Current Protocols in Immunology最新文献

{"title":"Issue Information TOC","authors":"","doi":"10.1002/cpim.67","DOIUrl":"10.1002/cpim.67","url":null,"abstract":"<p><b>Cover</b>: In Geddes-McAlister and Gadjeva (https://doi.org/10.1002/cpim.87), Overview of workflow for MS-based proteomics profiling of neutrophils. Neutrophils are isolated and purified from bone marrow of 7- to 9-week-old male or female mice. The quality of the purification is assessed by FACS. Proteins are extracted by chemical (e.g., urea) and mechanical (e.g., sonication) cell disruption and enzymatically digested with Lys-C and trypsin proteases. The purified peptides are measured in the first MS scan and peptide fragmentation patterns are observed in the second MS scan (MS/;MS). The data is processed and analyzed using the publicly available MaxQuant and Perseus platforms. Figure generated with Biorender.com. See e87.\u0000\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture><p></p>\u0000 </div>\u0000 </figure></p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"126 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.67","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42830613","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Experimental Mouse Model of Bleomycin-Induced Skin Fibrosis","authors":"Przemysław Błyszczuk,&nbsp;Anastasiia Kozlova,&nbsp;Zhongning Guo,&nbsp;Gabriela Kania,&nbsp;Oliver Distler","doi":"10.1002/cpim.88","DOIUrl":"10.1002/cpim.88","url":null,"abstract":"<p>Systemic sclerosis (SSc) refers to an autoimmune disease, which is manifested by inflammation, vasculopathy, and fibrosis of the skin and internal organs. There are a number of different animal models recapitulating specific aspects of SSc. The experimental mouse model of bleomycin-induced skin fibrosis is commonly used to study the pathogenesis observed in SSc. In this model, repetitive intradermal injections of the cytotoxic agent bleomycin trigger progressive skin thickening, associated with excessive accumulation of collagen, infiltration of immune cells, and formation of α-smooth muscle actin (α-SMA)-positive myofibroblasts. In this article, we provide a detailed protocol for the induction of skin fibrosis in experimental mice by bleomycin. Moreover, we describe procedures for processing and analyzing affected skin tissue, provide troubleshooting, highlight advantages and limitations of the presented model, and critically discuss representative results. © 2019 by John Wiley &amp; Sons, Inc.</p><p><b>Basic Protocol 1</b>: Intradermal bleomycin injections to induce skin fibrosis in mice</p><p><b>Support Protocol</b>: Mouse tissue collection for fibrosis evaluation and for other molecular assays</p><p><b>Basic Protocol 2</b>: Evaluation of mouse skin thickness using Masson's trichrome staining</p><p><b>Basic Protocol 3</b>: Measurement of hydroxyproline content in skin tissue using a colorimetric assay</p><p><b>Basic Protocol 4</b>: Evaluation of myofibroblasts in mouse skin by immunohistochemistry</p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"126 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.88","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42484366","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mass Spectrometry-Based Quantitative Proteomics of Murine-Derived Polymorphonuclear Neutrophils","authors":"Jennifer Geddes-McAlister,&nbsp;Mihaela Gadjeva","doi":"10.1002/cpim.87","DOIUrl":"10.1002/cpim.87","url":null,"abstract":"<p>Polymorphonuclear cells (PMNs or neutrophils) are the most abundant leukocyte in humans and represent an essential component of the innate immune system. The ability of neutrophils to initiate an immediate and non-specific host response against invading microbial species is the key to determining the outcome of infection. Neutrophils produce and secrete a plethora of immunomodulatory proteins, including major granule proteins and cytokines, as well as various enzymes, which regulate adherence, phagocytosis, chemotaxis, and cell survival. Historically, characterization of neutrophils and their roles during infection have relied on genetic and phenotypic analyses, as well as biochemical assays. However, recent advances in mass spectrometry-based proteomic workflows and technological platforms have supported the comprehensive profiling of neutrophil-associated immune responses in consideration of cellular factors and secreted proteins. Given the critical role of neutrophils in maintaining and regulating innate immune function, comprehensive profiling of their response to infection is imperative to ensuring host survival. Here, we briefly discuss the role of neutrophils in host-defense and describe methods to purify neutrophils from murine samples and comprehensively profile their proteomes. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"126 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.87","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42027687","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Production and Thermal Exchange of Conditional Peptide-MHC I Multimers","authors":"Jolien J. Luimstra,&nbsp;Kees L. M. C. Franken,&nbsp;Malgorzata A. Garstka,&nbsp;Jan W. Drijfhout,&nbsp;Jacques Neefjes,&nbsp;Huib Ovaa","doi":"10.1002/cpim.85","DOIUrl":"10.1002/cpim.85","url":null,"abstract":"<p>Cytotoxic CD8⁺ T cells mediate cellular immunity through recognition of specific antigens presented by MHC class I on all nucleated cells. Studying T cell interactions and responses provides invaluable information on infection, autoimmunity and cancer. Fluorescently labeled multimers of MHC I can be used to quantify, characterize, and isolate specific CD8⁺ T cells by flow cytometry. Here we describe the production and use of conditional MHC I multimers that can be loaded with peptides of choice by incubating them at a defined temperature. Multimers are folded with a template peptide that forms a stable complex at low temperature, but dissociates at a defined elevated temperature. Using this technology multiple MHC I multimers can be generated in parallel, to allow staining and isolation of large sets of antigen-specific CD8⁺ T cells, especially when combined with barcoding technologies. © 2019 The Authors.</p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"126 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.85","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44912683","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterization of Immune Cells from Adipose Tissue","authors":"Sagar P. Bapat,&nbsp;Yuqiong Liang,&nbsp;Ye Zheng","doi":"10.1002/cpim.86","DOIUrl":"10.1002/cpim.86","url":null,"abstract":"<p>Adipose tissue (AT) serves a crucial role in maintaining organismal metabolic homeostasis. Studies have demonstrated that AT is populated with a diverse array of immune cells that coordinate and regulate AT function. This adipo-immune system is highly dynamic, reflecting the physiologic state of the organism (e.g., obese, lean, aged, or young) as well as the constant physiologic remodeling of AT associated with the daily rhythms of fasting and feeding. Many of the adaptive and maladaptive functional changes of AT are regulated by changes in the quantity and quality of distinct sets of AT-resident immune cells. Here we present protocols to assess the dynamic state of the immune system within AT by constructing censuses of adipose-resident immune cells (macrophages, dendritic cells, neutrophils, eosinophils, NK cells, innate lymphocytes, T cells, and B cells, etc.) based on flow cytometry, which we term adipo-immune profiles (AIPs). Constructing AIPs can be an integral part of assessment for AT health and function. This article describes the protocols to generate such AIPs. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"126 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.86","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44221513","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Using Evans Blue Dye to Determine Blood-Brain Barrier Integrity in Rodents","authors":"Mariana Pereira de Souza Goldim,&nbsp;Amanda Della Giustina,&nbsp;Fabricia Petronilho","doi":"10.1002/cpim.83","DOIUrl":"10.1002/cpim.83","url":null,"abstract":"<p>The blood-brain barrier (BBB) is an active and selective barrier that shields the brain from endogenous and exogenous insults. Different stimuli may lead to the disruption of this barrier, including inflammation and trauma. Several methods are used to evaluate BBB disruption. The most widely used method is Evans blue (EB) dye extravasation. EB cannot normally pass through the BBB and thus its presence in brain tissue indicates alterations in permeability. This protocol details the steps of EB extravasation in rodents. Important aspects regarding critical steps and advantages are also provided. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"126 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.83","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45519510","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genome-Wide Measurement and Computational Analysis of Transcription Factor Binding and Chromatin Accessibility in Lymphocytes","authors":"M. Firas Sadiyah,&nbsp;Rahul Roychoudhuri","doi":"10.1002/cpim.84","DOIUrl":"10.1002/cpim.84","url":null,"abstract":"<p>Cells of the adaptive immune system, including CD4⁺ and CD8⁺ T cells, as well as B cells, possess the ability to undergo dynamic changes in population size, differentiation state, and function to counteract diverse and temporally stochastic threats from the external environment. To achieve this, lymphocytes must be able to rapidly control their gene-expression programs in a cell-type-specific manner and in response to extrinsic signals. Such capacity is provided by transcription factors (TFs), which bind to the available repertoire of regulatory DNA elements in distinct lymphocyte subsets to program cell-type-specific gene expression. Here we provide a set of protocols that utilize massively parallel sequencing–based approaches to map genome-wide TF-binding sites and accessible chromatin, with consideration of the unique aspects and technical issues facing their application to lymphocytes. We show how to computationally validate and analyze aligned data to map differentially enriched/accessible sites, identify enriched DNA sequence motifs, and detect the position of nucleosomes adjacent to accessible DNA elements. These techniques, when applied to immune cells, can enhance our understanding of how gene-expression programs are controlled within lymphocytes to coordinate immune function in homeostasis and disease. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"126 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.84","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43613099","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Protocols for the Analysis of microRNA Expression, Biogenesis, and Function in Immune Cells","authors":"Nannan Zhang,&nbsp;Guowu Hu,&nbsp;Timothy G. Myers,&nbsp;Peter R. Williamson","doi":"10.1002/cpim.78","DOIUrl":"10.1002/cpim.78","url":null,"abstract":"<p>MicroRNAs (miRNAs) are short (19- to 25-nucleotide) noncoding RNA molecules that target mRNAs to repress gene expression and that play important roles in regulating many fundamental biological functions including cell differentiation, development, growth, and metabolism. They are well conserved in eukaryotic cells and are considered essential ancient elements of gene regulation. miRNA genes are transcribed by RNA polymerase II to generate primary miRNAs (pri-miRNAs), which are cleaved by microprocessor complex in the nucleus to generate stem-loop structures known as pre-miRNAs. Pre-miRNAs are translocated to the cytoplasm and cleaved by Dicer to form the mature miRNAs, which mediate mRNA degradation through their loading to the RNA-induced silencing complex (RISC) and binding to complementary sequences within target mRNAs to repress their translation by mRNA degradation and/or translation inhibition. Because ∼1900 miRNA genes are reported in the human genome, many associated with disease, appropriate methods to study miRNA expression and regulation under physiological and pathological conditions have become increasingly important to the study of many aspects of human biology, including immune regulation. As with small interfering RNA (siRNA), the mechanism of miRNA-mediated targeting has been used to develop miRNA-based therapeutics. For a complete and systematic analysis, it is critical to utilize a variety of different tools to analyze the expression of pri-mRNAs, pre-miRNAs, and mature miRNAs and characterize their targets both <i>in vitro</i> and <i>in vivo</i>. Such studies will facilitate future novel drug design and development. This unit provides six basic protocols for miRNA analysis, covering next-generation sequencing, quantitative real-time PCR (qRT-PCR), and digoxigenin-based expression analysis of pri-mRNAs, pre-miRNAs, and mature miRNAs; mapping of pri-miRNA and their cleavage sites by rapid amplification of cDNA ends (RACE); electrophoretic mobility shift assays (EMSAs) or biotin-based nonradioactive detection of miRNA-protein complexes (miRNPs); and functional analysis of miRNAs using miRNA mimics and inhibitors. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"126 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.78","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47787406","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Issue Information TOC","authors":"","doi":"10.1002/cpim.66","DOIUrl":"10.1002/cpim.66","url":null,"abstract":"<p><b>Cover</b>: In Singh et al. (https://doi.org/10.1002/cpim.70), Hematoxylin- and eosin-stained sections of ear skin. Hematoxylin- and eosin-stained sections of untreated (left), rIL-23-treated (middle), and Aldara/IMQ-treated (right) ears. Acanthosis, thickening of the epidermis; hyperkeratosis, thickening of the stratum corneum; parakeratosis, retention of nucleated keratinocytes in the stratum corneum; micro-abscesses, neutrophil aggregates in the stratum corneum can be observed in these images. See e71.\u0000\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture><p></p>\u0000 </div>\u0000 </figure></p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"125 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.66","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48109026","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Fast and Reliable Method to Isolate Human Placental Macrophages","authors":"Soraya Mezouar,&nbsp;Amira Ben Amara,&nbsp;Céline Chartier,&nbsp;Laurent Gorvel,&nbsp;Jean-Louis Mege","doi":"10.1002/cpim.77","DOIUrl":"10.1002/cpim.77","url":null,"abstract":"<p>Macrophages are specialized cells involved in recognition, uptake, and destruction of microorganisms. Human placental macrophages are poorly investigated because of the lack of a convenient protocol for their isolation. Here, we present a straightforward and reliable method to isolate macrophages from full-term human placentas. After enzymatic digestion of placental tissue and centrifugation of the cell suspension on a Ficoll cushion, placental macrophages are selected using magnetic beads coated with anti-CD14 antibodies. Isolated cells are characterized by flow cytometry. Ninety eight percent of isolated CD14⁺ placental macrophages also express the macrophage marker CD68. Thus, this efficient and reliable method yields placental macrophages at high purity and sufficient quantity for functional studies. © 2019 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":10733,"journal":{"name":"Current Protocols in Immunology","volume":"125 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpim.77","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37269119","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}