4

nowrt of L&s. Avery r;xL EurtitiLll (tLasitited by l&s. ~am&Binkley,
Curnen, Goub~l, t;:cCtrty, iuiirick, Ptirlm;n, stillmi;n, anti Ziegler).

     Ltu:.y on the chemicnl n;.ture of the subtitcnce iniUCing tranuformti-

tion of buucific types of 0neumococcus (Avery LLr-iC dcC;kzty).  i3iologie ts, es-

ptrci&lly the gcn6ticists, h&vo long sttempte:'l by cher,Lc;i mans to inc;uce in

higher organisms prec.ict;,51e &ni specific chagcs which thereafter coulli be

transmit&C in tierics trs herutiittiry charzturs.  Among microorg#.nisms the

most titriking anC yurftips the only known ex;.wplo of inhcrittiblc nnci specific

iLt2r~tion0 in c&l structure tini! i'unct.ion thc+t cxn be tixperimtint2lly inoucod
LnG thr.t ix3 ruyrowt:i.Llt-t uniter ViCll wfinw tnd &quLtel;r controlicc. con-

c.itionr; is the trtnsforolltion of spcifis typis of' Pneumococcuti.   This phen-

o&r&n w&;i first c.c:;cribal ij;{ Griffith who ~UCC~~I.CC: in irr~.nsforning tin

sttonul;Lcc! and nun-onclysuLtc;c (1,) v:.ri::nt tleriv& from une sp,scific tyde

into fully enct..ytiulhtti(? LnL. virult;nt (b) cells of i. hzterclogbus specific

type of Amuwcoccus.  H typiccl instr;nce will buffice to illusLrr.te the

ttichniques: origirally used mci serve to incAiwta tho wide vcziety of Was-

formations thut iire possible within the &ctqritil species. For example,
Griffith found that mice injectad s&cutaneously with c small &mount.of a

living & culture aerivsci frun i?neumococcus Type II together with L: large
inoculum of heart kill& l'ype III (S) cells frequently succumbed to infection

an& thcrt heat'& blood of these wlimuls yiol&d Type III p%nococci in pure'

culture.  The fat th&t the ri s&An W(LI #*virulent tnci incapable by itself

of c&using fcrtal septicemia und the ncUtionc1 fact that the ho&ted suspen-

sion of Type III cells cc,ntsin& no vitible orgclniums brought convincing

evicance thtit the H forms &Towing uncer these cofiditions herd newly acquired

the cc+suLr abructure ani; biA&cul spcfficity of Type III yneumococci.


1
! i

boyunc suggesting that the cie~*c: btictcria in the inoculum might furui%b SOIIE
s+cific prutein thot encbles the ii forms to manuf6cturc ii capsulr;r CC&W-

hyar;Ptc of t hc same sgecific type LS thz.t of the 3 cells which sorve~ ~p3

initi:.i LLUYZ~ of th*; incucing subs'vilnca.

     i,ithough the ex+rirn~nt~ in mice: VJCZ'~ succosjlful, Griffith ~8
un&bib to r'bprucuco the ghe:numt;ndn withuut muse p,bSSJ~ge Lno on the: 'bzsis
of nrgc-tivc results conciuucci that incu'aticn of the h&tc?rial mixture

in vitrb failtld to iniuca tr:.nsf3rm:itii)n.
--
     Those vrigirlL.l cb;crv:Atibns v:ere Lter confirmed by i~eufcld hnd
Levinthci JX-...a~. ano by L'r..rtson ano ailcway in this labcrr;Lry. subsequent-

ly bi wbn WLY the first  t u succecti in incucing transf~,rmation in vitro. This
                                                                     --

he acc~mplishu~ by grdiing h ctills in ti fluiL moiium containing tLnti-ri sorum

I*nci hci.t iriiluu cnci:ysti:,too ;i colis.  in the: test tube I*Y in the aniutal

&ooy hc sr~cvfeci that trJ+nsftirmation c&n bo solcctively uetormincd c+enuing
bn the ty,~a specificity of the ti cells ubed in the reacting system.  Later

aliovuy was Lblt?  t \: c&use bpctcific trkcsformLtion in vitro using sterile
                                                        --
oxtriLcts sf rj cells frum which ~~11 form& elements and celiular debris had

been rcwva~ by Btirkefeld filtrtltion,  He thus showed t&t crtie exticucts con-

taining the &CtiVd materilil in soluble formclre effective, intiucing the SkUW
specific trtineformationu as do the intact cells from which the extracts were

    The pessnt report is cuncurnec with a m&o wtuilod analysis of

this #onUmen\m.  c)ur mt+jor interest hr;o centored in attempts tu isol&te the

ilctivc princi& from crutie oxtrc&cts c.no to itientify if Ixissibie its chcmictl

nature d at least tu chtLractoriz0 it ~;s belonging to ;i gonoral. @oUp of

PJnorm chtjmicdl subst;inces. For purpose jf study, the typical exrqle of

trLnsform;tion cho,stin L;S t vrbrking mvuol WAS the roncting system with which


                                              145

we hl+ve hi,&a most experience ;.na which consequently seem& best uuitea t'or

further knnlysis.  T!,is pi.rticuler ex;z&e retiresents the trLnsformc;tion of

Type II to 'i'yG;e III ~neumococcus through the intermekzy K form.  E'..ch

constituent of the ret.cting system grescnteti probioms which requirea clui-

:.
Y

f'ic&tion before it wtAs gossibiti to obkin consistent tinc. rcprociucible re-

suits.  The various comgontint:, ot' the system will Lx briufiy c;iscusbei; in

the foilwing ox-tier:  1) Lulture mtiium;  2) 'i'he n-strain;  3) Yiepr:tion

of trLns:`orming extracts of Type III cells;  4) Purification of the active

mttcrihlj  5) ChemicLl nt,ture ;:na biologic[A 4,ctivity of the $rifiod sub-

iulture meaium:  'i'ho z-sic mkium convists of' the sttinc~rd nei;t

infusion oro'uh routinely uscc. in th: cultivttion of gneumococci.  Girlier

stuaies ruvutlec; chtt in oracr to oi?ii,in trknsform:;tton it is neccssar;J to

~,cti buruiil to th:: &sic moaiu:;,. Hnti-11 ri.bbit :;erm ws usoa in tho first

succussful experiments in vitro.
                         --  it satin bcci.:mc ttviacnt, hoaovcr, t1Lt the

ei'factiveriess of aiffsrent lots of ~rum vLri8u i;nci tktilt the cifferencos

observe6 were not nocusskrily deyenaent upon the content of H untit%lies,

since winy 8era of high titer were fcsund to be inca;wblc of supporting

trbnsfcrfition.  This suggestou t&t f&ctor& other thkn H antibodies ~8

involvoa in the system.

      I&cLoti first showed t&t trcrk from various r&ma1 species ir-

rtisductive of their immune jzopertios, &nd inaeed norraul sera of divarse

11
("

origin cat&n tn enzyue.ccrt>nble of uetitrciying tho k&n&forming principle in      1

potent extracts.  (The na.ture of this enzyme cnti the spucific ~Ubtitrabe Upon

which it acts vrill bc referred to later in this ra&rt).  It w~is found t&t
this enzyme w~is intictiv;ltod by heating the serum ht 600-65%. and k&t tiera

urcviously heated tt tcmger~.turoo known to destrtiy thci enzyme were then


'or&h conttiining Type II LLntissrum for 36 serial pssugca and isolating the

h w&at thuu intruced.  The strtiin (~36H) iw lost the type-asecific chwac-
tcribtics of tho parent culture anti conuists only of tttenuuted, non-crimp-

suiikiGt% h ceilu.  The chmge S+ri is often u ruverrsible one, provided the

ri celis LTB not too fkr %iegrsded8t.  Tht: reversion of the h form to it6

ori&niii s-pecific t;rpv my i!requmtly be axom@ished by buccestjivr: ani.m.1

p.sstt~cs vr by repcatud growth in anti-h serum. Under these ca&itions,
hopaver, the ri cuitura ixm;ric&~ rr)verta to thti saae s+cific type i;ti thut

frolii wnich it WE clerivc%.  ;itr&in rt36~ h&b tmxme rci&tively fixeti in the

ft phLcbe encl bus never been i'wnc to revert spontaneously to tho Type II S

i'wm. .


i

.                                                      II!+7

1
u

    C;onversion oi 3--$ r( ;>.nt.. vice vers?. within the liaits of L given

t/>e is quite i. c.,if`fsrent iai.tter thdl 1 `Y the tr::nd'orrstion of orgmisms Of

one specific type into those of ,nother specific type.  `i'hc Ltter hi;s never

ueen o!jjbbri'eL to occur stjont;deousiy ant; inc.t?ec. cm iw incuced exyerirtien thlly


otllj' by Liie specii.1 tcchniqut2s outlinei~ emlier in this report.  Uncrcr these

contiitions, Lhe enzimtic s:.Tthesis of L chedcdly atid irnnunologicdLy
c.il'r'srel;t ci;+u~iir >oljmcch~d-icle is sp2cificallf oricnteu mu selectively
c~terwined c;eperdi.n& on the tfge tigecificity of the L cells used ns the

source of' the trtnsforming drincigie.

      Tn thr? course of Lhc present stuuy it was notm thtit the btock

CULLLU~ 01' 1~36 on Sbri:i:L trxnsf'er s in blo;3<: broth udergoes SgOnthneOUS

ai~~~,(:ii.t.im ami givsti rise to i nwlber of other ii vl:ri;nts uhic;h can be

c:isG.l?&bhm one from anti iher cl;' codmy i'orm.  The significCj.nce of this in

the &tsarlt instmce lies in the r';ct tbt of L, uifferunt v&ritints iboic*ted

l'ro111 the G&rent cuLture only one strdn, Lesignbteb h3bi, is susceytiblc to

the tr;ntiLorrning bction of potent extrc;icts, whlie the others Laiied to

rebycm2 tncl htrve since rcinainec inactive in this regard.  `l'he knowledge of

Ufferences In the responsiveness or' Different h vwiants to the same spep-

ific stimulus emphasizes the cbre th&t mutrt be exercised in the'selection of

ti aui%bie ti variant for use in trrnaformetion experiments.
     Although the significance of the following fact will become

apparent later on, it must be mentioned here that pneumococcctl cells possess

an enzyme cs*ble of destroying the rsctivity of transforming extracts. In-
deed, ciutolysetes of d36~ we highly active in this respect.  EArlier workers

found thut it is essential to use u s;ntili inoculunt of young md rcctively

ero.wing ir cultures in trtnsformtltion tests.  This rtquirement m&y berit be ex-

yltiineti on the t;bsumption th;t when l&rge inoculti irre used encrugh enzyme my
be relzs:sed bj' autolysis to inactivate the tk-aforming principle present in


/                                                         148

thr? system.

    i+cwrttion of tr&nsforminE extracts fiord Two III 0neUGoCocCi:

The prustz~t 1:rcthod of prspr;ration of extracts from yneumococc&l cells rep%-

SorrtS tht3 and r~sulss of nurirarous axperinontr;. .: t+pror;ches ckrried out over

i yariod c;f yazs.  The proccdurtiti usud in uxtrirction Lna +rii'icrticrn hrrve

been evolvd gr;,duily L:, evkunce ht& Lean rjbkinod concerning the nsture

tnti pro+rti.i'b of the c.ctivu SU'bSti.IlCf:.
     FJi2a III colib i.rti i;rown in ic*rgc qzntitios (50-75 liters) i..nd

ccGit;cteo by c;ontrifuC;kti~n in (. ~,t,titt~ arivan ;Ih,q.Lk;s cc;ntrit`ugti.  The

mkteriirl incurrac; in the VJWhing ~rcjcess is slight cotlparcd to the @lrificc;-

{
i ,

tiun r;chieveu.  'Ihe vrrrshtid calls; &ro then extrc;cte$ twice by shaking in Saline

con"&ining 0.5 pr oont souium deuoxychtilrzto.  Extracts yrtqared in *his

manner tifter remcrval crf the doeoxychol~te by &lcohol CuntcLin the major por-

tion of the'active princi&.e titi~metsurer: by their c&p&city to in&we trans-

r'oriwtitin. .

i [ i
1                l?urifict:tiun pi' tictivo Yubtii&ncc: nqrecipi.ttLtion of the cru&o

i         oxtrtrct with 4 vcjluws tf alcoh~rl flocigitatorr the uctSve mcWri&l L&A re-      .

wvas the last trtLcbb of dttsowclGktrJ vrhich rewin soluble in the t;lctihc;l.

'I'hs t)l'ecigitute is rouiuwivod in btiino rAnti the grctit buik tif ;Jrutein is


i          bicillus captlble of dcconpoving 'i'y~ III polysaccharide is then employed

8
5
f

j

tti digest the ctipsular pulysaccharide remaining in the extract. After di-

gesti;n of' ctpsulur pdySECCh~iriW3 is c~npletec! 68 c,etormincd by loss tif

reactivity vrith Ty+ III Lntib0dy klutL>n, the cxtrcl.ct is &g&in tr&.ted

by the bcvq metha to rcmovc the enzyme grotoin ;.nd any remaining pneu-

mococccJ protein.  This pr~ccss is rcyeatud until the sugernattnt fails to

give the biuret reaction. The finril ate2 in prificatiou iti the reieated

peciLAkti.on of the extract by the drop&e ai<ition of CL'9 - 1.0 volume

of abtolute ethyl &lCGhd with CcJnstclnt Stirring.  At this critical concen-

trutiun of tlctihoi, the active materitl seycsutes out in long, axtremely

fine fibers which collect on the stirring rod. Iiigestion 2rociucts of the

cqsuiar >olysacchr,rido together with t`ne somrLtic C carb&ydrate, ribs-

nucleic acid and remaining traces of protein t.re loft in the residue.  nfter

several repecipitatiuns, the material is finally aissulved in distilled

water and Dialyzed against distilled water to remove free stilts and other

ciiffusible substances.

      Chemical nature and biologic&l activity of the mrif%& &ubH&nce:

In tho oourse of chemical fractionation of active marteritrl it WCS reyeatedly

found th&t biological activity was always aseociatad with that frkctlon which

gave a strongly positive reaction in the diphewlwnine test, sugge8ting
that the active substance might be &esoxyribonucleic acid (tbyztu8 ty+).

Further evidence for this view was found on elementary analysis of several

preprations isolated by the .pocedures outlined above.  In the following

table analytical figures on two highly purified prepartitions are compared

with the theoretical values for the sodium salt of deso-xyribonuclei~ rcid

on the basis of the totrtinucleotiue structure of the mdocule.


                       Theory for des~x~- i5i,

Yreslirttion A(37   i?re&ration rir42   ribotetranucleotide

phoadhorous   8.57 per cent    9.U4 tier cent    9.05 per cent

Nitrcr;i'on    U.21 ,*r cent   15.36 per cent   15.32 per, cent

N/P ratio     1.66         1.69          1.69         ,


Carbon      34.27 ;rcr cent   35.50 pr cent    34.20 ller cent

Hydrogen     3.89 tJer cent    3.76 lxr cent    3.21 tier cent

                                                                           '
tect frorJ theory.  These figures by thclmaclvcs co not establish that the

substw~~ isolirted is t @rc chemictil entity.  However, on the basis of the

h/p rirtio it WUUh hype&r th:,t littlo prutt;in c)r other substances containing

r&tia ~ulcl be conoiuerz&Ay altered.

     l)n enzyWtic an&lysio of the active tiatorial it was found that
crystalline tql2sin or cmotrydsin ant! combinutirms'of both causad no

atipruciob10 1os.s in cicti.vitiJ.  Moreover, digestion with crystalline ribo-

nucloase duos not iq+ir biological rctivity. Amung various crude 2re+ra-

tions of enz;yoles derived from sciurces such as bone, kidney, ~eum~cocccrl

cells, pancretitin and'uerit only thuse which atttsck authentic ssPryle8 of

desoxyrihonucleic acid have been.found to cause w Getecttble loss in thu

activity of potent peprations of the transforming princi&~. This Ah    .
                                                                           . _'

further evitience of the chemical relationship suggested bjr the analytical

figures ubve.

     Preliminary inveotigatiuns in collaboration with Dr.T Rothen

irLict;te that the substince in highly purified form is ;Jrc)&Itiiy highly
&ymerizcsd anti has a nol,ecuLr weight of ~~iJjmxc~tely 0.5 - 1 million.
Uther stuuies of the &pica1 chemical pru,Jerties of the active substance

ure now in progress.


St clhould also be pointed out that as the crude extracts are puri-

fied, serological activity with Type III antiserum progressively deoreasee.
Solution6 of the purified substance itself give only faint trace reaction6

with high titer Type III antipneumococcus rabbit serum.  In view of the fact

that the Type III capsular polysacchsride and the somatic C carbohydrate
react with homologous antisera in &ilutions as high tis 1 ytrt in 5 million,

this loss of bcrologicel activity reflects the almost complete removql of

all sorologic&liy reactive substances from the final preparations.

The fuct th&t the transforming substance in purified skte 8x-

hibits iittle or no immunological reactivity is in titriking contrbet to its

biologi&i function in inducing highly specific changes in living pnoutno-

cocci1 cells.  ke little as 0.02 clgr., representing a final dilution in

the reacting system of 1 prt in 1oO,OOO,OOO has sufficed to bring about the

trtinsformcition of tho b variant (H36k) into un~qsulirtcd Type III pneumococci.

Assuming thLt the sodium desoxyribonuclefc acid and the active

principle are one irnd the sqne substr;nca, thon the trun+formation frpm

H--)SIII represents a change that is chemically induced anti specifically
direoted hy a known chemical compound.  Lloroover, this substsnco selectively

determines a differentiation of cellular function and structUre corresponding

in type to that of the 8 organisms from which the agent was derived; The
interaction between the R cell and the transforming principle initiatoa a
series of complex reactions which c&ninate in the synthesis of the Type III

capsular polysaccharide. Thus, the transforming principle - a nucleic acid -

and the end product of the synthesis it evokes - the Type III pcrlysnccharicie

- are each chemici;lly distinct and both ark requisite in the type specific

differentiation of the ceil of which they form a psrt. The.former has been

likened tu a gene, the latter to a gene product, the accession of which is

mediated thruugh enzymatic synthesis.  The go&tic interpretation of this


L



                                                              1%
       phenomenon is supported by the fact thzLt once trhnaformttion is induced,
        theretifter without further addition of the inciting agent both capsule forma-
       tion Lnd the gene-like substance are reduplicated in the daughter cells.
        The ci,cnges inuuced are therefore not bansient nooific&tions but c;re

+a       transmitted through innumerable trcnsfers in ordinary cuiture media.
                             ,
             Ii' the present studies are confirmed and the biologically active
       substance isolated in highly purified form L;S the sodium salt of dcsoxy-
       ribonucleic acid actually proves to be the transforming principle, ati the

         avhiltble evidence now suggests, than nucleic acids of this type must be

       rogttrood not merely as structurally important but as functionally active in
       determining the biochomicol activities and specific ch&ractcristics of

         pneumococcul cells.
             Leathes has pointed out that in the chemical make up of proto-
                                                                           .
       plasm proteins are not the only essentiz.1 s?Jbstsnces. Among the other con-
        stituents he ranks the nucleic acids ts preeminent, and reforring to their
       occurrence as major comp&onts of nuclear chromcrsomes he raises the question
        "whether the virtuas of nucleic acids may not rival the amino acid chains
                                                                          e.
         in their vital importance."
                                   .
             btwiies on nrimay t;t~icUl DIMBIW~~~ (Horsfall, Curnen, Mirick,

        and Ziegler). In 1930 it was first recugnised cletily that there &fisted L
       common clinic&l form of yneumuniu which differed from.the usual bacterial

*        pneumonias.  In the intcxvenirg yetas this illness, nuw termed "priapary

         ktypical,pneumonian, has been encountered with incretising frequency. Inter-
        est in the condition stems frcru the-ftats tb:t it is, L;t tho present time,

         about as often seen as is birctrjrial pneumonic tend that the cause .or ~8~808

         of it hbvo not been definitely oslxblished.
             Thore is good reason to believe that thiv clinical syndrome lo


                                       153

not a new disease.  It W&8 probablg recognised OCCa8iOlMd~ during the 8OC-

ond half of the last century and undoubtedly was observed not infrequently

8           during each of the first three decacres of this century.

`l'he recent marke& increase in the use of the x-ray in acute

reb&%tory aiseaties, the establishment of active full-time heaith units in

some schools, colleges and camps and the ineffectiveness of sulfonamide

chemotherapy in the illness seem to have been the most important factOr

in bringing this syndrome into c+zar relief.

      There can `be no doubt that this condition, ciuring the past three

.

years, has been incret;oing in incioencs more r&pidly than can be accounted

for on the basis merely of increased Lwareness and recognition of it by

physicians.  In certr;in Army camps in the continental United titates the

incidence of the illness has been GS high ati 1.3 per cent of the total


commana.  It is recognized both bjr the hrmy end the Navy as res~nsible for

T[rore mtm-days lost from duty thun almost my other acute infectious disease.
The frequency with which the illness occurs r;Idong civiliana cannot now be

estimated due to the fact that, with the exception only of New York City,

the disease is not reportable.

      Almost all investigators who have studied ctr8ek of primary criiypiosl.

pneumonia think that the illness Lo not the result of bacterial. infection.

/            &me workm8 have &uggeoted the pss$bility that the syndrome was caused hq
e        a virus and as a result the term %irus pneumonia" has come into conmon         ,'

U8ii@.

      There is cviaence that the syndrome is not a singlo disease en-

tity.  At lelist three different infectious agents have such been shown to

be otiologically reli+tod to certain small groups of ~0~8. Those arc the

i.WithCOSiS group of viruses, Hickettsia di:iRoriCa, and & viru8 infG&ticu8


.

154

for the mongoom.  &iditiont;l infectious ogbnts have bcon suggested LB

posscbsing ;r CLUX,~ r&&tionship to other c&se3 although the evitience upon
which th&c; suggestions hLve been 'b~iscd scdm8 insufficient to Frmit of

crl.*,Ackl assousment.

thio hirspitcrl one ytxz &go.  The primary objoctivcs of the study were two-

fcrld;  firstly, t dsttiiled tituay of all the clinic&l manifuatutions of the
iU.neus, &nd secondly, an investig&tion of the nrcture of the infectious

egunts reeponsiblo for'thc ayndromc.  During this year 112 patients .wero

&droittod tc, this hospittil with auto respiratory dibeirses.  Of these pktiento

80 wtxc found to lava primzy atypical pneumtinilr. Specimens obtained from
thbbe ijutient3 cunstituted the ~mrct: mtori&l fur the l&borLitory 8tUdie8~ -
In uaiitim, syticimms wart ~leo obthined from 511 pf.ti~nts with the syn-

drmo in other ha@tuls, hth civil&n Lnd military. nil trpecinens have

J

been stxed at -70%. mc consequently ure c~n3tantly available for bttiy.
This large Library of ptitcntially infectious SVUTCB W~tcrial ha3 blI%aCy

proven cd grunt value.
     The Laboratory stu&ie,s were faci.litated Iy fund8 ovrde uvrrilable

through G contract b3~weea the ciffice of Mientific tieSearCh and Deve+p-
ment tnd The Htickefellcr Institute. Comylete laboratories and animal quart-
er3 SUitible for the Vtudy of Virus di&8aS88 were f&tted GUt Wld t@Ed.&~

equipped for this investigntiofi.  kdditimrrl technic% iresisbnts were

       .,
engtgso.                                                                        .
       (;lini.cr;l studies.  As ii result of the detailed studies of 80

patients vfith @mry L;typ.ic&i pneumcinis it WLY possible to forimll.ato a
f&irly ticcur&tc clinical piCtIII?e of the syndrrjras.  The illnass ciffered

strikingiy frum thu bttctorial @neumonins.  The clinictil hfst&y, pwtlic~l


b

0

examination, chest x-ray, hnd clinicei course had hut little in common with

those encountered in the bacterkl pneumonias.  The most common finding6 were
fever, rel;rtive bradycnrdia, cough, headache, blight tlterntion in the phys-

ictrl signs over the chest, d.ef initt x-r&y evicence of consolidk tion, a

norral luucocyte count und normA urine.  Four chaructarivtic observations


were common;  (1) tht: patient selaom ayge~rou ;rb ii1 ab the: chut would lend

one tc expect,  (2) the gul, se rctf.2 wt~s s~ldor:~ i:s rrcpio (:s the temperature

wouiu incfikte, (3) the &r;icti cxkLline.tion r&rely suggested thitt conboli-
Ltion ~1' the extent dcmoraLrt'ole by x-ri.y WLIS prcsc)nt, hnd (4) the clinical
sovurit;r of the illness w&s ofton unrel&tai tu tho extent of puinmnriry in-

volvaent.

       hn knaiybis of the clinic:..1 dak reveCloci the f ollcwing cvorirge

fiaiings; i'cver $erbidtc?c for 10 (iCAys :,nd Wiichad 103.50F., the- pulse rrzte
was 23 beats por r&nub sluwur tha the tempaature inciict$ed, thu retlpira-

tory rtte wi.8 25 *r minute, lacocytta aere,7,800 per cu. ma. md the    .

erythrocyte sedimcnktion r;Lte W&Y 38 mm. pur hour.  In 75 per cent of

ins;taces the consolidation occurred in the lower lobes, tnd more cowreonly

on the left thm the right side.  In 70 per cent of instances only one lobe
was consolidated. LVldence of beginning rosoiution of the pneumonic process
could be discerned by x-r&y on the 10th tiy but complete resolution did not

vccur until the 17th c&y.  CLmpILications were uncommon, although recrudes-

ceqcc or' fever ffcs not infrequent.  Either extoncian of the cmsuiicktim or

recurrence of it uccurred in sWati ckeeb.  Pleuritis associated with small

collections or` i'luid anti a mild fbrm bf meningismus, or transient encephalitis,

cl*ch occurred in &+ut 5 per cznt of cases.  Slight splenomegaly, arthrtclgia
and tranbi,mt Clttz&tias in the electrocarcii~grc wore each ftiunii in, &me


CEsab.  This group of unus~l $hcnaena ir;d to the h;'p&hesis th;Lt the


ti.,n.  c,uiI',m.dib~ chal;iothorr;py seened nut to ;;ltor the clinical course of

the illness.  The wrtelity rtto was zero.

     fLchenicr+l studies w yrimry atypical pneumonia (B&son, Haag-
ltnd, Curnon, tiirick and Ziegler).  Through the cGoporution of other services
in the hoa&tal numertius biochemical stuoios were ccirricd out on aolectod

p;ltients.  It was found that tho icteric indicts, prothrcmbin lavels, serum
CC, and serum chloride levels wore within normal limits. Plasma carotene
anti vitamin A lt~els were reduced but only to an extant commonly encountered
in many other tcute infectious diseases as well.  Plasma 2 amino acfds urre

within nurmal limits and chloride balance studios inAcr,t.eG that there was

no striking abnormality in chloride metabolism.  These latter findings are
notliworthy since they serve uuditianclly to cifferenticte this syndrome from
those associated with the bacteritl pneumonits. As is well known, the
plasma g amino acid levels are Gofinitely roducod in bacterial pneumonia and
chloritie metabolism is distinctly abnormal in moot cases.

Bacteriological studies on nrimary atydcal cnewnia+ (Curnen,

Mirick, Ziegler, &cl McCarty).  The bacterial flora of the nose, throat and

sputum was studied in each case and an effort was made'to obtain both quali-
dative and qu&itative data.  The sputa usually contained relatively few

btLct;eria and in no instance was it possible to determine the type of pneu-
mococcus by the direct Quellung technique.  &waver, by mouse inoculation
one or another type of pneumococcus was obtained from 60 per cent of the

sputa.  It is noteworthy that neither Type I nor Type II Pneumococcus was

isolated.  The clistribution of pneumococcuo types was not dissimilar to
that which has boen found in the throats of healthy individuals. & hemolytic

strqptococci, hemolytic staphylococci and Ii. influenzae, though occasionally


i57

found were nut encountered with suffi'cient frequency or in numbers sufficient
to make them seem of etiological significance. None of the strains of fl`
influenzae isolatti were smooth and none could be typed. Numerous other

species of bacterial micro-organisms were also isolated from specimens ob-
twined from the respiratory tracts of patients. All of these organism8
are known to be present commonly in the upper respiratory tract of healtw

persons.  in no instance was any evidence obtained which suggested that any

of tha bucterial species isolated/were causally related to the illness.

Blood cultures were done on alb patients and none showed bxterial growth.

tioruover, pleural fluid and cerebrospinal fluid obtained from patients
faiieci to revetl the presence of btrctoria on culture.
     Sorologicol studies on nrimary atypical mewnonia (Horsfall,
Thorms, Curnon, rilirick and Ziegler).  Acute phase tilxi convalescent 8erb from

patients were tested for the presence of antibodios against a numbor of

viruses known to be caprble'of inciting acute respiratory disease in human

beings.  The agents selected for those tests were, psittacosis virus,

lymphocytic choriomeningiiis virus, influenza A virus, influenza B virus and

swine influenza virus.  In no instance was a significant increase in specPfic
antibodies against any of theve viruses demonstrable during convalescence
of patients admitted to this hospital.

           Acute phase and convalescent sera from patients were also teeted
       for the presence of the so-called C reactive protein. It will be,recalled
a      that notcrbly in pnmmococcus pnmnonie but also In other acute infectious
      diseases there is present in the serum Wring the acute phase of the ill-

ness an abnormal protein which has the peculiar property of reacting with
the C or somatic polysaccharicie of pneumococcus to form TV precipitate. It

was found that this protein was almost invariably present in the acute

1         phase sera of patients with primly atypical pneumonia and t&t it disl;ppeared


during early convalescence.

      In the course of certain serological tests a peculiar and unex-

pected phenomenon was encountered.  It was found thz.t approximately 35 per

cent of yatiants with primary atypical pneumonic cevelopcd in their S~IWII

during early convalescence the caLyt.city to fi,x complement with a wide variety

of apparently unrelated antigens.  The cunvclesccnt serum in high dilution

of some patients ratcted with many of those untiguns cLlthough the &cute p&se

serum from the same patients did not.  Similar reactions were not observed

when convalescent serc from other acute infectious diseases, either of bac-

terial or virus etiology, wr6 tested.  The reaction could bo domunstratad

with antigens prcparec. frc;m a vcriaty of organs obtuined from L number of

unreltited species ant it is not yet apparent what cr;mpcnent common to these

antigens m&y cxylitin the yhenomcnun.  In general, tissues infectec with

one tir kinither virus provkod mtire cctive antigens tin dia normal tiasuesi

This ghcnomenon seriously complicates tho interprut+tLn of complement fixa-

tion tests in this sync&me. aoraover, it serves.to cast s&me ti.oubt on the

significance of certain studies which have been based wholly or largely upon

the results obtiined rith the complement fixation technique in this illness.

      Etiolonical studies on nrimary atynical nneumoni.8 (Horsfall,

C-en, Miriok, Thomas, Ziegler). Specimens of sputum, throat washings,

plasma, pleural fluid and cerebra-spinal fluid obtained during the acute
phase of ilhese from a number of patients with prm atypic& pneumonia

were testsd for the presence of infectious agents.  In numerous instances
.these specimens were inoculated in mice, guinea-pigs, hamsters, rabbits,

cotton rats, white rets, hooded rats and monkeys by a variety of routes

incluciing the intranasal, intrutracheal, intraperitoneal, intravenous, sub-

cutaneous and intracerebral.  Many specimens were also inoculated in chick-

embryos of various ages by the following routes;  intra-.


i5'3

allantoic, intraamniotic, and on the chorio-allantoic membrane.  berial

passages were carried out in all species with the exception of monkeys. In

no instance were obvious signs of infection produced which could be repro-

duced on transmission in -series.  Indeed in only two of 21 sputa tested w&s

pulmonary consolidation grociuced repe&tedly in cotton rcrts following prim-

ury intrtlnasai inocuilition.  However, it wu8 not possible :n either insknce

to reproduce this lesion on serial passage in this species 'despite r%ny

varittions in experiment&l procedure.

Although no obvious signs of infection, trsnsmissible in series,

were encountered in eny of the species tested, it wus diocovertid that ani-

~1s inocultted with certain specimens or with p~~usi~ge material derived

from thetie specimen s subsequently developed in their serum, tintibodies which

vrem ccp&ble of specific&lly n3utrcilizing a hetorologous virus.  This hetero-
logous virus was pruviouvly ehv.vn to be present, though latent, in the lungs

of mny apparently healthy mice and hiis been termed "pneumonic, virus of mice".

B&uve of this unexpected obsorv&tion that an-18 inoculated with speci-   .

mens obtained from cusos of priw atypicel pneumonia produced antibodies

which completely n+utrsliaod large &mounts of u heterologoua virus derived

from the lungs of nrice, the possibility tirose that thare might be in the
agent recovered from patients and in the QxwaatonicL vizws of mice" minor

common antigens.  Moreovar, 3.f such trll stccidental antigenic relation&&p

actu&Lly existed, means would become avaLl.able of detecting the presence of
the hum&n cigont even though it were not cawble of producing ObVioUS Si@M

of infection in &viilable c;;nimcrl ciyecies.  This hypothesis has proven fruit-

ful hind h&s m&e possible the development of experimental procedures, brrsed
                                                                           .

almost entirely upon syacific immunologictil tests, by means of which certcrti

charccteristica of the human &gent hkive been defined.


Bvidenco has oeen obtained which in&icste& that 15 strclins of &

virus hz`ve been recovereii from ssecimeno ob+G&led f'rom a tot&l or" twenty-two

tj(Ltienty,  TWO CtrhXi WAY? recovered frOti thrO&t-WbhingS, eight f'rOIfl f3,~I.h

snti five i'rou citr~tocl pLi.Lsma.  sil 15 strbino eppcured to possasb: minor

ontigunic components; also prc;aent in the heterologouu pneumonii: virus of

mice, hereinkiter rcfbrrecl to t& PVti.. irlthough none of thitso Strains w:rS
cap2'bic of proaucing obvious signs of infection on sex%1 pWW.~go in t.ny
tnimcl byacie!s tested, neverthclctis, immunologictl tiviclence has been ob-
Wined th&t the ugant could bcr pascd in seristi in both chick embryos iind

cotton rtts.  It could clso be pased stiricilly in chick umbryo solid tissue

cUltUre:, was sntigunic for rabbits ana cf.utiod :a inapp;rent infection in

I&X?.  The virus ws filter&bla ttiough B.?rkcfelG V candles, ciici not lo&e

activity c.ftsr storqe c.t -73%. for morz th;a eight months, withstood rq)id

freezing Lnci thtiwing 10 tina, and uz..ti in;ctivLted by 56% for 30 minutos.
     Evidence inciicuting the prcacnce of this &gent in specimens from

patients with primary ~~tyyia.1 pneurdonic huu been obt&ined in t vtiriaty of


wuys .  Cotton ratv wore inoculutud and ri;bbits were injected with sputum,
throat-wtiehings, or plas~a~~ from yLtients t+s well GD with a&al or tissuk

culture -p&&sags mtericl derived from thob;e sr-pecimena.  Sera obtained front

theee cmimals before ancI nt interv&ls r+f'tur inoctition were tested for tlie

presence of neutrrlizing sntihudies ag;inst PVbi. Mice inoctitud with
                                                                         *

eimilr;r specimens were subsequently tasted for active immunity r@wt &I-

fection by PW.

     C)ne of three filtered throat wt;si;ings tested co&&nod an &gent
which followi& serial passage in chick abryos WCS cqabls of otimul.&ting

the production of trpocific neutrulizing antibodico aglrinst &%I in rsbbits.

c)f 15 acute pti;su pti~sraa toetod 5 wore fcund- to con&in tin a&gent which was


                                           ,.
.

c;+b.Le d stimulating the ~rA.xi;i\m of neutralizing mtibodias bgdnut

Pild either fd1wi.n~ inir~venous inj6ctJ.a of rtibbit.5 (jr intrkni.stl inocu-

?VIp ited.f it 'rr.,s fcunc; tht:t this virus Liii.;xd.i:toti the prductia uf neu-

~~UUYC virus thun riic; tither dce which ktiru givun the hutuzn iigmt. The

quuntitc;tive mcc ternpal ciiff&encorr in the mdfoet~tionti of immunity yY'o-

ducod by the&o two viruses intiicuted thtit trhereas they p~sessd,tintigen&~

in comma they were not'antigonicelly iauntic&l.

A aumber of etrtiincl uf the hum&n virus were p?daoC in series

either in cotton rut lungs or in ChiCK e~&rjws t.a rrell w on the chorio-

dluntoic mem'brma ur in tissue culture.  E@icrtaco for thu srttsonco uf the

agent in pmsup mterid WA ubttinbc; by the c.uaonstrutLn ~f.n~utrdizing


This I'inaing ww oT borne interest Yinco it seams prokbla that tha h-n

injection itoelr' is trtrnsmissible :.T contirct.  ili~t~olc~ictr 1 sections of

itli BC tt?ci rb t iUtlEY ;itlti itdec t@d egg uomtxxnes stuiwu by the Giemsc method

irncI impression i'iims tit&cd uy the kcchitiveilo or Cirsn method f&ilod to

rtses~l the grasace oi' dwtentary aooties, inclusion bwictb, tiLGke%tsitie or

bkcterit..

Thtl iuct that +,ho qent recoveroci from current cksc* of primary

atypicui pneillrl<initc bi.'geared 1.0 possess tntigenic components filso present in

the pneumoniti virus of mice srzvcd to confirm unpuuiishcd exyuriments

carried out (iidrbft 1.1.) in the Lt.borcttoriss oC thu Interntitionkl Ha~~lth

iounu thst rtrbbits itunmizdd with chick embryo tissue culture jx~~cga mtkter-

i& uarivad from i.. dsrkd'dti V filtrtri;c: of, LL tlEott wi.shing osL&nod r'rom u

source of thd first rticoverad str&in of i;ha mongoose infactiourj virus. In

the right o;r' the present findings it ww considered of importance to rustudy

this ktter agent.  A suspension of inr'octed mongoose lungs vkichl?+ been
                                 .
stored tit -70%. for over 2% years w~t9 Lvt~il+blc.  It WBY found thu,t the

&gent present in thib aspensIon could be pa61ad in beries `both in cotton

r&t iungs and on the chorioafltntoic membrane,  kaoreover, cotton riits inocu-

l&ted with phsstige mturiai. produced neutralizing antibodies hgainrt PVid

indic&ting that the moqgoose infectious virus tilso possesvad antigonic

components prevent in the virus lif mouse origin.
*


163

AJ.though these studies suggested that the agent recovered from cur-

rent cases was similar to the agent recovered from other cases three years

ago it.was of importtrnce to determine whether following atypicel pneumonia

there occurred in hman beings a. specific antibo&y response to this agent.

Neutralization touts with patients'  sera and tho human virus were difficult

to devise since the virus ftrilud to produce signs of' infection on p&soagc

in available animals.  However, advankge was taken of the fact that one

sputum known to conttin the &gent produced pulmontiry consolidation on

primary inoculation in cotton rats, though it dice not do so.on serial pas-

saga.  When mixtures of this sputum and either acute phase or convcrlescont

serum from patients with &typical pneumonia were inoculated in groups of

cotton r&b, it W&S found thtrt an increase in mtibodios was demonstrable

in the convalescent sora of 11 of a total of 18 patients teeted.  It seems

noteworthy that all 18 convalescent ser;+ completely neutrr;lised the ugont.

      Convaloecent hunan sere not only neutralized the egent respons-

ible-for pulmonary consolidation but also neutralized the agent responsible

for the stimulation of mtibodios against PVM in cotton rats. Moreover,

rats inoculatad with the hman virus produced antibodies which neutralieed

not only PVM but also the agent in the sputum responsible for consolkbtion

in rats. This evidence strongly suggbsted that pulmonary consolidation in

the cotton rat and the develoment of antibodids ag&inst PWA were the

result of infection by one and the OWN agent.

Acute phase &nd convqloscent sert: from these s&me patients were

tostea against mny hetsrologtius viruses mm of which me known to be

capable of inciting acute respiratory tii~oi~sos in hum&n beings. In no
instence was a significant increase against any of those other agents dedon-

strablc.  The evailtible evidence suggests that the mongovao infectious virus


.



                                                  iti

which'appeared to be etiologically related to certain cases of primary atypi-

cal pneumonic studied in 1939 is related antigenically to- the heterologous

virus of mouse origin, the lqpneumonia virus of mice". Since all 15 strains

of the agent recovered from current ceses of atypical pneumonia appear also

to be entigenically related to K5l, it seems reasonable to think that they

are either identical with the mcngoose infectious virus or are very c$osely

related to it both in antigenic composition and biological chclracteristics.

iuloreover, scrologiccl studies carried out with current cases indicate that

in L large proportion of instances there was a demonstrable increase in

antibodies against the human virus during convalescence.  This is strong

evidence that the agent actually b&d produced infection in these patients

and suggests that it txy hava been causally related to the illness.

      dost surprising perhaps WLS the fact that in some cases the

agent was recovered from the plasma.  This indicated that tha infection was

not confined to tho respiratory tract but instead was in certain instances

generalized.  It is well known that in almost all virus diseases in which e

systemic infection occurs, the immunity which follows such illnesses is

persistent.  By analogy, it also seems possiblo.that in prisnary atypical

pnemonie immunity may be enduring.  In this connection it may be of in-

terest thet so far bs is known, there have not yet been reported instances

of two attacks of this syndrome in one individual. If, as seems possible,

immunity foilowing this infection does persist in time, the lm$icatione

in relation to the pssibility of developing effective pr6phylrctic measures

are obvious.  Experiments designed to test the feasibility of actively

immunizing suticeptible anim&i.s using infected chick embryo material as a

vaccine are now under way.

humorous attempts have been made to devise a rapid and reliable


L

165

serological test for the presence of antibodies against the human virus in
order that an etiulogical diagnosis could be made more readily. Although
some suggzstivo preliminary results have been obtained Larry means of a care-
fully controlled complement fixation technique there are as yet insufficient
dctri to indicate that this promiure xi11 wAuz.lly serve to be useful in

solving the present diagnostic problem.

Sttiies on the intGrferonce phenomenon (Ziuglur, Mirick and

Hortifail).  It is well known that certain viruscv tire cuptble of rendering

an infected animal insusceptible to infection bjr a second and scmetinas

&ntigenichlly ImrelcLt6d virus.  This is termod the interference phenomenon.

Andmwcs hr~s rocontly reported thc.t a neurotroyic str&in of influenza A
virus uxl the parent pneumotropic strain from which it was derived h-ere
capble of reciprocally interfering with each other when tested in tissue

culture. The pssibility that influenza A and influenza B viruses, which
do not possess common antigenic components, might interfere reciprocally'

with each other in the chick embr,yo seamed worth investigating, mticu-

larly since it was considered possible that one or the other mi&t be
capable of interfering with the virus recovered from current cases of pri-

mary at~picol pneumonia.  Should the latter phenomenon be ,demonstrable
moans would become available of teeting readily and rapidly not only for the
presence of the human virus but also for antibodies against it. It has
beun.fou.nd that influenza A virus actually does titerfore in chick emwos

with subsequent infection by influenw B virus. Similarly, it has ben
found that iwluenzu B virus in the chick embryo interferes with subsequent

infection by influenza i virus.  &mall quantities of eitiher virus Snoculatod

in chick emtnyos are cr;pable of rcndering such embryos almost if not en-
tirely resistant to subsequent infection with the c&her virus even though

.


with suspensions of dead dysentery organisms will prevent the disease.  The

use of such vaccines is dangerous due to their great toxicity and the re-

actions to which they give rise.  Furthermore, the num'ber of different types
of entaric organisms which can cause the disease is considerable and any

effective vaccine must include organisms of ELU. the known types of dysentery.
     Dysentery bacilli isolated from the stools of patients in the

epidemics studied in this country during the past few years belong for the

                      I
                    i
                     !
                        i






                        ,




                       i





I i

very large amounts of the latter are given.

     So far it has not been possible to demonstrhte conclusively that
the virus recovered from cases of primary atypical pneumonia is ccrpable of
interfering %ith the infection of chick embryos by either of the two in-

fluenza viruses. However, it h&s been found that the human virus possesses
the capacity of interfering in mice with subsequent infection by the pneu-

monic virus of mice.  The available evidence indicates that this is an

additional instance of true interference, even though there exists an anti-
genie relationship between these two viruses, since when they are both

inoculsted simultaneously the phenometion still is repeatedly demonstrable.
     Antigens of the Flexncr group of dysentery bacilli (Goobel,

Binkiey and Perlman).  Epidemic dysentc?rf among mttmbers of the armed forces

and civilian population is at present I: problem of ~pocial concern to

public health officials and to military authorities. Since the outbreak of
the world conflict clinical bacilltry dysentery has been controlled .largely

through the use of the newt;r sulfonamide drugs. The problem of procuring
an efficacious prophylactic agent for the prevention of the disease among

closely grouped peoples where the general sanitary conditions cannot be
adequately controlled, has not been effectively solved. The reasons for

this are manifold.  There ia no assurance that the immunization of humans


!                                                                167

1         most ptrt to the Flexnor grcjup dthough Sonne, Newcastle, :;nd Schmitz in-

/

fsctitiris are froqui?ntly encountered.  The mLre virulent shiga biicillub h;l;s

Scan tiolaonstratari only infrcquontly ~1s the causal agent of dysentery out-

bresks hero in our tti:lnpsri,te ztino.

currently bolievcd thr.t this relatL~nship can bc ettritut& tu L; comkn

kntigcn.  'i'hc intiiviawl spucificitiss cxhibitui by msmh3rs of this group

arti thought tu `DC: duir to tin i:titiikitJnkl SilCCifiC r;nti@n pccUiir:r tc eLch

scrclugicLl type.

iarmunizing &gents r'or thi; prt'vantiun tif the disease in m&n.  Ha hove chusen

the V ntrain (bye I) i'or stuir;r bcctautit? LX tht: bru(d seroiogical crtiseing

which this strain exhibits with other maborb: tif'the Flaxnar grm$.

Ths antizen prepared with oiothylene alycol: a. Pre~trtiun of

the-c;ntiLen i&m the V strain of Shiga oa
--                   radysuntsriaa (Fioxnm~.  The type .

specific antigen of the Flexner V strain can be ubtaiaed in crude form by
extraction elf the organisns with dietbylene gQco1 following the technique

used w Morgan. Further fractfonatlon by vario& procedures has ytilded

I

a sufficient quantity of a highly purified material to charact&ize and to
learn much of its Mnunologicsl and chemical promties. We 1srt3 fortunate

in having at tar disposal the mo&ern techniques of eloctrophoresia ad the

photomutric quintithtive procipitin titration which have buen used very

uffectively in following the purificstion of the antigenic fractions. 1.6

43s. cf the specific antigen, derived from 300 gms.of dried dysentery

orgcinisms, has been obtained in nearly pure form.  The full~wing is an

accuunt c;f the chemical and immunological properties of this materi&


168

b.  Properties of the t.nti{;en.  The specific antigen of the V

c

strain of paradlsentery '&ciilus is a carbohyarate-protein-lipid complex

containing 4.5 per cent nitrogen, U, per cent yhospholipid, and approximately

6U per ce;tt carbohydrate.  Mhen examined by clectrop~lorcsis*t~le antigen is

essentially pure.  The substrlncc is powerfully antzgenic in rabbits. Three

intrhvunous injections of Ijo microgralas each on alturti te days SuffiWs to

evoke potent precipitating and agglutinating antisera in rabbits.  The intra-

dermL1 and aubcuttnoous routes of inoculation i:re equl;lly affective in

giving rise to crntibodios. I

The purifiad &ntiGen if; ilighly toxic, &bout 1000 mic~'ogrt~~M

suffice to kill both rabbits and mice.  On the basis of body wuight f+

rabbit is onu hwdred times more susceptible thcln is the mouse to the toxic

tction of the antigen.

        'JJe have trccurmiluted r, grckt ddal of evidence which indicates that

toxicity is an inherent quality of tho antigcnic molectie and that its toxic

propertics cannot be attributed to tin accompanying substance which is sop.r-

able bx chomic&l frtctionation.  Kor can the toxicity be attributed to the

phosyholipid const%tuent for when the latter is removed, neither the pre-

cipitability, antigenicity nor toxicity of the rosultiat crirbohydrgrte-

protein complcrx ir altered.

Preliminary attempts to alter or impair the toxicity of the anti*'

gen by chelnical mebns have been made, but thus far without SUCCESS. De&min-

ation of the protein cons%ituent with nitrow acid fails to destroy toxicity

nor does treatment with formaldehyde.  ijubstitution in tho tyrosin molecule

with diazonium salts likewise does not impair the toxic properties of the

antigen nor do mild oxidizing &gents.  The toxic antigen is singularly

resistant to chemical manipulation and only drtstic procedures appear to


       destroy its toxicity rind at the same time its capacity to elicit antibodies.

       Because of' the differential between the immunizing and toxic doses in exper-

       imental animls we .?zo of the opinion that the antigen US such may be safely

       used hs :L prophylsctic agent tnd we-~+ prepbring to conduct experiments on


b-r        hum&n beings with this end in view.

               c. Isolation of a tme specific oolyscLcchLcride.  Tha extraction

       of the V strtlin of Qsentory bacilli vrith dieth/lens glycol yiolde not O&y

        the E)ho~pho!..i:Iftl-c~.: bohydrcitti-protein complex but L second immunologic~tfly

          uctive constituent ins well.  The latter h&s been identifid as CI polysecchm-

       ide clnu is highly rc,:&ctivc in homologous antiserum. This polys~ccharide con-

        tctino niwogen prok.bly hl;xocjiains nitrogen and czlthough spocific~~lly rmc-

        tivo in high dilution in V Lntibtirum, it fails to evoke cntibodius or to

       exhibit toxic yrope;ties vrhcrr injected into r<.bbits and mica.  It i6 ix3-         .

       liewd thirt the polys~~hi-~idu is identiczl with the cLrbohydrt.te moiety

       of the antigenic corn&:x Lnd my occur free and uncombined in the hatiterial

          cell.



       tion of dyeentery bacilli with dietwlone glycol i8 not the most efficient

                                       Cells which have been extracted

      with this solvent yield on tryptic digestion large amounts of an anti&enic

        and serologically reactive product.  This materitll is toxic and evokes

      ' specific precipitins and ugglutinins when injected into rabbits. F'raction-

        ation of the substance yielda two major products which are essentially pure

        when examined by elsctruphwesis.  One fraction is G non-nitrogenws ad

        non-toxic polyeaccharide which is immunologically inert.  The other is a

        toxic protein-carbchydrato cauplex, freo of lipid which evokes in rabbits

       type sdocific agglutinins md precipitins. The ittter substance has both


chomiccrl and immunological properties which lsau. us to believe that it is
Vera similar to tinti ti~$e specific bntigcn obtrrined bjt diethylene glyc& ex-

tracticrn.  The tryptic zntigcn is loss toxic than the latter, however, iind

when inj5ct,cd into rabbits evokes precipitins c-tnd tiggiutinins.  The tinti-

bodies tire not qwA.iti~tivcly nor qwntitatively iaenticul with those evoked

either by the untri;trtcd cells cir try the diethylme glycul antigen.  It is

our tJfiiniu1~ that the antigen obtained by thu enzymcttic prclcddure reprcstints

EL tiegradation $rotiuct of the noturr;l mtigen in which the photipholipid has
been remcrved.by the lipc,sa or crude prepcrration of tho trypsin empluyod bind

in vihich ths i.'l-ott:in muiety hes beon +rtialiy dograded by hydrulysio.

AntiK;nic& 2nd toxicity t,f tha V strrAn kntigen. ks pcintod

rJut Lbuve, tcxicity r;ppCi<xs to be un inherent quality of tha V t;trr.in r:nti-

gen.  The most wnvincing aviaence in support d,f this view is the fact th&t

a lysogenic (Vh) strsin 0 f the p&rent organism obtained by second;ley growth
from ph:ige Qsates fails to snthesize any troceof the type specific toxic

antigen.  The organisms themselves exhibit no toxic properties when injected

into experimental animals.  Quantities as great as 30 mg. can be given to

mice intraperitoneally without effect, whereas 1 mg. of the parent strain

kills.

   `When the specific antigen derived from the V strain is adminiaterod 1
intr&ciermally in rabbits in amounts of 2000 micrograms (two lethal doses by
the intravonvus route) the aniuwls do not die. However, there is consider-

able local reaction characteriied by edema, hemorrhage and central necrosis.

This lesion persists for some ten days.  At the end of this period the sex.8
.
of such animals contain a high concentration of antibodies.

     If the ~lirncls me reinoculated at the end of the ten ciay period,

the dormal reaction is much more intense thiin tit first,  The freshly inocu-


171

luted area shows far greater indurcctior! and necrosis than the original.

Furthermore, the site of the first arcxi fLres up 2nd tht rextion persist6

for sweral days.

'iLc;i uifects 01 subcutc,neous inoculction of this antigen in rcb-

Ats is loss dramatic.  Whc:i ;;;030 LiicrObTamB are injected, there is no

lowlized w!cllction. Tne~t, is 6 syrjtemic reclction in the form of genornl

malaise snd a trcnsient rive in temperature.  On subsequent inoculation

these phenolnena do not mnifost themselves.  The animals immunizad by this

route develop in their SWUII poworful 3pecific sgglutinins &nd precipitins

in high titers.

      Isolirtion of a nolystccharide from the lysogonic (Vh) strain.

Thu tryptic diaostion of the orgcnisms derived from rncx~ cultures of the

lysogenic (VH) strcin followed by chemicrJ frtictionation of tho digest feils

to rcvetl any trace of the type 3pecific antigen. Furthermore, the Vtl

organisms do not ngglutinata inV clntiscrum nor do the V organism3 react in

anti VH serum.  From the enzymatic digest of the Vn strain u polysucchtride

has beon isolated which contQn3 neither phosphorous nor nitrogen, and which

posvesses no immunological activity.  The rsignificanco of this subskunce is

not known.  It appears to be i&ntical with the inert polysoccharide isola-

ted in larger qutintities from the tryptic digest of the V strain since both

substances have identical specific rotations and yield the same &wrcentags . .

4)        of.reducing sugars on hytiolysis.

                Immunological rel;LtionshSD of-Shiga uaradysenteriuo. As indicated

        .

previousPJ in this report member3 of the Flexner group arc) inmun~logic~ly

interrelated and it is believsd that their serological crowing can be
attributed to at least one common antigen, where&o their type specificities

are caused by an sdditionul specific :tntigenic constituent.  i&en the most

molcrn wrk, th;rt cjf Boyd in India, has not mtitorially modified 'the hypothe-


sis propounded by Imns and kndrens over twenty years ago.

     During the past few months we have prepared in highly purified

form the antigens from Flaxner 2 and Newcastle strains. Theae subs.tinces

are analogous to the antigen deriveci from the V strain i.e., they a-e

liyocurbohydrate-protein complexes which precipitete in homologous antiserum.

The antigens exhibit serologicai crossing in the ser& of rabbits immunizeed

to other Flexner typos.  When measured quantitatively by the photornatric

method the extent nnd degree of crossing is almost iacntic;A with thcrt

obstirvod by tho agglutination rctlction of heat killod organisms.  Cross

absorption exytirim*nts with the pure V ;:l?d 2 polysaccharides and their

rl<sp3ctive antisera show a ralktionship which is exact&j Enalogous to the

cross reaction of Tj*peS III and VIII pnsumocaccu6.  For e;&mgle, when V anti-

serum is ibaorbed with !~ornologous L)olyshcchtiriae, both precipitins and

sgglutinine for the homologous tind hetorologr>us antigens are removed.  Wien

the absorption is cr;lrriLd out with the hetorologous 2 polyssccharide, the

homologous antibodies remain and only the hoterologou3 agglutinins and pre-

cipitins are removed.  kiince these cell froc antigens represent pure or

nearly pure chemical entities, $t seems not unlikely that their Wological
crossing and that of the organisms from which they ark derived is not cautied

by a multiplicity of antigenic constituents but may be rrtbributed to Sin-

ilarities in the chemical constitution of the polysaccharide portion of the
respective bacterial antigens.  Bc hope to eskrblish this hypothesis by more

extensive work and tc( explain the immunologiccrl roktionship of the organisms
of the Flexner group strictly on the basis of chemical similurities of the

type q3ecific antigens.

Studies tin the bacterial flora in acute resDiratorv diseases

(~tillmnn).  In the lnstqanaemic of influenza the high mortality rate was


largely due to the frequent occurrence of highly fatul pleumonius caused

by pathogenic microorganioms which the mticnt had newly acquired or hap-

penod to be carrying ut the time in the upper ruspiratory tract. Those

opportunistic pathogens, notably the various typos of influenza bacilli,

hemolytic streptococci, and pncumococci, are also frequently found as

sectindury invaders in the purulcnt ccmplicttiwls asoociLtcd with the common

coid.  If infiusnza and tho common cold ;re in fact duo to viruses, it seems

not unlikely th.t these primary agents m&y so alter the defense reactions of

the patient as tc permit socondirry invasion by microorganisms which can be

carried with impunity by the healthy individual for considcrcblo priticifi of

time.  If this hypothesis is curroct then the Lctorial pneumonias and the

suppur&tivo lesions &scribed above must be rogardsc! not as primary disoases

but iis scconrisry comiAicutions 03 the initial virus infection.

      In a study ma& in 1920-21 un the occurrence of ti. influ,,neke
                                                                 -

in cuses of Qneumoccccal lobar pneumonia, this organism was WA&ted from 85

per cent of the piiticnts studied.  Although homophilic bacilli are only

rarely present in nasal cultures from healthy individuals, thuy have been

found in similar ckiltures in 23 per cent of cm388 of lohr pnaurwnfa. At

the time these studies were made the Uifferentiation between the encapsula-

ted (S) and the non-encapwltited (kc) forms of this group of ~giinism had nut
'been recognized and no serological ciassification of specific types h& b&n

developea.' When it was found that a large number of patients with atypical

(virus) pneumonia also harbor in their nasopharynx H. influenzae our in-

terest in this organism was reawakened, particularly since it is now

possible by means of immune sera to classify strains into known specific

types.  Type specific immune sera have been prepared by the immunization

of rabbits with strains of H. influensao of known specific types.  ii study


of the kcterii;l flora; of patients stifering from various mild upper resipir-

atory a&eases is being continued with the hope thtt the results may serve


us c. 'oxkgrodnd in the event of tin epidemic of rcspir;tory infection of viral

origin, complicated by secondaq~ bacterial invasion cs occurred in the last

p6.ndernic of influenza.  Speck1 attention is directed towtird &he occurrence
of type-specific strains of H. infiucnzas.

     Studies ire now in progress on experimentcl pneumonia induced
in mice by the mothod of inhal:Ation which simulates more closely the natuxl

pathwzy ol' infection.  It h:,s been found that pulmonccry consolidation

occtsionclly occurs in mice sprayed vGth H. influonzne followed by inhalation

of pnouinococci :.nri cht.t this lesion does not occur v;hen tither organism is

usoci clone.  Ex+rimonts :.rc being ctrried out to cietk:rmina whether the

combinttion of these two orgen,,, terns is the etscntirl fxtor or v;hether in

L:ssoci:-tion togcthcr they serve ks a provoct:tive stimuius to the pneumonia
virus of mice which is knovm to be htrbored in 1, l&tent statt: by many

animals of this species.

Publics tions

Adams, bn. H., The reaction between the enzyme tyrosinase and its specific
    antibody, J. Ike. i&d.. 1942, 76, 175.

Horsfall, F. L. Jr., The present sttrtus of the influenza problem, J. ml. Me&
     Assn., 1%2,~, 2&6.

Horsfall, F. L. Jr., Human influenza, Cornell University Press, New York, 1942

Horsfall, F. L. Jr., The effect of sulphonunides on vitience of pneumococc$.,.
    J. Clin. Invest.. 194.2, 21, 647 (abstr.)

Horsfal.1, F. L; Jr., Curnon, E. C., Mirick, G. S., Thorn&s, L., t;nci Ziegler, Jr
    A virW recovered from patients with priory utypic&l pneumonis.,
      SC ienca , 1943, 97, 289.

UacLeod, C. a., and PcCarty, ti., Helution of ii somatic factor to virulence
     of pneur;:ococci, J. Cl&. Invest., 1942, Xl., 647 (zbstr.)

                          o
? ?


,

1&+cL00a, C. Ml., ilnii Uirick, G. b., Qu.ntitc;ti.ve tiota!airation of thr? lxzter-
     iut&tic effect of thi: w.Lfunaii:e drugs on pneumwwxf, J. Net.,
     1542, d& 277.

blirick, G. b., Siocle of action of the s:d@~onc!slide drug6 in vitro,  3'. BiLC'ti.)
    1943, 45, 66 (ubutr.).

Lirick, C. b., Enzyi:rtitic i&ntifict.tiLin of p-t mint. tvnzoic cdic! (PAa) in
     cultl.lres of pneumocGccus ;.nil its reLatiun to ~~~~flon~~ni~e-f:stnous,
    J. (;lin. Iwest.,lY42, Zl., 6~8 (cbstr.)

Thaws, l,., Curtlcn, E. C,, dirick, G. k., Zicglcr, J. E., Jr., a-k Horuf;dl,
     F, L., Jr.,  Gcrmplemunt fixt&tion with dissimilrsr atigena in primuy
     atypical pncuwnie,  Proc. SW. Ekp. Mol. Mod., 1943, z, 121.