REPORT CX? TtlE DIRECI'OR tX? THE HDSPITAL
                   m---w.--
       October 19, 1923

REPORT OF TIE DIRIXI'Oii 3F "I-Z HOSPI?AL
October, 1923
Gentlemen:
    During the year no noteworthy change in the personnel of
the staff of the Hospital occurrod but word has just been received
of the appointment of Dr. Christen Lundsgaard as Frofcssor of Uedi-
tine in the University of Coponhagen, one of the first academic posts
in medicine in Europe, so that Dr. Lundsgaard will undoubtedly leave
before long to assume his new duties.  He first came to the Hospital
in 1917 as a Fellow, rermining but one year-   He returned in 1921 as
Aesboiafe' in Medicine and Resident Physician.  On July first of this
year he ma8 advanced to the rank of Associate Member. During his con-
nection :-Ath the HoBpital Dr. Lundsgaard has carried on important re-
searches in connection nith the physiology and pathology of respiration
and the 8ucce88 of this work here ha8 undoubtedly been largely instrU-
mental in causing him to, be appointed to the very honorable position in
Copenhagen.
His departure will leave a vacancy which should ba fill&d as
soon a8 poesible.  During the past decade there ha8 occurred a great
increase in interest in the study of diseases of disordered metabOliBm,
and there are noxr many more men trained in the application of the sci-
ence of chemistry to the study of diseases than were available v;hen
the Hospital was opened.  Consequently a constantly increasing share

of the Hospital resources are being devoted to this :rind of investi-
                         . .
gation.  It is therefore important that a clinician wit`n a thorough
trsining in chemistry shoul3 be secured to coa3lement and assist Dr.
                                                   _
Van Slyke in the investigation of Diseases ;;let:.bolic in nature.
     Of the Resident Staff, Dr. Boots ar.1 Pr. Dahl have re-
signed, and Dr. Crawford of Edinburgh and Dr. Andreas of London have
received appointments as Assistant Resident Physicians.
     It is gratifying to report that very sliostantial progress
has already been made in the bvo new undertakings first mentioned
in the last report.  Especially important have bezn the results
obtained by Dr. Rivers and Dr. Tillett
chicken-pox.  The details of this work
port.
in the investigation of
are given in Dr. Rivers ' re-
The division of the work in the FIoepital into m?Teral
parts, each carried on bp a group of workers aorking more or less
independently, has progressed and this is reflected in the present
report which is chiefly composed of the reports of the heads of the
various divisions.
Infectious Diseasee of the ResDirators Tract.
.
  The studies carried on have dealt chiefly with problems
^ ./                                                              .: , ., -`a
relating to pathogsneais and to problems relating to the biological
properties of pneumococcus.
Pathogenesis of Lu11p Infection%.
     Dr. Stillman has continued the study mentioned in the 2,;. -
report concerning the disposition of pneumococci and streptococci'
which have reached the lung following spraying.   The tentative re-
sults previously mentioned have been confirmed a:nd the conclusions

                        ::i ,i i
published.                                           116
     In a contil-mtion of this St&y it was found that if al-
cohol ia administersd to mice and the mice are then exposed to aft
containing pneumococcus in suspension, there occurs a delay in the
disappearance from the lungs of the inspired bacteria.   Al though
pneumococci will disappear fran the lungs of normal mice following
inhalation within four hours, in mice intoxicated by either i&ala-
tion of alcohol or by interperitoneal injection of alcohol, the
period of removal is delayed in most instances until after 24 hours.
Fur thermore, whereas the blood stream is seldom invaded in normal mice
f ollarving inhalation of pneumococci, about 4% of intoxicated mice
devalop a septicemia Y  In bvo instances pneumococci were found in the
heart's blood of intoxicated mice one hour after inhalation.
     Acute alcoholic intoxicatiOn also markedly increases the
mortality of mice following spraying.  Whereas only about 35 of normal
mice die from a pneumococcus septicemia following spraying with pneumo-
Cocci, mice intoxicated by interperitoneal injection of a lO$ solution
of aLcoho1 or by inhalation of alcohol have a mortality of 30$ and 5o;`j
respectively,  In order to determine whetiier the administration of al-
cohol is favorable to the in+asion of other bacteria, streptococcus
hemolyticus and B. influenzae were also used. The effect of intoxi-
cation is not as definite where these organisms are employed in the
experiment as when pneumococci sre.sprayed, probably because the bac-
ha mentioned are not removed as rapidly from the lungs of normal
mice.  However, even when these organisms are employed, septicemia is
definitely more frequently encountered in the intoxicated mice.
    Dr. Bratich'i~`making a histologkal s+Iy of the lungs of

the rr.ice which hrve br~r~ apl.iyeL 7~1f.h bas',c:ia, -;i;th a ~iex c? c'zitz,in-
lng infcxmation concerning the character of the earliest reaction which
occurs when bacteria are implanted in the lung by inhalation.
     Following the sI;rajring of nc.rmtil L;ics with pneumococci , in
no case hr?s a true pnsuxonis resulted.   In some of the intoxicated
mice, however, definite fibrinous pleurisy ar,d marked congestion of
the lung parenchyma have been found.  In certain instances mice have
been sprayed and then at a later date again sprayed either with or
without previous intoxication with alcohol.  In soni of these mice the
lungs have shown definite pneumonic patches, the sections having le-'
sions resembling red and grey hepatization.  This study is beicg con-
tinued with a view to studying the earliest pulmonary
lungs following infection, and also to study the role
munit:? in this process.
Vilvlence of Fneumoco~c~.
changes i-n the
of partial ix-
Studies concerning the properties of pneumonia upon which
virulence depend have been mde by Dr. Cole nnd Dr. Dahl.  It is gen-
erally belSeved that the properties of phagocytability and pathogeni-
city in pneumococci are directly related. An attempt is being made
to determine whether this is accurately true and if so whether a phago-
cytic test can be devised that &I permit measurements of pat'nogeni-
city.
     Changes in pathogenicity brought about by various procedures
are being investigated.  Dr. Dahl has studied further the changes 12
agglutinability md pathogenicity, brought about by growth irL irinnze
serum (first studied by Miss Stryker) and also by growth in gradxaI.l;-
increasing concentration of bile. k$ prolonged growth over rcmy gene-
rations it has been found t-hat pile fast strains may be obtained r:hich

                                       61 Q
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                                                     118
are aqj.ruJ.ent and which do not `oecome vi~"VlS!t eV3Il LftSr =fip t~`~fi;--
fers through animals.   This work is being ccntinued.
Studies on- the Xology or' tine Pneunococcns.
                     ,
     `pwo n&n lines of investigztioil hsve bean followsd: one
dealing with cermln physiological processes of the bacterial Cell;
the other with the immune-chemistr,- of the cell constituents.
peroxj.de Form;ttion brr Dacterin and the Effect of Vegetable Oxi&ses
                                            --.
gn 3% terial Grcmrtt\,.
Dr. Avery and Dr. Morgan.
      The studies have been the direct outcome of t:le earlter
observations on bacterial nutrition.  This work began with an analysts
of the accesaoI'y substances in blood essential for growth of the SO
called hemophillc bacilli.  This `knowledge has brought out cartair.
facts concerning the physiological activities of otE=er groups of bsc-
teria, particularly with referonce to the relation between grcnvth and
certain oxidative reactions of the cell.  In order to s?-ovt t-he contt-
nuity of the work and to correlate the facts recently acquired rtith
the observations previously reported, it seems desirable to review
briefly the study as a whole.
     Previous study of the growth requirements of 3. influenzae
h+s shown that the properties of blood upon which growth of this so
called hemoglobinophilic organism depends are related to two separable
and distinct substances.  The combined action of both of these substarc?
is essential, each being separably ineffective. On the basis of t%.l,*
relative resistance to heat these two factors can be differentiated ori'
from the other.  The so called I%" factor, which is associated ?vith the
pigment fraction of blood gives the peroxidase reac:ion and is destrocrn;
by moist heat at X3OtC.; while the second or so called V1 factor re-

sists boiling for short
t'ne hi$ler temperature.
perLo&, but is destroyed by exposure to
Because of 5his difference in susce$i-
bility to heat, blood can be deprived of the more labile substa:lce
(V) by autoclaving, and when so treated is no longer capable Of
supporting gro.vth cf B. Fnfluemae . Tine X substance under these
conditions, however, remains unAffected.  This Is proven by the
fact that the addition of boiled extract of yeast (V) can activate
nutoclaved blood (X) and render it again suitable for groivth. It
is interesting in this connection to note as IIop'kins, IdIeyerkof, nr.3
others have shown, that boiled extracts of plant and Animal tissues,
yeast and nn~scle, are of ph;wiologfcal inportnnce in Cell respirA-
tion.  This V substance which is capable of complemanting the X
factor of blood has been found in extracts of green vegetables Anl
yeast cells, and from the nature of its Action it IN;- be regarded
either as a coenzyme or as a vitamin-like substsncs.  The X fnctcr,
on the other hind, is found in hemoglobin And In certain of its de-
rivatives (hemin) and is always associated with the
the peroxidase reaction sri th benzidine .   Moreover,
is activs in such minute amounts as to suggest that
as a bio-catalyst.
fraction giving
the X substance
it functions
     Further study has confirmed the observation that the par-
ticular system represented by the combinatfon of the X and V factora
in blood has a complete analogue in the similar system of plant tis-
sue.  Unheated vegetable, for. instance potato, possesses substances
having the Ch!3racttWistics of both the X and V substances of blood.
Therefore, as might be expected on the basis of this analogy, veget-
able tissue can replace blood in the cultivation of g. $nfluenzas.

                                                         `j I?
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                                                   120
The mech%d.s,n ~3 the actjon of 71~~1~ tfe'lue In stimUlatin@; grmth
of the hemoph5li.c bacteria is not xholly clear; aralysis of the fac-
tors concerned, and the applzcat.!on of this tiovrletige to the growth
of other bacteria suggests that this mschsnism ap be related to the
physiological processes of cell respiration jn a ;;ray similar to that
of the substances described by Hopkins, idegerhof, and others.
    In discussing the growth stAndating action of plant tissue
upon bacteria, it is of interest to correlate these facts with the
effect of unheated vegetable tissue upon the growth of pneumococcus.
It is, of course, well recognized that if a fluid medium is inoculated
with an insufficient number of pneumococci little or no growth my
occur, although the culture fluid way be optima) for growth if larger
numbers of the organisms are introduced.  However, if to this medLUm
a piece of unheated potato is added, the minirmnn inoculum amply suf-
fices to initiate prompt and abundant growth. Furthermore. the pres-
ence of plant tissue in the bouillon effects a remarkable alteration
in the growth curve of pneumococcus.  The period of lag is eliminated,
the maxirmxn rate of cell division is proqtly initiated, and growth
reaohes $ts maximal development in the first eight hours of incubation.
Moreover, sensitive as pneumococcua ia to the iriitial reaction of the
medium, the Bone of optimal hydrogen ion concentration within which
growth ca.n be'iaitlatsd ia considerably extended both on the acid and
alkaline sides in broth containing unheated vegetable tissue (pabato~.
Vhile the seme mechanism which operates to facilitate growth of the
hemophilic bacilli in plant tissue medium may be involved in the growth,
acceleration of pneumococci, in the latter instance another fact has
been observed which may be of significance in eqlaining the action of
these accessory factors.  Whenever pneumococci are grown in bouillon

                             I@ ., q
to v;hich there is free access of air, hydrogen p?x.cx-Yde is foAmed ' ' T: :
in readily demons trable amount s  I&S pe;oxide is tcxic an1 cc-
cumulates in the culture fluid In co:~centrat:iorsw'.ziCt are Sacfer!.-
ocidal.  h the other hand, under id?ntii;al cond.itis:ls Of Oxygen
expc3'lre, hydrogen peroxide is not demo;?strable in cUltllr2S Of pneu-
mococci containing unheated Flar,t tissue (potato).   Tnc absence
of peroxide under :hese contitions is presmably due to the action
of the catalase and peroxidase 09 the potato which possess the Pro-
perty of decomposing this substance.  The presence of. t%ese enzymes,
therefore, p revents the accumulation of hydrcE;en peroxide in ,the CC+
dium.  The peroxidase in plant tissue is capable of actiag u?cn the
peroxide with the liberation of active oxygen which in turn an bring
about further oxidative reactions.  Plant tissue, therefore, :iay
exercise the dual function of providing aczessorU* growth factors, and
of destroying deleterious growth prodmts: perox.i<es.
    Finally, plant tissue makes possible the aerobic cultiva-
tion of arserobic bacteria,  The mec2-anlsm of aerobic ;groi-Jth of an-
serobic bacteria in aedia ccmtai,?iq ~ieses of unheated vegetable
SW?IIM to be related to the oxidation-reduction System present in plant
tissue.  The study of the formation of peroxide by pnemococci on ex-
posure to air and of the absence of this product when the organisms
are grown anaerobically suggested~the following hypot'aesis which finds
some justification in the facts presented.  Although no experimental
proof is yet available, it seems not unlj.kelg that anaero'oic organis:Ls
fail to groT4 in the presence of air net because atmospheric oxygen
as such .is a direct poison to the cell, but because of t-ke toxic
peroxides which are produced whenever the oxygen of the air combines
with autoxidizable substances of the cell.  Tnis process of auto-
XidatiOn gives rise finally to the formation of hy&:ogen Peroxide,
.._ .__.~_

which is known to be poisonous ta t>e `living proco$csr.~. Sines these
peroxides are formed only by the notjo3 of malesular oxygen on some
autoxidizable substance of t,he cell, the abserce of air precludes their
fomtion.  Under anaerobic conditions, therefore, the bacteria are
not exposed to the injurious action of the prod-lets cf autcxidation.
If this assumption is correct, t'nen the aerobic grazth of obldgsrte
anaerobcs in the presence of plant tissue finds partial eqlanation at
least in the fact that the peroxide forme b;- autoxidation is rqidly
broken up by the oxidizing enzymes of the plant fiarue. Under fher,e
cultmal conditions the sensitive cell is protected as ef;ectivelg
as if it were growing under anaerobic conditions .
StXldi0S
c;n.
        0 idatio
Dr. Avery and Dr. hTeil1.
     The fact that peroxide is forme?, and accumulates in reaiil;-
demonstrable amounts in cultural fluids in 3hich pneumococcus is gro;;-
ing, hae led to a further study of certain oxidation-rcducticn phenomc::::
of the pneumococcus cell.  To obtain exact havledge of these phenomena
unccnnplicated by the presence of cell grovJth, sterile extracts of pneu-
mococci have been employed in these studies.  In order to minimize the
accumulation of oxidation products,  the bacterial extracts tvere made
.
from suspensions of anaerobically grown cells, cart) baing taken to
exclude the action of molecular oxygen during the processes `of ex-
traction.  These sterile exfracts, prepared by repeated freszing and
thawing of pneumococci suspended in broth or phos+te solution;,
contain those active intracellular substances ?phich are ireed by dis-
ruption of the cell membrane, and which remain in the extraction
fluid after -prolonged centriiugation and Berkeleld filtration. `P.-r0
types of cell extracts *have been erqloged; one t:rpe prepared fror?,

umrashed bacteria concentrated and extracted directly in small amounts
Of the supernatant broth in which they had previously grown; another
type prepared from washed cells concentrated and extracted in phosphate
solutions of known reaction and salt content.  The sterile, cell-free
extracts of unwsshed pnwmococci represent what may be termed a "Ia,
pie te" oxidation-reduction system.   Extracts of this type are potent
and exhibit the follorling reactions of oxidation and reduction: 1. n-
pid formation of peroxide upon exposure to molecular oxygen; 2.  active
reduction of methylene blue; 3. consumption of dissolved, moleculq
oxygen1 . 4. destruction (oxidation) of pneurnococcus hemolssir.; 5. de-
struction of oxyhemoglobin (methemoglobin formation.)   On the other
hand, the s3cond type of cell extract, prepared from washed PneumocZi
suspended in phosphate solutions, is by itself wholly inactive, and
owing to the absence of activating substances, is incapable of bringing
about any of the osidation reactions characteristic of the extracts
of uxnvashed cells.  The "incompleteR system of this type of extract,
however, can be reactivated by the additi.on of boiled yeast extract,
muscle infusion, or the cell washings.  Then complemented in this man-
ner the extracts from washed batten `a also exhibit all the fzncticns COQ-
mon to the potent extracts from unwashed cells. Both yeast extract and
muscle infusion are known sources of a&oxidizable substance, and `it seem
probable that these are the constituents concerned in the %ompletionv
of the system.  The actual source of the activat5.n.g substances present
in the cell washings hai not been determined.  It my be that activa-
tion in this instance is due to the presence of traces of broth, Or of
certain autoxidizable substances of celi itself which have been re-
moved by washing.
In addition to other active intracellular agents, both t;-Ps
--

                                                                         --.
                                                                         2%
of extract contain the concentrated endo-hemolysin described in 1314
by Dr. Cole.  This hemolysin is exceedingly unstable and h&s been con-
sidered one of the most labile substances of ?neurnococcus origin.
In view of tte present work, it seems probable t-hat in many instances
this instability is due to oxidation.  For the hemolysic in both types
of extract has been found fairly stable if preserved under anaerobic
conditions .  However, if the type of extract containing the %ompletel'
oxidation-reduction system is exposed to molecular oxygen, the hemoly-
sin is very mpidly destroyed.  On the other hand, the henolysin m2y be
exposed to the scLIile oxygezr tension in the 9ncompleten or washed cell
extract with comparatively little effect.  In other words, aolecular
oxygex? and constituents of the washed pne-snor=occus cell cannot oxidize
the hemolysin molec=rle tc ncn-kemolytfc products in the absence of
these autolizahle subskncos which !-EL-IS been fa=md esser.tial in the
other oxidation and reduction activ:ties of sterile cell extracts.
     The destIvction of oxyhemoglobin (formation of me themoglobinj ,
one of the moat prominent characteristics of the living pne-~OCOCCUS
cell, is also an exceedingly active property of these sterile extracts.
Qusntitative measurements show that the hemoglobin destruction effected
by the reduced sterile extract is ach greater than Chat reported in
earlier experiments in which no attempt was made to prevent oxidation
of the pneumococcus cell pre;?arations.  In these studies it has been
found that as much as SO0 mg. of Hb@z may be destroyed by the addition of
O-5 cc. 0: the reduced extract to 4 cc. of blood. Here again, as in the
case 02 hemoglysin oxidation, a*Aoxidizable substances seem to be re-  7
         ----                                                             \
quired, as the constituents of the washed cell in the presence of mo-
lecular oxygen cannot convert oxyIhe~.~o,globin to methe~oglobin.  Tnrou&-
out the entire investigation there have 3een evident many close analogies

                                  .
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                                                   125
between the systems in the sterile extracts responsible for paroxide
production, me thylene blue red-action, and oxygen consuzption, End
those responsible for the oxidation of hemolysin and the destruction
of oxyhemoglobin (methemoglobin fonc3tion).  In fact, it seems likely
that all of these activities may be functions of the same, or quite
similar systems, not only in the sterile cell extract, but In the
living pneumococcus cell itself.
     These results are now in manuscript form, to be published
under the follov;iIIP; titles: Y&dies of Oxidation and Reduction by
Fn8umococcus":
    I.  The production of peroxide by anaerobically grcnvn pneumo-
      coccus cells under conditions not permitting active growth.
    II.  The production of peroxide by sterile extracts of pneumococcus.
  1x1. The reduction of m8thyl8ne blue by starile extracts of pneu-
            mococcus.
   IV.  The oxidation of hemolysin in sterile cell extracts of pneu-
            mococcus.
    V.  The destruction of oxyhemoglobin (nethemoglobin formation) by
       sterile extracts of pneumococcus.
The Soluble Snecific Substance of &l8umococcus.
Dr. Heidelberger and Dr. Aver>?.
     At the time gf filing the last report, prification of the
SO Called "soluble specific substance~l of Type II Pneumococcus had been
carried to a point at which the best preparation showed a nitrogen
content of 2 per cent, yield86 49 per cent of reducing sugars when sub-
jet ted to acid hydrolysis, and precipitated immune serum when present
at a dilution as high as ~:2,5OO,OOO.   The fact that the highest .
dilution reacting with immune serum also gave the Molisch test for
carbohydrates, taken together with the increase in optical rotation
and diminution of nitrogen content as successive preparation increased

in activity, was taken to indicate the probable carbohydrate nature
of the soluble substance itself.  Subsequent work has strengthened
thi3 viewpoint, an3. the data obtained have been published in the
Journal of Experimental. LIedicine for July, 1923.
      Refinements in the method of purification have led to
preparations more active than those previously reported upon. Alcohol
is substituted for acetone in the preparation of the concentrate from
broth cul turea of pneumococci , and the precipitate is centrifuged at
hi&h nysi. P. sspar:rt.tor. info t?res lcqers reeillts. Gf tteoo ti;e
top and bottom W$WS contain so Z.ittle active material t!mt they
can be discarded, and with thorn is eiiminated in the very first pre-.
cipitation, S!E greater par-S of the inactive substance in the bxth.
The r.ii d(fie hyd? , c", &pm?y cake, ccntains tL6 soluble specific i;ub-.
stmce, and is purified OS already described.  It has been found ad-
vaZItag8ous to add a final, third precipitation with solid arrrnonim
sulfate.
      A comparison is given of Preparations 17 and 21, which have
not yet been IWpOrt8d.  The fomer was obtained by the older method of
pxxrification, the latter by the improved process.
No.
17
21
It will thus be seen that in the more active preparation the
percentage of nitrogen has suxik to scarcely more than a tracer  The
percentage of the reducing sugars found on hydrolysis did not, however,

show an increase, and this will be investigated further as it mrSy
be due either to fnilcre to  reali-, e the optimum condition for the
hydrolysis, CT to the presence In the material of a non-polysac-
c-haride portion, either as an iqurity, or os a constituent of the
active mterirsl itself.
      Tne percentages of carbon 2nd hydrogen foxnd are not far
from the theoreticcl values for a poiysaccharide composed of hexose
units ) C, 44.4 per cant, R 6.2
the greater Fart of the active
purity is built. up is actually
the isolation of this sugar as
F;'I= Cent.  That the hexose from which
subst;:rGe in its preser.t state of
glu.co;;e `has been definitely shov;n by
the 0sazor.e frm the products of hy-
drolysis of 0.4 gm. of Prepzratlo:l 17.   3.063 gn. of purified
osazone were obtairied and identified as ElucosazJne both by the
melting ;3oixt and optical rotation.
`Gk%le it thus appsara that the soluble specific subst.ance
is actually a polysacchsrlde or glncoside, a@ not. merely asmciated
with a s*lbs tance of this type, the matter is lq? no means proved, and
three lines of investigation are now under way in the hope of de-
ciding the question and thus rendering a true contribution to the
chemical nature of bacterial specificity. These lines of work are:
    1.  Further attempts to purify the soluble substance by ch8micGl
and physical means.
   2.  Attqts to attack the carbohydrates by means of enzymes,
molds, and bacteria, and investigation of the fate of the soluble
substance Under th8S8 circumstances,
    3.  Pracipitation of the soluble SUbStanC8 with immune serum
and its recovery from the precipitate.
A comparison of the solublie specific substance of Types II
and III is now being made.

I28
Xature of the Alterations in Respiration tntieumonia.
Dr. Binger and Dr. Brow.
The NW Ox- Chamber.
     Building of the
the Sumner months - as it
during the busy period of
new Oxygen Chamber was not begun until
~3s not thought desirable to start work
the Hospital year.  The n&v chamber is
now completed - except for a few minor details.   It htLS not y8t
been tested, but should be entirely ready at the ConmTIencement of
the pn8UmOnia seasonr  The chamber hae been constructed with par-
ticular regard to safety from fire risk, tightness a.nd simplicity
and econcmy of operation.  To secure all these factors a considerable
initial outlay hae been necessitated.
    It is planned to use the chamber for the present for stud!*-
ing and treating cases of pneumonia. Of particular interest will
be an irmesti@tion of external and internal respirrstion in response
to oxygen therapy.  It is planned.to study the tension at which oxygen
exists in the arterial blood as well as the oxygen unsaturation.  The
general scheme will be to continU8 to look for physiological evidence
for
the
and
the efficacy or lack of efficacy of oxygen in pneumonia, to learn
d&age required and the optimum time for administering the drug
t0 E&CCUmUlate 8tati8tiCal evidence.
                     ,
The Lunn Yolume In Lobar Pneumonia.
Dr. Binger and Dr. Brow have completed a study of alteratiprts
in lung volwme in 10 cases of labar pneumonia. The mmuscript deezti-
bing the work has b88n submitted f9r publication to Yce Journal of
Experimental Medicine, they have measured the so called functional
resid'l air in a seri,ea of no-1 individuals and in IA-& Pneumonia
patients at different stages of the disease. This luag volum8 repre-

sents the volume of air enclosed in the lungs at the end of normal
sponbneow unforced sxpiration.  There were several reasons for
stuilying this prticulnr lung volume.  First, it could be determined
with no further cooperation on the part of sick ptients thar. simply
breathing through 2 rubber mouth piece with the nose Clipped. SedOnjly,
it obviated thB adventitious and unconttollsble circumctXc8S arising
from pleuritic pain, muscular weakness and psychic state when the me-
thods employed aim at d8teIlIIining the total lung czpacity I:vital capcG
city and residurrl airI , this method requiring forced rcxcim inspira-
tion or expiration.  Thirdlk, the functional residual air represents
the volume of air actually functioning in respiratory exchange and as
such mast bear a close relationshi? to the area of pulmonary opitheliu-
through which diffusion oi gases occurs.  It was soon learned that
fluctu2tioh.s in the volume of the functional r8SidUal air correspond
to alterations in the size of the functionally active lung as S~WJII by
radiographic pictures and by physical signs, and those fluctuatidi-.s oay
W811 therefore be interpreted as evidence of consolidation and reso?u-
tion.  For this reason the changes occurring at crisis were of especial
interest.  The general belief among clinical observers has been that
Crisis ifs not acco%npanied'by Stnnediate changes in the pulmonary lesion,
or, at least, that there is no prompt Wearing up" of the consolikted
..,
area. hdeed, one o? the striking c&racteristics of the change occur-
ring at this period has been considered to be the great decrease in
respiratory rate without any obvious change in the local lesion. It
has been faund, however, that drop in temper&are, cardiac and respfra-
tory rate is S'lwags accompanied by beginning increase in the volume of J
functional residual air.  hrthe?nom, it has been observed that in those
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                                               13c
cases in which fever, accelerated hevt rate, and `rapid braathing
is sustained there is prograssive decrease in the volume of the
functional residuai air.  A clJse coincidence has been observed
between these changes in symptmtology, lung volume, and the pres-
ence and disappearance of cyanosis.  The relationship discovered
is one of coincidence fn time and cannot serve as an explanatfon
of the mechanism of the 33-qtm5 irnoive&, thou@ it is conceimble
that the characteristic rapid and etillow breathing in pnuumonis
results from the heightened metabolic rate of the febrile state,
combined with a diminished diffusion area for gas exchange.  Th3t
the breathing of these patients is actually rapid and shallow has
been quantitatively established by many observations on respiratory
rate, tidal air, `and minute volume recorded by a graphic method.
The result of an au;gmented rate and Q diminished tidal air is usually
an increase in the minute volume of pulmonary ventilation.
&id Eese E;ruilibrium ir the Blood in Pneumonia.
This increase in minute vo'lume which oeours may explain the
loam CO; tension in the arterial blood of pneumonia patients durira
the febrile period which has been found to be present by Dr. Binger
in collaboration with Doctors Hastings, Nell1 and Morgan.
    In spite of the diminished tension of the CO2 in the ar-
terial blood of febrile pneumonia patients, no true acidosis occurs
in the sense of a change in pi3 4.  The hydrogen ion concentration is
maintained at its normal level end so we cannot expla3.n hy-perventik-
tion as the result c2 HC ions on the respiratory center. Indeed a
normal pH indicates a functionally efficient reqiratory center.
This function&l efficiency does not, however, signify the
fact that the res-&ratory center is not approaching fatime and ex-

\:. i
   , ,. ;
    .
       ?.3i
haustion, nor that the organism degs not eventually die from bi-nak
down of the respiratcry mechanism, b3 which we include both t1:c central
nervous and pulmcr~ry apparatus _
      Indeed, there is evidence v;hich points to just such an
eventual1 t:r.  The work of Neuburg, Means and Porter reveals the fact
that dogs with lobar pneumonia are unable to respond characteristically
to the a timulue of CO2 after they have suffered from the dyspnea of
experimental pneumonia.  Such dyspnea can be prevented by sectioning
the vngus nerves or blocking them with novocaine. Under these cir-
cumstances dyspnsa does not supervene and the respiratory respcnse
to a CO2 stimulus is a normal one.
Nervous h?echanfsm of Dysnnea.
    Dr. Binger and Dr. Brow are continuing studies in this
direction and have started an inquiry into the r~ture of the vagal
stimuli arising in the lungs, their origin and frequency, and the
means of blocking them.  The present working bypcthesis is that le-
sions of the Pulmonary capillaries are responsible for the stimuli.
    They have conducted a series of preliminary experiments
in dogs on the effect of acid inSection into the lung before and after
the va&otomy and a study of the resulting tachyPnea. The first Pro-
blem to be solved was to find a suitable anaast'netic - one which WO~AY
keep the aniroal
woUld not block
atten%ion.  All
in a relaxed a.@ insensitive state and still one which
the very impulees to which they were directing their
the -WUJ.~ volatile and non-volatile anesthetics wed
for laboratory animals failed.  They finally succeeded fn ackLieving
the necessary conditions by the use of Luminal given in small doses
by stomach tube.
Further work on this problem is in progress and together
. . - -.-._,

                                                                   L b . `i -1
                                                   ? !, 1 ~,
    .                                                    132
with the studies in the ox;rgen c'hambsr will occupy the major portion
of Dr. Binger's and Dr. Brow's time for the ensuing year.
studies on Chicken-pox.
Dr. Rivers 3rd Dr. Tillett.
      In October of 1922 we began our study of the contagious
diseases which occur most comonly in children.  This study was under-
taken in the hope of making observations Which would add to the general
knowledge of these diseases and which might eventually help in their
provention or cure.  Chicken-pox Is usurrlly considered a mild disorder,
but, nevertheless, for xmny reasons, it appeared to be a very suitable
one on which to begin a study of this group of diseases.
     The results of our year's work on the problem chosen for
study will be given under five headings:-
    A .-  Study of vaccinln in rabbits.
    B ."  Clinical study of cases or^ chicken-pox in the Hospital.
    C .-  Results of attempts to transmit chicken-pox to animals.
    D .- The effect of certain physical and chemical agents upon the
       virus recovered from varicella patients.
    E .-  Study of the immunity produced by the virus recovered from
       varicella patients .
          A.- Study of Vaccinia in Rabbits.
     VJe began immediately to work with vaccinia in order to learn
the proper methods of handling a virus which might be supposed to be
s-hat similar to the one causing chicken-pox.  This seems to have
been a wise step as we feel that the success we have had in our work
with varicella is due to t'ho knowledge acquired in this way.  We were
able to acquaint ourselves with the methods of studying the lesions
in the eye, skin and testicles when thase organisms were inoculated
with a virus.  Furthermore, it VELS Fossible. for us to confirm the

133
important observations G;` Calmette ant{ Gcerin nnd of Ohtawara. The
former workers demoLstratcd that vaccine virus injected intravenously
loccliz.es in the irri:nt.zd SkiA.  'ihe latter observer recovered vac-
cine virus from t:?e blood of rabbits previously vaccinated on the skin,
by injecting the blood into the testicles of normal rabbits and after
4 to 6 days removing the testicles, grinding them up, and testing for
the presence of the virus by inoculating the testicular emulsion on
the skin of other normal rabbits.   These observatioss hav\, a direc2
bonring upon our work in transmitting chicken-pox to rabbits and their
lmportancc will be seen in that part of our report.
B .- Clinical Stud-7 of Cases of Chicken-pox in the Hospital.
During the year 51 patients with chicken-pox were cared for
in the Hospital.  T?e encinu-&ered no instance of herpes zoster and
varicella occurring in the same fCy.;ily at or about the same time.
The blood picture was follunred in most of the patients. In the severe
cases a leukupenia was observed early in the disease but there was no
marked secondary leukocytosis similar to that occurring in smallpox.
`Phe majority of the p?,tients developed an eosinophilia during coma-
leacence.  No allergic rdsponse was demonstrated in patidnts recently
recovered f rem varfcella when fresh vesicle f kid was employed.  Vesicle
fluid was collected f mm `lesions at different stages of developznent
and a cytological study was made in fresh unstained specimens, in fresh
specimens stained with different vital *es and fat stains, and in
specimens f Axed and stained in many ways.  %a11 pieces of skin were
removed at various stages in the development of macules, papules and
vesicles, fixed, stained and studied in various ways.
Certain clinical observations made during the year helped us

                                                               a:,' 1
                                                   PZ.j 1
                                                   134
a great deal as they afforded indirect evidence that the virus is in
the blood of chicken-pox patients and that the amount there does not
uecessarily correspond to the number of lesions in the skin.  Fur the'&
more, they foc;rssed our attention upon the blood stream as a suitable
Place to recover the virus from patients. It was noticed that irri-
tation seemed to have a direct influence on the localization of the
vinrS in the skin, as evidenced by the appearance of an unusual
number of vesicles at the site of irritation.  The pit ture was parti-
cularly striking when the irritation Involved the skin of the face
and extremities, parts of the body usually least affected by the erup-
tion of chickerr-pox.  This effect of irritation on the lozalizaticn
of the virus in the skin was observed in 5 of 51 patjents.
        C .- Results of Attests to Transmit ChScken-dcx to Animals.
    At Pir'st our attearptti to transmit vzricella to Tnimals were
unaucceesful .  We had no definite idea of how to proceed other than
to inoculate different laboratory animals in various way$ with mate:is!.
collected from as mar@ geurces as poseible from chicken-@% patients.
It is unneccesaary to give the details of these negative results. Later,
hcywever, after oGti,&%t6nti& haa b&m f ocussed upon the .blood of patients
                    :
as a 8-e f&m which tb Wu&e~ 6 virus of Qariceih and after we
found b&t vaccine virus can be ddmonstrated in the bhd of tmzdinatsd
ratbits by injecting their blood into the testiclei' of n6r1.1~1 rabbits,
the work became more orderly and has yielded definst6 results.  It
mi&t be ~0x1 to give in detail tLe method emplb;jfeh and a descriptior!
of the results obtaitied.
   1, Method used in recovering a virus..from Varicelk 3tientS.
      Blood was dram from the patients usually during the first
24 hours after the appearbme of the eruption.  The blood was not Ci-

135
trated and before clotting occurred was injected in 2 cc. amounts
into each testfcle of no-1 rabbits (2000 gn.).   Large quantities
of blood were used intentionally. At the t!me of inoculation the
needle was Moved around to produce a certain amount of trauma.  Four
&ys later the testicles were removed, groad ap thoroughly with
sterile, chemically clean sand and mixed with 10 cc. of physiological
salt solution.  The mixture was allowed to stand until the sand set-
tled to the bottom.  Strict asepsis was observed throughout the work.
Portions of the testicular emulaioa were tested for the presence of '
ordinary bacteria by means of cultures on blmd agar, in broth and
in Saith-Noguohi tubes.  Other portions for future use were stored
on ice either in the original state or after the addition of equal
quantities of glycerine.  Then 1 cc. of the emuJ.sion was injected
into each testicle of normal rabbits. !I'wo plz~es in the rabbitst skin
were shaved and scarif ied.  One of the areas was smeared with the
emulsion, the other was used as a tiontrol. An eye of each rabbit
was also inoculated.  Both corneas were scarif ied with a cataract knife;
one was inoculated with the testicular emulsion, the other was used aa
a control.  The skin and cornea of each rabbit were inoculated to test
for scum visible r9etion of a virus that might be present in the ma-
terial injected into the testicles. The first few animals in each
series showed very little reaction other than that which might be ex-
pected to follow the trauma of Inoculation.  !&e skin and corneas healed
rapidly.  The scrotum was edermtous at times for 24 to, 48 hours,  The
tesficlles when removed, were slightly swollen. &crotic areas and oftsti
remains of the material injected studded the testicles in various places.
!&his reactionwas no more striking than that cmsed by the
of an eurtalsion of normal testicles.  The first few rabbits
injection
in each series

136
and all the rabbits in the serits f ram which no V&F.LS was recovered
served as excellent controls for the work.  After 4 to 8 passages,
however, in certain series, reactions were noticed ir. the inoculated
eyes, skin 2nd testicles which had not been seen in the enrlior ani-
mals and which did not occur In the controls.  A description of these
reactions will be given a littie irrter fn
     Using the method outlined above
f rorn 5 of li chicken-pox patients.  Three
which is
Faith the
rerained
    2.
(a)
now in the 45th generation, have
the report .
a virus has been recovered
of these otrains, one of
beon studied carefull;!.
exception of an occasional contamination all the cultures
free of ordinary bacteria
Deacriotlon of the reactions in rabbits inocuL?tec? with the
            a.
&Q:  Scarif ied corneas inoculated ivitt the virus shovzed
a reaction which was not present in the co:ltrols.  This reaction ~2s not
so severe as that caused by vaccine virus or the virus of herpes. It
appeared in 3 or 4 dnf~s and persisted 4 to 5 da;-s.  The cornea became
rough and opaqm, especially along the linee of scarification.. There
was increased lacrimation, photophobia and injection of the blood ves-
sels around the cornea. `Phe lesiona healed rapidly without permanent
WUti to the eye unless there was a secondary infection. A reaction
in the eye occurred less constantly than in the skin.  In sect ions
of the inoculated corneas, there was found alon= t!le lines of scari-
fication a type of degeneration involving the nucleus Ftnd the c$c-
plasm of the cells similar to the cellular changes f owd In :hickei+rs::
lesions in the human skin.  Occasionally small vesiclas were seen ir.
the thick layer of epithelial cells filling the defect in the comaa

                                                               P?, -'
                                                    r;*;i
                                                 139
produced by the scarification.
   (b)   5kk  Discrete l.esion:, f~llo'::~ at; tilt= inoculation Of
the: virus on the scarified skin, were smsl`l, supc:ficial, red pqluics
which might easily be overlooked.  Lhen th.2 ?;`-I 1s was concentrated
enough to cause confluent lesions alor thd eAzbrifications, however,
the reaction could hclrdly be missed.  The erythema caused by ths
scarifications in the control  ski n dioaTpezreT by the 3rd or 4th day
at least, while in the inoculnted skin the erjrthzra nnJ welling oi'
the tissues becamz more evident zt this time, lasted 3 to 6 tiys longer.
and then rapidly disappeared without leaving any scars.
     The intracutaneous inoculation of tiB virus g"vZ more ConstXit
results than smearing it on the scarified skin, especislly *Aen the
virus was dilute.  !Ihe virus for intracutaneous inoculation q.xs cenfi-
fuged and 0.1 cc. of the supernatant fluid was injected into the s:ci:;
by means of a tuberculin syringe.  The effects frcm the tram-a of in-
oculation usuGly disappeared within 48 hours and nothing further v&s
seen when the tests werd negative.  Vhen the tests were positive,
however, an area of the skin often larger than a silver dollar around
the point of inoculation became red and slight'& edematous. This fe-
action persisted 4 to `7 days and then disappeared without leaving any
scar.  Emulsions of normal testicles inoculated into the skin in a
similar manner gave no such reactions.
     Histological examinations of skin showing the different
types of lesions revealed a cellular infiltration of the corium, a
swelling and thickening of the epidermis and certain intracellular
changes similar to the ones seen in the peqular lesions of human chicken.
pox.

                                                 133
   (cl Tes'ticles : me pathological :hsnpes iii the testicles vfefe
dlsmgarded at first because of the nmcunt of Dteria!. injected.
Large qwtities wsre necessary to ad;zpt ths virus to m-hds. hteyt
ho.veve r a ;vhen the V:I-U; was more concent??.t3d., srf.di qU3ntitieS of
the supernatrrnt fluid from thoroughly centrifuged testicular emulsions
were injected.  Evdn after these small injections the testicles be-
came swollelt and tense in 3 to 4 days.  `Ihen the testicles were re-
moved for exnmination or for passages to other animls , they were
red, swollen, or edemtous with whitish areas ScAtterGd over the
surf ace.  Upon sectioning them the swollen condition was more evi-
dent as the parenchynu bulged out from the restraining capsule. This
reaction is similar to that caused by vaccine virus.
   (d) General reaction following intratesticular incculations
pf the virus: After the virus became adapted to rabbits, we often
observed a general reaction ih the animls in addition to the local
ones in the eye,  skin and testicles.  The rabbits locked sick, re-
fused to eat, lost weight, occasionally developed a diarrhea and of-
ten had a temperature of lW" to 507".  The animals did not always
have fever even when good local reactions occurred in the eye, skin
and testicles.  On the other hand, in some of the animals which had
high tearperatures, discrete lesions appeared in the control areas of
the skin or in the shaved skin of `rabbits inoculated only In the testi-
cles.  The lesions, red macules and papules of various sizes, a.p$aared
5 to `11 days after the inoculation, remained 3 to `I days, ajad. th&j
disappeared without scar ring.  Frequently a zone of erythema less
intense than that of the papules surrounded ea& lesion. These lesions
Se-d t0 indicate that some of the virus in the testicles `had bvzded

the blood stream and had localized in the irritated skin.  The da-
monstration of this invasim of the blood stream was accomplished by
removing blood fran the hearts of some of the rabbits and injecting
it into the testicies of normal rabbits.  Four days later an emulsirr:
of these testicles was tested on the skin of normal rabbits for tha
presence of the virus.
  (e) Effects of intranasal. intratracheal, intzavsno-a. and intm-
cerebral inoculationsof the virus:   No reactions have been observed
following the intranaaal, intravenous, and intratracheal inoculations
of the virus.  The animals Khich were inoculated intracerebrally were
sick and had hi& tmpratures for 5 or 6 days.  None of them died,
ha-lever.  These experiments are still in progess and a fir-al report
can not be given at this time, but it seems that although no siss
of infection follov: inoctiation by the four modes just mentioned
these inoculations Sive rise to an immune state.  We feel that in each
instance a true infection was set up by the inoculation of the virus
even though we were unable to detect evidences of it.
D,- EYfect of Certain Phswical and Chemical Agents Upon the
             YY..
1.  Ph~~~iolo~lcal salt solution:  Virus preserved on ice in
physiological salt solution deteriorates mpidl:i and after 4 days
ceases to give a visible
It is tiable under these
and can be recovered and
testicles of rabbits.
reaction when injected intracutanebusly.
conditions, however, at least up to 6 days
conuentrated by several passages through
   2.   Glycerine:  Virus preserved on ice in 50
also deteriorates rapidly. But although the virus
per cent glycerine
preserved in this
way produces no visible reaction when injected intracutaneously, it

is f reqndntly
thxt it still
p3sstLgos through ntimc~ls. To rccovcrod the virus from giyccrinc 4
times, once oath sftcr it hnd been in thz glycsrinc 7, 17, 29 and 33
days.
   3. Hcrr_t: I7c hsvc cvidcnca that the virus is very scnsitivc
to heat; sn cxposurc of 10 min. to o tcmp-raturc of 55OC. kills the
T<CUS, while on3 of 30 min. to CL tcmpcraturo of 45OC. is not sufficient
to dostroy it.
    4. Fj.ltratioq: We feel fairly certain that the virus can pass
an Ii filter in tho majority of instnnccs. `ilator suction wac used.
`%o filtration was not pomittcd to go on longer than 33 rr,in.   The
filters did net allow inf',uonzo bztll~i to 2x5 thro-ugh under thcsc
conditions. 'I213 virus has not bcon rccovcrcd from every filtmto,
and, when it wa&possiblc to demonstrate the prcecncc of the virus
in the filfzrcd-mstcriol, it was many times more dilute than in the
unfiltcrod.
I2 .-
The
Studv of tho Imrmiitv ?roduced by the TJirus.
time of appearance of on irrnunity to eny of the so called
filterable viruses probably varies inversely with the zunount of virus
in the immnizing dose. There are probably xany other factors which
also influence the time et which the ixnm~ity appears, such as the con-
dition of the virus snd the point of inoculation. As we had no wzy of
determining the xt,ml omount of virus used in our work we arbitrariQ
chose to test for tke presence of an immunity 2 to 3 woaks xftsr the
irtxm.nizing dose. A fresh virus well controlled wcs always used in
these tos ts .  In this way we were q.bl+ a to kaep at least two factors
fairly constmt. In these cxperimentsb the virus recovered from vard-
----

cella patients was ZllWClj~S inject?& intracutxxouaiy in amaints of
0.1 to 0.2 cc., and, the vaccine cirus was smcsred on the scarified
skin.  The iranuniziq strain was inoculated on one side of the aniW1
and tha test strzin on the other in order to be certain the anirrz~l
was irrmme to the former.
     Our work on tho mty produced by the vies recovered
from mricella patients can bc suunrarizcd as follows:
   1.  Three strains of the virus isolated from varicella patients
have been studied and each gives carrplete ;imrmnity to the other in
rabbito.  From this wo conclude they aro identical.
    2.   Rabbits immune to the virus from varicelln patients are
not immune to vaccine virzls and vice versa.
   3.   Rabbits can be ianzunized
cella patients into tie skin, into
into the veins.
   4.  Old glycerinated viruses
mu7lize animals.
by injecting the virus from vari-
the testicles, into the brain, or
which are still viable will im-
5.  Virus killed by heat does not immunize animals.
6.   Normal rabbits become ~XXUXX within 2 weeks after they re-
ceived axall quantities, 2 to 4 cc. of fresh blood from the hearts of
rabbits which had been inoculated intratesticutirly 4 day! previously
with the virus f tan vnricella patients.   This was considered active
immunization.
   7.  We were unrtble to demonstrate any passive imity to the
virus in rabbits which 24 hours -previously had received intr+Wenously
5 to 10 cc. of serum frca robbits irmrune to the virus, or had received
5 to 10 cc. of senam from convalescent chicken-pox patients.

                                                                           *' 4
                                                      P ,?. ; #I
                                                   142
    0.   Serum lrom immcL?e r.xbbits neuirniizcs the virus when it
is mixed :vith the V~YIIS and lcf t at 37OC. for 24 hours in the pres-
ence of compleme>:.  I~orm.zl rabbit serum Land norrxl huTan sem do
not xutralize the vir.x.  Strum collected from convalescent chickell-
pox patients nay have a little neutralizing power, but, because of
technical diff icultics 0~5r-q to the numerous titrztiono and controls
which we tXnk necconr.qr, WC r'oel a little hacitxcy in m-king rLny
definite statement about theso roaults.
      T7e believe that we are working with a virus .  Rurthexmore ,
wo are 30 certain as it is possible for us to be that the virus wa8
recovered from chicken-_nox patients under tha conditions outiinod.
It is not the vines of vaccinh or smallpox. VTnile the virus prcduces
lesions in robbits very much like those of hunnn chickox+pox, wo hove
not shown experimcnt~lly hhat tho virus is the etislogicnl agent of
chickon-pox.  As soon as we can get some fresh cases of chicken-pox
we will attempt the active ixnnunization of a number of rabbits by
injecting into them the blood of patients early in the disease. If
after 2 to 3 weeks these rabbits are iunnrne to cur virus we will be
warranted in ass-zning that me have been working with the etiological
agent of mricella and, furthermore, we will be warranted in using
the vines as o prophylactic mmsure against chicken-pox in children.
&g.z,di,es on Physiolo~~r and Patholos~~ of the Circulation.
Dr. Cohn, Dr. Mu.r~~y and Dr. StervcLrt .
Report by Dr. Cohn.
     In this lobcratory we have, during the past year, carried
on work in two dire&ions, first with tissue cultures mde of t'ne hemte
of chicken embryos t rrit'n the view to studying the behavior of heart

                                                          +\ I .)
                                                                         ` !*:
                                                  143
muscle cells or fibers; nnd second, 13th dogs, with the view to
studying the reaction of the muscle tissue of the heart in them to
certain injuries.
     The work on the hearte of chicken embryos was carried on
with Dr. Murray and with the assistance of Miss E\osenthnl. The work
was undert&en because it seemed essential from the point of view
of th$ study of disease, to turn to nn exdnation of the function of
contraction of heart rruscle.  In the paat twenty years, this study,
while not neglected, took h place second in interest to the analysis
of other functions 0;" muscle, nmely, those which involve the ir-
regularities to whit-n the heart is eubjact . This study was rewarded
with a large increase of success, but failad, I.think, to throw light
on tho nature of the processes which are involved when the heart
~t~~lly begins to fail.  There are several directions from which
ixrzestlgation of this problem may be appro%hed. For the time being
we have elected ta study it frcnn the point of view of the ageing of
such tissue.  We have chosen the hearts of chickan embryos bscau5e
this prq&zQtion can be very accuratb3y controlled oxperimentilly.
   Wa famd bzfore long that it WCS recessary to be pWticuL?.r
                                                     .
in the eggs we used. mith the cooperation of Mr. J. G. Webb, St be-
came possible for us to obta%n egga f ram CL r.ingle strain of hens Of
kmmn age; we know when the eggs were 1s;r.d and they are sent to US
daily under fairly uniform coaStions.  This McKee it possible to be
almost certain of the age of the eznbryos with which we experiment.
In order to have a measure with which to corqnre other fUn&ioIlS We
studied embryos of vtzriaus ages to 1era-r whether the tissues at sue--
ceeding ages shcrwed differences in their ability to grow.  After 3naking
CL mmber of attempts, we finally adopted a raethod by which we can plmt

conditions or, ,a singlo 1arcu mica. cover &LSS, covered by a Gabrit-
chcwski dish.  In preparations s-d& ns this , WC h:ive the f rcrpents
taken f ram r;ke ventricle, of the same si ze. p'.anted in the same me-
dium of exactly the szune Lamounts of blood ~~LLSKA-L and extract made
frca the juice of embryos. For CL single experiment we now proceed in
the following awnner.  jiist bcf ore the cul turns are made, o chicken
is bled and this plrrsrw is used for ~11 the cultures which are to be
made.  The culture medium, composed of plnsm~ nbd extract, or Ringers
solution, or plasma and Ringers solution and extract, is made with
calibmted pipettes.  Eggs are then t2ken containing embryos ranging
from 4 to 18 days; a number of fragments of tissue arc tzken from each
embryo ad planted on a eeparate cover glass.  All the cultures are
under these circumstances exposed to the same mediurri, the same tem-
perature and the ea.me at,mospherc. At stated periods, at 24, 48 and
96 hours, these cultures are magnified by LZ projectoscope and their
outlines are drawn.  The drawings nro than measured and the results
plotted.  Faith the date so obtained we find that WC cc?n construot a
curve of the rate of growth, which shows that the younger the embryo
from which the tissue is taken, the faster is its rate of growth. At
18 days the rate is strikingly slower than at 4 days (Figure 1).
The curve is smooth except at the points representing tissue
taken from embryos between the 8th znd the 10th days, where there is
a f la tt eaing of the curve.  We have for the moment no explanation to
offer of this phenomenon.  Over 2000 fragments have now been examined
and it is found that =L curve of this f om is always obtzined. The
points for cultures of 3 given age may deviate in different experiments

.
,
.
.  .
. .
*\
. I
.
*
. .
.
. .
       -.--
     . . . . . + .
-yJ -i
  .   * , , I . . .  i
                 . i `   I
. .   . , . . . . . . 4: 1
                                 i
. . . ` I . .
. . * 4s 1?1: Qroulth
                    I I
.........   . i I
........... I 1
...........    i
          .......  I  .
.............  i
l-
t
Age   4  5  6  7  8  9  10  11  12  I.3  14
           f'g. I

from those in othws, but t,ho differences are net material and do
not, we believe, disturb ths L;eneral conclusion to be drawn from the
experiments, namely, that the rate of growth of fragments depends on the
age of the f ragnents and that uhm the rates of the different ages are
plotted the points fall on a curve.  T;lis curve one night call "The
Curve of Potential Groyhh."
     Although the shape of `this curve is characteristic, it is
characteristic only for the conditions employed; if the conditions
are varied the precise, though not the genew.1, shape of the curve
is also varied.  Sken for instance, Ringers solution is added to the
plasma, it' is fcund that the r:.te of growth of tissue taken from the
older el:lbryos is accelerated.   It is of course possible by varying
the conditions in a number of ways to v:ry the curves obtained.   Eut
it does not seem important to us to do this, for the exact 1or::l of
the curve has no significance but only its ;encr:.l shape, and this we
believe :-le have demonstrated.  Embryos at succeeding ages have also
been weighed, and the percentage increases in neight have been plot-
ted (Fig.2).  It nil1 be seen that from the point of view of ageing,
the younger the embryos, the greater is the relative increase in aeight
and in a sense the greater is the rate of ageing; with increase in time
this rate slows down so that toward the end of this period, relative
incre:.se in weight and in the process of ageing are at a ninimum.
Ue have studies these cultures also' in other directions.
We have not iced a fact already pointed out by other observers, namely,
that after a certain latent period, the fragnents in the cultures begin
to beat regularly.  The fragments from younger 8mbryaS besin earlier.
Fragments older than 13 days were not observed to beat. The tissue so

.5Lv
.-Ku
300
.200
.150
.__ .,.. ---.._.---~.- --             _, --.,--
         ___. ___ ,_. - _ -..  .-- - ~. -
5   6   7   0   9   10  11  12  13  14  I.5  16  17  18  19  20
          R&2

                                                                           b' ;. ,
                                                             ,` .,--I
                                                      148
SO far stdiai f rem. this point of view was all ventricular; VJbether
auricular tissue behaves in a similar fashion is not yet ~I-DXI- It
is ho:Jcvsr IXJXI that in clnbr--ms of above !,C days, the auricles beat
fnst3r than the ventricles.  Y/bother there Is a differonce in the be-
havior of right. and left a?lricle:, wa do not yet know. Without chang-
ing the tedium, we have sem l.nd:viJua: fragments contract as long
as 18 days; usually- they cor,til:m to do SO fo= 6 to 8 days. We have
mado preliml~~ experiments also on the inf1~~1~e of temperature
on the rcte of contraction, but !;nvcr postA>oncd further cxyriments
q until wo can utilize tho constant temperature ram now building.
!I30 att3qt to mqgbgp 1 such e.qcrimcnts exce$ undor ,ro;Jerly con-
trolled conditions was too unsatisfactory. Xere it will also be
Gx3i3r for us to obtairr data on the r'lte of cardiac cor.trr,ction of
the heart in the intact embryos.
      Vfe have in progress eq Amsnts with chicken embryos in
other dIrections, reports of whichme prefer to pbstpone for the
present) these experiments incltide studies looking to the construction
o$ cunt& of dry a&I Ivet weight8 bf embeos witli advancing years,
     *
yielding data on the water content of the es'bryos; measurements of
the transfer duriq the course of emb$onic life of substances from
                        .
the egg to the b&y of the embryo; and measurements of the rate of
oxygen and carbon dioxide exchange.  !Ve mi<ght point out that in our
Plan the examination of these various functions is in each case con-
ceived from the point of view of learning the changes which occur
with increase of ago.  The dicta should therefore afford a basis for
describing the changes in ph;`siolom which occur with the process of
ageing.

149
     With Dr. Stewart im-estigation has been carried on to learn
what the effects on tho circulation arc of injury to the heart.  Dogs
have bscn used in these expcri:,lcnts. Tao first step has ccmisted
in deviskq mtimds.  Althou~h the production of val-.ulnr injuries
is not n-34, we are I:~J in a ;ositim to produce precisely the lesion
ne desire,  T?!e eccomplis:h tXs by inser%ir,.: vdithin the heart, a tur
                                                               .
bular instrument aimed at one md v.-ith a 1er.s and a knife. By look-
ing through the tube it io pc ssiblc to place the incision where we
desire.  Ve were about to proceed with zal~ng th2se injuries by an
older method, when the description 3i: a rror~ suitable ins timont by
Graham
at the
and Allen was published.  l%e!.r !r,etrunent was Tot obtainable
time so that we were obliged to conctrxt on2 oZ OCR own.
This we -have been able to do and have carried 0uG 0peratiol-s success-
fully with it.  The operation through the open pleural spnces is dif-
ficult and infection of the pleura is not unlikely.  But we have So
far operated on 8 dogs.  Three died ourin? the course of operation.
Of the remaining 5, 3 are alive after 7, 5 and 4 months.
     Althoxqh it is possible to produce valtvlar injuries, an
injured valve in dogs does not suffice to bring on a condition of heart
.failure, as the experiments of other observers `have shown. The animals
after operation either die of acute heart failure or live without any
sign of defective cfrculation.  In addition to the valvular defect it
is, therefore, necessaqy in some way to interfere t-ii th the integrity
of the `heart.
In the first experiments in this direction which we have
carried on, we employed diphtheria toxin.  This toxin, we have reason
to believe on the basis of clinical and of pathological eqerience,
effects the heart.  We have accordingly injected varying amounts of

and of losing weight, we soon learned that t?e hea:& of these dogs
decreased materially in size.  At first we thou&t that the loss in
size went parallel with the loss in weight, but in a series of dogs
which were first starved, then brought back to the initial y:eight and
subsequently injected, there was no decrease in the heart's size as
the result of starvation, comparable to that which occurred as the re-
sult of injectfon.  It was clear therefore that the diminution in size
had to do with the intoxication.  To account, for this action of the
toxin, a number of possibilities presented themselves.  The decrease
might be due (1) to destnu=tion of the substance of the heart; (2) to
destruction of the blood; (3) to decrease in the blood volume, `phe
attempt to relate decrease in the hearts* size with decrease in the
volume of the blood dr with destnrction of the blood failed. A change
in the siie and regularity of the red cells took place at least on one
occasion, but we do not know the significance of this change to the con-
dition we desired to explain. That diminution of the blood volume might
be expected to influence the size of the heart as seen in the X-ray is
shown by the results we obtained by bleeding a dog, for after withdraw-
ing 500 cc. from an animal the size of the heart's shadow decreased 25s.
  ,: -
  , . `; I 1;
150
toxin in different; Q'G:lps of dogs; tke amounts varying between .OOl cc.
and .00X8 cc.  The dogs -Jvhich received the larger doses died; v;ith
one exception, the dogs receil.-ing the smaT1e-c doses s-uvived.
      V/e devised a method for Tnezsclri.l;- ,I-e aixe of the hearts
by X-ra;rs, comparable to that Ireviousls er,p.lc~ed ir. human beings.
VJith this technique it is possible to ascert-in changes in cardiac
size during the life of the dogs.
     Aside from developing jaundice as t,L2 result of the injections,

Synchronously the count of the red cells decrcasad, from which :Ve infer
that the organism attempted promptly to make up the 1099 in blood v@l-
umc by di1utin.q the plasma.  `The blocd taken f ram the first animal was
transfused into a second dog, proper precautiuns as to agglutination
haviw, been taken. By injecting this amount or.3 would expect the heart
of the second dog to increase in size.  An increase did in fact take
place, to the extent of 9;;; but this half uf Ihe experiment has not
the validity of the first half for the body has facilities for storing
blood, whereas it has none for maL;ing good a rap+.& loss such as we
imposed. Although small changes in the histology of tile heart muscle
were found, the extent of this is not sufficient to account for the
decrease in size we detected, neither did the weight of the Whole
heart decrease beyond the extent anticipated by decrease in the body
weight.
    All these examinations leave us without an Odequate sxplana-
tion of what causes the decrease in heart size, lLvo other possibilities
present themselves which we have not explored; (1) the possibility
that the diphtheria toxin dilates the capillaries and that the blood
disappears Snto them with consequent decrease in the size of the heart;
and (2)
causing
the possibility that watervpasses out of the muscle tissue
the appearance we f tmnd.
Whatever the nature of the change, we have, it appears an
agent which if added to the valve lojury we can prodmoe, is capable
of decreasing the integrity of the muscle to the extent desirable in
Conduc tine these experiments. We ought accordingly nOtv to be able to
prepare dogs so that they may present the aspects of failing circula-
tions and make $t possible to study in detail the alterasons in the
circulation which occur under these circtnnstancea.

,Studi,es Carried on in the maim1
and Physical
    Chemistry- of the BlopxJ.
                         -
Dr. Van Slyke, Dr. Lundsgaard, Dr. Hzwtjng, I%, Salvesen, Dr. Linder,
Dr. Heidelberger, Dr. Neil1 and Miss Hiller.
Report by Dr. Van Slyke.
Cvanoai s .
     A review and theoretical consideration of the causes of
cyanosis has been prepared for publication by J.undsgaard and Van Slyke.
Lund sgaard had previously demonstrated that the factor responsible
for the production of cyanosis is the concentration of reduced hemo-
globin in the capillary blood, and our study resolved itself into an
investigation of the quantitativa effects of the factors contributory
thereto. The contributory factors are (l)>  , the degree of oxygen
unsaturation of the arterial blood coming from aerated lung areas;
(21% ) the proportion of blood passing from the right heart to the
left through unaerated channels; (3) 2   , the oxygen
the capillaries; (4) 1, the total hemoglobin content.
ness in causing th8 presence of reduced hemoglobin in
consumption in
In effective-
the capillary
blood, these four factors rank in the order named. For example an in-
crease of 50 per cent of the distance between nonxal and ~~~~im-um in
each factor increases the capillary reduced hemoglobin content from
t$e normal 2.6, expressed in grams per LOO cc. of blood, to 9.3, 6.4,
4.5, and 2.8, respectively, 4 to 6 being the range at which cyanosis
usually becomes visible.  The combined effect of the factors was found
to be expressible by the equation

  The min clirAca1 conditions in which cyanosis is a symptm havz
been considered in connection with the causative and mo3ifyir.F: factors
present, and ettmgts have been made  to estirmte the functional and
anatcmical significnnco of tile qanosis in certain of these conditions.
1Jophri t i 9.
     In ths nephritis wcrk Lizdc: and Lundsgaard have continued
thoir st-adios of blood motein and blood vo1u.e chcmtis. They find in
ylomerular nmhritis and neohrosis (terms used in the sense of Volhard
and Fahr) that the total protein content of the plasma is usually re-
duced belov: the normal; it varies from 3.5 to 5.5 gram per 100 cc.
compared with the norrxti 6 to 7.  In all the cases of the68 tvio types
studied a f.311 in the  albmlq   ratio below the normal occurred.
                7103ulin
Since albumin is the protein chiefly excreted in the urine, the readiest
explanation for its relative (and usually absolute) deficit in the
p&mm is direct loss bg excretion.  nether this is the entire expla-
ticn is uncertain,
the urine of these
view.
#   In cases
       \
however .  The excretion of albumin and globulin in
patients is beins studied with the above point of
of naohrosclerosis and functional albvtmimria the
plasma proteins were norrral.
    The cause of the lm concentration of proteins in the plasm
content observed in many nophritics hF.8~ been an object of speculation
since the time of Bright .'  There were two possibilities: either there
was a loss of protein f ram the body, or the blood ws diluted with

retained water (hydramic Piathora I .   In order to obtain data that
might lead to a decision, blooa v&lf~le determimtions by Keith,
xowntree, and Geraght;:`s Vital Red" method, repeated at intervals
over varyin? periods of time :have been performed on patients with
low plasm proteins.  In no instances, even when there was great
edema, has the blood volume been found abnomlly high.  r;Je have
thus far had no cases in which a plethoric hydremia existed.  The
low protein content appears due entirely to a diminution in the total
amount of plasma protcine in the bodr.  The latter, calculated from
the protein concentration and the plasm volume, have been found to
be about 8.5 grams per kilo body weight in no-1 subjects.   In the
nephritice with 1~: plasrz protein concentration the figure varzd
between 1.5 and 3.0 grams.
     In some cases a rise towards normal of the total plasma
proteins was observed to accwv clinical improvement. The increase
In plaslila proteins did not occur until eftex edema had disappeared.
It does not asear probable t-hat the low plasma protein content is
a factor in the production of edema.
                                                  1
Blood Gases.
With Dr. Neil1 the details have been perfected for,utili-
zing the nconstant ttolume" blood gas apparatus for all. blood ga&
analyses.  The gases are extracted in the same manner used with the
former "constant pressurel* apparatus. The measukement of the gas,
however, is based on the principle of reducing it to an arbitrarily
chosen, convenient, definite volume (5 cc-, 2 cc., or 0.5 CC.), and
measuring on a mercury manometsr the pressure which the gases exert
when compressed to that volume.  Tne amount of gas present is proport-
ional to the pressure.  In the former apparatus the pressure of meas-
,,
    j.
i ,:

155
urement was consmnG (at atmospheric), the mass of gas bo,ing meas-
ured by the vo1uz-1~.  In the present apparatus the volume is constant,
the i3ass of gas being measured by the pressure. A great gain in
accuracy is obtained, because we can now choose conditions of measure-
ment such that the error in measuring the volume of gas shall be no
greater than the error In measur1r.s the pressure, e.g. with the volume
of gas at 5 cc., as in our most accurate CO2 determinations, and the
pressure to be measured at 200 to 300 IXD. of mercury, both volume
and pressure can be measured to 1 part per 1000, and the sum of er-
rors in both measurements is only 2 parts per 1000. The maA.pAation
of the apparatus is so simple that this deplree of accuracy is, in
fact, attained in our routin determimtions. Refinement to this
point was forced upon us in order to obtain data from which we could
determine the effect of oxygenation and reduction on the base-binding
power of hemoglobin.
Physical Cllcmistrv of Hemoplobin.
     As reported in October, we found that e.t pH 7.4 the sodium
salt of oxyhemoglobin binds 2.15> reduced hemoglobin 1.47 equivalents
of Na per molecule of hemoglobin, the difference being 0.68 qi..v%-
lents of alkali; i.e. if reduced hemoglobin is oxygenated, its e.c.idit:'
Is augmented so that it binds at the same pH 0.68 more equivalents
of Na per molecule. However, we `found that in blood Lhe chnngs from
complete retiction to oxygenation increased the alkali bound by pro-
teins only by 0.56, instead of 0.68 equivalents per molecule of hemo-
globin.  ?YMs difference we were unable to e-lain at the time.
     me believe, however, that it is now completely explained.
Hastings, Neill, and Hatington have found that in hemolszed, blood

156
the same effect of oxygem.tion is obtained as in the solutions Of
pure sodium hemoglobinate.  Tae formerly observed difference between
the solution and the blood is therefore attributable to the confine-
ment of the hemolobin in th? ~11s of the blood.
Physical Chemistry of the Entire Blood.
     !I'he work done in Peking by Van Slyks , h, and McLean, and
here reported was I developmen: of the studies of the physical chemi-
Str:' Of 5!12 5l.C;O.l O`., 'rrtiiC.`i t!Iir 12CCr.iTfr)- iG ?l:r.S~~3,  It hsr i,eLx,
known th?t at normal p3 the chloride and bicarbonate in the cells are
only about half as concentr-ted as in the scrxn although both Cl and
HCCz pass readily through the cell q.valls.  On the other hand the hy-
drogell ion coxantration i3 greater in the celis than in the serum
althoq$ the cell cembranes appear pormaab',e to !I+ ions.  By some
mechanism iC, was lkno-in that the Cl and HC03 distribution is so regu-
lated that it embles the indiffusibLe cell buffers (hemoglobin) to
act throuF;fi the ce;l wall and assist in maintaining the neutrrtlity
of the sercUn with nearly the same degree of efficiency that the hemo-
globin would exert were it directly dissolved in the serum. We had
no explanations, other than vitalistic ones, for these
for the fact that rise of CO2 tension causes the cells
from the serum.  The discovery of the magnitude of the
phencxnena, nor
to take water
amounts of al-
kali with which hemoglobin
;H on the alkali combined,
explanation could be found
combines, and the great effect of changing
indicated the manner in which a comon
for the above phenomena, on the basis of
the known laws of sohitions. The problem was solved by combining the
three physicochemical laxs Tvhich govern the distribution of electro-
lytes between solutions at osmotic equilibrium on tvlo sides of a mem-
brace, which is permeable for only part of the ions present. These

. \ . . -..
,  , s i
157
laws rray be expressed, for t2.a conditions found in the blood as fol-
lows.
    1.  For electrical ne;ltralitv cations and anions must be equ31
at reactions as Rear the neutral point as the blood E+ and OH' ions
are negligible, and the base is Combined partly with monovalent anions
(Cf and HCOj ), partly with protein.  Pepresenting the concentntion
of total cell base as WC , that of the monovalent anions as c12,
that of the negatively ionized protein, or protein combined :vith al-
kali, as \.Pj
        C' and similar values in the serum as b'f , [A'f
                                                          8         , and
                                                               S
c J
P'
  9   , we have, nssumirq complete electrolyte dissociation,
LB+? = [A-i + p:!
         - -             .-
           C             C
LB?
   S = rg
(Pl
   II.  According to Donnan's law, when electrolyte solutions are
separated by a membrane permeable for only part of the ions, the mono-
                    .'
valent permeable ions so distribute themselves that the ratio Of the
concentration of any anion in.the cell to the concentration of-the same
aslion in the serum is the same as that of any other anion, and the re-
                            ,
ciprocal of that of any cation.  For the permeable ions in blood, there-
fore, we have the fQllQw%ng distribution:
the letter rrrlt
             _  being used to indicate the common ratio.
III. For osmotic esilalits between cell contents and serum the
concentration of osmotically;active ions and molecules in each must
.

approximate qqxa1.i tv _  IF/e fand tilat t,hz un? t of mmntic ~oriC=erLtrE~-
                                             ___-__--- 22-L-
-L;ub;tGGe ,
tiog is not the r3tia &x,sl&ed-_su&tancc   bu'> d'ssclvcd
                                                       1
                v0lUme Of SOiUt-iOTl             wc.tet
SO that i Cl], represents the cquivaltxts 3i :hlariJe 2er kilo 01' wvntsr
                                                              --
in the cells, not the chioride per liter oI cells.  The total con-
centration of the osmot ically active substtrxes in.the se- rrrry
be represented as 2 [R&L + (Bz.h, since the s?.lts represented b;r Ba,
arc dissociated into two omotic3lly active i0l.s C+ and A' ~ while
the protein aalt mPj dissociates into ions o_' wkic3 only one B+ ,
                   - -
has important osmotic activity, \Pl havir.g so little that it my be
neglected.  In the case of the cell contents we must add to the
osmotic activity of the electrolytes thcrt of the hemoglobin, which
is about l/IO of the total,  Therefore, if the osrrotic concentra-
tion 5n cells 2nd serum are even we have
The accuracy of this eqmtion was `demonstrated by analyses of cells
ana 6f2r~m.
By combining the above equationswe obtaSn finally one which
expresses the relatiorishiu. bdtween the electroMe distribution anQ
the'alkali. bound bsv the blood mote5ns'; via.    L              1 -
This equation was tested quantitatively by analysis of the
serum snd cells of blood after'it had been subjected to vaI?`ing CO 2
teasw.  IIt w8s..f oumi to agree nearly Vuithin the limit 43f exjjeri-
mental Crrar in H+ , @I' , 0~ HCO'
     \               3 ratios.

    The water distribut;on was cnlmlzted as follox:   the
equation of osmotically active substances may be mitten c&s
2,fB3. s - {Bp1'- - 2LB-j
               -9           C  - (Bfi
                                   -C  + CHbj C
or if we use (B)- to indicate the serum base,  (B), the cell base,
                 s
etc., u kilo OJ blood, we
   2 (Bls - (EfP)s
      &.o),
may write it as
= 2 (Bjc -  0') r + bib);   or
    (H$) c
(H20) s     2
        =   (B)s - (BP)s
--
(2 .o$   2 (B)
                 C - (BP+ ;
                                       C
     The distributbon of water between serum and cells was
found within the limits of analytical error to be related to the
mount of base bound by the cell proteins in the manner indicated by
the last of the above equations.
Electr0mfe Distribution and Osmotic Pressure between Blood Serum and.
Edam Flu&
The laws above discussed governing the distribution of salts
and water between serum and cells in the blood appear sufficient to
explain alao the distribution between the amum and edema fluid.  The
equation exp~esing the theoretical relationships differs from thEtt de-
veloped above, because not only the anions, but also the alkali cations
appear freely diffusible through the memberanes separating blood from
e&ma fluid.  ft is similar, however, in predicting a smaller Cl and
PC03 concenttiation and a greater Na concentration in the serum than in
the fluid.  Analyses of serum and fluid recently published by Atchley,
Loeb, and Palmer yielded results which agreed with the distribution

I
" ., ,ri
160
values predicyeo., as closely as the latter could be calculated
from the data. More complete analyses of serum and fluid from ed-
ematous patients have been made by Hastings and Salvesen, who find
that the agreement between the predicted and observed Cl and EC03
ratios is quantitative , and that between the Xa ratios is approximte.
    The osmotic pressure theoretically predicted cannot be
equal on both sides of a membrane when, as in this case, all the
anions and cations except protein fail Lo i.!.,.~.-;lns:`tl-FF_trtr' The yres-
sure must be towarde the solution with the greater protein content,
viz. the serum.  Dr. Hastings has prepared collodion membranes simu-
lating in permeability the body membrane s that separate serum and edema
fluid. With these membranes and a micro-osmometer he has devised, he
has determined that the osmotic pressure in the serum exceeds that in
the edema fluid by 15 to 20 ran. of mercury.
     The osmotic pressure is, therefore, in the direction that
would cause fluid to pass from the tissue spces into the serum. It
COnSequemly appears that forces other than csmotic pressure are in-
volved in the passage of fluid in the direction from the blood to the
serous cavities and intercellular spaces.
     Salvesen is studying the mineral and protein comuosition of
nephritic seq. He finds that among the mineral constituents, calcium
is peculiar in that it parallels the protein content.  It also exists
in higher concentration than it can attain in protein-free solutions
containing carbonate and phosphate concentration s equal to those in SSXTL~
At times in nephritis the calcium falls as low as in parathyroidectomized
dogs with tetany.  Yet the nephritic may Got have tetany. A preliminary
explangt%Qn of the phenomena is that a large part of the serum calcium
i
i
I
;;ii
j:, /
.Ilr
`;;!
::'
i,
:

iS combined in IlOn-iOsdzed fcm wi:h protein.  It is this part t?Dt
varies parallel to the protein content.  The ionized caiciillll, on the
other hand, r=ay be the prt that is lost by garathyroidectonlized zni-
axis, lack of it causing tetany.
    To test the probability that slightly ionized calcium pro-
tein compounds exist in the senxn, Hastings, !Jurray, and Miss Hiller
are engaged in preparing calcium salts of the separate serum proteins.
The degree of dissociation of these salts will be measured by means
of the solubility product method, and if pos&ble by the olectrzmetriz
method also.
     The method of Haidelbeaer for preparing
hemoglobin has been applied successfully by him to
globin from blocds of the guinea pig, rat, sheep,
Sttids of Rhqtic Fevex.
Dr. Swift, N. Miller and Dr. Boots.
Report by Dr. Swift .
crystalline horse
prevarins hemo-
and donkey.
tie clinical study of rheumatic fever has been continued
in the directions-previously outlined. Because of the close relation-
ship between this infection and chronic heart disease it is necessary
to follow all of our patients for long periods after recovery from the
acute attack.  The rual life history of the malady can, therefore, be
written only after mx.ny years, for only by repeated observation of the
same individuals can the influence of certain abnormalities in the firs
stages of the infection be correlated with later patiiological processes
Thus, for example, previous clinical studies indicated that f mm 30 per
cent to 50 per cent of patients with rh&matic fever develop signs
of endocarditis o.r chronic cardiac valvular disease.  The studies by
Dr. Cohn and myself, mentioned in previous reports but completed v;ith-

                                                                       6 .%  *)
                                                                       , i i ,.*
                                                  162
in the past year, shm~ >k.a; at least 50 ge? CCZAL of our patients had
abnormal electrocardiograms while under observation in the wards.
!Tkese results indicate either that the heart muscle, the impulse
conduction system CanJ branches of the coronary artery are affected
during the acute diaesse more cften tha:: is the lining meL:brane
of the heart, or 5iat our methods for detecting myocardial disturb-
ance are more acctrnte than those for detecting cndocardial injury.
These instrumental 3i@a of cardiac involvcsent Lre often only
temporary ; lasting from one day to A week, a period of time similar
in duration to that often oeen in a single i&lamed joint of a pa-
tient with rheumatic arthritis.  This stlF;;e6ts that some of the car-
Jiac lesions, at least, are similar in raturo to those present in
the joint tissuee.  The relation of these evidences of transitory
lesions to the more deep-eeated injuries fo& in chronic heart
disease is the subject of investigation at the present time.
     De histopathology of rheumatic fever is almost entirely
founded upon study of material obtained postmortem, and hence frcan
subjects who were victims of the chronic forms of the disease, especi-
ally those who had succumbed to heart failure.  The heart, therefore,
has been the principal organ studied.  fn recent years subcutaneous
fibroid nodules removed both postmortem and during life have been
studied microscopically.  Svo pathologists have feported upon the
investigation of joint tissues obtained postmo2tem. Because of the
desirability of knowing the nature of the earlier lesions we have,
during the past year, excised small portions of tissue from acutely
inflamed joints of patients in the early days of an attack of rheur
                                 .
matic fever and su?rnitted them to histological and cultural examin%
tion.  So far, the cultures have been sterile.   !l?he microscopic exEb-

                                                                         ` `.
                            0 4.;::
                                                 163
mination reveals cma! 1 foci of inflammation in all of the joint tis-
sues, but most marked in the synovF1 membranes. A very striking feature
is the involvement of ~~211 bi0d sessels sonsis:in:; at- times of oc-
clusion of the ver,seis v:ith proliferated lining cells, and at other
times of rrr.rlred accumulation of endotholiod cells together with poly-
morphonscloar leukocytes in the perivascular spaces.  This implica--
tion of thi: szxaller blood vessels is a feature of most of the lesions
of rheumatic fever,- whether in the brain, heart, subcutaneous tis-
S-JeE ) or joints, as a part of chorea, myocarditis, rheumatic no\iules,
or arthritis, respectively.  It ia probable that other structures
such as the muscles, and possibly the kidneys, are similarly affected
by the virus  of the disexe.
     Dr. Miller and I are attemptingto reproduce some of the les-
ions of rheumatic fever in rabbits, and are using; the type of reaction
above describad as the basis for cowsrison.  For inoculurn we have em-
ployed rheumatic heart valves, subcutaneous fibroid nodules, and joint
fluid.  V?e have introduced the inoculum into the veins, peritoneal ca-
vity, anterior chamber of the eyes, and into the testicles.  By in-
tratesticular inoculation a mild interstitial orchitis has been pro-
duced in certain series of rabbits and carried along through several
generat i&s. The op&nu~ cbhditions for the production of these'les-
ions are now under investigation anh several extensive skies of,con-
trols are under observation.  The optimum time for transfer seems to
be about a month.  Shorter periods have resulted in a failure to carry
on the peculiar lesions.  The resort of the results obtained so far
can only be considered preliminary in nature; but tti findings are suf-
ficiently encouraging to continue work along this line.'

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P :*, ,->
164
       Drs. Boots and Miller have completed a therapx.tfC; r,tud;,
of neocinchophen (ethyl ester of parame tl:yl phtinylcinc?unini c acid)
and novatcphan (et!lyl ester of phenylciwhoninic acid).  These twc
drugs are closely related; the former, also appearing under the t?jde
nazm of t,olysin, is joing extensively advertised as a noninjuYic.as
substitute for the salic:plates in tha treatment of rheumaticm.  Scxe
observers in toxicological studies with laboratory animals have re-
ported that only subtoxic doses are absored from the gastro-intestinal
tract.  Our studies with patients, on th6 other hand, indicate t-hat
doses of the drug sufficient to control complately the evidence of
arthritis and of general infection are not infreqqontly accompanied
by slight irritation of the kiiney, and occasionail;? by severe al-
tiimi&a and rmmerous casts.  Other mild toxic symptoms are also
sem.  Moreover, when the drug is discontinued a certain rrcmber of
patients have `had relapses in a manner similar to that Sean at times
following the withdrawal of salicylates. Occasionally a patient ia
encountered who does not respond well to neocinchophen but who shows
marked ef feet f ram the sallcylates; likewise, neocinchophen can some-
times be safely and efficiently used in patients with a marked i&o-
syncrasy to the salicylates.  There is no doubt that these nevJer drugs
are valuable additions to our therapeutic armaxnentarium; but, in so
far as we can determine, their action is similar to that of the older
salicylates in that they seem to have a favorable action upon certain
of the distressing manifestations of the
curative effect.
diset;se rather than a definite
      Dr. Boots and I have completed a study of joint sensitiza-
tion with nonhemolytic stre;?tococci. It has beer pseviously claimed
by Henry and by Faber that the jo$nbs of rabbits may be rendered `more

.", -
' :I ;- i
165
sensitive to invasion cf these cocci from the blood stream if the
joints were previously injected with small amounts of killed homo-
logous cocci or ex:racts of these micro-orgnr,isms. Upon the basis
of these expcrimentz they explained the pathogenesis of rheumatic
arthritis.  The recent work of Eesredka and of Gay, on the other hand,
would indicate that these tisseaes might be ircmmized by this local
treatment.  Our work `has failed to denonotrzte either a sensitizn-
tion or increased resistance to infection in joints injected. with
either vaccine or bacterial extract.  Zointn so treated were about
as liable to involvement as the other joints of the same animals
after inoculating them intravenously viith living streptococci. Tie,
therefore, do not think any eqeriments up to the present time prove
that the arthritis of rheTLx.ti,: fever is a phenomenon of hypersensi-
tisation.
     Mrs. Lancefield and I have been trying to demonstrate im-
mune bodies against nonhemolytic streptococci in the blood of rhemtic
fever patients.  As a starting point we used the results of Kinsella
tusd myself,
extensively
age'nts with
Viz: - (1) That certain nonhemolytic streptococci possess
reacting substances go that using extracts of them as re-
complement bincling tests the presence of imrmne bodies
in many rabbits inoculated with different strains of streptococci
Could be demonstrated.. VJe mipJlt designate such strains as "master
strains".  (2) In the blood of patients suffering from streptococcus
ViridanS endocarditis it was possible to demonstrate complement bind-
ing antibodies when'extracts of the homologous strain obtained in
blood cultures were used aa antigens.
     VJe bad never tested for cross fixation with the serum of
these patients and the `kaster strains" of streptococci; so that in

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                                               I.66
the past :-ear we, ob*;Liued serum ana blocd cultures f ram Fticnt:.; i:l
Mt. Sinai and St. L-&e' 3 `hospital for this purp%.e .  In spite oi
cor.tir:Jad efforts to find them, only a fe:J ca3es were available for
these test.;.  I:iueh :ime was consumed in preparing anticens that would
react saticfactorily with the serum of patiento.  The result3 with t5e
few cases available indicate that, with previously devised metlIoZ3,
complement fixin? antibodies could be demonstrated cnly a&aicst the
strain of streptococci pr3dueinq the endocardi tis in a given c=e,
but not against the %a3ter strain3."
    We then prepared extracts of nonheznclytic 3treptocccci by
the method recently derised by Avery and Heidelberyer for the demon-
stration of the nonspecific "PC1 cubctar.ce of pneumococci.  Thir; method
seems to give a more extensively reactin antigen, but we have not
had enough matarlal from ;?at:ents with streptococcus endocarditis to
teat It th.orou&lp.
     We have also testal the serum of a number of rheumatic
fever patients with ou;' "master strains", but up to the present time
have been unable to demonstrate any definite antibodies against Strep
tococci.  This failure may be due to (I.) insufficiently sensitive re-
agents; (2) absoiute specfficity of reaction between antibody and ho-
mologous antigen; or (3) to absence of antibody against streptococci
due to the possibility t&t these organisms are not the etiologic agents
in this disease.  Until our methods are more extensively tested we shall
be unable to decide w?Cc!l of these possibilities mzst be eliminated.
The Pathological Laboratory_.
Dra Branch,
     Derring the past year there have been 10 cases autopsied. S
of these cases came from the respiratory service, 5 from the rheumatic

                                                                          ,' -.,
                                                                     * :j f ,
                                                     167
3 from the cardiac, and one from the nephritic. Tney include:
       3 cases of ckonic valvular disease of rheumatic origin,
Aschaff Bodies having been found in all cases;
     1 of coron%-y artery thrombosis of non-specific ori&in;
       2 septicemias, one due to a meningococcus and the other to
a staphylococcus.  In both cf these there was a purulent arthritis
found.
      1 case cf subacute bacterial endocnrditio, the etiological
agent being a hemolytic hemophylic bacillus.
         2 cases cf lobar pne;unonia - pneznccoccus type I and III
being isolated, respectively.  The casa associated with the pneumo-
coccu6 type I was a serum treated individual in which an orgarking
pneumonia was present and an unusual form of pneumococcuo endocardi-
tis of the mitral vaive.
     1 case of pyelonephritis (staphylococcus) engrafted on a
nephrcsclerotic kidney.  !I!iAs latter conforms to the type of diffuse
sclerosis (arterio-capillary fibrosis (Gull and Sutton)or diffuse
hyper;plastic sclerosis (Evans) ) as vascular changes in spleen and
heart are also present as weli as those in the kidney.
      Two of the above cases have been writ ten up for publication:
one with Dr. Miller - V-ubacute Bacterial Endocarditis Dcle to a Haemo-
lytic Haemophilic Bacillus (Haemolytic Influenza Ekacillu~?)~, and the
other with Dr. Stewart - V&eumatic Pancarditis, with Calcff ication
of Myocardium and Mural Endocardium."
       Of 2 surgical specimens one, a gland from a case of infective
mononuc&eosis, was most interesting.
      Dr. Branch has also been engaged in stuUyi?% the pulnunzry

168
lasions product, in r+.ce lq- e-vcsinz tlw tic3 to atmspheres con-.
talning pneumococci in suspension.  This 3txxQ has been discussed
previously.