&port of Dr.. _ __.. .- VI --- .-- -. Avery_ with (.W%.- _- _ ____ Stil&nq.nL Till&t, Julianelle,~G-o-e~-e~~ - _ _--._ - ---.-.- I- .s - DJbos. Francis Kel. and Babers. m----L. - _ ..-.- a s-e Studies on Pneumococcus Infection and Immunity. --- --- --- -- - - -- - _ - ------- --- I. Further observations on Cutaneous Reactions in Pneumonia with the Specific Polysaccharides and Proteins of PneumococcusI IT. 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-I~unological Studies on Conjugated Carbohydrate-protein Antigens. 1. Synthetic sugar-protein antigens (Glucose and Calectose) 2. Synthetic polysaccharide-prot ein antigens. 3. The non-protein somatic ("C") fraction of Pnetrmococcus. 4. Demonstration of molecular sizes of type-specific polysaccharides, 5. Starches as haptens. 6. Relation between stereo-isomerism and specificity. v. Reactions of Rabbits to Injections of Pneunococci and their Products. 1. Antibody response* 2. Resistance t 0 inf e&ion. 3. Reaction at site of infection. 4. Development of skin reactivity. 5. Development of eye reactivity. 6. Hypersensitiveneas to Pneumococcue. VI. Immunity in Mice Induced by the Type-specific Polysaccharides. VII, Immunity Induced in Rabbits by Inhalation of Virulent Pneumococcus. VIII.Antipn8umoooccus protsctivs Action of Normal Pig %m.m. IX. Significance of Oxidation-reduction Processes in Bacterial Cells. x. Decomposition of Specific Polysaccharide of Type III Pneumococcus by a Bacterial Enzyme. XI. Publicat ions. I Further Observations on Cutaneous Reactions in 2--.-- ___--_,_-. . .- --.- .._---.-- a - --.. - Pneumonia with Polysaccharides and Proteins of Pneumococcus. -Be- -- (Dr. Till&t 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 followsa During the acute phase of pneumonia no reaction can be produced with either fraction. Following recovery, however, two responses are obtsined 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 obt.;ained following injections of the type-specific polysaccharides of Types I, II, and III, pre- sent several signif icant charact erist its. 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, ie "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 organism. For the polysac- charide reaction to occur it has been found that certain conditions are essential, namely, the ?atiest mutt have rec'vered fr::m ar. ilifec- tion with the hrmolcqus r.rgaziam a,d circulating t,y.Te-e3ecific anti- bc;dies fiust be wezer,t. Ir. c'.ztinuirg thi F Ftudv cf cuta;lerua regctirns, these lat- ter ncints have been given P;-ecial ccneideratipn because <\f the ?PF- sible qr8.ctical aqr)licatinnc!. &.e -f the -7rrctical a>cints is crn- netted with serum therauy in `?pe I qneurcc'ccus pneumfnfa. In9r.f ar as 3ur experience has gene, it ansears that Then a Tatient, receiv- ing semY, reacts tr the intradernal in3ectir.n r?f the Ty?e 1 carb:;- hydrate, it reans that recrvery has ,.ccurred and n mnre serum ia indicated. This rrinci->le has been fr llcwed in f!.urteen cases and has Seen found dqend.able. . Additirnsl evidence of the reliability rf the test 8.8 ax! index rf rec,.vsv is found in the Ty?e I cases which, though treated with sex-~, died, There have been f!jur in- stances rf this kind. Althr.ugh sufficient serum was given to Ipain- tain an excess r,f antibcdiea in the bleed rf those aatients, sBd althmgh in tfC: cases the teF,>crature was markedly redvced, at nn time was a ?Q,sitive cutaneous reactirln qrcduced by the T:rpe I car- . bohydrat e . in fcur co.808 pf P,Te I qncumr.nia, in yhich no s@rum .was given, a Wsitlve test was nbtsincd cnincidpnt aith rcc*ver.!r. Fran! the observatiene vade pn individwls ill rith !Ppe! I ?nnmo- ccccus nneum93ia, it seems juetifiable tc. cep,clude that skin tests lpith thr? Tyne I qnlysaccharidc are canablc of furnishing infcrma- t ion cf vract ical significs.nce, In cases of Tpe II and Ty?e III pcc~`ic: ecus iA?feCtiCn, the results pf skin tests aith hr;mrrlcgcus carbch:!Thrate sh: T that about fifty per cent * f the c~tscs react. The act; t:+m:ring t&l:: surraarizcs the results. The fpct thn.t P'*.P \ildividuals I'CC vcrcd frcm Tyoc II r Ty-?c III qnc~c~c* ecus inf2ctip.n failed tn rcnct in- trqdcrmally, although they n-,ssesscd circulating tpe-s::Ccif ic an- tibrbdics, indicatas that a.11 of the fsctprs invr\lvcd arc: nrt yet undcrstccd. Invcstigatinn rf these crnditirns is being c*xAinucd. I iNt . cBf capps ret Ipp) :! I , ~~TJC II : Tpe III ; GrriuTJ IV ' TYQC IIAi v ercd I 0 17 9 ! 13 ; 5 !N* . < ,)f c?.ses rrc*vored i (pivine qesitivc rcact1c.n I 18 i 10 4 i 0 I 0 , Fcr cent .-f nnsitive I 1 rcscticns I 100 : 58.8 i 44.4 i 0: 0, IN-. nf CaeCs n*-t rre.ctir,R I 4 I 0 7 t 14 1 5 _ _ IPnr crnt fntnl 1 100 I 12.5 I 28.5 i 7.1 I ; 3 ; I Fcr cent rccrvmrcd -se8 nrt resctine: . I 0 ! 87.5 I 62.5 ' 92.9 ; 100 1 111. The Dcvplr:rllant rf I?et,t?r`.l'?pus Tme-sn*cifiC htib;.dic:s &ing CC r.v?ln~~cc frr,m Fneum~i?.. (Dr. Tillctt e,-,d Dr. Frxcis) It is well knpwn th?t in >?ncumr.nia, qecific extibcdics `yaks their an?os.rnncct at crisis, the tina at which the oolysaccharide skin test is first deTcnstrable. I;? thp c.-ursc c.f `?~f r,bscrvq.ti?ns, it has been the cust,c'F to test the patients at rrl!c-ztcd intervo.le during the diee%Re and ccnvAloecance. Recently it has berm xtcd tha.t abnut tw* aecks after racf.very a ?eti\nnt may renct tc 0. noly- saccharido', hctcrr lo,o~s in t,;,Te to the nnPump.crccue causing tho disease. Tests :..f the serum at the tis, n f,f Euch a rcsr)nnsc h3,vc rtvcnled the presence vf specific antibcdies rcnctivc n-t cnly Tith the 3rimary causative organism but also with pnaumccclcci c"rrFsl)"nd- ing tpe tfi the %ew* enecific antibcdiP$. The cd r?eusmc- ded ir, norm&l salt solution 13 60 fold corccrtrstio::. The `c'cs.ct.-rio. nrr the= frozen: end tha.wcd pcvcral tima until disoolutio:? t?kcF illace. 0.3 to 0.5 cc. of normal acetic acid is s.ddl?d os.d the eolu- tiol boiled for 10 aicut es. The hoavy coegulum thus formd is -?rc- cipitsted by ceiltrifugatioz ar.d the water clear suoerr=atazt fluid is rmoved md rerdcrcd zcutrsl by the additicn of the ro *Tr FOu;:t of nomal NaOH. This fluid co;itsizs Fractioc I'C". That Tractis::. "C" i8 zot a tyoc-eoecific carboh.ydrste is indicated by the fact that it is derived from or,-t:?ne PTecific R strair.s of Pncmococcus; that it is not nuclco-oroteir. is indicated by the fact thet boiling the ma- terial with acid removes protein to such 3:; extent that the superna- tant fluid gives r,rne of the usual tests for uroteir. Although fin- al proof as to its oxact r,aturc awaits chenics.1 analysis, Ileverthe- less, cominci~g evidence of the eeoaratc identity of Fraction. "C* is brought out by serological reactiocs. Sera obtained at frequeilt il?tervals fror patients acutely ill vith or convalescent f roar ,weur?onia havo becn mixed nith vssying dilutions of Fraction 1lCs e;;d the .Zrescncc or absezco of precipita- tioz coted. It had bee-r found that sew derived fro? a wtiest during the acute stage of ptccu~ocia possesses a high titre of pre- cipitins for Fraction ItCtt. A day or two after recovery this pro- cipitstkg power abruptly and ~ermmtly disappears. The scra sf 50 patients have been tested at frequent intervals frolr: admission to the homitsl mtil several moathe after recovery. In every ic- stszlc0, the blood obtninod on admission has furnished sem cauable of precipitating Fraction sCs ic high titrc. This has been true even of patients i:: the fir(;t 24 to 36 hours after the onset of the icf ectim. I~idividuals who have succumbed to pzmmonia have rain- tsined antiJtCs grecipitins until exodus. Ir! a11 individual ill with pceumonia, cot only is the sudden appearance of the reactivity st&king,, but the rapid disap~esrance of the ohenorrmon coi:?cider,t with recovery is distinctive. A few days aftor the critical fall in tevoraturc, the nstieilt fp scrm fD.ils cor79lctcly to prcci?itntc Frnctio:: `Ctl. The ohemmeaon rray be further characterized by the fact th?.t it is urzclatcd to the tpne of Przeurnococcus causicg ir,fec- tio;l. The curve of the nrecipitii: titrc of Fraction I'C" is distinct- lp differmt from th?t obtained by the use of either the type-specific mrbohydrate or the mclco-protein fractions. V?ith scra obtained at frcquczt i::tervals during the course of nneumor.ia nr,d tosted with pr,eu- l-rococcus cucleo-orotcin, type-mecific carbohydrate, ar.d Frnctioz "C", three distir.ct curves of Frcci-@tin contcat may be derromtrated. An- tiprotcin ar-.tibodics do Lot vary r*%rkedl:r during the course of ?xeu- lPO?.iS. Type-specific antibodies are sbsect during the acute stage, apnesr at about the tim of crisis, ar.d are homologous to the tycm of the ir,fecting organisp. 011 the other hmd, anti-"Cl' precipitins are highest during the acute phase of the disease, disapaenr just of- ter the crisis, and are zot rols.ted to tyne-mccif icity. The resort so far has been limited to a oresentntion of results obtained with pticnts sufferirrg fror; pnemococcus irrfcctioi;. Pntierts hmizg pncmonia due to hmolytic stre@ococcus as well 8s individuals acutely ill r?ith other febrile discwses have beer. avail- able for `comarison. Patimts eufferizg fro? the folloring acute disenses hgvc beer, studied: Feasles, chicken-pox, acute rhcmatic favor, osteomyelitis (st?,ohylococcus) Talaria, typhoid fever, tubor- culosis, acute gonorrhea, md fevers of unknown origin. of this group the natior,ts afflicted with homolytic etrc~tococ~us DZeWnoni~., acute rhematic fever .a?d stauhylococcus osteomyelitis, have ?OS- seceed anti-llCtf -9reci~3itins in their serw when bled during neriods of acute infection. Tests ?,a& vit.h pzsr-u- fro..p t?:e other case: hwe .Tivm entirely iierative resu.ltp. Through t'*.e courtesy cf Dr. Srift serp r'Jtgiiled qt freou.ep.t irrtemyl? fro': fifteen cases 01 r?eWrttiC I ever have been availai:le. SiilCE?, in this dise?pe, there ?re re- :A? CA -?au;~d thmt r,reci ,lti::s for `JTr:3ctio;: C" exe ?reccr t durir.g xr- iode of fever but absent duriq re-:i?sions, These obscrva.tiens -.-s&z rjthA sere fro.%% cxes other then l.:eu'-ococcu3 infectioi3 indic.ete tkrt the reaction is not s9ecifi.c diseases associate? with ,FTTP >ositive COCCi. The si,qificence of the aerolo;`ical reactien ?phich has been described is not yet clear. However, its unumsl charsctcris- tice both AS to time of m%grance axd raTid disa?ue?.rarce follow- ing recovery, fndimte that &~1 unc7eretPnding of the factors concern- ed ix the sroductiol of this reaction ?ay thropr addit ions1 light on sop'e of tSe m031ema of acute bacterial infection. IV. C;lemo Iyrmolopical Stu+.ies OT: Cor..iumted Carbohsdmte-Froteix. (Dr. Goebel) 1. Sptkctic CarSohydrate (Hexose) Froteir! A;r.tigens:- 1~ the last rmort the synthesis of t,;le P-aTino-:ht?nol glucosides of glucose md galectose vas Described. It has ei2cs ben.n fourd t!la.t 14iien these tvo carbohydrate derivetives, whic5 Ciffer fro5 02e S>- other only in the s-s,ecial co;.f i-ration of the fourth carbon ato? 206 H li` of the sugar, ?rc bound to 3rotzin, thpso ;>rotoin-eugcr comnlexcs ,:a:/ function as cxc-llent antigens. It has bccn found, furthcrmorr, that Then thpse suger do- rivativcs arr bound to the ssmc nrotcin, tholr exhibit distinct im- rrunological s;ccificity. If, on the bthor hand, th3 same carbohy- drste radical is conjugated pith trro chemically diffcrpnt and scro- logically distinct r?rotcins both of the sugar wotcins thus formed acquire a comwn scrolagical soscificity. Tho nwly acquired S.X- cificity of t?-lre- artificially Trmarcd sugar nroteins is detcr?in- ed b:r the chcrricnl constitution of thr carbohydrate radical attach& to the trot.-in radical. Thue, sim~l:, diff Trcnccs in the rrolccular configuration of a hcxose suffices to orient nrotcin swcificity Then the corrcs?onding glucosides of the sugars arc counlcd to the same grdtein. The unconjugated glucosidcs, though thcmselvcs :lot ,?rcci;>itr.ble in homologous irlmuno scrwn, saocificnlly inhibit th? rcsction betaccn the homologous sugar .;?rotcin and its s?ccific an- tibody. Quinca t>ige have been naseivcly eensitizod +ith the serum of rabbits imwizcd with theao synthetic sugar proteins, It has born found thet they exhibit tynical en&Dhylactic stock ?hcn aubse- qwntlv inoculated dth the homologok eugsr combined dth n motd~ difforont from that used In immunizing the rabbit. The reactions erc in each inats.nco succific end denend for their snccificit:r on the carbohydrate r?dicnl and not on the orotcin molecule of the RI?I- thcsizcd conwound. The unconjug?,.ted glucotridcs, thoqh in themsolves incs.n- able of ir.ducing shock, inhibit the snanhylactic r?wtion *:hon in- -prot.zir.. I;: ardor to .>licit the ?-I~Eo-.-CLOL, tl:z ccrbo~ydr?,to ii:ust bo the s~.q~cl QS th9.t con;biT-c d i;-. the a.gar-nrot.:in coqlox. T:XP , for thr, First tilrc, it !??.s be-n shown by direct ?xsTGsri- mzctql cvidrr.cc t!lnt n.svrtry of the carbon a,toms in the supr rsd- ic?.l suffices to detclirir,c diffcrer.c?s iii the 9nccificity of rug?r- ';yTOtCiil X.tig?Xs. . ST? hc c ol:.vsncchqride - Frotci;: antiE?!=. For thr: $%kc of c_rry:ir;< t,hiR cor.ceqtio.,- i;:to th;c realm of bs.cttrisl nobly- sncchsridcs, rhcrc hc+rc %g%iz it ip bclicvcd th%t F .ccificity is dc- Tccder.t UFO;; the xrragcrcnt of the F.tom m;:d ~olcculcs v?lic? go to build UT? the comlcx nolys2cch?ridc, it YLS thou&t qoseiblo to corn&i - bino them lith forpie drotcin, z-d thus to rcndcr them Tntigcnic, n.ri! to elicit a:: s.r.tibodS rcFdonsc similsr to that obtained. by imp- izqtioi- T*ith the c:ic%npulntcd bs.cteriql bodies themselves. TL T?e III fnoumococcu.e soluble specific ~~olysacc5~ride CM cho~m for this etudr. 9s co~dei-.ei:q thie cor?ound pith TI-zitro beazgl bromide EG FthCr of the soluble substnncc ~9s obte.insd vr'nich, oil reduction Tith eodiwn lq+osul-?k;ite yielded the p~ino benzyl cthcr. WhCil thin comyouzd wa.e dfszotizcd. and thrri rvldod to ?a al- k2liile solutio:: of nroteic (pcrun\ globulin), the tiFo s-abstances con- bizcd. By chcmic-1 mctnitplrrtion it :`I&8 .I-oasible to separate the u.L- c'~~;:gcd -?rotci;-- fror t>e soluble cor~lcx zd thue to obt2irr n COL~U- g?tcd c%rbohsdratc-grot?in co;tDinizg neither m:boad ;?rotoir-,, nor uzbound saecif ic nolyFsc&sride, T%c chemical rcnctions mzy be re- Prcserited schsmaticnllp thus: ma H 12 I . . i . . --- --- C 0 `H + Bri CH2'(`-"`* ,/`I i 1 W- NO2 Nn.OHjo--- C --- 0 - CN2.w N82 Na~S~ I Soluble Substance p-nitrobeczgl bromide i 1 n-r.it ro bcr.zyl ether of the Soluble Subvtwcc I -`--I . . --- C --- . . cH2" \ ,`: NH2 HO1 `+ I Nn.HQ2 ' -n-sn;ir-obcr zyl cthcr of Soluble Subetnncc 1 ' .-- , --- ; --- , CH2 --`,) - N = Soluble Subs tlzncc PC nroteiz-cqrboQdrntc to bc insoluble ir. the wesc::cc of 1 /m . . --- C --- CH21,J `N=N?Cl f Protcir? ! rlilzanium dcrivqtivc of u-DFinobcnzyl cthcr of soluble substlrce N -+ Protcir I . ecrum globulin co~plcx t%us obt -ined "`?s f oui:d dilute minerr.1 acid, but soluble in dilute nlkali. It contRi;:ed q.bout 10 per cent of bomd cnrbo- hydrnt c. It rcncted snccif icnlly with w~ti~~cumococcus serum T;n?c III irL dilution of 1:5OO;OCO. This conjugrltod c?rbohydrstc-erotein hqs been uscll 9s an immunizing ngcnt end. it kl'ts been fouxd thnt FUC- cessivs dsils doecs of 2 mgs. of qntigsn given for fivo dxye, euff'ices to elicit 9 snccific nntibody reseonse. The scr? of r?bbite thus immunized, nrecioitqtc both the homologous antigen, and th.c oriino benzgl ether of the T;?o III r)newnococcus snecifio polyspechnridc in high dilutions, sgglutlnrtc snccif lc9ll:r !Q-w III nreumococci, and confer eweive 9rotection on vice apin& infection rith 1er.t oncumococci of the horrologoua tg->c. Thr! F,wc I soluble snocific subet.?r.cc of FY~I~~~cCUP vinl- is fir! axreholitc contnining both free wire ,x?. frw cwbox;rl groups. It has been found thn.t if X: ~lkn,line aqusous solution of this ?olylRac- chqride is FkqkcE 4th 3, solution of :~nitro beilzovl chloride in benzcnc , a dcrivrfitive of thie mlyezcchmido is formed, co::t?inin.g 3itrobeczoyl grou?p covcrkg the arino group. T?is derivative is spccific&llgi;*rc-ctive dth Tfle I wtipncumococcus aenm in dilutions of 1:5,000,300. When reduced thip dsriv7,tivc yield-s 9c amin.e -hi& cm be diazotizcd md couded to lmtCil:e. The chcaicql rc?ctions my be rcprePentcd thus: 1 C N . . --- --- ccc I 'NO2 .-> : . --- c --- m - CO l-' PO --`- ! L-1 * 2 N'IZ%?% .I Soluble Subetwca T-nit ro bcnzoyl v-citrobenzoyl chlori2.c aTide of Bolublt Sub9 t .qzce I i C . . --- --- NHCO !.%H ,-I NLI.NO, ~1 . . --- C --- NH - CO (`h&N-Cl + Protein I 2 l-xl" I .- J 9-n-in0 benzofl *miile of Soluble Subst nilcC* Di%zor.iwn o.C\lt I `-\ .' . . --- f --- DECO *I* _ -,\N;N-Protein ., ( Soluble Sub9 tmice) Ia EL ninilm %mEcr 020 :QY thue attnch the Type I soluble eubst wee of Pmumococcus to a,ny::pro t ei;! to yield F. conjugated csrbo- hydmte-srotein. This! dcrivotive All reset mith Typo I aztipnewo- COCCUR serum iz dilutions of 1:500,000. Animle we to be imunized 74th thin derivntive. ad the eerologlca1 findings ail1 be rc?orted lat cr. 3. Tho rrC" frwtion of Pncu~ococcus: - Wcn cells of 2 umncmsulnte~ R atmin of P~cmococcus %rcl brokm UTJ by frwzin$' m2cl tSwing, a.r.c? the remltir,g solution ie ?.c9tec? to 100o C. in the presence of `I alight excoRP of ncetic hci!!, the aomntic ccllul9.r grot.?iils coQulsto. In the filtrates from such coagula there re- Fains a substance, the serolrcigcal reactivity of which indicates zi 0 H i4 its se:,ari...te i<.3ntitzT fro... other fr.?ctiozs of ..:cv. occccus cells. By qreci.>ita.tion Fith nlc>hol in the `)repcnce of inore acid, a cc - ;ou~d has `xm ifoLo.tcd which givcc no;2o of t'?: neunl .rotyin re:>ctione. The -a.tcrio.l rotates tht: rlanc of ?olnrizcd lil;ht about 25* to the right. It is not e>rcci ?itPblc by the usual :,rotcin rc- agents. It gives only F very fpifit biumt test. The subetmcc coltqins about 5 .\er cent off nitr3rcn. It yioldcd 30 >-r cent of reducing suprs, calculated FR ~lucosc, or. hydrolvsis. It is not drstroyed bv ml,- olyt ic mz~/-rcs. Due to the lifEiculty in collect- irLc ?!or!cablc qumtit,icP, sxffici?nt dots `rms, not vst born pth~rcd to chsractrrizc this ht. +F mocies-snccif ic substencc. It aTSoars, `nowever, to &I a .Jolys*ccharidc. rurthcr invzstigntio;:. is hiilg carried oi? ix order to dctepino accurately the chmical nature cf' Fraction sCtt. 4. Det,er:inat,io:. of the olccular size of the soluble s xcif ic substnnccs: (Dr. Goebol rith Dr. %bcrs). - Ixncri~xents arc being carried out to dotcr-+ine the y -oleculnr size of the s.;.cci- f ic -~~olyeaccharide of T.~,POCOCCUB !&UC III, utilizing as a ;t,ethod the rata of diffusion of the carbohvdrate tbtosh -3orou.s r?zmbrpnce, end dotemining the Finute concentration of diffused polysacchmide colori~~ctrically. Although this research has not yet been corwleted, sufficient data ham, homver, been obtained to indicate that the oolysaccharide is of hig'n molecular reight, higher, probably, than vo9t votein. 5. Star&es as hmtene:- The fact that the tpe-s?ecific ,>olgsacchari&?s of `~jn~~~~c~ccu~ -ra,y be rendcrcd antigenic by cabin- ing thcrr aith foreign ??roteiE `ms led us to believe that cormon H 15 starches can also be rendered antigenic by cosbination with ,>retein carriers. The nitrobenzyl ethers of :>otets starch and of corn stnrcb have beoil ore?ered, but mfartur,e,telv these derivotivcs are totally P t insoluble in water and in the usual chppical solvents. It was thcqht, therefore, that the introduction of B carbopyl groun into the ring of the condeneiilg reagent citrobeazyl bromide, would render the cor- resrlondir.g starch ethers eolublo in Fa.tcr. Consequently A bra- o ni- trotoluic acid TRP pyuthotized ii* the f ol1onir.g - qn::sr fro- +toluil dine : 1 violet light BrCH2 BrCH d'\ HYO ,`. 2 r ,.,I igq `1. ,! No2 COdH COOH Brwn nitro tcluic acid This coqound has berz cszdensed with starch; . . --- C --- O:H + Br;CH2'._./COOH _, . . --- C --- CR.' i) COOH ! ; No2 i' I 2\. . . 4 NO 2 e to yield a stsrch ether readily soluble in dilute alkali, but ineol- uble in acid. By reducirg the nitro gram, to the arrir,o pou? and by diszitiziag the latter a.r.d counlim it with nrotein, R conjupted Ftarch-votein Aerivatiire hap %em meTared. Imimization of mi- -ale with this con.ju%ted. carbohydrate- -.)roteir. derivntive are to be cerried out to detep?ine v!het?lcr etnrchee -7p-y functior. a28 11s. tme. 6. The relatio~e3i-o hetveen stereo iro?erim (Eeo*etrical 1 0 ;.erlsm ) azd STecificitu: - In -?eragreoh 1 of this report it par shown that hexosides which differ fro;r: each other or,ly in the s;;ati- al c~~~figuretior. of one asymetric carbsn ato;.. elicit different anti- body res+`onses dee\)ite the fact that the cherrical configuratioc of the reaainder of the hexes molecule is iu each instance identical. Ir, o rder to secertsia whether this ohenoyenor! is cocfined to cc~??ouds csrtaicing only ammetric ca.rber. atoTs it was decided tc stud!7 the sntigenic res Jense elicited by ieorrers of the cls-trazs ty?e. H ti C s COCH -- H - C - COOV' Xaleric , I II .9Tld i cfumar c ii mids are the classics1 H - C - C*OH( I T-?ooc - c - B . . . exe-qle rf this tyne of *overierr, azd they vere chosen to serve as the hantens. In order to couole thcqe acids to motein it becave r.cceseery to eyr:thesize the ~030 vitro amlides of those acids. Thi s has beer. accor?liPhFd by tbr follwi;if syPC??ePis: F. . H ,~ COOH H co~$ I? ' I ;L' .C C' ' H "' O2 E ' c JO' ' so2 ' `-' (1) :I ECl, I ' + mm-2 / I! , C c\ qooc' . . \\ c cloc*c `H FOOC H Fwaric acid fuvaryl ch1orid.e + i:.itro analine (2) H ,) coop H, CO `C c `. zib~ "-' j. NO ~--E&--+> 0 + 2.s` 2 I C m-e-2 .I P `. ~ coov P;' `- co nitroanalice Yaleic acid ?baleic aLhydride ?I COONF~; N@2 c. t H c CooH Ii HOH ., it C .C H `. 11 `< Corn i .-. b, No2 n-nitroaxalize sslt of n-citroma,leanilic acid o-ni tro n-alemilic acid -- Those tvo C,:J'-`3Oui??C c!iffar OGC fro.- the other not in strut- turo but in the geo:.~tric?l rclatior:shi-9 of thclr nto:x. The iso.- crix cm best 3~ u;x-Izr~t:,od fro: the follo=*ing diogrm's: o-nitro mlcantlic a.cid p-nit ro fumsrenilic acid The nitro gram of thceo two isoTcrs, which contP.in no asymtric carbon p.to!*s, have been rcduccd pnd the corresi>onding ~.:-incs have be-n coupled to Trtitcinn. Animls arc to b? ivunitrd with L thcsc dcrivativcs to a,acortain ybethm gee-rtricsl &so~~.cris-: cm cf- feet s-ocific antibody rcs~msc. V. Poactions of Rcbbitn to fzlcctione of Fncu?ococci ?;:d their Prod.uCts. (Dr. Julinxllc). A study bee bcm T.M.c of the chxyze vbich trlkc ;-locc in rabbits followi,;g thz r:;r: *ted ii;tr~~cuts;.:~ouF i::.j.-,ctiot of EUS.: ?n- sions of hcg.t-killpd -;?cu?ococci. For the ank of co?>~risoi:, c!. eiV- ilsr study hne br, `z WGC ir? norr.a.1 rsbbite a;;d ii: rsbbit s tre?.ted nith pneu?ococci arid their ->roducts 13 various ways. The roactiona invos- tigs.ted have bee;l (1) the antibody res'orxe, (2) resistance to infec- tion, (3) the reactions at the site of injection, (4) the develo?ent of skin res.ctivity to derivntives of Pnemococcus, (5) the develo ment of eye reactivity, and (6) hypersensitivenens to Pneu:Tococcus ap.d its .9roduct s . Each of tlzese reactions is pm,:.?.rized in the Tresent resort. I'. The Antibody response. Sixty rabbits vere imunized by the repeated injections ij;,to the s.li;z of srall doses of heat-killed -meu- 21.4 B 18 mococci, Tyre I. Ir! the eera of 53 of the rabbita no tym-mecif ic antibodies were denorstrated, while in the sera of the reminiar TtiliTo.19, the titre of tvs:e-emecific ?stiboSieg F~F! vcr:; 10~. 17 3.71. cafes, however, t45 ccrq. --oFFeared hi?h titrcs of the = ccicr-o oci- fit sztibodicr. Forty-five rabbit e sicilnrly irzuzizcd by i-i?joctio,?s of ;Ao?t- `killed Tpe III ~;zcu:ococci .qlso f Tiled. to form ty:c-e xciiic 5\.:tl- bodies, but did. ~OIV c -"ci"F-a -rcif ic Fntibodics. Yor~ovzr, hc.?t- killed FUP cmime of R a::eu.-ococci or solutio;:s of t:? ,?ctcrial mbetq:.ceg wher: izjcctcd i:?ta the Fkin sti:.ulatcd the ,jroductioa of o;;lg F ccies-s !ccif ic a;;tibo?.icp. . Reeintsncr to infection. Folloving tho iztr?cut%eoue injections of he-t-killed S or R moulnococci, r*bbitzr mcquire P rwked degree of reeistarxx to intrwrxoue injcctione. of vitilect organic.*c, md this is true whether tile xxu~ococci izjcctad be of the SSITC ty% or of 3. differer-t tyx frolr thn.t onoloycd for i?ur,ization. on t?le other hand, regoated intrwutsnaeue injection of the aoluble moteins of the cell is not fflllored by an increased resietmce to infectioi3. The sera of both resistant nnd xor_-raPi!4wt Animalcr, in grneral, fail to woteat white mice against infection by or?miera of the ho~olo- gous ty;e. RoFcver, the scra of .nbout 20 ?cr cent of the rebbitn injected intracutaneously with me I Pnewococcus, were found to contain varyi;lg quantities of !>rotective ailtibodics. It is SCCi:, the*efore, thrrt while the antibody response to tSc int?Ct cell9 cL?=c eolutiacs of the cell is essentially tile sme (i.e. s:?CCi(!S-WCCifiC) the acquisition of resistance to icfection is characteristic of orly the acimalp receiving the intact calls, ., ;$ . e reaation at the Site of Injection. The ict racutan- * ,! eoue injection in nor.91 rabbits of 0.2 cc. of heated :`:eu ococ:i, re- , 1' oresenting the bacteria frolr 2 cc. of broth culture is folloaed by a :y ! ? 1" yy circumscribed, slightly raised, and indurated nodule, measuring about 1 cm. in diameter. Unon repeated injection of the eeae eeo;tnt of bac- terial sus:,eneion 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 CT. in diam- eter, and the ekin is markedly elevated and of a deen red to mrplish hue. The raised areas IP surrounded by an areole of erythc?. and out- side of this the skin may be edematous over a considereble area. Fre- quently necrosis of the skin occurs vith discharge of a sterile, puru- lent material. Then necrosis does not occur, the disa?Tearance of the lesions is delayed and the tive required for regression is related to the intensity of the reaction. This heightened skin reactivity to the bacteria is %wobably devendent upon some alteration of the tissues themselves since transfers of serum fror highly reactive to normal rabbits does not endow the latter vith the Tro?erty of increased ac- t;ivitv. It should be aointed out that frequently a secondary reec- tion ipay occur follorirg the disa~?earance of the wimary reaction to the first injection. This recrudescence occurs even without a second injection and is Trebably evidence of the develowent of hy- Dersensi t iveness. IV. me develoTent of skin resctivitv to derivatives Of . Pneurococcua. Folloffing a series of intracutaneoue injeCtionS Of heat-killed pneumococci, rabbits acquire an increased skin rc8CtiVitY (1) to the ;;vcleo .rotcin of Pxu. OC~JC:VF ,--C (2) to an extract of the becteri9. frnr which the acid-I-reci :itsble and heat ccagulable oroteics !tme been re-ovcd. III terx of bactariql swcificity thi9 Fkin reactivity cruet be consic'erod es s >eciee-s-ecific, snd it WI- !)ea.rs to be related to the .,rToenCe of circul3ti;;g F -ci:s-s*ecific antibcdias. A ei-ilar skin raqctivity has been weved to mzur ix r-b- bite fol1nwir.g the rex!ntcd a.d:-inistration by the intrqvenoua ?r ictracutanemus route. of the heat-killed bqctcrio or their ,rotein deriv+r tivog. The ~kir? reactivity, tborcforn, occum ir. both r-pie- tP.?it Qild i Oi>-r?piPt9llt *i?iy'FlP. V. The devoloq -:.ent of eve rcnctivitr to $criv*tivse of ,Eneumococcue. It vrfi.8 ~1180 f .u..:d thqt c*rt-.I of th? rx~.*Dbits bC- Cor*e c\re r.-xtive aftrr r.-Ceivir.,c i;=trPCut?L:-L:l:c i,..,j?CtiOi.;P Of i2- tact cells. If the cornpn. ip pcnrifiod ,-;I+ t:?c::. tl?o ZUC~CO :rotcii: of k:eu-COCOCCUP i? .>lac.:d iyL the co-..&ctivEl FAC, i:; shout 6G .xr cent of the rabbi te an rye reaction R 3Tenre within 24 hours xd / then increaeoe ir, ir-toneity for varying ?criodq, The rco.ctlor: COP- / eista of congcetion of the. cmjunctiva and the qmarazce of &t- ea ca9illnrie9 at t!m sclero-comes1 mar&n. In POW rabbite, the a" comea ia also iwolved and them is the develouccnt of turbidity ! ana, lees frequently, the f omet ion of a mnnus. The eye reaction `. 31~0 is excite-aycific. It hae been found thmt not infrequently, the iztrwezoue i?lSectior; of zucleomotein, 8fter 2.11 eviderace of the eye reactiu:: have comr)letely dipaT+eared, my cause the rem- yearance of th? -7~ reaction.. or. CortradiFctizctioc to the ekir! reaction to nrotcir., the eye rc?actio;le. do not occur iz rabbits fol- . Uizati .n by 9r.y route with solutions ;,$ ,rGteia~ o:? tile celi.. SO t:13t V:hile s?rin renctiozc occur in .?O~-re~iSt,?iit n.:.d resistant ani- 3.17 slike, t?le eye re-?ction, o;^ t're ot&r 3~6, occurs only i-.. t:le resiPtact rabbits. VI. FvereeneitivenePs t:, Fneu OC~CCUB n:-d its derivptives. There vere certain repso;?* fs.,r belicvicp t3t t'ne ekin rePctivitp to the bacterial `3rl>tein resembles the Arthus reaction, nhile the eye reactivity see-e to denend WOI? so-e fectorq which are different fro.-. those o-`,emtive in tSe skin rea.ctivity. It see..od ndvisable, there- f?re, to study simultaneously the Arthus reactioiz to egg? albulrin, the develr?ment of increased skin reDct,ivity to the bacteria themselves and to the Trotein of Pnemococcue, and the dcvelo -7.ent of eye rest-- , I tivity. A aum~ry of thie' comnarativo ptudv ip -,-resented in the ac- I I i coIxanying table. (See Table I). The iauortant noints in this table /i not already ,y*cntioned are (1) that the ,ckin reactivity t 0 ~?LlLncWTOCOCCUS Ii xotein can be transferred by serum fro:-- 9 renctive to a norral rab- i' bit; (2) on t'.le other hand, the eye reactivity cannot be thus trnne- f erred. In other, words, the skin reactivity a;s:;ears t0 be aZ 8Xm:~le of the Arthus reaction to a bacteria.1 protein, while the eye reactiv- \ ity aTgears to Se a special ty?t of sensitivity. VI. Active Imunituto Pn eumococcu8 Infection FollOWiiIE Inlections of the Soluble S~cific Sub~t.a.nce. (Dr. Julianelle). Caagar and Schiernpnn originally called attention to tigonicitv of the Soluble Suecific Subetance of Fneuvococcus. the an- In R mare recent co-Yunic?tion, Schiemann repeated the study of the immun- ?eac t! irs - Reaction at oite of each pollawiuq repeated izjections &llbwinq rsreated injections Follotig repeated ir,jections of ecus tro tein heat&i lled pnemnoc occi (Tyte I Intravenous Intracutaneous I nlmma ous 1st injed.+ of egg albumin ! of Pneurcoco( `In tracutaneous Intravenous Intracutaneow 1st 3 inject.- 1st 3 injec.-= 4th - 10th "+ 4th - lOth"+ Increase in intencity unti: 4th-6th injed. Then ds crease inj ec tion Table I. * Reactions of Hnersensitireness in Rabbits Dew lop- ment of circulat- ing anti- bodies ( 4 me- opecif ic (b)species sl;ecif ic Active Resistance Increased semi tititg (a) skin (b) em Transfer of wnsi tivi tS (a) skin + + + + + + + I + + + + + + + + + + hdicates presence of reaction - indicates absence of reaction Y I ity of ::icc to `lccu-ococcus infecti:n follming injcctionc of th? cqr- bohvdrnte derived fror Pneu-ococcue, Ty-oe II. F'e Toi,,ted out t!:-t the ii%?-unity develon.-d only when very? c--n11 qu*ntitics of thF ~l=ncchqr- idcc p.re ir,.jrctrfl q:ld the rcculti;lp i?rrunity is ty--e-p Tccific. Exm?ri~mr~ts hnve been unc?ortnksn to dotoF*ine, thcrefor-, yvhe thl?r thr! ~olv?~cch~rid~s of the three ty,sr.s of Fn"mococcuc ore- qn- tigpnic, in th; FC;IPC th?t thiy will stirrulst,? .m nctivc ic,:urity; 7~~- if so, uhnt quantity of the o )lys2cchCxrides is b?st for this ,`ur;os-. It 7P1F 4ol;nA thst t!x cprbohvdr?top +. riv,?d fro? Tpc I or Tyno III, irr-c `.-ctivc' of the .:othod choccn for in,jTctior, 2nd of t& qwntitiCs uecd, did not stimul?te in mitt ri etntc of rcaistnncc to Fncmococcus ipf Cctio:l. The total qusntitics of c?rboh;rdrstc studied vqricd ir. rnch C?P- f?0.. 5.3 tl, .00005 "$+. W.-n F- 7.- II .ol*~~.~cch~ridc VP crrmlov~d, it PR fmnd thrtt imunity to infection could br inducrd in ?icr nbm n-n11 qumtities of the s;llublc c>-cific substance were injected. The totnl qun.ntiti:s injxted v-ricd from 5.0 to .OOOl `FF., but only V?ounts of from .OO5 to .00005 ,`F?. etimulatcd mny dcgrcc of innunity. TIIP o)timm do?ngc wag .OCl 7gT. In thlT last instqnce, thn imitv induced was co?mar- able to thnt induc**d by the injection of heat-killrd T~QF II ~CW'O- cocci. The I .-- uzi t-T, Tor"ovcr, wq.g highly tv?.--s*>ccif ic. IPbbit~ azxi guincn Cge verc ale+ injected with diffcrmt quantities of `WC II .olvFpcch?ridc, but it TV-Q not nopeible to ahoa th*t cither of those two s-,xcics hsd ncquir:d ty >e-s?ecif ic i*. unity. In conclusion, it Faybbe sq.id thnt with the quantities em- ?lo,r d , no imunitl: wq.8 demozetratcd follovfing injectiots in e II pneu?ococcus Rhich has been rendered rough and avirulent, rabbits are repeatedI? sprayed vith a slight1.y virulent ?neu?ococcus Tyne II S. orotective antibodies only mmar in the blood. If a rab- bit virulent Type II pneu*ococcus, however, is used for inhalation both agqlutinins and protective bodies "ay develop. I!`+Jlloffing inha- lation of either a non-virulent or a highl? rabbit virulent strsin of Type III pneu-ococci, neither agglutinins nor -rotcctive aztibod- ies a?ocar in the mbbit 1s blood. The sern. of 2 rabbits unhich have survived treat-lent aad are now living, al-.est 4 years (1310 and 1360) days after their last ex- sosure to Tpe I meu-ococci, still orotoct .rice against infection of large doses of a virulent culture of the sme tne as that with which they were originally smsyed. VIII. Antime\rococcus Protective Actien of Nova.1 Pig Serm, (Dr. Kelley) Bull and WEee have reported experiments in which rice and @;minea pigs kere protected against "any ti?cs the lethal dose of pne~ococci by the injection of nopal chicken serum. It apTcared that this protective action of the chicken sem ras asscciated Fith the scrub globulin. fn studyi;ig -,?hagocytosis of ?neucococci by eeruy-lcucocyte -1xtures Robertson e=d Sia observed a mrkcd obsozic activit? of thC sera of imturally resistmt 2nim315~ Thie ~9s fouzd to be es:`:ocinl- lg characteristic of the sem of the. -*lg. Sia obscrvcd that cor.e.1 pig scruy would also co;lfer on :ice e. ronarkable degree of ~?essive -9rotection ag-inst pncu?ococcus infection. Ir- cases of Type I M II ?ixl~`OCOCCUR, -Irotectioc npinst 10,000 "301 doses 9pa.p 0btqinc.d. h'e also de?onstmted by abeomtio.7. ox-orimxlts that this wotoctivs action w!: s,xcif ic for c-?ch tyx of Fn~w~ococcue. At varisnco llVith the :` i Y c l;- ecc.qgtcd cc-llul.3.r th-ory of x?t.urql i;-..:unity, h-rc `r'c i;l- stances of a na.tursll;r occurric$ hmorn.1 defense wChwiF9. T?z? i .- ~-lxl~ .yro:7ortios of the 90~~ ar- -~qe~iv~lv tracPforab?o Yd. -. -nrr to bc tyax-wccif'ic in action. Si-?. xd to furthor 5nelyzs tho -rch=mis7- of tl:c --rotectivr n.ctio;? o< .f .,; I.`. $:,, t-j l.?ie SCIYXC. Tbc pcnm is obtninod fro*. the blood of rior ?I .Qn COG- lcctcd at slaughter, nxd is starilizod by Borkfcld Filtretioa. A4 faux? 5-y Si?., thr fresh s:nm fiv-n iztr? :-rite:: -.,:`I.-- r,rotcctP aicc rr?1lsrl:- qnixiet infection with 1,000 to 10,000 r'mtq1 dot2ce of TJ=X I ~3 3 Ty,o II gncmococci. !FJ~ s..c:rcc of ,:rot :ctioil varies eor:tcThrt vith the i3dividuq.l lots of s.:ru.`. 7ith t>U? o-r? so f?.r studied the greotcst dcp-o of r?FiPt?i:CC to infoctioi: hae b*C,l conferred on mice against infection rith P~~U~OCOCCU~ Tp" II. Tkis protective c&s.city diminishes gradurrlly on staading SO tk3.t ii1 the lots tcstod it was com?lotcly lost rrftrr 6 to 12 rocks. Them is 0. suggestion thet thn wotcctivo .:rowrtv is ,cons.x'vcd by storing the smmi at a low twper~ture nnd by covcricg it rith a vmrlinc swl, mhcthor the motectivn powers of t>- ~crum against nil thr difforcnt t;r;yec. of ,?rm@ococci disao-war sitiltnneouelv hw not vet be'n dctcr- --inod. %,tinz the gcrw nt 60-62oC. &cst,roye .n.ll, or very nmrly all, of its 7rotcctivc a.ction. V3d.mr th? c.7,~~ condition9 Dntiiqncu- The ?.ddition of 10 per cmt untrclted `,`ig scrw, .xcviously iwcti- v?tcd 1;;~ hvtia?, f:yilo to rcstom t?ic: motxtiv2 xtion. I 223 1: 27 The optimal protective dose of the oig serm seem to lie betareen 0.75 cc. and 1 cc. As Sia reTorted. the greatest degree of ?rotection to wice is afforded when the senm is injected intraner- itoi?eally 4 hours before the infecting dose of Pneur?ococcus culture ie Fiv?;1. The ?rotectior!, though quite distinct, is not of the ~1.`c degree vhen the sex is given simltancounly with the culture. The observations that the .-rotcctive factor is associated -7ith the globulis fraction of the TQ serm has been tZonf ixxcd. -0 5 ap;?reciable amotit of this ca,-?acit, 1' seem to be lost in the Trocess of se:oaration of the glo'oulin by :Jreci?itation with a3oniux sul?hate. In our exS>eriTqent 8 so far, absomtior, with virulent pnewo- cocci of a given ty-oe has re;?loved the xoperty of ;wotectiiw zice against infectloll with the ho!?ologous ty?e. In addition, the degree of motec'tion afforded by the absorbed senx against heterofogous tnes of oneuz!ococci is decreased. A sumrisi~gly few T)nemococci are required for the absor$ion. The time rwceasary is likewise short. There is a suggestion that Trolonged absoI+ion with a large cumber of virulent or avirulent pneumococci may co?letely deprive the serum of its ?ower to protect mice against infection `with either homologous or heterolmw ty?ee OS hreumococcus. PreliMn&y studies in which Pnemococcus polysaccharide was added to-the serun 24 hours before the orotection tests were made indicate that the sem has not hem alter- ed in its 2rotective Tower by this treat-ent. Agglutination and grotection tests have been xde with a serm which Trotects rrice against 10,000 lethal doses of me I or Ty-ge II prmmococci. Desnite its protective action, the senm fe.iled to cause agglutination of Ty-oe I or Type II pnemococcus. The precip- itic tests, using the uneu.ococcus ~~~olysaccherides derived froT Type I ?x II .+xu.:ococci, h"vc likmicc b,y n ncgstivr. So fqr, attc?-3ts to deqrsdc a virdcnt s str?ir of bcu?OcOccu? an avirulent R for by gr0v:t.n in ?ig scru: have not `;?.x succeseful. ,rrucocci sensitized by the pzrw- she:-r XG loss of virulimx. It I-z%? : LCCII learned 7hethc.r or not ccnsitization of an-urococci ir, #Tit* c,:r- iins a.p effect on their agglutinability in anti~xcuVxcoccus horac s;rur. 1~. vim of the fact that, certqin Sub~tPi~c'C, of th~:-l~clvcs zot 1 i. ;-: c n i c , ~I?cr, injcctcd intravznouslg ir.to n:>i--31s along vith llig screw * sti-ulatc nntibody resnonsc, it ip thoxht dc?irn'slr to learn if such "Sh1cT18'~ action -ay be obt?incd. ir! t?+: c9ss of :m~u--ococ7uc *olyen.c- . ,ridcs. Rabbits arc `acing i-,mnized sit3 ::ixtures of ,;ig sent.. a;;d the ,j;rsaccheride of Tyoc II -mcu-ococcue to dctcr .inc *;:hcthcr, mdcr t!iCsc ,nditions, t'r.c no~-~i~tig~nic sugars :`PJ' st,i*:ulate the for-atioit- of tJr?o- ,-, 2 cif ic antibodies. & Siz:nificailce of Oxidstion-reduction FhCnOmena iC the Sactorial Cell. (Dr. Dubos) 1. The role of Debtone and .?lucose in the izitietion of nrowth ;)f Pneumococci:- Preliminaryr anal,ysea of tho oxidation-reduction Wetem 3f the Fneumococcus cell, and ef the oxidation-reduction characteristics of sterile bacteriolosical redia, have `oeen discussed in a orevious reTor%. On the basis of tb.e data obtained wd of certain growth emer- iments in aedia treated in various ~aye, it IVBS suggeded that: (a) the growth of Pnemococcus ia conditioned by the existence in the Tedium `of a certain condition of reduction, (b) there are present in ordinary Fe- dia certain q?oducts of oxidation which have a bacteriostatic action on Fnemococcus. It was found that t3is bacteriostatic action cm be over- COTS by vmioue methods; addition of reducizc substaxes, incubation T.:r:l.er anaerobic col:.?.itions, eddit ioil of blood, he?tizg tf?c broth ?re- vious to isocul?tion 4A. h wir?f .= lerp inoculw. The `er cent beef serm, or 5 eer cent horse -plasma, 1te.s `freen observed to undergo dieso&tion, roost of the orgxisns chxlg- ing to R form, Q after 2 weeks incubstion. The s.3.m culture, i:: the se"?e medim, et the smc tcmeraturc, but under vqscline seal, rr-sin- ed unchanged (100 per cent S) after the fame length of tim. On sovcral occaeione, ze have be:n able to revert ii: R cd- turc derived frow l'ym III P~~~J~ococc~~, to a typical, cncax?ulatcd. acd virulent S culture on rcveatnd trsnsfcrc iz thy follo?4n? -:diw, . plain broth f .3 ncr cent glucoec + .05 wr cxt thioglvcollic acid, wovided the? culture bF: kept, u&cr vre?lico 9~~1. ly defined and Eva arc r.ot fient s . However, there is tion of the rrcdiw is axe The conc?itior;s rcquircd for rcvereion 0.r~ not, as yzt clrar- alqrays successful in rtmca.ti::.rs thos:: cxwri- ;10 doubt thst thz state of oxidatioz-rcduc- of the. factors imotvod iz the rcvcrsiblc cheago frm the cwsulated, virul;xt, svoot!; Pr.cu~ococcu~ to the R v.arient . J . The Decomositioz of thy S-tecific Polmacchwide of Tmo III Pncurr~coccu~ bv a Bacterial fizmcL Previous studies frop this dopartn??xt ha.ve csteblished that the specific oolyeaccharidce of Pnemrococcus are rot dccomosed by any of the body enzyms, nor aro they attacked by the cornon bacterial, ac- t inomycca and .Eolds. Theso polysaccharidce havo in particular be?n found resistant to all known carbohydrates mlitting enzymes. Q!c have succeeded, however, in isolating a rricroorg?niem thet decomposes the smcific pclyaaccharide of Type III Pmu~ococcus in a sediw contain- 1 ing only rind%,1 salt a. This organism ir! e minute, wore fomize, grw negative bacillus, which aac;ses through a Borkefeld V filter. Its rctior. is very mccific. It attacke only th? emcific rSolysscchar- ide of Tpe III Pneumococcus, but r:ot, t hpt of T;me I azd Typo I I, asd does not fement ordinary sugars. Thip ricroor&.nis?: gives riee to a;l extracellular enzyim which also decor ?oses the mecific ;olysaccharide. The decoVolvsac- charide with specific antisera following chemical hydrolysis. T?ut this treatment we.s of course a. very drastic one and could have affec- ted at the same time the hp>othetical i?Frities. It is not likely that such an objection would be justified in the caee of the Tuch 1-Dilder action of the enz?e. It will be interesting to detex6ne whether the adc'ition of the hydrolyeing anzyme to a lrediw seeded with encapsulated mewococci will affect the fomation of the cAr)sule (which is known to consist . largely of the soecif ic >olyss.ccharide) . le are also considering eqeriaents to determine whether the injection of the e?ecific enzyme into suece?tlble animals rrill increase their resistance to infection with Type III Pneulrococcus by rendering the cells Tore vulnerable to .nhaFocytosie. Av.2ry, O.T. xd Goi?'o,?l, `F.F., Chwo-i>--urologicnl studios o:- cozju- ptcd c?rbohydr*,t c?-Trotcizs. II. Inu~ological suxif i- city of eTriZthctic sug.ar-protclz tmtigax. J. Ex?, Xrd., 1929, 50, 521. DwFo~., uzrtir. I?. , Tao trxxforw.tioz of nmurococcnl tpra. I. The cozvcreioa of R f o=s of ~~~U~OCOCCUQ itio S fame or t'a- ho:ologow tgqe. J. `Em. XYL, 1930, a, 99. ??.-.n trY.sforF3tion of YPu.-ococcal typ?s. II. T3r irt?rco:wcrtibilitg of tvx-wncific S Wi?u?COCCi. kbor , RS-r.c, The rol,? of cq.rbo'Z.rdr?tes i,. T biologic;?1 oxidatioi: e;:d r,-ductio:-.f, ?mcril-pr:t.e r!ith ~ZCU~OCOCCUF. .J. Xx?. '::cd., 1429, 50, 143. Go~:5,:1, ;P.F., n,d Aver;, O.T., C?ic-.0-i' u;;ologic~l studics 0;: co:..- jugptcd c~rjo~dr-rt~--?rot;i-~. I. !@a sptcxis of' ;;-ezi- cophcnol R-glucosidc ,-a i:-.o+~-;;ol 3-gnlactoside, .x:d their cou$i:-g Tit!1 szruz globulin. ,I. Xx.2. Yad., 1929, 5Q, 533. :l'cid.-:lbcrg:?r, :!. , Avery, O.T. P;-.d Goobel, S.F., A eolublc awcific .wb~txcc dcrivod fro.? gu-r wzbic. ,J. Ex::,, WI., 1929, 49. Julizcllc, L.A., Rcacti0r.s of rnbbits to injcctio;:c of ~i.CUZOCOCCi ?r,d th.zir products. I. The xtibody rcsrJo;.cc. 3. EXT. Xcd. 1930, 5l, 441. II. RcPist?.zcc to iilf?ctio:. 3s n,,l. ?(?a. 1930, a, 449. III. RC'~ctio=P ~.t thy sit.- Of i~~jcctio~. .'. 'm. aid.) 1930, li.L, 463. IV. The devclo?:ct,t of ski= reactivity to dcrivstives of Pxu~ococcus. J. Exn. bEcd, 1930, a, 625. V. The dcveloo?crt of "ye rccctivit!; to d?rivn.tives of' Paeu~ococcus. J. Exn. lh$. 1930, 2, 633. VI. ?@pereersi t ivcnese to p~~~~ococci end their moduct e. J. Ex? . 8cd. , 1930, a, 643. Stillpac, ErncCt G., I-qzity reF?orclc of rabbits izoculntcd rvith Tpe I Fmu~ococcus by various routns. J. Ex-,. Ycd., 1930, md Brmch, A., Ewlv oul?ron?ry leaiozs iz mrtiQlly i;:;?une ?.lcoholized mice follo~*izg i-;lhllstio;l- oi viru mw~ococci. J. X:x?. ?`cd. 1930, a, 275. Tiilctt , W. S., &cry, O.T., wd Gocbcl, W.F., Chxo-i*mmologice.l studies on conjup,tcd cwbo'nydmte-protcizs. i~;=d :xmsivc ,xr?.?hylcxis with syathctic sug?r-?rotoiils. J. E&L !!cd., 1929, f?p, 551.