Macrophage/microglial cells infiltrate the Purkinje cell layer after cell death has already begun.

<p>The Purkinje cell layer is shown at three points in time. Cells of macrophage/monocyte lineage were not detected within the Purkinje cell layer at 48 hours, or at 72 hours, when cell death was already apparent. Macrophage/mononuclear infiltration did not begin until 96 hours (data not shown) and was more extensive at 120 hours. These data indicate that initiation of Purkinje cell death did not involve antibody-mediated cellular cytotoxicity. Scale bar = 20μ.</p

Comparison of uptake and cytotoxicity of anti-Yo antibody versus anti-Purkinje cell cytoplasmic antibodies obtained from three neurologically normal ovarian carcinoma patients whose sera did not react with Yo antigens.

<p>Uptake and accumulation of IgG (red) is seen with anti-Yo IgG and with IgGs from all three neurologically normal patients. Entry of SYTOX green into Purkinje cells containing IgG (yellow), indicative of cell membrane injury and death, is seen only in cultures incubated with anti-Yo IgGs for 96 hours (examples shown by arrows) but not with IgGs from neurologically normal Patients 1, 2, or 3. Uptake of normal IgG is not seen at confocal gain settings used for optimal visualization of anti-Yo antibody accumulation (13). Images shown are representative of three experiments. By the end of incubation (140 hours) staining for SYTOX green, indicative of necrotic cell death, was observed in less than 10% of cells incubated with sera from Patients 1, 2, and 3, an amount of background cell death staining similar to that seen in cultures incubated with control sera (data not shown). Parallel cultures incubated with anti-Yo sera or sera from Patients 1–3 sera remained negative by FLICA staining as compared to controls, indicating that with exposure to these sera did not result in apoptotic cell death (data not shown). Scale bar = 20μ.</p

Quantitative analysis of Purkinje cell death in rat cerebellar slice cultures incubated with native anti-Yo serum (native serum), sham adsorbed serum, and serum adsorbed with the 62 kDa major Yo antigen.

<p>The per cent of Purkinje cells which contained IgG and stained with SYTOX green was counted from eight fields similar to that seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123446#pone.0123446.g001" target="_blank">Fig 1</a>. Adsorption with the 62 kDa Yo expression protein effectively abolished Purkinje cell death compared to untreated serum or sham-adsorbed serum circulated through a nickel column with bound vector lacking Yo antigen.</p

Adsorption of the 62 kDa major anti-Yo antibody abolishes intracellular accumulation of anti-Yo IgG within Purkinje cells and IgG-mediated cytotoxicity.

<p>In this figure the upper row shows merged images demonstrating both IgG accumulation (red) and entry of SYTOX dyes indicative of cell death (green; yellow indicates colocalization of IgG and SYTOX); the bottom row shows staining with SYTOX death markers only. Rat cerebellar slice cultures were incubated with either 1) patient serum containing anti-Yo antibodies (Native serum); 2) the same serum passed over a nickel column bound with lysates from bacteria containing a control vector which lacked the His-tag Yo antigen (Sham adsorbed); or 3) a nickel column with bound His-tagged 62 kDa Yo expression protein (Anti-Yo adsorbed). Cultures were evaluated at intervals through 72 hours for IgG accumulation within Purkinje cells and for Purkinje cell death. Incubation of cultures with untreated (Native) anti-Yo antibodies for 72 hours resulted in antibody accumulation within virtually all Purkinje cells (red) and in Purkinje cell death as indicated by intracellular penetration of SYTOX green (green; co-labeling appears yellow). Antibody uptake and killing were essentially identical in cultures incubated with native anti-Yo serum and sham adsorbed serum (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123446#pone.0123446.g002" target="_blank">Fig 2</a>). In contrast, adsorption of sera with the 62 kDa Yo expression protein essentially abolished both antibody accumulation and cell death, indicating that Purkinje cell antibody accumulation and death are due specifically to interaction of anti-Yo antibodies with the 62 kDa cytoplasmic Purkinje cell protein. Scale bar = 20μ.</p

Comparison of uptake and cytotoxicity of anti-Yo antibody versus three other antibodies specific for the intracellular Purkinje cell proteins: calbindin, calmodulin, and PCP-2.

<p>The top row of panels demonstrates uptake and accumulation of IgG (red) within Purkinje cells after 96 hours in cultures incubated with anti-calmodulin, anti-calbindin, anti PCP-2, or anti-Yo IgGs. Entry of SYTOX green into Purkinje cells containing IgG, indicative of cell membrane injury and death (yellow), was seen in only in cultures incubated with anti-Yo IgGs (examples shown by arrows). The lower panels show only SYTOX green staining of Purkinje cells in cultures incubated with the antibodies indicated. In the culture incubated with anti-PCP-2. SYTOX staining indicative of cell death is seen in a single cell outside the Purkinje cell layer (asterisk) but not in Purkinje cells. Cultures were followed through 144 hours. There was progression of cell death in cultures incubated with anti-Yo antibodies. In contrast, cultures incubated calbindin, calmodulin, and PCP-2 did not exhibit Purkinje cell death above background levels seen in controls incubated with normal human IgG (data not shown). Scale bar = 20μ.</p

Rat cerebellar slice culture incubated with serum from Patient 3, a neurologically normal individual with mixed mesodermal ovarian sarcoma.

<p><b>Panels A and B</b> demonstrate. extensive accumulation of IgG not only within Purkinje cells (arrow) but also in other neurons within and near the Purkinje cell layer (asterisks). <b>Panels C and D</b>: Purkinje and other neurons exhibiting Cy5 labeling for human IgG do not contain SYTOX green, indicating that IgG uptake had occurred in viable cells. Scale bars = 20μ.</p

Comparison of Purkinje cell cytotoxicity produced by anti-Yo antibodies with cytotoxicity produced by commercial and patient antibodies reactive with other Purkinje cell cytoplasmic proteins.

<p>Rat cerebellar slice cultures were incubated for 72 hours with either 1) sera from patients with anti-Yo antibodies; 2) commercial antibodies to calbindin, calmodulin, or PCP-2/L7; 3) anti-Purkinje cells antibodies from the three neurologically normal patients studied, whose sera labeled Purkinje cell cytoplasm but did not react with Yo antigens; or 4) normal human IgG. Cultures were quantified for cell death as indicated by uptake of SYTOX dyes. Extensive Purkinje cell death was seen in cultures incubated with all 4 anti-Yo sera (data for 2 anti-Yo sera not shown). In contrast, cell death was not observed in cultures incubated with any of the three commercially obtained antibodies reactive with intracellular anti-Purkinje cell proteins nor with sera from Patients 1–3, despite extensive Purkinje cell uptake. Cultures incubated with normal human IgG exhibited only faint antibody accumulation by Purkinje cells and no detectable Purkinje cell death. Statistical significance between groups was determined by non-parametric Mann-Whitney ANOVA. Death in cultures incubated with anti-Yo antisera was statistically significantly greater than that seen in cultures incubated with commercial antisera, sera from patients 1–3, or normal human IgG.</p