William S. Tillett

The intravenous infusion of the streptococcal fibrinolytic principle (streptokinase) into patients.

The streptococcal fibrinolytic principle (streptokinase) has been demonstrated to be an effective agent, in vivo, in patients as a means of mediating the relatively rapid liquefaction of extravascular fibrin coagulums (1). Streptokinase (referred to in this article as SK) has also been shown to be useful as a therapeutic reagent, by which massive extravascular clots may be eradicated by liquefaction and aspiration (2). These findings have received extensive confirmation. (See Footnote 12 to the list of References.) It is the purpose of this article to describe the results of studies of the intravenous administration of SK into patients. The ultimate objective of the study is directed toward attempting to determine whether or not clots occurring within the vascular system as a result of disease may be subject to

Presence and Significance of Desoxyribose Nucleoprotein in the Purulent Pleural Exudates of Patients.

Summary Desoxyribose nucleoprotein has been identified as a significant constituent of purulent and inflammatory exudates derived from patients. Its chemical characteristics have been described and it has been isolated in quantities ranging from 30 to 70% of the total purulent sediment. Its physical characteristics of stringiness, viscidity, and coagulated gel, indicate its significance in contributing to the sedimented elements of exudations. Protein-free desoxy-ribonucleic acid has been derived from the nucleoprotein. The liquefaction of both the nucleoprotein and the nucleic acid has been found to occur following the addition of beef desoxyribonuclease and preparations of hemolytic streptococcal filtrates containing desoxyribonuclease,. By the Feulgen method of staining the abundance of desoxyribose nucleoprotein in exudates has been demonstrated microscopically.

Streptococcal Desoxyribonuclease Significance in Lysis of Purulent Exudates and Production by Strains of Hemolytic Streptococci.

Summary In experiments in which the following substrates were employed: 1) specimens of purulent exudations from patients, 2) desoxyribose nucleoprotein isolated from the exudates and, 3) desoxyribose nucleic acid derived from nucleoprotein, striking and rapid lytic changes were demonstrable in the coarse sediment of substrate 1, in the fibrous material of substrate 2, and in the gel of substrate 3, following the addition of concentrated filtrates obtained from cultures of hemolytic streptococci. The lytic action was found to be due to the presence of desoxyribonuclease which is elaborated during the growth of strains of hemolytic streptococci and is freely demonstrable in the fluid medium in which the organisms are cultivated. Among the strains tested, hemolytic streptococci of Group A which also possessed fibrinolytic properties were uniformly potent in the production of desoxyribonuclease. By the methods of simple testing that were employed, strains of streptococcus viridans and pneumococcus were not found to possess a comparable property. Desoxyribonuclease and streptokinase, produced by the same strains were found to be different substances. When streptococcal concentrates were added to whole samples of inflammatory exudates, the rapid lysis of both fibrin and desoxyribose nucleoprotein occurred.

THE OCCURRENCE OF ANTIFIBRINOLYTIC PROPERTIES IN THE BLOOD OF PATIENTS WITH ACUTE HEMOLYTIC STREPTOCOCCUS INFECTIONS

The rapid dissolution of human fibrin clot by Streptococcus hemolyticus of the beta type is dependent upon the presence in cultures of an enzymic substance which is excreted by the organisms (1, 2). The bacterial product, which induces fibrinolysis, is characteristically elaborated by strains of hemolytic streptococci which cause acute infection in patients. Furthermore, the cultures, obtained from patients suffering with very severe types of illness, have been found to exhibit, with greatest frequency, the most potent fibrinolytic activity (3). It has also been demonstrated that patients convalescent from acute infection due to hemolytic streptococci often develop a humoral property which is effectively antagonistic to the fibrin dissolving action of cultures of the infecting organisms (4).

Effects in Patients of Intravenous Infusions of Purified Streptokinase Preparations.

Summary 1. The clinical effects of specially purified streptokinase preparations have been determined in man. Streptokinase, 6-7 fold purer than that previously available, has been shown to have a greatly diminished toxicity, when administered by the intravenous route. The best of these preparations have only 1/20th of the toxicity of the previous preparations. 2. Systemic fibrinolysis has been induced in man by infusion of these preparations, unprotected by reaction suppressing drugs. A comparison between those patients who showed peripheral blood fibrinolysis, and those who did not, indicated that this state is asymptomatic in the human. 3. 165 electrocardiograms taken on 60 patients, before, during and after the infusion of streptokinase showed few changes and these were of minor degree. Since they followed no consistent pattern it was not posible to ascribe the changes to the effect of the infusion.

SKIN HOMOGRAFT SENSITIVITY CROSS REACTIONS IN MAN

The genetic disparity encountered in the random populations of normal human subjects employed in previous studies concerned with the mechanisms of homograft rejection in man has introduced inescapable variables into interpretations of the observed results.lS2 Since there is no large series surveying the specificity of the homograft response in comparable to the array of data on this subject available in experimental animals,6-10 we have been prompted to study further this aspect of homograft sensitivity in man. The application of a skin homograft to a normal recipient results in its initial survival, followed within 8 to 12 days by a characteristic pattern of rejection which has been termed the homograft rejection phenomenonP These changes are associated with the development in the host of a state of generalized sensitivity to further skin homografts obtained from the same donor. If the host is challenged with a subsequent skin homograft from the same donor, he will exhibit either the accelerated rejection reaction, with ensuing hemorrhagic necrosis of the graft within the first 4 to 5 postoperative days, or the white graft reaclion, characterized by complete nonvascularization and a parchment white appearance of the graft.5 The duration of the latent period allowed between rejection of the first-set graft and application of the following graft from the same donor conditions the type of response observed. A short latent period of 1 to 5 days results in a white graft, while a longer period of 10 to 26 days elicits the accelerated rejection reaction. The development of objective criteria for the evaluation of skin homograft reactions in man has made possible an attempt to evaluate the influence of individual specificity on the response of the host to skin homografts. This paper presents data collected on the fate of 147 skin homografts performed in normal human subjects in the course of a comprehensive study of the immunology of skin homograft reactions in man, with particular reference to possible means of detecting individual or group specific factors concerned with homograft sensitization in man.

Hydrogen ion Concentration and Anticoagulating and Fibrinolytic Action of Cultures of Streptococci and Pneumococci

Recently Neter and Witebsky 1 , 2 have reported that many strains of Streptococcus viridans and pneumococcus were found to be fibrin-olytic for human fibrin provided the organisms with which the tests were performed were grown in broth containing 2% dextrose. Tunnicliff 3 reported that strains of Streptococcus viridans were incapable of dissolving normal fibrin clot but that cultures of smooth forms inhibited coagulation. The latter author used for liquid culture medium meat extract broth containing one percent dextrose. Dennis and Berberian 4 reported that some strains of Streptococcus hemolyticus and viridans inhibited the formation of fibrin in plasma. They stated that 2% dextrose broth gave more active cultures than did 0.5% dextrose broth. Because of the experience in this laboratory, using culture media containing 0.05% dextrose, that neither strains of Streptococcus viridans nor Pneumococcus have been encountered which were capable of dissolving human fibrin under the experimental conditions previously described, 5 additional observations have been made with special consideration being given to the dextrose content of the culture media in which the organisms were cultivated in relation to anticoagulating and fibrinolytic activity of strains of Streptococcus hemolyticus, Streptococcus viridans, and Pneumococcus. Culture Media. Plain meat infusion broth containing one percent neo-peptone but no buffer, has been employed. Before use, the pH was adjusted to 7.4-7.6. Dextrose has been added to make concentration of 0.05%, 1.0%, and 2%. Strains: Streptococcus hemolyticus. Ten strains derived from patients suffering from acute infections were employed. These strains were all known to be actively fibrinolytic. Streptococcus viridans. Thirty-three strains, all of which were derived from patients with endocarditis, were tested. Pneumococcus. Ten strains, all derived from patients with lobar pneumonia, were used. All the cultures were approximately 18 to 24 hours old when used.

Bactericidal Action of Human Serum on Hemolytic Streptococci. Active Principle Obtained by Fractionation of Sera

The present report is part of an investigation concerning the capacity of serum from patients who are acutely ill to destroy hemolytic streptococci of the beta type. Previous reports 1 have demonstrated that the bactericidal property under consideration was demonstrable in the serum derived from patients during the period of active disease due to a variety of infections, but that following recovery from illness the streptococcidal activity was greatly diminished, as measured by the methods which were employed. Furthermore, normal sera have been found to be essentially devoid of streptococcidal activity and have served as controls throughout the observations. The previous articles have described the technical procedures. In the present study a strain of Streptococcus hemolyticus, which has been found to be uniformly highly sensitive to the killing effect of patients sera, has been used in all of the experiments. The samples of sera were derived from patients who were acutely, and usually severely ill from diseases such as pneumonia, or pyogenic infections due to different kinds of organisms. Studies have been carried out in an attempt to isolate the active principle in serum which is responsible for the streptococcidal activity. Observations have been made with a protein-fraction and a non-protein constituent. The materials have been used separately and in combination. The protein-fraction most regularly employed in the tests has been obtained by precipitation of serum at low temperatures with alcohol according to the method described by Felton. 2 Sufficient 95 % alcohol was added to serum to make a final concentration of 20%. The material was kept in the icebox overnight in order to obtain the maximal yield and was then spun down in a cold centrifuge.