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1. Based upon quantitative estimations of the factors that promote or retard the development and activity of the streptococcal fibrinolytic phenomenon, an active lytic system was developed within the circulating blood stream of rabbits following the intravenous infusion of streptokinase. During the infusion of adequate doses of SK, an active lytic system was observed to be present within 30 minutes following the beginning of the experiment and remained present for periods of time ranging from 1 to 20 hours depending primarily upon the concentration of SK and the duration of the infusion. Some of the biochemical changes accompanying the lytic system in vivo were: (a) a striking fall in the plasminogen; (b) a moderate fall in the serum inhibitor and the fibrinogen; and, (c) a rise in the acid-soluble nitrogen. These changes were usually self-terminating within 24 hours following the infusion of SK. In earlier studies similar trends were observed in the chest fluid of patients with hemothorax and empyema treated locally with streptokinase (22). 2. Intravascular clots, induced artificially by local applications of sodium morrhuate within the ear vein of the rabbits, were observed to liquefy and disappear in 23 of 25 rabbits during the intravenous infusion of SK into the opposite ear. The average quantity of SK necessary to effect an active lytic system was found to be about 40,000 units per kilo per hour. The clot in the ear vein was observed to be lysed completely in periods of time ranging from 3 to 7 hours after the infusion of SK was begun. Maintenance of an active lytic system for 3 to 4 additional hours was required to prevent re-formation of the clots at the original site. In 3 of 4 rabbits in which clots did reform, ACTH had been given to combat the toxic effects of the streptococcal concentrates. 3. The toxicity of the unusually large dose of the streptococcal concentrates, containing measured amounts of SK, along with other identifiable and unknown substances, was considerable, inasmuch as 8 animals eventually died. No evidence of pulmonary infarction was observed at autopsy. The administration of ACTH appeared to prevent a fatal outcome in at least 3 of the rabbits. Further studies of toxicity are in progress.

Aerobic Tests. Changes in pH Which Occurred during Incubation.—(a) The pH of sterile specimens of both normal and patients' sera increased, during 24 hours incubation, from 8.0–8.8 up to 9.2–9.6. (b) The pH of patients' sera, inoculated with hemolytic streptococci, progressed in the alkaline direction as did the sterile specimens. (c) The pH of normal sera, inoculated with hemolytic streptococci, pursued a variable course at lower levels than the sterile specimens. The differences in the changes in pH which occurred in streptococcidal sera and in normal controls were dependent upon and secondary to the presence or absence of killing action in the specimens. Aerobic Tests. Effect of Different Levels of Hydrogen Ion Concentration, Adjusted at Beginning of Experiment, but Uncontrolled during Period of Incubation.—(a) The streptococcidal activity of patients' sera was inactivated when the hydrogen ion concentration of the specimens was adjusted to levels ranging from 6.4 to 7.4. The inactivation of highly potent samples of sera required a greater reduction in pH than did specimens of moderate killing activity. (b) Normal sera, adjusted to pH 9.2 or higher, exhibited a relatively slowly acting bactericidal process, which seemed to represent the toxic effect of alkalinity. Aerobic Tests. Effect of Glutathione and Sodium Ascorbate.—The streptococcidal action of patients' sera was markedly impaired by the addition of reducing agents. The inactivation was, in general, comparable to that previously obtained under conditions of anaerobiosis. The results, suggesting that some factor pertaining to biological oxidation-reduction may influence the streptococcidal action of sera, have been discussed. Tests Performed in Closed Jars by Means of Which Relatively Constant pH Was Maintained during the Period of Incubation.—Under the experimental conditions which were employed, the effectiveness of the streptococcidal action of patients' sera was maintained only in alkaline ranges. The most potent specimens of sera were active at lower levels of pH (7.9–8.2) than were other samples (8.4–8.8). All of the observations reported in this article were made with a strain of hemolytic streptococcus of the beta type, which is highly sensitive to the destructive action of serum from acutely ill patients.

1. Immunization of rabbits with Type III pneumococci is effective in producing active immunity against infection with a virulent strain of the homologous organism. 2. Immunization of rabbits with Type I or II pneumococci, and with R forms derived from any of the fixed types, is equally effective in producing active immunity against Type III infection. 3. Immunization of rabbits with nucleoprotein or with desoxycholate solutions of heterologous pneumococci, under the experimental conditions described, appears to be ineffective in producing active immunity against Type III infection.

The observations recorded in this paper on the infectivity of Type III pneumococci for rabbits may be summarized as follows: 1. Of eleven strains of Type III isolated from human sources, ten were found to possess low virulence for rabbits. This was true despite the fact that all the strains tested possessed large capsules and a high degree of virulence for mice. 2. One strain of Type III pneumococcus was rendered highly virulent for rabbits. Since it possessed no other biological property demonstrably different from the other strains, its virulence must reside in some additional property. 3. An initial decrease in the number of circulating organisms following the injection of virulent bacteria is a well known occurrence, and it was observed in rabbits injected with the rabbit virulent strain of Type III. However, the extent of the reduction was in inverse proportion to the degree of virulence of the strain; a fact which makes mechanical explanations of the phenomenon insufficient. 4. The bacteremia produced in rabbits by Type III pneumococci, avirulent for this species, runs a characteristic course. It differs from that produced by non-encapsulated R forms of pneumococci although in both instances survival of the infected animal ensues. This is evidence that the mechanism of resistance against encapsulated and non-encapsulated pneumococci is not identical. 5. Phagocytosis of Type III pneumococci by circulating rabbit leucocytes was not demonstrable by a vital stain technique, whereas under the same conditions the ingestion of non-encapsulated R forms occurred. This is further evidence that the process whereby non-encapsulated pneumococci are disposed of, is insufficient to explain the natural resistance of rabbits to infection with encapsulated Type III pneumococci.

1. Type III pneumococci fail in the majority of instances to stimulate the production of anti-S antibodies. (Type specific agglutinins, type specific precipitins, and antibodies affording type specific protection in mice.) 2. Type III pneumococci are effective in the stimulation of the production of anti-P antibodies (agglutinins for R strains of pneumococci and precipitins for pneumococcus nucleoprotein). These antibodies are ineffectual in the passive protection of mice.

I. a. Pneumococcus polysaccharides, when injected intradermally into patients convalescent from pneumonia, are capable of eliciting a response. The polysaccharide inducing a cutaneous reaction was found always to be homologous in type to that of the pneumococcus causing the infection. b. The character of the reaction incited by the protein-free bacterial sugars is of the immediate wheal and erythema type. c. A patient's capacity to react was found to be intimately associated both with recovery from infection and with the presence of type specific antibodies in the circulating blood. II. a. The so-called nucleo-protein of pneumococcus, when injected intradermally, also causes a local cutaneous reaction in patients during convalescence from lobar pneumonia. b. The local lesion resulting from the injection of protein is tuberculin-like in character, and differs from that evoked by the type-specific polysaccharides in gross appearance, time of development, and duration. c. Individuals, acutely ill with and convalescent from pneumococcus pneumonia, possess in their circulating blood, precipitins reactive with pneumococcus protein. In the observations recorded, the concentration of anti-protein antibodies in the blood serum did not seem to influence the patient's capacity to react to intradermal injection of the protein.

1. The type-specific carbohydrates (haptens) of Pneumococcus Types I, II and III, when isolated in protein-free form, are devoid of the property of inducing active anaphylactic sensitization in guinea pigs. 2. The bacterial carbohydrates of Pneumococcus, of which the Type II and Type III substances are nitrogen-free, produce rapid and fatal anaphylactic shock in guinea pigs passively sensitized with the precipitating serum of rabbits immunized with pneumococci of the homologous type; the reactions induced are type-specific. 3. In contrast to the positive results with immune rabbit serum, there is a complete absence of anaphylactic response to pneumococcus carbohydrate in guinea pigs passively sensitized with antipneumococcus horse serum.

1. Rabbits, vaccinated by repeated intravenous injections of suspensions of heat-killed R pneumococci, acquire a marked degree of active immunity to infection with the virulent S forms of Pneumococcus Types I and II. Previously (1) it was shown that the immunization of rabbits with R cells induces active resistance to Type III infection. This immunity is effective when the infecting organisms are injected either intravenously, intraperitoneally, or intradermally. 2. Whole citrated blood or serum of rabbits immunized with R pneumococci, under the experimental conditions described, is capable of passively protecting normal rabbits against Type I and Type III infection. Whole blood appears to be more effective than an equivalent amount of serum. 3. Passive protection of mice by the use of whole blood or serum of the immune rabbits has been entirely ineffectual. This is in striking contrast to the results obtained with type-specific immune serum. 4. This form of acquired resistance to pneumococcus infection, elicited by R organisms which are devoid of type specificity, and exemplified in animals whose sera possess no demonstrable type-specific antibodies, has many characteristics strongly suggesting that the underlying mechanism differs from that concerned in type-specific immunity.

Pneumococci contain a non-protein constituent which, on the basis of its chemical and immunological properties, appears to be a carbohydrate distinct from the type-specific carbohydrate and common to the species.

1. Sera from individuals acutely ill with lobar pneumonia possess the capacity to precipitate in high titre a non-protein somatic fraction derived from pneumococci (Fraction C). Following crisis the reaction is no longer demonstrable. 2. Sera obtained from cases of pneumococcus pneumonia during illness and convalescence have been tested for antibodies specifically reactive with three chemically distinct constituents of Pneumococcus. The results, when correlated with the course of disease, demonstrate differences in the occurrence of each qualitatively distinct antibody. 3. The precipitation of pneumococcus Fraction C is not limited to the sera of individuals ill with pneumococcus infection. But in the few other cases available for comparative tests, definite reactions have been obtained only in streptococcus and staphylococcus infections and in acute rheumatic fever.

The majority of patients convalescent from pneumonia due to Types I, II and III Pneumococcus develop at the time of recovery circulating antibodies for the homologous type of organisms. At the same time an immediate wheal and erythema reaction followed the intradermal injection of the homologous type-specific polysaccharide in 100 per cent of Type I patients, 58.8 per cent of Type II patients, and 44 per cent of Type III patients. In a group of 18 patients repeatedly tested with the type-specific polysaccharides, 10 developed in the second or third week of convalescence circulating antibodies for one or more heterologous types. In none of 21 control patients was this phenomenon observed. It is suggested that the development of circulating antibodies for heterologous types of Pneumococcus was associated with the previous intradermal injections of the type-specific polysaccharides.

1. Guinea pigs passively sensitized with the serum of rabbits immunized with an artificially prepared sugar-protein (gluco-globulin) exhibit typical anaphylactic shock when subsequently inoculated with gluco-albumin; the serum of rabbits immunized with a second synthetic sugar-protein (galacto-globulin) similarly sensitizes guinea pigs to galacto-albumin. The reactions, in each instance, are specific and depend for their specificity on the carbohydrate component, and not on the protein fraction of the synthesized sugar-protein. 2. Guinea pigs actively sensitized with gluco-globulin or galacto-globulin are similarly subject to anaphylactic shock, when injected, after 21 days, with sugar-proteins containing carbohydrate identical with that present in the sensitizing antigen, regardless of the kind of protein with which it is combined. 3. The unconjugated glucosides, although themselves not capable of inducing shock, inhibit the anaphylactic reaction when injected immediately prior to the introduction of the toxigenic sugar-protein. The protective action of the glucosides disappears within two hours after injection. In order to elicit the phenomenon, the carbohydrate must be the same as that combined in the sugar-protein complex. 4. Anaphylactic shock may be induced by uncombined globulin in guinea pigs passively sensitized with either antigluco-globulin serum or antigalacto-globulin serum; globulin is similarly effective in animals actively sensitized with gluco-globulin or galacto-globulin. The reactions elicited by globulin alone are dependent upon the common protein present in the antigens, and exhibit only species specificity.

Small areas of skin infiltrated with immune serum proved refractory to the injurious action of a filterable rabbit virus inoculated 24 to 48 hours later. Infiltrations of normal serum did not appreciably protect the skin against the virus. The local protection afforded by the infiltrations of immune serum appears to constitute an example of local passive immunity. Small areas of skin infiltrated with either normal serum or meat infusion broth proved more refractory than normal skin to infection with hemolytic streptococci inoculated 24 hours later. The refractory state induced in the tissues was non-specific in character. A greater amount of local protection against hemolytic streptococci was afforded by infiltrations with a homologous immune serum than by infiltrations with normal serum. The difference in the amount of protection afforded by immune serum over that induced by normal serum may be thought of as representative of the degree of local passive immunity conferred.

The pathological changes produced by the virus under investigation are similar to those deemed characteristic of a certain well known group of filterable viruses.

1. The intradermal method of inoculating Virus III, a hitherto unknown filterable virus producing lesions in rabbits, gives more reliable results than those obtained by smearing the virus on the scarified skin. 2. Virus III, heated 10 minutes at 55°C., will not produce visible reactions in the skin of rabbits. 3. Virus III passes through Berkefeld N and V filters. 4. The data obtained so far indicate that the best method of preserving Virus III in an active state is to filter the testicular emulsions containing the virus, add glycerol to the filtrate up to 40 per cent of the total volume, seal with vaseline, and store on ice. 5. Viable Virus III produces a definite immunity in rabbits which persists for at least 6 months. The immunity follows intradermal, intratesticular, intravenous, intracerebral, or intranasal inoculations of the virus. 6. A single intradermal injection of Virus III, which has been killed by heat, will not produce a demonstrable immunity in rabbits. 7. No passive immunity to Virus III could be demonstrated in rabbits which had received intravenous injections of 5 to 10 cc. of immune rabbit serum 24 hours previously. 8. Immune rabbit serum neutralizes Virus III either in vitro, or locally in a rabbit's skin when the immune serum and the virus are injected into the same part of the skin at or about the same time. 9. Three strains of the virus under investigation are immunologically identical. 10. Virus III and vaccine virus are immunologically distinct. 11. Virus III and the virus of symptomatic herpes are immunologically distinct. 12. No passive immunity to Virus III could be demonstrated in rabbits which had received intravenous injections of 5 to 10 cc. of serum or whole blood from patients convalescent from varicella. 13. Sera from two normal adults and from fourteen patients convalescent from varicella did not neutralize Virus III in vitro. 14. Rabbits could not be actively immunized against Virus III by injections of whole blood, vesicle fluid, or nasal washings from patients with varicella. 15. Four of twenty sera collected from stock rabbits of different ages, 20 per cent, neutralized Virus III in vitro. The animals whose sera neutralized Virus III failed to show a reaction at the site of intradermal inoculations with the same virus. About 15 per cent of 200 young stock rabbits (1,800 gm.) used in routine transfers were found to be refractory to Virus III, as evidenced by a failure to react to intradermal inoculations of the virus. 16. No susceptibility to Virus III was observed in guinea pigs, mice, or monkeys. 17. A volunteer who had never suffered from varicella experienced no general reaction and only a mild local one following an intradermal inoculation of Virus III. A volunteer who had had chicken-pox in childhood experienced a moderate general action, viz., fever, headache, backache, and general malaise, and also a moderate local reaction, viz., redness, swelling, tenderness, and pain, following an intradermal inoculation of Virus III. 18. The study of the immunological reactions has failed to bring any evidence that the virus under investigation bears an etiologic relationship to varicella.

1. The localization of the virus of varicella in the human skin is influenced by irritation. This is indirect evidence that the virus is in the blood. 2. Rabbits are susceptible to a virus recovered from the blood of varicella patients. 3. Testicular emulsions containing the virus are free from ordinary aerobic and anaerobic bacteria, and produce lesions in the cornea, skin, and testicles of rabbits. 4. The virus can be transmitted indefinitely from rabbit to rabbit by means of testicular inoculation, and can be preserved 29 days in 50 per cent glycerol at a low temperature.

The fibrinolysin of hemolytic streptococci exerts no hydrolytic action upon casein, gelatin, or peptone. The action on solid human fibrin is characterized by a small and gradual increase in the amino nitrogen content of the solution. The specific and special enzymatic action of fibrinolysin is contrasted with trypsin and with streptococcal peptase (4). Solutions of human fibrinogen, after brief incubation with fibrinolysin, lose the capacity to form fibrin. Solutions of rabbit fibrinogen, on the other hand, retain the property of transformation into fibrin, even after prolonged exposure to fibrinolysin. Qualitative tests, with solutions resulting from the action of streptococcal fibrinolysin on human fibrin, indicate that the end-product may be protein and that the degradation of the molecule is not great.

The active fibrinolytic principle present in cultures of hemolytic streptococci can be isolated in stable form, and partially purified by the following methods: 1. Precipitation of culture filtrate with 3 volumes of 95 per cent ethyl alcohol. 2. Adsorption upon polyaluminum hydroxide B of Willstätter (5) followed by elution with M/10 sodium phosphate buffer, pH 7.3. Concentration can be best accomplished by vacuum dialysis (4) of either culture filtrates or preparations obtained by adsorption and elution. The streptococcal fibrinolysin is characterized by the following properties: 1. It may resist heating to 100°C. for 60 minutes; variations in thermal resistance are described. 2. Partially purified preparations give positive tests for protein. Activity is rapidly and completely destroyed by trypsin or papain. 3. The active principle is demonstrable in dissolved fibrin even after 18 hours incubation.

Broth cultures of hemolytic streptococci derived from patients are capable of rapidly liquefying normal human fibrin clot. The active fibrinolytic principle is also contained in sterile, cell-free filtrates of broth cultures. The degree of activity of filtrates parallels the activity of whole broth cultures sufficiently closely to indicate that large amounts of the fibrinolytic substance are freely excreted into the surrounding medium and pass readily through Berkefeld V, Seitz, and Chamberland filters. The occurrence of fibrinolysis is most strikingly observed when plasma or fibrinogen is mixed with active cultural material before clot formation is effected. Under the standard experimental conditions described, complete dissolution of human plasma clot (whole oxalated plasma + CaCl2) occurs in about 10 minutes; complete dissolution of human fibrinogen clot (chemically isolated fibrinogen + thrombin) takes place in about 2 minutes. Titration of filtrate activity is recorded in Table IV. Twenty-eight strains of Streptococcus hemolyticus, isolated from patients suffering from various manifestations of streptococcus infection, have been tested for the capacity to liquefy fibrin clot. Broth cultures of all of the strains induced fibrinolysis. Of 18 strains of Streptococcus hemolyticus of animal origin, only three were capable of causing dissolution of clot. Completely negative results were obtained with 38 strains of other bacterial species. The list is presented on pages 492 and 493. The plasma of many patients recovered from acute hemolytic streptococcus infections, when clotted in the presence of active cultures, is highly resistant to fibrinolysis. Furthermore, serum, derived from patients whose plasma clot is resistant, often confers on normal plasma clot an antifibrinolytic property. One example of the resistance possessed by the blood of convalescent patients is presented in this report. A second paper, now in preparation, will give in detail a large number of observations on the relation of infection to the development of resistance to the fibrinolytic activity of hemolytic streptococci. In contrast to the susceptibility of normal human fibrin clot to liquefaction by active culture, normal rabbit fibrin clot is totally resistant to dissolution when tested under comparable conditions. The insusceptibility of rabbit fibrin clot is manifest provided the coagulum is composed of rabbit constituents. When human thrombin is used to clot rabbit plasma or fibrinogen in the presence of active cultures, fibrinolysis is not prohibited. The rôle of thrombin in determining the resistance or susceptibility of rabbit fibrin to dissolution offers a suggestive approach to problems relating to the underiving mechanism.

The injection of the type-specific capsular polysaccharides of Pneumococcus Types I, II and III into the skin of rabbits, actively or passively immunized to one of these types of Pneumococcus, elicits a type-specific cutaneous reaction. The form of reaction resembles that described by Arthus. The reaction is produced only when type-specific precipitins for the homologous polysaccharide are demonstrable in the blood of the rabbit. In 84 per cent of actively immunized rabbits, the serum of which contained type-specific precipitins, a reaction was elicited. A positive result was obtained in 100 per cent of rabbits passively immunized with antipneumococcus horse serum whereas, attempts passively to transfer reactivity from immune rabbit to normal rabbit were unsuccessful. The recipients, in the latter group, possessed no demonstrable circulating type-specific precipitins. The reaction produced by specific capsular carbohydrates is always associated with a well grounded type-specific immunity. A brief summary of the relation of hypersensitiveness and immunity to pneumococcus is given.

Sera obtained from patients at the time of acute active infections were found, in every instance, to be bactericidal for hemolytic streptococci. The observations were made with sera from twenty-five patients. The group consisted of cases of pneumococcus, hemolytic streptococcus, staphylococcus, meningococcus, tubercle bacillus, and malarial infections; the etiology of the diseases in other patients was either uncertain or may have been a mixed infection. In sera taken from the same group of patients, soon after recovery or marked improvement had taken place, the streptococcidal property was absent or greatly diminished in potency. By comparative tests made with sera from twenty healthy adults, the streptococcidal action was not demonstrable. Three different strains of Streptococcus hemolyticus of the beta type were employed in the experiments. Differences in susceptibility of the strains to the killing power of the serum were noted.

Although sera derived from patients at the time of acute, active infection were found to be capable of destroying hemolytic streptococci under aerobic conditions, the organisms retained viability when the tests were performed in the environment of anaerobiosis afforded by a vaseline seal or an anaerobic jar. Within the limitations of the experimental procedures which were employed, the aerobic or anaerobic effect was found to be a reversible reaction. Heating sera at 60°C. for 1 hour inactivated the streptococcidal element in most instances, but not in every case; heating at 56°C. for 1 hour impaired the killing power of half of the specimens which were tested. Sera retained the capacity to destroy hemolytic streptococci when kept in the ice box for 3 weeks; a slight diminution in killing power was noted after 4 weeks. By the methods which were employed, the streptococcidal property of sera could not be correlated with either true antibacterial agglutination or with pseudo-agglutination. The significance of the findings as a basis for analyzing the mechanism of the streptococcidal phenomenon is discussed.

1. In rabbits receiving subcutaneous injections of benzene, the simultaneous administration of sulfapyridine, per os, prevented the development of leukopenia. The sparing effect of sulfapyridine on the intoxication of the leukopoietic tissue by benzene was demonstrated not only by the range of daily leukocyte counts but also by microscopic examination of the bone marrow of treated animals. 2. The administration of para-aminobenzoic acid failed to inhibit the leukotoxic action of benzene. 3. The administration of sulfapyridine alone to rabbits was followed by daily variation in the number of leukocytes in the circulating blood but persistent leukocytosis was not observed. The inhibiting action of sulfapyridine was not found to be referable to the development of an overcompensating leukocytosis. 4. In preliminary experiments, the excretion of phenols by rabbits receiving sulfapyridine together with benzene differed from that observed in animals receiving benzene alone. In the former group, the rise in the excretion of total phenols was not so high as in the latter group, but the percentage of combined phenols was greater. The possible significance of the findings has been discussed.