15 research outputs found

    1B2.1E12 anti-TNP

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    19650650

    Chromatin Remodeling, Cell Proliferation and Cell Death in Valproic Acid-Treated HeLa Cells

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    Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Background: Valproic acid (VPA) is a potent anticonvulsant that inhibits histone deacetylases. Because of this inhibitory action, we investigated whether VPA would affect chromatin supraorganization, mitotic indices and the frequency of chromosome abnormalities and cell death in HeLa cells. Methodology/Principal Findings: Image analysis was performed by scanning microspectrophotometry for cells cultivated for 24 h, treated with 0.05, 0.5 or 1.0 mM VPA for 1-24 h, and subjected to the Feulgen reaction. TSA-treated cells were used as a predictable positive control. DNA fragmentation was investigated with the TUNEL assay. Chromatin decondensation was demonstrated under TSA and all VPA treatments, but no changes in chromosome abnormalities, mitotic indices or morphologically identified cell death were found with the VPA treatment conditions mentioned above, although decreased mitotic indices were detected under higher VPA concentration and longer exposure time. The frequency of DNA fragmentation identified with the TUNEL assay in HeLa cells increased after a 24-h VPA treatment, although this fragmentation occurred much earlier after treatment with TSA. Conclusions/Significance: The inhibition of histone deacetylases by VPA induces chromatin remodeling in HeLa cells, which suggests an association to altered gene expression. Under VPA doses close to the therapeutic antiepileptic plasma range no changes in cell proliferation or chromosome abnormalities are elicited. The DNA fragmentation results indicate that a longer exposure to VPA or a higher VPA concentration is required for the induction of cell death.612Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2010/50015-6, 2009/11763-0]CNPq [471303/2009-7, 301943/2009-5]CNPq [301943/2009-5, 132345/2010-2

    Anti-TNP monoclonal antibodies as reagents for enzyme immunoassay (ELISA)

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    The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugated and used for the detection of antigen-antibody reactions, in which the antigen specific-antibody had been previously bound to trinitrophenyl (TNP). For hybridoma production, SP2/0-Ag14 cells were fused with spleen cells from mice previously immunized with TNP-ovalbumin (TNP-OVA). After 10 days, enzyme-linked immunoadsorbent assay (ELISA) was used to detect anti-TNP antibodies in the supernatants, and five cultures were found to be strictly positive for TNP. Three of these were subsequently cloned by limiting dilution, and 15 clones were chosen for expansion based on the criterion of high reactivity against TNP. Anti-TNP MAbs produced by those clones were isotyped as IgG(1), and purified by Sepharose-protein G affinity cromatography from ascites developed in BALB/c mice. Two purified MAbs (1B2.1B6 and 1B2.1E12) were coupled to horseradish peroxidase (HRPO). The resulting conjugates were evaluated in ELISA tests for interferon-gamma and interleukin-4 detection, in which the secondary anti-cytokine antibodies were coupled either to TNP or biotin. The performance of anti-TNP conjugates in these assays were compared with a biotin-streptavidin/peroxidase system. Both types of conjugates were similarly able to detect cytokines with r(2) (linear correlation coefficient) close to unity value. Growth studies of one of those hybridomas (1B2.1B6) yielded a specific growth rate of 0.042 h(-1) and a doubling time of 16.5 h. Data discussed here show that at least two MAbs against TNP raised in this work can be used as a reagent for enzyme immunoassays.19647347

    Yacon (Smallanthus sonchifolius)-derived fructooligosaccharides improves the immune parameters in the mouse

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    Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Owing to its high contents of fructooligosaccharides (FOSS), the yacon (Smallanthus sonchifolius) root is used in traditional Andean medicine as a substitute for cane sugar in diabetes and for obesity prevention. This study was designed to test the hypothesis that regular consumption of yacon works to improve the immune system. BALB/c mice were fed with the AIN-93 diet supplemented with 5% commercial FOS or either 3% or 5% yacon FOS for 30 consecutive days. Animals in the control group were fed with nonsupplemented ration. Food intake; weight gain; serum levels of IgA, IgM, and IgG; levels of fecal IgA, production of nitric oxide by peritoneal macrophages, frequencies of T and B lymphocytes in the spleen and peripheral blood, T-cell proliferation, and cytokine production were evaluated in all groups. No significant differences were observed in food intake and weight gain when the experimental and control groups were compared. Also, serum levels of IgA, IgM, and IgG; nitric oxide production in peritoneal macrophages; frequencies of T and B lymphocytes in the spleen and peripheral blood; T-cell proliferation; and production of interleukin (IL)-4, interferon-gamma, IL-10, and tumor necrosis factor a did not differ in the different groups. The intake of FOS, however, led to a significant reduction of the proinflammatory cytokine IL-1 beta in macrophage cultures and elevation of the levels of fecal IgA. Together, these results indicate that the daily consumption of yacon does not exert negative effects on the immune system, helps to preserve an anti-inflammatory state in phagocytic cells, and improves mucosal immunity, possibly preventing the risks associated with autoimmune and metabolic diseases. (c) 2012 Elsevier Inc. All rights reserved.3211884892Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

    Induction of systemic tolerance in normal but not in transgenic mice through continuous feeding of ovalbumin

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    The ingestion of most dietary protein can cause systemic tolerance, and such tolerance is easier to induce in younger than in older mice. In this study, we examined whether oral tolerance to ovalbumin (OVA) could be induced in OVA-T-cell receptor (OVA-TCR)-specific transgenic mice. Continuous feeding or gavage with OVA induced tolerance, measured as reduced antibody production, in young and aged BALB/c mice, in a dose-dependent manner, but this effect was not observed in transgenic mice. Once BALB/c mice became tolerant, this state was maintained for over 44 weeks, although the tolerant state could be reversed by adoptive cell transfer. DO11.10 mice did not become tolerant upon continuous feeding with OVA, and the adoptive transfer of naive cells increased the levels of specific antibodies in their sera after antigenic challenge. The immunization schedule used here leads to a Th2-dependent antibody response in normal BALB/c mice. However, the same schedule induced both Th1- and Th2-antibody responses in transgenic mice. Dendritic cells (DC) from tolerant BALB/c mice were less efficient in the induction of the proliferation of cocultured T cells from both BALB/c and DO11.10 mice, as well as Th1 [interleukin (IL)-2 and interferon (IFN)-gamma] and Th2 (IL-4 and IL-10) cytokine production. The DC from DO11.10 transgenic mice were equally efficient in the induction of T-cell proliferation in both normal and transgenic mice, as well as in the induction of Th1 and Th2 cytokines, whether or not the mice consumed OVA. Transforming growth factor (TGF)-beta secretion was significantly lower in the supernatants of T cells from both normal and transgenic mice cocultured with DC from DO11.10 mice that had consumed OVA, while it was significantly higher in the presence of DC from normal tolerant mice, thus implicating TGF-beta as a regulatory cytokine in oral tolerance in the murine model.60325726

    Synthesis of porous macrospheres from amino-polymers

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    Porous macrospheres with controlled diameter were produced using N-methylolacrylamide and urea-formaldehyde resins synthesized in our laboratories. Hot silicon oil was used to form the porous macrospheres which were characterized by using infrared spectroscopy (FTIR), scanning electron microscopy, mercury porosimetry and surface area determination (BET). Porous macrospheres of amino-polymers were used as support in immunoassays type ELISA (enzyme linked immunosorbent assay) in which mouse immunoglobulin (IgG) was immobilized, and detected by rabbit anti-mouse immunoglobulin (a-IgG) labeled with horseradish peroxidase. Responses were obtained up to a dilution ratio of 1:4000 of the conjugate standard (1 mg/ml) and 10 mug/ml of IgG solution to recover the spheres in pH 4.5. (C) 2001 Elsevier Science Ltd. All rights reserved.381576

    Evaluation of immobilized metal membrane affinity chromatography for purification of an immunoglobulin G(1) monoclonal antibody

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    The large scale production of monoclonal antibodies (McAbs) has gaining increased relevance with the development of the hybridoma cell culture in bioreactors creating a need for specific efficient bioseparation techniques. Conventional fixed bead affinity adsorption commonly applied for McAbs purification has the drawback of low flow rates and colmatage. We developed and evaluated a immobilized metal affinity chromatographies (IMAC) affinity membrane for the purification of anti-TNP IgG(1) mouse McAbs. We immobilized metal ions on a poly(ethylene vinyl alcohol) hollow fiber membrane (Me2+-IDA-PEVA) and applied it for the purification of this McAbs from cell culture supernatant after precipitation with 50% saturation of ammonium sulphate. The purity of IgG(1) in the eluate fractions was high when eluted from Zn2+ complex. The anti-TNP antibody could be eluted under conditions causing no loss of antigen binding capacity. The purification procedure can be considered as an alternative to the biospecific adsorbent commonly applied for mouse IgG(1) purification, the protein G-Sepharose. (C) 2004 Elsevier B.V. All rights reserved.8164167125926
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