H Eagle’s research while affiliated with Johns Hopkins Medicine and other places

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Publications (19)


The pharmacologic basis for the widely varying toxicity of arsenicals
  • Article

January 1944

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25 Citations

Journal of Pharmacology and Experimental Therapeutics

R.B. Hogan

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H. Eagle


ON THE PRESENCE IN SYPHILITIC SERUM OF ANTIBODIES TO SPIROCHETES, THEIR RELATION TO SO CALLED WASSERMANN REAGIN, AND THEIR SIGNIFICANCE FOR THE SERODIAGNOSIS OF SYPHILIS.
  • Article
  • Full-text available

January 1940

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12 Reads

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28 Citations

1. In confirmation of Gaehtgens, syphilitic human sera give positive complement fixation with cultures of so called T. pallidum (Reiter strain). Syphilitic rabbit sera are equally reactive. Syphilitic human and rabbit sera agglutinate these cultures, often in high titre (Beck). 2. Normal rabbit sera react weakly with the culture to give both agglutination and complement fixation in low titre. Normal human sera, despite the fact that they contain agglutinins in low titre, fail to fix complement with the Reiter strain of cultured spirochetes. Confirming Gaehtgens, the latter reaction is therefore of practical utility for the serum diagnosis of syphilis. 3. When syphilitic serum is heated at 63 degrees C., there is no demonstrable difference in the thermolability of the antibody to spirochetes, and of the reagin which determines the Wassermann and flocculation tests. 4. (a) The absorption of syphilitic serum by spirochetal suspensions removes all reactivity, not only for the spirochetes, but for tissue lipoids (alcoholic beef heart extract) as well; the sera become Wassermann- and flocculation-negative. (b) Absorption of syphilitic serum with tissue lipoids renders the Wassermann and flocculation tests negative, but does not demonstrably change the reactivity of the serum with spirochetes. (c) Rabbits immunized to beef heart lipoid develop spirochetal agglutinins and complement-fixing antibodies (Reiter strain) in high titre. 5. It is concluded that these cultured spirochetes contain antigenic material serologically related to a substance present in mammalian tissue, as well as other antigenic factors not present in such extracts, but equally reactive with syphilitic serum. 6. These findings support the thesis that the primary serologic change in syphilis is the development of antibodies to T. pallidum. The Wassermann and flocculation tests would be explained on the basis that the tissue extracts used as "antigen" in these tests contain one or more substances serologically related to antigenic components of T. pallidum. Similarly, the cultured Reiter strain of spirochete is apparently sufficiently close serologically to T. pallidum to be agglutinated by and to give complement fixation with the antibodies to T. pallidum present in syphilitic serum. 7. Since suspensions of cultured spirochetes contain antigenic factors which react specifically with syphilitic serum, some of which are not present in ordinary Wassermann and flocculation "antigens," they may prove even more valuable than those tissue extracts in the serodiagnosis of syphilis.

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SOME EFFECTS OF FORMALDEHYDE ON HORSE ANTIPNEUMOCOCCUS SERUM AND DIPHTHERIA ANTITOXIN, AND THEIR SIGNIFICANCE FOR THE THEORY OF ANTIGEN-ANTIBODY AGGREGATION.

March 1938

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10 Reads

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12 Citations

Journal of Experimental Medicine (JEM)

Small amounts of formaldehyde inhibited the precipitating activity of horse diphtheria antitoxin with toxin and of horse antipneumococcus serum with the homologous capsular carbohydrate. Approximately 1 part of commercial formaldehyde to 1000 parts of serum, acting for 24 hours, inhibited the flocculating activity completely. In both cases, the combining affinity of the treated antibody for the corresponding antigen was not demonstrably affected, as determined both by in vitro experiments and by animal protection. More intensive treatment of the antipneumococcus serum caused an apparent loss of its bacterial agglutinating activity, but on centrifugation the organisms cohered: combination had occurred, and only the spontaneous aggregation was prevented. These effects are the same as those previously described for diazo compounds, and have been qualitatively reproduced with acetaldehyde, benzaldehyde and butyraldehyde. The quantitative relationships suggest that only a few groups in the antibody molecule need be modified by formaldehyde in order to prevent aggregation; and it is probable that these are some of the free NH(2) groups of the antibody protein. In marked contrast, the combining affinity of both antipneumococcus antibody and diphtheria antitoxin for the corresponding antigens was only slightly affected by amounts of formaldehyde which sufficed to block the free NH(2) groups rapidly and almost completely. Similarly, this amount of treatment did not affect the reactivity of these two antisera acting as antigen with a rabbit antiserum versus horse serum. The integrity of the NH(2) groups is apparently not essential for the activity of these sera acting either as antigen or as antibody; and the slow disappearance of their activity in concentrated HCOH is apparently to be ascribed to some secondary reaction other than the simple addition of HCOH to free NH(2) groups. The present experiments do not support the theory that antigen-antibody aggregates are lattice-like structures built up from elementary antigen-antibody compounds because of residual specific combining groups. The aggregating activity of both antipneumococcus serum and diphtheria antitoxin was completely inhibited by procedures which did not demonstrably affect their combining power with antigen. This suggests that the aggregation of antigen-antibody compounds is a secondary, non-specific reaction. It is perhaps significant that the amount of formaldehyde which just sufficed to prevent aggregation also caused a marked increase in the solubility of the pneumococcus antibody, which could then no longer be precipitated at serum pH by dilution with water or by dialysis. This strongly suggests that the loss of precipitating activity is actually due to the increased solubility of the antibody and supports the hypothesis that the primary cause of specific antigen-antibody aggregation is the relative insolubility of the bound antibody.


The coagulation of blood by snake venoms and its physiologic significance

April 1937

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44 Reads

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121 Citations

Nine of the 17 venoms here tested were found capable of coagulating citrated blood or plasma. As has been believed by most workers in the field, 7 of these 9 coagulant venoms convert fibrinogen to an insoluble modification resembling fibrin (Bothrops atrox, Bothrops jararaca, Bothrops nummifera, Crotalus adamanteus, Crotalus horridus, Crotalus terrificus basiliscus, Crotalus terrificus terrificus). The optimum pH for this coagulation was determined for 3 of these, and was found in each case to be approximately pH 6.5, the same as that for the action of thrombin on fibrinogen. Unlike thrombin, however, the fibrinogen-coagulating activity of the venoms was unaffected by the antithrombin elaborated in the course of anaphylactic shock. In addition to coagulating fibrinogen directly, 3 of these venoms (Bothrops atrox, Bothrops jararaca, and to a less extent, Crotalus terrificus basiliscus) acted on prothrombin to convert it to thrombin, without the necessary intervention of either calcium or platelets. Finally, 2 venoms (Notechis scutatus, and to a slight extent, a mixed Micrurus venom), which had no demonstrable effect on purified fibrinogen, nevertheless converted prothrombin to thrombin. Unlike the reaction between the venoms and fibrinogen, this activation of prothrombin has no definite pH optimum, but takes place over a wide zone (pH 5.6-8.3). In the case of Bothrops atrox, there was some indication that the initial velocity of the reaction increased with increasing alkalinity, but that the amount of thrombin ultimately formed decreased. Extraordinarily minute quantities of some of these venoms sufficed to produce a demonstrable activation of prothrombin. Thus, the fer de lance (Bothrops atrox) venom was active in a 1:25,000,000 dilution, and that of the Australian tiger snake (Notechis scutatus) was active in a 1:4,000,000 dilution. The thrombin formed was indistinguishable from that produced by the action of calcium + platelets on prothrombin. Like the latter type of thrombin, and unlike venoms which act directly on fibrinogen, thrombin formed from prothrombin by venom was inhibited by antithrombin. Every one of the 9 non-coagulant venoms in this series destroyed prothrombin; and 5 of these destroyed fibrinogen as well. As is discussed in the text, there is reason to believe that these several properties of the venoms (coagulation and destruction of fibrinogen; activation and destruction of prothrombin) depend on the proteolytic enzymes which they were found to contain. These observations lend further support to the thesis that, in the course of physiological coagulation, (a) calcium plus platelets (or tissue derivative) constitute an enzyme system which reacts with prothrombin to form thrombin, and which is thus analogous to trypsin and to several of the proteolytic venoms here discussed, and (b) the thrombin so formed is itself a proteolytic enzyme which, like papain and the majority of the coagulant and proteolytic snake venoms here studied, reacts with fibrinogen to form a fibrillar gel, fibrin.


Studies in blood coagulation

March 1937

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15 Reads

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82 Citations

Crude or crystalline trypsin in proper concentration causes the blood or plasma of human beings, dogs, rabbits, guinea pigs, and horses to coagulate. It does not clot the fibrinogen directly, but reacts with prothrombin to form thrombin. Since trypsin thus has the same effect as the physiological system Ca plus platelets (or Ca plus tissue extracts), it is suggested as a tentative working hypothesis that the latter system contains a proteolytic enzyme with a specific affinity for prothrombin. Other implications of this trypsin effect with respect to the mechanism of physiological coagulation are discussed in the text (pages 557–558). The proteolytic enzyme papain also coagulates blood. In this case the enzyme does not activate prothrombin, but acts directly on fibrinogen to form a fibrillar gel resembling fibrin. If one admits this clot to be fibrin, this constitutes strong evidence that thrombin, the physiological coagulant, is also a proteolytic enzyme with a specific action on fibrinogen.


STUDIES IN BLOOD COAGULATION: V. THE COAGULATION OF BLOOD BY PROTEOLYTIC ENZYMES (TRYPSIN, PAPAIN)

March 1937

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165 Reads

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74 Citations

Journal of General Physiology (JGP)

Crude or crystalline trypsin in proper concentration causes the blood or plasma of human beings, dogs, rabbits, guinea pigs, and horses to coagulate. It does not clot the fibrinogen directly, but reacts with prothrombin to form thrombin. Since trypsin thus has the same effect as the physiological system Ca plus platelets (or Ca plus tissue extracts), it is suggested as a tentative working hypothesis that the latter system contains a proteolytic enzyme with a specific affinity for prothrombin. Other implications of this trypsin effect with respect to the mechanism of physiological coagulation are discussed in the text (pages 557-558). The proteolytic enzyme papain also coagulates blood. In this case the enzyme does not activate prothrombin, but acts directly on fibrinogen to form a fibrillar gel resembling fibrin. If one admits this clot to be fibrin, this constitutes strong evidence that thrombin, the physiological coagulant, is also a proteolytic enzyme with a specific action on fibrinogen.


The Non-Identity of the Antigenic and Antitoxic Groups in Diphtheria Antitoxin

April 1936

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2 Reads

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2 Citations

The Journal of Immunology

Diphtheria antitoxin (horse) saturated with a large excess of toxin can nevertheless combine with a rabbit antiserum against horse serum protein. Conversely, antitoxin precipitated by a large excess of the antiserum and presumably saturated with precipitin is almost unaffected as regards its ability to combine with toxin. In confirmation of Smith and Marrack, there are apparently different combining sites in the antitoxin molecule for toxin and for precipitin.


The Effect of Combination with Diazo Compounds on the Immunological Reactivity of Antibodies

April 1936

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9 Reads

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19 Citations

Sufficient coupling with any of five different diazo compounds eventually destroyed the reactivity of all the antisera here studied. The rates of inactivation varied considerably among the several antisera. By stopping the reaction at intervals, it was possible to prepare partially inactivated antibodies of peculiarly modified reactivity. Thus, the flocculating activity of diphtheria antitoxin with toxin was completely destroyed long before there was any demonstrable impairment of its protective titer in vivo. The first change induced in antipneumococcus horse sera was the apparently complete loss of reactivity with the capsular carbohydrate at a time when the agglutinating, animal-protecting and complement-fixing activity of the sera were only slightly affected. On further coupling, the sera no longer caused visible agglutination; but aggregation of the serum-treated bacteria could be induced by centrifugation. Still further coupling destroyed all antibody activity. Rabbit antisera to egg albumen and horse serum no longer precipitated the homologous antigen after treatment with diazo compounds, probably due to their failure to combine with the antigen. The hemolytic, complement-fixing and lipoid-flocculating activity of coupled rabbit antisera to sheep red blood cells fell off in parallel; the hemagglutinin seemed somewhat more resistant. The reagin of syphilitic serum was destroyed almost instantaneously by comparatively small amounts of diazo compounds. Finally, in the case of antityphoid agglutinin, the isoelectric point of the coupled antibody, measured on the surface of specifically sensitized bacteria, was found to shift from an original value of pH 4.7 to one of less than pH 2.7 as progressively more sulfanilic acid radicals added on to the antibody molecule. The groups in protein which participate in its reaction with diazo compounds probably include aliphatic amines, the imidazole ring of histidine, the indole group of tryptophane, the NH of proline and hydroxyproline and the phenyl group of tyrosine. Although it has been possible to modify antibodies chemically so that they combine with the corresponding antigens without causing their aggregation, the experiments here described furnish no indication as to which of these groups in antibody protein are primarily concerned in the antigen-antibody reaction, and which are responsible for the secondary flocculation. Such localization awaits the development of a technic for attacking individual groups in the protein molecule.


Reactions Between Lipoids and Antibodies I. The Isoelectric Point and Composition of the Aggregates Obtained on Adding Beef-Heart Lipoid to Syphilitic Serum

December 1935

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1 Read

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3 Citations

The Journal of Immunology

No analytically demonstrable protein is adsorbed from normal serum by the lipoid crystals used as antigen in the diagnostic tests for syphilis. However, their cataphoretic isoelectric point after being placed in normal serum and washed free of excess protein is found to be changed from an original value of pH 1.9 to approximately pH 3.5. This increase is probably to be ascribed to the retention of minute quantities of adsorbed protein on the surface of the particles, too small to be detected analytically by the methods used. The isoelectric point of the particles after similar sensitization with syphilitic serum rises as high as pH 4.85, the exact value obtained depending on the reagin content of the particular serum used. Even this is probably not the maximum value obtainable, as no serum has yet been found with a sufficiently high reagin titer to make the isoelectric point of the lipoid-reagin aggregates reach a definite maximum. The striking change in cataphoretic mobility caused by syphilitic serum is probably due to the deposition of the specific reactive protein, reagin, on the surface of the lipoid particles. The isoelectric point of reagin itself in the antigen-reagin complex is probably greater than pH 4.9, and thus falls into the same zone as the specific antibodies to bacteria, proteins and red blood cells, further evidence for the thesis that reagin, despite its apparent non-specificity, is a true antibody. The determination of its exact isoelectric point when in solution awaits its dissociation in quantity from the lipoid-reagin complex studied in the present paper. Quantitative analysis of the lipoid-reagin aggregates for nitrogen shows protein to constitute up to 6 per cent of the compound. The theoretical value calculated on the assumption that the reagin is deposited as a unimolecular layer of closely packed spherical molecules seven μμ in diameter, is on the order of 10 per cent. It is therefore suggested as a working hypothesis that the reagin protein of syphilitic serum is deposited on the surface of the antigen particles as a unimolecular layer which does not completely cover the individual particles. The actual concentration of reagin protein in syphilitic serum necessary to give a positive diagnostic test is estimated to be on the order of 0.3 mgm. per 100 cc. The average syphilitic serum, containing 5 to 50 such units, thus contains 1.5 to 15 mgm. per cent of the reactive factor. It is estimated, with the reservations entailed by the necessary assumptions, that approximately one-twentieth of the surface of the lipoid particles here studied must be covered with reagin to cause visible aggregation.


Citations (12)


... Azoproteins were prepared by standard methods as previously described (12,13, 14). Diazotates were obtained from the following substances: arsanilic acid, p-aminopheaylarseae oxide, sulfanilic acid, p-aminobeazoic acid, p-pheayleaediamine (as described by Sannders, reference 15), and aniline. ...

Reference:

THE ANTICOAGULANT AND ANTILYMPHOMA PROPERTIES OF ARSENIC AZOPROTEINS
On the nature of the reaction between diazotized sulfanilic acid and proteins
  • Citing Article

Journal of Biological Chemistry

... It is toxic to the animals due to the presence of some arsenical compounds. Cymelarsan showed irritating effect on the tissues of Nubian goats due to accumulation of high levels of arsenic in the muscles at the site of injection [49]. It is mainly used in camels to control surra via deep intramuscular injection at a dose rate of 0.25 mg/kg b wt [44], 0.25-0.5 mg/kg b wt in horses, 0.5 mg/kg b wt in cattle, and 0.75 mg/kg b wt in buffaloes [50][51][52]. ...

The pharmacologic basis for the widely varying toxicity of arsenicals
  • Citing Article
  • January 1944

Journal of Pharmacology and Experimental Therapeutics

... Papain is also regarded as having significant pharmacological potential, with drug-like properties that might aid in the treatment of atherosclerosis and related illnesses. However, the early discovery of papain's influence on the transition from fibrinogen to fibrin shed light on the fibrinolytic properties of papain, providing a fundamental comprehension of this enzyme's involvement in the coagulation process [35]. ...

Studies in blood coagulation
  • Citing Article
  • March 1937

... Papain, the first cysteine protease isolated from Papaya latex is one of the most studied peptidases has extensive applications across various industries; including food, medicine, pharmaceuticals and diagnostics as well as in clinical and laboratory [119,120]. Papain and papain-like cysteine proteases from Papaya (chymopapain, caricain and glycyl endopeptidases) are essential in the defense mechanism of the Papaya plant [121,122]. The discovery and characterization of these peptidases from latex sources underscore their role not only in plant defense mechanisms but also in medicinal applications. ...

STUDIES IN BLOOD COAGULATION: V. THE COAGULATION OF BLOOD BY PROTEOLYTIC ENZYMES (TRYPSIN, PAPAIN)

Journal of General Physiology (JGP)

... A group of globular proteins is called globulin [11,12]. Some globulins are produced in the liver and some in the immune system [13,14]. The main proteins in the blood include globulins, fibrinogen, and albumin. ...

THE IMMUNOLOGICAL SPECIFICITY OF THE EUGLOBULIN AND PSEUDOGLOBULIN FRACTIONS OF HORSE AND H;MAN SERUM.

Journal of General Physiology (JGP)

... Eagle studied patients with this disorder and showed that the platelet function in those patients was normal but that there was still a deficiency in prothrombin conversion [6]. While studies on deficient plasmas had established what the important components were, the mechanisms of action and the interactions between these components were not immediately clear. ...

Studies on blood coagulation. IV. The nature of the clotting deficiency in hemophilia

... To attribute multiple biological activities to the 3D object, different enzymes were directly integrated during the 3D printing process: alkaline phosphatase, chosen for its capacity to trigger calcification reaction [10][11][12] and thrombin, chosen for its ability to trigger fibrin network formation. [13][14][15] This approach will allow us to demonstrate that 4D printing is the groundbreaking approach toward the achievement of complex bioactive 3D printed materials. ...

Studies on blood coagulation. II. The formation of fibrin from thrombin and fibrinogen

... During granule implantation at the defect site, the FNG-BCP granules could naturally form a fibrin clot by reacting with blood discharged from the tissues at the surgical site. When a wound is created, pro-thrombin is activated to generate thrombin (Eagle 1935), and the activated thrombin converts FNG to fibrin, leading to the formation of fibrin clots (Wolberg 2007). These facts demonstrate that the FNGadsorbed BCP granules could rapidly form fibrin clots at the surgical site. ...

Studies on blood coagulation. I. The role of prothrombin and of platelets in the formation of thrombin

Journal of General Physiology (JGP)

... As the limitations imposed on the classical serological tests by the antigen used gradually emerged, the need for tests using treponema-specific antigens became more urgent. quently demonstrated that this antigen contains only a lipid component, which is also responsible for the classical non-treponema! tests (Eagle and Hogan, 1940). In an attempt to improve the specificity of antigens Banffer et al. (1974Banffer et al. ( , 1975 used the Reiter antigen in a counter-immunoelectrophoresis technique. ...

ON THE PRESENCE IN SYPHILITIC SERUM OF ANTIBODIES TO SPIROCHETES, THEIR RELATION TO SO CALLED WASSERMANN REAGIN, AND THEIR SIGNIFICANCE FOR THE SERODIAGNOSIS OF SYPHILIS.

... In 1937, Harry Eagle described two dissociable activities of anti-pneumococcal horse serum: aggregation of bacteria vs. protection in animal models of sepsis [2]. As early efforts in vaccine design appropriately focused on preventing sepsis, which was reflected in OPK activity, agglutinating activity was rarely studied. ...

SOME EFFECTS OF FORMALDEHYDE ON HORSE ANTIPNEUMOCOCCUS SERUM AND DIPHTHERIA ANTITOXIN, AND THEIR SIGNIFICANCE FOR THE THEORY OF ANTIGEN-ANTIBODY AGGREGATION.

Journal of Experimental Medicine (JEM)