qroteins. 1. ,i'he specificity of antigens pre.,ared by combining \Xana e glucosides of glucose Fith protein. i, 2. The s;ntAesis of glucosides of the disaccharides, cello5iose and maltose, IV. Studies on the interconvertibility of tne specific types L of Pneumococcus, H 37 Report of Dr. Averywith Drs. St illmn, Goebel, Dubos, -- _--_----m---e ----------------em Francis iJa\ers .__--- Goodner, -L--`---I-,-----.- H-_-v-- ,------,,--1 Allowax and ,ierrell I. The 6ecomposltion of the canslllar polysaccharide of r, ryne 111 Pne,Jmococcus by a bacterial enzy--e. 1. l!et:lads of purification and concentrat i-on 0 s?ecif ic enzjr:-.e, f ti,e 2. Protective action of the envy-:e in derma.1 infection of rabbits with Type III Pne-umococcus. 3. Production of Pneumococcus Pneumonia (Tvpe III) in :,ionkey 9. II. Isolation of microorganisms decomposing the can?ular polysaccharides of other t,:t.oes of Pneumococcus, III, Chemo-im:tiunological studies on conjugated sugar- V. The ciegree and duration of active and nassive im?runitp s to Fneumococcus in rabbits, VI, Pu:?licat ions. 228 H 30 I. The decomposition of the cansulnr Eolxsaccharide ---------- ---------------Lo----- __ e-----_--e of Tx~e III Pneumococcus by ---w --------e-------m- a specific bacterial enzyme. ---- ------------------ (Drs-. AverxLI Dubos,-Goodner and FrancisL) --- -e---m 1. Methods of Eurificntion and concentration of -------WV__ -----__I_-__________________I__ enzyme. we have described the isolation --- --- In previous reports of a microorganism capable of decomposing the ca.psular poly- saccharide of Pneumococcus Tyne III, and the extraction from cultures of this bacillus of the active enzyme rcsnonsible for this action on the polysnccharide. This enz;rc,c is not only active in vitro but retains -----em its specific activity in vivo as damonstrated by the protective action afforded -e-m--- mice infected vith Pneumococcus Type III. The work of this year has been concerned chiefly with a study of tho action of the enzyme on the course of an experimental disease induced by infecting rabbits intra- dermally with a rabbit virulent strain of Pneumococcus Type III. However, before describing this study, modifica- .tions in the technique of preparation of the enzyme will be given. It will be recalled that the yields of enzyme were much increased by the addition of small amounts of yeast extract to the synthetic mineral medium originally used. Unfortunately, the preparations thus obtained exhibited a definite primary toxicity expressing itself by a sharp rise in temperature of the injected animals and at times death within a ferf minutes after injection. The production of non-toxic preparations has been attempted along three different lines: (a) the amount of yeast extract in the medium has been reduced to the minimum concentrat?on comnat- ible with good yields of enzyme (.03 per cent); (b) toluene, which had been a,.ded to some cultures in order to achieve a more rapid and more complete extraction of the enzyme, has been completely eliminated from tl?e operation; (c) the auto- lysate is now purified by a process of differential adsorption on aluminum hydroxide; this process is such that, under well defined conditions, the toxic princinle is adsorbed on the aluminu;n gel while the enzyme remains in solution, Concentration of the enzyme preparations has been achieved by two different techniques: (a) distillation under reduced pressures at temperatures not exceeding 35OC; (5) ultrafiltration through a cellulose acetate ultrafilter; in this case the enzyme remains in the filter while most of the irrelevant material is eliminated with the filtrate. Throughout the steps of preparation, purification and concentration of the enzyme, the operations have been controlled by quantitative determinations of activity by the titration method.described in the previous report. Te have- found it convenient to express the potency of the preparations in terms of units. The unit is defined as 100 times the minimal amount of enzyme required for the decomposition of . 01 mg. of chemical ly purified capsular polysaccharide in 18 hours at 37b C. The purified, concent rated enz:`me preparations exhibit little if any toxicity for normal rabbits when in- jected intravenously. Xormal monkeys which received very , HZW large amounts of these preparations by tke intravenous route exhibited no reaction recognizable by change in the t~~mpCra.- ture or behavior of the anim;ll. However, the study of the blood picture revealed a sharp rise in the white cell count for a few hours following injection. Whatever the nature of the leucocytic reaction, we are inclined to consider it as beneficial in view of t;?e essential part played by phagocytosis in the course of events following enzyme treatment of infected animals. 2 The protective 2------ action of tke snecific enzyme in ----------__--------_-e-_---e--- ----- dermal infection of rabbits with Tzne III Pneumococcus. In a ___--_-___--_-___--_-------------- . ..------------------- previous report it has been shown that the enzyme which snec- ifically decornooses the capsular polysaccharide of Type III Pneumococcus has a marked protective act!.on In mice infected intraperitonc~~lly with this organism and is capable of bring- ing about the recovery of these animals V"nen given even after the infection is already esta31ishe.d. The intraneritoneal injection of mice with pneumococci leads quickly to a general- ized infection. On the other hand, the dermal infection of the rabbit, previously studied by one of us. in detail, gives rise to a disease which is primarily localized and in which the infec.ting organisms in the local lesion are less accessi?le to the action of an agent administered intravenously; Hamever, the specific enzyme has a marked curative action in the disease brought about by infecting rabbits intraders?ally with a highly virulent strain of Tyne III Pneumococcus. The use of this enzyme in suitable amounts in the infected animal promntly frees the blood stream of bacteria, leads to a 231. H 41 . sterilization of the focal area of infection, and a conseauent complete cessation of t.:e disease process. A mortality rate of 5 per cent in treated animals is in sharp contrast to a mortality rate of 95 per cent in untreated rabbits. The curative action of the enzyme is type-soecific. Its activity is ciestroyed by heat. The results of this study yield further evidence that the capsular .substance is an important condition- ing factor in pneumococcal infection, since, in so far as `xno wn , the only action of which the snecific enzyme is capable is that of decomposing the capsular polysaccharide. Animals which have recovered following enzyme injections within 24 to 48 hours after infective inoculation show no active immunity against subsequent massive reinfection, On the other hand, animals which were treated reneatedly over a period of days with smal 1 amounts of enzyme and in which the disease was not arrested until the fifth to ninth days, show a high degree of active immunity against reinfection. An elaboration of one phase of these studies has dealt `with the question of the minimal amounts of enzyme necessary to bring about recovery of the infected rabbit and with the correlation of certain factors involved in these quantitative relationships, Drs. Dubos and Avery have shown, by in,s&4,r~ experiments, that, after a ciefinite incubation neriod, the total amount of specific substrate decomposed bears a quantitative relationship to t:;le concentration and activity of tne enz.,ime TreBarat ion used. If it were possible to amply 232 H 42 this finding to the experimental infection, t::e minimal amount of enzyme necessary to bring about recovery should have a quantitative relationship to the amount of specific capsular polysaccharide in the animal bo<.y. This substance Is of course associated with the oneumococci but may also be present in soluble form entirely free from tne mlcroor&anisms. It is technically Impossible to deterrilne the total amount of the specif lc polysaccharlde in the infected animal or even to esti- . mate the total. number of ;>neumococcl. The nearest aoproxima- tion Is the determination of t..e number of via3le pneumococci in the circulating blood. Other factors the same, this lnd.ex should, on theoret ical grounds, bear some quantitative relation- shin with the amount of enz;`me necessary to bring about . recovery. A series of 85 rabbits has given definite evidence that taia is actually the case. For exnmnle, 2.5 units of enzyme were sufficient to bring a3out recovery when the bacterlemla amounted to 3 pneumococcl per cc. of blood, 6.6 units sufficed with 15 organisms per cc., 20 units were re- quired with bacteriemias between 100 and 1,000, 50 units sufficed with bacteriemias between 1,000 and 5,000. However, 100 unit a failed to bring about recovery when the number of pneumococci per cc. of blood exceeded 20,000. , The results Indicate that as the bacteremia in- `creases the amount of- enzyme required to bring about recovery is greater although the two ratio between the two factors is not :?roportlonal. Studies of the s>eciflc action of the enzymes on Type III infections in mice and rabbits have furnished con- vincing evidence that this agent exerts a favorable influence on the course of the oisease in these animals. `Interesting as these results are, it is at once aypal-ent, however, that the experimental conditions under tvllich the enzyme has thus far been tested are not entirely comparable to those prevailing in pneumococcus pneumonia. A crucial experiment has therefore been undertalren to determine whether with this particular tyne of Pneumococcus an experimental pneuzmonla can be produced in monkeys which in clinical course and p,qthology more closely resembles the se>ontaneous disease in man; and finally, whether the enzyme would be effective under tne more aromylicated con- ditions of an actual consolidation of the lung. For this study, tr.e Java (I?, Cynomologos) monkey was selected; since other workers in attempting to produce exper- imental pneumonia, have found the more common I!. Rhesus and the Cebus species rather unsatisfactory. In addition, the Java monkey has proven to be somewhat hardier and rarely in- fected with tuberculosis. A strain of TyQe If1 Pneumococcus, virulent for rabblto, was chosen for the original exoeriment; and this strain as recovered from an infeated monkey` has been reinoculated serially into the succeeding animals. The use of morphine, suggested by Robertson and his co-workers in the production of experimental pneumonia in dogs, has been adopted, for it was thought that morphine would abolish coughing, perhaps reduce the resistance of the animal temp- orarily and hence allow the organisms to gain a foothold in the lung. The drug is administered 2-3 hours before inocula- i t ion. Roentgenogr&ns of the chest are taken before inoculation : as control, and at least once daily thereafter, i: The technique of the inoculation is as follows: The animal is anaesthetized with ether. Yhile the jaws are held widely open, the tongue is drawn forward with lingual forceps, the epiglottis grasped with plain forceps, and the larynx thus made clearly visible. A h?.rd rubber catheter is passed into the trachea until resistance is encountered. The organisms centrifugated from a fresh culture and resuspended in 0.5 cc. of soluble starch solution are introduced from a syringe fitted .- ,: into an adaptor in the end of the catilcter. The material is ' ,' :, ? ii 4 allowed to flow into the trachea while the animal is held up- right. An equal volume of air is then slowly injected to ensure the emptying of the catheter. Care is taken at all . times to avoid contamination with material from the mouth. Following the inoculation, daily blood cultures, blood counts, and .tempcrature records are made in addition to the x-rays. By thia method it has been possible to produce a pneumonia which is dtstinctly lobar in distribution and tends progressively `t`o iriv'olve `Sore `than .`one lobe. ,Fhe-:sp`re&d df *I; the lesion `hiAs `been `clearly followed by means `of the roentgenograms, Ab autopsy the gross pathological findings are quite comparable to t:le typical lobar pneumonia in man. This similarity has been further confirmed by the microsconic exa=lnation of histological sections. Although the deaonstration of lobnr Dneumonia in monkeys has been successfully accom.nlished, certain obstecles have been encountered mhich have delayed the study of the therapeutic capacity of tke enzyme. These are primarily the variations in the resistance of t--e individual monlceys, and, consequently, the difficulty in establishing a ;?redictable course of disease with a minimal standard dose of micro- organisms. Within a narrow range of dosage, 0.2 - 0.4 cc., one animal may develon pneumonia and die in 48 hours, with massive pulmonary involvement and overwhelming septicemia, whereas another JULY survive a well-developed, progressing pneumonia, with or without septicemia, after 7 - 8 days. With tile repeated passages of the culture and the use of morphine, however, it is possible to produce a fatal pneumonia in practically all cases. Since the purnose of the experiment3 is to attempt an evaluation of the effect of enzyme treatment on the pneumonia, it is highly desirable to be able to induce infec- tion which will run a uniform course of 4 to 5 days, termin- at ing fatally when untreated. This would enable one to have fairly accurate information of tile animal's condition before ' any therapeutic interference was' attempted. At present, the effort is directed toward balancing the host factors and those of the invading organism to tnfs end, It is thought that these factors can be fairly well controlled and that the study may soon be carried to a definite conclusion, H 46 236 , II. Isolation of microorganisms decos73osing the ------------------- ----------------- -.--- caosular nolysaccbaride of different tees of Pneumococcus. ---------e-- -----------.--- --------.--- _--------.---- m4--__ (Drs Dubos and Goodner.) The search for other microorganisms -- L--------A---------- deco-nosing t-ie specific polysaccharides of PneuT;ococcus Ty.?es I and II is being continued. In principle, the mot-hod remains t`ne same as that elaborated two years ago for the Isolation of t3e bacillus capable of decomposing the Tyco III capsular uolysaccharfdc. The method consists in tile use of very specific media contain- ing the substance to be decomposed as sole source of enorgv; these media are incubated under a great variety of conditions so as to bring out as many as uossible of t2e organisms potontially capable of decomposing tile substance in question. In fact, several cultures (at least 5) have been obtained thus far which are canable of decomposing the Tyoe II spccif ic substance, but none of then has been sufffciently purified to warrant a statement as to the nature of the active organis:?. The work has been rendered difficult by the fact that all these cultures decompose the polysaccharide only very slowly in liquid media, although some `of the? are quite active in sand and soil media. An interesting lead has becn t`ne successful preparation during the past few weeks of catalysts which, when added in small amounts to tLc liquid medium, greatly increase the rate of decoriposition. Concerning the nature of these catalysts, we have no real knowledge, but there is some suggestion t'hat they may be soluble organic compounds of iron. H 7 ii37 III Che30-imnunolo~ical studies on conlusa4atd- -,`-------.--v.-e.---- _____I.*___________ cnrbohvdratc-uroteins I------&----L--z (Drs. ivc rlv GocScl and. i3ab?rs.) ------ ---,,-,,--,,,,,-,-,----,---------- 1. TLC sT>ccif icitx of antigens Ere3arcd bx cor*.biniz,:, ---------A--- -__-_-__ --__ ___-_____ ,,,,-,,--2 CL and -P ~lucosidc of ~lucosc VithJrotein. -----_ ,,a-,,-,-,,---- _-_________ _-_____ The study of antigens prepared by c'ne;l,icalljt combining sugar ierivatlvcs with protein has revL>alcd t-c fact that carbohydrates exert a de ter:.ining inf luoncc on t;ie in?unological specificity of compounds of xLich they form a part. In preceding studies evidence has been acquired that mere diffcrenccs in the structural conf lgurat ion of relatively s.zall grouw in the hcxose noleculo may deterline the serological snecif icit:: of tro sugar derivatives icient ical gave in this one respect. As proviously shown, t'nef -glucosides of glucose and galactose differing from each otncr only in tho interchange of the I! and OH groups on a single carbon atom, exhibit an individual specificity irrespective of the nrotein to n>ich they are A attached. These, earlier studies concerned the?,selvca with the s?ecificity of sinilsr glUCoEide8 of two different hexoses, glUCOS0 and galactoso.: J4 paper now in press deal8 Vith the specificity -of diffcr,ent glUCOBide8 of the 883318 sugar, namely the I'X and B form8 of gluCo80 coupled to globulin. `In both the Wand e antigenaft thus formed the glucosidic linkages to the protein differ from each other only in their snatial relations to tse rest of t.-.e antigenic molecule. Since the ultimate co::positioq of both antigens Is the same, the differences observed in their immunological specificity are referable only to known differences in the nolecular structure of each. The results show that the mere chmgo in the sl?atial ---- .-- H 48 23R arrangement of the grourrs on the first carbon atorn in these two derivatives of glucose suffice to confer on each antigen a ;;lnrked degree of dlffcrcntial s->ccificlt;r. These structural chenges are so sharply reflected in serologicnl. s?ocificitg that it is possible by means of i73une serum to diffcrentip.to select ivcly betucen the tW0 iSo?~ric glucoeides of the sare sugar. However, tjno antibodies present in both 1.X and p antisera cross react with the hcterologous test antigen. Recognizing tho diffarenco in the chani cal structure of the two glucos idc's it becomes necessary to account for the over- lapping specificity of the two 8UbBtanCes. The chemical basis. for the immunological crossing a-3nears to lie in the fact that the spatial arrangement of the polar groups on the remaining five carbon atoms 18 identical in both glucosides. This partial similarity of molecular groupings night then account for the degree of inxunological likeness oxhibitod by the two substances. Although In a portion of the molecule of both glucosides the structural relationship is identical, noverthe- less they behave' c,hezically as separate chemical entities and posseae eerolo~gically a separate and distinct specificity. * For, if complete reciprocal Inhibition of precipitins i's " `, accepted as the criterion of serological Identity, then the failure of both glucosides to exhibit this capacity may be taken as further evidence of differences in the immunological specificity of each. The lack of reciprocal absorption of agglutinins by two organisms mutually agglutinnblo in the ix.une serum of ---_ 239 H 49 c?.ch is generally conceded to indicate a lnc?.c of ix-?unologicr!l ider,tity. Relationships of this order are !rnov!n to exist bctnocn Pncuxococcus T;pc II and Fricdl&nder bacillus Type 3. In both instr?nccs the rcactivc substance has been identified chenicnlly as t-lc specific polysnccharidc oeculiar to the cn~sulo of each organisz. While tse structural constitution of those co';lnlejt: sugars is not as yet fully known, considerable ?rnor-rledg;o has bocn g;nincd conccrnlng their cholicnl pro?crt ies. For exnrpla, it is knosn that the Type II palysacch>ride is built up of ~lucosc units aEd that che.`.~lcally it bears a close rosazblanco to tho ?olgsacchnrids rocovercd fro:-1 the Tync 3 Friedl#nder bacillus. Zonevcr, the two substances are not che:ically identical, although tile sirjilarity between the'?. is sufficient to result in a certain likeness in ia-unological spccif icity. In the absence of precise knowledge of tae structural relations of the two polysaccharides, it seem reasonable to assuze taat both contain in a portion of the cortplex aolecule t0e `sar,e or a closely sinilnr configuration of atoms, This sicilarity of molocul~r grouping 2ight then account for t:le in:~unological eiailnrity of the tmo eubstances, Considerable evidence for this point of view is found in the reeults of the present study concerning the snecificity of the ti and p glucosides of glucose; A comparison of th'e serological relationships between the two isogeric derivatives of glucose nnd the capsular polysaccharidcs of the two organ- 163s in question is presented in the foll.o:ring Tn'?le. i , 240 c H 50'. Corwz2son of tile _______ p,----~--- ____ -_-__--___--_--- Serolo-ical Rela.tionshin betneen Iso~~r~._c_ --- -_------L- Derivatives of Glucose and the Cal)sular Polysaccharide ,f -________-__-----_-_- ---.--_-e-------e- -____ > ____-_ ------ TYO Unrelated Ssecies of Bacteria. ,`-----------------------`---`- --------------..-- i 0 I , !-lucoside j test test 16 1 antigen A---- /=z-~====5-------- !antiizsL-- --==L;-== ___--- i l#nder --- a------ $y addf- I tion of -------- P ;lucoside : --w-m--. CL glUCO8ide ._----~ i ited for ; for i for -------c- ----- -------- -I .Fried-L Pneumo- , j I coccus ; i I Tyne 3 i Ty?e II, i I 1 B.Zried- ! I I I ant i&ens I ! Tyne B -e--u-- I- I -----v c- 43 Tneumo - r A,--- m-u----- Pneumo - Friedl#nde I , test ? ???? ? o COCCUS I 1 COCCUS Tyne B antigen 1 antigen _IType II 1 Tyne II i I .w-- U-L-CL-e d--B -c-e- -,,-,-- ! ; lander 1 L-L- 1 i k/The test antigens in. all instance8 were prepared by combining the respective glucoside with a protein biologically unrelated to that in the imsunizing antigen, Analogous relationshiDs extending even to the cross i:,:munity reactions and t.le lack of reciprocal absorntion and inhibition of antibodies are evident in both the grouas of simple and co?piex carbohydrates. While the comparison is instructive it is not necessarily valid in the case of the capsular polysacc.lar ides, for ti,e final internretation of these relationships must await further ?:norvledge of the structural relations of tile more complex bacterial sugars. A possible explanation of the cross relationships between the Ty-)e II Pneumococcus and the Tyne B Friedlander bacillus has been advanced by Enders in his recent study on the presence in Pneunococcus of a type-specific agglutinogen unrelated to t'ne specific carbohydrate. In the case of the synthetic antigens containing OiCand ' P compounds oI` glucose alone, the evidence indicates that the iwtiunological specificity of tile reactive glucosides is determined by known variations in chemical constitution and is independent df tile protein to which they are attached. In view of these findings it seems not unlikely that in the case of the polysaccharides because of their mare complicated structure and the greater nossibility for variation in molecular configuratian, here may be found many instances of a similar overlalqing specificity among `carbohydrates of unrelated origin. In attempting to interpret the relationship between the chemical constitution and t:;e serological svecificity of carbohydrates, especially those of bacterial origin, it is of obvious advantage to begin with a study of the more simple --.- ._ mono and disaccharides. In these sugars both the chemical composition and t11e spatial arrangement of the groups Within the molecule are definitely known. YIith this end in vie; the fr>llowing study of disaccharides is in progress. 2. The sxnthcsis of zlucosides of tile di,s,acchnrides ------- -,,,--,-,-P ---_____________,________,___,) cellobiose and maltose --,-,,---,-------.--A (Drs. Goebel and Sabers ) The ------ --.- -------- m--,-L opportunity for inter-molecular differences in structure is almost infinite in complex pol;sacchnrides such PS t3e tyne- specific ca?suler polysaccharide of pneumococci. These ooly- saccharides are made UT? of disaccLarido units which are in turn composed of a simple hexose and a hexose uranic acid. Very little is known concerning the manner of lin!:Rge of these disaccharides or the units colO>oslng them. Still less is known of tLe effect w;,ich internal differences In lin?cage may have on tile immunological specificity of t5e Tolysacchnride. It seems, tileref ore, quite important to determine what effect differences in linkage of tne `nexose units, would ~ have on tile `immunological relationshins of two disaccharides of known struct'ure. A&tos~s, ` the diiacciaride ob'talned by `the partial hydrolysfs of starch, and cellobiose, that obtained in a similar manner from` cellulose ,. have been shown to be composed of two glucose units, tse aldehyde groun of one being linked to the fourt3 carbon atom of the other hexose. Thus maltose and cellobiose .are each glucosides of glucose linked through the same carbon atom. These disaccharides differ, however, in that maltose is an alpha glucoside of glucose while ---- cellobiose is a beta glucoside. This difference is best shosn by tile following structural formulae: Cellobiose The free reducing group of each disaccharide is indicated ny an asterisk*. The para aninqphenol glucosides of maltose and cellobiose havobeen pre?ared, diszotized and coupled to protein and tile resulting antigens used to immunize rabbits. T'iiie iz.:;unological work is now being carried out. IV Studies on t:,e interconvertibility of the --L-e ----------_-_-___-__---------- _______ gecific tpues of Pneumococcus, (Dr. Allomax.) Studies have -------- -----c----.-------- ----dI--- been made during the past few months on .the nature of the , phenomenon first described by Griffith in 1928, and subse- quently investigated by Neufeld, Dawson, and Sia, dealing with the transformation of R organisms derived from one type of Pneumococcus into S forms of a different type. These in- vest igat ors induced the change through the use of intact bodies of heat-killed S pneumococci either injected Sub- cutaneously into mice, * together with a a+z~ill InoculurL of living R organistls, or used in vitro together with anti-R --e--e- se rux in a medium Inoculated with R forms, The R cells assumed the properties of trle heat-killed S organism used in the culture rcediux. ----.--.----.._ - -_ 244 H 54 The phenomenon is of unusual biological significance, for it is a unique example of a complete transforzation of the biological characters of a yicroorganisn by senns of a specific sticu1us. An organism thus transforrjed not only acquires a type-specificity not present in the R cell, but it undergoes definite mornhologlcal changes, develops a high degree of virulence, and possesses antigenic propcrt ies which differ markedly from those present in tile original bacteriu?,. These properties, once acquired, are retained by the organism indefinitely under suitable conditions of gromth. The acquisition of these new characteristics is always associated with the regaining of t:?e function of producing the snecific capsular polysaccharide. It seemed worth while to attempt an analysis of the substance present in the heat-killed organism essential in the reaction, and to learn more of its general nature. Atten- tion has been directed, therefore, towards the separation in a flltrable, cell-free state, of. the substance active in effecting transformation. Studies already published have established several facts. Cell-free extracts which are atitive in causing transformation in type may be obtained from S pneumococci dierunted by repeated freezing and thawing. Such extracts under suitable conditions may be filtered through Berkefeld candles, and thus separated from all intact organ- isns. Avirulent R pneu?ococci derived from S forms of a specific type may be changed by growth in broth containing ant i-R serum and the filtered extract of S nneu%ococci of a different type into virulent S organisrqs identical in tyue with that of the bacteria extracted. The constituents of the extract suqply an 3ct ivat ing stimulus nf a snccific nature in that the R pneunococci acquire t53 capacity of claiornt ing t:;lc capsular material peculiar to t-le organisr:s extracted. Subsequent studies have shown t.`r.ot sti.11 further purif icnt ion can be ncco:~plisLcd by t:;e adsornt ion of pneuyo- coccus extracts on alu?.inlu-7 gels. Yost of t e protein and lipoid constituents in the extract are carried dolrn nith the aluziniu3, the active substance reT,aining in the su:>crnatnnt fluid. This supernatnnt, a mater-clear fluid, is snccif ically as effective in inducing transfer-ation in tyue as are tile suspensions of whole, heat-killed organisms. It is hoped that by further procedures such as selective adsorption, selective dialysis, and cLer.:ical analyses, more exact knowledge of t.?o active substance may be acquired, and an explanation provided for a phenomenon which possesses aide biological significance. V. The De&gee and Duration of Active and Passive -e-w--- ------------------------______cI_ Immunity to Pneumococcqe in Rabbits (Dr Ernest Stillnan.) ------ --B------Y- ,-,~-----.mP eMA-------------e-w . ( The experiments, `previouely reported, on the persistence of pneumococcus antibodies in the sera of rabbits following various innunizafion procedures is still in progress. The trend of this work Is illustrated by the fact that the serum of one rabbit, last exposed to inhalation of virulent Type I pneunococci 6 years ago, still contains sufficient nnti- bodies to protect mice against large doses of pneumococci. In colo.borr!t ion ?:it.-! Dr. Good.-cr, c l:>r;e series of rabbits have been given identical courses of injections of a sus.pension of heat-killed pneumococci, containing a mixture of equal amounts of Pneumococcus Types I, II and III. At monthly intervals groups of these animals are being examined as re- gards the agglutinin titer and mouse protective value of their serum, and as to actual active Immunity or~suscepti3ility to : `: `3 ! L: / !.I ts I, 1 I infection by the dermal route. The dermal infection perTi of a thorough evaluation of the active immunity since the results do not depend so much on survival or death as they on the clinical findings. do This investigation has now been under way for four months. While it is yet too early to predict the duration of immunity it has becdme apparent that it persists beyond the time of the disappearance of circulating antibodies. Further- more, it has been found that tLe immunity against the Type III Pneumococcus is the first to disappear, and the indications are that the -immunity`to Type 1 will have the longest duration, Production of &xgkrimental- pneumonia in animals. ---------- --y* ------ ----------&---- The'development of'a>,ttchnic.for' the T)roduction of experimental .,.i i.- ,,_. _ , lobar pneumonia"in labi;atoky `anikale.`is' stlii :biiiig"ktt;`ktiit`id;' ., Although by the'inhalatlon'nethod pneumonia may occa~ionslli' ' be produced In partially immunized mice under the influence of alcohol, these animals are too small for detailed study. Ex- periments have shown that partially immunized rabbits, whether infected by inhalation or by nasal inoculation, fail to local- ize the infection In the lungs, Although guinea pigs are subject to spontaneous epidemics of pneumococcus lobar pneu- mania, they are highly resistant to exnerimental infection with this organism. Numerous atteqts to raise the virulence of pneumococci for guinea pigs have failed. Various methods for lowering the natural resistance of guinea oigs are now being employed with the hope that these animals may become more susceptible to pneumococcus infection. VI. Publications -------,-----em' Alloway, 5. L. The transformation in vitro of R pneunococci ------- into S forms of different specific types by the use of filtered pneunococcus extracts. J. Ex? Yed ,-----z,,-,'* 1932, 55 91. --) Avery, 0. T, and Dubos, R. The protective action of a specific enzyse against Type III pneunococcus infection in mice. cl,-Exp,E&_d., 1931, 54, 471. Avery, 0. T. and Dubos, R. The soecific action of a bacterial enzyme on Type III pneumococci. Trans. Assoc. ------------- Amer. Phys., 1931, 4&, 216. Avery, 0. T. and Goebel, Wd'F, Che,To-immunological studies on conjugated carbohydrate proteins. V. The immunolog- ical specificity of an antigen prepared by combining the capsular polyaaccharide of Type III Pneumococcus with foreign protein. J. EXQ. ,L, 1931, 24, 437. Ye d Avery, 0. T. and Goebel, W. F., and Babere, F. Chemo- , immunoiogical studies on conjugated carbohydrate*' proteins. VII. Immunological specificity of antigen6 prepared by combining the ti and p-glucosides of glucose to protein. J, EXJ% Iled., 1932, (in press). -m-w Dubos, R. and Avery, 0. T. Decomposition of the capsular polysaccharide of Pneusococcus Type III by a bacterial enzyme, J. Exp, Hed., 1931, 55, 51. -m-e --- Dubos, R. Factors affecting yield of specific enzyme in cultures of the bacillus decomposing the capsular polysaccharide of Type III Pneusococcus. J. Ex-3. I'ed., 1932, 55 ----------- -- Dubo s , Ft. and Baudisch, 0. Uber Katalasemirkung von Eisenverbindungen in KulturTyedien. Bio che- 7eit -------L-k,--' 1 1932, (in press). Finkle, P. Eetabolis?? of S and R for+:s of Pneumococcus. J Exn 1.`~ d -`-e-i-L-s--' * 1931, 53 --@ 551. Francis, T. Jr. and Tillett, iY. S. Cutaneous reactions in raibits to the type-specific capsulnr polysaccharides of Pnouxococcus. J 2x33. ired. -L------z-- ) 1931, 54 587. --* Francis, T. Jr. and Tillett, `ii. S, The significance of the type-snecific skin test in the seru.2 treatTent of nneu?onia, Type I. .J. Clin. InvL, 1931, N, 659. Francis, Tho.l:as Jr. The identity of the Techanisms of tjrne- specific agglutinin and precipitln reactions aith ?neuTococcus. J. 9x9. i'ed. ----,-,,,-,) 1932, 55 55. --I Goebel, Y?. F. and Avery, 0. T. Che-zo-iT,aunologi cal studies on conjugated carbohydrate nroteins. IV. The synthesis of the p-aminobenzyl et.ler of the soluble specific substance of T;?e III Pneuaococcus and its coupling with protein. J. Exp. ,L, 1931, 54,481. He d ---- Goebel, V. F., Babers, F. and Aver;?, 0. T. Che.-o-itn-unolog- ical studies on conjugated carbohydrate proteins. VI. Synthesis of p-a-ninophenol ui glucoside of glucose vith cou?ling to protein. J. 3x1). , , , l!ed 1932, (in press). Goodner, K. The development and localization of the dermal pneumococcic lesion in the rabbit. J. EXE. Med. ---- ,,,---* 1931, 54 --* 847, Goodner, K., Dubos, R. and Avery, 0. T. The action of a specific eneyme in Type III pneunococcus deraal infect ion in rabbits. J. EXE. tbed. --we -_---- * 1932, 55, 393. Goodner, K., Dubos, R. and Avery, 0. T. Studies on the quantitative action of a specific enzyme in Type III pneunococcus dermal infection in rabbit. (In manuscriotj Kelley, W. Antipneunococcus immune reactions of nortial ewine serum. (In rzanuscript,) Rhoads, C. P, and Goodnor, K. The pat,lology of experimental dermal pneumococcus infection in the rabbit, J. EXE. Med. ---- ,,----) 1931) 54, 41. Stillnan, 3. G. Duration of de-?onstral?le antibodies in the serax of rabbits in -unized with heat-`-illed T>:>e I nne-mococci. J. 3x~. I!ed., 1531, 55, ----- 515. Still-;an, 2. G. anb aranch, A. Locali?a,tion of pneu9ococci in tic lungs of partially i.r;-?unize& zice f;;i;sing inkalation of pneil:-:oCocCi. JJ E:x:>. ,--L, xe 6 I 24, 323 .