t 131 Xeoort of Dr. Avery (assisted by Drs, Adams, Bttaon, Cattanao, Curnen, Daddl, Dublin, Duboa, Goebel,. Goodner. Heuglt, Hot&kiss, C, MI MacLeod, Mirlck, and Stillman), Studies on chemotherPnbyt Trentmtnt of pneumonls pith sulfanyrldlne alone and in combination with antlpneumococcal serum (MacLeoh, Mlrick and Curnen) , The earlier obecrvntions on the use of sulfar,yrldlne In the treat- ment of lober pneumonia have been confirmed and extended. In pneumonlae due to pneumococcl other than Type III the drug hae been uetd aa the sole specific theraztutlc went, except ln a few instances ae in oatienta of advanced age or where the infection hae been of great severity. In these caaea type-specific antlpntumococcal serum has been used In addition, Be- cause of the toxic effects incident to the use bf eulfaryrldlne an attempt hae been made to treat each aatient with RB small p. dally doe4ga of the drug ~a le coneletent with the malntainenct of an effective COnCentrRtiOn In the blood, and to discontinue ite admlnletrstion promntly follorlncz a fevorablc thtreueut lc rteConat, The total amount of drW admlnletered. per pntlent has been dtcremaed to an averaRe of approxtittly 15 mama nnd cessation of treatment hao been possible a.l.moet lnverlnbly after three ti,ysr Ulth bhle BChWUe of therapy, rtlnptar of the pneumonis hee not occurred except in one natltnt with Type If1 infection in whom the dfeenet wa8 com- plicated by asthma and bronchitctaeie, In the clinical treatment of the diseaee the use of~~ulfapyrldlne in lower doawe har decreased the lnci- dence of severe toxic effects without BaCrifiCing in any ray the theraptutla value of the dnrg, From the comparative result6 obtained in mice Infected with Pneurnococcus Type III and treated with sulfayyridine or a combination of the drw and antlpneumococcua serum, it wae felt that the combined form of 132 therapy milzht be more efficacious than either agent alone In the treatment of patients with Type III pneumonia. This opinion was based an the ob- servation that sulfapyridine or Immune serum when administered singly Is relatively ineffective whereas a combination of serum and drug; Is synerrris- tic and hiirhly effective In the treatment of experimental Type III infection in mice. Of 20 patients with Type III pneumonia. all but one were treated with both serum and sulfanyridlne. Following admission to the hospital sulfaoyrldine has been administered for a nerlod of twelve to elEhteen hours before begInnInE serum thqrany. Despite the presence of an adequate blood concentration of the drv? durlnq this Initial uerlod, definite evldancc of progression of the disease has been observed in five patients. However, in all but one of these a favorable outcome has resulted followinfc treatment with serum In dosage sufficient to product an excess of clrculatlnR Type III antibody, The mortality of patients with Type III pneumonia treated with a combination of serum and sulfapyridine has been much lower than that ob- served In patients receiving serum alone. During the years lc'36-1938, 26 patients with Type fIZ pneumonla were treated with concentrated antlpneu- mococcal rabhlt serum alone, and 7 of these died, a mortality of 2'7 per cent, In the past two gears only one patient has died in a groun of 20 treated with a combination of serum and sulfapyridine, a mortality of 5 per cpnt, EdetabOlism of a ~lsulfapyridlnc-fastn strain of Pneumococcus Type z (MacLtod). The experimental production of `Vsulfanvridlne-fastness" In a shin of Pneumococcue `Pyle I WAS described in the prPcedin+! rqort. "Fastness" has been shown to be associated with a relatively atRble RltPrA- tlon in metabolism without chances In mornholoRy, tvne-sptclflclty, or virulence of the pneumococcus. Studies of certain metabolic activities of the parent and drue;-fest strains have revealed differences which may be significant in relation to the mechanism Thereby sulfapyridine Is able to exert a bacteriostAtic action on the growth of these microorganisms. Hydroeen peroxide is formed in culture6 of pneumococcus as a product of tterobic metabolism, Whan 6rown in a shallow layer of broth exposed to air, the wrent strain produces an abundance of hydrogen peroxide while the dru&-facet strain under the same condltlone forms only a small mount. The relation of peroxide production to cRrbohydrete metabolism is unknown; however, the decreased formation of Drroxide in the d-fast strain is associated with chances in Its dehydropenase activity. The dehydrogen~ee activity of the two stralna was studied by ,_ : .i : : determinlnk the Pblllty of cell suspensions to reduce methylcne blue in the : presence of various subetrstee. In the preparation of the bacterial sus- pensions Care must be taken to minimize exposure to air In order to pre- serve the dshydroeanase activity of the cells, Of the substrates tested, glucose Is an active hydrosen donator in the presence of both strains of pneumococcua and no difference fs ob- served ln the time required for the reduction of methylone blue. On the other hand, the d-fast stmln shcwe little dehydrogenaee activity for glycerol, lactate or pyruvnte, wherema the mrent strnln dehydrogenntes these subotancek a& iv&y, : In the above experimenta the dehydrommase activity of the cell suspensions wrr.r tested In the nbeence of sulfspyrldlne. When eulfapyrldlne r le added to the renctlng eystem, there la no Inhibition of the glucose i dehydrogennse wtlvlty of either strain. However, the drug greatly in- hibit. the dehydrocenatlon of glycerol, lwtate, Rnd p,yruvate by the parent celle. It Would sppew, therefore, that the bWterio6tntlc action of 134 sulfepyridine may denend in part upon the inhibition of the enzyme Systems associated with the dehydrogenation of glycerol, lactate, and ovruvete, Furthermore, when a strain of pneumococcus becomes "fast6 to sulfanvidine the dehydrogenation of the same a-carbon compounds is greatly decreased. These system6 no longer function as actively In the metabolic processes of the cells of the "fast6 strain as they do in those of the oarent strain. The occurrence and nature of a substance which annuls the bacter- lostatic action of sulfonamide conr\vounds (Macteod and Mirlck). The presence of a substance in peptone which greatly diminishes the bacteriostatic action of sulfonamide compourds in vitro has been observed by many investigators. -- The occurrence of this material in the pentones used in the preparation of the usual bacteriological media has made comparative bscterioetatic test6 dlff icult to interpret, since different lots of media prenared under apparcnt- ly identical conditions may contain different amounts of the "sulfonamide inhibItor", However, the presence of this substance Is not restricted to peptones, since It has now been found to exist in certain body tiSSUI2S particularly in those In which autolytic changes have taken place, Purulent exudates are rich in ths substance as are also tissues containing necrotic lesions, This inhibitor is, of Importance, therefore, not only with respect to the lnvitro bsoteriostatlc tests but also In rslation to the lnck of -- bacterloststlo actlon of these drws frequently observed cllnlcally In the presence of purulent lesions, 13. coli has been used as R test orftenism for the detection of the inhibiting substance, since It will grow In R sinmle synthetic medium of which all the constituents are known. In this svnthetic medium certain of the sulfonamide derivatives exert a pronounced bscteriostatic effect on B, ~01i; however, if mRteriRls containing the inhibitor are added the F 135 bacteriostatic effect is either partially or completely annulled. On the other hand, complex culture media containing muscle infusion and oeptone are customarily used In the cultivation of pneumococcus and streptococcus hemolyt icus. Since both of theae ingredients contain varying amounts of the sulfonamide inhibitor an accurate determination of the becteriostetic effect of sulfonamide oompounds on organisms in nutrient broth is difficult and oft en misle4ding. It has been found, that fresh liver is free of the inhibiting substance and that an infusion of fresh liver which has been prepared rith- out heating on the alkaline side will support the growth of pneumococcus in the absence of peutone. Moreover, the inoculum necessary for the in- itiation of growth In this medium is much less thRn thAt required in the usual media, In liver infusion, sulfanllamlde in a dilution as great 9s 1:1,000,000 exerts a pronounced bectcriost%tic effect upon Streptococcus hemolyticus, and sulfapyridine in the se.me dilution Is bacteriostetic for pneumoc occus, On the other hand, In certain lots of peptone-containing media only slight bacterioetatic effect may be observed when these drugs are used In concentr&tions s`s high as 1:10,000. Up to the preoent time the results of becteriostatic tests in which a liver `infusion medium wae used hRve been highly consistent, and this technique is being used to determine the possible aoquisltion of drug- fastness in stralnm of pneumococcus Isolated from patients before rend after treatment with eulfapyridine, The presence of the inhibiting factor Is associated with the occurrence of autolyeis in tissues or exudates. Thus, fresh muscle contains only a small amount of the substance, but if autolysis takes place a great increase in inhibitor occurs. Pancrea,e and spleen are rich in the material, 136 but none is demonstrable in fresh liver. As previously mentioned, purulent exudates obtained from patient.8 with staphylococcal, pneumococcal, *.nd streptococcal Infections, as well aa freeh guinea pie: liver containing CAS~OUB tuberculous lesions have been found to yield 1Arqe amounts of the drug-inhibiting substance. Because of the importance of these facts in relation to the bacterloatatic effect of sulfonamide compounds both in vitro and in vivo -- -- nn investigation of the biochemical properties of the inhIbitinK substAnce has been undertaken, The active principle is readily dlalyaeble throwh cellophane; it io heat-stable at both alkaline and acid reactions but lta deetroyed on prolonaed heatirye: with atrow acid or alkali. It is soluble in ethyl alcohol, butpl alcohol, and acetone but Insoluble in ether. On the ncld eide the material can be readily absorbed from aqueous eolution with chnrcoal. The active subetance can be eluted from the charcoal with hot alcohol Rnd pyridine, Purther purification of the elate can be effected by precipitation of inert materials with isobutyl alcohol and acetone. Studies on a bactericidal agent extrscted from cultures of a eporulatiw behillue (kbos, Cattnaeo and Hotchkioe). It wao reported lnat year that a baqterlcidal princlplehad been extracted from wlturee of a sporulstlng bnclllus ieolated from eoil, The prolent report deocriber the methoda of purification of thin baotericidal agent, rtudier on ita ohemioal nature end propertier, and borne obeervatione on the mechanism of lte action on euscwt ible bncteria. Preparation of cryetalline substances which exhibit bsctericidal activity: The active princ$ple ie releneed In solution in autolysates of peptone cultures of the soil bacillus: it Is senarated from the culture 137 media by precipitation at pH 4.7. Extraction of the acid precipits.te with alcohol, acetone, or dioxane yields a fraction soluble in these organic solvents which cprries all the bactericidal activity of the orWna1 mat- erial. The bactericidal nrinciple present In the alcohol or acetone solu- tion ie practically insoluble in water in the presence of electrolytes; it is precipitated by diluting the alcohol or acetone solution in 10 volumes of aoueou6 saline. The precipitate thus obtained is free of protein; it carries the bactericidal activity of the or-i&al culture and can be desiccated without 1066 of activity. Further purification is obtained essentially by addin& ether to an alcoholic solution of the protein-free product to nreclpltate selected fractions. It is necessary to receat the preclpitntions a few times before the fraction6 represent entirely distinct grows of substances. This ie true becauec some of the substances present can modify considerably the solublllties of others. Inert material la found in fractions soluble in ether (fatty acid6 and waxes) and in fraction6 insoluble in absolute alcohol, The bactericidal material is collected in two fractions; 1) material insoluble In a mixture of one volume of alcohol and 15 volume6 of ether, and 2) ,: material soluble in the same m$xture but insoluble in pure ether, l?rom fraction (1) there were isolated by cryetallizat.ion from alcohol two crystalline aaidic substancee, which have been designated &mink acid and firamidinlc acid, Prom acetone aalutione of fraction (2) there ~88 isolated a crystalline neutral substance which ha6 been named gramicidin. From every one hundred liters of culture there were obtained about 10 m-6 of protein-free product which yielded &pproxima.tely 6 grams of mixed gramlnic md Kramidinic acids and 1.0 to 1.5 grfuna of eramicidin, 138 Bactericidal w2tivity of different crystAlline frs.ctionsc. As ststed above three fractions endowed with bnctericidal activity have been obtained in crystalline form. 0,005 to 0.01 mk;. of these substances is sufficient to kill in vitro 10' pneumococcl ot Group A streptococci In 2 -- hours at 37OC; grnmicidin 1s probnbly twice 68 active (per weight) as either gramlnic or gramldinic acid. Still smaller amount6 of either fraction in- hibit the growth of Gram positive bacteria in nutrient broth, %iS i6 particularly striking in the case of pneumococci which failed to mow in nutrient broth containing o. dilution of 1r1,000,000,000 of the active sub- stsnce. The standard test used for estimating the activity in vivo of -- the preparations of bactericidal substance has consisted In determinine the minimal amounts of substnnce which, when inJected intraperitoneally within 30 minutes after Infection, will protect mice against 10,000 fatal dose6 of T.ype I pneumococci, In spite of the great bactericidal activity which graminic and gramidinic acid exhibit in vitro, these SUbStanCe appear to be ineffective. in vivo~ On the contrary, one.ain&le dO6e of 0,001 to 0,002 mg, of gramici- w- din, injected into thf peritoneal cavity, is capable of protecting mice .) aeainat 10,000 fatal dOSe6 qf pneumococci or hemolytic streptococci, The . material has been fouui equal& effective a(rRinst infection with five differ- ent types of pnewnococcus, eJPven t;ypee of Oroq A etreptococoua, md five strains of Group C streutococcuet Bi usin@ lnraer doses of the bnctericiclal aaent and repeatinrr the treatment on three consecutive days, we have been able to protect mice aminst l,OOO,OOO fatal doses of pneumococcus and to cure mice of a ne~~-e6tahli6hed Infection, even when treatment WRS odmin- istered 6, 12, and 17 hours after inJection of the infective inoculum, 139 Marked protection has also been obtslned arrainst infection with a mouse virulent strain of staphylococcus. Finally, preliminary results obtained in colle.boration with Dr. R B. Little indicate that gramicidin also exhibits some effectiveness when lnjeettd into the udder of cows suf- ferlncz from Group C streptococcus mastitis. Extraction from cultures of the soil bAcillus of a water-soluble form of the bftctericiti.1 substance, The results which have just been re- ported demonstrate thst gramicidin when injected into the perltonenl cavity I of mice is very effective against Infection with pneumococci and streutococci. However, the same substance, when Injected intravenously, subcutaneously, or intramuscularly, fails to protect mice aEP.inst infection with the same organisms. Several possibilities may be invoked to accolmt for this failure; a) the active substance, when Introduced into the lreneral circuls.tion, may be elimlnnted so fast thnt It never reeches the effective concentre.tion; b) it may be Inactivated In some tissues, for instance, by hydrolysis, oxidation, or conjugation; c) the active substance is known to be very insoluble in aqueous media and may therefore fail to diffuse and reach the different foci of infection, In any case, it Is obvious that the inoolubllity of the material in aqueous media lta i `@eat handicap both for experiment81 studies and for possible usa in therapy,. It has been recently found that a number of bit& ' * persing agenti- &h La& &lphketeb an8:`rdp&L' _.. , "... _ `2.i y `? ._' 8 l,i' I ~ I' p, :,_ ,,, ; ' .% .T amata, since it ia known that paptides of dextro-amino sofdo we' not 1 4. > +: _. I > hydrolyted w moet of the &own peptidnseo and proteinseer, i. > FPL i d t;, * ./ I .-e.v'*-~L.m.ar. Irl., _ .., 1 ..:&t,F.,,,;`2: 2, .:' 7, . .:, ..y; ..,C'.. ,,;.", . . . . .I.> / . >_ : _L ,:*.--L'..l_?-: _ ,,"pur ,I * / ;. `3 ,* .?-.,`. 3' J,"*", ,&& y&g& - _-. _ I: 1 i..., 9, ' , Toxlclts for domi of a Prauaration of the bacterlcldal eubotanoe .,.a:{: ` `: 1 + The toxicity of a pur5fled protsin-ires I v [' 1.. 1" :t * `,, ,. m _ : `,I' .' .`>.*:= preparation of the bactericidal substance derived from (I o&l bacillus by h $ ~(-`-q ,; ,`.. Duboa wae tested in young, healthy, short-harisd doqe aeighine: frbm R to &" kgr, An alcoholic solutfon of the substance was diluted in 20 cc, of a 5 per cent solution of e;lucoee and inJected intravenously in daily doses va.ry- inu from 0.5 to 2.0 mg./kg, of body weight. The inJect ions were cant inued `4 /Q 145 for 10 days in the dogs which survived. Clinical records were keot and ppthological studies of the organs were made at autopsy. Seven of the R animals, which received from 0.4 to 2.0 m&/kg. died ~8 A result of the Injections and in 6 of these death occurred before the course of 10 dally inJectiona was completed, !l!he remaining doga were sacrificed from 23 to 54 daya after the course of injections wae begun, The more prominent elens of toxicity durinq life were lose of weight, anorexia, fever, anemia, henvnturia and the excretion of bile in the urine. In the nnlmale which died #cutely marked conaestion of the viscera wae preeent and petechlnl hemorrhaaae were observed in the hesrt, lun.ss,and kidneya, The livers showed acute central necroeis associated with hemorrhage . and dilatation of the elnusoide. The spleens were hemorrhR.?lc and pronounced pbRocytosie of erythrocytce by the mcroplmree WRB preeent. In the anlmlr which received daily 0.3 m&/kg. or more and which did not die acutely, the chentles In the organs were of a more chronic nature. The liver cells showed fatty degeneration which was most marked at the center of the lobules. In these area8 there WR.B an increase in reticular tissue but cirrhotic chanrree were not pronounced, Aacites was present In two of these animals, The only chance noted.in the organeof the animele recetvlng 0,2 ~&/kg, or lese .._. ,. writ a slight degree of fatty degensrnt~on of the liver, / `3: .? `5. 2: 3, " a? t-3 "! Effeut of the Imcterlcidal subetaxme on experimental PQ eumococcu~ : :< -,:;a+ r: _1 .~>g& ' pneumonis in do88 ()lncLeod, ,.Curaen, ~a.1 Mirick). ., The, bactetlcldnl eubatance' `%z was tested in dop;e for it-e: effica.cy la the treatment, of experimental pneu- monia, The disease was produced in morphWized animals by the intrnbronchial `. Instillation of :-Type I pneumococci and mucln According to the method of Robert son. Clinical studies comprising bacterioloeicnl, hem4toloaicPl and X-ray examinations and the pntholoaical study of the orPans removed at autopsy have been carried out in all cases. Over R period of several weeks ! Id6 groups of dons were Infected, half of the animale in eech erouD receiviw treatment end half servinfl RS controls. Therapy was carried out by the intravenous injection of the bPctericida1 substance In a 5 per cent plucose solution. The amount of bPctericidp.1 substance Riven per dp.y vwF)ed from 0.09 to 0.3 mg./kg. of body weicht, administered In divided do8es. Treat- ment WAS begun from 4 to 24 hours after infection extent In one qrouc of animele where 0.2 m&/kg, was adminletered twice during the 24 hours prior to infection and continued dally theretiter. !Pwenty-three docls with exoerimentnl pneumonia were trented end an eqml number served aa controla. TAO Incidence of bacteremia In the treated do&e WRE 78 per cent And 1P died, A mortf%lity of 78 per cent. six of the treated an-18 developed becteremla Rfter tree,tment WRB be-. The incidence of bactercmia in the untreated dose was R3 oer cent and 16 died, a mortality of 70 ner cent. The averace beriod of survlv~l in the treAted does was 77 hours and in the controls 71 hours. Under the conditions described, treatment with R aurif led nre- parRtion of the bfictericldnl substance ehowed no evidence of theraneutic effect upon en;erlmentRl Type I paeunmia in dew, _: (`,* % , r. Isduced rraturR1 reoiotancr (Qoodnsr), .3; 4ertnin enidemioloctlcnl 1 1 :. .I aspects of. p%eurdoni&,. m.xq?est the possibility of R%.relation between, dietary.: habite end ,the incidence. or severity ,of the diswae,, TYio ew?eetian,,,,.sly though v&e,. and. until now swther remote, has grunptsd :exgloration into : : ., that field of,`inmnznolo#y which Is not concerried with sxntibodiee or wit'h . . the specific character of resi'etance to.infeqtioue disease. Thle field occupies a heterodoxical poeition except $n 80 .far as all workers recognize the existence of a natural resietance not explicable in terms of the anti- body theory. In a rather renernl sense the protective mechanism is 147 conceived as having a physiological character but the biological mechanisms concerned have not been identified, As a preliminary test of this proposition, mice were given un- accustomed diets, that is, certain vee;etables or fruits were offered as the sole source of nourishment. After a period of a few days the normal diet was restored. The animals were then tested for resistance to Type I pneu- mococcue infection, 300 minimal infective doses being given by the intra- abdominal route. Some of these animals survived althounh the control ani- . mals lnvariebly died, Moreover, It was noted that survival was not associa- ted with all fruits end veaetablee but was sharply limited to certain particular items, These results suggested the poeefbility that the animals had acquired substances from the foods which had enhanced resistance, Since the results did not correspond with the incidence of well recognized vitamins, attempts were then made to extract the substances responsible, Infusions of the various materials were clarified and injected subcutaneously. In many instances subsequent Infection did not lead to death, These extracts could not be shown to be either bacteriostatic or bacterici&$,, indeed, it could be~shown~.that,.even i~,ssimals which survived, the pnemococci _. persisted for several dayo, often rith,increaeed nmbeFe.k llre physiological mrohan~um of the mouse: oimply. eoemrd to ,. ignore the presence of the pneuekc-- . -: aocoil .no orllular. rtmtioa,.waa prqduced, nor were the anlmala Ill, ,.Thia e ,a corresponds well with our kncrle&e, of ,natural resistance, For example, the rabbit'cennot be "infected" with human tubercle baoilli. It is ?cnown, however, that these orcrprlisme survive in the rabbit -- the eimnle fact is that this animal does not react, does not form tubercles, end therefore does not develop tuberculosis. Present Absent or relatively. so Beet tops Beet roots Vegetables Lettuce Celery Parsley Green beans Spinach Tomatoes Orange (Cnlifornia). I Orange (Florida) Fruits Grapefruit Apples Ore eng;sm plume Grape e Corn* Alfalfa .Gralne Oat e* tdillc t Wheat* ape Boneeet Elder flowers Herbs Chamomile Mustard, ground De lphin ium Plantain Horehound Saeefif rae * Not present in most of the edible derivatives 148 A survey as to the presence of these nrotectlve principles in nature has been extended to over 200 sources. By way of example a few instances may be cited: Early attempts at isolation of the active material eliminated the possibility of proteins end fate. Active fractions always gave 8. positive Molisch reaction and some reducing sugar after acid hydrolysis. This was euggeetive of the qluaoeidee, Many of the eourue materials were known to yield ~glucoeidee of definite composition, A comparison of oqr results with , i_ . I ~. . the available lnfcrmation browht cut,tho eumestive fact that the agtive II 1 *.i :. _.. . /. ,. , ,.. .,*. extracts wgre fronimateriale known to yield flavonee, flsvnnole, and fl8van- , ones (~collectivsly`known as anthoxanthins) with hydroxg groune in the 6,7. a' . positions on the benecpvrone ring structure, Isolatione based on this de- : ' ductlon have substantiated the aeneral premise although much remains to be . done along this line to show that other substances may ,not possess this biological property, With feeding, and with the ink&ton of `crude extracts, it has never been possible to protect all mice in any grour). The reasons for 149 this are not known but it has been noted that, as purification proceeds, the results have become more consistent. Pneumococcus heterophile antigen (Goebel). In 1911 Forssman observed that emulsions of guinea pig tissue stimulated the production of lysins for sheep red corpuscles when Injected into rabbits just,as did sheep erythrocytes themselves, Since this important discovery there have been many investigations concerning the P`orssman phenomenon, The `iPorssman antieen has been found widely distributed in nature, not only in the tissues of animals, but in many microorganisms and in plants as well. The chemical nature of the true Forssman hapten, however, has never been fully elucidated. In a previous report evidence was presented which established a direat chemicnl relationship between the blood moup specific substsnces and the capsular polysaccharide of Type XIV Pneumococcus, Throuirh this study it was possible to explain the remson underlying the untoward and frequently fatal reactions accompanying therapy with TyDe XIV antipneumococcus horse serum. Certain diseases in man notably infectious mononucleosis and serum sickness following therapy in acute lobar pneumonia are accompanied by a marked increase in the hemolytic titre of the serum, Some of the obscure phenomena of intoxication durihe; the acute phase of the infection may i:. r : ., ,i .._ I _ ,I. i 1 ,,, Y., .._ :, . L possibly be related to the presence of Foresman antigen in the infectious r . .., L.: agent, It seems not unlikely therefor: that-a thorough investigation of " I :. .: . :+ G -.* L : a ,, L i!". _' .~ .- - .I .; Forssman substances aa they occur in tissue and In microorganisms &RX lead _I .: _ .>`,>,? a. $I 7 2.24 t,., TI. to an explanation of their role `in Infect-ion' `B;;b &ei&tsnce, At 1 Se Y& . . ,.,: ), . ; time the results of such II study may reveal the chemical basis unde&ing ,_ - the phenomena of tissue and blood croup speclf icity. We have chosen therefore to invest iqate the chemical nature of the Foresman haptsn in the nvlrulent or R strain derived from Type I Rneu- mococcus. The results of the investleations are briefly ns follows; The 150 Borssman antieen as it occurs in pneumococci is thermostPble and resistant to .4uto1.vsis, The hApten portion of the antioen is partially liberated during the autolytic process, but filtrates of Putolvsed cells contain little or no intact antigen. The latter a~peers to be bound to the cell detritus and in this form it is still fully antireenic. The Porssm8,n hqten is not identicrrl with the pnewnococcus "Cn or species sp'cif ic golgsacchsride, for the residue or cell bodies after autolvsis Rive rise in rsbbits to potent hemolytlc Pntisera which are devoid of precipltins for the rC" sub- stsnce. The cellular detritus of autolvsed pneumococci is A comclex mix- ture of protein, carbohydrate, and lipids. The protein may be dirested away with trypein leaving sn insoluble mated&l which is rich in Porssman sub- stance. .There is no loss in Forssman hapten following tryptic diqestion. The residue obtained from the enzymatic hydrolysis is composed of an un- characterized material and some fifty ner cent of soluble lipids, which czul be extracted with boiling alcohol and ether. In so doing there occurs a dimfnut ion in the active Forssman, hapten, but this loss cannot be accounted for by the oreaenos.of the rubetance in the lipid extract. The residue left after extraction with the ormic eolvtnte ie, however, still rich in Tores- man hadten;`: ~-The latter can be separated by extraction with aqueotir pvridine, In this Pa&ion` a?rolutfon of -the Rordman haptexi hae been obtnined,' -Thik'* material ib hirrhlg actbe and nppenre to be ereentially'free from"protein and rioh In `cnrbohydrafe. Further work directed fowird'the chemcterisstion of this substnnce ie in progress* `. Synthetic antinenar InvestiRntlons carried out in this labora- tory on the chemical and immunolo~lcal properties of bacterial polvsacche.rides and artificial antigens containing simple sacchnrides of known constitution 151 have revealed many of the factors which govern the lnuuunoloaical specificify of encapsulated microorganisms. As our knowledge in this field has sdvanced we have come to believe that it should be possible to incite antibacterial. Immunity in experimental animals with artificially compounded antigens con- taining immune specific qrouns of synthetic origin rather than with anti- genitally complex bacterial cells as they naturally occur. In the report of last year It was shown that sera of rabbits immunized with an artificial antigen containing the naturally occurring aldo- bionic acid 4-&qlucuronosldo glucose (cellobiuronic acid) agglutinate T,ype III pneumococci and confer passive lrmnunity on mice against infection with multiple lethal doses of virulent Types II, III, and VIII organisms, A similar antigen containing the synthetic isomeric aldobionic acid S-,4- glucuronosldo Prlucose (eentlobluronic acid) Palled however to evoke in rabbits agglutlnins for Type III pneumococci or protectfve antibodies for Types III and VIII ormlsms. Thus It was proved that the chemical con- stitution of the aldobionlc acid determines the capacity of the corresponding artlflclal antIRon to Incite antibacterial immune bodies specifically directed toward pneumococcl of Types III and VIII. Although &tiobiuro'&z acid entieerum fniled to proteot mice . ," 1 ..I . ,. ,. ! ,. 1,. against infection with either Types. If1 or VIII organisma It has `now been _ .z ,I 1 ,' a _. . : ;.`% (r found to affdrd pzm&t'i&~&5.nst' `&f&ion with is much' as lOO,O&`minimnl . .,~ @ ' L. lethal doses of -Typa II pneumococclr , %, .._. I Since this p&&t;l'~la shared by _: .,I i, ,i, . :.. ,_*,' `. . `.... cellobfuronic acid antiserum it is evident that the differen& `In ihe later- molecular structure of the two aldoblonic acids is unimportant in determin- ing the specificity of antibodies evoked by the corresponding antigens to Type II pneumococcal infect Ion in mice. It hae been possible to show that the protective action of I gentiobiuronlc and cellobiuronic acid antisera to Type II nneumococml in- 9 I 152 fection is due to an antibody directed toward the Elucuronic acid con- stituent common to the aldobionic acid antigens. Two wtif icial antiaens have been prepared; one contAininR the AZO benzyl elycoside of qlucuronic acid and the other thst of the isomer, ep.lscturonic acid, These two hexose uronlc acids differ only in the confiqration of the fourth carbon atom where the position of the H and OH group is interchanged. COOH paaminobenzyl