Acetylcholine receptor alpha peptide binding to survivin positive cells in myasthenics by five color FACS analysis.

<p>A) FITC labeled AChR-alpha subunit peptide binding to total myasthenic PBMC (0.44%), B) Excess unlabeled AChR-alpha subunit peptide followed by competitive binding of FITC-AChR peptide (0.03%). C) Control PBMC, subject number 19, (SVN+/CD38+/CD138+/CD20+ gated sub-population) showing FITC-AChR-alpha subunit peptide binding (0.33% of gated cells). D–F; Myasthenic patient PBMC (SVN+/CD38+/CD138+/CD20+ gated sub-population) showing FITC-AChR-alpha subunit peptide binding (1.55% (pt#10), 6.54% (pt#15) and 2.50% (pt#14) of total gated cells; <i>n</i> = 3).</p

Survivin as a Potential Mediator to Support Autoreactive Cell Survival in Myasthenia Gravis: A Human and Animal Model Study

<div><p>The mechanisms that underlie the development and maintenance of autoimmunity in myasthenia gravis are poorly understood. In this investigation, we evaluate the role of survivin, a member of the inhibitor of apoptosis protein family, in humans and in two animal models. We identified survivin expression in cells with B lymphocyte and plasma cells markers, and in the thymuses of patients with myasthenia gravis. A portion of survivin-expressing cells specifically bound a peptide derived from the alpha subunit of acetylcholine receptor indicating that they recognize the peptide. Thymuses of patients with myasthenia gravis had large numbers of survivin-positive cells with fewer cells in the thymuses of corticosteroid-treated patients. Application of a survivin vaccination strategy in mouse and rat models of myasthenia gravis demonstrated improved motor assessment, a reduction in acetylcholine receptor specific autoantibodies, and a retention of acetylcholine receptor at the neuromuscular junction, associated with marked reduction of survivin-expressing circulating CD20+ cells. These data strongly suggest that survivin expression in cells with lymphocyte and plasma cell markers occurs in patients with myasthenia gravis and in two animal models of myasthenia gravis. Survivin expression may be part of a mechanism that inhibits the apoptosis of autoreactive B cells in myasthenia gravis and other autoimmune disorders.</p></div

Survivin expression in myasthenia gravis patients (1–15) and control (16–25) PBMCs.

<p><b>A,B</b>) Representative FACS analysis of total survivin expression of (A) myasthenic patient (7) and (B) control (24) PBMC. <b>C–H</b>) PBMC were stained with antibodies to indicated markers. Data reflect the percentage of total cells staining positive for: <b>C</b>) Survivin (SVN) (p = 0.010), <b>D</b>) CD20+/SVN+ (p = 0.0009), <b>E</b>) CD38+/SVN+ (p = 0.02), <b>F</b>) CD27+/SVN+ (p = 0.05), <b>G</b>) CD138+/SVN+ (p = 0.01) and <b>H</b>) CD8+/SVN+ (p = 0.4). Mean values of patients and controls were compared using two way ANOVA.</p

Demographic and treatment characteristics of myasthenic patients (1–15) and controls (16–25) consisting of individuals with non-myasthenic neurologic conditions.

<p>Demographic and treatment characteristics of myasthenic patients (1–15) and controls (16–25) consisting of individuals with non-myasthenic neurologic conditions.</p

Survivin expression in the thymus.

<p>Representitive images from an analysis of eleven thymuses from MG patients were analyzed for survivin expression (immunosuppression treated, <i>n</i> = 7; immnosuppression naïve, <i>n</i> = 4). <b>A–D</b>) EOMG thymus from a 24-year-old woman who had clinical symptoms for 2 years and an AChR antibody level of 19.3 nmol and had never received immunosuppression or prednisolone showed a well developed cortex (C) and medulla (M), lymphofollicular hyperplasia with a germinal center (GC) close to Hassall's corpuscle (HC). <b>B,D</b>) High number of survivin positive cells in the cortex (C), cortico-medullary junction and the GC. <b>E–H</b>) EOMG thymus from a AChR antibody positive 21-year-old female with lymphofollicular hyperplasia after long-term immunosuppression showing cortical atrophy and a slightly regressive germinal center (GC) close to a Hassall's corpuscle (HC). <b>F,H</b>) Low number of survivin positive lymphocytes in the remnant cortical area (C), and the germinal center (GC) (A,B,E,F, ×100; C,D,G,H ×200).</p

SVN53-67/M57-KLH post-treatment on active EAMG in the mouse model.

<p><b>A</b>) Animals were injected with SVN53-67/M57-KLH at times marked by red arrows. Black arrows mark injection times of tAChR. Grip strength was monitored throughout experiment. Mice were treated with SVN53-67/M57-KLH (circles, <i>n</i> = 9), or PBS (squares, <i>n</i> = 10) (* denotes <i>P</i><0.05, t-test). <b>B</b>) Table shows clinical scores of mice treated with SVN53-67/M57-KLH were significantly stronger than PBS treated mice (* denotes <i>P</i><0.05, t-test).</p

Therapeutic effect of pre-treatment of SVN53-67/M57-KLH on active EAMG.

<p>Rats were pre-treated with SVN53-67/M57-KLH (circles) or PBS (squares). <b>A</b>) Animals were injected with SVN53-67/M57-KLH at times marked by red arrows. Black arrows mark injection time of tAChR. Grip strength was measured throughout experiment and values are a mean of biweekly measurements. (* denotes <i>P</i><0.02, t-test) <b>B</b>) Table of clinical scores based on rats at day 47. <b>C</b>) Levels of total IgG, IgG1 and IgG2b were significantly higher in the PBS control group (<i>n</i> = 7) compared to SVN53-67/M57-KLH treated animals (<i>n</i> = 7). (<i>P</i><0.02 for all, t-test) <b>D</b>) SVN53-67/M57-KLH treated rats demonstrated statistically increased amount of AChR (red) at the NMJs compared to PBS-treated rats. <b>E</b>) Pixel density of the labeled AChR by Alexa Fluor 594 -bungarotoxin at the NMJ in SVN53-67/M57-KLH treated animals (<i>n</i> = 5) compared to PBS treated controls (<i>n</i> = 5) (<i>P</i> = 0.013, t-test).</p