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COMPARISON OF GLUCAN AND MURAMYL DIPEPTIDE IN STIMULA-
TION OF PARTICLE CLEARANCE AND HOST DEFENSE IN MICE

presented by

Di rk Warren Sprenger

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COMPARISON OF GLUCAN AND MURAMYL DIPEPTIDE IN STIMULATION

OF PARTICLE CLEARANCE AND HOST DEFENSE IN MICE

BY

Dirk Warren Sprenger

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Pathology

1980

‘3" Wit; It:

ABSTRACT

COMPARISON OF GLUCAN AND MURAMYL DIPEPTIDE IN STIMULATION
OF PARTICLE CLEARANCE AND HOST DEFENSE IN MICE

BY

Dirk Warren Sprenger

The purpose was to determine if there is a positive correlation
between the ability of 2 immunostimulants, Glucan and muramyl
dipeptide (MDP), to enhance the rate of intravascular particle
clearance in mice and to protect them against infections. Glucan
treatment consisted of i.v. injections given 7, 4 and 1 days prior
to the determination of carbon clearance rate. The MDP was given
i.v. 1 day before rate determination. The dose that induced the
greatest increase in carbon clearance rate was 40 mg/kg for Glucan
and 8 mg/kg for MDP. These doses were used to pretreat mice that*weree
inoculated i.v. with Candida albicans or Staphylococcus aureus. The
survival time of MDP-treated mice, but not Glucan-treated mice, was
significantly greater than untreated mice even though Glucan enhanced

carbon clearance more than MDP.

ACKNOWLEDGMENTS

I am grateful for the guidance of John E. Lund, my mentor, and
the financial support of The Upjohn Company. I thank Gary D. Gray,
Karen Knight, Paul Skinner, W. Pat Brown, and Lori Harris for tech—
nical assistance. My guidance committee, Martha Thomas, Charles
Whitehair, George Padgett, and Ronald Patterson, gave me valuable

advice.

ii

INTRODUCTION

TABLE

LITERATURE REVIEW. . . . . . . .

OBJECTIVES

MATERIALS AND METHODS. . . . . .

I.

II.

III.

RESULTS.

I.

II.

III.

DISCUSSION

SUMMARY.

APPENDIX .

REFERENCES

VITA

OF CONTENTS

Effect of Glucan and MDP Treatment on

Reticuloendothelial

System. . .

Experimental Infections . . . .

Interferon. . . . .

Effect of Glucan and MDP Treatment on

Reticuloendothelial

System. . .

Experinental Infections . . . .

Interferon. . . . .

iii

Page

10

10
12
14
16
16
23
28
34
39
4O
46

51

LIST OF TABLES
Table Page

1 Serum interferon sample times of mice treated with
Glucan or Lactated Ringer's . . . . . . . . . . . . . . . 32

2 Carbon clearance half times on day 0 of mice treated
with Glucan and MDP . . . . . . . . . . . . . . . . . . . 41

3 Serum lysozyme on day 0 of mice treated with Glucan
and mp O O O O O O O I O O O O O O O O O O O O O O 0 O O 42

4 Spleen weights on day 0 of mice treated with Glucan
and MDP O O O O O O O O O O O O O O O O O O O O O O O I O 4 3

5 Liver weights on day 0 of mice treated with Glucan
and MDP O O O O O O O O O O O O O O O O O O O 0 O O O O O 44

6 Interferon assay plaque counts. . . . . . . . . . . . . . 45

iv

Figure

10

ll

12

Effect of MDP and

Effect of MDP and
First experiment.

Effect of MDP and
Second experiment

Effect of MDP and
First experiment.

Effect of MDP and
Second experiment

First C. albicans
day, given on day

LIST OF FIGURES

Glucan on

Glucan on

Glucan on

Glucan on

carbon clearance in mice.

spleen weight in mice.

spleen weight in.ndce.

liver weight in mice.

liver weight in mice.

experiment.

Effect of MDP, 8 mg/kg/
-1, day -2, or by minipump on days
-2 through 4, on survival of mice inoculated with

6 x 107 CFU/kg on day 0 . . . . . . . . . . . . . . . .

First C. albicans experiment.
40 mg/kg, on days -7, -4, and -1, on survival of mice

Effect of Glucan,

inoculated with 6 x 107 CFU/kg on day 0 . . . . . . . .

Second C. albicans experiment.
on day -l, on survival of mice inoculated with 6 x 10

CFU/kg on day 0 .

Second C. albicans experiment.
40 mg/kg, on days -7, -4, and -1, on survival of mice

Effect of Glucan,

Effect of MDP, 8 mg/k

gr

inoculated with 6 x 107 CPU/kg on day 0 . . . . . . . .

First S. aureus experiment.

bacteria/kg on day 0. . . .

First S. aureus experiment.

lated with 5 x 1010 bacteria/kg on day 0. . . .

Second S. aureus experiment.
on day -l or day -2, on survival of mice inoculated
bacteria/kg on day 0.

with 5 x 1010

Effect of MDP, 8 mg/kg,
on day -1, on survival of mice inoculated with 5 x 10

Effect of MDP, 8 mg/kg,

Effect of Glucan, 40 mg/kg,
on days -7, -4, and -1, on the survival of mice inocur

Page

17

19

20

21

22

24

25

26

27

29

3O

31

Figure Page

13 Standard curve for determining S. aureus concentration. . 40

vi

INTRODUCT ION

Agents that modulate host defense are of great importance because
they may be useful in the prevention and/or treatment of infectious
diseases or neoplasia. Glucan and muramyl dipeptide (MDP) are two
compounds which have been reported to activate macrophages and to be
effective in treating various experimental infections (1,2). They are:
considered to be good candidates for immunotherapy because they are
non-antigenic and have low toxicity (1,3). Both Glucan and MDP increase
the rate of intravascular carbon clearance in mice. The rate of cartxnu
clearance is determined by administering carbon particles intravenouslyr
and measuring the relative carbon concentration in serial blood
samples (4). The enhancement of carbon clearance may indicate thera—
peutic potential because carbon particles are primarily sequestered
by macrophages in the liver and spleen and increased uptake is associ-
ated with activation of the macrophages.

The purpose of this research was to determine if there is a
positive correlation between the ability of an agent to enhance the
rate of carbon clearance and to protect the host against bacterial
and mycotic infectiOns. The dose-response relationship of Glucan and
MDP treatment of mice to the rate of carbon clearance was determined.
The protective activity of Glucan and MDP was determined by inoculating;
treated and untreated mice with Staphylococcus aureus and Candida

albicans. The value of drug treatment was correlated with carbon

2
clearance stimulation. Other host defense factors were examined to
further characterize the response to MDP and Glucan treatment. These
included the serum levels of lysozyme and interferon and the gross

and histologic appearance of the liver and spleen.

LITERATURE REVIEW

Muramyl dipeptide and Glucan are structurally related to the
peptidoglycan cell wall of microorganisms. Like certain bacterial
preparations, they have been reported to stimulate nonspecific
resistance to infection in animals (2,5). Glucan has also been
effective in the treatment of syngeneic tumors in animals (6) and
metastatic tumors in man (7). Both have low toxicity and therefore
are considered to be potential therapeutic agents (1,7). Although
previous research has not evaluated these agents simultaneously, it
has revealed many important similarities and differences.

Muramyl dipeptide is a water-soluble glycopeptide (N—acetylmuramyl—
L-alanyl-D—isoglutamine) that was synthesized to identify the active
structure in Freund's complete adjuvant (8). Glucan is an insoluble
polysaccharide {8-1.3 polyglucose) that was extracted from Saccharo-
myces cerevisiae and identified as the component of zymosan which
produced hyperplasia and hyperfunction of the reticuloendothelial
system (RES) (9). Treatment with MDP has produced hyperfunction of
the RES without inducing hepatomegaly and splenomegaly (10). Hyper-
function of the RES refers to increased rate of clearance of foreign
particles. Colloidal carbon has been used to measure the rate of
clearance, which purportedly indicates the phagocytic state of the
RES (11), although clearance rate may also reflect blood flow and

platelet function (12). Glucan has been reported to increase the

4
rate of carbon clearance 10-fold (11). The greatest increase reported
for MDP was 4-fold above that for untreated animals; however, doses
in excess of 200 ug/mouse were not examined (13). The clearance rate
remained elevated for 3 days following MDP treatment and 15 days
following Glucan treatment.

Glucan treatment enhanced the primary and secondary antibody
response to sheep erythrocytes in mice (14). This adjuvant activity
was assumed to be mediated by macrophages because of the effects of
Glucan on the RES.

The adjuvant activity of MDP has been more thoroughly studied
(1). In a water~in-oil emulsion, MDP was as active as whole myco-
bacteria in the induction of delayedrtype hypersensitivity and experi-
mental autoimmune diseases. Primary and secondary antibody responses
were increased when MDP was administered in saline both parenterally
and orally. Many analogs of MDP have been synthesized and examined
for adjuvant activity. Small structural changes can render the mole-
cule inactive (8).

The adjuvant activity of MDP in various in vitro systems indicated
that it acted directly on T and B lymphocytes (15). Although some
workers have concluded that macrophages were not the target cell for
the adjuvant activity of MDP (15), others have demonstrated otherwise
(16).

Macr0phages are important for natural resistance to infection.
The enhanced nonspecific resistance to infections in animals with
acquired cellular immunity is mediated by macrophages that have an
increased micrdbicidal capacity (17). Treating macrophages in vitro
with MDP increased phagocytosis and killing of bacteria as well as

adherence to glass and cell spreading, phenomena associated with

increased killing capacity (18,19). In vitro stimulation of peri-
toneal macrOphages with MDP increased colony stimulating activity,
collagenase production, prostaglandin production, and intracellular
cyclic AMP (l). Monocytes and macrophages secreted endogenous pyrogens
and lymphocyte activating factor in cultures treated with MDP (1).
Macrophages exposed to MDP in vitro, but not in vivo, inhibited the
growth of mastocytoma cells (20) and the presence of MDP increased
the cytolytic activity of macrophages from established cell lines
and from murine sarcoma virus induced tumors (21).

The effects of Glucan on macrophages have been determdned by
examining cells from Glucan-treated animals. For example, the
effect of Glucan on Kupffer's cells was examined histologically in
rats treated with Glucan (22). Three days after 5 daily i.v. injec-
tions many Kupffer's cells had vacuoles containing Glucan. Kupffer's
cells were increased in size and number and many granulomas were
present in the hepatic sinusoids. Peritoneal macrophages from mice
treated with Glucan intraperitoneally have also been examined (23,24).
They exhibited increased size, spreading, adherence, chemotactic
activity, acid phosphatase production, and antitumor cytotoxicity.

Lysozyme, an enzyme which lyses certain bacteria, is produced
by macrophages. Glucan treatment has been reported to double serum
lysozyme levels in mice (25). Although it has been reported that
endotoxin stimulation of macrOphages has a small effect on in vitro
lysozyme production (26), the determination of serum lysozyme levels
was suggested to be useful for monitoring in vivo macrophage stimu-
lation and depression (25). Serum lysozyme values were interpreted

as an expression of macrophage activity rather than macrophage number

6
because methyl palmitate, an RES depressant, inhibited the Glucan-
induced increase in serum lysozyme (25).

In view of the ability of MDP and Clucan to stimulate intra-
vascular particle clearance, immune responses, and macrophage
activities, it is not surprising that these agents enhance nonspecific
resistance to infection. Treatment with MDP increased host resistance
to Candida albicans (27,28), Klebsiella pneumoniae (S), and Trypanosomwi
cruzi (29) [MDP has been used as an adjuvant in vaccines for Plasmo-
dium falciparum (30) and influenza (31)]. The results of one study
indicated that the best treatment regimen was 4 daily injections of
MDP, 80 mg/kg, prior to an i.v. injection Of a lethal dose of C.
albicans (28). In another study MDP was most effective when given
once or twice several hours before inoculation or when given concur-
rent with C. albicans (27). In the latter study, when mice were given
an LDlOO of C. albicans, 50% of the MDP-treated mice survived; when
given an LDSO nearly 100% of the treated mice survived. Pretreating
mice with MDP protected them against an i.v. challenge of 103 K-
pneumoniae (5). Mice that were treated on day -l with MDP received
either 10, 100, or 1000 pg/mouse i.v. or 2000 pg/mouse orally. 0n the
eighth day following inoculation, 33% of the mice were still alive in
the group given 10 pg, 73% were alive in the group given 100 pg, 88%
in the group given 1000 pg, and 63% in the group that was treated
orally with 2000 pg of MDP. The percent of mice that survived did not
change after day 8. Ten percent of the untreated control mice were
alive on the eighth day.

Although treatment with MDP on the day prior to infection was
effective in protecting mice against Klebsiella infection, it did not

enhance resistance to Trypanosome cruzi in a study where 7 different

7
MDP treatment regimens were employed (29). Pretreatment on day -2
was effective and continuous infusion via subcutaneous osmotic mini-
pumps during days -2 through +4 was also effective.

Research on the fate of ll‘C-MDP administered i.v. indicated that;
75-95% is excreted in the urine unchanged within 2 hours after injec-
tion (32). This suggested that pretreatment induced changes in host
defense systems that developed and endured after most of the MDP had
been eliminated.

Glucan therapy enhanced nonspecific resistance to C. albicans
(2), Staphylococcus aureus (33,34), Francisella tularensis (35),
Leishmania donovani (36), and Sporothrix schenckii (37). Glucan
pretreatment, but not post-treatment, reduced mortality in mice that
were given 3 x 106 C. albicans i.v. In 12 days 10% of the pretreated
mice died and 50% of the untreated control mice died. Pretreatment
consisted of an i.v. injection of Glucan particles suspended in
saline, 0.45 mg/mouse, on days -7, -4, and -1.

Experimental S. aureus infections in normal, leukemic, and
cyclophosphamide-treated mice have been beneficially altered by
Glucan pretreatment (33,34,38). In 3 experiments with normal mice,
100% of the untreated mice were dead by 6-14 days. In each experi-
ment approximately 60% of the Glucan-treated mice were alive when
the last untreated mouse died. Two i.v. pretreatment regimens were
used: 0.45 mg/mouse on days -10, -7, -4, and —l; and 1.0 mg/mouse
on days -7 and -4, with the challenge of 109 S. aureus i.v. on day 0.
The beneficial effect of Glucan treatment was not absolute. Death
loss continued in the treated groups until all mice were dead on

day 26.

8

Glucan and MDP can stimulate immune responses, macrophage
activities, and resistance to infection, but the composition and
kinetics of the two drugs are different. These potentially thera-
peutic compounds cannot be compared using literature reports because
studies used different strains of mice or infectious agents. Addi-
tional research is needed to compare these compounds and to determine
the relationship of their protective activity to their ability to

stimulate the rate of carbon clearance.

OBJECTIVES

The objectives of this research were:

1. To determine the optimum dose for enhancing carbon clearance
in CF-l mice with MDP and Glucan.

2. To determine the effect of the optimum dose on the survival
of mice inoculated with C. albicans and S. aureus.

3. To determine the serum lysozyme value for mice treated with

Glucan and MDP.

4. To determine the effect of drug treatments on weight and
histological appearance of liver and spleen.

5. To determine if interferon is induced by Glucan treatment.

MATERIALS AND METHODS

I. Effect of Glucan and MDP Treatment on
the Reticuloendothelial System

Materials

Mice: Female CF-l mice were obtained from Charles River Breeding
Laboratories (Portage, MI). They were 6-10 weeks old (15-24 g) and
were fed Rodent Laboratory Chow 5001 (Ralston Purina Co., St. Louis,

MO) and water ad libitum.

Glucan: Sterile Glucan, 10 mg/ml in 5% dextrose water, lot E0737
(Accurate Chemical and Scientific Corp., Hicksville, NY) was diluted
to the desired concentration with Lactated Ringer's (Abbott Labora-
tories, Chicago, IL) and sonified l min with-a Branson Sonifier set

at power level 1 (Model 5125 with standard tapered microtip).

Muramyl dipeptide: Lyophilized MDP (Groupment d'Interet Economique -
Institut pour la Recherche et la Production d'Immunostimulants, Paris),

lot 79001, was dissolved in Lactated Ringer's.

Colloidal carbon: Special ink, 140 mg/ml, lot Cll/l431A (Gunther
Wagner, Hannover, Germany). was diluted to a concentration of 16

mg/ml with Lactated Ringer's.

10

11
Protease solution: Thermolysin, 64 units/mg, lot 570-0116
(Sigma Chemical Co., St. Louis, MO), was dissolved in water at a

concentration of 63 pg/ml.

Lysozyme: The standard was egg white lysozyme, grade I, lot
57C-8025 (Sigma Chemical Co.) dissolved in phosphate buffer, pH 6.3,
to make 5 standard solutions ranging from 0-20 pg/ml. Micrococcus
lysodeikticus, lot 107C-006l (Sigma Chemical Co.), was suspended at

100 UQ/ml in phosphate buffer, pH 6.3 (KZHPOQ, 0.036M; KH2POp, 0.114M).

Spectrophotometry: For the carbon clearance test a Coleman
Junior III spectrophotometer was used, wavelength 620 nm, with round,
lO-mm cuvettes. For the serum lysozyme assay, a Beckman DU spectro-

photometer was used, wavelength 540 nm, with square, lO-mm cuvettes.

Methods
All injections were given in the lateral tail vein at a constant

volume of 0.01 ml/g body weight.

Carbon clearance test: The method of Halpern et a1. (4) was
modified by using protease solution instead of 0.1% Na2C03 to lyse
blood samples. Colloidal carbon, 160 mg/kg, was injected and at four
2.5 minute intervals blood was collected from the retro-orbital venous
plexus using heparinized microhematocrit capillary tubes (Dade,

Miami, FL). Occasionally fibrin clots formed while the sample was
being transferred from the collection tube to a 50 p1 capillary
pipette (Drummond, Broomall, PA) prior to diluting. By using 2.5 ml
of protease solution to dilute each sample, fibrin clots which may
have formed were degraded. The absorbance of-each of the 4 diluted

samples was determined and plotted against time on semilog paper to

12
determine the half time of clearance, T/2. The clearance rate
. l 2
constant, K, was calculated by the equation: K = -¥3§-. Clearance

was expressed as the mean rate constant, 2, of each group divided by

the mean rate constant of the control group, KC.

Serum lysozyme: Serum or lysozyme standard, 0.1 ml, was mixed
with 0.5 m1 of the Micrococcus suspension at 25 C and the absorbance
at 540 nniwas recorded at 15 sec and 135 sec (39). The decrease in
absorbance was calculated and converted to lysozyme concentration with

a curve based on the standards.

Histology: Livers and spleens were weighed with an Arbor balance
(model 306) and fixed in 10% neutral buffered formalin. Paraffin

sections were prepared and stained with hematoxylin and eosin.

Statistics: Results were analyzed with the Student's t-test;

P<0.05 was considered significant.
II. Experimental Infections
Materials

Pathogens: Staphylococcus aureus was obtained from Drs. N. R.
Di Luzio and D. L. Williams (Tulane University School of Medicine,
New Orleans, LA) and was assigned number UC6966 in The Upjohn Company
Culture Collection (Kalamazoo, MI). Candida albicans, UC7165, The
Upjohn Company Culture Collection, was originally isolated from a

patient at Bronson Methodist Hospital (Kalamazoo, MI).

Phosphate Buffered Saline (PBS): The PBS, pH 7.2, contained
NaCl, 0.137M; KZHOPQ, 0.007M; and KHZPOp, 0.003M. It was sterilized

by autoclaving for 20 minutes at 121 C.

13
Osmotic Minipumps: Alzet pumps (model 2001) were obtained from

Alza Corp., Palo Alto, CA.
Methods

Minipump Implantation: The pumps were filled aseptically with
0.2 ml of MDP solution and were implanted subcutaneously in the
scapular region in mice anesthetized with methoxyfluorane. Wounds

were closed.with metal clips.

Preparation of Inocula: Candida albicans was grown in Nutrient
Broth (Difco, Detroit, MI) containing glucose (10 9/1) at 37 C for 18
hours on a shaker (200 rpm) (40). This yielded cultures containing
1-2 x 108 yeast/ml. Purity of the culture was verified by Gram's
stain. Two milliliters of the culture was mixed.with 10 ml PBS and
centrifuged at 570 G for 15 minutes. The supernatant was discarded
and the button was resuspended in PBS to a concentration of 6 x 106
Colony Forming Units/m1 (CPU/ml). The CFU/ml was determined by diluting
samples, in duplicate, to a concentration of approximately 7 x 103
yeast/nu with PBS and plating 0.1 m1 on Sabouraud's Dextrose Agar
(BBL, Cockeysville, MD) (40). After incubation at 37 C for 24 hours,
the number of colonies on each plate was counted with an American
Optical Colony Counter (Model 3327).

Staphylococcus aureus suspensions were prepared by the method of
Di Luzio and Williams (33), except that the concentration of bacteria
was determined from the absorbance of the suspension using a standard
curve (Figure 13, Appendix). The curve was established with dilutions
of 2 stock S. aureus suspensions in which the number of bacteria had

been determined with a Petroff—Hausser counting chamber (Hausser

14
Scientific, Blue Bell, PA) using standard technique. The cultures
were grown in Trypticase Soy Broth (BBL) for 18 hours at 37 C on a
shaker at 200 rpm. This yielded cultures containing 1-3 x 109 bac-
teria/ml. Purity of the culture was verified by Gram's stain. The
culture was washed 3 times with PBS in 15-ml tubes; centrifugation was
570 G for 20 minutes. After the first wash a sample was used to
determine the bacterial concentration and after the third wash the
cells were resuspended in PBS to a concentration of 5 x 109

bacteria/ml.

Mean survival time and statistics: Mean survival time was
expressed as the Harmonic mean (Hun which was calculated.by the
following equation: .Hm = n/(l/d1 + l/d2 + ... + l/dh), where n is
the number of mice in the group and d1, d2, ... dn are days of death
for individual mice (29). For surviving animals l/d = 0. Mortality
data were analyzed by the Mann-Whitney U test (two-tailed). Differ-

- ences were considered to be significant if P<0.05.
III. Interferon
Materials

Cells: Mouse fibroblast L929, American Type Culture Collection

(ATCC).

Virus: Vesicular Stomatitis Virus (VSV), ATCC, Indiana strain,

was diluted to 250 plaque-forming units per ml with culture media.

Culture media: Minimum Essential Medium (MEM) with Earle's
balanced salt solution (Micrdbiological Associates, Walkersville, MD)

was used with the following added: 10% fetal bovine serum (Sterile

15
Systems, Logan, UT), 100 p/ml Penicillin (Squibb, Princeton, NJ),
100 pg/ml Streptomycin Sulfate (Pfizer, New York, NY), and 5 pg/ml

Amphotericin B (Squibb).

Interferon standard: A 100 p/ml interferon standard was Obtained

from Dr. D. A. Stringfellow (The Upjohn Company) (41).

Agar: Equal volumes of a 1.5% solution of Noble agar (Difco)

and a double-strength solution of culture media were mixed at 47 C.
Neutral red saline: NaCl, 0.16M; neutral red, 3 g/l.

Dulbecco's phosphate buffered saline (PBS), pH 7.2, was Obtained

from Grand Island Biological Co. (Grand Island, NY).

Method

Interferon was assayed by viral plaque reduction (41,42). TWO
milliliters of fibrOblasts, 105 cells/ml, were put in each 35 nmlwell
in culture plates (3506, Costar, Cambridge, MA) and grown to confluence.
Six dilutions of each serum sample ranging from 1:10 to 1:5000 were
prepared in MEM. The interferon standard was diluted 1:50, 1:100,
and 1:500. Each dilution was incubated on 2 fibrOblast monolayers,
l ml/well, at 37 C with 5% C02. MEM was incubated on 6 monolayers as
a control. After 24 hours of incubation the mediumwwas removed and
the cells were washed with 1 ml of PBS. Two-tenths milliliter of VSV
were pipetted onto each monolayer and allowed to absorb for 1 hour.
Agar was poured on the monolayers, 2 ml/well, and they were incubated
2 days and then stained with neutral red to facilitate plaque counting.
Units/m1 of interferon was defined as the reCiprocal of the dilution
which had half of the number of plaques that were present in the control

wells.

RES ULTS

I. Effect of Glucan and MDP Treatment
on the Reticuloendothelial System

Carbon clearance was determined on day 0 in mice, 5-6 per group,
that were injected with MDP on day -l or with Glucan on days -7, -4,
and -l. The doses were 0, 0.8, 2.5, 8.0, and 25 mg/kg for MDP and
0, 4, 13, 40, and 80 mg/kg for Glucan. Control mice (dose = 0) were
given Lactated Ringer's. Clearance (Eyié) increased as the MDP dose
increased up to 8 mg/kg, where the maximum clearance was Observed,
2.1 i 0.7 (approximately twice the clearance of control mice and sta-
tistically significant). The clearance at the highest MDP dose was
1.5 r 0.4 (Figure 1). The clearance in mice treated with Glucan
increased to a maximum of 4.9 i 1.8 at the 40 mg/kg dose and
declined to 1.5 i 0.4 at the 80 mg/kg dose. The increase in clear-
ance that occurred with Glucan doses of 13 mg/kg and 40 mg/kg was
significant, compared to the control (Figure 1). The half times are
listed in Table 2 in the Appendix.

Serum lysozyme was determined on day 0 in mice, 5 per group, that
were injected with MDP on day -l or with Glucan on days -7, -4, and
-1. The doses were 0, 8.0 and 25 mg/kg for MDP and 0, 40, and 80 mg/kg
for Glucan. Serum lysozyme was not significantly different in groups
treated with MDP and Glucan than it was in control groups. Each group
had approximately 1.5 pg/ml (Table 3, Appendix). The change in absorb-

ance for the 20 pg/ml lysozyme standard in the assay was 0.050.

16

17

GLUCAN mg/kg

 

 

 

 

 

 

0 1 5 1o 20 l l 5101 I I
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9’1
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MDP mg/kg

Figure 1. Effect of MDP and Glucan on carbon clearance in
mice. Clearance was determined on day 0. The MDP was given on
day -1. Glucan'was given on days -7, -4, and -l. N = 3-6.

18

Four to six mice from each group in the carbon clearance experi—
ment and the lysozyme experiment were killed immediately after blood
samples were obtained and their livers and spleens were weighed. The
comparison of organ weights of groups used for the carbon clearance
test is termed "First experiment" and that of groups used for the
lysozyme assay is termed "Second experiment." The MDP treatment
resulted in significantly increased spleen weight in the first experi-
ment only; at the dose of 8 mg/kg the spleen weight was 1.5 times that
of the control. With Glucan treatment, spleen weights increased as
the dose increased, and at the highest dose in the first and second
experiments they were 3 and 2 times the control spleen weights,
respectively (Figures 2 and 3). The increases Observed in spleen
weight with Glucan doses of 40 mg/kg and 80 mg/kg were statistically
significant.

Liver weight increased to approximately 1.3 times the liver
weight of control mice as the Glucan dose increased to 40 mg/kg, and
it remained at that level at the highest Glucan dose, 80 mg/kg, in
both experiments. The increase was statistically significant. The
MDP treatment did not alter liver weight (Figures 4 and 5). Organ
weights are listed in Tables 4 and 5 in the Appendix.

Livers and spleens from mice used in the lysozyme experiment were
fixed in formalin and processed for histological examination. Many
granulomas were observed in the livers of mice that were given Glucan
doses of 40 mg/kg or 80 mg/kg on days -7, -4, and -1. Numerous
granulomas were also observed in the spleens of mice treated at the
highest Glucan dose. The Spleens of mice that were given 40 mg/kg
doses of Glucan had very few granulomas, but compared to controls they

contained an increased number of vacuolated macrophages in the marginal

SPLEEN WEIGHT mg

19

GLUCAN mg/kg

 

 

are, ,§fl,,19 2." ..59...
200'-
150-
100

50

 

 

 

MDP mg/kg

Figure 2. Effect of MDP and Glucan on spleen weight in mice.

First experiment. Spleens were weighed on day 0. The MDP was
given on day -1. Glucan was given on days -7, -4, and -1.
= 4-6.

N

20

 

 

 

 

 

 

GLUCAN mg/kg
0 . 20
"—'l—" '1 I I I I I I I
200 -
B!
E
E
g 150 ~
I.”
3
E
Ii; 100 -
(I)
50 ~
6 " '5 5 i 10 20
MDP mg/kg

Figure 3. Effect of MDP and Glucan on Spleen weight in mice.
Second experiment. Spleens were weighed on day 0. The MDP was
given on day -1. Glucan was given on days -7, -4, and -1. N = 5.

21

GLUCAN mg/kg

 

 

 

 

 

 

 

0 fr 5 10 2'0 I I 5I0 I I I
p
1.4L
°’ ’ \\
s 6“”
2 1.2»
LU
3 .
a: ' i
'4‘
:1 1.0- ]’ M1")
1 _
- i
08(-
T’T i 2 5 1o 20
MDP mg/kg

Figure 4. Effect of MDP and Glucan on liver weight in mice.
First experiment. Livers were weighed on day 0. The MDP was given
on day -l. Glucan was given on days -7, -4, and -1. N = 4—6.

22

 

 

 

 

 

 

 

 

 

GLUCAN mg/kg
‘—9'_/ r‘ 210 l I 5I0 I I I
1.6 - ..
a 1.4 -
.—
I
52
S
a; 1.2 -
Lu
2
...-l
1.0 -
0' ';___ g l L l l 110 20
MDP mg/kg

Figure 5. Effect of MDP and Glucan on liver weight in mice.
Second experiment. Livers were weighed on day 0. The MDP was given
on day -1. Glucan was given on days -7, -4, and -l. N = 5.

23

area of splenic nodules. The spleen and liver from mice treated with

MDP appeared similar to the spleen and liver from control mice.

II. Experimental Infections

Two experiments were performed by pretreating mice and inoculating
them on day 0 with 6 x 107 CPU/kg of c. albicans.

In the first experiment there were 5 groups of 10 mice. The
control mice, group 1; were treated with Lactated Ringer's on day -2.
The mice in group 2 were treated with Glucan, 40 mg/kg, on days -7,

-4, and -1. Muramyl dipeptide, 8 mg/kg, was given to the mice in
group 3 on day -2 and to the mice in group 4 on day -l. The mice in
group 5 were implanted with Alzet minipumps on day -2. Each pump
contained 200 p1 of MDP at a concentration of 6.9 mg/ml. The delivery
rate was calculated to be 8 mg/kg/day for 7 days. Only MDP, given on
day —1 or by minimpumps, significantly increased survival (Figures 6
and 7). The Harmonic mean survival time (Hm) for groups 1, 2, 3, 4,
and 5 was 4.5 days, 9.0 days, 11.5 days, 19.0 days, and 19.7 days,
respectively.

To confirm the observation that MDP treatment on day -1 increased
the survival of mice inoculated with C. albicans and the Glucan treat-
ment did not, a second experiment was conducted with separate control
groups for MDP and Glucan. Control mice, 10 per group, were given
injections of Lactated Ringer's on day -l (MDP control) or on days -7,
—4, and -l (Glucan control). Ten mice were given Glucan injections,

40 mg/kg, on days -7, -4, and -l, and 20 mice were given MDP injections,
8 mg/kg, on day -1. Only MDP significantly increased survival (Figures
8 and 9). The Hntwas 4.7 days for the MDP control group and 16.6

days for the MDP treated group. The Hm for the Glucan control group

was 14.9 days and for the Glucan treated group it was 6.4 days.

24

 

 

 

100 -— BINGERS
--- MDP —2
—-- MDP —1
80
----- MDP PUMPS
..l
<
z
a 60
D
m .
'- a
35 40 '
D "--.-..."--. _
a: “ N — 10
a .,
\. ~,
20 1‘ ‘\ ...... ‘
‘\. \“
...x. --
o l l \. I I
20 25 30 35

 

Figure 6. First C. albicans experiment. Effect of MDP,
8 mg/kg/day, given on day -1, day -2, or by minipump on days -2
through 4, on survival of mice inoculated with 6 x 107 CFU/kg
on day 0.

 

25

100

 
 
  

 

—— BINGERS
80 ---- GLUCAN
—-I
:5
5:60
E
=:
U)
.—
EB
L,40
C
E

N
C

 

 

Figure 7. First C. albicans experiment. Effect of Glucan,
40 mg/kg, on days —7, -4, and -l, on survival of mice inoculated
with 6 x 107 CPU/kg on day 0.

100 -----

a GI on
o G o
l I I

PERCENT SURVIVAL

N
O
I

 

26

‘----- ——- RINGERS

\
\ ---- MDP
\

 

 

Figure 8.

 

Second C. albicans experiment. Effect of MDP,

8 mg/kg, on day -l, on survival of mice inoculated with 6 x 107

CPU/kg on day 0.

27

 

 

 

 

 

100
— BINGEHS

80 ""- GLUCAN
.1
§5
'5 60
c:
:3
on
.—
2:
2:40 N==10
c:
a:

i
20[- \
i
----- W
\
0 l l l l \\ I l I
0 5 10 15 20 25 30 35

Figure 9. Second C. albicans experiment. Effect of Glucan,

40 mg/kg, on days -7, -4, and -1, on survival of mice inoculated
with 6 x 107 CFU/kg on day 0.

28

Two experiments were performed by inoculating mice in groups of
20 with 5 x 1010 S. aureus/kg after pretreatment with Glucan or MDP.

In the first experiment mice in 2 groups were treated on days
-7, —4, and -1 with either Lactated Ringer's or Glucan, 40 mg/kg.
Mice in 2 other groups were treated on day -1 with Lactated Ringer's
or MDP, 8 mg/kg. MDP treatment increased survival time significantly
whereas Glucan treatment did not (Figures 10 and 11). The Hm‘was 4.4
days for the MDP control group, 23.3 days for the MDP treated group,
3.9 days for the Glucan control group, and 2.5 days for the Glucan
treated group.

To determine if MDP given 2 days before inoculation would modify
S. aureus infection, a second experiment was performed. Mice in 2
groups were treated on day -2 with Lactated Ringer's or MDP, 8 mg/kg.
Mice in another group were treated on day -1 with MDP, 8 mg/kg. Both
day -2 and day —1 MDP treatments significantly increased survival
time (Figure 12). The Hm was 1.8 days for the control group, 4.1 days
for the group treated on day -1, and 3.6 days for the group treated

on day -2.

III. Interferon

Mice were bled at various times during Glucan treatment for serum
interferon determination. Glucan, 40 mg/kg, was administered i.v. at
time 0 and every 72 hours until mice were decapitated at the time
indicated in Table 1. Lactated Ringer's was administered to control
mice. Serum from 5 mice in each group was pooled and assayed for
interferon. None of the sera from treated and control nice contained
detectable levels (310 units/ml) of interferon. However, the interferon

level for the standard was less than 50 units/ml instead of the

29

 

 

 

RINGERS -—

100‘ ---
_, 80
«t
2!
>-
a:
a
,_ so 20
z:
“I
e:
E
a 40

20'

5 10 15
DAYS

Figure 10. First S. aureus experiment.

Effect of MDP, 8 mg/kg,

on day -l, on survival of mice inoculated with 5 x 1010 bacteria/kg
on day 0.

30

 

 

 

 

 

100
means ——
GLUCAN -- -

so»
-I
5‘
s so-
=
=
m
p—
5
,_, 40'
C
m
m

20» u - 20

N =- 19
\ __________________
' I 10 15

Figure 11. First S. aureus experiment. Effect of Glucan,
40 mg/kg, on days -7, -4, and -l, on the survival of mice inocu-
lated with 5 x 1010 bacteria/kg on day 0.

31

 

 

 

100 means —
MDP —1 ---
MDP -2 -----
so
..l
(
=_-
a so
=
U)
'z'
\-
§ 40 \\ 20
a: x. _____ N:= .......
20 \\
........... 8.119 .........
l l l l J L l l l L I
5 1o 15
DAYS

Figure 12. Second S. aureus experiment. Effect of MDP,
8 mg/kg, on day -1 or day -2, on survival of mice inoculated
with 5 x 1010 bacteria/kg on day 0.

32

Table 1. Serum interferon sample times of mice treated with Glucan
or Lactated Ringer's

 

 

Time of Time of

a b Sample Sample

Group [Treatment (Hours) Group Treatment (Hours)
1 Glucan 3 12 Glucan 84
2 Glucan 6 13 Ringer's 84
3 Ringer's 6 14 Glucan 96
4 Glucan 12 15 Glucan 120
5 Ringer's 12 16 Glucan 144
6 Glucan 24 17 Glucan 147
7 Glucan 48 18 Glucan 150
8 Glucan 72 19 Ringer's 150
9 Glucan 75 20 Glucan 156
10 Glucan 78 21 Ringer's 156
11 Ringer's 78 22 Glucan 168

 

a . .
Five mice per group.

bTreatment began at time 0 and consisted of 1 i.v. injection
every 72 hours until sample time.

33

expected value of 100 units/ml. To insure that the apparent lack of
interferon in the serum samples was not the result of a decreased
sensitivity of the assay, 4 serum samples that had reduced the plaque
count by at least one-third were retested. Plaque counts again indi-
cated that the pooled serum samples from Glucan-treated mice did not
contain detectable levels of interferon. The value for the interferon
standard in the second test was 100 units/ml, as expected. The plaque

counts for these assays are listed in Table 6 in the Appendix.

DISCUSSION

The carbon clearance experiment indicated that the optimum
Glucan dose for stimulating carbon clearance was 40 mg/kg. The large
number of Glucan particles in the 80 mg/kg dose may have impaired
phagocytosis. A S-fold increase, observed at the 40 mg/kg dose, was
also observed by Wooles and Di Luzio after 40 mg/kg doses were
administered on days -3, -2, and —1 (11). They did not examine higher
doses. We used different timing because our treatment was based on
the report of Glucan treatment to protect mice against C. albicans
by Williams et a1. (2).

The optimum dose for stimulating carbon clearance with MDP was
8 mg/kg. The stimulation of carbon clearance with MDP at doses of 0
to 8 mg/kg has been reported before with results similar to ours (43).

The increased liver and spleen weight and granuloma formation
observed in Glucan-treated mice confirmed reports that Glucan induced
hypertrophy of the RES (11). It was reported that spleen and liver
weight was not increased in mice treated.with less than 200 ug of
MDP (ca. 8 mg/kg) (10). In our research, liver weight was not sig-
nificantly altered by MDP treatment. The effect of MDP treatment on
spleen weight was not consistent. Spleen weight was significantly
increased following 8 mg/kg of MDP in the First experiment. However,‘
increased spleen weight was not observed in the Second experiment at
MDP doses up to 25 mg/kg. Spleen weight may have been affected by

carbon injections in the first experiment, but carbon was administered

34

 

35
to all grOUps and the weight of carbon injected was less than one-
tenth of the spleen weight. Since increased spleen weight was not
observed in the Second experiment, it is most likely that MDP treat-
ment does not alter spleen weight.

In previous research, increased serum lysozyme was correlated
with increased carbon clearance in rats and mice that were treated
with Glucan (25). However, in this research with treatment methods
that stimulated carbon clearance, neither MDP nor Glucan increased
serum lysozyme levels. This discrepancy may have resulted from dif-
ferences in the Glucan or the mouse strain used. Di Luzio used
C57Bl/6J mice and 7.5 ug/ml was the normal serum lysozyme value for
these mice (25). The serum lysozyme control value for the CF—l mouse
was 1.5 pg/ml, which may indicate decreased ability for lysozyme
production and release in this strain. The source of commercially
available Glucan is Di Luzio's laboratory, which uses a modification
of the method of Hassid et al. to prepare Glucan (34,44). It was
reported that the antitumor activity of Glucan from this source varies
from batch to batch (45) and presumably other properties could also
vary. The lot we used, E0737, may be unable to induce an increase in
serum lysozyme. HoWever, treatment with this lot did increase liver
and spleen weight and carbon clearance. Di Luzio's hypothesis that
serum lysozyme is a good index of macr0phage stimulation is not
supported by our research, which demonstrates that increased particle
clearance can occur without an elevation in serum lysozyme.

Interferon levels also did not reflect macrophage stimulation.
We postulated that Glucan treatment may induce interferon because
Glucan is a polysaccharide that affects viral host defense. Poly-

saccharides have been reported to induce interferon which mediates

36

host defense against viruses and may also affect host defense against non-
viral infections and neoplasia (46,47). However, interferon induction
was not observed under the conditions of our research.

Glucan treatment did not significantly increase the survival
time of infected mice even though our infection models were similar
to those used by Williams et al. and Kokoshis et a1. when they
reported the protective effects of Glucan (2,38). The survival curves
of control mice in our studies were similar to theirs and the dose,
culture conditions and strain of S. aureus that we used were the same
as theirs. The principal differences between our research and theirs
were the lot and total amount of Glucan given and the strain of mouse
used.

The potential differences in Glucan lots were discussed above.
The total dose of Glucan in our studies was 120 mg/kg (3 x 40 mg/kg),
the dose at which carbon clearance was maximally stimulated. A total
Glucan dose of approximately 60 mg/kg (20 mg/kg on days -7, -4, and
-1) was reported to increase the survival of mice inoculated with
C. albicans (2). Protection against S. aureus was observed with total
Glucan doses of approximately 80 mg/kg (34,38). The amount of Glucan
that we administered may have been sufficient to "blockade" the RES
and thereby eliminate some of the protective effects of Glucan.
Although the carbon clearance data indicate that the dose would stimu-
late rather than "blockade" the RES, it is possible that Glucan treat-
ment enhanced carbon clearance by mechanisms other than phagocytosis
(12). The dose we used may have adversely affected bacterial uptake
and killing.

It is possible that we did not observe enhanced host defense in

Glucan-treated mice because we used a strain of mouse different than

37
those used in previous studies. The lysozyme results suggest that
there is a strain difference in the response to Glucan treatment.

The MDP treatment which induced a 2-fold increase in carbon
clearance always increased survival time significantly. This treat-
ment, 8 mg/kg 24 hours before inoculation, was more effective against
S. aureus than it was against C. albicans in terms of percent survival.
Administering MDP 48 hours before inoculation instead of 24 hours
reduced its effect against both infectious agents, especially C.
albicans. This correlates with the reported observation that carbon
clearance was elevated more one day after MDP treatment than it was
at two days (43). The finding that resistance to T. cruzi was
increased when MDP was given 48 hours prior to inoculation but not
24 hours (29) does not agree with our findings and suggests that
defense mechanisms function differently in protozoal infections.

Administering MDP via minipumps (8 mg/kg/day, days -2 through 4)
was no more effective than 8 mg/kg on day -1 against C. albicans.

This agrees with a report that noted prolonged MDP treatment was not
advantageous in C. albicans infection (27).

The mechanism of enhanced resistance to infection induced by MDP
is not known. Presumably the compound interacts with macrophages
directly to enhance phagocytosis and intracellular killing of micro-
organisms (19). Amplification of antibody responses by MDP may con-
tribute to the destruction of any microorganisms that evade nonspecific
defenses.

The rate of particle clearance is often used as an index of the
phagocytic activity of reticuloendothelial cells (12,43). Agents that
increase the rate of particle clearance are assumed to activate macro—

phages. They may increase the phagocytic rate by enhancing the

38
attachment phase or the ingestion phase of particle phagocytosis (48).
However, macrophage number and other factors that contribute to
particle clearance, such as particle aggregation, attachment of
particles to platelets, and blood flow, may be affected by agents
that increase the clearance rate (48).

Our research indicates that particle clearance is not useful for
screening compounds to find agents that can stimulate nonspecific
resistance to infections. Enhancement of the rate of carbon clearance
did not corrleate with the status of host defense. Glucan treatment
failed to increase resistance to infection even though it increased
carbon clearance more than MDP treatment. The increased survival of
MDP-treated mice demonstrates that the experimental infections used
are susceptible to treatment and that host defense can be increased
without inducing splenomegaly and hepatomegaly. Our results also
indicate that serum lysozyme levels to not cOrrelate with host defense

status because MDP treatment did not increase serum lysozyme.

 

S UMMARY

The optimum dose of MDP and Glucan for enhancing the intravascular?
clearance of colloidal carbon injected in mice was determined. The
effect of the treatments on resistance to infection was determined by
inoculating mice with large doses, i.v., of C. albicans and S. aureus,
after drug treatments, and observing their survival time. Serum
lysozyme and interferon were assayed and liver and spleen were examined!
in treated mice.

For stimulating carbon clearance, the optimum Glucan dose was
40 mg/kg, i.v. (3 times), and the optimum MDP dose was 8 mg/kg, i.v.
(1 time). Carbon clearance was increased S-fold by the Glucan treat—
ment and 2-fold by the MDP treatment. The survival time of MDP—
treated mice, but not Glucanétreated mice, was significantly greater
than control mice in 2 experiments with each infectious agent. The
MDP was less effective when given 2 days prior to inoculation instead
of 1 day. Serum lysozyme was not increased by either treatment.
Glucan did not induce interferon but it did induce granulomatous
hypertrophy of liver and spleen.

The status of host defense against bacteria and yeast did not
correlate with the rate of carbon clearance or the concentration of
serum lysozyme in mice. This indicates that the carbon clearance
test and the measurement of serum lysozyme would not be useful for

screening compounds for host defense-stimulating activity.

39

APPENDIX

 

4O

ABSORBANCE 450NM

 

l l l

.1 .2 .3 .4 .5 .6
BACTERIA/Ml. x 109

I
h

'41.

Figure 13. Standard curve for determining S. aureus concen-
tration. Coleman Junior III spectrophotometer with 19 x 75 mm
cuvette.

41

Table 2. Carbon clearance half times on day 0 of mice treated with
Glucan and MDP

 

 

 

 

Treatment Clearance K/K___
(mg/kg) Half Times (Minutes) Mean S.DI
Glucan

days -7,-4,-l

0 8.2 9.9 6.8 6.4 8.0 1.00 0.17

4 7.3 8.5 7.9 6.2 5.1 1.14 0.24

13 1.9 2.8 2.6 * * 3.24 0.69

40 3.7 1.1 2.1 1.4 1.5 1.3 4.87 1.75

80 6.0 6.2 5.4 3.7 * 1.51 0.39

MDP

day -1

0 8.6 8.0 10.1 6.2 6.8 1.00 0.19

0.8 5.9 8.8 8.7 7.8 8.2 4.5 1.12 0.34

2.5 4.0 6.5 6.2 6.9 4.5 4.6 1.48 0.34

8.0 2.3 5.6 4.2 4.0 3.3 4.2 2.12 0.68

25.0 7.6 4.3 6.3 4.1 4.4 ~ 1.52 0.41

 

*
Blood samples were not obtained.

 

42

Table 3. Serum lysozyme on day 0 of mice treated with Glucan and MDP

Treatment
(mg/kg) Lysozyme (pg/ml) Mean S.D.

 

Glucan
days —7,-4,-1

0 5.0 1.6 1.2 1.6 0.0 1.9 1.9
40 0.0 1.6 1.2 1.2 1.2 1.0 0.6
80 0.8 2.3 1.2 0.0 0.8 1.0 0.8
MDP
day -l
0 2.7 0.4 0.4 1.6 1.2 1.3 1.0
8.0 1.6 0.8 1.9 1.6 0.0 1.2 0.8
25.0 1.6 0.0 2.7 1.6 0.4 1.3 1.1

 

43

Table 4. Spleen weights on day 0 of mice treated with Glucan and MDP

 

Treatment
(mg/kg) Spleen Weight (mg) Mean S.D.

 

First experiment
Glucan
days -7,-4,-1

0 50 4O 50 40 ‘45 10
4 50 50 50 50 50 0
13 50 100 80 90 80 2O
40 90 120 100 110 105 10
80 200 150 120 90 140 140 40
MDP
day -1
0 50 40 60 43 50 55 50 7
0.8 60 50 56 50 40 50 51 7
2.5 60 60 50 70 56 70 61 8
8.0 90 60 65 73 79 63 72 11
25.0 45 60 60 50 60 73 58 10

Second experiment
Glucan
days -7,-4,-l

0 75 90 64 89 63 76 13
40 106 72 127 108 132 109 24
80 143 190 147 178 177 167 21
MDP
day -1
0 88 81 67 48 82 73 16
8.0 96 114 58 96 47 82 28

25.0 80 74 107 65 80 81 16

 

44

 

 

Table 5. Liver weights on day 0 of mice treated with Glucan and MDP
Treatment
(mg/kg) Liver Weight (9) Mean S.D.
First experiment
Glucan
days -7,-4,-l
0 1.00 0.99 1.02 0.78 0.95 0.11
4 1.17 1.06 0.90 1.05 1.05 0.11
13 1.13 1.20 1.08 1.03 1.11 0.07
40 1.28 1.37 1.24 1.35 1.31 0.06
80 1.41 1.42 1.36 1.16 1.17 1.30 0.13
MDP
day -1
0 1.09 0.98 1.05 0.93 1.06 1.30 1.07 0.13
0.8 1.12 0.87 1.05 0.88 0.82 1.15 0.98 0.14
2.5 0.90 0.85 0.99 0.99 0.96 1.02 0.95 0.06
8.0 1.08 0.91 0.96 0.98 0.96 1.06 0.99 0.06
25.0 0.88 1.01 1.02 0.82 1.05 0.85 0.94 0.10
Second experiment
Glucan
days -7'-4'-1
0 1.24 1.12 1.30 1.34 1.08 1.22 0.11
40 1.44 1.20 1.52 1.50 1.59 1.45 0.15
80 1.75 1.58 1.29 1.46 1.32 1.48 0.19
MDP
day -1
o 1.21 1.07 1.12 0.91 1.16 1.09 0.12
8.0 1.12 1.20 1.03 1.20 0.92 1.09 0.12
25.0 1.24 1.18 0.90 1.15 1.21 1.14 0.14

 

45

Table 6. Interferon assay plaque counts*

 

First Assay;

Second Assay

 

 

 

Group Plaques/Well Group Plaques/Well
1:10 dilution 1:10 dilution
1 20 32 6 24 34
2 27 36 7 33 34
3 32 33 8 25 32
4 26 31 9 30 31
5 31 37
6 18 27 1:50 dilution
7 20 29
8 17 23 Standard 10 13
9 19 23
10 29 31 1:100 dilution
11 24 29
12 19 32 Standard 19 20
13 26 23
14 32 31 Virus
15 28 36 Control 48,40,37
16 27 24 35,37
17 33 32
18 23 24 Control
19 24 27 Mean 39
20 30 34
21 27 28
22 25 36
1:50 dilution
Standard 19 20
Virus
Control 35,38,34,
24,30,31
Control
Mean 32

 

*
If a dilution had more than 1/2 the number of plaques in the
control mean, higher dilutions were not counted.

 

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VITA

 

VITA

I lived on a farm in Midland County, Michigan, from my birth, in
1951, until after I graduated from Coleman High School. I began
studies in the School of Literature Science and Arts at the University
of Michigan in 1969- Majoring in Medical Technology, I received
clinical training at University Hospital. In 1973 I received my
B.S. degree and passed the Medical Technologist Registry Examination
of the American Society of Clinical Pathologists. During the next
five years I was employed at various hospitals and clinical labora—
tories in Michigan. I was admitted to the Clinical Laboratory Science
program at Michigan State University and received an Upjohn Fellowship
from the Department of Pathology in 1978. My research was conducted
at The Upjohn Company, Kalamazoo, Michigan, and I will receive my

M.S. degree August 1980.

51