK cells. These class I-recognizing receptors contribute to the acquisition of function by a dynamic process known as NK cell education or licensing. In the past, NK cells were assumed to be short lived, but more recently NK cells have been shown to mediate immunologic memory to secondary exposures to cytomegalovirus infection. Because of their ability to lyse tumors with aberrant MHC class I expression and to produce cytokines and chemokines upon activation, NK cells may be primed by many stimuli, including viruses and inflammation, to contribute to a graft-versus-tumor effect. In addition, interactions with other immune cells support the therapeutic potential of NK cells to eradicate tumor and to enhance outcomes after hematopoietic cell transplantation. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Chip based electroanalytical systems for cell analysis

DEFF Research Database (Denmark)

Spegel, C.; Heiskanen, A.; Skjolding, L.H.D.

2008-01-01

' measurements of processes related to living cells, i.e., systems without lysing the cells. The focus is on chip based amperometric and impedimetric cell analysis systems where measurements utilizing solely carbon fiber microelectrodes (CFME) and other nonchip electrode formats, such as CFME for exocytosis...

Targeting Cell Polarity Machinery to Exhaust Breast Cancer Stem Cells

Science.gov (United States)

2017-10-01

AWARD NUMBER: W81XWH-15-1-0644 TITLE: Targeting Cell Polarity Machinery to Exhaust Breast Cancer Stem Cells PRINCIPAL INVESTIGATOR: Chun-Ju...Targeting Cell Polarity Machinery to Exhaust Breast Cancer Stem Cells 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-15-1-0644 5c. PROGRAM ELEMENT...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Cancer stem cells (CSCs), a cell population with acquired perpetuating self-renewal properties which

Cell-specific targeting by heterobivalent ligands.

Science.gov (United States)

Josan, Jatinder S; Handl, Heather L; Sankaranarayanan, Rajesh; Xu, Liping; Lynch, Ronald M; Vagner, Josef; Mash, Eugene A; Hruby, Victor J; Gillies, Robert J

2011-07-20

Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach--to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle(4), dPhe(7)]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20-50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH(2). Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging.

Protective CD8+ T-cell responses to cytomegalovirus driven by rAAV/GFP/IE1 loading of dendritic cells

Directory of Open Access Journals (Sweden)

Dalle-Donne Isabella

2008-10-01

Full Text Available Abstract Background Recent studies demonstrate that recombinant adeno-associated virus (rAAV-based antigen loading of dendritic cells (DCs generates in vitro, significant and rapid cytotoxic T-lymphocyte (CTL responses against viral antigens. Methods We used the rAAV system to induce specific CTLs against CVM antigens for the development of cytomegalovirus HCMV gene therapy. As an extension of the versatility of the rAAV system, we incorporated immediate-early 1 (IE1, expressed in HCMV. Our rAAV vector induced a strong stimulation of CTLs directed against the HCMV antigen IE1. We then investigated the efficiency of the CTLs in killing IE1 targeted cells. Results A significant MHC Class I-restricted, anti-IE1-specific CTL killing was demonstrated against IE1 positive peripheral blood mononuclear cells (PBMC after one, in vitro, stimulation. Conclusion In summary, single PBMC stimulation with rAAV/IE1 pulsed DCs induces strong antigen specific-CTL generation. CTLs were capable to lyse low doses of peptides pulsed into target cells. These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against HCMV antigens.

Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip

Science.gov (United States)

Li, Ren; Zhou, Mingxing; Li, Jine; Wang, Zihua; Zhang, Weikai; Yue, Chunyan; Ma, Yan; Peng, Hailin; Wei, Zewen; Hu, Zhiyuan

2018-03-01

EGFR mutations companion diagnostics have been proved to be crucial for the efficacy of tyrosine kinase inhibitor targeted cancer therapies. To uncover multiple mutations occurred in minority of EGFR-mutated cells, which may be covered by the noises from majority of un-mutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multi-mutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cells were easily identified. By in situ lysing single cells, the cell lysates of EGFR-expressed cells were retrieved without cross-contamination. Benefited from excluding the noise from cells without EGFR expression, the simple and cost-effective Sanger's sequencing, but not the expensive deep sequencing of the whole cell population, was used to discover multi-mutations. We verified the new method with precisely discovering three most important EGFR drug-related mutations from a sample in which EGFR-mutated cells only account for a small percentage of whole cell population. The microfluidic chip is capable of discovering not only the existence of specific EGFR multi-mutations, but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. This microfluidic chip constitutes a promising method to promote simple and cost-effective Sanger's sequencing to be a routine test before performing targeted cancer therapy.[Figure not available: see fulltext.

Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma

DEFF Research Database (Denmark)

Drent, Esther; Groen, Richard W. J.; Noort, Willy A. Noort

2016-01-01

Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody...... sequences to generate second-generation retroviral CD38- chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence......, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite...

EWS/FLI-l peptide-pulsed dendritic cells induces the antitumor immunity in a murine Ewing's sarcoma cell model.

Science.gov (United States)

Peng, Wei; Huang, Xunwu; Yang, Dazhi

2014-08-01

An increasing number of T-cell epitopes derived from various tumor-associated antigens have been reported, and they proved to play significant roles for tumor rejection both in vivo and in vitro. Over 85% of Ewing's sarcoma family of tumors (ESFTs) express tumor-specific chimeric protein EWS/FLI-1, making it an attractive target for therapeutic cytotoxic T-lymphocyte responses. Here, we identified a novel peptide epitope derived from the EWS/FLI-1 protein and demonstrated that effectors induced by the peptide could specifically secrete IFN-γ and lyse the tumor cell line of EWS/FLI-1-positive and HLA-matched cells. In addition, mice treated with dendritic cells pulsed with the EWS/FLI-1 epitope were able to reject a lethal tumor inoculation of the Ewing's sarcoma A673 cells. Therefore, these data provide evidence for the use of the EWS/FLI-l peptide epitope in T cell-based immunotherapeutic concepts against Ewing's sarcoma cell in vitro and in vivo. Copyright © 2014 Elsevier B.V. All rights reserved.

Improving efficacy of cancer immunotherapy by genetic modification of natural killer cells.

Science.gov (United States)

Burga, Rachel A; Nguyen, Tuongvan; Zulovich, Jane; Madonna, Sarah; Ylisastigui, Loyda; Fernandes, Rohan; Yvon, Eric

2016-11-01

Natural killer (NK) cells are members of the innate immune system that recognize target cells via activating and inhibitory signals received through cell receptors. Derived from the lymphoid lineage, NK cells are able to produce cytokines and exert a cytotoxic effect on viral infected and malignant cells. It is their unique ability to lyse target cells rapidly and without prior education that renders NK cells a promising effector cell for adoptive cell therapy. However, both viruses and tumors employ evasion strategies to avoid attack by NK cells, which represent biological challenges that need to be harnessed to fully exploit the cytolytic potential of NK cells. Using genetic modification, the function of NK cells can be enhanced to improve their homing, cytolytic activity, in vivo persistence and safety. Examples include gene modification to express chemokine, high-affinity Fc receptor and chimeric antigen receptors, suicide genes and the forced expression of cytokines such as interleukin (IL)-2 and IL-15. Preclinical studies have clearly demonstrated that such approaches are effective in improving NK-cell function, homing and safety. In this review, we summarize the recent advances in the genetic manipulations of NK cells and their application for cellular immunotherapeutic strategies. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Cell lysis and superoxide dismutase activities of highly radioresistant bacteria

Energy Technology Data Exchange (ETDEWEB)

Yoshinaka, T; Yano, K; Yamaguchi, H [Tokyo Univ. (Japan). Faculty of Agriculture

1976-01-01

The highly radioresistant bacterium, Arthrobacter radiotolerans, has been isolated from the radioactive hot spring of Misasa, and it does not sporulate, it is Gram-positive, and its color is pink to red. This bacterium shows the highest resistance to gamma-ray among Gram-positive resistants, but the lytic enzyme capable of lysing the cells of strong radioresistants and the surface structure of the cells are little known except those about Micrococcus radiodurans. The cells of the M. radiodurans can be lysed by Achramobacter lyticus enzyme, and electron microscopic observation and chemical analysis revealed the mutilayered surface structure of the cells consisting of an inner membrane, a mucopeptide wall layer and a very outer layer. The superoxide dismutase (SOD) activity of aerobic and anaerobic bacteria was studied, and the relatively high SOD activity of the M. radiodurans was found. The SOD function acts against the threat posed by the reactive superoxide radical being generated biologically, photochemically and radiochemically in the presence of molecular oxygen. In this paper, it is reported that the lytic enzyme No.2 obtained from Cytophaga sp., containing N-acetyl-muramyl-L-alanine amidase, peptidase and endopeptidase, and showing broad lytic spectra, was able to lyse the cells of A. radiotolerans and four radioresistant micrococci, and the radioresistant bacteria showedrelatively high SOD activity except M. sp. 248. It is well known that superoxide anions are generated by aerobic irradiation, and are toxic to microbial cells.

Cell lysis and superoxide dismutase activities of highly radioresistant bacteria

International Nuclear Information System (INIS)

Yoshinaka, Taeko; Yano, Keiji; Yamaguchi, Hikoyuki

1976-01-01

The highly radioresistant bacterium, Arthrobacter radiotolerans, has been isolated from the radioactive hot spring of Misasa, and it does not sporulate, it is Gram-positive, and its color is pink to red. This bacterium shows the highest resistance to gamma-ray among Gram-positive resistants, but the lytic enzyme capable of lysing the cells of strong radioresistants and the surface structure of the cells are little known except those about Micrococcus radiodurans. The cells of the M. radiodurans can be lysed by Achramobacter lyticus enzyme, and electron microscopic observation and chemical analysis revealed the mutilayered surface structure of the cells consisting of an inner membrane, a mucopeptide wall layer and a very outer layer. The superoxide dismutase (SOD) activity of aerobic and anaerobic bacteria was studied, and the relatively high SOD activity of the M. radiodurans was found. The SOD function acts against the threat posed by the reactive superoxide radical being generated biologically, photochemically and radiochemically in the presence of molecular oxygen. In this paper, it is reported that the lytic enzyme No.2 obtained from Cytophaga sp., containing N-acetyl-muramyl-L-alanine amidase, peptidase and endopeptidase, and showing broad lytic spectra, was able to lyse the cells of A. radiotolerans and four radioresistant micrococci, and the radioresistant bacteria showed relatively high SOD activity except M. sp. 248. It is well known that superoxide anions are generated by aerobic irradiation, and are toxic to microbial cells. (Kako, I.)

Microfluidic systems and methods for transport and lysis of cells and analysis of cell lysate

Science.gov (United States)

Culbertson, Christopher T [Oak Ridge, TN; Jacobson, Stephen C [Knoxville, TN; McClain, Maxine A [Knoxville, TN; Ramsey, J Michael [Knoxville, TN

2008-09-02

Microfluidic systems and methods are disclosed which are adapted to transport and lyse cellular components of a test sample for analysis. The disclosed microfluidic systems and methods, which employ an electric field to rupture the cell membrane, cause unusually rapid lysis, thereby minimizing continued cellular activity and resulting in greater accuracy of analysis of cell processes.

Radiation responses of stem cells: targeted and non-targeted effects

International Nuclear Information System (INIS)

Kavanagh, J.N.; Waring, E.J.; Prise, K.M.

2015-01-01

Stem cells are fundamental to the development of any tissue or organism via their ability to self-renew, which is aided by their unlimited proliferative capacity and their ability to produce fully differentiated offspring, often from multiple lineages. Stems cells are long lived and have the potential to accumulate mutations, including in response to radiation exposure. It is thought that stem cells have the potential to be induced into a cancer stem cell phenotype and that these may play an important role in resistance to radiotherapy. For radiation-induced carcinogenesis, the role of targeted and non-targeted effects is unclear with tissue or origin being important. Studies of genomic instability and bystander responses have shown consistent effects in haematopoietic models. Several models of radiation have predicted that stem cells play an important role in tumour initiation and that bystander responses could play a role in proliferation and self-renewal. (authors)

Immune Response to Recombinant Adenovirus in Humans: Capsid Components from Viral Input Are Targets for Vector-Specific Cytotoxic T Lymphocytes

Science.gov (United States)

Molinier-Frenkel, Valérie; Gahery-Segard, Hanne; Mehtali, Majid; Le Boulaire, Christophe; Ribault, Sébastien; Boulanger, Pierre; Tursz, Thomas; Guillet, Jean-Gérard; Farace, Françoise

2000-01-01

We previously demonstrated that a single injection of 109 PFU of recombinant adenovirus into patients induces strong vector-specific immune responses (H. Gahéry-Ségard, V. Molinier-Frenkel, C. Le Boulaire, P. Saulnier, P. Opolon, R. Lengagne, E. Gautier, A. Le Cesne, L. Zitvogel, A. Venet, C. Schatz, M. Courtney, T. Le Chevalier, T. Tursz, J.-G. Guillet, and F. Farace, J. Clin. Investig. 100:2218–2226, 1997). In the present study we analyzed the mechanism of vector recognition by cytotoxic T lymphocytes (CTL). CD8+ CTL lines were derived from two patients and maintained in long-term cultures. Target cell infections with E1-deleted and E1-plus E2-deleted adenoviruses, as well as transcription-blocking experiments with actinomycin D, revealed that host T-cell recognition did not require viral gene transcription. Target cells treated with brefeldin A were not lysed, indicating that viral input protein-derived peptides are associated with HLA class I molecules. Using recombinant capsid component-loaded targets, we observed that the three major proteins could be recognized. These results raise the question of the use of multideleted adenoviruses for gene therapy in the quest to diminish antivector CTL responses. PMID:10906225

Human infektionsimmunologi. Basalvidenskabelige opdagelser og kliniske konsekvenser

DEFF Research Database (Denmark)

Kemp, M; Pedersen, B K; Theander, T G

2000-01-01

diseases such as malaria and tuberculosis. The discovery of T-cell responses to protein-free fractions of mycobacteria and Leishmania parasites has lead to the identification of non-protein T-cell antigens. T-cells that express the CD4 receptor on their surface and are capable of lysing infected target...

Trichomonas vaginalis Contact-Dependent Cytolysis of Epithelial Cells

Science.gov (United States)

Lustig, Gila; Ryan, Christopher M.; Secor, W. Evan

2013-01-01

Trichomonas vaginalis is an extracellular protozoan parasite that binds to the epithelium of the human urogenital tract during infection. In this study, we examined the propensities of 26 T. vaginalis strains to bind to and lyse prostate (BPH-1) and ectocervical (Ect1) epithelium and to lyse red blood cells (RBCs). We found that only three of the strains had a statistically significant preference for either BPH-1 (MSA1103) or Ect1 (LA1 and MSA1123). Overall, we observed that levels of adherence are highly variable among strains, with a 12-fold range of adherence on Ect1 cells and a 45-fold range on BPH-1 cells. Cytolysis levels displayed even greater variability, from no detectable cytolysis to 80% or 90% cytolysis of Ect1 and BPH-1, respectively. Levels of adherence and cytolysis correlate for weakly adherent/cytolytic strains, and a threshold of attachment was found to be necessary to trigger cytolysis; however, this threshold can be reached without inducing cytolysis. Furthermore, cytolysis was completely blocked when we prevented attachment of the parasites to host cells while allowing soluble factors complete access. We demonstrate that hemolysis was a rare trait, with only 4 of the 26 strains capable of lysing >20% RBCs with a 1:30 parasite/RBC ratio. Hemolysis also did not correlate with adherence to or cytolysis of either male (BPH-1)- or female (Ect1)-derived epithelial cell lines. Our results reveal that despite a broad range of pathogenic properties among different T. vaginalis strains, all strains show strict contact-dependent cytolysis. PMID:23429535

Leukocyte function-associated antigen-1-dependent lysis of Fas+ (CD95+/Apo-1+) innocent bystanders by antigen-specific CD8+ CTL.

Science.gov (United States)

Kojima, H; Eshima, K; Takayama, H; Sitkovsky, M V

1997-09-15

Exquisite specificity toward Ag-bearing cells (cognate targets) is one of the most important properties of CD8+ CTL-mediated cytotoxicity. Using highly Ag-specific CD8+ CTL lines and clones, which spare noncognate, Ag-free targets, we found that in the presence of Ag-bearing targets the CTL acquire the ability to lyse noncognate target cells (bystanders). It is shown that the unexpectedly rapid and efficient lysis of bystanders by Ag-activated CTL is mediated by a Fas ligand (FasL)/Fas-based mechanism and does not depend on perforin. The CTL lysed Fas-expressing bystanders, but spared the Fas-negative or anti-Fas mAb-resistant bystander cells. Accordingly, the FasL-deficient gld/gld CTL did not kill bystanders, while perforin-deficient CTL did. Unlike anti-Fas mAb-induced cell death, the lysis of bystanders was not only FasL/Fas dependent but also required adhesion molecule LFA-1 on the surface of the activated CTL. Lysis of bystanders is viewed as acceptable "collateral" damage, but the persistent presence of activated CTL could result in immunopathologies involving functional Fas-expressing tissues.

Buoyancy-activated cell sorting using targeted biotinylated albumin microbubbles.

Directory of Open Access Journals (Sweden)

Yu-Ren Liou

Full Text Available Cell analysis often requires the isolation of certain cell types. Various isolation methods have been applied to cell sorting, including fluorescence-activated cell sorting and magnetic-activated cell sorting. However, these conventional approaches involve exerting mechanical forces on the cells, thus risking cell damage. In this study we applied a novel isolation method called buoyancy-activated cell sorting, which involves using biotinylated albumin microbubbles (biotin-MBs conjugated with antibodies (i.e., targeted biotin-MBs. Albumin MBs are widely used as contrast agents in ultrasound imaging due to their good biocompatibility and stability. For conjugating antibodies, biotin is conjugated onto the albumin MB shell via covalent bonds and the biotinylated antibodies are conjugated using an avidin-biotin system. The albumin microbubbles had a mean diameter of 2 μm with a polydispersity index of 0.16. For cell separation, the MDA-MB-231 cells are incubated with the targeted biotin-MBs conjugated with anti-CD44 for 10 min, centrifuged at 10 g for 1 min, and then allowed 1 hour at 4 °C for separation. The results indicate that targeted biotin-MBs conjugated with anti-CD44 antibodies can be used to separate MDA-MB-231 breast cancer cells; more than 90% of the cells were collected in the MB layer when the ratio of the MBs to cells was higher than 70:1. Furthermore, we found that the separating efficiency was higher for targeted biotin-MBs than for targeted avidin-incorporated albumin MBs (avidin-MBs, which is the most common way to make targeted albumin MBs. We also demonstrated that the recovery rate of targeted biotin-MBs was up to 88% and the sorting purity was higher than 84% for a a heterogenous cell population containing MDA-MB-231 cells (CD44(+ and MDA-MB-453 cells (CD44-, which are classified as basal-like breast cancer cells and luminal breast cancer cells, respectively. Knowing that the CD44(+ is a commonly used cancer-stem-cell

Radioprotection of targeted and bystander cells by methylproamine

International Nuclear Information System (INIS)

Burdak-Rothkamm, Susanne; Smith, Andrea; Lobachevsky, Pavel; Martin, Roger; Prise, Kevin M.

2015-01-01

Radioprotective agents are of interest for application in radiotherapy for cancer and in public health medicine in the context of accidental radiation exposure. Methylproamine is the lead compound of a class of radioprotectors which act as DNA binding anti-oxidants, enabling the repair of transient radiation-induced oxidative DNA lesions. This study tested methylproamine for the radioprotection of both directly targeted and bystander cells. T98G glioma cells were treated with 15 μM methylproamine and exposed to 137 Cs γ-ray/X-ray irradiation and He 2+ microbeam irradiation. Radioprotection of directly targeted cells and bystander cells was measured by clonogenic survival or γH2AX assay. Radioprotection of directly targeted T98G cells by methylproamine was observed for 137 Cs γ-rays and X-rays but not for He 2+ charged particle irradiation. The effect of methylproamine on the bystander cell population was tested for both X-ray irradiation and He 2+ ion microbeam irradiation. The X-ray bystander experiments were carried out by medium transfer from irradiated to non-irradiated cultures and three experimental designs were tested. Radioprotection was only observed when recipient cells were pretreated with the drug prior to exposure to the conditioned medium. In microbeam bystander experiments targeted and nontargeted cells were co-cultured with continuous methylproamine treatment during irradiation and postradiation incubation; radioprotection of bystander cells was observed. Methylproamine protected targeted cells from DNA damage caused by γ-ray or X-ray radiation but not He 2+ ion radiation. Protection of bystander cells was independent of the type of radiation which the donor population received. (orig.) [de

T-cell epitopes from viral and tumor associated antigens: induction and analysis of antigen-specific T cells

OpenAIRE

Nastke, Maria-Dorothea

2005-01-01

T cells are important effectors in the defense of human pathogens entering the organism. CD8+ T cells recognize peptides which are presented by MHC class I molecules and lyse cells which are infected by virus or intracellular pathogens. Moreover, they are able to destroy cancer cells. CD4+ T cells recognize peptides from exogenous proteins acquired by endocytosis or from internalized plasma membrane proteins which are presented on MHC class II. CD4+ T cells play an important role in the defen...

Osteosarcoma: Cells-of-Origin, Cancer Stem Cells, and Targeted Therapies

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Ander Abarrategi

2016-01-01

Full Text Available Osteosarcoma (OS is the most common type of primary solid tumor that develops in bone. Although standard chemotherapy has significantly improved long-term survival over the past few decades, the outcome for those patients with metastatic or recurrent OS remains dismally poor and, therefore, novel agents and treatment regimens are urgently required. A hypothesis to explain the resistance of OS to chemotherapy is the existence of drug resistant CSCs with progenitor properties that are responsible of tumor relapses and metastasis. These subpopulations of CSCs commonly emerge during tumor evolution from the cell-of-origin, which are the normal cells that acquire the first cancer-promoting mutations to initiate tumor formation. In OS, several cell types along the osteogenic lineage have been proposed as cell-of-origin. Both the cell-of-origin and their derived CSC subpopulations are highly influenced by environmental and epigenetic factors and, therefore, targeting the OS-CSC environment and niche is the rationale for many recently postulated therapies. Likewise, some strategies for targeting CSC-associated signaling pathways have already been tested in both preclinical and clinical settings. This review recapitulates current OS cell-of-origin models, the properties of the OS-CSC and its niche, and potential new therapies able to target OS-CSCs.

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells.

Science.gov (United States)

Herce, Henry D; Schumacher, Dominik; Schneider, Anselm F L; Ludwig, Anne K; Mann, Florian A; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M Cristina; Hackenberger, Christian P R

2017-08-01

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells

Science.gov (United States)

Herce, Henry D.; Schumacher, Dominik; Schneider, Anselm F. L.; Ludwig, Anne K.; Mann, Florian A.; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M. Cristina; Hackenberger, Christian P. R.

2017-08-01

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

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

Directory of Open Access Journals (Sweden)

Aileen G Rowan

2016-11-01

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

Development of Anti-Human Mesothelin-Targeted Chimeric Antigen Receptor Messenger RNA-transfected Peripheral Blood Lymphocytes for Ovarian Cancer Therapy.

Science.gov (United States)

Hung, Chien-Fu; Xu, Xuequn; Li, Linhong; Ma, Ying; Jin, Qiu; Viley, Angelia; Allen, Cornell; Natarajan, Pachai; Shivakumar, Rama; Peshwa, Madhusudan V; Emens, Leisha A

2018-04-02

CD19-targeted chimeric antigen receptor (CAR) engineered T/natural killer (NK)-cell therapies can result in durable clinical responses in B-cell malignancies. However, CAR-based immunotherapies have been much less successful in solid cancers, in part due to "on-target off-tumor" toxicity related to expression of target tumor antigens on normal tissue. Based on preliminary observations of safety and clinical activity in proof-of-concept clinical trials, tumor antigen-specific messenger RNA (mRNA) CAR transfection into selected, activated, and expanded T/NK cells may permit prospective control of "on-target off-tumor" toxicity. To develop a commercial product for solid tumors, mesothelin was selected as an antigen target based on its association with poor prognosis and overexpression in multiple solid cancers. It was hypothesized that selecting, activating, and expanding cells ex vivo prior to mRNA CAR transfection would not be necessary, thus simplifying the complexity and cost of manufacturing. Now, the development of anti-human mesothelin mRNA CAR transfected peripheral blood lymphocytes (CARMA-hMeso) is reported, demonstrating the manufacture and cryopreservation of multiple cell aliquots for repeat administrations from a single human leukapheresis. A rapid, automated, closed system for cGMP-compliant transfection of mRNA CAR in up to 20 × 10 9 peripheral blood lymphocytes was developed. Here we show that CARMA-hMeso cells recognize and lyse tumor cells in a mesothelin-specific manner. Expression of CAR was detectable over approximately 7 days in vitro, with a progressive decline of CAR expression that appears to correlate with in vitro cell expansion. In a murine ovarian cancer model, a single intraperitoneal injection of CARMA-hMeso resulted in the dose-dependent inhibition of tumor growth and improved survival of mice. Furthermore, repeat weekly intraperitoneal administrations of the optimal CARMA-hMeso dose further prolonged disease control and survival

The quest for targets executing MYC-dependent cell transformation

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Markus eHartl

2016-06-01

Full Text Available MYC represents a transcription factor with oncogenic potential converting multiple cellular signals into a broad transcriptional response, thereby controlling the expression of numerous protein-coding and non-coding RNAs important for cell proliferation, metabolism, differentiation, and apoptosis. Constitutive activation of MYC leads to neoplastic cell transformation, and deregulated MYC alleles are frequently observed in many human cancer cell types. Multiple approaches have been performed to isolate genes differentially expressed in cells containing aberrantly activated MYC proteins leading to the identification of thousands of putative targets. Functional analyses of genes differentially expressed in MYC-transformed cells had revealed that so far more than forty upregulated or downregulated MYC targets are actively involved in cell transformation or tumorigenesis. However, for determination which of the known, or yet unidentified targets are responsible for processing the oncogenic MYC program, further systematic and selective approaches are required. The search for critical targets in MYC-dependent tumor cells is exacerbated by the fact that during tumor development, cancer cells progressively evolve in a multistep process thereby acquiring their characteristic features in an additive manner. Functional expression cloning, combinatorial gene expression and appropriate in vivo tests could represent adequate tools for dissecting the complex scenario of MYC-specified cell transformation. In this context, the central goal is to identify a minimal set of targets that suffices to phenocopy oncogenic MYC. Recently developed genomic editing tools could be employed to confirm the requirement of crucial transformation-associated targets.Knowledge about essential MYC regulated genes is beneficial to expedite the development of specific inhibitors to interfere with growth and viability of human tumor cells in which MYC is aberrantly activated

Targeting therapy-resistant cancer stem cells by hyperthermia

DEFF Research Database (Denmark)

Oei, A L; Vriend, L E M; Krawczyk, P M

2017-01-01

Eradication of all malignant cells is the ultimate but challenging goal of anti-cancer treatment; most traditional clinically-available approaches fail because there are cells in a tumour that either escape therapy or become therapy-resistant. A subpopulation of cancer cells, the cancer stem cells...... are limited. Here, we argue that hyperthermia - a therapeutic approach based on local heating of a tumour - is potentially beneficial for targeting CSCs in solid tumours. First, hyperthermia has been described to target cells in hypoxic and nutrient-deprived tumour areas where CSCs reside and ionising...

Targeting human breast cancer cells by an oncolytic adenovirus using microRNA-targeting strategy.

Science.gov (United States)

Shayestehpour, Mohammad; Moghim, Sharareh; Salimi, Vahid; Jalilvand, Somayeh; Yavarian, Jila; Romani, Bizhan; Mokhtari-Azad, Talat

2017-08-15

MicroRNA-targeting strategy is a promising approach that enables oncolytic viruses to replicate in tumor cells but not in normal cells. In this study, we targeted adenoviral replication toward breast cancer cells by inserting ten complementary binding sites for miR-145-5p downstream of E1A gene. In addition, we evaluated the effect of increasing miR-145 binding sites on inhibition of virus replication. Ad5-control and adenoviruses carrying five or ten copies of miR145-5p target sites (Ad5-5miR145T, Ad5-10miR145T) were generated and inoculated into MDA-MB-453, BT-20, MCF-7 breast cancer cell lines and human mammary epithelial cells (HMEpC). Titer of Ad5-10miR145T in HMEpC was significantly lower than Ad5-control titer. Difference between the titer of these two viruses at 12, 24, 36, and 48h after infection was 1.25, 2.96, 3.06, and 3.77 log TCID 50 . No significant difference was observed between the titer of both adenoviruses in MDA-MB-453, BT-20 and MCF-7 cells. The infectious titer of adenovirus containing 10 miR-145 binding sites in HMEpC cells at 24, 36, and 48h post-infection was 1.7, 2.08, and 4-fold, respectively, lower than the titer of adenovirus carrying 5 miR-145 targets. Our results suggest that miR-145-targeting strategy provides selectivity for adenovirus replication in breast cancer cells. Increasing the number of miRNA binding sites within the adenoviral genome confers more selectivity for viral replication in cancer cells. Copyright © 2017. Published by Elsevier B.V.

Cell cycle-tailored targeting of metastatic melanoma: Challenges and opportunities.

Science.gov (United States)

Haass, Nikolas K; Gabrielli, Brian

2017-07-01

The advent of targeted therapies of metastatic melanoma, such as MAPK pathway inhibitors and immune checkpoint antagonists, has turned dermato-oncology from the "bad guy" to the "poster child" in oncology. Current targeted therapies are effective, although here is a clear need to develop combination therapies to delay the onset of resistance. Many antimelanoma drugs impact on the cell cycle but are also dependent on certain cell cycle phases resulting in cell cycle phase-specific drug insensitivity. Here, we raise the question: Have combination trials been abandoned prematurely as ineffective possibly only because drug scheduling was not optimized? Firstly, if both drugs of a combination hit targets in the same melanoma cell, cell cycle-mediated drug insensitivity should be taken into account when planning combination therapies, timing of dosing schedules and choice of drug therapies in solid tumors. Secondly, if the combination is designed to target different tumor cell subpopulations of a heterogeneous tumor, one drug effective in a particular subpopulation should not negatively impact on the other drug targeting another subpopulation. In addition to the role of cell cycle stage and progression on standard chemotherapeutics and targeted drugs, we discuss the utilization of cell cycle checkpoint control defects to enhance chemotherapeutic responses or as targets themselves. We propose that cell cycle-tailored targeting of metastatic melanoma could further improve therapy outcomes and that our real-time cell cycle imaging 3D melanoma spheroid model could be utilized as a tool to measure and design drug scheduling approaches. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Advanced cell therapies: targeting, tracking and actuation of cells with magnetic particles.

Science.gov (United States)

Connell, John J; Patrick, P Stephen; Yu, Yichao; Lythgoe, Mark F; Kalber, Tammy L

2015-01-01

Regenerative medicine would greatly benefit from a new platform technology that enabled measurable, controllable and targeting of stem cells to a site of disease or injury in the body. Superparamagnetic iron-oxide nanoparticles offer attractive possibilities in biomedicine and can be incorporated into cells, affording a safe and reliable means of tagging. This review describes three current and emerging methods to enhance regenerative medicine using magnetic particles to guide therapeutic cells to a target organ; track the cells using MRI and assess their spatial localization with high precision and influence the behavior of the cell using magnetic actuation. This approach is complementary to the systemic injection of cell therapies, thus expanding the horizon of stem cell therapeutics.

NK-cell-dependent killing of colon carcinoma cells is mediated by natural cytotoxicity receptors (NCRs) and stimulated by parvovirus infection of target cells

International Nuclear Information System (INIS)

Bhat, Rauf; Rommelaere, Jean

2013-01-01

Investigating how the immune system functions during malignancies is crucial to developing novel therapeutic strategies. Natural killer (NK) cells, an important component of the innate immune system, play a vital role in immune defense against tumors and virus-infected cells. The poor survival rate in colon cancer makes it particularly important to develop novel therapeutic strategies. Oncolytic viruses, in addition to lysing tumor cells, may have the potential to augment antitumor immune responses. In the present study, we investigate the role of NK cells and how parvovirus H-1PV can modulate NK-cell mediated immune responses against colon carcinoma. Human NK cells were isolated from the blood of healthy donors. The cytotoxicity and antibody-mediated inhibition of NK cells were measured in chromium release assays. Phenotypic assessment of colon cancer and dendritic cells was done by FACS. The statistical significance of the results was calculated with Student’s t test (*p <0.05; **, p < 0.01; ***, p < 0.001). We show that IL-2-activated human NK cells can effectively kill colon carcinoma cells. Killing of colon carcinoma cells by NK cells was further enhanced upon infection of the former cells with parvovirus H-1PV. H-1PV has potent oncolytic activity against various tumors, yet its direct killing effect on colon carcinoma cells is limited. The cytotoxicity of NK cells towards colon carcinoma cells, both mock- and H-1PV-infected, was found to be mostly mediated by a combination of natural cytotoxicity receptors (NCRs), namely NKp30, 44, and 46. Colon carcinoma cells displayed low to moderate expression of NK cell ligands, and this expression was modulated upon H-1PV infection. Lysates of H-1PV-infected colon carcinoma cells were found to increase MHC class II expression on dendritic cells. Altogether, these data suggest that IL-2-activated NK cells actively kill colon carcinoma cells and that this killing is mediated by several natural cytotoxicity receptors

B cells as a target of immune modulation

Directory of Open Access Journals (Sweden)

Hawker Kathleen

2009-01-01

Full Text Available B cells have recently been identified as an integral component of the immune system; they play a part in autoimmunity through antigen presentation, antibody secretion, and complement activation. Animal models of multiple sclerosis (MS suggest that myelin destruction is partly mediated through B cell activation (and plasmablasts. MS patients with evidence of B cell involvement, as compared to those without, tend to have a worse prognosis. Finally, the significant decrease in new gadolinium-enhancing lesions, new T2 lesions, and relapses in MS patients treated with rituximab (a monoclonal antibody against CD20 on B cells leads us to the conclusion that B cells play an important role in MS and that immune modulation of these cells may ameliorate the disease. This article will explore the role of B cells in MS and the rationale for the development of B cell-targeted therapeutics. MS is an immune-mediated disease that affects over 2 million people worldwide and is the number one cause of disability in young patients. Most therapeutic targets have focused on T cells; however, recently, the focus has shifted to the role of B cells in the pathogenesis of MS and the potential of B cells as a therapeutic target.

Oncolytic Viruses-Interaction of Virus and Tumor Cells in the Battle to Eliminate Cancer.

Science.gov (United States)

Howells, Anwen; Marelli, Giulia; Lemoine, Nicholas R; Wang, Yaohe

2017-01-01

Oncolytic viruses (OVs) are an emerging treatment option for many cancer types and have recently been the focus of extensive research aiming to develop their therapeutic potential. The ultimate aim is to design a virus which can effectively replicate within the host, specifically target and lyse tumor cells and induce robust, long lasting tumor-specific immunity. There are a number of viruses which are either naturally tumor-selective or can be modified to specifically target and eliminate tumor cells. This means they are able to infect only tumor cells and healthy tissue remains unharmed. This specificity is imperative in order to reduce the side effects of oncolytic virotherapy. These viruses can also be modified by various methods including insertion and deletion of specific genes with the aim of improving their efficacy and safety profiles. In this review, we have provided an overview of the various virus species currently being investigated for their oncolytic potential and the positive and negative effects of a multitude of modifications used to increase their infectivity, anti-tumor immunity, and treatment safety, in particular focusing on the interaction of tumor cells and OVs.

Targeting Stromal Recruitment by Prostate Cancer Cells

Science.gov (United States)

2006-03-01

Ensinger, C., Tumer , Z., Tommerup, N. et al.: Hedgehog signaling in small-cell lung cancer : frequent in vivo but a rare event in vitro. Lung Cancer , 52...W81XWH-04-1-0157 TITLE: Targeting Stromal Recruitment by Prostate Cancer Cells PRINCIPAL INVESTIGATOR: Jingxian Zhang, Ph.D...DATES COVERED (From - To) 15 Feb 2004 – 14 Feb 2006 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Targeting Stromal Recruitment by Prostate Cancer

Broad target cell selectivity of Kaposi's sarcoma-associated herpesvirus glycoprotein-mediated cell fusion and virion entry

International Nuclear Information System (INIS)

Kaleeba, Johnan A.R.; Berger, Edward A.

2006-01-01

The molecular mechanism of Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus 8) entry is poorly understood. We tested a broad variety of cell types of diverse species and tissue origin for their ability to function as targets in a quantitative reporter gene assay for KSHV-glycoprotein-mediated cell fusion. Several human, non-human primate, and rabbit cell lines were efficient targets, whereas rodent and all human lymphoblastoid cell lines were weak targets. Parallel findings were obtained with a virion entry assay using a recombinant KSHV encoding a reporter gene. No correlation was observed between target cell activity and surface expression of α3β1 integrin, a proposed KSHV receptor. We hypothesize that target cell permissiveness in both the cell fusion and virion entry assays reflects the presence of a putative KSHV fusion-entry receptor

Magnetic stem cell targeting to the inner ear

Science.gov (United States)

Le, T. N.; Straatman, L.; Yanai, A.; Rahmanian, R.; Garnis, C.; Häfeli, U. O.; Poblete, T.; Westerberg, B. D.; Gregory-Evans, K.

2017-12-01

Severe sensorineural deafness is often accompanied by a loss of auditory neurons in addition to injury of the cochlear epithelium and hair cell loss. Cochlear implant function however depends on a healthy complement of neurons and their preservation is vital in achieving optimal results. We have developed a technique to target mesenchymal stem cells (MSCs) to a deafened rat cochlea. We then assessed the neuroprotective effect of systematically delivered MSCs on the survival and function of spiral ganglion neurons (SGNs). MSCs were labeled with superparamagnetic nanoparticles, injected via the systemic circulation, and targeted using a magnetized cochlea implant and external magnet. Neurotrophic factor concentrations, survival of SGNs, and auditory function were assessed at 1 week and 4 weeks after treatments and compared against multiple control groups. Significant numbers of magnetically targeted MSCs (>30 MSCs/section) were present in the cochlea with accompanied elevation of brain-derived neurotrophic factor and glial cell-derived neurotrophic factor levels (p < 0.001). In addition we saw improved survival of SGNs (approximately 80% survival at 4 weeks). Hearing threshold levels in magnetically targeted rats were found to be significantly better than those of control rats (p < 0.05). These results indicate that magnetic targeting of MSCs to the cochlea can be accomplished with a magnetized cochlear permalloy implant and an external magnet. The targeted stem cells release neurotrophic factors which results in improved SGN survival and hearing recovery. Combining magnetic cell-based therapy and cochlear implantation may improve cochlear implant function in treating deafness.

Surface-modified gold nanorods for specific cell targeting

Science.gov (United States)

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

2012-05-01

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

Autologous bone marrow purging with LAK cells.

Science.gov (United States)

Giuliodori, L; Moretti, L; Stramigioli, S; Luchetti, F; Annibali, G M; Baldi, A

1993-12-01

In this study we will demonstrate that LAK cells, in vitro, can lyse hematologic neoplastic cells with a minor toxicity of the staminal autologous marrow cells. In fact, after bone marrow and LAK co-culture at a ratio of 1/1 for 8 hours, the inhibition on the GEMM colonies resulted to be 20% less compared to the untreated marrow. These data made LAK an inviting agent for marrow purging in autologous bone marrow transplantation.

Curcumin targets fibroblast–tumor cell interactions in oral squamous cell carcinoma

International Nuclear Information System (INIS)

Dudás, József; Fullár, Alexandra; Romani, Angela; Pritz, Christian; Kovalszky, Ilona; Hans Schartinger, Volker; Mathias Sprinzl, Georg; Riechelmann, Herbert

2013-01-01

Co-culture of periodontal ligament fibroblasts (PDLs) and SCC-25 oral squamous carcinoma cells (OSCC) results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs) and induces epithelial-to mesenchymal transition (EMT) of OSCC tumor cells. We hypothesized that Curcumin targets this dynamic mutual interaction between CAFs and tumor cells. Normal and 2 μM Curcumin-treated co-culture were performed for 4 days, followed by analysis of tumor cell invasivity, mRNA/protein expression of EMT-markers and mediators, activity measure of matrix metalloproteinase 9 (MMP-9), and western blot analysis of signal transduction in tumor cells and fibroblasts. In Curcumin-treated co-culture, in tumor cells, the levels of nuclear factor κB (NFκBα) and early response kinase (ERK)—decreased, in fibroblasts, integrin αv protein synthesis decreased compared to corresponding cells in normal co-culture. The signal modulatory changes induced by Curcumin caused decreased release of EMT-mediators in CAFs and reversal of EMT in tumor cells, which was associated with decreased invasion. These data confirm the palliative potential of Curcumin in clinical application. - Graphical abstract: Co-culture of periodontal ligament fibroblasts (PDLs) and SCC-25 oral squamous carcinoma cells (OSCC) results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs) and induces epithelial-to mesenchymal transition (EMT) of tumor cells. Curcumin targets this dynamic mutual interaction between CAFs and tumor cells by inhibiting the production of EMT mediators in CAFs and by modification of intracellular signaling in tumor cells. This causes less invasivity and reversal of EMT in tumor cells. Highlights: ► Curcumin targets tumor–fibroblast interaction in head and neck cancer. ► Curcumin suppresses mediators of epithelial–mesenchymal transition. ► Curcumin decreases the invasivity of tumor cells

Curcumin targets fibroblast–tumor cell interactions in oral squamous cell carcinoma

Energy Technology Data Exchange (ETDEWEB)

Dudás, József, E-mail: jozsef.dudas@i-med.ac.at [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Fullár, Alexandra, E-mail: fullarsz@gmail.com [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, 1085 Budapest (Hungary); Romani, Angela, E-mail: angela.romani@i-med.ac.at [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Pritz, Christian, E-mail: christian.pritz@i-med.ac.at [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Kovalszky, Ilona, E-mail: koval@korb1.sote.hu [1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, 1085 Budapest (Hungary); Hans Schartinger, Volker, E-mail: volker.schartinger@i-med.ac.at [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Mathias Sprinzl, Georg, E-mail: georg.sprinzl@i-med.ac.at [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Riechelmann, Herbert, E-mail: herbert.riechelmann@i-med.ac.at [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria)

2013-04-01

Co-culture of periodontal ligament fibroblasts (PDLs) and SCC-25 oral squamous carcinoma cells (OSCC) results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs) and induces epithelial-to mesenchymal transition (EMT) of OSCC tumor cells. We hypothesized that Curcumin targets this dynamic mutual interaction between CAFs and tumor cells. Normal and 2 μM Curcumin-treated co-culture were performed for 4 days, followed by analysis of tumor cell invasivity, mRNA/protein expression of EMT-markers and mediators, activity measure of matrix metalloproteinase 9 (MMP-9), and western blot analysis of signal transduction in tumor cells and fibroblasts. In Curcumin-treated co-culture, in tumor cells, the levels of nuclear factor κB (NFκBα) and early response kinase (ERK)—decreased, in fibroblasts, integrin αv protein synthesis decreased compared to corresponding cells in normal co-culture. The signal modulatory changes induced by Curcumin caused decreased release of EMT-mediators in CAFs and reversal of EMT in tumor cells, which was associated with decreased invasion. These data confirm the palliative potential of Curcumin in clinical application. - Graphical abstract: Co-culture of periodontal ligament fibroblasts (PDLs) and SCC-25 oral squamous carcinoma cells (OSCC) results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs) and induces epithelial-to mesenchymal transition (EMT) of tumor cells. Curcumin targets this dynamic mutual interaction between CAFs and tumor cells by inhibiting the production of EMT mediators in CAFs and by modification of intracellular signaling in tumor cells. This causes less invasivity and reversal of EMT in tumor cells. Highlights: ► Curcumin targets tumor–fibroblast interaction in head and neck cancer. ► Curcumin suppresses mediators of epithelial–mesenchymal transition. ► Curcumin decreases the invasivity of tumor cells.

Selective tumor cell targeting by the disaccharide moiety of bleomycin.

Science.gov (United States)

Yu, Zhiqiang; Schmaltz, Ryan M; Bozeman, Trevor C; Paul, Rakesh; Rishel, Michael J; Tsosie, Krystal S; Hecht, Sidney M

2013-02-27

In a recent study, the well-documented tumor targeting properties of the antitumor agent bleomycin (BLM) were studied in cell culture using microbubbles that had been derivatized with multiple copies of BLM. It was shown that BLM selectively targeted MCF-7 human breast carcinoma cells but not the "normal" breast cell line MCF-10A. Furthermore, it was found that the BLM analogue deglycobleomycin, which lacks the disaccharide moiety of BLM, did not target either cell line, indicating that the BLM disaccharide moiety is necessary for tumor selectivity. Not resolved in the earlier study were the issues of whether the BLM disaccharide moiety alone is sufficient for tumor cell targeting and the possible cellular uptake of the disaccharide. In the present study, we conjugated BLM, deglycoBLM, and BLM disaccharide to the cyanine dye Cy5**. It was found that the BLM and BLM disaccharide conjugates, but not the deglycoBLM conjugate, bound selectively to MCF-7 cells and were internalized. The same was also true for the prostate cancer cell line DU-145 (but not for normal PZ-HPV-7 prostate cells) and for the pancreatic cancer cell line BxPC-3 (but not for normal SVR A221a pancreas cells). The targeting efficiency of the disaccharide was only slightly less than that of BLM in MCF-7 and DU-145 cells and comparable to that of BLM in BxPC-3 cells. These results establish that the BLM disaccharide is both necessary and sufficient for tumor cell targeting, a finding with obvious implications for the design of novel tumor imaging and therapeutic agents.

An innovative pre-targeting strategy for tumor cell specific imaging and therapy.

Science.gov (United States)

Qin, Si-Yong; Peng, Meng-Yun; Rong, Lei; Jia, Hui-Zhen; Chen, Si; Cheng, Si-Xue; Feng, Jun; Zhang, Xian-Zheng

2015-09-21

A programmed pre-targeting system for tumor cell imaging and targeting therapy was established based on the "biotin-avidin" interaction. In this programmed functional system, transferrin-biotin can be actively captured by tumor cells with the overexpression of transferrin receptors, thus achieving the pre-targeting modality. Depending upon avidin-biotin recognition, the attachment of multivalent FITC-avidin to biotinylated tumor cells not only offered the rapid fluorescence labelling, but also endowed the pre-targeted cells with targeting sites for the specifically designed biotinylated peptide nano-drug. Owing to the successful pre-targeting, tumorous HepG2 and HeLa cells were effectively distinguished from the normal 3T3 cells via fluorescence imaging. In addition, the self-assembled peptide nano-drug resulted in enhanced cell apoptosis in the observed HepG2 cells. The tumor cell specific pre-targeting strategy is applicable for a variety of different imaging and therapeutic agents for tumor treatments.

Single-Cell RNA Sequencing of Glioblastoma Cells.

Science.gov (United States)

Sen, Rajeev; Dolgalev, Igor; Bayin, N Sumru; Heguy, Adriana; Tsirigos, Aris; Placantonakis, Dimitris G

2018-01-01

Single-cell RNA sequencing (sc-RNASeq) is a recently developed technique used to evaluate the transcriptome of individual cells. As opposed to conventional RNASeq in which entire populations are sequenced in bulk, sc-RNASeq can be beneficial when trying to better understand gene expression patterns in markedly heterogeneous populations of cells or when trying to identify transcriptional signatures of rare cells that may be underrepresented when using conventional bulk RNASeq. In this method, we describe the generation and analysis of cDNA libraries from single patient-derived glioblastoma cells using the C1 Fluidigm system. The protocol details the use of the C1 integrated fluidics circuit (IFC) for capturing, imaging and lysing cells; performing reverse transcription; and generating cDNA libraries that are ready for sequencing and analysis.

Single cell biochemistry to visualize antigen presentation and drug resistance

NARCIS (Netherlands)

Griekspoor, Alexander Christiaan

2006-01-01

Many cellular processes are studied by biochemical techniques. Usually, this involves experiments where large number of cells are lysed, protein content is subsequently isolated and studied using antibodies to detect changes in protein levels, post-translational modifications, pairing with partner

Potential targets for lung squamous cell carcinoma

Science.gov (United States)

Researchers have identified potential therapeutic targets in lung squamous cell carcinoma, the second most common form of lung cancer. The Cancer Genome Atlas (TCGA) Research Network study comprehensively characterized the lung squamous cell carcinoma gen

Cytotoxic T lymphocyte recognition of HLA-A/B antigens introduced into EL4 cells by cell-liposome fusion.

Science.gov (United States)

Engelhard, V H; Powers, G A; Moore, L C; Holterman, M J; Correa-Freire, M C

1984-01-01

HLA-A2 and -B7 antigens were introduced into EL4 (H-2b) cells by cell-liposome fusion and were used as targets or stimulators for cytotoxic T lymphocytes (CTL) generated in C57B1/6 (H-2b) mice. It was found that such EL4-HLA cells were not recognized by CTL that had been raised against either a human cell line bearing these HLA antigens or the purified HLA-A2 and -B7 antigens reconstituted into liposomes. In addition, EL4-HLA cells were not capable of inducing CTL that could recognize a human cell line bearing HLA-A2 and -B7 antigens. Instead, EL4-HLA cells induced CTL that specifically lysed EL4-HLA cells and not human cells expressing HLA-A2 and -B7. CTL recognition required the presence of HLA antigens on the EL4 cell surface and was inhibited by antibodies against either H-2b or HLA-A/B. Monoclonal antibody binding studies showed that the expected polymorphic determinants of the HLA-A2 and -B7 antigens were still present on EL4-HLA cells. However, the specificity of CTL or their precursors that are capable of recognizing HLA-A2 or -B7 was altered after these antigens became associated with the EL4 surface. Possible explanations for these results are discussed.

GUCY2C-directed CAR-T cells oppose colorectal cancer metastases without autoimmunity.

Science.gov (United States)

Magee, Michael S; Kraft, Crystal L; Abraham, Tara S; Baybutt, Trevor R; Marszalowicz, Glen P; Li, Peng; Waldman, Scott A; Snook, Adam E

2016-01-01

Adoptive T-cell therapy (ACT) is an emerging paradigm in which T cells are genetically modified to target cancer-associated antigens and eradicate tumors. However, challenges treating epithelial cancers with ACT reflect antigen targets that are not tumor-specific, permitting immune damage to normal tissues, and preclinical testing in artificial xenogeneic models, preventing prediction of toxicities in patients. In that context, mucosa-restricted antigens expressed by cancers exploit anatomical compartmentalization which shields mucosae from systemic antitumor immunity. This shielding may be amplified with ACT platforms employing antibody-based chimeric antigen receptors (CARs), which mediate MHC-independent recog-nition of antigens. GUCY2C is a cancer mucosa antigen expressed on the luminal surfaces of the intestinal mucosa in mice and humans, and universally overexpressed by colorectal tumors, suggesting its unique utility as an ACT target. T cells expressing CARs directed by a GUCY2C-specific antibody fragment recognized GUCY2C, quantified by expression of activation markers and cytokines. Further, GUCY2C CAR-T cells lysed GUCY2C-expressing, but not GUCY2C-deficient, mouse colorectal cancer cells. Moreover, GUCY2C CAR-T cells reduced tumor number and morbidity and improved survival in mice harboring GUCY2C-expressing colorectal cancer metastases. GUCY2C-directed T cell efficacy reflected CAR affinity and surface expression and was achieved without immune-mediated damage to normal tissues in syngeneic mice. These observations highlight the potential for therapeutic translation of GUCY2C-directed CAR-T cells to treat metastatic tumors, without collateral autoimmunity, in patients with metastatic colorectal cancer.

Calcein AM release-based cytotoxic cell assay for fish leucocytes.

Science.gov (United States)

Iwanowicz, Luke R; Densmore, Christine L; Ottinger, Christopher A

2004-02-01

A non-specific cytotoxic cell assay for fish is presented that is based on the release of the activated fluorochrome calcein AM from lysed carp epithelioma papulosum cyprini (EPC) cells. To establish the suitability of treating EPC cells with calcein AM the uptake and spontaneous release of the calcein AM by the EPC cells was evaluated. Incubation of 5 microM calcein AM in culture medium with 1x10(5)EPC cells well(-1)for a minimum of 3 h provided sufficient labelling. Spontaneous release of fluorescence from the labelled EPC cells during 10 h of post labelling incubation ranged from 30 to 39% of the total observed fluorescence. Cytotoxic activity of trout leucocytes was evaluated at three leucocyte to target cell ratios (10:1, 2:1 and 1:1) following incubation (4, 6, 8, and 10 h) with calcein AM-labelled EPC cells at 15 degrees C. In some instances, the monoclonal antibody specific for the NCC surface receptor NCCRP-1 (MAb5C.6) was included in the cultures. The activity of NCC cells was significantly inhibited in the presence of 0.25 microg well(-1)of MAb5C.6 relative to no antibody (Pcell activity of approximately 18% was observed following 8 h of incubation at the 2:1 and 1:1 leucocyte to target cell ratios. Percent cytotoxic cell activity using calcein AM was similar to values reported for rainbow trout leucocytes using the 51Cr-release assay.

Targeting the bone marrow: applications in stem cell transplantation

International Nuclear Information System (INIS)

Orchard, K.; Cooper, M.

2004-01-01

Therapeutic doses of radiation cab be selectively directed to the bone marrow either directly using vectors that bind to myeloid and/or lymphoid specific antigens or indirectly by targeting bone matrix. The combination of an accessible target tissue and relatively radiation sensitive malignant cells favours the use of targeted radiotherapy in the treatment of haematopoietic malignancies. Dose escalation of targeted radiation can increase tumour cell destruction and has led to the use of myelosuppressive and possibly myeloablative doses of targeted radiation. A natural development has been the use of targeted radiation in conditioning prior to haematopoietic stem cell transplantation (HSCT). Several groups are actively exploring the use of targeted radiotherapy in the context of HSCT as treatment for haematological malignancies. Although no randomised trials using targeted radiotherapy in HSCT have been published, phase I and II trials have shown very encouraging results stimulating further clinical research in this field. After more than a decade of translational research the optimal combination of therapeutic radioisotope and vector has not been determined. This review summarises the clinical experience of targeted radiotherapy in HSCT and discusses the problems that still need to be solved to maximise the potential of this new treatment modality in HSCT

Targeting dendritic cells in vivo for cancer therapy

Directory of Open Access Journals (Sweden)

Irina eCaminschi

2012-02-01

Full Text Available Monoclonal antibodies that recognise cell surface molecules have been used deliver antigenic cargo to dendritic cells (DC for induction of immune responses. The encouraging anti-tumour immunity elicited using this immunisation strategy suggests its suitability for clinical trials. This review discusses the complex network of DC, the functional specialisation of DC-subsets, the immunological outcomes of targeting different DC-subsets and their cell surface receptors, and the requirements for the induction of effective anti-tumour immunity. Finally, we review preclinical experiments and the progress towards targeting human DC in vivo.

Targeting cancer stem cells in hepatocellular carcinoma

Directory of Open Access Journals (Sweden)

He AR

2014-12-01

Full Text Available Aiwu Ruth He,1 Daniel C Smith,1 Lopa Mishra2 1Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, 2Department of Gastroenterology, Hepatology, and Nutrition, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Abstract: The poor outcome of patients with hepatocellular carcinoma (HCC is attributed to recurrence of the disease after curative treatment and the resistance of HCC cells to conventional chemotherapy, which may be explained partly by the function of liver cancer stem cells (CSCs. Liver CSCs have emerged as an important therapeutic target against HCC. Numerous surface markers for liver CSCs have been identified, and include CD133, CD90, CD44, CD13, and epithelial cell adhesion molecules. These surface markers serve not only as tools for identifying and isolating liver CSCs but also as therapeutic targets for eradicating these cells. In studies of animal models and large-scale genomic analyses of human HCC samples, many signaling pathways observed in normal stem cells have been found to be altered in liver CSCs, which accounts for the stemness and aggressive behavior of these cells. Antibodies and small molecule inhibitors targeting the signaling pathways have been evaluated at different levels of preclinical and clinical development. Another strategy is to promote the differentiation of liver CSCs to less aggressive HCC that is sensitive to conventional chemotherapy. Disruption of the tumor niche essential for liver CSC homeostasis has become a novel strategy in cancer treatment. To overcome the challenges in developing treatment for liver CSCs, more research into the genetic makeup of patient tumors that respond to treatment may lead to more effective therapy. Standardization of HCC CSC tumor markers would be helpful for measuring the CSC response to these agents. Herein, we review the current strategies for developing treatment to eradicate liver CSCs and to improve the outcome for patients with

Breast cancer stem cells, EMT and therapeutic targets

Energy Technology Data Exchange (ETDEWEB)

Kotiyal, Srishti; Bhattacharya, Susinjan, E-mail: s.bhattacharya@jiit.ac.in

2014-10-10

Highlights: • Therapeutic targeting or inhibition of the key molecules of signaling pathways can control growth of breast cancer stem cells (BCSCs). • Development of BCSCs also involves miRNA interactions. • Therapeutic achievement can be done by targeting identified targets in the BCSC pathways. - Abstract: A small heterogeneous population of breast cancer cells acts as seeds to induce new tumor growth. These seeds or breast cancer stem cells (BCSCs) exhibit great phenotypical plasticity which allows them to undergo “epithelial to mesenchymal transition” (EMT) at the site of primary tumor and a future reverse transition. Apart from metastasis they are also responsible for maintaining the tumor and conferring it with drug and radiation resistance and a tendency for post-treatment relapse. Many of the signaling pathways involved in induction of EMT are involved in CSC generation and regulation. Here we are briefly reviewing the mechanism of TGF-β, Wnt, Notch, TNF-α, NF-κB, RTK signalling pathways which are involved in EMT as well as BCSCs maintenance. Therapeutic targeting or inhibition of the key/accessory players of these pathways could control growth of BCSCs and hence malignant cancer. Additionally several miRNAs are dysregulated in cancer stem cells indicating their roles as oncogenes or tumor suppressors. This review also lists the miRNA interactions identified in BCSCs and discusses on some newly identified targets in the BCSC regulatory pathways like SHIP2, nicastrin, Pin 1, IGF-1R, pro-inflammatory cytokines and syndecan which can be targeted for therapeutic achievements.

Multimodal Nanomedicine Strategies for Targeting Cancer Cells as well as Cancer Stem Cell Signalling Mechanisms.

Science.gov (United States)

Kanwar, Jagat R; Samarasinghe, Rasika M; Kamalapuram, Sishir K; Kanwar, Rupinder K

2017-01-01

Increasing evidence suggests that stem cells, a small population of cells with unique selfrenewable and tumour regenerative capacity, are aiding tumour re-growth and multidrug resistance. Conventional therapies are highly ineffective at eliminating these cells leading to relapse of disease and formation of chemoresistance tumours. Cancer and stem cells targeted therapies that utilizes nanotherapeutics to delivery anti-cancer drugs to specific sites are continuously investigated. This review focuses on recent research using nanomedicine and targeting entities to eliminate cancer cells and cancer stem cells. Current nanotherapeutics in clinical trials along with more recent publications on targeted therapies are addressed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

In vitro effects of PCDDs/Fs on NK-like cell activity of Eisenia andrei earthworms

Directory of Open Access Journals (Sweden)

Hayet Belmeskine

2012-02-01

Full Text Available In this study, we assessed in vitro the effects of PCDD/Fs on the NK-like cell activity in Eisenia andrei earthworms using flow cytometry for analysis. NK-like coelomocytes isolated from E. andrei and used as effectors were exposed to various concentrations of PCDDs/Fs mixture, C1 (6.25x10-3 ng 2378- TCDD/mL, C2 (12.5x10-3 ng 2378-TCDD/mL and C3 (25x10-3 ng 2378-TCDD/mL, before adding them to human tumoral cells (K562 used as targets. We evaluated the percentage of targets lysed by Nk-like cells. The results showed a significant stimulation of the NKlike activity at C3 when PCDD/Fs were not removed from effectors before contact with targets, while no effects were noted when the effectors were washed (PCDD/Fs removed or fixed. Assessment of the viability of the targets (K562, exposed alone and separately from effectors, to the three concentrations of PCDD/Fs, C1, C2 and C3, showed that all these concentrations were cytotoxic for K562. Results suggest that PCDD/Fs concentrations tested in this assay may be considered too low to induce suppressive effects on the immune function such as the NK-like activity in E. andrei earthworms.

Cell-density-dependent lysis and sporulation of Myxococcus xanthus in agarose microbeads.

OpenAIRE

Rosenbluh, A; Nir, R; Sahar, E; Rosenberg, E

1989-01-01

Vegetative cells of Myxococcus xanthus were immobilized in 25-microns-diameter agarose microbeads and incubated in either growth medium or sporulation buffer. In growth medium, the cells multiplied, glided to the periphery, and then filled the beads. In sporulation buffer, up to 90% of the cells lysed and ca. 50% of the surviving cells formed resistant spores. A strong correlation between sporulation and cell lysis was observed; both phenomena were cell density dependent. Sporulation proficie...

Targeting myeloid cells using nanoparticles to improve cancer immunotherapy.

Science.gov (United States)

Amoozgar, Zohreh; Goldberg, Michael S

2015-08-30

While nanoparticles have traditionally been used to deliver cytotoxic drugs directly to tumors to induce cancer cell death, emerging data suggest that nanoparticles are likely to generate a larger impact on oncology through the delivery of agents that can stimulate antitumor immunity. Tumor-targeted nanocarriers have generally been used to localize chemotherapeutics to tumors and thus decrease off-target toxicity while enhancing efficacy. Challengingly, tumor heterogeneity and evolution render tumor-intrinsic approaches likely to succumb to relapse. The immune system offers exquisite specificity, cytocidal potency, and long-term activity that leverage an adaptive memory response. For this reason, the ability to manipulate immune cell specificity and function would be desirable, and nanoparticles represent an exciting means by which to perform such manipulation. Dendritic cells and tumor-associated macrophages are cells of the myeloid lineage that function as natural phagocytes, so they naturally take up nanoparticles. Dendritic cells direct the specificity and potency of cellular immune responses that can be targeted for cancer vaccines. Herein, we discuss the specific criteria needed for efficient vaccine design, including but not limited to the route of administration, size, morphology, surface charge, targeting ligands, and nanoparticle composition. In contrast, tumor-associated macrophages are critical mediators of immunosuppression whose trans-migratory abilities can be exploited to localize therapeutics to the tumor core and which can be directly targeted for elimination or for repolarization to a tumor suppressive phenotype. It is likely that a combination of targeting dendritic cells to stimulate antitumor immunity and tumor-associated macrophages to reduce immune suppression will impart significant benefits and result in durable antitumor responses. Copyright © 2014 Elsevier B.V. All rights reserved.

Liposomes to target peripheral neurons and Schwann cells.

Directory of Open Access Journals (Sweden)

Sooyeon Lee

Full Text Available While a wealth of literature for tissue-specific liposomes is emerging, optimal formulations to target the cells of the peripheral nervous system (PNS are lacking. In this study, we asked whether a novel formulation of phospholipid-based liposomes could be optimized for preferential uptake by microvascular endothelia, peripheral neurons and Schwann cells. Here, we report a unique formulation consisting of a phospholipid, a polymer surfactant and cholesterol that result in enhanced uptake by targeted cells. Using fluorescently labeled liposomes, we followed particle internalization and trafficking through a distinct route from dextran and escape from degradative compartments, such as lysosomes. In cultures of non-myelinating Schwann cells, liposomes associate with the lipid raft marker Cholera toxin, and their internalization is inhibited by disruption of lipid rafts or actin polymerization. In contrast, pharmacological inhibition of clathrin-mediated endocytosis does not significantly impact liposome entry. To evaluate the efficacy of liposome targeting in tissues, we utilized myelinating explant cultures of dorsal root ganglia and isolated diaphragm preparations, both of which contain peripheral neurons and myelinating Schwann cells. In these models, we detected preferential liposome uptake into neurons and glial cells in comparison to surrounding muscle tissue. Furthermore, in vivo liposome administration by intramuscular or intravenous injection confirmed that the particles were delivered to myelinated peripheral nerves. Within the CNS, we detected the liposomes in choroid epithelium, but not in myelinated white matter regions or in brain parenchyma. The described nanoparticles represent a novel neurophilic delivery vehicle for targeting small therapeutic compounds, biological molecules, or imaging reagents into peripheral neurons and Schwann cells, and provide a major advancement toward developing effective therapies for peripheral

Targeting regulatory T cells in cancer.

LENUS (Irish Health Repository)

Byrne, William L

2012-01-31

Infiltration of tumors by regulatory T cells confers growth and metastatic advantages by inhibiting antitumor immunity and by production of receptor activator of NF-kappaB (RANK) ligand, which may directly stimulate metastatic propagation of RANK-expressing cancer cells. Modulation of regulatory T cells can enhance the efficacy of cancer immunotherapy. Strategies include depletion, interference with function, inhibition of tumoral migration, and exploitation of T-cell plasticity. Problems with these strategies include a lack of specificity, resulting in depletion of antitumor effector T cells or global interruption of regulatory T cells, which may predispose to autoimmune diseases. Emerging technologies, such as RNA interference and tetramer-based targeting, may have the potential to improve selectivity and efficacy.

The mechanism of gene targeting in human somatic cells.

Directory of Open Access Journals (Sweden)

Yinan Kan

2014-04-01

Full Text Available Gene targeting in human somatic cells is of importance because it can be used to either delineate the loss-of-function phenotype of a gene or correct a mutated gene back to wild-type. Both of these outcomes require a form of DNA double-strand break (DSB repair known as homologous recombination (HR. The mechanism of HR leading to gene targeting, however, is not well understood in human cells. Here, we demonstrate that a two-end, ends-out HR intermediate is valid for human gene targeting. Furthermore, the resolution step of this intermediate occurs via the classic DSB repair model of HR while synthesis-dependent strand annealing and Holliday Junction dissolution are, at best, minor pathways. Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector. Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process. Finally, we demonstrate that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations significantly advance our understanding of HR and gene targeting in human cells.

Nipah virus infection and glycoprotein targeting in endothelial cells

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Maisner Andrea

2010-11-01

Full Text Available Abstract Background The highly pathogenic Nipah virus (NiV causes fatal respiratory and brain infections in animals and humans. The major hallmark of the infection is a systemic endothelial infection, predominantly in the CNS. Infection of brain endothelial cells allows the virus to overcome the blood-brain-barrier (BBB and to subsequently infect the brain parenchyma. However, the mechanisms of NiV replication in endothelial cells are poorly elucidated. We have shown recently that the bipolar or basolateral expression of the NiV surface glycoproteins F and G in polarized epithelial cell layers is involved in lateral virus spread via cell-to-cell fusion and that correct sorting depends on tyrosine-dependent targeting signals in the cytoplasmic tails of the glycoproteins. Since endothelial cells share many characteristics with epithelial cells in terms of polarization and protein sorting, we wanted to elucidate the role of the NiV glycoprotein targeting signals in endothelial cells. Results As observed in vivo, NiV infection of endothelial cells induced syncytia formation. The further finding that infection increased the transendothelial permeability supports the idea of spread of infection via cell-to-cell fusion and endothelial cell damage as a mechanism to overcome the BBB. We then revealed that both glycoproteins are expressed at lateral cell junctions (bipolar, not only in NiV-infected primary endothelial cells but also upon stable expression in immortalized endothelial cells. Interestingly, mutation of tyrosines 525 and 542/543 in the cytoplasmic tail of the F protein led to an apical redistribution of the protein in endothelial cells whereas tyrosine mutations in the G protein had no effect at all. This fully contrasts the previous results in epithelial cells where tyrosine 525 in the F, and tyrosines 28/29 in the G protein were required for correct targeting. Conclusion We conclude that the NiV glycoprotein distribution is responsible for

Major histocompatibility complex-unrestricted cytolytic activity of human T cells: analysis of precursor frequency and effector phenotype

International Nuclear Information System (INIS)

Patel, S.S.; Thiele, D.L.; Lipsky, P.E.

1987-01-01

The frequency and phenotype of human T cells that mediate major histocompatibility complex (MHC)-unrestricted cytolysis were analyzed. T cell clones were generated by culturing adherent cell-depleted peripheral blood mononuclear cells at a density of 0.3 cell/well with phytohemagglutinin, recombinant interleukin 2 (rIL-2), and irradiated autologous peripheral blood mononuclear cells and/or Epstein-Barr virus-transformed lymphoblastoid cell lines. All of the 198 clones generated by this method were T cells (CD2 + , CD3 + , CD4 + or CD2 + , CD3 + , CD8 + ) that possessed potent lytic activity against K562, an erythroleukemia line sensitive to lysis by human natural killer cells, and Cur, a renal carcinoma cell line resistant to human natural killer activity. Cytolysis, measured by 51 Cr release, was MHC-unrestricted, since the clones were able to lyse MHC class I or class II negative targets, as well as MHC class I and class II negative targets. Although the clones produced tissue necrosis factor/lymphotoxin-like molecules, lysis of Cur of K562 was not mediated by a soluble factor secreted by the clones. These data indicate that the capacity for MHC-unrestricted tumoricidal activity and expression of NKH1 and CD11b, but not CD 16, are properties common to all or nearly all human peripheral blood-derived T cell clones regardless of CD4 or CD8 phenotype

Prodrug strategy for cancer cell-specific targeting: A recent overview.

Science.gov (United States)

Zhang, Xian; Li, Xiang; You, Qidong; Zhang, Xiaojin

2017-10-20

The increasing development of targeted cancer therapy provides extensive possibilities in clinical trials, and numerous strategies have been explored. The prodrug is one of the most promising strategies in targeted cancer therapy to improve the selectivity and efficacy of cytotoxic compounds. Compared with normal tissues, cancer cells are characterized by unique aberrant markers, thus inactive prodrugs targeting these markers are excellent therapeutics to release active drugs, killing cancer cells without damaging normal tissues. In this review, we explore an integrated view of potential prodrugs applied in targeted cancer therapy based on aberrant cancer specific markers and some examples are provided for inspiring new ideas of prodrug strategy for cancer cell-specific targeting. Copyright © 2017. Published by Elsevier Masson SAS.

DNA fragmentation: manifestation of target cell destruction mediated by cytotoxic T-cell lines, lymphotoxin-secreting helper T-cell clones, and cell-free lymphotoxin-containing supernatant

International Nuclear Information System (INIS)

Schmid, D.S.; Tite, J.P.; Ruddle, N.H.

1986-01-01

A Lyt-2 + , trinitrophenyl-specific, lymphotoxin-secreting, cytotoxic T-cell line, PCl 55, mediates the digestion of target cell DNA into discretely sized fragments. This phenomenon manifests itself within 30 min after effector cell encounter as measured by the release of 3 H counts from target cells prelabeled with [ 3 H]deoxythymidine and occurs even at very low effector to target cell ratios (0.25:1). A Lyt-1 + , ovalbumin-specific, lymphotoxin-secreting T-helper cell clone, 5.9.24, is also able to mediate fragmentation of target cell DNA over a time course essentially indistinguishable from the cytotoxic T lymphocyte-mediated hit. Cell-free lymphotoxin-containing supernatants also cause release of DNA from targets, although they require a longer time course, on the order of 24 hr. In contrast, lysis of cells by antibody plus complement or Triton X-100 does not result in DNA release even after extended periods of incubation (24 hr). All three treatments that result in the release of DNA from cells cause fragmentation of that DNA into discretely sized pieces that are multiples of 200 base pairs. The results thus suggest that cytotoxic T cells, lymphotoxin-secreting helper clones with cytolytic activity, and lymphotoxin all effect target cell destruction by means of a similar mechanism and that observed differences in time course and the absence of target cell specificity in killing mediated by lymphotoxin may simply reflect differences in the mode of toxin delivery

Targeting vaccines to dendritic cells

DEFF Research Database (Denmark)

Foged, Camilla; Sundblad, Anne; Hovgaard, Lars

2002-01-01

delivery systems (DDS) with adjuvant effect that target DC directly and induce optimal immune responses. This paper will review the current knowledge of DC physiology as well as the progress in the field of novel vaccination strategies that directly or indirectly aim at targeting DC....... to be far superior to that of B-cells and macrophages. DC are localized at strategic places in the body at sites used by pathogens to enter the organism, and are thereby in an optimal position to capture antigens. In general, vaccination strategies try to mimic the invasiveness of the pathogens. DC...

Trichomonas vaginalis and Tritrichomonas foetus: interaction with fibroblasts and muscle cells - new insights into parasite-mediated host cell cytotoxicity

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Ricardo Chaves Vilela

2012-09-01

Full Text Available Trichomonas vaginalis and Tritrichomonas foetus are parasitic, flagellated protists that inhabit the urogenital tract of humans and bovines, respectively. T. vaginalis causes the most prevalent non-viral sexually transmitted disease worldwide and has been associated with an increased risk for human immunodeficiency virus-1 infection in humans. Infections by T. foetus cause significant losses to the beef industry worldwide due to infertility and spontaneous abortion in cows. Several studies have shown a close association between trichomonads and the epithelium of the urogenital tract. However, little is known concerning the interaction of trichomonads with cells from deeper tissues, such as fibroblasts and muscle cells. Published parasite-host cell interaction studies have reported contradictory results regarding the ability of T. foetus and T. vaginalis to interact with and damage cells of different tissues. In this study, parasite-host cell interactions were examined by culturing primary human fibroblasts obtained from abdominal biopsies performed during plastic surgeries with trichomonads. In addition, mouse 3T3 fibroblasts, primary chick embryo myogenic cells and L6 muscle cells were also used as models of target cells. The parasite-host cell cultures were processed for scanning and transmission electron microscopy and were tested for cell viability and cell death. JC-1 staining, which measures mitochondrial membrane potential, was used to determine whether the parasites induced target cell damage. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling staining was used as an indicator of chromatin damage. The colorimetric crystal violet assay was performed to ana-lyse the cytotoxicity induced by the parasite. The results showed that T. foetus and T. vaginalis adhered to and were cytotoxic to both fibroblasts and muscle cells, indicating that trichomonas infection of the connective and muscle tissues is likely to occur; such

Pros and Cons of Antigen-Presenting Cell Targeted Tumor Vaccines

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Cleo Goyvaerts

2015-01-01

Full Text Available In therapeutic antitumor vaccination, dendritic cells play the leading role since they decide if, how, when, and where a potent antitumor immune response will take place. Since the disentanglement of the complexity and merit of different antigen-presenting cell subtypes, antitumor immunotherapeutic research started to investigate the potential benefit of targeting these subtypes in situ. This review will discuss which antigen-presenting cell subtypes are at play and how they have been targeted and finally question the true meaning of targeting antitumor-based vaccines.

Oncolytic viral therapy: targeting cancer stem cells

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Smith TT

2014-02-01

Full Text Available Tyrel T Smith,1 Justin C Roth,1 Gregory K Friedman,1 G Yancey Gillespie2 1Department of Pediatrics, The University of Alabama at Birmingham, Birmingham, AL, USA; 2Department of Neurosurgery, The University of Alabama at Birmingham, Birmingham, AL, USA Abstract: Cancer stem cells (CSCs are defined as rare populations of tumor-initiating cancer cells that are capable of both self-renewal and differentiation. Extensive research is currently underway to develop therapeutics that target CSCs for cancer therapy, due to their critical role in tumorigenesis, as well as their resistance to chemotherapy and radiotherapy. To this end, oncolytic viruses targeting unique CSC markers, signaling pathways, or the pro-tumor CSC niche offer promising potential as CSCs-destroying agents/therapeutics. We provide a summary of existing knowledge on the biology of CSCs, including their markers and their niche thought to comprise the tumor microenvironment, and then we provide a critical analysis of the potential for targeting CSCs with oncolytic viruses, including herpes simplex virus-1, adenovirus, measles virus, reovirus, and vaccinia virus. Specifically, we review current literature regarding first-generation oncolytic viruses with their innate ability to replicate in CSCs, as well as second-generation viruses engineered to enhance the oncolytic effect and CSC-targeting through transgene expression. Keywords: oncolytic virotherapy, cancer stem cell niche

Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

Science.gov (United States)

Cha, Jaehyun; Kwon, Inchan

2018-02-27

Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Studies on ADCC (antibody-dependent cell-mediated cytotoxicity) using sheep red blood cells as target cells, 2

International Nuclear Information System (INIS)

Ichikawa, Yukinobu; Takaya, Masatoshi; Arimori, Shigeru

1979-01-01

A non-specific cytotoxic mediator from effector cells (human peripheral blood leukocytes) was investigated in the ADCC (antibody-dependent cell-mediated cytotoxicity) system using antibody-coated sheep red blood cells (SRBC) as target cells. 51 Cr-labelled homologous (sheep) or heterologous (human) red blood cells were used as adjacent cells. Either crude lymphocyte fraction, phagocyte depleted fraction or granulocyte rich fraction separated from human peripheral leukocytes showed moderate cytotoxic effect on homologous adjacent cells, however no cytotoxic activity on heterologous adjacent cells was demonstrated in any leukocyte fraction. This suggests that the cytotoxic effects on homologous adjacent cells were resulted from the translocation of antibody molecules to adjacent cells from antibody-coated target cells. We concluded that the cytotoxic mechanism in this ADCC system was not mediated by non-specific soluble factors released from either human peripheral lymphocytes, monocytes or granulocytes. (author)

The polarized double cell target of the SMC

International Nuclear Information System (INIS)

Adams, D.; Adeva, B.; Arik, E.; Arvidson, A.; Badelek, B.; Ballintijn, M.K.; Bardin, G.; Baum, G.; Berglund, P.; Betev, L.; Bird, I.G.; Birsa, R.; Bjoerkholm, P.; Bonner, B.E.; Botton, N. de; Boutemeur, M.; Bradamante, F.; Bravar, A.; Bressan, A.; Bueltmann, S.; Burtin, E.; Cavata, C.; Crabb, D.; Cranshaw, J.; Cuhadar, T.; Torre, S. Dalla; Dantzig, R. van; Derro, B.; Deshpande, A.; Dhawan, S.; Dulya, C.; Dyring, A.; Eichblatt, S.; Faivre, J.C.; Fasching, D.; Feinstein, F.; Fernandez, C.; Forthmann, S.; Frois, B.; Gallas, A.; Garzon, J.A.; Gaussiran, T.; Gilly, H.; Giorgi, M.; Goeler, E. von; Goertz, S.; Gracia, G.; Groot, N. de; Perdekamp, M. Grosse; Guelmez, E.; Haft, K.; Harrach, D. von; Hasegawa, T.; Hautle, P.; Hayashi, N.; Heusch, C.A.; Horikawa, N.; Hughes, V.W.; Igo, G.; Ishimoto, S.; Iwata, T.; Kabuss, E.M.; Kageya, T.; Karev, A.; Kessler, H.J.; Ketel, T.J.; Kiryluk, J.; Kishi, A.; Kisselev, Yu.; Klostermann, L.; Kraemer, D.; Krivokhijine, V.; Kroeger, W.; Kurek, K.; Kyynaeraeinen, J.; Lamanna, M.; Landgraf, U.; Layda, T.; Le Goff, J.M.; Lehar, F.; Lesquen, A. de; Lichtenstadt, J.; Lindqvist, T.; Litmaath, M.; Lowe, M.; Magnon, A.; Mallot, G.K.; Marie, F.; Martin, A.; Martino, J.; Matsuda, T.; Mayes, B.; McCarthy, J.S.; Medved, K.; Meyer, W.; Middelkoop, G. van; Miller, D.; Miyachi, Y.; Mori, K.; Moromisato, J.; Nassalski, J.; Naumann, L.; Neganov, B.; Niinikoski, T.O.; Oberski, J.E.J.; Ogawa, A.; Ozben, C.; Parks, D.P.; Pereira, H.; Penzo, A.; Perrot-Kunne, F.; Peshekhonov, D.; Piegaia, R.; Pinsky, L.; Platchkov, S.; Plo, M.; Pose, D.; Postma, H.; Pretz, J.; Pussieux, T.; Pyrlik, J.; Raedel, G.; Reyhancan, I.; Reicherz, G.; Rieubland, J.M.; Rijllart, A.; Roberts, J.B.; Rock, S.; Rodriguez, M.; Rondio, E.; Rosado, A.; Roscherr, B.; Sabo, I.; Saborido, J.; Sandacz, A.; Savin, I.; Schiavon, P.; Schiller, A.; Schueler, K.P.; Segel, R.; Seitz, R.; Semertzidis, Y.; Sever, F.; Shanahan, P.; Sichtermann, E.P.; Simeoni, F.; Smirnov, G.I.; Staude, A.; Steinmetz, A.; Stiegler, U.; Stuhrmann, H.; Szleper, M.; Teichert, K.M.; Tessarotto, F.; Thers, D.; Tlaczala, W.; Trentalange, S.; Tripet, A.; Unel, G.; Velasco, M.; Vogt, J.; Voss, R.; Weinstein, R.; Whitten, C.; Windmolders, R.; Willumeit, R.; Wislicki, W.; Witzmann, A.; Zanetti, A.M.; Zaremba, K.; Zhao, J.

1999-01-01

The polarized target of the Spin Muon Collaboration at CERN was used for deep inelastic muon scattering experiments during 1993-1996 with a polarized muon beam to investigate the spin structure of the nucleon. Most of the experiments were carried out with longitudinal target polarization and 190 GeV muons, and some were done with transverse polarization and 100 GeV muons. Protons as well as deuterons were polarized by dynamic nuclear polarization (DNP) in three kinds of solid materials -- butanol, ammonia, and deuterated butanol -- with maximum degrees of polarization of 94%, 91% and 60%, respectively. Considerable attention was paid to the accuracies of the NMR polarization measurements and their analyses, the accuracies achieved were between 2.0% and 3.2%. The SMC target system with two cells of opposite polarizations, each cell 65 cm long and 5 cm in diameter, constitutes the largest polarized target system ever built and facilitates accurate spin asymmetry measurements. The design considerations, construction and performance of the target are reviewed

T-REX on-demand redox targeting in live cells.

Science.gov (United States)

Parvez, Saba; Long, Marcus J C; Lin, Hong-Yu; Zhao, Yi; Haegele, Joseph A; Pham, Vanha N; Lee, Dustin K; Aye, Yimon

2016-12-01

This protocol describes targetable reactive electrophiles and oxidants (T-REX)-a live-cell-based tool designed to (i) interrogate the consequences of specific and time-resolved redox events, and (ii) screen for bona fide redox-sensor targets. A small-molecule toolset comprising photocaged precursors to specific reactive redox signals is constructed such that these inert precursors specifically and irreversibly tag any HaloTag-fused protein of interest (POI) in mammalian and Escherichia coli cells. Syntheses of the alkyne-functionalized endogenous reactive signal 4-hydroxynonenal (HNE(alkyne)) and the HaloTag-targetable photocaged precursor to HNE(alkyne) (also known as Ht-PreHNE or HtPHA) are described. Low-energy light prompts photo-uncaging (t 1/2 <1-2 min) and target-specific modification. The targeted modification of the POI enables precisely timed and spatially controlled redox events with no off-target modification. Two independent pathways are described, along with a simple setup to functionally validate known targets or discover novel sensors. T-REX sidesteps mixed responses caused by uncontrolled whole-cell swamping with reactive signals. Modification and downstream response can be analyzed by in-gel fluorescence, proteomics, qRT-PCR, immunofluorescence, fluorescence resonance energy transfer (FRET)-based and dual-luciferase reporters, or flow cytometry assays. T-REX targeting takes 4 h from initial probe treatment. Analysis of targeted redox responses takes an additional 4-24 h, depending on the nature of the pathway and the type of readouts used.

Risky business: target choice in adoptive cell therapy.

Science.gov (United States)

Morgan, Richard A

2013-11-14

In this issue of Blood, Casucci et al present an elegant study that describes a potential new target for adoptive cell transfer (ACT), in this case CD44 splice variant 6 (CD44v6), and detail why it may be a good target for ACT and how to manage expected off-tumor/on-target toxicities.

Targeting senescence cells in pancreatic cancer | IDRC ...

International Development Research Centre (IDRC) Digital Library (Canada)

Targeting senescence cells in pancreatic cancer. Cellular senescence is a programmed response to oncogenic (tumour-causing) stress that aims to halt the expansion of cells with malignant potential. It does this by stopping the proliferation of pre-cancerous lesions and recruitment of the immune system for their elimination.

The cancer cell adhesion resistome: mechanisms, targeting and translational approaches.

Science.gov (United States)

Dickreuter, Ellen; Cordes, Nils

2017-06-27

Cell adhesion-mediated resistance limits the success of cancer therapies and is a great obstacle to overcome in the clinic. Since the 1990s, where it became clear that adhesion of tumor cells to the extracellular matrix is an important mediator of therapy resistance, a lot of work has been conducted to understand the fundamental underlying mechanisms and two paradigms were deduced: cell adhesion-mediated radioresistance (CAM-RR) and cell adhesion-mediated drug resistance (CAM-DR). Preclinical work has evidently demonstrated that targeting of integrins, adapter proteins and associated kinases comprising the cell adhesion resistome is a promising strategy to sensitize cancer cells to both radiotherapy and chemotherapy. Moreover, the cell adhesion resistome fundamentally contributes to adaptation mechanisms induced by radiochemotherapy as well as molecular drugs to secure a balanced homeostasis of cancer cells for survival and growth. Intriguingly, this phenomenon provides a basis for synthetic lethal targeted therapies simultaneously administered to standard radiochemotherapy. In this review, we summarize current knowledge about the cell adhesion resistome and highlight targeting strategies to override CAM-RR and CAM-DR.

Low-Cost Energy-Efficient 3-D Nano-Spikes-Based Electric Cell Lysis Chips

KAUST Repository

Riaz, Kashif; Leung, Siu; Fan, Zhiyong; Lee, Yi-Kuen

2017-01-01

Electric cell lysis (ECL) is a promising technique to be integrated with portable lab-on-a-chip without lysing agent due to its simplicity and fast processing. ECL is usually limited by the requirements of high power/voltage and costly fabrication

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

Directory of Open Access Journals (Sweden)

Purnima Bhat

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

Glypican-3 Targeting of Liver Cancer Cells Using Multifunctional Nanoparticles

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James O. Park

2011-01-01

Full Text Available Imaging is essential in accurately detecting, staging, and treating primary liver cancer (hepatocellular carcinoma [HCC], one of the most prevalent and lethal malignancies. We developed a novel multifunctional nanoparticle (NP specifically targeting glypican-3 (GPC3, a proteoglycan implicated in promotion of cell growth that is overexpressed in most HCCs. Quantitative real-time polymerase chain reaction was performed to confirm the differential GPC3 expression in two human HCC cells, Hep G2 (high and HLF (negligible. These cells were treated with biotin-conjugated GPC3 monoclonal antibody (αGPC3 and subsequently targeted using superparamagnetic iron oxide NPs conjugated to streptavidin and Alexa Fluor 647. Flow cytometry demonstrated that only GPC3-expressing Hep G2 cells were specifically targeted using this αGPC3-NP conjugate (fourfold mean fluorescence over nontargeted NP, and magnetic resonance imaging (MRI experiments showed similar findings (threefold R2 relaxivity. Confocal fluorescence microscopy localized the αGPC3 NPs only to the cell surface of GPC3-expressing Hep G2 cells. Further characterization of this construct demonstrated a negatively charged, monodisperse, 50 nm NP, ideally suited for tumor targeting. This GPC3-specific NP system, with dual-modality imaging capability, may enhance pretreatment MRI, enable refined intraoperative HCC visualization by near-infrared fluorescence, and be potentially used as a carrier for delivery of tumor-targeted therapies, improving patient outcomes.

Chemosensitization of cancer cells by siRNA using targeted nanogel delivery

International Nuclear Information System (INIS)

Dickerson, Erin B; Blackburn, William H; Smith, Michael H; Kapa, Laura B; Lyon, L Andrew; McDonald, John F

2010-01-01

Chemoresistance is a major obstacle in cancer treatment. Targeted therapies that enhance cancer cell sensitivity to chemotherapeutic agents have the potential to increase drug efficacy while reducing toxic effects on untargeted cells. Targeted cancer therapy by RNA interference (RNAi) is a relatively new approach that can be used to reversibly silence genes in vivo by selectively targeting genes such as the epidermal growth factor receptor (EGFR), which has been shown to increase the sensitivity of cancer cells to taxane chemotherapy. However, delivery represents the main hurdle for the broad development of RNAi therapeutics. We report here the use of core/shell hydrogel nanoparticles (nanogels) functionalized with peptides that specially target the EphA2 receptor to deliver small interfering RNAs (siRNAs) targeting EGFR. Expression of EGFR was determined by immunoblotting, and the effect of decreased EGFR expression on chemosensitization of ovarian cancer cells after siRNA delivery was investigated. Treatment of EphA2 positive Hey cells with siRNA-loaded, peptide-targeted nanogels decreased EGFR expression levels and significantly increased the sensitivity of this cell line to docetaxel (P < 0.05). Nanogel treatment of SK-OV-3 cells, which are negative for EphA2 expression, failed to reduce EGFR levels and did not increase docetaxel sensitivity (P > 0.05). This study suggests that targeted delivery of siRNAs by nanogels may be a promising strategy to increase the efficacy of chemotherapy drugs for the treatment of ovarian cancer. In addition, EphA2 is a viable target for therapeutic delivery, and the siRNAs are effectively protected by the nanogel carrier, overcoming the poor stability and uptake that has hindered clinical advancement of therapeutic siRNAs

MicroRNA-944 Affects Cell Growth by Targeting EPHA7 in Non-Small Cell Lung Cancer

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Minxia Liu

2016-09-01

Full Text Available MicroRNAs (miRNAs have critical roles in lung tumorigenesis and development. To determine aberrantly expressed miRNAs involved in non-small cell lung cancer (NSCLC and investigate pathophysiological functions and mechanisms, we firstly carried out small RNA deep sequencing in NSCLC cell lines (EPLC-32M1, A549 and 801D and a human immortalized cell line 16HBE, we then studied miRNA function by cell proliferation and apoptosis. cDNA microarray, luciferase reporter assay and miRNA transfection were used to investigate interaction between the miRNA and target gene. miR-944 was significantly down-regulated in NSCLC and had many putative targets. Moreover, the forced expression of miR-944 significantly inhibited the proliferation of NSCLC cells in vitro. By integrating mRNA expression data and miR-944-target prediction, we disclosed that EPHA7 was a potential target of miR-944, which was further verified by luciferase reporter assay and microRNA transfection. Our data indicated that miR-944 targets EPHA7 in NSCLC and regulates NSCLC cell proliferation, which may offer a new mechanism underlying the development and progression of NSCLC.

MicroRNA-944 Affects Cell Growth by Targeting EPHA7 in Non-Small Cell Lung Cancer.

Science.gov (United States)

Liu, Minxia; Zhou, Kecheng; Cao, Yi

2016-09-26

MicroRNAs (miRNAs) have critical roles in lung tumorigenesis and development. To determine aberrantly expressed miRNAs involved in non-small cell lung cancer (NSCLC) and investigate pathophysiological functions and mechanisms, we firstly carried out small RNA deep sequencing in NSCLC cell lines (EPLC-32M1, A549 and 801D) and a human immortalized cell line 16HBE, we then studied miRNA function by cell proliferation and apoptosis. cDNA microarray, luciferase reporter assay and miRNA transfection were used to investigate interaction between the miRNA and target gene. miR-944 was significantly down-regulated in NSCLC and had many putative targets. Moreover, the forced expression of miR-944 significantly inhibited the proliferation of NSCLC cells in vitro. By integrating mRNA expression data and miR-944-target prediction, we disclosed that EPHA7 was a potential target of miR-944, which was further verified by luciferase reporter assay and microRNA transfection. Our data indicated that miR-944 targets EPHA7 in NSCLC and regulates NSCLC cell proliferation, which may offer a new mechanism underlying the development and progression of NSCLC.

Cell targeting peptides as smart ligands for targeting of therapeutic or diagnostic agents: a systematic review.

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Mousavizadeh, Ali; Jabbari, Ali; Akrami, Mohammad; Bardania, Hassan

2017-10-01

Cell targeting peptides (CTP) are small peptides which have high affinity and specificity to a cell or tissue targets. They are typically identified by using phage display and chemical synthetic peptide library methods. CTPs have attracted considerable attention as a new class of ligands to delivery specifically therapeutic and diagnostic agents, because of the fact they have several advantages including easy synthesis, smaller physical sizes, lower immunogenicity and cytotoxicity and their simple and better conjugation to nano-carriers and therapeutic or diagnostic agents compared to conventional antibodies. In this systematic review, we will focus on the basic concepts concerning the use of cell-targeting peptides (CTPs), following the approaches of selecting them from peptide libraries. We discuss several developed strategies for cell-specific delivery of different cargos by CTPs, which are designed for drug delivery and diagnostic applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Aptamer-Mediated Polymeric Vehicles for Enhanced Cell-Targeted Drug Delivery.

Science.gov (United States)

Tan, Kei X; Danquah, Michael K; Sidhu, Amandeep; Yon, Lau Sie; Ongkudon, Clarence M

2018-02-08

The search for smart delivery systems for enhanced pre-clinical and clinical pharmaceutical delivery and cell targeting continues to be a major biomedical research endeavor owing to differences in the physicochemical characteristics and physiological effects of drug molecules, and this affects the delivery mechanisms to elicit maximum therapeutic effects. Targeted drug delivery is a smart evolution essential to address major challenges associated with conventional drug delivery systems. These challenges mostly result in poor pharmacokinetics due to the inability of the active pharmaceutical ingredients to specifically act on malignant cells thus, causing poor therapeutic index and toxicity to surrounding normal cells. Aptamers are oligonucleotides with engineered affinities to bind specifically to their cognate targets. Aptamers have gained significant interests as effective targeting elements for enhanced therapeutic delivery as they can be generated to specifically bind to wide range of targets including proteins, peptides, ions, cells and tissues. Notwithstanding, effective delivery of aptamers as therapeutic vehicles is challenged by cell membrane electrostatic repulsion, endonuclease degradation, low pH cleavage, and binding conformation stability. The application of molecularly engineered biodegradable and biocompatible polymeric particles with tunable features such as surface area and chemistry, particulate size distribution and toxicity creates opportunities to develop smart aptamer-mediated delivery systems for controlled drug release. This article discusses opportunities for particulate aptamer-drug formulations to advance current drug delivery modalities by navigating active ingredients through cellular and biomolecular traffic to target sites for sustained and controlled release at effective therapeutic dosages while minimizing systemic cytotoxic effects. A proposal for a novel drug-polymer-aptamer-polymer (DPAP) design of aptamer-drug formulation with

Therapeutic targeting strategies using endogenous cells and proteins.

Science.gov (United States)

Parayath, Neha N; Amiji, Mansoor M

2017-07-28

Targeted drug delivery has become extremely important in enhancing efficacy and reducing the toxicity of therapeutics in the treatment of various disease conditions. Current approaches include passive targeting, which relies on naturally occurring differences between healthy and diseased tissues, and active targeting, which utilizes various ligands that can recognize targets expressed preferentially at the diseased site. Clinical translation of these mechanisms faces many challenges including the immunogenic and toxic effects of these non-natural systems. Thus, use of endogenous targeting systems is increasingly gaining momentum. This review is focused on strategies for employing endogenous moieties, which could serve as safe and efficient carriers for targeted drug delivery. The first part of the review involves cells and cellular components as endogenous carriers for therapeutics in multiple disease states, while the second part discusses the use of endogenous plasma components as endogenous carriers. Further understanding of the biological tropism with cells and proteins and the newer generation of delivery strategies that exploits these endogenous approaches promises to provide better solutions for site-specific delivery and could further facilitate clinical translations. Copyright © 2017 Elsevier B.V. All rights reserved.

An assay system for factors involved in mammalian DNA replication

International Nuclear Information System (INIS)

Reinhard, P.; Maillart, P.; Schluchter, M.; Gautschi, J.R.; Schindler, R.

1979-01-01

An assay for cellular factors stimulating DNA synthesis by partially lysed CHO cells is presented. The assay is based on the observation that in highly lysed cells, DNA synthesis, as determined by [ 3 H]dTTP incorporation, was only 2-5% of that in gently lysed cells, and that this low level of DNA synthesis could be increased by a factor of approx. 50 by the addition of CHO cell extract (i.e. supernatant of a cell homogenate subjected to high-speed centrifugation.) (Auth.)

Enhancing oral vaccine potency by targeting intestinal M cells.

Directory of Open Access Journals (Sweden)

Ali Azizi

2010-11-01

Full Text Available The immune system in the gastrointestinal tract plays a crucial role in the control of infection, as it constitutes the first line of defense against mucosal pathogens. The attractive features of oral immunization have led to the exploration of a variety of oral delivery systems. However, none of these oral delivery systems have been applied to existing commercial vaccines. To overcome this, a new generation of oral vaccine delivery systems that target antigens to gut-associated lymphoid tissue is required. One promising approach is to exploit the potential of microfold (M cells by mimicking the entry of pathogens into these cells. Targeting specific receptors on the apical surface of M cells might enhance the entry of antigens, initiating the immune response and consequently leading to protection against mucosal pathogens. In this article, we briefly review the challenges associated with current oral vaccine delivery systems and discuss strategies that might potentially target mouse and human intestinal M cells.

Cell cycle and anti-estrogen effects synergize to regulate cell proliferation and ER target gene expression.

Directory of Open Access Journals (Sweden)

Mathieu Dalvai

Full Text Available Antiestrogens are designed to antagonize hormone induced proliferation and ERalpha target gene expression in mammary tumor cells. Commonly used drugs such as OH-Tamoxifen and ICI 182780 (Fulvestrant block cell cycle progression in G0/G1. Inversely, the effect of cell cycle stage on ER regulated gene expression has not been tested directly. We show that in ERalpha-positive breast cancer cells (MCF-7 the estrogen receptor gene and downstream target genes are cell cycle regulated with expression levels varying as much as three-fold between phases of the cell cycle. Steroid free culture conditions commonly used to assess the effect of hormones or antiestrogens on gene expression also block MCF-7 cells in G1-phase when several ERalpha target genes are overexpressed. Thus, cell cycle effects have to be taken into account when analyzing the impact of hormonal treatments on gene transcription. We found that antiestrogens repress transcription of several ERalpha target genes specifically in S phase. This observation corroborates the more rapid and strong impact of antiestrogen treatments on cell proliferation in thymidine, hydroxyurea or aphidicolin arrested cells and correlates with an increase of apoptosis compared to similar treatments in lovastatin or nocodazol treated cells. Hence, cell cycle effects synergize with the action of antiestrogens. An interesting therapeutic perspective could be to enhance the action of anti-estrogens by associating hormone-therapy with specific cell cycle drugs.

ATR-dependent bystander effects in non-targeted cells

International Nuclear Information System (INIS)

Burdak-Rothkamm, S.

2007-01-01

Complete text of publication follows. Radiation induced non-targeted bystander effects have been reported for a range of endpoints including the induction of γH2AX foci which serve as a marker for DNA double strand breaks. We have recently reported the induction of γH2AX foci in non-targeted bystander cells up to 48 hours after irradiation and the involvement of reactive oxygen species (ROS) and TGF-beta 1 in the induction of γH2AX foci (Oncogene (2007) 26:993-1002). Here, we wanted to determine the role of the PI3-like kinases ATM, ATR and DNA-PK in DNA damage signalling in bystander cells. Conditioned medium from T98G cells irradiated with 2 Gy of X-rays was transferred onto non-irradiated cells that were subsequently analysed for the induction of γH2AX, ATR and 53BP1 foci as well as clonogenic survival. Irradiated T98G glioma cells generated signals that induced γH2AX and 53BP1 foci in cells treated with the conditioned medium from irradiated cells. These foci co-localised with ATR foci. Inhibition of ATM and DNA-PK could not suppress the induction of bystander γH2AX foci whereas the mutation of ATR in Seckel cells abrogated bystander foci induction. A restriction of bystander foci to the S-phase of the cell cycle both in T98G cells and in ATR- proficient fibroblasts was observed. These results identify ATR as a central player within the bystander signalling cascade leading to γH2AX and 53BP1 foci formation, and suggest a mechanism of DNA damage induction in non-targeted cells. Further investigations have shown decreased clonogenic cell survival in bystander T98G and ATR wild-type fibroblasts. ATR mutated Seckel cells and also ATM-/- fibroblasts were resistant to this effect suggesting a role for both ATR and ATM in the bystander signalling cascade with regard to cell survival. Taken together, these observations support a hypothesis of DNA damage-induced accumulation of stalled replication forks in bystander cells which are subsequently processed by

A new prospect in cancer therapy: targeting cancer stem cells to eradicate cancer.

Science.gov (United States)

Chen, Li-Sha; Wang, An-Xin; Dong, Bing; Pu, Ke-Feng; Yuan, Li-Hua; Zhu, Yi-Min

2012-12-01

According to the cancer stem cell theory, cancers can be initiated by cancer stem cells. This makes cancer stem cells prime targets for therapeutic intervention. Eradicating cancer stem cells by efficient targeting agents may have the potential to cure cancer. In this review, we summarize recent breakthroughs that have improved our understanding of cancer stem cells, and we discuss the therapeutic strategy of targeting cancer stem cells, a promising future direction for cancer stem cell research.

The polarized double cell target of the SMC

CERN Document Server

Adams, D; Adeva, B; Arik, E; Arvidson, A; Badelek, B; Ballintijn, M K; Bardin, G; Baum, G; Berglund, P; Betev, L; Bird, I G; Birsa, R; Björkholm, P; Bonner, B E; De Botton, N R; Boutemeur, M; Bradamante, Franco; Bravar, A; Bressan, A; Bültmann, S; Burtin, E; Cavata, C; Crabb, D; Cranshaw, J; Çuhadar-Dönszelmann, T; Dalla Torre, S; Van Dantzig, R; Derro, B R; Deshpande, A A; Dhawan, S K; Dulya, C M; Dyring, A; Eichblatt, S; Faivre, Jean-Claude; Fasching, D; Feinstein, F; Fernández, C; Forthmann, S; Frois, Bernard; Gallas, A; Garzón, J A; Gaussiran, T; Gilly, H; Giorgi, M A; von Goeler, E; Görtz, S; Gracia, G; De Groot, N; Grosse-Perdekamp, M; Gülmez, E; Haft, K; Von Harrach, D; Hasegawa, T; Hautle, P; Hayashi, N; Heusch, C A; Horikawa, N; Hughes, V W; Igo, G; Ishimoto, S; Iwata, T; Kabuss, E M; Kageya, T; Karev, A G; Kessler, H J; Ketel, T; Kiryluk, J; Kishi, A; Kiselev, Yu F; Klostermann, L; Krämer, Dietrich; Krivokhizhin, V G; Kröger, W; Kurek, K; Kyynäräinen, J; Lamanna, M; Landgraf, U; Layda, T; Le Goff, J M; Lehár, F; de Lesquen, A; Lichtenstadt, J; Lindqvist, T; Litmaath, M; Loewe, M; Magnon, A; Mallot, G K; Marie, F; Martin, A; Martino, J; Matsuda, T; Mayes, B W; McCarthy, J S; Medved, K S; Meyer, W T; Van Middelkoop, G; Miller, D; Miyachi, Y; Mori, K; Moromisato, J H; Nassalski, J P; Naumann, Lutz; Neganov, B S; Niinikoski, T O; Oberski, J; Ogawa, A; Ozben, C; Parks, D P; Pereira, H; Penzo, Aldo L; Perrot-Kunne, F; Peshekhonov, V D; Piegaia, R; Pinsky, L; Platchkov, S K; Pló, M; Pose, D; Postma, H; Pretz, J; Pussieux, T; Pyrlik, J; Rädel, G; Reyhancan, I; Reicherz, G; Rijllart, A; Roberts, J B; Rock, S E; Rodríguez, M; Rondio, Ewa; Rosado, A; Roscherr, B; Sabo, I; Saborido, J; Sandacz, A; Savin, I A; Schiavon, R P; Schiller, A; Schüler, K P; Segel, R E; Seitz, R; Semertzidis, Y K; Sever, F; Shanahan, P; Sichtermann, E P; Simeoni, F; Smirnov, G I; Staude, A; Steinmetz, A; Stiegler, U; Stuhrmann, H B; Szleper, M; Teichert, K M; Tessarotto, F; Thers, D; Tlaczala, W; Trentalange, S; Tripet, A; Ünel, G; Velasco, M; Vogt, J; Voss, Rüdiger; Weinstein, R; Whitten, C; Windmolders, R; Willumeit, R; Wislicki, W; Witzmann, A; Zanetti, A M; Zaremba, K; Zhao, J

1999-01-01

The polarized target of the Spin Muon Collaboration at CERN was used for deep inelastic muon scattering experiments during 1993 to 1996 with a polarized muon beam to investigate the spin structure of the nucleon. Most of the experiments were carried out with longitudinal target polarization and 190 GeV muons, and some were done with transverse polarization and 100 GeV muons. Protons as well as deuterons were polarized by dynamic nuclear polarization (DNP) in three kinds of solid materials $-$ butanol, ammonia, and deuterated butanol, with maximum degrees of polarization of 94, 91, and 60 \\%, respectively. Considerable attention was paid to the accuracies of the NMR polarization measurements and their analyses. The achieved accuracies were between 2.0 and 3.2 \\%. The SMC target system with two cells of opposite polarizations, each cell 65 cm long and 5 cm in diameter, constitutes the largest polarized target system ever built and facilitates accurate spin asymmetry measurements. The design considerations, the ...

Endothelial Cell-Targeted Adenoviral Vector for Suppressing Breast Malignancies

National Research Council Canada - National Science Library

Huang, Shuang

2004-01-01

.... Our proposal is designed to develop an endothelial cell-targeted adenoviral vector and to use the targeted vector to express high levels of anticancer therapeutic genes in the sites of angiogenenic...

Luteolin suppresses cancer cell proliferation by targeting vaccinia-related kinase 1.

Directory of Open Access Journals (Sweden)

Ye Seul Kim

Full Text Available Uncontrolled proliferation, a major feature of cancer cells, is often triggered by the malfunction of cell cycle regulators such as protein kinases. Recently, cell cycle-related protein kinases have become attractive targets for anti-cancer therapy, because they play fundamental roles in cellular proliferation. However, the protein kinase-targeted drugs that have been developed so far do not show impressive clinical results and also display severe side effects; therefore, there is undoubtedly a need to investigate new drugs targeting other protein kinases that are critical in cell cycle progression. Vaccinia-related kinase 1 (VRK1 is a mitotic kinase that functions in cell cycle regulation by phosphorylating cell cycle-related substrates such as barrier-to-autointegration factor (BAF, histone H3, and the cAMP response element (CRE-binding protein (CREB. In our study, we identified luteolin as the inhibitor of VRK1 by screening a small-molecule natural compound library. Here, we evaluated the efficacy of luteolin as a VRK1-targeted inhibitor for developing an effective anti-cancer strategy. We confirmed that luteolin significantly reduces VRK1-mediated phosphorylation of the cell cycle-related substrates BAF and histone H3, and directly interacts with the catalytic domain of VRK1. In addition, luteolin regulates cell cycle progression by modulating VRK1 activity, leading to the suppression of cancer cell proliferation and the induction of apoptosis. Therefore, our study suggests that luteolin-induced VRK1 inhibition may contribute to establish a novel cell cycle-targeted strategy for anti-cancer therapy.

ErbB-targeted CAR T-cell immunotherapy of cancer.

Science.gov (United States)

Whilding, Lynsey M; Maher, John

2015-01-01

Chimeric antigen receptor (CAR) based immunotherapy has been under development for the last 25 years and is now a promising new treatment modality in the field of cancer immunotherapy. The approach involves genetically engineering T cells to target malignant cells through expression of a bespoke fusion receptor that couples an HLA-independent antigen recognition domain to one or more intracellular T-cell activating modules. Multiple clinical trials are now underway in several centers to investigate CAR T-cell immunotherapy of diverse hematologic and solid tumor types. The most successful results have been achieved in the treatment of patients with B-cell malignancies, in whom several complete and durable responses have been achieved. This review focuses on the preclinical and clinical development of CAR T-cell immunotherapy of solid cancers, targeted against members of the ErbB family.

Modelling T4 cell count as a marker of HIV progression in the absence of any defence mechanism

Directory of Open Access Journals (Sweden)

VSS Yadavalli

2010-12-01

Full Text Available The T4 cell count, which is considered one of the markers of disease progression in an HIV infected individual, is modelled in this paper. The World Health Organisation has recently advocated that countries encourage HIV infected individuals to commence antiretroviral treatments once their T4 cell count drops below 350 cells per ml of blood (this threshold was formerly 200 cells per ml of blood. This recommendation is made because when the T4 cell count is low, the T4 cells are unable to mount an effective immune response against antigens and any such foreign matters in the body, and consequently the individual becomes susceptible to opportunistic infections and lymphomas. A stochastic catastrophe model is developed in this paper to obtain the mean, variance and covariance of the uninfected, infected and lysed T4 cells. The amount of toxin produced in an HIV infected person from the time of infection to a later time may also be obtained from the model. Numerical illustrations of the correlation structures between uninfected and infected T4 cells, and between the infected and lysed T4 cells are also presented.

GEM-loaded magnetic albumin nanospheres modified with cetuximab for simultaneous targeting, magnetic resonance imaging, and double-targeted thermochemotherapy of pancreatic cancer cells.

Science.gov (United States)

Wang, Ling; An, Yanli; Yuan, Chenyan; Zhang, Hao; Liang, Chen; Ding, Fengan; Gao, Qi; Zhang, Dongsheng

2015-01-01

Targeted delivery is a promising strategy to improve the diagnostic imaging and therapeutic effect of cancers. In this paper, novel cetuximab (C225)-conjugated, gemcitabine (GEM)-containing magnetic albumin nanospheres (C225-GEM/MANs) were fabricated and applied as a theranostic nanocarrier to conduct simultaneous targeting, magnetic resonance imaging (MRI), and double-targeted thermochemotherapy against pancreatic cancer cells. Fe3O4 nanoparticles (NPs) and GEM co-loaded albumin nanospheres (GEM/MANs) were prepared, and then C225 was further conjugated to synthesize C225-GEM/MANs. Their morphology, mean particle size, GEM encapsulation ratio, specific cell-binding ability, and thermal dynamic profiles were characterized. The effects of discriminating different EGFR-expressing pancreatic cancer cells (AsPC-1 and MIA PaCa-2) and monitoring cellular targeting effects were assessed by targeted MRI. Lastly, the antitumor efficiency of double/C225/magnetic-targeted and nontargeted thermochemotherapy was compared with chemotherapy alone using 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and flow cytometry (FCM) assay. When treated with targeted nanospheres, AsPC-1 cells showed a significantly less intense MRI T2 signal than MIA PaCa-2 cells, while both cells had similar signal strength when incubated with nontargeted nanospheres. T2 signal intensity was significantly lower when magnetic and C225 targeting were combined, rather than used alone. The inhibitory and apoptotic rates of each thermochemotherapy group were significantly higher than those of the chemotherapy-alone groups. Additionally, both MTT and FCM analysis verified that double-targeted thermochemotherapy had the highest targeted killing efficiency among all groups. The C225-GEM/MANs can distinguish various EGFR-expressing live pancreatic cancer cells, monitor diverse cellular targeting effects using targeted MRI imaging, and efficiently mediate double-targeted thermochemotherapy

Phenotypic high-throughput screening elucidates target pathway in breast cancer stem cell-like cells.

Science.gov (United States)

Carmody, Leigh C; Germain, Andrew R; VerPlank, Lynn; Nag, Partha P; Muñoz, Benito; Perez, Jose R; Palmer, Michelle A J

2012-10-01

Cancer stem cells (CSCs) are resistant to standard cancer treatments and are likely responsible for cancer recurrence, but few therapies target this subpopulation. Due to the difficulty in propagating CSCs outside of the tumor environment, previous work identified CSC-like cells by inducing human breast epithelial cells into an epithelial-to-mesenchymal transdifferentiated state (HMLE_sh_ECad). A phenotypic screen was conducted against HMLE_sh_ECad with 300 718 compounds from the Molecular Libraries Small Molecule Repository to identify selective inhibitors of CSC growth. The screen yielded 2244 hits that were evaluated for toxicity and selectivity toward an isogenic control cell line. An acyl hydrazone scaffold emerged as a potent and selective scaffold targeting HMLE_sh_ECad. Fifty-three analogues were acquired and tested; compounds ranged in potency from 790 nM to inactive against HMLE_sh_ECad. Of the analogues, ML239 was best-in-class with an IC(50)= 1.18 µM against HMLE_sh_ECad, demonstrated a >23-fold selectivity over the control line, and was toxic to another CSC-like line, HMLE_shTwist, and a breast carcinoma cell line, MDA-MB-231. Gene expression studies conducted with ML239-treated cells showed altered gene expression in the NF-κB pathway in the HMLE_sh_ECad line but not in the isogenic control line. Future studies will be directed toward the identification of ML239 target(s).

Detecting drug-target binding in cells using fluorescence-activated cell sorting coupled with mass spectrometry analysis

Science.gov (United States)

Wilson, Kris; Webster, Scott P.; Iredale, John P.; Zheng, Xiaozhong; Homer, Natalie Z.; Pham, Nhan T.; Auer, Manfred; Mole, Damian J.

2018-01-01

The assessment of drug-target engagement for determining the efficacy of a compound inside cells remains challenging, particularly for difficult target proteins. Existing techniques are more suited to soluble protein targets. Difficult target proteins include those with challenging in vitro solubility, stability or purification properties that preclude target isolation. Here, we report a novel technique that measures intracellular compound-target complex formation, as well as cellular permeability, specificity and cytotoxicity-the toxicity-affinity-permeability-selectivity (TAPS) technique. The TAPS assay is exemplified here using human kynurenine 3-monooxygenase (KMO), a challenging intracellular membrane protein target of significant current interest. TAPS confirmed target binding of known KMO inhibitors inside cells. We conclude that the TAPS assay can be used to facilitate intracellular hit validation on most, if not all intracellular drug targets.

Targeted cancer cell death induced by biofunctionalized magnetic nanowires

KAUST Repository

Contreras, Maria F.; Ravasi, Timothy; Kosel, Jü rgen

2014-01-01

Magnetic micro and nanomaterials are increasingly interesting for biomedical applications since they possess many advantageous properties: they can become biocompatible, they can be functionalized to target specific cells and they can be remotely manipulated by magnetic fields. The goal of this study is to use antibody-functionalized nickel nanowires (Ab-NWs) as an alternative method in cancer therapy overcoming the limitations of current treatments that lack specificity and are highly cytotoxic. Ab-NWs have been incubated with cancer cells and a 12% drop on cell viability was observed for a treatment of only 10 minutes and an alternating magnetic field of low intensity and low frequency. It is believed that the Ab-NWs vibrate transmitting a mechanical force to the targeted cells inducing cell death. © 2014 IEEE.

Targeted cancer cell death induced by biofunctionalized magnetic nanowires

KAUST Repository

Contreras, Maria F.

2014-02-01

Magnetic micro and nanomaterials are increasingly interesting for biomedical applications since they possess many advantageous properties: they can become biocompatible, they can be functionalized to target specific cells and they can be remotely manipulated by magnetic fields. The goal of this study is to use antibody-functionalized nickel nanowires (Ab-NWs) as an alternative method in cancer therapy overcoming the limitations of current treatments that lack specificity and are highly cytotoxic. Ab-NWs have been incubated with cancer cells and a 12% drop on cell viability was observed for a treatment of only 10 minutes and an alternating magnetic field of low intensity and low frequency. It is believed that the Ab-NWs vibrate transmitting a mechanical force to the targeted cells inducing cell death. © 2014 IEEE.

HMGB1 Is Involved in IFN-α Production and TRAIL Expression by HIV-1-Exposed Plasmacytoid Dendritic Cells: Impact of the Crosstalk with NK Cells.

OpenAIRE

Héla Saïdi; Marlène Bras; Pauline Formaglio; Marie-Thérèse Melki; Bruno Charbit; Jean-Philippe Herbeuval; Marie-Lise Gougeon

2016-01-01

Plasmacytoid dendritic cells (pDCs) are innate sensors of viral infections and important mediators of antiviral innate immunity through their ability to produce large amounts of IFN-?. Moreover, Toll-like receptor 7 (TLR7) and 9 (TLR9) ligands, such as HIV and CpG respectively, turn pDCs into TRAIL-expressing killer pDCs able to lyse HIV-infected CD4+ T cells. NK cells can regulate antiviral immunity by modulating pDC functions, and pDC production of IFN-? as well as cell?cell contact is requ...

Profiling the Targets of Protective CD8+ T Cell Responses to Infection

Directory of Open Access Journals (Sweden)

Joseph T. Bruder

2017-12-01

Full Text Available T cells are critical effectors of host immunity that target intracellular pathogens, such as the causative agents of HIV, tuberculosis, and malaria. The development of vaccines that induce effective cell-mediated immunity against such pathogens has proved challenging; for tuberculosis and malaria, many of the antigens targeted by protective T cells are not known. Here, we report a novel approach for screening large numbers of antigens as potential targets of T cells. Malaria provides an excellent model to test this antigen discovery platform because T cells are critical mediators of protection following immunization with live sporozoite vaccines and the specific antigen targets are unknown. We generated an adenovirus array by cloning 312 highly expressed pre-erythrocytic Plasmodium yoelii antigens into adenovirus vectors using high-throughput methodologies. The array was screened to identify antigen-specific CD8+ T cells induced by a live sporozoite vaccine regimen known to provide high levels of sterile protection mediated by CD8+ T cells. We identified 69 antigens that were targeted by CD8+ T cells induced by this vaccine regimen. The antigen that recalled the highest frequency of CD8+ T cells, PY02605, induced protective responses in mice, demonstrating proof of principle for this approach in identifying antigens for vaccine development.

Neuraminidase treatment of respiratory syncytial virus-infected cells or virions, but not target cells, enhances cell-cell fusion and infection

International Nuclear Information System (INIS)

Barretto, Naina; Hallak, Louay K.; Peeples, Mark E.

2003-01-01

Respiratory syncytial virus (RSV) infection of HeLa cells induces fusion, but transient expression of the three viral glycoproteins induces fusion poorly, if at all. We found that neuraminidase treatment of RSV-infected cells to remove sialic acid (SA) increases fusion dramatically and that the same treatment of transiently transfected cells expressing the three viral glycoproteins, or even cells expressing the fusion (F) protein alone, results in easily detectable fusion. Neuraminidase treatment of the effector cells, expressing the viral glycoproteins, enhanced fusion while treatment of the target cells did not. Likewise, infectivity was increased by treating virions with neuraminidase, but not by treating target cells. Reduction of charge repulsion by removal of the negatively charged SA is unlikely to explain this effect, since removal of negative charges from either membrane would reduce charge repulsion. Infection with neuraminidase-treated virus remained heparan-sulfate-dependent, indicating that a novel attachment mechanism is not revealed by SA removal. Interestingly, neuraminidase enhancement of RSV infectivity was less pronounced in a virus expressing both the G and the F glycoproteins, compared to virus expressing only the F glycoprotein, possibly suggesting that the G protein sterically hinders access of the neuraminidase to its fusion-enhancing target

Evaluation of Cytochalasin B-Induced Membrane Vesicles Fusion Specificity with Target Cells

Directory of Open Access Journals (Sweden)

Marina Gomzikova

2018-01-01

Full Text Available Extracellular vesicles (EV represent a promising vector system for biomolecules and drug delivery due to their natural origin and participation in intercellular communication. As the quantity of EVs is limited, it was proposed to induce the release of membrane vesicles from the surface of human cells by treatment with cytochalasin B. Cytochalasin B-induced membrane vesicles (CIMVs were successfully tested as a vector for delivery of dye, nanoparticles, and a chemotherapeutic. However, it remained unclear whether CIMVs possess fusion specificity with target cells and thus might be used for more targeted delivery of therapeutics. To answer this question, CIMVs were obtained from human prostate cancer PC3 cells. The diameter of obtained CIMVs was 962,13 ± 140,6 nm. We found that there is no statistically significant preference in PC3 CIMVs fusion with target cells of the same type. According to our observations, the greatest impact on CIMVs entry into target cells is by the heterophilic interaction of CIMV membrane receptors with the surface proteins of target cells.

MiR-223 suppresses cell proliferation by targeting IGF-1R.

Directory of Open Access Journals (Sweden)

Cheng You Jia

Full Text Available To study the roles of microRNA-223 (miR-223 in regulation of cell growth, we established a miR-223 over-expression model in HeLa cells infected with miR-223 by Lentivirus pLL3.7 system. We observed in this model that miR-223 significantly suppressed the proliferation, growth rate, colony formation of HeLa cells in vitro, and in vivo tumorigenicity or tumor formation in nude mice. To investigate the mechanisms involved, we scanned and examined the potential and putative target molecules of miR-223 by informatics, quantitative PCR and Western blot, and found that insulin-like growth factor-1 receptor (IGF-1R was the functional target of miR-223 inhibition of cell proliferation. Targeting IGF-1R by miR-223 was not only seen in HeLa cells, but also in leukemia and hepatoma cells. The downstream pathway, Akt/mTOR/p70S6K, to which the signal was mediated by IGF-1R, was inhibited as well. The relative luciferase activity of the reporter containing wild-type 3'UTR(3'untranslated region of IGF-1R was significantly suppressed, but the mutant not. Silence of IGF-1R expression by vector-based short hairpin RNA resulted in the similar inhibition with miR-223. Contrarily, rescued IGF-1R expression in the cells that over-expressed miR-223, reversed the inhibition caused by miR-223 via introducing IGF-1R cDNA that didn't contain the 3'UTR. Meanwhile, we also noted that miR-223 targeted Rasa1, but the downstream molecules mediated by Rasa1 was neither targeted nor regulated. Therefore we believed that IGF-1R was the functional target for miR-223 suppression of cell proliferation and its downstream PI3K/Akt/mTOR/p70S6K pathway suppressed by miR-223 was by targeting IGF-1R.

High frequency of cytolytic 21-hydroxylase-specific CD8+ T cells in autoimmune Addison's disease patients.

Science.gov (United States)

Dawoodji, Amina; Chen, Ji-Li; Shepherd, Dawn; Dalin, Frida; Tarlton, Andrea; Alimohammadi, Mohammad; Penna-Martinez, Marissa; Meyer, Gesine; Mitchell, Anna L; Gan, Earn H; Bratland, Eirik; Bensing, Sophie; Husebye, Eystein S; Pearce, Simon H; Badenhoop, Klaus; Kämpe, Olle; Cerundolo, Vincenzo

2014-09-01

The mechanisms behind destruction of the adrenal glands in autoimmune Addison's disease remain unclear. Autoantibodies against steroid 21-hydroxylase, an intracellular key enzyme of the adrenal cortex, are found in >90% of patients, but these autoantibodies are not thought to mediate the disease. In this article, we demonstrate highly frequent 21-hydroxylase-specific T cells detectable in 20 patients with Addison's disease. Using overlapping 18-aa peptides spanning the full length of 21-hydroxylase, we identified immunodominant CD8(+) and CD4(+) T cell responses in a large proportion of Addison's patients both ex vivo and after in vitro culture of PBLs ≤20 y after diagnosis. In a large proportion of patients, CD8(+) and CD4(+) 21-hydroxylase-specific T cells were very abundant and detectable in ex vivo assays. HLA class I tetramer-guided isolation of 21-hydroxylase-specific CD8(+) T cells showed their ability to lyse 21-hydroxylase-positive target cells, consistent with a potential mechanism for disease pathogenesis. These data indicate that strong CTL responses to 21-hydroxylase often occur in vivo, and that reactive CTLs have substantial proliferative and cytolytic potential. These results have implications for earlier diagnosis of adrenal failure and ultimately a potential target for therapeutic intervention and induction of immunity against adrenal cortex cancer. Copyright © 2014 by The American Association of Immunologists, Inc.

Mitochondria-targeted vitamin E analogs inhibit breast cancer cell energy metabolism and promote cell death

International Nuclear Information System (INIS)

Cheng, Gang; Zielonka, Jacek; McAllister, Donna M; Mackinnon, A Craig Jr; Joseph, Joy; Dwinell, Michael B; Kalyanaraman, Balaraman

2013-01-01

Recent research has revealed that targeting mitochondrial bioenergetic metabolism is a promising chemotherapeutic strategy. Key to successful implementation of this chemotherapeutic strategy is the use of new and improved mitochondria-targeted cationic agents that selectively inhibit energy metabolism in breast cancer cells, while exerting little or no long-term cytotoxic effect in normal cells. In this study, we investigated the cytotoxicity and alterations in bioenergetic metabolism induced by mitochondria-targeted vitamin E analog (Mito-chromanol, Mito-ChM) and its acetylated ester analog (Mito-ChMAc). Assays of cell death, colony formation, mitochondrial bioenergetic function, intracellular ATP levels, intracellular and tissue concentrations of tested compounds, and in vivo tumor growth were performed. Both Mito-ChM and Mito-ChMAc selectively depleted intracellular ATP and caused prolonged inhibition of ATP-linked oxygen consumption rate in breast cancer cells, but not in non-cancerous cells. These effects were significantly augmented by inhibition of glycolysis. Mito-ChM and Mito-ChMAc exhibited anti-proliferative effects and cytotoxicity in several breast cancer cells with different genetic background. Furthermore, Mito-ChM selectively accumulated in tumor tissue and inhibited tumor growth in a xenograft model of human breast cancer. We conclude that mitochondria-targeted small molecular weight chromanols exhibit selective anti-proliferative effects and cytotoxicity in multiple breast cancer cells, and that esterification of the hydroxyl group in mito-chromanols is not a critical requirement for its anti-proliferative and cytotoxic effect

Designing and modeling a centrifugal microfluidic device to separate target blood cells

Science.gov (United States)

Shamloo, Amir; Selahi, AmirAli; Madadelahi, Masoud

2016-03-01

The objective of this study is to design a novel and efficient portable lab-on-a-CD (LOCD) microfluidic device for separation of specific cells (target cells) using magnetic beads. In this study the results are shown for neutrophils as target cells. However, other kinds of target cells can be separated in a similar approach. The designed microfluidics can be utilized as a point of care system for neutrophil detection. This microfluidic system employs centrifugal and magnetic forces for separation. After model validation by the experimental data in the literature (that may be used as a design tool for developing centrifugo-magnetophoretic devices), two models are presented for separation of target cells using magnetic beads. The first model consists of one container in the inlet section and two containers in the outlets. Initially, the inlet container is filled with diluted blood sample which is a mixture of red blood cells (RBCs) plus neutrophils which are attached to Magnetic beads. It is shown that by using centrifugal and magnetic forces, this model can separate all neutrophils with recovery factor of ~100%. In the second model, due to excess of magnetic beads in usual experimental analysis (to ensure that all target cells are attached to them) the geometry is improved by adding a third outlet for these free magnetic beads. It is shown that at angular velocity of 45 rad s-1, recovery factor of 100% is achievable for RBCs, free magnetic beads and neutrophils as target cells.

Systematic identification of combinatorial drivers and targets in cancer cell lines.

Directory of Open Access Journals (Sweden)

Adel Tabchy

Full Text Available There is an urgent need to elicit and validate highly efficacious targets for combinatorial intervention from large scale ongoing molecular characterization efforts of tumors. We established an in silico bioinformatic platform in concert with a high throughput screening platform evaluating 37 novel targeted agents in 669 extensively characterized cancer cell lines reflecting the genomic and tissue-type diversity of human cancers, to systematically identify combinatorial biomarkers of response and co-actionable targets in cancer. Genomic biomarkers discovered in a 141 cell line training set were validated in an independent 359 cell line test set. We identified co-occurring and mutually exclusive genomic events that represent potential drivers and combinatorial targets in cancer. We demonstrate multiple cooperating genomic events that predict sensitivity to drug intervention independent of tumor lineage. The coupling of scalable in silico and biologic high throughput cancer cell line platforms for the identification of co-events in cancer delivers rational combinatorial targets for synthetic lethal approaches with a high potential to pre-empt the emergence of resistance.

Systematic identification of combinatorial drivers and targets in cancer cell lines.

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Tabchy, Adel; Eltonsy, Nevine; Housman, David E; Mills, Gordon B

2013-01-01

There is an urgent need to elicit and validate highly efficacious targets for combinatorial intervention from large scale ongoing molecular characterization efforts of tumors. We established an in silico bioinformatic platform in concert with a high throughput screening platform evaluating 37 novel targeted agents in 669 extensively characterized cancer cell lines reflecting the genomic and tissue-type diversity of human cancers, to systematically identify combinatorial biomarkers of response and co-actionable targets in cancer. Genomic biomarkers discovered in a 141 cell line training set were validated in an independent 359 cell line test set. We identified co-occurring and mutually exclusive genomic events that represent potential drivers and combinatorial targets in cancer. We demonstrate multiple cooperating genomic events that predict sensitivity to drug intervention independent of tumor lineage. The coupling of scalable in silico and biologic high throughput cancer cell line platforms for the identification of co-events in cancer delivers rational combinatorial targets for synthetic lethal approaches with a high potential to pre-empt the emergence of resistance.

High frequency of cytolytic 21-Hydroxylase specific CD8+ T cells in autoimmune Addison’s disease patients1

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Dawoodji, Amina; Chen, Ji-Li; Shepherd, Dawn; Dalin, Frida; Tarlton, Andrea; Alimohammadi, Mohammad; Penna-Martinez, Marissa; Meyer, Gesine; Mitchell, Anna L; Gan, Earn H; Bratland, Eirik; Bensing, Sophie; Husebye, Eystein; Pearce, Simon H.; Badenhoop, Klaus; Kämpe, Olle; Cerundolo, Vincenzo

2016-01-01

The mechanisms behind the destruction of the adrenal glands in autoimmune Addison’s disease remain unclear. Autoantibodies against steroid 21-hydroxylase, an intracellular key enzyme of the adrenal cortex, are found in over 90% of patients, but these autoantibodies are not thought to mediate the disease. Here we demonstrate highly frequent 21-hydroxylase specific T cells detectable in 20 patients with Addison’s disease. Using overlapping 18aa peptides spanning the full length of 21-hydroxylase, we identified immunodominant CD8+ and CD4+ T cell responses in a large proportion of Addison’s patients both ex-vivo and after in-vitro culture of peripheral blood lymphocytes up to 20 years after diagnosis. In a large proportion of patients, CD8+ 21-hydroxylase specific T cells and CD4+ 21-hydroxylase specific T cells were very abundant and detectable in ex-vivo assays. HLA class-I tetramer-guided isolation of 21-hydroxylase specific CD8+ T cells showed their ability to lyse 21-hydroxylase positive target cells, consistent with a potential mechanism for disease pathogenesis. These data indicate strong cytotoxic T lymphocyte responses to 21-hydroxylase often occur in-vivo, and that reactive cytotoxic T lymphocytes have substantial proliferative and cytolytic potential. These results have implications for earlier diagnosis of adrenal failure and ultimately a potential target for therapeutic intervention and induction of immunity against adrenal cortex cancer. PMID:25063864

Preparing a suitable solid target for generating copper-64 using a biomedical cyclotron

International Nuclear Information System (INIS)

Jeffery, Charmaine; Cryer, David; Chan, Sun; Asad, Ali; Fleming, Adam; Hubble, Lee

2009-01-01

Full text: Radiopharmaceutical research at SCGH has been advancing in the production of copper-64, a promising radionuclide for PET. Production has commenced using a self-manufactured solid target with alBA 18/9 cyclotron, via the 6 4 N i(p,n) 6 4 C u reaction pathway. One aspect of the project has been the preparation of a suitable solid target for irradiation. The chosen production method involves electrolysis of a solution of nickel ammonium sulphate in a self-manufactured electroplating cell, using a gold disk as the cathode for deposition of nickel metal. Various defects in the nickel surface were observed ∼ including cracks, formation of pits and inclusions, loose powder-like plating, lack of metallic lustre and lifting of the plated nickel. Several variables were investigated - including adjustment of the anode-cathode distance, anode composition, solution composition, and voltage/current settings. A suitable method that produced acceptable plating was achieved - the surface was then analysed to ensure there were no underlying defects. Three analytical techniques were used - AFM, SEM and optical profilometry. Two disks were ana lysed (approx. 3 0 m g of natNi plated). The depth of plating, evenness of plating and surface uniformity were of particular interest. Conclusions: Analysis revealed that the surface was not completely uniform (thinner at edges, 'well' in centre, with inclusions on the surface more prevalent than pitting), and required more nickel to be plated to reach optimum thickness. Final target specifications are still being optimised, however test irradiation of a solid target ( 3 1 n ickel, I l0 m g) proved that a sound solid target can be reliably produced and irradiated.

Microchimeric cells in systemic lupus erythematosus: targets or innocent bystanders?

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Stevens, A M

2006-01-01

During pregnancy maternal and fetal cells commute back and forth leading to fetal microchimerism in the mother and maternal microchimerism in the child that can persist for years after the birth. Chimeric fetal and maternal cells can be hematopoietic or can differentiate into somatic cells in multiple organs, potentially acting as targets for 'autoimmunity' and so have been implicated in the pathogenesis of autoimmune diseases that resemble graft-versus-host disease after stem cell transplantation. Fetal cells have been found in women with systemic lupus erythematosus, both in the blood and a target organ, the kidney, suggesting that they may be involved in pathogenesis. Future studies will address how the host immune system normally tolerates maternal and fetal cells or how the balance may change during autoimmunity.

Myeloid derived suppressor cells as therapeutic target in hematological malignancies

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Kim eDe Veirman

2014-12-01

Full Text Available Myeloid derived suppressor cells (MDSC are a heterogeneous population of immature myeloid cells that accumulate during pathological conditions such as cancer and are associated with a poor clinical outcome. MDSC expansion hampers the host anti-tumor immune response by inhibition of T cell proliferation, cytokine secretion and recruitment of regulatory T cells. In addition, MDSC exert non-immunological functions including the promotion of angiogenesis, tumor invasion and metastasis. Recent years, MDSC are considered as a potential target in solid tumors and hematological malignancies to enhance the effects of currently used immune modulating agents. This review focuses on the characteristics, distribution, functions, cell-cell interactions and targeting of MDSC in hematological malignancies including multiple myeloma, lymphoma and leukemia.

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

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

2008-01-01

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

Targeting of porous hybrid silica nanoparticles to cancer cells

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Rosenholm, J.M.; Meinander, A.; Peuhu, E.; Niemi, R.; Eriksson, J.E.; Sahlgren, C.; Lindén, M.

2009-01-01

Mesoporous silica nanoparticles functionalized by surface hyperbranching polymerization of polyethylene imine), PEI, were further modified by introducing both fluorescent and targeting moieties, with the aim of specifically targeting cancer cells. Owing to the high abundance of folate receptors in

Liver cell-targeted delivery of therapeutic molecules.

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Kang, Jeong-Hun; Toita, Riki; Murata, Masaharu

2016-01-01

The liver is the largest internal organ in mammals and is involved in metabolism, detoxification, synthesis of proteins and lipids, secretion of cytokines and growth factors and immune/inflammatory responses. Hepatitis, alcoholic or non-alcoholic liver disease, hepatocellular carcinoma, hepatic veno-occlusive disease, and liver fibrosis and cirrhosis are the most common liver diseases. Safe and efficient delivery of therapeutic molecules (drugs, genes or proteins) into the liver is very important to increase the clinical efficacy of these molecules and to reduce their side effects in other organs. Several liver cell-targeted delivery systems have been developed and tested in vivo or ex vivo/in vitro. In this review, we discuss the literature concerning liver cell-targeted delivery systems, with a particular emphasis on the results of in vivo studies.

Activation of the human immune system by chemotherapeutic or targeted agents combined with the oncolytic parvovirus H-1

International Nuclear Information System (INIS)

Moehler, Markus; Sieben, Maike; Roth, Susanne; Springsguth, Franziska; Leuchs, Barbara; Zeidler, Maja; Dinsart, Christiane; Rommelaere, Jean; Galle, Peter R

2011-01-01

Parvovirus H-1 (H-1PV) infects and lyses human tumor cells including melanoma, hepatoma, gastric, colorectal, cervix and pancreatic cancers. We assessed whether the beneficial effects of chemotherapeutic agents or targeted agents could be combined with the oncolytic and immunostimmulatory properties of H-1PV. Using human ex vivo models we evaluated the biological and immunological effects of H-1PV-induced tumor cell lysis alone or in combination with chemotherapeutic or targeted agents in human melanoma cells +/- characterized human cytotoxic T-cells (CTL) and HLA-A2-restricted dendritic cells (DC). H-1PV-infected MZ7-Mel cells showed a clear reduction in cell viability of >50%, which appeared to occur primarily through apoptosis. This correlated with viral NS1 expression levels and was enhanced by combination with chemotherapeutic agents or sunitinib. Tumor cell preparations were phagocytosed by DC whose maturation was measured according to the treatment administered. Immature DC incubated with H-1PV-induced MZ7-Mel lysates significantly increased DC maturation compared with non-infected or necrotic MZ7-Mel cells. Tumor necrosis factor-α and interleukin-6 release was clearly increased by DC incubated with H-1PV-induced SK29-Mel tumor cell lysates (TCL) and was also high with DC-CTL co-cultures incubated with H-1PV-induced TCL. Similarly, DC co-cultures with TCL incubated with H-1PV combined with cytotoxic agents or sunitinib enhanced DC maturation to a greater extent than cytotoxic agents or sunitinib alone. Again, these combinations increased pro-inflammatory responses in DC-CTL co-cultures compared with chemotherapy or sunitinib alone. In our human models, chemotherapeutic or targeted agents did not only interfere with the pronounced immunomodulatory properties of H-1PV, but also reinforced drug-induced tumor cell killing. H-1PV combined with cisplatin, vincristine or sunitinib induced effective immunostimulation via a pronounced DC maturation, better cytokine

Enhancing Natural Killer Cell Mediated Targeting and Responses to Myeloid Leukemias

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2017-10-01

AWARD NUMBER: W81XWH-16-1-0380 TITLE: Enhancing Natural Killer Cell Mediated Targeting and Responses to Myeloid Leukemias PRINCIPAL...2017 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Enhancing Natural Killer Cell Mediated Targeting and Responses to Myeloid Leukemias 5b. GRANT NUMBER...leukemias still have poor prognosis, particularly in the elderly, and require hematopoietic cell transplants to fully kill the tumor, which is both

Radiobiological study on DNA strand breaks and repair using single cell gel electrophoresis

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Ikushima, Takaji

1994-01-01

Single cell gel electrophoresis (SCGE) provides a novel method to measure DNA damage in individual cells and more importantly, to assess heterogeneity in response within a mixed population of cells. Cells embedded in agarose are lysed, subjected to electrophoresis, stained with a fluorescent DNA-specific dye, and viewed under a fluorescence microscope. Damaged cells display 'comets', broken DNA migrating farther to the anode in the electric field. We have previously used this technique to quantify DNA damage induced by moderate doses of low and high LET radiations in cultured Chinese hamster cells. The assay has been optimized in terms of lysing and electrophoresis conditions, and applied to analyse the DNA strand breaks, their repair kinetics and heterogeneity in response in individual Chinese hamster cells exposed to gamma-rays, and to KUR thermal neutrons with and without 10 B or to KEK PF monochromatic soft X-rays as well as to a radio-mimetic agent, neocarzinostatin. The DNA double-strand breaks induced by boron-neutron captured reactions were repaired at a slower rate, but a heterogeneity in response might not contribute to the difference. The neocarzinostatin-induced DNA damage were efficiently repaired in a dose-dependent fashion. The initial amount of gamma-ray induced DNA double-strand breaks was not significantly altered in cells pre-exposed to very low adapting dose. (author)

Stem Cell-Based Cell Carrier for Targeted Oncolytic Virotherapy: Translational Opportunity and Open Questions

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Janice Kim

2015-11-01

Full Text Available Oncolytic virotherapy for cancer is an innovative therapeutic option where the ability of a virus to promote cell lysis is harnessed and reprogrammed to selectively destroy cancer cells. Such treatment modalities exhibited antitumor activity in preclinical and clinical settings and appear to be well tolerated when tested in clinical trials. However, the clinical success of oncolytic virotherapy has been significantly hampered due to the inability to target systematic metastasis. This is partly due to the inability of the therapeutic virus to survive in the patient circulation, in order to target tumors at distant sites. An early study from various laboratories demonstrated that cells infected with oncolytic virus can protect the therapeutic payload form the host immune system as well as function as factories for virus production and enhance the therapeutic efficacy of oncolytic virus. While a variety of cell lineages possessed potential as cell carriers, copious investigation has established stem cells as a very attractive cell carrier system in oncolytic virotherapy. The ideal cell carrier desire to be susceptible to viral infection as well as support viral infection, maintain immunosuppressive properties to shield the loaded viruses from the host immune system, and most importantly possess an intrinsic tumor homing ability to deliver loaded viruses directly to the site of the metastasis—all qualities stem cells exhibit. In this review, we summarize the recent work in the development of stem cell-based carrier for oncolytic virotherapy, discuss the advantages and disadvantages of a variety of cell carriers, especially focusing on why stem cells have emerged as the leading candidate, and finally propose a future direction for stem cell-based targeted oncolytic virotherapy that involves its establishment as a viable treatment option for cancer patients in the clinical setting.

Concise Review: Cell Surface N-Linked Glycoproteins as Potential Stem Cell Markers and Drug Targets.

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Boheler, Kenneth R; Gundry, Rebekah L

2017-01-01

Stem cells and their derivatives hold great promise to advance regenerative medicine. Critical to the progression of this field is the identification and utilization of antibody-accessible cell-surface proteins for immunophenotyping and cell sorting-techniques essential for assessment and isolation of defined cell populations with known functional and therapeutic properties. Beyond their utility for cell identification and selection, cell-surface proteins are also major targets for pharmacological intervention. Although comprehensive cell-surface protein maps are highly valuable, they have been difficult to define until recently. In this review, we discuss the application of a contemporary targeted chemoproteomic-based technique for defining the cell-surface proteomes of stem and progenitor cells. In applying this approach to pluripotent stem cells (PSCs), these studies have improved the biological understanding of these cells, led to the enhanced use and development of antibodies suitable for immunophenotyping and sorting, and contributed to the repurposing of existing drugs without the need for high-throughput screening. The utility of this latter approach was first demonstrated with human PSCs (hPSCs) through the identification of small molecules that are selectively toxic to hPSCs and have the potential for eliminating confounding and tumorigenic cells in hPSC-derived progeny destined for research and transplantation. Overall, the cutting-edge technologies reviewed here will accelerate the development of novel cell-surface protein targets for immunophenotyping, new reagents to improve the isolation of therapeutically qualified cells, and pharmacological studies to advance the treatment of intractable diseases amenable to cell-replacement therapies. Stem Cells Translational Medicine 2017;6:131-138. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

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Unzueta, Ugutz; Céspedes, María Virtudes; Ferrer-Miralles, Neus; Casanova, Isolda; Cedano, Juan; Corchero, José Luis; Domingo-Espín, Joan; Villaverde, Antonio; Mangues, Ramón; Vázquez, Esther

2012-01-01

Background Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4) is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing. Results Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer. Conclusion Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles, and imaging agents. PMID:22923991

MUC16 provides immune protection by inhibiting synapse formation between NK and ovarian tumor cells

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Migneault Martine

2010-01-01

Full Text Available Abstract Background Cancer cells utilize a variety of mechanisms to evade immune detection and attack. Effective immune detection largely relies on the formation of an immune synapse which requires close contact between immune cells and their targets. Here, we show that MUC16, a heavily glycosylated 3-5 million Da mucin expressed on the surface of ovarian tumor cells, inhibits the formation of immune synapses between NK cells and ovarian tumor targets. Our results indicate that MUC16-mediated inhibition of immune synapse formation is an effective mechanism employed by ovarian tumors to evade immune recognition. Results Expression of low levels of MUC16 strongly correlated with an increased number of conjugates and activating immune synapses between ovarian tumor cells and primary naïve NK cells. MUC16-knockdown ovarian tumor cells were more susceptible to lysis by primary NK cells than MUC16 expressing controls. This increased lysis was not due to differences in the expression levels of the ligands for the activating receptors DNAM-1 and NKG2D. The NK cell leukemia cell line (NKL, which does not express KIRs but are positive for DNAM-1 and NKG2D, also conjugated and lysed MUC16-knockdown cells more efficiently than MUC16 expressing controls. Tumor cells that survived the NKL challenge expressed higher levels of MUC16 indicating selective lysis of MUC16low targets. The higher csMUC16 levels on the NKL resistant tumor cells correlated with more protection from lysis as compared to target cells that were never exposed to the effectors. Conclusion MUC16, a carrier of the tumor marker CA125, has previously been shown to facilitate ovarian tumor metastasis and inhibits NK cell mediated lysis of tumor targets. Our data now demonstrates that MUC16 expressing ovarian cancer cells are protected from recognition by NK cells. The immune protection provided by MUC16 may lead to selective survival of ovarian cancer cells that are more efficient in

Targeting cancer stem cells: emerging role of Nanog transcription factor

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Wang ML

2013-09-01

Full Text Available Mong-Lien Wang,1 Shih-Hwa Chiou,2,3 Cheng-Wen Wu1,4–61Institute of Biochemistry and Molecular Biology, 2Institute of Pharmacology, National Yang Ming University, Taipei, Taiwan; 3Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan; 4Institute of Microbiology and Immunology, 5Institute of Clinical Medicine, National Yang Ming University, Taipei, Taiwan; 6Institute of Biomedical Science, Academia Sinica, Taipei, TaiwanAbstract: The involvement of stemness factors in cancer initiation and progression has drawn much attention recently, especially after the finding that introducing four stemness factors in somatic cells is able to reprogram the cells back to an embryonic stem cell-like state. Following accumulating data revealing abnormal elevated expression levels of key stemness factors, like Nanog, Oct4, and Sox2, in several types of cancer stem cells; the importance and therapeutic potential of targeting these stemness regulators in cancers has turned to research focus. Nanog determines cell fate in both embryonic and cancer stem cells; activating Nanog at an inappropriate time would result in cancer stem cells rather than normal pluripotent stem cells or differentiated somatic cells. Upregulated Nanog is correlated with poor survival outcome of patients with various types of cancer. The discoveries of downstream regulatory pathways directly or indirectly mediated by Nanog indicate that Nanog regulates several aspects of cancer development such as tumor cell proliferation, self-renewal, motility, epithelial-mesenchymal transition, immune evasion, and drug-resistance, which are all defined features for cancer stem cells. The current review paper illustrates the central role of Nanog in the regulatory networks of cancer malignant development and stemness acquirement, as well as in the communication between cancer cells and the surrounding stroma. Though a more defined model is needed to test the

Designing and modeling a centrifugal microfluidic device to separate target blood cells

International Nuclear Information System (INIS)

Shamloo, Amir; Selahi, AmirAli; Madadelahi, Masoud

2016-01-01

The objective of this study is to design a novel and efficient portable lab-on-a-CD (LOCD) microfluidic device for separation of specific cells (target cells) using magnetic beads. In this study the results are shown for neutrophils as target cells. However, other kinds of target cells can be separated in a similar approach. The designed microfluidics can be utilized as a point of care system for neutrophil detection. This microfluidic system employs centrifugal and magnetic forces for separation. After model validation by the experimental data in the literature (that may be used as a design tool for developing centrifugo-magnetophoretic devices), two models are presented for separation of target cells using magnetic beads. The first model consists of one container in the inlet section and two containers in the outlets. Initially, the inlet container is filled with diluted blood sample which is a mixture of red blood cells (RBCs) plus neutrophils which are attached to Magnetic beads. It is shown that by using centrifugal and magnetic forces, this model can separate all neutrophils with recovery factor of ∼100%. In the second model, due to excess of magnetic beads in usual experimental analysis (to ensure that all target cells are attached to them) the geometry is improved by adding a third outlet for these free magnetic beads. It is shown that at angular velocity of 45 rad s −1 , recovery factor of 100% is achievable for RBCs, free magnetic beads and neutrophils as target cells. (paper)

Identification of human embryonic progenitor cell targeting peptides using phage display.

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Paola A Bignone

Full Text Available Human pluripotent stem (hPS cells are capable of differentiation into derivatives of all three primary embryonic germ layers and can self-renew indefinitely. They therefore offer a potentially scalable source of replacement cells to treat a variety of degenerative diseases. The ability to reprogram adult cells to induced pluripotent stem (iPS cells has now enabled the possibility of patient-specific hPS cells as a source of cells for disease modeling, drug discovery, and potentially, cell replacement therapies. While reprogramming technology has dramatically increased the availability of normal and diseased hPS cell lines for basic research, a major bottleneck is the critical unmet need for more efficient methods of deriving well-defined cell populations from hPS cells. Phage display is a powerful method for selecting affinity ligands that could be used for identifying and potentially purifying a variety of cell types derived from hPS cells. However, identification of specific progenitor cell-binding peptides using phage display may be hindered by the large cellular heterogeneity present in differentiating hPS cell populations. We therefore tested the hypothesis that peptides selected for their ability to bind a clonal cell line derived from hPS cells would bind early progenitor cell types emerging from differentiating hPS cells. The human embryonic stem (hES cell-derived embryonic progenitor cell line, W10, was used and cell-targeting peptides were identified. Competition studies demonstrated specificity of peptide binding to the target cell surface. Efficient peptide targeted cell labeling was accomplished using multivalent peptide-quantum dot complexes as detected by fluorescence microscopy and flow cytometry. The cell-binding peptides were selective for differentiated hPS cells, had little or no binding on pluripotent cells, but preferential binding to certain embryonic progenitor cell lines and early endodermal hPS cell derivatives. Taken

Identification and Regulation of c-Myb Target Genes in MCF-7 Cells

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O'Rourke John P

2011-01-01

Full Text Available Abstract Background The c-Myb transcription factor regulates differentiation and proliferation in hematopoietic cells, stem cells and epithelial cells. Although oncogenic versions of c-Myb were first associated with leukemias, over expression or rearrangement of the c-myb gene is common in several types of solid tumors, including breast cancers. Expression of the c-myb gene in human breast cancer cells is dependent on estrogen stimulation, but little is known about the activities of the c-Myb protein or what genes it regulates in estrogen-stimulated cells. Methods We used chromatin immunoprecipitation coupled with whole genome promoter tiling microarrays to identify endogenous c-Myb target genes in human MCF-7 breast cancer cells and characterized the activity of c-Myb at a panel of target genes during different stages of estrogen deprivation and stimulation. Results By using different antibodies and different growth conditions, the c-Myb protein was found associated with over 10,000 promoters in MCF-7 cells, including many genes that encode cell cycle regulators or transcription factors and more than 60 genes that encode microRNAs. Several previously identified c-Myb target genes were identified, including CCNB1, MYC and CXCR4 and novel targets such as JUN, KLF4, NANOG and SND1. By studying a panel of these targets to validate the results, we found that estradiol stimulation triggered the association of c-Myb with promoters and that association correlated with increased target gene expression. We studied one target gene, CXCR4, in detail, showing that c-Myb associated with the CXCR4 gene promoter and activated a CXCR4 reporter gene in transfection assays. Conclusions Our results show that c-Myb associates with a surprisingly large number of promoters in human cells. The results also suggest that estradiol stimulation leads to large-scale, genome-wide changes in c-Myb activity and subsequent changes in gene expression in human breast cancer

Identification and Regulation of c-Myb Target Genes in MCF-7 Cells

International Nuclear Information System (INIS)

Quintana, Anita M; Liu, Fan; O'Rourke, John P; Ness, Scott A

2011-01-01

The c-Myb transcription factor regulates differentiation and proliferation in hematopoietic cells, stem cells and epithelial cells. Although oncogenic versions of c-Myb were first associated with leukemias, over expression or rearrangement of the c-myb gene is common in several types of solid tumors, including breast cancers. Expression of the c-myb gene in human breast cancer cells is dependent on estrogen stimulation, but little is known about the activities of the c-Myb protein or what genes it regulates in estrogen-stimulated cells. We used chromatin immunoprecipitation coupled with whole genome promoter tiling microarrays to identify endogenous c-Myb target genes in human MCF-7 breast cancer cells and characterized the activity of c-Myb at a panel of target genes during different stages of estrogen deprivation and stimulation. By using different antibodies and different growth conditions, the c-Myb protein was found associated with over 10,000 promoters in MCF-7 cells, including many genes that encode cell cycle regulators or transcription factors and more than 60 genes that encode microRNAs. Several previously identified c-Myb target genes were identified, including CCNB1, MYC and CXCR4 and novel targets such as JUN, KLF4, NANOG and SND1. By studying a panel of these targets to validate the results, we found that estradiol stimulation triggered the association of c-Myb with promoters and that association correlated with increased target gene expression. We studied one target gene, CXCR4, in detail, showing that c-Myb associated with the CXCR4 gene promoter and activated a CXCR4 reporter gene in transfection assays. Our results show that c-Myb associates with a surprisingly large number of promoters in human cells. The results also suggest that estradiol stimulation leads to large-scale, genome-wide changes in c-Myb activity and subsequent changes in gene expression in human breast cancer cells

Neuroblastoma cell lines contain pluripotent tumor initiating cells that are susceptible to a targeted oncolytic virus.

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Yonatan Y Mahller

Full Text Available Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers.Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice.These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.

Neutron activation analysis of antimony in chromatin and nucleoids of HeLa cells

International Nuclear Information System (INIS)

Ashry, H.A.; Topaloglou, A.; Altmann, H.

1988-02-01

Antimony seems to be cancerogenic in men. In the present investigations we tried to find out if Sb +++ are also bound to the cell nucleus. HeLa cells were incubated with SbCl 3 and after a 18 h incubation time cells were lysed and crude chromatin isolated. In this preparation Sb was determined by neutron activation analysis. From the same cell culture nucleoids were prepared by ultracentrifugation and also Sb detected in these structures. 12 refs., 2 tabs. (Author)

Targeted delivery of celastrol to mesangial cells is effective against mesangioproliferative glomerulonephritis.

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Guo, Ling; Luo, Shi; Du, Zhengwu; Zhou, Meiling; Li, Peiwen; Fu, Yao; Sun, Xun; Huang, Yuan; Zhang, Zhirong

2017-10-12

Mesangial cells-mediated glomerulonephritis is a frequent cause of end-stage renal disease. Here, we show that celastrol is effective in treating both reversible and irreversible mesangioproliferative glomerulonephritis in rat models, but find that its off-target distributions cause severe systemic toxicity. We thus target celastrol to mesangial cells using albumin nanoparticles. Celastrol-albumin nanoparticles crosses fenestrated endothelium and accumulates in mesangial cells, alleviating proteinuria, inflammation, glomerular hypercellularity, and excessive extracellular matrix deposition in rat anti-Thy1.1 nephritis models. Celastrol-albumin nanoparticles presents lower drug accumulation than free celastrol in off-target organs and tissues, thereby minimizing celastrol-related systemic toxicity. Celastrol-albumin nanoparticles thus represents a promising treatment option for mesangioproliferative glomerulonephritis and similar glomerular diseases.Mesangial cell-mediated glomerulonephritis is a frequent cause of kidney disease. Here the authors show that celastrol loaded in albumin nanoparticles efficiently targets mesangial cells, and is effective in rat models.

Comparison of human memory CD8 T cell responses to adenoviral early and late proteins in peripheral blood and lymphoid tissue.

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Amita Joshi

Full Text Available Treatment of invasive adenovirus (Ad disease in hematopoietic stem cell transplant (SCT recipients with capsid protein hexon-specific donor T cells is under investigation. We propose that cytotoxic T cells (CTLs targeted to the late protein hexon may be inefficient in vivo because the early Ad protein E3-19K downregulates HLA class I antigens in infected cells. In this study, CD8+ T cells targeted to highly conserved HLA A2-restricted epitopes from the early regulatory protein DNA polymerase (P-977 and late protein hexon (H-892 were compared in peripheral blood (PB and tonsils of naturally infected adults. In tonsils, epitope-specific pentamers detected a significantly higher frequency of P-977+CD8+ T cells compared to H-892+CD8+ T cells; this trend was reversed in PB. Tonsil epitope-specific CD8+ T cells expressed IFN-γ and IL-2 but not perforin or TNF-α, whereas PB T cells were positive for IFN-γ, TNF-α, and perforin. Tonsil epitope-specific T cells expressed lymphoid homing marker CCR7 and exhibited lower levels of the activation marker CD25 but higher proliferative potential than PB T cells. Finally, in parallel with the kinetics of mRNA expression, P-977-specific CTLs lysed targets as early as 8 hrs post infection. In contrast, H-892-specific CTLs did not kill unless infected fibroblasts were pretreated with IFN-γ to up regulate HLA class I antigens, and cytotoxicity was delayed until 16-24 hours. These data show that, in contrast to hexon CTLs, central memory type DNA polymerase CTLs dominate the lymphoid compartment and kill fibroblasts earlier after infection without requiring exogenous IFN-γ. Thus, use of CTLs targeted to both early and late Ad proteins may improve the efficacy of immunotherapy for life-threatening Ad disease in SCT recipients.

Identification and validation nucleolin as a target of curcumol in nasopharyngeal carcinoma cells.

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Wang, Juan; Wu, Jiacai; Li, Xumei; Liu, Haowei; Qin, Jianli; Bai, Zhun; Chi, Bixia; Chen, Xu

2018-06-30

Identification of the specific protein target(s) of a drug is a critical step in unraveling its mechanisms of action (MOA) in many natural products. Curcumol, isolated from well known Chinese medicinal plant Curcuma zedoary, has been shown to possess multiple biological activities. It can inhibit nasopharyngeal carcinoma (NPC) proliferation and induce apoptosis, but its target protein(s) in NPC cells remains unclear. In this study, we employed a mass spectrometry-based chemical proteomics approach reveal the possible protein targets of curcumol in NPC cells. Cellular thermal shift assay (CETSA), molecular docking and cell-based assay was used to validate the binding interactions. Chemical proteomics capturing uncovered that NCL is a target of curcumol in NPC cells, Molecular docking showed that curcumol bound to NCL with an -7.8 kcal/mol binding free energy. Cell function analysis found that curcumol's treatment leads to a degradation of NCL in NPC cells, and it showed slight effects on NP69 cells. In conclusion, our results providing evidences that NCL is a target protein of curcumol. We revealed that the anti-cancer effects of curcumol in NPC cells are mediated, at least in part, by NCL inhibition. Many natural products showed high bioactivity, while their mechanisms of action (MOA) are very poor or completely missed. Understanding the MOA of natural drugs can thoroughly exploit their therapeutic potential and minimize their adverse side effects. Identification of the specific protein target(s) of a drug is a critical step in unraveling its MOA. Compound-centric chemical proteomics is a classic chemical proteomics approach which integrates chemical synthesis with cell biology and mass spectrometry (MS) to identify protein targets of natural products determine the drug mechanism of action, describe its toxicity, and figure out the possible cause of off-target. It is an affinity-based chemical proteomics method to identify small molecule-protein interactions

Targeting nanoparticles to dendritic cells for immunotherapy.

NARCIS (Netherlands)

Cruz, L.J.; Tacken, P.J.; Rueda, F.; Domingo, J.C.; Albericio, F.; Figdor, C.G.

2012-01-01

Dendritic cells (DCs) are key players in the initiation of adaptive immune responses and are currently exploited in immunotherapy for treatment of cancer and infectious diseases. Development of targeted nanodelivery systems carrying vaccine components, including antigens and adjuvants, to DCs in

Radioprotection of targeted and bystander cells by methylproamine

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Burdak-Rothkamm, Susanne [Queen' s University Belfast, Centre for Cancer Research and Cell Biology, Belfast (United Kingdom); Oxford University Hospitals, Cellular Pathology, Oxford (United Kingdom); Smith, Andrea; Lobachevsky, Pavel; Martin, Roger [Peter MacCallum Cancer Centre, Molecular Radiation Biology Laboratory, Melbourne (Australia); University of Melbourne, The Sir Peter MacCallum Department of Oncology, Melbourne (Australia); Prise, Kevin M. [Queen' s University Belfast, Centre for Cancer Research and Cell Biology, Belfast (United Kingdom)

2014-09-23

Radioprotective agents are of interest for application in radiotherapy for cancer and in public health medicine in the context of accidental radiation exposure. Methylproamine is the lead compound of a class of radioprotectors which act as DNA binding anti-oxidants, enabling the repair of transient radiation-induced oxidative DNA lesions. This study tested methylproamine for the radioprotection of both directly targeted and bystander cells. T98G glioma cells were treated with 15 μM methylproamine and exposed to {sup 137}Cs γ-ray/X-ray irradiation and He{sup 2+} microbeam irradiation. Radioprotection of directly targeted cells and bystander cells was measured by clonogenic survival or γH2AX assay. Radioprotection of directly targeted T98G cells by methylproamine was observed for {sup 137}Cs γ-rays and X-rays but not for He{sup 2+} charged particle irradiation. The effect of methylproamine on the bystander cell population was tested for both X-ray irradiation and He{sup 2+} ion microbeam irradiation. The X-ray bystander experiments were carried out by medium transfer from irradiated to non-irradiated cultures and three experimental designs were tested. Radioprotection was only observed when recipient cells were pretreated with the drug prior to exposure to the conditioned medium. In microbeam bystander experiments targeted and nontargeted cells were co-cultured with continuous methylproamine treatment during irradiation and postradiation incubation; radioprotection of bystander cells was observed. Methylproamine protected targeted cells from DNA damage caused by γ-ray or X-ray radiation but not He{sup 2+} ion radiation. Protection of bystander cells was independent of the type of radiation which the donor population received. (orig.) [German] Radioprotektive Agenzien sind sowohl in der Strahlentherapie von Krebserkrankungen als auch im Strahlenschutz im Zusammenhang mit akzidenteller Exposition von Bedeutung. Methylproamine ist die Leitsubstanz einer Klasse von

Death receptor pathways mediate targeted and non-targeted effects of ionizing radiations in breast cancer cells

International Nuclear Information System (INIS)

Luce, A.; Courtin, A.; Levalois, C.; Altmeyer-Morel, S.; Chevillard, S.; Lebeau, J.; Romeo, P.H.

2009-01-01

Delayed cell death by mitotic catastrophe is a frequent mode of solid tumor cell death after γ-irradiation, a widely used treatment of cancer. Whereas the mechanisms that underlie the early γ-irradiation-induced cell death are well documented, those that drive the delayed cell death are largely unknown. Here we show that the Fas, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and tumor necrosis factor (TNF)-α death receptor pathways mediate the delayed cell death observed after γ-irradiation of breast cancer cells. Early after irradiation, we observe the increased expression of Fas, TRAIL-R and TNF-R that first sensitizes cells to apoptosis. Later, the increased expression of FasL, TRAIL and TNF-α permit the apoptosis engagement linked to mitotic catastrophe. Treatments with TNF-α, TRAIL or anti-Fas antibody, early after radiation exposure, induce apoptosis, whereas the neutralization of the three death receptors pathways impairs the delayed cell death. We also show for the first time that irradiated breast cancer cells excrete soluble forms of the three ligands that can induce the death of sensitive bystander cells. Overall, these results define the molecular basis of the delayed cell death of irradiated cancer cells and identify the death receptors pathways as crucial actors in apoptosis induced by targeted as well as non-targeted effects of ionizing radiation. (authors)

Targeting NK cells for anti-cancer immunotherapy: clinical and pre-clinical approaches

Directory of Open Access Journals (Sweden)

Sebastian eCarotta

2016-04-01

Full Text Available The recent success of checkpoint blockade has highlighted the potential of immunotherapy approaches for cancer treatment. While the majority of approved immunotherapy drugs target T cell subsets, it is appreciated that other components of the immune system have important roles in tumor immune-surveillance as well and thus represent promising additional targets for immunotherapy. Natural killer cells are the body’s first line of defense against infected or transformed cells as they kill target cells in an antigen-independent manner. Although several studies have clearly demonstrated the active role of NK cells in cancer-immune surveillance, only few clinically approved therapies currently exist that harness their potential. Our increased understanding of NK cell biology over the past few years has renewed the interest in NK cell based anti-cancer therapies, which has lead to a steady increase of NK cell based clinical and pre-clinical trials. Here, the role of NK cells in cancer immunesurveillance is summarized and several novel approaches to enhance NK cell cytotoxicity against cancer are discussed.

Therapeutic targeting of the p53 pathway in cancer stem cells

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Prabhu, Varun V.; Allen, Joshua E.; Hong, Bo; Zhang, Shengliang; Cheng, Hairong; El-Deiry, Wafik S.

2013-01-01

Introduction Cancer stem cells are a high profile drug target for cancer therapeutics due to their indispensable role in cancer progression, maintenance, and therapeutic resistance. Restoring wild-type p53 function is an attractive new therapeutic approach for the treatment of cancer due to the well-described powerful tumor suppressor function of p53. As emerging evidence intimately links p53 and stem cell biology, this approach also provides an opportunity to target cancer stem cells. Areas covered Therapeutic approaches to restore the function of wild-type p53, cancer and normal stem cell biology in relation to p53, and the downstream effects of p53 on cancer stem cells. Expert opinion The restoration of wild-type p53 function by targeting p53 directly, its interacting proteins, or its family members holds promise as a new class of cancer therapies. This review examines the impact that such therapies may have on normal and cancer stem cells based on the current evidence linking p53 signaling with these populations. PMID:22998602

Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells.

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Sato, Hiroshi; Kato, Hiroki; Yamaza, Haruyoshi; Masuda, Keiji; Nguyen, Huong Thi Nguyen; Pham, Thanh Thi Mai; Han, Xu; Hirofuji, Yuta; Nonaka, Kazuaki

2017-01-01

Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk) gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV). Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

A drug development perspective on targeting tumor-associated myeloid cells.

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Majety, Meher; Runza, Valeria; Lehmann, Christian; Hoves, Sabine; Ries, Carola H

2018-02-01

Despite decades of research, cancer remains a devastating disease and new treatment options are needed. Today cancer is acknowledged as a multifactorial disease not only comprising of aberrant tumor cells but also the associated stroma including tumor vasculature, fibrotic plaques, and immune cells that interact in a complex heterotypic interplay. Myeloid cells represent one of the most abundant immune cell population within the tumor stroma and are equipped with a broad functional repertoire that promotes tumor growth by suppressing cytotoxic T cell activity, stimulating neoangiogenesis and tissue remodeling. Therefore, myeloid cells have become an attractive target for pharmacological intervention. In this review, we summarize the pharmacological approaches to therapeutically target tumor-associated myeloid cells with a focus on advanced programs that are clinically evaluated. In addition, for each therapeutic strategy, the preclinical rationale as well as advantages and challenges from a drug development perspective are discussed. © 2017 Federation of European Biochemical Societies.

Glycan Markers as Potential Immunological Targets in Circulating Tumor Cells.

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Wang, Denong; Wu, Lisa; Liu, Xiaohe

2017-01-01

We present here an experimental approach for exploring a new class of tumor biomarkers that are overexpressed by circulating tumor cells (CTCs) and are likely targetable in immunotherapy against tumor metastasis. Using carbohydrate microarrays, anti-tumor monoclonal antibodies (mAbs) were scanned against a large panel of carbohydrate antigens to identify potential tumor glycan markers. Subsequently, flow cytometry and fiber-optic array scanning technology (FAST) were applied to determine whether the identified targets are tumor-specific cell-surface markers and are, therefore, likely suitable for targeted immunotherapy. Finally, the tumor glycan-specific antibodies identified were validated using cancer patients' blood samples for their performance in CTC-detection and immunotyping analysis. In this article, identifying breast CTC-specific glycan markers and targeting mAbs serve as examples to illustrate this tumor biomarker discovery strategy.

Targeting Myeloid-Derived Suppressor Cells to Bypass Tumor-Induced Immunosuppression

Directory of Open Access Journals (Sweden)

Viktor Fleming

2018-03-01

Full Text Available The immune system has many sophisticated mechanisms to balance an extensive immune response. Distinct immunosuppressive cells could protect from excessive tissue damage and autoimmune disorders. Tumor cells take an advantage of those immunosuppressive mechanisms and establish a strongly immunosuppressive tumor microenvironment (TME, which inhibits antitumor immune responses, supporting the disease progression. Myeloid-derived suppressor cells (MDSC play a crucial role in this immunosuppressive TME. Those cells represent a heterogeneous population of immature myeloid cells with a strong immunosuppressive potential. They inhibit an antitumor reactivity of T cells and NK cells. Furthermore, they promote angiogenesis, establish pre-metastatic niches, and recruit other immunosuppressive cells such as regulatory T cells. Accumulating evidences demonstrated that the enrichment and activation of MDSC correlated with tumor progression, recurrence, and negative clinical outcome. In the last few years, various preclinical studies and clinical trials targeting MDSC showed promising results. In this review, we discuss different therapeutic approaches on MDSC targeting to overcome immunosuppressive TME and enhance the efficiency of current tumor immunotherapies.

GEM-loaded magnetic albumin nanospheres modified with cetuximab for simultaneous targeting, magnetic resonance imaging, and double-targeted thermochemotherapy of pancreatic cancer cells

Directory of Open Access Journals (Sweden)

Wang L

2015-03-01

Full Text Available Ling Wang,1 Yanli An,2 Chenyan Yuan,3 Hao Zhang,2 Chen Liang,2 Fengan Ding,2 Qi Gao,1 Dongsheng Zhang4 1Department of Ultrasonography, Zhong Da Hospital, Medical School, Southeast University, Nanjing, People’s Republic of China; 2Medical School, Southeast University, Nanjing, People’s Republic of China; 3Department of Clinical Laboratory, Zhong Da Hospital, Medical School, Southeast University, Nanjing, People’s Republic of China; 4Jiangsu Key Laboratory for Biomaterials and Devices, Medical School, Southeast University, Nanjing, People’s Republic of China Background: Targeted delivery is a promising strategy to improve the diagnostic imaging and therapeutic effect of cancers. In this paper, novel cetuximab (C225-conjugated, gemcitabine (GEM-containing magnetic albumin nanospheres (C225-GEM/MANs were fabricated and applied as a theranostic nanocarrier to conduct simultaneous targeting, magnetic resonance imaging (MRI, and double-targeted thermochemotherapy against pancreatic cancer cells. Methods: Fe3O4 nanoparticles (NPs and GEM co-loaded albumin nanospheres (GEM/MANs were prepared, and then C225 was further conjugated to synthesize C225-GEM/MANs. Their morphology, mean particle size, GEM encapsulation ratio, specific cell-binding ability, and thermal dynamic profiles were characterized. The effects of discriminating different EGFR-expressing pancreatic cancer cells (AsPC-1 and MIA PaCa-2 and monitoring cellular targeting effects were assessed by targeted MRI. Lastly, the antitumor efficiency of double/C225/magnetic-targeted and nontargeted thermochemotherapy was compared with chemotherapy alone using 3-(4, 5-dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide (MTT and flow cytometry (FCM assay. Results: When treated with targeted nanospheres, AsPC-1 cells showed a significantly less intense MRI T2 signal than MIA PaCa-2 cells, while both cells had similar signal strength when incubated with nontargeted nanospheres. T2 signal

PEGylated anticancer-carbon nanotubes complex targeting mitochondria of lung cancer cells

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Kim, Sang-Woo; Lee, Yeon Kyung; Lee, Jong Yeon; Hong, Jeong Hee; Khang, Dongwoo

2017-11-01

Although activating apoptosis in cancer cells by targeting the mitochondria is an effective strategy for cancer therapy, insufficient targeting of the mitochondria in cancer cells restricts the availability in clinical treatment. Here, we report on a polyethylene glycol-coated carbon nanotube (CNT)-ABT737 nanodrug that improves the mitochondrial targeting of lung cancer cells. The polyethylene glycol-coated CNT-ABT737 nanodrug internalized into the early endosomes via macropinocytosis and clathrin-mediated endocytosis in advance of early endosomal escape and delivered into the mitochondria. Cytosol release of the nanodrug led to apoptosis of lung cancer cells by abruption of the mitochondrial membrane potential, inducing Bcl-2-mediated apoptosis and generating intracellular reactive oxygen species. As such, this study provides an effective strategy for increasing the anti-lung cancer efficacy by increasing mitochondria accumulation rate of cytosol released anticancer nanodrugs.

Targeted DNA vaccines for enhanced induction of idiotype-specific B and T cells

International Nuclear Information System (INIS)

Fredriksen, Agnete B.; Sandlie, Inger; Bogen, Bjarne

2012-01-01

Background: Idiotypes (Id) are antigenic determinants localized in variable (V) regions of Ig. Id-specific T and B cells (antibodies) play a role in immunotherapy of Id + tumors. However, vaccine strategies that enhance Id-specific responses are needed. Methods: Id + single-chain fragment variable (scFv) from multiple myelomas and B cell lymphomas were prepared in a fusion format that bivalently target surface molecules on antigen-presenting cells (APC). APC-specific targeting units were either scFv from APC-specific mAb (anti-MHC II, anti-CD40) or chemokines (MIP-1α, RANTES). Homodimeric Id-vaccines were injected intramuscularly or intradermally as plasmids in mice, combined with electroporation. Results: (i) Transfected cells secreted plasmid-encoded Id + fusion proteins to extracellular fluid followed by binding of vaccine molecules to APC. (ii) Targeted vaccine molecules increased Id-specific B and T cell responses. (iii) Bivalency and xenogeneic sequences both contributed to enhanced responses. (iv) Targeted Id DNA vaccines induced tumor resistance against challenges with Id + tumors. (v) Human MIP-1α targeting units enhanced Id-specific responses in mice, due to a cross reaction with murine chemokine receptors. Thus, targeted vaccines designed for humans can be quality tested in mice. (vi) Human Id + scFv from four multiple myeloma patients were inserted into the vaccine format and were successfully tested in mice. (vii) Human MIP-1α vaccine proteins enhanced human T cell responses in vitro. (viii) A hypothetical model for how the APC-targeted vaccine molecules enhance Id-specific T and B cells is presented. Conclusion: Targeted DNA Id-vaccines show promising results in preclinical studies, paving the way for testing in patients.

Targeted DNA vaccines for enhanced induction of idiotype-specific B and T cells

Energy Technology Data Exchange (ETDEWEB)

Fredriksen, Agnete B.; Sandlie, Inger; Bogen, Bjarne, E-mail: bjarne.bogen@medisin.uio.no [Centre for Immune Regulation, Institute of Immunology, University of Oslo and Oslo University Hospital, Oslo (Norway)

2012-10-30

Background: Idiotypes (Id) are antigenic determinants localized in variable (V) regions of Ig. Id-specific T and B cells (antibodies) play a role in immunotherapy of Id{sup +} tumors. However, vaccine strategies that enhance Id-specific responses are needed. Methods: Id{sup +} single-chain fragment variable (scFv) from multiple myelomas and B cell lymphomas were prepared in a fusion format that bivalently target surface molecules on antigen-presenting cells (APC). APC-specific targeting units were either scFv from APC-specific mAb (anti-MHC II, anti-CD40) or chemokines (MIP-1α, RANTES). Homodimeric Id-vaccines were injected intramuscularly or intradermally as plasmids in mice, combined with electroporation. Results: (i) Transfected cells secreted plasmid-encoded Id{sup +} fusion proteins to extracellular fluid followed by binding of vaccine molecules to APC. (ii) Targeted vaccine molecules increased Id-specific B and T cell responses. (iii) Bivalency and xenogeneic sequences both contributed to enhanced responses. (iv) Targeted Id DNA vaccines induced tumor resistance against challenges with Id{sup +} tumors. (v) Human MIP-1α targeting units enhanced Id-specific responses in mice, due to a cross reaction with murine chemokine receptors. Thus, targeted vaccines designed for humans can be quality tested in mice. (vi) Human Id{sup +} scFv from four multiple myeloma patients were inserted into the vaccine format and were successfully tested in mice. (vii) Human MIP-1α vaccine proteins enhanced human T cell responses in vitro. (viii) A hypothetical model for how the APC-targeted vaccine molecules enhance Id-specific T and B cells is presented. Conclusion: Targeted DNA Id-vaccines show promising results in preclinical studies, paving the way for testing in patients.

Protective effect of grape seed extracts on human lymphocytes: a preliminary study.

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Szeto, Yim Tong; Lee, Kit Yee; Kalle, Wouter; Pak, Sok Cheon

2013-03-01

Grape seed extracts (GSEs) possess a broad spectrum of antioxidative properties that protects various cells from free radicals and oxidative stress. In this study, the genoprotective effect of GSE on human lymphocytic DNA was studied using standard and lysed cell comet assays. Lymphocytes from 5 healthy subjects were pretreated with GSE in different concentrations. The standard and lysed cell comet assays were performed on treated, untreated, challenged, and unchallenged cells in parallel. Cells were then subjected to an oxidant challenge induced with 5-min exposures to hydrogen peroxide. In the standard comet assay, GSE significantly diminished hydrogen-peroxide-induced DNA damage in a dose-dependent manner. In the lysed cell assay, however, the antioxidant effect was diminished at a higher GSE concentration. Data indicate that the cell membrane might play a role in limiting cellular access to antioxidants, which directly affects the genoprotective or potential pro-oxidant effect of antioxidants on human DNA. Using both standard and lysed cell comet assays in parallel could be a useful way to elucidate the mechanism of protection or damage by antioxidants.

Single-cell analysis of targeted transcriptome predicts drug sensitivity of single cells within human myeloma tumors.

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Mitra, A K; Mukherjee, U K; Harding, T; Jang, J S; Stessman, H; Li, Y; Abyzov, A; Jen, J; Kumar, S; Rajkumar, V; Van Ness, B

2016-05-01

Multiple myeloma (MM) is characterized by significant genetic diversity at subclonal levels that have a defining role in the heterogeneity of tumor progression, clinical aggressiveness and drug sensitivity. Although genome profiling studies have demonstrated heterogeneity in subclonal architecture that may ultimately lead to relapse, a gene expression-based prediction program that can identify, distinguish and quantify drug response in sub-populations within a bulk population of myeloma cells is lacking. In this study, we performed targeted transcriptome analysis on 528 pre-treatment single cells from 11 myeloma cell lines and 418 single cells from 8 drug-naïve MM patients, followed by intensive bioinformatics and statistical analysis for prediction of proteasome inhibitor sensitivity in individual cells. Using our previously reported drug response gene expression profile signature at the single-cell level, we developed an R Statistical analysis package available at https://github.com/bvnlabSCATTome, SCATTome (single-cell analysis of targeted transcriptome), that restructures the data obtained from Fluidigm single-cell quantitative real-time-PCR analysis run, filters missing data, performs scaling of filtered data, builds classification models and predicts drug response of individual cells based on targeted transcriptome using an assortment of machine learning methods. Application of SCATT should contribute to clinically relevant analysis of intratumor heterogeneity, and better inform drug choices based on subclonal cellular responses.

Tributyltin (TBT) and Dibutyltin (DBT) Alter Secretion of Tumor Necrosis Factor Alpha (TNFα) from Human Natural Killer (NK) Cells and a Mixture of T cells and NK Cells

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Hurt, Kelsi; Hurd-Brown, Tasia; Whalen, Margaret

2012-01-01

Butyltins (BTs) have been in widespread use. Tributyltin (TBT) has been used as a biocide in a variety of applications and is found in human blood samples. Dibutyltin (DBT) has been used as a stabilizer in polyvinyl chloride plastics and as a de-worming agent in poultry. DBT, like TBT, is found in human blood. Human natural killer (NK) cells are the earliest defense against tumors and viral infections and secrete the cytokine tumor necrosis factor (TNF) alpha (α). TNFα is an important regulator of adaptive and innate immune responses. TNFα promotes inflammation and an association between malignant transformation and inflammation has been established. Previously, we have shown that TBT and DBT were able to interfere with the ability of NK cells to lyse tumor target cells. Here we show that BTs alter cytokine secretion by NK cells as well as a mixture of T and NK lymphocytes (T/NK cells). We examined 24 h, 48 h, and 6 day exposures to TBT (200- 2.5 nM) and DBT (5- 0.05 µM) on TNFα secretion by highly enriched human NK cells and T/NK cells. The results indicate that TBT (200 - 2.5 nM) decreased TNFα secretion from NK cells. In the T/NK cells 200 nM TBT decreased secretion while 100-5 nM TBT increased secretion of TNFα. NK cells or T/NK cells exposed to higher concentrations of DBT showed decreased TNFα secretion while lower concentrations showed increased secretion. The effects of BTs on TNFα secretion are seen at concentrations present in human blood. PMID:23047847

Glycoengineering of Human Cell Lines Using Zinc Finger Nuclease Gene Targeting

DEFF Research Database (Denmark)

Steentoft, Catharina; Bennett, Eric Paul; Clausen, Henrik

2013-01-01

Lectin affinity chromatography is a powerful technique for isolation of glycoproteins carrying a specific glycan structure of interest. However, the enormous diversity of glycans present on the cell surface, as well as on individual proteins, makes it difficult to isolate an entire glycoproteome...... with one or even a series of lectins. Here we present a technique to generate cell lines with homogenous truncated O-glycans using zinc finger nuclease gene targeting. Because of their simplified O-glycoproteome, the cells have been named SimpleCells. Glycoproteins from SimpleCells can be isolated...... in a single purification step by lectin chromatography performed on a long lectin column. This protocol describes Zinc finger nuclease gene targeting of human cells to simplify the glycoproteome, as well as lectin chromatography and isolation of glycopeptides from total cell lysates of SimpleCells....

Splenocytes cultured in low concentrations of IL-2 generate NK cell specificities toward syngenic and allogenic targets

DEFF Research Database (Denmark)

Nissen, Mogens Holst; Jeppesen, M; Claesson, M H

2000-01-01

Splenocytes cultured in the presence of 30-60 units/ml IL-2 for 5 days develop natural killer activity toward syngeneic and allogeneic tumor cell targets. The IL-2 activated splenocytes, themselves, are partially resistant, whereas concanavalin A-activated T blast cells are completely resistant...... to killing. Surprisingly, major histocompatibility complex (MHC)-I-negative target cells are also resistant to natural killer (NK)-cell-mediated killing. Cells resistant to killing were unable to block NK-cell-mediated killing of sensitive targets as judged from cold target cell inhibition experiments......, and one type of target cells sensitive to killing did generally not cross-block killing of other killing-sensitive target cell types. Alloantigen exposure of splenocytes, i.e., one-way mixed lymphocyte cultures, partially prevents the development of NK-cell activity. Our data suggest that target...

Orchestration of transplantation tolerance by regulatory dendritic cell therapy or in-situ targeting of dendritic cells.

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Morelli, Adrian E; Thomson, Angus W

2014-08-01

Extensive research in murine transplant models over the past two decades has convincingly demonstrated the ability of regulatory dendritic cells (DCregs) to promote long-term allograft survival. We review important considerations regarding the source of therapeutic DCregs (donor or recipient) and their mode of action, in-situ targeting of DCregs, and optimal therapeutic regimens to promote DCreg function. Recent studies have defined protocols and mechanisms whereby ex-vivo-generated DCregs of donor or recipient origin subvert allogeneic T-cell responses and promote long-term organ transplant survival. Particular interest has focused on how donor antigen is acquired, processed and presented by autologous dendritic cells, on the stability of DCregs, and on in-situ targeting of dendritic cells to promote their tolerogenic function. New evidence of the therapeutic efficacy of DCregs in a clinically relevant nonhuman primate organ transplant model and production of clinical grade DCregs support early evaluation of DCreg therapy in human graft recipients. We discuss strategies currently used to promote dendritic cell tolerogenicity, including DCreg therapy and in-situ targeting of dendritic cells, with a view to improved understanding of underlying mechanisms and identification of the most promising strategies for therapeutic application.

Therapeutic Approaches to Target Cancer Stem Cells

International Nuclear Information System (INIS)

Diaz, Arlhee; Leon, Kalet

2011-01-01

The clinical relevance of cancer stem cells (CSC) remains a major challenge for current cancer therapies, but preliminary findings indicate that specific targeting may be possible. Recent studies have shown that these tumor subpopulations promote tumor angiogenesis through the increased production of VEGF, whereas the VEGF neutralizing antibody bevacizumab specifically inhibits CSC growth. Moreover, nimotuzumab, a monoclonal antibody against the epidermal growth factor receptor (EGFR) with a potent antiangiogenic activity, has been shown by our group to reduce the frequency of CSC-like subpopulations in mouse models of brain tumors when combined with ionizing radiation. These studies and subsequent reports from other groups support the relevance of approaches based on molecular-targeted therapies to selectively attack CSC. This review discusses the relevance of targeting both the EGFR and angiogenic pathways as valid approaches to this aim. We discuss the relevance of identifying better molecular markers to develop drug screening strategies that selectively target CSC

Killing cancer cells by targeted drug-carrying phage nanomedicines

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Yacoby Iftach

2008-04-01

Full Text Available Abstract Background Systemic administration of chemotherapeutic agents, in addition to its anti-tumor benefits, results in indiscriminate drug distribution and severe toxicity. This shortcoming may be overcome by targeted drug-carrying platforms that ferry the drug to the tumor site while limiting exposure to non-target tissues and organs. Results We present a new form of targeted anti-cancer therapy in the form of targeted drug-carrying phage nanoparticles. Our approach is based on genetically-modified and chemically manipulated filamentous bacteriophages. The genetic manipulation endows the phages with the ability to display a host-specificity-conferring ligand. The phages are loaded with a large payload of a cytotoxic drug by chemical conjugation. In the presented examples we used anti ErbB2 and anti ERGR antibodies as targeting moieties, the drug hygromycin conjugated to the phages by a covalent amide bond, or the drug doxorubicin conjugated to genetically-engineered cathepsin-B sites on the phage coat. We show that targeting of phage nanomedicines via specific antibodies to receptors on cancer cell membranes results in endocytosis, intracellular degradation, and drug release, resulting in growth inhibition of the target cells in vitro with a potentiation factor of >1000 over the corresponding free drugs. Conclusion The results of the proof-of concept study presented here reveal important features regarding the potential of filamentous phages to serve as drug-delivery platform, on the affect of drug solubility or hydrophobicity on the target specificity of the platform and on the effect of drug release mechanism on the potency of the platform. These results define targeted drug-carrying filamentous phage nanoparticles as a unique type of antibody-drug conjugates.

Killing cancer cells by targeted drug-carrying phage nanomedicines

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Bar, Hagit; Yacoby, Iftach; Benhar, Itai

2008-01-01

Background Systemic administration of chemotherapeutic agents, in addition to its anti-tumor benefits, results in indiscriminate drug distribution and severe toxicity. This shortcoming may be overcome by targeted drug-carrying platforms that ferry the drug to the tumor site while limiting exposure to non-target tissues and organs. Results We present a new form of targeted anti-cancer therapy in the form of targeted drug-carrying phage nanoparticles. Our approach is based on genetically-modified and chemically manipulated filamentous bacteriophages. The genetic manipulation endows the phages with the ability to display a host-specificity-conferring ligand. The phages are loaded with a large payload of a cytotoxic drug by chemical conjugation. In the presented examples we used anti ErbB2 and anti ERGR antibodies as targeting moieties, the drug hygromycin conjugated to the phages by a covalent amide bond, or the drug doxorubicin conjugated to genetically-engineered cathepsin-B sites on the phage coat. We show that targeting of phage nanomedicines via specific antibodies to receptors on cancer cell membranes results in endocytosis, intracellular degradation, and drug release, resulting in growth inhibition of the target cells in vitro with a potentiation factor of >1000 over the corresponding free drugs. Conclusion The results of the proof-of concept study presented here reveal important features regarding the potential of filamentous phages to serve as drug-delivery platform, on the affect of drug solubility or hydrophobicity on the target specificity of the platform and on the effect of drug release mechanism on the potency of the platform. These results define targeted drug-carrying filamentous phage nanoparticles as a unique type of antibody-drug conjugates. PMID:18387177

Tumor initiating cells and chemoresistance: which is the best strategy to target colon cancer stem cells?

Science.gov (United States)

Paldino, Emanuela; Tesori, Valentina; Casalbore, Patrizia; Gasbarrini, Antonio; Puglisi, Maria Ausiliatrice

2014-01-01

There is an emerging body of evidence that chemoresistance and minimal residual disease result from selective resistance of a cell subpopulation from the original tumor that is molecularly and phenotypically distinct. These cells are called "cancer stem cells" (CSCs). In this review, we analyze the potential targeting strategies for eradicating CSCs specifically in order to develop more effective therapeutic strategies for metastatic colon cancer. These include induction of terminal epithelial differentiation of CSCs or targeting some genes expressed only in CSCs and involved in self-renewal and chemoresistance. Ideal targets could be cell regulators that simultaneously control the stemness and the resistance of CSCs. Another important aspect of cancer biology, which can also be harnessed to create novel broad-spectrum anticancer agents, is the Warburg effect, also known as aerobic glycolysis. Actually, little is yet known with regard to the metabolism of CSCs population, leaving an exciting unstudied avenue in the dawn of the emerging field of metabolomics.

Lipid extraction from microalgae using a single ionic liquid

Science.gov (United States)

Salvo, Roberto Di; Reich, Alton; Dykes, Jr., H. Waite H.; Teixeira, Rodrigo

2013-05-28

A one-step process for the lysis of microalgae cell walls and separation of the cellular lipids for use in biofuel production by utilizing a hydrophilic ionic liquid, 1-butyl-3-methylimidazolium. The hydrophilic ionic liquid both lyses the microalgae cell walls and forms two immiscible layers, one of which consists of the lipid contents of the lysed cells. After mixture of the hydrophilic ionic liquid with a suspension of microalgae cells, gravity causes a hydrophobic lipid phase to move to a top phase where it is removed from the mixture and purified. The hydrophilic ionic liquid is recycled to lyse new microalgae suspensions.

Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells

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Hiroshi Sato

2017-01-01

Full Text Available Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV. Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

Towards The Generation of Functionalized Magnetic Nanowires to Target Leukemic Cells

KAUST Repository

Alsharif, Nouf

2016-01-01

. In addition the NWs can be coated and functionalized to target cells of interest and, upon exposure to an alternating magnetic field, have been shown to induce cell death on several types of adherent cells, including several cancer cell types. For suspension

Targeting cancer cells using 3-bromopyruvate for selective cancer treatment

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Hussam H Baghdadi

2017-01-01

Full Text Available Cancer treatment deserves more research efforts despite intensive conventional treatment modalities for many types of malignancies. Metastasis and resistance to chemotherapy and radiotherapy receive a lot of global research efforts. The current advances in cancer biology may improve targeting the critical metabolic differences that distinguish cancer cells from normal cells. Cancer cells are highly glycolytic for energy production, exhibit the Warburg effect, establish aggressive acidic microenvironment, maintain cancer stem cells, exhibit resistance to chemotherapy, have low antioxidant systems but different ΔΨm (delta psi, mitochondrial transmembrane potential, express P-glycoprotein for multidrug resistance, upregulate glucose transporters and monocarboxylate transporters and are under high steady-state reactive oxygen species conditions. Normal cells differ in all these aspects. Lactate produced through the Warburg effect helps cancer metastasis. Targeting glycolysis reactions for energy production in cancer cells seems promising in decreasing the proliferation and metastasis of cancer cells. 3-bromopyruvate makes use of cancer biology in treating cancer cells, cancer stem cells and preventing metastasis in human cancer as discussed in this review. Updated advances are analyzed here, which include research analysis of background, experience, readings in the field of cancer biology, oncology and biochemistry.

Slp-76 is a critical determinant of NK cell-mediated recognition of missing-self targets

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Lampe, Kristin; Endale, Mehari; Cashman, Siobhan; Fang, Hao; Mattner, Jochen; Hildeman, David; Hoebe, Kasper

2015-01-01

Absence of MHC class I expression is an important mechanism by which NK cells recognize a variety of target cells, yet the pathways underlying “missing-self” recognition, including the involvement of activating receptors, remain poorly understood. Using ENU mutagenesis in mice, we identified a germline mutant, designated Ace, with a marked defect in NK cell-mediated recognition and elimination of “missing-self” targets. The causative mutation was linked to chromosome 11 and identified as a missense mutation [Thr428Ile] in the SH2 domain of Slp-76—a critical adapter molecule downstream of ITAM-containing surface receptors. The Slp-76 Ace mutation behaved as a hypomorphic allele—while no major defects were observed in conventional T cell development/function, a marked defect in NK cell-mediated elimination of β2-Microglobulin (β2M)-deficient target cells was observed. Further studies revealed Slp-76 to control NK cell receptor expression and maturation, however, activation of Slp-76ace/ace NK cells through ITAM-containing NK cell receptors or allogeneic/tumor target cells appeared largely unaffected. Imagestream analysis of the NK-β2M−/− target cell synapse, revealed a specific defect in actin recruitment to the conjugate synapse in Slp-76ace/ace NK cells. Overall these studies establish Slp-76 as a critical determinant of NK cell development and NK cell-mediated elimination of missing-self target cells. PMID:25929249

Slp-76 is a critical determinant of NK-cell mediated recognition of missing-self targets.

Science.gov (United States)

Lampe, Kristin; Endale, Mehari; Cashman, Siobhan; Fang, Hao; Mattner, Jochen; Hildeman, David; Hoebe, Kasper

2015-07-01

Absence of MHC class I expression is an important mechanism by which NK cells recognize a variety of target cells, yet the pathways underlying "missing-self" recognition, including the involvement of activating receptors, remain poorly understood. Using ethyl-N-nitrosourea mutagenesis in mice, we identified a germline mutant, designated Ace, with a marked defect in NK cell mediated recognition and elimination of "missing-self" targets. The causative mutation was linked to chromosome 11 and identified as a missense mutation (Thr428Ile) in the SH2 domain of Slp-76-a critical adapter molecule downstream of ITAM-containing surface receptors. The Slp-76 Ace mutation behaved as a hypomorphic allele-while no major defects were observed in conventional T-cell development/function, a marked defect in NK cell mediated elimination of β2-microglobulin (β2M) deficient target cells was observed. Further studies revealed Slp-76 to control NK-cell receptor expression and maturation; however, activation of Slp-76(ace/ace) NK cells through ITAM-containing NK-cell receptors or allogeneic/tumor target cells appeared largely unaffected. Imagestream analysis of the NK-β2M(-/-) target cell synapse revealed a specific defect in actin recruitment to the conjugate synapse in Slp-76(ace/ace) NK cells. Overall these studies establish Slp-76 as a critical determinant of NK-cell development and NK cell mediated elimination of missing-self target cells in mice. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Human tonsillar IgE biosynthesis in vitro. I. Enhancement of IgE and IgG synthesis in the presence of pokeweed mitogen by T-cell irradiation

International Nuclear Information System (INIS)

Ohta, K.; Manzara, T.; Harbeck, R.J.; Kirkpatrick, C.H.

1982-01-01

A study of the events regulating human IgE biosynthesis in vitro was undertaken with tonsillar lymphocytes. IgG synthesis was also studied to evaluate the specificity of our observations. T-cell irradiation significantly enhanced synthesis of IgE by pokeweed mitogen (PWM)-stimulated B cells from 12 of 18 donors and IgG in all 18 donors. This enhancement was the result of de novo immunoglobulin synthesis, since the amount of IgE and IgG spontaneously released from lysed and lysed-and-cultured mononuclear cells was significantly less than that detected in the cell cultures, and the augmentation was completely ablated by the treatment of the cells with cycloheximide or mitomycin C. Enhancement was also dependent on the presence of PWM; T-cell irradiation did not enhance IgE synthesis in unstimulated cultures. Moreover, this enhancement was also observed in the co-cultures of B cells and allogeneic irradiated T cells. These observations suggest that radiosensitive T cells exert a suppressive activity that contributes to regulation of human IgE and IgG synthesis and that the suppressor function as well as the helper function can overcome allogeneic disparities

Targeting of follicle stimulating hormone peptide-conjugated dendrimers to ovarian cancer cells

Science.gov (United States)

Modi, Dimple A.; Sunoqrot, Suhair; Bugno, Jason; Lantvit, Daniel D.; Hong, Seungpyo; Burdette, Joanna E.

2014-02-01

Ovarian cancer is the most lethal gynecological malignancy. Current treatment modalities include a combination of surgery and chemotherapy, which often lead to loss of fertility in premenopausal women and a myriad of systemic side effects. To address these issues, we have designed poly(amidoamine) (PAMAM) dendrimers to selectively target the follicle stimulating hormone receptor (FSHR), which is overexpressed by tumorigenic ovarian cancer cells but not by immature primordial follicles and other non-tumorigenic cells. Fluorescein-labeled generation 5 (G5) PAMAM dendrimers were conjugated with the binding peptide domain of FSH (FSH33) that has a high affinity to FSHR. The targeted dendrimers exhibited high receptor selectivity to FSHR-expressing OVCAR-3 cells, resulting in significant uptake and downregulation of an anti-apoptotic protein survivin, while showing minimal interactions with SKOV-3 cells that do not express FSHR. The selectivity of the FSH33-targeted dendrimers was further validated in 3D organ cultures of normal mouse ovaries. Immunostaining of the conjugates revealed their selective binding and uptake by ovarian surface epithelium (OSE) cells that express FSHR, while sparing the immature primordial follicles. In addition, an in vivo study monitoring tissue accumulation following a single intraperitoneal (i.p.) injection of the conjugates showed significantly higher accumulation of FSH33-targeted dendrimers in the ovary and oviduct compared to the non-targeted conjugates. These proof-of-concept findings highlight the potential of these FSH33-targeted dendrimers to serve as a delivery platform for anti-ovarian cancer drugs, while reducing their systemic side effects by preventing nonspecific uptake by the primordial follicles.Ovarian cancer is the most lethal gynecological malignancy. Current treatment modalities include a combination of surgery and chemotherapy, which often lead to loss of fertility in premenopausal women and a myriad of systemic side

Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment.

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Ruben R Bender

2016-06-01

Full Text Available Receptor-targeted lentiviral vectors (LVs can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance. Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4 was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs.

Targeting poly (ADP-ribose polymerase partially contributes to bufalin-induced cell death in multiple myeloma cells.

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He Huang

Full Text Available Despite recent pharmaceutical advancements in therapeutic drugs, multiple myeloma (MM remains an incurable disease. Recently, ploy(ADP-ribose polymerase 1 (PARP1 has been shown as a potentially promising target for MM therapy. A previous report suggested bufalin, a component of traditional Chinese medicine ("Chan Su", might target PARP1. However, this hypothesis has not been verified. We here showed that bufalin could inhibit PARP1 activity in vitro and reduce DNA-damage-induced poly(ADP-ribosylation in MM cells. Molecular docking analysis revealed that the active site of bufalin interaction is within the catalytic domain of PAPR1. Thus, PARP1 is a putative target of bufalin. Furthermore, we showed, for the first time that the proliferation of MM cell lines (NCI-H929, U266, RPMI8226 and MM.1S and primary CD138(+ MM cells could be inhibited by bufalin, mainly via apoptosis and G2-M phase cell cycle arrest. MM cell apoptosis was confirmed by apoptotic cell morphology, Annexin-V positive cells, and the caspase3 activation. We further evaluated the role of PARP1 in bufalin-induced apoptosis, discovering that PARP1 overexpression partially suppressed bufalin-induced cell death. Moreover, bufalin can act as chemosensitizer to enhance the cell growth-inhibitory effects of topotecan, camptothecin, etoposide and vorinostat in MM cells. Collectively, our data suggest that bufalin is a novel PARP1 inhibitor and a potentially promising therapeutic agent against MM alone or in combination with other drugs.

Mechanoresponsive stem cells to target cancer metastases through biophysical cues.

Science.gov (United States)

Liu, Linan; Zhang, Shirley X; Liao, Wenbin; Farhoodi, Henry P; Wong, Chi W; Chen, Claire C; Ségaliny, Aude I; Chacko, Jenu V; Nguyen, Lily P; Lu, Mengrou; Polovin, George; Pone, Egest J; Downing, Timothy L; Lawson, Devon A; Digman, Michelle A; Zhao, Weian

2017-07-26

Despite decades of effort, little progress has been made to improve the treatment of cancer metastases. To leverage the central role of the mechanoenvironment in cancer metastasis, we present a mechanoresponsive cell system (MRCS) to selectively identify and treat cancer metastases by targeting the specific biophysical cues in the tumor niche in vivo. Our MRCS uses mechanosensitive promoter-driven mesenchymal stem cell (MSC)-based vectors, which selectively home to and target cancer metastases in response to specific mechanical cues to deliver therapeutics to effectively kill cancer cells, as demonstrated in a metastatic breast cancer mouse model. Our data suggest a strong correlation between collagen cross-linking and increased tissue stiffness at the metastatic sites, where our MRCS is specifically activated by the specific cancer-associated mechano-cues. MRCS has markedly reduced deleterious effects compared to MSCs constitutively expressing therapeutics. MRCS indicates that biophysical cues, specifically matrix stiffness, are appealing targets for cancer treatment due to their long persistence in the body (measured in years), making them refractory to the development of resistance to treatment. Our MRCS can serve as a platform for future diagnostics and therapies targeting aberrant tissue stiffness in conditions such as cancer and fibrotic diseases, and it should help to elucidate mechanobiology and reveal what cells "feel" in the microenvironment in vivo. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Crispr-mediated Gene Targeting of Human Induced Pluripotent Stem Cells.

Science.gov (United States)

Byrne, Susan M; Church, George M

2015-01-01

CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem or induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, we describe an optimized protocol for genome engineering of human iPSCs using a simple transient transfection of plasmids and/or single-stranded oligonucleotides. With this protocol, we achieve transfection efficiencies greater than 60%, with gene disruption efficiencies from 1-25% and gene insertion/replacement efficiencies from 0.5-10% without any further selection or enrichment steps. We also describe how to design and assess optimal sgRNA target sites and donor targeting vectors; cloning individual iPSC by single cell FACS sorting, and genotyping successfully edited cells.

Targeted destruction of murine macrophage cells with bioconjugated gold nanorods

Science.gov (United States)

Pissuwan, Dakrong; Valenzuela, Stella M.; Killingsworth, Murray C.; Xu, Xiaoda; Cortie, Michael B.

2007-12-01

Gold nanorods manifest a readily tunable longitudinal plasmon resonance with light and consequently have potential for use in photothermal therapeutics. Recent work by others has shown how gold nanoshells and rods can be used to target cancer cells, which can then be destroyed using relatively high power laser radiation (˜1×105 to 1×1010 W/m2). Here we extend this concept to demonstrate how gold nanorods can be modified to bind to target macrophage cells, and show that high intensity laser radiation is not necessary, with even 5×102 W/m2 being sufficient, provided that a total fluence of ˜30 J/cm2 is delivered. We used the murine cell line RAW 264.7 and the monoclonal antibody CD11b, raised against murine macrophages, as our model system and a 5 mW solid state diode laser as our energy source. Exposure of the cells labeled with gold nanorods to a laser fluence of 30 J/cm2 resulted in 81% cell death compared to only 0.9% in the control, non-labeled cells.

Myeloid Conditioning with c-kit-Targeted CAR-T Cells Enables Donor Stem Cell Engraftment.

Science.gov (United States)

Arai, Yasuyuki; Choi, Uimook; Corsino, Cristina I; Koontz, Sherry M; Tajima, Masaki; Sweeney, Colin L; Black, Mary A; Feldman, Steven A; Dinauer, Mary C; Malech, Harry L

2018-05-02

We report a novel approach to bone marrow (BM) conditioning using c-kit-targeted chimeric antigen receptor T (c-kit CAR-T) cells in mice. Previous reports using anti-c-kit or anti-CD45 antibody linked to a toxin such as saporin have been promising. We developed a distinctly different approach using c-kit CAR-T cells. Initial studies demonstrated in vitro killing of hematopoietic stem cells by c-kit CAR-T cells but poor expansion in vivo and poor migration of CAR-T cells into BM. Pre-treatment of recipient mice with low-dose cyclophosphamide (125 mg/kg) together with CXCR4 transduction in the CAR-T cells enhanced trafficking to and expansion in BM (c-kit + population (9.0%-0.1%). Because congenic Thy1.1 CAR-T cells were used in the Thy1.2-recipient mice, anti-Thy1.1 antibody could be used to deplete CAR-T cells in vivo before donor BM transplant. This achieved 20%-40% multilineage engraftment. We applied this conditioning to achieve an average of 28% correction of chronic granulomatous disease mice by wild-type BM transplant. Our findings provide a proof of concept that c-kit CAR-T cells can achieve effective BM conditioning without chemo-/radiotherapy. Our work also demonstrates that co-expression of a trafficking receptor can enhance targeting of CAR-T cells to a designated tissue. Published by Elsevier Inc.

Targeting Gallium to Cancer Cells through the Folate Receptor

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Nerissa Viola-Villegas

2008-01-01

Full Text Available The development of gallium(III compounds as anti-cancer agents for both treatment and diagnosis is a rapidly developing field of research. Problems remain in exploring the full potential of gallium(III as a safe and successful therapeutic agent or as an imaging agent. One of the major issues is that gallium(III compounds have little tropism for cancer cells. We have combined the targeting properties of folic acid (FA with long chain liquid polymer poly(ethylene glycol (PEG 'spacers’. This FA-PEG unit has been coupled to the gallium coordination complex of 1,4,7,10-tetraazacyclo-dodecane-N, N′, N′, N′′-tetraacetic acid (DOTA through amide linkages for delivery into target cells overexpressing the folate receptor (FR. In vitro cytotoxicity assays were conducted against a multi-drug resistant ovarian cell line (A2780/AD that overexpresses the FR and contrasted against a FR free Chinese hamster ovary (CHO cell line. Results are rationalized taking into account stability studies conducted in RPMI 1640 media and HEPES buffer at pH 7.4.

Targeting Gallium to Cancer Cells through the Folate Receptor

Directory of Open Access Journals (Sweden)

Nerissa Viola-Villegas

2008-01-01

Full Text Available The development of gallium(III compounds as anti-cancer agents for both treatment and diagnosis is a rapidly developing field of research. Problems remain in exploring the full potential of gallium(III as a safe and successful therapeutic agent or as an imaging agent. One of the major issues is that gallium(III compounds have little tropism for cancer cells. We have combined the targeting properties of folic acid (FA with long chain liquid polymer poly(ethylene glycol (PEG ‘spacers’. This FA-PEG unit has been coupled to the gallium coordination complex of 1,4,7,10-tetraazacyclo-dodecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA through amide linkages for delivery into target cells overexpressing the folate receptor (FR. In vitro cytotoxicity assays were conducted against a multi-drug resistant ovarian cell line (A2780/AD that overexpresses the FR and contrasted against a FR free Chinese hamster ovary (CHO cell line. Results are rationalized taking into account stability studies conducted in RPMI 1640 media and HEPES buffer at pH 7.4.

Evaluating Cytotoxicity of Hyaluronate Targeted Solid Lipid Nanoparticles of Etoposide on SK-OV-3 Cells

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Parviz Mohammadi Ghalaei

2014-01-01

Full Text Available The epithelial ovarian carcinoma is one of the most fatal gynecological cancers. Etoposide is used in treating platinum-resistant ovarian cancer. Sodium hyaluronate is a substance that binds to the CD44 receptors overexpressed in SK-OV-3 cells of epithelial ovarian carcinoma. The aim of the present work was to study the cytotoxicity effect of hyaluronate targeted solid lipid nanoparticles (SLNs of etoposide on SK-OV-3 cells. The cytotoxicity of the targeted and nontargeted SLNs of etoposide was compared to free drug on the SK-OV-3 cells by MTT assay method. The cellular uptake of the targeted and nontargeted nanoparticles containing sodium fluorescein was also studied. The difference of cell vitality between nontargeted nanoparticles and also targeted nanoparticles with free drug was significant. Targeted nanoparticles also caused more toxicity than nontargeted nanoparticles (P<0.05. After 4 hours of incubating, the fluorescence was remarkably higher in the cells treated by targeted SLNs rather than nontargeted ones, and there was no observable fluorescence in cells incubated with pure sodium fluorescein. Hyaluronate targeted SLNs containing etoposide increased the cytotoxicity of etoposide on SK-OV-3 cells which may be a worthwhile potential method for reducing the prescribed dose and systemic side effects of this drug in epithelial ovarian carcinoma.

Gene targeting and cloning in pigs using fetal liver derived cells.

Science.gov (United States)

Waghmare, Sanjeev K; Estrada, Jose; Reyes, Luz; Li, Ping; Ivary, Bess; Sidner, Richard A; Burlak, Chris; Tector, A Joseph

2011-12-01

Since there are no pig embryonic stem cells, pig genetic engineering is done in fetal fibroblasts that remain totipotent for only 3 to 5 wk. Nuclear donor cells that remain totipotent for longer periods of time would facilitate complicated genetic engineering in pigs. The goal of this study was to test the feasibility of using fetal liver-derived cells (FLDC) to perform gene targeting, and create a genetic knockout pig. FLDC were isolated and processed using a human liver stem cell protocol. Single copy α-1,3-galactosyl transferase knockout (GTKO) FLDCs were created using electroporation and neomycin resistant colonies were screened using PCR. Homozygous GTKO cells were created through loss of heterozygosity mutations in single GTKO FLDCs. Double GTKO FLDCs were used in somatic cell nuclear transfer (SCNT) to create GTKO pigs. FLDCs grew for more than 80 population doublings, maintaining normal karyotype. Gene targeting and loss of heterozygosity mutations produced homozygous GTKO FLDCs. FLDCs used in SCNT gave rise to homozygous GTKO pigs. FDLCs can be used in gene targeting and SCNT to produce genetically modified pigs. The increased life span in culture compared to fetal fibroblasts may facilitate genetic engineering in the pig. Copyright © 2011 Elsevier Inc. All rights reserved.

Chimeric Antigen Receptor (CAR) T Cells: Lessons Learned from Targeting of CD19 in B-Cell Malignancies.

Science.gov (United States)

Hay, Kevin A; Turtle, Cameron J

2017-03-01

Adoptive immunotherapy with chimeric antigen receptor-modified (CAR)-T cells is a rapidly growing therapeutic approach to treating patients with refractory cancer, with over 100 clinical trials in various malignancies in progress. The enthusiasm for CAR-T cells has been driven by the clinical success of CD19-targeted CAR-T cell therapy in B-cell acute lymphoblastic leukemia, and the promising data in B-cell non-Hodgkin's lymphoma and chronic lymphocytic leukemia. Despite the success of targeting CD19 with CAR-T cells in early clinical studies, many challenges remain to improve outcomes, reduce toxicity, and determine the appropriate settings for CAR-T cell immunotherapy. Reviewing the lessons learned thus far in CD19 CAR-T cell trials and how some of these challenges may be overcome will help guide the development of CAR-T cell therapy for malignancies of B-cell origin, as well as for other hematopoietic and non-hematopoietic cancers.

Near-IR laser-triggered target cell collection using a carbon nanotube-based cell-cultured substrate.

Science.gov (United States)

Sada, Takao; Fujigaya, Tsuyohiko; Niidome, Yasuro; Nakazawa, Kohji; Nakashima, Naotoshi

2011-06-28

Unique near-IR optical properties of single-walled carbon nanotube (SWNTs) are of interest in many biological applications. Here we describe the selective cell detachment and collection from an SWNT-coated cell-culture dish triggered by near-IR pulse laser irradiation. First, HeLa cells were cultured on an SWNT-coated dish prepared by a spraying of an aqueous SWNT dispersion on a glass dish. The SWNT-coated dish was found to show a good cell adhesion behavior as well as a cellular proliferation rate similar to a conventional glass dish. We discovered, by near-IR pulse laser irradiation (at the laser power over 25 mW) to the cell under optical microscopic observation, a quick single-cell detachment from the SWNT-coated surface. Shockwave generation from the irradiated SWNTs is expected to play an important role for the cell detachment. Moreover, we have succeeded in catapulting the target single cell from the cultured medium when the depth of the medium was below 150 μm and the laser power was stronger than 40 mW. The captured cell maintained its original shape. The retention of the genetic information of the cell was confirmed by the polymerase chain reaction (PCR) technique. A target single-cell collection from a culture medium under optical microscopic observation is significant in wide fields of single-cell studies in biological areas.

Mechanosensation Dynamically Coordinates Polar Growth and Cell Wall Assembly to Promote Cell Survival.

Science.gov (United States)

Davì, Valeria; Tanimoto, Hirokazu; Ershov, Dmitry; Haupt, Armin; De Belly, Henry; Le Borgne, Rémi; Couturier, Etienne; Boudaoud, Arezki; Minc, Nicolas

2018-04-23

How growing cells cope with size expansion while ensuring mechanical integrity is not known. In walled cells, such as those of microbes and plants, growth and viability are both supported by a thin and rigid encasing cell wall (CW). We deciphered the dynamic mechanisms controlling wall surface assembly during cell growth, using a sub-resolution microscopy approach to monitor CW thickness in live rod-shaped fission yeast cells. We found that polar cell growth yielded wall thinning and that thickness negatively influenced growth. Thickness at growing tips exhibited a fluctuating behavior with thickening phases followed by thinning phases, indicative of a delayed feedback promoting thickness homeostasis. This feedback was mediated by mechanosensing through the CW integrity pathway, which probes strain in the wall to adjust synthase localization and activity to surface growth. Mutants defective in thickness homeostasis lysed by rupturing the wall, demonstrating its pivotal role for walled cell survival. Copyright © 2018 Elsevier Inc. All rights reserved.

Integrin Targeting and Toxicological Assessment of Peptide-Conjugated Liposome Delivery Systems to Activated Endothelial Cells

DEFF Research Database (Denmark)

Kermanizadeh, Ali; Villadsen, Klaus; Østrem, Ragnhild Garborg

2017-01-01

constructed with the aim of targeting integrins (i.e. vitronectin and/or fibronectin receptors) on activated endothelial cells. The peptide-conjugated liposomes induced only cytotoxicity at the highest concentration in non-activated or activated endothelial cells, as well as in co-culture of endothelial cells...... and macrophages. There was unaltered secretion of cytokines following exposure of peptide-conjugated liposomes to endothelial cells, indicating that the materials were not inflammogenic. Liposomes with a peptide targeting the fibronectin receptor (integrin α5β1) were more effective in targeting of activated....... Therefore, this study demonstrates the feasibility of constructing a peptide-conjugated cationic liposome, which displays targeting to activated endothelial cells at concentrations that are not cytotoxic or inflammogenic to the cells....

The cell's nucleolus: an emerging target for chemotherapeutic intervention.

Science.gov (United States)

Pickard, Amanda J; Bierbach, Ulrich

2013-09-01

The transient nucleolus plays a central role in the up-regulated synthesis of ribosomal RNA (rRNA) to sustain ribosome biogenesis, a hallmark of aberrant cell growth. This function, in conjunction with its unique pathohistological features in malignant cells and its ability to mediate apoptosis, renders this sub-nuclear structure a potential target for chemotherapeutic agents. In this Minireview, structurally and functionally diverse small molecules are discussed that have been reported to either interact with the nucleolus directly or perturb its function indirectly by acting on its dynamic components. These molecules include all major classes of nucleic-acid-targeted agents, antimetabolites, kinase inhibitors, anti-inflammatory drugs, natural product antibiotics, oligopeptides, as well as nanoparticles. Together, these molecules are invaluable probes of structure and function of the nucleolus. They also provide a unique opportunity to develop novel strategies for more selective and therefore better-tolerated chemotherapeutic intervention. In this regard, inhibition of RNA polymerase-I-mediated rRNA synthesis appears to be a promising mechanism for killing cancer cells. The recent development of molecules targeted at G-quadruplex-forming rRNA gene sequences, which are currently undergoing clinical trials, seems to attest to the success of this approach. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Targeting Gas6/TAM in cancer cells and tumor microenvironment.

Science.gov (United States)

Wu, Guiling; Ma, Zhiqiang; Cheng, Yicheng; Hu, Wei; Deng, Chao; Jiang, Shuai; Li, Tian; Chen, Fulin; Yang, Yang

2018-01-31

Growth arrest-specific 6, also known as Gas6, is a human gene encoding the Gas6 protein, which was originally found to be upregulated in growth-arrested fibroblasts. Gas6 is a member of the vitamin K-dependent family of proteins expressed in many human tissues and regulates several biological processes in cells, including proliferation, survival and migration, by binding to its receptors Tyro3, Axl and Mer (TAM). In recent years, the roles of Gas6/TAM signalling in cancer cells and the tumour microenvironment have been studied, and some progress has made in targeted therapy, providing new potential directions for future investigations of cancer treatment. In this review, we introduce the Gas6 and TAM receptors and describe their involvement in different cancers and discuss the roles of Gas6 in cancer cells, the tumour microenvironment and metastasis. Finally, we introduce recent studies on Gas6/TAM targeting in cancer therapy, which will assist in the experimental design of future analyses and increase the potential use of Gas6 as a therapeutic target for cancer.

Chimeric Antigen Receptor (CAR) T cells: Lessons Learned from Targeting of CD19 in B cell malignancies

Science.gov (United States)

Hay, Kevin A; Turtle, Cameron J

2017-01-01

Adoptive immunotherapy with chimeric antigen receptor-modified T (CAR-T) cells is a rapidly growing therapeutic approach to treating patients with refractory cancer, with over 100 clinical trials in various malignancies in progress. The enthusiasm for CAR-T cells has been driven by the clinical success of CD19-targeted CAR-T therapy in B-cell acute lymphoblastic leukemia, and the promising data in B-cell non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. Despite the success of targeting CD19 with CAR-T cells in early clinical studies, many challenges remain to improve outcomes, reduce toxicity, and determine the appropriate settings for CAR-T cell immunotherapy. Reviewing the lessons learned thus far in CD19 CAR-T cell trials and how some of these challenges may be overcome will help guide the development of CAR-T cell therapy for malignancies of B-cell origin, as well as for other hematopoietic and non-hematopoietic cancers. PMID:28110394

MicroRNA-145 targets YES and STAT1 in colon cancer cells

DEFF Research Database (Denmark)

Gregersen, Lea H; Jacobsen, Anders B; Frankel, Lisa

2010-01-01

miRNA overexpression. Gene Ontology analysis showed an overrepresentation of genes involved in cell death, cellular growth and proliferation, cell cycle, gene expression and cancer. A number of the identified miRNA targets have previously been implicated in cancer, including YES, FSCN1, ADAM17, BIRC2......, VANGL1 as well as the transcription factor STAT1. Both YES and STAT1 were verified as direct miR-145 targets. CONCLUSIONS/SIGNIFICANCE: The study identifies and validates new cancer-relevant direct targets of miR-145 in colon cancer cells and hereby adds important mechanistic understanding of the tumor......BACKGROUND: MicroRNAs (miRNAs) have emerged as important gene regulators and are recognized as key players in tumorigenesis. miR-145 is reported to be down-regulated in several cancers, but knowledge of its targets in colon cancer remains limited. METHODOLOGY/PRINCIPAL FINDINGS: To investigate...

Method and apparatus for iterative lysis and extraction of algae

Science.gov (United States)

Chew, Geoffrey; Boggs, Tabitha; Dykes, Jr., H. Waite H.; Doherty, Stephen J.

2015-12-01

A method and system for processing algae involves the use of an ionic liquid-containing clarified cell lysate to lyse algae cells. The resulting crude cell lysate may be clarified and subsequently used to lyse algae cells. The process may be repeated a number of times before a clarified lysate is separated into lipid and aqueous phases for further processing and/or purification of desired products.

Membrane Targeting of P-type ATPases in Plant Cells

International Nuclear Information System (INIS)

Harper, Jeffrey F.

2004-01-01

How membrane proteins are targeted to specific subcellular locations is a very complex and poorly understood area of research. Our long-term goal is to use P-type ATPases (ion pumps), in a model plant system Arabidopsis, as a paradigm to understand how members of a family of closely related membrane proteins can be targeted to different subcellular locations. The research is divided into two specific aims. The first aim is focused on determining the targeting destination of all 10 ACA-type calcium pumps (Arabidopsis Calcium ATPase) in Arabidopsis. ACAs represent a plant specific-subfamily of plasma membrane-type calcium pumps. In contrast to animals, the plant homologs have been found in multiple membrane systems, including the ER (ACA2), tonoplast (ACA4) and plasma membrane (ACA8). Their high degree of similarity provides a unique opportunity to use a comparative approach to delineate the membrane specific targeting information for each pump. One hypothesis to be tested is that an endomembrane located ACA can be re-directed to the plasma membrane by including targeting information from a plasma membrane isoform, ACA8. Our approach is to engineer domain swaps between pumps and monitor the targeting of chimeric proteins in plant cells using a Green Fluorescence Protein (GFP) as a tag. The second aim is to test the hypothesis that heterologous transporters can be engineered into plants and targeted to the plasma membrane by fusing them to a plasma membrane proton pump. As a test case we are evaluating the targeting properties of fusions made between a yeast sodium/proton exchanger (Sod2) and a proton pump (AHA2). This fusion may potentially lead to a new strategy for engineering salt resistant plants. Together these aims are designed to provide fundamental insights into the biogenesis and function of plant cell membrane systems

Targeting myeloid cells to the brain using non-myeloablative conditioning.

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Chotima Böttcher

Full Text Available Bone marrow-derived cells (BMDCs are able to colonize the central nervous system (CNS at sites of damage. This ability makes BMDCs an ideal cellular vehicle for transferring therapeutic genes/molecules to the CNS. However, conditioning is required for bone marrow-derived myeloid cells to engraft in the brain, which so far has been achieved by total body irradiation (TBI and by chemotherapy (e.g. busulfan treatment. Unfortunately, both regimens massively disturb the host's hematopoietic compartment. Here, we established a conditioning protocol to target myeloid cells to sites of brain damage in mice using non-myeloablative focal head irradiation (HI. This treatment was associated with comparatively low inflammatory responses in the CNS despite cranial radiation doses which are identical to TBI, as revealed by gene expression analysis of cytokines/chemokines such as CCL2, CXCL10, TNF-α and CCL5. HI prior to bone marrow transplantation resulted in much lower levels of blood chimerism defined as the percentage of donor-derived cells in peripheral blood ( 95% or busulfan treatment (> 50%. Nevertheless, HI effectively recruited myeloid cells to the area of motoneuron degeneration in the brainstem within 7 days after facial nerve axotomy. In contrast, no donor-derived cells were detected in the lesioned facial nucleus of busulfan-treated animals up to 2 weeks after transplantation. Our findings suggest that myeloid cells can be targeted to sites of brain damage even in the presence of very low levels of peripheral blood chimerism. We established a novel non-myeloablative conditioning protocol with minimal disturbance of the host's hematopoietic system for targeting BMDCs specifically to areas of pathology in the brain.

Characterization of aldehyde dehydrogenase 1 high ovarian cancer cells: Towards targeted stem cell therapy.

Science.gov (United States)

Sharrow, Allison C; Perkins, Brandy; Collector, Michael I; Yu, Wayne; Simons, Brian W; Jones, Richard J

2016-08-01

The cancer stem cell (CSC) paradigm hypothesizes that successful clinical eradication of CSCs may lead to durable remission for patients with ovarian cancer. Despite mounting evidence in support of ovarian CSCs, their phenotype and clinical relevance remain unclear. We and others have found high aldehyde dehydrogenase 1 (ALDH(high)) expression in a variety of normal and malignant stem cells, and sought to better characterize ALDH(high) cells in ovarian cancer. We compared ALDH(high) to ALDH(low) cells in two ovarian cancer models representing distinct subtypes: FNAR-C1 cells, derived from a spontaneous rat endometrioid carcinoma, and the human SKOV3 cell line (described as both serous and clear cell subtypes). We assessed these populations for stem cell features then analyzed expression by microarray and qPCR. ALDH(high) cells displayed CSC properties, including: smaller size, quiescence, regenerating the phenotypic diversity of the cell lines in vitro, lack of contact inhibition, nonadherent growth, multi-drug resistance, and in vivo tumorigenicity. Microarray and qPCR analysis of the expression of markers reported by others to enrich for ovarian CSCs revealed that ALDH(high) cells of both models showed downregulation of CD24, but inconsistent expression of CD44, KIT and CD133. However, the following druggable targets were consistently expressed in the ALDH(high) cells from both models: mTOR signaling, her-2/neu, CD47 and FGF18/FGFR3. Based on functional characterization, ALDH(high) ovarian cancer cells represent an ovarian CSC population. Differential gene expression identified druggable targets that have the potential for therapeutic efficacy against ovarian CSCs from multiple subtypes. Copyright © 2016 Elsevier Inc. All rights reserved.

A Phenotypic Cell-Binding Screen Identifies a Novel Compound Targeting Triple-Negative Breast Cancer.

Science.gov (United States)

Chen, Luxi; Long, Chao; Youn, Jonghae; Lee, Jiyong

2018-06-11

We describe a "phenotypic cell-binding screen" by which therapeutic candidate targeting cancer cells of a particular phenotype can be isolated without knowledge of drug targets. Chemical library beads are incubated with cancer cells of the phenotype of interest in the presence of cancer cells lacking the phenotype of interest, and then the beads bound to only cancer cells of the phenotype of interest are selected as hits. We have applied this screening strategy in discovering a novel compound (LC129-8) targeting triple-negative breast cancer (TNBC). LC129-8 displayed highly specific binding to TNBC in cancer cell lines and patient-derived tumor tissues. LC129-8 exerted anti-TNBC activity by inducing apoptosis, inhibiting proliferation, reversing epithelial-mesenchymal transition, downregulating cancer stem cell activity and blocking in vivo tumor growth.

Lyn kinase is activated following thrombopoietin stimulation of the megakaryocytic cell line B1647

DEFF Research Database (Denmark)

Santini, Valeria; Scappini, Barbara; Gozzini, Antonella

2002-01-01

BACKGROUND AND OBJECTIVES: B1647 is a cell line derived from bone marrow cells of a patient with acute myeloid leukemia (M2) with a complete erythro-megakaryocytic phenotype and bears both k and p isoforms of c-mpl. Interestingly, spontaneous B1647 cell proliferation is significantly potentiated...... by thrombopoietin (TPO). DESIGN AND METHODS: We aimed to evaluate the proliferative signal transduction events following the activation of c-mpl and we stimulated B1647 cells with TPO 40 ng/mL for 3, 7, 15 and 30 minutes; cells were then lysed and whole lysates were immunoprecipitated with anti...

Protocells and their use for targeted delivery of multicomponent cargos to cancer cells

Science.gov (United States)

Brinker, C Jeffrey; Ashley, Carlee Erin; Jiang, Xingmao; Liu, Juewen; Peabody, David S; Wharton, Walker Richard; Carnes, Eric; Chackerian, Bryce; Willman, Cheryl L

2015-03-31

Various embodiments provide materials and methods for synthesizing protocells for use in targeted delivery of cargo components to cancer cells. In one embodiment, the lipid bilayer can be fused to the porous particle core to form a protocell. The lipid bilayer can be modified with targeting ligands or other ligands to achieve targeted delivery of cargo components that are loaded within the protocell to a target cell, e.g., a type of cancer. Shielding materials can be conjugated to the surface of the lipid bilayer to reduce undesired non-specific binding.

Targeting tissue factor on tumour cells and angiogenic vascular endothelial cells by factor VII-targeted verteporfin photodynamic therapy for breast cancer in vitro and in vivo in mice

International Nuclear Information System (INIS)

Hu, Zhiwei; Rao, Benqiang; Chen, Shimin; Duanmu, Jinzhong

2010-01-01

The objective of this study was to develop a ligand-targeted photodynamic therapy (tPDT) by conjugating factor VII (fVII) protein with photosensitiser verteporfin in order to overcome the poor selectivity and enhance the effect of non-targeted PDT (ntPDT) for cancer. fVII is a natural ligand for receptor tissue factor (TF) with high affinity and specificity. The reason for targeting receptor TF for the development of tPDT is that TF is a common but specific target on angiogenic tumour vascular endothelial cells (VEC) and many types of tumour cells, including solid tumours and leukaemia. Murine factor VII protein (mfVII) containing a mutation (Lys341Ala) was covalently conjugated via a cross linker EDC with Veterporfin (VP) that was extracted from liposomal Visudyne, and then free VP was separated by Sephadex G50 spin columns. fVII-tPDT using mfVII-VP conjugate, compared to ntPDT, was tested in vitro for the killing of breast cancer cells and VEGF-stimulated VEC and in vivo for inhibiting the tumour growth of breast tumours in a mouse xenograft model. We showed that: (i) fVII protein could be conjugated with VP without affecting its binding activity; (ii) fVII-tPDT could selectively kill TF-expressing breast cancer cells and VEGF-stimulated angiogenic HUVECs but had no side effects on non-TF expressing unstimulated HUVEC, CHO-K1 and 293 cells; (iii) fVII targeting enhanced the effect of VP PDT by three to four fold; (iii) fVII-tPDT induced significantly stronger levels of apoptosis and necrosis than ntPDT; and (iv) fVII-tPDT had a significantly stronger effect on inhibiting breast tumour growth in mice than ntPDT. We conclude that the fVII-targeted VP PDT that we report here is a novel and effective therapeutic with improved selectivity for the treatment of breast cancer. Since TF is expressed on many types of cancer cells including leukaemic cells and selectively on angiogenic tumour VECs, fVII-tPDT could have broad therapeutic applications for other solid cancers

Analysis of the role of homology arms in gene-targeting vectors in human cells.

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Ayako Ishii

Full Text Available Random integration of targeting vectors into the genome is the primary obstacle in human somatic cell gene targeting. Non-homologous end-joining (NHEJ, a major pathway for repairing DNA double-strand breaks, is thought to be responsible for most random integration events; however, absence of DNA ligase IV (LIG4, the critical NHEJ ligase, does not significantly reduce random integration frequency of targeting vector in human cells, indicating robust integration events occurring via a LIG4-independent mechanism. To gain insights into the mechanism and robustness of LIG4-independent random integration, we employed various types of targeting vectors to examine their integration frequencies in LIG4-proficient and deficient human cell lines. We find that the integration frequency of targeting vector correlates well with the length of homology arms and with the amount of repetitive DNA sequences, especially SINEs, present in the arms. This correlation was prominent in LIG4-deficient cells, but was also seen in LIG4-proficient cells, thus providing evidence that LIG4-independent random integration occurs frequently even when NHEJ is functionally normal. Our results collectively suggest that random integration frequency of conventional targeting vectors is substantially influenced by homology arms, which typically harbor repetitive DNA sequences that serve to facilitate LIG4-independent random integration in human cells, regardless of the presence or absence of functional NHEJ.

PET imaging of T cells: Target identification and feasibility assessment.

Science.gov (United States)

Auberson, Yves P; Briard, Emmanuelle; Rudolph, Bettina; Kaupmann, Klemen; Smith, Paul; Oberhauser, Berndt

2018-06-01

Imaging T cells using positron emission tomography (PET) would be highly useful for diagnosis and monitoring in immunology and oncology patients. There are however no obvious targets that can be used to develop imaging agents for this purpose. We evaluated several potential target proteins with selective expression in T cells, and for which lead molecules were available: PKC , Lck, ZAP70 and Itk. Ultimately, we focused on Itk (interleukin-2-inducible T cell kinase) and identified a tool molecule with properties suitable for in vivo imaging of T cells, (5aR)-5,5-difluoro-5a-methyl-N-(1-((S)-3-(methylsulfonyl)-phenyl)(tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazol-4-yl)-1,4,4a,5,5a,6-hexahydro-cyclopropa[f]-indazole-3-carboxamide (23). While not having the optimal profile for clinical use, this molecule indicates that it might be possible to develop Itk-selective PET ligands for imaging the distribution of T cells in patients. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tumor Initiating Cells and Chemoresistance: Which Is the Best Strategy to Target Colon Cancer Stem Cells?

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Emanuela Paldino

2014-01-01

Full Text Available There is an emerging body of evidence that chemoresistance and minimal residual disease result from selective resistance of a cell subpopulation from the original tumor that is molecularly and phenotypically distinct. These cells are called “cancer stem cells” (CSCs. In this review, we analyze the potential targeting strategies for eradicating CSCs specifically in order to develop more effective therapeutic strategies for metastatic colon cancer. These include induction of terminal epithelial differentiation of CSCs or targeting some genes expressed only in CSCs and involved in self-renewal and chemoresistance. Ideal targets could be cell regulators that simultaneously control the stemness and the resistance of CSCs. Another important aspect of cancer biology, which can also be harnessed to create novel broad-spectrum anticancer agents, is the Warburg effect, also known as aerobic glycolysis. Actually, little is yet known with regard to the metabolism of CSCs population, leaving an exciting unstudied avenue in the dawn of the emerging field of metabolomics.

Nitric oxide mediated bystander responses induced by microbeam targeted cells

International Nuclear Information System (INIS)

Shao, C.; Prise, K.M.; Folkard, M.; Michael, B.D.

2003-01-01

Considerable evidence has recently been accumulated in support of the existence of a 'bystander effect', which cells having received no irradiation show biological consequences from their vicinal irradiated cells. The application of microbeams is providing new insights into the radiation-induced bystander effect. The present study found that when a fraction of radioresistant human glioblastoma cells were individually targeted with a precise number of helium ions generated from the Gray Cancer Institute Charged Particle Microbeam, micronucleus (MN) induction significantly exceeded the expected value that was calculated from the number of MN observed when all of the cells were targeted assuming no bystander effect occurring. Even when only a single cell within a population was hit by one helium ion, the MN induction in the population could be increased by 16%. These results provide direct evidence of radiation-induced bystander effect. Moreover, MN was effectively induced in the unirradiated primary human fibroblasts and glioblastoma cells either co-cultured with irradiated cells or treated with the medium harvested from irradiated cells, indicating a signal molecule was produced from the irradiated cells. However, when c-PTIO, a nitric oxide (NO)-specific scavenger, was present in the medium during and after irradiation until MN analysis, the production of MN in all of the above cases was reduced to low levels. Consequently, NO plays an important role in the radiation-induced bystander effect

Targeted destruction of murine macrophage cells with bioconjugated gold nanorods

Energy Technology Data Exchange (ETDEWEB)

Pissuwan, Dakrong [University of Technology Sydney, Institute for Nanoscale Technology (Australia); Valenzuela, Stella M. [University of Technology Sydney, Department of Medical and Molecular Biosciences (Australia)], E-mail: stella.valenzuela@uts.edu.au; Killingsworth, Murray C. [Sydney South West Pathology Service (Australia)], E-mail: murray.killingsworth@swsahs.nsw.gov.au; Xu, Xiaoda; Cortie, Michael B. [University of Technology Sydney, Institute for Nanoscale Technology (Australia)], E-mail: michael.cortie@uts.edu.au

2007-12-15

Gold nanorods manifest a readily tunable longitudinal plasmon resonance with light and consequently have potential for use in photothermal therapeutics. Recent work by others has shown how gold nanoshells and rods can be used to target cancer cells, which can then be destroyed using relatively high power laser radiation ({approx}1x10{sup 5} to 1x10{sup 10} W/m{sup 2}). Here we extend this concept to demonstrate how gold nanorods can be modified to bind to target macrophage cells, and show that high intensity laser radiation is not necessary, with even 5x10{sup 2} W/m{sup 2} being sufficient, provided that a total fluence of {approx}30 J/cm{sup 2} is delivered. We used the murine cell line RAW 264.7 and the monoclonal antibody CD11b, raised against murine macrophages, as our model system and a 5 mW solid state diode laser as our energy source. Exposure of the cells labeled with gold nanorods to a laser fluence of 30 J/cm{sup 2} resulted in 81% cell death compared to only 0.9% in the control, non-labeled cells.

Targeted destruction of murine macrophage cells with bioconjugated gold nanorods

International Nuclear Information System (INIS)

Pissuwan, Dakrong; Valenzuela, Stella M.; Killingsworth, Murray C.; Xu, Xiaoda; Cortie, Michael B.

2007-01-01

Gold nanorods manifest a readily tunable longitudinal plasmon resonance with light and consequently have potential for use in photothermal therapeutics. Recent work by others has shown how gold nanoshells and rods can be used to target cancer cells, which can then be destroyed using relatively high power laser radiation (∼1x10 5 to 1x10 10 W/m 2 ). Here we extend this concept to demonstrate how gold nanorods can be modified to bind to target macrophage cells, and show that high intensity laser radiation is not necessary, with even 5x10 2 W/m 2 being sufficient, provided that a total fluence of ∼30 J/cm 2 is delivered. We used the murine cell line RAW 264.7 and the monoclonal antibody CD11b, raised against murine macrophages, as our model system and a 5 mW solid state diode laser as our energy source. Exposure of the cells labeled with gold nanorods to a laser fluence of 30 J/cm 2 resulted in 81% cell death compared to only 0.9% in the control, non-labeled cells

Multifunctional Polymer Nanoparticles for Dual Drug Release and Cancer Cell Targeting

Directory of Open Access Journals (Sweden)

Yu-Han Wen

2017-06-01

Full Text Available Multifunctional polymer nanoparticles have been developed for cancer treatment because they could be easily designed to target cancer cells and to enhance therapeutic efficacy according to cancer hallmarks. In this study, we synthesized a pH-sensitive polymer, poly(methacrylic acid-co-histidine/doxorubicin/biotin (HBD in which doxorubicin (DOX was conjugated by a hydrazone bond to encapsulate an immunotherapy drug, imiquimod (IMQ, to form dual cancer-targeting and dual drug-loaded nanoparticles. At low pH, polymeric nanoparticles could disrupt and simultaneously release DOX and IMQ. Our experimental results show that the nanoparticles exhibited pH-dependent drug release behavior and had an ability to target cancer cells via biotin and protonated histidine.

Designing nanoconjugates to effectively target pancreatic cancer cells in vitro and in vivo.

Directory of Open Access Journals (Sweden)

Jameel Ahmad Khan

Full Text Available Pancreatic cancer is the fourth leading cause of cancer related deaths in America. Monoclonal antibodies are a viable treatment option for inhibiting cancer growth. Tumor specific drug delivery could be achieved utilizing these monoclonal antibodies as targeting agents. This type of designer therapeutic is evolving and with the use of gold nanoparticles it is a promising approach to selectively deliver chemotherapeutics to malignant cells. Gold nanoparticles (GNPs are showing extreme promise in current medicinal research. GNPs have been shown to non-invasively kill tumor cells by hyperthermia using radiofrequency. They have also been implemented as early detection agents due to their unique X-ray contrast properties; success was revealed with clear delineation of blood capillaries in a preclinical model by CT (computer tomography. The fundamental parameters for intelligent design of nanoconjugates are on the forefront. The goal of this study is to define the necessary design parameters to successfully target pancreatic cancer cells.The nanoconjugates described in this study were characterized with various physico-chemical techniques. We demonstrate that the number of cetuximab molecules (targeting agent on a GNP, the hydrodynamic size of the nanoconjugates, available reactive surface area and the ability of the nanoconjugates to sequester EGFR (epidermal growth factor receptor, all play critical roles in effectively targeting tumor cells in vitro and in vivo in an orthotopic model of pancreatic cancer.Our results suggest the specific targeting of tumor cells depends on a number of crucial components 1 targeting agent to nanoparticle ratio 2 availability of reactive surface area on the nanoparticle 3 ability of the nanoconjugate to bind the target and 4 hydrodynamic diameter of the nanoconjugate. We believe this study will help define the design parameters for formulating better strategies for specifically targeting tumors with nanoparticle

Simultaneous targeting of prostate stem cell antigen and prostate-specific membrane antigen improves the killing of prostate cancer cells using a novel modular T cell-retargeting system.

Science.gov (United States)

Arndt, Claudia; Feldmann, Anja; Koristka, Stefanie; Cartellieri, Marc; Dimmel, Maria; Ehninger, Armin; Ehninger, Gerhard; Bachmann, Michael

2014-09-01

Recently, we described a novel modular platform technology in which T cell-recruitment and tumor-targeting domains of conventional bispecific antibodies are split to independent components, a universal effector module (EM) and replaceable monospecific/monovalent target modules (TMs) that form highly efficient T cell-retargeting complexes. Theoretically, our unique strategy should allow us to simultaneously retarget T cells to different tumor antigens by combining the EM with two or more different monovalent/monospecific TMs or even with bivalent/bispecific TMs, thereby overcoming limitations of a monospecific treatment such as the selection of target-negative tumor escape variants. In order to advance our recently introduced prostate stem cell antigen (PSCA)-specific modular system for a dual-targeting of prostate cancer cells, two additional TMs were constructed: a monovalent/monospecific TM directed against the prostate-specific membrane antigen (PSMA) and a bivalent/bispecific TM (bsTM) with specificity for PSMA and PSCA. The functionality of the novel dual-targeting strategies was analyzed by performing T cell activation and chromium release assays. Similar to the PSCA-specific modular system, the novel PSMA-specific modular system mediates an efficient target-dependent and -specific tumor cell lysis at low E:T ratios and picomolar Ab concentrations. Moreover, by combination of the EM with either the bispecific TM directed to PSMA and PSCA or both monospecifc TMs directed to either PSCA or PSMA, dual-specific targeting complexes were formed which allowed us to kill potential escape variants expressing only one or the other target antigen. Overall, the novel modular system represents a promising tool for multiple tumor targeting. © 2014 Wiley Periodicals, Inc.

Nanoscale mapping and organization analysis of target proteins on cancer cells from B-cell lymphoma patients

Energy Technology Data Exchange (ETDEWEB)

Li, Mi [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Xiao, Xiubin [Department of Lymphoma, Affiliated Hospital of Military Medical Academy of Sciences, Beijing 100071 (China); Liu, Lianqing, E-mail: lqliu@sia.cn [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); Xi, Ning, E-mail: xin@egr.msu.edu [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); Department of Mechanical and Biomedical Engineering, City University of Hong Kong, Hong Kong (China); Wang, Yuechao; Dong, Zaili [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); Zhang, Weijing, E-mail: zhangwj3072@163.com [Department of Lymphoma, Affiliated Hospital of Military Medical Academy of Sciences, Beijing 100071 (China)

2013-11-01

CD20, a membrane protein highly expressed on most B-cell lymphomas, is an effective target demonstrated in clinical practice for treating B-cell non-Hodgkin's lymphoma (NHL). Rituximab is a monoclonal antibody against CD20. In this work, we applied atomic force microscopy (AFM) to map the nanoscale distribution of CD20 molecules on the surface of cancer cells from clinical B-cell NHL patients under the assistance of ROR1 fluorescence recognition (ROR1 is a specific cell surface marker exclusively expressed on cancer cells). First, the ROR1 fluorescence labeling experiments showed that ROR1 was expressed on cancer cells from B-cell lymphoma patients, but not on normal cells from healthy volunteers. Next, under the guidance of ROR1 fluorescence, the rituximab-conjugated AFM tips were moved to cancer cells to image the cellular morphologies and detect the CD20-rituximab interactions on the cell surfaces. The distribution maps of CD20 on cancer cells were constructed by obtaining arrays of (16×16) force curves in local areas (500×500 nm{sup 2}) on the cell surfaces. The experimental results provide a new approach to directly investigate the nanoscale distribution of target protein on single clinical cancer cells. - Highlights: • Cancer cells were recognized from healthy cells by ROR1 fluorescence labeling. • The nanoscale distribution of CD20 on cancer cells was characterized. • The distribution of CD20 was non-uniform on the surface of cancer cells.

Targeting of phage particles towards endothelial cells by antibodies selected through a multi-parameter selection strategy.

Science.gov (United States)

Mandrup, Ole A; Lykkemark, Simon; Kristensen, Peter

2017-02-10

One of the hallmarks of cancer is sustained angiogenesis. Here, normal endothelial cells are activated, and their formation of new blood vessels leads to continued tumour growth. An improved patient condition is often observed when angiogenesis is prevented or normalized through targeting of these genomically stable endothelial cells. However, intracellular targets constitute a challenge in therapy, as the agents modulating these targets have to be delivered and internalized specifically to the endothelial cells. Selection of antibodies binding specifically to certain cell types is well established. It is nonetheless a challenge to ensure that the binding of antibodies to the target cell will mediate internalization. Previously selection of such antibodies has been performed targeting cancer cell lines; most often using either monovalent display or polyvalent display. In this article, we describe selections that isolate internalizing antibodies by sequential combining monovalent and polyvalent display using two types of helper phages, one which increases display valence and one which reduces background. One of the selected antibodies was found to mediate internalization into human endothelial cells, although our results confirms that the single stranded nature of the DNA packaged into phage particles may limit applications aimed at targeting nucleic acids in mammalian cells.

Target-specific delivery of doxorubicin to human glioblastoma cell ...

Indian Academy of Sciences (India)

Abdullah Tahir Bayraç

2018-01-29

Jan 29, 2018 ... was previously selected for specific recognition of glioblastoma and represented many advantageous ... antigens, receptors or any 3-D structure on the target cells ..... both PSMA (?) and PSMA (-) prostate cancers.

Imaging and Targeting of Hypoxic Tumor Cells with Use of HIF-1-2

International Nuclear Information System (INIS)

Kizaka-Kondoh, Shinae; Harada, Hiroshi; Tanaka, Shotaro; Hiraoka, Masahiro

2006-01-01

This paper describes imaging (visualization) of transplanted tumor cells under hypoxia in vivo and molecular targeting to kill those cells by inducing their apoptosis. HIF (hypoxia inducible factor) concerned with angiogenesis is induced specifically in hypoxic tumor cells and its activity can be visualized by transfection of reporter vector construct of fluorescent protein GFP or luciferase. Authors established the transfected tumor cells with the plasmid p5HRE-luciferase and when transplanted in the nude mouse, those cells emitted light dependently to their hypoxic conditions, which could be visualized by in vivo imaging system (IVIS) with CCD camera. Authors prepared the oxygen-dependent degradation-procaspase 3-fusion protein (TOP3) to target the hypoxic tumor cells for enhancing their apoptotic signaling, whose apoptosis was actually observed by the IVIS. Reportedly, radiation transiently activates HIF-1 and combination treatment of radiation and TOP3 resulted in the enhanced death of tumor cells. Interestingly, the suppression of tumor growth lasted longer than expected, probably due to inhibition of angiogenesis. Authors called this anti-tumor strategy as the micro-environmental targeting. (T.I.)

Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

Directory of Open Access Journals (Sweden)

Unzueta U

2012-08-01

Full Text Available Ugutz Unzueta,1–3 María Virtudes Céspedes,3,4 Neus Ferrer-Miralles,1–3 Isolda Casanova,3,4 Juan Cedano,5 José Luis Corchero,1–3 Joan Domingo-Espín,1–3 Antonio Villaverde,1–3 Ramón Mangues,3,4 Esther Vázquez1–31Institut de Biotecnologia i de Biomedicina, 2Departamento de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, 3CIBER en Bioingeniería, Biomateriales y Nanomedicina, Bellaterra, Barcelona, 4Oncogenesis and Antitumor Drug Group, Biomedical Research Institute Sant Pau, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 5Laboratory of Immunology, Regional Norte, Universidad de la Republica, Salto, UruguayBackground: Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4 is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing.Results: Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer.Conclusion: Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles

Odin (ANKS1A is a Src family kinase target in colorectal cancer cells

Directory of Open Access Journals (Sweden)

Feller Stephan M

2008-10-01

Full Text Available Abstract Background Src family kinases (SFK are implicated in the development of some colorectal cancers (CRC. One SFK member, Lck, is not detectable in normal colonic epithelium, but becomes aberrantly expressed in a subset of CRCs. Although SFK have been extensively studied in fibroblasts and different types of immune cells, their physical and functional targets in many epithelial cancers remain poorly characterised. Results 64 CRC cell lines were tested for expression of Lck. SW620 CRC cells, which express high levels of Lck and also contain high basal levels of tyrosine phosphorylated (pY proteins, were then analysed to identify novel SFK targets. Since SH2 domains of SFK are known to often bind substrates after phosphorylation by the kinase domain, the LckSH2 was compared with 14 other SH2s for suitability as affinity chromatography reagent. Mass spectrometric analyses of LckSH2-purified pY proteins subsequently identified several proteins readily known as SFK kinase substrates, including cortactin, Tom1L1 (SRCASM, GIT1, vimentin and AFAP1L2 (XB130. Additional proteins previously reported as substrates of other tyrosine kinase were also detected, including the EGF and PDGF receptor target Odin. Odin was further analysed and found to contain substantially less pY upon inhibition of SFK activity in SW620 cells, indicating that it is a formerly unknown SFK target in CRC cells. Conclusion Rapid identification of known and novel SFK targets in CRC cells is feasible with SH2 domain affinity chromatography. The elucidation of new SFK targets like Odin in epithelial cancer cells is expected to lead to novel insight into cancer cell signalling mechanisms and may also serve to indicate new biomarkers for monitoring tumor cell responses to drug treatments.

MicroRNA-429 induces tumorigenesis of human non-small cell lung cancer cells and targets multiple tumor suppressor genes

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Lang, Yaoguo; Xu, Shidong; Ma, Jianqun; Wu, Jun [Department of Thoracic Surgery, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, Heilongjiang 150081 (China); Jin, Shi; Cao, Shoubo [Department of Medical Oncology, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, Heilongjiang 150081 (China); Yu, Yan, E-mail: yuyan@hrbmu.edu.cn [Department of Medical Oncology, Harbin Medical University Cancer Hospital, 150 Haping Road, Harbin, Heilongjiang 150081 (China)

2014-07-18

Highlights: • MiR-429 expression is upregulated in non-small cell lung cancer (NSCLC). • MiR-429 inhibits PTEN, RASSF8 and TIMP2 expression. • MiR-429 promotes metastasis and proliferation. • We report important regulatory mechanisms involved in NSCLC progression. • MiR-429 is a potential therapeutic target and diagnostic marker. - Abstract: Lung cancer is the major cause of cancer death globally. MicroRNAs are evolutionally conserved small noncoding RNAs that are critical for the regulation of gene expression. Aberrant expression of microRNA (miRNA) has been implicated in cancer initiation and progression. In this study, we demonstrated that the expression of miR-429 are often upregulated in non-small cell lung cancer (NSCLC) compared with normal lung tissues, and its expression level is also increased in NSCLC cell lines compared with normal lung cells. Overexpression of miR-429 in A549 NSCLC cells significantly promoted cell proliferation, migration and invasion, whereas inhibition of miR-429 inhibits these effects. Furthermore, we demonstrated that miR-429 down-regulates PTEN, RASSF8 and TIMP2 expression by directly targeting the 3′-untranslated region of these target genes. Taken together, our results suggest that miR-429 plays an important role in promoting the proliferation and metastasis of NSCLC cells and is a potential target for NSCLC therapy.

Robotic Automation of In Vivo Two-Photon Targeted Whole-Cell Patch-Clamp Electrophysiology.

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Annecchino, Luca A; Morris, Alexander R; Copeland, Caroline S; Agabi, Oshiorenoya E; Chadderton, Paul; Schultz, Simon R

2017-08-30

Whole-cell patch-clamp electrophysiological recording is a powerful technique for studying cellular function. While in vivo patch-clamp recording has recently benefited from automation, it is normally performed "blind," meaning that throughput for sampling some genetically or morphologically defined cell types is unacceptably low. One solution to this problem is to use two-photon microscopy to target fluorescently labeled neurons. Combining this with robotic automation is difficult, however, as micropipette penetration induces tissue deformation, moving target cells from their initial location. Here we describe a platform for automated two-photon targeted patch-clamp recording, which solves this problem by making use of a closed loop visual servo algorithm. Our system keeps the target cell in focus while iteratively adjusting the pipette approach trajectory to compensate for tissue motion. We demonstrate platform validation with patch-clamp recordings from a variety of cells in the mouse neocortex and cerebellum. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Intracellular Delivery of Nanobodies for Imaging of Target Proteins in Live Cells.

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Röder, Ruth; Helma, Jonas; Preiß, Tobias; Rädler, Joachim O; Leonhardt, Heinrich; Wagner, Ernst

2017-01-01

Cytosolic delivery of nanobodies for molecular target binding and fluorescent labeling in living cells. Fluorescently labeled nanobodies were formulated with sixteen different sequence-defined oligoaminoamides. The delivery of formulated anti-GFP nanobodies into different target protein-containing HeLa cell lines was investigated by flow cytometry and fluorescence microscopy. Nanoparticle formation was analyzed by fluorescence correlation spectroscopy. The initial oligomer screen identified two cationizable four-arm structured oligomers (734, 735) which mediate intracellular nanobody delivery in a receptor-independent (734) or folate receptor facilitated (735) process. The presence of disulfide-forming cysteines in the oligomers was found critical for the formation of stable protein nanoparticles of around 20 nm diameter. Delivery of labeled GFP nanobodies or lamin nanobodies to their cellular targets was demonstrated by fluorescence microscopy including time lapse studies. Two sequence-defined oligoaminoamides with or without folate for receptor targeting were identified as effective carriers for intracellular nanobody delivery, as exemplified by GFP or lamin binding in living cells. Due to the conserved nanobody core structure, the methods should be applicable for a broad range of nanobodies directed to different intracellular targets.

Targeting melanoma stem cells with the Vitamin E derivative δ-tocotrienol.

Science.gov (United States)

Marzagalli, Monica; Moretti, Roberta Manuela; Messi, Elio; Marelli, Marina Montagnani; Fontana, Fabrizio; Anastasia, Alessia; Bani, Maria Rosa; Beretta, Giangiacomo; Limonta, Patrizia

2018-01-12

The prognosis of metastatic melanoma is very poor, due to the development of drug resistance. Cancer stem cells (CSCs) may play a crucial role in this mechanism, contributing to disease relapse. We first characterized CSCs in melanoma cell lines. We observed that A375 (but not BLM) cells are able to form melanospheres and show CSCs traits: expression of the pluripotency markers SOX2 and KLF4, higher invasiveness and tumor formation capability in vivo with respect to parental adherent cells. We also showed that a subpopulation of autofluorescent cells expressing the ABCG2 stem cell marker is present in the A375 spheroid culture. Based on these data, we investigated whether δ-TT might target melanoma CSCs. We demonstrated that melanoma cells escaping the antitumor activity of δ-TT are completely devoid of the ability to form melanospheres. In contrast, cells that escaped vemurafenib treatment show a higher ability to form melanospheres than control cells. δ-TT also induced disaggregation of A375 melanospheres and reduced the spheroidogenic ability of sphere-derived cells, reducing the expression of the ABCG2 marker. These data demonstrate that δ-TT exerts its antitumor activity by targeting the CSC subpopulation of A375 melanoma cells and might represent a novel chemopreventive/therapeutic strategy against melanoma.

Pressure-mediated reduction of ultrasonically induced cell lysis

International Nuclear Information System (INIS)

Ciaravino, V.E.; Miller, M.W.; Carstensen, E.L.

1981-01-01

Chinese hamster V-79 cells, exposed in polystyrene tubes for 5 min to 1-MHz continuous-wave ultrasound, were lysed more by a 10 than a 5 W/cm 2 intensity. Higher atmospheric pressure was needed to eliminate lysis with the former relative to the latter intensity, but lysis by 10 W/cm 2 was completely climinated with 2 atm of hydrostatic pressure. The reduction in lysis per unit increase in atmospheric pressure was comparable for both ultrasound intensities

Low-Dose Irradiation Enhances Gene Targeting in Human Pluripotent Stem Cells.

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Hatada, Seigo; Subramanian, Aparna; Mandefro, Berhan; Ren, Songyang; Kim, Ho Won; Tang, Jie; Funari, Vincent; Baloh, Robert H; Sareen, Dhruv; Arumugaswami, Vaithilingaraja; Svendsen, Clive N

2015-09-01

Human pluripotent stem cells (hPSCs) are now being used for both disease modeling and cell therapy; however, efficient homologous recombination (HR) is often crucial to develop isogenic control or reporter lines. We showed that limited low-dose irradiation (LDI) using either γ-ray or x-ray exposure (0.4 Gy) significantly enhanced HR frequency, possibly through induction of DNA repair/recombination machinery including ataxia-telangiectasia mutated, histone H2A.X and RAD51 proteins. LDI could also increase HR efficiency by more than 30-fold when combined with the targeting tools zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats. Whole-exome sequencing confirmed that the LDI administered to hPSCs did not induce gross genomic alterations or affect cellular viability. Irradiated and targeted lines were karyotypically normal and made all differentiated lineages that continued to express green fluorescent protein targeted at the AAVS1 locus. This simple method allows higher throughput of new, targeted hPSC lines that are crucial to expand the use of disease modeling and to develop novel avenues of cell therapy. The simple and relevant technique described in this report uses a low level of radiation to increase desired gene modifications in human pluripotent stem cells by an order of magnitude. This higher efficiency permits greater throughput with reduced time and cost. The low level of radiation also greatly increased the recombination frequency when combined with developed engineered nucleases. Critically, the radiation did not lead to increases in DNA mutations or to reductions in overall cellular viability. This novel technique enables not only the rapid production of disease models using human stem cells but also the possibility of treating genetically based diseases by correcting patient-derived cells. ©AlphaMed Press.

Mitochondrial targets of photodynamic therapy and their contribution to cell death

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Oleinick, Nancy L.; Usuda, Jitsuo; Xue, Liang-yan; Azizuddin, Kashif; Chiu, Song-mao; Lam, Minh C.; Morris, Rachel L.; Nieminen, Anna-Liisa

2002-06-01

In response to photodynamic therapy (PDT), many cells in culture or within experimental tumors are eliminated by apoptosis. PDT with photosensitizers that localize in or target mitochondria, such as the phthalocyanine Pc 4, causes prompt release of cytochrome c into the cytoplasm and activation of caspases-9 and -3, among other caspases, that are responsible for initiating cell degradation. Some cells appear resistant to apoptosis after PDT; however, if they have sustained sufficient damage, they will die by a necrotic process or through a different apoptotic pathway. In the case of PDT, the distinction between apoptosis and necrosis may be less important than the mechanism that triggers both processes, since critical lethal damage appears to occur during treatment and does not require the major steps in apoptosis to be expressed. We earlier showed, for example, that human breast cancer MCF-7 cells that lack caspase-3 are resistant to the induction of apoptosis by PDT, but are just as sensitive to the loss of clonogenicity as MCF-7 cells stably expressing transfected procaspase-3. Many photosensitizers that target mitochondria specifically attack the anti-apoptotic protein Bcl-2, generating a variety of crosslinked and cleaved photoproducts. Recent evidence suggests that the closely related protein Bcl-xL is also a target of Pc 4-PDT. Transient transfection of an expression vector encoding deletion mutants of Bcl-2 have identified the critical sensitive site in the protein that is required for photodamage. This region contains two alpha helices that form a secondary membrane anchorage site and are thought to be responsible for pore formation by Bcl-2. As specific protein targets are identified, we are becoming better able to model the critical events in PDT-induced cell death.

MiRNA-125a-5p inhibits glioblastoma cell proliferation and promotes cell differentiation by targeting TAZ

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Yuan, Jian; Xiao, Gelei [Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); The Institute of Skull Base Surgery & Neuro-oncology at Hunan, Changsha, Hunan 410008 (China); Peng, Gang [Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); Liu, Dingyang [Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); The Institute of Skull Base Surgery & Neuro-oncology at Hunan, Changsha, Hunan 410008 (China); Wang, Zeyou [Cancer Research Institute, Central South University, Changsha, Hunan 410008 (China); Liao, Yiwei; Liu, Qing [Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); The Institute of Skull Base Surgery & Neuro-oncology at Hunan, Changsha, Hunan 410008 (China); Wu, Minghua [The Institute of Skull Base Surgery & Neuro-oncology at Hunan, Changsha, Hunan 410008 (China); Cancer Research Institute, Central South University, Changsha, Hunan 410008 (China); Yuan, Xianrui, E-mail: xry69@163.com [Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha, Hunan 410008 (China); The Institute of Skull Base Surgery & Neuro-oncology at Hunan, Changsha, Hunan 410008 (China)

2015-02-06

Highlights: • Expression of miR-125a-5p is inversely correlated with that of TAZ in glioma cells. • MiR-125a-5p represses TAZ expression in glioma cells. • MiR-125a-5p directly targets the 3′ UTR of TAZ mRNA and promotes its degradation. • MiR-125a-5p represses CTGF and survivin via TAZ, and inhibits glioma cell growth. • MiR-125a-5p inhibits the stem cell features of HFU-251 MG cells. - Abstract: Glioblastoma (GBM) is the most lethal brain tumor due to the resistance to conventional therapies, such as radiotherapy and chemotherapy. TAZ, an important mediator of the Hippo pathway, was found to be up-regulated in diverse cancers, including in GBM, and plays important roles in tumor initiation and progression. However, little is known about the regulation of TAZ expression in tumors. In this study, we found that miR-125a-5p is an important regulator of TAZ in glioma cells by directly targeting the TAZ 3′ UTR. MiR-125a-5p levels are inversely correlated with that of TAZ in normal astrocytes and a panel of glioma cell lines. MiR-125a-5p represses the expression of TAZ target genes, including CTGF and survivin, and inhibits cell proliferation and induces the differentiation of GBM cells; whereas over-expression of TAZ rescues the effects of miR-125a-5p. This study revealed a mechanism for TAZ deregulation in glioma cells, and also demonstrated a tumor suppressor role of miR-125a-5p in glioblastoma cells.

MiRNA-125a-5p inhibits glioblastoma cell proliferation and promotes cell differentiation by targeting TAZ

International Nuclear Information System (INIS)

Yuan, Jian; Xiao, Gelei; Peng, Gang; Liu, Dingyang; Wang, Zeyou; Liao, Yiwei; Liu, Qing; Wu, Minghua; Yuan, Xianrui

2015-01-01

Highlights: • Expression of miR-125a-5p is inversely correlated with that of TAZ in glioma cells. • MiR-125a-5p represses TAZ expression in glioma cells. • MiR-125a-5p directly targets the 3′ UTR of TAZ mRNA and promotes its degradation. • MiR-125a-5p represses CTGF and survivin via TAZ, and inhibits glioma cell growth. • MiR-125a-5p inhibits the stem cell features of HFU-251 MG cells. - Abstract: Glioblastoma (GBM) is the most lethal brain tumor due to the resistance to conventional therapies, such as radiotherapy and chemotherapy. TAZ, an important mediator of the Hippo pathway, was found to be up-regulated in diverse cancers, including in GBM, and plays important roles in tumor initiation and progression. However, little is known about the regulation of TAZ expression in tumors. In this study, we found that miR-125a-5p is an important regulator of TAZ in glioma cells by directly targeting the TAZ 3′ UTR. MiR-125a-5p levels are inversely correlated with that of TAZ in normal astrocytes and a panel of glioma cell lines. MiR-125a-5p represses the expression of TAZ target genes, including CTGF and survivin, and inhibits cell proliferation and induces the differentiation of GBM cells; whereas over-expression of TAZ rescues the effects of miR-125a-5p. This study revealed a mechanism for TAZ deregulation in glioma cells, and also demonstrated a tumor suppressor role of miR-125a-5p in glioblastoma cells

Novel targets for sensitizing breast cancer cells to TRAIL-induced apoptosis with siRNA delivery.

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Thapa, Bindu; Bahadur Kc, Remant; Uludağ, Hasan

2018-02-01

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in variety of cancer cells without affecting most normal cells, which makes it a promising agent for cancer therapy. However, TRAIL therapy is clinically not effective due to resistance induction. To identify novel regulators of TRAIL that can aid in therapy, protein targets whose silencing sensitized breast cancer cells against TRAIL were screened with an siRNA library against 446 human apoptosis-related proteins in MDA-231 cells. Using a cationic lipopolymer (PEI-αLA) for delivery of library members, 16 siRNAs were identified that sensitized the TRAIL-induced death in MDA-231 cells. The siRNAs targeting BCL2L12 and SOD1 were further evaluated based on the novelty and their ability to sensitize TRAIL induced cell death. Silencing both targets sensitized TRAIL-mediated cell death in MDA-231 cells as well as TRAIL resistant breast cancer cells, MCF-7. Combination of TRAIL and siRNA silencing BCL2L12 had no effect in normal human umbilical vein cells and human bone marrow stromal cell. The silencing of BCL2L12 and SOD1 enhanced TRAIL-mediated apoptosis in MDA-231 cells via synergistically activating capsase-3 activity. Hence, here we report siRNAs targeting BCL2L12 and SOD1 as a novel regulator of TRAIL-induced cell death in breast cancer cells, providing a new approach for enhancing TRAIL therapy for breast cancer. The combination of siRNA targeting BCL2L12 and TRAIL can be a highly effective synergistic pair in breast cancer cells with minimal effect on the non-transformed cells. © 2017 UICC.

Vibrio cholerae infection, novel drug targets and phage therapy.

Science.gov (United States)

Fazil, Mobashar Hussain Urf Turabe; Singh, Durg V

2011-10-01

Vibrio cholerae is the causative agent of the diarrheal disease cholera. Although antibiotic therapy shortens the duration of diarrhea, excessive use has contributed to the emergence of antibiotic resistance in V. cholerae. Mobile genetic elements have been shown to be largely responsible for the shift of drug resistance genes in bacteria, including some V. cholerae strains. Quorum sensing communication systems are used for interaction among bacteria and for sensing environmental signals. Sequence analysis of the ctxB gene of toxigenic V. cholerae strains demonstrated its presence in multiple cholera toxin genotypes. Moreover, bacteriophage that lyse the bacterium have been reported to modulate epidemics by decreasing the required infectious dose of the bacterium. In this article, we will briefly discuss the disease, its clinical manifestation, antimicrobial resistance and the novel approaches to locate drug targets to treat cholera.

Dendritic cell based PSMA immunotherapy for prostate cancer using a CD40-targeted adenovirus vector.

Directory of Open Access Journals (Sweden)

Briana Jill Williams

Full Text Available Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs with prostate specific membrane antigen (PSMA have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells. To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ. Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.

PSMA-targeted bispecific Fab conjugates that engage T cells.

Science.gov (United States)

Patterson, James T; Isaacson, Jason; Kerwin, Lisa; Atassi, Ghazi; Duggal, Rohit; Bresson, Damien; Zhu, Tong; Zhou, Heyue; Fu, Yanwen; Kaufmann, Gunnar F

2017-12-15

Bioconjugate formats provide alternative strategies for antigen targeting with bispecific antibodies. Here, PSMA-targeted Fab conjugates were generated using different bispecific formats. Interchain disulfide bridging of an αCD3 Fab enabled installation of either the PSMA-targeting small molecule DUPA (SynFab) or the attachment of an αPSMA Fab (BisFab) by covalent linkage. Optimization of the reducing conditions was critical for selective interchain disulfide reduction and good bioconjugate yield. Activity of αPSMA/CD3 Fab conjugates was tested by in vitro cytotoxicity assays using prostate cancer cell lines. Both bispecific formats demonstrated excellent potency and antigen selectivity. Copyright © 2017. Published by Elsevier Ltd.

Rapid Extraction of Genomic DNA from Medically Important Yeasts and Filamentous Fungi by High-Speed Cell Disruption

OpenAIRE

Müller, Frank-Michael C.; Werner, Katherine E.; Kasai, Miki; Francesconi, Andrea; Chanock, Stephen J.; Walsh, Thomas J.

1998-01-01

Current methods of DNA extraction from different fungal pathogens are often time-consuming and require the use of toxic chemicals. DNA isolation from some fungal organisms is difficult due to cell walls or capsules that are not readily susceptible to lysis. We therefore investigated a new and rapid DNA isolation method using high-speed cell disruption (HSCD) incorporating chaotropic reagents and lysing matrices in comparison to standard phenol-chloroform (PC) extraction protocols for isolatio...

Pretargeting vs. direct targeting of human betalox5 islet cells subcutaneously implanted in mice using an anti-human islet cell antibody

International Nuclear Information System (INIS)

Liu Guozheng; Dou Shuping; Akalin, Ali; Rusckowski, Mary; Streeter, Philip R.; Shultz, Leonard D.; Greiner, Dale L.

2012-01-01

Introduction: We previously demonstrated MORF/cMORF pretargeting of human islets and betalox 5 cells (a human beta cell line) transplanted subcutaneously in mice with the anti-human islet antibody, HPi1. We now compare pretargeting with direct targeting in the beta cell transplant model to evaluate the degree to which target/non-target (T/NT) ratios may be improved by pretargeting. Methods: Specific binding of an anti-human islet antibody HPi1 to the beta cells transplanted subcutaneously in mice was examined against a negative control antibody. We then compared pretargeting by MORF-HPi1 plus 111 In-labeled cMORF to direct targeting by 111 In-labeled HPi1. Results: HPi1 binding to betalox5 human cells in the transplant was shown by immunofluorescence. Normal organ 111 In backgrounds by pretargeting were always lower, although target accumulations were similar. More importantly, the transplant to pancreas and liver ratios was, respectively, 26 and 10 by pretargeting as compared to 9 and 0.6 by direct targeting. Conclusions: Pretargeting greatly improves the T/NT ratios, and based on the estimated endocrine to exocrine ratio within a pancreas, pretargeting may be approaching the sensitivity required for successful imaging of human islets within this organ.

CD19-Targeted CAR T Cells as Novel Cancer Immunotherapy for Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia

OpenAIRE

Davila, Marco L.; Brentjens, Renier J.

2016-01-01

Immunotherapy has demonstrated significant potential for the treatment of patients with chemotherapy-resistant hematologic malignancies and solid tumors. One type of immunotherapy involves the adoptive transfer of T cells that have been genetically modified with a chimeric antigen receptor (CAR) to target a tumor. These hybrid proteins are composed of the antigen-binding domains of an antibody fused to T-cell receptor signaling machinery. CAR T cells that target CD19 recently have made the ju...

Induction of cell-mediated immunity against B16-BL6 melanoma in mice vaccinated with cells modified by hydrostatic pressure and chemical crosslinking.

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Eisenthal, A; Ramakrishna, V; Skornick, Y; Shinitzky, M

1993-05-01

In the preceding paper we have demonstrated an increase in presentation of both major histocompatibility complex antigens (MHC) and a tumor-associated antigen of the weakly immunogenic B16 melanoma by a straight-forward technique. The method consists in modulating the tumor cell membrane by hydrostatic pressure and simultaneous chemical crosslinking of the cell-surface proteins. In B16-BL6 melanoma, the induced antigenic modulation was found to persist for over 48 h, which permitted the evaluation of the ability of modified B16-BL6 cells to induce immunity against unmodified B16-BL6 cells. In the present study, we have shown that a significant systemic immunity was induced only in mice that were immunized with modified B16-BL6 melanoma cells, whereas immunization with unmodified B16-BL6 cells had only a marginal effect when compared to the results in control sham-immunized mice. The induced immunity was specific since a single immunization affected the growth of B16-BL6 tumors but had no effect on MCA 106, an antigenically unrelated tumor. The addition of interleukin-2 to the immunization regimen had no effect on the antitumor responses induced by the modified B16-BL6 cells. The cell-mediated immunity conferred by immunization with treated B16-BL6 cells was confirmed in experiments in vitro where splenocytes from immunized mice could be sensitized to proliferate by the presence of B16-BL6 cells. In addition, the altered antigenicity of these melanoma cells appeared to correlate with their increased susceptibility to specific effectors. Thus, 51Cr-labeled B16-BL6 target cells, modified by pressure and crosslinking, in comparison to control labeled target cells, were lysed in much greater numbers by effectors such as lymphokine-activated killer cells and allogeneic cytotoxic lymphocytes (anti-H-2b), while such cells remained resistant to lysis by natural killer cells. Our findings indicate that the physical and chemical modifications of the tumor cells that are

Engineered Proteins Program Mammalian Cells to Target Inflammatory Disease Sites.

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Qudrat, Anam; Mosabbir, Abdullah Al; Truong, Kevin

2017-06-22

Disease sites in atherosclerosis and cancer feature cell masses (e.g., plaques/tumors), a low pH extracellular microenvironment, and various pro-inflammatory cytokines such as tumor necrosis factor α (TNFα). The ability to engineer a cell to seek TNFα sources allows for targeted therapeutic delivery. To accomplish this, here we introduced a system of proteins: an engineered TNFα chimeric receptor (named TNFR1chi), a previously engineered Ca 2+ -activated RhoA (named CaRQ), vesicular stomatitis virus glycoprotein G (VSVG), and thymidine kinase. Upon binding TNFα, TNFR1chi generates a Ca 2+ signal that in turn activates CaRQ-mediated non-apoptotic blebs that allow migration toward the TNFα source. Next, the addition of VSVG, upon low pH induction, causes membrane fusion of the engineered and TNFα source cells. Finally, after ganciclovir treatment cells undergo death via the thymidine kinase suicide mechanism. Hence, we assembled a system of proteins that forms the basis of engineering a cell to target inflammatory disease sites characterized by TNFα secretion and a low-pH microenvironment. Copyright © 2017 Elsevier Ltd. All rights reserved.

MicroRNA-424 suppresses estradiol-induced cell proliferation via targeting GPER in endometrial cancer cells.

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Zhang, H; Wang, X; Chen, Z; Wang, W

2015-11-30

Endometrial carcinoma (EC) is the most common gynecologic malignancy with increasing morbidity in recent years. MicroRNAs (miRNAs), a type of non-coding RNA, have been proven to be critical in the process of tumorigenesis. miR-424 has been reported to play a protective role in various type of cancer including endometrial carcinoma. It has been reported that high levels of estrogen increase morbidity of EC by promoting cell growth ability. The current research was designed to delineate the mechanism of miR-424 in regulating E2 (17β-estradiol)-induced cell proliferation in endometrial cancer. In this study, we confirmed that cell proliferation is increased significantly in E2-treated endometrial cancer cell lines. Moreover, miR-424 overexpression dramatically decreased E2-induced cell proliferation, indicating a pivotal role in endometrial cancer cell growth. In addition, the results suggest that miR-424 up-regulation inactivated the PI3K/AKT signaling, which was mediated by G-protein-coupled estrogen receptor-1 (GPER) in endometrial cancer. Furthermore, the luciferase report confirmed the targeting reaction between miR-424 and GPER. After transfection with the GPER overexpression vector into E2-induced endometrial cancer cells, we found that GPER significantly attenuated the inhibition effect of miR-424 in E2-induced cell growth in EC. Taken together, our study suggests that increased miR-424 suppresses E2-induced cell growth, and providing a potential therapeutic target for estrogen-associated endometrial carcinoma.

How immunoglobulin G antibodies kill target cells: revisiting an old paradigm.

Science.gov (United States)

Biburger, Markus; Lux, Anja; Nimmerjahn, Falk

2014-01-01

The capacity of immunoglobulin G (IgG) antibodies to eliminate virtually any target cell has resulted in the widespread introduction of cytotoxic antibodies into the clinic in settings of cancer therapy, autoimmunity, and transplantation, for example. More recently, it has become apparent that also the protection from viral infection via IgG antibodies may require cytotoxic effector functions, suggesting that antibody-dependent cellular cytotoxicity (ADCC) directed against malignant or virally infected cells is one of the most essential effector mechanisms triggered by IgG antibodies to protect the host. A detailed understanding of the underlying molecular and cellular pathways is critical, therefore, to make full use of this antibody effector function. Several studies over the last years have provided novel insights into the effector pathways and innate immune effector cells responsible for ADCC reactions. One of the most notable outcomes of many of these reports is that cells of the mononuclear phagocytic system rather than natural killer cells are critical for removal of IgG opsonized target cells in vivo. © 2014 Elsevier Inc. All rights reserved.

Immune Suppression in Tumors as a Surmountable Obstacle to Clinical Efficacy of Cancer Vaccines

International Nuclear Information System (INIS)

Wieërs, Grégoire; Demotte, Nathalie; Godelaine, Danièle; Bruggen, Pierre van der

2011-01-01

Human tumors are usually not spontaneously eliminated by the immune system and therapeutic vaccination of cancer patients with defined antigens is followed by tumor regressions only in a small minority of the patients. The poor vaccination effectiveness could be explained by an immunosuppressive tumor microenvironment. Because T cells that infiltrate tumor metastases have an impaired ability to lyse target cells or to secrete cytokine, many researchers are trying to decipher the underlying immunosuppressive mechanisms. We will review these here, in particular those considered as potential therapeutic targets. A special attention will be given to galectins, a family of carbohydrate binding proteins. These lectins have often been implicated in inflammation and cancer and may be useful targets for the development of new anti-cancer therapies

Fuel cell and hydrogen R and D targets and funding : a comparative analysis

International Nuclear Information System (INIS)

Adamson, K.A.; Jollie, D.; Baker, A.

2005-01-01

Substantial research and development is needed if fuel cells and hydrogen are to become a mass market reality. Setting research and development targets are central to the long term development of the market. An overview of fuel cell research in the United States, the European Union, and parts of Asia was presented. Research and development targets were analyzed, as well as funding levels for fuel cells and hydrogen. The time frames of targets were considered, as well as the levels of ambition and overall program goals of various countries. Funding barriers and challenges were also considered. It was noted that some governments, such as Japan and Korea, have set a number of very ambitious, highly focused long term targets with substantial funding. The European Union has taken a more integrated approach, wrapping fundamental research and development into large integrated projects which run in combination with a number of other market aspects, such as public acceptance and roadmapping. The United States has a number of long term programmes and targets, but levels of funding are set annually with the passing of each year's Fiscal Budget. The overall goal of the paper was to provide a clearer picture of regional fuel cell research in order to discover areas for potential international collaboration

Targeting neuroblastoma stem cells with retinoic acid and proteasome inhibitor.

Directory of Open Access Journals (Sweden)

Barbara Hämmerle

Full Text Available Neuroblastma cell lines contain a side-population of cells which express stemness markers. These stem-like cells may represent the potential underlying mechanism for resistance to conventional therapy and recurrence of neuroblastoma in patients.To develop novel strategies for targeting the side-population of neurobastomas, we analyzed the effects of 13-cis-retinoic acid (RA combined with the proteasome inhibitor MG132. The short-term action of the treatment was compared with effects after a 5-day recovery period during which both chemicals were withdrawn. RA induced growth arrest and differentiation of SH-SY5Y and SK-N-BE(2 neuroblastoma cell lines. Inhibition of the proteasome caused apoptosis in both cell lines, thus, revealing the critical role of this pathway in the regulated degradation of proteins involved in neuroblastoma proliferation and survival. The combination of RA with MG132 induced apoptosis in a dose-dependent manner, in addition to promoting G2/M arrest in treated cultures. Interestingly, expression of stem cell markers such as Nestin, Sox2, and Oct4 were reduced after the recovery period of combined treatment as compared with untreated cells or treated cells with either compound alone. Consistent with this, neurosphere formation was significantly impaired by the combined treatment of RA and MG132.Given that stem-like cells are associated with resistant to conventional therapy and are thought to be responsible for relapse, our results suggest that dual therapy of RA and proteasome inhibitor might be beneficial for targeting the side-population of cells associated residual disease in high-risk neuroblastoma.

Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death.

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Clark, Amy L; Kanekura, Kohsuke; Lavagnino, Zeno; Spears, Larry D; Abreu, Damien; Mahadevan, Jana; Yagi, Takuya; Semenkovich, Clay F; Piston, David W; Urano, Fumihiko

2017-07-17

Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.

Chlorin e6 Conjugated Interleukin-6 Receptor Aptamers Selectively Kill Target Cells Upon Irradiation

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Sven Kruspe

2014-01-01

Full Text Available Photodynamic therapy (PDT uses the therapeutic properties of light in combination with certain chemicals, called photosensitizers, to successfully treat brain, breast, prostate, and skin cancers. To improve PDT, current research focuses on the development of photosensitizers to specifically target cancer cells. In the past few years, aptamers have been developed to directly deliver cargo molecules into target cells. We conjugated the photosensitizer chlorin e6 (ce6 with a human interleukin-6 receptor (IL-6R binding RNA aptamer, AIR-3A yielding AIR-3A-ce6 for application in high efficient PDT. AIR-3A-ce6 was rapidly and specifically internalized by IL-6R presenting (IL-6R+ cells. Upon light irradiation, targeted cells were selectively killed, while free ce6 did not show any toxic effect. Cells lacking the IL-6R were also not affected by AIR-3A-ce6. With this approach, we improved the target specificity of ce6-mediated PDT. In the future, other tumor-specific aptamers might be used to selectively localize photosensitizers into cells of interest and improve the efficacy and specificity of PDT in cancer and other diseases.

Target cell cyclophilins facilitate human papillomavirus type 16 infection.

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Bienkowska-Haba, Malgorzata; Patel, Hetalkumar D; Sapp, Martin

2009-07-01

Following attachment to primary receptor heparan sulfate proteoglycans (HSPG), human papillomavirus type 16 (HPV16) particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB) facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV-induced diseases.

Target cell cyclophilins facilitate human papillomavirus type 16 infection.

Directory of Open Access Journals (Sweden)

Malgorzata Bienkowska-Haba

2009-07-01

Full Text Available Following attachment to primary receptor heparan sulfate proteoglycans (HSPG, human papillomavirus type 16 (HPV16 particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV-induced diseases.

Lipoproteins tethered dendrimeric nanoconstructs for effective targeting to cancer cells

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Jain, Anupriya; Jain, Keerti; Mehra, Neelesh Kumar; Jain, N. K.

2013-10-01

In the present investigation, poly (propylene imine) dendrimers up to fifth generation (PPI G5.0) were synthesized using ethylene diamine and acrylonitrile. Lipoproteins (high-density lipoprotein; HDL and low-density lipoprotein; LDL) were isolated from human plasma by discontinuous density gradient ultracentrifugation, characterized and tethered to G5.0 PPI dendrimers to construct LDL- and HDL-conjugated dendrimeric nanoconstructs for tumor-specific delivery of docetaxel. Developed formulations showed sustained release characteristics in in vitro drug release and in vivo pharmacokinetic studies. The cancer targeting potential of lipoprotein coupled dendrimers was investigated by ex vivo cytotoxicity and cell uptake studies using human hepatocellular carcinoma cell lines (HepG2 cells) and biodistribution studies in albino rats of Sprague-Dawley strain. Lipoprotein anchored dendrimeric nanoconstructs showed significant uptake by cancer cells as well as higher biodistribution of docetaxel to liver and spleen. It is concluded that these precisely synthesized engineered dendrimeric nanoconstructs could serve as promising drug carrier for fighting with the fatal disease, i.e., cancer, attributed to their defined targeting and therapeutic potential.

Lipoproteins tethered dendrimeric nanoconstructs for effective targeting to cancer cells

Energy Technology Data Exchange (ETDEWEB)

Jain, Anupriya; Jain, Keerti, E-mail: keertijain02@gmail.com; Mehra, Neelesh Kumar, E-mail: neelesh81mph@gmail.com; Jain, N. K., E-mail: dr.jnarendr@gmail.com [Dr. H. S. Gour University, Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences (India)

2013-10-15

In the present investigation, poly (propylene imine) dendrimers up to fifth generation (PPI G5.0) were synthesized using ethylene diamine and acrylonitrile. Lipoproteins (high-density lipoprotein; HDL and low-density lipoprotein; LDL) were isolated from human plasma by discontinuous density gradient ultracentrifugation, characterized and tethered to G5.0 PPI dendrimers to construct LDL- and HDL-conjugated dendrimeric nanoconstructs for tumor-specific delivery of docetaxel. Developed formulations showed sustained release characteristics in in vitro drug release and in vivo pharmacokinetic studies. The cancer targeting potential of lipoprotein coupled dendrimers was investigated by ex vivo cytotoxicity and cell uptake studies using human hepatocellular carcinoma cell lines (HepG2 cells) and biodistribution studies in albino rats of Sprague-Dawley strain. Lipoprotein anchored dendrimeric nanoconstructs showed significant uptake by cancer cells as well as higher biodistribution of docetaxel to liver and spleen. It is concluded that these precisely synthesized engineered dendrimeric nanoconstructs could serve as promising drug carrier for fighting with the fatal disease, i.e., cancer, attributed to their defined targeting and therapeutic potential.

Targeting Aberrant Glutathione Metabolism to Eradicate Human Acute Myelogenous Leukemia Cells*

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Pei, Shanshan; Minhajuddin, Mohammad; Callahan, Kevin P.; Balys, Marlene; Ashton, John M.; Neering, Sarah J.; Lagadinou, Eleni D.; Corbett, Cheryl; Ye, Haobin; Liesveld, Jane L.; O'Dwyer, Kristen M.; Li, Zheng; Shi, Lei; Greninger, Patricia; Settleman, Jeffrey; Benes, Cyril; Hagen, Fred K.; Munger, Joshua; Crooks, Peter A.; Becker, Michael W.; Jordan, Craig T.

2013-01-01

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34+) leukemic versus normal specimens. Our data indicate that CD34+ AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34+ AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34+ cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34+ AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34+ cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells. PMID:24089526

Engineering Multi-Walled Carbon Nanotube Therapeutic Bionanofluids to Selectively Target Papillary Thyroid Cancer Cells.

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Idit Dotan

Full Text Available The incidence of papillary thyroid carcinoma (PTC has risen steadily over the past few decades as well as the recurrence rates. It has been proposed that targeted ablative physical therapy could be a therapeutic modality in thyroid cancer. Targeted bio-affinity functionalized multi-walled carbon nanotubes (BioNanofluid act locally, to efficiently convert external light energy to heat thereby specifically killing cancer cells. This may represent a promising new cancer therapeutic modality, advancing beyond conventional laser ablation and other nanoparticle approaches.Thyroid Stimulating Hormone Receptor (TSHR was selected as a target for PTC cells, due to its wide expression. Either TSHR antibodies or Thyrogen or purified TSH (Thyrotropin were chemically conjugated to our functionalized Bionanofluid. A diode laser system (532 nm was used to illuminate a PTC cell line for set exposure times. Cell death was assessed using Trypan Blue staining.TSHR-targeted BioNanofluids were capable of selectively ablating BCPAP, a TSHR-positive PTC cell line, while not TSHR-null NSC-34 cells. We determined that a 2:1 BCPAP cell:α-TSHR-BioNanofluid conjugate ratio and a 30 second laser exposure killed approximately 60% of the BCPAP cells, while 65% and >70% of cells were ablated using Thyrotropin- and Thyrogen-BioNanofluid conjugates, respectively. Furthermore, minimal non-targeted killing was observed using selective controls.A BioNanofluid platform offering a potential therapeutic path for papillary thyroid cancer has been investigated, with our in vitro results suggesting the development of a potent and rapid method of selective cancer cell killing. Therefore, BioNanofluid treatment emphasizes the need for new technology to treat patients with local recurrence and metastatic disease who are currently undergoing either re-operative neck explorations, repeated administration of radioactive iodine and as a last resort external beam radiation or chemotherapy, with

Organelle targeting: third level of drug targeting

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Sakhrani NM

2013-07-01

Full Text Available Niraj M Sakhrani, Harish PadhDepartment of Cell and Molecular Biology, BV Patel Pharmaceutical Education and Research Development (PERD Centre, Gujarat, IndiaAbstract: Drug discovery and drug delivery are two main aspects for treatment of a variety of disorders. However, the real bottleneck associated with systemic drug administration is the lack of target-specific affinity toward a pathological site, resulting in systemic toxicity and innumerable other side effects as well as higher dosage requirement for efficacy. An attractive strategy to increase the therapeutic index of a drug is to specifically deliver the therapeutic molecule in its active form, not only into target tissue, nor even to target cells, but more importantly, into the targeted organelle, ie, to its intracellular therapeutic active site. This would ensure improved efficacy and minimize toxicity. Cancer chemotherapy today faces the major challenge of delivering chemotherapeutic drugs exclusively to tumor cells, while sparing normal proliferating cells. Nanoparticles play a crucial role by acting as a vehicle for delivery of drugs to target sites inside tumor cells. In this review, we spotlight active and passive targeting, followed by discussion of the importance of targeting to specific cell organelles and the potential role of cell-penetrating peptides. Finally, the discussion will address the strategies for drug/DNA targeting to lysosomes, mitochondria, nuclei and Golgi/endoplasmic reticulum.Keywords: intracellular drug delivery, cancer chemotherapy, therapeutic index, cell penetrating peptides

Magnetic targeting as a strategy to enhance therapeutic effects of mesenchymal stromal cells.

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Silva, Luisa H A; Cruz, Fernanda F; Morales, Marcelo M; Weiss, Daniel J; Rocco, Patricia R M

2017-03-09

Mesenchymal stromal cells (MSCs) have been extensively investigated in the field of regenerative medicine. It is known that the success of MSC-based therapies depends primarily on effective cell delivery to the target site where they will secrete vesicles and soluble factors with immunomodulatory and potentially reparative properties. However, some lesions are located in sites that are difficult to access, such as the heart, spinal cord, and joints. Additionally, low MSC retention at target sites makes cell therapy short-lasting and, therefore, less effective. In this context, the magnetic targeting technique has emerged as a new strategy to aid delivery, increase retention, and enhance the effects of MSCs. This approach uses magnetic nanoparticles to magnetize MSCs and static magnetic fields to guide them in vivo, thus promoting more focused, effective, and lasting retention of MSCs at the target site. In the present review, we discuss the magnetic targeting technique, its principles, and the materials most commonly used; we also discuss its potential for MSC enhancement, and safety concerns that should be addressed before it can be applied in clinical practice.

Emerging Therapeutic Strategies for Targeting Chronic Myeloid Leukemia Stem Cells

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Ahmad Hamad

2013-01-01

Full Text Available Chronic myeloid leukemia (CML is a clonal myeloproliferative disorder. Current targeted therapies designed to inhibit the tyrosine kinase activity of the BCR-ABL oncoprotein have made a significant breakthrough in the treatment of CML patients. However, CML remains a chronic disease that a patient must manage for life. Although tyrosine kinase inhibitors (TKI therapy has completely transformed the prognosis of CML, it has made the therapeutic management more complex. The interruption of TKI treatment results in early disease progression because it does not eliminate quiescent CML stem cells which remain a potential reservoir for disease relapse. This highlights the need to develop new therapeutic strategies for CML to achieve a permanent cure, and to allow TKI interruption. This review summarizes recent research done on alternative targeted therapies with a particular focus on some important signaling pathways (such as Alox5, Hedgehog, Wnt/b-catenin, autophagy, and PML that have the potential to target CML stem cells and potentially provide cure for CML.

Targeting nanoparticles to M cells with non-peptidic ligands for oral vaccination.

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Fievez, Virginie; Plapied, Laurence; des Rieux, Anne; Pourcelle, Vincent; Freichels, Hélène; Wascotte, Valentine; Vanderhaeghen, Marie-Lyse; Jerôme, Christine; Vanderplasschen, Alain; Marchand-Brynaert, Jacqueline; Schneider, Yves-Jacques; Préat, Véronique

2009-09-01

The presence of RGD on nanoparticles allows the targeting of beta1 integrins at the apical surface of human M cells and the enhancement of an immune response after oral immunization. To check the hypothesis that non-peptidic ligands targeting intestinal M cells or APCs would be more efficient for oral immunization than RGD, novel non-peptidic and peptidic analogs (RGD peptidomimitic (RGDp), LDV derivative (LDVd) and LDV peptidomimetic (LDVp)) as well as mannose were grafted on the PEG chain of PCL-PEG and incorporated in PLGA-based nanoparticles. RGD and RGDp significantly increased the transport of nanoparticles across an in vitro model of human M cells as compared to enterocytes. RGD, LDVp, LDVd and mannose enhanced nanoparticle uptake by macrophages in vitro. The intraduodenal immunization with RGDp-, LDVd- or mannose-labeled nanoparticles elicited a higher production of IgG antibodies than the intramuscular injection of free ovalbumin or intraduodenal administration of either non-targeted or RGD-nanoparticles. Targeted formulations were also able to induce a cellular immune response. In conclusion, the in vitro transport of nanoparticles, uptake by macrophages and the immune response were positively influenced by the presence of ligands at the surface of nanoparticles. These targeted-nanoparticles could thus represent a promising delivery system for oral immunization.

Targeted radiosensitization of cells expressing truncated DNA polymerase {beta}.

NARCIS (Netherlands)

Neijenhuis, S.; Verwijs-Janssen, M.; Broek, Bart van den; Begg, A.C.; Vens, C.

2010-01-01

Ionizing radiation (IR) is an effective anticancer treatment, although failures still occur. To improve radiotherapy, tumor-targeted strategies are needed to increase radiosensitivity of tumor cells, without influencing normal tissue radiosensitivity. Base excision repair (BER) and single-strand

Metastasis Targeted Therapies in Renal Cell Cancer

OpenAIRE

K. Fehmi Narter; Bora Özveren

2018-01-01

Metastatic renal cell cancer is a malignant disease and its treatment has been not been described clearly yet. These patients are generally symptomatic and resistant to current treatment modalities. Radiotherapy, chemotherapy, and hormonal therapy are not curative in many of these patients. A multimodal approach consisting of cytoreductive nephrectomy, systemic therapy (immunotherapy or targeted molecules), and metastasectomy has been shown to be hopeful in prolonging the survival and improvi...

CD19-Targeted CAR T cells as novel cancer immunotherapy for relapsed or refractory B-cell acute lymphoblastic leukemia.

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Davila, Marco L; Brentjens, Renier J

2016-10-01

Immunotherapy has demonstrated significant potential for the treatment of patients with chemotherapy-resistant hematologic malignancies and solid tumors. One type of immunotherapy involves the adoptive transfer of T cells that have been genetically modified with a chimeric antigen receptor (CAR) to target a tumor. These hybrid proteins are composed of the antigen-binding domains of an antibody fused to T-cell receptor signaling machinery. CAR T cells that target CD19 recently have made the jump from the laboratory to the clinic, and the results have been remarkable. CD19-targeted CAR T cells have induced complete remissions of disease in up to 90% of patients with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL), who have an expected complete response rate of 30% in response to chemotherapy. The high efficacy of CAR T cells in B-ALL suggests that regulatory approval of this therapy for this routinely fatal leukemia is on the horizon. We review the preclinical development of CAR T cells and their early clinical application for lymphoma. We also provide a comprehensive analysis of the use of CAR T cells in patients with B-ALL. In addition, we discuss the unique toxicities associated with this therapy and the management schemes that have been developed.

Engineering tolerance using biomaterials to target and control antigen presenting cells.

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Tostanoski, Lisa H; Gosselin, Emily A; Jewell, Christopher M

2016-05-01

Autoimmune diseases occur when cells of the adaptive immune system incorrectly recognize and attack "self" tissues. Importantly, the proliferation and differentiation of these cells is triggered and controlled by interactions with antigen presenting cells (APCs), such as dendritic cells. Thus, modulating the signals transduced by APCs (e.g., cytokines, costimulatory surface proteins) has emerged as a promising strategy to promote tolerance for diseases such as multiple sclerosis, type 1 diabetes, and lupus. However, many approaches have been hindered by non-specific activity of immunosuppressive or immunoregulatory cues, following systemic administration of soluble factors via traditional injections routes (e.g., subcutaneous, intravenous). Biomaterials offer a unique opportunity to control the delivery of tolerogenic signals in vivo via properties such as controlled particle size, tunable release kinetics, and co-delivery of multiple classes of cargo. In this review, we highlight recent reports that exploit these properties of biomaterials to target APCs and promote tolerance via three strategies, i) passive or active targeting of particulate carriers to APCs, ii) biomaterial-mediated control over antigen localization and processing, and iii) targeted delivery of encapsulated or adsorbed immunomodulatory signals. These reports represent exciting advances toward the goal of more effective therapies for autoimmune diseases, without the broad suppressive effects associated with current clinically-approved therapies.

Early T Cell Recognition of B Cells following Epstein-Barr Virus Infection: Identifying Potential Targets for Prophylactic Vaccination.

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Jill M Brooks

2016-04-01

Full Text Available Epstein-Barr virus, a B-lymphotropic herpesvirus, is the cause of infectious mononucleosis, has strong aetiologic links with several malignancies and has been implicated in certain autoimmune diseases. Efforts to develop a prophylactic vaccine to prevent or reduce EBV-associated disease have, to date, focused on the induction of neutralising antibody responses. However, such vaccines might be further improved by inducing T cell responses capable of recognising and killing recently-infected B cells. In that context, EBNA2, EBNA-LP and BHRF1 are the first viral antigens expressed during the initial stage of B cell growth transformation, yet have been poorly characterised as CD8+ T cell targets. Here we describe CD8+ T cell responses against each of these three "first wave" proteins, identifying target epitopes and HLA restricting alleles. While EBNA-LP and BHRF1 each contained one strong CD8 epitope, epitopes within EBNA2 induced immunodominant responses through several less common HLA class I alleles (e.g. B*3801 and B*5501, as well as subdominant responses through common class I alleles (e.g. B7 and C*0304. Importantly, such EBNA2-specific CD8+ T cells recognised B cells within the first day post-infection, prior to CD8+ T cells against well-characterised latent target antigens such as EBNA3B or LMP2, and effectively inhibited outgrowth of EBV-transformed B cell lines. We infer that "first wave" antigens of the growth-transforming infection, especially EBNA2, constitute potential CD8+ T cell immunogens for inclusion in prophylactic EBV vaccine design.

Genomic targets of Brachyury (T in differentiating mouse embryonic stem cells.

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Amanda L Evans

Full Text Available The T-box transcription factor Brachyury (T is essential for formation of the posterior mesoderm and the notochord in vertebrate embryos. Work in the frog and the zebrafish has identified some direct genomic targets of Brachyury, but little is known about Brachyury targets in the mouse.Here we use chromatin immunoprecipitation and mouse promoter microarrays to identify targets of Brachyury in embryoid bodies formed from differentiating mouse ES cells. The targets we identify are enriched for sequence-specific DNA binding proteins and include components of signal transduction pathways that direct cell fate in the primitive streak and tailbud of the early embryo. Expression of some of these targets, such as Axin2, Fgf8 and Wnt3a, is down regulated in Brachyury mutant embryos and we demonstrate that they are also Brachyury targets in the human. Surprisingly, we do not observe enrichment of the canonical T-domain DNA binding sequence 5'-TCACACCT-3' in the vicinity of most Brachyury target genes. Rather, we have identified an (AC(n repeat sequence, which is conserved in the rat but not in human, zebrafish or Xenopus. We do not understand the significance of this sequence, but speculate that it enhances transcription factor binding in the regulatory regions of Brachyury target genes in rodents.Our work identifies the genomic targets of a key regulator of mesoderm formation in the early mouse embryo, thereby providing insights into the Brachyury-driven genetic regulatory network and allowing us to compare the function of Brachyury in different species.

Simultaneous Vascular Targeting and Tumor Targeting of Cerebral Breast Cancer Metastases Using a T-Cell Receptor Mimic Antibody

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2014-05-01

in May 2013, the difference between nude mice (which lack T- cells , but still have a partially functional adaptive and innate immune system) and NSG...Mangada J, Greiner DL, Handgretinger R. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human...Targeting of Cerebral Breast Cancer Metastases Using a T- Cell Receptor Mimic Antibody PRINCIPAL INVESTIGATOR: Ulrich Bickel

Inhibition of mesothelin as a novel strategy for targeting cancer cells.

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Kun Wang

Full Text Available Mesothelin, a differentiation antigen present in a series of malignancies such as mesothelioma, ovarian, lung and pancreatic cancer, has been studied as a marker for diagnosis and a target for immunotherapy. We, however, were interested in evaluating the effects of direct targeting of Mesothelin on the viability of cancer cells as the first step towards developing a novel therapeutic strategy. We report here that gene specific silencing for Mesothelin by distinct methods (siRNA and microRNA decreased viability of cancer cells from different origins such as mesothelioma (H2373, ovarian cancer (Skov3 and Ovcar-5 and pancreatic cancer (Miapaca2 and Panc-1. Additionally, the invasiveness of cancer cells was also significantly decreased upon such treatment. We then investigated pro-oncogenic signaling characteristics of cells upon mesothelin-silencing which revealed a significant decrease in phospho-ERK1 and PI3K/AKT activity. The molecular mechanism of reduced invasiveness was connected to the reduced expression of β-Catenin, an important marker of EMT (epithelial-mesenchymal transition. Ero1, a protein involved in clearing unfolded proteins and a member of the ER-Stress (endoplasmic reticulum-stress pathway was also markedly reduced. Furthermore, Mesothelin silencing caused a significant increase in fraction of cancer cells in S-phase. In next step, treatment of ovarian cancer cells (OVca429 with a lentivirus expressing anti-mesothelin microRNA resulted in significant loss of viability, invasiveness, and morphological alterations. Therefore, we propose the inhibition of Mesothelin as a potential novel strategy for targeting human malignancies.

Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells.

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Gaëlle Gonzalez

Full Text Available Cell microparticles (MPs released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5, and serotype 35 (HAdV35, respectively. We found that MPs derived from CHO cells (MP-donor cells constitutively expressing CAR (MP-CAR or CD46 (MP-CD46 were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins.

Accurate measurement of peripheral blood mononuclear cell concentration using image cytometry to eliminate RBC-induced counting error.

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Chan, Leo Li-Ying; Laverty, Daniel J; Smith, Tim; Nejad, Parham; Hei, Hillary; Gandhi, Roopali; Kuksin, Dmitry; Qiu, Jean

2013-02-28

Peripheral blood mononuclear cells (PBMCs) have been widely researched in the fields of immunology, infectious disease, oncology, transplantation, hematological malignancy, and vaccine development. Specifically, in immunology research, PBMCs have been utilized to monitor concentration, viability, proliferation, and cytokine production from immune cells, which are critical for both clinical trials and biomedical research. The viability and concentration of isolated PBMCs are traditionally measured by manual counting with trypan blue (TB) using a hemacytometer. One of the common issues of PBMC isolation is red blood cell (RBC) contamination. The RBC contamination can be dependent on the donor sample and/or technical skill level of the operator. RBC contamination in a PBMC sample can introduce error to the measured concentration, which can pass down to future experimental assays performed on these cells. To resolve this issue, RBC lysing protocol can be used to eliminate potential error caused by RBC contamination. In the recent years, a rapid fluorescence-based image cytometry system has been utilized for bright-field and fluorescence imaging analysis of cellular characteristics (Nexcelom Bioscience LLC, Lawrence, MA). The Cellometer image cytometry system has demonstrated the capability of automated concentration and viability detection in disposable counting chambers of unpurified mouse splenocytes and PBMCs stained with acridine orange (AO) and propidium iodide (PI) under fluorescence detection. In this work, we demonstrate the ability of Cellometer image cytometry system to accurately measure PBMC concentration, despite RBC contamination, by comparison of five different total PBMC counting methods: (1) manual counting of trypan blue-stained PBMCs in hemacytometer, (2) manual counting of PBMCs in bright-field images, (3) manual counting of acetic acid lysing of RBCs with TB-stained PBMCs, (4) automated counting of acetic acid lysing of RBCs with PI-stained PBMCs

Single agent- and combination treatment with two targeted suicide gene therapy systems is effective in chemoresistant small cell lung cancer cells

DEFF Research Database (Denmark)

Michaelsen, Signe R; Christensen, Camilla L; Sehested, Maxwell

2012-01-01

Transcriptional targeted suicide gene (SG) therapy driven by the insulinoma-associated 1 (INSM1) promoter makes it possible to target suicide toxin production and cytotoxicity exclusively to small cell lung cancer (SCLC) cells and tumors. It remains to be determined whether acquired chemoresistance......, as observed in the majority of SCLC patients, desensitizes SCLC cells to INSM1 promoter-driven SG therapy....

Regulatory B cells: an exciting target for future therapeutics in transplantation

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Alexandre eNouël

2014-01-01

Full Text Available Transplantation is the preferred treatment for most end-stage solid organ diseases. Despite potent immunosuppressive agents, chronic rejection remains a real problem in transplantation. For many years, the predominant immunological focus of research into transplant rejection has been T cells. The pillar of immunotherapy in clinical practice is T cell-directed, which efficiently prevents acute T cell-mediated allograft rejection. However, the root of late allograft failure is chronic rejection and the humoral arm of the immune response now emerges as an important factor in transplantation. Thus, the potential effects of Abs and B cell infiltrates on transplants have cast B cells as major actors in late graft rejection. Consequently, a number of recent drugs target either B cells or plasma cells. However, immunotherapies, such as the anti-CD20 B cell-depleting Ab, can generate deleterious effects on the transplant, likely due to the deletion of beneficial population. The positive contribution of regulatory B (Breg cells -or B10 cells- has been reported in the case of transplantation, mainly in mice models and highlights the primordial role that some populations of B cells can play in graft tolerance. Yet, this regulatory aspect remains poorly characterized in clinical transplantation. Thus, total B cell depletion treatments should be avoided and novel approaches should be considered that manipulate the different B cell subsets. This article provides an overview of the current knowledge on the link between Breg cells and grafts, and reports a number of data advising Breg cells as a new target for future therapeutic approaches.

WRN-targeted therapy using inhibitors NSC 19630 and NSC 617145 induce apoptosis in HTLV-1-transformed adult T-cell leukemia cells

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R. Moles

2016-11-01

Full Text Available Abstract Background Human T-cell leukemia virus type 1 (HTLV-1 infection is associated with adult T-cell leukemia/lymphoma (ATLL, a lymphoproliferative malignancy with a dismal prognosis and limited therapeutic options. Recent evidence shows that HTLV-1-transformed cells present defects in both DNA replication and DNA repair, suggesting that these cells might be particularly sensitive to treatment with a small helicase inhibitor. Because the “Werner syndrome ATP-dependent helicase” encoded by the WRN gene plays important roles in both cellular proliferation and DNA repair, we hypothesized that inhibition of WRN activity could be used as a new strategy to target ATLL cells. Methods Our analysis demonstrates an apoptotic effect induced by the WRN helicase inhibitor in HTLV-1-transformed cells in vitro and ATL-derived cell lines. Inhibition of cellular proliferation and induction of apoptosis were demonstrated with cell cycle analysis, XTT proliferation assay, clonogenic assay, annexin V staining, and measurement of mitochondrial transmembrane potential. Results Targeted inhibition of the WRN helicase induced cell cycle arrest and apoptosis in HTLV-1-transformed leukemia cells. Treatment with NSC 19630 (WRN inhibitor induces S-phase cell cycle arrest, disruption of the mitochondrial membrane potential, and decreased expression of anti-apoptotic factor Bcl-2. These events were associated with activation of caspase-3-dependent apoptosis in ATL cells. We identified some ATL cells, ATL-55T and LMY1, less sensitive to NSC 19630 but sensitive to another WRN inhibitor, NSC 617145. Conclusions WRN is essential for survival of ATL cells. Our studies suggest that targeting the WRN helicase with small inhibitors is a novel promising strategy to target HTLV-1-transformed ATL cells.

Estrogen enhanced cell-cell signalling in breast cancer cells exposed to targeted irradiation

International Nuclear Information System (INIS)

Shao, Chunlin; Folkard, Melvyn; Held, Kathryn D; Prise, Kevin M

2008-01-01

Radiation-induced bystander responses, where cells respond to their neighbours being irradiated are being extensively studied. Although evidence shows that bystander responses can be induced in many types of cells, it is not known whether there is a radiation-induced bystander effect in breast cancer cells, where the radiosensitivity may be dependent on the role of the cellular estrogen receptor (ER). This study investigated radiation-induced bystander responses in estrogen receptor-positive MCF-7 and estrogen receptor-negative MDA-MB-231 breast cancer cells. The influence of estrogen and anti-estrogen treatments on the bystander response was determined by individually irradiating a fraction of cells within the population with a precise number of helium-3 using a charged particle microbeam. Damage was scored as chromosomal damage measured as micronucleus formation. A bystander response measured as increased yield of micronucleated cells was triggered in both MCF-7 and MDA-MB-231 cells. The contribution of the bystander response to total cell damage in MCF-7 cells was higher than that in MDA-MB-231 cells although the radiosensitivity of MDA-MB-231 was higher than MCF-7. Treatment of cells with 17β-estradiol (E2) increased the radiosensitivity and the bystander response in MCF-7 cells, and the effect was diminished by anti-estrogen tamoxifen (TAM). E2 also increased the level of intracellular reactive oxygen species (ROS) in MCF-7 cells in the absence of radiation. In contrast, E2 and TAM had no influence on the bystander response and ROS levels in MDA-MB-231 cells. Moreover, the treatment of MCF-7 cells with antioxidants eliminated both the E2-induced ROS increase and E2-enhanced bystander response triggered by the microbeam irradiation, which indicates that ROS are involved in the E2-enhanced bystander micronuclei formation after microbeam irradiation. The observation of bystander responses in breast tumour cells may offer new potential targets for radiation

Photochemical Targeting Of Phagocytic Trabecular Meshwork Cells Using Chlorin E6 Coupled Microspheres

Science.gov (United States)

Latina, M. A.; Kobsa, P. H.; Rakestraw, S. L.; Crean, E. A.; Hasan, T.; Yarmush, M. L.

1989-03-01

We have investigated a novel and efficient delivery system utilizing photosensitizer-coupled-latex microspheres to photochemically target and kill phagocytic trabecular meshwork (TM) cells. TM cells are the most actively phagocytic cells within the anterior chamber of the eye and are located within an optically accessible discrete band. This delivery system, along with the property of cell photocytosis, will achieve double selectivity by combining preferential localization of the photosensitizer to the target cells with spatial localization of illumination on the target cells. All experiments were performed with preconfluent bovine TM cells, 3rd to 4th passage, plated in 15 mm wells. Chlorin e6 monoethylene diamine monoamide was conjugated to the surface of 1.0 Am MX Duke Scientific fluorescent latex microspheres. Spectroscopic analysis revealed an average of 1.3 x 10 -17 moles of chlorin e6 per microsphere. TM cells were incubated for 18 hours with 5 x 10 7 microspheres/ml in MEM with 10% FCS, washed with MEM, and irradiated through fresh media using an argon-pumped dye laser emitting .2 W at 660 nm. A dose-survival study indicated that energy doses of 10 J/cm2 or greater resulted in greater than 95% cell death as determined by ethidium bromide exclusion. Cell death could be demonstrated as early as 4 hours post-irradiation. TM cells incubated with a solution of chlorin e6 at a concentration equal to that conjugated to the microspheres showed no cell death. Unirradiated controls also showed no cell death.

Multistage model for the action of cytotoxic T lymphocytes in multicellular conjugates

International Nuclear Information System (INIS)

Macken, C.A.; Perelson, A.S.

1984-01-01

The authors propose a multistage stochastic model to explain data on the kinetics of target cell lysis by cytotoxic T lymphocytes in multicellular conjugates. A novel feature of this model is that the authors explicitly consider both the lethal hitting stage and the target cell disintegration stage of the cytolytic process. Further, the authors allow for the possibility that target cell disintegration is itself a complex process composed of many events. The comparison of this model with the data of other investigators suggests that cytotoxic T cells deliver lethal hits at random to undamage target cells. Having received a lethal hit, the target cell disintegrates over a variable length of time. The disintegration times of target cells from different conjugates appear to be randomly distributed and to be consistent with a model in which disintegration occurs by at least two major, sequential, rate-limiting events. For conjugates containing one lymphocyte and multiple target cells, the mean rate at which a lethally hit target cell disintegrates is found to be independent of the total number of target cells in the conjugate. This model predicts that in such multicellular conjugates, individual target cells lyse one by one, on average at approximately 30-min intervals, thus agreeing closely with previously reported experimental observations. 35 references, 3 figures, 2 tables

miR-340 alleviates chemoresistance of osteosarcoma cells by targeting ZEB1.

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Yan, Haibin; Zhang, Bingyun; Fang, Chongbin; Chen, Liqiu

2018-06-01

Chemoresistance during treatment of osteosarcoma (OS) is attracting more and more attention as the main clinical obstacle. The purpose of this study was to elucidate the role of miR-340 in chemoresistance of OS. Plasmid construction and transfection, miRNA arrays, PCR analyses, and western blot analysis, as well as MTT, apoptosis, and luciferase assays were carried out in MG-63 cells and MG-63/cisplatin (DDP)-resistant cells. The results showed that miR-340 was downregulated in OS tissues and drug-resistant OS cells. Moreover, a negative correlation was observed between miR-340 and ZEB1 expression in OS tissues. Forced expression of miR-340 in drug-resistant OS cells significantly reduced multidrug resistance-1 and P-gp expression. Overexpression of miR-340 enhanced sensitivity to DDP by inhibiting viability and promoting apoptosis. The luciferase assay and western blot analysis identified ZEB1 as a direct target of miR-340, and miR-340 negatively regulated ZEB1 expression. Ectopic expression of ZEB1 reversed the effects of miR-340 on P-gp expression, cell viability, and apoptosis. miR-340 alleviated chemoresistance of OS cells by targeting ZEB1. Our results indicate that targeting miR-340 may be a potential therapeutic approach to treat drug-resistant OS.

Magnetic Nanoparticles for Targeting and Imaging of Stem Cells in Myocardial Infarction

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Michelle R. Santoso

2016-01-01

Full Text Available Stem cell therapy has broad applications in regenerative medicine and increasingly within cardiovascular disease. Stem cells have emerged as a leading therapeutic option for many diseases and have broad applications in regenerative medicine. Injuries to the heart are often permanent due to the limited proliferation and self-healing capability of cardiomyocytes; as such, stem cell therapy has become increasingly important in the treatment of cardiovascular diseases. Despite extensive efforts to optimize cardiac stem cell therapy, challenges remain in the delivery and monitoring of cells injected into the myocardium. Other fields have successively used nanoscience and nanotechnology for a multitude of biomedical applications, including drug delivery, targeted imaging, hyperthermia, and tissue repair. In particular, superparamagnetic iron oxide nanoparticles (SPIONs have been widely employed for molecular and cellular imaging. In this mini-review, we focus on the application of superparamagnetic iron oxide nanoparticles in targeting and monitoring of stem cells for the treatment of myocardial infarctions.

A targeted ferritin-microplasmin based thrombolytic nanocage selectively dissolves blood clots.

Science.gov (United States)

Seo, Junyoung; Al-Hilal, Taslim A; Jee, Jun-Goo; Kim, Yong-Lim; Kim, Ha-Jeong; Lee, Byung-Heon; Kim, Soyoun; Kim, In-San

2018-04-01

The use of thrombolytic therapies is limited by an increased risk of systemic hemorrhage due to lysis of hemostatic clots. We sought to develop a plasmin-based thrombolytic nanocage that efficiently dissolves the clot without causing systemic fibrinolysis or disrupting hemostatic clots. Here, we generated a double chambered short-length ferritin (sFt) construct that has an N-terminal region fused to multivalent clot targeting peptides (CLT: CNAGESSKNC) and a C-terminal end fused to a microplasmin (μPn); CLT recognizes fibrin-fibronectin complexes in clots, μPn efficiently dissolves clots, and the assembly of double chambered sFt (CLT-sFt-μPn) into nanocage structure protects the activated-μPn from its circulating inhibitors. Importantly, activated CLT-sFt-μPn thrombolytic nanocage showed a prolonged circulatory life over activated-μPn and efficiently lysed the preexisting clots in both arterial and venous thromboses models. Thus, CLT-sFt-μPn thrombolytic nanocage platform represents the prototype of a targeted clot-busting agent with high efficacy and safety over existing thrombolytic therapies. Copyright © 2018 Elsevier Inc. All rights reserved.

Relationship of DNA repair processes to mutagenesis and carcinogenesis in mammalian cells. Three-year report, February 1, 1981-September 30, 1983

International Nuclear Information System (INIS)

Evans, H.H.

1983-01-01

Mutant strains were selected which are deficient in various DNA repair pathways and these were studied with regard to (1) the nature of the defect in repair, and (2) the mutability and transformability of the defective cells by various agents as compared to the wild type parental cells. Lightly mutagenized wild-type cells were infected with irradiated herpes simplex virus (HSV). Cells which repair HSV are lysed so the surviving population is enriched in repair-deficient cells. Six strains which survived two rounds of infection were characterized with respect to their radiosensitivity

Characterization of novel bacteriophage phiC119 capable of lysing multidrug-resistant Shiga toxin-producing Escherichia coli O157:H7

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Luis Amarillas

2016-09-01

Full Text Available Background Shiga toxin-producing Escherichia coli (STEC is one of the most common and widely distributed foodborne pathogens that has been frequently implicated in gastrointestinal and urinary tract infections. Moreover, high rates of multiple antibiotic-resistant E. coli strains have been reported worldwide. Due to the emergence of antibiotic-resistant strains, bacteriophages are considered an attractive alternative to biocontrol pathogenic bacteria. Characterization is a preliminary step towards designing a phage for biocontrol. Methods In this study, we describe the characterization of a bacteriophage designated phiC119, which can infect and lyse several multidrug-resistant STEC strains and some Salmonella strains. The phage genome was screened to detect the stx-genes using PCR, morphological analysis, host range was determined, and genome sequencing were carried out, as well as an analysis of the cohesive ends and identification of the type of genetic material through enzymatic digestion of the genome. Results Analysis of the bacteriophage particles by transmission electron microscopy showed that it had an icosahedral head and a long tail, characteristic of the family Siphoviridae. The phage exhibits broad host range against multidrug-resistant and highly virulent E. coli isolates. One-step growth experiments revealed that the phiC119 phage presented a large burst size (210 PFU/cell and a latent period of 20 min. Based on genomic analysis, the phage contains a linear double-stranded DNA genome with a size of 47,319 bp. The phage encodes 75 putative proteins, but lysogeny and virulence genes were not found in the phiC119 genome. Conclusion These results suggest that phage phiC119 may be a good biological control agent. However, further studies are required to ensure its control of STEC and to confirm the safety of phage use.

Targeting nanoparticles to M cells with non-peptidic ligands for oral vaccination

OpenAIRE

Fievez, Virginie; Plapied, Laurence; des Rieux, Anne; Pourcelle, Vincent; Freichels, Hélène; Wascotte, Valentine; Vanderhaegen, Marie-Lyse; Jérôme, Christine; Vanderplasschen, Alain; Marchand-Brynaert, Jacqueline; Préat, Véronique

2009-01-01

The presence of RGD on nanoparticles allows the targeting of β1 integrins at the apical surface of human M cells and the enhancement of an immune response after oral immunization. To check the hypothesis that non-peptidic ligands targeting intestinal M cells or APCs would be more efficient for oral immunization than RGD, novel non-peptidic and peptidic analogs (RGD peptidomimitic (RGDp), LDV derivative (LDVd) and LDV peptidomimetic (LDVp)) as well as mannose were grafted on the PEG chain of P...

Stem cell targets and dosimetry for radiation-induced leukaemia and bone cancer

International Nuclear Information System (INIS)

Richardson, R.B.

2007-01-01

The ICRP are proposing changes to the assumed targets for the induction of bone cancer and leukaemias as described by Harrison et al in an accompanying article. This study of radiation targets in the skeleton finds that the endosteum of the long bone medullary cavities is not an important target, especially in the adult, as it supports a very low stem cell population associated with high adiposity, whereas the periosteum has a strong mesenchymal stem cell population throughout lifetime. Quiescent stem cells are found to be preferentially located close to the trabecular bone surface in the osteoblastic niche, whereas progenitors of stem cells prefer to reside in perivascular niches. Evidence is given in support of the suggestion that the absence of excess bone-cancer in atomic bomb survivors may be related to the extremely low prevalence of Paget's disease in Japan. The hypoxic conditions of the endosteum adjacent to quiescent bone surfaces provide a radioprotective stem cell microenvironment by a factor of 2-3 fold, whereas greater radiosensitivity is prevalent in the young and individuals with benign diseases of bone. Increasing the volume of the bone cancer target from a 10 μm thick endosteum to a 50 μm peripheral marrow layer will result in an approximately three-fold decline in the mean dose from alpha-emitters in bone. These new observations are shown to go some way in explaining the low incidences for leukaemia and especially bone cancer in radium dial painters, Thorotrast patients and Mayak nuclear workers. (author)

Targeted genome editing in human repopulating haematopoietic stem cells

NARCIS (Netherlands)

P. Genovese (Pietro); G. Schiroli (Giulia); G. Escobar (Giulia); T. Di Tomaso (Tiziano); C. Firrito (Claudia); A. Calabria (Andrea); D. Moi (Davide); R. Mazzieri (Roberta); C. Bonini (Chiara); M.V. Holmes (Michael); P.D. Gregory (Philip); M. van der Burg (Mirjam); B. Gentner (Bernhard); E. Montini (Eugenio); A. Lombardo (Angelo); L. Naldini (Luigi)

2014-01-01

textabstractTargeted genome editing by artificial nucleases has brought the goal of site-specific transgene integration and gene correction within the reach of gene therapy. However, its application to long-term repopulating haematopoietic stem cells (HSCs) has remained elusive. Here we show that

Pancreatic cancer cell detection by targeted lipid microbubbles and multiphoton imaging

Science.gov (United States)

Cromey, Benjamin; McDaniel, Ashley; Matsunaga, Terry; Vagner, Josef; Kieu, Khanh Quoc; Banerjee, Bhaskar

2018-04-01

Surgical resection of pancreatic cancer represents the only chance of cure and long-term survival in this common disease. Unfortunately, determination of a cancer-free margin at surgery is based on one or two tiny frozen section biopsies, which is far from ideal. Not surprisingly, cancer is usually left behind and is responsible for metastatic disease. We demonstrate a method of receptor-targeted imaging using peptide ligands, lipid microbubbles, and multiphoton microscopy that could lead to a fast and accurate way of examining the entire cut surface during surgery. Using a plectin-targeted microbubble, we performed a blinded in-vitro study to demonstrate avid binding of targeted microbubbles to pancreatic cancer cells but not noncancerous cell lines. Further work should lead to a much-needed point-of-care diagnostic test for determining clean margins in oncologic surgery.

Isthmin targets cell-surface GRP78 and triggers apoptosis via induction of mitochondrial dysfunction.

Science.gov (United States)

Chen, M; Zhang, Y; Yu, V C; Chong, Y-S; Yoshioka, T; Ge, R

2014-05-01

Isthmin (ISM) is a secreted 60-kDa protein that potently induces endothelial cell (EC) apoptosis. It suppresses tumor growth and angiogenesis in mice when stably overexpressed in cancer cells. Although αvβ5 integrin serves as a low-affinity receptor for ISM, the mechanism by which ISM mediates antiangiogenesis and apoptosis in ECs remain to be fully resolved. In this work, we report the identification of cell-surface glucose-regulated protein 78 kDa (GRP78) as a high-affinity receptor for ISM (Kd=8.6 nM). We demonstrated that ISM-GRP78 interaction triggers apoptosis not only in activated ECs but also in cancer cells expressing high level of cell-surface GRP78. Normal cells and benign tumor cells tend to express low level of cell-surface GRP78 and are resistant to ISM-induced apoptosis. Upon binding to GRP78, ISM is internalized into ECs through clathrin-dependent endocytosis that is essential for its proapoptotic activity. Once inside the cell, ISM co-targets with GRP78 to mitochondria where it interacts with ADP/ATP carriers on the inner membrane and blocks ATP transport from mitochondria to cytosol, thereby causing apoptosis. Hence, ISM is a novel proapoptotic ligand that targets cell-surface GRP78 to trigger apoptosis by inducing mitochondrial dysfunction. The restricted and high-level expression of cell-surface GRP78 on cancer cells and cancer ECs make them uniquely susceptible to ISM-targeted apoptosis. Indeed, systemic delivery of recombinant ISM potently suppressed subcutaneous 4T1 breast carcinoma and B16 melanoma growth in mice by eliciting apoptosis selectively in the cancer cells and cancer ECs. Together, this work reveals a novel ISM-GRP78 apoptosis pathway and demonstrates the potential of ISM as a cancer-specific and dual-targeting anticancer agent.

Isthmin targets cell-surface GRP78 and triggers apoptosis via induction of mitochondrial dysfunction

Science.gov (United States)

Chen, M; Zhang, Y; Yu, V C; Chong, Y-S; Yoshioka, T; Ge, R

2014-01-01

Isthmin (ISM) is a secreted 60-kDa protein that potently induces endothelial cell (EC) apoptosis. It suppresses tumor growth and angiogenesis in mice when stably overexpressed in cancer cells. Although αvβ5 integrin serves as a low-affinity receptor for ISM, the mechanism by which ISM mediates antiangiogenesis and apoptosis in ECs remain to be fully resolved. In this work, we report the identification of cell-surface glucose-regulated protein 78 kDa (GRP78) as a high-affinity receptor for ISM (Kd=8.6 nM). We demonstrated that ISM-GRP78 interaction triggers apoptosis not only in activated ECs but also in cancer cells expressing high level of cell-surface GRP78. Normal cells and benign tumor cells tend to express low level of cell-surface GRP78 and are resistant to ISM-induced apoptosis. Upon binding to GRP78, ISM is internalized into ECs through clathrin-dependent endocytosis that is essential for its proapoptotic activity. Once inside the cell, ISM co-targets with GRP78 to mitochondria where it interacts with ADP/ATP carriers on the inner membrane and blocks ATP transport from mitochondria to cytosol, thereby causing apoptosis. Hence, ISM is a novel proapoptotic ligand that targets cell-surface GRP78 to trigger apoptosis by inducing mitochondrial dysfunction. The restricted and high-level expression of cell-surface GRP78 on cancer cells and cancer ECs make them uniquely susceptible to ISM-targeted apoptosis. Indeed, systemic delivery of recombinant ISM potently suppressed subcutaneous 4T1 breast carcinoma and B16 melanoma growth in mice by eliciting apoptosis selectively in the cancer cells and cancer ECs. Together, this work reveals a novel ISM-GRP78 apoptosis pathway and demonstrates the potential of ISM as a cancer-specific and dual-targeting anticancer agent. PMID:24464222

Plasmonic nanodiamonds: targeted core-shell type nanoparticles for cancer cell thermoablation.

Science.gov (United States)

Rehor, Ivan; Lee, Karin L; Chen, Kevin; Hajek, Miroslav; Havlik, Jan; Lokajova, Jana; Masat, Milan; Slegerova, Jitka; Shukla, Sourabh; Heidari, Hamed; Bals, Sara; Steinmetz, Nicole F; Cigler, Petr

2015-02-18

Targeted biocompatible nanostructures with controlled plasmonic and morphological parameters are promising materials for cancer treatment based on selective thermal ablation of cells. Here, core-shell plasmonic nanodiamonds consisting of a silica-encapsulated diamond nanocrystal coated in a gold shell are designed and synthesized. The architecture of particles is analyzed and confirmed in detail using electron tomography. The particles are biocompatibilized using a PEG polymer terminated with bioorthogonally reactive alkyne groups. Azide-modified transferrin is attached to these particles, and their high colloidal stability and successful targeting to cancer cells overexpressing the transferrin receptor are demonstrated. The particles are nontoxic to the cells and they are readily internalized upon binding to the transferrin receptor. The high plasmonic cross section of the particles in the near-infrared region is utilized to quantitatively ablate the cancer cells with a short, one-minute irradiation by a pulse 750-nm laser. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tracking targeted bimodal nanovaccines: immune responses and routing in cells, tissue, and whole organism.

Science.gov (United States)

Cruz, Luis J; Tacken, Paul J; Zeelenberg, Ingrid S; Srinivas, Mangala; Bonetto, Fernando; Weigelin, Bettina; Eich, Christina; de Vries, I Jolanda; Figdor, Carl G

2014-12-01

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs), involved in the induction of immunity and currently exploited for antitumor immunotherapies. An optimized noninvasive imaging modality capable of determining and quantifying DC-targeted nanoparticle (NP) trajectories could provide valuable information regarding therapeutic vaccine outcome. Here, targeted poly(d,l-lactide-co-glycolide) nanoparticles (PLGA NPs) recognizing DC receptors were equipped with superparamagnetic iron oxide particles (SPIO) or gold nanoparticles with fluorescently labeled antigen. The fluorescent label allowed for rapid analysis and quantification of DC-specific uptake of targeted PLGA NPs in comparison to uptake by other cells. Transmission electron microscopy (TEM) showed that a fraction of the encapsulated antigen reached the lysosomal compartment of DCs, where SPIO and gold were already partially released. However, part of the PLGA NPs localized within the cytoplasm, as confirmed by confocal microscopy. DCs targeted with NPs carrying SPIO or fluorescent antigen were detected within lymph nodes as early as 1 h after injection by magnetic resonance imaging (MRI). Despite the fact that targeting did not markedly affect PLGA NP biodistribution on organism and tissue level, it increased delivery of NPs to DCs residing in peripheral lymph nodes and resulted in enhanced T cell proliferation. In conclusion, two imaging agents within a single carrier allows tracking of targeted PLGA NPs at the subcellular, cellular, and organismal levels, thereby facilitating the rational design of in vivo targeted vaccination strategies.

Communicating the non-targeted effects of radiation from irradiated to non-irradiated cells

International Nuclear Information System (INIS)

Laiakis, E.C.; Morgan, W.F.

2005-01-01

For many years, the central dogma in radiobiology has been that energy deposited in the cell nucleus is responsible for the biological effects associated with radiation exposure. However, non-targeted and delayed effects of radiation have shifted this belief. The studies of radiation-induced genomic instability, the bystander and abscopal effects, clastogenic factors, and the Death Inducing Effect have dominated the interest of the radiobiology field of late. The passing of signals from irradiated to non-irradiated cells can be accomplished through cell-to-cell gap junction communication or secretion of molecules, which in turn can elicit a response through activation of signal transduction pathways. Proposed mediators of this phenotype include proteins involved with inflammation. Given their size and connection with oxidative stress, cytokines are an attractive candidate as mediators of the induction of the non-targeted effects of radiation. Here we review the evidence for a possible connection between these delayed non-targeted effects of radiation and the cytokine cascades associated with inflammation. (author)

CD25 targeted therapy of chemotherapy resistant leukemic stem cells using DR5 specific TRAIL peptide

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Jayaprakasam Madhumathi

2017-03-01

Full Text Available Chemotherapy resistant leukemic stem cells (LSCs are being targeted as a modern therapeutic approach to prevent disease relapse. LSCs isolated from methotrexate resistant side population (SP of leukemic cell lines HL60 and MOLT4 exhibited high levels of CD25 and TRAIL R2/DR5 which are potential targets. Recombinant immunotoxin conjugating IL2α with TRAIL peptide mimetic was constructed for DR5 receptor specific targeting of LSCs and were tested in total cell population and LSCs. IL2-TRAIL peptide induced apoptosis in drug resistant SP cells from cell lines and showed potent cytotoxicity in PBMCs derived from leukemic patients with an efficacy of 81.25% in AML and 100% in CML, ALL and CLL. IL2-TRAIL peptide showed cytotoxicity in relapsed patient samples and was more effective than TRAIL or IL2-TRAIL proteins. Additionally, DR5 specific IL2-TRAIL peptide was effective in targeting and killing LSCs purified from cell lines [IC50: 952 nM in HL60, 714 nM in MOLT4] and relapsed patient blood samples with higher efficacy (85% than IL2-TRAIL protein (46%. Hence, CD25 and DR5 specific targeting by IL2-TRAIL peptide may be an effective strategy for targeting drug resistant leukemic cells and LSCs.

Nanobiotechnology meets plant cell biology: Carbon nanotubes as organelle targeting nanocarriers

KAUST Repository

Serag, Maged F.; Kaji, Noritada; Habuchi, Satoshi; Bianco, Alberto; Baba, Yoshinobu

2013-01-01

For years, nanotechnology has shown great promise in the fields of biomedical and biotechnological sciences and medical research. In this review, we demonstrate its versatility and applicability in plant cell biology studies. Specifically, we discuss the ability of functionalized carbon nanotubes to penetrate the plant cell wall, target specific organelles, probe protein-carrier activity and induce organelle recycling in plant cells. We also, shed light on prospective applications of carbon nanomaterials in cell biology and plant cell transformation. © 2013 The Royal Society of Chemistry.

MicroRNA-1185 Induces Endothelial Cell Apoptosis by Targeting UVRAG and KRIT1

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Haoyuan Deng

2017-04-01

Full Text Available Background/Aims: Atherosclerosis is a multifactorial chronic disease and is the main cause of death and impairment in the world. Endothelial injury and apoptosis play a crucial role in the onset and development of atherosclerosis. MicroRNAs (miRNAs have been proven to be involved in the pathogenesis of atherosclerosis. However, studies of the functional role of apoptosis-related miRNAs in the endothelium during atherogenesis are limited. Methods: Cell injury and apoptosis were measured in five types of cells transfected with miR-1185 or co-transfected with miR-1185 and its inhibitor. Bioinformatics analysis and a luciferase reporter assay were used to confirm the targets of miR-1185. The effects of the targets of miR-1185 on endothelial apoptosis were determined using small-interfering RNA. Results: In this study, we first report that miR-1185 significantly promoted apoptosis in endothelial cells but not in vascular smooth muscle cells and macrophages. A mechanistic analysis showed that ultraviolet irradiation resistance-associated gene (UVRAG and krev1 interaction trapped gene 1 (KRIT1, targets of miR-1185, mediated miR-1185-induced endothelial cell apoptosis. Conclusion: The results revealed the impact of miR-1185 on endothelial apoptosis, suggesting that miR-1185 may be a potential target for the prevention and treatment of atherosclerosis.

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

Science.gov (United States)

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

2010-11-01

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

Proteasomal targeting and minigene repetition improve cell-surface presentation of a transfected, modified melanoma tumour antigen

DEFF Research Database (Denmark)

Rasmussen, A B; Zocca, M-B; Bonefeld, C M

2004-01-01

Melanoma antigen recognized by T cell 1 (MART-1) is regarded as a candidate peptide for vaccination against malignant melanoma, and it is of importance to develop strategies to improve the vaccine-elicited T-cell activation towards MART-1. T-cell activation is, among other determinants, dependent...... on the density of specific major histocompatibility complex-peptide complexes on the surface of the antigen-presenting cell. In this study, we explored the cell-surface presentation of a substituted MART-1 peptide encoded by transfected minigenes. We investigated the potential of proteasomal targeting compared...... to non-proteasomal targeting of the epitope to increase its cell-surface presentation. Furthermore, we explored the potential of incorporating multiple minigenes instead of one to increase cell-surface presentation. We show that both proteasomal targeting and repetition of the minigene increase cell...

Rationally engineered nanoparticles target multiple myeloma cells, overcome cell-adhesion-mediated drug resistance, and show enhanced efficacy in vivo

International Nuclear Information System (INIS)

Kiziltepe, T; Ashley, J D; Stefanick, J F; Qi, Y M; Alves, N J; Handlogten, M W; Suckow, M A; Navari, R M; Bilgicer, B

2012-01-01

In the continuing search for effective cancer treatments, we report the rational engineering of a multifunctional nanoparticle that combines traditional chemotherapy with cell targeting and anti-adhesion functionalities. Very late antigen-4 (VLA-4) mediated adhesion of multiple myeloma (MM) cells to bone marrow stroma confers MM cells with cell-adhesion-mediated drug resistance (CAM-DR). In our design, we used micellar nanoparticles as dynamic self-assembling scaffolds to present VLA-4-antagonist peptides and doxorubicin (Dox) conjugates, simultaneously, to selectively target MM cells and to overcome CAM-DR. Dox was conjugated to the nanoparticles through an acid-sensitive hydrazone bond. VLA-4-antagonist peptides were conjugated via a multifaceted synthetic procedure for generating precisely controlled number of targeting functionalities. The nanoparticles were efficiently internalized by MM cells and induced cytotoxicity. Mechanistic studies revealed that nanoparticles induced DNA double-strand breaks and apoptosis in MM cells. Importantly, multifunctional nanoparticles overcame CAM-DR, and were more efficacious than Dox when MM cells were cultured on fibronectin-coated plates. Finally, in a MM xenograft model, nanoparticles preferentially homed to MM tumors with ∼10 fold more drug accumulation and demonstrated dramatic tumor growth inhibition with a reduced overall systemic toxicity. Altogether, we demonstrate the disease driven engineering of a nanoparticle-based drug delivery system, enabling the model of an integrative approach in the treatment of MM

Impact of 2-bromopropane on mouse embryonic stem cells and ...

African Journals Online (AJOL)

EWOMAZINO

medium containing various concentrations of 2-BP at 37°C in a CO2 incubator for 24 h. Cells were then washed twice with ice-cold PBS and lysed on ice for 10 min in 400 μl lysis buffer (20 mM Tris-HCl,. pH 7.4, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, ...

Targeting to carcinoma cells with chitosan- and starch-coated magnetic nanoparticles for magnetic hyperthermia.

Science.gov (United States)

Kim, Dong-Hyun; Kim, Kyoung-Nam; Kim, Kwang-Mahn; Lee, Yong-Keun

2009-01-01

The delivery of hyperthermic thermoseeds to a specific target site with minimal side effects is an important challenge in targeted hyperthermia, which employs magnetic method and functional polymers. An external magnetic field is used to control the site-specific targeting of the magnetic nanoparticles. Polymer-coated magnetic nanoparticles can confer a higher affinity to the biological cell membranes. In this study, uncoated, chitosan-coated, and starch-coated magnetic nanoparticles were synthesized for use as a hyperthermic thermoseed. Each sample was examined with respect to their applications to hyperthermia using XRD, VSM, and FTIR. In addition, the temperature changes under an alternating magnetic field were observed. As in vitro tests, the magnetic responsiveness of chitosan- and starch-coated magnetite was determined by a simple blood vessel model under various intensities of magnetic field. L929 normal cells and KB carcinoma cells were used to examine the cytotoxicity and affinity of each sample using the MTT method. The chitosan-coated magnetic nanoparticles generated a higher DeltaT of 23 degrees C under an AC magnetic field than the starch-coated magnetite, and the capturing rate of the particles was 96% under an external magnetic field of 0.4 T. The highest viability of L929 cells was 93.7%. Comparing the rate of KB cells capture with the rate of L929 cells capture, the rate of KB cells capture relatively increased with 10.8% in chitosan-coated magnetic nanoparticles. Hence, chitosan-coated magnetic nanoparticles are biocompatible and have a selective affinity to KB cells. The targeting of magnetic nanoparticles in hyperthermia was improved using a controlled magnetic field and a chitosan-coating. Therefore, chitosan-coated magnetic nanoparticles are expected to be promising materials for use in magnetic targeted hyperthermia. 2008 Wiley Periodicals, Inc.

Epithelial cell adhesion molecule aptamer functionalized PLGA-lecithin-curcumin-PEG nanoparticles for targeted drug delivery to human colorectal adenocarcinoma cells

Science.gov (United States)

Li, Lei; Xiang, Dongxi; Shigdar, Sarah; Yang, Wenrong; Li, Qiong; Lin, Jia; Liu, Kexin; Duan, Wei

2014-01-01

To improve the efficacy of drug delivery, active targeted nanotechnology-based drug delivery systems are gaining considerable attention as they have the potential to reduce side effects, minimize toxicity, and improve efficacy of anticancer treatment. In this work CUR-NPs (curcumin-loaded lipid-polymer-lecithin hybrid nanoparticles) were synthesized and functionalized with ribonucleic acid (RNA) Aptamers (Apts) against epithelial cell adhesion molecule (EpCAM) for targeted delivery to colorectal adenocarcinoma cells. These CUR-encapsulated bioconjugates (Apt-CUR-NPs) were characterized for particle size, zeta potential, drug encapsulation, stability, and release. The in vitro specific cell binding, cellular uptake, and cytotoxicity of Apt-CUR-NPs were also studied. The Apt-CUR-NP bioconjugates exhibited increased binding to HT29 colon cancer cells and enhancement in cellular uptake when compared to CUR-NPs functionalized with a control Apt (P<0.01). Furthermore, a substantial improvement in cytotoxicity was achieved toward HT29 cells with Apt-CUR-NP bioconjugates. The encapsulation of CUR in Apt-CUR-NPs resulted in the increased bioavailability of delivered CUR over a period of 24 hours compared to that of free CUR in vivo. These results show that the EpCAM Apt-functionalized CUR-NPs enhance the targeting and drug delivery of CUR to colorectal cancer cells. Further development of CUR-encapsulated, nanosized carriers will lead to improved targeted delivery of novel chemotherapeutic agents to colorectal cancer cells. PMID:24591829

Selective Inhibition of Tumor Growth by Clonal NK Cells Expressing an ErbB2/HER2-Specific Chimeric Antigen Receptor

Science.gov (United States)

Schönfeld, Kurt; Sahm, Christiane; Zhang, Congcong; Naundorf, Sonja; Brendel, Christian; Odendahl, Marcus; Nowakowska, Paulina; Bönig, Halvard; Köhl, Ulrike; Kloess, Stephan; Köhler, Sylvia; Holtgreve-Grez, Heidi; Jauch, Anna; Schmidt, Manfred; Schubert, Ralf; Kühlcke, Klaus; Seifried, Erhard; Klingemann, Hans G; Rieger, Michael A; Tonn, Torsten; Grez, Manuel; Wels, Winfried S

2015-01-01

Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy. PMID:25373520

A high content, high throughput cellular thermal stability assay for measuring drug-target engagement in living cells.

Science.gov (United States)

Massey, Andrew J

2018-01-01

Determining and understanding drug target engagement is critical for drug discovery. This can be challenging within living cells as selective readouts are often unavailable. Here we describe a novel method for measuring target engagement in living cells based on the principle of altered protein thermal stabilization / destabilization in response to ligand binding. This assay (HCIF-CETSA) utilizes high content, high throughput single cell immunofluorescent detection to determine target protein levels following heating of adherent cells in a 96 well plate format. We have used target engagement of Chk1 by potent small molecule inhibitors to validate the assay. Target engagement measured by this method was subsequently compared to target engagement measured by two alternative methods (autophosphorylation and CETSA). The HCIF-CETSA method appeared robust and a good correlation in target engagement measured by this method and CETSA for the selective Chk1 inhibitor V158411 was observed. However, these EC50 values were 23- and 12-fold greater than the autophosphorylation IC50. The described method is therefore a valuable advance in the CETSA method allowing the high throughput determination of target engagement in adherent cells.