bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of the HIV Promoter/Enhancer
Authors: Palmieri Camillo , Trimboli Francesca , Scala Giuseppe , Quinto Ileana , Bressler Peter B. 
Published: June 2004

AbstractThis unit describes adaptations of two molecular techniques that can be used to study the regulation of HIV expression. The first two protocols describe the chloramphenicol acetyltransferase (CAT) assay, in which the CAT reporter gene is attached to an HIV‐1 promoter and CAT activity is measured as an indication of the promoter's activity. The basic protocol is rapid, simple, and suited to analyzing multiple samples. An alternate protocol describes an assay for CAT function that involves separating the reaction products by thin‐layer chromatography (TLC). The second basic protocol describes an electrophoretic mobility shift assay for detecting proteins present in cell extracts that can bind to the HIV‐1 LTR (long terminal repeat). Such studies are central to current HIV research because it is important to know what agents induce and inhibit (or “down‐regulate”) HIV transcription.
In Situ Hybridization for Detection of HIVRNA
Authors: Fox Cecil H. , Cottler‐Fox Michele 
Published: June 2004

AbstractIn HIV studies, in situ hybridization can be used for identifying virion RNA, mRNA being produced for virion packaging, and proviral DNA in the cytoplasm or integrated in the nucleus. This unit focuses primarily on identifying virion RNA, because this is the most sensitive means by which in situ hybridization can be employed to detect HIV expression. In situ hybridization, as developed for HIV RNA detection, involves several protocols: (1) preparation of a radioactive or nonradioactive RNA probe; (2) in situ hybridization of probe to cells and paraffin sections of tissue; (3) detection of radiolabeled probe by emulsion autoradiography; (4) development, staining, and mounting of slides; and finally (5) examination of slides by bright‐field, dark‐field, specular reflectance, or laser‐scanning confocal microscopy. The protocols presented in this unit describe a setup involving up to 150 slides.
In Vitro Evaluation of Experimental Agents for Anti‐HIV Activity
Authors: Richman Douglas D. , Johnson Victoria A. , Mayers Douglas M. , Shirasaka Takuma , O'Brien Mary C. , Mitsuya Hiroaki 
Published: June 2004

AbstractThis unit presents an assay that has proven useful as an initial screening test is an HIV cytopathic effect (CPE) inhibition assay in which immortalized T cell lines (e.g., ATH8 or MT2) that are profoundly sensitive to the cytopathic effect of certain strains of HIV are utilized as target cells. Additional protocols assess the anti‐HIV activity of certain candidate agents by measuring inhibition of syncytium formation or p24 gag protein production by ELISA. Calculation of the 50% inhibitory concentration (IC50) is also presented.
Vaccinia‐Based Reporter Gene Cell‐Fusion Assays to Quantitate Functional Interactions of HIV Envelope Glycoprotein with Receptors
Authors: Dey Barna , Berger Edward A. 
Published: June 2004

AbstractThis unit describes quantitation of functional interactions between HIV envelope glcoprotein and target cell receptors, using assay of cell fusion‐dependent reporter gene activation. The method is particularly useful since it isolates the fusion reaction from the rest of the HIV replication cycle, and obviates the need for infectious HIV particles.Reporter Gene Cell Fusion Assays to Quantitate Functional Interactions of HIV Envelope Glycoprotein with Receptors
Classical Pathway Evaluation
Authors: Giclas Patricia C. 
Published: June 2004

AbstractThis unit describes several assay methods that can be used to determine the functional status of the classical pathway of complement and to quantitate its component proteins. The classical pathway includes C1qrs, C2, C4, C3, C5, C6, C7, C8, and C9, listed in the order in which they interact. Two CH50 assay procedures are presented that test total classical pathway function; one is carried out in test tubes, whereas the alternate protocol describes a variation that is carried out in 96‐well microtiter plates. Three support protocols describe preparing serum and erythrocyte‐antibody complexes (EA) for use in CH50 assays,and titrating of hemolysin for use in EA preparation. As an alternative to functional assays, radial immunodiffusion (RID) methods are also presented. These can be used to measure the concentrations of most circulating complement proteins and to evaluate the functional status of C1‐esterase inhibitor. A fourth support protocol provides a method to determine antibody concentrations for RID assays.
Isolation and Purification of C3 from Human Plasma
Authors: O'Rear Lynda D. , Ross Gordon D. 
Published: June 2004

AbstractThe alternative pathway of complement shares its terminal components (C3 and C5 through 9) with the classical pathway, but has several unique components, including factors D, B, and P (properdin). This unit presents methods for assaying total alternative pathway activity and the activity of factors B and D. Radial immunodiffusion (RID) can also be used to measure factor D, B, and P concentrations.
Isolation of Murine Macrophages
Authors: Fortier Anne H. , Falk Lydia A. 
Published: June 2004

AbstractMacrophages isolated from murine peritoneal cavity, bone marrow, and spleen are suitable samples for studying the activation properties of this immunologically important cell type. This unit describes the isolation of murine macrophages from the peritoneal cavity under inflammatory and noninflammatory conditions. To investigate macrophage activation using a more homogeneous population of cells, macrophages derived from immature progenitor cells in bone marrow can be studied. A second protocol describes the isolation of bone marrow‐derived progenitor cells and propagation of these immature macrophages by CSFs or IL‐3.
Characterization of Murine Macrophages
Authors: Riedy Mary C. , Stewart Carleton C. 
Published: June 2004

AbstractThis unit describes the phenotypic characterization of macrophages through the use of fluorescence‐labeled monoclonal antibodies (MAbs) that have specificity for certain extracellular membrane proteins expressed by macrophages. Although macrophages have no distinctive cell‐surface antigen that allows for their enrichment, a specific set of markers can be used to distinguish macrophages from other cell types in a heterogeneous population of cells. The method can also be easily applied to cells from other sources, e.g., peritoneal exudates or spleen. Through the use of image or flow cytometry, these cells can be identified and (with flow cytometry) sorted out from a heterogeneous population for further culture or analysis.
Characterization of Human Monocytes/Macrophages
Authors: Riedy Mary C. , Stewart Carleton C. 
Published: June 2004

AbstractHuman monocytes/macrophages display a variety of cell‐surface determinants to which monoclonal antibodies (MAbs) have been produced. These MAbs can be used to characterize phenotypic profiles of monocytes/macrophages to gain insight into their developmental and activation status. The basic and alternate protocols in this unit describe flow cytometry methods for measuring the expression of membrane determinants using directly conjugated or unconjugated MAbs, respectively. Because cells of this lineage display characteristic membrane marker patterns, combinations of MAbs are used in three‐color analysis to facilitate immunophenotypic characterization. Detailed information and commercial sources of fluorochrome‐labeled MAbs are provided, and suggested combinations of these MAbs for three‐color flow cytometry analysis of cell suspensions are discussed.
Activation of Murine Macrophages
Authors: Fortier Anne H. 
Published: June 2004

AbstractMacrophages can act as potent effector cells of the immune response. Cytokine signals generated during an immune response activate macrophages in a complex cascade of events. Activated macrophages are qualitatively different from mature resident tissue macrophages and from immature inflammatory macrophages. Activated macrophages acquire new abilities to eliminate intracellular pathogens (bacterial or parasitic) and extracellular tumor or parasite targets. This unit describes the production of macrophages that display cytotoxicity against intracellular parasites, starting from resident peritoneal macrophages. An describes the generation of macrophages with tumoricidal activity, starting from inflammatory peritoneal macrophages.
Human Natural Killer Receptors and Their Ligands
Authors: Biassoni Roberto , Bottino Cristina , Cantoni Claudia , Moretta Alessandro 
Published: June 2004

AbstractHuman Natural Killer Receptors and Their Ligands (Roberto Biassoni and Cristina Bottino, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; Claudia Cantoni, Universita degli Studi di Genova, Istituto Giannina Gaslini, Genova, Italy; Alessandro Moretta, Universita degli Studi di Genova, Genova, Italy). Natural killer (NK) cells are a lymphocyte subpopulation that are important effectors of innate immune responses against infectious pathogens. They are thought to play an important role in host defense, not only against virally infected cells, but also in killing of tumor cells. Recent progress indicates that NK cells express an array of receptors, some of them clonally distributed, able to modulate the natural cytotoxicity. Three NK‐specific activating receptors have been characterized; they belong to a novel receptor family called natural cytotoxicity receptors (NCR) and are represented by NKp46, NKp44, and NKp30. These receptors, upon engagement by their specific ligands, induce a strong activation of NK‐mediated cytotoxic activity. This overview discusses the receptors (both activating and inhibitory) expressed by NK cells and their ligands. Finally, the dysfunction of one of these molecules occurring in a genetically inherited immunodeficiency is discussed.
Isolation and Functional Use of Human NKT Cells
Authors: Exley Mark A. , Balk Steven P. , Wilson S. Brian 
Published: June 2004

AbstractIsolation and Functional Use of Human NK T Cells (Mark A. Exley andSteven P. Balk, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts; S. Brian Wilson, Massachusetts General Hospital, Harvard Medical School, Cambridge, Massachusetts). This unit describes methods for the isolation and functional analysis of human invariant NK T cells. The first protocol uses standard multicolor flow cytometry methods and staining reagents specific for V to generate invariant NK T cell clones. Support protocols for secondary stimulations and for rapid expansion of invariant NK T cell clones and functional analysis of these cells are also included.
Collagen‐Induced Arthritis
Authors: Rosloniec Edward F. , Cremer Michael , Kang Andrew , Myers Linda K. 
Published: June 2004

AbstractCollagen‐induced arthritis (CIA) is an experimental autoimmune disease that can be elicited in susceptible strains of rodents (rat and mouse) and nonhuman primates by immunization with type II collagen (CII), the major constituent protein of articular cartilage. Because of the important similarities between CIA and rheumatoid arthritis, this experimental model of autoimmune arthritis has been the subject of extensive investigation in several laboratories. Protocols for CIA are described in this unit for both the mouse model and the rat model. In addition, protocols are included for the purification of CII from bovine articular joints and chicken sternums, for the purification of collagen a1(II) chains, and for the purification of fragments of these chains following cyanogen bromide (CNBr) digestion. The preparation of CII is a time‐consuming procedure but is usually required because of the scarcity and expense of commercial sources of purified native CII. In addition, support protocols are provided for assessing the severity of inflammation following CIA and for measuring B and T cell responses to CII.
The NOD Mouse: A Model for Insulin‐Dependent Diabetes Mellitus
Authors: Leiter Edward H. 
Published: June 2004

AbstractNonobese diabetic (NOD) mice spontaneously develop autoimmune T cell‐mediated insulin‐dependent diabetes mellitus (IDDM). This unit presents a protocol for maintaining NOD mice under conditions permissive to full expression of their autoimmune potential. Methods are also described for diagnosing IDDM on the basis of glycosuria and glycosemia as well as for the semiquantitation of insulitis, a valuable subphenotype diagnostic of prediabetes in these mice, including a procedure for aldehyde fuchsin staining to identify β granules in β islet cells for diagnostic purposes. An adoptive‐transfer method is also included in which leukocytes, purified T cells, or T cell infiltrates obtained from the insulitic pancreas tissue of NOD mice are injected into prediabetic NOD or diabetes‐resistant F1 mice, which then develop disease in an accelerated fashion. This protocol also includes alternative steps in which bone‐marrow cells from NOD mice are transferred to syngeneic, irradiated NOD mice, allowing for reconsistution with a diabetogenic immune system. Steps for isolating pancreatic islet cells, which can then be used for a variety of purposes (e.g., as a source of islet antigens to establish and maintain autoreactive T cell lines) are included. Finally, steps are outlined that can be used to introduce transgenes into NOD mice. This protocol also discusses important considerations for introduction of targeted mutations produced in embryonic stem cells derived from other inbred strains, or introduction of other genes from non‐diabetes‐prone strains.
Induction of Autoimmune Disease by Depletion of Regulatory T Cells
Authors: Simmonds Steve , Mason Don 
Published: June 2004

AbstractOrgan‐specific autoimmune diseases can be induced in rodents that do not normally spontaneously develop autoimmunity by using procedures that render the animals partially T cell deficient. Using a protocol of adult thymectomy followed by four doses of sublethal gamma irradiation, insulin‐dependent diabetes can be induced in normal PVG.RT1u rats, an inbred congenic strain that has the same major histocompatibility complex (MHC) allotype as the spontaneously diabetic BB rat. Onset of the disease ranges from 3 to 18 weeks after the final dose of irradiation, with 98% of male and 70% of female animals becoming diabetic. This unit describes the induction of insulin‐dependent diabetes in the rat. A modified protocol allows for the induction of a more severe form of the disease.
Mercuric Chloride–Induced Autoimmunity
Authors: Badou A. , Saoudi A. , Dietrich G. , Druet E. , Druet P. , Pelletier L. 
Published: June 2004

AbstractThis unit describes methods for inducing autoimmune disease in Brown Norway rats through HgCl2 injections as well for assessing parameters that characterize the disease by serum IgE concentration assays, anti‐laminin antibody measurement, and renal immunofluorescence studies to detect autoantibodies. Also covered are disease induction using autoreactive CD4+ TH2 anti‐self MHC class II molecules and preparation of T cell lines. IL‐4 is produced very early after the first HgCl2 injection (beginning at day 3, peaking at day 14, and continuing up to day 30). Thus, IL‐4 mRNA expression may be detected in spleen and lymph nodes from HgCl2‐injected BN rats. The fact that HgCl2 induces in vitro mRNA IL‐4 gene expression in normal BN T cells but not in LEW T cells is probably crucial to susceptibility to the development of autoimmunity in the sense that it may condition the development of autoreactive T cells into pathogenic TH2 cells; a test for this condition is therefore also included.
The Day‐3 Thymectomy Model for Induction of Multiple Organ‐Specific Autoimmune Diseases
Authors: Suri‐Payer Elisabeth , Wei Kevin , Tung Kenneth 
Published: June 2004

AbstractAutoimmune diseases of the ovary and the stomach are major components of the murine autoimmune polyendocrinopathy syndrome that occurs following experimental perturbation of the normal immune system. Several methods of perturbation can lead to autoimmune disease, and the disease occurs in multiple organs in association with autoantibodies to tissue‐specific antigen of the respective organs. The autoimmune diseases develop in a variety of laboratory mouse strains that are not prone to spontaneous autoimmune diseases. This unit describes the induction of autoimmune disease of the ovary and the stomach in mice by thymectomy at the age of day 3 (day‐3 thymectomy) and methods for evaluating disease histologically and serologically.
Animal Models for SLE
Authors: Cohen Philip L. , Maldonado Michael A. 
Published: June 2004

AbstractSystemic lupus erythematosus (SLE) in humans is characterized by inflammatory lesions in skin, joints, kidneys, the central nervous system (CNS), and elsewhere. The clinical manifestations are accompanied by autoantibodies to diverse self antigens, mainly but not exclusively derived from the cell nucleus. Autoantibodies are generally believed to cause most of the tissue damage, although direct injury via cell‐mediated immunity is also important. This unit describes protocols for the quantitation of SLE‐associated autoantibody levels in mouse serum: detection of anti‐chromatin antibodies, detection of anti‐single and anti‐double‐stranded DNA antibodies, and detection of rheumatoid factor. Support protocols for preparing chicken chromatin and dsDNA are included. Also included is an immunoglobulin allotype‐specific adaptation of the basic autoantibody ELISA which is useful for measuring antibody production in chimeric animals. The unit includes an ELISPOT protocol for quantitating cells producing anti‐chromatin, as well as a method for genotyping mice for the faslpr and fasLgld mutations.
Choosing and Preparing Antigen‐Presenting Cells
Authors: Harding Clifford V. 
Published: June 2004

AbstractThe first issue in many antigen‐processing experiments is to choose and obtain appropriate antigen‐presenting cells (APCs). Macrophages have characteristics that dictate their use in certain antigen‐processing experiments, so this unit describes preparation of activated macrophages, which involves eliciting peritoneal macrophages from mice by infection with Listeria monocytogenes. This method is often preferred because the resulting macrophages are more consistently activated to a high degree, but macrophages can also be obtained (as described here) by sequential injection of L. moncytogenes and proteose peptone, by induction with concanavalin A (Con A), or by isolation from bone marrow. Lipopolysaccharide‐stimulated B lymphoblasts can also be used as APCs. Protocols are included for preparation of L. moncytogenes stocks and preparation of L929‐conditioned medium which is used for culturing bone marrow‐derived macrophages.
Bacteriophage Library Construction and Selection of Recombinant Antibodies
Authors: Galanis Maria , Irving Robert A. , Hudson Peter J. 
Published: June 2004

AbstractThis unit describes the use of E. coli and bacteriophages to display a diverse library of antibody fragments equivalent in complexity to the mammalian immune repertoire, and subsequent screening of the library for antibody fragments with specific binding affinities. The methods are also used for affinity enhancement (maturation), through the display and selection of improved affinity mutants derived from a single parent antibody. This unit discusses the following key components needed in library construction technology: a repertoire of antibody genes, typically amplified by polymerase chain reaction (PCR) technology; construction of scFv genes by PCR assembly; a method for producing a stable library, using bacteriophage that can both display individual antibodies on the viral surface and carry the gene encoding the antibody; a method of growing phage for selection; a method of selecting the highest‐affinity antibody from the phage library; a method for monitoring progress of phage selection; an affinity‐enhancement strategy for improving and manipulating the selected antibody; and expression of affinity‐enhanced antibodies.
MHC‐Peptide Tetramers to Visualize Antigen‐Specific T Cells
Authors: Altman John D. , Davis Mark M. 
Published: June 2004

AbstractMature T lymphocytes of the CD8 or CD4 classes bear αβ T cell receptors (TCR) that are specific for a molecular complex consisting of a major histocompatibility complex class I or II (MHC class I or II) molecule bound to a unique self or foreign peptide. Until recently, methods for monitoring the T cell immune response to a viral or tumor antigen were restricted primarily to functional assays based on limiting dilution analysis, because the lack of specific molecular reagents to identify clonal T cells obviated approaches to identify and enumerate specific T cells. Development of efficient methods to express and refold MHC class I molecules with synthetic peptides coincided with identification of specific protein sequences that provide the substrate for enzymatic biotinylation. This combination has led to the development of a straightforward method for generating synthetic TCR ligands, making them tetravalent to provide increased avidity, and labeling them through a streptavidin moiety with useful fluorescent tags such as fluorescein or phycoerythrin. This unit describes the preparation of MHC class I/peptide tetramers in detail, including bacterial expression and refolding of the MHC class I light chain, β2‐microglobulin (β2m), as well as the formation of a complex consisting of the MHC class I heavy chain of interest, β2m, and a chosen peptide.
Binding of Biotinylated Peptides to MHC Class II Proteins on Cell Surfaces
Authors: Rothbard Jonathan B. , Busch Robert 
Published: June 2004

AbstractThis unit describes a simple, reproducible, and semiquantitative assay for measuring ligand binding to cell surface receptors. This approach is useful as a quick screen for peptide binding to MHC class II proteins that avoids the need to purify the MHC class II molecules and that uses small numbers of cells. The basic protocol describes procedures for incubating cells expressing the MHC molecule of interest with a biotinylated peptide, washing off excess peptide, and detecting the bound peptides by staining with fluorescently labeled avidin. Bound fluorescence is then quantitated by flow cytometry. An alternate protocol provides a more sensitive method of measuring the peptide‐receptor complexes, and is useful when receptor density or binding site availability become limiting factors in the basic protocol. The alternate protocol uses an avidin/anti‐avidin/avidin sandwich, which has been found to increase sensitivity substantially without sacrificing specificity. Support protocols are provided for biotinylation of synthetic peptides in solution and for preparation of peptide stock solutions for use in the binding assays.
Detection of Low‐Affinity Ligand‐Receptor Interactions at the Cell Surface with Fluorescent Microspheres
Authors: Brown Marion H. 
Published: June 2004

AbstractA method of producing highly avid multivalent ligand binding reagents for detecting low‐affinity interactions at the cell surface is described in this unit. The principle is to immobilize multiple copies of extracellular regions of cell‐surface molecules on plastic fluorescent beads, to present the coated beads to cells, and to analyze binding in a quantitative manner by flow cytometry. The method of attaching proteins to the beads is designed to maximize display of the ligand‐binding region. The approach is applicable to immobilization of fusion proteins on a variety of beads, and can also be adapted for use with native proteins. A protocol for the biotinylation of MAbs is also presented.