H Qs " i ‘ 4 A EE MRT Organi ne Aa PRR le, Gosbel, Report_of Dr. Avery with (Drs. Stillman, Tillett, Juliane, Dubos, Francis, Kelley, and Babers. if . . RY Studies on Pneumococeus Infection and Immunity. out I. Further observations on Cutaneous Reactions in Pneumonia with the a Specific Polysaccharides and Proteins of Pneumococcus. iM II, Development of Hoterologous Type-specific Antibodies during Convalescence from Pneumonia. III. Serological Reactions in Pneumonia with a Non-protein Somatic Fraction ("C") of Pneumococcus. IV. Chemo-Immunological Studies on Conjugated Carbohydrate-protein Antigens. 1. Synthetic sugar-protein antigens (Glucose and Galectose) 2. Synthetic polysaccharide~-protein antigens. 3. The non-protein somatic ("C") fraction of Pneumococcus. 4, Demonstration of molecular sizes of type-specific polysaccharides. 5. Starches as haptens. 6. Relation between stereo-isomerism and specificity. Vv. Reactions of Rabbits to Injections of Pneumococci and their Products. 1. Antibody response. 2. Resistance to infection. 3, Reaction at site of infection. 4, Development of ekin reactivity. 5. Development of eye reactivity. 6. Hypersensitiveness to Pneumococcus. VI. Immunity in Mice Induced by the Type-specific Polysaccharides. VII, Immunity Induced in Rabbits by Inhalation of Virulent Pneumococcus. VIII. Antipneumococcus protective Action of Normal Pig Serum. IX. Significance of Oxidation-reduction Processes in Bacterial Cells. X. Decomposition of Specific Polysaccharide of Type IIE Pneumococcus by a Bactorial Enzyme. XI. Publications. il x's of DO ew wD Sine ee eee me le eee ete cee I. Further Observations on Cutaneous Reactions in Pneumonia with Polysaccharides and Proteins of Pneumococcus. (Dr. Tillett and Dr. Francis) . In a recent article observations were reported concerning cutaneous reactions obtained in pneumonia following intradermal in- jections of the type-specific polysaccharides and proteins of Pneu- mococcus. The results may be summarized as follows? During the acute phase of pneumonia no reaction can be produced with either fraction. Following recovery, however, two responses are obtained of entirely different character. The protein reaction, which reaches its height in twenty-four hours and gradually fades away in a few days, is tuberculin-like. It was elicited in most of the convalescents and its presence or absence appeared to bear no rela- tion to the titre of anti-protein precipitins present in the serum of the patient. On the other hand, reactions obtained following injections of the type-specific polysaccharides of Types I, II, and III, pre- sent several significant characteristics. In the first place, it is a particularly interesting fact that these bacterial sugars, protein- free, are capable of causing definite reactions in humans. In the second place, the character of the reaction is unique in that it ap~ pears within a few minutes after injection, is "wheal and erythema" in type and fades away in a few hours. When a positive response was obtained it was always induced by the Polysaccharide homolo- gous in type to that of the infecting organiem. For the polysac- charide reaction to occur it has been found that certain conditions ce Ea ta ES SEES 4 Bs cf are essential, namely, the catient muct have rec vered from ar. infec- tion with the hemalcgous crganism avd circulating tyoe-erecific anti- bcedies must be present. Ir. ecntinuing this study «f cutanerus reacticns, these lat- ter orints have been given s“ecial coneideraticn because of the nre- gible ~ractical anonlicatione. One -f the -ractical »cints is ecn- nected with serum theravy in Tyre I nneurcc’ccus pneumnia. Inecfar as sur exmerience has Erne, it anneare that when a ratient, receiv- ing serum, reacts t< the intraderral injection of the Tyne I carbs- hydrate, it reans that reccevery has -ccurred and n° were serur is indicated. This crincinle has been frlicwed in f..urteen cases and has >vcen found devendable. . Additicnsl evidence of the reliability -f the test as an index -f rec very is found in the Tyne I cases which, theugh treated with serum, died, There have oeen four in- stances ef this kind. Althcugh sufficient seruy was given to rain- tain an excees cf antibedies in the bleed -f these natients, end althcugh in twe cages the terocrature was markedly reduced, at no time was a nositive cuteneous reaction oreduced by the Type I car- bohydrate. In four casos “f Tyne I nncumenia, in which no serum -was given, 4 oreitive test was obtained ccincident with recovery. Frem the observations vade n individuels ill vith Tyne I pnoume- erecus oneumenia, it eeems juetifiable tc eenclude that skin tests with the Type I oelysaccharide are canable of furnishing inferma- tion of vractical significance. In cases of Tyne II and Type III pneurnc: ccus infection, the results “f skin tests with homelegceus cerbchvdrate sh: w that <9 about fifty ner cent -f the cascs react. The ace noanying table surmarizes the results. The fact that se jadividuals rec vered frem Tyoe II or Tyyve ITI vneumec’ ccus infecticn failed t” roret in- tradermally, although they n-ssesscd circulating tyne-svccific an- tibsedies, indicates that all of the facters invelved are net yet understood. Investigation «f these ernditicns is being ec ntinued. ; ityoo 1 Tyne 11 ‘Tyre JIL, Group JV "Tyne ITA ine of gascs ree vered 18 17 9 : 413i 2 Ine. nf cases ree-~vered j . . giving positive reacticn } 18 } 19 4 4 oy 9 Fer cont -f ongsitive \ : : reacticns ‘100 __: 58.8} __44.4 , —_ Q Ne. of oneeg nt reacting | 4 4, 8 74 14 5 Per cent fetal 100.1 .12.5 | 28.5 | 7. 9! Fer cent reervered \ ‘ : | | cases nct reacting o | a7.5 | 62.5 | 92.9 ' 100 Il. The Develewrent cf Heter-lorous Tyne-snecific An cdic (Dr. Tillett and Dr. Francis) It ie well known thot in oncumenia, enecific antibdedies rake their annearance at crisis, tac time at which the onlysaccharide skin test ie first dercnatrable. In the cvurec cf cur cbservoeticns, it has becn the custem to teet the vatients et rejoated intervols during the dieease and ecnvaleecence. Recently it hag been actcd that about two weeks after reervery 3 mationt may react te a noly- saccharide, heterclegeus in tyme to the oneumccecue cr2using the disease. Tests .f the serum at the time of such a resnense have revealed the oresonce -f snecific antibedics reactive nt only with the vrimary causative orgoniasr but aleo with oneumcescel corresynend- ing tyoe to the ‘newt anecific antibedier. The curse rf events is j (2041. HS more clearly demenetrated by the scecmanrving table: Patient: Rocche. Orgarnierm derived frem sputum cs adriseicn: Trpe Til. | Nurber c.f days aftcr crisis Type-soecific Agelutinins | Crisis 2 4 6 8 10 2 14 16 18-20 — 22 284 Tyne I - - - 7 Hebi T:-96e_ IT - - - Adofofe ttt | Tye Il] 1 tt ttt tet eet teh This patient euffered free Tyne III oneun ec ccus pacurcnin. At crisis her scrum sh-ved cnly Tyne III agglutinins. Sixtecn days later she orercascd dem natrabdle antibedics agninet Tyoo IT pneum erc- ci ng well ae Tyne IIT. Trenty-f-ur days after rec very there was resent in her serum agglutinins and sassive or’ tective antibodies for each »f the three fixed tyves. Since observations «f this character were firet made, sever- teen patients have been fre- civitated by centrifugation end the water clear suvernatant fluid is removed and rendered scutral by the additier. ef the roccF erourt of normal NaOH. This fluid contains Fraction "C". That Fractie. "Cc" is not a tyne-srecific carbohydrate is indicated by the fact that it is derived from nen-tyne enecific R strains of Pneumococcus; that it is not nucleo-vrotein is indicated by the fact that boiling the ma- terial with acid removes vrotein to such an cxtent that the superna- tant fluid gives nene of the usual tests for vrotein. Although fin- al vroof as to its oxact nature awaits chemical analysis, neverthe- less, convincing evidence of the scvarate identity of Fraction "Cc" is brought out by scrological reactions. Sera obtained at frequent intervals fror vatients acutely {ll with or convalescent fror oneumonia have been mixed with varying dilutions of Fraction "C" and the -resence or absence of precipita- tion noted. It had been found that serum derived fror a patient during the acute stage of preuronia possesses & high titre of pre- cinitins for Fraction "C". A day or two after recovery this pro- cipitating power abruptly and verranetly disappears. The sera ef 50 patients have been tested at frequent intervals fror adrission to the hosvitsl pntil several monthe after recovery. In every in- stance, the bload obtained on admission has furnished serum capable of precipitating Froction "C" in high titre. This has been truc even of oatients in the first 24 to 36 hours after the onset of the infectien. Individuals who have succumbed to pneumonia have main- tained anti-"C" precipitins until exodus. In an individual i11 with pneumonia, not only is the sudden anvearance of the reactivity styiking, but the ranid disspnearance of the vhenoreron coincident Te as ee anos apg with recovery is distinctive. A fow days after the critical fall in ternerature, the patient's serum fails commletcly to precivitate Fraction "C". The nhenomemon may be further characterized by the fact thot it is unrelatcd to the tyne of Preumococcus causing irfec- tion. The curve of the precipitin titre of Frection "C" is distinct- ly differert from that obtained by the use of cither the tync-specific carbehydrate or the rucleo-protein fractions. With sera obtsincd at frequent intervals during the course of vneumonia and tested with pneu- roceccus rucleo-nrotein, tyne-svecific carbohydrate, and Fraction wot, three distinct curves of vrecioitin content may be demorstrated. An- tiprotein antibodies do not vary msarkedlv during the course of nneu- monia. Type-svecific antibodies are absert during the acute stage, apnear at about the tire of crisis, ard are homologous to the type of the infecting organism. On the other hand, anti-"C" precipitins are highest during the acute phase of the disease, disapoear just af- ter the crisis, and are not related to tyne-svecificity. The renort eo far has been limited to 2 oresentotion of results ebtained with patients suffering frox pneumococcus infcctiox. Pationts having pneumonia duc to herolytic streptococcus 4s well as individuals acutcly ill with other febrile diseases have been avail- able for cormarison. Patients euffering fro the following acute disenses have been studied: mensles, chicken-nox, ocute rheuratic fever, osteoryelitis (stwohylococeus) valaria, tyohoid fever, tuber- culosis, acute gonorrhea, and fevers of unkmown origin. Of this group the vatients afflicted with hemolytic streptococcus pneumonias, acute rheumatic fever and staphylococcus osteomyelitis, have pos- sessed anti-"C" »recinitins in their serur when bled during vericds of acute infectien. Tests “ade with seruy frov the other cases have given entirely negative results. Tnrovgh te courtesy cf Dr. Swift ecra evtrined at frecvent intervals froxy fifteen cases of rreuwvatic jyever nave been availatle. Siiuce, in this disease, tnuere ere re- larses, these cares neve furnished instances of inter ittent feorile ane afebrile states. Sv tertine cera sbtrines frov sucn cases it ‘nae seen found thet vreci itins for torection CY are iresent during %er- jode of fever but adsent during revissions, These observatiens “ade vith sera fro engses other then saeu-ococeus infection indicate thet the reaction is not snecific for yneumococeus ~neuronia. It smerrs, however, to be livited to diseases associated with rraé yogitive eneci. The significance of the serological reactien which has been described is not yet clear. However, its unusval characteris- tice both as to time of an vearance ard ranid disapnearance follow- ing recovery, indicate that an unceretending of the factors concern- ed in the »roduction of this reaction may throw ad@itional light on sone of the vrobleme of acute bacterial infection. IV. Chemo I-vmnological Studies on Conjugated Carbohydérate-Froteiss. (Dr. Goebel) 1. Syuthetic Carvonycrate (Hexose) Frotein Antigens:- In the last renort the synthesis of tre P-arinovhenol slucosides of glucose and galactose ras Gescribded. It has since ‘Seen found tnat when these two carbohydrate cerivatives, which Giffer froy one an- other only in the ssecial configuration of the fourth caroon atom Regie mag Sr coe Stati: of the sugar, 2re beund to >rotcin, these ~rotcin-suger comnlexcs say function as cxecllent antigens. It has been found, furthermore, that when these suger de- rivatives are bound to the same protein, they exhibit distinct im- monological ssccificity. If, on the ethor hand, ths same carbohy- drate radical is conjugated vith tro chemically different and scro- logically distinct protcins both of the sugar »rotcins thus formed acquire a commen scrolegical svecificity. The nevly acquired s%c- cificity of theses artificially orepared suger nroteins is determin- ed by the chericnl] constitution of the carbohydrate radical attached to the nrotcin radical. Thus, simple differences in the rolecular configuration of a hexose suffices to oricnt vrotecin snecificity vhen the corresvonding glucosides of the sugars are coupled to the same vrdtein. The unconjugated glucosides, though themselves not ‘yrocinitable in homologous immune scrum, snocifically inhibit the reaction between the homologous sugar orotein and its specific an- tibody. @uinca cige have been naseivoly sensitized with the serum of rabbits imrunized with theso synthetic sugar proteins. It has bron found thet they exhibit tyoical anaphylactic stock rhen subse quently inoculated vith the homologous sugar combined with » vrotein different from that used in immunizing the rabbit. Tho reactions nro in each instance svecific snd devend for their soccificity on the carbohydrate radical and not on the orotein molecule of the s:n- thesizod compound. The unconjugrted glucoeides, though in themselves incan- able of inducing shock, inhibit the anaphylactic reaction “her in- 207 H 1 joeted immediately vrior to tie iztroduction o* the toxogerie eug3r- orotein. In order to alicit the “Aenorcion, tis carbohydrate must be the gave as thet combined in the eugar-orotcin complex. Tae, for the firet timo, it has becn shown by direct cxmeri- mental oviderce thet rasyretry of the carbon atoms in the sugar rad- ical suffices to detercine differerecs in tne enceificity of sugar- protein antigens. 2. Synthetic Polvsnccharide - Frotcin antigens. For the eAako of carrvine this corcentior 1..to the roalm of bactcrial noly- saccharides, where here again it is belicved that e ecificity isa de- pendent uoon the arrangement of the atoms and roleculcs whick go to build un the complex nolysacchtridc, it Tas thought vossibdle to com='° * pime them with foreign vrotcia, ard thus to ronder ther antigenic, ard to clicit an antibody reesvonse similar to that obteined by immun- izetior with the oneaneulated bacterin] bodics themaclves. Tic Trme III Fneumococcue eoluble specific nolysacchsride was choren for this study. By condensing this corpound with »-ritro benzyl bromide au ether of the soluble eubetance roe obteined woich, on reduction 7rith sodium hydrosulghite yielded the p-arino benzyl ether. When thir compound was d@iazotized and then added to an a1- kaline solution of protein (scrum globulin), the two substances com- binee. By chemicel manipulation it vas rosrible to separate the ui- changed orotein from the soluble cornlex and thus to obtnin a corju- gated carbohydrate-protzin containing neither wbourd srotein, nor wibound snecific nolyraccharide. The chemical rosctions may be re- oresented schematically thus: 208 H 12 a, ‘ : : / | at Na s 0 --- ' --- OF + Br CHa \_* Mg No.OH° --~ ' --- 0 - CHo\ / NOy SBgvat a> Soluble Substance v-nitrobenzyl . n-nitro benzyl cther bromide , of the Soluble Subst2nce ‘ | “7 | I. _o-- C --- .. CHA’ ‘(NE H6l_. .. --- © --- CHA\ /N=NeC1 + Protein 2 2 WHO > | Newt | 2 | n-aminober zyl ethor of Ainzoniur derivative Soluble Substence of p-eminobonzyl cther of scluble eubstar.ce --- GC --- CH, --4 \- N= N—1- Protein ? | ~/ ; Soluble Substance ecrum globulin Thc nrotein-carbohydrate complex tus obtrined “1s founc to be insoluble iz the vxresence of dilute minernl acid, but soluble in dilute nlkali. It contaiced about 10 ver cent of bound c*rbo- hydrate. It reacted snecifically vith »rtipscumococcus serum Tyne III in dilution of 1:500;0C0. This conjugated exrbohydrate-vrotein has been used as an imrunizing sgent end it his been found that suc- cessive daily doeor of 2 wes. of sntigen givon for fivo days, suffices to elicit a snecific antibody resvonse. The sera of rabbits thus immunized, orecivitate both the homologous antiger, and the arino benzyl ether of the Tyne III wneumococcus svecific polysrechsride in high dilutione, agelutinate seccifically Tyo> IIT oreumococci, and confer naseive »rotectior on mice agsinet infection with viru- lent oneurocecei of the horologous tyne. Thin Tyne I soluble enccific eubstonee of Paournccecus is an amoholite containing both free wrire wt fre> cnirboxyl groups. It has been found thet if an clknline aqueous sclution of thie polysac- cheride is shokern vith » solution of »-nitro benzoyl chloride in benzene, a acrivative of this volveacchoride is formed, co:teining nitrobenzorl grouse covering the arino grouns. Tris derivative is specificahly;revctive rith Type I nntipneumococcus serum in dilutions of 1:5,000,)00. When reduced thie derivative yields ar amine which can be diazotized and covvled to vrotcins. The chemics] rerctions may be reprerentcd thus: veces ¢ --- Me + Cito. NO, ——y:. --- C --- MH - cox HO, NagS20q | : a oe | Soluble Substance n-nitro benzoyl v-nitrobenzoyl chloride aride of Boluble Subetonce | . “ w= C --- / --- G --- - ON. NeN- oe C NHCO/ NH, Nano, ee C NH co, \NeN Cl + Protein } HC1 \ n-nrino benzoyl Diazorium erlt amide of Soluble Substance. . we -e- OC --- NECO’ ‘\NEN-Protecin (Soluble Subetance) In a similar manner one “ay thus attach the Type I soluble substance of Preumococcue to any:proteiz to yield » conjugated carbo- hydrate-nrotein. Thir derivative vill react with Tyne I antipneuro- coceus serum in dilutions of 1:500,000. Animals are to be immnized with this derivative and the serological findinge will be revorted later. 3. The "C" fraction of Pneumococcus: - When cells of on unencangulateé R strain of Pneumococcus are broken un by freezing and thawing, and the resulting solution ie heated to 100° C. in the presence of 2 slight excoss of neetic acid, the somatic cellular proteins congulate. In the filtrates from such coagula there re- mains a substance, the serolecigceal reactivity of which indicates ite sevarate identity fro” other fractions or .Eu oececus cells. By noreciszitation with mleshol in the >resence 2f inerpl acid, 4 cov sound has “cen isolatcd which gives none of the ueurl crotein reactions. The aterial rotates the plane of nolarized light about 25° to the right. It is not »recizitable by the usual »rotcin re- agents. It gives only © vory faint biuret test. The substance contains abdeut 5 ver cent of nitroren. It yielded 30 “"r cent of reducing eugors, calculatcd as glucose, on hydrolvsis. It is not destroyed bv rolcolvtic onzy~ce. Doc to the @ifficulty in collect- ing workable quantitics, euvficiont data hes not vot bern gathered to charactcrize this nor enccies-enceific substance. It ar-oars, nowever, to ve a xolyerccharidce. Turther investigatior is tcing carried on in order to determine accurately tho chemical nature of Fraction "C". 4, Detervination: of the oiccular size of the soluble (Dr. Goodel vith Dr. Raboers). - Bxnerivents are being carricd out to dotermine the solecular size of the s.cci- fic »olysaccharide of ~newrococeus Tyve III, utilizing as a vethod the rato of diffusion of the carbonvdrate through ~orove nembranes , end determining the imate concentration of diffused polysaccharide coloriretrically. Although thie research has not yet been cormmleted, sufficient data have, however, beon obtsined to indicete that the polysaccharide is of high molecular weight, higher, probably, than most orotein. 5. Starches as haptens:- The fact that the tyne-snecific aolysaccharides of Pneumococcus ‘ay be rendored antigenic by cowbin- ine them with foreign ~rotein hag led us to beliove that common 4. E15 starches can ale be rendered antigenic by combination with »retein carriers. The nitrobenzyl ethers of wotato starch and of corn eterch have decn ore .sred, but unfortunately these derivatives are totally insoluble in water and in the usual cherical solvents. It was thought, therefere, that the introduction of a carboxyl groun into the ring of the condeneaing reagent ritrobenzyl bromide, would render the cor- resconding starch ethers soluble in rater. Coresequently a bro-o ni- trotolvic acid wae evnthetized ir the following -enner fror n-tolui-+ dine: = CH, CH, CE CH | oS BIO. =o) Gud. eS EO. Bra WH HC1 N:NC1 ‘Oh 2 GooH ultra violet light BrcH, BrH, an ENO ‘x a uel Hpst, | 2! Me COOH COOH Brem nitro teluic acid This compound has beon condensed with starch; Lo . a * my _ nee C --- O'H + Br'CH,-_. / COOH 2... --- C --- CH). 8 NO, é ot me No 2 > COOH to yield a starch ether readily soluble in dilute alkali, but insol- uwble in acid. By reducirg the nitro grow) to the arino grou and by diazitizing the latter ard counlineg it with nrotein, 9 conjugated etarch-»rotein derivative has teen vrenared. Inmunization of ani- ~ale with thie conjugated carbonvdrate- rotein derivative are to be carried out to dGetervine whether stsrches vay function ae ha tenes. 6. The relationship between stereo jeorerisr (georetrical - In »eragranh | of this renort it war shown that hexosidee which differ frox each other only in the sati- ne eee iy aeancaalan actrees, LON Seo ny Mec ae ‘3 fond +“ = & + e np al configuration of one asymetric carben ato: elicit different anti- body res‘onses desvite the fact that the chemical configuration of the rerainder of the hexos molecule is in each instance identical. In erder to sscertain whether this nhenortenon is confined to ccmmnounds cortaining only saymetric carben atoms it was decided te study the antigenic res .ense elicited by isomers of the cis-trane tyne. “H = © = COOH | H - C - COOP! Maleric , and ; i fumaric il acids are the classical H - C - COOH: Fooc - C - ¥ —_— exa~nle ef this tyne of #eorerier, and they were chosen to serve as the hantens. In order to cowle these acids to vrotein it becare necessary to esyr.thesize the ~vono nitro aralides of these acids. This has been accormliched by the following synthesis: . E, . H COOH EH co'cl y/ 80, E BO’ |) 30, ~ c i C Ne H C wd (1) ‘| Fol. l. + ---3. ' _C. o~ ¢ Cc. 4O0C “Es cloc 68 BOOC H Furaric acid furaryl chloride + nitro aneline (2) H. _(COOF H. co —_ Cc Cc 2cH. = NO , a: 2 ' P.0, ay i Oo + Wo LG, ====5 F * C004 EF * CO nitroanaline Maleic acid “aleic anhydride Hq _COONF,. NO H COOH . RA . . C {i HOW . i| ¢ . _¢. on ; . . — H COBH , 1 MOQ E CONH ’ a s-nitroanaline salt of n-nitromaleanilic acid no-nitro talesnilic acid 2493 H 17 These tro comounés diffor one frov the other not in struc- ture but in the geo.ctricel rclationshin of their atovws. The iso.- crise can best be understood fro: the following dissrars: ¥ COUR H COOz 1 ‘ NZ 7 ~ / eof ys - ° zt 7 ~ Ny “ N , he ° “Ve Be CORR”, 80, NO, _ 7 SECO £ 5 n-nitro taleantlic acid p-nitro furaranilic acid The nitro grow of thceo two isomcre, which contain no asymetric carbon ators, have been reduced and the corresvonding avincs have beon counled to orotcins. Animale arc to bo immunized with these derivatives to aecortain shotuer georotrical teorcris’ can of - fect srocific antibody resmonsc. V. Reactions of Rabbits to Injections of Fneuzococei and their Froéuctes. (Dr. Julianclic). A study hee tecn wade of the chonece which take ~lece in rabbits followig the rovested introcuts.cour injzetion of sus.en- sions of heat-killed cneowvococci. For the ealo of cov perison, 5: sin- {lar etudy hae boon meds in noreal rebbite and in rabbits trested with pneurococei and their -roducts in various wave: The reactions invcos~ tigated have been (1) the antibody res -onre, (2) resistance to infec- tion, (3) the reactions at the site of injection, (4) the develoorent of skin reactivity £0 derivatives of Pneumococcus, (5) the develo went of eye reactivity, and (6) hynersensitiveness to Pnevrococeus and its -products. Bach of tnese reactions is summarized in the nresent revort. L. The Antibody response. Sixty rabbits were imrunized by the repeated injections into the skin of srall doses of heat-killed »neu- 214 E 18 mococci, Tyne I. In the sera of 53 of the rabbits no tyne-sxecific antibodies were deronstrated, while in the sera of the revwsining anitala, the titre of tv-e-enecific 2atitodies was very lox, Iz a1) cases, however, the cern correrred hich titres of the © ccics- eci- fic antibodice. Forty-five rabbits sirdlerly irzunized by injections of ieat- killed Tyxe III -neusococci -leo friled to form ty e-srccific a.ti- iit bodies, but did form e-scioe-s-ccific pntibodics. “orsover, nest- 1 ti killed sue ensions of R szev-ococet or solutions of the vectcrial ik eubst?zces when injected inte the rkin stiv-ulatced the sroduction of ouly s-ecies-siecific axtibtodics. I]. Resistance to infection. Folloving the intrscutsneous injections of heat-killed S or R mneumococei, revbite sequire a varked degree of resistance to intravenous injections of virblent organises, and thie is true whether the »neurococei injected be of the sare tye or of 2 differert tyxe fror that eroloved for ivrunization. On the other hand, revoated intracutanesue injection of the soluble nrotcins of the cell in not fellowed by an increased resistance to infection. The sera of both resigtant and non-resistant animale, in gcneral, fail to nroteet white mice against infection by organiers of the hovolo- gous ty:e. However, the aera of about 20 ver cent of the rabbits injected intracutaneously with Tyne I Pnewrococcus, rere found to contain varying quantities of »rotective antibdocics. It is secn, therefore, that while the antibody resyonse to the intact celle anc solutiens of the cell is essentially tne sare (i.e. enccies-s-ccific) the acquisition of resistance to infection is characteristic of oly the animale receiving the intact cells, : fy iE ® B 1 wat i<@} III. The reagtion at the Site of Injection. The intracutan- eous injection in nor al rabbits of 0.2 cc. of heated ‘eu ococci, re- voresenting the bacteria from 2 cc. of broth culture is followed by a circumscrided, slightly raised, and indurated nodule, measuring about lcm. in diameter. Upon repeated injection of the sare arount of bac- terial susvension at weekly intervals, the reaction beco-es more in- tense in character and greater in size until 4 to 6 injections have been made, after which the reactions become increasingly “ilder. In the more intense reactions, the size increases to 4-6 cm. in diam- eter, and the ekin is marvedly elevated and of a deen red to surplish hue. The raised areas ir eurrounded by an areole of erytheva ard out- side of this the skin ray be ederatous over a considerable area. Fre- quently necrosis of the ekin occurs with cischarge of a sterile, puru- lent raterial. ‘hen necrosis does not occur, the disaymearance of the lesions ig delayed and the tire required for regression ie related to the intensity of the reaction. This heightened skin reactivity to the bacteria is ~robably devendent uvon some alteration of the tissues themselves since transfers of eerur from highly reactive to norral rabbits does not endow the latter with the proverty of increased ac- tivity. It should be vointed out that freovently a secondary reac- tion ray occur following the disavz>earance of the ».rimary reaction to the first injection. “This recrudescence occure even without a second injection and is vrebably evidence of the develonment of hy- persensitiveness. IV. The develonrent of skin reactivity to derivatives of Pneurococcus. Following a series of intracutaneous injections of heat-killed pneumococci, rabbits acquire an increased skin reactivity (1) to the sveleo rotein of Paev aesnecus » € (2) to an extract of the bacteria fror which the acid-nreciitsble and heat ccagulable vroteing have been revroved. In terzvs of bacterinl snecificity this skin reactivity must be consicered 98 svecies-s~ecific, snd it sa- nears to be related to the »rcsence of circulating s ccics-s"ecific antibcdies. A si-ilar skin revctivity has been »reved to occur in reb- bite followirg the reverted ad-inistration by the intrnvenous °r intracutaneeus route. of the heat-killed bactrris or their -rotein derivatives. The skin reactivity, therefore, occurs in both r-eis- tent and :on-rceisteant onirels. V. The develov:ent of ove renctivity to derivotives of Fneumococcue. It was slso f.vu.d thet evrtci of the reovits be- core cye recetive after receiving intrecutonceve i. joctiour of in- tact celle. If the cornen ie ecarifiod ead tren tre rucleoo-roteia of Fneurococcus ie .laccd ix the co..juictival sec, in about 60 scr cent of the rabbits an cye reaction ao ears within 24 houre ad then increasos in intoneity for varying 3criods, The resctioz con- sists of congestion of the conjunctiva and the ogsearance of éilet- ed canillaries at tie sclero-correal margin. In fore rabbita, the cornea is also involved and there igs the develouront of turbidity and, less frequently, the forrmetion of ® oennus. The cye reaction. algo is e.ccies-sneeific. It has been found thet not infrequently, the intravenous injection of nucleonrotein, after all evidence of the eye reactios have cormletely dieawmeared, may cause the rean- nearance of the ove reaction. In cortradisctinction to the skin reaction to nrotein, the eye reactions do not occur in rabbits fol- x Lorine intravenove i ‘wmization wit? the ictact cell, or following iv- “unization by eny route with solutions «f roteine of tne cell. So tiet while svin resctiozs occur in -or-resistent acd resistant ani - ‘ale alike, the eye reaction, oF the otuer hendc, occurs only i. toe resistant rabvits. V1. yneresensitiveners t Fneu_ oc accus avd its derivetives. There were certain reasons for believing thot the ekin resctivity to the bacterial orotein resembles the Arthus reaction, while the eye reactivity seeve to denend won sovTe factors which are different fro~ those o-erative in tbe skin reactivity. It see-od advisable, there- fare, to study simultaneously the Arthus reaction to egg albumin, the develenment of increased skin reactivity to the vacteria therselves and to the -rotein of Pnewrococcue, and the develo vent of eye reac- tivity. A sum-ory of thie corvarative study is ‘srresented in the ac- corpanying table. (See Table I). The imvortont noints in thie table not already ‘ventioned are (1) that the skin resctivity to mneurococcus srotein can be tranefcrred by serum fro~ 9 reactive to a norval rab- bit; (2) on the other hand, the eye reactivity cannot ve thue trans- t sears to oe ax. exan le ferred. In other words, the ekin reactivity 2% of the Arthus reaction to a pacterial protein, while the eve reactiv- ity ar nears to be a soecial tyne of gensitivity. — - } V1. Active Immunity to Pnevmococcus Infection Followias Injections of the Soluble Svecific Substance. (Dr. Julianelle). Caspar and Schiemann originally called attention to the en- tigenicity of the Soluble Snecific Subetance of Pneumococcus. In a more recent corvunicstion, Schierann repeated the study of the immun- Table ot xX Reactions of Hypersensitiveness in Rabbits Q) _ Follawing repeated irjections Hollowing repeated injections Following repeated injections of Teactiors L_ of _ege albumin of Pneumococcus protein heat-killed pneumococci (Type I Intracutaneous Intravenous | Intracutaneous}| Intravenous|_ Intracutaneous Intrave ous | Reaction lst 3 inject.- lst 3 injec.- lst injed.+ at alte 4th - loth "+ 4th - lOth"+ Increase in of eack intensity until injection 4th-6th injed. Then de crease in reaction Develop~ ment of circulat— ing anti- bodies - (a) type— specific (ov) species specific + + + Active Resistance + Increased sensitivity (a) skin + + + (o) eye - - + Transfer of] sensitivity (a) skin + + + (bo) eye _ = | - = + indicates presence of reaction — indicates absence of reaction ity cf mice to ~ncu-ococcus infecti:n folloving injections of the cor- bohvdrate derived from Pneusococcus, Tyne II. He roiated out thet the imrunity develoned only when very s~»ll quentities of the “clvencchor- ides are injiceted ond the reeulting ivmunity is tye-s .ccific. Exverivcnte have been undertaken to detervinc, therefor-, whether the ‘olveaccharidce of the three tyocs of Fneurococcus ere an- tigenic, in the scnso thet they will stitulete on active iswunity; wwe if so, what quantity of the polysaccharides 1s best for thie cursos". It wre foun? thet the crrbohvdratce 4 rivod fro Tyoc I or Tyno ITI, irr-e-sctive of the vethod chosen for injection and of tac quantitics uscd, did net stinulrte in mice nan atate of resistance to Enewnococeus infretion. The toval quantitics of cxarbohydratc studied voried in cach eter fr0° 5.0 to .00005 "2. Whan Tran Il olvenecharide wes emmloved, it vas found that immunity to infection could be indueed in micc waoen avfll quantities of the soluble s>-cific substance were injected. The totnl quontitics injoeted vericd from 5.0 to .0001 rear., but only wrounte of from .005 to .00005 ror. atimulated -ny degree of immunity. The ortimum dosage was .OCl vgn. In the lest inetonce, the ime~unity induced was cornar- able to thst induced by the injection of heat-killed Tyne IT vneuro- cocci. The i-runity, vtoreover, wae highly tuare-asceific. Re obits and guinen vige rere alee injected with diffcrent quantities of Tyne Il -olveeechrride, but it wee not nessible to shor thot cither of these two s.ccice had required ty 2e-svecific ies unity. In conclusion, it vaysbe sid thot with the quantities er- slov ad, no irvunity wos demonstrated following injections in ice of 220 BH 24 Type I or Type III carbohydrates. When injections were «ade with min- ute quantities of Tyve II volvsgaccharide; a definite degree of itpun- ity was demonstrated in tice to infection by orgnniers of the hovolo- gous tyve. Irrunity was not demonstrated in rabbits and fuinea vies, following the injection of Type II soluble soecific substance. VI]. Immunity Induced in Rabbits by Inhalation of Virulent Fneurococci. (Dr. Stillman) Studies nlanned to determine the relation of virulence to susvevtibility of rabbite to infectien ani to determine the nature and character of antibody reenonse following inhalation of live pncu- wococei are eatill in vregrese. Rabbits are suscentible to fatal in- foctions following inhnlation of Ty-e I pneurococci. Tyne II »oneu- mococei whose virulence for rabbits has been increased by aniral sassage, and by certain naturallyrabbit virulent strains of Tyre II nneumococci. Rabbits are not subject to fatal infection following snraying with the usual strains of Type II pneumococcus, or with the rajority of straine of Tyre III oneurococcus. The results of the in- fectione following inhalation of oneumocoeci vary in direct oropor- tion to the virulence for rabbits of the strain used. Sore rabbits will even recover from a transient septicemia due to virulent Type I or Tyve II pneumococci, but in the case of rabbits exposed to in- halation of virulent Tyne III pneurococci, if the blood is once in- vaded, a fatal septicevia always ensues. Following inhalation of Tyne I oneumococci, agglutinins and orotective antibodies are ant to annear in the rabvit's sere. gape ous ae we OE? of. E 25 After spraying with a strain of Tyme II pneurococcus which has been rendered rough and avirulent, neither of these antibodies occur. If rabbits are reveatedly sprayed with a slightly virulent nneurococcus Tyne II S. vrotective antibodies only a nnear in the blood. If a rab- bit virulent Tyne II pneurococcus, however, is used for inhalation both agelutinins and protective bodies way develop. Fallowing inha- lation of either a non-virulent or a highly rabbit virulent strain of Type III pnewrococci, neither agglutinins nor srotective antibod- ies anoear in the rabbit's pleod. The sera of 2 rabbits which have survived treatvent and are now living, al-est 4 years (1310 and 1360) days after their last ex- nogure to Tyne I ~neu-ococci, etill vrotect vice against infection of large doses of a virulent culture of the sare type as that with which they were originally sprayed. VIII. Antinnew-ococcus Protective Action of Norval Pig Serur. (Dr. Kelley) Bull and MéKee have reported experiments in which “ice and gaoinea pigs were protected against Tany times the lethal dose of pneurococci by the injection of normal chicken serum. It apocared that this vrotective action of the chicken serum ras associated rith the serum globulin. In studying ~hagocytosis of nneurococci by eerut-loucocyte ~ixtures Robertson snd Sia ebeerved 2 rarked ovronie activity of the sera of naturally resistant anivrals. Thie was found to be egnocirl- ly characteristic of the serum of the “1g. Sia observed that nor.al pig scrury would also confer on “ice 9. romarkable degree of -sassive nrotection against pneurococcus infection. In cases of Type I and II Taowrococeus, rotection arsinet 10,000 **t#l doses was obtsinsé. He aleo deronstrated by absormtion ex-oriments thet this nrotective action was sorecifie for etch ty xe of Fnewnococcus. At variance with the vicely seerpted ecllular theory of natural imcunity, hore sre in- etances of a naturally occurring huroral defense nechenies. Too isp “yan aroverties of the serum arr ~acsivelv transferable and 7 °7Fr to be tyne-enecific in action. The stuev in -rogrees is an etter ot to roncat tis rork of Sin and to further anelyze the ~cechanis™ of tho “roteetive action of nig ecrun, The ecrum is obtained from the blood of uor 71 jigs col- lected at slaughter, and is sterilized by Berkfold filtretion. Aa found by Sin, the frosh scrum sivon intrescoritor:c il nmrotects mice recularly againet infection with 1,000 to 10,000 zatrl doscs of Tyne I and Tyoe II pneurococci. Ti- aceren of “rotcction varios somerhst with the individual lote of scru-. Sith the sora so far studied the greatest degree of resistonec to infection hae ven conferred on ~ice against infection vith Pnrumococeue Tyne II. Thuis protective capacity diminishes gradually on standing so tant in the lots tested it was commlotely loet after 6 to 12 weeks. There is % suggestion thet the »srotective srovoerty is conssrved by storing the scrum at a low temperature ond by covering it with a vascline seal, Toether the nrotective powers of th~ serum agrinst all the aifforent tyes of nncunococci cdisanmear simultaneously has not yet bern deter- ~ined. eating the serum at 60-62°C. dacetrove sll, or very nearly all, of its nrotcetive action. Unéer the same conditions ontioneu- macoccus horse serur snows £0 anarecinble loss of »rotoctive “ower. The addition of 10 per cent untreated “ig scrun, yreviously inacti- vated by honting, feils to rostore tho nrotective action. 203 om - & The ontimal protective dose of the nig serur seers to lie between 0.75 cc. and lec. As Sia revorted, the greatest degree of mrotection to mice is afforded when the serum is injected intraner- itoneally 4 hours before the infecting dose of Pneunococcus culture is given. The nrotection, though quite distinct, is not of the sv-e aegree when the seruz is given sirultancously with the culture. The observations that the -rotective factor is associated vith the globulin fraction of the nig serum has been €onfiried. No aporeciable amouht of thie capacity seems to be lost in the »rocess of senaration of the globulin oy xrecinitation with armoniu: sul shate. In our exnrerivents go far, eabsaoration with virulent oneuto- cocei of a given tyve has removed the oroperty of -rotecting mice against infection with the horologour ty%e. In addition, the degree of vrotection afforded by the absorbed serum against heterologous types of wnewrococci ie decreased. A surprisingly few pneumococci are required for the absorption. The time mecessary is likewiee short. There is a suggestion that prolonged absorotion with a large number of virulent or avirulent pneumococci may commletely deprive the serum of ite vower to vrotect mice against infection with either homologous or heterologous tyves of Fneumococecus. Preliminary studies in which Fneumococeus volysaccharide was added to- the serur 24 hours before the protection tests were made indicate thet the seru™ hae not been alter- ed in ite ~rotective nower by this treat~ent. Agglutination and protection tests have been wade with a seri which protects rice against 10,000 lethal doses of Tyve I or Type II yneurococci. Desnite its nrotective action, the serum failed to cause agglutination of Tyoe I or Type II pneumococcus. The precip- itin tests, using the vneumococcus ‘nolysaccharides derived from Type I 224 H 28 rie Tl “nevrocecci, neve likewiec be n ncogstive. So far, atternts to degradsc a virulent § strain of Pnucumococcus - an avirulent R form by growth in oig scrum have not ceon successful. yrocoeei sensitized by tho scrum show no loss of virulence. It Kas boen learned Thether or not eensitizetion of pneuroeseci in if scT- nas aav effect on their agflutinability in antinnounococcus horse scrur. In view of the fact that certein substarcss, of theveclves not Lisenie, Then injected intravenously into anivals along with vig seru~ , stimulate antibody resnronsc, it is thought desirsble to lcarn if such 'Shlenne" action “ay be obtained in the case of anszw-ococtus -olyenc- ariaes. Rabbits are being j-munized wita mixtures of cig seru: and the Jjysaccharide of Tyoe II »neuvococcus to detorine whether, under these nditions, the non-antigenic sugars “Ay etivulate the forvation of type- socific antibodies. 1X. Significance of Oxidstion-rcduction Fhenomena in the Bacterial Cell. (Dr. Dubos) 1. The role of ventone and silucose in the initiation of growth of _Pneumocogei: = Preliminary analyses of the oxidsation-reduction system of the Fneumococcus cell, and ef the oxidation-reduction characteristics of sterile pacteriolozical redia, have veen diecussed in a arevious renort. On the basis of the data obtained and of certain growth exver- iments in media treated in various ways, it was suggested that: (a) the growth of Pnewrococcus is conditioned by the existence in the ~edium ‘of a certain condition of reduction, (bv) there sre present in ordinary Te- dia certain products of oxidation which have & bacteriostatic action on Fneumococcus. It was found that this bacteriostatic action can be over- cove by various methods; adcition of reducing substances, incubation 225 EH 29 ynéer anaerobic conditions, addition of blood, heating the broth ore- vious to inoculstion avd ov usinc > large inoculum. Tre work of the ast year has confirced a..d extecded these observetions. It annears that, when solutions of neotones are ke vt vader aerobic conditions, tney becove pacteriostatic for Freurococcus. This bacteriostatic nower does “ot develon wnen pertone solutions are kent under vaseline seal. The vacteriostatic vower variee with daif- ferent nentones; it is for instance, 3 to 4 tines greater with Fair- child's than with Witte's vevtone. The bacteriostatic action of the ventene solutions tay be vrevented by the adéition of emall amounts of reduced thio-acids, by heating in the orescence of glucose, by the adcGition of heated glucose, or by incubatien in the »resence of glu- cose under anagrobic conditiens. Studies concerning the ~echanism of the action of glucose are still in vorogress, they seem to indicate, that under the condi- tions of eur experiments, the reducing nroperties of the glucose so- lution are much increased, as a reeult of a rearrangement of the glu- cose molecule or the formation of new substances fror it. These experiments indicate that the beneficial action of heating vroth previous to jnoculation with anaerobes is due vot only to the sechanical reroval of the oxygen in solution but also to the action of the glucose on the peotone. | 2, The role of carbohydrate in biological oxidations and reductions. Uxoeriments with Pneumococeus. We have been that a large inoculum of Pneumococcus culture can overcome the vacteriostatic action of pentones. This can be accounted for. by the actively reduc- ing system which is formed when Pneumococcus cells are placed in the 2226 H 30 sresence of vlain broth. An enalysis of this sytem has given tho fol- lowing results. The washed colls of Pneumococcus are able to reduce the var- ious indicators of oxidation-reduction potentials in the “resence of glucose. Oxidized thiol commounds (glutathione, cystine, oxidized thioglycollic acid) are likewise ranidly reduced by glucose in the ‘resence of washed cells of Pneurococcus. The Pnewrococcus-glucose system is able to form neroxide under aerobic conditions. Thoee eubetances *hich form yeroxide in the »resence of Pneurmococcus cells are also the ones which are active in changing hemoglobin into methemoglobin under the same conditions. The vower of washed cells of Pneurococcur to reduce methylene blue in the »resence of glucose is dependent on at least 2 constituents of the cell. One of these can be readily removed from the cell by wash- ing. The other ie removed or inactivated much more slowly by the proc- ese of washing and is destroyed by heating for 10 minutes at 55° C. These observations indicate that the expression “reducing oower of bacterial culture" rust be used cautiously, since this reduc- ing nower is dependent not only on the nature of the bacterial species, but algo on the »resence of definite metabolites. They also indicate that the cell-glucose system can act as a reducing agent, which can correct the bacteriostatic action of the nev- tone, thus germitting the growth of Pneurococcus even in an unfavorable redium, provided the {noculur be large. 3, The role of oxidation-reduction processes in bacterial veriation. A culture of Type III Pnewrococeus, maintained at 39° C. ee in media containing 5 ver cent beef serum, or 5 per cent horse plasma, 2, wey PA has been observed to undergo dissodation, most of the orgaisms cheag- ing to R forms after 2 weeks incubation. The sarc culture, in tue same mediur, at the sarc tommerature, but under vesecline ecal, romsain- ed unchanged (100 ver cent S$) after the same length of tinc. On several occasions, we have becn able to revert a R ecul- ture derived from Tyve III Pneurococcus, to a tynvical, eneaesulatcod. and virulent $ culture on reveated transfore in th- following ~} XII. Public-tions. 229 T.F., Choro-isvunologicsl studics ox conju- gated csrbohydreto-orotcins. IT. Irvunological soecifi- city of evnthctic sugar-protcin antigens. J. Exn. lied., 1929, 50, 521. Daveon, Martin B., The transformation of mneurococcal tyocs. I. The co-vereiorn of R forees of nreurococcus ixtbo § forzs or ta- hozologous tyne. J. Exp. Med, 1930, Sl, 99. M- tprreforration of wreuvococcal tyres. IT. Thre iztorconvertibility of tyte-r-ecific S nacwococci. J. Sx>, Ved., Dubos, Rene, The role of 1930, 5), 123. errbowwdratre in biological oxidatior end reductiovs., txneriverte vith oncurococcus. J. Ux). sede, 1929, 50, 143. Gocdol, W.F., and Avery, O.T., Cno-o-i: usological studics o: co:.- jugated earvohydrets- orotcins. I. The srnthceis of w-asi- nophenol B-glucoside )-2 {nonh-nol B-galactoeidc, and their counlicg rith Zoidalberger, “., Avery, sorur globulin. J, Bx. Mod., 1929, 50, 535. 0.1. 9:4 Goebel, 7.F., A soluble sxccific subetazee dcrived froz gu: arabic. J. Exp. Mod., 1929, 49, Julinnzelle, L.A., Reneti one of rebbite to injectiors of orcusococci end thoir producte. I. The santibody roenorsc. J. Exp. Med. 1930, 51, 441. II. Resistance III. Reretionrs Bl, 463. to infection. J, tm. Mcd. 1930, Bl, 449. at the eit: of injection. 1. "x9. Yed., 1930, IV. The develonzent of ekir renctivity to derivatives of Pueurococcus. J. Bxo, Mod. 1930, SL, 625. V. The develonrent of ~ye renetivity to drrivetives of Pneurococcus. VI. Yyvvereersi J, Exo. Yed. 1930, 51, 633. tiveneas to prowrococci end their nroducte. J, Exo. Med., 1930, Sl, 643. Stillman, Brncet G., Ivmunity rernorac of rabbits inoculated rith Tyne IT Pureuroc immane alcohol oceus by various routes. J. Bx. Med., 1930, and Brench, A., Barly oulronsry lesions in mw rtielly ized rice following inholation of virulent pneumococci. J. Exn. Ved. 1930, 51, 275. Tillett, W.S., Avery, O.T., and Goebel, F.F., Chero-ivmunological atudies on conjugated carbohydrote-proteins. III. Active and wassive an J. Ben. ticde, and Franci avhylaxis with synthetic suger-protcins. 1929, 50, 551. s, T., Jr., Cutrncous reretions to th: poly erccharides -nd »roteins of pnewrococeus in lobar pneuvonia. J Theray MAA ej ° le ors , 192°, 50, 687.