George A. Feigen

Experimental cardiac anaphylaxis: Physiologic, pharmacologic and biochemical aspects of immune reactions in the isolated heart

Abstract Experimental cardiac anaphylaxis is initiated by a specific reaction between antigen and antibody, one of which has to be fixed to susceptible cells in the cardiac tissue. The biochemical steps initiated by the reaction appear eventually to release a variety of agents which, either by their direct effect or by their indirect effect as enzymes acting on plasma substrates, exert pharmacologic actions on the heart, producing a variety of irregularities. In most animals, and particularly in the guinea pig, histamine is the principal agent liberated. It is especially abundant in the mast cells of the right atrium, and its release accounts for most of the increased rate and amplitude of contraction, coronary constriction and atrioventricular block observed in the perfused heart. The chronotropic and inotropic responses in anaphylaxis are demonstrated equally well in the isolated atria and they are indistinguishable, electrophysiologically, from similar changes produced by histamine. The intensity of the reaction, as demonstrated in experiments utilizing passive sensitization, depends on the load of immunologic reactant fixed to the tissue and the concentration of the agent, either antigen or antibody, used to evoke it. The character of the vasoactive agent released is, additionally, determined by the electrophoretic class of antibody used for sensitization. Finally, bacterial toxins such as streptolysin can simultaneously produce a double insult to the heart of an immunized animal: They evoke anaphylaxis by the specific reaction of the antigenic determinant with sensitized cells at the same time that they produce damage to other regions of the heart through the toxic group of the molecule.

Histamine Release and Intracellular Potentials During Anaphylaxis in the Isolated Heart

The response of the sensitized guinea pig heart to perfusion, with an effective dose of ovalbumin, was found to consist of an acceleration of the rate, an increase in the amplitude of contraction, and a decrease in coronary flow, confirming the earlier observations of Wilcox and Andrus. The characteristic mechanical reaction of the isolated atria was an increase in amplitude and frequency of contraction, the more intense effects resulting in fibrillation. Electrophysiologically, atrial activity was demonstrable by bursts of action potentials, recurring as soon as the membrane had repolarized sufficiently to be re-excited. The resting potential was reduced, and the overshoot and the rate of rise of the action potential were diminished. All of the mechanical and electrical events noted in the Langendorff heart and the atrial preparations during anaphylaxis could be reproduced precisely by an appropriate dose of histamine. Evidence for the release of histamine by both the perfused heart and the isolated atria was first obtained by pharmacologic methods and then confirmed by paper chromatography of butanolic extracts.* Histamine was quantitatively estimated by a bioassay method which is discussed in detail in this report.

Sex Hormones and the Immune Response

The administration of 75 microgram/kg of estradiol 17beta at successively later stages in the immune response of female guinea pigs to penicilloyl-coupled cavian globulins showed that this steroid reduces the rate of attainment of the maximum titer, the magnitude of the titer achieved, and the rate of titer decay. Control titers maximized at the third experimental week and diminished to one third the peak value by the 6th week. When steroid treatment was begun coincidentally with inoculation (week 0), the peak titer was delayed by 3 weeks, and by 2 weeks when hormone priming was begun at week 1 or 2. The highest antibody titers achieved in the presence of estrogen were 25-30% lower than those of sesame oil controls. The greatest immunosuppressive effect was observed when estradiol was given at the peak of the immune response, the titer dropping by 50% and remaining at that level for the next 4 weeks in spite of continued antigen inoculation and steroid treatment. Titer decay after the end of the inoculation course was prevented by estradiol but not by progesterone, CHP, or these same oil vehicle.

Isolation and characterization of sea urchin toxin

Abstract From the present results it is evident that the active principle of sea urchin toxin is a non-dialyzable, thermolabile protein. It is pH-stable, almost completely soluble in distilled water, and can be precipitated at relatively high potency in the presence of 2/3 saturated ammonium sulfate in a yield amounting to 26 per cent of the dry weight of the starting material. The potency tends to decline slowly at −20° with a half-life of approximately 3 years. Absorption peaks at 278 mμ and 323 mμ are reduced by maneuvers which remove inert materials. On the basis of an ultracentrifugal analysis, the most active fraction, which has approximately 60 per cent of the lethal toxicity of the parent material, appears to be a single molecular species having a sedimentation coefficient of 2.6 Svedberg units at 20°.

Studies on the mode of action of sea urchin toxin—I. Conditions affecting release of histamine and other agents from isolated tissues☆

Direct tests with non-dialysable preparations of the pedicellarial toxin of Tripneustes gratilla L. elicited prolonged contractions of isolated guinea pigs ileum. Preliminary pharmacological experiments ruled out acetylcholine but suggested that histamine and several other agents might be released. Quantitative dose-response analysis showed that the activity of the toxin was reduced by gentle heating, and that carbon treatment and ammonium sulfate precipitation could affect the potency. Chemical evidence was obtained for the release of histamine from the ileal, cardiac, and pulmonary tissues of the guinea pig as well as from colonic and pulmonary tissues of the rat. The release of histamine was shown to be quantitatively dependent on the concentration of toxin acting upon the tissue.

The Effect of Impure Tetanus Toxin on the Frequency of Miniature End-Plate Potentials

SUMMARY: A partially purified preparation of tetanus toxin raised the frequency of the random appearance of miniature end-plate potentials in the isolated hemithorax of the mouse. The ratio of the peripheral to the central activity was increased by removing the greater part of the centrally acting tetanospasmin by specifically adsorbing the tetanospasmin out on protagon. The activation energy of the peripheral effect was 7 kcal. higher in the presence of toxin than in its absence. The peripheral effect can be neutralized with antitoxin.

Adsorption of antibody in vitro and magnitude of the Schultz-Dale reaction of guinea-pig ileum.

Studies show that the adsorption of rabbit antiovalbumin-I131 on guinea-pig ileum conforms to a Langmuirian isotherm, and that the physiological response to challenge with antigen depends upon the amount of antibody bound to the tissue.

Quantitative Adsorption of Antibody by the Isolated Heart and the Intensity of Cardiac Anaphylaxis

The present experiments show that the adsorption of antibodies to the guinea pig heart can be achieved by perfusing the heart with rabbit antiovalbumin solutions. The amount of radioactive antibody remaining on the tissue is a definite function of the concentration of antibody in the bulk phase instilled into the coronary circulation. When the antibody-loaded organ is challenged with ovalbumin, the efflux rate of antibody is increased; it is also increased in response to such nonspecific agents as histamine and epinephrine. Since the output rate is not affected when the heart fails to react to rechallenge with antigen, the release of antibody may be entirely dependent upon the mechanical response. The maximal amount.it of histamine-like material that can be produced by the heart undergoing anaphylaxis was found to be 1 × 10−s moles/Gm./min., estimated as histamine; the minimal amount of antibody necessary to release this quantity was calculated to be 0.343 μg. of specific antiovalbumin/100 mg. of heart. This load can be achieved by perfusing the heart with an antibody solution containing 30 μg. of specific γ-globulin. Perfusion with an antibody solution containing 50 μg./ml. showed the amount of histamine subsequently liberated by the same dose of ovalbumin to be reduced by 50 per cent. The rate and amplitude of contraction were increased to the same absolute values in all cases in which either ovalbumin or histamine was employed to set off the reaction, suggesting that the release of active material at even the low sensitizing doses of antibody used exceeded the ceiling of the physiological response. The decrease in coronary flow was inversely related to the dose of sensitizing antibody and bore little relation to the changes in this parameter induced by the subsequent administration of histamine.

Studies on the mode of action of sea urchin toxin—II Enzymatic and immunological behavior

Abstract The interaction of several sea urchin toxin preparations with certain substrates in mammalian serum produces dialyzable, heat-stable substances which cause contractions of the guinea pigs ileum and rats uterus. Evidence is presented to show that the toxin acts kinetically as an enzyme and that one of the substrates is electrophoretically pure human α 2 -macroglobulin. Crude toxin preparations were also shown to be kininolytic with respect to the dialyzable reaction-product as well as to synthetic bradykinin. Rabbit antibodies to formalinized toxoids quantitatively precipitated active toxin, fixed complement, and protected mice against lethal intravenous doses in the usual quantitative protection and neutralization tests. Immunoelectrophoretic analysis brought out the existence of two distinct antigenic determinants, and additional serological tests showed that antibodies directed against the pedicellarial toxins of Tripneustes gratilla cross-reacted with test proteins of Strongylocentrotus purpuratus .

Serological identification and physical-chemical properties of the non-spasmogenic principle (NSP) in tetanus toxin

Abstract Previous studies had shown that crude tetanus toxin had at least two distinct physiological actions, one due to tetanospasmin which acts on the central nervous system, and the other due to a separate principle which acts on the peripheral neuromuscular junctions. In this paper we report the isolation of the non-spasmogenic principle by a combination of techniques involving (a) the removal of the centrally-acting material by adsorption on a cerebroside-ganglioside complex, (b) the fractionation of the supernate with ammonium sulfate, and (c) gel-filtration of the active fraction on Sephadex. The material thus prepared gave a single component in immunoelectrophoresis against equine antitoxin; this was separate and distinct from that given by the centrally-active fraction. Since the two activities could be independently concentrated, the peripheral-to-central ratio of potency could be used as a guide to the efficiency of purification. Accordingly, these ratios were: parent toxin, 1·2 × 10 2 ; peripheral factor, 1·2 × 10 10 ; and lethal factor, 2.2 × 10 −4 . The non-spasmogenic material gave a single peak in the analytical ultracentrifuge, corresponding to a Svedberg coefficient of 2·2.