$p-J H 82 Rcnort of Dr. Avex with Drs. StillTan Gocbel ----1,------A Dubo s ---tie--------- -------- I-- ---,-A Francis ---- L Babers, Goodner, Terre11 and Rogers. ------------- ---- I. Type III pneumococcus pneumonia in monkeys, (Drs. Francis and Torrell.) 1. Experimental production of disease, 2. Treatment with specific enzyme, 3. Pathology. II. Nature of lysis of Pneumococcus by nutolysis, bacteriolgtic enzyme and bile. (Dr. Dubos.) III. Study of the antigcnic comulex of tyne-specific pneumo7 cocci. (Drs. Avery and Dubos.) IV. Methods of purification of enzyme decomposing the capsular polysacchnride of Tyae III Pneumococcus. (Dr. Dubos.) V. Isolation of pure cultures of microorganisms decomposing the capsular polysaccharide of Type II Pneumococcus. (Drs. Dubos and Goodner.) VI. Pneumococcus dcrnal infect ion in rabbits. (Dr. Goodner, ) 1, !Chern.peutic action of ant$pneumococcus sera. 3 4. Deter-inatfon of active immunity by dermal infection, 3. Effect of pneunococcus autolysates unon pneumo- coccus dermal infection in rabbits. , . VII. Study of pneumococcus infection and immunity in rabbits i " by the inhalation method. 1. Pulmonary lesions produced by inhalation method. 2. Duration of de.i:onstrable antibodies in serum of rabbits following inhalation of Pneu?lococcus. 3. Active and passive immunity in rabbits. VIII. InterconvertiSilitp of specific types of Pneumococcus. (Dr. Rogers. ) H 83 IX. Study of the chemical nature of antibodies in antipneumo- coccus serus. (Drs. Goebel and Avery.) X. Derivatives of glucuronic acid and glucuron. (Drs. Goebel and Sabers, ) XI. Synthesis of p-amino phezol glucosides of lactose and gentiobiose. (Drs. Goebel and sabers. ) XII. 5ibllography. ---ooo--- I. The Eroduction and treatzent of Txqe III ljncuzococcus ----A-- ---------------- ----- ------------------ pneumonia in monlcexs. -------I----- (Drs Francis and Terre11 1 ---`,,,,,,-,-,-,-------L In their first papers, describing the action of an enzyme of brcterial origin upon the cansular nolysaccharide of Type III Pneumococcus, Avery and Dubos also presented convincing evidence t'nat this enzyme is capable of decomposing the same substance when encountered in the animal body. Mice Vere infected with many lethal doses of T.:pe III pneumococci. It was:found that as late as 12 to 18 hours after the infection / . . 1 was established intraperitoneally, the administration of the enzyme brought about recovery in most instances. Furthermore, 1_ when the enzyme was given 24 to 48 hours preceding the injection ., < `, ,. `. ! `, of many lethal doses of Type III organisms into mice, it was found to have a marked protectire action resulting in recovo'ry of most of the animals. T-he mecllanis% of thn .tberapeutic effect was shown to bc due to its action on the Capsular poly- saccharide, decomposing it, and thereby rendering the bacterial cells vulnerable to attack by the phagocytic cells of the animal body. This stud3 was limited to effect of the enzyme on Ty~c III Pneumococcus in aq experimental infection primnrily septicomic in nature. Later, Goodner, Dubos and Avery studied the action of the e n z y i.: e upon the localized infection of the s`cin of rabbits (terineci by Goodner "dermal pnaumonia") nroduced by a virulent strain of Tybe III PneumococcuF. In this form of infection, ti?e lesion is primarily localized and is accorpanied >y scoticemia of varying degree in different aniaals. Here agair., A definite therapeutic effect of the enz:me p?as demonstrable as shown by procpt subsitience of the cutaneous lesion, and sterilization of the blooo. %owever, with the a-rounte of enzyme used in the studies, tnere was found t 7 `le a degrre of scpticemin above which tile enzyzlr was unable to control the infection, alt'n.ough the duration of lit'e of the rabbits might be prolonged over that of control animals. Since excellent results had been obtained in the treatment of septicemic and local infections of the smaller animals, it seemed advisable to study the effect of the enzyme under conditions in which the disease process more close- ly resembled that in tho human patient suffering fron pneumo- coccus lobar pneur;l.onia. In this disease, the organ involved is highly vital, the course is comparatively constant, the pathology typical kid recovery nroblcmatic. Consequently, an atte.,pt was rade to produce in monkeys a disease resembling lobar pneumonia in man, which could then be subjected to treat- ment with the specific enz:*me under conditions more closely aDpron.ching t:lose encountered. with Datients in the hospital wartir I The Java monkey (?`. cynomolgas) was selected for the purpose, and t;:is cnoice has proved to ba a fortunate one, since . the K. rhesus is much more resistant to the strain of Type III Pneumococcus employed. In the first part of the study the animals were given morI>hine and then under ether anesthesia a small catheter VJRS inserted into the trachea; the inoculum, consisting of organisms centrifuged from a given amount of culture and resuspended in 0.5 cc. of soluble starch was allowed to flow into the lungs. During the procedure the animal was tipped to t,i,e rig,:lt with the hops of localizing toe material in the right lower lobe. Tnis technic was, in the -naJority of instsnces, successful. Eowever, the location of the infection was not always predictable, since the exact site of the catheter at the time of inJection was not known. At tines, too, during the recovery from ether anesthesia, the monkey often went through a stage of excitement, wit:? hyperventilnt ion and exaggerated physical activit), or the reverse condition with cessation of breathing which required art if ic ial respiration. Such incidents apparently resulted in pneuaonia scattered diffusely through the lungs and accompanied by a high septicesia. In spite of these variable factors, localized lobar pneumonia was Droduccd, in a high proportion of the animals. , In the latter part of the study, a more accurate method has been employed using the technic described by Terre11 and Robertson. With this method, the animal is given a dose of morphine sufficient to make it ouiet. The animal ls placed in the supine posit ion, and a s-1, 11 radio-opaque catheter is, inserted into the trachea. Yith the aid of the fluoroscope the catheter is tien guided with direct visual control into the -223 H 86 bronchioles of the lob6: or lobules, in which the infection is desired. When the catheter is so placed, the organisms sus- pendcd in 0.5 cc. of 5 per cent cornstarch emulsion nrc injected. The method has several advantages: First, it eliminates general. anesthesia; second, the site of inJection is known accura.cl;:; third, with better localization the number of organisms requirrd for infection is somewhat smaller and the scpt iceaia conseouent- ly tends to increase more gradually. Throu;hout the study, temperatures were recorded 3 or 4 timts daily and the course of the pneu;:onia was followed by daily roentgcnograms. Daily blood counts and blood cultures were mnde. In general, within 18 hours after infection, n definite well localized pneumonic consolidation can be seen in the X-rn;. Concomitantly, a gradually rising fever is noted. The white blood cells may be increased in number, or, if septicemia is present, the:r rnaJ' have docreased. The lesion continues to spraad so as to involve the entira lobe and may extend to other lobes. T5e temperature remains elevated, the septicemia increases, and the leu!iocyte count tends to fall progressivel;. Death may follon'in 4 to 7 days in the typical in a certain number of instarices' I casa. On ,the other hand, associated'at times with either a diffuse infection of the .lungs or with the well localized lesion, there is an immediat.e fall' ` in leukocytes, a heavy seTticea$e and a tendency to subnormal temperature with a rapidly snreading-lesion and an early fatal termination. There is also a grouo of monke.ys in which recovery takes Flace spontaneously after 3 to 7 days. In these, sent icey;ie X `1 y bo absent or slight in dogrec, although in two instances the .jnctcre:lia hns Seen comparatively high. There is a tendency of + <. . . is group to rps-Jond with oefinite fever, leu%ocytosis and a :,t?ll localized lesion, but with relatively less spread of the pneumonic process. The spontaneous recoveries occurring in, this ;:roup make it difficult to sp~a'c of control ani-:als in the usunl :;ensB, since the factor of individual variation, so far as ae have been able to discover, cannot be eliminated or allowed for. This is especially true when the purqose of tko experiment makes a comparatively long, but gradually progressive and fatal course t`ne most dcsirs.ble, From the point of view of the production of pneumonia, of course, it does not matter, but when an attcnpt to evaluate a therapeutic agent is made, the ideal is to annroach absnlute comparisons of treated and untreated animals. This has not been possible. The studies to date include the results in 82 monkeys with definite pneumonic lobar consolidation of various degrees. Of these, 20 animala, or 24.4 per cent, had no septicemia and all recovered. Wenty-three animals, 28 per cent, had rapidly %.I mounting `septicemia greater than 5,000 colonies per 1 cc. of \ L , blood in the first three' days. All of -these animals died. The remaining 'animals Were `subdivided in-to two groups' on' the. basis ; .I of the degroe of septicemia preaknt: (1)' those' in' which 8eQtiCenfa~ was present but not greater than 250 colonies per 1 cc. of blood in the first 3 days; (?) th ose in which the septicemia ranged between 2$CI and 2,000 colonies in the first 3 days.' The first of these two groups co?rprises 2i animals, or 31.9 por cent, and cernna 14.3 per cent, tho It is in *hpcfi ___^___.. -. .-. --- l_- -------_____2_ --- ._ two grouts that the conditions seem most fnvornblc to study the )ossib1c effect of the enz;`~e uyon t:qe course of tbc exoe:rilri-nt- nl LisePsc. The results o3tnined b;r trentmlcnt pre t?lbulnted b c` 1 o 1.1 , co+norins tLC Tr:ortnlit;J rntc in R serits of untrcqted flnimals wit?. t`nnt of si -,il:>r nni7als ri*CCiVing trc~twnt with the T';rnC 111 Dol~SrCCil. qride-splitting enzyr?c. The ?*ei-ison for ciividing t:;1: ;rou38 on the bns!.s of t:le first 3 cja~;s is t:lpt a11 of tL-,e a.ti~inls trant+:d, received t.ic o~z:~l-*f: in the first 3 days nitcr infection. Anirnrils Flth pnt?u:.oni:> and seyticerlie loss tilr,n 250 col. /cc. in first 3 dn:rs. jni 7:tls with 3ncu.:onin Fnti se~ticenin fro7 250 to 2600 col./cc. in first 3 days. --e-v- --_------_----------------- ------ -M---e- ----------------- Su*xAnr:* of both groups. I I The reaulte on the bn8i8 of coz'ynr~tive mortnlitlr figures I are striking. fn the animals of thoee groups which received treatment there have been no fatalities, whcreaa, well over 50 per cent of the untreated animals hznve succumbed. Five rrore animals nere treated on tillc first dag and. one on the second d.ay . after infection. In all of t'::esc a d&finite consolidat Ion VRS noted, in severe.1 t::r fever had renched 1050 to 1060, and the:r -- - H 89 seemed on a clinical basis to be suitable cases for the thera- peutic test; however. at the time of treatment their blood cultures were sterile and remained so. They all recovered. Six others rith very hi:;h initial septiceTias were treated and al- though they all died, one treated on the first da.., with a septi- cemia of lS,OOQ colonies per cc. at that time dici not oie until the fifth day, and then r*lth 2,000 colonies of Type III PneuTo- coccus per cc. Another with 25,OCO colonies per cc. on the second day of disease lived until the fourth da;,, end at the time of death the septicemia hPd been reduceo. to 3,700 colonies per cc. This mar-red reduction of the number of organisms in the circulating blood has been noted in others of the treated group in which deat;i occurred enrl,-. In addition to the efiect of the enzrvme on the sentice-ip, there is usually seen in the X-rays a definite nrompt effect unon the pulmonic consolidation. TSe spread of the lesion is limited and the advancing margin begins to clear. At the same time, the fever tends to subside and the animal is alert and active, and takes interest in his food and surroundings. The immediate constitutional effect of the enzyrre has varied with . different preparations and with the eeverity of the disease, , ,, Some preparations have caused a fall in temperature and, white blood count and have mad.e. the animals appear sicker. This ie esnecially noticed when the monkey is already extremel:? sick. In animals moderately sick there is a tendency to a febrile rise following treatment. It ma: be that the fall of the blood. count is partially due to an increased 705ilizat ion of tile leukocytes at the site of infection. .2g H 90 The treatment has usunlly consisted in the intravenous administra.tion of 10 Cc. of en2 -Kk?, var ,ing frnn 2.5 to 20 units per cc. Following tAc origin?1 t'rcatment , hlood cultures and white counts are -Lade a-d further treatment has been bqsec! on the results of' these stud.ies in conjunction with the general clinical nS>pr:arance and tLe X-ray cvidcnce. In some instances three treat-ilenta hove been given in a period. of 24 hours. In ti-ie later treatments the intrapcriton.al route has sometimes been emnloycc with satisfactory results, and p?rhar,s some rc- duction in the amount of general reaction. Al thougr. t!ic stud-, must be carried further, the results so far appear nromising. Becently, it has been our privilege to move tne monkey clinic to nuarters which combine man:' features that tend to ex9eoita the actual war":. It is honed that with more experimental ani-;als, and with further i-provemeqt in technical procedures of concentration ard purification of the enzgme, more potent and highly purified preaarations may be availablo and that unequivocal results of its theraaeutic effect may be obtained. Pathols1 As a side-product of the primary clinical study of - --- experimental pneumonia in monkeys, an excellent opportunity has: i been presented for the study of the pathalogg of the experikenti r* al disease in aniy:als dying at different times wi+.h various degrees and stages' of involvement. The microscopic preparations have baen made with the kind cooperation of Jr. Rhoads and Yrs. Whitehousc. TAe description of the microsconic lesions is limited tq sections obtained fro.-. the lungs of mon!ze-:s infected by allowing t-;e inoculu:: to flow b;; gravity to a given site. fJ$e H 91 In the animals dying early in the disease there has been an accoypanying heavy septicenia. AS early as 24 hours an ontire lobe may be inv>lvcd. Its color at this time is a dark bluish- red. It is large, firm and girlcas, except, pi;rhaes, at the .- extreme margins. There is usually a coderate degree of fibrinous pleurisy, sometimes a small amount of serous fluid. The sentuv between it anil adjacent 103es is obliterated by adhesions, On cut section, the involved lobe is comnnro,tivcly dry, but a viscous fluid, typical of Tyno III pneumococcus pneumonia, may be scraped off. t~lcroscopically, the most advanced nrocess may be seen in tAc portion nearest the root of the lobe, while extending peripherally different stages may be encountered. In the region of the main vessels .?nd bronchi :there ma;' already be seen a marked cellular exudate primarily composed of leukocytes. Fibrin has appeared in different parts. The alveolar ~~11s are moderately swollen and their canillaries are engorged. Ordinar- ily t the number of red blood cells in the alveolar exudate is not striking. The lymphatics of t:?e larger bronchi and bronchioles, although dilated, show little evidence of infection. TLe walls of tL;e' `bronchioles show little Infiltration, a.lthough L much exudate i& `present in their lumina. * The walls `of the blood 1 ,.: vessels `do `not ab+ear to b'e in'vo'lved. In this area" co&ara't'ively : f.ew organisms are seen. Extending peripherally in a gradual transition from the preceding phase, there is seen marked engorgement of the alveolar canillaries with swelling and desqunmation of the alveolar eplthelium. The alveoli are filled with exudate CO?- posed of edema fluid, moderate numbers of polymorphonuclear leukocytes and erythrocytes usually in small numbers. There ray be a few deposits of fibrin spicules and organisms Rre numerous in the exudate, but not notable in the walls of the alveoli. At the margins of the lesion the predominant features are the filling of the r-.spiratorg 103ules with edem,? fluid contDining I great numbers of brcteria and co:Dar?tively few cells. There may be extreme congestion of the alveolar capillaries so that the walls are outlined by erjthrocytes filling the CaoillariPs. The alveolar nails are swollen, but the outstanding feature is that, in snite of the numbcr of organisms in the material filling the nlveoli, the:f are infrequent in the nails of the blood vessels, the bronchioles nnd alveoli. In animals dying on the fourth to seventh days, one may find scvcrnl lobes completely involved, and the whole lung covered with fibrous pleurisy and adharent to the thoracic wall. The lobe which was the site of the original nneumonic consolidation is a slate grey in appearance, or bluish grey. It is large, fir3 and airless. At times a local area, red in appearnrlce, may be seen, The lung on cross section is reddish grey and ver:. ilry with rough, granular surf ace. Microscopically, most of the lobe will be seen to be in an advanced stage of the pneumonic nrocess. The alveolar ~~11s are poorl>: distinguished 4 because of the dense character of the exudate and the large amounts of fibrin. The alveolar caoillaries contain erythrocytes but are not prominent . In many phases there is a shrinkage of the exudate away from the wells of the sly-call, but strands of fibrin can be seen extending from one alveolus to another. In the terminal bronchioles the Trocess is auite marked. In the . areas of dense involvement, rsnecially where f i3rin is abundant, 3mJ H 93 fev organisms are seen .qnd tile number of leukocytes is 17~s. T&IF: fibrin Rnpen.rs to be ;ost aor':ed in nrc;7s adjncent to the blooc; vessels. There is practically no evidence to su.:gcst vnsculnr thrombi, and the recalls of the vessels are sur3risingly free of evidences of infection. In the larser bronchi, much exudate ma: be seen in t=lc lumen, but there is little infiltra- tion of the 19.~11~ even adjacent to nrens of marked nneuxonin. T:?c lymn_nhet.ics do not popcar to be infected. In certain ereos, peraeps most frequcntl; in the neripheral portions of the lobe so involved, one notes the npncnranco of large yollo~ish cells in the alvooli. Where the;? arc present the fi4rin is disapnonr- ing and tile pol;~norphonuclear leu!:ocy tes are such dir?inishcd. These cella are tkc tynical for.18 seen in resolution, and ere apparently actively phngoc::tic. In the other lobes, depending upon the durr?tion of involve- ment, the type of lesion apntars to vary nfth the age. In some instances there seeys to be an area of direct extension from lobe to lobe, but ueually the,oldest lesion is seen about the hilus, nkile in tne lobe in which the. process is Fast recent : : : the periphe-ral portions may show engorgeaent of the alveolar capillaries with much ede.Gcn fluid an'd many organisms in the .I , alveoli typifying the advancing m.argin of the spreading infection, In animals sacrificed after spontaneous recovery has begun, there is froauently a persistence of fibrous uleural adhesions. The involved lobe is s~~allcr thnn normnl, has a rubbery consist- ency, and Its color is R rather prrle greenish yellow. The surface may be lr*rin!~led. On cutting the lung, it is rather firr7, i.ml H 94 dry, but flabby. Vf.r; little air is present. In the micro- the scoplc section the striking feature is the predo-.:inance of large yellowish phagocytic cells. Certain alveoli contain numbers of the., with few pol>norphonuclear leukocytes and large less fibrin. `Q is the ease with which it undergoes autolysis and the readiness with which it is dissolved by 3110 and bile salts. These two properties serve to differentiate pneunococci from other Gran- positive cocci; they are also of sane interest in the study of bacterial variation in the Pneur;ococcus group, since the R variants are more resistant both to autolysis and bile lysis Elsewhere the alveoli are clear but for a. ~::a11 number of these cells; the alveolar walls are becoming less swollen; the epithelial cells are being replaced. The capillaries are per- haps more dilated than in tile stage of dense consolidation.' Scat tercd throughout the section are portions in which fibrous tissue is making its aopenrsnce and beginning organization. At times tne plugs of exudate nre coated with a la1;er of epitheliun and almost suggest the organization of a throabus with ~~a.11 new vascular channels making their a?peara.nce. The conplcte clearing of ti;e infection is comparatively rapid, although just as the laying down of fibrin is rr,arked in the acute nrocsss, so is the amount of fibrous tissue in tile reparative process. ---ooo--- II. Nature 6f Lysls of Pneumococcus by Autolysie; Bactoriol~t~c ----I------ ----------m---w --------- Enzxne and Bile. @r. Dubosd -- -------- -_----- Among the interesting properties of the Pneumococcus cell H 95 than the S forxs. Finally, these two processes result in the loss of tXe ch rn.cteristic antlgenic property of the S cell, i.e., its power to stimulate the production of tile type-specific capsular antibodies. What is t:le nature of the phenomena which cause such striki modif ications of the cell? It has long been realized that auto- lysis is the result of the breakdown of some cell constituents by the so-called autolytic enzymes. Cell-free preparations of these enzymes have the propert.: of changing heat-killed pneumo- cocci into A mass of Gram-negative de3ris; this action is fe.irl;r limited to the Pneumococcus group, but, within this group, both R and S forms, irrespective of type, are eaually affected. A very satisfactory method of preparation of the bacterio- phenomenon, most investigators have felt that bile lysis was of a more physical na.ture, particularly because of its amazing lytic enzyme is as follows: A chilled sus.pension of pneumo- coccus cells is thrown into 15 volumes of c:-.illed acetone, the precipitate separated by centrifugatlon and dried as a grey powder. When resuspended in a medium in which the cellular enzymes cannot function, this material may be shown to consist of a*>parently normal Gram-positive cells. If, however, the cells are resuspended in saline they undergo lysis instantan- --------- eouslxL leaving only a mass of Gram-negative debris. Outside --- of the interest attached to this phenomenon of flash lysis, the technique provides a rapid method of obtaining a solution which In turn is very active in causing lysis of heat-killed pneumococci. Although autolysis was soon recognized an an enzymatic z\yz H 96 rapidity, e'ven at ice box temperature. It was also felt that the property of undergoin.? lysis in bile was in some wny associated with t.le life nnd death of the cell, since a.ny procedure which killed the orgnnism would also render it bile in- soluble. Howcvcr, the following experiments, very briefly out- lined, SCCT~ to indicate that bile lysis is only an activation of the autolyt ic enzymes, nnd that it does not occur when these enzymes are inhibited or destroyed. When pneumococcus cells are suspended in citric acid, for lnstnncc, they slowly die but remsin Gram-positive and retain their morphology. If thesc dend culls ore then suspended in a buffer solution at nH 7.0, they undergo lysis at a normal rate, they are found to be bile soluble, nnd when subjected to f rcczing and thawing give an active preparntion of bnctcriolytic onzyine. This experiment SLOWS that dead cells remain bile soluble when their enzymes hnve not been dustrqycd, but hpve been only placed under conditions where they cannot function. When, however, the ~011s are killed 3y agents such as heat, mineral ncids,enlts of heavy metals, which not only inh'ibit but also destroy the bncteriolytic enzymes, they are no longer bile soluble. A direct ovidcnce of the ectiwting effect of bile. on lysis by the bnctariolytic enzyme is sunplicd by the fact thnt the rate of breakdown of hcst-killed cells by the enzyme is ver!; much accelerated in the presence of bile or bile salts. The enzymatic nature of bile lysis is also indicated by the influence of temperature on tae phenomenon. The rate of bile lysis was measured at 00 C., 5O C., 20o C., 37O C., 45O C., - -- 53O C. and 60o C. Between 00 C. and 450 C. the phenomenon was found to h.yve a tempcr,q.turc coefficient of 2 to 3. At 53O C. lysis began very rapidly but soon stopped and never WCLS com- pleted, suggosting that the enzyme itsolf hqd become inis report, tllnse three for38 of lysis are only different exprcs?ions of the ,qctirity of the aam lyt lc enz~m2s. We m3? say, thcrcforc, that the capsular pblysaccharide antigen Is rendered ineffective by the action of the cells1 own onzyles. When rabbits are inzxanized with a suspensI.on of heat-killed Type III organisrr.8, only A few animals respond rPith the produc- tion of type-specific antibodies. IThen, however, iodized cells are used for irxunleation, practically all anlma.18 exhibit the for?latlan of type-specific preclpltins, agglutinins and nrotect- ive antibodies. As has been stated earlier, iddized cells are, easily attacked by t:le becteriolytic enzyroes and thus changed to a mass of `Cliarh-liegitive debris; `Bow it has beon'found that t.rAatlient `iii& t&e :bsctdr~o~ytic"e~zn~~ doee' ii6t".s$om"ti, mddify the Ytigfi6-tip&tii'ii'c nhti,ben` oiY:tk;t?i fodisba"celis. ' fi,`fatit, . ..I'*`. $&bit@, im&nf#&& `-wf,i,, t'~~.~:&r~Pn6~at~v~.~~d~~ri~?.~btain~d'b~ di:gdating% kddiz&d* ceils r`fth !tKe `~bic~erioiyfic :enzytie gave aa good type-specific precipitins knd Frote~t~vo.antibodies as ' ' ' animals treated with undigested iodized cells. It is hoped ,that this observation may be naoe use of in the study of the chet?istrg of the capsular polysncchsride antigen. 2 Seconda= tue-specific antigen. 2 ----_--_- -- ------ .--m-w- -we AS stated before, mice i.~munized with the Graz-negative material. obtnint>d by digesting z,YJy H 100 hcnted Type I cells with the bncteriolytic enzyme exhibit anrkod resistnnco against infection with the honologous orgnnis:, but no rosistanca against Typo II. The se.~.? degree of active izmunity can be induced in --ice with sutolyzed cells or cells dissolved in bile. Besides being resistant to the Pneunococcus cnzyrms, this afitigon exhibits certi-bin re--..?.rknblc properties which have .z*qde possible sane progress in its purificrtion; it is insoluble in 95 per cent nlcohol, but soluble in 70 per cent nlcohol; it is soluble in acetic acid; it is not innctivated by crystalline trypsin; it go06 through Perkefeld filters. Anti- genie n3terial thus purified rencts soecificnlly in scra which have been freed of antibodies ngninst the type-specific poly- sacchpride. ---ooo--- IV Methods of Prcparetion of the Enzym Deco-*posing-the --L--,,,-----s-s-- ,-2,------------ ------ --- CaESUlar Polxsncch3ride of Tw III Pneu!3ococcus. 1Dr. Du3os.l -- ------ --------- -------1------ ------e-e The methods of propnrstion, concentration and purification of the bacterial enzy:ge c&Fable of dcco?posing the cnosulnr polysacchnride of Type III Pncuzococcus heve been described in pre'vioua reports. These methods' have been simplified in some details. In particular, the ar?.ount of yeast extract in the culture nediurn used for grow'ing the SIII bacillus has been reduced to ,005 per cent; this hns somewhat decrensed the ylsld of enzyne, but has also reduced the toxicity of the preoara- t ions. (It xi11 be rccnlled thet the toxicity ha6 been found to be due partly to the r+ction of the SIII b*-cillus on yen,st extract.) As a reeult, it hqs been found possible to eliminate one step in the purification process, i.e., the adsorption on J&3@ H 101 nluninun gel. We have also modified the composition of the ultrafilters used for concentration, employing for the membrane a Solution Of 3.5 per Cent CellUlOSe acetate instead of 5% per cent as previously used. This has resulted in the elimination by filtration of a greater part of irrelevant substances, and also in a more ranid concentration. i 7 1 i i .' !/ At the present time, the main difficulty seems to 3e in the great losses which occur at the time of concentration, probably by adsorption on the ultrafilter mem~rancs. Precipita- tion of the enzyme with acetone or alcohol ha6 given some successful but irrogulsr results. It i6 obvious that the tion and pur- methods can handling of the enzyme at this stage of concentra ificatlon will require much more study before the be completely standardized. ---ooo--- - V Isolation of Pure Cultures -I-,,,,,----~-------------- of Organisms Decomposing the ---- ------------ Cacsular PolEaccharide of Qne II Pneumococcus. IDr. Dubos and -k------ --w--m---- e-------------- ---------- Dr --A Goodner.). -y--v The work on cultures capable of decomposing tho capsular polysaccharido of Type II Pneumococcua `was continued during the spring and `the summer. Several cultures have been obtained pure, some of them in o. very active`form and soluble enzy'mes extracted from them in a few cases. Unfortunately, during the fall, the cultures bogan to lose 't&ir activity and at t`he preeent time wo have only two left which are capable of decoanosing the Type If polysaccharide. Furthermore, the activity of these two cultures has become so attenuated that it is now impossible to obtain from them active enzyme preparations. Time pcrmitt ine it is plnnned to resume the work this spring. ZG$j H 102 VI PneUmOCOCCus DermTl Infection in Rabbits fir. Goodncr.1 --`-----------------~,-,,,,-,,-,,,-,,,I,, --------- In the symptom-complex which follows the intrndcrmal in- joction of pnoumococci in rabbits, the number of organisms in the blood stream nt a given time seema to be en accurate index :t of the relative severity of the disease. The data which have accumulated during the past seven years are being an:?lysed to learn if this indox may be corrolntcd with any determinable conatitutionnl fectors. So far, this has not been possible, but it is apparent that the white cell count pt tLa time of infectivr inoculnt ion is signif icant. In those instances in which the! initial white counts dovinto moat greatly from the average, the Intensity of tho bsctoricmin 24 hours nftnr infective inocula- tion is thr greattat. This is eapecinlly true in rrtbbita which have high init in1 white counts, for these invrrria.bly show a massive blood fnvnaion--- a rather pnradoxicnl finding. Whon so-called flaub-effective~~ nmounts of specific rinti- serum are injected intravenously 24 hours after infective inocu- lation with Type I nneumococci, some animals survive while othcra die. If a definite "sub-effective" `qount of the sRme aerum is used in a large series of rabbita there is apparently no single indicator of what result may be expected. `Some animals with e h very high grade of bncterieaia survive, rhile others with R 16W degree of ,bnctcriemis die. However, if the bpcteriemla .nt' the time of tre?tment Is plottod against the white cell count at that time with respect to the end result it is found that anilrel:; which survive occupy one section of the figure while those which die are oppositely plnceh. With 9 conatnnt amount of serum the number of white cells must be greater in pronortion to the de:rF:r of bnctcriemin if the n,ni;3al is to survive. Increasing amounts of antiserum appear somewhat to compensate for a la&-: of white blood ~011s. The Dcterminnt ion of Active Immunity by Dermnl Infect ion --a- -.----------------------- -----,----,---,,A A project is being cp.rried out (with Dr. Stillman) to ascertain the durntion of nctive immunity na determined by intrncutaneous infection of rabbits which hnve previously been immunized by Intravenous inject ions of hent-klllod pneumococcl, Typos I, II pnd III. Each succeeding month nnimn.ls, not previously teatod, are given IntrndermAl injections of living virulent pneumococcl of the vnrioua types, and the reactions to these inject ions studied. At the time of infection, determlnn- tiona Tre also mpde of the'presence of circulating antibodies. This study hoa non reached the tenth month, and will be con- cluded shortly. It may be tentntively stated that there appears to be no close correlation between the degree of active immunity and the presence and titer of circulnting specific antibodies, An analysis of an extena,ive series of der>al infections carried.out to detexine if rabbita possess active immunity; haa made it poseISle to devise R significant scale by which the :. degree of active -1mmunfty may be judged. It Is believed that / the commonly ncce`:>ted ond-point of immunity, viz., survival and denth, does not give a very accurate picture of immunity, for careful study reveals that there are differences in the rate with which animals die, and certainly even greater differences in response to infection among those which survive. For example, an animal16 immunity may be of such a character as to bring about recovery, but the animal may nevertheless suffer from R Severe and protracted infection differing in no eaaontial respect from that occurring in one which may succumb. On the other hand, the degree of resistance may be such that the animal may develop only a alight locnlizod area of infection and ahow no temperature elovstion. Thus, while it is possible arbitrarily to state that animals which die are non-immune, and t'noae m;lich survive are immune, it would seem more reasonable to hold tha.t survival and death merely divide the gradient of response to infection into two phases, in ench of which considerable differences may be encountered. The Effect of Pneumococcua Autolyaatea unon PneuDococcus ---e-e p-w---------- --------m------- Dermal Infection in the Rabbit. ------------------- ---- It has previously been reported that the edema fluid removed from the lesion during the earlier phases of the dermR1 infection in rabbits possesses the property of inhibiting the coagulation of normal rabbit blood, Pneumo - coccus autolyaatea possess the same property. It hna been suggested that, in the course of tile infective process, some of the less resistant microorganisms may autolyse and thus, through the action of their end-products, pave the way for the invasion of the few which survive. Experiments hRve been undertaken to deteraine if bacterial autolysates might ,enhance the virulence `of Pneumococ*cu~. Sterile autolyaates of virulent or non-virulent strains do enhance the invasiveness of pneumococci. Thus, R strain of Type III Pneumococcus (PH) which is capable of bringing about the death of a rabbit when given IntradnrTally in certain amounts becomes capable, when autolysate is added, of producing the se:* result in much smaller nToUnt3. On the other hand, 3 are A..& . H 105 another Type III Pneumococcus (A 63) which does not inherently possess a lethal capacity does not acquire t.lis property from the autolysntc, but does seem capnblo of greater invasiveness. ---o()o--- VII ---l------L----------------,-~-,,_,-,,__- Study of Pneumococcus Infection and Immunity in Rabbits --------we 1~ the Inhalation Method ------,---,---------,,`,, 1Dr. Still-.lan.l --------- Pulmonnz - ----- lesions produced Q inhalntion method m-e-- we--- -----------,----z The problem of the production of expcrimentnl Pneumococcus lobnr pneu-ionin in small laboratory animals is still under study. All of the ~18uccessfu1 rosultsll of the experimental production of the disease reported in rabbits have been obtained by the intrn- thoracic or intrntrachenl inoculation. As lobar nneumonia may frequently be produced in mice by the artificial inhalation method, this method has been a3plied to partially immunized rabbits. Unfortunately , however, rabbits failed to show any evidence of pulmonary localization of the infection. This failure may be due to the arrangement of tke lymnhatics in the rabbit I s lung. The infection early reaches the pleura and the pericardium and as a result the animals succumb before lesions develop in the lungs. A study has also been made of the pulmonary reactions of rabbits following intranasal instilla- tion of pneumococci. It was found that the mortality of rabbits so infected variod in direct proportion to the number of pneurno- cocci instilled into the nose. Although no lesions of pneumonia were produced, characteristic vascular and lymphatic inflammatory changes were found in the lungs of rabbits infected by the nasal route, 312 H 106' Duration of demonstrable antibodies in serum of rabbits --L-----------"------------------------*--------- following ------- inhalation of Eneumococci. The duration of tyne- ---C-----.-- ---I------ specific agglutinins and orotective antibodies in the serum of rabbits follo!?ing inhalation of 1iLv ing: >neu=ococci has been st-adied. Type-specific I?.,~ rlutinins could be demonstrated or?l;r for a short period after the course of ,exnos=lres WEB ternin*>ted. EIowever. Ty/ne I protective antibodies nera still detertable in the sera of these animals yenrs after their final exposure to :>neum.ococci. `L'l>e protective antibodies dcvelonirg after expos- ure to inhalations of Tyne II pneunococci, on the other hand, remaineo de9onstra>le in tkeir sarum for only A relatively snort period. Active ard Rassive i.?;*r.u;lt~ in rabbits. The duration of ------;-- -e---.-v---- -------m- type-gnecific antibodies in ra5bit.s following inoculation of suspensions of bent-killed pneunococci is still under investiga- tion, In conjunction wit;1 Dr. Goodner, the active im7,unity of theso rabbits as detcr~~inod. 3:, ii~:r~~al infecti.on and the correla- t ion of this with tiio prc:sencc of tync-spccif ic antibodies in the serum is baini: studied. -- -ooo--- VIII fntcrconvertihil~ of Specific Taos of Pneumococcus. -e--L----- ----- ---- --w---q -----------c--w- LDr. Rogersa -_Lc -- It Is an eatabliahed although poorly undorstood fact that rough (R) pneumococci may be experimentally ,restored to smooth forms of the same type as thst from w'nich tbeg were derived, and that they may be l'transfor:uedlf into smooth pneunococci of a serological. type difft.rent from that of their origin. Since the initial work of Griffith subseouent studies by Neufeld and ST& H 107 Levinthal, Dawson and sia, and more recently by Alloway have not only confirmed the occurrence of t!:is phenomenon, but have added much to our knowledge and technical control of it. Tkc transformation of pneu~~ococci from one type to another, which at first could only be accomnlishcd by an ir, vivo --e--v- tochniout employing pncumococcus 'fVaCClncsfJ as the transforming agent , can now be carried out in vitro using sterile, cell-free ------ pneumococcus t3XtrACtS in the nl~ce of vaccines. It has, furthcr- more, been shown thst tke organisms thus converted from one Typo into anothor undergo R rcnl an& permanent change, acaulrlng all the binlogical chnractcristice of the n1.w type, i.e., virulence, typo-specificity, tyne-stability unon rcpcated sub- culture, Rnd snccific antigenicit;, -$llonay was the first to succeed in using filtered cell- free extracts of pncnmococci as the transforming agent. These extracts were obteincd by dissolving virulent cultures with sodium desoxycholata, precinitating the bacterial solutions in absolute alcohol, extracting the precipitate with salt solution and furtbcr,purifying by charcoal adsorption and Rerkefeld fil- tration. (J. Exp. 12od., 1932, 55, 91; 1933, 51, 265.) The : extract of type-specific pnoumococci., so prepared, containing the .so-called Ittransforming factor ,n was added to small ouan- tities of broth enriched by the addition of 25 per cent ascitic or chest fluid, and to the whole wrs added a very small inoculum of living nR" cells to be transforned. Trnnsf ormation usually occurred after from one to four subcultures hsd been made. Alloway found that although these extracts are rich In specific soluble substance the degree of transforming activity H 108 of an extract is not necessarily correlated with the alyount of t ::,.c specific capsular pol2sacchsride prcsent. ?;oreovor, the cilemically isolctad and purified snccific soluble substance possessed no transforming qualities. It has also bc en shown t;lat tile transforming factor lncks the thcr~ostabilit~: l,nsscssed by the purified carbohydrate. So far, it nqn,c;ars that the transforming factor romnins closeJy nr.socintcd with the specific soluble substance, thoug'h trio:, qr`c not identical in tbcir phys- ical proportica in so fnr ns we hpvo been able to test them. The work of Alloway has been confirmed in our recant studies, and further efforts at J)uri.ficntion of the transforming factor Rre being node at the prcrent tir,e. It has been found that by careful adjustment of the final pH of the mcdla em:jloyed In obtaining transformations, sufficient growth occurred to permit OS mnny as three transfers within a twenty-four hour period. This not only alfds greatly to the rapidity with which transformations may be obtnined, but also seems to produce more const,nnt resulte. X few experiments unon the so-called "serum factor"," which is an essential ad,junct in the cultural technique, would seem to indicate that the import- ance of this substance is dependent u?on a certain minimal content of anti-R pneumococcus antibodies, perhaps even more than upon its nutrient value. Active extracts, in addition to being thermolabile, lose their activity u'>on ageing for e.ven so 8:; I' 1 short a tima as three or four weeks in the ice box. The nature i j' /i, t; ,: ,i b of this degradation is being investigated. I Still further purification is being Rtteiauted e-nploying various enzymes, adsorbents, imaunological and chemical me thod.s , i rj # G?! gj v, 326 H lo%- but as yet the data are insufficient to warrant conclug~ong, It is desired, by t-18 methods outlined, to reduce the trWISfOrming factor to its simplest and most active form in order to acquire knowledge of it8 biological and chemical Drop- erties, and 80 to arrive at a better understanding of this most unusual and significant phenomenon. ---ago--- IX Study of the Chemical Nature of Antibodies in AntiE~eUmo= --r-e--- m--------e -- ----L----------------- coccu8 Serum. ADr8. Goebcl and Averp,l ------------ ---------I_ -m--w The investigations of Northrup concerning the nature of pepein and trypsin have led to the isolation of crystalline protein8 poesessing unique chemical properties and biological activity. The 8ucce88 of the88 investigationa, and the fact that these crystalline proteins have properties which dlffaren- tiate them sharply from other biologically inert proteins, have led u8 to believe that the immune protein of antipneuaococcus horse serum might, by mean8 of some unusual property, likewise be separated and differuntlated ,from ,the ACCompanYing inert / ,_ , 8erum protoias. , As a starting material for this investigation . we chose, that fraction. of antipneumococcue Berum (Type 1) which precioitatee..from whole :serua on the addition .of large quan- k, ; .' 7 tities of water,., This fraction,: which 1s insoluble in water, contain8 practically all of the antibodiee present in the original serum. It was found that this matnrial could be further separated,into an inert water insoluble englobulin, an inert water soluble pseudo-globulin, and a second Water soluble pseudo-globulin which contained all of the antipn8UmOCOCCU8 antibodies. This biological specific protein nO888sSe8 CertRin unusual chemical properties which permit it8 separation, 86 Cell a8 it8 differentiation, from the other inert serum proteins. This substance ha8 a lower total nitrogen content, and a much higher amino nitrogen content than the proteins of normal serum A chemical analysis by tile Van Slyke method reveal8 that the distribution of basic amino acids is different from that of . normal horee Berum proteins. An electrometric titration of this unusual protein ha8 shown that its isoelectric point lies at pfl 7.6. The protein is soluble in distilled water on either side Of it8 iSOeleCtriC point, but is precinitated from water at pH 7.6. The protein Is extremely active 3iologically; a solution containing as little nitrogen a8 0.007 mgs. per cc. causes specific ,agglutination of pneumococci. The protein contain8 all of the various antinneumococcus antibodies, i.e., it Contain8 the type-8p8CifiC and 8p8Cie8-8p8CifiC antibodies. Attempt8 have been made to separate these antibodies, but their ch8mical'prop8rtiea are 80 closely related, d88pite the fact that their immuno~dgical properties are sharply differenti- Studlee on the nrotdctive present being carried out. ---ooo--- X Derivative8 of Glucuronic Acid and Glucuron, lDr8 Goebel -A,----,,--- -------------------a-- ---A---wa.- ------me-- and Babers.) The presence of glucuronic acid in the specific carbo- hydrate8 of Types II and III Pneumococcus, in those of Type8 --- 33g iI 111 A, i3 and C Friedlinder bacillus, and in the specifically react- ing carbohydrate derived from gum acacia, has led to the belief that this sugar acid has en important function in directing the specific immunological properties of the poIysaccherides of which it forms a part. A study of the chemistry of glucuronic acid, and its lactone, glucuron, Is in progress. The ultimate objective of these studies is to prepare a glycoside of glucuronic acid which may be chemically combined with pkotein, The immunological behavior of the resulting complex may then be directly cbmpared with the responses elicited by the hexose Rnd disaccharide antlgena which have already bee* studied. An excellent source of glucuronlc acid is t-:e urine of dogs fed on a diet of d-borneol. Glucuronic acid is isolated directly and easily frorr; the urine as the zinc salt of bornyl glucuronlde. The latter compound yield.6 a mixture of glucuron and glucuronic acid on hydrolysis. It is not possible to separate glucuron from glucuronic acid by fractional crystallie- at ion. It has, however'; been found poasible to convert this 1 . mixture quantitat'ively Znto' glucuron, as well a0 `to reconvert glucuron quantitatively into pure glucuronlc acid by mild ( hydrolysis with b`ari-urn hydroxide. .,, . . . . -' ' ! .I . 0 The comparative reducing values of glucuronio acid and glucuron have for the first time been ouantifatively determined. i .I > 1 A report of this study is now in press. In a previous report (1931) there was described the preparation of& and B triacotyl glucuron. These derivatives have now been secured in optically pure forms. They show a difference in molecular rotation of 36,400, a value which is 399 H 112 in excellent agreement with tllc knovndifferences in molecular rotations of tie pentacetL.1 hexoses. A crystallographic study of these two derivatives is being made by Dr. Ralph Wylroff. Vhcn either a or 9 triacctyl glucuron is treated with ncetyl chloride and hydrogen c:lloride, an aceto chloro derivative is formed which is pro3ably a mixture of d and 6 . 1-chlor 2, 4 diacetyl glucuron. This compound serves as t>e source materiel for the syntnesis of glucuronides. When ncpto chloro glucuron is dissolved in moist ether and shaken with silver carbonate, tne chlorine atom is replaced by hydroxyl yielding a crystalline derivative which nopears to be 1 hydroxyl, 2, 4 dincetyl glucuron, We have prepared a non-crystalline methyl diacetyl glucuronide from diecrtyl chloro glucuron, and we have succeeded in synthesizing A smnll quantity of n crystalline diaoetyl p-nitrobeMy1 glucuronide. The condcnsat ion of diacetyl chloro glucuron with phenolic derivatives is very difficult to (pffect. It is necessary to overcome the difficult- ies before the synthesis of glucuronides can be profitably attempted, and we are still making serious efforts to do so. b I ---ooo-..- XI. The aynthesie of-aminophenol glucosides of lactose and --1--m -w- -I-- --------------m 4 gent iobiose. ----- -9 The result of the studies of the immunological behavior of synthetic carbohydrate protein antigens has proven to be so instructive that we have extended this investigation to the disaccharides. The factors which control the immunological specificity of carbohydrates anpear to be the pos+.tion of intermolecular linkages end differences in intra-molecular H 113 stereochemical arrangements. The disacchrrides, whose chemical constitutions have been well established, are ideal substances for investigating theeffect of such changes on imcunologica.1 response. The four disacch-rides which are being studied are represented graphically below. The differences in suatial constitution and inter-molecular linkage are readily discernable from these granhic formulae. In order to combine chemically a sugar with a protein it is' necessary to synthesize the p-aminophenol glucoside. Those derivatives are then combined by diazotization of t5e amino groun, and coupling the diazonium glyc'osido with protein in alkaline medium, Ye have synthesized tile heptmetyl 6 -n-nltrophenol, the ,p -p-nitrophenol and the fi -p-aninophenol glycosides of maltose, lactose, cellobiose, and gentlobiose. These glycosides have been combined with protein and a study of their antigenic properties is nearing completion. H 114 PUBLICATIOXS ,,,,,-4----z. Avery, 0. T. The role of specific carbohydrates in Pneumo- coccus infection and immunity. Annals Int. Ifed., 1932, 2, 1. (John Phillips 1!ernor~i-%!~~~~~--- Avery, 0. T., Goebel, W. F., and Babers, F. `s. Chemoimmunolog- ical studies on conjugated carbohydrate-proteins. VII. I-munological snecificity of Rntigens nrepared by combiningN and6 glucosides of glucone vith proteins. J. Exn E'ed., 1932, 22, 769. ----LA-.- Alloway, J, L. Further observations on the use of Pneumococcus extracts in effecting transformation of Type in vitro. ----e-m J. Exp. Med., 1933, 57, 265. Baudisch, 0.. and Dubos, R. fiber Katalaseairkung von Eisen- verbindungen in Kulturmedien. Biochem. Zeit., 1932, - --w-e---- 345 --- * 298. Francis, T. The value of the a!:in test with tyne-specific capsular polysaccharide in the serum treatment of Tlr>e I Pneumococcus pneumonis. J. Exn Med. ,---,L,,, ' 1533, (April number). Goebel, TI. If., Babers, F, H., and Avery, 0. T, Che?o-immuno- logical studies on conjugated carbohydrate-proteins, VI. The synthesis of p-aminophenol CL -glucoside and its coupling wit:1 protein. J. Exu Ned ,-,-L1--2,' 1932, 53, 751. Goebel, W. F. and Babers, F. U. Derivatives of glucuronic acid. I.The preparation of glucuronic acid from glucuron and the estimation of their reducing values. J: Biol Chem -w---A---L* 1933. (In press.) Goebel, 8. F. and Babers, F. H. Derivatives of glucuronic acid. II. The acetylation of glucuron. J. Biol Chem., 1933. -~se.AC-e-- (In press.) Goodner, E. A test for the therapeutic value of' anti-pneumo- coccus serum. (Presented before tie Amer. Public Health Service, Was.iington, D. C., October 24, 1932.) J. Immunol., 1933. m-p- (In press.) Goodner, K, and Dubos, R. Studies on the quantitative action of a specific enzyme in Type III Pneumococcus dermal infection in rabbits. J. Lxn. Yed., 1932, 35, 521. ----i--A--- Julianelle, I,. A. and Rhoads, C. P, Reactions of rabbits to intracutaneous injections of pneumococci and their products. VII. The relation of hypersensitiveness to lesions in the lungs of rabbits infected with pneumococci. J. Exp.Med. 55 ----- ,-,-* --* 797, (1932). Kelley, W. H. The antipneumococcus properties of normal swine serum. J. Exn ---M-w . Fed. , 1932, 55, 877. Stillman, Ernest G. React ion of rabbits following inhalation of Typo III pneumococci. J Inf -L-e Dis `--e-L * 1932, 50 e-9 542. In manuscript: ---------- -- Goodnor, K, and Stillman, 2. G. The evaluation of active immunity to Pneumococcus infection in rabbits. Goodner, K. The effect of Pneumococcus autolysate on intra- dermal infection in rabbits. J. Exn. Vcd., 1933. --_--A_- (In press.) Stillman, E. G. The react ion of partially Immunized rabbits to inhalation of Tyue I oneumococci. Stillman, Y. G. The reaction of normal and partially immunized rabbits to intranasnl instillation of Type I pneumococci. Stillman, Ed G. Duration of tyne-specific agglutinins and protective antibodies in rabbits following inhala- tion of living pneumococci, Types I and 11,