$p-J

H 82

Rcnort of Dr.  Avex with Drs. StillTan   Gocbel
                                             ----1,------A  Dubo s
---tie--------- -------- I--                  ---,-A
  Francis
    ---- L Babers, Goodner, Terre11 and Rogers.
                                     ------------- ----

I.  Type III pneumococcus pneumonia in monkeys, (Drs. Francis

  and Torrell.)

       1. Experimental production of disease,

       2. Treatment with specific enzyme,

       3. Pathology.

II.  Nature of lysis of Pneumococcus by nutolysis, bacteriolgtic

  enzyme and bile. (Dr. Dubos.)

III.  Study of the antigcnic comulex of tyne-specific pneumo7
  cocci. (Drs.  Avery and Dubos.)

IV.  Methods of purification of enzyme  decomposing the capsular

  polysacchnride of Tyae III Pneumococcus. (Dr. Dubos.)

V.  Isolation of pure cultures of microorganisms decomposing

  the capsular polysaccharide of Type II Pneumococcus.

  (Drs. Dubos and Goodner.)

VI.  Pneumococcus dcrnal infect ion in rabbits. (Dr. Goodner, )

       1, !Chern.peutic action of ant$pneumococcus sera.

3 4.  Deter-inatfon of active immunity by dermal infection,

3. Effect of pneunococcus autolysates unon pneumo-

coccus dermal infection in rabbits.

, .

VII.  Study of pneumococcus  infection and immunity in rabbits    i "

by the inhalation method.

    1.  Pulmonary lesions produced by inhalation method.

     2.  Duration of de.i:onstrable antibodies in serum of

     rabbits following inhalation of Pneu?lococcus.

3. Active and passive immunity in rabbits.

VIII.  InterconvertiSilitp of specific types of Pneumococcus.
   (Dr. Rogers. )


H 83

IX.  Study of the chemical nature of antibodies in antipneumo-

  coccus serus. (Drs.  Goebel and Avery.)

X.  Derivatives of glucuronic acid and glucuron. (Drs.

Goebel and Sabers, )

XI.  Synthesis of p-amino phezol glucosides of lactose and
  gentiobiose. (Drs.  Goebel and sabers. )

XII.  5ibllography.
                      ---ooo---

I.   The Eroduction and treatzent of Txqe III ljncuzococcus
----A--  ---------------- -----  ------------------

  pneumonia in monlcexs.
        -------I----- (Drs  Francis and Terre11 1
                                ---`,,,,,,-,-,-,-------L

   In their first papers,  describing the action of an enzyme
of brcterial origin upon the cansular nolysaccharide of Type

III Pneumococcus, Avery and Dubos also presented convincing
evidence t'nat this enzyme is capable of decomposing the same

substance when encountered in the animal body.   Mice Vere
infected with many lethal doses of T.:pe III pneumococci. It

was:found that as late as 12 to 18 hours after the infection
                             /
                  . . 1
was established intraperitoneally, the administration of the

enzyme brought about recovery in most instances.  Furthermore,
                     1_                                
when the enzyme was given 24 to 48 hours preceding the injection
     ., <    `,     ,. `. !    `,
of many lethal doses of Type III organisms into mice, it was
found to have a marked protectire action resulting in recovo'ry

of most of the animals.  T-he mecllanis% of thn .tberapeutic

effect was shown to bc due to its action on the Capsular poly-

saccharide, decomposing it, and thereby rendering the bacterial

cells vulnerable to attack by the phagocytic cells of the

animal body.  This stud3 was limited to effect of the enzyme
                                         


on Ty~c III Pneumococcus in aq experimental infection primnrily

septicomic in nature.

    Later,  Goodner,  Dubos and Avery studied the action of the

e n z y i.: e upon the localized infection of the s`cin of rabbits

(terineci by Goodner "dermal pnaumonia") nroduced by a virulent

strain of Tybe III PneumococcuF.  In this form of infection, ti?e

lesion is primarily localized and is accorpanied >y scoticemia

of varying degree in different aniaals.  Here agair., A definite

therapeutic effect of the enz:me p?as demonstrable as shown by

procpt subsitience of the cutaneous lesion,  and sterilization of

the blooo.   %owever,  with the a-rounte of enzyme used in the

studies,  tnere was found t 7 `le  a degrre of scpticemin above

which tile enzyzlr was unable to control the infection, alt'n.ough

the duration of lit'e of the rabbits might be prolonged over that

of control animals.  Since excellent results had been obtained

in the treatment of septicemic and local infections of the

smaller animals,  it seemed advisable to study the effect of the

enzyme under conditions  in which the disease process more close-

ly resembled that in tho human patient suffering fron pneumo-

coccus lobar pneur;l.onia.   In this disease,  the organ involved

is highly vital,  the course is comparatively constant, the
                                              

pathology typical kid recovery nroblcmatic.  Consequently, an

atte.,pt was rade to produce in monkeys a disease resembling

lobar pneumonia in man,  which could then be subjected to treat-

ment with the specific enz:*me under conditions more closely

aDpron.ching t:lose encountered. with Datients in the hospital wartir
I

   The Java monkey (?`.  cynomolgas) was selected for the

purpose, and t;:is cnoice has proved to ba a fortunate one,  since

.


the K.  rhesus is much more resistant to the strain of Type III

Pneumococcus employed.  In the first part of the study the

animals were given morI>hine and then under ether anesthesia a

small catheter VJRS inserted into the trachea; the inoculum,

consisting of organisms centrifuged from a given amount of

culture and resuspended in 0.5 cc. of soluble starch was

allowed to flow into the lungs.  During the procedure the animal

was tipped to t,i,e rig,:lt with the hops of localizing toe material

in the right lower lobe.  Tnis technic was,  in the -naJority of

instsnces,  successful.   Eowever,  the location of the infection

was not always predictable,  since the exact site of the catheter

at the time of inJection was not known.  At tines, too, during

the recovery from ether anesthesia, the monkey often went through

a stage of excitement,  wit:? hyperventilnt ion and exaggerated

physical activit),  or the reverse condition with cessation of

breathing which required art if ic ial respiration.   Such incidents

apparently resulted in pneuaonia scattered diffusely through the

lungs and accompanied by a high septicesia.  In spite of these

variable factors, localized lobar pneumonia was Droduccd, in a

high proportion of the animals.   ,
   In the latter part of the study, a more accurate method has
been employed using the technic described by Terre11 and

Robertson.   With this method,  the animal is given a dose of

morphine sufficient to make it ouiet.  The animal ls placed in

the supine posit ion,  and a s-1, 11 radio-opaque catheter is,

inserted into the trachea.  Yith the aid of the fluoroscope the

catheter is tien guided with direct  visual control into the


                             -223

                                                 H 86

bronchioles of the lob6: or lobules, in which the infection is

desired.  When the catheter is so placed, the organisms sus-

pendcd in 0.5 cc. of 5 per cent cornstarch emulsion nrc injected.

The method has several advantages:   First,  it eliminates general.

anesthesia;  second,  the site of inJection is known accura.cl;:;

third, with better localization the number of organisms requirrd

for infection is somewhat  smaller and the scpt iceaia conseouent-

ly tends to increase more gradually.
   Throu;hout the study, temperatures were recorded 3 or 4

timts daily and the course of the pneu;:onia was followed by

daily roentgcnograms.  Daily blood counts and blood cultures

were mnde.  In general,  within 18 hours after infection, n

definite well localized pneumonic consolidation can be seen in

the X-rn;.   Concomitantly, a gradually rising fever is noted.

The white blood cells may be increased in number, or, if
septicemia is present, the:r rnaJ' have docreased.   The lesion

continues to spraad so as  to involve the entira lobe and may

extend to other lobes.  T5e temperature remains elevated, the

septicemia increases, and the leu!iocyte count tends to fall

progressivel;.  Death may follon'in 4 to 7 days in the typical
                in a certain number of instarices'   I
casa.  On ,the other hand,
associated'at times with either a diffuse infection of the .lungs
or with the well localized lesion, there is an immediat.e fall' `

in leukocytes, a heavy seTticea$e and a tendency to subnormal

temperature with a rapidly snreading-lesion and an early fatal

termination.

   There is also a grouo of monke.ys in which recovery takes

Flace spontaneously after 3 to 7 days.   In these,  sent icey;ie


X `1 y bo absent or slight in dogrec, although in two instances the
.jnctcre:lia hns Seen comparatively high.  There is a tendency of

+
<. . . is group to rps-Jond with oefinite fever, leu%ocytosis and a

:,t?ll localized lesion, but with relatively less spread of the

pneumonic process.  The spontaneous recoveries occurring in, this

;:roup make it difficult to sp~a'c of control ani-:als in the usunl

:;ensB, since the factor of individual variation,  so far as ae

have been able to discover, cannot be eliminated or allowed for.

This is especially true when the purqose of tko experiment makes
a comparatively long, but gradually progressive and fatal course

t`ne most dcsirs.ble,  From the point of view of the production of
pneumonia, of course, it does not matter, but when an attcnpt to

evaluate a therapeutic agent is made, the ideal is to annroach

absnlute comparisons of treated and untreated animals.   This has

not been possible.

The studies to date include the results in 82 monkeys with

definite pneumonic lobar consolidation of various degrees. Of

these,  20 animala, or 24.4 per cent, had no septicemia and all

recovered.  Wenty-three animals, 28 per cent, had rapidly
                                          %.I

mounting `septicemia greater than 5,000 colonies per 1 cc. of
                          \

L ,

blood in the first three' days.     
                  All of -these animals died.   The

remaining 'animals Were `subdivided in-to two groups' on' the. basis       ;
                           .I
of the degroe of septicemia preaknt:  (1)' those' in' which 8eQtiCenfa~
was present but not greater than 250 colonies per 1 cc. of blood

in the first 3 days; (?)  th ose in which the septicemia ranged

between 2$CI and 2,000 colonies in the first 3 days.'  The first

of these two groups co?rprises 2i animals, or 31.9 por cent,  and

     cernna                  14.3 per cent,
tho                                   It is in *hpcfi

  ___^___.. -. .-. --- l_-
-------_____2_

---
                        ._


two grouts that the conditions seem most fnvornblc to study the

)ossib1c effect of the enz;`~e uyon t:qe course of tbc exoe:rilri-nt-

nl LisePsc.  The results o3tnined b;r trentmlcnt pre t?lbulnted

b c` 1 o 1.1 ,  co+norins tLC Tr:ortnlit;J rntc in R serits of untrcqted

flnimals wit?. t`nnt of si -,il:>r nni7als ri*CCiVing trc~twnt with

the T';rnC 111 Dol~SrCCil. qride-splitting enzyr?c.   The ?*ei-ison for

ciividing t:;1: ;rou38 on the bns!.s of t:le first 3 cja~;s is t:lpt

a11 of tL-,e a.ti~inls trant+:d,  received t.ic o~z:~l-*f: in the first

3 days nitcr infection.

Anirnrils Flth pnt?u:.oni:> and seyticerlie loss
tilr,n 250 col. /cc. in first 3 dn:rs.

jni 7:tls with 3ncu.:onin Fnti se~ticenin fro7
250 to 2600 col./cc. in first 3 days.

--e-v-

--_------_-----------------
------ -M---e- -----------------
         Su*xAnr:* of both groups.

                                                                           I
      I
  The reaulte on the bn8i8 of coz'ynr~tive mortnlitlr figures
                                         I
are striking.  fn the animals of thoee groups which received
                  

treatment there have been no fatalities, whcreaa, well over

50 per cent of the untreated animals hznve succumbed.   Five rrore

animals nere treated on tillc first dag and. one on the second d.ay
         .

after infection.   In all of t'::esc a d&finite consolidat Ion VRS

noted, in severe.1 t::r fever had renched 1050 to 1060, and the:r

--

-


H 89

seemed on a clinical basis to be suitable cases for the thera-

peutic test; however.  at the time of treatment their blood

cultures were sterile and remained so.   They  all recovered.   Six

others rith very hi:;h initial septiceTias were treated and al-

though they all died,  one treated on the first da.., with a septi-
cemia of lS,OOQ colonies per cc. at that time dici not oie until

the fifth day,  and then r*lth 2,000 colonies of Type III PneuTo-

coccus per cc.  Another with 25,OCO colonies per cc. on the
second day of disease lived until the fourth da;,, end at the
time of death the septicemia hPd been reduceo. to 3,700 colonies

per cc.  This mar-red reduction of the number of organisms in
the circulating blood has been noted in others of the treated

group in which deat;i occurred enrl,-.

   In addition to the efiect of the enzrvme on the sentice-ip,
there is usually seen in the X-rays a definite nrompt effect

unon the pulmonic consolidation.  TSe spread of the lesion is

limited and the advancing margin begins to clear.   At the same

time,  the fever tends to subside and the animal is alert and

active, and takes interest in his food and surroundings.   The

immediate constitutional effect of the enzyrre has varied with .
different preparations and with the eeverity of the disease, , ,,
Some preparations have caused a fall in temperature and, white
blood count and have mad.e. the animals appear sicker.  This ie

esnecially noticed when the monkey is already extremel:? sick.

In animals moderately sick there is a tendency to a febrile rise

following treatment.  It ma: be that the fall of the blood. count

is partially due to an increased 705ilizat ion of tile leukocytes

at the site of infection.


                                  .2g
                                                        H 90

         The treatment has usunlly consisted in the intravenous

     administra.tion of 10 Cc. of en2 -Kk?, var ,ing frnn 2.5 to 20

      units per cc.  Following tAc origin?1 t'rcatment , hlood cultures

        and white counts are  -Lade a-d further treatment has been bqsec!

     on the results of' these stud.ies in conjunction with the general

      clinical nS>pr:arance and tLe X-ray cvidcnce.   In some instances

      three treat-ilenta hove been given in a period. of 24 hours. In

      ti-ie later treatments the intrapcriton.al route has sometimes

     been emnloycc with satisfactory results, and p?rhar,s some rc-

      duction in the amount of general reaction.

          Al thougr.  t!ic stud-, must be carried further, the results so

      far appear nromising.  Becently,  it has been our privilege to

      move tne monkey clinic to nuarters which combine man:' features

       that tend to ex9eoita the actual war":.   It is honed that with

      more experimental ani-;als,  and with further i-provemeqt in

     technical procedures of concentration ard purification of the

        enzgme, more potent and highly purified preaarations may be

     availablo and that unequivocal results of its theraaeutic

      effect may be obtained.

      Pathols1  As a side-product of the primary clinical study of
            - ---
   experimental pneumonia in monkeys, an excellent opportunity has:

                       i
      been presented for the study of the pathalogg of the experikenti
r*     al disease in aniy:als dying at different times wi+.h various

      degrees and stages' of involvement.   The microscopic preparations

     have baen made with the kind cooperation of Jr. Rhoads and Yrs.

        Whitehousc.  TAe description of the microsconic lesions is

      limited tq sections obtained fro.-. the lungs of mon!ze-:s infected

     by allowing t-;e inoculu:: to flow b;; gravity to a given site.


                              fJ$e
                                             H 91

   In the animals dying early in the disease there has been an

accoypanying heavy septicenia.  AS early as 24 hours an ontire

lobe may be inv>lvcd.  Its color at this time is a dark bluish-

red.  It is large, firm and girlcas, except, pi;rhaes, at the
                                                       .-

extreme margins.  There is usually a coderate degree of fibrinous

pleurisy,  sometimes a small amount of serous fluid.   The sentuv

between it anil adjacent 103es is obliterated by adhesions, On

cut section,  the involved lobe is comnnro,tivcly dry, but a

viscous fluid, typical of Tyno III pneumococcus pneumonia, may

be scraped off.  t~lcroscopically,  the most advanced nrocess may

be seen in tAc portion nearest the root of the lobe, while

extending peripherally different stages may be encountered.   In

the region of the main vessels .?nd bronchi :there ma;' already be
seen a marked cellular exudate primarily composed of leukocytes.

Fibrin has appeared in different parts.   The alveolar ~~11s are

moderately swollen and their canillaries are engorged.   Ordinar-

ily t the number of red blood cells in the alveolar exudate is

not striking.  The lymphatics of t:?e larger bronchi and

bronchioles,  although dilated,  show little evidence of infection.

TLe walls of tL;e' `bronchioles show little Infiltration, a.lthough

                                          L
much exudate i& `present in their lumina.                               *
                     The walls `of the blood

                                                            1                 ,.:
vessels `do `not ab+ear to b'e in'vo'lved.  In this area" co&ara't'ively

          :                                                             
f.ew organisms are seen.
   Extending peripherally in a gradual transition from the

preceding phase, there is seen marked engorgement of the

alveolar canillaries with swelling and desqunmation of the

alveolar eplthelium.  The alveoli are filled with exudate CO?-

posed of edema fluid, moderate numbers of polymorphonuclear

leukocytes and erythrocytes usually in small numbers.  There ray


      be a few deposits of fibrin spicules and organisms Rre numerous

       in the exudate,  but not notable in the walls of the alveoli. At

      the margins of the lesion  the predominant features are the

     filling of the r-.spiratorg 103ules with edem,? fluid contDining

I     great numbers of brcteria and co:Dar?tively few cells.   There

     may be extreme congestion of the alveolar capillaries so that

     the walls are outlined by erjthrocytes filling the CaoillariPs.

       The alveolar nails are swollen,  but the outstanding feature

       is that,  in snite of the numbcr of organisms in the material

      filling the nlveoli,  the:f are infrequent in the nails of the

       blood vessels,  the bronchioles nnd alveoli.

         In animals dying on the fourth to seventh days, one may

     find scvcrnl lobes completely involved, and the whole lung

     covered with fibrous pleurisy and adharent to the thoracic

        wall.  The lobe which was the site of the original nneumonic

     consolidation is a slate grey in appearance, or bluish grey.

       It is large,  fir3 and airless.  At times a local area, red in

       appearnrlce,  may be seen,  The lung on cross section is reddish

      grey and ver:. ilry with rough,  granular surf ace.  Microscopically,

      most of the lobe will be seen to be in an advanced stage of the

      pneumonic nrocess.  The alveolar ~~11s are poorl>: distinguished

4      because of the dense character of the exudate and the large

        amounts of fibrin.  The alveolar caoillaries contain erythrocytes

      but are not prominent .  In many phases there is a shrinkage of
     the exudate away from the wells of the sly-call, but strands of
     fibrin can be seen extending from one alveolus to another. In

      the terminal bronchioles the Trocess is auite marked.   In the .

       areas of dense involvement, rsnecially where f i3rin is abundant,


3mJ
H 93

fev organisms are seen .qnd tile number of leukocytes is 17~s.

T&IF: fibrin Rnpen.rs to be ;ost aor':ed in nrc;7s adjncent to the

blooc; vessels.  There is practically no evidence to su.:gcst

vnsculnr thrombi,  and the recalls of the vessels are sur3risingly

free of evidences of infection.  In the larser bronchi, much

exudate ma: be seen in t=lc lumen,  but there is little infiltra-

tion of the 19.~11~ even adjacent to nrens of marked nneuxonin.

T:?c lymn_nhet.ics do not popcar to be infected.   In certain ereos,

peraeps most frequcntl; in the neripheral portions of the lobe

so involved, one notes the npncnranco of large yollo~ish cells

in the alvooli.  Where the;? arc present the fi4rin is disapnonr-

ing and tile pol;~norphonuclear leu!:ocy tes are such dir?inishcd.

These cella are tkc tynical for.18 seen in resolution, and ere

apparently actively phngoc::tic.

In the other lobes, depending upon the durr?tion of involve-

ment, the type of lesion apntars to vary nfth the age.   In some

instances there seeys to be an area of direct extension from

lobe to lobe, but ueually the,oldest lesion is seen about the

hilus, nkile in tne lobe in which the. process is Fast recent
                                                                          : :
                :

the periphe-ral portions may show engorgeaent of the alveolar

capillaries with much ede.Gcn fluid an'd many organisms in the    .I
                            ,

alveoli typifying the advancing m.argin of the spreading infection,
                                                                           

In animals sacrificed after spontaneous recovery has begun,

there is froauently a persistence of fibrous uleural adhesions.

The involved lobe is s~~allcr thnn normnl,  has a rubbery consist-

ency,  and Its color is R rather prrle greenish yellow.   The

surface may be lr*rin!~led.  On cutting the lung, it is rather firr7,


i.ml
H 94

dry,  but flabby.   Vf.r;  little air is present.   In the micro-

                                                         the

scoplc section the striking feature is the predo-.:inance of

large yellowish phagocytic cells.   Certain alveoli contain

numbers of the., with few pol>norphonuclear leukocytes and

large

less

fibrin.

`Q     is the ease with which it undergoes autolysis and the readiness

      with which it is dissolved by 3110 and bile salts.   These two

     properties serve to differentiate pneunococci from other Gran-

      positive cocci;  they are also of sane interest in the study of

      bacterial variation in the Pneur;ococcus group, since the R
      variants are more resistant both to autolysis and bile lysis

    Elsewhere the alveoli are clear   but for  a. ~::a11 number of

these cells;  the alveolar walls are becoming less swollen; the

epithelial cells are being replaced.  The capillaries are per-

haps more dilated than in tile stage of dense consolidation.'

Scat tercd throughout the section are portions in which fibrous

tissue is making its aopenrsnce and beginning organization. At

times tne plugs of exudate nre coated with a la1;er of epitheliun

and almost suggest the organization of a throabus with ~~a.11 new

vascular channels making their a?peara.nce.  The conplcte clearing

of ti;e infection is comparatively rapid, although just as the

laying down of fibrin is rr,arked in the acute nrocsss, so is the

amount of fibrous tissue in tile reparative process.

---ooo---

II. Nature 6f Lysls of Pneumococcus by Autolysie; Bactoriol~t~c
----I------ ----------m---w                 ---------

Enzxne and Bile. @r. Dubosd
-- -------- -_-----
  Among the interesting properties of the Pneumococcus cell


H 95

than the S forxs.  Finally,  these two processes result in the
loss of tXe ch rn.cteristic antlgenic property of the S cell,

i.e.,  its power to stimulate the production of tile type-specific

capsular antibodies.

   What is t:le nature of the phenomena which cause such striki

modif ications of the cell?  It has long been realized that  auto-

lysis is the result of the breakdown of some cell constituents

by the so-called autolytic enzymes.  Cell-free preparations of

these enzymes have the propert.: of changing heat-killed pneumo-

cocci into A mass of Gram-negative de3ris;  this action is fe.irl;r

limited to the Pneumococcus group, but, within this group, both

R and S forms,  irrespective of type, are eaually affected.

   A very satisfactory method of preparation of the bacterio-

phenomenon, most investigators have felt that bile lysis was of

a more physical na.ture, particularly because of its amazing

lytic enzyme is as follows:  A chilled sus.pension of pneumo-

coccus cells is thrown into 15 volumes of c:-.illed acetone, the

precipitate separated by centrifugatlon and dried as a grey

powder.  When resuspended in a medium in which the cellular

enzymes cannot function,  this material may be shown to consist

of a*>parently normal Gram-positive cells.  If, however, the

cells are resuspended in saline they undergo lysis instantan-
                                                                    ---------

eouslxL leaving only a mass of Gram-negative debris.   Outside
---

of the interest attached to this phenomenon of flash lysis,

the technique provides a rapid method of obtaining a solution

which In turn is very active in causing lysis of heat-killed

pneumococci.

Although autolysis was  soon recognized an an enzymatic


                                 z\yz
                                                H 96

rapidity,  e'ven at ice box temperature.   It was also felt that

the property of undergoin.? lysis in bile was in some wny

associated with t.le life nnd death of the cell, since a.ny

procedure which killed the orgnnism would also render it bile in-

soluble.   Howcvcr,  the following experiments, very briefly out-

lined,  SCCT~ to indicate that bile lysis is only an activation

of the autolyt ic enzymes,  nnd that it does not occur when these

enzymes are inhibited or destroyed.

   When pneumococcus cells are suspended in citric acid, for

lnstnncc,  they slowly die but remsin Gram-positive and retain

their morphology.  If thesc dend culls ore then suspended in a

buffer solution at nH 7.0,  they undergo lysis at a normal rate,

they are found to be bile soluble,  nnd when subjected to

f rcczing and thawing give an active preparntion of bnctcriolytic

onzyine.  This experiment SLOWS that dead cells remain bile

soluble when their enzymes hnve not been dustrqycd, but hpve

been only placed under conditions where they cannot function.

When,  however,  the ~011s are killed 3y agents such as heat,

mineral ncids,enlts of heavy metals,  which not only inh'ibit but

also destroy the bncteriolytic enzymes,  they are no longer bile

soluble.

   A direct ovidcnce of the ectiwting effect of bile. on

lysis by the bnctariolytic enzyme  is sunplicd by the fact thnt

the rate of breakdown of hcst-killed cells by the enzyme is ver!;

much accelerated in the presence of bile or bile salts.

    The enzymatic nature of bile lysis  is also indicated by the

influence of temperature on tae phenomenon.   The rate of bile

lysis was measured at 00 C., 5O C., 20o C., 37O C., 45O C.,


-                                                                         --

53O C. and 60o C.  Between 00 C. and 450 C. the phenomenon was

found to h.yve a tempcr,q.turc coefficient of 2 to 3.   At 53O C.

lysis began very rapidly but soon stopped and never WCLS com-

pleted,  suggosting that the enzyme itsolf hqd become in<Tctivatcd.

At 60o C.,  no lytic action could be detected.

   All these fncts seen to indicate the enzy?matic nature of

bilo 1~~1s.  The following observations also tend t? prove that

nutoljsis,  bile lysis and lysis of he-rt -killed cells by sterile

prcpnrations of bscteriol;rtic enz;/mes are fundamentally the same

phenomenon;  all 3 processes have R  temperature optimum at 450 C.

and are inhibited at temperatures greater than 520 C.; the range

of pH lies between 5.5 and 8 .2 with an optimum near the neutral

point;  the rate of all three processes becomes exceedingly slow

in distilled water,  but Increases with salt concentration, at

least up to 5 per cent NaCl; finally, in all three cases, lysis

does not occur In the presence of agents known to inhibit enzymes

   It seems that the foundations have now been placed on which

to build a study of t!le mechanism of lysis.   Four observations

are being studied as possible leads towards an understanding

of thie mechanism.  1, The fact already referred to that. cells

preaipitated with acetone exhibit flash lysia.   2. Activation

of lysis by bile and bile salts.  3. The rate of autolysis and

of lysis of heat-killed cells by bacteriolytic enzymes is very

much activated by small amounts of iodine.  This activation,

however,  can be recognized only within A narrow rn.nge of iodine

concentrrit ion.  Above a certain concentration the enzymes are

completely inhibited by iodine,  the cells revain permanently

Gram-positive and are no longer solu31e in bile.  It may be


I:

H 98

mentioned hero thrt cells thus  trented nith #an excess .and then

washed free of iodine nro still susceptible to lysis by the

bn.ctcriolytic cnz-rmes.

It has bet?n mc:?tioned early in t3i.s discussion th.qt ,711

heat-lcillad pncumococci, irr2spcctive of tyner, ore susccnti?lc

to lysis by tile bacteriolytic cnzy3e.   It should bc nddcd here,

however, that fron t?,is point of view the Tyne I cells are by far

the most resistant, and  the Ty?le III the least resistant.   It is

perhaps more than a coincidence that the Type I con:ains also

the most stable type-snecific antigenic coTnlex, and T;:ne III

the least stable.

---ooo---

LLL---~~~ ~~-`--,--a------------
   Study-of the AntiPenic Complex of Tzne-specific Pneu-o-
                                                        e---------------e

cocci IDr
-d--u-    Avery and Dr. Du'?oa.l
           ,-`---- ----e------e

    Hlhen virulent, encapsulated pneumococci are subjected to any

form of lysis, they lose their property of stimulating type-

specific antigenic resnonse in rabbits,  Such preparations of

lysed cells, however,  are still capable of immunizing mice agains:

infection with virulent pneumococci of the ho-ologous type.

This observation suggeets the existence in the intact virulent

Pneumococcus cell of two distinct antigenic functions, both of

q "  which are type-specific.

              1  Cssular Eolysacaharide anti&;sn,
                   A- ----         ---.---I_--     This antigen is con-
     cerned with the production in the imcunized animal of a type-
     specific ant ibody directed against the polysaccharide constit-

uent of the Pncuaococcus capsule.  Practicall;l all rabbits

immunized with suspensions of intact heat-killed Type I pneuno-

cocci respond :aith the production of type-specific pracinitins,

-


agglutinins and protective antibodies.  If, however, these sane

cells are first allowed to undergo autolysis, or arc dissolved

by bile,  or their hco.t-killed bodios are diges*od by the addi-

t ion of bacterial-:tic cnz;;7es,  and these preparations are in-

jected into rabbits,  tze animals fail to exhibit t:vne-specific

response.  Aa shonn t`arli~`]: in t>is report, tllnse three for38

of lysis are only different exprcs?ions of the ,qctirity of the

aam lyt lc enz~m2s.  We m3? say, thcrcforc, that the capsular

pblysaccharide antigen Is rendered ineffective by the action of

the cells1  own onzyles.

   When rabbits are inzxanized with a suspensI.on of heat-killed

Type III organisrr.8, only A few animals respond rPith the produc-

tion of type-specific antibodies.  IThen,  however,  iodized cells

are used for irxunleation, practically all anlma.18 exhibit the

for?latlan of type-specific preclpltins, agglutinins and nrotect-

ive antibodies.  As has been stated earlier, iddized cells are,

easily attacked by t:le becteriolytic enzyroes and thus changed to
a mass of `Cliarh-liegitive debris;  `Bow it has beon'found that
t.rAatlient `iii& t&e :bsctdr~o~ytic"e~zn~~ doee' ii6t".s$om"ti, mddify
the Ytigfi6-tip&tii'ii'c nhti,ben` oiY:tk;t?i fodisba"celis.  ' fi,`fatit, . ..I'*`.
$&bit@, im&nf#&& `-wf,i,, t'~~.~:&r~Pn6~at~v~.~~d~~ri~?.~btain~d'b~
di:gdating% kddiz&d* ceils r`fth !tKe `~bic~erioiyfic :enzytie gave aa
good type-specific precipitins knd Frote~t~vo.antibodies as ' ' '
animals treated with undigested iodized cells.   It is hoped ,that

this observation may be naoe use of in the study of the chet?istrg

of the capsular polysncchsride antigen.

     2  Seconda= tue-specific antigen.
       2 ----_--_- -- ------ .--m-w- -we  AS stated before, mice

i.~munized with the Graz-negative material. obtnint>d by digesting


                             z,YJy

                                              H 100

hcnted Type I cells with the bncteriolytic enzyme exhibit anrkod
resistnnco against infection with the honologous orgnnis:, but

no rosistanca against Typo II.  The se.~.? degree of active

izmunity can be induced in --ice with sutolyzed cells or cells

dissolved in bile.  Besides being  resistant to the Pneunococcus

cnzyrms, this afitigon exhibits certi-bin re--..?.rknblc properties

which have .z*qde possible sane progress in its purificrtion; it

is insoluble in 95 per cent nlcohol,  but soluble in 70 per cent

nlcohol;  it is soluble in acetic acid;  it is not innctivated by

crystalline trypsin; it go06 through Perkefeld filters.   Anti-

genie n3terial thus purified rencts soecificnlly in scra which

have been freed of antibodies ngninst the type-specific poly-

sacchpride.
                       ---ooo---

IV  Methods of Prcparetion of the Enzym Deco-*posing-the
--L--,,,-----s-s--  ,-2,------------ ------ ---
CaESUlar Polxsncch3ride of Tw III Pneu!3ococcus. 1Dr. Du3os.l
-- ------  --------- -------1------ ------e-e

   The methods of propnrstion,  concentration and purification

of the bacterial enzy:ge c&Fable of dcco?posing the cnosulnr
polysacchnride of Type III Pncuzococcus heve been described in

pre'vioua reports.  These methods' have been simplified in some

details.  In particular, the ar?.ount of yeast extract in the
culture nediurn used for grow'ing the SIII bacillus has been
reduced to  ,005 per cent; this hns somewhat decrensed the ylsld

of enzyne,  but has also reduced the toxicity of the preoara-

t ions.  (It xi11 be rccnlled thet the toxicity ha6 been found

to be due partly to the r+ction of the SIII b*-cillus on yen,st

extract.)  As a reeult,  it hqs been found possible to  eliminate

one step in the purification process, i.e., the adsorption on


J&3@
H 101

nluninun gel.  We have also modified the composition of the

ultrafilters used for concentration,  employing for the membrane
a Solution Of 3.5 per Cent CellUlOSe acetate instead of 5% per

cent as previously used.   This has resulted in the elimination

by filtration of a greater part of irrelevant substances, and

also in a more ranid concentration.

             i
7
         1
         i i
  .' !/

   At the present time,  the main difficulty seems to 3e in

the great losses which occur at the time of concentration,
probably by adsorption on the ultrafilter mem~rancs.  Precipita-

tion of the enzyme with acetone or alcohol ha6 given some

successful but irrogulsr results.   It i6 obvious that the

                                    tion and pur-

                                              methods can

handling of the enzyme at  this stage of concentra

ificatlon will require much more study before the

be completely standardized.

                      ---ooo---

-

V Isolation of Pure Cultures
-I-,,,,,----~-------------- of Organisms Decomposing the
                                        ----   ------------
Cacsular PolEaccharide of Qne II Pneumococcus. IDr. Dubos and
-k------   --w--m---- e-------------- ----------

Dr
--A Goodner.).
    -y--v
   The work on cultures capable of decomposing tho capsular

polysaccharido of Type II Pneumococcua `was continued during the

spring and `the summer.  Several cultures have been obtained pure,
some of them in o. very active`form and soluble enzy'mes extracted

from them in a few cases.  Unfortunately,  during the fall, the
cultures bogan to lose 't&ir activity and at t`he preeent time

wo have only two left which are capable of decoanosing the

Type If polysaccharide.   Furthermore,  the activity of these

two cultures has become so attenuated that it is now impossible

to obtain from them active enzyme preparations.  Time pcrmitt ine
it is plnnned to resume the work this spring.


                                  ZG$j

                                                         H 102

       VI   PneUmOCOCCus DermTl Infection in Rabbits  fir. Goodncr.1
         --`-----------------~,-,,,,-,,-,,,-,,,I,,   ---------

         In the symptom-complex which follows the intrndcrmal in-

      joction of pnoumococci in rabbits,  the number of organisms in

      the blood stream nt a given  time seema to be en accurate index

:t       of the relative  severity of the disease.   The data which have

      accumulated during the past seven years are being an:?lysed to

      learn if this indox may be corrolntcd with any determinable

       conatitutionnl fectors.   So far,  this has not been possible, but

     it is apparent that the white cell count pt tLa time of infectivr

      inoculnt ion is signif icant.   In those instances in which the!

      initial white counts dovinto moat greatly from the average, the

      Intensity of tho bsctoricmin 24 hours nftnr infective inocula-

      tion is thr greattat.  This is eapecinlly true in rrtbbita which

      have high init in1 white counts,  for these invrrria.bly show a

       massive blood fnvnaion--- a rather pnradoxicnl finding.

            Whon so-called flaub-effective~~ nmounts of specific rinti-

     serum are injected intravenously 24 hours after infective inocu-

      lation with Type I nneumococci,  some animals survive while othcra

        die.  If a definite "sub-effective" `qount of the sRme aerum is

     used in a large series of rabbita there is apparently no single
    indicator of what result may be expected. `Some animals with e

h      very high grade of bncterieaia survive, rhile others with R 16W

     degree of ,bnctcriemis die.   However,  if the bpcteriemla .nt' the

     time of tre?tment Is plottod against the white cell count at

      that time with respect to the  end result it is found that anilrel:;

     which survive occupy one section of the figure while those which

      die are oppositely plnceh.  With 9 conatnnt amount of serum the

      number of white cells must be greater in pronortion to the de:rF:r


of bnctcriemin if the n,ni;3al is to survive.  Increasing amounts

of antiserum appear somewhat to compensate for a la&-: of white

blood ~011s.

   The Dcterminnt ion of Active Immunity by Dermnl Infect ion
      --a- -.-----------------------       -----,----,---,,A

   A project is being cp.rried out (with Dr. Stillman) to

ascertain the durntion of nctive immunity na determined by

intrncutaneous infection of rabbits which hnve previously been

immunized by Intravenous  inject ions of hent-klllod pneumococcl,

Typos I,  II pnd III.  Each succeeding month nnimn.ls, not

previously teatod,  are given IntrndermAl injections of living

virulent pneumococcl of the vnrioua types, and the reactions to

these inject ions studied.  At the time of infection, determlnn-
tiona Tre also mpde of the'presence of circulating antibodies.

This study hoa non reached the tenth month, and will be con-

cluded shortly.  It may be tentntively stated that there appears
to be no close correlation between the degree of active immunity

and the presence and titer of circulnting specific antibodies,

   An analysis of an extena,ive series of der>al infections

carried.out to detexine if rabbita possess active immunity; haa

made it poseISle to devise R significant scale by which the :.

degree of active -1mmunfty may be judged.   It Is believed that /

the commonly ncce`:>ted ond-point of immunity, viz., survival and

denth,  does not give a very accurate picture of immunity, for

careful study reveals that there are differences in the rate

with which animals die,  and certainly even greater differences

in response to infection among those which survive.  For example,

an animal16 immunity may be of such a character as to bring

about recovery,  but the animal may nevertheless suffer from R


Severe and protracted infection differing in no eaaontial

respect from that occurring in one which may succumb.   On the

other hand,  the degree of resistance may be such that the animal

may develop only a alight locnlizod area of infection and ahow no

temperature elovstion.   Thus, while it is possible arbitrarily to

state that animals which die are non-immune, and t'noae m;lich

survive are immune,  it would seem more reasonable to hold tha.t

survival and death merely divide the gradient of response to

infection into two phases,  in ench of which considerable

differences may be encountered.

   The Effect of Pneumococcua Autolyaatea unon PneuDococcus
       ---e-e p-w----------  --------m-------

Dermal Infection in the Rabbit.
------------------- ----  It has previously been reported

that the edema fluid removed from the lesion during the earlier

phases of the dermR1 infection in rabbits possesses the property

of inhibiting the coagulation of normal rabbit blood,   Pneumo -

coccus autolyaatea possess the same property.   It hna been

suggested that,  in the course of tile infective process, some of
the less resistant microorganisms may autolyse and thus, through
the action of their end-products, pave the way for the invasion

of the few which survive.  Experiments hRve been undertaken to

deteraine if bacterial autolysates might ,enhance the virulence

`of Pneumococ*cu~.

   Sterile autolyaates of virulent or non-virulent strains do

enhance the invasiveness of pneumococci.   Thus,  R strain of

Type III Pneumococcus (PH) which is capable of bringing about

the death of a rabbit when given IntradnrTally in certain

amounts becomes capable,  when autolysate is added, of producing

the se:* result in much smaller nToUnt3.  On the other hand,


   3 are
       A..&
.    H 105

another Type III Pneumococcus (A 63) which does not inherently

possess a lethal capacity does not acquire t.lis property from

the autolysntc,  but does seem capnblo of greater invasiveness.

                        ---o()o---

VII

---l------L----------------,-~-,,_,-,,__-
   Study of Pneumococcus Infection and Immunity in Rabbits
                                                                   --------we

1~ the Inhalation Method
  ------,---,---------,,`,, 1Dr. Still-.lan.l
                                     ---------

   Pulmonnz
       - -----   lesions produced Q  inhalntion method
                  m-e-- we---   -----------,----z  The

problem of the production of expcrimentnl Pneumococcus lobnr

pneu-ionin in small laboratory animals is still under study.   All

of the  ~18uccessfu1 rosultsll of the experimental production of

the disease reported in rabbits have been obtained by the intrn-

thoracic or intrntrachenl inoculation.  As lobar nneumonia may

frequently be produced in mice by the artificial inhalation

method,  this method has been a3plied to partially immunized

rabbits.  Unfortunately , however,  rabbits failed to show any

evidence of pulmonary localization of the infection.   This

failure may be due to the arrangement of tke lymnhatics in the

rabbit I s lung.  The infection early reaches the pleura and the

pericardium and as a result the animals succumb before lesions

develop in the lungs.  A study has also been made of the

pulmonary reactions of rabbits following intranasal instilla-

tion of pneumococci.   It was found that the mortality of rabbits

so infected variod in direct proportion to the number of pneurno-

cocci instilled into the nose.  Although no lesions of pneumonia

were produced,  characteristic vascular and lymphatic inflammatory

changes were found in the lungs of rabbits infected by the

nasal route,


312
H 106'

  Duration of demonstrable antibodies in serum of rabbits
     --L-----------"------------------------*---------
following
-------  inhalation of Eneumococci.  The duration of tyne-
            ---C-----.-- ---I------

specific agglutinins and orotective antibodies in the serum of

rabbits follo!?ing inhalation of 1iLv ing: >neu=ococci has been

st-adied.  Type-specific I?.,~ rlutinins could be demonstrated or?l;r
for a short period after the course of ,exnos=lres WEB ternin*>ted.

EIowever. Ty/ne I protective antibodies nera still detertable in
the sera of these animals yenrs after their final exposure to

:>neum.ococci.  `L'l>e protective antibodies dcvelonirg after expos-
ure to inhalations of Tyne II pneunococci, on the other hand,
remaineo de9onstra>le in tkeir sarum for only A relatively

snort period.

   Active ard Rassive i.?;*r.u;lt~  in rabbits.   The duration of
       ------;-- -e---.-v----       -------m-
type-gnecific antibodies in ra5bit.s following inoculation of
suspensions of bent-killed pneunococci is still under investiga-

tion,  In conjunction wit;1 Dr. Goodner,  the active im7,unity of

theso rabbits as detcr~~inod. 3:, ii~:r~~al infecti.on and the correla-

t ion of this with tiio prc:sencc of tync-spccif ic antibodies in
the serum is baini: studied.

-- -ooo---

VIII  fntcrconvertihil~ of Specific Taos of Pneumococcus.
-e--L----- -----    ---- --w---q -----------c--w-

LDr. Rogersa
-_Lc --

  It Is an eatabliahed although poorly undorstood fact that
rough (R) pneumococci may be experimentally ,restored to smooth

forms of the same type as thst from w'nich tbeg were derived, and
that they may be l'transfor:uedlf into smooth pneunococci of a
serological. type difft.rent from that of their origin.   Since
the initial work of Griffith subseouent studies by Neufeld and


                              ST&
                                            H 107

Levinthal, Dawson and sia,  and more recently by Alloway have

not only confirmed the occurrence of t!:is phenomenon, but have

added much to our knowledge and technical control of it.

   Tkc transformation of pneu~~ococci from one type to another,

which at first could only be accomnlishcd by an ir, vivo
                                                                --e--v-

tochniout employing pncumococcus  'fVaCClncsfJ as the transforming

agent ,  can now be carried out in vitro using sterile, cell-free
                                        ------

pneumococcus t3XtrACtS in the nl~ce of vaccines.   It has,  furthcr-

more,  been shown thst tke organisms thus converted from one
Typo into anothor undergo R rcnl an& permanent change, acaulrlng

all the binlogical chnractcristice of the n1.w type, i.e.,

virulence,  typo-specificity,  tyne-stability unon rcpcated sub-

culture,  Rnd snccific antigenicit;,

   -$llonay was the first to succeed in using filtered cell-

free extracts of pncnmococci as the transforming agent.   These

extracts were obteincd by dissolving virulent cultures with

sodium desoxycholata, precinitating the bacterial solutions in

absolute alcohol, extracting the precipitate with salt solution

and furtbcr,purifying by charcoal adsorption and Rerkefeld fil-

tration.  (J. Exp. 12od., 1932, 55, 91; 1933, 51, 265.) The :
extract of type-specific pnoumococci., so prepared, containing
the .so-called Ittransforming factor ,n was added to small ouan-

tities of broth enriched by the addition of 25 per cent ascitic
or chest fluid, and to the whole wrs added a very small  inoculum

of living nR" cells to be transforned.  Trnnsf ormation usually

occurred after from one to four subcultures hsd been made.

    Alloway found that although  these extracts are rich In

specific soluble substance the degree of transforming activity


H 108

of an extract is not necessarily correlated with the alyount of

t ::,.c specific capsular pol2sacchsride prcsent.   ?;oreovor, the

cilemically isolctad and purified snccific soluble substance

possessed no transforming qualities.   It has also bc en shown

t;lat tile transforming factor lncks the thcr~ostabilit~: l,nsscssed

by the purified carbohydrate.   So far,  it nqn,c;ars that the

transforming factor romnins closeJy nr.socintcd with the specific

soluble substance, thoug'h trio:,  qr`c not identical in tbcir phys-

ical proportica in so fnr ns we  hpvo been able to test them.

The work of Alloway has been  confirmed in our recant studies,

and further efforts at J)uri.ficntion of the transforming factor

Rre being node at the prcrent tir,e.

It has been found that by careful adjustment of the final

pH of the mcdla em:jloyed In obtaining transformations, sufficient

growth occurred to permit OS mnny as three transfers within a

twenty-four hour period.  This not only alfds greatly to the

rapidity with which transformations may be obtnined, but also

seems to produce more const,nnt resulte.  X few experiments unon

the so-called "serum factor"," which is an essential ad,junct in

the cultural technique,  would seem to indicate that the import-

ance of this substance is dependent u?on a certain minimal

content of anti-R pneumococcus antibodies, perhaps even more

than upon its nutrient value.  Active extracts, in addition to

being thermolabile,  lose their activity u'>on ageing for e.ven so          8:;
                                                                           I' 1

short a tima as three or four weeks in the ice box.   The nature   i j'
                                                                           /i,
                                                                           t; ,:
                                                                           ,i b

of this degradation is being investigated.                     I

   Still further purification is being Rtteiauted e-nploying

various enzymes, adsorbents,  imaunological and chemical me thod.s ,        i
                                                      rj
                                  #
                                                                        G?!
                                                                   gj
                                                                           


v,

                                 326
                                      H lo%-

    but as yet the data are insufficient to warrant conclug~ong,

       It is desired, by t-18 methods outlined, to reduce the

    trWISfOrming factor to its simplest and most active form in
    order to acquire knowledge of it8 biological and chemical Drop-

     erties,  and 80 to arrive at a better understanding of this most

    unusual and significant phenomenon.

                            ---ago---

      IX  Study of the Chemical Nature of Antibodies in AntiE~eUmo=
        --r-e--- m--------e -- ----L-----------------
   coccu8 Serum. ADr8. Goebcl and Averp,l
         ------------ ---------I_ -m--w
     The investigations of Northrup concerning the nature of

    pepein and trypsin have led to the isolation of crystalline
   protein8 poesessing unique chemical properties and biological

     activity.  The 8ucce88 of the88 investigationa, and the fact
   that these crystalline proteins have properties which dlffaren-
   tiate them sharply from other biologically inert proteins, have
    led u8 to believe that the immune protein of antipneuaococcus

     horse serum might, by mean8 of some unusual property, likewise
   be separated and differuntlated ,from ,the ACCompanYing inert / ,_ ,
    8erum protoias. , As a starting material for this investigation
                                                                             .
   we chose, that fraction. of antipneumococcue Berum (Type 1) which
    precioitatee..from whole :serua on the addition .of large quan-   k, ;
                                                                           .' 7
    tities of water,., This fraction,: which 1s insoluble in water,
   contain8 practically all of the antibodiee present in the

     original serum.  It was found that this matnrial could be
   further separated,into an inert water insoluble englobulin, an
   inert water soluble pseudo-globulin, and a second Water soluble
   pseudo-globulin which contained all of the antipn8UmOCOCCU8

      antibodies.  This biological specific protein nO888sSe8 CertRin


      unusual chemical properties which permit it8 separation, 86 Cell

       a8 it8 differentiation,  from the other inert serum proteins.

       This substance ha8 a lower total nitrogen content, and a much

      higher amino nitrogen content than the proteins of normal serum

      A chemical analysis by tile  Van Slyke method reveal8 that the

        distribution of basic amino acids is different from that of        .

       normal horee Berum proteins.   An electrometric titration of

      this unusual protein ha8 shown that its isoelectric point lies

       at pfl 7.6.  The protein is soluble in distilled water on either

        side Of it8 iSOeleCtriC point,  but is precinitated from water

       at pH 7.6.  The protein Is extremely active 3iologically; a

      solution containing as little nitrogen a8 0.007 mgs. per cc.

      causes specific ,agglutination of pneumococci.  The protein

       contain8 all of the various antinneumococcus antibodies, i.e.,

      it Contain8 the type-8p8CifiC and 8p8Cie8-8p8CifiC antibodies.

      Attempt8 have been made to separate these antibodies, but

      their ch8mical'prop8rtiea are 80 closely related, d88pite the
     fact that their immuno~dgical properties are sharply differenti-

                                   Studlee on the nrotdctive

       present being carried out.

                                 ---ooo---

      X Derivative8 of Glucuronic Acid and Glucuron, lDr8   Goebel
             -A,----,,--- -------------------a-- ---A---wa.-

             ------me--
       and Babers.)

         The presence of glucuronic acid in the specific carbo-

      hydrate8 of Types II and III Pneumococcus, in those of Type8

---


                                  33g
                                        iI 111

    A, i3 and C Friedlinder bacillus,  and in the specifically react-

     ing carbohydrate derived from gum acacia, has led to the belief

     that this sugar acid has en important function in directing the

     specific immunological properties of the poIysaccherides of

      which it forms a part.  A study of the chemistry of glucuronic

      acid,  and its lactone, glucuron,  Is in progress.   The ultimate

     objective of these studies is to prepare a glycoside of

     glucuronic acid which may be chemically combined with pkotein,

     The immunological behavior of the resulting complex may then be

     directly cbmpared with the responses  elicited by the hexose Rnd

     disaccharide antlgena which have already bee* studied.

         An excellent source of glucuronlc acid is t-:e urine of

      dogs fed on a diet of d-borneol.   Glucuronic acid is isolated

      directly and easily frorr; the urine as  the zinc salt of bornyl

      glucuronlde.  The latter compound yield.6 a mixture of glucuron

      and glucuronic acid on hydrolysis.   It is not possible to

     separate glucuron from glucuronic acid by fractional crystallie-

      at ion. It has, however'; been found poasible to convert this
                                           1
              .
   mixture quantitat'ively Znto' glucuron, as well a0 `to reconvert

     glucuron quantitatively into pure glucuronlc acid by mild (
    hydrolysis with b`ari-urn hydroxide.                                .,,
                                                                        . .               . . -'
                                                                           ' !      .I
                                                                           .

0         The comparative reducing values of glucuronio acid and
    glucuron have for the first time been ouantifatively determined.
                                                                           i .I
                                            >

                                                                           1
      A report of this study is now in press.

        In a previous report (1931) there was described the
     preparation of& and B triacotyl glucuron.   These derivatives

      have now been secured in optically pure forms.  They show a

      difference in molecular rotation of 36,400, a value which is


                             399
                                                H 112

in excellent agreement with tllc knovndifferences in molecular

rotations of tie pentacetL.1 hexoses.  A crystallographic study

of these two derivatives is being made by Dr. Ralph Wylroff.
  Vhcn either a or 9 triacctyl glucuron is treated with
ncetyl chloride and hydrogen c:lloride,  an aceto chloro derivative
is formed which is pro3ably a mixture of d and 6 .   1-chlor

2, 4 diacetyl glucuron.  This compound serves as t>e source

materiel for the syntnesis of glucuronides.   When ncpto chloro

glucuron is dissolved in moist ether and shaken with silver

carbonate, tne chlorine atom is replaced by hydroxyl yielding

a crystalline derivative which nopears to be 1 hydroxyl, 2,

4 dincetyl glucuron,  We have prepared a non-crystalline methyl
diacetyl glucuronide from diecrtyl chloro glucuron, and we have
succeeded in synthesizing A smnll quantity of n crystalline
diaoetyl p-nitrobeMy1 glucuronide.  The condcnsat ion of

diacetyl chloro glucuron with phenolic derivatives is very

difficult to (pffect.  It is necessary to overcome the difficult-
ies before the synthesis of glucuronides can be profitably

attempted, and we are still making serious efforts to do so.

                            b I     ---ooo-..-
XI. The aynthesie of-aminophenol glucosides of lactose and
--1--m -w-      -I--                   --------------m
                                                             4
gent iobiose.
  ----- -9
   The result of the studies of the immunological behavior
of synthetic carbohydrate protein antigens has proven to be so
instructive that we have extended this investigation to the

disaccharides.  The factors which control the immunological
specificity of carbohydrates anpear to be the pos+.tion of
intermolecular linkages end differences in intra-molecular


H 113

stereochemical arrangements.  The disacchrrides, whose chemical

constitutions have been well established, are ideal substances

for investigating theeffect of such changes on imcunologica.1

response.  The four disacch-rides which are being studied are

represented graphically below.  The differences in suatial

constitution and inter-molecular linkage are readily discernable

from these granhic formulae.

In order to combine chemically a sugar with a protein it is'

necessary to synthesize the p-aminophenol glucoside.   Those

derivatives are then combined by diazotization of t5e amino groun,

and coupling the diazonium glyc'osido with protein in alkaline

medium,  Ye have synthesized tile heptmetyl 6 -n-nltrophenol, the

,p -p-nitrophenol and the fi -p-aninophenol glycosides of maltose,

lactose,  cellobiose,  and gentlobiose.  These glycosides have
been combined with protein and a study of their antigenic
properties is nearing completion.


H 114

PUBLICATIOXS
,,,,,-4----z.

Avery, 0. T.  The role of specific carbohydrates in Pneumo-
       coccus infection and immunity.  Annals Int. Ifed.,
       1932, 2, 1.  (John Phillips 1!ernor~i-%!~~~~~---

Avery, 0. T., Goebel, W. F., and Babers, F. `s.  Chemoimmunolog-
      ical studies on conjugated carbohydrate-proteins.
         VII. I-munological snecificity of Rntigens nrepared
     by combiningN and6 glucosides of glucone vith
       proteins.  J. Exn E'ed., 1932, 22, 769.
                         ----LA-.-

Alloway, J, L.  Further observations on the use of Pneumococcus
       extracts in effecting transformation of Type in vitro.
                                                                         ----e-m
     J. Exp. Med., 1933, 57, 265.

Baudisch, 0.. and Dubos, R.  fiber Katalaseairkung von Eisen-
      verbindungen in Kulturmedien.   Biochem. Zeit., 1932,
                                                        - --w-e----
         345
             --- *  298.

Francis, T.  The value of the a!:in test with tyne-specific
      capsular polysaccharide  in the serum treatment of
      Tlr>e I Pneumococcus pneumonis.   J. Exn   Med.
                                                    ,---,L,,, '  1533,
      (April number).

Goebel, TI. If., Babers, F, H., and Avery, 0. T,   Che?o-immuno-
     logical studies on conjugated carbohydrate-proteins,
     VI. The synthesis of p-aminophenol CL -glucoside and
      its coupling wit:1 protein.   J. Exu   Ned
                                            ,-,-L1--2,'  1932,
      53, 751.

Goebel, W. F. and Babers, F. U.  Derivatives of glucuronic
         acid.  I.The preparation of glucuronic acid from
     glucuron and the estimation of their reducing
        values.  J: Biol   Chem
                      -w---A---L* 1933. (In press.)

Goebel, 8. F. and Babers, F. H.  Derivatives of glucuronic
         acid.  II. The acetylation of glucuron.
        J. Biol  Chem., 1933.
           -~se.AC-e--      (In press.)

Goodner, E.  A test for the therapeutic value of' anti-pneumo-
          coccus serum.  (Presented before tie Amer. Public
    Health Service, Was.iington, D. C., October 24, 1932.)
      J. Immunol., 1933.
             m-p-       (In press.)

Goodner, K, and Dubos, R.  Studies on the quantitative action
    of a specific enzyme in Type III Pneumococcus dermal
     infection in rabbits. J. Lxn. Yed., 1932, 35, 521.
                                            ----i--A---

Julianelle,  I,. A. and Rhoads, C. P,  Reactions of rabbits to
      intracutaneous injections of pneumococci and their
       products.  VII. The relation of hypersensitiveness
      to lesions in the lungs of rabbits infected with
       pneumococci.  J. Exp.Med. 55
                               ----- ,-,-* --*  797, (1932).


Kelley, W. H.  The antipneumococcus properties of normal swine
           serum.   J. Exn
                        ---M-w . Fed. , 1932, 55, 877.

Stillman, Ernest G.  React ion of rabbits following inhalation
      of Typo III pneumococci.   J Inf
                                                -L-e    Dis
                                                  `--e-L *  1932, 50
                                                                           e-9  542.

In manuscript:
---------- --

Goodnor,  K, and Stillman, 2. G.   The evaluation of active
      immunity to Pneumococcus infection in rabbits.

Goodner, K.  The effect of Pneumococcus autolysate on intra-
       dermal infection in rabbits.
       J. Exn. Vcd., 1933.
             --_--A_-       (In press.)

Stillman, E. G.  The react ion of partially Immunized rabbits
      to inhalation of Tyue I oneumococci.

Stillman, Y. G.  The reaction of normal and partially immunized
      rabbits to intranasnl instillation of Type I
       pneumococci.

Stillman, Ed G.  Duration of tyne-specific agglutinins and
     protective antibodies in rabbits following inhala-
     tion of living pneumococci, Types I and 11,