STUDIES ON THE BIOLOGY OF STREPTOCOCCUS : I. ANTIGENIC RELATIONSHIPS BETWEEN STRAINS OF STREPTOCOCCUS H?MOLYTICUS.

1. Immunological differences have been shown to exist between strains of Streptococcus hœmolyticus of the human type. 2. Four biological types have been identified by means of the reactions of agglutination and protection. 3. At least two other types have been encountered and the indications are that more exist.     

STUDIES ON THE BIOLOGY OF STREPTOCOCCUS : III. AGGLUTINATION AND ABSORPTION OF AGGLUTININ WITH STREPTOCOCCUS SCARLATIN?.

1. Strains of hemolytic streptococci from cases of scarlet fever occurring in New York, San Francisco, Chicago, Baltimore, and Copenhagen, Denmark, all interagglutinate with immune sera prepared with these strains. 2. Sera prepared with these strains do not agglutinate pyogenic streptococci or strains isolated from cases of septic sore throat. 3. The strains obtained from the throats of patients from an epidemic of scarlet fever and the strain from the milk responsible for this epidemic fall into the scarlatinal group according to these agglutination tests. 4. Absorption tests can be carried out with these strains and sera under proper conditions. 5. A group of hemolytic streptococci biologically distinct from streptococci from other sources than scarlet fever is constantly associated with scarlatina. They constitute a group of closely related streptococci which may be identified by agglutination tests.     

THE ANTIGENIC COMPLEX OF STREPTOCOCCUS H?MOLYTICUS : I. DEMONSTRATION OF A TYPE-SPECIFIC SUBSTANCE IN EXTRACTS OF STREPTOCOCCUS H?MOLYTICUS.

1. Hydrochloric acid extracts of Streptococcus hæmolyticus contain type-specific, as well as non-type-specific, substances. The precipitates formed by these crude extracts with homologous antibacterial serum are flocculent, while those obtained with heterologous serum are usually disc-like. 2. The type-specific substance may be detected by the use of anti-bacterial sera absorbed with heterologous strains of hemolytic streptococci. Such absorbed sera are type-specific: they are precipitated only by extracts of strains of the homologous type. 3. Any heterologous strain of hemolytic streptococcus absorbs the antibodies for all other heterologous strains, but homologous strains absorb type-specific antibodies as well. Numerous repeated absorptions with heterologous hemolytic streptococci tend to lower the titer of the type-specific antibody. A possible explanation of this fact is suggested. 4. Three strains did not yield a type-specific substance; and it seems probable that they had lost this function because of long continued cultivation in artificial media. 5. Classification based on the precipitin test with absorbed serum agrees with that previously determined by agglutination and protection tests. The method is, therefore, applicable to the problem of classification of the hemolytic streptococci.     

STUDIES ON THE BIOLOGY OF STREPTOCOCCUS : IV. THE OCCURRENCE OF STREPTOCOCCUS SCARLATIN? IN CONVALESCENCE AND IN THE COMPLICATIONS OF SCARLET FEVER.

1. A group of streptococci which agglutinate with scarlatinal immune sera has been isolated from the throats of 65 per cent of cases of scarlet fever during the 1st week of the disease. 2. Strains of hemolytic streptococci belonging to this group have been isolated from the throats of patients at the termination of quarantine (30 days). 3. Hemolytic streptococci are found most frequently in the throat in convalescent patients in whom the tonsillar inflammation has not entirely subsided. 4. The complications occurring in scarlet fever may be due to the original scarlatinal strain or may be the result of a secondary infection with pyogenic strains of streptococci.     

THE ANTIGENIC COMPLEX OF STREPTOCOCCUS H?MOLYTICUS : V. ANAPHYLAXIS WITH THE TYPE-SPECIFIC SUBSTANCE.

1. Type-specific anaphylactic shock was produced with HCI extracts of Streptococcus hæmolyticus in guinea pigs passively sensitized with antibacterial sera. 2. With occasional sera and certain HCl extracts, type-specific shock was not produced unless the serum was first absorbed with heterologous bacteria or unless the guinea pigs were desensitized with heterologous extracts before testing with the homologous extract. The findings indicated that this was due to the presence of the non-type-specific substance which has been provisionally designated as Y. 3. Tryptic digestion destroyed the ability of HCI extracts containing the type-specific M substance and the non-type-specific Y substance to produce anaphylactic shock in passively sensitized guinea pigs. 4. Active sensitization was not accomplished with the type-specific M. It seems probable, therefore, that this substance is a hapten, reacting with antibodies but not stimulating their production after separation from the bacterial cell.     

THE ANTIGENIC COMPLEX OF STREPTOCOCCUS H?MOLYTICUS : IV. ANAPHYLAXIS WITH TWO NON-TYPE-SPECIFIC FRACTIONS.

The anaphylactic reactions of two non-type-specific fractions of hemolytic streptococcus extracts parallel the precipitin reactions. The nucleoprotein, P, is a true antigen, in that it stimulates antibody production in rabbits, as shown before, and produces anaphylactic shock in guinea pigs actively as well as passively. The probable carbohydrate, C, on the other hand, does not induce antibody formation in rabbits, so far as known at present, but does produce typical anaphylactic shock in guinea pigs passively sensitized with antibacterial serum provided the serum shows a high titer of C precipitins. This is an instance of a hapten, probably carbohydrate in nature, causing anaphylactic shock in passively sensitized guinea pigs.     

THE USE OF THE FINAL HYDROGEN ION CONCENTRATION IN DIFFERENTIATION OF STREPTOCOCCUS H?MOLYTICUS OF HUMAN AND BOVINE TYPES

1. Under the conditions of these experiments, there appears to be a distinct and constant difference in the final hydrogen ion concentration of Streptococcus hæmolyticus from human and bovine sources. 2. Of 124 strains of Streptococcus hæmolyticus from known human origin, 116 reached a final hydrogen ion concentration of from pH 5.0 to 5.3. Only 8 reached a pH more acid than 5.0 and none more acid than pH 4.8. 3. Of 45 strains of Streptococcus hæmolyticus from bovine sources, including 26 strains isolated from milk and the udder of cows, and 19 from cream cheese, 40 reached a final hydrogen ion concentration of pH 4.3 to 4.5. Of the remaining 5 which reached a pH of 5.0 to 5.2, two were of known human type and three of uncertain diagnosis. 4. A rapid and practical application of this method is proposed as a presumptive test in the differentiation of human and bovine types of Streptococcus hæmolyticus.     

PATHOLOGY OF THE EXPERIMENTAL PNEUMONIAS IN MICE FOLLOWING INHALATION OF STREPTOCOCCUS H?MOLYTICUS,OF FRIEDL?NDER'S BACILLUS,AND OF PNEUMOCOCCUS

1. Pulmonary consolidation may be produced in normal mice by the inhalation of Friedländer''s bacillus or Streptococcus hæmolylicus. 2. The initial lesion is in both instances interstitial in character and the spread is by way of the interstitial tissue, though the ultimate consolidation may come to resemble in the gross a lobar pneumonia. 3. The lesions grossly and microscopically may be distinguished from the lobar consolidation associated with pneumococcus infection of the lung. 4. Friedländer''s bacillus and Streptococcus hæmolyticus give rise to pneumonia in mice previously normal, whereas the pneumococcus produces pulmonary lesions only under special circumstances, as when the animals are alcoholized after partial immunization.     

EXPERIMENTAL PNEUMONIA IN MICE FOLLOWING THE INHALATION OF STREPTOCOCCUS H?MOLYTICUS AND OF FRIEDL?NDER'S BACILLUS

The results of these experiments support the previous observations that mice react in different ways to the inhalation of different bacteria. If normal mice are sprayed with hemolytic streptococci or Friedländer''s bacillus, these organisms may be recovered from the lungs and heart''s blood for a number of days after exposure. A large number of mice so exposed die of a septicemia during the 30 days following spraying. Consolidation of one or more lobes is not rare. If normal mice are exposed to an atmosphere of pneumococci, however, the organisms rapidly disappear from the lungs and only an occasional mouse succumbs to pneumococcus septicemia. In order to cause the pneumococcus to invade, the mice must first be alcoholized. If mice are first alcoholized and then exposed to a pneumococcus atmosphere, the pneumococci persist for a longer time in the lungs and a number will die of septicemia, a state of affairs which resembles that seen after the administration to normal mice of the other two organisms studied. In order to produce pneumococcus lobar consolidation, however, the mice must be first partially immunized and then allowed to inhale the organisms while under the influence of alcohol. A large percentage of both the normal and immune mice so alcoholized die within 5 days of exposure. There also appears to be a definite relation between the mortality and the length of time inspired bacteria persist in the lungs. Whether this lag in removal allows multiplication of the bacteria and massive infection to occur has not been determined. Following inhalation of pneumococcus, the organisms were either removed rapidly and without apparent detriment to the animal, or, as in the case of the alcoholized mice, invasion occurred, in which event the mouse usually succumbed rapidly, localization in the lung only occurring in partially immune animals. In the case of the hemolytic streptococci and Bacillus friedlænderi, however, the whole process of removal and infection seems to be slower. Infection and pneumonia occurred more often. These organisms seem to possess much greater invasive power when inhaled than the pneumococcus, though on intraperitoneal or subcutaneous inoculation, pneumococcus is highly virulent for normal mice.     

STUDIES ON EXPERIMENTAL PNEUMONIA : VIII. EXPERIMENTAL STREPTOCOCCUS H?MOLYTICUS PNEUMONIA IN MONKEYS.

1. Pneumonia has been consistently produced in normal monkeys by intratracheal injection of Streptococcus hæmolyticus. 2. The pneumonia produced has been shown to be comparable with hemolytic streptococcus pneumonia in man with respect to its clinical features, complications, and pathology. 3. Two pathologic types of the disease have occurred, interstitial pneumonia and confluent lobular pneumonia. Both types have been found in the same animal. 4. The type of pneumonia has appeared to be dependent upon the amount of streptococcus culture injected, interstitial pneumonia following the injection of small amounts and being an expression of considerable resistance, confluent lobular pneumonia following the injection of large amounts and being an expression of comparative lack of resistance. 5. Study of the distribution of streptococci in the lungs and of the character of the lesions in early stages of the disease has shown that streptococci may primarily invade the pulmonary tissue by penetration of the walls of the larger bronchial branches and that they are distributed from the points of invasion by way of the peribronchial, perivascular, and septal interstitial tissue and lymphatics. Infection of the alveoli is likewise primarily an interstitial invasion of the alveolar walls by streptococci. 6. In one experiment it was found that preliminary injury to the respiratory tract by gassing with chlorine and that lowering of resistance by a preceding intraperitoneal injection of Bacillus influenza without local injury to the respiratory tract greatly facilitated invasion of the lungs by Streptococcus hæmolyticus. 7. A normal monkey inoculated in the nose and throat with Streptococcus hæmolyticus failed to develop pneumonia and showed no evidence of infection of the upper respiratory tract.     

STUDIES ON THE BACTERIOPHAGE OF D'HéRELLE : IX. EVIDENCE OF HYDROLYSIS OF BACTERIAL PROTEIN DURING LYSIS.

1. During the process of lysis by bacteriophage, there is an appreciable increase in the amount of free amino acid present in the culture. 2. The increase of free amino acid is due to hydrolysis of bacterial protein.     

STUDIES ON THE BACTERIOPHAGE OF D'HéRELLE : II. EFFECT OF ALCOHOL ON THE BACTERIOPHAGE OF D'HéRELLE.

When bacteriophage is precipitated by alcohol at room temperature its activity rapidly and progressively decreases until it is totally destroyed, between 6 and 24 hours after exposure. If the percipitation is carried out at 7°C. the destruction of lytic activity is considerably slower; measurable traces may be detected even after 4 weeks exposure to alcohol. Although the major portion of the lytic activity is found in the precipitate, the supernatant alcohol carries a measurable amount of lytic principle which remains active for several days. In all cases the residual lytic activity was found to be transmissible in series. In no instance were we able to observe the non-transmissible action ascribed by d''sHérelle to the enzyme. The persistence of traces of active principle after many weeks of exposure to alcohol at low temperature is not found to be due to the existence in the original filtrate of a fraction relatively resistant to the effect of alcohol. The inactivation of bacteriophage by alcohol seems, therefore, analogous to the alcoholic inactivation of certain enzymes and toxins.     

STUDIES OF CALCIUM AND PHOSPHORUS METABOLISM : V. A STUDY OF THE BONE TRABECUL? AS A READILY AVAILABLE RESERVE SUPPLY OF CALCIUM.

1. Bone trabeculae are easily depleted by the prolonged administration of parathormone, long continued negative calcium balance and growth. 2. A long continued high calcium diet results in a rapid accumulation of the trabeculae. 3. Alizarin red, as has previously been shown in the literature, is deposited in newly formed bone. Its use has made clear that easily mobilizable calcium is not deposited in the shafts of adult animals, but in the trabeculae of bone. 4. The bone trabeculae therefore serve as the storehouse of readily available calcium. 5. The shafts have a slow progressive exchange of inorganic salts and are not influenced except in the case of unusual body demands. 6. It is suggested that the absence of trabeculae in premature infants and their depletion at the end of 4 months in a normal baby might well be an etiological factor in rickets. 7. In our observations, parathormone administration to growing or adult cats has been without effect. 8. Daily injections of parathormone in growing rats results in an increased number of trabeculae and smaller bones.     

THE SOLUBLE SPECIFIC SUBSTANCE OF FRIEDL?NDER'S BACILLUS : PAPER II. CHEMICAL AND IMMUNOLOGICAL RELATIONSHIPS OF PNEUMOCOCCUS TYPE II AND OF A STRAIN OF FRIEDL?NDER'S BACILLUS.

The chemical and immunological properties of the soluble specific substances of a strain of Friedländer''s bacillus and Pneumococcus Type II are described and correlated, and the serological and antigenic similarity of these biologically unrelated organisms is discussed as an example of heterogenetic specificity among bacteria.     

STUDIES ON EXPERIMENTAL PNEUMONIA : X. PATHOLOGY OF EXPERIMENTAL INFLUENZA AND OF BACILLUS INFLUENZ? PNEUMONIA IN MONKEYS.

1. Virulent influenza bacilli, when injected into the nose and throat of monkeys (Cebus capucinus and Macacus syrichtus), excite an acute inflammation of the upper respiratory tract, characterized by swelling and hyperemia of the mucous membrane, infiltration of the mucosa and subrnucosa with leucocytes, desquamation of epithelial cells, and the production of a mucopurulent exudate. The accessory sinuses are often implicated in the infection. 2. Experimental Bacillus influenzæ infections of the upper respiratory tract are frequently accompanied or followed by bronchiolitis, peribronchial infiltration, and bronchopneumonia with hemorrhage and edema in the early stage, emphysema and bronchiectasis in the later stages. In general, the process closely resembles uncomplicated Bacillus influenzæ pneumonia in man. 3. The injection of virulent influenza bacilli directly into the trachea of monkeys induces in them an experimental bronchiolitis and hemorrhagic bronchopneumonia, similar in all respects to spontaneous Bacillus influenzæ pneumonia. 4. In experimental Bacillus influenzæinfections of either the upper or lower respiratory tract the influenza bacillus can usually be recovered during .the acute stage by culture, either pure or in association with other bacteria. 5. In experimental Bacillus influenzæ infections in monkeys characteristic changes occur in the thymus gland—hyperplasia of the follicles, distention of the lymphatic channels, and infiltration of the parenchyma with leucocytes. This enlargement appears to be merely part of a general hyperplasia of the lymphoid structures in the cervical and thoracic regions.     

STUDIES UPON EXPERIMENTAL PNEUMONIA IN RABBITS : V. THE R?LE OF THE LEUCOCYTE IN EXPERIMENTAL PNEUMONIA. THE RELATION OF THE NUMBER OF ORGANISMS INJECTED TO THE MORTALITY.

1. The importance of the leucocyte in the resistance of animals to experimental pneumonia is emphasized by the fact that animals treated with benzol, a leucotoxic substance, rapidly succumb to the disease, while animals treated in like manner with toluol, a very similar chemical substance causing no leucopenia, show no decreased resistance. 2. The rôle of the leucocyte in the resistance of animals to experimental pneumonia is further emphasized by the fact that animals that respond to the pneumococcus infection with a leucocytosis, as occurs after the repeated injection of toluol, are more resistant to the pneumonia. Further, the hyperleucocytosis produced by repeated injection of nutrose before the production of pneumonia likewise seems to increase the resistance of the animals. 3. Experimental pneumonia is not necessarily fatal in rabbits. The factors determining the outcome of the disease are numerous; among these is the number of bacteria inoculated. Animals receiving small doses usually survive, while those receiving comparatively large numbers usually succumb.     

STUDIES ON THE METABOLISM OF CELLS IN VITRO : I. THE TOXICITY OFα-AMINO-ACIDS FOR EMBRYONIC CHICKEN CELLS.

Summing up these results, we found that all the ten α-amino-acids used inhibited the growth of the cells and finally killed the cultures. This inhibition is preceded by a short period of activity. The typical effect on the cells is shown in Figs. 1 and 2. The first (Fig. 1) is a control culture showing the usual growth of cells and their typical spindle shape form. The second (Fig. 2) is a culture in plasma plus asparagine showing the cells rounded off and beginning to undergo dissolution. We do not wish to draw too extensive conclusions from these experiments, but we believe that toxicity of α-amino-acids towards growing cells has been shown beyond a reasonable doubt; while we have found that compounds of higher molecular weight, namely, the peptones of egg yolk and proteins, are non-toxic. This toxicity depends upon the concentration and the time that the cells are exposed to their action. As these factors are reduced, the toxicity is decreased. In this respect, these substances are similar to all cell poisons. Applying these results to the work done on the intravenous injection of digestion mixtures, we believe that we have found a reason for the death of the experimental animals when the hydrolyzed proteins were injected too rapidly. Buglia found that large amounts of α-amino-acids could be injected into the circulation without causing deep-seated changes in the renal and intestinal functions, provided they were injected slowly enough; in fact, that enough of these mixtures could be injected in this way to cover the nitrogen consumption of the body. This injection, however, was always accompanied by an α-amino excretion through the urine and an increase of the peristalsis, of the intestine, with resultant liquid stools. As is well known, a sudden great concentration of these substances in the blood of an animal causes death. These results agree with our findings Folin and Denis demonstrated the fact that α-amino-acids probably pass into the circulation through the intestines. Van Slyke and Meyer, by means of Van Slyke''s nitrogen method, have practically proven this, and Abel, Rowntree, and Turner, and Abderhalden have lately succeeded in obtaining α-amino-acids in crystalline form from the blood. Van Slyke and Meyer have shown that the tissues take up α-amino-acids to a certain point, but that after this the limit of saturation is reached. This is not so in the liver, which continually desaturates itself by metabolizing the α-amino-acids that it has absorbed, and consequently maintains indefinitely its power of removing them from the circulation, as long as they enter it no faster than the liver can metabolize them. Marshall and Rowntree have shown that there is an increase of the α-amino-acid concentration in the blood after injuries to the liver, which have caused deep-seated anatomical changes. Our experiments prove that tissue cells in general are unable to live in the presence of any great concentration of these acids. At the present time we do not feel able to give an explanation of the significance of this evident toxicity. However, the fact in itself seems to indicate that we should expect stimulation from a certain increase of the α-amino-acid concentration in the body, or the concentration of any one of the acids, while a greater increase would lead to marked disturbances of the metabolism.     

THE R?LE OF THE RETICULO-ENDOTHELIAL SYSTEM IN IMMUNITY : I. THE R?LE OF THE RETICULO-ENDOTHELIAL SYSTEM IN THE PRODUCTION OF DIPHTHERIA ANTITOXIN.

1. Following massive doses of India ink injected intravenously into guinea pigs before a subcutaneous injection of diphtheria toxin-antitoxin mixture, no antitoxin was found in the blood serum for 3 weeks, as indicated by intracutaneous tests, whereas an appreciable amount could be detected in non-blocked, immunized control animals. 2. During the 4th week following immunization, the titer of the serum of blocked animals equaled that of non-blocked controls within the limits of the intracutaneous test dose. 3. The smaller doses of India ink used in these experiments, given before immunization, had no stimulating effect on the production of diphtheria antitoxin but, on the contrary, also inhibited the appearance of this antibody, although to a less extent.     

THE SOLUBLE SPECIFIC SUBSTANCE OF A STRAIN OF FRIEDL?NDER'S BACILLUS : PAPER I.

1. A method is given for the isolation of a specifically reacting nitrogen-free polysaccharide from the so called E strain of Friedländer''s bacillus. 2. The properties of this polysaccharide are described.     

THE R?LE OF THE RETICULO-ENDOTHELIAL SYSTEM IN IMMUNITY : V. PHENOMENA OF PASSIVE IMMUNITY IN BLOCKED AND SPLENECTOMIZED MICE.

1. Blockade of the reticulo-endothelial system by means of one intravenous injection of India ink as well as splenectomy did not alter the course of either Pneumococcus Type I infection or tetanus intoxication in mice. 2. The protective action of Antipneumococcus Type I serum against the corresponding infection, as determined by the injection of in vitro prepared mixtures of serum and culture, was definitely lower in mice which had received one blocking injection of India ink shortly before the test. 3. Titration of tetanus toxin and antitoxin in blocked and splenectomized mice gave results identical with those obtained in normal mice, if in vitro prepared and incubated toxin-antitoxin mixtures were injected. The degree of protection, however, conferred by a preceding dose of antitoxin against subsequent intoxication, was markedly lower in blocked mice than in normal control animals, this difference becoming more pronounced with the increase of the time interval.