18

show very clearly that the elementary bodies of vaccinia both in the Ti-
selius apparatus and the analytical centrifuge act in the same way as

other particles do.  This was demonstrated by making collodion and glass
particles of the same size as elementary bodies, coating them with various

proteins, including specific ones from vaccinia, and investigating their
behavior in the two apparatuses when mixed with elementary bodies of vac-

cinia.

    During the course of the chemical studies of vaccinia made by
Dr. Hoagland it was necessary to devise micromethods for the accurate es-

timation of certain substances,  As a result of this necessity micromethods
are now available for the determination of phosphate, sulfate and magne-

SiUl5.  Only 0.5 microgram of phosphorus is required for an accurate de-

termination.  As little as 20 micrograms of sulfate can now be determined

accurately.  Ten micrograms of magnesium yield an error less than 2 per

cent with the new method.

      PNEUSI0NI.A

Dr. Avery and Associates.

    Dr. Avery and his associates have continued their work on pneu-

monia and the pnewnococcus.  Some of the work described in this report is

a continuation of that reported last year , at which time it uas noted that
drugs of the sulfonamide group had to a large extent replaced the use of

antipneumococcus serum.  It has been previously shown that dnrg-fast (re-
sistant to the drug) strains of pneumococci can be developed in the labora-

tory.  Inasmuch as all patients receiving sulfonamide drugs do not recover
and since some recover more promptly than others, it was thought advisable
to determine whether there is a correlation betTeen clinical results in the

i

i
1


19

use of drugs and degrees of drug fastness in the organisms isolated from

the patients at different times in the course of the disease.  Twenty-

two patients with pneumonia were investigated in this manner. A compari-

son of the results obtained in these patients showed that there is a cor-

relation.  Furthor studies arc plannod to dotormino the conditions undor
which pncumococci dovolop resistance to the sulfonamide drugs and the na-

ture of the alterations brought about in the microorganisms when this

phenomenon occurs.

     In last year's report it was stated that in certain materials,
particularly in pus, there are inhibitors to the bacteriostatic (power to
prevent gronth) action of the sulfonamide drugs and that one of the in-

hibitors had been isolated and partially purified.  In the meantime, re-

ports from England have stated that p-aminobenzoic acid acts as a power-

ful sulfonamide inhibitor.  Vorkers in Dr. Avery's laboratory in attempt-

ing to correlate these findings with their own and to determine as accu-
rately as possible whether p-aminobenzoic acid alone is responsible for

all inhibition, have carried on work similar to that of Dr. Dubos in the

development of adaptive snsymes.  As a result of these efforts, Dr. Mirick

has obtained from a soil bacillus an enzyme which will more or less spe-

cifically modify p-aminobeneoic acid so that it will no longer act as a

sulfonamide inhibitor.  The use of this adaptive enzyme has clearly ShOm

that some inhibitors are of the general nature of p-aminobenzoic acid,

while others certainly,are not.  This enzyme obviously affords a very use-

ful tool for the further study of the nature and action of the sulfonamide

inhibitors and for the detection of minute amounts of p-aminobenzoic acid.

     Since the introduction of the sulfonamide drugs into the arma-

mentarium of the physician, arguments regarding how these chemical agents

---  -- --- ---~-- --...-_ --.-____ -._ __..-                       ___~ .-.--..-


20

effect a cure have arisen.  Some contend that the drugs have a direct ac-

tion on the pnewnococcus, while others hold that immunological forces ac-

tually bring about the cure, eliminating pneumococci held in check by the

bacteriostatic action of the drugs.  This latter view is held by Dr. Avery

and his associates, and during the year certain experimental findings in
rabbits give furthor support to the idea that in man effective sulfonamide
drug therapy is intimately associated with the development of active im-

muni ty.

     The work of Dr. Dubos and Dr. Hot&kiss concerning bactericidal
eubatancea obtained from a soil bacillus (B. brevis) has kept the Hospi-

tal in the forefront in the field of chemotherapy.  Remarks concerning

these activities were made last year;  since then the work has continued

actively and two substances, gramicidin and the hydrochloride of tyroci-
dine,have been obtained in crystalline and in highly purified form. Both

of these substances are polypeptides;  the former ie active against all
Gram-positive bacteria so far tested except the tubercle bacillus, while
the latter is active against both Gram-positive and Gram-negative bacteria.
Further work is in progress to ascertain fuller knowledge concerning the
structure of these two substances and the manner in which they act to kill

microorganisms.  Early in the work it was found that tyrocidine would not
act in viva (in the living body), and that gramicidin had a limited action
Because of the
in vivo, particularly against blood-stream infections.
toxicity of these compounds and their limited value under certain condi-

tions, they have not been employed in the treatment of pneumonia in human

beings.  Recently an explanation has been found of why gramicidin probably
is ineffective against Gram-negative organisms and why it is impotent

against Gram-positive organisms in the blood stream.  Dr. Folch-Pi on Dr.


21

Van Slyke's service has discovered a new type of cephalin (belonging to

the family of fats) which is non knovm as l'amino acid cephalin" and has

demonstrated the presence of this substance in the blood stream. During

the course of Rome work Dr. Dubos and Dr. Coburn shoved that the l'amino

acid cephalin" strongly inhibits the action of gramicidin.  It is also

known that Gram-negative organisms, against nhich gramlcidin is impotent,

contain a polypeptide-polysaccharide-phospholipid complex.  This complex

obtained from dysentery organiams and colon bacilli Is very active in in-
hibiting the effect of gramicidin on microorganisms. Whether this inhibi-
tory action is due to the presence of the phospholipid cephalin in the com-
plex removed from the Gram-negative organisms cannot be considered as es-
tablished, but it is interesting to note that the cellular structure which

remaine after removal of the complex is without effect upon the action of

gramicidin.

     A number of years ago Dr. Avery and his associates found in the
blood of patients acutely ill with pneumonia a protein which was desig-
nated as C-reactive (C is a sugar found in bodiele of pneumococci) protein

and which is not present in the blood of normal individuals.  The protein

is an albumin -- this differentiates it from ordinary antibodies which are

globulin -- and is demonstrable by precipitation when it is mixed with the
C-polysaccharide obtained from pneumococci. Later it was found that C-
protein occurs not only in. pneumonia patients, but in the blood of individ-

uals suffering from a number of acute and chronic infections.  In addition,

they showed that a specific antiserum can be made against the C-protein
and that through the use of this antiserum much smaller quantities of it
can be detected than by means of the precipitation reaction in which the C-

I

poiysaccharide 15 employed.  During the past year, using an antiserum against  i
                1
                                                                           ! *


22

the C-protein for its detection in the serum of patients, workers in Dr.
Avery's labosatory were able to show that there is a closer correlation
betrveen the presence of C-protein and the clinical condition of the pa-
tient than thero is between the clinical condition of the patient and the

mhite blood cell count or the sedimentation rate of the red blood tolls.

All of this may sound very technical, but it means that there has been
discovered in the blood serum of patients suffering from infectious dis-

eases another substance which was previously unknown and which is not

demonstrable in the blood of normal individuals. This discovery offers
another tool for the study of what goes on in the body of a person suffer-

ing from an infection, something in which all research physicians are

greatly interested.

    The capacity of pneumococcl to grow and produce disease in the

animal body is conditioned by, if not wholly dependent upon, the activity
of the particular group of enzymes concerned in the smthesis of the cell

capsule.  Capsular synthesis is most highly developed end the product of
its activity most pronounced in cells best adapted to growth in the animal

                                    I
body.  The presence in the capsule of a chemically unique and serological-
ly reactive polysaccharide (complex eugar) confers upon the co11 a highly
selective specificity which makee possible the differentiation of sharply
defined and specific types within the species. The enzymes responsible
for capsular synthesis can be reversibly inactivated by known changes in
environmental conditions without impairing the viability of the microor-

ganisms .  The selective inactivation of this particular function results

in the loss of capsules, together with the consequent loss of type speci-
ficity and disease-producing properties. Under these conditions highly         *

pathogenic microorganisms are reduced to a state in which they are no longer


                                                   23

capable of inducing disease in animals highly susceptible to fatal infec-

tion with Ae oriL,inarlji encaijsalated parent strain,

     Important and essential as capsular synthesis is to the disease-
producing properties of pneumococci in the living host, this function is
not vital to the life and growth of the microorganisms outside the animal

body, since cells in rvhich the formation of capsules has been inhibited
are still capable of carrying on the vegetative processes of metabolism

and multiplication in artificial media.  Capsule formation may be regarded,
therefore, as an adaptive mechanism whereby the bacterial cell seeks to

protect itself against the defense reactions of the host.
     For a number of years it has been known that by experimental
means a pneumococcus that is virulent and surrounded by a capsule made up
of complex sugars can be induced to lose its capsule and become avirulent.

Furthermore, it has been possible by the proper means to induce a Type I

Pneumococcus to become a Type III Pneumococcus.  Much of this work has been

done in Dr. Avery's laboratory.  He has always been interested in the trans-
formation of one type of pneumococcus into another and from time to time

has sought, along with his associates, the substance responsible for this

tranef ozmation.  Progreea hae been made gradually, and within the last year
Dr. &cLeod and Dr. Avery have been able to obtain from cell-free extracts
of encapsulated pneumococci a substance which seems to be an ester, and
which in very sm~I.1 quantities Ss active in bringing about transformation

of one type of pneumococcus to another.
    Tne study is being continued with the hope that knowledge of this
important cellular mechanism may lead to a better understanding of the prin-
ciples involved in certain induced variations of living cells, not only Of
the pneumococcus, but also those of other biological systems. Furthermore,


                                                         24

it is possible that knowledge pertaining to the nature of the substances

which serve as activators and inhibitors of the capsule-producing enzymes

might afford a specific approach to the suppression of the capsular func-

tion, upon the activity of which the disease-producing properties of the

pneumococcus depends.  Such a statement might be interpreted as dealing

with a new approach to the prevention and cure of pneumonia.

      For a number of years Dr. Goebel and his associates have been

studying the chemical structure of the complex sugars that make up the cap-

sules of pneumococci.  In a previous report it was stated that the strut-

ture of the Type III polysaccharide had been determined.  Dr. Goebel was

not satisfied with the evidence and proceeded to obtain confirmation Of

his findings by a different chemical approach. This work has been com-

pleted and his previous findings have boen fully substantiated, and now

the structure of at least one of the complex capsular sugars of tho pneumo-

coccus is firmly eatablishod.

      In 1911 Forssman showed that in certain animal tissues, for in-

stance horse kidney, a substance or antigen occurs which when injected in-

to rabbits produces antibodies that agglutinate sheep red cells. This an-

tigen has come to be known as the Forssman or heterophile antigen. Some

years after this discovery, it was shown that pneumococcus cells and cer-

tain other bacteria contain the antigen.  Inasmuch as the workers in Dr.

Avery's laboratory have always been interested in studying all of the

components of the pneumococcus, Dr. Goebel undertook further investigation

of the heterophile antigen of this organism and has been able to obtain it

in a relatively pure state.  Early in the work he had difficulty in sepa-

rating the antigen from tne complex sugar of the body of the pneumococcus

which has been designated as C-polysaccharide.  He finally devised a method


of separating the two substances and now believes that the Forssman anti-

gen is a cotlplex sugar somem&t similar to the C-polysaccharide, yet dif-

fering from the latter in that it contains a chemical grouping not pos-

sessed by the former.  Further rrork will be carried on with this substance


in the hope of obtaining a better understanding of the pneumococcus cell.

  RHEUMAT I C FE'@R

Dr. Swift and Associates.

of the

year ' 8

Dr. %ift and his associates have continued their investigations

ralation of homolytic streptococci to rheumatic fever.  In last

report it was statod that attempts had been made to confirm obser-

vations of British workers to the effect that hetnolytic streptococci are

obtained at autopsy from heart valves injured by rheumatic fever.  The

evidence at that time seemed fairly conclusive that the British observers

were in error and that the streptococci obtained by them were not of etio-

logical significance but contaminants encountered because of a lack of suf-

ficient aseptic technique in the performance of the autopsies. This work

has beon continued with an improvement in tho technique of porforming au-

topsies;  in fact, Dr. Swift and his associates now employ strict SW&~

technique in the performance of autopsies. The results of this additional

work substantiate their previous findings, because in the presonce of prop-

er technique hemolytic streptococci have not been found in rheumatic heart

valves.

     The negative findings just mentioned do not invalidate the con-

ception that the hemolytic streptococcus is the cause of rheumatic fever.

In fact, most workers recognize that this organism is in some way associ-

ated with t`he disease.  However, the nature of the association is not

clear.  In order to throw light upon this problem, Dr. Swift and his asso-

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