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Sample records for mdv-derived lymphoblastoid cell

  1. Restricted Replication of Vesicular Stomatitis Virus in Human Lymphoblastoid Cells

    PubMed Central

    Nowakowski, Maja; Bloom, Barry R.; Ehrenfeld, Ellie; Summers, Donald F.

    1973-01-01

    Replication of vesicular stomatitis virus (VSV) is restricted in one human lymphoblastoid cell line (Raji), but not in another similar cell line (Wil-2), compared with growth in HeLa cells. This restriction is characterized by a low proportion of cells yielding infectious virus and is associated with limited production of 42S virion RNA. Primary transcription of 13S and 26S VSV-specific RNA is not restricted in Raji cells, and the 13S RNA produced contains adenylate-rich sequences. This suggests that the block in Raji cells involves some step required for the replication of virion RNA. PMID:4357508

  2. [Production of a dialysable transfer factor of cell mediated immunity by lymphoblastoid cells in continuous proliferation].

    PubMed

    Goust, J M; Viza, D; Moulias, R; Trejdosiewicz, L; Lesourd, B; Marescot, M R; Prévot, A

    1975-01-20

    Four lymphoblastoid cell lines tested in this work contain normally a dialysable moiety having by ultraviolet spectroscopy, column chromatography (Biogel P 10) and chemically the same properties than human dialysable Transfer Factor (TFd), but unable to transfer cell mediated immune response against common antigens. Two of them are able to do so after incubation with minimal amounts of TFd. Production of a molecule identical to human TFd is possible in some lymphoblastoid cell lines after induction with TFd. PMID:808340

  3. Molecular signatures in response to Isoliquiritigenin in lymphoblastoid cell lines

    SciTech Connect

    Lee, Jae-Eun; Hong, Eun-Jung; Nam, Hye-Young; Hwang, Meeyul; Kim, Ji-Hyun; Han, Bok-Ghee; Jeon, Jae-Pil

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer We identified the inhibitory effect of ISL on cell proliferation of LCLs. Black-Right-Pointing-Pointer We found ISL-induced genes and miRNAs through microarray approach. Black-Right-Pointing-Pointer ISL-treated LCLs represented gene expression changes in cell cycle and p53 pathway. Black-Right-Pointing-Pointer We revealed 12 putative mRNA-miRNA functional pairs associated with ISL effect. -- Abstract: Isoliquiritigenin (ISL) has been known to induce cell cycle arrest and apoptosis of various cancer cells. However, genetic factors regulating ISL effects remain unclear. The aim of this study was to identify the molecular signatures involved in ISL-induced cell death of EBV-transformed lymphoblastoid cell lines (LCLs) using microarray analyses. For gene expression and microRNA (miRNA) microarray experiments, each of 12 LCL strains was independently treated with ISL or DMSO as a vehicle control for a day prior to total RNA extraction. ISL treatment inhibited cell proliferation of LCLs in a dose-dependent manner. Microarray analysis showed that ISL-treated LCLs represented gene expression changes in cell cycle and p53 signaling pathway, having a potential as regulators in LCL survival and sensitivity to ISL-induced cytotoxicity. In addition, 36 miRNAs including five miRNAs with unknown functions were differentially expressed in ISL-treated LCLs. The integrative analysis of miRNA and gene expression profiles revealed 12 putative mRNA-miRNA functional pairs. Among them, miR-1207-5p and miR-575 were negatively correlated with p53 pathway- and cell cycle-associated genes, respectively. In conclusion, our study suggests that miRNAs play an important role in ISL-induced cytotoxicity in LCLs by targeting signaling pathways including p53 pathway and cell cycle.

  4. Getting personal: Endogenous adenosine receptor signaling in lymphoblastoid cell lines.

    PubMed

    Hillger, J M; Diehl, C; van Spronsen, E; Boomsma, D I; Slagboom, P E; Heitman, L H; IJzerman, A P

    2016-09-01

    Genetic differences between individuals that affect drug action form a challenge in drug therapy. Many drugs target G protein-coupled receptors (GPCRs), and a number of receptor variants have been noted to impact drug efficacy. This, however, has never been addressed in a systematic way, and, hence, we studied real-life genetic variation of receptor function in personalized cell lines. As a showcase we studied adenosine A2A receptor (A2AR) signaling in lymphoblastoid cell lines (LCLs) derived from a family of four from the Netherlands Twin Register (NTR), using a non-invasive label-free cellular assay. The potency of a partial agonist differed significantly for one individual. Genotype comparison revealed differences in two intron SNPs including rs2236624, which has been associated with caffeine-induced sleep disorders. While further validation is needed to confirm genotype-specific effects, this set-up clearly demonstrated that LCLs are a suitable model system to study genetic influences on A2AR response in particular and GPCR responses in general. PMID:27297283

  5. Estrogen treatment induces MLL aberrations in human lymphoblastoid cells

    PubMed Central

    Schnyder, Sabine; Du, Nga T.; Le, Hongan B.; Singh, Sheetal; Loredo, Grace A.; Vaughan, Andrew T.

    2009-01-01

    Epidemiological data indicates increased risk of infant acute leukemia involving MLL gene aberrations with use of oral contraceptives. To determine whether estrogens might be implicated, we examined the effect of estradiol (E2) or 4-OH-E2 in an in vitro model of translocation susceptibility. Genomic DNA from the TK6 human lymphoblastoid cell line was screened by ligation mediated PCR and inverse PCR at a rearrangement hot spot within the MLL breakpoint cluster region to detect DNA aberrations. An increase in DNA double strand breaks was observed within this region after exposure to either E2 or 4-OH-E2. An increase in the frequency of MLL translocations was only found after exposure to E2. Induction of cleavage due to increased activation of apoptotic nucleases was excluded by pre-treatment with the pancaspase inhibitor, zVAD.fmk. We conclude that concentrations of E2 and 4-OH-E2 that may occur during pregnancy, or during use of oral contraceptives, can cause aberrations of the MLL gene and could thus be a factor in the early events of leukemogenesis occurring in utero. PMID:19264358

  6. Different toxic effects of YTX in tumor K-562 and lymphoblastoid cell lines

    PubMed Central

    Fernández-Araujo, Andrea; Sánchez, Jon A.; Alfonso, Amparo; Vieytes, Mercedes R.; Botana, Luis M.

    2015-01-01

    Yessotoxin (YTX) modulates cellular phosphodiesterases (PDEs). In this regard, opposite effects had been described in the tumor model K-562 cell line and fresh human lymphocytes in terms of cell viability, cyclic adenosine 3',5'-cyclic monophosphate (cAMP) production and protein expression after YTX treatment. Studies in depth of the pathways activated by YTX in K-562 cell line, have demonstrated the activation of two different cell death types, apoptosis, and autophagy after 24 and 48 h of treatment, respectively. Furthermore, the key role of type 4A PDE (PDE4A) in both pathways activated by YTX was demonstrated. Therefore, taking into account the differences between cellular lines and fresh cells, a study of cell death pathways activated by YTX in a non-tumor cell line with mitotic activity, was performed. The cellular model used was the lymphoblastoid cell line that represents a non-tumor model with normal apoptotic and mitotic machinery. In this context, cell viability and cell proliferation, expression of proteins involved in cell death activated by YTX and mitochondrial mass, were studied after the incubation with the toxin. Opposite to the tumor model, no cell death activation was observed in lymphoblastoid cell line in the presence of YTX. In this sense, variations in apoptosis hallmarks were not detected in the lymphoblastoid cell line after YTX incubation, whereas this type I of programmed cell death was observed in K-562 cells. On the other hand, autophagy cell death was triggered in this cellular line, while other autophagic process is suggested in lymphoblastoid cells. These YTX effects are related to PDE4A in both cellular lines. In addition, while cell death is triggered in K-562 cells after YTX treatment, in lymphoblastoid cells the toxin stops cellular proliferation. These results point to YTX as a specific toxic compound of tumor cells, since in the non-tumor lymphoblastoid cell line, no cell death hallmarks are observed. PMID:26136685

  7. Perspectives on fast-neutron mutagenesis of human lymphoblastoid cells.

    PubMed

    Kronenberg, A

    1991-10-01

    The effects of low-fluence exposures to (Pu, Be) neutrons (En = 4.2 MeV) have been studied in a sensitive human B-lymphoblastoid cell line, TK6. Mutations were scored for two genetic loci, hypoxanthine phosphoribosyltransferase (hgprt) and thymidine kinase (tk), as a function of dose and dose rate. For exposures limited to less than one cell cycle, the mutation frequency for the hgprt locus was 1.92 X 10(-7)/cGy. When exposures were protracted over multiple cell generations, mutation yields were increased to 6.07 X 10(-7)/cGy. Similar yields were obtained for the induction of tk-deficient mutants with a normal cell generation time (tk-ng) when exposures were carried out at very low dose rates over multiple cell generations. In the series of data presented here, the results obtained for short-duration neutron exposures are compared with data obtained for monoenergetic heavy charged particles of defined linear energy transfer (LET) produced at the BEVALAC accelerator at Lawrence Berkeley Laboratory. TK6 cells have been exposed to beams ranging in atomic number from 20Ne to 40Ar over an energy range from 330 to 670 MeV/amu. Mutation induction was evaluated for both loci for a subset of these beams. The results obtained with 20Ne ions of 425 MeV/amu (LET = 32 keV/microns) and 28Si ions of 670 MeV/amu (LET = 50 keV/microns) closely resemble the mutation yields obtained for brief exposures to (Pu, Be) neutrons. The nature of alterations in DNA structure induced within the tk locus of tk-ng mutants is reviewed for a series of neutron-induced mutants and a series of mutants induced by exposure to 40Ar ions (470 MeV/amu, LET = 95 keV/microns). The mutational spectra for these two types of mutants were similar and were dominated by allele loss mutations. Multilocus deletions inclusive of the c-erbA1 locus were common among tk-deficient mutants induced by these densely ionizing radiations. For the mutants induced by 40Ar ions, it is likely that the mutations were produced by

  8. Network signatures of cellular immortalization in human lymphoblastoid cell lines

    SciTech Connect

    Shim, Sung-Mi; Jung, So-Young; Nam, Hye-Young; Kim, Hye-Ryun; Lee, Mee-Hee; Kim, Jun-Woo; Han, Bok-Ghee; Jeon, Jae-Pil

    2013-11-15

    Highlights: •We identified network signatures of LCL immortalization from transcriptomic profiles. •More than 41% of DEGs are possibly regulated by miRNAs in LCLs. •MicroRNA target genes in LCLs are involved in apoptosis and immune-related functions. •This approach is useful to find functional miRNA targets in specific cell conditions. -- Abstract: Human lymphoblastoid cell line (LCL) has been used as an in vitro cell model in genetic and pharmacogenomic studies, as well as a good model for studying gene expression regulatory machinery using integrated genomic analyses. In this study, we aimed to identify biological networks of LCL immortalization from transcriptomic profiles of microRNAs and their target genes in LCLs. We first selected differentially expressed genes (DEGs) and microRNAs (DEmiRs) between early passage LCLs (eLCLs) and terminally differentiated late passage LCLs (tLCLs). The in silico and correlation analysis of these DEGs and DEmiRs revealed that 1098 DEG–DEmiR pairs were found to be positively (n = 591 pairs) or negatively (n = 507 pairs) correlated with each other. More than 41% of DEGs are possibly regulated by miRNAs in LCL immortalizations. The target DEGs of DEmiRs were enriched for cellular functions associated with apoptosis, immune response, cell death, JAK–STAT cascade and lymphocyte activation while non-miRNA target DEGs were over-represented for basic cell metabolisms. The target DEGs correlated negatively with miR-548a-3p and miR-219-5p were significantly associated with protein kinase cascade, and the lymphocyte proliferation and apoptosis, respectively. In addition, the miR-106a and miR-424 clusters located in the X chromosome were enriched in DEmiR–mRNA pairs for LCL immortalization. In this study, the integrated transcriptomic analysis of LCLs could identify functional networks of biologically active microRNAs and their target genes involved in LCL immortalization.

  9. Integrity of genome-wide genotype data from low passage lymphoblastoid cell lines.

    PubMed

    McCarthy, Nina S; Allan, Spencer M; Chandler, David; Jablensky, Assen; Morar, Bharti

    2016-09-01

    We compared genotype data from the HumanExomeCore Array in peripheral blood mononuclear cells and low passage lymphoblastoid cell lines from the same 24 individuals to test for genotypic errors caused by the Epstein-Barr Virus transformation process. Genotype concordance across the 24 comparisons was 99.57% for unfiltered genotype data, and 99.63% following standard genotype quality control filters. Mendelian error rates and levels of heterozygosity were not significantly different between lymphoblastoid cell lines and their parent peripheral blood mononuclear cells. These results show that at low passage numbers, genotype discrepancies are minimal even before stringent quality control, and extend current evidence qualifying the use of low-passage lymphoblastoid cell lines as a reliable DNA source for genotype analysis. PMID:27330997

  10. [Antiviral activity of interferon and its inducers in human lymphoblastoid and somatic cells].

    PubMed

    Novokhatskiĭ, A S; Labzo, S S; Tsareva, A A

    1979-04-01

    The antiviral effect of interferon inductors, such as poly-I--poly-C, phage f2 RNA replicative form and low molecular inductor GSN and their influence on cellular DNA synthesis were studied in the cultures of lymphoblastoid (inplanting lines Raji Namalva) and somatic human cells. The Semliki forest virus used as the test organism multiplicated well in cells Raji accumulating up to 9 lg BOU/ml. The two-strand RNA was less active in the lymphoid cells than in the somatic ones. GSN was 10 times more active and less toxic in cells Raji as compared to the fibroblasts. The lymphoblastoid interferon had higher antiviral activity as compared to the fibroblast interferon in the system of Raji--Semliki forest virus than in the system of the human embryon fibroblast--Venezuela Horse Encephalytic Virus. Romantadin actively inhibited (100 times) production of the alfavirus in both the somatic and lymphoblastoid cells. PMID:220908

  11. Use of lymphoblastoid cell lines to evaluate the hypersensitivity to ultraviolet radiation in Cockayne syndrome

    SciTech Connect

    Otsuka, F.; Tarone, R.E.; Cayeux, S.; Robbins, J.H.

    1984-05-01

    Cockayne syndrome (CS) is a rare autosomal recessive disease characterized by acute sun sensitivity, cachectic dwarfism, and neurologic and skeletal abnormalities. Cultured skin fibroblasts from patients with this disease are known to be hypersensitive to the lethal effects of 254-nm UV radiation. The authors have studied the sensitivity of 254-nm UV radiation of lymphoblastoid lines derived from 3 typical CS patients, 1 atypical CS patient who had a very late age of onset of clinical manifestations, 2 patients who had both xeroderma pigmentosum (XP) and typical CS, and 3 heterozygous parents of these patients. Post-UV survival was determined by the trypan-blue dye-exclusion method. The lymphoblastoid lines from the 3 typical CS patients, the atypical CS patient, and the 2 patients with both CS and XP had decreased post-UV viability in comparison with lines from normal donors. Lines from the heterozygous parents had normal post-UV viability. The post-UV viability of the typical CS lines was similar to that of a XP complementation group C line. The relative post-UV viability of lymphoblastoid lines from the typical CS patients was similar to the relative post-UV survival of their fibroblast lines. The lymphoblastoid line from the atypical CS patient had a post-UV viability similar to that of the typical CS patients. Thus, the relative hypersensitivity of CS patients cells in vitro does not reflect the severity or age of onset of the patients clinical manifestations. The lymphoblastoid lines from the 2 patients who had both CS and XP were significantly more sensitive to the UV radiation than those from patients with only CS. Our studies demonstrate that lymphoblastoid lines from patients with CS are appropriate and useful cell lines for the study of the inherited hypersensitivity to UV radiation.

  12. Utilization of Lymphoblastoid Cell Lines as a System for the Molecular Modeling of Autism

    ERIC Educational Resources Information Center

    Baron, Colin A.; Liu, Stephenie Y.; Hicks, Chindo; Gregg, Jeffrey P.

    2006-01-01

    In order to provide an alternative approach for understanding the biology and genetics of autism, we performed statistical analysis of gene expression profiles of lymphoblastoid cell lines derived from children with autism and their families. The goal was to assess the feasibility of using this model in identifying autism-associated genes.…

  13. Impairment of natural killer functions by interleukin 6 increases lymphoblastoid cell tumorigenicity in athymic mice.

    PubMed Central

    Tanner, J; Tosato, G

    1991-01-01

    Expression of the human IL-6 gene in EBV-immortalized normal human B lymphocytes following retroviral-mediated transduction rendered these cells highly tumorigenic in athymic mice. The tumors were lymphomas composed of the originally inoculated human lymphoblastoid cells. Co-injection of IL-6 expressing EBV-immortalized cells with IL-6 nonexpressing control cells resulted in increased tumorigenicity of the IL-6 nonexpressing cells. The lymphoblastoid cells expressing IL-6 were indistinguishable from parental cell lines in morphology and in a variety of cell surface characteristics, and did not exhibit growth advantage over parental cell lines in vitro, such that increased tumorigenicity is unlikely to depend upon a direct oncogenic effect of IL-6 on the B cells. Rather, at high concentrations, IL-6 markedly inhibits human lymphoblastoid cell killing by IL-2-activated murine splenocytes in vitro, suggesting that IL-6-related tumorigenicity might depend upon IL-6 inhibiting cytotoxicity at the tumor site. Thus, production of IL-6 by tumor cells that results in natural killer cell dysfunctions illustrates a novel mechanism of tumor cell escape from immune surveillance. Images PMID:1647416

  14. [Functional activity of lymphoblastoid cells infected by human adenovirus type 2 and Epstein-Barr virus].

    PubMed

    Povnitsa, O Iu; Diachenko, N S; Nosach, L N; Olevinskaia, Z M; Zhovnovataia, V L; Polishchuk, V N; Spivak, N Ia

    2005-01-01

    The paper deals with the influence of the adenovirus (Ad) and Epstein-Barr virus (EBV) on functional activity of lymphocytes, in particular, the production of alpha- and gamma-interferons, tumor necrosis factor (TNF) in conditions of mono- or double infection of B- and T-phenotype (CEM) lymphoblastoid cells. It is shown, that Ad, EBV or both viruses induce high enough levels of interferon on both lines of cells and in control epithelial cells. The lymphoblastoid cells infected by viruses deep ability to synthesize alpha- and gamma-interferons under the influence of the corresponding inducers (Newcastle disease virus and hemagglutinine). Nevertheless, the levels of their formation are not high. Rather high parameters of activity of the tumor necrosis factor (TNF) were revealed during a day in the initial B95-8 cells and superinfected Ad after the effect of LPS of E. coli. Their activity in CEM cells also did not depend on the infection type. PMID:16018208

  15. Investigation of the apoptotic way induced by digallic acid in human lymphoblastoid TK6 cells

    PubMed Central

    2012-01-01

    Background The digallic acid (DGA) purified from Pistacia lentiscus. L fruits was investigated for its antiproliferative and apoptotic activities on human lymphoblastoid TK6 cells. Methods We attempt to characterize the apoptotic pathway activated by DGA. Apoptosis was detected by DNA fragmentation, PARP cleavage and by evaluating caspase activities. Results The inhibition of lymphoblastoid cell proliferation was noted from 8.5 μg/ml of DGA. The induction of apoptosis was confirmed by DNA fragmentation and PARP cleavage. We have demonstrated that DGA induces apoptosis by activating the caspase-8 extrinsic pathway. Caspase-3 was also activated in a dose dependent manner. Conclusion In summary, DGA exhibited an apoptosis inductor effect in TK6 cells revealing thus its potential as a cancer-preventive agent. PMID:22686580

  16. Lymphoblastoid Cell Lines as a Tool to Study Inter-Individual Differences in the Response to Glucose

    PubMed Central

    Grassi, Michael A.; Rao, Vidhya R.; Chen, Siquan; Cao, Dingcai; Gao, Xiaoyu; Cleary, Patricia A.; Huang, R. Stephanie; Paterson, Andrew D.; Natarajan, Rama; Rehman, Jalees; Kern, Timothy S.

    2016-01-01

    Background White blood cells have been shown in animal studies to play a central role in the pathogenesis of diabetic retinopathy. Lymphoblastoid cells are immortalized EBV-transformed primary B-cell leukocytes that have been extensively used as a model for conditions in which white blood cells play a primary role. The purpose of this study was to investigate whether lymphoblastoid cell lines, by retaining many of the key features of primary leukocytes, can be induced with glucose to demonstrate relevant biological responses to those found in diabetic retinopathy. Methods Lymphoblastoid cell lines were obtained from twenty-three human subjects. Differences between high and standard glucose conditions were assessed for expression, endothelial adhesion, and reactive oxygen species. Results Collectively, stimulation of the lymphoblastoid cell lines with high glucose demonstrated corresponding changes on molecular, cellular and functional levels. Lymphoblastoid cell lines up-regulated expression of a panel of genes associated with the leukocyte-mediated inflammation found in diabetic retinopathy that include: a cytokine (IL-1B fold change = 2.11, p-value = 0.02), an enzyme (PKCB fold change = 2.30, p-value = 0.01), transcription factors (NFKB-p50 fold change = 2.05, p-value = 0.01), (NFKB-p65 fold change = 2.82, p-value = 0.003), and an adhesion molecule (CD18 fold change = 2.59, 0.02). Protein expression of CD18 was also increased (p-value = 2.14x10-5). The lymphoblastoid cell lines demonstrated increased adhesiveness to endothelial cells (p = 1.28x10-5). Reactive oxygen species were increased (p = 2.56x10-6). Significant inter-individual variation among the lymphoblastoid cell lines in these responses was evident (F = 18.70, p < 0.0001). Conclusions Exposure of lymphoblastoid cell lines derived from different human subjects to high glucose demonstrated differential and heterogeneous gene expression, adhesion, and cellular effects that recapitulated features found in

  17. Autophagy is the predominant process induced by arsenite in human lymphoblastoid cell lines

    SciTech Connect

    Bolt, Alicia M.; Byrd, Randi M.; Klimecki, Walter T.

    2010-05-01

    Arsenic is a widespread environmental toxicant with a diverse array of molecular targets and associated diseases, making the identification of the critical mechanisms and pathways of arsenic-induced cytotoxicity a challenge. In a variety of experimental models, over a range of arsenic exposure levels, apoptosis is a commonly identified arsenic-induced cytotoxic pathway. Human lymphoblastoid cell lines (LCL) have been used as a model system in arsenic toxicology for many years, but the exact mechanism of arsenic-induced cytotoxicity in LCL is still unknown. We investigated the cytotoxicity of sodium arsenite in LCL 18564 using a set of complementary markers for cell death pathways. Markers indicative of apoptosis (phosphatidylserine externalization, PARP cleavage, and sensitivity to caspase inhibition) were uniformly negative in arsenite exposed cells. Interestingly, electron microscopy, acidic vesicle fluorescence, and expression of LC3 in LCL 18564 identified autophagy as an arsenite-induced process that was associated with cytotoxicity. Autophagy, a cellular programmed response that is associated with both cellular stress adaptation as well as cell death appears to be the predominant process in LCL cytotoxicity induced by arsenite. It is unclear, however, whether LCL autophagy is an effector mechanism of arsenite cytotoxicity or alternatively a cellular compensatory mechanism. The ability of arsenite to induce autophagy in lymphoblastoid cell lines introduces a potentially novel mechanistic explanation of the well-characterized in vitro and in vivo toxicity of arsenic to lymphoid cells.

  18. Cytotoxic effect of anti-idiotype antibody-chlorambucil conjugates against human lymphoblastoid cells.

    PubMed

    Tung, E; Goust, J M; Chen, W Y; Kang, S S; Wang, I Y; Wang, A C

    1983-09-01

    The secreted IgMs of two human lymphoblastoid cell lines, RPMI-6410 and RPMI-8392, were purified. Antisera against these two IgMs were raised in rabbits and made idiotypically specific to the respective antigens through various absorption procedures. By immunofluorescence and radioimmunoassay techniques, the purified anti-idiotype antibodies were found to react also with the membrane Igs of the respective cell lines, but not with those of other cell lines. The purified anti-idiotype antibodies were then coupled with Chlorambucil to form antibody-drug conjugates, whose effectiveness in the in-vitro killing of target cells was evaluated by a chromium-release cytotoxicity assay. The results showed that these anti-idiotype antibody-Chlorambucil conjugates were specifically cytotoxic to lymphoblastoid cells that bore membrane Igs carrying the respective idiotypic determinant(s). Furthermore, the conjugates were far more effective in causing cytolysis to the target cells than either Chlorambucil or the anti-idiotype antibodies alone. PMID:6350169

  19. Cytotoxic effect of anti-idiotype antibody-chlorambucil conjugates against human lymphoblastoid cells.

    PubMed Central

    Tung, E; Goust, J M; Chen, W Y; Kang, S S; Wang, I Y; Wang, A C

    1983-01-01

    The secreted IgMs of two human lymphoblastoid cell lines, RPMI-6410 and RPMI-8392, were purified. Antisera against these two IgMs were raised in rabbits and made idiotypically specific to the respective antigens through various absorption procedures. By immunofluorescence and radioimmunoassay techniques, the purified anti-idiotype antibodies were found to react also with the membrane Igs of the respective cell lines, but not with those of other cell lines. The purified anti-idiotype antibodies were then coupled with Chlorambucil to form antibody-drug conjugates, whose effectiveness in the in-vitro killing of target cells was evaluated by a chromium-release cytotoxicity assay. The results showed that these anti-idiotype antibody-Chlorambucil conjugates were specifically cytotoxic to lymphoblastoid cells that bore membrane Igs carrying the respective idiotypic determinant(s). Furthermore, the conjugates were far more effective in causing cytolysis to the target cells than either Chlorambucil or the anti-idiotype antibodies alone. PMID:6350169

  20. Induction of apoptosis by epigallocatechin-3-gallate in human lymphoblastoid B cells

    SciTech Connect

    Noda, Chiseko He, Jinsong; Takano, Tomoko; Tanaka, Chisato; Kondo, Toshinori; Tohyama, Kaoru; Yamamura, Hirohei; Tohyama, Yumi

    2007-11-03

    (-)-Epigallocatechin-3-gallate (EGCG), a major constituent of green tea polyphenols, has been shown to suppress cancer cell proliferation and induce apoptosis. In this study we investigated its efficacy and the mechanism underlying its effect using human B lymphoblastoid cell line Ramos, and effect of co-treatment with EGCG and a chemotherapeutic agent on apoptotic cell death. EGCG induced dose- and time-dependent apoptotic cell death accompanied by loss of mitochondrial transmembrane potential, release of cytochrome c into the cytosol, and cleavage of pro-caspase-9 to its active form. EGCG also enhanced production of intracellular reactive oxygen species (ROS). Pretreatment with diphenylene iodonium chloride, an inhibitor of NAD(P)H oxidase and an antioxidant, partially suppressed both EGCG-induced apoptosis and production of ROS, implying that oxidative stress is involved in the apoptotic response. Furthermore, we showed that combined-treatment with EGCG and a chemotherapeutic agent, etoposide, synergistically induced apoptosis in Ramos cells.

  1. Selective production of interferon-alpha subtypes by cultured peripheral blood mononuclear cells and lymphoblastoid cell lines.

    PubMed Central

    Greenway, A L; Overall, M L; Sattayasai, N; Rowley, M J; Hertzog, P J; McMullen, G L; Cheetham, B F; Marzuki, S

    1992-01-01

    The biological significance of the existence of multiple interferon-alpha (IFN-alpha) subtypes is unknown but may represent a finely tuned mechanism whereby different subtypes are produced in response to different stimuli. To investigate the expression of individual IFN-alpha subtypes, polyclonal antipeptide antisera designed to react with all IFN-alpha subtypes, or with a particular subtype, IFN-alpha 2 or IFN-alpha 4, have been produced. In this study we demonstrate the utility of these antisera for the detection, using indirect immunofluorescence staining, of intracellular IFN-alpha produced by human peripheral blood mononuclear cells (PBMC) and lymphoblastoid cells. Secreted IFN-alpha was also investigated by bioassay and a sandwich radioimmunoassay (RIA), using two monoclonal antibodies (mAb) and specific for IFN-alpha 4. The PBMC were shown to produce IFN reactive with all three polyclonal antisera, after stimulation with Sendai virus. The lymphoblastoid cells also produced IFN, including IFN-alpha 2, but IFN-alpha 4 was not detected either intracellularly, by immunofluorescence, or in the medium, by sandwich RIA. The immunofluorescence studies also demonstrate that in the absence of viral stimulation IFN-alpha is found in the cytoplasm of PBMC and lymphoblastoid cells but not secreted in detectable levels. The finding that two lymphoblastoid cell lines do not produce the subtype IFN-alpha 4 raises important questions as to whether other cell lines and cell types produce IFN-alpha subtypes selectively, and whether individual IFN-alpha subtypes have different roles in human physiology and pathology. Images Figure 1 Figure 2 Figure 3 PMID:1537595

  2. The influence of various bovine sera on the maintenance of Theileria parva lymphoblastoid cell culture.

    PubMed

    Siddig, H A; Franssen, F F; Spanjer, A A; Jongejan, F; Uilenberg, G

    1982-01-01

    Theileria parva infected lymphoblastoid bovine cells were grown in a medium based on HEPES-buffered RPMI 1640 with glutamine and antibiotics, supplemented with bovine serum. There were no significant differences in growth rate, viability, and percentage of infected cells when the substrate contained 10 or 20 per cent of either commercially available newborn calf serum of serum prepared from adult non-infected Friesian cattle or of serum prepared from a Friesian calf immunised against East Coast fever and having a high titre of antibodies to T. parva antigen in the indirect fluorescent antibody test. If studies showing that newborn calf serum gives results in the establishment and maintenance of T. parva cell culture similar to those of foetal calf serum are confirmed, this finding could mean an appreciable saving in the cost of in vitro work on this parasite. PMID:6815877

  3. Fast neutrons-induced apoptosis is Fas-independent in lymphoblastoid cells

    SciTech Connect

    Fischer, Barbara; Benzina, Sami; Jeannequin, Pierre; Dufour, Patrick; Bergerat, Jean-Pierre; Denis, Jean-Marc; Gueulette, John; Bischoff, Pierre L. . E-mail: Pierre.Bischoff@ircad.u-strasbg.fr

    2005-08-26

    We have previously shown that ionizing radiation-induced apoptosis in human lymphoblastoid cells differs according to their p53 status, and that caspase 8-mediated cleavage of BID is involved in the p53-dependent pathway. In the present study, we investigated the role of Fas signaling in caspase 8 activation induced by fast neutrons irradiation in these cells. Fas and FasL expression was assessed by flow cytometry and by immunoblot. We also measured Fas aggregation after irradiation by fluorescence microscopy. We found a decrease of Fas expression after irradiation, but no change in Fas ligand expression. We also showed that, in contrast to the stimulation of Fas by an agonistic antibody, Fas aggregation did not occur after irradiation. Altogether, our data strongly suggest that fast neutrons induced-apoptosis is Fas-independent, even in p53-dependent apoptosis.

  4. Sorting of chromosome 13 from lymphoblastoid cell lines derived from patients with Wilson disease

    SciTech Connect

    Nasedkina, T.V.; Polesskaya, A.N.; Surkov, S.A.; Poletaev, A.I. ); Aksenov, N.; Zenin, V.V. )

    1993-01-01

    Lymphoblastoid cell lines were established from patients with Wilson disease (WD) which maps to human chromosome 13 and served as a source of chromosomes. The authors used a modified isolation procedure to increase the yield of metaphase chromosomes and additional purification of the chromosome suspension on Percoll gradient to achieve more stable sorting conditions. Vibariate flow analysis using dual laser cell-sorter, ATC-3000, showed a sufficient resolution of the flow karyotype and a low level of debris. They sorted chromosome 13 at a speed of up to 5,000 chr/sec, providing about 2 million chromosomes per day. The purity of the sorted fraction was about 90%. The fractions will be further used to construct cosmid libraries to facilitate studies of the WD locus.

  5. The TP53 dependence of radiation-induced chromosome instability in human lymphoblastoid cells

    NASA Technical Reports Server (NTRS)

    Schwartz, Jeffrey L.; Jordan, Robert; Evans, Helen H.; Lenarczyk, Marek; Liber, Howard

    2003-01-01

    The dose and TP53 dependence for the induction of chromosome instability were examined in cells of three human lymphoblastoid cell lines derived from WIL2 cells: TK6, a TP53-normal cell line, NH32, a TP53-knockout created from TK6, and WTK1, a WIL2-derived cell line that spontaneously developed a TP53 mutation. Cells of each cell line were exposed to (137)Cs gamma rays, and then surviving clones were isolated and expanded in culture for approximately 35 generations before the frequency and characteristics of the instability were analyzed. The presence of dicentric chromosomes, formed by end-to-end fusions, served as a marker of chromosomal instability. Unexposed TK6 cells had low levels of chromosomal instability (0.002 +/- 0.001 dicentrics/cell). Exposure of TK6 cells to doses as low as 5 cGy gamma rays increased chromosome instability levels nearly 10-fold to 0.019 +/- 0.008 dicentrics/cell. There was no further increase in instability levels beyond 5 cGy. In contrast to TK6 cells, unexposed cultures of WTK1 and NH32 cells had much higher levels of chromosome instability of 0.034 +/- 0.007 and 0.041 +/- 0.009, respectively, but showed little if any effect of radiation on levels of chromosome instability. The results suggest that radiation exposure alters the normal TP53-dependent cell cycle checkpoint controls that recognize alterations in telomere structure and activate apoptosis.

  6. Phorbol diesters and transferrin modulate lymphoblastoid cell transferrin receptor expression by two different mechanisms

    SciTech Connect

    Alcantara, O.; Phillips, J.L.; Boldt, D.H.

    1986-12-01

    Expression of transferrin receptors (TfR) by activated lymphocytes is necessary for lymphocyte DNA synthesis and proliferation. Regulation of TfR expression, therefore, is a mechanism by which the lymphocyte's proliferative potential may be directed and controlled. The authors studied mechanisms by which lymphoblastoid cells modulate TfR expression during treatment with phorbol diesters or iron transferrin (FeTf), agents which cause downregulation of cell surface TfR. Phorbol diester-induced TfR downregulation occurred rapidly, being detectable at 2 min and reaching maximal decreases of 50% by 15 min. It was inhibited by cold but not by agents that destabilize cytoskeletal elements. Furthermore, this downregulation was reversed rapidly by washing or by treatment with the membrane interactive agent, chlorpromazine. In contrast, FeTf-induced TfR downregulation occurred slowly. Decreased expression of TfR was detectable only after 15 min and maximal downregulation was achieved after 60 min. Although FeTf-induced downregulation also was inhibited by cold, it was inhibited in addition by a group of microtubule destabilizing agents (colchicine, vinblastine, podophyllotoxin) or cytochalasin B, a microfilament inhibitor. Furthermore, FeTf-induced downregulation was not reversed readily by washing or by treatment with chlorpromazine. Phorbol diesters cause TfR downregulation by a cytoskeleton-independent mechanism. These data indicate that TfR expression is regulated by two independent mechanisms in lymphoblastoid cells, and they provide the possibility that downregulation of TfR by different mechanisms may result in different effects in these cells.

  7. Modulation of tubulin mRNA levels by interferon in human lymphoblastoid cells.

    PubMed Central

    Fellous, A; Ginzburg, I; Littauer, U Z

    1982-01-01

    Blot hybridization with labeled tubulin cDNA showed that treatment of Ramos cells, a human cell line of lymphoblastoid origin, with either alpha or beta interferon (IFN) induced a marked increase in the amount of tubulin mRNA sequences. The level of tubulin mRNA sequences increased rapidly after exposure of cells to IFN-alpha and reached a maximum after 1 h of treatment, which was four times the control level. Treatment with IFN-beta induced a maximal increase after 4 h; the amount of tubulin mRNA sequences was seven times higher than the control level. The mRNA extracted from IFN-treated and nontreated cells was translated in vitro in a reticulocyte lysate cell-free system containing [35S]methionine. Electrophoretic analysis of the labeled cell-free products showed an increase in the amount of translatable tubulin mRNA that parallels the time course of induction of tubulin mRNA sequences. Two-dimensional gel electrophoresis of the labeled protein products directed by mRNA indicates that IFN caused a more pronounced increase in the level of alpha-tubulin than beta-tubulin mRNA. Treatment with colchicine, which disrupts the cell microtubules, caused a marked decrease in the tubulin mRNA content. Concomitant treatment of the cells with colchicine and IFN abolished the interferon-dependent induction of tubulin mRNA. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:6964957

  8. Different Mechanisms of Regulation of the Warburg Effect in Lymphoblastoid and Burkitt Lymphoma Cells

    PubMed Central

    Mushtaq, Muhammad; Darekar, Suhas

    2015-01-01

    Background The Warburg effect is one of the hallmarks of cancer and rapidly proliferating cells. It is known that the hypoxia-inducible factor 1-alpha (HIF1A) and MYC proteins cooperatively regulate expression of the HK2 and PDK1 genes, respectively, in the Burkitt lymphoma (BL) cell line P493-6, carrying an inducible MYC gene repression system. However, the mechanism of aerobic glycolysis in BL cells has not yet been fully understood. Methods and Findings Western blot analysis showed that the HIF1A protein was highly expressed in Epstein–Barr virus (EBV)-positive BL cell lines. Using biochemical assays and quantitative PCR (Q-PCR), we found that—unlike in lymphoblastoid cell lines (LCLs)—the MYC protein was the master regulator of the Warburg effect in these BL cell lines. Inhibition of the transactivation ability of MYC had no influence on aerobic glycolysis in LCLs, but it led to decreased expression of MYC-dependent genes and lactate dehydrogenase A (LDHA) activity in BL cells. Conclusions Our data suggest that aerobic glycolysis, or the Warburg effect, in BL cells is regulated by MYC expressed at high levels, whereas in LCLs, HIF1A is responsible for this phenomenon. PMID:26312753

  9. Cytogenetic characterization of low-dose hyper-radiosensitivity in Cobalt-60 irradiated human lymphoblastoid cells.

    PubMed

    Joshi, Gnanada S; Joiner, Michael C; Tucker, James D

    2014-12-01

    The dose-effect relationships of cells exposed to ionizing radiation are frequently described by linear quadratic (LQ) models over an extended dose range. However, many mammalian cell lines, when acutely irradiated in G2 at doses ≤0.3Gy, show hyper-radiosensitivity (HRS) as measured by reduced clonogenic cell survival, thereby indicating greater cell lethality than is predicted by extrapolation from high-dose responses. We therefore hypothesized that the cytogenetic response in G2 cells to low doses would also be steeper than predicted by LQ extrapolation from high doses. We tested our hypothesis by exposing four normal human lymphoblastoid cell lines to 0-400cGy of Cobalt-60 gamma radiation. The cytokinesis block micronucleus assay was used to determine the frequencies of micronuclei and nucleoplasmic bridges. To characterize the dependence of the cytogenetic damage on dose, univariate and multivariate regression analyses were used to compare the responses in the low- (HRS) and high-dose response regions. Our data indicate that the slope of the response for all four cell lines at ≤20cGy during G2 is greater than predicted by an LQ extrapolation from the high-dose responses for both micronuclei and bridges. These results suggest that the biological consequences of low-dose exposures could be underestimated and may not provide accurate risk assessments following such exposures. PMID:25771872

  10. Effects of Simulated Microgravity on the Expression Profile of Microrna in Human Lymphoblastoid Cells

    NASA Astrophysics Data System (ADS)

    Zhang, Ye; Wu, Honglu; Ramesh, Govindarajan; Rohde, Larry; Story, Michael; Mangala, Lingegowda

    2012-07-01

    EFFECTS OF SIMULATED MICROGRAVITY ON THE EXPRESSION PROFILE OF MICRORNA IN HUMAN LYMPHOBLASTOID CELLS Lingegowda S. Mangala1,2, Ye Zhang1,3, Zhenhua He2, Kamal Emami1, Govindarajan T. Ramesh4, Michael Story 5, Larry H. Rohde2, and Honglu Wu1 1 NASA Johnson Space Center, Houston, Texas, USA 2 University of Houston Clear Lake, Houston, Texas, USA 3 Wyle Integrated Science and Engineering Group, Houston, Texas, USA 4 Norfolk State University, Norfolk, VA, USA 5 University of Texas, Southwestern Medical Center, Dallas, Texas, USA This study explores the changes in expression of microRNA (miRNA) and related genes under simulated microgravity conditions. In comparison to static 1g, microgravity has been shown to alter global gene expression patterns and protein levels in cultured cells or animals. miRNA has recently emerged as an important regulator of gene expression, possibly regulating as many as one-third of all human genes. However, very little is known about the effect of altered gravity on miRNA expression. To test the hypothesis that the miRNA expression profile would be altered in zero gravity resulting in altered regulation of gene expression leading to metabolic or functional changes in cells, we cultured TK6 human lymphoblastoid cells in a High Aspect Ratio Vessel (HARV; bioreactor) for 72 h either in the rotating condition to model microgravity in space or in the static condition as a control. Expression of several miRNA was changed significantly in the simulated microgravity condition including miR-150, miR-34a, miR-423-5p, miR-22 and miR-141, miR-618 and miR-222. To confirm whether this altered miRNA expression correlates with gene expression and functional changes of the cells, we performed DNA microarray and validated the related genes using q-RT PCR. Network and pathway analysis of gene and miRNA expression profiles indicates that the regulation of cell communication and catalytic activities, as well as pathways involved in immune response_IL-15

  11. The impact of FANCD2 deficiency on formaldehyde-induced toxicity in human lymphoblastoid cell lines

    PubMed Central

    Ren, Xuefeng; Ji, Zhiying; McHale, Cliona M.; Yuh, Jessica; Bersonda, Jessica; Tang, Maycky; Smith, Martyn T.; Zhang, Luoping

    2015-01-01

    Formaldehyde (FA), a major industrial chemical and ubiquitous environmental pollutant, has recently been classified by the International Agency for Research on Cancer as a human leukemogen. The major mode of action of FA is thought to be the formation of DNA-protein crosslinks (DPCs). Repair of DPCs may be mediated by the Fanconi anemia pathway; however, data supporting the involvement of this pathway is limited, particularly in human hematopoietic cells. Therefore, we assessed the role of FANCD2, a critical component of the Fanconi anemia pathway, in FA-induced toxicity in human lymphoblast cell models of FANCD2-deficiency (PD20 cells) and FANCD2-sufficiency (PD20-D2 cells). After treatment of the cells with 0-150 μM FA for 24 hours, DPCs were increased in a dose-dependent manner in both cell lines, with greater increases in FANCD2-deficient PD20 cells. FA also induced cytotoxicity, micronuclei, chromosome aberrations, and apoptosis in a dose-dependent manner in both cell lines, with greater increases in cytotoxicity and apoptosis in PD20 cells. Increased levels of γ-ATR and γ-H2AX in both cell lines suggested the recognition of FA-induced DNA damage; however, the induction of BRCA2 was compromised in FANCD2-deficient PD20 cells, potentially reducing the capacity to repair DPCs. Together, these findings suggest that FANCD2 protein and the Fanconi anemia pathway are essential to protect human lymphoblastoid cells against FA toxicity. Future studies are needed to delineate the role of this pathway in mitigating FA-induced toxicity, particularly in hematopoietic stem cells, the target cells in leukemia. PMID:22872141

  12. Proliferation-dependent positioning of individual centromeres in the interphase nucleus of human lymphoblastoid cell lines

    PubMed Central

    Ollion, Jean; Loll, François; Cochennec, Julien; Boudier, Thomas; Escudé, Christophe

    2015-01-01

    The cell nucleus is a highly organized structure and plays an important role in gene regulation. Understanding the mechanisms that sustain this organization is therefore essential for understanding genome function. Centromeric regions (CRs) of chromosomes have been known for years to adopt specific nuclear positioning patterns, but the significance of this observation is not yet completely understood. Here, using a combination of fluorescence in situ hybridization and immunochemistry on fixed human cells and high-throughput imaging, we directly and quantitatively investigated the nuclear positioning of specific human CRs. We observe differential attraction of individual CRs toward both the nuclear border and the nucleoli, the former being enhanced in nonproliferating cells and the latter being enhanced in proliferating cells. Similar positioning patterns are observed in two different lymphoblastoid cell lines. Moreover, the positioning of CRs differs from that of noncentromeric regions, and CRs display specific orientations within chromosome territories. These results suggest the existence of not-yet-characterized mechanisms that drive the nuclear positioning of CRs and therefore pave the way toward a better understanding of how CRs affect nuclear organization. PMID:25947134

  13. Expression of genes and proteins in human cultured lymphoblastoid cells during spaceflight

    NASA Astrophysics Data System (ADS)

    Takahashi, Akihisa; Suzuki, Hiromi; Omori, Katsunori; Seki, Masaya; Hashizume, Toko; Shimazu, Toru; Ishioka, Noriaki; Ohnishi, Takeo

    2012-07-01

    The space environment contains two major biologically significant influences: space radiations and microgravity. The aim of this study was to clarify the biological effects of space radiations, microgravity and a space environment on the gene and protein expression. Space experiments were performed with human cultured lymphoblastoid cell lines at the first life science experiment to be conducted on the Japanese Experimental Module "Kibo" of the International Space Station (ISS). Under one gravity or microgravity condition, the cells were grown in the cell biology experimental facility (CBEF) of the ISS for 8 days without experiencing the stress during launching and landing because the cells were frozen during these periods. Ground control samples also were cultured for 8 days in the CBEF on the ground during the spaceflight. Gene and protein expression was analyzed by using DNA chip (a 44k whole human genome microarray, Agilent Technologies Inc.) and protein chip (Panorama ^{TM} Ab MicroArray, Sigma-Aldrich Co.), respectively. We already reported the behavior of p53-dependent regulated genes and proteins after exposure to space radiations, microgravity, and the space environment during spaceflight. Next stage, we will profile the expression except for the p53 gene status and discuss the biological meaning during spaceflight

  14. Humic acids reduce the genotoxicity of mitomycin C in the human lymphoblastoid cell line TK6.

    PubMed

    Ferrara, G; Loffredo, E; Senesi, N; Marcos, R

    2006-01-31

    The antimutagenic/desmutagenic activity of a leonardite humic acid (LHA) and a soil humic acid (SHA) was studied in the cultured human lymphoblastoid cell line TK6 treated with mitomycin C (MMC) as reference mutagen by evaluating the induction of micronuclei (MN). Two different concentrations of HA were used, 2.5 and 10 microg/ml, in three different treatments: (1) HA alone (genotoxic test); (2) HA after 2-h pre-incubation with 0.3 microM of MMC (desmutagenic test) and (3) combinations of HA and MMC at 0.3 microM without pre-incubation (antimutagenic test). Neither of the HA used alone did produce genotoxic effects, but both HAs reduced significantly the frequencies of MN induced by MMC, especially in the desmutagenic test. A slight cell-protective effect against the cytotoxicity of MMC was also exhibited by the two HAs in the desmutagenic test. The LHA showed a desmutagenic/antimutagenic activity that was more pronounced than that of SHA, which is possibly related to the higher carboxylic group content and lower phenolic group content of LHA. These results confirm the antigenotoxic action exerted by HAs in human cells, similarly to what has been previously observed in various plant species. PMID:16386451

  15. Estimation of Mutation Rates Based on the Analysis of Polypeptide Constituents of Cultured Human Lymphoblastoid Cells

    PubMed Central

    Chu, EHY.; Boehnke, M.; Hanash, S. M.; Kuick, R. D.; Lamb, B. J.; Neel, J. V.; Niezgoda, W.; Pivirotto, S.; Sundling, G.

    1988-01-01

    A subclone of a human diploid lymphoblastoid cell line, TK-6, with consistently high cloning efficiency has been used to estimate the rates of somatic mutations on the basis of protein variation detected by two-dimensional polyacrylamide gel electrophoresis. A panel of 267 polypeptide spots per gel was screened, representing the products of approximately 263 unselected loci. The rate of human somatic mutation in vitro was estimated by measuring the proportion of protein variants among cell clones isolated at various times during continuous exponential growth of a TK-6 cell population. Three mutants of spontaneous origin were observed, giving an estimated spontaneous rate of 6 X 10(-8) electrophoretic mutations per allele per cell generation (i.e., 1.2 X 10(-7) per locus per cell generation). Following treatment of cells with N-ethyl-N-nitrosourea, a total of 74 confirmed variants at 54 loci were identified among 1143 clones analyzed (approximately 601,000 allele tests). The induced variants include 65 electromorphs which exhibit altered isoelectric charge and/or apparent molecular weight and nine nullimorphs for each of which a gene product was not detected at its usual location on the gel. The induced frequency for these 65 structural gene mutants is 1.1 X 10(-4) per allele. An excess of structural gene mutations at ten known polymorphic loci and repeat mutations at these and other loci suggest nonrandomness of mutation in human somatic cells. Nullimorphs occurring at three heterozygous loci in TK-6 cells may be caused by genetic processes other than structural gene mutation. PMID:3402732

  16. No defect in G1/S cell cycle arrest in irradiated Li-Fraumeni lymphoblastoid cell lines.

    PubMed Central

    Williams, K. J.; Heighway, J.; Birch, J. M.; Norton, J. D.; Scott, D.

    1996-01-01

    The radiation response of Epstein-Barr virus (EBV)-immortalised lymphoblastoid cell lines derive from Li-Fraumeni syndrome (LFS) and LFS-like individuals was investigated. Cells from all LFS and LFS-like cases showed an accumulation of p53 protein following 137Cs gamma-irradiation, which was associated with cell cycle arrest at the G1/S border. This response was indistinguishable from that seen in cells derived from normal individuals, and occurred in cases with missense mutations in the TP53 gene at codons 175, 180, 220 and 248 and also in two LFS-like individuals with no TP53 mutation. Previous studies using lymphocytes and fibroblasts from LFS individuals have demonstrated abnormal radiation responses in these cells. This suggest cell type specificity in the contribution of a mutant p53 protein to phenotype. Images Figure 1 Figure 4 PMID:8795570

  17. Increased Susceptibility to Ethylmercury-Induced Mitochondrial Dysfunction in a Subset of Autism Lymphoblastoid Cell Lines

    PubMed Central

    Wynne, Rebecca; Frye, Richard E.; Melnyk, Stepan; James, S. Jill

    2015-01-01

    The association of autism spectrum disorders with oxidative stress, redox imbalance, and mitochondrial dysfunction has become increasingly recognized. In this study, extracellular flux analysis was used to compare mitochondrial respiration in lymphoblastoid cell lines (LCLs) from individuals with autism and unaffected controls exposed to ethylmercury, an environmental toxin known to deplete glutathione and induce oxidative stress and mitochondrial dysfunction. We also tested whether pretreating the autism LCLs with N-acetyl cysteine (NAC) to increase glutathione concentrations conferred protection from ethylmercury. Examination of 16 autism/control LCL pairs revealed that a subgroup (31%) of autism LCLs exhibited a greater reduction in ATP-linked respiration, maximal respiratory capacity, and reserve capacity when exposed to ethylmercury, compared to control LCLs. These respiratory parameters were significantly elevated at baseline in the ethylmercury-sensitive autism subgroup as compared to control LCLs. NAC pretreatment of the sensitive subgroup reduced (normalized) baseline respiratory parameters and blunted the exaggerated ethylmercury-induced reserve capacity depletion. These findings suggest that the epidemiological link between environmental mercury exposure and an increased risk of developing autism may be mediated through mitochondrial dysfunction and support the notion that a subset of individuals with autism may be vulnerable to environmental influences with detrimental effects on development through mitochondrial dysfunction. PMID:25688267

  18. Genotoxicity of microcystin-LR in human lymphoblastoid TK6 cells.

    PubMed

    Zhan, Li; Sakamoto, Hiroko; Sakuraba, Mayumi; Wu, De Sheng; Zhang, Li Shi; Suzuki, Takayoshi; Hayashi, Makoto; Honma, Masamitsu

    2004-01-10

    Toxic cyanobacteria (blue-green algae) water blooms have become a serious problem in several industrialized areas of the world. Microcystin-LR (MCLR) is a cyclic heptapeptidic toxin produced by the cyanobacteria. In the present study, we used human lymphoblastoid cell line TK6 to investigate the in vitro genotoxicity of MCLR. In a standard 4h treatment, MCLR did not induce a significant cytotoxic response at <80 microg/ml. In a prolonged 24h treatment, in contrast, it induced cytotoxic as well as mutagenic responses concentration-dependently starting at 20 microg/ml. At the maximum concentration (80 microg/ml), the micronucleus frequency and the mutation frequency at the heterozygous thymidine kinase (TK) locus were approximately five-times the control values. Molecular analysis of the TK mutants revealed that MCLR specifically induced loss of heterozygosity at the TK locus, but not point mutations or other small structural changes. These results indicate that MCLR had a clastogenic effect. We discuss the mechanisms of MCLR genotoxicity and the possibility of its being a hepatocarcinogen. PMID:14706513

  19. Transcriptomic variation of pharmacogenes in multiple human tissues and lymphoblastoid cell lines

    PubMed Central

    Chhibber, Aparna; French, Courtney E.; Yee, Sook Wah; Gamazon, Eric R.; Theusch, Elizabeth; Qin, Xiang; Webb, Amy; Papp, Audrey C.; Wang, Ann; Simmons, Christine Q.; Konkashbaev, Anuar; Chaudhry, Amarjit S.; Mitchel, Katrina; Stryke, Doug; Ferrin, Thomas E.; Weiss, Scott T.; Kroetz, Deanna L.; Sadee, Wolfgang; Nickerson, Deborah A.; Krauss, Ronald M.; George, Alfred L.; Schuetz, Erin G.; Medina, Marisa W.; Cox, Nancy J.; Scherer, Steven E.; Giacomini, Kathleen M.; Brenner, Steven E

    2015-01-01

    Variation in the expression level and activity of genes involved in drug disposition and action (“pharmacogenes”) can affect drug response and toxicity, especially when in tissues of pharmacological importance. Previous studies have relied primarily on microarrays to understand gene expression differences, or have focused on a single tissue or small number of samples. The goal of this study was to use RNA-seq to determine the expression levels and alternative splicing of 389 PGRN pharmacogenes across four tissues (liver, kidney, heart and adipose) and lymphoblastoid cell lines (LCLs), which are used widely in pharmacogenomics studies. Analysis of RNA-seq data from 139 different individuals across the 5 tissues (20–45 individuals per tissue type) revealed substantial variation in both expression levels and splicing across samples and tissue types. This in-depth exploration also revealed 183 splicing events in pharmacogenes that were previously not annotated. Overall, this study serves as a rich resource for the research community to inform biomarker and drug discovery and use. PMID:26856248

  20. Effects of simulated microgravity on expression profile of microRNA in human lymphoblastoid cells.

    PubMed

    Mangala, Lingegowda S; Zhang, Ye; He, Zhenhua; Emami, Kamal; Ramesh, Govindarajan T; Story, Michael; Rohde, Larry H; Wu, Honglu

    2011-09-16

    This study explores the changes in expression of microRNA (miRNA) and related genes under simulated microgravity conditions. In comparison with static 1 × g, microgravity has been shown to alter global gene expression patterns and protein levels in cultured cells or animals. miRNA has recently emerged as an important regulator of gene expression, possibly regulating as many as one-third of all human genes. However, very little is known about the effect of altered gravity on miRNA expression. To test the hypothesis that the miRNA expression profile would be altered in zero gravity resulting in altered regulation of gene expression leading to metabolic or functional changes in cells, we cultured TK6 human lymphoblastoid cells in a high aspect ratio vessel (bioreactor) for 72 h either in the rotating condition to model microgravity in space or in the static condition as a control. Expression of several miRNAs was changed significantly in the simulated microgravity condition including miR-150, miR-34a, miR-423-5p, miR-22, miR-141, miR-618, and miR-222. To confirm whether this altered miRNA expression correlates with gene expression and functional changes of the cells, we performed DNA microarray and validated the related genes using quantitative RT-PCR. Expression of several transcription factors including EGR2, ETS1, and c-REL was altered in simulated microgravity conditions. Taken together, the results reported here indicate that simulated microgravity alters the expression of miRNAs and genes in TK6 cells. To our knowledge, this study is the first to report the effects of simulated microgravity on the expression of miRNA and related genes. PMID:21775437

  1. Induction of centrosome amplification by formaldehyde, but not hydroquinone, in human lymphoblastoid TK6 cells.

    PubMed

    Ji, Zhiying; McHale, Cliona M; Bersonda, Jessica; Tung, Judy; Smith, Martyn T; Zhang, Luoping

    2015-07-01

    Benzene and formaldehyde (FA) are important industrial chemicals and environmental pollutants that cause leukemia by inducing DNA damage and chromosome aberrations in hematopoietic stem cells (HSC), the target cells for leukemia. Our previous studies showed that workers exposed to benzene and FA exhibit increased levels of aneuploidy in their blood cells. As centrosome amplification is a common phenomenon in human cancers, including leukemia, and is associated with aneuploidy in carcinogenesis, we hypothesized that benzene and FA would induce centrosome amplification in vitro. We treated human lymphoblastoid TK6 cells with a range of concentrations of hydroquinone (HQ, a benzene metabolite) or FA for 24 h, allowed the cells to recover in fresh medium for 24 h, and examined centrosome amplification; chromosomal gain, loss, and breakage; and cytotoxicity. We included melphalan and etoposide, chemotherapeutic drugs that cause therapy-related acute myeloid leukemia and that have been shown to induce centrosome amplification as well as chromosomal aneuploidy and breakage, as positive controls. Melphalan and etoposide induced centrosome amplification and chromosome gain and breakage in a dose-dependent manner, at cytotoxic concentrations. HQ, though cytotoxic, did not induce centrosome amplification or any chromosomal aberration. FA-induced centrosome amplification and cytotoxicity, but did not induce chromosomal aberrations. Our data suggest, for the first time, that centrosome amplification is a potential mechanism underlying FA-induced leukemogenesis, but not benzene-induced leukemogenesis, as mediated through HQ. Future studies are needed to delineate the mechanisms of centrosome amplification and its association with DNA damage, chromosomal aneuploidy and carcinogenesis, following exposure to FA. PMID:25821186

  2. Pro-oxidant Induced DNA Damage in Human Lymphoblastoid Cells: Homeostatic Mechanisms of Genotoxic Tolerance

    PubMed Central

    Seager, Anna L.

    2012-01-01

    Oxidative stress contributes to many disease etiologies including ageing, neurodegeneration, and cancer, partly through DNA damage induction (genotoxicity). Understanding the i nteractions of free radicals with DNA is fundamental to discern mutation risks. In genetic toxicology, regulatory authorities consider that most genotoxins exhibit a linear relationship between dose and mutagenic response. Yet, homeostatic mechanisms, including DNA repair, that allow cells to tolerate low levels of genotoxic exposure exist. Acceptance of thresholds for genotoxicity has widespread consequences in terms of understanding cancer risk and regulating human exposure to chemicals/drugs. Three pro-oxidant chemicals, hydrogen peroxide (H2O2), potassium bromate (KBrO3), and menadione, were examined for low dose-response curves in human lymphoblastoid cells. DNA repair and antioxidant capacity were assessed as possible threshold mechanisms. H2O2 and KBrO3, but not menadione, exhibited thresholded responses, containing a range of nongenotoxic low doses. Levels of the DNA glycosylase 8-oxoguanine glycosylase were unchanged in response to pro- oxidant stress. DNA repair–focused gene expression arrays reported changes in ATM and BRCA1, involved in double-strand break repair, in response to low-dose pro-oxidant exposure; however, these alterations were not substantiated at the protein level. Determination of oxidatively induced DNA damage in H2O2-treated AHH-1 cells reported accumulation of thymine glycol above the genotoxic threshold. Further, the H2O2 dose-response curve was shifted by modulating the antioxidant glutathione. Hence, observed pro- oxidant thresholds were due to protective capacities of base excision repair enzymes and antioxidants against DNA damage, highlighting the importance of homeostatic mechanisms in “genotoxic tolerance.” PMID:22539617

  3. Effects of Modeled Microgravity on Expression Profiles of Micro RNA in Human Lymphoblastoid Cells

    NASA Technical Reports Server (NTRS)

    Mangala, Lingegowda S.; Emami, Kamal; Story, Michael; Ramesh, Govindarajan; Rohde, Larry; Wu, Honglu

    2010-01-01

    Among space radiation and other environmental factors, microgravity or an altered gravity is undoubtedly the most significant stress experienced by living organisms during flight. In comparison to the static 1g, microgravity has been shown to alter global gene expression patterns and protein levels in cultured cells or animals. Micro RNA (miRNA) has recently emerged as an important regulator of gene expression, possibly regulating as many as one-third of all human genes. miRNA represents a class of single-stranded noncoding regulatory RNA molecules ( 22 nt) that control gene expressions by inhibiting the translation of mRNA to proteins. However, very little is known on the effect of altered gravity on miRNA expression. We hypothesized that the miRNA expression profile will be altered in zero gravity resulting in regulation of the gene expression and functional changes of the cells. To test this hypothesis, we cultured TK6 human lymphoblastoid cells in Synthecon s Rotary cell culture system (bioreactors) for 72 h either in the rotating (10 rpm) to model the microgravity in space or in the static condition. The cell viability was determined before and after culturing the cells in the bioreactor using both trypan blue and guava via count. Expressions of a panel of 352 human miRNA were analyzed using the miRNA PCRarray. Out of 352 miRNAs, expressions of 75 were significantly altered by a change of greater than 1.5 folds and seven miRNAs were altered by a fold change greater than 2 under the rotating culture condition. Among these seven, miR-545 and miR-517a were down regulated by 2 folds, whereas miR-150, miR-302a, miR-139-3p, miR-515-3p and miR-564 were up regulated by 2 to 8 folds. To confirm whether this altered miRNA expression correlates with gene expression and functional changes of the cells, we performed DNA Illumina Microarray Analysis and validated the related genes using q-RT PCR.

  4. Arsenite-induced autophagy is associated with proteotoxicity in human lymphoblastoid cells

    SciTech Connect

    Bolt, Alicia M.; Zhao, Fei; Pacheco, Samantha; Klimecki, Walter T.

    2012-10-15

    Epidemiological studies of arsenic-exposed populations have provided evidence that arsenic exposure in humans is associated with immunosuppression. Previously, we have reported that arsenite-induced toxicity is associated with the induction of autophagy in human lymphoblastoid cell lines (LCL). Autophagy is a cellular process that functions in the degradation of damaged cellular components, including protein aggregates formed by misfolded or damaged proteins. Accumulation of misfolded or damaged proteins in the endoplasmic reticulum (ER) lumen causes ER stress and activates the unfolded protein response (UPR). In an effort to investigate the mechanism of autophagy induction by arsenite in the LCL model, we examined the potential contribution of ER stress and activation of the UPR. LCL exposed to sodium arsenite for 8-days induced expression of UPR-activated genes, including CHOP and GRP78, at the RNA and the protein level. Evidence for activation of the three arms of the UPR was observed. The arsenite-induced activation of the UPR was associated with an accumulation of protein aggregates containing p62 and LC3, proteins with established roles in the sequestration and autophagic clearance of protein aggregates. Taken together, these data provide evidence that arsenite-induced autophagy is associated with the generation of ER stress, activation of the UPR, and formation of protein aggregates that may be targeted to the lysosome for degradation. -- Highlights: ► Arsenite induces endoplasmic reticulum stress and the unfolded protein response. ► Arsenite induces the formation of protein aggregates that contain p62 and LC3-II. ► Time-course data suggests that arsenite-induced autophagy precedes ER stress.

  5. Oxidative stress induces mitochondrial dysfunction in a subset of autistic lymphoblastoid cell lines.

    PubMed

    Rose, S; Frye, R E; Slattery, J; Wynne, R; Tippett, M; Melnyk, S; James, S J

    2014-01-01

    There is an increasing recognition that mitochondrial dysfunction is associated with autism spectrum disorders. However, little attention has been given to the etiology of mitochondrial dysfunction and how mitochondrial abnormalities might interact with other physiological disturbances such as oxidative stress. Reserve capacity is a measure of the ability of the mitochondria to respond to physiological stress. In this study, we demonstrate, for the first time, that lymphoblastoid cell lines (LCLs) derived from children with autistic disorder (AD) have an abnormal mitochondrial reserve capacity before and after exposure to reactive oxygen species (ROS). Ten (44%) of 22 AD LCLs exhibited abnormally high reserve capacity at baseline and a sharp depletion of reserve capacity when challenged with ROS. This depletion of reserve capacity was found to be directly related to an atypical simultaneous increase in both proton-leak respiration and adenosine triphosphate-linked respiration in response to increased ROS in this AD LCL subgroup. In this AD LCL subgroup, 48-hour pretreatment with N-acetylcysteine, a glutathione precursor, prevented these abnormalities and improved glutathione metabolism, suggesting a role for altered glutathione metabolism associated with this type of mitochondrial dysfunction. The results of this study suggest that a significant subgroup of AD children may have alterations in mitochondrial function, which could render them more vulnerable to a pro-oxidant microenvironment as well as intrinsic and extrinsic sources of ROS such as immune activation and pro-oxidant environmental toxins. These findings are consistent with the notion that AD is caused by a combination of genetic and environmental factors. PMID:24690598

  6. Linking short tandem repeat polymorphisms with cytosine modifications in human lymphoblastoid cell lines.

    PubMed

    Zhang, Zhou; Zheng, Yinan; Zhang, Xu; Liu, Cong; Joyce, Brian Thomas; Kibbe, Warren A; Hou, Lifang; Zhang, Wei

    2016-02-01

    Inter-individual variation in cytosine modifications has been linked to complex traits in humans. Cytosine modification variation is partially controlled by single nucleotide polymorphisms (SNPs), known as modified cytosine quantitative trait loci (mQTL). However, little is known about the role of short tandem repeat polymorphisms (STRPs), a class of structural genetic variants, in regulating cytosine modifications. Utilizing the published data on the International HapMap Project lymphoblastoid cell lines (LCLs), we assessed the relationships between 721 STRPs and the modification levels of 283,540 autosomal CpG sites. Our findings suggest that, in contrast to the predominant cis-acting mode for SNP-based mQTL, STRPs are associated with cytosine modification levels in both cis-acting (local) and trans-acting (distant) modes. In local scans within the ±1 Mb windows of target CpGs, 21, 9, and 21 cis-acting STRP-based mQTL were detected in CEU (Caucasian residents from Utah, USA), YRI (Yoruba people from Ibadan, Nigeria), and the combined samples, respectively. In contrast, 139,420, 76,817, and 121,866 trans-acting STRP-based mQTL were identified in CEU, YRI, and the combined samples, respectively. A substantial proportion of CpG sites detected with local STRP-based mQTL were not associated with SNP-based mQTL, suggesting that STRPs represent an independent class of mQTL. Functionally, genetic variants neighboring CpG-associated STRPs are enriched with genome-wide association study (GWAS) loci for a variety of complex traits and diseases, including cancers, based on the National Human Genome Research Institute (NHGRI) GWAS Catalog. Therefore, elucidating these STRP-based mQTL in addition to SNP-based mQTL can provide novel insights into the genetic architectures of complex traits. PMID:26714498

  7. Characterization of DNA methylation and its association with other biological systems in lymphoblastoid cell lines

    PubMed Central

    Zhang, Zhe; Liu, Jinglan; Kaur, Maninder; Krantz, Ian D.

    2016-01-01

    Lymphoblastoid cell line (LCL) is a common tool to study genetic disorders. However, it has not been fully characterized to what degree LCLs preserve the in vivo status of non-genetic biological systems, such as DNA methylation and gene transcription. We previously reported that DNA methylation in LCLs is highly variable in a data set of ~27,000 CpG dinucleotide sites around transcription start site (TSS) and 63 human subjects including healthy controls and probands of genetic disorders. Disease-causing mutations are linked to differential methylation at some CpG sites, but account for a small proportion of the total variance. In this study, we repeated the experiments to ensure that the high variance is not due to technical error and scrutinized the characteristics of DNA methylation and its association with other biological systems. Using sequence information and ChIP-seq data, we conclude that local CpG density and histone modifications not only correlate to baseline methylation level, but also affect the direction of methylation change in LCLs. Integrative analysis of gene transcription and DNA methylation data of the same subjects shows that medium or high methylation around TSS blocks the transcription while low methylation is a necessary, but not sufficient condition of downstream gene transcription. We utilized epigenetic information around TSS to predict active gene transcription via logistic regression models. The multivariate model using DNA methylation, eight histone modifications, and two regulatory protein complexes (CTCF and cohesin) as predictors has better performance (accuracy = 95.1%) than any univariate models of single predictors. Linear regression analysis further shows that the transcriptional levels predicted by epigenetic markers have significant correlation to microarray measurements (p = 2.2e-10). This study provides new insights into the epigenetic systems of LCLs and suggests that more specifically designed experiments are needed to

  8. Effect of cell-derived growth factors and cytokines on the clonal outgrowth of EBV-infected B cells and established lymphoblastoid cell lines.

    PubMed

    Ifversen, P; Zhang, X M; Ohlin, M; Zeuthen, J; Borrebaeck, C A

    1993-07-01

    Epstein-Barr virus (EBV) is a potent inducer of polyclonal B lymphocyte proliferation and is widely used as a tool for the establishment of B cell lines producing human monoclonal antibodies. However, because of low transformability, low clonability, and the inherent instability of EBV-infected B cells, valuable antibody-producing B cells are often lost during this procedure. We have here examined various cell-derived cytokines for their ability to enhance both the cellular outgrowth of newly infected B cells and the clonability of infected B cells and lymphoblastoid cell lines. Our results show that the murine thymoma cell line EL-4 is superior to peripheral blood mononuclear cells in both cellular outgrowth and cloning experiments, whereas monocyte-derived factors and monocyte cell lines were less capable than peripheral blood mononuclear cells in enhancing cellular outgrowth and cloning. Furthermore, the human T cell hybridoma cell line MP6 that secretes a B cell growth and differentiation factor, recently identified as an isoform of thioredoxin, is also capable of stimulating EBV-infected B cells and lymphoblastoid cell lines. Co-cultivation of EBV-infected B cells with MP6 cells significantly enhanced the cloning efficiency at the 1 cell/well level. The present results also suggest that one potential role of the MP6-derived thioredoxin could be the up regulation of IL-6 receptor expression in EBV-infected B cells. PMID:8395232

  9. Identification of low-dose responsive metabolites in X-irradiated human B lymphoblastoid cells and fibroblasts

    PubMed Central

    Tsuyama, Naohiro; Mizuno, Hajime; Katafuchi, Atsushi; Abe, Yu; Kurosu, Yumiko; Yoshida, Mitsuaki; Kamiya, Kenji; Sakai, Akira

    2015-01-01

    Ionizing radiation (IR) induces cellular stress responses, such as signal transduction, gene expression, protein modification, and metabolite change that affect cellular behavior. We analyzed X-irradiated human Epstein-Barr virus-transformed B lymphoblastoid cells and normal fibroblasts to search for metabolites that would be suitable IR-responsive markers by Liquid Chromotography–Mass spectrometry (LC–MS). Mass spectra, as analyzed with principal component analysis, showed that the proportion of peaks with IR-induced change was relatively small compared with the influence of culture time. Dozens of peaks that had either been upregulated or downregulated by IR were extracted as candidate IR markers. The IR-changed peaks were identified by comparing mock-treated groups to 100 mGy-irradiated groups that had recovered after 10 h, and the results indicated that the metabolites involved in nucleoside synthesis increased and that some acylcarnitine levels decreased in B lymphoblastoids. Some peaks changed by as much as 20 mGy, indicating the presence of an IR-sensitive signal transduction/metabolism control mechanism in these cells. On the other hand, we could not find common IR-changed peaks in fibroblasts of different origin. These data suggest that cell phenotype-specific pathways exist, even in low-dose responses, and could determine cell behavior. PMID:25227127

  10. Effects of soluble and particulate Cr(VI) on genome-wide DNA methylation in human B lymphoblastoid cells.

    PubMed

    Lou, Jianlin; Wang, Yu; Chen, Junqiang; Ju, Li; Yu, Min; Jiang, Zhaoqiang; Feng, Lingfang; Jin, Lingzhi; Zhang, Xing

    2015-10-01

    Several previous studies highlighted the potential epigenetic effects of Cr(VI), especially DNA methylation. However, few studies have compared the effects of Cr(VI) on DNA methylation profiles between soluble and particulate chromate in vitro. Accordingly, Illumina Infinium Human Methylation 450K BeadChip array was used to analyze DNA methylation profiles of human B lymphoblastoid cells exposed to potassium dichromate or lead chromate, and the cell viability was also studied. Array based DNA methylation analysis showed that the impacts of Cr(VI) on DNA methylation were limited, only about 40 differentially methylated CpG sites, with an overlap of 15CpG sites, were induced by both potassium dichromate and lead chromate. The results of mRNA expression showed that after Cr(VI) treatment, mRNA expression changes of four genes (TBL1Y, FZD5, IKZF2, and KIAA1949) were consistent with their DNA methylation alteration, but DNA methylation changes of other six genes did not correlate with mRNA expression. In conclusion, both of soluble and particulate Cr(VI) could induce a small amount of differentially methylated sites in human B lymphoblastoid cells, and the correlations between DNA methylation changes and mRNA expression varied between different genes. PMID:26433257

  11. Identification and localization of huntingtin in brain and human lymphoblastoid cell lines with anti-fusion protein antibodies.

    PubMed Central

    Gutekunst, C A; Levey, A I; Heilman, C J; Whaley, W L; Yi, H; Nash, N R; Rees, H D; Madden, J J; Hersch, S M

    1995-01-01

    The Huntington disease (HD) phenotype is associated with expansion of a trinucleotide repeat in the IT15 gene, which is predicted to encode a 348-kDa protein named huntington. We used polyclonal and monoclonal anti-fusion protein antibodies to identify native huntingtin in rat, monkey, and human. Western blots revealed a protein with the expected molecular weight which is present in the soluble fraction of rat and monkey brain tissues and lymphoblastoid cells from control cases. In lymphoblastoid cell lines from juvenile-onset heterozygote HD cases, both normal and mutant huntingtin are expressed, and increasing repeat expansion leads to lower levels of the mutant protein. Immunocytochemistry indicates that huntingtin is located in neurons throughout the brain, with the highest levels evident in larger neurons. In the human striatum, huntingtin is enriched in a patch-like distribution, potentially corresponding to the first areas affected in HD. Subcellular localization of huntingtin is consistent with a cytosolic protein primarily found in somatodendritic regions. Huntingtin appears to particularly associate with microtubules, although some is also associated with synaptic vesicles. On the basis of the localization of huntingtin in association with microtubules, we speculate that the mutation impairs the cytoskeletal anchoring or transport of mitochondria, vesicles, or other organelles or molecules. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7568002

  12. B lymphoblastoid cell lines as efficient APC to elicit CD8+ T cell responses against a cytomegalovirus antigen.

    PubMed

    Sun, Q; Burton, R L; Dai, L J; Britt, W J; Lucas, K G

    2000-10-01

    Potent and readily accessible APC are critical for development of immunotherapy protocols to treat viral disease and cancer. We have shown that B lymphoblastoid cell lines (BLCL) that stably express CMV phosphoprotein 65 (BLCLpp65), as a result of retroviral transduction, can be used to generate ex vivo CTL cultures that possess cytotoxicity against CMV and EBV. In this report, we demonstrate that the EBV-specific cytotoxicity in the BLCLpp65-primed culture had a spectrum of EBV-Ag recognition similar to that of the BLCL-primed counterpart, suggesting that retroviral transduction and expression of the CMV Ag would not compromise the Ag-presenting capacity of BLCL. In addition, BLCLpp65 appeared to present multiple natural pp65 epitopes, because pp65-specific CTL, which recognized different CMV clinical isolates, were generated in BLCLpp65-primed cultures from individuals with various HLA backgrounds. Consistent with a polyclonal expansion of virus-specific CTL, T cell lines established from the BLCLpp65-primed CTL cultures expressed different TCR-Vbeta Although most of the virus-specific T cell isolates were CD8+, EBV-specific CD4+ lines were also established from BLCLpp65-primed cultures. Western blot analysis revealed that the CD8+ lines, but not the CD4+ line, expressed granzyme B, consistent with features of classic CTL. Thus, our results suggested that BLCL stably expressing a foreign Ag might be used as a practical APC to elicit CD8+ T cell responses. PMID:11034422

  13. Protein degradation in a LAMP-2-deficient B-lymphoblastoid cell line from a patient with Danon disease.

    PubMed

    Sánchez-Lanzas, Raul; Alvarez-Castelao, Beatriz; Bermejo, Teresa; Ayuso, Teresa; Tuñón, Teresa; Castaño, José G

    2016-08-01

    Danon disease, a condition characterized by cardiomyopathy, myopathy, and intellectual disability, is caused by mutations in the LAMP-2 gene. Lamp-2A protein, generated by alternative splicing from the Lamp-2 pre-mRNA, is reported to be the lysosomal membrane receptor essential for the chaperone-mediated autophagic pathway (CMA) aimed to selective protein targeting and translocation into the lysosomal lumen for degradation. To study the relevance of Lamp-2 in protein degradation, a lymphoblastoid cell line was obtained by EBV transformation of B-cells from a Danon patient. The derived cell line showed no significant expression of Lamp-2 protein. The steady-state mRNA and protein levels of alpha-synuclein, IΚBα, Rcan1, and glyceraldehyde-3-phosphate dehydrogenase, four proteins reported to be selective substrates of the CMA pathway, were similar in control and Lamp-2-deficient cells. Inhibition of protein synthesis showed that the half-life of alpha-synuclein, IΚBα, and Rcan1 was similar in control and Lamp-2-deficient cells, and its degradation prevented by proteasome inhibitors. Both in control and Lamp-2-deficient cells, induction of CMA and macroautophagy by serum and aminoacid starvation of cells for 8h produced a similar decrease in IΚBα and Rcan1 protein levels and was prevented by the addition of lysosome and autophagy inhibitors. In conclusion, the results presented here showed that Lamp-2 deficiency in human lymphoblastoid cells did not modify the steady-state levels or the degradation of several protein substrates reported as selective substrates of the CMA pathway. PMID:27130438

  14. Targeting Epstein-Barr virus-transformed B lymphoblastoid cells using antibodies with T-cell receptor-like specificities.

    PubMed

    Lai, Junyun; Tan, Wei Jian; Too, Chien Tei; Choo, Joanna Ai Ling; Wong, Lan Hiong; Mustafa, Fatimah Bte; Srinivasan, Nalini; Lim, Angeline Pei Chiew; Zhong, Youjia; Gascoigne, Nicholas R J; Hanson, Brendon J; Chan, Soh Ha; Chen, Jianzhu; MacAry, Paul A

    2016-09-01

    Epstein-Barr virus (EBV) is an oncovirus associated with several human malignancies including posttransplant lymphoproliferative disease in immunosuppressed patients. We show here that anti-EBV T-cell receptor-like monoclonal antibodies (TCR-like mAbs) E1, L1, and L2 bound to their respective HLA-A*0201-restricted EBV peptides EBNA1562-570, LMP1125-133, and LMP2A426-434 with high affinities and specificities. These mAbs recognized endogenously presented targets on EBV B lymphoblastoid cell lines (BLCLs), but not peripheral blood mononuclear cells, from which they were derived. Furthermore, these mAbs displayed similar binding activities on several BLCLs, despite inherent heterogeneity between different donor samples. A single weekly administration of the naked mAbs reduced splenomegaly, liver tumor spots, and tumor burden in BLCL-engrafted immunodeficient NOD-SCID/Il2rg(-/-) mice. In particular, mice that were treated with the E1 mAb displayed a delayed weight loss and significantly prolonged survival. In vitro, these TCR-like mAbs induced early apoptosis of BLCLs, thereby enhancing their Fc-dependent phagocytic uptake by macrophages. These data provide evidence for TCR-like mAbs as potential therapeutic modalities to target EBV-associated diseases. PMID:27338099

  15. Gene expression analysis of whole blood, peripheral blood mononuclear cells, and lymphoblastoid cell lines from the Framingham Heart Study

    PubMed Central

    Joehanes, Roby; Johnson, Andrew D.; Barb, Jennifer J.; Raghavachari, Nalini; Liu, Poching; Woodhouse, Kimberly A.; O'Donnell, Christopher J.; Munson, Peter J.

    2012-01-01

    Despite a growing number of reports of gene expression analysis from blood-derived RNA sources, there have been few systematic comparisons of various RNA sources in transcriptomic analysis or for biomarker discovery in the context of cardiovascular disease (CVD). As a pilot study of the Systems Approach to Biomarker Research (SABRe) in CVD Initiative, this investigation used Affymetrix Exon arrays to characterize gene expression of three blood-derived RNA sources: lymphoblastoid cell lines (LCL), whole blood using PAXgene tubes (PAX), and peripheral blood mononuclear cells (PBMC). Their performance was compared in relation to identifying transcript associations with sex and CVD risk factors, such as age, high-density lipoprotein, and smoking status, and the differential blood cell count. We also identified a set of exons that vary substantially between participants, but consistently in each RNA source. Such exons are thus stable phenotypes of the participant and may potentially become useful fingerprinting biomarkers. In agreement with previous studies, we found that each of the RNA sources is distinct. Unlike PAX and PBMC, LCL gene expression showed little association with the differential blood count. LCL, however, was able to detect two genes related to smoking status. PAX and PBMC identified Y-chromosome probe sets similarly and slightly better than LCL. PMID:22045913

  16. A genome-wide association analysis of temozolomide response using lymphoblastoid cell lines reveals a clinically relevant association with MGMT

    PubMed Central

    Brown, Chad C.; Havener, Tammy M.; Medina, Marisa Wong; Auman, J. Todd; Mangravite, Lara M.; Krauss, Ronald M.; McLeod, Howard L.; Motsinger-Reif, Alison A.

    2013-01-01

    Recently, lymphoblastoid cell lines (LCLs) have emerged as an innovative model system for mapping gene variants that predict dose response to chemotherapy drugs. In the current study, this strategy was expanded to the in vitro genome-wide association approach, using 516 LCLs derived from a Caucasian cohort to assess cytotoxic response to temozolomide. Genome-wide association analysis using approximately 2.1 million quality controlled single-nucleotide polymorphisms (SNPs) identified a statistically significant association (p < 10−8) with SNPs in the O6-methylguanine–DNA methyltransferase (MGMT) gene. We also demonstrate that the primary SNP in this region is significantly associated with differential gene expression of MGMT (p< 10−26) in LCLs, and differential methylation in glioblastoma samples from The Cancer Genome Atlas. The previously documented clinical and functional relationships between MGMT and temozolomide response highlight the potential of well-powered GWAS of the LCL model system to identify meaningful genetic associations. PMID:23047291

  17. Effects of glucocorticoids on the interaction of lymphoblastoid cells with human endothelial cells in vitro.

    PubMed

    Maca, R D; Fry, G L; Hakes, A D

    1978-08-01

    The adhesive characteristics of cultured acute lymphocytic leukemia cells (CCRF-CEM), lymphoma cells (Raji), and freshly isolated acute lymphocytic leukemia cells to human cultured endothelial cells were studied. An assay system was used whereby these neoplastic cells were allowed to interact with endothelial cells while being continuously agitated on a rocking platform. All cell lines adhered significantly to the endothelium monolayers. This process appeared not to be dependent upon intact microtubular or microfilament function. Likewise, removing surface sialic acid from either cell type did not alter this process. In contrast incubating the endothelial cells for 24 or 48 hr with dexamethasone decreased adhesiveness of either CCRF-CEM or Raji cells to the endothelial cells by approximately 40%. Incubating these cells with hydrocortisone instead of dexamethasone for 48 hr was equally as effective in altering the endothelial cell adhesiveness. The decreased adhesiveness could be blocked by cycloheximide, indicating that this altered adhesiveness of the endothelial cells involves protein synthesis, presumably of a surface protein. We suggest that this assay system may provide a means to evaluate other agents that can alter the surface characteristics of endothelial cells, which may have important implications in various disease states such as inflammation, thrombogenesis, and metastatic disease. PMID:276420

  18. [The reproductive characteristics of human adenovirus type 2 in cultures of lymphoblastoid cells with B and T phenotypes].

    PubMed

    Povnitsa, O Iu; Diachenko, N S; Chernomaz, A A; Nosach, L N; Rybalko, S L; Gritsak, T F; Beregovenko, V N; Diadiun, S T

    1997-01-01

    A comparative characteristic of the reproduction process of type 2 human adenovirus in several lines of lymphoblastoid cells of B- and T-phenotype is presented. Formation of hexone and infectious virus in the cells of Jurkat, MT4, Raji lines was rather intensive and approached to that in the culture of the permissive epithelium cells Hep-2. These indices were much lower in the cultures of cells B 95-8 and MT4/BIII LBK which were chronically infected by VEB and HIV, accordingly and produced them that can evidence for the interference of Ad and VEB or Ad and HIV under superinfection of cells. Cells of SEM line possessing T-phenotype, were apparently semi-permissive for Ad h2, though the low almost unchanged content of hexone and infectious virus remains in them for a rather long time: about 15 days. Thus, obtained data within analyzed series of experiments expand the present ideas about lymphotropicity of Ad as their important property realized at the level of cell and infected macroorganism. PMID:9511371

  19. The effect of uranyl acetate on human lymphoblastoid cells (RPMI 6410) and HeLa cells.

    PubMed Central

    Ghadially, F. N.; Yang-Steppuhn, S. E.; Lalonde, J. M.

    1982-01-01

    RPMI 6410 cells and HeLa cells were exposed to uranyl acetate. In RPMI 6410 cell cultures this produced single-membrane-bound presumably lysosomal bodies (called "uraniosomes") containing electron-dense crystals in the cultured cells and crystalline deposits in extracellular locations. Neither uraniosomes nor extracellular uranium deposits were found in HeLa cell cultures. All uraniosomes and extracellular uranium deposits analysed by electron-probed X-ray analysis were found to contain uranium, potassium and phosphorus. Traces of sulphur were detected in some but not all uraniosomes and extracellular uranium deposits. Traces of calcium were found in all extracellular uranium deposits and in some uraniosomes also. Images Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7093141

  20. Size-dependent cytotoxicity and genotoxicity of ZnO particles to human lymphoblastoid (WIL2-NS) cells.

    PubMed

    Yin, Hong; Casey, Philip S; McCall, Maxine J; Fenech, Michael

    2015-12-01

    The relationship between particle size and cytogenotoxicity of ZnO particles was systematically studied in vitro using WIL2-NS human lymphoblastoid cells. Before toxicity measurements, the ZnO particles of three different sizes (26 nm, 78 nm, and 147 nm) were well characterized for their physical and chemical properties to ensure that variations in other properties including surface chemistry and particle shape, which also may influence particle toxicity, were minimal. Cell viability testing showed that increasing cytotoxicity was associated with decreasing particle size. Both the dissolution kinetics of ZnO particles in supplemented cell culture medium and the apparent numbers of ZnO particles internalized by cells were size dependent and showed strong correlation with cytotoxicity. Genotoxicity, as measured by micronucleus formation, was significantly enhanced in the presence of the medium-sized and large-sized particles. The observation that necrosis increased with smaller- sized particles but micronuclei were present to a greater extent with larger- sized particles suggests that different mechanisms of cell damage induction or susceptibilities are operating depending on particle size. PMID:26248212

  1. Establishment of clival chordoma cell line MUG-CC1 and lymphoblastoid cells as a model for potential new treatment strategies

    PubMed Central

    Gellner, Verena; Tomazic, Peter Valentin; Lohberger, Birgit; Meditz, Katharina; Heitzer, Ellen; Mokry, Michael; Koele, Wolfgang; Leithner, Andreas; Liegl-Atzwanger, Bernadette; Rinner, Beate

    2016-01-01

    Chordomas are rare malignant tumors that develop from embryonic remnants of the notochord and arise only in the midline from the clivus to the sacrum. Surgery followed by radiotherapy is the standard treatment. As chordomas are resistant to standard chemotherapy, further treatment options are urgently needed. We describe the establishment of a clivus chordoma cell line, MUG-CC1. The cell line is characterized according to its morphology, immunohistochemistry, and growth kinetics. During establishment, cell culture supernatants were collected, and the growth factors HGF, SDF-1, FGF2, and PDGF analyzed using xMAP® technology. A spontaneous lymphoblastoid EBV-positive cell line was also developed and characterized. MUG-CC1 is strongly positive for brachyury, cytokeratin, and S100. The cell line showed gains of the entire chromosomes 7, 8, 12, 13, 16, 18, and 20, and high level gains on chromosomes 1q21–1q24 and 17q21–17q25. During cultivation, there was significant expression of HGF and SDF-1 compared to continuous chordoma cell lines. A new, well-characterized clival chordoma cell line, as well as a non-tumorigenic lymphoblastoid cell line should serve as an in vitro model for the development of potential new treatment strategies for patients suffering from this disease. PMID:27072875

  2. Biomarkers of Exposure and Effect in Human Lymphoblastoid TK6 Cells Following [13C2]-Acetaldehyde Exposure

    PubMed Central

    Swenberg, James A.

    2013-01-01

    The dose-response relationship for biomarkers of exposure (N2-ethylidene-dG adducts) and effect (cell survival and micronucleus formation) was determined across 4.5 orders of magnitude (50nM–2mM) using [13C2]-acetaldehyde exposures to human lymphoblastoid TK6 cells for 12h. There was a clear increase in exogenous N 2-ethylidene-dG formation at exposure concentrations ≥ 1µM, whereas the endogenous adducts remained nearly constant across all exposure concentrations, with an average of 3.0 adducts/107 dG. Exogenous adducts were lower than endogenous adducts at concentrations ≤ 10µM and were greater than endogenous adducts at concentrations ≥ 250µM. When the endogenous and exogenous adducts were summed together, statistically significant increases in total adduct formation over the endogenous background occurred at 50µM. Cell survival and micronucleus formation were monitored across the exposure range and statistically significant decreases in cell survival and increases in micronucleus formation occurred at ≥ 1000µM. This research supports the hypothesis that endogenously produced reactive species, including acetaldehyde, are always present and constitute the majority of the observed biological effects following very low exposures to exogenous acetaldehyde. These data can replace default assumptions of linear extrapolation to very low doses of exogenous acetaldehyde for risk prediction. PMID:23425604

  3. Host Genetic Variants and Gene Expression Patterns Associated with Epstein-Barr Virus Copy Number in Lymphoblastoid Cell Lines

    PubMed Central

    Houldcroft, Charlotte J.; Petrova, Velislava; Liu, Jimmy Z.; Frampton, Dan; Anderson, Carl A.; Gall, Astrid; Kellam, Paul

    2014-01-01

    Lymphoblastoid cell lines (LCLs) are commonly used in molecular genetics, supplying DNA for the HapMap and 1000 Genomes Projects, used to test chemotherapeutic agents, and informing the basis of a number of population genetics studies of gene expression. The process of transforming human B cells into LCLs requires the presence of Epstein-Barr virus (EBV), a double-stranded DNA virus which through B-cell immortalisation maintains an episomal virus genome in every cell of an LCL at variable copy numbers. Previous studies have reported that EBV alters host-gene expression and EBV copy number may be under host genetic control. We performed a genome-wide association study of EBV genome copy number in LCLs and found the phenotype to be highly heritable, although no individual SNPs achieved a significant association with EBV copy number. The expression of two host genes (CXCL16 and AGL) was positively correlated and expression of ADARB2 was negatively correlated with EBV copy number in a genotype-independent manner. This study shows an association between EBV copy number and the gene expression profile of LCLs, and suggests that EBV copy number should be considered as a covariate in future studies of host gene expression in LCLs. PMID:25290448

  4. Cellular factors associated with latency and spontaneous Epstein-Barr virus reactivation in B-lymphoblastoid cell lines.

    PubMed

    Davies, Michael L; Xu, Shushen; Lyons-Weiler, James; Rosendorff, Adam; Webber, Steven A; Wasil, Laura R; Metes, Diana; Rowe, David T

    2010-04-25

    EBV-immortalized B-lymphoblastoid cell lines are used as models for cellular transformation and as antigen-presenting cells in immunological assays. LCLs vary in surface markers and other phenotypic properties, but it is not known how this heterogeneity relates to the EBV life cycle. To explore correlations, we examined 62 LCLs for cellular and viral phenotypes. LCLs generated from pediatric and adult donors could similarly be categorized as either low in EBV copy number or fluctuating within a high range. High-copy status accompanied higher lytic viral gene expression and lower latent gene expression. Inhibiting lytic EBV replication did not affect cellular phenotype or lytic switch protein expression, indicating that an LCL's lytic permissivity was a stable property. Among the cellular genes overexpressed in permissive LCLs were unfolded protein response genes and plasma cell markers. Among genes overexpressed in non-permissive LCLs were transcription factors involved in maintaining B cell lineage, in particular EBF1. This study suggests previously undetected mechanisms by which cellular pathways influence the lytic reactivation of EBV. PMID:20153012

  5. Constant splice-isoform ratios in human lymphoblastoid cells support the concept of a splico-stat.

    PubMed

    Kramer, Marcel; Huse, Klaus; Menzel, Uwe; Backhaus, Oliver; Rosenstiel, Philip; Schreiber, Stefan; Hampe, Jochen; Platzer, Matthias

    2011-03-01

    Splicing generates mature transcripts from genes in pieces in eukaryotic cells. Overwhelming evidence has accumulated that alternative routes in splicing are possible for most human and mammalian genes, thereby allowing formation of different transcripts from one gene. No function has been assigned to the majority of identified alternative splice forms, and it has been assumed that they compose inert or tolerated waste from aberrant or noisy splicing. Here we demonstrate that five human transcription units (WT1, NOD2, GNAS, RABL2A, RABL2B) have constant splice-isoform ratios in genetically diverse lymphoblastoid cell lines independent of the type of alternative splicing (exon skipping, alternative donor/acceptor, tandem splice sites) and gene expression level. Even splice events that create premature stop codons and potentially trigger nonsense-mediated mRNA decay are found at constant fractions. The analyzed alternative splicing events were qualitatively but not quantitatively conserved in corresponding chimpanzee cell lines. Additionally, subtle splicing at tandem acceptor splice sites (GNAS, RABL2A/B) was highly constrained and strongly depends on the upstream donor sequence content. These results also demonstrate that unusual and unproductive splice variants are produced in a regulated manner. PMID:21220357

  6. Utility of Lymphoblastoid Cell Lines for Induced Pluripotent Stem Cell Generation

    PubMed Central

    Kumar, Satish; Curran, Joanne E.; Glahn, David C.; Blangero, John

    2016-01-01

    A large number of EBV immortalized LCLs have been generated and maintained in genetic/epidemiological studies as a perpetual source of DNA and as a surrogate in vitro cell model. Recent successes in reprograming LCLs into iPSCs have paved the way for generating more relevant in vitro disease models using this existing bioresource. However, the overall reprogramming efficiency and success rate remain poor and very little is known about the mechanistic changes that take place at the transcriptome and cellular functional level during LCL-to-iPSC reprogramming. Here, we report a new optimized LCL-to-iPSC reprogramming protocol using episomal plasmids encoding pluripotency transcription factors and mouse p53DD (p53 carboxy-terminal dominant-negative fragment) and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate using this optimized protocol. Further, we investigated the transcriptional changes in mRNA and miRNA levels, using FC-abs ≥ 2.0 and FDR ≤ 0.05 cutoffs; 5,228 mRNAs and 77 miRNAs were differentially expressed during LCL-to-iPSC reprogramming. The functional enrichment analysis of the upregulated genes and activation of human pluripotency pathways in the reprogrammed iPSCs showed that the generated iPSCs possess transcriptional and functional profiles very similar to those of human ESCs. PMID:27375745

  7. Treatment of metastatic renal cell carcinoma with a combination of human lymphoblastoid interferon-alpha and cimetidine.

    PubMed

    Kotake, T; Kinouchi, T; Saiki, S; Kuroda, M; Miki, T; Kiyohara, H; Usami, M

    1991-02-01

    Human lymphoblastoid interferon-alpha was administered intramuscularly at a dose of 5 x 10(6) units/day to 20 metastatic renal cell carcinoma patients. For potentiating the antitumor effect of interferon, cimetidine was also given to them orally at a dose of 800 mg/day. The combination therapy obtained a complete response in three patients (15%) and a partial response in three (15%). Nine patients (45%) had stable disease and five (25%), progressive disease. All six patients who responded to the combination therapy had been nephrectomized and had pulmonary metastases. Two of them also had metastases to other sites (mediastinal lymph nodes and bone). The pulmonary metastases were significantly more receptive to interferon therapy than those at the other sites. The average times before a response was obtained were 2.2 months for a minor response, 2.7 months for a partial response and 3.0 months for a complete response, and the average duration of response was 26 months. The six patients who responded survived for a significantly longer period than the 14 non-responding patients treated with interferon in combination with cimetidine. The major toxicities encountered were fever, fatigue and anorexia due to interferon, and the combination therapy was well tolerated except in three patients. The results suggest that interferon-alpha and cimetidine combination therapy may be of use in the management of patients with metastatic renal cell carcinoma. PMID:2067120

  8. Membrane permeation characteristics of abacavir in human erythrocytes and human T-lymphoblastoid CD4+ CEM cells: comparison with (-)-carbovir.

    PubMed

    Mahony, William B; Domin, Barbara A; Daluge, Susan M; Zimmerman, Thomas P

    2004-11-01

    Abacavir, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol, is a novel purine carbocyclic nucleoside analogue that has been approved by the FDA for the treatment of HIV (as Ziagen trade mark [abacavir sulfate]). Chemically, abacavir and (-)-carbovir (CBV) differ only at the 6-position of the purine ring; abacavir contains a cyclopropylamino moiety in place of the 6-lactam functionality of CBV. Intracellularly both are ultimately metabolized to CBV triphosphate. We compared the membrane permeation characteristics of these two compounds at 20 degrees C in human erythrocytes and in human T-lymphoblastoid CD4+ CEM cells, using a "papaverine-stop" assay. In erythrocytes, abacavir influx was rapid, nonsaturable (rate constant=200 pmol/s/mM/microl cell water), and unaffected by inhibitors of nucleoside or nucleobase transport. CBV influx was slow, saturable, strongly inhibited by adenine or hypoxanthine, and occurred via both the nucleobase carrier (Vmax=0.67 pmol/s/microl cell water; Km=50 microM) and the nucleoside carrier (Vmax=0.47 pmol/s/microl cell water; Km=440 microM). Similar qualitative results were obtained with CD4+ CEM cells, although CBV influx rates were somewhat higher and abacavir influx rates lower, compared to the corresponding rates in erythrocytes. Equilibrium studies further revealed that both compounds are concentrated intracellularly, but nonmetabolically, in both cell types, apparently due to cytosolic protein binding (absent in erythrocyte ghosts). We conclude that, in both cell types, while CBV influx is slow and carrier-dependent, abacavir influx occurs rapidly by nonfacilitated diffusion. The membrane permeation characteristics of abacavir are consistent with its superior oral bioavailability and its impressive ability to penetrate the central nervous system. PMID:15450945

  9. Human pathogenic Mycoplasma species induced cytokine gene expression in Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines.

    PubMed

    Schäffner, E; Opitz, O; Pietsch, K; Bauer, G; Ehlers, S; Jacobs, E

    1998-04-01

    We addressed the question whether the in vitro interaction of two Epstein-Barr virus (EBV)-genome-positive B cell lines (EB-3 and HilB-gamma) with either Mycoplasma pneumoniae or M. hominis, with the mycoplasma species (M. fermentans, M. fermentans subsp. incognitus, M. penetrans, M. genitalium) or with mycoplasma species known to be mere commensals of the respiratory tract (M. orale and M. salivarium) would result in expression of mRNAs for IL-2, IL-2R, IL-4 and IL-6 as determined by reverse transcriptase (RT)-PCR after 4 and 24 h of cocultivation. The pattern of cytokine gene expression observed depended on (i) the origin of the transformed cell line, (ii) the pathogenicity of the Mycoplasma species, and (iii) the length of cocultivation. The EBV-immortalized lymphoblastoid cell line HilB-gamma showed mRNA expression for IL-2, IL-2-receptor, IL-4 and IL-6 peaking 24 h after stimulation with M. pneumoniae and all AIDS-related mycoplasma species tested. The Burkitt lymphoma cell line EB-3 showed a distinct and isolated strong II-2/IL-2 R-mRNA expression within 4 h after contact with the pathogenic and all of the AIDS related mycoplasma species. In neither EBV-containing cell line cytokine was gene expression detectable after stimulation with the commensal mycoplasma species, M. orale and M. salivarium, indicating species differences in the ability of mycoplasmas to interact with and stimulate B-cell lines. Our data suggest that some mcyoplasma species may act as immunomodulatory cofactors by eliciting inappropriate cytokine gene expression in B cells latently infected with EBV. Therefore, this cultivation model may prove useful in evaluating the pathogenetic potential of novel isolated mycoplasma species. PMID:9533897

  10. Noninvolvement of the X chromosome in radiation-induced chromosome translocations in the human lymphoblastoid cell line TK6

    SciTech Connect

    Jordan, R.; Schwartz, J.L. )

    1994-03-01

    Fluorescence in situ hybridization procedures were used to examine the influence of chromosome locus on the frequency and type of chromosome aberrations induced by [sup 60]Co [gamma] rays in the human lymphoblastoid cell line TK6. Aberrations involving the X chromosome were compared to those involving the similarly sized autosome chromosome 7. When corrected for DNA content, acentric fragments were induced with equal frequency in the X and 7 chromosomes. Dose-dependent increases in chromosomal interchanges involving chromosome 7 were noted, and the frequencies of balanced translocations and dicentrics produced were approximately equal. Chromosome interchanges involving the X chromosome were rare and showed no apparent dose dependence. Thus, while chromosomes 7 and X are equally sensitive to the induction of chromosome breaks, the X chromosome is much less likely to interact with autosomes than chromosome 7. The noninvolvement of the X chromosome in translocations with autosomes may reflect a more peripheral and separate location for the X chromosome in the mammalian nucleus. 20 refs., 2 figs., 1 tab.

  11. Expression of LFA-1 by a lymphoblastoid cell line from a patient with monosomy 21: effects on intercellular adhesion.

    PubMed Central

    Taylor, G M; Braddock, D; Robson, A J; Fergusson, W D; Duckett, D P; D'Souza, S W; Brenchley, P

    1990-01-01

    Monosomy 21 (M21) is a rare aneuploid condition which in certain cases leads to reduced levels of chromosome 21 gene products. We have prepared an Epstein-Barr virus lymphoblastoid cell-line (LCL) from patient with M21 who has immunological abnormalities, and analysed the expression of lymphocyte function-associated antigen-1 (LFA-1). This heterodimeric leucocyte integrin consists of CD11a (alpha) subunits non-covalently associated with CD18 (beta) subunits coded, respectively, by genes on chromosomes 16 and 21. To determine whether monosomy 21 results in decreased expression of LFA-1, monoclonal antibodies were used to compare the expression of CD11a and CD18 on the M21 LCL with LCL from trisomy 21 (Down's syndrome, T21), normal controls and a possible case of leucocyte adhesion deficiency. In addition, phorbol-ester-induced homotypic adhesion, an LFA-1-mediated effect, was compared in these LCLs. The results are consistent with a gene dosage mediated reduction of LFA-1 expression by the M21 LCL. Images Fig. 2 PMID:1975779

  12. Proteomic Analysis of Lymphoblastoid Cells from Nasu-Hakola Patients: A Step Forward in Our Understanding of This Neurodegenerative Disorder

    PubMed Central

    Giuliano, Serena; Agresta, Anna Maria; De Palma, Antonella; Viglio, Simona; Mauri, Pierluigi; Fumagalli, Marco; Iadarola, Paolo; Montalbetti, Lorenza

    2014-01-01

    Nasu-Hakola disease (NHD) is a recessively inherited rare disorder characterized by a combination of neuropsychiatric and bone symptoms which, while being unique to this disease, do not provide a rationale for the unambiguous identification of patients. These individuals, in fact, are likely to go unrecognized either because they are considered to be affected by other kinds of dementia or by fibrous dysplasia of bone. Given that dementia in NHD has much in common with Alzheimer’s disease and other neurodegenerative disorders, it cannot be expected to achieve the differential diagnosis of this disease without performing a genetic analysis. Under this scenario, the availability of protein biomarkers would indeed provide a novel context to facilitate interpretation of symptoms and to make the precise identification of this disease possible. The work here reported was designed to generate, for the first time, protein profiles of lymphoblastoid cells from NHD patients. Two-dimensional electrophoresis (2-DE) and nano liquid chromatography-tandem mass spectrometry (nLC-MS/MS) have been applied to all components of an Italian family (seven subjects) and to five healthy subjects included as controls. Comparative analyses revealed differences in the expression profile of 21 proteins involved in glucose metabolism and information pathways as well as in stress responses. PMID:25470616

  13. In vitro evaluation of human hybrid cell lines generated by fusion of B-lymphoblastoid cells and ex vivo tumour cells as candidate vaccines for haematological malignancies.

    PubMed

    Mohamed, Yehia S; Dunnion, Debbie; Teobald, Iryna; Walewska, Renata; Browning, Michael J

    2012-10-12

    Fusions of dendritic cells (DCs) and tumour cells have been shown to induce protective immunity to tumour challenge in animal models, and to represent a promising approach to cancer immunotherapy. The broader clinical application of this approach, however, is potentially constrained by the lack of replicative capacity and limited standardisation of fusion cell preparations. We show here that fusion of ex vivo tumour cells isolated from patients with a range of haematological malignancies with the human B-lymphoblastoid cell line (LCL), HMy2, followed by chemical selection of the hybridomas, generated stable, self-replicating human hybrid cell lines that grew continuously in tissue culture, and survived freeze/thawing cycles. The hybrid cell lines expressed HLA class I and class II molecules, and the major T-cell costimulatory molecules, CD80 and CD86. All but two of 14 hybrid cell lines generated expressed tumour-associated antigens that were not expressed by HMy2 cells, and were therefore derived from the parent tumour cells. The hybrid cell lines stimulated allogeneic T-cell proliferative responses and interferon-gamma release in vitro to a considerably greater degree than their respective parent tumour cells. The enhanced T-cell stimulation was inhibited by CTLA4-Ig fusion protein, and by blocking antibodies to MHC class I and class II molecules. Finally, all of five LCL/tumour hybrid cell lines tested induced tumour antigen-specific cytotoxic T-cell responses in vitro in PBL from healthy, HLA-A2+ individuals, as detected by HLA-A2-peptide pentamer staining and cellular cytotoxicity. These data show that stable hybrid cell lines, with enhanced immunostimulatory properties and potential for therapeutic vaccination, can be generated by in vitro fusion and chemical selection of B-LCL and ex vivo haematological tumour cells. PMID:22939910

  14. Analysis of cellular response by exposure to acute or chronic radiation in human lymphoblastoid TK-6 cells

    NASA Astrophysics Data System (ADS)

    Ohnishi, T.; Yasumoto, J.; Takahashi, A.; Ohnishi, K.

    To clarify the biological effects of low-dose rate radiation on human health for long-term stay in space, we analyzed the induction of apoptosis and apoptosis-related gene expression after irradiation with different dose-rate in human lymphoblastoid TK-6 cells harboring wild-type p53 gene. We irradiated TK-6 cells by X-ray at 1.5 Gy (1 Gy/min) and then sampled at 25 hr after culturing. We also irradiated by gamma-ray at 1.5 Gy (1 mGy/min) and then sampled immediately or 25 hr after irradiation. For DNA ladder analysis, we extracted DNA from these samples and electrophoresed with 2% agarose gel. In addition, we extracted mRNA from these samples for DNA-array analysis. mRNA from non-irradiated cells was used as a control. After labeling the cDNA against mRNA with [α -33P]-dCTP and hybridizing onto DNA array (Human Apoptosis Expression Array, R&D Systems), we scanned the profiles of the spots by a phosphorimager (BAS5000, FUJI FILM) and calculated using a NIH Image program. The data of each DNA-array were normalized with eight kinds of house keeping genes. We analyzed the expression level of apoptosis-related genes such as p53-related, Bcl-2 family, Caspase family and Fas-related genes. DNA ladders were obviously detected in the cells exposed to a high dose-rate radiation. We detected the induction of the gene expression of apoptosis-promotive genes. In contrast, almost no apoptosis was observed in the cells exposed to the chronic radiation at a low dose-rate. In addition, we detected the induction of the gene expression of apoptosis-suppressive genes as compared with apoptosis promotive-genes immediately after chronic irradiation. These results lead the importance of biological meaning of exposure to radiation at low dose-rate from an aspect of carcinogenesis. Finally, the effects of chronic irradiation become a highly important issue in space radiation biology for human health.

  15. Sodium arsenite induces apoptosis and Epstein-Barr virus reactivation in lymphoblastoid cells.

    PubMed

    Zebboudj, Abderezak; Maroui, Mohamed Ali; Dutrieux, Jacques; Touil-Boukoffa, Chafia; Bourouba, Mehdi; Chelbi-Alix, Mounira K; Nisole, Sébastien

    2014-12-01

    Epstein-Barr virus (EBV) is associated with several malignancies, including carcinomas, such as nasopharyngeal carcinoma, and lymphomas, such as Burkitt's lymphoma and Hodgkin's lymphoma. The Latent Membrane Protein 1 (LMP1) is the major oncogene protein of EBV as its expression is responsible for the induction of cell transformation, immortalization and proliferation. Arsenic trioxide was shown to induce a cytotoxic effect on nasopharyngeal cancer cells associated with LMP1 down-regulation. However, the effect of arsenic on EBV-associated lymphoproliferative malignancies has been less studied. We investigated the effect of two different arsenical compounds, arsenic trioxide (As2O3) and sodium arsenite (NaAsO2) on the induction of cell death in P3HR1 cells, an Epstein-Barr virus-positive Burkitt lymphoma derived cell line. Both compounds inhibited cell growth and induced cell death. By flow-cytometry and Western blot analysis, we provide evidence that NaAsO2 induced caspase-dependent apoptosis whereas As2O3 triggered autophagic cell death. Furthermore, we show that NaAsO2 treatment led to a dramatic decrease of the expression level of LMP1 and the cellular protein PML. Importantly, this down-regulation was associated with a reactivation of EBV lytic cycle through the induction of immediate-early proteins Zta and Rta. These results are in agreement with a model in which LMP1 maintains EBV in a latent state by stabilizing PML expression. Altogether, our results suggest that NaAsO2 would represent a better therapeutic candidate than As2O3 in EBV-induced B lymphoma for its capacity to promote viral reactivation. PMID:25241256

  16. Radiosensitivity in lymphoblastoid cell lines derived from Shwachman-Diamond syndrome patients.

    PubMed

    Morini, J; Babini, G; Mariotti, L; Baiocco, G; Nacci, L; Maccario, C; Rößler, U; Minelli, A; Savio, M; Gomolka, M; Kulka, U; Ottolenghi, A; Danesino, C

    2015-09-01

    Shwachman-Diamond syndrome is an autosomal-recessive disorder characterised by bone marrow failure and a cumulative risk of progression to acute myeloid leukaemia. The Shwachman-Bodian-Diamond syndrome (SBDS) gene, the only gene known to be causative of the pathology, is involved in ribosomal biogenesis, stress responses and DNA repair, and the lack of SBDS sensitises cells to many stressors and leads to mitotic spindle destabilisation. The effect of ionising radiation on SBDS-deficient cells was investigated using immortalised lymphocytes from SDS patients in comparison with positive and negative controls in order to test whether, in response to ionising radiation exposure, any impairment in the DNA repair machinery could be observed. After irradiating cells with different doses of X-rays or gamma-rays, DNA repair kinetics and the residual damages using the alkaline COMET assay and the γ-H2AX assay were assessed, respectively. In this work, preliminary data about the comparison between ionising radiation effects in different patients-derived cells and healthy control cells are presented. PMID:25870433

  17. Leukemia patient-derived lymphoblastoid cell lines exhibit increased induction of leukemia-associated transcripts following high-dose irradiation.

    PubMed

    Spencer, A; Granter, N

    1999-09-01

    Improvement in diagnostic cytogenetic techniques has led to the recognition of an increasing number of leukemia-associated chromosomal translocations and inversions. These genetic lesions frequently are associated with the disruption of putative transcription factors and the production of hybrid transcripts that are implicated in leukemogenesis. Epidemiologic evidence suggests that some, but not all, individuals with a history of gamma-irradiation exposure are at increased risk of developing chronic myeloid leukemia (CML). CML is characterized by the Philadelphia chromosome and transcription of the resulting hybrid BCR-ABL gene. Utilizing the leukemia-associated BCR-ABL p210 transcript as a marker, we sought differences in the induction of illegitimate genetic recombination following high-dose gamma-irradiation of karyotypically normal lymphoblastoid cell lines (LCL) derived from individuals with and without a history of myeloid leukemias. Six LCL [4 leukemia patient derived [2 acute myeloid leukemia and 2 CML] and 2 from normal individuals were analyzed with reverse transcriptase polymerase chain reaction for BCR-ABL under stringent conditions following exposure to 0, 50, or 100 Gy of LET gamma-irradiation delivered via a Varian linear accelerator at 4 MV. Transcripts identical to disease-associated b2a2 and b3a2 transcripts were detected both spontaneously (background illegitimate genetic recombination) and following gamma-irradiation. Background BCR-ABL positivity was demonstrable in 4 of the 6 LCL, with no significant difference in detection between leukemic- and nonleukemic-derived LCL. Overall, increasing gamma-irradiation dose resulted in an increased frequency of BCR-ABL transcript detection (0 Gy vs 50 Gy vs 100 Gy,p = 0.0023, Chi-square test). Within the leukemic- but not the nonleukemic-derived LCL there was significantly greater BCR-ABL positivity after gamma-irradiation compared to unirradiated equivalents. Furthermore, the BCR-ABL positivity of both

  18. Effects of cell cycle position on ionizing radiation mutagenesis. I. Quantitative assays of two genetic loci in a human lymphoblastoid cell line

    SciTech Connect

    Chuang, Yao-Yu; Liber, H.L.

    1996-11-01

    Relatively little work has been done on the influence of the position of the cell in the cell cycle on ionizing radiation-induced mutagenesis. We synchronized WTK1 human lymphoblastoid cells with 200 {mu}M lovastatin for 48 h; under these conditions more than 80% of the cells were arrested in G{sub 1} phase. Upon release, there was a 12-15-h lag followed by movement of a large fraction into S phase. We irradiated cells with either 1.5 Gy X rays at 1, 15, 18, 21 or 24 h or 1.5 Gy {gamma} rays at 1, 5, 10, 15 or 24 h after release from lovastatin. We showed that WTK1 cells were most sensitive to ionizing radiation-induced toxicity in G{sub 1} and into S phase, and more resistant in mid to late S and G{sub 2}/M phase. Somewhat surprisingly, we found that the two different gene loci had different sensitivities to radiation-induced mutation through the cell cycle. Cells in late G{sub 1} through mid-S phase were most sensitive to radiation-induced mutations at the autosomal thymidine kinase (TK) locus, whereas G{sub 1} phase was the most sensitive phase at the X-linked hypoxanthine guanine phosphoribosyl transferase (HPRT) locus. 29 refs., 6 figs., 1 tab.

  19. Studying the protein expression in human B lymphoblastoid cells exposed to 1.8-GHz (GSM) radiofrequency radiation (RFR) with protein microarray.

    PubMed

    Zhijian, Chen; Xiaoxue, Li; Wei, Zheng; Yezhen, Lu; Jianlin, Lou; Deqiang, Lu; Shijie, Chen; Lifen, Jin; Jiliang, He

    2013-03-29

    In the present study, the protein microarray was used to investigate the protein expression in human B-cell lymphoblastoid cells intermittently exposed to 1.8-GHz GSM radiofrequency radiation (RFR) at the specific absorption rate (SAR) of 2.0 W/kg for 24 h. The differential expression of 27 proteins was found, which were related to DNA damage repair, apoptosis, oncogenesis, cell cycle and proliferation (ratio >1.5-fold, P<0.05). The results validated with Western blot assay indicated that the expression of RPA32 was significantly down-regulated (P<0.05) while the expression of p73 was significantly up-regulated in RFR exposure group (P<0.05). Because of the crucial roles of those proteins in DNA repair and cell apoptosis, the results of present investigation may explain the biological effects of RFR on DNA damage/repair and cell apoptosis. PMID:23454122

  20. [The viral genome status studied under the conditions of a mixed infection in lymphoblastoid cells by adenovirus and the Epstein-Barr virus].

    PubMed

    Nosach, L N; Diachenko, N S; Povnitsa, O Iu; Smirnova, I A; Kishinskaia, E G; Butenko, Z A; Panasenko, G V

    1998-01-01

    Some indices have been studied which characterized the state of Epstein-Barr virus genome and adenovirus in the implanted lines of lymphoblastoid cells of B and T phenotype under the mixed or monoinfection. It has been shown that super infection by type 2 adenovirus rather sharply affects the state of Epstein-Barr virus genome in the Raji cells containing integrated Epstein-Barr virus genome. The state of adenovirus genome in the studied cells is less subject to changes. Its early area is revealed by hybridization using DNA-DNA method in a form of two fragments of different intensity which is maximum in the Raji and Jurkat cells, which evidences for the more expressivity of adenovirus genome in these cells. PMID:9813890

  1. Apoptotic death induced by the cyclophosphamide analogue mafosfamide in human lymphoblastoid cells: Contribution of DNA replication, transcription inhibition and Chk/p53 signaling

    SciTech Connect

    Goldstein, Michael; Roos, Wynand P. Kaina, Bernd

    2008-05-15

    Cyclophosphamide is one of the most often used anticancer drugs. Although DNA interstrand cross-links are considered responsible for its cytotoxicity, the mechanism of initiation and execution of cell death is largely unknown. Using the cyclophosphamide analogue mafosfamide, which does not need metabolic activation, we show that mafosfamide induces apoptosis dose and time dependently in lymphoblastoid cells, with clearly more apoptosis in p53{sup wt} cells. We identified two upstream processes that initiate apoptosis, DNA replication blockage and transcriptional inhibition. In lymphoblastoid cells, wherein DNA replication can be switched off by tetracycline, proliferation is required for inducing apoptosis at low dose mafosfamide. At high dose, transcriptional inhibition also contributes to cell death. The RNA synthesis inhibitor {alpha}-amanitin induced similar to mafosfamide more apoptosis in p53{sup wt} than in p53{sup mt} cells. In combination with mafosfamide, however, {alpha}-amanitin had no additive effect. Mafosfamide caused p53 stabilization by phosphorylation of Ser15, 20 and 37, and activation of ATM/ATR and Chk1/Chk2. Inhibition of ATM/ATR, PI3-kinase and Chk1/Chk2 by CGK733, wortmannin and DBH, respectively, attenuated the apoptotic response in p53{sup wt} but not p53{sup mt} cells. Mafosfamide induced caspase dependent apoptosis and, for low dose treated cells, caspases were preferentially activated in the S-phase, whereas at high dose caspases were activated in all cell cycle stages. These data support the conclusion that at low dose level of mafosfamide, DNA replication blockage is the dominant apoptosis-inducing event, while at high dose, transcriptional inhibition comes into play. The data provide a mechanistic explanation of why cyclophosphamide applied at therapeutic doses preferentially kills replicating tumor cells.

  2. Use of lymphoblastoid cells for the estimation of environmental insults to DNA. Comprehensive report of the overall activities of the contract during the past three years. Progress report, August 1, 1978-June 31, 1981

    SciTech Connect

    1981-01-01

    Research progress is reported on a study to detect chronic low-level exposure of individuals to polycyclic aromatic hydrocarbons by analysis of DNA in cells with low turnover rates. The technique used was to measure the level of excision repair activity in lymphoblastoid and lymphoma cell lines. (ACR)

  3. Expression of p53-regulated genes in human cultured lymphoblastoid TSCE5 and WTK1 cell lines after spaceflight in a frozen state

    NASA Astrophysics Data System (ADS)

    Takahashi, A.; Suzuki, H.; Omori, K.; Seki, M.; Hashizume, T.; Shimazu, T.; Ishioka, N.; Ohnishi, T.

    2011-03-01

    The 53 kDa tumor suppressor protein p53 is generally thought to contribute to the genetic stability of cells and to protect cells from DNA damage through the activity of p53-centered signal transduction pathways. To clarify the effect of space radiation on the expression of p53-dependent regulated genes, gene expression profiles were compared between two human cultured lymphoblastoid cell lines: one line (TSCE5) has a wild-type p53 gene status, and the other line (WTK1) has a mutated p53 gene status. Frozen human lymphoblastoid cells were stored in a freezer in the International Space Station (ISS) for 133 days. Gene expression was analyzed using DNA chips after culturing the space samples for 6 h on the ground after their return from space. Ground control samples were also cultured for 6 h after being stored in a frozen state on the ground for the same time period that the frozen cells were in space. p53-Dependent gene expression was calculated from the ratio of the gene expression values in wild-type p53 cells and in mutated p53 cells. The expression of 50 p53-dependent genes was up-regulated, and the expression of 94 p53-dependent genes was down-regulated after spaceflight. These expression data identified genes which could be useful in advancing studies in basic space radiation biology. The biological meaning of these results is discussed from the aspect of gene functions in the up- and down-regulated genes after exposure to low doses of space radiation.

  4. Lymphoblastoid cell supernatants increase expression of C3b receptors on human polymorphonuclear leucocytes: direct binding studies with 125I-C3b.

    PubMed Central

    Berger, M; Cross, A S

    1984-01-01

    Human PMN incubated in culture supernatants of the Raji long-term human lymphoblastoid cell line showed increased rosette formation with sheep erythrocytes coated with C3b (EIgM C4b3b) but no change in rosette formation with IgG-coated erythrocytes. This suggested a specific increase in cell surface C3b receptors, which was further investigated using 125I-C3b for direct binding studies. The results confirmed that specific binding of 125I-C3b to PMN incubated in culture supernatants increased up to three- to four-fold over binding to PMN incubated in control media alone. Scatchard analysis revealed that the apparent Ka for supernatant-treated cells, 3.36 +/- 0.89 X 10(7) L/M did not differ from the Ka for cells incubated in control media, 3.76 +/- 0.75 X 10(7) L/M, suggesting an increase in a single class of C3b receptors. Kinetic studies revealed that the active factor was present within 24 hr of culture of the Raji cells, and that neutrophils incubated in culture supernatants increased their C3b receptors continuously for up to 4 hr, the longest interval tested. The effect of the culture supernatant was lost with dilution beyond eight- to 10-fold. The results suggest that culture supernatants of this long-term lymphoblastoid cell line contain soluble factors that induce increased expression of C3b receptors on PMN and may thus serve as a model for study of important physiologic effects of lymphocyte products on PMN in vivo. PMID:6230308

  5. Host cell reactivation of uv- and X-ray-damaged herpes simplex virus by Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines

    SciTech Connect

    Henderson, E.E.; Long, W.K.

    1981-12-01

    The efficacy of using an infected centers assay, employing herpes simplex virus-infected, Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) as components, to study host cell reactivation has been explored. Herpes simplex virus type 1 (HSV-1) was shown through the infected centers assay to have detectable but varying ability to lytically infect LCLs established from chromosomal breakage syndromes or closely related genetic disorders. The rate of HSV inactivation by ultraviolet (uv) irradiation was faster in LCLs established from Cockaynes's syndrome than in normal LCLs, and faster still in LCLs established from xeroderma pigmentosum. These results indicate that Cockayne's syndrome, while having what appears to be quantitatively normal levels of uv-induced DNA repair replication, shows decreased ability to host cell reactivated uv-damaged HSV. In direct contrast, X-irradiated HSV showed identical survival when assayed on normal LCLs or LCLs established from ataxia telangiectasia showing increased sensitivity to X irradiation as measured by colony formation. Through the infected centers assay, it has also been possible to demonstrate low levels of multiplicity reactivation of mutagen-damaged HSV in permanently proliferating LCLs.

  6. The human T-cell leukemia virus type 1 Rex regulatory protein exhibits an impaired functionality in human lymphoblastoid Jurkat T cells.

    PubMed Central

    Hamaia, S; Cassé, H; Gazzolo, L; Duc Dodon, M

    1997-01-01

    The Rex protein of human T-cell leukemia virus type 1 (HTLV-1) intervenes in the posttranscriptional regulation of proviral gene expression. Its binding to the Rex response element (XRE) present in the 3' long terminal repeat ensures the coordinate cytoplasmic accumulation of spliced and unspliced forms of viral messengers. Consequently, synthesis of viral structural and enzymatic proteins is strictly dependent on the Rex posttranscriptional activity. Here we report that synthesis of HTLV-1 envelope glycoproteins by Jurkat T cells could be detected only when they were regulated in a Rex-independent manner. Indeed, Jurkat T cells transfected with a Rex-dependent env expression vector (encompassing both the env and pX open reading frames) do not produce significant levels of envelope glycoproteins despite the production of significant amounts of Rex protein. The analysis of levels and distribution patterns of the unspliced env and of the singly spliced tax/rex transcripts suggests that the failure in envelope glycoprotein synthesis may be ascribed to a deficiency of Rex in mediating the nucleocytoplasmic transport of unspliced env RNAs in these cells. Furthermore, despite the synthesis of regulatory proteins, HTLV-1 structural proteins were not detected in Jurkat T cells transfected with an HTLV-1 infectious provirus. Conversely, and as expected, structural proteins were produced by Jurkat cells transfected by a human immunodeficiency virus type 1 (HIV-1) infectious provirus. This phenotype appeared to be linked to a specific dysfunction of Rex, since the functionally equivalent Rev protein of HIV-1 was shown to be fully efficient in promoting the synthesis of HTLV-1 envelope glycoproteins in Jurkat cells. Therefore, it seems likely that the block to Rex function in these lymphoblastoid T cells is determined by inefficient Rex-XRE interactions. These observations suggest that the acquisition of this Rex-deficient phenotype by in vivo-infected HTLV-1 T cells may

  7. Failure to detect DUP25 in lymphoblastoid cells derived from patients with panic disorder and control individuals representing European and American populations.

    PubMed

    Zhu, Guanshan; Bartsch, Oliver; Skrypnyk, Cristina; Rotondo, Alessandro; Akhtar, Longina A; Harris, Claudia; Virkkunen, Matti; Cassano, Giovanni; Goldman, David

    2004-06-01

    Investigation of the co-occurrence of panic and phobic disorders with joint laxity led to the identification of interstitial duplications involving human chromosome 15q24-26 (named 'DUP25') in a Spanish population. DUP25 was observed in 97% of patients and in 7% of control individuals. In the present study, we used two different methods to detect DUP25: high-throughput molecular gene dosage analysis and fluorescence in situ hybridization (FISH). We evaluated 56 lymphoblastoid cell lines derived from 26 unrelated patients with panic disorder obtained from several European and American populations and 30 normal controls. We could not find any cell line showing a result consistent with DUP25. These data do not support any association of DUP25 with panic disorder. PMID:15054397

  8. The EBNA3 Family of Epstein-Barr Virus Nuclear Proteins Associates with the USP46/USP12 Deubiquitination Complexes to Regulate Lymphoblastoid Cell Line Growth

    PubMed Central

    Calderwood, Michael A.; Lai, Chiou-Yan; Krastins, Bryan; Sarracino, David; Johannsen, Eric

    2015-01-01

    The Epstein-Barr virus (EBV) nuclear proteins EBNA3A, EBNA3B, and EBNA3C interact with the cell DNA binding protein RBPJ and regulate cell and viral genes. Repression of the CDKN2A tumor suppressor gene products p16INK4A and p14ARF by EBNA3A and EBNA3C is critical for EBV mediated transformation of resting B lymphocytes into immortalized lymphoblastoid cell lines (LCLs). To define the composition of endogenous EBNA3 protein complexes, we generated lymphoblastoid cell lines (LCLs) expressing flag-HA tagged EBNA3A, EBNA3B, or EBNA3C and used tandem affinity purification to isolate each EBNA3 complex. Our results demonstrated that each EBNA3 protein forms a distinct complex with RBPJ. Mass-spectrometry revealed that the EBNA3A and EBNA3B complexes also contained the deubquitylation complex consisting of WDR48, WDR20, and USP46 (or its paralog USP12) and that EBNA3C complexes contained WDR48. Immunoprecipitation confirmed that EBNA3A, EBNA3B, and EBNA3C association with the USP46 complex. Using chromatin immunoprecipitation, we demonstrate that WDR48 and USP46 are recruited to the p14ARF promoter in an EBNA3C dependent manner. Mapping studies were consistent with WDR48 being the primary mediator of EBNA3 association with the DUB complex. By ChIP assay, WDR48 was recruited to the p14ARF promoter in an EBNA3C dependent manner. Importantly, WDR48 associated with EBNA3A and EBNA3C domains that are critical for LCL growth, suggesting a role for USP46/USP12 in EBV induced growth transformation. PMID:25855980

  9. A Single Amino Acid in EBNA-2 Determines Superior B Lymphoblastoid Cell Line Growth Maintenance by Epstein-Barr Virus Type 1 EBNA-2

    PubMed Central

    Tzellos, Stelios; Correia, Paulo B.; Karstegl, Claudio Elgueta; Cancian, Laila; Cano-Flanagan, Julian; McClellan, Michael J.; West, Michelle J.

    2014-01-01

    ABSTRACT Sequence differences in the EBNA-2 protein mediate the superior ability of type 1 Epstein-Barr virus (EBV) to transform human B cells into lymphoblastoid cell lines compared to that of type 2 EBV. Here we show that changing a single amino acid (S442D) from serine in type 2 EBNA-2 to the aspartate found in type 1 EBNA-2 confers a type 1 growth phenotype in a lymphoblastoid cell line growth maintenance assay. This amino acid lies in the transactivation domain of EBNA-2, and the S442D change increases activity in a transactivation domain assay. The superior growth properties of type 1 EBNA-2 correlate with the greater induction of EBV LMP-1 and about 10 cell genes, including CXCR7. In chromatin immunoprecipitation assays, type 1 EBNA-2 is shown to associate more strongly with EBNA-2 binding sites near the LMP-1 and CXCR7 genes. Unbiased motif searching of the EBNA-2 binding regions of the differentially regulated cell genes identified an ETS-interferon regulatory factor composite element motif that closely corresponds to the sequences known to mediate EBNA-2 regulation of the LMP-1 promoter. It appears that the superior induction by type 1 EBNA-2 of the cell genes contributing to cell growth is due to their being regulated in a manner different from that for most EBNA-2-responsive genes and in a way similar to that for the LMP-1 gene. IMPORTANCE The EBNA-2 transcription factor plays a key role in B cell transformation by EBV and defines the two EBV types. Here we identify a single amino acid (Ser in type 1 EBV, Asp in type 2 EBV) of EBNA-2 that determines the superior ability of type 1 EBNA-2 to induce a key group of cell genes and the EBV LMP-1 gene, which mediate the growth advantage of B cells infected with type 1 EBV. The EBNA-2 binding sites in these cell genes have a sequence motif similar to the sequence known to mediate regulation of the EBV LMP-1 promoter. Further detailed analysis of transactivation and promoter binding provides new insight into the

  10. Low Dose Radiation Response Curves, Networks and Pathways in Human Lymphoblastoid Cells Exposed from 1 to 10 cGy of Acute Gamma Radiation

    SciTech Connect

    Wyrobek, A. J.; Manohar, C. F.; Nelson, D. O.; Furtado, M. R.; Bhattacharya, M. S.; Marchetti, F.; Coleman, M.A.

    2011-04-18

    We investigated the low dose dependency of the transcriptional response of human cells to characterize the shape and biological functions associated with the dose response curve and to identify common and conserved functions of low dose expressed genes across cells and tissues. Human lymphoblastoid (HL) cells from two unrelated individuals were exposed to graded doses of radiation spanning the range of 1-10 cGy were analyzed by transcriptome profiling, qPCR and bioinformatics, in comparison to sham irradiated samples. A set of {approx}80 genes showed consistent responses in both cell lines; these genes were associated with homeostasis mechanisms (e.g., membrane signaling, molecule transport), subcellular locations (e.g., Golgi, and endoplasmic reticulum), and involved diverse signal transduction pathways. The majority of radiation-modulated genes had plateau-like responses across 1-10 cGy, some with suggestive evidence that transcription was modulated at doses below 1 cGy. MYC, FOS and TP53 were the major network nodes of the low-dose response in HL cells. Comparison our low dose expression findings in HL cells with those of prior studies in mouse brain after whole body exposure, in human keratinocyte cultures, and in endothelial cells cultures, indicates that certain components of the low dose radiation response are broadly conserved across cell types and tissues, independent of proliferation status.

  11. Infection of simian B lymphoblastoid cells with simian immunodeficiency virus is associated with upregulation of CD23 and CD40 cell surface markers.

    PubMed

    Titti, Fausto; Zamarchi, Rita; Maggiorella, Maria Teresa; Sernicola, Leonardo; Geraci, Andrea; Negri, Donatella Rita Maria; Borsetti, Alessandra; Menin, Chiara; D'Andrea, Emma; Modesti, Andrea; Masuelli, Laura; Verani, Paola; Chieco-Bianchi, Luigi; Amadori, Alberto

    2002-09-01

    Simian immunodeficiency virus (SIV) as well as human immunodeficiency virus (HIV) induce polyclonal B-cell activation and are associated with the appearance of lymphomas in their respective hosts in either the presence or the absence of other co-infecting viruses such as Epstein-Barr virus (EBV). However, the pathogenic role of these retroviruses in the development of lymphoproliferative disorders remains poorly understood. To explore the virus-B-cell interactions, two immortalized lymphoblastoid B-cell lines (SL-P1 and SL-691) were established from cynomolgus monkeys that were naturally co-infected with a simian type D retrovirus-2 (SRV-2) and with the herpes virus Macaca fascicularis (HVMF-1). We addressed their susceptibility to SIV infection and the phenotypic modifications associated with SIV infection. In response, both cell lines (1) were co-infected with HVMF-1 (latent infection) and with SRV-2 (productive infection), (2) had a transformed phenotype because they did not require exogenous growth factors, and (3) when injected into mice with severe combined immunodeficiency (SCID), generated serially transplantable tumors. The B-cell origin of SL cells was demonstrated by the presence of rearrangements of the IgH gene and by the expression of typical B-cell lineage markers, such as CD20. SL-P1 and SL-691 could be discriminated on the basis of different expressions of CD23 and CD40 and of kappa- and lambda-chains. Most importantly, SL-691 cells, but not SL-P1 cells, were susceptible to chronic noncytolytic SIV infection. This infection occurred in a CD4/CCR5/CXCR4-independent manner and was associated with the upregulated expression of CD23 and CD40 cell surface markers. In addition, CD20 expression, which progressively disappeared in SL-691 noninfected cells, was maintained in the SIV-infected counterpart. These findings support the hypothesis that SIV induce phenotypic perturbations in B cells that might eventually contribute to the development of

  12. Studying the protein expression in human B lymphoblastoid cells exposed to 1.8-GHz (GSM) radiofrequency radiation (RFR) with protein microarray

    SciTech Connect

    Zhijian, Chen; Institute of Environmental Health, Medical College, Zhejiang University, Hangzhou 310058, Zhejiang ; Xiaoxue, Li; Wei, Zheng; Yezhen, Lu; Jianlin, Lou; Deqiang, Lu; Shijie, Chen; Lifen, Jin; Jiliang, He

    2013-03-29

    Highlights: ► Protein microarray shows the differential expression of 27 proteins induced by RFR. ► RPA32 related to DNA repair is down-regulated in Western blot. ► p73 related to cell genome stability and apoptosis is up-regulated in Western blot. -- Abstract: In the present study, the protein microarray was used to investigate the protein expression in human B-cell lymphoblastoid cells intermittently exposed to 1.8-GHz GSM radiofrequency radiation (RFR) at the specific absorption rate (SAR) of 2.0 W/kg for 24 h. The differential expression of 27 proteins was found, which were related to DNA damage repair, apoptosis, oncogenesis, cell cycle and proliferation (ratio >1.5-fold, P < 0.05). The results validated with Western blot assay indicated that the expression of RPA32 was significantly down-regulated (P < 0.05) while the expression of p73 was significantly up-regulated in RFR exposure group (P < 0.05). Because of the crucial roles of those proteins in DNA repair and cell apoptosis, the results of present investigation may explain the biological effects of RFR on DNA damage/repair and cell apoptosis.

  13. Marek's disease virus undergoes complete morphogenesis after reactivation in a T-lymphoblastoid cell line transformed by recombinant fluorescent marker virus.

    PubMed

    Denesvre, Caroline; Rémy, Sylvie; Trapp-Fragnet, Laetitia; Smith, Lorraine P; Georgeault, Sonia; Vautherot, Jean-François; Nair, Venugopal

    2016-02-01

    T-lymphocytes are central targets of Marek's disease, a major chicken disease induced by the oncogenic alphaherpesvirus Marek's disease virus (MDV). T-lymphocyte infection is also associated with immunosuppression and virus latency. To decipher viral morphogenesis in T-lymphocytes, we used the recombinant vRB-1B 47EGFP marker virus to generate a new lymphoblastoid cell line, 3867K, that exhibited typical properties of other MDV-transformed chicken cell lines in term of cell markers, reactivation rate and infectivity. Examination of reactivating EGFP-positive 3867K cells by transmission electron microscopy revealed the presence of most types of herpesvirus particles inside the cells but no extracellular ones. Quantification of virion types indicated only 5% cytoplasmic particles, with 0.5% being mature. This study demonstrated that MDV morphogenesis is complete upon reactivation in T-lymphocytes, albeit with poor efficiency, with a defect in the exit of virions from the nucleus and secondary envelopment, as occurs in infected fibroblasts. PMID:26612074

  14. Cytoskeletal proteins from human skin fibroblasts, peripheral blood leukocytes, and a lymphoblastoid cell line compared by two-dimensional gel electrophoresis

    SciTech Connect

    Giometti, C.S.; Willard, K.E.; Anderson, N.L.

    1982-04-01

    Differences in proteins between cells grown as suspension cultures and those grown as attached cultures were studied by comparing the proteins of detergent-resistant cytoskeletons prepared from peripheral blood leukocytes and a lymphoblastoid cell line (GM607) (both grown as suspension cultures) and those of human skin fibroblasts (grown as attached cultures) by two-dimensional gel electrophoresis. The major cytoskeletal proteins of the leukocytes were also present in the protein pattern of GM607 cytoskeletons. In contrast, the fibroblast cytoskeletal protein pattern contained four groups of proteins that differed from the patterns of the leukocytes and GM607. In addition, surface labeling of GM607 and human fibroblasts with /sup 125/I demonstrated that substantial amounts of vimentin and actin are exposed at the surface of the attached fibroblasts, but there is little evidence of similar exposure at the surface of the suspension-grown GM607. These results demonstrate some differences in cytoskeletal protein composition between different types of cells could be related to their ability or lack of ability to grow as attached cells in tissue culture.

  15. Microwave electromagnetic field regulates gene expression in T-lymphoblastoid leukemia CCRF-CEM cell line exposed to 900 MHz.

    PubMed

    Trivino Pardo, Juan Carlos; Grimaldi, Settimio; Taranta, Monia; Naldi, Ilaria; Cinti, Caterina

    2012-03-01

    Electric, magnetic, and electromagnetic fields are ubiquitous in our society, and concerns have been expressed regarding possible adverse effects of these exposures. Research on Extremely Low-Frequency (ELF) magnetic fields has been performed for more than two decades, and the methodology and quality of studies have improved over time. Studies have consistently shown increased risk for childhood leukemia associated with ELF magnetic fields. There are still inadequate data for other outcomes. More recently, focus has shifted toward Radio Frequencies (RF) exposures from mobile telephony. There are no persuasive data suggesting a health risk, but this research field is still immature with regard to the quantity and quality of available data. This technology is constantly changing and there is a need for continued research on this issue. To investigate whether exposure to high-frequency electromagnetic fields (EMF) could induce adverse health effects, we cultured acute T-lymphoblastoid leukemia cells (CCRF-CEM) in the presence of 900 MHz MW-EMF generated by a transverse electromagnetic (TEM) cell at short and long exposure times. We evaluated the effect of high-frequency EMF on gene expression and we identified functional pathways influenced by 900 MHz MW-EMF exposure. PMID:22332889

  16. Zinc carnosine protects against hydrogen peroxide-induced DNA damage in WIL2-NS lymphoblastoid cell line independent of poly (ADP-Ribose) polymerase expression.

    PubMed

    Ooi, Theng Choon; Mohammad, Nur Hafiza; Sharif, Razinah

    2014-12-01

    The aim of this study is to investigate the ability of zinc carnosine to protect the human lymphoblastoid (WIL2-NS) cell line from hydrogen peroxide-induced DNA damage. Cells were cultured with medium containing zinc carnosine at the concentrations of 0.4, 4, 16 and 32 μM for 9 days prior to treatment with 30 μM of hydrogen peroxide (30 min). Zinc carnosine at the concentration 16 μM was optimal in protecting cells from hydrogen peroxide-induced cytotoxicity and gave the lowest percentage of apoptotic and necrotic cells. Results showed that zinc carnosine was able to induce glutathione production and protect cells from hydrogen peroxide-induced oxidative stress at all concentration and the highest protection was observed at 32-μM zinc carnosine culture. Cytokinesis-block micronucleus cytome assay showed that cells cultured with 4-32 μM of zinc carnosine showed significant reduction in micronuclei formation, nucleoplasmic bridges and nuclear bud frequencies (p < 0.05), suggesting that these concentrations maybe optimal in protecting cells from hydrogen peroxide-induced DNA damage. However, after being challenged with hydrogen peroxide, no increase in poly(ADP-ribose) polymerase expression was observed. Thus, results from this study demonstrate that zinc carnosines possess antioxidant properties and are able to reduce hydrogen peroxide-induced DNA damage in vitro independent of poly(ADP-ribose) polymerase. Further studies are warranted to understand the mechanism of protection of zinc carnosine against hydrogen peroxide-induced damage. PMID:25326781

  17. Induction of epstein-barr virus (EBV) lytic cycle in vitro causes lipid peroxidation, protein oxidation and DNA damage in lymphoblastoid B cell lines

    PubMed Central

    2011-01-01

    Background We investigated the oxidative modifications of lipids, proteins and DNA, potential molecular targets of oxidative stress, in two lymphoblastoid cell lines: B95-8 and Raji, after EBV lytic cycle induction. Conjugated dienes level was measured as biomarker of lipid peroxidation. Malondialdehyde adduct and protein carbonyl levels, as well as protein thiol levels were measured as biomarkers of protein oxidation. DNA fragmentation was evaluated as biomarker of DNA oxidation. Results After 48 h (peak of lytic cycle), a significant increase in conjugated dienes level was observed in B95-8 and Raji cell lines (p = 0.0001 and p = 0.019 respectively). Malondialdehyde adduct, protein carbonyl levels were increased in B95-8 and Raji cell lines after EBV lytic cycle induction as compared to controls (MDA-adduct: p = 0.008 and p = 0.006 respectively; Carbonyl: p = 0.003 and p = 0.0039 respectively). Proteins thiol levels were decreased by induction in B95-8 and Raji cell lines (p = 0.046; p = 0.002 respectively). DNA fragmentation was also detected in B95-8 and Raji cell lines after EBV lytic cycle induction as compared to controls. Conclusion The results of this study demonstrate the presence of increased combined oxidative modifications in lipids, proteins in B95-8 and Raji cells lines after EBV lytic cycle induction. These results suggest that lipid peroxidation, protein oxidation and DNA fragmentation are generally induced during EBV lytic cycle induction and probably contribute to the cytopathic effect of EBV. PMID:21722381

  18. Insulin-like Growth Factor 1 Differentially Affects Lithium Sensitivity of Lymphoblastoid Cell Lines from Lithium Responder and Non-responder Bipolar Disorder Patients.

    PubMed

    Milanesi, Elena; Hadar, Adva; Maffioletti, Elisabetta; Werner, Haim; Shomron, Noam; Gennarelli, Massimo; Schulze, Thomas G; Costa, Marta; Del Zompo, Maria; Squassina, Alessio; Gurwitz, David

    2015-07-01

    Bipolar disorder (BD) is a chronic psychiatric illness with an unknown etiology. Lithium is considered the cornerstone in the management of BD, though about 50-60 % of patients do not respond sufficiently to chronic treatment. Insulin-like growth factor 1 (IGF1) has been identified as a candidate gene for BD susceptibility, and its low expression has been suggested as a putative biomarker for lithium unresponsiveness. In this study, we examined the in vitro effects of insulin-like growth factor 1 (IGF-1) on lithium sensitivity in lymphoblastoid cell lines (LCLs) from lithium responder (R) and non-responder (NR) bipolar patients. Moreover, we evaluated levels of microRNA let-7c, a small RNA predicted to target IGF1. We found that exogenous IGF-1 added to serum-free media increased lithium sensitivity selectively in LCLs from NR BD patients. However, no significant differences were observed when comparing let-7c expression in LCLs from R vs. NR BD patients. Our data support a key role for IGF-1 in lithium resistance/response in the treatment of bipolar disorder. PMID:25740013

  19. Growth of diploid, Epstein-Barr virus-carrying human lymphoblastoid cell lines heterotransplanted into nude mice under immunologically privileged conditions.

    PubMed

    Giovanella, B; Nilsson, K; Zech, L; Yim, O; Klein, G; Stehlin, J S

    1979-07-15

    Human Epstein-Barr virus-carrying lymphoid cell lines which have been classified on the basis of studies on clonality and morphological, chromosomal and functional parameters as lymphoblastoid cell lines (LCL) of presumed non-neoplastic origin were inoculated intracerebrally into nude mice. All eighteen of them grew, killing the host mice within 7 to 25 days, except for 2 which grew more slowly. At autopsy, the brain of the nudes was found to be invaded by infiltrating lymphomas. Sixteen of these lymphomas, when recultured in vitro, gave rise to cell lines with growth properties and morphology indistinguishable from those of the inoculated LCL. Chromosomal examinations showed that 3/7 cell lines injected, which grew as lymphomas in the brain, were still normal diploid on reexplantation whereas the remaining four had become aneuploid. Four lines derived from intracerebral lymphomas (2 diploid, 1 aneuploid and 1 untested) were inoculated subcutaneously into adult nude mice. None of them grew. When the corresponding four original LCL lines were inoculated subcutaneously into newborn nude mice, they grew rapidly, but failed to do so in newborn normal mice or intracerebrally in adult normal mice. One such line, U-1450, was treated with anti-lymphocyte serum (ALS). Small nodules developed at the site of inoculation. From one nodule a cell line was cultured, 1450 ALSAD. It was morphologically indistinguishable from the line of origin. The lines obtained from nude mice inoculated with polyclonal LCL seem to have a restricted clonal representation, but were not monoclonal, as evidenced by analyses of their pattern of immunoglobulin synthesis. PMID:225282

  20. Whole genome and normalized mRNA sequencing reveal genetic status of TK6, WTK1, and NH32 human B-lymphoblastoid cell lines.

    PubMed

    Revollo, Javier; Petibone, Dayton M; McKinzie, Page; Knox, Bridgett; Morris, Suzanne M; Ning, Baitang; Dobrovolsky, Vasily N

    2016-01-01

    Closely related TK6, WTK1, and NH32 human B-lymphoblastoid cell lines differ in their p53 functional status. These lines are used frequently in genotoxicity studies and in studies aimed at understanding the role of p53 in DNA repair. Despite their routine use, little is known about the genetic status of these cells. To provide insight into their genetic composition, we sequenced and analyzed the entire genome of TK6 cells, as well as the normalized transcriptomes of TK6, WTK1, and NH32 cells. Whole genome sequencing (WGS) identified 21,561 genes and 5.17×10(6) small variants. Within the small variants, 50.54% were naturally occurring single nucleotide polymorphisms (SNPs) and 49.46% were mutations. The mutations were comprised of 92.97% single base-pair substitutions and 7.03% insertions or deletions (indels). The number of predicted genes, SNPs, and small mutations are similar to frequencies observed in the human population in general. Normalized mRNA-seq analysis identified the expression of transcripts bearing SNPs or mutations for TK6, WTK1, and NH32 as 2.88%, 2.04%, and 1.71%, respectively, and several of the variant transcripts identified appear to have important implications in genetic toxicology. These include a single base deletion mutation in the ferritin heavy chain gene (FTH1) resulting in a frame shift and protein truncation in TK6 that impairs iron metabolism. SNPs in the thiopurine S-methyltransferase (TPMT) gene (TPMT*3A SNP), and in the xenobiotic metabolizing enzyme, NADPH quinine oxidoreductase 1 (NQO1) gene (NQO1*2 SNP), are both associated with decreased enzyme activity. The clinically relevant TPMT*3A and NQO1*2 SNPs can make these cell lines useful in pharmacogenetic studies aimed at improving or tailoring drug treatment regimens that minimize toxicity and enhance efficacy. PMID:26774668

  1. Effect of oxidative stress on DNA damage and beta-amyloid precursor proteins in lymphoblastoid cell lines from a Nigerian population.

    PubMed

    Lahiri, D K; Xu, Y; Klaunig, J; Baiyewu, O; Ogunniyi, A; Hall, K; Hendrie, H; Sahota, A

    1999-01-01

    The epsilon 4 allele of apolipoprotein E (APOE) is strongly associated with late-onset Alzheimer's disease (AD) in Caucasian populations, but our studies suggest that APOE epsilon 4 is not a risk factor for AD in Nigerian blacks and is a weak risk factor in African-Americans. The prevalence of AD is lower in Nigerians than in African-Americans. Increased oxidative damage to macromolecules in brain tissue by reactive oxygen species (ROS) has been reported in AD. Here we examined the effects of endogenous and induced oxidative stress on total (nuclear and mitochondrial) DNA damage in lymphoblastoid cell lines (5 probable AD and 3 controls) from Ibadan, Nigeria. Cells were exposed to 200 microM t-butyl peroxide (a generator of ROS) for 4 hours. Total DNA was isolated and digested with nuclease P1 and alkaline phosphatase. DNA fragments were separated by HPLC and the levels of 8-hydroxy-2'-deoxyguanosine (OH8dG, an indicator of DNA damage) and deoxyguanosine (dG) determined. We did not detect a significant difference in the OH8dG/dG ratio in untreated or treated cell lines in the two groups, and this was independent of APOE genotype. We also examined, by Western blotting, the level of beta-amyloid precursor protein (APP) which is involved in AD. The level of the heat shock protein (HSP-70) was examined as a control. There was a slight decrease in levels of APP and HSP-70 following treatment. Studies in cell lines from Caucasian subjects have shown an increase in mitochondrial DNA damage following oxidative challenge. Our preliminary results suggest that African populations are less vulnerable to chemical-induced oxidative DNA damage. PMID:10672260

  2. Human B lymphoblastoid cells contain distinct patterns of cathepsin activity in endocytic compartments and regulate MHC class II transport in a cathepsin S-independent manner.

    PubMed

    Lautwein, Alfred; Kraus, Marianne; Reich, Michael; Burster, Timo; Brandenburg, J; Overkleeft, Herman S; Schwarz, Gerold; Kammer, Winfried; Weber, Ekkehard; Kalbacher, Hubert; Nordheim, Alfred; Driessen, Christoph

    2004-05-01

    Endocytic proteolysis represents a major functional component of the major histocompatibility complex class II antigen-presentation machinery. Although transport and assembly of class II molecules in the endocytic compartment are well characterized, we lack information about the pattern of endocytic protease activity along this pathway. Here, we used chemical tools that visualize endocytic proteases in an activity-dependent manner in combination with subcellular fractionation to dissect the subcellular distribution of the major cathepsins (Cat) CatS, CatB, CatH, CatD, CatC, and CatZ as well as the asparagine-specific endoprotease (AEP) in human B-lymphoblastoid cells (BLC). Endocytic proteases were distributed in two distinct patterns: CatB and CatZ were most prominent in early and late endosomes but absent from lysosomes, and CatH, CatS, CatD, CatC, and AEP distributed between late endosomes and lysosomes, suggesting that CatB and CatZ might be involved in the initial proteolytic attack on a given antigen. The entire spectrum of protease activity colocalized with human leukocyte antigen-DM and the C-terminal and N-terminal processing of invariant chain (Ii) in late endosomes. CatS was active in all endocytic compartments. Surprisingly and in contrast with results from dendritic cells, inhibition of CatS activity by leucine-homophenylalanine-vinylsulfone-phenol prevented N-terminal processing of Ii but did not alter the subcellular trafficking or surface delivery of class II complexes, as deferred from pulse-chase analysis in combination with subcellular fractionation and biotinylation of cell-surface protein. Thus, BLC contain distinct activity patterns of proteases in endocytic compartments and regulate the intracellular transport and surface-delivery of class II in a CatS-independent manner. PMID:14966190

  3. Necrosis is increased in lymphoblastoid cell lines from children with autism compared with their non-autistic siblings under conditions of oxidative and nitrosative stress.

    PubMed

    Main, Penelope A E; Thomas, Philip; Esterman, Adrian; Fenech, Michael F

    2013-07-01

    Autism spectrum disorders are a heterogeneous group of neurodevelopmental conditions characterised by impairments in reciprocal social interaction, communication and stereotyped behaviours. As increased DNA damage events have been observed in a range of other neurological disorders, it was hypothesised that they would be elevated in lymphoblastoid cell lines (LCLs) obtained from children with autism compared with their non-autistic siblings. Six case-sibling pairs of LCLs from children with autistic disorder and their non-autistic siblings were obtained from the Autism Genetic Resource Exchange (AGRE) and cultured in standard RPMI-1640 tissue culture medium. Cells were exposed to medium containing either 0, 25, 50, 100 and 200 µM hydrogen peroxide (an oxidative stressor) or 0, 5, 10, 20 and 40 µM s-nitroprusside (a nitric oxide producer) for 1h. Following exposure, the cells were microscopically scored for DNA damage, cytostasis and cytotoxicity biomarkers as measured using the cytokinesis-block micronucleus cytome assay. Necrosis was significantly increased in cases relative to controls when exposed to oxidative and nitrosative stress (P = 0.001 and 0.01, respectively). Nuclear division index was significantly lower in LCLs from children with autistic disorder than their non-autistic siblings when exposed to hydrogen peroxide (P = 0.016), but there was no difference in apoptosis, micronucleus frequency, nucleoplasmic bridges or nuclear buds. Exposure to s-nitroprusside significantly increased the number of micronuclei in non-autistic siblings compared with cases (P = 0.003); however, other DNA damage biomarkers, apoptosis and nuclear division did not differ significantly between groups. The findings of this study show (i) that LCLs from children with autism are more sensitive to necrosis under conditions of oxidative and nitrosative stress than their non-autistic siblings and (ii) refutes the hypothesis that children with autistic disorder are abnormally

  4. Necrosis is increased in lymphoblastoid cell lines from children with autism compared with their non-autistic siblings under conditions of oxidative and nitrosative stress

    PubMed Central

    Fenech, Michael F.

    2013-01-01

    Autism spectrum disorders are a heterogeneous group of neurodevelopmental conditions characterised by impairments in reciprocal social interaction, communication and stereotyped behaviours. As increased DNA damage events have been observed in a range of other neurological disorders, it was hypothesised that they would be elevated in lymphoblastoid cell lines (LCLs) obtained from children with autism compared with their non-autistic siblings. Six case–sibling pairs of LCLs from children with autistic disorder and their non-autistic siblings were obtained from the Autism Genetic Resource Exchange (AGRE) and cultured in standard RPMI-1640 tissue culture medium. Cells were exposed to medium containing either 0, 25, 50, 100 and 200 µM hydrogen peroxide (an oxidative stressor) or 0, 5, 10, 20 and 40 µM s-nitroprusside (a nitric oxide producer) for 1h. Following exposure, the cells were microscopically scored for DNA damage, cytostasis and cytotoxicity biomarkers as measured using the cytokinesis-block micronucleus cytome assay. Necrosis was significantly increased in cases relative to controls when exposed to oxidative and nitrosative stress (P = 0.001 and 0.01, respectively). Nuclear division index was significantly lower in LCLs from children with autistic disorder than their non-autistic siblings when exposed to hydrogen peroxide (P = 0.016), but there was no difference in apoptosis, micronucleus frequency, nucleoplasmic bridges or nuclear buds. Exposure to s-nitroprusside significantly increased the number of micronuclei in non-autistic siblings compared with cases (P = 0.003); however, other DNA damage biomarkers, apoptosis and nuclear division did not differ significantly between groups. The findings of this study show (i) that LCLs from children with autism are more sensitive to necrosis under conditions of oxidative and nitrosative stress than their non-autistic siblings and (ii) refutes the hypothesis that children with autistic disorder are abnormally

  5. The effect of zinc sulphate and zinc carnosine on genome stability and cytotoxicity in the WIL2-NS human lymphoblastoid cell line.

    PubMed

    Sharif, Razinah; Thomas, Philip; Zalewski, Peter; Graham, Robin D; Fenech, Michael

    2011-02-28

    Zinc (Zn) is an essential cofactor required by numerous enzymes that are essential for cell metabolism and the maintenance of DNA integrity. We investigated the effect of Zn deficiency or excess on genomic instability events and determined the optimal concentration of two Zn compounds that minimize DNA-damage events. The effects of Zn sulphate (ZnSO(4)) and Zn carnosine (ZnC) on cell proliferation were investigated in the WIL2-NS human lymphoblastoid cell line. DNA damage was determined by the use of both the comet assay and the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. Zn-deficient medium (0μM) was produced using Chelex treatment, and the two Zn compounds (i.e. ZnSO(4) and ZnC) were tested at concentrations of 0.0, 0.4, 4.0, 16.0, 32.0 and 100.0μM. Results from an MTT assay showed that cell growth and viability were decreased in Zn-depleted cells (0μM) as well as at 32μM and 100μM for both Zn compounds (P<0.0001). DNA strand-breaks, as measured by the comet assay, were found to be increased in Zn-depleted cells compared with the other treatment groups (P<0.05). The CBMN-Cyt assay showed a significant increase in the frequency of both apoptotic and necrotic cells under Zn-deficient conditions (P<0.0001). Elevated frequencies of micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBuds) were induced in Zn-depleted cells (P<0.0001), whereas genome damage was reduced in supplemented cultures for both Zn compounds at 4μM and 16μM, possibly suggesting that these concentrations may be optimal for genome stability. The potential protective effect of ZnSO(4) and ZnC was also investigated following exposure to 1.0Gy γ-radiation. Culture in medium containing these compounds at 4-32μM prior to irradiation displayed significantly reduced frequencies of MNi, NPBs and NBuds compared with cells maintained in 0μM medium (P<0.0001). Expression of γ-H2AX and 8-oxoguanine glycosylase measured by western blotting was increased in Zn

  6. Ciprofloxacin-induced G2 arrest and apoptosis in TK6 lymphoblastoid cells is not dependent on DNA double-strand break formation

    PubMed Central

    Smart, Daniel J.; Halicka, H. Dorota; Traganos, Frank; Darzynkiewicz, Zbigniew; Williams, Gary M.

    2008-01-01

    Drugs developed for the treatment of conditions other than neoplasia can also show promise as potential antitumor agents. The fluoroquinolone antibiotic ciprofloxacin (CPFX) is known to modulate cycle cell progression and apoptosis in cancer cells, and is thought to induce DNA double-strand breaks (DSBs) via topoisomerase II (topo II) inhibition and stabilized cleavage complex (SCC) formation. DSBs trigger Ser-139 phosphorylation of histone H2AX (γH2AX) by PI-3-like kinases including ATM; γH2AX can serve as a marker of DNA damage when measured in situ using immunocytochemistry and flow cytometry. The aim of the present study was to investigate the relationship between CPFX-mediated DNA damage and induction of apoptosis in human lymphoblastoid cells and phytohaemagglutinin (PHA)-stimulated lymphocytes (Lymphs). Treatment of TK6 cells (wild-type p53) with 100 µg/ml CPFX for 2-10 h produced no increase in γH2AX; to the contrary, its level in S phase cells was reduced at 10 h compared to controls. Nevertheless, stabilization of topo IIα, ATM Ser-1981 phosphorylation and G2 arrest was observed in TK6 cells exposed to CPFX for ≥4 h. However, following 24 h treatment, γH2AX was dramatically increased in a sub-population of cells indicating the onset of apoptosis (confirmed by presence of activated caspase 3). CPFX had a similar lack of effect on induction of γH2AX at early time points in WTK1 and NH32 cells (devoid of functional p53) and proliferating Lymphs, however, induction of apoptosis was less pronounced than in TK6 cells. Formation of SCC and activation of ATM (but lack of γH2AX induction) indicates topo II-mediated chromatin or DNA changes in the absence of DSBs; ATM activation apparently triggers the G2M checkpoint leading to G2 arrest. The subsequent induction of apoptosis appears to be facilitated by functional p53. CPFX may therefore have a potential use as a chemotherapeutic agent in the treatment of lymphoblast-derived cancer. PMID:18059176

  7. A Study of Alterations in DNA Epigenetic Modifications (5mC and 5hmC) and Gene Expression Influenced by Simulated Microgravity in Human Lymphoblastoid Cells.

    PubMed

    Chowdhury, Basudev; Seetharam, Arun; Wang, Zhiping; Liu, Yunlong; Lossie, Amy C; Thimmapuram, Jyothi; Irudayaraj, Joseph

    2016-01-01

    Cells alter their gene expression in response to exposure to various environmental changes. Epigenetic mechanisms such as DNA methylation are believed to regulate the alterations in gene expression patterns. In vitro and in vivo studies have documented changes in cellular proliferation, cytoskeletal remodeling, signal transduction, bone mineralization and immune deficiency under the influence of microgravity conditions experienced in space. However microgravity induced changes in the epigenome have not been well characterized. In this study we have used Next-generation Sequencing (NGS) to profile ground-based "simulated" microgravity induced changes on DNA methylation (5-methylcytosine or 5mC), hydroxymethylation (5-hydroxymethylcytosine or 5hmC), and simultaneous gene expression in cultured human lymphoblastoid cells. Our results indicate that simulated microgravity induced alterations in the methylome (~60% of the differentially methylated regions or DMRs are hypomethylated and ~92% of the differentially hydroxymethylated regions or DHMRs are hyperhydroxymethylated). Simulated microgravity also induced differential expression in 370 transcripts that were associated with crucial biological processes such as oxidative stress response, carbohydrate metabolism and regulation of transcription. While we were not able to obtain any global trend correlating the changes of methylation/ hydroxylation with gene expression, we have been able to profile the simulated microgravity induced changes of 5mC over some of the differentially expressed genes that includes five genes undergoing differential methylation over their promoters and twenty five genes undergoing differential methylation over their gene-bodies. To the best of our knowledge, this is the first NGS-based study to profile epigenomic patterns induced by short time exposure of simulated microgravity and we believe that our findings can be a valuable resource for future explorations. PMID:26820575

  8. A Study of Alterations in DNA Epigenetic Modifications (5mC and 5hmC) and Gene Expression Influenced by Simulated Microgravity in Human Lymphoblastoid Cells

    PubMed Central

    Wang, Zhiping; Liu, Yunlong; Lossie, Amy C.; Thimmapuram, Jyothi; Irudayaraj, Joseph

    2016-01-01

    Cells alter their gene expression in response to exposure to various environmental changes. Epigenetic mechanisms such as DNA methylation are believed to regulate the alterations in gene expression patterns. In vitro and in vivo studies have documented changes in cellular proliferation, cytoskeletal remodeling, signal transduction, bone mineralization and immune deficiency under the influence of microgravity conditions experienced in space. However microgravity induced changes in the epigenome have not been well characterized. In this study we have used Next-generation Sequencing (NGS) to profile ground-based “simulated” microgravity induced changes on DNA methylation (5-methylcytosine or 5mC), hydroxymethylation (5-hydroxymethylcytosine or 5hmC), and simultaneous gene expression in cultured human lymphoblastoid cells. Our results indicate that simulated microgravity induced alterations in the methylome (~60% of the differentially methylated regions or DMRs are hypomethylated and ~92% of the differentially hydroxymethylated regions or DHMRs are hyperhydroxymethylated). Simulated microgravity also induced differential expression in 370 transcripts that were associated with crucial biological processes such as oxidative stress response, carbohydrate metabolism and regulation of transcription. While we were not able to obtain any global trend correlating the changes of methylation/ hydroxylation with gene expression, we have been able to profile the simulated microgravity induced changes of 5mC over some of the differentially expressed genes that includes five genes undergoing differential methylation over their promoters and twenty five genes undergoing differential methylation over their gene-bodies. To the best of our knowledge, this is the first NGS-based study to profile epigenomic patterns induced by short time exposure of simulated microgravity and we believe that our findings can be a valuable resource for future explorations. PMID:26820575

  9. Oxidative stress induces mitochondrial dysfunction in a subset of autism lymphoblastoid cell lines in a well-matched case control cohort.

    PubMed

    Rose, Shannon; Frye, Richard E; Slattery, John; Wynne, Rebecca; Tippett, Marie; Pavliv, Oleksandra; Melnyk, Stepan; James, S Jill

    2014-01-01

    There is increasing recognition that mitochondrial dysfunction is associated with the autism spectrum disorders. However, little attention has been given to the etiology of mitochondrial dysfunction or how mitochondrial abnormalities might interact with other physiological disturbances associated with autism, such as oxidative stress. In the current study we used respirometry to examine reserve capacity, a measure of the mitochondrial ability to respond to physiological stress, in lymphoblastoid cell lines (LCLs) derived from children with autistic disorder (AD) as well as age and gender-matched control LCLs. We demonstrate, for the first time, that LCLs derived from children with AD have an abnormal mitochondrial reserve capacity before and after exposure to increasingly higher concentrations of 2,3-dimethoxy-1,4-napthoquinone (DMNQ), an agent that increases intracellular reactive oxygen species (ROS). Specifically, the AD LCLs exhibit a higher reserve capacity at baseline and a sharper depletion of reserve capacity when ROS exposure is increased, as compared to control LCLs. Detailed investigation indicated that reserve capacity abnormalities seen in AD LCLs were the result of higher ATP-linked respiration and maximal respiratory capacity at baseline combined with a marked increase in proton leak respiration as ROS was increased. We further demonstrate that these reserve capacity abnormalities are driven by a subgroup of eight (32%) of 25 AD LCLs. Additional investigation of this subgroup of AD LCLs with reserve capacity abnormalities revealed that it demonstrated a greater reliance on glycolysis and on uncoupling protein 2 to regulate oxidative stress at the inner mitochondria membrane. This study suggests that a significant subgroup of AD children may have alterations in mitochondrial function which could render them more vulnerable to a pro-oxidant microenvironment derived from intrinsic and extrinsic sources of ROS such as immune activation and pro

  10. Analysis of the tumorigenic potential of common marmoset lymphoblastoid cells expressing a constitutively activated c-myc gene.

    PubMed Central

    Hotchin, N. A.; Wedderburn, N.; Roberts, I.; Thomas, J. A.; Bungey, J. A.; Naylor, B.; Crawford, D. H.

    1993-01-01

    The respective roles of Epstein-Barr virus (EBV) and c-myc in the pathogenesis of endemic Burkitt's lymphoma (BL) are unclear. In order to help resolve the question whether constitutive expression of the c-myc gene in an EBV-immortalised B cell is sufficient to induce a tumorigenic phenotype, B cells from a common marmoset (Callithrix jacchus) were immortalised with EBV, transfected with a constitutively activated c-myc gene and inoculated into the host animals. Despite the cell line transfected with c-myc displaying enhanced growth characteristics, in vitro and in vivo experiments demonstrated that this was not sufficient to induce a tumorigenic phenotype. This supports our previous findings with EBV-immortalised human B cells transfected with an activated c-myc gene (Hotchin et al., 1990). Images Figure 1 Figure 2 Figure 4 PMID:8388232

  11. Effects of maglev-spectrum magnetic field exposure on CEM T-lymphoblastoid human cell growth and differentiation

    SciTech Connect

    Groh, K.R.; Chubb, C.B.; Collart, F.R.; Huberman, E.

    1992-01-01

    Exposure to magnetic fields similar to those produced by maglev vehicles (combined ac and dc components) was studied for the ability to alter cell growth and chemically induced cellular differentiation processes in cultured human CEM Tlymphoblastoid leukemia cells. A series of continuous and intermittent magnetic field (MF) exposures for varying lengths of time were tested at intensities up to 7-fold greater than that produced by the German TR07 maglev vehicle. Phorbol 12-myristate 13-acetate or mycophenolic acid were used to induce cell differentiation. Changes in cell number, morphology, and fluorescence expression of antigenic markers of differentiation were monitored. The results indicated that maglev-spectrum magnetic field exposures up to 2 gauss had little effect on culture growth or chemically induced cellular differentiation when exposed to maglev-spectrum magnetic fields compared to chemically treated but MF-unexposed controls.

  12. Interferon-dependent induction of mRNA for the major histocompatibility antigens in human fibroblasts and lymphoblastoid cells.

    PubMed Central

    Fellous, M; Nir, U; Wallach, D; Merlin, G; Rubinstein, M; Revel, M

    1982-01-01

    In human cells treated with interferons, there is an increase in the amount of HLA-A,B,C and beta 2-microglobulin exposed on the cell surface. We have used a cloned HLA-A,B,C cDNA probe to demonstrate by molecular hybridization that this effect of interferon is preceded by a large increase in the amount of HLA mRNA in the cell. This effect was found in five different human cell lines, with purified leukocyte and fibroblast interferons. The increase in HLA mRNA is comparable in its kinetics and dose-response to the induction of (2'-5') oligo(A) synthetase mRNA by interferons. Therefore, interferons seem to activate at least two cellular genes which have different biochemical functions. Images PMID:6179076

  13. Mutagenicity and clastogenicity of extracts of Helicobacter pylori detected by the Ames test and in the micronucleus test using human lymphoblastoid cells.

    PubMed

    Arimoto-Kobayashi, Sakae; Ohta, Kaori; Yuhara, Yuta; Ayabe, Yuka; Negishi, Tomoe; Okamoto, Keinosuke; Nakajima, Yoshihiro; Ishikawa, Takeshi; Oguma, Keiji; Otsuka, Takanao

    2015-07-01

    Epidemiological studies have demonstrated a close association between infection with Helicobacter pylori (H.pylori) and the development of gastric carcinoma. Chronic H.pylori infection increases the frequency of mutation in gastric epithelial cells. However, the mechanism by which infection of H.pylori leads to mutation in gastric epithelial cells is unclear. We suspected that components in H.pylori may be related to the mutagenic response associated with DNA alkylation, and could be detected with the Ames test using a more sensitive strain for alkylating agents. Our investigation revealed that an extract of H.pylori was mutagenic in the Ames test with Salmonella typhimurium YG7108, which is deficient in the DNA repair of O(6)-methylguanine. The extract of H.pylori may contain methylating or alkylating agents, which might induce O (6)-alkylguanine in DNA. Mutagenicity of the alkylating agents N-methyl-N-nitrosourea (MNU) and N-methyl-N'-nitro-N-nitrosoguanidine in the Ames test with S.typhimurium TA1535 was enhanced significantly in the presence of the extract of H.pylori. The tested extracts of H.pylori resulted in a significant induction of micronuclei in human-derived lymphoblastoid cells. Heat instability and dialysis resistance of the extracts of H.pylori suggest that the mutagenic component in the extracts of H.pylori is a heat-unstable large molecule or a heat-labile small molecule strongly attached or adsorbed to a large molecule. Proteins in the extracts of H.pylori were subsequently fractionated using ammonium sulphate precipitation. However, all fractions expressed enhancing effects toward MNU mutagenicity. These results suggest the mutagenic component is a small molecule that is absorbed into proteins in the extract of H.pylori, which resist dialysis. Continuous and chronic exposure of gastric epithelial cells to the alkylative mutagenic component from H.pylori chronically infected in the stomach might be a causal factor in the gastric carcinogenesis

  14. Induction of anti-EBNA-1 protein by 12-O-tetradecanoylphorbol-13-acetate treatment of human lymphoblastoid cells

    SciTech Connect

    Wen, Longthung; Tanaka, Akiko; Nonoyama, Meihan )

    1989-08-01

    Binding of the Epstein-Barr virus (EBV) nuclear antigen (EBNA-1) to BamHI-C DNA was studied by affinity column chromatography followed by immunoblotting with human serum specific for EBNA-1. Two species of EBNA-1 (68 and 70 kilodaltons) were identified in nuclear extracts of the EBV-positive Burkitt's lymphoma cell line Raji and not in nuclear extracts of the EBV-negative Burkitt's lymphoma cell line BJAB. Both EBNA-1s bound specifically to the region required for EBV plasmid DNA maintenance (oriP) located in the BamHI-C fragment. Upon treatment with 12-O-tetradecanoylphorbol-13-acetate, which activates latent EBV genome in Raji cells, the 68-kilodalton EBNA-1 was uncoupled from binding to EBV oriP. Nuclear extracts from 12-O-tetradecanoylphorbol-13-acetate-treated BJAB cells also uncoupled the binding of both EBNA-1s to oriP. DNA-cellulose column chromatography identified two protein species which competed for and uncoupled the binding of EBNA-1 to oriP. The two cellular competitors the authors called anti-EBNA-1 proteins had molecular masses of 60 and 40 kilodaltons, respectively. They were not found in nuclear extracts of BJAB cells not activated by 12-O-tetradecanoylphorbol-13-acetate.

  15. Epitope analysis of peanut allergen Ara h1 with oligoclonal IgM antibody from human B-lymphoblastoid cells.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To analyze epitopes of peanut allergen Ara h1, Epstein-Barr virus-transformed human peripheral oligoclonal B-cells were cultured to obtain antibodies to Ara h1. The combined reaction pattern with six oligoclonal antibodies showed there were six antibody binding areas named a to f in Ara h1. We found...

  16. Epstein-Barr Viruses (EBVs) Deficient in EBV-Encoded RNAs Have Higher Levels of Latent Membrane Protein 2 RNA Expression in Lymphoblastoid Cell Lines and Efficiently Establish Persistent Infections in Humanized Mice

    PubMed Central

    Gregorovic, Goran; Boulden, Elizabeth A.; Bosshard, Rachel; Elgueta Karstegl, Claudio; Skalsky, Rebecca; Cullen, Bryan R.; Gujer, Cornelia; Rämer, Patrick; Münz, Christian

    2015-01-01

    Functions of Epstein-Barr virus (EBV)-encoded RNAs (EBERs) were tested in lymphoblastoid cell lines containing EBER mutants of EBV. Binding of EBER1 to ribosomal protein L22 (RPL22) was confirmed. Deletion of EBER1 or EBER2 correlated with increased levels of cytoplasmic EBV LMP2 RNA and with small effects on specific cellular microRNA (miRNA) levels, but protein levels of LMP1 and LMP2A were not affected. Wild-type EBV and EBER deletion EBV had approximately equal abilities to infect immunodeficient mice reconstituted with a human hematopoietic system. PMID:26339045

  17. Epstein-Barr Viruses (EBVs) Deficient in EBV-Encoded RNAs Have Higher Levels of Latent Membrane Protein 2 RNA Expression in Lymphoblastoid Cell Lines and Efficiently Establish Persistent Infections in Humanized Mice.

    PubMed

    Gregorovic, Goran; Boulden, Elizabeth A; Bosshard, Rachel; Elgueta Karstegl, Claudio; Skalsky, Rebecca; Cullen, Bryan R; Gujer, Cornelia; Rämer, Patrick; Münz, Christian; Farrell, Paul J

    2015-11-01

    Functions of Epstein-Barr virus (EBV)-encoded RNAs (EBERs) were tested in lymphoblastoid cell lines containing EBER mutants of EBV. Binding of EBER1 to ribosomal protein L22 (RPL22) was confirmed. Deletion of EBER1 or EBER2 correlated with increased levels of cytoplasmic EBV LMP2 RNA and with small effects on specific cellular microRNA (miRNA) levels, but protein levels of LMP1 and LMP2A were not affected. Wild-type EBV and EBER deletion EBV had approximately equal abilities to infect immunodeficient mice reconstituted with a human hematopoietic system. PMID:26339045

  18. Cis-regulation of IRF5 expression is unable to fully account for systemic lupus erythematosus association: analysis of multiple experiments with lymphoblastoid cell lines

    PubMed Central

    2011-01-01

    Introduction Interferon regulatory factor 5 gene (IRF5) polymorphisms are strongly associated with several diseases, including systemic lupus erythematosus (SLE). The association includes risk and protective components. They could be due to combinations of functional polymorphisms and related to cis-regulation of IRF5 expression, but their mechanisms are still uncertain. We hypothesised that thorough testing of the relationships between IRF5 polymorphisms, expression data from multiple experiments and SLE-associated haplotypes might provide useful new information. Methods Expression data from four published microarray hybridisation experiments with lymphoblastoid cell lines (57 to 181 cell lines) were retrieved. Genotypes of 109 IRF5 polymorphisms, including four known functional polymorphisms, were considered. The best linear regression models accounting for the IRF5 expression data were selected by using a forward entry procedure. SLE-associated IRF5 haplotypes were correlated with the expression data and with the best cis-regulatory models. Results A large fraction of variability in IRF5 expression was accounted for by linear regression models with IRF5 polymorphisms, but at a different level in each expression data set. Also, the best models from each expression data set were different, although there was overlap between them. The SNP introducing an early polyadenylation signal, rs10954213, was included in the best models for two of the expression data sets and in good models for the other two data sets. The SLE risk haplotype was associated with high IRF5 expression in the four expression data sets. However, there was also a trend towards high IRF5 expression with some protective and neutral haplotypes, and the protective haplotypes were not associated with IRF5 expression. As a consequence, correlation between the cis-regulatory best models and SLE-associated haplotypes, regarding either the risk or protective component, was poor. Conclusions Our analysis

  19. Different capacities for recombination in closely related human lymphoblastoid cell lines with different mutational responses to X-irradiation.

    PubMed Central

    Xia, F; Amundson, S A; Nickoloff, J A; Liber, H L

    1994-01-01

    WIL2-NS and TK6 are two distinct human lymphoblast cell lines derived from a single male donor. WIL2-NS cells are significantly more resistant to the cytotoxic effects of X-irradiation but considerably more sensitive to induced mutation. In an effort to determine the mechanistic basis for these differences, we analyzed the physical structures of thymidine kinase (tk)-deficient mutants isolated after X-ray treatment of tk heterozygotes derived from TK6 and the more mutable WIL2-NS. Southern analysis showed that while 84% of TK6-derived mutants had arisen by loss of heterozygosity (LOH), all 106 mutants from WIL2-NS derivatives arose with LOH at tk and all but one showed LOH at other linked loci on chromosome 17. We adapted a fluorescence in situ hybridization technique to distinguish between LOH due to deletion, which results in retention of only one tk allele, and LOH due to a mechanism involving the homologous chromosome (e.g., recombination), which results in the retention of two alleles. Among the LOH mutants derived that were analyzed in this way, 9 of 26 from WIL2-NS and 11 of 17 from TK6 cell lines arose by deletion. The remaining mutants retained two copies of the tk gene and thus arose by a mechanism involving the homologous allele. Since many of these mutants arising by a homologous mechanism retained partial heterozygosity of chromosome 17, they must have arisen by recombination or gene conversion, and not chromosome loss and reduplication. Finally, the recombinational capacities of WIL2-NS and TK6 were compared in transfection assays with plasmid recombination substrates. Intermolecular recombination frequencies were greater in WIL2-NS than in TK6. These data are consistent with a model suggesting that a recombinational repair system is functioning at a higher level in WIL2-NS than in TK6; the greater mutability of the tk locus in WIL2-NS results from more frequent inter- and intramolecular recombination events. Images PMID:8065318

  20. The NRF2-KEAP1 Pathway Is an Early Responsive Gene Network in Arsenic Exposed Lymphoblastoid Cells

    PubMed Central

    Córdova, Emilio J.; Martínez-Hernández, Angélica; Uribe-Figueroa, Laura; Centeno, Federico; Morales-Marín, Mirna; Koneru, Harsha; Coleman, Matthew A.; Orozco, Lorena

    2014-01-01

    Inorganic arsenic (iAs), a major environmental contaminant, has risen as an important health problem worldwide. More detailed identification of the molecular mechanisms associated with iAs exposure would help to establish better strategies for prevention and treatment. Although chronic iAs exposures have been previously studied there is little to no information regarding the early events of exposure to iAs. To better characterize the early mechanisms of iAs exposure we conducted gene expression studies using sublethal doses of iAs at two different time-points. The major transcripts differentially regulated at 2 hrs of iAs exposure included antioxidants, detoxificants and chaperones. Moreover, after 12 hrs of exposure many of the down-regulated genes were associated with DNA replication and S phase cell cycle progression. Interestingly, the most affected biological pathway by both 2 or 12 hrs of iAs exposure were the Nrf2-Keap1 pathway, represented by the highly up-regulated HMOX1 transcript, which is transcriptionally regulated by the transcription factor Nrf2. Additional Nrf2 targets included SQSTM1 and ABCB6, which were not previously associated with acute iAs exposure. Signalling pathways such as interferon, B cell receptor and AhR route were also responsive to acute iAs exposure. Since HMOX1 expression increased early (20 min) and was responsive to low iAs concentrations (0.1 µM), this gene could be a suitable early biomarker for iAs exposure. In addition, the novel Nrf2 targets SQSTM1 and ABCB6 could play an important and previously unrecognized role in cellular protection against iAs. PMID:24516582

  1. Impaired 2',3'-dideoxy-3'-thiacytidine accumulation in T-lymphoblastoid cells as a mechanism of acquired resistance independent of multidrug resistant protein 4 with a possible role for ATP-binding cassette C11.

    PubMed Central

    Turriziani, O; Schuetz, J D; Focher, F; Scagnolari, C; Sampath, J; Adachi, M; Bambacioni, F; Riva, E; Antonelli, G

    2002-01-01

    Cellular factors may contribute to the decreased efficacy of chemotherapy in HIV infection. Indeed, prolonged treatment with nucleoside analogues, such as azidothymidine (AZT), 2',3'-deoxycytidine or 9-(2-phosphonylmethoxyethyl)adenine, induces cellular resistance. We have developed a human T lymphoblastoid cell line (CEM 3TC) that is selectively resistant to the antiproliferative effect of 2',3'-dideoxy-3'-thiacytidine (3TC) because the CEM 3TC cells were equally sensitive to AZT, as well as the antimitotic agent, vinblastine. The anti-retroviral activity of 3TC against HIV-1 was also severely impaired in the CEM 3TC cells. Despite similar deoxycytidine kinase activity and unchanged uptake of nucleosides such as AZT and 2'-deoxycytidine, CEM 3TC had profoundly impaired 3TC accumulation. Further studies indicated that CEM 3TC retained much less 3TC. However, despite a small overexpression of multidrug resistance protein (MRP) 4, additional studies with cells specifically engineered to overexpress MRP4 demonstrated there was no impact on either 3TC accumulation or efflux. Finally, an increased expression of the MRP5 homologue, ATP-binding cassette C11 (ABCC11) was observed in the CEM 3TC cells. We speculate that the decreased 3TC accumulation in the CEM 3TC might be due to the upregulation of ABCC11. PMID:12133003

  2. Inhibition of cell proliferation by interferons. 2. Changes in processing and stability of newly synthesized DNA in human lymphoblastoid (Daudi) cells.

    PubMed

    Moore, G; Gewert, D R; Clemens, M J

    1984-03-15

    The inhibition of proliferation of Daudi cells in culture by human interferons is characterized by a change in the kinetics of labelling of different size classes of newly synthesized DNA. Initially, labelled precursors are incorporated exclusively into small DNA (Okazaki fragments) in both control and interferon-treated cells, as revealed by alkaline sucrose gradient centrifugation. In the interferon-treated cells, there is enhanced labelling of this small DNA after short periods of incorporation and slower conversion to larger DNA size classes, in comparison with the DNA of control cells. This effect is apparent after 12 h of interferon treatment, coincident with the onset of the inhibition of cell proliferation. It becomes progressively more marked up to 4 days, by which time cell growth has ceased completely. Experiments using bromodeoxyuridine as a density label and analysis of radioactive DNA on caesium chloride/caesium sulphate gradients also reveal that some newly replicated DNA may be unstable and may turn over within a few hours of its synthesis. The label derived from DNA breakdown is efficiently reincorporated into newly synthesized molecules. It is suggested that interferon treatment inhibits DNA replication by activating DNA turnover rather than by directly inhibiting synthesis. This effect, together with the progressive retardation of conversion of Okazaki fragments to larger DNA, may lead to the eventual failure of cell proliferation. PMID:6698030

  3. Epstein-Barr virus nuclear protein 3C binds to the N-terminal (NTD) and beta trefoil domains (BTD) of RBP/CSL; Only the NTD interaction is essential for lymphoblastoid cell growth

    SciTech Connect

    Calderwood, Michael A.; Lee, Sungwook; Holthaus, Amy M.; Blacklow, Stephen C.; Kieff, Elliott; Johannsen, Eric

    2011-05-25

    Association of EBV nuclear proteins EBNA2, EBNA3A and EBNA3C with RBP/CSL, is essential for lymphoblastoid cell line (LCL) proliferation. Conserved residues in the EBNA3 homology domain, required for RBP/CSL interaction, lack the W{Phi}P motif that mediates EBNA2 and Notch binding to the RBP/CSL beta-trefoil domain (BTD). We map RBP/CSL interacting residues within EBNA3A(aa128-204) and EBNA3C(aa211-233). The EBNA3A results are consistent with an earlier report (aa125-222), but the EBNA3C domain is unexpectedly small and includes a 'WTP' sequence. This EBNA3C WTP motif confers RBP/CSL binding in vitro, in yeast, and in mammalian cells. Further, an EBNA3C WTP {yields} STP(W227S) mutation impaired BTD binding whereas EBNA3 homology domain mutations disrupted RBP/CSL N-terminal domain (NTD) binding. WTP was not essential for EBNA3C repression of EBNA2 in reporter assays or for maintenance of LCL growth. Our results indicate that EBNA3 proteins interact with multiple RBP/CSL domains, but only NTD interactions are required for LCL growth.

  4. Combination of SAHA and bortezomib up-regulates CDKN2A and CDKN1A and induces apoptosis of Epstein-Barr virus-positive Wp-restricted Burkitt lymphoma and lymphoblastoid cell lines.

    PubMed

    Hui, Kwai Fung; Leung, Yvonne Y; Yeung, Po L; Middeldorp, Jaap M; Chiang, Alan K S

    2014-12-01

    Epstein-Barr virus (EBV) latent proteins exert anti-apoptotic effects on EBV-transformed lymphoid cells by down-regulating BCL2L11 (BIM), CDKN2A (p16(INK4A) ) and CDKN1A (p21(WAF1) ). However, the potential therapeutic effects of targeting these anti-apoptotic mechanisms remain unexplored. Here, we tested both in vitro and in vivo effects of the combination of histone deacetylase (HDAC) and proteasome inhibitors on the apoptosis of six endemic Burkitt lymphoma (BL) lines of different latency patterns (types I and III and Wp-restricted) and three lymphoblastoid cell lines (LCLs). We found that the combination of HDAC and proteasome inhibitors (e.g. SAHA/bortezomib) synergistically induced the killing of Wp-restricted and latency III BL and LCLs but not latency I BL cells. The synergistic killing was due to apoptosis, as evidenced by the high percentage of annexin V positivity and strong cleavage of PARP1 (PARP) and CASP3 (caspase-3). Concomitantly, SAHA/bortezomib up-regulated the expression of CDKN2A and CDKN1A but did not affect the level of BCL2L11 or BHRF1 (viral homologue of BCL2). The apoptotic effects were dependent on reactive oxygen species generation. Furthermore, SAHA/bortezomib suppressed the growth of Wp-restricted BL xenografts in nude mice. This study provides the rationale to test the novel application of SAHA/bortezomib on the treatment of EBV-associated Wp-restricted BL and post-transplant lymphoproliferative disorder. PMID:25155625

  5. Characterization of a lymphoblastoid line deleted for lambda immunoglobulin genes

    SciTech Connect

    Hough, C.A., White, B.N., Holden, J.A.

    1995-04-01

    While characterizing the cat eye syndrome (CES) supernumerary chromosome for the presence of {lambda} immunoglobulin gene region sequences, a lymphoblastoid cell line from one CES patient was identified in which there was selection of cells deleted from some IGLC and IGLV genes. Two distinct deletions, one on each chromosome 22, were identified, presumably arising from independent somatic recombination events occurring during B-lymphocyte differentiation. The extent of the deleted regions was determined using probes from the various IGLV subgroups and they each covered at least 82 kilobases. The precise definition of the deletions was not possible because of conservation of some restriction sites in the IGLV region. The cell line was used to map putative IGLV genes within the recombinant phage {lambda}V{lambda}135 to the distal part of the IGLV gene region. 35 refs., 4 figs.

  6. Vitamin B12 and methionine deficiencies induce genome damage measured using the cytokinesis-block micronucleus cytome assay in human B lymphoblastoid cell lines.

    PubMed

    Wu, Xiayu; Cheng, Jiaoni; Lu, Lin

    2013-01-01

    One-carbon metabolism is a network of interrelated biochemical reactions that has 2 major functions: DNA methylation and DNA synthesis. Methionine (Met), an essential amino acid, is converted to S-adenosyl-methionine (SAM), the body's main methyl group donor, which is converted to S-adenosylhomocysteine during methylation reactions. Vitamin B12 (B12) acts as a coenzyme of methionine synthase, which is required for the synthesis of Met and SAM. To determine the effects of Met and B12, we used the cytokinesis-block micronucleus assay in GM13705 and GM12593 cell line cultures exposed to 13 unique combinations of B12 and Met concentrations over 9 days. The nutrient levels chosen span the normal physiological ranges in humans. The Met-B12 concentration significantly and negatively correlated with all markers of genotoxicity in the 2 cell lines tested. In both cell lines, all markers of genotoxicity were significantly higher when treated with 15 μM Met than when treated with 50 μM Met, regardless of the B12 treatment level. Genotoxicity was significantly reduced in the group treated with 50 μM Met and 600 pM B12. Concentrations of 50 μM Met and 600 pM B12 are an optimal combination for stabilizing the genome. It is advisable to acquire adequate amounts of Met and B12 for maintaining genome stability. PMID:23909731

  7. Induction of selective cytotoxicity and apoptosis in human T4-lymphoblastoid cell line (CEMss) by boesenbergin a isolated from boesenbergia rotunda rhizomes involves mitochondrial pathway, activation of caspase 3 and G2/M phase cell cycle arrest

    PubMed Central

    2013-01-01

    Background Boesenbergia rotunda (Roxb.) Schlecht (family zingiberaceae) is a rhizomatous herb that is distributed from north-eastern India to south-east Asia, especially in Indonesia, Thailand and Malaysia. Previous research has shown that the crude extract of this plant has cytotoxic properties. The current study examines the cytotoxic properties of boesenbergin A isolated from Boesenbergia rotunda. Methods MTT assay was used to check the cytotoxicity of boesenbergin A. The morphological assessment of apoptosis was monitored using normal and fluorescence microscopy. The early and late phase of apoptosis was investigated using annexin V and DNA laddering assays, respectively. The mitochondrial membrane potential (MMP) was assessed by fluorescence microscopy. Human apoptosis proteome profiler assays were performed to investigate the mechanism of cell death. In addition, the protein levels of Bax, Bcl2 and HSP 70 were also analyzed using western blot. Assays of caspase =-3/7, -8 and =-9 were carried out in order to test for induction during treatment. Lastly, cell cycle progression was analyzed using flow cytometry. Results Boesenbergin A was found to have the highest toxicity towards CEMss cancer cells (IC50 = 8 μg/ml). The morphology of CEMss cells after treatment showed evidence of apoptosis that included blebbing and chromatin condensation. The annexin V assay revealed that early apoptosis is induced after treatment. The DNA laddering assay confirmed that DNA fragmentation had occurred during late apoptosis. The cell cycle analysis indicated that boesenbergin A was able to induce G2/M phase arrest in CEMss cells. The activity of caspases -3/7, -8 and -9 was increased after treatment which indicates both intrinsic and extrinsic pathways are induced during apoptosis. The involvement of mitochondria was established by increased mitochondrial membrane potential and up and down regulation of Bcl2 and Bax proteins as well as HSP70. Conclusion In conclusion, the

  8. [Combination therapy with natural type human tumor necrosis factor (MHR-24) and human lymphoblastoid interferon-alpha (MOR-22) against renal cell carcinoma--a multiclinic cooperative, early phase II study. Subcommittee on Urogenital Malignancy, Committee on MHR-24 against Tumors].

    PubMed

    Niijima, T; Akaza, H; Koyanagi, T; Togashi, M; Kumamoto, Y; Funyu, T; Suzuki, T; Orikasa, S; Yoshikawa, K; Koiso, K

    1992-10-01

    The combination therapy with natural type human tumor necrosis factor (n-TNF; MHR-24) and human lymphoblastoid interferon-alpha (n-IFN-alpha; MOR-22) was investigated for antitumor effect against renal cell carcinoma in a multiclinic cooperative study throughout Japan. The "Response criteria of Japan Society for Cancer Therapy" were followed for the handling of subjects and the evaluation of antitumor effect. MHR-24 was administered at a daily dosage of 5,000-10,000 JRU by intravenous drip and MOR-22 at a dosage of 5,000,000 IU daily was administered intramuscularly at the same time. Both drugs were administered for 4 weeks or longer. A total of 36 patients were enrolled as subjects in the study. None of them were classified as ineligible. Five patients, were classified as imperfectly evaluable, and 31, as evaluable for the results of treatment. The responses in the evaluable patients were partial response (PR) in 4 patients, minor response (MR) in 3 patients, no change (NC) in 14 patients and progressive disease (PD) in 10 patients, with a response rate of 12.9%. Adverse reactions to the therapy were investigated in all 36 patients. The frequent subjective and objective reactions that occurred were fever, rigors and shivering, anorexia, and generalized malaise, and the frequent abnormal laboratory findings were leukopenia, thrombocytopenia, elevation of GOT, and elevation of GPT. PMID:1481783

  9. Distinct patterns of somatic alterations in a lymphoblastoid and a tumor genome derived from the same individual

    PubMed Central

    Galante, Pedro A. F.; Parmigiani, Raphael B.; Zhao, Qi; Caballero, Otávia L.; de Souza, Jorge E.; Navarro, Fábio C. P.; Gerber, Alexandra L.; Nicolás, Marisa F.; Salim, Anna Christina M.; Silva, Ana Paula M.; Edsall, Lee; Devalle, Sylvie; Almeida, Luiz G.; Ye, Zhen; Kuan, Samantha; Pinheiro, Daniel G.; Tojal, Israel; Pedigoni, Renato G.; de Sousa, Rodrigo G. M. A.; Oliveira, Thiago Y. K.; de Paula, Marcelo G.; Ohno-Machado, Lucila; Kirkness, Ewen F.; Levy, Samuel; da Silva, Wilson A.; Vasconcelos, Ana Tereza R.; Ren, Bing; Zago, Marco Antonio; Strausberg, Robert L.; Simpson, Andrew J. G.; de Souza, Sandro J.; Camargo, Anamaria A.

    2011-01-01

    Although patterns of somatic alterations have been reported for tumor genomes, little is known on how they compare with alterations present in non-tumor genomes. A comparison of the two would be crucial to better characterize the genetic alterations driving tumorigenesis. We sequenced the genomes of a lymphoblastoid (HCC1954BL) and a breast tumor (HCC1954) cell line derived from the same patient and compared the somatic alterations present in both. The lymphoblastoid genome presents a comparable number and similar spectrum of nucleotide substitutions to that found in the tumor genome. However, a significant difference in the ratio of non-synonymous to synonymous substitutions was observed between both genomes (P = 0.031). Protein–protein interaction analysis revealed that mutations in the tumor genome preferentially affect hub-genes (P = 0.0017) and are co-selected to present synergistic functions (P < 0.0001). KEGG analysis showed that in the tumor genome most mutated genes were organized into signaling pathways related to tumorigenesis. No such organization or synergy was observed in the lymphoblastoid genome. Our results indicate that endogenous mutagens and replication errors can generate the overall number of mutations required to drive tumorigenesis and that it is the combination rather than the frequency of mutations that is crucial to complete tumorigenic transformation. PMID:21493686

  10. [Clinical effect of human lymphoblastoid interferon in patients with multiple myeloma].

    PubMed

    Yoshida, M; Ohta, M; Muroi, K; Takeda, K; Kitagawa, S; Tsuboyama, A; Sakamoto, S; Miura, Y; Mutoh, Y

    1984-07-01

    Five patients with multiple myeloma were treated with human lymphoblastoid interferon (HLBI). HLBI, 3 X 10(6) IU/day, was administered daily for more than two weeks by intramuscular injection. Out of four evaluable patients, a minor response was obtained in 3 patients. In these responders, one patient developed pleural effusion due to the infiltration of myeloma cells during the administration of HLBI, and drug resistance was observed in another patient during the re-administration of HLBI. Therefore, out of six evaluable courses, a minor response was obtained in 3 courses of HLBI treatment. No severe side effects were observed. Thrombocytopenia, general malaise, liver dysfunction and anorexia were the main reasons for discontinuation of HLBI administration. On the basis of the preliminary study, it is concluded that HLBI is worth trying in the management of refractory multiple myeloma. PMID:6742866

  11. Reduction of immunoglobulin G secretion in vitro following long term lymphoblastoid interferon (Wellferon) treatment in multiple sclerosis patients.

    PubMed Central

    O'Gorman, M R; Oger, J; Kastrukoff, L F

    1987-01-01

    Pokeweed-mitogen-induced IgG secretion, Con A suppression and T cell surface markers were measured in 30 chronic progressive multiple sclerosis (MS) patients and 21 healthy controls. Mean IgG secretion was higher in the MS patients than in the controls (2392 +/- 270 vs 1499 +/- 243); Con A suppression was lower (4 +/- 5% vs 24 +/- 4%) and the CD4/CD8 ratio was higher (4.1 +/- 0.4 vs 2.9 +/- 0.4). The above assays were used in vitro to monitor the effects of Wellferon (lymphoblastoid interferon) injections on this group of MS patients. Before treatment the INF-group (n = 14) did not differ from the PLA-group (n = 16). After 1 week of daily injections the level of IgG secreted was dramatically reduced in the INF group (629 +/- 96 ng/ml) compared to the PLA-group (1756 +/- 319 ng/ml). There was no change in either Con A suppression or T cell surface markers. IgG secretion remained lower in the INF-group for the 6 month treatment period. Following cessation of the injections and a 6 month washout period, IgG secretion in the INF-group rose and was equivalent to that observed in the PLA-group. A series of lymphocyte subset mixing experiments implicates the B lymphocyte subset as being directly affected by interferon injections in vitro. PMID:2957131

  12. [Clinical study on the effect of human lymphoblastoid interferon in multiple myeloma].

    PubMed

    Takeyama, H; Yano, K; Kataoka, T; Yamamoto, M; Emi, N; Kodera, Y; Kawashima, K; Ohno, R; Yokomaku, S; Kobayashi, M

    1983-09-01

    Ten patients with multiple myeloma were treated with human lymphoblastoid interferon (HLBI). The dosages used were 3 X 10(6) IU to 6 X 10(6) IU of HLBI intramuscularly daily. Out of eight evaluable patients, one partial remission and 3 minor response were observed. More than half patients experienced fever exceeding 38 degrees C and mild myelosuppression. Other toxic effects consisted of anorexia, malaise, liver dysfunction and skin rash. On the basis of our preliminary study, we conclude that HLBI is an effective agent against multiple myeloma. PMID:6614939

  13. FMLP- and TNF-stimulated monoclonal Lym-1 antibody-dependent lysis of B lymphoblastoid tumour targets by neutrophils.

    PubMed

    Ottonello, L; Morone, P; Mancini, M; Amelotti, M; Dapino, P; Dallegri, F

    1999-05-01

    Human neutrophils, incubated with Cr51-labelled B lymphoblastoid Raji cells in the presence of the anti-target monoclonal antibody (mAb) Lym-1 plus formyl-methionyl-leucyl-phenylalanine (FMLP) or tumour necrosis factor alpha (TNF-alpha), were found to induce significant C51 release, i.e. significant cytolysis. The lytic process was inhibited by mAb IV.3, specific for the Fcgamma receptor (FcgammaR) type II. The mAb 3G8, which reacts with FcgammaR type III, was ineffective. Moreover, the lysis was inhibited by the anti-CD18 mAb MEM-48. These data suggest that FMLP/Lym-1 as well as TNF-alpha/Lym-1 cytolytic systems strictly require FcgammaRII and CD18 integrins. As the lysis induced by TNF-alpha/Lym-1 was prevented by pertussis toxin (PT), PT-sensitive G-proteins are likely to intervene in post-FcgammaRII signal transduction. Both the FMLP- and the TNF-alpha-dependent systems were also found to be equally susceptible to inhibition by various inhibitors of kinases (genistein, staurosporin, 1-(5-isoquinolinnylsulphonyl)-2-methylpiperazine and wortmannin). On the contrary, an inhibitor of protein kinase C (bis-indolyl-maleimide, BIM) was effective only in the FMLP/Lym-1 cytolytic system. Therefore, it appears that signals delivered by FMLP or TNF-alpha, BIM-sensitive and insensitive respectively, converge and synergize with those from G-protein-coupled FcgammaRII and, probably, CD18-integrins to promote the expression of the neutrophil cytolytic potential. PMID:10408834

  14. FMLP- and TNF-stimulated monoclonal Lym-1 antibody-dependent lysis of B lymphoblastoid tumour targets by neutrophils

    PubMed Central

    Ottonello, L; Morone, P; Mancini, M; Amelotti, M; Dapino, P; Dallegri, F

    1999-01-01

    Human neutrophils, incubated with Cr51-labelled B lymphoblastoid Raji cells in the presence of the anti-target monoclonal antibody (mAb) Lym-1 plus formyl-methionyl-leucyl-phenylalanine (FMLP) or tumour necrosis factor alpha (TNF-α), were found to induce significant Cr51 release, i.e. significant cytolysis. The lytic process was inhibited by mAb IV.3, specific for the Fcγ receptor (FcγR) type II. The mAb 3G8, which reacts with FcγR type III, was ineffective. Moreover, the lysis was inhibited by the anti-CD18 mAb MEM-48. These data suggest that FMLP/Lym-1 as well as TNF-α/Lym-1 cytolytic systems strictly require FcγRII and CD18 integrins. As the lysis induced by TNF-α/Lym-1 was prevented by pertussis toxin (PT), PT-sensitive G-proteins are likely to intervene in post-FcγRII signal transduction. Both the FMLP- and the TNF-α-dependent systems were also found to be equally susceptible to inhibition by various inhibitors of kinases (genistein, staurosporin, 1-(5-isoquinolinnylsulphonyl)-2-methylpiperazine and wortmannin). On the contrary, an inhibitor of protein kinase C (bis-indolyl-maleimide, BIM) was effective only in the FMLP/Lym-1 cytolytic system. Therefore, it appears that signals delivered by FMLP or TNF-α, BIM-sensitive and insensitive respectively, converge and synergize with those from G-protein-coupled FcγRII and, probably, CD18-integrins to promote the expression of the neutrophil cytolytic potential. © 1999 Cancer Research Campaign PMID:10408834

  15. [Phase I study of human lymphoblastoid alpha-interferon on malignant tumor].

    PubMed

    Furue, H

    1986-04-01

    A phase I study with human lymphoblastoid alpha-interferon (IFN-alpha) was conducted in 31 patients with malignant tumors. IFN-alpha was administered by intravenous drip infusion, intramuscular injection or local injection. In each patient, the dose was increased in 6 steps from 3 X 10(6) IU/body up to 54 X 10(6) IU/body for the purpose of investigating the safety, optimal regimen, pharmacokinetics and antitumor effect. The following findings were obtained: 1) Fever as a side effect was most frequently (in about 80%) found. However, the temperature did not exceed 40 degrees C in most cases and, on the next day, spontaneously fell to normal. 2) The dose-limiting factors (DLF) may include the subjective symptoms of anorexia, general fatigue and nausea/vomiting and the objective symptom of pancytopenia. 3) The maximum tolerated dose (MTD) was estimated to be between 36 X 10(6) and 54 X 10(6) IU/body per dose. 4) As for the route of administration, the intramuscular one was considered most suitable on the basis of the plasma concentration profile of INF-alpha. It was therefore concluded that the drug may be further submitted to a phase II study which is to be conducted with due consideration of its safety. PMID:3963861

  16. Genetic instability on chromosome 16 in a Human B lymphoblastoid cell line

    SciTech Connect

    Smith, L.E.; Grosovsky, A.J. )

    1993-11-01

    Mutagenesis at the aprt locus in TK6 human lymphoblasts has been found to occur at an unusually high rate (1.2 [times] 10[sup [minus]9]) for a homozygous diploid locus. Evaluation of linked microsatellite polymorphisms demonstrated that loss of heterozygosity (LOH) accompanies conventional intragenic sequence alterations in each APRT[sup [minus

  17. In vitro sensitization of human lymphocytes to a myeloma cell-related antigen

    SciTech Connect

    Whitson, M.E.; Griffin, G.D.; Novelli, G.D.; Solomon, A.

    1981-01-01

    Peripheral blood lymphocytes from normal human donors were cocultivated with cells from two established human multiple myeloma cell lines, RPMI 8226 and K-737, and with lymphoblastoid cells from a third B cell line, RAMM. After a comparison of three methods of lymphocyte sensitization, a 6-day incubation protocol with equal numbers of normal lymphocytes and mitomycin C-treated tumor cells was selected. Cells fom the RPMI 8226 myeloma line stimulated the differentiation of lymphocytes into cytotoxic effector cells as measured by /sup 51/Cr release from labeled target cells. The RPMI 8226-sensitized lymphocytes were cytotoxic for myeloma cells (RPMI 8226 and K-737) and for lymphoblastoid cells (RAMM) but not for cells from human lung tumor lines (A549, A427, MB9812), a breast carcinoma line (ALAB), a normal diploid fibroblast line (HSBP), or normal lymphocytes.

  18. Preclinical development of hybrid cell vaccines for multiple myeloma.

    PubMed

    Walewska, Renata; Teobald, Iryna; Dunnion, Debbie; Abdulmajed, Hind; Aldred, Micheala; Sadler, Jean; Chapman, Claire; Browning, Michael

    2007-01-01

    Immunotherapy may provide alternative or supplementary treatment of multiple myeloma (MM). We propose that hybrid cells, formed by fusing professional antigen-presenting cells with malignant plasma cells, would induce immune responses capable of mediating tumour regression. The human B-lymphoblastoid cell line, HMy2, was fused in vitro with CD138+ bead-separated myeloma plasma cells from five patients with MM. The hybrid cell lines generated in these studies grew stably in tissue culture, and maintained their phenotypic and functional characteristics, providing self-renewing cell lines with potential for therapeutic vaccination. The hybrid cells stimulated allogeneic and autologous T-cell proliferative responses in vitro to a considerably greater degree than their respective parent myeloma plasma cells, and directly activated both CD4+ and CD8+ T-cell responses. The enhanced T-cell stimulation correlated with expression of CD80 on the hybrid cells, and was inhibited by CTLA4-Ig fusion protein. The hybrid cell lines expressed several tumour-associated antigens known to be expressed in myeloma. These data show that self-replicating cell lines with enhanced immunostimulatory properties and potential for therapeutic vaccination can be generated by in vitro fusion of ex vivo myeloma cells and B-lymphoblastoid cell lines. PMID:17302859

  19. Diverse delayed effects in human lymphoblastoid cells surviving exposure to high-LET (56)Fe particles or low-LET (137)Cs gamma radiation

    NASA Technical Reports Server (NTRS)

    Evans, H. H.; Horng, M. F.; Ricanati, M.; Diaz-Insua, M.; Jordan, R.; Schwartz, J. L.

    2001-01-01

    To obtain information on the origin of radiation-induced genomic instability, we characterized a total of 166 clones that survived exposure to (56)Fe particles or (137)Cs gamma radiation, isolated approximately 36 generations after exposure, along with their respective control clones. Cytogenetic aberrations, growth alterations, responses to a second irradiation, and mutant frequencies at the Na(+)/K(+) ATPase and thymidine kinase loci were determined. A greater percentage of clones that survived exposure to (56)Fe particles exhibited instability (defined as clones showing one or more outlying characteristics) than in the case of those that survived gamma irradiation. The phenotypes of the unstable clones that survived exposure to (56)Fe particles were also qualitatively different from those of the clones that survived gamma irradiation. A greater percentage (20%) of the unstable clones that survived gamma irradiation than those that survived exposure to (56)Fe particles (4%) showed an altered response to the second irradiation, while an increase in the percentage of clones that had an outlying frequency of ouabain-resistant and thymidine kinase mutants was more evident in the clones exposed to (56)Fe particles than in those exposed to gamma rays. Growth alterations and increases in dicentric chromosomes were found only in clones with more than one alteration. These results underscore the complex nature of genomic instability and the likelihood that radiation-induced genomic instability arises from different original events.

  20. Xenohybridization of Epstein-Barr virus-transformed cells for the production of human monoclonal antibodies.

    PubMed

    Tiebout, R F; Stricker, E A; Oosterhof, F; van Heemstra, D J; Zeijlemaker, W P

    1985-12-01

    Transformation of human B lymphocytes, obtained from hyperimmune donors with Epstein-Barr virus, yields polyclonal cell populations in which a minority of cells produce IgG antibodies of predetermined specificity, whereas the majority of cells produce 'non-specific' immunoglobulin (mainly of the IgM class). Such lymphoblastoid cell lines can be easily propagated in high-density cultures. Because cloning at 1 cell per well is not possible, stabilization of lymphoblastoid cell lines by limiting dilution is not feasible and most newly established lines cease to produce specific antibody within a few weeks. Xenohybrids, resulting from fusion of Epstein-Barr virus-transformed cells with NS1 mouse plasmacytoma cells, can be cloned at 1 cell per well. Stable xenohybridoma subclones, producing antibody of the desired specificity, can be isolated after a series of limiting dilutions. In a model system, we have studied the efficiency of xenohybridization of human lymphoblastoid cells. Using this system, we have constructed IgG anti-tetanus-toxoid- and IgG anti-HBsAg-producing cell lines. Next, we investigated whether transformation with Epstein-Barr virus is essential in such a two-step procedure or whether a polyclonal stimulator, such as pokeweed mitogen, could also be used. It was found that antibody-producing xenohybrids can be obtained after stimulation with pokeweed mitogen. However, this latter system is subject to more variations and lacks the advantage of pre-selection of antibody-producing cells as compared to xenohybridization after transformation. PMID:3003887

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

    NASA Technical Reports Server (NTRS)

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

    1999-01-01

    BACKGROUND: Data currently available on HIV-1-induced cytopathology is unclear regarding the mechanism of cell killing. OBJECTIVE: To clarify the extent to which apoptosis or necrosis is involved in HIV-1-induced cell death in view of conflicting existing data. METHODS: T lymphoblastoid cells or peripheral blood mononuclear cells were infected by various strains of HIV-1 and the numbers of apoptotic or necrotic cells were quantified at various times after infection using video-image analysis techniques; the results were compared with the amount of fragmented DNA using a quantitative method. Measurement of mitochondrial transmembrane potential (deltapsi(m)) and intracellular calcium concentrations [Ca2+]i was performed with fluorescent probes and fluorescence concentration analysis (FCA). RESULTS: Although lymphoblastoid and monocytoid cells acutely infected by HIV-1 had increased levels of fragmented DNA, a marker of apoptotic cell death, few (<12%) had condensed chromatin and fragmented nuclei, the morphological features of apoptosis. The predominant alterations in acutely infected cells were distended endoplasmic reticulum and abnormal mitochondria; these ultrastructural changes are consistent with necrosis, although some infected cells simultaneously displayed features of both necrosis and apoptosis. Viability of cells persistently infected by HIV-1 was only minimally reduced from that of uninfected cells. This reduction was accounted for by an increased propensity of the persistently infected cells to die by apoptosis. Alterations in [Ca2+]i and deltapsi(m) occurred in both acutely and persistently infected cells. CONCLUSION: Both necrosis and apoptosis contribute to HIV-1-induced killing of CD4 cells.

  2. Epstein-Barr virus (EBV) induces expression of B-cell activation markers on in vitro infection of EBV-negative B-lymphoma cells.

    PubMed Central

    Calender, A; Billaud, M; Aubry, J P; Banchereau, J; Vuillaume, M; Lenoir, G M

    1987-01-01

    A set of B-cell activation markers, including the EBV/C3d receptor [complement receptor type 2 (CR2) (CD21)], the 45-kDa lymphoblastoid cell-associated (Blast-2) antigen (CD23), and the B-cell restricted activation (Bac-1) antigen (which was recently identified as a potential B-cell growth factor receptor) can be turned on by infecting lymphoma cells that are genome negative for Epstein-Barr virus (EBV) with the B95-8 immortalizing strain of the virus. The nonimmortalizing EBV variant, strain P3HR-1, which possesses a deletion within the BamHI WYH region of the genome containing the coding sequence for the EBV-determined nuclear antigen 2, does not induce expression of these markers. Other lymphoblastoid cell-associated antigen markers can be activated by infection with either immortalizing or nonimmortalizing viruses. These results suggest that the immortalizing potential of EBV is correlated with its ability to induce expression of B-cell activation markers, which are suspected to play a major role in the physiological pathway leading to lymphoid cell proliferation. The viral genomic region deleted in the nonimmortalizing strain of EBV seems to be required for activation of some of these markers. Human lymphoma cell lines, such as those used in this study, can thus help identify the specific EBV genes involved in lymphoid B-cell proliferation and the mechanism of action of these genes. PMID:2825176

  3. Mechanisms of lymphocyte adhesion to endothelial cells: studies using a LFA-1-deficient cell line.

    PubMed Central

    Haskard, D O; Strobel, S; Thornhill, M; Pitzalis, C; Levinsky, R J

    1989-01-01

    In order to investigate the role of lymphocyte function-associated antigen 1 (LFA-1) in lymphocyte adhesion to endothelial cells (EC), we have studied the adhesion of a LFA-1-deficient lymphoblastoid cell line, ICH-KM, which has < 10% of the cell surface LFA-1 expressed on a normal lymphoblastoid cell line, ICH-BJ. The adhesion of ICH-KM cells to unstimulated EC was 49.9 +/- 8.6% (mean +/- SD) that of ICH-BJ cells. Moreover, phorbol ester-stimulated ICH-KM cells showed a considerably weaker increase in adhesion to unstimulated EC compared with ICH-BJ cells (mean +/- SD increase in percentage adhesion, 3.8 +/- 2.3 compared with 18.5 +/- 8.0; P<0.025). In contrast, there was no significant difference between the enhanced adhesion of ICH-KM cells and ICH-BJ cells to interleukin-1 (IL-1)-stimulated EC. Thus ICH-KM cells showed a 22.7 +/- 11.0 (mean +/- SD) increase in percentage adhesion to IL-1-stimulated EC compared with the 24.8 +/- 8.5 increase in percentage adhesion of ICH-BJ cells. Anti-LFA-1 monoclonal antibodies had no effect on the enhanced adhesion of ICH-KM and ICH-BJ cells to IL-1-stimulated EC but abolished the differences in adhesion between the two cell lines. The study therefore indicates that although a major part of unstimulated and phorbol ester-stimulated lymphocyte-EC adhesion is dependent upon LFA-1, the enhanced adhesion due to stimulation of EC with IL-1 is not dependent upon this molecule. The data therefore supports the existence of cytokine-inducible LFA-1-independent adhesion molecules for lymphocytes on EC. PMID:15493272

  4. IDS transfer from overexpressing cells to IDS-deficient cells.

    PubMed

    Millat, G; Froissart, R; Maire, I; Bozon, D

    1997-02-01

    Iduronate sulfatase (IDS) is responsible for mucopolysaccharidosis type II, a rare recessive X-linked lysosomal storage disease. The aim of this work was to test the ability of overexpressing cells to transfer IDS to deficient cells. In the first part of our work, IDS processing steps were compared in fibroblasts, COS cells, and lymphoblastoid cell lines and shown to be identical: the two precursor forms (76 and 90 kDa) were processed by a series of intermediate forms to the 55- and 45-kDa mature polypeptides. Then IDS transfer to IDS-deficient cells was tested either by incubation with cell-free medium of overexpressing cells or by coculture. Endocytosis and coculture experiments between transfected L beta and deleted fibroblasts showed that IDS transfer occurred preferentially by cell-to-cell contact as IDS precursors are poorly secreted by transfected L beta. The 76- and 62-kDa IDS polypeptides transferred to deleted fibroblasts were correctly processed to the mature 55- and 45-kDa forms. L beta were not able to internalize the 90-kDa phosphorylated precursor forms excreted in large amounts in the medium of overexpressing fibroblasts. Enzyme transfer occurred only by cell-to-cell contact, but the precursor forms transferred in L beta after cell-to-cell contact were not processed. This absence of maturation was probably due to a mistargeting of IDS precursors in these cells. PMID:9024795

  5. Selective activation of functional suppressor cells by human seminal fluid.

    PubMed Central

    Witkin, S S

    1986-01-01

    The ability of seminal fluid (SF) to induce suppressor cell activity from peripheral blood mononuclear cells (PBMN) was examined. PBMN were incubated with SF for 48 h, washed to remove SF components, treated with mitomycin C (mit C) and co-cultured with Raji cells, a lymphoblastoid cell line. Raji cell proliferation was inhibited by SF-treated PBMN proportionally to SF concentration. SF (50-200 micrograms), mit C-treated Raji cells or mit C-treated PBMN pre-incubated with phytohaemagglutinin were without effect on Raji cell growth. Suppressor T lymphocytes generated by incubation of PBMN with concanavalin A inhibited Raji cells to the same extent as did SF-treated PBMN. All activity was lost following heating at 56 degrees C for 30 min; freezing and thawing reduced the ability of SF to induce suppression by 50%. Dialysis of SF or treatment with antibody to prostaglandin E2 led to a 50% reduction in suppression. PMID:2943541

  6. Increased initial levels of chromosome damage and heterogeneous chromosome repair in ataxia telangiectasia heterozygote cells.

    PubMed

    Pandita, T K; Hittelman, W N

    1994-10-01

    Individuals heterozygous for ataxia telangiectasia (AT) appear clinically normal but have a 2-3-fold overall excess risk of cancer. Various approaches have been used to identify AT heterozygotes, however, the results are ambiguous. We recently reported that AT homozygotes exhibit more initial chromosome damage after irradiation than normal cells despite identical levels of DNA double strand breaks (DSBs) as well as a reduced fast repair component at both the DNA and chromosome levels. To determine whether AT heterozygotes exhibit the AT or normal cellular phenotype, we compared four AT heterozygote lymphoblastoid cell lines with normal control and AT homozygote lymphoblastoid cells with regard to cell survival, initial levels of damage, and repair at the DNA and chromosome levels after gamma-irradiation in G1, S, and G2 phase (estimated by neutral DNA filter elution and premature chromosome condensation). There was no significant difference in survival, induction and repair of DNA DSBs, or chromosome repair between AT heterozygote and normal cells. In contrast, all four AT heterozygote cell lines showed increased levels of chromosome damage; G1 phase cells showed intermediate levels and G2 phase cells showed levels equivalent to the AT homozygote phenotype. These results suggest that premature chromosome condensation may be useful for detecting AT heterozygotes. PMID:7523872

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

    SciTech Connect

    Kaleeba, Johnan A.R.; Berger, Edward A. . E-mail: edward_berger@nih.gov

    2006-10-10

    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 {alpha}3{beta}1 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.

  8. Expansion of NK Cells Using Genetically Engineered K562 Feeder Cells.

    PubMed

    Phan, Minh-Trang Thi; Lee, Seung-Hwan; Kim, Sang-Ki; Cho, Duck

    2016-01-01

    Natural killer (NK) cells can be expanded upon activation by proliferative cytokines (such as IL-2 and IL-15). The NK cell expansion can be greatly enhanced by proteins from feeder cells such as tumor cell lines or PBMCs. Therefore, coculture systems of irradiated feeder cells and NK cells in media containing IL-2 and IL-15 have been developed to generate large numbers of NK cells, although NK cell expansion protocol using anti-CD3 antibody (OKT-3) without feeder cells has also been developed. Commonly used feeder cell lines are RPMI8866, Epstein-Barr lymphoblastoid cell line (EBV-LCL), and K562. Stimulation with NK-sensitive K562 cells is known to augment NK cell proliferation to IL-2, IL-15, and IL-21 in combination.Recently, remarkable NK cell-expansion rates are achieved when genetically engineered (GE) feeder cells are used. Dr. Dario Campana's group found that membrane-bound IL-15 and 4-1BBL, coexpressed by K562 cells, acted synergistically to augment K562-specific NK stimulatory capacity, resulting in vigorous expansion of peripheral blood CD56(+) CD3(-) NK cells without concomitant growth of T lymphocytes. Here, we describe an in vitro expansion method of human NK cells among PBMCs by coculturing with GE_K562 cells. PMID:27177665

  9. Monoclonal Lym-1 antibody-dependent lysis of B-lymphoblastoid tumor targets by human complement and cytokinine-exposed mononuclear and neutrophilic polymorphonuclear leukocytes.

    PubMed

    Ottonello, L; Morone, P; Dapino, P; Dallegri, F

    1996-06-15

    Lym-1 is a murine IgG2a monoclonal antibody that recognizes a polymorphic variant of HLA-DR antigens on malignant B cells, with minimal cross-reactivity with normal tissues. Because it can be safely administered in vivo, a detailed knowledge of its ability to recruit and trigger the antitumor immune effector systems is required to optimize potential serotherapeutic approaches in B-lymphoma patients. By using Raji cells as a model of B-lymphoma targets, we found that Lym-1 activates complement-mediated lysis efficiently. Moreover, Lym-1 was capable of triggering the antibody-dependent cellular cytolysis (ADCC) by peripheral blood mononuclear cells (MNCs). On the contrary, it failed to trigger neutrophilic polymorphonuclear leukocyte (PMN)-mediated ADCC activity. In an attempt to enhance Lym-1 ADCC by MNCs and PMNs, nine biologic response modifiers were tested. MNC-mediated Lym-1 ADCC was significantly stimulated by interleukin-2 (IL-2) and unaffected by other mediators, including gamma-interferon (gamma-IFN), tumor necrosis factor a (TNFalpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF). On the other hand, PMN-mediated Lym-1 ADCC was induced or significantly augmented by various cytokines, such as GM-CSF, TNFalpha, and gamma-IFN, and chemotaxins, such as formyl peptides (FMLP), complement fragment C5a, and IL-8. Both MNC- and PMN-mediated ADCC was unaffected by granulocyte colony-stimulating factor (G- CSF) and insulin-like growth factor-1 (IGF-1). Finally, only GM-CSF and TNFalpha augmented the number of PMNs actually engaged in the binding of Raji target cells. The findings presented here, in particular those showing stimulatory activity of biologic response modifiers, may inspire new attempts for developing Lym-1 antibody-based approaches to the therapy of B lymphomas. PMID:8652830

  10. Role of interleukin in human natural killer cell proliferation

    SciTech Connect

    London, L.; Perussia, B.; Trinchieri, G.

    1986-03-01

    Human NK cells, defined by the antibody B73.1, can be induced to proliferate in vitro in the presence of an IL-2 containing conditioned medium (CM) and an irradiated lymphoblastoid line, Daudi. Proliferating NK cells maintain phenotypic and functional characteristics of resting NK cells while newly expressing surface activation antigens (HLA-DR, transferrin receptor, and IL-2 receptor recognized by anti-TAC antibody). A goat anti-IL-2 antiserum and the anti-TAC monoclonal antibody completely block /sup 3/H-TdR incorporation in NK cells stimulated with CM alone or with irradiated Daudi cells. Inhibition is also observed when the antibodies are added up to day 4 of culture, indicating that IL-2 is required for both initiation and maintenance of proliferation. Human recombinant IL-2, either alone or with irradiated lymphoblastoid cells, replaces the CM in initiating /sup 3/H-TdR incorporation. In limiting dilution analysis the frequency of B73.1 (+) cells responding to rIL-2 is approximately 1/2000 and it is increased ten to thirty fold with the addition of irradiated Daudi cells to the cultures. Cultures stimulated with rIL-2 in the presence of colchicine, show a significant proportion of B73.1 + cells entering cycle each day during the first 3 days. These data show that a significant proportion of resting NK cells are capable of responding to IL-2 and that this response can occur over a period of several days after initiation of cultures.

  11. Lymphocyte culture: induction of colonies by conditioned medium from human lymphoid cell lines.

    PubMed

    Galbraith, R M; Goust, J M; Fudenberg, H H

    1977-12-01

    The presence of phytohemagglutinin or pokeweed mitogen in cultures of human peripheral blood mononuclear cells in agar is known to stimulate the formation of lymphoid colonies. We now report that similar colonies can be induced in the absence of plant lectins upon addition of filtered and ultracentrifuged conditioned medium (CM) obtained from certain human lymphoblastoid cell lines. Colony formation required at least 6 X 10(5) mononuclear cells per milliliter, and optimum results were obtained at concentrations of 1 X 10(6) cells/ml in the presence of 20% CM (50-500 colonies per 10(6) cells cultured). Individual cells within colonies displayed uniform morphological characteristics of lymphoid cells, and the majority formed rosettes with sheep erythrocytes, suggesting that they were of T-cell type. PMID:303689

  12. Detection of high affinity receptor sites for IL 1. beta. on a human B lymphoblastoid line which fail to recognize IL 1. cap alpha

    SciTech Connect

    Chin, J.; Cameron, P.; Sigal, N.H.; Schmidt, J.A.

    1986-03-05

    A large number of EBV-transformed human B lines were screened for their ability to bind human pI 6.8 IL 1 (IL 1..beta..) which was labeled to high specific radioactivity with Bolton-Hunter reagent. One of these, designated 2C2, bound (/sup 125/)I-IL 1 in a saturable dose-dependent fashion. Scatchard analysis of direct binding data obtained at equilibrium suggested a single family of receptor sites, at approx. 10,000 sites per cell, with a K/sub d/ = 1.5 +/- 0.2 (+SD) nM. Competition experiments with cold pI 6.8 IL 1 gave a K/sub i/ = 1.0 +/- 0.3 nM. No competition was seen with a 20-fold molar excess of human IL 2, human gamma-INF, or the pI 5.2 and pI 5.4 species of human IL 1. These anionic species of IL 1 have recently been purified to homogeneity by us from monocyte culture supernatants. Amino acid sequence analysis of the pI 5.4 species demonstrates that it is encoded by the recently reported IL 1..cap alpha.. cDNA. Cross linking of pI 6.8 (/sup 125/)I-IL 1 to intact 2C2 cells with increasing amounts of cross linker revealed a single band with a MW congruent to 80,000. Cross-linking was totally abolished by excess unlabeled pI 6.8 IL 1 but not by excess pI 5.4 IL 1. These results show that the receptor for IL 1..beta.. on 2C2 cells is highly specific for one species of human IL 1 and raises the possibility that IL 1..cap alpha.. and IL 1..beta.., though very similar in their biological properties, have separate receptor sites.

  13. Development of a cell microarray chip for detection of circulating tumor cells

    NASA Astrophysics Data System (ADS)

    Yamamura, S.; Yatsushiro, S.; Abe, K.; Baba, Y.; Kataoka, M.

    2012-03-01

    Detection of circulating tumor cells (CTCs) in the peripheral blood of metastatic cancer patients has clinical significance in earlier diagnosis of metastases. In this study, a novel cell microarray chip for accurate and rapid detection of tumor cells from human leukocytes was developed. The chip with 20,944 microchambers (105 μm diameter and 50 μm depth) was made from polystyrene, and the surface was rendered to hydrophilic by means of reactive-ion etching, which led to the formation of mono-layers of leukocytes on the microchambers. As the model of CTCs detection, we spiked human bronchioalveolar carcinoma (H1650) cells into human T lymphoblastoid leukemia (CEM) cells suspension and detected H1650 cells using the chip. A CEM suspension contained with H1650 cells was dispersed on the chip surface, followed by 10 min standing to allow the cells to settle down into the microchambers. About 30 CEM cells were accommodated in each microchamber, over 600,000 CEM cells in total being on a chip. We could detect 1 H1650 cell per 106 CEM cells on the microarray by staining with fluorescence-conjugated antibody (Anti-Cytokeratin) and cell membrane marker (DiD). Thus, this cell microarray chip has highly potential to be a novel tool of accurate and rapid detection of CTCs.

  14. From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing.

    PubMed

    Marinov, Georgi K; Williams, Brian A; McCue, Ken; Schroth, Gary P; Gertz, Jason; Myers, Richard M; Wold, Barbara J

    2014-03-01

    Single-cell RNA-seq mammalian transcriptome studies are at an early stage in uncovering cell-to-cell variation in gene expression, transcript processing and editing, and regulatory module activity. Despite great progress recently, substantial challenges remain, including discriminating biological variation from technical noise. Here we apply the SMART-seq single-cell RNA-seq protocol to study the reference lymphoblastoid cell line GM12878. By using spike-in quantification standards, we estimate the absolute number of RNA molecules per cell for each gene and find significant variation in total mRNA content: between 50,000 and 300,000 transcripts per cell. We directly measure technical stochasticity by a pool/split design and find that there are significant differences in expression between individual cells, over and above technical variation. Specific gene coexpression modules were preferentially expressed in subsets of individual cells, including one enriched for mRNA processing and splicing factors. We assess cell-to-cell variation in alternative splicing and allelic bias and report evidence of significant differences in splice site usage that exceed splice variation in the pool/split comparison. Finally, we show that transcriptomes from small pools of 30-100 cells approach the information content and reproducibility of contemporary RNA-seq from large amounts of input material. Together, our results define an experimental and computational path forward for analyzing gene expression in rare cell types and cell states. PMID:24299736

  15. Human gamma delta T-cell recognition of Yersinia enterocolitica.

    PubMed Central

    Young, J L; Goodall, J C; Beacock-Sharp, H; Gaston, J S

    1997-01-01

    We have studied the human gamma delta T-cell response to Yersinia enterocolitica, a facultative intracellular bacterium which causes gastroenteritis and, particularly in human leucocyte antigen (HLA)-B27+ individuals, reactive arthritis (ReA). A marked proliferation of that cytotoxic gamma delta T cells is seen when Yersinia-infected lymphoblastoid cell lines or fixed intact Yersinia are added to cultures of mononuclear cells derived from the synovial fluid of ReA patients or from the peripheral blood of healthy donors. In contrast, heat-inactivated Yersinia fail to stimulate the gamma delta T-cell response. The gamma delta T-cell lines generated killed both autologous and allogeneic infected cell lines. Interestingly, a T-cell line generated from synovial fluid mononuclear cells (SFMC) killed infected autologous cell lines and a cell line matched for HLA-B27 less well than infected allogeneic target cells. gamma delta T-cell clones isolated from this line were found to express V gamma 9V delta 2 T-cell receptor (TCR) and also killed infected mismatched cells more efficiently than autologous targets. Moreover, from experiments using major histocompatability complex (MHC)-deficient cell lines, it was apparent that target cell recognition was MHC independent. Our results suggest that gamma delta T cells can be involved in immunity to Yersinia enterocolitica and should be taken into account when considering immunopathological mechanisms leading to reactive arthritis. PMID:9378487

  16. Comparison of DNA fragmentation and color thresholding for objective quantitation of apoptotic cells

    NASA Technical Reports Server (NTRS)

    Plymale, D. R.; Ng Tang, D. S.; Fermin, C. D.; Lewis, D. E.; Martin, D. S.; Garry, R. F.

    1995-01-01

    Apoptosis is a process of cell death characterized by distinctive morphological changes and fragmentation of cellular DNA. Using video imaging and color thresholding techniques, we objectively quantitated the number of cultured CD4+ T-lymphoblastoid cells (HUT78 cells, RH9 subclone) displaying morphological signs of apoptosis before and after exposure to gamma-irradiation. The numbers of apoptotic cells measured by objective video imaging techniques were compared to numbers of apoptotic cells measured in the same samples by sensitive apoptotic assays that quantitate DNA fragmentation. DNA fragmentation assays gave consistently higher values compared with the video imaging assays that measured morphological changes associated with apoptosis. These results suggest that substantial DNA fragmentation can precede or occur in the absence of the morphological changes which are associated with apoptosis in gamma-irradiated RH9 cells.

  17. Expression of gamma-glutamyl transpeptidase protects ramos B cells from oxidation-induced cell death.

    PubMed

    Karp, D R; Shimooku, K; Lipsky, P E

    2001-02-01

    The ectoenzyme, gamma-glutamyl transpeptidase (GGT, EC ) cleaves glutathione (GSH) to facilitate the recapture of cysteine for synthesis of intracellular GSH. The impact of GGT expression on cell survival during oxidative stress was investigated using the human B cell lymphoblastoid cell line, Ramos. Ramos cells did not express surface GGT and exhibited no GGT enzyme activity. In contrast, Ramos cells stably transfected with the human GGT cDNA expressed high levels of surface GGT and enzymatic activity. GGT-transfected Ramos cells were protected from apoptosis when cultured in cyst(e)ine-deficient medium. The GGT-expressing cells also had lower levels of intracellular reactive oxygen species (ROS). Homocysteic acid and alanine, inhibitors of cystine and cysteine uptake, respectively, caused increased ROS content and diminished viability of GGT expressing cells. Exogenous GSH increased the viability of the GGT-transfected cells more effectively than that of control cells, whereas the products of GSH metabolism prevented death of both the control and GGT-transfected cells comparably. These data indicate that GGT cleavage of GSH and the subsequent recapture of cysteine and cystine allow cells to maintain low levels of cellular ROS and thereby avoid apoptosis induced by oxidative stress. PMID:11080500

  18. Assay of immunoglobulins in supernatants of lymphoid cell lines by conventional laser nephelometry.

    PubMed

    Virella, G; Muñoz, J; Robinson, J E; Goust, J M

    1979-03-01

    An adaptation of the nephelometric assay for serum immunoglobulins has been developed for detection and quantitation of extracellular immunoglobulins in cultures of lymphoblastoid cell lines. This assay employs the standard equipment for laser nephelometry and commercial reagents for immunoglobulin quantitation. By adjusting dilutions of controls and sample volumes of culture supernatants, amounts of IgG and IgM below 1 microgram/ml can be detected in culture supernatants. At concentrations between 1 and 4 microgram/ml, day-to-day and within-run variations for IgM assays were 16 and 11% respectively. The possibility of measuring immunoglobulins secreted by cell lines by conventional laser nephelometry opens several areas of application in the study of the functional activity of B cells and of cell-cell interactions. PMID:313634

  19. The clastogenicity of 4NQO is cell-type dependent and linked to cytotoxicity, length of exposure and p53 proficiency.

    PubMed

    Brüsehafer, Katja; Manshian, Bella B; Doherty, Ann T; Zaïr, Zoulikha M; Johnson, George E; Doak, Shareen H; Jenkins, Gareth J S

    2016-03-01

    4-Nitroquinoline 1-oxide (4NQO) is used as a positive control in various genotoxicity assays because of its known mutagenic and carcinogenic properties. The chemical is converted into 4-hydroxyaminoquinoline 1-oxide and gives rise to three main DNA adducts, N-(deoxyguanosin-8-yl)-4AQO, 3-(desoxyguanosin-N (2)-yl)-4AQO and 3-(deoxyadenosin-N (6)-yl)-4AQO. This study was designed to assess the shape of the dose-response curve at low concentrations of 4NQO in three human lymphoblastoid cell lines, MCL-5, AHH-1 and TK6 as well as the mouse lymphoma L5178Y cell line in vitro. Chromosomal damage was investigated using the in vitro micronucleus assay, while further gene mutation and DNA damage studies were carried out using the hypoxanthine-guanine phosphoribosyltransferase forward mutation and comet assays. 4NQO showed little to no significant increases in micronucleus induction in the human lymphoblastoid cell lines, even up to 55±5% toxicity. A dose-response relationship could only be observed in the mouse lymphoma cell line L5178Y after 4NQO treatment, even at concentrations with no reduction in cell viability. Further significant increases in gene mutation and DNA damage induction were observed. Hence, 4NQO is a more effective point mutagen than clastogen, and its suitability as a positive control for genotoxicity testing has to be evaluated for every individual assay. PMID:26362870

  20. The clastogenicity of 4NQO is cell-type dependent and linked to cytotoxicity, length of exposure and p53 proficiency

    PubMed Central

    Brüsehafer, Katja; Manshian, Bella B.; Doherty, Ann T.; Zaïr, Zoulikha M.; Johnson, George E.; Doak, Shareen H.; Jenkins, Gareth J. S.

    2016-01-01

    4-Nitroquinoline 1-oxide (4NQO) is used as a positive control in various genotoxicity assays because of its known mutagenic and carcinogenic properties. The chemical is converted into 4-hydroxyaminoquinoline 1-oxide and gives rise to three main DNA adducts, N-(deoxyguanosin-8-yl)-4AQO, 3-(desoxyguanosin-N 2-yl)-4AQO and 3-(deoxyadenosin-N 6-yl)-4AQO. This study was designed to assess the shape of the dose–response curve at low concentrations of 4NQO in three human lymphoblastoid cell lines, MCL-5, AHH-1 and TK6 as well as the mouse lymphoma L5178Y cell line in vitro. Chromosomal damage was investigated using the in vitro micronucleus assay, while further gene mutation and DNA damage studies were carried out using the hypoxanthine–guanine phosphoribosyltransferase forward mutation and comet assays. 4NQO showed little to no significant increases in micronucleus induction in the human lymphoblastoid cell lines, even up to 55±5% toxicity. A dose–response relationship could only be observed in the mouse lymphoma cell line L5178Y after 4NQO treatment, even at concentrations with no reduction in cell viability. Further significant increases in gene mutation and DNA damage induction were observed. Hence, 4NQO is a more effective point mutagen than clastogen, and its suitability as a positive control for genotoxicity testing has to be evaluated for every individual assay. PMID:26362870

  1. [Comparative analysis of natural and synthetic antimutagens as regulators of gene expression in human cells under exposure to ionizing radiation].

    PubMed

    Mikhailov, V F; Shishkina, A A; Vasilyeva, I M; Shulenina, L V; Raeva, N F; Rogozhin, E A; Startsev, M I; Zasukhina, G D; Gromov, S P; Alfimov, M V

    2015-02-01

    This paper studies the effect of plant peptides of thionine Ns-W2 extracted from seeds of fennel flower (Nigella sativa) and β-purothionine from wheat germs (Triticum kiharae), as well as a synthetic antimutagen (crown-compound), on the expression of several genes involved in the.control of cellular homeostasis, processes of carcinogenesis, and radiation response in human rhabdomyosarcoma cells (RD cells), T-lymphoblastoid cell line Jurkat, and blood cells. All of these agents acted as antimutagens-anticarcinogens, reducing the expression of genes involved in carcinogenesis (genes of families MMP, TIMP, and IAP and G-protein genes) in a tumor cell. A pronounced reduction in the mRNA level of these genes was caused by thionine Ns-W2, and the least effect was demonstrated by β-purothionine. Antimutagens had very little effect on the mRNA levels of the several studied genes in normal blood cells. PMID:25966580

  2. Validation of the cell cycle G2 delay assay in assessing ionizing radiation sensitivity and breast cancer risk

    PubMed Central

    Hill, Jeff W; Tansavatdi, Kristina; Lockett, Kristin L; Allen, Glenn O; Takita, Cristiane; Pollack, Alan; Hu, Jennifer J

    2009-01-01

    Genetic variations in cell cycle checkpoints and DNA repair genes are associated with prolonged cell cycle G2 delay following ionizing radiation (IR) treatment and breast cancer risk. However, different studies reported conflicting results examining the association between post-IR cell cycle delay and breast cancer risk utilizing four different parameters: cell cycle G2 delay index, %G2–M, G2/G0–G1, and (G2/G0–G1)/S. Therefore, we evaluated whether different parameters may influence study results using a data set from 118 breast cancer cases and 225 controls as well as lymphoblastoid and breast cancer cell lines with different genetic defects. Our results suggest that cell cycle G2 delay index may serve as the best parameter in assessing breast cancer risk, genetic regulation of IR-sensitivity, and mutations of ataxia telangiectasia mutated (ATM) and TP53. Cell cycle delay in 21 lymphoblastoid cell lines derived from BRCA1 mutation carriers was not different from that in controls. We also showed that IR-induced DNA-damage signaling, as measured by phosphorylation of H2AX on serine 139 (γ-H2AX) was inversely associated with cell cycle G2 delay index. In summary, the cellular responses to IR are extremely complex; mutations or genetic variations in DNA damage signaling, cell cycle checkpoints, and DNA repair contribute to cell cycle G2 delay and breast cancer risk. The cell cycle G2 delay assay characterized in this study may help identify subpopulations with elevated risk of breast cancer or susceptibility to adverse effects in normal tissue following radiotherapy. PMID:21188122

  3. DNA damage in dihydroartemisinin-resistant Molt-4 cells.

    PubMed

    Park, Jungsoo; Lai, Henry C; Sasaki, Tomikazu; Singh, Narendra P

    2015-03-01

    Artemisinin generates carbon-based free radicals when it reacts with iron, and induces molecular damage and apoptosis. Its toxicity is more selective toward cancer cells because cancer cells contain a higher level of intracellular free iron. Dihydroartemisinin (DHA), an analog of artemisinin, has selective cytotoxicity toward Molt-4 human lymphoblastoid cells. A major concern is whether cancer cells could develop resistance to DHA, thus limiting its therapeutic efficacy. We have developed a DHA-resistant Molt-4 cell line (RTN) and found out that these cells exhibited resistance to DHA but no significant cross- resistance to artemisinin-tagged holotransferrin (ART-TF), a synthetic artemisinin compound. In the present study, we investigated DNA damage induced by DHA and ART-TF in both Molt-4 and RTN cells using the comet assay. RTN cells exhibited a significantly lower level of basal and X-ray-induced DNA damage compared to Molt-4 cells. Both DHA and ART-TF induced DNA damage in Molt-4 cells, whereas DNA damage was induced in RTN cells by ART-TF, and not DHA. The result of this study shows that by the cell selection method, it is possible to generate a Molt-4 cell line which is not sensitive to DHA, but sensitive to ART-TF, as measured by DNA damage. PMID:25750283

  4. Molecular Mechanisms of Radiation-Induced Genomic Instability in Human Cells

    SciTech Connect

    Howard L. Liber; Jeffrey L. Schwartz

    2005-10-31

    There are many different model systems that have been used to study chromosome instability. What is clear from all these studies is that conclusions concerning chromosome instability depend greatly on the model system and instability endpoint that is studied. The model system for our studies was the human B-lymphoblastoid cell line TK6. TK6 was isolated from a spontaneously immortalized lymphoblast culture. Thus there was no outside genetic manipulation used to immortalize them. TK6 is a relatively stable p53-normal immortal cell line (37). It shows low gene and chromosome mutation frequencies (19;28;31). Our general approach to studying instability in TK6 cells has been to isolate individual clones and analyze gene and chromosome mutation frequencies in each. This approach maximizes the possibility of detecting low frequency events that might be selected against in mass cultures.

  5. Noncytotoxic T cell clones obtained from a human mixed leukocyte culture.

    PubMed

    Chu, M H; Wee, S L; Bach, F H

    1990-02-01

    Peripheral blood mononuclear cells from a DQW-1 homozygous individual were cocultured with irradiated lymphoblastoid cell line from a DQW-1 homozygous unrelated donor bearing BW35-DW1 haplotype. From T cell cloning of primary and twice-stimulated mixed leukocyte cultures (MLC), 7 and 11 T cell clones were obtained respectively. None of the 18 clones showed specific cytotoxic activity against the alloantigen of the stimulator cell as well as natural killer (NK)-like activity against K562 cells. However, most T cell clones from both primary and re-stimulated MLC demonstrated moderate cytotoxic activity in lectin-dependent cell-mediated cytolysis (LDCC) assay. Screening assay for cell-mediated lympholysis (CML) performed on growing microcultures obtained from restimulated MLC cloning confirmed the non-cytotoxic status of these T cell clones by showing that 41 out of 44 growing microcultures were not cytotoxic against the stimulator cell; the other 3 clones lyzed the target cell mildly. The cells from all 5 T cell clones detected for indirect fluorescence expressed CD3 and CD4 surface markers. Taken together, the results suggested that proliferation-regulating T cell subsets or factor(s) may be generated during the course of MLCs under the present responder-stimulator combination, and may suppress the development of alloreactive cytotoxic T cells and NK-like cells. PMID:2144231

  6. Assay for mutagenesis in heterozygous diploid human lymphoblasts

    DOEpatents

    Skopek, Thomas R.; Liber, Howard L.; Penman, Bruce W.; Thilly, William G.; Hoppe, IV, Henry

    1981-01-01

    An assay is disclosed for determining mutagenic damage caused by the administration of a known or suspected mutagen to diploid human lymphoblastoid cell lines. The gene locus employed for this assay is the gene for thymidine kinase, uridine kinase, or cytidine deaminase. Since human lymphoblastoid cells contain two genes for these enzymes, heterozygotes of human lymphoblastoid cells are used in this assay.

  7. Reprogramming human B cells into induced pluripotent stem cells and its enhancement by C/EBPα.

    PubMed

    Bueno, C; Sardina, J L; Di Stefano, B; Romero-Moya, D; Muñoz-López, A; Ariza, L; Chillón, M C; Balanzategui, A; Castaño, J; Herreros, A; Fraga, M F; Fernández, A; Granada, I; Quintana-Bustamante, O; Segovia, J C; Nishimura, K; Ohtaka, M; Nakanishi, M; Graf, T; Menendez, P

    2016-03-01

    B cells have been shown to be refractory to reprogramming and B-cell-derived induced pluripotent stem cells (iPSC) have only been generated from murine B cells engineered to carry doxycycline-inducible Oct4, Sox2, Klf4 and Myc (OSKM) cassette in every tissue and from EBV/SV40LT-immortalized lymphoblastoid cell lines. Here, we show for the first time that freshly isolated non-cultured human cord blood (CB)- and peripheral blood (PB)-derived CD19+CD20+ B cells can be reprogrammed to iPSCs carrying complete VDJH immunoglobulin (Ig) gene monoclonal rearrangements using non-integrative tetracistronic, but not monocistronic, OSKM-expressing Sendai Virus. Co-expression of C/EBPα with OSKM facilitates iPSC generation from both CB- and PB-derived B cells. We also demonstrate that myeloid cells are much easier to reprogram than B and T lymphocytes. Differentiation potential back into the cell type of their origin of B-cell-, T-cell-, myeloid- and fibroblast-iPSCs is not skewed, suggesting that their differentiation does not seem influenced by 'epigenetic memory'. Our data reflect the actual cell-autonomous reprogramming capacity of human primary B cells because biased reprogramming was avoided by using freshly isolated primary cells, not exposed to cytokine cocktails favoring proliferation, differentiation or survival. The ability to reprogram CB/PB-derived primary human B cells offers an unprecedented opportunity for studying developmental B lymphopoiesis and modeling B-cell malignancies. PMID:26500142

  8. Interaction of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) with owl monkey kidney cells in enhancing the yields of Herpesvirus saimiri (HVS) and its antigens

    SciTech Connect

    Faggioni, A.; Ablashi, D.V.; Dahlberg, J.; Armstrong, G.; Sundar, S.K.

    1984-05-01

    Pre- and posttreatment with N-methyl-N'-nitro-nitrosoguanidine (MNNG) of owl monkey kidney (OMK) cells infected with Herpesvirus saimiri (HVS) resulted in one to three logs higher yields of virus, depending upon the dose of MNNG. A higher percentage of cells also showed HVS early antigen (EA) and late antigen (LA) by immunofluorescence when OMK cells infected with HVS were fed with medium containing MNNG. The high yields of HVS were also observed by electron microscopy. MNNG did not induce HVS-EA in HVS nonproducer lymphoblastoid T cells, nor did it enhance TPA-induced EA to LA. The data suggest that MNNG could be useful in obtaining high yields of virus and/or antigen-producing cells for immunofluorescence or other biomedical experiments, especially from those strains of HVS which grow poorly in vitro. The interaction of MNNG and HVS could also be useful for in vitro transformation or in vivo enhancement of the malignant process.

  9. Identification of a novel gene expressed in activated natural killer cells and T cells

    SciTech Connect

    Dahl, C.A.; Schall, R.P.; He, H.; Cairns, J.S. )

    1992-01-15

    The authors have isolated a cDNA clone from a human activated NK cell-derived cDNA library that identifies a transcript [NK4] that is selectively expressed in lymphocytes. The expression of this transcript is increased after activation of T cells by mitogens or activation of NK cells by IL-2 (lymphokine-activated killer cells). The transcript levels demonstrated by Northern blot analysis increase by 12 h after activation, remain high for at least 48 h, and require protein synthesis for expression. Southern blot analysis of B lymphoblastoid lines derived from 18 unrelated individuals reveal variable banding patterns suggestive of polymorphism within the NK4 gene. No homology was found between the sequence of the coding region of this transcript and any sequences in the GenBank data base. Sequence homology to the U1 small nuclear RNA was found within the 3[prime] untranslated region immediately upstream of the site of polyadenylation, suggesting a possible role for U1 in the polyadenylation process. Sequence analysis indicates the transcript would encode a protein having a mass of 27 kDa. The presence of a signal sequence and lack of a transmembrane region suggests that the protein is secreted. In addition, the protein contains an RGD sequence that may be involved in cellular adhesion. This transcript appears to encode a novel product common to the activation pathways of both NK cells and T cells. 50 refs., 8 figs.

  10. Mutagenic Effects of Iron Oxide Nanoparticles on Biological Cells

    PubMed Central

    Dissanayake, Niluka M.; Current, Kelley M.; Obare, Sherine O.

    2015-01-01

    In recent years, there has been an increased interest in the design and use of iron oxide materials with nanoscale dimensions for magnetic, catalytic, biomedical, and electronic applications. The increased manufacture and use of iron oxide nanoparticles (IONPs) in consumer products as well as industrial processes is expected to lead to the unintentional release of IONPs into the environment. The impact of IONPs on the environment and on biological species is not well understood but remains a concern due to the increased chemical reactivity of nanoparticles relative to their bulk counterparts. This review article describes the impact of IONPs on cellular genetic components. The mutagenic impact of IONPs may damage an organism’s ability to develop or reproduce. To date, there has been experimental evidence of IONPs having mutagenic interactions on human cell lines including lymphoblastoids, fibroblasts, microvascular endothelial cells, bone marrow cells, lung epithelial cells, alveolar type II like epithelial cells, bronchial fibroblasts, skin epithelial cells, hepatocytes, cerebral endothelial cells, fibrosarcoma cells, breast carcinoma cells, lung carcinoma cells, and cervix carcinoma cells. Other cell lines including the Chinese hamster ovary cells, mouse fibroblast cells, murine fibroblast cells, Mytilus galloprovincialis sperm cells, mice lung cells, murine alveolar macrophages, mice hepatic and renal tissue cells, and vero cells have also shown mutagenic effects upon exposure to IONPs. We further show the influence of IONPs on microorganisms in the presence and absence of dissolved organic carbon. The results shed light on the transformations IONPs undergo in the environment and the nature of the potential mutagenic impact on biological cells. PMID:26437397

  11. Gene expression in cell lines from propionic acidemia patients, carrier parents, and controls.

    PubMed

    Chapman, Kimberly A; Bush, William S; Zhang, Zhe

    2015-08-01

    Propionic acidemia (PA) is an inborn of metabolism which usually presents with metabolic acidosis and accumulation of 3-hydroxypropionate among other toxins. Examining the gene expression in lymphoblastoid cell lines (LCLs) from PA patients, their carrier parents and age/sex-matched controls at normal glucose and low glucose growth conditions demonstrated differences among and between these groups. Using three-way ANOVA analysis, four DAVID clusters of response were identified of which three of the four clusters showed that LCLs from carrier parents had an intermediate response between healthy controls and PA patients. These differences included changes in expression of cell cycle regulatory genes, mitochondrial related genes, and transcriptional regulation. In addition, differences also were observed in expression of genes involved in transendothelial migration and focal adhesion at normal growth conditions when comparing the LCLs from PA patients and controls. These studies demonstrate transcriptional differences between LCLs from PA patients, their parents and biochemically normal controls. PMID:25963861

  12. [Intracellular immunoglobulins in Namalwa and U266 cells co-cultivated with mesenchymal stromal cells].

    PubMed

    Aĭzenshtadt, A A; Ivanova, N A; Bagaeva, V V; Smolianinov, A B; Pinevich, A A; Samoĭlovich, M P; Klimovich, V B

    2014-01-01

    There are contradictory data concerning the influence of mesenchymal stromal cells (MSC) on immunoglobulin (Ig) production. Most of them were obtained using MSC from bone marrow. Properties of MSC from other tissues are elusive. In the present work MSC cultures were derived from umbilical cord, adipose tissue, and bone marrow of healthy donors, as well as from bone marrow of patients with autoimmune diseases. MSC from all these sources had similar surface markers phenotype. The influence of co-cultivation with MSC at exponential or stationary phase on IgM and IgE content in Namalva and U266 cells was evaluated. MSC from bone marrow of healthy donors had no effect on IgM and IgE production. Proliferating MSC obtained from patients with Crohn's disease and multiple sclerosis stimulated Ig production. Exponentially growing MSC derived from umbilical cord and adipose tissue also stimulated Ig synthesis. MSC at stationary cultures amplified IgM production in Namalva cells and suppressed IgE synthesis in U266. Thus, MSC with similar phenotype but derived from different sources differ in their capacity to modulate Ig production in B-lymphoid cells. The effect of MSC depends on their growth stage and may differ for lymphoblastoid and myeloma cells. PMID:25509151

  13. Long Noncoding RNA Expression Profiling in Normal B-Cell Subsets and Hodgkin Lymphoma Reveals Hodgkin and Reed-Sternberg Cell-Specific Long Noncoding RNAs.

    PubMed

    Tayari, Mina Masoumeh; Winkle, Melanie; Kortman, Gertrud; Sietzema, Jantine; de Jong, Debora; Terpstra, Martijn; Mestdagh, Pieter; Kroese, Frans G M; Visser, Lydia; Diepstra, Arjan; Kok, Klaas; van den Berg, Anke; Kluiver, Joost

    2016-09-01

    Hodgkin lymphoma (HL) is a malignancy of germinal center (GC) B-cell origin. To explore the role of long noncoding RNAs (lncRNAs) in HL, we studied lncRNA expression patterns in normal B-cell subsets, HL cell lines, and tissues. Naive and memory B cells showed a highly similar lncRNA expression pattern, distinct from GC-B cells. Significant differential expression between HL and normal GC-B cells was observed for 475 lncRNA loci. For two validated lncRNAs, an enhanced expression was observed in HL, diffuse large B-cell lymphoma, and lymphoblastoid cell lines. For a third lncRNA, increased expression levels were observed in HL and part of Burkitt lymphoma cell lines. RNA fluorescence in situ hybridization on primary HL tissues revealed a tumor cell-specific expression pattern for all three lncRNAs. A potential cis-regulatory role was observed for 107 differentially expressed lncRNA-mRNA pairs localizing within a 60-kb region. Consistent with a cis-acting role, we showed a preferential nuclear localization for two selected candidates. Thus, we showed dynamic lncRNA expression changes during the transit of normal B cells through the GC reaction and widely deregulated lncRNA expression patterns in HL. Three lncRNAs showed a tumor cell-specific expression pattern in HL tissues and might therefore be of value as a biomarker. PMID:27423697

  14. Human clusterin gene expression is confined to surviving cells during in vitro programmed cell death.

    PubMed Central

    French, L E; Wohlwend, A; Sappino, A P; Tschopp, J; Schifferli, J A

    1994-01-01

    Clusterin is a serum glycoprotein endowed with cell aggregating, complement inhibitory, and lipid binding properties, and is also considered as a specific marker of dying cells, its expression being increased in various tissues undergoing programmed cell death (PCD). However, no study has so far directly shown that cells expressing clusterin in these tissues are actually apoptotic as defined by morphological and biochemical criteria. We have studied cellular clusterin gene expression in vitro using three different models of PCD: (a) ultraviolet B (UV-B) irradiation of human U937, HeLa, and A431 cell lines, (b) in vitro aging of human peripheral blood neutrophils (PMNs), and (c) dexamethasone-induced cell death of the human lymphoblastoid cell line CEM-C7. In all three models, the classical morphological and biochemical features of PCD observed did not correlate with an increase, but with either a marked decrease or an absence of clusterin gene expression as assessed by Northern blot analysis. In situ hybridization of U937 and A431 cells after UV-B irradiation revealed, in addition, that only morphologically normal cells that are surviving continue to express the clusterin gene. Our results demonstrate that in the human myeloid, lymphoid, and epithelial cell types studied, clusterin gene expression is not a prerequisite to their death by apoptosis. In addition, and most interestingly, in situ hybridization of U937 and A431 cells revealed that only surviving cells express the clusterin gene after the induction of PCD, thus providing novel evidence suggesting that clusterin may be associated with cell survival within tissues regressing as a consequence of PCD. Images PMID:8113419

  15. Proteomics Based Identification of Proteins with Deregulated Expression in B Cell Lymphomas.

    PubMed

    Wu, Rui; Nijland, Marcel; Rutgers, Bea; Veenstra, Rianne; Langendonk, Myra; van der Meeren, Lotte E; Kluin, Philip M; Li, Guanwu; Diepstra, Arjan; Chiu, Jen-Fu; van den Berg, Anke; Visser, Lydia

    2016-01-01

    Follicular lymphoma and diffuse large B cell lymphomas comprise the main entities of adult B cell malignancies. Although multiple disease driving gene aberrations have been identified by gene expression and genomic studies, only a few studies focused at the protein level. We applied 2 dimensional gel electrophoresis to compare seven GC B cell non Hodgkin lymphoma (NHL) cell lines with a lymphoblastoid cell line (LCL). An average of 130 spots were at least two folds different in intensity between NHL cell lines and the LCL. We selected approximately 38 protein spots per NHL cell line and linked them to 145 unique spots based on the location in the gel. 34 spots that were found altered in at least three NHL cell lines when compared to LCL, were submitted for LC-MS/MS. This resulted in 28 unique proteins, a substantial proportion of these proteins were involved in cell motility and cell metabolism. Loss of expression of B2M, and gain of expression of PRDX1 and PPIA was confirmed in the cell lines and primary lymphoma tissue. Moreover, inhibition of PPIA with cyclosporine A blocked cell growth of the cell lines, the effect size was associated with the PPIA expression levels. In conclusion, we identified multiple differentially expressed proteins by 2-D proteomics, and showed that some of these proteins might play a role in the pathogenesis of NHL. PMID:26752561

  16. Gammaherpesvirus Infection of Human Neuronal Cells

    PubMed Central

    Jha, Hem Chandra; Mehta, Devan; Lu, Jie; El-Naccache, Darine; Shukla, Sanket K.; Kovacsics, Colleen; Kolson, Dennis

    2015-01-01

    ABSTRACT Gammaherpesviruses human herpesvirus 4 (HHV4) and HHV8 are two prominent members of the herpesvirus family associated with a number of human cancers. HHV4, also known as Epstein-Barr virus (EBV), a ubiquitous gammaherpesvirus prevalent in 90 to 95% of the human population, is clinically associated with various neurological diseases such as primary central nervous system lymphoma, multiple sclerosis, Alzheimer’s disease, cerebellar ataxia, and encephalitis. However, the possibility that EBV and Kaposi’s sarcoma-associated herpesvirus (KSHV) can directly infect neurons has been largely overlooked. This study has, for the first time, characterized EBV infection in neural cell backgrounds by using the Sh-Sy5y neuroblastoma cell line, teratocarcinoma Ntera2 neurons, and primary human fetal neurons. Furthermore, we also demonstrated KSHV infection of neural Sh-Sy5y cells. These neuronal cells were infected with green fluorescent protein-expressing recombinant EBV or KSHV. Microscopy, genetic analysis, immunofluorescence, and Western blot analyses for specific viral antigens supported and validated the infection of these cells by EBV and KSHV and showed that the infection was efficient and productive. Progeny virus produced from infected neuronal cells efficiently infected fresh neuronal cells, as well as peripheral blood mononuclear cells. Furthermore, acyclovir was effective at inhibiting the production of virus from neuronal cells similar to lymphoblastoid cell lines; this suggests active lytic replication in infected neurons in vitro. These studies represent a potentially new in vitro model of EBV- and KSHV-associated neuronal disease development and pathogenesis. PMID:26628726

  17. Development of a dihydroartemisinin-resistant Molt-4 leukemia cell line.

    PubMed

    Park, Jungsoo; Lai, Henry C; Singh, Mallika; Sasaki, Tomikazu; Singh, Narendra P

    2014-06-01

    Artemisinin generates cytotoxic free radicals when it reacts with iron. Its toxicity is more selective toward cancer cells because cancer cells contain a higher level of intracellular-free iron. We previously reported that dihydroartemisinin (DHA), an active metabolite of artemisinin, has selective cytotoxicity toward Molt-4 human lymphoblastoid cells. A concern is whether cancer cells could develop resistance to DHA after repeated administration, thus limiting its therapeutic efficacy. In the present study, we developed a DHA-resistant Molt-4 cell line (RTN) by exposing Molt-4 cells to gradually increasing concentrations of DHA in vitro. The half-maximal inhibitory concentration (IC50) of DHA for RTN cells is 7.1-times higher than that of Molt-4 cells. RTN cells have a higher growth rate than Molt-4 cells. In addition, we investigated the toxicities of two more potent synthetic artemisinin compounds, artemisinin dimer-alcohol and artemisinin-tagged holotransferrin toward RTN cells; RTN cells showed no significant cross-resistance to these compounds. PMID:24922643

  18. CD4+ T-cell clones recognizing human lymphoma-associated antigens: generation by in vitro stimulation with autologous Epstein-Barr virus-transformed B cells.

    PubMed

    Long, Heather M; Zuo, Jianmin; Leese, Alison M; Gudgeon, Nancy H; Jia, Hui; Taylor, Graham S; Rickinson, Alan B

    2009-07-23

    Epstein-Barr virus (EBV)-specific T-cell preparations, generated by stimulating immune donor lymphocytes with the autologous virus-transformed B-lymphoblastoid cell line (LCL) in vitro, can be used to target EBV-positive malignancies. Although these preparations are enriched for EBV antigen-specific CD8(+) T cells, most also contain a CD4(+) T-cell population whose specificity is unknown. Here, we show that, although CD4(+) T-cell clones derived from such cultures recognize HLA class II-matched LCLs but not mitogen-activated B lymphoblasts, many (1) do not map to any known EBV antigen, (2) can be raised from EBV-naive as well as EBV-immune persons, and (3) can recognize a broad range of human B lymphoma-derived cell lines irrespective of EBV genome status, providing those lines to express the relevant HLA class II-restricting allele. Importantly, such CD4(+) clones not only produce IFNgamma but are also cytotoxic and can control the outgrowth of HLA-matched lymphoma cells in cocultivation assays. We infer that such CD4(+) T cells recognize cellular antigens that are preferentially up-regulated in EBV-transformed but not mitogen-activated B lymphoblasts and that are also expressed in a range of B-cell malignancies. Such antigens are therefore of potential value as targets for CD4(+) T cell-based immunotherapy. PMID:19443664

  19. Caffeic Acid Phenylethyl Ester and MG-132 Have Apoptotic and Antiproliferative Effects on Leukemic Cells But Not on Normal Mononuclear Cells12

    PubMed Central

    Cavaliere, Victoria; Papademetrio, Daniela L; Lorenzetti, Mario; Valva, Pamela; Preciado, María Victoria; Gargallo, Patricia; Larripa, Irene; Monreal, Mariela B; Pardo, María Laura; Hajos, Silvia E; Blanco, Guillermo AC; Álvarez, Élida MC

    2009-01-01

    Chemotherapy aims to limit proliferation and induce apoptotic cell death in tumor cells. Owing to blockade of signaling pathways involved in cell survival and proliferation, nuclear factor κB (NF-κB) inhibitors can induce apoptosis in a number of hematological malignancies. The efficacy of conventional chemotherapeutic drugs, such as vincristine (VCR) and doxorubicine (DOX), may be enhanced with combined therapy based on NF-κB modulation. In this study, we evaluated the effect of caffeic acid phenylethyl ester (CAPE) and MG-132, two nonspecific NF-κB inhibitors, and conventional chemotherapeutics drugs DOX and VCR on cell proliferation and apoptosis induction on a lymphoblastoid B-cell line, PL104, established and characterized in our laboratory. CAPE and MG-132 treatment showed a strong antiproliferative effect accompanied by clear cell cycle deregulation and apoptosis induction. Doxorubicine and VCR showed antiproliferative effects similar to those of CAPE and MG-132, although the latter drugs showed an apoptotic rate two-fold higher than DOX and VCR. None of the four compounds showed cytotoxic effect on peripheral mononuclear cells from healthy volunteers. CAPE- and MG-132-treated bone marrow cells from patients with myeloid and lymphoid leukemias showed 69% (P < .001) and 25% decrease (P < .01) in cell proliferation and 42% and 34% (P < .01) apoptosis induction, respectively. Overall, our results indicate that CAPE and MG-132 had a strong and selective apoptotic effect on tumor cells that may be useful in future treatment of hematological neoplasias. PMID:19252751

  20. Rescue of “crippled” germinal center B cells from apoptosis by Epstein-Barr virus

    PubMed Central

    Mancao, Christoph; Altmann, Markus; Jungnickel, Berit; Hammerschmidt, Wolfgang

    2005-01-01

    Epstein-Barr virus (EBV) is associated with B-cell lymphomas such as Hodgkin lymphoma, Burkitt lymphoma, and post-transplantation lymphoma, which originate from clonal germinal center (GC) B cells. During the process of somatic hypermutation, GC B cells can acquire deleterious or nonsense mutations in the heavy and light immunoglobulin genes. Such mutations abrogate the cell surface expression of the B-cell receptor (BCR), which results in the elimination of these nonfunctional B cells by immediate apoptosis. EBV encodes several latent genes, among them latent membrane protein 1 (LMP1) and LMP2A, which are regularly expressed in EBV-positive Hodgkin lymphoma and posttransplantation lymphomas. Since LMP1 and LMP2A mimic the function of 2 key receptors on B cells, CD40 and BCR, respectively, we wanted to learn whether EBV infection can rescue proapoptotic GC B cells with crippling mutations in the heavy chain immunoglobulin locus from apoptosis. We show here that BCR-negative GC B cells readily enter the cell cycle upon infection with EBV in vitro and yield clonal lymphoblastoid cell lines that are incapable of expressing a functional BCR because the rearranged and formerly functional heavy chain immunoglobulin alleles carry deleterious mutations. Our findings imply an important role for EBV in the process of lymphomagenesis in certain cases of Hodgkin lymphoma and posttransplantation lymphomas. PMID:16076866

  1. Internalization of anti-nucleolin antibody into viable HEp-2 cells.

    PubMed

    Deng, J S; Ballou, B; Hofmeister, J K

    1996-01-01

    Anti-nucleolin antibodies have been detected in patients with systemic connective tissue diseases (SCTD) including systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). In vivo bound autoantibodies to nucleoli of epidermal keratinocytes have been demonstrated in skin from patients with SCTD. In this study, monoclonal antibody to nucleolin (D-3) was used to determine the distribution of nucleolin in different culture cells including HEp-2, HepG2, HRCC, Molt-4 and Wil2 cells. Nucleolin was found to be present on the surface of HEp-2 and HepG2 cells, but not on the surface of HRCC and lymphoblastoid (Molt-4 and Wil2) cells; in contrast, nucleolin was detected in the nucleoli of all permeabilized cells examined. In immunoprecipitation, using extracts from 32P-labeled HEp-2 cells as antigenic source, cell membrane as well as nuclear nucleolins were found to be phosphorylated with a molecular weight of 105 kDa. Viable HEp-2 and HepG2 cells were cocultured with IgG fraction of D-3 in a CO2 incubator for 1 to 24 h, and then permeabilized with acetone followed by immunofluorescence staining with FITC-labeled goat anti-mouse IgG antibodies. Nucleolar staining was observed in cells after 10 h or longer of coculture. These data indicated that D-3 antibody reacted with cell membrane nucleolin and subsequently gain access into cells in a process related to pinocytosis. PMID:9112228

  2. Studies of EBV-lymphoid cell interactions in two patients with the X-linked lymphoproliferative syndrome: normal EBV-specific HLA-restricted cytotoxicity.

    PubMed

    Rousset, F; Souillet, G; Roncarolo, M G; Lamelin, J P

    1986-02-01

    Two X-linked lymphoproliferative syndrome (XLP) patients with the hypogammaglobulinemia phenotype were investigated at a time remote from their primary infection with the Epstein-Barr virus (EBV). The lymphoblastoid cell lines derived from these patients expressed the phenotypic markers characteristic of normal mature B lymphocytes and produced normal levels of immunoglobulins (Ig). These observations imply that at least some of their B cells are phenotypically normal. The natural killer (NK) activity of the two patients was low. In one patient, activated lymphocyte killer (ALK) activity was inefficient. These two XLP patients expressed a normal EBV-specific, HLA-restricted cytotoxic activity. It thus appears, from the present findings and those in cases published previously (6/11 patients expressing normal EBV-specific cytotoxic activity), that the notion of poor specific T cell memory for EBV may not be as pivotal ass suggested or, alternatively, that this defect may not be common in hypogammaglobulinemic survivors. PMID:3009061

  3. {sub p}53-Dependent Adaptive Responses in Human Cells Exposed to Space Radiations

    SciTech Connect

    Takahashi, Akihisa; Su Xiaoming; Suzuki, Hiromi; Omori, Katsunori; Seki, Masaya; Hashizume, Toko; Shimazu, Toru; Ishioka, Noriaki; Iwasaki, Toshiyasu; Ohnishi, Takeo

    2010-11-15

    Purpose: It has been reported that priming irradiation or conditioning irradiation with a low dose of X-rays in the range of 0.02-0.1 Gy induces a p53-dependent adaptive response in mammalian cells. The aim of the present study was to clarify the effect of space radiations on the adaptive response. Methods and Materials: Two human lymphoblastoid cell lines were used; one cell line bears a wild-type p53 (wtp53) gene, and another cell line bears a mutated p53 (mp53) gene. The cells were frozen during transportation on the space shuttle and while in orbit in the International Space Station freezer for 133 days between November 15, 2008 and March 29, 2009. After the frozen samples were returned to Earth, the cells were cultured for 6 h and then exposed to a challenging X-ray-irradiation (2 Gy). Cellular sensitivity, apoptosis, and chromosome aberrations were scored using dye-exclusion assays, Hoechst33342 staining assays, and chromosomal banding techniques, respectively. Results: In cells exposed to space radiations, adaptive responses such as the induction of radioresistance and the depression of radiation-induced apoptosis and chromosome aberrations were observed in wtp53 cells but not in mp53 cells. Conclusion: These results have confirmed the hypothesis that p53-dependent adaptive responses are apparently induced by space radiations within a specific range of low doses. The cells exhibited this effect owing to space radiations exposure, even though the doses in space were very low.

  4. Identification of a Cell Surface Protein, p97, in Human Melanomas and Certain Other Neoplasms

    NASA Astrophysics Data System (ADS)

    Woodbury, Richard G.; Brown, Joseph P.; Yeh, Ming-Yang; Hellstrom, Ingegerd; Hellstrom, Karl Erik

    1980-04-01

    BALB/c mice were immunized with a human melanoma cell line, SK-MEL 28, and their spleen cells were fused with mouse NS-1 myeloma cells. Hybrid cells were tested in an indirect 125I-labeled protein A assay for production of antibodies that bound to surface antigens of SK-MEL 28 melanoma cells but not to autologous skin fibroblasts. One hybridoma, designated 4.1, had the required specificity. It was cloned and grown in mice as an ascites tumor. The monoclonal IgG1 antibody produced by the hybridoma was purified from the ascites fluid and labeled with 125I. The labeled antibody bound, at significant levels, to approximately 90% of the melanomas tested and to approximately 55% of other tumor cells, but not to three B-lymphoblastoid cell lines or to cultivated fibroblasts from 15 donors. Immunoprecipitation and sodium dodecyl sulfate gel electrophoresis were used to detect the target antigen in 125I-labeled cell membranes of both cultivated cells and tumor biopsy samples. A protein with a molecular weight of 97,000 was identified. This protein, designated p97, was present in both cultured cells and biopsy material from melanomas and certain other tumors, but it was not detected in eight different samples of normal adult epithelial or mesenchymal tissues obtained from five donors.

  5. Histone deacetylase inhibitor abexinostat affects chromatin organization and gene transcription in normal B cells and in mantle cell lymphoma.

    PubMed

    Markozashvili, Diana; Pichugin, Andrei; Barat, Ana; Camara-Clayette, Valerie; Vasilyeva, Natalia V; Lelièvre, Hélène; Kraus-Berthier, Laurence; Depil, Stéphane; Ribrag, Vincent; Vassetzky, Yegor

    2016-04-15

    Mantle cell lymphoma (MCL) is a rare lymphoma caused by the t(11:14) juxtaposing the cyclin D1 (CCND1) locus on chromosome 11 and the immunoglobulin heavy chain (IgH) locus on chromosome 14. Several new treatments are proposed for MCL, including histone deacetylase inhibitors (HDACi). We have studied gene expression and chromatin organization in the translocated 11q13 locus in MCL cells as compared to lymphoblastoid cell lines as well as the effect of HDACi abexinostat on chromatin organization and gene expression in the 11q13 locus. We have identified a cluster of genes overexpressed in the translocation region on chromosome 11 in MCL cells. Abexinostat provokes a genome-wide disaggregation of heterochromatin. The genes upregulated after the t(11;14) translocation react to the HDACi treatment by increasing their expression, but their gene promoters do not show significant alterations in H3K9Ac and H3K9me2 levels in abexinostat-treated cells. PMID:26774800

  6. KIR2DS1-dependent acquisition of CCR7 and migratory properties by human NK cells interacting with allogeneic HLA-C2+ DCs or T-cell blasts.

    PubMed

    Marcenaro, Emanuela; Pesce, Silvia; Sivori, Simona; Carlomagno, Simona; Moretta, Lorenzo; Moretta, Alessandro

    2013-04-25

    Natural killer (NK) cells may capture the CCR7 chemokine receptor from allogeneic CCR7(+) cells by trogocytosis and acquire migrating properties in response to lymph node chemokines. This event is negatively regulated by inhibitory killer Ig-like receptors (KIRs) and NKG2A. In this study, we analyzed the role of the HLA-C2-specific activating receptor KIR2DS1 in the process of CCR7 uptake by NK cells interacting with different allogeneic CCR7(+) cells. Co-incubation of KIR2DS1(+) fresh NK cells or NK-cell clones with HLA-C2(+) CCR7(+) lymphoblastoid cell lines resulted in increased CCR7 uptake. Remarkably, KIR2DS1 expression represented a major advantage for acquiring CCR7 from HLA-C2(+) allogeneic dendritic cells (DCs) and T-cell blasts. These findings have important implications in haploidentical hematopoietic stem cell transplantation in which donor-derived (alloreactive) KIR2DS1(+) NK cells, upon CCR7 acquisition, become capable of migrating toward lymph nodes, where they may kill patient DCs and T cells, preventing graft-versus-host and host-versus-graft reactions. PMID:23449637

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

    SciTech Connect

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

    1987-12-01

    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/sup +/, CD3/sup +/, CD4/sup +/ or CD2/sup +/, CD3/sup +/, CD8/sup +/) 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 /sup 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.

  8. Accessing Epstein-Barr Virus-Specific T-Cell Memory with Peptide-Loaded Dendritic Cells

    PubMed Central

    Redchenko, I. V.; Rickinson, A. B.

    1999-01-01

    The conventional means of studying Epstein-Barr virus (EBV)-induced cytotoxic T-lymphocyte (CTL) memory, by in vitro stimulation with the latently infected autologous lymphoblastoid cell line (LCL), has important limitations. First, it gives no information on memory to lytic cycle antigens; second, it preferentially amplifies the dominant components of latent antigen-specific memory at the expense of key subdominant reactivities. Here we describe an alternative approach, based on in vitro stimulation with epitope peptide-loaded dendritic cells (DCs), which allows one to probe the CTL repertoire for any individual reactivity of choice; this method proved significantly more efficient than stimulation with peptide alone. Using this approach we first show that reactivities to the immunodominant and subdominant lytic cycle epitopes identified by T cells during primary EBV infection are regularly detectable in the CTL memory of virus carriers; this implies that in such carriers chronic virus replication remains under direct T-cell control. We further show that subdominant latent cycle reactivities to epitopes in the latent membrane protein LMP2, though rarely undetectable in LCL-stimulated populations, can be reactivated by DC stimulation and selectively expanded as polyclonal CTL lines; the adoptive transfer of such preparations may be of value in targeting certain EBV-positive malignancies. PMID:9847337

  9. Effect of caffeine on radiation-induced apoptosis in TK6 cells

    SciTech Connect

    Zhen, W.; Vaughan, A.T.M.

    1995-02-01

    Apoptosis has been measured in cells of the human TK6 lymphoblastoid cell line by recording the release of endonuclease-digested DNA from affected cells using flow cytometry. In asynchronously dividing cells, DNA degradation characteristic of apoptosis was first seen 12 h after irradiation as a defined DNA fluorescent peak of sub-G{sub 1}-phase content, reaching a maximum of 30-50% of the population by 24-72 h. Treating cells with 2 mM caffeine either before or up to 3 h after irradiation eliminated the degradation of DNA entirely. In addition, the percentage of cells in which apoptosis could be detected microscopically decreased from 62.4 {+-} 0.95% to 16.7 {+-} 1.5% 72 h after caffeine treatment. Delaying caffeine treatment for 12 h after irradiation reduced DNA degradation by approximately 50% compared to cells receiving radiation alone. DNA degradation induced by serum deprivation was unaffected by caffeine treatment. These data support the contention that irradiation of TK6 cells produces a long-lived cellular signal which triggers apoptosis. Apoptosis produced by serum deprivation does not operate through the same pathway. 36 refs., 5 figs.

  10. Two classes of single-stranded regions evident in deproteinized preparations of replicating DNA isolated from mammalian cells

    SciTech Connect

    Stewart, B.W.; Kavallaris, M.; Catchpoole, D.; Norris, M.D. )

    1991-02-01

    In DNA isolated from proliferating human lymphoblastoid CCRF-CEM cells which had been pulse-labeled by exposure to (3H)thymidine for periods from 30 s to 10 min, single-stranded regions were analyzed by caffeine-gradient elution from benzoylated DEAE-cellulose. Two classes of structural defect were evident. Some replicating DNA exhibited single-stranded regions of approximately 200 nucleotides, while most newly incorporated radioactivity was associated with DNA containing single-stranded regions from 900 to approximately 4000 nucleotides. The distribution of thymidine-derived radioactivity did not suggest sequential or preferential labeling of these DNA fractions as the incorporation time was varied. The findings may be correlated with recent proposals regarding the structural basis of eukaryotic DNA replication.

  11. Epstein-Barr virus-carrying B cells are large, surface IgM, IgD-bearing cells in normal individuals and acute malaria patients.

    PubMed Central

    Lam, K M; Whittle, H; Grzywacz, M; Crawford, D H

    1994-01-01

    In this study the presence of Epstein-Barr virus (EBV) carrying B lymphocytes in different B-cell subpopulations from peripheral blood was determined by spontaneous outgrowth which gives rise to lymphoblastoid cell lines. In healthy seropositive adults, the EBV-carrying B cell was predominantly within the IgM- and IgD-positive but not the IgG-positive subpopulations. Furthermore, these B lymphocytes were in the low-density (large cell) Percoll fraction. The IgM- and IgD-positive B cell phenotype suggests the EBV-carrying B cells to be circulating virgin B cells recently released from the bone marrow. These B cells have an estimated life span of only 6-8 weeks suggesting that long-term EBV persistence in the body may be the result of infection of a more primitive B-cell type. Similar experiments were carried out in children with acute malaria from the Gambia, West Africa, where Burkitt lymphoma (BL) is endemic in order to determine whether a population of EBV-carrying B cells could be identified which had a similar phenotype to the BL cell. The EBV-carrying B cells in this patient group were also found in the IgM-positive, IgG-negative B-cell subpopulation. The majority of these cells were found in the low-density (large cell) Percoll fraction although in some patients a proportion was derived from the high-density (small cell) fraction. This cellular phenotype is not representative of a BL cell. PMID:7959872

  12. B-cell proliferation and induction of early G1-regulating proteins by Epstein-Barr virus mutants conditional for EBNA2.

    PubMed

    Kempkes, B; Spitkovsky, D; Jansen-Dürr, P; Ellwart, J W; Kremmer, E; Delecluse, H J; Rottenberger, C; Bornkamm, G W; Hammerschmidt, W

    1995-01-01

    Infection of primary B-lymphocytes by Epstein-Barr virus (EBV) leads to growth transformation of these B-cells in vitro. EBV nuclear antigen 2 (EBNA2), one of the first genes expressed after EBV infection of B-cells, is a transcriptional activator of viral and cellular genes and is essential for the transforming potential of the virus. We generated conditional EBV mutants by expressing EBNA2 as chimeric fusion protein with the hormone binding domain of the estrogen receptor on the genetic background of the virus. Growth transformation of primary normal B-cells by mutant virus resulted in estrogen-dependent lymphoblastoid cell lines expressing the chimeric EBNA2 protein. In the absence of estrogen about half of the cells enter a quiescent non-proliferative state whereas the others die by apoptosis. EBNA2 is thus required not only for initiation but also for maintenance of transformation. Growth arrest occurred at G1 and G2 stages of the cell cycle, indicating that functional EBNA2 is required at different restriction points of the cell cycle. Growth arrest is reversible for G1/G0 cells as indicated by the sequential accumulation and modification of cell cycle regulating proteins. EBV induces the same cell cycle regulating proteins as polyclonal stimuli in primary B-cells. These data suggest that EBV is using a common pathway for B-cell activation bypassing the requirement for antigen, T-cell signals and growth factors. PMID:7828599

  13. Skewed T cell receptor repertoire of Vδ1+ γδ T lymphocytes after human allogeneic haematopoietic stem cell transplantation and the potential role for Epstein–Barr virus-infected B cells in clonal restriction

    PubMed Central

    Fujishima, N; Hirokawa, M; Fujishima, M; Yamashita, J; Saitoh, H; Ichikawa, Y; Horiuchi, T; Kawabata, Y; Sawada, K-I

    2007-01-01

    The proliferation of Vδ1+ γδ T lymphocytes has been described in various infections including human immunodeficiency virus (HIV), cytomegalovirus (CMV) and malaria. However, the antigen specificity and functions of the human Vδ1+ T cells remain obscure. We sought to explore the biological role for this T cell subset by investigating the reconstitution of T cell receptor (TCR) repertoires of Vδ1+ γδ T lymphocytes after human allogeneic haematopoietic stem cell transplantation (HSCT). We observed skewed TCR repertoires of the Vδ1+ T cells in 27 of 44 post-transplant patients. Only one patient developed EBV-associated post-transplant lymphoproliferative disorder in the present patient cohort. The -WGI- amino acid motif was observed in CDR3 of clonally expanded Vδ1+ T cells in half the patients. A skew was also detected in certain healthy donors, and the Vδ1+ T cell clone derived from the donor mature T cell pool persisted in the recipient's blood even 10 years after transplant. This T cell clone expanded in vitro against stimulation with autologous EBV–lymphoblastoid cell lines (LCL), and the Vδ1+ T cell line expanded in vitro from the same patient showed cytotoxicity against autologous EBV–LCL. EBV-infected cells could also induce in vitro oligoclonal expansions of autologous Vδ1+ T cells from healthy EBV-seropositive individuals. These results suggest that human Vδ1+ T cells have a TCR repertoire against EBV-infected B cells and may play a role in protecting recipients of allogeneic HSCT from EBV-associated disease. PMID:17425654

  14. Effects of biomaterials for Lab-on-a-chip production on cell growth and expression of differentiated functions of leukemic cell lines.

    PubMed

    Destro, Federica; Borgatti, Monica; Iafelice, Bruno; Gavioli, Riccardo; Braun, Tanja; Bauer, Jörg; Böttcher, Lars; Jung, Erik; Bocchi, Massimo; Guerrieri, Roberto; Gambari, Roberto

    2010-09-01

    The rapid increase of the applications for Lab-on-a-chip devices has attracted the interest of researchers and engineers on standard process of the electronics industry for low production costs and large scale development, necessary for disposable applications. The printed circuit board technology could be used for this purpose, in particular for the wide range of materials available. In this paper, assays on biocompatibility of materials used for Lab-on-a-chip fabrication has been carried out using two tumor cell lines growing in suspension, the human chronic myelogenous leukemia K562 cell line, able to undergo erythroid differentiation when cultured with chemical inducers, and the lymphoblastoid cell line (LCL), extensively used for screening of cytotoxic T-lymphocytes (CTLs). We have demonstrated that some materials strongly inhibit cell proliferation of both the two cell lines to an extent higher that 70-75%, but only after a prolonged exposure of 3-6 days (Copper, Gold over Nickel, Aramid fiber filled epoxy uncured, b-stage epoxy die attach film, Tesa 4985 adhesive tape, Pyralux uncured, Copper + 1-octodecanethiol). However, when experiments were performed with short incubation time (1 h), only Aramid fiber filled epoxy uncured was cytotoxic. Variation of the results concerning the other materials was appreciable when the experiments performed on two cell lines were compared together. Furthermore, the effects of the materials on erythroid differentiation and CTL-mediated LCL lysis confirmed, in most of the cases, the data obtained in cytotoxic and antiproliferative tests. PMID:20625835

  15. EBV latent membrane proteins (LMPs) 1 and 2 as immunotherapeutic targets: LMP-specific CD4+ cytotoxic T cell recognition of EBV-transformed B cell lines.

    PubMed

    Haigh, Tracey A; Lin, Xiaorong; Jia, Hui; Hui, Edwin P; Chan, Anthony T C; Rickinson, Alan B; Taylor, Graham S

    2008-02-01

    The EBV-latent membrane proteins (LMPs) 1 and 2 are among only three viral proteins expressed in EBV-associated Hodgkin's lymphoma and nasopharyngeal carcinoma. Since these tumors are HLA class I and class II-positive, the LMPs could serve as both CD8+ and CD4+ T cell targets. In contrast to CD8 responses, very little is known about CD4 responses to LMPs. In this study, we describe CD4+ T cell clones defining four LMP1- and three LMP2-derived peptide epitopes and their restricting alleles. All clones produced Th1-like cytokines in response to peptide and most killed peptide-loaded target cells by perforin-mediated lysis. Although clones to different epitopes showed different functional avidities in peptide titration assays, avidity per se was a poor predictor of the ability to recognize naturally infected B lymphoblastoid cell lines (LCLs) expressing LMPs at physiologic levels. Some epitopes, particularly within LMP1, consistently mediated strong LCL recognition detectable in cytokine release, cytotoxicity, and outgrowth inhibition assays. Using cyclosporin A to selectively block cytokine release, we found that CD4+ T cell cytotoxicity is the key effector of LCL outgrowth control. We therefore infer that cytotoxic CD4+ T cells to a subset of LMP epitopes could have therapeutic potential against LMP-expressing tumors. PMID:18209060

  16. ERAP1 regulates natural killer cell function by controlling the engagement of inhibitory receptors.

    PubMed

    Cifaldi, Loredana; Romania, Paolo; Falco, Michela; Lorenzi, Silvia; Meazza, Raffaella; Petrini, Stefania; Andreani, Marco; Pende, Daniela; Locatelli, Franco; Fruci, Doriana

    2015-03-01

    The endoplasmic reticulum aminopeptidase ERAP1 regulates innate and adaptive immune responses by trimming peptides for presentation by MHC class I (MHC-I) molecules. Herein, we demonstrate that genetic or pharmacological inhibition of ERAP1 on human tumor cell lines perturbs their ability to engage several classes of inhibitory receptors by their specific ligands, including killer cell Ig-like receptors (KIR) by classical MHC-I-peptide (pMHC-I) complexes and the lectin-like receptor CD94-NKG2A by nonclassical pMHC-I complexes, in each case leading to natural killer (NK) cell killing. The protective effect of pMHC-I complexes could be restored in ERAP1-deficient settings by the addition of known high-affinity peptides, suggesting that ERAP1 was needed to positively modify the affinity of natural ligands. Notably, ERAP1 inhibition enhanced the ability of NK cells to kill freshly established human lymphoblastoid cell lines from autologous or allogeneic sources, thereby promoting NK cytotoxic activity against target cells that would not be expected because of KIR-KIR ligand matching. Overall, our results identify ERAP1 as a modifier to leverage immune functions that may improve the efficacy of NK cell-based approaches for cancer immunotherapy. PMID:25592150

  17. Enkephalins stimulate leukemia cell migration and surface expression of CD9.

    PubMed Central

    Heagy, W; Duca, K; Finberg, R W

    1995-01-01

    Opioid peptides have been implicated in the regulation of tumor growth and biology; however, little attention has been given to the mechanisms that are involved. In this study we show that physiological concentrations of the endogenous opioid neuropeptide methionine-enkephalin (MET-ENK) and the synthetic enkephalins D-Ala2, Me-Phe4, Gly(ol)5 and D-Ala2, D-Leu5 are stimulants for the in vitro migration of pre-B acute lymphoblastoid leukemia (ALL) cells. Activation of the human pre-B ALL cell lines NALM 6 and LAZ 221 with MET-ENK resulted in both an increase in their migration and an augmentation in the surface expression of the leukemia cell marker CD9. The opiate receptor antagonist naloxone reversed these enkephalin-induced effects on the leukemia cells. When the pre-B ALL cells were preincubated with an anti-CD9 mAb before challenge with MET-ENK their migration to the enkephalin was markedly reduced. These studies show that endogenous and synthetic opioid peptides are stimulants for pre-B ALL cell migration and suggest that CD9 is important in the regulation of leukemia cell motility. Images PMID:7657811

  18. Integrative "omic" analysis for tamoxifen sensitivity through cell based models.

    PubMed

    Weng, Liming; Ziliak, Dana; Lacroix, Bonnie; Geeleher, Paul; Huang, R Stephanie

    2014-01-01

    It has long been observed that tamoxifen sensitivity varies among breast cancer patients. Further, ethnic differences of tamoxifen therapy between Caucasian and African American have also been reported. Since most studies have been focused on Caucasian people, we sought to comprehensively evaluate genetic variants related to tamoxifen therapy in African-derived samples. An integrative "omic" approach developed by our group was used to investigate relationships among endoxifen (an active metabolite of tamoxifen) sensitivity, SNP genotype, mRNA and microRNA expressions in 58 HapMap YRI lymphoblastoid cell lines. We identified 50 SNPs that associate with cellular sensitivity to endoxifen through their effects on 34 genes and 30 microRNA expression. Some of these findings are shared in both Caucasian and African samples, while others are unique in the African samples. Among gene/microRNA that were identified in both ethnic groups, the expression of TRAF1 is also correlated with tamoxifen sensitivity in a collection of 44 breast cancer cell lines. Further, knock-down TRAF1 and over-expression of hsa-let-7i confirmed the roles of hsa-let-7i and TRAF1 in increasing tamoxifen sensitivity in the ZR-75-1 breast cancer cell line. Our integrative omic analysis facilitated the discovery of pharmacogenomic biomarkers that potentially affect tamoxifen sensitivity. PMID:24699530

  19. A modified host-cell reactivation assay to quantify DNA repair capacity in cryopreserved peripheral lymphocytes.

    PubMed

    Mendez, Pedro; Taron, Miquel; Moran, Teresa; Fernandez, Marco A; Requena, Gerard; Rosell, Rafael

    2011-06-10

    The host-cell reactivation assay (HCRA) is a functional assay that allows the identification of the genes responsible for DNA repair-deficient syndromes, such as Xeroderma pigmentosum, by cross-complementation experiments. It has also been used in molecular epidemiology studies to correlate the low nucleotide excision repair pathway function in peripheral blood lymphocytes with an increased risk of bladder, head and neck, skin and lung cancers. Herein, we present the technical validation of a newly modified HCRA, where nucleofection is used for the transfection of the pmaxGFP plasmid into cryopreserved peripheral blood lymphocytes (PBLs) or lymphoblastoid cell lines. In each sample, 20-24h after transfection, the relative DNA repair capacity (DRC) was quantified by flow cytometry, comparing the transfection efficiency of nucleoporated cells with undamaged plasmid to those transfected with UV-light damaged plasmid in the seven cell lines that were characterized by different DNA repair phenotypes. Dead cells were excluded from the analysis. We observed a high reproducibility of the relative DRC, transfection efficiency and cell viability. The inter-experimental normalization of the flow cytometry resulted in an increased data accuracy and reproducibility. The amount of cells required for each transfection reaction was reduced fourfold, without affecting the final relative DRC. Furthermore, our HCRA demonstrated strong discrimination power in the UV-light dose-response, both in lymphoblastoid cell lines and cryopreserved PBLs. We also observed a strong correlation of the relative DRC data, when samples were measured against two independent batches of both damaged and undamaged plasmid DNA. The relative DRC variable shows a normal distribution when analyzed in the cryopreserved PBLs from a cohort of 35 lung cancer patients and a 5.59-fold variation in the relative DRC is identified among our patients. The mitotic dynamic was discarded as a confounding factor for the

  20. DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

    SciTech Connect

    Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D. )

    1991-07-01

    Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links.

  1. The Sonoda–Tajima Cell Collection: A Human Genetics Research Resource with Emphasis on South American Indigenous Populations

    PubMed Central

    Danjoh, Inaho; Saijo, Kaoru; Hiroyama, Takashi

    2011-01-01

    The Sonoda–Tajima Cell Collection includes cell samples obtained from a range of ethnic minority groups across the world but in particular from South America. The collection is made all the more valuable by the fact that some of these ethnic populations have since died out, and thus it will be impossible to prepare a similar cell collection again. The collection was donated to our institute, a public cell bank in Japan, by Drs Sonoda and Tajima to make it available to researchers throughout the world. The original cell collection was composed of cryopreserved peripheral blood samples that would obviously have been rapidly exhausted if used directly. We, therefore, immortalized some samples with the Epstein–Barr virus and established B-lymphoblastoid cell lines (B-LCLs). As there is continuing controversy over whether the B-LCL genome is stably maintained, we performed an array comparative genomic hybridization (CGH) analysis to confirm the genomic stability of the cell lines. The array CGH analysis of the B-LCL lines and their parental B cells demonstrated that genomic stability was maintained in the long-term cell cultures. The B-LCLs of the Sonoda–Tajima Collection will therefore be made available to interested scientists around the world. At present, 512 B-LCLs have been developed, and we are willing to increase the number if there is sufficient demand. PMID:21383383

  2. Genetic Regulation of Charged Particle Mutagenesis in Human Cells

    NASA Technical Reports Server (NTRS)

    Kronenberg, Amy; Gauny, S.; Cherbonnel-Lasserre, C.; Liu, W.; Wiese, C.

    1999-01-01

    Our studies use a series of syngeneic, and where possible, isogenic human B-lymphoblastoid cell lines to assess the genetic factors that modulate susceptibility apoptosis and their impact on the mutagenic risks of low fluence exposures to 1 GeV Fe ions and 55 MeV protons. These ions are representative of the types of charged particle radiation that are of particular significance for human health in the space radiation environment. The model system employs cell lines derived from the male donor WIL-2. These cells have a single X chromosome and they are hemizygous for one mutation marker, hypoxanthine phosphoribosyltransferase (HPRT). TK6 and WTK1 cells were each derived from descendants of WIL-2 and were each selected as heterozygotes for a second mutation marker, the thymidine kinase (TK) gene located on chromosome 17q. The HPRT and TK loci can detect many different types of mutations, from single basepair substitutions up to large scale loss of heterozygosity (LOH). The single expressing copy of TK in the TK6 and WTKI cell lines is found on the same copy of chromosome 17, and this allele can be identified by a restriction fragment length polymorphism (RFLP) identified when high molecular weight DNA is digested by the SacI restriction endonuclease and hybridized against the cDNA probe for TK. A large series of polymorphic linked markers has been identified that span more than 60 cM of DNA (approx. 60 megabasepairs) and distinguish the copy of chromosome 17 bearing the initially active TK allele from the copy of chromosome 17 bearing the silent TK allele in both TK6 and WTKI cells. TK6 cells express normal p53 protein while WTKI cells express homozygous mutant p53. Expression of mutant p53 can increase susceptibility to x-ray-induced mutations. It's been suggested that the increased mutagenesis in p53 mutant cells might be due to reduced apoptosis.

  3. Induction of heat shock proteins in B-cell exosomes.

    PubMed

    Clayton, Aled; Turkes, Attilla; Navabi, Hossein; Mason, Malcolm D; Tabi, Zsuzsanna

    2005-08-15

    Exosomes are nanometer-sized vesicles secreted by a diverse range of live cells that probably have physiological roles in modulating cellular immunity. The extracellular factors that regulate the quantity and phenotype of exosomes produced are poorly understood, and the properties of exosomes that dictate their immune functions are not yet clear. We investigated the effect of cellular stress on the exosomes produced by B-lymphoblastoid cell lines. Under steady-state conditions, the exosomes were positive for hsp27, hsc70, hsp70 and hsp90, and other recognised exosome markers such as MHC class I, CD81, and LAMP-2. Exposing cells to heat stress (42 degrees C for up to 3 hours), resulted in a marked increase in these heat shock proteins (hsps), while the expression of other stress proteins such as hsp60 and gp96 remained negative, and other exosome markers remained unchanged. Stress also triggered a small increase in the quantity of exosomes produced [with a ratio of 1.245+/-0.07 to 1 (mean+/-s.e.m., n=20) of 3-hour-stress-exosomes to control-exosomes]. Flow-cytometric analysis of exosome-coated beads and immuno-precipitation of intact exosomes demonstrated that hsps were located within the exosome lumen, and not present at the exosome-surface, suggesting that such exosomes may not interact with target cells through cell-surface hsp-receptors. Functional studies further supported this finding, in that exosomes from control or heat-stressed B cells did not trigger dendritic cell maturation, assessed by analysis of dendritic-cell-surface phenotype, and cytokine secretion profile. Our findings demonstrate that specific alterations in exosome phenotype are a hitherto unknown component of the cellular response to environmental stress and their extracellular function does not involve the direct activation of dendritic cells. PMID:16046478

  4. Role of EBNA-3 Family Proteins in EBV Associated B-cell Lymphomagenesis

    PubMed Central

    Bhattacharjee, Shaoni; Ghosh Roy, Shatadru; Bose, Priyanka; Saha, Abhik

    2016-01-01

    Epstein-Barr virus (EBV) is highly ubiquitous in human population and establishes a lifelong asymptomatic infection within the infected host unless the immune system is compromised. Following initial infection in the oropharyngeal epithelial cells, EBV primarily infects naive B-lymphocytes and develops a number of B-cell lymphomas particularly in immune-deficient individuals. In vitro, EBV can also infect and subsequently transform quiescent B-lymphocytes into continuously proliferating lymphoblastoid cell lines (LCLs) resembling EBV-induced lymphoproliferative disorders in which a subset of latent transcripts are detected. Genetic studies revealed that EBNA-3 family comprising of three adjacent genes in the viral genome—EBNA-3A and -3C, but not -3B, are critical for B-cell transformation. Nevertheless, all three proteins appear to significantly contribute to maintain the overall proliferation and viability of transformed cells, suggesting a critical role in lymphoma development. Apart from functioning as important viral transcriptional regulators, EBNA-3 proteins associate with many cellular proteins in different signaling networks, providing a suitable platform for lifelong survival of the virus and concurrent lymphoma development in the infected host. The chapter describes the function of each these EBV nuclear antigen 3 proteins employed by the virus as a means to understand viral pathogenesis of several EBV-associated B-cell malignancies. PMID:27092119

  5. Induction of heme oxygenase: A general response to oxidant stress in cultured mammalian cells

    SciTech Connect

    Applegate, L.A.; Luscher, P.; Tyrrell, R.M. )

    1991-02-01

    Accumulation of heme oxygenase mRNA is strongly stimulated by treatment of cultured human skin fibroblasts with ultraviolet radiation, hydrogen peroxide, or the sulfhydryl reagent sodium arsenite. Since this will result in a transient reduction in the prooxidant state of cells, the phenomenon may represent an important inducible antioxidant defense mechanism. To examine the generality of the response, we have measured the accumulation of the specific mRNA in a variety of human and mammalian cell types after inducing treatments. Induction by sodium arsenite is observed in all additional human cell types tested. This includes primary epidermal keratinocytes and lung and colon fibroblasts as well as established cell lines such as HeLa, TK6 lymphoblastoid, and transformed fetal keratinocytes. Strong induction of heme oxygenase mRNA is also observed following sodium arsenite treatment of cell lines of rat, hamster, mouse, monkey, and marsupial origin. The agents which lead to induction in cultured human skin fibroblasts fall into two categories: (a) those which are oxidants or can generate active intermediates (ultraviolet A radiation, hydrogen peroxide, menadione, and the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate); (b) agents which are known to interact with or modify cellular glutathione levels (buthionine sulfoximine, sodium arsenite, iodoacetamide, diamide, and cadmium chloride). These observations strongly support the hypothesis that induction of the enzyme is a general response to oxidant stress in mammalian cells and are consistent with the possibility that the cellular redox state plays a key role.

  6. Nitroxide TEMPO: a genotoxic and oxidative stress inducer in cultured cells.

    PubMed

    Guo, Xiaoqing; Mittelstaedt, Roberta A; Guo, Lei; Shaddock, Joseph G; Heflich, Robert H; Bigger, Anita H; Moore, Martha M; Mei, Nan

    2013-08-01

    2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) is a low molecular weight nitroxide and stable free radical. In this study, we investigated the cytotoxicity and genotoxicity of TEMPO in mammalian cells using the mouse lymphoma assay (MLA) and in vitro micronucleus assay. In the absence of metabolic activation (S9), 3mM TEMPO produced significant cytotoxicity and marginal mutagenicity in the MLA; in the presence of S9, treatment of mouse lymphoma cells with 1-2mM TEMPO resulted in dose-dependent decreases of the relative total growth and increases in mutant frequency. Treatment of TK6 human lymphoblastoid cells with 0.9-2.3mM TEMPO increased the frequency of both micronuclei (a marker for clastogenicity) and hypodiploid nuclei (a marker of aneugenicity) in a dose-dependent manner; greater responses were produced in the presence of S9. Within the dose range tested, TEMPO induced reactive oxygen species and decreased glutathione levels in mouse lymphoma cells. In addition, the majority of TEMPO-induced mutants had loss of heterozygosity at the Tk locus, with allele loss of ⩽34Mbp. These results indicate that TEMPO is mutagenic in the MLA and induces micronuclei and hypodiploid nuclei in TK6 cells. Oxidative stress may account for part of the genotoxicity induced by TEMPO in both cell lines. PMID:23517621

  7. Zinc deficiency enhanced inflammatory response by increasing immune cell activation and inducing IL6 promoter demethylation

    PubMed Central

    Wong, Carmen P.; Rinaldi, Nicole A.; Ho, Emily

    2015-01-01

    Scope Zinc deficiency results in immune dysfunction and promotes systemic inflammation. The objective of this study was to examine the effects of zinc deficiency on cellular immune activation and epigenetic mechanisms that promote inflammation. This work is potentially relevant to the aging population given that age-related immune defects, including chronic inflammation, coincide with declining zinc status. Methods and results An in vitro cell culture system and the aged mouse model were used to characterize immune activation and DNA methylation profiles that may contribute to the enhanced proinflammatory response mediated by zinc deficiency. Zinc deficiency up-regulated cell activation markers ICAM1, MHC class II, and CD86 in THP1 cells, that coincided with increased IL1β and IL6 responses following LPS stimulation. A decreased zinc status in aged mice was similarly associated with increased ICAM1 and IL6 gene expression. Reduced IL6 promoter methylation was observed in zinc deficient THP1 cells, as well as in aged mice and human lymphoblastoid cell lines derived from aged individuals. Conclusion Zinc deficiency induced inflammatory response in part by eliciting aberrant immune cell activation and altered promoter methylation. Our results suggested potential interactions between zinc status, epigenetics, and immune function, and how their dysregulation could contribute to chronic inflammation. PMID:25656040

  8. The nature of the natural killer (NK) cell of human intestinal mucosa and mesenteric lymph node.

    PubMed Central

    Gibson, P R; Jewell, D P

    1985-01-01

    The relationship of the mononuclear cell (MNC) from human intestinal mucosa and mesenteric lymph node mediating anti-K-562 activity with that of peripheral blood has been assessed. Depletion of macrophages did not alter the measured cytotoxicity confirming that the effector cells were lymphocytes. Complement lysis of Leu 7 and Leu 11b coated cells reduced intestinal natural killer (NK) activity by a similar degree to that of peripheral blood but mesenteric lymph node NK activity was affected to a lesser extent. The response in NK activity of mucosal and nodal MNC to short incubation with lymphoblastoid interferon was similar to that for peripheral blood MNC. Twenty-four hours incubation of MNC with low concentrations of purified interleukin-2 (IL-2) consistently augmented intestinal and nodal NK activity but failed to augment that of peripheral blood MNC. No differences between the inhibitory effects of cAMP and prostaglandin E2 on NK activity from the three sites were seen. In addition, inhibition of cyclo-oxygenase activity with indomethacin had no effect on NK activity of intestinal and peripheral blood MNC while the lipoxygenase inhibitor, nordihydroguaiaretic acid, suppressed intestinal and peripheral blood NK activity similarly. In conclusion, anti-K-562 activity by intestinal MNC is mediated by NK cells with similar phenotypic and functional properties to those of peripheral blood. However, the increased sensitivity of mucosal NK cells to IL-2 suggests that higher proportions of NK cell precursors may be present in intestinal MNC populations. PMID:2412737

  9. Targeted activation of human Vγ9Vδ2-T cells controls epstein-barr virus-induced B cell lymphoproliferative disease.

    PubMed

    Xiang, Zheng; Liu, Yinping; Zheng, Jian; Liu, Ming; Lv, Aizhen; Gao, Yulong; Hu, Huaidong; Lam, Kowk-Tai; Chan, Godfrey Chi-Fung; Yang, Yuanzhong; Chen, Honglin; Tsao, George Sai-Wah; Bonneville, Marc; Lau, Yu-Lung; Tu, Wenwei

    2014-10-13

    Epstein-Barr virus-induced lymphoproliferative disease (EBV-LPD) after transplantation remains a serious and life-threatening complication. Herein we showed that the aminobisphosphonate pamidronate-expanded human Vγ9Vδ2-T cells efficiently killed EBV-transformed autologous lymphoblastoid B cell lines (EBV-LCL) through γ/δ-TCR and NKG2D receptor triggering and Fas and TRAIL engagement. By inoculation of EBV-LCL in Rag2(-/-)γc(-/-) mice and humanized mice, we established lethal EBV-LPD with characteristics close to those of the human disease. Adoptive transfer of pamidronate-expanded Vγ9Vδ2-T cells alone effectively prevented EBV-LPD in Rag2(-/-)γc(-/-) mice and induced EBV-LPD regression in EBV(+) tumor-bearing Rag2(-/-)γc(-/-) mice. Pamidronate treatment inhibited EBV-LPD development in humanized mice through selective activation and expansion of Vγ9Vδ2-T cells. This study provides proof-of-principle for a therapeutic approach using pamidronate to control EBV-LPD through Vγ9Vδ2-T cell targeting. PMID:25220446

  10. The effect of puerarin against IL-1β-mediated leukostasis and apoptosis in retinal capillary endothelial cells (TR-iBRB2)

    PubMed Central

    Zhu, Xue; Wang, Ke; Zhang, Kai; Gao, Yun; Zhu, Ling; Zhou, Fanfan

    2014-01-01

    Purpose Blood–retinal barrier (BRB) breakdown, the early hallmark of diabetic retinopathy (DR), is thought to depend on retinal inflammation and cell damage. The proinflammatory factor interleukin-1β (IL-1β) was demonstrated to cause inflammation as well as cell apoptosis during the process of BRB breakdown. This study extensively evaluated the protective effect of puerarin, a major active component extracted from the traditional herb Radix puerariae, against IL-1β-induced cell dysfunction in TR-iBRB2 cells, a retinal capillary endothelial cell line. Methods TR-iBRB2 cells were pretreated with IL-1β (10 ng/ml) for 24 h and then exposed to puerarin (0, 10, 25, and 50 μM) for another 24 h. Leukocyte endothelial adhesion was assessed through a cell-based assay using lymphoblastoid cells. Cell apoptosis was evaluated with flow cytometry, and the expression of adhesion molecules and apoptosis-related molecules was assessed with western blot analysis. Results Our data showed that puerarin attenuated IL-1β-mediated leukostasis and cell apoptosis in TR-iBRB2 cells. Furthermore, puerarin strikingly prevented IL-1β-induced molecular events of the upstream and downstream signaling pathways involved in this cellular process. Conclusions These findings may significantly contribute to better understanding of the protective effect of puerarin, in particular for DR, as well as provide novel insights into the potential application of this compound in DR therapy. PMID:25593509

  11. Human Leukocyte Antigen (HLA) A*1101-restricted Epstein-Barr Virus-specific T-cell Receptor Gene Transfer to Target Nasopharyngeal Carcinoma

    PubMed Central

    Zheng, Yong; Parsonage, Greg; Zhuang, Xiaodong; Machado, Lee R; James, Christine H.; Salman, Asmaa; Searle, Peter F.; Hui, Edwin P.; Chan, Anthony T.C.; Lee, Steven P.

    2015-01-01

    Infusing virus-specific T cells is effective treatment for rare Epstein-Barr virus (EBV)-associated post-transplant lymphomas and more limited success has been reported using this approach to treat a far more common EBV-associated malignancy, nasopharyngeal carcinoma (NPC). However, current approaches using EBV-transformed lymphoblastoid cell lines to reactivate EBV-specific T cells for infusion take 2 to 3 months of in vitro culture and favour outgrowth of T cells targeting viral antigens expressed within EBV+ lymphomas but not in NPC. Here we explore T-cell receptor (TCR) gene transfer to rapidly and reliably generate T cells specific for the NPC-associated viral protein LMP2. We cloned a HLA A*1101-restricted TCR, which would be widely applicable since 40% of NPC patients carry this HLA allele. Studying both the wild-type and modified forms we have optimised expression of the TCR and demonstrated high avidity antigen-specific function (proliferation, cytotoxicity, cytokine release) in both CD8+ and CD4+ T cells. The engineered T cells also inhibited LMP2+ epithelial tumour growth in a mouse model. Furthermore, transduced T cells from patients with advanced NPC lysed LMP2-expressing NPC cell lines. Using this approach, within a few days large numbers of high avidity LMP2-specific T cells can be generated reliably to treat NPC, thus providing an ideal clinical setting to test TCR gene transfer without the risk of autoimmunity through targeting self-antigens. PMID:25711537

  12. Loss of APC protein expressed by human colonic epithelial cells and the appearance of a specific low-molecular-weight form is associated with apoptosis in vitro.

    PubMed

    Browne, S J; Williams, A C; Hague, A; Butt, A J; Paraskeva, C

    1994-10-01

    APC (adenomatous polyposis coli) protein is differentially expressed in the normal colonic crypt and believed to be involved in colonic cell maturation. In this work we investigated whether expression of the APC protein is associated with cell death in colonic epithelial cells. We have previously reported an in vitro system to study apoptosis. Briefly, cells attached to the flask have a low frequency of apoptosis (1-3%), whereas cells that detach from the flask and float in the medium have a high proportion of apoptotic cells (36-96% depending on the cell line). The full-length 300-kDa or truncated APC protein, normally expressed by the attached cells (detected using the FE9 antibody), was found to be lost in the floating apoptotic cells in 8/11 colon tumour cell lines examined. In addition, the APC antibody FE9 detected a 90-kDa protein in the floating apoptotic cells of all cell lines investigated, which was not present in attached cells. Furthermore, loss of full-length APC and gain of the 90-kDa protein was observed in the apoptotic cells of 2 cell lines derived from other tissues: the SV40-transformed fibroblast cell line CMSV40fib and the lymphoblastoid B-cell line BJA-B. In cells repeatedly frozen and thawed, believed to induce necrotic cell death, full-length or truncated APC was also lost, though a 95-kDa protein distinct from that in apoptotic cells was observed. Specific loss of full-length or truncated APC (resulting in a 90-kDa protein in apoptotic cells but a 95-kDa protein in necrotic cells) is therefore associated with cell death. Our findings suggest a possible role for APC in cell survival. PMID:7927905

  13. Abnormal T cell subpopulations and circulating immune complexes in the Guillain-Barré syndrome and multiple sclerosis.

    PubMed

    Goust, J M; Chenais, F; Carnes, J E; Hames, C G; Fudenberg, H H; Hogan, E L

    1978-05-01

    Immunologic studies were performed in 21 patients with multiple sclerosis (MS) and 16 with the Guillain-Barré syndrome (GBS). Levels of thymus-derived (T) cells measured by "total" and "active" rosette formation between sheep erythrocytes and peripheral blood mononuclear cells (TEt, TEa) were within normal limits in all the patients, with the exception of four GBS patients, including one who also had received chemotherapy for lymphoma and three who were receiving steroids. When lymphocytes from the 21 patients were incubated with the bone-marrow-derived (B) lymphoblastoid cell line PGLC-33H, there were, for 12 of 18 MS patients and 11 of 16 GBS patients, significant decreases in a subpopulation of peripheral blood T lymphocytes that form "PGLC rosettes" (PGR) with the PGLC-33H cells. (Peripheral blood T cells from normal individuals formed PGR with 23.9 +/- 3.8 percent of PGLC-33H cells.) Using the 125l-C1q binding assay, immune complexes were detected in the serum of 14 of 19 MS patients and 15 of 16 GBS patients. An association between increased C1q binding and decreased PGR values was found in 10 of 18 MS patients and 12 of 17 GBS patients. The results suggest that in both diseases the etiology may involve a decrease in the subset of T cells that bind to the IgM-producing cell line PGLC-33H, in association with the appearance of circulating immune complexes containing the infectious viral agent. PMID:306075

  14. Quantitative proteomic analysis of HIV-1 infected CD4+ T cells reveals an early host response in important biological pathways: Protein synthesis, cell proliferation, and T-cell activation

    SciTech Connect

    Navare, Arti T.; Sova, Pavel; Purdy, David E.; Weiss, Jeffrey M.; Wolf-Yadlin, Alejandro; Korth, Marcus J.; Chang, Stewart T.; Proll, Sean C.; Jahan, Tahmina A.; Krasnoselsky, Alexei L.; Palermo, Robert E.; Katze, Michael G.

    2012-07-20

    Human immunodeficiency virus (HIV-1) depends upon host-encoded proteins to facilitate its replication while at the same time inhibiting critical components of innate and/or intrinsic immune response pathways. To characterize the host cell response on protein levels in CD4+ lymphoblastoid SUP-T1 cells after infection with HIV-1 strain LAI, we used mass spectrometry (MS)-based global quantitation with iTRAQ (isobaric tag for relative and absolute quantification). We found 266, 60 and 22 proteins differentially expressed (DE) (P-value{<=}0.05) at 4, 8, and 20 hours post-infection (hpi), respectively, compared to time-matched mock-infected samples. The majority of changes in protein abundance occurred at an early stage of infection well before the de novo production of viral proteins. Functional analyses of these DE proteins showed enrichment in several biological pathways including protein synthesis, cell proliferation, and T-cell activation. Importantly, these early changes before the time of robust viral production have not been described before.

  15. An Efficient Method for Electroporation of Small Interfering RNAs into ENCODE Project Tier 1 GM12878 and K562 Cell Lines

    PubMed Central

    Muller, Ryan Y.; Hammond, Ming C.

    2015-01-01

    The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion. PMID:26543439

  16. An Efficient Method for Electroporation of Small Interfering RNAs into ENCODE Project Tier 1 GM12878 and K562 Cell Lines.

    PubMed

    Muller, Ryan Y; Hammond, Ming C; Rio, Donald C; Lee, Yeon J

    2015-12-01

    The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion. PMID:26543439

  17. Effects of toxic chemicals on the release of pyrimidine compounds in cell culture.

    PubMed

    Uziel, M; Butler, A; Owen, B

    1987-07-01

    Exposure of hamster embryo cells and BF lymphoblastoid cells to 18 known toxic substances and four nominally nontoxic substances results in the release of pyrimidines (and their nucleosides) into the culture medium. The extent of release is dependent on the specific chemical and the specific cells present in the assay. BF cells are not affected by exposure to benzo(a)pyrene, while the hamster embryo cells exhibit enhanced excretion on exposure to benzo(a)pyrene. This difference in response may be due to the difference in endogenous aryl hydrocarbon hydroxylase (BaP) activity. In contrast, diethylstilbestrol, which is metabolized by a peroxidase-mediated enzyme system, causes enhanced excretion in both cell types. Direct alkylating agents and Ni(+2) salts also cause enhanced excretion in both cell types. We have used concentrations of chemicals that give a 5% enhanced excretion as the criterion of low-dose response. Within the range of concentrations tested, chromate induces enhanced excretion in BF cells but not the HEC cells, and Pb(+2) induces enhanced excretion in HEC cells but not the BF cells. Benzene, dimethylnitrosamine, and Mg(+2) did not affect either cell type. 7,12-Dimethylbenzo(a)anthracene, anthracene, benzo(a)anthracene, phenylazoaniline, N-methyl, N-nitroso, N'-nitroguanidine, dioxane, and pyrene cause enhanced excretion in the hamster embryo cells while benzo(e)pyrene, ZnSO4 and cholesterol do not cause enhanced excretion in the hamster embryo cells. Of those chemicals causing enhanced excretion, the concentration range bracketing 5% enhanced excretion approximated low-dose exposures reported to result in toxic responses like cancer, teratogenesis or pulmonary disease. PMID:3662812

  18. Static magnetic field of 6 mT induces apoptosis and alters cell cycle in p53 mutant Jurkat cells.

    PubMed

    Ahmadianpour, Mohammad Reza; Abdolmaleki, Parviz; Mowla, Seyed Javad; Hosseinkhani, Saman

    2013-03-01

    This study aimed to investigate the effect of 6 milliTesla (mT) static magnetic field (SMF) on apoptosis induction and cell cycle alteration in T-lymphoblastoid Jurkat E6.1 cells. Exposure of human p53 mutant Jurkat cells to 6 mT SMF resulted in apoptosis, which was detected by luminometric and flow cytometric analysis also, phosphorylated ATM and E2F1 proteins were detected by western blot analysis. Based on luminescence detection data, apoptosis initiated 36 h after exposure to 6 mT SMF. Apoptosis also reached its maximum rate 48 h after treatment. Flow cytometric analysis revealed a temporary G2 arrest after exposure to 6 mT SMF. Indeed, cellular population of S and G2 phases was increased. Based on reports of other investigations on the effect of magnetic fields on Ca2+flux changes in cell membranes and the effect of MFs on free radical formation, it can be suggested that the magnetic fields may induce the apoptosis and alter the cell population in different cell cycle phases of Jurkat cells via changing the Ca2+fluxes through cell membranes and playing a role in free radical formation. Western blot analysis showed that the amount of phosphorylated ATM and E2F1 proteins were increased in treated cells. The results of luminometric and flow cytometric detection did not show a significant difference in the apoptosis rate between 6 h-treated and 24 h-treated cells by 6 mT SMF. Thus, 6 mT SMF can induce apoptosis and alter cell cycle in Jurkat cells via a p53-independent pathway. PMID:23320430

  19. Tetravalent anti-CD20/CD3 bispecific antibody for the treatment of B cell lymphoma.

    PubMed

    Lu, Chia-Yen; Chen, Gregory J; Tai, Pei-Han; Yang, Yu-Chen; Hsu, Yu-Shen; Chang, Mingi; Hsu, Chuan-Lung

    2016-05-13

    Bispecific antibodies (bsAbs) are second generation antibodies for therapeutic application in immunotherapy. One of the major strategies of the bsAb platform is the recruitment of immune effector T cells by incorporating an anti-CD3 domain. A bispecific T-cell engager (BiTE), with one end having an affinity for CD3 and the other end with affinity for CD19, has been approved in the US and Europe for the treatment of acute lymphoblastic leukemia. However, due to their small size and lack of Fc region, these single-chain variable fragment (scFv) bsAbs have short half-lives in vivo. Additionally, poor solubility, structural instability, and low production yields have also become major challenges in the bulk production process. To overcome these challenges, we have engineered a tetravalent bsAb with bivalent binding specificity for the CD20 and CD3 antigen in an immunoglobulin G (IgG) format. The fusion of the anti-CD3 scFvs to the CD20 antibody via a linker-hinge domain (LHD) results in improved antibody stabilization and properties. Here we demonstrate this antibody's highly efficient cancer cell elimination in a dose-dependent manner in a CD20-expressing B lymphoblastoid cell line in vitro. Our data suggest the potential clinical application of this bsAb for the treatment of CD20-expressing B cell malignancies. PMID:27040766

  20. Constituents of French Marigold (Tagetes patula L.) Flowers Protect Jurkat T-Cells against Oxidative Stress.

    PubMed

    Chkhikvishvili, Irakli; Sanikidze, Tamar; Gogia, Nunu; Enukidze, Maia; Machavariani, Marine; Kipiani, Nana; Vinokur, Yakov; Rodov, Victor

    2016-01-01

    The flowers of French marigold (Tagetes patula L.) are widely used in folk medicine, in particular for treating inflammation-related disorders. However, cellular mechanisms of this activity demand further investigation. In the present work, we studied the potential of T. patula compounds to alleviate the oxidative stress in hydrogen peroxide-challenged human lymphoblastoid Jurkat T-cells. Crude extracts of marigold flowers and purified fractions containing flavonoids patuletin, quercetagetin, and quercetin and their derivatives, as well as the carotenoid lutein, were brought in contact with Jurkat cells challenged with 25 or 50 μM H2O2. Hydrogen peroxide caused oxidative stress in the cells, manifested as generation of superoxide and peroxyl radicals, reduced viability, arrested cell cycle, and enhanced apoptosis. The stress was alleviated by marigold ingredients that demonstrated high radical-scavenging capacity and enhanced the activity of antioxidant enzymes involved in neutralization of reactive oxygen species. Flavonoid fraction rich in quercetin and quercetagetin showed the highest cytoprotective activity, while patuletin in high dose exerted a cytotoxic effect associated with its anticancer potential. T. patula compounds enhanced the production of anti-inflammatory and antioxidant interleukin-10 (IL-10) in Jurkat cells. Both direct radical-scavenging capacity and stimulation of protective cellular mechanisms can underlay the anti-inflammatory properties of marigold flowers. PMID:27433287

  1. A nonpromoting phorbol from the samoan medicinal plant Homalanthus nutans inhibits cell killing by HIV-1.

    PubMed

    Gustafson, K R; Cardellina, J H; McMahon, J B; Gulakowski, R J; Ishitoya, J; Szallasi, Z; Lewin, N E; Blumberg, P M; Weislow, O S; Beutler, J A

    1992-05-29

    Extracts of Homalanthus nutans, a plant used in Samoan herbal medicine, exhibited potent activity in an in vitro, tetrazolium-based assay which detects the inhibition of the cytopathic effects of human immunodeficiency virus (HIV-1). The active constituent was identified as prostratin, a relatively polar 12-deoxyphorbol ester. Noncytotoxic concentrations of prostratin from greater than or equal to 0.1 to greater than 25 microM protected T-lymphoblastoid CEM-SS and C-8166 cells from the killing effects of HIV-1. Cytoprotective concentrations of prostratin greater than or equal to 1 microM essentially stopped virus reproduction in these cell lines, as well as in the human monocytic cell line U937 and in freshly isolated human monocyte/macrophage cultures. Prostratin bound to and activated protein kinase C in vitro in CEM-SS cells and elicited other biochemical effects typical of phorbol esters in C3H10T1/2 cells; however, the compound does not appear to be a tumor promoter. In skin of CD-1 mice, high doses of prostratin induced ornithine decarboxylase only to 25-30% of the levels induced by typical phorbol esters at doses 1/30 or less than that used for prostratin, produced kinetics of edema formation characteristic of the nonpromoting 12-deoxyphorbol 13-phenylacetate, and failed to induce the acute or chronic hyperplasias typically caused by tumor-promoting phorbols at doses of 1/100 or less than that used for prostratin. PMID:1597853

  2. Constituents of French Marigold (Tagetes patula L.) Flowers Protect Jurkat T-Cells against Oxidative Stress

    PubMed Central

    Chkhikvishvili, Irakli; Sanikidze, Tamar; Gogia, Nunu; Enukidze, Maia; Machavariani, Marine; Kipiani, Nana; Vinokur, Yakov; Rodov, Victor

    2016-01-01

    The flowers of French marigold (Tagetes patula L.) are widely used in folk medicine, in particular for treating inflammation-related disorders. However, cellular mechanisms of this activity demand further investigation. In the present work, we studied the potential of T. patula compounds to alleviate the oxidative stress in hydrogen peroxide-challenged human lymphoblastoid Jurkat T-cells. Crude extracts of marigold flowers and purified fractions containing flavonoids patuletin, quercetagetin, and quercetin and their derivatives, as well as the carotenoid lutein, were brought in contact with Jurkat cells challenged with 25 or 50 μM H2O2. Hydrogen peroxide caused oxidative stress in the cells, manifested as generation of superoxide and peroxyl radicals, reduced viability, arrested cell cycle, and enhanced apoptosis. The stress was alleviated by marigold ingredients that demonstrated high radical-scavenging capacity and enhanced the activity of antioxidant enzymes involved in neutralization of reactive oxygen species. Flavonoid fraction rich in quercetin and quercetagetin showed the highest cytoprotective activity, while patuletin in high dose exerted a cytotoxic effect associated with its anticancer potential. T. patula compounds enhanced the production of anti-inflammatory and antioxidant interleukin-10 (IL-10) in Jurkat cells. Both direct radical-scavenging capacity and stimulation of protective cellular mechanisms can underlay the anti-inflammatory properties of marigold flowers. PMID:27433287

  3. Cytological and molecular studies of chromosomal radiosensitivity in Down Syndrome cells

    SciTech Connect

    MacLaren, R.A.

    1988-01-01

    Molecular, cellular and cytogenetic studies were conducted to determine if altered levels of poly(ADP-ribose) polymerase, a DNA repair-related enzyme, is responsible for the reported formation of excess X-ray induced chromosome aberrations in cells derived from Down Syndrome (DS) patients. Nonstimulated lymphocytes from normal and DS subjects were pretreated with 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, for 30 minutes before exposure to X-rays and the levels of induced chromosome aberrations were determined in mitotic cells. DS lymphocytes exhibited significantly higher frequencies of chromosome aberrations in the presence of 3-aminobenzamide that normal lymphocytes. No difference was observed in the absence of 3-aminobenzamide. Additional studies were done using normal and DS lymphoblastoid cell lines which exhibited a similar response at the DNA level as the lymphocytes. Analysis of poly(ADP-ribose) polymerase activity based on incorporation of the substrate, NAD{sup +}, into acid insoluble materials, revealed that there was no significant difference in the ability to form poly (ADP-ribose) in the DS or normal cells. 3-aminobenzamide effectively inhibited poly(ADP-ribose) polymerase in both the normal and DS cells.

  4. Molecular cloning of cDNA encoding a novel platelet-endothelial cell tetra-span antigen, PETA-3.

    PubMed

    Fitter, S; Tetaz, T J; Berndt, M C; Ashman, L K

    1995-08-15

    Platelet-endothelial cell tetra-span antigen (PETA-3) was originally identified as a novel human platelet surface glycoprotein, gp27, which was detected by a monoclonal antibody (MoAb), 14A2.H1. Although this glycoprotein is present in low abundance on the platelet surface, MoAb 14A2.H1 stimulates platelet aggregation and mediator release. We now report isolation of a cDNA clone encoding PETA-3 from a library derived from the megakaryoblastic leukemia cell line MO7e. The clone encodes an open reading frame of 253 amino acids that displays 25% to 30% amino acid sequence identity with several members of the newly defined Tetraspan, or Transmembrane 4 superfamily. These proteins consist of four conserved putative transmembrane domains with a large divergent extracellular loop between the third and fourth membrane-spanning regions. PETA-3 has a single consensus sequence for N-linked glycosylation located in this extracellular loop. A single PETA-3 RNA transcript (1.6 kb) was detected in RNA isolated from MO7e cells, bone marrow stromal cells, the C11 endothelial cell line, and several myeloid leukemia cell lines. No transcript was detected in the lymphoblastoid cell lines MOLT-4 and BALM-1. This pattern correlates well with previous protein expression data. Northern blot analysis of RNA from a range of human tissues indicated that the transcript was present in most tissues, the notable exception being brain. PMID:7632941

  5. Natural killer cell stimulatory factor (NKSF) augments natural killer cell and antibody-dependent tumoricidal response against colon carcinoma cell lines.

    PubMed

    Lieberman, M D; Sigal, R K; Williams, N N; Daly, J M

    1991-04-01

    The therapy of colorectal cancer may be improved by biologic response modifiers that enhance natural killer (NK) cell and antibody-dependent tumoricidal mechanisms. This study examined the effect of a recently discovered cytokine purified from the supernatant of an Ebstein-Barr virus-transformed B-lymphoblastoid cell line (RPMI-8866), natural killer cell stimulatory factor (NKSF), on NK and antibody-dependent cellular cytotoxicity (ADCC) of human colon adenocarcinoma cell lines. Human peripheral blood lymphocytes were cultured for 24 hr in the presence or absence of NKSF (3.6 pM) or interleukin-2 (1 nM). The cultured lymphocytes were analyzed for lytic potential toward chromium-51-labeled colon carcinoma targets SW 1116, 498 LI, and WC 1. ADCC was measured by incubating chromium-51-labeled SW 1116 or WC 1 targets with the monoclonal antibody CO17-1A, an IgG2a antibody reactive with gastrointestinal cancer-associated cell antigen, or control mouse IgG prior to testing NKSF-treated or control PBL effectors in a 6-hr cytotoxicity assay. NKSF significantly enhanced NK cytolysis of colon carcinoma and NK-resistant lymphoma cell lines, and on a molar basis was approximately 300 times more potent than interleukin-2 in generating NK cytotoxicity. Furthermore, NKSF significantly augmented lymphocyte-mediated ADCC against colon carcinoma targets, and the combination of NKSF with the antibody CO17-1A had an additive effect on lymphocyte tumoricidial capacity. Thus, NKSF may have a potential role in the treatment of colon cancer. PMID:1673486

  6. Development of an IFNγ ELISPOT for the analysis of the human T cell response against mumps virus.

    PubMed

    Han, Wanda G H; Emmelot, Maarten E; Jaadar, Haziz; Ten Hulscher, Hinke I; van Els, Cécile A C M; Kaaijk, Patricia

    2016-04-01

    In the last decade, mumps virus (MuV) causes outbreaks in highly vaccinated populations. Sub-optimal T cell immunity may play a role in the susceptibility to mumps in vaccinated individuals. T cell responses to mumps virus have been demonstrated, yet the quality of the MuV-specific T cell response has not been analyzed using single cell immunological techniques. Here we developed an IFNγ ELISPOT assay to assess MuV-specific T cell responses in peripheral blood mononuclear cells (PBMC) of healthy (vaccinated) donors and mumps patients. Various in vitro MuV-specific stimulation methods of PBMC were compared, using either live or inactivated MuV alone or MuV-infected autologous antigen presenting cells, i.e. Epstein Barr Virus-transformed B lymphoblastoid cell lines (EBV-BLCL) or (mitogen pre-activated) PBMC, for their ability to recall IFNγ-producing responder cells measured by ELISPOT. For the detection of MuV-specific T cell responses, direct exposure (24h) to live MuV was the preferred stimulation method when assay sensitivity and practical reasons were considered. Notably, flowcytometric confirmation of data revealed that primarily T cells and NK cells produce IFNγ upon live MuV stimulation. Depleting PBMC from CD56(+) NK cells prior to stimulation with live MuV led to the enumeration of MuV-specific T cell responses by ELISPOT. Our assay constitutes a tool to evaluate memory MuV-specific T cell responses in MuV vaccinated or infected persons. Furthermore, this study provides evidence that live MuV not only induces IFNγ production by T cells, but also by NK cells. PMID:26872407

  7. An association between antibodies specific for endothelial cells and renal transplant failure.

    PubMed

    Perrey, C; Brenchley, P E; Johnson, R W; Martin, S

    1998-06-01

    Human leucocyte antigen (HLA)-specific antibodies, present at the time of transplant, cause renal transplant rejection but cases of rejection of HLA-identical renal transplants indicate that antibodies to non-HLA antigens may also be detrimental. There is increasing evidence that antibodies to antigens present on endothelial cells and monocytes, and on endothelial cells alone, are associated with transplant rejection. We investigated 105 patients with failed renal transplants for the presence of endothelial cell reactive antibodies and compared them with 94 successful transplant patients to determine the role of non-HLA antibodies in transplant failure. Patient sera were tested by enzyme-linked immunoabsorbent assay (ELISA) using as a target fixed cells either from the endothelial/epithelial cell line EAHy.926 or primary cultures of human umbilical vein endothelial cells. Antibody binding was detected using an alkaline phosphatase-conjugated anti-human immunoglobulin G (IgG) antibody. Fourteen of the 105 failed transplant patients had endothelial cell-reactive antibodies as compared with only three of the 94 patients with successful transplants (Fisher's exact test, p = 0.02). Antibody-positive sera were absorbed with the epithelial cell line A549 to remove antibodies directed against the epithelial component of EAHy.926 and with a pool of lymphoblastoid cell line cells to remove HLA-specific antibodies. Absorption did not reduce antibody activity showing the antibodies to be directed against endothelial cell determinants. Antibody-positive sera were also tested by flow cytometry against the monocyte cell line THP-1 and 13 of the 14 patients were negative. In conclusion, we have demonstrated the presence of IgG antibodies directed against endothelial cell determinants in renal transplant recipients in association with renal transplant failure. PMID:9777698

  8. Genes related to growth regulation, DNA repair and apoptosis in an oestrogen receptor-negative (MDA-231) versus an oestrogen receptor-positive (MCF-7) breast tumour cell line.

    PubMed

    Skog, Sven; He, Qimin; Khoshnoud, Reza; Fornander, Tommy; Rutqvist, Lars-Erik

    2004-01-01

    The molecular mechanism(s) behind the development of endocrine resistance in breast cancer remains controversial. Here, we compare the capability of oestrogen receptor (ER)-negative cells (MDA-231) versus ER-positive tamoxifen-sensitive cells (MCF-7) to handle DNA repair, transmit signals from damaged DNA, initiate cell death via apoptosis, and then to control transmitted signals from the cell cycle and to synthesize growth factors and receptors. Genes related to these events were studied by cDNA micro-array. Normal human breast cells (H2F) and human lymphoblastoid tumour cells (CEM) were used as controls. Of the 18 genes investigated, 10 genes showed differences in their expression between the cell types. The ER-negative cells showed higher expressions of BRCA1, BRCA2, cdc2, cyclin B1, cyclin D1, cyclin E, IGFBP-3, TGF-alpha, TGF beta 2 and a lower expression of TGF beta R1. No differences in the expressions of bax, bcl-2, p53, p21 and GADD45 were found between the two cell lines. We found that the ER-negative cells were characterized by: (1) a stimulated expression of growth factors and cell cycle regulation compounds, (2) improved DNA repair capacity, but (3) no change in DNA damage signals and apoptotic pathways. Improved DNA repair capacity of ER-negative cells would have a growth advantage over ER-positive tumours when receiving antitumour therapy. PMID:15192311

  9. The in vitro generation of multi-tumor antigen-specific cytotoxic T cell clones: Candidates for leukemia adoptive immunotherapy following allogeneic stem cell transplantation.

    PubMed

    Mohamed, Yehia S; Bashawri, Layla A; Vatte, Chittibabu; Abu-Rish, Eman Y; Cyrus, Cyril; Khalaf, Wafaa S; Browning, Michael J

    2016-09-01

    Adoptive T-cell immunotherapy is a promising approach to manage and maintain relapse-free survival of leukemia patients, especially following allogeneic stem cell transplantation. Post-transplant adoptive immunotherapy using cytotoxic T lymphocytes (CTLs) of the donor origin provide graft-versus-tumor effects, with or without graft-versus-host disease. Myeloid leukemias express immunogenic leukemia associated antigens (LAAs); such as WT-1, PRAME, MAGE, h-TERT and others, most of them are able to induce specific T cell responses whenever associated with the proper co-stimulation. We investigated the ability of a LAA-expressing hybridoma cell line to induce CTL clones in PBMCs of HLA-matched healthy donors in vitro. The CTL clones were induced by repetitive co-culture with LAAs-expressing, HLA-A*0201(+) hybrid cell line, generated by fusion of leukemia blasts to human immortalized APC (EBV-sensitized B-lymphoblastoid cell line; HMy2). The induced cytotoxic T cell clones were phenotypically and functionally characterized by pentamer analysis, IFN-γ release ELISPOT and cellular cytotoxicity assays. All T cell lines showed robust peptide recognition and functional activity when sensitized with HLA-A*0201-restricted WT-1235-243, hTERT615-624 or PRAME100-108 peptides-pulsed T2 cells, in addition to partially HLA-matched leukemia blasts. This study demonstrates the feasibility of developing multi-tumor antigen-specific T cell lines in allogeneic PBMCs in vitro, using LAA-expressing tumor/HMy2 hybrid cell line model, for potential use in leukemia adoptive immunotherapy in partially matched donor-recipient setting. PMID:27490939

  10. Array-Based Platform To Select, Release, and Capture Epstein-Barr Virus-Infected Cells Based on Intercellular Adhesion.

    PubMed

    Attayek, Peter J; Hunsucker, Sally A; Wang, Yuli; Sims, Christopher E; Armistead, Paul M; Allbritton, Nancy L

    2015-12-15

    Microraft arrays were developed to select and separate cells based on a complex phenotype, weak intercellular adhesion, without knowledge of cell-surface markers or intracellular proteins. Since the cells were also not competent to bind to a culture surface, a method to encapsulate nonadherent cells within a gelatin plug on the concave microraft surface was developed, enabling release and collection of the cells without the need for cell attachment to the microraft surface. After microraft collection, the gelatin was liquified to release the cell(s) for culture or analysis. A semiautomated release and collection device for the microrafts demonstrated 100 ± 0% collection efficiency of the microraft while increasing throughput 5-fold relative to that of manual release and collection. Using the microraft array platform along with the gelatin encapsulation method, single cells that were not surface-attached were isolated with a 100 ± 0% efficiency and a 96 ± 4% postsort single-cell cloning efficiency. As a demonstration, Epstein-Barr virus-infected lymphoblastoid cell lines (EBV-LCL) were isolated based on their intercellular adhesive properties. The identified cell colonies were collected with a 100 ± 0% sorting efficiency and a postsort viability of 87 ± 3%. When gene expression analysis of the EBV latency-associated gene, EBNA-2, was performed, there was no difference in expression between blasting or weakly adhesive cells and nonblasting or nonadhesive cells. Microraft arrays are a versatile method enabling separation of cells based on complicated and as yet poorly understood cell phenotypes. PMID:26558605

  11. Sequence and chromatin determinants of cell-type-specific transcription factor binding.

    PubMed

    Arvey, Aaron; Agius, Phaedra; Noble, William Stafford; Leslie, Christina

    2012-09-01

    Gene regulatory programs in distinct cell types are maintained in large part through the cell-type-specific binding of transcription factors (TFs). The determinants of TF binding include direct DNA sequence preferences, DNA sequence preferences of cofactors, and the local cell-dependent chromatin context. To explore the contribution of DNA sequence signal, histone modifications, and DNase accessibility to cell-type-specific binding, we analyzed 286 ChIP-seq experiments performed by the ENCODE Consortium. This analysis included experiments for 67 transcriptional regulators, 15 of which were profiled in both the GM12878 (lymphoblastoid) and K562 (erythroleukemic) human hematopoietic cell lines. To model TF-bound regions, we trained support vector machines (SVMs) that use flexible k-mer patterns to capture DNA sequence signals more accurately than traditional motif approaches. In addition, we trained SVM spatial chromatin signatures to model local histone modifications and DNase accessibility, obtaining significantly more accurate TF occupancy predictions than simpler approaches. Consistent with previous studies, we find that DNase accessibility can explain cell-line-specific binding for many factors. However, we also find that of the 10 factors with prominent cell-type-specific binding patterns, four display distinct cell-type-specific DNA sequence preferences according to our models. Moreover, for two factors we identify cell-specific binding sites that are accessible in both cell types but bound only in one. For these sites, cell-type-specific sequence models, rather than DNase accessibility, are better able to explain differential binding. Our results suggest that using a single motif for each TF and filtering for chromatin accessible loci is not always sufficient to accurately account for cell-type-specific binding profiles. PMID:22955984

  12. 8-Hydroxy-2'-deoxyguanosine as a biomarker of oxidative DNA damage induced by perfluorinated compounds in TK6 cells.

    PubMed

    Yahia, Doha; Haruka, Igarashi; Kagashi, Yae; Tsuda, Shuji

    2016-02-01

    8-Hydroxy-2'-deoxyguanosine (8-OHdG) is the most common biomarker of oxidative DNA damage, it is formed by chemical carcinogens and can be measured in any species. Perfluorooctanoic acid (PFOA) and perfluorononanoic acid (PFNA) are suspected genotoxic carcinogens through induction of reactive oxygen species that are responsible for oxidative DNA damage. This study was conducted to investigate the in vitro genotoxicity of PFOA and PFNA in human lymphoblastoid (TK6) cell line. TK6 cells were exposed to PFOA at 0, 125, 250, and 500 ppm and PFNA at 125 and 250 ppm for 2 h. Single cell gel electrophoresis (comet assay) was used to measure DNA damage; at least 50 cells per sample were analyzed using comet Assay Software Project (CASP). 8-OHdG was measured in DNA of exposed cells using high-performance liquid chromatography (HPLC)-mass spectrometry (MS)/MS. Results showed that both PFOA and PFNA induced DNA damage indicated by increased tail length (DNA migration). The level of 8-OHdG was increased in a dose-dependent manner in both PFOA and PFNA exposure. We concluded that PFOA and PFNA induced DNA damage and the biomarker of oxidative DNA damage (8-OHdG) could be measured by HPLC-MS/MS. In addition, PFNA produced high level of 8-OHdG at concentrations lower than PFOA, this may indicate that PFNA is more potent genotoxicant for TK6 cells than PFOA. PMID:25113910

  13. Rosmarinic Acid-Rich Extracts of Summer Savory (Satureja hortensis L.) Protect Jurkat T Cells against Oxidative Stress

    PubMed Central

    Chkhikvishvili, Irakli; Sanikidze, Tamar; Gogia, Nunu; Mchedlishvili, Tamar; Enukidze, Maia; Machavariani, Marine; Vinokur, Yakov

    2013-01-01

    Summer savory (Satureja hortensis L., Lamiaceae) is used in several regions of the world as a spice and folk medicine. Anti-inflammatory and cytoprotective effects of S. hortensis and of its rosmarinic acid-rich phenolic fraction have been demonstrated in animal trials. However, previous studies of rosmarinic acid in cell models have yielded controversial results. In this study, we investigated the effects of summer savory extracts on H2O2-challenged human lymphoblastoid Jurkat T cells. LC-MS analysis confirmed the presence of rosmarinic acid and flavonoids such as hesperidin and naringin in the phenolic fraction. Adding 25 or 50 µM of H2O2 to the cell culture caused oxidative stress, manifested as generation of superoxide and peroxyl radicals, reduced cell viability, G0/G1 arrest, and enhanced apoptosis. This stress was significantly alleviated by the ethanolic and aqueous extracts of S. hortensis and by the partially purified rosmarinic acid fraction. The application of an aqueous S. hortensis extract doubled the activity of catalase and superoxide dismutase in the cells. The production of IL-2 and IL-10 interleukins was stimulated by H2O2 and was further enhanced by the addition of the S. hortensis extract or rosmarinic acid fraction. The H2O2-challenged Jurkat cells may serve as a model for investigating cellular mechanisms of cytoprotective phytonutrient effects. PMID:24349613

  14. Signal-peptide-peptidase-like 2a is required for CD74 intramembrane proteolysis in human B cells

    PubMed Central

    Schneppenheim, Janna; Hüttl, Susann; Kruchen, Anne; Fluhrer, Regina; Müller, Ingo; Saftig, Paul; Schneppenheim, Reinhard; Martin, Christa L; Schröder, Bernd

    2015-01-01

    The invariant chain (CD74) mediates targeting of the MHCII complex to endosomal compartments, where CD74 undergoes degradation allowing MHCII to acquire peptides. We demonstrated recently that intramembrane proteolysis of the final membrane-bound N-terminal fragment (NTF) of CD74 is catalysed by Signal-peptide-peptidase-like 2a (SPPL2a) and that this process is indispensable for development and function of B lymphocytes in mice. In SPPL2a−/− mice, homeostasis of these cells is disturbed by the accumulation of the unprocessed CD74 NTF. So far, evidence for this essential role of SPPL2a is restricted to mice. Nevertheless, inhibition of SPPL2a has been suggested as novel approach to target B cells for treating autoimmunity. Here, we characterize human B cell lines with a homozygous microdeletion on chromosome 15. We demonstrate that this deletion disrupts the SPPL2a genomic locus and leads to loss of SPPL2a transcript. Lymphoblastoid cell lines from patients with this deletion exhibit absence of SPPL2a at the protein level and show an accumulation of the CD74 NTF comparable to B cells from SPPL2a−/− mice. By this means, we present evidence that the role of SPPL2a in CD74 proteolysis is conserved in human B cells and provide support for modulation of SPPL2a activity as a therapeutic concept. PMID:25035924

  15. Detection of Chromosomal Structural Alterations in Single Cells by SNP Arrays: A Systematic Survey of Amplification Bias and Optimized Workflow

    PubMed Central

    Iwamoto, Kazuya; Bundo, Miki; Ueda, Junko; Nakano, Yoko; Ukai, Wataru; Hashimoto, Eri; Saito, Toshikazu; Kato, Tadafumi

    2007-01-01

    Background In single-cell human genome analysis using whole-genome amplified product, a strong amplification bias involving allele dropout and preferential amplification hampers the quality of results. Using an oligonucleotide single nucleotide polymorphism (SNP) array, we systematically examined the nature of this amplification bias, including frequency, degree, and preference for genomic location, and we assessed the effects of this amplification bias on subsequent genotype and chromosomal copy number analyses. Methodology/Principal Findings We found a large variability in amplification bias among the amplified products obtained by multiple displacement amplification (MDA), and this bias had a severe effect on the genotype and chromosomal copy number analyses. We established optimal experimental conditions for pre-screening for high-quality amplified products, processing array data, and analyzing chromosomal structural alterations. Using this optimized protocol, we successfully detected previously unidentified chromosomal structural alterations in single cells from a lymphoblastoid cell line. These alterations were subsequently confirmed by karyotype analysis. In addition, we successfully obtained reproducible chromosomal copy number profiles of single cells from the cell line with a complex karyotype, indicating the applicability and potential of our optimized workflow. Conclusions/Significance Our results suggest that the quality of amplification products should be critically assessed before using them for genomic analyses. The method of MDA-based whole-genome amplification followed by SNP array analysis described here will be useful for exploring chromosomal alterations in single cells. PMID:18074030

  16. A peptide-based inhibitor for prevention of B cell hyperproliferation induced by Epstein-Barr virus.

    PubMed

    Knight, Jason S; Lan, Ke; Bajaj, Bharat; Sharma, Nikhil; Tsai, Donald E; Robertson, Erle S

    2006-10-10

    Epstein-Barr virus (EBV) infects and transforms resting B lymphocytes in vitro. The virus can also cause B cell lymphomas in immunosuppressed humans. Indeed, EBV-mediated post-transplant lymphoproliferative disease causes significant complications in transplant recipients, including loss of the transplanted organ and even death. The limited treatment options include, nonspecific targeting of B cell surface antigens with monoclonal antibodies or withdrawal of immunosuppression. These therapies fail in approximately 50% of patients. Clearly, treatments that specifically target EBV-infected cells are desirable. The EBV antigen EBNA3C regulates cell cycle by targeting critical cellular complexes such as cyclin A/cdk2, SCF(Skp2), and Rb. Here, we use a 20-amino-acid EBNA3C-derived peptide, fused to an HIV TAT tag for efficient delivery, to disrupt cell cycle regulation by EBNA3C. The peptide inhibited hyperproliferation of EBV-infected B cell lines and reduced in vitro immortalization of primary B lymphocytes by EBV. Importantly, the peptide inhibited lymphoblastoid outgrowth from the blood of an EBV-positive transplant patient in vitro. PMID:16876848

  17. Mutation induction by 125iodoacetylproflavine, a DNA-intercalating agent, in human cells.

    PubMed

    Whaley, J M; Kassis, A I; Kinsey, B M; Adelstein, S J; Little, J B

    1990-06-01

    Survival and the induction of mutations at the hprt and tk loci were measured in TK6 human lymphoblastoid cells following treatment with the DNA-intercalating agent 125iodoacetylproflavine (125IAP). 125IAP was readily taken up into the cells, was localized to the nucleus, and was released rapidly following resuspension of the cells in fresh medium. Treatment with 125IAP for 24 h yielded a D0 of 110 decays/cell and an induced mutant fraction of 0.13 x 10(-6) per decay at the hprt locus and 0.4 x 10(-6) per decay at the tk locus. Molecular analyses of 125IAP-induced hprt mutants by Southern blot revealed a high proportion of large-scale changes at this locus. When these results are compared with those observed with 125IdUrd, 125IAP shows a reduced effectiveness per decay, related perhaps to the non-covalent nature of intercalator binding, resulting in reduced energy deposition in the DNA. PMID:1971836

  18. Overexpression of the human BCL-2 gene product results in growth enhancement of Epstein-Barr virus-immortalized B cells

    SciTech Connect

    Tsujimoto, Yoshihide )

    1989-03-01

    The biological activity of the human BCL-2 gene product was analyzed in an Epstein-Barr virus (EBV)-infected human lymphoblastoid B-cell line transfected with BCL-2 sequences driven by the simian virus 40 promoter and enhancer. Overproduction of the BCL-2 protein conferred a selective growth advantage to the EBV-infected B cells as compared with control transfectants in low-serum medium and also after seeding at limiting dilution but did not render the cells tumorigenic in athymic nude mice. This growth enhancement was also seen in cells transfected with the BCL-2 gene with its own promoter juxtaposed to the immunoglobulin heavy chain gene enhancer, which represents the translocated form of the BCL-2 gene observed in follicular lymphomas with the t(14;18) translocation. The growth advantage of EBV-infected B cells overproducing the BCL-2 protein is neither due to the enhanced growth factor production nor due to an enhanced sensitivity of the BCL-2 transfectants to interleukins 1 or 6, although both lymphokines are known to stimulate proliferation of EBV-infected B-cell lines. The growth advantage of EBV-infected B-cell lines. The growth advantage of EBV-infected B cells by overproduction of the BCL-2 protein suggests the direct involvement of the BCL-2 gene product in the pathogenesis of follicular lymphoma.

  19. Similarity of spontaneous germinal and in vitro somatic cell mutation rates in humans: implications for carcinogenesis and for the role of exogenous factors in "spontaneous" germinal mutagenesis.

    PubMed Central

    Kuick, R D; Neel, J V; Strahler, J R; Chu, E H; Bargal, R; Fox, D A; Hanash, S M

    1992-01-01

    The rate of spontaneous mutation resulting in electrophoretic variants per cell generation in a human lymphoblastoid cell line, on the basis of experiments described in this paper, is found to be 7.2 x 10(-8) per locus. A review of similar data on electrophoretic variants resulting from spontaneous mutation in the human germ line leads to an estimate of 3.3 x 10(-8) per locus per cell generation. It is argued that the similarity of these two estimates, despite an average cell generation time of 18.5 hr for the cultured somatic cells but about 26 days in the germ line, suggests that spontaneous mutation involving nucleotide substitutions is much more dependent on cell generation than on time. This finding permits the inference that environmental (exogenous) variables make a relatively small contribution to the rate of this type of human germinal spontaneous mutation. While in vitro somatic-cell mutation rates, such as derived in this study, provide a basis for modeling the contribution of nucleotide substitutions in multihit/clonal theories of carcinogenesis, it is also argued that the complex of events involved in carcinogenesis, including chromosomal rearrangements and mitotic recombination, could have very different individual probabilities. Estimates for the rates of these other types of mutation are needed to provide a better understanding of the manner in which multiple mutations accumulate in malignant cells. Images PMID:1495998

  20. Interchromosomal gene conversion at an endogenous human cell locus.

    PubMed Central

    Quintana, P J; Neuwirth, E A; Grosovsky, A J

    2001-01-01

    To examine the relationship between gene conversion and reciprocal exchange at an endogenous chromosomal locus, we developed a reversion assay in a thymidine kinase deficient mutant, TX545, derived from the human lymphoblastoid cell line TK6. Selectable revertants of TX545 can be generated through interchromosomal gene conversion at the site of inactivating mutations on each tk allele or by reciprocal exchange that alters the linkage relationships of inactivating polymorphisms within the tk locus. Analysis of loss of heterozygosity (LOH) at intragenic polymorphisms and flanking microsatellite markers was used to initially evaluate allelotypes in TK(+) revertants for patterns associated with either gene conversion or crossing over. The linkage pattern in a subset of convertants was then unambiguously established, even in the event of prereplicative recombinational exchanges, by haplotype analysis of flanking microsatellite loci in tk(-/-) LOH mutants collected from the tk(+/-) parental convertant. Some (7/38; 18%) revertants were attributable to easily discriminated nonrecombinational mechanisms, including suppressor mutations within the tk coding sequence. However, all revertants classified as a recombinational event (28/38; 74%) were attributed to localized gene conversion, representing a highly significant preference (P < 0.0001) over gene conversion with associated reciprocal exchange, which was never observed. PMID:11404339

  1. Positively selected Leu-11a (CD16+) cells require the presence of accessory cells or factors for the lysis of herpes simplex virus-infected fibroblasts but not herpes simplex virus-infected Raji.

    PubMed

    Fitzgerald-Bocarsly, P; Feldman, M; Curl, S; Schnell, J; Denny, T

    1989-08-15

    Previous studies from our laboratory indicated that human NK activity against HSV-infected fibroblasts (HSV-Fs) but not K562 targets was sensitive to treatment with anti-HLA-DR plus C. In the current study, we have selected Leu-11a+ (CD-16) cells by fluorescence activated cell sorting and found that although Leu-11a enriched populations lysed K562 targets in 14-h 51Cr-release assays, they were unable to kill HSV-Fs targets unless a Leu-11a-depleted population was added back to the effectors or unless known activators of NK cells (IFN-alpha or IL-2) were added to the assays. In contrast, Leu-11a-enriched populations were able to mediate ADCC against HSV-Fs in the presence of sera from HSV-seropositive individuals without the requirement for accessory cells. We have begun preliminary characterization of the accessory cells which allow lysis of HSV-Fs by NK cells: they are HLA-DR+ cells which enrich in the light density fractions of Metrizamide density gradients. They need be present in very small numbers for lysis to take place and are not MHC restricted in that heterologous add-backs between anti-HLA-DR plus C and anti-Leu-11b plus C-treated populations are capable of target cell lysis at levels similar to those achieved with the autologous add-backs. Further, the levels of lysis in heterologous add-back experiments reflected the lytic potential of the effector rather than the accessory cell donor. Finally, although the requirement for accessory cells for NK lysis has been demonstrated for fibroblasts infected with HSV-1, CMV, and VZV, lysis of HSV-infected Raji lymphoblastoid cells is relatively accessory-cell independent, indicating that the requirement for accessory cells for lysis by NK cells is not a property of all herpesvirus-infected targets. PMID:2526183

  2. Activity-based ubiquitin-specific protease (USP) profiling of virus-infected and malignant human cells

    PubMed Central

    Ovaa, Huib; Kessler, Benedikt M.; Rolén, Ulrika; Galardy, Paul J.; Ploegh, Hidde L.; Masucci, Maria G.

    2004-01-01

    The family of ubiquitin (Ub)-specific proteases (USP) removes Ub from Ub conjugates and regulates a variety of cellular processes. The human genome contains many putative USP-encoding genes, but little is known about USP tissue distribution, pattern of expression, activity, and substrate specificity. We have used a chemistry-based functional proteomics approach to identify active USPs in normal, virus-infected, and tumor-derived human cells. Depending on tissue origin and stage of activation/differentiation, different USP activity profiles were revealed. The activity of specific USPs, including USP5, -7, -9, -13, -15, and -22, was up-regulated by mitogen activation or virus infection in normal T and B lymphocytes. UCH-L1 was highly expressed in tumor cell lines of epithelial and hematopoietic cell origin but was not detected in freshly isolated and mitogen-activated cells. Up-regulation of this USP was a late event in the establishment of Epstein–Barr virus-immortalized lymphoblastoid cell lines and correlated with enhanced proliferation, suggesting a possible role in growth transformation. PMID:14982996

  3. Protective activity of C-geranylflavonoid analogs from Paulownia tomentosa against DNA damage in 137Cs irradiated AHH-1 cells.

    PubMed

    Moon, Hyung-In; Jeong, Min Ho; Jo, Wol Soon

    2014-09-01

    Radiotherapy is an important form of treatment for a wide range of cancers, but it can damage DNA and cause adverse effects. We investigated if the diplacone analogs of P. tomentosa were radio-protective in a human lymphoblastoid cell line (AHH-1). Four geranylated flavonoids, diplacone, 3'-O-methyl-5'-hydroxydiplacone, 3'-O-methyl-5'-O-methyldiplacone and 3'-O-methyldiplacol, were tested for their antioxidant and radio-protective effects. Diplacone analogs effectively scavenged free radicals and inhibited radiation-induced DNA strand breaks in vitro. They significantly decreased levels of reactive oxygen species and cellular DNA damage in 2 Gy-irradiated AHH-1 cells. Glutathione levels and superoxide dismutase activity in irradiated AHH-1 cells increased significantly after treatment with these analogs. The enhanced biological anti-oxidant activity and radioprotective activity of diplacone analogs maintained the survival of irradiated AHH-1 cells in a clonogenic assay. These data suggest that diplacone analogs may protect healthy tissue surrounding tumor cells during radiotherapy to ensure better control of radiotherapy and allow higher doses of radiotherapy to be employed. PMID:25918796

  4. Retroviral insertional activation of the c-myb proto-oncogene in a Marek's disease T-lymphoma cell line.

    PubMed Central

    Le Rouzic, E; Perbal, B

    1996-01-01

    Marek's disease virus (MDV) is an avian herpesvirus that causes, in chickens, a lymphoproliferative disease characterized by malignant transformation of T lymphocytes. The rapid onset of polyclonal tumors indicates the existence of MDV-encoded oncogenic products. However, the molecular basis of MDV-induced lymphoproliferative disease and latency remains largely unclear. Several lines of evidence suggest that MDV and Rous-associated virus (RAV) might cooperate in the development of B-cell lymphomas induced by RAV. Our present results indicate for the first time that MDV and RAV might also act synergistically in the development of T-cell lymphomas. We report an example of an MDV-transformed T-lymphoblastoid cell line (T9) expressing high levels of a truncated C-MYB protein as a result of RAV integration within one c-myb allele. The chimeric RAV-c-myb mRNA species initiated in the 5' long terminal repeat of RAV are deprived of sequences corresponding to c-myb exons 1 to 3. The attenuation of MDV oncogenicity has been strongly related to structural changes in the MDV BamHI-D and BamHI-H DNA fragments. We have established that both DNA restriction fragments are rearranged in the T9 MDV-transformed cells. Our results suggest that retroviral insertional activation of the c-myb proto-oncogene is a critical factor involved in the maintenance of the transformed phenotype and the tumorigenic potential of this T-lymphoma cell line. PMID:8892859

  5. The c-Jun N-terminal kinase pathway is critical for cell transformation by the latent membrane protein 1 of Epstein-Barr virus

    SciTech Connect

    Kutz, Helmut; Reisbach, Gilbert; Schultheiss, Ute; Kieser, Arnd

    2008-02-20

    The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) transforms cells activating signal transduction pathways such as NF-{kappa}B, PI3-kinase, or c-Jun N-terminal kinase (JNK). Here, we investigated the functional role of the LMP1-induced JNK pathway in cell transformation. Expression of a novel dominant-negative JNK1 allele caused a block of proliferation in LMP1-transformed Rat1 fibroblasts. The JNK-specific inhibitor SP600125 reproduced this effect in Rat1-LMP1 cells and efficiently interfered with proliferation of EBV-transformed lymphoblastoid cells (LCLs). Inhibition of the LMP1-induced JNK pathway in LCLs caused the downregulation of c-Jun and Cdc2, the essential G2/M cell cycle kinase, which was accompanied by a cell cycle arrest of LCLs at G2/M phase transition. Moreover, SP600125 retarded tumor growth of LCLs in a xenograft model in SCID mice. Our data support a critical role of the LMP1-induced JNK pathway for proliferation of LMP1-transformed cells and characterize JNK as a potential target for intervention against EBV-induced malignancies.

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

    PubMed Central

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

    2016-01-01

    Cancer chemotherapy is characterized by an elevated intrinsic toxicity and the development of drug resistance. Thus, there is a compelling need for new intervention strategies with an improved therapeutic profile. Immunogenic cell death (ICD) represents an innovative anticancer strategy where dying cancer cells release damage-associated molecular patterns promoting tumor-specific immune responses. The roots of Withania somnifera (W. somnifera) are used in the Indian traditional medicine for their anti-inflammatory, immunomodulating, neuroprotective, and anticancer activities. The present study is designed to explore the antileukemic activity of the dimethyl sulfoxide extract obtained from the roots of W. somnifera (WE). We studied its cytostatic and cytotoxic activity, its ability to induce ICD, and its genotoxic potential on a human T-lymphoblastoid cell line by using different flow cytometric assays. Our results show that WE has a significant cytotoxic and cytostatic potential, and induces ICD. Its proapoptotic mechanism involves intracellular Ca2+ accumulation and the generation of reactive oxygen species. In our experimental conditions, the extract possesses a genotoxic potential. Since the use of Withania is suggested in different contexts including anti-infertility and osteoarthritis care, its genotoxicity should be carefully considered for an accurate assessment of its risk–benefit profile. PMID:27187469

  7. Missense Mutations in Fumarate Hydratase in Multiple Cutaneous and Uterine Leiomyomatosis and Renal Cell Cancer

    PubMed Central

    Alam, N. Afrina; Olpin, Simon; Rowan, Andrew; Kelsell, David; Leigh, Irene M.; Tomlinson, Ian P. M.; Weaver, Todd

    2005-01-01

    Heterozygous germline mutations in fumarate hydratase (FH) predispose to the multiple cutaneous and uterine leiomyomatosis syndrome (MCUL), which, when co-existing with renal cancer, is also known as hereditary leiomyomatosis and renal cell cancer. Twenty-seven distinct missense mutations represent 68% of FH mutations reported in MCUL. Here we show that FH missense mutations significantly occurred in fully conserved residues and in residues functioning in the FH A-site, B-site, or subunit-interacting region. Of 24 distinct missense mutations, 13 (54%) occurred in the substrate-binding A-site, 4 (17%) in the substrate-binding B-site, and 7 (29%) in the subunit-interacting region. Clustering of missense mutations suggested the presence of possible mutational hotspots. FH functional assay of lymphoblastoid cell lines from 23 individuals with heterozygous FH missense mutations showed that A-site mutants had significantly less residual activity than B-site mutants, supporting data from Escherichia coli that the A-site is the main catalytic site. Missense FH mutations predisposing to renal cancer had no unusual features, and identical mutations were found in families without renal cancer, suggesting a role for genetic or environmental factors in renal cancer development in MCUL. That all missense FH mutations associating with MCUL/hereditary leiomyomatosis and renal cell cancer showed diminished FH enzymatic activity suggests that the tumor suppressor role of fumarate hydratase may relate to its enzymatic function. PMID:16237213

  8. Genome-wide DNA methylation analysis in cohesin mutant human cell lines

    PubMed Central

    Liu, Jinglan; Zhang, Zhe; Bando, Masashige; Itoh, Takehiko; Deardorff, Matthew A.; Li, Jennifer R.; Clark, Dinah; Kaur, Maninder; Tatsuro, Kondo; Kline, Antonie D.; Chang, Celia; Vega, Hugo; Jackson, Laird G.; Spinner, Nancy B.; Shirahige, Katsuhiko; Krantz, Ian D.

    2010-01-01

    The cohesin complex has recently been shown to be a key regulator of eukaryotic gene expression, although the mechanisms by which it exerts its effects are poorly understood. We have undertaken a genome-wide analysis of DNA methylation in cohesin-deficient cell lines from probands with Cornelia de Lange syndrome (CdLS). Heterozygous mutations in NIPBL, SMC1A and SMC3 genes account for ∼65% of individuals with CdLS. SMC1A and SMC3 are subunits of the cohesin complex that controls sister chromatid cohesion, whereas NIPBL facilitates cohesin loading and unloading. We have examined the methylation status of 27 578 CpG dinucleotides in 72 CdLS and control samples. We have documented the DNA methylation pattern in human lymphoblastoid cell lines (LCLs) as well as identified specific differential DNA methylation in CdLS. Subgroups of CdLS probands and controls can be classified using selected CpG loci. The X chromosome was also found to have a unique DNA methylation pattern in CdLS. Cohesin preferentially binds to hypo-methylated DNA in control LCLs, whereas the differential DNA methylation alters cohesin binding in CdLS. Our results suggest that in addition to DNA methylation multiple mechanisms may be involved in transcriptional regulation in human cells and in the resultant gene misexpression in CdLS. PMID:20448023

  9. Investigation of micronucleus induction in MTH1 knockdown cells exposed to UVA, UVB or UVC.

    PubMed

    Fotouhi, Asal; Cornella, Nicola; Ramezani, Mehrafarin; Wojcik, Andrzej; Haghdoost, Siamak

    2015-11-01

    The longer wave parts of UVR can increase the production of reactive oxygen species (ROS) which can oxidize nucleotides in the DNA or in the nucleotide pool leading to mutations. Oxidized bases in the DNA are repaired mainly by the DNA base excision repair system and incorporation of oxidized nucleotides into newly synthesized DNA can be prevented by the enzyme MTH1. Here we hypothesize that the formation of several oxidized base damages (from pool and DNA) in close proximity, would cause a high number of base excision repair events, leading to DNA double strand breaks (DSB) and therefore giving rise to cytogenetic damage. If this hypothesis is true, cells with low levels of MTH1 will show higher cytogenetic damage after the longer wave parts of UVR. We analyzed micronuclei induction (MN) as an endpoint for cytogenetic damage in the human lymphoblastoid cell line, TK6, with a normal and a reduced level of MTH1 exposed to UVR. The results indicate a higher level of micronuclei at all incubation times after exposure to the longer wave parts of UVR. There is no significant difference between wildtype and MTH1-knockdown TK6 cells, indicating that MTH1 has no protective role in UVR-induced cytogenetic damage. This indicates that DSBs induced by UV arise from damage forms by direct interaction of UV or ROS with the DNA rather than through oxidation of dNTP. PMID:26520386

  10. Epstein-Barr Virus oncoprotein super-enhancers control B cell growth

    PubMed Central

    Zhou, Hufeng; Schmidt, Stefanie CS; Jiang, Sizun; Willox, Bradford; Bernhardt, Katharina; Liang, Jun; Johannsen, Eric C; Kharchenko, Peter; Gewurz, Benjamin E; Kieff, Elliott; Zhao, Bo

    2015-01-01

    Summary Super-enhancers are clusters of gene-regulatory sites bound by multiple transcription factors that govern cell transcription, development, phenotype, and oncogenesis. By examining Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs), we identified four EBV oncoproteins and five EBV-activated NF-κB subunits co-occupying ~1800 enhancer sites. Of these, 187 had markedly higher and broader histone H3K27ac signals characteristic of super-enhancers, and were designated “EBV super-enhancers”. EBV super-enhancer-associated genes included the MYC and BCL2 oncogenes, enabling LCL proliferation and survival. EBV super-enhancers were enriched for B cell transcription factor motifs and had a high co-occupancy of the transcription factors STAT5 and NFAT. EBV super-enhancer-associated genes were more highly expressed than other LCL genes. Disrupting EBV super-enhancers by the bromodomain inhibitor, JQ1 or conditionally inactivating an EBV oncoprotein or NF-κB decreased MYC or BCL2 expression and arrested LCL growth. These findings provide insight into mechanisms of EBV-induced lymphoproliferation and identify potential therapeutic interventions. PMID:25639793

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

    PubMed

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

    2016-01-01

    Cancer chemotherapy is characterized by an elevated intrinsic toxicity and the development of drug resistance. Thus, there is a compelling need for new intervention strategies with an improved therapeutic profile. Immunogenic cell death (ICD) represents an innovative anticancer strategy where dying cancer cells release damage-associated molecular patterns promoting tumor-specific immune responses. The roots of Withania somnifera (W. somnifera) are used in the Indian traditional medicine for their anti-inflammatory, immunomodulating, neuroprotective, and anticancer activities. The present study is designed to explore the antileukemic activity of the dimethyl sulfoxide extract obtained from the roots of W. somnifera (WE). We studied its cytostatic and cytotoxic activity, its ability to induce ICD, and its genotoxic potential on a human T-lymphoblastoid cell line by using different flow cytometric assays. Our results show that WE has a significant cytotoxic and cytostatic potential, and induces ICD. Its proapoptotic mechanism involves intracellular Ca(2+) accumulation and the generation of reactive oxygen species. In our experimental conditions, the extract possesses a genotoxic potential. Since the use of Withania is suggested in different contexts including anti-infertility and osteoarthritis care, its genotoxicity should be carefully considered for an accurate assessment of its risk-benefit profile. PMID:27187469

  12. Epstein-Barr virus oncoprotein super-enhancers control B cell growth.

    PubMed

    Zhou, Hufeng; Schmidt, Stefanie C S; Jiang, Sizun; Willox, Bradford; Bernhardt, Katharina; Liang, Jun; Johannsen, Eric C; Kharchenko, Peter; Gewurz, Benjamin E; Kieff, Elliott; Zhao, Bo

    2015-02-11

    Super-enhancers are clusters of gene-regulatory sites bound by multiple transcription factors that govern cell transcription, development, phenotype, and oncogenesis. By examining Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs), we identified four EBV oncoproteins and five EBV-activated NF-κB subunits co-occupying ∼1,800 enhancer sites. Of these, 187 had markedly higher and broader histone H3K27ac signals, characteristic of super-enhancers, and were designated "EBV super-enhancers." EBV super-enhancer-associated genes included the MYC and BCL2 oncogenes, which enable LCL proliferation and survival. EBV super-enhancers were enriched for B cell transcription factor motifs and had high co-occupancy of STAT5 and NFAT transcription factors (TFs). EBV super-enhancer-associated genes were more highly expressed than other LCL genes. Disrupting EBV super-enhancers by the bromodomain inhibitor JQ1 or conditionally inactivating an EBV oncoprotein or NF-κB decreased MYC or BCL2 expression and arrested LCL growth. These findings provide insight into mechanisms of EBV-induced lymphoproliferation and identify potential therapeutic interventions. PMID:25639793

  13. Virus and Autoantigen-Specific CD4+ T Cells Are Key Effectors in a SCID Mouse Model of EBV-Associated Post-Transplant Lymphoproliferative Disorders

    PubMed Central

    Linnerbauer, Stefanie; Behrends, Uta; Adhikary, Dinesh; Witter, Klaus; Bornkamm, Georg W.; Mautner, Josef

    2014-01-01

    Polyclonal Epstein-Barr virus (EBV)-infected B cell line (lymphoblastoid cell lines; LCL)-stimulated T-cell preparations have been successfully used to treat EBV-positive post-transplant lymphoproliferative disorders (PTLD) in transplant recipients, but function and specificity of the CD4+ component are still poorly defined. Here, we assessed the tumor-protective potential of different CD4+ T-cell specificities in a PTLD-SCID mouse model. Injection of different virus-specific CD4+ T-cell clones showed that single specificities were capable of prolonging mouse survival and that the degree of tumor protection directly correlated with recognition of target cells in vitro. Surprisingly, some CD4+ T-cell clones promoted tumor development, suggesting that besides antigen recognition, still elusive functional differences exist among virus-specific T cells. Of several EBV-specific CD4+ T-cell clones tested, those directed against virion antigens proved most tumor-protective. However, enriching these specificities in LCL-stimulated preparations conferred no additional survival benefit. Instead, CD4+ T cells specific for unknown, probably self-antigens were identified as principal antitumoral effectors in LCL-stimulated T-cell lines. These results indicate that virion and still unidentified cellular antigens are crucial targets of the CD4+ T-cell response in this preclinical PTLD-model and that enriching the corresponding T-cell specificities in therapeutic preparations may enhance their clinical efficacy. Moreover, the expression in several EBV-negative B-cell lymphoma cell lines implies that these putative autoantigen(s) might also qualify as targets for T-cell-based immunotherapy of virus-negative B cell malignancies. PMID:24853673

  14. Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells.

    PubMed

    Iordanskiy, Sergey; Van Duyne, Rachel; Sampey, Gavin C; Woodson, Caitlin M; Fry, Kelsi; Saifuddin, Mohammed; Guo, Jia; Wu, Yuntao; Romerio, Fabio; Kashanchi, Fatah

    2015-11-01

    The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4(+) T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4(+) T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4(+) T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the "Shock and Kill" strategy for latently HIV-1 infected cells. PMID:26184775

  15. Dynamic Epstein-Barr virus gene expression on the path to B-cell transformation.

    PubMed

    Price, Alexander M; Luftig, Micah A

    2014-01-01

    Epstein-Barr virus (EBV) is an oncogenic human herpesvirus in the γ-herpesvirinae subfamily that contains a 170-180kb double-stranded DNA genome. In vivo, EBV commonly infects B and epithelial cells and persists for the life of the host in a latent state in the memory B-cell compartment of the peripheral blood. EBV can be reactivated from its latent state, leading to increased expression of lytic genes that primarily encode for enzymes necessary to replicate the viral genome and structural components of the virion. Lytic cycle proteins also aid in immune evasion, inhibition of apoptosis, and the modulation of other host responses to infection. In vitro, EBV has the potential to infect primary human B cells and induce cellular proliferation to yield effectively immortalized lymphoblastoid cell lines, or LCLs. EBV immortalization of B cells in vitro serves as a model system for studying EBV-mediated lymphomagenesis. While much is known about the steady-state viral gene expression within EBV-immortalized LCLs and other EBV-positive cell lines, relatively little is known about the early events after primary B-cell infection. It was previously thought that upon latent infection, EBV only expressed the well-characterized latency-associated transcripts found in LCLs. However, recent work has characterized the early, but transient, expression of lytic genes necessary for efficient transformation and delayed responses in the known latency genes. This chapter summarizes these recent findings that show how dynamic and controlled expression of multiple EBV genes can control the activation of B cells, entry into the cell cycle, the inhibition of apoptosis, and innate and adaptive immune responses. PMID:24373315

  16. Dynamic Epstein-Barr Virus Gene Expression on the Path to B-Cell Transformation

    PubMed Central

    Price, Alexander M.; Luftig, Micah A.

    2016-01-01

    Epstein-Barr Virus is an oncogenic human herpesvirus in the γ-herpesvirinae sub-family that contains a 170–180 kb double stranded DNA genome. In vivo, EBV commonly infects B and epithelial cells and persists for the life of the host in a latent state in the memory B cell compartment of the peripheral blood. EBV can be reactivated from its latent state leading to increased expression of lytic genes that primarily encode for enzymes necessary to replicate the viral genome as well as structural components of the virion. Lytic cycle proteins also aid in immune evasion, inhibition of apoptosis, and the modulation of other host responses to infection. In vitro, EBV has the potential to infect primary human B cells and induce cellular proliferation to yield effectively immortalized lymphoblastoid cell lines, or LCLs. EBV immortalization of B cells in vitro serves as a model system for studying EBV-mediated lymphomagenesis. While much is known about the steady state viral gene expression within EBV immortalized LCLs and other EBV-positive cell lines, relatively little is known about the early events after primary B-cell infection. It was previously thought that upon latent infection EBV only expressed the well-characterized latency associated transcripts found in LCLs. However, recent work has characterized the early, but transient, expression of lytic genes necessary for efficient transformation as well as delayed responses in the known latency genes. This review summarizes these recent findings that show how dynamic and controlled expression of multiple EBV genes can control the activation of B cells, entry into the cell cycle, inhibition of apoptosis, and control of innate and adaptive immune responses. PMID:24373315

  17. Early gene expression changes by Epstein-Barr virus infection of B-cells indicate CDKs and survivin as therapeutic targets for post-transplant lymphoproliferative diseases.

    PubMed

    Bernasconi, Michele; Ueda, Seigo; Krukowski, Patricia; Bornhauser, Beat C; Ladell, Kristin; Dorner, Marcus; Sigrist, Juerg A; Campidelli, Cristina; Aslandogmus, Roberta; Alessi, Davide; Berger, Christoph; Pileri, Stefano A; Speck, Roberto F; Nadal, David

    2013-11-15

    Lymphoproliferative diseases (LPDs) associated with Epstein-Barr virus (EBV) infection cause significant morbidity and mortality in bone marrow and solid organ transplant recipients. To gain insight into LPD pathogenesis and to identify potential effective therapeutic approaches, we investigated early molecular events leading to B-cell transformation by gene expression profiling of EBV-infected B-cells from tonsils by Affymetrix microarray 72 hr postinfection when the B-cells hyperproliferation phase starts. Cell cycle and apoptosis were the most significantly affected pathways and enriched gene sets. In particular, we found significantly increased expression of cyclin-dependent kinase (CDK)1 and CCNB1 (cyclin B1) and of one of their downstream targets BIRC5 (survivin). Importantly, the strong upregulation of the antiapoptotic protein survivin was confirmed in lymphoblastoid cell lines (LCLs) and 71% of EBV-positive post-transplant EBV-LPD lesions scored positive for survivin. The validity of early transforming events for the identification of therapeutic targets for EBV-LPD was confirmed by the marked antiproliferative effect of the CDK inhibitor flavopiridol on LCLs and by the strong induction of apoptosis by survivin inhibition with YM155 or terameprocol. Our results suggest that targeting of CDKs and/or survivin in post-transplant EBV-LPD by specific inhibitors might be an important approach to control and eliminate EBV-transformed B-cells that should be further considered. PMID:23640782

  18. Multiple Sclerosis Risk Allele in CLEC16A Acts as an Expression Quantitative Trait Locus for CLEC16A and SOCS1 in CD4+ T Cells

    PubMed Central

    Gustavsen, Marte W.; Bjølgerud, Anja; Brorson, Ina S.; Celius, Elisabeth G.; Spurkland, Anne; Bos, Steffan D.; Harbo, Hanne F.; Berge, Tone

    2015-01-01

    For multiple sclerosis, genome wide association studies and follow up studies have identified susceptibility single nucleotide polymorphisms located in or near CLEC16A at chromosome 16p13.13, encompassing among others CIITA, DEXI and SOCS1 in addition to CLEC16A. These genetic variants are located in intronic or intergenic regions and display strong linkage disequilibrium with each other, complicating the understanding of their functional contribution and the identification of the direct causal variant(s). Previous studies have shown that multiple sclerosis-associated risk variants in CLEC16A act as expression quantitative trait loci for CLEC16A itself in human pancreatic β-cells, for DEXI and SOCS1 in thymic tissue samples, and for DEXI in monocytes and lymphoblastoid cell lines. Since T cells are major players in multiple sclerosis pathogenesis, we have performed expression analyses of the CIITA-DEXI-CLEC16A-SOCS1 gene cluster in CD4+ and CD8+ T cells isolated from multiple sclerosis patients and healthy controls. We observed a higher expression of SOCS1 and CLEC16A in CD4+ T cells in samples homozygous for the risk allele of CLEC16A rs12927355. Pair-wise linear regression analysis revealed high correlation in gene expression in peripheral T cells of CIITA, DEXI, CLEC16A and SOCS1. Our data imply a possible regulatory role for the multiple sclerosis-associated rs12927355 in CLEC16A. PMID:26203907

  19. A predictive toxicogenomics signature to classify genotoxic versus non-genotoxic chemicals in human TK6 cells

    PubMed Central

    Williams, Andrew; Buick, Julie K.; Moffat, Ivy; Swartz, Carol D.; Recio, Leslie; Hyduke, Daniel R.; Li, Heng-Hong; Fornace, Albert J.; Aubrecht, Jiri; Yauk, Carole L.

    2015-01-01

    Genotoxicity testing is a critical component of chemical assessment. The use of integrated approaches in genetic toxicology, including the incorporation of gene expression data to determine the DNA damage response pathways involved in response, is becoming more common. In companion papers previously published in Environmental and Molecular Mutagenesis, Li et al. (2015) [6] developed a dose optimization protocol that was based on evaluating expression changes in several well-characterized stress-response genes using quantitative real-time PCR in human lymphoblastoid TK6 cells in culture. This optimization approach was applied to the analysis of TK6 cells exposed to one of 14 genotoxic or 14 non-genotoxic agents, with sampling 4 h post-exposure. Microarray-based transcriptomic analyses were then used to develop a classifier for genotoxicity using the nearest shrunken centroids method. A panel of 65 genes was identified that could accurately classify toxicants as genotoxic or non-genotoxic. In Buick et al. (2015) [1], the utility of the biomarker for chemicals that require metabolic activation was evaluated. In this study, TK6 cells were exposed to increasing doses of four chemicals (two genotoxic that require metabolic activation and two non-genotoxic chemicals) in the presence of rat liver S9 to demonstrate that S9 does not impair the ability to classify genotoxicity using this genomic biomarker in TK6cells. PMID:26425668

  20. DNA Electrophoretic Migration Patterns Change after Exposure of Jurkat Cells to a Single Intense Nanosecond Electric Pulse

    PubMed Central

    Romeo, Stefania; Zeni, Luigi; Sarti, Maurizio; Sannino, Anna; Scarfì, Maria Rosaria; Vernier, P. Thomas; Zeni, Olga

    2011-01-01

    Intense nanosecond pulsed electric fields (nsPEFs) interact with cellular membranes and intracellular structures. Investigating how cells respond to nanosecond pulses is essential for a) development of biomedical applications of nsPEFs, including cancer therapy, and b) better understanding of the mechanisms underlying such bioelectrical effects. In this work, we explored relatively mild exposure conditions to provide insight into weak, reversible effects, laying a foundation for a better understanding of the interaction mechanisms and kinetics underlying nsPEF bio-effects. In particular, we report changes in the nucleus of Jurkat cells (human lymphoblastoid T cells) exposed to single pulses of 60 ns duration and 1.0, 1.5 and 2.5 MV/m amplitudes, which do not affect cell growth and viability. A dose-dependent reduction in alkaline comet-assayed DNA migration is observed immediately after nsPEF exposure, accompanied by permeabilization of the plasma membrane (YO-PRO-1 uptake). Comet assay profiles return to normal within 60 minutes after pulse delivery at the highest pulse amplitude tested, indicating that our exposure protocol affects the nucleus, modifying DNA electrophoretic migration patterns. PMID:22164287

  1. Spontaneous expression of a low affinity Fc receptor for IgA (Fc alpha R) on human B cell lines.

    PubMed Central

    Millet, I; Briere, F; Vincent, C; Rousset, F; Andreoni, C; De Vries, J E; Revillard, J P

    1989-01-01

    Expression of receptors for IgA (Fc alpha Rs) was investigated on a panel of 35 human B cell lines by labelling with human secretory IgA (0.5 mg/ml) and flow cytometry analysis after staining with fluoresceinated goat anti-human secretory component and/or anti-alpha chain F(ab')2 fragments. Receptors for IgA could be demonstrated on one out of nine Burkitt's lymphoma cell lines, three out of five myeloma cell lines and five out of 21 lymphoblastoid cell lines. The percentage of Fc alpha R-positive cells within the same B cell line varied upon repeated examination. Human dimeric IgA1 lambda myeloma protein revealed the same number of IgA receptor positive cells as did secretory IgA, whereas monomeric IgA did not bind to Fc alpha R. Detection of Fc alpha R was not inhibited when the tests were carried out in the presence of human dimeric IgG, IgM, asialo-orosomucoid, and secretory component but it was abrogated by pre-treatment of the cells with trypsin. The binding characteristics of Fc alpha Rs were studied on the myeloma cell line Esteve, using 125I-labelled human dimeric IgA and secretory IgA. The binding was dose-dependent with rapid kinetics and specific inhibition by unlabelled secretory IgA. Scatchard plot analysis resulted in an equilibrium constant K ranging from 3.2 to 4.7 x 10(6) M/l. No correlation was observed between Fc alpha R expression and differentiation stage, monoclonality, polyclonality of the cell lines, or Ig class produced by the B cells. PMID:2788048

  2. Fatal B-cell lymphoproliferative syndrome in allogeneic marrow graft recipients. A clinical, immunobiological and pathological study.

    PubMed

    Simon, M; Bartram, C R; Friedrich, W; Arnold, R; Schmeiser, T; Hampl, W; Müller-Hermelink, H K; Heymer, B

    1991-01-01

    We have studied four cases of fatal B-cell lymphoproliferative syndrome (LPS) developing among 333 patients (incidence 1.2%) treated with allogeneic bone marrow transplantation (BMT). All four patients had received a T-cell depleted graft. Onset of the first clinical symptoms (palpable lymph node enlargement in three and IgA-lambda paraproteinemia in two patients) occurred between 41 and 188 days post-BMT (median 76 days). The course of the LPS was rapidly progressive in all cases, leading to death in 2-5 weeks. The peripheral blood showed progressive pancytopenia with disproportionally high numbers of activated NK cells, apparently compensating for the T-cell deficiency. Post-mortem histological studies disclosed polymorphic B-cell proliferations, most pronounced in the lymph nodes, spleen, liver, lungs and kidneys. Lymphohemopoietic cells were of donor origin in three patients. In the fourth patient, graft failure suggested a host origin for the proliferating cells. Immunophenotyping and gene rearrangement analysis revealed polyclonal proliferation in one patient, monoclonal proliferation in another patient, and an oligoclonal pattern in the other two patients. The clinical behavior of the LPS was independent of clonality. Immunohistologically, the proliferating cells showed characteristics of relatively mature B-cells in three cases, and pre-B-cell features in one case. Epstein Barr virus (EBV) serology indicated seroconversion (primary infection) in one child, and chronic active EBV infection in both adults. EBV DNA as well as EBV nuclear antigen (EBNA) were detected in infiltrated tissues of all four patients. The labeling pattern on in situ hybridization suggested a replicative EBV infection comparable to that in lymphoblastoid cell lines. We conclude that EBV-associated LPS developing as a result of post-transplant immunodeficiency is a distinct clinicopathologic entity, differing from non-Hodgkin's lymphoma (including Burkitt's lymphoma) and infectious

  3. Polyphenolic Profile and Targeted Bioactivity of Methanolic Extracts from Mediterranean Ethnomedicinal Plants on Human Cancer Cell Lines.

    PubMed

    Pollio, Antonino; Zarrelli, Armando; Romanucci, Valeria; Di Mauro, Alfredo; Barra, Federica; Pinto, Gabriele; Crescenzi, Elvira; Roscetto, Emanuela; Palumbo, Giuseppe

    2016-01-01

    The methanol extracts of the aerial part of four ethnomedicinal plants of Mediterranean region, two non-seed vascular plants, Equisetum hyemale L. and Phyllitis scolopendrium (L.) Newman, and two Spermatophyta, Juniperus communis L. (J. communis) and Cotinus coggygria Scop. (C. coggygria), were screened against four human cells lines (A549, MCF7, TK6 and U937). Only the extracts of J. communis and C. coggygria showed marked cytotoxic effects, affecting both cell morphology and growth. A dose-dependent effect of these two extracts was also observed on the cell cycle distribution. Incubation of all the cell lines in a medium containing J. communis extract determined a remarkable accumulation of cells in the G2/M phase, whereas the C. coggygria extract induced a significant increase in the percentage of G1 cells. The novelty of our findings stands on the observation that the two extracts, consistently, elicited coherent effects on the cell cycle in four cell lines, independently from their phenotype, as two of them have epithelial origin and grow adherent and two are lymphoblastoid and grow in suspension. Even the expression profiles of several proteins regulating cell cycle progression and cell death were affected by both extracts. LC-MS investigation of methanol extract of C. coggygria led to the identification of twelve flavonoids (compounds 1-11, 19) and eight polyphenols derivatives (12-18, 20), while in J. communis extract, eight flavonoids (21-28), a α-ionone glycoside (29) and a lignin (30) were found. Although many of these compounds have interesting individual biological activities, their natural blends seem to exert specific effects on the proliferation of cell lines either growing adherent or in suspension, suggesting potential use in fighting cancer. PMID:27023497

  4. Involvement of recombination in x-ray mutagenesis of human cells

    SciTech Connect

    Amundson, S.A. ); Xia, F.; Liber, H.L. )

    1993-01-01

    Closely related human lymphoblastoid cell lines derived from WI-L2 differ greatly in their responses to X-irradiation. Compared with TK6 (ATCC CRL 8015), WI-L2-NS (ATCC CRL 8155) has an enhanced X-ray survival. The induction of mutation by X-rays is also markedly different. The hemizygous hprt locus is slightly more mutable in WI-L2-NS than in TK6, and the dose response fits best to a linear-quadratic curve rather than the linear fit of TK6X-ray induced mutation at the autosomal tk locus in heterozygotes derived from WI-L2-NS is 20-50 fold higher than in heterozygotes derived from TK6. A larger proportion of WI-L2-NS mutants had lost heterozygosity compared with mutants of TK6. , Fluorescence in situ hybridization indicated that loss of heterozygosity was due almost uniformly to deletion of an allele in mutants of TK6, and to recombination or gene conversion in mutants of WI-L2-NS. These results indicate that recombinational repair contributes to both cell survival and mutation following exposure to ionizing radiation.

  5. Proteomic profiling of ATM kinase proficient and deficient cell lines upon blockage of proteasome activity☆

    PubMed Central

    Marzano, Valeria; Santini, Simonetta; Rossi, Claudia; Zucchelli, Mirco; D'Alessandro, Annamaria; Marchetti, Carlo; Mingardi, Michele; Stagni, Venturina; Barilà, Daniela; Urbani, Andrea

    2012-01-01

    Ataxia Telangiectasia Mutated (ATM) protein kinase is a key effector in the modulation of the functionality of some important stress responses, including DNA damage and oxidative stress response, and its deficiency is the hallmark of Ataxia Telangiectasia (A-T), a rare genetic disorder. ATM modulates the activity of hundreds of target proteins, essential for the correct balance between proliferation and cell death. The aim of this study is to evaluate the phenotypic adaptation at the protein level both in basal condition and in presence of proteasome blockage in order to identify the molecules whose level and stability are modulated through ATM expression. We pursued a comparative analysis of ATM deficient and proficient lymphoblastoid cells by label-free shotgun proteomic experiments comparing the panel of proteins differentially expressed. Through a non-supervised comparative bioinformatic analysis these data provided an insight on the functional role of ATM deficiency in cellular carbohydrate metabolism's regulation. This hypothesis has been demonstrated by targeted metabolic fingerprint analysis SRM (Selected Reaction Monitoring) on specific thermodynamic checkpoints of glycolysis. This article is part of a Special Issue entitled: Translational Proteomics. PMID:22641158

  6. Bioenergetic and Antiapoptotic Properties of Mitochondria from Cultured Human Prostate Cancer Cell Lines PC-3, DU145 and LNCaP

    PubMed Central

    Panov, Alexander; Orynbayeva, Zulfiya

    2013-01-01

    The purpose of this work was to reveal the metabolic features of mitochondria that might be essential for inhibition of apoptotic potential in prostate cancer cells. We studied mitochondria isolated from normal prostate epithelial cells (PrEC), metastatic prostate cancer cell lines LNCaP, PC-3, DU145; and non-prostate cancer cells - human fibrosarcoma HT1080 cells; and normal human lymphoblastoid cells. PrEC cells contained 2 to 4 times less mitochondria per gram of cells than the three PC cell lines. Respiratory activities of PrEC cell mitochondria were 5-20-fold lower than PC mitochondria, depending on substrates and the metabolic state, due to lower content and lower activity of the respiratory enzyme complexes. Mitochondria from the three metastatic prostate cancer cell lines revealed several features that are distinctive only to these cells: low affinity of Complex I for NADH, 20-30 mV higher electrical membrane potential (ΔΨ). Unprotected with cyclosporine A (CsA) the PC-3 mitochondria required 4 times more Ca2+ to open the permeability transition pore (mPTP) when compared with the PrEC mitochondria, and they did not undergo swelling even in the presence of alamethicin, a large pore forming antibiotic. In the presence of CsA, the PC-3 mitochondria did not open spontaneously the mPTP. We conclude that the low apoptotic potential of the metastatic PC cells may arise from inhibition of the Ca2+-dependent permeability transition due to a very high ΔΨ and higher capacity to sequester Ca2+. We suggest that due to the high ΔΨ, mitochondrial metabolism of the metastatic prostate cancer cells is predominantly based on utilization of glutamate and glutamine, which may promote development of cachexia. PMID:23951286

  7. Measurement of CD8+ and CD4+ T Cell Frequencies Specific for EBV LMP1 and LMP2a Using mRNA-Transfected DCs.

    PubMed

    Sohn, Dae-Hee; Sohn, Hyun-Jung; Lee, Hyun-Joo; Lee, Seon-Duk; Kim, Sueon; Hyun, Seung-Joo; Cho, Hyun-Il; Cho, Seok-Goo; Lee, Suk-Kyeong; Kim, Tai-Gyu

    2015-01-01

    An EBV-specific cellular immune response is associated with the control of EBV-associated malignancies and lymphoproliferative diseases, some of which have been successfully treated by adoptive T cell therapy. Therefore, many methods have been used to measure EBV-specific cellular immune responses. Previous studies have mainly used autologous EBV-transformed B-lymphoblastoid cell lines (B-LCLs), recombinant viral vectors transfected or peptide pulsed dendritic cells (DCs) as stimulators of CD8(+) and CD4(+) T lymphocytes. In the present study, we used an interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay by using isolated CD8(+) and CD4(+) T cells stimulated with mRNA-transfected DCs. The frequency of latent membrane protein 1 (LMP1)-specific IFN-γ producing CD4(+) T cells was significantly higher than that of LMP2a. The frequency of IFN-γ producing CD4(+) T cells was significantly correlated with that of CD8(+) T cells in LMP1-specific immune responses (r = 0.7187, Pc < 0.0001). To determine whether there were changes in LMP1- or LMP2a-specific immune responses, subsequent peripheral blood mononuclear cells (PBMCs) samples were analyzed. Significant changes were observed in 5 of the 10 donors examined, and CD4(+) T cell responses showed more significant changes than CD8(+) T cell responses. CD8(+) and CD4(+) T cells from EBV-seropositive donors secreted only the Th1 cytokines IFN-γ, TNF-α, and IL-2, while Th2 (IL-4) and Th17 (IL-17a) cytokines were not detected. CD4(+) T cells secreted significantly higher cytokine levels than did CD8(+) T cells. Analysis of EBV-specific T cell responses using autologous DCs transfected with mRNA might provide a comprehensive tool for monitoring EBV infection and new insights into the pathogenesis of EBV-associated diseases. PMID:26023769

  8. Rare Circulating Cells in Familial Waldenström Macroglobulinemia Displaying the MYD88 L265P Mutation Are Enriched by Epstein-Barr Virus Immortalization

    PubMed Central

    Pertesi, Maroulio; Galia, Perrine; Nazaret, Nicolas; Vallée, Maxime; Garderet, Laurent; Leleu, Xavier; Avet-Loiseau, Hervé; Foll, Matthieu; Byrnes, Graham; Lachuer, Joel; McKay, James D.; Dumontet, Charles

    2015-01-01

    The MYD88 L265P is a recurrent somatic mutation in neoplastic cells from patients with Waldenström Macroglobulinemia (WM). We identified the MYD88 L265P mutation in three individuals from unrelated families, but its presence did not explain the disease segregation within these WM pedigrees. We observed the mutation in these three individuals at high allele fractions in DNA extracted from EBV-immortalized Lymphoblastoid cell lines established from peripheral blood (LCL), but at much lower allele fractions in DNA extracted directly from peripheral blood, suggesting that this mutation is present in a clonal cell subpopulation rather than of germ-line origin. Furthermore, we observed that the MYD88 L265P mutation is enriched in WM families, detected in 40.5% of patients with familial WM or MGUS (10/22 WM, 5/15 MGUS), compared to 3.5% of patients with familial MM or MGUS (0/72 MM, 4/41 MGUS) (p = 10−7). The mutant allele frequency increased with passages in vitro after immortalization with Epstein-Barr virus (EBV) consistent with the MYD88 L265P described gain-of-function proposed for this mutation. The MYD88 L265P mutation appears to be frequently present in circulating cells in patients with WM, and MGUS, and these cells are amenable to immortalization by EBV. PMID:26352266

  9. A cellular genome-wide association study reveals human variation in microtubule stability and a role in inflammatory cell death

    PubMed Central

    Salinas, Raul E.; Ogohara, Cassandra; Thomas, Monica I.; Shukla, Kajal P.; Miller, Samuel I.; Ko, Dennis C.

    2014-01-01

    Pyroptosis is proinflammatory cell death that occurs in response to certain microbes. Activation of the protease caspase-1 by molecular platforms called inflammasomes is required for pyroptosis. We performed a cellular genome-wide association study (GWAS) using Salmonella typhimurium infection of human lymphoblastoid cell lines as a means of dissecting the genetic architecture of susceptibility to pyroptosis and identifying unknown regulatory mechanisms. Cellular GWAS revealed that a common human genetic difference that regulates pyroptosis also alters microtubule stability. An intergenic single-nucleotide polymorphism on chromosome 18 is associated with decreased pyroptosis and increased expression of TUBB6 (tubulin, β 6 class V). TUBB6 is unique among tubulin isoforms in that its overexpression can completely disrupt the microtubule network. Cells from individuals with higher levels of TUBB6 expression have lower microtubule stability and less pyroptosis. Reducing TUBB6 expression or stabilizing microtubules pharmacologically with paclitaxel (Taxol) increases pyroptosis without affecting the other major readout of caspase-1 activation, interleukin-1β secretion. The results reveal a new role for microtubules and possibly specific tubulin isoforms in the execution of pyroptosis. Furthermore, the finding that there is common diversity in TUBB6 expression and microtubule stability could have broad consequences for other microtubule-dependent phenotypes, diseases, and pharmacological responses. PMID:24173717

  10. Epstein-Barr virus nuclear protein EBNA3C directly induces expression of AID and somatic mutations in B cells.

    PubMed

    Kalchschmidt, Jens S; Bashford-Rogers, Rachael; Paschos, Kostas; Gillman, Adam C T; Styles, Christine T; Kellam, Paul; Allday, Martin J

    2016-05-30

    Activation-induced cytidine deaminase (AID), the enzyme responsible for induction of sequence variation in immunoglobulins (Igs) during the process of somatic hypermutation (SHM) and also Ig class switching, can have a potent mutator phenotype in the development of lymphoma. Using various Epstein-Barr virus (EBV) recombinants, we provide definitive evidence that the viral nuclear protein EBNA3C is essential in EBV-infected primary B cells for the induction of AID mRNA and protein. Using lymphoblastoid cell lines (LCLs) established with EBV recombinants conditional for EBNA3C function, this was confirmed, and it was shown that transactivation of the AID gene (AICDA) is associated with EBNA3C binding to highly conserved regulatory elements located proximal to and upstream of the AICDA transcription start site. EBNA3C binding initiated epigenetic changes to chromatin at specific sites across the AICDA locus. Deep sequencing of cDNA corresponding to the IgH V-D-J region from the conditional LCL was used to formally show that SHM is activated by functional EBNA3C and induction of AID. These data, showing the direct targeting and induction of functional AID by EBNA3C, suggest a novel role for EBV in the etiology of B cell cancers, including endemic Burkitt lymphoma. PMID:27217538

  11. Cytotoxicity and cell cycle effects of novel indolo[2,3-b]quinoline derivatives.

    PubMed

    Humeniuk, Rita; Kaczmarek, Lukasz; Peczyńska-Czoch, Wanda; Marcinkowska, Ewa

    2003-01-01

    Cellular effects of novel indolo[2,3-b]quinoline derivatives were studied. These compounds are synthetic analogs of plant alkaloid neocryptolepine (5-methyl-5H-indolo[2,3-b]quinoline) present in extracts from Cryptolepis sanguinolenta. They are traditionally used in natural medicine in Central and West Africa. Previous molecular and computational studies indicated that these compounds were DNA intercalators and inhibitors of topoisomerase II. We have extended our studies on their mode of action to the cellular level. Past experiments have shown that these compounds were active in vitro against cell lines derived from solid tumors, so for the present studies we selected leukemic cell lines. Jurkat acute T cell, CCRF-CEM T lymphoblastoid, THP-1 acute monocytic, HL-60 acute promyelocytic leukemias, and HL-60/MX2 subline with reduced expression of topoisomerase II were used. We evaluated the cytotoxicity and cell cycle effects of the indolo[2,3-b]quinoline compounds. We also tested if these compounds were able to induce apoptosis in the cells. Our studies revealed that novel indolo[2,3-b]quinoline derivatives were more cytotoxic to all cell lines than etoposide (used as a reference topoisomerase II inhibitor), and that their cytotoxicity depended on the substituents introduced to the indolo[2,3-b]quinoline core. Surprisingly, our studies have shown that HL-60/MX2 cell line and also THP-1 cell line, resistant to etoposide, were susceptible to methyl- and methoxy-substituted indolo[2,3-b]quinoline derivatives. In parallel to the evaluation of cytotoxicity we studied cell cycle effects of these compounds. Treatment of HL-60 cells with etoposide in subcytotoxic concentrations resulted in a massive accumulation of the cells in the G2/M phase of the cell cycle. When we used subcytotoxic concentrations of our novel indolo[2,3-b]quinoline derivatives the cell cycle progression of HL-60 cells was not affected. Moreover, the cell cycle of HL-60/MX2 cells was not influenced by

  12. Human B-cell TNF-beta microheterogeneity.

    PubMed

    Benjamin, D; Kofler, G; Tschachler, E

    1992-02-01

    The production of TNF-alpha and TNF-beta by human B-cell lines was studied at both the molecular and biological levels. The 24 B-cell lines studied included EBV+ cell lines (n = 13), EBV- cell lines (n = 8), and AIDS-associated B-cell lines (AABCL) (n = 3) which are EBV+/HIV-. Whereas radioimmunoprecipitation using TNF-alpha antisera detected 17-kDa TNF-alpha as expected, similar studies with anti-TNF-beta antisera revealed TNF-beta microheterogeneity. In the AABCL three bands with approximate MW of 26, 24, and 22 kDa were detected under reducing conditions, and in the non-AABCL, two bands only with 26 and 22 kDa were observed. To determine whether the size heterogeneity of TNF-beta is due to glycosylation, TNF-beta deglycosylation studies were done in two AABCL (PA682BM-2, PA682PE-1) and one non-AABCL (IM-1178). As control, the normal lymphoblastoid B-cell line RPMI-1788, which is known to secrete TNF-beta with MW 25 and 20 kDa, has been used. Deglycosylation studies using N-glycanase + neuraminidase + O-glycanase reduced the various bands in all cell lines to one band with 18.6 kDa, which is compatible with the TNF-beta backbone. In attempt to determine whether the differential glycosylation of TNF has any functional significance, all 24 cell lines were studied for TNF secretion and for TNF neutralization by monoclonal antibodies and polyclonal antibodies to TNF-alpha and TNF-beta. Constitutive secretion of TNF-alpha and TNF-beta has been detected only in the three AABCL. Following activation with the tumor promoter teleocidin, the secretion of both TNFs has been triggered in 2/8 EBV- cell lines and in 8/13 EBV+ non-AABCL. Using rabbit polyclonal antibodies to human TNF-alpha and to human TNF-beta, only little if any neutralization of these TNFs has been shown. Our data suggest that the differences in glycosylation of B-cell-derived TNFs may account for the incomplete neutralization, and may influence the cytotoxic biological activity of this lymphokine. PMID

  13. CD19 regulation of human B cell responses. B cell proliferation and antibody secretion are inhibited or enhanced by ligation of the CD19 surface glycoprotein depending on the stimulating signal used.

    PubMed

    Callard, R E; Rigley, K P; Smith, S H; Thurstan, S; Shields, J G

    1992-05-15

    The regulation of human B cell proliferation and differentiation by the CD19 surface glycoprotein was investigated. As expected, proliferation induced by costimulation with anti-IgM plus IL-4 or IL-2, or with G28.8 antibody plus IL-4 was inhibited by antibody ligation of CD19. In contrast, proliferation of tonsillar B cells to mitogenic doses of PMA (5 ng/ml) or to EBV were enhanced, and proliferation of B cell lines to BCGF(low) was unaffected. Similarly, specific antibody responses by tonsillar B cells to influenza virus, and Ig secretion by the CESS lymphoblastoid cell line in response to IL-6 were inhibited, whereas polyclonal Ig production in response to EBV was enhanced. These results show that human B cell responses may be inhibited or enhanced by CD19 depending on the stimulating signal used. The difference in response to CD19 ligation did not depend on whether proliferation or differentiation was being measured, or whether stimulation was by surface Ig. In experiments using PMA as a T cell independent mitogen, it was found that ligation of CD19 inhibited proliferation of B cells costimulated with low doses of PMA plus G28.5 (CD40) antibody, but enhanced the response to higher (mitogenic) doses with or without costimulation with G28.5. The change from inhibition to enhancement occurred over a very small increase in PMA dose (0.5-1.0 ng/ml) that corresponded exactly to the lowest dose required for mitogenic activity. Finally, we showed that CD19 ligation inhibited the increase in surface expression of CD23, but not IgM, induced by IL-4, showing that CD19 ligation can have opposed effects on different responses to the same signal. Together our results suggest that CD19 activation of human B cells interacts with other signaling events to enhance or inhibit the subsequent response. PMID:1374445

  14. Gamma radiation alters cell cycle and induces apoptosis in p53 mutant E6.1 Jurkat cells.

    PubMed

    Ahmadianpour, Mohammad Reza; Abdolmaleki, Parviz; Mowla, Seyed Javad; Hosseinkhani, Saman

    2013-01-01

    This study aimed at investigating the effect of gamma radiation with 1.5, 3.0 and 7.5 Gy doses on apoptosis induction, cell cycle alteration and increment of amount of p-ATM (phosphorylated ATM) and p-E2F1 (phosphorylated E2F1) proteins in Jurkat T-lymphoblastoid E6.1 cells. Exposure of human p53 mutant Jurkat cells to gamma radiation resulted in apoptosis, which was detected by luminometric and flow cytometric analysis. Also, phosphorylated ATM (ataxia telangiectasia mutated) and E2F1 (elongation factor) proteins were detected by western blot analysis. Based on luminescence detection data the lethal dose of 7.5 Gy induced cell death 12 h after exposure (p<0.05) while sub-lethal doses of 1.5 and 3.0 Gy induced apoptosis 18 h after exposure (p<0.05). Flow cytometric analysis revealed a G2 arrest 24h after exposure to 3.0 and 7.5 Gy. This arrest was accompanied by cell death with an increasing rate of occurrence up to 72 h after exposure. Western blot analysis showed that 1 h after cell irradiation by 1.5, 3.0 and 7.5 Gy, the amount of p-ATM increased to its maximum rate and remained constant up to 6 h, and then it decreased. Moreover, the amount of phosphorylated E2F1 (Ser-31) increased 2 h after exposure to the same doses and remained constant up to 12 h after irradiation. Survival and cell division of treated Jurkat cells showed a decrease compared to the control group. We believe that ionizing radiation-induced DNA damage activates a p53-independent apoptosis pathway via back-up systems in which the phosphorylation of ATM and E2F1 proteins was involved. Thus, gamma radiation can induce apoptosis and cell cycle alteration in Jurkat cells via a P53-independent pathway. PMID:23079488

  15. Epstein-Barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells.

    PubMed Central

    Margolskee, R F; Kavathas, P; Berg, P

    1988-01-01

    Efficient transfection and expression of cDNA libraries in human cells has been achieved with an Epstein-Barr virus-based subcloning vector (EBO-pcD). The plasmid vector contains a resistance marker for hygromycin B to permit selection for transformed cells. The Epstein-Barr virus origin for plasmid replication (oriP) and the Epstein-Barr virus nuclear antigen gene have also been incorporated into the vector to ensure that the plasmids are maintained stably and extrachromosomally. Human lymphoblastoid cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10(6) to 10(7) independent transformants. Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells. Furthermore, since EBO-pcD plasmids are maintained as episomes at two to eight copies per cell, intact cDNA clones can be readily isolated from transformants and recovered by propagation in Escherichia coli. By using such vectors, human cells have been stably transformed with EBO-pcD-hprt to express hypoxanthine-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2 (CD8). Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10(6) total clones or one clone of EBO-pcD-Leu-2 per 2 x 10(4) total clones can be recovered intact from the transformed cells. The ability to directly select for expression of very rare EBO-pcD clones and to then recover these episomes should make it possible to clone certain genes where hybridization and immunological screening methods are not applicable but where a phenotype can be scored or selected in human cell lines. Images PMID:2841588

  16. NBS1 knockdown by small interfering RNA increases ionizing radiation mutagenesis and telomere association in human cells

    NASA Technical Reports Server (NTRS)

    Zhang, Ying; Lim, Chang U K.; Williams, Eli S.; Zhou, Junqing; Zhang, Qinming; Fox, Michael H.; Bailey, Susan M.; Liber, Howard L.

    2005-01-01

    Hypomorphic mutations which lead to decreased function of the NBS1 gene are responsible for Nijmegen breakage syndrome, a rare autosomal recessive hereditary disorder that imparts an increased predisposition to development of malignancy. The NBS1 protein is a component of the MRE11/RAD50/NBS1 complex that plays a critical role in cellular responses to DNA damage and the maintenance of chromosomal integrity. Using small interfering RNA transfection, we have knocked down NBS1 protein levels and analyzed relevant phenotypes in two closely related human lymphoblastoid cell lines with different p53 status, namely wild-type TK6 and mutated WTK1. Both TK6 and WTK1 cells showed an increased level of ionizing radiation-induced mutation at the TK and HPRT loci, impaired phosphorylation of H2AX (gamma-H2AX), and impaired activation of the cell cycle checkpoint regulating kinase, Chk2. In TK6 cells, ionizing radiation-induced accumulation of p53/p21 and apoptosis were reduced. There was a differential response to ionizing radiation-induced cell killing between TK6 and WTK1 cells after NBS1 knockdown; TK6 cells were more resistant to killing, whereas WTK1 cells were more sensitive. NBS1 deficiency also resulted in a significant increase in telomere association that was independent of radiation exposure and p53 status. Our results provide the first experimental evidence that NBS1 deficiency in human cells leads to hypermutability and telomere associations, phenotypes that may contribute to the cancer predisposition seen among patients with this disease.

  17. Cell Type-Dependent Changes in CdSe/ZnS Quantum Dot Uptake and Toxic Endpoints

    PubMed Central

    Soenen, Stefaan J.; Al-Ali, Abdullah; Brown, Andy; Hondow, Nicole; Wills, John; Jenkins, Gareth J. S.; Doak, Shareen H.

    2015-01-01

    Toxicity of nanoparticles (NPs) is often correlated with the physicochemical characteristics of the materials. However, some discrepancies are noted in in-vitro studies on quantum dots (QDs) with similar physicochemical properties. This is partly related to variations in cell type. In this study, we show that epithelial (BEAS-2B), fibroblast (HFF-1), and lymphoblastoid (TK6) cells show different biological responses following exposure to QDs. These cells represented the 3 main portals of NP exposure: bronchial, skin, and circulatory. The uptake and toxicity of negatively and positively charged CdSe:ZnS QDs of the same core size but with different surface chemistries (carboxyl or amine polymer coatings) were investigated in full and reduced serum containing media following 1 and 3 cell cycles. Following thorough physicochemical characterization, cellular uptake, cytotoxicity, and gross chromosomal damage were measured. Cellular damage mechanisms in the form of reactive oxygen species and the expression of inflammatory cytokines IL-8 and TNF-α were assessed. QDs uptake and toxicity significantly varied in the different cell lines. BEAS-2B cells demonstrated the highest level of QDs uptake yet displayed a strong resilience with minimal genotoxicity following exposure to these NPs. In contrast, HFF-1 and TK6 cells were more susceptible to toxicity and genotoxicity, respectively, as a result of exposure to QDs. Thus, this study demonstrates that in addition to nanomaterial physicochemical characterization, a clear understanding of cell type-dependent variation in uptake coupled to the inherently different capacities of the cell types to cope with exposure to these exogenous materials are all required to predict genotoxicity. PMID:25601991

  18. Cell type-dependent changes in CdSe/ZnS quantum dot uptake and toxic endpoints.

    PubMed

    Manshian, Bella B; Soenen, Stefaan J; Al-Ali, Abdullah; Brown, Andy; Hondow, Nicole; Wills, John; Jenkins, Gareth J S; Doak, Shareen H

    2015-04-01

    Toxicity of nanoparticles (NPs) is often correlated with the physicochemical characteristics of the materials. However, some discrepancies are noted in in-vitro studies on quantum dots (QDs) with similar physicochemical properties. This is partly related to variations in cell type. In this study, we show that epithelial (BEAS-2B), fibroblast (HFF-1), and lymphoblastoid (TK6) cells show different biological responses following exposure to QDs. These cells represented the 3 main portals of NP exposure: bronchial, skin, and circulatory. The uptake and toxicity of negatively and positively charged CdSe:ZnS QDs of the same core size but with different surface chemistries (carboxyl or amine polymer coatings) were investigated in full and reduced serum containing media following 1 and 3 cell cycles. Following thorough physicochemical characterization, cellular uptake, cytotoxicity, and gross chromosomal damage were measured. Cellular damage mechanisms in the form of reactive oxygen species and the expression of inflammatory cytokines IL-8 and TNF-α were assessed. QDs uptake and toxicity significantly varied in the different cell lines. BEAS-2B cells demonstrated the highest level of QDs uptake yet displayed a strong resilience with minimal genotoxicity following exposure to these NPs. In contrast, HFF-1 and TK6 cells were more susceptible to toxicity and genotoxicity, respectively, as a result of exposure to QDs. Thus, this study demonstrates that in addition to nanomaterial physicochemical characterization, a clear understanding of cell type-dependent variation in uptake coupled to the inherently different capacities of the cell types to cope with exposure to these exogenous materials are all required to predict genotoxicity. PMID:25601991

  19. Epstein-Barr virus infection of naïve B cells in vitro frequently selects clones with mutated immunoglobulin genotypes: implications for virus biology.

    PubMed

    Heath, Emily; Begue-Pastor, Noelia; Chaganti, Sridhar; Croom-Carter, Debbie; Shannon-Lowe, Claire; Kube, Dieter; Feederle, Regina; Delecluse, Henri-Jacques; Rickinson, Alan B; Bell, Andrew I

    2012-01-01

    Epstein-Barr virus (EBV), a lymphomagenic human herpesvirus, colonises the host through polyclonal B cell-growth-transforming infections yet establishes persistence only in IgD⁺ CD27⁺ non-switched memory (NSM) and IgD⁻ CD27⁺ switched memory (SM) B cells, not in IgD⁺ CD27⁻ naïve (N) cells. How this selectivity is achieved remains poorly understood. Here we show that purified N, NSM and SM cell preparations are equally transformable in vitro to lymphoblastoid cells lines (LCLs) that, despite upregulating the activation-induced cytidine deaminase (AID) enzyme necessary for Ig isotype switching and Ig gene hypermutation, still retain the surface Ig phenotype of their parental cells. However, both N- and NSM-derived lines remain inducible to Ig isotype switching by surrogate T cell signals. More importantly, IgH gene analysis of N cell infections revealed two features quite distinct from parallel mitogen-activated cultures. Firstly, following 4 weeks of EBV-driven polyclonal proliferation, individual clonotypes then become increasingly dominant; secondly, in around 35% cases these clonotypes carry Ig gene mutations which both resemble AID products and, when analysed in prospectively-harvested cultures, appear to have arisen by sequence diversification in vitro. Thus EBV infection per se can drive at least some naïve B cells to acquire Ig memory genotypes; furthermore, such cells are often favoured during an LCL's evolution to monoclonality. Extrapolating to viral infections in vivo, these findings could help to explain how EBV-infected cells become restricted to memory B cell subsets and why EBV-driven lymphoproliferative lesions, in primary infection and/or immunocompromised settings, so frequently involve clones with memory genotypes. PMID:22589726

  20. Epstein-Barr Virus Infection of Naïve B Cells In Vitro Frequently Selects Clones with Mutated Immunoglobulin Genotypes: Implications for Virus Biology

    PubMed Central

    Croom-Carter, Debbie; Shannon-Lowe, Claire; Kube, Dieter; Feederle, Regina; Delecluse, Henri-Jacques; Rickinson, Alan B.; Bell, Andrew I.

    2012-01-01

    Epstein-Barr virus (EBV), a lymphomagenic human herpesvirus, colonises the host through polyclonal B cell-growth-transforming infections yet establishes persistence only in IgD+ CD27+ non-switched memory (NSM) and IgD− CD27+ switched memory (SM) B cells, not in IgD+ CD27− naïve (N) cells. How this selectivity is achieved remains poorly understood. Here we show that purified N, NSM and SM cell preparations are equally transformable in vitro to lymphoblastoid cells lines (LCLs) that, despite upregulating the activation-induced cytidine deaminase (AID) enzyme necessary for Ig isotype switching and Ig gene hypermutation, still retain the surface Ig phenotype of their parental cells. However, both N- and NSM-derived lines remain inducible to Ig isotype switching by surrogate T cell signals. More importantly, IgH gene analysis of N cell infections revealed two features quite distinct from parallel mitogen-activated cultures. Firstly, following 4 weeks of EBV-driven polyclonal proliferation, individual clonotypes then become increasingly dominant; secondly, in around 35% cases these clonotypes carry Ig gene mutations which both resemble AID products and, when analysed in prospectively-harvested cultures, appear to have arisen by sequence diversification in vitro. Thus EBV infection per se can drive at least some naïve B cells to acquire Ig memory genotypes; furthermore, such cells are often favoured during an LCL's evolution to monoclonality. Extrapolating to viral infections in vivo, these findings could help to explain how EBV-infected cells become restricted to memory B cell subsets and why EBV-driven lymphoproliferative lesions, in primary infection and/or immunocompromised settings, so frequently involve clones with memory genotypes. PMID:22589726

  1. Enhanced B Cell Alloantigen Presentation and Its Epigenetic Dysregulation in Liver Transplant Rejection.

    PubMed

    Ningappa, M; Ashokkumar, C; Higgs, B W; Sun, Q; Jaffe, R; Mazariegos, G; Li, D; Weeks, D E; Subramaniam, S; Ferrell, R; Hakonarson, H; Sindhi, R

    2016-02-01

    T cell suppression prevents acute cellular rejection but causes life-threatening infections and malignancies. Previously, liver transplant (LTx) rejection in children was associated with the single-nucleotide polymorphism (SNP) rs9296068 upstream of the HLA-DOA gene. HLA-DOA inhibits B cell presentation of antigen, a potentially novel antirejection drug target. Using archived samples from 122 white pediatric LTx patients (including 77 described previously), we confirmed the association between rs9296068 and LTx rejection (p = 0.001, odds ratio [OR] 2.55). Next-generation sequencing revealed that the putative transcription factor (CCCTC binding factor [CTCF]) binding SNP locus rs2395304, in linkage disequilibrium with rs9296068 (D' 0.578, r(2) = 0.4), is also associated with LTx rejection (p = 0.008, OR 2.34). Furthermore, LTx rejection is associated with enhanced B cell presentation of donor antigen relative to HLA-nonidentical antigen in a novel cell-based assay and with a downregulated HLA-DOA gene in a subset of these children. In lymphoblastoid B (Raji) cells, rs2395304 coimmunoprecipitates with CTCF, and CTCF knockdown with morpholino antisense oligonucleotides enhances alloantigen presentation and downregulates the HLA-DOA gene, reproducing observations made with HLA-DOA knockdown and clinical rejection. Alloantigen presentation is suppressed by inhibitors of methylation and histone deacetylation, reproducing observations made during resolution of rejection. Enhanced donor antigen presentation by B cells and its epigenetic dysregulation via the HLA-DOA gene represent novel opportunities for surveillance and treatment of transplant rejection. PMID:26663361

  2. Characterization of Epstein-Barr virus-induced lymphoproliferation derived from human peripheral blood mononuclear cells transferred to severe combined immunodeficient mice.

    PubMed

    Okano, M; Taguchi, Y; Nakamine, H; Pirruccello, S J; Davis, J R; Beisel, K W; Kleveland, K L; Sanger, W G; Fordyce, R R; Purtilo, D T

    1990-09-01

    Mice with severe combined immunodeficiency (SCID) received 6 X 10(7) fresh human peripheral blood mononuclear cells (PBMC) intraperitoneally from Epstein-Barr virus (EBV)-seropositive and -seronegative donors. B95-8 EBV was inoculated intraperitoneally and intravenously to the mice 6 weeks after transfer of seronegative PBMC. Three of four mice transferred with PBMC from two EBV-seropositive donors and two of four mice from two EBV-seronegative donors inoculated with EBV developed fatal EBV-induced lymphoproliferative disease within 6 to 10 weeks. These tumors were oligoclonal or polyclonal by cytoplasmic immunoglobulin expression. Furthermore no consistent clonal chromosomal abnormalities were shown. Cell lines established from these tumors showed low cloning efficiency in soft agarose. In addition, latent membrane protein, B-lymphocyte activation antigen (CD23), and cell-adhesion molecules (ICAM-1, CD18) all were expressed in the EBV-positive infiltrating lymphoproliferative lesions in each mouse. These results suggest that lymphoid tumors are comparable to lymphoblastoid cell lines immortalized by EBV and are not malignant lymphomas such as Burkitt's lymphoma. This model may be useful for investigating mechanisms responsible for the growing numbers of lymphoproliferative diseases that are occurring in patients with inherited or acquired immunodeficiencies. PMID:1975985

  3. Endogenous DNA Damage and Risk of Testicular Germ Cell Tumors

    SciTech Connect

    Cook, M B; Sigurdson, A J; Jones, I M; Thomas, C B; Graubard, B I; Korde, L; Greene, M H; McGlynn, K A

    2008-01-18

    Testicular germ cell tumors (TGCT) are comprised of two histologic groups, seminomas and nonseminomas. We postulated that the possible divergent pathogeneses of these histologies may be partially explained by variable endogenous DNA damage. To assess our hypothesis, we conducted a case-case analysis of seminomas and nonseminomas using the alkaline comet assay to quantify single-strand DNA breaks and alkali-labile sites. The Familial Testicular Cancer study and the U.S. Radiologic Technologists cohort provided 112 TGCT cases (51 seminomas & 61 nonseminomas). A lymphoblastoid cell line was cultured for each patient and the alkaline comet assay was used to determine four parameters: tail DNA, tail length, comet distributed moment (CDM) and Olive tail moment (OTM). Odds ratios (OR) and 95% confidence intervals (95%CI) were estimated using logistic regression. Values for tail length, tail DNA, CDM and OTM were modeled as categorical variables using the 50th and 75th percentiles of the seminoma group. Tail DNA was significantly associated with nonseminoma compared to seminoma (OR{sub 50th percentile} = 3.31, 95%CI: 1.00, 10.98; OR{sub 75th percentile} = 3.71, 95%CI: 1.04, 13.20; p for trend=0.039). OTM exhibited similar, albeit statistically non-significant, risk estimates (OR{sub 50th percentile} = 2.27, 95%CI: 0.75, 6.87; OR{sub 75th percentile} = 2.40, 95%CI: 0.75, 7.71; p for trend=0.12) whereas tail length and CDM showed no association. In conclusion, the results for tail DNA and OTM indicate that endogenous DNA damage levels are higher in patients who develop nonseminoma compared with seminoma. This may partly explain the more aggressive biology and younger age-of-onset of this histologic subgroup compared with the relatively less aggressive, later-onset seminoma.

  4. Translation of branched-chain aminotransferase-1 transcripts is impaired in cells haploinsufficient for ribosomal protein genes.

    PubMed

    Pereboom, Tamara C; Bondt, Albert; Pallaki, Paschalina; Klasson, Tim D; Goos, Yvonne J; Essers, Paul B; Groot Koerkamp, Marian J A; Gazda, Hanna T; Holstege, Frank C P; Costa, Lydie Da; MacInnes, Alyson W

    2014-05-01

    Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome linked to mutations in ribosomal protein (RP) genes that result in the impaired proliferation of hematopoietic progenitor cells. The etiology of DBA is not completely understood; however, the ribosomal nature of the genes involved has led to speculation that these mutations may alter the landscape of messenger RNA (mRNA) translation. Here, we performed comparative microarray analysis of polysomal mRNA transcripts isolated from lymphoblastoid cell lines derived from DBA patients carrying various haploinsufficient mutations in either RPS19 or RPL11. Different spectrums of changes were observed depending on the mutant gene, with large differences found in RPS19 cells and very few in RPL11 cells. However, we find that the small number of altered transcripts in RPL11 overlap for the most part with those altered in RPS19 cells. We show specifically that levels of branched-chain aminotransferase-1 (BCAT1) transcripts are significantly decreased on the polysomes of both RPS19 and RPL11 cells and that translation of BCAT1 protein is especially impaired in cells with small RP gene mutations, and we provide evidence that this effect may be due in part to the unusually long 5'UTR of the BCAT1 transcript. The BCAT1 enzyme carries out the final step in the biosynthesis and the first step of degradation of the branched-chain amino acids leucine, isoleucine, and valine. Interestingly, several animal models of DBA have reported that leucine ameliorates the anemia phenotypes generated by RPS19 loss. Our study suggests that RP mutations affect the synthesis of specific proteins involved in regulating amino acid levels that are important for maintaining the normal proliferative capacity of hematopoietic cells. PMID:24463277

  5. Hematopoietic gene therapy restores thymidine phosphorylase activity in a cell culture and a murine model of MNGIE.

    PubMed

    Torres-Torronteras, J; Gómez, A; Eixarch, H; Palenzuela, L; Pizzorno, G; Hirano, M; Andreu, A L; Barquinero, J; Martí, R

    2011-08-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in the TYMP gene, which encodes thymidine phosphorylase (TP). TP dysfunction results in systemic thymidine (dThd) and deoxyuridine (dUrd) overload, which selectively impair mitochondrial DNA replication. Allogeneic hematopoietic transplantation has been used to treat MNGIE patients; however, this approach has serious adverse effects, including the toxicity of myeloablative conditioning, graft rejection and graft-versus-host disease. With the aim of testing the feasibility of gene therapy for MNGIE, we transduced TP-deficient B-lymphoblastoid cells from two MNGIE patients, with lentiviral vectors carrying a functional copy of the human TYMP DNA coding sequence. This restored TP activity in the cells, which reduced the excretion of dThd and dUrd and their concentrations when added in excess. Additionally, lentiviral-mediated hematopoietic gene therapy was used in partially myeloablated double Tymp/Upp1 knockout mice. In spite of the relatively low levels of molecular chimerism achieved, high levels of TP activity were observed in the peripheral blood of the transplanted mice, with a concomitant reduction of nucleoside concentrations. Our results suggest that hematopoietic gene therapy could be an alternative treatment for this devastating disorder in the future. PMID:21451581

  6. Neutron Exposures in Human Cells: Bystander Effect and Relative Biological Effectiveness

    PubMed Central

    Seth, Isheeta; Schwartz, Jeffrey L.; Stewart, Robert D.; Emery, Robert; Joiner, Michael C.; Tucker, James D.

    2014-01-01

    Bystander effects have been observed repeatedly in mammalian cells following photon and alpha particle irradiation. However, few studies have been performed to investigate bystander effects arising from neutron irradiation. Here we asked whether neutrons also induce a bystander effect in two normal human lymphoblastoid cell lines. These cells were exposed to fast neutrons produced by targeting a near-monoenergetic 50.5 MeV proton beam at a Be target (17 MeV average neutron energy), and irradiated-cell conditioned media (ICCM) was transferred to unirradiated cells. The cytokinesis-block micronucleus assay was used to quantify genetic damage in radiation-naïve cells exposed to ICCM from cultures that received 0 (control), 0.5, 1, 1.5, 2, 3 or 4 Gy neutrons. Cells grown in ICCM from irradiated cells showed no significant increase in the frequencies of micronuclei or nucleoplasmic bridges compared to cells grown in ICCM from sham irradiated cells for either cell line. However, the neutron beam has a photon dose-contamination of 5%, which may modulate a neutron-induced bystander effect. To determine whether these low doses of contaminating photons can induce a bystander effect, cells were irradiated with cobalt-60 at doses equivalent to the percent contamination for each neutron dose. No significant increase in the frequencies of micronuclei or bridges was observed at these doses of photons for either cell line when cultured in ICCM. As expected, high doses of photons induced a clear bystander effect in both cell lines for micronuclei and bridges (p<0.0001). These data indicate that neutrons do not induce a bystander effect in these cells. Finally, neutrons had a relative biological effectiveness of 2.0±0.13 for micronuclei and 5.8±2.9 for bridges compared to cobalt-60. These results may be relevant to radiation therapy with fast neutrons and for regulatory agencies setting standards for neutron radiation protection and safety. PMID:24896095

  7. Gene conversion is strongly induced in human cells by double-strand breaks and is modulated by the expression of BCL-XL

    SciTech Connect

    Wiese, Claudia; Pierce, Andrew J.; Gauny, Stacey S.; Jasin, Maria; Kronenberg, Amy

    2001-09-25

    Homology-directed repair (HDR) of DNA double-strand breaks (DSBs) is a well-established mechanism that contributes to the maintenance of genomic stability in rodent cells, and it has been assumed that HDR is of similar importance in the repair of DSBs in human cells. However, in addition to promoting genomic stability, some outcomes of homologous recombination can be deleterious, suggesting that factors exist to regulate HDR. We previously demonstrated that overexpression of BCL-2 or BCL-xL enhanced the frequency of x-ray-induced mutations involving the TK1 locus, including loss of heterozygosity (LOH) events presumed to arise by mitotic recombination. The present study was designed to test whether HDR is a prominent DSB repair pathway in human cells, and to directly determine whether ectopic expression of BCL-xL affects HDR. We used the B-lymphoblastoid cell line TK6, which expresses wild-type TP53 and resembles normal lymphocytes in undergoing apoptosis following! genotoxic stress. U sing isogenic derivatives of TK6 cells (TK6-neo, TK6-bcl-xL), we find that a DSB in an integrated HDR reporter stimulates gene conversion 40-50-fold in TK6-neo cells, demonstrating that a DSB can be efficiently repaired by gene conversion in human cells. Significantly, DSB-induced gene conversion events are 3- to 4-fold more frequent in BCL-xL overexpressing cells. The results demonstrate that HDR plays an important role in maintaining genomic integrity in human cells and that ectopic expression of BCL-xL enhances HDR of DSBs. To our knowledge, this is the first study to highlight a function for BCL-xL in modulating DSB repair in human cells.

  8. Space experiment "Rad Gene"-report 1; p53-Dependent gene expression in human cultured cells exposed to space environment

    NASA Astrophysics Data System (ADS)

    Takahashi, Akihisa; Ohnishi, Takeo; Suzuki, Hiromi; Omori, Katsunori; Seki, Masaya; Hashizume, Toko; Shimazu, Toru; Ishioka, Noriaki

    The space environment contains two major biologically significant influences: space radiations and microgravity. A p53 tumor suppressor protein plays a role as a guardian of the genome through the activity of p53-centered signal transduction pathways. The aim of this study was to clarify the biological effects of space radiations, microgravity and a space environment on the gene and protein expression of p53-dependent regulated genes. Space experiments were performed with two human cultured lymphoblastoid cell lines: one cells line (TSCE5) bears a wild-type p53 gene status, and another cells line (WTK1) bears a mutated p53 gene status. Un-der one gravity or microgravity condition, the cells were grown in the cell biology experimental facility (CBEF) of the International Space Station (ISS) for 8 days without experiencing the stress during launching and landing because the cells were frozen during these periods. Ground control samples also were cultured for 8 days in the CBEF on the ground during the same periods as space flight. Gene and protein expression was analyzed by using DNA chip (a 44k whole human genome microarray, Agilent Technologies Inc.) and protein chip (PanoramaTM Ab MicroArray, Sigma-Aldrich Co.), respectively. In addition, we analyzed the gene expression in cultured cells after space flight during 133 days with frozen condition. We report the results and discussion from the viewpoint of the functions of the up-regulated and down-regulated genes after an exposure to space radiations and/or microgravity. The initial goal of this space experiment was completely achieved. It is expected that data from this type of work will be helpful in designing physical protection from the deleterious effects of space radiations during long term stays in space.

  9. Interferon-induced 56,000 Mr protein and its mRNA in human cells: molecular cloning and partial sequence of the cDNA.

    PubMed Central

    Chebath, J; Merlin, G; Metz, R; Benech, P; Revel, M

    1983-01-01

    Treatment of responsive cells by interferons (IFNs) induces within a few hours a rise in the concentration of several proteins and mRNAs. In order to characterize these IFN-induced mRNA species, we have cloned in E. coli the cDNA made from a 17-18S poly(A)+ RNA of human fibroblastoid cells (SV80) treated with IFN-beta. We describe here a pBR322 recombinant plasmid (C56) which contains a 400 bp cDNA insert corresponding to a 18S mRNA species newly induced by IFN. The C56 mRNA codes for a 56,000 dalton protein easily detectable by hybridization-translation experiments. The sequence of 66 of the carboxy-terminal amino-acids of the protein can be deduced from the cDNA sequence. IFNs-alpha, beta or gamma are able to activate the expression of this gene in human fibroblasts as well as lymphoblastoid cells. The mRNA is not detectable without IFN; it reaches maximum levels (0.1% of the total poly(A)+ RNA) within 4-8 hrs and decreases after 16 hrs. Images PMID:6186990

  10. Receptors for B cell stimulatory factor 2. Quantitation, specificity, distribution, and regulation of their expression

    SciTech Connect

    Taga, T.; Kawanishi, Y.; Hardy, R.R.; Hirano, T.; Kishimoto, T.

    1987-10-01

    B cell stimulatory factor 2 receptors (BSF-2-R) were studied using radioiodinated recombinant BSF-2 with a specific activity of 6.16 X 10(13) cpm/g. Kinetic studies showed that binding of /sup 125/I-BSF-2 to CESS cells reached maximum level within 150 min at 0 degrees C. There was a single class of receptors with high affinity (Kd 3.4 X 10(-10) M) on CESS, and the number of receptors was 2700 per cell. Binding of /sup 125/I-BSF-2 to CESS was competitively inhibited by unlabeled BSF-2 but not by IL-1, IL-2, IFN-beta, IFN-gamma, and G-CSF, indicating the presence of the receptors specific for BSF-2. EBV-transformed B lymphoblastoid cell lines (CESS, SKW6-CL4, LCL13, and LCL14) expressed BSF-2-R, whereas Burkitt's lines did not. EBV or EBNA2 did not induce the expression of the receptors on Burkitt's cells. The plasma cell lines (ARH-77 and U266) expressed BSF-2-R, fitting the function of BSF-2 as plasma cell growth factor. Several other cell lines, the histiocytic line U937, the promyelocytic line HL60, the astrocytoma line U373 and the glioblastoma line SK-MG-4, in which BSF-2 was inducible with IL-1 or TPA, displayed BSF-2-R with Kd in the range of 1.3-6.4 X 10(-10) M, suggesting the autocrine mechanism in BSF-2 function. The four T cell lines (CEM, HSB, Jurkat, and OM 1) did not express a detectable number of receptors, but normal resting T cells expressed 100-1000 receptors per cell. BSF-2-R were not present on normal resting B cells but expressed on activated B cells with a Kd of 3.6-5.0 X 10(-10) M, fitting the function of BSF-2, which acts on B cells at the final maturation stage to induce immunoglobulin production.

  11. Identification of candidate genes involved in coronary artery calcification by transcriptome sequencing of cell lines

    PubMed Central

    2014-01-01

    Background Massively-parallel cDNA sequencing (RNA-Seq) is a new technique that holds great promise for cardiovascular genomics. Here, we used RNA-Seq to study the transcriptomes of matched coronary artery disease cases and controls in the ClinSeq® study, using cell lines as tissue surrogates. Results Lymphoblastoid cell lines (LCLs) from 16 cases and controls representing phenotypic extremes for coronary calcification were cultured and analyzed using RNA-Seq. All cell lines were then independently re-cultured and along with another set of 16 independent cases and controls, were profiled with Affymetrix microarrays to perform a technical validation of the RNA-Seq results. Statistically significant changes (p < 0.05) were detected in 186 transcripts, many of which are expressed at extremely low levels (5–10 copies/cell), which we confirmed through a separate spike-in control RNA-Seq experiment. Next, by fitting a linear model to exon-level RNA-Seq read counts, we detected signals of alternative splicing in 18 transcripts. Finally, we used the RNA-Seq data to identify differential expression (p < 0.0001) in eight previously unannotated regions that may represent novel transcripts. Overall, differentially expressed genes showed strong enrichment (p = 0.0002) for prior association with cardiovascular disease. At the network level, we found evidence for perturbation in pathways involving both cardiovascular system development and function as well as lipid metabolism. Conclusions We present a pilot study for transcriptome involvement in coronary artery calcification and demonstrate how RNA-Seq analyses using LCLs as a tissue surrogate may yield fruitful results in a clinical sequencing project. In addition to canonical gene expression, we present candidate variants from alternative splicing and novel transcript detection, which have been unexplored in the context of this disease. PMID:24628908

  12. c-myc activation renders proliferation of Epstein-Barr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1.

    PubMed Central

    Polack, A; Hörtnagel, K; Pajic, A; Christoph, B; Baier, B; Falk, M; Mautner, J; Geltinger, C; Bornkamm, G W; Kempkes, B

    1996-01-01

    Two genetic events contribute to the development of endemic Burkitt lymphoma (BL) infection of B lymphocytes with Epstein-Barr virus (EBV) and the activation of the protooncogene c-myc through chromosomal translocation. The viral genes EBV nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for transformation of primary human B cells by EBV in vitro; however, these genes are not expressed in BL cells in vivo. To address the question whether c-myc activation might abrogate the requirement of the EBNA2 and LMP1 function, we have introduced an activated c-myc gene into an EBV-transformed cell line in which EBNA2 was rendered estrogen-dependent through fusion with the hormone binding domain of the estrogen receptor. The c-myc gene was placed under the control of regulatory elements of the immunoglobulin kappa locus composed a matrix attachment region, the intron enhancer, and the 3' enhancer. We show here that transfection of a c-myc expression plasmid followed by selection for high MYC expression is capable of inducing continuous proliferation of these cells in the absence of functional EBNA2 and LMP1. c-myc-induced hormone-independent proliferation was associated with a dramatic change in the growth behavior as well as cell surface marker expression of these cells. The typical lymphoblastoid morphology and phenotype of EBV-transformed cells completely changed into that of BL cells in vivo. We conclude that the phenotype of BL cells reflects the expression pattern of viral and cellular genes rather than its germinal center origin. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8816814

  13. Measurement of DNA damage and apoptosis in Molt-4 cells after in vitro exposure to radiofrequency radiation.

    PubMed

    Hook, Graham J; Zhang, Peng; Lagroye, I; Li, Li; Higashikubo, Ryuji; Moros, Eduardo G; Straube, William L; Pickard, William F; Baty, Jack D; Roti Roti, Joseph L

    2004-02-01

    To determine whether exposure to radiofrequency (RF) radiation can induce DNA damage or apoptosis, Molt-4 T lymphoblastoid cells were exposed with RF fields at frequencies and modulations of the type used by wireless communication devices. Four types of frequency/modulation forms were studied: 847.74 MHz code-division multiple-access (CDMA), 835.62 MHz frequency-division multiple-access (FDMA), 813.56 MHz iDEN(R) (iDEN), and 836.55 MHz time-division multiple-access (TDMA). Exponentially growing cells were exposed to RF radiation for periods up to 24 h using a radial transmission line (RTL) exposure system. The specific absorption rates used were 3.2 W/kg for CDMA and FDMA, 2.4 or 24 mW/kg for iDEN, and 2.6 or 26 mW/kg for TDMA. The temperature in the RTLs was maintained at 37 degrees C +/- 0.3 degrees C. DNA damage was measured using the single-cell gel electrophoresis assay. The annexin V affinity assay was used to detect apoptosis. No statistically significant difference in the level of DNA damage or apoptosis was observed between sham-treated cells and cells exposed to RF radiation for any frequency, modulation or exposure time. Our results show that exposure of Molt-4 cells to CDMA, FDMA, iDEN or TDMA modulated RF radiation does not induce alterations in level of DNA damage or induce apoptosis. PMID:14731070

  14. Inhibitory Effect of Individual or Combinations of Broadly Neutralizing Antibodies and Antiviral Reagents against Cell-Free and Cell-to-Cell HIV-1 Transmission

    PubMed Central

    Kolodkin-Gal, Dror; Eslamizar, Leila; Owuor, Joshua O.; Mazzola, Emanuele; Gonzalez, Ana M.; Korioth-Schmitz, Birgit; Gelman, Rebecca S.; Montefiori, David C.; Haynes, Barton F.; Schmitz, Joern E.

    2015-01-01

    ABSTRACT To date, most therapeutic and vaccine candidates for human immunodeficiency virus type 1 (HIV-1) are evaluated preclinically for efficacy against cell-free viral challenges. However, cell-associated HIV-1 is suggested to be a major contributor to sexual transmission by mucosal routes. To determine if neutralizing antibodies or inhibitors block cell-free and cell-associated virus transmission of diverse HIV-1 strains with different efficiencies, we tested 12 different antibodies and five inhibitors against four green fluorescent protein (GFP)-labeled HIV-1 envelope (Env) variants from transmitted/founder (T/F) or chronic infection isolates. We evaluated antibody/inhibitor-mediated virus neutralization using either TZM-bl target cells, in which infectivity was determined by virus-driven luciferase expression, or A3R5 lymphoblastoid target cells, in which infectivity was evaluated by GFP expression. In both the TZM-bl and A3R5 assays, cell-free virus or infected CD4+ lymphocytes were used as targets for neutralization. We further hypothesized that the combined use of specific neutralizing antibodies targeting HIV-1 Env would more effectively prevent cell-associated virus transmission than the use of individual antibodies. The tested antibody combinations included two gp120-directed antibodies, VRC01 and PG9, or VRC01 with the gp41-directed antibody 10E8. Our results demonstrated that cell-associated virus was less sensitive to neutralizing antibodies and inhibitors, particularly using the A3R5 neutralization assay, and the potencies of these neutralizing agents differed among Env variants. A combination of different neutralizing antibodies that target specific sites on gp120 led to a significant reduction in cell-associated virus transmission. These assays will help identify ideal combinations of broadly neutralizing antibodies to use for passive preventive antibody administration and further characterize targets for the most effective neutralizing antibodies

  15. Constitutive Interferon-Inducible Protein 16-Inflammasome Activation during Epstein-Barr Virus Latency I, II, and III in B and Epithelial Cells

    PubMed Central

    Ansari, Mairaj Ahmed; Singh, Vivek Vikram; Dutta, Sujoy; Veettil, Mohanan Valiya; Dutta, Dipanjan; Chikoti, Leela; Lu, Jie; Everly, David

    2013-01-01

    Epstein-Barr virus (EBV), etiologically linked with human B-cell malignancies and nasopharyngeal carcinoma (NPC), establishes three types of latency that facilitate its episomal genome persistence and evasion of host immune responses. The innate inflammasome responses recognize the pathogen-associated molecular patterns which lead into the association of a cytoplasmic sensor such as NLRP3 and AIM2 proteins or nuclear interferon-inducible protein 16 (IFI16) with adaptor ASC protein (apoptosis-associated speck-like protein with a caspase recruitment domain) and effector procaspase-1, resulting in active caspase-1 formation which cleaves the proforms of inflammatory interleukin-1β (IL-1β), IL-18, and IL-33 cytokines. Whether inflammasome responses recognize and respond to EBV genome in the nuclei was not known. We observed evidence of inflammasome activation, such as the activation of caspase-1 and cleavage of pro-IL-1β, -IL-18, and -IL-33, in EBV latency I Raji cells, latency II NPC C666-1 cells, and latency III lymphoblastoid cell lines (LCL). Interaction between ASC with IFI16 but not with AIM2 or NLRP3 was detected in all three latencies and during EBV infection of primary human B cells. IFI16 and cleaved caspase-1, IL-1β, IL-18, and IL-33 were detected in the exosomes from Raji cells and LCL. Though EBV nuclear antigen 1 (EBNA1) and EBV-encoded small RNAs (EBERs) are common to all forms of EBV latency, caspase-1 cleavage was not detected in cells expressing EBNA1 alone, and blocking EBER transcription did not inhibit caspase-1 cleavage. In fluorescence in situ hybridization (FISH) analysis, IFI16 colocalized with the EBV genome in LCL and Raji cell nuclei. These studies demonstrated that constant sensing of latent EBV genome by IFI16 in all types of latency results in the constitutive induction of the inflammasome and IL-1β, IL-18, and IL-33 maturation. PMID:23720728

  16. The in vitro PIG-A gene mutation assay: glycosylphosphatidylinositol (GPI)-related genotype-to-phenotype relationship in TK6 cells.

    PubMed

    Krüger, Christopher T; Fischer, Bettina M; Armant, Olivier; Morath, Volker; Strähle, Uwe; Hartwig, Andrea

    2016-07-01

    In our previous work, we established an in vitro variant of the currently developed in vivo PIG-A assay as promising mutagenicity test system. We applied the human B-lymphoblastoid cell line TK6 for the in vitro assay development, which is based on the cellular glycosylphosphatidylinositol (GPI) status. At least 22 genes are involved in GPI biosynthesis, leading to the complex situation that, in principle, multiple genes could induce a GPI-deficient phenotype by acquiring inactivating mutations. However, only the PIG-A gene is located on the X-chromosome, rendering PIG-A more sensitive compared to autosomal linked, GPI-relevant genes. In this work, we investigated the GPI-related genotype-to-phenotype relationship in TK6 cells. By a next-generation sequencing approach, we identified a heterozygous chromosomal deletion on chromosome 17, where the PIG-L gene is located. In the analyzed TK6 cell clones, the GPI-deficient phenotype was induced either by mutations in PIG-A, by the complete absence of PIG-A mRNA, or by deletions in the remaining functional PIG-L gene, causing loss of heterozygosity. The identified PIG-L heterozygosity could also be responsible for the increased sensitivity toward mutagenic ethyl methanesulfonate or UV-C treatments of p53-proficient TK6 compared to the TK6-related, but p53-deficient WI-L2-NS cell line. Moreover, the WI-L2-NS cell line was found to exhibit a much lower number of GPI-deficient mutant cells in the purchased cell batch, and WI-L2-NS exerted a lower spontaneous rate of GPI deficiency compared to TK6 cells. PMID:27100116

  17. Constitutive interferon-inducible protein 16-inflammasome activation during Epstein-Barr virus latency I, II, and III in B and epithelial cells.

    PubMed

    Ansari, Mairaj Ahmed; Singh, Vivek Vikram; Dutta, Sujoy; Veettil, Mohanan Valiya; Dutta, Dipanjan; Chikoti, Leela; Lu, Jie; Everly, David; Chandran, Bala

    2013-08-01

    Epstein-Barr virus (EBV), etiologically linked with human B-cell malignancies and nasopharyngeal carcinoma (NPC), establishes three types of latency that facilitate its episomal genome persistence and evasion of host immune responses. The innate inflammasome responses recognize the pathogen-associated molecular patterns which lead into the association of a cytoplasmic sensor such as NLRP3 and AIM2 proteins or nuclear interferon-inducible protein 16 (IFI16) with adaptor ASC protein (apoptosis-associated speck-like protein with a caspase recruitment domain) and effector procaspase-1, resulting in active caspase-1 formation which cleaves the proforms of inflammatory interleukin-1β (IL-1β), IL-18, and IL-33 cytokines. Whether inflammasome responses recognize and respond to EBV genome in the nuclei was not known. We observed evidence of inflammasome activation, such as the activation of caspase-1 and cleavage of pro-IL-1β, -IL-18, and -IL-33, in EBV latency I Raji cells, latency II NPC C666-1 cells, and latency III lymphoblastoid cell lines (LCL). Interaction between ASC with IFI16 but not with AIM2 or NLRP3 was detected in all three latencies and during EBV infection of primary human B cells. IFI16 and cleaved caspase-1, IL-1β, IL-18, and IL-33 were detected in the exosomes from Raji cells and LCL. Though EBV nuclear antigen 1 (EBNA1) and EBV-encoded small RNAs (EBERs) are common to all forms of EBV latency, caspase-1 cleavage was not detected in cells expressing EBNA1 alone, and blocking EBER transcription did not inhibit caspase-1 cleavage. In fluorescence in situ hybridization (FISH) analysis, IFI16 colocalized with the EBV genome in LCL and Raji cell nuclei. These studies demonstrated that constant sensing of latent EBV genome by IFI16 in all types of latency results in the constitutive induction of the inflammasome and IL-1β, IL-18, and IL-33 maturation. PMID:23720728

  18. Cloning and partial nucleotide sequence of human immunoglobulin mu chain cDNA from B cells and mouse-human hybridomas.

    PubMed Central

    Dolby, T W; Devuono, J; Croce, C M

    1980-01-01

    Purified mRNAs coding for mu and kappa human immunoglobulin polypeptides were translated in vitro and their products were characterized. The mu-specific mRNAs, derived from both human lymphoblastoid cells (GM607) and from a mouse-human somatic cell hybrid secreting human mu chains (alpha D5-H11-BC11), were copied into cDNAs and inserted into the plasmid pBR322. Several recombinant cDNAs that were obtained were identified by a combination of colony hybridization with labeled probes, in vitro translation of plasmid-selected mu mRNAs, and DNA nucleotide sequence determination. One recombinant DNA, for which the sequence has been partially determined, contains the codons for part of the C3 constant region domain through the carboxy-terminal piece (155 amino acids total) as well as the entire 3' noncoding sequence up to the poly(A) site of the human mu mRNA. The sequence A-A-U-A-A occurs 12 nucleotides prior to the poly(A) addition site in the human mu mRNA. Considerable sequence homology is observed in the mouse and human mu mRNA 3' coding and noncoding sequences. Images PMID:6777778

  19. Decreased heat shock protein 27 expression and altered autophagy in human cells harboring A8344G mitochondrial DNA mutation.

    PubMed

    Chen, Chin-Yi; Chen, Hsueh-Fu; Gi, Siao-Jhen; Chi, Tang-Hao; Cheng, Che-Kun; Hsu, Chi-Fu; Ma, Yi-Shing; Wei, Yau-Huei; Liu, Chin-Shan; Hsieh, Mingli

    2011-09-01

    Mitochondrial DNA (mtDNA) mutations are responsible for human neuromuscular diseases caused by mitochondrial dysfunction. Myoclonus epilepsy associated with ragged-red fibers (MERRF) is a maternally inherited mitochondrial encephalomyopathy with various syndromes involving both muscular and nervous systems. The most common mutation in MERRF syndrome, A8344G mutation in mtDNA, has been associated with severe defects in protein synthesis. This defect impairs assembly of complexes in electron transport chain and results in decreased respiratory function of mitochondria. In this study, we showed a significant decrease of the heat shock protein 27 (Hsp27) in lymphoblastoid cells derived from a MERRF patient and in cybrid cells harboring MERRF A8344G mutation. However, normal cytoplasmic distributions of Hsp27 and normal heat shock responses were observed in both wild type and mutant cybrids. Furthermore, overexpression of wild type Hsp27 in mutant MERRF cybrids significantly decreased cell death under staurosporine (STS) treatment, suggesting a protective function of Hsp27 in cells harboring the A8344G mutation of mtDNA. Meanwhile, reverse transcriptase PCR showed no difference in the mRNA level between normal and mutant cybrids, indicating that alterations may occur at the protein level. Evidenced by the decreased levels of Hsp27 upon treatment with proteasome inhibitor, starvation and rapamycin and the accumulation of Hsp27 upon lysosomal inhibitor treatment; Hsp27 may be degraded by the autophagic pathway. In addition, the increased formation of LC3-II and autophagosomes was found in MERRF cybrids under the basal condition, indicating a constitutively-activated autophagic pathway. It may explain, at least partially, the faster turnover of Hsp27 in MERRF cybrids. This study provides information for us to understand that Hsp27 is degraded through the autophagic pathway and that Hsp27 may have a protective role in MERRF cells. Regulating Hsp27 and the autophagic pathway

  20. Physical mapping of new DNA probes near the fragile X mutation (FRAXA) by using a panel of cell lines

    PubMed Central

    Suthers, G. K.; Hyland, V. J.; Callen, D. F.; Oberle, I.; Rocchi, M.; Thomas, N. S.; Morris, C. P.; Schwartz, C. E.; Schmidt, M.; Ropers, H. H.; Baker, E.; Oostra, B. A.; Dahl, N.; Wilson, P. J.; Hopwood, J. J.; Sutherland, G. R.

    1990-01-01

    The fragile X syndrome is a very common disorder, but there has been little progress toward isolating the fragile X mutation (FRAXA). We describe a panel of 14 somatic cell hybrid lines, lymphoblastoid cell lines, and peripheral lymphocytes with X-chromosome translocation or deletion breakpoints near FRAXA. The locations of the breakpoints were defined with 16 established probes between pX45d (DXS100) and St14–1 (DXS52). Seven of the cell lines had breakpoints between the probes RN1 (DXS369) and U6.2 (DXS304), which flank FRAXA at distances of 3–5 centimorgans. The panel of cell lines was used to localize 16 new DNA probes in this region. Six of the probes–VK16, VK18, VK23, VK24, VK37, and VK47–detected loci near FRAXA, and it was possible to order both the X-chromosome breakpoints and the probes in relation to FRAXA. The order of probes and loci near FRAXA is cen–RN1,VK24–VK47–VK23–VK16,FRAXA–VK21A–VK18–IDS–VK37–U6.2-qter. The breakpoints near FRAXA are sufficiently close together that probes localized with this panel can be linked on a large-scale restriction map by pulsed-field gel electrophoresis. This panel of cell lines will be valuable in rapidly localizing other probes near FRAXA. ImagesFigure 2 PMID:2378346

  1. Frozen human cells can record radiation damage accumulated during space flight: mutation induction and radioadaptation.

    PubMed

    Yatagai, Fumio; Honma, Masamitsu; Takahashi, Akihisa; Omori, Katsunori; Suzuki, Hiromi; Shimazu, Toru; Seki, Masaya; Hashizume, Toko; Ukai, Akiko; Sugasawa, Kaoru; Abe, Tomoko; Dohmae, Naoshi; Enomoto, Shuichi; Ohnishi, Takeo; Gordon, Alasdair; Ishioka, Noriaki

    2011-03-01

    To estimate the space-radiation effects separately from other space-environmental effects such as microgravity, frozen human lymphoblastoid TK6 cells were sent to the "Kibo" module of the International Space Station (ISS), preserved under frozen condition during the mission and finally recovered to Earth (after a total of 134 days flight, 72 mSv). Biological assays were performed on the cells recovered to Earth. We observed a tendency of increase (2.3-fold) in thymidine kinase deficient (TK(-)) mutations over the ground control. Loss of heterozygosity (LOH) analysis on the mutants also demonstrated a tendency of increase in proportion of the large deletion (beyond the TK locus) events, 6/41 in the in-flight samples and 1/17 in the ground control. Furthermore, in-flight samples exhibited 48% of the ground-control level in TK(-) mutation frequency upon exposure to a subsequent 2 Gy dose of X-rays, suggesting a tendency of radioadaptation when compared with the ground-control samples. The tendency of radioadaptation was also supported by the post-flight assays on DNA double-strand break repair: a 1.8- and 1.7-fold higher efficiency of in-flight samples compared to ground control via non-homologous end-joining and homologous recombination, respectively. These observations suggest that this system can be used as a biodosimeter, because DNA damage generated by space radiation is considered to be accumulated in the cells preserved frozen during the mission, Furthermore, this system is also suggested to be applicable for evaluating various cellular responses to low-dose space radiation, providing a better understanding of biological space-radiation effects as well as estimation of health influences of future space explores. PMID:21161544

  2. Acetaminophen-NAPQI Hepatotoxicity: A Cell Line Model System Genome-Wide Association Study

    PubMed Central

    Moyer, Ann M.; Fridley, Brooke L.; Jenkins, Gregory D.; Batzler, Anthony J.; Pelleymounter, Linda L.; Kalari, Krishna R.; Ji, Yuan; Chai, Yubo; Nordgren, Kendra K. S.; Weinshilboum, Richard M.

    2011-01-01

    Acetaminophen is the leading cause of acute hepatic failure in many developed nations. Acetaminophen hepatotoxicity is mediated by the reactive metabolite N-acetyl-p-benzoquinonimine (NAPQI). We performed a “discovery” genome-wide association study using a cell line–based model system to study the possible contribution of genomics to NAPQI-induced cytotoxicity. A total of 176 lymphoblastoid cell lines from healthy subjects were treated with increasing concentrations of NAPQI. Inhibiting concentration 50 values were determined and were associated with “glutathione pathway” gene single nucleotide polymorphisms (SNPs) and genome-wide basal messenger RNA expression, as well as with 1.3 million genome-wide SNPs. A group of SNPs in linkage disequilibrium on chromosome 3 was highly associated with NAPQI toxicity. The p value for rs2880961, the SNP with the lowest p value, was 1.88 × 10−7. This group of SNPs mapped to a “gene desert,” but chromatin immunoprecipitation assays demonstrated binding of several transcription factor proteins including heat shock factor 1 (HSF1) and HSF2, at or near rs2880961. These chromosome 3 SNPs were not significantly associated with variation in basal expression for any of the genome-wide genes represented on the Affymetrix U133 Plus 2.0 GeneChip. We have used a cell line–based model system to identify a SNP signal associated with NAPQI cytotoxicity. If these observations are validated in future clinical studies, this SNP signal might represent a potential biomarker for risk of acetaminophen hepatotoxicity. The mechanisms responsible for this association remain unclear. PMID:21177773

  3. Novel Insights into the Regulatory Architecture of CD4+ T Cells in Rheumatoid Arthritis

    PubMed Central

    Aterido, Adrià; Palacio, Carlos; Marsal, Sara; Ávila, Gabriela; Julià, Antonio

    2014-01-01

    Rheumatoid arthritis (RA) is the most frequent autoimmune chronic inflammatory disease of the joints and it is characterized by the inflammation of the synovial membrane and the subsequent destruction of the joints. In RA, CD4+ T cells are the main drivers of disease initiation and the perpetuation of the damaging inflammatory process. To date, however, the genetic regulatory mechanisms of CD4+ T cells associated with RA etiology are poorly understood. The genome-wide analysis of expression quantitative trait loci (eQTL) in disease-relevant cell types is a recent genomic integration approach that is providing significant insights into the genetic regulatory mechanisms of many human pathologies. The objective of the present study was to analyze, for the first time, the genome-wide genetic regulatory mechanisms associated with the gene expression of CD4+ T cells in RA. Whole genome gene expression profiling of CD4+ T cells and the genome-wide genotyping (598,258 SNPs) of 29 RA patients with an active disease were performed. In order to avoid the excessive burden of multiple testing associated with genome-wide trans-eQTL analysis, we developed and implemented a novel systems genetics approach. Finally, we compared the genomic regulation pattern of CD4+ T cells in RA with the genomic regulation observed in reference lymphoblastoid cell lines (LCLs). We identified a genome-wide significant cis-eQTL associated with the expression of FAM66C gene (P = 6.51e−9). Using our new systems genetics approach we identified six statistically significant trans-eQTLs associated with the expression of KIAA0101 (P<7.4e−8) and BIRC5 (P = 5.35e−8) genes. Finally, comparing the genomic regulation profiles between RA CD4+ T cells and control LCLs we found 20 genes showing differential regulatory patterns between both cell types. The present genome-wide eQTL analysis has identified new genetic regulatory elements that are key to the activity of CD4+ T cells in RA. PMID:24959711

  4. DICER1 mutations in Familial Multi-Nodular Goiter with and without Ovarian Sertoli-Leydig Cell Tumors

    PubMed Central

    Frio, Thomas Rio; Bahubeshi, Amin; Kanellopoulou, Chryssa; Hamel, Nancy; Niedziela, Marek; Sabbaghian, Nelly; Pouchet, Carly; Gilbert, Lucy; O’Brien, Paul K.; Serfas, Kim; Broderick, Peter; Houlston, Richard S.; Lesueur, Fabienne; Bonora, Elena; Muljo, Stefan; Schimke, R. Neil; Soglio, Dorothée Bouron-Dal; Arseneau, Jocelyne; Schultz, Kris Ann; Priest, John R.; Nguyen, Van-Hung; Harach, H. Ruben; Livingston, David M.; Foulkes, William D.; Tischkowitz, Marc

    2012-01-01

    Context Non-toxic multinodular goiter (MNG) is frequently observed in the general population, but little is known about the underlying genetic susceptibility to this disease. Familial cases of MNG have been reported and there are five such published families which also contain individuals with Sertoli-Leydig cell tumors of the ovary (SLCT). Germline mutations in DICER1, a gene that codes for an RNase III endoribonuclease, have recently been identified in families affected pleuropulmonary blastoma (PPB), some of whom include cases of MNG and gonadal tumors such as SLCT. Objective To determine whether familial MNG with or without SLCT in the absence of PPB was caused by mutations in DICER1. Design, Setting and Patients From September 2009 to September 2010, we studied two MNG families and three MNG/SLCT families. We screened affected probands for mutations in the DICER1 gene. We investigated blood lymphocytes, MNG and SLCT tissue from family members for loss of the wild-type allele (loss of heterozygosity), DICER1 expression and microRNA dysregulation. Main Outcome Measure(s) Detection of germline DICER1 gene mutations in familial MNG with and without SLCT. Results We identified and characterized germline DICER1 mutations in all five families. Molecular analysis of the three SLCTs showed no loss of heterozygosity at DICER1, and IHC analysis in two available samples showed strong expression of DICER1 in Sertoli cells, but weak staining of Leydig cells. MicroRNA profiling of RNA derived from lymphoblastoid cell lines from both affected and unaffected members of the familial MNG cases revealed miRNA perturbations in DICER1 mutation carriers. Conclusions DICER1 mutations predispose to both familial MNG and MNG with SLCT, independent of PPB and germline DICER1 mutations lead to dysregulation of miRNA. This could be investigated further as a possible novel mechanism of tumorigenesis. PMID:21205968

  5. Stem Cells

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    Stem cells are cells with the potential to develop into many different types of cells in the body. ... the body. There are two main types of stem cells: embryonic stem cells and adult stem cells. Stem ...

  6. Cell Structure

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  7. Stem Cells

    MedlinePlus

    Stem cells are cells with the potential to develop into many different types of cells in the body. They serve as a repair ... body. There are two main types of stem cells: embryonic stem cells and adult stem cells. Stem ...

  8. CD4(+)and CD8(+)T-cell reactions against leukemia-associated- or minor-histocompatibility-antigens in AML-patients after allogeneic SCT.

    PubMed

    Steger, Brigitte; Milosevic, Slavoljub; Doessinger, Georg; Reuther, Susanne; Liepert, Anja; Braeu, Marion; Schick, Julia; Vogt, Valentin; Schuster, Friedhelm; Kroell, Tanja; Busch, Dirk H; Borkhardt, Arndt; Kolb, Hans-Jochem; Tischer, Johanna; Buhmann, Raymund; Schmetzer, Helga

    2014-04-01

    T-cells play an important role in the remission-maintenance in AML-patients (pts) after SCT, however the role of LAA- (WT1, PR1, PRAME) or minor-histocompatibility (mHag, HA1) antigen-specific CD4(+) and CD8(+)T-cells is not defined. A LAA/HA1-peptide/protein stimulation, cloning and monitoring strategy for specific CD8(+)/CD4(+)T-cells in AML-pts after SCT is given. Our results show that (1) LAA-peptide-specific CD8+T-cells are detectable in every AML-pt after SCT. CD8(+)T-cells, recognizing two different antigens detectable in 5 of 7 cases correlate with long-lasting remissions. Clonal TCR-Vβ-restriction exemplarily proven by spectratyping in PRAME-specific CD8(+)T-cells; high PRAME-peptide-reactivity was CD4(+)-associated, as shown by IFN-γ-release. (2) Two types of antigen-presenting cells (APCs) were tested for presentation of LAA/HA1-proteins to CD4(+)T-cells: miniEBV-transduced lymphoblastoid cells (B-cell-source) and CD4-depleted MNC (source for B-cell/monocyte/DC). We provide a refined cloning-system for proliferating, CD40L(+)CD4(+)T-cells after LAA/HA1-stimulation. CD4(+)T-cells produced cytokines (GM-CSF, IFN-γ) upon exposure to LAA/HA1-stimulation until after at least 7 restimulations and demonstrated cytotoxic activity against naive blasts, but not fibroblasts. Antileukemic activity of unstimulated, stimulated or cloned CD4(+)T-cells correlated with defined T-cell-subtypes and the clinical course of the disease. In conclusion we provide immunological tools to enrich and monitor LAA/HA1-CD4(+)- and CD8(+)T-cells in AML-pts after SCT and generate data with relevant prognostic value. We were able to demonstrate the presence of LAA-peptide-specific CD8(+)T-cell clones in AML-pts after SCT. In addition, we were also able to enrich specific antileukemic reactive CD4(+)T-cells without GvH-reactivity upon repeated LAA/HA1-protein stimulation and limiting dilution cloning. PMID:24315637

  9. Long-term preservation of antibody-dependent cellular cytotoxicity (ADCC) of natural killer cells amplified in vitro from the peripheral blood of breast cancer patients after chemotherapy.

    PubMed

    Clémenceau, Béatrice; Gallot, Géraldine; Vivien, Régine; Gaschet, Joëlle; Campone, Mario; Vié, Henri

    2006-01-01

    Twenty percent of breast cancer adenocarcinomas overexpress the oncogene c-erb-2 that is recognized by the humanized anti-Her2/neu monoclonal antibody Herceptin. Results from clinical studies suggest that antibody-dependent cellular cytotoxicity (ADCC) is involved in the clinical response of Herceptin-treated patients. The purpose of the current study was to evaluate the possibility of amplifying in vitro the CD3-/CD16+ natural killer (NK) cell subset that mediates ADCC from breast cancer patients after chemotherapy. Peripheral blood mononuclear cells from six breast cancer patients taken 2 months after chemotherapy completion were co-cultured with an autologous irradiated Epstein-Barr virus-transformed B-lymphoblastoid cell line (LCL) in the presence of interleukin-2 (IL-2) for 4-6 weeks. These LCL + IL2 activated cultures (ACs) were tested for ADCC potential, and their CD3/CD16 NK proportion was quantified. Among the ACs, the proportion of CD3-/CD16+ NK cells increased up to 64% over the first 2 weeks of culture and the ACs continued to expand for 1 month thereafter. Control and patient ACs displayed ADCC activity (tested in the presence of Rituximab against the autologous LCL to take into account any possible effect of inhibitory NK receptors) as well as against the MCF-7(Her2/neu) breast cancer cell line in the presence of Herceptin. This ADCC activity was maintained during the entire culture period. In conclusion, chemotherapy in breast cancer patients does not obviate the possibility of amplifying in vitro the NK cell subset that mediates ADCC. Consequently, adoptive transfer of lymphocytes mediating ADCC can be considered using this protocol to test its benefit in patients under Herceptin treatment. PMID:16365600

  10. Mechanisms of acquired resistance to the quinazoline thymidylate synthase inhibitor ZD1694 (Tomudex) in one mouse and three human cell lines.

    PubMed Central

    Jackman, A. L.; Kelland, L. R.; Kimbell, R.; Brown, M.; Gibson, W.; Aherne, G. W.; Hardcastle, A.; Boyle, F. T.

    1995-01-01

    Four cell lines, the mouse L1210 leukaemia, the human W1L2 lymphoblastoid and two human ovarian (CH1 and 41M) cell lines, were made resistant to ZD1694 (Tomudex) by continual exposure to incremental doses of the drug. A 500-fold increase in thymidylate synthase (TS) activity is the primary mechanism of resistance to ZD1694 in the W1L2:RD1694 cell line, which is consequently highly cross-resistant to other folate-based TS inhibitors, including BW1843U89, LY231514 and AG337, but sensitive to antifolates with other enzyme targets. The CH1:RD1694 cell line is 14-fold resistant to ZD1694, largely accounted for by the 4.2-fold increase in TS activity. Cross-resistance was observed to other TS inhibitors, including 5-fluorodeoxyuridine (FdUrd). 41M:RD1694 cells, when exposed to 0.1 microM [3H]ZD1694, accumulated approximately 20-fold less 3H-labelled material over 24 h than the parental line. Data are consistent with this being the result of impaired transport of the drug via the reduced folate/methotrexate carrier. Resistance was therefore observed to methotrexate but not to CB3717, a compound known to use this transport mechanism poorly. The mouse L1210:RD1694 cell line does not accumulate ZD1694 or Methotrexate (MTX) polyglutamates. Folylpolyglutamate synthetase substrate activity (using ZD1694 as the substrate) was decreased to approximately 13% of that observed in the parental line. Cross-resistance was found to those compounds known to be active through polyglutamation. PMID:7537518

  11. SPIB and BATF provide alternate determinants of IRF4 occupancy in diffuse large B-cell lymphoma linked to disease heterogeneity

    PubMed Central

    Care, Matthew A.; Cocco, Mario; Laye, Jon P.; Barnes, Nicholas; Huang, Yuanxue; Wang, Ming; Barrans, Sharon; Du, Ming; Jack, Andrew; Westhead, David R.; Doody, Gina M.; Tooze, Reuben M.

    2014-01-01

    Interferon regulatory factor 4 (IRF4) is central to the transcriptional network of activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), an aggressive lymphoma subgroup defined by gene expression profiling. Since cofactor association modifies transcriptional regulatory input by IRF4, we assessed genome occupancy by IRF4 and endogenous cofactors in ABC-DLBCL cell lines. IRF4 partners with SPIB, PU.1 and BATF genome-wide, but SPIB provides the dominant IRF4 partner in this context. Upon SPIB knockdown IRF4 occupancy is depleted and neither PU.1 nor BATF acutely compensates. Integration with ENCODE data from lymphoblastoid cell line GM12878, demonstrates that IRF4 adopts either SPIB- or BATF-centric genome-wide distributions in related states of post-germinal centre B-cell transformation. In primary DLBCL high-SPIB and low-BATF or the reciprocal low-SPIB and high-BATF mRNA expression links to differential gene expression profiles across nine data sets, identifying distinct associations with SPIB occupancy, signatures of B-cell differentiation stage and potential pathogenetic mechanisms. In a population-based patient cohort, SPIBhigh/BATFlow-ABC-DLBCL is enriched for mutation of MYD88, and SPIBhigh/BATFlow-ABC-DLBCL with MYD88-L265P mutation identifies a small subgroup of patients among this otherwise aggressive disease subgroup with distinct favourable outcome. We conclude that differential expression of IRF4 cofactors SPIB and BATF identifies biologically and clinically significant heterogeneity among ABC-DLBCL. PMID:24875472

  12. MicroRNAs 221 and 222 target p27Kip1 in Marek's disease virus-transformed tumour cell line MSB-1.

    PubMed

    Lambeth, Luke S; Yao, Yongxiu; Smith, Lorraine P; Zhao, Yuguang; Nair, Venugopal

    2009-05-01

    MicroRNAs (miRNAs) are a class of short RNAs that function as post-transcriptional suppressors of protein expression and are involved in a variety of biological processes, including oncogenesis. Several recent studies have implicated the involvement of miR-221 and miR-222 in tumorigenesis as these miRNAs are upregulated in a number of cancers and affect the expression of cell cycle regulatory proteins such as the cyclin-dependent kinase (cdk) inhibitor p27(Kip1). Marek's disease virus (MDV) is a highly oncogenic herpesvirus that affects poultry, causing acute neoplastic disease with lymphomatous lesions in several organs. MDV-encoded oncogenes such as Meq are directly implicated in the neoplastic transformation of T cells and have been well studied. More recently, however, the involvement of both host and virus-encoded miRNAs in the induction of MD lymphomas is being increasingly recognized. We analysed the miRNA expression profiles in the MDV-transformed lymphoblastoid cell line MSB-1 and found that endogenous miRNAs miR-221 and miR-222 were significantly upregulated. Demonstration of the conserved binding sites for these miRNAs in the chicken p27(Kip1) 3'-untranslated region sequence and the repression of luciferase activity of reporter constructs indicated that miR-221 and miR-222 target p27(Kip1) in these cells. We also found that overexpression of miR-221 and miR-222 decreased p27(Kip1) levels and that treatment with retrovirally expressed antagomiRs partially alleviated this suppression. These data show that an oncogenic herpesvirus, as in the case of many cancers, can exploit the miRNA machinery for suppressing cell cycle regulatory molecules such as p27(Kip1) in the induction and progression of T-cell lymphomas. PMID:19264608

  13. SPIB and BATF provide alternate determinants of IRF4 occupancy in diffuse large B-cell lymphoma linked to disease heterogeneity.

    PubMed

    Care, Matthew A; Cocco, Mario; Laye, Jon P; Barnes, Nicholas; Huang, Yuanxue; Wang, Ming; Barrans, Sharon; Du, Ming; Jack, Andrew; Westhead, David R; Doody, Gina M; Tooze, Reuben M

    2014-07-01

    Interferon regulatory factor 4 (IRF4) is central to the transcriptional network of activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), an aggressive lymphoma subgroup defined by gene expression profiling. Since cofactor association modifies transcriptional regulatory input by IRF4, we assessed genome occupancy by IRF4 and endogenous cofactors in ABC-DLBCL cell lines. IRF4 partners with SPIB, PU.1 and BATF genome-wide, but SPIB provides the dominant IRF4 partner in this context. Upon SPIB knockdown IRF4 occupancy is depleted and neither PU.1 nor BATF acutely compensates. Integration with ENCODE data from lymphoblastoid cell line GM12878, demonstrates that IRF4 adopts either SPIB- or BATF-centric genome-wide distributions in related states of post-germinal centre B-cell transformation. In primary DLBCL high-SPIB and low-BATF or the reciprocal low-SPIB and high-BATF mRNA expression links to differential gene expression profiles across nine data sets, identifying distinct associations with SPIB occupancy, signatures of B-cell differentiation stage and potential pathogenetic mechanisms. In a population-based patient cohort, SPIBhigh/BATFlow-ABC-DLBCL is enriched for mutation of MYD88, and SPIBhigh/BATFlow-ABC-DLBCL with MYD88-L265P mutation identifies a small subgroup of patients among this otherwise aggressive disease subgroup with distinct favourable outcome. We conclude that differential expression of IRF4 cofactors SPIB and BATF identifies biologically and clinically significant heterogeneity among ABC-DLBCL. PMID:24875472

  14. Are there x-ray induced signature mutations in human cells?

    SciTech Connect

    Nelson, S.L.; Giver, C.R.; Grosovsky, A.J.

    1994-12-31

    Investigations of mutational spectra generally have two primary objectives: identification of mutagen specific hallmark mutations, and insight into mutagenic mechanisms. In order to address these two crucial issues we have examined the spectrum of 116 x-ray induced, and 78 spontaneous, HPRT{sup -} mutants derived from the human B lymphoblastoid cell line TK6. Multiplex PCR analysis demonstrated that the overall representation of large deletions was not significantly different in the 2 spectra, although highly significant differences were observed for specific deletion types. Total gene deletions represented 41/78 (.53) of x-ray induced, but only 7/43 (.16) of spontaneous, deletions (p < .0001). In contrast, 5{prime} terminal deletions were significantly more common among spontaneous (17/43, .40) than x-ray induced (13/78, .17) large deletions (p=.0079). Chromosomal scale investigation of x-ray induced and spontaneous hprt total deletion mutants was also performed using cytogenetic examination, and X-linked PCR probes. The types of point mutations induced by x-ray exposure were very diverse, including all classes of transitions and transversions, tandem base substitutions, frameshifts, and a deletion/insertion compound mutation. Compared to spontaneous data, radiation induced point mutations exhibited a significantly reduced number of transitions, and an increased representation of small deletions. Small deletions were uniformly surrounded by direct sequence repeats. The distribution of x-ray induced point mutations was characterized by a cluster of 8 mutants within a 30 base region of exon 8. Thirteen HPRT{sup -} point mutants exhibited aberrant splicing, 4 of which were attributable to coding sequence alterations within exons 4 and 8. These results suggest that it may be possible to identify hallmark mutations associated with x-ray exposure of human cells.

  15. Oxidative stress enables Epstein-Barr virus-induced B-cell transformation by posttranscriptional regulation of viral and cellular growth-promoting factors.

    PubMed

    Chen, X; Kamranvar, S A; Masucci, M G

    2016-07-21

    Infection of human B lymphocytes by Epstein-Barr virus (EBV) leads to the establishment of immortalized lymphoblastoid cell lines (LCLs) that are widely used as a model of viral oncogenesis. An early consequence of infection is the induction of DNA damage and activation of the DNA damage response, which limits the efficiency of growth transformation. The cause of the DNA damage remains poorly understood. We have addressed this question by comparing the response of B lymphocytes infected with EBV or stimulated with a potent B-cell mitogen. We found that although the two stimuli induce comparable proliferation during the first 10 days of culture, the EBV-infected blasts showed significantly higher levels of DNA damage, which correlated with stronger and sustained accumulation of reactive oxygen species (ROS). Treatment with ROS scavengers decreased DNA damage in both mitogen-stimulated and EBV-infected cells. However, while mitogen-induced proliferation was slightly improved, the proliferation of EBV-infected cells and the establishment of LCLs were severely impaired. Quenching of ROS did not affect the kinetics and magnitude of viral gene expression but was associated with selective downregulation of the viral LMP1 and phosphorylated cellular transcription factor STAT3 that have key roles in transformation. Analysis of the mechanism by which high levels of ROS support LMP1 expression revealed selective inhibition of viral microRNAs that target the LMP1 transcript. Our study provides novel insights into the role of EBV-induced oxidative stress in promoting B-cell immortalization and malignant transformation. PMID:26592445

  16. The protective roles of phosphorylated heat shock protein 27 in human cells harboring myoclonus epilepsy with ragged-red fibers A8344G mtDNA mutation.

    PubMed

    Chen, Hsueh-Fu; Chen, Chin-Yi; Lin, Ting-Hui; Huang, Zhao-Wei; Chi, Tang-Hao; Ma, Yi-Shing; Wu, Shi-Bei; Wei, Yau-Huei; Hsieh, Mingli

    2012-08-01

    Mitochondrial DNA (mtDNA) mutations are associated with a large number of neuromuscular diseases. Myoclonus epilepsy with ragged-red fibers (MERRF) syndrome is a mitochondrial disease inherited through the maternal lineage. The most common mutation in MERRF syndrome, the A8344G mutation of mtDNA, is associated with severe defects in mitochondrial protein synthesis, which impair the assembly and function of the respiratory chain. We have previously shown that there is a decreased level of heat shock protein 27 (HSP27) in lymphoblastoid cells derived from a MERRF patient and in cytoplasmic hybrids (cybrids) harboring the A8344G mutation of mtDNA. In the present study, we found a dramatic decrease in the level of phosphorylated HSP27 (p-HSP27) in the mutant cybrids. Even though the steady-state level of p-HSP27 was reduced in the mutant cybrids, normal phosphorylation and dephosphorylation were observed upon exposure to stress, indicating normal kinase and phosphatase activities. To explore the roles that p-HSP27 may play, transfection experiments with HSP27 mutants, in which three specific serines were replaced with alanine or aspartic acid, showed that the phosphomimicking HSP27 desensitized mutant cybrids to apoptotic stress induced by staurosporine (STS). After heat shock stress, p-HSP27 was found to enter the nucleus immediately, and with a prolonged interval of recovery, p-HSP27 returned to the cytoplasm in wild-type cybrids but not in mutant cybrids. The translocation of p-HSP27 was correlated with cell viability, as shown by the increased number of apoptotic cells after p-HSP27 returned to the cytoplasm. In summary, our results demonstrate that p-HSP27 provides significant protection when cells are exposed to different stresses in the cell model of MERRF syndrome. Therapeutic agents targeting anomalous HSP27 phosphorylation might represent a potential treatment for mitochondrial diseases. PMID:22742457

  17. Translocation of a hydrocarbon fluorescent probe between Epstein-Barr virus and lymphoid cells: an assay for early events in viral infection.

    PubMed

    Rosenthal, K S; Yanovich, S; Inbar, M; Strominger, J L

    1978-10-01

    Translocation of the hydrocarbon fluorescent probe diphenylhexatriene (DPH) between membranes was studied by fluorescence polarization (P) analysis. First, using a model system, the high P value (0.324) of DPH-labeled cholesterol/phosphatidylcholine liposomes and the low P value (0.157) of DPH-labeled phosphatidylcholine liposomes allowed detection of DPH translocation between interacting liposomes. This was monitored by the change in P in either direction. Early events during cell-virus interactions were similarly studied by monitoring DPH translocation. The P value of DPH-labeled Epstein-Barr Virus (EBV) was significantly higher (0.350-0.392) than the P value of DPH-labeled lymphoid cells (0.238-0.289). Hence, DPH translocation could be detected by changes in P following incubation of DPH-labeled EBV and nonlabeled cells. A marked decrease in P was observed after incubation of DPH-labeled EBV with either nonlabeled lymphoblastoid Raji cells or fresh human B lymphocytes. However, only a slight decrease in P was obtained when DPH-labeled EBV was incubated with either nonlabeled fresh human T lymphocytes or fresh T or B rabbit lymphocytes. Moreover, incubation of fresh human B lymphocytes with the purified C3 component of complement (a putative inhibitor for the EBV receptor) prior to the addition of DPH-labeled EBV abolished the observed decrease in the P value. Most of these experiments were carried out with both the P3HR-1 and the B95-8 strains of EBV. DPH translocation, as determined by fluorescence polarization analysis, is, therefore, measuring some early event during interaction of this enveloped virus and mammalian cells. The potential applicability of this technique to other viruses is illustrated by an experiment with Semliki Forest virus. PMID:217012

  18. An in vitro study of liposomal curcumin: stability, toxicity and biological activity in human lymphocytes and Epstein-Barr virus-transformed human B-cells.

    PubMed

    Chen, Changguo; Johnston, Thomas D; Jeon, Hoonbae; Gedaly, Roberto; McHugh, Patrick P; Burke, Thomas G; Ranjan, Dinesh

    2009-01-21

    Curcumin is a multi-functional and pharmacologically safe natural agent. Used as a food additive for centuries, it also has anti-inflammatory, anti-virus and anti-tumor properties. We previously found that it is a potent inhibitor of cyclosporin A (CsA)-resistant T-cell co-stimulation pathway. It inhibits mitogen-stimulated lymphocyte proliferation, NFkappaB activation and IL-2 signaling. In spite of its safety and efficacy, the in vivo bioavailability of curcumin is poor, and this may be a major obstacle to its utility as a therapeutic agent. Liposomes are known to be excellent carriers for drug delivery. In this in vitro study, we report the effects of different liposome formulations on curcumin stability in phosphate buffered saline (PBS), human blood, plasma and culture medium RPMI-1640+10% FBS (pH 7.4, 37 degrees C). Liposomal curcumin had higher stability than free curcumin in PBS. Liposomal and free curcumin had similar stability in human blood, plasma and RPMI-1640+10% FBS. We looked at the toxicity of non-drug-containing liposomes on (3)H-thymidine incorporation by concanavalin A (Con A)-stimulated human lymphocytes, splenocytes and Epstein-Barr virus (EBV)-transformed human B-cell lymphoblastoid cell line (LCL). We found that dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) were toxic to the tested cells. However, addition of cholesterol to the lipids at DMPC:DMPG:cholesterol=7:1:8 (molar ratio) almost completely eliminated the lipid toxicity to these cells. Liposomal curcumin had similar or even stronger inhibitory effects on Con A-stimulated human lymphocyte, splenocyte and LCL proliferation. We conclude that liposomal curcumin may be useful for intravenous administration to improve the bioavailability and efficacy, facilitating in vivo studies that could ultimately lead to clinical application of curcumin. PMID:18840516

  19. Preliminary results of space experiment: Implications for the effects of space radiation and microgravity on survival and mutation induction in human cells

    NASA Astrophysics Data System (ADS)

    Yatagai, F.; Honma, M.; Ukai, A.; Omori, K.; Suzuki, H.; Shimazu, T.; Takahashi, A.; Ohnishi, T.; Dohmae, N.; Ishioka, N.

    2012-02-01

    In view of the concern for the health of astronauts that may one day journey to Mars or the Moon, we investigated the effect that space radiation and microgravity might have on DNA damage and repair. We sent frozen human lymphoblastoid TK6 cells to the International Space Station where they were maintained under frozen conditions during a 134-day mission (14 November 2008 to 28 March 2009) except for an incubation period of 8 days under 1G or μG conditions in a CO2 incubator. The incubation period started after 100 days during which the cells had been exposed to 54 mSv of space radiation. The incubated cells were then refrozen, returned to Earth, and compared to ground control samples for the determination of the influence of microgravity on cell survival and mutation induction. The results for both varied from experiment to experiment, yielding a large SD, but the μG sample results differed significantly from the 1G sample results for each of 2 experiments, with the mean ratio of μG to 1G being 0.55 for the concentration of viable cells and 0.59 for the fraction of thymidine kinase deficient (TK-) mutants. Among the mutants, non-loss of zygosity events (point mutations) were less frequent (31%) after μG incubation than after 1G incubation, which might be explained by the influence of μG on cellular metabolic or physiological function. Additional experiments are needed to clarify the effect of μG interferes on DNA repair.

  20. Human B-cell interleukin-10: B-cell lines derived from patients with acquired immunodeficiency syndrome and Burkitt's lymphoma constitutively secrete large quantities of interleukin-10.

    PubMed

    Benjamin, D; Knobloch, T J; Dayton, M A

    1992-09-01

    A recent addition to the lymphokine network is human IL-10 (hIL-10). This novel lymphokine has striking homology to BCRF1 protein, the product of a previously uncharacterized open-reading frame in the Epstein-Barr virus (EBV) genome. To date, IL-10 expression has been described in several T clones induced with anti-CD3 and phorbol myristate acetate (PMA), in monocytes stimulated with lipopolysaccharide (LPS), and in murine B-cell lymphomas. We sought to determine whether human B cells express hIL-10 and, if so, its relationship to EBV and to other B-cell lymphokines. We studied 21 EBV-positive B-cell lines derived from patients with acquired immunodeficiency syndrome (AIDS) and Burkitt's lymphoma (n = 6), American Burkitt's (n = 3), African Burkitt's (n = 5), and normal lymphoblastoid cell lines (n = 7), in comparison with seven EBV-negative cell lines. All cell lines were activated with the tumor promoters PMA and teleocidin and were studied by Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), and enzyme-linked immunoadsorbent assay (ELISA). We demonstrated that EBV-positive cell lines derived from patients with American Burkitt's lymphoma, and especially those from patients with AIDS, constitutively express large quantities of hIL-10 by Northern blot analysis and ELISA (range, 3,101 to 25,915 pg/mL), and that both teleocidin and PMA induce hIL-10 in these cell lines. In contrast, six of seven EBV-negative cell lines did not express hIL-10 even by RT-PCR, and hIL-10 was not triggered by PMA or teleocidin. To assure that the 350 bp amplified by PCR was hIL-10 and not BCRF1, we used PCR primers, which do not amplify a fragment from plasmid templates containing BCRF1. Cloning and sequencing of the 350 bp product also demonstrated that B-cell IL-10 is identical to hIL-10 from the T-cell clone B21. Correlation of hIL-10 with other B-cell lymphokines secreted by these B-cell lines demonstrated that hIL-10 secretor cell lines also

  1. Identification and Validation of Genetic Variants that Influence Transcription Factor and Cell Signaling Protein Levels

    PubMed Central

    Hause, Ronald J.; Stark, Amy L.; Antao, Nirav N.; Gorsic, Lidija K.; Chung, Sophie H.; Brown, Christopher D.; Wong, Shan S.; Gill, Daniel F.; Myers, Jamie L.; To, Lida Anita; White, Kevin P.; Dolan, M. Eileen; Jones, Richard Baker

    2014-01-01

    Many genetic variants associated with human disease have been found to be associated with alterations in mRNA expression. Although it is commonly assumed that mRNA expression changes will lead to consequent changes in protein levels, methodological challenges have limited our ability to test the degree to which this assumption holds true. Here, we further developed the micro-western array approach and globally examined relationships between human genetic variation and cellular protein levels. We collected more than 250,000 protein level measurements comprising 441 transcription factor and signaling protein isoforms across 68 Yoruba (YRI) HapMap lymphoblastoid cell lines (LCLs) and identified 12 cis and 160 trans protein level QTLs (pQTLs) at a false discovery rate (FDR) of 20%. Whereas up to two thirds of cis mRNA expression QTLs (eQTLs) were also pQTLs, many pQTLs were not associated with mRNA expression. Notably, we replicated and functionally validated a trans pQTL relationship between the KARS lysyl-tRNA synthetase locus and levels of the DIDO1 protein. This study demonstrates proof of concept in applying an antibody-based microarray approach to iteratively measure the levels of human proteins and relate these levels to human genome variation and other genomic data sets. Our results suggest that protein-based mechanisms might functionally buffer genetic alterations that influence mRNA expression levels and that pQTLs might contribute phenotypic diversity to a human population independently of influences on mRNA expression. PMID:25087611

  2. Influence of low-dose and low-dose-rate ionizing radiation on mutation induction in human cells

    NASA Astrophysics Data System (ADS)

    Yatagai, F.; Umebayashi, Y.; Suzuki, M.; Abe, T.; Suzuki, H.; Shimazu, T.; Ishioka, N.; Iwaki, M.; Honma, M.

    This is a review paper to introduce our recent studies on the genetic effects of low-dose and low-dose-rate ionizing radiation (IR). Human lymphoblastoid TK6 cells were exposed to γ-rays at a dose-rate of 1.2 mGy/h (total 30 mGy). The frequency of early mutations (EMs) in the thymidine kinase ( TK) gene locus was determined to be 1.7 × 10 -6, or 1.9-fold higher than the level seen in unirradated controls [Umebayashi, Y., Honma, M., Suzuki, M., Suzuki, H., Shimazu, T., Ishioka, N., Iwaki, M., Yatagai, F., Mutation induction in cultured human cells after low-dose and low-dose-rate γ-ray irradiation: detection by LOH analysis. J. Radiat. Res., 48, 7-11, 2007]. These mutants were then analyzed for loss of heterozygosity (LOH) events. Small interstitial-deletion events were restricted to the TK gene locus and were not observed in EMs in unirradated controls, but they comprised about half of the EMs (8/15) after IR exposure. Because of the low level of exposure to IR, this specific type of event cannot be considered to be the direct result of an IR-induced DNA double strand break (DSB). To better understand the effects of low-level IR exposure, the repair efficiency of site-specific chromosomal DSBs was also examined. The pre γ-irradiation under the same condition did not largely influence the efficiency of DSB repair via end-joining, but enhanced such efficiency via homologous recombination to an about 40% higher level (unpublished data). All these results suggest that DNA repair and mutagenesis can be indirectly influenced by low-dose/dose-rate IR.

  3. T Cells

    MedlinePlus

    ... or turn off the immune response. Cytotoxic or “killer” T cells directly attack and destroy cells bearing ... involve selective activation of helper T cells and killer T cells, with a corresponding decrease in regulatory ...

  4. Cell division

    MedlinePlus Videos and Cool Tools

    ... hours after conception, the fertilized egg cell remains a single cell. After approximately 30 hours, it divides ... 3 days, the fertilized egg cell has become a berry-like structure made up of 16 cells. ...

  5. DY determinants, possibly associated with novel class II molecules, stimulate autoreactive CD4+ T cells with suppressive activity

    PubMed Central

    1988-01-01

    A set of T cell clones (TCC) isolated from HLA-DR-, Dw-, DQ-matched allogeneic MLCs was found to proliferate autonomously when stimulated with cells carrying a wide range of class I or II specificities. This apparently unrestricted proliferation was relatively weak, and only low levels of IL-2 were present in the supernatants of stimulated cells. Autologous as well as allogeneic PBMC and B lymphoblastoid cell lines (B-LCL) were capable of stimulating such clones, which were also restimulated by suppressive, but not by helper, TCC. Moreover, such clones displayed the unusual property of autostimulation. mAb inhibition experiments suggested that class II- or class II-restricted antigens were involved in stimulation. Thus, certain "broad" mAbs (TU39, SG520) reacting with multiple locus products inhibited activation of these reagents, but none of those reacting more specifically with DR (TU34, TU37, L243, Q2/70, SG157), DQ (TU22, SPV- L3, Leu 10), or DP (B7/21), or mixtures of these mAbs, were able to do so. Evidence from sequential immunoprecipitation experiments suggested that mAb TU39 bound class II-like molecules other than DR, DQ, and DP on TCC and B-LCL, and it is therefore proposed that such putative novel class II-like molecules may carry the stimulating determinants for these autoreactive clones. DY-reactive clones lacked helper activity for B cells but mediated potent suppressive activity on T cell proliferative responses that was not restricted by the HLA type of the responding cells. Suppressive activity was induced in normal PBMC by such clones, as well as by independent suppressive clones, which was also inhibited only by mAb TU39. These findings lead to the proposal that DY-reactive autostimulatory cells may constitute a self- maintaining suppressive circuit, the level of activity of which would be regulated primarily by the availability of IL-2 in the microenvironment. PMID:2450156

  6. Indole-3-carbinol induces cMYC and IAP-family downmodulation and promotes apoptosis of Epstein-Barr virus (EBV)-positive but not of EBV-negative Burkitt's lymphoma cell lines.

    PubMed

    Perez-Chacon, Gema; de Los Rios, Cristobal; Zapata, Juan M

    2014-11-01

    Indole-3-carbinol (I3C) is a natural product found in broadly consumed plants of the Brassica genus, such as broccoli, cabbage, and cauliflower, which exhibits anti-tumor effects through poorly defined mechanisms. I3C can be orally administered and clinical trials have demonstrated that I3C and derivatives are safe in humans. In this study we show that I3C efficiently induces apoptosis in cell lines derived from EBV-positive Burkitt's lymphomas (virus latency I/II), while it does not have any cytotoxic activity against EBV-negative Burkitt's lymphomas and immortalized EBV-infected lymphoblastoid cell lines (virus latency III). The effect of I3C in EBV-positive Burkitt's lymphoma is very specific, since only I3C and its C6-methylated derivative, but not other 3-substituted indoles, have an effect on cell viability. I3C treatment caused apoptosis characterized by loss of mitochondria membrane potential and caspase activation. I3C alters the expression of proteins involved in the control of apoptosis and transcription regulation in EBV-positive Burkitt's lymphoma cell lines. Among those, cMYC, cIAP1/2 and XIAP downmodulation at mRNA and protein level precede apoptosis induction, thus suggesting a role in I3C cytotoxicity. We also showed that I3C and, more particularly, its condensation dimer 3,3'-diindolylmethane (DIM) prolonged survival and reduced tumor burden of mice xenotransplanted with EBV-positive Burkitt's lymphoma Daudi cells. In summary these results, together with previous reports from clinical trials indicating the lack of toxicity in humans of I3C and derivatives, support the use of these compounds as a new therapeutic approach for treating patients with endemic (EBV-positive) Burkitt's lymphoma. PMID:25180456

  7. The A-myb gene is preferentially expressed in tonsillar CD38+, CD39-, and sIgM- B lymphocytes and in Burkitt's lymphoma cell lines.

    PubMed

    Golay, J; Erba, E; Bernasconi, S; Peri, G; Introna, M

    1994-07-15

    The A-myb gene is structurally related to the c-mby proto-oncogene, a transcription factor involved in the regulation of hemopoietic proliferation and differentiation. Recent evidence has shown that A-myb also functions as a transcriptional activator. We have previously demonstrated that A-myb RNA is not expressed in most mature human leukocytes at rest or after mitogenic or functional activation. We show here, by using cell sorting, PCR, and Western analyses that A-myb is most highly expressed in the subsets of human tonsillar B lymphocytes with the phenotypes CD38+, CD39-, and SIgM-. The preferential expression of A-myb in these populations was seen at both the RNA and protein levels. CD38 was consistently best at separating high from low A-myb-expressing cells, whereas other markers (CD10, 22, 23, 77, 11a, and 49d) did not correlate with A-myb expression. The CD38+ population expressing the highest levels of A-myb was shown to contain mostly cycling cells inasmuch as more than 95% were in the late G1, S, G2, and M phases of the cell cycle. In addition, A-myb expression always correlated with the percentage of cells in S/G2/M in the populations sorted with either CD38, CD39, or sIgM. Small resting tonsillar B lymphocytes induced to proliferate in vitro by several different polyclonal B cell activators did not, however, express detectable levels of A-myb, although these cells were demonstrated to express CD38 and enter the S/G2/M phases of the cell cycle. These data suggest that A-myb is a marker of in vivo-activated but not in vitro-activated B lymphocytes. Finally, A-myb was also found to be highly expressed in five of seven Burkitt's lymphoma lines and in none of three EBV lymphoblastoid cell lines. This finding is in agreement with the phenotype of the normal B cells that express high levels of A-myb in vivo and suggests that A-myb may be specifically induced within germinal center B cells. PMID:8021494

  8. Polymerase ε (POLE) ultra-mutated tumors induce robust tumor-specific CD4+ T cell responses in endometrial cancer patients

    PubMed Central

    Bellone, Stefania; Centritto, Floriana; Black, Jonathan; Schwab, Carlton; English, Diana; Cocco, Emiliano; Lopez, Salvatore; Bonazzoli, Elena; Predolini, Federica; Ferrari, Francesca; Silasi, Dan-Arin; Ratner, Elena; Azodi, Masoud; Schwartz, Peter E.; Santin, Alessandro D.

    2015-01-01

    Objective Around 7–10% of endometrial carcinomas are characterized by Polymerase-ε-(POLE) exonuclease-domain-mutations, an ultra-mutated-phenotype and a favorable prognosis. It is currently unknown whether POLE ultra-mutated-tumors are more immunogenic when compared to the other groups of endometrial cancers. Methods We used autologous-dendritic-cells (DC) pulsed with whole-tumor-extracts to assess the level of CD8+ and CD4+ T-cell-activation induced by POLE-ultramutated (+) and POLE wild-type (−) endometrial cancer cells in vitro. T-lymphocyte-proliferations were evaluated using CFSE and/or [3H]thymidine-incorporation-assays while the ability to specifically kill autologous-tumor-cells by cytotoxic-T-lymphocyte (CTL) was tested in standard 4-hr-51Cr-cytotoxicity-assays. In order to correlate cytotoxic activity and proliferation by CD4+ and CD8+ T-lymphocytes, respectively, with a particular lymphoid subset, two-color-flow-cytometric analysis of intracellular-cytokine-expression (IFN-γ vs IL-4) at the single cell level, was also performed. Results DC-pulsed with tumor extracts were able to induce CTL-responses against autologous-tumor-cells in both POLE (+) and POLE (−) cancer patients (P=0.305). These CD8+ T-cell-populations were cytotoxic against tumor-cells but they did not lyse PHA-stimulated-autologous-lymphocytes or autologous-EBV-transformed-lymphoblastoid-control-cell-lines. In contrast, only POLE (+) tumor-lysate-pulsed-DC were able to induce significant proliferation and high IFN-γ expression (i.e., Th1-cytokine-bias) in autologous in vitro DC-stimulated CD4+ T-cells as well as naïve CD4+ and CD8+ T-cells from patients-peripheral-blood (P < 0.05). Conclusions POLE ultra-mutated-tumors are significantly more immunogenic when compared to POLE (−) tumors, in particular to the helper arm of the immune system. These data lend support to the hypothesis that the better prognosis of patients with POLE (+) tumors may at least in part be linked to their

  9. Correction of both spontaneous and DEB-induced chromosome instability in Fanconi anemia FA-C cells by FACC cDNA

    SciTech Connect

    Stavropoulos, D.J.; Tomkins, D.J.; Allingham-Hawkins, D.J.; Buchwald, M.

    1994-09-01

    Cells from all four Fanconi anemia complementation groups show hypersensitivity to cell-killing by mitomycin C (MMC), diepoxybutane (DEB) and other DNA cross-linking agents, and increased spontaneous and DEB-induced chromosome aberrations (CA). The extent of these phenotypes varies between lymphoblastoid cell lines from different complementation groups. Our data showed that the difference in MMC hypersensitivity and DEB-CA was not always coupled. While 230N (FA-B) had higher DEB-induced CA/cell than 536N (FA-C) (7.42 vs. 4.46 respectively), that latter was much more sensitive to cell-killing by MMC (dose at 10% survival, D{sub 10}: 5.2 vs. 1.2 ng/ml respectively). Strathdes et al. (1992) cloned a cDNA Fanconi anemia complementation group C (FACC) which complemented the hypersensitivity to MMC and DEB cell-killing of FA-C cells (536N) but not cells from the other three complementation groups. The present study was initiated to determine whether chromosome instability in 536N is also complemented by the FACC (FAC3) cDNA. The pREP4-FAC3 vector was transfected into 536N and transfectants selected with hygromycin B. The DEB D{sub 10} of 536N (1.0 {mu}M) was corrected to the control level (16.2 {mu}M for 3TO) by FACC (15.1 {mu}M for 536N-FACC), as previously demonstrated. Chromosome instability (cab, cse, ctb, cte) was determined without and with 0.1 {mu}g/ml DEB treatment. Spontaneous CA of 536N (0.30 aberrations/cell) was corrected to the control level (0.04 for 3TO) by FACC (0.06 for 536N-FACC). Similarly, the DEB-induced CA was corrected (2.74 for 536N vs. 0.06 and 0.02 for 3TO and 536N-FACC respectively). Thus, at least for FA complementation group C, hypersensitivity to cell-killing and chromosome instability are not dissociated and are most likely caused by the same gene defect.

  10. Heterogeneity of multifunctional IL-17A producing S. Typhi-specific CD8+ T cells in volunteers following Ty21a typhoid immunization.

    PubMed

    McArthur, Monica A; Sztein, Marcelo B

    2012-01-01

    Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, continues to cause significant morbidity and mortality world-wide. CD8+ T cells are an important component of the cell mediated immune (CMI) response against S. Typhi. Recently, interleukin (IL)-17A has been shown to contribute to mucosal immunity and protection against intracellular pathogens. To investigate multifunctional IL-17A responses against S. Typhi antigens in T memory subsets, we developed multiparametric flow cytometry methods to detect up to 6 cytokines/chemokines (IL-10, IL-17A, IL-2, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1β (MIP-1β)) simultaneously. Five volunteers were immunized with a 4 dose regimen of live-attenuated S. Typhi vaccine (Ty21a), peripheral blood mononuclear cells (PBMC) were isolated before and at 11 time points after immunization, and CMI responses were evaluated. Of the 5 immunized volunteers studied, 3 produced detectable CD8+ T cell responses following stimulation with S. Typhi-infected autologous B lymphoblastoid cell lines (B-LCL). Additionally, 2 volunteers had detectable levels of intracellular cytokines in response to stimulation with S. Typhi-infected HLA-E restricted cells. Although the kinetics of the responses differed among volunteers, all of the responses were bi- or tri-phasic and included multifunctional CD8+ T cells. Virtually all of the IL-17A detected was derived from multifunctional CD8+ T cells. The presence of these multifunctional IL-17A+ CD8+ T cells was confirmed using an unsupervised analysis program, flow cytometry clustering without K (FLOCK). This is the first report of IL-17A production in response to S. Typhi in humans, indicating the presence of a Tc17 response which may be important in protection. The presence of IL-17A in multifunctional cells co-producing Tc1 cytokines (IL-2, IFN-γ and TNF-α) may also indicate that the distinction between Tc17 and Tc1

  11. Cell counting.

    PubMed

    Phelan, M C; Lawler, G

    2001-05-01

    This unit presents protocols for counting cells using either a hemacytometer or electronically using a Coulter counter. Cell counting with a hemacytometer permits effective discrimination of live from dead cells using trypan blue exclusion. In addition, the procedure is less subject to errors arising from cell clumping or size heterogeneity. Counting cells is more quickly and easily performed using an electronic counter, but live-dead discrimination is unreliable. Cell populations containing large numbers of dead cells and/or cell clumps are difficult to count accurately. In addition, electronic counting requires resetting of the instrument for cell populations of different sizes; heterogeneous populations can give rise to inaccurate counts, and resting and activated cells may require counting at separate settings. In general, electronic cell counting is best performed on fresh peripheral blood cells. PMID:18770655

  12. Tumor cell lysis by activated human neutrophils: analysis of neutrophil-delivered oxidative attack and role of leukocyte function-associated antigen 1.

    PubMed

    Dallegri, F; Ottonello, L; Ballestrero, A; Dapino, P; Ferrando, F; Patrone, F; Sacchetti, C

    1991-02-01

    The lysis of tumor cells, and other nucleated mammalian cells, by neutrophilic polymorphonuclear leukocytes (PMNs) triggered by phorbol myristate acetate (PMA) represents a widely used model system to dissect the PMN cytolytic armamentarium, potentially responsible for the cell damage at tissue sites of PMN activation. Although oxidants are generally considered to be instrumental in the target lysis by PMNs, the mediators actually involved remain a matter of controversy. Moreover, other factors potentially crucial to the lysis have not been clearly identified. In order to reexamine the determinants of the cytolytic process, we studied the events underlying the PMA-triggered PMN-delivered attack against two different targets, selected on the basis of preliminary experiments (B lymphoblastoid Daudi cells and erythroleukemic K 562 cells). The results suggest that the lysis is promoted by hypochlorous acid (HOCl) or a compound with characteristics very similar to HOCl itself. No evidence was obtained for the intervention or contribution of hydrogen peroxide (H2O2), hydroxyl (OH.) radicals, and the major HOCl-derived chloramines. PMNs appeared to use 35% of the generated H2O2 to produce HOCl, while the remainder appears to be consumed by PMNs themselves and target cells as well. Moreover, PMNs and target cells coaggregated at an early step of the cytolytic reaction, through a process efficiently prevented by a monoclonal antibody (MoAb J-90) directed against leukocyte function-associated antigen-1 (LFA-1). The inhibition of the PMN-target aggregation by the MoAb J-90 resulted in the impairment of the lysis, despite a normal generation of HOCl. Thus, the data demonstrate that the PMA-triggered lysis of tumor target cells by PMNs requires at least two events, occurring simultaneously: the LFA-1-mediated effector-target adherence and the PMN production of HOCl. The intervention of the LFA-1-mediated PMN-target adherence in the PMA-triggered lysis is likely to allow PMNs to

  13. Reduced DNA double-strand break repair capacity and risk of squamous cell carcinoma of the head and neck-A case-control study.

    PubMed

    Liu, Zhensheng; Liu, Hongliang; Gao, Fengqin; Dahlstrom, Kristina R; Sturgis, Erich M; Wei, Qingyi

    2016-04-01

    Tobacco smoke and alcohol use play important roles in the etiology of squamous cell carcinoma of the head and neck (SCCHN). Smoking causes DNA damage, including double-strand DNA breaks (DSBs), that leads to carcinogenesis. To test the hypothesis that suboptimal DSB repair capacity is associated with risk of SCCHN, we applied a flow cytometry-based method to detect the DSB repair phenotype first in four EBV-immortalized human lymphoblastoid cell lines and then in human peripheral blood T-lymphocytes (PBTLs). With this blood-based laboratory assay, we conducted a pilot case-control study of 100 patients with newly diagnosed, previously untreated SCCHN and 124 cancer-free controls of non-Hispanic whites. We found that the mean DSB repair capacity level was significantly lower in cases (42.1%) than that in controls (54.4%) (P<0.001). When we used the median DSB repair capacity level in the controls as the cutoff value for calculating the odds ratios (ORs) with adjustment for age, sex, smoking and drinking status, the cases were more likely than the controls to have a reduced DSB repair capacity (adjusted OR=1.93; 95% confidence interval, CI=1.04-3.56, P=0.037), especially for those subjects who were ever drinkers (adjusted OR=2.73; 95% CI=1.17-6.35, P=0.020) and had oropharyngeal tumors (adjusted OR=2.17; 95% CI=1.06-4.45, P=0.035). In conclusion, these findings suggest that individuals with a reduced DSB repair capacity may be at an increased risk of developing SCCHN. Larger studies are warranted to confirm these preliminary findings. PMID:26963119

  14. An open study of human lymphoblastoid interferon and oral acyclovir in chronic hepatitis B virus infection.

    PubMed

    Guarascio, P; De Felici, A P; Migliorini, D; Alexander, G J; Fagan, E A; Visco, G

    1986-01-01

    Ten patients were entered into an open study of interferon (IFN) 'induction' and oral acyclovir (ACV) 'maintenance' therapy. They received 5 Mega units (Mu)/m2 IFN by intramuscular injection daily for 3 days, followed by 7.5 Mu/m2 IFN daily for 7 days. IFN therapy was then discontinued and a 6-week course of oral ACV at a dose of 800 mg 4 times daily commenced. At 6 months, 2 patients had become HBeAg-negative and 1 had developed anti-HBe. Elimination of HBeAg in these patients was accompanied by return of serum liver function tests to normal. There was a statistically significant inhibition of DNA polymerase levels after the 1st week of IFN therapy, which then slowly increased to pretreatment values over 8 weeks. There were no significant adverse effects of ACV therapy, while fever, 'flu-like illness', fatigue, anorexia, and leucopenia were the main side-effects observed during the course of IFN which necessitated dose reduction in 7 patients. Combination therapy appears to effectively inhibit viral replication, although the 'maintenance' effect of oral ACV is minimal. A more effective drug to combine with IFN is needed. PMID:3598155

  15. Galvanic Cells

    ERIC Educational Resources Information Center

    Young, I. G.

    1973-01-01

    Many standard physical chemistry textbooks contain ambiguities which lead to confusion about standard electrode potentials, calculating cell voltages, and writing reactions for galvanic cells. This article shows how standard electrode potentials can be used to calculate cell voltages and deduce cell reactions. (Author/RH)

  16. Cell Biochips

    NASA Astrophysics Data System (ADS)

    Pioufle, B. Le; Picollet-D'Hahan, N.

    A cell biochip is a microsystem, equipped with electronic and microfluidic functions, designed to manipulate or analyse living cells. The first publications in this emerging area of research appeared toward the end of the 1980s. In 1989 Washizu described a biochip designed to fuse two cells by electropermeabilisation of the cytoplasmic membrane [1]. Research centers have devised a whole range of cell chip structures, for simultaneous or sequential analysis of single cells, cell groups, or cell tissues reconstituted on the chip. The cells are arranged in a square array on a parallel cell chip for parallel analysis, while they are examined and processed one by one in a microchannel in the case of a series cell chip. In contrast to these biochips for high-throughput analysis of a large number of cells, single-cell chips focus on the analysis of a single isolated cell. As in DNA microarrays, where a large number of oligonucleotides are ordered in a matrix array, parallel cell chips order living cells in a similar way. At each point of the array, the cells can be isolated, provided that the cell type allows this, e.g., blood cells, or cultivated in groups (most adhesion cells can only survive in groups). The aim is to allow massively parallel analysis or processing. Le Pioufle et al. describe a microdevice for the culture of single cells or small groups of cells in a micropit array [2]. Each pit is equipped to stimulate the cell or group of cells either electrically or fluidically. Among the applications envisaged are gene transfer, cell sorting, and screening in pharmacology. A complementary approach, combining the DNA microarray and cell biochip ideas, has been put forward by Bailey et al. [3]. Genes previously arrayed on the chip transfect the cultured cells on the substrate depending on their position in the array (see Fig. 19.1). This way of achieving differential lipofection on a chip was then taken up again by Yoshikawa et al. [4] with primary cells, more

  17. Cell division

    MedlinePlus Videos and Cool Tools

    ... structure made up of 16 cells. This structure is called a morula, which is Latin for mulberry. The cells continue to divide ... days following conception into a blastocyst. Although it is only the size of a pinhead, the blastocyst ...

  18. Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV): implications for the immune control of EBV-positive malignancies.

    PubMed

    Murray, R J; Kurilla, M G; Brooks, J M; Thomas, W A; Rowe, M; Kieff, E; Rickinson, A B

    1992-07-01

    Epstein-Barr virus (EBV), a human herpes virus with oncogenic potential, persists in B lymphoid tissues and is controlled by virus-specific cytotoxic T lymphocyte (CTL) surveillance. On reactivation in vitro, these CTLs recognize EBV-transformed lymphoblastoid cell lines (LCLs) in an HLA class I antigen-restricted fashion, but the viral antigens providing target epitopes for such recognition remain largely undefined. Here we have tested EBV-induced polyclonal CTL preparations from 16 virus-immune donors on appropriate fibroblast targets in which the eight EBV latent proteins normally found in LCLs (Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 3B, 3C, leader protein [LP], and latent membrane protein [LMP] 1 and 2) have been expressed individually from recombinant vaccinia virus vectors. Most donors gave multicomponent responses with two or more separate reactivities against different viral antigens. Although precise target antigen choice was clearly influenced by the donor's HLA class I type, a subset of latent proteins, namely EBNA 3A, 3B, and 3C, provided the dominant targets on a range of HLA backgrounds; thus, 15 of 16 donors gave CTL responses that contained reactivities to one or more proteins of this subset. Examples of responses to other latent proteins, namely LMP 2 and EBNA 2, were detected through specific HLA determinants, but we did not observe reactivities to EBNA 1, EBNA LP, or LMP 1. The bulk polyclonal CTL response in one donor, and components of that response in others, did not map to any of the known latent proteins, suggesting that other viral target antigens remain to be identified. This work has important implications for CTL control over EBV-positive malignancies where virus gene expression is often limited to specific subsets of latent proteins. PMID:1319456

  19. Application of the TGx-28.65 transcriptomic biomarker to classify genotoxic and non-genotoxic chemicals in human TK6 cells in the presence of rat liver S9.

    PubMed

    Yauk, Carole L; Buick, Julie K; Williams, Andrew; Swartz, Carol D; Recio, Leslie; Li, Heng-Hong; Fornace, Albert J; Thomson, Errol M; Aubrecht, Jiri

    2016-05-01

    In vitro transcriptional signatures that predict toxicities can facilitate chemical screening. We previously developed a transcriptomic biomarker (known as TGx-28.65) for classifying agents as genotoxic (DNA damaging) and non-genotoxic in human lymphoblastoid TK6 cells. Because TK6 cells do not express cytochrome P450s, we confirmed accurate classification by the biomarker in cells co-exposed to 1% 5,6 benzoflavone/phenobarbital-induced rat liver S9 for metabolic activation. However, chemicals may require different types of S9 for activation. Here we investigated the response of TK6 cells to higher percentages of Aroclor-, benzoflavone/phenobarbital-, or ethanol-induced rat liver S9 to expand TGx-28.65 biomarker applicability. Transcriptional profiles were derived 3 to 4 hr following a 4 hr co-exposure of TK6 cells to test chemicals and S9. Preliminary studies established that 10% Aroclor- and 5% ethanol-induced S9 alone did not induce the TGx-28.65 biomarker genes. Seven genotoxic and two non-genotoxic chemicals (and concurrent solvent and positive controls) were then tested with one of the S9s (selected based on cell survival and micronucleus induction). Relative survival and micronucleus frequency was assessed by flow cytometry in cells 20 hr post-exposure. Genotoxic/non-genotoxic chemicals were accurately classified using the different S9s. One technical replicate of cells co-treated with dexamethasone and 10% Aroclor-induced S9 was falsely classified as genotoxic, suggesting caution in using high S9 concentrations. Even low concentrations of genotoxic chemicals (those not causing cytotoxicity) were correctly classified, demonstrating that TGx-28.65 is a sensitive biomarker of genotoxicity. A meta-analysis of datasets from 13 chemicals supports that different S9s can be used in TK6 cells, without impairing classification using the TGx-28.65 biomarker. Environ. Mol. Mutagen. 57:243-260, 2016. © 2016 Her Majesty the Queen in Right of Canada. Environmental and

  20. Solar cells

    NASA Astrophysics Data System (ADS)

    Cuquel, A.; Roussel, M.

    The physical and electronic characteristics of solar cells are discussed in terms of space applications. The principles underlying the photovoltaic effect are reviewed, including an analytic model for predicting the performance of individual cells and arrays of cells. Attention is given to the effects of electromagnetic and ionizing radiation, micrometeors, thermal and mechanical stresses, pollution and degassing encountered in space. The responses of different types of solar cells to the various performance-degrading agents are examined, with emphasis on techniques for quality assurance in the manufacture and mounting of Si cells.

  1. Types of Stem Cells

    MedlinePlus

    ... PDF) Download an introduction to stem cells and stem cell research. Stem Cell Glossary Stem cell terms to know. ... stem cells blog from the International Society for Stem Cell Research. Learn About Stem Cells From Lab to You ...

  2. Electrolytic cell

    NASA Astrophysics Data System (ADS)

    Bullock, J. S.; Hale, B. D.

    1984-09-01

    An apparatus is described for the separation of the anolyte and the catholyte during electrolysis. The electrolyte flows through an electrolytic cell between the oppositely charged electrodes. The cell is equipped with a wedge-shaped device, the tapered end is located between the electrodes on the effluent side of the cell. The wedge diverts the flow of the electrolyte to either side of the wedge, substantially separating the anolyte and the catholyte.

  3. Cell Chauvinism

    ERIC Educational Resources Information Center

    Keller, Dolores Elaine

    1972-01-01

    Indicates that biological terminology, such as mother cell'' and labels of sex factors in bacteria, reflect discrimination against females by reinforcing perpetuation of stereotyped gender roles. (AL)

  4. Cell migration.

    PubMed

    Trepat, Xavier; Chen, Zaozao; Jacobson, Ken

    2012-10-01

    Cell migration is fundamental to establishing and maintaining the proper organization of multicellular organisms. Morphogenesis can be viewed as a consequence, in part, of cell locomotion, from large-scale migrations of epithelial sheets during gastrulation, to the movement of individual cells during development of the nervous system. In an adult organism, cell migration is essential for proper immune response, wound repair, and tissue homeostasis, while aberrant cell migration is found in various pathologies. Indeed, as our knowledge of migration increases, we can look forward to, for example, abating the spread of highly malignant cancer cells, retarding the invasion of white cells in the inflammatory process, or enhancing the healing of wounds. This article is organized in two main sections. The first section is devoted to the single-cell migrating in isolation such as occurs when leukocytes migrate during the immune response or when fibroblasts squeeze through connective tissue. The second section is devoted to cells collectively migrating as part of multicellular clusters or sheets. This second type of migration is prevalent in development, wound healing, and in some forms of cancer metastasis. PMID:23720251

  5. Cell Migration

    PubMed Central

    Trepat, Xavier; Chen, Zaozao; Jacobson, Ken

    2015-01-01

    Cell migration is fundamental to establishing and maintaining the proper organization of multicellular organisms. Morphogenesis can be viewed as a consequence, in part, of cell locomotion, from large-scale migrations of epithelial sheets during gastrulation, to the movement of individual cells during development of the nervous system. In an adult organism, cell migration is essential for proper immune response, wound repair, and tissue homeostasis, while aberrant cell migration is found in various pathologies. Indeed, as our knowledge of migration increases, we can look forward to, for example, abating the spread of highly malignant cancer cells, retarding the invasion of white cells in the inflammatory process, or enhancing the healing of wounds. This article is organized in two main sections. The first section is devoted to the single-cell migrating in isolation such as occurs when leukocytes migrate during the immune response or when fibroblasts squeeze through connective tissue. The second section is devoted to cells collectively migrating as part of multicellular clusters or sheets. This second type of migration is prevalent in development, wound healing, and in some forms of cancer metastasis. PMID:23720251

  6. Cell Trivision of Hyperploid Cells

    PubMed Central

    Nagy, Gabor; Kiraly, Gabor; Turani, Melinda

    2013-01-01

    Malignant transformation is likely to render cells hyperploid, primarily tetraploid. We have measured the frequency of division into three rather than two daughter cells as a function of ploidy. Such trivisions were followed in near-tetraploid uveal melanoma (UM), hypotetraploid HaCaT (<4 N), hypertriploid HeLa (>3 N), and in near-diploid (∼2 N) lung epithelial cell lines by time-lapse image analyses. A stepwise analysis of cytokinesis revealed higher frequency of cell trivisions relative to divisions in hyperploid HeLa (1:24, 4%), HaCaT (1:126, 8%), and UM (1:186, 0.5%) cells. The occurrence of trivision was significantly lower in near-diploid endothelial cells (1:1400, 0.07%). We have previously observed the phenomenon of trivision in HaCaT cells treated with heavy metal lead, and here we describe that trivision is a spontaneous process taking place without genotoxic treatment. Beside re-diploidization by trivision, the hyperploid state decreases the cell size of the daughter cells and is likely to increase the time of cytokinesis. On the basis of the results, it is hypothesized that among other cancer-related causes, hyperploidy could be related to cell trivision, could cause random aneuploidy, and could generate new cancer-specific karyotypes. PMID:24093497

  7. Photovoltaic cell

    SciTech Connect

    Bronstein-Bonte, I.Y.; Fischer, A.B.

    1986-12-16

    This patent describes a product comprising a photovoltaic cell including a luminescent dye which will absorb radiation at a wavelength to which the cell is not significantly responsive and emit radiation at a higher wavelength at which it is responsive. The improvement described here is wherein the dye comprises a lepidopterene.

  8. Fuel Cells

    ERIC Educational Resources Information Center

    Hawkins, M. D.

    1973-01-01

    Discusses the theories, construction, operation, types, and advantages of fuel cells developed by the American space programs. Indicates that the cell is an ideal small-scale power source characterized by its compactness, high efficiency, reliability, and freedom from polluting fumes. (CC)

  9. Cell Lines

    PubMed Central

    Cherbas, Lucy; Gong, Lei

    2014-01-01

    We review the properties and uses of cell lines in Drosophila research, emphasizing the variety of lines, the large body of genomic and transcriptional data available for many of the lines, and the variety of ways the lines have been used to provide tools for and insights into the developmental, molecular, and cell biology of Drosophila and mammals. PMID:24434506

  10. Host cells and cell banking.

    PubMed

    Stacey, Glyn N; Merten, Otto-Wilhelm

    2011-01-01

    Gene therapy based on the use of viral vectors is entirely dependent on the use of animal cell lines, mainly of mammalian origin, but also of insect origin. As for any biotechnology product for clinical use, viral -vectors have to be produced with cells derived from an extensively characterized cell bank to maintain the appropriate standard for assuring the lowest risk for the patients to be treated. Although many different cell types and lines have been used for the production of viral vectors, HEK293 cells or their derivatives have been extensively used for production of different vector types: adenovirus, oncorectrovirus, lentivirus, and AAV vectors, because of their easy handling and the possibility to grow them adherently in serum-containing medium as well as in suspension in serum-free culture medium. Despite this, these cells are not necessarily the best for the production of a given viral vector, and there are many other cell lines with significant advantages including superior growth and/or production characteristics, which have been tested and also used for the production of clinical vector batches. This chapter presents basic -considerations concerning the characterization of cell banks, in the first part, and, in the second part, practically all cell lines (at least when public information was available) established and developed for the production of the most important viral vectors (adenoviral, oncoretroviral, lentiviral, AAV, baculovirus). PMID:21590393

  11. Fuel cells 101

    SciTech Connect

    Hirschenhofer, J.H.

    1999-07-01

    This paper discusses the various types of fuel cells, the importance of cell voltage, fuel processing for natural gas, cell stacking, fuel cell plant description, advantages and disadvantages of the types of fuel cells, and applications. The types covered include: polymer electrolyte fuel cell, alkaline fuel cell, phosphoric acid fuel cell; molten carbonate fuel cell, and solid oxide fuel cell.

  12. Cell polarity

    PubMed Central

    Romereim, Sarah M

    2011-01-01

    Despite extensive genetic analysis of the dynamic multi-phase process that transforms a small population of lateral plate mesoderm into the mature limb skeleton, the mechanisms by which signaling pathways regulate cellular behaviors to generate morphogenetic forces are not known. Recently, a series of papers have offered the intriguing possibility that regulated cell polarity fine-tunes the morphogenetic process via orienting cell axes, division planes and cell movements. Wnt5a-mediated non-canonical signaling, which may include planar cell polarity, has emerged as a common thread in the otherwise distinct signaling networks that regulate morphogenesis in each phase of limb development. These findings position the limb as a key model to elucidate how global tissue patterning pathways direct local differences in cell behavior that, in turn, generate growth and form. PMID:22064549

  13. 9. ENGINE TEST CELL BUILDING INTERIOR. CELL ACCESS ELEVATOR, CELLS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    9. ENGINE TEST CELL BUILDING INTERIOR. CELL ACCESS ELEVATOR, CELLS 2 AND 4, BASEMENT LEVEL. LOOKING SOUTHEAST. - Fairchild Air Force Base, Engine Test Cell Building, Near intersection of Arnold Street & George Avenue, Spokane, Spokane County, WA

  14. Squamous cell skin cancer

    MedlinePlus

    ... cell; NMSC - squamous cell; Squamous cell skin cancer; Squamous cell carcinoma of the skin ... squamous cell cancer is called Bowen disease (or squamous cell carcinoma in situ). This type does not spread to ...

  15. Electrochemical cell

    DOEpatents

    Redey, L.I.; Vissers, D.R.; Prakash, J.

    1996-07-16

    An electrochemical cell is described having a bimodal positive electrode, a negative electrode of an alkali metal, and a compatible electrolyte including an alkali metal salt molten at the cell operating temperature. The positive electrode has an electrochemically active layer of at least one transition metal chloride at least partially present as a charging product, and additives of bromide and/or iodide and sulfur in the positive electrode or the electrolyte. Electrode volumetric capacity is in excess of 400 Ah/cm{sup 3}; the cell can be 90% recharged in three hours and can operate at temperatures below 160 C. There is also disclosed a method of reducing the operating temperature and improving the overall volumetric capacity of an electrochemical cell and for producing a positive electrode having a BET area greater than 6{times}10{sup 4}cm{sup 2}/g of Ni. 6 figs.

  16. Electrochemical cell

    DOEpatents

    Redey, Laszlo I.; Vissers, Donald R.; Prakash, Jai

    1994-01-01

    An electrochemical cell having a bimodal positive electrode, a negative electrode of an alkali metal, and a compatible electrolyte including an alkali metal salt molten at the cell operating temperature. The positive electrode has an electrochemically active layer of at least one transition metal chloride at least partially present as a charging product, and additives of bromide and/or iodide and sulfur in the positive electrode or the electrolyte. Electrode volumetric capacity is in excess of 400 Ah/cm.sup.3 ; the cell can be 90% recharged in three hours and can operate at temperatures below 160.degree. C. There is also disclosed a method of reducing the operating temperature and improving the overall volumetric capacity of an electrochemical cell and for producing a positive electrode having a BET area greater than 6.times.10.sup.4 cm.sup.2 /g of Ni.

  17. Electrochemical cell

    DOEpatents

    Redey, Laszlo I.; Vissers, Donald R.; Prakash, Jai

    1996-01-01

    An electrochemical cell having a bimodal positive electrode, a negative electrode of an alkali metal, and a compatible electrolyte including an alkali metal salt molten at the cell operating temperature. The positive electrode has an electrochemically active layer of at least one transition metal chloride at least partially present as a charging product, and additives of bromide and/or iodide and sulfur in the positive electrode or the electrolyte. Electrode volumetric capacity is in excess of 400 Ah/cm.sup.3 ; the cell can be 90% recharged in three hours and can operate at temperatures below 160.degree. C. There is also disclosed a method of reducing the operating temperature and improving the overall volumetric capacity of an electrochemical cell and for producing a positive electrode having a BET area greater than 6.times.10.sup.4 cm.sup.2 /g of Ni.

  18. Electrochemical cell

    DOEpatents

    Redey, L.I.; Vissers, D.R.; Prakash, J.

    1994-02-01

    An electrochemical cell is described having a bimodal positive electrode, a negative electrode of an alkali metal, and a compatible electrolyte including an alkali metal salt molten at the cell operating temperature. The positive electrode has an electrochemically active layer of at least one transition metal chloride at least partially present as a charging product, and additives of bromide and/or iodide and sulfur in the positive electrode or the electrolyte. Electrode volumetric capacity is in excess of 400 Ah/cm[sup 3]; the cell can be 90% recharged in three hours and can operate at temperatures below 160 C. There is also disclosed a method of reducing the operating temperature and improving the overall volumetric capacity of an electrochemical cell and for producing a positive electrode having a BET area greater than 6[times]10[sup 4] cm[sup 2]/g of Ni. 8 figures.

  19. Dry cell battery poisoning

    MedlinePlus

    Batteries - dry cell ... Acidic dry cell batteries contain: Manganese dioxide Ammonium chloride Alkaline dry cell batteries contain: Sodium hydroxide Potassium hydroxide Lithium dioxide dry cell batteries ...

  20. Load cell

    DOEpatents

    Spletzer, Barry L.

    2001-01-01

    A load cell combines the outputs of a plurality of strain gauges to measure components of an applied load. Combination of strain gauge outputs allows measurement of any of six load components without requiring complex machining or mechanical linkages to isolate load components. An example six axis load cell produces six independent analog outputs which can be combined to determine any one of the six general load components.

  1. Load cell

    DOEpatents

    Spletzer, B.L.

    1998-12-15

    A load cell combines the outputs of a plurality of strain gauges to measure components of an applied load. Combination of strain gauge outputs allows measurement of any of six load components without requiring complex machining or mechanical linkages to isolate load components. An example six axis load cell produces six independent analog outputs, each directly proportional to one of the six general load components. 16 figs.

  2. Load cell

    DOEpatents

    Spletzer, Barry L.

    1998-01-01

    A load cell combines the outputs of a plurality of strain gauges to measure components of an applied load. Combination of strain gauge outputs allows measurement of any of six load components without requiring complex machining or mechanical linkages to isolate load components. An example six axis load cell produces six independent analog outputs, each directly proportional to one of the six general load components.

  3. Electrochemical cell

    DOEpatents

    Redey, Laszlo I.; Vissers, Donald R.; Prakash, Jai

    1994-01-01

    An electrochemical cell having an alkali metal negative electrode such as sodium and a positive electrode including Ni or transition metals, separated by a .beta." alumina electrolyte and NaAlCl.sub.4 or other compatible material. Various concentrations of a bromine, iodine and/or sulfur containing additive and pore formers are disclosed, which enhance cell capacity and power. The pore formers may be the ammonium salts of carbonic acid or a weak organic acid or oxamide or methylcellulose.

  4. Cell sealant

    SciTech Connect

    Markin, C.; Book, R.J.; James, D.A.

    1988-04-26

    An electrochemical cell is described comprising an anode, a cathode and an electrolyte disposed within an open ended cylindrical metallic cell container, with an insulative cell top member being positioned within the open end of a sealant at the interface between the cell top member and the metallic cell container. The sealant is a mixture of a Type 2 BUR asphalt and an elastomeric material selected from the group consisting of (cis-1,4-polyisoprene), styrene-butadiene copolymer (SBR), cis-1,4-polybutadiene and styrene butadiene styrene (SBS), styrene isoprene styrene (SIS), neoprene (poly-chloprene), acrylonitrile-butadiene copolymer (NBR), ethylene-propylene elastomers (EPR), butyl rubber (copolymers of isobutylene), urethane, nitrile (polymers of butadiene and acrylonitrile), polysulfide, polyacrylate, silicone, chlorosulfonated polyethylene, and EPDM (terpolymers of ethylene, propylene and diene monomers), and mixtures thereof, and wherein the elastomeric material is substantially inert to the electrolyte and is present in an amount between 0.5% to 10% by weight of the asphalt.

  5. Electrochemical cell

    DOEpatents

    Redey, Laszlo I.; Myles, Kevin M.; Vissers, Donald R.; Prakash, Jai

    1996-01-01

    An electrochemical cell with a positive electrode having an electrochemically active layer of at least one transition metal chloride. A negative electrode of an alkali metal and a compatible electrolyte including an alkali metal salt molten at cell operating temperature is included in the cell. The electrolyte is present at least partially as a corrugated .beta." alumina tube surrounding the negative electrode interior to the positive electrode. The ratio of the volume of liquid electrolyte to the volume of the positive electrode is in the range of from about 0.1 to about 3. A plurality of stacked electrochemical cells is disclosed each having a positive electrode, a negative electrode of an alkali metal molten at cell operating temperature, and a compatible electrolyte. The electrolyte is at least partially present as a corrugated .beta." alumina sheet separating the negative electrode and interior to the positive electrodes. The alkali metal is retained in a porous electrically conductive ceramic, and seals for sealing the junctures of the electrolyte and the adjacent electrodes at the peripheries thereof.

  6. Electrochemical cell

    DOEpatents

    Redey, L.I.; Myles, K.M.; Vissers, D.R.; Prakash, J.

    1996-07-02

    An electrochemical cell is described with a positive electrode having an electrochemically active layer of at least one transition metal chloride. A negative electrode of an alkali metal and a compatible electrolyte including an alkali metal salt molten at cell operating temperature is included in the cell. The electrolyte is present at least partially as a corrugated {beta}{double_prime} alumina tube surrounding the negative electrode interior to the positive electrode. The ratio of the volume of liquid electrolyte to the volume of the positive electrode is in the range of from about 0.1 to about 3. A plurality of stacked electrochemical cells is disclosed each having a positive electrode, a negative electrode of an alkali metal molten at cell operating temperature, and a compatible electrolyte. The electrolyte is at least partially present as a corrugated {beta}{double_prime} alumina sheet separating the negative electrode and interior to the positive electrodes. The alkali metal is retained in a porous electrically conductive ceramic, and seals for sealing the junctures of the electrolyte and the adjacent electrodes at the peripheries thereof. 8 figs.

  7. Electrochemical cell

    DOEpatents

    Nagy, Zoltan; Yonco, Robert M.; You, Hoydoo; Melendres, Carlos A.

    1992-01-01

    An electrochemical cell has a layer-type or sandwich configuration with a Teflon center section that houses working, reference and counter electrodes and defines a relatively narrow electrolyte cavity. The center section is surrounded on both sides with thin Teflon membranes. The membranes are pressed in place by a pair of Teflon inner frames which are in turn supported by a pair of outer metal frames. The pair of inner and outer frames are provided with corresponding, appropriately shaped slits that are in plane generally transverse to the plane of the working electrode and permit X-ray beams to enter and exit the cell through the Teflon membranes that cover the slits so that the interface between the working electrode and the electrolyte within the cell may be analyzed by transmission geometry. In one embodiment, the center section consists of two parts, one on top of the other. Alternatively, the center section of the electrochemical cell may consist of two intersliding pieces or may be made of a single piece of Teflon sheet material. The electrolyte cavity is shaped so that the electrochemical cell can be rotated 90.degree. in either direction while maintaining the working and counter electrodes submerged in the electrolyte.

  8. Electrochemical cell

    DOEpatents

    Nagy, Z.; Yonco, R.M.; You, H.; Melendres, C.A.

    1992-08-25

    An electrochemical cell has a layer-type or sandwich configuration with a Teflon center section that houses working, reference and counter electrodes and defines a relatively narrow electrolyte cavity. The center section is surrounded on both sides with thin Teflon membranes. The membranes are pressed in place by a pair of Teflon inner frames which are in turn supported by a pair of outer metal frames. The pair of inner and outer frames are provided with corresponding, appropriately shaped slits that are in plane generally transverse to the plane of the working electrode and permit X-ray beams to enter and exit the cell through the Teflon membranes that cover the slits so that the interface between the working electrode and the electrolyte within the cell may be analyzed by transmission geometry. In one embodiment, the center section consists of two parts, one on top of the other. Alternatively, the center section of the electrochemical cell may consist of two intersliding pieces or may be made of a single piece of Teflon sheet material. The electrolyte cavity is shaped so that the electrochemical cell can be rotated 90[degree] in either direction while maintaining the working and counter electrodes submerged in the electrolyte. 5 figs.

  9. Cell Phones

    PubMed Central

    Sansone, Lori A.

    2013-01-01

    Cell phones are a relatively novel and evolving technology. While the potential benefits of this technology continue to emerge, so do the potential psychosocial risks. For example, one psychosocial risk is user stress, which appears to be related to feeling compelled to promptly respond to cell-phone activity in order to maintain spontaneity and access with others. Other potential psychosocial risks include disruptions in sleep; the user’s risk of exposure to cyberbullying, particularly the unwanted exposure of photographs and/or videos of the victim; and overuse, particularly among adolescents. With regard to the latter phenomenon, the boundaries among overuse, misuse, dependence, and addiction are not scientifically clear. Therefore, while cell phones are a convenient and expedient technology, they are not without their potential psychosocial hazards. PMID:23439568

  10. Solar cells

    NASA Astrophysics Data System (ADS)

    Treble, F. C.

    1980-11-01

    The history, state of the art, and future prospects of solar cells are reviewed. Solar cells are already competitive in a wide range of low-power applications, and during the 1980's they are expected to become cheaper to run than diesel or gasoline generators, the present mainstay of isolated communities. At this stage they will become attractive for water pumping, irrigation, and rural electrification, particularly in developing countries. With further cost reduction, they may be used to augment grid supplies in domestic, commercial, institutional, and industrial premises. Cost reduction to the stage where photovoltaics becomes economic for large-scale power generation in central stations depends on a technological breakthrough in the development of thin-film cells. DOE aims to reach this goal by 1990, so that by the end of the century about 20% of the estimated annual additions to their electrical generating capacity will be photovoltaic.

  11. Electrochemical cell

    DOEpatents

    Kaun, T.D.

    An improved secondary electrochemical cell is disclosed having a negative electrode of lithium aluminum, a positive electrode of iron sulfide, a molten electrolyte of lithium chloride and potassium chloride, and the combination that the fully charged theoretical capacity of the negative electrode is in the range of 0.5 to 1.0 that of the positive electrode. The cell thus is negative electrode limiting during discharge cycling. Preferably, the negative electrode contains therein, in the approximate range of 1 to 10 volume % of the electrode, an additive from the materials of graphitized carbon, aluminum-iron alloy, and/or magnesium oxide.

  12. Electrochemical cell

    DOEpatents

    Redey, L.I.; Vissers, D.R.; Prakash, J.

    1994-08-23

    An electrochemical cell is described having an alkali metal negative electrode such as sodium and a positive electrode including Ni or transition metals, separated by a [beta] alumina electrolyte and NaAlCl[sub 4] or other compatible material. Various concentrations of a bromine, iodine and/or sulfur containing additive and pore formers are disclosed, which enhance cell capacity and power. The pore formers may be the ammonium salts of carbonic acid or a weak organic acid or oxamide or methylcellulose. 6 figs.

  13. Cell Libraries

    NASA Technical Reports Server (NTRS)

    1994-01-01

    A NASA contract led to the development of faster and more energy efficient semiconductor materials for digital integrated circuits. Gallium arsenide (GaAs) conducts electrons 4-6 times faster than silicon and uses less power at frequencies above 100-150 megahertz. However, the material is expensive, brittle, fragile and has lacked computer automated engineering tools to solve this problem. Systems & Processes Engineering Corporation (SPEC) developed a series of GaAs cell libraries for cell layout, design rule checking, logic synthesis, placement and routing, simulation and chip assembly. The system is marketed by Compare Design Automation.

  14. Electrochemical cell

    DOEpatents

    Kaun, Thomas D.

    1984-01-01

    An improved secondary electrochemical cell is disclosed having a negative electrode of lithium aluminum, a positive electrode of iron sulfide, a molten electrolyte of lithium chloride and potassium chloride, and the combination that the fully charged theoretical capacity of the negative electrode is in the range of 0.5-1.0 that of the positive electrode. The cell thus is negative electrode limiting during discharge cycling. Preferably, the negative electrode contains therein, in the approximate range of 1-10 volume % of the electrode, an additive from the materials of graphitized carbon, aluminum-iron alloy, and/or magnesium oxide.

  15. Stem Cell Basics

    MedlinePlus

    ... stem cells? What are the potential uses of human stem cells and the obstacles that must be overcome before ... two kinds of stem cells from animals and humans: embryonic stem cells and non-embryonic "somatic" or "adult" stem cells . ...

  16. Stem Cell Information: Glossary

    MedlinePlus

    ... based therapies Cell culture Cell division Chromosome Clone Cloning Cord blood stem cells Culture medium Differentiation Directed ... Pluripotent Polar body Preimplantation Proliferation Regenerative medicine Reproductive cloning Signals Somatic cell Somatic cell nuclear transfer (SCNT) ...

  17. Learn About Stem Cells

    MedlinePlus

    ... PDF) Download an introduction to stem cells and stem cell research. Stem Cell Glossary Stem cell terms to know. ... ISSCR Get Involved Media © 2015 International Society for Stem Cell Research Terms of Use Disclaimer Privacy Policy

  18. Photoelectrodialytic cell

    DOEpatents

    Murphy, G.W.

    1983-09-13

    A multicompartment photoelectrodialytic demineralization cell is provided with a buffer compartment interposed between the product compartment and a compartment containing an electrolyte solution. Semipermeable membranes separate the buffer compartment from the product and electrolyte compartments. The buffer compartment is flushed to prevent leakage of the electrolyte compartment from entering the product compartment. 3 figs.

  19. Photovoltaic cell

    DOEpatents

    Gordon, Roy G.; Kurtz, Sarah

    1984-11-27

    In a photovoltaic cell structure containing a visibly transparent, electrically conductive first layer of metal oxide, and a light-absorbing semiconductive photovoltaic second layer, the improvement comprising a thin layer of transition metal nitride, carbide or boride interposed between said first and second layers.

  20. Nonaqueous cell

    SciTech Connect

    Kalnoki-kis, T.

    1981-07-07

    A nonaqueous cell is disclosed that utlizes an active metal anode, such as lithium, a cathode collector and an ionically conductive cathode electrolyte comprising a solute dissolved in a liquid cathode, such as an oxyhalide, and wherein a vinyl polymer is dissolved in the cathode-electrolyte.

  1. Potent Cells

    ERIC Educational Resources Information Center

    Liu, Dennis

    2007-01-01

    It seems hard to believe that Dolly the cloned sheep was born 10 years ago, kindling furious arguments over the prospects and ethics of cloning a human. Today, the controversy over cloning is entwined, often confused, with concerns over the use of human embryonic stem cells. Most people are unclear what cloning is, and they know even less when it…

  2. 19. Oblique, typical cell (south cells) from rear of cell; ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    19. Oblique, typical cell (south cells) from rear of cell; view to north, 65mm lens with electronic flash illumination. - Tule Lake Project Jail, Post Mile 44.85, State Route 139, Newell, Modoc County, CA

  3. Deviation from additivity in mixture toxicity: relevance of nonlinear dose-response relationships and cell line differences in genotoxicity assays with combinations of chemical mutagens and gamma-radiation.

    PubMed Central

    Lutz, Werner K; Vamvakas, Spyros; Kopp-Schneider, Annette; Schlatter, Josef; Stopper, Helga

    2002-01-01

    Sublinear dose-response relationships are often seen in toxicity testing, particularly with bioassays for carcinogenicity. This is the result of a superimposition of various effects that modulate and contribute to the process of cancer formation. Examples are saturation of detoxification pathways or DNA repair with increasing dose, or regenerative hyperplasia and indirect DNA damage as a consequence of high-dose cytotoxicity and cell death. The response to a combination treatment can appear to be supra-additive, although it is in fact dose-additive along a sublinear dose-response curve for the single agents. Because environmental exposure of humans is usually in a low-dose range and deviation from linearity is less likely at the low-dose end, combination effects should be tested at the lowest observable effect levels (LOEL) of the components. This principle has been applied to combinations of genotoxic agents in various cellular models. For statistical analysis, all experiments were analyzed for deviation from additivity with an n-factor analysis of variance with an interaction term, n being the number of components tested in combination. Benzo[a]pyrene, benz[a]anthracene, and dibenz[a,c]anthracene were tested at the LOEL, separately and in combination, for the induction of revertants in the Ames test, using Salmonella typhimurium TA100 and rat liver S9 fraction. Combined treatment produced no deviation from additivity. The induction of micronuclei in vitro was investigated with ionizing radiation from a 137Cs source and ethyl methanesulfonate. Mouse lymphoma L5178Y cells revealed a significant 40% supra-additive combination effect in an experiment based on three independent replicates for controls and single and combination treatments. On the other hand, two human lymphoblastoid cell lines (TK6 and WTK1) as well as a pilot study with human primary fibroblasts from fetal lung did not show deviation from additivity. Data derived from one cell line should therefore

  4. Reprogrammed pluripotent stem cells from somatic cells.

    PubMed

    Kim, Jong Soo; Choi, Hyun Woo; Choi, Sol; Do, Jeong Tae

    2011-06-01

    Pluripotent stem cells, such as embryonic stem (ES) cells, can differentiate into all cell types. So, these cells can be a biological resource for regenerative medicine. However, ES cells known as standard pluripotent cells have problem to be used for cell therapy because of ethical issue of the origin and immune response on the graft. Hence, recently reprogrammed pluripotent cells have been suggested as an alternative source for regenerative medicine. Somatic cells can acquire the ES cell-like pluripotency by transferring somatic cell nuclei into oocytes, by cell fusion with pluripotent cells. Retroviral-mediated introduction of four factors, Oct4, Sox2, Klf4 and c-Myc can successfully reprogram somatic cells into ES cell-like pluripotent stem cells, known as induced pluripotent stem (iPS) cells. These cells closely resemble ES cells in gene expression pattern, cell biologic and phenotypic characteristics. However, to reach the eventual goal of clinical application, it is necessary to overcome the major drawbacks such as low reprogramming efficiency and genomic alterations due to viral integration. In this review, we discuss the current reprogramming techniques and mechanisms of nuclear reprogramming induced by transcription factor transduction. PMID:24298328

  5. Ghost cell lesions

    PubMed Central

    Rajesh, E.; Jimson, Sudha; Masthan, K. M. K.; Balachander, N.

    2015-01-01

    Ghost cells have been a controversy for a long time. Ghost cell is a swollen/enlarged epithelial cell with eosnophilic cytoplasm, but without a nucleus. In routine H and E staining these cells give a shadowy appearance. Hence these cells are also called as shadow cells or translucent cells. The appearance of these cells varies from lesion to lesion involving odontogenic and nonodontogenic lesions. This article review about the origin, nature and significance of ghost cells in different neoplasms. PMID:26015694

  6. Crystalline Silicon Solar Cells

    NASA Astrophysics Data System (ADS)

    Green, Martin A.

    2015-10-01

    The following sections are included: * Overview * Silicon cell development * Substrate production * Cell processing * Cell costs * Opportunities for improvement * Silicon-supported thin films * Summary * Acknowledgement * References

  7. Red blood cells, sickle cell (image)

    MedlinePlus

    Sickle cell anemia is an inherited blood disease in which the red blood cells produce abnormal pigment (hemoglobin). ... abnormal hemoglobin causes deformity of the red blood cells into crescent or sickle-shapes, as seen in this photomicrograph.

  8. Red blood cells, sickle cells (image)

    MedlinePlus

    These crescent or sickle-shaped red blood cells (RBCs) are present with Sickle cell anemia, and stand out clearly against the normal round RBCs. These abnormally shaped cells may become entangled and ...

  9. Red blood cells, sickle cell (image)

    MedlinePlus

    Sickle cell anemia is an inherited blood disease in which the red blood cells produce abnormal pigment (hemoglobin). The abnormal hemoglobin causes deformity of the red blood cells into crescent or sickle-shapes, as seen in this photomicrograph.

  10. Red blood cells, multiple sickle cells (image)

    MedlinePlus

    Sickle cell anemia is an inherited disorder in which abnormal hemoglobin (the red pigment inside red blood cells) is produced. The abnormal hemoglobin causes red blood cells to assume a sickle shape, like the ones seen in this photomicrograph.

  11. Robotic adherent cell injection for characterizing cell-cell communication.

    PubMed

    Liu, Jun; Siragam, Vinayakumar; Gong, Zheng; Chen, Jun; Fridman, Michael D; Leung, Clement; Lu, Zhe; Ru, Changhai; Xie, Shaorong; Luo, Jun; Hamilton, Robert M; Sun, Yu

    2015-01-01

    Compared to robotic injection of suspended cells (e.g., embryos and oocytes), fewer attempts were made to automate the injection of adherent cells (e.g., cancer cells and cardiomyocytes) due to their smaller size, highly irregular morphology, small thickness (a few micrometers thick), and large variations in thickness across cells. This paper presents a robotic system for automated microinjection of adherent cells. The system is embedded with several new capabilities: automatically locating micropipette tips; robustly detecting the contact of micropipette tip with cell culturing surface and directly with cell membrane; and precisely compensating for accumulative positioning errors. These new capabilities make it practical to perform adherent cell microinjection truly via computer mouse clicking in front of a computer monitor, on hundreds and thousands of cells per experiment (versus a few to tens of cells as state of the art). System operation speed, success rate, and cell viability rate were quantitatively evaluated based on robotic microinjection of over 4000 cells. This paper also reports the use of the new robotic system to perform cell-cell communication studies using large sample sizes. The gap junction function in a cardiac muscle cell line (HL-1 cells), for the first time, was quantified with the system. PMID:25073160

  12. Sickle cell test

    MedlinePlus

    The sickle cell test looks for the abnormal hemoglobin in the blood that causes the disease sickle cell anemia . ... if a person has abnormal hemoglobin that causes sickle cell disease and sickle cell trait. Hemoglobin is a ...

  13. Sickle cell anemia

    MedlinePlus

    Anemia - sickle cell; Hemoglobin SS disease (Hb SS); Sickle cell disease ... Sickle cell anemia is caused by an abnormal type of hemoglobin called hemoglobin S. Hemoglobin is a protein inside red blood cells ...

  14. Stem cell glycolipids.

    PubMed

    Yanagisawa, Makoto

    2011-09-01

    Glycolipids are compounds containing one or more monosaccharide residues bound by a glycosidic linkage to a hydrophobic moiety. Because of their expression patterns and the intracellular localization patterns, glycolipids, including stage-specific embryonic antigens (SSEA-3, SSEA-4, and possibly SSEA-1) and gangliosides (e.g., GD3, GD2, and A2B5 antigens), have been used as marker molecules of stem cells. In this review, I will introduce glycolipids expressed in pluripotent stem cells (embryonic stem cells, induced pluripotent stem cells, very small embryonic-like stem cells, amniotic stem cells, and multilineage-differentiating stress enduring cells), multipotent stem cells (neural stem cells, mesenchymal stem cells, fetal liver multipotent progenitor cells, and hematopoietic stem cells), and cancer stem cells (brain cancer stem cells and breast cancer stem cells), and discuss their availability as biomarkers for identifying and isolating stem cells. PMID:21161592

  15. Murine Mueller cells are progenitor cells for neuronal cells and fibrous tissue cells

    SciTech Connect

    Florian, Christian; Langmann, Thomas; Weber, Bernhard H.F.; Morsczeck, Christian

    2008-09-19

    Mammalian Mueller cells have been reported to possess retinal progenitor cell properties and generate new neurons after injury. This study investigates murine Mueller cells under in vitro conditions for their capability of dedifferentiation into retinal progenitor cells. Mueller cells were isolated from mouse retina, and proliferating cells were expanded in serum-containing medium. For dedifferentiation, the cultured cells were transferred to serum-replacement medium (SRM) at different points in time after their isolation. Interestingly, early cell passages produced fibrous tissue in which extracellular matrix proteins and connective tissue markers were differentially expressed. In contrast, aged Mueller cell cultures formed neurospheres in SRM that are characteristic for neuronal progenitor cells. These neurospheres differentiated into neuron-like cells after cultivation on laminin/ornithine cell culture substrate. Here, we report for the first time that murine Mueller cells can be progenitors for both, fibrous tissue cells and neuronal cells, depending on the age of the cell culture.

  16. Electrorefining cell evaluation

    SciTech Connect

    Bronson, M.C.; Thomas, R.L.

    1989-04-14

    Operational characteristics of the LANL electrorefining cell, a modified LANL electrorefining cell, and an advanced electrorefining cell (known as the CRAC cell) were determined. Average process yields achieved were: 75% for the LANL cell, 82% for the modified LANL cell, and 86% for the CRAC cell. All product metal from the LANL and modified LANL cells was within foundry specifications. Metal from one run in the CRAC cell exceeded foundry specifications for tantalum. The LANL and modified LANL cells were simple in design and operation, but product separation was more labor intensive than with the CRAC cell. The CRAC cell was more complicated in design but remained relatively simple in operation. A decision analysis concluded that the modified LANL cell was the preferred cell. It was recommended that the modified LANL cell be implemented by the Plutonium Recovery Project at Rocky Flats and that development of the CRAC cell continue. 8 refs., 22 figs., 12 tabs.

  17. Leydig cells: From stem cells to aging.

    PubMed

    Chen, Haolin; Ge, Ren-Shan; Zirkin, Barry R

    2009-07-10

    Leydig cells are the testosterone-producing cells of the testis. The adult Leydig cell population ultimately develops from undifferentiated mesenchymal-like stem cells present in the interstitial compartment of the neonatal testis. Four distinct stages of adult Leydig cell development have been identified and characterized: stem Leydig cells, progenitor Leydig cells, immature Leydig cells and adult Leydig cells. The stem Leydig cells are undifferentiated cells that are capable of indefinite self-renewal, differentiation, and replenishment of the Leydig cell niche. Progenitor Leydig cells are derived from the stem Leydig cells. These spindle-shaped cells are luteinizing hormone (LH) receptor positive, have high mitotic activity, and produce little testosterone but rather testosterone metabolites. The progenitor Leydig cells give rise to immature Leydig cells which are round, contain large amounts of smooth endoplasmic reticulum, and produce some testosterone but also very high levels of testosterone metabolites. A single division of these cells produces adult Leydig cells, which are terminally differentiated cells that produce high levels of testosterone. As men age, serum testosterone levels decline, and this is associated with alterations in body composition, energy level, muscle strength, physical, sexual and cognitive functions, and mood. In the Brown Norway rat, used extensively as a model for male reproductive aging, age-related reductions in serum testosterone result from significant decline in the ability of aged Leydig cells to produce testosterone in response to LH stimulation. This review describes Leydig cell development and aging. Additionally, the molecular mechanisms by which testosterone synthesis declines with aging are discussed. PMID:19481681

  18. Fuel cells: A handbook

    NASA Astrophysics Data System (ADS)

    Kinoshita, K.; McLarnon, F. R.; Cairns, E. J.

    1988-05-01

    The purpose of this handbook is to present information describing fuel cells that is helpful to scientists, engineers, and technical managers who are not experienced in this technology, as well as to provide an update on the current technical status of the various types of fuel cells. Following the introduction, contents of this handbook are: fuel cell performance variables; phosphoric acid fuel cell; molten carbonate fuel cell; solid oxide fuel cell; alternative fuel cell technologies; fuel cell systems; and concluding remarks.

  19. CORONAL CELLS

    SciTech Connect

    Sheeley, N. R. Jr.; Warren, H. P. E-mail: harry.warren@nrl.navy.mil

    2012-04-10

    We have recently noticed cellular features in Fe XII 193 A images of the 1.2 MK corona. They occur in regions bounded by a coronal hole and a filament channel, and are centered on flux elements of the photospheric magnetic network. Like their neighboring coronal holes, these regions have minority-polarity flux that is {approx}0.1-0.3 times their flux of majority polarity. Consequently, the minority-polarity flux is 'grabbed' by the majority-polarity flux to form low-lying loops, and the remainder of the network flux escapes to connect with its opposite-polarity counterpart in distant active regions of the Sun. As these regions are carried toward the limb by solar rotation, the cells disappear and are replaced by linear plumes projecting toward the limb. In simultaneous views from the Solar Terrestrial Relations Observatory and Solar Dynamics Observatory spacecraft, these plumes project in opposite directions, extending away from the coronal hole in one view and toward the hole in the other view, suggesting that they are sky-plane projections of the same radial structures. We conclude that these regions are composed of closely spaced radial plumes, extending upward like candles on a birthday cake and visible as cells when seen from above. We suppose that a coronal hole has this same discrete, cellular magnetic structure, but that it is not seen until the encroachment of opposite-polarity flux closes part or all of the hole.

  20. Molluscan cells in culture: primary cell cultures and cell lines

    PubMed Central

    Yoshino, T. P.; Bickham, U.; Bayne, C. J.

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

  1. Indium phosphide solar cells

    NASA Technical Reports Server (NTRS)

    Weinberg, Irving

    1991-01-01

    The direction for InP solar cell research; reduction of cell cost; increase of cell efficiency; measurements needed to better understand cell performance; n/p versus p/n; radiation effects; major problems in cell contacting; and whether the present level of InP solar cell research in the USA should be maintained, decreased, or increased were considered.

  2. Endothelial cell Ca2+ increases upon tumor cell contact and modulates cell-cell adhesion.

    PubMed Central

    Pili, R; Corda, S; Passaniti, A; Ziegelstein, R C; Heldman, A W; Capogrossi, M C

    1993-01-01

    The signal transduction mechanisms involved in tumor cell adhesion to endothelial cells are still largely undefined. The effect of metastatic murine melanoma cell and human prostate carcinoma cell contact on cytosolic [Ca2+] of bovine artery endothelial cells was examined in indo-1-loaded endothelial cell monolayers. A rapid increase in endothelial cell [Ca2+] occurred on contact with tumor cells, but not on contact with 8-microns inert beads. A similar increase in endothelial cell [Ca2+] was observed with human neutrophils or monocyte-like lymphoma cells, but not with endothelial cells, red blood cells, and melanoma cell-conditioned medium. The increase in endothelial cell [Ca2+] was not inhibited by extracellular Ca2+ removal. In contrast, endothelial cell pretreatment with thapsigargin, which releases endoplasmic reticulum Ca2+ into the cytosol and depletes this Ca2+ store site, abolished the cytosolic [Ca2+] rise upon melanoma cell contact. Endothelial cell pretreatment with the membrane-permeant form of the Ca2+ chelator bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid blocked the increase in cytosolic [Ca2+]. Under static and dynamic flow conditions (0.46 dyn/cm2) bis-(O-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid pretreatment of bovine pulmonary artery endothelial cell monolayers inhibited melanoma cell adhesion to the endothelial cells. Thus, tumor cell contact with endothelial cells induces a rapid Ca2+ release from endothelial intracellular stores, which has a functional role in enhancing cell-cell adhesion. Images PMID:8254056

  3. DNA-cell conjugates

    DOEpatents

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2016-05-03

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  4. Induction of apoptosis in Epstein-Barr virus-infected B-lymphocytes by the NF-kappaB inhibitor DHMEQ.

    PubMed

    Miyake, Ariko; Dewan, Md Zahidunnabi; Ishida, Takaomi; Watanabe, Mariko; Honda, Mitsuo; Sata, Tetsutaro; Yamamoto, Naoki; Umezawa, Kazuo; Watanabe, Toshiki; Horie, Ryouichi

    2008-06-01

    Epstein-Barr virus (EBV) causes EBV-associated lymphoproliferative diseases in patients with profound immune suppression. Most of these diseases are life-threatening and the prognosis of AIDS-associated lymphomas is extremely unfavorable. Polyclonal expansion of virus infected B-cell predisposes them to transformation. We investigated the possibility of nuclear factor kappa B (NF-kappaB) inhibition by dehydroxymethylepoxyquinomicin (DHMEQ) for the treatment and prevention of EBV-associated lymphoproliferative diseases. We examined the effect of DHMEQ on apoptosis induction in four EBV-transformed lymphoblastoid cell lines as well as peripheral blood mononuclear cells infected with EBV under immunosuppressed condition. DHMEQ inhibits NF-kappaB activation in EBV-transformed lymphoblastoid cell lines and induces apoptosis by activation of mitochondrial and membranous pathways. Using an in vivo NOD/SCIDgammac mouse model, we showed that DHMEQ has a potent inhibitory effect on the growth of lymphoblastoid cells. In addition, DHMEQ selectively purges EBV-infected cells expressing latent membrane protein (LMP) 1 from peripheral blood mononuclear cells and inhibits the outgrowth of lymphoblastoid cells. These results suggest that NF-kappaB is a molecular target for the treatment and prevention of EBV-associated lymphoproliferative diseases. As a potent NF-kappaB inhibitor, DHMEQ is a potential compound for applying this strategy in clinical medicine. PMID:18538617

  5. Cell-cell interactions on solid matrices.

    PubMed

    Louis, Nancy A; Daniels, Dionne; Colgan, Sean P

    2006-01-01

    Models to study molecular, biochemical, and functional responses in vitro generally incorporate an individual cell type or group of cells organized in a random fashion. Normal physiological responses in vivo require that individual cell types be oriented in an organized fashion with three-dimensional architecture and appropriately positioned cellular interfaces. Much recent progress has been made in the development and implementation of models to study cell-cell contact using substrate grown cells. Here, we summarize the use of membrane permeable supports to study functional responses in appropriately positioned cell types. These models incorporate two or more different cells cultured in physiologically positioned locales on solid substrates. Models incorporating nonadherent cells (e.g., leukocytes) in co-culture with such models also are discussed. Such models have been used extensively to discovery both cell-bound as well as soluble mediators of physiological and pathophysiological processes. PMID:16799188

  6. Photovoltaic cell

    SciTech Connect

    Jordan, J.F.; Lampkin, C.M.

    1981-12-08

    A photovoltaic cell has: an electrically conductive substrate, which may be glass having a film of conductive tin oxide; a first layer containing a suitable semiconductor, which layer has a first component film with an amorphous structure and a second component film with a polycrystalline structure; a second layer forming a heterojunction with the first layer; and suitable electrodes where the heterojunction is formed from a solution containing copper, the amorphous film component is superposed above an electrically conductive substrate to resist permeation of the copper-containing material to shorting electrical contact with the substrate. The penetration resistant amporphous layer permits a variety of processes to be used in forming the heterojunction with even very thin layers (1-6 mu thick) of underlying polycrystalline semi-conductor materials. In some embodiments, the amorphous-like structure may be formed by the addition of aluminum or zirconium compounds to a solution of cadmium salts sprayed over a heated substrate.

  7. Photoelectrochemical cell

    DOEpatents

    Rauh, R. David; Boudreau, Robert A.

    1983-06-14

    A photoelectrochemical cell comprising a sealed container having a light-transmitting window for admitting light into the container across a light-admitting plane, an electrolyte in the container, a photoelectrode in the container having a light-absorbing surface arranged to receive light from the window and in contact with the electrolyte, the surface having a plurality of spaced portions oblique to the plane, each portion having dimensions at least an order of magnitude larger than the maximum wavelength of incident sunlight, the total surface area of the surface being larger than the area of the plane bounded by the container, and a counter electrode in the container in contact with the electrolyte.

  8. Integrated circuit cell library

    NASA Technical Reports Server (NTRS)

    Whitaker, Sterling R. (Inventor); Miles, Lowell H. (Inventor)

    2005-01-01

    According to the invention, an ASIC cell library for use in creation of custom integrated circuits is disclosed. The ASIC cell library includes some first cells and some second cells. Each of the second cells includes two or more kernel cells. The ASIC cell library is at least 5% comprised of second cells. In various embodiments, the ASIC cell library could be 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more comprised of second cells.

  9. Monitoring cell growth.

    PubMed

    Strober, W

    2001-05-01

    This appendix provides two protocols for monitoring cell growth. Counting cells using a hemacytometer is tedious but it allows one to effectively distinguish live cells from dead cells (using Trypan Blue exclusion). In addition, this procedure is less subject to errors due to cell clumping or heterogeneity of cell size. The use of an electronic cell counter is quicker and easier than counting cells using a hemacytometer. However, an electronic cell counter as currently constructed does not distinguish live from dead cells in a reliable fashion and is subject to error due to the presence of cell clumps. Overall, the electronic cell counter is best reserved for repetitive and rapid counting of fresh peripheral blood cells and should be used with caution when counting cell populations derived from tissues. PMID:18432653

  10. Automated Cell-Cutting for Cell Cloning

    NASA Astrophysics Data System (ADS)

    Ichikawa, Akihiko; Tanikawa, Tamio; Matsukawa, Kazutsugu; Takahashi, Seiya; Ohba, Kohtaro

    We develop an automated cell-cutting technique for cell cloning. Animal cells softened by the cytochalasin treatment are injected into a microfluidic chip. The microfluidic chip contains two orthogonal channels: one microchannel is wide, used to transport cells, and generates the cutting flow; the other is thin and used for aspiration, fixing, and stretching of the cell. The injected cell is aspirated and stretched in the thin microchannel. Simultaneously, the volumes of the cell before and after aspiration are calculated; the volumes are used to calculate the fluid flow required to aspirate half the volume of the cell into the thin microchannel. Finally, we apply a high-speed flow in the orthogonal microchannel to bisect the cell. This paper reports the cutting process, the cutting system, and the results of the experiment.

  11. Eukaryotic Cells and their Cell Bodies: Cell Theory Revised

    PubMed Central

    BALUŠKA, FRANTIŠEK; VOLKMANN, DIETER; BARLOW, PETER W.

    2004-01-01

    • Background Cell Theory, also known as cell doctrine, states that all eukaryotic organisms are composed of cells, and that cells are the smallest independent units of life. This Cell Theory has been influential in shaping the biological sciences ever since, in 1838/1839, the botanist Matthias Schleiden and the zoologist Theodore Schwann stated the principle that cells represent the elements from which all plant and animal tissues are constructed. Some 20 years later, in a famous aphorism Omnis cellula e cellula, Rudolf Virchow annunciated that all cells arise only from pre‐existing cells. General acceptance of Cell Theory was finally possible only when the cellular nature of brain tissues was confirmed at the end of the 20th century. Cell Theory then rapidly turned into a more dogmatic cell doctrine, and in this form survives up to the present day. In its current version, however, the generalized Cell Theory developed for both animals and plants is unable to accommodate the supracellular nature of higher plants, which is founded upon a super‐symplasm of interconnected cells into which is woven apoplasm, symplasm and super‐apoplasm. Furthermore, there are numerous examples of multinucleate coenocytes and syncytia found throughout the eukaryote superkingdom posing serious problems for the current version of Cell Theory. • Scope To cope with these problems, we here review data which conform to the original proposal of Daniel Mazia that the eukaryotic cell is composed of an elemental Cell Body whose structure is smaller than the cell and which is endowed with all the basic attributes of a living entity. A complement to the Cell Body is the Cell Periphery Apparatus, which consists of the plasma membrane associated with other periphery structures. Importantly, boundary stuctures of the Cell Periphery Apparatus, although capable of some self‐assembly, are largely produced and maintained by Cell Body activities and can be produced from it de novo. These

  12. Photovoltaic cell

    SciTech Connect

    Jordan, J. F.; Lampkin, C. M.

    1981-02-03

    A photovoltaic cell is disclosed having an electrically conductive substrate, which may be glass having a film of conductive tin oxide. A first layer contains a suitable semiconductor, which layer has a first component film with an amorphous structure and a second component film with a polycrystalline structure a second layer forms a heterojunction with the first layer suitable electrodes are provided where the heterojunction is formed from a solution containing copper, and the amorphous film component is superposed above an electrically conductive substrate to resist permeation of the copper-containing material to shorting electrical contact with the substrate. The penetration resistant amorphous layer permits a variety of processes to be used in forming the heterojunction with even very thin layers (1-6 mu thick) of underlying polycrystalline semi-conductor materials. In some embodiments, the amorphous-like structure may be formed by the addition of aluminum or zirconium compounds to a solution of cadmium salts sprayed over a heated substrate.

  13. Fuel cell arrangement

    DOEpatents

    Isenberg, Arnold O.

    1987-05-12

    A fuel cell arrangement is provided wherein cylindrical cells of the solid oxide electrolyte type are arranged in planar arrays where the cells within a plane are parallel. Planes of cells are stacked with cells of adjacent planes perpendicular to one another. Air is provided to the interior of the cells through feed tubes which pass through a preheat chamber. Fuel is provided to the fuel cells through a channel in the center of the cell stack; the fuel then passes the exterior of the cells and combines with the oxygen-depleted air in the preheat chamber.

  14. Fuel cell arrangement

    DOEpatents

    Isenberg, A.O.

    1987-05-12

    A fuel cell arrangement is provided wherein cylindrical cells of the solid oxide electrolyte type are arranged in planar arrays where the cells within a plane are parallel. Planes of cells are stacked with cells of adjacent planes perpendicular to one another. Air is provided to the interior of the cells through feed tubes which pass through a preheat chamber. Fuel is provided to the fuel cells through a channel in the center of the cell stack; the fuel then passes the exterior of the cells and combines with the oxygen-depleted air in the preheat chamber. 3 figs.

  15. Squamous cell carcinoma —

    Cancer.gov

    The hallmarks of squamous cell carcinoma are the differentiation features of the squamous epithelium: keratinization and intercellular bridges. Large central masses of keratin, individual cell keratinization, and/or keratin pearls may form. Necrosis of tumor cell nests and accumulation of acute inflammatory cells are frequent features of poorly differentiated squamous cell carcinoma.

  16. Sickle Cell Anemia

    MedlinePlus

    Sickle cell anemia is a disease in which your body produces abnormally shaped red blood cells. The cells are shaped like a crescent or sickle. They don' ... problem causes sickle cell anemia. People with the disease are born with two sickle cell genes, one ...

  17. Sickle Cell Anemia

    MedlinePlus

    Sickle cell anemia is a disease in which your body produces abnormally shaped red blood cells. The cells are shaped like a crescent or sickle. They ... last as long as normal, round red blood cells. This leads to anemia. The sickle cells also ...

  18. Virus Cell-to-Cell Transmission▿

    PubMed Central

    Mothes, Walther; Sherer, Nathan M.; Jin, Jing; Zhong, Peng

    2010-01-01

    Viral infections spread based on the ability of viruses to overcome multiple barriers and move from cell to cell, tissue to tissue, and person to person and even across species. While there are fundamental differences between these types of transmissions, it has emerged that the ability of viruses to utilize and manipulate cell-cell contact contributes to the success of viral infections. Central to the excitement in the field of virus cell-to-cell transmission is the idea that cell-to-cell spread is more than the sum of the processes of virus release and entry. This implies that virus release and entry are efficiently coordinated to sites of cell-cell contact, resulting in a process that is distinct from its individual components. In this review, we will present support for this model, illustrate the ability of viruses to utilize and manipulate cell adhesion molecules, and discuss the mechanism and driving forces of directional spreading. An understanding of viral cell-to-cell spreading will enhance our ability to intervene in the efficient spreading of viral infections. PMID:20375157

  19. Cell culture purity issues and DFAT cells

    SciTech Connect

    Wei, Shengjuan; Bergen, Werner G.; Zan, Linsen; Dodson, Michael V.

    2013-04-12

    Highlights: •DFAT cells are progeny cells derived from dedifferentiated mature adipocytes. •Common problems in this research is potential cell contamination of initial cultures. •The initial cell culture purity is crucial in DFAT cell research field. -- Abstract: Dedifferentiation of mature adipocytes, in vitro, has been pursued/documented for over forty years. The subsequent progeny cells are named dedifferentiated adipocyte-derived progeny cells (DFAT cells). DFAT cells are proliferative and likely to possess mutilineage potential. As a consequence, DFAT cells and their progeny/daughter cells may be useful as a potential tool for various aspects of tissue engineering and as potential vectors for the alleviation of several disease states. Publications in this area have been increasing annually, but the purity of the initial culture of mature adipocytes has seldom been documented. Consequently, it is not always clear whether DFAT cells are derived from dedifferentiated mature (lipid filled) adipocytes or from contaminating cells that reside in an impure culture.

  20. Deformability of Tumor Cells versus Blood Cells.

    PubMed

    Shaw Bagnall, Josephine; Byun, Sangwon; Begum, Shahinoor; Miyamoto, David T; Hecht, Vivian C; Maheswaran, Shyamala; Stott, Shannon L; Toner, Mehmet; Hynes, Richard O; Manalis, Scott R

    2015-01-01

    The potential for circulating tumor cells (CTCs) to elucidate the process of cancer metastasis and inform clinical decision-making has made their isolation of great importance. However, CTCs are rare in the blood, and universal properties with which to identify them remain elusive. As technological advancements have made single-cell deformability measurements increasingly routine, the assessment of physical distinctions between tumor cells and blood cells may provide insight into the feasibility of deformability-based methods for identifying CTCs in patient blood. To this end, we present an initial study assessing deformability differences between tumor cells and blood cells, indicated by the length of time required for them to pass through a microfluidic constriction. Here, we demonstrate that deformability changes in tumor cells that have undergone phenotypic shifts are small compared to differences between tumor cell lines and blood cells. Additionally, in a syngeneic mouse tumor model, cells that are able to exit a tumor and enter circulation are not required to be more deformable than the cells that were first injected into the mouse. However, a limited study of metastatic prostate cancer patients provides evidence that some CTCs may be more mechanically similar to blood cells than to typical tumor cell lines. PMID:26679988

  1. Deformability of Tumor Cells versus Blood Cells

    PubMed Central

    Shaw Bagnall, Josephine; Byun, Sangwon; Begum, Shahinoor; Miyamoto, David T.; Hecht, Vivian C.; Maheswaran, Shyamala; Stott, Shannon L.; Toner, Mehmet; Hynes, Richard O.; Manalis, Scott R.

    2015-01-01

    The potential for circulating tumor cells (CTCs) to elucidate the process of cancer metastasis and inform clinical decision-making has made their isolation of great importance. However, CTCs are rare in the blood, and universal properties with which to identify them remain elusive. As technological advancements have made single-cell deformability measurements increasingly routine, the assessment of physical distinctions between tumor cells and blood cells may provide insight into the feasibility of deformability-based methods for identifying CTCs in patient blood. To this end, we present an initial study assessing deformability differences between tumor cells and blood cells, indicated by the length of time required for them to pass through a microfluidic constriction. Here, we demonstrate that deformability changes in tumor cells that have undergone phenotypic shifts are small compared to differences between tumor cell lines and blood cells. Additionally, in a syngeneic mouse tumor model, cells that are able to exit a tumor and enter circulation are not required to be more deformable than the cells that were first injected into the mouse. However, a limited study of metastatic prostate cancer patients provides evidence that some CTCs may be more mechanically similar to blood cells than to typical tumor cell lines. PMID:26679988

  2. Red blood cells, multiple sickle cells (image)

    MedlinePlus

    ... inherited disorder in which abnormal hemoglobin (the red pigment inside red blood cells) is produced. The abnormal hemoglobin causes red blood cells to assume a sickle shape, like the ones seen in this photomicrograph.

  3. Fuel cell-fuel cell hybrid system

    DOEpatents

    Geisbrecht, Rodney A.; Williams, Mark C.

    2003-09-23

    A device for converting chemical energy to electricity is provided, the device comprising a high temperature fuel cell with the ability for partially oxidizing and completely reforming fuel, and a low temperature fuel cell juxtaposed to said high temperature fuel cell so as to utilize remaining reformed fuel from the high temperature fuel cell. Also provided is a method for producing electricity comprising directing fuel to a first fuel cell, completely oxidizing a first portion of the fuel and partially oxidizing a second portion of the fuel, directing the second fuel portion to a second fuel cell, allowing the first fuel cell to utilize the first portion of the fuel to produce electricity; and allowing the second fuel cell to utilize the second portion of the fuel to produce electricity.

  4. Cell Membrane Softening in Cancer Cells

    NASA Astrophysics Data System (ADS)

    Schmidt, Sebastian; Händel, Chris; Käs, Josef

    Biomechanical properties are useful characteristics and regulators of the cell's state. Current research connects mechanical properties of the cytoskeleton to many cellular processes but does not investigate the biomechanics of the plasma membrane. We evaluated thermal fluctuations of giant plasma membrane vesicles, directly derived from the plasma membranes of primary breast and cervical cells and observed a lowered rigidity in the plasma membrane of malignant cells compared to non-malignant cells. To investigate the specific role of membrane rigidity changes, we treated two cell lines with the Acetyl-CoA carboxylase inhibitor Soraphen A. It changed the lipidome of cells and drastically increased membrane stiffness by up regulating short chained membrane lipids. These altered cells had a decreased motility in Boyden chamber assays. Our results indicate that the thermal fluctuations of the membrane, which are much smaller than the fluctuations driven by the cytoskeleton, can be modulated by the cell and have an impact on adhesion and motility.

  5. Kidney cell electrophoresis

    NASA Technical Reports Server (NTRS)

    Todd, P.

    1980-01-01

    The following aspects of kidney cell electrophoresis are discussed: (1) the development and testing of electrophoresis solutions; (2) optimization of freezing and thawing; (3) procedures for evaluation of separated kidney cells; and (4) electrophoretic mobility characterization of kidney cells.

  6. Kidney cell electrophoresis

    NASA Technical Reports Server (NTRS)

    Todd, P.

    1979-01-01

    A kidney cell electrophoresis technique is described in four parts: (1) the development and testing of electrophoresis solutions; (2) optimization of freezing and thawing; (3) procedures for evaluation of separated kidney cells; and (4) electrophoretic mobility characteristics of kidney cells.

  7. Lung cancer - small cell

    MedlinePlus

    Cancer - lung - small cell; Small cell lung cancer; SCLC ... About 15% of all lung cancer cases are SCLC. Small cell lung cancer is slightly more common in men than women. Almost all cases of SCLC ...

  8. Sickle Cell Information Center

    MedlinePlus

    ... change Sickle Cell News from Around the Web Google Custom Search – sickle cell Our healthcare system abandons ... professor of ... NYT, Nature, Wash Post, SciAm, CNN - Google Custom Search Sickle Cell Anemia News -- ScienceDaily January ...

  9. Lung cancer - small cell

    MedlinePlus

    Cancer - lung - small cell; Small cell lung cancer; SCLC ... About 15% of all lung cancer cases are SCLC. Small cell lung cancer is slightly more common in men than women. Almost all cases of SCLC are ...

  10. Fuel cells: A survey

    NASA Technical Reports Server (NTRS)

    Crowe, B. J.

    1973-01-01

    A survey of fuel cell technology and applications is presented. The operating principles, performance capabilities, and limitations of fuel cells are discussed. Diagrams of fuel cell construction and operating characteristics are provided. Photographs of typical installations are included.

  11. Advances in cell culture

    SciTech Connect

    Maramorosch, K. )

    1987-01-01

    This book presents papers on advances in cell culture. Topics covered include: Genetic changes in the influenza viruses during growth in cultured cells; The biochemistry and genetics of mosquito cells in culture; and Tree tissue culture applications.

  12. Sickle cell anemia - resources

    MedlinePlus

    Resources - sickle cell anemia ... The following organizations are good resources for information on sickle cell anemia : American Sickle Cell Anemia Association -- www.ascaa.org National Heart, Blood, and Lung Institute -- www. ...

  13. Reprogramming of somatic cells.

    PubMed

    Rajasingh, Johnson

    2012-01-01

    Reprogramming of adult somatic cells into pluripotent stem cells may provide an attractive source of stem cells for regenerative medicine. It has emerged as an invaluable method for generating patient-specific stem cells of any cell lineage without the use of embryonic stem cells. A revolutionary study in 2006 showed that it is possible to convert adult somatic cells directly into pluripotent stem cells by using a limited number of pluripotent transcription factors and is called as iPS cells. Currently, both genomic integrating viral and nonintegrating nonviral methods are used to generate iPS cells. However, the viral-based technology poses increased risk of safety, and more studies are now focused on nonviral-based technology to obtain autologous stem cells for clinical therapy. In this review, the pros and cons of the present iPS cell technology and the future direction for the successful translation of this technology into the clinic are discussed. PMID:22917226

  14. Inside the Cell

    MedlinePlus

    ... Business Basics Describes functions shared by virtually all cells: making fuel and proteins, transporting materials and disposing of wastes. » more Chapter 3: On the Job: Cellular Specialties Explains how cells specialize. Features a number of cell types: nerves, ...

  15. Closed Large Cell Clouds

    Atmospheric Science Data Center

    2013-04-19

    article title:  Closed Large Cell Clouds in the South Pacific     ... unperturbed by cyclonic or frontal activity. When the cell centers are cloudy and the main sinking motion is concentrated at cell ...

  16. Pancreatic islet cell tumor

    MedlinePlus

    Islet cell tumors; Islet of Langerhans tumor; Neuroendocrine tumors ... In the healthy pancreas, cells called islet cells produce hormones that regulate a several bodily functions. These include blood sugar level and the production of ...

  17. Sickle Cell Disease Quiz

    MedlinePlus

    ... False: People with sickle cell disease cannot get malaria. A True B False 4. True or False: ... False: People with sickle cell disease cannot get malaria. False People with sickle cell disease can get ...

  18. Identification of a second T-cell antigen receptor in human and mouse by an anti-peptide gamma-chain-specific monoclonal antibody.

    PubMed Central

    Ioannides, C G; Itoh, K; Fox, F E; Pahwa, R; Good, R A; Platsoucas, C D

    1987-01-01

    We developed a monoclonal antibody (mAb) (9D7) against a synthetic peptide (P13K) selected from the deduced amino acid sequence of the constant region of the gamma chain of the murine T-cell antigen receptor (TCR) (amino acids 118-130). Using this mAb, we identified a putative second TCR expressed on peripheral blood lymphocytes from a patient with severe combined immunodeficiency (SCID) that were propagated in culture with recombinant interleukin 2 (rIL-2) and Con A. This mAb immunoprecipitated two polypeptide chains of 40 and 58 kDa under nonreducing conditions and of 40 and 56 kDa under reducing conditions from 125I-labeled denatured lysates of T3+ WT31- lymphocytes expanded in culture from a SCID patient. These polypeptide chains were not disulfide linked and were not present on human peripheral blood lymphocytes from normal donors cultured for 5 days with phytohemagglutinin or for 2 weeks with rIL-2 and polyclonal activators or on cells of the Jurkat lymphoblastoid human T-cell line. Chemical crosslinking of 125I-labeled cells followed by immunoprecipitation with anti-Leu-4 mAb under nonreducing or reducing conditions revealed that the 40- and 56-kDa polypeptide chains were associated with the T3 differentiation antigen. These results were confirmed by sequential immunoprecipitation with anti-Leu-4 mAb followed by 9D7 anti-P13K mAb. The 9D7 anti-P13K mAb immunoprecipitated two polypeptide chains of 43 and 64 kDa from denatured lysates of lymphocytes from a patient with severe common variable immunodeficiency (CVI) that were expanded in culture with rIL-2 and Con A. Thus, this second TCR may be composed of two polypeptide chains (gamma gamma'), both of which appear to be the product of the gamma-chain gene. These experiments were done with polyclonal cell populations. Cloned T3+ WT31- cell populations are required to determine whether this TCR contains two gamma polypeptide chains. In contrast, only one polypeptide chain of 56 kDa was immunoprecipitated by the

  19. Glial cells: Old cells with new twists

    PubMed Central

    Ndubaku, Ugo; de Bellard, Maria Elena

    2008-01-01

    Summary Based on their characteristics and function – migration, neural protection, proliferation, axonal guidance and trophic effects – glial cells may be regarded as probably the most versatile cells in our body. For many years, these cells were considered as simply support cells for neurons. Recently, it has been shown that they are more versatile than previously believed – as true stem cells in the nervous system – and are important players in neural function and development. There are several glial cell types in the nervous system: the two most abundant are oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system. Although both of these cells are responsible for myelination, their developmental origins are quite different. Oligodendrocytes originate from small niche populations from different regions of the central nervous system, while Schwann cells develop from a stem cell population (the neural crest) that gives rise to many cell derivatives besides glia and which is a highly migratory group of cells. PMID:18068219

  20. CellFinder: a cell data repository

    PubMed Central

    Stachelscheid, Harald; Seltmann, Stefanie; Lekschas, Fritz; Fontaine, Jean-Fred; Mah, Nancy; Neves, Mariana; Andrade-Navarro, Miguel A.; Leser, Ulf; Kurtz, Andreas

    2014-01-01

    CellFinder (http://www.cellfinder.org) is a comprehensive one-stop resource for molecular data characterizing mammalian cells in different tissues and in different development stages. It is built from carefully selected data sets stemming from other curated databases and the biomedical literature. To date, CellFinder describes 3394 cell types and 50 951 cell lines. The database currently contains 3055 microscopic and anatomical images, 205 whole-genome expression profiles of 194 cell/tissue types from RNA-seq and microarrays and 553 905 protein expressions for 535 cells/tissues. Text mining of a corpus of >2000 publications followed by manual curation confirmed expression information on ∼900 proteins and genes. CellFinder’s data model is capable to seamlessly represent entities from single cells to the organ level, to incorporate mappings between homologous entities in different species and to describe processes of cell development and differentiation. Its ontological backbone currently consists of 204 741 ontology terms incorporated from 10 different ontologies unified under the novel CELDA ontology. CellFinder’s web portal allows searching, browsing and comparing the stored data, interactive construction of developmental trees and navigating the partonomic hierarchy of cells and tissues through a unique body browser designed for life scientists and clinicians. PMID:24304896

  1. Snail modulates cell metabolism in MDCK cells

    SciTech Connect

    Haraguchi, Misako; Indo, Hiroko P.; Iwasaki, Yasumasa; Iwashita, Yoichiro; Fukushige, Tomoko; Majima, Hideyuki J.; Izumo, Kimiko; Horiuchi, Masahisa; Kanekura, Takuro; Furukawa, Tatsuhiko; Ozawa, Masayuki

    2013-03-22

    Highlights: ► MDCK/snail cells were more sensitive to glucose deprivation than MDCK/neo cells. ► MDCK/snail cells had decreased oxidative phosphorylation, O{sub 2} consumption and ATP content. ► TCA cycle enzyme activity, but not expression, was lower in MDCK/snail cells. ► MDCK/snail cells showed reduced PDH activity and increased PDK1 expression. ► MDCK/snail cells showed reduced expression of GLS2 and ACLY. -- Abstract: Snail, a repressor of E-cadherin gene transcription, induces epithelial-to-mesenchymal transition and is involved in tumor progression. Snail also mediates resistance to cell death induced by serum depletion. By contrast, we observed that snail-expressing MDCK (MDCK/snail) cells undergo cell death at a higher rate than control (MDCK/neo) cells in low-glucose medium. Therefore, we investigated whether snail expression influences cell metabolism in MDCK cells. Although gylcolysis was not affected in MDCK/snail cells, they did exhibit reduced pyruvate dehydrogenase (PDH) activity, which controls pyruvate entry into the tricarboxylic acid (TCA) cycle. Indeed, the activity of multiple enzymes involved in the TCA cycle was decreased in MDCK/snail cells, including that of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase (IDH2), succinate dehydrogenase (SDH), and electron transport Complex II and Complex IV. Consequently, lower ATP content, lower oxygen consumption and increased survival under hypoxic conditions was also observed in MDCK/snail cells compared to MDCK/neo cells. In addition, the expression and promoter activity of pyruvate dehydrogenase kinase 1 (PDK1), which phosphorylates and inhibits the activity of PDH, was increased in MDCK/snail cells, while expression levels of glutaminase 2 (GLS2) and ATP-citrate lyase (ACLY), which are involved in glutaminolysis and fatty acid synthesis, were decreased in MDCK/snail cells. These results suggest that snail modulates cell metabolism by altering the expression and activity of

  2. Sertoli cells as biochambers

    NASA Technical Reports Server (NTRS)

    Cameron, Don F. (Inventor); Sanberg, Paul R. (Inventor); Saporta, Samuel (Inventor); Hushen, Joelle J. (Inventor)

    2004-01-01

    According to the present invention, there is provided a biological chamber system having a biochamber defined by outer walls of Sertoli cells. Also provided is a transplantation facilitator including a biochamber. A method of making biochambers by co-culturing facilitator cells and therapeutic cells and then aggregating the facilitator celes is also provided. Also provided is a method of transplanting cells by incorporating transplant cells into a biochamber and transplanting the biochamber containing the transplant cells.

  3. Stem Cell Research

    SciTech Connect

    Verfaillie, Catherine

    2009-01-23

    We have identified a population of primitive cells in normal human post-natal bone marrow that can, at the single cell level, differentiate in many ways and also proliferate extensively. These cells can differentiate in vitro into most mesodermal cell types (for example, bone cells, and others), as well as cells into cells of the nervous system. The finding that stem cells exist in post-natal tissues with previously unknown proliferation and differentiation potential opens up the possibility of using them to treat a host of degenerative, traumatic or congenital diseases.

  4. Heterostructure solar cells

    NASA Technical Reports Server (NTRS)

    Chang, K. I.; Yeh, Y. C. M.; Iles, P. A.; Morris, R. K.

    1987-01-01

    The performance of gallium arsenide solar cells grown on Ge substrates is discussed. In some cases the substrate was thinned to reduce overall cell weight with good ruggedness. The conversion efficiency of 2 by 2 cm cells under AMO reached 17.1 percent with a cell thickness of 6 mils. The work described forms the basis for future cascade cell structures, where similar interconnecting problems between the top cell and the bottom cell must be solved. Applications of the GaAs/Ge solar cell in space and the expected payoffs are discussed.

  5. Stem Cell Research

    SciTech Connect

    Verfaillie, Catherine

    2002-01-23

    We have identified a population of primitive cells in normal human post-natal bone marrow that can, at the single cell level, differentiate in many ways and also proliferate extensively. These cells can differentiate in vitro into most mesodermal cell types (for example, bone cells, and others), as well as cells into cells of the nervous system. The finding that stem cells exist in post-natal tissues with previously unknown proliferation and differentiation potential opens up the possibility of using them to treat a host of degenerative, traumatic or congenital diseases.

  6. Gaucher cell, photomicrograph (image)

    MedlinePlus

    Gaucher's disease is called a "lipid storage disease" where abnormal amounts of lipids called "glycosphingolipids" are stored in special cells called reticuloendothelial cells. Classically, the nucleus is ...

  7. Cell heterogeneity during the cell cycle

    SciTech Connect

    Darzynkiewicz, Z.; Crissman, H.; Traganos, F.; Steinkamp, J.

    1982-12-01

    Using flow cytometry, populations of Chinese hamster ovary cells, asynchronous and synchronized in the cycle, were measured with respect to cellular RNA- and protein-content, as well as cell light scatter properties. Heterogeneities of cell populations were expressed as coefficients of variation (c.v.) in percent of the respective mean values. Populations of cells immediately after mitosis have about 15% higher c.v. than mitotic cell populations, regardless of whether RNA, proteins, or light scatter are measured. These data indicate that cytoplasmic constituents are unequally distributed into the daughter cells during cytokinesis and that unequal cytokinesis generates intercellular metabolic variability during the cycle. An additional increase in heterogeneity, although of smaller degree, occurs during G/sub 2/ phase. Populations of S-phase cells are the most uniform, having 20-30% lower c.v. than the postmitotic cells. Cell progression through S does not involve any significant increase in intercellular variability with respect to RNA or protein content. In unperturbed exponentially growing cultures a critical RNA content is required for G/sub 1/ cells prior to their entrance into S. The cell residence times in the equalization compartments are exponentially distributed, which may reflect the randomness generated by the uneven division of metabolic constituents to daughter cells during cytokinesis. The cell heterogeneities were presently estimated at two metabolic levels, transcription (RNA content) and translation (proteins). The most uniform were populations stained for RNA and the highest variability was observed after staining of proteins. This suggests that the regulatory mechanisms equalizing cells in the cell cycle may operate primarily at the level of DNA transcription.

  8. Stem cells supporting other stem cells

    PubMed Central

    Leatherman, Judith

    2013-01-01

    Adult stem cell therapies are increasingly prevalent for the treatment of damaged or diseased tissues, but most of the improvements observed to date are attributed to the ability of stem cells to produce paracrine factors that have a trophic effect on existing tissue cells, improving their functional capacity. It is now clear that this ability to produce trophic factors is a normal and necessary function for some stem cell populations. In vivo adult stem cells are thought to self-renew due to local signals from the microenvironment where they live, the niche. Several niches have now been identified which harbor multiple stem cell populations. In three of these niches – the Drosophila testis, the bulge of the mammalian hair follicle, and the mammalian bone marrow – one type of stem cell has been found to produce factors that contribute to the maintenance of a second stem cell population in the shared niche. In this review, I will examine the architecture of these three niches and discuss the molecular signals involved. Together, these examples establish a new paradigm for stem cell behavior, that stem cells can promote the maintenance of other stem cells. PMID:24348512

  9. Cell and tissue mechanics in cell migration

    PubMed Central

    Lange, Janina R.; Fabry, Ben

    2013-01-01

    Migrating cells generate traction forces to counteract the movement-resisting forces arising from cell-internal stresses and matrix adhesions. In the case of collective migration in a cell colony, or in the case of 3-dimensional migration through connective tissue, movement-resisting forces arise also from external stresses. Although the deformation of a stiffer cell or matrix causes larger movement-resisting forces, at the same time a larger stiffness can also promote cell migration due to a feedback between forces, deformations, and deformation speed that is mediated by the acto-myosin contractile machinery of cells. This mechanical feedback is also important for stiffness sensing, durotaxis, plithotaxis, and collective migration in cell colonies. PMID:23664834

  10. Cell and tissue mechanics in cell migration.

    PubMed

    Lange, Janina R; Fabry, Ben

    2013-10-01

    Migrating cells generate traction forces to counteract the movement-resisting forces arising from cell-internal stresses and matrix adhesions. In the case of collective migration in a cell colony, or in the case of 3-dimensional migration through connective tissue, movement-resisting forces arise also from external stresses. Although the deformation of a stiffer cell or matrix causes larger movement-resisting forces, at the same time a larger stiffness can also promote cell migration due to a feedback between forces, deformations, and deformation speed that is mediated by the acto-myosin contractile machinery of cells. This mechanical feedback is also important for stiffness sensing, durotaxis, plithotaxis, and collective migration in cell colonies. PMID:23664834

  11. Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

    SciTech Connect

    Felthaus, O.; Ettl, T.; Gosau, M.; Driemel, O.; Brockhoff, G.; Reck, A.; Zeitler, K.; Hautmann, M.; Reichert, T.E.; Schmalz, G.; Morsczeck, C.

    2011-04-01

    Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

  12. Specific cell cycle synchronization with butyrate and cell cycle analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Synchronized cells have been invaluable for many kinds of cell cycle and cell proliferation studies. Butyrate induces cell cycle arrest and apoptosis in MDBK cells. To explore the possibility of using butyrate-blocked cells to obtain synchronized cells, we investigated the property of the cell cyc...

  13. Stem cell therapy without the cells

    PubMed Central

    Maguire, Greg

    2013-01-01

    As an example of the burgeoning importance of stem cell therapy, this past month the California Institute for Regenerative Medicine (CIRM) has approved $70 million to create a new network of stem cell clinical trial centers. Much work in the last decade has been devoted to developing the use of autologous and allogeneic adult stem cell transplants to treat a number of conditions, including heart attack, dementia, wounds, and immune system-related diseases. The standard model teaches us that adult stem cells exists throughout most of the body and provide a means to regenerate and repair most tissues through replication and differentiation. Although we have often witnessed the medical cart placed in front of the scientific horse in the development of stem cell therapies outside of academic circles, great strides have been made, such as the use of purified stem cells1 instead of whole bone marrow transplants in cancer patients, where physicians avoid re-injecting the patients with their own cancer cells.2 We most often think of stem cell therapy acting to regenerate tissue through replication and then differentiation, but recent studies point to the dramatic effects adult stem cells exert in the repair of various tissues through the release of paracrine and autocrine substances, and not simply through differentiation. Indeed, up to 80% of the therapeutic effect of adult stem cells has been shown to be through paracrine mediated actions.3 That is, the collected types of molecules released by the stem cells, called the secretome, or stem cell released molecules (SRM), number in the 100s, including proteins, microRNA, growth factors, antioxidants, proteasomes, and exosomes, and target a multitude of biological pathways through paracrine actions. The composition of the different molecule types in SRM is state dependent, and varies with cell type and conditions such as age and environment. PMID:24567776

  14. Sickle Cell Disease

    MedlinePlus

    ... sickle cell disease? Sickle cell disease, also called sickle cell anemia, is a hereditary condition (which means it runs ... or blocks blood and oxygen reaching nearby tissues. Sickle cell disease ... the whites of the eyes) Anemia (the decreased ability of the blood to carry ...

  15. Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

    SciTech Connect

    Varga, Nora; Vereb, Zoltan; Rajnavoelgyi, Eva; Nemet, Katalin; Uher, Ferenc; Sarkadi, Balazs; Apati, Agota

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

  16. SMOOTH MUSCLE STEM CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vascular smooth muscle cells (SMCs) originate from multiple types of progenitor cells. In the embryo, the most well-studied SMC progenitor is the cardiac neural crest stem cell. Smooth muscle differentiation in the neural crest lineage is controlled by a combination of cell intrinsic factors, includ...

  17. Nanocomposite Photoelectrochemical Cells

    NASA Technical Reports Server (NTRS)

    Narayan, Sri R.; Kindler, Andrew; Whitacre, Jay F.

    2007-01-01

    Improved, solid-state photoelectrochemical cells for converting solar radiation to electricity have been proposed. (In general, photoelectrochemical cells convert incident light to electricity through electrochemical reactions.) It is predicted that in comparison with state-of-the-art photoelectrochemical cells, these cells will be found to operate with greater solar-to-electric energy-conversion efficiencies.

  18. Fluorescence activated cell sorting.

    NASA Technical Reports Server (NTRS)

    Bonner, W. A.; Hulett, H. R.; Sweet, R. G.; Herzenberg, L. A.

    1972-01-01

    An instrument has been developed for sorting biological cells. The cells are rendered differentially fluorescent and incorporated into a small liquid stream illuminated by a laser beam. The cells pass sequentially through the beam, and fluorescent light from the cells gives rise to electrical signals. The stream is broken into a series of uniform size drops downstream of the laser. The cell signals are used to give appropriate electrostatic charges to drops containing the cells. The drops then pass between two charged plates and are deflected to appropriate containers. The system has proved capable of providing fractions containing large numbers of viable cells highly enriched in a particular functional type.

  19. Molten carbonate fuel cell

    DOEpatents

    Kaun, T.D.; Smith, J.L.

    1986-07-08

    A molten electrolyte fuel cell is disclosed with an array of stacked cells and cell enclosures isolating each cell except for access to gas manifolds for the supply of fuel or oxidant gas or the removal of waste gas. The cell enclosures collectively provide an enclosure for the array and effectively avoid the problems of electrolyte migration and the previous need for compression of stack components. The fuel cell further includes an inner housing about and in cooperation with the array enclosure to provide a manifold system with isolated chambers for the supply and removal of gases. An external insulated housing about the inner housing provides thermal isolation to the cell components.

  20. Molten carbonate fuel cell

    DOEpatents

    Kaun, Thomas D.; Smith, James L.

    1987-01-01

    A molten electrolyte fuel cell with an array of stacked cells and cell enclosures isolating each cell except for access to gas manifolds for the supply of fuel or oxidant gas or the removal of waste gas, the cell enclosures collectively providing an enclosure for the array and effectively avoiding the problems of electrolyte migration and the previous need for compression of stack components, the fuel cell further including an inner housing about and in cooperation with the array enclosure to provide a manifold system with isolated chambers for the supply and removal of gases. An external insulated housing about the inner housing provides thermal isolation to the cell components.

  1. Jameson cell coal flotation

    SciTech Connect

    Manlapig, E.V.; Jackson, B.R.; Harbort, G.J.; Cheng, C.Y.

    1993-12-31

    Jameson cells have been applied to coal flotation recent years. Some coal mines have Jameson cells in operation, some are having Jameson cells installed and others are considering to use Jameson cells in their flotation circuits. As an example, the application of Jameson cells in Newlands Coal Mine is described and the performance of the Jameson Cell is summarized. It has been demonstrated that the Jameson Cell performs well with minimum supervision and maintenance. The test work conducted at the University of Queensland and Blackwater Coal Mine has showed that the Jameson Cell performed consistently well at all testing conditions. The parallel tests have demonstrated that the Jameson Cell is superior over the conventional cells.

  2. Modeling collective cell motility

    NASA Astrophysics Data System (ADS)

    Rappel, Wouter-Jan

    Eukaryotic cells often move in groups, a critical aspect of many biological and medical processes including wound healing, morphogenesis and cancer metastasis. Modeling can provide useful insights into the fundamental mechanisms of collective cell motility. Constructing models that incorporate the physical properties of the cells, however, is challenging. Here, I discuss our efforts to build a comprehensive cell motility model that includes cell membrane properties, cell-substrate interactions, cell polarity, and cell-cell interaction. The model will be applied to a variety of systems, including motion on micropatterned substrates and the migration of border cells in Drosophila. This work was supported by NIH Grant No. P01 GM078586 and NSF Grant No. 1068869.

  3. Fuel cells seminar

    SciTech Connect

    1996-12-01

    This year`s meeting highlights the fact that fuel cells for both stationary and transportation applications have reached the dawn of commercialization. Sales of stationary fuel cells have grown steadily over the past 2 years. Phosphoric acid fuel cell buses have been demonstrated in urban areas. Proton-exchange membrane fuel cells are on the verge of revolutionizing the transportation industry. These activities and many more are discussed during this seminar, which provides a forum for people from the international fuel cell community engaged in a wide spectrum of fuel cell activities. Discussions addressing R&D of fuel cell technologies, manufacturing and marketing of fuel cells, and experiences of fuel cell users took place through oral and poster presentations. For the first time, the seminar included commercial exhibits, further evidence that commercial fuel cell technology has arrived. A total of 205 papers is included in this volume.

  4. Kidney cell electrophoresis

    NASA Technical Reports Server (NTRS)

    Todd, P. W.

    1985-01-01

    Tasks were undertaken in support of two objectives. They are: (1) to carry out electrophoresis experiments on cells in microgravity; and (2) assess the feasibility of using purified kidney cells from embryonic kidney cultures as a source of important cell products. Investigations were carried out in the following areas: (1) ground based electrophoresis technology; (2) cell culture technology; (3) electrophoresis of cells; (4) urokinase assay research; (5) zero-g electrophoresis; and (6) flow cytometry.

  5. Stem cell biobanks.

    PubMed

    Bardelli, Silvana

    2010-04-01

    Stem cells contribute to innate healing and harbor a promising role for regenerative medicine. Stem cell banking through long-term storage of different stem cell platforms represents a fundamental source to preserve original features of stem cells for patient-specific clinical applications. Stem cell research and clinical translation constitute fundamental and indivisible modules catalyzed through biobanking activity, generating a return of investment. PMID:20560026

  6. Technology Status: Fuel Cells and Electrolysis Cells

    NASA Technical Reports Server (NTRS)

    Mcbryar, H.

    1978-01-01

    The status of the baselined shuttle fuel cell as well as the acid membrane fuel cell and space-oriented water electrolysis technologies are presented. The more recent advances in the alkaline fuel cell technology area are the subject of a companion paper. A preliminary plan for the focusing of these technologies towards regenerative energy storage applications in the multi-hundred kilowatt range is also discussed.

  7. Screening of solar cells

    NASA Technical Reports Server (NTRS)

    Appelbaum, J.; Chait, A.; Thompson, D. A.

    1993-01-01

    Because solar cells in a production batch are not identical, screening is performed to obtain similar cells for aggregation into arrays. A common technique for screening is based on a single operating point of the I-V characteristic of the cell, usually the maximum power point. As a result, inferior cell matching may occur at the actual operating points. Screening solar cells based on the entire I-V characteristic will inherently result in more similar cells in the array. An array consisting of more similar cells is likely to have better overall characteristics and more predictable performance. Solar cell screening methods and cell ranking are discussed. The concept of a mean cell is defined as a cell 'best' representing all the cells in the production batch. The screening and ranking of all cells are performed with respect to the mean cell. The comparative results of different screening methods are illustrated on a batch of 50 silicon cells of the Space Station Freedom.

  8. Optimizing stem cell culture.

    PubMed

    van der Sanden, Boudewijn; Dhobb, Mehdi; Berger, François; Wion, Didier

    2010-11-01

    Stem cells always balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical and need to be continuously refined according to progress in our stem cell biology understanding and the latest technological developments. In the past few years, major efforts have been made to define more precisely the medium composition in which stem cells grow or differentiate. This led to the progressive replacement of ill-defined additives such as serum or feeder cell layers by recombinant cytokines or growth factors. Another example is the control of the oxygen pressure. For many years cell cultures have been done under atmospheric oxygen pressure which is much higher than the one experienced by stem cells in vivo. A consequence of cell metabolism is that cell culture conditions are constantly changing. Therefore, the development of high sensitive monitoring processes and control algorithms is required for ensuring cell culture medium homeostasis. Stem cells also sense the physical constraints of their microenvironment. Rigidity, stiffness, and geometry of the culture substrate influence stem cell fate. Hence, nanotopography is probably as important as medium formulation in the optimization of stem cell culture conditions. Recent advances include the development of synthetic bioinformative substrates designed at the micro- and nanoscale level. On going research in many different fields including stem cell biology, nanotechnology, and bioengineering suggest that our current way to culture cells in Petri dish or flasks will soon be outdated as flying across the Atlantic Ocean in the Lindbergh's plane. PMID:20803548

  9. Screening of solar cells

    SciTech Connect

    Appelbaum, J.; Chait, A.; Thompson, D.A.

    1993-07-01

    Because solar cells in a production batch are not identical, screening is performed to obtain similar cells for aggregation into arrays. A common technique for screening is based on a single operating point of the I-V characteristic of the cell, usually the maximum power point. As a result, inferior cell matching may occur at the actual operating points. Screening solar cells based on the entire I-V characteristic will inherently result in more similar cells in the array. An array consisting of more similar cells is likely to have better overall characteristics and more predictable performance. Solar cell screening methods and cell ranking are discussed. The concept of a mean cell is defined as a cell 'best' representing all the cells in the production batch. The screening and ranking of all cells are performed with respect to the mean cell. The comparative results of different screening methods are illustrated on a batch of 50 silicon cells of the Space Station Freedom.

  10. Analytical pyrolysis of cells and cell fragments

    SciTech Connect

    Faix, O.; Bertelt, E.

    1995-12-01

    Wood of spruce, beech and birch was disintegrated without chemical pretreatment after 10 minutes of steaming at 110{degrees}C in a laboratory defibrator. Fibers, vessels, and fragments of secondary wall were separated by wet screening. A hydrocylon was used for separation of middle lamellae. By using analytical pyrolysis-GC/MS, parenchymatic cells were found to be richer in lignin than the other cells. The lignin content of middle lamellae was 35% (beech, spruce) and 39% (birch). In agreement with the literature, the S/G ratios of the vessels and middle lamellae was lower than those of the other cells and cell fragments.

  11. The cell biology of planar cell polarity

    PubMed Central

    2014-01-01

    Planar cell polarity (PCP) refers to the coordinated alignment of cell polarity across the tissue plane. Key to the establishment of PCP is asymmetric partitioning of cortical PCP components and intercellular communication to coordinate polarity between neighboring cells. Recent progress has been made toward understanding how protein transport, endocytosis, and intercellular interactions contribute to asymmetric PCP protein localization. Additionally, the functions of gradients and mechanical forces as global cues that bias PCP orientation are beginning to be elucidated. Together, these findings are shedding light on how global cues integrate with local cell interactions to organize cellular polarity at the tissue level. PMID:25349257

  12. Mast cells enhance T cell activation: Importance of mast cell-derived TNF

    NASA Astrophysics Data System (ADS)

    Nakae, Susumu; Suto, Hajime; Kakurai, Maki; Sedgwick, Jonathon D.; Tsai, Mindy; Galli, Stephen J.

    2005-05-01

    Mast cells are not only important effector cells in immediate hypersensitivity reactions and immune responses to pathogens but also can contribute to T cell-mediated disorders. However, the mechanisms by which mast cells might influence T cells in such settings are not fully understood. We find that mast cells can enhance proliferation and cytokine production in multiple T cell subsets. Mast cell-dependent enhancement of T cell activation can be promoted by FcRI-dependent mast cell activation, TNF production by both mast cells and T cells, and mast cell-T cell contact. However, at high concentrations of cells, mast cells can promote T cell activation independent of IgE or TNF. Finally, mast cells also can promote T cell activation by means of soluble factors. These findings identify multiple mechanisms by which mast cells can influence T cell proliferation and cytokine production. allergy | asthma | autoimmunity | cytokines | immune response

  13. AB241. Cancer stem cell-like side population cells in clear cell renal cell carcinoma cell line 769P

    PubMed Central

    Huang, Bin; Wang, Dao-Hu; Chen, Jun-Xing; Qiu, Shao-Peng

    2016-01-01

    Background Although cancers are widely considered to be maintained by stem cells, the existence of stem cells in renal cell carcinoma (RCC) has seldom been reported, in part due to the lack of unique surface markers. We here identified cancer stem cell-like cells with side population (SP) phenotype in five human RCC cell lines. Methods We here identified cancer stem cell-like cells with side population (SP) phenotype in five human RCC cell lines. Results Flow cytometry analysis revealed that 769P, a human clear cell RCC cell line, contained the largest amount of SP cells among five cell lines. These 769P SP cells possessed characteristics of proliferation, self-renewal, and differentiation, as well as strong resistance to chemotherapy and radiotherapy that were possibly related to the ABCB1 transporter. In vivo experiments with serial tumor transplantation in mice also showed that 769P SP cells formed tumors in NOD/SCID mice. Conclusions Taken together, these results indicate that 769P SP cells have the properties of cancer stem cells, which may play important roles in tumorigenesis and therapy-resistance of RCC.

  14. Single cell mechanics of keratinocyte cells.

    PubMed

    Lulevich, Valentin; Yang, Hsin-ya; Isseroff, R Rivkah; Liu, Gang-yu

    2010-11-01

    Keratinocytes represent the major cell type of the uppermost layer of human skin, the epidermis. Using AFM-based single cell compression, the ability of individual keratinocytes to resist external pressure and global rupturing forces is investigated and compared with various cell types. Keratinocytes are found to be 6-70 times stiffer than other cell types, such as white blood, breast epithelial, fibroblast, or neuronal cells, and in contrast to other cell types they retain high mechanic strength even after the cell's death. The absence of membrane rupturing peaks in the force-deformation profiles of keratinocytes and their high stiffness during a second load cycle suggests that their unique mechanical resistance is dictated by the cytoskeleton. A simple analytical model enables the quantification of Young's modulus of keratinocyte cytoskeleton, as high as 120-340 Pa. Selective disruption of the two major cytoskeletal networks, actin filaments and microtubules, does not significantly affect keratinocyte mechanics. F-actin is found to impact cell deformation under pressure. During keratinocyte compression, the plasma membrane stretches to form peripheral blebs. Instead of blebbing, cells with depolymerized F-actin respond to pressure by detaching the plasma membrane from the cytoskeleton underneath. On the other hand, the compression force of keratinocytes expressing a mutated keratin (cell line, KEB-7) is 1.6-2.2 times less than that for the control cell line that has normal keratin networks. Therefore, we infer that the keratin intermediate filament network is responsible for the extremely high keratinocyte stiffness and resilience. This could manifest into the rugged protective nature of the human epidermis. PMID:20728993

  15. Plant stem cell niches.

    PubMed

    Aichinger, Ernst; Kornet, Noortje; Friedrich, Thomas; Laux, Thomas

    2012-01-01

    Multicellular organisms possess pluripotent stem cells to form new organs, replenish the daily loss of cells, or regenerate organs after injury. Stem cells are maintained in specific environments, the stem cell niches, that provide signals to block differentiation. In plants, stem cell niches are situated in the shoot, root, and vascular meristems-self-perpetuating units of organ formation. Plants' lifelong activity-which, as in the case of trees, can extend over more than a thousand years-requires that a robust regulatory network keep the balance between pluripotent stem cells and differentiating descendants. In this review, we focus on current models in plant stem cell research elaborated during the past two decades, mainly in the model plant Arabidopsis thaliana. We address the roles of mobile signals on transcriptional modules involved in balancing cell fates. In addition, we discuss shared features of and differences between the distinct stem cell niches of Arabidopsis. PMID:22404469

  16. Microscale Fuel Cells

    SciTech Connect

    Holladay, Jamie D.; Viswanathan, Vish V.

    2005-11-03

    Perhaprs some of the most innovative work on fuel cells has been the research dedicated to applying silicon fabrication techniques to fuel cells technology creating low power microscale fuel cells applicable to microelectro mechanical systems (MEMS), microsensors, cell phones, PDA’s, and other low power (0.001 to 5 We) applications. In this small power range, fuel cells offer the decoupling of the energy converter from the energy storage which may enable longer operating times and instant or near instant charging. To date, most of the microscale fuel cells being developed have been based on proton exchange membrane fuel cell technology (PEMFC) or direct methanol fuel cell (DMFC) technology. This section will discuss requirements and considerations that need to be addressed in the development of microscale fuel cells, as well as some proposed designs and fabrication strategies.

  17. Tetraspanins in Cell Migration

    PubMed Central

    Jiang, Xupin; Zhang, Jiaping; Huang, Yuesheng

    2015-01-01

    Tetraspanins are a superfamily of small transmembrane proteins that are expressed in almost all eukaryotic cells. Through interacting with one another and with other membrane and intracellular proteins, tetraspanins regulate a wide range of proteins such as integrins, cell surface receptors, and signaling molecules, and thereby engage in diverse cellular processes ranging from cell adhesion and migration to proliferation and differentiation. In particular, tetraspanins modulate the function of proteins involved in all determining factors of cell migration including cell–cell adhesion, cell–ECM adhesion, cytoskeletal protrusion/contraction, and proteolytic ECM remodeling. We herein provide a brief overview of collective in vitro and in vivo studies of tetraspanins to illustrate their regulatory functions in the migration and trafficking of cancer cells, vascular endothelial cells, skin cells (keratinocytes and fibroblasts), and leukocytes. We also discuss the involvement of tetraspanins in various pathologic and remedial processes that rely on cell migration and their potential value as targets for therapeutic intervention. PMID:26091149

  18. Bacterial Cell Wall Components

    NASA Astrophysics Data System (ADS)

    Ginsberg, Cynthia; Brown, Stephanie; Walker, Suzanne

    Bacterial cell-surface polysaccharides cells are surrounded by a variety of cell-surface structures that allow them to thrive in extreme environments. Components of the cell envelope and extracellular matrix are responsible for providing the cells with structural support, mediating intercellular communication, allowing the cells to move or to adhere to surfaces, protecting the cells from attack by antibiotics or the immune system, and facilitating the uptake of nutrients. Some of the most important cell wall components are polysaccharide structures. This review discusses the occurrence, structure, function, and biosynthesis of the most prevalent bacterial cell surface polysaccharides: peptidoglycan, lipopolysaccharide, arabinogalactan, and lipoarabinomannan, and capsular and extracellular polysaccharides. The roles of these polysaccharides in medicine, both as drug targets and as therapeutic agents, are also described.

  19. Aquaporins and cell migration.

    PubMed

    Papadopoulos, M C; Saadoun, S; Verkman, A S

    2008-07-01

    Aquaporin (AQP) water channels are expressed primarily in cell plasma membranes. In this paper, we review recent evidence that AQPs facilitate cell migration. AQP-dependent cell migration has been found in a variety of cell types in vitro and in mice in vivo. AQP1 deletion reduces endothelial cell migration, limiting tumor angiogenesis and growth. AQP4 deletion slows the migration of reactive astrocytes, impairing glial scarring after brain stab injury. AQP1-expressing tumor cells have enhanced metastatic potential and local infiltration. Impaired cell migration has also been seen in AQP1-deficient proximal tubule epithelial cells, and AQP3-deficient corneal epithelial cells, enterocytes, and skin keratinocytes. The mechanisms by which AQPs enhance cell migration are under investigation. We propose that, as a consequence of actin polymerization/depolymerization and transmembrane ionic fluxes, the cytoplasm adjacent to the leading edge of migrating cells undergoes rapid changes in osmolality. AQPs could thus facilitate osmotic water flow across the plasma membrane in cell protrusions that form during migration. AQP-dependent cell migration has potentially broad implications in angiogenesis, tumor metastasis, wound healing, glial scarring, and other events requiring rapid, directed cell movement. AQP inhibitors may thus have therapeutic potential in modulating these events, such as slowing tumor growth and spread, and reducing glial scarring after injury to allow neuronal regeneration. PMID:17968585

  20. Induction of Functional Hair-Cell-Like Cells from Mouse Cochlear Multipotent Cells

    PubMed Central

    Liu, Quanwen; Shen, Yi; Chen, Jiarong; Ding, Jie; Tang, Zihua; Zhang, Cui; Chen, Jianling; Li, Liang; Chen, Ping; Wang, Jinfu

    2016-01-01

    In this paper, we developed a two-step-induction method of generating functional hair cells from inner ear multipotent cells. Multipotent cells from the inner ear were established and induced initially into progenitor cells committed to the inner ear cell lineage on the poly-L-lysine substratum. Subsequently, the committed progenitor cells were cultured on the mitotically inactivated chicken utricle stromal cells and induced into hair-cell-like cells containing characteristic stereocilia bundles. The hair-cell-like cells exhibited rapid permeation of FM1-43FX. The whole-cell patch-clamp technique was used to measure the membrane currents of cells differentiated for 7 days on chicken utricle stromal cells and analyze the biophysical properties of the hair-cell-like cells by recording membrane properties of cells. The results suggested that the hair-cell-like cells derived from inner ear multipotent cells were functional following differentiation in an enabling environment. PMID:27057177

  1. Lithium cell test results

    NASA Technical Reports Server (NTRS)

    Bragg, B. J.

    1977-01-01

    Three lithium SO2 cells, two lithium CF cells, and a vinyl chloride cell, all with crimped seals, and all strictly experimental, were independently discharged on resistors. Three temperatures were used and several different storage temperatures. Discharge rate generally on the nominal discharges were 0.1 amp, 0.5 amp, and 1 amp. Tests results show that the crimp seals are inadequate, especially for the SO2 cells. Normal discharges present no hazards. All cells discharge to zero. The problem of lithium cell explosions, such as occurred during off-limits testing, is discussed.

  2. Cell adhesion force microscopy

    PubMed Central

    Sagvolden, G.; Giaever, I.; Pettersen, E. O.; Feder, J.

    1999-01-01

    The adhesion forces of cervical carcinoma cells in tissue culture were measured by using the manipulation force microscope, a novel atomic force microscope. The forces were studied as a function of time and temperature for cells cultured on hydrophilic and hydrophobic polystyrene substrates with preadsorbed proteins. The cells attached faster and stronger at 37°C than at 23°C and better on hydrophilic than on hydrophobic substrates, even though proteins adsorb much better to the hydrophobic substrates. Because cell adhesion serves to control several stages in the cell cycle, we anticipate that the manipulation force microscope can help clarify some cell-adhesion related issues. PMID:9892657

  3. Hydrogen/bromine cell

    SciTech Connect

    Hohne, K.; Starbeck, G.

    1985-05-28

    Described herein is an energy storage device which utilizes a hydrogen/bromine cell. The cell includes a bromine electrode and a hydrogen electrode. The cell is light weight, resists corrosion caused by bromine or hydrobromic acid and uses both an electrolysis and a fuel cell reaction to store or discharge electrical energy. The cell frame is made of graphite and has a pyrographite coating on at least the portion facing the bromine electrode. This cell is therefore very useful in matching varying energy supplies with varying energy demands and allows for decentralization of energy storage.

  4. On cells and size

    NASA Astrophysics Data System (ADS)

    Boudaoud, Arezki

    2002-03-01

    The growth of isolated cells is studied. They are modeled as growing elastic shells submitted to an internal pressure. A scaling law for a preferred radius of curvature is derived. It is in agreement with compiled experimental data over more than orders of magnitude in cell radia (for elongated cells from bacteria to giant algae cells). The physical picture is that a cell grow spherically until the preferred radius and then elongate into a cylindrical tube to keep the preferred curvature, except if a cell division occurs. A simplification of the model is investigated more quantitavely.

  5. Solar cell activation system

    SciTech Connect

    Apelian, L.

    1983-07-05

    A system for activating solar cells involves the use of phosphorescent paint, the light from which is amplified by a thin magnifying lens and used to activate solar cells. In a typical system, a member painted with phosphorescent paint is mounted adjacent a thin magnifying lens which focuses the light on a predetermined array of sensitive cells such as selenium, cadmium or silicon, mounted on a plastic board. A one-sided mirror is mounted adjacent the cells to reflect the light back onto said cells for purposes of further intensification. The cells may be coupled to rechargeable batteries or used to directly power a small radio or watch.

  6. Assessment of pancreas cells

    NASA Technical Reports Server (NTRS)

    Vanoss, C. J.

    1978-01-01

    Pancreatic islets were obtained from guinea pig pancreas by the collagenase method and kept alive in tissue culture prior to further studies. Pancreas cell morphology was studied by standard histochemical techniques using light microscopy. Preparative vertical electrophoresis-levitation of dispersed fetal guinea pig pancreas cells was conducted in phosphate buffer containing a heavy water (D20) gradient which does not cause clumping of cells or alter the osmolarity of the buffers. The faster migrating fractions tended to be enriched in beta-cell content. Alpha and delta cells were found to some degree in most fractions. A histogram showing the cell count distribution is included.

  7. Are mesenchymal stromal cells immune cells?

    PubMed

    Hoogduijn, Martin J

    2015-01-01

    Mesenchymal stromal cells (MSCs) are considered to be promising agents for the treatment of immunological disease. Although originally identified as precursor cells for mesenchymal lineages, in vitro studies have demonstrated that MSCs possess diverse immune regulatory capacities. Pre-clinical models have shown beneficial effects of MSCs in multiple immunological diseases and a number of phase 1/2 clinical trials carried out so far have reported signs of immune modulation after MSC infusion. These data indicate that MSCs play a central role in the immune response. This raises the academic question whether MSCs are immune cells or whether they are tissue precursor cells with immunoregulatory capacity. Correct understanding of the immunological properties and origin of MSCs will aid in the appropriate and safe use of the cells for clinical therapy. In this review the whole spectrum of immunological properties of MSCs is discussed with the aim of determining the position of MSCs in the immune system. PMID:25880839

  8. Germ cell binding to rat Sertoli cells in vitro

    SciTech Connect

    DePhilip, R.M.; Danahey, D.G.

    1987-12-01

    The interaction between male germ cells and Sertoli cells was studied in vitro by co-incubation experiments using isolated rat germ cells and primary cultures of Sertoli cells made germ cell-free by the differential sensitivity of germ cells to hypotonic shock. The germ cell/Sertoli cell interaction was examined morphologically with phase-contrast and scanning electron microscopy and