151

Report Of Dr. Avery (assisted by Drs. Adams, Beeson, Cattaneo,       '


Daddi, Dublin, Dubos, Coebel, Goodner, Heggie, Howland, fiotchkiss

            MacLeod, Reeves and Stillman.

   Treatment of pneumonia with antipneumococcal rabbit serum (MacLeod,

Beeson, Dublin, and Heggie).  One hundred patients suffering from pneumonia

due to pneumococci comprising 12 different types, have been treated in the

Hospital with unconcentrated antlpneumococcal rabbit serum since this thera-

peutic agent was first used three years ago.  The total mortality was 11 per

cent, but if pneumonia due to Pneumococcus Type III is excluded, the mortality

is only 5.4 per cent of 74 cases in which the infeotion was caused by other

types of pneumococcus~

   Unconcentrated antipneumococcal rabblt serum is a very satisfactory

and effective therapeutic agent except In the cases of Type III pneumococcal

pneumonia.  26 patients who suffered from Type III pneumonia were treated

with serum, and of these 7 died.  Of the patients witk Type III pneumonia who

died only three could be considered as having received adequate dosage of

serum by our present standards, but the fact that these patients died despite

Intensive traatment, indicates that specific antiserum alone is not a4 en-

.

tlrely adequate therapeutic agent in Type III pneumonlar

   The patients who died, other than those in the `typo III group, nm

bered ozlly 4.  In two of thsee empyema was present associated with severe

bacteremis;  one patient had meningitis, a?d the fourth patient was an elder-

ly man with Type II pneumonia who had involvement of four pulmonary lobes

when admitted to hospital.

   During tne past 2 years a.zl attempt -has been made in each case t0

administer the required amount of antibody within as short a period as pos-

sible.  This has resulted in more rapid defervescence in the patients 80


                                                    152

treated, and is a more advantageous method of treatment than that of giving

the serum in divided doses over a 12 to 24 hour period,  16 patients with

Type I pneumonia were given the required amount of serum within a 2 hour

period.  In these patients, the time required for defervescence, as calculated

from the time serum treatment was begun, was only 9.3 hours.  This Is less

than half the time required for defervescence in Type I patients who were
treated wl'th concentrated holse serum in divided doses (22.6 hours).

    The administration of serum has been greatly facilitated by the use

of the skin teat with the homologous type specific polysaccharides. The

teat has been used a8 a guide to dosage of both horse and rabbit antipneumo-

coccal serum in patients with pneumonia caused by Pneumococcus Types I, II,

III, v, VII, VIII.  In 77 per cent of the 104 patient8 upon whom skin tests

were done, the reaction was found to be entirely satisfactory in determining

when a sufficient amount of serum had been given.  12.5 per cent of the

patient8 showed a positive alcinreact~on before the administration Of specific

antiserum and at a time when the disease was advancing.  In this group the

teat could not be used as a guide to therap,,.  Patients who die of the in-

fection (19 per cent of this group) do not develop a positfve akin reaction

despite the presence of specific antibody in their circulating blood, In

such individuals the &in test cannot .be `used as a guide to serum dosage, but

is a vahable prognostic aid.

   The skin test 16 of greatest value in patient6 whe show a negatfve

reaction before serum adminlatration, and fn whom serum is effective in

initiating recovery, for on the appearsice of a positive *eat, serum can be

safely discontinued.

   Treatment of pneumococcal infectionr with sulfapyridine. 2-(para
aminobenzenesulfonamido)-pyridine or sulfapyridlno has been advocated recently

as a therapeutic agent in pneumococcal infections.  Preliminary observation8


                                                     153

indicated a definite protective action against intraperitoneal infection6 of

mice with pneumococci Types I, II, III, V, VII and VIII, and a curative

effect in patient6 suffering from cneumOcocca1 infectiOns.  Consequently, the
study Of the action of this new sulfanilamide derivative was undertaken both
in the laboratory and in the clinic patient6 with pneumonia.
  Effect & vitro of sulfapyridine on the Pneumococcus (MacLeod and Daddi)
Sulfapyridine exerts a definite bacteriostatic effect on the Pneumococcus &

vitro in high dilutions.  The lag phase is prolonged and growth is less pro-

fuse than in the absence of the drug,  Higher concentrations of drug may

inhibit growth entirely.  However, if transferred repeatedly in serum broth
containing sulfapyridine, Pneumococcus Type I will tolerate gradually in-
creasing concentrations of the drug until finally it is able to grow in con-
centrations which inhibit entirely the growth of organisms not so accustomed.
   The sulfapyridine-fast Pneumococcus is encapsulated, end retains its

virulence for the mouse.  Indeed, salfapyridine in amounts sufficient to pro-
tect mice against infection wit`n Type I pneumococci fails to protect mice
infected with the drug-fast strain derived from the parent culture. Prelimin-
ary Ob6ervation6 indicate that the type specific serolOgica and antigenic
properties of the drug-fast organisms are not altered. The Neufeld reactilon

i6 positive, Indicating that the function of capsule formation is not impaired
and Type I serum protects mice infected with the strain resistant to the ac-

tion of the drug,  Investigation of the biochemical activity of this dru&

fast strain fs being undertaken.
   Effect of sulfapgridine on pneumococcal infections Of mice& The

effect of sulfapyridine on the outcome of infection induced in mice with

Pneumococcus Types I, II, am3 III differs markedly in each instance,
   Mice Infected intraperitoneally with l,OOO,OOO lethal doses of
Pneumococcus Type I respond well to sulfapyridine administered by stomach tube.


154

Practically all of the treated mice survive infection if the administration

of the drug is continued over a two day period.  The mice which survive are

found to be immune to re-infection with Pnemococcus Type I. With the same

infecting dose of Pneumococcus Type II, even though treatment is prolonged

for 4 days, approximately only 60 per cent of the mice will survive.  In the

case of Type III Pneumococcus, the survtval rate is very low even though

treatment is carried out for as long as seven days.

   If aormal mice are immunfzed fntraperftoneally with a single injection

of heat-killed pneumococci of these three types , and infected four days later

with the homologous organism the survival rate is approximately the same as

in the above experiments where sulfapyrldine was used.  In other words, the

survival rate in mice Infected wlthPneumococcus Types I, II and III and

treated with sulfapyridine appears to be related to known differences in the

anti-genie potency of these three type8 of pneumocOccu8r  the better the

immune response to the infecting inoculum, the higher the survival rate.

    In the case of infection of mice with !&pe III, synergism exists
                                   .-.

between the action of specific antiserum and sulfapyridine, Amounts Of serum

and drug, which by themeelvee fail to protect mice, exert a high degree of

protective action when used in combinatloti

   Treatment of pneumon$a with sulfapgrldiae (MacZeod, Beeson, Dublin and

Heggie).  On the basis of experimental observations the treatment of pneumo-

coccal pneumonia wttii sulfapyridine haa been undertaken,  With all infections

other than those due to Pneumococcne Type III, treatment has been carried out

wltin the drug alone, if t&s was possi\ile, but serum has been used as an

adjunct when the outcome of the infection has appeared doubtAL However,

in the case Of Type III pneumo;lia, treatment with the drug alone has not been

considered to be justified, so that In these Infections a combination of

eulfapyrldine and Type III unconcentrated antipneumococcal rabbit serum has


                                                   155


been used.

    A beneficial action of the drug on the outcome of pneumococcal pneu-

monia seems undoubted.  Crisis within 24 hours from the beginning of drug

administration has occurred in the majority of patients even though treated

at all staC?;es of the disaase and despite the prcsenco Of bacteramia.

     In the casa of Type III pneumonia, synergism appears to exist between

the action of sulfapyrldino and specific antiserum.  I:1 three instances

sulfapyridine alone has failed to izduce sterilization of the blood of the

invading orga~lsms, but this has occurrad promptly with subsequent serum

treatment.  However, the amount of serum used ha5 been considerably less than

has been necessary previously in treating patients with Type III pneumonia.

    Nausea and vomiting have been an almost invariable accompaniment Of

sulfapyridine administration, and have been severe s-lough to necessitate

the parenteral administration of fluid in about one half of the patients so

affected,

    Hemolytic anemia has occurred in three patients, all of whom subse-

quently recovered.  This phase of the toxicity of sulfapyridine has been

investigated in collaboration with Dr. C. P. Rhoad's department. Ia the

case8 studted, the occurrence of hemolyela, as measured by the excretion of

urobilinogen, has not occurred unless a total dosage of 20 gms. of sulfa-

. pyrldine has been gives

     Aoute asotemia has occurred in three patients, in one of whom acute

hemorrhagic Bright's disease has supervene&  !Che problem of the effect of

sulfapyridine on renal pl~~iology is being investigated in collaboration with

Dr. D. D, Van Slyke'5 department.

     The primary toxicity of the compound may limit seriously it8 use

unless means are found of reducing the nocuous effects.


156

The conversion of creatine into creatinine by a bacterial e:lzyme

(Dubos) .  A bacterial enzyme capable of oxidizing creatinine to urea, end the
use of this enzyme a5 a specific reagent for the quantitative analysis of

creatinine, have been described in earlier reports.  It has now been found
that the same bacterial species which produces this oxidizing enzyme also
produces another enzyme which converts creatine into creatinine. This
reaction offers an opportunity for the study in vitro of (a) the enzymatic pro-
                                                  --
duction of a biologically important cyclic compound from an aliphatic one, and
(b) the enzymatic combination of an amino and a carboxyl group to form the

important CO-NH llnkage.

   The new enzyme has been obtained in aqueous solution, free of the cellu-

lar s true ture.  It exhibits a high degree of specificity for creatine, since
it does not convert *he closely related compound glycocyemine fnto glycocy-

ami dine, nor does it change hydantoic acid into its anhydride, hydantoin.
   The enzyme 15 not produced when the bacterial cells are grown in a

medium which does not contain creatine or creatinine;  it belonss, therefore,

to the group of "adaptive enzymes" which are produced only in the presence Of
their specific sub$trate, or related compounds.  The mechanism of the forma-
tion of adaptive en@nes ha5 often been discussed in the past and it ha5 been
suggested in particular that the adaptation serves a useful purpose to the     :

organism, The fact that creatinine - the end product of* the reaction - is as
effective as creatine in stimulating the formation of the enzyme under con-
sideration is convincing evidence that the "adaptive" response cannot bo ex-

plained on teleological grounds.  It is apparent that t#e presence in the
culture medium of some linkage or structure common to both creatine and
creatinine is necessary and sufficient to cause the formation by the micro-
organism of the enzyme which catalyses the reaction creatlne - creatinine,
irrespective of whether or not this reaction is of any use to the organism.


                                                    157

    Properties of a nucleohistone antigen extracted from pneumococci

(Dubos).  The preparation of a nucleohistone fraction, present in R and S

pneumococci, has been described in the preceding report.  The serum of rab-

bits immunized with this soluble fraotion is capable of protecting mice against

infection with virulent pneumococci.  Although the protective action is not

entirely specific since it is effective against several types of pneumococci,

it is always of a much higher degree against the type of pneumococcus from

which the antigen has been prepared.

    In recent experiments, the nucleohlstone antigen has been prepared frcm

R pneumcoccl derived from Type II.  The sera of rabbits immunized with this

antigen sometimes protect mice against small doses of Type I pneumococci ;

the same sera, On the other hand, always protect against large doses Of Type

II organisms.  Interestingly enough, these sera fafl to agglutinate suspen-

sions of pneumococci {living or heat-killed) of any type tested, nor do they

precipitate soluble fractions extracted from them, not even the nucleohistone

used as antigen in the preparation of the immune sera.  The sera do not appear

to exert any bacteriostatic or bactericidal effect on pneumococci and in fact,

the mechanism of the protection which they induce is still unexplained.

    It is usually considered that, among proteins, histones and proteminea

are peculiar in not being anti~~alc.  It is true that the Pneumococcus nucleo-

histone considered fn the present report fails to produce any antibody

detectable by the preclpitln reaction, but the protective effect exhibited by

the sera of rabbits immunleed tith this subetance seems to indicate that the

latter possesses antlgenic activity. It appears possible, therefore, that

like the pneumococcus nucleohistones other histones and protamines m8y in

reality act as antigens, but that the entibodies to which they give rise are

not detectable by the classical precipitin reaction.


158

Bactericidal effect of an extract of a soil bacillus (Dubos).  It is

well tiown that most living cells are resistant to the action of common

enzymes;  tllis is true in particular of pathogenic microorganisms which are

not affected by any of the known tissue enzymes,

   During the past few years we have made a number Of attempts to dis-

cover whether certain microbial species are endowed with enzymes which per-

mit them to cause the lysis of the living cells of Gram positive COCCia TO

this end, suspensions of living pneumococci, streptococci end staphylococci

were repeatedly added to different samples of soil over a period Of three

years in the hope that a soil flora would develop, capable of specifically

attacking the living cells of the same bacterial specie8 repeatedly added as

specific substrate.  From these SOILS, an unidentified spore bearing bacillus

has recently been isolated which is capable of causin$ the lysis of living

Gram positive cocci;  the lysls takes place through the agency of a soluble

substance which is produced by the soil bacillus in the course of its growth.

The soluble factor has been separated from the bacterial cells and can be
purified by precipitating it at its isoelectric point (pE 4.6).

   The purified bacterial extract exerts a bactericidal effect on all

Gram positive cocci so far tested and prevents their growth in laboratory

media.  The phenomenon of l$sis, however, ia not observed with all the

species which are killed by the extract;  many species of streptococci, for

instance, fall to undergo lysis in the preeence of amount0 Of extract far

in excess of the amount required to destroy their viability.  In fact it

appears that , la the caees where it is observed, lysls is only a secondary
phenomenon due to the action of the autolytic enzymes Of the affected Cell8

following the primary injury inflicted upon the living bacteria Although

it is not yet possible to form an opinion as to the nature of this primary

                         -.


                                                 159

injury, it has been found that the glucose de-hydrogenase of the bacterial
cell is inactivated by the extract before any morphological alteration Can

be recognized.

   The extract is effective in vivo as well as in vitro and protects mice
                                  --              --

against infection with virulent pneumococci and streptococci.  For ins tazce,

a daily dose of 1 mgm. administered by the intraabdominal route on three
consecutive days protects mice against 10 million fatal doses of pneumococci.

The extract also exerts a curative effect when administered several hours

after infection,  The degree of protection afforded, and the minimal amount
of extract required, are the same irrespective of the type of ~XI~U~OCOCCUS

used as infective agents;  experiments have so far been conducted with

pneumococci of Types I, II, III, V, and VIII.

    Tests carried out in collaboration with Dr. R. C. Lancefield have

shown that the protective effect of the extract egainst virulent hemolytic

streptococci is of the same order as that against pneumococci;  experiments

have been instituted with 10 strains of group A and 2 strains Of group C

hemolytic streptococcus, all with euccesafkl results.

   It ia worth emphasizing again that the amounts of extract required
for protection against infection are the same Irrespective of the types of
pneumococcl or hemolytic streptococci used as infective agents, lt is clear,
therefore, that the extraat exert6 its effect by altering some stl?ucturs, Or
interfering with some M&ion which 1s common to all Gram positive COcci and

which is essential for maintaining their metabolic activity.
   Finally it may be mentioned that, in spite of many attempts, it haa
been impossible to observe any effect of the extract against any of the

Gram negative bacilli so far tested.


                                                      160

   On the nature and behavior of antibodies (Goodner). It now appears

that several different serum proteins may have tine properties of antibodies;

indeed it seems certain that in some immune sera one antigen may react with

more then one variety of antibody protein.  Moreover, within any one class Of

serum proteins, there appear to be antibodies having varying affinities for

a single antigen.  Precise chemical analyses have shown that, with some

antigen--antibody systema, the addition of increasing amounts of antigen gives

results in terms of antibody precipitated which do not form a curve but which

give a figure of ascending straight lines.  With the simplest possible system
only one ascending phase (facet) is obtained.  Other systems have given

examples of two, three and four ascending phases. Each of these phases of

constant antigen-antibody ratios appears to be related to the participation
of a single regional or spatial configuration of the antie;en,each of these

being dominated by a chemical combining group.  `ihe inference would be that

a single antigen forms a series of antibodies of sharp specificity and that

these antibodies react with the antigen in a perfectly definite order, this

order being determined by Mass Action equilibria.  The antibody which forms

the least soluble compound with the antigen is first precipitated, the next

least soluble compound second, etc.  For any one system the ratios for any
one phase are definite but are altered by alteration af the system as by the

removal of certain of these hig3y specific a;ltibodiea.  In other words, the

equilibria are altered depending upon the other reactive components of the

sys tern.
   If large excesses of antigen axe added to antisera the well known

phenouenon of prozone is obtained.  In this the anti.~en-antibody combination

is perfectly soluble.  Space limitation does :lot permit the explanation of

t-his matter but it may be said that from the chemical point of view t'nosa
antibodies which are first precipitated by small amounts of antigen are the


                                                    161

first to form soluble compounds on the addition of excessive amounts Of anti-


gen.  Th8 order of precipitatio:l and of inhibition of precipitation are the


same.

   In simpler systems the region of maximum precipitation is characterized

by an absolute plateau.  In more complex systems the antibody first precipita-

ted may be involved in an inhibitory phase even before the last reactive
component has been precipitated and the configuration may aTpear t0 be that

of a curvtl duo to the general complt;xity.

    General significzlce of these filldin,gs:

     1.  A single eutigen does not give rise to a single antibody as has

loug been assumed, but el:enders, rather, a series of altibodics each sharply

specific for a particular c'hemical region of the antigen.

     2.  Each combination of a sil?le antibody with its related region

of the antigen follows the simple laws of classical chemistry.

     3.  On the basis of these findings it is possible to offer simple and

clear tixplanations for such immunological puzzles as:  Cross reactions, the

Danysz phenomenon, the prozone, and ma?y others.
   A specific protein found in the blood during infection (Avery, MacLeod

and Ho t&kiss).  As prsviously reported the blood serum of a patient acutely
ill with pneumococcus pneumonia or with an infection due to organisms other

than pneumococci yields a precipitate when a dilute solution of the pneuma,
COCCUS sCs polysaccharide is added to it.  The serum of normal individuals

fails to react when similarly tested.  Moreover, ILO other polysacoharide has

as yet been feud Which yields a precipitate with sera klown to contain the

sC1l-reactive substance.  Patfents, wllose serum gives a positive precipitation
test, develop a local and typical skin reaction at the site of Injection of

0.1 mg. of the test carbohydrate.  Both the precipitation test and the skin


,
    162

reaction are positive durin., P the acute phase of the infection and Secome

negative when the patient recovers.  The early appearance and the subsequent

disappearance of the reactive substance in the blood, as determined by the

results of these tests, parallel closely the clinical course of the infection;

both reactions remaining positive with conti;lued activity of lesion, and

becoming negative with termination of the infectious process.

    In collaboration with Dr. Hotcnkiss considerable progress has been

made in the isolation and identification of the W'-reactive substance. It

has been recovered in purified form from the blood of fatal cases of pneumo-

coccus pneumonia , and nas been shown to be a protein possessing certain chem-

ical and immunological properties which distinguish it sharply from the

normal serum proteins.  Tnis protein has been separated by fractional precipi-

tation of the serum with aodium or ammonium sulphate;  the active protein

flocculates from

separation thus

the presence of

of serum lipids

     The

protein is

to calcium

and in the

was made


with the

reacting

role

the albumin fraction on dialysis against tap water,  The

effected has been found to depend on two factors, namely,

small amounts of calcium in tap water and the occurrence

in the albumin fraction.

of calciwn ions in relation to certain properties of this

of special interest;  the fact that the active protein is sensitive

has been utilized in the process of isolation and purification,

study of its reactivity with the ICN polysaccharids- Reference

In the preceding report to the fact that the pretiipitation reaction

test carbohydrate is conditioned by the presence of calcium in the

system; for example, 20 visible precipitate forms when vC" is added

to reactive serum from which the calcium has first been removed. However,

the re-addition of a mere trace of calcium chloride suffices to cause pre-

cipi tation,  Moreover, the W" precipitate from reactive serum is dissolved


163

when the calcium ions are removed by adding oxalate or citrate.  It is now

kno~m that in the absence of other electrolytes, calcium chloride at the

concentration five ten-thousandths molar, is sufficient to precipitate the

protein.  For this reason the active protein separates out of solution when

the albumin fraction of l'pathologicald serum is dialyzed against ordinary tap

water.  Dialysis against distilled water at the same pH does not result in

precipitation. However, with the concentration of electrolytes in physio-

logical saline solutions, a considerably higher concentration of calcium

chloride, about two-tiu-ldredths molar, is required to bring the protein out

of solution.

    The reactivity of the protein with calcium under these conditions

appears to be due to the prGsence of a lipoid substance.  When lipids are

removed from the serum by extraction at low temperatures with alcohol and

e.t:er, the active protein ca? no longer be precipitated from the mixture by

calckum i on9 ,  The preoipitability by oalcium can be restored by adding the

albumin fraction of a serum contaid.>g lipids, such as normal human serum or


normal rabbit serum.  The precipitation reaction with NC" polysaccharide,

however, is not affected by extracting the lipids from the serum or by de-

fatting the protein after isolation from reactive sezllIIL

   It is evident that the laterplay of calcium and a lipid or lipids

present in reactive serum determines the unique insolubility of the active

protein by virtue of which it can be separated in relatively pure form from

the accompanying inert serum albumin.  While calcium is essential in the

precipitation reaction nitii the "Cs polysacc-harfde, the lipoid material is     :


not necessary.  Since both calcium and ttie lipold material have been demOnstraW

ted in 110rmal `inman serum zeit`ner of these components cm be considered as

as being a specific result of the infection, whereas the presence of the


164

"Cl'--reactive protein bears a direct relationship to the infectious process.

Moreover, normal serum does not react with the flCY polysacchaPide, nor has it
been possible by the methods described to demonstrate in normal blood a
substance having the unique chemical and immunological properties mi:ich
characterize the active protein found In the serum of patients during the

acute stego of bacterial infectiori.

   The active protein Isolated from pattiological serum is relatively
resistant to the action of such proteolytic enzymes as crystalline trypsin,,
ch~otrypslu and crude commercial preparations of the pancreatic enzyme.. How-

ever, the crude ensyme preparation slowly destroys the activity of the protein.

    Immunological propertiea.  The "C"-reactive protein in pifled form
is markedly antigenic giving rise in rabbits to antibodies which ere speclf%-
cr-lip directed against this particular protein.  Rabbits immunized with the
,rC't precipitate containing the active protein produce antibodies wiiich
specifically react with Rpatiolo&cal" sera and with solutions of the active

protein prepared from them.  Absorption of rabbit antiserum with normal human
serum removes all precipitins for the latter but leavee undiminished in titre
the precipitating antibodies reactive with patients' serum and with the
specific protein isolated therefrom.

Through the courtesy of `Dr. Goodner, complement fixatfon tests have

been carried

normal human

antiserum to

the presence

4ut using antisera of rabbit8 fmuniaed with the proteins Oi
serum or with the active protein from patient serum  Rabbf. t
the normal proteins of human blood failed to fix complement in
of the specific protein Isolated from patients'ti serum.  On the

other hand, rabbit antiserum to the specific protein from "pathological"  serum
fixed complement with the sme antipn in dilutions as high as 1:5OO,OOO.
   By precipitin and complement fixation reactions it 1s possible, there-
fore, to differentiate specifically the ;lormal serum proteins from the flClt-


                                                      165

reactive protein which circulates in the blood during the acute phase of

bacterial infection.

   Immune-chemistry of.carbohydrates of synthetic and bacterial origin

(Goebel, Beeson end Adams).  During the past year coneiderable progress has

been made in the major problem under investigation in this laboratory, namely,

the %le of carbohydrates in infection and resistance.  This problem has been

approached in two diatixct ways,  First, artificial antigens have been pre-

pared containing simple carbohydrates of known chemical structure. The
immunological properties of these antigens have been studied and correlated

with changes in chemical oonstitution.  These studies have revealed that the

stereochemical configuration of simple hexoses, the configuration and position

of intramolecular li&sgee of disaccharides, as well as the conversion of the

primary alcohol group of mono- and disaccharides to the carboxyl group are

all important determinants in orfenting the specificities of simple carbo-

hydrates.  A comprehensive understanding of the sharply defined specifioitles

exhibited by escapeulated microorganisme cannot be attained until the exact

chemical structures of the capsular polysaccharides themselves have been

ascertained.  Our second approach, therefore, has been a detailed study of

chemical constitution of the specific polyeacckarides of certain types of the

pneumococci.                             

     Synthetic antiRem:  A. Cellobiuronic a&d. In the previous report

the preparation and serological properties of two artificial antigens, one

containing the disaccharide cellobiose, and the other cellobiuronic acid. the

building stone of the !Pype III pneumococous polysaccharide, were describe&

Cellobiose and cellobiuronlc acid differ only in the nature of the group

occupying the 12th position as can be seen from tile following graphic

formulae.


H   ?H        CHj.jOH

CdOH           kI Bo

Cellobiuronic Acid

Celloblase

   In the disaccharide cellobiose this grouping is a primary alcohol
(CH2OH) and in the aldobionic acid a carbowl group (COOH) occupies this

position,  This slight difference in chemical constitution suffices however,
to confer upon each antigen entirely different iannunological properties.  The

serum of rabbits immunized with an antigen containing the asobenzyl glycoside
of cellobiuronic acid agglutinates in high dilution Type III pneumococci and
causes typical swelling of the capsule.  This serum also precipitates !Pype
III pneumococcus specific polysaccharide and confers passive protection on
mice against isfeation with multiple lethal doses of virulent Type II, III

and VIII organlams.  Following inuaunization with this antigen, the rabbit8
themselves are actively resietant to dermal infection with !&pe III pneumo--

cocci*  On tne other hand, the sora of rabbits i~ized with the antigon

containing the corresponding glycoside of cellobiose exhibit none of these

phenomena.

     B,  Gentiobiuronic acid.  Gontiobiuronlc acid, first prepared syn-

thetically in this laboratory, is a? isomer of cellobiurozxic acid. Both


167

uranic acids are constituted from a molecule of glucuronic acid linked in

glycosidic union to a molecule of glucose,  the configuration of the inter-
molecular glycosidic linkage is in each instance the same*  The two aldo-

bionic acids diffar only in the position of glycosidic linkage. In cello-

biuronic acid this linkage is through the 4th carbon atom of the glucose

molecule, and in gentiobluronic acid the 11-e is through the 6th carbon

atom.  The structural relationships of these two aidobionic acids is

repreaented by the following graphic formulae.

H    OH        OH

CAlobiwoxiic acid

Qantiobiuronic acid

During the past year the syntheule of gentiobiuronic acid from

1:2r3:4 tetracetyl glucose and a-bromotriacetyl glucuronic acid methyl ester

has been perfected.  The corresp0ndi.q heptaacatyl gentiobiuronic acid met&y%

ester has been converted to a-bromohexacetyl derivative and from this various

gentlobiuronic acid glycosides have been synthestzed, among them the p-

aminobenzyl gentiobiuronide.  The latter derivative has been coupltid with

serum globulln to yield an artificial antigen contatning the asobenzyl

glycoside of the uranic acid.


                                                  168

    This artificial antigen, containing a carbahydrate radical of synthetic

origin, exhibits certain of the serological properties of pneumococcus poly-

saccharides.  It precipitates in high dilutions in antipneumococcal horse

sera of !i!ypes II, III, V, and VIII.  A corresponding antigen containing the

disaccharide, gentiobiose. has likewise been prepared. When rabbits are

immunized ri th these antigens,  the antibodies evoked are in each instance

specific and selectively differentiate between the disaccharide and the

uranic acid derivative.  Furthermore, the antibodies to the gentiobiuronic

acid antigen are serologically different and distinct from those obtained by

immunization with the Isomer, cellobiuronic acid. Moreover, unlike the

latter Immune serum, gentiobiuronlc acid antiserum contain8 no agglutinins,

precipitins, or protective antibodies for Type III pneumococci.  The striking

differencoa in protective action against Type III infection of the antisera

to these two closely related biuranides demonstrates that the aldobiuronic

acid conetituent of the antigen must posses8 an exact stereochemical pattern

in order to evoke antibacterial immunity.

    Structure of.Type III pneumococcus polysaccharlde (Goebel, Bdams and

ReeVes).  Because of the importance of carbohydrates in determining the type

specificity of encapsulated pathogens an extensive investigation is being

carried out on the chemical constitution of certain of the bacterial

specific polysaccharldes.  We have chosen the specific polyaaccharide Of

Type III Pneumococcus for investigation6

    The capsular polysaccharide of !Fype III Pneumococcue is oonstituted
from units of cellobiuronic acid (4+`-glucuronosido-glucose) joined in

glycosidic Union to form a IIUvX'OmQleCUl%e  We are at present engaged in the

problem of determining the position of the gly~osidic union between the

aldobionic acid units in the intact polysaccharlde molecule.


169

   On reducing the esterified acid groups of the fully methylated capsular

polysaccharide by catalytic hyeogenation, a new polysaccharlde is obtained

in which the acid groups of the aldobionic acid residues are replaced by

primary alcohol groups.  On acid hydrolysis the reduced polysaccharide

yields the kown 2:3:6 trimethyl glucose (derived from the methylated glucose

constituent of the original aldobionic acid unit) and an unknown dimethyl

glucose (obtained from the reduced glucuronic acid portion of the cello-

biuronfc acid).  We are at present attempting a rigorous synthetic proof Of

the structura of this derivative, which we have reason to believe is 2:4

dlmu thy1 glucose.  When tiiie synthesis Is completed we -hall know the

entire chemical gtructure and hence shall be in a position to define the

exact chemical basis for the unique specifiolty exhibited by the Type III

polysaccharide,

   The capsular po$ysaccharide of Type XIY Pneumocoocus and the relation-

ship to the group specific substances of human blood (Goebel, Beeson and

Hoagland) .  It has been found that Type XIV dntlpneumococcus horse serum

contains agglutinins for

Since this phenomenon is

that an investigati0a Of

human erythrocytes of the different blood groups.

peculiar to Type XIV antiserum, it was thought

the propersies of the capsular polysaccharide of

Type XIV Pneumococcus might reveal points of similarity with the blood

group specific substances, and at the same time shed light upon the mechanism

whereby antipneumococoal serum of thie particular type

erythrocytes.

The capsular polysaccharide has, therefore, been

agglutinates human

isolated from Type

XIV pneumococci.  The products of hydrolysis of the carbohydrate have been

investigated.  The polysacchar$de has been found to be constituted from one

molecule of acetyl glucoseamine and tiiree molecules of galactose linked in

glycosidic union to form a non+diffusable macromoleCQ.le. This plysac-


                                                    170

charide is unique in that it is the first type specific carbohydrate of
bacterial origin we have isolated which does not contain uranic acids within

the molecule.  The substance resembles both In its chemical and immunological

properties the blood group A specific substance isolated and identified
during the course of the past year from different brands of commercial

peptone.
   Type XIV antlpneumococcus horse serum precipitates both the homologous
pOlySa&UWide apd the blood group A specific substance in high dilution.
When the antiserum is absorbed with the bacterial polysaccharide not only
are all homologous precipitlns and agglutfnins removed, but agglutination Of
human erythx'ocytee and precipitation of the blood group specific A substance

no longer occurs with the absorbed serum.  Absorption of the serum with the

A substance or with erythrocytes of A, B, and 0 groups removes the agglutin-

Ins for all blood groups, but not the antibodies which react with Typo XIV

Fneumococci or with the capsular polysaccharide derived from them,
   0 thers have reTorted that administration of me XIV antipneumococcus
horse serum to pneumonia patients has in some instances been followed by

hemoglobinuria and death.  !Pheee untoward reactions can now be attributed

to a similarity in structure of the Type XIV specific polyeacoharlde and

the blood groqz aubatanceo gs reflected in the 3xxzk.i~ serum*

      e a th ala of
   w gn e _ a&dobionidee and the relatlonship.behween the molecu-
lar rotation of derlvatfvss of acetylated aldoser and uranic acids (Goebel

and Reevea).  In continuing the chemical and immunological Investigations on

artificial antigens it has become desirable to pxpre azo protefns con@

tafning aldobionic acids.  A general method for the synthesis of the glycc-

sides Of aldobfonic acids has therefore been devised.  This method consists

In converting the aldobfonic acid to the methyl ester *hlch is in turn

acetylated to yield isomeria he>taacetates.  These subatancer are then


                                                     171

converted to the %-bromo derivative, from which any desired glycoside can

be obtained by the conveutional method.  In the course of our studies on

uranic acids we have been struck by the fact that a correlation 8881118 to

exist between the values for tine molecular rotation 0f certain mono- and
disaccharides and their correspOndlAg uranic acid herivativee. A comparison
has therefore been made of the molecular rotations of various acetylated

derivatives of these saccharides in which a~ acetyl, metho-1, or halogen
group has been substituted OA the first or aldehydic carbon atom.  It has

been observed that in all instances the values for the molecular rotation
of each saccharide and the CorreepoAdiAg uranic acid derivative differs by

a small and approximately constant amouAt.  It is amarent, therefore, that

a change i4 molecular rotation, the value of which approximates a constant,
accompanies the coAverslon of the terminal acetylated primary alcohol group
(CHzOH) to the carboxymethyl group (C6C)Me). This relationship can be cx-
pressed by the equation - w,l - LqD2 u K nhere @j3, is the molecular
rotation of the hexose uranic or aldobionic acid derivati:r? and &f],
the molecular rotation of the hexose or disaccharide derivative. We
consider this newly disaovered rule to oe of Value In I)rognosticatln$ the
configuration of the derivatives of hexose-uronlo and aldobionlc acids.
   Difference8 in viru$enCe of titrarlW3 tmse 0f.~neumoc0ccl for mice

( stillman). Most experimental work of late gears hae been carried out rriih
strains of pneumococci selected because of their paVioge2icity for certain

laboratory aaima$s.  Many of these strains have been under artificial

cultivation for long periods of time, and most of them have been repeatedly

passed through mice, so that their virulence for t`nis particular qetiee

of animals has been selectively increased.  Since pneumococci of the dif-
ferent 8Fecific type8 are associated with IAfections of varying severity in
mew it was of interest to determine whetker stralAs of this ~rganisn freshly


                                                  172

isolated from human sources varied in their initial VirdenCe for mice*

   Two different methods have teen used for determining the primary

Infectivity of pneumococci in mice,  In all instances the cultures have

been tested as soon as possible after direct isolation from patients, in

order to minimize possible changes resulting from artificial cultivation.

Studies have been made of the virulence of these microorganisms as measured

by their capacity to invade the animal's tissuea when the living bacteria

are inhaled and thus implanted on the uninjured respiratory mucoea of mice.

The results obtained by the inhalation method have been compared with those

following the Injection of these same organisms through vctured tissues

Into the peritoneal cavity.  The former method more closely resembles the

natural route by which these organisms gain entrance to the respiratory

tract of man and serves as a measure of their ability to penetrate the

defensive tissues of the normal mucous membranea.

   From this study it is .aqarent that there exist great differences In

the virulence of freshly isolated strains of pneumococcl when tested by
direct inhalation method and by interperitoned fnjeotfon. Although the

number of atrai3.a of pneumococcl aad the nulnber of dce expose& to each

strain are too fen to draw any definite dedxltMonr, the results indicate
that !&pe I pnkmooocci have a relatlvrly low virulence for mice irreqtective

of the route by which they gain entrance to the animal body. Type II

organlutmcr have higher virulence as judged by the number of fatal lnfectfona
induced in mios folloting Inhalation or interperitoneal iajebkion.  Under

the same experimental conditions, `pype III and Type VIII orgahisms both

possess a much higher degree of virulence than do the strains Of me I and

Type II which have been tested.  The lncId.ence of fatal septicemia is

highest In mice exposed to infection with Type III peumOcocdi.


173

Publications

Downie, A. 1,.  Antipneumococcus species immunity, J. Hyg., 1938, xxxvii$.,292

Downie, A..W.,  Antigenic activity of extracts of pneumococci, J. Ryg.,
         1938, xxxviil, 279

Dubos, R.J.,   Immunization of experimental animals with a soluble antigen
        extracted from pneumococci, J. Ex& Med. , 1938, 67, 799

Dubos, R.J.,

The bacterlcidel effect of an extract of a soil bacillus on
Gram positive cocci,  P* 1939, 40,311

Dubos, R.J., and MacLeod, C.Y.,  The effect of a tissue enzyme upon pneumo-
         cocci, J. Exp. Med., 1938, 67, 791

Dubos, R.J., end Miller, BIB., A bacterial enzyme which converts creatine
         Into its anhydride creatlnine, Proc. Sot. Exp. Biol, & Mod.,
        1936, 39, 66

Dubos, R.J., end Thompson, R.H. SI ,  The decompositlan of yeast nucleic acid
        by a heat-resistant enzyme, J. Biol. Chem., 1938, 124, 501

Goebel, W. F. ,   The isolation of the blood group A specific substance from
         conznerclal peptone, J. E~P. Med., 1938, 68, 221

Goebel, V.F. ,   Chemo-immunological studies on conjugated carbohydrats-
         pro telns. XII.  The immunological properties of an arti-
         fleial antigen containing cellobiuronic acid, J. Exp. Med.,
         1938, 66, 469

Goebel, W.F.,   Immunity to experimental pneumococcus infection with an
          artificial antigen,  Nature, 1938, &43, 77

Goebel, W.F. , end Reeves, R.E.,  Derivatives of glucuronio acid. IX. The
        s~thesfs of aldobionides and the relationship between the
        molecular rotation of derivatives of acetylated aldoses and
          ur0nLc acids,  J. Biol. Chem, , 1938, 124, 207

Gooduer, K. B Horsfall, F.L. Jr., end Bauer, J.H., Some factors which affect
        the ultrafiltration of autipneumococcal sera) J. Immunol.,
        1938, 35, 439

Hoe&and, C.L. , Beeson, P.B. , end Goebel, U.F., The capsular polysaccharide
        Of the ryPe XIV Pneumococaus and its relationship to the
        specific substances of human blood, Science, 1938, 88, 261

Horsfall, F,L, Jr.,  The characteristics of entipneumococcus sera produced
        by various animal species, J. Bact., 1938, 35, 207

!

,









I
!
,

Lavin, 0. I., Th0nIp8On, R.H.S., and Dubos, R. J., The ultraviolet absorption
        spectra of fractions isolated from pneupx)cocci,  J. Biol.
         Chem. c 1938, 125, 75


174

EdacLeod, C.M. ,  Treatment of pneumonia with antipneumocbccal rabbit serum,
        Bull. N.Y. Aoad. Med., 1939, l5, 116

MacLeod, CM. , Hoagland, C, L. , and Beeson, P.B. ,  The use of the skin test
         with the type spooific polyaaccharides in the control of
         serum dosage in pneutnococcal pneumonia,  J. Clin. Invest.,
          1938, xvii, 739

S tillmen, E. 0. , Viability of pneu~~ococcl In dried sputum, J. Infect. Die. t
          1938, 63, 340
                         -

Thomspon, R. H, S. , and Duboe, R,J. , Production of experimental osteomyelitis
         in rabbit8 by intravenous injection of staphylococcus aureus,
          J. Exp. Med., 1938, 68, 191

Thanpeon, R.H.S., and Dubos, R.J., The isolation of nucleic acid and nucleo-
        protein fractions from pneumococci, J. Blol. Chem., 1938
         m, 66

Is Press

Dubos, R.J.,

Enzymatic analysis of the antigenic structure of pnetio-
cocci, ~.@&A. Bxzymforach. , 1939, 2

IA Preparation

Beeaon, P.B., and Boa&and, C.L.,  The use of calcium chloride IA the
            treatment of chills.

Goodner , R.. MacLeod, C,M. , Beeson, P.B. , snd Ho&and, 0. L. , S tudiee on
         host fgctors in pneumOcoccus infectionfr~ ISI. C8rtaiA
         factom involved in the therapeutic action Of Type I anti-
        ~neumococcus 88IlfIp iA Type s lobar pneumonia

StIllman, E.G., snd Schulr , R. 2. ,  Difference in virubnce Of V~iOUS types
         of pneumecocci for mice,