GRANULOCYTOPOIETIC FRACTION OF
YELLOW BONE MARROW

THESIS

Respectfully Submitted to the
Graduate School of Michigan State College
in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy

By

Joseph Zichis

ProQuest Number: 10008243

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Thm euthor wishes to «^re«tt S&s -t&enke
to Dr* G* Am Hopport end Dip* C* W* Goiter for
their internet is this work end for their help*
£©I edrie© end eritSel*n* end to Frederlok
Stearns end Company* fdr the flwmoiel support
of the problem.

331677

XSTROmiCtlQ*
In recent years the condition known

cub

granulocytopenia has

attained a prominent position i s the medical world*

While the

etiology of the disease still remains obscure* the many agents
for its treatment here n e t with varying degrees o f suoeess*
(1*8*3)*

Of these therapeutic agents* yellow bona marrow has

attracted m u c h attention*
M M a s

(4} has obtained encouraging result* by oral ad*

ministration of bene narrow at the onset of the disease*

B e gave

daily dcera of approximately 00 to 120 g m * o f fresh yellow bone
marrow* obtained free beef * until the blood assumed a normal
picture* and smaller doses for seserel wraths later*

Sines large

amounts of narrow were necessary to obtain a change in the
loukooytio picture* and sines I n osrtaln rases the patient w a s un*
able to digest and assimilate gush large quantities* the use o f the
fresh marrow for this purpose was considered undesirable*

Aooord*

ingly* a study was undertaken to separate the gramlooytopoiatie
fraetion from yellow bora aarrow* end to determine its activity o n
experimental animals*

gQURCg OF BOffiB MAffiGW * The bone marrow which an* used is
these studio* was obtained from fresh long braes of cattle* It
was fkeed of blood* boxy and fibrous material and ground*
p*$r i
ISBfaDD Of mEPtRIISKJ GOSGraPEAMS
SXTRACTXQKS % to 1& *”«•* 10 X@b* Of bone marrow was dehydrated
by thoroughly miwing it with 10 Eg©* of plaster of Paris* Tie
•
■
*
-W ■
■
mixture was allowed to dry over~stight* and then broken, up into
small particles* 2 E@&* of this mixture was placed lute each of 10
percolator* sod extracted at roan temperature with the

1} Acetone
2) ievbn*
S) (Merefens
4]
Absolute
Absolute ethyl alcohol

8

Carbon tetrachloride
10
Each of the above solvents dissolved such a
fat fr«a the bene marrow that It era found impossible to determine
their hematopoietic properties* Due to the lack of concentration
this method sac considered useless and discarded*
FREPAR&TIQH 1* (B»l)

2 £gn* of brae marrow was saponified

by belling with 4 liters of $$S alcoholic potassium hydroxide and 4

m$m

liter* of 95$ aleohbl for *0 minutes voider * reflex condenser#
^hea the sapwiiftcatioa m s complete and while the solution mas
still hot# it m e filtered through e large fluted filter# Hie
residue consisted mostly of fibrous sad bony structure*. To the
fillets 3 liters of distilled ester m e added# sad hosted to
boiling* The tohxtiei m e soiled# sad 1 liter portions were ear*
tracted by ahsfcliig thoroughly with 400 ee# of petroleum ether .
(B#P# 40° <* 70° 0#)* la order to prevent the formation of an
emulsion 50 es* of.'distilled water m e added to each 1 liter
portion# .After the separation of the petroleum ether layer the
underlying soap solution m s dram off. The other solution m e
washed 5 times with 50 ee* of 40$ alcohol* Hun ell the saponify*
lag alcoholic solution was thus estreated the petroleum ether
solution was collected sad the ether removed by heating over a
sbem*heth# A yield of 4*8 gm* of the nortsaponifi&ble materiel
was obtained#
The substance was a dark yellow# oily liquid# having a slight
acrid odor and m oily after""tewfee* The specific gravity was
.8940 and it gave a negative test for sterols. The substance m s
soluble in 95$ alcohol# ether# pebrelsm ether and chloroform* «foen
administered orally to animals in which a condition of granule*
oytopenle had been produced# It shewed no ipriciulGoytopeietic activity*
Hue results of the biological test are given In Table X#
g%BB&8AfXoH 2# (B*g> m * S Kgm* of b m sssrrew m i saponified
with 6 liters of %% alcoholic potassium hydroxide at room temperature
far 24 hours* Tbs mpe*sl&*a&ie& m s aided by mixing the solution
with a mechnioal stirrer* At the wad of 24 horns the solution was
filtered through a large fluted filter# Hie residue consisted chiefly

4* 4 m
of Insoluble bony and fibrous tissues* An equal volume of distilled
meter m s added to the filtrate whloh separated late two layers* the
upper layer containing the ndnmpenlftsbXe fraction m s saved end the
lover soapy layer discarded* In order to free the concentrate free
alkali It m s seabed 5 time with 50 ee* of distilled water and 5
times with 50

go*

of 40$ alcohol* From the original 2 Kgm* of bone

marrow* 100 @8* of the concentrate m s obtained*
The produst thus obtained m s a yellow* oily liquid# being
nearly odorless and having a slightly oily after^tasbe* It had a
neutral reaction to phenolphthaleia$ gave a negative test for sterols
and hada specific gravity of *3620* It m s soluble in 95$ alcohol#
ether* petroleum ether# chloroform* and only slightly soluble in
methyl alcohol* This concentrate dsn administered orally to animals
having the gram&ecytopeaio condition ^ ios^I marked granulocytopoietic
activity*
His results are shown in Table 5*
PREPARATION 8. (F^S) <»*•* 2 Kga* of bene marrow sag' saponified
end filtered in exactly the same manner as under Preparation 2* 1
liter of distilled water m e added be the filtrate* After shaking
thoroughly* 1 liber portions of the filtrate were extracted with 500
oc* of petroleum ether (B*P* 4O°~S0°c*)* After the ether layer
separated out the underlying soap solution was dram off* llhea all
the filtrate m s thus extracted* the petroleum ether solution was
resaponified with 5 liters of 5$ alcoholic potassium hydroxide at
row twperature f w 24 hours* The soap formed by this procedure
m e insoluble in the liquid medium end was easily separated by
filtration aided by suotion* The soap was washed 5 times with 50 as*
portions of petroleum ether* The addition of % liter of distilled

water to the filtrate caused the formation of Z layers. The upper layer
wee sewed end the lower discarded* 1 liter portions of the petrelem
ether solution ware washed 0 time with 100 co* of 40$ alcohol* Ukum
ell of the ether solution wee thus washed* It was oolleoted end distill*
«d off wader diminished pressure «l 30°C, The resulting product had a
slight cloudiness# hut after filtering through a layer of asbestos it
remained clear* 20 gm. of the concentrate was obtained from the original
Z Kgzas* of boas marrow*
This concentrate was a brilliant amber* oily liquid# helm odorless
and imparting a feint oily taste* The specific gravity was *9270* It
gave a neutral reaction to phenolphthalein sad a negative test for
sterols* said it was soluble in 95$ alcohol* ether* petroleum ether and
chloroform. Hhen this substance m s administered intramuscularly to
animals which showed a condition of gramlooytopenia# a lasrked granule*
activity was observed within 4 hours*
The results ere given in Tables 5 end 6*

m 0m
max it
m n m x m of ( m m s m t x o m w L

Siam the 41«m

m

gnade*

eybopeixl* t u first described in Seraasay in 1922 (5) sad la the
United States in 1324 (6)* isfcay off«*t* <6#7#3#9*10#U*12#13#14*15J
ham been mode to determine Its oaaxae* Ks»y theories of the etiology
of this disease ham been advanced* namely* dietary deficiency (7*8)*
hoopaonio dysfunction

(9*10)*

nee of certain coal-tar derivatives

(11*12*15)* and bsetarlal infection (6*14*15)*
The first theory is supported by the eerie of l^eaageton and Hay
(7) who were able to demonstrate profound leukopenia in monkey* by
feeding these a diet deficient la Vitamin 0* However* thee# animals
alee shewed deficiencies in red blood oalla and Mood platelets* la
addition* Miller and Bho&ds {8} produced a similar Mood picture by
feeding doge a diet used to produce Mack tongue*
The second theory is upheld by the studies of Britton and
Corey (9) who demonstrated leukopenic conditions In earn which were
qdrcttalcotomlr.qd* .Others also (10*11*12) a re o f the opinion that
the condition nay he associated with ecsae harmonic dysfunction* be*
sense la many instance* the onset of the disease ooourred during the

(18) supports the ehemleel etiology by showing that
the a<fo&oistration of small doses of bsnsaas* to rabbits caused a
dselded leukopenic condition* Furthermore* Smoke (14) by injecting
intravenously small dome of benseae Into rabbits produced profound
granulocytopenia* and free ihis he concluded that benssne has a
selective action on the leaScopoietic system# This theory Is also up­
held by clinicians (15*10*17) Who Mata that the disease frequently
develop* as a result of the use of certain ooaX-tar derivatives*

m 7 «#

The bacterial etiology was first demonctrutedby Lovett (8}

who produced leukopenic conditions in guinea-pig* by injecting
culture* *f Bacillus pyocyeneua itttraperitauealXy# In addition#
Banal# (18} found Hint Staphyloooooua tsvus or Streptoeeeeua
he*nolytioae*whon placed Into a rabbit in ouch a manner a* to
liberate toxins by dialysis* without causing a general bacterial
Infection* developed a severe type of leukopenia* More recently*
Felsen (IS) described $ eases of sente baoillery dysentery infeetlea ^ioh terminated la the production of profound granulooytopenia*
Moreover* Shell (20) described a ease of granulocytopenia in a pig
which upon autopsy shooed a severely Infected gall bladder*
m a m of

frgduguig

GMmoesropEsxe co^moas

A8XM&L3 *mm Healthy, jftaXX-growa rabbit and rats were used
and swob sex mss represented In about equal numbers* The animals
were kept on a normal diet* Blood was obtained from the marginal
vain of the ear in the ease of the rabbits* and from the tail in
the ease of wmbe* The blood smear* were stained with Wright** Stain*
i* m m m m * mrnms
«*••» *1 **» of bensene w e administered orally to
each of 24 rata and 1 ee* to each of 8 rabbits daily for 10 weeks*
Another group of 3 rabbit* received daily intravenous injections of
*05 es* of banswas into each for S weeks.

The nativity of the benssne on the leukopoietic system was
determined in the rabbit* by making daily total and differential
leukocytic aouate# and in the rate by making weekly counts*

8-

The result# of this study did not show may changes in the blood
picture# end for this reason only the average values are given mm
folloess
Total leukocytes
a. Polynuolosr neutrophils*
3* Momeuyte*

Rabbits
V
*

4* Transitional sells

8500,
K0J

Bate 14600
#
19*

1*

n

1*

2?t

«»

3#

5. Waophila*

«

25*

*t

3* Basophils*

w

®£

4»

m

7* Snail lymphocyte*

It

84$J

n

50*

3* Large lymphoeytes

«

4#

«

26*

(b) AMIBQFtRlHE — Gaily doses of a 100 agm* of amidopyrine per
£$a* of feody-weighfc were fMbalslstered eubewtaneeusly to each of 12
rabbits for 12 seeks*
Hie leufcopoletie activity of this compound warn determined by ob­
taining total and differential leskoeytle counts daily for the first 5
weeks and weekly during the remaining time#
The results demonstrated no changes in the blood picture* Has
average values are as follows*
1* Total leukocyte*
2* Polymiolear neutropMIe*
5# Monocytes
4» Transitional cells

6* Baeophilee

S9G0
49$
1*8$
*5$

2$

7* Snail lymphocytes

56$

8* large lymphocytes

9$

«» 9 »
id)

—

25 ngn* of hydretjuinon* was ad*

ainiatersd intravenously to eaofc of 2 rabbit#* The animal* were
male* and each weighed approximately 2 &gn* In addition 20 rata#
(19 male* and 19 females} weighing about 290 gta* eaeh* were kept
6 nonth* cm a steak diet containing 2$ hydroquinon«»
Total and differential leukocytic count# were made la order
to determine the effeet of Hie substance* in the rabbit# daily
counts were made and In the rote weekly counts for the first six
week# and thereafter one# every 3 weeks*
9a the 6th day of the experiment one rabbit died without
showing any changes In the blood picture* Pereas the other did not
shew may 111 effect# after 20 day*. The blood counts on the rata
did not show any changes* For this reason* only the average values
are given*
1* Total leukocyte*
2*

Bolynaelear neutropMles

Bat*

Babbits

1SQ0O

8S86

20$

51$

3* Monocytes

1$

2*6$

4* Transitional Celia

2$

1*6$

6*

-

1$

7* Small lymphooytas

50$

32$

6*

26$

6$

Basophils*

Large lymphocyte*

.2* aACHBRIAl. METHOD —

The procedure used was a modification

of the method described by Dennis (16)* A #000 gelatin capsule
Seated with collodion was filled with a 24-hour culture of
Staphylococcus mstmm by mesas of a hypodenaio needle end syringe.
The capsule was sealed with a drop of collodion* and sterilised by
Ixssfftrsing in a 1$ solution of HgC12 6 tines at 5-281nute intervals*

*» 10 •

Later this jroeedune was modified by replacing the collodion
coated gelatin capsule* with Saturalamb gkin eace and by intredneleg a larger volume of the orgenima* 10 ee* of a 24-hour culture of
the organism was pieced into the «ae# the open end of which wae
sealed by tying with a linen thread* The sac m m sterilised by
several tines in 70$ alcohol*
total said differential leukocytic counts were de­
termined om each animal* The rabbits were starved for 20 hours be-'
fere the operation* A light ether anesthesia was administered to
the animals# and the peritoneal cavity was exposed by incision# care
being exercised to maintain aseptic conditions* 3 capsules or %
Naturalamb skin see ware placed into, the 1ewer part of tbs peritoneal
cavity* The peritoneum and abdominal wall were sutured with plain eat
gut* Metal clips were used on the skin* A bandogs was applied and
held la position by a binder*
The gzwiHilecytepeietie effect obtained by this procedure was
determined by making dally total and differential lenkceytio counts*
The results of Hie total end differential leukocytic counts were used
its the criterion of the effects produced on the granulocytopoietic
system by the bene marrow concentrates*
46 rabbits were prepared according to this method* Of this
number 17 developed a condition of gramilooytopenia* 9 died without
sheering way change In the blood picture# and 20 recovered without
showing any 111 effects* The 17 animals that developed the granuleoytcpenic condition were weed In the following experiments*

- li -

BABBITS I and 8 — Female rabbits each weighing 3 Kgp* were
prepared by placing 3 collodion seated capsules filled with a 24hour suitors of Staphylococcus aureus Into sash animal as described*
His rabbits developed the gramilooytopssdo condition* and although
10 as* of &•& was administered by naans of a atonmoh tube both

sssim&l* 4%e*P*
Tbs data are given in Tah&s 1*

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In bhooe Tobies the cella tensed ** Polyzmoleftr
Degenerated* were poXynuelear neutropbiXe*
oimrao'torlood by disintegration of tbe eell membrane end cytoplaeci
(pro*m m of oneoeloo) and a distortion of the nucleus* Baa* eoll*
atedned very poorly*
IUBBIT5 3*4*6 and 6. —

Babbit* 3 and 4 (male*) and 5 and 6

(female*} <aoh weighing apprcadlaatoly 2«5 Egra* oare pr«|*ered in
the same aatsmor a* the firat 2* Kaoh animal developed the
granulocytopenic condition, 10 gm* of fresh yellow tone auto*
imm melted art 40°C*, strained through silfcolina and we* administered
to each animal by wmwm of a stomach tube* Babbit* 0*4 end 6 diet
on th* fallowisg day without showing «sy sign* of recovery* Babbit
8 resolved 5 dose* of the melted marrow* but showed no recovery,
and diod on tfee 4feh day*
fhe data are given la Table 8*

5ABUS

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- 16 -

RABBITS T eat 8 —

Male rehMte* *e*3* neighing 6 % b*«

map* prepnarmd aeeordlsg t* the m m procedure* M l enHsel
developed * eondltion of grerealQeytopenie* end died heifer*
therapy eotald he instituted*

The det* ere zfaam in Teble 6*

♦ *16-

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*17 •
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Eftbbit* 9 and 10 (®ale») f0fed U

and 12 {faciala*) «ftQk weighing ftbonfc 2*fi Xg?a* m n prepared ft* th*
prftftftdliig onas* All developed t&a granulocytopenic coaditia** and
Tm&mermI upon daily oral adirdni*tra£i<ra of F*4U
Tha data are given In Table 4*

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* 19 *

gsmysgiog m t m A s m m r n m m $ m m * eb&gtioii of p-s ~ ~ 12
Mil grown rat# end 3 Mil grows rabbits were U89d* The right leg of
each animal m m sheared and the skin m e cleaned thoroughly with so*?
sad water* tJader aseptic conditions *2 os* of B#3 wee injected into
the asuade of the leg daily for 4 day»• B*Sh injection m

made at

the sms sits*
The injections did set muse say local reaction as manifested by
the absence of redneea* swelling e d restlessness in the aniraala* 4 days
after the lest Injections were made the enimale w « killed and upas
autopsy the muscles Into which the preparation m e injected showed no
gross abnormality, This indicates definitely th&t B*s ie easily ab*
sorbed when injected into an s M

intrmusenlarly*

EABBXTS 13, 14 and 15 *** fmale rabbits^ each weighing approx*
iiostely 2*0 $ga** a m prepared according to the method described* and
* j^turalasab Skin ass containing 10 so* of * 24-hour culture of
Staphylococcus aureus was placed into the peritoneal cavity of each
animal* Babbit 13 did not develop a typical condition of granule*
oytopenia* The second day after the operation it lad a total leukocyte
aewssb of 31000* and norma! pdymelmr neutrophilee oould not be found*
The animal was givea *loo» of B*3 intranueeulerly daily far 4 day*# At
the end of this period* the blood picture returned to normal and ths
rabbit showed apparent recovery* 48 hours alter the operation rabbits
14 and 10 showed a condition of granulocytopenia* Babbit 14 m s
gisen *3 os* and Babbit 10* *1 os* of B*8 intramuscularly daily for
3 days* At the end of the 3*day period both animals showed a normal
blood picture and both appeared to be in good health*
The data are shown in Table 3*

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R&SS1TS 18*17 «••* Beraeles, each weighing 3 Kgft* ware used*
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of e 24*hour culture of Streptococcus pyogenes gram in serum broth
m e placed into the i M i t skis n««* Babbit 18 developed the
granulocytopenic condition* and after ediaAnietering *1 os* of iws
intr*jnu*ova*rly for 4 eonseeutiv* days* recovered. Babbit 17 did not
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2$ •
m s c s s s io n

Flra» these studies* it 1* evident that by ©e&d sepeaifioetlon
it 1* possible t© Isolate Proa yellow bon© narrow © nonaaponifiable
fraction aMsh appears to possess pfiomXooytopoietie properties* The
forego!ng data ale© shew that thla fraetion 1# &ot£v© vfoen administered
either orally or introa^ooalorly to aaimela in which a condition of
granulocytopenia m m produced experimentally* The data in Table 2 show
that if tfcesapo&ifioetiott £« carried out ot S0° to 100° C* tfeo fraotion
Baa no aetivity# 3a addition* Hi# data in Table 3 show that granule-*

©vho'&aiehle ootivi'tsf dcmid not bo demonstrated -on atolt ywiXgi? bone
o
marrow* ©Mob had been melted at 40 0* Thio may bo duo to the foot
tbit the sssmImmbi amount of sserrew that could bo id&didoto^idi to

rabbits w m insufficient to produce a stimulating effect m m the
groiidoo^P^^tio system* The fob solvents
% to 10 did net offord a

in Extractions

of asparatioii* because as ststod each

tobr^st dissolved too jauoh of iha fats- thus preventing a ©onoontrati©*u
Althoyghji a fsy highly active

m © jwepered*. it wilt bo

necessary to conduct further studios os tbs partfieatieB end m s *
centration* before « © suggestion of M e d

oompooitlos can be sods*

Is this wit, ottewpi| to produce granulocytopenic M i t t s u
is rabbits sad rots by the use of bensone* a^dcpyrine* #o4-hydro**
quinone sms unsuccessful* M » e p N « % # th» method employed by
Dennis (26) sss used sad proved to be satisfactory* Although* tbs
condition obtained by th£© procedure m y sot be identical with that
is humans* it affords s eonvanlseb laboratory method of demonstrating
the greaa2©cytepoietfo activity of yellow boss marrow eonoentr&tians*

•M

emawtm

vwwn prepared fmse yd lew bene iswmssi*

*■ 33 *

BIBLIOGBAFHT
1* Uinter, M«M.i The Treatment of Agranulocytic
Angina. Southwest Insi Medicine 2s8 (April) 1935.
2. Harris, W.H., and Sehattewberg, H.J.* So-called
Agranulocytic Angina with Special Consideration
of the Caned Agent* Hew Orleans Med* and 3arg. J.
881283 (Mew.) 1935.
3* Burcky, F.W,* Agranulocytosis. Illinois Med. J.
67*59 (Jan.) 1935.
4. Watkins, C.W.s Personal Goasssaieatlon to the
Anther in November 2924.
5* Sehult*,f.i Bdber elgeuartlge Halserkrankttngeiu
Deutsche Mod* 9 s M r . 48s1494 (Her. 2) 2922.
6. Lovett, B.R.t Agranulocytic Angina. J.A.M.A.
83*1498 (How. 3) 2924*
?4 Langston, W.C., and Bay, P„* Cited by Xraoke, E.H.,
and Parker, f.P.i The Relationship of Brag Therapy
to Agranulocytosis* J.A.M.A. 105:900 (Sept. 22) 1935*
8* Miller, B.K., and Hhoada, C.P.s Experimental
Production In Bogs of Acute Stomatitis, Associated
with Leukopenia and a Maturation Defect of the
Myeloid Sleme&ts of the Bone Marrow. J. Kxper. Med.
61:173 (Feb.) 1936.
9. Britton,S.W., and Corey, 2.L.* Blood Cellular
Changes la Adrenal Insufficiency and the Effects
of Cortleo-Adrenal Extract. Am. J* Jhyeiol. 202*699
(Boo.) 1932.
10. Thompson, W.P.t 0bserrations on the Possible
Halation Between Agranulocytosis and Menstruation,
with Further Studies on a Case of Cyclic
Heutropenia. Hew England J. Med. 202*176 (Jan.) 1934.
11. Jackson, H. Jr., Merrill, B«, and Buane, M.s
Agranulocytic Angina Associated with Menstrual
Cycle. Hew Hug. J. Med. 210*276 (Jan.) 2934.
22m Hutledge, B.E., Hansen-Prusa, C.C., and Thayer, W.S.
Beeurrent Agranulocytosis. Boll. Johns Hopkins Hosp.
461369 (June) 1930.
23. telshotten, H.0. The Action of Bensols The Bormal Life
Span of Heuirophile (Aaphophlle) Leukocyte (Babbit).
Am. J. Path. 6*183 (March) 2930.

14* XnMde>f IUB»i Sb« Experimental Production of
Agranulocytosis* Am.
Clio* Path* 2s11 (Jan.) 1232.
15* Madison* ?.f., and SQOler, f.X*t fbo Etiology of
Primary Granulocytopenia. J.A.M.A. 102:755 (Mave& 10)
1234.

16* Hoffaam* A.M. v Butt, B.M*t and Hickey, 8.G.: Benxtropenia
following Amidopyrines Preliminary Beport. J.A.M.A.
102s1213 (April 14) 1334.
17* Special Beporte of the Council on Pharmacy and Chemistry,
A*M*A«s The Bsl&fcion of Amidopyrine and the Barbituric
Acid Derivatives to Granulocytopenia* J.A.M.A. * 102:2183
(June 30) 1334*
18. Bennie. 3*3.s Experimental Granulepenia Boo to
Bacterial foxims Elaborated to Tiro, J* Bxper. Hod*

67s993 (June) 1233*
It. Poison, Jf*t Baeillasy Dysentery * Acute folmtnattnfe
TTpo eiiti Marked Toxic Beutropenia. Her Tork State J*
Sled. 35:103? (Dot* 15) 1235*
20* Sioll, &2*i

Personal Goamaaioation to the Anther in