Measuring the killing of intracellular pathogens: Leishmania.
Authors: Stenger S, van Zandbergen G
Abstract
Macrophages are professional phagocytes serving as a first line of defence against pathogenic organisms. Macrophages are equipped with efficient effector functions to kill invading microorganisms. The first important mechanism of macrophage host-defence is phagocytosis of pathogens. Subsequently, internalized pathogens are targeted for destruction in maturating phagolysosomal compartments. This process is mediated by lysosomal proteases and an acidified compartment. To investigate macrophages' killing potential in this chapter, we describe an assay based on hum...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Measuring matrix metalloproteinase activity in macrophages and polymorphonuclear leukocytes.
Authors: Kessenbrock K, Brown M, Werb Z
Abstract
Macrophages and polymorphonuclear cells (PMNs) represent an essential part of the innate immune system. These cells mediate a wide spectrum of immunological functions including bacterial defense, immune modulation, and inflammation; they are necessary for tissue homeostasis and also contribute to pathologies such as malignancy, autoimmunity, and chronic inflammation. Both macrophages and PMNs express a set of matrix metalloproteinases (MMPs), zinc-dependent endopeptidases that are involved in a variety of biological functions such as the turnover of extracel...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Measurement of macrophage-mediated killing of intracellular bacteria, including Francisella and mycobacteria.
Authors: Elkins KL, Cowley SC, Conlan JW
Abstract
Macrophages activated by T cell cytokines are a critical defense mechanism against intracellular bacterial pathogens. This unit presents two general methods for assessing the capacity of mouse macrophages, activated with either soluble cytokines or whole immune T lymphocytes, to control or reduce numbers of intracellular bacteria residing within them. "Measurement of killing" is inferred from a reduction in the number of colony-forming units (cfu) of bacteria at the end of a culture period, compared to the input numbers of cfu at initiation of culture, to t...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Infection of mice with Francisella as an immunological model.
Authors: Conlan JW, Chen W, Bosio CM, Cowley SC, Elkins KL
Abstract
This unit describes the utility of various mouse models of infection for studying pathogenesis and adaptive immune responses to the facultative intracellular bacteria pathogen Francisella tularensis. By judicious use of different combinations of mouse and bacterial strains, as well as different routes of infection, murine tularemia models may be used to explore a complete picture of F. tularensis infection and immunity. Moreover, studies using Francisella, particularly the Live Vaccine Strain (LVS), serve as a convenient and tractable mode...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Experimental asexual blood stage malaria immunity.
Authors: Amante FH, Engwerda CR, Good MF
Abstract
Immunity to asexual blood stages of malaria is complex, involving both humoral and cell-mediated immune mechanisms. The availability of murine models of malaria has greatly facilitated the analysis of immune mechanisms involved in resistance to the asexual blood stages. This unit details the materials and methods required for inducing protective immunity toward experimental blood stage malaria parasites by vaccination, repeated infection, and drug cure, as well as adoptive transfer of antigen-specific T cells.
PMID: 21462169 [PubMed - indexed for ME...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Unbiased molecular analysis of T cell receptor expression using template-switch anchored RT-PCR.
Authors: Quigley MF, Almeida JR, Price DA, Douek DC
Abstract
A detailed knowledge of the principles that guide clonal selection within the memory and effector T cell pools is essential to further our understanding of the factors that influence effective T cell-mediated immunity and has direct implications for the rational design of vaccines and immunotherapies. This unit provides methods for the unbiased quantification and characterization of all expressed T cell receptor (TCR) gene products within any defined T cell population. The approach is based on a template-switch anchored reverse transcription-poly...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Animal models of mycobacteria infection.
Authors: Ordway DJ, Orme IM
Abstract
This unit describes the infection of mice and guinea pigs with mycobacteria via various routes, as well as necropsy methods for the determination of mycobacterial loads within target organs. Additionally, methods for cultivating mycobacteria and preparing stocks are described. The protocols outlined are primarily used for M. tuberculosis, but can also be used for the study of other non-tuberculosis mycobacterial species.
PMID: 21809318 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Measurement of proliferative responses of cultured lymphocytes.
Authors: Muul LM, Heine G, Silvin C, James SP, Candotti F, Radbruch A, Worm M
Abstract
Measurement of proliferative responses of human lymphocytes is a fundamental technique for the assessment of their biological responses to various stimuli. Most simply, this involves measurement of the number of cells present in a culture before and after the addition of a stimulating agent. This unit contains several different prototype protocols to induce proliferation in lymphocytes following exposure to mitogens, antigens, allogeneic or autologous cells, or soluble factors. Each of these protocols can be used in conj...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation of human B cell populations.
Authors: Heine G, Sims GP, Worm M, Lipsky PE, Radbruch A
Abstract
To study the function and biology of human B cells, it is necessary to isolate pure populations. Historically, B cells were enriched by the sequential depletion of monocytes, natural killer cells, and T cells. However, this time-consuming process has been superseded by sorting methods using specific antibodies, targeting, in negative-selection strategies, unwanted cell types, or, in positive-selection strategies, B cell markers such as CD19. Here we describe in detail four methods for isolating B cells from human blood or mononuclear cells, ...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Metabolomics-lipidomics of eicosanoids and docosanoids generated by phagocytes.
Authors: Yang R, Chiang N, Oh SF, Serhan CN
Abstract
Lipid mediators derived from essential fatty acids, such as arachidonic acid, play important roles in physiologic and pathophysiologic processes. Prostaglandins, thromboxane, and leukotrienes are well-known eicosanoids that play critical roles in hemodynamics and inflammation. New families of mediators were recently uncovered that constitute a new genus stimulating resolution of acute inflammation, and are organ-protective. These include the resolvins (E-series and D-series), protectins (neuroprotectin D1/protectin D1), and maresins biosynthesized from o...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Measuring opsonic phagocytosis via Fcγ receptors and complement receptors on macrophages.
Authors: Mosser DM, Zhang X
Abstract
Phagocytosis is a cellular process that plays crucial roles in the removal of dead or dying cells, tissue remodeling, and host defense against invading pathogens. Most eukaryotic cells are decorated with glycoproteins containing terminal sialic acids, whose negative charges tend to repel cells, making so-called "nonspecific" phagocytosis a relatively inefficient process. Professional phagocytes are so designated because they express two major classes of receptors on their surfaces that are primarily involved in phagocytosis. Paradoxically, these receptors do not recogni...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Experimental autoimmune myasthenia gravis in the mouse.
Authors: Wu B, Goluszko E, Huda R, Tüzün E, Christadoss P
Abstract
Myasthenia 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...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Immortalization of human and rhesus macaque primary antigen-specific T cells by retrovirally transduced telomerase reverse transcriptase.
Authors: Barsov EV
Abstract
Human and rhesus macaque primary antigen-specific T cells derived from infected or immunized individuals or animals are a valuable material with which to study cellular immune responses against pathogens and tumors. Antigen-specific T cells can be expanded in vitro but have a finite proliferative life span. After a limited period in culture, primary T cells undergo replicative senescence and stop dividing. This restricts their applicability to short-term experiments and complicates their use in adoptive immunotherapy. The proliferative life span of primary human and rhesus macaq...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Analysis of gene expression and gene silencing in human macrophages.
Authors: Martinez FO
Abstract
This unit describes how to execute a gene expression study with human macrophages. It includes protocols for human macrophage preparation, RNA extraction, real-time PCR analysis, and microarray analysis. The unit also includes a protocol for gene silencing in human macrophages. Altering gene expression can be useful to study the contribution of the gene to macrophage function or even expression of other genes.
PMID: 22314831 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Mouse models of atherosclerosis.
Authors: Maganto-Garcia E, Tarrio M, Lichtman AH
Abstract
Genetically altered mice carrying mutations of genes encoding crucial components of the immune system and lipid metabolism have been widely used to study the role of immune responses and inflammation in atherosclerosis. These mice are often fed a diet, with a high content of cholesterol and saturated fat in order to induce hypercholesterolemia and arterial lesions. We review the different mouse models of atherosclerosis, type of diets, and techniques to measure lipid deposition and lesion size in the arterial walls. Moreover, the methods used to det...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
