124

 

REPORT OF THE DIRECTOR OF Tif HOSPITAL

April 21, 1923

 

 
 

 

LAG

REPORT OF THE WORK OF THE HOSPITAL.

April 1923.

During the period covered by this report patients suffering from
the following named diseasos have been admitted to the hospital and
various problems relating to these diseases have been studied: Heart
Disease, Nephritis, Acute Rheumatic Fever, Chicken-pox, Acute Respi-
ratory Infections.

Hea ease. (Report of Dr. Cohn.)
Dr. Cohn, Dr. Stewart and Dr, Murray.

With Dr. Murray, a study of muscle contraction has been begun.

It is the general object of this research to arrive at some description
of the behavior of muscle relative to certain changes in the life history
of this tissue and to the nature of its environment under various cir~
cumatances. As criteria of behavior we count on its ‘reaction in terms” of
of growth, of rate and of contractile power. Me method of approaching
this problem hos given us much concern for, so far as “human “physiology
in especial is’ ‘concerned, muscle may be regarded from “two aspects; ‘firet
its relation to the process of ageing and senescence; and'second its
relation to the processes of disease. It is increased information on
these matters in particular at which we wish to arrive. In the second

case there are included at least two phases; that involved in hypertrophy

 

 

 

 

 
P25
118

of the fibre ond that involved in incraase of its length.

It seemed desirable to study first the relation to ageing.
ror this purpose it is advantageous to confine the investigation especi-
ally at first to a single species, such as the fowl, since material is
readily available throughout embryonic as well as in post-embryonic life.
We were glod to mke this choice not only on this account but because of
the great experience with it which Dr. Carrel has accwmlated and which
he has plinced unreservedly at our disposal.

We have, in the first place, devoted considerable time to
becoming familiar with our material. In choosing the direction of our
work we thousht it important first to learn the influence of age on the
rate of growth of tissue. In a sense this is net unlike that aspect
of the subject which Dr. Carrel has developed in studying the inhibiting
influence on growth of plasm taken from fowl of increasing ages. In our
worl, we are on the other hand, studying the rate of growth of which the
tissue is capable at various life periods. With this, we hope to be able
to compare certain other properties of heart muscle, more directly con-
cerned with contradtion,

‘In’ our early experiments we planted the tissue in hanging drops
composed of chicken plasm. and of extract made of chicken embryos, each
fragment being placed on mica covers closed in by separate hollow ground
Glass cells. Later, so that wo might assume a like influence of a mumber
of variables, we planted a mumber af fragments (5 to 20) in the same medium
on large mica govers, supported on large dishes (Gabritschewski). This
gave us the opportuni ty of averaging the performance of a mumber of frag-
mente and of studying the deviation of each from this average. We found
that, although this method lent itself te the coristrustion of a curve of

growth, the rate of growth decreaging with the age of ‘che embryo, the de~

 

 

 
br

119
viations of individual experiments from the average were greater than we
supposed could be accourited for by varintions in technique from one to
another experiment.

Tae basis of the differences appears to lie in the nature of
the eggs with which we were supplied. Yle hove accordingly soughtand ob-
teined the cordial cooperation of tir. J. G. Webd, who has supplied us with
the eggs we require. ‘ith this help we have succeeded in eliminating
certain errors. Ve now know exactly the histor: and age of the hens, the
eggs of which we examine; we xmow the time at which tho eggs are laid; we
recoive the eass daily, within e4 nours of the time they are laid. These
are the oggs we are now utilizing but they have not been available long
enough to permit us to report the results.

Directly conmectad with this study, we are estimating the
water content of the tissue through emoryonis life, The embryos are
weighed before and after drving. In measurements already made, it was
found, just as in the case of tissue growth, that a curve could be con-
structed showing a relation between age and growth of the embryo as a
whole. But here again it was thought desirable to carry on the work with
more accurately Imown uatarial. For this reason the work was postponed
until now, when the eggs already referred to supplied by the Webb Fara
have become available.

We hope to relate these studies to the histology of muscle -

at different ages and also to certain other functions. We have theres °°."

fore begun to study rhythnic contraction. For tnis we have preparations

of two sorts. _ In the first it was our object to learn whether under the
‘conditions of tissue culture a preparation could be obtained to serve as
a reliable control. Fragments of tissue were taken from the hearts of

enbrvos at various ages, and were planted in a medium consisting of chicken

Ba Adiga oe ie ee

 

Nine

 

 
 

120

plcsma and ombrvonic tissue extract. We have so far not cxperinented
with types of media, of irrigation, or of variations in oxgven tension
ror with the results of transplaniing the tissue frequently to new media.
We do not kmow how long tissus myr live when these factors arc taken into
account. Fragnents of tissue huvs deen toatson from the two auricles and
from the ventricles, esnecisll; the left one. In these sulturss, cer-
tain frapments, usually more than kaif Fron each ci thess localities, con-
tracted; thay contract for pericds of wina ranging from one to fifteen
days. It is usual for them to contirue to jo eo for six to eight days;
it is wmsuol for them to do so for a less period. Usunliy the fragments
contract as soon as they ave planted; sometimes they ao not begin until
after a lapse of two to three days. Often the rate slows after a fev
days, but later rises again. We have roticed thet the rate at which
auricular tissue contracts is greater than that talen from the ventricles.
There soems also to be a difference in the rate of contraction between
right and left auricles. We have done a few experiments to observe the
effect of alteration of temperature on rate and have seen that in these.
fragments temperature exerts an influence on rate, an effect frequently _.
observed by others in other preparations. We are, however, not yet able
to say whether in our preparations the rate increases with temperature...
according to the laws of a simple chemical reaction. The differences in
behavior of avricles and ventricles, and the possiole dirference noticed .
between the tvo auricles, suggest either that these differances indicate
differences in the structure of the nmuscle taken from various regions of
the heart, or that tha differences observed depend on the presence of other
2ssues such as ganglion cells or nerve tissue incorporated in the muscular
structure. These are matters for future study. They recall those observa-

tions in embrrology bearing on the \mwandering of ganglion cells into the

 

 

 
 

2)

121
heart muscle. Ag to the eZfert of the praseace of these cells on the func-
tion of the heart muscle <3 apart from ths fonmation of eleccracardiograms,
little or nothing is lnom.

In cddition w2 vellevs i% tu bs impeviait for ue vo study the
rate of the wnole heart in tne invace mmeryo whize this is 35/1, widisturbed
in the egg or, at least, disturted us little as nossible. Yar this we ate
employing the gelvanometzi: methoc cf registering the t.eart yeots. Tne
method has been used before now, bvt 1% hi been vead te staiy che form of
the electric curve which is obtained. Listie att antion nas been attached
to the question of rate. But it is this function: of ‘he muscle which to
us ia ismortant in the atterpt to estabiisn those retations to other func-
tions, scch as metabolic rate, to which reference has alrecdy been mide in
discussing growth, age and tissue density.

The subjects which I have mentioned cover a wider field then can
be investigated immediately. I have described them so as to indicate the
point of view from which at the present time we are approaching this problem.
Having arrived at these general views and explored the possibilities afforded
to us by our waterial, we intend first to make the measurements which we hope
will permit us to construct grovth curves} next to learn the natural rate of
the heart during embryonic life; and next to ascertain the yalidity of those
preliminary observations, already ‘refsrred to, on rate exhibited by tiesue

from various portions of the heart, and the influence of temperature upon it.

With Doctor Stewart experiments are in progress on dogs with
the view to producing valvular lesions in ther so tnat information on certain
circulatory problems connected with the volume and minute outputs and with
the rate of flow, may be gained. Lesions of the valves in dogs that survive
“ave of course been made, notably with such pieces of apparatus as the va)vu-

lotome, It was our intention to use this instrument, although the precise

 

 

 

 

 

 
 EGAee

lesions to be obtained with it could not be predicted. Meaywhile, during the
autum (1922), a notice appeared in the Journal of the American Medical As-~
sociation by Graham and Allen stating that they had made and used an apparatus
not unlike 2 cystoscope with which it is possible, when introduced into the
heart through one or the other auricle, to cut the valves and to see precisely
what one is cutting. The instrument used by them was not available at the
time. Accordingly we set to work to devise one here and have now succeeded in
producing one which meets our requirements, giving a large field of vision in

the area of operation. So far Doctor Stewart has succeeded in operating

 

successfully on two dogs which have survived. The risks of the operation
are great as Graham and Allen reported. We also found this to be true during
the tims when the intracardioscope was being perfected. Since then. subse~
quent operations have shown that the dangers appear not to be unduly great.
The aseptic technique is, as is well known, difficult of attainment because

of the prolonged and wide exposure of the pleural cavity in the approach to
the heart. The dogs so far operated on have survived 10 and 7 days. There

4s reason to believe that wa shall succeed in preparing the sort of deg re-

quired for our subsequent studies.

 

It 4s however not enough merely to produce 4 valvular defect in
order to bring about a state ‘of ‘chronic heart failure as other experimenters,

notably Cushing, ‘have ‘pointed out. le je the usual experiance, and it wos

ming here, that ‘the degree of failure ‘after injuring the valve either je. eXx~

4

 

tia a

treme resulting promptly’ dn death ‘or ‘that ‘heart failure: fais entirely to oP
pear. in view of the fact that this is still likely to be our experience, \ wo
mean to resort to the device of subjecting the animals which survive the op-
erations to one of several procedures, notably to work on a treadmill. But
meanwhile we have studied the effect of another method. We plan, in addition

to producing valve lesions, to cause the muscle of the dogs to degenerate.

 
I

In rabbits a veriety of agents injure the heart muscle, hut there is as yet
little experience in this direction in dogs.

"fe have resorted in the first instonce to the use of diphtheria
toxin, and have met with cesults we did not anticipate. A number of control
measurements were first obtained such as electrocardiograms, X-ray photo-
graphs and the body waight. It vas found that if 0.0016 cc. por kilo of
body weight of diphtheria toxin vas injected intravenously tho dogs became
ill, failed to eat, lost weight and died. The duration of life after in-
jection was from 34 to 73 days. During this time it was noticed that the
skin of the dogs became discolored, the urine took on a yellow-brown color
due to bile, the red cells underwent changes so that in the smear they pre-
sented marked variation in shcpe and size. Very striking was the reduction
in the size of the hearts. This was determined from X-ray plates by meagur-
ing the size of the hearts with the plenimeter. In yicw of the fact that
after the injections the animals failed to eat, it was thought necessary to
exclude the possibility that the reduction in size was due to starvation.
With this phenomenon we were familiar from studies made in cases of diabetes
mellitus treated by the method of undernourishment. ‘We starved dogs accord-
ingly for the same period of time (4 days) in the course of whieh reduotion
in size after injection had been observed, and found that starvation of this
duration produced no greater change in the size of the hearts than could be
accounted for by the inaccuracy of the method, which Dr.Levy pointed out.::
The noarte of the same dogs subsequently showed reduction in size when toxin
was injetted. Other dogs then received diphtheria toxin 0.001 cc.per kilo
and survived. These dogs failed to show those changes in the size of the
cardiac silhouette which have already been described in the case of the dogs
which died. ‘le have in progress experiments designed to determine whether

in dogs receiving doses of diphtheria toxin large enough to causs death,

 

 

 
the change in the heart cream is due to a chu:mge in blood volume, to blcot

destruction, or to change in the heart muscle itself.

Autopsies were made of the dogs, the hearts and kidneys being
taken for further studies. The hearts were dissected and weighed by
the method of Miller with the modifications recommended by Wilson and
Herrmann. Ve thought it of value to do this first to complete the
measurements bearing cn the reduction in the size of the hearts in con-
parison with the body weight and second to learn whether the changes
found in certain of the electrocardiograms denoted an alteration in the
relative weights of the two ventricles. These studies as well as the
study of the microscopic anatomy of the heart muscle and of the kidneys
are not yet complete.

In addition to these methods (valve injury and diphtheria in-
toxication) ef bringing about changes in the heart and consequently in
the circulation, we design to excise portions of the kidney. In this
phase of our study we think it necessary to come to definite conclusions
on the relative value of various methodsof taking blood pressure im dogs
by indirect. means... After many experiments with varieties of. apparatus.
applied both to the carotid artery, by the method of Van Leersum, and to
“the femoral artery, we have finally concluded to abandon the method. of, |.
Van Leersum and to adopt: the method. of indirect measurement of blood pres~ = 7
sure in the femoral artery. - These methods have of course been compared
with the direct method. In taking the pressure of the femoral artery we”
find the method of Kolls the most satisfactory. The method is essentially
the method of Erlanger. There is added a record of the levels of the
mercury column and a modification of the device which magnifies the ex-
cursions of the vessel wall. It may be pointed out that any method of tak-

ing blood pressure in dogs, requires careful interpretation because of the

 

 
ner cna nnn aeniamanaagereretan

fluctuations which are known to occur from day to day.

Ye are accordingly in the early stage of this study. What
has so far been accomplished is {n the direction of disposing of cer-
tain preliminary technical difficulties.

We have admitted patients to the hospital and have been im
terested in studying tuem from to points of view. Tt was shown by
Cohn and Levy, that in dogs dosee of digitalis preswmod. to be com-
parable to doses used in the clinic, doses which in many case do not
kill intact dogs, were nevertheless sufficient to increase the degres
of contraction of the ventricular muscle, when this is measured by
the myocardiograph of Roy and Adami. To learn whether an effect on
the contraction of the ventricular muscle occurs also in man under
therapeutic conditions 48 a mitter of importance for its own sake, bat
more especially since thé view is commonly held that this action does
not take place, that the use of dizitalis in man is confined to its
effect on auriculo-ventricular ecnduction, an effect which is seen in
the reduction of the ventricular rate in fibrillation of the auricles.
To test this matter of action on the miscle in man is difficult. We
report now a plan we have adopted in order to test this possibility.

By reans of the X~ray the left border of the left ventricle can be pho-

- tographed.. “If in a single record the excursion of this bordér from:

the @lastolic to the systolic position could de photographed and mea
sured, béth in the control period and in the period when the heart is
under the influence of digitalis, a compirison between the two measure-
ments should indicate whether the drug has produced an effect. There
ave naturally precautions to be taken in estimating the value of such
measurements, for digitalis may have an influence on cardinc rate, and

alterations in pate are ., as is well kmown, of importance in bringing

 

 
 

about changes in the volume output per beat of the heart and consequently

in the excursion of any given point in the cardiac surface.

In attempting to make such measurements we have followed the pian
of Goett in constructing a. moving X-ray camera (Fig. 1). Between the pa~
tient and the moving plate there is a lead screen. In this screen there
is cut a slit 1.0 en. niga and about 17 to 18 cm. wide. By this means
there is exposed to the moving plate only 1.0 cm. of the left border of
the left ventricle ani 1.0 cn. of the right border of the right auricle.
We photograph only the shadows which pass through the 1.0 cm. slot. ‘The
exact position whith is photographed in this mumner is mown by photo-~
Graphing the whole chest at the same time in the usual way, (Fig.2), this .
plate being placed between the individual and the lead screen. The X-ray
plate is drawn upward by & motor behind the slit. The excursions of
single portions of about 1.0 em. of the two cardiac borders then appear
a3 saw-toothed or wavy lines (Fig.3), the outer points representing the
Position of the portion exposed in diastole; the base of the trough its
position in systole. The difference between these points is the neasure
of the systolic excursion of this fraction of the heart's border. In
these curves can be seen the rate of change of position both of the sys~
tolic and diastolic processes at the point studied. We record both the
time and the phases of respiration by photographing the movements of le~
vers hung opposite the Slot, between the X-ray tube and the lead screen.
The shadows of these levers appear in the form of continuous curves. We
are now making successful photographs by this method. We have ag yet no |
data to report dealing with our problem.

A second study concerns the inability of digitalis in certain
cases of heart failure in which there is edema to relieve this condition.

These are cases in which the kidneys are active, for by means of diuretics,

 

 

 

 
 

 

 

 
 

126 b

 

 
Ps
227

such as theocin, marked increase in she output of urine is obtsired.,
though the effect is temporary; and in which the heart, or at least 0.
inhibitory apparatus is affected by digitalis action, as is evident by
the contimued effect of this drug on the rate of the ventricles when the
auricles aro fibrillating. The failure is apparently due to the absence
of muscular action of the drug. ‘The group of patients which corresponds
to this description is large and the reasons for the failure of digitalis

$o affect them is unknown. In this study we are attempting to understard

this group and to learn tha nature of the factors which are involved.

ca
te

vesen, Hastings,
Ne and M Hi

A review and theoretical consideration of the causes of cyanosis
has been prepared for publication by Lundsgaard and Van Slyke. lundsgaard
had previously demonstrated that the factor responsible for the production
of cyanosis is the concentration of reduced hemoglobin in the capillary
blood, and our study resolved itself into an investigation of the quanti-
tative effects of the factors contributory thereto. The contributory
factors are (1) L » the degree of oxygen wsaturation of the arterial...
blood coming from aerated lung areas, (2) & , the proportion of blood
passing from the right heart to the left through unaerated channels, (3)
D, the oxygen consurption in the capillaries, (4) 2 , the total hemo-
globin content. In effectiveness in causing the presence of reduced
hemoglobin in the capillary blood, these four factors rank in the order
named. For example an increase of 50 per cent of the distance between

normal and maximum in each factor increases the capillary reduced hemo-

 

 

 
flobin content from the normal, expr2ssed in grems per 100 cc. of blood,
to 9.3, 6.4, 4.5, and 2.8, respectively, @ to 6 being the range au wii.t..
cyanosis usually becomes visible. The combined effect of the factors

was found to be expressibie by the equation

C = Le + _& DPD

ee

li &

There are various other factors which modify the coloration. Such are
local skin vascularity, pigmentation, thickness of epidermis.

The main clinical conditions in which cyanosis is a symptom
have been considered in connection with the causative and modifying fac-
tors present, and attempts have been made to estimate the functional and
anatomical significance of the cyanosis in certain of these conditions.
Nephritis.

In the nephritis work Linder and Lundsgaard have contimed

their studies of blcod protein and blood volume changes. They find in

‘curitis and nsvhrosis (terms used in the sense of Volhard

 

and Fahr) that the total protein content of the plasma is usually reduced
below the normal; it varies from 3.5 to 5.5 grams per 100 cc. compared

with the normal 6. to 7. In all the cases of thase two types studied a ~
fall in the Cee ratio below the normal occurred. Since albumin

is the protein chiefly excreted in the urine, the readiest explanation for
its relative (and usually absolute) deficit in the plasma is direct loss

by excretion. Whether this is the entire explanation is uncertain, how-
ever. The excretion of albumin and globumin in thé urine of these patients
is being studied with the above point in view.

In cases of nephrosclerosis and functional albuminuria the plasma

prateins were normal.

tg. Ree

Dota Rn ae :
pet Me ge ob toumasun che ARE eS

 

 

a ewan abit

 

esta nana Rae a a ata ali densioumd ants
SR cy aie

wenn Sah itn Dente reese oe nee ve caer as oatanee Pe
Ret

   
    

 
 

15>
125

The cause of the lov concentration of proteins in the rlacm
content observed in many nephritics has been an object of speculation
ginca tne time of Brisht. There were two possibilities: either there
was a loss of protein from ths body, or tne Dlood was diluted with re-
tained water (hydremic plethora). In order to obtain data that might
lead to m decision, blood volume deterinations, oy Keith, Rowntree,
and Geraghty's "Vital Red" method repeated at intervals over varying
periods of time have been performed on patients with low plasma pro-
teins. In no instances, even wnen there was great ederm, has the blood
volise been founi abnormally high. We have thus far had no cases in
wanich a plethoric hydremla existed. The low protein content appears
duc entirely to a diminution in the total amount of plasma proteins in
the bod. Toe latter, calculated from the protein concentration and
the plasma volume, have been found to be about 3.5 grams per kilo body
weight in nermal subjects. In the nephritics with low plasm protein
concentration the figure varied between 1.5 and 3.0 grams.

In some cases a rise towards normal of the total plasma pro-
teins was observed to accompany clinical imprevement. The increase in
plasma proteins did not occur until after edema had disappeared. It
_doss not appear provable that the low plasma protein content is a factor
in the production ox edema.

Chemistry of the Blood.

With Dr. Neill the details have been perfected for utilizing
the "constant volume" blood gas apparatus for all blood gas analivses,
re gases are extracted in the same manner used with the former "constant
pressure" apparatus. Ths measurenent of the gas, however, is based on
the principle of redvcing it to an aroitrarily chosen, convenient, defi-

nite voluse (5 CC., 2 cc., Or 0.5 cc.), and measuring on a mercury mano~

 

 
130
meter the pressure which the gases 2xert when comprecsud to that volume.
Ths amount of gas present is proportional to the pressure. In the forme.
apparatus the pressure of measurement was constant (at atmospheric), the
mass of gas being measured by the volume. In the present apparatus the
volumes is constant, the mass of gas being measured by the pressure. A
great gair. in accurac: is obtained, because we can now choose conditions
of measurement such tnat the error in measuring the volume of gas shall
be no greater than the error in measuring the pressure, &.g- with the
volume of gas at 5 cc., as in our most accurate CO9 determinations, and
the pressure to be measured at 300 to 300 rm. of mercury, both volume and
pressure can be measured to 1 part per 1000, and the sum of errors in
doth measurements is only 2 parts per 1000. The manipulation of the ap-
parctus is so simple that this degree of accuracy is, in fact, attained |
4n our routine determinations. Refinement to this point was forced upon
us in order to obtain data from which we could determine the effeot of
oxygenation and reduction on the base binding power of hemoglobin.

As reported in October, we found that at 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 equivalents of alkali;

-4.e. if reduced hemoglobin is oxygenated, its acidity je augmented so that

%
ot

it binds: at the same pH 0.68 more equivalents of Na'per molecule. However,”
we found that in blood the change from complete reduction to oxygenation
increased the allali bound by proteins only by 0.56, instead of 0.68 equi~ -
valents per molecule of hemoglobin. This difference we were unable to
explain at the time.

We believe, however, that it is now completely explained. Hast«

4ngs, Neill, and Harrington have found that in hemolyzed blood the same

Fg Ot ES RSA SES I SES

effect of oxygenation is odtained as in the solutions of pure sodium hemo-

 
giobinate. The formerly 2ocsrved differencs Ssivreen the solution and toe’
blood is tuerefore attributeadle to the conzineasnt of the hemoglobin ina
tne cells of the blood.

Ven Slyke, Va, and ilcLean in Pexing (ir work reported below)
shoved taat when the seraa pH cf blood is 7.4C the cell pE is only 7.26.
And at 7.23 the oxygemtion of pure soaiium hemoglobin-te increases the
Nal by only 0.62 instead of ©.$8 equivalents. This accounted for half
the observed difference between whole blooé ard pure solutions. The other
helf was due to some other factors, ané of knowm differences between cell
contents and Wald solutions the most striking is the fact that all the
alkali in the cells is K rather than Na (in henolyzed blood the alkali.
is 2/3 Va). We accordingly determined the effect of oxygenation in so-
lutions of potcesiwa hemoglobinate and found the same value as in blood,
when the pH differance between cells and serum is included in the calcula-
tion. The effect of oxygenation and yeduction on the acid-base valance of

the bloca is one move physiological phenomenon which has become explain-

i
.
pae
i
iif
ith
i
4
¢
t° Sy
re
i ram
| a .
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iy
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able on a physicochemical basis.

mene aan:

The work done in Peking was 4 development of the work on the
physical chemistry of the blood on which this laboratory is engaged. It
has been know that at normal pH the chloride and bicarbonate in the cells —
are only about halt as concentrated as in the serum although both Cl’ and
‘COZ pass readily through the cell walls. On the other hand the hydrogen
ion concentration is greater in the cells than in the serum although the
cell membranes appear permeable to Ht ions. By some mechanism it was
kmown that the Cl and HCO, distribution is so regulated that it enables the
inaifeusible cell buffers (hemoglobin) to act through the cell wall and as-
sist in maintaining the neutrality of the serum with nearly the same degree

of efficiency that the hemoglobin would exert were it directly dissolved in

clear PANES el Ee eeeranr

 
fa:

132
the seraa, “e had no explcnations, other thon vitalistic ones, for these
pocnomena, nor for the fact that use of C0. tensions caused the cells to take
water from the serum. The discovery of the magnitude of the smounts ef al-
kali with which hemoglobin combines, and the creat effect of changing pH on
the alkali combined, indicnted the manner in which a cormon explanation could
be found for the above phenomena, on the basis of the lmovwn lews of solutions.
The solution was obtained br comoining the three physicochemical laws which
Govern the distribution of electrolytes between solutions at osmotic equili-
trium on two sides of a membrane, waich is permeable for only part of the
ions present. These laws ray be expressed, for the conditions found in the
bloci, as follows.

'. For ejeztrical noutralitv cations and anions must de equal at re-
actions as near the neutral point as the blood 2!* and M3’ ions are negli-
gible, and the base 1s combined ncartly with monovalent anions (Cl”° and HCO, ),
partly with protein. Represonting the conec2ntration of total cell vase
as B+ o! that of the monovalent anions as iA | ec that of the negatively
ionized protein,or protein combined with alkali, as [P'1, , and similar values
in the serun as 13") 1 [Ay g? anc iP" 3? we have, assuming complete electro-

lytic dissociation,

Bis = lfl, + [FI

II. According to Dognan's law, when electrolyte solutions are separated
by a membrane permeadla for only part of the ions, the monovalent permeable
‘ions so distribute themselves that the ration of the concentration oF any anion
in the cell to the concentration of the sams anion in the serum is the same as
that of any other anion, and the rsciprocal of that or any cation. For the

 

 

 
permeable ions in blood therefore, we have the following distribution

~ yt “ar
HW] 5 (SF l fC [AT eee,

a ee vo
{Ho (Ply HCO; | 5 (ay s

 

the letter r being used to indicate the common ratio.

TII. For osmotic equaljty between cell contents and serum the con

     

centrations of osmotically active ions and molecules in each mast approxi-

mate quality. We found that the ynit of osmotic. cnngentration is not

the ratio: dissolved substance , . dissolved substance so that [cg]

represents the equivalents of chloride ner kilo oF water in the cells,

 

 

not the chloride per liter of cells. The total concentration of the
osmotically active substances in the serum way be represented as 2 Bay
+ (BP. since the salts represented by Ba, are dissociated into two
osmotically active fons BY end AX , while the protein salt [BP[ dis-
sociatesa into ions of which only one [3t} , has important osmotic acti-
vity, FP f. having so ‘ittle that it may be neglected. In the case of the
call contents we mist add to the osmotic activity of the. dlectrelytes that
of the hemoglobin, which is about afro of the total. Therefore, iz the

bamdtic concentrations iA cells and: sérum ave aven we have

2 tml, + fe] = 2 2m, + iF, Dy

- -o

The atcuracy of this equation was demonstrated by analyses of celle and
serum.
By combining the above equations we obtain finally one which

. expresses the relationship between the electrolyte distrisution and the
2 i ad bv the dlocd >roteins, viz.
L1

 

 

“134
Sytr Ti or tie “T _ ney =e
vag 102) ¢ HOO [BP |, + [HDI (BP | S
r= = = oc 1 -_ ve te ce ne er erent
art. —  * 7 -
THe = [1p (HeOyr s 6 - BP s)

This equation was tested quantitatively by analysis of the serum
and cells of blood after it had been subjected to varying CO. “tensions. It
was found to agree nearly within the limit of experimental error in Hq ,
Cl ,» and HCO, ratios.

The water distribution was calculated as follws: the equation

cf osmotically active subst-nces may be written cs

2 ear - (PP; a Wwe a (BP), * (Hoy
or if we use (B) to indicate the serum base, (3). the cell base, etc.,
per kilo of bleod, we may write it as

2 (3),- CP), 2 (3), - (BP), + Ge)

c , or
WO) tT
(2,0) | (220) |

(#50), 2 (3). (BP)

OR, 2 (8), - (iP), + (i),

The distribution of water between serum and cells was found within the .
limits of analytical error to be related to the amount of basd bound by .
the cell proteins in the manner indicated by the last of the above equa-
tions. |

It appears probable that the Jaws governing the distribution of
salts and water between intracellular and extracellular fluids in the blood
also govern the distribution in other parts of the body, between tissue

cells and exudate, for exaxxple. The study is being extended in this direc-

 

ERE Po tie peepee ak ET a

wp giana eet:
Pet vat alan ers o ea

ee

ESSE aN ce tat
Fone ahr

 
fa
135

tion, ani it is hoped that it my assist in explaining the physicochemical

disturbances that underlie such conditions as edem.

ficoute Respiratory Diseases.

Drs. Cole, Avery, Morgan, Dahl and Stillman.

The admission of patients has not been confined to those suffer-
ing from lobar pneumonia but patients sufferirg from various types of the
milder acute respiratory infections hove also teer. admitted for study.
Through a combination of the clinical study of these patients and more in-
tensive laboratory investigations an attempt is Seing made to differentiate
more clearly the various types of infection of the respiratory tract and to
learn more concerning the nature of the infective agents concerned in the
primary locus of infection and the mode of invanion of the lungs. Further
analysis is being made of the phenomena of infection with pnevmococei and
the process of recovery.

Bacterial Incitant of Acute Upoer Respiratory Tract Infection.
Dr. Morgan and Dr. Avery,

Studies directed toward the isolation of 35. pnewnosintes or other
bacteria of this type from the nasal secretions have been continued. The
technic followed in this study has been essentially that descrived by Ole
itsky and Gates in their investigations on the bacterivlogy of epidemic: -
influenza. In al] 54 speoimens ef naso pharyngeal secretions from 45
individuals have heen examined by cultural and animal procedures. In 17
instances the washings came from the nose and throat of patients in whom
the diagnosis of clinical influenza seemed certain or highly probable.

The naso pharyngeal’ washings from 16 individuals suffering from acute co.
ryza, simsitis or bronchitis were similarly studied and of the remaining
individuals investigated 5 were suffering from lobar pnewmonia and 7 were

fres from respiratory infection.

 

 

 

 
fj
133

from none of these 45 individuals has b. pneumosintes beer re~

covered. On the other hand, during the study of these 54 specimens of

filtered naso pharyngeal secretions, 23 strains of small Gram negative,

anaerobic bacilli have been isolated. From ons case of probable influenza

an identical organism was isolated on four successive examinations at
weekly intervals. All strains recovered thus far apparently belong to
a heterogenous group of organisms which are closely allisd to B. pnewae~
sintes but are serologically distinct.

Of the 23 strains of these pnewnosirtes-like organisms, 135 were
optained from the 17 cases of supposed influenza; 6 strains from the 16
pationts with "colds", (coryza simsitis, etc.) ani 4 strains from the 7
normal individuals; from the filtered spatun and naso pharyngeal washings
from S patients with lobar pneumonia no organisms of this type were isce
lated.

At present the significance of these organisms in the causation
of mild infeations of the upper respiratory tract must de regarded as un-

certain. Their relation to each other and to B. pnevumosintes is not

clear. Experiments to determine their antigenic relationships are being

carried out. Serological investigations are exceedingly difficult within

‘this group because of .the tendency of these bacteria to agglutinate spon<

taneously in normal serum and in salt mixtures.

Chemical Na and I ological Proverties of Svecific Substances ‘o
D. us Origin.
Dr. Avery and Dr, Heidelberger. .

In 1917 Dr. Dochez and Dr. Avery shoved that whenever pnewmo-

cocci are grown in fluid media, there is present in the culture fluid, even

during the early hours of growth, a substance which reacts specifically with

antipneumococcus serun of the homologous type. This (selssla) substanca
“> ad

 

 

 
137

is demonstrnbls in culture filtrates Guriay the initial grovth
phase of the Organisas, that ie, furing the period of their maxinurn
rate of multiplication when little or no ce)l death and disintegra.
tion is cccurring. The formtion of this soluble specific material
by pneumococci on Browth in vitro, suggssted the prooability of
an analogous substance being: formed on growth of the organism in
the animal body. Examination of the blood and urine of experi-~
mentally infected animals gave proos of the presence of this sub—
Stance in considerable quantities in the bedy fluids, Tollowine
intraperitoneal infection with Pneumoceccus. In other words, this
soluble material Slaborated at the focus of the disease readily
diffuses throughout the body, is taken up in the blood, passes the
kidney and appears in the urine unchanged in Specificit:. Simic
larly a Study of the serum of patients suffering from lobar pneu-
monia has revealec a suostance of like nature in the circulating
blood during the Courss of the disease in man. Furthermore, exami-~
nation of the urine of patients having pneumonia due tO pneumococcus
types I, II ang III has shown that in approximately two thirds of
the cases, this specifically reacting bedy 1s excreted inthe urine
in quantities Yeadily demonstrable by precipitin tests, and it has -:.
also been found that -the -amgunt of precipitable substance in the
urine séems to be a measure of the severity of the infection

In the earlier studies by Docheg and Avery certain fects
were ascertained concerning the chemical Characteristics of this
substance. It wag found that the Specific substance is not destrovred
by dodling; that it is Peadily soluble in water, and precipitable in
acetone, alcohol and ether; that it 4s Precivitated by colloidal dror

and does not dialyze throuch parchment; that the Serolorical reagtic:s

 

 

 
138

of the substance are not affected by proteolytic digestion by trypsin.
Since this substance is easily soluble, thermostable and type specific
it seered an ideal basis for the beginninz of a study of the relation
between bacterial specificity and chemical constitution. The present
report deals with the work done in this direction.

As the most abundant sources of the soluble specific substance
appeared to be an 8 day old, autolyzed broth culture, this material
has been used for most of the work. The organisn used was the Type II
pneumococcus. It was found that when the broth was concentrated on
the water bath to one tenth of its volume and then treated with alco-
hol or acetone, a separation into two layers occurred. by addition
of the precipitating agent until the upper layer no longer gave a tur-
bidity with imcune serum, the specific soluble substance could be con-
centrated in the lower layer, leaving in the upper layer a large pro-
portion of the coloring matter, peptones, and other impurities, der? ved
from the broth. In this way the active material from 75 to 100 liters
of broth culture could be concentrated into a veiuma of about 1.5 liters.
Further purification consisted in dilution and repeated precipitation
with acetone or alcohol, first from the neutral solution, and finally
after acidification with acetic acid. Careful fractional precipitation
in the later stages resulted in the removal of inactive fractions, as
it was a simple matter to redissolwe a portion of each precipitate and
test with immme serum. In this way it was possible in several instances,
without the use of other reagents, to isolate small amounts of highly
reactive material which no longer gave the biuret reaction and no longer
precipitated phosphotungstic acid, one of the best precipitants for
uitrogenous compounds. Under the most favorable conditions the yield

was 10 milligrams of dried active substance per liter of broth used.

 

 
 

1og

In other cases, however, in order to obtain products of the same degree
of pvrity it was found necessary to precipitate the nitrogenous inmpuci-
ties first with phosphotungstic acid, mercuric choleride, or neutral
lead acetate, or to leave them behind by procipitating the soluble sub-
stance (and other gums) by saturation with ammoniun sulfate.

The best products obtiinod in this way caused a precipitate
to form in the humologuus ircune serwa when the dried substance in a
solution of as great as 1 part to 2,500,000 was added to the serm.
On the oths1 hand, no trace of clouding occurred when the substance,
even in high consentration, was added to the heterologous serum. This
conoentrated and purified substance tx refore is exquisitoly type spec-
ific. The further study of this purified substance showed it to be free
from substances giving the biurat rerction, it rotated the plane of
polcrized light about 31° to the right, gavo the Molisch test for car-
bohydrates in the highest dilution at which specific precipitation oc-
curred, (a point near the limit of dslicacy for the Molisch reaction, )
and yielded reducing sugars on hydrojysis with acid. The findings

are summarized in the accompanying table:

 

 
Sumary of the Properties of Various Preparations of the Soluble

Table I.

Specific Substance of Pneumecoccus Type If.

 

 

 

 

 

|
| Hydrolysis __
Prepn. | Total
Vo. | a. WHO N | j Red!
2 53 &
1% % ih, sugars.
o/o
a ¢ [6.2 [3.5
At | 4.7 63.
9 6.6
il 2.1 0.9 1.3
15 2.0 49.0
{
|
{

 

 

 

 

 

1.8

Spec
ific
Rotation

[<}>
foo dark
-20.6°
+19.8°
-8.6°
+51.6°

+30 .8°

 

Pptn.with
Immune

serum

80,000
640,000
1,250,000
640 ,000
2,500,000

2,500,000

 

 

{From urine.

7)Prepn. 4 repurified.
mFrom dissolved pneumococci.

 

Mo lisch
reaction

1,250 ,000

1,250,000

 

 
Preparation 4 A was obtained fron thr urine of a case of Type IT prev nwria.
While Preparation 8 was Csrivec from washed Type IT pnewmocecei whic. hae
been dissclved in antiformin. TL also yielaca vecucing substances cu
hydrolysis.

Since the specific scluble sicstancs ef the Type II pnewmococcus
appears, in its present stata o? purity, to consist largsly of a nolysac-
Charide, it was of intevest te dste.mine, if possible, the sugar from
which the complex molecula is built up. fccerdingly *00 mg. of Prepara-
tion 15 wore aydrolyzed by heatirg 7 hours on the water bath in 0.5 i
hydrochloride acid, Although the activa material Wao Wisitored dy the
acid at room temperature for 36 hours, heating o: the water vath re-~
sulted in the disappearance of the specific reaction with immune serum,
and the appearance of reducing sugars. The hydrolyzed. solution was
treated in the cold with phenylhydrazine and sodiw acetato, and as no
hydrazone separated, was then haated on tho water bath. Ina very shor
tima a crystalline osazone Separated. Although the amount was very small,
it was possible to recrystallize the osazone. Its melting point was then
190°, showing that it was the osazone of a hexose. When dissolved in.
Pyridine and alcohol it rotated the plane of polarized light to the left,.,

a phenomenon characteristic of the osazone of glucose. Unfortunately the
anount of substance was too small to perwit the quantitative estimation

of the optical activity, and conclusive proof that glucose is the sugar

from which the polysaccharide is built up must await the purification of nore
material, which is now under way.

It will also be attempted to carry the purification of the solubie
substance still further, in order to establish with certaint- whether the
carbohydrate reactions are due to the soluble substance itself, as now seems

highly probable, or are simply due to an admixed impurity.

 
 

142 ' 4 }
Studies have also been undertaken to learn more concerning the
chemical and immunological properties of the protein constitutents of the
pneumococeus cells. To obtain the bacterial protein as free as possible
from *he other constitutents of the cell, including the type specific
non- protein soluble substance, the following method has been employed.
From bouillon cultures of actively growing pneumococci, the
cells are removed by centrifugation and resuspended in 1/10 volume of
salt solution. Solution of bacterial bodies is immediately effected by
the addition of the minimum lytic amount of bile and autolysis reduced by
carrying out the reaction in the cold. From the clear, slightly alkaline
solution, frecd frama cell detritus by filtration or centrifugation the
protoin is pracipitated by dilute acetic acid, the white floculent pre-
cipitate is washed with distilled water several times, redissolved in
weak alkaline solution and again precipitated by acid. The process is
repeated several times to rid the protein of the soluble substance al~
ready referred to, and the final precipitate rapidly washed with acetona
and then dry ether and dried in vacuo. The prodact thus obtained ina
form of a white powder, is readily solubie in dilute alkali and gives
qualitatively all the protein reactions: Biuret, Millon, phosphorus, etc.
The quanti tative analysis of this substance is being undertaken as rapidly
as sufficient purified material is available. The immunological atudy of
this substance, while it is not yet compicte, has yielded most interesting
results. It has been found that when a solution of this substance is added
to anti-pneumococcus immune serum precipitation occurs. No precipitation
occurs with anti-serun Broduced by immunization with bacteria other than :
pneumococcus. It is, therefore, a specific pneumococcus substance. The
remarkable fact, however, has now developed that this precipitation occurs
no matter which type of anti-pnewumococous serum is employed. It is there-

fore, although species specific, apparently not type spacific. Tmmninza-

 

 

 
tion of animals with this pneumococcus protein substance is now under wey
but conclusive results have not deen obtained.

Tuese studies have resulted in observations which indicate that
immunity to pneusococcus is related to two entirely distinct bacterial
substances. One of them is 2 protein, immnity to which is very specific
as regards pnevmecoccus but is entirely non-specific as regards type. The
second substance is non-protein in mature, in its prosent state of puri-
fication possesses the propertics of a carbohydrate, and is to © very
high dogree type specific. This second sudstance when injected alone is
apparently non-antigenic. These facts have sutzested entirely new con-
ceptions concerning pnevmococcus immunity which may explain a number of
obscure facts, includihg the changes of specificity of pnewnzococcus wita
changes of virulerce. The further study of this problem is now in pro-
gress.

Lunes as Portals of Entry for Bacteria in Infection in Mice.
Dr. Ernest Stillman.

In the previous report it was stated that when mice are 6x~
posed to an atmosphere containing cultures of bacteria in the form of a
fine mist, the bacteria readily penetrates into the lower respiratory
tract. Pnevmococei which have reached the lungs as a result of this pro-
cedure usually disappear within a few hours and give rise to no infection. °
Attempts to cause infection to occur more frequently by means such as
chilling and exposure to cold were without result. It was then attemptsd
to lower local resistance by inducing slight injuries to the mucous memorane
by inhaling ether, alcohol, and atmospheres containing finely divided qaarte

sand or tale in the form of dust. The procedure, however, did not increase
the frequency of infection, It was found that mice could bé easily intoxi-

cated hy breathing an atmosphere containing alcohol and that, if the mice,

 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
  
  
 
 

 

 

 
 

144

while under tne influence of clcvuhol, were allowed to inhale an atmosphe-«

   

conteining pneumococci in suspension there was a marked rise in the inciaenve
of oneunococcus septicemin. As tne direct innalation of an alcoholic at-
mosphere might alter the respiratory rmcosa and this might explain the in-

crcased froquency of infection, mice were intoxicated by injecting a 10%

 

 

alconolic solution intraperitoneally. When mice were intoxicated by this
means and were then exposed to an atmosphere containing pneumococci in sus-
pension there was a definite increase in ths incidence of fatal sopticonia,
but not as great an increase as in the series of mice wnich had inhaled
alcohol. In other experiments mice were first intoxicated by an injection
of alcohol intrapcritoneally; they were then exposed to an atmosphcre con-

taining B. influonz:e in suspension, and then they were allowed te inhale

 

air containing pnewumococcus culture in the form cf spray. In those mico
tho incidencs of pnoumococcus infection was almost as hich as in the mice
intoxicated ov inhalation of alcohc} ond thon exposed to the air containing
pneumococci. A studs of the histological changes which occur in the lungs
following tha inhalation of an atmosphere containing influenza bacilli and
pneumococci has alse besn undertaken. Apparently definite pathological

changes in the lunes occur as a result of the inhalation of 3. influenzae.

ing _in Inrmne Serum and

 

be Growing in Media Containing Bile.
Dr. Dahl.

It was previously demonstrated by Miss Strwker working in this
laboratory that when pneumococe are cultivated repeatedly in media contain-
ing homologous immine serum the bacteria decrease markedly in virulence and
they also become much less sepeific in their immmity reactions. Dr. Aver:

has also shown that when pneumococci are grown in media containing minute

amew.ts of bile the bacteria becomes resistant to the bile, and by gradux! lr
arercasing tae amount of bile esntsinoa “n the nedia ths bacteria may =.
rendcrec highly vesistant to tho blie. ho virulone> ¢f theses bacteric

‘as also found to ba markedly dininisned. Recunbiy Josnioxa worxing with
wouleld has shovm that a sindlar modiftoation eon Le brought asout by grov-
ing Fnewmococci at an increasod temperature, It has furthermore been claimed
by this observer and also by otners that tho immunity response in animals
Following the injection of theao modified bactoria differs from that fol-
lowing the injection of virulent pneunococc!. 4 stuiy has bean undertarcn
for the purpose of extonding those observetions sand if possible of axsinin-
ing the rosults obtained. The observations of Miss Stryker have deen cor
sirmod, and br growth in immune se>un, cultsros of pneumococci bave zy baer
obtained which possess almost no viruierce for miso and which sho litsle
type specificity.

By repeatad cultivation in media containing bile cultures of
preumocceci have also deen obtained waiien nor grow readily in 903 bile.
These cultures have also practically lost their virulence ani tevpe speci.
Ticity.

_ Animals are now { being immmmnized to these minds t ted cultures and
studies are being nade of the properties of the 6 serun, The results of these

studies are not yet conclusive,

Acute Rhoumatic Fever. (Report of Dr. Swift.)

Dr t Boot : Miller.

The types of rheumatic fever studied auring the past six monthe
have been mostly of the subencute and chronic forms; and practically all
of the patients have suffered from carditis. As mentioned in previous re-
ports the chronic form of the disease seens to be demanding more and mor

of our attention,

 

 

 
fot

146
Interesting clinical manifestations, not previously encountered

by us, have been skin rashes. Four patients with skin lesions, commonly
classified under the term, Erythema miltiforne, have been seen recently.
In two instances, these rashes have almost constantly been observed for
two or three months; this wo consider another indication of the per-
Sistence of the infection. In another patient the relapses have been
accompanied by recurrences of the erythema. Three or four patients have
had mumerous subcutaneous nodules which have furnished us mterial for
histopathological studies as well as for ineculation. We are now ree
Moving small pieces of the joint capsule and periarticular tissue for
histopathological study. Wea have also written to various pathologists
in England and in this country requesting material from fatal cases of
rheumatic fever and chorea.

As for as I know, there ig nd comprehensive study of the pa-
thology of rheumatic fever. Phe histopathology of the myocarditis is
well known. The pathology of the subcutaneous nodules is fairly well
Btudied; that of the jointa during sicute asthritis and of the brain in
chorea patients is meagerly desersbea, , as we believe, all of those
lesions are duc to a common ottologta ogsit, there mat ‘be some borrele~
tion betveen the tissue respérises ie the various organs. We fool that, ;
aiready a more or less wnifora type of response has been detected. a. |
is ‘Probable that the aniualising of the rhewmatie. fever virus can 1 only
be determined oy hi etopatholegical studies. |

Observations on relation to x Leukocytosis to tho activity of
infection in these pationts have been contimed. The group of rheumatic
fever patients seen in the winter of 1922~2922 has been followed this
winter. Last year the patients were suffering from aétive infection;

this year they were nearly all apparently well. Our results indicate

 

 
fo
ated
127

that when the white blood count remains consistently below 8,000 the
prognosis is emch more favorable than when it is above this figure.
The leukecyte curve is, therefore, a waluable aid in prognosis: in the
acute infection if leukocytosis persists while the patient is under the
influence of anti-rheumatic drugs it serves as an indication for the
contimiation ef these remedies even though the patient is free fron
symptoms; and in the sub-acute and chronic forms of the disease a per-
sistent leukocytosis serves as an indication for contimued rest, and
supportive treatment. We feel that the results of our investigations
along this line will be helpful to practitioners in treating rheumatic
fever patients. °

The clinical study of neocinchophen (or tolysin) bas been
contimed. There is no doubt that this drug is a valuable addition to
our therapeutic armamentarium, because in most instances marked anti-~
pyretic and anti-arthritic effects can be obtained with doses that are
practically nun-toxic. The recent amouncemont of Barbour and Lozensky
that enormous doses of tolysin could be given to dogs and other labora-
tory animals without producing toxic symptoms may lead practitioners to.
administer too large amounts to patients. We have occasionally observed
distinet evidence of renal irritation in patients; so the same care must
‘be observed with the use of this drug as with the ‘older and cheaper
galicylates.

Dr. Boots and I have completed a series of experiments upon
the subject of joint sensitization. Herry and Faber have previously
claimed that the joints of animals may be rendered more sensitive to
invasion of bacteria fram the blood stream if the joints are previously
injected with small amounts of killed streptococci or with extracts

prepared from these microorganisms. The recent work of Desredka and of

 

 
148
Gay indicates that immunization of tissues may result from local treatment
with small amounts of vaccine. This is the opposite of Harry's and
Faber's point of view. Qur work has failed to demonstrate any hyper~
sensitiveness of joints injected with either vaccine or bacterial ex-
tracts. Joints so treated were about as liable to involvement as the other
joints of the game animal following the intravenous inoculation of these
animals with living streptococci.

Mrs. Lancefield and I have been trying to determine the presence
of immune bodies against non-hemolytic streptococci in the blood of rheu-
matic fever patients and comparing them with similar reaction in patients
suffering from streptococcus viridans endocarditis. This work is still
in the preliminary stage as the methods previously developed de not seem
to be sensitive enoush to detect low concentrations of immune bodies, such
as possibly exist in patients with the first-named disease. We are also
trying to develop methods for the demonstration of antigenic substances

in exudates. If such antizenic substances could be demonstrated in the

arthritic fluid and pleural exudates, we would have a substance with which |

to test for immune bodies in the serum of patients with more doubtful
conditions, and to demonstrate the unity or . plurality of. the rheumatic
infection.

Dr. Miller and I are continuing our efforte ‘to animalize the
virus of. rheumatic fever. As sources of virus we have used (1) the heart
valves from a fatal case; (2) the subcutaneous nodules excised from a
patient with arthritis, chorea and extensive subcutaneous nodules ; (3) the
joint fluid obtained from the knees of patients during the early. stages
of the disease. Rabbits have been the test animals. First the eyes were
used; but it has been impossible consistently to obtain lesions differing

very markedly from those incited in controls.

 

 
Recently, attempts have been made to carry the infection through
successive inoculations of rabbits! testicles. In several instances diffuse
testicular lesions have been induced in series of rabbits with material far
enough removed from the original human source to insure us that tissue from

a haterologous species was not responsible for the injury.

 

 

An extensive series of controls is now being observed, paralleling
those inoculated with rhevwastic material. While it is too early to report
any conclusive results, we can state that in several instances we have ob=
tained microscopic lesions mach more severe in the "rheumatic series"

than in the controls.

 

Chicken-pox. (Report of Dr. Rivers.)
Dr, Rivers and Dr, Tillett:

In undertaking a study of the contagious diseases occurring in
children, varicella was chosen as the one with which to begin for the fol-
lowing reasons: |

1. If produced in animals, there would be a more or less character-
istic lesion to identify it.

 

2. ‘There are at least three other diseases, voccinia, variola and
alastrim, with which it can be compared.

 

3. Relatively little. attention has previously been given to the
disease.

4. There is at present a great deal of interest in ell diseases that
produce vesicles in the skin. _

5. I! spite. of ite mildness this dibeave may hold ‘the key to the
understanding of more serious diseases.

The report will be made under three heads:
A. Study of vaccinia in rabbits.
B. Clinical study of cases of varicella in the hospital.
C. Results of attempts to produce varicella in animals.
A. 5 £ Vac its.

The study of vaccink was undertaken in order to learn the proper

 
150 Py
methods of handling a virus which .. the one causing varicella
might be expected to resemble. It was attempted to answer the following
questions: 1.~ Can vaccine virus injected intravenously be mde to lo-
calize in the skin by shaving ond irritation? Calmette and Guerin
claimed it could be done. We have confirmed their observations. 2.- Does
vaccine virus get into the blood when an animal is vaccinated on the skin
and, if it dees, can it be recovered from the bloed in demonstrable am-
ounts? Ohtawara says the virus gets into the blood and that he was able
to recover it by putting the blood of a vaccinated rabbit into the testi-
cles of a normal rabbit and after four to six days removing the testicles,
grinding them up and testing for the virus on the skin of a normal rabbit.
We have confirmed and extended this work. 3.- What is the appearance
of the cell inclusions in waccinmia? Rabbits’ eves were inoculated with
vaccine virus and removed at different times, ranginz fron seventeen nours
to fourteen days. Various fixatives and staining methods have been em~
ployed, including vitel dyes. We are now familiar with what have been
called inclusions or vaccine bodies. It seems from our observations that
there are several classes of inclusions arising in different ways and that
the inclusions seen under certain conditions of fixation and staining are
not always the sane ae those seen after other fixativea and stains have
been used or the same as those seen in fresh unstained cells, or in fresh
cells stained with vital dyes. Conclusions frém studies employing vital
dyes in fresh normal tissues carmmot be applied to pathological conditions,
such as the vaccinated cornea. Under these circumstances diseased or dead
cells are present even in the freshest specimens collected under the best
conditions. For instance, mitochondria in normal cells do not stain with

brilliant cresyl blue (in proper dilution) but do stain a3 soon as the

cell dies. This dye also reacts differently to fat droplets and vacuoles,

 

 

 

 
ye
depending upon whether the cells are dead or alive Such differences
also occur when Nile blue sulphate is wai. (See Lewis? article in
Am. Jour. Amat.) Mitochondria, apparently undergo marked changes in
the cells of a vaccinated cornea. They become swolien, vesiculated and
clumped in various parts of the cell, especially around the nucleus or
around certain inclusions. This has been demonstrated with brilliant
cresyl blue and jamus green in fresh specimens and with anilin fuchsin
and methyl green after proper fixation.

>. Clinical Study of Cases of Varicella in the Hospits}.

 

Twenty-seven patients with varicella have been aduitted since
the ward was opened. No instance of herpes zoster and varicella oc-
curring in the same family at or about the same time has been noted.
The clinical studies can be discussed under the following headings.

1. The blood picture.

2. Effect of trauma or hyperemia on the localization of the erupt-
ions in the skin.

3. Allergy.

4. The skin lesions.
1.~ The blood picture was followed in twenty-two cases and the

findings are sumsarized as follows:

a.  Leukopenia (below 7,000) occurred in four cases.

bd. White blood cells were normal (7,000 to 9,000) in seven cases,

c. A leukocytosis (above 9,000) occurred in eleverl cases. This
was never marked, The highest count was 14,000; the average
was between 10,000 and 11,000.

a. Twelve patients had an eosinophilia of 4 per cent or more.
The ecsinophiles reached 12 per cent in one instance.
The increase of these cells came during convalescence. The
stools were examined but no parasites or ova were found.

a. e effect of Tra y Hyperemia the Localization t

eruption, The localization of the erustion was certainly influenced by

 

 

 

 

 
me.
ee

1.52 co
- haat]

trauma or hyperemia in three cases. One patient had an almost conflu-
ent eruption pensath adhesive plaste> applied to the ankles to relieve
the pain of a chronic arthritis. The pavient had had a Schick test
recently and the eruptéon appeared earlier at the site of injection
and the vesicles were much larger and more numerous than elsewhere.
In another patient the eruntion was most marked beneath the napkin which
gave evidence of not being changed very frequently. Ina third patien’,
a boy who wore soft collers with his necktie dravm very tignt, there
wos a rarrow band of vesicles around his neck just beneath the tie.
These observations seem to indicate clearly that the virus is dissemina-
tea through the blood stream and localizes especially where the skin
4g irritated. Similar observations have been mentioned casually by a
few writers in regard to varicella and particularly in regard to measles
by Pirquet and in regard to variola by Schick and others. Attempts
were made to induce this localization after patients were admitted to
the hospital by irritating the skin in certain areas. It has been in-
possible, however, to induce this localization under these conditions.
It seema that the irritation must be present before the rash makes its
appearance.

These facts may have some relationship to the occurrence of
herpes zoster and varicella in the same patient at the same tima. They
may also have a bearing ori the cases reported in the literature in which
following attempts to yabdinate against varicella with fresh vesicle
lymph, vesicles have later appeared at the site of vaccination. In
the majority of these cases & mild general eruption appeared at the same
time.

3.- Allergy. No allergic response has been demonstrated in
patients recently recovered from varicella following the application of

fresh vesicle fluid to the scarified skin. Tais may indicate tact tne

 

 

 

 
 

lod

virus is not very concentrated in the vesicle fliia.

4.—- Stidy of tro skin lesicns. -

 

a, Vesicle fluid. Vesicle fluid was co.lacted from lesions at

different stages of development and a cytological siudy was made in fresh
unstained specimens, in fresh specimens stained with different vital dyes
and fat stains, and in sveoimens fixed and stained in many ways. There
are only a few cells in the fluid from young vesicles and these consist
mostly of swollen epithelial cells and only an occasional giant cell.
Only a few inclusions are seén at this time. Later the cells become more
munerovs and are of various kinds. Many are undergoing dJeceneration
characterized by fatty or lipeitdal changes and coagulation necrosis.
Tho mitechondria exhibit a change in size, shape and location in many of
the cells that show degeneration. At this time many s0~ 2iled inclusions
can be made out. In addition to the cytoplasmic changes, there are also
profound alterations in the miclet.

b. Skin. Small piecés of skin have been removed at various stages
jn the development of macules, papules and vesicles, and have been fixed,
stained, and studied in many ways. The following facts are worth noting:

(1) There occurs a definite reaction around the small blood vessels
in the corium just beneath the epidermis. Tis is prooably the earliest
lesion. _ | . | . -

(2) There is a marked fatty ar lipoidal degeneration of the epi-
dermal cells as shown by sudan iii and osmic acid methods. Apparently no
one has stressed this type of degeneration of the cells in the diseases in
which 80 many cell inclusions are found, certainly not in varicella.

(3) There are changes in the mitochondria, 46 swelling, vesiculation
and clumping.

(4) There are cell inclusions, which, after certain fixatives and

 

 

 

 

  
 

stains resemble vaccine bodies. These are net 80 “waerous, however, as
in vaccinia. Some of the grarules and bodies scen in degensrating cells
after treatment with certain fixatives and siains may be directly related
to fatty or lipoidal changes or they may even represent altered mitochon-
dria.

C. Attempts to Transmit Varicella.

Most of the experimenta} work with varicella has been limited
to attempts to vaccinate normal children with fresh vesicle lymph. In
the majority of instances the results have been negative or, when posi-~-
tive, open to criticism because of improper controls. Mm all previous
attexpts to transcit the disease to animals the results ray be considered
negative. In these experiments vesicle lymmh was employed and the skin
or eye of the animal used as the place of inoculation.

In undertaking the present attempts to transmit varicella to
animals we were guided by the following considerations.

It has seemed that the virus might be present in at least ons of
the following locations. It might be present in the vesicle lymph, in the
haso-gharyngeal secretions (especially early in the disease) in the spinal
fluid, or in the ‘blood | “Raperinents: were therefore made with material from
all of these sourtes and notikeys ‘and rabbite were used for the tests. More-
over, it seemed certain that the site of injection of the infected material
would be of great importance. Therefore the virus was injected into the
animals in various places and in various ways. The following are some of
the procedures carried out:

1. Unfiltered nasopharyngeal washings were injected intratracheally
into monkeys. The skin over the abdomen was shaved shortly afterwards.

2, Vesicle fluid was scratched into the eyes and the skinof young

rabbits and young monkeys.

 

 

 

    
   
   

fe aa
3. Vesicle fluid was injected into the brains cf young rabvits and
monkeys and into the trachea of monkevs.

4. Spinal fluid from a patient with vericeila was injected subara-
chnoidally and intravenously into a morkey. The skin over the abdomen was
shaved and scarified.

5. Unfiltered nasal washings were injected into the trachea of
monkeys and blood was injected into the veins at the same time. The skin
over the abdomen was shaved and irritated.

6.  Bloecd from varicella patients was injected intracerebrally into
rabbits.

7. 10 cc. of uncitrated blood from varicella natients was injected
intravenously into rabbits. The skin was shaved and scarified in places
immediately after the injection.

In all of these experiments the results were negative or very
doubtful, It seemed inadvisable, therefcre, to proceed further along these
lines.

Certain clinical observations previously mentioned, however, gave
a ciuo which when followed up have yielded more successful results. It has
been stated that in certain patients the localization of the lesion has
strongly indicated that the virus is present in the blood in considerable
concentration, at least early in the disease. Moreover, the extent of the
eruption may bear no relation to the concentration of the virus in the blood.
On the other hand, all the experiments previously made suggest that the vi-
rus may be in very small amounts in the vesicles.

| Experiments with vaccinia have indicated that the testicles of
rabbits is an excellent place for propagating a foreign virus of this type.

In view of these considerations it was decided to make a serious

attempt to transmit the virus by injeating considerable amounts of blood

 

 

 
trectly from the pationts into the testicles ot rurbits and to persist

in the attempts to arimelize the virus by reneated transfer, even though
no lesions were evident on the first teansfer. Blood waa therafore cbs
tainod from a patient early in the disease anc ocfore coagulation occurred
two cubic centimeters were injected into cach testicle of a raboit. fn
immediate reaction occurred and the swolling persisted aia the testicles
became hard and firm. After five days these tosticles were removed, ground
up in salt solution and 0.5 ce. to 1.5 cc. of the enmision was injected
Ante the testicles of other rabbits. In further transfers inoculation

of the testicular material was sade not only inte the testicles but on

the scarified skin and scarified cornea of rabbits. The results were
quite strikin;. Lesions much resembling those of vaccinia ware opivined
in a mumber of rabbits, both in the akin and in She eye.

A considerable number of experiments similar to the av,ove ve
now boon carricd out. Matorial has boon obtainad from five peticnts and
positive rosults havo now boon obtained by this method with tho material
from throo of the casos. It is ovident tat if the virus of varicella
has boon obtained it closéiy roscmblos that of vaccinia and variola.

Exporimonts to study tho occurrence of immunity and the relation
ef this immunity to that of vaccinia arco now wandor way but dofinits con-
clusions cannot be draw at tho present timo. It is indendod to continue
this study, to learn more concerning the naturo of the virus and tho
characteriatic lesions produced. Many possible applications of this
now imowledgs, such as vaccination against the discaso in children, are,
of courso, obvious. Tae possibility of application of a similar mothod
in the study of other infections discases, such ag alastrim and even

measles, suggosts itself.

 

 

 

 

 

 

 
 

1s

Oxygen Tucrapy in Pnownonis. (Ranuct by or. Binger.)
Dr. Bingor and Dr. brow.

Tho work on oxygon thorapy in ynewalria was anterrapted or the
unfortunate accident in which ths chamocy wis dGestrovcd, Dr. Dingor svarved
at once to plan a now chamber =< tho plans for which ars now somplste. The
chambor is to bo built of anglo iron with glass and shoot iron sides. Ts
is to be locatod on tho 4th floor of the hospital in room 405, fomorly
uscd for hydrotherapy oquipmont. A small 2 cubie/hbacl of tho chamber
has boon built and tostod and found sufficiently leak tight. Tho now
chanbor is to bo fire proof end as loak tight es it can bo made. This
should considcrably rcduco the cost of operation. All wiring and othor
sources of onergy will bo kopt outside the oxygen rich ataospnerc. This
has involved numorous now problems and explein:tns delay in starting op-
crations. Tho chambershould bo completed by spring.

Wo have been fortunate this yrar in having vory four cases of
p neumonia with cyanosis ~ the ones in which oxygen therapy appcars to be
of so much benefit. A fow cases hava beon treated by oxygen insufflation
with a nasal cathoter. This method, while it sometimes gives good practical
'yesults, does not serve as a substitute for tho oxygen chamber and affords
no opportunitr for studying the optiznr: dosage of oxygen.

Experiments have been made with a bed tent constructed of rub=
berized canvas such as used for the construction of air-craft. This tent
is easily portable and should prove to b2 of uso in homes and in such |
hospitals as aro unable to havo chambers. The tent is not yot ready for
use. The construction of it has ocen undertaken by the Connecticut Air
craft Company under the direction of Dr. singer.

Acid=eDase Equilibrium in Pneumonia.

Closcly related to the problem of disturbed respiratory function

 

 
156

in pnoumonia is tho question of the statc of the Vlocd and the pscsonce
or absence of acidosis. Cortain investigators (leans st a1) bolicve
thoy have domonst ted the existenee of acidosis ix ~nowsenia and have
recommended the routine uss of sodium bicarbomts for the purpose of
increasing the C0, binding powor of tho blood. These worcers conssructed
COg absorpticn curve: ani calculated the pil of the blood Sy using an
assumod constant. Binger, Hastings snd Noill hava recsntly rublished
(Arch. of Int lod. Jan,]963,) an articls dosertomg tho untowrd rosults
which may follow the indiscriminats uso of sciiua bicarbonats. Tha pare
ticular caso was one in which the kKidnoys could nct sxcrete bicarbonate.
Thore was rotontion of the salt and o concanifent and serious rotention
of wator resulting in cdema of tho lungs, hydrothorax and gonerslised
afasarca = with profound cyanosis and rapid and shallow breathing. The
caso was brought to tho hospital in this state. Investigation of ‘ne
artcrial blood showed a pronounced alkalosis and anoxemia. Tho pationt
Was trcated in the oxygen chamber and bicarbonate administration was of
course stopped. There was almost i:ediato recovary. The patient is now
well.

This pointed to the desirsbility of reinvestig-ting the condition
of acidebase b-.lnneo in pnoumornia hy more direct mothods. Hastings and -
Neill, Morgan and Binger have studied the arterial blood of a seriss of
cases of pneumonia ~ making direct observations of oxygen and Oly content
and combining power and hydrogen ion concentration by both the eloctro-
metric and coloremetric methods. Noa evidence of ucidosis nos been fount.
lung Volume Studiss.

Method: (a) Tae method for studying lung volume in individurls sufferirs
from shortness of breath has been doscribed and the report will sacti;

appear in the Journal of Experiments] Medicine.

 
(b) The studies on patients with heart diseas2 are completed and

an article on this work has bagn submitted for publication in tac Journal

 

 

of Experimental Medicine. In brief the findings are as folloys: The
reduction in tho vital capaicty of thc lungs obszrvod in conditions of
heart failure is gonorally associutca with a reduction in the total lung
capacity and in the so-called mid-capacity 23 vi2ll. No svidence of om
physema has beon found - a condition described in tho foreign literaturc.
In the carly cases whore reduction of vital capncity is slignt there

ie an inercase in residual air - often sufficicns to oxplain the dim-
intshed vital cxpacity. In tho cases of long strrnding with martcd ovi-

dence of congestion in the pulnon-ry circulation there is usanlly a roe

 

 

' duction in residual air as wcoll. Tho cause and signifionee of onch

onc of tnoso volumo chanzos has been fully discussed and the relations

 

which they boar to the gcneral problem of dyspne-x.
Vv ic Studics Lob. nin.
bi na Dr. 3
The intimate relation which appeared to exist between reduction
of lung volums and dyspnea indicated the desirability of studying this ,

i condition in lobar pneumonia where we know that a good deal of the air

 

content of the lung my be roplaced by solid mitcrial and where the surface
through which. the : respiratory gasos diffiuso is discased. Ta study a con
9° dition in which tho. disonsed. stato gradually disappears and reverts to -
normal seomed an admirable opportunity for loarning same exact facts about
the pathological physiology of respiration.

Now a study of respiration in the pneumonia patient is an oie
tromoly difficult affair as any obstruction or resistance immedictcly
varies the typo of breathing. Furthermore, it was necessary to g2%

graphic curves of respirations while the patient was brorthing atmospheric

 
f

is gy,

air. By modifying 2 large 90 Liter Mssot Spironster and providing for
contimous removal of CO, and supply of 05 at the rate of consumption,

4t was possible to securd such tracings. This mado it feasible to meas-
wre tho voluns of air in the lungs at cny given phase of respiration.
Becauso of the relative constancy of the cxpiratory position as compared
to the inspiratory - lung volum?s have been measured at this position i.c.
the volume of air romiining in the lungs at the end of 4 quict expir-tory
acursion is being studied. Thero appears to be 4 definite relation bet-
ween this valuo and the type of breathing and perhaps - though this is
not yet certain - to the appearance of anoxemio and cycnosis. These
studics are boing mde throughout the course of tho discise and the changes
during resolution and convalescence aro being noted. Tho work is not

yet sufficiently advinced to permit of gonoraliz:tions.

A series of normal individuals are being studicd as well. The
mothod has new beon perfected to a vory satisfactory degroc of accuracy.
Deviations of 100 cc. in 2 litcrs are maximal - ropresonting o maxim:
error of 5%. The semo rolations appear, to obtain in normals i.o. the depth
and rate of pulmomary ventilation seems in some way related to tho volume
of air in tho lig at the end of norma] expiration ~ which, of course, is
a function of the surface arca of respiratory opitheliun. . |

An effort is being made to correlate the size of the resting
expiratory lung volume with some other physical measurement such as sur-
face area (from the DuSois Hoight, Weight formla) or chest mocsuremont -

in order to bs able to predict what a lung volume should 62 for any given

individual,

Report of the Pathologist. (Dr. Sranch)

The routine pathological work has included 9 autopsies, the

y
w

4

 

 

 
 

reorgenization of the Museum and the running of a pathological laboratory _

2s a unit including the training of a technicinn. One of tne autopsics,
a case of subacute bocterial endocarditis due to a hemolytic hemophylic
bicillus, hos been written up for publication with br. Miller.

Apart from the Above work the chiof interest has centered around
the carly pathological lcsions of pnouriceccous prouronia ,

In ordor to obtain som? insizht into the cellular reactions pro-
duced by this organism under vorying conditions, 2 mmbor of guinza pigs
woro first used. Subcutancous injection vas employed on the assumption
that pneumonia is a collulitis. By this method also dosage can be accur.—
tely measured, time periods readily taken into account, and tho sin easily
renoved at differont intervals for histological examin.tion and impression
smears. The difference of reaction in intonsity per timo period in immuno
and older animals could be readily compared with stocx ond young snim-ls.

With this background wo have prococded to study the early his-
tological losions in the lung. Material usod here was obtained from nice
sprayed by Dr. Stillman in his work including those sprayed with pneumo-
cocci, alcohol and pneumococci, influenzs bacillus andi pneumococci, etc.
Further mterial was also obtained from Drs. Avery and Morgan in thelr work
on intra-tracheal injection of rabbits with misopharyngeal washings and

suspensions of infected crushed rabbit lungs. This study is still in

progress.