Origins of the Study of Immunological Features of Myasthenia GravisIn 1954, prompted by certain clinical observations1" and the findings of other investigators,3.' Nastuk, Strauss, and Osserman undertook studies to determine what role, if any, humoral inhibitors of neuromuscular transmission might play in the pathogenesis of myasthenia gravis. Their findings have been reported in detail e l~e w h e r e .~'~ In early experiments, an isolated frog sartorius muscle was indirectly stimulated via its attached motor nerve and measurements were made of the muscle tension developed upon exposure of the muscle to serum diluted to isotonicity with frog plasma. I t was found that serums from a few of the myasthenic patients studied caused reduction in the muscle tension output and cytolytic destruction of fibers lying on the surface of the sartorius muscle. After a period of latency of 7 to 10 minutes with the most active samples, there appeared a wrinkling of the membranes of surface muscle fibers, a clouding of these cells, and shortly thereafter destruction of membranes and retraction of the contractile elements. Larger numbers of myasthenic and normal serums were studied for these effects. Of 36 patients with myasthenia gravis, 16 (44 per cent) had serums with cytolytic activity. Of 36 normal individuals, eight (22 per cent) had serums which showed cytolytic properties. No titrations were performed, however, to determine the cytolytic potency of individual serums in the myasthenic and control groups. On the other hand, the heat lability of the cytolytic function of myasthenic serum (inactivated by heating to 56°C. for 30 minutes), and its reconstitution by mixture with an equal volume of fresh noncytolytic normal serum suggested the participation of serum complement in these effects. This led Nastuk, Plescia, and O~s e r m a n~.~ to study the total serum complement (C') activity of myasthenia gravis patients and normal controls. Serum complement activity did not vary greatly in normal individuals. All determinations were within A 20 per cent of the mean value. In patients with myasthenia gravis, serum C' activity varied much more widely with values
Summary A panel of sera from 90 persons was studied with respect to sites of binding of γ-globulins to striated muscle in vitro. The indirect and direct fluorescent antibody techniques were employed and compared. Sera from 51 individuals including 47 selected patients with myasthenia gravis, when tested with the indirect technique, reacted consistently with skeletal muscle I-bands in cryostat sections and myofibril suspensions of bovine, human and rabbit origin in a titer range 1:120 to 1:1920. H-zone (M-line) staining was a frequent associated finding. Sera from 49 control individuals reacted with the same morphologic loci, within limits determinable by light microscopic techniques, in a titer range 1:15 to 1:30. Individual and pooled serum globulins from patients with myasthenia gravis, which had been conjugated directly with fluorescein isothiocyanate, also reacted with I-bands and H-zones in a titer range 1:40 to 1:80. Directly conjugated serum globulins from normal controls failed to produce striational staining. The pattern of staining seen with both indirect and direct fluorescent antibody techniques strongly resembles that produced upon treatment of striated muscle with fluorescent antibodies against purified actin and tropomyosin. The present findings strongly suggest that A-band and Z-line staining with human sera on a specific immunologic basis, as described or inferred in previous reports, is an uncommon effect, if indeed it occurs at all. It is more likely that previous descriptions or interpretations of A-band staining by sera from patients with myasthenia gravis can be explained on the basis of procedural flaws and limitations. The possible staining of narrow zones of A-bands immediately adjacent to stained I-bands cannot be totally excluded because of the limits of resolution of the fluorescent antibody technique.
Summary Diethylaminoethyl (DEAE)-cellulose chromatography of whole human serum, and sodium sulfate precipitated serum globulins in Trisphosphate at pH 8.6, 0.005 M phosphate was used to obtain highly purified human γ-globulins. A 14-fold increase in yield of purified γ-globulin was obtained for a given column size when sodium sulfate precipitated globulins were used. γ-Globulins so derived were incorporated in Freund's adjuvant and injected repeatedly into rabbits over many months. The hyperimmune rabbit serum contained antibodies directed solely against the γ-globulins of human serum and cross-reactive with β2A and β2M globulins when tested by immunodiffusion techniques. Sustained immunization in several animals has been stressed as a more critical procedure for assessing purity of a γ-globulin preparation than initial in vitro immunodiffusion studies. The use and merits of specific and potent anti-human γ-globulin serums in the preparation of immunohistologic reagents has been discussed. Chromatographically prepared γ-globulins have been studied by immunodiffusion, electrophoretic and analytical ultracentrifugal techniques. Fragmentation was found repeatedly in preparations derived from sodium sulfate precipitated globulins which had been allowed to dialyze under sterile conditions for long periods against the chromatography buffer. The fragmentation is presumably different from the produced by papain because both electrophoretically slow and fast fragments retained the ability to bind antigens.
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