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Immunological changes in mesothelioma patients and their experimental detection.

Maeda M, Miura Y, Nishimura Y, Murakami S, Hayashi H, Kumagai N, Hatayama T, Katoh M, Miyahara N, Yamamoto S, Fukuoka K, Kishimoto T, Nakano T, Otsuki T - Clin Med Circ Respirat Pulm Med (2008)

Bottom Line: We focused on the immunological effects of asbestos and silica on the human immune system.In this brief review, we present immunological changes in patients with MM and outline their experimental detection.Analysis of the immunological effects of asbestos may help our understanding of the biological effects of asbestos.

View Article: PubMed Central - PubMed

Affiliation: Department of Hygiene, Kawasaki Medical School, 577 Matsushima, Kurashiki 7010192, Japan.

ABSTRACT
It is common knowledge that asbestos exposure causes asbestos-related diseases such as asbestosis, lung cancer and malignant mesothelioma (MM) not only in people who have handled asbestos in the work environment, but also in residents living near factories that handle asbestos. These facts have been an enormous medical and social problem in Japan since the summer of 2005. We focused on the immunological effects of asbestos and silica on the human immune system. In this brief review, we present immunological changes in patients with MM and outline their experimental detection. For example, there is over-expression of bcl-2 in CD4+ peripheral T-cells, high plasma concentrations of interleukin (IL)-10 and transforming growth factor (TGF)-ß, and multiple over-representation of T cell receptor (TcR)-Vß in peripheral CD3+ T-cells found in MM patients. We also detail an experimental long-term exposure T-cell model. Analysis of the immunological effects of asbestos may help our understanding of the biological effects of asbestos.

No MeSH data available.


Related in: MedlinePlus

Comparison of bcl-2 relative expression ratio vs. gapdh plasma concentrations in anti-inflammatory cytokines in MM patients and healthy volunteers (HV), and bcl-2 expression and secretion of these cytokines from experimental low-dose and long-term exposed T-cell models to asbestos (MT-2Org and MT-2Rst, see text for details).Panel A shows the relative expression ratio of bcl-2 in peripheral blood CD4+ cells (upper panel) from MM patients and HV, or in cultured MT-2Org and MT-2Rst cells (lower panel). Panels B and C show the plasma concentrations of IL-10 (B) and TGF-ß (C) from MM patients and HV (upper panels), or the concentrations in culture supernatants of IL-10 (B) and TGF-ß (C) from MT-2Org and MT-2Rst cells (lower panels).Peripheral blood mononuclear cells (PBMCs) were isolated from the heparinized blood of healthy donors and MM patients using a Ficoll-Hypaque density gradient (Separate-L®, Muto Pure Chemicals Co. Ltd., Tokyo, Japan). For the isolation of CD4+ T cells, PBMCs were further separated using Magnetic Cell Separation (MACS) CD4 MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The enriched cells were >90% pure as determined by flow cytometry. Specimens were taken from healthy volunteers and patients from whom informed consent had been obtained. The Institutional Ethics Committee of Kawasaki Medical School, Hyogo College of Medicine, and Okayama Rosai Hospital approved the project. A fluorescence thermocycler (Mx3000P® QPCR System, Stratagene Corporation, La Jolla, CA) was used for real-time RT-PCR experiments by following the instructions of the manufacturer. The fluorescence-labeled amplification product is measured continuously with this technique. Total RNA obtained from CD4+ T cells isolated from peripheral CD4+ T cells was extracted using an RNA Bee kit (Tel-Test, Inc., Friendswood, Texas), and 5 μg of RNA was reverse-transcribed with standard methods using a RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas International Inc., Ontario, Canada). An amount of cDNA equivalent to 50 ng of RNA served as the template for PCR in a volume of 20 μl (each primer and SYBER Premix Ex Taq, TaKaRa). The primers for bcl-2 and gapdh were added to the same reaction tube at the optimal concentration for each primer set and PCR was performed. Primers were as follows: bcl-2; 5′-TGATGTGAGTCTGGGCTGAG-3′ (Forward: Fw) and 5′-GAACGCTTTGTCCAGAGGAG-3′ (Reverse: Rv), Bax; 5′-AGTAACATGGAGCTGCAGAGG-3′ (Fw) and 5′-ATGGTTCTGATCAGTTCCGG-3′ (Rv), gapdh; 5′-GAGTCAACGGATTTGGTCGT-3′ (Fw) and 5′-TTGATTTTGGAGGGATCTCG-3′ (Rv).The relative expression of various target genes such as bcl-2 was calculated as follows when real-time RT-PCR was performed: [A: number of PCR cycles required to reach a certain intensity of fluorescence for the gapdh product. B: number of PCR cycles required to reach the same fluorescent intensity for the target gene product (bcl-2) derived from the same sample.] The relative level of the target gene is expressed as 1/2[B-A], with gapdh expression being 1.0. PCR products were confirmed to be successfully amplified by standard agarose gel electrophoresis and staining with ethidium bromide. Comparisons of the results for relative gene expression and proliferation assayed by real-time RT-PCR were analyzed using the Mann-Whitney U-test.Cytokines in plasma from MM patients and HV and culture supernatant were measured using an ELISA kit (Quantikine® Human TGF-ß1 (or IL-10) Immunoassay; R&D Systems) and the Cytometric Bead Array of Human Th1/Th2 cytokine kit II (CBA, BD Bioscience, San Jose, CA, U.S.A.), and measurements were made using FACSCalibur flow-cytometry (BD Bioscience) according to the manufacturer’s instructions.
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f1-ccrpm-2008-011: Comparison of bcl-2 relative expression ratio vs. gapdh plasma concentrations in anti-inflammatory cytokines in MM patients and healthy volunteers (HV), and bcl-2 expression and secretion of these cytokines from experimental low-dose and long-term exposed T-cell models to asbestos (MT-2Org and MT-2Rst, see text for details).Panel A shows the relative expression ratio of bcl-2 in peripheral blood CD4+ cells (upper panel) from MM patients and HV, or in cultured MT-2Org and MT-2Rst cells (lower panel). Panels B and C show the plasma concentrations of IL-10 (B) and TGF-ß (C) from MM patients and HV (upper panels), or the concentrations in culture supernatants of IL-10 (B) and TGF-ß (C) from MT-2Org and MT-2Rst cells (lower panels).Peripheral blood mononuclear cells (PBMCs) were isolated from the heparinized blood of healthy donors and MM patients using a Ficoll-Hypaque density gradient (Separate-L®, Muto Pure Chemicals Co. Ltd., Tokyo, Japan). For the isolation of CD4+ T cells, PBMCs were further separated using Magnetic Cell Separation (MACS) CD4 MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The enriched cells were >90% pure as determined by flow cytometry. Specimens were taken from healthy volunteers and patients from whom informed consent had been obtained. The Institutional Ethics Committee of Kawasaki Medical School, Hyogo College of Medicine, and Okayama Rosai Hospital approved the project. A fluorescence thermocycler (Mx3000P® QPCR System, Stratagene Corporation, La Jolla, CA) was used for real-time RT-PCR experiments by following the instructions of the manufacturer. The fluorescence-labeled amplification product is measured continuously with this technique. Total RNA obtained from CD4+ T cells isolated from peripheral CD4+ T cells was extracted using an RNA Bee kit (Tel-Test, Inc., Friendswood, Texas), and 5 μg of RNA was reverse-transcribed with standard methods using a RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas International Inc., Ontario, Canada). An amount of cDNA equivalent to 50 ng of RNA served as the template for PCR in a volume of 20 μl (each primer and SYBER Premix Ex Taq, TaKaRa). The primers for bcl-2 and gapdh were added to the same reaction tube at the optimal concentration for each primer set and PCR was performed. Primers were as follows: bcl-2; 5′-TGATGTGAGTCTGGGCTGAG-3′ (Forward: Fw) and 5′-GAACGCTTTGTCCAGAGGAG-3′ (Reverse: Rv), Bax; 5′-AGTAACATGGAGCTGCAGAGG-3′ (Fw) and 5′-ATGGTTCTGATCAGTTCCGG-3′ (Rv), gapdh; 5′-GAGTCAACGGATTTGGTCGT-3′ (Fw) and 5′-TTGATTTTGGAGGGATCTCG-3′ (Rv).The relative expression of various target genes such as bcl-2 was calculated as follows when real-time RT-PCR was performed: [A: number of PCR cycles required to reach a certain intensity of fluorescence for the gapdh product. B: number of PCR cycles required to reach the same fluorescent intensity for the target gene product (bcl-2) derived from the same sample.] The relative level of the target gene is expressed as 1/2[B-A], with gapdh expression being 1.0. PCR products were confirmed to be successfully amplified by standard agarose gel electrophoresis and staining with ethidium bromide. Comparisons of the results for relative gene expression and proliferation assayed by real-time RT-PCR were analyzed using the Mann-Whitney U-test.Cytokines in plasma from MM patients and HV and culture supernatant were measured using an ELISA kit (Quantikine® Human TGF-ß1 (or IL-10) Immunoassay; R&D Systems) and the Cytometric Bead Array of Human Th1/Th2 cytokine kit II (CBA, BD Bioscience, San Jose, CA, U.S.A.), and measurements were made using FACSCalibur flow-cytometry (BD Bioscience) according to the manufacturer’s instructions.

Mentions: As shown in the upper panel of Figure 1-A, peripheral CD4+ T cells from MM patients showed a significantly higher expression of bcl-2 compared to that of healthy volunteers (Miura et al. 2006). This may suggest that the over-expression of bcl-2 in peripheral CD4+ T cells is one of the markers for the occurrence of MM, although it should be determined whether many cancer-bearing patients respond in a similar manner. The experimental background of this finding is as follows.


Immunological changes in mesothelioma patients and their experimental detection.

Maeda M, Miura Y, Nishimura Y, Murakami S, Hayashi H, Kumagai N, Hatayama T, Katoh M, Miyahara N, Yamamoto S, Fukuoka K, Kishimoto T, Nakano T, Otsuki T - Clin Med Circ Respirat Pulm Med (2008)

Comparison of bcl-2 relative expression ratio vs. gapdh plasma concentrations in anti-inflammatory cytokines in MM patients and healthy volunteers (HV), and bcl-2 expression and secretion of these cytokines from experimental low-dose and long-term exposed T-cell models to asbestos (MT-2Org and MT-2Rst, see text for details).Panel A shows the relative expression ratio of bcl-2 in peripheral blood CD4+ cells (upper panel) from MM patients and HV, or in cultured MT-2Org and MT-2Rst cells (lower panel). Panels B and C show the plasma concentrations of IL-10 (B) and TGF-ß (C) from MM patients and HV (upper panels), or the concentrations in culture supernatants of IL-10 (B) and TGF-ß (C) from MT-2Org and MT-2Rst cells (lower panels).Peripheral blood mononuclear cells (PBMCs) were isolated from the heparinized blood of healthy donors and MM patients using a Ficoll-Hypaque density gradient (Separate-L®, Muto Pure Chemicals Co. Ltd., Tokyo, Japan). For the isolation of CD4+ T cells, PBMCs were further separated using Magnetic Cell Separation (MACS) CD4 MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The enriched cells were >90% pure as determined by flow cytometry. Specimens were taken from healthy volunteers and patients from whom informed consent had been obtained. The Institutional Ethics Committee of Kawasaki Medical School, Hyogo College of Medicine, and Okayama Rosai Hospital approved the project. A fluorescence thermocycler (Mx3000P® QPCR System, Stratagene Corporation, La Jolla, CA) was used for real-time RT-PCR experiments by following the instructions of the manufacturer. The fluorescence-labeled amplification product is measured continuously with this technique. Total RNA obtained from CD4+ T cells isolated from peripheral CD4+ T cells was extracted using an RNA Bee kit (Tel-Test, Inc., Friendswood, Texas), and 5 μg of RNA was reverse-transcribed with standard methods using a RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas International Inc., Ontario, Canada). An amount of cDNA equivalent to 50 ng of RNA served as the template for PCR in a volume of 20 μl (each primer and SYBER Premix Ex Taq, TaKaRa). The primers for bcl-2 and gapdh were added to the same reaction tube at the optimal concentration for each primer set and PCR was performed. Primers were as follows: bcl-2; 5′-TGATGTGAGTCTGGGCTGAG-3′ (Forward: Fw) and 5′-GAACGCTTTGTCCAGAGGAG-3′ (Reverse: Rv), Bax; 5′-AGTAACATGGAGCTGCAGAGG-3′ (Fw) and 5′-ATGGTTCTGATCAGTTCCGG-3′ (Rv), gapdh; 5′-GAGTCAACGGATTTGGTCGT-3′ (Fw) and 5′-TTGATTTTGGAGGGATCTCG-3′ (Rv).The relative expression of various target genes such as bcl-2 was calculated as follows when real-time RT-PCR was performed: [A: number of PCR cycles required to reach a certain intensity of fluorescence for the gapdh product. B: number of PCR cycles required to reach the same fluorescent intensity for the target gene product (bcl-2) derived from the same sample.] The relative level of the target gene is expressed as 1/2[B-A], with gapdh expression being 1.0. PCR products were confirmed to be successfully amplified by standard agarose gel electrophoresis and staining with ethidium bromide. Comparisons of the results for relative gene expression and proliferation assayed by real-time RT-PCR were analyzed using the Mann-Whitney U-test.Cytokines in plasma from MM patients and HV and culture supernatant were measured using an ELISA kit (Quantikine® Human TGF-ß1 (or IL-10) Immunoassay; R&D Systems) and the Cytometric Bead Array of Human Th1/Th2 cytokine kit II (CBA, BD Bioscience, San Jose, CA, U.S.A.), and measurements were made using FACSCalibur flow-cytometry (BD Bioscience) according to the manufacturer’s instructions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2990230&req=5

f1-ccrpm-2008-011: Comparison of bcl-2 relative expression ratio vs. gapdh plasma concentrations in anti-inflammatory cytokines in MM patients and healthy volunteers (HV), and bcl-2 expression and secretion of these cytokines from experimental low-dose and long-term exposed T-cell models to asbestos (MT-2Org and MT-2Rst, see text for details).Panel A shows the relative expression ratio of bcl-2 in peripheral blood CD4+ cells (upper panel) from MM patients and HV, or in cultured MT-2Org and MT-2Rst cells (lower panel). Panels B and C show the plasma concentrations of IL-10 (B) and TGF-ß (C) from MM patients and HV (upper panels), or the concentrations in culture supernatants of IL-10 (B) and TGF-ß (C) from MT-2Org and MT-2Rst cells (lower panels).Peripheral blood mononuclear cells (PBMCs) were isolated from the heparinized blood of healthy donors and MM patients using a Ficoll-Hypaque density gradient (Separate-L®, Muto Pure Chemicals Co. Ltd., Tokyo, Japan). For the isolation of CD4+ T cells, PBMCs were further separated using Magnetic Cell Separation (MACS) CD4 MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The enriched cells were >90% pure as determined by flow cytometry. Specimens were taken from healthy volunteers and patients from whom informed consent had been obtained. The Institutional Ethics Committee of Kawasaki Medical School, Hyogo College of Medicine, and Okayama Rosai Hospital approved the project. A fluorescence thermocycler (Mx3000P® QPCR System, Stratagene Corporation, La Jolla, CA) was used for real-time RT-PCR experiments by following the instructions of the manufacturer. The fluorescence-labeled amplification product is measured continuously with this technique. Total RNA obtained from CD4+ T cells isolated from peripheral CD4+ T cells was extracted using an RNA Bee kit (Tel-Test, Inc., Friendswood, Texas), and 5 μg of RNA was reverse-transcribed with standard methods using a RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas International Inc., Ontario, Canada). An amount of cDNA equivalent to 50 ng of RNA served as the template for PCR in a volume of 20 μl (each primer and SYBER Premix Ex Taq, TaKaRa). The primers for bcl-2 and gapdh were added to the same reaction tube at the optimal concentration for each primer set and PCR was performed. Primers were as follows: bcl-2; 5′-TGATGTGAGTCTGGGCTGAG-3′ (Forward: Fw) and 5′-GAACGCTTTGTCCAGAGGAG-3′ (Reverse: Rv), Bax; 5′-AGTAACATGGAGCTGCAGAGG-3′ (Fw) and 5′-ATGGTTCTGATCAGTTCCGG-3′ (Rv), gapdh; 5′-GAGTCAACGGATTTGGTCGT-3′ (Fw) and 5′-TTGATTTTGGAGGGATCTCG-3′ (Rv).The relative expression of various target genes such as bcl-2 was calculated as follows when real-time RT-PCR was performed: [A: number of PCR cycles required to reach a certain intensity of fluorescence for the gapdh product. B: number of PCR cycles required to reach the same fluorescent intensity for the target gene product (bcl-2) derived from the same sample.] The relative level of the target gene is expressed as 1/2[B-A], with gapdh expression being 1.0. PCR products were confirmed to be successfully amplified by standard agarose gel electrophoresis and staining with ethidium bromide. Comparisons of the results for relative gene expression and proliferation assayed by real-time RT-PCR were analyzed using the Mann-Whitney U-test.Cytokines in plasma from MM patients and HV and culture supernatant were measured using an ELISA kit (Quantikine® Human TGF-ß1 (or IL-10) Immunoassay; R&D Systems) and the Cytometric Bead Array of Human Th1/Th2 cytokine kit II (CBA, BD Bioscience, San Jose, CA, U.S.A.), and measurements were made using FACSCalibur flow-cytometry (BD Bioscience) according to the manufacturer’s instructions.
Mentions: As shown in the upper panel of Figure 1-A, peripheral CD4+ T cells from MM patients showed a significantly higher expression of bcl-2 compared to that of healthy volunteers (Miura et al. 2006). This may suggest that the over-expression of bcl-2 in peripheral CD4+ T cells is one of the markers for the occurrence of MM, although it should be determined whether many cancer-bearing patients respond in a similar manner. The experimental background of this finding is as follows.

Bottom Line: We focused on the immunological effects of asbestos and silica on the human immune system.In this brief review, we present immunological changes in patients with MM and outline their experimental detection.Analysis of the immunological effects of asbestos may help our understanding of the biological effects of asbestos.

View Article: PubMed Central - PubMed

Affiliation: Department of Hygiene, Kawasaki Medical School, 577 Matsushima, Kurashiki 7010192, Japan.

ABSTRACT
It is common knowledge that asbestos exposure causes asbestos-related diseases such as asbestosis, lung cancer and malignant mesothelioma (MM) not only in people who have handled asbestos in the work environment, but also in residents living near factories that handle asbestos. These facts have been an enormous medical and social problem in Japan since the summer of 2005. We focused on the immunological effects of asbestos and silica on the human immune system. In this brief review, we present immunological changes in patients with MM and outline their experimental detection. For example, there is over-expression of bcl-2 in CD4+ peripheral T-cells, high plasma concentrations of interleukin (IL)-10 and transforming growth factor (TGF)-ß, and multiple over-representation of T cell receptor (TcR)-Vß in peripheral CD3+ T-cells found in MM patients. We also detail an experimental long-term exposure T-cell model. Analysis of the immunological effects of asbestos may help our understanding of the biological effects of asbestos.

No MeSH data available.


Related in: MedlinePlus