A new method to induce myasthenia gravis models and the protective effect of soluble decay accelerating factors.

It is expensive to induce experimental autoimmune myasthenia gravis (EAMG) by active immunity, and difficult to obtain natural acetylcholine receptor (AChR). We sought a new method of inducing EAMG by immunizing rats with artificially synthesized AChR. The AChR mRNA in TE671 cells was extracted and reverse transcribed. The inclusion body was purified and protein concentration was determined, and the EAMG animal model was used for induction. The serum was extracted from rat blood. The antibody titer was determined using enzyme-linked immunosorbant assay (ELISA). The concentration of decay accelerating factor (DAF) in the rat serum was determined by ELISA, and the metabolism of serum rDAF was determined by western blot. We evaluated the inhibition of rDAF by determining the 50% complement hemolysis unit in the rat serum. The extracellular domain (ECD) nucleotide sequence clone produced by polymerase chain reaction was completely consistent with that in the human gene bank; it was induced by isopropyl ß-D-1-thiogalactopyranoside to express the protein after insertion into vector pET16b. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the inclusion body protein was the exact target. The ECD protein was able to bind with mAb35 after dialysis and renaturation, which demonstrated protein activity. The soluble ECD protein was used to immunize rats and obtain the EAMG models. The inhibitory effect of the complement was unsatisfactory owing to high decay rate after rDAF injection into the EAMG models. It is easy to induce the EAMG model by obtaining the AChRTEα1 subunit ECD protein using the substitution method.

Beneficial effect of crotamine in the treatment of myasthenic rats.

INTRODUCTION: Crotamine is a basic, low-molecular-weight peptide that, at low concentrations, improves neurotransmission in isolated neuromuscular preparations by modulating sodium channels. In this study, we compared the effects of crotamine and neostigmine on neuromuscular transmission in myasthenic rats. METHODS: We used a conventional electromyographic technique in in-situ neuromuscular preparations and a 4-week treadmill program. RESULTS: During the in-situ electromyographic recording, neostigmine (17 µg/kg) caused short-term facilitation, whereas crotamine induced progressive and sustained twitch-tension enhancement during 140 min of recording (50 ± 5%, P < 0.05). On the treadmill evaluation, rats showed significant improvement in exercise tolerance, characterized by a decrease in the number of fatigue episodes after 2 weeks of a single-dose treatment with crotamine. CONCLUSIONS: These results indicate that crotamine is more efficient than neostigmine for enhancing muscular performance in myasthenic rats, possibly by improving the safety factor of neuromuscular transmission.