qroteins.


              1.  ,i'he specificity of antigens pre.,ared by combining
       \Xana e glucosides of glucose Fith protein.


i,        2. The s;ntAesis of glucosides of the disaccharides,

             cello5iose and maltose,


         IV. Studies on the interconvertibility of tne specific types



L          of Pneumococcus,

H 37

Report of Dr. Averywith Drs. St illmn, Goebel, Dubos,
-- _--_----m---e ----------------em

    Francis   iJa\ers
       .__---         Goodner,
              -L--`---I-,-----.-  H-_-v-- ,------,,--1
                    Allowax and ,ierrell

I.  The 6ecomposltion of the canslllar polysaccharide of

r, ryne 111 Pne,Jmococcus by a bacterial enzy--e.

  1. l!et:lads of purification and concentrat i-on 0

   s?ecif ic enzjr:-.e,

f ti,e

2. Protective action of the envy-:e in derma.1 infection

  of rabbits with Type III Pne-umococcus.

3. Production of Pneumococcus Pneumonia (Tvpe III)

       in :,ionkey 9.


II.  Isolation of microorganisms decomposing the can?ular

  polysaccharides of other t,:t.oes of Pneumococcus,

III, Chemo-im:tiunological studies on conjugated sugar-

V.  The ciegree and duration of active  and nassive im?runitp
                                                                s

to Fneumococcus in rabbits,

VI,  Pu:?licat ions.


228

H 30

         I.  The decomposition of the cansulnr Eolxsaccharide
             ----------   ---------------Lo----- __  e-----_--e

of Tx~e III Pneumococcus by
---w --------e-------m-  a specific bacterial enzyme.
                                    ----  ------------------
(Drs-. AverxLI Dubos,-Goodner and FrancisL)
--- -e---m

      1. Methods of Eurificntion and concentration of
                 -------WV__ -----__I_-__________________I__

enzyme.                   we have described the isolation
--- ---  In previous reports

of a microorganism capable of decomposing the ca.psular poly-

saccharide of Pneumococcus Tyne III, and the extraction from

cultures of this bacillus of the active enzyme rcsnonsible

for this action on the polysnccharide.  This enz;rc,c is not

only active in vitro but retains
                -----em          its specific activity

in vivo as damonstrated by the protective action afforded
-e-m---

mice infected vith Pneumococcus Type III.

      The work of this year has been concerned chiefly
with a study of tho action of the enzyme on the course of

an experimental disease induced by infecting rabbits intra-

dermally with a rabbit virulent  strain of Pneumococcus

Type III.

        However, before describing this study, modifica-

.tions in the technique of preparation of the enzyme will be

given.  It will be recalled that the yields of enzyme were

much increased by the addition of small amounts of yeast

extract to the synthetic mineral medium originally used.

Unfortunately, the preparations thus obtained exhibited a

definite primary toxicity expressing itself by a sharp rise

in temperature of the injected animals and at times death

within a ferf minutes after injection.  The production of

non-toxic preparations has been attempted along three


different lines:  (a) the amount of yeast extract in the
medium has been reduced to the minimum concentrat?on comnat-
ible with good yields of enzyme (.03 per cent); (b) toluene,

which had been a,.ded to some cultures  in order to achieve a
more rapid and more complete extraction of the enzyme, has
been completely eliminated from tl?e operation; (c) the auto-
lysate is now purified by a process of differential adsorption
on aluminum hydroxide; this process is such that, under well

defined conditions, the toxic princinle is adsorbed on the

aluminu;n gel while the enzyme  remains in solution,
      Concentration of the enzyme preparations has been

achieved by two different techniques:  (a) distillation under
reduced pressures at temperatures not exceeding 35OC;
(5) ultrafiltration through a cellulose acetate ultrafilter;
in this case the enzyme remains  in the filter while most of

the irrelevant material is eliminated with the filtrate.
     Throughout the steps of preparation, purification

and concentration of the enzyme, the operations have been
controlled by quantitative determinations of activity by the
titration method.described in the previous report. Te have-
found it convenient to express the potency of the preparations

in terms of units.  The unit is defined as 100 times the

minimal amount of enzyme required for the decomposition of

. 01 mg. of chemical ly purified capsular polysaccharide in 18

hours at 37b C.

       The purified,  concent rated enz:`me preparations
exhibit little if any toxicity for normal rabbits when in-

jected intravenously.  Xormal monkeys which received very


,

HZW

large amounts of these preparations by tke intravenous route

exhibited no reaction recognizable by change in the t~~mpCra.-

ture or behavior of the anim;ll.   However,  the study of the

blood picture revealed a sharp rise in the white cell count

for a few hours following injection.   Whatever the nature of

the leucocytic reaction,  we are inclined to consider it as

beneficial in view of t;?e essential part played by phagocytosis

in the course of events following enzyme treatment of infected

animals.


          2   The protective
             2------        action of tke snecific enzyme in
                          ----------__--------_-e-_---e---  -----

dermal infection of rabbits with Tzne III Pneumococcus.    In a
___--_-___--_-___--_-------------- . ..-------------------

previous report it has been shown that the enzyme which snec-

ifically decornooses the capsular polysaccharide of Type III

Pneumococcus has a marked protective act!.on In mice infected

intraperitonc~~lly with this organism and is capable of bring-

ing about the recovery of these animals V"nen given even after

the infection is already esta31ishe.d.   The intraneritoneal

injection of mice with pneumococci leads quickly to a general-

ized infection.  On the other hand,  the dermal infection of

the rabbit, previously studied by one of us. in detail, gives
rise to a disease which is primarily localized and in which

the infec.ting organisms  in the local lesion are less accessi?le

to the action of an agent administered intravenously;  Hamever,

the specific enzyme has a marked curative action in the

disease brought about by infecting rabbits intraders?ally with

a highly virulent strain of Tyne III Pneumococcus.   The use

of this enzyme in suitable amounts in the infected animal

promntly frees the blood stream of bacteria, leads to a


          231.
            H 41

.

sterilization of the focal area of infection, and a conseauent

complete cessation of t.:e disease process.  A mortality rate

of 5 per cent in treated animals  is in sharp contrast to a

mortality rate of 95 per cent  in untreated rabbits.   The

curative action of the enzyme is type-soecific.  Its activity

is ciestroyed by heat.  The results of this study yield further

evidence that the capsular .substance  is an important  condition-

ing factor in pneumococcal infection, since, in so far as

`xno wn , the only action of which the snecific enzyme is capable

is that of decomposing the capsular polysaccharide.

       Animals which have recovered following enzyme

injections within 24 to 48 hours after infective inoculation

show no active immunity against subsequent massive  reinfection,

On the other hand, animals  which were treated reneatedly over

a period of days with smal 1 amounts of enzyme and in which the

disease was not arrested until the fifth to ninth days, show

a high degree of active  immunity against reinfection.

       An elaboration of one phase of these studies has

dealt `with the question of the minimal amounts of enzyme
necessary to bring about recovery of the infected rabbit and

with the correlation of certain factors involved in these

quantitative relationships,

         Drs.  Dubos and Avery have shown, by in,s&4,r~

experiments, that,  after a ciefinite incubation neriod, the

total amount of specific substrate decomposed bears a

quantitative relationship  to t:;le concentration and activity

of tne enz.,ime TreBarat ion used.  If it were possible to amply


232
H 42

this finding to the experimental infection, t::e minimal amount

of enzyme necessary to bring about recovery should have a

quantitative relationship to  the amount of specific capsular

polysaccharide in the animal bo<.y.   This substance Is of course

associated with the oneumococci but may also be present in

soluble form entirely free from tne mlcroor&anisms.   It is

technically Impossible to deterrilne the total amount of the

specif lc polysaccharlde  in the infected animal or even to esti-

                             .

mate the total. number of ;>neumococcl.   The nearest aoproxima-

tion Is the determination of t..e number of via3le pneumococci

in the circulating blood.  Other factors the same, this lnd.ex

should,  on theoret ical grounds,  bear some quantitative relation-

shin with the amount of enz;`me necessary to bring about

                     .
recovery.

A series of 85 rabbits has given definite evidence

that taia is actually the case.   For exnmnle,  2.5 units of

enzyme were sufficient to bring a3out recovery when the

bacterlemla amounted to 3 pneumococcl per cc. of blood, 6.6

units sufficed with 15 organisms per cc., 20 units were re-

quired with bacteriemias between 100 and 1,000, 50 units

sufficed with bacteriemias between 1,000 and 5,000.   However,

100 unit a failed to bring about recovery when the number of

pneumococci per cc.  of blood exceeded 20,000.
                                               ,

The results Indicate that as the bacteremia in-

`creases the amount of- enzyme required to bring about recovery

is greater although the  two ratio between the two factors is

not :?roportlonal.

Studies of the s>eciflc action of the enzymes on


Type III infections  in mice and rabbits have furnished con-

vincing evidence that this agent exerts a favorable influence

on the course of the oisease in these   animals.   `Interesting

as these results are,  it is at once aypal-ent, however, that

the experimental conditions under tvllich the enzyme has thus

far been tested are not entirely comparable to those prevailing

in pneumococcus pneumonia.  A crucial experiment has therefore

been undertalren to determine whether with this particular tyne

of Pneumococcus an experimental pneuzmonla can be produced in

monkeys which in clinical course and p,qthology more closely

resembles the se>ontaneous disease in man; and finally, whether

the enzyme would be effective under  tne more aromylicated con-

ditions of an actual consolidation of the lung.

      For this study, tr.e Java (I?, Cynomologos) monkey was

selected;  since other workers in attempting to produce exper-

imental pneumonia,  have found the more common I!. Rhesus and

the Cebus species rather unsatisfactory.  In addition, the

Java monkey has proven to be somewhat hardier and rarely in-

fected with tuberculosis.  A strain of TyQe If1 Pneumococcus,

virulent for rabblto, was chosen for the original exoeriment;
and this strain as recovered from an infeated monkey` has been

reinoculated serially into the succeeding animals.   The use

of morphine, suggested by Robertson and his co-workers in the

production of experimental pneumonia in dogs, has been
adopted, for it was thought that morphine would abolish

coughing, perhaps reduce  the resistance of the animal temp-

orarily and hence allow the organisms to gain a foothold in

the lung.  The drug is administered 2-3 hours before inocula-


i

t ion.  Roentgenogr&ns of the chest are taken before inoculation              :

as control,  and at least once daily thereafter,                                    

                                                                           i:

      The technique of the inoculation is as follows:   The

animal is anaesthetized with ether.  Yhile the jaws are held

widely open,  the tongue is drawn forward with lingual forceps,

the epiglottis grasped with plain forceps, and the larynx thus

made clearly visible.  A h?.rd rubber catheter is passed into

the trachea until resistance is encountered.  The organisms

centrifugated from a fresh culture and resuspended in 0.5 cc.

of soluble starch solution are introduced from a syringe fitted      .- ,:

into an adaptor in the end of the catilcter.   The material is              ' ,' :,
                                                                           ?
                                                                           ii
                                                                           4

allowed to flow into the trachea while the animal is held up-

right.  An equal volume of air is then slowly injected to

ensure the emptying of the catheter.  Care is taken at all .

times to avoid contamination with material from the mouth.

      Following the inoculation, daily blood cultures,

blood counts, and .tempcrature records are made in addition to

the x-rays.

     By thia method it has been possible to produce a
pneumonia which is dtstinctly lobar in distribution and tends
progressively `t`o iriv'olve `Sore `than .`one lobe. ,Fhe-:sp`re&d df *I;
the lesion `hiAs `been `clearly followed by means `of the

roentgenograms,  Ab autopsy the gross pathological findings
are quite comparable to t:le typical lobar pneumonia in man.
This similarity has been further confirmed by the microsconic

exa=lnation of histological sections.

Although the deaonstration of lobnr Dneumonia in

monkeys has been successfully accom.nlished, certain obstecles


have been encountered mhich have delayed the study of the

therapeutic capacity of tke enzyme.  These are primarily the

variations in the resistance of t--e individual monlceys, and,

consequently,  the difficulty in establishing a ;?redictable

course of disease with a minimal standard dose of micro-

organisms.  Within a narrow range of dosage, 0.2 - 0.4 cc.,

one animal may develon pneumonia and die in 48 hours, with
massive pulmonary involvement and overwhelming septicemia,

whereas another JULY survive a well-developed, progressing

pneumonia,  with or without septicemia, after 7 - 8 days.

With tile repeated passages of the culture and the use of

morphine, however,  it is possible to produce a fatal pneumonia

in practically all cases.

      Since the purnose of the experiment3 is to attempt

an evaluation of the effect of enzyme treatment on the

pneumonia, it is highly desirable to be able to induce infec-

tion which will run a uniform course of 4 to 5 days, termin-

at ing fatally when untreated.   This would enable one to have

fairly accurate information of tile animal's condition before '
any therapeutic interference was' attempted.  At present, the

effort is directed toward balancing the host factors and

those of the invading organism to tnfs end,   It is thought

that these factors can be fairly well controlled and that the

study may soon be carried to a definite conclusion,


H 46
236

,

         II.  Isolation of microorganisms  decos73osing the
                    ------------------- -----------------  -.---

caosular nolysaccbaride of different tees of Pneumococcus.
---------e--  -----------.--- --------.---   _--------.---- m4--__

(Drs  Dubos and Goodner.)  The search for other microorganisms
--  L--------A----------

deco-nosing t-ie specific polysaccharides of PneuT;ococcus

Ty.?es I and II is being continued.

       In principle,  the mot-hod remains t`ne same as that

elaborated two years ago for the  Isolation of t3e bacillus

capable of decomposing the Tyco III capsular uolysaccharfdc.

The method consists in tile use of very specific media contain-

ing the substance  to be decomposed as sole source of enorgv;

these media are incubated under a great variety of conditions

so as to bring out as many as uossible of t2e organisms

potontially capable of decomposing tile substance in question.

        In fact, several cultures (at least 5) have been

obtained thus far which are canable of decomposing the Tyoe II

spccif ic substance,  but none of then has been sufffciently

purified to warrant a statement as to the nature of the active

organis:?.  The work has been rendered difficult by the fact



that all these cultures decompose the polysaccharide only very
slowly in liquid media, although some `of the? are quite active

in sand and soil media.  An interesting lead has becn t`ne

successful preparation during the past few weeks of catalysts

which,  when added in small amounts to tLc liquid medium,

greatly increase the rate of decoriposition.  Concerning the

nature of these catalysts,  we have no real knowledge, but

there is some suggestion t'hat they may be soluble organic

compounds of iron.


                                               H 7
                    ii37




         III   Che30-imnunolo~ical studies on conlusa4atd-
            -,`-------.--v.-e.---- _____I.*___________

cnrbohvdratc-uroteins
       I------&----L--z   (Drs.  ivc rlv  GocScl and. i3ab?rs.)
------                           ---,,-,,--,,,,,-,-,----,----------

          1.  TLC sT>ccif icitx of antigens Ere3arcd bx cor*.biniz,:,
              ---------A--- -__-_-__ --__ ___-_____ ,,,,-,,--2
CL and -P  ~lucosidc of ~lucosc VithJrotein.
-----_ ,,a-,,-,-,,---- _-_________ _-_____   The study of
antigens prepared by c'ne;l,icalljt combining sugar ierivatlvcs

with protein has revL>alcd t-c fact that carbohydrates exert a

de ter:.ining inf luoncc on t;ie in?unological specificity of

compounds of xLich they form a part.  In preceding studies
evidence has been acquired that mere diffcrenccs in the
structural conf lgurat ion of relatively s.zall grouw in the

hcxose noleculo may deterline the serological snecif icit:: of

tro sugar derivatives icient ical gave in this one respect. As

proviously shown, t'nef -glucosides of glucose and galactose
differing from each otncr only in tho interchange of the I! and
OH groups on a single carbon atom, exhibit an individual
specificity irrespective of  the nrotein to n>ich they are
                                            A

attached.

     These, earlier studies concerned the?,selvca with the
s?ecificity of sinilsr glUCoEide8 of two different hexoses,
glUCOS0 and galactoso.: J4 paper now in press deal8 Vith the
specificity -of diffcr,ent glUCOBide8 of the 883318 sugar, namely

the I'X and B form8 of gluCo80 coupled to globulin.  `In both

the Wand e antigenaft thus formed the glucosidic linkages to
the protein differ from each other only in their snatial
relations to tse rest of t.-.e antigenic molecule.   Since the
ultimate co::positioq of both antigens Is the same, the
differences observed in their immunological specificity are

referable only to known differences  in the nolecular structure

of each.  The results show that the mere chmgo in the sl?atial

---- .--


H 48
23R

arrangement of the grourrs on the first carbon atorn in these two

derivatives of glucose suffice to confer on each antigen a

;;lnrked degree of dlffcrcntial s->ccificlt;r.   These structural

chenges are so sharply reflected in serologicnl. s?ocificitg

that it is possible by means of i73une serum to diffcrentip.to

select ivcly betucen the tW0 iSo?~ric glucoeides of the sare


sugar.   However, tjno antibodies present in both 1.X and p

antisera cross react with the hcterologous test antigen.

Recognizing tho diffarenco  in the chani cal structure of the

two glucos idc's it becomes necessary to account for the over-

lapping specificity of the two 8UbBtanCes.   The chemical basis.

for the immunological crossing a-3nears  to lie in the fact that

the spatial arrangement of the polar groups on the remaining
five carbon atoms 18 identical in both glucosides.   This

partial similarity of molecular groupings night then account
for the degree of inxunological likeness oxhibitod by the two

substances.  Although In a portion of the molecule of both
glucosides the structural relationship is identical, noverthe-
less they behave' c,hezically as separate chemical entities and
posseae eerolo~gically a separate and distinct specificity. *

For, if complete reciprocal Inhibition of precipitins i's " `,
accepted as the criterion of serological Identity, then
the failure of both glucosides to exhibit this capacity may
be taken as further evidence of differences in the immunological

specificity of each.

The lack of reciprocal absorption of agglutinins by

two organisms mutually agglutinnblo in the ix.une serum of

---_


239
H 49

c?.ch is generally conceded to  indicate a lnc?.c of ix-?unologicr!l

ider,tity.  Relationships of this order are !rnov!n to exist
bctnocn Pncuxococcus T;pc II and Fricdl&nder bacillus Type 3.
In both instr?nccs the rcactivc substance has been identified
chenicnlly as t-lc specific polysnccharidc oeculiar to the

cn~sulo of each organisz.   While  tse structural constitution

of those co';lnlejt: sugars is not as yet fully known, considerable
?rnor-rledg;o has bocn g;nincd conccrnlng their cholicnl pro?crt ies.

For exnrpla, it is knosn that the Type II palysacch>ride is
built up of ~lucosc units aEd that che.`.~lcally it bears a close
rosazblanco to tho ?olgsacchnrids rocovercd fro:-1 the Tync 3

Friedl#nder bacillus.   Zonevcr,  the two substances are not

che:ically identical, although tile sirjilarity between the'?. is
sufficient to result in a certain likeness in ia-unological

spccif icity.  In the absence of precise knowledge of tae
structural relations of the two polysaccharides, it seem
reasonable to assuze taat both contain in a portion of the

cortplex aolecule t0e `sar,e or a closely sinilnr configuration

of atoms,  This sicilarity of molocul~r grouping 2ight then
account for t:le in:~unological eiailnrity of the tmo eubstances,

      Considerable evidence for this point of view is found
in the reeults of the present study concerning the snecificity
of the ti and p glucosides of glucose;  A comparison of th'e
serological relationships between the two isogeric derivatives

of glucose nnd the capsular polysaccharidcs of the two organ-
163s in question is presented in the foll.o:ring Tn'?le.

i ,


   240
c    H 50'.

Corwz2son  of tile _______ p,----~--- ____ -_-__--___--_---
       Serolo-ical Rela.tionshin betneen Iso~~r~._c_
---       -_------L-

Derivatives of Glucose and the Cal)sular Polysaccharide ,f
-________-__-----_-_- ---.--_-e-------e- -____ > ____-_ ------

TYO Unrelated Ssecies of Bacteria.
,`-----------------------`---`-
                                     --------------..--

                                                                           i
                                                                           0
I                                                                           ,

!-lucoside j test       test
16       1 antigen
            A----
/=z-~====5--------  !antiizsL--
               --==L;-== ___---

i l#nder

              --- a------

$y addf-
I tion of
--------
P
;lucoside
:

--w-m--.
  CL
glUCO8ide

._----~
      i ited for      ; for    i for
                             -------c- ----- --------
                     -I
              .Fried-L Pneumo- ,     j
               I  coccus ;        i

I Tyne 3 i Ty?e II, i
I      1  B.Zried- !

I
I
I

ant i&ens  I

                 ! Tyne B
               -e--u-- I-                     I
                               -----v c-
         43       Tneumo - r    A,--- m-u-----
                           Pneumo -   Friedl#nde
              I ,
test    ? ???? ? o COCCUS
      I     1 COCCUS   Tyne B
antigen  1 antigen _IType II 1 Tyne II i                I
.w-- U-L-CL-e d--B  -c-e- -,,-,--

!

; lander 1

L-L-

1

i

k/The test antigens in. all instance8 were prepared by combining
the respective glucoside with a protein biologically unrelated

to that in the imsunizing antigen,


     Analogous relationshiDs extending even to the cross
i:,:munity reactions and t.le lack of reciprocal absorntion and
inhibition of antibodies are evident in both the grouas of

simple and co?piex carbohydrates.  While the comparison is

instructive it is not necessarily valid in the case of the

capsular polysacc.lar ides,  for ti,e final internretation of
these relationships must await further ?:norvledge of the
structural relations of tile more complex bacterial sugars.
     A possible explanation of the cross relationships

between the Ty-)e II Pneumococcus and the Tyne B Friedlander

bacillus has been advanced by Enders  in his recent study on
the presence in Pneunococcus of a type-specific agglutinogen
unrelated to t'ne specific carbohydrate.
     In the case of the synthetic antigens containing

OiCand ' P compounds oI` glucose alone, the evidence indicates
that the iwtiunological specificity of tile reactive glucosides
is determined by known variations  in chemical constitution

and is independent df tile protein to which they are attached.
In view of these findings it seems not unlikely that in the

case of the polysaccharides because of their mare complicated

structure and the greater nossibility for variation in
molecular configuratian, here may be found many instances
of a similar overlalqing specificity among `carbohydrates of

unrelated origin.

      In attempting to interpret  the relationship between
the chemical constitution and t:;e serological svecificity of

carbohydrates, especially those of bacterial origin, it is of
obvious advantage to begin with a study of the more simple

--.-                                                              ._


mono and disaccharides.  In these sugars both the chemical

composition and t11e spatial arrangement of the groups Within

the molecule are definitely known.  YIith this end in vie; the

fr>llowing study of disaccharides is in progress.

          2.  The sxnthcsis of  zlucosides of tile di,s,acchnrides
             ------- -,,,--,-,-P ---_____________,________,___,)

cellobiose and maltose
--,-,,---,-------.--A  (Drs.  Goebel and Sabers )    The
                                   ------ --.- -------- m--,-L

opportunity for inter-molecular differences in structure is

almost infinite in complex pol;sacchnrides such PS t3e tyne-
specific ca?suler polysaccharide of pneumococci.  These ooly-

saccharides are made UT? of disaccLarido units which are in

turn composed of a simple hexose  and a hexose uranic acid.

Very little is known concerning the manner of lin!:Rge of these

disaccharides or the units colO>oslng them.   Still less is

known of tLe effect w;,ich internal differences In lin?cage may
have on tile immunological specificity of t5e Tolysacchnride.

         It seems,  tileref ore, quite important to determine
what effect differences in linkage of tne `nexose units, would

                    ~

have on tile `immunological relationshins of two disaccharides

of known struct'ure.

    A&tos~s, ` the diiacciaride ob'talned by `the partial
hydrolysfs of starch, and cellobiose, that obtained in a
similar manner from` cellulose ,. have been shown to be composed

of two glucose units, tse aldehyde groun of one being linked

to the fourt3 carbon atom of the other hexose.   Thus maltose
and cellobiose .are each glucosides of glucose linked through

the same carbon atom.  These disaccharides differ, however,

in that maltose is an alpha glucoside of glucose while

----


cellobiose is a beta glucoside.  This difference is best

shosn by tile following structural formulae:

Cellobiose

The free reducing group of each  disaccharide is indicated

ny an asterisk*.

The para aninqphenol glucosides of maltose and

cellobiose havobeen pre?ared,  diszotized and coupled to
protein and tile resulting antigens used to immunize rabbits.

T'iiie iz.:;unological work is now being carried out.

        IV  Studies on t:,e interconvertibility of the
            --L-e ----------_-_-___-__---------- _______

gecific tpues of Pneumococcus, (Dr. Allomax.)   Studies have
  -------- -----c----.-------- ----dI---

been made during the past few months on .the nature of the
,

phenomenon first described by Griffith in 1928, and subse-
quently investigated by Neufeld, Dawson, and Sia, dealing with
the transformation of R organisms derived from one type of
Pneumococcus into S forms of a different type.  These in-

vest igat ors induced the change through the use of intact
bodies of heat-killed S pneumococci either injected Sub-

cutaneously into mice,  * together with a a+z~ill InoculurL of

living R organistls, or used in vitro together with anti-R
                                     --e--e-


se rux in a medium Inoculated with R forms,   The R cells

assumed the properties of trle heat-killed S organism used in

the culture rcediux.

----.--.----.._ -                       -_


244
H 54

     The phenomenon is of unusual biological significance,

for it is a unique example of a complete transforzation of the
biological characters of a yicroorganisn by senns of a

specific sticu1us.  An organism thus transforrjed not only
acquires a type-specificity not present in the R cell, but it
undergoes definite mornhologlcal changes, develops a high

degree of virulence, and possesses antigenic propcrt ies which
differ markedly from those present in tile original bacteriu?,.
These properties, once acquired,  are retained by the organism
indefinitely under suitable conditions of gromth. The

acquisition of these new characteristics is always associated

with the regaining of t:?e function of producing the snecific

capsular polysaccharide.

     It seemed worth while to attempt an analysis of the
substance present in the heat-killed organism essential in

the reaction, and to learn more of its general nature. Atten-
tion has been directed, therefore, towards the separation in

a flltrable,  cell-free state,  of. the substance active in

effecting transformation.  Studies already published have

established several facts.  Cell-free extracts which are atitive
in causing transformation in type may be obtained from S
pneumococci dierunted by repeated freezing and thawing.   Such
extracts under suitable conditions may be filtered through

Berkefeld candles, and thus separated from all intact organ-


isns.  Avirulent R pneu?ococci derived from S forms of a
specific type may be changed by growth in broth containing
ant i-R serum and the filtered extract of S nneu%ococci of a


different type into virulent S organisrqs identical in tyue

with that of the bacteria extracted.   The constituents of the

extract suqply an 3ct ivat ing  stimulus nf a snccific nature

in that the R pneunococci acquire t53 capacity of claiornt ing

t:;lc capsular material peculiar to t-le organisr:s extracted.

      Subsequent studies  have shown t.`r.ot sti.11 further

purif icnt ion can be ncco:~plisLcd by t:;e adsornt ion of pneuyo-

coccus extracts on alu?.inlu-7 gels.  Yost of t e protein and

lipoid constituents  in the extract are carried dolrn nith the

aluziniu3,  the active substance reT,aining in the su:>crnatnnt

fluid.  This supernatnnt, a mater-clear fluid, is snccif ically
as effective in inducing transfer-ation in tyue as are tile

suspensions of whole,  heat-killed organisms.

      It is hoped that by further procedures such as

selective adsorption, selective dialysis, and cLer.:ical

analyses, more exact knowledge of t.?o active substance may

be acquired, and an explanation provided for a phenomenon

which possesses aide biological significance.

     V. The De&gee and Duration of Active and Passive
             -e-w--- ------------------------______cI_
Immunity to Pneumococcqe in Rabbits (Dr Ernest Stillnan.)
------ --B------Y- ,-,~-----.mP eMA-------------e-w    .
                            (

The experiments, `previouely reported, on the persistence of
pneumococcus antibodies in the sera of rabbits following

various innunizafion procedures is still in progress.   The

trend of this work Is illustrated by the fact that the serum

of one rabbit,  last exposed to inhalation of virulent Type I

pneunococci 6 years ago,  still contains sufficient nnti-

bodies to protect mice against large doses of pneumococci.


In colo.borr!t ion ?:it.-! Dr. Good.-cr, c l:>r;e series of

rabbits have been given identical courses of injections of a

sus.pension of heat-killed pneumococci,  containing a mixture of

equal amounts of Pneumococcus Types I, II and III.  At monthly

intervals groups of these animals are being examined as re-
gards the agglutinin titer and mouse protective value of their

serum,  and as to actual active Immunity or~suscepti3ility to                 : `:
                                                                           `3 !
                                                                           L:
                                                                      / !.I
                                                              ts                  I, 1
                                                                           I

infection by the dermal route.  The dermal infection perTi

of a thorough evaluation of the active immunity since the
results do not depend so much on survival or death as they

on the clinical findings.

do

This investigation has now been under way for four

months.  While it is yet too early to predict the duration of

immunity it has becdme apparent that it persists beyond the

time of the disappearance of circulating antibodies.   Further-


more,  it has been found that tLe immunity against the Type III

Pneumococcus is the first to disappear, and the indications
are that the -immunity`to Type 1 will have the longest duration,

     Production of &xgkrimental- pneumonia in animals.
             ---------- --y*  ------ ----------&----
The'development of'a>,ttchnic.for' the T)roduction of experimental
                  .,.i i.- ,,_. _       ,
lobar pneumonia"in labi;atoky `anikale.`is' stlii :biiiig"ktt;`ktiit`id;'
                                                      .,
Although by the'inhalatlon'nethod pneumonia may occa~ionslli' '

be produced In partially immunized mice under the influence of

alcohol,  these animals are too small for detailed study. Ex-

periments have shown that partially immunized rabbits, whether
infected by inhalation or by nasal inoculation, fail to local-

ize the infection In the lungs,  Although guinea pigs are

subject to spontaneous epidemics of pneumococcus lobar pneu-


mania,  they are highly resistant  to exnerimental infection

with this organism.  Numerous atteqts to raise the virulence

of pneumococci for guinea pigs have failed.   Various methods

for lowering the natural resistance of guinea oigs are now

being employed with the hope that these animals may become

more susceptible to pneumococcus infection.

VI. Publications
-------,-----em'

Alloway, 5. L.  The transformation in vitro of R pneunococci
                                              -------
       into S forms of different specific types by the use
      of filtered pneunococcus extracts.   J. Ex?  Yed
                                                            ,-----z,,-,'*
       1932, 55 91.
                     --)

Avery,  0. T, and Dubos, R.  The protective action of a specific
      enzyse against Type III pneunococcus infection in
         mice.  cl,-Exp,E&_d., 1931, 54, 471.

Avery, 0.  T. and Dubos, R.  The soecific action of a bacterial
      enzyme on Type III pneumococci.   Trans. Assoc.
                                                         -------------
      Amer. Phys., 1931, 4&, 216.

Avery, 0. T. and Goebel, Wd'F,  Che,To-immunological studies on
      conjugated carbohydrate proteins.  V. The immunolog-
     ical specificity of an antigen prepared by combining
      the capsular polyaaccharide of Type III Pneumococcus
       with foreign protein.   J. EXQ.
                      ,L, 1931, 24, 437.
                                   Ye d

Avery, 0. T. and Goebel, W. F., and Babere, F.   Chemo-               ,
      immunoiogical studies on conjugated carbohydrate*'
       proteins.   VII.  Immunological specificity of antigen6
     prepared by combining the ti and p-glucosides of
      glucose to protein.  J, EXJ% Iled., 1932, (in press).
                                         -m-w

Dubos, R.  and Avery, 0. T.  Decomposition of the capsular
      polysaccharide of Pneusococcus Type III by a
       bacterial enzyme,   J. Exp, Hed., 1931, 55, 51.
                                      -m-e   ---

Dubos, R.  Factors affecting yield of specific enzyme in
      cultures of the bacillus decomposing the capsular
      polysaccharide of Type III Pneusococcus.
       J. Ex-3. I'ed., 1932, 55
             -----------             --


Dubo s ,  Ft. and Baudisch, 0.  Uber Katalasemirkung von
       Eisenverbindungen in KulturTyedien.   Bio che-   7eit
                                                         -------L-k,--' 1
      1932, (in press).

Finkle, P.   Eetabolis?? of S and R for+:s of Pneumococcus.
        J Exn   1.`~ d
             -`-e-i-L-s--' *  1931, 53
                                        --@  551.

Francis, T. Jr. and Tillett, iY. S.   Cutaneous reactions in
      raibits to the type-specific capsulnr polysaccharides
         of Pnouxococcus. J   2x33. ired.
                                    -L------z-- )  1931, 54 587.
                                                                --*

Francis, T. Jr. and Tillett, `ii. S,  The significance of the
      type-snecific skin test in the seru.2 treatTent of
      nneu?onia, Type I. .J. Clin. InvL, 1931, N, 659.

Francis,  Tho.l:as Jr.  The identity of the Techanisms of tjrne-
     specific agglutinin and precipitln reactions aith
          ?neuTococcus.   J. 9x9. i'ed.
                                 ----,-,,,-,)  1932, 55 55.
                                                            --I

Goebel, Y?. F. and Avery, 0. T.  Che-zo-iT,aunologi cal studies
      on conjugated carbohydrate nroteins.   IV. The
      synthesis of the p-aminobenzyl et.ler of the soluble
      specific substance of T;?e III Pneuaococcus and its
       coupling with protein.  J. Exp. ,L, 1931, 54,481.
                                            He d
                                             ----

Goebel, V. F., Babers, F. and Aver;?, 0. T.  Che.-o-itn-unolog-
      ical studies on conjugated carbohydrate proteins.
         VI.  Synthesis of p-a-ninophenol ui glucoside of
      glucose vith cou?ling to protein. J. 3x1). , , ,
                                                l!ed
      1932, (in press).

Goodner, K.  The development and localization of the dermal
      pneumococcic lesion in the rabbit.  J. EXE. Med.
                                                             ---- ,,,---*
       1931, 54
                     --*  847,

Goodner, K., Dubos, R. and Avery, 0. T.   The action of a
     specific eneyme in Type III pneunococcus deraal
        infect ion in rabbits.  J. EXE. tbed.
                                           --we -_---- * 1932, 55, 393.

Goodner, K., Dubos, R. and Avery, 0. T.   Studies on the
     quantitative action of a specific enzyme in Type III
    pneunococcus dermal infection in rabbit. (In manuscriotj

Kelley, W.  Antipneunococcus immune reactions of nortial ewine
           serum.  (In rzanuscript,)

Rhoads,  C. P, and Goodnor, K.  The pat,lology of experimental
      dermal pneumococcus infection in the rabbit,
      J. EXE. Med.
             ---- ,,----)  1931)  54, 41.


Stillnan, 3. G.  Duration of de-?onstral?le antibodies in the
    serax of rabbits in -unized with heat-`-illed
    T>:>e I nne-mococci. J. 3x~. I!ed., 1531, 55,
                                         ----- 515.

Still-;an, 2. G. anb aranch, A.  Locali?a,tion of pneu9ococci
   in tic lungs of partially i.r;-?unize& zice f;;i;sing
     inkalation of pneil:-:oCocCi. JJ E:x:>. ,--L,
                                       xe 6           I
       24,  323 .