Abstract: BACKGROUND: This prospective, randomized, double-blind, placebo-controlled, phase III trial assessed the efficacy, safety, and tolerability of mycophenolate mofetil (MMF) as a steroid-sparing agent in patients with myasthenia gravis (MG). METHODS: Patients with acetylcholine receptor antibody-positive class II-IVa MG (MG Foundation of America [MGFA] criteria) taking corticosteroids for at least 4 weeks were randomized to MMF (2 g/day) or placebo for 36 weeks. The primary endpoint was a composite measure defined as achievement of minimal manifestations or pharmacologic remission (MGFA post-intervention status), with reduction of corticosteroid dose on a set schedule. Secondary endpoints included disease severity, quality-of-life scores, and safety. RESULTS: A total of 44% of MMF-treated (n = 88) and 39% of placebo-receiving (n = 88) patients achieved the primary endpoint (p = 0.541). Improvements in mean quantitative MG, MG activities of daily living, and 36-item Short-Form health survey scores were similar in both groups. Numbers of adverse events were similar in both groups. The most commonly reported adverse events in the MMF-treated group were headache (12.5%) and worsening of MG (11.4%), and in the placebo group, worsening of MG (20.5%) and diarrhea (10.2%). CONCLUSIONS: Initiation of mycophenolate mofetil (MMF) treatment was not superior to placebo in maintaining myasthenia gravis (MG) control during a 36-week schedule of prednisone tapering. There were no significant differences in the primary or secondary endpoints between the study groups. MMF was well tolerated and adverse events were consistent with previous studies. Experience from this large, international, multicenter, phase III study employing full MG Foundation of America guidelines will aid the design of future MG studies.
Abstract: Objectives - The use of telencephalin as a possible marker for altered cortical function as demonstrated by functional MRI was investigated in a pilot study with 16 patients with localization-related epilepsy and secondarily generalized seizures. Materials and methods - Functional MRI of verbal working memory performance (Sternberg paradigm) and self-regulatory control processes (Stroop paradigm) was used to examine cortical activation in 16 patients with localization-related epilepsy and secondarily generalized seizures. Additionally, blood serum concentrations of soluble telencephalin (marker for neuronal damage) were determined. Results - In three patients (one temporal and two frontal focus), telencephalin was detected. All three patients had lower functional MRI activation in the frontotemporal region (P = 0.04), but not in other regions (P > 0.35) compared with patients without detectable telencephalin. Additionally, an association of levetiracetam and frontotemporal activation was observed. Conclusions - These preliminary data in a heterogeneous group suggest an association between decreased frontotemporal activation on fMRI and both detectable telencephalin serum levels and levetiracetam use. Future longitudinal studies with larger patient groups are required to confirm these observations. It is hypothesized that altered local function of the frontotemporal cortex in localization-related epilepsy might be better predicted by the biochemical marker telencephalin than epilepsy characteristics such as seizure focus.
Abstract: The primary autoantigen in myasthenia gravis, the acetylcholine receptor (AChR), is clustered and anchored in the postsynaptic membrane of the neuromuscular junction by rapsyn. Previously, we found that overexpression of rapsyn by cDNA transfection protects AChRs in rat muscles from antibody-mediated loss in passive transfer experimental autoimmune myasthenia gravis (EAMG). Here, we determined whether rapsyn overexpression can reduce or even reverse AChR loss in muscles that are already damaged by chronic EAMG, which mimics the human disease. Active immunization against purified AChR was performed in female Lewis rats. Rapsyn overexpression resulted in an increase in total muscle membrane AChR levels, with some AChR at neuromuscular junctions but much of it in extrasynaptic membrane regions. At the ultrastructural level, most endplates in rapsyn-treated chronic EAMG muscles showed increased damage to the postsynaptic membrane. Although rapsyn overexpression stabilized AChRs in intact or mildly damaged endplates, the rapsyn-induced increase of membrane AChR enhanced autoantibody binding and membrane damage in severe ongoing disease. Thus, these results show the complexity of synaptic stabilization of AChR during the autoantibody attack. They also indicate that the expression of receptor-associated proteins may determine the severity of autoimmune diseases caused by anti-receptor antibodies.
Abstract: Plasmid-based gene delivery to muscle is a treatment strategy for many diseases with potential advantages above viral-based gene delivery methods, however, with a relative low transfection efficiency. We compared two physical methods - electroporation and ultrasound - that facilitate DNA uptake into cells. Mice (C57Bl/6) were injected intramuscular using plasmid DNA encoding an intracellular protein (p53) followed by electroporation or ultrasound. Then 48 hr after the injections the mice were sacrificed. The parameter for transfection efficiency was the area of muscle expressing the transgene. The p53 expression plasmid showed a 36-fold increase (p = 0.015) in transfection efficiency with electroporation compared to ultrasound. Compared with ultrasound, electroporation significantly improves transfection efficiency of naked plasmid DNA transfer into skeletal muscle.
Abstract: INTRODUCTION: The generation of antibodies against G protein-coupled receptors (GPCRs) can be technically challenging. A modified DNA immunization protocol was employed in order to generate polyclonal antibodies against two herpes virus-encoded GPCRs, i.e. Epstein-Barr virus (EBV) pBILF1 and rat cytomegalovirus (RCMV) pR78. METHODS: pBILF1 and pR78 expression plasmids were first injected into the tibialis anterior muscle of rats and rabbits, respectively. Subsequently, the uptake of plasmids by the muscle cells was facilitated through in vivo electroporation. RESULTS: Potent antisera against both vGPCRs were obtained, as determined by immunoblot analysis and immunofluorescence. By using the antisera, we were able to show that the EBV BILF1 protein is expressed as a 45-kD, glycosylated protein, and that it is localized in the cytoplasmic membrane of EBV-infected cells. Interestingly, we found the R78-encoded vGPCRs to have unusual perinuclear localization in both R78-transfected and RCMV-infected cells. DISCUSSION: The in vivo DNA electroporation method is a useful technique for generating antibodies against GPCRs.
Abstract: Antibodies play a central role in immunity by forming an interface with the innate immune system and, typically, mediate proinflammatory activity. We describe a novel posttranslational modification that leads to anti-inflammatory activity of antibodies of immunoglobulin G, isotype 4 (IgG4). IgG4 antibodies are dynamic molecules that exchange Fab arms by swapping a heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule, which results in bispecific antibodies. Mutagenesis studies revealed that the third constant domain is critical for this activity. The impact of IgG4 Fab arm exchange was confirmed in vivo in a rhesus monkey model with experimental autoimmune myasthenia gravis. IgG4 Fab arm exchange is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 antibodies.
