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Recording Phagosome Maturation Through the Real-Time, Spectrofluorometric Measurement of Hydrolytic Activities

The efficient degradation of internalized particulate matter is a principal objective of the macrophage’s phagosome. Assessment of the true hydrolytic capacity within the phagosomal lumen is often difficult as it is subject to many factors beyond recruitment of lysosomal hydrola ...

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Genetic Modification of Murine Dendritic Cells by RNA Transfection

The ability to manipulate in vitro cultured dendritic cells (DCs) by transfection represents an attractive strategy to load these antigen-presenting cells with genetic material encoding various immunogenic epitopes. The gene transfer approach can also be applied to DCs with the aim ...

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Tracing Tolerance and Immunity In Vivo by CFSE-Labeling of Administered Cells

Tracking antigen-specific cytotoxic T lymphocyte (CTL) function in vivo can be difficult due to the need to monitor the presence and subsequent destruction of antigen-bearing target cells. In this report, we describe a simple method using the fluorescent dye 5-(and 6-) carboxyfluoresce ...

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Intravital Two-Photon Imaging of T-Cell Priming and Tolerance in the Lymph Node

Two-photon microscopy makes it possible to image in real-time fluorescently labeled cells located in deep tissue environments. We describe a procedure to visualize the behavior of lymph node T cells during either priming or tolerance, in live, anesthetized mice. Intravital imaging of T ly ...

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Quantification of Immunotoxin Number for Complete Therapeutic Response

Radiolabeled antibodies are commonly administered to patients, and their biodistribution to tumor and normal tissues is routinely quantitated by nuclear medicine scanning for dosimetry (1). Only a few studies have examined the uptake of radiolabeled immunotoxins into tumors or n ...

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Tracing the Route Taken by Peptides and Major Histocompatibility Complex Class I Molecules in Presentation of Exogenous Antigens

Since the discovery of cross priming by Bevan (1) nearly thirty years ago, a large amount of work has focused on defining the mechanisms that account for this in vivo phenomenon. Following the discovery that the majority of major histocompatibility complex (MHC-I)-bound peptides are derived f ...

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Thymic Involution: Implications for Self-Tolerance

The thymus contributes to the regulation of tolerance and the prevention of autoimmunity at many levels. First, auto-reactive CD4+ and CD8+ T cells are clonally deleted during negative selection in the thymus, establishing central tolerance. The unique expression of the AIRE (autoimmu ...

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Inducing Mixed Chimerism and Transplantation Tolerance Through Allogeneic Bone Marrow Transplantation With Costimulation Blockade

Induction of mixed chimerism (i.e., coexistence of donor and recipient hematopoietic cells) through transplantation of allogeneic donor bone marrow under appropriate host conditioning, is one of the most reliable strategies to induce transplantation tolerance. Robust toler ...

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Induction of Dominant Tolerance Using Monoclonal Antibodies

Monoclonal antibodies (MAb) have been shown to be effective in inducing immune tolerance in transplantation and autoimmunity. Several different MAb have tolerogenic properties and their effect has been studied in a range of experimental animal models and, in some cases, in clinical tri ...

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Induction of Tolerance by Adoptive Transfer of Treg Cells

Naturally arising CD4+CD25+ regulatory T (Treg) cells can be exploited to establish immunologic tolerance to allogeneic transplants. In vivo exposure of CD4+CD25+ T cells from normal na�ve mice to alloantigen in a T cell-deficient environment elicits spontaneous expansion of alloan ...

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Modulation of the Immune Response Using Dendritic Cell-Derived Exosomes

Initial studies in our laboratory were focused on the use of dendritic cells (DC) genetically modified to express Th2-derived cytokines (i.e., interleukin -4 and IL-10) or apoptotic proteins (i.e., Fas Ligand ) to reduce inflammation in a mouse model of experimentally induced arthritis. Exo ...

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Breaking Self-Tolerance to Tumor-Associated Antigens by In Vivo Manipulation of Dendritic Cells

Dendritic cells (DC) are extremely potent antigen-presenting cells, which can prime both na�ve CD4+ and CD8+ T lymphocytes. In their immature state, DC continuously sample and process antigens from the surrounding environment, but only mature DC express sufficient levels of costimula ...

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Identification of Tumor-Associated Autoantigens With SEREX

Serological analysis of tumor antigens by recombinant cDNA expression cloning (SEREX) allows the systematic cloning of tumor antigens recognized by the spontaneous autoantibody repertoire of cancer patients. For SEREX, cDNA expression libraries are constructed from fresh t ...

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Dendritic Cells in Clinical Trials for Multiple Myeloma

Due to the existence of the truly specific tumor antigen idiotype in multiple myeloma and based on encouraging data from dendritic cell vaccinated B-cell non-Hodgkin’s lymphoma (NHL) patients, dendritic cell-based vaccination was first initiated in myeloma patients in 1995. This ove ...

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Application of Proteomics and Protein Analysis for Biomarker and Target Finding for Immunotherapy

Regulatory T-cells play a central role in the maintenance of the immunological balance and are powerful inhibitors of T-cell activation both in vivo and in vitro. The enhancement of suppressor-cell function might be a target for immunotherapeutic approaches for the treatment of immune- ...

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Isolation and Expansion of Tumor-Reactive Cytotoxic T-Cell Clones for Adoptive Immunotherapy

Attempts to treat patients with tumor-reactive cytotoxic T-lymphocytes (CTL) have been limited. This is due to the difficulty of isolating and expanding functionally active T-cells, which are present at extremely low frequencies in the peripheral blood. Recently developed multim ...

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Tracking Adoptively Transferred Antigen-Specific T-Cells With Peptide/MHC Multimers

Advances in immunological monitoring provide the means to track tumor antigen-specific cytotoxic T-lymphocytes (CTL) in humans after adoptive transfer with greater specificity and sensitivity than before. Novel tools can be used not only to detect antigen-specific CTL, but also to e ...

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Gene Transfer of MHC-Restricted Receptors

Adoptive therapy with allogeneic or tumor-specific T-cells has shown substantial clinical effects for several human tumors, but the widespread application of this strategy remains a daunting task. The antigen specificity of T-lymphocytes is solely determined by the T-cell recept ...

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Immunotherapy With CTL Restricted by Nonself MHC

In the past years a number of target antigens recognized by cytotoxic T-lymphocytes (CTL) have been identified in human malignancies. In most cases, the CTL-recognized antigens did not arise from mutations, but were instead encoded by genes that were identical in normal and tumor cells. Gene ex ...

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Designing TCR for Cancer Immunotherapy

Reprogramming T-cell populations by T-cell receptor (TCR) gene transfer is a new therapeutic tool for adoptive tumor immunotherapy. Gene transfer of human leukocyte antigen (HLA)-transgenic mice-derived TCR into human T-cells allows the circumvention of tolerance to tumor-ass ...

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