1 Anti-rhGAA antibody titers in GAA KO mice treated with three different regimens. ERT in patients with Pompe disease, leading to improved clinical outcomes. and 4?C followed by re-suspension of the pellet in PBS. Each SVP-Rapa injection consisted of ~?50?g of rapamycin. Methotrexate Alvimopan dihydrate was purchased from Calbiochem (San Diego, CA, USA). Diphenhydramine was purchased from Baxter Healthcare Corporation (Deerfield, IL, USA). 2.2. Mice and treatment Homozygous GAA-KO mice Mmp28 (6gene [20], were used in this study. A total of 15 male mice were utilized for ERT with weekly intravenous injections of 20?mg/kg rhGAA. For each mouse, pretreatment with 15?mg/kg diphenhydramine by intraperitoneal (IP) injection was performed 10C15?min prior to intravenous (IV) administration of rhGAA to prevent anaphylactic reactions [21]. The ERT was initiated at age of 10?weeks (set as ERT week 0) and ended at age of 22?weeks (ERT week 12) and mice received 13 injections of rhGAA in total. These mice were randomly divided into 3 groups (n?=?5 each) for different adjunct treatments as explained below. Group 1 (Empty NP group): 4?ml/kg vacant NP was mixed with rhGAA for injection in ERT weeks 0, 1, and 2; Group 2 (SVP-Rapa group): 4?ml/kg SVP-Rapa was mixed with rhGAA for injection in ERT weeks 0, 1, and 2. Group 3 (MTX group): 3 consecutive IP injections of MTX (10?mg/kg) were given at 0, 24, and 48?h after IV injection of rhGAA in each of week 0, 1, and 2 of ERT, as previously described [21]. All animal experiments were approved by the Institutional Animal Care and Use Committee of Duke University or college, and following local and national guidelines and regulations. 2.3. Sample collection and analyses Plasma samples were obtained every two weeks 4C6?days following rhGAA administration and stored at ??80?C for later analysis of anti-rhGAA antibody titer. Urine samples were collected prior to ERT and after 12?weeks of ERT. Total urinary hexose tetrasaccharide (Glca1-6Glca1-4Glca1-4Glc (Glc4), Hex4) assessments were performed for therapeutic responses by liquid chromatography-stable isotope dilution tandem mass spectrometry (LC-MS/MS) as explained [22]. Rota-rod assessments were performed every 4?weeks to determine motor balance, strength, and coordination [23]. Mice were euthanized 48?h after the last rhGAA injection following overnight fasting. All tissues were kept frozen for evaluating glycogen content and GAA activity as explained [23]. 2.4. Measurement of anti-rhGAA IgG antibody The anti-rhGAA antibody titer was measured by enzyme linked immunosorbent assay (ELISA) as Alvimopan dihydrate explained [24]. Briefly, 96-well plates (Corning Inc., Corning, NY, USA) were coated immediately at 4?C with 100?l per well 5?g/ml rhGAA. Following washing with 0.05% Tween 20 in PBS, 100?l per well diluted serum (1:200) were added in duplicates to rhGAA-coated plates and incubated at 37?C for 1?h. The plates were washed, and alkaline phosphatase-conjugated goat anti-mouse IgG secondary Ab (Cat # 115-055-205, Jackson ImmunoReasearch Laboratory Inc., West Grove, PA, USA) was added and allowed to incubate for 1?h at 37?C. Following a final wash, 4-Nitronphenyl phosphate disodium salt hexahydrate (Sigma-Aldrich Co., St. Louis, MO, USA) was added and allowed to develop for 20?min at room heat. Absorbance at 405?nm was read on a VICTOR X Multilabel Plate Reader (PerkinElmer Corporation, Waltham, MA, USA). 2.5. Statistical analysis One-way ANOVA with post hoc test (Tukey) was performed to analyze the differences among the three groups. If the Alvimopan dihydrate data did not meet the Shapiro-Wilk test for normality, the Kruskal-Wallis test and Mann-Whitney test were performed for nonparametric data. Data in graphs were offered as mean??standard deviation (SD) or standard errors of mean (SEM) as indicated. The urinary Hex4 levels prior to and post ERT were compared using paired em t- /em test. Data analyses were conducted using SPSS version 20.0 for Windows (IBM Corp, Armonk, NY, USA), and p? ?0.05 was considered significant. 3.?Results 3.1. Immune tolerance induction against rhGAA Co-administration of SVP-Rapa with the first three doses of rhGAA effectively prevented anti-rhGAA antibody development throughout the 12-week study period except for ERT week 12 (Fig. 1). After 12?weeks on ERT, two of the five mice in the SVP-Rapa group showed an increase of anti-rhGAA antibody, while the remaining three animals showed no sign of antibody formation. The empty NP co-treatment did not show any suppressive effect on anti-rhGAA antibody response, as the kinetics of.