Methods in Molecular Biology

Division of Allergy, Immunology, and Rheumatology, Department of Medicine, University of Rochester Medical Center, Rochester, NY, USA.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2012; 900:109-34. DOI: 10.1007/978-1-60761-720-4_6
Source: PubMed


B lymphocyte involvement in systemic lupus erythematosus has been recognized for several decades, mainly in the context of autoantibody production. Both mouse and human studies reveal that different types of antibody responses, as well as antibody-independent effector functions can be ascribed to distinct subpopulations (subsets) of circulating B cells. Characterizing human B cell subsets can advance the field of autoimmunity even further by establishing B cell signatures associated with disease severity, progression, and response-to-treatment. For this purpose, we have developed specialized B cell reagent panels for multiparameter flow cytometry, and combine their use with advanced bioinformatics strategies that together will likely be advantageous for improving the characterization, prognosis, and for possibly improving treatment regimens of chronic inflammatory diseases such as lupus.

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    • "These elements include single-color instrument compensation controls, fluorescence-minus-one controls that help determine the lower-limits of a positive stain when gating, and monitoring and documenting instrument set-up and performance on different procedure days of a study. Monitoring instrument performance as well as staining consistency is also facilitated by having a designated biological sample that is aliquoted and frozen for staining with each run (Kaminski et al., 2012). The source of this control may either be a pool of healthy control PBMC or of hemochromatosis-patient PBMC, known to be highly enriched for B lymphocytes without excessive perturbances in known constituent subsets. "
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    ABSTRACT: To advance our understanding and treatment of disease, research immunologists have been called-upon to place more centralized emphasis on impactful human studies. Such endeavors will inevitably require large-scale study execution and data management regulation ("Big Biology"), necessitating standardized and reliable metrics of immune status and function. A well-known example setting this large-scale effort in-motion is identifying correlations between eventual disease outcome and T lymphocyte phenotype in large HIV-patient cohorts using multiparameter flow cytometry. However, infection, immunodeficiency, and autoimmunity are also characterized by correlative and functional contributions of B lymphocytes, which to-date have received much less attention in the human Big Biology enterprise. Here, we review progress in human B cell phenotyping, analysis, and bioinformatics tools that constitute valuable resources for the B cell research community to effectively join in this effort.
    Full-text · Article · Oct 2012 · Frontiers in Immunology
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    ABSTRACT: B cells are central pathogenic players in systemic lupus erythematosus and multiple other autoimmune diseases through antibody production as well as antibody independent function. At the same time, B cells are known to play important regulatory functions that may protect against autoimmune manifestations. Yet, the functional role of different B cell populations and their contribution to disease remain to be understood. The advent of agents that specifically target B cells, in particular anti-CD20 and ant-BLyS antibodies, have demonstrated the efficacy of this approach for the treatment of human autoimmunity. The analysis of patients treated with these and other B cell agents provides a unique opportunity to understand the correlates of clinical response and the significance of different B cell subsets. Here, we discuss this information and how it could be used to better understand SLE and improve the rational design of B cell-directed therapies in this disease.
    No preview · Article · Apr 2014 · Seminars in Immunopathology
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    ABSTRACT: Background: Atopic dermatitis (AD) and psoriasis pathogeneses involve skin barrier impairment and immune dysregulation; however, the contribution of B-cell imbalances to these diseases has not yet been determined. Objective: We sought to quantify B-cell populations and antibody-secreting cells in the blood of patients with AD, patients with psoriasis, and control subjects. Methods: We studied 34 adults with moderate-to-severe AD (mean SCORAD score, 65), 24 patients with psoriasis (mean Psoriasis Area and Severity Index score, 16), and 27 healthy subjects using an 11-color flow cytometric antibody panel. IgD/CD27 and CD24/CD38 core gating systems were used to determine frequencies of plasmablasts and naive, memory, transitional, and activated B cells. Results: We measured increased CD19 1 CD20 1 B-cell counts in the skin and blood of patients with AD (P < .01). Significantly higher frequencies of chronically activated CD27 1 memory and nonswitched memory B cells were observed in patients with AD
    Full-text · Article · Oct 2015 · Journal of Allergy and Clinical Immunology
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