NRG in the presence of ACh induces a significant decrease in secreted TNF-α and IL-6 levels in LPS challenged microglia.

<p>Media from untreated (control), NRG, LPS, or both NRG and LPS, in the presence or absence of ACh was analyzed for TNF-α and IL-6 concentrations using a commercially available ELISA kit. BV-2 cells pretreated with ACh and then treated with LPS showed a concentration of 428.02±70.24pg/mL in TNF-α compared to untreated cells while BV-2 cells pretreated with ACh then treated with NRG and LPS showed a concentration of 287.80±47.69pg/mL (n=7, <i>p</i><0.01, Student’s t-test). Interlukin-6 levels significantly decreased with neuregulin treatment with (0.67±0.17pg/mL, n=3, <i>p</i><0.0001, ANOVA) or without ACh (3.01±0.12pg/mL, n=3, <i>p</i><0.05). Taken together our results indicate that BV-2 cells in the presence of ACh and NRG and immunologically challenged with LPS result in a 32.76% decrease in TNF-α and a 87.73% decrease in IL-6 compared to BV-2 cells pretreated with ACh and challenged with LPS alone.</p

NRG increases α7nAChR expression in microglia and macrophages.

<p>Microglia (BV-2) and macrophage (RAW) cell cultures were treated overnight with NRG (N) or left untreated (C). LPS (L) was added to activate cells four hours prior to extraction. Protein extracts were then precipitated with biotin-conjugated α-BTX and streptavidin agarose beads. Panel A is a representative immunoblot probed with anti-α7nAChR antisera. The α7nAChR band is shown at approximately 55kDa. Subsequent densitometric analysis of immunoblots shows a 1.53 ± 0.42 fold increase in α7nAChRs over control in NRG treated BV-2 cells (n=3, <i>p</i> <0.05, t-test). BV-2 cells appeared to show a decrease in α7nAChR expression when treated with LPS, 0.76 ± 0.47 compared to untreated cells, however LPS effect on BV-2 cells was variable. RAW264.7 cells treated with NRG showed a 1.11± 0.49 fold increase over untreated cells. Taken together our results indicate that NRG increases α7nAChR expression in BV-2 cells but have little effect in RAW264.7 cells. Values are means ± SEM. Error bars represent SEM.</p

ErbB2-4 receptor expression and phosphotyrosine activity in microglial and macrophage cell lines.

<p>Immunoblots of whole cell lysates (panel A) or isolates from immunoprecipitations using anti-ErbB2, anti-ErbB3 and anti-ErbB4 antibodies (panel B) from microglial cell lines (EOC and BV-2) and macrophage (RAW) were probed with antibodies specific for each ErbB receptor. Arrows to the right of each panel indicate the position of each expected immunoreactive species and its mass (185 kDa). Whole lysates from EOC-20, BV-2 and RAW264.7 cell lines immunoblotted with the anti-ErbB2, anti-ErbB3 and anti-ErbB4 antibodies showed no detectable levels of these proteins in each of these cell line (A). However, immunoprecipitated samples from the same cell lines indicate that ErbB4 is expressed (IP, bottom panel B) and that ErbB4 phosphotyrosylation in these cells increased with neuregulin treatment (4G10, bottom panel B). Positive control cell lines were C2C12 for ErbB2 and 3 and HeLa S3 (H) for ErbB4. Secondary controls (shown at right) were probed with secondary antibody only. Mouse anti-β-tubulin antibody indicates equal loading of cell extracts indicated by the band at the bottom of each ErbB panel at 50kDa. Lack of detectable banding in EOC, BV-2 and RAW may indicate no or low levels of the ErbB receptors.</p

Genes induced during the clearance phase of self-limited infection.

a<p>Expression levels of genes induced above the confidence interval at week 13 but below the confidence interval at weeks 4, 6 & 40 of X0190.</p><p>The bold and italicized values represent data above the 99% confidence interval as described in the text.</p

Genes induced during the early phase of self-limited and persistent infections.

a<p>Expression levels of genes induced above the confidence interval at weeks 4 & 6 but below the confidence interval at weeks 13 & 40 in X0190.</p><p>The bold and italicized values represent data above the 99% confidence interval as described in the text.</p

Real-time PCR quantification of candidate genes involved in viral clearance and persistence.

<p>TaqMan real-time PCR was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003442#s4" target="_blank">Materials and Methods</a>. The y-axis shows the relative unit of a given gene normalized to GAPDH and 18s rRNA. Data are expressed as means±SEM. In all cases, average values obtained during the first eight weeks of infection were compared between recovered (X0190) and chronically infected chimpanzees (X 0234, X0142 and X6412). * <i>P<0.05</i>, ** <i>P<0.01</i>, *** <i>P<0.01</i>, **** <i>P<0.005</i>.</p

Genes induced during the early phase of self-limited infection as defined by average expression values.

a<p>Average expression values of biopsies taken during the 1^st 8 weeks of infection.</p><p>Bold-face genes are also in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003442#pone-0003442-t001" target="_blank">Table 1</a>.</p