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 nom been reached where it is hoped that it will be

possible to investigate the relationships of two variables - bacterial hyper-

sensitivity and true immunity, in experimental stroptococcal diseases and in

rheumatic fever and other human infectione.

Pneumonia

Dr. C,ole, Dr, Avow and thoir 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 ward8
of the hospital, and to Investigations In the laboratories on biological and
chemical problems relating to pneumococcus infection and immunity. The
r8COrd8 of the clinical 8tUdie8 provide a wealth of source material for study-
ing the natural hi8tOI-y of the disease and form a most valuable aeeet, which
is constantly being omployed in the formulation of now work and in checking
the results of special investigations, Tho two contributions by Drs. Goodner
and Horsfall and by Dr. Dubos,mhich are given in dctail later, emphasize the
mutual advantages of laboratory activities carried on in close contact with
clinical work, for, in both instancas the results of laboratory experimenta-

tion have`found direct and practical applioation in tho clinic.

In addition to the general remark8 concerning activities in the

wards and laboratories of the hoepital it seems advisable to present a more
detailed account of some work that hae been done, (1) by Dr. Goodner and Dr.
, Horsfall, associates of Dr. Avery, (2) by Dr. Dubos also an assooiate of Dr.
Avery, and (3) by Dr. Smadel and Dr. Farr, associates of Dr. Swift and Dr.

Van Slyke respectively.


312

36

     Tme specific antipneumococcus rabbit serum in the treatment of lo-

bar pneumonia.  The past quarter of a century ha8 seen enormous advances in

the understanding of lobar pneumonia.  It is now recognized that lobar pneu-

monia is not one dieease, but a long series of maladies .preeenting similar
symptoms caused by a number of different, although related, bacteria.  Much

is now known of these bacteria, the pneumococci, and of tho way in which ex-
traordinarily severs infections are produced by them. Much also has been

learnod of mothode of specific therapy, for it was coon recognized that each
of thasa forms of pneumonia must bo troatod with a special agont, each agent
being specifically diroctod against tho particular variety of pnoumococcus

causing the infection.  Tho dcvolopmont of specific sorum therapy by.Cole and
his associates forms ono of tho 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, it is almost
invariably found that at the time of recovery there appear in the patients'
blood new substances called antibOdi88, which are capable of reacting with
the particular microorganism that caused the infection. 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 soUrcei  many patients

die before they can develop their own immUne SUbBtaWe8,  In order to t88t
this possibility, horses were immunized with dead pneumococci, since these
serve a8 well as living, and the blood serum of these horses wae administered

to patients.  This form of therapy, if 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 nrith these four types, but even her8 the treatment left much to

be desired.

._-- -....-"....V1C.
                ..I ._ ,._ L __ _.,.-   _,         --- .- _--__


     As a natural part in the development of specific therapeutic measures

came the question of whether or not the horse was the moat desirable source of

the antibodies.  It has only recently been recognized that the immune serum

from one animal spccios may possess properties remarkably dissimilar to those

of the immune strum from anothor spocios.  Most of these differonces reside in

the antibodies thornselves.  Ono may take as an oxamplo tho immune swum from

thu horse and the rabbit.  Both possess antibadios which have the common
property of reacting with the pneumococcus and its chemical constituents. Bo-

pond that, all similarity is lost,  The numbor of differences nom rccognizcd

is ovor thirty,  In considorlng these differences In antibodfos it became ap-

parent, in thoory at least, that tho immune rabbit serum should serve as a

much more efficient therapeutic agent than is imnuno horse swum in treating

human lobar pneumonia.  This subject is sonewhat technical and it is unneces-

sary that it be discussed at length in this place, but as a simple example the

implications of one of the differences may be xentionod.  The antibody in im-

mune rabbit serum has a diemotor one-third the length of that of the antibody

of immune horse serum.  Because of this smaller `size, it was reasoned, the

rabbit antibodies might bo botter able to penetrate the tissues and reach the

site of infection more readily than would the horse antibodies.  Since pneu-

monic consolidation is primarily an extravascular lesion, as too are most Of
the various complications resulting from it, any agent which is to act local-

ly on the lesion or its cause must do so after penetrating tissues.
     These theoretical deductions were exceedingly intereating, but the

effort to prove their validity by clinical demonstration has involved 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 prohibitively expensive to use the serum


.i' small animals, but as the work progressed it became apparent that the im-

,:ln.e rabbit serum could be produced at a comparatively low cost.  This was an

r,;lportant point, since the cost of concentrated horse serum has made the treat-

.!<:nt 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

cioaling with this problem;  methods have been devised for reducing or eliminat-
ing this toxic quality, and methods of testing for its presence have been made

availabla.

    The results of the clinical application of type specific immune 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 pnewnococcus Typos 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 serum

has not proven to be uniformly successful, but with certain improvements in
the quality of the serum and in the methods of administration the therapeutic
possibilities with this type have become very promising,
     In evaluating the advantages of antipneumococcus rabbit serum as a
therapeutic agent one must consider not only the mortality rates but what ac-

tually happens in individual cases.  With antipneumococcus horse serum great


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 nith immune rabbit serum in this hospital, the average time from the

first injection of strum until the crisis was less than nine hours.  In many

patients normal temperature, pulse, and rospiration were regained in as short

a time as fivo hours after strum was administorcd.  To one familiar with the

results obtained with immune horse serum these results aro striking.

     Another rosult which had bcon anticipated from thcorotical grounds

is that the numbor of complications has bcon vury low in this sorics of treat-

ed patients.  Empyema, the accumulation of pus in the pleural cavity, has OC-
curred only once and this nae in an individual with defective circulation. It
has been possible to demonstrate that the specific antibodies of the immune
rabbit 8erum penetrate into the infected fluids which lead to this complica-

tion and bring about sterilization.

    An acquaintance with the details of this subject tempts the conclu-
sion that considerable advance has been nade in the troatnent of an infectious
diseaso, lobar pneumonia, which is ono of the greatest causes of death,

The Production pf Specific Bacterial Enzymes

    Organic compounds exhibit a remarkable,specificity in their physio-

logical action.  Unfortunately the chemical methods e,vailable for the ihenti-
fication and quantitative analysis of these compounds are on the contrary very

non-specific.  Many of these methods utilize reagent8 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 mothode

has therefore rendered more difficult the study of physiologically active

substances, especially when they are present in small amounts in complex tis-


sues and biological fluids.

     It is known that the lower forms of life (bacteria and mold8 in par-

ticular) manifest a great specificity in their ability to decompose different

organic substances.  Since practically all organic matter in nature eventually

undergo88 microbial d8COmpO8itiOn, and in view of the fact that microorganisms

act through the agency of their enzymes, ono may infer that with proper tech-
niques it should be poasible to find in the microbial world a number of enzymes

adapted to the decomposition of most types of organic compounds.
     Two entirely different problems have offered the opportunity of
demonstrating that bacterial enzymes specifically directed against given sub-
stances can indeed bo produced and that these enzymes can be usod to great ad-

vantage as reagents in biological studios.

      A fen years ago, an onzymo capable of decomposing the capsular
polysaccharido of Typo III pnoumococcus was extracted from the cells of an

unrelated bacterial spccics.  The enzyme is very specific in its action

against the polysaccharidc, and was used in N? analysis of the rolo played by

this cell constituent in dotermining tho immunolo&.c~l specificity and in con-

ditioning the virulence of cncapsulatcd pncumococci.
     baore recently, in the course of studies on renal function, it be-
came necessary to develop a method for the quantitative estimation of the
very small amounts of creatinine present in blood. The identification and
analysis of creatinine in biological fluids have chiefly depended on colori-
metric methods which are so non-specific, that many authors dew the very

presence of creatinfne in the circulating blood.  In an attempt to solee this
problem, two bacterial enzymes have been propared which exhibit a remarkable

specificity for croatinine, 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 those enzymes, it


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 ana urine of normal individuals and nephritic patients have also
led to the conclusion that other substances (probably phenols), often confused

with creatlnine 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 lmpairmont In the
ranal oxcrotion of toxic compounds and may afford a tost of the efficloncy of

kidney function.

    The tmo examples presented above illustrate the possible applica-
tions of specific bacterial enzymes in the study of biological problems. The
principles of the methods usod In the preparation of these enzymes will now be

considered, with special reference to creatinine.

     No animal or plant tissue is known to docompose croatinino.  It is

obvious, however, that creatinine does not accumulate in nature and therefore
must undergo decomposition probably through, the agency of microorganisms. To

test this assumption, creatinine was added to samples of a number of different
experimental conditions obtained by varying systematically the factors of tem-

perature, humidity, aoration, reaction, etc.  It was found that creatinine

was rapidly decomposed in certain samples of soil incubated aerobically at

37OC and at neutral and acid reactions.  In order to favor the gronth of the
microbial species specifically adapted to the decomposition of croatinino,
the mixtures in which this material had undorgono docomposition were inocu-
lated into a variety of liquid media in which creatlnine wan the sole organic

00mp0~a present.  Again, croatinino was aOOOmpOsOa in some of these prspara-
tions and by repeated transfers in the same nedia, four active bacterial
species mcro ovantually isolated in pure culture.  Only tao of these cultures

(NC and HR) have boen carefully studied.

" I._-  -. ..I)
              --


42

It was found that these two cultures grow well on common peptone

media;  but, whereas the creatinine splitting enzyme nas readily formed when
the organisms were compelled to use creatlnine in the course of their growth,
only little or no specific enzyme appeared under other conditions.  A similar

situation had already been observed in the case of the bacterium which decom-

poses the capsular polyaaccharide of Type III Pnoumococcus.  These so-called

"adaptive enzymes", which are formed only as a response to the presence Of a
definite substrate in the medium, appear to oxhibit a romarkablo degree of

specificity in thoir action.  This is Illustrated in tho few follorring oxam-

ples.  Creatinino is 3-Methyl glycocyamidino. The mero prossnce 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 poeition 5 retards considerably the action of
enzyme NC and completely inhibits enzyme HR.  Finally, substances which give

the same color reactions as creatinlne but are otherwise unrelated in struc-

turo are not attacked at all by either enzyme.
    The product&on of a definite enzyme by bacteria as a response to

the presence of the corresponding substrate in the medium is a striking exam-

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 tp the study of many

types of organic compounds.

Nochri ti s

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