Muscle glycogen synthase protein is elevated in GAA^−/− mice.

<p>Tissue lysates were prepared for the indicated tissues from each mouse strain. Lysates from 5 mice were pooled and 100 µg of protein from the pooled lysate was analyzed by Western blot analysis. All of the blots were probed with an anti-GAPDH antibody (∼37 kDa in all panels) to verify equal protein loading of all the test samples. (A) An anti-muscle glycogen synthase antibody was used to probe the blots containing samples from the heart, triceps, and quad. An anti-liver glycogen synthase antibody was used to probe the blot containing samples from the liver. (B) An anti-phospho-glycogen synthase (Ser641) antibody was used to probe the blots for phosphorylated GS. (C) Glycogen synthase transcript levels are not elevated in GAA^−/− mice compared to wild type animals. RNA was isolated from triceps and heart of C57Bl/6 and GAA^−/− mice and probed with qPCR primers as described under “Experimental Procedures.” Values shown are means ± SEM.</p

G6P (A) and hexokinase (B) levels are elevated in GAA^−/− mice compared to wild type (C57/Bl6) and reduced by rhGAA treatment.

<p>GAA^−/− mice were dosed with rhGAA as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056181#pone-0056181-g004" target="_blank">Figure 4</a>. G6P and hexokinase was quantified in heart and triceps homogenates from the strains indicated. Values are means ± SEM. Data was analyzed by one-way ANOVA followed by Newman-Keuls comparing groups. ***<i>P</i><0.001.</p

Glycogenin levels are dysregulated in GAA^−/− mice and normalized by rhGAA treatment.

<p>Homogenates from triceps were prepared for the indicated strains. Lysates from 5 mice for each strain were pooled and 100 µg of protein analyzed by Western blot. A monoclonal antibody to glycogenin was used to probe the blots. (<i>A</i>) lysates not treated with amyloglucosidase. (<i>B)</i>, treated with amyloglucosidase. (<i>C</i>) GAA^−/− mice were dosed with rhGAA as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056181#pone-0056181-g004" target="_blank">Figure 4</a>. Lysates from the strains indicated in this panel were not treated with amyloglucosidase.</p

rhGAA treatment normalizes glycogen synthase protein and activity levels in heart of GAA^−/− mice.

<p>Groups (n = 5) of 3 to 4-month old mice of the indicated strains were dosed with rhGAA (100 mg/kg) weekly for 4 weeks by tail vein injection. Muscle homogenates were prepared, pooled and analyzed by: (A) Western blot using an anti-GS antibody and (B) densitometry analysis of the western blots (with GS normalized to GAPDH)., Samples were also processed for measurements of (C) glycogen synthase activity (D) tissue glycogen levels. Values are means ± SEM. Values are means ± SEM. Data was analyzed by one-way ANOVA followed by Newman-Keuls comparing groups. ***<i>P</i><0.001.</p

Glycogen is not reduced in GAA^−/− mice lacking S6K1 and S6K2.

<p>Glycogen was quantified in tissue lysates collected from groups (n = 10) of 3–4 month old mice of the indicated strains. Values shown are means ± SEM. Data was analyzed by one-way ANOVA followed by Newman-Keuls comparing all groups to C57Bl/6. ***<i>P</i><0.001.</p

ITI treatment regimen which includes rituximab, methotrexate and intravenous immunoglobulin (IVIG).

<p>This short course of ITI regimen (5 weeks) needs to be started together with the first dose of ERT. IVIG is administered on a monthly basis for a period of 5–6 months.</p

Representative Western gel blot showing CRIM negative status of four patients (lanes 3–6).

<p>Lane 1- protein magic marker; lane 8 -CRIM negative control cell line; lane 10 - normal human fibroblast (NHF) control; Lanes 2, 7 and 9 - left empty. 20 ug of skin fibroblast cell protein extract was loaded for each patient lane and 2.5 ug protein was loaded for NHF. Western blot was probed with anti-GAA antibody and ß-Actin was used as a protein loading control.</p

An algorithm for the management of cross-reactive immunologic material (CRIM)-negative (CN) infantile Pompe disease patients.

<p>^*Institutional review board (IRB) approved study (NCT01665326; <a href="http://www.clinicaltrials.gov" target="_blank">www.clinicaltrials.gov</a>) for rapid determination of CRIM status and long-term follow-up of response to treatment and ITI in Pompe disease. ^†CN status determination from an established CRIM negative mutation database, which allows prediction of CN status in more than 90% cases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone.0067052-Bali1" target="_blank">[15]</a>. ^‡ITI regimen is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone-0067052-g002" target="_blank">Figure 2</a>. ^§Based on the literature antibody titers sustained at ≥6,400 results in a suboptimal therapeutic response to ERT. For that reason, 6,400 was used a cutoff for further intervention <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone.0067052-Banugaria1" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone.0067052-Abbott1" target="_blank">[19]</a>. **Based on the half-life of rituximab, CD19% recovery is typically noted around 5 months. ^††The decision to repeat the same ITI regimen (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone-0067052-g003" target="_blank">figure 3</a>) or to administer ITI with a plasma-cell-targeting agent <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone.0067052-Banugaria4" target="_blank">[20]</a> should be based on multiple factors including, but not limited to, patients clinical status, CD19% and Fc_γ receptor polymorphism. ^‡‡ITI regimen with plasma cell targeting agent such as bortezomib has been described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067052#pone.0067052-Banugaria4" target="_blank">[20]</a>.</p

Clinical parameters.

<p>Clinical parameters.</p

Comparison of median left ventricular mass index (LVMI) values seen over time in CRIM-negative (CN) ERT monotherapy (n = 11) versus CN ERT+ITI (n = 7) treated patients.

<p>The upper limit of normal LVMI is 64 g/m² (represented by a horizontal dashed line).</p