Four patients with molecularly proven HIBM were enrolled in a protocol approved by the NHGRI Institutional Review Board, and gave written, informed consent. Baseline evaluations at the NIH Clinical Center consisted of a complete history and physical examination, laboratory studies including quantitative immune globulins, a cardiac evaluation with a chest radiograph, electrocardiogram and echocardiogram, an ophthalmologic examination, and a neuromuscular evaluation that included an MRI of the calf and/or thigh. A baseline Rehabilitation Medicine evaluation included quantitative muscle strength testing, i.e., isometric fixed dynamometry  of 10 muscle groups, and measurements of tongue strength [32,33]. Strength was tested with subjects positioned supine and sitting on a treatment table, with stabilization provided by belts or examiners. Subjects performed two maximal voluntary isometric contractions of 10 muscle groups bilaterally separated by at least 30 s of rest. Joint angles were kept constant throughout the study via goniometric measurements. Cuffs were placed around the desired limb and were attached to a force transducer fixed to a stationary bed frame. The means of two trials were used for data analyses. Functional testing included a modified barium swallow evaluation, functional reach tests , timed up and go , and a 6-minute walk test to measure endurance . A quadriceps muscle biopsy was taken for histology, immunohistochemistry and immunoblotting. Pulmonary function testing was performed in patients 3 and 4.
After baseline testing, patients received a loading dose (1 g/kg) of immune globulin (Octagam 5% or Gamunex 10%) by continuous intravenous infusion on two consecutive days. Patients were hydrated before and after the infusions with 500 mL of either normal saline or half-normal saline. They were also premedicated with 50 mg of IV diphenhydramine and either 650 mg of acetaminophen or 250 mg of naproxen. One week after the two loading doses, the patients began receiving weekly maintenance doses of IVIG at 400 mg/kg for a total of 3 maintenance infusions. Hydration and premedication were also provided for these infusions. Evaluations of quantitative muscle testing, grip and pinch strength, 6-minute walk test and functional reach studies were performed directly after the loading dose and at the end of the study. Tongue strength measurements, a modified barium swallow, an ophthalmologic evaluation and a muscle biopsy were repeated at the end of the study. Laboratory testing was performed after each infusion and at the end of the study to monitor for safety and to follow levels of immune globulins.
Open quadriceps muscle biopsies were performed before and 3–4 days after the last dose of IVIG. Specimens were processed for muscle enzyme histochemistry, immunocytochemistry, and immunoblotting as described below.
Alpha-dystroglycan and NCAM immunocytochemistry
Coded serial cryosections (5 μm each), obtained before and after IVIG treatment from the patients, one normal control (N1), and one individual with sporadic IBM (N2), were fixed in acetone for 5 min and pre-incubated with phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA). The sections were then incubated overnight at 4°C with antibodies against α-dystroglycan (VIA4-1 and IIH6, Upstate, Lake Placid, NY, USA) diluted at 1:100, or anti-NCAM (123C3, Santa Cruz Biotechnologies, CA, USA) diluted at 1:200. Immunoreactivity was detected using FITC-labeled goat anti-mouse IgG or IgM (Molecular Probes, Eugene, OR) at a dilution of 1:500 for 1 h. Negative controls included the omission of each primary antibody in every staining. The sections were mounted in 30% glycerol-based medium and viewed using fluorescent microscopy.
Muscle tissues were homogenized in 200 μL of CelLytic (CelLytic, Sigma, St Louis, MO, USA), consisting of a mild detergent, bicine buffer and 150 mM NaCl, with protease inhibitors (Complete Mini®, Roche, Mannheim, Germany). The lysates were sonicated and microfuged at 8000 g for 10 min at 4°C. The protein concentration of the supernatant was determined using the BCA protein assay (Pierce, Rockford, IL, USA) and equal amounts of protein (25–50 μg) were loaded and electrophoresed on 4–12% Tris-Glycine gels (Novex, Invitrogen, Carlsbad, CA, USA). Resolved proteins were electrophoretically transferred to a 0.45 μm Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, IL, USA). Subsequently, membranes were blocked with 10% fat-free milk in PBS with 0.1% Tween 20 (PBST) for 30 min and then incubated with antibodies against NCAM (123C3, Santa Cruz Biotechnologies, CA, USA), α-dystroglycan (clone IIH6C4, 05–593, Upstate, Lake Placid, NY, USA) or β-actin (AAN01, Cytoskeleton, Denver, CO, USA) followed by horseradish peroxidase-conjugated secondary antibodies in PBST containing 3% fat-free milk. Immunoreactive bands were visualized by enhanced chemiluminescence using the Amersham ECL Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK). Density analysis was performed on digital images obtained with the Kodak Image station and software (Perkin Elmer, Boston, MA, USA). The protein levels were normalized to those of β-actin to correct for differences in protein loading and/or transfer.
For enzyme treatment experiments, protein samples (25 μg) were incubated with neuraminidase (N-6514, Sigma, St Louis, MO, USA), 1 mU/μg of protein, for 30 min at 37°C. Undigested and digested samples were analyzed by immunoblotting.
For isoelectric focusing (IEF) of transferrin, whole serum was iron-saturated and isofocused on pH4-6.5 PhastGels (Amersham Biosciences, Piscataway, NJ, USA) as described [36,37]. Transferrin isoforms were visualized by incubation with rabbit anti-human transferrin antibody (Dakocytomation, Denmark) followed by silver staining (Amersham Biosciences, Piscataway, NJ, USA). For IEF of apolipoprotein C-III (Apo C-III), whole serum proteins were acetone-delipidated and resuspended in 8 M urea. Subsequently, Apo C-III was isolated and concentrated by centrifugation through centricon filters (Millipore, Bedford, MA, USA) and isofocused on PhastGels pH4-6.5 (Amersham Biosciences, Piscataway, NJ, USA). The proteins were transferred by diffusion blotting onto nitrocellulose membranes and visualized by incubation with goat anti-Apo CIII antibodies (Chemicon, Temecula, CA, USA) and Enhanced ChemiLuminescence detection (Amersham Biosciences, Piscataway, NJ, USA) as described [37,38].