Figure 8

Correction of GAA activity in Pompe disease fibroblasts. (A) To study the time course of GAA correction, fibroblasts from Pompe disease (PD) patients were incubated in the presence of 50 μg/ml rhGAA for 2, 4 and 6 days. The medium and the enzyme were refreshed every other day. After the times indicated the cells were harvested and GAA activity measured. Untreated fibroblasts were cultured and harvested in parallel. Complete correction of GAA activity in severe PD fibroblasts requires 6 days and is slower than in the other cell lines. (B) CI-MPR intracellular distribution in untreated severe fibroblasts and severe fibroblasts treated for 6 days with rhGAA. The cells were fixed, permealized and stained with mouse monoclonal antibody to CI-MPR and Alexa 488-conjugated donkey anti-mouse IgG secondary antibody. Stained cells were visualized by fluorescence microscopy. * = p ≤ 0.000003, chi-square test. The data were analyzed using EpiInfo (version 3.3, 2004). (C) CI-MPR distribution was scored by two independent researchers. The analysis was performed on 10 images, each containing 40 cells. After rhGAA treatment for 6 days the rate of PD cells showing a reduced and/or mislocalized signal is decreased to 40%, as compared with untreated cell (90%). p ≤ 0.026