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Incidence involving Strongyloidiasis inside a Cohort associated with Migrants in Italia as well as Precision of an Novel ELISA Analysis regarding Ersus. stercoralis An infection, the Cross-Sectional Research.
Transforming growth factor (TGF)-β1 is one of the regulatory cytokines produced by B cells and has a pivotal role in intestinal homeostasis. TGF-β1 can determine the fate of naive T cells, such as differentiation, proliferation, and apoptosis, which are relevant to the pathogenesis of autoimmunity, infection, inflammation, allergy, and cancer. Here, we describe detailed methods for detection and quantification of TGF-β1 secreted by B cells using ELISA, flow cytometry, and real-time PCR.With the ever-increasing understanding of the roles of B cells in immune response and autoimmune pathogenesis, various techniques have been optimized for the detection of IL-10 production in B cells. In this chapter, we describe several commonly used methods for the effective detection of IL-10 in B cells at both mRNA and protein levels, including quantitative PCR analysis, intracellular staining of IL-10 in live B cells by flow cytometry, ELISA for secreted IL-10 detection, and ELISPOT assay for enumerating IL-10-producing B cells. We have further co-stained IL-10 with other cytokines and examined the staining efficiency. Moreover, we provide a detailed protocol for the detection of IL-10-producing B cells in situ by immunofluorescence microscopy. Since emerging evidence has suggested the promising strategy of cell therapy, we also provide a protocol to determine CD19+CD1dhiCD5+ B-cell distribution upon adoptive transfer using tile-scan imaging. Together, the application of the described methods for the detection of IL-10 will facilitate the characterization of B-cell subsets with regulatory functions and enhance our current understanding of the critical roles of B cells in immune response and autoimmune development.Regulatory B cells (Bregs) have immunosuppressive capacity, primarily via the production of IL-10. IL-10 expression and immunosuppression have been described in a number of human B cell subsets, two of which include the CD19+CD24hiCD38hi and CD19+CD24hiCD27+ populations. In this chapter, we describe how to identify and isolate these subsets from peripheral blood B cells via flow cytometry. We also explain how to expand Bregs in culture and how to identify them based on intracellular expression of IL-10.IL-10 is the best known and most studied anti-inflammatory cytokine and, in the last 20 years, it has acquired even greater fame as it has been associated with the regulatory phenotype of B cells. Indeed, although great efforts have been made to find a unique marker, to date IL-10 remains the main way to follow both murine and human regulatory B cells, hence the need of precise and reproducible methods to identify and purify IL-10-producing B cells for both functional and molecular downstream assays. In this chapter, we present our protocols to isolate these cells from the murine spleen and peritoneum and from human peripheral blood. Since the production of IL-10 by B cells is not only a weapon to counteract the adverse effect of pro-inflammatory cytokines but also a response to cellular activation, we focused on those B cells that are prone to IL-10 production and detectable following a short-term stimulation with phorbol-12-myristate-13-acetate, ionomycin, and lipopolysaccharide (murine system) or CpG (human system).B cells are primarily known for their capacity to differentiate into antibody-secreting cells (ASCs). ASCs are usually viewed as terminally differentiated cells sharing a unique phenotype. However, it lately became evident that ASCs exist in a variety of subsets differing by their lifespan, anatomic location, and immunological function. Thus, ASCs can exist as long-lived plasma cells (LLPC) that can persist for years in a nonproliferating state within particular niches in the bone marrow (BM), or as short-lived plasma cells (SLPC) that are primarily found in secondary lymphoid organs or inflamed tissues and wane upon the termination of the associated immune response. Another layer of ASC diversity was uncovered with the discovery of their capacity to produce various pro- or anti-inflammatory cytokines. Notably, a subset of natural regulatory plasma cells characterized by the distinctive expression of the inhibitory receptor lymphocyte activation gene (LAG)-3 and a unique capacity to produce interleukin (IL)-10 upon stimulation was recently identified. Here, we describe how to immunophenotypically characterize murine plasma cells as well as how to isolate them using cell sorting, with a special focus on these recently described natural regulatory plasma cells.B-1 cells are fetal-origin B lymphocytes with unique developmental and functional characteristics that can generate natural, polyreactive antibodies with important functions in tissue homeostasis and immune defense. While B-1 cell frequencies in bone marrow and secondary lymphoid tissues are low, relative high frequencies exist within peritoneal and pleural cavities of mice, including both CD5+ and CD5- B-1 cells. see more These cells represent B-1 reservoirs that, when activated, migrate to lymphoid tissues to secrete antibodies and/or cytokines. Here, we outline efficient methods for the extraction and magnetic isolation of CD5+ B-1 cells from the peritoneal and pleural cavities as well as the separation and phenotypic characterization of CD5+ and CD5- B-1 cells by flow cytometry.The spleen is the second major reservoir of B cells in the adult. In the spleen, cells, generated in the bone marrow, are selected, mature, and become part of the peripheral B-cell pool. Murine spleen comprises several B-cell subsets representing various maturation stages and/or cell functions. The spleen is a complex lymphoid organ organized into two main structures with different functions the red and white pulp. The red pulp is flowed with blood while the white pulp is organized in primary follicles, with a B-cell area composed of follicular B cells and a T-cell area surrounding a periarterial lymphatic sheath. The frontier between the red and white pulp is defined as the marginal zone (MZ) and contains the MZ B cells. Because B cells, localized in different areas, are characterized by distinct expression levels of B-cell receptor (BCR) and of other surface markers, splenic B-cell subsets can be easily identified and purified by flow cytometry analyses and fluorescence-activated cell sorting (FACS).Here, we will focus on MZ B cells and on their precursors, giving some experimental hints to identify, generate, and isolate these cells.
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