The Experts below are selected from a list of 1935 Experts worldwide ranked by ideXlab platform

Tjakko Abee - One of the best experts on this subject based on the ideXlab platform.

  • a novel method for continuous determination of the intracellular ph in bacteria with the internally conjugated fluorescent probe 5 and 6 Carboxyfluorescein Succinimidyl Ester
    Applied and Environmental Microbiology, 1996
    Co-Authors: Pieter Breeuwer, Frank M. Rombouts, Jean-louis Drocourt, Tjakko Abee
    Abstract:

    A novel method based on the intracellular conjugation of the fluorescent probe 5 (and 6-)-Carboxyfluorescein Succinimidyl Ester (cFSE) was developed to determine the intracellular pH of bacteria. cFSE can be taken up by bacteria in the form of its diacetate Ester, 5 (and 6-)-Carboxyfluorescein diacetate Succinimidyl Ester, which is subsequently hydrolyzed by Esterases to cFSE in the cytoplasm. When Lactococcus lactis cells were permeabilized with ethanol, a significant proportion of cFSE was retained in the cells, which indicated that cFSE was bound intracellularly. Unbound probe could be conveniently extruded by a short incubation of the cells in the presence of a fermentable sugar, most likely by exploiting an active transport system. Such a transport system for cFSE was identified in L. lactis, Listeria innocua, and Bacillus subtilis. The intracellular pH in bacteria can be determined from the ratio of the fluorescence signal at the pH-sensitive wavelength (490 nm) and the fluorescence signal at the pH-insensitive wavelength (440 nm). This cFSE ratio method significantly reduced problems due to the efflux of fluorescent probe from the cells during the measurement. Moreover, the method described was successfully used to determine the intracellular pH in bacteria under stress conditions, such as elevated temperatures and the presence of detergents.

Atsuo Urisu - One of the best experts on this subject based on the ideXlab platform.

  • allergen specific t cell response in patients with phenytoin hypersensitivity simultaneous analysis of proliferation and cytokine production by Carboxyfluorescein Succinimidyl Ester cfse dilution assay
    Allergology International, 2007
    Co-Authors: Ikuya Tsuge, Akihisa Okumura, Yasuto Kondo, Seiko Itomi, Michiko Kakami, Makiko Kawamura, Yoichi Nakajima, R Komatsubara, Atsuo Urisu
    Abstract:

    ABSTRACT Background Phenytoin can induce diversified adverse reactions including generalized eruptions and the hypersensitivity syndrome. Delayed-type allergic mechanisms have been postulated to underlie these reactions. The tests most widely used to detect T-cell sensitization to drugs are the patch test and the lymphocyte transformation test (LTT), but their sensitivity is not sufficient. Simultaneous assessment of both the frequencies and the cytokine-producing phenotypes of allergen-specific T cells has become possible with the recently introduced Carboxyfluorescein Succinimidyl Ester (CFSE) assay. Methods Seven patients who presented with phenytoin-induced maculopapular exanthema with and without fever were included in this study. Peripheral blood mononuclear cells (PBMCs) were labeled with CFSE and cultured with phenytoin for seven days. The cells were stained with anti-CD4 and cytokine-specific monoclonal antibodies (MoAbs), and analyzed with FACSCalibur. Results The phenytoin-specific proliferation of CD4 + cells in patients was significantly higher than in the four controls exposed to phenytoin, and in seven healthy children with no previous phenytoin intake. A significant difference in the percentages of CD4 + IFN-γ + cells between patients and the seven healthy children was observed. The sensitivity and specificity of proliferation were 100% and 90.9%, and those of IFN-γ secretion were 71.4% and 100%, respectively. Conclusions Phenytoin-specific proliferation may be detected with greater sensitivity by the CFSE dilution assay than the conventional LTT. The assay revealed that both CD4 + and CD4 − T cells proliferated and produced IFN-γ and TNF-α after stimulation with phenytoin. The CFSE dilution assay might be useful for the diagnosis and understanding of drug hypersensitivity.

  • allergen specific helper t cell response in patients with cow s milk allergy simultaneous analysis of proliferation and cytokine production by Carboxyfluorescein Succinimidyl Ester dilution assay
    Clinical & Experimental Allergy, 2006
    Co-Authors: Ikuya Tsuge, Yasuto Kondo, Michiko Kakami, Makiko Kawamura, Yoichi Nakajima, R Komatsubara, Reiko Tokuda, K Yamada, Atsuo Urisu
    Abstract:

    Summary Background The role of antigen-specific T cells in the allergic reaction to cow's milk or in tolerance induction is not yet fully understood. Objective This study was designed to analyse both cow's milk protein (CMP)-specific T cell proliferation and cytokine production simultaneously in children with cow's milk allergy (CMA) in comparison with subjects with various allergic backgrounds. Methods Carboxyfluorescein Succinimidyl Ester was used to detect cow's milk-specific T cells by flow cytometry. The intra-cytoplasmic cytokine production of these antigen-specific T cells was also analysed. Results Significant differences of both CMP-specific CD4+ cell proliferation and cytokine production between CMA and non-allergic children were observed. While the proliferative responses of children who recently outgrew CMA were not significantly different from those of patients, the patterns of cytokine production were similar to those of non-allergic children. Conclusion These results suggest that the presence of CMP-specific T cell clones per se does not produce CMA, but that the T-helper type 2-skewed pattern of those T cells is associated with adverse reactions. Although it is not possible to distinguish between individual patients with and without CMA on the basis of CFSE assays, these results contribute to the understanding of the pathogenesis and tolerance induction of CMA.

Ikuya Tsuge - One of the best experts on this subject based on the ideXlab platform.

  • Phenytoin desensitization monitored by antigen specific T cell response using Carboxyfluorescein Succinimidyl Ester dilution assay.
    European journal of paediatric neurology : EJPN : official journal of the European Paediatric Neurology Society, 2007
    Co-Authors: Akihisa Okumura, Ikuya Tsuge, Tetsuo Kubota, Hirokazu Kurahashi, Jun Natsume, Tamiko Negoro, Kazuyoshi Watanabe
    Abstract:

    We evaluated drug-specific T cell responses in a patient with refractory partial seizures and paroxysmal kinesigenic choreoathetosis successfully treated with clinical desensitization to phenytoin. Drug-induced lymphocyte transformation test before desensitization was negative with a stimulation index of 130%. The frequencies and cytokine-producing phenotypes of phenytoin-specific T cells were examined simultaneously by using a Carboxyfluorescein Succinimidyl Ester (CFSE) dilution assay. Before desensitization, the proportion of CFSElow CD4+ cells in whole CD4+ was 3.09%; 13.6% of CFSElow CD4+ cells were stained with anti-interferon gamma antibody. After desensitization, phenytoin-specific CFSElow CD4+ cells decreased to background level. These results indicate that CFSE dilution assay will be useful for the diagnosis and monitoring of drug hypersensitivity.

  • allergen specific t cell response in patients with phenytoin hypersensitivity simultaneous analysis of proliferation and cytokine production by Carboxyfluorescein Succinimidyl Ester cfse dilution assay
    Allergology International, 2007
    Co-Authors: Ikuya Tsuge, Akihisa Okumura, Yasuto Kondo, Seiko Itomi, Michiko Kakami, Makiko Kawamura, Yoichi Nakajima, R Komatsubara, Atsuo Urisu
    Abstract:

    ABSTRACT Background Phenytoin can induce diversified adverse reactions including generalized eruptions and the hypersensitivity syndrome. Delayed-type allergic mechanisms have been postulated to underlie these reactions. The tests most widely used to detect T-cell sensitization to drugs are the patch test and the lymphocyte transformation test (LTT), but their sensitivity is not sufficient. Simultaneous assessment of both the frequencies and the cytokine-producing phenotypes of allergen-specific T cells has become possible with the recently introduced Carboxyfluorescein Succinimidyl Ester (CFSE) assay. Methods Seven patients who presented with phenytoin-induced maculopapular exanthema with and without fever were included in this study. Peripheral blood mononuclear cells (PBMCs) were labeled with CFSE and cultured with phenytoin for seven days. The cells were stained with anti-CD4 and cytokine-specific monoclonal antibodies (MoAbs), and analyzed with FACSCalibur. Results The phenytoin-specific proliferation of CD4 + cells in patients was significantly higher than in the four controls exposed to phenytoin, and in seven healthy children with no previous phenytoin intake. A significant difference in the percentages of CD4 + IFN-γ + cells between patients and the seven healthy children was observed. The sensitivity and specificity of proliferation were 100% and 90.9%, and those of IFN-γ secretion were 71.4% and 100%, respectively. Conclusions Phenytoin-specific proliferation may be detected with greater sensitivity by the CFSE dilution assay than the conventional LTT. The assay revealed that both CD4 + and CD4 − T cells proliferated and produced IFN-γ and TNF-α after stimulation with phenytoin. The CFSE dilution assay might be useful for the diagnosis and understanding of drug hypersensitivity.

  • allergen specific helper t cell response in patients with cow s milk allergy simultaneous analysis of proliferation and cytokine production by Carboxyfluorescein Succinimidyl Ester dilution assay
    Clinical & Experimental Allergy, 2006
    Co-Authors: Ikuya Tsuge, Yasuto Kondo, Michiko Kakami, Makiko Kawamura, Yoichi Nakajima, R Komatsubara, Reiko Tokuda, K Yamada, Atsuo Urisu
    Abstract:

    Summary Background The role of antigen-specific T cells in the allergic reaction to cow's milk or in tolerance induction is not yet fully understood. Objective This study was designed to analyse both cow's milk protein (CMP)-specific T cell proliferation and cytokine production simultaneously in children with cow's milk allergy (CMA) in comparison with subjects with various allergic backgrounds. Methods Carboxyfluorescein Succinimidyl Ester was used to detect cow's milk-specific T cells by flow cytometry. The intra-cytoplasmic cytokine production of these antigen-specific T cells was also analysed. Results Significant differences of both CMP-specific CD4+ cell proliferation and cytokine production between CMA and non-allergic children were observed. While the proliferative responses of children who recently outgrew CMA were not significantly different from those of patients, the patterns of cytokine production were similar to those of non-allergic children. Conclusion These results suggest that the presence of CMP-specific T cell clones per se does not produce CMA, but that the T-helper type 2-skewed pattern of those T cells is associated with adverse reactions. Although it is not possible to distinguish between individual patients with and without CMA on the basis of CFSE assays, these results contribute to the understanding of the pathogenesis and tolerance induction of CMA.

Pieter Breeuwer - One of the best experts on this subject based on the ideXlab platform.

  • a novel method for continuous determination of the intracellular ph in bacteria with the internally conjugated fluorescent probe 5 and 6 Carboxyfluorescein Succinimidyl Ester
    Applied and Environmental Microbiology, 1996
    Co-Authors: Pieter Breeuwer, Frank M. Rombouts, Jean-louis Drocourt, Tjakko Abee
    Abstract:

    A novel method based on the intracellular conjugation of the fluorescent probe 5 (and 6-)-Carboxyfluorescein Succinimidyl Ester (cFSE) was developed to determine the intracellular pH of bacteria. cFSE can be taken up by bacteria in the form of its diacetate Ester, 5 (and 6-)-Carboxyfluorescein diacetate Succinimidyl Ester, which is subsequently hydrolyzed by Esterases to cFSE in the cytoplasm. When Lactococcus lactis cells were permeabilized with ethanol, a significant proportion of cFSE was retained in the cells, which indicated that cFSE was bound intracellularly. Unbound probe could be conveniently extruded by a short incubation of the cells in the presence of a fermentable sugar, most likely by exploiting an active transport system. Such a transport system for cFSE was identified in L. lactis, Listeria innocua, and Bacillus subtilis. The intracellular pH in bacteria can be determined from the ratio of the fluorescence signal at the pH-sensitive wavelength (490 nm) and the fluorescence signal at the pH-insensitive wavelength (440 nm). This cFSE ratio method significantly reduced problems due to the efflux of fluorescent probe from the cells during the measurement. Moreover, the method described was successfully used to determine the intracellular pH in bacteria under stress conditions, such as elevated temperatures and the presence of detergents.

Jean-louis Drocourt - One of the best experts on this subject based on the ideXlab platform.

  • a novel method for continuous determination of the intracellular ph in bacteria with the internally conjugated fluorescent probe 5 and 6 Carboxyfluorescein Succinimidyl Ester
    Applied and Environmental Microbiology, 1996
    Co-Authors: Pieter Breeuwer, Frank M. Rombouts, Jean-louis Drocourt, Tjakko Abee
    Abstract:

    A novel method based on the intracellular conjugation of the fluorescent probe 5 (and 6-)-Carboxyfluorescein Succinimidyl Ester (cFSE) was developed to determine the intracellular pH of bacteria. cFSE can be taken up by bacteria in the form of its diacetate Ester, 5 (and 6-)-Carboxyfluorescein diacetate Succinimidyl Ester, which is subsequently hydrolyzed by Esterases to cFSE in the cytoplasm. When Lactococcus lactis cells were permeabilized with ethanol, a significant proportion of cFSE was retained in the cells, which indicated that cFSE was bound intracellularly. Unbound probe could be conveniently extruded by a short incubation of the cells in the presence of a fermentable sugar, most likely by exploiting an active transport system. Such a transport system for cFSE was identified in L. lactis, Listeria innocua, and Bacillus subtilis. The intracellular pH in bacteria can be determined from the ratio of the fluorescence signal at the pH-sensitive wavelength (490 nm) and the fluorescence signal at the pH-insensitive wavelength (440 nm). This cFSE ratio method significantly reduced problems due to the efflux of fluorescent probe from the cells during the measurement. Moreover, the method described was successfully used to determine the intracellular pH in bacteria under stress conditions, such as elevated temperatures and the presence of detergents.