_ rrr eee

O11

35
streptococci, a system that is being adopted in all parts of the world. The
second has been the guiding motive in investigating sensitive tissues and
cells; and tests have been carried out both in the intact animal and in tis-
sue cultures. A point has now been reached where it is hoped that {t will be
possible to investigate the relationships of two variables - pacterial hyper-
sensitivity and true 4mmunity, in experimental streptococcal diseases and in

rheumatic fever and other human infections.

Pneumonl

Dr. Cole, Dr, Avery and their Associates.

Dr. Cole, Dr. Avery and their associates have devoted a large amount

of time to the clinical study and treatment of pneumonia patients in the wards

of the hospital, and to investigations 4n the laboratories on biological and

chemical problems relating to pneumococcus infection and {mmanity. The

records of the clinical studies provide a wealth of source material for study-

ing the natural history of the disease and form a most valuable asset, which
4s constantly being employed in the formulation of new work and in checking

the results of special investigationss Tho two contributions by Drs. Goodner
and Horsfall and py Dr. Dubos, which are given in detail later, emphasize the
mutual advantages of laboratory activities carried on in close contact with

clinical work, for, in both instances the results of laboratory experimenta~
tion have found direct and practical application in tho clinic.

In addition to the general remarks concerning activities in the
wards and laboratories of the hospital it seems advisable to present a more
detailed account of some work that has been done, (1) by Dr. Goodner and Dr.
Horsfall, associates of Dr. Avery, (2) by Dr. Dubos also an associate of Dr.
Avery, and (3) by Dr. Smadel and Dr. Farr, associates of Dr. Swift and Dr.

Van Slyke respectively.

 

 

 

eetbee et

 

 

 

 

 

 
36

Type specific antipneumococcus rabbit serum in the treatment of lo-
bar pneumonia. The past quarter of a century has seen enormous advances in
the understanding of lobar pneumonia. It is now recognized that lobar pneu-
monia is not one disease, but a long series of maladies presenting similar
symptoms caused by a number of different, although related, bacteria. Much
is now known of these bactoria, the pneumococel, and of tho way jn which ex-
traordinarily severe infections are produced by then. Much also has been
learned of mothods of specific therapy, for it was soon recognized that each
of these forms of pneumonia must bo troated with a special agent, each agent
peing gpocifically directod against the particular variety of pneumococcus
causing the infection. Tho dcvelopment of specific sorum therapy by. Cole and
his associates forms one of the most important chapters in the history of this
hospital.

Specific serum therapy is based essentially on the following obser-
vation: If patients spontaneously recover from lobar pneumonia, 4t is almost
invariably found that at the time of recovery there appear in the patients!
blood new substances called antibodies, which are capable of reacting with
the particular microorganism that caused the anfection. It was reasoned from
this observation that recovery might be hastened and the disease aborted if
some of these antibodies could be supplied from another source; many patients
aie before they can develop their own immune substances, In order to test
thie possibility, horses were {mmunized with dead pneumococci, since these
serve as well as living, and the blood serum of these horses was administered
to patients. This forn of therapy, 4f administered early in the course of the
disease, proved remarkably successful in infections caused by four of the 32
varieties of the pneumococcus. The mortality rates were definitely lowered

in infection with these four types, but even here the treatment left much to

be desired.

 

 

 

 

 

 
313
37

As a natural part in the development of specific therapeutic measures
came the question of whether or not the horse was the most desirable source of
the antibodies. It has only recently been recognized that the immune serum
from one animal species may possess propertics remarkably dissimilar to those
of the immuno scrum from another apocios. Most of these differonces reside in
the antibodies thomselves. Ono may take as an oxamplo tho immune scrum fron
the horse and the rabbit. Both possess antibodios which have the common
property of reacting with the pneumococcus and its chemical constituents. Bo-
yond that, all similarity is lost, The number of differences now rocognized
ig over thirty. In considoring these differences in antibodics it becane ap~
parent, in theory at least, that tho inmune rabbit serun should sorve as &
much nore efficient thorapeutic agent than is immuno horse scrun in treating
hunan lobar pneunonia. This subject is gsonewhat technical and it is unneces-
sary that it be discussed at length in this place, put as a simple exanple the
implications of one of the differences may be nentioned. The antibody in in-
mune rabbit serun has a dianetor one-third the length of that of the antibody
of immune horse serum. Because of this snaller size, it was reasoned, the
rabbit antibodies night be better able to ponetrate the tissues and reach the
site of infection nore readily than would the horse antibodies. Since pneu-
nonic consolidation 49 prinarily an extravascular lesion, as too are nost of
the various complications resulting fron it, any agont which is to act local-
ly on the lesion or {ts cause must do 80 after penetrating tissues.

These theoretical deductions were exceedingly interesting, but the
effort to prove their validity by clinical demonstration has 4nvolved many
problems. For example, it has long been known that rabbits are readily im-
munized by means of injections of pneumococci, but the use of rabbit serum for
the treatment of human lobar pneumonia demanded an intensive investigation.

It might at first sight appear to be pronibitively expensive to use the serum

 

watgng
ats
 

_f small animals, but as the work progressed it became apparent that the im-
une rabbit serum could be produced at a comparatively low cost. This was an
vaportant point, since the cost of concentrated horse serum has made the treat-
cunt of lobar pneumonia somewhat impractical from an economic standpoint.

A second problem of considerable importance had to do with the
toxicity of immune rabbit serum, for with the treatment of the first patient
it was recognized that this material in a raw or untreated form could produce
the most alarming chill reactions. Considerable progress has been made in
dealing with this problen; methods have been devised for reducing or eliminat-
ing this toxic quality, and methods of testing for ite prosence have been made
available.

The results of the clinical application of type specific jmmine rab-
bit serum in the treatment of lobar pneumonia have justified the hopes gained
from the theoretical studies which preceded its adoption. Sera have been pro-
duced against nine of the more common varieties of pneumococci and these sera
have been used in the treatment of about 70 patients suffering with lobar
pneumonia. The results have been good. Among more than fifty patients suf
foring with lobar pneumonias due to pneumococcus Types I, II, v, VI, VII,
VIII, XIV, XVIII, there has been but one death and this occurred in a patient
five weeks convalescent from pneumonia, In untreated patients with similar
type distribution the death rate would have amounted to about 34 per cent.

With lobar pneumonia due to Pneumococcus Type III the immune rabbit serun

 

has not proven to be wnifornmly successful, but with certain {improvements in

 

the quality of the serum and in the methods of administration the therapeutic
possibilities with this type have becone very pronising.

In evaluating the advantages of antipneumococcus rabbit serum as 4 |
therapeutic agent one mast consider not only the mortality rates but what ac-

tually happens in individual cases. With antipneumococcus horse serun great

 

eT

 

 

 
a

315
39

stress was placed on the possibility of lowering the mortality rates. With
this new serum, so successful has been its application that stress has come
to be placed on the matter of prompt recovery. In the last several cases
treated with imaune rabbit serum in this hospital, the average time from the
first injection of serum until the crisis was less than nine hours. In many
patients normel temperature, pulse, and respiration were regained in as short
a timc as five hours after serum wos administcred. To one familiar with the
results obtained with immune horse serum these results are striking.

Another rosult which had been anticipated from theoretical grounds
4g that the numbor of complications has been very lov in this sories of treat-
ed patients. Empyema, the accumulation of pus in the pleural cavity, has oc-
curred only once and this was in an individual with defective circulation. It
has been possible to demonstrate that the specific antibodies of the immune
rabbit serum penetrate into the infected fluids which lead to this complica-
tion and bring about sterilization.

An acquaintance with the details of this subject tompts the conclu-
sion that considerable advance has been nade in the troatment of an infectious

disease, lobar pneumonia, which is one of the greatest causes of death.

The Production of Specific Bacterial Enzymes e
Organic compounds exhibit a remarkable specificity in their physio-

logical action. Unfortunately the chemical methods evailable for the identi-

 

fication and quantitative analysis of these compounds are on the contrary very
non-specific. Many of these methods utilize reagents developed for certain

chemical groups of general occurrence and which may be common to many differ-

ent substances otherwise unrelated. The lack of specific chemical mothods
has therefore rendered more aifficult the study of physiologically active

substances, especially when they are present in gmall amounts in complex tis-
ee] EES

40
316

sues and biological fluids.

It is known that the lower forms of life (bacteria and molds in par-
ticular) manifest a great specificity in their ability to decompose different
organic substances. Since practically all organic matter in nature eventually
undergoes microbial decomposition, and in view of the fact that microorganisms
act through the agency of their enzymes, one may infer that with proper tech-
niques it should be possible to find jn the microbial world a number of enzymes
adapted to the decomposition of most types of organic compounds.

Tyo entirely different problems have offered the opportunity of
demonstrating that bacterial enzymes specifically directed against given sub-
stances can indeed be produced and that these enzymes can be used to great ad-
vantage as reagents in biological studios.

A few years ago, an onzyme capabla of decomposing the capsular

polysaccharide of Typo III pnoumococcus was extracted from the cells of an
unrelated bacterial specics. The enzyme is very specific in its action
against the polysaccharide, and was used in an analysis of the rolo played by
this cell constitucnt in dotermining thao {mmunological specificity and in con=-
ditioning the virulence of cncapsulated pneumococci.

More recently, in the course of studies on renal function, it be- !
came necessary to develop 4 method for the quantitative estimation of the
very small amounts of creatinine present in biood. The identification and }

|

analysis of creatinine in biological fluids have chiefly depended on colori-

metric methods which are 80 non-specific, that many authors deny the very
presence of creatinine in the circulating blood. In an attempt to solve this
problem, two bacterial enzymes have been prepared which exhibit a remarkable
specificity for creatinine, decomposing it completely after a short incuba-

tion, but failing to attack other substances which are closely related to it

and which give the same color reactions. With the holp of these enzymes, it
_._———————————_——sts—t—<‘“i—i—s—s—s—s—sS

Ol?

41
has been demonstrated that creatinine is indeed present in blood plasma and in
the erythrocytes. Quantitative studies of the amounts of creatinine present
in the blood and urine of normal individuals and nephritic patients have also
led to the conclusion that other substances (probably phenols), often confused
with creatinine because they give the same test with the non-specific colori-
metric method so far employed, accumulate in the blood during uremia and fail
to appear in the urine. This observation indicates a marked impairment in the
ronal oxeretion of toxic compounds and may afford a test of the efficiency of
kidney function.

The two examples presented above illustrate the possible applica-
tions of specific bacterial enzymes in the study of biological problems. The
principles of the methods used in the preparation of these enzymes will now be
considered, with special reference to creatinine.

No animal or plant tissue 4g known to decompose creatinine. It is
obvious, however, that creatinine does not accumulate in nature and therefore
mist undergo decomposition probably through the agency of microorganisms. To
test this assumption, creatinine was added to samples of a number of aifferent
experimental conditions obtained by varying systomatically the factors of tem~
perature, hunidity, aeration, reaction, etc. It waa found that creatinine
was rapidly decomposed in certain samples of soil incubated acrobically at
37°C and at neutral and acid reactions. In order to favor the growth of the
microbial species specifically adapted to the decomposition of croatinino,
the mixtures in which this material had undergone decomposition were inocu-
lated into a variety of liquid media in which creatinine was the sole organic
compound present. Again, croatinine was aecomposed in some of these prepara
tions and by repeated transfers in the sane media, four activo bacterial
species were eventually isolated in pure culture. Only two of these cultures

(NC and HR) have been carefully studied.
318

42

It was found that these two cultures grow well on common peptone
media; but, whereas the creatinine splitting enzyme was readily formed when
the organisms were compelled to use creatinine in the course of their growth,
only little or no specific enzyme appeared under other conditions. A similar
situation had already been observed jn the case of the bacterium ahich decom-
poses the capsular polysaccharide of Type III Pneumococcus. These so-called
"adaptive enzymes", which are formed only as a response to the presence of a
definite substrate in the medium, appear to exhibit a romarkable degree of
specificity in their action. This is illustrated in the few following cxam—
ples. Creatinine is 3-Methyl glycocyamidine. Tho mere presence of an addi-
tional methyl or acetyl group in the molecule completely inhibits both enzyme
NC and HR. The removal of the methyl group in position 3 {leaving glycocyami-
dine) decreases the activity of both enzymes by 90 per cent. The shift of the
methyl group from position 3 to position 5 retards considerably the action of
enzyme NC and completely inhibits enzyme BR. Finally, substances which give
the same color reactions as creatinine but are otherwise unrelated in struc-
ture are not attacked at all by either enzyme.

The production of a definite enzyme by bacteria as 4 response to
the presence of the corresponding substrate in the medium 4s a striking exan-
ple of specific adaptation, and may eventually be used in an analysis of this
phenomenon, But in the meantime it may also offer to the biochemist a method
for the preparation of reagents specifically adapted to the study of many

types of organic compounds.

Nephritis
Dr. Van Slyke and Associates.
The work of Dr. Van Slyke and his associates is centered chiefly on

problems arising from a study of nephritis. These involve: (1) Clinical work