Abstract：
BACKGROUND:CNS inflammation resulting from infection, injury, or neurodegeneration leads to accumulation of diverse B cell subsets. Although antibody secreting cells (ASC) within the inflamed CNS have been extensively examined, memory B cell (Bmem) characterization has been limited as they do not secrete antibody without stimulation. Moreover, unlike human Bmem, reliable surface markers for murine Bmem remain elusive. NEW METHOD:Using a viral encephalomyelitis model we developed a modified limiting dilution in vitro stimulation assay to convert CNS-derived virus specific Bmem into ASC. COMPARISON WITH EXISTING METHODS:Stimulation methods established for lymphoid tissue cells using prolonged stimulation with viral lysate resulted in substantial ASC loss and minimal Bmem to ASC conversion of CNS-derived cells. By varying stimulation duration, TLR activators, and culture supplements, we achieved optimal conversion by culturing cells with TLR7/8 agonist R848 in the presence of feeder cells for 2days. RESULTS:Flow cytometry markers CD38 and CD73 characterizing murine Bmem from lymphoid tissue showed more diverse expression patterns on corresponding CNS-derived B cell subsets. Using the optimized TLR7/8 stimulation protocol, we compared virus-specific IgG Bmem versus pre-existing ASC within the brain and spinal cord. Increasing Bmem frequencies during chronic infection mirrored kinetics of ASC. However, despite initially similar Bmem and ASC accumulation, Bmem prevailed in the brain, but were lower than ASC in the spinal cord during persistence. CONCLUSION:Simultaneous enumeration of antigen-specific Bmem and ASC using the Bmem assay optimized for CNS-derived cells enables characterization of temporal changes during microbial or auto-antigen induced neuroinflammation.
journal_name
J Neurosci Methodsjournal_title
Journal of neuroscience methodsauthors
DiSano KD,Stohlman SA,Bergmann CCdoi
10.1016/j.jneumeth.2017.05.011subject
Has Abstractpub_date
2017-06-15 00:00:00pages
58-68eissn
0165-0270issn
1872-678Xpii
S0165-0270(17)30130-9journal_volume
285pub_type
杂志文章abstract：BACKGROUND:Normal defaecation involves activation of anorectal mechanoreceptors responsive to pressure and stretch. The aim of this study was to develop selective anal and rectal mucosal light-touch stimulation suitable for measurement of cortical evoked potentials (EPs) in order to explore the sensory arm of these pat...
journal_title：Journal of neuroscience methods
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journal_title：Journal of neuroscience methods
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abstract：BACKGROUND:The accuracy of source reconstruction depends on the spatial configuration of the neural sources underlying encephalographic signals, the temporal distance of the source activity, the level and structure of noise in the recordings, and - of course - on the employed inverse method. This plenitude of factors r...
journal_title：Journal of neuroscience methods
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journal_title：Journal of neuroscience methods
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journal_title：Journal of neuroscience methods
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journal_title：Journal of neuroscience methods
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journal_title：Journal of neuroscience methods
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journal_title：Journal of neuroscience methods
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journal_title：Journal of neuroscience methods
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