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Tsuguhiro Kaneda - One of the best experts on this subject based on the ideXlab platform.

  • New estimation method for highly sensitive quantitation of human immunodeficiency virus type 1 DNA and its application.
    Journal of Virological Methods, 2005
    Co-Authors: Hiromi Nagai, Kaoru Wada, Takayuki Morishita, Makoto Utsumi, Yukihiro Nishiyama, Tsuguhiro Kaneda
    Abstract:

    Abstract A new estimation method for quantitation of HIV-1 DNA was established by introducing a pre-quantitation polymerase chain reaction (PCR) before conventional real-time PCR. Two alternative methods for estimating the copy number can be used: the first method utilizes the rate of β2-microglobulin (β2M) gene amplification during the pre-quantitation PCR, and the second utilizes a calibration curve of the crossing point of real-time PCR versus the standard HIV-1-plasmid concentration. These methods could be used to reproducibly and accurately detect a provirus density down to five copies/106 cells (for methods 1 and 2, inter-assay CV = 17 and 16% and accuracy = 81 and 92%, respectively). The levels of HIV-1 DNA could be measurable using as little as 100 μl of whole blood or buffy coat cells. Using a combination of a conventional and highly sensitive methods, we found that the amount of HIV-1 DNA ranged from 2 to 5960 copies/106 cells (median of 830 copies/106 cells) in CD4-positive T lymphocytes isolated from 30 patients responding well to highly active antiretroviral therapy (HAART). Thus, the highly sensitive method developed in this study allows estimation of the HIV-1 reservoirs in peripheral CD4-positive T lymphocytes of patients responding well to HAART.

  • New estimation method for highly sensitive quantitation of human immunodeficiency virus type 1 DNA and its application.
    Journal of Virological Methods, 2005
    Co-Authors: Hiromi Nagai, Kaoru Wada, Takayuki Morishita, Makoto Utsumi, Yukihiro Nishiyama, Tsuguhiro Kaneda
    Abstract:

    A new estimation method for quantitation of HIV-1 DNA was established by introducing a pre-quantitation polymerase chain reaction (PCR) before conventional real-time PCR. Two alternative methods for estimating the copy number can be used: the first method utilizes the rate of beta2-microglobulin (beta2M) gene amplification during the pre-quantitation PCR, and the second utilizes a calibration curve of the crossing point of real-time PCR versus the standard HIV-1-plasmid concentration. These methods could be used to reproducibly and accurately detect a provirus density down to five copies/10(6) cells (for methods 1 and 2, inter-assay CV=17 and 16% and accuracy=81 and 92%, respectively). The levels of HIV-1 DNA could be measurable using as little as 100 microl of whole blood or buffy coat cells. Using a combination of a conventional and highly sensitive methods, we found that the amount of HIV-1 DNA ranged from 2 to 5960 copies/10(6) cells (median of 830 copies/10(6) cells) in CD4-positive T lymphocytes isolated from 30 patients responding well to highly active antiretroviral therapy (HAART). Thus, the highly sensitive method developed in this study allows estimation of the HIV-1 reservoirs in peripheral CD4-positive T lymphocytes of patients responding well to HAART.

Ira Mellman - One of the best experts on this subject based on the ideXlab platform.

  • mhc class ii distribution in dendritic cells and b cells is determined by ubiquitin chain length
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Jessica K, Mia Y Platt, Jeffrey Easthamanderson, Jeoungsook Shin, Ira Mellman
    Abstract:

    Abstract Dendritic cells (DCs) and B cells present antigen-derived peptides bound to MHC class II (MHC II) molecules for recognition by CD4-positive T lymphocytes. DCs control the intracellular traffic of peptide–MHC II complexes by regulating the ubiquitination of MHC II. In resting or “immature” DCs, ubiquitinated MHC II molecules are targeted to lysosomes, but upon pathogen-induced “maturation,” ubiquitination is down-regulated and MHC II can accumulate on the plasma membrane of mature DCs. Although B cells constitutively ubiquitinate their MHC II, it unexpectedly remains at the surface. We find that DCs and B cells differ in MHC II-conjugated ubiquitin (Ub) chain length: four to six Ub in immature DCs vs. two to three in B cells. In both cell types, experimentally increasing Ub chain length led to efficient lysosomal transport of MHC II, whereas MHC II with fewer than two Ubs did not reach lysosomes. Thus, Ub chain length plays a crucial role in regulating the intracellular fate and function of MHC II in DCs and B cells.

Peter Cresswell - One of the best experts on this subject based on the ideXlab platform.

  • MHC class II-restricted presentation of the major house dust mite allergen Der p 1 Is GILT-dependent: implications for allergic asthma.
    PLoS ONE, 2013
    Co-Authors: Laura Ciaccia West, Jeff E. Grotzke, Peter Cresswell
    Abstract:

    Gamma-interferon-inducible lysosomal thiol reductase (GILT) is known to reduce disulfide bonds present in proteins internalized by antigen presenting cells, facilitating optimal processing and presentation of peptides on Major Histocompatibility Complex class II molecules, as well as the subsequent activation of CD4-positive T lymphocytes. Here, we show that GILT is required for class II-restricted processing and presentation of immunodominant epitopes from the major house dust mite allergen Der p 1. In the absence of GILT, CD4-positive T cell responses to Der p 1 are significantly reduced, resulting in mitigated allergic airway inflammation in response to Der p 1 and house dust mite extracts in a murine model of asthma.

  • Defective Antigen Processing in GILT-Free Mice
    Science, 2001
    Co-Authors: Maja Maric, Balasubramanian Arunachalam, Uyen T. Phan, Chen Dong, Wendy S. Garrett, Kurt S. Cannon, Christopher Alfonso, Lars Karlsson, Richard A. Flavell, Peter Cresswell
    Abstract:

    Processing of proteins for major histocompatibility complex (MHC) class II-restricted presentation to CD4-positive T lymphocytes occurs after they are internalized by antigen-presenting cells (APCs). Antigenic proteins frequently contain disulfide bonds, and their reduction in the endocytic pathway facilitates processing. In humans, a gamma interferon-inducible lysosomal thiol reductase (GILT) is constitutively present in late endocytic compartments of APCs. Here, we identified the mouse homolog of GILT and generated a GILT knockout mouse. GILT facilitated the processing and presentation to antigen-specific T cells of protein antigens containing disulfide bonds. The response to hen egg lysozyme, a model antigen with a compact structure containing four disulfide bonds, was examined in detail.

  • formation of a nine subunit complex by hla class ii glycoproteins and the invariant chain
    Nature, 1991
    Co-Authors: Michael S Marks, Paul A Roche, Peter Cresswell
    Abstract:

    HLA CLASS II molecules are heterodimeric transmembrane glycoproteins that bind and present processed antigenic peptides to CD4-positive T lymphocytes. Intracellularly, class II molecules associate with a third subunit termed the invariant (I) chain. Here we describe the physical characteristics of the intracellular class II αβI complex. Chemical crosslinking, size exclusion chromatography and sedimentation velocity studies demonstrate that the αβI complex is a nine-subunit transmembrane protein that contains three αβ dimers associated with an I chain trimer. The organization of class II α-and β-subunits in such a multimer may have a role in the documented ability of the I chain to inhibit peptide binding to class II molecules1–3. In addition, the formation of the nine-chain complex may induce the structural changes necessary to overcome the cytoplasmic retention signal responsible for the localization of free I chain in the endoplasmic reticulum, releasing class II-I chain complexes for transport to endosomes4–6.

Paul A Roche - One of the best experts on this subject based on the ideXlab platform.

  • formation of a nine subunit complex by hla class ii glycoproteins and the invariant chain
    Nature, 1991
    Co-Authors: Michael S Marks, Paul A Roche, Peter Cresswell
    Abstract:

    HLA CLASS II molecules are heterodimeric transmembrane glycoproteins that bind and present processed antigenic peptides to CD4-positive T lymphocytes. Intracellularly, class II molecules associate with a third subunit termed the invariant (I) chain. Here we describe the physical characteristics of the intracellular class II αβI complex. Chemical crosslinking, size exclusion chromatography and sedimentation velocity studies demonstrate that the αβI complex is a nine-subunit transmembrane protein that contains three αβ dimers associated with an I chain trimer. The organization of class II α-and β-subunits in such a multimer may have a role in the documented ability of the I chain to inhibit peptide binding to class II molecules1–3. In addition, the formation of the nine-chain complex may induce the structural changes necessary to overcome the cytoplasmic retention signal responsible for the localization of free I chain in the endoplasmic reticulum, releasing class II-I chain complexes for transport to endosomes4–6.

A Potena - One of the best experts on this subject based on the ideXlab platform.

  • CD4-positive T-Lymphocytes infiltrate the bronchial mucosa of patients with Sjögren's syndrome.
    American journal of respiratory and critical care medicine, 1997
    Co-Authors: S A Papiris, M Saetta, G Turato, R La Corte, L Trevisani, C E Mapp, P Maestrelli, L M Fabbri, A Potena
    Abstract:

    To investigate the degree and the type of inflammation in the bronchial mucosa in patients with Sjögren's syndrome, we examined lobar bronchial biopsies obtained from 10 patients with Sjögren's syndrome (six with primary and four with secondary) and eight control subjects. Histochemistry with hematoxylin-eosin was performed both to identify the number of mononuclear cells and eosinophils and to measure the thickness of the basement membrane. Immunohistochemistry was performed to identify neutrophils (neutrophil-elastase), macrophages (CD68), and T-lymphocyte subpopulations (CD4 and CD8) in the submucosa. Subjects with Sjögren's syndrome presented a greater number of CD4-positive T-Lymphocytes than did the normal control subjects (p = 0.0129). Instead, eosinophils, neutrophils, macrophages, CD8 positive T-Lymphocytes, and basement membrane thickness were similar in the two groups. There were no differences in cell counts between patients with primary and those with secondary Sjögren's syndrome and between symptomatic and asymptomatic patients. No correlation was found between cell counts, symptoms, lung volumes, and disease duration. This study has shown that patients with Sjögren's syndrome have an increased number of CD4 positive T-Lymphocytes in the bronchial mucosa outside of the bronchial glands, supporting the concept that, in the airways. Sjögren's syndrome involves also extraglandular tissues.

  • CD4-positive T-Lymphocytes Infiltrate the Bronchial Mucosa of Patients with Sjögren's Syndrome
    American Journal of Respiratory and Critical Care Medicine, 1997
    Co-Authors: S A Papiris, M Saetta, G Turato, R La Corte, L Trevisani, C E Mapp, P Maestrelli, L M Fabbri, A Potena
    Abstract:

    To investigate the degree and the type of inflammation in the bronchial mucosa in patients with Sjogren's syndrome, we examined lobar bronchial biopsies obtained from 10 patients with Sjogren's syndrome (six with primary and four with secondary) and eight control subjects. Histochemistry with hematoxylin-eosin was performed both to identify the number of mononuclear cells and eosinophils and to measure the thickness of the basement membrane. Immunohistochemistry was performed to identify neutrophils (neutrophil-elastase), macrophages (CD68), and T-lymphocyte subpopulations (CD4 and CD8) in the submucosa. Subjects with Sjogren's syndrome presented a greater number of CD4-positive T-Lymphocytes than did the normal control subjects (p = 0.0129). Instead, eosinophils, neutrophils, macrophages, CD8 positive T-Lymphocytes, and basement membrane thickness were similar in the two groups. There were no differences in cell counts between patients with primary and those with secondary Sjogren's syndrome and between ...