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T H 5518

6-1

This is to certify that the

thesis entitled

Fiological Activity of the Lipids
From Escherichia Coli and Shigella Dysenteriae
and their Characterization by ”eans of Infrare Spectro-
photometry

presented by

Thomas R. ifeblett

has been accepted towards fulfillment
of the requirements for

Ph. D. degree in Elicrobiology

 

Date I'ay 17, 1957

 

0-169

BIOLOGICAL ACTIVITY OF THE LI PI 05 F ROM
ESCTIIER IC H ILA ( OLI. A ND SH IGELLA DYS ENTER I’AE
AND THE I. R CHARACTER I ZATI ON BY MEANS OF

I. X F RA RED SPECTROPHOTOMETRY

I);

THOMAS RANDOLPH, NEBLETT

A TIIES IS

SLJISMITTED TO THE SCHOOL OF ADVANCED GRADUATE STUDIES
OF MICHIGAN STATE LNIVERSITY OF AC‘rRICIJLTURE
AND APPLIED SC IENCE
IN- PARTIAL FLLFILLMENT OF THE REQUIREMENTS OF THE
DEGREE OF

DOCTOR OF PHILOSOPHY

DEPARTMENT OF MICROBIOLOGY AND PUBLIC HEALTH

1957

Wax-'5‘?
C” 455:?

ABSTRACT

The lipids from two enteric gram negatix‘e organisms,
Escherichia coli , considered to be non-pathogenic, and
Shifiella d3senteriae, an intestinal pathogen, were extracted
and injected intraperitoneally into laborator} mice in an
effort to learn if they possessed stimulatory activity for
the. reticulo-endothelial system.

[3} use of radioactive colloidal chromium phosphate
and colloidal carbon preparations as: indicators of the rate
of phagoc5tosis of particulate matter from active circula-

tion it was found that the lipids from S. (llsent‘eriae

 

possessed marked power to stimulate phagocyt‘i c actix it)‘ to
hyperfunction, while those of E. coli and its sub-fractions
did similarly to a lesser degree.

Partial characterization of the sub-fractions from
E. col'i indicated that certain of them. exhibited characteris-
tics of phospholipids, since themost‘ active fractions
possessed an absorption band in the infrared region not
shared by less active ones. The same band was observed in
a known phosphatide, and it was. tentatively identified as

that produced b} the phosphorous-containing group found in

phosphat ide structures .

AC K N OW LE DG E ME NTS

Few pieces of graduate level scientific investiga-
tion have ever been accomplished without close practical,
and academic supervision, or without collaboration, advice,

and assistance of persons allied elsewhere with the field

or within scientific disciplines other than the investigator's

own. Such collaboration and assistance from sources other
than those of Michigan State University have contributed
toward, and made possible the bulk of this paper.

The author wishes gratefully to acknowledge the pro—
vision of equipment, materials, guidance, and devotion of
personal time by the Director and staff of the New England
Institute for Medical Research, Ridgef‘ield, Connecticut.

John H. Heller, M. D., as Executive Director made
it possible for the author to undertake this work as a part
of an investigative program at the New England institute in
the role of Pro-Doctoral Research Fellow. The author was
under the direct supervision and personal guidance of A. K.
Bicknell, Ph. D., Chief of Di\ision of Microbiology, and
recei\ed guidance and technical assistance from R. Turner,
Ph. D., Director of Laboratories; J. Blizzard, Ph. D., of ‘
Nuclear Physics, and R. Meier, M. D., of Physiology, were
responsible for the supervision of isotope handling and

counting in the phagocytic velocity measurements.

D. A. Boroff, Ph. D., provided the author with
large quantities of lyophilized Stlmella dysenterizg: cells.

The infrared spectra of lipid fractions which are
included in this work were obtained through the courtesy of
the Perkin-Elmer Instrument (‘orporat ion of Norwalk,
Connecticut, and the direct efforts of Mrs. H. Sternglantz
of the Perkin-Elmer demonstration laboratory.

W. L. Mal lmann, Ph. D., Professor of Microbiology
and Public Health, Michigan State University, was the
author's major professor and directed all aspects of the
work on the trniversity campus. Dr. Mallmann's cooperation
and willingness toward such an endeavor made it possible
for the author to avail himself of the opportunity offered
in the Pre—Doctoral Fellowship. Dr. Mallmann further aided
the author with encouragement and the philosophy that a
major provision in the course of a graduate training program

\

is the proper environment in which to work.

iv

\ITA

THOMAS RANDOLPH NEBLETT
CANDIDATE FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

Final Examination: May 17, 1957

Dissertation: BIOLOGICAL ACTIVITY OF THE LIPIDS FROM
ESCHERICHIA COLI AND SHIGELLA DYSENTERIAE
AND THEIR CHARACTERIZATION BY MEANS OF
INFRARED SPECTROPHOTOMETRY.

Outline of Studies:
Majn I‘ Subj C(‘ t‘: Mi (,‘l‘nb'lifl. (fig)
Minor Subjects: Biological Chemistry,
Physical Chemistry"

I3ir4§raifl1i(111 I tenra:
Born: June LL, 1928, Lexington, Kentucky
High School: Thomas M. Cool ey' High School,
De troi t , Michigan
Graduated June, 1937 _
Undergraduate Studies: Michigan State College, B. A. 1931
I University of Louisville, 1951—1952
Wayne University, 1952-1953
Graduate Studies: Wayne University, 1953-195u, M. S. 1955
Major Professor: A. K. Bieknell, Ph. D.
Thesis Title: A CHEMICAL AND
BACTERIOLOGICAL’TNVESTI—
GATION OF SODIUM LAURYL
SULFATE AND ITS EFFECT
ON THE GROWTH AND ISOLA-
TION OF COLIFORM
ORGAN I SMS .
Michigan State Uni\ersity, 195u-1957,
Ph. D. 1957
Major Professor: W. L. Mallmann, Ph. D.
Fellowship: Pre—Doetoral Research
Fellow, New England
Institute for Medical
iReseaiwfli, Ridgefield,
Connecticut, 1955-1936.
Affiliations:
Society of American Bacteriologists
The Society of Sigma Xi

TABLE OF CONTENTS

ACKNOWLEDGEMENTS . . . . . .

INTI-{ODL'C-T [ON AND H I STOR I (A L REV I EW

METHODS AND PROCEDURE . . .

(ul t i \ at .i on of Organisms;
and Fractionation . . . .

Measurement of Phagocy tic Velocit

Characterization of Lipids

RESUIATS o o o o o o o o o 0
Figure l .

ELAPS ED TIME .
Control

Escherichia col i

Figure 2 .

ELAPSED TI MP. .
Control

Shigella Lipid

Figure '3 .

E LAPSED TIME .
Control

Fraction 'I\ .

Figure. LI.

PLOT or P12 ACTIVITY
OF 5 LAMBDA BLOOD SAMPLES

PLOT OF P32 ACTIVIT\
OF 5 LAMBDA BLOOD

PLOT OF P'32 ACTIVITY
OF 5 LAMBDA BLOOD SAMPLES

Lipid

I N C PM
SAMP LES

I N C" PM

OF 5 LAMILBDA BLOOD SAMPLES

LLAPSED T I Mb .
Con trol.
Fraction I I .

Figure '3 .

. . . . . .

Extraction

. . . . . .

ies

o . . o .

. . . . . .
IN

C' PM

PLOT OF P32 ACTIVITY IN CPM

\S'

S

\S'.

‘ S

PLOT OF OPTICAL DENSITY OF CARBON

FROM 20 LAMISDA BLOOD SAMPLES vs.

ELAPSED TIME.
Control

Shiggl lg; lipid

V

Page

19

19

2'4

'30

'i '3

'iLI

'36

77

Figure 6 .

Figure 7 .

Figure 8 .

Tabl e

Tabl e

l .

Infrared

PLOT OF OPTICAL DENSITY OF CARBON
FROM 20 LAMBDA BLOOD SAMPLES vs.
ELAPSED TIME.

Control

E. coli l.i_p'id . . . . . . . . . .

PLOT OF OPTICAL DENSITY OF CARBON
FROM 20 LAMBDA BLOOD SAMPLES vs.
EIAPSED TIME.

Control

Fraction IV . . . . . . . . . .

PLOT OF OPTICAL DENSITY OF CARBON
FROM 20 LAMBDA BLOOD SAMPLES vs.
ELAPSED TIME.

Control

Fraction II . . . . . . . . . .

MEAN T/2 viLULs FOR THE VARIOUS
LIPID FRACTIONS . . . . . . . . .

ABSORPTION BANDS OF FRACTIONS COMMON
TO ’\ KNOWN C'EPIIALIN PHOSPHATIDE. .

Spectra .
C(lIJIIa].j.II o o o o o o o o o o o o
FI‘aC‘tloll [\i o o o o o o o o o o 0

Fraction I I . . . . . . . . . . .
Fraction V I I . . . . . . ~. . . . .
Fraction III . . . . . . . . . . .
,Fra ct ion \9 . . . . . . . . I. . .
Fract .i. on I . . . . . . . . . . .
F ra ct ion \' I I I . . . . . . . . . . .
Fraction ”IX . . . . . . . . . . .

Fraction VI . . . . . . . . . . .

Page

'38

39

U0

LII

DISCUSSION
S I I MMARX .
(”-ONCLL'S ION

BIBLIOGRAPHY

VIII

IN’I‘RODIITION AND HISTORICAL RE\ IEW

The primary topic of this paper is a description
of the physiological elfects of cell—free bacterial lipid
extracts and their partial characteiization by physical
Ineans. The author explains the actionol~ UH? total ex-

tracteuule, FMW)tOIn-IJTW‘ Iipitlifl)tairunl flinn Escluu ichia

 

coli and Shigella dysenteriae, and the lipid sub-fractions
OI3taIJ10(I II onI E. C()II. llies(‘ uwnrc> Itiurmi t() st.inuilat1> tlIe
rate of phagocytosis of a negathely charged colloidal
particle from the circulating blood of laboratory mice.
Infrared spectrophotometric analysis has been carried out
IIptHl ea<fl1 cu” tIIOS(‘ SIH)SI£in(W?S 1J1 OITIPI‘ to (lOHHHfiStIEfitO

the uniqueness.of each.

The data are presented in this paper to illustrate
Uiat thc‘.res[n)ns(\ of tJIe rW‘ti(WIIo-cuulot|u>lial :systtun as
indicated by" an elexation of phagocytic velocity of tracer
particles withiri<flJXWIHating blood is rdiyiificantly
stimulated by administration of lipids Irom the non-
pathogenic Escherichia coli and by the total lipid from
the enteric pathogen, Shigella dysenteriae.

The antigenicity of lipids has been almost
uniyersaily negated among bacteriologists and .immunologists.
The stimulation of antibody production is commonly accepted
as the criterion of antigenicity; howe\er, particular

2
att<uitiori is gixrni to ()thOI‘ Iactrnws whixli may'13e siinilarlyr
applicable as criteria also. One criterion which includes
antibody pIOdIKWjJfll is the ability (H‘ U10 foreign sub-
starKW‘ to OV(H\O a (tirectly'IneasIuifl)Ie Ln‘ obseiwefl)le rfmu‘tion
within a host organism. The antigen—antibody reaction is
one siudi phcwnnnenoriwfliich is INT“! obse1wzfl>le arKlIneaslnffl)Ie
in tin? laboréitory, \dIile arI(‘le\atth opstniic iruhax arulzan
inciwuased IYitO (H‘ifiiago(y%4nsis alt“ two ()thers (ihasely
related whi<l1€1hso CUHLUIHII()IJH‘ ciiterion. MHiiIe all of
these functions belong to the reticqu-endothelial system
(yr a11~ \ery'( losely relzited ti) its zactiyi.ty, tlufi stinuila—
IIJHW of pfluugocyti<r VOI(H‘Ity OI‘(‘I0\at(WI ops(nric HKM‘\ are
not commonly regarded singly as criteria of antigenicity.

Phagocytic \'(‘I()(“,IIy changes cannot be relied upon
5H)|ely as ziineans IH‘ eyalimating FOtIJWIIO-Ond0tlHTLU3I func—
tion; howe\er, a measure of phagocytic rate Changes giyes a
\ery good indication of body del‘ensixe acti\ ity. Although
other acti\ities exist which are less well understood, the
rate of disappearance of negati\ely charged colloid
partix‘les Idwnn actiyté ciIIWIlatiini and.(£q)illary wahs may

IN? considered.1ntha Iacie e\idCWKW‘(if changes tinting place

 

IWKMH‘ certaiii stinnLIi. llris pagHU‘InakcwsIMJ clainr.in Ia\in‘.
of tlniauitigenicity‘(if lipflths. Howexcu‘, r80t(n"4tflllfh
stimulatcilfiiagocytix‘\(Wlocity airiifl7 consideralflm\_interest
becatuwi Hiey a11\(wmxflule of ele\atiing one actiyity [Manse

of a very \ital system.

3

C(nisitleiéiti(n1 slniulcl be InaCHF ol‘ th(\ facét tlult (lie
physiological activity of bacterial lipids as described in
this work fits at least one of the criteria accepted by
some persons as indicati\e of antigenicity. Since the
Inecharnann for tlufi obSOIWTWl actiy;ity descuflibed Imuwain is ru3t
[Orescuitly lvnowri, CTH1SiCUTratltH1 of Inactcueial. lipicbs an(l |i1)id
fractions as haptens cannot be validly claimed either.

The ability of certain cells within human and
animal bodies to engulf particulate matter and other cells
has been observed and recorded for many years. )bll(ny
(IOO) 'in l€398 (“uservcwi that lunnan (nuiotluilial («fills (HT
lymph nodes exhibited phagocytic powers and underwent rapid
proliferation to fOIm observable lesions found in autopsies
()f cathiveiws recruitly (lead (1L tyifiioid li‘ver. Cellss ol‘ U10
splenic pulp, blood vessels, and intestinal lining behaved
in a similar fashion during progress of the disease. He
des(W‘ibed IIH‘ esscuitial lesi<ni in tyifiioitlzas a (tiffusC‘
proliferation of endothelial cells giving rise to larger
epithelioid cells having marked phagocytic properties.

The organism responsible for the disease was cuItured from
all organs checked for such lesions: liver, spleen, blood
vessels, and lymph nodes.

In an investigation of pneumococcus antiserum Bull
(23) described a clumping of injected cells within the
hlcx)d \(Mssel:a arKl raI)id ITWHU\EII oI' Uieswi cIIunps l)y (walls ()I
the spleen, liver, lungs, and possible other organs. An

examination of stairuwl thsue sections revealtwl UIe en-

{gulfed ba< teria. He described phagocytic cells as the
chief defensive agents against infection (2’4). E\‘(‘essi-\e
doses of the serum caused large clumps to form which,

when taken up phagotytical ly , impeded. circulation in lung
capillaries. in another investigation Hull (2%) [ound that
typhoid bacilli were agglutinated and removed by' poly—
morphonuclear leucocytes in the spleen, liver, and lungs
and. were digested. and destroyed by the phagnwytes. Blood
drawn from the heart following intravenous injections into
the rabbits produced cultures of successively fewer colonies
until within a period of l3—20 minutes after initial in-
JOCtiOl’l the blood was \ery frequently sterile. No more
than one colony per ml was ever produced by blood culture
at this period.

Hopkins, (79) studying the effects of .inJections
of hemolytic streptococci into rabbits, found that sub—
lethal dos-es disappeared completely from the bloodstream
within a few hours, while in lethal injections ninety
percent of the organisms disappeared within a few minutes,
and. blood culture minima were obtained .in two hours.
However, in the latter case the engulfed organisms began
to ,re-appear in the circulation from U to 6 hours following
injection. lle attributed the removal of bacteria to fixed
endothelial cells. Some were. reported removed by" leucocytes,
but sessile macrophages possessed the greatest removal

axftivi ty, largyfily iii the lilng, livcu‘, arKl splewni. Ehnne

.41

phagocytic activity was reported in th‘lxnuilmartow, lymph
nodes, and kidneys to a lesser degree. The phagocytosed
bacteria were reported killed in 5 to 8 hours, and fatal
septicemiae were indicated as probably not attributable to
saturation of the macrophages, but rather to a physical.
lavage of the infected tissues.

Aschoff (6) in l92U was the first to group all the
activities of phagocytic cells previously described
togetlunf for (WNhfiidOrathfl1 as a rdiugle systan. He (walled
it the reticulo-endothelial metabolic apparatus, or Just
tlu?iretixWIIo-cwuiotluxlial :systcnn, irl()rdOT' to lJ1CllKh? all
cells suiting'au; phagocyist irrespectiw(>()l their ltxiation
within the body. The system included reticulum cells of
U10 spleen in the pulp and malphigian bodies, and those
of tJie lynnfilatixz glaruks arKl lynnfiioit tissvu‘. [t Ellso jiv-
cluded the endothelial cells which line the lymph sinuses
(3f lymph glaIKhs, sinusoidal ifltnxi capillarirwatif the liver,
bone maiixnv, supraxwwurl glands aruliaituitary glawul. The
lvuprW‘ cell:e ol‘ U10 l_iver‘vyerr‘(les(uxibed.ahs beirw; thc‘rnost
act ive (if tJres(\. liie sywstenizils() incliuled U10 annocdxiid
wandering macrophages and the mononuclear cells found in
the splenic pulp and occasionally in the blood of intes—
tinal organs.

Since the introduction of the concept of the
reticulo-endothelial system several decades ago a nearly

ccnitiruiolhs effkgrt luas lnéen {Hit lkrrtliluy nuwlicai arul

6
luioltggical irnwxstigattnws to stiuiy arKlanaSlUW? its [inn tions.
lfiy turnisvu‘enu‘nt:s ()f th(‘ a(‘tiv.iti(‘s of‘ tlie raysateni, i-ts
normalcy and integral function may be ascertained. Measure-
xnents of tlu‘ functions (d‘ U10 sy+dan have presrwnxxi special
difficulties because the component parts are spread
(lifflesely/ tln‘oiqfliovit tJle l)ody arui Initt.le (win lve 'learnned
through organ extirpation. Removal of a lung or liver
usually resulted in death or gross abnormality of a labora-
tory animal; removal of the spleen alone gave little
.infornuation alxnrt the finu tions (H‘<Jther [EH‘tS of tin? systenh
As a result, most approaches to measurement of reticulo-
endothelial activity have involved use of the propensity of
the system's cells to selectively engulf particulate matter
of appropriate size and charge. Techniques of study: have
included the intravenous introduction of vital dyes, living
organisms, inert colloids, ‘arbon black and India ink, and
in tiK‘IHOFO ”unharn and s(ufl1isticat(wl reseaiwiv efforts iéniio-
activm‘ tra(Wirs havci beervlised.

Drinker (42) and also Lund, et al. (98) utilized
particles of manganese dioxide for intravenous administra-
tion to laboratory animals in order to determine its fate
within the syrdan. Manganese dioxide was insoluble in
tisslu‘ fluids. :[t wa>;])repaiwnl for iJiJectirn1_in a snuspen—
sion of 0.4 percent acacia solution. Animals were given
l.u mg per kg of body weight. All particles greater than

one micron in size were removed by selective centrifugation

of the preparation. The rate of removal from the blood was
determined by performing gravimetric analyses on successively
drawn 10 ml samples. All injected material disappeared from
“no blood within l8 minutes. Of the total particulate matter
injected 90 percent went to the lung, iiver, and spleen;

l0 pelwwnit werH. to varitnus of otlwu~ bod) (Hiyans. llu‘inan—

\
ganese dioxide was recoverable from these sites quantita-
tiv<1ly \vitliin {in INJUF fol lowii1g iiijcu‘ti(n1. Di inkcn“ c<n1ct1uted
that the phagocvtic power of the vascular endothelium for
[filftiXHliatCFIHaitl‘r was; resqaonsii)le {Rrr tiH‘ resuilts.

Lund, et al. worked with numerous other species of
’laboratory animals and found that for all the manganese
>(iiuxilhfi wrnit unlintv to tlu‘ li\TU‘. 'Hiey ailso [M)int(wi out
that the phagocytic effect was the same if an inert materiai
such as manganese dioxide were used or if a protein sub-
stance wwnww SUhSiiiJliOd.

Cappell (27,28) did extensive work using vital dyes,
and he included. studies of phagocvltic uptake of these
materials within animal systems. He used dves such as
trvixan l)lu(\, liiJiitmi caiwniru‘, (W)Hg() rebl, ath a liost. of
otluars. No zatt<nnpt \yas Inadt? to cletcninin(\ the (unarfl.itat ive
Iiitt‘s ()f (iyr\ l”)tEU\f‘, init lie (lid. ptrint Otlt tile tixsslu‘s ()f
.Astfliof‘f'rs syssteni wliicli tin)k lip (iycue at. a inaiive(l iritc‘. lle
also indicated that phagocytosed dyes remained for long
iJPJZi0(Ls (if tiine vvitliiri FC‘til‘Ui()-(H1dl)til@i.ia.l <~el ls if‘ tlie

nm1terial.vyere ruui-toxi(‘, norrwtiffusalfl(~, and (wnild YN3t be

solubilized by the cell fluids.

Administration of carbon black or India ink dilu-
tions via the intravenous route has been another method of
measuring the phagocv tic velocity rate of endothelial cells.
[lalpern and his co-workers have been the chief advocates of
this method, and their work has spanned more than two
decades. The method consisted of injecting a known amount
of carbon per unit of body weight and determining quanti-
tatively the amount removed from (irculation as a function
of time (l9, ()9, 70). llalpern, et al. (7|) found that
India ink which contained shell-ac slowed the rate of
phagocvtosis, and that an excess of carbon, i.e., greater
than 2’4 mg per [00 gm of bod) weight in rats, caused a
liberation of thromolmplastin. This tended to exhaust the
reserves of blood anticoagulants and to cause the carbon
to precipitate. Administration of thrombin or thrombo-
plastin intravenously caused the rate of carbon disappearance
to increase and modified the organ distribution of
phagocvtosed particles. Biozzi et al. (.l8) reported that
the quantity of carbon injected and the degree of satura—
tion of Rh cells influenced the rate of phagocytosis of
subsequent doses. The uptake under these conditions was
found to be inverselv related to the amount of carbon.

The granulopectic activity was also found to be directl)
related to the cube of the relative weights of the liver

and spleen. An equation was established which related. to

specwl of inatakc‘ to lite auuiunt -inj<u_ted:

- 3% r 0.06(l- go) in which c z the concentration
in the blood at any one time, (0 : the initial concentra-
tion of‘<%irbon in tlu\l)lood, I" the anunuit'phagocyixrsed
by the RES, and T V the time from initial injection. They

also established the concept of a granulopectic index:
-kT

(' 1: ((J lO ill ulii< h h .is tlie {fiifinlll0[)0(“tt(“ 01‘
phagocy'tic index. Optimum carbon concentration for maximum
uptake rate was found to ‘be 8 mg per 100 gm of body weight.
Benacerraf et al. (l6) found that saturation of the system
with excess carbon could effectively block all response.
'll1(w 2111 iinzi I (‘()ll lCt l (‘(‘t)\ (‘1‘ l‘r*()ni :1 l)l (>< iii fl{§ <l«)s<(\ l)llt thcx
presence of shellac in india ink preparations retarded the
recovery making it four times the normal rate.

Jones et al. (8l) were the first to use radioacti\e
i.st)t()[)e t 121(‘e1‘ lut‘tlitidrs tt)!‘ tlle nua€1s1|r(‘nie11t ()f rt‘tfi(‘ltl()-
cuidiitliel ia I {\Inr tlt)n. 1i 1‘a<li<na(‘tiv e c()ll(ii(1al (flirtnnitun
phosphate was prepared fronidi-wuxlhun phosphate containing

P72.

The particle size of the colloid was less than one
micron , a nd lO m icrocur i.es of act iv i. t y" were injected int 0
“nice iii a {gltuwise ruispxnisitni. IAJcal.izal ion ()f tlie irsotcnae
was 90 percent in the liver, and most of the rest was
found in the spleen , with the combined radioactivity of

these two organs being lOO times that of the remaining

tissue. Preparation of a non-radioactive colloid by the

. l0
same method resulted in material which stayed in the .l iv er
and spleen cells for over a year.

Gabrielli (53) used Jones' radioactive colloid to
[HOarfllIC‘ thc‘tratx? al. wllicll digsa[n3ea1%1n(wa fiwnn tlie l)lo(ml-
stream took place. He used biological half-life as a
criterion of phagocytic velocity. This was defined as the
amount of time in which half the activity of the colloid
preparation had disappeared from circulation. The maximum
Tf2 valiu~lh)r rats was linuul to be l.23 HHJNltOS, with the~
mean being 0.87 minutes. He established an equation
siinilal‘ to lJIat (3f Halrnfirn's (Md-wcnlters: 'Ax ~T.AU((\ ”\Wd
in vfliicfl1.Ax (Nlualr4 act ivit) irl the l3lO(Kl at zany tinu\’T,
Ao equals the radioactivity in all the blood, and. \ equals
the v1~locit5’ of {fluago(3xtosiae. (kfl)riel Ii alsc) revcmiled
that [XH‘tiCIO size iiu"hiences tlH‘JTitO of phagocyt(nsLs and
the site of localization. When the particle size was
13-20 microns the lungs were found to absorb most of the
'(w)lloid', vfliile tlu‘ liver iwnnoved,nurst partixrles witJl a
size of around one micron. Phagocytosis of the larger
pai‘ticltvs war; sltnver tluln {in‘ tin; smalQler (n1es ( 36).
Galn‘ielli,inadr~ the ElShCU‘tiUrl that the cListriJnltitn1 of
'raciioac‘tiv1a (l1r(nnilun [fliospfllat€> wi.thii1 tile ziniinali sy5<ten1
is so constant that .it seems the' best possible agent to
us(\ fei‘ stluly (1f 11*ti<wnlo—cwulotluilial. ratcns arul kiru‘ti(ws.

The mechanisms involved in phagocytosis and some

()f tin) StH38tEU1CO>§ arul ctnutitixjns vvhi<fl1 aC(W3lOI£ltO ()r Jw‘tairt

it
its progress have been investigated by numerous workers.
Ponder (Ill) found that phagocytosis is favored
by a decrease in the surface energy of the cell or particle
and an increase in the surface energy of the particle about
to become phagocytosed. if the expression --

(SE particle) -(Sh cell particle) has a value greater
(SE cell)

 

than l.0, tlun1 phagocytix~ ingestitni is favored. 'Hiis was
thc~ bafivis lkir a [wiper l)y [Harry (1?) in vfliicll svnifacc>
activr>zuyfiits were linuui to favcn‘ U10 in leJW) ingesti(n1
of bacteria by human neutrophiles. Surface active agents
decrease the surface energy of the cell and phagocytosis
is tlurs erflmnficed. lierryrzand acssocijites (mn1tendxwt that
bacteria are more readily ingested in the presence of
serum because ol an increase in their surface energy and a
drufreascs in tluat ol‘ the rN‘UtllnfililKF. A.ru3t dcwxreasc> in
free energy was tin‘iwwuilttdescribed when bacteria were
phagocy tosed.

Gasselin (58) using radioactive colloidal gold

I98

described the removal of Au by rabbit peritoneal cavity
macrophages in vitro as taking place in two stages. The
gold colloid particles were reversibly adsorbed onto the
InacIIJpha{y‘ cel I sulliice. flngcwetiorltif tin? stuffatw‘ botfiKl
particle into the cell became irreversible. The rate at
which these two processes occurred was proportional to the

amount (H” colloiCtauLsorbed.t4) the cell :nirface wlflxfli could

be in turn related to the-concentration in the extra-

cellular fluid by the adsorption isotherm. The only

limit which was noted was that imposed by the theoretical
adsorption capacity of the external surface of the macro-
phage. Tn Gasselin's experiments less than one percent of
the total surface was ever covered at any one time. He
described no limit to the capacity of the macrophages to
Continue the reaction. At 37 C and pH 7.H, two to 20
percent of the adsorbed gold particles were ingested with-
in one minute.

Waddell, et al. (ITQ) noted the localization of
fats-dyed with Sudan .1:\ and Sudan Blaclx I”. in the spleens
and livers of laboratory animals (rats). llowev er, when the
RF. activity was blocked by excessive administration of
carbon black, trypan blue, or lithium carmine, the fats
did not localize as before. However, they continued to
disappear from the bloodstream at the same rate as in un-
treated animals. Examination revealed concentrations of
the colored fats in the hepatic parenchy-ma and splenic
pulp.

According to Heller (73) the reticulo-endothel.ial
system has several v ital roles including p1 oduction of
antibodies, phagocytosis of negatively charged particles,
and intermediary metabolism of fats. Because of the vital
nature of this system a substance or condition which can
elevate the system to a state of hy/perfunction is to be

Considered of the utmost importance. The number of such

l3
substances or conditixnrs is, up to this period, linutcwh
TWICW‘OfXJrf‘, ziny’ ncwr rétilnlllatl)Fy inal.erfial:s liax e 23 [H1i(“10
hnpordinice.

hlotz (88) stated that fat administered intraven-
ously to rabbits was filtered out by lung capillary beds.
Fats given intravenously‘tended to form emboli which
stimulated IUT)|if0f8titN1()r endothelial (walls having
‘phagocyli(‘zactivity qun1 thenh ine vas<w1hrr endotlH‘liaI
rdiagyocy‘ti(‘ aC‘tivxity“ of‘ t|u> sq)lcu~n, acircnial glanrwls, arul
arteries was stimulated by administration of cholesterin
(c‘hcllerrte11)l) . .Dtu‘infg plulgtufyttisi s ()f (Wiol(‘st(7riii fiat
there was an associated proliferative response which tended
t() dirst()rt tlua v(‘ss(‘l lunufin.

The cytotoxic sera produced by Hogomoletz (2i,
lOl,‘l26) have a pronounced elevating effect on the
reticulo-endothelial system in ver; minute quantities.
lhfil lei 's wv>rlv (‘itt‘d pi'ev ignis ly ilfid,ie€lt(‘d tluit cliol irie .is
(wapai)le (3f irroru)uru*ed. HtiJnHiéitltln (if INlQ§flJC§ tic‘ Icwspcnis(>,
and (kfl)riell i (3U) (108(W‘ih0d lJle activ ity ol liistanihie as
i)0]lfighfiiJHilEile* stiinulzttOIy . llie l‘OSfN)ns(‘ ol‘ th(\ sysstehi
can also be depressed by various means. Whole body irradia-
ti(n1 (37) and.£uhnh1istratitni(fl‘(Wirtisone (7o) aux? capable
()f stud] elW‘ect¢s. i

Ideas of looking toward cell-free products of
bacdxaria aiw‘ not rhuv witli resrxnjt to ifliysi(rlogirzil activ ity.

foley (1%) successfully treated cases of malignancy with

l u

{Drepaliltions ()f the t1>xins liwnn Stiwuptoctuwuis ergssipeléitis.

 

fln<~ludtul auuing thf‘rsUC(W‘Ssldlll)' trfuatecl casw‘s \V0l(‘ sevt‘ral,
of reticulum cell saicoma of bone. Not until late in the
(%)HIW%O ()f (tile) 's \VOllx “zis i t 11‘a|_i1(wl_tliat orué of‘ illO
efltw ts (>f irriectixuns ()f stmdv toxilis ths an (‘l0\£iii(nl of
the leticulo—endothelial system and phagocytic activity.
1W1e littrratlrre (W)ntE\lHlJIfi rw‘ptn’ts ()f (tiley'ss “wirk is too
ext<u1sivz‘ t()lH0f(‘ thanmalvcx brit‘f nunwtiorilielwfi, lnnt it
servrml to ix)int (Hli tlmlt batlwarial toximis arm] perlmqps
otliel' bac‘tei‘ial, pitMJu(‘ts \VOJT‘ inqxirtauit it} tllis I‘OHLX‘Ci. I

Girard £HM1 Muriay (CH , 62) havta revealed tluit the
t()tal lir>id~; exl.ra<‘tedl frtnn a liatluggcw1ic l‘ist(‘ria

monocytogenes can elicit certain of the responses charac—

 

teristically observed in infections caused by this organism.
Their MFA (monocy te producing agent) was a chloroform
extract of protein-free total cell lipids. When inJected

with sodium lauryl sulfate as an emulsifying agent, the MPA

 

Wiveri rcu icwrirug tlve vvoiivs ()f lfiogyanu)l(»tz £1n(l (t)l0}'
one must be aware that their publications in this country
l121\ (‘ 511‘()v1s~(*(l (‘()l15 i<l(\1‘:1l)l (\ (‘()l1 L] L)\ (‘1 svy zalnt)11g; HIC‘Cli (‘61 l ElriCl
lyhnlogical reseaiwfluuws. Many investigfilhors feel that nmrit
exists in each, and that they may provide groundwork'for
future approaches to malignancy therapy. Others disagree
and regard these as perhaps representing a bizarre type of

;researw%i haviru; littl(‘ valixlity (H';[Opr(KhJCilH lit}. Iln
citing these works as part of this review the author does
ruit vvisli i() svibst‘ril)e to (fiitllei‘ scdlotyl tu‘ tl10tqflit. live

puiij)os(‘ 111 illOlI” (nitEiti()n is to inclicaxte tha t tile}: {(rpiwé-
s(u1t tJVO irulivichual I_ines <3f apuyroa<41 t(> the ritudy of
methods of reticulo-endothelial stimulation

caused monocytosis which persisted in rabbits for 20 days
or longer. Animals immunized against typhoid and
staphylococcus toxins gave higher titers during monocyto-
sis than did controls. This was the type of situation
observed by these workers during actual. infections by the
organism. Girard and Murray reported the MFA to be non-
toxic and non-antigenic.

Stanley (122) preceded Girard and Murray with an
MFA substance, but he reported that the MPA was probably
bound to a polysaccharide within the cell. When the poly-
saccharide was precipitated independently of the MPA it
was found not to have the same monocyte stimulating capacity
as the lipids. Stanley later reported that lipids from

Aspergil lus fumigatii and Listeria monocytogenes when mixed

 

 

with lecithin greatly augmented the antibody production to

Salmonella typhimurium when all substances were inJO(‘t(‘(i

 

simultaneously (l23).
The pigment complex, v‘iolacein, obtained by ethanol.

extraction of (hromobacterium violaceum, has been used also

 

to stimulate the rate of phagocytic velocity in Laboratory
mice. (tnpubl ished data from the New England 'l‘nstitute for
Medical Research). This substance has been shown to possess
infra-red absorption bands which are identical to certain
of those found in bacterial lipid preparations.

The lipids of. certain bacteria have been extracted,

isolated, and characterized by several workers, but none

lo

seem to have used them directly as agents to experimentally
StlJmJiatC‘.fOtl(Wllu-OrKh)thOliiil activ ity.

Nicolle and Allilaire (IO3) grew entel ic bacteria
in large (unmitities and stlwhtwl their 'lipids. hfluu1 grown
on ix>hato agal'zit 37 ( for 2leunrrs they'ith3rted tlu‘ colon
bacillus as having 9 to lo percent total extractable lipid.
They seem to have been the first to report phospholipids
from this group of organisms. Dawson (38) grew E. coli
on a peptone~meat extract medium and. reported a lipid
content of u to 3 percent on the basis of cell dry weight.
.Addition ol‘(nué‘percent gly<wuw>l to the nuwihun resulted in
an increase to 8 percent lipid.

Eklustein and SovH(\ U46) reported tlu‘ lipids of
IL. («)li to l)e (fliarélctcu‘izcwl by low .iodll10 Iiunu3er>s arwl by
having fatty acids more saturated than oleic acid. When
organisms were grown on a medium containing a small quantity
(if alaniru‘ Urey welr\anlnd to Imux‘za greatly‘tflevat(wt rate
of phospholipid synthesis. The phosphatide was reduced to
a very low amount when cysteine was substituted for alanine.
'Hie itxline rnnM>er ltn" total lipiths was 235.6, udrile tluat fol~
fatty acid fraction was 37.4. The saponification number
was 2th. Negative reactions were obtained for the
[.ielnrrmarur-Bulwflialrl arul Salliowsdti t€v<ts liar ritertyls.

Arwlers(n1 arui hi:s felltnv wcndveiws (l., 2, 73, U, l06,
i133) dixl extcwrsive wunlt on nunnbers ()f tlu? Mycolmu‘teria.

They 1wumlrted.'H3.7 pelanit phospflmitide {Winn the avi£u1

1?
tubercle bacillus which contained mainly stearic and pal-
nu tic acdxhs. Data: for ()ther huunbers ()f tlufi grolq)vyere
esscuitially' Ute s%une witlvlninor (flvararleristix? variiitions.
llowcutir, lJie l(n)rosy bax~illtu4 wars fourul to (THTtairl only!
2.25 percent phosphol ipids.
Williams et al.(l36) cultivated 9 strains of enteric

organisms including Shigella, Escherichia, and Aerobacter

 

 

on a peptone—beef extract medium. Shigella paradysenteriae

 

was found to have a total lipid content of 5.5 percent of
.its (iry thiglit, vvitli 7'3 pcu‘c(u1t tu" tluat l)eirug [flvosrfilol ipixl.
in. col i. was found to have 8 percent total lipid and ()0
percent of the amount was phospholipid. iodine numbers
were found to vary between 42 and 82, tests for sterols were
negative, and P:N ratios were approximately J:l for the
phospholipid fractions.

Folclv (30) des(1'ibed a nudluxi for tlu? sepaiéition of
vzirious {flursphatithfi fractiinrs fimnn braiii tiSStK?. lh) requg-
n‘i zed cephalin as a complex of several fractions, and he
U5(Ki a<watoru3 arul etluanol foi‘ theiJfi Sfflfllratilnl by (liffeimuitial.
precipitatiiui. Levene arullh)lf (93) adefl)(jOSCfib0d a
method for the separation and purification of lecithin using
tllO instrlulrili ty ()f ltré C(Hle(§X uritll ca<bnivun (lilolxidc> as; a
lxasis lku“ sepaifltion.

Dawson, (39) and Lea, et al. (9i) described tech-
niques for chromatographic separations of phospholipids.

Lea and his associates used silicated papers as an aid to

luettcn‘ selmrratiinfis, zand. U10}Iila\(‘ repinfted EllHOiiHKi of
fractixnuation tluwaugh a (irhunn usiru; a fifu‘tion (willector
which would tend to eliminate error and loss through separa-
tion by routine chemical means.

The characterization and identification of complex
nuileciiles l‘roni b3(ft0fiii arul e\xan (if win>le («\ils lunxe lM‘en
successfully undertaken by se\eral jn\estigators. Le\ine
et al. (96) applied the infra-red spectrophotometric method
for the identification of glycogen within whole bacterial
cells, while Shirk and Greathouse (119) did essentially the
sanu\ Uiing [YH‘zn determmruition CH” Uie prcwunice of lxutterial
cellulose. Stevenson and Bolduan (l25) were among those
losing tln> infiir—red fVDGCtldun [in' the i<lentil‘icatitni of
whole bacteria. (omplex polymers such as glycogen, cellu-
lose, protein, and complexes found in cells present some
degree of difficulty in infra-red determinations not ex-
perienced with simpler substances, yet the results obtained

by th08H? wculters :seeni to luaxe ‘iustil‘ied iJlOiI‘llsO t)f tlu?

tecfliniiuie.

METHODS AND PROCEDtRE

(tiltivatjiniiaf Orgarnénns; Extrfuhtion arml Fracticnuition

E. coli received the greatest emphasis in this
:invtfist.ig:1ti()n. llie sstriliri icse(l wras ()n(* jx<0iilt(%i l’rtnn iwaw
sewage which gave typical coliform reactions: acid and gas
[qrodlu'tiori froni lathJse l)rotl1, arKl a (fliarax‘tericstic ( t) (+)
(-) (—) reaction to the lM\i( test. The S. dysenteriae
used was Dubos' strain 2308.

The lipid content of W00 gm of dried shigella cells
wals ev.triict«)d. urving; a.lHi.XitlF(‘ (if th) rxart-s (Wilt)rt)f()rni t() 0110
part methanol. The extraction was conducted passively,

i .(W. , 1)) slialvi ng: (‘c'll s ill tl1r> r)r(‘s(‘r1c(‘ ()f tlie s()lv'erit
without mechanical rupture for about I20 hours.

E. coli was grown on a chemical I} defined medium

containing the following per liter:

Natl ................ 3.0 gm
KZHPOL, ......'......... l.O gm
(NH )ZHPoh ........... l.O gm
MgS h ................ [.0 gm
glucose............... 2.0 gm
glycerol.............. 2.0 ml

These constituents were incorporated into agar or
titilized as l hiuid nuwlium as (humund qulHITYi. Petrd.<tishes,
l30 mm in diameter, were used in early phases of the work
for (Wiltivat.ion t)f Uifylnirflnfi. .[n0(dliatiJHTfiIWTW‘0 nmtha by

swabbing from broth starter cultures, and growth was removed

I9

2O

l)y wardring illtO tlué chchXJfornrqnetharn)t solv(uit. lknw?

rapid production of cells was achieved later when a super
centrifuge became available. It was then possible to
cultivate cells in large containers of aerated liquid and to
recover them by centrifugation.

tse of an anhydrous, water-immiscible solvent such
as chloroform or ethyl ether alone for extraction purposes
was unsatisfactory. The presence of a solvent miscible with
water was necessary in the system to break weakly bonded
complexes in order to release lipid material.

Several methods of treatment of E. coli cells were
followed. Extraclicnrevwere conducted (H1IMJLHt and dry
cellrs, witliznmd witlunit me<flwniical iflipttnti. Exrxntience
'indicated that violent methods such as grinding produced
large iuwu1tities of tH)JectiinuH)le denatvuwul prottdrivfliich
had to be eliminated. Extraction of moist cells in the
presence of a water-miscible solvent and chloroform (mis-
cible) seemed to give more rapid removal of lipids.

Following passive extraction of cells with chloro-
forwn-wnetlvaruil :soliitj()n, wilter* “was ( a1w>fvilly' l21y(\re(t ov<fir
the extracted l iquid. Shaking and agitation had to be
zavoixled iM‘CaIDSO (if tlua prcusen<w1 of‘ s U‘fa(w* a(‘tiv(‘ SlU)SiaJH“PS
iii tlie (‘xtrfiact. wlricli terulecl tti forin rstal)le (HntliSiJDHF; wi.th
watCH‘. A lzlrge, rflfllfitl<Yii tij\ separiitory [Rupiel udiich
gave maximum interface area was used for this purpose.

Successive separations against water in the cold (U to 7 C)

were conducted. until the extract no longer gave positive
reactions for protein or ‘arbohydrate. Most of the methanol
passed into the water phase on the first separation.

The chloroform phase, containing the lipid extract,
was Concentrated by vacuum distillation at temperatures not.
exceeding U0 (. Total crude lipid left after removal, of
solvtu1t was (hdiydirlted iii vacwu) ovcuh sullWU‘ic atfitl.

Extracts lrom both organisms were brown oils at
tlris :staige. lh)tli exl rac‘ts wenx it‘sttKi l1)r :stiinulz1t(rry
effects in animals. The Shifella extract was not fraction—
ated further.

The crude extract from E. coli was dissolved in
armalytitfvl grath‘ re-disd.illed (fllhiroformi. Arwn1)ximat(*ly
l0 nul [501‘ {gni ol‘ (firtul(\ nElt(\rjill wx‘rt‘ llS(Hi. 'fo tliis wzis
added a double volume of acetone. tpon standing, small
amounts of a yellowish-white precipitate formed. Two dis-
tiiict liiiuict lziycu s \MOIT‘ a Ist) ev idcuit. llie ])rr‘cii)itzitt‘ “Q15
separated by centrifugation at 7300 RPM for 20 minutes at
U (w 7H10 SHpOlTKIUEHt was (men1led and infitained. 'Hie pre-
(fi{)itéit(‘ Wias (lirss()lv(wfl tin 5 INT, ol‘ IV?-(li>%til le(l (liltiriifornn
arul trianlerrrcwl t() a \Matxwig|£1ss fol‘ evzlporzltitni router r1itiwngtu1.
'tt was tinten l”) in IQ nH (3f anhydiinbs ethyl (‘HIOI arul Uien
transferred to a tared glass vial. The ether was removed

under a reduced atmosphere of nitrogen, and the sample was

 

'lzil)(* l it‘Ci l:r‘£1(‘ t i()Il :[ . '

’fhc> siu3er11atiant liiniitl ccnitCILniiig lit)id.zan(l s()lv(3nt

22

was (wHMcentratCKi by vacwunn disti llatitni to a v hscous liipiid
vfliich was dissuflvtul in 25 HH ()f re—distiJthl(diloroforniznul
transferred to a small separatory funnel. To the funnel was
auidcwi 2 31ml (if Eibfit)lut(‘ etluanol , arxl it. wa+; al lowcwi t() stanfid
lin' 2U lunirs {it 4 it) 7 (. TWt) laycnws wer1\ visilvle ats the
end tfl‘ this ixwriod. 'Hie TUHTU‘ layer “mus carefvulyx<lrained.
from the funnel. Its volume was l8 ml, and upon standing
several hours at u C it separated into two phases in the
approximate ratio of 1.3:1. (entrifugation at 14000 RPM at

21 temperaturt1 of -23 ('IWJr U0 nHJuites separatmwl Hie lower
layer into a solid sediment. The supernatant layer was

lal>el lcwl l:ra(‘tii)n .[l, arul tlie rual.ei\ial s()Ii(l at. —225 ( “11s

lal)el le<l Flilvt,i0fl II t.

 

- 'fhe [Mirtitni renniiniiug iri the ssepaliltory' fvuuiel vvas
£1 l l()vv(~(l t () r%t_6111(i iii order~ t() (icit (‘l'nli ll(‘ i_f‘ ni()1‘£3 l £1y=(\1‘i.r1;§
would take place. None did in 2Ll hours. More absolute
ethanol was added, and the mixture was again allowed to
stainl 2Q THJUIH Eli 4 (‘. A sunni-stilid nEiterizal fcnwned (Hi the
surface of the liquid mixture. The liquid was drained
away, and the floating layer was recovered in ethyl ether.

lt vvass lal)el led Fra<~titn1 l\ . llve quviid {fiias(‘ fixnn tlie

 

funnel was concentrated by v acuum distillation to approxi-
umatc*ly haill‘ \()ilHHC‘. iXdLiiti_or1 ol‘ al)St)ItltC‘ ellialiol. tt) tliis
ITUHlltOd irizi layer linnning whixdi had a ngfller viscwrsity.
This separated out as a solid when centrifuged at -23 C.

The supernatant was decanted and retained. The fraction

1‘»
a.)

solid at -23 ( only partially dissolved in eth)l ether. The

portion completely dissolving was label led Fraction \, and

 

the white portion hav ing: no ether solubility but dissolving:

in clrlorol‘ornrwas label led Fraction \I.

 

An excess of acetone was added to the supernatant
From the. previous procedure, and the tube was stored under
desiccation at ~20 C. A small amount ol‘ white precipitate
was formed on standing '36 hours .i n the. cold. The liquid was
decanted while still cold, and the solid phase was dissolved
in ethyl ether and transferred to a tared vial. The latter

was labelled Fraction \[ll. The supernatant was concen-

 

trated by vacuum distillation to a viscous oil which was
taken up in ether, transferred to a tared vial and labelled

Fraction IX .

 

Fraction II separated into two distinct layers upon

 

standing at room temperature For 8 days. The upper lay er
was much darker than the lower. (entril‘ugat ion at -2 3 (‘
caused the lower layer to solidify. The supernatant was

retained as Fraction ll, while the phase which sol id il‘ied

 

at reduced tenu.)erature was dissolved in ether, transl‘erred

to a tared vial, and labelled Fraction \ll.

 

\‘ials containing); the Fractions were stored ,in a
desiccator Jar at a reduced atmosphei e ol‘ ni tlogjen pend inn
animal in Ject i on and analytical pi ()(‘Ot'llll'OS .

Two extractions were carried out with E. coli.

cells, while only one was performed upon S. dysenteriae cells.

 

N
.4?

Measurement of Phagocytic \e‘locities

The stimulatory activ it} of the lipid fractions

from E. coli and the lipid extract fiom S. dysenteriae

 

upon phagocvtic velocity as an indication of reticulo—
endothelizil anCtitHl\W1s measiuwxi by two [HWHTKUIFOS3

Tin thci fiJ st [)FtH‘OdtllO rnal(* win te ini(w\, (H)talI10d
from Armer Farms, (roton Falls, New York, 90 to ICC days of
age, averaging 30 gm wwflgflit, received via intraperitoneal
injection 0.2 ml of the undiluted fraction whose activity
wars to l)e nn‘asiuxed. Ir1trav<uloihs tl1J0(‘ti(HiS ()f i ipixls
caused l‘at emboli and frequentl) death of the animal. All
li[)i(1 {d a(‘ti oris \VOJ‘O oi Is: at. l)ocly tt‘nq30113tllff‘ wi.tl1 orie
e\(w‘ptii)n. A.:senii-stilid l ra< titfll “11s (iisstrlvcwl iri fUlU‘
parts;t)f sesauu‘ oil lkn‘ inicw tion. IX SOSanK‘(Jli (WHWLIOI
“(is riecw\ssziry fiir {iniina ls I ec(‘i\iiig thj.s [)repfilrzrti(n1.
(ommercial cephalin and the E. coli extract were eiiiulsil‘ied
in sodiiun lauryl :uilfate s(Hiition also {Yn‘ iniectimni. These
wcw e [)reixrrecl bv' siuspcw1di11g ().2 lHi (if lJie :saanIe in IQ lnl t)f
a ().l per(ww1t aqiuuius s()thior1()f sodiJun latnq l sillfate.

After a period of 72 hours following; injections of
lirnibs, each arHrmxl was giv(u1;an thOCtitfli()f l0 ndcuwuwrries
a1<‘ t i v i t v t) l‘ 1 21(1 i_()a1(‘ t i_v (‘ (‘() l l t)i (151 l (‘il l'()llli_tllil [>l1():s'1)l1£1 t (‘
according to the method of Jones (lOO). Phosphorous :32 in
tlie foiwn ()f (ii-uso<liun1 plu)s]fl1at1> “(is zndcled- t() ari e<pial anuiurit

of soditmnl>icarbonate soliditni,:nmi an excess of (Wuxnnic

25

nitrate was added. The precipitate which formed was insol-
uble in cold water; but it was soluble in hot water, acids,
and bases. .[t was washed and centrifuged twice in cold
water and dried at llO C. After drying it was heated at
600 C for l2 hours in an electric furnace. The amorphous
product which resulted was insoluble in aqua regia, strong
acids, and alkali. The precipitate was transferred to a
heavy-walled glass-stoppered flask half full of glass beads
and rotated for l2 hours with one to two ml of isotonic
glucose per mg of material. This procedure gave a particle
size of approximately one micron, and the predmninating
charge was negative.‘ The radioactivity of an accurately
measured a'liquant was determined by evaporation under an
infra-red heat lamp and counting under a Geiger tube. Di--
lutj4)ns intci gliuwuse vverc‘inad(\ ac(W)rdiruj t()'radi(nactiw ity
level. This preparation was insoluble in tissue fluids.

Injections were made via the caudal vein, and the
experimenter cut the tip of the tail. exactly 30 seconds
following injection of the last bit of colloid. Five

- -6 .
lambda (3 x l0 liter) blood. samples were drawn by means of
micro-pipettes each '30 seconds for a period of three minutes.
These blood samples were quickly blown onto filter paper
planchets and their radioactivity was determined under the
Geiger tube of anhlvB (Sweden) automatic Robot Scaler.
(ontrol animals received only the radioactive colloid except

in the case of the sample containing sesame oil.

26

A 13lot ()f izulioa<d ive (wJunt vMas nmuie (n1 a lcug scaICF
vs. a linear scale of elapsed. time. The slope of the
resulting lines was used to determine T,2, the biological
lialf lilW‘ value. 'Hiis was tiu‘ thne in udm(d1 half"Uie ac-
tivity had disappeared from the blood.

In the second procedure the sequence of events was
essentially the same except that a preparation of colloidad
carbon was injected instead of the radioactive colloid..The
anunuit of caiixni.suspensiinivflrich each armnm1l receiv(w1vfl1s
determined on the basis of body weight. No animal was given
more than 8 mg of carbon per lOO gm of body weight. A 30 gm
arrimal winild lve ggiveri no rnorc‘ thari 2.(§6 nwg by :sucii a [)rocwwl-
tire. Samples wrum3<irawn by nwwup<<3finicro-pipettes lawn“
the tip of th- cut tail every 20 minutes. The sample
representing zero time was taken 30 seconds after completion
of injection. This was repeated until 6 samples had been
drawn. Tlm~vw)hune of O&(dl\fi35 20 landxhhs, and tlu> first
sample was diluted and lysed in 0.l percent sodium carbonate
solution such that its optical density read on a Coleman Jr.
spectrophotometer at ()00 mmu was not greater than l.O.

Lysed control blood was used as a standard for setting the
instrument. Other samples drawn successively were given
parallel dilution, and the optical density was plotted
against time in minutes.

in each of the procedures a minimum of 6 animals

was used for each of the fractions tested. Six controls
were used for establishment of normal T 2 value. "In many
cases more animals were used because of the difficulties
eXpericnced in making caudal. vein injections without damage
to the vessel wall. Animals were kept in restraint by
means of a specially constructed tubular cage which per-
mitted only the tail to protrude- over an illuminated slit
for in‘ject ion.

The carbon used was obtained from National Midland
Company of lvatonah, New York, and it was prepared and
stabilized in gelatin solution according to the methods of
Menaceriaf, et al. (lb). The particle size was claimed by

the manufacturer to average 0.3 micron.

28
(haracterization of Lipids

'Hie irilia—iiwl siijtra. incltuled iii this [yaper'vvere
inadc~ini a Ikuivin—Fdiner numlel 2l Chnuble luumn recwnmling iiifra-
l‘Cd sq)ectiwn)hottnnet(>r at the INirkirr-ElHMUf plEUlt ill N01wu3lk,
(inn1ecticwit. Spuw tra wruwa run l1)r Pa(dl fractiini at (W‘ll
spacings of 0.] mm and 25 microns respectively. The thinner
films gave greater detail than was observed .in spectra ob-
tained at the larger spacing. This was especially true in
the case of several of the fractions which were much more
()})E1(lllf‘ tlizlri titli(‘i‘s .

Only‘ the erW‘tra ol‘ Hie istHzated {dru‘tions uxuw‘
(H)taiiied. Thirs was: by aulvi(w\ of‘ the igersuni s(%1nniiu§ tluw
spectra. Tlu1(q)hiion was (Mdkuxwl that, bOFalhK‘()f Uie nature
of the mixture present in the whole cell lipid extracts, any
:spectiwnn le<di ndgflit be (fldtairuwl wotflci not ru‘cesrfiirily (und-
\ey any useful information. A rapid scan of the sample of

total lipid from E. coli and that from S. dysenteriae

 

reer1led v<~ry li ttle (iiffOITWiCO lx‘twecw1 thcmi, so it.vwas
decided to abandon the attempt to determine characteristic
spectra.

l5ra<‘ti(n1s 'TT, '[ll , T\', arui \iII \VOIY‘ sid3Je( ted. to
(iiromatiuzraphi(‘:separatitni by ascwwuling tcufliniqtu~ using
silica treated Whatman #l paper. Spots containing l0
lambdas of the fractions were placed two inches above the

S()vaH1t li‘ved , arKl al‘ter‘ 7O lniletOF4 eqiiilil3rat.ior1 irl a (”dc

liter glass-stoppered mixing cylinder irrigation was begun.
(lllorolknwn-metluanol 83:2 wilil two FHH‘COYH.EM1U001H§ sodiiun
a<wrhate-ax%‘tic acid.lnllfer at {fit 1.5 ad<hw1 was thwwl as a
s()l\ erlt. P;th)*lcn1e {jl)(‘0l nu)n(nneti1)l etJlel‘ (WJIItaiJIllMZ 10
percent of the buffer was also used. The buffer was added
to stop objectionable Lrailing e\ident in pre\ious chromato-
grams. Commercial cephalin was chromatographed .in each of
the sol\ents.

Spots were stained for location using Sudan I\,
Slmknn lilacflx li, 01 l Rrwl 0, 21nd lU1odeunine (3 will] [H traxix)let
\ieuflJug. Sudartlilack Liljro\ed nunrt USOldll. tsirug \aldtflJH
lipid samples, it was iflhfiiu3l0 to detect ll'lO3 lipid dilution
on paper by means of Sudan Black B. Tests for choline were
‘perlknnned acwwirdiru; to (luirgali‘, et a1 . (32). iPapewrstyere
heated at 93 ( for 30 to 60 minutes, dipped into two percent
ffl1()s[)htinu)l yl)d.i(‘ z1( it] s()ltlt i()rl, ilHHlOl‘S(‘(l .irl ri-l)tltzar10 l f()r 5
minutes, washed in tap water, and dipped into fresh O.h
percent stannous chloiide in 3X Htl. Drying by heating
pr<n1uccxl hllH‘ spnits irulicratiru: ch()|inc‘. ld‘ee zuuiru) gtmulps.
were detected. by spin) ing‘papers with 0.1 percent ninhydrin

in n-butanol and heating at 95 ( for 3 to lfi minutes.

RESL LTS ‘

IJisaInJeaxznice [Tito ()f tlH‘ inJCM‘ted nMitOFijll fimnn
peripheral circulation is used as a measure of phagocytic
\elocity. in plots of sample optical density \s. time the
result is a straight line obeying the Beer-Lambert law. A
stizaigflit lir1e 211st) Iw‘stllts fttnn a. piiit ()f tradixiacrti\ e (rotu1t
\s. time. The disappearance rate of radioactivity from
the bloodstream produces an exponential function. In both
methods of representing data the lines haxe the character-
istic function Y * MX + H which possesses \alidity for any
linear graphic plot. Rather than use iine slope as a
unwaslHXW of .in<w*eas(* or (teciwaas(* of 13ha{u)cyt.ic Iqatakf‘, corr-
sideration of the biological half—life or T 2 \alue was
regarded. as being more conrenient. This \alue represents
tllO tilne Elt wirich 30 [)eiww\nt ()f tile nuat(uhiai injcw‘ted
initially'IHAs disappealwxl from actista circulatiinr. Specifi—
call)/, it is the anuuuit of time wfilii respect to tiu‘ first
sample drawn that the concentration or actix ity of the
particles has decreased to 30 percent of the initial lexel.
An elexated phagocytic rate was evidenced by a plot line of
greater slope; a decreased T12 value gaxe the same evidence.
In references to these results it is perhaps more meaningful
t() us(‘ 15 2 \zaltles 01‘ bii)lcq§i(%1l l1al.f~l.i\(‘s 21s (‘fiilOIVia ,in
experiments of this type rather than line slope.

3O

'3 l

The \alues for T, 2 demonstrated. graphically or
presented in tabular form have been determined against
(natical (lensity Caiilnfitlon (w1r\es iJ1.the c1hse of‘ U10 ca1-
bon suspension or against corrected count rate for the P32
acti\.ity. ZIn tlu‘ latt(U‘ casc‘, the (X)unt liate ars indixiited
on the Y axis was obtained from the number of disintegra-
tions picked up by the Geiger tube, corrected by a factor
detcuwnined {kn the {yeometig'ijf tin? partiJWJlar <wnu1ting
castlc‘. Thc‘IMIrposC‘()f Sludl a (WJFIOCliAHW was lJ) gi\(‘ so
11031‘ as possibltx tlua at tual Iaclioa(ftiyi ty (H‘ a l i\C\ ianflxfia
~+z1111})'| (\ () l‘ l) l t)()(i .

\altles (H‘rnearl'f.2 irKhlced.l)y tlufi yard<3us l ipid
fractiinrs are tabulatcwl.h1<flecreasing tuxhu‘()f phagocytic
stimulatixni, and tlu* shortest nmwu1'fg2 \altunszare also
quvresentcwl{graphically . Rankirugtaf these \EHlies detcuwnined
by (withcn‘ the (firborl tecluihiue (H‘ thP raxlioa<dai\e [T32 lIYEPCF
technique is parallei.

The S. dysenteriae lipid extract gi\es the shortest

 

[nearl'f 2 \ElluO. Fracti4n1s T1 aux] IV aiw>rnore an ti\c> than
the total extract from E. coli, while both the total ex—
tract (Wmllsifi(%1.in soditun lauryl rulllate arr>rmrre active
than the rest of the fractions. Emulsification with the
surface acti\e agent seems to ha\e reduced the acti\ity of
the extract from E. coli.

ExanflJuition ol‘ U10 imifra-rwwl SpOCtlYl re\(%1ls a

small and somewhat weak absorption band ranging from

2700 cm- to 2830 cm—l e\ ident in Fractions II, III, ]:\,

and \II. This band is also present on the spectrum of

. . -| -l .
commercial cephalin at 2720 cm .((m is l .)

Wax<Tlengtl1.in micrxnrs
idiO lPSEi 8(‘ll\(‘ fiiictiiins (io 11ot shcnv tlu‘ sanu‘ alM<OITDtiUTI
chaifu‘terirstics Ell thi~s wa\(‘ nunflnar.

The data in Table II rexeal that the actixe frac-
titnis rlt)ni E. c(r|i llax e [”01 e l3a1uls irl ctnnnu)n \ritJI tile
s[)e(‘tiw1ni ol‘ a lgn(nvn (‘erfllal iri tluln zlny of‘ tile ()tllOI‘ fd"a(‘ticn1s.
Sey en bands are shared with the cephalin spectrum: “2'30 cm-‘l ,

-l _[ -l -l H -i
2900 cm , 2700 cm , 1730 cm , IUOO cm , IO/Q cm , and
720 cm . An exception is noted in that Fraction II does

. . . -|
110i (‘Xhll3ll EH1 alruirptitni barul at.'720 (fin .

IBra<‘tii)n Tl liacl arr RI‘ \axltua ()f ().9l iri cl1l011)f01wn-
methanol, and 0.86 in buffered ceillosolre. It reacted
posititely to a phosphomolybdic acid—stannous chloride test.
A spot which migrated poorly and diffusely indicated the
r)rescn1ce (if inunnrit.ies. Fra(‘tior1 l\ \vas {Riund to {giye £n1
lif \allu\()f 0.80 in. the (11h)rofoinrum>thanol ini\tlHT‘, and
0 .7(3 l)y tliO ii‘i'igfiiti or] i n 'btifl‘ei‘e(i (‘el l()s()l\(‘. 'llu\ ina‘jol'

})(> r t i.()ri 1'(‘a1<‘ ttacl 1)t):s i t i,\ (‘I yr t t) l)()l l1 r1i Iiliy (l: i.11 l‘(‘€1{{(‘li t 2111ci tl1(‘
phosphomolybdic acid-stannous chltudxh‘ test. Impurities
were exident. Fractions III and \II both gate data similar
t () t ll(‘ za_l)()\ (‘ . ( ()liflll(‘ l‘(‘i :1 l (‘f‘IDliEl l i_11 ~s li()\V(‘(i {111 I? l‘ \‘El lllt“ ()l‘
().79 iJ1 chl(rrof(nin-nu‘tharu)l, i)ut ,it nfl{§rat(w1 \eiy' pUtH ly

and gaxe incOnclus'n e results in the buffered cellosohe

mixture. It reacted positixely to ninhydrin reagent.

'3?
F if: U RE I

PLOT OF P32 ACTlVlTY lN
5 LAMBDA BLOOD SAMPLES VS.

  
   

 

 

ELAPSED TIME
F
Control T/2 *4 1.2 min.
2000 '-°. Tota] E. coli extract
T/2 s 0.80 min.
1000 p.
‘2‘
‘3.
>
i'.‘
E
{\I
0".
Q-i
100
r
l l ‘I
0 1 * 2 3

ELAPSED TIME TN [\Ith TBS

27CH) cm"I it) 28330 (WM—l exiclerH. iii FIYJCli1)nS :EI, TlIli, I\ ,
and VII. This band .is also present on the spectrum of

. "l -l
commercial cephalin at 2720 cm .((m is l J

Waxeiength in microns
The less actixe fractitnb<<ha not show the same absorption
(filaram‘teri:<ti<wa at thi~s watt‘ n1mfl>er.

'fhe datrl in Talfl(~ff[ reveal that line actiu1 frac-
ticnfis fi(;ni R. ctili liay e inoi e l)a1uls iri cinnnu)n \vitJl tilO
S[)0(‘l1WJHl ol‘ a 1tn(nvn (ferflial iri tluln z1ny of‘ tlie ()tllei‘ fxiact.icn1s.
Seven bands are shared with the cephalin spectrum: H230 cm_J,
2900 cm_l, 2700 cm_l, I770 cm-l, IUOO cm_l, 1070 cm-l, and

-l .
72C) cni . Ari ex<w3ptit)n i:s ru>ted in tilat lirac‘tiorl iI (hoes

. . . -l
[NJi exlni>nt an alhflifplltni band Ell 720 (m1 .

FraX‘tiorl TI lmui arllif \altu> of ().9l in (41l011)fornr-
HH‘lhfliH)l, 23nd ().8(> in l)ufl1*1ed (‘ell(>sol\13. fit imwlcttwl
1)osi ti\(‘ly ii) a {filosifiionuilylxlic Elviti-SlEHWHOths cliloiixie tt‘st.
A spot which migrated poorly and diffusely indicated the
[Jrescnfice ()f iinptu'iti(‘s. Fra(‘tiori I\ \vas {Krund to §;i\e EU]

Rf \alue of 0.80 by the chloroform-methanol mixture, and
().7C> by th(\ ilw'igal.iori in lnnflirreCl cel los()l\(‘. 'Hie Inakhyr
yyrrrl.i()r1 r e21(~t<‘cl 1)t)s it ix e lyr it) l)()tl1 i1i11liycli Ii] r(\ag{(\rit aiicl tllO
phosphomolybdic acid-stannous chloride test. Impurities
were exident. Fractions III and \II both gate data similar
t() tlie al)()\(\. (i)nun(‘r(~i:1l (‘eralunl i11 ,sli(nvcui a11 lif‘ \’a llie ()f
0.79 in chloroform-methanol , but it migrated \ery poorly

and ga\e inconclusi\e results in the buffered cellosolte

mixture. It reacted positiyely to ninhydrin reagent.

'3 '%
F l’ G U RE [

PLOT OF P32 ACTIViTY 1N
5 LAMBDA BLOOD SAMPLES \s

  
   

 

 

ELAPSED TIME
Control T/Z 3‘4 1.2 min.
2000 -; Total E. coli extract
T/2 * 0.80 min,
1000 _
>—
E:
>
E
E
N
C"
a.
100
r—
l I ~1
o 1 2 3

ELAPSED TIME IN MlNLTES

2000

1000

P12 ACTIVITY

100

 

'% 14

\

FIGURE 2

PLOT or P'32 ACTIVITY IN CPU
or 5 LAMHDA BLOOD SAMPLES \S.
hLAPShD TIME

 

 

PJA\PSFA)‘TI\H§

p—
(‘ontrol T/2 7-“ 1.2 min.
Total Shigella lipid
T/2 - 0.65 min.
b
l-
l _ I
2 W

lN WlNLTES

OPTICAL DENS [TY

 

27
FIGURE 5
PLOT OF OPTICAL DhNSlTY or

CARBON FROM 20 LAMBDA BLOOD
SAMPLES vs. ELAPSED TIME,

Control T/2 * 51.9 min.

 

’ Shigella lipid T/2 r 22.0 min.
0
o
o
O
l l l
20 no 60

2OOO

1000

P72 ACTlVITY

100

 

16
FIGURE u

PLOT OF P72 ACTIVITY IN CPM
OF 5 LAMHOA BLOOD SAMPLES \s.

 

l: LAPSED T I Mb.
(ontrol T/2 ' 1.2 min.
- Fraction II T/2 m 0.78 min.
p
—
O
O
O
b
P
O
l l
O 2 3

ELAPSED TIME IN MINLTES

DENS lTY

OPT l' (' /\ l,

 

'3 9
r I (It, R P, 7
PLOT or OPTrCAL DhNSlT\ or

CARBON FROM 20 LAMBDK BLOOD
SAMPLES \‘s. hLAPSED Tle

Control T/Z '3 {31.9 min.

Fraction lV T/2 3 25.h min.

 

 

O
l 1
no 60

Ul

DhNSlT\

OPTICAL

 

#0
1“ [GURE 8

PLOT OF OPTICAL DENSIT\ OF
(”\RHON FROM 20 LAMBDAI BLOOD
SAMPLES \s. hLAPShD Tth

Control T/2 T 5|.9 min.

‘ . l , ~ , , .
Fraction II T/z." 28.2 min.

 

MO 60

DhNSlT\

OPTlCAL

 

F lGUR la. 8
PLOT OF OPTlCAL DENSITY OF

CARBON FROM 20 LAMBDA BLOOD
SAMPLES rs. LLAPSLD Tth

Control T/2 * 51.9 min.

Fraction ll T/2 : 28.2 min.

 

no 60

141

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

977;...

...:: CqNN ...:: wdlo ...:...qu :_~,;.fl,_.:m ~ ..38
1...... 3.4%. N II. C: Nndo >_ .2... 7.1:“;
J ...: fiwN .: ...: wqu __ 12.. 7.2.;
dS ...: filiow. .: ...:. Ofldq 7.5.. ./m ...:; la radish.
J: ...:. 0.4.4. I: m... _O.Q mam 2. 7.5.. .7... ...:; lump 45.3%
41...: w.Nm. 11...... no.0 mqm ._.. ._....5:aj
lql: ...E Oaflfl dZ ...: mOlO __> .23.. TQM—n—
.....E N.Om. 1...: mo.o :— 23.7.1.9;
...:? mlwm .......: No.0 .2 .2... 7:9...
#1... E OWNS J. ..E 0040 _ ddm 71.5..
«...:. film... 4.. ...: meO A— ..C ...:afilm Mi. ___> :C.. 7.5.:
l 1...... qujl .:,..:. mole l x. ..OHTZVT.
1...... quJ 4: ...: _al_ _.,. 1.: .93....
3:: fig: J. ...: —. _. T... ...:; _...c a..::1...m
J. ...: mt—m q: ...: N._ 4d,. ...dlv
.2:.me zoiz<. mwddzz..e m e_m_a
N».
szTfirvfz...‘ 2 7&1. .r....O_x<> ...::t :0; IFS/f) N\.,__. 2(2?

'C-\l3LI3 I l

M2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

AISSORPTION HANDS OI“ FRACTIONS (OMMON TO .1\ KNOWN (bPIIALI V PIIOSPIIATI IJI‘.
—] --1 -] -l —l

(cphalin 3210 cm 3710 cm 2900 cm 2700 cm 1210 cm

‘Fraction l l;) (-l (+) I-) (—l

F‘ract ion [1* LT) (-) Cl”) (+) (“31

Fractipu lll"= is) t-) (4) 1+1 J-PL‘

Fract ion IV"" (7) L—L (’31 (I) i!) __

FractltNI V it) til, ._t+l., i—) (+)

Fraetlcnl VI (T) .t-) it) t-) LiL

Fracti(nl Vllk ~_L{) (-) (+) (+) L?)

Fraction \2' 1 I I (..) (+1 1+) hi 1+;

.Fractlxnl IX (ll, Lil (ii. *_ L:l_, (TJ._'
—l ' —1 —l -l

(ephalin Lfik0,cm 1400 cm lO7QLLm, 720 cm

FractiOn l til (+) (-l Li)

Fraction II” l-) (ii Lt) .isl.

Fractiou_lll”‘ t-I (+J (bl Lil,

Isract uln I\‘ "'"' t-) I '1) (‘7‘) (:L

Fractign_fi .Lrl (+1. Lrl (+1

Fraction,\l .Lrl, (41_ l-) 0+)

Fraction \II* i4 (4-) (+1 (4.-)

Fraction VIII (+1. til t-) {-1

Fraction IX Dial, (+) I-) t—)

*Drun)tes Durst actl\¢‘ Fractirnns

(+v) I:nli<~at.e+< I)alld III (‘onrn(nl \vit h c<‘plu11 III

(-I 1|Hll(%1t()fi I)a1HI lH)t sIHaTW\d xvitIT (ufiplulllll

ITEM 0. number In cm. _1 I S the reciprocal of wave] engt h in mi c rons .

Q3

Infrared Spectra

On the following pages are infrared Spectra

of the fractions listed in order of decreas-

ing biological activit}. These spectra were
obtained on a Perkin—Elmer model 2] recording
spectropho'tmeter with ce] ]. spacingS as indicated.

M U

3.

72CLL...E

: m

S...w..cd.c>m.s

m

EH .. A

to . ahead 2: mo

: ..i. amp—QC b

 

- d

 

 

 

 

 

 

 

 

3H

NH

$.27TO.2

:_

S.m._o.mc>w3

mzmomflm :Chc.2 mm
>s :ceaoahm

 

 

 

 

 

 

 

 

 

 

 

Ll C)

...H

1.27.9.2

2.

£336 ac>m3

\O

®:_odnm
_r_

:35. ...E m N
..CAaCGLA

 

 

 

 

Cl

 

 

d

 

 

 

 

.r...

N—

127... ..2

OH

:_..

S. ...:... .Hc .ZmB

m.._...cw;m
._ _ .2

28.7. «E m N
:3 .. ,. Lag;

 

 

 

 

 

 

 

 

 

’48

7.27.92).

2.

2.3.... _c>d3

w

m... .3157“
I.

23,... ..S m. N
2: .. ...mhh

 

 

4?

 

« J

 

 

 

 

 

‘

 

 

 

 

u 0

N—

12399 :2.

O.

2.x..0 7. 2a.;

m

c

.w: .. LmLJ.

2

......L :2. rm.
::_.Lagn

 

 

 

 

 

 

 

 

 

 

 

 

.1.

N.

1.277;:

OH

2..

S . 3.... T. 253

..L.._..,.c:m
_

22.7. ...E m.
:3_. drvw.~,v_

N

N

 

 

 

 

 

 

 

 

 

 

 

,3]

a.

my

[ZSLL :4

0..

5.3.... 7.2.3:

...:...Odlm
___>

23.9 _. E

m

N

..C .. .. Oman.

 

 

‘l

 

 

 

 

 

pl.

1......12

iii... .91....»

...:...LGQJ. 23.7....2 .N

‘

A. ...:. «,3»... .._

N

G 3

 

 

 

 

 

 

 

 

,3

I

N

1.2.7... ..2

O—

:—

S . 3.... T. 3.3

...:—9611 ......,._.E .N

c \l

__/ I... ...5...

C J.

N

 

 

 

 

 

 

 

 

 

 

 

D'ILS( t SSION

The results obtained ie\eal that the tendency _-t.()wz.1r(t
ele\ation oI‘ [:)l1a{’;ocyt.ic \elocity by each ol‘ the I‘ia(tions
ran paral lel whether the determination was made by means of
the carbon technique or by using colloidal radioacthe
chromium phosphate as an indicator. The stimulation brought
about by Fractions l\ and [I was {greater than that ol‘ the
total lipid l‘rori It. coli, while other fractions, although
l‘airly stimulatory, were less so than the total extract.

The reason to: this is not known. The stimulatory power or

the lipid extract I‘rom S. dysenteriae was noticeably greater

 

than that From the other enteri c organism. One could per-
haps ini‘ei some relationship between the degree ol‘ stimula-
tion oI‘ phagocytic acti\ i ty/ and the pathogenicity. It. coli
is regarded as non-patlurgeni c to mildly pathogenic, with
some few strains being rather \ irulent, especially in
infants. The Shiga bacillus is usually regarded with a
{great deal oI respect so tar as .i.t.s.capac.ity’ to cause .in-
testinal disorders is concerned. No e'\ idence has been
presented to support such an inference, but it is an
interesting point ol‘ consideration which more complete
experimentation in a similar \ei.n with other enter ic

pathogens and null—[)3tIlUngHs‘ may either tend to conl‘irm or

negate. II‘ such a relationship were to hold we might ha\e

Sh

Vt

some idea as to what the mechanisms l'or phagocytic procession
are.

The data gained by comparison of inl‘ra-red spectra
indicate that the most actixe Fractions From E... coli ha\e a
band at 2700 cm-' which is not present in other I‘ractions.
All other Fractions were less acti\e and did not possess
this band. 'lhis particular band, though weak, may hold
some l‘acto'r responsible For the difference in actir lty. It
may be presumed with some \alidity that this is the al)sorp--
tion due to the phosphate group within the phosphatide
structure. Bellamy (IN) reports absorption or the phosphate
group in cephalin at 2700 cm-I. The method ol‘ separation ol‘
the I‘ractions does not permit one to make any statement on
the homogeneity of the preparations other than they are
components partially separated from the total lipid extra(t.
it was the opinion ol‘ the Perkin-Elmer spectroscopists that
all of these lrartions were mixtures of at least two or more
components. Due to the presence ol‘ mixtures and the masking
and shifting: ol‘ absorption in the inl‘ra-ied region there may
exist a reason [or the weak bands obserr ed at 2700 cur-l.

'l‘he inl‘ra-red ,spectrophotometer .is based in its
I‘unction upon the response of‘ a molecule to- interaction with
radiant energy in the particular -'portioi'i Or the spectrum
between 2 microns and I”) to l8 microns. The dil‘l‘erent mole—

cular \ ibrations and rotational changes ol‘ the molecule

taking place absorb energy and ghe identity to ind i.\_idua|

molecules. In order to arcurately make qualitathe identi-
l'ical ion ol' molecular structure the compound under scrutiny

must be a pure one. This was not the case with the lipid

A

fl‘i.t( t ions . Phosphol ipids , especially ‘ephal ins , tend to
complex aiiiol‘ig’; themsel\es and with other molecules, and they
present unique difficulties of separation. The use of the
infra-red spertrophotometer in this work was not for
qualitathe identification of these materials. Rather, .its
use was intended to ser\ e much the same lunction as that ol'
the in\ estiga tors who used it for characterization of whole
bacterial cells and complex substances within the cell such
as cellulose and glycogen. The purpose in this work was to
gain an idea ol the characteristics which tended to make.
one fraction distinct from another, and .if possible, to
show absorption band differences and relationships among
them.

.'Indi\ idual groups or’radicles, such as the phosphate
oi phosphoryl of phosphatides will tend to exert their ab-
sorptix e influences in the region of the spectrum below 7
microns (Ill/30 cm-I), whi Ie the \ ibrations of whole molecules
tend to exert their characteristic influences in the longer
wruelengths. (omplex and large molecules tend to be \ery

sluggish and good response to the infra-red beam .is not

achim ed in the longer waxelengths. This is especially true
it contaminating substances are present. For this reason,

particular attention has been gix en to the bands in the

shorter regions of the spectrum, although the bands beyond
7 microns ha\e been tabulated for their similarities of
characteristics.

The reticulo-endothelial system functions as a
main bulwark for defense of the animal mey, and. one of its
primary functions is that of phagocytosis of foreign matter
twithin the \ascular system. PJust exactly why the system
responds by supplying increased numbers of wandering
macrophages to localized lesions and general, systemic in-
fections alike is not known. One explanation found largely
in. textbooks, which offers partial clarifiration -is that of
chemotaxis. llowe\ er, discrepancies exist which are not
irrade entirely clear by the theory of chemotaxis. it does
not explain the simultaneous propensity of sessile macro-
phages lor .remo\a| of material from the \ascular stream;
neither does it gire any indication why the phagocytes tend
to engulf inert, insoluble substances, such as radiocol—
loids and, carbon particles known to haxe no chemical by—
produr ts or secretions comparable to bacteria and poly—
morphonuclear cells. Because the increased actiyity of
phagocytosis is extremely important any substance not
harmful or non—toxic to the host which can stimulate phago—
cy-tic acti\ity has tremendous importance. An understanding
of the stimulation and depression of phagocytic response is
fundannei'ital in the host-'pathogen relationship in infection.

The monocyte is one of the reticulo--en(lothelial

cells, of particular importance because it has the lunction
of phagocytosis. Tompkins (l28, l29) has reported that
phospholipids stimulate an increase in numbers of monocytes
in circulation and, that monocytes are phagocytic toward
such lipid moieties and metabolize them. If the fractions
which ha\e been shown to possess the highest actix ity as
described herein can, be conclusixely identified as parts

of the cell phospholipid complex, a partial explanation of
the mechanism of phagocytic stimulation may be at hand.
Such a partial solution seems to fit in well with the
theory of chemotaX’is.

Lipids hax e been considered as haptens by immunolo-
gists. According: to the concept held, they are thought to
combine with substances already forle within the system
to produce antibodies. ’l‘he data presented. in this paper
support the contention that lipids ha\e the acti\ ity
necessary to fulfill at least one of the criteria prescribed
for an antigen: they stimulate phagocytic Velocity to
hyperfunct ion. Said stimulation is without the presence of
a li\ inf; bacterial cell.

lf the theory of chemotaxis were to be accepted as
applicable to the results described her e, one might tend
to {gather that the responsible factorts) were coming from
the cell of the. pathogen rather than being; liberated by the
tissue at the site of an infection or lesion. Except for

the fact that the lipids used in this series of experiments

vir‘r (‘ (1(lnri rri s tr‘r‘r‘cl i_r1t rzir)r‘r i t()rrr\;1l l y , tlrr‘ tilt‘()l‘i (\s ili\ r)| \ (NJ
in chomotaxis might well apply. Early attempts at infirect ion
()f tlre l i[)i(l iritr11\ errorrs ly nret. \vi.tlr li tt lr\ ,iur‘cr‘srs, sr) rro
data ha\e been gathered. for comparatixe stimulator} rates of
the fractions introduced by the two routes. Thus, if chemo-
tzrxi¢s i s (le[)erchw1t ur)or1 a. sricr et_irn1 ()f rnr\tal)ol ic by -r)rrulru t
irr tlre \ZIscrrlar‘ str eaur frn‘ st.imrrlat()ry (\ffcw t rqporr phanto-
cytosis and attractiinrr>frnobile macrophages, little

(‘rirrs i<1(\r art i()i1 ( arr] l)(> {ti \ (\rr i.t for~ {1})r)l’ir‘:1l)i l i t y lrr‘r (x.

Arn/ agrnit wirich (THW stinnrlat(\ retirwrlo-(nulotlu‘lial
[11nr‘tirni Li) a ruarixecl degrer1 luls [dotr‘nt.ial as; a tlu‘rai)errti(‘
agent with a number of possible applications. Many of the
})er+u)ns (lerr)ted to rw‘searwdr (n1 tlu~ ret icrHr)-erulotlu‘lial
'syst(nn recrqniize tlurt tlu‘ furu tions (if tlu‘ systrwn are
essfflitial ly tlu‘ sanu> whether~ it ln‘ a (Vlso (if ruw)pltustir‘
disease or a microbial infection. The system is thought to
hate a limiting acti\ity upon neoplastic cells, and in
r“ x [)(‘i‘ irrrr\rr t ar l t rrrnr) l‘-i)(‘El r i rig: {111 i nrer l s: :1 r)r r) l i.l‘(\r'21 t i.r)ri () l‘ I] i r; t i.()-
(y tes, aunt kru)fer <%\lls of tlu‘ |i\(u , and rubleni(‘lrypertrmufl1y
lr£1\ (‘ i)(‘(‘li (l(‘~4(‘r‘ il)(‘r1.. ESrrr‘lr ril)s<(‘r \ (“(l ii,i s:t.()| r>g§i (‘ })lr(‘rir)nr(‘rrrl
may perhaps be interpreted. as the manifestation of a
physiological defense mechanism on the part of the host.

One of the possible applications of a reticulo-endothelial
stinuflzating zqyfint, sruir as tlre bactru ial lir)id, (inrld lu\
its rrse zis :1 clnnuotlu‘raru\uti<‘ agu‘nt .in tlre tr eatrnent, of

neoplastic disease. Further dexelopment of such a

()0

possibility would require a {treat deal of refined experi—
mentation, but the basic principle outlined in this work
might serxe as a beginning); point.

The author has a great deallof interest in intesti-
gations of cell-free extracts. Progress has been made in
past years in the areas of general, rrrorphological , and
taxonomic microbiology, and newer problems are being
\ isualized e\ my day. Many of these, including certain
ones of pathogenesis and immunity, lie within the scope of
fundziunental cell chemistry and physiology. lSy' cell chemistry
is meant the investigations of reactions and phenomena which
are related to products obtained from the li_\ in“; cell. The
isolation and study of enzyme acti\ities and kinetics tall
in this category; the knowledge of cell enterotoxins and
certain metabolic identities ha\e been arr ixed at by in—
\estigation of these products apart from the cell. The
research upon cell lipids in other than analytical fashion
represents another form of effort del\ inp; into the complexi-
ties of the cell. The disciplines of biological and physical
chemistry are \ery closely related. to microbiolrugy at this
le\el, and microbiologists could do \ery little without
knowledge of them.

Trnestigatfion of cell products represents a newer
approach to study of rrricrobiologi ‘al probi ems. Findings
from cell extract research indicate that certain cell

products can be used to artificially induce phenorrrena which

t)t

ha\ e been normally associated with the presence of a micro-
0' {’jani sm. The .‘lPA substance of Ci rard and Mur r a)’ ) and
Stanley is an example . The mono cy tos is produced by admi n is-

tration of the total cell lipi'd contents is the same as that

produced by an infection of Listeria monocytogenes. The

 

.w

cel l. lipid Work began as a source of this paper is perhaps.
another such example. tse of bacterial cell products as
described here may also ha\ e introduced a new concept of
therapy where the tissues are stimulated to greater anti-
bacterial actitity as compared with the use of an antibiotic
agent to combat the organism.

While such study of cell-free products may represent
a departure from classical microbiological procedures, its
methods may possess shortcomings which could ser\ e t'o offset
some of its positi\e attributes. Any treatment of cells,
regardless of tissue identity, which is disintegrath e in
its action creates gross abnorrrralities from which only data
ol questionable reliability may be obtained. The irnestigator

may not be certain that he is reco\ er ing the desired fraction
\

intact in a technique which utilizes blendors, homogenixers,
or colloid mills. Doctor Erwin (hargaff of (olumbia tni-
\ersity enlarged this point at length while conducting a
series of lectures in fundamental cell chemistry. He
expressed an opinion that any resemblance to the original
state after such drastic means of fraction separation was

entirely a matter of chance, and that the milder the treat-

 

ment the more likely the in\est_igator was to obtain a re-
co\ered product near its nati\e state.' The author has
tried to make this clear in the section describing methods
of extraction as a reason for carrying out passi\e extrac—
tions .in preference to more \ iolent treatment.

Future work upon bacterial lipids would- necessitate
much more refined and extensi\ e techniques than employed
in this .in\ estigation. For example, a major problem ,in
biochemical work with lipids is their oxygen ability.
lla‘ndtingirf all stages of the extraction and. separation
procedures in an inert atmosphere would greatly enhance
accuracy, \alidity, and reproducibilit‘y. Tlt was not possible
to protect all stages of the work from oxidatire changes
either at the New England 'l'nstitute or at the lnixersity‘
because of limitations of materials and space.

Fractionation of'crude lipid by column chroma-
tography using a fraction collector would seem to be superior
to the method of precipitation by chemical agents. The loss
incurred in handling and repeated transfers would thus be.
minimized, and the accuracy of analytical determinations
would. tlnrs be enhanced. This should. be particularly empha—
sized .in future work, because it will be extremely impor tant-
to identity the fractions more precisely than was done here.
liy use of the column and fraction collecting technique one
can obtain cur\ es by p'lottirg sample \olume, recorery time,

or sample number against the percentage of a giren component

sub‘iect to analysis. These curres show maxima, and by
obser\ ing such maxima one can determine which of the frtac—
tions collected, or what \olume of eluat‘e contains the
greatest percentage; thus, one could determine where the
greatest concentration of phosphorous was, similarly for
nitrogen, etc.

A major handicap throughout the entire inxestigation
was that of insufficient lipid with which to work. Large
quantities of cells were required to pro’\ ide sufficient
extracts, and the time required for arcumulation ol such
large quantities was the predominant prohibitire factor.
Growth of cells in small quantities presented problems of
storage until sufficient amounts were accumulated.
Lyophilization in tissue containers seemed the best method,
and it seems doubtful if l'uture procedures would offer a
great deal of impro\ement. Separation of large quantities
of cells without a super-centrifuge is almost impossible.

The decision to use 0.2 ml as .inJection dose of
lipid. was an arbitrary one which resulted in the expenditure
of (Her a milliliter of a lipid fraction with only () or 7
anirrrals. The size of a minimal stimulatory dose should be
determined as a part of future work. There is a distinct l
possibility that less lipid than was used in this series of
experiments would demonstrate the same degree of actir i ty
and yet enable experimentation to be carried on with {greater

consenation of the acti\'e substance.

()Li

‘\i) (a \ r)l zrrrzi t i t)il () r (‘()Hl[) l(‘ t (\ ll)”[)() tl lt‘ssi rs zr~s ti) t l1(‘
lll(‘( l rcrrr i ‘4lll () l‘ l i r) i (l ~< t i lllll l :r t i i) ii lizr s» l)(‘ (\rr () l l‘(‘ r (\(I ll (‘1 (r . 'l llt‘
tlrs(‘ ol~ l:r l)t‘l l (\(l f‘r‘zrr‘ t i t)l]f\ irr l tll Ill (\ \V() rl\ riii {glr t l)(‘ i) f l)(‘rr(\ f i t
i rr {iii i rrr (‘rs t i {:51 t i irrr () l t liifi lll(‘(‘llLtll irsiii. ( ii I t i rirr t i (rrr ()i
l)z1<‘ t (‘l i :1 (iii lll(‘(l izr (‘()Iii :r irr i rr{j l (r(| i ()i ><ir t irr)(\~< \f’i t Ir t |r(\ :1 irrr irl‘
producing label led. fractions which could be followed in the
animal system would be irnaluable as a technique to permit
determination of the fate or site of localization of the
l i1)i(l fr ai‘t l()n>$. \i tr‘rq§(‘n l 3 irr‘ rflrirsiihirrirrrs 'i2 wwrrrlcl lil\i~ly
l)(‘ t ll(‘ i -ir t () [)(rrs () l‘ (‘l l() i ('(‘ . ( :1 r l)() ii l 1+ vvir til (I })(‘ r l121 [)rs [)r t) \ i (l t‘
i ii f‘()l‘lll{tt i (>11 () l‘ t lit‘ l ()(‘Et | i 7.z1 t i ()rr () l‘ l‘r (1(‘ t i irrrrs () tl 1(‘1 t lizrrr
phospholipids, but it would also present certain problems
of counting technique not inherent with the use of the
tither' isotrn)es.

In expanded future work in\esligation of possibility
() f r'(‘l zit i irrr~4lri r) l)c~t\V(a(~rr [3;] tlrirggi‘rri ('i t y irf‘ tllt‘ l>L1(‘t c-r iiirn zlrril
st.inurlati)ry' ac‘tix ity' ol‘ ilrs l ipiil l ra(~ti(nrs ripinr [fliagurcy tic‘
,,.. ‘ ‘ d”
\ (a l L)(‘-i t yr rslri) lll (l l)(\ <‘(311:s i (l(‘ r (‘(l i nr})(‘ r‘zl t i \ (‘ . 1\:s >4 t £1 t (‘(l (\zrr‘ | i.(‘i* ,
t lrf‘ (la) til [)r‘(\-(‘rr t(‘tl li(‘l‘(‘ >+(‘(‘rrr t t) i lltl i (‘51 t (\ e1 r‘(‘ l;) t i.t)ll (lt‘[)(‘ri<l(‘ri t
rrr)irrr {)1} t lit){g(‘ri.i (‘i t y () l‘ t lr(\ ()r‘{§£1r1 irsiri, l)tl t t lr(\ (\r i(l (\r1(‘(‘ irs
irisrd‘fii‘ierrt ti) drvlw {lny \al id ( oru‘lrnsiini. Pll‘pnliliit)n irf
t lri‘ t () t I] l (i‘\ t.r'za (‘t :1 l) l(‘ l i r) i (l (Irril i t >< l‘r :1 c‘ t i ()Il s l‘ l‘()lli ><(\ \ (N [‘11 l
organisrrrs representing pathogens and non—pathogens Would be

necessary.

S t M i\'l/'\ R V

'rhe lipid»; from two enteric. {gram negrg'at i\e
organisms" Escherichia coli, considered to he. non-pathogonic,
and Shigellu dysonteriae, an intestinal pathogen, were
e\tracted'zlnd injected intraperit()ir<\al..,l).’ into lahonatoi‘y
mice in an effort to learn if they possessed stimulatory
acti\ity for the reticulo—eudothelial system.

l3)! use of radioactix‘e coll o I'dal chromium phosphate
and colloidal ca‘rhon suspensions as indicators of the rate
of phagocytosis of particulate matter from actixe circula—
tion it was found that the lipids from S, dysenteriae
Pfisfls‘t‘ss’t‘d marked power to stimulate phagocytic act'iVity to
hyperfunction, while those of E. coli and the sub—fractions
did similarly to a lesser degree.
Pcutizil characterization of the sul)-fracti_ous from
la”. col ,i indicated that they e\hil)ited man)" of the attributes
of phospholipids, since the most active fractions possessed
an absorption hand in the infra—led ‘r'eggion not shared h).
.Iess acti\e ones. The same hand was ol)ser\e.d in a known
phosphatide, and it was tentat'irei)‘ identified as that ‘
produced by the phosphorous containing: group found in

phosphat i de structures.

(OVt LtIS l ON

The total e\tractnh.l e lipids from laschericliin coli
and Shinellzi (lysentei iae and four of nine suh—firictions
from h, coli found to possess Ph}*ical characteristics of
phosphzit ides are decidedly stimulatory to the phagocytii
fun<tiou of the I‘eticuflo-endothelinl system of lalnnator}
mice {is measured h) the Uptake of ladioacth'e chromium

phosphate and colloidal curhon from the bloodstream.

()()

().

8M

10.

11.

Ill BLI OI:II2.\PIIY

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OXVGEN AHSORPTION HARD IN THE lNFRA-RED SPECTRUM OF
ALCOHOLS. Journal of tho Amonican (homical Society,
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