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This is to certify that the
thesis entitled

THE EFFECT OF DIETARY DEOXYNIVALENOL ON
SERUM IMMUNOGLOBULIN A IN THE MOUSE

presented by

Mark Alan Moorman

has been accepted towards fulfillment
of the requirements for

M . S . d . FSHN
egree 1n

 

 

”BRA“
loan Ste.
University

 

Major professor

Date 91 19/ 88

0-7639 MS U is an Aflinnau've Action/Equal Opportunity Institution

 

 

 

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MSU Is An Affirmative Action/Equal Opportunity Institution

_%——M—

THE EFFECT OF DIETARY DEOXYNIVALENOL ON SERUM

IMMUNOGLOBULIN A IN THE MOUSE

BY

Mark Alan Moorman

A THESIS

Submitted to
Michigan State University
in partial fullfilment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1988

ABSTRACT

THE EFFECT OF DIETARY DEOXYNIVALENOL ON SERUM

IMMUNOGLOBULIN A IN THE MOUSE

BY

Mark Alan Moorman

Fusarium mycotoxins are commonly found in cereal grains
and when consumed are frequently associated with serious
animal health problems. Recently the consumption of
mycotoxins has been associated with immunosuppression. The
objective of this research was to examine the effect of the
trichothecene mycotoxin deoxynivalenol (DON) on total and
specific immunoglobulin in the mouse. Exposure to 25 ppm
dietary deoxynivalenol caused a decrease in serum
immunoglobulin G (IgG) and immunoglobulin M (IgM) but
increased immunoglobulin A (IgA) up to 17-fold after 24 weeks
of feeding. Serum from mice fed deoxynivalenol and
challenged with cholera toxin showed decreased cholera toxin-
specific serum IgG but not IgA. Casein specific serum 19A
from these mice was increased whereas casein-specific serum
IgG was decreased. Isolated splenocytes from mice fed
deoxynivalenol exhibited increased IgA in mitogen and non-
mitogen stimulated cultures. These results suggest that
dietary exposure to deoxynivalenol alters regulation of IgA

production.

This thesis is dedicated to the memory of my father.

Eugene Frank Moorman

ACKNOWLEDGMENTS

The author is deeply indebted to his major professor,
Dr. James J. Pestka for his guidance, understanding and
encouragement throughout this work.

Appreciations are expressed to Drs. Kathy Brooks, Debbie
Dixon-Holland, and John Linz for their helpful suggestions
and as members of the guidance committee.

I would also like to thank Mary Witt for the
deoxynivalenol and Roscoe Warner for his help with the
immunoassays.

iv

TABLE OF CONTENTS

LITERATURE REVIEWOOOOOOOOOOOOOOOO00......OOOOOOOOOOOOOOOOOOO
MATERIALS AND METHODS.OOOOOCOOOOOOOOOOOOOOOOOOOOOOOOO0......

General experimental design.........................
DeoxynivalenOI0.0.0.0...OOOOOOOOOOOCOOOOOOOOO0......

Diet.00.000.000.00...0.0....0.000000000000000...0.0.

Animals.............................................
Feeding protocol....................................
Sample collection...................................
Immunoglobulin quantitation.........................
Cholera toxin and casein specific immunoglobulin

quantitation........................................

RESULTS.0.000000000COOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO....0.

Serum immunoglobulin profile in mice following
dietary DON exposure-0.0.0....OOOOOIOOOOOOOOOOOOOOO.

Salivary IgA concentrations in mice following
dietary DON exposure...OOOOOCOOOOOOOOOOOOO0.0...O...

Intestinal IgA levels following dietary
DON exposure...00......OOOOOOOOIOOOOOOOOOOO0.0....0.

The effect of dietary DON on the total and
antigen-specific serum immunoglobulin response......

£13 vitro immunoglobulin production by isolated
splenocytes following dietary DON exposure..........

DISCUSSION....0...00......OOOOOOOOOOOOOOOOOOOOOOO. ...... O...
SUMY.‘0.0000000000000000.00.00.00.00.00.00...OOOOOOOOOOO.

LITERATURE CITEDOOOOOOO ..... .0. ........... 0.. ...... 000......

PP

17

24

28

28

43
54
63

65

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

10.

11.

12.

13.

14.

LIST OF TABLES

Serum IgA concentration in mice following
dietary exposure to 2 and 10 ppm DON.H.".”..

Serum immunoglobulin.concentrations in mice
following dietary exposure to 10 ppm DON.......

Serum immunoglobulin concentrations in mice
following dietary DON exposure.................

Serum IgA concentration in mice following
dietary exposure to 25 ppm DON

Salivary IgA concentration in mice following
dietary exposure to 2 and 10 ppm DON.".".H..

Serum IgA in mice following exposure to
10 ppm DON and oral cholera toxin challenge....

Intestinal 19A of mice following dietary
DON and oral cholera toxin challenge.u.u.u..

Serum 19A in mice following exposure to
25 ppm dietary DON and oral cholera toxin
Challenge.OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Cholera toxin and casein-specific IgA
following dietary DON and oral cholera
toxin challenge................................

Cholera and casein-specific serum IgG
following 8 weeks of dietary DON and
oral cholera toxin challenge..................

Total and antigen-specific intestinal
IgA following 22 weeks of dietary DON
and oral cholera toxin challengeu.u.u.n.n

Effect of dietary DON on in vitro IgA
production by isolated splenic lymphocytesn..

Effect of dietary DON on in vitro IgG
production by isolated splenic lymphocytesu..

Effect of dietary DON on in vitro IgM
production by isolated splenic lymphocytes"..

vi

18
20
21
25
29
31

32

33

35

41

42 I
44
48

51

Figure 1 .

Figure 2.

Figure 3 .

Figure 4 .

LIST OF FIGURES

The chemical structure of deoxynivalenol. The
epoxide structure is essential for toxic action
(Ueno, 1983).

Serum immunoglobulin isotype profile following
dietary DON exposure. Mice were fed 25 ppm DON in
AIM-76A diet for 12 weeks. Data shown are geometric
means : S.D. for groups of 6 ad lib control and 8
treated and restricted control mice. Difference
between treatment and ad lib control determined by
Dunnett's t test. Difference between treatment and
restricted or restricted and ad lib control
determined by the improved Bonferroni t test.
Treatment and control groups with identical letters
are significantly different.

Serum IgA following long term dietary DON exposure.
Mice were fed 25 ppm DON in AIN-76A diet for 24
weeks. Data shown are geometric means tug/ml) i
S.D. for groups of 13 mice. Difference between
treatment and ad lib control group determined by
Dunnettls t test. Treatment and control groups are
significantly different from week 4 to week 24
(p<o.01).

Cholera toxin -specific equivalent IgA and IgG
concentrations following dietary DON and cholera
toxin challenge. Mice were fed 25 ppm DON for 24
weeks and challenged with 10 ug cholera toxin/mouse
at wk 5, and boosted at wks 7 and 21. Values
determined by ELISA, Absorbances for immunoglobulin
specific antibody were equated to absorbances from
dilutions of mouse reference serum run concurrently
and equivalent concentration values were assigned.
Data shown are geometric mean (ng/ml) : SJL for
groups of 13 mice. The cholera-25 ppm DON group had
14 mice“ Difference between treatment and ad lib
control determined by Dunnett's t test. Difference
between DON treatment and matched control
determined by Bonferroni t test. Bars with
identical letters are significantly different
(p<0.01).

vii

Figure 5 .

Figure 6 .

Figure 7 .

Casein -specific equivalent IgA and IgG
concentrations following dietary DON and cholera
toxin challenge. Mice were fed 25 ppm DON for 24
weeks and challenged with 10 ug cholera toxin/mouse
at wk 5, and boosted at wks 7 and 21. Values
determined by ELISA. Absorbances for immunoglobulin
specific antibody were equated to absorbances from
dilutions of mouse reference serum run concurrently
and equivalent concentration values were assigned.
Data shown are geometric mean (ng/ml) : SJL for
groups of 13 mice. The cholera-25 ppm DON group had
14 mice. Difference between treatment.and ad lib
control determined by Dunnetts't test. Difference
between DON treatment and matched control
determined by Bonferroni t test. Bars with
identical letters are significantly different
(p<0.01).

Effect of dietary DON on in vitro IgA production by
isolated splenocytes. BGC3F1 mice (2/trial) were
fed 25 ppm DON for 21 weeks. Restricted groups were
fed AIN—76A diet at level equivalent to mean intake
of 25 ppm DON group. Spleen cell suspensions (5 x
105 cells/well) were prepared in 20% RPMI-164O and
exposed to LPS (20 ug/ml), Con A (10 ug/ml), Con A-
LPS (10 and 20 ug/ml) or water for seven days at
37°C-5% C02. Supernatants were analyzed for IgA by
ELISA. Data shown are mean (ug/ml) : S.E.. Groups
with identical letters are significantly different
(p<0.01). Data for figure from trial 2 of table 12.

Effect of dietary DON on in vitro IgG production by
isolated splenocytes. BGC3F1 mice (2/trial) were
fed 25 ppm DON or 21 weeks. Spleen cell
suspensions (5 x 10 cells/well) were prepared in
20% RPMI-164O and exposed to LPS (20 ug/ml), Con A
(10 ug/ml), Con A-LPS (10 and 20 ug/ml) or water
for seven days at 37°C-5% CO . Supernatants were
analyzed for IgG by ELISA. ata shown are mean
(ug/ml) : S.E.. Groups with identical letters are
significantly different (p<0.01). Data for figure
from trial 2 of table 13.

viii

Figure 8. Effect of dietary DON on in vitro IgM production by
isolated splenocytes. B6C3F1 mice (2/trial) were
fed 25 ppm DON or 21 weeks. Spleen cell
suspensions (5 x 10 cells/well) were prepared in
20% RPMI-1640 and exposed to LPS (20 ug/ml), Con A
(10 ug/ml), Con A-LPS (10 and 20 ug/ml) or water
for seven days at 37 °C-5% CO Supernatants were
analyzed for IgM by ELISA. ata shown are mean
(ug/ml) + S. E.. Groups with identical letters are
significantly different (p<0. 01). Data for figure
from trial 2 of table 14.

Figure 9.Major organs involved in an immune response and the

route of circulation of lymphocytes between the
tissues (Male, 1986).

ix

Introduction

Mycotoxins are secondary metabolites produced by certain
fungi (molds) that when consumed can cause significant health
effects in humans and animals. There have been many reported
outbreaks of mycotoxicoses due to the consumption of
mycotoxins in humans dating back to the occurence of ergotism
or "holy fire" during the middle ages (Davis and Diener,
1987). Humans consuming bread prepared from rye infected

with Claviceps purpurea were affected with either convulsive

 

or gangrenous ergotism (Floss and Anderson, 1980). The
patient experienced violent, burning pains in extremities
(hence the Fire of St. Anthony). The part gradually became
numb, and gangrenous.

Two other historic outbreaks of mycotoxins pointed out
their importance to human and animal health. One came about
in England in the 1960's with the death of more than 100,000
turkey poults in what is called the Turkey-x disease (Davis
and Diener, 1987; CAST Report, 1979). The causative agent

was found to be aflatoxin produced by Aspergillus flavus in

 

the peanut meal fraction of the feed. More recently it has
been shown that of the aflatoxins present (El, 32' G1, Gz),
aflatoxin Bl is one of the most carcinogenic naturally
occuring substances known (CAST Report, 1979). Alimentary

Toxic Aleukia (ATA) was caused by the consumption of Fusarium

contaminated grain resulting in severe outbreaks between 1930
and 1950. A 1932 outbreak.of ATA.in one district of Russia
resulted in mortality of 62% of those afflicted (Davis and
Diener, 1987). Consumption of the spoiled grains resulted in
severe bone marrow suppression, anaemia, bleeding, and immune
suppression with the infectious complications leading to
death after several weeks (Bunner et al., 1985). It was
later determined that the tricothecene T-2 was the principal
toxin involved.

The focus of this thesis research is on one chronic
effect of tmdchothecene ‘mycotoxins, namely,
immunosuppression. The trichothecene mycotoxins are a
specific group of fungal metabolites based on their common
trichothecane structure. The skeleton structure includes a
six-membered oxygen containing ring, an epoxide group in the
12, 13 position, and an olefinic bond in the 9,10 position.
The chemical structure of the trichothecene mycotoxin
deoxynivalenol is presented in Fig 1. These naturally
occuring compounds are produced by various genera of fungi
such as 251222222222. Irisagéerae. Exrefgesiga.

Cephalosporium, Fusarium, Stachybotrys, Verticimonosporium,

 

 

 

and Cylindocarpon. The 45 known trichothecenes can be

 

categorized into groups A,B,C, or D according to their
chemical characters and the particular producing fungi (Ueno,
1983). Since the first isolation of a trichothecene
mycotoxin in 1949 these compounds have postulated to be
etiological agents in a variety of human and animal disease

outbreaks. Japan has been especially afflicted with this

Figure 1. The chemical structure of deoxynivalenol. The
epoxide structure is essential for toxic action
(Ueno, 1983).

problem dating back to 1890 with what is called the Red Mold
Disease. The Fusarium genus is responsible for these
outbreaks when it grows in wheat, barley, oats, rye, rice and
other crops primarily during heavy raining seasons. When
humans or farm animals consume these commodities, nausea,
vomiting, diarrhea, feed refusal, congestion or hemorrhage in
the lung, adrenals, intestine, uterus, vagina and brain, and
destruction of the bone marrow are the characteristic
symptoms (Ueno, 1983).

Deoxynivalenol (DON or vomitoxin) is a trichothecene

mycotoxin produced by Fusarium graminearum and is a common

 

 

contaminant of cereal grains worldwide (Mirocha et a1”

1976). Infection of corn and cereal grains by Fusarium

 

graminearum and concomitant DON production occur most

 

frequently in years that are particularly cool and wet at
times of harvest (Cote et al., 1984). DON produced in
feedstuffs has been shown to survive processing involved in
manufacture of the commodity (Greenhalgh et al., 1984) to the
extent that in an FDA market survey, 60% of breakfast cereal
tested contained DON at an average level of contamination of
approximately 100 ppb (Trucksess et al., 1986).

Farm animals that consume corn and cereal grains
containing DON develop various health problems such as feed
refusal, reduced weight gain, reproductive problems,
diarrhea, emesis, and death. Swine are especially
susceptible to the effects of the toxin giving DON the name
vomitoxin due to its emetic activity (Forsyth et al., 1977).

In 1982, the Centralia Diagnostic Laboratory and the

laboratory at the University of Illinois found that 80% of
the feed samples tested that were suspected of causing or
contributing to animal health problems contained DON at a
concentration ranging from 0.1 to 41.6 ppm (mean 3.1 ppm)
(Cote et al., 1984). In 1982, 33 samples of wheat from
Kansas and Nebraska were analyzed for the mycotoxins DON, T-2
toxin, zearalenone, and aflatoxin. DON was identified in 31
of 33 samples (Hagler et al., 1984).

Trichothecenes inhibit protein and DNA synthesis and are
therefore generally considered to have immunosuppressive
properties (Miller'and.Atkinson, 1987). Pier et a1. (1980)
noted that subacute mycotoxin exposure can lead to impairment
of immunogenesis and native mechanisms of resistance thus
leading to secondary mycotoxin diseases. Both humoral and
cellular immunity can be affected by small quantities of
mycotoxins which can eventually lead to profound alterations
in the defense mechanisms of the body against infection.
Experimentally, acute doses of DON have resulted in extensive
necrosis of the gastro-intestinal tract, bone marrow and
lymphoid tissues, and focal lesions in kidney and cardiac
tissue (Forsell et al. 1987). Acute and chronic toxicities
of trichothecenes include depletion of lymphoid tissues,
which strongly suggest that the trichothecenes can alter

immune responses (Ueno 1983). ‘When Listeria monocytogenes

 

infection is used as a measure of immune resistance, dietary
DON causes a decreased time-to-death interval of mice
(Tryphonas et al., 1986). Using a similar model, Pestka et

al., (1986) demonstrated that 25 ppm DON induced higher

splenic Listeria monocytogenes counts than the ad lib control

 

indicating a decreased ability to fight disease.

The specific effects of trichothecenes on the immune
system have been studied by examining the cellular responses
of lymphocytes to these toxins. Lymphocyte blastogenesis is
one valid in yitgg model for determining the effect of
environmental contaminants on immunocompetent cells (Forsell
and Pestka, 1985). Lafarge-Frayssinet et al. (1979) observed
a decrease in mitogen-stimulated blastogenesis of B and T

cells after experimental treatment of mice with Fusarium

 

extracts containing T-2 toxin and diacetoxyscirpenol.
Mitogen-induced blastogenesis in human peripheral blood
lymphocytes is inhibited by 50% with approximately'115 ng/ml
T-2 toxin (Cooray, 1984; Forsell et al., 1985; Tomar et al.,
1988) Robbana-Barnat et al. (1988) showed that 131 ng/ml DON
decreased murine splenocyte blastogenesis by 50%. Assuming
equivalent sensitivity of human and murine lymphocytes, these
experiments indicate the greater toxicity of T-2 toxin and
demonstrates the extreme potency of these toxins.

There is some conflicting evidence for stimulation or
increased blastogenesis in lymphocytes treated with low
concentrations of trichothecene mycotoxins. Lafarge-
Frayssinet et al. (1979) observed an increase in
blastogenesis in lymphocytes of mice treated with low doses
of Fusarium extracts containing T-2 toxin and

diacetoxyscirpenol. Forsell et al. (1985) also observed a

slight stimulation of lymphoblastogenesis with low (5 x 10"4

to 5 x 10'3 ng/ml) T-2 toxin concentrations. Robbana—Barnat

et al.(1988) could.not.duplicate these results with low in
gitrg doses of DON.

Robbana-Barnat et al. (1988) examined the effects of DON,
the most commonly occuring trichothecene, on murine lymphoid
organs and specifically on humoral immunity. Orally
administered DON causes significant reduction of thymus and
splenic weights and the authors observed that sensitivity to
DON was greater with lymphocytes than fibroblastic cells.
Sheep red blood cells (sRBC) are T-cell dependent for immune
response and are commonly used to study humoral immunity.
Oral DON has been shown to lower serum antibody responses to
sheep red blood cells (Robbana-Barnat et al., 1988),
specifically decreasing serum IgM (Tryphonas et al., 1984).
Robbana -Barnat et a1. (1988) speculated that DON could
directly impair antibody formation to sheep red blood cells
by adversely affecting the functional integrity of a subset
of the T-lymphocyte, possibly the helper T-cell. This
conclusion is supported by DON-induced decreased thymus
weights (Tryphonas et al., 1984; Robbana-Barnat et al., 1988;
Pestka et al, 1986). Forsell et al. (1986) also observed a
dose -dependent decrease in serum IgM upon dietary DON
exposure but, in contrast a dose-dependent increase in serum
IgA. The IgA concentration was maximal at 10 ppm DON
decreasing at the 25 ppm level. Consistent with this was the
observation of Cooray and Lindahl-Kiessling (1988) that there
was a dose-dependent increase in the number of spontaneous
antibody secreting cells from the spleen of mice treated with

T-2 toxin. Based on these and previous results (Otakawa et

al. (1979)), the authors speculated that the increase in
antibody producing cells could be due to the selective
destruction of T suppressor cells. In conflict to this
hypothesis, Tomar et al. (1988) demonstrated that T-2 toxin
did not induce the generation of suppressor cells by the T
cell mitogen concanavalin A.

IgA serves a very important role at mucosal surfaces
where it binds potentially infectious agents and prevents
their absorption. The dose-dependent increase in serum IgA
noted by Forsell et al., (1986) at DON levels found
frequently in agricultural commodities was unexpected. The
objective of this project was to further characterize the
effect of dietary DON on serum IgA. Particular attention was
placed on evaluating the dose effects, and.determining the
kinetics at the optimal dose. The effect of dietary DON on
mitogen induced splenic lymphocyte immunoglobulin production
was evaluated to determine if the spleen was involved in the
elevation of serum IgA. It is anticipated that these studies
will result in a model that can be used to study mechanisms

by which dietary DON alters serum immunoglobulin levels.

Materials and Methods

 

General Experimental Design. The research in this
thesis was designed to develop a model to study the effects
of dietary DON on serum and mucosal immunoglobulin
production. The mice were fed diet that contained DON and
the serum and mucosal compartments were analyzed for total
and antigen specific immunoglobulin. Based on the results of
these feeding studies, the model was extended to evaluating
the immunoglobulin response of mitogen-stimulated splenocytes
frommice.

Deoxynivalenol. Deoxynivalenol was produced in

Fusarium graminearum R6576 cultures and purified by water-

 

saturated silica gel chromatography (Witt et al. 1985)

Diet. Mice were fed powdered AIN-76A semi-purified
diet (ICN Nutritional Biochemicals, Cleveland, OH) to
minimize non-specific immune effects caused by nutrient
variability. Purified DON was incorporated into the diet as
described by Forsell et al. (1986). Briefly, DON was first
dissolved in ethanol and added to the powdered diet to yield
a 1000 ppm concentrate. After being mechanically mixed £0 10
minutes and dried overnight under a fume hood, the
concentrate was mixed with diet to obtain the desired
mycotoxin levels. During experiments all diets were stored
in the dark at 10°C.

Animals B6C3F1, weanling female mice purchased from

10

11

Harlan Sprague Dawley, Inc. were housed individually in
protected environment cages. Each cage unit included a
transparent poly carbonate body with filter cover, stainless-
steel wire lid, and a raised cage floor over treated hardwood
chips. iMice were given distilled water ad libitum.

Feeding Protocol. IExperiments consisted of a DON
treatment group and control groups. Treatment groups
received fresh DON diet every 3 days with the control group
receiving control diet every 3 days. Experiments with
restricted control groups received control feed at a dosage
equivalent to the previous 3 day mean intake for the DON
treatment group (Pestka et al., 1987).

Sample Collection. Blood was obtained from the retro-
bulbar plexus of ether-anesthetized mice by using non-
heparinized capillary tubes. After centrifugation of
samples, serum was removed and stored.at -20°C until assay.

Saliva was obtained from mice after intraperitoneal
(i.p.) injection with 1 mg pilocarpine (Sigma Chemical) in
0a2 ml water (Challacombe and Tomasi, 1980). After 2 minutes
saliva was collected in small glass tube, diluted 1:1 with
1:1000 ThimersolR (Sigma Chemical) and stored at -20°C until
assay.

Intestinal contents were collected by the procedure of
Snider and Underdown (1986) with minor modifications. In
short, mice were deprived of food for 2-4 hours prior to
intestine removal. After determining body weight, mice were
sacrificed in a C02 chamber; The mouse abdomen was washed

with 70% ethanol, and the whole small intestine was removed

12

below the stomach and ligated within 4 cm of the end of the
ileum. Ice cold trypsin inhibitor solution (1.5 ml) was
injected into the duodenal end and the intestine was massaged
for 1 minute. Trypsin inhibitor solution was composed of 0.1
M phosphate buffered saline (PBS, pH 7.2) , 5mM
ethylenediaminetetraacetic acid trisodium salt (EDTA), 2mM
phenylmethysulfonylfluoride (PMSF) (Sigma Chemical) and 0.05
U aprotinin (trypsin inhibitor) (Sigma Chemical). Contents
were drained into a sterile petri dish and transfered to a 15
ml conical tube. Supernatants were collected after
centrifugation at 800 x g for 10 minutes at 4°C and then
frozen at -20°C until assayed.

Immunoglobulin quantitation. Total serum immunoglobulin
was quantitated by radial immunodiffusion.(RID) according to
the method of Luster et al. (1982). RID-agarose gel for 1
liter consisted of 6.98 g sodium barbital, 6.0 g sodium
chloride and 0.1 g ThimersolR. After adjusting pH to 7.6
with concentrated (38%) HCL, 10 g agarose (Bethesda Research
Laboratories, Gaithersburg MD) was added and dispensed into 9
ml tubes. RID plates (Miles Scientific, Naperville IL) were
prepared by adding goat antimouse immunoglobulin (Cooper
Biomedical, West Chester PA) to 9 ml of tempered RID agarose
gel (62°C) and pouring onto RID plates. Optimized dilutions
were as follows: goat anti-mouse IgA (alpha chain specific),
1:60; IgM (mu chain specific), 1:70; and IgG (gamma chain
specific), 1:50. After allowing plates to solidify for 1
hr at 25°C, 3 mm holes were cut with a template (Miles

Scientific) and 10 ul of serum sample or mouse reference

13

serum (ICN ImmunoBiologicals, Lisle, IL) were added. IgA
serum samples were diluted 1:1 in (0.85%) saline, IgM samples
1:3 and IgG samples 1:6. RID plates were incubated in a
humid chamber for 48 hours (IgG and IgM plates for 24 hours)
at 4°C. The resultant precipitin bands were measured with a
vernier caliper and immunoglobulin concentrations were
determined based on the precipitin bands obtained with
reference serum (Miles Scientific, Naperville IL).

Salivary IgA was estimated by the enzyme linked
immunosorbent assay (ELISA). For this assay, goat-antimouse
IgA (alpha chain specific) was diluted to 10 ug/ml in 0.1 M
bicarbonate buffer (pH 9.6) and 100 ul incubated in Immulon 2
Removawell Microtiter Strips (Dynatech Laboratories,
Chantilly, VA) overnight at 4°C. Coated plates were then
washed 3 times with 0.1 M phosphate buffered saline (pH 7.2:
PBS) containing 0.2% Tween 20 (PBS-Tween). To reduce non-
specific protein binding, 0.3 ml 1% (w/v) bovine serum
albumin (BSA) in PBS were added to each well. The plates
were incubated at 37°C for 30 min. and then washed 4 times
with PBS-Tween. One hundred microliter aliquots of saliva or
immunoglobulin reference serum both diluted in 1% BSA-PBS
were added to appropriate wells and incubated for 1 hr at
37°C. After washing 5 times with PBS-Tween, 0.1 ml goat
antimouse IgA peroxidase (alpha chain specific, Cooper
Biomedical) diluted 1:500 in 1% BSA-PBS was added to each
well. After 30 minutes the plate was washed 8 times. Bound
peroxidase was determined by incubating 100 ul ABTS-HZOZ

substrate in each well for 5-30 minutes, and the reaction was

14

terminated with 100 ul citric acid stopping solution (Pestka
et al, 1980). Absorbance at 405 nm was determined on an EIA
Minireader 2 (Dynatech Product, Alexandria VA). IgA was
quantitated by extrapolation from reference curves.
Intestinal IgA was quantitated by the same ELISA
protocol with a few modifications. The sample and reference
serum volume added to the wells was 50 ul with the sample
diluted 1:2. Goat antimouse IgA peroxidase was diluted
1:3000 in 1% BSA-PBS with a 50 ul volume added to the well.
Cholera toxin and casein specific immunoglobulin
quantitation. One (1) mg of cholera toxin from Vibrio
cholerae (Sigma Chemical, ST. Louis MO) was dissolved in 1.0
m1 sterile distilled water. After dilution and filter
sterilization, 0.2 ml was gavaged orally with 20 gauge
intubation needle at a dose of 10 ug/mouse. Cholera toxin
and casein antibody titers were measured by ELISA. In this
method, 50 ul of 10 ug/ml cholera toxin, casein, or goat
antimouse immunoglobulin (alpha or gamma chain specific)
diluted in 0.1 M bicarbonate buffer (pH 9.6) were adsorbed
onto microtiter strips (Dynatech laboratories, Chantilly, VA)
by incubation overnight at 4°C. Following incubation plates
were washed 3 times with PBS-Tween and then blocked with 1%
ovalbumin in PBS (OA—PBS) for 1 hour at 37°C to prevent non-
specific binding. After washing 4 times, 50 ul of serum
sample diluted 1:10 in 0.1 M PBS were added to the wells
coated with cholera toxin or casein. At this same time, 50
ul of serially diluted reference serum were added to anti-

immunoglobulin wells which were flanked by the cholera toxin

15

and casein wells. These plates were incubated 1 hour at
37°C. After washing 5 times with PBS-Tween, 50 ul of goat
anti-mouse immunoglobulin peroxidase conjugate diluted in 1%
OA-PBS (IgA : 1:500; IgG : 1:750) were added to all wells of
plate and incubated 30 minutes at 37°C. The plates were then
washed 6 times. Bound peroxidase was determined by
incubating 100 ul ABTS-HZOZ substrate in each well for 5-30
minutes, and the reaction was terminated with 100 ul stopping
solution (Pestka et al, 1980). Absorbance at 405 nm was
determined on an EIA Minireader 2 (Dynatech Product,
Alexandria, VA). Immunoglobulin was quantitated by
extrapolation from standard reference curves.

In vitro immunoglobulin production. Single cell spleen
suspensions were prepared after passing the spleen through an
85 mesh screen tissue grinder (Thomas Scientific, Swedesboro
NJ) into a petri dish. The screen was then washed with RPMI-
1640 (Sigma Chemical Company, St. Louis MO) supplemented with
20% fetal calf serum (FCS) (Gibco Laboratories), 1% sodium
pyruvate (Sigma Chemical), 2 mM L-Glutamine (Sigma Chemical),
5 x 10-5 M 2-mercaptoethanol (Sigma Chemical), 1% non-
essential amino acids (Sigma Chemical), and Penicillin (100
U/ml)-Streptomycin (100 microgram/ml) (Gibco Laboratories).
Cells were collected and placed on ice for 10 minutes to let
debris settle. The supernatant was then washed twice by
centrifugation at 450 x g for 8 minutes. Viable cells were
enumerated with a hemocytometer by trypan blue exclusion.
Spleen cell concentrations were adjusted to 2.5 x 106

cells/ml. The cell suspension (200 111) was transferred to

16

wells of flat bottomed 96-well tissue culture plates (Costar,
Cambridge MA). Mitogen (10 ul) was added to all wells except
the spontaneous control which received an equivalent volume
of sterile distilled water. The final concentration of
mitogen used per ml of culture was as follows: LPS, 20 ug/ml;
Con A, 10 ug/ml: and Con A + LPS, 10 and 20 ug/ml,
respectively. Cultures were incubated for 7 days in a 5% C02
incubator.

In Egg production of IgA, IgG, and IgM was measured by
ELISA. For this assay, 100 ul of 10 ug/ml goat antimouse
immunoglobulin (alpha, gamma, mu specific) diluted in 0.1 M
bicarbonate buffer (pH 9.6) were coated onto microtiter
strips overnight at 4°C. Following incubation, coated plates
were washed 3 times with PBS-Tween and then blocked with OMB
ml 1% OA-PBS (Sigma Chemical) for 30 minutes at 37°C. .After
washing 4 times with PBS-tween, 50 ul of supernatant or
reference serum both diluted in 20% FCS-RPMI-1640 were
incubated for 1 hour at 37°C. IgA supernatants were diluted
1:2 with IgG and IgM supernatants diluted 1:4. After washing
5 times, 50 ul of goat anti-mouse IgA.peroxidase conjugate
(diluted 1:500 in 20% FCS—RPMI-1640) was added for 30 minutes
at 37°C. Goat antimouse IgG and IgM peroxidase diluted 1:750
in 20% FCS-RPMI-1640. After washing 8 times, total

immunoglobulins were determined as described above.

RESULTS

Serum immunoglobulin profile in mice following dietary
deoxynivalenol exposure. Exposure to dietary DON for 8 weeks
has been previously shown to cause a dose-dependent decrease
in serum IgM and a dose-dependent increase in serum IgA
(Forsell et al., 1986). The minimum DON concentration to
increase serum IgA was 2 ppm, maximum IgA at 10 ppm, and
decreasing at 25 ppm DON. The first objective of this project
was to further characterize the effect of dietary DON on
serum immunoglobulin levels. Particular attention was placed
on replicating the dose effect, determining the kinetics at
the optimal dose, and evaluating the possible effect DON-
induced feed refusal has on serum immunoglobulin. To measure
this latter effect, a "restricted" control group which
received clean AIN-76A diet at levels equivalent to the mean
intake of dietary DON for the treatment group was included.

The effect of 2 and 10 ppm dietary DON on serum IgA over
an 8 week period is presented in Table 1 (Exp. 1). Serum IgA
in the 10 ppm DON group was significantly increased relative
to the restricted and ad lib control groups at weeks 2 and 4
(p<0.05). This group was elevated when compared to the
restricted and ad lib control groups at weeks 6 and 8 but the
difference was not significant due to the large amount of
variation. IgA was not significantly affected in the 2 ppm

DON group.

17

1
Table 1. Serum lgA concentrations in mice fo lowing dietary exposure to 2 and 10 ppm DON.a

b
IgA Log1otug/ml)

AA

Treatment wk 2 wk 4 wk 6 wk 8

 

2 ppm DON 2.63 s 0.23 (93) 2.66 t 0.09 (102) 2.80 s 0.11 (118) 2.88 s 0.16 (107)
2 Restricted 2.66 z 0.17 (96) 2.60 s 0.10 (91) 2.7% s 0.13 (103) 2.80 s 0.07 (85)

10 ppm DON 2.77 : 0.89d(122) 2.76 t 0.20e(161) 2.85 t 0.13 (130) 2.98 s 0.20 (141)

10 Restricted 2.63 t 0.09d(90) 2.62 t 0.06e(94) 2.76 s 0.10 (106) 2.88 s 0.22 (114)

ad lib control 2.68 t 0.09 2.64 t 0.06 2.73 t 0.12 2.85 t 0.10

a8603F1 treatment mice were fed 2 or 10 ppm DON in AIM-76A diet. (Exp 1)
bbeta shown are geometric mean a standard deviation for groups of 22 controls
and 10 treated mice. Numbers in parentheses are (ratio of means of treatment to ad lib)
x 100. Difference between treatment groups and ad lib control determined by Dunnett's
t test. Difference between treatment groups and their restricted controls determined by
the improved Bonferroni t test.

cNumbers with a superscript c are significantly different than the ad lib control

(p<0.05).

-e
Groups with identical letters are significantly different.

19

A second experiment (Exp 2) was initiated to
examine serum IgA, IgG, and IgM at 10 ppm DON using a larger
number of animals (n=16) than the previous experiment (n=10).
The 10 ppm restricted control was omitted from this
experiment since IgA in the restricted group was not elevated
in the prior experiment suggesting that decreased feed intake
did not effect serum IgA concentrations at 10 ppm dietary
DON. Table 2 presents the serum immunoglobulin
concentrations over 8 wks following 10 ppm DON exposure (Exp
2). Serum IgA was significantly elevated in the 10 ppm DON
group at weeks 4 and 8 (p<0.01) and at week 6(p<0.05) while
no effect was seen on serum IgM. Serum IgG was significantly
decreased (p<0.05) in the 10 ppm DON group at week 8.

A third study (Exp 4) was initiated to determine the
effects of higher DON levels (25 and 50 ppm) on serum
immunoglobulin concentration over time (Table 3). Serum IgA
was significantly elevated relative to its restricted control
from weeks 2 through 12 (p<0.05) and significantly elevated
relative to the ad lib control at weeks 10, and 12 (p<0.05).
Figure 2 presents the serum immunoglobulin.concentrations
after 12 weeks of 25 ppm dietary DON.

Mortality was not observed.in mice.fed.25 ppm whereas
all animals recieving 50 ppm died before wk 4 of dietary DON
feeding. This represents a toxic level of DON.

Serum IgM in 25 ppm DON treatment mice was
significantly decreased relative to its restricted and ad lib
controls from wk 4 to wk 12 (p<0.05). Serum IgG at 25 ppm

DON was significantly decreased when compared to the

20
Table 2. Serun immoglobulin concentrations in mice following dietary exposure to 10 ppm DON.a

v—v

lumunoglobulin concentration Logmwg/ml)b

.1

 

 

 

 

 

Treatment wk 0 wk 2 wk 4 wk 6 wk 8

19A
10 ppm DON 2.87 s 0.51 (138) 2.76 s 0.12 (112) 2.86 : 0.10d(126) 2.77 s 0.10c(115) 3.02 : 0.19dt145)
ad lib control 2.73 z 0.08 2.71 s 0.08 2.76 z 0.03 2.71 z 0.04 2.86 z 0.09

196
10 ppm DON 3.70 s 0.24 (126) 3.66 z 0.18 (107) 3.59 z 0.18 (91) 3.62 s 0.16 (81) 3.65 : 0.12c(81)
ad lib control 3.60 s 0.11 3.63 t 0.10 3.63 s 0.11 3.71 s 0.11 3.74 s 0.12

19H
10 ppm DON 2.30 s 0.15 (120) 2.26 z 0.10 (89) 2.32 z 0.12 (95) 2.36 s 0.10 (93) 2.39 s 0.13 (138)
ad lib control 2.22 s 0.11 2.31 s 0.10 2.34 s 0.08 2.39 s 0.10 2.25 z 0.59

v-

aNice were fed 10 ppm DON in AIM-76A diet for 8 weeks. (Exp 2)

Data shown are geometric mean 2 standard deviation for groups of 16 mice. Difference between 10 ppm DON

and ad lib control groups determined by Dunnett's t test. Values in parentheses are percent change relative to
the ad lib control.

csignificantly different than ad lib control (p<0.05)

dSignificantly different than ad lib control (p<0.01)

Table 3. Serum immnoglobulin concentrations in mice following dietary DON exposure.3

21

 

 

 

 

 

 

immunoglobulin concentration Log10(ug/ml)b

Treatment wk 0 wk 2 wk 4 wk 6 wk 8 wk 12

19A
25 ppm 0011 2.83 s 0.15 2.89 a 0.17.“ 2.86 : o.15° 2.86 z 0.22: 2.98 : o.25° 3.19 a mild 3.40 t 0.11.d
25 Restricted 2.75 s 0.09 2.69 : 0.13d 2.67 s 0.15 2.66 s 0.08 2.63 s 0.12 2.69 s 0.16 2.71 s 0.13 '
ad lib control 2.86 s 0.12 2.75 2 0.13 2.74 s 0.10 2.72 z 0.08 2.76 s 0.11 2.70 s 0.15 2.77 z 0.15

196
25 ppm 0011 3.61 s 0.17 .— 3.33 2 mild — 3.1.2 : 0.12d 3.31. s of?“

. d d d

25 Restricted 3.65 s 0.08 —— 3.45 s 0.10 --— 3.60 z 0.13 3.52 1: 0.11
ad lib control 3.68 s 0.11 --— 3.67 z 0.11 —-—. 3.84 z 0.16 3.83 z 0.18

19H

d

25 ppm 0011 2.23 s 0.17 —— 2.17 : 0.14d —— 2.17 2 0.15 2.18 : 0.17d
25 Restricted 2.18 a 0.17 —- 2.37 s 0.16 -—-- 2.44 z 0.13 2.43 z 0.21
ad lib control 2.12 s 0.11 --- 2.50 z 0.20 -—- 2.48 z 0.09 2.58 a 0.15

f

3Mice were fed 25 ppm DON in AlN-76A diet for twelve weeks. (Exp 4)

boata shown are geouetric means 2 standard deviation for groups of 6 control and 8 treated and restricted
control mice. Differences between treatment groups and ad lib control determined by Dmnett's t test. Differences
between treatment groups and their restricted controls determined by the inproved Bonferroni t test.

CSignificantly different than restricted control within immunoglobulin (p<0.05).

c‘Significantly different than ad lib control within immnoglobulin (p<0.05)

Figure

22

2.Serum immunoglobulin isotype profile following

dietary DON exposure. Mice were fed 25 ppm DON in
AIN-76A diet for 12 weeks. Data shown are geometric
means I SAL fer groups of 6 ad lib control and 8
treated and restricted control mice. Difference
between treatment and ad lib control determined by
Dunnett's t test. Difference between treatment and
restricted or restricted and ad lib control
determined by the improved Bonferroni t test.
Treatment and control groups with identical letters
are significantly different.

 

23

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24

restricted and ad lib controls from wk 4 to wk 12 (p<0.05).
Serum IgG was also significantly decreased (p<0.01) in the 25
restricted group when it is compared to the ad lib control
from wk 4 to wk 12. This indicates that depressed IgG and
IgM levels could result from decreased feed intake whereas
the elevated IgA was a specific DON-related effect.

The potent effect of 25 ppm DON on serum immunoglobulin
was replicated in a later experiment (Table 4). This
experiment (exp 5) contained a greater number of replicates
for statistical power than the previous 25 ppm experiment.
These animals also served as sources of spleens for the in
m experiments which will be discussed later. Here the 25
ppm DON group was significantly elevated (p<0.01) when
compared to the restricted and ad lib control groups at weeks
4 through 12. The DON treatment group after 12 weeks had a
serum IgA concentration that was elevated over 4-fold
relative to the ad lib control.

A fourth study (Exp 6) was initiated that measured the
long term effects of exposure to 25 ppm DON. Table 8
presents the serum IgA concentrations following 6 months of
25 ppm DON exposure. The 25 ppm DON and ad lib control
groups were present as controls for the cholera toxin
experiments which will be discussed in the antigen specific
section. The 25 ppm DON group was significantly elevated
(p<0.01) from week 4 to week 24. Serum IgA concentrations
steadily increased over time (Fig 3). After 24 weeks the 25
ppm DON group was elevated over 17-fold relative to the ad

1 ib control.

25

Table 4. Serum IgA concentration in mice following dietary
exposure to 25 ppm DON

b
ISA Losmws/ml)

 

Treatment wk 0 wk 4 wk 8 wk 12

 

25 ppm 0011 2.80 z 0.15 3.00 z 0.13“ 3.22 : 0.57.d 3.38 : 0.23.5d
25 Restricted 2.75 s 0.07 2.72 s 0.08 2.69 s 0.04 2.89 z 0.09

ad lib control 2.78 s 0.11 2.76 s 0.09 2.68 s 0.08 2.77 s 0.09

 

aNice were fed 25 ppm DON in AlN-76A diet for 8 weeks. (Exp 5)

bData shown are geometric mean 2 standard deviation

for groups of 11 ad lib control, 12 treated, and 11 restricted
control mice. Difference between treatment and ad lib control
determined by Dunnett's t test, and difference between treatment

and restricted controls determined by the improved Bonferroni t test.

cSignificantly different than restricted control (p<0.01).

dSignificantly different than ad lib control (ps0.01).

26

Figure 3. Serum IgA following long term dietary DON exposure.
Mice were fed 25 ppm DON in AIN-76A diet for 24
weeks. Data shown are geometric means (ug/ml) :
S.D. for groups of 13 mice. Difference between
treatment and ad lib control group determined.by
Dunnettfls t test. Treatment and control groups are

significantly different from week 4 to week 24
(p<0.01).

27

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28

Salivary IgA concentrations in mice following dietary
deoxynivalenol exposure. Since IgA serves a critical role as
a mucosal immunoglobulin, it was of interest to know the
salivary IgA concentrations following exposure to this
dietary antigen. The immunoglobulin concentrations were
measured in the saliva of Exp. 1 animals following 2 and 10
ppm dietary DON and are presented in Table 5. The 2 ppm DON
group was significantly elevated (p<0.05) compared to its
restricted control after 6 weeks of feeding. The 2 and 10
ppm DON groups were elevated relative to their restricted and
ad lib controls at weeks 2, and 8 but were not significant
due to the large amount of variation.

The effect of dietary DON on total and antigen-specific
serum immunoglobulin response. Two studies were performed to
determine if dietary DON could increase the antigen-specific
IgA concentrations using cholera toxin and casein. Cholera
toxin has been found to be an extremely potent immunogen for
mucosal IgA responses when administered orally (Elson and
Ealding, 1984). Casein was the protein source in the AIN-76A
diet that the animals received for the duration of the study.

In the first antigen challenge study (Exp 3), mice were
fed 10 ppm DON for 14 days and then orally challenged with 10
ug of cholera toxin. Animals were continued on the DON diet
and boosted with 10 ug cholera toxin on days 28 and 41. At
day 48, animals were sacrificed and serum and intestinal
contents were collected. Table 6 presents the serum IgA

concentrations in mice following 10 ppm DON and cholera toxin

29

Table 5. Salivary IgA concentration in mice following dietary exposure to 2 and 10 ppm DON.a

 

 

b
IgA L09 (US/ml)
10
Treatment wk 2 wk 4 wk 6 wk 8
2 ppm DON 1.09 s 0.36 (182) 0.77 s 0.35 (105) 1.05 s 0.326(195) 1.15 s 0.43 (129)

2 Restricted 0.86 s 0.37 (107) 0.64 a 0.40 (78) 0.75 s 0.30c(98) 0.91 s 0.25 (74)
10 ppm DON 0.95 s 0.36 (132) 0.74 s 0.32 (98) 0.79 s 0.32 (107) 1.09 s 0.21 (112)
10 Restricted _ 0.77 s 0.32 (87) 0.52 s 0.15 (59) 0.74 s 0.34 (95) 0.94 s 0.31 (79)

ad lib control 0.83 z 0.24 0.75 a 0.27 0.76 s 0.30 1.04 s 0.36

aD6C3F1 treatment mice were fed 2 or 10 ppm DON in AlN-76A diet. (Exp 1)
bData shown are geometric mean 2 standard deviation for groups of 22 control and
10 treated mice. Numbers in parentheses are (ratio of means of treatment to ad lib) x 100.
Difference between treatment groups and ad lib control determined by Dunnett's t test.
Difference between treatment groups and their restricted controls determined by the
improved Bonferroni t test. -

cGroups with identical letters are significantly different (p<0.05).

30

Table 6. Serum IgA in mice following exposure to 10 ppm DON and oral
cholera toxin challenge.

b
1911 1.0910 (us/ml)

—v

 

Treatment Day 0 Day 14 Day 35 Day 41 Day 48
ad lib control 2.72 s 0.07 2.71 s 0.10 2.68 s 0.05 2.68 z 0.11 2.74 s 0.08
10 ppm 0011 2.69 a 0.08 2.79 : 0.10° 2.79 : 0.15c 2.77 s 0.12 2.77 z 0.05

cholera control 2.68 e 0.09 2.68 s 0.05 2.75 s 0.06 2.70 a 0.09 2.85 s 0.15

cholera-10 ppm DON 2.74 s 0.08 2.82 s 0.89d 2.81 s 0.14 2.70 s 0.12 2.89 s 0.15

w

'Nice were fed 10 ppm DON DON in ATN-76A diet for 48 days. Cholera toxin treatment
animals were gavaged with 10 ug cholera toxin at day 14, and boosted on
days 28, and 41. (Exp 3)

bData shown are geometric mean values a standard deviation for groups of

13 control, 12 10 ppm, 10 cholera control, and 15 cholera-10 ppm DON mice.
Difference between treatment and control groups determined by Dunnett's t test.
Difference between treatment groups and their matched controls determined by the
Bonferronit test.

cSignificantly different than ad lib control (p<0.05).

d
Significantly different than matched cholera toxin control (p<0.05).

31

challenge. Unfortunately the DON effect on serum IgA was
minimal in this experiment. The 10 ppm DON group was
significantly increased (p<0.05) relative to the ad lib
control at days 14 and 35. IgA concentrations in the cholera-
10 ppm DON group were higher (p<0.01) than the ad lib control
at days 14,35, and 48 and higher than the cholera control at
day 14 (p<0.05). Table 7 presents the intestinal IgA
concentrations ‘following 10 ppm DON and cholera toxin
challenge. Intestinal IgA concentrations were higher in the
cholera-10 ppm DON group than the cholera control however
this increase was not significant due to the large amount of
variation.

Serum.IgA concentrations were measured in mice that were
fed 25 ppm DON and challenged with cholera toxin (Exp 6).
Mice were fed DON for 5 weeks and then challenged with
cholera toxin at wk 5, and boosted at weeks 7, and 21. Table
8 reveals that serum IgA was elevated (p<0.01) in the
cholera-DON group relative to the cholera control and to the
ad lib control at weeks 4, 8, and 12. Cholera challenge alone
did not significantly alter the total serum IgA
concentration, nor did it act synergistically with DON to
increase the serum IgA concentration.

Cholera toxin and casein-specific serum IgA were
measured in the 25 ppm DON-cholera toxin experiment (Exp 6).
Table 9 and Fig. 4 present the ELISA titers for cholera toxin
-specific IgA at weeks 8 and 21. Cholera toxin-specific
antibody was measured at week 20 for the ad lib control and

25 ppm DON group, and at week 22 for the cholera toxin and

32

Table 7. Total intestinal 19A of mice following dietary DON and oral
cholera toxin challenge.

b
IgA Log1otug/ml)

Treatment control 10 ppm DON cholera control cholera-10 ppm DON

4—

Day 48 1.29 s 0.48 1.22 s 0.39 1.13 s 0.53 1.39 s 0.44

___V_

.Mice were fed 10 ppm DON in AlN-76 diet for 48 days. Cholera toxin treatment
animals were gavaged with 10 ug cholera toxin on day 14, and boosted on
days 28, and 41. (Exp 3)

b

Data shown are geometric mean a standard deviation for groups of

13 control, 12 10 ppm, 10 cholera control, and 15 cholera-10 ppm DON mice.
Difference between groups determined by Student's t test.

33

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Figure 4. Cholera toxin -specific equivalent IgA and IgG
concentrations following dietary DON and cholera
toxin challenge. Mice were fed 25 ppm DON for 24
weeks and challenged with 10 ug cholera toxin/mouse
at wk 5, and boosted at wks 7 and 21. Values
determined by ELISA. Absorbances for immunoglobulin
specific antibody were equated to absorbances from
dilutions of mouse reference serum run concurrently
and equivalent concentration values were assigned.
Data shown are geometric mean (ng/ml) _-I_- S.D. for
groups of 13 mice. The cholera-25 ppm DON group had
14 mice. Difference between treatment and ad lib
control determined by Dunnett's t test. Difference
between DON treatment and matched control
determined by Bonferroni t test. Bars with
identical letters are significantly different
(p<0.01).

36

 

 

 

 

 

 

 

 

 

 

 

m
4 - 1
"u“ , P N
"M“ 1 .4.
.4. .4. w
.4. .4.
.4. .4.
.4. .4.
.4. .4.
x x H N
4 .4
x a a .1...
.4. c .4.
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. . .4. r... 1
.M. .4. u o m n
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V.‘ V b
.M. m o. o m
.4. .
m a L.’ § r1
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1 m
o m _ 1 .1
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11.0.91. 11
1 N.

200 E
on
. Amwwummmoo. 9.6.0
2%... 820m0 g
0 as .w 8
.888 M

 

(101/60) 0L6o sad
'1 61 01p s 13
O

37

cholera toxin-25 ppm DON group. The means of these groups
were statistically analyzed by Dunnett's t test and grouped
together as week 21 in figure 5. Cholera toxin specific
IgA levels in the immunized DON treated animals were elevated
(p<0.01) 5 fold at week 21 when compared to the ad lib
control. Interestingly, cholera toxin specific IgA at this
time was not elevated in the DON group that was challenged
with cholera toxin when compared to the cholera control.
Both cholera toxin groups were elevated (p<0.01) relative to
the ad lib control at weeks 8 and 21. The cholera toxin-
specific IgA titer in the unimmunized control fed 25 ppm DON
was significantly elevated relative to the ad lib control.
Casein-specific IgA data were pooled at weeks 20 and 22
in the same manner as the cholera toxin specific IgA (Fig.5).
Following 21 weeks of dietary DON, casein-specific IgA was
significantly elevated (p<0.01) in the 25 ppm DON treatment
groups relative to their ad lib controls for both cholera
toxin immunized and unimmunized groups (Table 9 and Fig. 5).
A 32-fold difference in DON-treated animals was observed in
immunized animals relative to the ad lib control. This same
profile was seen at week 8 in the immunized groups. The
antigen-specific equivalent IgA level and total IgA
concentration in the intestine are presented in Table 11.
The casein-specific equivalent IgA level and the total IgA
concentration were increased (p<0.05) in the cholera toxin-25
ppm DON group, however the latter was not significant.
Cholera toxin-specific equivalent IgA level was decreased in

mice fed 25 ppm DON and challenged with cholera toxin

38

Figure 5. Casein -specific equivalent IgA and IgG

concentrations following dietary DON and cholera
toxin challenge. Mice were fed 25 ppm DON for 24
weeks and challenged with 10 ug cholera toxin/mouse
at wk 5, and boosted at wks 7 and 21. Values
determined by ELISA. Absorbances for immunoglobulin
specific antibody were equated to absorbances from
dilutions of mouse reference serum run concurrently
and equivalent concentration values were assigned.
Data shown are geometric mean (ng/ml) : S.D. for
groups of 13 mice. The cholera-25 ppm DON group had
14 mice. Difference between treatment and ad lib
control determined by Dunnetts't test. Difference
between DON treatment and matched control
determined by Bonferroni t test. Bars with
identical letters are significantly different
(p<0.01).

39

9.602,

 

 

 

 

 

 

 

 

 

 

 

a?
(lw/6u)°L60'| 6| omoeds ugesog

 

zoo 5% 81:98 8295 8
Coo £on 2225 a

zoo 8%. mm a

68:8 flu

40

Table 10. Cholera and Casein-specific serua 196 following 8 weeks of
dietary DON and oral cholera toxin challenge.

\

. . b
Antigen specific 19!) Logmms/ml)

7w—

 

Treatment Cholera toxin Casein

ad lib control 3.65 s 0.18 6.11 s 0.62

25 ppm 0011 3.48 s 0.08 4.92 s 0.94°

cholera control 5.43 s 1.09° 5.93 s 0.86
d 85",

cholera-25 ppa DON 3.59 s 0.20 5.26 s 0.

 

.Mice were fed 25 ppm 0011 in AlN-76A diet for 8 weeks. Cholera toxin treatment
animals were gavaged with 10 ug cholera toxin at week 5, and boosted at week 7. (Exp 6)

bData shown are geometric mean a standard deviation for grows of 13 mice.

The cholera-25 ppm DON groq: had 14 mice. Values determined by ELISA. Absorbances
for igG specific antibody were equated to absorbances from mouse reference serua
and equivalent values were assigned. Difference between treatment and ad lib control
determined by Dmtt' s t test. Difference between DON treatment and matched control
determined by Bonferroni t test.

cSignificantly different than ad lib control (p<0.01).

d
Significantly different than matched cholera toxin control (p<0.01).

41

Table 11. Total and antigen-specific intestinal lgA following 22 weeks of
dietary DON and oral cholera toxin challenge.

 

 

 

b

Antigen specific lgA-Log1o(ng/ml)
Treatment Cholera toxin Casein ‘ Total TgA
cholera control 3.68 s 1.18 1.70 s 0.00c 2.51 3 0.22
cholera-25 ppm pm 2.45 s 0.71 1.99 a 033° 2.66 3 0.21.

~
r—v

.aNice were fed 25 ppm DON in AlN-76A diet for 8 weeks. Cholera toxin treatment
animals were gavaged with 10 ug cholera toxin at week 5, and boosted at week 7 and 21. (Exp 6)

bData shown for antigen specific lgA are geometric mean a standard deviation
for groups of 13 mice. The cholera-25 ppm DON group had 14 mice. Values determined by
ELISA. Absorbances for lgG specific antibody were equated to absorbances from mouse
reference serum and equivalent values were assigned. Values for total lgA were
determined by ELTSA with concentrations determined from mouse reference serua.
Difference between treatments determined by Students t test

cValues with identical letters are significantly different (p<0.05)

42

relative to the cholera toxin control but this was not
significant due to the large amount of variation.

Cholera toxin and casein-specific IgA responses
correspond to the total IgA profile generated upon chronic
dietary DON exposure. Serum was analyzed for cholera toxin
and casein specific IgG at week 8 to see if this
immunoglobulin also mimics the decrease in total levels found
following DON exposure (Table 10, Fig.4,5). Cholera toxin
challenge elevates cholera toxin specific IgG (p<0.01) as has
been described previously (Elson and Balding, 1984). This
effect was suppressed when animals were fed 25 ppm DON and
challenged with cholera toxin (p<0.01). Casein-specific IgG
concentrations were also significantly decreased (p<0.05) in
animals that received dietary DON regardless of cholera toxin
challenge. Casein specific IgG levels were the same in the
cholera toxin control and in the ad lib control. These
results suggest that the dietary DON is responsible for the
decrease in casein specific IgG and not due to the co-
administration of DON and cholera toxin.

To validate the specificity of the antigenic-specific
ELISA's, fractions of serum were pooled from mice at week 22
that were treated with 25 ppm DON and challenged with cholera
toxin. Absorbance decreased 67% with 100 ug/ml free cholera
toxin in the assay. Fractions of serum were likewise pooled
after 20 weeks of 25 ppm DON and tested for casein specific
IgA. Absorbance was decreased 59% with 500 ug/ml free
casein.

I}; vitro immunoglobulin production by isolated

43

splenocytes following dietary DON exposure. In M
immunoglobulin production by isolated splenocytes was
evaluated to assess the contribution of the systemic
compartment to elevated serum IgA following DON exposure The
objective of these _i_n li_t_rg studies were to demonstrate that
the spleen was involved with the elevated serum IgA presented
in earlier in 2139 studies.

Mitogens are commonly used to induce lymphocyte
activation. LPS and Con A are B and T cell lymphocyte
mitogens respectively and were employed to stimulate the
lymphocytes and induce immunoglobulin secretion. Table 12
indicates that IgA production by isolated splenocytes was
markedly affected by DON exposure. Of 5 trials performed to
demonstrate the _i_n E1319 immunoglobulin production, trial 2
was selected as most representative of the Q y_i_trg studies
and is presented in figure 6. Here LPS stimulated
lymphocytes from DON -treated animals exhibited a 400%
increase in IgA production relative to the restricted and ad
lib control lymphocytes. This same profile was seen in the
Con A and Con A-LPS stimulated lymphocytes. Interestingly,
there was an even greater total increase in IgA production
from DON treated animals compared to the restricted and ad
lib control in spontaneous (non-mitogen stimulated)
lymphocytes. This is shown by the stimulation index in table
12 which describes the IgA produced by the combined effect of
mitogen and/or treatment relative to the spontaneous ad lib
control.

Table 13 shows in vitro IgG production by isolated

411

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soda) am. on «neocoucoomxoa.o> «coeuoocu u a._.mv woos. co_ua.:e_umo

.mco_uac_ecouop e .6 case «neonates. undo) .ouoo_oo:p c. po>ammo aces .amcu zone soc.

ozooz m soc. «accusatooam .m use .4 .n m.o_cu :— .aco_uoc_5copop a so come mucomocooc o:.a> ..a_cu some so.»
m..os e co. ounu_.o:p c_ postsccoo <m_om .~ use — e.a_cu :— ..o.m « Ao—ooov some o.cuosoou can cacao conga

~ .<m_om so (a. co. oouzdaca ace: mucouoccooam

. oo am.oo~m co memo co>oo co. coco: co .oexo: o~ no. ope mc3-< coo ...e\u: ope < coo ...e\m: o~a me.
o. oooooxo uco 84o.-_za¢-mo. uo~ c. cocoooco oco: .ooo:\_c\o..oo o. x m..aco_acooooa .ooo «oo_om
..m.o.n _m_co. coco: - co .~.. .oaca. oooo: —~ co. ao_o coo_o co zoo coo mm no. cap: ._o_co\~c oo_a .mooo

 

 

 

 

 

 

Am~.o-v Aep.~v Ase.o—v .~—.pv ahc.ne A—o.ov Apn.onv
-.o « -.~ pm—.o « a~.n no.o « o—.n pop.o « os.n w—.o « ~o.~ pmo.o « ’n.n pp.o « ~h.n poo.o « mn.e m
anm.mv A—c.pv A-.¢v amp.—v Aoo.nv Aoo.nv “om.hv
up.o « up.» poo.o « po.n mo.o « nn.n p~o.o « —Q.n p~.o « n~.n no.o « s~.n -.o « o~.n -.o « oo.e e
«~m.ov “No.-v Ae~.hv n~o.~—. “05.0. “~—.ov Aoo.pov
op.o « —m.~ po-.o « n¢.n o—.o « om.n up.o « sn.n oe.o « om.m n—.o « on.n up.o « ~¢.n poo.o « on.¢ n
An—.opv Amm.ov zoo.nv Aon.ov n¢~.nv zoo.~v Awo.opv
o~.o « mm.n pop.o « n¢.¢ n~.o « so.m pm—.o « ~o.n e—.o w po.~ pmo.o « «o.n on.o « '5.n poo.o « oo.¢ ~
. Ao~.m~v ahn.o~ aco.o—v n—n.oO n—e.hv Ano.~v. awe.n~v ua._.mv
em.o « oo.~ poo.o « on.¢ s..o « be.~ pop.o « no.n o—.o « on.~ opp.o « p~.n oo.o «.pn.n p~p.o « o~.¢ —
am. on zoo o_. no zoo o.. no zoo n_. on zoo .a_cp
11111mseocoucoom111111 mo41< cou1111111. 11111111< sou 1 moo
338.28. 8:22.88 s:

o
amouxoocos>d u_co.oo pouadom_ zo co_uu:p6co (a. ocu_> c_ co zoo >couozp co uuowmm .NF o.oop

C...
1 4 .

.9.

45

Figure 6. Effect of dietary DON on in vitro IgA production by
isolated splenocytes. B6C3F1 mice (2/trial) were
fed 25 ppm DON for 21 weeks. Restricted groups were
fed AIN—76A diet at level equivalent to mean intake
of 25 ppm DON group. Spleen cell suspensions (51:
105 cells/well) were prepared in 20% RPMI-1640 and
exposed to LPS (20 ug/ml), Con A (10 ug/ml), Con A-
LPS (10 and 20 ug/ml) or water for seven days at
37°C-5% C02. Supernatants were analyzed for IgA by
ELISA. Data shown are mean (ug/ml) : S.E.. Groups
with identical letters are significantly different
(p<0.01). Data for figure from trial 2 of table 12.

46

mnj

maoocoucoom L... :00 < :00 .

 

 

ma:

 

e... 1.3a: we:

to o

 

 

o

.988 so
.9560 popotbmoc mm a
zoo Eoo mm D

m

 

 

.9
.8
.mm
on
on
-9.
en.

 

-OP

(lw/6n) v6]

47

lymphocytes. Three experiments were analyzed for IgG with
trial 2 being most representative and presented in figure 7.
As expected, LPS- activated lymphocytes produced the largest
amount of immunoglobulin. With all the mitogens and the
spontaneous control, no significant difference was seen
between lymphocytes from DON treated animals and the ad lib
control. Contrary to the IgA profile, spontaneous IgG
production was very small.

In gInng IgM production by isolated lymphocytes is
presented in table 14. Three experiments were measured for
IgM with trial 2 shown in figure 8. IgM production was
significantly decreased in single mitogen stimulated cultures
from DON treated animals relative to the ad lib control
(p<0.05). .Again, very little IgM was produced in the

spontaneous cultures.

48

Amo.ovov .ocucou o_. to comou_e-acucm coca «cocommmp >.ucao_~_co_mp

03d.) Dmd U0 §u§m\gd0> ucguflflhu 1|. Ao—amv KO‘: Smuadgwumu

.mco_uoc_scouoo n we case as» uc_ucomocaoc oa.o> ecu :u_z poaaco>o ecu: mouoo_.a:o

.m..os n co» ouuu.oa:o c. ooscoccoo >amoa .n use ~ u_o_cu c. .acomuac_eceuop n .6 cans «accustom. o:.o>
.m..o3 e to. ouau_.a:p c. ooscowcoa zooms .- .a_cu c~ ..o.m « “owned. cane u.cuosoou ace cacao conga

~ .<m_.m >3 um~ cow po~>.ncn aces «accusatooam

. 8 smock so or... coaoo co. coco: .3 333: 8 as. o: 2.-.. :8 .359 o: < :8 .253 2.... m...
3 B893 2.. o3.-§s..8. en E 8.82.. 22. 3.2.2328 2 x .8 8388a... :8 «8.8
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“eh.ov “Fo.—u Awn.—v “No.pv Apn.ov An~.0v Amn.m—v
~F.o « mh.~ «n.o « ~o.~ co.o « no.n ¢~.o « Fo.n s—.o « po.n c—.o « oo.~ mo.o « oh.~ Np.o « mo.n n

Aoo.wv goo.nv a—n.nv aho.pv Aop.—v “oh.vpv A0¢.mpv
op.o « ce.~ mo.o « ve.~ -.o « no.~ mo.o « oa.~ op.o « s¢.~ oo.o « ce.~ ~o.o w po.n o~.o « no.n ~

 

 

 

 

 

 

 

Aom.ov A...oe .~..oe «mo.ne Apo.nv 6....mv
~m.o « so.n —o.o « eo.~ 11111 11111 n~.o « -.~ oo.o « oe.~ oo.o « mo.n no.o « bu.n p
o_. no zoo a_. on zoo o_. no zoo o_. no zOo .o_cp
maoocmucoom 11 ma.-< coo < coo 1mm.
AALexocvo—ooa co.uocucoucou on.

amou>665os>. o_co.om pouaooam zn co_uu:poco on. ocu_> cm co zoo zcoue_p we nooccm .n— o.oap

49

Figure 7. Effect of dietary DON on In vitro IgG production by
isolated splenocytes. B6C3F1 mice (2/trial) were
fed 25 ppm DON or 21 weeks. Spleen cell
suspensions (5 x 10 cells/well) were prepared in
20% RPMI-1640 and exposed to LPS (20 ug/ml), Con A
(10 ug/ml), Con A-LPS (10 and 20 ug/ml) or water
for seven days at 37°C-5% CO . Supernatants were
analyzed for IgG by ELISA. Data shown are mean
(ug/ml) : S.E.. Groups with identical letters are
significantly different (p<0.01). Data for figure
from trial 2 of table 13.

 

50

'90 (119/ ml)

 

 

 

 

 

.1974. 3% 11.2.

 

rt

m/

00: > oo: >l mooofioomocm
Cum

 

51.

Amo.ovav dagucou n_. an caucuma-oguc_ cusu acogow*_u >ducnumwmcmmmu

03;) nm— E §u§m\gdfl> HEUOOBU l Aonomv Rug: Smufldgmumu

.mco_uac_agouuu n *0 came 0:» oc_ucomogaog o:.u> as» :u_3 vomago>a «Lo: monoumdaso

.m.do: n Lo. ouaumdaju cm vaEgo*goa >ammo .n was ~ m.a_gu :— .mco_uac_agouou o vo came uucomogaou o:.a>
.m..az ¢ go» ouau__a:u c. uoELo*Loa >omma .— domgu c. ..o.m « Ac—nodv cane u_guosoou ago csozm ganja

m .<m_4m >3 to. go» no~>.aco one: mucauQCguaam

. cu am-uo~n an axon co>au Lo. Luau: Lo ..a\ua o~ ac. °.. mad-< cog ...e\ma o.. < cog ...e\u: o~v mm;
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a—m.0v Aon.o~v aom.~pv akm.~v AnF.—v Anm.p~v AnN.N—v
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Ano.ov A—o.-v Am~.F—v Ao~.ov n—o.—v a¢n.~nv A—m.ov
p..o « uo.~ no.9 « so.~ mo.c « no.q uno.o « an.n o~.o « om.n uno.o « mo.~ oo.o « n—.c unp.o « oo.n ~

A.~.cv .oo.o. Aw—.ov o~.¢v Aq~.n. ua._.m.
om.o « ~o.n o~.o « se.n III». .0511. Fn.o « mm.~ No.0 « o~.~ oo.o « on.¢ oc.c « mp.e p

a_. u. zoo n_. u. :09 n_. no zoo 3.. no zoa .._g»
Illllmaoocnucoam 'r, wa4-< con «¢ < coul14 mag-
o,
A.e\ucv no. co_uagucoucou aa—

a
amouxuogasx. u_co.aa touadoom xn cowuunuOQQ zu— Ogum> cm :0 20° >gauo.v we uuom*w .e— 0.50»

52

Figure 8. Effect of dietary DON on _i_p_ vitrg IgM production by
isolated splenocytes. B€C3F mice (2/trial) were
fed 25 ppm DON or 21 weeks. Spleen cell
suspensions (5 x 10 cells/well) were prepared in
20% RPMI-164O and exposed to LPS (20 ug/ml), Con A
(10 ug/ml), Con A-LPS (10 and 20 ug/ml) or water
for seven days at 37°C-5% CO . Supernatants were
analyzed for IgM by ELISA. ata shown are mean
(ug/ml) : S.E.. Groups with identical letters are
significantly different (p<0.01). Data for figure
from trial 2 of table 14.

 

53

 

mm

Con A- Spontaneous

LPS

 

ppm DON .

 

”WA
”*l

Control

 

[:125
m
g

Con A

”C

 

~—"’f’/////////////////////////
“H

 

 

 

lllllllrl
(DCDV'NOCDCOV'NO

(lw/bn) W5l

LPS

DISCUSSION

Immunoglobulin is a major weapon of the immune response
to infection. Immunoglobulin responses involve binding and
neutralization of infectious agents as well as interaction
with other cell types that initiate various immune reactions.
Since the greatest exposure the host has to foreign antigens
occurs in the gut, specialized lymphoid tissues and cell
networks are located here to respond to antigens thus
preventing harm to the host. Figure 9 demonstrates the major
organs involved in an immune response, and the route of
circulation of lymphocytes between the tissues (Male, 1986).
Peyer's patches are specialized pools of lymphocytes that
line the small intestine. Here specialized M cells within
the Peyer's patches function to sample antigen absorbed
through the intestine. This antigen, still fully
immunogenic, is exposed to the underlying B and T cells of
the Peyer's patch. A significant percentage of B cells in
the Peyer's patch are committed to IgA production. The
antigen-stimulated B cells migrate from the Peyer's patch
through the mesenteric lymph nodes to the thoracic duct and
ultimately into circulation. During this time, the B cells
mature to IgA producing plasma cells or memory cells which
eventually will "home" to distant mucosal tissues. Here they
produce IgA specific for the antigen that caused stimulation

in the Peyer's patch.

54

55

Figure 9.Major organs involved in an immune response and the
route of circulation of lymphocytes between the
tissues (Male, 1986).

56

 

 
 
  

Q o ' '

cl >
“u“ .--§y

d. a,

a ”I ,' '. . ‘ _ . . . ,. -"-"T.'." .
49:33....3 ;*;;§€$e.condary lymphoud organs.

 

lymph

Waldeyer's nodes.
ring tonsils and
adenoids

thymus

 

lymph
nodes

bone marrow

 

 

spleen

 

mesenteric
lymph nodes
Peyefs
patch

lymph

nodes

 

 

 

tp/

 

 

 

 

57

Immunoglobulin A (IgA) is the major immunoglobulin
at mucosal surfaces where it functions as a first line of
defense. Here it binds and neutralizes antigen before it
can be absorbed and cause harm. The function of IgA in serum
is however unclear relative to its critical role in
secretions. Immunoglobulin G (IgG) and immunoglobulin M
(IgM) are the major serum immunoglobulins whereas IgA
accounts for only 15-20% of total immunoglobulin (Roit et
al., 1985). In humans the majority of serum IgA does not
enter external secretions in significant amounts. It has
been hypothesized that IgA in serum exists only to bind
absorbed antigen that wasnfit cleared at the mucosal surfaces.

The work.presented here provides strong evidence that
DON exerts a specific effect on IgA. The decrease in serum
IgG and IgM observed here and previously (Forsell et al.,
1986) upon dietary DON exposure is consistent with a potent
protein synthesis inhibitor. The inclusion of restricted
controls demonstrated that the decrease in serum IgG can be
partially explained by decreased feed intake caused by DON.

Work performed by Forsell et alu.(1986) demonstrated
that 8-week dietary DON causes a dose-dependent increase in
serum IgA. The threshold DON level for this increase was 2
ppm, whereas maximum IgA was found at 10 ppm. IgA at 25 ppm
was significantly greater than the control but less than at
10 ppm. The purpose of this thesis was to develop a model
that could be used to study mechanisms by which dietary DON
alters serum immunoglobulin levels. The first experiment did

not show elevated IgA following 8 weeks of 2 ppm dietary DON

58

although 10 ppm DON did significantly elevate serum IgA in
experiment 2 from weeks 4 through 8. This verified the work
of Forsell et al. (1986) and demonstrated that the increase
in IgA can be seen as early as 4 weeks of dietary DON.
Subsequent experiments demonstrated an even greater increase
(17 fold at 24 weeks) in serum IgA at 25 ppm DON which
expands upon the previous study. There is a possible
explanation for the difference between DON levels where
maximum serum IgA were found in the earlier work (10 ppm) by
Forsell and that (25 ppm) reported here. The treatment
animals in the Forsel study were less robust and appeared
"more sickly" than animals in these studies (MQF. Witt,
personal communication). A prior or ongoing mucosal
infection in the animals of the Forsell study could have
conceivably caused a much larger IgA response thus making the
animals more sensitive to DON effects. IgA levels in the
control mice from the Forsell study were 2-fold higher than
an average of the control mice reported in this thesis.
Although serum IgA from the previous study was quantitated by
the ELISA method, these levels were again verified in frozen
serum samples by the RID procedure performed for this thesis.
In a preliminary experiment not reported in this thesis,
animals were challenged with sheep red blood cells and
keyhole limpet hemocyanin concurrently with dietary DON in an
attempt to increase the control levels of serum IgA. ‘Ehis
protocol was unsuccessful in elevating the control levels of
serum IgA to those reported in the Forsell study.

To better assess the affect of DON on the antigen-

59

specific IgA response, cholera toxin and dietary casein were
used as model antigens. Cholera toxin has been found to be
an extremely potent immunogen for mucosal IgA responses when
administered orally. When fed 10 ug cholera toxin, mice
exhibit cholera toxin-specific IgA in intestinal secretions
and substantial increases in cholera toxin-specific plasma
IgG and IgA levels (Elson and Ealding, 1984). When the mice
in this thesis were fed DON for 21 weeks and simultaneously
challenged with cholera toxin, there was similar elevation in
cholera toxin-specific serum IgA, but no difference was
detectable between the cholera toxin or cholera toxin-25 ppm
DON groups. It is thus apparent that the ability of cholera
toxin to elicit a serum IgA response was not effected by DON.
However cholera toxin-specific IgG concentrations were
dramatically decreased in animals receiving dietary DON.
This is likely a reflection of DON induced reduction of total
IgG.

Total IgA concentrations and antigen-specific equivalent
IgA levels in the intestine correspond to those reported in
the serum. There was a trend toward higher casein-specific
as well as total IgA in the cholera toxin-DON treated mice
than in mice treated with cholera toxin only. However this
was not significant due to the large amount of variation.
Nevertheless the results do suggest that DON treatment does
not decrease gut IgA.

Caseinrspecific serum IgA concentrations were sharply
increased in animals receiving DON. 'The opposite profile was

observed with casein specific IgG. Casein is normally

60

digested in the gastrointestinal tract and absorbed as small
peptides. These small peptides are then further metabolized
to carbon and nitrogen sources for metabolic activities in
the cell. Forsell et al., (1987) showed that acute doses of
DON resulted.in.extensive necrosis of thelgastrointestinal
tract. It is possible that increased.absorption of casein
due to DON induced increased intestinal permeation is
responsible for an increased IgA response. This is
consistent with the total IgA profile generated upon dietary
DON exposure. The decrease in casein-specific serum IgG also
parallels the total IgG profile, and like cholera toxin may
be explained as a reflection of the DON induced decrease in
total IgG concentration. Tryphonas et al. (1986) showed that
certain blood proteins were non-specifically decreased in
animals consuming DON, and one could speculate that IgG was
similarly reduced. .Again these observations may also beta
secondary effect of DON-induced feed refusal.

The observation that DON feeding increased in yitgg IgA
production by splenocytes was important because it favors the
possibility that DON affected synthesis of IgA rather than
catabolism. A large amount of IgA produced is catabilized by
the liver. Vaerman et al., (1978) proposed that in rodents,
the liver functions as an "IgA pump" that regulates serum
levels of IgA by transporting circulating polymeric IgA into
the bile. Thus blockage of catabolism might be explained to
cause elevated serum IgA. However the observation that
spleen cell cultures from DON treated mice produced elevated

IgA favors the possibility that DON was affecting the

61

regulation of IgA synthesis.

Mitogen stimulated splenic lymphocytes from mice fed
dietary DON produced much larger amounts of IgA in yitgg than
their respective restricted and ad lib controls (Fig. 6).
The total IgA produced in the DON groups relative to its
controls was larger in the spontaneous than in the LPS
stimulated group. LPS is a polyclonal B cell mitogen. One
would expect LPS-stimulated B cells to produce larger amounts
of immunoglobulin than in non-mitogen stimulated
(spontaneous) controls. It is possible splenic IgA
lymphocytes from DON treated animals were already stimulated
and did not require mitogen to stimulate B cells to produce
immunoglobulin. This could be argued by observations in the
Con A and Con A-LPS mitogen stimulated IgA cultures. Elson
et al., (1979) demonstrated that Con A-pulsed spleen T cells
added to fresh LPS-stimulated B cell cultures induced the
suppression of IgA, IgG, and IgM synthesis. The authors
point out that this suppression is mediated by T cells. Fig.
6 shows that IgA produced in Con A—LPS stimulated cultures
from DON treated animals were similarly less than that
produced in LPS-stimulated cultures from these same animals.
The suppression exhibited by Con A alone in these cultures
further argues for prior stimulation of lymphocytes in DON
treated animals. Although the preparation of spleen cell
cultures in this thesis did not involve the separation of B
and T cells as was done by Elson et al., the results of the
in 31359 studies using crude splenocyte cultures support the

observation of Elson et al. and suggests prior stimulation of

62

splenic lymphocytes from DON treated animals. A further
argument for lymphocyte stimulation by DON can be made with
the antigen-specific response data. Non-immunized mice fed
dietary DON exhibited an increased cholera toxin-specific
equivalent IgA level (Fig 4). This suggests that dietary DON
by causing an increased IgA response in a non-immunized host
might be acting as a polyclonal stimulator of IgA production.

There was no significant difference in IgG production in
cultures between DON-treated or control mice. In contrast to
IgA, there is very little IgG produced in the non-mitogen
stimulated cultures from DON treated animals. This was also
true for IgM production from spontaneous cultures of DON
treated animals. There was a trend toward decreased IgM
production in mitogen stimulated cultures from DON treated
animals but these results were significant in only one of
three experimental trials (table 13). In general these
results support the concept of isotype-specific dysregulation
of immunoglobulin production caused by DON.

The major success of the research in this thesis was the
development of a model for studying trichothecene-induced
dysregulation of IgA synthesis. Since much of the
information is preliminary it is not possible to make
specific conculusions on the mechanistic basis for this
effect. However it is possible to speculate as to possible
causes for the observations and suggest further experiments.
First, the possibility of increased permeation of the dietary
protein casein eliciting an IgA immune response was addressed

earlier in this thesis. Future experiments should further

63

investigate antigen-specific IgA production in isolated
splenic lymphocytes from the spleen and Peyer‘s patches.
This would help to determine if the DON effect represents an
antigen specific response rather than a polyclonal effect. A
second potential explanation for the increase in serum IgA
would be an increase in the number of IgA producing cells.
There are many potential ways that this could occur. One
would be through a direct effect of DON; IRecently Miller and
Atkinson (1986) demonstrated that low DON concentrations
induced the release of the lymphokine interleukin 1 (IL-1)
from peritoneal macrophage. IL-l release from antigen
presenting cells such as macrophage could initiate a cascade
of T cell activity involving the further release of
lymphokines to mediate immune activity. Murray et al.,
(1987) demonstrated that the lymphokine IL-S produced by T
cells causes secretion of IgA by B cells and that this
secretion is magnified two to threefold in combination with
IL-4. IL-4 and IL-5 are produced by the T helper 2 (Th2)
subset of T cells. Gamma interferon is produced by the Th1
subset of T cells and has been shown to down-regulate IL-S
stimulation of IgA secretion. The authors suggest that a
higher proportion of Th2 relative to Th1 cells reside in the
Peyem"s patches. The effects of trichothecenes on lymphocyte
stimulation suggest that DON may be affecting T cells and
accessory cells such as macrophage. This could ultimately
involve interleukins at the Peyer's patch level which could
theoretically alter IgA regulation. Future experiments

should examine spleen and Peyer"s patch cultures from DON

64

treated animals for an increase in interleukin activity,
specifically IL-4 and IL-5.

In summary, the data presented here demonstrate that
exposure to dietary DON increases serum IgA and concurrently
decreases IgG and IgM. The antigen-specific immunoglobulin
response to cholera toxin and casein reflects the total
immunoglobulin profile generated upon dietary DON exposure.
Splenocytes from mice fed dietary DON produced increased IgA
in mitogen stimulated and spontaneous cultures. These
results suggest that dietary DON alters the regulation of IgA
production.

The dysregulation of IgA production has been associated
with a common form of glomerulonephritis known as IgA
nephropathy. This nephropathy is characterized by
accumulation of IgA in the mesangial region of the kidney
glomerulus. IgA has also been shown to inhibit in yitrg
natural killer cell activity which attack tumor, virus-
infected, and undifferentiated normal cells. This research
demonstrated a dysregulation of IgA production in the
systemic compartment be dietary DON and provides a model for
further study of the effects of mycotoxins on the immune

system.

LITERATURE CITED

65

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