In Partial Fulfillment of the Requirements For the Degree of
MASTER OF SCIENCE WITH A MAJOR IN MICROBIOLOGY . '
In the Graduate College
THE UNIVERSITY OF ARIZONA
19 6 9
STATEMENT BY AUTHOR
This thesis has been -submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited' in the University Library to be made available to borrowers under rules of the Library,
Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship, In all other instances, however, permission must be obtained from the author.
s IGNED: (7
APPROVAL BY THESIS DIRECTOR
•This thesis has been approved on the date shown below:
WAYBURNxJyf JETER • Professor of Microbiology.
Date
ACKNOWLEDGMENTS
With gratitude to Dr, Wayburn S, Jeter for his guidance and
understanding during this study; in sincere appreciation to my wife
for her help and encouragement; with thanks to Mr, Aftab Ansari for
' SERUM SUGAR AND KETONE LEVELS OF NORMAL AND ALLOXAN DIABETIC RATS IN PHAGOCYTIC AND BACTERICIDAL STUDIES USING STAPHYLOCOCCUS EPIDERMIDIS AND DIPLOCOCCUS PNEUMONIAE TYPE 1 WITH RABBIT ANTISERUM „ „ „ „ „ „ „ „
PHAGOCYTIC ACTIVITY OF NEUTROPHILES FROM NORMAL AND ALLOXAN DIABETIC RATS IN STUDIES USING STAPHYLOCOCCUS EPIDERMIDIS AND DIPLOCOCCUS PNEUMONIAE TYPE 1 WITH RABBIT ANTISERUM . . „ „ . „ „ „ . . „ . . . „
BACTERICIDAL ACTIVITY OF NEUTROPHILES FROM NORMAL AND ALLOXAN DIABETIC RATS IN STUDIES USING STAPHYLO-. COCCUS EPIDERMIDIS AND DIPLOCOCCUS PNEUMONIAE TYPE 1WITH RABBIT ANTISERUM . . . .......
SERUM SUGAR, KETONE, AND AGGLUTININ LEVELS OF NORMAL ■'AND ALLOXAN DIABETIC RATS IMMUNIZED WITH DIPLOCOCCUSPNEUMONIAE TYPE I VACCINE . . . . . . „ , . . , , . „ „
SERUM SUGAR AND KETONE LEVELS OF NORMAL AND ALLOXAN DIABETIC RATS IN PHAGOCYTIC AND BACTERICIDAL STUDIES USING DIPLOCOCCUS PNEUMONIAE TYPE 1 WITH NORMAL AND . DIABETIC RAT ANTISERA . . . .. „ . „ , „ „ „ . V
• PHAGOCYTIC ACTIVITY OF NEUTROPHILES FROM NORMAL AND ALLOXAN DIABETIC RATS IN STUDIES USING DIPLOCOCCUS
, PNEUMONIAE TYPE 1 WITH NORMAL AND DIABETIC RAT ANTI-S ERA o" a o e o o o o o o o o o o o • o o o e o o o o o o
BACTERICIDAL ACTIVITY OF NEUTROPHILES FROM NORMAL AND ALLOXAN DIABETIC RATS IN STUDIES USING DIPLOCOCCUS PNEUMONIAE TYPE I WITH NORMAL AND DIABETIC RAT ANTI-S ER-A: ' o - O - . O- 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 - 4 4 4
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. LIST OF ILLUSTRATIONS .
Figure Page
lo Experimental procedure employed for phagocytic andbactericidal studies using Staphylococcus epidermidis and Diplococcus pneumoniae type 1 with rabbit anti-pneumococcal serum „ „ „ „ , „ „ o „ „ 14
2o Experimental procedure employed for phagocytic andbactericidal studies using Diplococcus pneumoniae type 1 with normal and diabetic rat anti-pneumococcalS e r a o o o o o o o o o o o o o o o o o o o o o o o o o o 13
ABSTRACT
Diabetes was induced in male Sprague-Dawley rats by intraven
ous injection of alloxan in order to study possible factors contribut
ing to the lowered resistance to infection prevalent in this disease0
The phagocytic and bactericidal action of neutrophiles from ketotic
diabetics on Staphylococcus epidermidis and Diplococcus pneumoniae
type 1 was found to be the same as that of cells from normal animals0
The antibody response of normal and diabetic rats to immunization with
Do pneumoniae vaccine was also comparable0 Pneumococci opsonized with
diabetic rat antibody were submitted to the phagocytic and bactericidal
action of normal neutrophiles6 The results did not differ from those
obtained using normal rat antibody in the same systems Use of neutro
philes from diabetic animals in place of normal cells did not alter the
results o
INTRODUCTION
It has long been recognized that individuals with poorly con
trolled diabetes mellitus are particularly susceptible to various
pyogenic infections (1) as well as tuberculosis (2)0 When earlier
workers attempted to study the reasons for this increased susceptibil
ity using animals5 they produced experimental diabetes by total or par
tial pancreatectomy (3) <, Loss of other pancreatic secretions besides
insulin introduced marked nutritive changes,, however, which complicated
the picture (2)« Controlled conditions for investigation were finally
made possible in 1943 when Dunn, Sheehan, and McLetchie (4) discovered
that Intravenous injection of alloxan into rabbits caused selective ne
crosis of the islets of Langerhans in the pancreas»
Alloxan is the ureide of mesoxalic acid„ It is a component of
the uric acid molecule and can be obtained from it by oxidation with
nitric acid (3).
: Jacobs (5), in 1937, was the first to report that intravenous
injection of alloxan into rabbits produced a fatal hypoglycemia0 In
1943, Dunn et ale (4, 6) reported that intravenous injection of alloxan
into rabbits caused an initial hyperglycemia with subsequent hypogly
cemia and selective acute necrosis of the pancreatic islet tissue,
Bailey and Bailey (7) confirmed this work and were also the first to
show that if the animals were kept alive during the acute hypoglycemic
phase by glucose administration, extreme hyperglycemia subsequently
developed« They indicated that their rabbits developed signs comparable to those seen in human diabetics„
Refuting the idea of Dunn et al„ (6) that the diabetogenic ac
tion of alloxan was due to exhaustion of overstimulated islet tissue,
Gomori and Goldner (8y 9) presented experiments to show that the toxic
effect of alloxan is limited to a few minutes immediately following in
jection and that it exerts its effect by direct damage to the pancre
atic beta cells * Ridout, Ham, and Wrenshall (10) and other workers
(11, 12) proved histologically that the transitory alloxan hypoglycemia
is produced by insulin liberated from degenerating pancreatic islet
cells rather than by excess insulin from overactive cells (6)0
While the features of alloxan-induced beta cell damage have
been described repeatedly from observations with the light microscope,
Wellmann, Volk, and Lazarus (13) used electron microscopy to study it.
They indicated that the sequence of beta cell changes observed suggested
that alloxan exerts its principal damaging action upon the paramembran-
ous portion of beta cell cytoplasm with ultimate disintegration of the
cell. ' ' ' -
. The first successful results of experimentation with dosages
permitting animals to survive alloxan diabetes were reported in 1943 by
Dunn and McLetchie (14) in the rat. They induced diabetes by repeated
subcutaneous injections of alloxan0 Gomori and Goldner (15) independ
ently reported the same results, but their rats had been given a single
in trap er i tone a1 injection of 200 mg alloxan/kg body weight. Subse
quently, optimal intravenous dosages were worked out and the course of
.permanent alloxan diabetes was described for rats (16), rabbits (17),
dogs (18), and mice (19), The way was now open for controlled study
of the susceptibility of diabetic animals to infection.
Among the various defense mechanisms of the body against infec
tion, the most logical ones to consider first in a situation of obvious
breakdown in resistance would be antibody production and phagocytic
function, .
The ability of the diabetic to produce adequate antibody titers
was questioned in the report by Moen and Reimann (20) that uncontrolled
diabetics produced distinctly weaker agglutinin titers to typhoid vac
cine than did normal individuals or controlled diabetics, Bsites and
Weiss (21) found this situation to be the same with antibody production
against staphylococcus toxin in diabetic children, Wohl et al, (22)
also reported diminished antibody production in diabetics but indi
cated that this decreased capacity was not related to hyperglycemia
but to hypoproteinemia,
When Payne and Cruickshank (23) immunized normal and uncon- .
trolled alloxan diabetic rabbits with crystalline egg albumin, they
found the precipitin titers of both groups to be the same, Beattie,
(24) also found no significant impairment in the primary and secondary
immune responses of alloxan diabetic rats to injection with Salmonella
typfrpsa.; : 7:; v: ; . y7’ . ; % ■Altered activity of the phagocytes in diabetes could be due
either to primary or to secondary causes, \
As a direct result of the lack of insulin (primary cause), it
has been reported that glucose utilization (25, 26) and lactic acid
z-
production (25) are lower in leukocytes from diabetics than in those
from normal individuals» A decrease in glycogen synthesis and a lower
glycogen content have also been reported (27)* Since the efficient and.
continuing ingestion of bacteria by neutrophiles has been shown to re
quire energy and to be dependent on glycolysis (28-32), the ability of
neutrophiles from diabetic individuals to phagocytize would be expected
to be depressed * .The hydrolyzing and oxidizing activities of the vari
ous antimicrobial agents found in neutrophiles (33) are also energy-
dependent (32) and this, along with decreased lactic acid production,
would mean decreased bactericidal activity* -
Secondary causes would result from.the deranged metabolism of
the diabetic* The high sugar concentrations of the blood and tissues
and,the increased levels of ketone bodies could affect the phagocytic
activity of the neutrophiles *
. The situation, as shown in the following reports, has been
found to be more complicated than these theoretical considerations
would suggest, however*
Cruickshank (34) felt that severe ketosis may devitalize tis
sues sufficiently to favor growth of invading bacteria* He injected .
virulent and avirulent pileumococci and virulent staphylococci into
nonketotic diabetic rabbits and failed to find an impairment of resist
ance* Sheldon and Bauer (35) reactivated healing mucormycotic granulo
ma t a in rabbits by inducing alloxan diabetes, but this occurred only in
animals that became ketotic*
Subsequent studies by many workers intimated that ketosis and
acidosis seemed to be responsible for a delayed and reduced inflamma
tory reaction in diabetics. This was reported in rabbits (36, 37?' 38),
rats. (39), and human, beings (40) 0 Others (41, 42), however, had not
observed this to be the case. 'Dubosf work (43) showed that lactic acid antagonized and ketones
favored the survival of staphylococci and tubercle bacilli in the type
of acid environment thought to exist within the phagocyte. Thus, the
possibility existed that ketone bodies could neutralize, in part at
least, the antimicrobial effect of lactic acid produced by the neutro-
philes.
The experiments of Cruickshank and Payne (44) suggested that
the leukocytes from alloxan diabetic rabbits had normal phagocytic ac
tivity but reduced bactericidal power. Others (45) indicated that this
was possibly also the situation in the pathogenesis of experimental
Rhizopus oryzae (mucormycosis) infection. But Wertman and Henney (46)
reported that alloxan diabetic rats had a lower than normal percentage
of active neutrophile.s and that the phagocytic activity of these cells
was reduced.
Bybee and Rogers (47) found that leukocytes from nonacidotic
diabetic patients exhibited a normal capacity to ingest and destroy
pathogenic and nonpathogenic staphylococci whether suspended in dia
betic or normal serum. On the other hand, leukocytes from ketoaci-
dotic diabetics showed a significant decrease in uptake of pathogenic
staphylococci. This defect disappeared upon correction of the acidotic
state but was still present if cells from acidotic diabetics were sus
pended in normal serume However9 no abnormality of phagocytic capacity
was observed in normal cells suspended in acidotic serum0 They felt
that these studies clearly demonstrated a defect in the leukocytes of
the diabetice
Briscoe and Allison (48) used peritoneal exudative phagocytes
from nonketotic alloxanized rats and found the phagocytosis and killing
of Diplococcus pneumoniae type 1 to be essentially the same for cells
from normal and diabetic animals» They felt that this supported the
idea that neutrophifes from diabetics are not altered0 They favored
the osmotic effects of hyperglycemia as being possibly important to the
general problem of infection. This work was therefore extended by
Crosby and Allison (49) in nonketotic diabetic human beings. They. i
still could find no impairment in the phagocytic and bactericidal func
tion of the neutrophiles. They then added glucose to the sera to ob
tain concentrations in the range of 1050-1620 mg% which corresponded to
serum osmolarity levels of 358-373 mOsm/liter. Even in this extreme
environment) cellular function was found to be completely unaltered.
Unfortunately, they did not study leukocytes from patients with ketosis.
Dr.achman (50) and Drachman, Root, and Wood (51) reported on a
whole series of experiments using nonketotic diabetic rats and D. pneu-
mpniae type 25. Histologic studies of experimental pneumococcal pneu
monia indicated no impairment of the.inflammatory response but rather a
failure of the mobilized leukocytes to ingest the invading organisms.
A similar depression of in vivo phagocytosis occurred in preformed
: ''; V ■ " . ■' '' ' ■ 7 peritoneal exudates«' Subsequent in vitro experiments indicated to them
that the principal defect here resided in the serum rather than in the
neutrophiles, and. they identified this as the abnormally high concen
tration of glucose. Contrary to others (49), they found that addition
of large amounts of several different sugars, including glucose, de
pressed the phagocytic ability of normal cells suspended in normal
serum. This suggested that the effect was osmotic rather than meta
cerning impairment of the antibody and cellular defense mechanisms of
diabetic individuals and concerning ketosis-as a contributing factor.
Consequently, the purpose of this study was to re-examine some of the
factors that may contribute to the lowered resistance to infection of
individuals with diabetes mellitus. Alloxan diabetic rats were com
pared to normal rats with regard to: 1) the phagocytic and bacteri
cidal activity of their, neutrophiles, 2) the ability of the animals to
form antibody to D, pneumoniae type 1 vaccine, 3) the activity of this
antibody in effectively preparing virulent D, pneumoniae type 1 for
phagocytosis, and 4) the effect of ketosis on the aforementioned fac
tors.
MATERIALS AND METHODS
Experimental Animals
Male albino Sprague-Dawley rats obtained from the Holtzman Co0
(Madisona Wisconsin) were used throughout this investigation. They
were housed individually in metal cages with wire mesh bottoms9 main
tained on Purina laboratory chow, and provided with water ad libitum.
Their body.weights ranged from 295 to 469 grams. In all cases blood
was obtained from. these animals under ether anesthesia by cardiac punc
ture (52) using a 20 gauge, needle.
Induction of Diabetes
A 57o solution of alloxan in 0,15 M saline was freshly prepared
and'stexllized by filtration through an 0,45u Mi11ipore membrane filter
each time injections were made. Two milliliters of blood were drawn
from each animal for a base line sugar determination, and 40 mg
alloxan/kg body weight were injected into the tail vein with a 25 gauge
needle. This dosage was chosen on the basis of reports (16, 469 53)
that this was the least amount.of the chemical that would consistently
cause a diabetic condition in rats with no significant renal damage.
In 7-10 days after the alloxan injection) 2 ml of blood were
drawn again for sugar determinations. Rats with serum sugar levels
greater than 300 mg/100 ml were considered to be diabetic.
8
Determination of Serum Sugar and Ketone Levels
Blood drawn for sugar and ketone determinations was allowed to
clot at room temperature. The serum was separated and stored at -20 C
until all base bleedings and subsequent bleedings from the same animal
could be analyzed together, -
The sera to be tested were thawed and protein-free filtrates
•were prepared according to the method of Folin and Malmros (54), Sugar
determinations were made in duplicate on these filtrates using the an-
throne method (55)0 Optical densities were measured at a wavelength of
620 mu on a Coleman.Jr, spectrophotometer and were compared against a
blank containing only distilled water and anthrone reagent. Glucose
standards ranging from 50 mg/100 ml to 500 mg/100 ml were treated the
same as the serum samples and were run simultaneously, A standard
curve was prepared each time from which serum sugar values were read as
mg/100 ml serum, ' - :
. The presence of serum ketones was determined by adding 2 drops
of serum to an Acetest nitroprusside tablet (Ames Co,, Inc,, Elkhart,
Indiana) that had been crushed to;insure intimate contact of serum and
chemicals, Development of a color change indicated the presence of ke
tone bodies. This was estimated semi-quantitatively by scoring the
reactions as 0 to 4+ for no color to a deep purple.
A culture of Staphylococcus epidermfdis was used and grown on
• trypticase soy agar plates,. It was transferred every 48 hr and main
tained at room temperature between subcultures, For phagocytic and
: 10. bactericidal studies.,' the organism was grown at 37 C for 18 hr in tryp-ticase soy brotha
Diplococcus pneumoniae type 1 was also employed« It was passed■ „6. through Swiss albino mice until 0„ 1 ml of a 10 dilution of an 8 hr
broth culture killed a mouse in 18 hr (56)„ Its identity and encapsu
lation were confirmed, by the Quellung reaction using rabbit anti-
pneumococcal type 1 serumo This organism was also transferred: every 48
hr and maintained at room temperature between subcultures on 5% sheep
blood agar plateso Virulence was kept high by passages through mice
: twice weeklyo For phagocytic and bactericidal studies,. D, pneumoniae
was grown at 37 C for 8 hr in brain-heart infusion broth containing 1%
glucose and 10% sterile rat serum0
After the growth period, both organisms were centrifuged at
350Q x g for 15 min at 4C in a model PR-2 International refrigerated
centrifugeo They were then washed and resuspended in sterile 0015 M
saline to a concentration equivalent to the MacFarland no0 4 nephel-
ometer tube (57) for use in the phagocytic and bactericidal experi-
• men ts 6
Type 1 Do pneumoniae Vaccine ,
; Type 1 Do pneumoniae vaccine was prepared according to the
method of Goodner, Horsfall, and Dubos (58) 0 The 0017o formalinized
saline stock suspension of organisms was adjusted to a MacFarland no0 2
nephelometer tube with sterile 0o15 M saline for injection into rabbits
... and to a MacFarland no0 3 tube for injection into rats0
Preparation of Antisera
Rabbit antibody to Do penumoniae type 1 was prepared using the
method of Goodner et al* (58)* The agglutinin titer of the antiserum
was 128, and a 1:100 dilution of this was used in the phagocytic and
bactericidal experiments0 Normal rabbit serum was tested and found to
be negative for agglutinins to type 1 D„ pneumoniae. All rabbit sera
were adsorbed with kaolin (58) to reduce any. possible toxicity.
Rat antibody to D. pneumoniae type 1 was prepared by injection
of vaccine, into the tail vein of the animals according to the following
schedule: ,
Day 1 0.1 ml Day 15 0.2 ml
3 0.1 ml 17 0.4 ml
5 0.1 ml 19 0.4 ml
7 0.1 ml 21 0.4 ml -
: 9 0.2 ml 23 0.4 ml
11 0.2 ml _ 25 0.8 ml
13 0.2 ml 32 test bleed
Rats in both the normal and diabetic groups were immunized according to
this same schedule, starting 10 days after the injection of alloxan
into the animals of the diabetic group.
A pool of normal rat antisera and a pool of diabetic rat anti
sera were prepared. The agglutinin titer of each pool was eight, and
each was used at a 1:6 dilution in the phagocytic and bactericidal
studies. Serum obtained from the normal and diabetic rats before im- .
munization was found to be negative for agglutinins to type 1 D. pneu-
moniae.
Rat S erurn P o o 1 s Use d for Leukocyte Resuspens ion
Approximately 2 weeks before each group of normal and diabetic
rats was to be used for experimentation, 4 ml of blood were drawn from
the animals in each group, and allowed to clot* The sera were used to
form.normal and diabetic pools which were divided into 1*5 ml aliquots
and stored at -60 C to preserve the complement activity* These pools
were used for resuspension of the leukocytes in the phagocytic and bac
tericidal studies *
Phagocytic Experiments
Phagocytic studies were performed using a modification of the
method reported by Jeter, McKee, and Mason (59)* From each rat 4 ml of
blood were collected in an equal volume of sterile Alsever1s solution
in a siliconized centrifuge tube» After centrifugation at 3500 x g. for
15 min at 20 C, the supernatant fluid was discarded and the buffy coat
drawn off* The buffy coat was washed .once in sterile 0*15 M saline and
resuspended in 1*4 ml of pooled rat serum (either normal rat cells in
normal rat serum or diabetic rat cells in diabetic rat serum) * This
approximately 3-fold concentration of leukocytes was necessary because
of the low percentage of neutrophiles in rat blood (60)* Use of this
procedure also provided for the addition of complement to the system*
A leukocyte count was performed on this concentrated cell sus
pension, and in each case the number, of leukocytes was adjusted with
the appropriate serum to a range of 24,800-30,300 cells/mm serum* The
suspension was then incubated at 37 C for 30 min before being used *
■ - ■ / ; ; . : - w:Leukocyte suspensions, organisms, and sera were mixed as indid
eated in Figsp 1 and 2 in 10 x 35 mm siliconized tubes and stoppered
with corks dipped in melted paraffin. The tubes were then placed in a
rotating box (8 rpm) at 37 C for 30 min after which they were removed
and plunged into an ice bath to stop phagocytosis. Smears were made
and stained with Giemsa stain (61)e Duplicate slides were read and 100
unclumped, unbroken neutrophiles were examined on each slide for: 1)
the percentage of neutrophiles active in phagocytosis, and 2) the aver
age number of cocci ingested by 10.0 neutrophiles.
Bactericidal Experiments
Bactericidal studies were carried out using the same protocol
as that in the phagocytic experiments. After the bacterial cells were
diluted to a turbidity comparable to the MacFarland no. 4 tube, a 0.1
ml sample, was .immediately removed, diluted in 10-fold increments, and
the dilutions used to make spread plates in triplicate. A 0.05 ml
sample of this bacterial suspension was also put into tubes contain
ing the leukocyte suspensions as shown in Figs. 1 and 2* After the 30
min incubation in the rotator, 0.1 ml samples were removed from these
tubes, diluted in 10-fold increments, and the dilutions again used to
make.spread plates in triplicate. Trypticase soy agar plates were used ,
for"So: epidermidis and 5% sheep blood agar plates were used for type 1
P. pneumoniae., Colonies were counted after 48 hr incubation of the
plates at 37 C.
14
•LEUKOCYTE SUSPENSION-
0.5 ml 0.5 ml 0.5 ml
0.20 ml saline 0.20 ml RAS 0.20 ml NRS
0.05 ml S.e.C 0.05 ml D.p.^ 0.05 ml D.p.
30 min rotation (8 rpm) at 37 C
smears (phagocytic) or dilutions (bactericidal)
Figure 1. Experimental procedure employed for phagocytic and bactericidal studies using Staphylococcus epidermidis and Diplo- coccus pneumoniae type 1 with rabbit anti-penumococcal serum.
a. Rabbit anti-pneumococcal serum (dil. 1:100)
b. Normal rabbit serum (dil. 1:100)
c. Staphylococcus epidermidis (conc. comparable to Mac. #4)
d. Diplococcus pneumoniae type 1 (conc. comparable to Mac. #4)
15
0.5 ml
0.20 ml NRAC
0.05 ml D.p.
-LEUKOCYTE SUSPENSION-
0.5 ml
0.20 ml DRAb
0.05 ml D.p.
0.5 ml
0.20 ml C0NC
0.05 ml D.p.
30 min rotation (8 rpm) at 37 C
smears (phagocytic) or dilutions (bactericidal)
Figure 2. Experimental procedure employed for phagocytic and bactericidal studies using Diplococcus pneumoniae type 1 with normal and diabetic rat anti-pneumococcal sera.
a. Normal rat anti-pneumococcal serum (dil. 1:6)
b. Diabetic rat anti-pneumococcal serum (dil. 1:6)
c. Non-immune rat serum (dil. 1:6)
d. Diplococcus pneumoniae type 1 (conc. comparable to Mac. #4)
EXPERIMENTAL RESULTS
Serum sugar and ketone levels were obtained initially for base
line values on all normal groups of animals, and were determined again
at the time that blood was drawn for a phagocytic or bactericidal,ex
periment e r For the diabetic groups, initial serum sugar and ketone
levels were also'obtained« These were determined again at 7-10 days
•after injection of alloxan to confirm the induction of diabetes and at
3-4 weeks after injection of alloxan when blood was collected for ex
perimentation e
For the phagocytic studies using S epidermidis and type 1 De
pneumoniae, Table 1 shows that after 1 week all of the rats injected
with alloxan had serum sugar values in excess of 500 mg/100 ml, . All
but two had 2+ or greater ketonemia. In addition, all exhibited ruf
fled fur, polyphagia, polydipsia, polyuria, and glycosuriao Ketonuria
was detected but not regularlye Lipemia was observed and the extent
correlated well with the amount of ketonemia found. .
By the time the phagocytic experiments were performed, all of
the diabetic animals were, ketotic, and their cells were suspended in
ketotic, diabetic pooled serum. Cells from nonketotic normal animals
were,: suspended in nonketotic, normal pooled serum. The sugar .and ke
tone levels of these serum pools are shown also in Table 1.
An analysis, of /variance (95% confidence limits) of the data
shown in Table 2 indicated that the percentage of active neutrophiles
,,
TABLE 1
SERUM SUGAR AND KETONE LEVELS OF NORMAL AND ALLOXAN DIABETIC RATS IN PHAGOCYTIC AND BACTERICIDAL STUDIES USING
STAPHYLOCOCCUS EPIDERMIDIS AND DIPLOCOCCUS PNEUMONIAE TYPE 1 WITH RABBIT ANTISERUM
GroupTotalno.
Initial 7-10 days after alloxan Finala
rats Range Aver. Range Aver. Range Aver.
A. Phagocytic study
sb 94-126 Ill —• — 95-123 110Normal 9
Kc 0-0 0 - - 0-0 0
S 90-155 112 540-747 625 544-732 622Diabetic 7
K 0-0 0 0-4+ 2+ 1+- 4+ 2+
Normal rat serum pool: S = 107; K = 0Diabetic rat serum pool: S = 663; K = 2+
B. Bactericidal study
NormalSc 107-126 117 - - 106-131 118
3K 0-0 0 - - 0-0 0
DiabeticS 90-155 120 560-747 629 536-744 629
5K 0-0 0 0-3+ 2+ 1+-3+ 2+
Normal rat serum pool: S = 108; K = 0Diabetic rat serum pool: S = 582; K = 2+
a. At the time of the phagocytic or bactericidal study
b. Sugar in mg/100 ml serum
c. Ketonemia scored as 0-4+
TABLE 2
PHAGOCYTIC ACTIVITY OF NEUTROPHILES FROM NORMAL AND ALLOXAN DIABETICRATS IN STUDIES USING STAPHYLOCOCCUS EPIDERMIDIS AND DIPLOCOCCUS
PNEUMONIAE TYPE 1 WITH RABBIT ANTISERUM
GroupTotal % active n0i neutrophilesrats
No. cocci per 100
neutrophiles
Aver. no. cocci per active
neutrophileRange Aver. Range Aver. Range Aver.
A. Staphylococcus epidermidis'
Normal3 9 - 64-86 76 382-677 520 5.9-7.9 6.8
Diabetic3 7 - 61-82 71 387-593 496 6.2-8.0 7.0
B. Diplococcus pneumoniae type 1
Normal3Tb 76-99 91
9525-1079 774 6.9-10.9 8.5
CC 2-9 4 4-17 9 1.8-2.6 2.1
DiabeticT 71-99 90
3 7438-1322 796 6.2-13.4 8.7
C 1-6 3 2-12 6 1.5-2.0 1.8
3a. Leukocyte counts in range of 24,800-30,300 cells/mm serum
b. Organisms and rabbit anti-pneumococcal serum
c. Organisms and normal rabbit serum
and the phagocytic activity of these cells from normal and diabetic
animals were not significantly.different0 This was so whether or not
the system used was one requiring specific antibodyo
On the basis of these results, bactericidal studies were per
formed to see if a difference could be detected. Again, Table 1 shows
that, at the time the experiments were, carried out, the diabetic ani
mals providing the cells were all ketotic. They also exhibited the
signs described aboveo The cells from these ketotic animals were sus
pended in ketotic, diabetic pooled serum.
It was found that, under the conditions of these experiments,
the bactericidal power of the leukocytes from diabetic animals was not
any less than that of the leukocytes from normal animals as indicated
in Table 3. In fact, inspection of the total number of surviving or-
ganisms shows that fewer survived in the leukocyte suspensions from the
diabetics than in those from the normal animals. It may be that the
ketone bodies in the pooled serum of the diabetics had some adverse ef
fect on the organisms. Of course, such a situation would not be in
line with Dubosf findings (43).
It can also be seen in Table 3 that a large number of pneumo
cocci were killed in the controls. Since the phagocytic experiments
showed that only about 3% of the neutrophiles in the control system had
phagocytized.organisms, this would seem to indicate the presence of a
bactericidin either in the rat serum or added with the rabbit serum.
Myrvik (62), in his studies on serum bactericidins against gram-
positive bacteria, found large amounts in rat and rabbit sera.
20
TABLE 3
BACTERICIDAL ACTIVITY OF NEUTROPHILES FROM NORMAL AND ALLOXAN DIABETICRATS IN STUDIES USING .STAPHYLOCOCCUS EPIDERMIDIS AND DIPLOCOCCUS
PNEUMONIAE TYPE, 1 WITH RABBIT ANTISERUM
A„ Staphylococcus epidermidis
Eat no. Total no. of orgs. added (x 10 )
Total no. surviving
of orgs. (x 10b)
Normal3 Diabetic3Normal Diabetic
1 1 2.5 9.5 8.8
2 2 1.1 4.4 3.4
3 3 ; 1.2 6.4 4.3 .
■/ 4 ;;; 4 0.55 4.0 3.4
5 5 1.8 6.8 4.2 :
B. Diplococcus pneumoniae type 1- f
Total no. of orgs.Rat no. Total no. of ofgs. surviving (x 10S)
Normal Diabetic3 added (x 10^) Normal DiabeticTb CG T C
' 1 1 1.4 1.4 2.1 1.2 1.7
; 2 r 2 0.55 0.96 1.3 0.50 1.0
3 3 0.65 1.1 1.6 0.57 . 1.2
4 4 2.0 2.9 , 4.4 2.5 3.8
' 5 5 1.2 2.1 3.8 1.8 3.5■ 2 a* Leukocyte co.unts in range of 249800~309300 cells/mm serum
b0 Organisms and rabbit anti-pneumococcal serumCo Organisms and normal rabbit serum -
- ' - - - / \ - / . . - - - - ' ^ 21 Nevertheless,, a leukocytic effect can still be detected here because
the numbers of surviving organisms in the test systems were signifi
cantly lower than those in the control systems.
The agglutinin titers shown in Table 4 indicate that, in gen
eral, rats do not make very high titers of antibody against type 1 D0
pneumoniaeo Although all of the animals had extremely low titers, it
probably can still be stated that the immunologic response of the dia
betics was comparable to that of the normal animals, which is in agree
ment with previous work (23, 24). The presence or absence of ketosis
in these animals did not affect their ability to form antibody.
Even though the diabetic rats seemed capable of producing anti
body titers as high as those of normal rats, the possibility existed
that this antibody might not be as effective as antibody from normal
animals in preparing encapsulated pneumococci for phagocytosis0 Phago
cytic and bactericidal studies were undertaken to investigate this. It
can be seen in Table.5, however, that this time not all of the diabetic
rats were, ketotic at the time of the experiment. Two of the six ani
mals were not ketotic while the others exhibited ketonemia ranging.from
14- to 44-o
The data in Table 6 show that, when normal cells suspended in
normal pooled serum were used, pneumococci coated with antibody made- by
diabetic rats were phagocytized just as efficiently as those coated
with antibody from normal rats. ■
In order to expose any possible depressive synergistic inter
action between the neutrophiles and the antibody of the diabetic
22
TABLE 4
SERUM SUGAR, KETONE,' AND AGGLUTININ LEVELS OF NORMAL AND . ALLOXAN'DIABETIC RATS IMMUNIZED WITH DIPLOCOCCUS
- PNEUMONIAE TYPE 1 VACCINE '
Ratno.
Initial 7-10after
daysalloxan Agglutinin titer
Sugar mg/100 ml
Ketones O'-4+
Sugar mg/100 ml
Ketones0-4+
Beforeimmuniz.
After immuniz.
A. Normal rats
1 ' : 103 0 - - P ' 82 84 0 - : o - 4
3 95 0 - - 0 44 84 0 . - 0 8
5 112 0 0 4
Normal rat antiserum pool 8
B. Diabetic rats
1 1X5 0 513 1+ 0 8
2 : 133 0 442 4+ 0 4
3 11.6 0 490 . 2+ 0 8
4 103 0 612 + 0 16
5 109 0 389 0 0 4 .
6 153 0 520 0 0 4
Diabetic rat antiserum pool 8
TABLE 5
SERUM SUGAR AND KETONE LEVELS OF NORMAL AND ALLOXAN DIABETIC RATSIN PHAGOCYTIC AND BACTERICIDAL STUDIES USING DIPLOCOCCDSPNEUMONIAE TYPE 1 WITH NORMAL AND DIABETIC RAT ANTISERA
GroupTotal no. .
Initial 7-10 days after alloxan Final3 '
rats Range Aver o Range Aver. Range Aver o
Normal 6sh 82-128 109 89-129 110
KC 0-0 0 0-0 0
Diabetic 6S 107-155 132 417-601 539 400-575 513
K 0-0 0 0-1+ ± 0-4+ 2+
. Normal rat serum pools S = 119; K = 0Diabetic rat serum pools S = 536; K = 1+
a. At the time of the phagocytic and bactericidal studies
b» Sugar in mg/100 ml. serum
Co Ketpnemia scored as 0-4+
TABLE 6
PHAGOCYTIC ACTIVITY OF NEUTROPHILES FROM NORMAL AND ALLOXAN DIABETIC RATS IN STUDIES.USING DIPLOCOCCUS PNEUMONIAE TYPE I WITH NORMAL AND DIABETIC RAT ANTISERA
GroupTotalno.rats
7o active neutrophiles
No. cocci per 100
neutrophiles
Aver. No. cocci per active
neutrophile
Range Aver. Range . ' Aver o • Range Aver.'' b NRA 87-99 94 15 15 - 25 25 1982 17.2-26.0 ■ 20.9
a. Leukocyte counts in range of 24,800- • 330,300 cells/mm serum
b. Organisms and normal rat anti-pneumococcal serum
Co Organisms and diabetic rat anti-pneumococcal serum
do Organisms and non-immune rat serum
- . . ■■ ' ■ v . ■ ■ : ' y' y " 25-animals, leukocytes from ketotic diabetics were suspended in ketotic,
diabetic pooled serum and were also used with these antisera,■ Inspec
tion of Table 6 shows that there was no statistically significant dif
ference in phagocytic activity whether diabetic rat antibody was used
in a normal or a diabetic system. As already mentioned, with two of
the animals there was a situation of cells from nonketotic diabetics
suspended in ketotic, diabetic pooled serum, but this did not alter the
results o
Table 7 presents data indicating that the bactericidal -activity
of cells from normal and diabetic animals against organisms coated with
either normal or diabetic rat antibody was comparable under the condi-
• tions of the experiments. The use of cells from the two nonketotic
diabetics made no difference here either.y . ' . . y - iAs was seen in Table 3 also, the number of surviving organisms
was generally less in the leukocyte suspensions from the diabetics than
in those from the normal animals although the difference was not as
pronounced this time. It may be noted that the ketone level of the
diabetic pooled serum was not as high either.
- The fact that the number of organisms surviving in the test and
control systems was more when rat antibody and control sera replaced
that of rabbit would seem to indicate that the rabbit serum was donat
ing the bactericidal factor, but not all, however, since the non-immune
rat serum controls still showed a high bactericidal activity.
26
- TABLE 7
BACTERICIDAL ACTIVITY OF NEUTROPHlLES FROM NORMAL AND ALLOXAN DIABETICRATS IN STUDIES USING DIPLOCQCCUS PNEUMONIAE TYPE 1
WITH NORMAL AND-DIABETIC RAT ANTISERA
Rat
Normal^
no.
Diabetic3
Total no. of orgs. added(x 1010)
Total no. of orgs. surviving (x 10 )
NRAbNormal
. DRAC CONd
Diabetic
NRA DRA CON
' 1 1 . 2.3 1.9 1.9 7.1 1.8 1.8 6.9
. 2 1.8 1.8 1.6 4.8 . 1.5 1.6 4.6
3 ;\Y;. 3 1.8 1.9 . 2.0 4.5 1.8 1.8 4.0
4 4 2.1 1.8 1.6 6.0 1.8 1.6 6.0
5 5 2.3 1.7 2.0 7.3 1.6 1.9 6.9
6 6 1.6 1.8 1.7 4.3 1.6 1.4 4.0
3a0 Leukocyte counts in range of 24,800-30,300 cells/mm serum
b0 Organisms and normal rat anti-pneumococcal serum
Co Organisms and diabetic rat anti-pneumococcal serum
do Organisms and non-immune rat serum
DISCUSSION
Wertman and Henney (46) reported that the phagocytic capability
of leukocytes from diabetic rats'was depressed., but they made no men
tion of the presence or absence of ketosis in their animals0 z Some in
vestigators. (47, 48, 49) subsequently reported results indicating that
the leukocytes of nonketoacidotic diabetics were in fact normal in
their ability to phagocytize and kill ingested organisms0 They sug
gested (47) that only in ketoacidosis are the leukocytes depressed in
their activity0 Others (50, 51), however, have reported finding de
pressed phagocytic activity in nonketotic diabetic animals6 In the
studies reported here, the polymorphonuclear leukocytes from alloxan
diabetic rats exhibited normal phagocytic and bactericidal activity 1 even though the animals were ketotic*
The occurrence of ketosis in rats does not seem to be as prev
alent as it is in rabbits, although it has been reported (63, 64, 65)0
This is probably only an apparent situation since attempts are usually
made to detect ketonuria and not ketonemia. In this investigation ke-
tonuria was detected but not with any regularity even though the ani
mals had 1+ to 4-f ketonemiao A high renal threshold in rats for ketone
bodies could account for this0 It could also lead to the false conclu
sion that there is no ketosis if only urine ketones are tested for *
The presence of even moderate ketosis may not necessarily mean
that acidosis exists (66), and it is possible that the animals in this
27 ' ' T
■■■■ ■■ : ' - : . V-V -. ^study 9 although :ketotics were not actually acidotic0 Unf or tuna te ly 9 blood pH values were not determined in order to ascertain this, ' But
numerous experiments have demonstrated that severe ketosis is accompan
ied invariably by high concentrations of plasma fatty acids9 fatty
acids being the precursors of ketone bodies (67). Since extreme lac-
tescence was observed in the sera of rats also possessing 3+ to 4+ ke-
tonemia, it would seem that these animals would have had a severe
enough ketosis to be at least mildly ketoacidotic.
Reasons for many of the conflicting results reported to date in
this area can probably be traced to the fact that different phagocytic
systems and different sources of cells have been used. Leukocytes have
been suspended in various percentages of serum and put into rotating
tubes for varying lengths of time (469 47, 49); they have been suspended
in saline and allowed to phagocytize on moist filter paper (48); and
they have been suspended in serum and allowed to phagocytize on glass
slides (51), In this investigationj washed leukocytes were suspended
in 100% serum with complement activity and placed in rotating tubes.
Since the necessity of complement and the existence of other serum fac
tors that may play a role in the phagocytic process are unknown and
still under study (595 68-74)9 it seemed desirable to have these pre
served in the system.
The commonest sources of leukocytes have been peripheral blood
and .peritoneal exudates. While the activity of inflammatory exudative
cells should be studied9 these exudates have always been elicited with
starch-aleuronat which itself could possibly alter cell activity. For
: v ; ; v ■ ■ ■ v - : 25example9 Drachman (5.0) found that addition of .large amounts of glucose
to normal exudative leukocytes suspended in normal serum depressed the
phagocytic activity of these cells6 Peripheral blood leukocytes, how
ever, have not been found to be sensitive to this effect (49, 50)»
This would seem to indicate that the phagocytic cells from the exudate
were altered in their sensitivity to osmotic effectse But was this due
to the movement of these cells out of the capillaries or to the starch-
aleuronat itself?
On the basis of previous reports and of the results obtained in
this investigation, the following points can be made:
1) The data obtained in this study support the notion that, in
general, ketotic diabetics are capable of producing normal titers of .
antibody (23), although severe diabetes with hypoproteinemia may result
in depressed titers (22), Previous reports on the opsonic activity of
antibody from diabetics could not be found, but the data in this report
indicate that this antibody is normal in its ability to prepare bacteria
for phagocytosis.
2) It seems to be fairly conclusive that the peripheral blood
neutrophiles of nonketotic diabetics are as active in phagocytizing .and
destroying bacteria as those of normal individuals (47, 48, 49)* How
ever, the results obtained in the investigation reported here do not
support the suggestion (47) that a condition of ketosis or ketoacidosis
alters this0
3) Thus, the answer to the problem of a reduced response to in
fection may lie in the effect of ketosis on such mechanisms as the
' -- / 3Pinflammatory response, which is reported to be normal in nonketotic di
abetics (51) but to be depressed in ketoacidotic individuals (36-40) <>
In addition, there is the possibility that the cells of these inflamma
tory exudates are more sensitive to the osmotic effect of high glucose
concentrations and thus have depressed phagocytic capabilities (50, 51)0
SUMMARY
Diabetes was induced in rats by intravenous injection of al
loxan which produced, in addition to hyperglycemia and a number of
other signs5 a 1+ to 44- ketosis in all but four of the animals0 In
order to study possible factors contributing to the lowered.resistance
to infection of individuals with diabetes mellitus, these alloxan dia
betic rats were compared to normal animals with regard to: 1) phago
cytic and bactericidal activity of neutrophiles, 2) competency of
antibody response to D0 pneumoniae vaccine9 3) opsonic activity of
antibody, and 4) effect of ketosis on the aforementioned factors0 The
. following results were obtained:
1) Alloxan diabetes and ketosis had no effect on the ability of •
polymorphonuclear leukocytes to ingest and destroy S_0 epidermidis and
Do pneumoniae type lo
2) The antibody response of ketotic and nonketotic diabetics to
injection with D0 pneumoniae type 1 vaccine was comparable to that of
normal animals0
3) The opsonic activity of anti-pneumococcal antibody produced
by ketotic diabetic animals was normal, and no depressive synergistic
effect was noted when diabetic rat phagocytes ingested organisms opson
ized with diabetic rat antibody.
31
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