Heymann nephritis in lewis rats.
Authors: Wang YM, Lee VW, Wu H, Harris DC, Alexander SI
Abstract
Human membranous nephritis is a major cause of end-stage kidney disease. Active Heymann nephritis (HN) is an auto-immune model of membranous nephritis induced in Lewis rats by immunization with a crude renal tubular antigen (Fx1A) or megalin (gp330). The pathogenesis of HN is through the binding of anti-Fx1A autoantibodies to the auto-antigen expressed on glomerular epithelial cells, resulting in severe glomerular injury and proteinuria. The pathological features of HN include immune deposits in glomeruli and infiltration of glomeruli and the...
Source: Current Protocols in Immunology - April 9, 2015 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Use of proteasome inhibitors.
Authors: Downey SL, Florea BI, Overkleeft HS, Kisselev AF
Abstract
Proteasome inhibitors are indispensable research tools in immunology and cell biology. With numerous proteasome inhibitors available commercially, choosing the appropriate compound for a biological experiment may be challenging, especially for a novice. This unit provides an overview of the proteasome inhibitors commonly used in research. It discusses how to select an appropriate highly specific inhibitor, its concentration, and length of exposure for mammalian cell culture experiments. In addition, assays that can be used to confirm protea...
Source: Current Protocols in Immunology - April 9, 2015 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Adriamycin nephropathy in BALB/c mice.
Authors: Wang YM, Wang Y, Harris DC, Alexander SI, Lee VW
Abstract
Chronic proteinuric renal injury is a major cause of end stage renal disease. Adriamycin nephropathy (AN) is a murine model of chronic proteinuric renal disease whereby chemical injury is followed by immune and structural changes that mimic human disease. This unit describes the method of AN induced by a single injection of adriamycin (ADR) in BALB/c mice. After the initial toxic injury, an immune-mediated chronic proteinuric renal disease that resembles human focal segmental glomerulosclerosis develops. The clinic pathological features of ...
Source: Current Protocols in Immunology - February 6, 2015 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Animal models for the analysis of immune responses to leishmaniasis.
Authors: Sacks DL, Melby PC
Abstract
This unit focuses on the murine model of cutaneous leishmaniasis and models of visceral leishmaniasis in mice and hamsters. Each basic protocol describes the methods used to inoculate parasites and to evaluate infections with regard to lesion progression and visceralization, and quantification of parasite load. © 2015 by John Wiley & Sons, Inc.
PMID: 25640990 [PubMed - in process] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - February 6, 2015 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Mouse model for pre-clinical study of human cancer immunotherapy.
Authors: Ya Z, Hailemichael Y, Overwijk W, Restifo NP
Abstract
This unit describes protocols for developing tumors in mice, including subcutaneous growth, pulmonary metastases of B16 melanoma, and spontaneous melanoma in B-Raf V600E/PTEN deletion transgenic mouse models. Two immunization methods to prevent B16 tumor growth are described using B16.GM-CSF and recombinant vaccinia virus. A therapeutic approach is also included that uses adoptive transfer of tumor antigen-specific T cells. Methods including CTL induction, isolation, testing, and genetic modification of mouse T cells for adoptive transfer by us...
Source: Current Protocols in Immunology - February 6, 2015 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
ELISPOT Assay for Measurement of Antigen-Specific and Polyclonal Antibody Responses.
Authors: Lycke N, Coico R
Abstract
The enzyme-linked immunospot (ELISPOT) assay for detection of antigen-specific and polyclonal antibody responses by single antibody-secreting cells has become the method of choice due to its cell-based quantitative value. Antigen stability and specificity and the diversity of antigens that can be used in the assay have contributed to the translational application of ELISPOT as demonstrated by many FDA-approved clinical tests that employ this technique. In addition, the ELISPOT assay can be used to detect two antigenically different secreted antibodies simultaneously by tw...
Source: Current Protocols in Immunology - February 6, 2015 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
The immune response to tumors.
Authors: Dougan M, Dranoff G
Abstract
The immune response to tumors is complex. Cells of the immune system can inhibit tumor growth and progression through the recognition and rejection of malignant cells, a process referred to as immunoediting. Yet, immune responses can also promote tumor cell growth, survival, and angiogenesis through the induction of oncogenic inflammation. Immunodeficiency can predispose to the development of spontaneous and virally induced cancer, and established tumors often generate immunosuppressive microenvironments that can block productive antitumor immunity, serving as a substa...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation of whole mononuclear cells from peripheral blood and cord blood.
Authors: Fuss IJ, Kanof ME, Smith PD, Zola H
Abstract
Peripheral blood is the primary source of lymphoid cells for investigation of the human immune system. Its use is facilitated by Ficoll-Hypaque density gradient centrifugation-a simple and rapid method of purifying peripheral blood mononuclear cells (PBMC) that takes advantage of the density differences between mononuclear cells and other elements found in the blood sample. Thus, cells are distributed in the solution in layers based on the differences in their density/size. Additional purification methods can be employed as the mononuclear cell sample c...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Immunomagnetic purification of T cell subpopulations.
Authors: Horgan K, Shaw S, Boirivant M
Abstract
There are two types of magnetic cell isolation technologies, one column-based and the other tube-based. The column-based technology utilizes nano-sized particles that need to pass through a ferromagnetic spheres column to increase cell-capture capacity. The tube-based system utilizes micron-sized beads that can be selected using a magnet applied to the tube. The beads are used for direct or indirect labeling of cells. Direct labeling is achieved with antibodies coupled to magnetic particles directly added to the cell suspension. For indirect labeling the cell...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation of dendritic cells.
Authors: Inaba K, Swiggard WJ, Steinman RM, Romani N, Schuler G, Brinster C
Abstract
This unit presents two methods for preparing dendritic cells (DCs), a highly specialized type of antigen-presenting cell (APC). The first method involves the isolation of DCs from mouse spleen, resulting in a cell population that is highly enriched in accessory cell and APC function. A support protocol for collagenase digestion of splenocyte suspensions is described to increase the yield of dendritic cells. The second method involves generating large numbers of DCs from mouse bone marrow progenitor cells. In that technique...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation of mononuclear cells from tonsillar tissue.
Authors: Johnston A, Sigurdardottir SL, Ryon JJ
Abstract
Located on the inside of the throat, the paired palatine tonsils form part of the first major barrier protecting the digestive and respiratory tracts from potentially invading microorganisms. The tonsils have a surface of stratified squamous epithelium that extends into deep and branched crypts lined by reticulated epithelium, which in parts may only be one cell thick. Organized in the sub-epithelial space are B cell rich lymphoid follicles. T cells are mostly located in the extra-follicular spaces with a very high CD4:CD8 T cell ratio. In addition t...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Phage display selection, analysis, and prediction of B cell epitopes.
Authors: Freund NT, Enshell-Seijffers D, Gershoni JM
Abstract
Combinatorial phage display libraries of random peptides can be used to discover the epitopes of antibodies through a procedure termed "biopanning." The affinity isolation of phage-displayed epitope peptidomimetics allows molecular definition of the epitopes of monoclonal antibodies (MAbs). Panels of MAb-specific peptides allow computational prediction of B cell epitopes. Epitope profiles recognized by polyclonal serum samples can also be generated. Detailed step by step protocols and discussion of applications are provided.
PMID: 196532...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Phosphoinositide and inositol phosphate analysis in lymphocyte activation.
Authors: Sauer K, Huang YH, Lin H, Sandberg M, Mayr GW
Abstract
Lymphocyte antigen receptor engagement profoundly changes the cellular content of phosphoinositide lipids and soluble inositol phosphates. Among these, the phosphoinositides phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) play key signaling roles by acting as pleckstrin homology (PH) domain ligands that recruit signaling proteins to the plasma membrane. Moreover, PIP2 acts as a precursor for the second messenger molecules diacylglycerol and soluble inositol 1,4,5-trisphosphate (IP3), essential m...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
TRAF-mediated TNFR-family signaling.
Authors: Ha H, Han D, Choi Y
Abstract
The tumor necrosis factor (TNF) superfamily consists of a wide variety of cell-bound and secreted proteins that regulate numerous cellular processes. In particular, TNF-family proteins regulate the proliferation and death of tumor cells, as well as activated immune cells. This overview discusses the mammalian TNF receptor-associated factors (TRAFs), of which TRAF1, 2, 3, 5, and 6 have been shown to interact directly or indirectly with members of the TNF receptor superfamily. Structural features of TRAF proteins are described along with a discussion of TRAF-interacting ...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Measuring autophagy in macrophages.
Authors: Harris J, Hanrahan O, De Haro SA
Abstract
Macroautophagy is a conserved intracellular homeostatic mechanism for the degradation of cytosolic constituents. Autophagy can promote cell survival by providing essential amino acids from the breakdown of macromolecules during periods of nutrient deprivation, and can remove damaged or excess organelles, such as mitochondria and peroxisomes. More recently, autophagy has been shown to play an important role in innate and adaptive immune responses to pathogenic bacteria in macrophages and dendritic cells. This unit presents protocols for the measurement of a...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
