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The results of the present study indicate that by means of serological reactions it is possible to differentiate selectively the p-aminophenol glycosides of maltose, cellobiose, gentiobiose, and lactose. The immunological specificity of disaccharide-protein antigens prepared from these derivatives, irrespective of the nature of the conjoined protein, is determined by (1) the glycoside molecule as a whole, (2) the configuration of the terminal hexose molecule, and (3) the position of linkage of the two hexose units in the carbohydrate radical. The specificity of the antibodies induced by the disaccharide antigens appears to be more sharply defined when the configuration of the terminal hexose is of the ß rather than of the α type.

1. Methods are given for the isolation of specifically reacting nitrogen-free polysaccharides from Type A and Type C Friedländer's bacillus. 2. The properties of these specific carbohydrates have been outlined.

1. At least three subgroups of Pneumococcus Type II may be recognized by specific immune reactions. They have been called Subgroups II A, II B, and II X. 2. That the organisms of these three subgroups are biologically related to Pneumococcus Type II is shown by the following facts: (a) Agglutination with Antipneumococcus Serum II. (b) Protection with Antipneumococcus Serum II, except Subgroup II X. (c) Absorption of Antipneumococcus Serum II with typical Type II pneumococcus removes the antibodies for all subgroups, (d) Absorption of Antipneumococcus Serum II with a member of Subgroups II A or II B removes only the antibodies for the homologous subgroup. Absorption of Antipneumococcus Serum II with any given member of Subgroup II X removes the antibodies for that particular strain only. 3. That the three subgroups, although biologically related to Pneumococcus Type II, possess, nevertheless, specific differential characterswhich separate them one from another, is evidenced by thefollowing facts: (a) The organisms of any subgroup are not agglutinated bythe antisera of the other two subgroups. (b) They are not protected against by the sera of the other subgroups. (c) They do not absorb from Antipneumococcus Serum II the specific immune bodies of the other subgroups. 4. Subgroups II A and II B are characterized by immunity reactions identical within the respective group. 5. Subgroup II X consists of heterogeneous strains which do not cross in their immunity reaction with each other or with Subgroups II A or II B.

The immune bodies of antipneumococcus serum are completely precipitated by 38 to 42 per cent. saturation with ammonium sulphate. They are incompletely precipitated by (a) ammonium sulphate in less than 38 per cent. saturation, (b) saturation with sodium chloride, (c) dilution and saturation with carbon dioxide, (d) removal of crystalloids by dialysis. The immune bodies of antipneumococcus serum are, therefore, associated or combined with that fraction of the globulins precipitated by 38 to 42 per cent. saturation with ammonium sulphate. The immune body fraction does not correspond exactly with the ordinary euglobulin (one-third saturation with ammonium sulphate or complete saturation with sodium chloride) or with the insoluble globulins precipitated by carbon dioxide or dialysis. These fractions carry with them only a part of the immune bodies. Neither the albumin nor that fraction of the globulin not precipitated by 38 to 42 per cent. saturation of ammonium sulphate contain any of the demonstrable antibodies. The most promising method for the practical purification of the immune bodies occurring in antipneumococcus serum appears to be precipitation by 38 to 42 per cent. saturation with ammonium sulphate.

It has been demonstrated that many multiples of minimal doses of Bacillus typhosus reacting factors can be neutralized by specific immune sera. The potency of a given serum can be conveniently titrated against increasing amounts of reacting factors. If the immune serum is diluted or if the amount of the reacting factors is too large for a given amount of serum, there is obtained neutralization but only irregularly. Normal and heterologous sera (therapeutic meningococcus and erysipelas horse sera) free of normal agglutinins or possessing normal agglutinins of a low titer (1:16) for Bacillus typhosus are not able to neutralize the reacting factors. There is obtained questionable neutralization with a serum possessing normal Bacillus typhosus agglutinins in dilution 1:64. The titer of the neutralizing antibodies increases in the course of immunization. Immune sera exercise a definite protection against the mortality induced by intravenous injection of Bacillus typhosus culture filtrates.

1. By partial acid hydrolysis a specific carbohydrate may be isolated from gum arabic (gum acacia). This carbohydrate is comparable in its precipitating activity for Type II (and Type III) antipneumococcus serum with the bacterial soluble specific substances themselves. 2. On hydrolysis this fraction yields galactose and two or more complex sugar acids, one of which appears to be a disaccharide add comparable with those isolated from the specific polysaccharides of the Type III pneumococcus and the Type A Friedländer bacillus. 3. The significance of these findings is discussed.

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. Autolysis of Pneumococcus is accompanied by proteolysis, which results in an increase in amino and non-coagulable nitrogen. 2. Autolysis of Pneumococcus is accompanied by lipolysis during which there is a liberation of ether-soluble fatty acids. 3. When extracts containing the active intracellular enzymes are added to heat-killed pneumococci, lysis of the cells occurs and there is an increase in the non-coagulable and amino nitrogen, comparable to the changes accompanying spontaneous autolysis. 4. When extracts containing the active intracellular enzymes are added to emulsions of the alcohol-soluble lipoids extracted from pneumococci, an increase in the ether-soluble fatty acid occurs. 5. Sodium desoxycholate in excess inhibits the action of pneumococcus protease; it does not inhibit the action of pneumococcus lipase. 6. When suspensions of pneumococci are cooled to 0°C., a temperature at which the rate of enzyme action is greatly retarded, the organisms go into solution rapidly when sodium desoxycholate is added, but this process is not accompanied by lipolysis or proteolysis. It does not seem probable, therefore, that the "bile" solution of pneumococci is identical with the phenomenon of autolysis as ordinarily understood and measured. 7. The relation of enzyme action to antigenic dissociation is discussed.

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.

1. The synthesis of p-aminophenol α-glucoside has been described. This glucoside can be coupled to any protein to yield a synthetic α-glucoside-protein complex. 2. A synthetic ß-glucoside-protein complex has also been prepared. 3. These synthetic sugar-protein complexes have been used as immunizing antigens in order to ascertain whether α- and ß-glucosidic unions influence the specificity of the immune response in animals. The results of the immunological studies are presented in the following paper.

The chemical and immunological properties of the p-aminophenol α- and ß-glucosides of glucose are described and correlated. The results are discussed with reference to their possible bearing on the chemo-immunological nature of the specific polysaccharides of bacterial origin.

The action of the enzyme which specifically decomposes the capsular polysaccharide of Type III Pneumococcus has been tested in Type III pneumococcus dermal infections in rabbits. When injected in sufficient amounts, this enzyme is capable of bringing about a favorable and early termination of the experimental disease which ordinarily is fatal in nearly all instances. The results of the present study yield further evidence that the capsular substance is of great importance in pneumococcus infection, since, in so far as known, the only action of which the specific enzyme is capable is that of decomposing the capsular polysaccharide.

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. When two chemically different carbohydrate derivatives are bound to the same protein, the newly formed antigens exhibit distinct immunological specificity. 2. When the same carbohydrate radical is conjugated with two chemically different and serologically distinct proteins both of the sugar-proteins thus formed acquire a common serological specificity. 3. The newly acquired specificity of the artificially prepared sugar-proteins is determined by the chemical constitution of the carbohydrate radical attached to the protein molecules. Simple differences in the molecular configuration of the two isomers,—glucose and galactose—suffice to orientate protein specificity when the corresponding glucosides of the two sugars are coupled to the same protein. 4. The unconjugated glucosides, although themselves not precipitable in immune serum, inhibit the reaction between the homologous sugar-protein and its specific antibody. The inhibition test is specific. 5. The sugar derivatives unattached to protein exhibit the properties of carbohydrate haptens; they are non-antigenic but specifically reactive, as shown by inhibition tests, with antibodies induced by proteins containing the homologous diazotized glucoside. 6. The specificity of artificially prepared sugar-proteins is discussed with reference to the chemo-immunological nature of the bacterial antigens containing complex sugars.

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.

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.

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 data presented in this paper clearly indicate that the isolated carbohydrate and nucleoprotein constituents of Pneumococcus differ both serologically and antigenically one from the other. Moreover, each of these fractions of the cell separately exhibits immunological properties distinct from those manifested by the whole organism of which they form a part. The carbohydrate is a protein-free polysaccharide and as such is devoid of the property of stimulating antibodies. Although in the free state, dissociated from other cellular substances, it is non-antigenic, in this form it still retains the property of reacting specifically in anti-pneumococcus serum of the homologous type. Further, this nonprotein constituent is not reactive with antiprotein serum. In other words, neither pneumococcus carbohydrate nor protein as separate antigen gives rise to antibodies with specific affinities for the carbohydrate or so called soluble specific substance of Pneumococcus. The nucleoprotein of Pneumococcus, on the other hand, is antigenic. Immunization with this cell constituent gives rise to immune serum which precipitates solutions of pneumococcus protein without regard to the type from which it is derived. The interrelations of the carbohydrate and protein of Pneumococcus as they exist in the intact cell to form the complete antigen, and the interpretation of the differences in the antigenic properties of the whole bacterium as contrasted with those of its component parts are reserved for discussion in a subsequent paper.

1. Intact pneumococci, possessing specific antigenic powers unimpaired by cultural or other procedures, give rise to agglutinins for organisms of the homologous type and to precipitins for the type-specific carbohydrate derived from them. 2. Solutions of pneumococci free of all formed elements, but containing the carbohydrate and protein of the original cell, fail to stimulate the formation of type-specific antibodies. Sera prepared in this manner do not react with the carbohydrate constituent of the cell and do not agglutinate organisms of the homologous type. The loss of this antigenic function is related to changes incurred during dissolution of the bacterial cell. 3. Solutions of the cellular substances of Pneumococcus, although lacking the specific antigen of the whole cell, induce the formation of antibodies reactive with pneumococcus protein regardless of the type from which the latter is derived.

In this paper the general immunological significance of the intact pneumococcus cell and of its protein and carbohydrate components is discussed.

1. Refinements in the methods for the isolation of the soluble specific substances of Types II and III pneumococcus are described. These improvements have resulted in the isolation of the end-products in a form free from nitrogen and of enhanced activity with immune serum. 2. The soluble specific substance of Type I pneumococcus is described and shown to differ sharply from the corresponding substances of the other two types, each of which, in turn, differs from the other. 3. Progress is reported on the identification of the sugar units from which the polysaccharides are built up. 4. The evidence so far accumulated is believed to favor strongly the view that the polysaccharides isolated are the actual specific substances of Pneumococcus.

1. The systems responsible for oxidation and reduction in the pneumococcus cell have been shown to consist of two components, the nature of which has been discussed. 2. The oxidizing and reducing power of active extracts of pneumococci is inactivated by exposure to air. This loss in activity is due to an inactivation of the thermolabile cellular component consequent on secondary reactions analogous to those which under similar conditions destroy the hemotoxin and enzymes in pneumococcus extracts.

Sterile filtrates of autolyzed anaerobic cultures of Pneumococcus contain much higher concentrations of active endocellular enzymes than do the filtrates of autolyzed aerobic cultures. These differences in the enzyme activity of the filtrates of aerobic and anaerobic cultures may be explained by a destruction of the formed enzymes by oxidation reactions analogous to the destruction previously observed during the oxidation of sterile broth extracts of unwashed pneumococcus cells. The occurrence of more complete destruction of enzyme activity in autolyzed aerobic cultures than in "oxidized" sterile cell extracts is probably due to the longer exposure to oxidation products.

1. Certain enzymes of Pneumococcus are destroyed by oxidizing agents formed when sterile extracts of the cellular substances are exposed to air. The carbohydrate-hydrolyzing enzymes (sucrase, raffinase, inulase, and amylase) are the most easily inactivated under these conditions, although the bacteriolytic enzyme is also reduced in activity. Similar treatment is without effect upon the active concentration of pneumococcus lipase and peptonase. 2. The enzymes which are destroyed during the oxidation of unwashed cell extracts are themselves non-reactive with molecular oxygen. The reactions by which they are destroyed seem to represent oxidations of a type similar to those proposed in previous papers for the oxidation of hemotoxin and of hemoglobin. 3. A study has been made of the relative resistance of different pneumococcus enzymes to heat and to the action of hydrogen peroxide. 4. The various enzymes may be arranged in the same order of relative resistance whether the rating be made from the standpoint of resistance to heat or of resistance to oxidation. Nevertheless, it appears that by a proper regulation of conditions of oxidation, certain labile constituents of a mixture of cellular enzymes may be inactivated with less effect upon the activity of other constituents of the mixture than when inactivation is brought about by heat.

1. The method for the concentration and purification of the soluble specific substance of Pneumococcus has been improved. 2. Highly purified specific substance of Type II pneumococcus of polysaccharide nature is shown to be recovered essentially unchanged after precipitation by immune serum, by uranyl nitrate, by basic lead acetate, or by safranine. 3. Marked chemical differences are shown to exist between the specific substances of Type II and Type III pneumococcus, although both react as polysaccharides. 4. The weight of evidence is considered to be in favor of the view that the specific substances of Pneumococcus Types II and III are actually polysaccharide derivatives. 5. The immunological significance of the foregoing view is discussed.

In the present paper methods have been described for the preparation of sterile extracts of pneumococci. These extracts may be obtained by dissolving the bacteria in broth cultures by means of bile, or by extraction of the cellular substances by repeated freezing and thawing of broth or saline suspensions of unwashed cells. Under special precautions these extracts may be passed through Berkefeld filters without loss of potency. In this procedure, as in all other manipulations incident to their preparation, the extracts should be protected as far as possible from contact with air. All extracts were proved sterile by cultural and animal tests. Sterile extracts of unwashed pneumococcus cells promptly form peroxide on exposure to air. Peroxide formation is almost as active in extracts aerated at 2°C. as in those exposed to the air at room temperature. Detectable amounts of peroxide may be produced by these cell extracts within the reaction range of pH 5 to 9, the optimal zone lying at reactions less acid than pH 6. The peroxide-forming activity of the extracts is gradually diminished by prolonged exposure to 55°C., and is completely destroyed by heating at 65°C. for 5 minutes. Cell extracts of pneumococci which have been thoroughly washed prior to extraction in salt or phosphate solutions exhibit no peroxide-forming activity. These extracts of washed cells may be activated by the addition of the cell washings, yeast extract, or muscle infusion.

In the present work on oxidation and reduction by sterile extracts of pneumococcus, the preparations employed contain among other constituents, a hemolytic substance the properties of which have been described by Cole (1, 2) in his studies on pneumococcus hemotoxin. Pneumococcus extracts prepared by the methods described are actively hemolytic, 0.005 cc. of extract causing complete lysis of 2.5 cc. of a 1 per cent suspension of red cells from rabbit blood. This hemolytic property of pneumococcus extracts is destroyed by 10 minutes exposure to 55°C. When pneumotoxin-containing extracts are protected from the action of molecular oxygen, their hemolytic activity remains unimpaired for considerable periods of time. In the presence of air, on the other hand, the stability of the hemolytic substance depends upon whether the particular type of extract contains a "complete" or "incomplete" oxidation-reduction system. Sterile broth extracts of unwashed pneumococci are reactive with molecular oxygen, and as a result of this union peroxide is formed whenever these extracts are exposed to air. The hemolytic activity of "complete" extracts of this type is rapidly decreased and finally destroyed in the presence of molecular oxygen. On the other hand, the "incomplete" type of extract prepared by saline extraction of washed pneumococci may be exposed to air with little or no loss of hemolytic power. This "incomplete" washed cell extract, unless reactivated, does not undergo autoxidation in the presence of air; under these circumstances peroxide is not formed and the hemolytic activity of this type of extract is not impaired by exposure to air. The stability of the hemolytic agent in the "incomplete" type of extract is evidence that this substance is itself not reactive with or affected by molecular oxygen, even in the presence of the cell enzymes. The destruction of the same hemolytic substance in extracts capable of undergoing autoxidation may be ascribed to the action of some peroxide formed by the union of molecular oxygen with easily oxidized or autoxidizable substances of the extract. It is now known that a peroxide, having the reactions of hydrogen peroxide, accumulates in sterile pneumococcus extracts during oxidation. It has been shown in the present study that the addition of preformed hydrogen peroxide destroys the hemolytic activity of pneumococcus extracts, although higher concentrations were required than were detected in oxidized extracts themselves. These facts and the known action of superoxides in analogous types of reaction make it seem not unlikely that the active agent in the destruction of pneumotoxin in oxidized cell extracts may be a peroxide; either hydrogen peroxide or some higher organic peroxide formed during autoxidation of the extract.

1. Anaerobically grown pneumococci rapidly form peroxide upon exposure to molecular oxygen. 2. The peroxide-forming activity of pneumococci varies with different strains, and with the age of the cell. 3. Peroxide production by pneumococcus can proceed under conditions of reaction and temperature that do not permit active growth and multiplication of the cell.

1. Sterile broth extracts of unwashed pneumococci actively destroy hemoglobin to methemoglobin and lower degradation products. 2. Sterile saline extracts of washed pneumococci do not by themselves form methemoglobin; extracts of this type may be completed or activated by the addition of certain complementary substances such as meat infusion and yeast extract. 3. The hemoglobin-destroying activity of pneumococcus extract is lost by exposure to 65°C. for 10 minutes. 4. The properties of an extract upon which these blood changes depend are related to other known oxidation-reduction functions of the same extract. 5. Oxyhemoglobin is converted to methemoglobin only by cell extracts in the reduced form; completely oxidized extracts are inactive in the presence of blood. The action of hydrogen peroxide and the influence of blood catalase on these reactions are discussed. 6. During the reaction between oxyhemoglobin and pneumococcus extract oxygen is consumed. 7. The mechanism of methemoglobin formation by pneumococcus is interpreted as an oxidation process in which deoxygenation of oxyhemoglobin and peroxide formation occur as intermediary reactions. The active agent of the oxidation of hemoglobin is considered to be a peroxide of bacterial origin.

The known factors concerned in the autoinhibition of growth of pneumococcus are: 1. The accumulation of acid products of metabolism, resulting in unfavorable reaction changes in the medium. 2. The exhaustion of the nutritive substances of the medium. 3. Under certain cultural conditions the formation and accumulation of peroxide in the medium.

1. Conditions which favor the formation and accumulation of peroxide in broth cultures of pneumococcus are free access of air, and the absence of catalase, peroxidase, and other catalysts capable of decomposing this compound. Under these favorable conditions peroxide becomes demonstrable in the culture fluid during the logarithmic phase of growth and persists for a period of at least 6 to 12 days. 2. In the absence of these favorable conditions the formation of peroxide is inhibited. In a culture with deficient oxygen exposure the accumulation of peroxide is delayed; when anaerobic conditions are maintained the substance is not formed. In the presence of active catalysts, peroxide does not accumulate in the medium in quantities sufficient to give a positive reaction. The accumulation of peroxide in pneumococcus cultures is dependent upon the balance between the amount produced by the microorganisms and the amount destroyed by substances in the medium. 3. The peroxide formed in pneumococcus cultures is unstable. It gradually disappears during prolonged incubation at 37°C.; it is less stable in alkaline than in neutral or acid media. It is destroyed in culture filtrates exposed to the temperature of boiling water for 15 minutes, and to that of steam under pressure (15 pounds) for 10 minutes. 4. Peroxide formation occurred early in broth cultures of the seven strains of pneumococcus and of the six strains of non-hemolytic streptococci studied. Fifteen of twenty-three strains of Streptococcus hæmolyticus and one of three strains of Streptococcus mucosus formed peroxide. In the positively reacting cultures of Streptococcus hæmolyticus and Streptococcus mucosus the presence of peroxide was not demonstrable until the 3rd to 5th day of incubation. Peroxide could not be detected at any time during growth of the two strains of Staphylococcus aureus.

1. Certain anaerobic bacteria grow under aerobic conditions in plain broth in the presence of sterile unheated plant tissue. 2. Aerobic growth of anaerobic bacteria under the above conditions may be related to the presence of growth-accessory substances, and to the action of oxidizing-reducing systems of plant tissue in the destruction of toxic peroxides of bacterial origin.

1. An improved method is outlined for the isolation and purification of the pneumococcal transforming substance. This method makes use of the fact that citrate inhibits the destructive action of the enzyme, desoxyribonuclease, which is released together with the active material during lysis of the living bacterial cells. A fivefold greater yield of purified transforming agent is obtained by the present method than by the procedure previousiy described. 2. The specific transforming substance has been isolated from pneumococci of types II and VI, in addition to Type III. In each instance the biologically active material has been found to consist of desoxyribonucleic acid.

1. From Type III pneumococci a biologically active fraction has been isolated in highly purified form which in exceedingly minute amounts is capable under appropriate cultural conditions of inducing the transformation of unencapsulated R variants of Pneumococcus Type II into fully encapsulated cells of the same specific type as that of the heat-killed microorganisms from which the inducing material was recovered. 2. Methods for the isolation and purification of the active transforming material are described. 3. The data obtained by chemical, enzymatic, and serological analyses together with the results of preliminary studies by electrophoresis, ultracentrifugation, and ultraviolet spectroscopy indicate that, within the limits of the methods, the active fraction contains no demonstrable protein, unbound lipid, or serologically reactive polysaccharide and consists principally, if not solely, of a highly polymerized, viscous form of desoxyribonucleic acid. 4. Evidence is presented that the chemically induced alterations in cellular structure and function are predictable, type-specific, and transmissible in series. The various hypotheses that have been advanced concerning the nature of these changes are reviewed.

1. Sterile broth extracts of unwashed pneumococci, entirely free from living or intact cells, actively reduce methylene blue. 2. Sterile extracts prepared by extracting washed pneumococci in phosphate solution are unable by themselves to reduce methylene blue. Upon the addition of meat infusion or yeast extract, these washed cell extracts actively reduce methylene blue. 3. The system or systems in pneumococcus extracts responsible for methylene blue reduction are destroyed by exposure to temperatures practically identical with those which have been previously found to destroy the peroxide-forming activity of the same extracts. 4. It is suggested that peroxide formation and methylene blue reduction by pneumococcus extracts are functions of the same or closely related systems, the particular reaction induced depending upon whether molecular oxygen or methylene blue serves as hydrogen acceptor or oxygen donator.

The chemical and immunological properties of the acetylated and unacetylated forms of the p-aminophenol ß-glucoside of glucose have been described. The serological specificity of these ß-glucosides in combination with protein has been correlated with known changes in chemical structure and has been compared with the immunological properties of the α-glucoside of the same hexose.

The effects of specific enzyme therapy upon experimental Type III pneumococcus pneumonia in monkeys were studied by comparing the course and outcome of the disease in treated animals with that in animals which received no therapeutic aid. Enzyme treatment was found to exert a distinctly favorable influence upon the experimental pneumonia. Treatment was followed by cessation of spread of the pneumonic lesion, sterilization of the blood, and early recovery, except in animals in which the severity of the disease was extreme. While in the untreated animals a high incidence of empyema and pericarditis was observed, suppurative sequelae were apparently prevented by adequate enzyme therapy. The limitations of the therapeutic action of the specific enzyme in the presence of marked depression of the cellular reaction in infected animals are again emphasized.

The soluble specific substance of Pneumococcus Type I has been chemically isolated from the bacterial cells and from autolyzed cultures as an acetyl polysaccharide. So far as could be determined by the methods employed, the acetyl polysaccharide in highly purified form absorbs from Type I antipneumococcus serum all demonstrable type-specific precipitins, agglutinins and protective antibodies. Mice injected intraperitoneally with minute quantities of the acetyl polysaccharide develop active immunity to subsequent infection with Pneumococcus Type I. The immunity thus induced is type-specific. In several instances purpura has been observed in mice following the injection of larger amounts of the acetyl polysaccharide. Under the experimental conditions of this study, no type-specific precipitins, agglutinins or protective antibodies were demonstrable in the serum of rabbits following repeated intravenous injections of the Type I acetyl polysaccharide. The treated rabbits were not immune to subsequent infection with Pnemnococcus Type I. The acetyl polysaccharide is readily converted into its deacetylated derivative by treatment with dilute alkali. The chemical and immunological properties of the deacetylated polysaccharide are identical with those of the soluble specific substance in the chemical form in which it was originally isolated; the deacetylated form of the specific carbohydrate is non-antigenic, does not produce purpura in mice, and only incompletely absorbs the type-specific antibodies from Type I antipneumococcus serum. The immunological significance of the acetyl polysaccharide and its possible relationship to the specific substances isolated from Pneumococcus Type I by other workers are discussed.

1. An organism has been isolated from peat soil which decomposes the specific capsular polysaccharide of Type III Pneumococcus. 2. The isolation has been made possible by the use of a synthetic mineral medium containing the specific polysaccharide as sole source of carbon. By repeated transfers in this medium the potential capacity of the organism to decompose the specific substance has been progressively increased. 3. The organism is a pleomorphic bacillus, motile and spore-bearing, exhibiting metachromatic granules; its reaction to the Gram stain varies according to the medium on which it is grown. It is strictly aerobic and grows well in plain broth and peptone solutions; it does not produce gas in any media and it forms small amounts of acid only on dextrin, galactose, lactose, salicin, and trehalose; its growth is inhibited by glucose. 4. The organism decomposes the capsular polysaccharide of Type III Pneumococcus aerobically, between pH 6.2 and 7.8, at room temperature and at 37.5°C., but not at 54°C. The decomposition of the specific substance is inhibited by the presence in the medium of other nutrients, such as peptones, which act as a more readily available source of energy. The action of the organism is specific; it does not attack the soluble specific substance of Type I or Type II Pneumococcus, nor any of the other bacterial polysaccharides thus far tested. 5. The organism possesses an endocellular enzyme. This enzyme has been extracted by autolysis of the bacterial cells; in sterile solution it exhibits the same specific action as do the organisms from which it is derived, decomposing only the capsular polysaccharide of Type III Pneumococcus. 6. This enzyme decomposes the Type III specific polysaccharide under anaerobic as well as under aerobic conditions; it is inactivated at 60–65°C.; the rate of decomposition of the specific substance is not affected by the presence of normal serum. 7. There exists a quantitative relationship between the total amount of specific substance decomposed and the amount of enzyme preparation used; the existence of this relation makes it possible to express the activity of a given enzyme preparation in terms of the minimal amount required for the complete decomposition of a given amount of specific substance. 8. The specific decomposition of the capsular polysaccharide of Type III Pneumococcus, by the organism as well as by the enzyme it produces, illustrates once more the specificity of the types of Pneumococcus and confirms the fact that the capsular polysaccharides, and not some impurities carried along with them, are responsible for type specificity.

1. Type-specific antipneumococcus immunity has been induced in rabbits by immunization with antigen prepared by combining a specific derivative of the capsular polysaccharide of Type III Pneumococcus with globulin from horse serum. 2. Rabbits immunized with this antigen acquire active immunity against infection with virulent Type III pneumococci. 3. The sera of the immune rabbits contain type-specific antibodies which precipitate the Type III capsular polysaccharide, agglutinate Type III pneumococci, and specifically protect mice against Type III infection. 4. The experimental data are discussed with reference to: (1) the concurrence in the immune sera of type-specific antibodies for Pneumococcus and precipitins for horse globulin; (2) the determining influence of the capsular polysaccharide on the specificity of the antigen as a whole; (3) the unity of the type-specific precipitins, agglutinins, and protective antibodies induced by a single component of the pneumococcus in chemical union with an unrelated, animal protein.

1. The p-amino and p-nitromonobenzyl ethers of the specific polysaccharide of Type III Pneumococcus have been prepared. 2. The diazonium ether of the specific polysaccharide has been coupled with serum globulin to yield a specific polysaccharide-protein complex and this complex has been used for immunization. The results of the immunological studies are presented in the following paper.

The bacterial enzyme which decomposes the purified capsular polysaccharide of Type III Pneumococcus in vitro also destroys the capsules of the living organisms growing in media and in the animal body. Potent preparations of this same enzyme protect mice against infection with virulent Type III Pneumococcus. The protective action is type-specific. The protective activity of the specific enzyme is destroyed by heat (70°C. for 10 minutes). The enzyme remains in an effective concentration 24 to 48 hours after its injection into normal mice. The enzyme has been found to exert a favorable influence on the outcome of an infection already established at the time of treatment. A definite relationship has been found to exist between the activity of the enzyme in vitro and its protective power in the animal body. The mechanism of the protective action is discussed with special reference to the relation between the decapsulation of the bacteria by the enzyme and the phagocytic response of the host.

It has been shown that extremely minute amounts of purified preparations of desoxyribonuclease are capable of bringing about the complete and irreversible inactivation of the transforming substance of Pneumococcus Type III. The significance of the effect of the enzyme, and its bearing on the chemical nature of the transforming substance, together with certain considerations concerning the biological specificity of desoxyribonucleic acids in general, are discussed.

Methods are described for isolating a protein commonly present in the blood of patients during the acute phase of various infections which, unlike the normal serum proteins, is precipitable by the C polysaccharide of Pneumococcus. The reactive protein is present in the fraction of serum albumin precipitated by either ammonium or sodium sulfate between 50 and 75 per cent saturation. From this fraction the reactive protein separates out on dialysis against tap water. Following removal of the alcohol-ether-soluble lipids from acute phase serum the reactive protein becomes soluble in tap water, and is no longer precipitable by traces of calcium but still retains its precipitability with the C polysaccharide.

The C-reactive protein present in the albumin fraction of the serum of patients during certain acute bacterial infections is highly antigenic upon injection into rabbits. The antiserum thus prepared reacts specifically with this protein and does not react with the proteins of normal human serum. Immunological specificity has been demonstrated by both precipitin and complement-fixation tests. Antiserum prepared in rabbits to the C-reactive protein from human sources also reacts specifically with the similar protein in the serum of monkeys acutely ill with experimental pneumococcus infection. By means of immunological reactions it is possible to detect amounts of reactive protein which are too small to yield a visible precipitate in tests with the C polysaccharide. Certain of the properties are discussed which distinguish the C-reactive protein from the proteins of normal human serum.

The serum obtained from human beings and monkeys during the acute phase of diverse infections contains a protein which is precipitable by the C polysaccharide of pneumococcus. The distribution of this protein in acute phase serum has been studied, and the effect of calcium on the precipitation reaction with the C polysaccharide is described. Other distinctive features of this reaction are discussed. 1. When heated above 65°C., serum obtained from patients during certain acute infections loses the property of reacting in precipitation tests with the C polysaccharide of pneumococcus. The loss of activity under these conditions occurs at temperatures known to denature many proteins. 2. The reactive component in "acute phase" serum which precipitates with the C polysaccharide is tentatively regarded as a protein. 3. The reactive substance is associated with the albumin fraction of serum. 4. In the reaction between patients' serum and C polysaccharide, flocculation is conditioned by the presence of calcium ions. 5. The following distinctive features of the C-reaction are discussed with reference to known characteristics of antigen-antibody phenomena: (a) the occurrence of the reactive component in blood only during the acute stage of the infection; (b) the lack of specificity of the reaction with respect to the inciting cause of the disease; (c) the presence of the active substance in the albumin fraction of the serum; (d) the action of calcium in producing flocculation.

The discovery by Schaudinn and Hoffmann1 in 1905 of a spirillum in various luetic lesions marked the beginning of a decided advance in our knowledge of the etiology of syphilis. The announcement of the discovery, while most conservative in its assertions, gave a fresh impetus to the study of the many phases presented in the parasitology and pathology of this disease. A wide-spread interest was awakened and many investigators directed their attention to the problems in this new field of research. From the results of many of their observations we have already gained a new conception of the cause, course and treatment of lues.The organism found by Schaudinn and Hoffmann was first described by them under the name of Spirochæta pallida, but a more intimate study of its morphology led them to forsake its classification under the spirochætæ and to rename it the Treponema pallidum. Much

In the preceding communications, the intracutaneous vaccination of rabbits with Pneumococcus (S forms) has been shown to give rise chiefly to the formation of the antiprotein rather than the type specific antibodies, and to the development of an increased resistance to infection with organisms of homologous and heterologous types. The present paper describes briefly the development of an altered tissue reaction to Pneumococcus and its protein derivatives in rabbits which have been inoculated repeatedly into the skin with heat killed suspensions of R and S pneumococci.Mackenzie and Woo1have shown that guinea pigs, injected intracutaneously with an alkaline extract of Pneumococcus, develop an allergic reaction in the skin to the bacterial protein; Zinsser and Grinnell2have produced allergic sensitization to pneumococcus autolysates in guinea pigs previously injected intradermally or intraperitoneally with the same material. Bull and McKee3have recently shown that rabbits, after recovery from infection induced by intranasal inoculation of pneumococci, are highly skin-sensitive to pneumococcus autolysate.The present observations were made in the course of a study of the antibody response and the immunity developed as a result of intracutaneous injection of rabbits. The intracutaneous injection in normal rabbits of 0.2 cc. of a heated vaccine, representing the bacteria from 2 cc. of broth culture, is followed by the appearance locally of a circumscribed slightly raised and indurated nodule, reddish in color, and measuring about 1 cm. in diameter. Upon repeated injection at weekly intervals the reaction changes in character; the size increases, often reaching a maximum of 4 to 6 cm. in diameter, accompanied by a spreading edema and purplish discoloration. The maximum reaction is generally reached after 6 to 8 injections and thereafter each successive lesion tends to, become less intense but to persist longer, often breaking down with the discharge of sterile necrotic material.