bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation and Functional Assay of the Membrane Complement Inhibitors CD55 (DAF) and CD59 (MIRL)
Authors: Parker Charles J. 
Published: June 2004

AbstractThe complement system is the primary effector of humoral immunity. Because of its enormous destructive capacity, mechanisms for confining the activity of the system to the desired target and elaborate safeguards for protecting self against complement‐mediated injury have evolved. Human cells, particularly those found at sites of inflammation (e.g., hematopoietic and endothelial cells), express highly specialized membrane constituents that act independently or in concert with plasma regulatory proteins to inhibit the functional activity of complement. Decay‐accelerating factor (DAF), or CD55, directly inhibits the formation and stability of the amplification C3 and C5 convertases of both the classical and the alternative pathways. Failure of a cell to regulate the amplification C3 and C5 convertases allows the generation of the potentially cytolytic membrane attack complex (MAC), or C5b‐9 (consisting of the complement components C5b, C6, C7, C8, and C9). The primary cellular regulator of the MAC is the membrane inhibitor of reactive lysis (MIRL), or CD59, which restricts complement‐mediated lysis by blocking assembly of the MAC (primarily at the stage of C9 binding and polymerization). This unit provides a basic protocol for isolating CD55 and CD59, along with two support protocols describing separate functional assays for CD59 and CD55.
Oxidative Metabolism of Murine Macrophages
Authors: Green Shawn J. , Aniagolu Jacinta , Raney Jennifer J. 
Published: June 2004

AbstractThis unit describes two simple and straightforward microassays that can be used to measure the levels of NO2− and O2−, respectively that are generated by a small number of immunologically‐stimulated macrophages. Detection of these products may be used to identify cytokine(s), microbe(s), or microbial products(s) that regulate oxidative metabolism and effector activity. Although a number of other reliable and sensitive methods are available for assaying these two oxidative metabolites, the microassays described here require little time, technical expertise, or materials. It is not clear at present whether human monocytes/macrophages can also produce NO2−. These protocols are therefore restricted to mouse macrophages.
Assay for Macrophage‐Mediated Anti‐Tumor Cytotoxicity
Authors: Cox George W. 
Published: June 2004

AbstractThis unit describes a procedure for determining ability of mouse macrophages to lyse tumor cells in vitro. The outlines a three‐stage assay that includes: (1) culture of macrophages (freshly explanted from mice or grown from a cell line) with suspected activating reagents; (2) extensive washing of the cultured macrophages to remove residual reagents, followed by incubation with [111In]‐labeled tumor cells to allow lysis to occur; and (3) collection of cell‐free culture supernatants and measurement of cytolytic activity as a function of 111In released from tumor cells destroyed by activated macrophages. The outlines a method for radiolabeling tumor cells with [111In]oxine.
Experimental Autoimmune Encephalomyelitis in the Mouse
Authors: Miller Stephen D. , Karpus William J. 
Published: June 2004

AbstractThis unit details the materials and methods required for both active induction and adoptive transfer of experimental autoimmune encephalomyelitis (EAE) in the SJL mouse strain using intact proteins or peptides from the two major myelin proteins: proteolipid protein (PLP) and myelin basic protein (MBP). Detailed materials and methods required for the purification of both PLP and MBP are also described. Modifications of the specified protocols may be necessary for efficient induction of active or adoptive EAE in other mouse strains.
Experimental Autoimmune Encephalomyelitis in the Rat
Authors: Swanborg Robert H. , Stepaniak Jolie A. 
Published: June 2004

AbstractThis unit details the materials and methods required for both active induction and adoptive transfer of experimental autoimmune encephalomyelitis (EAE) in the SJL mouse strain using intact proteins or peptides from the two major myelin proteins: proteolipid protein (PLP) and myelin basic protein (MBP). Detailed materials and methods required for the purification of both PLP and MBP are also described. Modifications of the specified protocols may be necessary for efficient induction of active or adoptive EAE in other mouse strains.
The BB Rat as a Model of Human Insulin‐Dependent Diabetes Mellitus
Authors: Whalen Barbara J. , Mordes John P. , Rossini Aldo A. 
Published: June 2004

AbstractUse of the BioBreeding (BB) rat to model human insulin‐dependent diabetes mellitus (IDDM) is useful in that characteristics of diabetes in the BB rat closely parallel those observed in human IDDM. Diabetic animals can be biopsied, autopsied, and bred to study the genetic basis of IDDM. The genetic, immunological, and environmental components of the disease can all be investigated under controlled conditions. Two inbred lines of BB have been used in the majority of published studies using this model system; these rats are designated as diabetes prone (DP‐BB/Wor) and diabetes resistant (DR‐BB/Wor). This unit presents two protocols for the diagnosis and prevention of spontaneous IDDM in DP‐BB/Wor rats. Two alternate protocols are given for the induction of autoimmune disease in DR‐BB/Wor rats and for the adoptive transfer of autoimmunity into histocompatible athymic WAG
nu/nu recipient rats. Support protocols are also given for diagnosis of insulitis, treatment of diabetic rats with insulin, and serological analysis of blood samples from sentinel rats to monitor the pathogen status of the colony.
Adjuvant Arthritis in the Rat
Authors: van Eden Willem , Wagenaar‐Hilbers Josee P.A. , Wauben Marca H.M. 
Published: June 2004

AbstractAdjuvant arthritis (AA) is an induced form of (sub)chronic arthritis. Strains of rats have a varying genetic susceptibility to AA, whereas mice generally are not susceptible. AA is most easily induced with mycobacteria suspended in oil, although in some strains of rats it can be induced with oily adjuvants in the absence of mycobacteria. The disease is a T cell‐mediated autoimmune arthritis that is frequently used to study immunological aspects of rheumatoid arthritis (RA) and other arthritic or inflammatory diseases in humans. Furthermore, it is used as a model for developing and testing antiinflammatory drugs. There are no particularly well‐defined autoantigens in AA; in this respect, the model resembles spontaneous arthritic diseases in humans. In all susceptible rat strains, the inflammatory process of AA is self remitting, although usually the disease is severe and leads to permanent joint malformations, including ankylosis; a time line for AA development is presented. This unit describes the induction and evaluation of AA and the preparation of adjuvant used to induce AA.
Experimental Autoimmune Uveoretinitis in the Rat and Mouse
Authors: Caspi Rachel R. 
Published: June 2004

AbstractExperimental autoimmune uveoretinitis (EAU) in rats and mice is a prototypic T cell‐mediated autoimmune disease that targets the neural retina and related tissues. The model is used to represent human sight‐threatening inflammatory eye diseases that are believed to have an autoimmune etiology, and to study basic mechanisms of tolerance and autoimmunity to organ‐specific antigens from immunologically privileged sites. In this unit, EAU is induced in rats and mice by immunization with uveitogenic peptide antigens emulsified in complete Freund's adjuvant (CFA). Clinical onset is observed by both external and microscopic examination. A protocol is provided for preparation of tissue sections of affected eyes for microscopic analysis. EAU can also be induced in the rat (as described) by adoptive transfer of lymphocytes from uveitic rats into unimmunized recipients, which obviates the use of CFA. To induce EAU in mice, Bordetella pertussis toxin (PTX) is included to overcome immunological resistance mechanisms.
Experimental Autoimmune Thyroiditis in the Mouse
Authors: Kong Yi‐chi M. 
Published: June 2004

AbstractExperimental autoimmune thyroiditis (EAT) in mice is an excellent model for Hashimoto's thyroiditis (HT). It is induced with thyroglobulin (Tg), a known thyroid autoantigen that is common to both mouse and human and for which several conserved, thyroiditogenic epitopes have been identified. This unit describes induction and evaluation of EAT using thyroid histology and in vitro proliferative response assays. An ELISA is presented to detect thelevel of antibody to mouse thyroglobulin (MTg). To induce EAT, either bacterial lipopolysaccharide (LPS) or supplemented complete Freund's adjuvant (CFA) can be used as adjuvant. A support protocol for preparing MTg is included. The T cell proliferation assay can be used to examine the antigenicity of synthetic peptides derived from MTg or heterologous Tg. EAT can be adoptively transferred utilizing cells that have been expanded in vitro, as described. A protocol is provided for inducing tolerance using deaggregated MTg; induction of tolerance requires larger amounts of MTg but efficiently suppresses EAT development.
Experimental Autoimmune Myasthenia Gravis in the Mouse
Authors: Wu Bo , Goluszko Elzbieta , Christadoss Premkumar 
Published: June 2004

AbstractMyasthenia gravis (MG) is a T cell‐dependent antibody‐mediated autoimmune neuromuscular disease. Antibodies to the nicotinic acetylcholine receptor (AChR) destroy the AChR, thus leading to defective neuromuscular transmission of electrical impulse and to muscle weakness. This unit is a practical guide to the induction and evaluation of experimental autoimmune myasthenia gravis (EAMG) in the mouse, the animal model for MG. Protocols are provided for the extraction and purification of AChR from the electric organs of Torpedo californica, or eel. The purified receptor is used as an immunogen to induce autoimmunity to AChR, thus causing EAMG. The defect in neuromuscular transmission can also be measured quantitatively by electromyography, as described here. In addition, EAMG is frequently characterized by the presence of antibodies to AChR, which are measured by radioimmunoassay and by a marked antibody‐mediated reduction in the number of muscle AChRs. AChR extracted from mouse muscle is used in measuring serum antibody levels and for quantifying muscle AChR content.
Streptococcal Cell Wall Arthritis
Authors: Wilder Ronald L. 
Published: June 2004

AbstractStreptococcal cell wall (SCW) arthritis in rats is an experimentally‐induced inflammatory model with many features that resemble rheumatoid arthritis (RA) in humans. In this unit, Lewis rats are injected with an aqueous suspension of Group A SCW streptococcal cell wall peptidoglycan‐polysaccharide polymers (SCW PG‐PS) and observed for the development of arthritis. The resulting arthritis is biphasic. An acute phase typically develops within 48 hr, followed 10 to 21 days later by a chronic phase which persists for months. Support protocols are included for preparing Group A PG‐PS and measuring PG‐PS concentration.
Induction of Inflammatory Bowel Disease in Immunodeficient Mice by Depletion of Regulatory T Cells
Authors: Read Simon , Powrie Fiona 
Published: June 2004

AbstractThe severe combined immunodeficiency (SCID) model of colitis shares many features of idiopathic inflammatory bowel disease (IBD) in humans. The SCID model is highly reproducible and easily manipulated, and as such provides a useful tool for studying mucosal immune regulation as it relates to the pathogenesis and treatment of IBD in humans. This unit describes a cell transfer system in which SCID mice are reconstituted with CD4+ CD45RBhigh cells to induce IBD. The CD4+ cells are isolated by immunomagnetic negative selection using monoclonal antibodies, and are then separated by fluorescent cell sorting into CD45RBhigh and CD45RBlow cells. The former population reproducibly induces disease in recipients. A support protocol describes methods to monitor disease progression, which is characterized by weight loss, loose stools, and histologically assessed lesions in the colon.
Autoimmune Myocarditis
Authors: Smith Stacy C. 
Published: June 2004

AbstractMyosin‐induced autoimmune myocarditis (EAM) is a model of inflammatory heart disease initiated by CD4+ T cells. It is a paradigm of the immune‐mediated cardiac damage believed to play a role in the pathogenesis of a subset of postinfectious human cardiomyopathies. Myocarditis is induced in susceptible mice by immunization with purified cardiac myosin or specific peptides derived from cardiac myosin, or by adoptive transfer of myosin‐reactive T cells. Myocarditis is induced in Lewis rats by immunization with purified cardiac myosin or by adoptive transfer of T cells stimulated by specific peptides derived from cardiac myosin. Myocarditis begins 12 to 14 days after the first immunization, and is maximal after 21 days. In addition to the protocols used to induce EAM in mice and rats, support protocols are included for preparing purified cardiac myosin using mouse or rat heart tissue, preparing purified cardiac myosin for injection, and collecting and assessing hearts by histopathological means.
Induction of TNBS Colitis in Mice
Authors: Scheiffele Frank , Fuss Ivan J. 
Published: June 2004

AbstractThe animal model described in this unit is one of several that have been used to study the immunopathogenesis of inflammatory bowel disease. The model employs the use of 2,4,6‐trinitrobenzenesulfonic acid (TNBS), which induces severe colonic inflammation when administered intrarectally in SJL/J mice. The colitis which results from this procedure presents clinical and histopathological findings that resemble those seen in Crohn's disease. The unit describes the critical parameters needed for successful induction of TNBS‐colitis as well methods for monitoring and grading disease levels. A support protocol for isolating lamina propria mononuclear cells from mouse colons is also included.
Monitoring Antigen‐Specific T Cells Using MHC‐Ig Dimers
Authors: Schneck Jonathan P. , Slansky Jill E. , O'Herrin Sean M. , Greten Tim F. 
Published: June 2004

AbstractThe lack of high affinity reagents has made distinguishing T cells on the basis of antigen specificity difficult to accomplish. This unit provides protocols that utilize innovations in molecular design to permit construction of soluble multivalent MHC complexes (MHC‐Ig dimers) with high avidity for cognate T cell receptors. MHC‐Ig dimers display stable binding properties when they interact with antigen‐specific T cells thus allowing their use in the staining of antigen‐specific T cells by flow cytometry. Methods for constructing and detecting these MHC‐Ig dimers are included along with protocols for applying their use for the quantitation of antigen‐specific T cells.
Biotin Labeling and Quantitation of Cell‐Surface Proteins
Authors: Turvy Diane N. , Blum Janice S. 
Published: June 2004

AbstractCell surface receptors, such as transferrin receptors and MHC molecules, are internalized into the endocytic pathway and recycled to the plasma membrane. Previous assays used to measure endocytosis and recycling were cumbersome and often required radioactive reagents. This unit describes protocols that employ the combination of a cleavable biotin reagent to label surface molecules and a capture ELISA to detect these molecules allowing for rapid and safe quantitation of cell surface protein expression, endocytosis, and recycling.
Fluorescence Resonance Energy Transfer: Techniques for Measuring Molecular Conformation and Molecular Proximity
Authors: Edidin Michael 
Published: June 2004

AbstractThis overview unit focuses on the basics of fluorescence and of the FRET phenomenon, and on methods for detecting FRET and data interpretation. FRET is very versatile and there are more application of these basics than can be covered in a single overview. However, some examples are given of applications of various FRET techniques.
Animal Models for Toxoplasma gondii Infection
Authors: Subauste Carlos , Remington Jack 
Published: June 2004

AbstractToxoplasma gondii is an obligate intracellular protozoan that commonly infects mammals and birds throughout the world. This unit describes murine models of acute T. gondii infection and toxoplasmic encephalitis. T. gondii infection in severe combined immunodeficient (SCID) mice, which lack T and B cells, has allowed for the study of T cell‐independent mechanisms of defense against intracellular organisms, as described here. The establishment of temperature‐sensitive mutant strains of T. gondii has allowed adoptive‐transfer experiments without the concern for the transfer of the parasite at the same time. The temperature‐sensitive mutant ts‐4 strain disappears from tissues of immunocompetent mice without forming tissue cysts and induces protection against challenge with virulent strains of the parasite, and a protocol is provided for infection with this mutant strain. Support protocols present methodology for evaluation of progression of infection and immune response to the parasite, maintenance of T. gondii tissue cysts and tachyzoites, as well as preparation of T. gondii lysate antigens.
B16 as a Mouse Model for Human Melanoma
Authors: Overwijk Willem W. , Restifo Nicholas P. 
Published: June 2004

AbstractThis unit details protocols for in vivo models of subcutaneous growth and pulmonary metastases of B16 melanoma. Therapeutic approaches include the use of B16.GM‐CSF and rVVmTRP‐1 to induce autoimmune vitiligo and tumor protection. The induction and use of gp100‐specific therapeutic cytotoxic T lymphocytes (CTL) are discussed. Methods are also included for CTL induction, isolation and testing, CTL maintenance, and adoptive transfer. Support protocols detail the testing of mouse sera for presence of MDA‐specific antibodies by immunoblotting and ELISA, respectively. Additional sections, including growing B16 melanoma, enumerating pulmonary metastases, and use of recombinant viruses for vaccination, are discussed together with safety concerns.
Mouse SaI Sarcoma Tumor Model
Authors: Ostrand‐Rosenberg Suzanne 
Published: June 2004

AbstractSaI is an A/J‐derived (H‐2KkDd) transplantable mouse fibrosarcoma that produces a solid tumor when inoculated subcutaneously, intradermally, or intramuscularly, and an ascites tumor when inoculated intraperitoneally into syngeneic mice. This unit describes the establishment of primary solid SaI/N tumor, the establishment of SaI ascites tumor, and the removal for sampling of in vivo‐passaged SaI ascites tumor cells in mice.
Models for Lymphoma
Authors: Biragyn Arya , Kwak Larry W. 
Published: June 2004

AbstractThis unit describes experimental procedures for development of therapeutic vaccines, particularly “second‐generation” recombinant vaccines. Specifically, a general procedure for handling and culturing lymphoma cell lines in vitro and their subsequent challenge into syngeneic mice is described. Several protocols describe the production of various idiotype (Id)‐based or cellular lymphoma vaccine formulations. In particular, the novel approach of rendering nonimmunogenic lymphoma‐derived scFv or Id immunogenic by fusing it with a chemokine moiety is described. As an alternative, a protocol for expression and purification of these chemokine‐fusion proteins from E. coli is included. A general procedure is included for cloning of cytokine genes, for example, murine GM‐CSF, in A20 lymphoma cells. Support protocols are presented for chemically conjugating intact Ig protein with KLH to produce a prototype protein vaccine and using the Helios Gene Gun System to immunize mice with recombinant DNA tumor vaccines.
Immunofluorescence Staining
Authors: Donaldson Julie G. 
Published: June 2004

AbstractThe light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications (e.g., brightfield, phase‐contrast, differential interference contrast, and fluorescence epi‐illumination), and how it should be maintained as required to obtain maximum image‐forming capacity and resolution. The principles of microscopy and step‐by‐step alignment and adjustment procedures are described in this unit.
Mouse Endogenous Superantigens: Ms and Mls‐like Determinants Encoded by Mouse Retroviruses
Authors: Hodes Richard J. , Abe Ryo 
Published: June 2004

AbstractCommonly used inbred mouse strains express different combinations of integrated mouse mammary tumor proviruses (MMTV). This appendix summarizes the proviruses that have been detected. The reported functional properties of those MMTV proviral products which have been identified as superantigens are also summarized, including the ability to elicit primary or secondary T cell responses and to induce Vb‐specific clonal deletion during T cell differentiation. In addition, the amino acid sequences of putative ORF gene products of different MMTV are compared.
Overview of Protein Folds in the Immune System
Authors: Sun Peter D. , Boyington Jeffrey C. 
Published: June 2004

AbstractThe rapid advancement of X‐ray crystallography and nuclear magnetic resonance techniques in recent years has resulted in the solution of macromolecular structures at an unprecedented rate. This review aims at providing a comprehensive description of structures and folds related to the function of the immune system. Focus is placed on immunologically relevant proteins such as immunoreceptors and major histocompatibility complexes. Information is also provided regarding protein structure data banks.
Large‐Scale Cell Culture
Authors: Bleckwenn Nicole A. , Shiloach Joseph 
Published: June 2004

AbstractMammalian cells offer a good production system for complex biologic products that require proper post‐translational processing and folding. These products can be endogenous proteins, recombinant proteins, or recombinant antibodies, which are often needed in large quantities for clinical evaluations and structural studies. Many cell lines are available for production purposes and are derived from various sources, therefore, exhibiting a wide variety of growth and production characteristics. In general, cell types can be divided into two major categories: those that are able to grow in suspension (anchorage‐independent) and those that require a physical support for growth (anchorage‐dependent). In either case, the products produced can be secreted into the production medium or can be accumulated inside the cells. The properties of the cells and the location of the produced product dictate the method used for growth and production, and consequently the selection of a suitable type of bioreactor. This unit focuses on the methods used to cultivate large amounts of cells for the purpose of obtaining an endogenous or recombinant product.
Trypan Blue Exclusion Test of Cell Viability
Authors: Strober Warren 
Published: June 2004

AbstractThe dye exclusion test is used to determine the number of viable cells present in a cell suspension. It is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue, Eosin, or propidium, whereas dead cells do not. In this test, a cell suspension is simply mixed with dye and then visually examined to determine whether cells take up or exclude dye. In the protocol presented here, a viable cell will have a clear cytoplasm whereas a nonviable cell will have a blue cytoplasm.
Obtaining Human Peripheral Blood Cells
Authors: Strober Warren 
Published: June 2004

AbstractPeripheral blood is the chief source of mononuclear cells (lymphocytes and monocytes) for immunologic studies of humans. This appendix presents protocols for obtaining blood by simple venipuncture when relatively small amounts of blood (10 to 100 ml) are necessary or by lymphapheresis when large amounts (300 to 5000 ml) are necessary. Cells collected by these procedures can be further separated by techniques described in Chapter 7.