THVH 256 Chemo-inmunological Studies on Pneumococcus. Renort of Dr. Avery with Drs. Tillett, Julianelle, Goebel, Dubos and Dawson. I. Theory of Antigenic Dissociation. Brief statement of theory. Attempts to prevent antigenic dissociation py cell fixation. Action of selective agents; Iodine, Formalin. II. Avutolysis: Chemical study of Enzyme Action. Relation to proteolysis, lipolvsis. Reaction accompanied by antigenic dissociation, inactivation of enzymes, oxidation of hemolysin, and formation of purpura producing substance. Previous studies of intracellular enzymes on foreign substrates ; what action have they on native protein, carbohydrate and lin- oids of cell itself? III. Bacterial Variation. 1) Pneumococcus. a. Interconvertibility of R and § forms. b. Conversion of S -+ R. c. Reversion of R -+ S. 2) Bffect of adaptation to growth at high temperatures. a. Attenuation of virulence. b. Production of variants. c. Differences in suscentibdility of different tynes. 3) Friedldnder's Bacillus. a. Occurrence of variants. bd. Different forcs of variants. c. Distribution of specific types and variants in human and animal infections. 257 IV. Studies on Natural Resistance and Acquired Immunity to Fneumococcus:-: Natural resistance and immune response of normal rabbits to Tyne III. Non-type svecific resistance induced by "R" forms of Pneurococcus. Passive transfer of acquired resistance. Vv. Anaphylaxis with Pneumococcus Polysaccharides. a. Nature of Pneumococcus haptens (protein-free polysaccharides). b. Active sensitization. c. Passive sensitization. d. Fatal anaphylactic shock. VI. Are the Svecific Precipitable Substances of Pneumococcus, Faotens? a. Investigations at Foch Institute. >. Experimental evidence of non-antigenic nature. VII. Immune Resvonse of Rabbits to Intracutaneous Vaccination. VIII. Studies on Oxidation and Reduction by Fneumococcus: * 1) Bacterial antagonism between Fneumococcus and Stanhylococcus aureu a. Antagonism in vresence of oxygen. b. Symb'osis in absence of oxygen. c. Reaction of phenomenon to cellular oxidation. 2) Relation of oxidation prooceses to viability. 3) Relation of oxidation processes to initiation of growth. | 4) Importance of oxidation - reduction potential of media. IX. Nature and Duration of Immunity Induced by Inhalation Method . X. Publications. I. Antigenic Dissociation. In the last renort the theory of mtigenic dissociation and its significance in Pneumococcus immunity was discussed in detail. During the past year new facts in support of this theory have been ac- quired and the principles involved have been exoerimentally applied in the study of active and passive immunity. In order to establish the sequence of thought between the work vreviously recorded and that to be discussed in the present renort, the results of recont chemo-immn~ ological studies may be briefly summarized as follows: Chemically the bacterial coll is composed, among other things, of two constituents each belonging to two wholly different classes of substances and both forming togethor the major part of the cell as a whole. Those two components, - one carbohydrate and the other protein - constitute a unique system which determines both the type-soccificity and the anti- genicity of the cell. The protein is tne somatic substance racially common to all pneumococci, while the carbohydrate, the capsular ma- terial, is chemically different and serologically specific for each type. In this system the protein is constantly present as an essent- dal constituent of the cell protoplasm; the carbohydrate, on the other hand, is the synthetic product of a specialized function which is independent of the purely vegetative processes of cell reproduc- tion and growth. By reason of the chemical nature of the substance elaborated, this function bestows upon the cell highly differential properties which biologically endow it with type-specificity and morphologically distinguish it with a well defined capsule. This special function is inhibited or suppressed whonover the cell is sub- jected to an untoward cultural environment, and is enhanced whenever oye) a yup 259 the organism is grown in the animal body. The presence or absence of carbohydrates determines the immunological specificity of the cell and modifies its antigenic nature; for, pneumococct which by chemical or biological methods have suffered loss of this substance no longer exhibit the specific reattions of the encapsulated forms from which they were originally derived. In more recent studies on pacterial variation, to be discussed later in this report, the appearance of variants in a given culture is shown to be intimately, sf not casual- ly, related to the loss of the physiological function of elaborating this specific substance. Apart from the importance of the carbohydrate as the de- terminative substance of type specificity, this constituent is unique in that when chemically isolated in protein-free form, it still re- tains the capacity to react with antibodics induced by immunization with the whole cell but Leses completely tho power to stimulate these same antibodies when jnjectod by Atself into the animal body - The polysaccharides of pmeumococcus, therefore, belong to the group of {mmunologically 4mportant substances which Landsteiner has called haptens. In cellular union these substances constitute the type- specific antigen, separatoly they are devoid of antigenic function. . Bvidently, their antigenicity is dependent upon the presence in the cell of some other substance from which they are easily dissociated ' in the form of haptens. Upon conditions detormining the physical or sely the more eas- iu i 5; chemical stability of this combination rests the antigenic effective- if ness of any given type of pneumococcus, and conver | i ily dissociable this complex is, the less efficient it is as antigen; ‘ that is, the antigenic potency of any given type of pneurococcus is f 260 inversely proportional to tho rate and extent of dissociation of the haptaphoro groun. This, in brief, is the essence of the theory of antigenic dissociation. Its significance in the problems of antipneumocaccus immunity at once becomes clear when it is realized that tho only specific therapy of the disease at present available is dependent upon the use of typo-specific serum, the potency of which is in turn directly relatod to the antigenic quality of the orgmisms used in its production. As pointed out ir the last report the titre of antipneumococcus serum in terms of type specific antibodies is con- ditioned by a balance between those factors of the aniral body which bring about mtigen cleavage and those chemical properties of the bacterial coll which determine the stability of the specific anti- genic complex of each type. The factors relating to the animal body will be discussed in the experimental work on the "Natural resistance and acquired im- munity". The relation between chemical constitution ard antigenic stability is being studied from two viewpoints: - 1) Selective fix- ation of antigen by chemical agents, and 2) Chemical changes occurr~ ing during autolysis. 1) Selective fixation. Assuming that the union between pneumococcus hapton (polysaccharide) and its antigenic activator is a labile one, a search is being made for chemical roagents whicn might "fix" this linkage and by stabilizing the compound prevent or {inhibit subsequent dissociation. One of the most promising agents employed thus far in an attempt to bring about atigenic fixation is Iodine. Quantitative estimation has shovn that living ew .occcel suspended in a standard pty oe 261 ~ solution of iodine absorb surprisingly large amounts of the reagent. After saturation with iodine the cells are killed and remain intact, well formed and Gram positive for ronths. They undergo no autolysis and the processes of oxidation cease. The inactivation of lytic en- zynes, the suppression of oxidative reactions and the preservation of morphological integrity are of distinct advantage in conserving the antigen. Comparison of heat killed and iodized cells shows that in the former instance, death resulting from exposure to 56° - 60°C. is soon followed by cell disintegration with the appearance of shrunken, Gram negative and shadow forms, due in part perhaps to incomplete i.- netivation of cellular enzymes at this temperature and in vart to cx traction which subsequently goes on in the saline suspension. On the pther hand, the unheated, iodized celle prepared from the same mass culture romain intact and retain indefinitely their form, size and staining reactions. Moreover, the iodized vaccine is non-toxic, and animals treated for weoks with these preparations show 20 411 effects. Immunological investigations by other workers have demon- stratod that iodized proteins are altered in their antigenic specific- ity. Tho type-specificity of pneumococcus antigen, however, is de- termined not by the bacterial protein but by the carbohydrate subst auce which envelopes the cell. Moreover, the polysaccharides of the three specific types differ from the etarch-glycogen group of carbohydrates in giving no color reaction with iodine. Such combination as iodine rakes with the coll, therefore, does not cherically alter this imoort- ant and dotcrminative constituent of the antigen. That the specific- ity of the iodized ooll remains entigenicolly waoltered is shown by the fact that sorur of irpunized aniztals agglutirates equally well both the treated and untreatod organisms and protects mice against infection with virulont orgaonisrs of the homologous type. The use of formalin as fixative and the increased produc- tion of antibodies when formolized cells of Type I are used as anti- gon, was discussed in the preceding report. Experiments now in pro- gress will shortly yield results whereby the comparative rerits of the various fixatives for the different bacterial types may pe fir- ally detervined. Apart from the intercating thooretieal considcera- tions of the relatior of these observations to the actual chemical constituents of the irmunizing antigen, the methods evolved may eventually come to have practical value in the production of potent antisera for therapeutic purposes. II. Autolysis. (Dr. Goebel) As part of the problem of determining the nature and chemical structure of the immunizing antigen of pneumococcus, 4 study is being made of cell autolysis. During spontancous dissolu- tion of pnewnococcl there is an accompanying diesociation by the ef- fective type-antigen. Therefore, to know whether proteolytic and lipolytic cleavage of cell substances occurs during autolysis, and to what extent, if any, these changes are associated with the loss of antigenicity of the autolysate, would, indirectly at least, af- ford some clue to the rechanism of antigenic dissociation and to the nature of the linkage between the carbohydrate and the sub- stance which confers antigenicity unon it. Fro~ the experimental evidence already available, it is apparent that autolysis is not to be confused with proteolysis, since only slight increase in amino nitrogen occurs and the cell protein after prolonged autolysis still retains its antigenicity. The ratios of total nitroger. to non-conguable and amino nitrogen are also being determined since it is known that during outolysis sone degradation product is released, probably in the form of 3 protcose, which gives rise to the purpura producing substance in pneumococcus autolysates. So far, little or no approciable hydrolysis of the lip- oidal substance of the cell has been determined, although final judgment on this phase of the autolytic process must await the use of the more exact methods now being perfected. The outcome of these studies on autolysis, whethor they throw light on the processes of antigenic dissociation or not, will nt lonst add to the knowledge of the action of the intracellular enzymes on tho native constituents of the cell itself and supplerent our earlier studies on the action of these sane ferments on forcign substrates. III. Bacterial Variation: 1) Pneuroccccus. a. Interconvertibility of wR and "sg" forms - (Dr. Dawson) _ (Conversion of "S" to wR.) Tho process of conversion of ee panne i peer a nme att aint virulont, type-specific, capsulated gt types of pneurmococcus into avirulent, non-type specific, non-capsulated wR forms has bocr stud- fed in further dotail. The most effective renns of bringing about this change is by growth in homologous immune sera and differences have been found to exist in tie readiness with which the transform.- tion can be effected in the various typos. Type 1 "st has beer. found to be the most difficult to convert to the "R" forms requiring ten to fifteen transfers in serum dilutions of 1:2 or 1:4. Even after this number of transfers tantermediate" colonies exist which readily revert to the "S'"' type. On the other hand, Types II "S" and III "si are readily converted to the UR" variety, and in each instance the change is more abrupt and complete than in the case of Type I "5S". (Reversion of "R" to ns"). Reversion of "R" forms to their homologous "S" type has been effected both by an in vivo and an in vitro method. (a) In Vivo. By animal passage "R" forms, de- rived respectively from Types I, II, and III "S", have been trans- formed into the "S" type. Differences have been found to exist in the constancy of the "R" variant. Some "R" forms readily revert to the virulent "S" type while one strain has been studied which has remained completely refractory to all attempts to effect the trans- formation. In the earlier experiments the intraperitoncal route was used exclusively but. more recently the subcutancous injections of large amounts of "R" cultures has given a much greater number of positive results. Single-cell as well as mass cultures have been employed in all experiments and in every instance the single cell cultures have reacted in the same manner as the mass cultures from which they were derived. (b) In Vitro. The in vitro method of effecting the "R" -+ tg treneformation ig by growth of the RN forms in anti-"R" sera, the optimal concentration of which is 10 per cent. As in the in vivo experiments both mass and single-cell cultures were employed. Sucha finding argues against the hypoth- esis that the virulence of a culture depends upon the relative num- ber of "RY and "st forms of which it is commosed. ewe 265 In all instances in which roversion has been effected both in vivo and in vitro the "R" forms have reverted to the "§" type from which they were originally derived. Roversion of "R" to "S" is alvays accompanied by the ac- quisition of all the charactoristics of the "§" type, including max- imal virulence. 2) Effect of Adaptation to Growth at Higher Terperatures. Prolirinary observations have been made on the crowth of Typos I and III pnewnococcus at higher temperntures (39°C). The re- sults of carly investigations have been such as to suggest that fur- ther work may roveal significant differences in the behavior of the various types. After fiftcon transfers at 30°C the strain of Type I "S" employed had completely chanced to the "R" form, while after thirty transfers at the sare temperature a Type IIT ig strain. still produced only "S" colonies. Further vork is now being undertaken to determine the effect of growth at 39°C on a variety of serological types. 3) Friedlander’s Bacillus. (Dr. Julianelle). During the course of studies on the biological and immnological properties of Friedlunder's bacillus, three sharply defined types were found to exist among different strains of this organisr. The types were de- signated A, B, C, and into one Group, X, were placed several hetero- genous strains. Later ‘studies revealed that, under certain condi- tions, variant forms may be induced in cultures of Friedldnder's bac- jllus. The typical colonies of the organism’ are identified as Smooth (s) and the variant colonies as Rough (R). The "S" strains produce capsules, soluble specific substance, are virulent and type specific. aoe ea oan aan PR RES PST ee Ae ee er ie ead on egg SO The R strains, on the other har, produce no capsules, nor soluble specific substance, are not pathogenic and are group specific; that is, they are serologically undifferentiated regardless of their type derivation. Further study has since disclosed that there exist among the "R" variants of Friedldnder's bacillus additional forms which exhibit definite differences in morphology and antigenicity. Three different forms of R colonies have been studied and designated as Ry> Ro: Ro: The Re variety is extremely unstable and since it was never obtained in pure culture, it was studied only morphologically. Ry and Ro» however, have been observed in greater detail and they have becn found to differ both grossly by colony formation, and mi- croscopically by the size and arrangement of the individual cells. Both variants (R, and Ry) moreover, may be differentiated further by serological reactions. Both forms are agglutinated in antisera ' prepared by the injections of rabbits with either strain, but they lack the capacity of complete reciprocal agglutinin adsorption. Bach variant adsorbs from the homologous antiserum agglutinins for both homologous and heterologous organisms; from the heterologous serum, however, antibody is removed only for the strain employed in the adsorption. The R forms differ considerably from their ant - ecedent "S" strain in colony appearance morphology, virulence and antigenicity. A number of methods have been adopted to induce reversion of the R forms to the parent "S" type. However, whether the tech- nique or its application was inadequate, the results were uniformly negutive. This does not mean that all "R" cells of Friedlander's bacillus are irreversible, but that under the conditions of the Foe 4 25'7 “a experiment, the method employed did not supply the proper stinrulus. to reversion. The spontaneous development of "RY variants in "S" cul- ture has been found to accompany the process of acing. Growth in homologous immone sera in vitro also converts the "§" colls into "R" forms. That variation, however, is more than in vitro or cul- tural decradation gains support from the fact that "R" forms have been found in cultures taken directly from foci of infection in the animal body. In fact, in 17 culturas from different sources WRI forms werc found in 5 instanccs. Interestingly enough, the rR variants were found only in chronic infections and always in con- junction with "S" forms. Further Observations on the Occurrence of Specific Types of Fried- lHnder's Bacillus in Disease. (Dr. Julianelle). In the preceding report, & summary Was presented of the distribution and relative frequency of occurrence of spacific types in infections associated with Vriedlunder's bacillus. Up to that time, abservations were made upon 39 strains; since thon, the number hos been increased to 68. In the following table the distribution of the specific types ia summarized to date. Total number of | Type | Type Type | Group Strains studied A B C. X 62 32 9 7 13 Type A - 32 etrains. . 24 strains from Pneumonia in man, 2 from adenoid tissue, 2 from throat cultures, 2 from fatal abscesses in guines pies, 1 from liver abscess in man, l from cystitis. ERI 268 Type B - 9 Strains. 4 strains from fatal Pneumonia in guinea pigs, 3 from Pneumonia in man, 2 from genito-urinary infection in horses. Type C - 7 Strains. 2 stroins from Pneumonia in man,.1 from sputum of Pneumococcus pneumonia, 1 from subacute sinusitis of antrum, 1 from nose (sinus infection), 2 source unknown. Group X - 13 Strains. 6 strains from Pneumonia in man, 2 from fatal infection in guinea pigs,l from cystitis, 1 from feces (Pellagra),1 from livor abscess in man, 1 from lung ab- scesa, 1 from throat culture. Although the total numbor of strains of Friedlinder's bacillus studied is still inevufficient to furnish conclusive data on the relative frequency and distribution of types, nevertheless, it is interesting to observe that of 35 strains isolated from pneu- monia in man, 29 or 83 per cent belonged to one or other of the three fixed types, The frequency of Typo A infection in Friedl&nd- er'gs pneumonia in man is shown by the fact that organisms of this type were isolated from 24 (68 per cent) of 35 casea studied. It is remarkable that these percentages are the same now with a total of 62 strains,as they were upon the former analysis based on 39 strains. Since there seers to be great confusion as to what de-- finitely constitutes a Friedldnder's bacillus, and whnt sugars are fermented by organisms of this group, the reactions of 45 strains have been studied in redia containing the differont carbohydrates, dextrose, lnctose, sucrose, mannite and maltose. my 269 ° The results of the ferrent>ztion studies revenl that, in general, there is creat variability naong the different strains and that the adaptation of such reactions for a classification of this svecics offers more confusion than syster. The formentation reactions not only do not aid in the classification of types but offer no as- sistance in differentiating the Friedldnder's bacillus from closely allied organisms. The one striking fact brought out in this study ig that whereas the majority of strains in Typee B and © and Group X ferrent lactose, 61 per cent of the strains of Type A produce neither acid nor gas from lactose. IV. Natural Resistance and Acquired Imrunity to Pneumococcus (Dr. Tillett‘ In three previous papers, experimental studies on natural and acquired immunity of rabbits against pneuwmococecus smucosus have been described. Since an interpretation of the results reported in the published experirents has formed the basis of subsequent work, a brief review of the earlier studies will be given. fTrom observations on the results of injection of Tyne III pnewrococci into normal rabbits, two rain facts have accrued which seer. to be related. First, it was found that immunization of rabbits with heat killed nype III vaccine or living orgenisne failed to elicit specific antibody response; secondly, that injection of living, encsp- sulated, mouse-virulent, Type III organisms in large doses into rabbits did not produce fatal infection. These two results led to the devolco- ment of the hypothesis that normal rabbits possess some mechanism which is capable of severely injuring the capsular corponent of the Type III bacterial cell. On the basis of this conception the absence of type specific agglutinins in the serum of immunized rabbits is explained as oft being due to the dissociation of the type specific comporent of the anticen; when living pneurococci ore injected, injury of a sirilar character is inflicted on that part of tho ecll which is intirately associated with virulence, namely, the capsular fraction, and, in this instance, recovery of the infected animal is brought about. In the previous published experiments, which concerned acquired re- sistance it has been shown that rabbits when immunized eithor with non-type specific, avirulent, R forms of pneumococci or with the fixed types themselves, are resistant to infection with a strain of Type III, which had been sade virulent for rabbits. It has also been subsequently found that immunization with R forms of pneumococci induces active immunity in rabbits against infection with Type I or Type II organisms as well as Type III. Since when R organisms are used for immunization the production of type specific antibodies is excluded, it has been necessary to seek elsewhere for a correct in- terpretation of this form of active immunity. It has seemed possble that the factors of natural resistance previously discussed might play a part in this non-type specific immunity and that the increas- ed resistance of rabbits immunized with R pneumococel might repres- ent an exaltation of the mechanism normally present. Consequently, the work of the past year has been devoted to a study of this form of specific immunity. ‘It seemed of first importance to determine whether the acquired immunity was due to protective substances present in the circulating blood, in the fixed tissucs, or both. Consequently, experiments have been carried out to Getermine wheth- er this form of resistance may be passively transferred to normal animals by means of the blood or serum of resistant rabbits. Prev- Sager ar enn a Oe ee TT ET ee NETS er ae eps SSSR TE OTR COMER ARTE EON FSO co bad fous attorpts to nrotcct ~ice passively by the use of serum of rab- bits immunized with R pneumococci have been uniformly negative. Repetitions of protection tests of this kind using both whole blood and serum in relatively large amounts and varying the time of serun injection with respect to time of infection, have always resulted in failure. However, in contrast to the failure of passive transfer- ence in mice, it has been found that the blood and serum of anti-R rabbits does nassively protect normal rabbits to a marked degree against infection with any of the fixed types of pneumococci. The transference of protection, successfully accomplished by the use of whole citrated blood and by serum, demonstrates that the protective substances are present in the circulating blood. Following the es- tablishment of this fact, it became necessary to determine how mach blood or serum was required to furnish maximum protection; the de- gree of protection in terms of the infecting dose of culture; and the duration of the protection this afforded. It has been found that 15 to 20 cc. of blood or 10 to 15 cc. of serum afford great- est protection and that this amount constantly bestows a solid im- munity against 10,000 lethal doses and in many instances against 100,000 lethal doses of virulent pneumococci of any type. Jt has also been determined that the passive protection afforded by whole blood endures for as long os three weeks after transfusion - long- er periods have not been tested. Experiments planned to define more clearly the nature of the protective substances in the blood of rabbits immunized with R organisms are at present in progress but the results are not as yet complete enough to warrant reporting. The nature of the investiga- Sage 272 0°" tions at present under way include attempts to absorb from immune serum, by means of pneumococcal cells, the active »rinciples; to neutralize them with specific carbohydrates derived from pneumo- cocci; to determine their heat lability; to determine their influ- ence on growth and viability of the organisms jn vitro. It is 4 striking fact that although immunization of rabbits with degraded, avirulent pneumococci stimulates no demonstrable type specific pce ate ER EY antibodies, these animals posses ahigh degree of active immunity pus smperr masses we HE. “REISE ES ieee Te " to virulent pneumococci of all types. Purthermorc, the plood of rabbits so immunized although unable to protect mice, does afford nowy passive protection to normal rabbits. Work 4g being carried on to define more clearly the actual nature of this form of acquired re- sistance which is effective in the absence of type specific anti- bodies and which appears to invotte principles other than those usually associated with type-specific immunity. V. Specific Anaphylaxis with Pneumococcus Polysaccharide. (Dr. Tillett) Previous studies on bacterial anaphylaxis with Pneume- coccus have dealt with the action of cell solutions and autolytic extracts containing unavoidable admixtures of protein and other goluble products which are often primarily toxic, As the result of recent chemical studies, there are now available in highly purified form specific carbohydrate derivates of Pneumococcus which are devoid of toxic properties and free from protein degrad- ation products. In view of the increasing significance of these specific solysaccharides in the processes of immunity, it seemed important to determine whether these chemically purified and pro- tein-free substances isolated from type strains of pneumococci 273 actively participate in the reactions of bacterial anaphylaxis. The results may be briefly summarized as follows: Pneumococeus polysaccharides isolated in »rotein-free form from Pneumococcus Tynes I, II, and III are devoid of the function of inducing active anaphylactic sensitization in guinea digs. This lack of sensitizing power adds further evidence in support of the view that these substances, when chemically purified, are devoid of true antigenic function. The fact, however, that in the dis- sociated form they retain, as haptens, the property of combining specifically with antibacterial antibodies led to atternts to test their capacity to induce anaphylactic shock in passively sensitiz- ed animals. Guinea pigs may be rendered naseively anaphylactic to the epecific carbohydrates with the orecinitating sera of izvmune rabbits. Animals passively sensitized in this manner die in three to four minutes with acute shock following an injection of amounts as small as 0.005 mg. of protein-free polysaccharides of the homol- ogous type. The symptomalogy and pathology of the animals are iden- tical in every way ‘with those of true protein anavhylaxis. The re- actions are type-specific. VI. Are the Specific Precipitable Substances of Pnowrococcus, Hapt ens? (Dr. julianelle)_- | rhis is the title of a paper recently published by Schiemann and Casper of the Koch Institute in which, on the basis of their experimental results thoy answer this question in the neg- ative. They isolated carbohydrate substances from the specific types of pneumococci and purified then by chomicnl methods until they no longer gave the ~rotein color tests. In one instance, by repented purification the matorial was rendered nitrogen-free. They found that mice injected with theso »reparations in repeated srall doses, acquired a considerable degree of active irrunity against in- fection with virwlent pneurococci of the horologous tyne. By reason of the inmr-unity induced, they conclude that the specific nrecipitsdle substances are true antigens and not haptens. These results were apparently so definite, and at the same time so contrary to our own conception of the inrmunological nature of the specific polysaccharides, that confirmation of one or the other noint of view seemed essontial. Fortunately, we had at our disvosal highly purified pre- parations of the specific carbohydrates which by chemical test were lmown to be free of protein and in the case of the Type II and Type III substance to be also nitrogen-free. The Type I substance differs from the other two in containing nitrogen as an appa rently essential component of a nitrogenous sugar compound; despite the occurrence of nitrogen in this preparation it fails to give any of the protein color tests. To test the antigenic and immunizing power of these sub- stances, mice were given five injections intraperitoneally at four day intervals of a solution containing weighed amounts of carbohy- hydrate of each of the three types. The total amounts of substance given the various groups of mice comprising 96 animals in all, rang- ed from 5 mgs. to 0.5 mgs, which in terms of bacterial equivalent represents a large amount and a wide range of dosage. Nine days aftcr the last injection the animals vere infected with graded doses of virulent pnoumococei of the corresponding typos. TPEITY 2750 The results of these exneriments were decisive. Under the conditions employed, repeated injections of the purified, pro- tein-free polysaccharides failed to induce in mice any measurable immunity against infection with virulent pneumococci; the animals died of an overwholming septicemia following an infecting dose as small as 0.000001 cc. of virulent culture. These results, together with the previous failure to de- monstrate the presence of antibodies in the serum of rabbits re- peatedly injected with larger amounts of these substances, and our recent failure to induce active sensitization in guinea pigs after the administration of relatively enormous amounts of those same preparations, convince us of the validity of the views that pneu- mococcus polysaccharides when sufficiently purified are non-anti- genic substances which conform in all respects to the immunologi- cal principles governing haptens in general. VII. Immune Response of Rabbits to intracutaneous Vaccination. (Dr. Julianelle). Rabbits have been- injected intracutaneously at weckly in- tervals with suspension.of heat killed pneumococci and the charact- er of the local reaction, the antibody response and the development of active immunity have been followed over a considerable period in a large series of animals. The injection of the dead bacterial bod- ies into the skin gives rise to a local reaction which increases in size and intensity for the first four or five injections, then grad- vally diminishes but never completely disappears. Accompanying these reactions the rabbits acquire a marked resistance to intra- venous injection of virulent, living organisms; the active immunity PUA inernS UNINC 8 IERIE TS femme he ecrmegat ac 5-5 sialic i erence ee senate feed =: Sprsaele ce LOWES Nay aE acquired in this manner is sufficiently solid and broad to protect the animal against infection vith hetcrologous types of pneumococci. This form of actively acquired immunity, moreover, is built up in tho absence of demonstrable type-specific antibodics; for rarely do rabbits vaccinated by the intracutaneous route, even after prolong- ed treatment, develop detectable traces of type-specific immune sub- stances in their blood. This is the more striking since in the case of pneumococcus, Type I, at least, commarable amounts of heated cells injected intravenously, invariably stimulates the formation of tripe specific antibodies in readily demonstrable quantities. During cu- taneous vaccination, but often only late in the process, the so-cali- ed secondary antiprotein antibodies appear in considerable titre iz the blood. However, antibodies of this variety are unrelated to the type of pneumococci used in immunization and represent the respoise to bacterial protein without references to type specificity. Although animals cutaneously vaccinated are themsclves actively imne to in- fection, their serum, in the absence of type-specific antibodies, fails to confer passive protection on mice against infection with pneumococci. The relation of these results to prohpylactic vaccination, to bacterial allergy and acquired resistance offers an intoresting field for further investigation. VIII. Studies on Oxidation and Reduction by Pnewnococcus (Dr. Dubos) 1) Bacterial Antagonism. The antagonistic action of Pneu- mococcus unor. tho growth of Staphylococcus aurcus was first noted by Alivasotos who recorded his observations without attempting to ex- plain tho ~echanis:, sinco sone of the reasons usually ascribed ade- quately explained the phenomenon he observed. Whenever pneumococci rene Sun and staphylococci are seeded together or the surface of an ascitic agar plate, the latter organisms fail to develop and are oftcn com pletely suppressed in the areas whereas growth of pneumococcus is most abundant. It is known that during aerobic growth, pneumococcus cells combine with oxygen and that as a result of this union there is form- ed a substance, having the reactions of hydrogen perotide, which ac- cumulates in considerable concentration in the medium. The presence of this agent in aerobic cultures has in earlier experiments been shorm to account for many of the phenomena associated with the life and death of the bacterial cell. The formation of methomoglobin, the destruction of the endocéllular herolysin, the inactivation of the cell enzymes, are in ench instance reactions dependent upon ox~ {dation processes, brought about through the action of pneumococcus peroxide. It has now been demonstrnted that the mechanisin of the bacterial antagonism between pneurococci and staphylococci is direct- ly related to the presence and accurmiation of this same peroxide, to the toxic action of which stapltylococcus is particularly suscept - ible. Proof that the mechanism is one of cellular oxidation lies in tho fact, thot these same two species, between which there exists such marked antagonism on aerobic cultivation, grow together in per- fect symbiosis when air is excluded from, the culture or when a cat- alyst is added to the ~edium; that is, under conditions which inhibit the for.ation or prevent the acecurulnation of neroxide. 2) Relation of Oxidatior processes to froth and vinbility. of Preumococcus. In recent exvcriments it has boon found tht the 278 Tes processes of cellular oxidation workedly influences the death rate of pnewsococci during artificial cultivation. Gells suspended in vroth or buffer solution exposed in shallow layers to the action of nir fort: large aounts of peroxide and die” within 24 hours. Tuc nddition to these bacteri2l suspensions of substances, such as yeast extract rhich are known to activate oxidation reactions, increases the forsation of peroxide and lessen tho viability of the cells; while on the other hand, the mere addition of organic or jnorgsnic catalase, in the form of plant tissue or active iron salts, prevents the accumulation of peroxide and correspondingly increases the via- bility of the cells. In the latter instance, the cells are protected fetch almost as effectively as if they were placed under anaerobic condi- ee eee ee tions and the bacteria rerain viable for weeks. Sarkar The influence of oxidation processes upon cell rultiplic.a- tion and growth is being studied particularly with reference to the PEE! Peaeitaan nner effect upon the initiation of grovth in media of different oxidation - meaner ones reduction potentials. IX. Nature and Duration of Inmunity Induced by Inhalation Method. (Dr. Stillman). | Studies on the nature of the immune response of rabbits to repeated inhalations of living pneumococci have been continued. It has already been shown that the serum of rabbits which have been repeatodly eprayed with Type I pneumococci developed in their serum agglutinins and protective antibodies. After the inhnlations of pneumococci are discontimed, the agel utinins rapidly disappeared. The protective antibodies, however, persist for long poriods. The sera of certain rabbits, which survived treatment ond are living 640 days after thoir last exposure to 42 atmospner of sprayed pnev- 7 mococci, still continue to protect mice ngninst infection with vir- ulent pneumococci. In order to compare the nature ond duration of the immune response of rabbits sprayed with living pneumococci with that of ani- mals vaccinated by different routes with dead organisms, 2 gerics of rabbits were injected with graded amounts of heat killed pueumococcus Type I, intravenously, intraperitoneally, intramoscularly, and sub- cutancously. The sera of 80 per cent of the intravenously vaccinated rabbits contained agdutinins and oll showed protective ontibodics. The sera of 60 per cent of the intraperitoneally vaccinated rnovits contained agglutinins and all showed protective antibodies. The sera of 33 per cent of the’ intramuscularly vaccinated rabbits contained agglutinins and 86 per cent also showed protective antibodies. None of the sera of the subcutaneously vaccinated rabbits contained agelu- tinins although protective antibodies were present in 71 per cent. From these experiments it would appear that although there is @ close relationship between the presence of agglutinirs and protect- ive antibodies in a given {mmane serum, they do not run parallel. The duration of immunity following vaccination is also being studied. It ie apparent that the duration of immunity bears a close relationship to the amount of vaccine administered. Whereas, the serum of a rabbit which has received but 1 cc. of vaccine passive- ly protects na mouse against 0.01 cc- of a virulent pneumococcus 10 days following the last vaccination, ofter the lapss of 30 days more, the scrum of this rabbit has lost its capacity to confer passive protection. On the other hand, the sera of rabbits which received Ree ee MaRS ss Sonoran 12 or 15 ce. of vaccine, after m interval of as lone as 570 days, still continue to confer upon mice a high degree of protection. its actively immuniz- he duration of the {mmunity in rabb ed by the inhalation method and by vaccination is being follorec. The immunological response of rabbits sprayed with other g being gatudied. The results, thus far, sec types of pneumococci 4 of immnity induced by to indicate that the character and duration these methods varies with the type of pneumococci used. ng songeagtes rpm (tennneac ee eee aes Publications Millett, W. $., Studies on immunity to pneumococcus mucosus. I. Antibody response of rabbits to. Type III Pneumococcus, Journal Experimental Medicine, 1927, xlv, 713. Tillett, W. S., Studies on immunity to pneumococcus (Type III). If. The infectivity of Type III pneumococcus for rabbits. Journal Experimental Medicine, 1927, xlv, 1093. Tillett, W. S., Studies on immunity to pneumococcus mucosus (Type III) III. Increased resistance to Type III infection in- duced in rabbits by immunization with "R" and "5S" forms of pnevmococcus. Journal Expperimental Medicine, 1927, xlvi, 343. Goebel, Walther F., The soluble specific substance of Friedlindcr's bacillus. IV. On the nature of the hydrolytic products of the specific carbohydrate from Type II. Journal Biological Chemistry, 1927, lxxiv, 619. Heidelberger, M. and Goebel, W. F., The soluble specific substance of pneumococcus. V. On the chemical nature of the aldo- bionic acid from the specific polysaccharide of Tyne III pneumococcus. Journal Biological Chemistry, 1927, lxxiv, 613. Goebel, Walther F. and Avery, 0. T., The soluble specific substance of Friedldnder's bacillus. III. On the isolation and properties of the specific carbohydrates from Types A and C Friedl&nder's bacillus, J-Exp.Med. 1927,xlvi,601. Julianelle, L. A. and Reimann, H. A,, The production of purpura by derivatives of pneumococcus. III. Further studies on the nature of the purpura producing principle. Journal Experimental Medicine, 1927, xlv, 609. Dawson, M.H. and Avery, O.T., Reversion of avirulent "rough" forms of pneumococcus to virulent "Smooth" types. Proc. Soc. Exp. Biol. and Med., 1927, xxiv, 943. Goebel, Yalthor, F., The preparation of hexonic and bionic acids by oxidation of aldoses with barium hyporodite. Journal Biological Chemistry, 1927, ixxii, 809. Goebel, Walther, F., On the oxidation of glucose in alkaline solu- tions of iodine. Journal biological chemistry, 1927, lxxii, 801. 282 In Press n in cultures of Friedldnder's linnelle, L. A.. Bacterial variatio al Medicine. Ju bacillus. Journal Experiment of "R" and "s" forms of pneu- Intraconvertibility Experimental Medicine. Dawson, “M.H. . mococcus. Journal