Enhancing the Effects of Low Dose Doxorubicin Treatment by the Radiation in T47D and SKBR3 Breast Cancer Cells

PubMed Central

Aghaee, Fahimeh; Baradaran, Behzad; Mesbahi, Asghar; Mohammadzadeh, Mohammad; Jafarabadi, Mohammad Asghari

2013-01-01

Purpose Breast cancer is the most common malignancy of women worldwide. Radiotherapy consists of a vital element in the treatment of breast cancer but relative side effects and different radioactive responses are limiting factors for a successful treatment. Doxorubicin has been used to treat cancers for over 30 years and is considered as the most effective drug in the treatment of breast cancer. There are also many chronic side effects that limit the amount of doxorubicin that can be administered. The combined radio-drug treatment, with low doses, can be an approach for reducing side effects from single modality treatments instead of suitable cure rates. Methods We have studied the effect of 1, 1.5, and 2 Gy doses of 9 MV X-rays along with 1 µM doxorubicin on inducing cell death, apoptosis and also p53 and PTEN gene expression in T47D and SKBR3 breast cancer cells. Results Doxorubicin treatment resulted in upregulation of radiation-induced levels of p53 and downregulation of PTEN at 1 and 1.5 Gy in T47D breast cancer cells, as well as downregulation of p53 mRNA level of expression and upregulation of PTEN mRNA level of expression in SKBR3 breast cancer cell line. In addition, doxorubicin in combination with radiation decreased the viability of breast cancer cell lines in the both cell lines. Conclusion Low doses of doxorubicin, with least cell toxicity, may be an effective treatment for breast cancer when used in conjunction with ionizing radiation. PMID:23843848

Enabling screening in 3D microenvironments: probing matrix and stromal effects on the morphology and proliferation of T47D breast carcinoma cells.

PubMed

Montanez-Sauri, Sara I; Sung, Kyung Eun; Berthier, Erwin; Beebe, David J

2013-03-01

During breast carcinoma progression, the three-dimensional (3D) microenvironment is continuously remodeled, and changes in the composition of the extracellular matrix (ECM) occur. High throughput screening platforms have been used to decipher the complexity of the microenvironment and to identify ECM components responsible for cancer progression. However, traditional screening platforms are typically limited to two-dimensional (2D) cultures, and often exclude the influence of ECM and stromal components. In this work, a system that integrates 3-dimensional cell culture techniques with an automated microfluidic platform was used to create a new ECM screening platform that cultures cells in more physiologically relevant 3D in vitro microenvironments containing stromal cells and different ECM molecules. This new ECM screening platform was used to culture T47D breast carcinoma cells in mono- and co-culture with human mammary fibroblasts (HMF) with seven combinations of three different ECM proteins (collagen, fibronectin, laminin). Differences in the morphology of T47D clusters, and the proliferation of T47D cells were found in ECM compositions rich in fibronectin or laminin. In addition, an MMP enzyme activity inhibition screening showed the capabilities of the platform for small molecule screening. The platform presented in this work enables screening for the effects of matrix and stromal compositions and show promises for providing new insights in the identification of key ECM components involved in breast cancer.

Thiolated chitosan nanoparticles as a delivery system for antisense therapy: evaluation against EGFR in T47D breast cancer cells.

PubMed

Talaei, Fatemeh; Azizi, Ebrahim; Dinarvand, Rassoul; Atyabi, Fatemeh

2011-01-01

Thiolated chitosan has high transfection and mucoadhesive properties. We investigated the potential of two recently synthesized polymers: NAC-C (N-acetyl cysteine-chitosan) and NAP-C (N-acetyl penicillamine-chitosan) in anticancer drug delivery targeting epidermal growth factor receptor (EGFR). Doxorubicin (DOX) and antisense oligonucleotide (ASOND)-loaded polymer nanoparticles were prepared in water by a gelation process. Particle characterization, drug loading, and drug release were evaluated. To verify drug delivery efficiency in vitro experiments on a breast cancer cell line (T47D) were performed. EGFR gene and protein expression was analyzed by real time quantitative polymerase chain reaction and Western blotting, respectively. A loading percentage of 63% ± 5% for ASOND and 70% ± 5% for DOX was achieved. Drug release data after 15 hours showed that ASOND and DOX were completely released from chitosan-based particles while a lower and more sustained release of only 22% ± 8% was measured for thiolated particles. In a cytosol simulated release medium/reducing environment, such as found intracellularly, polymer-based nanoparticles dissociated, liberating approximately 50% of both active substances within 7 hours. ASOND-loaded polymer nanoparticles had higher stability and high mucoadhesive properties. The ASOND-loaded thiolated particles significantly suppressed EGFR gene expression in T47D cells compared with ASOND-loaded chitosan particles and downregulated EGFR protein expression in cells. This study could facilitate future investigations into the functionality of NAP-C and NAC-C polymers as an efficient ASOND delivery system in vitro and in vivo.

Thiolated chitosan nanoparticles as a delivery system for antisense therapy: evaluation against EGFR in T47D breast cancer cells

PubMed Central

Talaei, Fatemeh; Azizi, Ebrahim; Dinarvand, Rassoul; Atyabi, Fatemeh

2011-01-01

Thiolated chitosan has high transfection and mucoadhesive properties. We investigated the potential of two recently synthesized polymers: NAC-C (N-acetyl cysteine-chitosan) and NAP-C (N-acetyl penicillamine-chitosan) in anticancer drug delivery targeting epidermal growth factor receptor (EGFR). Doxorubicin (DOX) and antisense oligonucleotide (ASOND)-loaded polymer nanoparticles were prepared in water by a gelation process. Particle characterization, drug loading, and drug release were evaluated. To verify drug delivery efficiency in vitro experiments on a breast cancer cell line (T47D) were performed. EGFR gene and protein expression was analyzed by real time quantitative polymerase chain reaction and Western blotting, respectively. A loading percentage of 63% ± 5% for ASOND and 70% ± 5% for DOX was achieved. Drug release data after 15 hours showed that ASOND and DOX were completely released from chitosan-based particles while a lower and more sustained release of only 22% ± 8% was measured for thiolated particles. In a cytosol simulated release medium/reducing environment, such as found intracellularly, polymer-based nanoparticles dissociated, liberating approximately 50% of both active substances within 7 hours. ASOND-loaded polymer nanoparticles had higher stability and high mucoadhesive properties. The ASOND-loaded thiolated particles significantly suppressed EGFR gene expression in T47D cells compared with ASOND-loaded chitosan particles and downregulated EGFR protein expression in cells. This study could facilitate future investigations into the functionality of NAP-C and NAC-C polymers as an efficient ASOND delivery system in vitro and in vivo. PMID:21976973

Combination of low-concentration of novel phytoestrogen (8,9)-furanyl-pterocarpan-3-ol from Pachyrhizus erosus attenuated tamoxifen-associated growth inhibition on breast cancer T47D cells

PubMed Central

Nurrochmad, Arief; Lukitaningsih, Endang; Monikawati, Ameilinda; Septhea, Dita Brenna; Meiyanto, Edy

2013-01-01

Objective To investigate the estrogenic effect of (8,9)-furanyl-pterocarpan-3-ol (FPC) on growth of human breast cancer T47D cells and the interactions between the FPC and tamoxifen (TAM), on the growth of estrogen receptor-dependent breast cancer T47D cells. Methods The proliferation effect of FPC were conducted on T47D cells in vitro by MTT test. T47D cells were treated with FPC alone (0.01-200 µmol/L) or in combination with TAM 20 nmol/L. Furthermore, the expression of ERα or c-Myc were also determined by immunohistochemistry. Results The results indicated that administration of an anti-estrogen TAM showed growth inhibitory effect on T47D cells, wheraes co-administered with low concentration (less than 1 µmol/L) of FPC attenuated to promote cell proliferation. In contrast, the combination of TAM with higher doses (more than 20 µmol/L) of FPC showed growth inhibitory. This result was supported by immunocytochemistry studies that the administration of 20 nmol/L TAM down-regulated ER-α and c-Myc, but the combination of 20 nmol/L TAM and 1 µmol/L FPC robustly up-regulated expression of ER-α. Thus, the reduced growth inhibition of TAM 20 nmol/L by FPC 1 µmol/L on T47D cells may act via the modulation of ER-α. Conclusions The findings indicate and suggest that FPC had estrogenic activity at low concentrations and anti-estrogenic effect that are likely to be regulated by c-Myc and estrogen receptors. We also confirm that low concentration of FPC attenuated the growth-inhibitory effects of TAM on mammary tumor prevention. Therefore, the present study suggests that caution is warranted regarding the consumption of dietary FPC by breast cancer patients while on TMA therapy.

Opposing effects of estradiol- and testosterone-membrane binding sites on T47D breast cancer cell apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kampa, Marilena; Nifli, Artemissia-Phoebe; Charalampopoulos, Ioannis

Classical steroid mode of action involves binding to intracellular receptors, the later acting as ligand-activated nuclear transcription factors. Recently, membrane sites for different steroids have been also identified, mediating rapid, non-genomic, steroid actions. Membrane sites for estrogen and androgen have been found in a number of different cell types, bearing or not classical intracellular receptors. In the present study, with the use of radioligand binding, flow cytometry and confocal laser microscopy, we report that T47D human breast cancer cells express specific and saturable membrane receptors for both estrogen (K {sub D} 4.06 {+-} 3.31 nM) and androgen (K {sub D}more » 7.64 {+-} 3.15 nM). Upon activation with BSA-conjugated, non-permeable ligands (E{sub 2}-BSA and testosterone-BSA), membrane estrogen receptors protect cells from serum-deprivation-induced apoptosis, while androgen receptors induce apoptosis in serum-supplemented T47D cells. In addition, co-incubation of cells with a fixed concentration of one steroid and varying concentrations of the other reversed the abovementioned effect (apoptosis for androgen, and anti-apoptosis for E{sub 2}), suggesting that the fate of the cell depends on the relative concentration of either steroid in the culture medium. We also report the identification of membrane receptors for E{sub 2} and androgen in biopsy slides from breast cancer patients. Both sites are expressed, with the staining for membrane E{sub 2} being strongly present in ER-negative, less differentiated, more aggressive tumors. These findings suggest that aromatase inhibitors may exert their beneficial effects on breast cancer by also propagating the metabolism of local steroids towards androgen, inducing thus cell apoptosis through membrane androgen receptor activation.« less

Src Drives Growth of Antiestrogen Resistant Breast Cancer Cell Lines and Is a Marker for Reduced Benefit of Tamoxifen Treatment

PubMed Central

Larsen, Sarah L.; Laenkholm, Anne-Vibeke; Duun-Henriksen, Anne Katrine; Bak, Martin; Lykkesfeldt, Anne E.; Kirkegaard, Tove

2015-01-01

The underlying mechanisms leading to antiestrogen resistance in estrogen-receptor α (ER)-positive breast cancer is still poorly understood. The aim of this study was therefore to identify biomarkers and novel treatments for antiestrogen resistant breast cancer. We performed a kinase inhibitor screen on antiestrogen responsive T47D breast cancer cells and T47D-derived tamoxifen and fulvestrant resistant cell lines. We found that dasatinib, a broad-spectrum kinase inhibitor, inhibited growth of the antiestrogen resistant cells compared to parental T47D cells. Furthermore western blot analysis showed increased expression and phosphorylation of Src in the resistant cells and that dasatinib inhibited phosphorylation of Src and also signaling via Akt and Erk in all cell lines. Immunoprecipitation revealed Src: ER complexes only in the parental T47D cells. In fulvestrant resistant cells, Src formed complexes with the Human Epidermal growth factor Receptor (HER)1 and HER2. Neither HER receptors nor ER were co-precipitated with Src in the tamoxifen resistant cell lines. Compared to treatment with dasatinib alone, combined treatment with dasatinib and fulvestrant had a stronger inhibitory effect on tamoxifen resistant cell growth, whereas dasatinib in combination with tamoxifen had no additive inhibitory effect on fulvestrant resistant growth. When performing immunohistochemical staining on 268 primary tumors from breast cancer patients who had received tamoxifen as first line endocrine treatment, we found that membrane expression of Src in the tumor cells was significant associated with reduced disease-free and overall survival. In conclusion, Src was identified as target for treatment of antiestrogen resistant T47D breast cancer cells. For tamoxifen resistant T47D cells, combined treatment with dasatinib and fulvestrant was superior to treatment with dasatinib alone. Src located at the membrane has potential as a new biomarker for reduced benefit of tamoxifen. PMID

Effect of silibinin-loaded nano-niosomal coated with trimethyl chitosan on miRNAs expression in 2D and 3D models of T47D breast cancer cell line.

PubMed

Yazdi Rouholamini, Seyede Elmira; Moghassemi, Saeid; Maharat, Zahra; Hakamivala, Amirhossien; Kashanian, Susan; Omidfar, Kobra

2018-05-01

Silibinin is a natural flavonoid with a strong antioxidant property and weak cytotoxic activity. It has demonstrated anti-tumoural activity against many types of malignancies; however, due to its hydrophobic structure, it has poor water solubility, bioavailability and permeability across intestinal epithelial cells. To improve the effect of silibinin, we have vehiculated silibinin by a highly stable niosomal nanostructure based on a Span 60/cholesterol (CH)/N-trimethyl chitosan (TMC) system in order to study its potential application for the delivery of silibinin in T47D cultured under three-dimensional (3D) and two-dimensional (2D) conditions. To study the effect of nanodrug on miRNAs expression, we evaluated quantitative expression of miRNA-21 and miRNA-15a as well as miR-141 and miR-200c which act as oncogene and tumour suppressors by real-time PCR. Results demonstrated that the mechanism of nanodrug action as well as the response of tumour cells differed in 3D culture as compared to 2D. Delivery of silibinin-loaded niosomes coated with TMC was found to be more effective in inhibiting the growth of tumour cells and inducing apoptosis than free silibinin administration. In silibinin-treated cells, death occurred in a dose- and time- dependent manner by induction of apoptosis and alteration of the cell cycle. Real-time PCR analysis revealed a decrease in miR-21, miR-15a and miR-141while increase in miR-200c expression levels was observed in silibinin-treated cells relative to the levels in the untreated cells. The results show that nanodrug delivery was more effective than free silibinin administration in changing the level of miRNAs expression in cancer cells. Therefore, niosomal nanostructure with TMC could be a suitable vehicle for hydrophobic compounds, such as silibinin, by improving their action in cancer therapy.

Perfluorinated chemicals, PFOS and PFOA, enhance the estrogenic effects of 17β-estradiol in T47D human breast cancer cells.

PubMed

Sonthithai, Pacharapan; Suriyo, Tawit; Thiantanawat, Apinya; Watcharasit, Piyajit; Ruchirawat, Mathuros; Satayavivad, Jutamaad

2016-06-01

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are the two most popular surfactants among perfluorinated compounds (PFCs), with a wide range of uses. Growing evidence suggests that PFCs have the potential to interfere with estrogen homeostasis, posing a risk of endocrine-disrupting effects. This in vitro study aimed to investigate the estrogenic effect of these compounds on T47D hormone-dependent breast cancer cells. PFOS and PFOA (10(-12) to 10(-4)  M) were not able to induce estrogen response element (ERE) activation in the ERE luciferase reporter assay. The ERE activation was induced when the cells were co-incubated with PFOS (10(-10) to 10(-7)  M) or PFOA (10(-9) to 10(-7)  M) and 1 nM of 17β-estradiol (E2). PFOS and PFOA did not modulate the expression of estrogen-responsive genes, including progesterone (PR) and trefoil factor (pS2), but these compounds enhanced the effect of E2-induced pS2 gene expression. Neither PFOS nor PFOA affected T47D cell viability at any of the tested concentrations. In contrast, co-exposure with PFOS or PFOA and E2 resulted in an increase of E2-induced cell viability, but no effect was found with 10 ng ml(-1) EGF co-exposure. Both compounds also intensified E2-dependent growth in the proliferation assay. ERK1/2 phosphorylation was increased by co-exposure with PFOS or PFOA and E2, but not with EGF. Collectively, this study shows that PFOS and PFOA did not possess estrogenic activity, but they enhanced the effects of E2 on estrogen-responsive gene expression, ERK1/2 activation and the growth of the hormone-deprived T47D cells. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

[gammadelta T cells stimulated by zoledronate kill osteosarcoma cells].

PubMed

Jiang, Hui; Xu, Qiang; Yang, Chao; Cao, Zhen-Guo; Li, Zhao-Xu; Ye, Zhao-Ming

2010-12-01

To investigate the cytotoxicity of human γδT cells from PBMCs stimulated by zoledronate against osteosarcoma cell line HOS in vitro and in vivo and evaluate the relavent pathways. The peripheral blood mononuclear cells (PBMCs)of healthy donors were stimulated by single dose zoledronate and cultured in the present of IL-2 for two weeks, analysising the percentage of γδT cells on a FACSCalibur cytometer.Study the cytotoxicity of γδT cells against the osteosarcoma line HOS using LDH release assay kit. Pre-treatment of γδT cells with anti-human γδTCR antibody, anti-human NKG2D antibody and concanamycin A to bolck the relavent pathways for evaluating the mechenisms of its cytotoxicity. In vivo, BALB/c mice were inoculated subcutaneously osteosarcoma cell HOS for developing hypodermal tumors. And they were randomized into two groups: unteated group, γδT cell therapy group. Tumor volume and weight of the two groups were compared. After two weeks of culture, γδT cells from zoledronate-stimulated PBMCs could reach (95±3)%. When the E:T as 6:1, 12:1, 25:1, 50:1, the percentage of osteosarcoma cell HOS killed by γδT cells was 26.8%, 31.5%, 37.8%, 40.9%, respectively.When anti-huma γδTCR antibody, anti-human NKG2D antibody and concanamycin A blocked the relavent pathways, the percentage was 32.3%, 4.7%, 16.7% ( E:T as 25:1), respectively. In vivo, the tumor inhibition rate of the group of γδT cell therapy was 42.78%. γδT cells derived from PBMCs stimulated by zoledronate can acquired pure γδT cells. And they show strong cytoxicity against osteosarcoma cell line HOS in vitro and in vivo.

Cytotoxicity of fucosterol containing fraction of marine algae against breast and colon carcinoma cell line

PubMed Central

Khanavi, Mahnaz; Gheidarloo, Razieh; Sadati, Nargess; Ardekani, Mohammad Reza Shams; Nabavi, Seyed Mohammad Bagher; Tavajohi, Shohreh; Ostad, Seyed Nasser

2012-01-01

Context: Marine algae produce different secondary metabolites with a wide range of biological activities. Many studies have been achieved on the screening of biological effects of marine organisms and a lot of active compounds were isolated and characterized. Aims: In an attempt to find cytotoxic compound of hexane fraction, isolation, identification, and cytotoxicity of active compound of this fraction were performed. Materials and Methods: In this study, total methanolic (70%) extract and partition fractions of hexane, chloroform (CHCl3), ethyl acetate (EtOAc), and MeOH–H2O of Sargassum angustifolium, Chondria dasyphylla, and Ulva flexuosa, collected from coastlines of the Persian Gulf in south of Iran, were studied against colon carcinoma (HT-29), colorectal adenocarcinoma (Caco-2), breast ductal carcinoma (T47D), and Swiss mouse embryo fibroblast (NIH 3T3) cell lines by MTT assay. Statistical Analysis Used: IC50 (median growth inhibitory concentration) values were calculated by Sigmaplot (10) software. Results: Hexane fraction of Chondria dasyphylla (IC50 82.26 ± 4.09 μg/ml) and MeOH-H2O fraction of Ulva flexuosa (IC50 116.92 ± 8.58 μg/ml) showed cytotoxic activity against proliferation of T47D cells. Hexane fraction of Sargassum angustifolium was also observed for cytotoxicity against T47D and HT-29 cell lines (IC50 166.42 ± 26.7 and 190.24 ± 52.8 μg/ml), respectively. An investigation of a component from the hexane fraction of Sargassum angustifolium yielded a steroidal metabolite, fucosterol, with cytotoxicity in T47D and HT29 (IC50 27.94 ± 9.3 and 70.41 ± 7.5 μg/ml). Conclusions: These results indicated that fucosterol, the most abundant phytosterol in brown algae, is responsible for cytotoxic effect of this extract against breast and colon carcinoma cell lines. PMID:22438665

Characterization of a cultured human T-cell line with genetically altered ribonucleotide reductase activity. Model for immunodeficiency.

PubMed

Waddell, D; Ullman, B

1983-04-10

From human CCRF-CEM T-cells growing in continuous culture, we have selected, isolated, and characterized a clonal cell line, APHID-D2, with altered ribonucleotide reductase activity. In comparative growth rate experiments, the APHID-D2 cell line is less sensitive than the parental cell line to growth inhibition by deoxyadenosine in the presence of 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase. The APHID-D2 cell line has elevated levels of all four dNTPs. The resistance of the APHID-D2 cell line to growth inhibition by deoxyadenosine and the abnormal dNTP levels can be explained by the fact that the APHID-D2 ribonucleotide reductase, unlike the parental ribonucleotide reductase, is not normally sensitive to inhibition by dATP. These results suggest that the allosteric site of ribonucleotide reductase which binds both dATP and ATP is altered in the APHID-D2 line. The isolation of a mutant clone of human T-cells which contains a ribonucleotide reductase that has lost its normal sensitivity to dATP and which is resistant to deoxyadenosine-mediated growth inhibition suggests that a primary pathogenic target of accumulated dATP in lymphocytes from patients with adenosine deaminase deficiency may be the cellular ribonucleotide reductase.

Adoptive immunotherapy mediated by ex vivo expanded natural killer T cells against CD1d-expressing lymphoid neoplasms.

PubMed

Bagnara, Davide; Ibatici, Adalberto; Corselli, Mirko; Sessarego, Nadia; Tenca, Claudya; De Santanna, Amleto; Mazzarello, Andrea; Daga, Antonio; Corvò, Renzo; De Rossi, Giulio; Frassoni, Francesco; Ciccone, Ermanno; Fais, Franco

2009-07-01

CD1d is a monomorphic antigen presentation molecule expressed in several hematologic malignancies. Alpha-galactosylceramide (alpha-GalCer) is a glycolipid that can be presented to cytotoxic CD1d-restricted T cells. These reagents represent a potentially powerful tool for cell mediated immunotherapy. We set up an experimental model to evaluate the use of adoptively transferred cytotoxic CD1d-restricted T cells and alpha-GalCer in the treatment of mice engrafted with CD1d(+) lymphoid neoplastic cells. To this end the C1R cell line was transfected with CD1c or CD1d molecules. In addition, upon retroviral infection firefly luciferase was expressed on C1R transfected cell lines allowing the evaluation of tumor growth in xenografted immunodeficient NOD/SCID mice. The C1R-CD1d cell line was highly susceptible to specific CD1d-restricted T cell cytotoxicity in the presence alpha-GalCer in vitro. After adoptive transfer of CD1d-restricted T cells and alpha-GalCer to mice engrafted with both C1R-CD1c and C1R-CD1d, a reduction in tumor growth was observed only in CD1d(+) masses. In addition, CD1d-restricted T-cell treatment plus alpha-GalCer eradicated small C1R-CD1d(+) nodules. Immunohistochemical analysis revealed that infiltrating NKT cells were mainly observed in CD1d nodules. Our results indicate that ex vivo expanded cytotoxic CD1d-restricted T cells and alpha-GalCer may represent a new immunotherapeutic tool for treatment of CD1d(+) hematologic malignancies.

Long-term adaptation of breast tumor cell lines to high concentrations of nitric oxide.

PubMed

Vesper, Benjamin J; Elseth, Kim M; Tarjan, Gabor; Haines, G Kenneth; Radosevich, James A

2010-08-01

Nitric oxide (NO), a free radical, has been implicated in the biology of human cancers, including breast cancer, yet it is still unclear how NO affects tumor development and propagation. We herein gradually adapted four human breast adenocarcinoma cell lines (BT-20, Hs578T, T-47D, and MCF-7) to increasing concentrations of the NO donor DETA-NONOate up to 600 muM. The resulting model system consisted of a set of fully adapted high nitric oxide ("HNO") cell lines that are biologically different from the "parent" cell lines from which they originated. Although each of the four parent and HNO cell lines had identical morphologic appearance, the HNO cells grew faster than their corresponding parent cells and were resistant to both nitrogen- and oxygen-based free radicals. These cell lines serve as a novel tool to study the role of NO in breast cancer progression and potentially can be used to predict the therapeutic response leading to more efficient therapeutic regimens.

Generation and functional analysis of T cell lines and clones specific for schistosomula released products (SRP-A).

PubMed

Damonneville, M; Velge, F; Verwaerde, C; Pestel, J; Auriault, C; Capron, A

1987-08-01

Antigens present in the products released by the larval stage of schistosome (SRP-A) were shown to induce a strong cytotoxic and protective IgE response both in the rat and the monkey. T cell lines and clones specific for SRP-A or 26 kD antigens which are the main target of the cytotoxic IgE have been derived. The passive transfer of SRP-A specific T lymphocytes into infected rats led to an increase of the IgE response, conferring a significant level of protection to the rats. In coculture assays in vitro, these cell lines significantly enhanced the production of IgE by SRP-A sensitized rat spleen cells. This helper effect on the IgE response was confirmed with 26 kD T cell clone supernatants. Moreover, supernatants obtained after stimulation with phorbol myristate acetate were able to enhance the IgE production of a hybridoma B cell line (B48-14) producing a monoclonal IgE antibody, cytotoxic for the schistosomula.

Adoptive immunotherapy mediated by ex vivo expanded natural killer T cells against CD1d-expressing lymphoid neoplasms

PubMed Central

Bagnara, Davide; Ibatici, Adalberto; Corselli, Mirko; Sessarego, Nadia; Tenca, Claudya; De Santanna, Amleto; Mazzarello, Andrea; Daga, Antonio; Corvò, Renzo; De Rossi, Giulio; Frassoni, Francesco; Ciccone, Ermanno; Fais, Franco

2009-01-01

Background CD1d is a monomorphic antigen presentation molecule expressed in several hematologic malignancies. Alpha-galactosylceramide (α-GalCer) is a glycolipid that can be presented to cytotoxic CD1d-restricted T cells. These reagents represent a potentially powerful tool for cell mediated immunotherapy. Design and Methods We set up an experimental model to evaluate the use of adoptively transferred cytotoxic CD1d-restricted T cells and α-GalCer in the treatment of mice engrafted with CD1d+ lymphoid neoplastic cells. To this end the C1R cell line was transfected with CD1c or CD1d molecules. In addition, upon retroviral infection firefly luciferase was expressed on C1R transfected cell lines allowing the evaluation of tumor growth in xenografted immunodeficient NOD/SCID mice. Results The C1R-CD1d cell line was highly susceptible to specific CD1d-restricted T cell cytotoxicity in the presence α-GalCer in vitro. After adoptive transfer of CD1d-restricted T cells and α-GalCer to mice engrafted with both C1R-CD1c and C1R-CD1d, a reduction in tumor growth was observed only in CD1d+ masses. In addition, CD1d-restricted T-cell treatment plus α-GalCer eradicated small C1R-CD1d+ nodules. Immunohistochemical analysis revealed that infiltrating NKT cells were mainly observed in CD1d nodules. Conclusions Our results indicate that ex vivo expanded cytotoxic CD1d-restricted T cells and α-GalCer may represent a new immunotherapeutic tool for treatment of CD1d+ hematologic malignancies. PMID:19454494

Genetic heterogeneity of RPMI-8402, a T-acute lymphoblastic leukemia cell line

PubMed Central

STOCZYNSKA-FIDELUS, EWELINA; PIASKOWSKI, SYLWESTER; PAWLOWSKA, ROZA; SZYBKA, MALGORZATA; PECIAK, JOANNA; HULAS-BIGOSZEWSKA, KRYSTYNA; WINIECKA-KLIMEK, MARTA; RIESKE, PIOTR

2016-01-01

Thorough examination of genetic heterogeneity of cell lines is uncommon. In order to address this issue, the present study analyzed the genetic heterogeneity of RPMI-8402, a T-acute lymphoblastic leukemia (T-ALL) cell line. For this purpose, traditional techniques such as fluorescence in situ hybridization and immunocytochemistry were used, in addition to more advanced techniques, including cell sorting, Sanger sequencing and massive parallel sequencing. The results indicated that the RPMI-8402 cell line consists of several genetically different cell subpopulations. Furthermore, massive parallel sequencing of RPMI-8402 provided insight into the evolution of T-ALL carcinogenesis, since this cell line exhibited the genetic heterogeneity typical of T-ALL. Therefore, the use of cell lines for drug testing in future studies may aid the progress of anticancer drug research. PMID:26870252

Equol as a potent radiosensitizer in estrogen receptor-positive and -negative human breast cancer cell lines.

PubMed

Taghizadeh, Bita; Ghavami, Laleh; Nikoofar, Alireza; Goliaei, Bahram

2015-07-01

Breast cancer is the most common cause of cancer death among women worldwide, and diet plays an important role in its prevention and progression. Radiotherapy has a limited but important role in the management of nearly every stage of breast cancer. We studied whether equol, the major metabolite of the soybean isoflavone daidzein, could enhance radiosensitivity in two human breast cancer cell lines (T47D and MDA-MB-231). MTT assay was used to examine equol's effect on cell viability. Sensitivity of cells to equol, radiation and a combination of both was determined by colonogenic assays. Induction of apoptosis by equol, radiation and the combination of both was also determined by acridine orange/ethidium bromide double staining fluorescence microscopy. DNA strand breaks were assessed by Comet assay. MTT assay showed that equol (0.1-350 μM) inhibited MDA-MB-231 and T47D cell growth in a time- and dose-dependent manner. Treatment of cells with equol for 72 h (MDA-MB-231) and 24 h (T47D) was found to inhibit cell growth with IC50 values of 252 μM and 228 μM, respectively. Furthermore, pretreatment of cells with 50 μM equol for 72 h (MDA-MB-231) and 24 h (T47D) sensitized the cells to irradiation. Equol was also found to enhance radiation-induced apoptosis. Comet assay results showed that the radiosensitizing effect of equol was accompanied by increased radiation-induced DNA damages. These results suggest for the first time that equol can be considered as a radiosensitizing agent and its effects may be due to increasing cell death following irradiation, increasing the remaining radiation-induced DNA damage and thus reducing the surviving fraction of irradiated cells.

Generation and functional analysis of T cell lines and clones specific for schistosomula released products (SRP-A).

PubMed Central

Damonneville, M; Velge, F; Verwaerde, C; Pestel, J; Auriault, C; Capron, A

1987-01-01

Antigens present in the products released by the larval stage of schistosome (SRP-A) were shown to induce a strong cytotoxic and protective IgE response both in the rat and the monkey. T cell lines and clones specific for SRP-A or 26 kD antigens which are the main target of the cytotoxic IgE have been derived. The passive transfer of SRP-A specific T lymphocytes into infected rats led to an increase of the IgE response, conferring a significant level of protection to the rats. In coculture assays in vitro, these cell lines significantly enhanced the production of IgE by SRP-A sensitized rat spleen cells. This helper effect on the IgE response was confirmed with 26 kD T cell clone supernatants. Moreover, supernatants obtained after stimulation with phorbol myristate acetate were able to enhance the IgE production of a hybridoma B cell line (B48-14) producing a monoclonal IgE antibody, cytotoxic for the schistosomula. PMID:3498590

Nucleotide composition analysis of tRNA from leukemia patient cell samples and human cell lines.

PubMed Central

Agris, P F

1975-01-01

A technique developed for analysis of less than microgram quantities of tRNA has been applied to the study of human leukemia. Leucocytes from peripheal blood and bone marrow samples of six, untreated leukemia patients and cells of five different established human cell lines were maintained for 18 hours in media containing (32P)-phosphate. Incorporation of radioactive phosphate into the cells from the patient samples was slightly less than that of the cell lines. Likewise, incorporation of (32P)-phosphate into the tRNA of the patient samples (approximately 5 x 106 DPM/mug tRNA) was also less then that incorporated into the tRNA of the cell lines. The major and minor nucleotide compositions of the unfractionated tRNA preparations from each patient sample and each cell line were determined and compared. Similarities and differences in the major and minor nucleotide compositions of the tRNA preparations are discussed with reference to types of leukemia and the importance of patient sample analysis versus analysis of cultured human cells. PMID:1057159

Cdx2 Polymorphism Affects the Activities of Vitamin D Receptor in Human Breast Cancer Cell Lines and Human Breast Carcinomas

PubMed Central

Di Benedetto, Anna; Korita, Etleva; Goeman, Frauke; Sacconi, Andrea; Biagioni, Francesca; Blandino, Giovanni; Strano, Sabrina; Muti, Paola; Mottolese, Marcella; Falvo, Elisabetta

2015-01-01

Vitamin D plays a role in cancer development and acts through the vitamin D receptor (VDR). It regulates the action of hormone responsive genes and is involved in cell cycle regulation, differentiation and apoptosis. VDR is a critical component of the vitamin D pathway and different common single nucleotide polymorphisms have been identified. Cdx2 VDR polymorphism can play an important role in breast cancer, modulating the activity of VDR. The objective of this study is to assess the relationship between the Cdx2 VDR polymorphism and the activities of VDR in human breast cancer cell lines and carcinomas breast patients. Cdx2 VDR polymorphism and antiproliferative effects of vitamin D treatment were investigated in a panel of estrogen receptor-positive (MCF7 and T-47D) and estrogen receptor-negative (MDA-MB-231, SUM 159PT, SK-BR-3, BT549, MDA-MB-468, HCC1143, BT20 and HCC1954) human breast cancer cell lines. Furthermore, the potential relationship among Cdx2 VDR polymorphism and a number of biomarkers used in clinical management of breast cancer was assessed in an ad hoc set of breast cancer cases. Vitamin D treatment efficacy was found to be strongly dependent on the Cdx2 VDR status in ER-negative breast cancer cell lines tested. In our series of breast cancer cases, the results indicated that patients with variant homozygote AA were associated with bio-pathological characteristics typical of more aggressive tumours, such as ER negative, HER2 positive and G3. Our results may suggest a potential effect of Cdx2 VDR polymorphism on the efficacy of vitamin D treatment in aggressive breast cancer cells (estrogen receptor negative). These results suggest that Cdx2 polymorphism may be a potential biomarker for vitamin D treatment in breast cancer, independently of the VDR receptor expression. PMID:25849303

Binding of galectin-1 to breast cancer cells MCF7 induces apoptosis and inhibition of proliferation in vitro in a 2D- and 3D- cell culture model.

PubMed

Geiger, Pamina; Mayer, Barbara; Wiest, Irmi; Schulze, Sandra; Jeschke, Udo; Weissenbacher, Tobias

2016-11-08

Galectin-1 (gal-1) belongs to the family of β-galactoside-binding proteins which primarily recognizes the Galβ1-4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. The lectin recognizes correspondent glycoepitopes on human breast cancer cells. Galectin-1 is expressed both in normal and malignant tissues. Lymphatic organs naturally possessing high rates of apoptotic cells, express high levels of Galectin-1. Furthermore galectin-1 can initiate T cell apoptosis. Binding of galectin-1 to trophoblast tumor cells presenting the oncofetal Thomsen-Friedenreich (TF) carbohydrate antigen inhibits tumor cell proliferation. In this study we examined the impact galectin-1 has in vitro on cell proliferation, apoptotic potential and metabolic activity of MCF-7 and T-47D breast cancer cells in dependence to their expression of the Thomsen-Friedenreich (TF) tumor antigen. For proliferation and apoptosis assays cells were grown in presence of 10, 30 and 60 μg gal-1/ml medium. Cell proliferation was determined by a BrdU uptake ELISA. Detection of apoptotic cells was done by M30 cyto death staining, in situ nick translation and by a nucleosome ELISA method. Furthermore we studied the impact galectin-1 has on the metabolic activity of MCF-7 and T-47D cells in a homotypic three-dimensional spheroid cell culture model mimicking a micro tumour environment. Gal-1 inhibited proliferation of MCF-7 cells (strong expression of the TF epitope) but did not significantly change proliferation of T-47D cells (weak expression of the TF epitope). The incubation of MCF-7 cells with gal-1 raised number of apoptotic cells significantly. Treating the spheroids with 30 μg/ml galectin-1 in addition to standard chemotherapeutic regimes (FEC, TAC) resulted in further suppression of the metabolic activity in MCF-7 cells whereas T-47D cells were not affected. Our results demonstrate that galectin-1 can inhibit proliferation und metabolic cell activity and induce

Spontaneous pregnancy in a woman with 45,X/47,XXX mosaicism in both serum and germ cell lines. A case report.

PubMed

Eblen, Abby C; Nakajima, Steve T

2003-02-01

This is the first published case report of pregnancy in a women with 45, X/47, XXX mosaicism in both blood and germ cell lines. The patient conceived, and analysis of ovarian tissue confirmed a karyotype of 45, X/47, XXX. Women with a 45, X/47, XXX karyotype in the germ cell line can conceive, as this case demonstrates.

Structural effects of TiO2 nanoparticles and doxorubicin on DNA and their antiproliferative roles in T47D and MCF7 cells.

PubMed

Hekmat, Azadeh; Saboury, Ali Akbar; Divsalar, Adeleh; Seyedarabi, Arefeh

2013-07-01

The structural changes in DNA caused by the combined effects of TiO2 nanoparticles (TiO2 NPs) and doxorubicin (DOX) were investigated along with their corresponding inhibitory roles in the growth of T47D and MCF7 cells. The UV-visible titration studies showed that DOX+ TiO2 NPs could form a novel complex with DNA. The data also reveal that the TiO2-DOX complex forms through a 1:4 stoichiometric ratio in solution. The values of binding constants reveal that DOX+TiO2 NPs interact more strongly with DNA as compared to TiO2 NPs or DOX alone. CD data show that DOX+TiO2 NPs can noticeably cause disturbance on DNA structure compared to TiO2 NPs or DOX alone, considering that DNA is relatively thermally stable in the condition used. The anticancer property of 0.3 µM DOX+ 60 µM TiO2 NPs and 0.4 µM DOX+ 670 µM TiO2 NPs by MTT assay and DAPI stain demonstrates that this combination can tremendously diminish proliferation of T47D and MCF7cells compared to DOX or TiO2 NPs alone. The UV-Vis absorption spectroscopy, flow cytometry and fluorescence microscopy experiments show much more enhancement of DOX uptake through the use of TiO2 NPs. These results reveal that DOX+TiO2 NPs could proffer a novel strategy for the development of promising and efficient chemotherapy agents.

Antigen-specific T-cell lines transfer protective immunity against Trichinella spiralis in vivo.

PubMed Central

Riedlinger, J; Grencis, R K; Wakelin, D

1986-01-01

T-cell lines specific for infective muscle larvae antigens of the intestinal nematode Trichinella spiralis have been generated in vitro. These antigen-specific T-cell lines express the L3T4+ Ly2- phenotype and secrete the lymphokines IL-2, IL-3 and gamma-IFN. They are stable in culture for up to 15 weeks and are protective when adoptively transferred into naive recipients. As few as 2 x 10(5) T. spiralis-specific tract. In addition, intestinal mastocytosis and peripheral blood eosinophilia were accelerated after adoptive transfer of T. spiralis-specific T-cell lines. PMID:2423438

Frequent mutations in the p53 tumor suppressor gene in human leukemia T-cell lines.

PubMed Central

Cheng, J; Haas, M

1990-01-01

Human T-cell leukemia and T-cell acute lymphoblastic leukemia cell lines were studied for alterations in the p53 tumor suppressor gene. Southern blot analysis of 10 leukemic T-cell lines revealed no gross genomic deletions or rearrangements. Reverse transcription-polymerase chain reaction analysis of p53 mRNA indicated that all 10 lines produced p53 mRNA of normal size. By direct sequencing of polymerase chain reaction-amplified cDNA, we detected 11 missense and nonsense point mutations in 5 of the 10 leukemic T-cell lines studied. The mutations are primarily located in the evolutionarily highly conserved regions of the p53 gene. One of the five cell lines in which a mutation was detected possesses a homozygous point mutation in both p53 alleles, while the other four cell lines harbor from two to four different point mutations. An allelic study of two of the lines (CEM, A3/Kawa) shows that the two missense mutations found in each line are located on separate alleles, thus both alleles of the p53 gene may have been functionally inactivated by two different point mutations. Since cultured leukemic T-cell lines represent a late, fully tumorigenic stage of leukemic T cells, mutation of both (or more) alleles of the p53 gene may reflect the selection of cells possessing an increasingly tumorigenic phenotype, whether the selection took place in vivo or in vitro. Previously, we have shown that the HSB-2 T-cell acute lymphoblastic leukemia cell line had lost both alleles of the retinoblastoma tumor suppressor gene. Taken together, our data show that at least 6 of 10 leukemic T-cell lines examined may have lost the normal function of a known tumor suppressor gene, suggesting that this class of genes serves a critical role in the generation of fully tumorigenic leukemic T cells. Images PMID:2144611

Heparan Sulfate Modification of the Transmembrane Receptor CD47 Is Necessary for Inhibition of T Cell Receptor Signaling by Thrombospondin-1*

PubMed Central

Kaur, Sukhbir; Kuznetsova, Svetlana A.; Pendrak, Michael L.; Sipes, John M.; Romeo, Martin J.; Li, Zhuqing; Zhang, Lijuan; Roberts, David D.

2011-01-01

Cell surface proteoglycans on T cells contribute to retroviral infection, binding of chemokines and other proteins, and are necessary for some T cell responses to the matricellular glycoprotein thrombospondin-1. The major cell surface proteoglycans expressed by primary T cells and Jurkat T cells have an apparent Mr > 200,000 and are modified with chondroitin sulfate and heparan sulfate chains. Thrombospondin-1 bound in a heparin-inhibitable manner to this proteoglycan and to a soluble form released into the medium. Based on mass spectrometry, knockdown, and immunochemical analyses, the proteoglycan contains two major core proteins as follows: amyloid precursor-like protein-2 (APLP2, apparent Mr 230,000) and CD47 (apparent Mr > 250,000). CD47 is a known thrombospondin-1 receptor but was not previously reported to be a proteoglycan. This proteoglycan isoform of CD47 is widely expressed on vascular cells. Mutagenesis identified glycosaminoglycan modification of CD47 at Ser64 and Ser79. Inhibition of T cell receptor signaling by thrombospondin-1 was lost in CD47-deficient T cells that express the proteoglycan isoform of APLP2, indicating that binding to APLP2 is not sufficient. Inhibition of CD69 induction was restored in CD47-deficient cells by re-expressing CD47 or an S79A mutant but not by the S64A mutant. Therefore, inhibition of T cell receptor signaling by thrombospondin-1 is mediated by CD47 and requires its modification at Ser64. PMID:21343308

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system.

PubMed

Kutlesa, Snjezana; Zayas, Jennifer; Valle, Alexandra; Levy, Robert B; Jurecic, Roland

2009-08-01

Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Talpha, RAG-1, and T-cell receptor-Vbeta genes; and 3) produced interferon-gamma in response to T-cell receptor stimulation. These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation.

CD47-ligation induced cell death in T-acute lymphoblastic leukemia.

PubMed

Leclair, Pascal; Liu, Chi-Chao; Monajemi, Mahdis; Reid, Gregor S; Sly, Laura M; Lim, Chinten James

2018-05-10

CD47 is a cell-surface marker well recognized for its anti-phagocytic functions. As such, an emerging avenue for targeted cancer therapies involves neutralizing the anti-phagocytic function using monoclonal antibodies (mAbs) to enhance tumour cell immunogenicity. A lesser known consequence of CD47 receptor ligation is the direct induction of tumour cell death. While several mAbs and their derivatives with this property have been studied, the best characterized is the commercially available mAb B6H12, which requires immobilization for induction of cell death. Here, we describe a commercially available mAb, CC2C6, which induces T-cell acute lymphoblastic leukemia (ALL) cell death in soluble form. Soluble CC2C6 induces CD47-dependent cell death in a manner consistent with immobilized B6H12, which is characterized by mitochondrial deficiencies but is independent of caspase activation. Titration studies indicated that CC2C6 shares a common CD47-epitope with B6H12. Importantly, CC2C6 retains the anti-phagocytic neutralizing function, thus possessing dual anti-tumour properties. Although CD47-ligation induced cell death occurs in a caspase-independent manner, CC2C6 was found to stimulate increases in Mcl-1 and NOXA levels, two Bcl-2 family proteins that govern the intrinsic apoptosis pathway. Further analysis revealed that the ratio of Mcl-1:NOXA were minimally altered for cells treated with CC2C6, in comparison to cells treated with agents that induced caspase-dependent apoptosis which alter this ratio in favour of NOXA. Finally, we found that CC2C6 can synergize with low dose chemotherapeutic agents that induce classical apoptosis, giving rise to the possibility of an effective combination treatment with reduced long-term sequelae associated with high-dose chemotherapies in childhood ALL.

Acetonic Extract of Buxus sempervirens Induces Cell Cycle Arrest, Apoptosis and Autophagy in Breast Cancer Cells

PubMed Central

Ait-Mohamed, Ouardia; Battisti, Valentine; Joliot, Véronique; Fritsch, Lauriane; Pontis, Julien; Medjkane, Souhila; Redeuilh, Catherine; Lamouri, Aazdine; Fahy, Christine; Rholam, Mohamed; Atmani, Djebbar; Ait-Si-Ali, Slimane

2011-01-01

Plants are an invaluable source of potential new anti-cancer drugs. Here, we investigated the cytotoxic activity of the acetonic extract of Buxus sempervirens on five breast cancer cell lines, MCF7, MCF10CA1a and T47D, three aggressive triple positive breast cancer cell lines, and BT-20 and MDA-MB-435, which are triple negative breast cancer cell lines. As a control, MCF10A, a spontaneously immortalized but non-tumoral cell line has been used. The acetonic extract of Buxus sempervirens showed cytotoxic activity towards all the five studied breast cancer cell lines with an IC50 ranging from 7.74 µg/ml to 12.5 µg/ml. Most importantly, the plant extract was less toxic towards MCF10A with an IC50 of 19.24 µg/ml. Fluorescence-activated cell sorting (FACS) analysis showed that the plant extract induced cell death and cell cycle arrest in G0/G1 phase in MCF7, T47D, MCF10CA1a and BT-20 cell lines, concomitant to cyclin D1 downregulation. Application of MCF7 and MCF10CA1a respective IC50 did not show such effects on the control cell line MCF10A. Propidium iodide/Annexin V double staining revealed a pre-apoptotic cell population with extract-treated MCF10CA1a, T47D and BT-20 cells. Transmission electron microscopy analyses indicated the occurrence of autophagy in MCF7 and MCF10CA1a cell lines. Immunofluorescence and Western blot assays confirmed the processing of microtubule-associated protein LC3 in the treated cancer cells. Moreover, we have demonstrated the upregulation of Beclin-1 in these cell lines and downregulation of Survivin and p21. Also, Caspase-3 detection in treated BT-20 and T47D confirmed the occurrence of apoptosis in these cells. Our findings indicate that Buxus sempervirens extract exhibit promising anti-cancer activity by triggering both autophagic cell death and apoptosis, suggesting that this plant may contain potential anti-cancer agents for single or combinatory cancer therapy against breast cancer. PMID:21935420

Determining estrogenic activity in serum from ovariectomized rats treated with environmental compounds using an in vitro estrogen-mediated transcriptional activation assay (T47D-KBluc).

EPA Science Inventory

The use of cell-based assays to quantify low levels of estrogen in human serum is an accepted method. These assays are more sensitive but less specific than radioimmunoassays (RIA). Thus, we hypothesized that estrogen responsive T47D-KBluc cells would detect estrogenic activity i...

Determining estrogenic activity in serum from ovariectomized rats treated with environmental compounds using an in vitro estrogen-mediated transcriptional activation assay (T47D-KBluc)

EPA Science Inventory

The use of cell-based assays to quantify low levels of estrogen in human serum is an accepted method. These assays are more sensitive but less specific than radioimmunoassays (RIA). Thus, we hypothesized that estrogen responsive T47D-KBluc cells would detect estrogenic activity i...

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system

PubMed Central

Kutleša, Snježana; Zayas, Jennifer; Valle, Alexandra; Levy, Robert B.; Jurecic, Roland

2011-01-01

Objective Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. Materials and Methods EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. Results The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Tα, RAG-1, and T-cell receptor – Vβ genes; and 3) produced interferon-γ in response to T-cell receptor stimulation. Conclusions These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation. PMID:19447159

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

PubMed Central

Jinadasa, Rasika; Balmus, Gabriel; Gerwitz, Lee; Roden, Jamie; Weiss, Robert; Duhamel, Gerald

2011-01-01

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year. PMID:21490582

Human common acute lymphoblastic leukemia-derived cell lines are competent to recombine their T-cell receptor delta/alpha regions along a hierarchically ordered pathway.

PubMed

Hansen-Hagge, T E; Yokota, S; Reuter, H J; Schwarz, K; Bartram, C R

1992-11-01

Rearrangements of the T-cell receptor (TCR) delta locus are observed in the majority of human B-cell precursor acute lymphoblastic leukemias (ALL) with a striking predominance of V delta 2(D)D delta 3 recombinations in common ALL (cALL) patients. Recently, we and others showed that almost 20% of cALL cases are characterized by further recombination of V delta 2(D)D delta 3 segments to J alpha elements, thereby deleting the TCR delta locus in analogy to the delta Rec/psi J alpha pathway in differentiating alpha/beta-positive T cells. We report here that two human cALL-derived cell lines, REH and Nalm-6, are competent to recombine the TCR delta/alpha locus under standard tissue culture conditions. Analysis of different REH subclones obtained by limiting dilution of the initial culture showed a biased recombination of V delta 2D delta 3 to distinct J alpha elements. During prolonged tissue culture, a subclone acquired growth advantage and displaced parental cells as well as other subclones. Frequently, the DJ junctions of REH subclones contained extended stretches of palindromic sequences derived from modified D delta 3 coding elements. The other cell line, Nalm-6, started the TCR delta/alpha recombination with an unusual signal joint of a cryptic recombinase signal sequence (RSS) upstream of D delta 3 to the 3' RSS of D delta 3. The RSS dimer was subsequently rearranged in all investigated subclones to an identical J alpha element. Both cell lines might become valuable tools to unravel the complex regulation of TCR delta/alpha recombination pathways in malignant and normal lymphopoiesis.

Early exposure to interleukin-21 limits rapidly generated anti-Epstein-Barr virus T-cell line differentiation.

PubMed

Orio, Julie; Carli, Cédric; Janelle, Valérie; Giroux, Martin; Taillefer, Julie; Goupil, Mathieu; Richaud, Manon; Roy, Denis-Claude; Delisle, Jean-Sébastien

2015-04-01

The adoptive transfer of ex vivo-expanded Epstein-Barr virus (EBV)-specific T-cell lines is an attractive strategy to treat EBV-related neoplasms. Current evidence suggests that for adoptive immunotherapy in general, clinical responses are superior if the transferred cells have not reached a late or terminal effector differentiation phenotype before infusion. The cytokine interleukin (IL)-21 has shown great promise at limiting late T-cell differentiation in vitro, but this remains to be demonstrated in anti-viral T-cell lines. We adapted a clinically validated protocol to rapidly generate EBV-specific T-cell lines in 12 to 14 days and tested whether the addition of IL-21 at the initiation of the culture would affect T-cell expansion and differentiation. We generated clinical-scale EBV-restricted T-cell line expansion with balanced T-cell subset ratios. The addition of IL-21 at the beginning of the culture decreased both T-cell expansion and effector memory T-cell accumulation, with a relative increase in less-differentiated T cells. Within CD4 T-cell subsets, exogenous IL-21 was notably associated with the cell surface expression of CD27 and high KLF2 transcript levels, further arguing for a role of IL-21 in the control of late T-cell differentiation. Our results show that IL-21 has profound effects on T-cell differentiation in a rapid T-cell line generation protocol and as such should be further explored as a novel approach to program anti-viral T cells with features associated with early differentiation and optimal therapeutic efficacy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Influenza viruses production: Evaluation of a novel avian cell line DuckCelt®-T17.

PubMed

Petiot, Emma; Proust, Anaïs; Traversier, Aurélien; Durous, Laurent; Dappozze, Frédéric; Gras, Marianne; Guillard, Chantal; Balloul, Jean-Marc; Rosa-Calatrava, Manuel

2018-05-24

The influenza vaccine manufacturing industry is looking for production cell lines that are easily scalable, highly permissive to multiple viruses, and more effective in term of viral productivity. One critical characteristic of such cell lines is their ability to grow in suspension, in serum free conditions and at high cell densities. Influenza virus causing severe epidemics both in human and animals is an important threat to world healthcare. The repetitive apparition of influenza pandemic outbreaks in the last 20years explains that manufacturing sector is still looking for more effective production processes to replace/supplement embryonated egg-based process. Cell-based production strategy, with a focus on avian cell lines, is one of the promising solutions. Three avian cell lines, namely duck EB66®cells (Valneva), duck AGE.CR® cells (Probiogen) and quail QOR/2E11 cells (Baxter), are now competing with traditional mammalian cell platforms (Vero and MDCK cells) used for influenza vaccine productions and are currently at advance stage of commercial development for the manufacture of influenza vaccines. The DuckCelt®-T17 cell line presented in this work is a novel avian cell line developed by Transgene. This cell line was generated from primary embryo duck cells with the constitutive expression of the duck telomerase reverse transcriptase (dTERT). The DuckCelt®-T17 cells were able to grow in batch suspension cultures and serum-free conditions up to 6.5×10 6 cell/ml and were easily scaled from 10ml up to 3l bioreactor. In the present study, DuckCelt®-T17 cell line was tested for its abilities to produce various human, avian and porcine influenza strains. Most of the viral strains were produced at significant infectious titers (>5.8 log TCID50/ml) with optimization of the infection conditions. Human strains H1N1 and H3N2, as well as all the avian strains tested (H5N2, H7N1, H3N8, H11N9, H12N5) were the most efficiently produced with highest titre reached of 9

Cytotoxic human peripheral blood-derived γδT cells kill glioblastoma cell lines: implications for cell-based immunotherapy for patients with glioblastoma.

PubMed

Nakazawa, Tsutomu; Nakamura, Mitsutoshi; Park, Young Soo; Motoyama, Yasushi; Hironaka, Yasuo; Nishimura, Fumihiko; Nakagawa, Ichiro; Yamada, Shuichi; Matsuda, Ryosuke; Tamura, Kentaro; Sugimoto, Tadashi; Takeshima, Yasuhiro; Marutani, Akiko; Tsujimura, Takahiro; Ouji, Noriko; Ouji, Yukiteru; Yoshikawa, Masahide; Nakase, Hiroyuki

2014-01-01

Glioblastoma (GBM) is a highly aggressive brain tumor for which novel therapeutic approaches, such as immunotherapy, are urgently needed. Zoledronate (ZOL), an inhibitor of osteoclastic activity, is known to stimulate peripheral blood-derived γδT cells and sensitize tumors to γδT cell-mediated killing. To investigate the feasibility of γδT cell-based immunotherapy for patients with GBM, we focused on the killing of GBM cell lines by γδT cells and the molecular mechanisms involved in these cell-cell interactions. Peripheral blood mononuclear cells were expanded in ZOL and interleukin (IL)-2 for 14 days, and γδT cells were enriched in the expanded cells by the immunomagnetic depletion of αβT cells. Gliomas are resistant to NK cells but susceptible to lymphokine-activated killer cells and some cytotoxic T lymphocytes. When the γδT cell-mediated killing of three GBM cell lines (U87MG, U138MG and A172 cells) and an NK-sensitive leukemia cell line (K562 cells) were tested, 32% U87MG, 15% U138MG, 1% A172, and 50% K562 cells were killed at an effector:target ratio of 5:1. The γδT cell-mediated killing of all three GBM cell lines was significantly enhanced by ZOL and this ZOL-enhanced killing was blocked by an anti-T cell receptor (TcR) antibody. These results indicated that TcR γδ is crucial for the recognition of ZOL-treated GBM cells by γδT cells. Since the low level killing of GBM cells by the γδT cells was enhanced by ZOL, γδT cell-targeting therapy in combination with ZOL treatment could be effective for patients with GBM.

Induction of antigen-specific cytotoxic T-cell response by dendritic cells generated from ecto-mesenchymal stem cells infected with an adenovirus containing the MAGE-D4a gene.

PubMed

Hu, Shijie; Li, Bing; Shen, Xuefeng; Zhang, Rui; Gao, Dakuan; Guo, Qingdong; Jin, Yan; Fei, Zhou

2016-04-01

The present study aimed to investigate the feasibility of using ecto-mesenchymal stem cell (EMSC)-derived dendritic cells (DCs) for glioma immunotherapy following infection by a recombinant adenovirus containing the melanoma-associated antigen D4a (MAGE-D4a) gene. The ex vivo cultured EMSCs were infected by the adenoviral plasmid containing MAGE-D4a (pAd/MAGE-D4a). Efficiency of transfection was evaluated through the detection of green fluorescent protein-marked MAGE-D4a. The MAGE-EMSCs were induced to differentiate into DCs, termed as MAGE-EMSCs-DCs. The morphology was subsequently analyzed under a microscope, and methyl thiazolyl tetrazolium (MTT) and interferon-γ (IFN-γ) assays were performed to analyze the cytotoxicity of the MAGE-EMSC-DCs on the human glioma U251 cell line. Following purification by magnetic-activated cell sorting, the EMSCs grew into swirls, with a long spindle shape and were fibroblast-like. The gene transfected with recombinant adenovirus vectors maintained high and stable expression levels of MAGE-D4a, and its efficiency was increased in a multiplicity of infection-dependent manner. The results of the MTT assay indicated that the T cells, primed by the recombinant MAGE-D4a-infected EMSC-DCs in vitro , recognized MAGE-D4a-expressing tumor cell lines in a human leukocyte antigen class I-restricted manner, and evoked a higher cytotoxic T cell (CTL) response. The CTL response induced by the MAGE-EMSC-DCs, co-cultured with the U251 cells for 24 h, produced 765.0 pg/ml IFN-γ, which was significantly greater when compared to the control wells. T lymphocytes stimulated by MAGE-EMSC-DCs evoke a higher CTL response to human glioma cell lines, and may serve as a promising therapeutic modality for the treatment of MAGE-D4a-expressing glioma.

Macrophage cell lines derived from major histocompatibility complex II-negative mice

NASA Technical Reports Server (NTRS)

Beharka, A. A.; Armstrong, J. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1998-01-01

Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-gamma, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphlococcal enterotoxins A or B C2D(t) cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.

A novel 2,6-diisopropylphenyl-docosahexaenoamide conjugate induces apoptosis in T cell acute lymphoblastic leukemia cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Altenburg, Jeffrey D.; Harvey, Kevin A.; McCray, Sharon

2011-07-29

Highlights: {yields} 2,6-Diisopropylphenyl-docosahexaenoamide conjugates (DIP-DHA) inhibits the proliferation of T-cell leukemic cell lines. {yields} DIP-DHA resulted in increased activation of caspase-3, and caspase-7. {yields} DIP-DHA significantly downregulated CXCR4 surface expression. -- Abstract: We have previously characterized the effects of 2,6-diisopropylphenyl-docosahexaenoamide (DIP-DHA) conjugates and their analogs on the proliferation and progression of breast cancer cell lines. For this study, we investigated the effects of the DIP-DHA conjugate on 2 representative T cell acute lymphoblastic leukemia (T-ALL) cell lines: CEM and Jurkat. Treatment of both cell lines with DIP-DHA resulted in significantly greater inhibition of proliferation and induction of apoptosis than thatmore » of parent compounds, 2,6-diisopropylphenol (DIP) or docosahexaenoate (DHA). Treatment of the cells with DIP-DHA resulted in increased activation of caspase-3, and caspase-7. Furthermore, induction of apoptosis in both cell lines was reversed in the presence of a caspase family inhibitor. Treatment with DIP-DHA reduced mitochondrial membrane potential. These observations suggest that the effects are driven by intrinsic apoptotic pathways. DIP-DHA treatment also downregulated surface CXCR4 expression, an important chemokine receptor involved in cancer metastasis that is highly expressed in both CEM and Jurkat cells. In conclusion, our data suggest that the DIP-DHA conjugate exhibits significantly more potent effects on CEM and Jurkat cells than that of DIP or DHA alone. These conjugates have potential use for treatment of patients with T cell acute lymphoblastic leukemia.« less

Breast cancer cell-associated endopeptidase EC 24.11 modulates proliferative response to bombesin.

PubMed

Burns, D M; Walker, B; Gray, J; Nelson, J

1999-01-01

We have investigated the production, growth and inactivation of gastrin-releasing peptide (GRP)-like peptides in human breast cancer cell lines. Radioimmunoassay detected GRP-like immunoreactivity (GRP-LI) in T47D breast cancer cells but not in the conditioned medium, indicating rapid clearance. No GRP-LI was found in the ZR-75-1 or MDA-MB-436 cells or their conditioned medium. High-performance liquid chromatography (HPLC) analysis of the GRP-LI in the T47D cells revealed a major peak, which co-eluted with GRP(18-27), and a minor more hydrophilic peak. In vitro stimulation of T47D cell growth by bombesin (BN) was enhanced to 138% of control levels (bombesin alone) by the addition of the selective endopeptidase EC 3.4.24.11 inhibitor phosphoramidon (0.1 ng ml(-1)). Fluorogenic analysis using whole cells confirmed low levels of this phosphoramidon-sensitive enzyme on the T47D cells. This enzyme, previously unreported in human breast cancer cells, significantly modulates both T47D growth and its response to BN-induced growth.

Pirfenidone inhibits proliferation, arrests the cell cycle, and downregulates heat shock protein-47 and collagen type I in rat hepatic stellate cells in vitro.

PubMed

Xiang, Xian-Hong; Jiang, Tian-Peng; Zhang, Shuai; Song, Jie; Li, Xing; Yang, Jian-Yong; Zhou, Shi

2015-07-01

Pirfenidone (esbiret) is an established anti-fibrotic and anti-inflammatory drug used to treat idiopathic pulmonary fibrosis. In the present study, the dose-dependent effects of pirfenidone on the cell cycle, proliferation and expression of heat shock protein (HSP)-47 and collagen type I in a cultured rat hepatic stellate cell line (HSC-T6) were investigated. Following pirfenidone treatment, cell proliferation was determined using the cell counting kit-8 assay and the cell cycle was measured using flow cytometry. HSP-47 expression was estimated using western blot analysis and collagen type I mRNA was assessed using reverse transcription quantitative polymerase chain reaction. Pirfenidone induced significant dose-dependent inhibition of proliferation in HSC-T6 cells. Cell viability was unaffected by treatment with pirfenidone (0, 10 or 100 µM) for 24 and 72 h. However, after 24 h, HSC-T6 cells exhibited dose-dependent decreases in HSP-47 protein and collagen I mRNA levels. In conclusion, pirfenidone inhibited HSC-T6 cell proliferation, arrested the cell cycle and reduced the expression of HSP-47 and collagen type I, indicating that pirfenidone may be a promising drug in the treatment of liver fibrosis.

CRISPR/Cas9-generated p47phox-deficient cell line for Chronic Granulomatous Disease gene therapy vector development.

PubMed

Wrona, Dominik; Siler, Ulrich; Reichenbach, Janine

2017-03-13

Development of gene therapy vectors requires cellular models reflecting the genetic background of a disease thus allowing for robust preclinical vector testing. For human p47 phox -deficient chronic granulomatous disease (CGD) vector testing we generated a cellular model using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a GT-dinucleotide deletion (ΔGT) mutation in p47 phox encoding NCF1 gene in the human acute myeloid leukemia PLB-985 cell line. CGD is a group of hereditary immunodeficiencies characterized by impaired respiratory burst activity in phagocytes due to a defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In Western countries autosomal-recessive p47 phox -subunit deficiency represents the second largest CGD patient cohort with unique genetics, as the vast majority of p47 phox CGD patients carries ΔGT deletion in exon two of the NCF1 gene. The established PLB-985 NCF1 ΔGT cell line reflects the most frequent form of p47 phox -deficient CGD genetically and functionally. It can be differentiated to granulocytes efficiently, what creates an attractive alternative to currently used iPSC models for rapid testing of novel gene therapy approaches.

Development of Third-generation Cocal Envelope Producer Cell Lines for Robust Lentiviral Gene Transfer into Hematopoietic Stem Cells and T-cells.

PubMed

Humbert, Olivier; Gisch, Don W; Wohlfahrt, Martin E; Adams, Amie B; Greenberg, Phil D; Schmitt, Tom M; Trobridge, Grant D; Kiem, Hans-Peter

2016-08-01

Lentiviral vectors (LVs) pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G) have demonstrated great promise in gene therapy trials employing hematopoietic stem cell and T-cells. The VSV-G envelope confers broad tropism and stability to the vector but is toxic when constitutively expressed, which has impeded efforts to generate stable producer cell lines. We previously showed that cocal pseudotyped LVs offer an excellent alternative to VSV-G vectors because of their broad tropism and resistance to human serum inactivation. In this study, we demonstrate that cocal LVs transduce CD34(+) and CD4(+) T-cells more efficiently than VSV-G LVs and share the same receptor(s) for cell entry. 293T-cells stably expressing the cocal envelope produced significantly higher LV titers than VSV-G expressing cells. We developed cocal pseudotyped, third-generation, self-inactivating LV producer cell lines for a GFP reporter and for a WT1 tumor-specific T-cell receptor, which achieved concentrated titers above 10(8) IU/ml and were successfully adapted for growth in suspension, serum-free culture. The resulting LVs were at least as effective as standard LVs in transducing CD34(+) and CD4(+) T-cells. Our stable cocal LV producer cell lines should facilitate the production of large-scale, high titer clinical grade vectors.

Breast cancer cell-associated endopeptidase EC 24.11 modulates proliferative response to bombesin

PubMed Central

Burns, D M; Walker, B; Gray, J; Nelson, J

1999-01-01

We have investigated the production, growth and inactivation of gastrin-releasing peptide (GRP)-like peptides in human breast cancer cell lines. Radioimmunoassay detected GRP-like immunoreactivity (GRP-LI) in T47D breast cancer cells but not in the conditioned medium, indicating rapid clearance. No GRP-LI was found in the ZR-75-1 or MDA-MB-436 cells or their conditioned medium. High-performance liquid chromatography (HPLC) analysis of the GRP-LI in the T47D cells revealed a major peak, which co-eluted with GRP18–27, and a minor more hydrophilic peak. In vitro stimulation of T47D cell growth by bombesin (BN) was enhanced to 138% of control levels (bombesin alone) by the addition of the selective endopeptidase EC 3.4.24.11 inhibitor phosphoramidon (0.1 ng ml−;1). Fluorogenic analysis using whole cells confirmed low levels of this phosphoramidon-sensitive enzyme on the T47D cells. This enzyme, previously unreported in human breast cancer cells, significantly modulates both T47D growth and its response to BN-induced growth. © 1999 Cancer Research Campaign PMID:9888460

Development and characterization of Histoplasma capsulatum-reactive murine T-cell lines and clones

NASA Technical Reports Server (NTRS)

Deepe, George S., Jr.; Smith, James G.; Denman, David; Bullock, Ward E.; Sonnenfeld, Gerald

1986-01-01

Several Histoplasma capsulatum-reactive murine cloned T-cell lines (TCLs) were isolated from spleens of C57BL/6 mice immunized with viable H. capsulatum yeast cells, using the methodology of Kimoto and Fathman (1980). These T-cells were characterized phenotypically as Thy-1.2(+) Lyt-1(+) L3T4(+) Lyt-2(-), that is, as the helper/inducer phenotype. The cloned T cells proliferate in response to histoplasmin and, in some cases, to heterologous fungal anigens. Upon injection of mice with the antigen, the T-cells mediate local delayed-type hypersensitivity responses and, after stimulation, release regulatory lymphokines.

D-galactose induces necroptotic cell death in neuroblastoma cell lines.

PubMed

Li, Na; He, Yangyan; Wang, Ling; Mo, Chunfen; Zhang, Jie; Zhang, Wei; Li, Junhong; Liao, Zhiyong; Tang, Xiaoqiang; Xiao, Hengyi

2011-12-01

D-Galactose (D-gal) can induce oxidative stress in non-cancer cells and result in cell damage by disturbing glucose metabolism. However, the effect of D-gal on cancer cells is yet to be explored. In this study, we investigated the toxicity of D-gal to malignant cells specifically neuroblastoma cells. As the results, high concentrations of D-gal had significant toxicity to cancer cells, whereas the same concentrations of glucose had no; the viability loss via D-gal treatment was prominent to malignant cells (Neuro2a, SH-SY5Y, PC-3, and HepG2) comparing to non-malignant cells (NIH3T3 and LO(2)). Differing from the apoptosis induced by H(2) O(2), D-gal damaged cells showed the characters of necrotic cell death, such as trypan blue-tangible and early phase LDH leakage. Further experiments displayed that the toxic effect of D-gal can be alleviated by necroptosis inhibitor Necrostatin (Nec-1) and autophagy inhibitor 3-methyladenine (3-MA) but not by caspase inhibitor z-VAD-fmk. D-Gal treatment can transcriptionally up-regulate the genes relevant to necroptosis (Bmf, Bnip3) and autophagy (Atg5, TIGAR) but not the genes related to apoptosis (Caspase3, Bax, and p53). D-Gal did not activate Caspase-3, but prompted puncta-like GFP-LC3 distribution, an indicator for activated autophagy. The involvement of aldose reductase (AR)-mediated polyol pathway was proved because the inhibitor of AR can attenuate the toxicity of D-gal and D-gal treatment elevates the expression of AR. This study demonstrates for the first time that D-gal can induce non-apoptotic but necroptotic cell death in neuroblastoma cells and provides a new clue for developing the strategy against apoptosis-resistant cancers. Copyright © 2011 Wiley Periodicals, Inc.

Estradiol and corticosterone stimulate the proliferation of a GH cell line, MtT/S: Proliferation of growth hormone cells.

PubMed

Nogami, Haruo; Hiraoka, Yoshiki; Aiso, Sadakazu

2016-08-01

Estrogens are known as a potent growth-stimulator of the anterior pituitary cells such as prolactin cells and somatomammotroph cell lines, while glucocorticoids often inhibit cellular proliferation in the pituitary gland as well as in the extra-pituitary tissues. In this study, the involvement of these steroid hormones in the regulation of proliferation was examined in the MtT/S cells, secreting growth hormone (GH). Effects of estrogens and glucocorticoids were examined in MtT/S cells grown in the medium containing dextran-coated charcoal treated serum. The relative cell density after culture was estimated by the Cell Titer-Glo Luminescent Cell Viability Assay System, and the proliferation rate was determined by the BrdU incorporation method. The mRNA levels were determined by real-time PCR. Estradiol and the specific agonist for both estrogen receptor (ER) α and ERβ stimulated MtT/S growth at a dose dependent manner. The membrane impermeable estrogen, 17β-estradiol-bovine serum albumin conjugate also stimulated the MtT/S proliferation. The effects of all estrogens were inhibited by an estrogen receptor antagonist, ICI182780. Corticosterone stimulated the proliferation of MtT/S cells at doses lower than 10nM without stimulating GH gene transcription, whereas it did not change the proliferation rate at 1μM. The effects of corticosterone were inhibited by glucocorticoid receptor inhibitor, RU486, but not by the mineralocorticoid receptor antagonist, spironolactone. Both estrogens and glucocorticoids were found to stimulate the proliferation of MtT/S, increasing the mRNA expression of cyclins D1, D3, and E. The results suggest that estrogens and glucocorticoids may be involved in the mechanisms responsible for the proliferation of GH cells in the course of pituitary development, to maintain the population of GH cells in the adult pituitary gland, and also in the promotion of GH cell tumors. Copyright © 2016 Elsevier Ltd. All rights reserved.

LipiD-QuanT: a novel method to quantify lipid accumulation in live cells.

PubMed

Varinli, Hilal; Osmond-McLeod, Megan J; Molloy, Peter L; Vallotton, Pascal

2015-11-01

Lipid droplets (LDs) are the main storage organelles for triglycerides. Elucidation of lipid accumulation mechanisms and metabolism are essential to understand obesity and associated diseases. Adipogenesis has been well studied in murine 3T3-L1 and human Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cell lines. However, most techniques for measuring LD accumulation are either not quantitative or can be destructive to samples. Here, we describe a novel, label-free LD quantification technique (LipiD-QuanT) to monitor lipid dynamics based on automated image analysis of phase contrast microscopy images acquired during in vitro human adipogenesis. We have applied LipiD-QuanT to measure LD accumulation during differentiation of SGBS cells. We demonstrate that LipiD-QuanT is a robust, nondestructive, time- and cost-effective method compared with other triglyceride accumulation assays based on enzymatic digest or lipophilic staining. Further, we applied LipiD-QuanT to measure the effect of four potential pro- or antiobesogenic substances: DHA, rosiglitazone, elevated levels of D-glucose, and zinc oxide nanoparticles. Our results revealed that 2 µmol/l rosiglitazone treatment during adipogenesis reduced lipid production and caused a negative shift in LD diameter size distribution, but the other treatments showed no effect under the conditions used here. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

Effects of Long-Term 50Hz Power-Line Frequency Electromagnetic Field on Cell Behavior in Balb/c 3T3 Cells

PubMed Central

An, Guang-Zhou; Xu, Hui; Zhou, Yan; Du, Le; Miao, Xia; Jiang, Da-Peng; Li, Kang-Chu; Guo, Guo-Zhen; Zhang, Chen; Ding, Gui-Rong

2015-01-01

Power-line frequency electromagnetic field (PF-EMF) was reported as a human carcinogen by some epidemiological research, but the conclusion is lack of robust experiment evidence. To identify the effects of long-term PF-EMF exposure on cell behavior, Balb/c 3T3 cells in exponential growth phase were exposed or sham-exposed to 50 Hertz (Hz) PF-EMF at 2.3 mT for 2 hours (h) one day, 5 days every week. After 11 weeks exposure, cells were collected instantly. Cell morphology was observed under invert microscope and Giemsa staining, cell viability was detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, cell cycle and apoptosis was examined by flow cytometry, the protein level of Proliferating Cell Nuclear Antigen (PCNA) and CyclinD1 was detected by western blot, cell transformation was examined by soft agar clone assay and plate clone forming test, and cell migration ability was observed by scratch adhesion test. It was found that after PF-EMF exposure, cell morphology, apoptosis, cell migration ability and cell transformation didn’t change. However, compared with sham group, cell viability obviously decreased and cell cycle distribution also changed after 11 weeks PF-EMF exposure. Meanwhile, the protein level of PCNA and CyclinD1 significantly decreased after PF-EMF exposure. These data suggested that although long-term 50Hz PF-EMF exposure under this experimental condition had no effects on apoptosis, cell migration ability and cell transformation, it could affect cell proliferation and cell cycle by down-regulation the expression of PCNA and CyclinD1 protein. PMID:25695503

Effects of long-term 50Hz power-line frequency electromagnetic field on cell behavior in Balb/c 3T3 cells.

PubMed

An, Guang-Zhou; Xu, Hui; Zhou, Yan; Du, Le; Miao, Xia; Jiang, Da-Peng; Li, Kang-Chu; Guo, Guo-Zhen; Zhang, Chen; Ding, Gui-Rong

2015-01-01

Power-line frequency electromagnetic field (PF-EMF) was reported as a human carcinogen by some epidemiological research, but the conclusion is lack of robust experiment evidence. To identify the effects of long-term PF-EMF exposure on cell behavior, Balb/c 3T3 cells in exponential growth phase were exposed or sham-exposed to 50 Hertz (Hz) PF-EMF at 2.3 mT for 2 hours (h) one day, 5 days every week. After 11 weeks exposure, cells were collected instantly. Cell morphology was observed under invert microscope and Giemsa staining, cell viability was detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, cell cycle and apoptosis was examined by flow cytometry, the protein level of Proliferating Cell Nuclear Antigen (PCNA) and CyclinD1 was detected by western blot, cell transformation was examined by soft agar clone assay and plate clone forming test, and cell migration ability was observed by scratch adhesion test. It was found that after PF-EMF exposure, cell morphology, apoptosis, cell migration ability and cell transformation didn't change. However, compared with sham group, cell viability obviously decreased and cell cycle distribution also changed after 11 weeks PF-EMF exposure. Meanwhile, the protein level of PCNA and CyclinD1 significantly decreased after PF-EMF exposure. These data suggested that although long-term 50Hz PF-EMF exposure under this experimental condition had no effects on apoptosis, cell migration ability and cell transformation, it could affect cell proliferation and cell cycle by down-regulation the expression of PCNA and CyclinD1 protein.

LipiD-QuanT: a novel method to quantify lipid accumulation in live cells[S

PubMed Central

Varinli, Hilal; Osmond-McLeod, Megan J.; Molloy, Peter L.; Vallotton, Pascal

2015-01-01

Lipid droplets (LDs) are the main storage organelles for triglycerides. Elucidation of lipid accumulation mechanisms and metabolism are essential to understand obesity and associated diseases. Adipogenesis has been well studied in murine 3T3-L1 and human Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cell lines. However, most techniques for measuring LD accumulation are either not quantitative or can be destructive to samples. Here, we describe a novel, label-free LD quantification technique (LipiD-QuanT) to monitor lipid dynamics based on automated image analysis of phase contrast microscopy images acquired during in vitro human adipogenesis. We have applied LipiD-QuanT to measure LD accumulation during differentiation of SGBS cells. We demonstrate that LipiD-QuanT is a robust, nondestructive, time- and cost-effective method compared with other triglyceride accumulation assays based on enzymatic digest or lipophilic staining. Further, we applied LipiD-QuanT to measure the effect of four potential pro- or antiobesogenic substances: DHA, rosiglitazone, elevated levels of D-glucose, and zinc oxide nanoparticles. Our results revealed that 2 µmol/l rosiglitazone treatment during adipogenesis reduced lipid production and caused a negative shift in LD diameter size distribution, but the other treatments showed no effect under the conditions used here. PMID:26330056

[Autologous regulatory T cells can suppress the proliferation of lymphoma cell line in vitro].

PubMed

Ying, Zhi-Tao; Guo, Jun; Ren, Jun; Kong, Yan; Yuan, Zhi-Hong; Liu, Xi-Juan; Zhang, Chen; Zheng, Wen; Song, Yu-Qin; Zhang, Yun-Tao; Zhu, Jun

2009-06-01

This study was aimed to investigate the suppressive effect of regulatory T (Treg) cells on the T cell lymphoma EL4 cell line and to explore its mechanism. C57BL/6 Mouse Treg cells were isolated by MACS (magnetic cell sorting). The purity and the expression of Foxp3 were detected by flow cytometry. The suppressive effect of sorted Treg cells on EL4 cells was detected by MTT assay. The secretion of TGF-beta1 and IL-10 was examined by enzyme-linked immunosorbent assay (ELISA). The results showed that CD4(+)CD25(+) T cells could be successfully isolated by MACS with the purity reaching 91.6% and the expression level of Foxp3 was 78.9%. The ratio of viable cells was more than 95%. Regulatory T cells could suppress the proliferation of EL4 cells effectively in the presence of antigen presenting cells (APCs). And the suppressive effect was most significant at 1:1 ratio. In addition, the suppression still existed without APCs. TGF-beta1 and IL-10 could not be detected by ELISA. It is concluded that the Treg cells can suppress T lymphoma cell in vitro. The suppressive effect of Treg cells works in dose-dependent manner, but not in cytokine-dependent manner. The mechanism of this suppression may take effect through cell-cell contact.

Calbindin-D(28k) controls [Ca(2+)](i) and insulin release. Evidence obtained from calbindin-d(28k) knockout mice and beta cell lines

NASA Technical Reports Server (NTRS)

Sooy, K.; Schermerhorn, T.; Noda, M.; Surana, M.; Rhoten, W. B.; Meyer, M.; Fleischer, N.; Sharp, G. W.; Christakos, S.

1999-01-01

The role of the calcium-binding protein, calbindin-D(28k) in potassium/depolarization-stimulated increases in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release was investigated in pancreatic islets from calbindin-D(28k) nullmutant mice (knockouts; KO) or wild type mice and beta cell lines stably transfected and overexpressing calbindin. Using single islets from KO mice and stimulation with 45 mM KCl, the peak of [Ca(2+)](i) was 3.5-fold greater in islets from KO mice compared with wild type islets (p < 0.01) and [Ca(2+)](i) remained higher during the plateau phase. In addition to the increase in [Ca(2+)](i) in response to KCl there was also a significant increase in insulin release in islets isolated from KO mice. Evidence for modulation by calbindin of [Ca(2+)](i) and insulin release was also noted using beta cell lines. Rat calbindin was stably expressed in betaTC-3 and betaHC-13 cells. In response to depolarizing concentrations of K(+), insulin release was decreased by 45-47% in calbindin expressing betaTC cells and was decreased by 70-80% in calbindin expressing betaHC cells compared with insulin release from vector transfected betaTC or betaHC cells (p < 0.01). In addition, the K(+)-stimulated intracellular calcium peak was markedly inhibited in calbindin expressing betaHC cells compared with vector transfected cells (225 nM versus 1,100 nM, respectively). Buffering of the depolarization-induced rise in [Ca(2+)](i) was also observed in calbindin expressing betaTC cells. In summary, our findings, using both isolated islets from calbindin-D(28k) KO mice and beta cell lines, establish a role for calbindin in the modulation of depolarization-stimulated insulin release and suggest that calbindin can control the rate of insulin release via regulation of [Ca(2+)](i).

A boosted negative bit-line SRAM with write-assisted cell in 45 nm CMOS technology

NASA Astrophysics Data System (ADS)

Bhatnagar, Vipul; Kumar, Pradeep; Pandey, Neeta; Pandey, Sujata

2018-02-01

A new 11 T SRAM cell with write-assist is proposed to improve operation at low supply voltage. In this technique, a negative bit-line voltage is applied to one of the write bit-lines, while a boosted voltage is applied to the other write bit-line where transmission gate access is used in proposed 11 T cell. Supply voltage to one of the inverters is interrupted to weaken the feedback. Improved write feature is attributed to strengthened write access devices and weakened feedback loop of cell at the same time. Amount of boosting required for write performance improvement is also reduced due to feedback weakening, solving the persistent problem of half-selected cells and reliability reduction of access devices with the other suggested boosted and negative bit-line techniques. The proposed design improves write time by 79%, 63% and slower by 52% with respect to LP 10 T, WRE 8 T and 6 T cells respectively. It is found that write margin for the proposed cell is improved by about 4×, 2.4× and 5.37× compared to WRE8 T, LP10 T and 6 T respectively. The proposed cell with boosted negative bit line (BNBL) provides 47%, 31%, and 68.4% improvement in write margin with respect to no write-assist, negative bit line (NBL) and boosted bit line (BBL) write-assist respectively. Also, new sensing circuit with replica bit-line is proposed to give a more precise timing of applying boosted voltages for improved results. All simulations are done on TSMC 45 nm CMOS technology.

Establishment of cell lines from adult T-cell leukemia cells dependent on negatively charged polymers.

PubMed

Kagami, Yoshitoyo; Uchiyama, Susumu; Kato, Harumi; Okada, Yasutaka; Seto, Masao; Kinoshita, Tomohiro

2017-07-05

Growing adult T-cell leukemia/lymphoma (ATLL) cells in vitro is difficult. Here, we examined the effects of static electricity in the culture medium on the proliferation of ATLL cells. Six out of 10 ATLL cells did not proliferate in vitro and thus had to be cultured in a medium containing negatively charged polymers. In the presence of poly-γ-glutamic acid (PGA) or chondroitin sulfate (CDR), cell lines (HKOX3-PGA, HKOX3-CDR) were established from the same single ATLL case using interleukin (IL)-2, IL-4, and feeder cells expressing OX40L (OX40L + HK). Dextran sulfate inhibited growth in both HKOX3 cell lines. Both PGA and OX40L + HK were indispensable for HKOX3-PGA growth, but HKOX3-CDR could proliferate in the presence of CDR or OX40L + HK alone. Thus, the specific action of each negatively charged polymer promoted the growth of specific ATLL cells in vitro.

The PIKfyve–ArPIKfyve–Sac3 triad in human breast cancer: Functional link between elevated Sac3 phosphatase and enhanced proliferation of triple negative cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ikonomov, Ognian C., E-mail: oikonomo@med.wayne.edu; Filios, Catherine, E-mail: cfilios@med.wayne.edu; Sbrissa, Diego, E-mail: dsbrissa@med.wayne.edu

2013-10-18

Highlights: •We assess PAS complex proteins and phosphoinositide levels in breast cancer cells. •Sac3 and ArPIKfyve are markedly elevated in triple-negative breast cancer cells. •Sac3 silencing inhibits proliferation in triple-negative breast cancer cell lines. •Phosphoinositide profiles are altered in breast cancer cells. •This is the first evidence linking high Sac3 with breast cancer cell proliferation. -- Abstract: The phosphoinositide 5-kinase PIKfyve and 5-phosphatase Sac3 are scaffolded by ArPIKfyve in the PIKfyve–ArPIKfyve–Sac3 (PAS) regulatory complex to trigger a unique loop of PtdIns3P–PtdIns(3,5)P{sub 2} synthesis and turnover. Whereas the metabolizing enzymes of the other 3-phosphoinositides have already been implicated in breast cancer,more » the role of the PAS proteins and the PtdIns3P–PtdIns(3,5)P{sub 2} conversion is unknown. To begin elucidating their roles, in this study we monitored the endogenous levels of the PAS complex proteins in cell lines derived from hormone-receptor positive (MCF7 and T47D) or triple-negative breast cancers (TNBC) (BT20, BT549 and MDA-MB-231) as well as in MCF10A cells derived from non-tumorigenic mastectomy. We report profound upregulation of Sac3 and ArPIKfyve in the triple negative vs. hormone-sensitive breast cancer or non-tumorigenic cells, with BT cell lines showing the highest levels. siRNA-mediated knockdown of Sac3, but not that of PIKfyve, significantly inhibited proliferation of BT20 and BT549 cells. In these cells, knockdown of ArPIKfyve had only a minor effect, consistent with a primary role for Sac3 in TNBC cell proliferation. Intriguingly, steady-state levels of PtdIns(3,5)P{sub 2} in BT20 and T47D cells were similar despite the 6-fold difference in Sac3 levels between these cell lines. However, steady-state levels of PtdIns3P and PtdIns5P, both regulated by the PAS complex, were significantly reduced in BT20 vs. T47D or MCF10A cell lines, consistent with elevated Sac3 affecting

Human T lymphocytes express N-methyl-D-aspartate receptors functionally active in controlling T cell activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miglio, Gianluca; Varsaldi, Federica; Lombardi, Grazia

2005-12-30

The aim of this study was to investigate the expression and the functional role of N-methyl-D-aspartate (NMDA) receptors in human T cells. RT-PCR analysis showed that human resting peripheral blood lymphocytes (PBL) and Jurkat T cells express genes encoding for both NR1 and NR2B subunits: phytohemagglutinin (PHA)-activated PBL also expresses both these genes and the NR2A and NR2D genes. Cytofluorimetric analysis showed that NR1 expression increases as a consequence of PHA (10 {mu}g/ml) treatment. D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [(+)-MK 801], competitive and non-competitive NMDA receptor antagonists, respectively, inhibited PHA-induced T cell proliferation, whereas they did not affect IL-2 (10more » U/ml)-induced proliferation of PHA blasts. These effects were due to the prevention of T cell activation (inhibition of cell aggregate formation and CD25 expression), but not to cell cycle arrest or death. These results demonstrate that human T lymphocytes express NMDA receptors, which are functionally active in controlling cell activation.« less

Gomisin G Inhibits the Growth of Triple-Negative Breast Cancer Cells by Suppressing AKT Phosphorylation and Decreasing Cyclin D1.

PubMed

Maharjan, Sony; Park, Byoung Kwon; Lee, Su In; Lim, Yoonho; Lee, Keunwook; Kwon, Hyung-Joo

2018-05-01

A type of breast cancer with a defect in three molecular markers such as the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor is called triple-negative breast cancer (TNBC). Many patients with TNBC have a lower survival rate than patients with other types due to a poor prognosis. In this study, we confirmed the anti-cancer effect of a natural compound, Gomisin G, in TNBC cancer cells. Treatment with Gomisin G suppressed the viability of two TNBC cell lines, MDA-MB-231 and MDA-MB-468 but not non-TNBC cell lines such as MCF-7, T47D, and ZR75-1. To investigate the molecular mechanism of this activity, we examined the signal transduction pathways after treatment with Gomisin G in MDA-MB-231 cells. Gomisin G did not induce apoptosis but drastically inhibited AKT phosphorylation and reduced the amount of retinoblastoma tumor suppressor protein (Rb) and phosphorylated Rb. Gomisin G induced in a proteasome-dependent manner a decrease in Cyclin D1. Consequently, Gomisin G causes cell cycle arrest in the G1 phase. In contrast, there was no significant change in T47D cells except for a mild decrease in AKT phosphorylation. These results show that Gomisin G has an anti-cancer activity by suppressing proliferation rather than inducing apoptosis in TNBC cells. Our study suggests that Gomisin G could be used as a therapeutic agent in the treatment of TNBC patients.

Agarose hydrogel induced MCF-7 and BMG-1 cell line progressive 3D and 3D revert cultures.

PubMed

Subramaniyan, Aishwarya; Ravi, Maddaly

2018-04-01

3D culture systems have enhanced the utility of cancer cell lines as they are considered closer to the in vivo systems. A variety of changes are induced in cells cultured in 3D systems; an apparent and striking feature being the spontaneous acquisition of distinct morphological entities. 3D reverts (3DRs) can be obtained by introducing 3D aggregates in scaffold/matrix-free culture units. It could be seen that the two cell lines used in this study exhibited differences in 3DR structures, though both were cultured on agarose hydrogels. Also, differences in 3DR formation, growth and survival were different. While 3D aggregates of several cell lines have been reported for a variety of studies, there are no studies that describe or utilize 3DRs. 3DRs can provide insights into complex events that can occur in cancer cells; especially as material to study metastasis, migration, and invasion. © 2017 Wiley Periodicals, Inc.

DNA demethylation and histone H3K9 acetylation determine the active transcription of the NKG2D gene in human CD8+ T and NK cells

PubMed Central

Fernández-Sánchez, Alba; Baragaño Raneros, Aroa; Carvajal Palao, Reyes; Sanz, Ana B.; Ortiz, Alberto; Ortega, Francisco; Suárez-Álvarez, Beatriz; López-Larrea, Carlos

2013-01-01

The human activating receptor NKG2D is mainly expressed by NK, NKT, γδ T and CD8+ T cells and, under certain conditions, by CD4+ T cells. This receptor recognizes a diverse family of ligands (MICA, MICB and ULBPs 1–6) leading to the activation of effector cells and triggering the lysis of target cells. The NKG2D receptor-ligand system plays an important role in the immune response to infections, tumors, transplanted graft and autoantigens. Elucidation of the regulatory mechanisms of NKG2D is therefore essential for therapeutic purposes. In this study, we speculate whether epigenetic mechanisms, such as DNA methylation and histone acetylation, participate in NKG2D gene regulation in T lymphocytes and NK cells. DNA methylation in the NKG2D gene was observed in CD4+ T lymphocytes and T cell lines (Jurkat and HUT78), while this gene was unmethylated in NKG2D-positive cells (CD8+ T lymphocytes, NK cells and NKL cell line) and associated with high levels of histone H3 lysine 9 acetylation (H3K9Ac). Treatment with the histone acetyltransferase (HAT) inhibitor curcumin reduces H3K9Ac levels in the NKG2D gene, downregulates NKG2D transcription and leads to a marked reduction in the lytic capacity of NKG2D-mediated NKL cells. These findings suggest that differential NKG2D expression in the different cell subsets is regulated by epigenetic mechanisms and that its modulation by epigenetic treatments might provide a new strategy for treating several pathologies. PMID:23235109

Development of a Stable Cell Line, Overexpressing Human T-cell Immunoglobulin Mucin 1

PubMed Central

Ebrahimi, Mina; Kazemi, Tohid; Ganjalikhani-hakemi, Mazdak; Majidi, Jafar; khanahmad, Hossein; Rahimmanesh, Ilnaz; Homayouni, Vida; Kohpayeh, Shirin

2015-01-01

Background Recent researches have demonstrated that human T-cell immunoglobulin mucin 1 (TIM-1) glycoprotein plays important roles in regulation of autoimmune and allergic diseases, as well as in tumor immunity and response to viral infections. Therefore, targeting TIM-1 could be a potential therapeutic approach against such diseases. Objectives In this study, we aimed to express TIM-1 protein on Human Embryonic kidney (HEK) 293T cell line in order to have an available source of the TIM-1 antigen. Materials and Methods The cDNA was synthesized after RNA extraction from peripheral blood mononuclear cells (PBMC) and TIM-1 cDNA was amplified by PCR with specific primers. The PCR product was cloned in pcDNA™3.1/Hygro (+) and transformed in Escherichia coli TOP 10 F’. After cloning, authenticity of DNA sequence was checked and expressed in HEK 293T cells. Finally, expression of TIM-1 was analyzed by flow cytometry and real-time PCR. Results The result of DNA sequencing demonstrated correctness of TIM-1 DNA sequence. The flow cytometry results indicated that TIM-1 was expressed in about 90% of transfected HEK 293T cells. The real-time PCR analysis showed TIM-1 mRNA expression increased 195-fold in transfected cells compared with un-transfected cells. Conclusions Findings of present study demonstrated the successful cloning and expression of TIM-1 on HEK 293T cells. These cells could be used as an immunogenic source for production of specific monoclonal antibodies, nanobodies and aptamers against human TIM-1. PMID:28959306

Cooperation of p27Kip1 and p18INK4c in Progestin-Mediated Cell Cycle Arrest in T-47D Breast Cancer Cells

PubMed Central

Swarbrick, Alexander; Lee, Christine S. L.; Sutherland, Robert L.; Musgrove, Elizabeth A.

2000-01-01

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. The long-term effect of progestins on T-47D breast cancer cells is inhibition of cellular proliferation. This is accompanied by decreased G1 cyclin-dependent kinase (CDK) activities, redistribution of the CDK inhibitor p27Kip1 among these CDK complexes, and alterations in the elution profile of cyclin E-Cdk2 upon gel filtration chromatography, such that high-molecular-weight complexes predominate. This study aimed to determine the relative contribution of CDK inhibitors to these events. Following progestin treatment, the majority of cyclin E- and D-CDK complexes were bound to p27Kip1 and few were bound to p21Cip1. In vitro, recombinant His6-p27 could quantitatively reproduce the effects on cyclin E-Cdk2 kinase activity and the shift in molecular weight observed following progestin treatment. In contrast, cyclin D-Cdk4 was not inhibited by His6-p27 in vitro or p27Kip1 in vivo. However, an increase in the expression of the Cdk4/6 inhibitor p18INK4c and its extensive association with Cdk4 and Cdk6 were apparent following progestin treatment. Recombinant p18INK4c led to the reassortment of cyclin-CDK-CDK inhibitor complexes in vitro, with consequent decrease in cyclin E-Cdk2 activity. These results suggest a concerted model of progestin action whereby p27Kip1 and p18INK4c cooperate to inhibit cyclin E-Cdk2 and Cdk4. Since similar models have been developed for growth inhibition by transforming growth factor β and during adipogenesis, interaction between the Cip/Kip and INK4 families of inhibitors may be a common theme in physiological growth arrest and differentiation. PMID:10713180

Tissue Factor promotes breast cancer stem cell activity in vitro.

PubMed

Shaker, Hudhaifah; Harrison, Hannah; Clarke, Robert; Landberg, Goran; Bundred, Nigel J; Versteeg, Henri H; Kirwan, Cliona C

2017-04-18

Cancer stem cells (CSCs) are a subpopulation of cells that can self-renew and initiate tumours. The clotting-initiating protein Tissue Factor (TF) promotes metastasis and may be overexpressed in cancer cells with increased CSC activity. We sought to determine whether TF promotes breast CSC activity in vitro using human breast cancer cell lines. TF expression was compared in anoikis-resistant (CSC-enriched) and unselected cells. In cells sorted into of TF-expressing and TF-negative (FACS), and in cells transfected to knockdown TF (siRNA) and overexpress TF (cDNA), CSC activity was compared by (i) mammosphere forming efficiency (MFE) (ii) holoclone colony formation (Hc) and (iii) ALDH1 activity. TF expression was increased in anoikis-resistant and high ALDH1-activity T47D cells compared to unselected cells. FACS sorted TF-expressing T47Ds and TF-overexpressing MCF7s had increased CSC activity compared to TF-low cells. TF siRNA cells (MDAMB231,T47D) had reduced CSC activity compared to control cells. FVIIa increased MFE and ALDH1 in a dose-dependent manner (MDAMB231, T47D). The effects of FVIIa on MFE were abrogated by TF siRNA (T47D). Breast CSCs (in vitro) demonstrate increased activity when selected for high TF expression, when induced to overexpress TF, and when stimulated (with FVIIa). Targeting the TF pathway in vivo may abrogate CSC activity.

Hepatocytes and IL-15: a favorable microenvironment for T cell survival and CD8+ T cell differentiation.

PubMed

Correia, Margareta P; Cardoso, Elsa M; Pereira, Carlos F; Neves, Rui; Uhrberg, Markus; Arosa, Fernando A

2009-05-15

Human intrahepatic lymphocytes are enriched in CD1d-unrestricted T cells coexpressing NKR. Although the origin of this population remains controversial, it is possible to speculate that the hepatic microenvironment, namely epithelial cells or the cytokine milieu, may play a role in its shaping. IL-15 is constitutively expressed in the liver and has a key role in activation and survival of innate and tissue-associated immune cells. In this in vitro study, we examined whether hepatocyte cell lines and/or IL-15 could play a role in the generation of NK-like T cells. The results show that both HepG2 cells and a human immortalized hepatocyte cell line increase survival and drive basal proliferation of T cells. In addition, IL-15 was capable of inducing Ag-independent up-regulation of NKR, including NKG2A, Ig-like receptors, and de novo expression of CD56 and NKp46 in CD8(+)CD56(-) T cells. In conclusion, our study suggests that hepatocytes and IL-15 create a favorable microenvironment for T cells to growth and survive. It can be proposed that the increased percentage of intrahepatic nonclassical NKT cells could be in part due to a local CD8(+) T cell differentiation.

Assessment of cell line competence for studies of pharmacological GPR30 modulation.

PubMed

Sousa, Cátia; Ribeiro, Madalena; Rufino, Ana Teresa; Leitão, Alcino Jorge; Mendes, Alexandrina Ferreira

2017-04-01

Cell lines used to study the role of the G protein-coupled receptor 30 (GPR30) or G protein-coupled estrogen receptor (GPER) as a mediator of estrogen responses have yielded conflicting results. This work identified a simple assay to predict cell line competence for pharmacological studies of GPR30. The phosphorylation or expression levels of ERK1/2, Akt, c-Fos and eNOS were evaluated to assess GPR30 activation in response to known agonists (17β-estradiol and G-1) in MCF-7 and T-47D breast cancer cell lines and in bovine aortic endothelial cells. GPR30 expression was analyzed by qRT-PCR and Western blot with two distinct antibodies directed at its carboxy and amino terminals. None of the agonists, at any of the concentrations tested, activated any of those target proteins. Additional experiments excluded the disruption of the signaling pathway, interference of phenol red in the culture medium and constitutive proteasome degradation of GPR30 as possible causes for the lack of response of the three cell lines. Analysis of receptor expression showed the absence of clearly detectable GPR30 species of 44 and 50-55 kDa previously identified in cell lines that respond to 17β-estradiol and G-1. Cells that do not express the 44 and 50-55 kDa species do not respond to GPR30 agonists. Thus, the presence or absence of these GPR30 species is a simple and rapid manner to determine whether a given cell line is suitable for pharmacological or molecular studies of GPR30 modulation.

[Effects of fasudil on bleomycin-induced pulmonary fibrosis in mice and on the biological behaviors in NIH3T3 mouse fibroblast cell line].

PubMed

Jiang, Chunguo; Huang, Hui; Liu, Jia; Wang, Yanxun; Zhao, Yuyue; Xu, Zuojun

2014-09-01

To determine the beneficial effects and mechanisms of fasudil, a selective ROCK inhibitor, on bleomycin-induced pulmonary fibrosis in mice and to determine the effects and mechanisms of fasudil on the biological behaviors in NIH3T3 mouse fibroblast cell line. The BPF model was induced by a single dosage of 2.5 mg/kg bleomycin intratracheal injection in mice and fasudil intraperitoneal injection was given to the mice. The fibrosis degree was determined pathologically by using the Ashcroft scoring method and biochemically by hydroxyproline assay in lung tissue. NIH3T3 mouse fibroblast cell line was cultured in vitro and fasudil was given to the cell. The proliferation activity in NIH3T3 cells were detected by MTT assay and flat colony forming experiment. The migration activity in NIH3T3 cells were detected by scratch test and transwell chamber experiment. The expression of CyclinD1, MMP2 and TIMP1 mRNA in NIH3T3 cells was detected by RT-PCR. The expression of CyclinD1, MMP2 and TIMP1 protein and the level of MYPT1 phosphorylation in NIH3T3 cells was detected by Western blot. Compare to the mice administrated by bleomycin, the Ashcroft score and hydroxyproline content were significantly decreased in the mice administered fasudil. Administration of fasudil can reduce the ability of proliferation and migration in a dose-dependent manner in NIH3T3 cells. The effect of fasudil was possibly related to increase the production of TIMP1 and decrease the production of CyclinD1 and MMP2. Administration of fasudil can attenuate pulmonary fibrosis both in vivo and in vitro. These findings suggest that fasudil may be a potential therapeutic candidate for the treatment of pulmonary fibrosis.

Modulation of ceramide metabolism in T-leukemia cell lines potentiates apoptosis induced by the cationic antimicrobial peptide bovine lactoferricin.

PubMed

Furlong, Suzanne J; Ridgway, Neale D; Hoskin, David W

2008-03-01

Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that selectively induces apoptosis in several different types of human cancer cells. However, the potential use of LfcinB as an anticancer agent is presently limited by the need for relatively high concentrations of the peptide to trigger apoptosis. Ceramide is a membrane sphingolipid that is believed to function as a second messenger during apoptosis. In this study, we investigated the role of ceramide in LfcinB-induced apoptosis in CCRF-CEM and Jurkat T-leukemia cell lines. Exposure to LfcinB caused nuclear condensation and fragmentation, poly(ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation in CCRF-CEM and Jurkat T-cell acute lymphoblastic leukemia cell lines. Treatment with C6 ceramide, a cell-permeable, short-chain ceramide analog, also induced apoptotic nuclear morphology, PARP cleavage, and DNA fragmentation in T-leukemia cells. Although LfcinB treatment did not cause ceramide to accumulate in CCRF-CEM or Jurkat cells, the addition of C6 ceramide to LfcinB-treated T-leukemia cells resulted in increased DNA fragmentation. Furthermore, modulation of cellular ceramide metabolism either by inhibiting ceramidases with D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol or N-oleoylethanolamine, or by blocking glucosylceramide synthase activity with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, enhanced the ability of LfcinB to trigger apoptosis in both Jurkat and CCRF-CEM cells. In addition, LfcinB-induced apoptosis of T-leukemia cells was enhanced in the presence of the antiestrogen tamoxifen, which has multiple effects on cancer cells, including inhibition of glucosylceramide synthase activity. We conclude that manipulation of cellular ceramide levels in combination with LfcinB therapy warrants further investigation as a novel strategy for the treatment of T cell-derived leukemias.

Specific DNA binding activity of T antigen subclasses varies among different SV40-transformed cell lines.

PubMed

Burger, C; Fanning, E

1983-04-15

Large tumor antigen (T antigen) occurs in at least three different oligomeric subclasses in cells infected or transformed by simian virus 40 (SV40): 5-7 S, 14-16 S, and 23-25 S. The 23-25 S form is complexed with a host phosphoprotein (p53). The DNA binding properties of these three subclasses of T antigen from nine different cell lines and free p53 protein were compared using an immunoprecipitation assay. All three subclasses of T antigen bound specifically to SV40 DNA sequences near the origin of replication. However, the DNA binding activity varied between different cell lines over a 40- to 50-fold range. The 23-25 S and 14-16 S forms from most of the cell lines tested bound much less SV40 origin DNA than 5-7 S T antigen. The free p53 phosphoprotein did not bind specifically to any SV40 DNA sequences.

Functions of NKG2D in CD8+ T cells: an opportunity for immunotherapy.

PubMed

Prajapati, Kushal; Perez, Cynthia; Rojas, Lourdes Beatriz Plaza; Burke, Brianna; Guevara-Patino, Jose A

2018-02-05

Natural killer group 2 member D (NKG2D) is a type II transmembrane receptor. NKG2D is present on NK cells in both mice and humans, whereas it is constitutively expressed on CD8 + T cells in humans but only expressed upon T-cell activation in mice. NKG2D is a promiscuous receptor that recognizes stress-induced surface ligands. In NK cells, NKG2D signaling is sufficient to unleash the killing response; in CD8 + T cells, this requires concurrent activation of the T-cell receptor (TCR). In this case, the function of NKG2D is to authenticate the recognition of a stressed target and enhance TCR signaling. CD28 has been established as an archetype provider of costimulation during T-cell priming. It has become apparent, however, that signals from other costimulatory receptors, such as NKG2D, are required for optimal T-cell function outside the priming phase. This review will focus on the similarities and differences between NKG2D and CD28; less well-described characteristics of NKG2D, such as the potential role of NKG2D in CD8 + T-cell memory formation, cancer immunity and autoimmunity; and the opportunities for targeting NKG2D in immunotherapy.Cellular and Molecular Immunology advance online publication, 5 February 2018; doi:10.1038/cmi.2017.161.

A hybrid of B and T lymphoblastic cell line could potentially substitute dendritic cells to efficiently expand out Her-2/neu-specific cytotoxic T lymphocytes from advanced breast cancer patients in vitro.

PubMed

Chen, Sheng; Gu, Feifei; Li, Kang; Zhang, Kai; Liu, Yangyang; Liang, Jinyan; Gao, Wei; Wu, Gang; Liu, Li

2017-02-28

Adoptive transfer of cytotoxic T lymphocytes (CTLs) holds promises to cure cancer. However, this treatment is hindered by lacking a robust way to specifically expand out CTLs. Here, we developed a hybrid of B lymphoblastic cell line and T lymphoblastic cell line (T2 cells) as a substitute of dendritic cells, together with irradiated autologous peripheral blood mononuclear cell (PBMC) as feeder cells and rhIL-2, to activate and expand Her-2/neu-specific CD8 + T cells from human epidermal growth factor receptor 2 (Her-2/neu) and human leukocyte antigen (HLA)-A2 double positive advanced breast cancer patients in vitro. These Her-2/neu-loaded T2 cells reproducibly activated and expanded out Her-2/neu-specific CD8 + T cells to 10 7 in 8 weeks. Furthermore, these Her-2/neu-specific CD8 + T cells had good sensitivity of recognition and killing Her-2/neu-overexpressed breast cancer cell line SK.BR.3. This technique gives us another insight on how to rapidly obtain sufficient CTLs for adoptive cancer immunotherapy.

Breast implant-associated, ALK-negative, T-cell, anaplastic, large-cell lymphoma: establishment and characterization of a model cell line (TLBR-1) for this newly emerging clinical entity.

PubMed

Lechner, Melissa G; Lade, Stephen; Liebertz, Daniel J; Prince, H Miles; Brody, Garry S; Webster, Howard R; Epstein, Alan L

2011-04-01

Primary lymphomas of the breast are very rare (0.2-1.5% of breast malignancies) and the vast majority (95%) are of B-cell origin. Recently, 40 cases of clinically indolent anaplastic large-cell kinase (ALK)-negative, T-cell, anaplastic, non-Hodgkin lymphomas (T-ALCL) have been reported worldwide. A tumor biopsy specimen from a patient in this series was obtained for characterization. By using a human stromal feeder layer and IL-2, a novel cell line, TLBR-1, was established from this biopsy and investigated by using cytogenetics and various biomolecular methods. Immunoperoxidase staining of the tumor biopsy showed a CD30/CD8/CD4 coexpressing T-cell population that was epithelial membrane antigen (EMA)(+) and perforin(+) . Multiplex polymerase chain reaction (PCR) of TCRγ genes showed monoclonality that suggested a T-cell origin, yet pan-T markers CD2/5/7, anaplastic large-cell kinase (ALK)-1, pancytokeratins, CD20, CD56, and Epstein-Barr virus (EBV) by in situ hybridization (ISH) were negative. TLBR-1 is IL-2 dependent, has a relatively long doubling time (55 hours), and displays different cellular shapes in culture. Cytogenetic analysis of tumor and TLBR-1 cells confirmed a highly anaplastic cell population with a modal number of 47 chromosomes lacking t(2;5). PCR screens for EBV and human T-lymphotropic virus types 1 and 2 (HTLV-1/2) were negative. Fluorescence-activated cell-sorting (FACS) analysis showed strong positivity for CD4/8, CD30, CD71, and CD26 expression, and antigen presentation (HLA-DR(+) CD80(+) CD86(+) ), IL-2 signaling (CD25(+) CD122(+) ), and NK (CD56(+) ) markers, and Western blots demonstrated strong Notch1 expression. Severe combined immunodeficiency (SCID) mouse TLBR-1 heterotransplants recapitulated the histology and marker characteristics of the original tumor. TLBR-1, a novel ALK-negative, T-cell, anaplastic, large-cell lymphoma, closely resembles the original biopsy and represents an important tool for studying this newly recognized

Breast Implant-Associated, ALK-Negative, T-Cell, Anaplastic, Large-Cell Lymphoma: Establishment and Characterization of a Model Cell Line (TLBR-1) for This Newly Emerging Clinical Entity

PubMed Central

Lechner, Melissa G.; Lade, Stephen; Liebertz, Daniel J.; Prince, H. Miles; Brody, Garry S.; Webster, Howard R.; Epstein, Alan L.

2014-01-01

BACKGROUND Primary lymphomas of the breast are very rare (0.2–1.5% of breast malignancies) and the vast majority (95%) are of B-cell origin. Recently, 40 cases of clinically indolent anaplastic large-cell kinase (ALK)-negative, T-cell, anaplastic, non-Hodgkin lymphomas (T-ALCL) have been reported worldwide. METHODS A tumor biopsy specimen from a patient in this series was obtained for characterization. By using a human stromal feeder layer and IL-2, a novel cell line, TLBR-1, was established from this biopsy and investigated by using cytogenetics and various biomolecular methods. RESULTS Immunoperoxidase staining of the tumor biopsy showed a CD30/CD8/CD4 coexpressing T-cell population that was epithelial membrane antigen (EMA)+ and perforin+. Multiplex polymerase chain reaction (PCR) of TCRγ genes showed monoclonality that suggested a T-cell origin, yet pan-T markers CD2/5/7, anaplastic large-cell kinase (ALK)-1, pancytokeratins, CD20, CD56, and Epstein-Barr virus (EBV) by in situ hybridization (ISH) were negative. TLBR-1 is IL-2 dependent, has a relatively long doubling time (55 hours), and displays different cellular shapes in culture. Cytogenetic analysis of tumor and TLBR-1 cells confirmed a highly anaplastic cell population with a modal number of 47 chromosomes lacking t(2;5). PCR screens for EBV and human T-lymphotropic virus types 1 and 2 (HTLV-1/2) were negative. Fluorescence-activated cell-sorting (FACS) analysis showed strong positivity for CD4/8, CD30, CD71, and CD26 expression, and antigen presentation (HLA-DR+CD80+CD86+), IL-2 signaling (CD25+CD122+), and NK (CD56+) markers, and Western blots demonstrated strong Notch1 expression. Severe combined immunodeficiency (SCID) mouse TLBR-1 heterotransplants recapitulated the histology and marker characteristics of the original tumor. CONCLUSIONS TLBR-1, a novel ALK-negative, T-cell, anaplastic, large-cell lymphoma, closely resembles the original biopsy and represents an important tool for studying this

[The correlation of synuclein-γ and matrix metalloproteinase 9 in breast cancer].

PubMed

Chen, Jian; Huang, Shuo; Wu, Ke-jin; Wang, Yong-kun; Jia, Yi-jun; Lu, Yun-shu; Weng, Zi-yi

2013-07-01

To evaluate the expression of synuclein-γ (SNCG) and metalloproteinase 9 (MMP-9) both in the invasive ductal breast cancer samples and T47D and T47D(SNCG)- cell lines, to investigate the correlation between SNCG and MMP-9. Totally 96 invasive ductal breast cancer samples (female, mean age of (56 ± 8) years) were collected between June 2009 and June 2012. The expressions of SNCG and MMP-9 were investigated by immunohistochemistry. T47D and SNCG knock down T47D(SNCG)- cell lines were established and SNCG and MMP-9 protein expression were investigated by Western blot and gene expression by real-time PCR. Among 96 samples, 26 (27.1%) of them co-expressed SNCG and MMP-9, 30(31.2%) of them expressed neither SNCG nor MMP-9. The expression of SNCG was correlated with the expression of MMP-9 (r = 0.655, P = 0.000).SNCG mRNA level of T47D cell line was 13.5 fold of T47D(SNCG)- cell line and SNCG protein expression was 2.1 fold. While MMP-9 mRNA level of T47D cell line was 7.3 fold of T47D(SNCG)- cell line and MMP-9 protien expression was 1.6 fold.When SNCG was knocked down, the expression of MMP-9 decreased. SNCG and MMP-9 are significantly correlated with each other in breast cancer. SNCG may promote the invasion and metastasis of breast cancer mediated by up-regulating the expression of MMP-9.

Characterisation of adriamycin- and amsacrine-resistant human leukaemic T cell lines.

PubMed Central

Snow, K.; Judd, W.

1991-01-01

Cell lines resistant to adriamycin and amsacrine were derived from cloned sublines of the human T cell line Jurkat. Most of the lines resemble atypical MDR cells (Danks et al., 1987; Beck et al., 1987). Thus, resistant Jurkat sublines were cross resistant to several topoisomerase II inhibiting drugs but had low or no resistance to other classes of drugs, resistance was not reversed by verapamil, Pgp was not overexpressed, and drug accumulation was unaltered in resistant compared to parental (control) sublines. Other findings were that anthracycline metabolism differed between resistant and parental sublines, and that resistant sublines displayed altered expression of small polypeptides (less than 20K MW) and an 85K MW protein. Drug resistant cells showed resistance to the production of drug induced cytogenetic aberrations, DNA breaks, and protein-DNA complexes. Resistance was not mediated by altered binding of drugs to DNA or by increased repair of DNA damage. Indirect evidence suggests that the resistant cells had an altered drug-DNA-topoisomerase II association. The study highlights the complex relationships between DNA breaks, cytogenetic aberrations, protein-DNA complexes and drug cytotoxicity, and shows that the relationships differ for adriamycin and amsacrine, suggesting some differences in the modes of action and/or resistance for the drugs and cell lines. Images Figure 2 Figure 3 PMID:1989661

Mitochondrial cyclophilin D regulates T cell metabolic responses and disease tolerance to tuberculosis.

PubMed

Tzelepis, Fanny; Blagih, Julianna; Khan, Nargis; Gillard, Joshua; Mendonca, Laura; Roy, Dominic G; Ma, Eric H; Joubert, Philippe; Jones, Russell G; Divangahi, Maziar

2018-05-11

Mycobacterium tuberculosis ( Mtb ) is one of the most ancient human pathogens, yet the exact mechanism(s) of host defense against Mtb remains unclear. Although one-third of the world's population is chronically infected with Mtb , only 5 to 10% develop active disease. This indicates that, in addition to resistance mechanisms that control bacterial burden, the host has also evolved strategies to tolerate the presence of Mtb to limit disease severity. We identify mitochondrial cyclophilin D (CypD) as a critical checkpoint of T cell metabolism that controls the expansion of activated T cells. Although loss of CypD function in T cells led to enhanced Mtb antigen-specific T cell responses, this increased T cell response had no impact on bacterial burden. Rather, mice containing CypD-deficient T cells exhibited substantially compromised disease tolerance and succumbed to Mtb infection. This study establishes a mechanistic link between T cell-mediated immunity and disease tolerance during Mtb infection. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

GS-nitroxide (JP4-039)-mediated radioprotection of human Fanconi anemia cell lines.

PubMed

Bernard, Mark E; Kim, Hyun; Berhane, Hebist; Epperly, Michael W; Franicola, Darcy; Zhang, Xichen; Houghton, Frank; Shields, Donna; Wang, Hong; Bakkenist, Christopher J; Frantz, Marie-Celine; Forbeck, Erin M; Goff, Julie P; Wipf, Peter; Greenberger, Joel S

2011-11-01

Fanconi anemia (FA) is an inherited disorder characterized by defective DNA repair and cellular sensitivity to DNA crosslinking agents. Clinically, FA is associated with high risk for marrow failure, leukemia and head and neck squamous cell carcinoma (HNSCC). Radiosensitivity in FA patients compromises the use of total-body irradiation for hematopoietic stem cell transplantation and radiation therapy for HNSCC. A radioprotector for the surrounding tissue would therefore be very valuable during radiotherapy for HNSCC. Clonogenic radiation survival curves were determined for pre- or postirradiation treatment with the parent nitroxide Tempol or JP4-039 in cells of four FA patient-derived cell lines and two transgene-corrected subclonal lines. FancG(-/-) (PD326) and FancD2(-/-) (PD20F) patient lines were more sensitive to the DNA crosslinking agent mitomycin C (MMC) than their transgene-restored subclonal cell lines (both P < 0.0001). FancD2(-/-) cells were more radiosensitive than the transgene restored subclonal cell line (ñ = 2.0 ± 0.7 and 4.7 ± 2.2, respectively, P = 0.03). In contrast, FancG(-/-) cells were radioresistant relative to the transgene-restored subclonal cell line (ñ = 9.4 ± 1.5 and 2.2 ± 05, respectively, P = 0.001). DNA strand breaks measured by the comet assay correlated with radiosensitivity. Cell lines from a Fanc-C and Fanc-A patients showed radiosensitivity similar to that of Fanc-D2(-/-) cells. A fluorophore-tagged JP4-039 (BODIPY-FL) analog targeted the mitochondria of the cell lines. Preirradiation or postirradiation treatment with JP4-039 at a lower concentration than Tempol significantly increased the radioresistance and stabilized the antioxidant stores of all cell lines. Tempol increased the toxicity of MMC in FancD2(-/-) cells. These data provide support for the potential clinical use of JP4-039 for normal tissue radioprotection during chemoradiotherapy in FA patients.

Constitutively expressing cell lines that secrete a truncated bovine herpes virus-1 glycoprotein (gpI) stimulate T-lymphocyte responsiveness.

PubMed

Leary, T P; Gao, Y; Splitter, G A

1992-07-01

The desire to obtain authentically glycosylated viral protein products in sufficient quantity for immunological study has led to the use of eucaryotic expression vectors for protein production. An additional advantage is that these protein products can be studied individually in the absence of their native viral environment. We have cloned a complementary DNA (cDNA) encoding bovine herpes virus-1 (BHV-1) glycoprotein 1 (gpI) into the eucaryotic expression vector, pZipNeo SVX1. Since this protein is normally embedded within the membrane of BHV-1 infected cells, we removed sequences encoding the transmembrane domain of the native protein. After transfection of the plasmid construct into the canine osteosarcoma cell line, D17, or Madin-Darby bovine kidney (MDBK) cells, a truncated BHV-1 (gpI) was secreted into the culture medium as demonstrated by radioimmunoprecipitation and SDS-PAGE. Both a CD4+ T-lymphocyte line specific for BHV-1 and freshly isolated T lymphocytes could recognize and respond to the secreted recombinant gpI. Further, recombinant gpI could elicit both antibody and cellular responses in cattle when used as an immunogen. Having established constitutively glycoprotein producing cell lines, future studies in vaccine evaluation of gpI will be facilitated.

Establishment of a rat hepatoma-derived cell line proliferating in D-phenylalanine medium and expressing D-amino-acid oxidase.

PubMed

Yoda, N; Konno, R; Nagashima, S

2001-01-01

A cell line (R-Y121B.DF) has been established from a cell line (R-Y121B) derived from a rat hepatoma line (H4-II-E). The R-Y121B.DF cells have been continuously cultured in a serum-free modified Eagle's minimum essential medium in which L-phenylalanine was replaced by D-phenylalanine. They had D-amino-acid oxidase (DAO) activity which is essential for the growth in the medium containing D-amino acids. The enzyme activity of the R-Y121B.DF cells was approximately one-fourth of that of the rat liver. Northern hybridization using a DAO cDNA probe detected a hybridizing signal in the R-Y121B.DF cells and the rat liver but not in the parental R-Y121B and H4-II-E cells. Reverse transcription-polymerase chain reaction using DAO-specific primers amplified a DNA fragment of the expected size in the R-Y121B.DF cells but not in the R-Y121B and H4-II-E cells. This fragment was confirmed to be DAO cDNA by nucleotide sequencing. Western blotting showed that DAO protein was present in the R-Y121B.DF cells and the rat liver but not in the R-Y121B and H4-II-E cells. Southern hybridization showed that the DAO gene structure was not different among the R-Y121B.DF cells, R-Y121B cells, H4-II-E cells, and the rat liver. These results indicate that the R-Y121B.DF is a unique cell line which proliferates in the medium containing D-phenylalanine and explicitly expresses DAO. This line is useful for the study of DAO in vitro.

Involvement of Semaphorin (Sema4D) in T-Dependent Activation of B Cells.

PubMed

Kuklina, Е М; Nekrasova, I V; Valieva, Yu V

2017-08-01

The involvement of endogenous semaphorin (Sema4D) into the key stage of T-dependent differentiation of B cells, formation of plasmoblasts, was demonstrated in vitro in T/B cell co-culture under conditions of polyclonal activation of T cells. The effect of semaphorin was not associated with activation of high-affinity Sema4D receptor plexin B1, but involves lowaffinity receptor CD72. These data indicate that Sema4D-dependent signal regulates not only the initial stage of B-cell activation, proliferative response to the antigen, but also further differentiation of B cells into plasma cells.

Regulation and gene expression profiling of NKG2D positive human cytomegalovirus-primed CD4+ T-cells.

PubMed

Jensen, Helle; Folkersen, Lasse; Skov, Søren

2012-01-01

NKG2D is a stimulatory receptor expressed by natural killer (NK) cells, CD8(+) T-cells, and γδ T-cells. NKG2D expression is normally absent from CD4(+) T-cells, however recently a subset of NKG2D(+) CD4(+) T-cells has been found, which is specific for human cytomegalovirus (HCMV). This particular subset of HCMV-specific NKG2D(+) CD4(+) T-cells possesses effector-like functions, thus resembling the subsets of NKG2D(+) CD4(+) T-cells found in other chronic inflammations. However, the precise mechanism leading to NKG2D expression on HCMV-specific CD4(+) T-cells is currently not known. In this study we used genome-wide analysis of individual genes and gene set enrichment analysis (GSEA) to investigate the gene expression profile of NKG2D(+) CD4(+) T-cells, generated from HCMV-primed CD4(+) T-cells. We show that the HCMV-primed NKG2D(+) CD4(+) T-cells possess a higher differentiated phenotype than the NKG2D(-) CD4(+) T-cells, both at the gene expression profile and cytokine profile. The ability to express NKG2D at the cell surface was primarily determined by the activation or differentiation status of the CD4(+) T-cells and not by the antigen presenting cells. We observed a correlation between CD94 and NKG2D expression in the CD4(+) T-cells following HCMV stimulation. However, knock-down of CD94 did not affect NKG2D cell surface expression or signaling. In addition, we show that NKG2D is recycled at the cell surface of activated CD4(+) T-cells, whereas it is produced de novo in resting CD4(+) T-cells. These findings provide novel information about the gene expression profile of HCMV-primed NKG2D(+) CD4(+) T-cells, as well as the mechanisms regulating NKG2D cell surface expression.

Calcium signaling properties of a thyrotroph cell line, mouse TαT1 cells.

PubMed

Tomić, Melanija; Bargi-Souza, Paula; Leiva-Salcedo, Elias; Nunes, Maria Tereza; Stojilkovic, Stanko S

2015-12-01

TαT1 cells are mouse thyrotroph cell line frequently used for studies on thyroid-stimulating hormone beta subunit gene expression and other cellular functions. Here we have characterized calcium-signaling pathways in TαT1 cells, an issue not previously addressed in these cells and incompletely described in native thyrotrophs. TαT1 cells are excitable and fire action potentials spontaneously and in response to application of thyrotropin-releasing hormone (TRH), the native hypothalamic agonist for thyrotrophs. Spontaneous electrical activity is coupled to small amplitude fluctuations in intracellular calcium, whereas TRH stimulates both calcium mobilization from intracellular pools and calcium influx. Non-receptor-mediated depletion of intracellular pool also leads to a prominent facilitation of calcium influx. Both receptor and non-receptor stimulated calcium influx is substantially attenuated but not completely abolished by inhibition of voltage-gated calcium channels, suggesting that depletion of intracellular calcium pool in these cells provides a signal for both voltage-independent and -dependent calcium influx, the latter by facilitating the pacemaking activity. These cells also express purinergic P2Y1 receptors and their activation by extracellular ATP mimics TRH action on calcium mobilization and influx. The thyroid hormone triiodothyronine prolongs duration of TRH-induced calcium spikes during 30-min exposure. These data indicate that TαT1 cells are capable of responding to natively feed-forward TRH signaling and intrapituitary ATP signaling with acute calcium mobilization and sustained calcium influx. Amplification of TRH-induced calcium signaling by triiodothyronine further suggests the existence of a pathway for positive feedback effects of thyroid hormones probably in a non-genomic manner. Published by Elsevier Ltd.

Regulation and Gene Expression Profiling of NKG2D Positive Human Cytomegalovirus-Primed CD4+ T-Cells

PubMed Central

Jensen, Helle; Folkersen, Lasse; Skov, Søren

2012-01-01

NKG2D is a stimulatory receptor expressed by natural killer (NK) cells, CD8+ T-cells, and γδ T-cells. NKG2D expression is normally absent from CD4+ T-cells, however recently a subset of NKG2D+ CD4+ T-cells has been found, which is specific for human cytomegalovirus (HCMV). This particular subset of HCMV-specific NKG2D+ CD4+ T-cells possesses effector-like functions, thus resembling the subsets of NKG2D+ CD4+ T-cells found in other chronic inflammations. However, the precise mechanism leading to NKG2D expression on HCMV-specific CD4+ T-cells is currently not known. In this study we used genome-wide analysis of individual genes and gene set enrichment analysis (GSEA) to investigate the gene expression profile of NKG2D+ CD4+ T-cells, generated from HCMV-primed CD4+ T-cells. We show that the HCMV-primed NKG2D+ CD4+ T-cells possess a higher differentiated phenotype than the NKG2D– CD4+ T-cells, both at the gene expression profile and cytokine profile. The ability to express NKG2D at the cell surface was primarily determined by the activation or differentiation status of the CD4+ T-cells and not by the antigen presenting cells. We observed a correlation between CD94 and NKG2D expression in the CD4+ T-cells following HCMV stimulation. However, knock-down of CD94 did not affect NKG2D cell surface expression or signaling. In addition, we show that NKG2D is recycled at the cell surface of activated CD4+ T-cells, whereas it is produced de novo in resting CD4+ T-cells. These findings provide novel information about the gene expression profile of HCMV-primed NKG2D+ CD4+ T-cells, as well as the mechanisms regulating NKG2D cell surface expression. PMID:22870231

Vitamin D Actions on CD4+ T Cells in Autoimmune Disease

PubMed Central

Hayes, Colleen Elizabeth; Hubler, Shane L.; Moore, Jerott R.; Barta, Lauren E.; Praska, Corinne E.; Nashold, Faye E.

2015-01-01

This review summarizes and integrates research on vitamin D and CD4+ T-lymphocyte biology to develop new mechanistic insights into the molecular etiology of autoimmune disease. A deep understanding of molecular mechanisms relevant to gene–environment interactions is needed to deliver etiology-based autoimmune disease prevention and treatment strategies. Evidence linking sunlight, vitamin D, and the risk of multiple sclerosis and type 1 diabetes is summarized to develop the thesis that vitamin D is the environmental factor that most strongly influences autoimmune disease development. Evidence for CD4+ T-cell involvement in autoimmune disease pathogenesis and for paracrine calcitriol signaling to CD4+ T lymphocytes is summarized to support the thesis that calcitriol is sunlight’s main protective signal transducer in autoimmune disease risk. Animal modeling and human mechanistic data are summarized to support the view that vitamin D probably influences thymic negative selection, effector Th1 and Th17 pathogenesis and responsiveness to extrinsic cell death signals, FoxP3+CD4+ T-regulatory cell and CD4+ T-regulatory cell type 1 (Tr1) cell functions, and a Th1–Tr1 switch. The proposed Th1–Tr1 switch appears to bridge two stable, self-reinforcing immune states, pro- and anti-inflammatory, each with a characteristic gene regulatory network. The bi-stable switch would enable T cells to integrate signals from pathogens, hormones, cell–cell interactions, and soluble mediators and respond in a biologically appropriate manner. Finally, unanswered questions and potentially informative future research directions are highlighted to speed delivery of etiology-based strategies to reduce autoimmune disease. PMID:25852682

Lymphotoxin activation by human T-cell leukemia virus type I-infected cell lines: role for NF-kappa B.

PubMed

Paul, N L; Lenardo, M J; Novak, K D; Sarr, T; Tang, W L; Ruddle, N H

1990-11-01

Human T-cell leukemia virus type I (HTLV-I)-infected T-cell lines constitutively produce high levels of biologically active lymphotoxin (LT; tumor necrosis factor-beta) protein and LT mRNA. To understand the regulation of LT transcription by HTLV-I, we analyzed the ability of a series of deletions of the LT promoter to drive the chloramphenicol acetyltransferase (CAT) reporter gene in HTLV-I-positive MT-2 cells. The smallest LT promoter fragment (-140 to +77) that was able to drive CAT activity contained a site that was similar to the immunoglobulin kappa-chain NF-kappa B-binding site. Since the HTLV-I tax gene activates the nuclear form of NF-kappa B, this finding suggested a possible means of HTLV-I activation of LT production. We found that the LT kappa B-like site specifically formed a complex with NF-kappa B-containing nuclear extract from MT-2, C81-66-45, and other activated T cells. Mutation of the LT kappa B site in the context of the LT promoter (-293 to +77) (mutant M1) reduced the ability of the promoter to drive the CAT gene in HTLV-I-infected and noninfected human T-cell lines. These data suggest a general role for NF-kappa B activation in the induction of LT gene transcription. Activation of LT in HTLV-I-infected cells may explain the pathology associated with HTLV-I infection, including the hypercalcemia that is prevalent in adult T-cell leukemia.

Live attenuated yellow fever 17D infects human DCs and allows for presentation of endogenous and recombinant T cell epitopes.

PubMed

Barba-Spaeth, Giovanna; Longman, Randy S; Albert, Matthew L; Rice, Charles M

2005-11-07

The yellow fever (YF) 17D vaccine is one of the most successful live attenuated vaccines available. A single immunization induces both long-lasting neutralizing antibody and YF-specific T cell responses. Surprisingly, the mechanism for this robust immunity has not been addressed. In light of several recent reports suggesting flavivirus interaction with dendritic cells (DCs), we investigated the mechanism of YF17D interaction with DCs and the importance of this interaction in generating T cell immunity. Our results show that YF17D can infect immature and mature human DCs. Viral entry is Ca(2+) dependent, but it is independent of DC-SIGN as well as multiple integrins expressed on the DC surface. Similar to infection of cell lines, YF infection of immature DCs is cytopathic. Although infection itself does not induce DC maturation in vitro, TNF-alpha-induced maturation protects DCs from YF-induced cytopathogenicity. Furthermore, we show that DCs infected with YF17D or YF17D carrying a recombinant epitope can process and present antigens for CD8(+) T cell stimulation. These findings offer insight into the immunologic mechanisms associated with the highly capable YF17D vaccine that may guide effective vaccine design.

Megalin-Mediated Endocytosis of Vitamin D Binding Protein Correlates with 25-Hydroxycholecalciferol Actions in Human Mammary Cells1

PubMed Central

Rowling, Matthew J.; Kemmis, Carly M.; Taffany, David A.; Welsh, JoEllen

2007-01-01

The major circulating form of vitamin D is 25-hydroxycholecalciferol [25(OH)D3], which is delivered to target tissues in complex with the serum vitamin D binding protein (DBP). We recently observed that mammary cells can metabolize 25(OH)D3 to 1,25-dihydroxycholecalciferol [1,25(OH)2D3], the vitamin D receptor (VDR) ligand, and the objective of our study was to elucidate the mechanisms by which the 25(OH)D3-DBP complex is internalized by mammary cells prior to metabolism. Using fluorescent microscopy and temperature-shift techniques, we found that T-47D breast cancer cells rapidly internalize DBP via endocytosis, which is blunted by receptor-associated protein, a specific inhibitor of megalin-mediated endocytosis. Endocytosis of DBP was associated with activation of VDR by 25(OH)D3 but not 1,25(OH)2D3 (as measured by induction of the VDR target gene, CYP24). We also found that megalin and its endocytic partner, cubilin, are coexpressed in normal murine mammary tissue, in nontransformed human mammary epithelial cell lines, and in some established human breast cancer cell lines. To our knowledge, our studies are the first to demonstrate that mammary-derived cells express megalin and cubilin, which contribute to the endocytic uptake of 25(OH)D3-DBP and activation of the VDR pathway. PMID:17056796

The Role of SIRT1 in Breast Cancer Stem Cells

DTIC Science & Technology

2014-07-01

Stem cell markers, SOX-2 and Nanog, are significantly decreased in SIRT1 inhibitor treated cancer cells by qRT-PCR and western blot in T47D cell line...cells. Immunohistochemistry performed on breast cancer specimens shows the correlation between cancer stem cell markers and SIRT1 overexpression. SIRT1

Stem-Like Memory T Cells Are Discovered | Center for Cancer Research

Cancer.gov

T cells are the white blood cells that are the body’s first line of attack against foreign invaders.  When designing immunotherapies to treat cancer the goal is to prolong the immune response of T cells a bit beyond what the body normally does when a bacterium or a virus is encountered.   Nicholas P. Restifo, M.D., working with Luca Gattinoni, M.D., and other colleagues in

Characterization of a Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Cell Line CVG-1.

PubMed

Velásquez, Celestino; Amako, Yutaka; Harold, Alexis; Toptan, Tuna; Chang, Yuan; Shuda, Masahiro

2018-01-01

Merkel cell polyomavirus (MCV) plays a causal role in ∼80% of Merkel cell carcinomas (MCC). MCV is clonally integrated into the MCC tumor genome, which results in persistent expression of large T (LT) and small T (sT) antigen oncoproteins encoded by the early locus. In MCV-positive MCC tumors, LT is truncated by premature stop codons or deletions that lead to loss of the C-terminal origin binding (OBD) and helicase domains important for replication. The N-terminal Rb binding domain remains intact. MCV-positive cell lines derived from MCC explants have been valuable tools to study the molecular mechanism of MCV-induced Merkel cell carcinogenesis. Although all cell lines have integrated MCV and express truncated LT antigens, the molecular sizes of the LT proteins differ between cell lines. The copy number of integrated viral genome also varies across cell lines, leading to significantly different levels of viral protein expression. Nevertheless, these cell lines share phenotypic similarities in cell morphology, growth characteristics, and neuroendocrine marker expression. Several low-passage MCV-positive MCC cell lines have been established since the identification of MCV. We describe a new MCV-positive MCV cell line, CVG-1, with features distinct from previously reported cell lines. CVG-1 tumor cells grow in more discohesive clusters in loose round cell suspension, and individual cells show dramatic size heterogeneity. It is the first cell line to encode an MCV sT polymorphism resulting in a unique leucine (L) to proline (P) substitution mutation at amino acid 144. CVG-1 possesses a LT truncation pattern near identical to that of MKL-1 cells differing by the last two C-terminal amino acids and also shows an LT protein expression level similar to MKL-1. Viral T antigen knockdown reveals that, like other MCV-positive MCC cell lines, CVG-1 requires T antigen expression for cell proliferation.

Stem-Like Memory T Cells Are Discovered | Center for Cancer Research

Cancer.gov

T cells are the white blood cells that are the body’s first line of attack against foreign invaders.  When designing immunotherapies to treat cancer the goal is to prolong the immune response of T cells a bit beyond what the body normally does when a bacterium or a virus is encountered.   Nicholas P. Restifo, M.D., working with Luca Gattinoni, M.D., and other colleagues in CCR’s Surgery Branch recently discovered a subset within the human T cell population that may help clinicians to do just  this.

Lymphotoxin activation by human T-cell leukemia virus type I-infected cell lines: role for NF-kappa B.

PubMed Central

Paul, N L; Lenardo, M J; Novak, K D; Sarr, T; Tang, W L; Ruddle, N H

1990-01-01

Human T-cell leukemia virus type I (HTLV-I)-infected T-cell lines constitutively produce high levels of biologically active lymphotoxin (LT; tumor necrosis factor-beta) protein and LT mRNA. To understand the regulation of LT transcription by HTLV-I, we analyzed the ability of a series of deletions of the LT promoter to drive the chloramphenicol acetyltransferase (CAT) reporter gene in HTLV-I-positive MT-2 cells. The smallest LT promoter fragment (-140 to +77) that was able to drive CAT activity contained a site that was similar to the immunoglobulin kappa-chain NF-kappa B-binding site. Since the HTLV-I tax gene activates the nuclear form of NF-kappa B, this finding suggested a possible means of HTLV-I activation of LT production. We found that the LT kappa B-like site specifically formed a complex with NF-kappa B-containing nuclear extract from MT-2, C81-66-45, and other activated T cells. Mutation of the LT kappa B site in the context of the LT promoter (-293 to +77) (mutant M1) reduced the ability of the promoter to drive the CAT gene in HTLV-I-infected and noninfected human T-cell lines. These data suggest a general role for NF-kappa B activation in the induction of LT gene transcription. Activation of LT in HTLV-I-infected cells may explain the pathology associated with HTLV-I infection, including the hypercalcemia that is prevalent in adult T-cell leukemia. Images PMID:1976820

Antiestrogen Resistant Cell Lines Expressing Estrogen Receptor α Mutations Upregulate the Unfolded Protein Response and are Killed by BHPI

PubMed Central

Mao, Chengjian; Livezey, Mara; Kim, Ji Eun; Shapiro, David J.

2016-01-01

Outgrowth of metastases expressing ERα mutations Y537S and D538G is common after endocrine therapy for estrogen receptor α (ERα) positive breast cancer. The effect of replacing wild type ERα in breast cancer cells with these mutations was unclear. We used the CRISPR-Cas9 genome editing system and homology directed repair to isolate and characterize 14 T47D cell lines in which ERαY537S or ERαD538G replace one or both wild-type ERα genes. In 2-dimensional, and in quantitative anchorage-independent 3-dimensional cell culture, ERαY537S and ERαD538G cells exhibited estrogen-independent growth. A progestin further increased their already substantial proliferation in micromolar 4-hydroxytamoxifen and fulvestrant/ICI 182,780 (ICI). Our recently described ERα biomodulator, BHPI, which hyperactivates the unfolded protein response (UPR), completely blocked proliferation. In ERαY537S and ERαD538G cells, estrogen-ERα target genes were constitutively active and partially antiestrogen resistant. The UPR marker sp-XBP1 was constitutively activated in ERαY537S cells and further induced by progesterone in both cell lines. UPR-regulated genes associated with tamoxifen resistance, including the oncogenic chaperone BiP/GRP78, were upregulated. ICI displayed a greater than 2 fold reduction in its ability to induce ERαY537S and ERαD538G degradation. Progestins, UPR activation and perhaps reduced ICI-stimulated ERα degradation likely contribute to antiestrogen resistance seen in ERαY537S and ERαD538G cells. PMID:27713477

Presentation of antigen to T lymphocytes by non-immune B-cell hybridoma clones: evidence for specific and non-specific presentation

NASA Technical Reports Server (NTRS)

Cohly, H. H.; Morrison, D. R.; Atassi, M. Z.

1988-01-01

Non-immune SJL (H-2s) spleen cells were fused with (H-2d) Balb/c 653-myeloma cells and the hybridomas were cloned by two limiting dilutions. The resulting hybrid B- cell clones were tested for their antigen presentation capability to SJL T-cell lines that were specific for either lysozyme or myoglobin. In proliferative assays, 53% of the antigen presenting B-cell clones were able to present both myoglobin and lysozyme (general presenters) while the other 47% presented specifically either myoglobin or lysozyme (specific presenters). The ability to selectively present either myoglobin or lysozyme indicates that antigen presentation at the clonal level can be specific or non-specific depending on the particular B-cell clone.

Presentation of antigen to T lymphocytes by non-immune B-cell hybridoma clones: evidence for specific and non-specific presentation

NASA Technical Reports Server (NTRS)

Cohly, H. H.; Morrison, D. R.; Zouhair Atassi, M. Z.

1989-01-01

Non-immune SJL (H-2s) spleen cells were fused with non-secreting, non-antigen presenting (H-2d) Balb/c 653-myeloma cells and the hybridomas were cloned by two limiting dilutions. The resulting hybrid B-cell clones were tested for their antigen presentation capability to SJL T-cell lines that were specific for either lysozyme or myoglobin. In proliferative assays, 53% of the antigen presenting B-cell clones presented both myoglobin and lysozyme (general presenters) while the other 47% presented specifically either myoglobin or lysozyme (specific presenters). The ability to selectively present either myoglobin or lysozyme indicates that antigen presentation at the clonal level can be specific or non-specific depending on the particular B-cell clone.

Reversal of infectious mononucleosis-associated suppressor T cell activity by D-mannose

PubMed Central

1983-01-01

Epstein-Barr virus-induced infectious mononucleosis (IM) is associated with the activation of suppressor T lymphocytes that profoundly inhibit immunoglobulin (Ig) production in vitro. We have examined the nature of signals operating in the interaction between IM suppressor T cells and their targets, and explored the possibility that a lectin-like receptor molecule and its specific sugar might provide specificity to this interaction. When D-mannose or some of its derivatives, including alpha- methyl-D-mannoside, mannose-6-phosphate, and mannan, were added to suppressed cultures containing IM T lymphocytes and pokeweed mitogen (PWM)-stimulated normal mononuclear cells, a significant enhancement of Ig production was observed. These sugars had little or no effect on Ig production by the PWM-stimulated responder cells alone and thus the enhanced Ig production could be attributed to the reversal of suppression in the co-cultures by these sugars. This was further confirmed by the observation that the sugars were effective only if present during the first 24 h of culture, a time when IM suppressor T cells exert their principal effect. The effect of sugars on Ig production by suppressed cultures was similar to that achieved by decreasing by about fourfold the number of IM T cells in culture. The effect of the sugars is unlikely to represent a form of nonspecific toxicity, since inhibited cultures become responders in the presence of the sugar. Furthermore, toxicity restricted to the suppressor T cells is unlikely, since preincubation of the T cells with the sugars did not reduce their subsequent ability to suppress in secondary indicator cultures. In addition, there was no correlation between the effect of the sugars on T cell proliferation and their effect on T cell-mediated suppression. The reversal of suppression by sugars was dose dependent and demonstrated stereo-specificity in that L-mannose was without effect while D-mannose reversed suppression. These data indicate

Evaluation of accessory cell heterogeneity. I. Differential accessory cell requirement for T helper cell activation and for T-B cooperation.

PubMed

Ramila, G; Studer, S; Kennedy, M; Sklenar, I; Erb, P

1985-01-01

Several Ia+ tumor cell lines and peritoneal exudate macrophages were tested as accessory cells (AC) for the activation of antigen-specific T cells and for T-B cooperation. The macrophages and all the Ia+ tumor lines tested induced the release of lymphokines from T cells in a major histocompatibility complex (MHC)-restricted fashion and reconstituted the antibody responses of AC-depleted spleen cells or of purified T and B cells. However, only the normal macrophages but none of the tumor lines induced carrier-specific T helper (Th) cells which help B cells for specific antihapten antibody responses by linked recognition. For T-B cooperation accessory cells were also required, but in contrast to Th cell activation any type of Ia+ AC (e.g. macrophage or tumor line) was effective. Strong MHC-restriction between the lymphocytes and the AC was seen if antigen-pulsed AC were added into the AC-depleted T-B cooperation cultures. If the AC and antigen were concomitantly added to the AC-depleted T-B cultures, MHC-restriction was less obvious. Concanavalin A supernatant reconstituted the response of AC-depleted T-B cultures provided antigen-specific Th cells and the hapten-carrier conjugate were present. If, however, tumor line-activated T cells were added instead of macrophage-induced Th cells, no cooperation with B cells took place even in the presence of Con A supernatant. The results obtained demonstrate a differential AC requirement for the induction of Th cells depending on the differentiation stage of the Th cells.

Molecular Predictors of 3D Morphogenesis by Breast Cancer Cell Lines in 3D Culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Ju; Chang, Hang; Giricz, Orsi

Correlative analysis of molecular markers with phenotypic signatures is the simplest model for hypothesis generation. In this paper, a panel of 24 breast cell lines was grown in 3D culture, their morphology was imaged through phase contrast microscopy, and computational methods were developed to segment and represent each colony at multiple dimensions. Subsequently, subpopulations from these morphological responses were identified through consensus clustering to reveal three clusters of round, grape-like, and stellate phenotypes. In some cases, cell lines with particular pathobiological phenotypes clustered together (e.g., ERBB2 amplified cell lines sharing the same morphometric properties as the grape-like phenotype). Next, associationsmore » with molecular features were realized through (i) differential analysis within each morphological cluster, and (ii) regression analysis across the entire panel of cell lines. In both cases, the dominant genes that are predictive of the morphological signatures were identified. Specifically, PPAR? has been associated with the invasive stellate morphological phenotype, which corresponds to triple-negative pathobiology. PPAR? has been validated through two supporting biological assays.« less

GS-Nitroxide (JP4-039)-Mediated Radioprotection of Human Fanconi Anemia Cell Lines

PubMed Central

Bernard, Mark E.; Kim, Hyun; Berhane, Hebist; Epperly, Michael W.; Franicola, Darcy; Zhang, Xichen; Houghton, Frank; Shields, Donna; Wang, Hong; Bakkenist, Christopher J.; Frantz, Marie-Celine; Forbeck, Erin M.; Goff, Julie P.; Wipf, Peter; Greenberger, Joel S.

2011-01-01

Fanconi anemia (FA) is an inherited disorder characterized by defective DNA repair and cellular sensitivity to DNA crosslinking agents. Clinically, FA is associated with high risk for marrow failure, leukemia and head and neck squamous cell carcinoma (HNSCC). Radiosensitivity in FA patients compromises the use of total-body irradiation for hematopoietic stem cell transplantation and radiation therapy for HNSCC. A radioprotector for the surrounding tissue would therefore be very valuable during radiotherapy for HNSCC. Clonogenic radiation survival curves were determined for pre- or postirradiation treatment with the parent nitroxide Tempol or JP4-039 in cells of four FA patient-derived cell lines and two transgene-corrected subclonal lines. FancG–/– (PD326) and FancD2–/– (PD20F) patient lines were more sensitive to the DNA crosslinking agent mitomycin C (MMC) than their transgene-restored subclonal cell lines (both P < 0.0001). FancD2–/– cells were more radiosensitive than the transgene restored subclonal cell line (ñ = 2.0 ± 0.7 and 4.7 ± 2.2, respectively, P = 0.03). In contrast, FancG–/– cells were radioresistant relative to the transgene-restored subclonal cell line (ñ = 9.4 ± 1.5 and 2.2 ± 05, respectively, P = 0.001). DNA strand breaks measured by the comet assay correlated with radiosensitivity. Cell lines from a Fanc-C and Fanc-A patients showed radiosensitivity similar to that of Fanc-D2–/– cells. A fluorophore-tagged JP4-039 (BODIPY-FL) analog targeted the mitochondria of the cell lines. Preirradiation or postirradiation treatment with JP4-039 at a lower concentration than Tempol significantly increased the radioresistance and stabilized the antioxidant stores of all cell lines. Tempol increased the toxicity of MMC in FancD2–/– cells. These data provide support for the potential clinical use of JP4-039 for normal tissue radioprotection during chemoradiotherapy in FA patients. PMID:21939290

Estradiol impairs the antiproliferative and proapoptotic effect of Zoledronic acid in hormone sensitive breast cancer cells in vitro.

PubMed

Gschwantler-Kaulich, Daphne; Weingartshofer, Sigrid; Grunt, Thomas W; Mairhofer, Mario; Tan, Yen; Gamper, Jutta; Singer, Christian F

2017-01-01

Zoledronic acid (ZA) has antiresorptive effects and protects from bone metastasis in women with early breast cancer. In addition, in postmenopausal women with endocrine responsive breast cancer ZA prolongs DFS. The exact mechanism is still unclear. We have therefore investigated the effect of increasing concentrations of ZA in breast cancer cell lines in the absence or presence of estradiol to mimic the hormonal environment in vitro. Using assays for cell proliferation (EZ4U, BrdU) and cell death (Annexin/PI), we have analyzed the dose-dependent antiproliferative and pro-apoptotic effects of ZA in two hormone sensitive cell lines (MCF-7 and T47D) and a hormone insensitive, triple negative cell line (MDA-MB-231) in the presence of 0, 1 and 10 nM estradiol. In the absence of estradiol, ZA exerts dose-dependent antiproliferative and pro-apoptotic antitumor effects in both, hormone sensitive (MCF-7, T47D) and -insensitive (MDA-MB-231) breast cancer cell lines (p<0.0001). In the presence of estradiol, the antitumoral effect of ZA was significantly decreased only in the hormone sensitive MCF-7 and T47D cell lines (p = 0.0008 and p = 0.0008, respectively). We have demonstrated that estradiol impairs the antiproliferative and proapoptotic effect of ZA in hormone sensitive, but not in hormone insensitive breast cancer cell lines. Our findings provide a possible explanation for the differential effect of ZA on DFS in pre- and postmenopausal patients with hormone sensitive early breast cancer, which has been demonstrated clinically. We further hypothesize that endocrine insensitive tumors such as triple negative breast cancer (TNBC) should benefit from ZA irrespective of their menopausal status.

CD47 expression in Epstein-Barr virus-associated gastric carcinoma: coexistence with tumor immunity lowering the ratio of CD8+/Foxp3+ T cells.

PubMed

Abe, Hiroyuki; Saito, Ruri; Ichimura, Takashi; Iwasaki, Akiko; Yamazawa, Sho; Shinozaki-Ushiku, Aya; Morikawa, Teppei; Ushiku, Tetsuo; Yamashita, Hiroharu; Seto, Yasuyuki; Fukayama, Masashi

2018-04-01

Epstein-Barr virus-associated gastric carcinoma (EBVaGC) frequently harbors dense lymphocytic infiltration, suggesting a specific microenvironment allowing coexistence with tumor immunity. CD47, which mediates the "do not eat me" signal in innate immunity, is also important in adaptive anti-tumor immunity. We investigated the significance of CD47 in EBVaGC compared with EBV-negative gastric cancer and the correlation with various immune cells. By immunohistochemistry of CD47, high, low, and negative expression was observed in 24, 63, and 12% of EBVaGC (n = 41), while 11, 49, and 39% of EBV-negative gastric cancer (n = 262), respectively, indicating that high expression of CD47 in cancer cells was significantly frequent and increased in EBVaGC (P = 0.043). In contrast to EBV-negative gastric carcinoma in which no significant correlation was observed between CD47 and survival, high expression of CD47 correlated significantly with worse disease-specific survival (P = 0.011) and overall survival (P = 0.013) in EBVaGC. To further clarify the role of CD47 expression in EBVaGC, digital image analysis of immune cell infiltration revealed that high CD47 expression was correlated with a lower ratio of CD8 + /Foxp3 + T cells (P = 0.021), a sensitive indicator of tumor immunity. Thus, CD47 lowers anti-tumor immunity in EBVaGC by finely tuning profile of infiltrating T cells, suggesting that CD47 is an additional target for cancer immunotherapy against this virus-driven gastric cancer.

B cells as accessory cells in a Con A response of a T cell clone.

PubMed

Takeuchi, M; Kakiuchi, T; Taira, S; Nariuchi, H

1987-12-01

Accessory cell (AC) function of B cells was examined in Con A response of a cloned T cell line, 22-9D, which is Thy 1+,L3T4+,Lyt2-,H-2KbDb+ and I-Ab-.22-9D cells produced IL 2 in the presence of Con A without participation of AC. For the initiation of a proliferative response to Con A, the addition of spleen cells or spleen adherent cells was required. B cells as AC were unable to induce the proliferative response. In the presence of culture supernatant of spleen cells stimulated with Con A (CAS), 22-9D cells showed proliferative response to Con A with B cell AC. The response was inhibited by a relevant monoclonal anti-I-A antibody. Although irradiated spleen cells as AC induced IL 2 receptor expression of 22-9D cells in the presence of Con A, B cells were shown to require the addition of unknown factor(s) in CAS, which was suggested to be different from IL 1, IL 2, IL 3, or IFN-gamma, for the induction of the receptor expression on 22-9D cells.

CP-25 Attenuates the Activation of CD4+ T Cells Stimulated with Immunoglobulin D in Human.

PubMed

Wu, Yu-Jing; Chen, Heng-Shi; Chen, Wen-Sheng; Dong, Jin; Dong, Xiao-Jie; Dai, Xing; Huang, Qiong; Wei, Wei

2018-01-01

Researchers have shown that the level of immunoglobulin D (IgD) is often elevated in patients with autoimmune diseases. The possible roles of IgD on the function of human T cell activation are still unclear. Paeoniflorin-6'- O -benzene sulfonate (code: CP-25), the chemistry structural modifications of paeoniflorin, was a novel drug of anti-inflammation and immunomodulation. The aims of this study were to determine if human CD4 + T cells could be activated by IgD via the IgD receptor (IgDR)-Lck pathway and whether the novel compound CP-25 could affect the activation of T cells by regulating Lck. Human CD4 + T cells were purified from peripheral blood mononuclear cells using microbeads. T cell viability and proliferation were detected by Cell Counting Kit-8 and CFSE Cell Proliferation Kit. Cytokines secreted by T cells were assessed with the Quantibody Human Inflammation Array. The binding affinity and expression of IgDR on T cells were detected by flow cytometry, and protein expression of IgDR, Lck, and P-Lck were analyzed by western blot. IgD was shown to bind to IgDR on CD4 + T cells in a concentration-dependent manner and stimulate the activation and proliferation of these cells by enhancing phosphorylation of the activating tyrosine residue of Lck (Tyr 394 ). CP-25 inhibited the IgD-stimulated activation and proliferation of CD4 + T cells, as well as the production of inflammatory cytokines; it was thus suggested that this process might be related to the downregulation of Lck (Tyr 394 ) phosphorylation. These results demonstrate that IgD amplifies the activation of CD4 + T cells, which could be mediated by Lck phosphorylation. Further, CP-25, via its ability to modulate Lck, is a novel potential therapeutic agent for the treatment of human autoimmune diseases.

A differential role for CXCR4 in the regulation of normal versus malignant breast stem cell activity.

PubMed

Ablett, Matthew P; O'Brien, Ciara S; Sims, Andrew H; Farnie, Gillian; Clarke, Robert B

2014-02-15

C-X-C chemokine receptor type 4 (CXCR4) is known to regulate lung, pancreatic and prostate cancer stem cells. In breast cancer, CXCR4 signalling has been reported to be a mediator of metastasis, and is linked to poor prognosis. However its role in normal and malignant breast stem cell function has not been investigated. Anoikis resistant (AR) cells were collected from immortalised (MCF10A, 226L) and malignant (MCF7, T47D, SKBR3) breast cell lines and assessed for stem cell enrichment versus unsorted cells. AR cells had significantly higher mammosphere forming efficiency (MFE) than unsorted cells. The AR normal cells demonstrated increased formation of 3D structures in Matrigel compared to unsorted cells. In vivo, SKBR3 and T47D AR cells had 7- and 130-fold enrichments for tumour formationrespectively, compared with unsorted cells. AR cells contained significantly elevated CXCR4 transcript and protein levels compared to unsorted cells. Importantly, CXCR4 mRNA was higher in stem cell-enriched CD44+/CD24- patient-derived breast cancer cells compared to non-enriched cells. CXCR4 stimulation by its ligand SDF-1 reduced MFE of the normal breast cells lines but increased the MFE in T47D and patient-derived breast cancer cells. CXCR4 inhibition by AMD3100 increased stem cell activity but reduced the self-renewal capacity of the malignant breast cell line T47D. CXCR4+ FACS sorted MCF7 cells demonstrated a significantly increased MFE compared with CXCR4- cells. This significant increase in MFE was further demonstrated in CXCR4 over-expressing MCF7 cells which also had an increase in self-renewal compared to parental cells. A greater reduction in self-renewal following CXCR4 inhibition in the CXCR4 over-expressing cells compared with parental cells was also observed. Our data establish for the first time that CXCR4 signalling has contrasting effects on normal and malignant breast stem cell activity. Here, we demonstrate that CXCR4 signalling specifically regulates breast

Quantitation of CYP1A1 and 1B1 mRNA in polycyclic aromatic hydrocarbon-treated human T-47D and HepG2 cells by a modified bDNA assay using fluorescence detection.

PubMed

Wu, Susan J; Spink, David C; Spink, Barbara C; Kaminsky, Laurence S

2003-01-15

The quantitation of mRNA, essential for assessing mechanisms of enzyme regulation, is normally carried out using reverse transcriptase-polymerase chain reaction (RT-PCR). An alternative method uses a signal-amplification nucleic acid probe assay, which measures RNA directly by the QuantiGene Expression Kit and incorporates branched DNA technology from Bayer and luminometer-based readings of a chemilumigenic alkaline phosphatase substrate. To broaden the utility of this assay, we investigated substitution of a fluorescent substrate, 2'-(2-benzothiazol)-6'-hydroxybenzothiazole phosphate and a fluorometer, and applied the method to quantitation of CYP1A1 and 1B1 mRNA in human T-47D and HepG2 cells following induction by benzo[a]pyrene (B[a]P) and dibenzo[a,h]anthracene (DB[a,h]A). The fluorescence response increased linearly for 200 min without photobleaching and increased linearly (r2=0.997) up to at least 0.2 microg total RNA. The data revealed that at 0.5 and 1.0 microM inducing agent, the induction of CYP1A1 mRNA in HepG2 cells by DB[a,h]A exceeded that by B[a]P by 18- and 6-fold, respectively. In T-47D cells B[a]P induced CYP1A1 mRNA by 23-fold and CYP1B1 mRNA by 3.9-fold. A B[a]P cocontaminant in the environment, arsenite, did not affect B[a]P-induced levels of CYP1A1 or 1B1 mRNA in these cells. The modified analytical system provides a rapid-throughput, reproducible, and less labor-intensive method than RT-PCR for quantifying cellular mRNA levels.

Rapid obtention of stable, bioluminescent tumor cell lines using a tCD2-luciferase chimeric construct

PubMed Central

2011-01-01

Background Bioluminescent tumor cell lines are experimental tools of major importance for cancer investigation, especially imaging of tumors in xenografted animals. Stable expression of exogenous luciferase in tumor cells combined to systemic injection of luciferin provides an excellent signal/background ratio for external optical imaging. Therefore, there is a need to rationalize and speed up the production of luciferase-positive tumor cell lines representative of multiple tumor phenotypes. For this aim we have designed a fusion gene linking the luciferase 2 protein to the c-terminus of a truncated form of the rat CD2 protein (tCD2-luc2). To allow simultaneous assessment of the wild-type luciferase 2 in a context of tCD2 co-expression, we have made a bicistronic construct for concomitant but separate expression of these two proteins (luc2-IRES-tCD2). Both the mono- and bi-cistronic constructs were transduced in lymphoid and epithelial cells using lentiviral vectors. Results The tCD2-luc2 chimera behaves as a type I membrane protein with surface presentation of CD2 epitopes. One of these epitopes reacts with the OX34, a widely spread, high affinity monoclonal antibody. Stably transfected cells are sorted by flow cytometry on the basis of OX34 staining. In vitro and, moreover, in xenografted tumors, the tCD2-luc2 chimera retains a substantial and stable luciferase activity, although not as high as the wild-type luciferase expressed from the luc2-IRES-tCD2 construct. Expression of the tCD2-luc2 chimera does not harm cell and tumor growth. Conclusion Lentiviral transduction of the chimeric tCD2-luc2 fusion gene allows selection of cell clones with stable luciferase expression in less than seven days without antibiotic selection. We believe that it will be helpful to increase the number of tumor cell lines available for in vivo imaging and assessment of novel therapeutic modalities. On a longer term, the tCD2-luc2 chimera has the potential to be expressed from multi

Surfactant protein D delays Fas- and TRAIL-mediated extrinsic pathway of apoptosis in T cells.

PubMed

Djiadeu, Pascal; Kotra, Lakshmi P; Sweezey, Neil; Palaniyar, Nades

2017-05-01

Only a few extracellular soluble proteins are known to modulate apoptosis. We considered that surfactant-associated protein D (SP-D), an innate immune collectin present on many mucosal surfaces, could regulate apoptosis. Although SP-D is known to be important for immune cell homeostasis, whether SP-D affects apoptosis is unknown. In this study we aimed to determine the effects of SP-D on Jurkat T cells and human T cells dying by apoptosis. Here we show that SP-D binds to Jurkat T cells and delays the progression of Fas (CD95)-Fas ligand and TRAIL-TRAIL receptor induced, but not TNF-TNF receptor-mediated apoptosis. SP-D exerts its effects by reducing the activation of initiator caspase-8 and executioner caspase-3. SP-D also delays the surface exposure of phosphatidylserine. The effect of SP-D was ablated by the presence of caspase-8 inhibitor, but not by intrinsic pathway inhibitors. The binding ability of SP-D to dying cells decreases during the early stages of apoptosis, suggesting the release of apoptotic cell surface targets during apoptosis. SP-D also delays FasL-induced death of primary human T cells. SP-D delaying the progression of the extrinsic pathway of apoptosis could have important implications in regulating immune cell homeostasis at mucosal surfaces.

Expression of hepatitis B virus 1.3-fold genome plasmid in an SV40 T-antigen-immortalized mouse hepatic cell line

PubMed Central

Song, Xiu-Guang; Bian, Peng-Fei; Yu, Shu-Li; Zhao, Xiu-Hua; Xu, Wei; Bu, Xue-Hui; Li, Xia; Ma, Li-Xian

2013-01-01

AIM: To investigate the expression of the hepatitis B virus (HBV) 1.3-fold genome plasmid (pHBV1.3) in an immortalized mouse hepatic cell line induced by SV40 T-antigen (SV40T) expression. METHODS: Mouse hepatic cells were isolated from mouse liver tissue fragments from 3-5 d old Kunming mice by the direct collagenase digestion method and cultured in vitro. The pRSV-T plasmid was transfected into mouse hepatic cells to establish an SV40LT-immortalized mouse hepatic cell line. The SV40LT-immortalized mouse hepatic cells were identified and transfected with the pHBV1.3 plasmid. The levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the supernatant were determined by an electrochemiluminescence immunoassay at 24, 48, 72 and 96 h after transfection. The expressions of HBsAg and hepatitis B c antigen (HBcAg) in the cells were investigated by indirect immunofluorescence analysis. The presence of HBV DNA replication intermediates in the transfected cells and viral particles in the supernatant of the transfected cell cultures was monitored using the Southern hybridization assay and transmission electronic microscopy, respectively. RESULTS: The pRSV-T plasmid was used to immortalize mouse hepatocytes and an SV40LT-immortalized mouse hepatic cell line was successfully established. SV40LT-immortalized mouse hepatic cells have the same morphology and growth characteristics as primary mouse hepatic cells can be subcultured and produce albumin and cytokeratin-18 in vitro. Immortalized mouse hepatic cells did not show the characteristics of tumor cells, as alpha-fetoprotein levels were comparable (0.58 ± 0.37 vs 0.61 ± 0.31, P = 0.37). SV40LT-immortalized mouse hepatic cells were then transfected with the pHBV1.3 plasmid, and it was found that the HBV genome replicated in SV40LT-immortalized mouse hepatic cells. The levels of HBsAg and HBeAg continuously increased in the supernatant after the transfection of pHBV1.3, and began to decrease 72 h

Expression of hepatitis B virus 1.3-fold genome plasmid in an SV40 T-antigen-immortalized mouse hepatic cell line.

PubMed

Song, Xiu-Guang; Bian, Peng-Fei; Yu, Shu-Li; Zhao, Xiu-Hua; Xu, Wei; Bu, Xue-Hui; Li, Xia; Ma, Li-Xian

2013-11-28

To investigate the expression of the hepatitis B virus (HBV) 1.3-fold genome plasmid (pHBV1.3) in an immortalized mouse hepatic cell line induced by SV40 T-antigen (SV40T) expression. Mouse hepatic cells were isolated from mouse liver tissue fragments from 3-5 d old Kunming mice by the direct collagenase digestion method and cultured in vitro. The pRSV-T plasmid was transfected into mouse hepatic cells to establish an SV40LT-immortalized mouse hepatic cell line. The SV40LT-immortalized mouse hepatic cells were identified and transfected with the pHBV1.3 plasmid. The levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the supernatant were determined by an electrochemiluminescence immunoassay at 24, 48, 72 and 96 h after transfection. The expressions of HBsAg and hepatitis B c antigen (HBcAg) in the cells were investigated by indirect immunofluorescence analysis. The presence of HBV DNA replication intermediates in the transfected cells and viral particles in the supernatant of the transfected cell cultures was monitored using the Southern hybridization assay and transmission electronic microscopy, respectively. The pRSV-T plasmid was used to immortalize mouse hepatocytes and an SV40LT-immortalized mouse hepatic cell line was successfully established. SV40LT-immortalized mouse hepatic cells have the same morphology and growth characteristics as primary mouse hepatic cells can be subcultured and produce albumin and cytokeratin-18 in vitro. Immortalized mouse hepatic cells did not show the characteristics of tumor cells, as alpha-fetoprotein levels were comparable (0.58 ± 0.37 vs 0.61 ± 0.31, P = 0.37). SV40LT-immortalized mouse hepatic cells were then transfected with the pHBV1.3 plasmid, and it was found that the HBV genome replicated in SV40LT-immortalized mouse hepatic cells. The levels of HBsAg and HBeAg continuously increased in the supernatant after the transfection of pHBV1.3, and began to decrease 72 h after transfection. The

Establishment and characterization of a dairy goat mammary epithelial cell line with human telomerase (hT-MECs).

PubMed

Shi, Huaiping; Shi, Hengbo; Luo, Jun; Wang, Wei; Haile, Abiel B; Xu, Huifen; Li, Jun

2014-07-01

Although research on dairy goat mammary gland have referred extensively to molecular mechanisms, research on lines of dairy goat mammary epithelial cells (MECs) are still rare. This paper sought to establish an immortal MEC line by stable transfection of human telomerase. MECs from a lactating (45 days post-parturition) Xinong Saanen dairy goat were cultured purely and subsequently transfected with a plasmid carrying the sequence of human telomerase. Immortalized MECs by human telomerase (hT-MECs) exhibited a typical cobblestone morphology and activity and expression levels of telomerase resembled that of MCF-7 cells. hT-MECs on passage 42 grew vigorously and 'S' sigmoid curves of growth were observed. Moreover, hT-MECs maintained a normal chromosome modal number of 2n=60, keratin 8 and epithelial membrane antigen (EMA) were evidently expressed, and beta-casein protein was synthesized and secreted. Beta-casein expression was enhanced by prolactin (P<0.05). Lipid droplets were found in hT-MECs, and messenger RNA levels of PPARG, SREBP, FASN, ACC and SCD in hT-MECs (passage 40) were similar to MECs (passage 7). In conclusion, the obtained hT-MEC line retained a normal morphology, growth characteristics, cytogenetics and secretory characteristics as primary MECs. Hence, it can be a representative model cell line, for molecular and functional analysis, of dairy goat MECs for an extended period of time. © 2014 Japanese Society of Animal Science.

Identification of T-cell Receptors Targeting KRAS-mutated Human Tumors

PubMed Central

Wang, Qiong J.; Yu, Zhiya; Griffith, Kayla; Hanada, Ken-ichi; Restifo, Nicholas P.; Yang, James C.

2015-01-01

KRAS is one of the most frequently mutated proto-oncogenes in human cancers. The dominant oncogenic mutations of KRAS are single amino acid substitutions at codon 12, in particular G12D and G12V present in 60–70% of pancreatic cancers and 20–30% of colorectal cancers. The consistency, frequency, and tumor specificity of these “neo-antigens” make them attractive therapeutic targets. Recent data associates T cells that target mutated antigens with clinical immunotherapy responses in patients with metastatic melanoma, lung cancer, or cholangiocarcinoma. Using HLA-peptide prediction algorithms, we noted that HLA-A*11:01 could potentially present mutated KRAS variants. By immunizing HLA-A*11:01 transgenic mice, we generated murine T cells and subsequently isolated T-cell receptors (TCRs) highly reactive to the mutated KRAS variants G12V and G12D. Peripheral blood lymphocytes (PBLs) transduced with these TCRs could recognize multiple HLA-A*11:01+ tumor lines bearing the appropriate KRAS mutations. In a xenograft model of large established tumor, adoptive transfer of these transduced PBLs reactive with an HLA-A*11:01, G12D-mutated pancreatic cell line could significantly reduce its growth in NSG mice (P = 0.002). The success of adoptive transfer of TCR-engineered T cells against melanoma and other cancers support clinical trials with these T cells that recognize mutated KRAS in patients with a variety of common cancer types. PMID:26701267

Protective effect of nuclear factor E2-related factor 2 on inflammatory cytokine response to brominated diphenyl ether-47 in the HTR-8/SVneo human first trimester extravillous trophoblast cell line.

PubMed

Park, Hae-Ryung; Loch-Caruso, Rita

2014-11-15

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants, and BDE-47 is a prevalent PBDE congener detected in human tissues. Exposure to PBDEs has been linked to adverse pregnancy outcomes in humans. Although the underlying mechanisms of adverse birth outcomes are poorly understood, critical roles for oxidative stress and inflammation are implicated. The present study investigated antioxidant responses in a human extravillous trophoblast cell line, HTR-8/SVneo, and examined the role of nuclear factor E2-related factor 2 (Nrf2), an antioxidative transcription factor, in BDE-47-induced inflammatory responses in the cells. Treatment of HTR-8/SVneo cells with 5, 10, 15, and 20μM BDE-47 for 24h increased intracellular glutathione (GSH) levels compared to solvent control. Treatment of HTR-8/SVneo cells with 20μM BDE-47 for 24h induced the antioxidant response element (ARE) activity, indicating Nrf2 transactivation by BDE-47 treatment, and resulted in differential expression of redox-sensitive genes compared to solvent control. Pretreatment with tert-butyl hydroquinone (tBHQ) or sulforaphane, known Nrf2 inducers, reduced BDE-47-stimulated IL-6 release with increased ARE reporter activity, reduced nuclear factor kappa B (NF-κB) reporter activity, increased GSH production, and stimulated expression of antioxidant genes compared to non-Nrf2 inducer pretreated groups, suggesting that Nrf2 may play a protective role against BDE-47-mediated inflammatory responses in HTR-8/SVneo cells. These results suggest that Nrf2 activation significantly attenuated BDE-47-induced IL-6 release by augmentation of cellular antioxidative system via upregulation of Nrf2 signaling pathways, and that Nrf2 induction may be a potential therapeutic target to reduce adverse pregnancy outcomes associated with toxicant-induced oxidative stress and inflammation. Copyright © 2014 Elsevier Inc. All rights reserved.

Estradiol impairs the antiproliferative and proapoptotic effect of Zoledronic acid in hormone sensitive breast cancer cells in vitro

PubMed Central

Weingartshofer, Sigrid; Grunt, Thomas W.; Mairhofer, Mario; Tan, Yen; Gamper, Jutta; Singer, Christian F.

2017-01-01

Background Zoledronic acid (ZA) has antiresorptive effects and protects from bone metastasis in women with early breast cancer. In addition, in postmenopausal women with endocrine responsive breast cancer ZA prolongs DFS. The exact mechanism is still unclear. We have therefore investigated the effect of increasing concentrations of ZA in breast cancer cell lines in the absence or presence of estradiol to mimic the hormonal environment in vitro. Materials and methods Using assays for cell proliferation (EZ4U, BrdU) and cell death (Annexin/PI), we have analyzed the dose-dependent antiproliferative and pro-apoptotic effects of ZA in two hormone sensitive cell lines (MCF-7 and T47D) and a hormone insensitive, triple negative cell line (MDA-MB-231) in the presence of 0, 1 and 10 nM estradiol. Results In the absence of estradiol, ZA exerts dose-dependent antiproliferative and pro-apoptotic antitumor effects in both, hormone sensitive (MCF-7, T47D) and -insensitive (MDA-MB-231) breast cancer cell lines (p<0.0001). In the presence of estradiol, the antitumoral effect of ZA was significantly decreased only in the hormone sensitive MCF-7 and T47D cell lines (p = 0.0008 and p = 0.0008, respectively). Conclusion We have demonstrated that estradiol impairs the antiproliferative and proapoptotic effect of ZA in hormone sensitive, but not in hormone insensitive breast cancer cell lines. Our findings provide a possible explanation for the differential effect of ZA on DFS in pre- and postmenopausal patients with hormone sensitive early breast cancer, which has been demonstrated clinically. We further hypothesize that endocrine insensitive tumors such as triple negative breast cancer (TNBC) should benefit from ZA irrespective of their menopausal status. PMID:28945801

Multi-turn multi-gap transmission line resonators - Concept, design and first implementation at 4.7T and 7T.

PubMed

Frass-Kriegl, Roberta; Laistler, Elmar; Hosseinnezhadian, Sajad; Schmid, Albrecht Ingo; Moser, Ewald; Poirier-Quinot, Marie; Darrasse, Luc; Ginefri, Jean-Christophe

2016-12-01

A novel design scheme for monolithic transmission line resonators (TLRs) is presented - the multi-turn multi-gap TLR (MTMG-TLR) design. The MTMG-TLR design enables the construction of TLRs with multiple turns and multiple gaps. This presents an additional degree of freedom in tuning self-resonant TLRs, as their resonance frequency is fully determined by the coil geometry (e.g. diameter, number of turns, conductor width, etc.). The novel design is evaluated at 4.7T and 7T by simulations and experiments, where it is demonstrated that MTMG-TLRs can be used for MRI, and that the B 1 distribution of MTMG-TLRs strongly depends on the number and distribution of turns. A comparison to conventional loop coils revealed that the B 1 performance of MTMG-TLRs is comparable to a loop coil with the same mean diameter; however, lower 10g SAR values were found for MTMG-TLRs. The MTMG-TLR design is expected to bring most benefits at high static field, where it allows for independent size and frequency selection, which cannot be achieved with standard TLR design. However, it also enables more accurate geometric optimization at low static field. Thereby, the MTMG-TLR design preserves the intrinsic advantages of TLRs, i.e. mechanical flexibility, high SAR efficiency, mass production, and coil miniaturization. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Evaluation of the toxic effects of brominated compounds (BDE-47, 99, 209, TBBPA) and bisphenol A (BPA) using a zebrafish liver cell line, ZFL.

PubMed

Yang, Jie; Chan, King Ming

2015-02-01

The toxic effects of three polybrominated diphenyl ether (PBDE) congeners (BDE-47, -99, and -209), tetrabromobisphenol A (TBBPA) and bisphenol A (BPA), were evaluated by determining their 24h and 96 h median lethal concentrations using a zebrafish liver cell line, ZFL. It was found that BDE-47, BDE-99 and TBBPA showed comparative cytotoxicity within the range of 1.2-4.2 μM, and were more toxic than BPA (367.1 μM at 24 h and 357.6 μM at 96 h). However, BDE-209 induced only 15% lethality with exposures up to 25 μM. The molecular stresses of BDE-47, -99, TBBPA and BPA involved in thyroid hormone (TH) homeostasis and hepatic metabolism were also investigated. Using a reporter gene system to detect zebrafish thyroid hormone receptor β (zfTRβ) transcriptional activity, the median effective concentration of triiodothyronine (T3) was determined to be 9.2×10(-11) M. BDE-47, BDE-99, TBBPA and BPA alone, however, did not exhibit zfTRβ agonistic activity. BPA displayed T3 (0.1 nM) induced zfTRβ antagonistic activity with a median inhibitory concentration of 19.3 μM. BDE-47, BDE-99 and TBBPA displayed no antagonistic effects of T3-induced zfTRβ activity. Target gene expressions were also examined under acute exposures. The significant inhibition of different types of deiodinases by all of the test chemicals indicated TH circulation disruption. All four chemicals, especially BPA, were able to affect transcripts of phase II hepatic metabolizing enzymes (UGT2A1, SULT1) in vitro. In conclusion, the zfTRβ reporter gene system developed here helps delineate an in vitro model to enable the analysis of the TH disruption effects of environmental pollutants in fish. BPA and the brominated compounds tested were able to disrupt the TH system at the gene expression level, probably through the deiodination pathways. Copyright © 2014 Elsevier B.V. All rights reserved.

Expression of Master Regulators of T-cell, Helper T-cell and Follicular Helper T-cell Differentiation in Angioimmunoblastic T-cell Lymphoma.

PubMed

Matsumoto, Yosuke; Nagoshi, Hisao; Yoshida, Mihoko; Kato, Seiichi; Kuroda, Junya; Shimura, Kazuho; Kaneko, Hiroto; Horiike, Shigeo; Nakamura, Shigeo; Taniwaki, Masafumi

2017-11-01

Objective It has been postulated that the normal counterpart of angioimmunoblastic T-cell lymphoma (AITL) is the follicular helper T-cell (TFH). Recent immunological studies have identified several transcription factors responsible for T-cell differentiation. The master regulators associated with T-cell, helper T-cell (Th), and TFH differentiation are reportedly BCL11B, Th-POK, and BCL6, respectively. We explored the postulated normal counterpart of AITL with respect to the expression of the master regulators of T-cell differentiation. Methods We performed an immunohistochemical analysis in 15 AITL patients to determine the expression of the master regulators and several surface markers associated with T-cell differentiation. Results BCL11B was detected in 10 patients (67%), and the surface marker of T-cells (CD3) was detected in all patients. Only 2 patients (13%) expressed the marker of naïve T-cells (CD45RA), but all patients expressed the marker of effector T-cells (CD45RO). Nine patients expressed Th-POK (60%), and 7 (47%) expressed a set of surface antigens of Th (CD4-positive and CD8-negative). In addition, BCL6 and the surface markers of TFH (CXCL13, PD-1, and SAP) were detected in 11 (73%), 8 (53%), 14 (93%), and all patients, respectively. Th-POK-positive/BCL6-negative patients showed a significantly shorter overall survival (OS) than the other patients (median OS: 33.0 months vs. 74.0 months, p=0.020; log-rank test). Conclusion Many of the AITL patients analyzed in this study expressed the master regulators of T-cell differentiation. The clarification of the diagnostic significance and pathophysiology based on the expression of these master regulators in AITL is expected in the future.

3D printed lattices as an activation and expansion platform for T cell therapy.

PubMed

Delalat, Bahman; Harding, Frances; Gundsambuu, Batjargal; De-Juan-Pardo, Elena M; Wunner, Felix M; Wille, Marie-Luise; Jasieniak, Marek; Malatesta, Kristen A L; Griesser, Hans J; Simula, Antonio; Hutmacher, Dietmar W; Voelcker, Nicolas H; Barry, Simon C

2017-09-01

One of the most significant hurdles to the affordable, accessible delivery of cell therapy is the cost and difficulty of expanding cells to clinically relevant numbers. Immunotherapy to prevent autoimmune disease, tolerate organ transplants or target cancer critically relies on the expansion of specialized T cell populations. We have designed 3D-printed cell culture lattices with highly organized micron-scale architectures, functionalized via plasma polymerization to bind monoclonal antibodies that trigger cell proliferation. This 3D technology platform facilitate the expansion of therapeutic human T cell subsets, including regulatory, effector, and cytotoxic T cells while maintaining the correct phenotype. Lentiviral gene delivery to T cells is enhanced in the presence of the lattices. Incorporation of the lattice format into existing cell culture vessels such as the G-Rex system is feasible. This cell expansion platform is user-friendly and expedites cell recovery and scale-up, making it ideal for translating T cell therapies from bench to bedside. Copyright © 2017 Elsevier Ltd. All rights reserved.

Proresolving lipid mediators resolvin D1, resolvin D2, and maresin 1 are critical in modulating T cell responses.

PubMed

Chiurchiù, Valerio; Leuti, Alessandro; Dalli, Jesmond; Jacobsson, Anders; Battistini, Luca; Maccarrone, Mauro; Serhan, Charles N

2016-08-24

Resolution of inflammation is a finely regulated process mediated by specialized proresolving lipid mediators (SPMs), including docosahexaenoic acid (DHA)-derived resolvins and maresins. The immunomodulatory role of SPMs in adaptive immune cells is of interest. We report that D-series resolvins (resolvin D1 and resolvin D2) and maresin 1 modulate adaptive immune responses in human peripheral blood lymphocytes. These lipid mediators reduce cytokine production by activated CD8(+) T cells and CD4(+) T helper 1 (TH1) and TH17 cells but do not modulate T cell inhibitory receptors or abrogate their capacity to proliferate. Moreover, these SPMs prevented naïve CD4(+) T cell differentiation into TH1 and TH17 by down-regulating their signature transcription factors, T-bet and Rorc, in a mechanism mediated by the GPR32 and ALX/FPR2 receptors; they concomitantly enhanced de novo generation and function of Foxp3(+) regulatory T (Treg) cells via the GPR32 receptor. These results were also supported in vivo in a mouse deficient for DHA synthesis (Elovl2(-/-)) that showed an increase in TH1/TH17 cells and a decrease in Treg cells compared to wild-type mice. Additionally, either DHA supplementation in Elovl2(-/-) mice or in vivo administration of resolvin D1 significantly reduced cytokine production upon specific stimulation of T cells. These findings demonstrate actions of specific SPMs on adaptive immunity and provide a new avenue for SPM-based approaches to modulate chronic inflammation. Copyright © 2016, American Association for the Advancement of Science.

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

PubMed

Su, Shan; Cheng, Xinlai; Wink, Michael

2015-09-01

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

CD133 Is Not Suitable Marker for Isolating Melanoma Stem Cells from D10 Cell Line.

PubMed

Rajabi Fomeshi, Motahareh; Ebrahimi, Marzieh; Mowla, Seyed Javad; Firouzi, Javad; Khosravani, Pardis

2016-01-01

Cutaneous melanoma is the most hazardous malignancy of skin cancer with a high mortality rate. It has been reported that cancer stem cells (CSCs) are responsible for malignancy in most of cancers including melanoma. The aim of this study is to compare two common methods for melanoma stem cell enriching; isolating based on the CD133 cell surface marker and spheroid cell culture. In this experimental study, melanoma stem cells were enriched by fluorescence activated cell sorting (FACS) based on the CD133 protein expression and spheroid culture of D10 melanoma cell line,. To determine stemness features, the mRNA expression analysis of ABCG2, c-MYC, NESTIN, OCT4-A and -B genes as well as colony and spheroid formation assays were utilized in unsorted CD133(+), CD133(-) and spheroid cells. Significant differences of the two experimental groups were compared using student's t tests and a two-tailed value of P<0.05 was statistically considered as a significant threshold. Our results demonstrated that spheroid cells had more colony and spheroid forming ability, rather than CD133(+) cells and the other groups. Moreover, melanospheres expressed higher mRNA expression level of ABCG2, c-MYC, NESTIN and OCT4-A com- pared to other groups (P<0.05). Although CD133(+) derived melanoma cells represented stemness fea- tures, our findings demonstrated that spheroid culture could be more effective meth- od to enrich melanoma stem cells.

CD133 Is Not Suitable Marker for Isolating Melanoma Stem Cells from D10 Cell Line

PubMed Central

Rajabi Fomeshi, Motahareh; Ebrahimi, Marzieh; Mowla, Seyed Javad; Firouzi, Javad; Khosravani, Pardis

2016-01-01

Objective Cutaneous melanoma is the most hazardous malignancy of skin cancer with a high mortality rate. It has been reported that cancer stem cells (CSCs) are responsible for malignancy in most of cancers including melanoma. The aim of this study is to compare two common methods for melanoma stem cell enriching; isolating based on the CD133 cell surface marker and spheroid cell culture. Materials and Methods In this experimental study, melanoma stem cells were enriched by fluorescence activated cell sorting (FACS) based on the CD133 protein expression and spheroid culture of D10 melanoma cell line,. To determine stemness features, the mRNA expression analysis of ABCG2, c-MYC, NESTIN, OCT4-A and -B genes as well as colony and spheroid formation assays were utilized in unsorted CD133+, CD133- and spheroid cells. Significant differences of the two experimental groups were compared using student’s t tests and a two-tailed value of P<0.05 was statistically considered as a significant threshold. Results Our results demonstrated that spheroid cells had more colony and spheroid forming ability, rather than CD133+ cells and the other groups. Moreover, melanospheres expressed higher mRNA expression level of ABCG2, c-MYC, NESTIN and OCT4-A com- pared to other groups (P<0.05). Conclusion Although CD133+ derived melanoma cells represented stemness fea- tures, our findings demonstrated that spheroid culture could be more effective meth- od to enrich melanoma stem cells. PMID:27054115

Induction of CYP1A1 and CYP1B1 by benzo(k)fluoranthene and benzo(a)pyrene in T-47D human breast cancer cells: Roles of PAH interactions and PAH metabolites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spink, David C.; Wu, Susan J.; Spink, Barbara C.

2008-02-01

The interactions of polycyclic aromatic hydrocarbons (PAH) and cytochromes P450 (CYP) are complex; PAHs are enzyme inducers, substrates, and inhibitors. In T-47D breast cancer cells, exposure to 0.1 to 1 {mu}M benzo(k)fluoranthene (BKF) induced CYP1A1/1B1-catalyzed 17{beta}-estradiol (E{sub 2}) metabolism, whereas BKF levels greater than 1 {mu}M inhibited E{sub 2} metabolism. Time course studies showed that induction of CYP1-catalyzed E{sub 2} metabolism persisted after the disappearance of BKF or co-exposed benzo(a)pyrene, suggesting that BKF metabolites retaining Ah receptor agonist activity were responsible for prolonged CYP1 induction. BKF metabolites were shown, through the use of ethoxyresorufin O-deethylase and CYP1A1-promoter-luciferase reporter assays tomore » induce CYP1A1/1B1 in T-47D cells. Metabolites formed by oxidation at the C-2/C-3 region of BKF had potencies for CYP1 induction exceeding those of BKF, whereas C-8/C-9 oxidative metabolites were somewhat less potent than BKF. The activities of expressed human CYP1A1 and 1B1 with BKF as substrate were investigated by use of HPLC with fluorescence detection, and by GC/MS. The results showed that both enzymes efficiently catalyzed the formation of 3-, 8-, and 9-OHBKF from BKF. These studies indicate that the inductive effects of PAH metabolites as potent CYP1 inducers are likely to be additional important factors in PAH-CYP interactions that affect metabolism and bioactivation of other PAHs, ultimately modulating PAH toxicity and carcinogenicity.« less

Growth Inhibition of Osteosarcoma Cell Lines in 3D Cultures: Role of Nitrosative and Oxidative Stress.

PubMed

Gorska, Magdalena; Krzywiec, Pawel Bieniasz; Kuban-Jankowska, Alicja; Zmijewski, Michal; Wozniak, Michal; Wierzbicka, Justyna; Piotrowska, Anna; Siwicka, Karolina

2016-01-01

3D cell cultures have revolutionized the understanding of cell behavior, allowing culture of cells with the possibility of resembling in vivo intercellular signaling and cell-extracellular matrix interaction. The effect of limited oxygen penetration into 3D culture of highly metastatic osteosarcoma 143B cells in terms of expression of nitro-oxidative stress markers was investigated and compared to standard 2D cell culture. Human osteosarcoma (143B cell line) cells were cultured as monolayers, in collagen and Matrigel. Cell viability, gene expression of nitro-oxidative stress markers, and vascular endothelial growth factor were determined using Trypan blue assay, quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Three-dimensional environments modify nitro-oxidative stress and influence gene expression and cell proliferation of OS 143B cells. Commercial cell lines might not constitute a good model of 3D cultures for bone tissue engineering, as they are highly sensitive to hypoxia, and hypoxic conditions can induce oxidation of the cellular environment. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

MHC class II/ESO tetramer-based generation of in vitro primed anti-tumor T-helper lines for adoptive cell therapy of cancer.

PubMed

Poli, Caroline; Raffin, Caroline; Dojcinovic, Danijel; Luescher, Immanuel; Ayyoub, Maha; Valmori, Danila

2013-02-01

Generation of tumor-antigen specific CD4(+) T-helper (T(H)) lines through in vitro priming is of interest for adoptive cell therapy of cancer, but the development of this approach has been limited by the lack of appropriate tools to identify and isolate low frequency tumor antigen-specific CD4(+) T cells. Here, we have used recently developed MHC class II/peptide tetramers incorporating an immunodominant peptide from NY-ESO-1 (ESO), a tumor antigen frequently expressed in different human solid and hematologic cancers, to implement an in vitro priming platform allowing the generation of ESO-specific T(H) lines. We isolated phenotypically defined CD4(+) T-cell subpopulations from circulating lymphocytes of DR52b(+) healthy donors by flow cytometry cell sorting and stimulated them in vitro with peptide ESO(119-143), autologous APC and IL-2. We assessed the frequency of ESO-specific cells in the cultures by staining with DR52b/ESO(119-143) tetramers (ESO-tetramers) and TCR repertoire of ESO-tetramer(+) cells by co-staining with TCR variable β chain (BV) specific antibodies. We isolated ESO-tetramer(+) cells by flow cytometry cell sorting and expanded them with PHA, APC and IL-2 to generate ESO-specific T(H) lines. We characterized the lines for antigen recognition, by stimulation with ESO peptide or recombinant protein, cytokine production, by intracellular staining using specific antibodies, and alloreactivity, by stimulation with allo-APC. Using this approach, we could consistently generate ESO-tetramer(+) T(H) lines from conventional CD4(+)CD25(-) naïve and central memory populations, but not from effector memory populations or CD4(+)CD25(+) Treg. In vitro primed T(H) lines recognized ESO with affinities comparable to ESO-tetramer(+) cells from patients immunized with an ESO vaccine and used a similar TCR repertoire. In this study, using MHC class II/ESO tetramers, we have implemented an in vitro priming platform allowing the generation of ESO

A BCWD-resistant line of rainbow trout exhibits higher abundance of IgT+ B cells and heavy chain tau transcripts compared to a susceptible line following challenge with Flavobacterium psychrophilum.

PubMed

Zwollo, Patty; Hennessey, Erin; Moore, Catherine; Marancik, David P; Wiens, Gregory D; Epp, Lidia

2017-09-01

Bacterial Cold Water Disease (BCWD) is a common, chronic disease in rainbow trout, and is caused by the gram-negative bacterium Flavobacterium psychrophilum (Fp). Through selective breeding, the National Center for Cool and Cold Water Aquaculture has generated a genetic line that is highly resistant to Fp challenge, designated ARS-Fp-R (or R-line), as well as a susceptible "control" line, ARS-Fp-S (S-line). In previous studies, resistance to Fp had been shown to correlate with naive animal spleen size, and further, naïve R-line trout had been shown to have a lower abundance of IgM + and IgM ++ cells compared to S-line fish. Here we wished to first determine whether the abundance of IgT + and/or IgT ++ cells differed between the two lines in naïve fish, and if so, how these patterns differed after in vivo challenge with Fp. Fp challenge was by intramuscular injection of live Fp and tissue collections were on days 5, 6, and/or 28 post-challenge, in two independent challenge experiments. Flow cytometric and gene expression analyses revealed that naïve R-line fish had a higher abundance of IgT + B cells in their anterior kidney, spleen, and blood, compared to S line fish. Further, that after Fp challenge, this difference was maintained between the two lines. Lastly, abundance of IgT + B cells and expression of secHCtau correlated with lower Fp pathogen loads in challenged fish. In the anterior kidney, IgM + B cell abundance correlated with increased Fp loads. Together, these results suggest that IgT + B lineage cells may have a protective function in the immune response to Fp. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tax Protein-induced Expression of Antiapoptotic Bfl-1 Protein Contributes to Survival of Human T-cell Leukemia Virus Type 1 (HTLV-1)-infected T-cells*♦

PubMed Central

Macaire, Héloïse; Riquet, Aurélien; Moncollin, Vincent; Biémont-Trescol, Marie-Claude; Duc Dodon, Madeleine; Hermine, Olivier; Debaud, Anne-Laure; Mahieux, Renaud; Mesnard, Jean-Michel; Pierre, Marlène; Gazzolo, Louis; Bonnefoy, Nathalie; Valentin, Hélène

2012-01-01

Human T lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL). ATLL is a severe malignancy with no effective treatment. HTLV-1 regulatory proteins Tax and HTLV-1 basic leucine zipper factor (HBZ) play a major role in ATLL development, by interfering with cellular functions such as CD4+ T-cell survival. In this study, we observed that the expression of Bfl-1, an antiapoptotic protein of the Bcl-2 family, is restricted to HTLV-1-infected T-cell lines and to T-cells expressing both Tax and HBZ proteins. We showed that Tax-induced bfl-1 transcription through the canonical NF-κB pathway. Moreover, we demonstrated that Tax cooperated with c-Jun or JunD, but not JunB, transcription factors of the AP-1 family to stimulate bfl-1 gene activation. By contrast, HBZ inhibited c-Jun-induced bfl-1 gene activation, whereas it increased JunD-induced bfl-1 gene activation. We identified one NF-κB, targeted by RelA, c-Rel, RelB, p105/p50, and p100/p52, and two AP-1, targeted by both c-Jun and JunD, binding sites in the bfl-1 promoter of T-cells expressing both Tax and HBZ. Analyzing the potential role of antiapoptotic Bcl-2 proteins in HTLV-1-infected T-cell survival, we demonstrated that these cells are differentially sensitive to silencing of Bfl-1, Bcl-xL, and Bcl-2. Indeed, both Bfl-1 and Bcl-xL knockdowns decreased the survival of HTLV-1-infected T-cell lines, although no cell death was observed after Bcl-2 knockdown. Furthermore, we demonstrated that Bfl-1 knockdown sensitizes HTLV-1-infected T-cells to ABT-737 or etoposide treatment. Our results directly implicate Bfl-1 and Bcl-xL in HTLV-1-infected T-cell survival and suggest that both Bfl-1 and Bcl-xL represent potential therapeutic targets for ATLL treatment. PMID:22553204

Thymocyte Maturation Is Regulated by the Activity of the Helix-Loop-Helix Protein, E47

PubMed Central

Bain, Gretchen; Quong, Melanie W.; Soloff, Rachel S.; Hedrick, Stephen M.; Murre, Cornelis

1999-01-01

The E2A proteins, E12 and E47, are required for progression through multiple developmental pathways, including early B and T lymphopoiesis. Here, we provide in vitro and in vivo evidence demonstrating that E47 activity regulates double-positive thymocyte maturation. In the absence of E47 activity, positive selection of both major histocompatibility complex (MHC) class I– and class II–restricted T cell receptors (TCRs) is perturbed. Additionally, development of CD8 lineage T cells in an MHC class I–restricted TCR transgenic background is sensitive to the dosage of E47. Mice deficient for E47 display an increase in production of mature CD4 and CD8 lineage T cells. Furthermore, ectopic expression of an E2A inhibitor helix-loop-helix protein, Id3, promotes the in vitro differentiation of an immature T cell line. These results demonstrate that E2A functions as a regulator of thymocyte positive selection. PMID:10587351

T Cells Engineered With Chimeric Antigen Receptors Targeting NKG2D Ligands Display Lethal Toxicity in Mice

PubMed Central

VanSeggelen, Heather; Hammill, Joanne A; Dvorkin-Gheva, Anna; Tantalo, Daniela GM; Kwiecien, Jacek M; Denisova, Galina F; Rabinovich, Brian; Wan, Yonghong; Bramson, Jonathan L

2015-01-01

Ligands for the NKG2D receptor are overexpressed on tumors, making them interesting immunotherapy targets. To assess the tumoricidal properties of T cells directed to attack NKG2D ligands, we engineered murine T cells with two distinct NKG2D-based chimeric antigen receptors (CARs): (i) a fusion between the NKG2D receptor and the CD3ζ chain and (ii) a conventional second-generation CAR, where the extracellular domain of NKG2D was fused to CD28 and CD3ζ. To enhance the CAR surface expression, we also engineered T cells to coexpress DAP10. In vitro functionality and surface expression levels of all three CARs was greater in BALB/c T cells than C57BL/6 T cells, indicating strain-specific differences. Upon adoptive transfer of NKG2D-CAR-T cells into syngeneic animals, we observed significant clinical toxicity resulting in morbidity and mortality. The severity of these toxicities varied between the CAR configurations and paralleled their in vitro NKG2D surface expression. BALB/c mice were more sensitive to these toxicities than C57BL/6 mice, consistent with the higher in vitro functionality of BALB/c T cells. Treatment with cyclophosphamide prior to adoptive transfer exacerbated the toxicity. We conclude that while NKG2D ligands may be useful targets for immunotherapy, the pursuit of NKG2D-based CAR-T cell therapies should be undertaken with caution. PMID:26122933

Sensitization of breast cancer cells to doxorubicin via stable cell line generation and overexpression of DFF40.

PubMed

Bagheri, Fatemeh; Safarian, Shahrokh; Eslaminejad, Mohamadreza Baghaban; Sheibani, Nader

2015-12-01

There are a number of reports demonstrating a relationship between the alterations in DFF40 expression and development of some cancers. Here, increased DFF40 expression in T-47D cells in the presence of doxorubicin was envisaged for therapeutic usage. The T-47D cells were transfected with an eukaryotic expression vector encoding the DFF40 cDNA. Following incubation with doxorubicin, propidium iodide (PI) staining was used for cell cycle distribution analysis. The rates of apoptosis were determined by annexin V/PI staining. Apoptosis was also evaluated using the DNA laddering analysis. The viability of DFF40-transfected cells incubated with doxorubicin was significantly decreased compared with control cells. However, there were no substantial changes in the cell cycle distribution of pIRES2-DFF40 cells incubated with doxorubicin compared to control cells. The expression of DFF40, without doxorubicin incubation, had also no significant effect on the cell cycle distribution. There was no DNA laddering in cells transfected with the empty pIRES2 vector when incubated with doxorubicin. In contrast, DNA laddering was observed in DFF40 transfected cells in the presence of doxorubicin after 48 h. Also, the expression of DFF40 and DFF45 was increased in DFF40 transfected cells in the presence of doxorubicin enhancing cell death. Collectively our results indicated that co-treatment of DFF40-transfected cells with doxorubicin can enhance the killing of these tumor cells via apoptosis. Thus, modulation of DFF40 level may be a beneficial strategy for treatment of chemo-resistant cancers.

T Cell-Mediated Immunity towards Yellow Fever Virus and Useful Animal Models.

PubMed

Watson, Alan M; Klimstra, William B

2017-04-11

The 17D line of yellow fever virus vaccines is among the most effective vaccines ever created. The humoral and cellular immunity elicited by 17D has been well characterized in humans. Neutralizing antibodies have long been known to provide protection against challenge with a wild-type virus. However, a well characterized T cell immune response that is robust, long-lived and polyfunctional is also elicited by 17D. It remains unclear whether this arm of immunity is protective following challenge with a wild-type virus. Here we introduce the 17D line of yellow fever virus vaccines, describe the current state of knowledge regarding the immunity directed towards the vaccines in humans and conclude with a discussion of animal models that are useful for evaluating T cell-mediated immune protection to yellow fever virus.

Activation of T cells recognizing self 60-kD heat shock protein can protect against experimental arthritis

PubMed Central

1995-01-01

Lewis rats are susceptible to several forms of experimental arthritis- induced using heat-killed Mycobacterium tuberculosis (adjuvant arthritis, or AA), streptococcal cell walls, collagen type II, and the lipoidal amine CP20961. Prior immunization with the mycobacterial 65-kD heat shock protein (hsp65) was reported to protect against AA, and other athritis models not using M. tuberculosis, via a T cell-mediated mechanism. Hsp65 shares 48% amino acid identity with mammalian hsp60, which is expressed at elevated levels in inflamed synovia. Several studies have reported cross-reactive T cell recognition of mycobacterial hsp65 and self hsp60 in arthritic and normal individuals. We previously described nine major histocompatibility complex class II- restricted epitopes in mycobacterial hsp65 recognized by Lewis rat T cells. Of these only one, covering the 256-270 sequence, primed for cross-reactive T cell responses to the corresponding region of rat hsp60. Here we have tested each hsp65 epitope for protective activity by immunizing rats with synthetic peptides. A peptide containing the 256-270 epitope, which induced cross-reactive T cells, was the only one able to confer protection against AA. Similarly, administration of a T cell line specific for this epitope protected against AA. Preimmunization with the 256-270 epitope induced T cells that responded to heat-shocked syngeneic antigen-presenting cells, and also protected against CP20961-induced arthritis, indicating that activation of T cells, recognizing an epitope in self hsp60 can protect against arthritis induced without mycobacteria. Therefore, in contrast to the accepted concept that cross-reactive T cell recognition of foreign and self antigens might induce aggressive autoimmune disease, we propose that cross-reactivity between bacterial and self hsp60 might also be used to maintain a protective self-reactive T cell population. This discovery might have important implications for understanding T cell- mediated

Influence of calcitriol on prostaglandin- and vitamin D-metabolising enzymes in benign and malignant breast cell lines.

PubMed

Thill, Marc; Cordes, Tim; Hoellen, Friederike; Becker, Steffi; Dittmer, Christine; Kümmel, Sherko; Salehin, Darius; Friedrich, Michael; Diedrich, Klaus; Köster, Frank

2012-01-01

Cyclooxygenase-2 (COX-2) is a potential molecular prognostic factor for breast cancer, and calcitriol [1,25(OH)(2)D(3)], the biologically active form of vitamin D, is a promising target in breast cancer therapy. The influence of calcitriol on the proliferation and the effects of calcitriol on the expression of prostaglandin- and vitamin D-metabolising enzymes were examined in benign and malignant breast cells. Calcitriol inhibited the proliferation of MCF-10F and MCF-7 cells but not of invasive MDA-MB-231 cells and reduced the expression of COX-2 and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in the benign breast cell line MCF-10F. Furthermore, dysregulation in vitamin D-metabolising proteins was detected, especially in MDA-MB-231 cells. These results suggest dysregulation of vitamin D metabolism and a lack of a possible influence of calcitriol on the metabolism of prostaglandins in the malignant breast cell lines.

CAR T cell therapy for breast cancer: harnessing the tumor milieu to drive T cell activation.

PubMed

Bajgain, Pradip; Tawinwung, Supannikar; D'Elia, Lindsey; Sukumaran, Sujita; Watanabe, Norihiro; Hoyos, Valentina; Lulla, Premal; Brenner, Malcolm K; Leen, Ann M; Vera, Juan F

2018-05-10

The adoptive transfer of T cells redirected to tumor via chimeric antigen receptors (CARs) has produced clinical benefits for the treatment of hematologic diseases. To extend this approach to breast cancer, we generated CAR T cells directed against mucin1 (MUC1), an aberrantly glycosylated neoantigen that is overexpressed by malignant cells and whose expression has been correlated with poor prognosis. Furthermore, to protect our tumor-targeted cells from the elevated levels of immune-inhibitory cytokines present in the tumor milieu, we co-expressed an inverted cytokine receptor linking the IL4 receptor exodomain with the IL7 receptor endodomain (4/7ICR) in order to transform the suppressive IL4 signal into one that would enhance the anti-tumor effects of our CAR T cells at the tumor site. First (1G - CD3ζ) and second generation (2G - 41BB.CD3ζ) MUC1-specific CARs were constructed using the HMFG2 scFv. Following retroviral transduction transgenic expression of the CAR±ICR was assessed by flow cytometry. In vitro CAR/ICR T cell function was measured by assessing cell proliferation and short- and long-term cytotoxic activity using MUC1+ MDA MB 468 cells as targets. In vivo anti-tumor activity was assessed using IL4-producing MDA MB 468 tumor-bearing mice using calipers to assess tumor volume and bioluminescence imaging to track T cells. In the IL4-rich tumor milieu, 1G CAR.MUC1 T cells failed to expand or kill MUC1+ tumors and while co-expression of the 4/7ICR promoted T cell expansion, in the absence of co-stimulatory signals the outgrowing cells exhibited an exhausted phenotype characterized by PD-1 and TIM3 upregulation and failed to control tumor growth. However, by co-expressing 2G CAR.MUC1 (signal 1 - activation + signal 2 - co-stimulation) and 4/7ICR (signal 3 - cytokine), transgenic T cells selectively expanded at the tumor site and produced potent and durable tumor control in vitro and in vivo. Our findings demonstrate the feasibility of targeting breast

Phase II enzyme induction by a carotenoid, lutein, in a PC12D neuronal cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miyake, Seiji; Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582; Wakasa Seikatsu Co., Ltd., 134 Chudoujiminami-cho, Shimogyo-ku, Kyoto 600-8813

Highlights: • Lutein reduced ROS levels in a PC12D neuronal cell line. • Lutein induced mRNAs of phase II antioxidative enzymes in PC12D neuronal cells. • Lutein increased protein levels of HO-1, SOD2, and NQO-1 in PC12D neuronal cells. • Lutein had no effect on intranuclear Nrf2 levels in PC12D neuronal cells. • Lutein did not activate potential upstream Nrf2 nuclear translocation pathways. - Abstract: The mechanism by which lutein, a carotenoid, acts as an antioxidant in retinal cells is still not fully understood. Here, lutein treatment of a neuronal cell line (PC12D) immediately resulted in reduced intracellular ROS levels,more » implying that it has a direct role in ROS scavenging. Significantly, lutein treatment also induced phase II antioxidative enzyme expression, probably via a nuclear factor-like 2 (Nrf2) independent pathway. This latter mechanism could explain why lutein acts diversely to protect against oxidative/cytotoxic stress, and why it is physiologically involved in the human neural tissue, such as the retina.« less

Low-frequency, Raman-active vibrational modes of poly(dA).poly(dT)

NASA Astrophysics Data System (ADS)

Liu, C.; Edwards, G. S.; Morgan, S.; Silberman, E.

1989-12-01

The Raman activity of low-frequency (20-300 cm-1) vibrational modes of dehydrated, oriented fibers of the sodium salts of poly(dA).poly(dT) and random sequenced DNA have been measured. Distinct bands near 60, 75-100, and 125-140 cm-1 are resolved in poly(dA).poly(dT). The Raman activity of the two lowest bands correlate with the previously observed infrared activity of poly(dA).poly(dT). The apparent reduction in spectral line broadening for poly(dA).poly(dT), as demonstrated by this and previous measurements of a number of different polynucleotides, is considered as possible evidence for inhomogeneous line broadening.

3D printing of biomimetic microstructures for cancer cell migration.

PubMed

Huang, Tina Qing; Qu, Xin; Liu, Justin; Chen, Shaochen

2014-02-01

To understand the physical behavior and migration of cancer cells, a 3D in vitro micro-chip in hydrogel was created using 3D projection printing. The micro-chip has a honeycomb branched structure, aiming to mimic 3D vascular morphology to test, monitor, and analyze differences in the behavior of cancer cells (i.e. HeLa) vs. non-cancerous cell lines (i.e. 10 T1/2). The 3D Projection Printing system can fabricate complex structures in seconds from user-created designs. The fabricated microstructures have three different channel widths of 25, 45, and 120 microns wide to reflect a range of blood vessel diameters. HeLa and 10 T1/2 cells seeded within the micro-chip were then analyzed for morphology and cell migration speed. 10 T1/2 cells exhibited greater changes in morphology due to channel size width than HeLa cells; however, channel width had a limited effect on 10 T1/2 cell migration while HeLa cancer cell migration increased as channel width decreased. This physiologically relevant 3D cancer tissue model has the potential to be a powerful tool for future drug discoveries and cancer migration studies.

47 CFR 32.5040 - Private line revenue.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 47 Telecommunication 2 2014-10-01 2014-10-01 false Private line revenue. 32.5040 Section 32.5040 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES UNIFORM SYSTEM OF ACCOUNTS FOR TELECOMMUNICATIONS COMPANIES Instructions For Revenue Accounts § 32.5040 Private line revenue...

47 CFR 32.5040 - Private line revenue.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 47 Telecommunication 2 2011-10-01 2011-10-01 false Private line revenue. 32.5040 Section 32.5040 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES UNIFORM SYSTEM OF ACCOUNTS FOR TELECOMMUNICATIONS COMPANIES Instructions For Revenue Accounts § 32.5040 Private line revenue...

The T-cell-specific adapter protein family: TSAd, ALX, and SH2D4A/SH2D4B.

PubMed

Lapinski, Philip E; Oliver, Jennifer A; Bodie, Jennifer N; Marti, Francesc; King, Philip D

2009-11-01

Adapter proteins play key roles in intracellular signal transduction through complex formation with catalytically active signaling molecules. In T lymphocytes, the role of several different types of adapter proteins in T-cell antigen receptor signal transduction is well established. An exception to this is the family of T-cell-specific adapter (TSAd) proteins comprising of TSAd, adapter protein of unknown function (ALX), SH2D4A, and SH2D4B. Only recently has the function of these adapters in T-cell signal transduction been explored. Here, we discuss advances in our understanding of the role of this family of adapter proteins in T cells. Their function as regulators of signal transduction in other cell types is also discussed.

CD1d expression on chronic lymphocytic leukemia B cells affects disease progression and induces T cell skewing in CD8 positive and CD4CD8 double negative T cells.

PubMed

Zaborsky, Nadja; Gassner, Franz Josef; Asslaber, Daniela; Reinthaler, Petra; Denk, Ursula; Flenady, Sabine; Hofbauer, Josefina Piñón; Danner, Barbara; Rebhandl, Stefan; Harrer, Andrea; Geisberger, Roland; Greil, Richard; Egle, Alexander

2016-08-02

Chronic lymphocytic leukemia develops within a complex network driven by genetic mutations and microenvironmental interactions. Among the latter a complex interplay with the immune system is established by the clone. Next to a proposed recruitment of support from T and myeloid cells, potential anti-CLL immune reactions need to be subverted. By using TCL1 mice as a CLL model, we show that TCR-Vβ7+ NK1.1+ T cells are overrepresented in this disease model and constitute a main subset of peripheral CD3+ cells with biased TCR usage, showing that these cells account for a major part for T cell skewing in TCL1 mice. Moreover, we show that overrepresentation is dependent on CD1d expression in TCL1 mice, implicating that these cells belong to a NKT-like cell fraction which are restricted to antigen presented by the MHC-like surface marker CD1d. Accordingly, we observed a high fraction of CD161+ cells within overrepresented T cells in CLL patients and we found downregulation of CD1d on the surface of CLL cells, both in TCL1 mice and patients. Finally, we show that in TCL1 mice, CD1d deficiency resulted in shortened overall survival. Our results point to an interaction between CLL and CD161+ T cells that may represent a novel therapeutic target for immune modulation.

MELK as a potential target to control cell proliferation in triple-negative breast cancer MDA-MB-231 cells

PubMed Central

Li, Gang; Yang, Mei; Zuo, Li; Wang, Mei-Xing

2018-01-01

Maternal embryonic leucine zipper kinase (MELK) is an important regulator in tumorigenesis of human breast cancer, and if silenced leads to programmed cell death in specific breast cancer cell lines, including MDA-MB-231 cells. In the present study, RNA interference, proliferation assay and semi-quantification of cell cycle relative proteins were performed to determine the effects of MELK in human breast cancer cells. Data demonstrated that the highest level of MELK protein in the MDA-MB-231 cell line among eight breast cancer cell lines. The sensitivity of MELK small interfering-RNA varied in different breast cancer cell lines, but MELK silencing resulted in marked suppression of proliferation of triple-negative breast cancer (TNBC) and non-TNBC cells. Specific silencing of MELK caused G2 arrest in TNBC MDA-MB-231 and HCC1143 cells, and G1 arrest in non-TNBC T47D and MCF7 cells. Notably, the knockdown of MELK did not induce apoptosis in HCC1143 cells, indicated by the lack of caspase-3 expression. In addition, in response to MELK silencing, cyclin B and cyclin D1 were downregulated in four breast cancer cell lines. Furthermore, the silencing of MELK resulted in the upregulation of p21, p27 and phosphorylated (p)-c-Jun N-terminal kinase (JNK) in HCC1143 TNBC cells, and downregulation of p21 and p-JNK in T47D non-TNBC cells. Additionally, MELK protein was markedly suppressed in non-TNBC cells in response to estrogen deprivation. The findings from the present study suggested that MELK may be a potential target in MDA-MB-231 cells, although genetic knockdown of MELK resulted in inhibitory effects on proliferation of TNBC and non-TNBC cells. MELK exert its effect on different breast cancer cells via arrest of different cell cycle phases and therefore mediated by different mediators, which may be involved in the crosstalk with MELK signaling and with the estrogen receptor signaling pathway. PMID:29805690

Alkylphosphocholines and curcumin induce programmed cell death in cutaneous T-cell lymphoma cell lines.

PubMed

Yosifov, Deyan Y; Kaloyanov, Kaloyan A; Guenova, Margarita L; Prisadashka, Kamelia; Balabanova, Maria B; Berger, Martin R; Konstantinov, Spiro M

2014-01-01

While most patients with early-stage cutaneous T-cell lymphomas (CTCL) have a very good prognosis, the survival of patients with extensive tumour stage and visceral involvement remains extremely poor and necessitates the development of more effective treatment modalities. In this study, we evaluated the in vitro effects of two alkylphosphocholines (APCs, miltefosine and erufosine) and the polyphenolic compound curcumin on 5 human CTCL cell lines (Hut-78, HH, MJ, My-La CD4+ and My-La CD8+). All tested drugs showed considerable cytotoxic activity, as determined by the MTT dye reduction assay. The IC50 values of both APCs ranged from the low micromolar level (Hut-78 cells) to 60-80μM (HH cells). The IC50 values of curcumin ranged from 12 to 24μM. All tested drugs induced apoptosis, as ascertained by morphological changes, DNA fragmentation and activation of caspase cascades. Miltefosine and erufosine induced dephosphorylation of Akt in My-La CD8+ cells and phosphorylation of JNK in Hut-78 and My-La CD8+ cells. APCs increased the level of the autophagic marker LC3B in Hut-78 and MJ cells. Results from co-treatment with autophagy modulators suggested that the cytotoxicity of APCs in CTCL cells is mediated, at least in part, by induction of autophagy. Copyright © 2013 Elsevier Ltd. All rights reserved.

[The expression of Cyclin D1 modulated by somatotropin on human pancreas cancer cell lines Bxpc-3].

PubMed

Li, Fei; Liu, Da-chuan; Sun, Jia-bang

2004-04-07

To observe the growth effect of somatostapin on human pancreas cancer lines Bxpc-3. The Bxpc-3 pancreas cancer cells were treated with Somatotropin. The cells hyperplasia were detected by MTT and were observed apoptosis cells determinated quantitatively by TUNEL, quantify immune fluoresence double marked the proliferation cells and apoptosis cells, the expression of Cyclin D1 detected by immunohistochemical. The growth effect of pancrea cancer cells were limited by 10(-7) M, 10(-8) M, 10(-9) M Somatotropin on 2 day. The limited effect was decreased from 3 day. The cells proliferation were increased by somotostapin on 4day to 5day. The relationship between the expression of Cyclin D1 and apoptosis was negative correlation and the cells hyperplasia was positive correlation in Bxpc-3 cell line. From the cell study we knew the expression of Cyclin D1 reflected the prolefiration of pancreas cancer cells.

Protective effect of nuclear factor E2-related factor 2 on inflammatory cytokine response to brominated diphenyl ether-47 in the HTR-8/SVneo human first trimester extravillous trophoblast cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Hae-Ryung, E-mail: heaven@umich.edu; Loch-Caruso, Rita

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants, and BDE-47 is a prevalent PBDE congener detected in human tissues. Exposure to PBDEs has been linked to adverse pregnancy outcomes in humans. Although the underlying mechanisms of adverse birth outcomes are poorly understood, critical roles for oxidative stress and inflammation are implicated. The present study investigated antioxidant responses in a human extravillous trophoblast cell line, HTR-8/SVneo, and examined the role of nuclear factor E2-related factor 2 (Nrf2), an antioxidative transcription factor, in BDE-47-induced inflammatory responses in the cells. Treatment of HTR-8/SVneo cells with 5, 10, 15, and 20 μM BDE-47more » for 24 h increased intracellular glutathione (GSH) levels compared to solvent control. Treatment of HTR-8/SVneo cells with 20 μM BDE-47 for 24 h induced the antioxidant response element (ARE) activity, indicating Nrf2 transactivation by BDE-47 treatment, and resulted in differential expression of redox-sensitive genes compared to solvent control. Pretreatment with tert-butyl hydroquinone (tBHQ) or sulforaphane, known Nrf2 inducers, reduced BDE-47-stimulated IL-6 release with increased ARE reporter activity, reduced nuclear factor kappa B (NF-κB) reporter activity, increased GSH production, and stimulated expression of antioxidant genes compared to non-Nrf2 inducer pretreated groups, suggesting that Nrf2 may play a protective role against BDE-47-mediated inflammatory responses in HTR-8/SVneo cells. These results suggest that Nrf2 activation significantly attenuated BDE-47-induced IL-6 release by augmentation of cellular antioxidative system via upregulation of Nrf2 signaling pathways, and that Nrf2 induction may be a potential therapeutic target to reduce adverse pregnancy outcomes associated with toxicant-induced oxidative stress and inflammation. - Highlights: • BDE-47 stimulated ARE reporter activity and GSH production. • BDE-47 resulted in differential

T Cell-Mediated Immunity towards Yellow Fever Virus and Useful Animal Models

PubMed Central

Watson, Alan M.; Klimstra, William B.

2017-01-01

The 17D line of yellow fever virus vaccines is among the most effective vaccines ever created. The humoral and cellular immunity elicited by 17D has been well characterized in humans. Neutralizing antibodies have long been known to provide protection against challenge with a wild-type virus. However, a well characterized T cell immune response that is robust, long-lived and polyfunctional is also elicited by 17D. It remains unclear whether this arm of immunity is protective following challenge with a wild-type virus. Here we introduce the 17D line of yellow fever virus vaccines, describe the current state of knowledge regarding the immunity directed towards the vaccines in humans and conclude with a discussion of animal models that are useful for evaluating T cell-mediated immune protection to yellow fever virus. PMID:28398253

Retinoids Modulate Expression of the Endocytic Partners Megalin, Cubilin, and Disabled-2 and Uptake of Vitamin D-Binding Protein in Human Mammary Cells12

PubMed Central

Chlon, Timothy M.; Taffany, David A.; Welsh, JoEllen; Rowling, Matthew J.

2008-01-01

The major circulating form of vitamin D, 25-hydroxycholecalciferol (25D3), circulates bound to vitamin D-binding protein (DBP). Prior to activation to 1,25-dihydroxycholecalciferol in the kidney, the 25D3-DBP complex is internalized via receptor-mediated endocytosis, which is absolutely dependent on the membrane receptors megalin and cubilin and the adaptor protein disabled-2 (Dab2). We recently reported that mammary epithelial cells (T-47D) expressing megalin, cubilin, and Dab2 rapidly internalize DBP via endocytosis, whereas cells that do not express all 3 proteins (MCF-7) do not. The objectives of this study were to characterize megalin, cubilin, and Dab2 expression and transport of DBP in human mammary epithelial cells. Using immunoblotting and real-time PCR, we found that megalin, cubilin, and Dab2 were expressed and dose dependently induced by all-trans-retinoic acid (RA) in T-47D human breast cancer cells and that RA-treated T-47D cells exhibited enhanced DBP internalization. These are the first studies to our knowledge to demonstrate that mammary epithelial cells express megalin, cubilin, and Dab2, which are enhanced during differentiation and may explain, at least in part, our finding that receptor-mediated endocytosis of DBP is upregulated in differentiated mammary epithelial cells. PMID:18567755

Susceptibility to cytotoxic T cell lysis of cancer stem cells derived from cervical and head and neck tumor cell lines.

PubMed

Liao, Tian; Kaufmann, Andreas M; Qian, Xu; Sangvatanakul, Voramon; Chen, Chao; Kube, Tina; Zhang, Guoyou; Albers, Andreas E

2013-01-01

To explore cancer stem cell susceptibility to a host's cytotoxic T lymphocyte (CTL)-mediated immune response. We compared the susceptibility of putative CSC generated from cancer cell lines to immunologic recognition and killing by alloantigen-specific CD8(+) CTL. CSC-enriched spheroid culture-derived cells (SDC) exhibited higher expression of ALDH, ICAM1 and of stem/progenitor cell markers on all 3 tumor cell lines investigated and lower MHC class I on the cervical cancer cell line as compared to their monolayer-derived cells (MDC). The expression of ICAM1 and MHCI was upregulated by IFN-γ treatment. CSC populations were less sensitive to MHC class I-restricted alloantigen-specific CD8(+) CTL lysis as compared to matched MDC. IFN-γ pretreatment resulted in over-proportionally enhanced lysis of SDC. Finally, the subset of ALDH(high) expressing SDC presented more sensitivity toward CD8(+) CTL killing than the ALDH(low) SDC. Tumor therapy resistance has been attributed to cancer stem cells (CSC). We show in vitro susceptibility of CSC to CTL-mediated lysis. Immunotherapy targeting of ALDH(+) CSC may therefore be a promising approach. Our results and method may be helpful for the development and optimization of adjuvants, as here exemplified for INF-γ, for CSC-targeted vaccines, independent of the availability of CSC-specific antigens.

Spa47 is an oligomerization-activated type three secretion system (T3SS) ATPase from Shigella flexneri.

PubMed

Burgess, Jamie L; Jones, Heather B; Kumar, Prashant; Toth, Ronald T; Middaugh, C Russell; Antony, Edwin; Dickenson, Nicholas E

2016-05-01

Gram-negative pathogens often use conserved type three secretion systems (T3SS) for virulence. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into host cells. The protein Spa47 localizes to the base of the apparatus and is speculated to be an ATPase that provides the energy for T3SA formation and secretion. Here, we developed an expression and purification protocol, producing active Spa47 and providing the first direct evidence that Spa47 is a bona fide ATPase. Additionally, size exclusion chromatography and analytical ultracentrifugation identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer. An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47. Interestingly, the mutant maintained a similar oligomerization pattern as active Spa47, but was unable to restore invasion phenotype when used to complement a spa47 null S. flexneri strain. Together, these results identify Spa47 as a Shigella T3SS ATPase and suggest that its activity is linked to oligomerization, perhaps as a regulatory mechanism as seen in some related pathogens. Additionally, Spa47 catalyzed ATP hydrolysis appears to be essential for host cell invasion, providing a strong platform for additional studies dissecting its role in virulence and providing an attractive target for anti-infective agents. © 2016 The Protein Society.

Comparison of Cultivars and Seasonal Variation in Blueberry (Vaccinium Species) Leaf Extract on Adult T-Cell Leukemia Cell Line Growth Suppression.

PubMed

Kai, Hisahiro; Fuse, Takuichi; Kunitake, Hisato; Morishita, Kazuhiro; Matsuno, Koji

2014-06-30

The inhibitory effects of blueberry leaves on the proliferation of adult T-cell leukemia (ATL) cell lines have previously been reported. A comparison of blueberry leaf extracts from different cultivars and seasonal variation were investigated regarding their effects on ATL cell line proliferation. The inhibitory effects of 80% ethanol leaf extracts from different blueberry cultivars collected from April to December in 2006 or 2008 were evaluated using two ATL cell lines. The bioactivities of leaf extracts of rabbit-eye blueberry ( Vaccinium virgatum Aiton; RB species), southern highbush blueberry ( V. spp.; SB species), northern highbush blueberry ( V. corymbosum L.; NB species), and wild blueberry ( V. bracteatum Thunb.; WB species) were compared. Of these, leaves of the RB species collected in December showed a significantly stronger inhibitory effect in both cell lines than the SB, NB, or WB species. These results suggest elevated biosynthesis of ATL-preventative bioactive compounds in the leaves of the RB species before the defoliation season.

Comparison of Cultivars and Seasonal Variation in Blueberry (Vaccinium Species) Leaf Extract on Adult T-Cell Leukemia Cell Line Growth Suppression

PubMed Central

Kai, Hisahiro; Fuse, Takuichi; Kunitake, Hisato; Morishita, Kazuhiro; Matsuno, Koji

2014-01-01

The inhibitory effects of blueberry leaves on the proliferation of adult T-cell leukemia (ATL) cell lines have previously been reported. A comparison of blueberry leaf extracts from different cultivars and seasonal variation were investigated regarding their effects on ATL cell line proliferation. The inhibitory effects of 80% ethanol leaf extracts from different blueberry cultivars collected from April to December in 2006 or 2008 were evaluated using two ATL cell lines. The bioactivities of leaf extracts of rabbit-eye blueberry (Vaccinium virgatum Aiton; RB species), southern highbush blueberry (V. spp.; SB species), northern highbush blueberry (V. corymbosum L.; NB species), and wild blueberry (V. bracteatum Thunb.; WB species) were compared. Of these, leaves of the RB species collected in December showed a significantly stronger inhibitory effect in both cell lines than the SB, NB, or WB species. These results suggest elevated biosynthesis of ATL-preventative bioactive compounds in the leaves of the RB species before the defoliation season. PMID:28933373

A Dual-Modality Herpes Simplex Virus 2 Vaccine for Preventing Genital Herpes by Using Glycoprotein C and D Subunit Antigens To Induce Potent Antibody Responses and Adenovirus Vectors Containing Capsid and Tegument Proteins as T Cell Immunogens.

PubMed

Awasthi, Sita; Mahairas, Gregory G; Shaw, Carolyn E; Huang, Meei-Li; Koelle, David M; Posavad, Christine; Corey, Lawrence; Friedman, Harvey M

2015-08-01

We evaluated a genital herpes prophylactic vaccine containing herpes simplex virus 2 (HSV-2) glycoproteins C (gC2) and D (gD2) to stimulate humoral immunity and UL19 (capsid protein VP5) and UL47 (tegument protein VP13/14) as T cell immunogens. The HSV-2 gC2 and gD2 proteins were expressed in baculovirus, while the UL19 and UL47 genes were expressed from replication-defective adenovirus vectors. Adenovirus vectors containing UL19 and UL47 stimulated human and murine CD4(+) and CD8(+) T cell responses. Guinea pigs were either (i) mock immunized; (ii) immunized with gC2/gD2, with CpG and alum as adjuvants; (iii) immunized with the UL19/UL47 adenovirus vectors; or (iv) immunized with the combination of gC2/gD2-CpG/alum and the UL19/UL47 adenovirus vectors. Immunization with gC2/gD2 produced potent neutralizing antibodies, while UL19 and UL47 also stimulated antibody responses. After intravaginal HSV-2 challenge, the mock and UL19/UL47 adenovirus groups developed severe acute disease, while 2/8 animals in the gC2/gD2-only group and none in the combined group developed acute disease. No animals in the gC2/gD2 or combined group developed recurrent disease; however, 5/8 animals in each group had subclinical shedding of HSV-2 DNA, on 15/168 days for the gC2/gD2 group and 13/168 days for the combined group. Lumbosacral dorsal root ganglia were positive for HSV-2 DNA and latency-associated transcripts for 5/8 animals in the gC2/gD2 group and 2/8 animals in the combined group. None of the differences comparing the gC2/gD2-only group and the combined group were statistically significant. Therefore, adding the T cell immunogens UL19 and UL47 to the gC2/gD2 vaccine did not significantly reduce genital disease and vaginal HSV-2 DNA shedding compared with the excellent protection provided by gC2/gD2 in the guinea pig model. HSV-2 infection is a common cause of genital ulcer disease and a significant public health concern. Genital herpes increases the risk of transmission and

A Dual-Modality Herpes Simplex Virus 2 Vaccine for Preventing Genital Herpes by Using Glycoprotein C and D Subunit Antigens To Induce Potent Antibody Responses and Adenovirus Vectors Containing Capsid and Tegument Proteins as T Cell Immunogens

PubMed Central

Mahairas, Gregory G.; Shaw, Carolyn E.; Huang, Meei-Li; Koelle, David M.; Posavad, Christine; Corey, Lawrence; Friedman, Harvey M.

2015-01-01

ABSTRACT We evaluated a genital herpes prophylactic vaccine containing herpes simplex virus 2 (HSV-2) glycoproteins C (gC2) and D (gD2) to stimulate humoral immunity and UL19 (capsid protein VP5) and UL47 (tegument protein VP13/14) as T cell immunogens. The HSV-2 gC2 and gD2 proteins were expressed in baculovirus, while the UL19 and UL47 genes were expressed from replication-defective adenovirus vectors. Adenovirus vectors containing UL19 and UL47 stimulated human and murine CD4+ and CD8+ T cell responses. Guinea pigs were either (i) mock immunized; (ii) immunized with gC2/gD2, with CpG and alum as adjuvants; (iii) immunized with the UL19/UL47 adenovirus vectors; or (iv) immunized with the combination of gC2/gD2-CpG/alum and the UL19/UL47 adenovirus vectors. Immunization with gC2/gD2 produced potent neutralizing antibodies, while UL19 and UL47 also stimulated antibody responses. After intravaginal HSV-2 challenge, the mock and UL19/UL47 adenovirus groups developed severe acute disease, while 2/8 animals in the gC2/gD2-only group and none in the combined group developed acute disease. No animals in the gC2/gD2 or combined group developed recurrent disease; however, 5/8 animals in each group had subclinical shedding of HSV-2 DNA, on 15/168 days for the gC2/gD2 group and 13/168 days for the combined group. Lumbosacral dorsal root ganglia were positive for HSV-2 DNA and latency-associated transcripts for 5/8 animals in the gC2/gD2 group and 2/8 animals in the combined group. None of the differences comparing the gC2/gD2-only group and the combined group were statistically significant. Therefore, adding the T cell immunogens UL19 and UL47 to the gC2/gD2 vaccine did not significantly reduce genital disease and vaginal HSV-2 DNA shedding compared with the excellent protection provided by gC2/gD2 in the guinea pig model. IMPORTANCE HSV-2 infection is a common cause of genital ulcer disease and a significant public health concern. Genital herpes increases the risk of

Identification of TL-Om1, an Adult T-Cell Leukemia (ATL) Cell Line, as Reference Material for Quantitative PCR for Human T-Lymphotropic Virus 1

PubMed Central

Okuma, Kazu; Yamagishi, Makoto; Yamochi, Tadanori; Firouzi, Sanaz; Momose, Haruka; Mizukami, Takuo; Takizawa, Kazuya; Araki, Kumiko; Sugamura, Kazuo; Yamaguchi, Kazunari; Watanabe, Toshiki

2014-01-01

Quantitative PCR (qPCR) for human T-lymphotropic virus 1 (HTLV-1) is useful for measuring the amount of integrated HTLV-1 proviral DNA in peripheral blood mononuclear cells. Many laboratories in Japan have developed different HTLV-1 qPCR methods. However, when six independent laboratories analyzed the proviral load of the same samples, there was a 5-fold difference in their results. To standardize HTLV-1 qPCR, preparation of a well-defined reference material is needed. We analyzed the integrated HTLV-1 genome and the internal control (IC) genes of TL-Om1, a cell line derived from adult T-cell leukemia, to confirm its suitability as a reference material for HTLV-1 qPCR. Fluorescent in situ hybridization (FISH) showed that HTLV-1 provirus was monoclonally integrated in chromosome 1 at the site of 1p13 in the TL-Om1 genome. HTLV-1 proviral genome was not transferred from TL-Om1 to an uninfected T-cell line, suggesting that the HTLV-1 proviral copy number in TL-Om1 cells is stable. To determine the copy number of HTLV-1 provirus and IC genes in TL-Om1 cells, we used FISH, digital PCR, and qPCR. HTLV-1 copy numbers obtained by these three methods were similar, suggesting that their results were accurate. Also, the ratio of the copy number of HTLV-1 provirus to one of the IC genes, RNase P, was consistent for all three methods. These findings indicate that TL-Om1 cells are an appropriate reference material for HTLV-1 qPCR. PMID:25502533

Differential anti-tumor activity of coriolus versicolor (Yunzhi) extract through p53- and/or Bcl-2-dependent apoptotic pathway in human breast cancer cells.

PubMed

Ho, Cheong-Yip; Kim, Chi-Fai; Leung, Kwok-Nam; Fung, Kwok-Pui; Tse, Tak-Fu; Chan, Helen; Lau, Clara Bik-San

2005-06-01

Coriolus versicolor (CV), also called Yunzhi, has been demonstrated to exert anti-tumor effects on various types of cancer cells, but the underlying mechanism has not been fully elucidated. The present study aimed to evaluate the in vitro anti-tumor activity of a standardized aqueous ethanol extract prepared from CV on four breast cancer cell lines using MTT assay, and test whether the mechanism involves apoptosis induction and modulation of p53 and Bcl-2 protein expressions using cell death detection ELISA, p53 and Bcl-2 ELISAs respectively. Our results demonstrated that the CV extract dose-dependently suppressed the proliferation of three breast tumor cell lines, with ascending order of IC50 values: T-47D, MCF-7, MDA-MB-231, while BT-20 cells were not significantly affected. Tumoricidal activity of the CV extract was found to be comparable to a chemotherapeutic anti-cancer drug, mitomycin C. Nucleosome productions in apoptotic MDA-MB-231, MCF-7 and T-47D cells were significantly augmented in a time-dependent manner and paralleled the anti-proliferative activity of CV extract. Expression of p53 protein was significantly upregulated only in T-47D cells treated with the CV extract in a dose- and time-dependent fashion, but not in MCF-7 (except at 400 mug/ml after 16 h) and MDA-MB-231 cells. The CV extract significantly induced a dose-dependent downregulation of Bcl-2 protein expression in MCF-7 and T-47D cells, but not in MDA-MB-231 cells. These results suggested that apoptosis induction, differentially dependent of p53 and Bcl-2 expressions, might be the possible mechanism of CV extract-mediated cytotoxicity in human breast cancer cells in vitro.

C22-bronchial and T7-alveolar epithelial cell lines of the immortomouse are excellent murine cell culture model systems to study pulmonary peroxisome biology and metabolism.

PubMed

Karnati, Srikanth; Palaniswamy, Saranya; Alam, Mohammad Rashedul; Oruqaj, Gani; Stamme, Cordula; Baumgart-Vogt, Eveline

2016-03-01

In pulmonary research, temperature-sensitive immortalized cell lines derived from the lung of the "immortomouse" (H-2k(b)-tsA58 transgenic mouse), such as C22 club cells and T7 alveolar epithelial cells type II (AECII), are frequently used cell culture models to study CC10 metabolism and surfactant synthesis. Even though peroxisomes are highly abundant in club cells and AECII and might fulfill important metabolic functions therein, these organelles have never been investigated in C22 and T7 cells. Therefore, we have characterized the peroxisomal compartment and its associated gene transcription in these cell lines. Our results show that peroxisomes are highly abundant in C22 and T7 cells, harboring a common set of enzymes, however, exhibiting specific differences in protein composition and gene expression patterns, similar to the ones observed in club cells and AECII in situ in the lung. C22 cells contain a lower number of larger peroxisomes, whereas T7 cells possess more numerous tubular peroxisomes, reflected also by higher levels of PEX11 proteins. Moreover, C22 cells harbor relatively higher amounts of catalase and antioxidative enzymes in distinct subcellular compartments, whereas T7 cells exhibit higher levels of ABCD3 and plasmalogen synthesizing enzymes as well as nuclear receptors of the PPAR family. This study suggest that the C22 and T7 cell lines of the immortomouse lung are useful models to study the regulation and metabolic function of the peroxisomal compartment and its alterations by paracrine factors in club cells and AECII.

Theileria parva antigens recognized by CD8+ T cells show varying degrees of diversity in buffalo-derived infected cell lines.

PubMed

Sitt, Tatjana; Pelle, Roger; Chepkwony, Maurine; Morrison, W Ivan; Toye, Philip

2018-05-06

The extent of sequence diversity among the genes encoding 10 antigens (Tp1-10) known to be recognized by CD8+ T lymphocytes from cattle immune to Theileria parva was analysed. The sequences were derived from parasites in 23 buffalo-derived cell lines, three cattle-derived isolates and one cloned cell line obtained from a buffalo-derived stabilate. The results revealed substantial variation among the antigens through sequence diversity. The greatest nucleotide and amino acid diversity were observed in Tp1, Tp2 and Tp9. Tp5 and Tp7 showed the least amount of allelic diversity, and Tp5, Tp6 and Tp7 had the lowest levels of protein diversity. Tp6 was the most conserved protein; only a single non-synonymous substitution was found in all obtained sequences. The ratio of non-synonymous: synonymous substitutions varied from 0.84 (Tp1) to 0.04 (Tp6). Apart from Tp2 and Tp9, we observed no variation in the other defined CD8+ T cell epitopes (Tp4, 5, 7 and 8), indicating that epitope variation is not a universal feature of T. parva antigens. In addition to providing markers that can be used to examine the diversity in T. parva populations, the results highlight the potential for using conserved antigens to develop vaccines that provide broad protection against T. parva.

Generation of ΔF508-CFTR T84 cell lines by CRISPR/Cas9-mediated genome editing.

PubMed

Chung, Woo Young; Song, Myungjae; Park, Jinhong; Namkung, Wan; Lee, Jinu; Kim, Hyongbum; Lee, Min Goo; Kim, Joo Young

2016-12-01

To provide a simple method to make a stable ΔF508-CFTR-expressing T84 cell line that can be used as an efficient screening model system for ΔF508-CFTR rescue. CFTR knockout cell lines were generated by Cas9 with a single-guide RNA (sgRNA) targeting exon 1 of the CFTR genome, which produced indels that abolished CFTR protein expressions. Next, stable ΔF508-CFTR expression was achieved by genome integration of ΔF508-CFTR via the lentivirus infection system. Finally, we showed functional rescue of ΔF508-CFTR not only by growing the cells at a low temperature, but also incubating with VX-809, a ΔF508-CFTR corrector, in the established T84 cells expressing ΔF508-CFTR. This cell system provides an appropriate screening platform for rescue of ΔF508-CFTR, especially related to protein folding, escaped from endoplasmic-reticulum-associated protein degradation, and membrane transport.

High-dose vitamin D in Addison's disease regulates T-cells and monocytes: A pilot trial.

PubMed

Penna-Martinez, Marissa; Filmann, Natalie; Bogdanou, Dimitra; Shoghi, Firouzeh; Huenecke, Sabine; Schubert, Ralf; Herrmann, Eva; Koehl, Ulrike; Husebye, Eystein S; Badenhoop, Klaus

2018-05-01

On the basis of the immunomodulatory actions of vitamin D (VD), we investigated the effects of high-dose VD therapy over a 3 mo period on the immune response in patients with Addison's disease (AD). This randomized, controlled, crossover trial included 13 patients with AD who received either cholecalciferol (4000 IU/d) for 3 mo followed by 3 mo placebo oil or the sequential alternative placebo followed by verum. Glucocorticoid replacement doses remained stable. The primary outcome measures were changes in 25-hydroxyvitamin D3 (25(OH)D 3 ) levels and immune cells including T helper cells (Th; CD3 + CD4 + ), late-activated Th cells (CD3 + CD4 + HLA-DR + ), regulatory T cells (CD3 + CD4 + CD25 bright CD127 dim/neg ), cytotoxic T cells (Tc; CD3 + CD8 + ), late-activated Tc cells (CD3 + CD8 + HLA-DR + ), and monocytes. The explorative analysis included the correlation of changes with VD-related gene polymorphisms and 21-hydroxylase antibody titers. Ten of 13 patients (77%) were VD deficient. Median 25(OH)D 3 concentrations increased significantly to 41.5 ng/ml (median changes: 19.95 ng/ml; P = 0.0005) after 3 mo of cholecalciferol treatment. Within the T-cells, only the late-activated Th (median changes: 1.6%; P = 0.02) and late-activated Tc cells (median changes: 4.05%; P = 0.03) decreased, whereas monocytes (median changes: 1.05%; P = 0.008) increased after VD therapy. T-cell changes were associated with two polymorphisms (CYP27B1-rs108770012 and VDR-rs10735810), but no changes in the 21-hydroxylase antibody titers were observed. Three months of treatment with cholecalciferol achieved sufficient 25(OH)D 3 levels and can regulate late-activated T-cells and monocytes in patients with AD. Explorative analysis revealed potential genetic contributions. This pilot trial provides novel insights about immunomodulation in AD. Copyright © 2017 Elsevier Inc. All rights reserved.

A Recombinant Fragment of Human Surfactant Protein D induces Apoptosis in Pancreatic Cancer Cell Lines via Fas-Mediated Pathway

PubMed Central

Kaur, Anuvinder; Riaz, Muhammad Suleman; Murugaiah, Valarmathy; Varghese, Praveen Mathews; Singh, Shiv K.; Kishore, Uday

2018-01-01

Human surfactant protein D (SP-D) is a potent innate immune molecule, which is emerging as a key molecule in the recognition and clearance of altered and non-self targets. Previous studies have shown that a recombinant fragment of human SP-D (rfhSP-D) induced apoptosis via p53-mediated apoptosis pathway in an eosinophilic leukemic cell line, AML14.3D10. Here, we report the ability of rfhSP-D to induce apoptosis via TNF-α/Fas-mediated pathway regardless of the p53 status in human pancreatic adenocarcinoma using Panc-1 (p53mt), MiaPaCa-2 (p53mt), and Capan-2 (p53wt) cell lines. Treatment of these cell lines with rfhSP-D for 24 h caused growth arrest in G1 cell cycle phase and triggered transcriptional upregulation of pro-apoptotic factors such as TNF-α and NF-κB. Translocation of NF-κB from the cytoplasm into the nucleus of pancreatic cancer cell lines was observed via immunofluorescence microscopy following treatment with rfhSP-D as compared to the untreated cells. The rfhSP-D treatment caused upregulation of pro-apoptotic marker Fas, as analyzed via qPCR and western blot, which then triggered caspase cascade, as evident from cleavage of caspase 8 and 3 analyzed via western blot at 48 h. The cell number following the rfhSP-D treatment was reduced in the order of Panc-1 (~67%) > MiaPaCa-2 (~60%) > Capan-2 (~35%). This study appears to suggest that rfhSP-D can potentially be used to therapeutically target pancreatic cancer cells irrespective of their p53 phenotype.

A Recombinant Fragment of Human Surfactant Protein D induces Apoptosis in Pancreatic Cancer Cell Lines via Fas-Mediated Pathway.

PubMed

Kaur, Anuvinder; Riaz, Muhammad Suleman; Murugaiah, Valarmathy; Varghese, Praveen Mathews; Singh, Shiv K; Kishore, Uday

2018-01-01

Human surfactant protein D (SP-D) is a potent innate immune molecule, which is emerging as a key molecule in the recognition and clearance of altered and non-self targets. Previous studies have shown that a recombinant fragment of human SP-D (rfhSP-D) induced apoptosis via p53-mediated apoptosis pathway in an eosinophilic leukemic cell line, AML14.3D10. Here, we report the ability of rfhSP-D to induce apoptosis via TNF-α/Fas-mediated pathway regardless of the p53 status in human pancreatic adenocarcinoma using Panc-1 (p53 mt ), MiaPaCa-2 (p53 mt ), and Capan-2 (p53 wt ) cell lines. Treatment of these cell lines with rfhSP-D for 24 h caused growth arrest in G1 cell cycle phase and triggered transcriptional upregulation of pro-apoptotic factors such as TNF-α and NF-κB. Translocation of NF-κB from the cytoplasm into the nucleus of pancreatic cancer cell lines was observed via immunofluorescence microscopy following treatment with rfhSP-D as compared to the untreated cells. The rfhSP-D treatment caused upregulation of pro-apoptotic marker Fas, as analyzed via qPCR and western blot, which then triggered caspase cascade, as evident from cleavage of caspase 8 and 3 analyzed via western blot at 48 h. The cell number following the rfhSP-D treatment was reduced in the order of Panc-1 (~67%) > MiaPaCa-2 (~60%) > Capan-2 (~35%). This study appears to suggest that rfhSP-D can potentially be used to therapeutically target pancreatic cancer cells irrespective of their p53 phenotype.

47 CFR 69.120 - Line information database.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 47 Telecommunication 3 2011-10-01 2011-10-01 false Line information database. 69.120 Section 69...) ACCESS CHARGES Computation of Charges § 69.120 Line information database. (a) A charge that is expressed... from a local exchange carrier database to recover the costs of: (1) The transmission facilities between...

47 CFR 69.120 - Line information database.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 47 Telecommunication 3 2013-10-01 2013-10-01 false Line information database. 69.120 Section 69...) ACCESS CHARGES Computation of Charges § 69.120 Line information database. (a) A charge that is expressed... from a local exchange carrier database to recover the costs of: (1) The transmission facilities between...

47 CFR 69.120 - Line information database.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 47 Telecommunication 3 2014-10-01 2014-10-01 false Line information database. 69.120 Section 69...) ACCESS CHARGES Computation of Charges § 69.120 Line information database. (a) A charge that is expressed... from a local exchange carrier database to recover the costs of: (1) The transmission facilities between...

47 CFR 69.120 - Line information database.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 47 Telecommunication 3 2010-10-01 2010-10-01 false Line information database. 69.120 Section 69...) ACCESS CHARGES Computation of Charges § 69.120 Line information database. (a) A charge that is expressed... from a local exchange carrier database to recover the costs of: (1) The transmission facilities between...

47 CFR 69.120 - Line information database.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 47 Telecommunication 3 2012-10-01 2012-10-01 false Line information database. 69.120 Section 69...) ACCESS CHARGES Computation of Charges § 69.120 Line information database. (a) A charge that is expressed... from a local exchange carrier database to recover the costs of: (1) The transmission facilities between...

Tumor evasion of the immune system by converting CD4+CD25- T cells into CD4+CD25+ T regulatory cells: role of tumor-derived TGF-beta.

PubMed

Liu, Victoria C; Wong, Larry Y; Jang, Thomas; Shah, Ali H; Park, Irwin; Yang, Ximing; Zhang, Qiang; Lonning, Scott; Teicher, Beverly A; Lee, Chung

2007-03-01

CD4+CD25+ T regulatory (T(reg)) cells were initially described for their ability to suppress autoimmune diseases in animal models. An emerging interest is the potential role of T(reg) cells in cancer development and progression because they have been shown to suppress antitumor immunity. In this study, CD4+CD25- T cells cultured in conditioned medium (CM) derived from tumor cells, RENCA or TRAMP-C2, possess similar characteristics as those of naturally occurring T(reg) cells, including expression of Foxp3, a crucial transcription factor of T(reg) cells, production of low levels of IL-2, high levels of IL-10 and TGF-beta, and the ability to suppress CD4+CD25- T cell proliferation. Further investigation revealed a critical role of tumor-derived TGF-beta in converting CD4+CD25- T cells into T(reg) cells because a neutralizing Ab against TGF-beta, 1D11, completely abrogated the induction of T(reg) cells. CM from a nontumorigenic cell line, NRP-152, or irradiated tumor cells did not convert CD4+CD25- T cells to T(reg) cells because they produce low levels of TGF-beta in CM. Finally, we observed a reduced tumor burden in animals receiving 1D11. The reduction in tumor burden correlated with a decrease in tumor-derived TGF-beta. Treatment of 1D11 also reduced the conversion of CD4+ T cells into T(reg) cells and subsequent T(reg) cell-mediated suppression of antitumor immunity. In summary, we have demonstrated that tumor cells directly convert CD4+CD25- T cells to T(reg) cells through production of high levels of TGF-beta, suggesting a possible mechanism through which tumor cells evade the immune system.

Enterolactone: A novel radiosensitizer for human breast cancer cell lines through impaired DNA repair and increased apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bigdeli, Bahareh, E-mail: bhr.bigdeli@ut.ac.ir

Introduction: Radiotherapy is a potent treatment against breast cancer, which is the most commonly diagnosed cancer among women. However, the emergence of radioresistance due to increased DNA repair leads to radiotherapeutic failure. Applying polyphenols combined with radiation is a more promising method leading to better survival. Enterolactone, a phytoestrogenic polyphenol, has been reported to inhibit an important radioresistance signaling pathway, therefore we conjectured that enterolactone could enhance radiosensitivity in breast cancer. To assess this hypothesis, radiation response of enterolactone treated MDA-MB-231 and T47D cell lines and corresponding cellular mechanisms were investigated. Methods: Cytotoxicity of enterolactone was measured via MTT assay.more » Cells were treated with enterolactone before X-irradiation, and clonogenic assay was used to evaluate radiosensitivity. Cell cycle distribution and apoptosis were measured by flow cytometric analysis. In addition, DNA damages and corresponding repair, chromosomal damages, and aberrations were assessed by comet, micronucleus, and cytogenetic assays, respectively. Results: Enterolactone decreased the viability of cells in a concentration- and time dependent manner. Enterolactone significantly enhanced radiosensitivity of cells by abrogating G2/M arrest, impairing DNA repair, and increasing radiation-induced apoptosis. Furthermore, increased chromosomal damages and aberrations were detected in cells treated with enterolactone combined with X-rays than X-ray alone. These effects were more prominent in T47D than MDA-MB-231 cells. Discussion: To our knowledge, this is the first report that enterolactone is a novel radiosensitizer for breast cancer irrespective of estrogen receptor status. Authors propose enterolactone as a candidate for combined therapy to decrease the radiation dose delivered to patients and subsequent side effects. - Highlights: • Enterolactone is proposed to be a novel

Differential Effect of MyD88 Signal in Donor T Cells on Graft-versus-Leukemia Effect and Graft-versus-Host Disease after Experimental Allogeneic Stem Cell Transplantation.

PubMed

Lim, Ji-Young; Ryu, Da-Bin; Lee, Sung-Eun; Park, Gyeongsin; Choi, Eun Young; Min, Chang-Ki

2015-11-01

Despite the presence of toll like receptor (TLR) expression in conventional TCRαβ T cells, the direct role of TLR signaling via myeloid differentiation factor 88 (MyD88) within T lymphocytes on graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect after allogeneic stem cell transplantation (allo-SCT) remains unknown. In the allo-SCT model of C57BL/6 (H-2(b)) → B6D2F1 (H-2(b/d)), recipients received transplants of wild type (WT) T-cell-depleted (TCD) bone marrow (BM) and splenic T cells from either WT or MyD88 deficient (MyD88KO) donors. Host-type (H-2(d)) P815 mastocytoma or L1210 leukemia cells were injected either subcutaneously or intravenously to generate a GVHD/GVL model. Allogeneic recipients of MyD88KO T cells demonstrated a greater tumor growth without attenuation of GVHD severity. Moreover, GVHD-induced GVL effect, caused by increasing the conditioning intensity was also not observed in the recipients of MyD88KO T cells. In vitro, the absence of MyD88 in T cells resulted in defective cytolytic activity to tumor targets with reduced ability to produce IFN-γ or granzyme B, which are known to critical for the GVL effect. However, donor T cell expansion with effector and memory T-cell differentiation were more enhanced in GVHD hosts of MyD88KO T cells. Recipients of MyD88KO T cells experienced greater expansion of Foxp3- and IL4-expressing T cells with reduced INF-γ producing T cells in the spleen and tumor-draining lymph nodes early after transplantation. Taken together, these results highlight a differential role for MyD88 deficiency on donor T-cells, with decreased GVL effect without attenuation of the GVHD severity after experimental allo-SCT.

Selective bispecific T cell recruiting antibody and antitumor activity of adoptive T cell transfer.

PubMed

Kobold, Sebastian; Steffen, Julius; Chaloupka, Michael; Grassmann, Simon; Henkel, Jonas; Castoldi, Raffaella; Zeng, Yi; Chmielewski, Markus; Schmollinger, Jan C; Schnurr, Max; Rothenfußer, Simon; Schendel, Dolores J; Abken, Hinrich; Sustmann, Claudio; Niederfellner, Gerhard; Klein, Christian; Bourquin, Carole; Endres, Stefan

2015-01-01

One bottleneck for adoptive T cell therapy (ACT) is recruitment of T cells into tumors. We hypothesized that combining tumor-specific T cells, modified with a marker antigen and a bispecific antibody (BiAb) that selectively recognizes transduced T cells and tumor cells would improve T cell recruitment to tumors and enhance therapeutic efficacy. SV40 T antigen-specific T cells from T cell receptor (TCR)-I-transgenic mice were transduced with a truncated human epidermal growth factor receptor (EGFR) as a marker protein. Targeting and killing by combined ACT and anti-EGFR-anti-EpCAM BiAb therapy was analyzed in C57Bl/6 mice (n = six to 12 per group) carrying subcutaneous tumors of the murine gastric cancer cell line GC8 (SV40(+) and EpCAM(+)). Anti-EGFR x anti-c-Met BiAb was used for targeting of human tumor-specific T cells to c-Met(+) human tumor cell lines. Differences between experimental conditions were analyzed using the Student's t test, and differences in tumor growth with two-way analysis of variance. Overall survival was analyzed by log-rank test. All statistical tests were two-sided. The BiAb linked EGFR-transduced T cells to tumor cells and enhanced tumor cell lysis. In vivo, the combination of ACT and Biab produced increased T cell infiltration of tumors, retarded tumor growth, and prolonged survival compared with ACT with a control antibody (median survival 95 vs 75 days, P < .001). In human cells, this strategy enhanced recruitment of human EGFR-transduced T cells to immobilized c-Met and recognition of tyrosinase(+) melanoma cells by TCR-, as well as of CEA(+) colon cancer cells by chimeric antigen receptor (CAR)-modified T cells. BiAb recruitment of tumor-specific T cells transduced with a marker antigen to tumor cells may enhance efficacy of ACT. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Vitamin D fails to prevent serum starvation- or staurosporine-induced apoptosis in human and rat osteosarcoma-derived cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Witasp, Erika; Division of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm; Gustafsson, Ann-Catrin

2005-05-13

Previous studies have suggested that 1,25(OH){sub 2}D{sub 3}, the active form of vitamin D{sub 3}, may increase the survival of bone-forming osteoblasts through an inhibition of apoptosis. On the other hand, vitamin D{sub 3} has also been shown to trigger apoptosis in human cancer cells, including osteosarcoma-derived cell lines. In the present study, we show that 1,25(OH){sub 2}D{sub 3} induces a time- and dose-dependent loss of cell viability in the rat osteosarcoma cell line, UMR-106, and the human osteosarcoma cell line, TE-85. We were unable, however, to detect nuclear condensation, phosphatidylserine externalization, or other typical signs of apoptosis in thismore » model. Moreover, 1,25(OH){sub 2}D{sub 3} failed to protect against apoptosis induced by serum starvation or incubation with the protein kinase inhibitor, staurosporine. These in vitro findings are thus at variance with several previous reports in the literature and suggest that induction of or protection against apoptosis of bone-derived cells may not be a primary function of vitamin D{sub 3}.« less

4-1BB regulates NKG2D costimulation in human cord blood CD8+ T cells.

PubMed

Kim, Young-June; Han, Myung-Kwan; Broxmeyer, Hal E

2008-02-01

Ligation of NKG2D, a potent costimulatory receptor, can be either beneficial or detrimental to CD8(+) cytotoxic T cell (CTL) responses. Factors for these diverse NKG2D effects remain elusive. In this study, we demonstrate that 4-1BB, another costimulatory receptor, is an essential regulator of NKG2D in CD8(+) T cells. Costimulation of NKG2D caused down-modulation of NKG2D, but induced 4-1BB expression on the cell surface, even in the presence of TGF-beta1, which inhibits 4-1BB expression. Resulting NKG2D(-)4-1BB(+) cells were activated but still in an immature state with low cytotoxic activity. However, subsequent 4-1BB costimulation induced cytotoxic activity and restored down-modulated NKG2D. The cytotoxic activity and NKG2D expression induced by 4-1BB on NKG2D(+)4-1BB(+) cells were refractory to TGF-beta1 down-modulation. Such 4-1BB effects were enhanced by IL-12. In contrast, in the presence of IL-4, 4-1BB effects were abolished because IL-4 down-modulated NKG2D and 4-1BB expression in cooperation with TGF-beta1, generating another CD8(+) T-cell type lacking both NKG2D and 4-1BB. These NKG2D(-)4-1BB(-) cells were inert and unable to gain cytotoxic activity. Our results suggest that 4-1BB plays a critical role in protecting NKG2D from TGF-beta1-mediated down-modulation. Co-expression of NKG2D and 4-1BB may represent an important biomarker for defining competency of tumor infiltrating CD8(+) T cells.

Generation of integration-free induced pluripotent stem cells (GZHMUi001-A) by reprogramming peripheral blood mononuclear cells from a 47, XXX syndrome patient.

PubMed

Chen, Yuchang; Ou, Zhanhui; Song, Bing; Xian, Yexing; Ouyang, Shuming; Xie, Yuhuan; Xue, Yanting; Sun, Xiaofang

2017-08-01

47, XXX syndrome is one of several sex-chromosomal aneuploidies, and it has an incidence of approximately 1/1000 in newborn females. Because of heterogeneity in X-inactivation, these patients may exhibit a variety of clinical symptoms. Here, we report the generation of an integration-free human induced pluripotent stem cell line (GZHMUi001-A) by using Sendai virus to reprogram peripheral blood mononuclear cells from a 47, XXX syndrome patient with premature ovarian failure. This 47, XXX iPS cell line has characteristics of pluripotent stem cells and is a useful tool for the investigation of this X chromosome aneuploid disease. Copyright © 2017. Published by Elsevier B.V.

Safety and T Cell Modulating Effects of High Dose Vitamin D3 Supplementation in Multiple Sclerosis

PubMed Central

Smolders, Joost; Peelen, Evelyn; Thewissen, Mariëlle; Cohen Tervaert, Jan Willem; Menheere, Paul; Hupperts, Raymond; Damoiseaux, Jan

2010-01-01

Background A poor vitamin D status has been associated with a high disease activity of multiple sclerosis (MS). Recently, we described associations between vitamin D status and peripheral T cell characteristics in relapsing remitting MS (RRMS) patients. In the present study, we studied the effects of high dose vitamin D3 supplementation on safety and T cell related outcome measures. Methodology/Principal Findings Fifteen RRMS patients were supplemented with 20 000 IU/d vitamin D3 for 12 weeks. Vitamin D and calcium metabolism were carefully monitored, and T cell characteristics were studied by flowcytometry. All patients finished the protocol without side-effects, hypercalcaemia, or hypercalciuria. The median vitamin D status increased from 50 nmol/L (31–175) at week 0 to 380 nmol/L (151–535) at week 12 (P<0.001). During the study, 1 patient experienced an exacerbation of MS and was censored from the T cell analysis. The proportions of (naïve and memory) CD4+ Tregs remained unaffected. Although Treg suppressive function improved in several subjects, this effect was not significant in the total cohort (P = 0.143). An increased proportion of IL-10+ CD4+ T cells was found after supplementation (P = 0.021). Additionally, a decrease of the ratio between IFN-γ+ and IL-4+ CD4+ T cells was observed (P = 0.035). Conclusion/Significance Twelve week supplementation of high dose vitamin D3 in RRMS patients was well tolerated and did not induce decompensation of calcium metabolism. The skewing towards an anti-inflammatory cytokine profile supports the evidence on vitamin D as an immune-modulator, and may be used as outcome measure for upcoming randomized placebo-controlled trials. Trial Registration Clinicaltrials.gov NCT00940719 PMID:21179201

CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer

PubMed Central

Weiskopf, Kipp; Jahchan, Nadine S.; Schnorr, Peter J.; Ring, Aaron M.; Maute, Roy L.; Volkmer, Anne K.; Volkmer, Jens-Peter; Liu, Jie; Lim, Jing Shan; Yang, Dian; Seitz, Garrett; Nguyen, Thuyen; Wu, Di; Guerston, Heather; Trapani, Francesca; George, Julie; Poirier, John T.; Gardner, Eric E.; Miles, Linde A.; de Stanchina, Elisa; Lofgren, Shane M.; Vogel, Hannes; Winslow, Monte M.; Dive, Caroline; Thomas, Roman K.; Rudin, Charles M.; van de Rijn, Matt; Majeti, Ravindra; Garcia, K. Christopher; Weissman, Irving L.

2016-01-01

Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers. PMID:27294525

NKL homeobox gene activities in hematopoietic stem cells, T-cell development and T-cell leukemia.

PubMed

Nagel, Stefan; Pommerenke, Claudia; Scherr, Michaela; Meyer, Corinna; Kaufmann, Maren; Battmer, Karin; MacLeod, Roderick A F; Drexler, Hans G

2017-01-01

T-cell acute lymphoblastic leukemia (T-ALL) cells represent developmentally arrested T-cell progenitors, subsets of which aberrantly express homeobox genes of the NKL subclass, including TLX1, TLX3, NKX2-1, NKX2-5, NKX3-1 and MSX1. Here, we analyzed the transcriptional landscape of all 48 members of the NKL homeobox gene subclass in CD34+ hematopoietic stem and progenitor cells (HSPCs) and during lymphopoiesis, identifying activities of nine particular genes. Four of these were expressed in HSPCs (HHEX, HLX1, NKX2-3 and NKX3-1) and three in common lymphoid progenitors (HHEX, HLX1 and MSX1). Interestingly, our data indicated downregulation of NKL homeobox gene transcripts in late progenitors and mature T-cells, a phenomenon which might explain the oncogenic impact of this group of genes in T-ALL. Using MSX1-expressing T-ALL cell lines as models, we showed that HHEX activates while HLX1, NKX2-3 and NKX3-1 repress MSX1 transcription, demonstrating the mutual regulation and differential activities of these homeobox genes. Analysis of a public T-ALL expression profiling data set comprising 117 patient samples identified 20 aberrantly activated members of the NKL subclass, extending the number of known NKL homeobox oncogene candidates. While 7/20 genes were also active during hematopoiesis, the remaining 13 showed ectopic expression. Finally, comparative analyses of T-ALL patient and cell line profiling data of NKL-positive and NKL-negative samples indicated absence of shared target genes but instead highlighted deregulation of apoptosis as common oncogenic effect. Taken together, we present a comprehensive survey of NKL homeobox genes in early hematopoiesis, T-cell development and T-ALL, showing that these genes generate an NKL-code for the diverse stages of lymphoid development which might be fundamental for regular differentiation.

NKL homeobox gene activities in hematopoietic stem cells, T-cell development and T-cell leukemia

PubMed Central

Pommerenke, Claudia; Scherr, Michaela; Meyer, Corinna; Kaufmann, Maren; Battmer, Karin; MacLeod, Roderick A. F.; Drexler, Hans G.

2017-01-01

T-cell acute lymphoblastic leukemia (T-ALL) cells represent developmentally arrested T-cell progenitors, subsets of which aberrantly express homeobox genes of the NKL subclass, including TLX1, TLX3, NKX2-1, NKX2-5, NKX3-1 and MSX1. Here, we analyzed the transcriptional landscape of all 48 members of the NKL homeobox gene subclass in CD34+ hematopoietic stem and progenitor cells (HSPCs) and during lymphopoiesis, identifying activities of nine particular genes. Four of these were expressed in HSPCs (HHEX, HLX1, NKX2-3 and NKX3-1) and three in common lymphoid progenitors (HHEX, HLX1 and MSX1). Interestingly, our data indicated downregulation of NKL homeobox gene transcripts in late progenitors and mature T-cells, a phenomenon which might explain the oncogenic impact of this group of genes in T-ALL. Using MSX1-expressing T-ALL cell lines as models, we showed that HHEX activates while HLX1, NKX2-3 and NKX3-1 repress MSX1 transcription, demonstrating the mutual regulation and differential activities of these homeobox genes. Analysis of a public T-ALL expression profiling data set comprising 117 patient samples identified 20 aberrantly activated members of the NKL subclass, extending the number of known NKL homeobox oncogene candidates. While 7/20 genes were also active during hematopoiesis, the remaining 13 showed ectopic expression. Finally, comparative analyses of T-ALL patient and cell line profiling data of NKL-positive and NKL-negative samples indicated absence of shared target genes but instead highlighted deregulation of apoptosis as common oncogenic effect. Taken together, we present a comprehensive survey of NKL homeobox genes in early hematopoiesis, T-cell development and T-ALL, showing that these genes generate an NKL-code for the diverse stages of lymphoid development which might be fundamental for regular differentiation. PMID:28151996

Evaluating cell lines as tumour models by comparison of genomic profiles

PubMed Central

Domcke, Silvia; Sinha, Rileen; Levine, Douglas A.; Sander, Chris; Schultz, Nikolaus

2013-01-01

Cancer cell lines are frequently used as in vitro tumour models. Recent molecular profiles of hundreds of cell lines from The Cancer Cell Line Encyclopedia and thousands of tumour samples from the Cancer Genome Atlas now allow a systematic genomic comparison of cell lines and tumours. Here we analyse a panel of 47 ovarian cancer cell lines and identify those that have the highest genetic similarity to ovarian tumours. Our comparison of copy-number changes, mutations and mRNA expression profiles reveals pronounced differences in molecular profiles between commonly used ovarian cancer cell lines and high-grade serous ovarian cancer tumour samples. We identify several rarely used cell lines that more closely resemble cognate tumour profiles than commonly used cell lines, and we propose these lines as the most suitable models of ovarian cancer. Our results indicate that the gap between cell lines and tumours can be bridged by genomically informed choices of cell line models for all tumour types. PMID:23839242

Lessons From the First Comprehensive Molecular Characterization of Cell Cycle Control in Rodent Insulinoma Cell Lines

PubMed Central

Cozar-Castellano, Irene; Harb, George; Selk, Karen; Takane, Karen; Vasavada, Rupangi; Sicari, Brian; Law, Brian; Zhang, Pili; Scott, Donald K.; Fiaschi-Taesch, Nathalie; Stewart, Andrew F.

2008-01-01

OBJECTIVE—Rodent insulinoma cell lines may serve as a model for designing continuously replicating human β-cell lines and provide clues as to the central cell cycle regulatory molecules in the β-cell. RESEARCH DESIGN AND METHODS—We performed a comprehensive G1/S proteome analysis on the four most widely studied rodent insulinoma cell lines and defined their flow cytometric profiles and growth characteristics. RESULTS—1) Despite their common T-antigen–derived origins, MIN6 and BTC3 cells display markedly different G1/S expression profiles; 2) despite their common radiation origins, RINm5F and INS1 cells display striking differences in cell cycle protein profiles; 3) phosphorylation of pRb is absent in INS1 and RINm5F cells; 4) cyclin D2 is absent in RINm5F and BTC3 cells and therefore apparently dispensable for their proliferation; 5) every cell cycle inhibitor is upregulated, presumably in a futile attempt to halt proliferation; 6) among the G1/S proteome members, seven are pro-proliferation molecules: cyclin-dependent kinase-1, -2, -4, and -6 and cyclins A, E, and D3; and 7) overexpression of the combination of these seven converts arrested proliferation rates in primary rat β-cells to those in insulinoma cells. Unfortunately, this therapeutic overexpression appears to mildly attenuate β-cell differentiation and function. CONCLUSIONS—These studies underscore the importance of characterizing the cell cycle at the protein level in rodent insulinoma cell lines. They also emphasize the hazards of interpreting data from rodent insulinoma cell lines as modeling normal cell cycle progression. Most importantly, they provide seven candidate targets for inducing proliferation in human β-cells. PMID:18650366

Effect of brominated flame retardant BDE-47 on androgen production of adult rat Leydig cells.

PubMed

Zhao, Yan; Ao, Hong; Chen, Li; Sottas, Chantal M; Ge, Ren-Shan; Zhang, Yunhui

2011-08-28

As one of the most abundant polybrominated diphenylethers (PBDEs) detected in adipose tissue and breast milk of humans, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) is considered as a potential endocrine disruptor. The objective of this study is to explore whether environment-related level of BDE-47 could affect the androgen production in rat Leydig cells. Rat adult Leydig cells (ALCs) were treated with 10(-8) to 10(-4)M BDE-47 in vitro, the production of testosterone (T) and steroidogenic acute regulatory (StAR) protein level were determined. BDE-47 significantly increased basal T production and steroidogenic acute regulatory protein (StAR) level of ALCs after treatment with 10(-4)M BED-47. Overall, LH (0.1ng/ml) stimulated T production in ALCs by 6 folds, however it did not increase T production in BDE-47-treated ALCs when compared to untreated ALC. Both 8-Br-cAMP (for cAMP signaling) and 22R-hydroxycholesterol (22-diol, for P450 cholesterol side chain cleavage enzyme P450scc activity) significantly increased T production in ALCs treated with BDE-47 from 10(-7) to 10(-5)M. The results of this study indicate that environment-related level of BDE-47 in vitro increased T production in a dose-dependent manner. The stimulated effects of BDE-47 on StAR and P450scc might play key roles in BDE-47-mediated stimulation of T production. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Lawrence Transfer Factor: Transference of Specific Immune Memory by Dialyzable Leukocyte Extract from a CD8+ T Cell Line.

PubMed

Wang, Jason F; Park, Andrew J; Rendini, Tina; Levis, William R

2017-12-01

Lawrence transfer factor (TF) is defined as dialyzable leukocyte extract (DLE) that can transfer antigen-specific cell-mediated immunity from a person testing positive for the antigen in a delayed type hypersensitivity skin test manner to a person negative for the same antigen. A recent article by Myles et al1 has identified a DLE isolated from an established CD8+ T cell line capable of transferring antigen-specific immunity. The DLE contains a portion of the beta chain of the T cell receptor and additional nucleotide and protein factors that are being subjected to further modern biochemical analysis. After months of study that included interviews of TF physician-scientists, we conclude that an antigen-specific TF exists for most, if not all, antigens. By working from a CD8+ T cell line with modern biochemical technology, it should be possible to identify and patent products capable of treating infectious diseases, antigen-responsive cancers, and autoimmune disorders.

A prototype methodology combining surface-enhanced laser desorption/ionization protein chip technology and artificial neural network algorithms to predict the chemoresponsiveness of breast cancer cell lines exposed to Paclitaxel and Doxorubicin under in vitro conditions.

PubMed

Mian, Shahid; Ball, Graham; Hornbuckle, Jo; Holding, Finn; Carmichael, James; Ellis, Ian; Ali, Selman; Li, Geng; McArdle, Stephanie; Creaser, Colin; Rees, Robert

2003-09-01

An ability to predict the likelihood of cellular response towards particular chemotherapeutic agents based upon protein expression patterns could facilitate the identification of biological molecules with previously undefined roles in the process of chemoresistance/chemosensitivity, and if robust enough these patterns might also be exploited towards the development of novel predictive assays. To ascertain whether proteomic based molecular profiling in conjunction with artificial neural network (ANN) algorithms could be applied towards the specific recognition of phenotypic patterns between either control or drug treated and chemosensitive or chemoresistant cellular populations, a combined approach involving MALDI-TOF matrix-assisted laser desorption/ionization-time of flight mass spectrometry, Ciphergen protein chip technology and ANN algorithms have been applied to specifically identify proteomic 'fingerprints' indicative of treatment regimen for chemosensitive (MCF-7, T47D) and chemoresistant (MCF-7/ADR) breast cancer cell lines following exposure to Doxorubicin or Paclitaxel. The results indicate that proteomic patterns can be identified by ANN algorithms to correctly assign 'class' for treatment regimen (e.g. control/drug treated or chemosensitive/chemoresistant) with a high degree of accuracy using boot-strap statistical validation techniques and that biomarker ion patterns indicative of response/non-response phenotypes are associated with MCF-7 and MCF-7/ADR cells exposed to Doxorubicin. We have also examined the predictive capability of this approach towards MCF-7 and T47D cells to ascertain whether prediction could be made based upon treatment regimen irrespective of cell lineage. Models were identified that could correctly assign class (control or Paclitaxel treatment) for 35/38 samples of an independent dataset. A similar level of predictive capability was also found (> 92%; n = 28) when proteomic patterns derived from the drug resistant cell line MCF-7

Multiple discrete encephalitogenic epitopes of the autoantigen myelin basic protein include a determinant for I-E class II-restricted T cells

PubMed Central

1988-01-01

Immunization with the autoantigen myelin basic protein (MBP) causes experimental allergic encephalomyelitis (EAE). Initial investigations indicated that encephalitogenic murine determinants of MBP were located only within MBP 1-37 and MBP 89-169. Encephalitogenic T cell epitopes within these fragments have been identified. Each epitope is recognized by T cells in association with separate allelic I-A molecules. A hybrid I-E-restricted T cell clone that recognizes intact mouse (self) MBP has been examined. The epitope recognized by this clone includes MBP residues 35-47. When tested in vivo, p35-47 causes EAE. T cell recognition of p35-47 occurs only in association with I-E molecules. These results provide the first clear example that antigen-specific T cells restricted by I-E class II molecules participate in murine autoimmune disease. Furthermore, it is clear that there are multiple (at least three) discrete encephalitogenic T cell epitopes of this autoantigen, each recognized in association with separate allelic class II molecules. These results may be relevant to human autoimmune diseases whose susceptibility is associated with more than one HLA-D molecule. PMID:2459291

Androgen Triggers the Pro-Migratory CXCL12/CXCR4 Axis in AR-Positive Breast Cancer Cell Lines: Underlying Mechanism and Possible Implications for the Use of Aromatase Inhibitors in Breast Cancer.

PubMed

Azariadis, Kalliopi; Kiagiadaki, Fotini; Pelekanou, Vasiliki; Bempi, Vasiliki; Alexakis, Kostas; Kampa, Marilena; Tsapis, Andreas; Castanas, Elias; Notas, George

2017-01-01

Reports regarding the role of androgen in breast cancer (BC) are conflicting. Some studies suggest that androgen could lead to undesirable responses in the presence of certain BC tumor characteristics. We have shown that androgen induces C-X-C motif chemokine 12 (CXCL12) in BC cell lines. Our aim was to identify the mechanisms regulating the phenotypic effects of androgen-induced CXCL12 on Androgen Receptor (AR) positive BC cell lines. We analyzed the expression of CXCL12 and its receptors with qPCR and ELISA and the role of Nuclear Receptor Coactivator 1 (NCOA1) in this effect. AR effects on the CXCL12 promoter was studied via Chromatin-immunoprecipitation. We also analyzed publically available data from The Cancer Genome Atlas to verify AR-CXCL12 interactions and to identify the effect or Aromatase Inhibitors (AI) therapy on CXCL12 expression and disease progression in AR positive cases. CXCL12 induction occurs only in AR-positive BC cell lines, possibly via an Androgen Response Element, upstream of the CXCL12 promoter. The steroid receptor co-regulator NCOA1 is critical for this effect. Androgen only induced the motility of p53-mutant BC cells T47D cells via upregulation of CXCR4 expression while they had no effect on wild-type p53 MCF-7 cells. Loss of CXCR4 expression and depletion of CXCL12 abolished the effect of androgen in T47D cells while inhibition of p53 expression in MCF-7 cells made them responsive to androgen and increased their motility in the presence to androgen. Patients with estrogen receptor positive (ER+)/AR+ BC treated with AIs were at increased risk of disease progression compared to ER+/AR+ non-AI treated and ER+/AR- AI treated cases. AIs may lead to unfavorable responses in some ER/AR positive BC cases, especially in patients with AR+, p53 mutant tumors. © 2017 The Author(s). Published by S. Karger AG, Basel.

Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): New mechanistic insights

DOE Office of Scientific and Technical Information (OSTI.GOV)

Katika, Madhumohan R.; Department of Health Risk Analysis and Toxicology, Maastricht University; Netherlands Toxicogenomics Centre

Deoxynivalenol (DON) or vomitoxin is a commonly encountered type-B trichothecene mycotoxin, produced by Fusarium species predominantly found in cereals and grains. DON is known to exert toxic effects on the gastrointestinal, reproductive and neuroendocrine systems, and particularly on the immune system. Depending on dose and exposure time, it can either stimulate or suppress immune function. The main objective of this study was to obtain a deeper insight into DON-induced effects on lymphoid cells. For this, we exposed the human T-lymphocyte cell line Jurkat and human peripheral blood mononuclear cells (PBMCs) to various concentrations of DON for various times and examinedmore » gene expression changes by DNA microarray analysis. Jurkat cells were exposed to 0.25 and 0.5 μM DON for 3, 6 and 24 h. Biological interpretation of the microarray data indicated that DON affects various processes in these cells: It upregulates genes involved in ribosome structure and function, RNA/protein synthesis and processing, endoplasmic reticulum (ER) stress, calcium-mediated signaling, mitochondrial function, oxidative stress, the NFAT and NF-κB/TNF-α pathways, T cell activation and apoptosis. The effects of DON on the expression of genes involved in ER stress, NFAT activation and apoptosis were confirmed by qRT-PCR. Other biochemical experiments confirmed that DON activates calcium-dependent proteins such as calcineurin and M-calpain that are known to be involved in T cell activation and apoptosis. Induction of T cell activation was also confirmed by demonstrating that DON activates NFATC1 and induces its translocation from the cytoplasm to the nucleus. For the gene expression profiling of PBMCs, cells were exposed to 2 and 4 μM DON for 6 and 24 h. Comparison of the Jurkat microarray data with those obtained with PBMCs showed that most of the processes affected by DON in the Jurkat cell line were also affected in the PBMCs. -- Highlights: ► The human T cell line Jurkat and

Ligation of TLR7 on CD19(+) CD1d(hi) B cells suppresses allergic lung inflammation via regulatory T cells.

PubMed

Khan, Adnan R; Amu, Sylvie; Saunders, Sean P; Hams, Emily; Blackshields, Gordon; Leonard, Martin O; Weaver, Casey T; Sparwasser, Tim; Sheils, Orla; Fallon, Padraic G

2015-06-01

B cells have been described as having the capacity to regulate cellular immune responses and suppress inflammatory processes. One such regulatory B-cell population is defined as IL-10-producing CD19(+) CD1d(hi) cells. Previous work has identified an expansion of these cells in mice infected with the helminth, Schistosoma mansoni. Here, microarray analysis of CD19(+) CD1d(hi) B cells from mice infected with S. mansoni demonstrated significantly increased Tlr7 expression, while CD19(+) CD1d(hi) B cells from uninfected mice also demonstrated elevated Tlr7 expression. Using IL-10 reporter, Il10(-/-) and Tlr7(-/-) mice, we formally demonstrate that TLR7 ligation of CD19(+) CD1d(hi) B cells increases their capacity to produce IL-10. In a mouse model of allergic lung inflammation, the adoptive transfer of TLR7-elicited CD19(+) CD1d(hi) B cells reduced airway inflammation and associated airway hyperresponsiveness. Using DEREG mice to deplete FoxP3(+) T regulatory cells in allergen-sensitized mice, we show that that TLR7-elicited CD19(+) CD1d(hi) B cells suppress airway hyperresponsiveness via a T regulatory cell dependent mechanism. These studies identify that TLR7 stimulation leads to the expansion of IL-10-producing CD19(+) CD1d(hi) B cells, which can suppress allergic lung inflammation via T regulatory cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A T-cell-directed chimeric antigen receptor for the selective treatment of T-cell malignancies.

PubMed

Mamonkin, Maksim; Rouce, Rayne H; Tashiro, Haruko; Brenner, Malcolm K

2015-08-20

Options for targeted therapy of T-cell malignancies remain scarce. Recent clinical trials demonstrated that chimeric antigen receptors (CARs) can effectively redirect T lymphocytes to eradicate lymphoid malignancies of B-cell origin. However, T-lineage neoplasms remain a more challenging task for CAR T cells due to shared expression of most targetable surface antigens between normal and malignant T cells, potentially leading to fratricide of CAR T cells or profound immunodeficiency. Here, we report that T cells transduced with a CAR targeting CD5, a common surface marker of normal and neoplastic T cells, undergo only limited fratricide and can be expanded long-term ex vivo. These CD5 CAR T cells effectively eliminate malignant T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoma lines in vitro and significantly inhibit disease progression in xenograft mouse models of T-ALL. These data support the therapeutic potential of CD5 CAR in patients with T-cell neoplasms. © 2015 by The American Society of Hematology.

Correlation between erythropoietin receptor(s) and estrogen and progesterone receptor expression in different breast cancer cell lines.

PubMed

Trošt, Nina; Hevir, Neli; Rižner, Tea Lanišnik; Debeljak, Nataša

2013-03-01

Erythropoietin (EPO) receptor (EPOR) expression in breast cancer has been shown to correlate with the expression of estrogen receptor (ESR) and progesterone receptor (PGR) and to be associated with the response to tamoxifen in ESR+/PGR+ tumors but not in ESR- tumors. In addition, the correlation between EPOR and G protein-coupled estrogen receptor 1 [GPER; also known as G protein-coupled receptor 30 (GPR30)] has been reported, suggesting the prognostic potential of EPOR expression. Moreover, the involvement of colony stimulating factor 2 receptor, β, low‑affinity (CSF2RB) and ephrin type-B receptor 4 (EPHB4) as EPOR potential receptor partners in cancer has been indicated. This study analyzed the correlation between the expression of genes for EPO, EPOR, CSF2RB, EPHB4, ESR, PGR and GPER in the MCF-7, MDA-MB-361, T-47D, MDA-MB-231, Hs578Bst, SKBR3, MCF-10A and Hs578T cell lines. The cell lines were also treated with recombinant human EPO (rHuEPO) in order to determine its ability to activate the Jak/STAT5, MAPK and PI3K signaling pathways and modify cell growth characteristics. Expression analysis stratified the cell lines in 2 main clusters, hormone-dependent cell lines expressing ESR and PGR and a hormone-independent cluster. A significant correlation was observed between the expression levels of ESR and PGR and their expression was also associated with that of GPER. Furthermore, the expression of GPER was associated with that of EPOR, suggesting the connection between this orphan G protein and EPO signaling. A negative correlation between EPOR and CSF2RB expression was observed, questioning the involvement of these two receptors in the hetero-receptor formation. rHuEPO treatment only influenced the hormone-independent cell lines, since only the MDA-MB-231, SKBR3 and Hs578T cells responded to the treatment. The correlation between the expression of the analyzed receptors suggests that the receptors may interact in order to activate signaling pathways

Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways.

PubMed

Ying, Tsung-Ho; Chen, Chia-Wei; Hsiao, Yu-Ping; Hung, Sung-Jen; Chung, Jing-Gung; Yang, Jen-Hung

2013-10-01

Citric acid is an alpha-hydroxyacid (AHA) widely used in cosmetic dermatology and skincare products. However, there is concern regarding its safety for the skin. In this study, we investigated the cytotoxic effects of citric acid on the human keratinocyte cell line HaCaT. HaCaT cells were treated with citric acid at 2.5-12.5 mM for different time periods. Cell-cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, flow cytometry, western blot and confocal microscopy. Citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent manner, but also induced apoptosis and cell cycle-arrest at the G2/M phase (before 24 h) and S phase (after 24 h). Citric acid increased the level of Bcl-2-associated X protein (BAX) and reduced the levels of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-extra large (BCL-XL) and activated caspase-9 and caspase-3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathways. Citric acid also activated death receptors and increased the levels of caspase-8, activated BH3 interacting-domain death agonist (BID) protein, Apoptosis-inducing factor (AIF), and Endonuclease G (EndoG). Therefore, citric acid induces apoptosis through the mitochondrial pathway in the human keratinocyte cell line HaCaT. The study results suggest that citric acid is cytotoxic to HaCaT cells via induction of apoptosis and cell-cycle arrest in vitro.

Methods of Controlling Invasive Fungal Infections Using CD8+ T Cells.

PubMed

Kumaresan, Pappanaicken R; da Silva, Thiago Aparecido; Kontoyiannis, Dimitrios P

2017-01-01

Invasive fungal infections (IFIs) cause high rates of morbidity and mortality in immunocompromised patients. Pattern-recognition receptors present on the surfaces of innate immune cells recognize fungal pathogens and activate the first line of defense against fungal infection. The second line of defense is the adaptive immune system which involves mainly CD4 + T cells, while CD8 + T cells also play a role. CD8 + T cell-based vaccines designed to prevent IFIs are currently being investigated in clinical trials, their use could play an especially important role in acquired immune deficiency syndrome patients. So far, none of the vaccines used to treat IFI have been approved by the FDA. Here, we review current and future antifungal immunotherapy strategies involving CD8 + T cells. We highlight recent advances in the use of T cells engineered using a Sleeping Beauty vector to treat IFIs. Recent clinical trials using chimeric antigen receptor (CAR) T-cell therapy to treat patients with leukemia have shown very promising results. We hypothesized that CAR T cells could also be used to control IFI. Therefore, we designed a CAR that targets β-glucan, a sugar molecule found in most of the fungal cell walls, using the extracellular domain of Dectin-1, which binds to β-glucan. Mice treated with D-CAR + T cells displayed reductions in hyphal growth of Aspergillus compared to the untreated group. Patients suffering from IFIs due to primary immunodeficiency, secondary immunodeficiency (e.g., HIV), or hematopoietic transplant patients may benefit from bioengineered CAR T cell therapy.

Methods of Controlling Invasive Fungal Infections Using CD8+ T Cells

PubMed Central

Kumaresan, Pappanaicken R.; da Silva, Thiago Aparecido; Kontoyiannis, Dimitrios P.

2018-01-01

Invasive fungal infections (IFIs) cause high rates of morbidity and mortality in immunocompromised patients. Pattern-recognition receptors present on the surfaces of innate immune cells recognize fungal pathogens and activate the first line of defense against fungal infection. The second line of defense is the adaptive immune system which involves mainly CD4+ T cells, while CD8+ T cells also play a role. CD8+ T cell-based vaccines designed to prevent IFIs are currently being investigated in clinical trials, their use could play an especially important role in acquired immune deficiency syndrome patients. So far, none of the vaccines used to treat IFI have been approved by the FDA. Here, we review current and future antifungal immunotherapy strategies involving CD8+ T cells. We highlight recent advances in the use of T cells engineered using a Sleeping Beauty vector to treat IFIs. Recent clinical trials using chimeric antigen receptor (CAR) T-cell therapy to treat patients with leukemia have shown very promising results. We hypothesized that CAR T cells could also be used to control IFI. Therefore, we designed a CAR that targets β-glucan, a sugar molecule found in most of the fungal cell walls, using the extracellular domain of Dectin-1, which binds to β-glucan. Mice treated with D-CAR+ T cells displayed reductions in hyphal growth of Aspergillus compared to the untreated group. Patients suffering from IFIs due to primary immunodeficiency, secondary immunodeficiency (e.g., HIV), or hematopoietic transplant patients may benefit from bioengineered CAR T cell therapy. PMID:29358941

Special regulatory T-cell review: T-cell dependent suppression revisited.

PubMed

Basten, Antony; Fazekas de St Groth, Barbara

2008-01-01

The concept of T-cell dependent regulation of immune responses has been a central tenet of immunological thinking since the delineation of the two cell system in the 1960s. Indeed T-cell dependent suppression was discovered before MHC restriction. When reviewing the data from the original wave of suppression, it is intriguing to reflect not just on the decline and fall of suppressor T cells in the 1980s, but on their equally dramatic return to respectability over the past decade. Hopefully their resurgence will be supported by solid mechanistic data that will underpin their central place in our current and future understanding of the immune system. Cannon to right of them, Cannon to left of them, Cannon in front of them Volley'd and thunder'd Storm'd at with shot and shell, Boldly they rode and well, Into the jaws of Death, Into the mouth of Hell, Rode the six hundred (suppressionists). (Adapted from The Charge of the Light Brigade, Alfred, Lord Tennyson)

Vitamin D Status Is Positively Correlated with Regulatory T Cell Function in Patients with Multiple Sclerosis

PubMed Central

Smolders, Joost; Thewissen, Mariëlle; Peelen, Evelyn; Menheere, Paul; Cohen Tervaert, Jan Willem; Damoiseaux, Jan; Hupperts, Raymond

2009-01-01

Background In several autoimmune diseases, including multiple sclerosis (MS), a compromised regulatory T cell (Treg) function is believed to be critically involved in the disease process. In vitro, the biologically active metabolite of vitamin D has been shown to promote Treg development. A poor vitamin D status has been linked with MS incidence and MS disease activity. In the present study, we assess a potential in vivo correlation between vitamin D status and Treg function in relapsing remitting MS (RRMS) patients. Methodology/Principal Findings Serum levels of 25-hydroxyvitamin D (25(OH)D) were measured in 29 RRMS patients. The number of circulating Tregs was assessed by flow-cytometry, and their functionality was tested in vitro in a CFSE-based proliferation suppression assay. Additionally, the intracellular cytokine profile of T helper cells was determined directly ex-vivo by flow-cytometry. Serum levels of 25(OH)D correlated positively with the ability of Tregs to suppress T cell proliferation (R = 0.590, P = 0.002). No correlation between 25(OH)D levels and the number of Tregs was found. The IFN-γ/IL-4 ratio (Th1/Th2-balance) was more directed towards IL-4 in patients with favourable 25(OH)D levels (R = −0.435, P = 0.023). Conclusions/Significance These results show an association of high 25(OH)D levels with an improved Treg function, and with skewing of the Th1/Th2 balance towards Th2. These findings suggest that vitamin D is an important promoter of T cell regulation in vivo in MS patients. It is tempting to speculate that our results may not only hold for MS, but also for other autoimmune diseases. Future intervention studies will show whether modulation of vitamin D status results in modulation of the T cell response and subsequent amelioration of disease activity. PMID:19675671

Thy-1+ dendritic epidermal cells express T3 antigen and the T-cell receptor gamma chain.

PubMed Central

Stingl, G; Koning, F; Yamada, H; Yokoyama, W M; Tschachler, E; Bluestone, J A; Steiner, G; Samelson, L E; Lew, A M; Coligan, J E

1987-01-01

The murine epidermis is a heterogeneous epithelium composed of keratinocytes, melanocytes, Langerhans cells, and a recently described subpopulation (2-3%) of bone-marrow-derived leukocytes with a dendritic morphology and the cell surface phenotype Thy-1+, L3T4-, Lyt-2-. Previous studies have demonstrated that cell lines derived from freshly explanted Thy-1+ dendritic epidermal cells (DEC) have abundant mRNA for rearranged T-cell receptor (TCR) gamma-chain genes. Analysis of Thy-1+ DEC in situ, freshly isolated cell suspensions of Thy-1+ DEC, and long-term Thy-1+ DEC lines demonstrated that 100% of the Thy-1+ DEC reacted with a monoclonal antibody to the epsilon chain of the murine T3 complex and that 40-60% of resident Thy-1+ DEC were also reactive with an antiserum to the TCR gamma chain. Two Thy-1+ DEC lines expressed a disulfide-linked 70-kDa molecule that could be precipitated with an anti-gamma-chain antiserum and could be coprecipitated with an antiserum to the T3 delta chain; the molecule appeared as a single 34-kDa band under reducing conditions. The phenotype of Thy-1+ DEC (T3+, L3T4-, Lyt-2-, TCR gamma chain+) thus resembles that of the recently described subpopulation of murine and human lymphocytes that have been identified in the thymus, peripheral blood, and fetal blood. Images PMID:2885839

A Novel Bcl-x Isoform Connected to the T Cell Receptor Regulates Apoptosis in T Cells

PubMed Central

Yang, Xiao-Feng; Weber, Georg F.

2014-01-01

Summary We define a novel Bcl-x isoform, Bcl-xγ, that is generated by alternative splicing and characterized by a unique 47 amino acid C-terminus. Bcl-xγ is expressed primarily in thymocytes, where it may depend on an interaction between the TCR and host MHC products, and in mature T cells, where its expression is associated with ligation of the T cell receptor. Overexpression of Bcl-xγ in T cells inhibits activation-induced apoptosis; inhibition of Bcl-xγ, after stable expression of Bcl-xγ antisense cDNA, enhances activation-induced apoptosis. In contrast to other Bcl-x isoforms, cells that fail to express Bcl-xγ after CD3 ligation undergo programmed cell death, while activated T cells that express Bcl-xγ are spared. Identification of Bcl-xγ helps provide amolecular explanation of T cell activation and death after antigen engagement. PMID:9390687

Activation pathways of synovial T lymphocytes. Expression and function of the UM4D4/CDw60 antigen.

PubMed Central

Fox, D A; Millard, J A; Kan, L; Zeldes, W S; Davis, W; Higgs, J; Emmrich, F; Kinne, R W

1990-01-01

Accumulating evidence implicates a central role for synovial T cells in the pathogenesis of rheumatoid arthritis, but the activation pathways that drive proliferation and effector function of these cells are not known. We have recently generated a novel monoclonal antibody against a rheumatoid synovial T cell line that recognizes an antigen termed UM4D4 (CDw60). This antigen is expressed on a minority of peripheral blood T cells, and represents the surface component of a distinct pathway of human T cell activation. The current studies were performed to examine the expression and function of UM4D4 on T cells obtained from synovial fluid and synovial membranes of patients with rheumatoid arthritis and other forms of inflammatory joint disease. The UM4D4 antigen is expressed at high surface density on about three-fourths of synovial fluid T cells and on a small subset of synovial fluid natural killer cells; in synovial tissue it is present on more than 90% of T cells in lymphoid aggregates, and on approximately 50% of T cells in stromal infiltrates In addition, UM4D4 is expressed in synovial tissue on a previously undescribed population of HLA-DR/DP-negative non-T cells with a dendritic morphology. Anti-UM4D4 was co-mitogenic for both RA and non-RA synovial fluid mononuclear cells, and induced IL-2 receptor expression. The UM4D4/CDw60 antigen may represent a functional activation pathway for synovial compartment T cells, which could play an important role in the pathogenesis of inflammatory arthritis. Images PMID:2212003

Targeting CD47 Enhances the Efficacy of Anti-PD-1 and CTLA-4 in an Esophageal Squamous Cell Cancer Preclinical Model.

PubMed

Tao, Hua; Qian, Pudong; Wang, Feijiang; Yu, Hongliang; Guo, Yesong

2017-11-02

Esophageal squamous cell cancer is a highly aggressive cancer with a dismal 5-year survival rate. CD47 is a cell transmembrane protein that is involved in cell apoptosis, proliferation, adhesion, migration, and antigen presentation in the immune system. By interacting with signal regulatory protein-α expressed in antigen-presenting cells (APCs), CD47 acts as an antiphagocytic mechanism to inhibit APC-dependent antigen presentation. Overexpression of CD47 was found in various types of cancer. However, its role in esophageal squamous cell cancer is not yet clear. Anti-CD47 is an antagonist of CD47 signaling pathways by competing with its ligand. In the current study, we investigated the effects of anti-CD47 treatment on the antitumor immune response in an esophageal squamous cell cancer preclinical model. We found that anti-CD47 treatment enhanced proinflammatory responses and increased CD8+ T-cell infiltration in tumor tissue in the animal model. T cells in anti-CD47-treated tumors showed higher PD-1 and CTLA-4 expression, indicating T-cell activation and the rationale of combining anti-CD47 with anti-PD-1 and CLTA-4. The combinatory treatment showed the best antitumor response, implying a novel treatment strategy. The effects of anti-CD47 depended on dendritic cell function. In patient samples, expression of CD47 was negatively correlated with CD8+ T-cell infiltration in esophageal squamous cell cancer patients. Taken together, CD47 might be a novel target to enhance anti-PD-1 and CLTA-4 efficacy in esophageal squamous cell cancer.

Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medina, D.; Oborn, C.J.; Li, M.L.

1987-09-01

The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appearedmore » to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.« less

Human T-cell leukemia virus type 1 Tax oncoprotein represses the expression of the BCL11B tumor suppressor in T-cells

PubMed Central

Takachi, Takayuki; Takahashi, Masahiko; Takahashi-Yoshita, Manami; Higuchi, Masaya; Obata, Miki; Mishima, Yukio; Okuda, Shujiro; Tanaka, Yuetsu; Matsuoka, Masao; Saitoh, Akihiko; Green, Patrick L; Fujii, Masahiro

2015-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia (ATL), which is an aggressive form of T-cell malignancy. HTLV-1 oncoproteins, Tax and HBZ, play crucial roles in the immortalization of T-cells and/or leukemogenesis by dysregulating the cellular functions in the host. Recent studies show that HTLV-1-infected T-cells have reduced expression of the BCL11B tumor suppressor protein. In the present study, we explored whether Tax and/or HBZ play a role in downregulating BCL11B in HTLV-1-infected T-cells. Lentiviral transduction of Tax in a human T-cell line repressed the expression of BCL11B at both the protein and mRNA levels, whereas the transduction of HBZ had little effect on the expression. Tax mutants with a decreased activity for the NF-κB, CREB or PDZ protein pathways still showed a reduced expression of the BCL11B protein, thereby implicating a different function of Tax in BCL11B downregulation. In addition, the HTLV-2 Tax2 protein reduced the BCL11B protein expression in T-cells. Seven HTLV-1-infected T-cell lines, including three ATL-derived cell lines, showed reduced BCL11B mRNA and protein expression relative to an uninfected T-cell line, and the greatest reductions were in the cells expressing Tax. Collectively, these results indicate that Tax is responsible for suppressing BCL11B protein expression in HTLV-1-infected T-cells; Tax-mediated repression of BCL11B is another mechanism that Tax uses to promote oncogenesis of HTLV-1-infected T-cells. PMID:25613934

The lupus susceptibility gene Pbx1 regulates the balance between follicular helper T cell and regulatory T cell differentiation

PubMed Central

Choi, Seung-Chul; Hutchinson, Tarun E.; Titov, Anton A.; Seay, Howard R.; Li, Shiwu; Brusko, Todd M.; Croker, Byron P.; Salek-Ardakani, Shahram; Morel, Laurence

2016-01-01

Pbx1 controls chromatin accessibility to a large number of genes and is entirely conserved between mice and humans. The Pbx1-d dominant negative isoform is more frequent in the CD4+ T cells from lupus patients than from healthy controls. Pbx1-d is associated with the production of autoreactive T cells in mice carrying the Sle1a1 lupus susceptibility locus. Transgenic expression of Pbx1-d in CD4+ T cells reproduced the phenotypes of Sle1a1 mice, with increased inflammatory functions of CD4+ T cells and impaired regulatory T cell homeostasis. Pbx1-d Tg also expanded the number of follicular helper T cells in a cell-intrinsic and antigen-specific manner that was enhanced in recall responses, and resulted in TH1-biased antibodies. Moreover, Pbx1-d Tg CD4+ T cells upregulated the expression of miR-10a, miR-21 and miR-155, which have been implicated in Treg and TFH cell homeostasis. Our results suggest that Pbx1-d impacts lupus development by regulating effector T cell differentiation and promoting TFH cells at the expense of Treg cells. In addition, our results identify Pbx1 as a novel regulator of CD4+ T cell effector function. PMID:27296664

The lymphotoxin promoter is stimulated by HTLV-I tax activation of NF-kappa B in human T-cell lines.

PubMed

Paul, N L; Millet, I; Ruddle, N H

1993-07-01

The HTLV-I transcriptional activator tax was used to gain insight into the mechanism of lymphotoxin (LT; TNF-beta) gene induction. Tax-expressing cell lines produce LT biologic activity. An LT promoter (LT-293) CAT construct that contained an NF-kappa B site was active in the LT-producing C81-66-45 cell line, which contains defective HTLV-I but expresses tax. The observation that a mutated LT-kappa B construct (M1-CAT) was inactive in C81-66-45, confirmed the importance of NF-kappa B in LT gene expression. Tax was transfected into HTLV-I-negative human T-cell lines. Jurkat T cells stably expressing tax contained elevated levels of NF-kappa B that directly bound to the LT-kappa B site. Tax co-transfected with reporter constructs into Jurkat cells maximally activated HTLV-I-LTR-CAT and kappa B-fos-CAT and also activated LT-293 to a lesser extent. In JM T cells, tax induced LT-293 activity by two- to four-fold, though there was no induction of M1-CAT. The increase in LT-293 CAT activity mirrored the increase in LT biologic activity seen under these conditions. These studies, the first to demonstrate induction of LT promoter activity over basal levels, indicate that HTLV-I tax causes low-level activation of both endogenous LT and the LT promoter, at least in part through activation of NF-kappa B.

BAFF induces spleen CD4{sup +} T cell proliferation by down-regulating phosphorylation of FOXO3A and activates cyclin D2 and D3 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ji, Fang; Chen, Rongjing; Liu, Baojun

2012-09-07

Highlights: Black-Right-Pointing-Pointer Firstly analyze the mechanism of BAFF and anti-CD3 co-stimulation on purified mouse splenic CD4{sup +} T cells. Black-Right-Pointing-Pointer Carrying out siRNA technology to study FOXO3A protein function. Black-Right-Pointing-Pointer Helpful to understand the T cell especially CD4{sup +} T cell's role in immunological reaction. -- Abstract: The TNF ligand family member 'B cell-activating factor belonging to the TNF family' (BAFF, also called BLyS, TALL-1, zTNF-4, and THANK) is an important survival factor for B and T cells. In this study, we show that BAFF is able to induce CD4{sup +} spleen T cell proliferation when co-stimulated with anti-CD3. Expressionmore » of phosphorylated FOXO3A was notably down-regulated and cyclins D2 and D3 were up-regulated and higher in the CD4{sup +} T cells when treated with BAFF and anti-CD3, as assessed by Western blotting. Furthermore, after FOXO3A was knocked down, expression of cyclin D1 was unchanged, compared with control group levels, but the expression of cyclins D2 and D3 increased, compared with the control group. In conclusion, our results suggest that BAFF induced CD4{sup +} spleen T cell proliferation by down-regulating the phosphorylation of FOXO3A and then activating cyclin D2 and D3 expression, leading to CD4{sup +} T cell proliferation.« less

Direct trans-activation of the human cyclin D2 gene by the oncogene product Tax of human T-cell leukemia virus type I.

PubMed

Huang, Y; Ohtani, K; Iwanaga, R; Matsumura, Y; Nakamura, M

2001-03-01

Cyclins are one of the pivotal determinants regulating cell cycle progression. We previously reported that the trans-activator Tax of human T-cell leukemia virus type I (HTLV-I) induces endogenous cyclin D2 expression along with cell cycle progression in a resting human T-cell line, Kit 225, suggesting a role of cyclin D2 in Tax-mediated cell cycle progression. The cyclin D2 gene has a typical E2F binding element, raising the possibility that induction of cyclin D2 expression is a consequence of cell cycle progression. In this study, we examined the role and molecular mechanism of induction of the endogenous human cyclin D2 gene by Tax. Introduction of p19(INK4d), a cyclin dependent kinase (CDK) inhibitor of the INK4 family specific for D-type CDK, inhibited Tax-mediated activation of E2F, indicating requirement of D-type CDK in Tax-mediated activation of E2F. Previously indicated E2F binding element and two NF-kappaB-like binding elements in the 1.6 kbp cyclin D2 promoter fragment had little, if any, effect on responsiveness to Tax. We found that trans-activation of the cyclin D2 promoter by Tax was mainly mediated by a newly identified NF-kappaB-like element with auxiliary contribution of a CRE-like element residing in sequences downstream of -444 which were by themselves sufficient for trans-activation by Tax. These results indicate that Tax directly trans-activates the cyclin D2 gene, resulting in growth promotion and perhaps leukemogenesis through activation of D-type CDK.

Turned on by danger: activation of CD1d-restricted invariant natural killer T cells

PubMed Central

Lawson, Victoria

2012-01-01

CD1d-restricted invariant natural killer T (iNKT) cells bear characteristics of innate and adaptive lymphocytes, which allow them to bridge the two halves of the immune response and play roles in many disease settings. Recent work has characterized precisely how their activation is initiated and regulated. Novel antigens from important pathogens have been identified, as has an abundant self-antigen, β-glucopyranosylcaramide, capable of mediating an iNKT-cell response. Studies of the iNKT T-cell receptor (TCR)–antigen–CD1d complex show how docking between CD1d–antigen and iNKT TCR is highly conserved, and how small sequence differences in the TCR establish intrinsic variation in iNKT TCR affinity. The sequence of the TCR CDR3β loop determines iNKT TCR affinity for ligand–CD1d, independent of ligand identity. CD1d ligands can promote T helper type 1 (Th1) or Th2 biased cytokine responses, depending on the composition of their lipid tails. Ligands loaded into CD1d on the cell surface promote Th2 responses, whereas ligands with long hydrophobic tails are loaded endosomally and promote Th1 responses. This information is informing the design of synthetic iNKT-cell antigens. The iNKT cells may be activated by exogenous antigen, or by a combination of dendritic cell-derived interleukin-12 and iNKT TCR–self-antigen–CD1d engagement. The iNKT-cell activation is further modulated by recent foreign or self-antigen encounter. Activation of dendritic cells through pattern recognition receptors alters their antigen presentation and cytokine production, strongly influencing iNKT-cell activation. In a range of bacterial infections, dendritic cell-dependent innate activation of iNKT cells through interleukin-12 is the dominant influence on their activity. PMID:22734667

Cloning of an osteoblastic cell line involved in the formation of osteoclast-like cells.

PubMed

Yamashita, T; Asano, K; Takahashi, N; Akatsu, T; Udagawa, N; Sasaki, T; Martin, T J; Suda, T

1990-12-01

Experiments have been carried out to determine the mechanisms involved in the formation of osteoclast-like cells from spleen cells in mice. Osteoclasts were defined as tartrate-resistant acid phosphatase-positive multinucleated cells (TRACP-positive MNCs) in which specific calcitonin receptors were identified by autoradiography with labeled salmon calcitonin. Furthermore, cultures rich in these cells produced resorption pits when grown on dentine slices. Several clonal cell lines were obtained from fetal mouse calvariae and screened for their ability to induce TRACP-positive MNCs in response to 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25(OH)2D3] in co-cultures with spleen cells. A cell line, KS-4, was identified with the greatest potency in inducing osteoclast-like cell formation in co-culture with spleen cells. The capacity of KS-4 cells to produce this effect was much greater than that of two bone marrow-derived stromal cell lines (MC3T3-G2/PA6 and ST2 cells), which we have previously shown to be effective in this system but to require treatment with dexamethasone in addition to 1 alpha, 25(OH)2D3 (Udagawa et al.: Endocrinology 125:1805-1813, 1989). Parathyroid hormone (PTH) increased cAMP production in KS-4 cells, and PTH and interleukin-1 alpha also induced TRACP-positive MNCs in co-cultures with spleen cells. Contact between living KS-4 and spleen cells was necessary for osteoclast formation to take place, since this did not occur when the two populations were separated by a membrane filter, or when the KS-4 cells were killed by fixation. Separate cultures of either spleen cells or KS-4 cells formed no TRACP-positive MNCs. KS-4 cells synthesized predominantly type I collagen, formed bone nodules without added of beta-glycerophosphate in a long-term culture, and expressed increasing alkaline phosphatase activity after confluence in culture. These results indicate that the KS-4 cells have properties consistent with progression toward the osteoblast phenotype

Differential cytokine production in clonal macrophage and T-cell lines cultured with bifidobacteria.

PubMed

Marin, M L; Lee, J H; Murtha, J; Ustunol, Z; Pestka, J J

1997-11-01

When used in commercial fermented dairy products, bifidobacteria may enhance immunity by stimulating cytokine secretion by leukocytes. To assess whether interaction between bifidobacteria and leukocytes promote cytokine production, we cultured RAW 264.7 cells (macrophage model) and EL-4.IL-2 thymoma cells (helper T-cell model) in the presence of 14 representative strains of heat-killed bifidobacteria. In unstimulated RAW 264.7 cells, all bifidobacteria induced pronounced increases (up to several hundred-fold) in the production of tumor necrosis factor-alpha compared with that of controls. Interleukin-6 production by unstimulated cells also increased significantly, but less than did tumor necrosis factor-alpha. Upon concurrent stimulation of RAW 264.7 cells with lipopolysaccharide, production of tumor necrosis factor-alpha and interleukin-6 were both enhanced between 1.5- to 5.8-fold and 4.7- to 7.9-fold, respectively, when cultured with 10(8) bifidobacteria/ml. In unstimulated EL-4.IL-2 cells, bifidobacteria had no effect on the production of interleukin-2 or interleukin-5. Upon stimulation of EL-4.IL-2 with phorbol-12-myristate-13-acetate, there were variable increases in interleukin-2 secretion (up to 2.4-fold for 10(6) Bifidobacterium Bf-1/ml) and interleukin-5 secretion (up to 4.6-fold for 10(8) B. adolescentis M101-4). The results indicated that, even when variations among strains were considered, direct interaction of most bifidobacteria with macrophages enhanced cytokine production, but the effects on cytokine production by the T-cell model were less marked. Interestingly, the 4 bifidobacteria strains used commercially for diary foods showed the greatest capacity for cytokine stimulation. The in vitro approaches employed here should be useful in future characterization of the effects of bifidobacteria on gastrointestinal and systemic immunity.

Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

PubMed Central

Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

2015-01-01

Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

Ficus carica latex prevents invasion through induction of let-7d expression in GBM cell lines.

PubMed

Tezcan, Gulcin; Tunca, Berrin; Bekar, Ahmet; Yalcin, Murat; Sahin, Saliha; Budak, Ferah; Cecener, Gulsah; Egeli, Unal; Demir, Cevdet; Guvenc, Gokcen; Yilmaz, Gozde; Erkan, Leman Gizem; Malyer, Hulusi; Taskapilioglu, Mevlut Ozgur; Evrensel, Turkkan; Bilir, Ayhan

2015-03-01

Glioblastoma multiforme (GBM) is one of the deadliest human malignancies. A cure for GBM remains elusive, and the overall survival time is less than 1 year. Thus, the development of more efficient therapeutic approaches for the treatment of these patients is required. Induction of tumor cell death by certain phytochemicals derived from medicinal herbs and dietary plants has become a new frontier for cancer therapy research. Although the cancer suppressive effect of Ficus carica (fig) latex (FCL) has been determined in a few cancer types, the effect of this latex on GBM tumors has not been investigated. Therefore, in the current study, the anti-proliferative activity of FCL and the effect of the FCL-temozolomide (TMZ) combination were tested in the T98G, U-138 MG, and U-87 MG GBM cell lines using the WST-1 assay. The mechanism of cell death was analyzed using Annexin-V/FITC and TUNEL assays, and the effect of FCL on invasion was tested using the chick chorioallantoic membrane assay. To determine the effect of FCL on GBM progression, the expression levels of 40 GBM associated miRNAs were analyzed in T98G cells using RT-qPCR. According to the obtained data, FCL causes cell death in GBM cells with different responses to TMZ, and this effect is synergistically increased in combination with TMZ. In addition, the current study is the first to demonstrate the effect of FCL on modulation of let-7d expression, which may be an important underlying mechanism of the anti-invasive effect of this extract.

Biocompatibility of bio based calcium carbonate nanocrystals aragonite polymorph on NIH 3T3 fibroblast cell line.

PubMed

Kamba, Abdullahi Shafiu; Ismail, Maznah; Ibrahim, Tengku Azmi Tengku; Zakaria, Zuki Abu Bakar

2014-01-01

Currently, there has been extensive research interest for inorganic nanocrystals such as calcium phosphate, iron oxide, silicone, carbon nanotube and layered double hydroxide as a drug delivery system especially in cancer therapy. However, toxicological screening of such particles is paramount importance before use as delivery carrier. In this study we examine the biocompatibility of CaCO3 nanocrystal on NIH 3T3 cell line. Transmission and field emission scanning electron microscopy (TEM and FESEM) were used for the characterisation of CaCO3 nanocrystals. Cytotoxicity and genotoxic effect of calcium carbonate nanocrystals in cultured mouse embryonic fibroblast NIH 3T3 cell line using various bioassays including MTT, and Neutral red/Trypan blue double-staining assays. LDH, BrdU and reactive oxygen species were used for toxicity analysis. Cellular morphology was examined by scanning electron microscopy (SEM) and confocal fluorescence microscope. The outcome of the analyses revealed a clear rod-shaped aragonite polymorph of calcium carbonate nanocrystal. The analysed cytotoxic and genotoxicity of CaCO3 nanocrystal on NIH 3T3 cells using different bioassays revealed no significance differences as compared to control. A slight decrease in cell viability was noticed when the cells were exposed to higher concentrations of 200 to 400 µg/ml, while increase in ROS generation and LDH released at 200 and 400 µg/ml was observed. The study has shown that CaCO3 nanocrystal is biocompatible and non toxic to NIH 3T3 fibroblast cells. The analysed results offer a promising potential of CaCO3 nanocrystal for the development of intracellular drugs, genes and other macromolecule delivery systems.

47 CFR 15.113 - Power line carrier systems.

Code of Federal Regulations, 2012 CFR

2012-10-01

...-interference basis in accordance with § 15.5 of this part. If harmful interference occurs, the electric power... frequencies. (e) Power line carrier system apparatus shall conform to such engineering standards as may be... 47 Telecommunication 1 2012-10-01 2012-10-01 false Power line carrier systems. 15.113 Section 15...

Human T-cell leukemia virus type 1 Tax oncoprotein represses the expression of the BCL11B tumor suppressor in T-cells.

PubMed

Takachi, Takayuki; Takahashi, Masahiko; Takahashi-Yoshita, Manami; Higuchi, Masaya; Obata, Miki; Mishima, Yukio; Okuda, Shujiro; Tanaka, Yuetsu; Matsuoka, Masao; Saitoh, Akihiko; Green, Patrick L; Fujii, Masahiro

2015-04-01

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia (ATL), which is an aggressive form of T-cell malignancy. HTLV-1 oncoproteins, Tax and HBZ, play crucial roles in the immortalization of T-cells and/or leukemogenesis by dysregulating the cellular functions in the host. Recent studies show that HTLV-1-infected T-cells have reduced expression of the BCL11B tumor suppressor protein. In the present study, we explored whether Tax and/or HBZ play a role in downregulating BCL11B in HTLV-1-infected T-cells. Lentiviral transduction of Tax in a human T-cell line repressed the expression of BCL11B at both the protein and mRNA levels, whereas the transduction of HBZ had little effect on the expression. Tax mutants with a decreased activity for the NF-κB, CREB or PDZ protein pathways still showed a reduced expression of the BCL11B protein, thereby implicating a different function of Tax in BCL11B downregulation. In addition, the HTLV-2 Tax2 protein reduced the BCL11B protein expression in T-cells. Seven HTLV-1-infected T-cell lines, including three ATL-derived cell lines, showed reduced BCL11B mRNA and protein expression relative to an uninfected T-cell line, and the greatest reductions were in the cells expressing Tax. Collectively, these results indicate that Tax is responsible for suppressing BCL11B protein expression in HTLV-1-infected T-cells; Tax-mediated repression of BCL11B is another mechanism that Tax uses to promote oncogenesis of HTLV-1-infected T-cells. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Development of 3D imaging technique of reconstructed human epidermis with immortalized human epidermal cell line.

PubMed

Inoue, Yu; Hasegawa, Seiji; Miyachi, Katsuma; Yamada, Takaaki; Nakata, Satoru; Ipponjima, Sari; Hibi, Terumasa; Nemoto, Tomomi; Tanaka, Masahiko; Suzuki, Ryo; Hirashima, Naohide

2018-05-01

The epidermis, the outermost layer of the skin, retains moisture and functions as a physical barrier against the external environment. Epidermal cells are continuously replaced by turnover, and thus to understand in detail the dynamic cellular events in the epidermis, techniques to observe live tissues in 3D are required. Here, we established a live 3D imaging technique for epidermis models. We first obtained immortalized human epidermal cell lines which have a normal differentiation capacity and fluorescence-labelled cytoplasm or nuclei. The reconstituted 3D epidermis was prepared with these lines. Using this culture system, we were able to observe the structure of the reconstituted epidermis live in 3D, which was similar to an in vivo epidermis, and evaluate the effect of a skin irritant. This technique may be useful for dermatological science and drug development. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Electroporation enhances mitomycin C cytotoxicity on T24 bladder cancer cell line: a potential improvement of intravesical chemotherapy in bladder cancer.

PubMed

Vásquez, Juan L; Gehl, Julie; Hermann, Gregers G

2012-12-01

Intravesical mitomycin instillation combined with electric pulses is being used experimentally for the treatment of T1 bladder tumors, in patients unfit for surgery. Electroporation may enhance the uptake of chemotherapeutics by permeabilization of cell membranes. We investigated if electroporation improves the cytotoxicity of mitomycin. In two cell lines, T24 (bladder cancer cell line) and DC3F (Chinese hamster fibroblast), exposure to different concentrations of mitomycin (0.01-2000μM) was tested with and without electroporation (6 pulses of 1kV/cm, duration: 99μs, frequency: 1Hz). Cell viability was assessed by colorimetric assay (MTT). For both cell lines, mitomycin's IC_50 was approximately 1000μM in both pulsed and unpulsed cells. On T24 cells, electroporation and mitomycin caused (relative reduction) RR of survival of: 25%, 31% and 29%, by concentrations 0μM, 500μM and 1000μM respectively. For DC3F cells, the RRs of survival were: 28%, 29%, and 33%, by concentrations 0μM, 500μM and 1000μM respectively. In conclusion, electroporation and mitomycin together are about 30% more effective than mitomycin alone. The results help to elucidate the additive effect of mitomycin and electric pulses and support the use of this combination in the treatment of bladder cancer. Copyright © 2012 Elsevier B.V. All rights reserved.

Novel hydroxyamides and amides containing D-glucopyranose or D-fructose units: Biological assays in MCF-7 and MDST8 cell lines.

PubMed

Carreiro, Elisabete P; Costa, Ana R; Cordeiro, Maria M; Martins, Rute; Pires, Tiago O; Saraiva, Mafalda; Antunes, Célia M; Burke, Anthony J

2016-02-01

A novel library of 15 compounds, hydroxyamides and amides containing a β-D-glucopyranose (D-Gluc) or a β-D-fructose (D-Fruc) units was designed and synthesized for antiproliferative assays in breast (MCF-7) and colon (MDST8) cancer cell lines. Twelve of them were hydroxyamides and were successfully synthesized from β-D-glucuronic acid (D-GluA). Six of these hydroxyamides which were acetylated hydroxy-β-D-glucopyranuronamide 2a-2f (1st Family) and the other six were their respective isomers, that is, hydroxy-β-D-fructuronamide 3a-3f (2nd Family), obtained by acid-base catalyzed isomerization. These compounds have the general structure, D-Gluc-C=ONH-CHR-(CH2)n-OH and D-Fruc-C=ONH-CHR-(CH2)n-OH, where R=an aromatic, alkyl or a hydrogen substituent, with n=0 or 1. Eight of these contained a chiral aminoalcohol group. Three compounds were amides containing a D-glucopyranose unit (3rd Family). SAR studies were conducted with these compounds. Antiproliferative studies showed that compound 4a, the bromo-amide containing the β-D-glucopyranose ring, potently inhibits the proliferation of the MDST8 cells. Five compounds (2e, 2f, 3d, 3e, and 3f) were shown to potently selectively inhibit the proliferation of the MCF-7 cells. Compound 4b was the only one showing inhibition in both cell lines. In general, the more active compounds were the amides and hydroxyamides containing the β-D-fructose moiety, and containing an alkyl group or hydrogen. Half-inhibitory concentrations (IC50) of between 0.01 and 10 μM, were observed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Polymer encapsulated dopaminergic cell lines as "alternative neural grafts".

PubMed

Jaeger, C B; Greene, L A; Tresco, P A; Winn, S R; Aebischer, P

1990-01-01

Our preliminary findings (Jaeger et al., 1988; Aebischer et al., 1989; Tresco et al., 1989) and the studies in progress show that encapsulated dopaminergic cell lines survive enclosure within a semi-permeable membrane. The encapsulated cells remained viable for extended time periods when maintained in vitro. Moreover, encapsulated PC12 and T28 cells have the potential to survive following their implantation into the forebrain of rats. Cell lines are essentially "immortal" because they continue to divide indefinitely. This property allows perpetual "self-renewal" of a given cell population. However, the capacity of continuous uncontrolled cell division may also lead to tumor formation. This in fact is the case for unencapsulated PC12 cell implants placed into the brain of young Sprague Dawley rats (Jaeger, 1985). Cell line encapsulation has the potential to prevent tumor growth (Jaeger et al., 1988). Survival for 6 months in vitro suggests that encapsulation does not preclude long-term maintenance of an homogeneous cell line like PC12 cells. The presence of mitotic figures in the capsules further supports the likelihood of propagation and self renewal of the encapsulated population. Another significant property of cell lines is that they consist of a single, genetically homogeneous cell type. They do not require specific synaptic interactions for their survival. In the case of PC12 and T28 lines, the cells synthesize and release neurotransmitters. Our data show that PC12 and T28 cells continue to release dopamine spontaneously and to express specific transmitters and enzymes following encapsulation. Thus, cell lines such as these may constitute relatively simple "neural implants" exerting their function via humoral release.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor-specific delivery of biologics by a novel T-cell line HOZOT

PubMed Central

Onishi, Teppei; Tazawa, Hiroshi; Hashimoto, Yuuri; Takeuchi, Makoto; Otani, Takeshi; Nakamura, Shuji; Sakurai, Fuminori; Mizuguchi, Hiroyuki; Kishimoto, Hiroyuki; Umeda, Yuzo; Shirakawa, Yasuhiro; Urata, Yasuo; Kagawa, Shunsuke; Fujiwara, Toshiyoshi

2016-01-01

“Cell-in-cell” denotes an invasive phenotype in which one cell actively internalizes in another. The novel human T-cell line HOZOT, established from human umbilical cord blood, was shown to penetrate a variety of human cancer cells but not normal cells. Oncolytic viruses are emerging as biological therapies for human cancers; however, efficient viral delivery is limited by a lack of tumor-specific homing and presence of pre-existing or therapy-induced neutralizing antibodies. Here, we report a new, intriguing approach using HOZOT cells to transmit biologics such as oncolytic viruses into human cancer cells by cell-in-cell invasion. HOZOT cells were successfully loaded via human CD46 antigen with an attenuated adenovirus containing the fiber protein of adenovirus serotype 35 (OBP-401/F35), in which the telomerase promoter regulates viral replication. OBP-401/F35–loaded HOZOT cells were efficiently internalized into human cancer cells and exhibited tumor-specific killing by release of viruses, even in the presence of anti-viral neutralizing antibodies. Moreover, intraperitoneal administration of HOZOT cells loaded with OBP-401/F35 significantly suppressed peritoneally disseminated tumor growth in mice. This unique cell-in-cell property provides a platform for selective delivery of biologics into human cancer cells, which has important implications for the treatment of human cancers. PMID:27901098

S179D prolactin diminishes the effects of UV light on epidermal gamma delta T cells

PubMed Central

Guzmán, Esther A.; Langowski, John L.; De Guzman, Ariel; Konrad Muller, H.; Walker, Ameae M.; Owen, Laurie B.

2008-01-01

Epidermal gamma delta T cells (γδ T) and Langerhans cells (LC) are immune cells altered by exposure to ultraviolet radiation (UVB), a powerful stressor resulting in immune suppression. Prolactin (PRL) has been characterized as an immunomodulator, particularly during stress. In this study, we have asked whether separate administration of the two major forms of prolactin, unmodified and phosphorylated, to groups of 15 mice (3 experiments, each with 5 mice per treatment group) affected the number and morphology of these epidermal immune cells under control conditions, and following UV irradiation. Under control conditions, both PRLs reduced the number of γδ T, but a molecular mimic of phosphorylated PRL (S179D PRL) was more effective, resulting in a 30% reduction. In the irradiated group, however, S179D PRL was protective against a UV-induced reduction in γδ T number and change in morphology (halved the reduction and normalized the morphology). In addition, S179D PRL, but not unmodified (U-PRL), maintained a normal LC: γδ T ratio and sustained the dendritic morphology of LC after UV exposure. These findings suggest that S179D PRL may have an overall protective effect, countering UV-induced cellular alterations in the epidermis. PMID:17945411

Immunohistochemical Analysis Using Antipodocalyxin Monoclonal Antibody PcMab-47 Demonstrates Podocalyxin Expression in Oral Squamous Cell Carcinomas.

PubMed

Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Harada, Hiroyuki; Kato, Yukinari

2017-10-01

Podocalyxin is a CD34-related type I transmembrane protein that is highly glycosylated with N-glycan, O-glycan, and keratan sulfate. Podocalyxin was originally found in the podocytes of rat kidney and is reportedly expressed in many types of tumors, including brain tumors, colorectal cancers, and breast cancers. Overexpression of podocalyxin is an independent predictor of progression, metastasis, and poor outcome. We recently immunized mice with recombinant human podocalyxin, which was produced using LN229 glioblastoma cells, and produced a novel antipodocalyxin monoclonal antibody (mAb), PcMab-47, which reacts with endogenous podocalyxin-expressing cancer cell lines and normal cell lines independent of glycosylation in Western blot, flow cytometry, and immunohistochemical analyses. In this study, we performed immunohistochemical analysis against oral cancers using PcMab-47. PcMab-47-stained oral squamous cell carcinoma cells in a cytoplasmic pattern and detected 26/38 (68.4%) of oral squamous cell carcinoma cells on tissue microarrays. These results indicate that PcMab-47 is useful in detecting podocalyxin of oral cancers for immunohistochemical analysis.

IL-1R and MyD88 signalling in CD4+ T cells promote Th17 immunity and atherosclerosis.

PubMed

Engelbertsen, Daniel; Rattik, Sara; Wigren, Maria; Vallejo, Jenifer; Marinkovic, Goran; Schiopu, Alexandru; Björkbacka, Harry; Nilsson, Jan; Bengtsson, Eva

2018-01-01

The role of CD4+ T cells in atherosclerosis has been shown to be dependent on cytokine cues that regulate lineage commitment into mature T helper sub-sets. In this study, we tested the roles of IL-1R1 and MyD88 signalling in CD4+ T cells in atherosclerosis. We transferred apoe-/-myd88+/+ or apoe-/-myd88-/- CD4+ T cells to T- and B-cell-deficient rag1-/-apoe-/- mice fed high fat diet. Mice given apoe-/-myd88-/- CD4+ T cells exhibited reduced atherosclerosis compared with mice given apoe-/-myd88+/+ CD4+ T cells. CD4+ T cells from apoe-/-myd88-/- produced less IL-17 but similar levels of IFN-γ. Treatment of human CD4+ T cells with a MyD88 inhibitor inhibited IL-17 secretion in vitro. Transfer of il1r1-/- CD4+ T cells recapitulated the phenotype seen by transfer of myd88-/- CD4+ T cells with reduced lesion development and a reduction in Th17 and IL-17 production compared with wild type CD4+ T cell recipients. Relative collagen content of lesions was reduced in mice receiving il1r1-/- CD4+ T cells. We demonstrate that both IL1R and MyD88 signalling in CD4+ T cells promote Th17 immunity, plaque growth and may regulate plaque collagen levels. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions please email: journals.permissions@oup.com.

Rapid generation of combined CMV-specific CD4+ and CD8+ T-cell lines for adoptive transfer into recipients of allogeneic stem cell transplants.

PubMed

Rauser, Georg; Einsele, Hermann; Sinzger, Christian; Wernet, Dorothee; Kuntz, Gabriele; Assenmacher, Mario; Campbell, John D M; Topp, Max S

2004-05-01

Adoptive transfer of cytomegalovirus (CMV)-specific T cells can restore long-lasting, virus-specific immunity and clear CMV viremia in recipients of allogeneic stem cell transplants if CD4(+) and CD8(+) CMV-specific T cells are detected in the recipient after transfer. Current protocols for generating virus-specific T cells use live virus, require leukapheresis of the donor, and are time consuming. To circumvent these limitations, a clinical-scale protocol was developed to generate CMV-specific T cells by using autologous cellular and serum components derived from a single 500-mL blood draw. CMV-specific T cells were stimulated simultaneously with CMV-specific major histocompatibility complex class I (MHC I)- restricted peptides and CMV antigen. Activated T cells were isolated with the interferon-gamma (IFN-gamma) secretion assay and expanded for 10 days. In 8 randomly selected, CMV-seropositive donors, 1.34 x 10(8) combined CD4(+) and CD8(+) CMV-specific T cells, on average, were generated, as determined by antigen-triggered IFN-gamma production. CMV-infected fibroblasts were efficiently lysed by the generated T cells, and CMV-specific CD4(+) and CD8(+) T cells expanded if they were stimulated with natural processed antigen. On the other hand, CD4(+) and CD8(+) T cell-mediated alloreactivity of generated CMV-specific T-cell lines was reduced compared with that of the starting population. In conclusion, the culture system developed allowed the rapid generation of allodepleted, highly enriched, combined CD4(+) and CD8(+) CMV-specific T cells under conditions mimicking good manufacturing practice.

Dopamine D4 receptor, but not the ADHD-associated D4.7 variant, forms functional heteromers with the dopamine D2S receptor in the brain

PubMed Central

González, Sergio; Rangel-Barajas, Claudia; Peper, Marcela; Lorenzo, Ramiro; Moreno, Estefanía; Ciruela, Francisco; Borycz, Janusz; Ortiz, Jordi; Lluís, Carme; Franco, Rafael; McCormick, Peter J.; Volkow, Nora D.; Rubinstein, Marcelo; Floran, Benjamin; Ferré, Sergi

2011-01-01

Polymorphic variants of the dopamine D4 receptor have been consistently associated with attention-deficit hyperactivity disorder (ADHD). However the functional significance of the risk polymorphism (variable number of tandem repeats in exon 3) is still unclear. Here we show that whereas the most frequent 4-repeat (D4.4) and the 2-repeat (D4.2) variants form functional heteromers with the short isoform of the dopamine D2 receptor (D2S), the 7-repeat risk allele (D4.7) does not. D2 receptor activation in the D2S-D4 receptor heteromer potentiates D4 receptor-mediated MAPK signaling in transfected cells and in the striatum, which did not occur in cells expressing D4.7 or in the striatum of knock-in mutant mice carrying the 7 repeats of the human D4.7 in the third intracellular loop of the D4 receptor. In the striatum D4 receptors are localized in cortico-striatal glutamatergic terminals, where they selectively modulate glutamatergic neurotransmission by interacting with D2S receptors. This interaction shows the same qualitative characteristics than the D2S-D4 receptor heteromer-mediated MAPK signaling and D2S receptor activation potentiates D4 receptor-mediated inibition of striatal glutamate release. It is therefore postulated that dysfunctional D2S-D4.7 heteromers may impair presynaptic dopaminergic control of corticostriatal glutamatergic neurotransmission and explain functional deficits associated with ADHD. PMID:21844870

31P NMR spectroscopy studies of phospholipid metabolism in human melanoma xenograft lines differing in rate of tumour cell proliferation.

PubMed

Lyng, H; Olsen, D R; Petersen, S B; Rofstad, E K

1995-04-01

The concentration of phospholipid metabolites in tumours has been hypothesized to be related to rate of cell membrane turnover and may reflect rate of cell proliferation. The purpose of the study reported here was to investigate whether 31P NMR resonance ratios involving the phosphomonoester (PME) or phosphodiester (PDE) resonance are correlated to fraction of cells in S-phase or volume-doubling time in experimental tumours. Four human melanoma xenograft lines (BEX-t, HUX-t, SAX-t, WIX-t) were included in the study. The tumours were grown subcutaneously in male BALB/c-nu/nu mice. 31P NMR spectroscopy was performed at a magnetic field strength of 4.7 T. Fraction of cells in S-phase was measured by flow cytometry. Tumour volume-doubling time was determined by Gompertzian analysis of volumetric growth data. BEX-t and SAX-t tumours differed in fraction of cells in S-phase and volume-doubling time, but showed similar 31P NMR resonance ratios. BEX-t and WIX-t tumours showed significantly different 31P NMR resonance ratios but similar fractions of cells in S-phase. The 31P NMR resonance ratios were significantly different for small and large HUX-t tumours even though fraction of cells in S-phase and volume-doubling time did not differ with tumour volume. None of the 31P NMR resonance ratios showed significant increase with increasing fraction of cells in S-phase or significant decrease with increasing tumour volume-doubling time across the four xenograft lines.(ABSTRACT TRUNCATED AT 250 WORDS)

A CRISPR-Based Toolbox for Studying T Cell Signal Transduction

PubMed Central

Chi, Shen; Weiss, Arthur; Wang, Haopeng

2016-01-01

CRISPR/Cas9 system is a powerful technology to perform genome editing in a variety of cell types. To facilitate the application of Cas9 in mapping T cell signaling pathways, we generated a toolbox for large-scale genetic screens in human Jurkat T cells. The toolbox has three different Jurkat cell lines expressing distinct Cas9 variants, including wild-type Cas9, dCas9-KRAB, and sunCas9. We demonstrated that the toolbox allows us to rapidly disrupt endogenous gene expression at the DNA level and to efficiently repress or activate gene expression at the transcriptional level. The toolbox, in combination with multiple currently existing genome-wide sgRNA libraries, will be useful to systematically investigate T cell signal transduction using both loss-of-function and gain-of-function genetic screens. PMID:27057542

Establishment and characterization of feeder cell-dependent bovine fetal liver cell lines.