a ae

156

140
Report of Dr. Cole end Dr. Avery (essisted by Drs. Beeson, Dubos, Goebel, Good-
ner, Hoaglend, Horsfsll, Hotchkiss, “MecLeod, Reeves, Stillman end Thompson).
Serum trertment of pneumonia (“ecLeod, Goodner, Beeson, end Hoagland)
Since the use of unconcentreted entipneumococcus rabbit serum “es begun tro
‘ years ego, 75 cases comprising infections due to 11 tynes of  sneumococcus
heve been treated in the hosnitel. Of these 7 heve died, e mortelity rate of
9.2%. Until feirly recently edeauste rmounts of Tyve IIT entinneumococcus
rabbit serum hove not been eaveilable and if 211 ceases due to this tyne ere
excluded, the mortelity rate fells to 5.26%.

An attempt hes been mede to obtain a lerger number of Type III
petients in order to test the usefulness of entioneunococcus rabbit serum in
infections due to this type. It vould anpesr thet in e certein proportion of
ceses, adequate amounts of serum given early in the diserse “ill induce rapid
diseppearance of the acute symptoms in much the same way es occurs vith pneu-
monia due to other types. The greater veriebility in the course of Type IIT
pneumonia, however, perticulerly in respect to infections occurring in young
end old individuals mekes the results of therapy difficult to essess. Thus,
in cases occurring before the sixth decade, the disease tends naturelly to
run e much less melignent course,

The skin test vith the homologous type specific cepsular nolysac-
charide hes been extended to petients vith infection due to Pneumococcus
¢ Types II, III, and VIII in edition to Type I nd has oroved. in most instences
to be an accurate meens of determining when sufficient serum hes been given,
The difficulties essociated vith this test sre due to occasionel false vos-
itive or false negative rerctions. The confusion resulting from felse pos-
itive reactions cen be avoided, es ves previously found vith the Type I poly-

saccheride skin test, if the patient's resctivity to the intradermelly in-
157

141

jected polysaccharide is determined before the injection of serum is begun.
The information geined from frecuently reperted skin tests hes mde it 20s-
sible to give the optimum thereneutic dose of serum over a shorter veriod
following edmission then ‘es zossi*le vreviously.

The length of time consumed in :dministering the necessery thera-
peutic amount of serum hes e striking effect unon the duration of ecute symp-
toms, since termination of the diserse by crisis can occur only when suf-
ficient entibody has been given, or the patient's orn defense mechanisn hes
geined the uoper hand, The traditionel method of administering sntinneumo~
coccus serum hes been thet of divided doses over e variable neriod of time,
until evidences of subsidence of the acute nrocess hed occurred, or * nosi-
tive stin test to the homologous cepsuler nolyseccheride h-d » .nerred. Dur-
ing the pest yeer en ettenot hes been mede to rduinister the full theraneutic
dose of serum in e single intravenous injection ~s soon es jossible follor-
ing the edmission of the ortient to hosnitel. FO ecses, distributed among
nine types of »neumonie heve heen so trerted “ith unconcentrated entipneu-
mococcus rabbit serum, In these ceses the averege ele»sed time fron the
beginning of theraoy until the termination of the ecute symptoms hes been
only 8.6 hours. The uniform success of this method of serum rdministration
is conditioned by the need of accurete knorledge ‘of the sntibody content of
the serum used, end the severity end extent of the infection from which the
patient is suffering.

Antipneumococcus rabbit serum (Goodner). Reports submitted in
previous years have deslt vith the rationale of enti-meumococcus rabbit ser-
um in the therapy of human lobar pneumonia end with the first results of
clinical trial. The »vresent account mey he regarded as a nropress note,

The present status of the oroblems “hich ere nom in the foreground is con-

sidered “ith reference to chanses “hich have occurred “ithin the yeer.
 

158

142

Clinical results: Sere vreprred in this leboratory heve heen used
in the treatment of lobar oneumonies essocieted “ith Pneumococcus Types I, IT,
III, IV, V, VI, VII, VIII, IX, XIV, XVITI, XTX, snd XXYTIT, Sere for cer-
tein other types rre nov evcileble. Fxeent vith Toe III the results heve
been exceptionally good end heve justified the theoretical deductions “hich
led to its use.

Vath Type III meunonicrs the results ere still regerded as incon-
clusive. It is difficult to produce serum of good titer and the supnly hes
heen gomewhat limited. It is « curious contrediction thet certain ceses “ith
positive blood cultures have res onded in excellent frshion to the adminis-
tration of this serum while others edmitted to the hospitel vith negrtive
blood cultures have not resnonded and the course of the disease hes been
termineted by deeth. Certein of the-e therraneutic failures hrve been as-
sociated with conditions beyond the scope of serum theraoy but there re-
meined e curious paradox. Observrtions mede during the course of the pres-
ent pneumonia seeson may, hovever, furnish the clue necessery for the solu-
tion of this problem. A lerge number of persons are normel cerriers of this
type of pneumococcus and it seems to be associsrted with bronchitis in some
instences, It seemed not unlikely, therefore, that while this type of oneu-
mococcus might be predominant in the sputum snother type might serve as the
actual infecting agent. A somewhet similer instance hes been observed in
the experimental animal, viz.; Type IIT pneumococci are not highly virulent
for the rabbit but if the tissues ere preprred, es for example by extracts
of other types of pneumococci, the Type III becomes exceedingly virulent.
Consequently a vigorous seerch has been mede for type pneumococci other then
III in these clinicel cases. In seven instences, or epproximetely 75- per

cent of the so-called III ceses, enother type pneumococcus has been discovered

 

 

 

 

 

 
 

 

dj

and five of these cases heve been trerted as of the concomitant tyne. These

cases have recovered. Tyo cases of mixed infection were tre-ted es Type ITI
end both died. It is undoubtedly true that Type III can infect alone end
that it cen cause death but the foregoing observetions bid fair to shed con-
siderable light on the apparent failure of the snecific serum to benefit a
certain proportion of these cases. The solution probably resides in most
careful tyning and the appjicetion of this rule: When Type III end one other
pneumococcus type fre present in the s»xutum the other tvpe nneumococcus should
be regarded as the inciting agent.

Toxicity of rabbit serum: Methods have been develoved which greet-
ly reduce and in most instances entirely eliminate the toxicity essociated
with rav entipneumococcus rabbit serum. These methods ere simple end in-
expensive and it is pretifying to report thet other leboratories “hich have
employed these methods have been uniformly successful in the vroduction of
non-toxic serum.

Standerdizetion of serum: The question of standerdization of
antipneumococcus serum is much too complicated to eleborate in this revort.
It may be seid, however, that the only hope for logicel dosege is that a
satisfactory method of stendardization be achieved. Great progress has
been made in this direction and this laboratory is colleboreting with state
end federal egencies in e plen leeding to the adoption of suitable standerds
and methods for atanderdization. The sera in this leboratory rre eveluated
on the basis of ectual antibody content.

. Amounts of serum recuired to bring ebhout recovery: ith sccurate
evaluation of serum potency it is nov possible to study the factors vhich
in one patient recuire thet a very lerge emount of serum be used while in
enother nermit adequate therapy “ith smell emounts. Tt is obvious thet the

amount is determined partly by the severity of the infection but the cepadity
160

144

of the individuel to utilize the passively conferred antibodies is even more
important. This leboratory hes developed e considerrble experience in the
evaluation of the encalled host factors in ex»erimental infections. This
rork hed been underteken “ith the idee that it might some cay hecome useful
in the clinic. ‘uch to the satisfection of «11 concerned » direct annlice-
tion of these findings has heen discovered and it ray be expected thet “ithin
a fer veeks an accurate dosare scale vill be pvailable for use in most csses
of lobar pneumoniae.

The use of calcium chloride in the relief of chills folloving
entipneumococcus serum therapy (Beeson pnd Hoerlend). The occesionel oc-
currence of chills folloving serum edministretion hes to some extent inter-
fered vith the widesprerd adoption of serum thereov in pneumonis. Because of
the inadequacy of the psllistive measures employed in the trertment of such
chills, an ettempt was mede to find en agent which vould sllevirte them. In
® consideration of chemical substances ‘hich ight vorevent or elleviste
chills, celcium chloride, oing to its effect in relexing smooth muscle,
seemed a good possibility.

To date, 10% calcium chloride solution in amounts from 5 to 20 cc.
has been administered intravenously to 1@ vatients, during chills following
antipneumococcus serum therexy. The results obtrined heve been encoursging.
In 11 cases (61%) there wes complete relief, subjective end objective. In
7 cases (39%) there "as only pertiel, or trensient relief. Five of the pe-
tients in the letter group hed received epinephrine during the serum edmin-
istration and it is possible thet this sccounted in pert for the poor result
obtained with calcium therapy, since calcium end evinephrine ere knovm to be
antagonistic. Crlcium chloride is not considered ¢ dengerous thereveutic

agent, except in those patients who have received digitelis.

 
161

145

Studies are nov in progress as to the mechenism of this ection of
Calcium, also in regard to the possibility of a similer effect in chills due
to other causes.

Serum lipids in pneumococcal pneumonia (Horglend). Thet blood
lipids play en important pert in certain acute infectious processes is in-
creasingly evident as more exect technicel procedures for fat estimation be-
come available. Of these, cholesterol has received the most emphesis. Lete-
ly, however, varietion in the total lioid pettern in various diseeses hes
engaged considerable rttention.

Cholesterol hes long heen knorn to 20ssess nerked entihemolytic

properties egainst olestés, pneunococcus hematoxin and tetanolysin, and both

early and recent vorkers have found e merked droo in the cholesterol level
during acute infections, “ith return to normel vilues during early convel-
escence.

The early vork of Christian in showing vest stores of oleic fetty
acids in the stege of grey hepatization, end the work of Lamar in shoving
accelerated lysis by immune sere of pneumococci treeted “ith minute amounts
of sodium oleate, ettach considerable importance to the role of lipids in
the recovery from pneumococcal pneumonia,

The present study was undertaken in order to gein some idea of the

blood lipid pattern in pneumococcal pneumoniae, end to correlate, if possible,

the change in blood linids with the onset, course, and convalescence in the
disease.

Observations presented in this summary vere mede on the serum.
lipids of 25 patients with lobar pneunonie, rith enelyses at three day in-
tervals, covering the disease in most instances from its onset, through its
course to convalescence. Total linid carbon velues have been obteined in .

all cases, together with a smaller series in “hich the unsaturation, as

 

gE TT I TS on
Foren Spake aa One eRe ee
1pe

reflected by the serum iodine uotr’e, ond cholesterol v-lu's, free and com-
bined, have been determined.

“ero~easometric technicue, efter the iethoc of “irk, Prre, rnd
Ven Slyke, modified by Folch-Pi, ‘rs used throughout. Serum todine cbsorp-
tion determinetions ‘ere ccrried out :ccording to the tethod of Pare, Pes-
ternak rnd Burt.

bnelysis of dete reverls in all cases a nerved dron in serum
lipid cerbon, cholesterol end unsetursted fetty rcids during the onset of
pneumococcal oneumonir, with ¢ return to normel during convelescence. The
most merked dron romenrs to be in the cholesterol ester frection, “hich in
meny instences fells to less then helf the nornel or conve lescent level,

Free cholesterol values rre not sipnificrntly eltered, “hich is perticul:r-

“ly interesting in the light of recent vor by Snerry “ho wes phle to shor

nr remerkrhle constency in the retio hetween free rnc ester cholesterol in
normel individurls over an extenced period of time. The *e1l in serum
unsrturetion poocers to orrellel the dron observed in total liold carbon
end cholesterol ester, end to return ouite rs 2romotly to the conve lescent
level.

Although dete cre inconrlete et oresent, it ould annerr thrt
the neriod of hvpolipemis in meumonie is considerrbly shortened by the
eerly institution of serum thereny. |

| As en exeaple of the chenzes observed in the serur: linids during
the onset, course end convelescence of meumonie, ane lyses in tro erses

ere presented es follovs:

 
 

463

 

 

 

Case Dey of | Totel lisid Cholesterol Uns«ture tion

ddiseese cerbon Free Ester Total | "Ig. 1°/100 ec. serum
Ferly se- Pe 412 40 140 1A0 92
rum rdmin- 5 700 38 71 109 P40
istretion 8 51? 4? 180 PP %6°
} Ul 418 46 195 241 41?
Un- f+ 2 3e7 30 1-2 182 320
trerted 5 274 22 Qf 127 215
8 238 PR 70 98 ?08
11 277 2B lee 150 230

 

 

 

 

 

 

Certain proverties of the C-rerctive substence present in the
blood during pneumonie and other infectious diseases (fvery end MrcLeod).
Ferlier studies from this leboretory heve shorn that curing the active
stere of lobrr meunonie there 4s denonstrable in the blood, » substence
which yields a precinitete when er dilute solution of th: "C" cerbohvdrate
of Pneumococcus is added to prtients! serum. The nrecinitrble suhstence hes
been detected in serun obteined within 4 to 1? hours efter the initial
chill. The orecivzitstion reection renrins oesitive throughout the course
of ective diserse and becores negrtive ith the onset of recovery. Petients
in whose blood the reactive substance is denonstreble develop © chereacter-
istic skin resection folloving the intredernel injection of 0.1 mg. of the
test cerbohydrate. In a lerge series of petients studied, the results of
the cutaneous end precinitetion tests are in close agreement end both re-
actions prrallel in striking manner the clinicerl course end outcome of the
diseese. Thus, the oresence of this substence in the circulation during
the acute disease and its absence in convelescence are in turn correlated
with the activity end terninetion of the disease »rocess in “en.

It ves soon found, horever, that the orincinles involved in these
reactions ere not restricted nerely to oneumococerl infections. An identi-

cel substance, or at least one chemicelly so similer rs to be eouelly
 

.

464

reective with the seme test carbohydrete, is oresent in the blood during.
the ecute ovhese of certain infectious diseases caused by egents other then
Pneumococcus. For exemple, in rheumetic fever, bacteriel endocarditis,
oulmonrry tuberculosis end e number of other infections, there exists in

the blood r substence which is serologicelly the seme as thet found in pneu-
monie. It enenrs, therefore, thrt the reections described are not svec-
ific with reference to the etiological incitent of the infection but repre-
sent rather a nutuel interection of © selective cerbohydrete vith the re-
retive substence common to meny infections of unreleted etiology. This
substence possesses the »roperty of combining “ith the test cerbohydrete to
form e precipitate. No other becterial suger thus fer tested hes heen found
to precipitete the moteriel in reactive serum. It will be recalled thet the
"C" cerbohydrete is present in the cell hody of ell forms of meumococci
irresnective of their tyne derivetion. Althouch chemically less vell de-
fined, this carbohydrate differs mrredly from the ce psuler rolyseccharides
in conteining vhosnhorus end ventose but no uronic ecid constituent in the
molecule.

Attempts cre being mede to isolate the rerctive substance from
gerum end to determine its chemicel nature. On frectional precipitation of
patients! serum vith axmonium or sodium sulphate, the ective materiel is.
cerried along "ith the serum elbumin, the globulins being vholly inert.
From e solution of the elbumin it is precipitated together ith the portion
of normel albumin removed by 2/2 saturation. On removel of selts by dialysis
at neutral or slightly alkeline reaction, the ective substence vrecipitetes
from the solution of inert elbumin. The neteriel recovered is ective vith
"ot and hes the folloving proverties; it is soluble in dilute elkali end
physiologicel seline; insoluble in “eter end dilute ecids; it is reedily

denatured end inectivrted et hydrogen ion concentrations more scid then pH 6.

 

 
 

1465

The extreme lebility of the active substance differentirtes it fron the normal
blood vroteins but renders difficult its isoletion end vsurification in active
form.

From reective human serum the substence cen be ourntitetively
vreciziteted by the eddition of ontinel snounts of the cerbohy rete. On the
besis of its knorn proverties, tec'nioues rre being deviced to dissociete
the ective meteriel fron the "C" precinitnte. The formetion of © vrecivitete
is conditioned by the vresence of crlcium in the reection-systen, since no
precinitrtion occurs on rdding the errbohydrete to reective serum from “ich
the celcium hes first been removed. On the reeddition of minute treces of
CaCl, to such serum, the ective substence agrin orecizitetes in the presence
of the test suger.

The C-vrecipitoble substence hrs not yet been obteined in e suf-
ficiently ourified form to justify eny conclusion rs to its neture. Hor-
ever, on the basis of its nitrocen content (147) end certrin other vroper-
ties, it is tentetively regrrded es protein end one not normelly found in
the blood of heelthy individurls.

Vork on the nurificetion of this interesting substance end a
study of its chemicel end entigenic proverties pre being ectively cursued.
One would like to mov its origin end fete in the enimal body; its vossible-
significance in relation to certein menifestietion of diserse; end the
chemicel groupings involved in the precinitetion reection ‘ith "C". The
interpretetion of the precinitetion end svin rerctions in terms of rntigen-
entibody union is difficult for the folloving remsons: the rerctive sub-
stance is demonstrable not only in pneumonie but in diserses not rssocirted
“ith pneumococeus infection; it is present in the blood errlv in the course

of diseese, long before svecific rmntibodics rre denonstreble, end it disep-

 

|
/
i
!
j

 

 

 
166

150

peers from the circuletion durins convelescence, * time rhen entibodies are
usuelly found in highest concentration. Thus, the tine of eoverrence end
disenreerence of this substance vith respect to the occurrence of *“nov™n
pntibodies is comoletely reversed; this tosether vith the leck of snecifici-
ty, the essocistion of the remctive substance vith the albumin rether then
vith the globulins of serum, end the imoortence of celcium in the dorecirite-
tion reection ere facts not ersily reconciled vith the orthodox conceptions
of entizen-entibody phenomene.

Preumococcel cerrier stete in nenhrotic children (‘lecLeod rnd Farr)
Most of the deeths occurring in children vith nevhrosis rre due to pneumococ~
cel peritonitis. A study hrs heen mede of the mneumococeci cerried in the
neso-vhervnx of such ceses, in order to determine “hether veritonitis is
ceused by the strein or streins of »nneumococcus carried in the xetient's thrort,
or whether it is due to the ecouisition from e cerricr of en invrsive strein
of pneumococcus. The first sud .0sition hrs been found to be most generrlly
true.

In ell the ceses which develoved peritonitis the seme type of
pneumococcus hed been recovered from the thrort end the peritoneal cevity,
The longest period from the first isolation of © strein of oneumococcus from
the throet until. peritonitis due to the seme type occurred, vas three months,
| though children tho recover from peritonitis mey cerry | the ine
veding strein in ‘their ‘throat for Ag long eg © yerr afterrerd, in some in- -
‘stences specific agglutinins may not be demonstreble in the blood serum. Hor-
ever, the development of specific rgglutinins does not furrentee freedom from
neritonitis, since the serum of one patient who died shoved snecific agglutin-
ins egainst the inveding orgenism on the dey the fetel infection beren.

Children “ho do not develon reritonitis, end “ho recovor from

hephrosis, tend to shor more variation in the tnes of -neumococci crrried

poe

Si ee

 

B e

 
167
1§1
in the nasopharynx. Thus en rcouired strein wy be rether quickly lost or
revleced by e different strain.

The role of nucleic rcid in the structure end inmmolorice]l beherv-~
jor of the pneumococcus cell (Dubos end Thomison). The preperation of a ribo-
nucleese from animel tissues rnd its effect on heet-killed oneunococci. It
hes been described in e pnrevious renort thrt leucocytes conterin e hert resis-
tent enzyme cepeble of ettrcking villed meunococci. The enzvme hrs heen
surified by e technique besed on the folloving proverties; e¢) it is soluble
in 50” ecid ecetone, but precipitetes from this mediun on eddition of more
ecetone, b) it can be subjected to boiling temnerrture betreen nH @ end nH
6.5 without eny loss of ectivity. The vourest prenerrtion so frr evetlable
conteins 12% Nitroren rnd 0.58% Phosnhorus.

The only soluble substrrte found to be rttecked by the enzyme is
ribonucleic acid, which it devolymerizes.

“hen heat-killed oneunococei rre resusnenced et neutrel reaction
in the vresence of the enzyne, they slovlv lose their “ffinity for the basic
dyes elthough this chenge is not accompenied by rny evidence of celiuler dis-
integration. Under these conditions the killed cells of encepsuleted meu-
mococel lose the vorer to stimulete in rabbits the oroduction of the type
specific antibodies directed against the cerpsulrr »xolysrccheride of the cell:
in other words the ribonuclesse inectivetes the cepsuler rnticen of virulent.
pneumococci,

The extraction of ribonucleic ecid end of e ribonucleoprotein from
pneumococci. Although pneumococci sre killed within « fer seconds in an rce-
tate buffer at pH 4.2, they retein in this ecid medium their Grem nositive
character; ‘hen the same cells, hovever, sre senereted fron the acetrte sol-

ution end resusvended et neutrel reection, they slovly undergo r chenze from

 

 

 
i
if68
the Gram positive to the Gram negative stete end at the seme time lose their ,
effinity for the basic dyes. Two of the substences which ere released in sol-
ution in the course of this chenge, have been separated and identified. One
fraction is soluble in dilute ecetic ecid but insoluble in HCl- it contains
14% N and 7.75% P end is decomposed by the ribonuclease described in the
i preceding section of this report. This substance is a ribonucleic scid.

Another fraction is precinitsated et pH 4.° and remeins insoluble
in stronger acids; it conteins 14% N end 4% P. The high nhosphorus content
is due to the presence of ribonucleic acid in the molecule. This nucleic
acid is combined with » nrotein which is deficient in srometic amino-acids
end in sulfur and which msy thercfore belong to the group of vrotamincs or
histones.

The release in solution of nucleic ecid and of nucleoprotein, which
tekcs plece when pneumococci killed at pH 4.2 are rcsuspended in neutral
solution, is a phenomenon common to both R end S varients, irrespective of
type derivation. In the case of the encapsulated cells, there is observed
at the same time a loss of the ability to stimulate rabbits to produce the
specific antibodies directed ageinst the cepsuler polyseccharide. It ap-

pears, therefore, that when the ribonucleic acid of the Pneumococeus cell

 

is attecked by ribonuclense, or when en injury to the celluler structure el-
lows it to diffuse into the medium, the cepsuler rntigen is et the aeme time
inectiveted, Nothing is known, horever, of the neture of this reletionship,

¢ ' It mey be mentioned here thet e study is being mede of the immuno-
logicel properties of the nucleoprotein fraction relersed in solution when
the cell chenges from the Grem positive to the Grrm negative state; definite
evidence hes been obteined thet this frection, when injected into experimentel
enimels, crn produce immunity to infection "ith virulent pneunococel; this

protection is not type specific «nd the sntigen can indeed be preprred from

 
 

169

153

rough as well as encensuleted pneumococci. So little is known of this phe-
nomenon, however, that a detailed description is not warranted st the vresent
time.

Structure of Type ITI sneumococcus nolyseccharide and eldobionic
acid (Goebel, Reeves ond Hotchkiss). The capsuler nolysecchsride of Type
III Pneumococcus is e mecromolecule constituted from nits of an eldobionic
ecid linked in glycosidic union. In addition to its vroperty of conveying
virulence and type specificity upon the orgenism from which it is derived,
the capsular polysaccharides, when injected into certein species, induce an
active antipneumococcal immunity. Becruse of these important biologicel
properties we heve found it desirable to esteblish the constitution of the
pldobionic ecid from which the polysecchrride of Pneumococcus Type III is
constituted. In colleboretion with Dr. Hotchkiss we heve shorn thet the
rldobionic ecid is 4 4-8lucuronosido-glucose end hes the folloving struc-

turel formula:

OH OH

 

COOH f)

Since this sugar ecid is the uronic ecia of cellobiose, the dissc-
cheride unit from which cellulose is constituted, we have termed the Pneu-
mococeus Type III aldobionic ecid cellobiuronic ecid, The relationship of
the cepsuler polysaccheride to cellulose is obvious.

The position of glycosidic union of sldobioniec acid units to one
enother in the intect polysecchrride molecule is being investigated with the
assistance of Dr. Reeves. “hen the errbohydrrte is methylrted with methyl
sulfrte end elkeli, e fully methylated derivetive is obteined which still

reteins the property of rercting in rntinneumococcus serum. This fact

 

 

 
g
4 ee

1'70

154

reaffirms the concept exoressed in earlier renorts thet the union of poly-
saccharide to homologous antibody is orimarily through the cerboxyl group of
the former. The identificetion of the products of hydrolysis of the methyle-
ted polysaccharide is under investigation and will yield information definite-
4 ly establishing the complete structure of the specific polysecchrride itself. 7
Synthetic entigen containing cellobiuronic ecid (Goebel). Al-
though antipneumococcal immunity cen be induced in certein snecies, including
man, by immunization with the intect specific polyssacchrride, it is now
knovm thet immunity mey rlso be induced by chemically nodified end denoly-

merized forms of the csarbohydrete. It is of great intercst, therefore, to

 

escertrain whether immunity to Type III Pneumococcus infection con be induced

by the immunization of enimels with the aldobionic rcid unit of the poly-
saccharide molecule, Consequently * method for the synthcsis of eldobionides
hes been devised, end used in the successful synthesis of the p-reminobenzyl
glycoside of cellobiuronic ecid. This durivetive, when combined with pro-
tein by merns of the dirzo rerction has yiclded an ertificiel cellobiuronic

ecid-protein entigen which cxhibit: ‘aeny of the scrologicr] charecteristics

 

of the Type III polysecchrride itself. The antigcn precipitetes both in
entipncumococcus Type III horse end rebbit sere in high dilutions, but not
in the normel serr of these species. Experimentrl enimels erc being im-
munized with the antigen rnd the results will be renorted leter,

€ The neture of fowl prothrombin (Schoenhcyder rnd Goebel). De-
ficiency of vitamin K ceuses ¢ hemorrhegic dirthesis in chicks which is
secompenied by r disenperrence from the plisme of the clotting fector, pro-
thrombin. In colleboretion with Dr. Fritz Schocnheyder en investigetion con--
cerning the mechrnism underlying this elteration in »hysiologicel function

weg initisted lrst July through « study of the chemicrl nrture of the pro-

 

 
ws i771 |
thrombin of normel fowl plrsme end - comprrison of this plesme frercetion with
thet derived from the plesm of vitemin K deficient birds.

A quentitetive method for the cstimetion of prothrombin wes de-
vised, * procedure which wrs used to grent adventsge in studying the chemi-
eel frectionetion of the »ctive ¢gent. By frectionating fibrinogen-free
plesme ct 0° with emmonium sulfrte it is possible to obtrin the prothrombic
retivity in the fraction insoluble in 75-65 per cent srturetion. Further
frectionation vith neutrel srlt solutions is not fersible. However, ad-
sorption end cluetion of this mrtcrirl on colloidel gcls results in a sherp
scperetion of inert protein from the active principle. On the besis of ni-
trogen content it is possible to obtrin prothrombin solutions with en ec-
tivity nerrly one hundred fold grertcr then thet of the plesmr from which
it wes derived. Solutions of prothrombin thus isoleted rre auite strble
cbove pH 7.0 end very unstrble in the scid renee. Although forl prothrombin,
es we herve obteined it, is probrbly not + cure chenicel ontity, yet from
its chemicrl chrrrecteristics, its thernolrbility, ond its susceptibility to
enzymetic hydrolysis, the substrnce roperrs to be » protcin endowed vith
certpin unioue chemicrl rnd biologicrl cherecteristics which distinguish it
shrrply from other olesmr proteins, Dr. Schoenheyder's return to Denmerk
in Januery termineted the reserrch ct this juncture.

Preprretion end use of ferrous gluconrt: in hypochromic cnemir
(Reznikoff end Goebel). In colleborrtion with Dr, Poul Rezniloff of the
Denertment of Medicine, Cornell Medical College, we heve rttempted to pre-
pere weter soluble iron compound which cen be edministered prrenterrlly. A
therapeuticrlly effective end rerdily sssimileble comnound of iron is grert-
ly to be desired in conducting studtes on hemoglobin since prrenterrl rd-
ministrrtion eliminetes the uncertrinty of ebsorbtion regeneretion, It is

believed thrt the lecel tissuc necrosis crused by the injection of inorgrnic

 

 
172

156

selts, es vell rs the grstro intestinel distress which freouently eccomprnics
their orel rdministretion, mey be rttributed to their crpacity to act as pro-
tein precipitents. Ferrous gluconrte, the iron selt of the polyhydroxy orpen-
ic ecid glucuronic rcid, does not precipitr te protein becruse the iron is in
cP un'mizg-ble complex combinetion with the errboxvl end hydroxyl grouns of the
hexonic ccid. &# precticrble end incxpcnsive method for the vroduction of
erystrlline ferrous gluconrte hrs been devised. Ferrous gluconrte comnrres
very frvorrbly vith inorgrnic iron comnounds in giving renid :nd merked rc-
ticulocyte, red cell rnd hemoglobin responses both in enremic rlbino rets

rnd in humns suffering from hynpochromic enenmir. Prticnts showing toxic
rerctions to other iron compounds “cre cble to tolerrte ferrous gluconrte
without eny unduc distress. In prtients who reccive ferrous gluconrte intre-
musculerly no systemic end only rere end rild locrl resections occurred. In
view of the efficecy of the orrl rnd prrenterrl rdministretion of ferrous
gluconete end its Irck of toxicity the drug vossesses properties which ren~
der it of velue in the trestment of hypochromic eneate.

Virbility of mncumococci in dricd sxutum end blood (Stillmen) In
continuing the errlicr enidemiologicel studies on the mode of disseminetion
of the diserse~producing types of nmneumococcus, exveriments herve been crrried
out to determine whether these orgrnisms crn survive for long periods of time
in dried sputum under conditions simuleting those encountered outside the body.

Smell auentities of sputum freshly obtsined from petients with
known types of pneumonir vere placed in + scrics of test tubes stopnered
with cotton plugs; one sct of tubes vrs pleced rt room tenperrture in dif-
fuse deylight end the other in the icebox et r temperrture of rbout 40°F, At
intervels, duplicate svecimens of the snutun which hee dricd et different
temnerrturcs rnd under vrrying conditions of humidity end light were emulsi-

fied in beth end injected into mice. If the virulent nneumococci originslly

 

 

 
 

J73

present in the sputum remeined virble in the dricd specimen, the inoculrted
mouse died of infection end pneumococci of the homologous type "ere recovered
in cultures of the enimel's blood *t eutopsy.

In severel instences Type I pneumococci remeined vieble in dried
sputum efter exnosure for 16 ~eeks rt room temperrture: Type IT rftcr 17
weeks; Type III efter 20 “coks. Despite the grert verirtions in the number
or orgenisms present in individurl specimens of sputum, rnd the merked dif-
ferences in the vhysicrl end chenicel propertics of the verious semmles, it
mey be conservatively steted thet the everrge time of survivel of Type I
orgenisms in dricd sputum is 4 wecks, Type II 6 veeks and Type IIT 8 weeks.

The length of time during which pneumococci remeined virble in
specimens of sputum stored in the icchox is considersbly lonecr. Under
these conditions Type I pneumococei "ere reeavered from specimens rfter 15
weeks, Type II 26 veeks,end Type ITI 16 weeks. In one instrnce Type IT
pneumococci were found to be vieble in snecimens of sputum which hed been
stored in the icebox for 41 weeks.

The results indicrte thet under these experimentel eonditions
pneumococci cen remrin virble end virulent for long verinods of time in
dried sputum end the findings suggest that these orgonisms my survive and
retein their diserse-producing properties under the more rigorous conditions
of the outside world for longer periods of time then hes generrlly been
supposed.

Similer studies ere under way to determine the survivel end derth
rete of pneumococci in dried rebbit's blood. To defibrinrted blood, mer sured
quentities of pneumococci of the different types were rdded, end the inocu-
leted specimens vere rlloved to dry under conditicns similer to those used
in the experiments with sputum. Types I rnd If pneumococei hrve been re-

covered from specimens of dried bload “hich hed been kept for to months

 

 

 
—— eee

174
158

exposed to the air under the conditicns of tempcreture, light end humidity

 

prevailing in the leborrtory. However, specimens exemined after 2 months
feiled to yield vieble orgenisms. On the other hend Type III pneumococci
under identierl conditions heve remained virble rnd virulent up to the
present time, 8 months efter the initicl observetions.

R) The production of experimenter] sestcemyclitis in rcbbits by intre-
venous infection with Strphylocaccus eurcus (Thompsen end Dubos). In the
course of experiments dcsigned to comprre the virulence of different strrins
of Strphyloceccus in rebbits, it wes obscrved thet severel enimrls develnped “:
well cherectcrized bone lesions following the intrevennous injection of © cer-
thin strrin of Staphylocsccus rureus, In view of the ernflicting stetements
in the literature concerning the nossibility cf producing strnhyloencerl as-
teomyelitis in exncrimentrl enimels, en pttempt was mede t> understand the
conditions under which rebbits resnond to the injection of this strein of
stephylocoecus by the develooment of bone inflemmetion.

The virulence of the strein wes rriscd by enimel prssege end es-
pecielly by iscletion from - bone ebscess produced in + rebbit by the in-
jection of o lerge amount of culture, In mst %7f the cxperiments an ettempt
wes mede t> produce in the rabbits e 1» degree of immunity by injection of
suspensions of killed stenhylococei, prior to infection, in the hope of
thereby obteining e relrtively prolonged course of the infection so thet
macroseopicrlly recognisrble lesions might develop before the death of the

C enimel,.

 

Young rebbits were injeetod intrevenously with smrll doses cf

 

culture end were observed deily for the develonment of rny swelling, tender~
ness or loss of function in the limbs; in some of the expcriments g-rey
photogrephs were teken of the enimels before desth, end rlso of any inflemed

benes efter removal from the beady ct autonsy. At the eutrpsies perticuler

 
“

175

159

attention wes peid tm the conditien of the joints in the hone of determining
whether lesions in the meteohysis of the long bones were due t> + primery
osteomyelitis or to « sprerd from r primery purulent erthritis.

21 pnimels in ell hevce been used. 9 of these rnimels died shortly
after they herd been infected before sufficicnt time hed elapsed t> ellow the
development of bone lesinns reengnisrble with the neked eye. Of the 22 ani-
mels in which tho infection sssumed ¢ subecute course Irsting from lL to 3
weeks, 18 or 81.8% showed definite mecroscrpic evidence of bone inflemmsticn.
Tho bone lesirns vericd fr-m mere seftening ond hyperremir with multinle sub-
perinstenl bleeding prints, t+ sdvenced abscess fornrtion “ith suppuretion
pnd extensive bony destruction. In crses where frenk suppurrticn end bony
destruction were rbsent, micrrsernic exrminstion of strined films of the bone
merrow beneath the inflemed region showed in every case the presence of Grrm-
positive cncci end of en inflemmatory rerction. 55 bone lesicns were present
in the 18 enimels; of these 47 occurred in the metrphysis of the long bones,
yet only 8 were associcted with e purulent erthritis.

Severel prints of interest heve emerged suggesting - close simil-
arity between the experimentr1 ce ndition end strohylocnceal osteomyelitis in
children. Firstly, tho characteristic nredilectirn of the ergenism to ‘pro-
duce inflemmetion in the metephysis of the long benes; secondly, the rela-
tive infrequency °f spreed of f netavhysirl ebscess through the epiphysis
into the neighboring joint cevity end, thirdly, the pyrexiel course of the
infection in the neute stege, vith ep lecae into er chromic condition of pro-
gressive suppuretion, if the rnimel survive the scute strge,

The results -f the work indieste thet it is possible t+ produce ’
consistently inflemmetirn *f the benes “f rebbits by the mere intrevenrus
injection cf e suiteble strein cf stonhyloc-ccus without reserting to eny

elabcrate xperrtive technique cesigned t> lncelise the ~rgrnisms in the bones.

 

 

 
Beeson, Prul B.

Cule, Rufus.,

Dub:s, René J.,

176
160
Publics tions

end H-eglend, Cherles L.. Use -f crlcium chleride in relicf
“f chills following scru> cdministreticn, Proe. Soc. Exp.

Bicl. end Med., 1978, 78, 160,

Pessibilitics for meumni ccntr 1 es indierted by oresent
scientific kn-vlcege, The Militcry Surge-n, 1977, 81, ?4l.

The decrompssition -f yerst nucleic scie by » hert resistrnt
enzyme, Science, 1927, 85, 549,

Dub>s, René J. end MacLe-d, Colin, Effect -f + heet-resistent enzyme upon

the entigenicity of oncum-ercci, Proc. Soc. Fxp, Biol, end
Med., 1977, 26, 696.

Dubos, René J, rnd Miller, Benjemin, The production of boetericl enzymes

Dubos, René J,

Goebel, Welther

Goebel, Writher

Goebel, Wrlther

Goebel, Welther

erprble of cecsmpesing crertinine, J. Bicl, Chem., 1937,
121, 429.

The effect of formrldehyce on pneumoeecci, J. Fxn. Med.,
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F. The chemicrl constitution of benzyl glucuronic rcid,
Science, 1927, 86, 105,

F. end Hotchkiss, R.D., Chom>-immunolngicel stucies sn con-
jugrted cerbohydrrte-prsteins. XI. The snecificity of r25-
protein entigens esntrining glucurnnic rne gelrecturonic
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F., Reeves, Richere rnd H-tchkiss, Rellend D., The synthesis
of elanbionides, J. An. Chom, Soc., 1977, 59, 2748,

F., Derivetives of glucuronic rcid. ‘VIII. The structure of
benz-ylglucur-nic rcid, J. Binl. Chem., 1978, 1°2, 649,

Gondner, Kenneth end Horsfell, Frenk L,Jr., Properties of the type specific

proteins sf entipneumrerccus serc, 1. The mouse pr-tective
velue of Type I serr vith reference t= the precipitin ecn-
tent, J, Exp, Mec., 1977, 412, 66,

Goodner, Kenneth end Horsfell, Frenk L. Jr., Properties of the type specific

proteins of entipneumrenccus sere. II. Ivmun>vlogicel frec-
tionetion of Type I cntipneumoenccus horse anc rebbit scre,

I. Exp. Mec., 1927, 66, 425.

Geodner, Kenneth enc Horsfell, Frenk L, Jr., Properties of the type specific

proteins of entipneumrenccus sere. TIT. Immun::chemicel
fractionation of Type I entipneumoeoccus horse end rebbit

sera, J. Exp. Med., 1977, 66, 477.

Gondner, K. and Horsfrll, F. L. Jr., Prssive enephylectic sensitivity to pneu-

mocreerl cepsuler polysrccherices, J. Immunrl,, 1977, 23, 259,

 

 

 
17°7
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Gonener, K.,Horsfe}l, F.L. Jr., end Dubos, R., Type specific mntipneumocrccic
rebbit serum for therepcutic purposes. Proeucti-n, processing
pnd stendardizetion, J. Immunol. 1927, 27, R79.

Goocner, K.,Horsfell, F. L., dre, The purpuric reection produced. in animels
by derivetives of the Pneumeenccus, Proc, 8oc. Exp. Biol.
end Med,, 1927, 37, 178.

Horsfrll, Frenk L. Jr., The ecntrol of lobrr pneumanir, Cenecdien Public Heelth
J., 1927, October 476,

Horsfell, F. L., dr., Grocner, K., ene MecLeod, Colin M., Antipneumeecccus
rabbit serum rs r therrpeutic egent in Lobrr nmneumenie. II.
Additicnel cbservrticns in »neumoerccus pneumonirs of nine
differont tyoes, N.Y. Stete J. of Med., 1978, 28, 1.

Hotchkiss, Rollin D., ene Grebel, Welther F., Chem>-immunol-gicrl studies on
the scluble specific substrnce nf Pneumeerecus III. The
structure of the alebicnic rcid fr-m the Type ITI po lysrc-
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MecLeod, Colin M. end Ferr, Lee E., Relrticn of the cerrier stete t> pneume-
enceel perit«nitis in young children with the nephrstic syn-
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Miller, Benjemin F., end Dub»s, Pene, Studies ~n the presence of creatinine
in humen bloo, J. Bicl. Chem., 1927, 121, 448.

Miller, B. F. end Dubss, R., Determinetion by + specific, enzyme tic method
of the erertinine content of bloca end urine from normel end
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Regnikoff, Paul end Goebel, W. F., The use of ferrous gluconrte in the treet-
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In Press

Dubos, R.J. The cecomposition of ribonucleic ecid by « hert resistant
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oa

Dubos, RJ. and MecLend, C, M., The effect of F hert rosistrnt enzyme on’ °°

pneummercci, J. Exp. Med., 1928.
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