Nicotinic acetylcholine receptor (AChR) has been purified from Torpedo electric organ, foetal calf muscle, adult human leg muscle, and foetal human skeletal muscle, by extraction in non-ionic detergent followed by affinity purification on immobilised a-toxin. The purified AChR preparations were used to study cellular responses in vitro from patients with myasthenia gravis. In addition, several characterisation studies were carried out on the foetal calf AChR preparation. Purified foetal calf AChR was shown in isoelectric focussing experiments to focus as a single sharp peak at pH 5.2 +/- 0.1, and the receptor sedimented in sucrose density gradients as a single species with a sedimentation coefficient S20w= 9.35 +/- 0.10 S, when indirectly labelled with [125 I]-a-bungarotoxin. SDS-polyacrylamide gel electrophoresis of purified foetal calf AChR consistently showed five major protein bands with (Mr) 40, 44, 47, 52 and 57 K. The 57K protein sub-unit was always the most prominent band. A procedure by which to measure antigen-specific lymphocyte proliferation in vitro was developed and optimised in a system using tetanus toxoid and peripheral blood mononuclear leucocytes (PBL) from a normal donor recently immunised against tetanus. Conditions for the production of human T-cell growth factor (TCGF), or Interleukin 2 (IL-2) were optimised, and used in the tetanus toxoid system to develop a procedure by which antigen-specific reactive T-lymphocytes could be isolated, expanded into long-term lines, and ultimately cloned. Antigen and TCGF were required for the expansion and maintenance of the T-cell lines and clones. The AChR purification procedure was modified several times in order to obtain a workable, non-inhibitory AChR preparation for studies of myasthenia gravis in vitro. Lymphocytes, collected from myasthenic blood samples, were shown to proliferate weakly in response to purified AChR added in vitro, with a mean stimulation index (+/- SEM) of 1.72 +/- 0.12. The proliferative response did not appear to be related to the species of AChR employed, age, sex, or clinical classification of the patient, but higher stimulation indices were obtained from patients in a state of disease exacerbation at the time the sample was taken. T-cell fractionation, or reactive T-cell pre-selection steps, did not enhance the proliferative response of myasthenic lymphocytes to purified AChR in vitro, and attempts to isolate and expand AChR-autoreactive T-lymphocytes using antigen and IL-2 were unsuccessful.
|Date of Award||1985|