JOURNAL OF BACTERIOLOGY, July 1967, p. 6-12 Vol. 94, No.

1
Copyright'@ 1967 American Society for Microbiology Printed in U.S.A.

Relationships of X Irradiation to the Enhancement

of Candida albicans Infections
ROBERT S. GORDEE AND PATRICK J. SIMPSON
The Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, Indiana 46206
Received for publication 6 March 1967
Preirradiation significantly reduced the number of Candida albicans cells required
for the LD50 of experimentally infected mice. The start and extent of recovery of
total leukocytes of preirradiated infected mice were proportional to the dose of
X rays administered. During 10 days postinfection, heterophils of infected mice

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preirradiated with 400 R recovered to levels above unirradiated, uninfected controls
but did not exceed those of unirradiated, infected animals. On the other hand, the
more radiosensitive lymphocytes were depressed greatly, and a limited recovery
below normal values was obtained. The lymph to heterophil ratio of uninfected mice
irradiated with 400 R recovered to normal values by 11 days postirradiation. How-
ever, decreases in the ratio of unirradiated or X-irradiated infected mice showed
little recovery. During 6 to 10 days postinfection, a reduction in microhematocrit
values after a dose of 400 R alone was not observed when a C. albicans infection
was superimposed on X-irradiated mice. This difference was not due to changes in
the red blood cell volume which was decreased by 400 R with or without infection,
but was attributed to a greater decrease in plasma volume caused by a combination
of X irradiation and infection. Reticulocyte counts indicated that recovery from the
significant decrease in erythrocyte production caused by 400 R was retarded by a
progressive infection. Elevated total serum protein of unirradiated mice at 6 days
after infection was attributed to increases in a- and ,3-globulins. A dose of 400 R
limited but did not prevent an increase in ca- and (3-globulins upon subsequent infec-
tion. Candida species infection or 400 R or both did not greatly affect the concen-
tration of y-globulins. The albumin to globulin ratio of irradiated, infected mice
was intermediate between those ratios found after X irradiation or infection.

Enhancement of the pathogenicity of experi- by Emmons (5). Viable cell suspensions were stand-
mental Candida albicans infections has been ob- ardized by optical density determinations at 660 my
tained by depressing host responses. Various on a Coleman Model 6A spectrophotometer and were
agents such as mucin, cortisone, X irradiation, comparedCD1 with a standard curve to obtain uniform
and certain antibiotics have been used success- inocula. male albino mice, Caesarean-born and
barrier-sustained River Mouse Farms,
fully (2, 19, 27). Various mechanisms by which Wilmington, Mass.),(Charles were infected intravenously in a
antibiotics alter the host resistance to this path- lateral tail vein with 0.1 ml of freshly prepared
ogenic yeast have been reviewed (20, 21). Hasen- Candida species cell suspension.
clever and Mitchell (7) found that multiplication Values for LDso were determined by the method of
of Torulopsis glabrata was higher in mice treated Reed and Muench (18). Groups of 10 mice were sepa-
with cortisone, alloxan monohydrate, or X rately infected with 10-', 10-2, and 10-3 dilutions of
irradiation. The enhancement of bacterial infec- 7 X 107 viable C. albicans cells for virulence titration.
tions by X irradiation has been investigated (14, X-Ray X-irradiation techniques. A 250-kv Westinghouse
15). Unit at 15 ma with a 0.25-mm copper and
1-mm aluminum filter was the X-irradiation source.
The purpose of this study is to show the effec- The dose rate, determined by a Victoreen dosimeter,
tiveness of X irradiation in enhancing lethal was 40 to 45 R/min. There was an average variation
experimental C. albicans infections and to describe of 4 R from the center to the outer perimeter of the
the effects of X irradiation on leukocytes, eryth- X-irradiation field. Mice were placed 40 cm from the
rocytes, blood volume, and serum proteins during source in a sectioned plastic-screened chamber 35.6 by
an acute infection. 5 cm. The movement of the mice in the chamber was
restricted by filling each section to capacity. The
MATERIALS AND METHODS chamber was then attached to a turntable which ro-
Organism and experimental infections. C. albicans tated the mice 1 rev/min to insure a uniform exposure
A26 was grown at 30 C on Sabouraud agar as modified of X irradiation.
6
VOL. 94,1967 EFFECT OF X IRRADIATION ON C. ALBICANS INFECTION
Hematological techniques. A model A Coulter
Counter was used to count white blood cells. Count-
ing suspensions of whole blood in saline were cleared
of red blood cells by the lysing action of 1% saponin.
Mice were bled from the orbital sinus by use of
heparinized capillary tubes. The total number of leu-
kocytes was recorded as the average of duplicate
counts performed on each of three mice. Counts of
lymphocytes and heterophils were obtained by the
conventional method of differentiating leukocytes
from a Wright-stained blood smear. Microhemato-
crits were determined by centrifuging for 5 min dupli-
7
RESULTS
A dose of 100 R of X ray 24 hr preinfection did
not greatly affect the number of cells of Candida
species required for the 7-day LD5o (Fig. 1). How-
ever, 200, 300, 400, and 500 R were very effective
in decreasing the number of cells for the 50%
mortality end point.
Mice X irradiated with 100 or 200 R and
infected with 1 X 106 C. albicans A26 cells ex-
hibited increased leukocyte levels above normal

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cate heparinized capillary tubes of blood from each of counts by 5 days after X irradiation (Fig. 2). Re-
three mice in an International Microhematocrit covery was evident by day 3 postirradiation for
Centrifuge. Reticulocyte counts were carried out ac- a dose of 100 R, day 4 for 200 R, and day 5 for
cording to the procedures described by Frankel and 400 R. Recovery of leukocytes in uninfected mice
Reitman (6). irradiated with 100, 200, and 400 R did not com-
Radioactive tracer techniques and serum protein mence until 6 to 7 days after X irradiation.
analyses. Blood volume was measured by use of radio- A dramatic decrease in leukocytes was seen 24
chromate-51Cr (Na261CrO4) (17, 24). The specific ac- hr after X irradiation with 400 R (Fig. 3A).
tivity of the radioactive tracer containing 6 X 1O-4 mg When unirradiated mice were infected, the leuko-
of Cr per ml was 334 mc per mg of Cr. A 10-ml pool
of mouse blood was labeled in vitro by adding 10 ,uc of cyte count was significantly higher than that of
51Cr per ml to red blood cells suspended in saline and
then incubating the radioactive suspension at 37 C for 20 f
2 hr in a Dubnoff shaker incubator. The labeled cells
were washed three times with saline by centrifugation
at 1,000 X g and then resuspended in the original
plasma. Labeled red blood cells were prepared each
day determinations were made. Blood volumes for
each experimental parameter were expressed as abso- 0
lute values by use of the average of five mice. The
plasma volume was calculated as the difference be- -J 10
tween the total blood volume and the red blood cell
volume with a hematocrit value corrected for the un-
even distribution of red blood cells in total body blood 0 8
and venous blood. At prescribed intervals, mice were
weighed and injected intravenously with 0.2 ml of
51Cr-labeled blood. After a 5-min period for thorough 0 6k
mixing of blood, each mouse was anesthetized lightly
with ether and bled by opening the chest cavity and In
collecting blood with a Pasteur pipette from a pool 0
formed by severing the brachial vessels. In addition to
use of blood samples for microhematocrit determina-
tion and reticulocyte count, 0.5 ml of blood was
-

cm
4k
-
UJ
added to tubes containing 1 ml of Alsever's solution C-,
X
for counting by a Packard Auto-Gamma Spectrome-
ter model 410A. At 5% counting efficiency, approxi-
mately 30,000 counts per min was obtained from a 0

0.2-ml sample of freshly labeled blood counted each 0

day prior to injection.
Serum protein fractions were analyzed with a
IC.
2F
Spinco model R electrophoresis system by use of the
Beckman Procedure A staining method and the
Analytrol scanner. Blood samples were taken at 6, 8,
and 10 days postinfection. At each samphng, nine ani-
mals from each experimental group were randomly
divided into three sets. The blood from each set was 1
pooled and separated by centrifugation to obtain Or lOOr 300r 500r
serum. The three serum samples from each set were
then analyzed separately by paper electrophoresis, IRRADIATION (24 Hrs Preintection)
and the average values for the group were obtained.
Total serum protein was determined by the method of FIG. 1. Influence of X irradiation on the Candida al-
Lowry (13). bicans cell requirement for the 7-day LDMo of mice.
8 GORDEE AND SIMPSON J. BAmrRioL.

cells showed no recovery until 7 to 8 days post-

(CANDIDA INFECTION infection. The rate and extent of recovery of
With Ix l 0 6 Cells) lymphocytes was greater when mice were infected
with 106 cells rather than with 105 cells. A signifi-

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E x
E E

C.3

U'a
o
I-
C.,2
clo

w-
10
0:>
I0-
IJ
-A lOOr-o 100l
lOOr + Infection - -
200r- A
200r + Infection - A
400r-o E
E
400r + Infection - U 1
C.D
1
0 1 2 3 4 5 6 7
DAYS POST X-IRRADIATION
FIG. 2. Effect of X irradiation or Candida albicans
infection or both on the total leukocytes of mice.

control animals by 2 days postinfection, whereas

irradiated mice required 3 to 4 days postinfection
for leukocyte levels above those of mice receiving
400 R without infection. The leukocyte counts of
X-irradiated mice infected with 105 or 106 cells
proceeded to increase from day 2 postinfection.
Counts of the 105 challenge remained below con- C.,)
trol values, whereas those of the 106 challenge
recovered by 8 days postinfection to the values
found for unirradiated, infected mice. Mice re-
ceiving 400 R without infection showed depressed -1 0 2 4 6 8 10
leukocyte counts that did not start to recover until 40Cr -DAYS POSTINFECTION -
7 to 8 days postinfection.
After 24 hr of X irradiation with 400 R, an o UNINFECTED UNIRRADIATED CONTROLS
-

approximate 10-fold reduction in lymphocytes * I x 10 5 CANDIDA INFECTION

compared with heterophils was observed (Fig.
3B and C). As expected, infection of unirradiated
mice resulted in slight changes in the lymphocytes
and caused a marked increase in heterophils. An
increase in lymphocytes of irradiated mice in-
fected with 106 cells was observed by 3 to 4 days
postinfection, whereas those infected with 105
o I x 106 CANDIDA INFECTION
* UNINFECTED- IRRADIATED CONTROLS (400r)
A I X lO5 CANDIDA INFECTION + 400r
* I x 10 6 CANDIDA INFECTION + 400r
FIG. 3. Individual and combined effects of 400 R
and Candida albicans infection on mouse (A) total leu-
kocytes, (B) total heterophils, and (C) total lympho-
cytes.
VOL. 94,1967 EFFECT OF X IRRADIATION ON C. ALBICANS INFECTION 9
cant increase in lymphocytes of uninfected, irra-
diated animals was seen by 9 to 10 days post-
infection. Recovery of heterophils of irradiated
mice infected with both levels of C. albicans was +10
found by 2 days postinfection. Although levels of -

heterophils of uninfected, irradiated mice were 0

below normal and showed recovery by 6 days
0
postinfection, the infected, irradiated mice dem-
onstrated heterophil counts well above normal 0
counts.
Decrease of the lymph to heterophil ratio -10
I-
caused by 400 R was overcome, and recovery to * 400r CONTROL
normal ranges was observed by 11 days after X A 105 INFECTION
irradiation (Fig. 4). X-irradiated, infected mice 0D -20 A 105 INFECTION +400r
demonstrated depressed lymph to heterophil O 106 INFECTION

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ratios, showing little recovery for 10 days post- * 106 INFECTION + 400r
infection. 0 2 4 6 8 10
The microhematocrit values of either X-irra-
diated or unirradiated infected mice did not de- (24 Hrs Post 400r) DAYS POSTINFECTION
crease during 6 to 10 days postinfection to the FIG. 5. Individual and combined effects of 400 R and
extent of those of X-irradiated, uninfected ani- Candida albicans infection on the microhematocrit of
mals (Fig. 5). This relationship is seen for either mice.
105 or 106 Candida cells.
Chanyes in blood volume were determined bv
use of 51Cr during 6, 8, and 10 days postinfection X-irradiated, infected animals. The diminished
to compare the effect (of a Candida infection on blood volume on day 6 of mice which received
unirradiated and X-ir radiated mice (Table 1). 400 R only was attributed to a reduction in
During 6 to 10 days r)ostinfection, body weight erythrocytes, whereas the lowered blood volume
loss as a result of 400 R or C. albicans infection in X-irradiated, infected animals was a result of a
contributed to significatnt reductions in weight of decrease in both erythrocyte and plasma volumes.
By day 8, the reduction in blood volume in X-
irradiated, infected animals was mainly caused by
the significant effects of 400 R on cell and plasma
volumes. A decrease in blood volumes of X-
irradiated, infected, and both X-irradiated and
infected animals by day 10 was a consequence of
diminution of both erythrocyte and plasma vol-
umes.
The effect of acute doses of X irradiation on
C-)
plasma protein of various experimental animals
.t has been investigated (10, 11, 15, 16). Little
coo
0. change was seen in the total plasma protein of
s
infected, irradiated mice during 6 to 8 days post-
infection (Table 2). However, a concomitant
decrease in total plasma proteins of infected un-
irradiated and irradiated mice was seen 8 to 10
days postinfection. An increase in total proteins
caused by X irradiation alone appeared to limit
0.2
o UNINFECTED)-UNIRRADIATED CONTROLS the extent of the decrease in infected, irradiated
* IRRADIATED - 400r CONTROLS mice. The decrease in albumin of infected, irra-
l X 10 6 CANIDIDA CELLS diated mice was attributed to the Candida infec-
a IX106 CAN DIDA CELLS+ 400r tion. Elevated a-globulin fractions in infected,
0.1 I I irradiated mice followed infection. The extent of
0 2 4 6 8 10 increase, however, was mediated by the effects of
DAYS P'OSTINFECTION 400 R. Elevated A-globulin fractions of irradiated
FIG. 4. Effects of Cantdida albicans infection on the and unirradiated infected mice decreased con-
lymph to heterophil ratio of unirradiated and 24-hr pre- comitantly during 8 to 10 days postinfection.
irradiated mice. Overall, neither 400 R nor 106 Candida cells
10 GORDEE AND SIMPSON J. BAc1EioLF.

TABLE 1. Changes in the blood volume of mice 6, 8, and 10 days postinfection with Candida albicans
or after X irradiation or both
Expermental"
ExperimentalaI wtAvg
(g)body
t SE Total
(ml)blood
& SEvol Red Blood cell
vol (ml) =tSE Plasma
(ml) vol
SE
* Cell/
plasma Hematocritb
(%) * Reticulo-
sz(a ;

Day 6 postinfection
Unirradiated, un- 26 ± 0.10 1.95 ± 0.09 0.84 ± 0.05 1.11 ± 0.06 0.76 43 t 0.98 5.0
infected control
X-irradiated (400 R) 24 ± 0.63 1.57 ± 0.02 0.55 ± 0.01 1.02 ± 0.03 0.54 35 + 0.91 1.1
Infected (106 cells) 21 + 1.47 1.66 ±00.11 0.68 ± 0.05 0.98 ± 0.07 0.69 41 ± 0.89 2.4
X-irradiated (400 R), 17 ± 0.58 1.30 + 0.05 0.48 ± 0.03 0.82 ± 0.03 0.59 37 ± 1.66 1.5
infected (10' cells)
Day 8 postinfection

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Unirradiated, un- 26 ± 0.51 1.84 ± 0.04 0.77 ± 0.02 1.07 ± 0.04 0.72 42 ±t 1.45 5.0
infected controls
X-irradiated (400 R) 22 ± 2.50 1.33 ± 0.15 0.51 ± 0.08 0.82 + 0.08 0.62 38 4 1.00 1.4
Infected (106 cells) 21 ± 1.28 1.64 ± 0.09 0.71 ± 0.04 0.93 ± 0.06 0.76 43 ±t 0.74 2.7
X-irradiated (400 R), 15 ± 0.88 1.19 ±t 0.05 0.49 ± 0.02 0.70 ± 0.03 0.70 41 ± 1.12 1.1
infected (106 cells)
Day 10 postinfection
Unirradiated, un- 29 ± 0.19 2.05 ± 0.06 0.86 ± 0.04 1.19 ± 0.02 0.73 42 ± 0.66 5.0
infected control
X-irradiated (400 R) 25 + 0.50 1.52 + 0.05 0.58 ± 0.05 0.94 ± 0.00 0.62 38 ± 2.00 2.8
Infected (10' cells) 18 ± 0.91 1.57 ± 0.04 0.69 ± 0.02 0.88 ± 0.02 0.78 44 ± 0.51 1.9
X-irradiated (400 R), 13 ± 0.69 1.23 ± 0.05 0.50 ± 0.03 0.73 ± 0.04 0.68 41 ± 1.77 1.8
infected (106 cells)
Mice X-irradiated 24 hr prior to infection.
b Corrected by factor of 0.9.
TABLE 2. Influence of Candida albicans infection or X irradiation or both on serum proteins during 6, 8,
and 10 days postinfection
Concn (mg/ml)
~A/G
Experimental Total protein Albumin a-globulin | -globulin |y-globulin
6a 8 10 6 8 10 6 8 10 6 8 10 6 8 10 6 8 10

Unirradiated, un- 55.0 55.0 54.0 36.3 34.3 33.4 8.1 9.0 7.6 8.3 7.1 9.3 3.9 4.5 3.2 1.8 1.7 1.7
infected control
400 R (24 hr pre- 52.2 50.0 54 3 35.1 33.6|32.8 6.2 7.0 8.3 7.8 6.0 9.5 3.0 3.4 3.7 2.2 2.1 1.5
infection)
106 Candida cells 61.6 66.2 45.5 18.2 21.4 15.5 17.9 23.0 12.3 24.0 20.0 14.6 4.4 2.4 3.2 0.4 0.5 0.5
106 Candida cells 56.0 54.0 49.5 25.2 23.2 24.8 9.0 11.9 11.3 17.3 16.7 9.4 4.5 2.2 4.0 0.8 0.8 1.0
± 400R
*Refers to number of days postinfection.

greatly affected 'y-globulins; however, the decrease
on day 8 postinfection in unirradiated, infected
mice was also observed in irradiated, infected
animals.
Although 400 R did not greatly affect the
albumin to globulin (A/G) ratio, infection with
Candida species significantly reduced the ratio.
The A/G ratio values of X-irradiated, infected
mice were intermediate between those of X-irra-
diated or infected mice. It appears that a dose of
400 R moderated both the decrease in albumin
and the increase in a- and 3-globulins when
administered 24 hr prior to infection.
DIscussIoN
Miner, Hammond, and Anderle (15) found
that the LD50 of Pseudomonas organisms in irra-
diated mice was significantly lower than that in
unirradiated mice when challenged during the
first 3 days after exposure to 400 R. However,
enhancement of the susceptibility to bacterial in-
fection caused by X irradiation was not seen 17
VOL. 94,1967 EFFECT OF X IRRADIATION ON C. ALBICANS INFECTION
days postirradiation. Roth, Friedman, and Syver-
ton (19) showed a significant increase in mortality
of mice X irradiated with 200, 300, or 400 R 24
hr prior to C. albicans infection, which was propor-
tional to the dose of X irradiation. Furthermore,
lesions in X-irradiated animals were more general-
ized and developed faster than those found in
unirradiated, infected mice. The present study
demonstrates that, although 100 R showed little
effect, a dose of 200, 300, 400, and 500 R 24 hr
prior to infecting mice with C. albicans markedly
reduced the number of Candida cells required for
the 7-day LD50.
The effect of acute doses of X irradiation on
leukocytes has been reviewed (8). It has been
reported (22, 23) that survival of X-irradiated
mice infected with P. aeruginosa followed the
increase in leukocyte counts, and that granulo-
cytes played a more important role than lympho-
cytes in the survival of X-irradiated mice infected
with bacteria.
The total leukocyte count of X-irradiated mice
infected with C. albicans is influenced by non-
lethal doses of X ray as to when recovery starts
and the extent leukocytes are produced. When
X-irradiated mice were challenged with a 10-fold
difference in inocula of C. albicans, the extent of
recovery was proportional to the challenge.
Radiosensitive lymphocytes showed little re-
covery when a Candida infection was super-
imposed on mice receiving a dose of 400 R. On
the other hand, the comparatively radioresistant
heterophils demonstrated a significantly greater
recovery. It has been shown (1) that, although
600 R did not affect phagocytic activity of the
reticuloendothelial system, it did inhibit recovery
of normal phagocytic function after blockade
with carbon particles and it did prevent stimula-
tion of phagocytosis by zymosan. Phagocytosis of
Candida species by polymorphonuclear cells oc-
curs in the blood, but in the peritoneal cavity
these cells are not mobilized extensively, and
phagocytic activity there is accomplished by large
monocytes (28). It appears that susceptibility of
X-irradiated mice to Candida infection is due in
part to their inability to proliferate leukocytes and
other cells for such host responses as phagocytosis.
After nonlethal doses of X ray, there is a
temporal relationship between circulating lym-
phocytes and response of the rabbit to an antigen
(25). Both are intact immediately after irradia-
tion, but decrease rapidly thereafter, reaching
minimal levels in 24 to 48 hr. The lymphocyte
count and response to an antigen require 30 days
or longer for complete recovery, despite the fact
that lymphoid tissues and lymph nodes appear
to be fully recovered much earlier. It has been
suggested that lymphocytes are the site of anti-
II
body production (4), and that these cells are
antibody carriers (3). Therefore, a reduction of
the functional capacities of lymphocytes as a
result of X irradiation associated with host re-
sponses could contribute to a decrease in resist-
ance to C. albicans infection.
Erythroblasts are highly radiosensitive cells.
However, after exposure to doses of X ray in the
LD50 range, significant reduction in erhthrocyte,
hemoglobin, or hematocrit values is not seen until
approximately 6 days postirradiation (8). This
delayed effect is caused by the accumulating loss
of erythrocytes by normally aging cells without a
proportionate replacement and by latent damage
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to capillary walls that causes diversion of erythro-
cytes into tissue spaces and lymphatics.
A proportionate decrease in plasma volume
with a reduction in erythrocytes accounted for
the absence of lowered microhematocrit values
6 days after preirradiated mice were infected with
C. albicans. Decreases in blood volume seen in
uninfected, irradiated animals by 11 days post-
irradiation agree with those reported previously
(9). The rate of body weight loss of infected,
irradiated mice was significantly greater than the
decrease in total blood volume. Therefore, ex-
pression of blood volume data in terms of mil-
liliters per 100 g of body weight, as was done for
X-irradiated rats (24), would have shown mis-
leading increases instead of actual decreases in
blood volume. The role of malnutrition asso-
ciated with the combined effects of X irradiation
and infection is difficult to assess. However, no
change has been found to occur in plasma volume
in rats fasting for 7 days (24).
Mice exposed to 400 R showed the same re-
covery in reticulocytes 7 to 11 days after X
irradiation as that reported with rabbits exposed
to 800 R (8). Between 6 to 8 days postinfection, the
reticulocyte counts of infected, irradiated mice
showed the effects of 400 R. However, as the ef-
fects of 400 R were diminished during 8 to 10
days postinfection, the reticulocyte count was
influenced by the progressing Candida infection.
An inhibitory factor for C. albicans in blood
plasma has been reported by Louira and Brayton
(12). The fungicidal factor for Candida was found
primarily associated with the a-globulins and to a
lesser extent with (-globulins. Neither the albu-
min nor y-globulin factor demonstrated anti-
Candida activity. It is interesting that the elevated
total plasma protein on day 6 that decreased by
day 10 postinfection in infected, unirradiated
mice was attributed to changes primarily in the
a- and A-globulins. If a concomitant decrease in
the fungicidal plasma factor for Candida occurred
with the lower concentration of a- and (3-globulins
of infected, irradiated mice as compared with
 12 GORDEE AND SIMPSON
unirradiated, infected animals, then increased
susceptibility to a progressive infection could
ensue.
Since Candida infection does not cause a signifi-
cant increase in y-globulins as some infectious
microorganisms do, the effect of 400 R on the
biosynthesis of -y-globulins may not be a signifi-
cant contribution to the enhancement of a
Candida infection.
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