131 Report of Drs. Coie and Avery (assisted by Drs. Dubos, Goebel, Goodner, Horsfail, Hotcnkiss, MacLeod and Stillman) 4. The chemical and specific properties of pneumococcus anti- bodies. &. Antipneusococcus rabbit serun as a tnerapeutic agent in ober pneumonia. 5. Tne autolytic system of pneumococci and its reaction to tine antigenicity of tne ceiis. 4. The action of tissue enzymes upon bacterie. 5. The production of specific enzy.nes by bacteria as a tool in biochemical analysis. 6. The transformation of specific types of pncumococcus. 7. The antigenic mosaic of pnewnococcus - the so-calied Z sub- stance. 8. Comparative virulence of pneumococcus implanted on the mucous membranes of mice by the inhaiation method. 9. Conjugated carbohydrete-protein antigens containing g.ucuronic and galacturonic acids, The chemicas and specific properties of antibodies ana of tne fac- - tors wnicn condition their benaviour, (Goodner and Horsfali) A. The pro-. | tective action of antibodies. Tne work on this subject nas been greatuy stiaulated by the recognition that immune sers from aninals of different species, although produced witn the same antigen and reacting specificalty with tnis antigen, may differ greatly in tneir immunological properties. For instance, the antibodies present in antipneunococcus nurse serum differ in size, solubility, and degree of specificity from tnose in anti- pneumococc~ us rabbit serum. The optimum physical conditions for the interaction be- 146 132 tween antigen and antibody differ for the two kinds of sera and the immune precipitates formed with the specific poiysaccnarides are distinctive in appearance. Evidence has been presented wnicn suggests that the antibody in norse serum is a lecithoprotein, while tnat in immune rabbit serum is possibly a cephaloprotein. Indeed, it has been shown that tnese antisera aiffer in at least thirty respects. An intensive study has been carried out as to the mouse protective value of nine antipneumococcus sera with reference to their apparent anti- body content as determined by in vitro methods. The amounts of "antibody nitrogen" were determined by the method of Heideiberger und Kendall. The mouse protective values were obtained with the method of Kirkbride and Hendry, the results being enalysed according to the Meunch 50 per cent end- point system. From the dats summarized in Tabse T it wiil be noted that the results lend themsesves to an errangement by groups. Tebile I The Protective Vaiues of Antipneumococcus Seru with Reference to Antibody Content Group |Serum | Nature of Serum Protective Specificaily Protective sunite per | precipitabie junits per mg. cc. of nitrogen precipitabse serum mg/ce nitrogen... ..+ I 1 Horse - raw 440° 0,894 550 2 " tt 555 1.02 550 3 " 760 1.50 510 4 " concentrated | 3145 «275 550 II 1 " raw 1000 1.167 860 2 " concentrated | 6100 7.125 855 III | 1 |Rabbit - raw 1070 0.938 1140 2 " " 1830 1.40 1300 3 " " 1910 1.51 1265 247 In each of these groups are inciuded sera wnich have simiiar pro- tective properties with reference to the amount of antibody nitrogen. Tnaus Group I comprises those horse sera whicn snow approximatesy 55U protective units per mg. of syecificasty precipitabie nitrogen. In our experience tne majority of norse seru, botna raw ana concentrated, rais into tnis grou,. The two horse sera of the second group suow 4 restitivesy aign protective index and are to be regarded as exceptionas and aty pica. Tne inmune rabbit sera form a group unto themseives and are charecterizea by uigh protective index. It seems therefore, that the amount of protein precipitated by means of the specific pojysaccharide is not an accurate index of the amount of specific antibody. The amount of protein precipitated is dependent to some extent on physical conditions such as time, temperature, diiution, etc. Furthermore, just as it has been shown that iipids are readily adsorbed by these imnune precipitates, so too is it possible that various proteins, oth- er than the specific antibody, may also be adsorbed. It has been shown taut precipitates frequentiy contain not only the specific entibody but &.s0 other antibodies wnich happen to occur in tne serua. - '.. The higner protective ratios in the case of rubbit sera tre per- tiasiy expiicubse on tne basis that the antibodies in iimune norse serum are several times iurger than those in imaune rabbit serum end cnnsequentiy the proportion of surface to mass is much smcier. The immune sero may contuin mixtures of untibodies of various cap- abilities. This possibility is supported by tne foilowing points: a. From antipneumococcus horse serum there nas been isoisted in a stute of immunological purity, i.e., complete precipitability upon the addition of the capsular polysaccharide, « substance of the neture of u lipo-protein which gives a protective va.ue of 1550 units per mg. of 154 148 specificaily precipituble nitrogen. Tnis materiai contains 4.45 per cent lipid, has the properties of an eugiobuiin ind reucts ripidiy witn the poly- sacchuride in the formation of immune precipitutes. It wiii be noted tnet this protective value is about twice that of the antibody of the best un- purified horse serum and tnree times thut of tne usuai immune horse serum. b. In addition, a second form of antibody hus been obtained. Its precise nature is as yet uncertain because in the form now at hand it is only 92 per cent precipitable. However, the properties of this material are very different from those of the first isoluted entibody. Tnis second substance is water soluble (except ct its isoelectric point), beneves es 4 pseudo-giobulin toward ammonium suiphite, reacts sioway with toe poiysec- charide, in contrast to tne first described antibody, end nes & 1ipid con- tent of approximutery 5 per cent. Moreover its protective quasitics are sow, about 3U0U units per ag. of precipitaubse nitrogen. B. Antipneumococcus rabbit serum us 4 therupeutic agent in iobsr pneumonia. (Horsfail end Goodner) With increased knowieage of the con- trasting properties of antipneumococcus nurse und rabbit sere, it beceme apparent that the latter might prove the more usefui therapeutic ugent in human lobar pneumonia. The indications for its use were as foliows: l. The specific antibodies in antipneunococcus rabbit serum are much smaller than those in antipneumococcus horse serum us judged both by ultrefiltration and by ultracentrifugation. Since tne infective processes of lobar pneumonia and many of its complications ure chirccteristically of en extra-vascular order, it is necessary thet the antibodies pass through the vascular membranes. Other factors constent, tne smeiler the antibody tne nore effective shouid be its penetrstion into the tissues. 2. Under certain circuastences antipneumecoccus nurse serun con- fers no protection to mice. Taus with uwssive infecti.ns, if eacunts of 135 449 serum in excess of a definite optimum are used, the antibodies are in- effective. In proportion to body weight these amounts of serum are not greater than nave occasionaily been administered to ,atients. This prozone effect is not obtained with antipneumococcus rabbit serum. Secondiy, the protective action of imaune norse serua is compietery uborished by the ad- dition of smais amounts of eitner cno.esteros or cepusiin. Tnese sipids do not effect the protective action of tne rabbit wntibouy. It seemed possibie that this situation might be operative in the specific therapy of sober pneunonia in certain types of patients. 5. Immune rabbit sera are mucn more easisy produced. For any given type of pneumococcus an effective serum may be produced in rabbits in as short a period as four weeks wnereas many months ure required with the horse. Furthermore, the cost of producing rabbit serum is much less, (about one-fifth) than that of producing concentrated horse serum. Because raw immune rabbit sera possess high protective potencies, concentration is not so impprtant as in the case of horse serum. Practiceily all rabbits will produce serum of high potency wnile meny horses will not produce effective serum. Since there seemed to be no obvious contraindication to the use of type-specific antipneumococcus rabbit serum in numen lobar pneuaonic, this tnerapeutic agent was first used in those types witn which a cnnsid- erabie experience had been obtained with tne corresponding horse immune serum. Twenty-two patients with lobar pneumoniz have now been treatea. by types these ure divided cs foiiows? Type I, 10 putients; Type II, 4 pa- tients; Type VII, 3 patients; and Type VIII, 5 patients. In tweive of the 22 patients, poyitive blood cultures were wbtained before serum was administered, in seven there was consoiidution of two or more Lobes, und 30-90 in three, pleural exudates containing large numbers of pneumococci were pre- sent. Of these twenty-two patients, twenty-one recovered. In the fatal case, death occurred following rupture of tne aorta five weeks after the onset of the disease. Crisis occurred on an average <6 hours after the institution of serum therapy. With improvements in the techniyue of serum adininistration this time interval has become much shorter, the average for the isast five cases being J nours. The average totai amount of immune rabbit serum aduwinistered, ir- respective of tne type, was i5¥Y cc. per case. It is of interest that the average dosage in tne ten Type I paticnts was 144 cc. wnereus in the iast 437 patients treated with unconcentrated Type I horse serum, tne average dos- age was 440 cc. In the first cases treated, chill reaction not infrequintily followed the administration of rabbit serum. It was found, nowever, that the frequen- cy and severity of these reactions were much less if the serum employed hd been heated to 56° and adsorbed with sterile kaolin. Finally it has been found that the chills may be entirely prevented if acetylsalicylic acid be administered simultaneously with the first injection of this heeted and ad- sorbed serum. On the other hand acetylsalicylic acid is not able to prevent chills following the use of untreuted serum. In no cases have angphylactoid reactions occurred. By combination of the procedures mentioned, it has been possible to entirely eliminate any untoward symptoms in the recent cases. Recovery witnout operation occurred in two patients with grossiy infected pieural exudates, and it was possibie to demonstrate that the fiuid from these cases contained type specific antibody of .abbit origin. In one 137 154 patient, antibody was not demonstrable in the pleural fiuid, and empyema sub- sequently developed. Tnis brief summary of the resuits obtained in a snort series of Severe cuses does not adequately convey the favorable c1inicai impression which the use of entipneumococcus rabbit serum nus created. It is noped tnet additional experience will confirm tne impression now neid taat the empioyment of rabbit serum marks & distinct udvence over tne use of horse serum in tne treatient of lobar pneumonia. The autolytic system of pneumococci. (Dubos) (a) Reiation to the antigenicity of the ceils. Under eppropriete conditions, the immunization of rabbits with suspensions of virulent, encupsuteted pneumococci brings about the production of type specific antibodies directed egainst the capsular poly- saccharides of the organisms used zs antigens. However, it is known that when pneumococci are disintegrated by autolysis, by treutment with bile salts, or by freezing and thawing, they fail to incite rabbits to produce these partic- ular antibodies. It has been found that, to be soluble in bile, or to be liable to disintegration by repeated freezing end thawing, pneumococci, living or dead, must still possess in an active form some, at 1eest, of their auto- dytic enzymes; furthermore, the disintegration cun take pluice only when con- ditions are favorable for enzymatic action. It appeurs therefore that ail methods comaoniy used to achieve a dissoiution of pneusococci invoive as a necessary step the action of at least one or u few of tne enzymes which con- stitute the autoiytic system of the cells. A study nas been wade of tne action of purified preparations of the . autolytic enzymes upon pneusococci and other substrates. It has been found in particular that these enzyme preparations are capable of inactivating the capsular polysaccharide antigens of virulent pneuuococci. To achieve this inactivation, it is not necessary to bring ubout « compiete dissolution of 158 452 tne bacteria ceiis; it is mereiy sufficient to cause a iimited aiteration of the ceiiusar structure sucn tnat tne sutter nas .ost its abisity to re- tain the Gram strain, but stiii retains its essentias morpnoiogicas char- acteristics. The structure wnich carries tne caysular pouysaccnaride anti- gen wnich determines tue Graia positive reaction of tne ceis has been found to be destroyed by the bacterioisytic enzy ie but to be resistant to common proteolytic and lipolytic enzymes. It nus & 1ow nitrogen content (about 8%) and accounts for less than ¥5 per cent of tne originai weignt of the ceil. The bucteriolytic enzyme aiso possesses tne property of releasing reducing sugars from acetyl amino glucose giucuronides of animal and bac- terial origin wnich are otherwise very resistant to enzymatic action. It is hoped that a continued study of the mechanisa of action of bhe bacteriolytic enzyme may reveal information reiuted to tne antigenic structure of pneumococci. But in any case, tne fact tnat tnis enzyme is capable of destroying the type specific antigenicity of tnis group of micro- organisms does empnasize tne importance of minimizing tne cnances of e.ter- ations duc to enzymatic action in tne course of preparation of bacterias antigens. (b) Tne action of tissue enzymes upon bacteria (Dubos) The immunization of rabbits with neat kisled pneumococci by the intraderimas route fuils to incite the production of tne type specific carbohydrate antibodies which would follow immunization by tne intravenous route. Tunis lack of antigenic response suggested the existence in the skin of a mech- anism capable of inactivating the capsular polysaccharide antigen of pneu- mococci. In fact it has been possible to isolate from different tissues and from leucocytes an igent endowed with the foliowing activities; 6) it decomposes yeast nucseic acid without, however, relezssing free phosphoric 139153 acid in solution, b) it attacks heat killed bacteria (pneunococci, strep- tococci, staphylococci) and yeasts, destroying their affinity for the basic dyes but without causing a disintegration of the cellular structure, c) it renders ineffective in rabbits tne type specific antigen of pneumococci. The agent responsible for these different types of action appears to be a protein with enzyme-like properties wnich is rapidly inactivated by pepsin, and more slowly by trypsin. It is very resistant to heat, especially at acid reactions. The resistance to heating is explained by the fact that the agent is denatured at temperutures above 80°C, but thet the denaturation is rapidly reversed on cooling. It appears therefore thet tnere exist in tissues, agents of en enzyme-Like nature which sre next resistant, end capable of attacking and modifying the antigenicity of injected meterials. (c) Tne production of specific enzymes by becteria as a too1 in biochemical analysis. (Dubos) In previous years 4 technique has been des- cribed for the production of a bacterial enzyme capseble of decomposing the capsular polysaccharide of Type III pneumococcus. This enzyme exhibits 4 remarkable specificity since it does not decompose any other polysacchsride tested, not even the capsular polysaccharide of Type VIII pneumococcus which is very closely related to Type III polysaccharide both chemically and sero- logically. The production of: the enzyme also exhibits an interesting char- acter of specificity. Whereas the microorganism which produces it can grow readily end abundantly on all sorts of laboratory media, it fails to produce the specific enzyme except when compelled to utilize the Type III polysacch- aride in the course of its growth. These observations suggested the possibility of developing a method for the production of enzymes specificaily directed aguinst definite 140 454 There are no setisfactory chemicas wetaods for tne aetermination of creatinine in tissue end body fiuids. It wes taerefure of interest to ubtein an enzyme capubse of decomposing this substance. Tu tunis ena, four bucterita specics capebse of uttecking crextinine were obtsinea in pure Cui- ture. Curtures of one of these species ms yiesaced tn enzyie pre pure tion cu, D1 OF Avcomposing creatinine intu ure. ena prubi.bsy sercosine. Tue action of tais enzyme is specific. Of e11 tne otuer pubstrutes tested, ony creatine, giycocyamiuine und 5-wethy. zgiycocyuwidine, substances Civusesy re~- lated to creatinine, cre aiso attucked. The bucteriui culture grows reudisy on peptone soiutions but in- terestingly enougn it produccs the specific enzyme oniy when compelled to utilize creatinine in synthetic medie. Tne situution is taerefore cumper- able to that described for the productiun of the enzynie which decomposes the capsular polysaccheride of Type III pneumoccccus. It appeers therefore that a method is uvciirbse for the producticn of reagents specific for substances of bivlugice.s interest. In cullebore- tion with Dr. Mister, the new enzyne preperition is being empioyed for phys- ivivgicas studies on the presence uf creatinine in tissues end body fuuids, una for a weagure of rena. functicn. The transfurwetion of specific types vf pneulivcsccus. (Mucheod) In the reactiun system used in tne trensfcraution uf pneumucucca.s types in vitro, three compunents are required in addition tu tne broth used 8 culture medium; 1) siving R pneumcecci, 2) the specific transfurming ex- tract prepared from smooth pneumococcel ceils - the becteriel factur, and 3) serwa known to contain cn uccessory factor, serun factor. Further observetions aave been mude en some of tne properties uf the specific or bacterial fectur. It nes beon found tu be extremely subiile 141 155 if expused tu urtra-viciet rignt, but resists vxidution us by tne actin uf judine ur expusure tu atmespneric Gir in & sutsiew sayer £t S74, The spec~ ific agent is mure stubie tu neut at acid resection in 4 suiuticn conteining 50 per cent ethyl aicehul tnan if heated in aquevus sulutivn under the same cunditiuns. Enzyme preperativns kmuw ty be uign in pnusphatase ectivity, such «gs bune phosphatase preperati.ns, very quickiy inuctivete the specific becterial factor, but it is nut neld from the evidence that tne specific agent is necessarily vf the nature of & phusphuric ester, incsmuch 4s such enzyme preparations probubly contain uther enzymes than tne preduminant phosphatase. Further precipitation of bacterial extrects hus been accvn- plished by depruteinizaticn by the use of chioroform end amyl uiconol after tne method uf Sevag, fuilowed by repeated frectivnati.n in ascunsi. How- ever, nu selective means -f isoigting tne specific fraction nus yet been discuvered. Tne third cump nent ef tae reacti.n systeu used in tne trensf -rac- tion uf pneumucuccas types, numeiy tne Lccess-ry fuct.r present in serum, has been mure extensively studica. It nes been suvwn previvussay tnut rabbit serum, normal swine serum, ind numan ascitic ur pieurcl fiuid cuntein the necessary serum factor. However no serum oF fiuia nus yet been fvuund to function in the transfurming system that nas nut ecunteined uggsutinins fir R pneumococci. This fact suggests that the R antibodies are in some wey related to, if not responsible fur the accessory action of serum in the transforming process. When serum is fractionuted the accesscry factor and’ also the antihodies ure contained in the globulin fraction. Tnst complement igs not concemned in the reaction is show by the fact that heeting at 60YC for 30 minutes, which completely inactivates the cumplement, dves nut impair the capacity of serum to functicn 4s accessury substance. Under tnese con- ditiuns tne R entibsdies remain uncffected. Tnese substances and cise tne SER Go Dona: is Ae BAS zs ————— oe > ape i eR oa 4k 456 accessory factor are destroyed wnen serum is neated at 7U°C, a temperature at wnich tne serwn proteins are denatured. Altnough tne presence of R anti- bodies and the uccessory factor of seruo are intimate.iy essociatea, it does not necessarily follow tnat they are identical. Wnise it is true tnat no serum has functioned as accessory substance whicn hus not contained demon- strable R antibodies, the converse of this does not nold true, némeiy, that all sera containing these antibodies necessarily act in the transforming system. It has been of common occurrence, when uifferent sera of equal anti- R titers are tested,to find that one may function and another completeiy fail to act as accessory factor in the reaction of transformation. The evidence so far obtained therefore, indicates that the serum factor necessary in tne transformation reaction is contained in the giobuiin fracti.n, is not identical with comp.enent, and that it is reiativeiy stabse, resisting heating at 60° for 30 minutes. Ali sera, moreover, are not effective in tnis reaction. It nas been found that white sera of patients activesy ill wita pneumonia or other acute infections, may function as tne serum factor in tne transformation, sera from normal persons and persons during convaiescence from pneumonia fail to act, It has furtner been found that normal and convasescent sera not only fail to act, but, ‘when these sera are udded to the becterial trens- forming ugent, they so modify the latter that no transformation occurs even when serum known to contain the accessory factor is added. When normal or conve.lescent serum is heated to 60°C &t an aikaiine reaction, which temperuture does not destroy tne serun fuctor, the heated serum is as effective in promoting the trunsformetion reaction as is serum from acutely ill patients. It is evident, therefore, thut there is something in normal serum which destroys. or prevents the action of tne bacterial fac- tor and that this agent in normal serum :wy be destroyed by neuting at 60° * ‘aime NRE ASN OCT eIRONTNS et ee 143 157 at an alkaline reaction. It seems not improbable that this substance is of the nature of an enzyme. This enzymatic substance, however, my be isoleted from aii human serum, whether normal or serum obtained during the scute phese of pneumonie . Isolation is accomplished by aicohol fractionation of the serum. The iso- lated enzymatic fraction of acute phase serum inactivates the transforming principle &athough the wnole serum from whicn tne fraction was derived does not do so. Ais numan sera therefore, not oniy contain tne uccessory factor necessary for transformation, but they are &1i potentiaily cbse to destroy the specific bacterial factor. The reason that ecute phase serum is active in the transforming process wnile convalescent or normal serum is not, is therefore probably to be expluined by the presence in acute phase serum of an antiferment wnich is absent from normal serum. Studies on the antigenic mosuic of pneumococcus. (Avery, Geobel, and MacLeod). In the course of experiments on the nature of the capsular entigen of Type I pneumococcus, a hitherto unrecognized fraction of the cell has been isoluted. This material, designeted the "Z substance" is tentative- ly regarded as a protein which in meny of its chemicai properties resembles the alconol soiuble globuiins, the so-called protamines, of plant tissue. The Z substance in the present state of purity contains approx- imately 15 per cent nitrogen, % per cent suifur, but no phospnorus. In aqueous solution tnis substance gives the usuai protein color reactions, and is precipitated from soiution by trichioracetic acid, picric, phos pho- stungstic end eulfosalicytic acids; In the form of the nydrochioride it is soluble in 80-90 per cent aicoho. and as the picrate it is so.ubie in 70 per cent acetone. The solubility of 2 in aciduiated alcohol und its property of forming acetone-soluble picrates have been utilized in devising methods for pean ne ne Rn I a i44 158 extrecting the materiel froa the becterisi ceiis. The substance n&s & snavp isoesectric point, precipitating compictesy from so.utions udjusted to pH 4.7. Tno Z substance derived from Type I pneumococci is entigenic in rabbits end gives rise to tne formation of type specific entibodies. The serum of immunized rabbits agglutinutes Type I ce1iis und confers specific protection on mice against infection with viruient orgunisms of the noiuolo- gous type. Interestingly enough, however, tnis imaune serum does not react in precipitin and compiement fixc.tion reactions with Type I capsular poly- saccharide. Further work is in progress on the chemistry and immunology of this particular fraction which appeurs to be concerned in the type specific mechuniam of the cell. Comparative virusence of wneumococci impiintea on the respiratory mucous wembrenes of mice by tne inna1ution inetnod. (Stisiman). In order to determine what importance the viruience of tie infecting orgeunism piuys in tne severity of tne ectuei diseuise, tne resutive invesiveness of different strains of freshiy isotuted pneuinococci nis been tested in mice by the in- najation method. As soon as possibie ufter isoiution of & cuiture from a petient with lobar pneumonia, tne virulence of tne strain ues been tested by spraying 10 mice with the organisms. At tne same time virulence has been determined by injecting mice int“ruperitoneally witn smounts representing 10-5 and 10-6 ec. of the original culture. In the case of the mice exposed in a closed chamber to un atmosphere laden with Types I or II pneumococci, no infection resulted umless the enimeis were intoxicated with aicohol just prior to exposure. It hus been found tnet fresniy isolated Type I pneumo~ cocci have little invasive virulence for mice as tested by impiantation on the mucous membranes by inheletion. Indeed, even wnen injected intreperit- 445 159 oneally in the diiutions used oniy an occasionai strain proved fetai. Type II organisms, on the contrary, were quite invasive wnen tested by in- né.zation end reguigray Kissed iaice wien injected intrauyeritonezsiy. Tne Type III ana VIII streins showed gre.t variations. Aitnouga tney were uni- formy virusent waen tested intraperitonessiy, onay & few otruins were found to invade by way of the mucuus inumbrunes, toe aujority of cuitures fuising entireiy to incite infection when introducea by tais route. The ciinical course of the spuntancous disease in patients is be- ing correlated with the virutence of tne infecting pnewnocecci as tested in mice by the inhelation method. Specificity of conjugated curbouyarate-protein antigens contuining glucuronic and pulacturonic acids. (Goebel and Hutcnkiss). Tne uronic «cid constituent of the encapsulating polysszccaurides of virusent pneumococci appears to be of special significence in aetermining sero1ogicas properties. In certain of tneye type specific subst-nces, giucurunic ecid is fund es an integrait part of the molecuse wnite in others gasecturonic cid formas «€ part of the encupsuieting acterics. It ces of interest tncrefore to cum pare the immumoswgices properties cf urtificinsiy comp undea entigens con- tuining giucurunic und gusucturunic ccids, since tn. sterevcucaicaus config- uration of tnyse twu substunces is known. In tuis senner it snouid be pos- sible tu establish directly tne reiationsnip between tne cneaicas cunstitu- tion of these biologically iaportunt ccias and tue specificity vf the anti- bodies to which they give rise. Vinen rabbits are immunized with azo-protein antigens cunteining glucuronic and gulucturonic acids, tne antibodies elicited ure specific end show no serological crossing. Since tne configurction uf the urunic acid rudical of each antigen differs only by an intercnenge of tne H and OH groups on the fourth carbon atom, the sharply defined differences in inmun.- logical properties wust be directuy uttributabie t. this fundementei differ- ence in chemical ecnfigureticn. 14e 160 Fron a comparison of tne imaunosogicai proyerties of antigens con- taining tne benzy1i glycosides of y.sucose and gaiactose witn antigens con- taining tne nexose uronic acids, it nas now been found tuat each gives rise to antibodies wnich are specific and which show no sero1ogicas crossing. Differences in the immunological properties of these sugar and sugar acid antigens is consequently attributed to differences in polarity of the group- ing occupying the sixth position in each carbohydrate radical as weil as differences in configuration within the sugar molecule. In the giucoside and galactoside molecules this grouping is 4 primary alconol (CHzOH), whereas the polar carboxyl group (COOH) occupies tnis position in the giucuronide and galacturonide. Thus 4 new end important factor must be tuken into con- sideration in understanding tne specificity of simple tarbohydrztes and their uronic acid derivatives, nameiy the posterity of groups in the moecuie. Tne artificial azo-protein antigens contuining gaucuronic and gés- acturonic acids bear a striking iamumo.ogical resutionsnip to tne specific pneumococcus poiysaccnurides, for the antigens, in disutions cs igh as one part in a miiiion, precipitate in entipneuaococcus sera. Tne nexose enti- gens, containing glucose and gulactose, on the other nend show no seroiogicél | activity in these same antisera. It has been found by Dr. Hotchkiss that synthetic high molecuiar polymers bearing carboxyl and hydroxyl groups as substituents upon a Long chain of carbon atoms precipitate specifically in antipneumococcus horse serum Type III. The reaction stili occurs at a ditution 1:300,000 of the polymer, but there is no precipitation even in much nigher concentrations in antipneumococcus serum Type IIT previousiy absorbed with nomoiogous poiy- saccharide or in wabsorbed Types I and II immune sere. The reactive syn- tnetic substunces were prepared by tae poiymerizution of ucrysic acid with ia7 167 vinyl acetate, followed by soponification of the ecetyi groupings. These haptens, in contrast to the azo~-proteins, are entirely synthetic and their chemical constitution is quite wetl understood. The precipitation of uronic «cid azo-protein antigens end of syn- thetic acidic polymers in antiyneumococcus sera is beiieved to be caused by a neutralization of the charge of the basic groups of the antibody molecuse py the acidic group of the entigen. Tnis hypotaesis is in part substantia- ted by the fuct that when the simino groups of pneusococcus antibody. sre aucety- iated, the resu:ting acetyiated protein fuiis to precipitate eny of the ecid- ic antigens. This mecnenism is in purt believed to be tae reaction under- lying tne cowbination of antiboay with nomoiogous type specific pneumococcus polysaccnarides. The specificity of tais phenowenon my possibsy be gov- erned by the arrangement in spece of tne potser groups of tne nuyten mo1ecuie, wnich in turn conaition the spatizl pattern of tne reactive groups of tne antibody molecule. Publications Abernethy, T.J., Concentrated antipneumococcus serum in Type I pneumonia, Control of dosage by skin tests with type specific poly- sacchuride, N.Y. State J. of ifed., 1936, 56, 627 Abernethy, T.J., and Francis, T. Jr., Studies on the somatic "C" poiysacch- aride of pneumococcus I. Cutaneous and serological re- actions in pneumonia, J. Exp. Med., 1937, 65, 59 Abernethy, T.J., Studies on the somatic "C" poly saccnaride of pneumococcus II. The precipitation reaction in animsis with experinen- tally induced pneumococcic infection, J. Exp. ifed. 1Y57, 65, 75 CoLe, Rufue, The treatment of pnewnonie, Ann. Int. wed., 1936, 40, i Coie, Rufus, Pnewaonia - prevention, wanegement end serwn treetment, N.Y. State J. of ded., 1456, 56, 1699 Cole, Rufus, Recent advances in the controi of pneumonia, Am. J. of Pub. Heaith, i956, <6, le wud 62 Dubos, René aid Miller, Be «, Enzyme for decomposition of creatinine and its action on the "apparent creatinine” of blood. Proc. SOCe Exp. Biol. % Med., 1936, 35, 355 Dubos, René, weyer, K., ana Sayth, E.iil., Action of the lytic principle of pneumococcus on certein tissue posy secchariaes. Proc. Soc. Exp. Bios. & Med, 1946, 54, 616 Dubos, René, Meyer, K., and OGuytn, E.M., Tne nydroiysis of the poiysaccher- ide acia of vitreous hunors, of wabilicai cord, and of strep- tococcus by the autolytic enzyme of pneumccoccus. 3. Biot. Cuem., 1937, 118, TL Goebel, Walther F., Chemo-immunological studies on conjugated carbohydrate- proteins. X. The jumunologicai properties of an artificial antigen containing glucuronic acid, J. Exp. Med., 1936, 64, 29 Goebel, W.F., and Reznikoff, Paul, The preparation of ferrous gluconate and its use in the treatment of inypocnromic anemia in rats. J. Phar. & Exp. TFoerapy, 1957, 59, 16¢ : Goouner, K., Horsfall, F.L. & Bauer, J.H., Ultrafiltration of Type I pneu- mococcal sera. Proc. Soc. Exp. Biol. & Wed., 1936, $4, 617 Goodner, K., and Horsfall, F.L., The protective action of Type I antipneu- mococcus serum in mice. IV. The prozone. V. The effect of added lipids on the ppotective mechanism, J. Exp. Med., 1936, 64, 369 and 377 Goodner, K., and Horsfali, F.L., The complement fixation reaction with pneu- mococcus capsular poiy saccharide, J. Exp. ed., 1936, 64, <0L Horsfall, F. L., ana Goodner, K., wipids end jmmunosogical reactions. If. Furtner experiments on the relation of 1tipids to the type Spec- ific reactions of antipnewacococcus sera, J. Jinauno., 1936, 31, 135 Horsfall, F.L., and Goodner, K., Lipids and immuno.ogicas reactions. Ill. Lipid content of specific precipitates frou Type I antipneumoco- ccus sera, J. Exp. ifed., 1936, 64, 585 Horsfall, F.L., and Goodner, K., Lipids and immunological reactions IV. The lipid patterns of specific precipitetes from Type I antipneumo- coccus sera. J. Exp. Meds, 1936, 64, 855 Horsfall, F.L., Goodner, K., and MacLeod, C.M., Type specific antipneumo-— coccus rabbit serum, Science, 1936, 84, 579 Hotchkiss, R.D., and Goebel, W.F., The synthesis of the uldobionic acid of gum acecia. J. Am. Chem. Soc., 1936, 58, 858 Hotchkiss, R.D., and Goebel, W.F., The synthesis of the heptacetyl methyl ester of gentiobiuronic acid, Science, 1936, 83, 553 43° 163 uotehkiss, R.D. and Gvebes, W.F., Derivatives of giucuronic acid. VII. Tne syntaesis vf wsavbicnic ucids, J. Bios. Cueu., 186, 445, «85 Stiitnan, Ernest, Duretion cf demonstrebse antibvuies in the serua or reb- bits imuunizea witn neut Kisica Type II and III pneuncccci, J. Infec, Dis., 1955, 57, Kcd Stillman, Ernest, The reaction of pertisisy imuunized rabbits tu inntsatien uf Type I pneuaecocci, J. Infec. bis., 1955, 57, “£35 Stillmun, E., and Scnuiz, R.A., The reucticn of nurwat and vurtiessy inmu- nized rebbits to intrenausai: instisiation of Type I pnewavcocci J. Infac. Dis., 1455, 57, «58 In Press Dubos, René The autolytic system of pneumococci. Dubus, René Mechanism of the lysis of pneuavcucci by freezing and thew- ing bile and other -gents. Dubus, René The effect uf the bucterivsytic enzyme vf ,neumecoccus upn the untigenicity uf encapsuizted pyneunocucci. dursfall, F.L., Gooaner, K., mecuesd, C.d., end Harris, A.H.gnd., Antipneun- cuccus rabbit serua as a tnerepeutic ugent in icbur pynewanie.