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Mucus secretagogue activity in cecal contents of rabbits with experimentally-induced mucoid enteropathy

Material Information

Title:
Mucus secretagogue activity in cecal contents of rabbits with experimentally-induced mucoid enteropathy
Creator:
Hotchkiss, Charlotte Evans, 1961-
Publication Date:
Language:
English
Physical Description:
xii, 232 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Diseases ( jstor )
Glycoproteins ( jstor )
Intestines ( jstor )
Lectins ( jstor )
Ligation ( jstor )
Mucins ( jstor )
Mucus ( jstor )
Rabbits ( jstor )
Rats ( jstor )
Secretion ( jstor )
Department of Large Animal Clinical Sciences thesis Ph.D ( mesh )
Dissertations, Academic -- College of Veterinary Medicine -- Department of Large Animal Clinical Sciences -- UF ( mesh )
Gastric Mucosa ( mesh )
Gastrointestinal Diseases -- etiology ( mesh )
Gastrointestinal Diseases -- veterinary ( mesh )
Intestinal Mucosa ( mesh )
Ligation ( mesh )
Mucus ( mesh )
Rabbits ( mesh )
Research ( mesh )
Genre:
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1994.
Bibliography:
Bibliography: leaves 207-231.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Charlotte Evans Hotchkiss.

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University of Florida
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University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
001993049 ( ALEPH )
48625815 ( OCLC )
AKH0109 ( NOTIS )

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Full Text












MUCUS SECRETAGOGUE ACTIVITY IN CECAL CONTENTS
OF RABBITS WITH EXPERIMENTALLY-INDUCED
MUCOID ENTEROPATHY


















By

CHARLOTTE EVANS HOTCHKISS


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1994




MUCUS SECRETAGOGUE ACTIVITY IN CECAL CONTENTS
OF RABBITS WITH EXPERIMENTALLY-INDUCED
MUCOID ENTEROPATHY
By
CHARLOTTE EVANS HOTCHKISS
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1994


Copyright 1994
by
Charlotte Evans Hotchkiss


To Mark, Laura, and Arthur who never let me forget what
is really important.


ACKNOWLEDGMENTS
First and foremost, I would like to thank my advisor, Dr.
Merritt for his continuous advice and support. Special thanks
also go to Dr. Moreland for support in the laboratory animal
training program that allowed me to work for this degree. In
addition, my committee members whose scepticism inspired me to
prove my point were instrumental in completing this work. I
would like to thank Dr. Jacobson and Sylvia Tucker for
allowing me to use their laboratory, computer,
photomicroscope, and supplies. Mark Hotchkiss provided help
with the photography. Finally, thanks go to Dr. Davis and the
Animal Resources Department for diagnostic laboratory support,
and particularly to Dr. Schoeb for his help with the
histopathology.
IV


TABLE OF CONTENTS
ACKNOWLEDGMENTS iv
LIST OF FIGURES viii
KEY TO ABBREVIATIONS X
ABSTRACT xi
CHAPTERS
1. INTRODUCTION 1
2. LITERATURE REVIEW 6
Introduction 6
Characteristics of Mucus 7
Physical Characteristics 7
Production 16
Secretion 21
Degradation 3 0
Postulated Functions of Mucus 33
Lubrication 33
Cytoprotection 33
Protection from Infection 35
Nutrient for Flora 42
Diffusion Barrier 44
Creation of Microenvironment 46
Techniques Used to Study Mucus 4 8
Model Systems 4 8
Quantitation of Mucus 50
Secretion of Mucus 51
Composition of Mucus 52
Diffusion Through Mucus 53
Microbial Virulence 53
Theoretical Considerations 54
Lubrication 54
Cytoprotection 55
Protection from Infection 55
Nutrient for Flora 56
Diffusion Barrier 57
Creation of Microenvironment 58
Conclusions 59
v


3. PILOT STUDIES 6 0
Introduction 60
Cecal Filtrate Collection 60
Intestinal Explants 61
Enzyme-Linked Lectin Assay 62
Data Analysis 63
Cecal Ligation 64
4. REFINEMENT OF METHODS 73
Use of Soybean Agglutinin to Quantitate Mucus . 73
Introduction 73
Materials and Methods 73
Results 76
Discussion 78
Evaluation of Enzyme-Linked Lectin Assay 79
Introduction 79
Materials and Methods 80
Results 83
Discussion 85
Harvesting of Mucus from Explants 88
Introduction 88
Materials and Methods 88
Results 89
Discussion 89
5. CECAL LIGATION AS A MODEL OF MUCOID ENTEROPATHY . 95
Introduction 95
Materials and Methods 96
Results 98
Discussion 102
6. MUCUS SECRETION FROM INTESTINAL EXPLANTS 120
Introduction 120
Materials and Methods 120
Results 123
Discussion 124
7. COMPARISON OF ELLA AND IN VITRO LABELLING 128
Introduction 128
Materials and Methods 128
Results 131
Discussion 132
8. PHYSICAL CHARACTERISTICS OF MUCUS SECRETAGOGUE . 136
Introduction 136
Materials and Methods 136
Results 139
Discussion 141
9. CONCLUSIONS 152
Future Directions 155
vi


APPENDICES
A. DATA TABLES FOR CHAPTER 3 156
B. DATA TABLES FOR CHAPTER 5 165
C. DATA TABLES FOR CHAPTER 6 171
D. DATA TABLES FOR CHAPTER 7 (RADIOACTIVITY) 185
E. DATA TABLES FOR CHAPTER 7 (ELLA) 193
F. DATA TABLES FOR CHAPTER 8 (RADIOACTIVITY) 196
G. DATA TABLES FOR CHAPTER 8, PART A (ELLA) 2 00
H. DATA TABLES FOR CHAPTER 8, PART B (ELLA) 2 04
REFERENCE LIST 207
BIOGRAPHICAL SKETCH 232
Vll


LIST OF FIGURES
Figure page
3.1. Sources of intestinal explants 66
3.2. Standard curves (ELLA) 67
3.3. Serial dilutions of control filtrates 68
3.4. Serial dilutions of ME filtrates 69
3.5. Mucus in medium/filtrates (ELLA) 70
3.6. Mucus secretion from explants 71
3.7. Site of cecal ligation 72
4.1. 6% SDS-polyacrylamide gel 91
4.2. Western blot (lectin) 93
4.3. Explant treated with N-acetylcysteine 94
5.1. Excessive colonic mucus 106
5.2. Gross cecal necrosis 107
5.3. Weight gain or loss 108
5.4. Cecal necrosis and inflammation 109
5.5. Goblet cell hyperplasia Ill
5.6. Depletion of acidic mucin 114
5.7. Colonic inflammation 117
5.8. Characteristic of cecal contents 119
6.1. Mucus in medium/filtrates (ELLA) 126
6.2. Mucus secretion from explants 127
7.1. Mucus in medium/filtrates (ELLA) 134
viii


7.2. Comparison of ELLA and tracer secretion 135
8.1. Proportion of tracer in medium/filtrate 145
8.2. Precipitable secreted tracer 146
8.3. Mucus secretion, part A (ELLA) 147
8.4. Mucus secretion, part B (ELLA) 148
8.5. Western blot (lectin) 149
8.6. Western blot (lectin) 150
8.7. SDS-polyacrylamide gel 151
IX


KEY TO ABBREVIATIONS
BSA
Bovine serum albumin
CAMP
Cyclic adenosine monophosphate
EDTA
Ethylenediaminetetraacetic acid
ELISA
Enzyme-linked immunosorbent assay
ELLA
Enzyme-linked lectin assay
H&E
Hemotoxylin and eosin
HBSS
Hank's balanced salt solution
kDa
Kilodalton
ME
Mucoid enteropathy
NZW
New Zealand White
OPD
O-phenylene diamine
PBS
Phosphate-buffered saline
PBS-T20
Phosphate-buffered saline-tween 20
PMSF
Phenylmethylsulfonyl fluoride
SBA-HRP
Soybean agglutinin-horseradish peroxidase
SEM
Standard error of the mean
SPF
Specific pathogen-free
VIP
Vasoactive intestinal peptide
x


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
MUCUS SECRETAGOGUE ACTIVITY IN CECAL CONTENTS
OF RABBITS WITH EXPERIMENTALLY-INDUCED
MUCOID ENTEROPATHY
By
Charlotte Evans Hotchkiss
August 1994
Chairperson: Dr Alfred Merritt II
Major Department: Veterinary Medicine
Mucoid enteropathy is a disease of rabbits, characterized
by copious mucus secretion in the intestinal tract. The
etiology of this disease is unknown. This study uses an
adaptation of the cecal ligation model developed by Toofanian
and Targowski to induce experimental mucoid enteropathy.
Filtrates of cecal contents from these rabbits and from
control animals were prepared. Explants from healthy rabbits
were incubated with these filtrates, and mucus secretion was
measured using an enzyme-linked lectin assay, or by measuring
secretion of radiolabelled mucus from explants that had been
preincubated with 3H-glucosamine. The results demonstrate the
presence of a secretagogue for colonic mucus in the cecal
contents of rabbits with experimentally-induced mucoid
enteropathy. Pretreatment of filtrates demonstrates that this
secretagogue can be precipitated with ammonium sulfate, and is
xi


destroyed by heat (100C for 30 minutes) or strong acid (pH 1
for 30 minutes).
Xll


CHAPTER 1
INTRODUCTION
Mucoid enteropathy (ME) is a disease that affects only
rabbits. It primarily affects young animals, but it can occur
at any age (105) Mortality from mucoid enteropathy has been
reported as 1-2% (238) to 10-20% (105, 251) of rabbits
kindled. Signs associated with the disease include anorexia,
dehydration, depression, subnormal temperature, distended
abdomen, and the defecation of clear to yellow gelatinous
mucus in the feces (238) Afflicted rabbits may also have
diarrhea, or may pass nothing but mucus (208) There is
disagreement as to whether or not affected rabbits drink
water. In our experience, and in some reports (235) they are
adipsic; but many reports describe polydipsia (238, 251).
Clinically, the rabbits are dehydrated. Afflicted rabbits may
die acutely, or after several days of illness. Affected
rabbits may recover, with or without intervention (105).
At necropsy, the consistent finding is copious amounts of
clear to yellow gelatinous mucus in the colon. The small
intestine may contain fluid and/or mucus. The cecum is often
impacted with dry digesta, which may contain gas pockets. The
gallbladder is often distended. Histologically, there may be
goblet cell hyperplasia, most noticeable in the ileum but may
1


2
occur in any part of the gastrointestinal tract. There is
minimal histologic evidence of inflammation, with mild mixed
infiltrates seen inconsistently (142, 238, 251).
Some reports of "mucoid enteritis" (142, 163) describe
gross paintbrush hemorrhages of the large intestine, and
microscopic edema and inflammation. Such lesions are more
characteristic of clostridial enterotoxemia, a more recently
defined disease of young rabbits (20), which is caused by an
overgrowth of toxin-producing clostridial organisms, C.
spiroforme or C. difficile most commonly. This disease is
usually initiated by antibiotic administration or overeating
of a high calorie, low fiber diet. Clinical signs of
enterotoxemia include anorexia and dehydration, as in mucoid
enteropathy. However, rabbits with enterotoxemia usually have
watery diarrhea and tend to die acutely. Interestingly,
animals with enterotoxemia can also pass large quantities of
mucus, suggesting that mucoid enteropathy may be a syndrome
with many possible etiologies, rather than a specific disease.
Studies of the cause and treatment of mucoid enteropathy
has thus far been unrewarding. Several studies have
concentrated on potential infectious causes, especially since
the incidence of the disease occurs in "outbreaks." Greenham
(64) found that tetracycline decreased diarrhea and delayed
death, but did not prevent or cure the disease. Van
Kruiningen (238) created an oral inoculum of macerated
intestine and contents in an attempt to transmit the disease.


3
Only 5 out of 17 of these transfaunated rabbits became ill,
while 2 out of 17 rabbits fed a control inoculum also
developed mucoid enteropathy. Intestinal coliform
concentrations are often increased in animals with mucoid
enteropathy (64, 113, 207); however inoculation with E. coli
obtained from rabbits exhibiting diarrhea, or E. coli heat
labile enterotoxin (207) failed to induce disease. Lelkes and
Chang (114) found several differences between the cecal flora
in normal rabbits and in those with mucoid enteropathy, but
could not identify a single organism associated with the
disease. They also transfaunated cecal contents from sick
rabbits directly into the ceca of healthy fistulated rabbits
and were unable to cause increased mucus secretion.
Research on ME has generally focused on determining the
inciting cause. There could very well be more than one
etiology. Individual cases have been seen in adult rabbits
with debilitating and/or stressful conditions, such as
neoplasia, surgical stress, and septicemia (Hotchkiss,
unpublished observations) The present study was therefore
designed to focus on the direct cause of the increased mucus
secretion, the one consistent finding in all afflicted
animals.
In the past, studies of mucoid enteropathy have been
hampered by the lack of viable models of the disease. There
is now one model available: based on the hypothesis that
mucoid enteropathy is primarily due to constipation,


4
Sinkovics (207) was able to induce colonic mucus
hypersecretion by surgical ligation of the colon. Toofanian
and Targowski (235) found that ligation of the blind pouch of
the cecum, permitting the flow of ingesta from ileum to colon,
was just as effective, causing mucus hypersecretion in
approximately 70% of rabbits. Partial cecectomy did not
induce a mucoid enteropathy-like syndrome, while injection of
tetracycline into the ligated cecum prevented the development
of disease (235). Little work has been done with the cecal
ligation model, however. Toofanian's group found decreased
small intestinal disaccharidase activity in ligation-induced
sick rabbits (233) and also minor alterations in cecal
volatile fatty acid concentrations (234) They incubated
colonic explants with cecal contents from rabbits with
experimentally-induced mucoid enteropathy and reported goblet
cell hyperplasia (236) .
In the project described herein, methods have been
developed to test this finding in a quantitative manner.
Evidence is presented to support the hypothesis that cecal
contents from rabbits with mucoid enteropathy possess mucus
secretagogue activity. Preliminary tests have been carried
out to determine the nature of the secretagogue.
The importance of doing research on this disease is
twofold: First, little is known about it, considering that it
is one of the most important causes of morbidity and mortality
in rabbit production (113). Since rabbits are used in large


5
numbers in biomedical research, these losses can add up to
substantial costs, especially in situations where affected
animals have been injected for antibody production with very
precious and irreplaceable antigens. Second, this disease
provides a unique opportunity to study the control of mucus
secretion. Increased mucus secretion without inflammation can
be seen in enterotoxic diseases, such as cholera and E. coli
enterotoxemia, but there is no associated goblet cell
hyperplasia (96), and there are other confounding toxic
effects, notably a secretory diarrhea. On the other hand,
goblet cell hyperplasia with mucus hypersecretion can be seen
with some diseases and parasitisms (96), but because of the
associated inflammatory changes it is difficult to examine the
mucus related aspects alone.
Although the secretagogue effects of specific chemicals
such as acetylcholine and PGE2 are well known (160, 258),
their physiological importance is not always clear. This
study is intended to demonstrate the presence of a
secretagogue that is pathologically relevant, with few effects
other than the stimulation of mucus production. It may be a
microbial product or a host secretion; in either case it could
prove to be useful for studying the processes controlling
differentiation of goblet cells, as well as production and
secretion of intestinal mucus.


CHAPTER 2
LITERATURE REVIEW
Introduction
In studying a disease in which the most consistent sign
is mucus hypersecretion, it is necessary to examine the role
of mucus in the normal physiological state. This review
presents information on what mucus is, what controls its
synthesis, secretion, and degradation, and what the normal
functions of gastrointestinal mucus are.
The function of mucus has been a subject of study since
ancient times. The ancient Greeks considered mucus, commonly
translated as "phlegm", to be one of the four humors, along
with blood, yellow bile, and black bile. In the second
century Galen (56) stated, "The best physicians concur in the
opinion that if a considerable amount of phlegm accumulates on
account of some bad conditions of the body, the most serious
abdominal disorders ensue, intestinal obstruction, lientery,
and tenesmus". Although it is now generally believed that
mucus is involved in lubrication rather than obstruction,
there is no question that increased mucus production occurs in
certain disease states.
6


7
Characteristics of Mucus
Physical Characteristics
Initially, the term mucus was used to describe any
viscous material in the stomach and intestines. Primarily,
mucus was the jelly-like material which lined the
gastrointestinal tract. However, for a time, the material
which could be precipitated from gastric juice with suitable
agents was known as "dissolved mucin" (9) However, as it was
found that this material contained a heterogenous mixture of
substances, this terminology was dropped (59). Still, mucus
was variably described as clear and colorless, white and
cloudy, and occasionally yellow. Hollander et al. (81)
determined that these differences were due to the amount of
cells and debris dissolved in the mucus layer. They were able
to show that the irritants used to stimulate mucus secretion
resulted in desquamation of epithelial cells, associated with
inflammation (80).
Pilocarpine injected intravenously causes increased
secretion of mucus of egg-white consistency; however, an
enormous dose is required (44) In 1949, Morton and Stavraky
(153) found that injection of acetylcholine into the
mesenteric arteries caused secretion of mucus, while Janowitz
et al. (92) found that topical administration of acetylcholine
resulted in secretion of clear, cell-free mucus, facilitating
the study of "pure" mucus. It is now generally accepted


8
terminology that mucin is a specific glycoprotein, while mucus
is a heterogenous mixture containing mucin, protein, lipids,
nucleic acids, water, and electrolytes. Specifically, mucus
contains trefoil-type protein, kallikrein protease, lactose
binding lectins, vitamin B12 binding protein, and other
proteins which are secreted with mucin from the same granules
in the goblet cell (178). In the respiratory tract, most of
the lipid in the mucus is bound to the mucin glycoprotein
(157) Also, nucleic acids entwine other proteins with mucin,
making acidification and nuclease treatment necessary in the
purification of mucin (157). In the digestive tract, the
mucus layer also includes bacteria, food particles, and
products of digestion. In disease states, such as cystic
fibrosis, mucus also contains materials from degenerated
leukocytes, especially DNA and actin which contribute to its
viscosity, and interfere with clearance (239) .
The most striking feature of mucus is its jelly-like
nature. Early studies revealed that unlike most proteins,
which show minimum viscosity at the isoelectric point, gastric
mucin shows a maximum viscosity at its isoelectric point of
4.98 (144). As rheological studies have become more
sophisticated, it has been shown that mucus is a visco-elastic
gel; and that the elastic component is greater than the
viscous component over the entire range of frequency and
strain studies with native and reconstituted mucus (4). The
elastic properties will be decreased, allowing the gel to


9
collapse if the mucin is treated with proteolytic enzymes or
if disulfide bonds are reduced with mercaptoethanol (4). In
addition, proper hydration is necessary for maintenance of the
gel. Hydration is controlled by small polyionic proteins, pH,
and electrolytes within the gel (242). Since mucin is a
negatively charged glycoprotein, a high concentration of a
positively-charged ion such as calcium can result in
condensation of the mucus gel, with an increase in viscosity
and lack of elasticity.
The chemical properties of mucin have been slowly
elucidated over the past century. Mucins are a heterogeneous
group of glycoproteins, with an average subunit molecular
weight of 2 x 10s (220). Carbohydrate comprises approximately
80% of this molecular weight, with the rest being protein.
The protein forms a core, with linear and branched
carbohydrate side chains covalently attached to form a "bottle
brush" structure (126). As well as heavily glycosylated
areas, there are believed to be "naked" areas on the protein
core, which are subject to cleavage by proteolytic enzymes
(4). These regions also contain cysteine residues, allowing
for polymerization via disulfide bonds (160), and are the
major antigenic determinants of mucin (131). The glycosylated
portion of the protein core contains a high proportion of
serine and threonine, which are involved in oligosaccharide
linkage via hydroxyl groups (220). In addition, there is a


10
high percentage of proline, which prevents a-helix formation,
allowing for increased glycosylation and flexibility.
The oligosaccharide chains of mucin are attached to the
protein core via an O-glycosidic bond between serine or
threonine and N-acetylgalactosamine (220) These chains may
be linear or branched, and vary in length from 2 to 12
residues. Some chains will terminate in A, B, H, Lewis3, or
Lewis13 antigens, reflecting the blood group genes of the
individual (41) The backbone of each chain contains N-
acetylgalactosamine, N-acetylglucosamine, and galactose, which
may comprise I or i antigens (126) The chains often
terminate in a fucose, sialic acid, or ester sulfate residue.
The glycosylation pattern depends on genetics, the region of
the gastrointestinal tract, the age of the animal, and can
vary in certain disease states, as determined by the
glycosyltransferase activity levels (228) There is also a
great deal of heterogeneity in a single preparation, even
within a single granule of a goblet cell (220) .
The DNA sequences of several mucins have been determined
(116) These genes show a high degree of polymorphism due to
variable numbers of tandem repeats. The gene MUC1 encodes a
mucin-like cell surface glycoprotein which is present on many
epithelial cells, but MUC2 to MUC6 appear to encode secreted
mucins. MUC2 and MUC4 are associated with colonic mucins, and
MUC3 and MUC5 are associated with gastric mucins (116) .


11
For several years it was debated whether or not mucin
contains a "link" glycopeptide, of approximately 118 kDa
(160). Roberton et al. (188) demonstrated that this
glycoprotein is different from other mucin subunits, in that
it contains only 50% carbohydrate, and it contains mannose,
which is generally associated with N-linked glycosylation
(220) Amino acid composition, carbohydrate composition, and
immunological studies have indicated that the "link"
glycopeptide is actually a 118 kDa fragment of fibronectin
(210). However, the cDNA for this peptide has recently been
sequenced in rats (257) and humans (256) and the 118 kDa
glycopeptide appears to represent the cysteine-rich carboxyl
terminal of a much larger mucin-like peptide. It is now
generally accepted that proteolytic cleavage of the mucin core
peptide occurs during purification, and that reduction and
alkylation allows for separation of the larger N-terminal
fragment and the 118 kDa C-terminal fragment (262) .
In this chapter, there has been some generalization in
order to describe the overall functions of mucus. However, it
is important to recognize that mucin molecules are
heterogeneous, and some of the characteristics of mucus vary
along the length of the gastrointestinal tract, change during
development and maturation, and can be altered by disease
(160) In fact, a diurnal rhythm to mucus secretion has been
seen in rats and mice which were given access to food only in


12
the daytime: there was more cell proliferation, and more mucus
was made during the daytime (73).
The mucin produced in newborn rats is different from that
found in adults (160). Newborn mucin contains more protein
and less sugar, resulting in increased density (216) The
threonine content is increased, while serine and glutamic acid
content are decreased in newborn rats (160). Sulfation
decreases in the first 2 months of a rats life, with the major
change occurring at weaning; increased sulfation has also been
associated with mucus from "immature" cells (160) .
Correspondingly, the pH within the mucus layer in the colon is
lower in suckling rats than in weanling or adult rats (193) .
Aged (350 day old) rats also have increased sulfation of mucus
(225) .
Newborn rat mucin has fewer fucose and N-
acetylgalactosamine residues, corresponding to altered lectin
binding (216) This likely relates to developmental changes
in glycosyl-transferase activities (12). During the suckling
period, N-acetylgalactosaminyl-transferase activity remains
constant, sialyl-transferase activity decreases just prior to
weaning, while fucosyl-transferase activity decreases
gradually throughout the suckling period. Following weaning,
the activities of all three enzymes increase dramatically. In
a parallel manner, more sialyl-transferase can be detected
immunocytochemically in the mucus of mature goblet cells, when
compared to immature goblet cells in the colonic crypt (226) .


13
Sialylation of surface glycoproteins is replaced by
fucosylation at weaning, but sialylation of goblet cell mucus
continues (227).
The composition and structure of mucin varies along the
length of the gastrointestinal tract. The data on the actual
differences are sometimes contradictory, due to different
techniques, species differences, and the heterogeneous nature
of all mucins (160). In general, colonic mucin is more
aggregated, and contains more protein. It may not contain the
fragment that was formerly known as the "link" glycopeptide.
The concentration of acidic glycoproteins, containing sialic
acid and/or sulfate, relative to neutral glycoprotein
increases distally in the digestive tract. The gastric and
Brunner's gland secretions are largely neutral, lacking sialic
acid, but enriched in fucose, although in gastric mucin there
is a discrete subpopulation of highly sulfated mucin
associated with the mucous neck cells (90).
The viscosity of gastric mucus is greater than that of
duodenal mucus, while the elasticity is decreased (263) In
the stomach, there is a continuous layer of mucus, but the
thickness is variable, and tends to be thickest along the
lesser curvature (165). Mucus is present in the small
intestine, but mostly in the intervillous spaces, so that the
tips of the villi are sometimes visible. There is less mucus
in the ileum than in the duodenum and jejunum, so that most of
the villi are bare in vivo (165) although a layer 50-200¡jl


14
thick can be seen in explants (67) There is no distinct
mucus layer in the cecum (165, 194). In the proximal colon
there is a discontinuous layer of mucus, varying in
composition and thickness, while in the distal colon there is
a thin, compact, continuous mucus layer present (194, 225).
Mucus from the healthy colon is more hydrophobic than ileal
mucus; however, induced colonic inflammation decreases the
hydrophobicity (124).
Smectite is a cytoprotective agent that binds to mucus to
increase its barrier properties. It also causes gastric mucus
cells to produce mucus that contains more fucose and less
sulfate, as is seen with fasting (151) Dietary fiber has
been shown to increase the rate of turnover of intestinal
mucins, and also change their lectin-binding properties,
reflecting a change in composition (152). This is consistent
with the fact that alterations in glycosyltransferase
activities are seen with different diets (12).
Mucus can be abnormal in various disease states.
Although in cystic fibrosis the major mucus abnormalities are
likely decreased hydration (160, 242) and the presence of
degenerated leukocyte components (239), some investigators
have also found an increased fucose content (231) Celiac
disease (gluten-related enteropathy) results in a decreased
number of goblet cells, and a shift from neutral to acidic
mucin (160). Atrophic gastritis is associated with decreased
epithelial mucus, either due to decreased secretion or


15
increased degradation (135). In mucoid enteropathy of rabbits
there are increased numbers of immature colonic mucins with
heterogeneous lectin binding patterns, perhaps due to the
increased rate of turnover (89). Abnormal mucins are also
seen in infection of rats with Nippostrongylus brasiliensis
(88) .
Goblet cell numbers are markedly reduced in patients with
ulcerative colitis (160). In addition, there is a change in
the composition, with decreased fucosylation and sulfation,
and exposure of galactose residues. This, along with deficient
O-acetylation of sialic acid (94), is suggestive of incomplete
mucin glycosylation. Intestinal malignancy is also often
associated with loss of terminal sugars, decreased O-acylation
of sialic acid, and shortened oligosaccharide chains (160).
Consequently, the presence of small intestinal mucin antigen
(SIMA) in the colon, where there is normally masking of
antigens by sulfate, may reflect malignancy (43) SIMA can be
detected in the serum of 36% of colorectal cancer patients vs.
5% of controls, and may be useful as a screening test (179).
The abnormal glycosylation appears to be a clonal phenomenon,
reflective of somatic mutation (55). Gastric adenocarcinomas
are abnormally rich in sulfomucin and sialic acid, which may
reflect cellular dedifferentiation, or abnormalities in the
regulation of mucin biosynthesis (160) .


16
Production
Mucus is formed by several cells in the gastrointestinal
tract. In the stomach, mucus is secreted by surface cells,
the mucous neck cells of the fundus, and the mucous cells of
the cardiac and pyloric glands. In the duodenum, the mucus-
secreting cells are the Brunner's glands. Throughout the rest
of the intestine the goblet cells are the source of mucus
(47) Goblet cells are named for their characteristic goblet
shape seen in histologic section, but electron microscopic
studies on cryofixed tissues show that these cells are
columnar in vivo (84, 242).
Duthie (38) reported that the mucus granules are first
formed near the nucleus, then are transported to the Golgi
apparatus, where stainable mucus first appears. This is
consistent with current knowledge that the peptide backbone,
which comprises less than 50% of each mucin molecule, is
formed in the endoplasmic reticulum. Sugar residues are then
added by the linkage of N-acetylgalactosamine to serine or
threonine (19) Unlike N-linked glycosylation that is
initiated in the endoplasmic reticulum, 0-linked glycosylation
occurs entirely within the Golgi and condensing vacuoles (63).
The initial enzyme involved, al,3N-acetylgalactosaminyl-
transferase, is located in the Golgi apparatus (191). The
enzyme responsible for terminal sialylation, S-galactoside
oi2,6-sialyltransferase, is present in the Golgi apparatus, but
also in post-Golgi apparatus structures, including the mucus


17
droplets and the plasma membrane (228). The mucin granules
are stored in condensed form in membrane bound vesicles within
the goblet cells until the mucus is secreted. During goblet
cell migration along the middle half of the villus, the mucin
granules are apparently renewed twice (23). The composition
of mucin in the granules is different along the crypt-villus
axis (23, 160); perhaps there is continued sialylation of
mucin within the storage granules.
The rate of mucin synthesis depends on the rate of
protein synthesis within the cell, the concentration and
positioning within the cell of glycosyltransferases, and the
presence of amino acid and sugar precursors (16 0) Glutamine
is known to stimulate glycoprotein synthesis as the major
metabolic substrate in the intestinal epithelium (51), but
little is known about the control of internal cellular
factors.
Mucus synthesis can be decreased by factors which
interfere with protein or oligosaccharide incorporation.
Factors which interfere with protein synthesis will also
decrease mucus production. These include malnutrition, and
metabolic inhibitors such as cycloheximide and puromycin
(160) Dietary fatty acids, non-steroidal antiinflammatory
drugs, and zinc diminish sulfation or interfere with
incorporation of individual sugars (51, 160) and may thereby
alter the composition of mucus. Cysteamine, which is used to


18
experimentally induce duodenal ulcers, decreases glycoprotein
production in Brunner's glands (104).
S-Adrenergic drugs, cyclic AMP, and theophylline have
been shown to increase overall glycoprotein synthesis in rat
and rabbit intestine (50, 110). Adrenergic compounds
(dopamine, epinephrine, isoproterenol, phenylephrine) have no
direct effect on mucus secretion, although they may increase
fluid secretion, making the mucus blanket appear thicker
(160) Cholinergic drugs and cholera toxin increase both
synthesis and secretion of mucus (198). Reserpine, a
hypotensive agent and carcinogen, increases glycoprotein
synthesis; the mucin that is released is more viscous than
normal, so that reserpine-treated rats have been used as a
model for cystic fibrosis (169) Epidermal growth factor has
been shown to increase glycosaminoglycan synthesis, and has a
protective effect on the gastric mucosa, but its relationship
to mucus secretion has not been studied (209) .
Histamine increases mucin synthesis in the canine stomach
via H2 receptors, through activation of adenylate cyclase and
increases in cAMP (198). Gastrin increases mucus synthesis in
the corpus of the rat stomach, and this effect is not blocked
by H2 blockers (86). Conversely, the H2 blockers roxatidine
and FRG-8813, but not cimetidine or ranitidine, increase mucin
synthesis (87). Additionally, the H+,K+-ATPase inhibitor NC-
1300-0-3, but not omeprazole, stimulates mucin synthesis (87) .


19
Anatomic alterations which affect the number of goblet
cells present and/or the rate of development will also change
the amount of mucus synthesized. Duodenal epithelial cells
from chick embryos show an increased number of goblet cells
when cultured in vitro compared to the number seen in vivo
(13). This increase in goblet cells is accelerated by
thyroxine, but prevented by hydrocortisone. Vitamin A
deficiency causes atrophy of goblet cells in salivary glands,
trachea, and small intestine (30). Salmonella infection in
mice decreases the number of goblet cells, apparently via
tumor necrosis factor a (8) Feeding of dietary fiber has
been associated with increased turnover of jejunal mucins
(152) Some substances which damage the intestinal
epithelium, such as methotrexate, will decrease the total
number of goblet cells, and therefore decrease mucin synthesis
(95). Following radiation damage (154), or the radiomimetic
disease caused by canine or feline parvovirus infection (96),
there is an initial increase in the number of immature goblet
cells, followed by a relative decrease, and then a second
increase as the tissue recovers. Corresponding changes in
mucus production take place following radiation exposure
(201) A decreased proportion of goblet cells is seen in
lesions of transmissible colonic murine hyperplasia caused by
Citrobacter freundii biotype 4280, and is followed by goblet
cell hyperplasia during recovery (10).


20
Several organisms have been associated with goblet cell
hyperplasia leading to mucus hypersecretion, including
Treponema hyodysenteriae in swine, and Ostertagia and
Oesophagostomum species in ruminants (36, 96). These
organisms are also associated with a varying degree of
inflammation, so the hyperplasia may be a response to
inflammation, repair, or the organism.
Nippostrongylus braziliensis infection in rats causes
goblet cell hyperplasia as the worms are being expelled (147).
This appears to be associated with the immune response, since
hyperplasia will be seen earlier in immunized rats, and does
not occur if the rats are treated with antihelmintics early in
the course of infection. Trichinella spiralis, Nematodirus
bat tus, Trichostrongylus tenuis, and Hymenolepis dimunuta have
all been associated with goblet cell hyperplasia (111, 139).
Yersinia enterocolitica has been shown to cause goblet
cell hyperplasia and increased mucin synthesis throughout the
intestinal tract of rabbits (129) There is also a great deal
of inflammation associated with this disease. The goblet cell
hyperplasia develops more rapidly and to a greater extent in
those areas of the intestine where mucosal injury is most
severe, and consequently may be associated with injury and or
inflammation. Alternatively, the hyperplasia may be part of
a repair mechanism, since it persists as the mucosa recovers
morphologically.


21
Intestinal coccidiosis caused by Eimeria species has been
associated with inflammation and goblet cell hyperplasia in
rabbits (114). Mucoid enteropathy, the subject of this study,
is characterized by copious production of intestinal mucus,
where the major histologic change reported is goblet cell
hyperplasia, and inflammation is minimal (238) .
On the other hand, it is important to realize that goblet
cell hyperplasia in response to infection and/or inflammation
is not a universal phenomenon. Mucus secretion is decreased
in both small and large intestines during infection with
Isospora suis in piglets (106, 107), and numbers of colonic
goblet cells are decreased with Ehrlichia risticii in horses
(187). Clostridium difficile toxin A had no effect on goblet
cells in rabbits (118) .
Secretion
Mucus is secreted from intestinal goblet cells in two
ways. "Baseline", or constitutive, secretion, involving the
slow transport and secretion of glycoproteins that can be
documented by autoradiography, occurs in mucosal explants for
up to 24 hours in the absence of circulating factors or
enteric nerves. In fact, baseline secretion occurs even in a
cultured goblet cell line (173) While the bulk of the mucin
granules remain in the center of the goblet cell after
formation, there is constant formation of mucus at the
supranuclear region of the cell. This mucin migrates along


22
the periphery towards the apical border of the cell over the
course of 4 to 6 hours (184). Thus, some newly formed mucin
reaches the apical border and is secreted before older mucin
granules (23). A single granule will undergo exocytosis,
discharging mucus into the lumen. This process apparently
involves the cytoskeleton, as depolymerization of microtubules
with colchicine prevents this migration (219) However, once
the granule has reached the apical membrane, the act in
filaments localized there normally act as a barrier, since
depolymerization of actin filaments with cytochalasin D or
dihydrocytochalasin B results in increased baseline secretion
(167) Organelles are gradually shed from goblet cells as
mucin is secreted, so that the mean cell volume decreases
along the crypt-villus axis (184).
In the cases where it has been examined, stimulated
secretion has been shown to occur by compound exocytosis
(217) In this situation fusion of the initial mucin granule
with the plasma membrane is rapidly followed by tandem fusion
with the subjacent granules, allowing the contents of many
granules to exit through a single surface site. More recent
studies suggest that there is also fusion of granules within
the cell prior to exocytosis (84, 158) Cavitation in a
goblet cell that has just discharged its granules in this
manner can be recognized by electron, or even light,
microscopy (217). Following cholinergic stimulation,
cavitation is visible in crypt cells for 15-30 minutes; after


23
that it is difficult to distinguish mucin-depleted goblet
cells from epithelial cells (178). Cavitation may never be
apparent in villous goblet cells (178). In rabbit colonic
goblet cells stimulated by acetylcholine, compound exocytosis
is not inhibited by colchicine, implying that microtubules are
not necessary, since the granules are already at the site of
release (219). However, the protein synthesis inhibitor
cycloheximide, the microtubule inhibitor colchicine, and the
actin inhibitor cytochalasin B were all found to inhibit
cholera toxin stimulated secretion in rabbit ileal loops
(164) A jack-in-the-box mechanism for secretion has been
proposed (242) in which a small pore is opened over the
granule, water is allowed to enter and calcium can leave, with
the expansion of the granule contents causing release from the
cell.
It has been reported recently that mucus secretion is
stimulated independently by cAMP/protein kinase A and
increased intracellular calcium/protein kinase C mechanisms
(93), although previous reports state that cAMP does not
affect mucus secretion (161). In situ hybridization reveals
the calcium binding protein calcyclin preferentially expressed
in mucus-secreting cells, suggesting that calcyclin, in
conjunction with the p36 subunit of calpactin, is involved in
calcium-stimulated mucus secretion (232).
Several substances are known to stimulate mucus
secretion. Agents that disrupt the mucosal barrier, such as


24
mustard oil, alcohol (160), bile salts (117), and even
mechanical irritation (51) cause mucus release from surface
goblet cells. It has been shown that mustard oil causes
compound exocytosis (217). Florey (45) demonstrated that
secretion in response to mustard oil could be blocked with
cyanide, indicating that the process requires energy, and
secretion is not simply due to disruption of the plasma
membrane. Mechanical irritation has been shown to increase
levels of prostaglandins (26), which could be acting as
secondary messengers to stimulate secretion. Crypt goblet
cells are not affected, presumably because they do not come
into contact with the irritant. Proteolytic enzymes are known
to be mucus secretagogues in respiratory epithelium, but this
has not been shown in the intestine (22).
Stimulation of extrinsic autonomic nerves or electrical
field stimulation causes discharge of intestinal mucus in vivo
(175) This has been shown to be due to muscarinic
cholinergic innervation, as injection of pilocarpine
accelerates release of mucus by compound exocytosis. Vagotomy
or vagal stimulation does not affect mucus secretion in the
rabbit jejunum, implying that cholinergic control takes place
entirely within the enteric nervous system (65).
Acetylcholine-induced secretory events occur rapidly, and are
generally complete within 5 minutes (158, 178). It was long
thought that only crypt cells were susceptible to stimulation
(218), but it is now known that villous cells secrete a


25
significant amount of mucus (100), although they do not
demonstrate cavitation (178). In the rat, even crypt cells
are unresponsive to carbachol until 20 to 25 days of age,
corresponding to weaning (176). Acetylcholine (218),
pilocarpine (219), and carbachol (174) have also been shown to
stimulate mucus release in colonic explant systems.
Furthermore, it is fairly certain that the goblet cells
themselves possess muscarinic receptors, as secretion occurs
in a goblet cell line descended from a colonic adenocarcinoma
(173) The response is variable, but this may reflect the
variation of responsiveness in vivo between crypt cells and
the older surface or villous cells.
Prostaglandins, particularly PGE2, increase mucus
secretion, and perhaps synthesis (197, 198, 258). Non
steroidal antiinflammatory drugs and indomethacin decrease
mucus synthesis and secretion, and it has been proposed that
inhibition of prostaglandin synthesis may be responsible (164,
206). Glucocorticoids also decrease mucus secretion (141).
Low doses of nicotine greatly decrease rectal prostaglandin
levels and decrease the thickness of the mucus layer, while
high doses cause an increase in mucus thickness and decrease
prostaglandin concentrations less dramatically (266). Other
arachidonic acid metabolites (1eukotrienes,
hydroxyeicosatetranoic acids) act as mucus secretagogues in
respiratory epithelium (171), but do not affect rabbit colon
in vi tro (177) .


26
Gastrointestinal peptide hormones have also been
suggested as potential mucus secretagogues. Neutra et al.
(161) found no increase in compound exocytosis in rabbit
intestinal explants when stimulated with caerulein,
cholecystokinin, pentagastrin, secretin, somatostatin,
substance P, or vasoactive intestinal peptide (VIP). However,
some studies indicate that secretin increases mucus secretion
in the stomach (99), and that both secretin and the related
hormone VIP weakly stimulate colonic mucus secretion in vivo
(40) Recently, VIP receptors have been found on mucus-
secreting cells in culture (108) VIP alone does not
stimulate mucus secretion from these goblet-like cells, but
both VIP and cAMP potentiate the secretagogue effects of
carbachol. This potentiating effect of VIP has also been seen
in tracheal submucosal glands (204).
The vasoactive amine histamine causes increased colonic
mucus secretion (161), but only under nonphysiologic culture
conditions (160) Histamine has also been shown to increase
PGE2 levels, and so may act indirectly (247) A greater
amount of gastric mucus was recovered after stimulation with
serotonin (141); however, there was no morphologic evidence of
increased secretion in rabbit colon following serotonin
stimulation (160) .
Several inflammatory products of neutrophils,
macrophages, and mast cells have been shown to stimulate mucus
secretion from respiratory epithelium (70, 160) but they have


27
not been studied extensively in the intestine. There is now
increasing evidence that interleukin 1 (IL-1) increases
intestinal mucus release (27, 69) In addition, a macrophage
product, MMS-68, first isolated from the respiratory tract,
has been shown to stimulate intestinal mucus secretion (221).
Immune complexes have been shown to induce compound exocytosis
of mucin (246). Antigen challenge following oral, but not
intraperitoneal, inoculation increases mucus secretion,
suggesting involvement of mucosal immunity (109). When a
jejunal self-filling blind loop is created, causing bacterial
overgrowth, there is increased mucus secretion within the
loop, but it is decreased outside the loop (202) This
phenomenon could be the direct result of the organisms, or a
secondary response to inflammation.
Cryptosporidium parvum infection in mice resulted in an
increased amount of mucus in ileal washings (77). It is not
clear whether more mucus was actually secreted or if it was
simply dislodged more easily. If secretion was increased, it
is still not possible to tell if the effect is a direct result
of the organism, or secondary to some inflammatory mediator.
Virulent strains of Entamoeba histolytica have been shown
to increase secretion of preformed and newly synthesized mucus
glycoproteins, and also to increase mucin synthesis in rats,
in the absence of an inflammatory response (22). Intestinal
trematodes in dogs and cats have been associated with mucoid


28
inflammatory response, but no quantitative studies of mucus
secretion have been performed (96).
Cholera toxin and the heat labile toxin (LT) from
Escherichia coli both stimulate small intestinal mucus
secretion (160) This phenomenon can be separated from fluid
and electrolyte secretory effects, which are stimulated by
cyclic AMP (18 9), and blocked by tetrodotoxin (149) The
mechanisms for both increased fluid and mucus secretion are
complex. Lencer et al. (115) have shown that the B subunit of
cholera toxin binds to cloned human goblet cells in monolayer
culture, but there was no stimulation of mucus release,
suggesting that an indirect mechanism may be involved. It is
currently believed that binding of cholera toxin to
enterochromaffin cells results in increased intracellular cAMP
and secretion of serotonin, which in turn stimulates
cholinergic neurons (149). Both the fluid and mucus secretory
activities of cholera toxin are blocked by capsaicin,
supporting the involvement of the enteric nervous system
(149). However, fluid secretion is blocked by tetrodotoxin,
while mucus secretion is not, suggesting that mucus secretion
may be mediated by local effectors released by sensory
neurons. In addition, a portion of the capsaicin-sensitive
response is also atropine-sensitive, suggesting that there may
be a tetrodotoxin-insensitive interaction between cholinergic
and sensory nerve terminals occurring in the small intestine.


29
Although neither cyclic AMP or cyclic GMP alone affects
mucus secretion in explant systems (161), Jarry et al. (93)
found that cAMP directly stimulated both MUC2 gene expression
and mucus secretion from goblet cells in culture, in a protein
kinase A-dependent manner, which could account for the effects
of cholera toxin. This toxin has also been shown to increase
mucosal prostaglandin synthesis (183), and the effects of PGEX
and cholera toxin are qualitatively the same in in vivo rabbit
intestinal loops (164).
Mucus secretion has been shown to be increased in rats
with streptozotocin-induced diabetes, with less mucin present
in the tissue than in the luminal washings (134) Synthesis
of mucins relative to other glycoproteins is also increased.
Interestingly, the intestinal goblet cells from these diabetic
rats are no longer responsive to bethanechol or cholera toxin.
This phenomenon has not yet been confirmed, and may be an
artifact of collection, since fluid secretion is also
increased in diabetes, and may flush out the mucus present.
The antiulcer drugs zolimidine, carbenoxolone, quercetin,
and oral copper compounds have been reported to increase mucus
secretion, but the mechanisms are unknown (2, 3, 51).
Parathyroid hormone has been reported to increase gastric
mucus secretion (141) The carbonic anhydrase inhibitor,
acetazolamide, also is a mucin secretagogue (28) A new
antiinflammatory drug, SCH12223, which has protective effects
on the gastric epithelium increases gastric mucus content


30
(25) The amount of gastric mucus that can be aspirated in
vivo is increased after a meal (98) Feeding increases
intestinal mucus secretion, even when the duodenum has been
transplanted to a subcutaneous location and separated from its
nerve supply (47). Mucus secretion is increased during the
daytime in rats that are allowed food only during the daylight
hours [13) .
The synthetic opioid anti-diarrheal, loperamide, has been
shown to slightly decrease baseline mucus secretion, and
drastically reduce secretion stimulated by PGE2 or deoxycholic
acid in the rat colon (121). This may be related to neural
effects or calcium channel blocking activity (186) .
Cysteamine, which is used to induce duodenal ulcers
experimentally, also decreases mucus secretion (104).
Interferon-y does not affect baseline mucin synthesis or
secretion, but inhibits secretion stimulated by both cAMP and
calcium ionophores, apparently at the exocytotic step (93).
Degradation
Little mucus is excreted in the feces. It was found that
crude mucus undergoes a spontaneous loss of viscosity when
incubated overnight at body temperature (72, 91). This
phenomenon was accelerated by the addition of certain
proteases; different investigators described different
sensitivities. It has been shown that pepsin, but not HCl,
can dissolve the gastric mucus layer, and cause epithelial


31
damage (126) That some degradation occurs in vivo is
supported by the fact that secreted mucin has a lower
molecular weight than stored mucin (220) .
Although proteolytic enzymes can degrade mucin into
smaller glycosylated fragments, resulting in collapse of the
mucus gel, and loss of elasticity (4), they cannot completely
digest mucopolysaccharides (126) Both a- and S-glycosidases
are required for the removal of sugar residues from mucin
(126), and it has been found in humans that these enzymes are
primarily derived from Bifidobacterium and Ruminococcus
species (82) in the colon.
Gastrointestinal mucin digestion is markedly decreased in
germ-free rodents, due to a lack of glycosidases capable of
removing the oligosaccharide sidechains from the peptide
backbone (120, 126). This results in an increased mass of
mucus in the cecum, increased mucus excretion in the feces,
and retention of mucus within a thickened cecal wall. When
normal enteric bacterial flora are administered to germ-free
rats, excretion of glycoproteins increases for 2-3 days, then
drops to the level of conventional animals, confirming that
bacterial enzymes play a major role in mucin digestion (120).
As mentioned previously, pepsin partially degrades
gastric mucin, resulting in solubilization, and dissolution of
the mucus layer. It has been found that patients with peptic


32
ulcer disease have a higher ratio of pepsin 1 to pepsin 3 than
normal individuals, and pepsin 1 is much more efficient at
dissolving mucus. Consequently, ulceration may be secondary
to the breakdown of the protective mucus (126) .
It has been demonstrated in vivo that Helicobacter pylori
infection decreases the thickness of the gastric mucus layer
in humans (166, 196) This has been related to the production
of protease, lipase, phospholipase, and glycosulfatase by the
organisms which impair the protective mucus layer, and may
promote mucosal injury. Sulglycotide, a modified sulfomucin
gastroprotective agent, acts by inhibition of these enzymes,
along with aggregation of the organisms (156, 180) In
addition, Helicobacter produces urease, and the high levels of
ammonia and bicarbonate produced may impair the protection
afforded by mucus (35).
Vibrio cholerae contains a virulence factor which
consists of a "mucinase complex" (168) Since mucus can bind
and inhibit cholera toxin, the destruction of mucus by a
metalloproteinase allows the toxin to retain activity, as well
as permitting better access to the epithelial cells (32). In
addition, a neuraminidase may increase the amount of GM1
ganglioside available in the membranes for toxin binding.


33
Postulated Functions of Mucus
Lubrication
In 1800, Glover postulated that the mucus secretion of
the intestinal tract was involved in lubrication (79). Florey
(46) saw that particles were removed from the intestine by
entrapment in mucus, which was then pushed downstream by the
intestinal villi. Nondigestible solids that are emptied from
the stomach are entrapped in mucus plugs (66). Mucus has been
experimentally shown to aid in ciliary propulsion of objects
in tubes (255) The need for lubrication varies with the
segment of the intestinal tract discussed. There is little
mucus secretion in the small intestine where the contents are
fluid, and there is little need for lubrication (47) .
Conversely, there are many goblet cells within the colon where
lubrication is clearly needed for the passage of solid feces
(47) More recent studies have shown an incomplete mucus
layer in the proximal colon, where the contents still have a
high water content, but a thick, compact layer in the distal
colon, in order to facilitate the passage of feces (194, 225).
Cvtoprotection
In the same thesis, Glover proposed that mucus "must
likewise defend the internal surface of the stomach and
intestines, from the action of the gastric juice, and from the
acritude of bile when regurgitated" (79). Several theories


34
were put forth for the mechanism of protection. The simplest
was that of a diffusion barrier, which will be discussed in a
later section. It was believed for a period of time that
mucus had direct acid-neutralizing capacity (79). This was
first discussed by Pavlov, and studied in detail by Hollander
(79). He found a definite buffering capacity in the mucous
secretions from Heidenhain pouches of dogs. However, it was
shown conclusively by Heatley that mucin itself has minimal
buffering power against hydrochloric acid; rather the
buffering activity is due to the content of bicarbonate (47) .
On the other hand, the mucus is responsible for keeping that
bicarbonate next to the mucosal surface, as will be discussed
later in the section on creation of a microenvironment.
In 1855, Claude Bernard postulated that mucus has a
specific chemical or adsorbent action (79) At the beginning
of the twentieth century, the presence of a specific
antipepsin in mucus was reported (79). However, since 1914
there has been no further evidence of any enzymatic activity
of mucus. Bucher (17) showed adsorption of pepsin by the
mucus, and Zaus and Foskick (264) and Bradley and Hodges (15,
16) also documented antipeptic effects, however Heatley (74)
found that purified mucin does not directly inhibit peptic
digestion.
Hydrogen peroxide is rapidly degraded in porcine gastric
mucus in vitro (34). Although purified mucin is subject to
attack by reactive oxygen intermediates, it has been shown


35
that the lipids associated with native mucus have a protective
effect against these radicals (62). Consequently, one
function of mucus may be to protect the mucosa from attack by
reactive oxygen intermediates released by the host when
killing bacteria or as a response to toxins. However, mucus
does not affect the cytotoxic activity of Clostridium
difficile toxin A in rabbits (118).
It has been shown that mucus plays a major role in
"adaptive cytoprotection" (21). Following a mild epithelial
injury with oleic acid, the thickness of the mucus layer is
increased. This prevents injury from a second exposure by
delaying the passage of the irritant through the mucus to the
epithelial cells. Increased mucus secretion stimulated by the
antiulcer drugs carbenoxolone and quercetin protects gastric
epithelium from damage by ethanol, 0.6N HCl, or 30% NaCl (2,
3). When acidified ethanol is used for challenge to negate
antacid effects, the H2-blocker FRG-8813 still offers
significant cytoprotection through increased mucus production
and secretion (85).
Protection from Infection
Cramer (30) found that vitamin A deficient rats are more
susceptible to bacterial infections, and related this to the
decreased production of intestinal mucosubstance, secondary to
decreased numbers of goblet cells. This suggests that mucus
can act as a barrier to bacteria. Goldsworthy and Florey (61)


36
then demonstrated that intestinal mucus contains lysozyme, a
non-specific antibacterial enzyme. Specific immunity in the
intestinal tract is dependent on secretory IgA. slgA alone
does not prevent bacterial interaction with intestinal
epithelium (245), but rather appears to bind to mucin
glycoproteins through hydrogen or disulfide bonds (140),
allowing for aggregation and neutralization of bacteria.
Because mucus comprises a physical barrier, bacteria have
developed mechanisms to allow association with the underlying
epithelium. One of these mechanisms is motility. Flagella
allow motility of bacteria in aqueous solutions, but are
ineffective in viscous environments. However, Campylobacter
jejuni, although flagellated, acts in high viscosity solution
like a spirochete, relying on endocellular organelles for
locomotion, and shows an increase in motility related to
increased viscosity (42) Treponema hyodysenteriae, is
similarly highly motile in intestinal mucus (101). This may
provide a selective advantage to helical bacteria, such as
Campylobacter, Treponema, Vibrio, and Helicobacter species, in
penetrating mucus and colonizing the intestinal epithelium.
A lipopolysaccharide-deficient mutant of Salmonella
typhimurium is able to colonize mouse large intestine when
given alone, in combination with E. coli, or with low
concentrations of wild type Salmonella (162). However, if
high dose of wild type Salmonella is given concurrently, or if
both strains are allowed to multiply within the host for 8


37
days, the lipopolysaccharide-deficient mutant is eliminated.
This appears to be related to the fact that the mutant adhered
to cecal mucus far better, but penetrated mucus less well than
the wild-type, or even nonflagellated or nonchemotactic
transductants (137) This also supports the idea that
flagellar motility is not important in mobility within mucus.
The other major mechanism bacteria use to remain in the
intestinal tract is adhesion. Piliated Escherichia coli are
able to bind to sugar residues in the glycoproteins and
glycolipids of both epithelial cells and mucus. In
enterotoxigenic E. coli strains, K99 fimbriae bind to
galactose (155) and sialic acid residues (119), K88ab fimbriae
bind D-galactosamine residues (143), and the terminal subunit
of F17 fimbriae binds to undetermined carbohydrate sidechains
which are also present in cow plasma glycoproteins and hen egg
white (195) The F-18 colicin which is found in nonpathogenic
E. coli binds to mannose (243) Although E. coli lacking F-18
are able to colonize streptomycin-treated mouse large
intestine when given alone, they cannot compete with the
fimbriated bacteria in establishing infection when given
concurrently (244) .
Both piliated and non-piliated strains of
enteropathogenic E. coli adhere to mucus (259). Denaturation,
trypsinization, or removal of carbohydrate from the mucin all
decrease binding (248). More mucin bound to both
enteropathogenic and enterotoxigenic E. coli strains at pH 5.7


38
than at pH 7.4 (248) In vitro, the presence of mucus
competitively inhibits binding of E. coli to colonic
epithelium (125), suggesting a protective effect. On the
other hand, adherence to mucus may allow for the initial
colonization by bacteria. Hydrophobicity of the mannose-
resistant AF/R1 pilus of the enteropathogenic E. coli strain
RDEC plays a major role in bacterial binding to mucus and
membranes (37), and RDEC preferentially colonizes colons of
weanling rabbits, where mucus is more hydrophobic than in
sucklings or in the ileum (124). Additionally, more mucus
binding is seen in weanlings than in adolescents (248), which
may reflect the increased susceptibility of weanlings to
clinical disease.
Virulent strains of Yersinia enterocolitica are able to
colonize the intestine because the yadA gene on the virulence
plasmid codes for a high molecular weight outer membrane
protein which allows binding to mucin (170). However,
preincubation with mucus decreases the adherence of the
organism to brush border membranes (130). It appears that
coating with mucus changes the bacterial surface from
hydrophobic to hydrophilic, decreasing the interaction of the
organism with the epithelial surface (170). However, strains
containing the virulence plasmid are able to degrade mucin to
a greater extent than non-virulent strains (132), and thus
overcome the protection afforded by mucus.


39
Helicobacter pylori has also been shown to bind to mucin,
and the binding is decreased after the removal of sialic acid
(237). Electron microscopy shows that most strains of Vibrio
cholerae bind preferentially to mucus, rather than to the
epithelial surface (260, 261) Guinea pig mucus inhibits
invasion of epithelial cells by Shigella flexneri, but monkey
mucus does not (245) .
Intestinal mucins inhibit replication of rotavirus in
vitro (24, 262). The rotavirus vp4 protein, which also
mediates binding to cells, is involved in the binding (24);
however it is not clear if sialic acid residues or short
oligosaccharide chains act as the mucin receptor (262) .
Mucus also plays a significant role in parasitic
infections. Frick and Ackert (54) found that duodenal mucus
of adult chickens inhibits the growth of Ascaridia galli more
than that of young birds, and that this may play a role in age
resistance. A nematode which has been studied more
extensively is Nippostrongylus brasiliensis in rats.
Following a transient decrease, an increase in the number of
goblet cells is associated with an immune-mediated expulsion
of this worm. If the number of goblet cells is decreased
secondary to a protein deficient diet, the efficiency of
expulsion is decreased (249). Secretion of mucus is necessary
for the expulsion event: if mustard oil or a combination of
cysteine and papain is administered to immune rats to cause
secretion of stored mucin 1.5 hours prior to challenge, then


40
worm expulsion is inhibited (146) The role of prostaglandins
in this process is unclear. While some investigators have
found that administration of prostaglandins enhances
expulsion, others have been unable to confirm this (28). The
vehicles used (chloroform or alcohol), or the timing of
administration may be responsible for the discrepancy.
Some worms are entrapped by mucus prior to expulsion
(145) In addition, in a phenomenon called "immune
exclusion", worms are unable to penetrate the mucus of
immunized animals, while in naive animals, worms are able to
burrow through the mucus to the intervillous space (145) .
Similarly, it has been shown that mucus from infected sheep
inhibits the motility of Trichostrongylus colubriformis in
agar gel (103) Nippostrongylus is able to move through
viscous gels by forming a tight corkscrew (111); the presence
of coating antibodies may prevent this motion, resulting in
immune exclusion. It has also been shown that the worms
ingest mucin, and morphologic damage of the adult worm gut is
associated with the development of immunity (145) .
Trichinella spiralis is expelled from immunized rats in
a very similar manner, although Trichinellae enter the
epithelium, while Nippostrongylus remain between villi (145) .
It has been shown that antibody or complement coating of
Trichinella larvae allows entrapment in mucus (18, 145) This
may be important in the expulsion of worms from the organisms,
although the injection of antibody allows for the expulsion of


41
worms from the infected epithelium, prior to association with
mucus.
The expulsion of both Trichinella and Nippostrongylus is
an immune-mediated event. Transfer of thoracic duct
lymphocytes from immunized rats results in accelerated
expulsion of Nippostrongylus associated with early goblet cell
hyperplasia (147) and this was later shown to be T cell-
dependent (14 5) However, transfer of hyperimmune serum will
also result in rapid expulsion (145) Furthermore, at least
two steps are involved in worm expulsion (88) The first
involves T-cell dependent "damage" to the worms. These
damaged worms can then stimulate alterations in the terminal
sugar residues of mucus resulting in the selective expulsion
of damaged or healthy worms, even from athymic rats (88) .
There is also synergistic interaction between immune serum and
thoracic duct lymphocytes in the rapid expulsion of
Trichinella (1). In both worms there are increased
concentrations of leukotrienes and rat mast cell protease II
(145, 150) Corticosteroids, which inhibit the immune
response in many ways and also decrease mucus secretion (141),
delay expulsion of Nippostrongylus (145) as well as the rat
tapeworm Hymenolepis diminuta (139). Reserpine also alters
mucus secretion (169) and delays expulsion of Nippostrongylus
(145) .
On the other hand, mucus can apparently increase
pathogenicity of other parasites. In particular, it promotes


42
survival of Giardia lamblia in many ways. For example,
Giardia can be killed by the lipolytic products of milk, but
this killing is inhibited by the presence of intestinal mucus
(265) Mucus provides a nutrient source for the parasite
(57), and allows for its enhanced adherence to the intestinal
epithelium (145) This lectin-like adhesion to simple sugar
residues within mucus, especially sialic acid, is a
characteristic of several protozoa, including Entamoeba
histolytica (145) and Tritrichomonas mobilensis (33) The
yeast Candida albicans has also been shown to associate with
mucus, but the ability to colonize the intestinal mucosa is
dependent on the absence of normal flora (102) .
Nutrient for Flora
Evidence that mucus may aid the growth of bacteria was
demonstrated by Smith et al. (213, 214), who found increased
virulence of intraperitoneally injected bacteria
(Staphylococcus aureus and Streptococcus species) when mucus
was administered with the organism. This effect was partially
due to sequestration of the bacteria from the immune system.
However, the chemical components of the mucus itself increased
the growth of the organisms, presumably by serving as a
nutrient source. It has been shown in vitro that jejunal
mucus stimulates the growth of Giardia lamblia (57). Mucin
can also enhance growth of both virulent and avirulent
Yersinia enterocolitica, as well as serve as a nutrient source


43
for E. coli, Salmonella typhimurium, Clostridium perfringens,
Bacteroides sp, Shigella flexneri, Rumenococcus, and
Bifidobacterium (8, 132).
By comparing the cecal mucus of conventional and germfree
rats, Lindstedt et al. (120) were able to show that normal
flora degrade a significant amount of mucus. There was less
mucus in the cecum of conventional animals, and a higher
proportion of what was there was of lower molecular weight,
indicating partial degradation. Other investigators have
confirmed these findings (52) However in the colon,
Szentkuti et al. (225) found a thinner mucus layer in germfree
rats, associated with decreased mucosal thickness, and
decreased numbers of goblet cells.
As discussed previously, Rumenococcus and Bifidobacterium
species possess glycosidases that are responsible for the
degradation of mucin in humans. The monosaccharides released
from mucin by these glycosidases support growth of Bacteroides
and other fecal bacteria that lack such glycosidases (82).
However, a strain of Bacteroides vulgatus isolated from
patients with Crohn's disease is capable of degrading mucus
glycoproteins (192). The enzymes produced by normal flora may
also directly attack pathogenic bacteria (160) ; normal flora
certainly inhibit colonization by pathogens, since antibiotics
increase infection with pathogenic organisms (245) .


44
Diffusion Barrier
Much of the cytoprotective action of the mucus layer has
been attributed to the barrier properties of mucus (79) .
However, when this property was experimentally examined,
Heatley illustrated that mucus is not a barrier to the passage
of solutes, such as H+ ions and pepsin. Furthermore, when
studies were performed to determine the thickness of the
"unstirred water layer" in the intestine, the calculated value
for several solutes was similar, so it was proposed that mucus
merely acts as a support for the unstirred water layer (215,
250) However, more recent studies show that mucus does
retard diffusion of many molecules. For instance, it slows
the passage of H+ ions relative to their rate of diffusion in
water (172, 253), and Lucas (122) demonstrated that this
retardation is dependent on the concentration of mucus. The
mobilities of sodium, potassium, and chloride are also greatly
reduced in mucus (68) .
Mucus forms a polyanionic gel, and thus acts as an ion
exchange resin (48). In general, chloride is excluded,
whereas there is a high affinity for calcium and potassium
(67, 199). However, although the transport of chloride (and
other ions) is retarded, it is not prevented (68) .
Sequestration of potassium by mucus and cell surface
glycoproteins may be important in recycling of potassium by
the Na+/K+-ATPase. Calcium may be important in the


45
condensation of mucus into compact granules prior to
exocytosis (242) .
Mucus retards diffusion of molecules as well as ions.
Smith (212) found a diffusion coefficient for butyrate in
mucus to be 50-60% of that in water. Desai et al. (34)
determined the diffusion coefficients for a wide variety of
molecules in mucus and concluded that no consistent effect of
molecular weight was evident with regard to barrier properties
for the weight range tested (34-660 Da) Using cultured
goblet cells, it has been shown that the overlying mucus layer
is a significant barrier to the passive absorption of the
lipophilic and uncharged drug testosterone (97). Mucus has
been shown to trap iron, but it is not clear if this aids
(181), or prevents excess, (252) absorption. This trapping
may make iron more available to bacteria (51).
Since mucus is a polyelectrolyte gel, it behaves as a
Donnan system (67). Consequently, the hydration of mucus is
dependent on the ionic concentrations in the bathing solution.
As ionic strength is decreased, hydration is increased, and
noncovalent interactions between mucin subunits are fewer,
decreasing viscosity. Conversely, at high ionic strength,
anionic charges within the gel will be shielded, and hydration
and volume decreased. It has been postulated that lack of
functional chloride channels resulting in decreased chloride
movement in cystic fibrosis could alter ionic composition
resulting in abnormal mucus secretions in intestinal,


46
pancreatic, and respiratory tissues (138, 242). Mucus from
cystic fibrosis patients is hyperpermeable to small ions and
water, associated with an increased calcium content (58).
Furthermore, glucose absorption was enhanced in afflicted
patients, coinciding with a decreased thickness of the
calculated unstirred water layer (53).
Mucus may even have a "waterproofing" function (159) If
the external mucus layer is removed from an eel, the weight of
the animal will increase when the eel is placed in distilled
water. This property has not been examined in the intestine,
and would be difficult to address in light of the complex
absorptive and secretory processes involved in digestion and
absorption. However, it is interesting that Westergaard and
Dietschy (250) saw swelling of intestinal villi inversely
correlated the thickness of the unstirred water layer.
Creation of Microenvironment
A microenvironment occurs when the concentration of
solutes next to a membrane is different from that of the bulk
phase on either side of the membrane, as has been shown to
occur in mucus (67). In the gastrointestinal tract, the
presence of a microenvironment is important in two basic
areas. In the stomach, it is necessary to prevent direct
contact between the highly acidic bulk contents and the
mucosal surface. When Heatley (75) found that mucus did not
act as a barrier for hydrogen ions, he proposed a pH gradient


47
within the mucus layer. In his dynamic model, mucus and
bicarbonate are secreted at the mucosal surface, and then
migrate outward. As the mucus progresses into the lumen, it
becomes more hydrated and accumulates more hydrogen ions. The
viscosity thus decreases, until the outermost layer is shed
into the lumen. Therefore, some of the hydrogen ions will be
brought back into the lumen with the dissolving mucus, while
others will be neutralized by the bicarbonate within.
This "mucus-bicarbonate barrier" model has stood the test
of time very well (5, 31, 148, 205). With the development of
microelectrode techniques, pH gradients have been demonstrated
in rat (190), rabbit (254), and human (182) gastric mucus in
vivo. The pH gradient can be decreased with the addition of
compounds that dissolve mucus, such as N-acetylcysteine (190).
Mucus is responsible for the creation, as well as the
support, of a microenvironment in the intestine. Hogben et
al. (78) proposed that an acidic microenvironment could
influence the absorption of drugs from the intestinal tract.
Acidic drugs are absorbed more rapidly than would be expected
at neutral pH; however at acidic pH these acids would be
undissociated, and would readily cross the epithelial membrane
by nonionic diffusion. He calculated that the pH at the rat
jejunal surface must be 5.3 to account for measured drug
absorption rates. Surface pH measurements with
microelectrodes have revealed an acidic microenvironment, but
with pH 6-7 (123, 185, 203) .


48
Similarly, a slightly acidic microenvironment is present
in the colonic unstirred layer (185) This would allow for
the passive absorption of butyrate and other short chain fatty
acids in the undissociated form (7) Although there is
evidence for a bicarbonate gradient-dependent, carrier-
mediated anion exchange process for butyrate in the colon,
this cannot account for the rate of absorption measured
experimentally, nor could passive diffusion of the dissociated
ionized form of butyrate (136) In the rodent cecum, the
microenvironment is more basic than the luminal contents, and
the pH may have an effect on the virulence of Entamoeba
histolytica (112). This organism is subject to killing by
ammonia, and species with a higher cecal mucosal pH, such as
the rat, are less susceptible to infection than those with a
lower pH, such as the gerbil.
Techniques Used to Study Mucus
Model Systems
Intestinal loops. For in vivo experimentation of the
gastrointestinal tract, it is common to isolate the part of
the tract under study to minimize effects of the rest of the
system, and to allow for easy access. For the study of
gastric secretion, the first widely used technique was
formation of the Heidenhain pouch (76). Florey (44, 45, 46,
47) used a variety of intestinal pouches, blind loops, and
transplanted segments to examine movement of mucus within the


49
intestine, and the effects of feeding, neural stimulation, and
various chemicals on mucus secretion.
Explants. Mucosal samples (161) and full thickness
intestinal explants (50) have been used to measure
secretagogue activity in vitro. These tissue sections can be
maintained in culture medium for several hours, and mucus
production and secretion can be measured by several methods.
This technique allows for concurrent testing of several
different chemicals on tissues from a single animal. It has
even been used to demonstrate goblet cell secretion following
electrical field stimulation, due to intrinsic nerve
stimulation (175).
HT-29 cloned goblet cells. Until recently, it was
impossible to work with isolated goblet cells, because these
cells lose their polarity and ability to secrete when
separated from the mucosal epithelium (160) However, cell
culture systems are now available. The HT2 9 colon
adenocarcinoma cell line is undifferentiated under standard
culture conditions. However, substitution of glucose with
galactose (83), or long term treatment with butyrate (108)
results in differentiation of cells. Individual cells can be
selected for cloning. Several sublines, including HT29-18N2
and C1.16E, show characteristics of goblet cells, including
baseline mucus secretion, and the ability to respond to
cholinergic stimulation (108, 173).


50
Quantitation of Mucus
Biochemical techniques. Biochemical measurement of total
protein-bound hexose (258) acid-precipitable protein (50) ; or
glycoprotein (127) has been used to estimate mucus
concentration; however mucins are not the only glycoproteins
present in the intestine. Mucin can be separated from many
other glycoproteins by gel filtration on a Sepharose-4B
column, but many proteins remain associated with the sticky
mucin (160) .
Radioactivity incorporation. Glycoprotein synthesis has
been measured by determining the rate of 14C- or 3H-glucosamine
incorporation into intestinal slices (50) and secretion is
measured by counting precipitable radioactivity released into
the media (49) By fractionating the cell sap on a Sepharose-
4B column, and collecting the void volume, it is possible to
estimate the amount of mucin synthesized. It has also been
shown that labelled butyrate and acetate are incorporated into
mucus glycoproteins (29) However, absorptive cells
synthesize and release glycoproteins at a faster rate than
goblet cells (160).
Morphometry. Goblet cell hyperplasia, which is
associated with increased mucus secretion, can be quantitated
morphometrically (147). Compound exocytosis can be
visualized, and the percentage of cavitated goblet cells is
reflective of the amount of mucus secreted (217).


51
Immunologic techniques. Standard radioimmunoassay (131)
and enzyme-linked immunosorbant assay (ELISA) techniques (188)
have been developed, using antibodies to mucin. Polyclonal
antibodies react predominantly to the "naked" regions of
mucin, and therefore do not measure degraded mucin (131) .
Monoclonal antibodies have been made specific for both the
naked peptide backbone, and for intact mucin (210).
Antibodies have also been made which are specific for the
fragment known as the "link" glycopeptide (188) .
Lectin binding techniques. An enzyme-linked lectin assay
(ELLA), very similar to a standard ELISA, has been developed
(27, 69) The lectin soybean agglutinin preferentially binds
to N-acetylgalactosamine residues, which are abundant in mucin
but less common in other glycoproteins.
Secretion of Mucus
Light microscopy. A monoclonal antibody has been
developed which specifically labels goblet cells of the human
colon, appendix, and small intestine (240) This may be
useful in the diagnosis of disease states, in which the
quantity or quality of mucus is altered. Histologic
examination of the thickness of the mucus coating can be used
to evaluate changes in secretion (225) .
Morphometry has also been used to quantitate secretion by
comparing the amount of stained mucin in goblet cells before
and after addition of a secretagogue (100). Autoradiography


52
has been used to measure the rate of secretion (73).
Cavitation of goblet cells indicates that compound exocytosis
has taken place, and has been used to measure secretagogue
activity (176, 177, 217). Exocytosis has been directly
observed by video-enhanced light microscopy (230) .
Electron microscopy. The steps of the secretory process
can best be followed by electron microscopy (217) Since
conventional fixation techniques can fragment the limiting
membranes, cryofixation may allow for better evaluation of the
mechanism of secretion (84) The thickness and composition of
the mucus layer can also be examined by this technique (165) .
Composition of Mucus
Biochemistry. Standard biochemical techniques have been
used to determine the amino acid and sugar composition of
mucus (160) Glycosyl-transferase activities can be measured
by conventional biochemical assays (12, 226), providing clues
to changes in carbohydrate composition. Methods for measuring
the adhesiveness, plasticity, viscoelasticity, and
spinnability of mucus microsamples are also now available
(263) .
Histochemistry. Differential staining techniques can be
used to distinguish mucin characteristics. When sections are
stained with combined alcian blue and periodic acid Schiff
reagent, acidic mucins will appear blue, and neutral mucins
will appear red (225). High iron diamine will stain sulfated


53
sialomucins (266) Positive staining with mild periodic acid
Schiff (mPAS) indicates deficiency of O-acetylation of sialic
acid, as is seen in ulcerative colitis (94) Mucins of
different chemical composition can be distinguished by
differential lectin binding. Therefore, lectin histochemistry
can be used to identify abnormally glycosylated mucins in
goblet cells, which may be associated with disease (89, 241),
developmental changes (227), or diet (152) Finally, specific
antibodies can be used to distinguish small intestinal from
large intestinal mucins by indirect immunoperoxidase staining
(43) .
Diffusion Through Mucus
Mucus can be immobilized on a filter, or between two
filters, and the rate of diffusion through this compared to
the rate of diffusion through the filter alone (75) The
mathematical modeling of diffusion through mucus in this type
of apparatus has been described (172) Diffusion through
mucus overlying HT29 monolayers can also be measured (97).
Microbial Virulence
In vivo colonization assays, where the number of colony
forming units of bacteria recovered in the feces after
experimental infection is measured over time, reflect a
combination of virulence factors (162). Microbial adherence
can be evaluated by coating plates with mucus or brush border


54
membranes, and measuring the percentage of radiolabelled
bacteria which will then adhere (130). Competition for
binding can also be measured with this assay, by adding the
competitor along with the organism. Adhesion of organisms to
cells in culture can also be evaluated microscopically (33).
Penetration of organisms through mucus can be evaluated by the
passage of radiolabelled bacteria through a mucus gel (170),
or by direct observation of the organism (42, 111).
Theoretical Considerations
Lubrication
The idea that mucus is necessary for lubrication to aid
in the passage of feces has been mostly intuitive, with little
direct experimental support. However, there is one disease
state which illustrates the need for such lubrication quite
clearly. In cystic fibrosis, where mucus is abnormally
viscous and lacks elasticity, it is common for infants to
develop meconium ileus, and for adults to develop meconium
ileus equivalent (160). As indirect evidence, the most
compact, continuous mucus layer is in the distal colon, where
there is greatest need for lubrication for the passage of
formed feces (194) In addition, the fact that nondigestible
solids are coated in mucus when they are expelled from the
stomach suggests that mucus aids in lubrication to facilitate
their passage through the pyloric sphincter and expel them
from the body (66).


55
Cvtoprotection
Mucin itself has no direct buffering or antienzymatic
activities. However, its presence is necessary to protect
underlying cells. Its removal may result in increased gastric
or duodenal ulceration (104) Chronic inflammatory bowel
disease is associated with decreased mucus production, which
may help to perpetuate the disease (160) This protection is
partly due to the role of mucus as a diffusion barrier, and
the creation of a microenvironment, which will be discussed
below. In addition, it appears that mucus is able to detoxify
reactive oxygen intermediates (34, 62). Irritants of many
types stimulate the accelerated secretion of mucus; this may
be a protective mechanism to keep such irritants away from the
intestinal mucosa (21, 47).
Protection from Infection
The interactions of mucus and pathogenic organisms are
complex. It is perhaps easier to determine the protective
effects of mucus by looking at mechanisms that pathogens have
developed to overcome these effects. Mucus is a slippery
substance, and when the motility of the intestine is taken
into account, it is clear that attachment to mucus is
necessary if organisms are to avoid being flushed out. The
viscosity of mucus is also a barrier, and motility based on
internal structures rather than flagellae helps microorganisms


56
negotiate this barrier (42) That organisms have developed
mucinase enzymes indicates that mucus is a barrier that must
be negotiated (168) .
Mucus also contains antibacterial substances, in
particular lysozyme (61) and secretory IgA (14 0) Mucin bound
to IgA allows for entrapment of parasites such as
Nippostrongylus, so that the parasite is immobilized and swept
out of the body (145) The goblet cell hyperplasia seen with
many parasitic infections emphasizes the importance of mucus
in their expulsion. Mucus coating can also prevent attachment
of pathogens to the intestinal epithelium by competition (125)
or by changing bacterial surface properties (170) .
Pathogenic organisms have also been able to use mucus to
increase virulence. Within mucus, bacteria are "hidden" from
some of the defense mechanisms of the host (265) Also, mucus
can provide nutrients to these organisms, as discussed below.
Nutrient for Flora
Although the experimental evidence is incomplete, it
seems reasonable that degraded mucus provides nutrients for
bacterial flora, especially in times of fasting. The trapping
of iron by mucus (181, 252) allows the flora increased access
to this mineral which is so tightly controlled in the rest of
the body. It is important to remember that the normal flora
also provide a major defense against pathogenic bacteria (160,


57
245) so that in providing nutrients for flora, mucus is
actually protecting the host from infection.
Diffusion Barrier
The gastrointestinal mucus layer has been shown to delay
diffusion of a wide variety of molecules. In the intestine,
this layer is between 50 and 500 /m thick, depending on
species and site evaluated (34, 67) In general, the measured
unstirred water layer is slightly thicker. This may be due to
an unstirred layer overlying the mucus, but is more likely due
to the decreased diffusion of solutes in mucus leading to
erroneously large values for the unstirred water layer (215) .
Consequently the barrier to nutrient absorption is formidable,
although the calculated maximum allowable water barrier which
would allow physiological absorption of glucose and lipid is
4 0 jum (223) Furthermore, the mucus layer is not static;
there is constant renewal at the mucosal surface and
degradation at the luminal surface, resulting in a net flow of
mucus away from the mucosa.
It would seem that this unstirred mucus layer would make
absorption of nutrients impossible. But on further
examination, the term "unstirred" is also inappropriate. The
mixing caused by segmental and peristaltic contractions of the
intestine, with three dimensional shear forces, as well as
expansion and contraction of the mucus layer itself, is not
comparable to mixing by a stir bar in a beaker. Furthermore,


58
the intestinal villi move in and out of the mucus layer,
cleansing themselves of particulate matter, and moving the
entire mucus layer aborad (46) Villi bare of mucin are
present in the small intestine, particularly the ileum, in
vivo (165) The villi may also cause mixing within the mucus
layer, as speculated by Strocchi and Levitt (223).
The fluxes of water in and out of the intestine must also
be considered. Large volumes (approximately 1 liter/20cm/hr
in a human, 53) are secreted from the crypts and absorbed from
the villi. This could aid in absorption by solvent drag
effects, as well as contribute to mixing within the mucus
layer. And finally, the mucus may actually "trap" certain
molecules, such that the concentration within the mucus can
exceed the luminal concentration, leading to increased
absorption.
Creation of Microenvironment
The pH difference between the lumen and the mucosal
surface is most important in the stomach, where the presence
of a "mucus-bicarbonate" barrier is generally accepted. By
delaying the diffusion of H+ toward the epithelial cells, and
the diffusion of bicarbonate away from them, mucus plays a
vital role. The microenvironment may also play a role in the
selective absorption of drugs and nutrients from the
intestinal tract. In particular,
a slightly acidic


59
microenvironment would aid in colonic absorption of short
chain fatty acids (7) .
Conclusions
The differences in the mucus layer throughout the
gastrointestinal tract reflect the differing functions
required. In the stomach, the major function is
cytoprotection, by creation of a diffusion barrier and a less
acidic microenvironment. For this, the mucus is neutral
rather than acidic, and is more viscous. In the small
intestine, mucus must act as a diffusion barrier to
destructive enzymes and pathogenic organisms, yet provide a
microenvironment to allow for absorption of nutrients. The
fact that absorption occurs at the tips of the villi, which
stick up through the mucus layer, may be a physiologic
necessity, rather than a random fact for students to memorize.
In the proximal colon, the loose, discontinuous mucous
layer provides for the nutrition of friendly flora, and
creates a microenvironment which allows for absorption of the
short chain fatty acids produced by these bacteria. Finally,
in the distal colon, a thin, compact mucus layer provides
lubrication for the passage of feces. All in all, mucus aids
the host in a number of ways, and its importance in the
maintenance of good health is often overlooked.


CHAPTER 3
PILOT STUDIES
Introduction
The overall purpose of this project was to determine if
there is a mucus secretagogue in the cecal contents of rabbits
with mucoid enteropathy, as proposed by Toofanian and
Targowski (236). Therefore pilot studies were performed to:
1) explore the presence of such a secretagogue in the cecal
contents of a rabbit with naturally-occurring mucoid
enteropathy (ME); and, 2) confirm that the cecal ligation
model developed by Toofanian and Targowski (229, 235) would
reproducibly cause a mucoid enteropathy-like syndrome.
Cecal Filtrate Collection
Cecal contents were collected from a juvenile female New
Zealand White (NZW) rabbit (Oryctalagus cuniculus) with
naturally-occurring mucoid enteropathy, and also from two
healthy adult NZW rabbits. The affected rabbit also had a
severe intestinal Eimeria infestation. For each rabbit, the
volume of the contents was estimated, and an equal volume of
Hank's Buffered Salt Solution (HBSS) was added. The slurry
was centrifuged at 3000 rpm (700 x g) 4C for 15 minutes.
The supernatant was collected, and recentrifuged at 9500 rpm
60


61
(10,000 x g), 4C for 30 minutes. The resulting supernatant
was then filtered through a Whatman #50 paper filter, followed
by a 0.8 /xm membrane filter, and finally a 0.22 /xm sterilizing
membrane filter. The sterile filtrate was stored at -70C
until use.
Intestinal Explants
The cecal filtrates that were collected were tested for
mucus secretagogue activity in an in vitro intestinal explant
system. Five healthy adult NZW rabbits were sedated with
ketamine/xylazine, and killed with an overdose of barbiturate.
Intestinal segments were collected, opened along the
mesenteric border, and rinsed in HBSS, containing 100 /xg/ml
gentamicin, to remove ingesta. A 6 mm diameter Baker's biopsy
punch (Baker Cummins Pharmaceuticals, Inc., Miami, FL) was
used to take explants from the ileum and proximal colon
(approximately 10 cm from the cecocolic junction, at the point
where the number of longitudinal bands (teniae) decreases from
3 to 1, fig. 3.1). Longitudinally paired punches were taken
for control and experimental samples, as the concentration of
goblet cells varies along the length of the colon (236) .
Explants were incubated in 24 well polystyrene microtiter
plates containing 0.5 ml of Trowell's T8 medium (GIBCO BRL,
Gaithersburg, MD) with 100 /xg/ml gentamicin, and 0.5 ml of the
appropriate cecal filtrate in each well. The plates were
incubated for 1 hour at 37C in a 95%02/5%C02 humidified


62
environment. Following incubation, the culture medium was
removed and frozen, while the explants were fixed in 10%
neutral buffered formalin. Following routine processing and
sectioning, the tissues were examined histologically. After
1, 2, and 3 hours of incubation, the tissues appeared viable
in every respect.
Enzyme-Linked Lectin Assay
Quantitation of mucus release by the explants was
accomplished using an enzyme-linked lectin assay (ELLA) as
described by Cohan et al. (27). Ninety-six well polystyrene
microtiter plates were coated overnight at 4C with 100 /I of
serial dilutions of porcine gastric mucin standard or
medium/filtrate mixture following explant incubation in 0.5M
sodium carbonate buffer, pH 9.6. The plates were washed with
phosphate buffered saline containing 0.5% Tween-20 (PBS-T20),
and blocked with 5% fetal calf serum in PBS-T20 for 1 hour at
37C. The plates were again washed, and incubated for 1 hour
at 37C with 100 /1/well of a 10/xg/ml solution of a soybean
agglutinin (Glycine max)-horseradish peroxidase conjugate,
which specifically binds N-acetylgalactosamine residues.
Plates were then washed a third time, and the substrate o-
phenyl diamine (OPD) was added. After 10 minutes, the
reaction was stopped with 100 /I of 4N H2S04, and the optical
density at 492 nm was read. The standard curves for porcine


63
gastric mucin were consistent from plate to plate (figure
3.2).
Data Analysis
After the filtrates were incubated with paired explants,
the mucus secreted into the culture medium was quantitated by
the ELLA described above. The effect of sample dilution on
the amount of mucus in the medium/filtrate measured by the
ELLA is illustrated in figure 3.3 for a control rabbit, and in
figure 3.4 for the rabbit with naturally-occurring mucoid
enteropathy. At the lower dilutions (higher concentrations)
there were too many proteins in each sample competing with the
mucus for a limited number of binding sites on the plate,
resulting in falsely low mucus readings. At the higher
dilutions, there seemed to be some appropriate dilutional
effect; however the signal-to-noise ratio is decreased,
creating a larger variance. The 1:64 dilution was selected
for studies requiring quantitation of mucus.
Using the porcine gastric mucin standard curve for the
same plate, the optical density readings were converted to
apparent /xg mucus per ml of medium/filtrate (figure 3.5) The
amount of mucus in the medium/filtrate incubated without an
explant was subtracted from each value to give the amount of
mucus secreted. Since each explant was incubated in 1 ml
medium/filtrate, this number was equivalent to ng mucus
secreted per explant. Each value was then divided by the


64
weight of the explant to obtain nanograms mucus secreted per
milligram of tissue (figure 3.6).
The amount of mucus in the medium/filtrates initially and
following explant incubation varied with the source of the
cecal filtrate (figure 3.5; Appendix A). Secretion from both
ileal and colonic explants was significantly increased
compared to control (P < 0.05) when incubated with the cecal
filtrate from the rabbit with mucoid enteropathy (figure 3.6).
Cecal Ligation
To better study mucoid enteropathy, it is necessary to
have a reproducible experimental model of the disease. Cecal
ligation has been reported to cause a mucoid enteropathy-like
syndrome in rabbits (229, 235). Surgery was performed on one
adult, female, conventionally housed NZW rabbit (Oryctalagus
cuniculus). The procedure was approved by the University of
Florida Institutional Animal Care and Use Committee. The
rabbit was anesthetized with 35 mg/kg ketamine and 5 mg/kg
xylazine intramuscularly. A sterile field was prepared, and
the abdomen was opened. A window was made in the mesentery
adjacent to the cecum. Two adjacent ligatures of 2-0 silk
were placed around the cecum just distal to the sacculus
rotundus, preventing the flow of ingesta into and out of the
cecum, but not obstructing the flow from the ileum to the
colon (figure 3.7) The linea alba was closed with 3-0


65
chromic gut, and the skin was closed with 3-0 silk. Recovery
was uneventful.
The rabbit was observed twice daily. By the first
postoperative day the rabbit was eating small amounts, and
passing dry feces. However, appetite and fecal output
decreased on the second day. On the third day the rabbit was
anorectic, appeared painful, and had mucoid diarrhea.
Euthanasia and necropsy were performed.
At necropsy, there was perineal staining with greenish,
liquid fecal material. There were mild subcutaneous
hemorrhages at the incision site, but the sutures were intact,
and there was no gross inflammation. The cecal contents and
the body of the cecum distal to the sutures appeared normal.
The ampulla coli and proximal 2-3 cm of the colon were grossly
dilated with gas and fluid, although there was no mechanical
obstruction. The colon contained a small amount of greenish
fluid, with mucus adhering to the mucosa. The contents of the
ileum were similar. The jejunum contained yellow fluid, with
pockets of accumulation of clear, gelatinous mucus. On
histologic examination there appeared to be mild goblet cell
hyperplasia in the ileum and colon. These results are
consistent with those described by Toofanian (229, 235), and
suggest that this method provides a workable model to study
mucoid enteropathy.


66
Rabbit (OryctolaguM cuniculus)
Body Length :48 cm
i i i i i
O cm io
Figure 3.1. Sources of intestinal explants
Explants were taken from the distal ileum (closed arrow) and
the single-banded proximal colon (open arrow). Adapted from
reference 6 (p. 270). Used by permission of the publisher,
Cornell University Press.


67
Porcine Gastric Mucin (ng/ml)
Figure 3.2. Standard curves (ELLA).
Serial dilutions of porcine gastric mucin analyzed by ELLA.
The standard curves for the 5 plates used to collect data for
this chapter are shown.


68
Serial dilutions of medium/filtrate samples from a control
rabbit (P8) incubated in the absence of an explant (P8B) or
the presence of ileal explants from 3 rabbits (Rl, R2, and
R3), analyzed by ELLA.


69
Serial dilutions of medium/filtrate samples from a rabbit with
mucoid enteropathy (P4) incubated in the absence of an explant
(P4B) or the presence of ileal explants from 3 rabbits (Rl,
R2, and R3), analyzed by ELLA.


70
1.20
1.10
1.00
f 0.90
3 0.80
0.70
CO
^ 0.60
u.
E- 0.50
3
g 0.40
2
c 0.30
w 0.20
u
| 0.10
0.00
-0.10
-0.20
Explant
Blank
m Ileum
Colon
Control
(P3)
Control
(PS)
Filtrate
Mucoid
Enteropathy
(P4)
Figure 3.5. Mucus in medium/filtrates (ELLA).
Calculated mucus (porcine gastric mucin equivalents) in
medium/filtrates, measured by ELLA. Bars represent mean +
SEM. N=1 (P8 blank), 5 (P3, P4 blank), 6 (P8 ileum, colon),
7 (P3 ileum, colon), 10 (P4 colon), or 14 (P4 ileum).


71
Q)
3
GO
If
0.60
0.50
0.40
O)
O)
c
T3 0.30
(U
+J
0)
k_
o
a)
w
GO
3
o
3
0.20
0.10
0.00
Filtrate
Control
Mucoid Enteropathy
Ileum Colon
Explant
Figure 3.6. Mucus secretion from explants
Mucus secretion was determined by subtracting the amount of
mucus in the medium/filtrate incubated without explants (as
measured by ELLA) from the amount incubated with explants.
Values shown are relative to the wet weight of the explant.
Bars represent mean + SEM. N = 8 (ME) or 11 (control). =
significantly different from control (P < 0.05) .


72
The cecum was ligated distal to the sacculus rotundus (arrow),
allowing the flow of ingesta from ileum to colon. Adapted
from reference 235, with permission.


CHAPTER 4
REFINEMENT OF METHODS
Use of Soybean Agglutinin to Quantitate Mucus
Introduction
Soybean agglutinin, the lectin from Glycine max, specifically
binds to N-acetylgalactosamine residues, which are a major
constituent of the O-linked mucin glycoprotein, but are rare
in N-linked glycoproteins. A direct enzyme-linked assay using
this lectin for the measurement of mouse intestinal mucus has
been described (27). Its suitability for the measurement of
rabbit intestinal and colonic mucin was investigated using
Western blot techniques.
Materials and Methods
Purification of rabbit colonic mucin. Colonic mucus from
a rabbit with experimentally-induced mucoid enteropathy
(rabbit H5, see chapter 5) was isolated by the method of
Mantle and Allen (128) with minor modifications. Mucus gel
was removed from the colonic mucosa with forceps, and was
homogenized by hand in a Kontes tissue grinder with an equal
volume of 5 mM EDTA and 1 mM PMSF. The suspension was
centrifuged at 30,000 x g for 30 minutes. 8.1 g of cesium
73


74
chloride (CsCl) was added to 13 ml of the supernatant for a
final concentration of 0.6 g/ml. This solution was
centrifuged for 24 hours at 1.5 x 105 x g at 4C in a Beckman
L7 Ultracentrifuge with a 70.1T rotor. Eight 1.6 ml
fractions were collected, and 100 /I of each was evaluated for
carbohydrate content. The four densest fractions were pooled,
and the volume was brought up to 13.5 ml with 0.6 g/ml CsCl,
and the centrifugation was repeated, this time for 48 hours.
Following carbohydrate quantitation, fractions 2 to 5 (from
heaviest to lightest) were pooled, then concentrated and
desalted using a Centricon-100 concentrator (Amicon, Inc,
Beverly, MA) This solution was applied to a 2.6 x 14 cm
Sepharose 4B column. The fractions containing the void volume
were pooled, dialyzed against distilled water, and frozen at -
70C. Porcine gastric mucin (Sigma Chemical Co., St. Louis,
MO), was used as a mucin standard for comparative purposes.
Periodic acid-Schiff assay for carbohydrate quantitation.
The method used was that of Mantle and Allen (127) Periodic
acid solution was prepared by dissolving 25 mg of periodic
acid in 7% acetic acid (3.5 ml glacial acetic acid in 50 ml
distilled water. Each sample was brought up to a volume of
2.0 ml. 0.2 ml periodic acid solution was added to each
sample and incubated for 2 hours at 37C. Then 0.2 ml
Modified Schiff reagent (Fisher Chemical, Pittsburgh, PA) was
added, and samples were incubated 30 minutes at room
temperature. The optical density at 555 nm was read.


75
Protein concentration. A kit (Sigma Chemical Co., St.
Louis, MO) employing Peterson's modification of the micro
Lowry method was used to measure total protein concentration.
The kit was used according to the manufacturer's instructions.
Direct enzyme-linked lectin assay (ELLA). Porcine
gastric mucin standards, and serial dilutions of purified
rabbit colonic mucin in 100 /xl 0.5 M carbonate buffer (pH 9.6)
were coated onto duplicate 96-well polystyrene plates for 2
hours at room temperature. The plates were washed 4 times
with phosphate buffered saline containing 0.1% Tween-20 (PBS-
T20) using an automated plate washer. The plates were blocked
with 3% bovine serum albumin (BSA) in PBS-T20 for 1 hour at
37C. Following incubation, the plates were again washed, and
then incubated with 100 /il of a 1:200 dilution of a 1 mg/ml
solution of soybean agglutinin-horseradish peroxidase
conjugate (SBA-HRP) in PBS-T20 for 1 hour at 37C. Following
5 washes with PBS-T20, 85 ¡J.1 of the substrate 0.4 mg/ml o-
phenylene diamine in 0.05 M phosphate-citrate buffer (OPD) ,
was added to each well. After 10 minutes at room temperature,
the reaction was stopped with 85 /I of 4 N H2S04. The optical
density was read at 490 nm.
Western Blots. Six percent SDS-polyacrylamide gels were
prepared (71) Samples, up to 100 /l, were boiled 3 to 5
minutes in 0.25 M 2-mercaptoethanol and 0.1% SDS and applied
to gels. Following electrophoresis, gels were either stained
for protein with 0.25% Coomassie Blue, or samples were


76
transferred to nitrocellulose by wet electrophoretic transfer
for measurement of lectin-binding activity (mucus) of total
glycoprotein.
After transfer, blots to be labelled with lectin were
washed with PBS, and blocked in 2.5% casein or 3% BSA in PBS
for 1 hour at 37C. The blots were transferred to a solution
of 10 /g/ml SBA-HRP in blocking solution. Following a 1 hour
incubation at 37C, blots were washed 4 times for 5 minutes
each in PBS. The substrate solution of 300 mg/ml 4-chloro-l-
naphthol was added, and color was allowed to develop for 30
minutes.
Alternatively, transferred blots were stained for total
glycoprotein with periodic acid/Schiff (224) Membranes were
washed in distilled deionized water for 5 minutes, then
incubated in 1% periodic acid, 3% acetic acid for 15 minutes.
Following 15 minutes washing with several water changes,
modified Schiff's reagent (Fisher Chemical, Pittsburgh, PA)
was added, and the membrane was stained for 15 minutes in the
dark. The reaction was stopped by a 5 minute incubation in
0.5% sodium bisulfite solution, and the membrane was washed
and dried.
Results
Rabbit colonic mucin was successfully purified, as
evidenced by a lack of contaminating proteins on SDS-PAGE
(figure 4.1, lane 3), and a strong lectin-binding signal in


77
the stacking gel of a Western blot (figure 4.2, lane 3). No
band was seen at 118 kDa, corresponding to the "link"
glycopeptide (188) although the mucus was collected with
protease inhibitors. Using bovine serum albumin as a
standard, the concentration of protein in the purified mucus
solution was calculated to be 134 ¡xg/ml by a modified Lowry
assay. Using commercially obtained porcine gastric mucin as
a standard, the glycoprotein concentration for the same sample
was calculated to be 73 /xg/ml by the PAS method, but only 17
/ig/ml by ELLA.
Coomassie blue staining reveals a major band at 55 kDa in
all the cecal filtrates examined (figure 4.1, lanes 5-8),
along with a smear that may or may not contain minor bands.
This major band most likely represents serum albumin (65).
Mucin itself is not visible at these concentrations, in either
purified or crude samples. As would be expected, homogenates
of ileal and colonic explants demonstrate a large number of
protein bands.
For many cecal filtrate and medium/filtrate samples, the
only band detectable with soybean agglutinin was in the
stacking gel, and consistent with mucus (figure 4.2, lanes 5-
7) Other samples showed a smear of high molecular weight
material, with a major band at 144 kDa (lanes 8-11). PAS
staining of gels (data not shown) gave very similar results to
lectin staining. However, only the material in the stacking
gel representing mucus was visible in homogenates of ileal and


78
colonic explants (lanes 12-13), although there was a large
number of protein bands (figure 4.1, lanes 9-10).
Discussion
Soybean agglutinin binds to both rabbit colonic mucin and
porcine gastric mucin. There appear to be fewer available
binding sites on the rabbit mucin, as the amount of purified
rabbit colonic mucin calculated from the ELLA was much less
than was calculated by more conventional means. This is not
surprising, since colonic mucins are more highly sialylated
and sulfated than gastric mucins (160), making the internal N-
acetylgalactosamine residues less accessible. Therefore,
although porcine gastric mucin is used as a standard
throughout this study because of availability and consistency,
the actual concentrations of rabbit mucus are probably higher
than reported. However, the relationships between rabbit
samples will remain the same.
It is worth noting that this mucin was collected from a
rabbit with experimental mucoid enteropathy. A recent paper
(89) demonstrates changes in lectin-binding capacities in
mucins from rabbits with mucoid enteropathy. It indicates an
increase in binding to soybean agglutinin, as well as other
lectins with affinities for internal sugar residues,
consistent with the incomplete glycosylation characteristic of
many disease states.


79
The soybean agglutinin is not as specific a marker for
mucus as had been expected. However, when non-mucin bands are
present in cecal filtrates, they do not appear to change after
explant incubation, and can therefore be subtracted out.
Furthermore, they are not present in the homogenates of the
explants themselves, and so cannot be released into the
medium/filtrates following incubation.
Evaluation of Enzyme-Linked Lectin Assay
Introduction
Soybean agglutinin, the lectin used in the Western blots,
was also used in an ELLA (27). The mucus-containing sample
was used to coat polystyrene wells, and a SBA-HRP conjugate
was used to quantitate the amount of mucin bound.
However, a direct assay is generally not considered
appropriate for samples that contain only a small
concentration of the target molecule. A standard polystyrene
ELISA plate binds only 100 ng protein/well (71); the
medium/filtrate samples contain approximately 10 mg/ml.
Therefore, the majority of an undiluted sample does not bind
to the plate, and is removed in the first wash. Only when the
sample is diluted will a significant fraction of it bind.
Looking at the problem another way, if there is 1 /xg/ml mucus
in a sample that contains 10 mg/ml total protein, then mucus
constitutes 0.01% of that sample. If 100 ng protein binds to


80
the plate, then only 0.01 ng of mucus will bind, which is at
approximately the limit of detection (71).
Three types of modifications were made in an attempt to
develop a lectin-based assay that would be more sensitive.
One set of modifications was based on classic ELISA techniques
(71). Competition (indirect) assays and sandwich assays are
used to capture the antigen of interest out of contaminated
samples. Crude purification of the samples themselves was
also considered, and affinity chromatography and size
separation by ultrafiltration were performed. Finally, use of
a substrate that has a greater binding capacity (e.g.
nitrocellulose vs. polystyrene) allows for greater binding of
both contaminants and the molecule of interest, so that it can
be accurately quantitated without as great dilution of the
sample.
Materials and Methods
Unless otherwise noted, all reagents were obtained from
Sigma Chemical Co. (St. Louis, MO). The samples used for this
experiment were medium/filtrate samples following explant
incubation that had been obtained from pilot studies.
Competition assay. Plates were coated with porcine
gastric mucin (10 ng/well, 100 ng/well, or 1 /xg/well) for 2
hours at room temperature, and then blocked with 2.5% casein
for 1 hour at 37C. 100 /xl of serial dilutions of porcine
gastric mucin (100 ng/ml stock) or medium/filtrate sample were


81
added simultaneously with 100 /x 1 of 1, 2, 5, or 10 /xg/ml SBA-
HRP. Alternatively, mucin containing samples and SBA-HRP were
preincubated together for 1 hour at 37C, and then applied to
the plate. Plates were washed with PBS-T20, and 100 /x 1 of the
substrate OPD was added. After 10 minutes, the reaction was
stopped with 100 /xl of 4N H2S04, and the optical density was
read at 492 nm.
Sandwich assay. Plates were coated with 2 /xg/well of
unconjugated soybean agglutinin (Glycine max lectin), Wisteria
floribunda lectin, or Madura pomfera lectin for 2 hours at
room temperature, and then blocked with 2.5% casein for 1 hour
at 37C. Then 100 /x 1 of serial dilutions of porcine gastric
mucin (100 ng/ml stock) or medium/filtrate sample were added,
and incubated 1-2 hours at 37C. After multiple washes with
PBS-T20, 100 /xl of 10 /xg/ml SBA-HRP was added, and incubated
1 hour at 37C. Plates were washed with PBS-T20, and 100 /xl
of the substrate OPD was added. After 10 minutes, the
reaction was stopped with 100 /xl of 4N H2S04, and the optical
density was read at 490 nm.
Centricon filtration. Centricon-100 (Amicon, Inc.,
Beverly, MA) filters were rinsed with distilled water, and
used according to the manufacturer's instructions to separate
1 ml samples. Both retentates, which should contain only
mucus and other molecules > 100,000 Daltons, and filtrates,
which should contain molecules < 100,000 Daltons, were assayed
in direct ELLA. In addition, these samples were applied to a


82
6% SDS-polyacrylamide gel to determine the actual protein
compositions.
Affinity chromatography. One hundred /I of SBA-sepharose
beads were washed and resuspended in 100 /xl PBS, and added to
50 ill of sample. After 15 minutes of incubation at room
temperature with some mixing, the beads were removed by
centrifugation for 1 minute at 1000 x g. The supernatant was
retained as "wash 0", an indication of how much mucus did not
stick to the beads. The beads were washed 3 times with 1.4 ml
PBS. Free N-acetylgalactosamine (200 /xl of 10 mM, 0.1 M, 0.5
M, or 1.0 M) was added, and incubated for 15 minutes at room
temperature to elute the mucus. Following centrifugation, the
eluates were collected and dialyzed overnight vs. PBS. The
washes and eluates were analyzed for mucus concentration using
a direct ELLA.
Enzyme-linked lectin flow-through assay. An Easy-Titer
ELIFA apparatus (Pierce, Rockford, IL) was assembled according
to the manufacturer's instructions, using either
nitrocellulose or a Biodyne B membrane. The membrane was
coated with 100 /x 1 of serial dilutions of porcine gastric
mucin (10 /xg/ml stock) or medium/filtrate sample, which was
pulled through the membrane over a 5 minute period. A 2.5%
casein or 3% BSA solution was pulled through over a 10-15
minute period to block the membrane. One hundred /xl of 0.1,
0.25, 1, 2.5, or 10 /xg/ml SBA-HRP was added, and pulled
through in 5 minutes. Plates were washed 3 times with 200 fil


83
PBS, and 100 ¡jlI of the substrate OPD was pulled through as
quickly as possible. The reaction was stopped with 100 /xl of
4N H2S04, and the optical density was read at 490 nm.
Dot blots. Dot blots were performed using nitrocellulose
in a miniblot apparatus. Serial dilutions of mucus or samples
in PBS or 0.5 M carbonate buffer, pH 9.6, were used to coat
the nitrocellulose for 2-3 hours at room temperature. The
blots were removed from the apparatus, washed with PBS, and
blocked with 3% BSA for 1 hour at 25C. Five ig/ml SBA-HRP in
PBS was added, and incubated for 1 hour at 25C. As a control
for endogenous peroxidase activity, some blots were not
incubated with SBA-HRP, but rather remained in blocking
solution. Following four 5 minute washes with PBS, blots were
developed by addition of 0.3 g/ml of the substrate 4-chloro-l-
naphthol in 50 mM tris, pH 7.6. After 5-10 minutes, the blot
was washed with distilled water and dried. Densitometry was
performed to quantitate color development.
Results
In all competition assays the optical density reading
obtained was a direct reflection of the coating mucus
concentration, and SBA-HRP concentration. There was no
difference in optical density in the presence or absence of
competing mucus or filtrate sample.
In the sandwich assay, the optical density readings
depended solely on the type of lectin used for coating the


84
plate. With soybean agglutinin coating, the optical density
ranged from 1.8 to 2.5; for Wisteria floribunda the range was
2.1 to 2.7; for Madura pomifera it was 1.5 to 2.3. The
presence or absence of mucus had no effect.
Direct ELLA of retentates following Centricon-100
ultrafiltration did not demonstrate appropriate decreases in
optical density with serial dilution. SDS-PAGE demonstrated
that the banding pattern for these retentates was identical to
that for unfractionated medium/filtrate samples (figure 4.1).
No protein was visible by Coomassie blue staining of the
ultrafiltrates on SDS-PAGE.
Following affinity chromatography with soybean
agglutinin, the material eluted from the beads was calculated
to contain approximately 25 ng/ml mucus by direct ELLA.
However, the material in "wash 0" (that which did not bind to
the beads) contained approximately 250 ng/ml mucus, as
measured by ELLA.
With the Easy-Titer system, the optical density readings
were dependent on the SBA-HRP concentration used for
detection, but did not vary with mucus concentration.
Background readings (no mucus added) were 2.4-2.8 for 10 /g/ml
SBA, 2.1-2.5 for 2.5 /xg/ml, 1.9-2.3 for 1 /xg/ml, and 1.1-1.4
for 0.25 /xg/ml on Biodyne B membranes. The background values
on nitrocellulose were lower; however values for mucus coated
wells were also lower, and consequently meaningless.


85
In contrast, dot blots demonstrated appropriate
dilutional effects for both samples and mucus standards; wells
which did not contain mucus did not have color development.
However, dots containing medium/filtrate samples that were not
incubated with SBA-HRP demonstrated significant color
development, indicating the presence of endogenous peroxidase
activity within the samples. Subtraction of densitometry
values obtained in the absence of SBA-HRP from those obtained
in the presence of SBA-HRP led to inconsistent data that were
deemed unusable.
Discussion
Attempts to increase sensitivity and specificity of mucus
quantitation over the direct ELLA described by Cohan (27), and
used in the pilot studies were uniformly unsuccessful.
Competitive assays for the measurement of mucus have been
successful using an antibody-based system (133) However,
mucin in solution was not able to compete with bound mucin for
lectin binding. It may be that the lectin has a greater
affinity for mucin that is bound to the plate as opposed to
mucin that is free in solution. Attachment to the plate may
allow greater exposure of N-acetylgalactosamine sites to which
the lectin may bind. Alternatively, mucin may bind to the
lectin in solution, but not block all of the multiple binding
sites, thereby allowing the lectin to bind to bound mucus.


86
Two sets of lectins were used for sandwich assays. At
first, the same soybean agglutinin was used to coat the plate
and for detection. Soybean agglutinin itself contains N-
acetylgalactosamine residues (60), so it is no surprise that
once the plate was coated with lectin, the lectin bound
maximally, even in the absence of mucin. The other lectins
used for coating were Wisteria floribunda lectin, a
glycoprotein which does not contain N-acetylgalactosamine, and
has an affinity for N-acetylglucosamine; and Madura pomfera
lectin, a non-glycoprotein that has an affinity for N-
acetylgalactosamine (60). Unfortunately, the lectin used for
detection was again soybean agglutinin; so that each coating
lectin had a direct affinity for the detecting lectin. Use of
a different lectin for detection was not attempted, although
it might have been successful.
Ultrafiltration was performed to remove contaminants with
a molecular weight of < 100,000 Daltons. However, the
acrylamide gel demonstrates that essentially all proteins were
retained by the filter. Therefore, the contamination in the
sample was not reduced, and ultrafiltration was of little
benefit. It has been recently reported that nuclease
treatment and acidification are required to separate
extraneous proteins from mucin (157).
Affinity chromatography was successful in removing
contaminants from the final sample. Unfortunately, more mucus
was present in the initial wash than in the sample eluted from


87
the beads. This lack of affinity between the lectin and mucin
in solution may relate to the problems with the competition
assay.
Nitrocellulose was selected as a substrate with increased
binding capacity for the enzyme-linked lectin flow-through
assays and dot blots. This worked well for samples containing
just mucus, or even samples that had been "spiked" with a
known contaminant, such as BSA. Unfortunately, cecal contents
contain significant endogenous peroxidase activity.
Peroxidase was selected as an enzyme over alkaline
phosphatase, which is known to have significant endogenous
activity in the gut (71) In the polystyrene plate assay,
small amounts of endogenous peroxidase activity can be
detected, but these values are very small compared to the
mucus/lectin-associated activity. However, when measured by
densitometry, nitrocellulose blots incubated without
lectin/horseradish peroxidase conjugate often showed greater
activity than those incubated with the conjugate. It is not
clear why the endogenous peroxidase should bind with more
avidity to nitrocellulose than to polystyrene, but the fact
exists that it is not feasible to quantitate mucus bound to
nitrocellulose.
Therefore, it was not possible to improve upon the direct
ELLA using the above techniques. In order to use the direct
assay, it is necessary to perform serial dilutions to
ascertain that the values used for analysis are not


Full Text


MUCUS SECRETAGOGUE ACTIVITY IN CECAL CONTENTS
OF RABBITS WITH EXPERIMENTALLY-INDUCED
MUCOID ENTEROPATHY
By
CHARLOTTE EVANS HOTCHKISS
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1994

Copyright 1994
by
Charlotte Evans Hotchkiss

To Mark, Laura, and Arthur who never let me forget what
is really important.

ACKNOWLEDGMENTS
First and foremost, I would like to thank my advisor, Dr.
Merritt for his continuous advice and support. Special thanks
also go to Dr. Moreland for support in the laboratory animal
training program that allowed me to work for this degree. In
addition, my committee members whose scepticism inspired me to
prove my point were instrumental in completing this work. I
would like to thank Dr. Jacobson and Sylvia Tucker for
allowing me to use their laboratory, computer,
photomicroscope, and supplies. Mark Hotchkiss provided help
with the photography. Finally, thanks go to Dr. Davis and the
Animal Resources Department for diagnostic laboratory support,
and particularly to Dr. Schoeb for his help with the
histopathology.
IV

TABLE OF CONTENTS
ACKNOWLEDGMENTS iv
LIST OF FIGURES viii
KEY TO ABBREVIATIONS X
ABSTRACT xi
CHAPTERS
1. INTRODUCTION 1
2. LITERATURE REVIEW 6
Introduction 6
Characteristics of Mucus 7
Physical Characteristics 7
Production 16
Secretion 21
Degradation 3 0
Postulated Functions of Mucus 33
Lubrication 33
Cytoprotection 33
Protection from Infection 35
Nutrient for Flora 42
Diffusion Barrier 44
Creation of Microenvironment 46
Techniques Used to Study Mucus 4 8
Model Systems 4 8
Quantitation of Mucus 50
Secretion of Mucus 51
Composition of Mucus 52
Diffusion Through Mucus 53
Microbial Virulence 53
Theoretical Considerations 54
Lubrication 54
Cytoprotection 55
Protection from Infection 55
Nutrient for Flora 56
Diffusion Barrier 57
Creation of Microenvironment 58
Conclusions 59
v

3. PILOT STUDIES 6 0
Introduction 60
Cecal Filtrate Collection 60
Intestinal Explants 61
Enzyme-Linked Lectin Assay 62
Data Analysis 63
Cecal Ligation 64
4. REFINEMENT OF METHODS 73
Use of Soybean Agglutinin to Quantitate Mucus . . 73
Introduction 73
Materials and Methods 73
Results 76
Discussion 78
Evaluation of Enzyme-Linked Lectin Assay 79
Introduction 79
Materials and Methods 80
Results 83
Discussion 85
Harvesting of Mucus from Explants 88
Introduction 88
Materials and Methods 88
Results 89
Discussion 89
5. CECAL LIGATION AS A MODEL OF MUCOID ENTEROPATHY . . 95
Introduction 95
Materials and Methods 96
Results 98
Discussion 102
6. MUCUS SECRETION FROM INTESTINAL EXPLANTS 120
Introduction 120
Materials and Methods 120
Results 123
Discussion 124
7. COMPARISON OF ELLA AND IN VITRO LABELLING 128
Introduction 128
Materials and Methods 128
Results 131
Discussion 132
8. PHYSICAL CHARACTERISTICS OF MUCUS SECRETAGOGUE . . . 136
Introduction 136
Materials and Methods 136
Results 139
Discussion 141
9. CONCLUSIONS 152
Future Directions 155
vi

APPENDICES
A. DATA TABLES FOR CHAPTER 3 156
B. DATA TABLES FOR CHAPTER 5 165
C. DATA TABLES FOR CHAPTER 6 171
D. DATA TABLES FOR CHAPTER 7 (RADIOACTIVITY) 185
E. DATA TABLES FOR CHAPTER 7 (ELLA) 193
F. DATA TABLES FOR CHAPTER 8 (RADIOACTIVITY) 196
G. DATA TABLES FOR CHAPTER 8, PART A (ELLA) 2 00
H. DATA TABLES FOR CHAPTER 8, PART B (ELLA) 2 04
REFERENCE LIST 207
BIOGRAPHICAL SKETCH 232
Vll

LIST OF FIGURES
Figure page
3.1. Sources of intestinal explants 66
3.2. Standard curves (ELLA) 67
3.3. Serial dilutions of control filtrates 68
3.4. Serial dilutions of ME filtrates 69
3.5. Mucus in medium/filtrates (ELLA) 70
3.6. Mucus secretion from explants 71
3.7. Site of cecal ligation 72
4.1. 6% SDS-polyacrylamide gel 91
4.2. Western blot (lectin) 93
4.3. Explant treated with N-acetylcysteine 94
5.1. Excessive colonic mucus 106
5.2. Gross cecal necrosis 107
5.3. Weight gain or loss 108
5.4. Cecal necrosis and inflammation 109
5.5. Goblet cell hyperplasia Ill
5.6. Depletion of acidic mucin 114
5.7. Colonic inflammation 117
5.8. Characteristic of cecal contents 119
6.1. Mucus in medium/filtrates (ELLA) 126
6.2. Mucus secretion from explants 127
7.1. Mucus in medium/filtrates (ELLA) 134
viii

7.2. Comparison of ELLA and tracer secretion 135
8.1. Proportion of tracer in medium/filtrate 145
8.2. Precipitable secreted tracer 146
8.3. Mucus secretion, part A (ELLA) 147
8.4. Mucus secretion, part B (ELLA) 148
8.5. Western blot (lectin) 149
8.6. Western blot (lectin) 150
8.7. SDS-polyacrylamide gel 151
IX

KEY TO ABBREVIATIONS
BSA
Bovine serum albumin
CAMP
Cyclic adenosine monophosphate
EDTA
Ethylenediaminetetraacetic acid
ELISA
Enzyme-linked immunosorbent assay
ELLA
Enzyme-linked lectin assay
H&E
Hemotoxylin and eosin
HBSS
Hank's balanced salt solution
kDa
Kilodalton
ME
Mucoid enteropathy
NZW
New Zealand White
OPD
O-phenylene diamine
PBS
Phosphate-buffered saline
PBS-T20
Phosphate-buffered saline-tween 20
PMSF
Phenylmethylsulfonyl fluoride
SBA-HRP
Soybean agglutinin-horseradish peroxidase
SEM
Standard error of the mean
SPF
Specific pathogen-free
VIP
Vasoactive intestinal peptide
x

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
MUCUS SECRETAGOGUE ACTIVITY IN CECAL CONTENTS
OF RABBITS WITH EXPERIMENTALLY-INDUCED
MUCOID ENTEROPATHY
By
Charlotte Evans Hotchkiss
August 1994
Chairperson: Dr Alfred Merritt II
Major Department: Veterinary Medicine
Mucoid enteropathy is a disease of rabbits, characterized
by copious mucus secretion in the intestinal tract. The
etiology of this disease is unknown. This study uses an
adaptation of the cecal ligation model developed by Toofanian
and Targowski to induce experimental mucoid enteropathy.
Filtrates of cecal contents from these rabbits and from
control animals were prepared. Explants from healthy rabbits
were incubated with these filtrates, and mucus secretion was
measured using an enzyme-linked lectin assay, or by measuring
secretion of radiolabelled mucus from explants that had been
preincubated with 3H-glucosamine. The results demonstrate the
presence of a secretagogue for colonic mucus in the cecal
contents of rabbits with experimentally-induced mucoid
enteropathy. Pretreatment of filtrates demonstrates that this
secretagogue can be precipitated with ammonium sulfate, and is
xi

destroyed by heat (100°C for 30 minutes) or strong acid (pH 1
for 30 minutes).
Xll

CHAPTER 1
INTRODUCTION
Mucoid enteropathy (ME) is a disease that affects only
rabbits. It primarily affects young animals, but it can occur
at any age (105) . Mortality from mucoid enteropathy has been
reported as 1-2% (238) to 10-20% (105, 251) of rabbits
kindled. Signs associated with the disease include anorexia,
dehydration, depression, subnormal temperature, distended
abdomen, and the defecation of clear to yellow gelatinous
mucus in the feces (238) . Afflicted rabbits may also have
diarrhea, or may pass nothing but mucus (208) . There is
disagreement as to whether or not affected rabbits drink
water. In our experience, and in some reports (235) , they are
adipsic; but many reports describe polydipsia (238, 251).
Clinically, the rabbits are dehydrated. Afflicted rabbits may
die acutely, or after several days of illness. Affected
rabbits may recover, with or without intervention (105).
At necropsy, the consistent finding is copious amounts of
clear to yellow gelatinous mucus in the colon. The small
intestine may contain fluid and/or mucus. The cecum is often
impacted with dry digesta, which may contain gas pockets. The
gallbladder is often distended. Histologically, there may be
goblet cell hyperplasia, most noticeable in the ileum but may
1

2
occur in any part of the gastrointestinal tract. There is
minimal histologic evidence of inflammation, with mild mixed
infiltrates seen inconsistently (142, 238, 251).
Some reports of "mucoid enteritis" (142, 163) describe
gross paintbrush hemorrhages of the large intestine, and
microscopic edema and inflammation. Such lesions are more
characteristic of clostridial enterotoxemia, a more recently
defined disease of young rabbits (20), which is caused by an
overgrowth of toxin-producing clostridial organisms, C.
spiroforme or C. difficile most commonly. This disease is
usually initiated by antibiotic administration or overeating
of a high calorie, low fiber diet. Clinical signs of
enterotoxemia include anorexia and dehydration, as in mucoid
enteropathy. However, rabbits with enterotoxemia usually have
watery diarrhea and tend to die acutely. Interestingly,
animals with enterotoxemia can also pass large quantities of
mucus, suggesting that mucoid enteropathy may be a syndrome
with many possible etiologies, rather than a specific disease.
Studies of the cause and treatment of mucoid enteropathy
has thus far been unrewarding. Several studies have
concentrated on potential infectious causes, especially since
the incidence of the disease occurs in "outbreaks." Greenham
(64) found that tetracycline decreased diarrhea and delayed
death, but did not prevent or cure the disease. Van
Kruiningen (238) created an oral inoculum of macerated
intestine and contents in an attempt to transmit the disease.

3
Only 5 out of 17 of these transfaunated rabbits became ill,
while 2 out of 17 rabbits fed a control inoculum also
developed mucoid enteropathy. Intestinal coliform
concentrations are often increased in animals with mucoid
enteropathy (64, 113, 207); however inoculation with E. coli
obtained from rabbits exhibiting diarrhea, or E. coli heat
labile enterotoxin (207) failed to induce disease. Lelkes and
Chang (114) found several differences between the cecal flora
in normal rabbits and in those with mucoid enteropathy, but
could not identify a single organism associated with the
disease. They also transfaunated cecal contents from sick
rabbits directly into the ceca of healthy fistulated rabbits
and were unable to cause increased mucus secretion.
Research on ME has generally focused on determining the
inciting cause. There could very well be more than one
etiology. Individual cases have been seen in adult rabbits
with debilitating and/or stressful conditions, such as
neoplasia, surgical stress, and septicemia (Hotchkiss,
unpublished observations) . The present study was therefore
designed to focus on the direct cause of the increased mucus
secretion, the one consistent finding in all afflicted
animals.
In the past, studies of mucoid enteropathy have been
hampered by the lack of viable models of the disease. There
is now one model available: based on the hypothesis that
mucoid enteropathy is primarily due to constipation,

4
Sinkovics (207) was able to induce colonic mucus
hypersecretion by surgical ligation of the colon. Toofanian
and Targowski (235) found that ligation of the blind pouch of
the cecum, permitting the flow of ingesta from ileum to colon,
was just as effective, causing mucus hypersecretion in
approximately 70% of rabbits. Partial cecectomy did not
induce a mucoid enteropathy-like syndrome, while injection of
tetracycline into the ligated cecum prevented the development
of disease (235). Little work has been done with the cecal
ligation model, however. Toofanian's group found decreased
small intestinal disaccharidase activity in ligation-induced
sick rabbits (233) , and also minor alterations in cecal
volatile fatty acid concentrations (234) . They incubated
colonic explants with cecal contents from rabbits with
experimentally-induced mucoid enteropathy and reported goblet
cell hyperplasia (236) .
In the project described herein, methods have been
developed to test this finding in a quantitative manner.
Evidence is presented to support the hypothesis that cecal
contents from rabbits with mucoid enteropathy possess mucus
secretagogue activity. Preliminary tests have been carried
out to determine the nature of the secretagogue.
The importance of doing research on this disease is
twofold: First, little is known about it, considering that it
is one of the most important causes of morbidity and mortality
in rabbit production (113). Since rabbits are used in large

5
numbers in biomedical research, these losses can add up to
substantial costs, especially in situations where affected
animals have been injected for antibody production with very
precious and irreplaceable antigens. Second, this disease
provides a unique opportunity to study the control of mucus
secretion. Increased mucus secretion without inflammation can
be seen in enterotoxic diseases, such as cholera and E. coli
enterotoxemia, but there is no associated goblet cell
hyperplasia (96), and there are other confounding toxic
effects, notably a secretory diarrhea. On the other hand,
goblet cell hyperplasia with mucus hypersecretion can be seen
with some diseases and parasitisms (96), but because of the
associated inflammatory changes it is difficult to examine the
mucus related aspects alone.
Although the secretagogue effects of specific chemicals
such as acetylcholine and PGE2 are well known (160, 258),
their physiological importance is not always clear. This
study is intended to demonstrate the presence of a
secretagogue that is pathologically relevant, with few effects
other than the stimulation of mucus production. It may be a
microbial product or a host secretion; in either case it could
prove to be useful for studying the processes controlling
differentiation of goblet cells, as well as production and
secretion of intestinal mucus.

CHAPTER 2
LITERATURE REVIEW
Introduction
In studying a disease in which the most consistent sign
is mucus hypersecretion, it is necessary to examine the role
of mucus in the normal physiological state. This review
presents information on what mucus is, what controls its
synthesis, secretion, and degradation, and what the normal
functions of gastrointestinal mucus are.
The function of mucus has been a subject of study since
ancient times. The ancient Greeks considered mucus, commonly
translated as "phlegm", to be one of the four humors, along
with blood, yellow bile, and black bile. In the second
century Galen (56) stated, "The best physicians concur in the
opinion that if a considerable amount of phlegm accumulates on
account of some bad conditions of the body, the most serious
abdominal disorders ensue, intestinal obstruction, lientery,
and tenesmus". Although it is now generally believed that
mucus is involved in lubrication rather than obstruction,
there is no question that increased mucus production occurs in
certain disease states.
6

7
Characteristics of Mucus
Physical Characteristics
Initially, the term mucus was used to describe any
viscous material in the stomach and intestines. Primarily,
mucus was the jelly-like material which lined the
gastrointestinal tract. However, for a time, the material
which could be precipitated from gastric juice with suitable
agents was known as "dissolved mucin" (9) . However, as it was
found that this material contained a heterogenous mixture of
substances, this terminology was dropped (59). Still, mucus
was variably described as clear and colorless, white and
cloudy, and occasionally yellow. Hollander et al. (81)
determined that these differences were due to the amount of
cells and debris dissolved in the mucus layer. They were able
to show that the irritants used to stimulate mucus secretion
resulted in desquamation of epithelial cells, associated with
inflammation (80).
Pilocarpine injected intravenously causes increased
secretion of mucus of egg-white consistency; however, an
enormous dose is required (44) . In 1949, Morton and Stavraky
(153) found that injection of acetylcholine into the
mesenteric arteries caused secretion of mucus, while Janowitz
et al. (92) found that topical administration of acetylcholine
resulted in secretion of clear, cell-free mucus, facilitating
the study of "pure" mucus. It is now generally accepted

8
terminology that mucin is a specific glycoprotein, while mucus
is a heterogenous mixture containing mucin, protein, lipids,
nucleic acids, water, and electrolytes. Specifically, mucus
contains trefoil-type protein, kallikrein protease, lactose¬
binding lectins, vitamin B12 binding protein, and other
proteins which are secreted with mucin from the same granules
in the goblet cell (178). In the respiratory tract, most of
the lipid in the mucus is bound to the mucin glycoprotein
(157) . Also, nucleic acids entwine other proteins with mucin,
making acidification and nuclease treatment necessary in the
purification of mucin (157). In the digestive tract, the
mucus layer also includes bacteria, food particles, and
products of digestion. In disease states, such as cystic
fibrosis, mucus also contains materials from degenerated
leukocytes, especially DNA and actin which contribute to its
viscosity, and interfere with clearance (239) .
The most striking feature of mucus is its jelly-like
nature. Early studies revealed that unlike most proteins,
which show minimum viscosity at the isoelectric point, gastric
mucin shows a maximum viscosity at its isoelectric point of
4.98 (144). As rheological studies have become more
sophisticated, it has been shown that mucus is a visco-elastic
gel; and that the elastic component is greater than the
viscous component over the entire range of frequency and
strain studies with native and reconstituted mucus (4). The
elastic properties will be decreased, allowing the gel to

9
collapse if the mucin is treated with proteolytic enzymes or
if disulfide bonds are reduced with mercaptoethanol (4). In
addition, proper hydration is necessary for maintenance of the
gel. Hydration is controlled by small polyionic proteins, pH,
and electrolytes within the gel (242). Since mucin is a
negatively charged glycoprotein, a high concentration of a
positively-charged ion such as calcium can result in
condensation of the mucus gel, with an increase in viscosity
and lack of elasticity.
The chemical properties of mucin have been slowly
elucidated over the past century. Mucins are a heterogeneous
group of glycoproteins, with an average subunit molecular
weight of 2 x 10s (220). Carbohydrate comprises approximately
80% of this molecular weight, with the rest being protein.
The protein forms a core, with linear and branched
carbohydrate side chains covalently attached to form a "bottle
brush" structure (126) . As well as heavily glycosylated
areas, there are believed to be "naked" areas on the protein
core, which are subject to cleavage by proteolytic enzymes
(4). These regions also contain cysteine residues, allowing
for polymerization via disulfide bonds (160), and are the
major antigenic determinants of mucin (131). The glycosylated
portion of the protein core contains a high proportion of
serine and threonine, which are involved in oligosaccharide
linkage via hydroxyl groups (220). In addition, there is a

10
high percentage of proline, which prevents a-helix formation,
allowing for increased glycosylation and flexibility.
The oligosaccharide chains of mucin are attached to the
protein core via an O-glycosidic bond between serine or
threonine and N-acetylgalactosamine (220) . These chains may
be linear or branched, and vary in length from 2 to 12
residues. Some chains will terminate in A, B, H, Lewis3, or
Lewis13 antigens, reflecting the blood group genes of the
individual (41) . The backbone of each chain contains N-
acetylgalactosamine, N-acetylglucosamine, and galactose, which
may comprise I or i antigens (126) . The chains often
terminate in a fucose, sialic acid, or ester sulfate residue.
The glycosylation pattern depends on genetics, the region of
the gastrointestinal tract, the age of the animal, and can
vary in certain disease states, as determined by the
glycosyltransferase activity levels (228) . There is also a
great deal of heterogeneity in a single preparation, even
within a single granule of a goblet cell (220) .
The DNA sequences of several mucins have been determined
(116). These genes show a high degree of polymorphism due to
variable numbers of tandem repeats. The gene MUC1 encodes a
mucin-like cell surface glycoprotein which is present on many
epithelial cells, but MUC2 to MUC6 appear to encode secreted
mucins. MUC2 and MUC4 are associated with colonic mucins, and
MUC3 and MUC5 are associated with gastric mucins (116) .

11
For several years it was debated whether or not mucin
contains a "link" glycopeptide, of approximately 118 kDa
(160). Roberton et al. (188) demonstrated that this
glycoprotein is different from other mucin subunits, in that
it contains only 50% carbohydrate, and it contains mannose,
which is generally associated with N-linked glycosylation
(220) . Amino acid composition, carbohydrate composition, and
immunological studies have indicated that the "link"
glycopeptide is actually a 118 kDa fragment of fibronectin
(210). However, the cDNA for this peptide has recently been
sequenced in rats (257) and humans (256) , and the 118 kDa
glycopeptide appears to represent the cysteine-rich carboxyl
terminal of a much larger mucin-like peptide. It is now
generally accepted that proteolytic cleavage of the mucin core
peptide occurs during purification, and that reduction and
alkylation allows for separation of the larger N-terminal
fragment and the 118 kDa C-terminal fragment (262) .
In this chapter, there has been some generalization in
order to describe the overall functions of mucus. However, it
is important to recognize that mucin molecules are
heterogeneous, and some of the characteristics of mucus vary
along the length of the gastrointestinal tract, change during
development and maturation, and can be altered by disease
(160) . In fact, a diurnal rhythm to mucus secretion has been
seen in rats and mice which were given access to food only in

12
the daytime: there was more cell proliferation, and more mucus
was made during the daytime (73).
The mucin produced in newborn rats is different from that
found in adults (160). Newborn mucin contains more protein
and less sugar, resulting in increased density (216) . The
threonine content is increased, while serine and glutamic acid
content are decreased in newborn rats (160). Sulfation
decreases in the first 2 months of a rats life, with the major
change occurring at weaning; increased sulfation has also been
associated with mucus from "immature" cells (160).
Correspondingly, the pH within the mucus layer in the colon is
lower in suckling rats than in weanling or adult rats (193) .
Aged (350 day old) rats also have increased sulfation of mucus
(225) .
Newborn rat mucin has fewer fucose and N-
acetylgalactosamine residues, corresponding to altered lectin
binding (216) . This likely relates to developmental changes
in glycosyl-transferase activities (12). During the suckling
period, N-acetylgalactosaminyl-transferase activity remains
constant, sialyl-transferase activity decreases just prior to
weaning, while fucosyl-transferase activity decreases
gradually throughout the suckling period. Following weaning,
the activities of all three enzymes increase dramatically. In
a parallel manner, more sialyl-transferase can be detected
immunocytochemically in the mucus of mature goblet cells, when
compared to immature goblet cells in the colonic crypt (226) .

13
Sialylation of surface glycoproteins is replaced by
fucosylation at weaning, but sialylation of goblet cell mucus
continues (227).
The composition and structure of mucin varies along the
length of the gastrointestinal tract. The data on the actual
differences are sometimes contradictory, due to different
techniques, species differences, and the heterogeneous nature
of all mucins (160). In general, colonic mucin is more
aggregated, and contains more protein. It may not contain the
fragment that was formerly known as the "link" glycopeptide.
The concentration of acidic glycoproteins, containing sialic
acid and/or sulfate, relative to neutral glycoprotein
increases distally in the digestive tract. The gastric and
Brunner's gland secretions are largely neutral, lacking sialic
acid, but enriched in fucose, although in gastric mucin there
is a discrete subpopulation of highly sulfated mucin
associated with the mucous neck cells (90).
The viscosity of gastric mucus is greater than that of
duodenal mucus, while the elasticity is decreased (263). In
the stomach, there is a continuous layer of mucus, but the
thickness is variable, and tends to be thickest along the
lesser curvature (165). Mucus is present in the small
intestine, but mostly in the intervillous spaces, so that the
tips of the villi are sometimes visible. There is less mucus
in the ileum than in the duodenum and jejunum, so that most of
the villi are bare in vivo (165) , although a layer 50-200¡jl

14
thick can be seen in explants (67) . There is no distinct
mucus layer in the cecum (165, 194). In the proximal colon
there is a discontinuous layer of mucus, varying in
composition and thickness, while in the distal colon there is
a thin, compact, continuous mucus layer present (194, 225).
Mucus from the healthy colon is more hydrophobic than ileal
mucus; however, induced colonic inflammation decreases the
hydrophobicity (124).
Smectite is a cytoprotective agent that binds to mucus to
increase its barrier properties. It also causes gastric mucus
cells to produce mucus that contains more fucose and less
sulfate, as is seen with fasting (151) . Dietary fiber has
been shown to increase the rate of turnover of intestinal
mucins, and also change their lectin-binding properties,
reflecting a change in composition (152). This is consistent
with the fact that alterations in glycosyltransferase
activities are seen with different diets (12).
Mucus can be abnormal in various disease states.
Although in cystic fibrosis the major mucus abnormalities are
likely decreased hydration (160, 242) and the presence of
degenerated leukocyte components (239), some investigators
have also found an increased fucose content (231) . Celiac
disease (gluten-related enteropathy) results in a decreased
number of goblet cells, and a shift from neutral to acidic
mucin (160). Atrophic gastritis is associated with decreased
epithelial mucus, either due to decreased secretion or

15
increased degradation (135) . In mucoid enteropathy of rabbits
there are increased numbers of immature colonic mucins with
heterogeneous lectin binding patterns, perhaps due to the
increased rate of turnover (89). Abnormal mucins are also
seen in infection of rats with Nippostrongylus brasiliensis
(88) .
Goblet cell numbers are markedly reduced in patients with
ulcerative colitis (160). In addition, there is a change in
the composition, with decreased fucosylation and sulfation,
and exposure of galactose residues. This, along with deficient
O-acetylation of sialic acid (94) , is suggestive of incomplete
mucin glycosylation. Intestinal malignancy is also often
associated with loss of terminal sugars, decreased O-acylation
of sialic acid, and shortened oligosaccharide chains (160).
Consequently, the presence of small intestinal mucin antigen
(SIMA) in the colon, where there is normally masking of
antigens by sulfate, may reflect malignancy (43) . SIMA can be
detected in the serum of 36% of colorectal cancer patients vs.
5% of controls, and may be useful as a screening test (179).
The abnormal glycosylation appears to be a clonal phenomenon,
reflective of somatic mutation (55). Gastric adenocarcinomas
are abnormally rich in sulfomucin and sialic acid, which may
reflect cellular dedifferentiation, or abnormalities in the
regulation of mucin biosynthesis (160) .

16
Production
Mucus is formed by several cells in the gastrointestinal
tract. In the stomach, mucus is secreted by surface cells,
the mucous neck cells of the fundus, and the mucous cells of
the cardiac and pyloric glands. In the duodenum, the mucus-
secreting cells are the Brunner's glands. Throughout the rest
of the intestine the goblet cells are the source of mucus
(47) . Goblet cells are named for their characteristic goblet
shape seen in histologic section, but electron microscopic
studies on cryofixed tissues show that these cells are
columnar in vivo (84, 242).
Duthie (38) reported that the mucus granules are first
formed near the nucleus, then are transported to the Golgi
apparatus, where stainable mucus first appears. This is
consistent with current knowledge that the peptide backbone,
which comprises less than 50% of each mucin molecule, is
formed in the endoplasmic reticulum. Sugar residues are then
added by the linkage of N-acetylgalactosamine to serine or
threonine (19) . Unlike N-linked glycosylation that is
initiated in the endoplasmic reticulum, 0-linked glycosylation
occurs entirely within the Golgi and condensing vacuoles (63).
The initial enzyme involved, al,3N-acetylgalactosaminyl-
transferase, is located in the Golgi apparatus (191). The
enzyme responsible for terminal sialylation, S-galactoside
o;2,6 - sialy 1 transí erase, is present in the Golgi apparatus, but
also in post-Golgi apparatus structures, including the mucus

17
droplets and the plasma membrane (228). The mucin granules
are stored in condensed form in membrane bound vesicles within
the goblet cells until the mucus is secreted. During goblet
cell migration along the middle half of the villus, the mucin
granules are apparently renewed twice (23). The composition
of mucin in the granules is different along the crypt-villus
axis (23, 160); perhaps there is continued sialylation of
mucin within the storage granules.
The rate of mucin synthesis depends on the rate of
protein synthesis within the cell, the concentration and
positioning within the cell of glycosyltransferases, and the
presence of amino acid and sugar precursors (16 0) . Glutamine
is known to stimulate glycoprotein synthesis as the major
metabolic substrate in the intestinal epithelium (51), but
little is known about the control of internal cellular
factors.
Mucus synthesis can be decreased by factors which
interfere with protein or oligosaccharide incorporation.
Factors which interfere with protein synthesis will also
decrease mucus production. These include malnutrition, and
metabolic inhibitors such as cycloheximide and puromycin
(160) . Dietary fatty acids, non-steroidal antiinflammatory
drugs, and zinc diminish sulfation or interfere with
incorporation of individual sugars (51, 160) and may thereby
alter the composition of mucus. Cysteamine, which is used to

18
experimentally induce duodenal ulcers, decreases glycoprotein
production in Brunner's glands (104).
S-Adrenergic drugs, cyclic AMP, and theophylline have
been shown to increase overall glycoprotein synthesis in rat
and rabbit intestine (50, 110). Adrenergic compounds
(dopamine, epinephrine, isoproterenol, phenylephrine) have no
direct effect on mucus secretion, although they may increase
fluid secretion, making the mucus blanket appear thicker
(160) . Cholinergic drugs and cholera toxin increase both
synthesis and secretion of mucus (198). Reserpine, a
hypotensive agent and carcinogen, increases glycoprotein
synthesis; the mucin that is released is more viscous than
normal, so that reserpine-treated rats have been used as a
model for cystic fibrosis (169) . Epidermal growth factor has
been shown to increase glycosaminoglycan synthesis, and has a
protective effect on the gastric mucosa, but its relationship
to mucus secretion has not been studied (209) .
Histamine increases mucin synthesis in the canine stomach
via H2 receptors, through activation of adenylate cyclase and
increases in cAMP (198). Gastrin increases mucus synthesis in
the corpus of the rat stomach, and this effect is not blocked
by H2 blockers (86). Conversely, the H2 blockers roxatidine
and FRG-8813, but not cimetidine or ranitidine, increase mucin
synthesis (87). Additionally, the H+,K+-ATPase inhibitor NC-
1300-0-3, but not omeprazole, stimulates mucin synthesis (87) .

19
Anatomic alterations which affect the number of goblet
cells present and/or the rate of development will also change
the amount of mucus synthesized. Duodenal epithelial cells
from chick embryos show an increased number of goblet cells
when cultured in vitro compared to the number seen in vivo
(13). This increase in goblet cells is accelerated by
thyroxine, but prevented by hydrocortisone. Vitamin A
deficiency causes atrophy of goblet cells in salivary glands,
trachea, and small intestine (30). Salmonella infection in
mice decreases the number of goblet cells, apparently via
tumor necrosis factor a (8) . Feeding of dietary fiber has
been associated with increased turnover of jejunal mucins
(152) . Some substances which damage the intestinal
epithelium, such as methotrexate, will decrease the total
number of goblet cells, and therefore decrease mucin synthesis
(95). Following radiation damage (154), or the radiomimetic
disease caused by canine or feline parvovirus infection (96),
there is an initial increase in the number of immature goblet
cells, followed by a relative decrease, and then a second
increase as the tissue recovers. Corresponding changes in
mucus production take place following radiation exposure
(201) . A decreased proportion of goblet cells is seen in
lesions of transmissible colonic murine hyperplasia caused by
Citrobacter freundii biotype 4280, and is followed by goblet
cell hyperplasia during recovery (10).

20
Several organisms have been associated with goblet cell
hyperplasia leading to mucus hypersecretion, including
Treponema hyodysenteriae in swine, and Ostertagia and
Oesophagostomum species in ruminants (36, 96). These
organisms are also associated with a varying degree of
inflammation, so the hyperplasia may be a response to
inflammation, repair, or the organism.
Nippostrongylus braziliensis infection in rats causes
goblet cell hyperplasia as the worms are being expelled (147).
This appears to be associated with the immune response, since
hyperplasia will be seen earlier in immunized rats, and does
not occur if the rats are treated with antihelmintics early in
the course of infection. Trichinella spiralis, Nematodirus
bat tus, Trichostrongylus tenuis, and Hymenolepis dimunuta have
all been associated with goblet cell hyperplasia (111, 139).
Yersinia enterocolitica has been shown to cause goblet
cell hyperplasia and increased mucin synthesis throughout the
intestinal tract of rabbits (129) . There is also a great deal
of inflammation associated with this disease. The goblet cell
hyperplasia develops more rapidly and to a greater extent in
those areas of the intestine where mucosal injury is most
severe, and consequently may be associated with injury and or
inflammation. Alternatively, the hyperplasia may be part of
a repair mechanism, since it persists as the mucosa recovers
morphologically.

21
Intestinal coccidiosis caused by Eimeria species has been
associated with inflammation and goblet cell hyperplasia in
rabbits (114). Mucoid enteropathy, the subject of this study,
is characterized by copious production of intestinal mucus,
where the major histologic change reported is goblet cell
hyperplasia, and inflammation is minimal (238) .
On the other hand, it is important to realize that goblet
cell hyperplasia in response to infection and/or inflammation
is not a universal phenomenon. Mucus secretion is decreased
in both small and large intestines during infection with
Isospora suis in piglets (106, 107), and numbers of colonic
goblet cells are decreased with Ehrlichia risticii in horses
(187). Clostridium difficile toxin A had no effect on goblet
cells in rabbits (118).
Secretion
Mucus is secreted from intestinal goblet cells in two
ways. "Baseline", or constitutive, secretion, involving the
slow transport and secretion of glycoproteins that can be
documented by autoradiography, occurs in mucosal explants for
up to 24 hours in the absence of circulating factors or
enteric nerves. In fact, baseline secretion occurs even in a
cultured goblet cell line (173) . While the bulk of the mucin
granules remain in the center of the goblet cell after
formation, there is constant formation of mucus at the
supranuclear region of the cell. This mucin migrates along

22
the periphery towards the apical border of the cell over the
course of 4 to 6 hours (184). Thus, some newly formed mucin
reaches the apical border and is secreted before older mucin
granules (23). A single granule will undergo exocytosis,
discharging mucus into the lumen. This process apparently
involves the cytoskeleton, as depolymerization of microtubules
with colchicine prevents this migration (219) . However, once
the granule has reached the apical membrane, the act in
filaments localized there normally act as a barrier, since
depolymerization of actin filaments with cytochalasin D or
dihydrocytochalasin B results in increased baseline secretion
(167) . Organelles are gradually shed from goblet cells as
mucin is secreted, so that the mean cell volume decreases
along the crypt-villus axis (184).
In the cases where it has been examined, stimulated
secretion has been shown to occur by compound exocytosis
(217) . In this situation fusion of the initial mucin granule
with the plasma membrane is rapidly followed by tandem fusion
with the subjacent granules, allowing the contents of many
granules to exit through a single surface site. More recent
studies suggest that there is also fusion of granules within
the cell prior to exocytosis (84, 158) . Cavitation in a
goblet cell that has just discharged its granules in this
manner can be recognized by electron, or even light,
microscopy (217). Following cholinergic stimulation,
cavitation is visible in crypt cells for 15-30 minutes; after

23
that it is difficult to distinguish mucin-depleted goblet
cells from epithelial cells (178). Cavitation may never be
apparent in villous goblet cells (178). In rabbit colonic
goblet cells stimulated by acetylcholine, compound exocytosis
is not inhibited by colchicine, implying that microtubules are
not necessary, since the granules are already at the site of
release (219). However, the protein synthesis inhibitor
cycloheximide, the microtubule inhibitor colchicine, and the
actin inhibitor cytochalasin B were all found to inhibit
cholera toxin stimulated secretion in rabbit ileal loops
(164) . A jack-in-the-box mechanism for secretion has been
proposed (242) , in which a small pore is opened over the
granule, water is allowed to enter and calcium can leave, with
the expansion of the granule contents causing release from the
cell.
It has been reported recently that mucus secretion is
stimulated independently by cAMP/protein kinase A and
increased intracellular calcium/protein kinase C mechanisms
(93), although previous reports state that cAMP does not
affect mucus secretion (161). In situ hybridization reveals
the calcium binding protein calcyclin preferentially expressed
in mucus-secreting cells, suggesting that calcyclin, in
conjunction with the p36 subunit of calpactin, is involved in
calcium-stimulated mucus secretion (232).
Several substances are known to stimulate mucus
secretion. Agents that disrupt the mucosal barrier, such as

24
mustard oil, alcohol (160), bile salts (117), and even
mechanical irritation (51) cause mucus release from surface
goblet cells. It has been shown that mustard oil causes
compound exocytosis (217). Florey (45) demonstrated that
secretion in response to mustard oil could be blocked with
cyanide, indicating that the process requires energy, and
secretion is not simply due to disruption of the plasma
membrane. Mechanical irritation has been shown to increase
levels of prostaglandins (26), which could be acting as
secondary messengers to stimulate secretion. Crypt goblet
cells are not affected, presumably because they do not come
into contact with the irritant. Proteolytic enzymes are known
to be mucus secretagogues in respiratory epithelium, but this
has not been shown in the intestine (22).
Stimulation of extrinsic autonomic nerves or electrical
field stimulation causes discharge of intestinal mucus in vivo
(175). This has been shown to be due to muscarinic
cholinergic innervation, as injection of pilocarpine
accelerates release of mucus by compound exocytosis. Vagotomy
or vagal stimulation does not affect mucus secretion in the
rabbit jejunum, implying that cholinergic control takes place
entirely within the enteric nervous system (65) .
Acetylcholine-induced secretory events occur rapidly, and are
generally complete within 5 minutes (158, 178). It was long
thought that only crypt cells were susceptible to stimulation
(218), but it is now known that villous cells secrete a

25
significant amount of mucus (100), although they do not
demonstrate cavitation (178). In the rat, even crypt cells
are unresponsive to carbachol until 20 to 25 days of age,
corresponding to weaning (176). Acetylcholine (218),
pilocarpine (219), and carbachol (174) have also been shown to
stimulate mucus release in colonic explant systems.
Furthermore, it is fairly certain that the goblet cells
themselves possess muscarinic receptors, as secretion occurs
in a goblet cell line descended from a colonic adenocarcinoma
(173) . The response is variable, but this may reflect the
variation of responsiveness in vivo between crypt cells and
the older surface or villous cells.
Prostaglandins, particularly PGE2, increase mucus
secretion, and perhaps synthesis (197, 198, 258). Non¬
steroidal antiinflammatory drugs and indomethacin decrease
mucus synthesis and secretion, and it has been proposed that
inhibition of prostaglandin synthesis may be responsible (164,
206). Glucocorticoids also decrease mucus secretion (141).
Low doses of nicotine greatly decrease rectal prostaglandin
levels and decrease the thickness of the mucus layer, while
high doses cause an increase in mucus thickness and decrease
prostaglandin concentrations less dramatically (266). Other
arachidonic acid metabolites (1eukotrienes,
hydroxyeicosatetranoic acids) act as mucus secretagogues in
respiratory epithelium (171), but do not affect rabbit colon
in vi tro (177) .

26
Gastrointestinal peptide hormones have also been
suggested as potential mucus secretagogues. Neutra et al.
(161) found no increase in compound exocytosis in rabbit
intestinal explants when stimulated with caerulein,
cholecystokinin, pentagastrin, secretin, somatostatin,
substance P, or vasoactive intestinal peptide (VIP). However,
some studies indicate that secretin increases mucus secretion
in the stomach (99), and that both secretin and the related
hormone VIP weakly stimulate colonic mucus secretion in vivo
(40) . Recently, VIP receptors have been found on mucus-
secreting cells in culture (108) . VIP alone does not
stimulate mucus secretion from these goblet-like cells, but
both VIP and cAMP potentiate the secretagogue effects of
carbachol. This potentiating effect of VIP has also been seen
in tracheal submucosal glands (204).
The vasoactive amine histamine causes increased colonic
mucus secretion (161), but only under nonphysiologic culture
conditions (160) . Histamine has also been shown to increase
PGE2 levels, and so may act indirectly (247) . A greater
amount of gastric mucus was recovered after stimulation with
serotonin (141); however, there was no morphologic evidence of
increased secretion in rabbit colon following serotonin
stimulation (160) .
Several inflammatory products of neutrophils,
macrophages, and mast cells have been shown to stimulate mucus
secretion from respiratory epithelium (70, 160) , but they have

27
not been studied extensively in the intestine. There is now
increasing evidence that interleukin 1 (IL-1) increases
intestinal mucus release (27, 69) . In addition, a macrophage
product, MMS-68, first isolated from the respiratory tract,
has been shown to stimulate intestinal mucus secretion (221).
Immune complexes have been shown to induce compound exocytosis
of mucin (246). Antigen challenge following oral, but not
intraperitoneal, inoculation increases mucus secretion,
suggesting involvement of mucosal immunity (109). When a
jejunal self-filling blind loop is created, causing bacterial
overgrowth, there is increased mucus secretion within the
loop, but it is decreased outside the loop (202) . This
phenomenon could be the direct result of the organisms, or a
secondary response to inflammation.
Cryptosporidium parvum infection in mice resulted in an
increased amount of mucus in ileal washings (77). It is not
clear whether more mucus was actually secreted or if it was
simply dislodged more easily. If secretion was increased, it
is still not possible to tell if the effect is a direct result
of the organism, or secondary to some inflammatory mediator.
Virulent strains of Entamoeba histolytica have been shown
to increase secretion of preformed and newly synthesized mucus
glycoproteins, and also to increase mucin synthesis in rats,
in the absence of an inflammatory response (22). Intestinal
trematodes in dogs and cats have been associated with mucoid

28
inflammatory response, but no quantitative studies of mucus
secretion have been performed (96).
Cholera toxin and the heat labile toxin (LT) from
Escherichia coli both stimulate small intestinal mucus
secretion (160) . This phenomenon can be separated from fluid
and electrolyte secretory effects, which are stimulated by
cyclic AMP (189), and blocked by tetrodotoxin (149) . The
mechanisms for both increased fluid and mucus secretion are
complex. Lencer et al. (115) have shown that the B subunit of
cholera toxin binds to cloned human goblet cells in monolayer
culture, but there was no stimulation of mucus release,
suggesting that an indirect mechanism may be involved. It is
currently believed that binding of cholera toxin to
enterochromaffin cells results in increased intracellular cAMP
and secretion of serotonin, which in turn stimulates
cholinergic neurons (149). Both the fluid and mucus secretory
activities of cholera toxin are blocked by capsaicin,
supporting the involvement of the enteric nervous system
(149). However, fluid secretion is blocked by tetrodotoxin,
while mucus secretion is not, suggesting that mucus secretion
may be mediated by local effectors released by sensory
neurons. In addition, a portion of the capsaicin-sensitive
response is also atropine-sensitive, suggesting that there may
be a tetrodotoxin-insensitive interaction between cholinergic
and sensory nerve terminals occurring in the small intestine.

29
Although neither cyclic AMP or cyclic GMP alone affects
mucus secretion in explant systems (161), Jarry et al. (93)
found that cAMP directly stimulated both MUC2 gene expression
and mucus secretion from goblet cells in culture, in a protein
kinase A-dependent manner, which could account for the effects
of cholera toxin. This toxin has also been shown to increase
mucosal prostaglandin synthesis (183), and the effects of PGEX
and cholera toxin are qualitatively the same in in vivo rabbit
intestinal loops (164).
Mucus secretion has been shown to be increased in rats
with streptozotocin-induced diabetes, with less mucin present
in the tissue than in the luminal washings (134) . Synthesis
of mucins relative to other glycoproteins is also increased.
Interestingly, the intestinal goblet cells from these diabetic
rats are no longer responsive to bethanechol or cholera toxin.
This phenomenon has not yet been confirmed, and may be an
artifact of collection, since fluid secretion is also
increased in diabetes, and may flush out the mucus present.
The antiulcer drugs zolimidine, carbenoxolone, quercetin,
and oral copper compounds have been reported to increase mucus
secretion, but the mechanisms are unknown (2, 3, 51).
Parathyroid hormone has been reported to increase gastric
mucus secretion (141) . The carbonic anhydrase inhibitor,
acetazolamide, also is a mucin secretagogue (28) . A new
antiinflammatory drug, SCH12223, which has protective effects
on the gastric epithelium increases gastric mucus content

30
(25) . The amount of gastric mucus that can be aspirated in
vivo is increased after a meal (98) . Feeding increases
intestinal mucus secretion, even when the duodenum has been
transplanted to a subcutaneous location and separated from its
nerve supply (47). Mucus secretion is increased during the
daytime in rats that are allowed food only during the daylight
hours {13) .
The synthetic opioid anti-diarrheal, loperamide, has been
shown to slightly decrease baseline mucus secretion, and
drastically reduce secretion stimulated by PGE2 or deoxycholic
acid in the rat colon (121). This may be related to neural
effects or calcium channel blocking activity (186) .
Cysteamine, which is used to induce duodenal ulcers
experimentally, also decreases mucus secretion (104).
Interferon-y does not affect baseline mucin synthesis or
secretion, but inhibits secretion stimulated by both cAMP and
calcium ionophores, apparently at the exocytotic step (93).
Degradation
Little mucus is excreted in the feces. It was found that
crude mucus undergoes a spontaneous loss of viscosity when
incubated overnight at body temperature (72, 91). This
phenomenon was accelerated by the addition of certain
proteases; different investigators described different
sensitivities. It has been shown that pepsin, but not HCl,
can dissolve the gastric mucus layer, and cause epithelial

31
damage (126) . That some degradation occurs in vivo is
supported by the fact that secreted mucin has a lower
molecular weight than stored mucin (220) .
Although proteolytic enzymes can degrade mucin into
smaller glycosylated fragments, resulting in collapse of the
mucus gel, and loss of elasticity (4), they cannot completely
digest mucopolysaccharides (126) . Both a- and S-glycosidases
are required for the removal of sugar residues from mucin
(126), and it has been found in humans that these enzymes are
primarily derived from Bifidobacterium and Ruminococcus
species (82) in the colon.
Gastrointestinal mucin digestion is markedly decreased in
germ-free rodents, due to a lack of glycosidases capable of
removing the oligosaccharide sidechains from the peptide
backbone (120, 126). This results in an increased mass of
mucus in the cecum, increased mucus excretion in the feces,
and retention of mucus within a thickened cecal wall. When
normal enteric bacterial flora are administered to germ-free
rats, excretion of glycoproteins increases for 2-3 days, then
drops to the level of conventional animals, confirming that
bacterial enzymes play a major role in mucin digestion (120).
As mentioned previously, pepsin partially degrades
gastric mucin, resulting in solubilization, and dissolution of
the mucus layer. It has been found that patients with peptic

32
ulcer disease have a higher ratio of pepsin 1 to pepsin 3 than
normal individuals, and pepsin 1 is much more efficient at
dissolving mucus. Consequently, ulceration may be secondary
to the breakdown of the protective mucus (126) .
It has been demonstrated in vivo that Helicobacter pylori
infection decreases the thickness of the gastric mucus layer
in humans (166, 196) . This has been related to the production
of protease, lipase, phospholipase, and glycosulfatase by the
organisms which impair the protective mucus layer, and may
promote mucosal injury. Sulglycotide, a modified sulfomucin
gastroprotective agent, acts by inhibition of these enzymes,
along with aggregation of the organisms (156, 180) . In
addition, Helicobacter produces urease, and the high levels of
ammonia and bicarbonate produced may impair the protection
afforded by mucus (35).
Vibrio cholerae contains a virulence factor which
consists of a "mucinase complex" (168) . Since mucus can bind
and inhibit cholera toxin, the destruction of mucus by a
metalloproteinase allows the toxin to retain activity, as well
as permitting better access to the epithelial cells (32). In
addition, a neuraminidase may increase the amount of GM1
ganglioside available in the membranes for toxin binding.

33
Postulated Functions of Mucus
Lubrication
In 1800, Glover postulated that the mucus secretion of
the intestinal tract was involved in lubrication (79). Florey
(46) saw that particles were removed from the intestine by
entrapment in mucus, which was then pushed downstream by the
intestinal villi. Nondigestible solids that are emptied from
the stomach are entrapped in mucus plugs (66). Mucus has been
experimentally shown to aid in ciliary propulsion of objects
in tubes (255) . The need for lubrication varies with the
segment of the intestinal tract discussed. There is little
mucus secretion in the small intestine where the contents are
fluid, and there is little need for lubrication (47) .
Conversely, there are many goblet cells within the colon where
lubrication is clearly needed for the passage of solid feces
(47) . More recent studies have shown an incomplete mucus
layer in the proximal colon, where the contents still have a
high water content, but a thick, compact layer in the distal
colon, in order to facilitate the passage of feces (194, 225).
Cvtoprotection
In the same thesis, Glover proposed that mucus "must
likewise defend the internal surface of the stomach and
intestines, from the action of the gastric juice, and from the
acritude of bile when regurgitated" (79). Several theories

34
were put forth for the mechanism of protection. The simplest
was that of a diffusion barrier, which will be discussed in a
later section. It was believed for a period of time that
mucus had direct acid-neutralizing capacity (79). This was
first discussed by Pavlov, and studied in detail by Hollander
(79). He found a definite buffering capacity in the mucous
secretions from Heidenhain pouches of dogs. However, it was
shown conclusively by Heatley that mucin itself has minimal
buffering power against hydrochloric acid; rather the
buffering activity is due to the content of bicarbonate (47) .
On the other hand, the mucus is responsible for keeping that
bicarbonate next to the mucosal surface, as will be discussed
later in the section on creation of a microenvironment.
In 1855, Claude Bernard postulated that mucus has a
specific chemical or adsorbent action (79) . At the beginning
of the twentieth century, the presence of a specific
antipepsin in mucus was reported (79). However, since 1914
there has been no further evidence of any enzymatic activity
of mucus. Bucher (17) showed adsorption of pepsin by the
mucus, and Zaus and Foskick (264) and Bradley and Hodges (15,
16) also documented antipeptic effects, however Heatley (74)
found that purified mucin does not directly inhibit peptic
digestion.
Hydrogen peroxide is rapidly degraded in porcine gastric
mucus in vitro (34). Although purified mucin is subject to
attack by reactive oxygen intermediates, it has been shown

35
that the lipids associated with native mucus have a protective
effect against these radicals (62). Consequently, one
function of mucus may be to protect the mucosa from attack by
reactive oxygen intermediates released by the host when
killing bacteria or as a response to toxins. However, mucus
does not affect the cytotoxic activity of Clostridium
difficile toxin A in rabbits (118).
It has been shown that mucus plays a major role in
"adaptive cytoprotection" (21). Following a mild epithelial
injury with oleic acid, the thickness of the mucus layer is
increased. This prevents injury from a second exposure by
delaying the passage of the irritant through the mucus to the
epithelial cells. Increased mucus secretion stimulated by the
antiulcer drugs carbenoxolone and quercetin protects gastric
epithelium from damage by ethanol, 0.6N HCl, or 30% NaCl (2,
3). When acidified ethanol is used for challenge to negate
antacid effects, the H2-blocker FRG-8813 still offers
significant cytoprotection through increased mucus production
and secretion (85).
Protection from Infection
Cramer (30) found that vitamin A deficient rats are more
susceptible to bacterial infections, and related this to the
decreased production of intestinal mucosubstance, secondary to
decreased numbers of goblet cells. This suggests that mucus
can act as a barrier to bacteria. Goldsworthy and Florey (61)

36
then demonstrated that intestinal mucus contains lysozyme, a
non-specific antibacterial enzyme. Specific immunity in the
intestinal tract is dependent on secretory IgA. slgA alone
does not prevent bacterial interaction with intestinal
epithelium (245), but rather appears to bind to mucin
glycoproteins through hydrogen or disulfide bonds (140),
allowing for aggregation and neutralization of bacteria.
Because mucus comprises a physical barrier, bacteria have
developed mechanisms to allow association with the underlying
epithelium. One of these mechanisms is motility. Flagella
allow motility of bacteria in aqueous solutions, but are
ineffective in viscous environments. However, Campylobacter
jejuni, although flagellated, acts in high viscosity solution
like a spirochete, relying on endocellular organelles for
locomotion, and shows an increase in motility related to
increased viscosity (42) . Treponema hyodysenteriae, is
similarly highly motile in intestinal mucus (101). This may
provide a selective advantage to helical bacteria, such as
Campylobacter, Treponema, Vibrio, and Helicobacter species, in
penetrating mucus and colonizing the intestinal epithelium.
A lipopolysaccharide-deficient mutant of Salmonella
typhimurium is able to colonize mouse large intestine when
given alone, in combination with E. coli, or with low
concentrations of wild type Salmonella (162). However, if
high dose of wild type Salmonella is given concurrently, or if
both strains are allowed to multiply within the host for 8

37
days, the lipopolysaccharide-deficient mutant is eliminated.
This appears to be related to the fact that the mutant adhered
to cecal mucus far better, but penetrated mucus less well than
the wild-type, or even nonflagellated or nonchemotactic
transductants (137) . This also supports the idea that
flagellar motility is not important in mobility within mucus.
The other major mechanism bacteria use to remain in the
intestinal tract is adhesion. Piliated Escherichia coli are
able to bind to sugar residues in the glycoproteins and
glycolipids of both epithelial cells and mucus. In
enterotoxigenic E. coli strains, K99 fimbriae bind to
galactose (155) and sialic acid residues (119), K88ab fimbriae
bind D-galactosamine residues (143), and the terminal subunit
of F17 fimbriae binds to undetermined carbohydrate sidechains
which are also present in cow plasma glycoproteins and hen egg
white (195) . The F-18 colicin which is found in nonpathogenic
E. coli binds to mannose (243) . Although E. coli lacking F-18
are able to colonize streptomycin-treated mouse large
intestine when given alone, they cannot compete with the
fimbriated bacteria in establishing infection when given
concurrently (244) .
Both piliated and non-piliated strains of
enteropathogenic E. coli adhere to mucus (259). Denaturation,
trypsinization, or removal of carbohydrate from the mucin all
decrease binding (248). More mucin bound to both
enteropathogenic and enterotoxigenic E. coli strains at pH 5.7

38
than at pH 7.4 (248) . In vitro, the presence of mucus
competitively inhibits binding of E. coli to colonic
epithelium (125), suggesting a protective effect. On the
other hand, adherence to mucus may allow for the initial
colonization by bacteria. Hydrophobicity of the mannose-
resistant AF/R1 pilus of the enteropathogenic E. coli strain
RDEC plays a major role in bacterial binding to mucus and
membranes (37), and RDEC preferentially colonizes colons of
weanling rabbits, where mucus is more hydrophobic than in
sucklings or in the ileum (124). Additionally, more mucus
binding is seen in weanlings than in adolescents (248), which
may reflect the increased susceptibility of weanlings to
clinical disease.
Virulent strains of Yersinia enterocolitica are able to
colonize the intestine because the yadA gene on the virulence
plasmid codes for a high molecular weight outer membrane
protein which allows binding to mucin (170). However,
preincubation with mucus decreases the adherence of the
organism to brush border membranes (130). It appears that
coating with mucus changes the bacterial surface from
hydrophobic to hydrophilic, decreasing the interaction of the
organism with the epithelial surface (170). However, strains
containing the virulence plasmid are able to degrade mucin to
a greater extent than non-virulent strains (132), and thus
overcome the protection afforded by mucus.

39
Helicobacter pylori has also been shown to bind to mucin,
and the binding is decreased after the removal of sialic acid
(237). Electron microscopy shows that most strains of Vibrio
cholerae bind preferentially to mucus, rather than to the
epithelial surface (260, 261) . Guinea pig mucus inhibits
invasion of epithelial cells by Shigella flexneri, but monkey
mucus does not (245) .
Intestinal mucins inhibit replication of rotavirus in
vitro (24, 262). The rotavirus vp4 protein, which also
mediates binding to cells, is involved in the binding (24);
however it is not clear if sialic acid residues or short
oligosaccharide chains act as the mucin receptor (262) .
Mucus also plays a significant role in parasitic
infections. Frick and Ackert (54) found that duodenal mucus
of adult chickens inhibits the growth of Ascaridia galli more
than that of young birds, and that this may play a role in age
resistance. A nematode which has been studied more
extensively is Nippostrongylus brasiliensis in rats.
Following a transient decrease, an increase in the number of
goblet cells is associated with an immune-mediated expulsion
of this worm. If the number of goblet cells is decreased
secondary to a protein deficient diet, the efficiency of
expulsion is decreased (249) . Secretion of mucus is necessary
for the expulsion event: if mustard oil or a combination of
cysteine and papain is administered to immune rats to cause
secretion of stored mucin 1.5 hours prior to challenge, then

40
worm expulsion is inhibited (146) . The role of prostaglandins
in this process is unclear. While some investigators have
found that administration of prostaglandins enhances
expulsion, others have been unable to confirm this (28). The
vehicles used (chloroform or alcohol), or the timing of
administration may be responsible for the discrepancy.
Some worms are entrapped by mucus prior to expulsion
(145) . In addition, in a phenomenon called "immune
exclusion", worms are unable to penetrate the mucus of
immunized animals, while in naive animals, worms are able to
burrow through the mucus to the intervillous space (145) .
Similarly, it has been shown that mucus from infected sheep
inhibits the motility of Trichostrongylus colubriformis in
agar gel (103) . Nippostrongylus is able to move through
viscous gels by forming a tight corkscrew (111); the presence
of coating antibodies may prevent this motion, resulting in
immune exclusion. It has also been shown that the worms
ingest mucin, and morphologic damage of the adult worm gut is
associated with the development of immunity (145) .
Trichinella spiralis is expelled from immunized rats in
a very similar manner, although Trichinellae enter the
epithelium, while Nippostrongylus remain between villi (145) .
It has been shown that antibody or complement coating of
Trichinella larvae allows entrapment in mucus (18, 145) . This
may be important in the expulsion of worms from the organisms,
although the injection of antibody allows for the expulsion of

41
worms from the infected epithelium, prior to association with
mucus.
The expulsion of both Trichinella and Nippostrongylus is
an immune-mediated event. Transfer of thoracic duct
lymphocytes from immunized rats results in accelerated
expulsion of Nippostrongylus associated with early goblet cell
hyperplasia (147) , and this was later shown to be T cell-
dependent (14 5) . However, transfer of hyperimmune serum will
also result in rapid expulsion (145) . Furthermore, at least
two steps are involved in worm expulsion (88) . The first
involves T-cell dependent "damage" to the worms. These
damaged worms can then stimulate alterations in the terminal
sugar residues of mucus resulting in the selective expulsion
of damaged or healthy worms, even from athymic rats (88) .
There is also synergistic interaction between immune serum and
thoracic duct lymphocytes in the rapid expulsion of
Trichinella (1). In both worms there are increased
concentrations of leukotrienes and rat mast cell protease II
(145, 150) . Corticosteroids, which inhibit the immune
response in many ways and also decrease mucus secretion (141),
delay expulsion of Nippostrongylus (145) as well as the rat
tapeworm Hymenolepis diminuta (139). Reserpine also alters
mucus secretion (169) and delays expulsion of Nippostrongylus
(145) .
On the other hand, mucus can apparently increase
pathogenicity of other parasites. In particular, it promotes

42
survival of Giardia lamblia in many ways. For example,
Giardia can be killed by the lipolytic products of milk, but
this killing is inhibited by the presence of intestinal mucus
(265) . Mucus provides a nutrient source for the parasite
(57), and allows for its enhanced adherence to the intestinal
epithelium (145) . This lectin-like adhesion to simple sugar
residues within mucus, especially sialic acid, is a
characteristic of several protozoa, including Entamoeba
histolytica (145) and Tritrichomonas mobilensis (33) . The
yeast Candida albicans has also been shown to associate with
mucus, but the ability to colonize the intestinal mucosa is
dependent on the absence of normal flora (102) .
Nutrient for Flora
Evidence that mucus may aid the growth of bacteria was
demonstrated by Smith et al. (213, 214), who found increased
virulence of intraperitoneally injected bacteria
(Staphylococcus aureus and Streptococcus species) when mucus
was administered with the organism. This effect was partially
due to sequestration of the bacteria from the immune system.
However, the chemical components of the mucus itself increased
the growth of the organisms, presumably by serving as a
nutrient source. It has been shown in vitro that jejunal
mucus stimulates the growth of Giardia lamblia (57). Mucin
can also enhance growth of both virulent and avirulent
Yersinia enterocolitica, as well as serve as a nutrient source

43
for E. coli, Salmonella typhimurium, Clostridium perfringens,
Bacteroides sp, Shigella flexneri, Rumenococcus, and
Bifidobacterium (8, 132).
By comparing the cecal mucus of conventional and germfree
rats, Lindstedt et al. (120) were able to show that normal
flora degrade a significant amount of mucus. There was less
mucus in the cecum of conventional animals, and a higher
proportion of what was there was of lower molecular weight,
indicating partial degradation. Other investigators have
confirmed these findings (52). However in the colon,
Szentkuti et al. (225) found a thinner mucus layer in germfree
rats, associated with decreased mucosal thickness, and
decreased numbers of goblet cells.
As discussed previously, Rumenococcus and Bifidobacterium
species possess glycosidases that are responsible for the
degradation of mucin in humans. The monosaccharides released
from mucin by these glycosidases support growth of Bacteroides
and other fecal bacteria that lack such glycosidases (82) .
However, a strain of Bacteroides vulgatus isolated from
patients with Crohn's disease is capable of degrading mucus
glycoproteins (192). The enzymes produced by normal flora may
also directly attack pathogenic bacteria (160) ; normal flora
certainly inhibit colonization by pathogens, since antibiotics
increase infection with pathogenic organisms (245) .

44
Diffusion Barrier
Much of the cytoprotective action of the mucus layer has
been attributed to the barrier properties of mucus (79) .
However, when this property was experimentally examined,
Heatley illustrated that mucus is not a barrier to the passage
of solutes, such as H+ ions and pepsin. Furthermore, when
studies were performed to determine the thickness of the
"unstirred water layer" in the intestine, the calculated value
for several solutes was similar, so it was proposed that mucus
merely acts as a support for the unstirred water layer (215,
250) . However, more recent studies show that mucus does
retard diffusion of many molecules. For instance, it slows
the passage of H+ ions relative to their rate of diffusion in
water (172, 253), and Lucas (122) demonstrated that this
retardation is dependent on the concentration of mucus. The
mobilities of sodium, potassium, and chloride are also greatly
reduced in mucus (68) .
Mucus forms a polyanionic gel, and thus acts as an ion
exchange resin (48). In general, chloride is excluded,
whereas there is a high affinity for calcium and potassium
(67, 199). However, although the transport of chloride (and
other ions) is retarded, it is not prevented (68) .
Sequestration of potassium by mucus and cell surface
glycoproteins may be important in recycling of potassium by
the Na+/K+-ATPase. Calcium may be important in the

45
condensation of mucus into compact granules prior to
exocytosis (242) .
Mucus retards diffusion of molecules as well as ions.
Smith (212) found a diffusion coefficient for butyrate in
mucus to be 50-60% of that in water. Desai et al. (34)
determined the diffusion coefficients for a wide variety of
molecules in mucus and concluded that no consistent effect of
molecular weight was evident with regard to barrier properties
for the weight range tested (34-660 Da) . Using cultured
goblet cells, it has been shown that the overlying mucus layer
is a significant barrier to the passive absorption of the
lipophilic and uncharged drug testosterone (97). Mucus has
been shown to trap iron, but it is not clear if this aids
(181), or prevents excess, (252) absorption. This trapping
may make iron more available to bacteria (51).
Since mucus is a polyelectrolyte gel, it behaves as a
Donnan system (67). Consequently, the hydration of mucus is
dependent on the ionic concentrations in the bathing solution.
As ionic strength is decreased, hydration is increased, and
noncovalent interactions between mucin subunits are fewer,
decreasing viscosity. Conversely, at high ionic strength,
anionic charges within the gel will be shielded, and hydration
and volume decreased. It has been postulated that lack of
functional chloride channels resulting in decreased chloride
movement in cystic fibrosis could alter ionic composition
resulting in abnormal mucus secretions in intestinal,

46
pancreatic, and respiratory tissues (138, 242). Mucus from
cystic fibrosis patients is hyperpermeable to small ions and
water, associated with an increased calcium content (58).
Furthermore, glucose absorption was enhanced in afflicted
patients, coinciding with a decreased thickness of the
calculated unstirred water layer (53).
Mucus may even have a "waterproofing" function (159) . If
the external mucus layer is removed from an eel, the weight of
the animal will increase when the eel is placed in distilled
water. This property has not been examined in the intestine,
and would be difficult to address in light of the complex
absorptive and secretory processes involved in digestion and
absorption. However, it is interesting that Westergaard and
Dietschy (250) saw swelling of intestinal villi inversely
correlated the thickness of the unstirred water layer.
Creation of Microenvironment
A microenvironment occurs when the concentration of
solutes next to a membrane is different from that of the bulk
phase on either side of the membrane, as has been shown to
occur in mucus (67). In the gastrointestinal tract, the
presence of a microenvironment is important in two basic
areas. In the stomach, it is necessary to prevent direct
contact between the highly acidic bulk contents and the
mucosal surface. When Heatley (75) found that mucus did not
act as a barrier for hydrogen ions, he proposed a pH gradient

47
within the mucus layer. In his dynamic model, mucus and
bicarbonate are secreted at the mucosal surface, and then
migrate outward. As the mucus progresses into the lumen, it
becomes more hydrated and accumulates more hydrogen ions. The
viscosity thus decreases, until the outermost layer is shed
into the lumen. Therefore, some of the hydrogen ions will be
brought back into the lumen with the dissolving mucus, while
others will be neutralized by the bicarbonate within.
This "mucus-bicarbonate barrier" model has stood the test
of time very well (5, 31, 148, 205). With the development of
microelectrode techniques, pH gradients have been demonstrated
in rat (190), rabbit (254), and human (182) gastric mucus in
vivo. The pH gradient can be decreased with the addition of
compounds that dissolve mucus, such as N-acetylcysteine (190).
Mucus is responsible for the creation, as well as the
support, of a microenvironment in the intestine. Hogben et
al. (78) proposed that an acidic microenvironment could
influence the absorption of drugs from the intestinal tract.
Acidic drugs are absorbed more rapidly than would be expected
at neutral pH; however at acidic pH these acids would be
undissociated, and would readily cross the epithelial membrane
by nonionic diffusion. He calculated that the pH at the rat
jejunal surface must be 5.3 to account for measured drug
absorption rates. Surface pH measurements with
microelectrodes have revealed an acidic microenvironment, but
with pH 6-7 (123, 185, 203) .

48
Similarly, a slightly acidic microenvironment is present
in the colonic unstirred layer (185) . This would allow for
the passive absorption of butyrate and other short chain fatty
acids in the undissociated form (7) . Although there is
evidence for a bicarbonate gradient-dependent, carrier-
mediated anion exchange process for butyrate in the colon,
this cannot account for the rate of absorption measured
experimentally, nor could passive diffusion of the dissociated
ionized form of butyrate (136) . In the rodent cecum, the
microenvironment is more basic than the luminal contents, and
the pH may have an effect on the virulence of Entamoeba
histolytica (112). This organism is subject to killing by
ammonia, and species with a higher cecal mucosal pH, such as
the rat, are less susceptible to infection than those with a
lower pH, such as the gerbil.
Techniques Used to Study Mucus
Model Systems
Intestinal loops. For in vivo experimentation of the
gastrointestinal tract, it is common to isolate the part of
the tract under study to minimize effects of the rest of the
system, and to allow for easy access. For the study of
gastric secretion, the first widely used technique was
formation of the Heidenhain pouch (76). Florey (44, 45, 46,
47) used a variety of intestinal pouches, blind loops, and
transplanted segments to examine movement of mucus within the

49
intestine, and the effects of feeding, neural stimulation, and
various chemicals on mucus secretion.
Explants. Mucosal samples (161) and full thickness
intestinal explants (50) have been used to measure
secretagogue activity in vitro. These tissue sections can be
maintained in culture medium for several hours, and mucus
production and secretion can be measured by several methods.
This technique allows for concurrent testing of several
different chemicals on tissues from a single animal. It has
even been used to demonstrate goblet cell secretion following
electrical field stimulation, due to intrinsic nerve
stimulation (175).
HT-29 cloned goblet cells. Until recently, it was
impossible to work with isolated goblet cells, because these
cells lose their polarity and ability to secrete when
separated from the mucosal epithelium (160) . However, cell
culture systems are now available. The HT2 9 colon
adenocarcinoma cell line is undifferentiated under standard
culture conditions. However, substitution of glucose with
galactose (83), or long term treatment with butyrate (108)
results in differentiation of cells. Individual cells can be
selected for cloning. Several sublines, including HT29-18N2
and C1.16E, show characteristics of goblet cells, including
baseline mucus secretion, and the ability to respond to
cholinergic stimulation (108, 173).

50
Quantitation of Mucus
Biochemical techniques. Biochemical measurement of total
protein-bound hexose (258) , acid-precipitable protein (50) ; or
glycoprotein (127) has been used to estimate mucus
concentration; however mucins are not the only glycoproteins
present in the intestine. Mucin can be separated from many
other glycoproteins by gel filtration on a Sepharose-4B
column, but many proteins remain associated with the sticky
mucin (160) .
Radioactivity incorporation. Glycoprotein synthesis has
been measured by determining the rate of 14C- or 3H-glucosamine
incorporation into intestinal slices (50), and secretion is
measured by counting precipitable radioactivity released into
the media (49) . By fractionating the cell sap on a Sepharose-
4B column, and collecting the void volume, it is possible to
estimate the amount of mucin synthesized. It has also been
shown that labelled butyrate and acetate are incorporated into
mucus glycoproteins (29) . However, absorptive cells
synthesize and release glycoproteins at a faster rate than
goblet cells (160).
Morphometry. Goblet cell hyperplasia, which is
associated with increased mucus secretion, can be quantitated
morphometrically (147). Compound exocytosis can be
visualized, and the percentage of cavitated goblet cells is
reflective of the amount of mucus secreted (217).

51
Immunologic techniques. Standard radioimmunoassay (131)
and enzyme-linked immunosorbant assay (ELISA) techniques (188)
have been developed, using antibodies to mucin. Polyclonal
antibodies react predominantly to the "naked" regions of
mucin, and therefore do not measure degraded mucin (131).
Monoclonal antibodies have been made specific for both the
naked peptide backbone, and for intact mucin (210).
Antibodies have also been made which are specific for the
fragment known as the "link" glycopeptide (188) .
Lectin binding techniques. An enzyme-linked lectin assay
(ELLA), very similar to a standard ELISA, has been developed
(27, 69) . The lectin soybean agglutinin preferentially binds
to N-acetylgalactosamine residues, which are abundant in mucin
but less common in other glycoproteins.
Secretion of Mucus
Light microscopy. A monoclonal antibody has been
developed which specifically labels goblet cells of the human
colon, appendix, and small intestine (240) . This may be
useful in the diagnosis of disease states, in which the
quantity or quality of mucus is altered. Histologic
examination of the thickness of the mucus coating can be used
to evaluate changes in secretion (225) .
Morphometry has also been used to quantitate secretion by
comparing the amount of stained mucin in goblet cells before
and after addition of a secretagogue (100). Autoradiography

52
has been used to measure the rate of secretion (73).
Cavitation of goblet cells indicates that compound exocytosis
has taken place, and has been used to measure secretagogue
activity (176, 177, 217). Exocytosis has been directly
observed by video-enhanced light microscopy (230) .
Electron microscopy. The steps of the secretory process
can best be followed by electron microscopy (217) . Since
conventional fixation techniques can fragment the limiting
membranes, cryofixation may allow for better evaluation of the
mechanism of secretion (84) . The thickness and composition of
the mucus layer can also be examined by this technique (165) .
Composition of Mucus
Biochemistry. Standard biochemical techniques have been
used to determine the amino acid and sugar composition of
mucus (160) . Glycosyl-transferase activities can be measured
by conventional biochemical assays (12, 226), providing clues
to changes in carbohydrate composition. Methods for measuring
the adhesiveness, plasticity, viscoelasticity, and
spinnability of mucus microsamples are also now available
(263) .
Histochemistry. Differential staining techniques can be
used to distinguish mucin characteristics. When sections are
stained with combined alcian blue and periodic acid Schiff
reagent, acidic mucins will appear blue, and neutral mucins
will appear red (225). High iron diamine will stain sulfated

53
sialomucins (266) . Positive staining with mild periodic acid
Schiff (mPAS) indicates deficiency of O-acetylation of sialic
acid, as is seen in ulcerative colitis (94) . Mucins of
different chemical composition can be distinguished by
differential lectin binding. Therefore, lectin histochemistry
can be used to identify abnormally glycosylated mucins in
goblet cells, which may be associated with disease (89, 241),
developmental changes (227), or diet (152) . Finally, specific
antibodies can be used to distinguish small intestinal from
large intestinal mucins by indirect immunoperoxidase staining
(43) .
Diffusion Through Mucus
Mucus can be immobilized on a filter, or between two
filters, and the rate of diffusion through this compared to
the rate of diffusion through the filter alone (75) . The
mathematical modeling of diffusion through mucus in this type
of apparatus has been described (172) . Diffusion through
mucus overlying HT29 monolayers can also be measured (97).
Microbial Virulence
In vivo colonization assays, where the number of colony¬
forming units of bacteria recovered in the feces after
experimental infection is measured over time, reflect a
combination of virulence factors (162). Microbial adherence
can be evaluated by coating plates with mucus or brush border

54
membranes, and measuring the percentage of radiolabelled
bacteria which will then adhere (130). Competition for
binding can also be measured with this assay, by adding the
competitor along with the organism. Adhesion of organisms to
cells in culture can also be evaluated microscopically (33).
Penetration of organisms through mucus can be evaluated by the
passage of radiolabelled bacteria through a mucus gel (170),
or by direct observation of the organism (42, 111).
Theoretical Considerations
Lubrication
The idea that mucus is necessary for lubrication to aid
in the passage of feces has been mostly intuitive, with little
direct experimental support. However, there is one disease
state which illustrates the need for such lubrication quite
clearly. In cystic fibrosis, where mucus is abnormally
viscous and lacks elasticity, it is common for infants to
develop meconium ileus, and for adults to develop meconium
ileus equivalent (160). As indirect evidence, the most
compact, continuous mucus layer is in the distal colon, where
there is greatest need for lubrication for the passage of
formed feces (194) . In addition, the fact that nondigestible
solids are coated in mucus when they are expelled from the
stomach suggests that mucus aids in lubrication to facilitate
their passage through the pyloric sphincter and expel them
from the body (66).

55
Cvtoprotection
Mucin itself has no direct buffering or antienzymatic
activities. However, its presence is necessary to protect
underlying cells. Its removal may result in increased gastric
or duodenal ulceration (104) . Chronic inflammatory bowel
disease is associated with decreased mucus production, which
may help to perpetuate the disease (160) . This protection is
partly due to the role of mucus as a diffusion barrier, and
the creation of a microenvironment, which will be discussed
below. In addition, it appears that mucus is able to detoxify
reactive oxygen intermediates (34, 62). Irritants of many
types stimulate the accelerated secretion of mucus; this may
be a protective mechanism to keep such irritants away from the
intestinal mucosa (21, 47).
Protection from Infection
The interactions of mucus and pathogenic organisms are
complex. It is perhaps easier to determine the protective
effects of mucus by looking at mechanisms that pathogens have
developed to overcome these effects. Mucus is a slippery
substance, and when the motility of the intestine is taken
into account, it is clear that attachment to mucus is
necessary if organisms are to avoid being flushed out. The
viscosity of mucus is also a barrier, and motility based on
internal structures rather than flagellae helps microorganisms

56
negotiate this barrier (42) . That organisms have developed
mucinase enzymes indicates that mucus is a barrier that must
be negotiated (168) .
Mucus also contains antibacterial substances, in
particular lysozyme (61) and secretory IgA (14 0) . Mucin bound
to IgA allows for entrapment of parasites such as
Nippostrongylus, so that the parasite is immobilized and swept
out of the body (145) . The goblet cell hyperplasia seen with
many parasitic infections emphasizes the importance of mucus
in their expulsion. Mucus coating can also prevent attachment
of pathogens to the intestinal epithelium by competition (125)
or by changing bacterial surface properties (170) .
Pathogenic organisms have also been able to use mucus to
increase virulence. Within mucus, bacteria are "hidden" from
some of the defense mechanisms of the host (265) . Also, mucus
can provide nutrients to these organisms, as discussed below.
Nutrient for Flora
Although the experimental evidence is incomplete, it
seems reasonable that degraded mucus provides nutrients for
bacterial flora, especially in times of fasting. The trapping
of iron by mucus (181, 252) allows the flora increased access
to this mineral which is so tightly controlled in the rest of
the body. It is important to remember that the normal flora
also provide a major defense against pathogenic bacteria (160,

57
245) , so that in providing nutrients for flora, mucus is
actually protecting the host from infection.
Diffusion Barrier
The gastrointestinal mucus layer has been shown to delay
diffusion of a wide variety of molecules. In the intestine,
this layer is between 50 and 500 /¿m thick, depending on
species and site evaluated (34, 67). In general, the measured
unstirred water layer is slightly thicker. This may be due to
an unstirred layer overlying the mucus, but is more likely due
to the decreased diffusion of solutes in mucus leading to
erroneously large values for the unstirred water layer (215) .
Consequently the barrier to nutrient absorption is formidable,
although the calculated maximum allowable water barrier which
would allow physiological absorption of glucose and lipid is
4 0 jum (223) . Furthermore, the mucus layer is not static;
there is constant renewal at the mucosal surface and
degradation at the luminal surface, resulting in a net flow of
mucus away from the mucosa.
It would seem that this unstirred mucus layer would make
absorption of nutrients impossible. But on further
examination, the term "unstirred" is also inappropriate. The
mixing caused by segmental and peristaltic contractions of the
intestine, with three dimensional shear forces, as well as
expansion and contraction of the mucus layer itself, is not
comparable to mixing by a stir bar in a beaker. Furthermore,

58
the intestinal villi move in and out of the mucus layer,
cleansing themselves of particulate matter, and moving the
entire mucus layer aborad (46) . Villi bare of mucin are
present in the small intestine, particularly the ileum, in
vivo (165) . The villi may also cause mixing within the mucus
layer, as speculated by Strocchi and Levitt (223).
The fluxes of water in and out of the intestine must also
be considered. Large volumes (approximately 1 liter/20cm/hr
in a human, 53) are secreted from the crypts and absorbed from
the villi. This could aid in absorption by solvent drag
effects, as well as contribute to mixing within the mucus
layer. And finally, the mucus may actually "trap" certain
molecules, such that the concentration within the mucus can
exceed the luminal concentration, leading to increased
absorption.
Creation of Microenvironment
The pH difference between the lumen and the mucosal
surface is most important in the stomach, where the presence
of a "mucus-bicarbonate" barrier is generally accepted. By
delaying the diffusion of H+ toward the epithelial cells, and
the diffusion of bicarbonate away from them, mucus plays a
vital role. The microenvironment may also play a role in the
selective absorption of drugs and nutrients from the
intestinal tract. In particular,
a slightly acidic

59
microenvironment would aid in colonic absorption of short
chain fatty acids (7) .
Conclusions
The differences in the mucus layer throughout the
gastrointestinal tract reflect the differing functions
required. In the stomach, the major function is
cytoprotection, by creation of a diffusion barrier and a less
acidic microenvironment. For this, the mucus is neutral
rather than acidic, and is more viscous. In the small
intestine, mucus must act as a diffusion barrier to
destructive enzymes and pathogenic organisms, yet provide a
microenvironment to allow for absorption of nutrients. The
fact that absorption occurs at the tips of the villi, which
stick up through the mucus layer, may be a physiologic
necessity, rather than a random fact for students to memorize.
In the proximal colon, the loose, discontinuous mucous
layer provides for the nutrition of friendly flora, and
creates a microenvironment which allows for absorption of the
short chain fatty acids produced by these bacteria. Finally,
in the distal colon, a thin, compact mucus layer provides
lubrication for the passage of feces. All in all, mucus aids
the host in a number of ways, and its importance in the
maintenance of good health is often overlooked.

CHAPTER 3
PILOT STUDIES
Introduction
The overall purpose of this project was to determine if
there is a mucus secretagogue in the cecal contents of rabbits
with mucoid enteropathy, as proposed by Toofanian and
Targowski (236). Therefore pilot studies were performed to:
1) explore the presence of such a secretagogue in the cecal
contents of a rabbit with naturally-occurring mucoid
enteropathy (ME); and, 2) confirm that the cecal ligation
model developed by Toofanian and Targowski (229, 235) would
reproducibly cause a mucoid enteropathy-like syndrome.
Cecal Filtrate Collection
Cecal contents were collected from a juvenile female New
Zealand White (NZW) rabbit (Oryctalagus cuniculus) with
naturally-occurring mucoid enteropathy, and also from two
healthy adult NZW rabbits. The affected rabbit also had a
severe intestinal Eimeria infestation. For each rabbit, the
volume of the contents was estimated, and an equal volume of
Hank's Buffered Salt Solution (HBSS) was added. The slurry
was centrifuged at 3000 rpm (700 x g) , 4°C for 15 minutes.
The supernatant was collected, and recentrifuged at 9500 rpm
60

61
(10,000 x g), 4°C for 30 minutes. The resulting supernatant
was then filtered through a Whatman #50 paper filter, followed
by a 0.8 /xm membrane filter, and finally a 0.22 /xm sterilizing
membrane filter. The sterile filtrate was stored at -70°C
until use.
Intestinal Explants
The cecal filtrates that were collected were tested for
mucus secretagogue activity in an in vitro intestinal explant
system. Five healthy adult NZW rabbits were sedated with
ketamine/xylazine, and killed with an overdose of barbiturate.
Intestinal segments were collected, opened along the
mesenteric border, and rinsed in HBSS, containing 100 /xg/ml
gentamicin, to remove ingesta. A 6 mm diameter Baker's biopsy
punch (Baker Cummins Pharmaceuticals, Inc., Miami, FL) was
used to take explants from the ileum and proximal colon
(approximately 10 cm from the cecocolic junction, at the point
where the number of longitudinal bands (teniae) decreases from
3 to 1, fig. 3.1). Longitudinally paired punches were taken
for control and experimental samples, as the concentration of
goblet cells varies along the length of the colon (236) .
Explants were incubated in 24 well polystyrene microtiter
plates containing 0.5 ml of Trowell's T8 medium (GIBCO BRL,
Gaithersburg, MD) with 100 /xg/ml gentamicin, and 0.5 ml of the
appropriate cecal filtrate in each well. The plates were
incubated for 1 hour at 37°C in a 95%02/5%C02 humidified

62
environment. Following incubation, the culture medium was
removed and frozen, while the explants were fixed in 10%
neutral buffered formalin. Following routine processing and
sectioning, the tissues were examined histologically. After
1, 2, and 3 hours of incubation, the tissues appeared viable
in every respect.
Enzyme-Linked Lectin Assay
Quantitation of mucus release by the explants was
accomplished using an enzyme-linked lectin assay (ELLA) as
described by Cohan et al. (27). Ninety-six well polystyrene
microtiter plates were coated overnight at 4°C with 100 /¿I of
serial dilutions of porcine gastric mucin standard or
medium/filtrate mixture following explant incubation in 0.5M
sodium carbonate buffer, pH 9.6. The plates were washed with
phosphate buffered saline containing 0.5% Tween-20 (PBS-T20),
and blocked with 5% fetal calf serum in PBS-T20 for 1 hour at
37°C. The plates were again washed, and incubated for 1 hour
at 37°C with 100 /¿1/well of a 10/xg/ml solution of a soybean
agglutinin (Glycine max)-horseradish peroxidase conjugate,
which specifically binds N-acetylgalactosamine residues.
Plates were then washed a third time, and the substrate o-
phenyl diamine (OPD) was added. After 10 minutes, the
reaction was stopped with 100 /¿I of 4N H2S04, and the optical
density at 492 nm was read. The standard curves for porcine

63
gastric mucin were consistent from plate to plate (figure
3.2).
Data Analysis
After the filtrates were incubated with paired explants,
the mucus secreted into the culture medium was quantitated by
the ELLA described above. The effect of sample dilution on
the amount of mucus in the medium/filtrate measured by the
ELLA is illustrated in figure 3.3 for a control rabbit, and in
figure 3.4 for the rabbit with naturally-occurring mucoid
enteropathy. At the lower dilutions (higher concentrations)
there were too many proteins in each sample competing with the
mucus for a limited number of binding sites on the plate,
resulting in falsely low mucus readings. At the higher
dilutions, there seemed to be some appropriate dilutional
effect; however the signal-to-noise ratio is decreased,
creating a larger variance. The 1:64 dilution was selected
for studies requiring quantitation of mucus.
Using the porcine gastric mucin standard curve for the
same plate, the optical density readings were converted to
apparent /xg mucus per ml of medium/filtrate (figure 3.5) . The
amount of mucus in the medium/filtrate incubated without an
explant was subtracted from each value to give the amount of
mucus secreted. Since each explant was incubated in 1 ml
medium/filtrate, this number was equivalent to ng mucus
secreted per explant. Each value was then divided by the

64
weight of the explant to obtain nanograms mucus secreted per
milligram of tissue (figure 3.6).
The amount of mucus in the medium/filtrates initially and
following explant incubation varied with the source of the
cecal filtrate (figure 3.5; Appendix A). Secretion from both
ileal and colonic explants was significantly increased
compared to control (P < 0.05) when incubated with the cecal
filtrate from the rabbit with mucoid enteropathy (figure 3.6).
Cecal Ligation
To better study mucoid enteropathy, it is necessary to
have a reproducible experimental model of the disease. Cecal
ligation has been reported to cause a mucoid enteropathy-like
syndrome in rabbits (229, 235). Surgery was performed on one
adult, female, conventionally housed NZW rabbit (Oryctalagus
cuniculus). The procedure was approved by the University of
Florida Institutional Animal Care and Use Committee. The
rabbit was anesthetized with 35 mg/kg ketamine and 5 mg/kg
xylazine intramuscularly. A sterile field was prepared, and
the abdomen was opened. A window was made in the mesentery
adjacent to the cecum. Two adjacent ligatures of 2-0 silk
were placed around the cecum just distal to the sacculus
rotundus, preventing the flow of ingesta into and out of the
cecum, but not obstructing the flow from the ileum to the
colon (figure 3.7) . The linea alba was closed with 3-0

65
chromic gut, and the skin was closed with 3-0 silk. Recovery
was uneventful.
The rabbit was observed twice daily. By the first
postoperative day the rabbit was eating small amounts, and
passing dry feces. However, appetite and fecal output
decreased on the second day. On the third day the rabbit was
anorectic, appeared painful, and had mucoid diarrhea.
Euthanasia and necropsy were performed.
At necropsy, there was perineal staining with greenish,
liquid fecal material. There were mild subcutaneous
hemorrhages at the incision site, but the sutures were intact,
and there was no gross inflammation. The cecal contents and
the body of the cecum distal to the sutures appeared normal.
The ampulla coli and proximal 2-3 cm of the colon were grossly
dilated with gas and fluid, although there was no mechanical
obstruction. The colon contained a small amount of greenish
fluid, with mucus adhering to the mucosa. The contents of the
ileum were similar. The jejunum contained yellow fluid, with
pockets of accumulation of clear, gelatinous mucus. On
histologic examination there appeared to be mild goblet cell
hyperplasia in the ileum and colon. These results are
consistent with those described by Toofanian (229, 235), and
suggest that this method provides a workable model to study
mucoid enteropathy.

66
Rabbit (OryctolaguM cuniculus)
Body Length :48 cm
i i i i » i
O cm io
Figure 3.1. Sources of intestinal explants
Explants were taken from the distal ileum (closed arrow) and
the single-banded proximal colon (open arrow). Adapted from
reference 6 (p. 270). Used by permission of the publisher,
Cornell University Press.

67
Porcine Gastric Mucin (ng/ml)
Figure 3.2. Standard curves (ELLA).
Serial dilutions of porcine gastric mucin analyzed by ELLA.
The standard curves for the 5 plates used to collect data for
this chapter are shown.

68
Serial dilutions of medium/filtrate samples from a control
rabbit (P8) incubated in the absence of an explant (P8B) or
the presence of ileal explants from 3 rabbits (Rl, R2, and
R3), analyzed by ELLA.

69
Serial dilutions of medium/filtrate samples from a rabbit with
mucoid enteropathy (P4) incubated in the absence of an explant
(P4B) or the presence of ileal explants from 3 rabbits (Rl,
R2, and R3), analyzed by ELLA.

70
1.20
1.10
1.00
f 0.90
3 0.80
® 0.70
CO
^ 0.60
u.
E- 0.50
3
g 0.40
c 0.30
w 0.20
u
| 0.10
0.00
-0.10
-0.20
Explant
â–  Blank
m Ileum
â–¡ Colon
Control
(P3)
Control
(P8)
Filtrate
Mucoid
Enteropathy
(P4)
Figure 3.5. Mucus in medium/filtrates (ELLA).
Calculated mucus (porcine gastric mucin equivalents) in
medium/filtrates, measured by ELLA. Bars represent mean +
SEM. N=1 (P8 blank), 5 (P3, P4 blank), 6 (P8 ileum, colon),
7 (P3 ileum, colon), 10 (P4 colon), or 14 (P4 ileum).

71
0.60 -i
*
0.50
Q)
3
GO
«
o>
_E
O)
c
0.40
TJ
0)
0}
k_
o
a)
w
«
3
o
3
0.30
0.20
0.10
0.00
Filtrate
â–  Control
â–¡ Mucoid Enteropathy
Ileum Colon
Explant
Figure 3.6. Mucus secretion from explants.
Mucus secretion was determined by subtracting the amount of
mucus in the medium/filtrate incubated without explants (as
measured by ELLA) from the amount incubated with explants.
Values shown are relative to the wet weight of the explant.
Bars represent mean + SEM. N = 8 (ME) or 11 (control). * =
significantly different from control (P < 0.05) .

72
The cecum was ligated distal to the sacculus rotundus (arrow),
allowing the flow of ingesta from ileum to colon. Adapted
from reference 235, with permission.

CHAPTER 4
REFINEMENT OF METHODS
Use of Soybean Agglutinin to Quantitate Mucus
Introduction
Soybean agglutinin, the lectin from Glycine max, specifically
binds to N-acetylgalactosamine residues, which are a major
constituent of the O-linked mucin glycoprotein, but are rare
in N-linked glycoproteins. A direct enzyme-linked assay using
this lectin for the measurement of mouse intestinal mucus has
been described (27). Its suitability for the measurement of
rabbit intestinal and colonic mucin was investigated using
Western blot techniques.
Materials and Methods
Purification of rabbit colonic mucin. Colonic mucus from
a rabbit with experimentally-induced mucoid enteropathy
(rabbit H5, see chapter 5) was isolated by the method of
Mantle and Allen (128) with minor modifications. Mucus gel
was removed from the colonic mucosa with forceps, and was
homogenized by hand in a Kontes tissue grinder with an equal
volume of 5 mM EDTA and 1 mM PMSF. The suspension was
centrifuged at 30,000 x g for 30 minutes. 8.1 g of cesium
73

74
chloride (CsCl) was added to 13 ml of the supernatant for a
final concentration of 0.6 g/ml. This solution was
centrifuged for 24 hours at 1.5 x 105 x g at 4°C in a Beckman
L7 Ultracentrifuge with a 70.1TÍ rotor. Eight 1.6 ml
fractions were collected, and 100 /xl of each was evaluated for
carbohydrate content. The four densest fractions were pooled,
and the volume was brought up to 13.5 ml with 0.6 g/ml CsCl,
and the centrifugation was repeated, this time for 48 hours.
Following carbohydrate quantitation, fractions 2 to 5 (from
heaviest to lightest) were pooled, then concentrated and
desalted using a Centricon®-100 concentrator (Amicon, Inc,
Beverly, MA) . This solution was applied to a 2.6 x 14 cm
Sepharose 4B column. The fractions containing the void volume
were pooled, dialyzed against distilled water, and frozen at -
70°C. Porcine gastric mucin (Sigma Chemical Co., St. Louis,
MO), was used as a mucin standard for comparative purposes.
Periodic acid-Schiff assay for carbohydrate quantitation.
The method used was that of Mantle and Allen (127) . Periodic
acid solution was prepared by dissolving 25 mg of periodic
acid in 7% acetic acid (3.5 ml glacial acetic acid in 50 ml
distilled water. Each sample was brought up to a volume of
2.0 ml. 0.2 ml periodic acid solution was added to each
sample and incubated for 2 hours at 3 7°C. Then 0.2 ml
Modified Schiff reagent (Fisher Chemical, Pittsburgh, PA) was
added, and samples were incubated 30 minutes at room
temperature. The optical density at 555 nm was read.

75
Protein concentration. A kit (Sigma Chemical Co., St.
Louis, MO) employing Peterson's modification of the micro
Lowry method was used to measure total protein concentration.
The kit was used according to the manufacturer's instructions.
Direct enzyme-linked lectin assay (ELLA). Porcine
gastric mucin standards, and serial dilutions of purified
rabbit colonic mucin in 100 fil 0.5 M carbonate buffer (pH 9.6)
were coated onto duplicate 96-well polystyrene plates for 2
hours at room temperature. The plates were washed 4 times
with phosphate buffered saline containing 0.1% Tween-20 (PBS-
T20) using an automated plate washer. The plates were blocked
with 3% bovine serum albumin (BSA) in PBS-T20 for 1 hour at
37°C. Following incubation, the plates were again washed, and
then incubated with 100 /il of a 1:200 dilution of a 1 mg/ml
solution of soybean agglutinin-horseradish peroxidase
conjugate (SBA-HRP) in PBS-T20 for 1 hour at 37°C. Following
5 washes with PBS-T20, 85 ¡J.1 of the substrate 0.4 mg/ml o-
phenylene diamine in 0.05 M phosphate-citrate buffer (OPD) ,
was added to each well. After 10 minutes at room temperature,
the reaction was stopped with 85 /¿I of 4 N H2S04. The optical
density was read at 490 nm.
Western Blots. Six percent SDS-polyacrylamide gels were
prepared (71) . Samples, up to 100 /¿l, were boiled 3 to 5
minutes in 0.25 M 2-mercaptoethanol and 0.1% SDS and applied
to gels. Following electrophoresis, gels were either stained
for protein with 0.25% Coomassie Blue, or samples were

76
transferred to nitrocellulose by wet electrophoretic transfer
for measurement of lectin-binding activity (mucus) of total
glycoprotein.
After transfer, blots to be labelled with lectin were
washed with PBS, and blocked in 2.5% casein or 3% BSA in PBS
for 1 hour at 37°C. The blots were transferred to a solution
of 10 /¿g/ml SBA-HRP in blocking solution. Following a 1 hour
incubation at 37°C, blots were washed 4 times for 5 minutes
each in PBS. The substrate solution of 300 mg/ml 4-chloro-l-
naphthol was added, and color was allowed to develop for 30
minutes.
Alternatively, transferred blots were stained for total
glycoprotein with periodic acid/Schiff (224) . Membranes were
washed in distilled deionized water for 5 minutes, then
incubated in 1% periodic acid, 3% acetic acid for 15 minutes.
Following 15 minutes washing with several water changes,
modified Schiff's reagent (Fisher Chemical, Pittsburgh, PA)
was added, and the membrane was stained for 15 minutes in the
dark. The reaction was stopped by a 5 minute incubation in
0.5% sodium bisulfite solution, and the membrane was washed
and dried.
Results
Rabbit colonic mucin was successfully purified, as
evidenced by a lack of contaminating proteins on SDS-PAGE
(figure 4.1, lane 3), and a strong lectin-binding signal in

77
the stacking gel of a Western blot (figure 4.2, lane 3). No
band was seen at 118 kDa, corresponding to the "link"
glycopeptide (188) , although the mucus was collected with
protease inhibitors. Using bovine serum albumin as a
standard, the concentration of protein in the purified mucus
solution was calculated to be 134 ¡xg/ml by a modified Lowry
assay. Using commercially obtained porcine gastric mucin as
a standard, the glycoprotein concentration for the same sample
was calculated to be 73 /xg/ml by the PAS method, but only 17
/xg/ml by ELLA.
Coomassie blue staining reveals a major band at 55 kDa in
all the cecal filtrates examined (figure 4.1, lanes 5-8),
along with a smear that may or may not contain minor bands.
This major band most likely represents serum albumin (65).
Mucin itself is not visible at these concentrations, in either
purified or crude samples. As would be expected, homogenates
of ileal and colonic explants demonstrate a large number of
protein bands.
For many cecal filtrate and medium/filtrate samples, the
only band detectable with soybean agglutinin was in the
stacking gel, and consistent with mucus (figure 4.2, lanes 5-
7) . Other samples showed a smear of high molecular weight
material, with a major band at 144 kDa (lanes 8-11). PAS
staining of gels (data not shown) gave very similar results to
lectin staining. However, only the material in the stacking
gel representing mucus was visible in homogenates of ileal and

78
colonic explants (lanes 12-13), although there was a large
number of protein bands (figure 4.1, lanes 9-10).
Discussion
Soybean agglutinin binds to both rabbit colonic mucin and
porcine gastric mucin. There appear to be fewer available
binding sites on the rabbit mucin, as the amount of purified
rabbit colonic mucin calculated from the ELLA was much less
than was calculated by more conventional means. This is not
surprising, since colonic mucins are more highly sialylated
and sulfated than gastric mucins (160), making the internal N-
acetylgalactosamine residues less accessible. Therefore,
although porcine gastric mucin is used as a standard
throughout this study because of availability and consistency,
the actual concentrations of rabbit mucus are probably higher
than reported. However, the relationships between rabbit
samples will remain the same.
It is worth noting that this mucin was collected from a
rabbit with experimental mucoid enteropathy. A recent paper
(89) demonstrates changes in lectin-binding capacities in
mucins from rabbits with mucoid enteropathy. It indicates an
increase in binding to soybean agglutinin, as well as other
lectins with affinities for internal sugar residues,
consistent with the incomplete glycosylation characteristic of
many disease states.

79
The soybean agglutinin is not as specific a marker for
mucus as had been expected. However, when non-mucin bands are
present in cecal filtrates, they do not appear to change after
explant incubation, and can therefore be subtracted out.
Furthermore, they are not present in the homogenates of the
explants themselves, and so cannot be released into the
medium/filtrates following incubation.
Evaluation of Enzyme-Linked Lectin Assay
Introduction
Soybean agglutinin, the lectin used in the Western blots,
was also used in an ELLA (27). The mucus-containing sample
was used to coat polystyrene wells, and a SBA-HRP conjugate
was used to quantitate the amount of mucin bound.
However, a direct assay is generally not considered
appropriate for samples that contain only a small
concentration of the target molecule. A standard polystyrene
ELISA plate binds only 100 ng protein/well (71); the
medium/filtrate samples contain approximately 10 mg/ml.
Therefore, the majority of an undiluted sample does not bind
to the plate, and is removed in the first wash. Only when the
sample is diluted will a significant fraction of it bind.
Looking at the problem another way, if there is 1 /xg/ml mucus
in a sample that contains 10 mg/ml total protein, then mucus
constitutes 0.01% of that sample. If 100 ng protein binds to

80
the plate, then only 0.01 ng of mucus will bind, which is at
approximately the limit of detection (71).
Three types of modifications were made in an attempt to
develop a lectin-based assay that would be more sensitive.
One set of modifications was based on classic ELISA techniques
(71). Competition (indirect) assays and sandwich assays are
used to capture the antigen of interest out of contaminated
samples. Crude purification of the samples themselves was
also considered, and affinity chromatography and size
separation by ultrafiltration were performed. Finally, use of
a substrate that has a greater binding capacity (e.g.
nitrocellulose vs. polystyrene) allows for greater binding of
both contaminants and the molecule of interest, so that it can
be accurately quantitated without as great dilution of the
sample.
Materials and Methods
Unless otherwise noted, all reagents were obtained from
Sigma Chemical Co. (St. Louis, MO). The samples used for this
experiment were medium/filtrate samples following explant
incubation that had been obtained from pilot studies.
Competition assay. Plates were coated with porcine
gastric mucin (10 ng/well, 100 ng/well, or 1 /xg/well) for 2
hours at room temperature, and then blocked with 2.5% casein
for 1 hour at 37°C. 100 /xl of serial dilutions of porcine
gastric mucin (100 ng/ml stock) or medium/filtrate sample were

81
added simultaneously with 100 /x 1 of 1, 2, 5, or 10 /xg/ml SBA-
HRP. Alternatively, mucin containing samples and SBA-HRP were
preincubated together for 1 hour at 37°C, and then applied to
the plate. Plates were washed with PBS-T20, and 100 /xl of the
substrate OPD was added. After 10 minutes, the reaction was
stopped with 100 /xl of 4N H2S04, and the optical density was
read at 492 nm.
Sandwich assay. Plates were coated with 2 /xg/well of
unconjugated soybean agglutinin (Glycine max lectin), Wisteria
floribunda lectin, or Madura pomífera lectin for 2 hours at
room temperature, and then blocked with 2.5% casein for 1 hour
at 37°C. Then 100 /xl of serial dilutions of porcine gastric
mucin (100 ng/ml stock) or medium/filtrate sample were added,
and incubated 1-2 hours at 37°C. After multiple washes with
PBS-T20, 100 /xl of 10 /xg/ml SBA-HRP was added, and incubated
1 hour at 37°C. Plates were washed with PBS-T20, and 100 /xl
of the substrate OPD was added. After 10 minutes, the
reaction was stopped with 100 /xl of 4N H2S04, and the optical
density was read at 490 nm.
Centricon filtration. Centricon®-100 (Amicon, Inc.,
Beverly, MA) filters were rinsed with distilled water, and
used according to the manufacturer's instructions to separate
1 ml samples. Both retentates, which should contain only
mucus and other molecules > 100,000 Daltons, and filtrates,
which should contain molecules < 100,000 Daltons, were assayed
in direct ELLA. In addition, these samples were applied to a

82
6% SDS-polyacrylamide gel to determine the actual protein
compositions.
Affinity chromatography. One hundred /¿I of SBA-sepharose
beads were washed and resuspended in 100 /xl PBS, and added to
50 ill of sample. After 15 minutes of incubation at room
temperature with some mixing, the beads were removed by
centrifugation for 1 minute at 1000 x g. The supernatant was
retained as "wash 0", an indication of how much mucus did not
stick to the beads. The beads were washed 3 times with 1.4 ml
PBS. Free N-acetylgalactosamine (200 /xl of 10 mM, 0.1 M, 0.5
M, or 1.0 M) was added, and incubated for 15 minutes at room
temperature to elute the mucus. Following centrifugation, the
eluates were collected and dialyzed overnight vs. PBS. The
washes and eluates were analyzed for mucus concentration using
a direct ELLA.
Enzyme-linked lectin flow-through assay. An Easy-Titerâ„¢
ELIFA apparatus (Pierce, Rockford, IL) was assembled according
to the manufacturer's instructions, using either
nitrocellulose or a Biodyne® B membrane. The membrane was
coated with 100 /x 1 of serial dilutions of porcine gastric
mucin (10 /xg/ml stock) or medium/filtrate sample, which was
pulled through the membrane over a 5 minute period. A 2.5%
casein or 3% BSA solution was pulled through over a 10-15
minute period to block the membrane. One hundred /xl of 0.1,
0.25, 1, 2.5, or 10 /xg/ml SBA-HRP was added, and pulled
through in 5 minutes. Plates were washed 3 times with 200 /xl

83
PBS, and 100 ¡jlI of the substrate OPD was pulled through as
quickly as possible. The reaction was stopped with 100 /xl of
4N H2S04, and the optical density was read at 490 nm.
Dot blots. Dot blots were performed using nitrocellulose
in a miniblot apparatus. Serial dilutions of mucus or samples
in PBS or 0.5 M carbonate buffer, pH 9.6, were used to coat
the nitrocellulose for 2-3 hours at room temperature. The
blots were removed from the apparatus, washed with PBS, and
blocked with 3% BSA for 1 hour at 25°C. Five ¿ig/ml SBA-HRP in
PBS was added, and incubated for 1 hour at 25°C. As a control
for endogenous peroxidase activity, some blots were not
incubated with SBA-HRP, but rather remained in blocking
solution. Following four 5 minute washes with PBS, blots were
developed by addition of 0.3 g/ml of the substrate 4-chloro-l-
naphthol in 50 mM tris, pH 7.6. After 5-10 minutes, the blot
was washed with distilled water and dried. Densitometry was
performed to quantitate color development.
Results
In all competition assays the optical density reading
obtained was a direct reflection of the coating mucus
concentration, and SBA-HRP concentration. There was no
difference in optical density in the presence or absence of
competing mucus or filtrate sample.
In the sandwich assay, the optical density readings
depended solely on the type of lectin used for coating the

84
plate. With soybean agglutinin coating, the optical density
ranged from 1.8 to 2.5; for Wisteria floribunda the range was
2.1 to 2.7; for Madura pomifera it was 1.5 to 2.3. The
presence or absence of mucus had no effect.
Direct ELLA of retentates following Centricon®-100
ultrafiltration did not demonstrate appropriate decreases in
optical density with serial dilution. SDS-PAGE demonstrated
that the banding pattern for these retentates was identical to
that for unfractionated medium/filtrate samples (figure 4.1).
No protein was visible by Coomassie blue staining of the
ultrafiltrates on SDS-PAGE.
Following affinity chromatography with soybean
agglutinin, the material eluted from the beads was calculated
to contain approximately 25 ng/ml mucus by direct ELLA.
However, the material in "wash 0" (that which did not bind to
the beads) contained approximately 250 ng/ml mucus, as
measured by ELLA.
With the Easy-Titerâ„¢ system, the optical density readings
were dependent on the SBA-HRP concentration used for
detection, but did not vary with mucus concentration.
Background readings (no mucus added) were 2.4-2.8 for 10 /¿g/ml
SBA, 2.1-2.5 for 2.5 /xg/ml, 1.9-2.3 for 1 /xg/ml, and 1.1-1.4
for 0.25 /xg/ml on Biodyne® B membranes. The background values
on nitrocellulose were lower; however values for mucus coated
wells were also lower, and consequently meaningless.

85
In contrast, dot blots demonstrated appropriate
dilutional effects for both samples and mucus standards; wells
which did not contain mucus did not have color development.
However, dots containing medium/filtrate samples that were not
incubated with SBA-HRP demonstrated significant color
development, indicating the presence of endogenous peroxidase
activity within the samples. Subtraction of densitometry
values obtained in the absence of SBA-HRP from those obtained
in the presence of SBA-HRP led to inconsistent data that were
deemed unusable.
Discussion
Attempts to increase sensitivity and specificity of mucus
quantitation over the direct ELLA described by Cohan (27), and
used in the pilot studies were uniformly unsuccessful.
Competitive assays for the measurement of mucus have been
successful using an antibody-based system (133) . However,
mucin in solution was not able to compete with bound mucin for
lectin binding. It may be that the lectin has a greater
affinity for mucin that is bound to the plate as opposed to
mucin that is free in solution. Attachment to the plate may
allow greater exposure of N-acetylgalactosamine sites to which
the lectin may bind. Alternatively, mucin may bind to the
lectin in solution, but not block all of the multiple binding
sites, thereby allowing the lectin to bind to bound mucus.

86
Two sets of lectins were used for sandwich assays. At
first, the same soybean agglutinin was used to coat the plate
and for detection. Soybean agglutinin itself contains N-
acetylgalactosamine residues (60), so it is no surprise that
once the plate was coated with lectin, the lectin bound
maximally, even in the absence of mucin. The other lectins
used for coating were Wisteria floribunda lectin, a
glycoprotein which does not contain N-acetylgalactosamine, and
has an affinity for N-acetylglucosamine; and Madura pomífera
lectin, a non-glycoprotein that has an affinity for N-
acetylgalactosamine (60). Unfortunately, the lectin used for
detection was again soybean agglutinin; so that each coating
lectin had a direct affinity for the detecting lectin. Use of
a different lectin for detection was not attempted, although
it might have been successful.
Ultrafiltration was performed to remove contaminants with
a molecular weight of < 100,000 Daltons. However, the
acrylamide gel demonstrates that essentially all proteins were
retained by the filter. Therefore, the contamination in the
sample was not reduced, and ultrafiltration was of little
benefit. It has been recently reported that nuclease
treatment and acidification are required to separate
extraneous proteins from mucin (157).
Affinity chromatography was successful in removing
contaminants from the final sample. Unfortunately, more mucus
was present in the initial wash than in the sample eluted from

87
the beads. This lack of affinity between the lectin and mucin
in solution may relate to the problems with the competition
assay.
Nitrocellulose was selected as a substrate with increased
binding capacity for the enzyme-linked lectin flow-through
assays and dot blots. This worked well for samples containing
just mucus, or even samples that had been "spiked" with a
known contaminant, such as BSA. Unfortunately, cecal contents
contain significant endogenous peroxidase activity.
Peroxidase was selected as an enzyme over alkaline
phosphatase, which is known to have significant endogenous
activity in the gut (71) . In the polystyrene plate assay,
small amounts of endogenous peroxidase activity can be
detected, but these values are very small compared to the
mucus/lectin-associated activity. However, when measured by
densitometry, nitrocellulose blots incubated without
lectin/horseradish peroxidase conjugate often showed greater
activity than those incubated with the conjugate. It is not
clear why the endogenous peroxidase should bind with more
avidity to nitrocellulose than to polystyrene, but the fact
exists that it is not feasible to quantitate mucus bound to
nitrocellulose.
Therefore, it was not possible to improve upon the direct
ELLA using the above techniques. In order to use the direct
assay, it is necessary to perform serial dilutions to
ascertain that the values used for analysis are not

88
artifactually low. It is also desirable to select the lowest
dilution (highest concentration) possible to obtain the best
signal-to-noise ratio.
Harvesting of Mucus from Explants
Introduction
Mucus is a tenacious substance, and a problem in
quantitation is collection of the mucus from the tissue (160).
In fact, a specific gastric mucosal mucin receptor has been
described in the stomach (211). Different methods were
evaluated to see if an improvement could be made over simple
washing, without seriously damaging tissue and releasing
additional mucus. Since mucus is primarily held in
association by noncovalent mechanisms (160), acid and base
were added to alter pH. Because mucus contains mostly
negative charges, and relies on the presence of calcium,
magnesium, and other positive ions to maintain gel-forming
properties (67), EDTA was added in an attempt to chelate
cations, and disaggregate mucus. Finally, N-acetylcysteine,
a known mucolytic which breaks down disulfide bonds (200) was
added.
Materials and Methods
Explants were collected with a 6 mm Baker's biopsy punch,
and incubated in 0.5 ml Trowell's T8 medium with gentamicin
and 0.5 ml of a filtrate of cecal contents at 37°C in

89
95%02/5%C02 for 1, 2 or 3 hours. Following incubation, one of
the following was added: nothing, acid (10 /xl, 50 /xl, or 100
/x 1 concentrated HCl) , base (50 /xl 0.5N NaOH) , EDTA (1 mg/ml or
10 mg/ml), or N-acetylcysteine (0.1%). The medium/filtrate
solution was then collected with a pasteur pipette, after
aspirating and discharging five times. Direct ELLA and
Western blots were performed to determine the amount of mucus
collected. The explants were saved in 10% formalin, and
processed for histologic evaluation.
Results
Only the medium/filtrate solutions collected following
addition N-acetylcysteine appeared to contain more mucus than
those without any exogenous substances (figure 4.2, lane 7 vs.
lane 6) . Also, at the concentrations of acid used, tissue
damage could be seen grossly. Histologically, the explants
that had been incubated for 3 hours and collected with N-
acetylcysteine all looked healthy and normal (figure 4.3).
Discussion
N-acetylcysteine was deemed to be the best collection
method, as it either allows for collection of more mucus, or
perhaps just exposes more N-acetylgalactosamine sites on the
mucus that is washed off. One hour incubations were chosen
for the explants, since stimulated mucus secretion is apparent
within that time for several secretagogues, including

90
acetylcholine (217), histamine (161), mustard oil (161),
cholera toxin (189) , prostaglandin E2 (259), and interleukin-1
(27) .

91
123456789 10
Figure 4.1. 6% SDS-polyacrylamide gel.
Lanes containing: 1) molecular weight standards, 2) porcine
gastric mucin, 3) purified rabbit colonic mucin, 4) raw impure
rabbit colonic mucus, 5) filtrate from a control rabbit, 6)
same filtrate incubated with colon explant, 7) filtrate from
a rabbit with experimental ME, 8) same filtrate incubated with
colon explant, 9) homogenized ileal explant, and 10)
homogenized colonic explant stained for protein with Coomassie
blue.

Figure 4.2. Western blot (lectin).
Lanes containing: 1) molecular weight standards, 2) porcine
gastric mucin, 3) purified rabbit colonic mucin, 4) raw impure
rabbit colonic mucus, 5) filtrate from a rabbit with natural
ME, 6) same filtrate incubated with an ileal explant without
addition of N-acetylcysteine, 7) same collected with N-
acetylcysteine, 8) filtrate from a control rabbit, 9) same
filtrate incubated with colon explant, 10) filtrate from a
rabbit with experimental ME, 11) same filtrate incubated with
colon explant, 12) homogenized ileal explant, and 13)
homogenized colonic explant labelled with soybean agglutinin.
Since samples were not filtered prior to loading, aggregated
material is visible in the wells of lanes loaded with raw
mucus or homogenized explants.

93
MW
(kDa)
190-
125-
88-
56-
38-
Lane Number

94
Figure 4.3. Explant treated with N-acetylcysteine.
Colonic epithelium is intact, and cells appear viable. Goblet
cells containing mucin are visible in the crypts. H&E stain,
475x.

CHAPTER 5
CECAL LIGATION AS A MODEL OF MUCOID ENTEROPATHY
Introduction
Mucoid enteropathy is a serious disease of rabbits, for
which the cause is unknown. Research on the disease has been
hampered by a lack of good models. Pursuing the hypothesis
that mucus hypersecretion is secondary to constipation,
Sinkovics (207) was able to induce a mucoid enteropathy-like
illness by ligation of the colon. His success rate was
approximately 70%. Toofanian and Targowski (229, 235) found
that ligation of the cecum, but not cecectomy, also induced a
mucoid enteropathy-like syndrome in approximately 70% of
rabbits.
In this study specific pathogen-free (SPF, Pasteurella-
free) rabbits, with no evidence of coccidiosis or other
intestinal disease were used. Only 1 out of 8 rabbits
developed grossly evident mucus hypersecretion following
Toofanian and Targowski's protocol, in which only the cecum
was ligated, while vessels and nerves were spared. Therefore,
cecal ligations in which the ileocecocolic artery, vein and
nerve were incorporated into the ligature were also performed.
95

96
Materials and Methods
The experimental procedures were approved by the
University of Florida Institutional Animal Care and Use
Committee. Nineteen SPF male NZW rabbits, ranging in weight
from 1.3 to 2.3 kg, were used in this study. Fecal
examinations failed to reveal intestinal coccidia in any
rabbit. Five were assigned to be unoperated controls (group
1). Eight underwent surgery in which the cecum was ligated
without involving major blood vessels or nerves, as described
by Toofanian and Targowski (group 2) . Six underwent surgery
in which the distal branch of the ileocecocolic artery, vein,
and nerve were incorporated into the cecal ligature (group 3) .
This artery supplies the sacculus rotundus, the second and
third limbs of the cecum, and the distal ileum. The branches
to the ileum, sacculus rotundus, and third limb of the cecum
anastamose with branches of the proximal branch of the
ileocecocolic artery (11) . For comparison, four rabbits with
naturally-occurring mucoid enteropathy also underwent necropsy
(group 4).
The surgery was performed as follows. Anesthesia was
induced with 35 mg/kg ketamine and 5 mg/kg xylazine
intramuscularly and was maintained with additional injections
of ketamine, or with isoflurane administered by facemask. The
rabbits were positioned in dorsal recumbency, and the area
surrounding the umbilicus was shaved and prepared for aseptic
surgery. A 3-5 cm longitudinal incision was made at the

97
umbilicus. The base of the cecum was exteriorized. Windows
were created through both the ileocecal and cecocolic
mesenteries, distal to the sacculus rotundus. Double
ligatures of 2-0 silk were passed around the cecum, taking
care to either avoid or incorporate the major vessels.
Following ligation, ingesta was able to pass freely from the
ileum to the colon. The linea alba and then the subcuticular
tissues were closed with 3-0 chromic gut in an interrupted
pattern.
Clinical observations were made on the rabbits daily.
Those with clear mucus in the feces, or that appeared to be in
pain, were sacrificed immediately. Those that did not show
evidence of a mucoid enteropathy-like syndrome were sacrificed
4 to 10 days following surgery. Body weights were recorded at
time of surgery and at necropsy. Prior to euthanasia, rabbits
were anesthetized with ketamine/xylazine and blood was
collected from the cecal vein. Euthanasia was performed
immediately following by an overdose of pentobarbital.
Each rabbit underwent necropsy. Gross mucus
hypersecretion was defined as the presence of mucus in the
colon that could be lifted with forceps (figure 5.1) .
Portions of stomach, duodenum, ileum, cecum, proximal colon,
distal colon, mesenteric lymph node, kidney, liver, and lung
were preserved in 10% neutral buffered formalin. These were
processed and sectioned routinely, and slides were stained

98
with hemotoxylin and eosin. Selected slides were also double
stained with periodic acid/Schiff and alcian blue.
Cecal contents were collected and filtrates were prepared
for additional studies. The entire cecum was removed at the
ligature site and weighed. Fifty ml of sterile 0.9% saline
was infused, and the contents were massaged gently. The
contents were expressed into a sterile container and the empty
cecum was weighed. The volume of cecal contents in saline was
recorded. The slurry was centrifuged at 5000 rpm (3000 x g),
4 °C for 15 minutes. The supernatant was collected, and
centrifuged at 12,000 rpm (15,000 x g), 4°C for 30 minutes;
then at 18,000 rpm (30,000 x g) for 1 hour, and then 2 hours.
The resulting supernatant was then filtered through a 0.22
sterilizing membrane filter with a prefilter. The sterile
filtrates and sera from the cecal vein were stored at -70°C
until use. 1 ml aliquots were dried overnight at 90°C and
weighed.
Results
Surgery was uneventful in all rabbits. One rabbit (group
2) suffered dehiscence of the skin incision the day after
surgery. The skin was resutured under ketamine/xylazine
anesthesia, and there were no further problems. Food
consumption was decreased following surgery, but rabbits
continued to drink. All rabbits had a greatly decreased fecal
output following surgery. The day after surgery, all but one

99
of the group 2 rabbits had normal feces intermingled with soft
feces or diarrhea. The remaining rabbit passed small, dry-
pellets. All group 3 rabbits had diarrhea following surgery.
Six group 2 rabbits were sacrificed 4 days after surgery;
they had abnormal feces at the time of death. Two group 2
rabbits were sacrificed 10 days following surgery. They both
had normal feces from the fifth day following surgery until
death. Five group 3 rabbits passed copious amounts of mucus
in the feces on the third day following surgery, and were
sacrificed. The remaining rabbit did not excrete mucus, but
continued to have diarrhea, and was sacrificed on day 5.
Group one rabbits remained healthy throughout the study.
Table 5-1. Results of cecal ligation.
Group
Treatment
Number of
Animals
Mucus
Secretion
Cecal
Necrosis
1
Control
5
0
0
2
Ligation/
without
vessels
8
1
0
3
Ligation
incorporating
vessels
6
6
5
(3 seen
grossly)
4
Natural
disease
4
4
0
At necropsy (see table), all group 3 and 4 rabbits had
gross evidence of mucus hypersecretion in the colon (figure
5.1) . One group 2 rabbit had evidence of mucus hypersecretion
in the colon, while one had white chunks of inspissated mucus
in the cecum. Using a z-statistic to compare binomial

100
proportions, the difference in the percentage of rabbits
demonstrating mucus hypersecretion is statistically
significant (P < 0.003). In three out of 6 group 3 rabbits,
areas of the cecum (2 to 5 cm in diameter) appeared necrotic
grossly (figure 5.2), and there were adhesions of the cecum to
other organs in 4 of these rabbits. In one rabbit, the cecal
ligature was no longer present. This rabbit did not excrete
mucus antemortem, but mucus was present in the colon. These
abnormalities were not seen in group 1, 2 or 4 rabbits.
All rabbits that underwent surgery lost weight (figure
5.3; Appendix B), while control rabbits gained weight. Group
3 rabbits lost more weight than group 2 rabbits (P < 0.05).
The major histologic abnormality was segmental severe
full-thickness cecal necrosis with acute inflammation, edema,
and hemorrhage of the adjacent tissue in five out of six group
3 rabbits (figure 5.4). In one rabbit, the mucosa of a cecal
fold was necrotic, while surrounding tissue was normal. In
another, a cecal fold was spared while adjacent tissue was
necrotic. In the two group 3 rabbits without severe necrosis
there was hemorrhage and edema in the mucosal layer. One of
these also had acute peritonitis. No group 2 rabbit had full
thickness necrosis, but four out of eight had mild to moderate
peritonitis, and two had hemorrhages in the lamina propria.
These changes were apparently related to surgical technique.
In the sections taken from rabbits with the naturally-
occurring disease (group 4) , two rabbits had mild acute

101
inflammation of the cecal mucosa. Several stages of coccidia
and a few pinworms were present in one of these rabbits with
spontaneous ME. One rabbit in each of the four groups
appeared to have goblet cell hyperplasia (figure 5.5).
In the proximal colon, two group 2 rabbits, and two
rabbits with naturally-occurring disease demonstrated acute
mucosal inflammation. One group 3 rabbit had acute
peritonitis. Goblet cells appeared depleted of acidic (alcian
blue-staining) mucin in four group 3 rabbits, and in one
section from a group 4 rabbit (figure 5.6). In the distal
colon, four out of eight group 2 rabbits, four out of six
group 3 rabbits, and all four group 4 rabbits demonstrated
mucosal inflammation (figure 5.7). This was mild in most
animals, but was more severe than in any control animal. This
was usually characterized by heterophils, but in a few rabbits
plasma cells were predominant. Two group 2 rabbits, and one
rabbit each in group 3 and 4 had necrosis of the surface
epithelium with focal erosions.
In the small intestine, mild inflammatory infiltrates
were seen sporadically in all groups. Dilated lacteals were
seen in the one group 2 rabbit that secreted mucus, in three
group 3 rabbits, and in one group 4 rabbit. Villous atrophy
was present in the ileum of one group 3 rabbit. Villi
appeared shorter overall in group 3 rabbits, but quantitative
measurement was not attempted. Minimal goblet cell
hyperplasia was present in one rabbit each in groups 2, 3, and

102
4. There were no abnormal findings in the stomach of any
rabbit.
There were no significant findings in the lungs. The
mesenteric lymph nodes appeared congested in two group 3
rabbits. Hepatic congestion was seen in two group 3 rabbits.
These tissues had not been saved from group 4 rabbits.
Although the ratio of empty cecal weights relative to
body weights were similar for the three groups (figure 5.8),
the full cecal weights relative to body weight were
significantly higher in the control rabbits. This corresponds
to the finding that the volume and weight of cecal contents
were significantly decreased in group 2 and 3 rabbits. Other
parameters, such as the volume of filtrate collected relative
to the volume of cecal contents processed and the dry weight
of the final cecal filtrate, were not significantly different
between groups (Appendix B).
Discussion
Toofanian and coworkers (229, 233, 234, 235, 236)
reported a 70% incidence of mucoid enteropathy-like syndrome
following cecal ligation without incorporation of vessels or
nerves. The success rate in this study was substantially
lower (1 out of 8, or 12.5%). One possible explanation for
this is the quality of the rabbits used. No mention is made
of the status of the rabbits used in their experiments,
therefore it is likely that conventional rabbits were used.

103
On the other hand, we used SPF rabbits, which were free of
intestinal coccidia. Coccidia are known to increase mucus
secretion (114), and it is possible that their presence has a
synergistic effect with cecal ligation, resulting in a higher
incidence of mucus hypersecretion.
Incorporation of the ileocecocolic vessels into the cecal
ligature results in a more reproducible model of mucoid
enteropathy in the rabbit. It does have drawbacks, however.
Severe cecal necrosis was seen with this model, but has not
been reported in association with the natural disease (142,
238, 251). On the other hand, mild cecal necrosis has been
reported (238, 251) .
Histologically, there were many similarities between
ligation-induced mucoid enteropathy and the natural disease.
In both cases inflammatory changes were inconsistent, but
multifocal mild infiltrates of heterophils and mononuclear
cells were seen throughout the intestinal tracts of some
animals (142, 229, 235, 238, 251). Goblet cells were depleted
of acidic mucus in the colon (23 8) . Edema of intestinal
tissues or mild nephrosis was an occasional finding (142,
238) .
In this study the consistent goblet cell hyperplasia of
the small intestine that is supposed to be characteristic of
mucoid enteropathy (142, 229, 238, 251) was not observed.
However, the number of rabbits with goblet cell hyperplasia
was similar, whether or not the disease was natural or

104
experimentally induced. Cecal ligation may induce villous
atrophy in the ileum (229, 235), presumably due to small
intestinal bacterial overgrowth; this finding has not been
reported in the natural disease.
Although the cecum maintained its size in relation to the
weight of the animal following cecal ligation, the amount of
cecal contents, both weight and volume, was decreased. The
dry weights for the final cecal filtrates were not
significantly different for the three groups. Since the same
volume of saline (50 ml) was added to each cecum for the
collection of cecal contents, the cecal contents from rabbits
in Groups 2 and 3 were diluted more than the controls, and
therefore must have started out more concentrated. This is
consistent with reports that cecal contents from rabbits with
mucoid enteropathy are drier than normal (207, 238).
In two additional adult rabbits, only the ileocecocolic
vessels and nerve, but not the cecum, were ligated (data not
shown). Neither rabbit showed any sign of illness, or
exhibited any gross abnormalities at necropsy. Therefore, it
appears that there are two factors necessary for induction of
experimental mucoid enteropathy in SPF rabbits: 1) stasis of
cecal contents induced by cecal ligation, and 2) interference
with the blood and/or nerve supply.
Cecal ligation has also been performed on rabbits as part
of the reversible ileal tie adult rabbit diarrhea ("RITARD")
model for the study of cholera and enterotoxigenic E. coli

105
(222) . No diarrhea or illness was seen following cecal
ligation and temporary ileal occlusion in the absence of
pathogenic organisms. However, mucoid diarrhea was seen when
1010 Vibrio cholerae organisms were inoculated into the jejunum
concurrent with cecal ligation, as opposed to the watery
diarrhea that was seen when organisms were inoculated in
conjunction with ileal occlusion. This supports the finding
in this study that an additional insult is required along with
cecal ligation to stimulate mucus hypersecretion.

106
Figure 5.1. Excessive colonic mucus.
Single-banded proximal colon from group 3 rabbit #A7, opened
to reveal mucus.

107
Figure 5.2. Gross cecal necrosis.
Cecum from group 3 rabbit #A7. The base of the cecum is
severely congested. The necrotic area in the body of the
cecum ruptured during removal from the carcass.

108
100
Group
â–  Control
«i Cecal Ligation
â–¡ Cecal/Vessel Ligation
*
-200
Weight gain/day
Figure 5.3. Weight gain or loss.
Weight gain or loss per day following cecal ligation surgery,
in grams. Bars represent mean + SEM. N = 5 (control), 8
(cecal ligation), or 6 (cecal/vessel ligation). *
significantly different from control (P < 0.05) .

109
Figure 5.4. Cecal necrosis and inflammation.
At the left is relatively normal cecal tissue, however at the
right there is full thickness necrosis with a mixed
inflammatory infiltrate. H&E stain, 45x.

Figure 5.5. Goblet cell hyperplasia.

Ill
Figure 5.5a. Normal jejunum, stained for mucin with PAS/alcian
blue. Most goblet cells contain neutral mucin, staining with
PAS (arrow). 115x.

112
Figure 5.5b. Jejunum from rabbit with experimental mucoid
enteropathy, stained with PAS/alcian blue. Numerous goblet
cells contain acidic mucin, staining with alcian blue
(arrows). 115x.

Figure 5.6. Depletion of acidic mucin.

114
Figure 5.6a. Normal distal colon, stained for mucin with
PAS/alcian blue. Mucin-containing goblet cells are prominent
throughout the mucosa. Neutral (PAS-stained) mucins are
generally confined to deep in the crypts (arrow). 115x.

115
Figure 5.6b. Colon from a rabbit with experimental mucoid
enteropathy. Goblet cells contain little mucus, particularly
in the middle portion of the crypts. PAS/alcian blue stain,
115x.

Figure 5.7. Colonic inflammation.

117
Figure 5.7a. Distal colon from a group 2 rabbit. There is
loss of epithelium, and exudation of purulent material into
the intestinal lumen. Box indicates area shown in figure
5.7b. H&E stain, 115x.

118
Figure 5.7b. Higher magnification of colon in 5.7a.
Epithelial cells are flattened and numerous heterophils are
present in the lamina propria. H&E stain, 450x.

119
90
80
70
60
50
40
30
20
10
0
Group
â–  Control
¡s Cecal Ligation
â–¡ Cecal/Vessel Ligation
Weight
Volume
Full
Empty
(g)
(ml)
Cecum/Body
Cecum/Body
Weight
Ratio
Weight
Ratio
(g/kg)
(g/kg)
Figure 5.8. Characteristic of cecal contents.
Weight and volume of cecal contents collected, and the ratio
of full and empty cecal weights (in grams) to body weights (in
kg) . Bars represent mean + SEM. N = 5 (control) , 8 (cecal
ligation), or 6 (cecal/vessel ligation). * = significantly
different from control (P < 0.05).

CHAPTER 6
MUCUS SECRETION FROM INTESTINAL EXPLANTS
Introduction
It has been hypothesized that there is a substance in the
cecal contents of rabbits with experimental ME that causes
goblet cell hyperplasia (236). Pilot studies (chapter 3)
suggested that cecal contents from a rabbit with naturally-
occurring mucoid enteropathy stimulate mucus secretion from
intestinal explants. This study tests the hypothesis that
there is a mucus secretagogue within the luminal cecal
contents of rabbits with cecal ligation-induced mucoid
enteropathy.
Materials and Methods
Cecal contents were collected from control and
experimental rabbits, as described in chapter 5. Filtrates
from all 5 control rabbits (group 1) were used for this study.
Eight rabbits had undergone cecal ligation without ligation of
the ileocecocolic artery (group 2); however minor vessels had
been ligated in three of these rabbits. Such rabbits were
excluded, leaving 5 rabbits in group 2. In one of the 6
rabbits that had undergone cecal and vessel ligation (group
3) , the ligature was not present at necropsy; cecal contents
120

121
from this rabbit were not used, leaving 5 rabbits in this
group also.
Five male SPF NZW rabbits, weighing 1.8 to 2.3 kg were
used as explant donors. The rabbits were sedated with 3 5
mg/kg ketamine, and euthanasia was performed with
pentobarbital. The ileum and single-banded (aboral) portion
of each proximal colon was removed, and opened along the
mesenteric border. These intestinal sections were rinsed to
remove digesta, first in sterile 0.9% saline, and then in HBSS
containing 100 /¿g/ml gentamicin.
Fifteen explants were collected from each site in each
rabbit, using a 6 mm Baker's biopsy punch. To minimize the
effects of variation in mucus-secreting capacity along the
length of the gut, explants were collected in groups of 3; one
for each treatment group from comparable locations along the
length of the intestine. For each explant donor rabbit, the
filtrates from the three groups were matched in different
combinations. The explants were placed in wells containing
0.5 ml Trowell's T8 medium with 100 /xg/ml gentamicin and 0.5
ml of one of the cecal filtrates. The explants were incubated
1 hour at 37°C in 95% 02/5% C02. Additional wells containing
each medium/filtrate solution without explants, "blanks", were
also incubated.
Following incubation, 20 /xl of 5% N-acetylcysteine (final
concentration 0.1%) was added to each sample, and the
medium/filtrate solution was collected. The samples were

122
frozen at -70°C for enzyme-linked lectin assay (ELLA), and the
explants were weighed.
ELLA analyses were performed by modification of a
previously described technique (27). Porcine gastric mucin
standards, and 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640,
and 1:1280 dilutions of medium/filtrate samples in 100 /xl 0.5
M carbonate buffer (pH 9.6) were coated onto duplicate 96-well
polystyrene plates for 2 hours at room temperature. An
additional plate was coated in the same way to be used as an
endogenous peroxidase control plate. The plates were washed
4 times with PBS-T20 using an automated plate washer. The
plates were blocked with 3% BSA in PBS-T20 for 1 hour at 37°C.
Following incubation, the test plates were again washed, and
then incubated with 100 /ul of a 1:200 dilution of a 1 mg/ml
solution of SBA-HRP in PBS-T20 for 1 hour at 37°C. The
endogenous peroxidase control plate remained in the blocking
solution. Following 5 washes with PBS-T20, 85 /xl of the
substrate OPD was added to each well. After 10 minutes at
room temperature, the reaction was stopped with 85 /xl of 4 N
H2S04. The optical density was read at 490 nm.
Each sample was corrected for endogenous peroxidase
activity by subtracting the corresponding values on the
control plate (maximum = .032 optical density units). A
standard curve was prepared for each plate, and the
concentration of mucus in the undiluted sample (in relation to
porcine gastric mucus), was calculated for each well. The

123
1:80 dilution was selected for further analysis, as not having
artifactually low readings due to interference by
contaminating proteins, or too much variability. The amount
of mucus in the medium/filtrate in the absence of an explant
("blank") was subtracted from each sample to give the amount
of mucus secreted from the explant.
All results were subjected to analysis of variance using
a randomized block design. When significant main effects were
found, a Fisher's Least Significant Difference test was used
to determine differences between groups.
Results
In the absence of any explant the control medium/filtrate
mixtures contained more mucus than those of either of the
surgically ligated groups (7.09 /xg/ml for group 1 vs 0.32
¿ig/ml for group 2 and 0.4 7 ¡xg/ml for group 3; figure 6.1,
Appendix C) . When the outlier (rabbit A2) is removed from the
calculations, the mucus concentration for group 1 (1.31 /¿g/ml)
is significantly greater than for the ligated groups (P <
0.05) . There was a great deal of variation in mucus
concentration due to the explant source following incubation
with ileal explants, but no consistent differences due to
filtrates were seen (Appendix C) . On the other hand, there
was little evidence of secretion from colonic explants
incubated with control filtrates, while explants from
different rabbits were consistent in showing an increase

124
following incubation with filtrates from cecal-ligated
rabbits.
Significantly (P < 0.05) more mucus was secreted from
colonic explants when incubated with cecal filtrates from
rabbits that underwent cecal ligation than when incubated with
filtrates from control rabbits (figure 6.2). This effect was
more striking in the rabbits where only the cecum and not the
vessel was ligated. Most of this was due to the filtrate from
a single rabbit (A5), which had grossly visible mucus present
in the colon at necropsy. There was no difference between
groups in secretion from ileal explants.
Discussion
Cecal filtrates from control rabbits contain more mucus
than those from rabbits with experimentally-induced mucoid
enteropathy. At first this seems paradoxical, since mucus was
visible in the ceca of some of these rabbits. However, when
mucus is in high enough concentration locally (^25 mg/ml, 51) ,
it will form an insoluble polymeric gel which is unable to
pass through a .22 /xm filter, and is therefore removed from
the final filtrate. Alternatively, control filtrates may
contain a non-mucus substance that contains N-
acetylgalactosamine residues.
Once the baseline amount of mucus present in the filtrate
initially is subtracted, the results may be analyzed more
easily. No secretagogue effect is seen in the ileum in this

125
experiment. A major confounding factor was the wide variation
in measured mucus depending on the source of the explant.
This may be a real phenomenon, reflecting variation in ileal
mucus secretion based on individual factors such as vagal
tone, circadian rhythms, or the timing of the last meal (47,
73, 98) . However, another plausible explanation is that this
is a washing artifact. In the colon, there are no villi, so
removal of endogenous mucus from its surface (although not
from the crypts) should be more easily accomplished.
Therefore, the individual variations seen here in the amount
of mucus released from the ileal explants may just reflect the
difference in the amount of mucus trapped in the villi
following washing, which was then released into the
medium/filtrate during incubation.
The fact that filtrates from certain rabbits induced
mucus secretion more strongly than others could result from
the production of the secretagogue by microbial flora, which
can vary a great deal between individual rabbits; as opposed
to the secratagogue being a host product, such as an
inflammatory mediator, which one would expect to see in all
rabbits.

126
Calculated mucus (porcine gastric mucin equivalents) in
medium/filtrates, as measured by ELLA. Bars represent mean +
SEM. N=5 (blanks) or 25 (with explants).

127
Filtrate
-20 -1
Ileum Colon
Explant
Figure 6.2. Mucus secretion from explants.
Mucus secretion determined by subtracting the amount of mucus
in the medium/filtrate incubated without explants (as measured
by ELLA) from the amount incubated with explants. Values
shown are relative to the wet weight of the explant. Bars
represent mean + SEM. N = 25. * = significantly different
from control (P < 0.05).

CHAPTER 7
COMPARISON OF ELLA AND IN VITRO LABELLING
Introduction
Since the preceding data support, but do not prove, the
hypothesis that there is mucus secretagogue activity in the
luminal cecal contents of rabbits with experimentally-induced
mucoid enteropathy, further work was performed. In this
chapter two independent methods were used to measure mucus
secretion from intestinal explants; ELLA, and secretion of
mucus that had been radiolabelled in vitro. Two filtrates
from each group: control, cecal ligated, and cecal ligated
with gross mucus hypersecretion (including a rabbit that did
not undergo vessel ligation), were selected to form sample
pools to maximize the differences between the groups.
Materials and Methods
One male 2.0 kg SPF NZW rabbit was used. The rabbit was
sedated with 35 mg/kg ketamine, and euthanasia was performed
with pentobarbital. The single-banded (aboral) portion of the
proximal colon was removed, and opened along the mesenteric
border. This intestinal section was rinsed to remove digesta,
first in sterile 0.9% saline, and then in HBSS containing 100
fig/ml gentamicin.
128

129
Forty explants were collected, using a 6 mm Baker's
biopsy punch. To minimize the effects of variation in mucus-
secreting capacity along the length of the gut, explants were
collected in groups of 4; one for each treatment group taken
from comparable locations along the length of the intestine.
Each explant was placed in a polystyrene well containing 1 ml
Trowell's T8 medium with 100 /xg/ml gentamicin. Half of these
wells also contained 2 /xCi 3H-glucosamine. The explants were
incubated for 2 hours (pulse) at 37°C in 95% 02/5% C02.
Following this incubation, the explants were washed by
transferring them to individual wells containing 1 ml HBSS,
and the wash buffer was replaced 3 times. The explants were
then transferred to wells containing 0.5 ml Trowell's T8
medium with 100 /xg/ml gentamicin and 0.5 ml of one of the
following: control pool cecal filtrate (rabbits A1 and A12);
control pool cecal filtrate (rabbits A1 and A12) + 4 /xg/ml
bethanechol (final concentration = 10"6M); ligation/no mucus
pool (rabbits A6 and H2); or ligation/mucus pool (rabbits A5
and H7). Note that rabbit A5 underwent cecal ligation without
vessel ligation; however mucus hypersecretion was present at
necropsy, and filtrates from this rabbit were the most potent
stimulators of mucus secretion from explants. The explants
were incubated 1 hour at 37°C in 95% 02/5% C02. Additional
wells containing medium/filtrate solutions without explants,
"blanks", were also incubated.

130
Following incubation, 20 ¡i 1 of 5% N-acetylcysteine (final
concentration 0.1%) were added to each sample, and the
medium/filtrate solution was collected. The non-radioactive
samples were frozen at -70°C for enzyme-linked lectin assay
(ELLA).
Radioactive medium/filtrate samples and radioactive
explants were transferred to scintillation vials. One ml
water was added to each radioactive explant. Then, prior to
counting, 3.5 ml scintillation fluid (Scintiverse BD, Fisher
Scientific, Pittsburgh, PA) was added to each of the
radioactive medium/filtrate solutions and the radioactive
explants. Radioactive standards containing 0, 0.5, 1, and 2
/xCi of 3H-glucosamine in Trowell's T8 medium were also
counted.
Samples were counted on day 1, and recounted on day 4 for
verification. Counts were corrected for quenching using an
external standard ratio (ESR). Since the ESR were so low for
the medium/filtrate samples, suggesting extensive quenching,
100 ¡i! of each medium/filtrate/scintillation fluid mixture was
diluted into 3.5 ml additional scintillation fluid, and then
recounted.
ELLA analyses were performed as in chapter 6. All
results, both radioactive counts and mucus concentrations
calculated from ELLA, were subjected to analysis of variance
using a randomized block design. When significant main

131
effects were found, a Fisher's Least Significant Difference
test was used to determine differences between groups.
Results
Dilution of radioactive samples did reveal some quenching
of counts by the medium/filtrate solutions (Appendix D) .
However, following this dilution, the relationships between
the groups remained the same, although variability within the
groups was increased. The raw counts (corrected for
background and ESR) in the medium/filtrate, and the percent of
total counts in the medium/filtrate both demonstrated a
significant increase in radiolabelled mucus secretion from
explants incubated with cecal filtrates from rabbits with
experimentally-induced ME as compared with those incubated
with control filtrates. The explants incubated with either
bethanechol in the control filtrate or with filtrates from
rabbits that underwent cecal ligation without vessel ligation
did not secrete a significantly greater amount of mucus into
the medium.
As before, the ELLA demonstrated more mucus in the
control filtrates initially, but not following explant
incubation (figure 7.1). Following subtraction of the
"blanks", these results were similar to those derived from 3H-
glucosamine incorporation (figure 7.2). Again, significantly
more mucus was secreted from explants in the presence of cecal
filtrates from rabbits with cecal ligation and mucus

132
hypersecretion than in the presence of filtrates from control
rabbits (P < 0.05) . In addition, according to the ELLA
results, the increase seen with filtrates from cecal ligation
rabbits (without mucus hypersecretion) is significantly
greater than control (P < 0.05) .
Discussion
It is surprising that no increase was seen in response to
bethanechol, which has been shown to increase mucus secretion
in rabbit jejunum (65) and rat colon (14). Other cholinergic
agents, such as acetylcholine, also increase mucus secretion
in the rabbit colon (217). However, in a pilot study (data
not shown), explants were incubated with bethanechol in HBSS
and still showed no effect; therefore this lack of response is
probably not due to inhibitors in the cecal filtrate. Also,
the consistency between ELLA and radioactive labelling results
increases confidence in the ELLA model, and supports a lack of
bethanechol effect.
Two different methods were used to quantitate mucus
secretion in this study. The first involved incorporation of
radioactive glucosamine into mucus in vitro, followed by
stimulation of secretion into the medium/filtrate. The second
involved an enzyme-linked assay, depending on the specificity
of soybean agglutinin for N-acetylgalactosamine residues,
which are a major component of mucus. Although both methods
have several potential sources for error, there is good

133
agreement in the results obtained. The ELLA method showed
significance for more subtle differences. Together, the
results of these experiments support the hypothesis that there
is a mucus secretagogue present in the cecal contents of
rabbits with experimentally-induced mucoid enteropathy.

134
5.0
4.0
-1.0
Explant
â–  Blank
â–¡ Colon
X
I
Control Bethan- Cecal Mucus
echol Ligation Production
Filtrate
Figure 7.1. Mucus in medium/filtrates (ELLA).
Calculated mucus (porcine gastric mucin equivalents) in
medium/filtrates, as measured by ELLA. Bars represent mean +
SEM. N=1 (blanks) or 5 (with explants).

135
-2
ELLA Radioactivity
Analysis
Figure 7.2. Comparison of ELLA and tracer secretion.
Mucus secretion determined by measuring the increase in mucus
in the medium/filtrate following explant incubation (ELLA), or
by the percentage of counts released into the medium/filtrate
following incorporation of 3H-glucosamine into mucus
(Radioactivity) . Bars represent mean + SEM. N = 5. * =
significantly different from control (P < 0.05).

CHAPTER 8
PHYSICAL CHARACTERISTICS OF MUCUS SECRETAGOGUE
Introduction
The results obtained in Chapters 3, 6, and 7 provide
strong evidence for the presence of mucus secretagogue
activity in the cecal contents of rabbits with natural and
experimental mucoid enteropathy. The next question is
obvious: what is the physical nature of this secretagogue?
This chapter describes the first step in defining the
secretagogue: determination of protein or non-protein
properties. Another question that is raised by the previous
experiments is how a substance in the cecal contents can
affect the colon when the cecum has been ligated. Therefore,
since the most obvious route of transfer is through the blood,
serum collected from cecal vein blood was tested for
secretagogue activity.
Materials and Methods
Pooled cecal filtrate and serum samples were prepared.
The "control" pool contained 6 ml from rabbit A1, and 19.5 ml
from rabbit A12. The "ME" pool contained 8 ml from rabbit A5,
and 17 ml from rabbit H7. In addition, cecal vein serum pools
were prepared from 4.8 ml A1 and 5.5 ml A12 ("control"), and
136

137
5.5 ml A5 and 3.7 ml H7 ("ME") . Aliquots were taken from each
pool: 4 ml for part A, which were used immediately, and 3 ml
for part B, which were refrozen at -70°C. One aliquot for
each experiment was untreated. Ammonium sulfate (2.24 g) was
added to one 4 ml aliquot to give a final concentration of
85%; after 10 minutes the precipitate was collected by
centrifugation and resuspended in 4 ml HBSS (39). Ten units
of trypsin immobilized on beaded agarose (Sigma Chemical Co.)
and 10 units of similar chymotrypsin beads were washed with 5
ml HBSS, divided in two equal portions, and each was
resuspended in 0.2 ml HBSS, as described by the manufacturer.
Aliquots of cecal filtrates were added to each of these enzyme
preparations, and incubated at 25°C for 10 minutes. The beads
were removed by centrifugation. Aliquots of both pooled
filtrates and pooled serum were heated to 100°C for 30
minutes. The precipitated material was removed by
centrifugation. Finally, filtrates were adjusted to pH 1.0
with HCl and kept at 25°C for 30 minutes, then returned to pH
7.0 with NaOH.
Three male SPF NZW rabbits, each weighing 2.3 kg were
used as explant donors. In part A, two rabbits were
sacrificed, and the single-banded section of each proximal
colon was harvested. The intestinal segment was opened along
the mesenteric border, washed in saline followed by
HBSS/gentamicin, and 30 explants were collected from each
rabbit using a 6 mm Baker's biopsy punch. Paired explants

138
were assigned to "control" and "ME" groups. The explants were
incubated for 2 hours (pulse) at 37°C in 95% 02/5% C02 in one
ml Trowell's T8 medium containing 2 /xCi 3H-glucosamine and 100
/xg gentamicin.
Each aliquot of treated or untreated filtrate, or serum,
was added to an equal volume of Trowell's T8 medium containing
100 /xg/ml gentamicin. One ml aliquots were placed in labelled
polystyrene wells, including blank wells that would not
contain explants. Following the pulse incubation, the
explants from part A were washed by transferring them to
individual wells containing 3 ml HBSS, and replacing the wash
buffer 3 times. After washing, these explants were placed in
the wells containing the appropriate medium/filtrate solution
and incubated 1 hour at 37°C in 95% 02/5% C02. Explants from
the single rabbit in part B were collected from the colon in
the same manner as for part A, and were immediately incubated
with the corresponding medium/filtrate or medium/serum.
Then 20 /xl of 5% N-acetylcysteine (final concentration
0.1%) was added to each well, and the medium/filtrate was
collected. For part A, 100 fil of each medium/filtrate, as
well as each explant, were transferred to a scintillation
vial. Then 3.5 ml of scintillation fluid were added, and the
samples were counted in a LKB 1218 Rackbeta. The remainder of
each medium/filtrate from both parts was frozen in aliquots at
-70°C.

139
Finally, 200 /x 1 of each mediura/filtrate from part A was
precipitated by the addition of 0.112 g of ammonium sulfate
(final concentration = 85%). Following centrifugation, the
pellet was resuspended in 0.2 ml PBS and counted, to give a
better estimate of radioactivity that had been incorporated
into macromolecules. A direct ELLA was performed on each
sample in both parts as described in Chapter 6. Selected
samples were applied to 6% SDS-polyacrylamide gels, and either
stained for protein with 0.25% Coomassie blue or analyzed for
mucus using the Western blot technique described in Chapter 4 .
All results, both radioactive counts and mucus
concentrations calculated from ELLA, were subjected to
analysis of variance using a randomized block design. When
significant main effects were found, a Fisher's Least
Significant Difference test was used to determine differences
between groups. A Student's t test was used to compare serum
results.
Results
The data obtained by measuring radioactivity in the
medium/filtrate as a proportion of the total counts in both
the medium/filtrate and the explant (figure 8.1, Appendix F),
and that obtained by counting radioactivity that could be
precipitated from the medium/filtrate by ammonium sulfate
(figure 8.2) show similar overall trends. In the first case
the only significant difference between control filtrates and

140
those from rabbits with experimental ME can be seen in
untreated filtrates. On the other hand, for the precipitated
samples, reflecting only incorporated 3H-glucosamine and
excluding any unincorporated radioactivity, the difference
between control and experimental samples is significant for
ammonium sulfate-insoluble filtrate fractions and cecal vein
sera, but not the untreated filtrates. In neither case do the
trypsin or chymotrypsin-treated filtrates appear to stimulate
an increase in mucus secretion.
The results obtained by ELLA (figure 8.3, Appendix G) do
not fully coincide with those obtained by measuring secretion
of incorporated radioactivity. By this method, there is a
significant increase in mucus secretion associated with
filtrates from rabbits with experimental ME, whether the
entire filtrates, the ammonium sulfate-insoluble fractions, or
filtrates digested with trypsin or chymotrypsin are tested.
However, secretagogue activity was abolished by heat or acid
treatment (figure 8.4, Appendix H). When the data from part
A are taken alone and analyzed by t-test, serum from rabbits
with experimental ME appears to stimulate mucus secretion, but
in much lesser amounts. In part B, no secretagogue activity
was seen with either untreated or heat-treated serum.
Western blots (figure 8.5, lane 4) clearly demonstrated
lectin-binding material in the stacking gel, verifying that
the ELLA was measuring mucus. This material can be seen in
medium/filtrate samples from ME rabbits following explant

141
incubation (lane 4), but not in the absence of an explant
(lane 3). Material can be seen at approximately 144 kDa in
control filtrates (lanes 1-2), but this is unchanged following
explant incubation. There is minimal increase in staining of
the mucus band in the stacking gel following incubation of
control filtrates with colonic explants (lane 2).
Western blots further demonstrate that stimulation of
mucus secretion from the explants occurs following incubation
with trypsin and chymotrypsin-treated filtrates, as well as
untreated filtrate and the ammonium sulfate-insoluble filtrate
fraction (figure 8.6, lanes 6-9). No mucus could be seen in
the ME serum sample following explant incubation (lane 10).
Discussion
The treatments performed on the cecal filtrate samples
for this experiment were designed to determine whether or not
the mucus secretagogue is a protein. Ammonium sulfate
precipitates proteins, ribosomes, membrane fragments, and
denatured proteins (39). The fact that the secretagogue is
insoluble in ammonium sulfate suggests that it is a
macromolecule. However, smaller molecules may be aggregated
with larger molecules in the filtrate, so that they could also
precipitate. We do have evidence of aggregation in the
filtrates, in that the reténtate following ultrafiltration
with a Centricon®-100 showed the same banding pattern on an

142
SDS-polyacrylamide gel as the unfractionated filtrate (chapter
4) .
The results following trypsin or chymotrypsin treatment
were not consistent between the two assays. According to the
ELLA, the secretagogue activity remained following protease
treatment. This finding was supported by Western blot
analysis of the medium/filtrate following explant incubation.
Gels stained for protein with Coomassie blue revealed the same
banding pattern with or without protease treatment, suggesting
that in fact the proteases were not active in the
medium/filtrates. Therefore, we would predict activity in the
trypsin and chymotrypsin samples, whatever the nature of the
secretagogue. It is possible that the radioactivity
incorporation method was not sensitive enough to demonstrate
mucus secretion in these samples. This lack of sensitivity
has been reported previously (49).
Protease treatments were employed to determine whether or
not the secretagogue is a protein. Unfortunately, trypsin and
chymotrypsin appeared to have no effect on any of the proteins
in the cecal filtrates. In addition, incubation of filtrates
with 2 units/ml pronase for 30 minutes at 30°C had no effect
(figure 8.7), suggesting the presence of protease inhibitors
in the cecal filtrates. Consequently, it was not possible in
this study to state definitively that the secretagogue is a
protein. However, the secretagogue is both heat- and acid-

143
labile, giving further evidence that it is a large molecule,
and the most likely candidate is still a protein.
Cecal vein serum was also tested in the explant system,
to determine if the secretagogue is transmitted by serum, as
previously postulated (236). These results are ambiguous. An
increase in macromolecular radioactivity was seen in
medium/serum following explant incubation with serum from
rabbits with experimental ME. Using a t-test to evaluate the
ELLA, results separately from the filtrate-associated samples
also demonstrated an increase. However, the increase was a
degree of magnitude lower than that seen with the filtrate,
and the mucus concentration was below the limit of detection
of the Western blot. Following refreezing of the serum, even
that minor activity was eliminated. It is possible that a
very small amount of the secretagogue is present in the serum,
and that by chance the radioactivity results were able to
detect it. Alternatively, the radioactivity increase may be
associated with a glycoprotein other than mucus.
There is another explanation for these discrepancies that
may be considered. The radioactivity incorporation method
only measures release of newly synthesized mucin. On the
other hand, the ELLA measures release of all mucin--that which
is newly synthesized, and that which was formed prior to the
death of the animal and has been stored. It is possible that
two mechanisms of increased secretion are involved. One is
not protease-sensitive under these conditions, is not present

144
in the serum, and results in the accelerated release by
"compound exocytosis" of stored mucin in the manner of
acetylcholine and mustard oil (217). The other which is
trypsin- and chymotrypsin-sensitive, and is in the serum would
either accelerate the rate of "baseline" secretion (167, 184) ,
or would increase the rate of 3H-glucosamine incorporation
into mucin, such that a greater amount of the mucin that is
secreted in a baseline manner contains the radioactive label.
The increase in incorporation at first appears less likely,
since the explants were washed prior to incubation with the
medium/filtrates; but free glucosamine could still be present
and available for incorporation within the cells, or
associated with the explants.
From these experiments it is apparent that a large
macromolecule, most likely a protein, could be responsible for
the majority of the stimulated mucus secretion. The exact
nature of the secretagogue is still not certain, however, or
even if the secretagogue is a single entity. Alternative
techniques will have to be developed to distinguish mucus
synthesis from baseline secretion from compound exocytosis.

145
0.80 -i
A B C D E
Treatment Group
Figure 8.1. Proportion of tracer in medium/filtrate.
Following incubation with 3H-glucosamine-labelled explants,
counts in 100 /¿I of medium/filtrate were multiplied by 10 and
divided by this number plus the counts in the explant (total
counts). "Control" indicates filtrates collected from healthy
rabbits; "ME" indicates filtrates collected from rabbits with
experimental mucoid enteropathy. Treatment groups are A)
untreated filtrate, B) fraction of filtrate insoluble in 85%
ammonium sulfate, C) trypsin-treated filtrate, D)
chymotrypsin-treated filtrate, and E) cecal vein serum. Bars
represent mean + SEM. N = 6. * = significantly different
from control (P < 0.05).

146
160
140
o
O
O
Q.
£ 120
as
o
> 100
cd
80
Fíltrate
â–  Control
â–¡ ME
BCD
Treatment Group
Figure 8.2. Precipitable secreted tracer.
Macromolecules (ammonium sulfate insoluble material) in
medium/filtrates following incubation with ^-glucosamine-
labelled explants. Results are shown as percent of control
values. "Control" indicates filtrates collected from healthy
rabbits; "ME" indicates filtrates collected from rabbits with
experimental mucoid enteropathy. Treatment groups are A)
untreated filtrate, B) fraction of filtrate insoluble in 85%
ammonium sulfate, C) trypsin-treated filtrate, D)
chymotrypsin-treated filtrate, and E) cecal vein serum. Bars
represent mean + SEM. N = 6. * = significantly different
from control (P < 0.05).

147
ABODE
Treatment Group
Figure 8.3. Mucus secretion, part A (ELLA).
Mucus secretion determined by subtracting the amount of mucus
in the medium/filtrate incubated without explants from the
amount incubated with explants, as measured by ELLA.
"Control" indicates filtrates collected from healthy rabbits;
"ME" indicates filtrates collected from rabbits with
experimental mucoid enteropathy. Treatment groups are A)
untreated filtrate, B) fraction of filtrate insoluble in 85%
ammonium sulfate, C) trypsin-treated filtrate, D)
chymotrypsin-treated filtrate, and E) cecal vein serum. Bars
represent mean + SEM. N = 6. * = significantly different
from control (P < 0.05) by ANOVA; ** = significantly different
from control by t-test (P < 0.05) .

148
1 2 3 4 5
Treatment Group
Figure 8.4. Mucus secretion, part B (ELLA).
Mucus secretion determined by subtracting the amount of mucus
in the medium/filtrate incubated without explants from the
amount incubated with explants, as measured by ELLA.
"Control" indicates filtrates or sera collected from healthy
rabbits; "ME" indicates filtrates or sera collected from
rabbits with experimental mucoid enteropathy. Treatment
groups are 1) untreated filtrate, 2) filtrate heated to 100°C
for 30 minutes, 3) filtrate acidified to pH 1 for 30 minutes,
4) untreated cecal vein serum, and 5) cecal vein serum heated
to 100°C for 30 minutes. Bars represent mean + SEM. N = 5.
* = significantly different from control (P < 0.05).

149
Lane Number
12 3 4
Figure 8.5. Western blot (lectin).
Lanes containing: 1) pooled control medium/filtrate, 2) same
filtrate following incubation with colonic explant, 3) pooled
experimental ME medium/filtrate, and 4) same filtrate
following incubation with colonic explant labelled with
soybean agglutinin.

150
Figure 8.6. Western blot (lectin).
Lane 1 contains molecular weight standards. Lanes contain
control (2-5) or ME (6-9) medium/filtrates that are untreated
(2,6), insoluble in ammonium sulfate (3,7), trypsin-treated
(4,8), or chymotrypsin-treated (5,9); or ME cecal vein serum
(10), all following incubation with colonic explants, and
labelled with soybean agglutinin.

151
Figure 8.7. SDS-polyacrylamide gel.
Lanes containing: 1) molecular weight standards, 2)
experimental ME cecal filtrate, 3) same filtrate heated to
100°C for 30 minutes, and 4) same filtrate treated with 2
Units/ml pronase for 30 minutes stained for protein with
Coomassie blue.

CHAPTER 9
CONCLUSIONS
The goal of this project was to study the etiology of a
serious, not uncommon disease of rabbits. Mucoid enteropathy
is unique to the rabbit, and so has not been the subject of
intense interest. Therefore, the first task was to establish
a reproducible model of the disease to provide samples for
further study. The cecal ligation model developed by
Toofanian and Targowski (229, 235) was reported to induce a
mucoid enteropathy-like syndrome in 70% of rabbits. In this
present study the success rate was only 12.5% in SPF rabbits.
However, incorporation of the cecal vessels into the ligature
increased the incidence of gross mucus hypersecretion to 100%
in SPF rabbits. Yet, this also caused severe necrosis and
inflammation of the cecum, which is not seen in natural cases
of mucoid enteropathy.
The histologic changes seen following cecal ligation were
inconsistent, but they paralleled the inconsistent changes
reported for the natural disease. Inflammation of the distal
colon is seen in both natural and experimental disease, as is
mild necrosis of the cecum (142, 238). Therefore, cecal
ligation appears to represent a valid model of mucoid
enteropathy.
152

153
The cecal filtrates from rabbits with both natural and
experimental mucoid enteropathy induced mucus secretion from
colonic explants from healthy rabbits. Secretion from ileal
explants was also demonstrated following incubation with a
filtrate from a rabbit with the natural disease. However, the
variability between ileal explants obscured any potential
stimulation when the filtrates from rabbits with the
experimental disease were tested. Thus, ileal explants were
not used to assess secretagogue activity for these studies.
There was significant variation between the filtrates in
secretagogue activity. In particular, the filtrate from the
rabbit (A5) that developed gross mucus hypersecretion without
vessel ligation demonstrated the strongest activity. Two
explanations for this seem plausible: 1) if the secretagogue
is of host origin, this rabbit may have had a genetic
predilection to produce the secretagogue and develop the
disease; 2) if the secretagogue is of microbial origin, this
rabbit may have harbored more of the organism prior to
surgery, allowing for quicker overgrowth following even the
less radical surgery.
The secretagogue appears to be a macromolecule, most
likely a protein. Of the known intestinal mucus secretagogues
(chapter 2), the chemical properties are most consistent with
microbial enterotoxins, such as cholera and E. coli labile
toxin, or products of inflammation, such as interleukin-1,
immune complexes or MMS-68. Based on a comparison of

154
histology and secretagogue activity, it seems unlikely that
the secretagogue is an inflammatory mediator. There was no
apparent relationship between the degree of inflammation seen
following surgery, and the stimulation of mucus secretion from
explants. Furthermore, inflammation is not always present in
the natural disease.
On the other hand, cecectomy does not induce mucoid
enteropathy and instillation of tetracycline into the ligated
cecum prevents development of the disease, suggesting a
bacterial etiology (235). Cecal ligation in conjunction with
inoculation of Vibrio cholerae organisms results in mucoid
diarrhea in the RITARD model (222) . This demonstrates that
rabbits are susceptible to cholera toxin if the concentration
is high enough. Also, cholera toxin and E. coli LT are
stimulated, not destroyed, by trypsin. Therefore, resistance
of enterotoxins to digestive enzymes is not unexpected.
It appears that either very small amounts of the
secretagogue are carried in the serum, or that there is a
second factor in the serum that increases production or
baseline secretion of mucus. Thus, the serum is not a useful
fluid for study of the secretagogue, but it is possible that
the secretagogue present in the ligated cecum is transported
via the bloodstream to the colon to stimulate goblet cells
(236) .
This study has added to our understanding of mucoid
enteropathy of rabbits. The existence of a secretagogue has

155
been demonstrated, which can be the focus of further study.
This secretagogue is present in rabbits with the natural
disease, and can be induced experimentally. Physical
separation techniques can be used to identify it. Once it is
isolated, antibodies can be constructed and used to localize
its source. Once that is known, rational therapy may be
possible.
Future Directions
This study has only looked at mucus secretion, which may
be the last step in the pathogenesis of this disease. If this
is a microbial toxin, what is the microbe? How do the rabbits
become infected? What makes some animals resistant? Is the
microbe directly pathogenic, or does there have to be an
additional insult or disruption of microbial flora? If the
secretagogue is of host origin, from whence does it come?
What regulates it? What is the secretagogue of the
secretagogue? Although progress has been made, there is still
a great deal of work to be done to truly understand this
disease.

APPENDIX A
DATA TABLES FOR CHAPTER 3
in media/filtrates, measured by ELLA
(/xg/ml porcine gastric mucin equivalents)
Filtrate
Control P8 Mucoid enteropathy P4
Mucus
No explant:
Control P3
-0 ,
. 333
0 .
. 033
0 .
. 031
0 ,
.339
0 ,
. 144
Mean
0 ,
. 043
n
5
SEM
0
. 098
0.410 0.057
-0.202
-0.185
-0.032
0.041
0.410 -0.064
1 5
N/A 0.049
156

157
Mucus in media/filtrates, measured by ELLA
(ng/ml porcine
gastric mucin equivalents)
Ileum
explants:
Filtrate
Control P3
Control P8
Mucoid enteropathy
P8
0.733
2.148
P9
0.499
0.558
1.049
1.252
1.005
1.283
1.080
1.049
1.293
1.336
R1
0.429
0.749
0.566
0.666
0.502
R2
0.454
0.744
1.078
0.512
0.881
R3
0.376
0.571
0.646
0.476
0.585
Mean
0.649
0.620
1.014
n
7
6
14
SEM
0.245
0.044
0.115

158
Mucus in media/filtrates, measured by ELLA
(/¿g/ml porcine
gastric mucin equivalents)
Colon
explants:
Filtrate
Control P3
Control P8
Mucoid enteropathy
P8
0.078
0.084
P9
0.417
0.883
0.223
0.882
0.594
0.438
R1
0.505
0.454
0.317
0.581
0.366
R2
0.232
0.634
0.341
0.534
0.334
R3
0.302
0.683
0.566
0.410
0.454
Mean
0.336
0.549
0.467
n
7
6
10
SEM
0.063
0.039
0.075

159
Mucus secreted
into media/filtrate
(/¿g/ml)
Ileum
explants:
Filtrate
Control P3
Control P8 Mucoid
enteropathy
P8
1.066
2.091
P9
0.466
0.760
0.710
1.283
0.666
1.314
1.112
1.081
1.325
1.304
R1
0.285
0.339
0.524
0.256
0.461
R2
0.310
0.334
1.037
0.102
0.839
R3
0.232
0.161
0.605
0.066
0.544

160
Mucus secreted
into media/filtrate
(/¿g/ml)
Colon
explants:
Filtrate
Control P3
Control P8 Mucoid
enteropathy
P8
0.411
0.026
P9
0.384
1.085
0.192
1.068
0.563
0.624
R1
0.361
0.044
0.276
0.171
0.324
R2
0.088
0.224
0.300
0.124
0.293
R3
0.159
0.273
0.524
0.000
0.412

161
Explant weights (mg)
Filtrate
Control P3
Control P8
Mucoid enteropathy-
Ileum
explants:
P8
31.00
39 .00
P9
29.24
35.10
R1
21.67
22.37
19.83
18.55
28.46
R2
24.87
24.56
25.83
22.20
24.69
R3
19.76
13.59
18.30
21.79
18.00
Colon
explants:
P8
34.57
41.93
P9
35.54
42.87
R1
19.40
21.64
22.51
20.95
25.08
R2
25.16
18.78
21.27
19.40
19.59
R3
24.19
25.55
22.24
25.06
23.33

162
Mucus secreted (ng/mg explant tissue)
Ileum explants:
Filtrate
Control P3 Control P8 Mucoid enteropathy P4
P8
34.39
53.61
P9
15.93
21.65
R1
13.17
15.16
26.45
13.81
16.20
R2
12.46
13.61
40.14
4.61
33.99
R3
11.73
11.85
33.06
3.02
30.22
Mean
17.53
10.34
31.91
n
5
6
8
SEM
4.27
2.12
4.07
Control
Mucoid Enteropathy
Mean
13.61
31.91*
n
11
8
SEM
2.41
4.07
* t
3.86, P
0.002, significantly different from control

163
Mucus secreted (ng/mg explant tissue)
Colon explants:
Filtrate
Control P3
Control P8
Mucoid enteropathy P4
P8
11.89
0.63
P9
10.81
25.31
R1
18.61
2.03
12.25
8.15
12.94
R2
3.49
11.95
14.11
6.41
14.94
R3
6.55
10.69
23.58
0.00
17.67
Mean
10.27
6.54
15.18
n
5
6
8
SEM
2.17
1.93
2.69
Mean
n
SEM
* t = 2.21, P
Control
8.24
11
1.60
Mucoid Enteropathy
15.18*
8
2.69
0.05, significantly different from control

APPENDIX B
DATA TABLES FOR CHAPTER 5

165
Animal
Number
Survival
(days)
Initial
weight
(kg)
Final
weight
(kg)
Weight
gain
(kg)
Weight
gain/day
(kg)
Cecal
contents
(g)
Cecal
contents
(ml)
Filtrate
retained
(%)
Dry
weight
(mg/ml)
Full
cecum/body
wt(g/kg)
Empty
cecum/body
wt(g/kg)
Control
:
A01
2.0
2.015
0.0
0.005
64
50
35.0%
36.67
57.07
25.31
A02
1.9
1.978
0.1
0.019
103
100
26.7%
72.22
70.78
18.71
A04
2.1
2.368
0.3
0.027
49
30
37.5%
44.44
46.45
25.76
All
1.9
2.205
0.3
0.038
87
80
26.9%
48.00
73.47
34.01
Al 2
2.0
2.331
0.3
0.033
67
60
31.8%
36.67
63.92
35.18
Ligation/no vessel:
A03
10
2.0
1.857
-0.1
-0.014
18
20
50.0%
44.00
27.46
17.77
A05
4
1.9
1.563
-0.3
-0.084
37
40
27.8%
33.00
53.74
30.07
A06
4
1.7
1.512
-0.2
-0.047
28
30
45.0%
28.00
41.01
22.49
A10
10
2.2
1.890
-0.3
-0.031
43
40
27.8%
48.00
38.62
15.87
H1
4
1.40
1.092
-0.3
-0.077
23
20
42.9%
37.00
43.96
22.89
H2
4
1.32
1.289
-0.0
-0.008
42
40
22.2%
53.00
45.77
13.19
H3
4
1.50
1.370
-0.1
-0.032
41
40
22.2%
39.00
48.18
18.25
H4
4
1.56
1.456
-0.1
-0.026
36
30
43.8%
39.00
41.21
16.48

Animal
Number
Survival
(days)
Initial
weight
(kg)
Final
weight
(kg)
Weight
gain
(kg)
Weight
gain/day
(kg)
Cecal
contents
(g)
Cecal
contents
(ml)
Filtrate
retained
(%)
Dry
weight
(mg/ml)
Full
cecum/body
wt(g/kg)
Empty
cecum/body
wt(g/kg)
Ligation/vessel:
AO 7
3
1.7
1.692
-0.0
-0.049
58
50
25.0%
36.00
63.83
29.55
AO 8
3
2.0
1.615
-0.4
-0.128
51
50
35.0%
64.00
56.97
25.39
AO 9
5
1.7
1.475
-0.2
-0.178
15
15
61.5%
12.00
48.14
37.97
H05
3
2.30
1.905
-0.4
-0.132
32
30
40.0%
33.68
43.57
26.77
H06
3
2.08
1.786
-0.3
-0.098
33
35
41.2%
47.00
35.27
16.80
H07
3
2.12
1.821
-0.3
-0.100
31
30
43.8%
39.00
35.69
18.67
166

All animals
Initial
weight
(kg)
Final
weight
(kg)
Weight
gain
(kg)
Weight
gain/day
(kg)
Control
mean
1.98
2.18
0.20
0.025
std
0.08
0.18
0.14
0.013
Ligation
mean
1.70
1.50
-0.19
-0.040
(no vessel)
std
0.31
0.27
0.11
0.028
Ligation
mean
1.98
1.72
-0.27
-0.114
(vessel)
std
0.24
0.16
0.14
0.043
Ligation
mean
1.82
1.59
-0.23
-0.072
(both)
std
0.31
0.25
0.13
0.051
Study animals Initial Final Weight Weight
weight
(kg)
weight
(kg)
gain
(kg)
gain/day
(kg)
Control
mean
1.98
2.18
0.20
0.025
std
0.08
0.18
0.14
0.013
Ligation
mean
1.82
1.62*
-0.20*
-0.037**
(no vessel)
std
0.33
0.25
0.13
0.031
Ligation
mean
2.04
1.76*
-0.28*
-0.101*
(vessel)
std
0.22
0.11
0.16
0.033
* = significantly different from control (P < 0.05);
Cecal
Cecal
Filtrate
Dry
Full
Empty
contents
contents
retained
weight
cecum/body
cecum/body
(g)
(ml)
(%)
(mg/ml)
wt(g/kg)
wt(g/kg)
74.00
64.00
31.6%
47.60
62.34
27.79
21.12
27.02
4.8%
14.62
10.93
6.82
33.50
32.50
35.2%
40.13
42.49
19.63
9.40
8.86
11.3%
8.04
7.71
5.33
36.67
35.00
41.1%
38.61
47.24
25.86
15.47
13.42
12.0%
17.05
11.49
7.68
34.86
33.57
37.7%
39.48
44.53
22.30
11.93
10.64
11.6%
12.14
9.42
9.89
Cecal
Cecal
Filtrate
Dry
Full
Empty
contents
contents
retained
weight
cecum/body
cecum/body
(g)
(ml)
(%)
(mg/ml)
wt(g/kg)
wt(g/kg)
74.00
64.00
31.58%
47.60
62.34
27.79
21.12
27.02
4.81%
14.62
10.93
6.82
33.60*
34.00*
34.56%
41.20
41.32**
19.88
10.55
8.94
12.16%
10.43
12.99
7.53
41.00*
39.00*
36.99%
43.94
47.07
23.44
12.59
10.25
7.42%
12.29
12.84
5.46
** = (p
< 0.05)

168
ANALYSIS OF VARIANCE TABLES FOR CHAPTER 5
Initial
weight (kg)
Source
SS
df
MS
F
Between
0.124
2
0.062
1.12
Within
0.668
12
0.056
Total
0.793
14
Necropsy weight
(kg)
Source
SS
df
MS
F
LSD
Between
0.839
2
0.419
11.67*
0.26
Within
0.431
12
0.036
Total
1.270
14
Weight '
gain (kg)
Source
SS
df
MS
F
LSD
Between
0.654
2
0.327
16.13**
0.20
Within
0.243
12
0.020
Total
0.898
14
Weight <
gain/day
(kg)
Source
SS
df
MS
F
LSD
Between
0.0396
2
0.019788
26.90**
0.037
Within
0.0088
12
0.000736
Total
0.0484
14
* P < 0.005; **P < 0.001

169
Weight cecal contents collected (g)
Source
SS
df
MS
F
LSD
Between
4627
2
2313
9.70*
21.3
Within
2863
12
239
Total
7490
14
Volume of
cecal
contents
collected
(ml)
Source
SS
df
MS
F
LSD
Between
2583
2
1292
4.23***
24.1
Within
3660
12
305
Total
6243
14
Filtrate retained following processing of cecal contents (%)
Source
SS
df
MS
F
Between
0.00732
2
0.003662
0.49
Within
0.09042
12
0.007535
Total
0.09775
14
Dry weight (mg/ml)
Source
SS
df
MS
F
Between
103
2
52
0.33
Within
1895
12
158
Total
1998
14
* P < 0.005; ***P < 0.05

170
Full cecum/body weight ratio
Source
SS
df
MS
F
Between
1180
2
590
4.69***
Within
1511
12
126
Total
2691
14
Empty cecum/body
weight
ratio
Source
SS
df
MS
F
Between
157
2
79
1.96
Within
481
12
40
Total
638
14
***P < 0.05
LSD
15.5

APPENDIX C
DATA TABLES FOR CHAPTER 6
Mucus in undiluted media/filtrate (/¿g/ml)
Control filtrates: Ileum explants
A1
A2a
A4
All
A12
Mean
SEM
No explant
2.908
30.199
0.842
0.440
1.052
7.088
5.793
H8
3.837
29.578
3.837
2.975
2.190
8.484
5.283
H9
1.904
31.351
1.231
0.760
1.074
7.264
6.025
H10
2.131
27.470
3.893
1.779
2.143
7.485
5.010
Hll
3.943
30.514
4.447
5.157
3.734
9.559
5.245
H12
2.647
30.139
1.610
2.783
1.858
7.807
5.587
Mean
2.892
29.811
3.004
2.691
2.200
8.119
2.228
(explants)
SEM
0.425
0.652
0.658
0.733
0.432
Control filtrates
: Colon
explants
A1
A2a
A4
All
A12
Mean
SEM
No explant
2.908
30.199
0.842
0.440
1.052
7.088
5.793
H8
2.763
30.369
1.134
1.134
1.202
7.320
5.771
H9
2.545
29.612
0.952
0.789
1.048
6.989
5.664
H10
2.361
27.430
1.089
1.053
1.120
6.611
5.211
Hll
2.159
26.673
1.122
1.015
1.217
6.437
5.063
H12
1.920
31.978
1.212
0.843
1.376
7.466
6.130
Mean
2.350
29.212
1.102
0.967
1.193
6.965
2.280
(explants)
SEM
0.147
0.969
0.043
0.065
0.055
171

172
Mucus in undiluted media/filtrate (/xg/ml)
Ligation/no vessel filtrates: Ileum explants
A3
A5
A6
A10
H2
Mean
SEM
No explant
0.519
0.236
0.823
0.002
0.031
0.322
0.156
H8
2.129
2.344
1.994
0.545
1.620
1.726
0.318
H9
0.760
3.229
1.366
0.029
0.719
1.211
0.545
H10
0.955
2.344
1.095
0.265
1.150
1.162
0.335
Hll
1.487
3.038
2.378
1.627
3.976
2.501
0.462
H12
0.552
2.534
1.535
0.194
1.563
1.276
0.414
Mean
1.177
2.698
1.673
0.532
1.806
1.577
0.201
(explants)
SEM
0.284
0.184
0.229
0.286
0.566
Ligation/nc
i vessel
. filtrates: <
Solon explants
A3
A5
A6
A10
H2
Mean
SEM
No explant
0.519
0.236
0.823
0.002
0.031
0.322
0.156
H8
0.712
3.437
1.255
0.054
1.015
1.295
0.572
H9
0.530
5.616
1.417
0.103
1.013
1.736
0.995
H10
0.479
4.205
1.748
0.143
1.026
1.520
0.724
Hll
0.690
3.696
1.514
0.360
1.511
1.554
0.582
H12
0.489
3.208
1.822
0.200
0.943
1.332
0.543
Mean
0.580
4.032
1.551
0.172
1.102
1.487
0.289
(explants)
SEM
0.050
0.429
0.105
0.053
0.103

173
Mucus in undiluted media/filtrate (/xg/ml)
Ligation/vessel filtrates: Ileum explants
A7
A8
H5
H6
H7
Mean
SEM
No explant
1.897
0.137
0.152
0.031
0.122
0.468
0.358
H8
4.890
1.125
2.131
0.845
1.706
2.139
0.723
H9
1.689
0.174
0.279
0.294
0.654
0.618
0.280
H10
2.478
0.450
0.620
0.271
0.570
0.878
0.405
Hll
5.629
1.624
2.629
1.689
2.329
2.780
0.737
H12
2.748
0.195
1.119
0.520
1.274
1.171
0.440
Mean 3.487
(explants)
SEM 0.753
Ligation/vessel :
0.714
0.285
filtrates
1.355 0.724 1.307
0.446 0.262 0.330
i: Colon explants
1.517
0.279
A7
A8
H5
H6
H7
Mean
SEM
No explant
1.897
0.137
0.152
0.031
0.122
0.468
0.358
H8
2.217
0.323
0.393
0.437
1.215
0.917
0.363
H9
1.828
0.487
0.494
0.228
1.796
0.967
0.349
H10
1.859
0.572
0.417
0.336
2.317
1.100
0.411
Hll
1.973
0.572
0.530
0.510
2.123
1.142
0.371
H12
1.953
0.461
0.633
0.161
1.532
0.948
0.340
Mean
(explants)
SEM
1.966
0.068
0.483
0.046
0.494
0.043
0.334
0.064
1.797
0.198
1.015
0.151
a = not represented in figure 6.1.

174
Mucus
secreted
{fig/ml)
Control
filtrates
: Ileum
explants
Al
A2
A4
All
A12
H8
0.929
0.621
2.995
2.535
1.139
H9
-1.003
1.152
0.389
0.319
0.023
H10
-0.776
2.728
3.050
1.338
1.091
Hll
1.035
0.316
3.605
4.717
2.682
H12
-0.261
0.060
0.768
2.342
0.806
Control
filtrates
: Colon
explants
Al
A2
A4
All
A12
H8
-0.145
0.170
0.292
0.693
0.150
H9
-0.363
0.587
0.109
0.348
-0.003
H10
-0.547
2.769
0.247
0.613
0.068
Hll
-0.748
3.526
0.279
0.575
0.165
H12
-0.987
1.779
0.369
0.402
0.324

175
Mucus secreted (¿ig/ml)
Ligation/no mucus filtrates: Ileum explants
A3
A5
A6
A10
H2
H8
1.611
2.107
1.171
0.543
1.589
H9
0.241
2.993
0.543
0.027
0.688
H10
0.437
2.108
0.272
0.263
1.119
Hll
0.968
2.802
1.555
1.625
3.945
H12
0.033
2.297
0.712
0.192
1.532
Ligation/no mucus
! filtrates: Colon
explants
A3
A5
A6
A10
H2
H8
0.194
3.200
0.432
0.053
0.984
H9
0 . Oil
5.380
0.594
0.101
0.983
H10
-0.040
3.969
0.925
0.141
0.995
Hll
0.171
3.459
0.691
0.358
1.480
H12
-0.030
2.972
0.999
0.198
0.912

176
Mucus secreted (/¿g/ml)
Ligation/mucus
filtrates:
Ileum
explants
A7
A8
H5
H6
H7
H8
2.993
0.988
1.979
0.814
1.584
H9
-0.207
0.037
0.127
0.263
0.533
H10
0.581
0.313
0.468
0.240
0.448
Hll
3.732
1.487
2.477
1.658
2.208
H12
0.851
0.058
0.967
0.489
1.153
Ligation/mucus
filtrates:
Colon
explants
A7
A8
H5
H6
H7
H8
0.320
0.186
0.241
0.405
1.093
H9
-0.069
0.351
0.342
0.196
1.675
H10
-0.038
0.436
0.265
0.304
2.195
Hll
0.076
0.436
0.378
0.479
2.002
H12
0.056
0.324
0.481
0.130
1.411

177
Explant weights (mg)
Control
filtrates:
Ileum
explants
A1
A2
A4
All
A12
H8
22
22
21
21
19
H9
30
25
27
20
27
H10
25
19
29
31
21
Hll
25
20
20
24
27
H12
24
18
23
23
22
Control
filtrates:
Colon
A1
A2
H8
28
22
H9
16
22
H10
27
23
Hll
34
42
explants
A4
All
A12
28
47
24
26
30
22
30
27
25
29
26
32
27
31
22
H12
26
25

178
Explant weights
(mg)
Ligation/no
vessel filtrates:
Ileum
explants
A3
A5
A6
A10 H2
H8
25
23
24
28 21
H9
24
31
28
27 27
H10
23
20
26
32 23
Hll
30
22
26
27 22
H12
26
23
27
28 25
Ligation/
'no
vessel filtrates:
Colon
explants
A3
A5
A6
A10 H2
H8
30
23
24
24 26
H9
25
28
29
21 25
H10
25
27
24
28 26
Hll
31
32
27
33 27
H12
23
30
30
24 23

179
Explant
. weights (mg)
Ligation/vessel
filtrates:
Ileum
explants
A7
A8
H5
H6
H7
H8
32
22
24
23
29
H9
23
28
24
23
31
H10
29
25
13
20
29
Hll
23
27
24
19
24
H12
20
21
24
23
19
Ligation/vessel
filtrates:
Colon
explants
A7
A8
H5
H6
H7
H8
28
28
26
28
26
H9
26
23
27
29
22
H10
29
22
24
22
23
Hll
29
26
29
29
30
H12
31
27
31
26
30

180
Mucus secreted (ng/mg explant tissue)
Control filtrates: Ileum explants
A1
A2
A4
All
A12
Mean
SEM
H8
42.23
-28.21
142.62
120.71
59.92
67.45
30.27
H9
-33.45
46.07
14.41
15.96
0.84
8.77
12.88
H10
-31.06
-143.60
105.18
43.18
51.96
5.13
43.06
Hll
41.40
15.78
180.25
196.52
99.34
106.66
36.10
H12
-10.85
-3.33
33.38
101.85
36.64
31.54
19.98
Mean
1.65
-22.66
95.17
95.64
49.74
43.91
14.70
SEM
16.86
32.58
31.57
31.58
16.02
Control filtrates: <
Solon explants
A1
A2
A4
All
A12
Mean
SEM
H8
-5.17
7.74
10.41
14.75
6.26
6.80
3.32
H9
-22.67
-26.69
4.21
11.60
-0.16
-6.74
7.59
H10
-20.24
-120.40
8.23
22.70
2.73
-21.40
25.70
Hll
-22.01
-83.96
9.63
22.10
5.17
-13.81
18.96
H12
-37.98
71.16
13.68
12.98
14.74
14.92
17.27
Mean
-21.62
-30.43
9.23
16.83
5.75
-4.05
7.32
SEM
5.20
33.73
1.54
2.33
2.50

181
Mucus secreted (ng/mg explant tissue)
Ligation/no
vessel
filtrates:
Ileum
explants
A3
A5
A6
A10
H2
Mean
SEM
H8
64.42
91.62
48.79
19.38
75.66
59.97
12.33
H9
10.03
96.55
19.40
1.00
25.47
30.49
17.03
H10
18.98
105.41
10.46
8.22
48.66
38.35
18.25
Hll
32.27
127.34
59.79
60.19
179.34
91.79
26.92
H12
1.28
99.88
26.36
6.87
61.28
39.14
18.46
Mean
25.40
104.16
32.96
17.13
78.08
51.95
9.05
SEM
11.02
6.21
9.23
10.69
26.62
Ligation/no
vessel
filtrates:
Colon
explants
A3
A5
A6
A10
H2
Mean
SEM
H8
6.45
139.14
18.00
2.19
37.85
40.73
5.14
H9
0.46
192.15
20.49
4.80
39.30
51.44
7.83
H10
-1.60
147.00
38.55
5.05
38.26
45.45
23.36
Hll
5.53
108.11
25.60
10.84
54.81
40.98
16.23
H12
-1.29
99.06
33.30
8.27
39.65
35.80
13.24
Mean
1.91
137.09
27.19
6.23
41.97
42.88*
10.55
SEM
1.17
16.46
3.86
1.50
3.23
★ —
significantly different from control (P
<
0.05)

182
Mucus secreted (ng/mg explant tissue)
Ligation/vessel filtrates: Ileum explants
A7
A8
H5
H6
H7
Mean
SEM
H8
93.54
44.91
82.47
35.38
54.63
62.19
11.11
H9
-9.02
1.33
5.28
11.43
17.18
5.24
4.47
H10
20.04
12.54
35.99
11.98
15.46
19.20
4.43
Hll :
162.26
55.09
103.23
87.25
91.98
99.96
17.51
H12
42.57
2.78
40.29
21.26
60.68
33.52
9.90
Mean
61.88
23.33
53.45
33.46
47.99
44.02
8.14
SEM
30.19
11.17
17.49
14.13
14.40
Ligation/mucu
s filtrates: Colon explants
A7
A8
H5
H6
H7
Mean
SEM
H8
11.42
6.64
9.29
14.47
42.04
16.77
6.45
H9
-2.65
15.24
12.68
6.76
76.13
21.63
13.97
H10
-1.31
19.80
11.06
13.83
95.45
27.77
17.27
Hll
2.63
16.75
13.04
16.50
66.73
23.13
11.20
H12
1.82
12.00
15.53
4.98
47.03
16.27
8.07
Mean
2.38
14.09
12.32
11.31
65.47
21.11
4.99
SEM
2.46
2.25
1.04
2.28
9.75

183
ANALYSIS OF VARIANCE TABLES FOR CHAPTER
Ileum: Mucus secretion
(ng/mg
tissue)
Source
SS
df
MS
F
Main effects
Group
1062
2
531
0.138
Explant
73226
4
18306
4.781
Interactions
Group/exp
78733
8
9841
2.570
Error
229708
60
3828
Total
362729
74
Colon: Mucus secretion
. (ng/mg
tissue)
Source
SS
df
MS
F
Main effects
Group
27573
2
13786
7.504
Explant
447
4
111
0.060
Interactions
Group/exp
33123
8
4140
2.253
Error
110223
60
1837
Total
171366
74
LSD
24.25
* P < 0.005

APPENDIX D
DATA TABLES FOR CHAPTER 7 (RADIOACTIVITY)

185
Counts with background subtracted, and corrected for quenching by external standard ratio
Control filtrates:
Counts
in |
Calculated from
undiluted media|
1:45
dilution
Day 1
Day 4
Day 4
Day 5
Explant
1
1
2781
3056 |
5159
7515
2
2797
2913 |
4102
7245
3
2732
2929 |
5582
7875
4
2826
3026 |
6154
8820
5
3886
3828 |
8831
13635
Mean
3004
3151 |
5966
9018
SEM
221
172 1
791
1185
Explants | Counts in media/
total counts
Day 1
Day 4
1
Day 1
Day 4
Day 4
Day 5
1
(dil)
2295
3905
1
0.548
0.439
0.569
0.658
1454
2927
1
0.658
0.499
0.584
0.712
1981
3640
1
0.580
0.446
0.605
0.684
1444
3914
1
0.662
0.436
0.611
0.693
2720
5131
1
0.588
0.427
0.632
0.727
1979
3903
1
0.603
0.447
0.604
0.698
246
356
1
0.023
0.013
0 . Oil
0.012

Counts with background subtracted, and corrected for quenching by external standard ratio
Control filtrates with bethanachol:
Counts
in |
Calculated from |
Explants
1
Counts
in media/
undiluted media|
1:45
dilution
1
total counts
Day 1
Day 4 |
Day 4
Day 5 |
Day 1
Day 4
1
Day 1
Day 4
Day 4
Day 5
Explant
:
1
1
1
(dil)
1
3083
3209 |
7626
11655 |
1215
2810
1
0.717
0.533
0.731
0.806
2
2866
3153 |
6578
8550 |
1086
2683
1
0.725
0.540
0.710
0.761
3
3083
2993 |
6023
9495 |
1376
3096
1
0.691
0.492
0.660
0.754
4
2748
2763 |
6835
9765 |
2524
4252
1
0.521
0.394
0.617
0.697
5
2273
2331 |
5395
6885 |
1290
2674
1
0.638
0.466
0.669
0.720
Mean
2811
2890 |
6491
9270 |
1498
3103
1
0.652
0.482
0.677
0.749
SEM
149
160 1
376
781 1
261
297
1
0.038
0.027
0.020
0.019
186

Counts with background subtracted, and corrected for quenching by external standard ratio
Ligation/no vessel
, no mucus
hypersecretion
Counts
in |
Calculated from
undiluted media|
1:45
dilution
Day 1
Day 4 |
Day 4
Day 5
Explant
1
1
1957
2126 |
6438
8595
2
2329
2470 |
5789
8145
3
2550
2612 |
7292
9630
4
2587
2828 |
6181
10080
5
2974
2983 |
7236
14040
Mean
2480
2604 |
6587
10098
SEM
167
148 1
295
1045
filtrates:
| Explants | Counts in media/
total counts
Day 1
Day 4
1
Day 1
Day 4
Day 4
Day 5
1
(dil)
1071
2631
1
0.646
0.447
0.710
0.766
1032
2588
1
0.693
0.488
0.691
0.759
1718
3393
1
0.597
0.435
0.682
0.739
1460
2888
1
0.639
0.495
0.682
0.777
2998
6651
1
0.498
0.310
0.521
0.679
1656
3630
1
0.600
0.418
0.645
0.736
359
769
1
0.033
0.033
0.034
0.017
187

Counts with background subtracted, and corrected for quenching by external standard ratio
Ligation with mucus hypersecretion filtrates:
Counts
in |
Calculated from
undiluted media|
1:45
dilution
Day 1
Day 4
Day 4
Day 5
Explant
:
1
1
3402
3693 |
7278
9540
2
3549
3938 |
7785
12465
3
4018
4009 |
5649
9315
4
3285
3576 |
9298
12105
5
6562
7061 |
14349
19260
Mean
4163*
4455* |
8872
12537
SEM
613
656 1
1488
1799
* = significantly
different
from control (P
Explants | Counts in media/
total counts
Day 1
Day A
Day 1
Day 4
Day 4
Day 5
(dil)
1281
2699 |
0.726
0.578
0.729
0.779
1243
2653 |
0.741
0.597
0.746
0.825
2548
3834 |
0.612
0.511
0.596
0.708
1144
2763 |
0.742
0.564
0.771
0.814
3519
6017 |
0.651
0.540
0.705
0.762
1947
3593 |
0.681
0.554*
0.712
0.777*
470
644 1
0.027
0.015
0.030
0.021
< 0.05)
188

189
ANALYSIS OF VARIANCE TABLES, CHAPTER 7 (RADIOACTIVITY)
Counts in
undiluted
media
, day 1:
Source
SS
df
MS
F
LSD
Treatment
8035274
3
2678425
5.35*
976
Blocks
3469493
4
867373
1.73
Error
6013300
12
501108
Total
17518067
19
Counts in
undiluted
media
, day 4:
Source
SS
df
MS
F
LSD
Treatment
10038860
3
3346287
5.65*
1060
Blocks
3047190
4
761798
1.29
Error
7103036
12
591920
Total
20189087
19
Calculated from 1:45
dilution, day 4
:
Source
SS
df
MS
F
Treatment
25010161
3
8336720
2.56
Blocks
22349087
4
5587272
1.72
Error
39033051
12
3252754
Total
86392299
19
* P < 0.05

190
Calculated from 1:45 dilution, day 5:
Source
SS
df
MS
F
Treatment
38651074
3
12883691
2.16
Blocks
55272983
4
13818246
2.32
Error
71568158
12
5964013
Total
1.7E+08
19
Explants,
day 1:
Source
SS
df
MS
F
Treatment
808756
3
269585
0.67
Blocks
4760451
4
1190113
2.97
Error
4800961
12
400080
Total
10370168
19
Explants,
day 4 :
Source
SS
df
MS
F
Treatment
1664090
3
554696
0.63
Blocks
13852704
4
3463176
3.93
Error
10566780
12
880565
Total
26083573
19

191
Counts in
media/total
counts, day 1:
Source
SS
df
MS
F
Treatment
0.0248
3
0.00827
2.14
Blocks
0.0278
4
0.00696
1.80
Error
0.0463
12
0.00386
Total
0.0989
19
Counts in
media/total
counts, day 4:
Source
SS
df
MS
F
LSD
Treatment
0.0454
3
0.0151
7.64*
0.061
Blocks
0.0204
4
0.0051
2.57
Error
0.0237
12
0.0020
Total
0.0895
19
Counts in
media/total
counts, day 4,
1:45 dilution:
Source
SS
df
MS
F
Treatment
0.0315
3
0.01045
2.98
Blocks
0.0104
4
0.0026
0.74
Error
0.0422
12
0.0035
Total
0.0840
19
* P < 0.05

192
Counts in
media/total
counts
, day 5,
1:45 dilution:
Source
SS
df
MS
F
LSD
Treatment
0.0177
3
0.00592
3.79*
0.054
Blocks
0.0057
4
0.00143
0.92
Error
0.0187
12
0.00156
Total
0.0421
19
* P < 0.05

APPENDIX E
DATA TABLES FOR CHAPTER 7 (ELLA)
Mucus in media/filtrate, calculated from ELLA (¿¿g/ml)
Control
Bethanechol Lig/no mucus
Ligation/mucus
No explant 3.153
3.022
-0.402
-0.672
1
3.648
3.578
0.481
4.851
2
2.998
3.656
0.295
3.972
3
2.911
3.301
0.327
3.586
4
2.864
2.852
0.545
4.516
5
3.105
2.699
0.369
3.044
Explant mean 3.105
3.217
0.403
3.994
SEM
0.142
0.191
0.047
0.322
Mucus Secreted (/xg/ml)
Control Bethanechol
Lig/no mucus
Ligation/mucus
1
0.496
0.556
0.882
5.523
2
-0.154
0.634
0.697
4.644
3
-0.241
0.279
0.729
4.258
4
-0.289
-0.170
0.947
5.189
5
-0.047
-0.323
0.771
3.717
Mean
-0.047
0.196
0.805*
4.666**
SEM
0.142
0.191
0.047
0.322
* = significantly
different
from control (P
< 0.05)
** = significantly
different
from control (P
< 0.002)
193

194
ANALYSIS OF
VARIANCE
TABLE FOR CHAPTER 7
(ELLA)
Source
SS
df
MS
F
LSD 05
Treatment
72.834
3
24.278
166.847*
0.526
Blocks
1.508
4
0.377
2.591
Error
1.746
12
0.146
Total
76.088
19
LSD qo2
0.948
* P < 0.001

APPENDIX F
DATA TABLES FOR CHAPTER 8 (RADIOACTIVITY)

196
Counts in media/filtrate : Total counts
Untreated Precipitated
Control
Mucus
Control
Mucus
H15
1
0.682
0.785
0.739
0.779
2
0.728
0.700
0.752
0.757
3
0.657
0.666
0.702
0.687
H16
1
0.650
0.658
0.771
0.803
2
0.662
0.772
0.768
0.826
3
0.703
0.750
0.761
0.783
Mean
0.680
0.722*
0.749
0.773
SEM
0.012
0.022
0 . Oil
0.020
* = significantly different from control
Tryps
in
Chymo t ryp sin
Serum
Control
Mucus
Control
Mucus
Control
Mucus
0.712
0.746
0.723
0.767
0.771
0.790
0.688
0.681
0.723
0.710
0.768
0.819
0.614
0.642
0.656
0.683
0.716
0.747
0.695
0.697
0.682
0.676
0.760
0.765
0.710
0.728
0.710
0.693
0.800
0.815
0.739
0.743
0.741
0.730
0.773
0.794
0.693
0.706
0.706
0.710
0.765
0.788
0.017
0.017
0.013
0.014
0.011
0.012
(P < 0.05)

Radioactivity in ammonium sulfate-precipitated material
Average of triplicate counts with background subtracted
Untreated
Precipitated
Trypsin Chymotrypsin
Serum
Control
Mucus
Control
Mucus
Control
Mucus
Control
Mucus
Control
Mucus
H15
1
48.67
40.33
33.67
39.33
43.00
43.00
27.33
42.67
30.33
48.33
2
34.33
34.33
21.67
47.67
36.67
47.33
34.33
52.00
64.00
86.67
3
22.33
40.67
29.00
35.67
28.67
25.00
33.67
27.33
41.67
44.00
H16
1
21.67
32.67
33.67
46.33
30.00
45.67
44.33
46.67
36.00
39.00
2
39.67
46.67
26.33
45.67
31.00
30.67
32.00
31.00
68.33
80.67
3
26.00
39.67
20.33
31.33
49.00
48.33
41.00
34.33
56.33
75.33
Mean
32.11
39.06
27.44
41.00*
36.39
40.00
35.44
39.00
49.44
62.33*
SEM
4.39
2.05
2.35
2.70
3.33
3.99
2.53
3.93
6.38
8.51
* = significantly different from control (P < 0.05)
197

198
ANALYSIS OF VARIANCE TABLES, CHAPTER 8 (RADIOACTIVITY)
Proportion of counts in media/filtrate vs. total:
Source
SS
df
Treatment
0.073
9
Blocks
0.042
5
Error
0.028
45
Total
0.143
59
* P < 0.001
MS
F
LSD.os
0.0081
13.03*
0.029
0.0084
13.47
0.0006
Counts in media/filtrate precipitated with ammonium sulfate:
Source
SS
df
MS
F
LSD 05
Treatment
5064
9
562.70
5.19*
12.15
Blocks
1072
5
214.32
1.98
Error
4877
45
108.37
Total
11013
59
* P < 0.001

APPENDIX G
DATA TABLES FOR CHAPTER 8, PART A (ELLA)

200
Mucus in media/filtrate, calculated from ELLA (/xg/ml)
Untreated
Precipitated
Trypsin Chymotrypsin
Serum
H15 blank
1
2
3
H16 blank
1
2
3
Means:
Blanks
Explants
SEM:
Blanks
Control
Mucus
Control
Mucus
Control
Mucus
Control
Mucus
Control
1.927
0.148
1.610
0.405
1.809
0.423
1.678
0.321
0.049
2.076
2.943
1.485
4.514
1.927
2.718
1.902
2.613
0.049
1.746
2.565
1.759
3.662
1.790
2.853
1.865
2.235
-0.076
1.703
2.265
1.678
2.757
1.858
1.815
1.678
1.899
-0.101
1.112
0.069
0.992
-0.076
1.178
-0.003
1.288
-0.097
-0.077
1.264
2.012
1.312
2.521
1.421
2.063
1.436
1.889
0.063
1.283
1.520
1.312
2.657
1.312
1.604
1.493
1.876
-0.018
1.245
1.599
1.374
2.371
1.379
1.841
1.135
1.542
0.020
1.519
0.039
1.301
0.164
1.494
0.210
1.483
0.112
0.055
1.553
2.150
1.486
3.080
1.614
2.048
1.584
2.009
-0.025
0.407
0.108
0.308
0.241
0.315
0.213
0.195
0.209
0.014
0.139
0.226
0.078
0.341
0 . Ill
0.210
0.118
0.150
0.027
Mucus
0.507
0.555
0.609
0.891
0.008
0.023
0.071
0.145
0.265
0.454
0.250
Explants
0.144

Mucus Secreted (/xg/ml)
Untreated Precipitated Trypsin Chymotrypsin Serum
Control
Mucus
Control
Mucus
Control
Mucus
Control
Mucus
Control
Mucus
H15
1
0.149
2.795
-0.124
4.109
0.118
2.291
0.224
2.291
0
0.048
2
-0.180
2.417
0.149
3.257
-0.019
2.429
0.187
1.913
-0.124
0.102
3
-0.224
2.117
0.068
2.351
0.050
1.391
0
1.577
-0.149
0.384
H16
1
0.153
2.080
0.319
2.598
0.243
2.065
0.148
1.986
-0.014
0.015
2
0.172
1.588
0.319
2.733
0.133
1.606
0.205
1.973
-0.095
0.064
3
0.133
1.667
0.381
2.448
0.200
1.843
-0.153
1.639
-0.057
0.138
Mean
0.034
2.Ill*
0.186
2.916*
0.121
1.938*
0.102
1.897*
-0.073
0.125
SEM
0.075
0.186
0.079
0.272
0.039
0.163
0.061
0.106
0.024
0.055
* = significantly different from control (P < 0.05)

202
Source
SS
df
Treatment
69 ,
. 85
9
Blocks
1.
.20
5
Error
3 .
. 80
45
Total
74 .
. 85
59
* P < 0.001
MS
F
LSD 05
7.76
91.95*
0.339
0.24
2.85
0.084

APPENDIX H
DATA TABLES FOR CHAPTER 8, PART B (ELLA)

204
Mucus in media/filtrate, calculated from ELLA (/xg/ml)
Untreated-
Filtrate
Heat-Filtrate
Acid-Filtrate
Untreated
-Serum
Heat-
Serum
Control
Mucus
Control
Mucus
Control
Mucus
Control
Mucus
Control
Mucus
Blank
1.552
-0.433
1.941
-0.443
1.687
-0.357
-0.610
-0.328
-0.613
-0.279
1
1.434
0.975
1.671
-0.327
1.459
-0.414
-0.502
-0.316
-0.567
-0.321
2
1.401
1.055
1.754
-0.304
1.177
-0.388
-0.524
-0.244
-0.334
-0.112
3
1.629
1.085
2.613
-0.350
1.658
-0.304
-0.470
-0.391
-0.538
-0.280
4
0.894
0.643
1.691
-0.422
1.546
-0.445
-0.663
-0.391
-1.042
-0.716
5
1.588
0.997
2.186
-0.266
1.401
-0.239
-0.413
-0.348
-1.042
-0.716
Explant:
Mean
1.389
0.951
1.983
-0.333
1.448
-0.358
-0.515
-0.338
-0.705
-0.429
SEM
0.131
0.080
0.183
0.026
0.081
0.038
0.042
0.028
0.143
0.122

Mucus Secreted (/xg/ml)
Untreated-
Filtrate
Heat-Filtrate
Acid-Filtrate
Untreated
-Serum
Heat-Serum
Control
Mucus
Control
Mucus
Control
Mucus
Control
Mucus
Control
Mucus
1
-0.118
1.408
-0.270
0.116
-0.228
-0.057
0.107
0 . Oil
0.046
-0.042
2
-0.152
1.488
-0.187
0.139
-0.511
-0.031
0.086
0.084
0.279
0.167
3
0.077
1.519
0.673
0.093
-0.029
0.053
0.139
-0.064
0.075
-0.001
4
-0.658
1.076
-0.249
0.021
-0.141
-0.088
-0.054
-0.064
-0.429
-0.437
5
0.035
1.431
0.245
0.177
-0.286
0.118
0.197
-0.020
-0.429
-0.437
Mean
-0.163
1.384
0.042
0.110
-0.239
-0.001
0.095
-0.010
-0.091
-0.150
SEM
0.131
0.080
0.183
0.026
0.081
0.038
0.042
0.028
0.143
0.122
205

206
ANALYSIS OF
VARIANCE
TABLE FOR
CHAPTER
8, PART
B (ELLA)
Source
SS
df
MS
F
LSD 05
002
Treatment
9.79
9
1.09
27.61
254
417
Blocks
0.66
4
0.16
4.17
Brror
1.42
36
0.04
Total
11.86
49

REFERENCE LIST
1) Ahmad, A., C. H. Wang, M. Korenaga, R. G. Bell, and L. S.
Adams. 1990. Synergistic interaction between immune
serum and thoracic duct cells in the adoptive transfer of
rapid expulsion of Trichinella spiralis in adult rats.
Exp Parasitol 71:90-99.
2) Alarcón de la Lastra, C., A. López, and V. Motilva. 1993.
Gastroprotection and prostaglandin E2 generation in rats
by flavonoids of Dittrichia viscosa. Planta Med 59:497-
501.
3) Alarcón de la Lastra, C., M. J. Martin, and V. Motilva.
1994. Antiulcer and gastroprotective effects of
quercetin: a gross and histologic study. Pharmacology
48:56-62.
4) Allen, A., W. J. Cunliffe, J. P. Pearson, L. A. Sellers,
and R. Ward. 1984. Studies on gastrointestinal mucus.
Scand J Gastroent Suppl 93:101-113.
5) Allen, A., A. J. Leonard, and L. A. Sellers. 1988. The
mucus barrier. Its role in gastroduodenal protection. J
Clin Gastroenterol 10(Suppl 1):S93-S98.
6) Argenzio, R. A. 1985. Digestion, absorption, and
metabolism. In: Dukes' Physiology of Domestic Animals.
Tenth Edition. Eds. Swenson, M. J. and W. O. Reece.
Ithaca: Cornell University Press, p.262-277.
7) Argenzio, R. A., M. Southworth, J. E. Lowe, and C. E.
Stevens. 1977. Interrelationship of NA, HC03, and
volatile fatty acid transport by equine large intestine.
Am J Physiol 233 (Endocrinol Metab Gastrointest Physiol
2):E469-E478.
8) Arnold, J. W. , G. R. Klimpel, and D. W. Niesel. 1993.
Tumor necrosis factor (TNFo;) regulates intestinal mucus
production during salmonellosis. Cell Immunol 151:336-
344 .
9) Babkin, B. P. 1950. Secretory Mechanism of the Digestive
Glands. 2nd Ed. New York: Hoeber.
207

208
10) Barthold, S. W. , G. L. Coleman, R. O. Jacoby, E. M.
Livstone, and A. M. Jonas. 1978. Transmissible murine
colonic hyperplasia. Vet Pathol 15:223-236.
11) Bensley, B. A. 1938. Practical Anatomy of the Rabbit. 6th
ed. Philadelphia: P. Blaciston's Son + Co., Inc.
12) Biol, M. C., A. Martin, M. Richard, and P. Louisot. 1987.
Developmental changes in intestinal glycosyl-transferase
activities. Pediatr Res 22 : 250-256 .
13) Black, B. L. and F. Moog. 1977. Goblet cells in embryonic
intestine: accelerated differentiation in culture.
Science 197 : 368-370.
14) Bradbury, J. E., J. W. Black, and J. H. Wyllie. 1980.
Stimulation of mucus output from rat colon in vivo. Eur
J Pharmacol 68:417-25.
15) Bradley, H. C. 1933. Inhibition of pepsin by mucin. J
Biol Chem 100:xx.
16) Bradley, H. C. and M. Hodges. 1934. The effect of mucin
and mucinoids on peptic digestion. J Lab Clin Med 20:165-
169 .
17) Bucher, R. 1932. Das Wesen der Schutzwirkung des
Magenschleims. Deutsche Zeitschr Chir 236:515.
18) Carlisle, M. S., D. D. McGregor, and J. A. Appleton. 1990.
The role of mucus in antibody-mediated rapid expulsion of
Trichinella spiralis in suckling rats. Immunol 70:126-
132 .
19) Carlson, D. M. 1977. Mucous glycoproteins. Mod Probl
Paediat 19:1-10.
20) Carman, R. J. and R. H. Evans. 1984. Experimental and
spontaneous clostridial enteropathies of laboratory and
free living lagamorphs. Lab Anim Sci 34:443-452.
21) Cepinskas, G. , R. D. Specian, and P. R. Kvietys. 1993.
Adaptive cytoprotection in the small intestine: role of
mucus. Am J Physiol 264 (Gastrointest Liver Physiol
27):G921-G927.
22) Chadee, K., K. Keller, J. Forstner, D. J. Innes, and J. I.
Ravdin. 1991. Mucin and nonmucin secretagogue activity
of Entamoeba histolytica and cholera toxin in rat colon.
Gastroenterol 100:986-997.

209
23) Chambraud, L., A. Bernadac, J. P. Gorvel, and S. Maroux.
1989. Renewal of goblet cell mucus granules during the
cell migration along the crypt-villus axis in rabbit
jejunum: an immunolabeling study. Biol Cell 65:151-162.
24) Chen, C. C. , M. Baylor, and D. M. Bass. 1993. Murine
intestinal mucins inhibit rotavirus infection.
Gastroenterology 105:84-92.
25) Chiu, P. J., A. Barnett, M. Siegel, A. D. Brown, and C.
Gerhart. 1988. Protective action of SCH12223 against
experimentally induced gastric and intestinal lesions. J
Pharmacol Exp Ther 246:578-584.
26) Clarke, L. L. and R. A. Argenzio. 1990. NaCl transport
across equine proximal colon and the effect of endogenous
prostanoids. Am J Physiol 259 (Gastrointest Liver Physiol
22):G62-G69.
27) Cohan, V. L., A. L. Scott, C. A. Dinarello, and R. A.
Prendergast. 1991. Interleukin-1 is a mucus
secretagogue. Cell Immunol 136:425-434.
Condor,
G. A., L.
F.
Mayberry,
J. R. Bristol,
G.
A.
Castro,
B. L. Lee,
D.
D. Kratzer
, S. D. Folz, and
D.
L.
Rector.
1987.
Effects of
PGE, or PGE2
and/or
acetazolamide on expulsion of Nippostrongylus
braziliensis from rats. Prostaglandins 34:817-827.
29) Corfield, A. P., A. D. Casey, S. A. Wagner, J. Eldridge,
M. Cox, C. D. Corfield, and J. R. Clamp. 1987. Selection
of radioactive precursors for metabolic labelling of
mucus glycoproteins. Biochem Soc Trans 17:1037-1038.
30) Cramer, W. and A. N. Kingsbury. 1924. Local and general
deficiencies against infections, and the effect on them
of vitamin-deficiency. Brit J Exp Path 5.: 300.
31) Crampton, J. R. 1988. Gastroduodenal mucus and
bicarbonate: The defensive zone. Q J Med 252:269-272.
32) Crowther, R. S., N. W. Roomi, R. E. F. Fahim, and J. F.
Forstner. 1987. Vibrio cholerae metalloproteinase
degrades intestinal mucin and facilitates enterotoxin-
induced secretion from rat intestine. Biochim Biophvs
Acta 924:393-402.
Demes, P., F. F. Pindak, D. J. Wells, and W. A. Gardner
Jr. 1989. Adherence and surface properties of
Tri trichomonas mobilensis, an intestinal parasite of the
squirrel monkey. Parasitol Res 75:589-594.
33)

210
34) Desai, M. A., C. V. Nicholas, and P. Vadgama. 1991.
Electrochemical determination of the permeability of
porcine mucus to model solute compounds. J Pharm
Pharmacol 43 :124-127.
35) Desai, M. A. and P. M. Vadgama. 1993. An in vitro study
of enhanced H+ diffusion by urease action on urea. Scand
J Gastroenterol 28:915-919.
36) Dobson, C. 1967. Changes in the protein content of the
serum and intestinal mucus of sheep with reference to the
histology of the gut and immunological response to
Oesophagostomum columbianum infections. Parasitol 57:201-
219.
37) Drumm, B., A. W. Neumann, Z. Policova, and P. M. Sherman.
1989. Bacterial cell surface hydrophobicity properties
in the mediation of in vitro adhesion by the rabbit
enteric pathogen Escherichia coli strain RDEC-1. J Clin
Invest 84:1588-1594.
38) Duthie, E. S. 1933. Mucus formation in goblet cells.
Proc Roy Soc London Series B 113:459-463.
39) Englard, S. and S. Seifter. 1990. Precipitation
techniques. Methods Enzvmol 182:285-300.
40) Farack, U. M., J. Reiter, M. Gross, L. Moroder, E. Wünsch,
and K. Loeschke. 1987. Influence of vasoactive
intestinal peptide, secretin, and Ala4, Val5-secretin on
the net movements of electrolytes, fluid, and mucus in
the rat colon in vivo. Scand J Gastroenterol Suppl
139:32-36.
41) Feizi, T. 1982. Antigenicities of mucins - their
relevance to tumor associated and stage specific
embryonic antigens. Adv Exp Med Biol 144.: 29-37.
42) Ferrero, R. L. and A. Lee. 1988. Motility of
Campylobacter jejuni in a viscous environment: comparison
with conventional rod-shaped bacteria. J Gen Microbiol
134:53-59.
43) Filipe, M. I., A. Sandey, and J. Ma. 1988. Intestinal
mucin antigens in ulcerative colitis and their
relationship with malignancy. Hum Pathol 19:671-681.
44) Florey, H. 1930. The secretion of mucus by the colon.
Brit J Exp Path 11:348-361.

211
45) Florey, H. 1931. The mechanism of goblet cell secretion
in the mammal; the effects of cyanide. Brit J Exp Path
12:301-305.
46) Florey, H. W. 1933. Observations on the functions of
mucus and the early stages of bacterial invasion of the
intestinal mucosa. J Path Bacteriol 37:283-289.
47) Florey, H. 1955. Mucin and the protection of the body.
Proc Roy Soc London Series B 143 :147-158.
48) Forlong, C., C. Tasman-Jones, L. Thomsen, and J.
Clearwater. 1990. The Na+/H+ exchange and H+ diffusion
properties of human postmortem mucus: A comparison of
gastric antral, gastric body, jejunal, and ileal mucus.
J Clin Gastroenterol 12(Suppl i):S110-S115.
49) Forstner, J. F., N. W. Roomi, R. E. F. Fahim, and G. G.
Forstner. 1981. Cholera toxin stimulates secretion of
immunoreactive intestinal mucin. Am J Physiol 240
(Gastrointest Liver Physiol 3.) :G10-G16.
50) Forstner, G., M. Shih, and B. Lukie. 1973. Cyclic AMP and
intestinal glycoprotein synthesis: The effect of £-
adrenergic agents, theophylline, and dibutyryl cyclic
AMP. Can J Physiol Pharmacol 51:122-129.
51) Forstner, J. F. 1978. Intestinal mucins in health and
disease. Digestion 17:234-263.
52) Foster, H. L. 1980. Gnotobiology. In: The Laboratory Rat
Volume II Research Applications Ed. Baker, H. J., J. R.
Lindsey, and S. H. Weisbroth. New York:Academic Press,
p.43-59.
53) Frase, L. L., A. D. Strickland, G. W. Rachel, and G. J.
Krejs. 1985. Enhanced glucose absorption in the jejunum
of patients with cystic fibrosis. Gastroenterol 88:478-
484 .
54) Frick, L. P. and J. E. Ackert. 1948. Further studies on
duodenal mucus as a factor in age resistance of chickens
to parasitism. J Parasit 34 :192-206.
55) Fuller, C. E., R. P. Davies, G. T. Williams, and E. D.
Williams. 1990. Crypt restricted heterogeneity of goblet
cell mucus glycoprotein in histologically normal human
colonic mucosa: a potential marker of somatic mutation.
Br J Cancer 61:382-384.
56) Galen. 1968. On the Usefulness of the Parts of the Body.
Translated by M. T. May. Ithaca: Cornell Univ Press.

212
57) Gault, M. J. , F. D. Gillin, and A. J. Zenian. 1987.
Giardia lamblia: stimulation of growth by human
intestinal mucus and epithelial cells in serumfree
medium. Exp Parasitol 64:29-37.
58) Gibson, L. E., W. J. Matthews, P. T. Minihan, and J. A.
Patti. 1971. Relating mucus, calcium and sweat in a new
concept of cystic fibrosis. Pediatrics 48:695-710 .
59) Glass, G. B. J. 1953. Gastric mucin and its constituents:
Physico-chemical characteristics, cellular origin, and
physiological significance. Gastroenterol 23:636-658 .
60) Goldstein, I. J. and R. D. Poretz. 1986. Isolation,
physicochemical characterization, and charbohydrate-
binding specificity of lectins. In: The Lectins. Ed.
Leiner, I. E., N. Sharon, and I. J. Goldstein, Orlando:
Academic Press, p. 35-250.
61) Goldsworthy, N. E. and H. Florey. 1930. Some properties
of mucus, with special reference to its antibacterial
functions. Brit J Exp Path 11:192-208.
62) Gong, D. , B. Turner, K. R. Bhaskar, and J. T. Lamont.
1990. Lipid binding to gastric mucin: protective effect
against oxygen radicals. Am J Physiol 259 (Gastrointest
Liver Physiol 22):G681-G686.
63) Gorelick, F. S. and J. D. Jamieson. 1987. Structure-
function relationship of the pancreas. In Physiology of
the Gastrointestinal Tract. 2nd ed Ed. L. R. Johnson, New
York: Raven Press, p. 1089-1108.
64) Greenham, L. W. 1962. Some preliminary observations on
rabbit mucoid enteritis. Vet Rec 74:79-84.
65) Greenwood, B. and M. Mantle. 1992. Mucin and protein
release in the rabbit jejunum: effects of bethanechol and
vagal nerve stimulation. Gastroenterology 103 :496-505.
66) Gruber, P., A. Rubinstein, V. H. K. Li, P. Bass, and J. R.
Robinson. 1987. Gastric emptying of nondigestible solids
in the fasted dog. J Pharmaceut Sci 76:117-122.
67) Gupta, B. L. 1989. The relationship of mucoid substances
and ion and water transport, with new data on intestinal
goblet cells and a model for gastric secretion. Svmp Soc
Exp Biol 43:81-110.
Guth, D. and W. von Engelhardt. 1989. Is gastrointestinal
mucus an ion-selective barrier? Svmp Soc Exp Biol 43 :117-
121.
68)

213
69) Han, V. , J. Resau, R. Predergast, A. Scott, and D. A.
Levy. 1987. Interleukin-1 induces mucus secretion from
mouse intestinal explants. Int Archs Allergy appl Immun
82:364-365.
70) Harkema, J. R. and J. A. Hotchkiss. 1991. In vivo effects
of endotoxin on nasal epithelial mucosubstances:
quantitative histochemistry. Exp Lung Res 17:743-761
71) Harlow, E. and D. Lane. 1988. Antibodies A Laboratory
Manual. Cold Spring Harbor, New York: Cold Spring Harbor
Laboratories.
72) Hartiala, K. and M. I. Grossman. 1951. Studies on
chemical and physical changes in duodenal mucus. J Biol
Chem 195:251-256.
73) Hattori, T. and N. Arizono. 1988. Cell kinetics and
secretion of mucus in the gastrointestinal mucosa, and
their diurnal rhythm. J Clin Gastrenterol 10 (Suppl
1):SI-S6.
74) Heatley, N. G. 1956. Does mucin inhibit the action of
pepsin? Quart J Exp Physiol 41:25-30.
75) Heatley, N. G. 1959. Mucosubstance as a barrier to
diffusion. Gastroenterol 37:313-317.
76) Heidenhain, R. 1878. Ueber die Pepsinbildung in den
Pylorusdrüsen. Archiv für Physiologie (Pflügers) 18:169-
171.
77) Hill, B. D., D. A. Blewett, A. M. Dawson, and S. Wright.
1991. Cryptosporidium parvum: Investigation of
sporozoite excystation in vivo and the association of
merozoites with intestinal mucus. Res Vet Sci 51:264-267.
78) Hogben, C. A. M. , D. J. Tocco, B. R. Brodie, and L. S.
Schanker. 1959. On the mechanism of intestinal
absorption of drugs. J Pharm Exp Ther 125:275-282.
79) Hollander, F. 1954. The two-component mucus barrier.
Archs Intern Med 93:107-129.
80) Hollander, F., B. P. Sonnenblick, and H. A. Sober. 1947.
Experimental impairment of the gastric mucous barrier in
dogs. J Natl Cancer Inst 2:361-364.
81) Hollander, F., J. Stein, and F. U. Lauber. 1946. The
consistency, opacity, and columnar cell content of
gastric mucus secreted under the influence of several
mild irritants. Gastroenterol 6:576-595.

214
82) Hoskins, L. C., M. Agustines, W. B. McKee, E. T. Boulding,
M. Kriaris, and G. Niedermeyer. 1985. Mucin degradation
in human colon ecosystems. J Clin Invest 75:944-953.
83) Huet, C., C. Sahuquillo-Merino, E. Coudrier, and D.
Louvard. 1987. Absorptive and mucus-secreting subclones
isolated from a multipotent intestinal cell line (HT-29)
provide new models for cell polarity and terminal
differentiation. J Cell Biol 105:345-357.
84) Ichikawa, M., A. Ichikawa, and S. Kidokoro. 1987.
Secretory process of mucus-secreting cells in mouse
colonic mucosa studied by rapid freezing and freeze-
substitution. J Electron Microsc Tokyo 36:117-127.
85) Ichikawa, T., K. Ishihara, Y. Komuro, Y. Kojima, K.
Saigenji, and K. Hotta. 1994. Effects of the new
histamine H2 receptor antagonist, FRG-8813, on gastric
mucin in rats with or without acidified ethanol-induced
gastric damage. Life Sci 54:PL159-PL164.
86) Ichikawa, T. , K. Ishihara, K. Saigenji, and K. Hotta.
1993. Stimulation of mucus glycoprotein biosynthesis in
rat gastric mucosa by gastrin. Biochem Pharmacol 46:1551-
1557.
87) Ichikawa, T., K. Ishihara, K. Saigenji, and K. Hotta.
1994. Effects of acid-inhibitory antiulcer drugs on
mucin biosynthesis in the rat stomach. Eur J Pharmacol
251:107-111.
88) Ishikawa, N. , Y. Horii, T. Oinuma, T. Suganuma, and Y.
Nawa. 1994. Goblet cell mucins as the selective barrier
for the intestinal helminths: T-cell-independent
alteration of goblet cell mucins by immunologically
'damaged' Nippostrongylus brasiliensis worms and its
significance on the challenge infection with homologous
and heterologous parasites. Immunology 81:480-486.
89) Itagaki, S., C. J. Perfumo, M. A. Petruccelli, and K. Doi.
1994. Lectin histochemical changes of colon goblet cell
mucin in rabbit mucoid enteropathy. Lab Anim Sci 44:82-
84 .
90) Ito, S. 1987. Functional gastric morphology. In
Physiology of the Gastrointestinal Tract. 2nd ed Ed. L.
R. Johnson, New York: Raven Press, p. 817-851.
91) Janowitz, H. D. and F. Hollander. 1951. Some properties
of cell-free gastric mucus. Fed Proc 10:70.

215
92) Janowitz, H. D., F. Hollander, and C. Jackson. 1951.
Stimulation of cell-free gastric mucus by the topical
application of acetylcholine. Proc Soc Exp Biol Med
76:578-580.
93) Jarry, A., D. Merlin, A. Velcich, U. Hopfer, L. H.
Augenlicht, and C. L. Laboisse. 1994. Interferon-y
modulates cAMP-induced mucin exocytosis without
affecting mucin gene expression in a human colonic goblet
cell line. Eur J Pharmacol 267:95-103.
94) Jass, J. R., K. Sugihara, and S. B. Love. 1988. Basis of
sialic acid heterogeneity in ulcerative colitis. J Clin
Pathol 41:388-392.
95) Jeynes, B. J. and G. G. Altman. 1978. Light and scanning
electron microscopic observations of the effects of
sublethal doses of methotrexate on the rat small
intestine. Anat Rec 191:1-18.
96) Jubb, K. V. F., P. C. Kennedy, and N. Palmer. 1985.
Pathology of Domestic Animals 3rd ed. Vol 2. New York:
Academic Press.
97) Karlsson, J. , A. Wikman, and P. Artursson. 1993. The
mucus layer as a barrier to drug absorption in monolayers
of human intestinal epithelial HT29-H goblet cells. Int
J Pharm 99:209-218.
98) Kaufman, J. 1908. Lack of gastric mucus (amyxorrhea
gástrica) and its relation to hyperacidity and gastric
ulcer. Am J Med Sci 135:207-214.
99) Kaura, R., A. Allen, and B. H. Hirst. 1980. Mucus in the
gastric juice of cats during pentagastrin and secretin
infusions. The viscosity in relation to glycoprotein
structure and concentration. Biochem Soc Trans 8.: 52-53.
100) Kemper, A. C. and R. D. Specian. 1991. Rat small
intestinal mucins: a quantitative analysis. Anat Rec
229:219-226.
101) Kennedy, M. J. , D. K. Rosnick, R. G. Ulrick, and R. J.
Yancey Jr. 1988. Association of Treponema hyodysenteriae
with porcine intestinal mucosa. J Gen Microbiol 134:1565-
1576 .
102) Kennedy, M. J. , P. A. Volz, C. A. Edwards, and R. J.
Yancey. 1987. Mechanisms of association of Candida
albicans with intestinal mucosa. J Med Microbiol 24:333-
341.

216
103) Kimambo, A. E. and J. C. MacRae. 1988. Measurement in
vitro of a larval migration inhibitory factor in
gastrointestinal mucus of sheep made resistant to the
roundworm Trichostrongylus colubriformis. Vet Parasitol
2^:213-222.
104) Koga, K. , Y. Nonaka, T. Kawai, T. Seki, H. Kon, M.
Tsurui, H. Ikeda, Y. Harada, T Saito, and S. Ashizawa.
1989. Changes in levels of mucosal glycoprotein on
cysteamine-induced duodenal ulcers in rats. Scand J
Gastroenterol Suppl 162 :120-123.
105) Kraus, A. L., S. H. Weisbroth, R. E. Flatt, and N.
Brewer. 1984. Biology and diseases of rabbits. In:
Laboratory Animal Medicine. Ed. Fox J. G., B. J. Cohen,
& F. M. Loew, New York: Academic Press, p. 207-240.
106) Kudweis, M., Z. Lojda, J. Vitovec, and B. Koudela. 1989.
Mucus synthesis in the goblet cells of the small
intestine in experimental infection with the coccidium
Isospora suis in piglets. Vet Med Praha 43:727-734.
107) Kudweis, M., Z. Lojda, J. Vitovec, and B. Koudela. 1990.
Activity of mucosubstances and the goblet cell count in
the large intestine of piglets infected with the
coccidium, Isospora suis. Vet Med Praha 35:21-29.
108) Laburthe, M., C. Augeron, C. Rouyer-Fessard, I.
Roumagnac, J.-J. Maoret, E. Grasset, and C. Laboisse.
1989. Functional VIP receptors in the human mucus-
secreting colonic epithelial cell line CL.16E. Am J
Physiol 256 (Gastrointest Liver Physiol 19):G443-G450.
109) Lake, A. M., K. J. Bloch, M. R. Neutra, and W. A. Walker.
1979. Intestinal goblet cell mucus release. II. In vivo
stimulation by antigen in the immunized rat. J Immunol
122:834-837.
110) LaMont, J. T. and A. Ventola. 1977. Stimulation of
colonic glycoprotein synthesis by dibutyryl cyclic AMP
and theophylline. Gastroenterol 72:82-86.
111) Lee, D. L. and W. D. Biggs. 1990. Two- and three-
dimensional locomotion of the nematode Nippostrongylus
brasiliensis. Parasitol 101:301-308.
112) Leitch, G. J. 1988. Intestinal lumen and mucosal
microclimate H+ and NH3 concentrations as factors in the
etiology of experimental amebiasis. Am J Trop Med Hyq
38:480-486.

217
113) Lelkes, L. 1986. Overeating and microbial imbalance in
the development of mucoid enteropathy in rabbits. J Appl
Rabbit Res 9:148-151.
114) Lelkes, L. and C.-L. Chang. 1987. Microbial dysbiosis in
rabbit mucoid enteropathy. Lab Anim Sci 37:757-764.
115) Lencer, W. I., F. D. Reinhart, and M. R. Neutra. 1990.
Interaction of cholera toxin with cloned human goblet
cells in monolayer culture. Am J Physiol 258
(Gastrointest Liver Physiol 21):G96-G102.
116) Lesuffleur, T., N. Porchet, J.-P. Aubert, D. Swallow, J.
R. Gum, Y. S. Kim, F. X. Real, and A. Zweibaum. 1993.
Differential expression ofthe human mucin genes MUC1 to
MUC5 in relation to growth and differentiation of
different mucus-secreting HT-29 cell subpopulations. J
Cell Sci 106:771-783.
117) Lewin, M. R. , S. H. El-Masri, and C. G. Clark. 1979.
Effects of bile acids on mucus secretion in the dog
colon. Eur Sura Res 11:392-399.
118) Lima, A. A. M., D. J. Innes, K. Chadee, D. M. Lyerly, T.
D. Wilkins, and R. L. Guerrant. 1989. Clostridium
difficile toxin A. Interactions with mucus and early
sequential histopathologic effects in rabbit small
intestine. Lab Invest 61:419-425.
119) Lindahl, M. and I. Carlstedt. 1990. Binding of K99
fimbriae of enterotoxigenic Escherichia coli to pig small
intestinal mucin glycopeptides. J Gen Microbiol 136:1609-
1614 .
120) Lindstedt, G., S. Lindstedt, and B. E. Gustafsson. 1965.
Mucus in the intestinal contents of germfree rats. J Exp
Med 121:201-213.
121)Loeschke, K., T. Schmid, and U. M. Farack. 1989.
Inhibition by loperamide of mucus secretion in the rat
colon in vivo. Eur J Pharmacol 170:41-46.
122) Lucas, M. 1982. Restriction of hydrogen and sodium ion
diffusion in porcine gastric mucin: a concentration
dependent phenomenon. Adv Exp Med Biol 144:193-195.
123) Lucas, M. 1983. Determination of acid surface pH in vivo
in rat proximal jejunum. Gut 24:734-739.
124) Mack, D. R., W. Neumann, Z. Policova, and P. M. Sherman.
1992. Surface hydrophobicity of the intestinal tract. Am
J Physiol 262 (Gastrointest Liver Physiol 25.) :G171-177.

218
125) Mack, D. R. and P. M. Sherman. 1991. Mucin isolated from
rabbit colon inhibits in vitro binding of Escherichia
coli RDEC-1. Infect Immun 59:1015-1023.
126) Mantle, M. and A. Allen. 1989. Gastrointestinal mucus.
In: Gastrointestinal Secretion. Ed J. S. Davison. London:
Wright, p. 202-229.
127) Mantle, M. and A. Allen. 1978. A colorimetric assay for
glycoproteins based on the periodic acid/Schiff stain.
Biochem Soc Trans £:607-609.
128) Mantle, M. and A. Allen. 1981. Isolation and
characterization of the native glycoprotein from pig
small-intestinal mucus. Biochem J 195:267-275 .
129) Mantle, M., E. Atkins, J. Kelly, E. Thakore, A Buret, and
D. G. Gall. 1991. Effects of Yersinia enterocolitica
infection on rabbit intestinal and colonic goblet cells
and mucin: morphometries, histochemistry, and
biochemistry. Gut 32 :1131-1138.
130) Mantle, M., L. Basaraba, S. C. Peacock, and D. G. Gall.
1989. Binding of Yersinia enterocolitica to rabbit
intestinal brush border membranes, mucus, and mucin.
Infect Immun 57 : 3292-3299.
131) Mantle, M. , G. G. Forstner, and J. F. Forstner. 1984.
Antigenic and structural features of goblet-cell mucin of
human small intestine. Biochem J 217:159-167.
132) Mantle, M. and C. Rombough. 1993. Growth in and
breakdown of purified rabbit small intestinal mucin by
Yersinia enterocolitica. Infect Immun 61:4131-4138.
133) Mantle, M. and E. Thakore. 1988. Rabbit intestinal and
colonic mucins: isolation, partial characterization, and
measurement of secretion using an enzyme-linked
immunoassay. Biochem Cell Biol 66:1045-1054.
134) Mantle, M., E. Thakore, R. Mathison, and J. S. Davison.
1991. Intestinal mucin secretion in streptozotocin-
diabetic rats: lack of response to cholinergic
stimulation and cholera toxin. Dig Pis Sci 36 :1574-1581.
135) Masci, E., P. A. Testoni, L. Fanti, M. Guslandi, M. Zuin,
and A. Tittobello. 1987. Duodenogastric reflux:
correlations among bile acid pattern, mucus secretion,
and mucosal damage. Scand J Gastroenterol 22 :308-312.
136) Mascólo, N. , V. M. Rajendran, and H. J. Binder. 1991.
Mechanism of short-chain fatty acid uptake by apical

219
membrane vesicles of rat distal colon. Gastroenterol
101:331-338.
137) McCormick, B. A., B. A. Stocker, D. C. Laux, and P. S.
Cohen. 1988. Roles of motility, chemotaxis, and
penetration through and growth in intestinal mucus in the
ability of an avirulent strain of Salmonella typhimirium
to colonize the large intestine of streptomycin-treated
mice. Infect Immun 56 : 2209-2217.
138) McIntosh, I. and G. R. Cutting. 1992. Cystic fibrosis
transmembrane conductance regulator and the etiology and
pathogenesis of cystic fibrosis. FASEB J .6:2775-2782.
139) McKay, D. M., D. W. Halton, M. D. McCaigne, C. F.
Johnston, I. Fairweather, and C. Shaw. 1990. Hymenolepis
diminuta: intestinal goblet cell response to infection in
male mice. Exp Parasitol 71:9-20.
140) McSweegan, E., D. H. Burr, and R. I. Walker. 1987.
Intestinal mucus gel and secretory antibody are barriers
to Campylobacter jejuni adherence to INT 407 cells.
Infect Immun 55 :1431-1435.
141) Menguy, R. and A. E. Thompson. 1967. Regulation of
secretion of mucus from the gastric antrum. Ann NY Acad
Sci 140:797-803.
142) Meshorer, A. 1976. Histological findings in rabbits
which died with symptoms of mucoid enteritis. Laboratory
Animals 10:199-202.
143) Metcalfe, J. W. , K. A. Krogfelt, H. C. Krivan, P. S.
Cohen, and D. C. Laux. 1991. Characterization and
identification of a porcine small intestine mucus
receptor for the K88ab fimbrial adhesin. Infect Immun
59:91-96.
144) Miller, C. O. and J. M. Dunbar. 1933. Change in
viscosity of mucin with pH. Proc Soc Exp Biol Med
30 : 627-629.
145) Miller, H. R. P. 1987. Gastrointestinal mucus, a medium
for survival and for elimination of parasitic nematodes
and protozoa. Parasitol 94 Suppl:S77-S100.
146) Miller, H. R. P. and J. F. Huntley. 1982. Protection
against nematodes by intestinal mucus. Adv Exp Med Biol
144 :243-245.

220
147) Miller, H. R. P. and Y. Nawa. 1979. Nippos tr ongylus
brasiliensis: intestinal goblet-cell response in
adoptively immunized rats. Exp Parasitol 47:81-90.
148) Miller, T. A. 1988. Gastroduodenal mucosal defense:
Factors responsible for the ability of the stomach and
duodenum to resist injury. Surgery 103:389-397.
149) Moore, B. A., K. A. Sharkey, and M. Mantle. 1993. Neural
mediation of cholera toxin-induced mucin secretion in the
rat small intestine. Am J Physiol 265 (Gastrointest Liver
Phvsiol 28):G1050-G1056.
150) Moqbel, R., D. Wakelin, A. J. MacDonald, S. J. King, R.
K. Grencis, and A. B. Kay. 1987. Release of leukotrienes
during rapid expulsion of Trichinella spiralis from
immune rats. Immunol ¿0:425-430.
151) Moré, J., F. Bénazet, J. Fioramonti, and M.-T. Dray-
Lefaix. 1987. Effects of treatment with smectite on
gastric acid and intestinal glycoproteins in the rat: a
histochemical study. Histochem J 19:665-669.
152) Moré, J., L. Fioramonti, F. Bénazet, and L. Buéno. 1987.
Histochemical characterization of glycoproteins present
in jejunal and colonic goblet cell of pigs on different
diets. Histochemistry 87:189-194.
153) Morton, G. M. and G. W. Stavraky. 1949. A histo-
physiological study of the effect of intraarterial
injection of acetylcholine upon the gastric mucosa of the
dog. Gastroenterol 12:808-820.
154) Mottram, J. C. 1923. Some effects of exposure to radium
upon the alimentary canal. Proc R Soc Med 16:41.
155) Mouricout, M. A. and R. A. Julien. 1987. Pilus-mediated
binding of bovine enterotoxigenic Escherichia coli to
calf small intestinal mucins. Infect Immun 55:1216-1223.
156) Murty, V. L. N., J. Piotrowski, A. Czajkowski, A.
Slomiany, and B. L. Slomiany. 1993. Inhibition of
Helicobacter pylori glycosulf atase activity towards human
gastric sulfomucin by a gastroprotective agent,
sulglycotide. Gen Pharmacol 24:1463-1466.
157) Nadziejko, C. E., B. L. Slomiany, and A. Slomiany. 1993.
Most of the lipid in purulent sputum is bound to mucus
glycoprotein. Exp Lung Res 19:671-684.
158) Nakamura, A., T. Nakahari, T. Senda, and Y. Imai. 1993.
Exocytosis in the lingual mucus cells of Rana esculenta

221
evoked by acetylcholine: observation of living cells by
confocal laser scanning microscopy. Jap J Physiol 43:833-
846 .
159) Negus, V. 1967. The function of mucus: A hypothesis.
Proc Rov Soc Med 60:75-77.
160) Neutra, M. R. and J. F. Forstner. 1987. Gastrointestinal
mucus--synthesis, secretion, and function. In Physiology
of the Gastrointestinal Tract, 2^ ed Ed. L. R. Johnson,
New York: Raven Press, p.975-1009.
161) Neutra, M. R., L. J. O'Malley, and R. D. Specian. 1982.
Regulation of intestinal goblet cell secretion. II. A
survey of potential secretagogues. Am J Physiol 242
(Gastrointest Liver Physiol 5):G380-G387.
162) Nevóla, J. J., D. C. Laux, and P. S. Cohen. 1987. In
vivo colonization of the mouse large intestine and in
vitro penetration of intestinal mucus by an avirulent
smooth strain of Salmonella typhimirium and its
lipopolysaccharide-deficient mutant. Infect Immun
55:2884-2890.
163) Nikkels, R. J. , J. W. M. A. Mull ink, and J. C. J. Van
Vliet. 1976. An outbreak of rabbit enteritis:
Pathological and microbiological findings and possible
therapeutic regime. Lab Animals 10:195-198.
164) Njoku, O. 0. and G. J. Leitch. 1983. Separation of
cholera enterotoxin-induced mucus secretion from
electrolyte secretion in rabbit ileum by acetazolamide,
colchicine, cycloheximide, cytochalasin B and
indomethacin. Digestion 27:174-184.
165) Nunn, S., R. St. C. Gilmore, J. A. Dodge, and K. E. Carr.
1990. Exudate variation in the rabbit gastrointestinal
tract: a scanning electron microscope study. J Anat
170:87-98.
166) Oderda, G., M. D'Alessandro, P. Mariani, P. Lionetti, M.
Bonamico, D. Dell'Olio, and N. Ansaldi. 1993.
Prostaglandin E2 in gastric mucosa of children with
Helicobacter pylori gastritis: Relation to thickness of
mucus gel layer. J Clin Pathol 46:836-839.
167) Oliver, M. G. and R. D. Specian. 1990. Cytoskeleton of
intestinal goblet cells: role of actin filaments in
baseline secretion. Am J Physiol 259 (Gastrointest Liver
Phvsiol 22):G991-G997.

222
168) Ollar, R. A. and D. E. S. Stewart-Tull. 1987. Studies on
the Vibrio cholerae mucinase complex. II. Specific
neuraminidase activity measured histochemically in a
goblet cell assay. J Med Microbiol 23:227-232.
169) Park, C. M., P. E. Reid, D. A. Owen, J. M. Sanker, and D.
A. Applegarth. 1987. Morphological and histochemical
changes in intestinal mucosa in the reserpine-treated rat
model of cystic fibrosis. Exp Mol Pathol 47:1-12.
170) Paerregaard, A., F. Espersen, O. M. Jenson, and M.
Skurnik. 1991. Interactions between Yersinia
enterocolitica and rabbit ileal mucus: growth, adhesion,
penetration, and subsequent changes in surface
hydrophobicity and ability to adhere to ileal brush
border membrane vesicles. Infect Immun 59:253-260.
171) Peatfield, A. C., P. J. Piper, and P. S. Richardson.
1982. The effect of leukotriene C4 on mucin release into
the cat trachea in vivo and in vitro. Br J Pharmacol
77:391-393.
172) Pfeiffer, C. J. 1981. Experimental analysis of hydrogen
ion diffusion in gastrointestinal mucus glycoprotein. Ml
J Physiol 240 (Gastrointest Liver Physiol 3.) :G176-G182.
173) Phillips, T. E., C. Huet, P. R. Bilbo, D. K. Podolsky, D.
Louvard, and M. R. Neutra. 1988. Human intestinal goblet
cells in monolayer culture: characterization of a mucus
secreting subclone derived from the HT29 colon
adenocarcinoma cell line. Gastroenterol 94:1390-1403.
174) Phillips, T. E., T. H. Phillips, and M. R. Neutra. 1984.
Regulation of intestinal goblet cell secretion. III.
Isolated intestinal epithelium. Am J Physiol 247:G674-
G681.
175) Phillips, T. E., T. H. Phillips, and M. R. Neutra. 1984.
Regulation of intestinal goblet cell secretion. IV.
Electrical field stimulation in vitro. M J Physiol
24_7 : G682 - G687 .
176) Phillips, T. E., T. H. Phillips, and M. R. Neutra. 1989.
Cholinergic responsiveness of goblet cells during
intestinal maturation. Biol Neonate 55:197-203.
177) Phillips, T. E., W. F. Stenson, and M. R. Neutra. 1989.
Lipoxygenase metabolites of arachidonic acid do not
induce mucus secretion from rabbit intestinal goblet
cells in vitro. Prost Leuk Essent Fatty Acids 37:51-55.

223
178) Phillips, T. E. and J. Wilson. 1993. Morphometric
analysis of muous granule depletion and replenishment in
rat colon. Dig Pis Sci 38 :2299-2304 .
179) Pinczower, G. D., R. D. Gianello, R. P. W. Williams, B.
N. Preston, H. Preston, and A. W. Linnane. 1993.
Monoclonal antibody 4D3 detects small intestinal mucin
antigen (SIMA)-glycoprotein in the serum of patients with
colorectal cancer. Int J Cancer 54:391-396.
180) Piotrowski, J. , A. Czajkowski, F. Yotsumoto, A. Slomiany,
and B. L. Slomiany. 1994. Sulglycotide effect on the
proteolytic and lpolytic activities of Helicobacter
pylori toward gastric mucus. Am J Gastroenterol 89:232-
236 .
181) Quarterman, J. 1987. Metal absorption and the intestinal
mucus layer. Digestion 37:1-7.
182) Quigley, E. M. M. and L. A. Turnberg. 1987. pH of the
microclimate lining human gastric and duodenal mucosa in
vivo. Gastroenterol 92 :1876-1884 .
183) Rabbani, G. H. and W. B. Greenough. 1990. Cholera. In
Textbook of Secretory Diarrhea Ed. E. Lebenthal and M.
Duffey. New York:Raven Press, p. 233-254.
184) Radwan, K. A., M. G. Oliver, and R. D. Specian. 1990.
Cytoarchitectural reorganization of rabbit colonic goblet
cells during baseline secretion. Am J Anat 189:365-376.
185) Rechkemmer, G. , M. Wahl, W. Kuschinsky, and W. von
Engelhardt. 1986. pH-Microclimate at the luminal surface
of the intestinal mucosa of guinea pig and rat. Pflügers
Arch 407:33-40.
186) Reynolds, I. J. , R. J. Gould, and S. H. Snyder. 1984.
Loperamide: blockade of calcium channels as a mechanism
for antidiarrheal effects. J Pharmacol Exp Ther 231:628-
632 .
187) Rikihisa, Y., G. C. Johnson, Y.-Z. Wang, S. M. Reed, R.
Fertel, and H. J. Cooke. 1992. Loss of absorptive
capacity for sodium and chloride in the colon causes
diarrhoea in Potomac horse fever. Res Vet Sci 52:353-362.
188)Roberton, M., M. Mantle, R. E. F. Fahim, R. D. Specian,
A. Bennick, S. Kawagishi, P. Sherman, and J. F. Forstner.
1989. The putative 'link' glycopeptide associated with
mucus glycoproteins. Biochem J 261:637-647.

224
189) Roomi, N., M. Laburthe, N. Fleming, R. Crowther, and J.
Forstner. 1984. Cholera-induced mucin secretion from rat
intestine: lack of effect of cAMP, cycloheximide, VIP,
and colchicine. Am J Physiol 247 .-G140-G148 .
190) Ross, I. N., H. M. M. Bahari, and L. A. Turnberg. 1982.
Studies of the pH gradient across the mucus on rat
gastric mucosa in vivo and across mucus on human gastric
mucosa in vitro. Adv Exp Med Biol 144:189-191.
191) Roth, J., P. Greenwell, and W. M. Watkins. 1988.
Immunolocalization of blood group A gene specified al,3N-
acetylgalactosaminyl-transferase and blood group A
substance in the trans-tubular network of the Golgi
apparatus and mucus of intestinal goblet cells. Eur J
Cell Biol 46:105-112.
192) Ruseler van Embden, J. G., R. van der Helm, and L. M. van
Lieshout. 1989. Degradation of intestinal glycoproteins
by Bacteroides vulgatus. FEMS Microbiol Lett 49:37-41.
193) Said, H. M., R. Smith, and R. Redha. 1987. Studies on
the intestinal surface acid microclimate: developmental
aspects. Pediatr Res 22:497-499.
194) Sakata, T. and W. von Engelhardt. 1981. Luminal mucin in
the large intestine of mice, rats, and guinea pigs. Cell
Tissue Res 219:629-635.
195) Sanchez, R. , L. Kanarek, J. Koninkx, H. Hendriks, P.
Lintermans, A. Bertels, G. Charlier, and E. Van
Driessche. 1993. Inhibition of adhesion of
enterotoxigenix Escherichia coli cells expressing F17
fimbriae to small intestinal mucus and brush-border
membranes of young calves. Microbial Path 15:407-419.
196) Sarosiek, J., B. J. Marshall, D. A. Peura, S. Hoffman, T.
Feng, and R. W. McCallum. 1991. Gastroduodenal mucus gel
thickness in patients with Helicobacter pylori: a method
for assessment of biopsy specimens. Am J Gastroenterol
86=729-734.
197) Satchithanandam, S., M. M. Cassidy, A. T. Kharroubi, R.
J. Calvert, A. R. Leeds, and G. V. Vahouny. 1990.
Alterations in rat intestinal mucin patterns following
luminal infusion of acetylsalicylic acid and
prostaglandin derivatives. Digest Pis Sci 35 :1518-1527.
198) Scheiman, J. M. , E. R. Kraus, and C. R. Boland. 1992.
Regulation of canine gastric mucin synthesis and
phospholipid secretion by acid secretagogues.
Gastroenterology 103:1842-1850.

225
199) Schlichter, L. C. 1982. Unstirred mucus layers: ion
exchange properties and effect on ion regulation in
Lymnaea stagnalis. J Exp Biol 98:363-372.
200) Schrager, J. and M. D. G. Oates. 1974. The isolation and
partial characterization of a glycoprotein isolated from
human gastric aspirates and from extracts of gastric
mucosae. Biochim Biophvs Acta 372:183-195.
201) Shea-Donohue, T., E. Danquechin-Dorval, E. Montcalm, H.
El-Bayer, A. Durakovic, J. J. Conklin, and A. Dubois.
1985. Alterations in gastric mucus secretion in rhesus
monkeys after exposure to ionizing radiation.
Gastroenterol 88 : 685-690.
202) Sherman, P., N. Fleming, J. Forstner, N. Roomi, and G.
Forstner. 1987. Bacteria and the mucus blanket in
experimental small bowel bacterial overgrowth. Am J
Pathol 126:527-534.
203) Shimada, T. 1987. Factors affecting the microclimate pH
in rat jejunum. J Physiol 392:113-127.
204) Shimura, S., T. Sasaki, K. Ikeda, H. Sasaki, and T.
Takishima. 1988. VIP augments cholinergic-induced
glycoconjugate secretion in tracheal submucosal glands.
J AppI Physiol £5:2537-2544.
205) Shorrock, C. J. and D. W. Rees. 1988. Overview of
gastroduodenal mucosal protection. Am J Med 84(Suppl
2A):25-34.
206) Shorrock, C. J. and D. W. Rees. 1989. Mechanisms of
gastric damage by non-steroidal antiinflammatory drugs.
Scand J Rheumatol Suppl 78:5-11.
207) Sinkovics, G. 1976. Intestinal flora studies in rabbit
mucoid enteritis. Vet Rec 98:151-152.
208) Sinkovics, G. 1978. Rabbit dysentery: 3 Diagnostic
differentiation. Vet Rec 103:331-332.
209) Skov Olson, P. 1988. Role of epidermal growth factor in
gastroduodenal mucosal protection. J Clin Gastroenterol
10 (Suppl 1):S146-S151.
210) Slomiany, A., K. Okazaki, S. Tamura, and B. L. Slomiany.
1991. Identity of mucin's "118-kDa link protein" with
fibronectin fragment. Arch Biochem Biophvs 286:383-388.

226
211) Slomiany, B. L., J. Piotrowski, A. Czajkowski, V. L. N.
Murty, and A. Slomiany. 1993. Characterization of gastric
mucosal mucin receptor. Biochem Mol Biol Int 31:745-753.
212) Smith, G. W., P. M. Wiggins, S. P. Lee, and C. Tasman-
Jones. 1986. Diffusion of butyrate through pig colonic
mucus in vitro. Clin Sci 90:271-276.
213) Smith, H. 1953. Factors involved in the virulence¬
enhancing action of mucin. Proc Roy Soc Med 46:787-790.
214) Smith, H., H. T. Zwartow, R. C. Gallop, and P. W. Harris-
Smith. 1953. The virulence-enhancing factor of mucins.
7. The remaining components of the "third factor"
involved in virulence enhancement. Biochem J 53:673-678.
215) Smithson, K. W., D. B. Millar, L. R. Jacobs, and G. M.
Gray. 1981. Intestinal diffusion barrier: Unstirred
water layer of membrane surface mucous coat? Science
214:1241-1244.
216) Snyder, J. D. and W. A. Walker. 1987. Structure and
function of intestinal mucin: developmental aspects. Int
Arch Allergy Appl Immunol 82:351-356.
217) Specian, R. D. and M. R. Neutra. 1980. Mechanism of
rapid mucus secretion in goblet cells stimulated by
acetylcholine. J Cell Biol 85:626-640.
218) Specian, R. D. and M. R. Neutra. 1982. Regulation of
intestinal goblet cell secretion. I. Role of
parasympathetic stimulation. Am J Physiol 242
(Gastrointest Liver Physiol 5.) :G370-G379.
219) Specian, R. D. and M. R. Neutra. 1984. Cytoskeleton of
intestinal goblet cells in rabbit and monkey. The theca.
Gastroenterol 87:1313-1325.
220) Specian, R. D. and M. G. Oliver. 1991. Functional
biology of intestinal goblet cells. Ml J Physiol 260
(Cell Phvsiol 29): C183-C193.
221) Sperber, K. , S. Ogata, C. Sylvester, J. Aisenberg, A.
Chen, L Mayer, and S. Itzkowitz. 1993. A novel human
macrophage-derived intestinal mucin secretagogue:
implications for the pathogenesis of inflammatory bowel
disease. Gastroenterology 104:1302-1309.
222) Spira, W. M., R. B. Sack, and J. L. Froehlich. 1981.
Simple adult rabbit model for Vibrio cholerae and
enterotoxigenic Escherichia coli diarrhea. Infect Immun
32:739-747.

227
223) Strocchi, A. and M. D. Levitt. 1991. A reappraisal of
the magnitude and implications of the intestinal
unstirred layer. Gastroenterol 101:843-847.
224) Strómqvist, M. and H. Gruffman. 1992. Periodic
acid/Schiff staining of glycoproteins immobilized on a
blotting matrix. Biotechniaues 13:744-749.
225) Szentkuti, L., H. Riedesel, M.-L. Enss, K. Gaertner, and
W. von Engelhardt. 1990. Pre-epithelial mucus layer in
the colon of conventional and germ-free rats. Histochem
J 22 : 491-497.
226) Taatjes, D. J. and J. Roth. 1988. Alteration in
sialyltransferase and sialic acid expression accompanies
cell differentiation in rat intestine. Eur J Cell Biol
4_6 : 289-298 .
227) Taatjes, D. J. and J. Roth. 1990. Selective loss of
sialic acid from rat small intestinal epithelial cells
during postnatal development: demonstration with lectin-
gold techniques. Eur J Cell Biol 53:255-266.
228) Taatjes, D. J., J. Roth, J. Weinstein, and J. C. Paulson.
1988. Post-golgi apparatus localization and regional
expression of rat intestinal sialyltransferase detected
by immunoelectron microscopy with polypeptide epitope-
purified antibody. J Biol Chem 263:6302-6309.
229) Targowski, S. and F. Toofanian. 1982. Induction of mucoid
enteritis in rabbits by ligation of the cecum or colon.
J Ml Vet Med Assoc 181:1378-1380.
230) Terakawa, S. and Y. Suzuki. 1991. Exocytosis in colonic
goblet cells visualized by video-enhanced light
microscopy. Biochem Biophvs Res Commun 176:466-472.
231) Thiru, S., G. Devereux, and A. King. 1990. Abnormal
fucosylation of ileal mucus in cystic fibrosis: I A
histochemical study using peroxidase labelled lectins. J
Clin Pathol 43 :1014-1018.
232) Timmons, P. M. , C.-T. J. Chan, P. W. J. Rigby, and F.
Poirier. 1993. The gene encoding the calcium binding
protein calcyclin is expressed at sites of exocytosis in
the mouse. J Cell Sci 104:187-196.
233) Toofanian, F. 1985. Intestinal disaccharidase and
alkaline phosphatase activities in experimental rabbit
mucoid enteropathy. Lab Animal Sci 35:624-626.

228
234) Toofanian, F. and D. W. Hamar. 1986. Cecal short-chain
fatty acids in experimental rabbit mucoid enteropathy.
Am J Vet Res 47:2423-2425.
235) Toofanian, F. and S. Targowski. 1983. Experimental
production of rabbit mucoid enteritis. Am J Vet Res
44:705-708.
236) Toofanian, F. and S. Targowski. 1986. Stimulation of
colonic goblet cells by cecal filtrates from rabbits with
experimental mucoid enteropathy. Lab Anim Sci 36:157-
160.
237) Tzouvelekis, L. S., A. F. Mentis, A. M. Makris, C.
Spiliadis, C. Blackwell, and D. M. Weir. 1991. In vitro
binding of Helicobacter pylori to human gastric mucin.
Infect Immun 59:4252-4254.
238) van Kruiningen, H. J. and C. B. Williams. 1972. Mucoid
enteritis of rabbits. Vet Path 9:53-77.
239) Vasconcellos, C. A., P. G. Allen, M. E. Wohl, J. M.
Drazen, P. A. Janmey, and T. P. Stossel. 1994. Reduction
in viscosity of cystic fibrosis sputum in vitro by
gelsolin. Science 263 : 969-971.
240) Vecchi, M., S. Sakamaki, B. Diamond, A. B. Novikoff, P.
M. Novikoff, and K. M. Das. 1987. Development of a
monoclonal antibody specifically reactive to
gastrointestinal goblet cells. Proc Natl Acad Sci USA
84:3425-3429.
241) Vecchi, M., G. Torgano, M. Monti, E. Berti, D. Agape, M
Primignani, G. Ronchi, and R. de Franchis. 1987.
Evaluation of structural and secretory glycoconjugates in
normal human jejunum by means of lectin histochemistry.
Histochem 86 : 359-364.
242) Verdugo, P. 1990. Goblet cells secretion and
mucogenesis. Ann Rev Physiol 52:157-176.
243) Wadolkowski, E. A., D. C. Laux, and P. S. Cohen. 1988.
Colonization of the streptomycin-treated mouse large
intestine by a human fecal Escherichia coli strain: role
of adhesion to mucosal receptors. Infect Immun 56:1036-
1043 .
244) Wadolkowski, E. A., D. C. Laux, and P. S. Cohen. 1988.
Colonization of the streptomycin-treated mouse large
intestine by a human fecal Escherichia coli strain: role
of growth in mucus. Infect Immun 56:1030-1035.

229
245) Walker, R. I. and R. L. Owen. 1990. Intestinal barriers
to bacteria and their toxins. Ann Rev Med 41:393-400.
246) Walker, W. A., M. Wu, and K. J. Bloch. 1977. Stimulation
by immune complexes of mucus release from goblet cells of
the rat small intestine. Science 197:370-371.
247) Wang, Y.-Z., H. J. Cooke, H.-C. Su, and R. Fertel. 1990.
Histamine augments colonic secretion in guinea pig distal
colon. Am J Physiol 258 (Gastrointest Liver Physiol
21):G432-G439.
248) Wanke, C. A., S. Cronan, C. Goss, K. Chadee, and R. L.
Guerrant. 1990. Characterization of binding of
Escherichia coli strains which are enteropathogens to
small-bowel mucin. Infect Immun 58:794-800.
249) Wells, P. D. 1963. Mucin-secreting cells in rats
infected with Nippostrongylus brasiliensis. Exp Parasitol
14:15-22.
250) Westergaard, H. and J. M. Dietschy. 1974. Delineation of
the dimensions and permeability characteristics of the
two major diffusion barriers to passive mucosal uptake in
the rabbit intestine. J Clin Invest 54:718-732.
251) Whitney, J. C. 1976. A review of non-specific enteritis
in the rabbit. Laboratory Animals 10:209-221.
252) Wien, E. M. and D. R. Van Campen. 1991. Mucus and iron
absorption regulation in rats fed various levels of
dietary iron. J Nutr 121:92-100.
253) Williams, S. E. and L. A. Turnberg. 1980. Retardation of
acid diffusion by pig gastric mucus: a potential role in
mucosal protection. Gastroenterol 79:299-304.
254) Williams, S. E. and L. A. Turnberg. 1981. Demonstration
of a pH gradient across mucus adherent to rabbit gastric
mucosa: evidence for a "mucus-bicarbonate" barrier. Gut
22:94-96.
255) Winet, H. 1976. Ciliary propulsion of objects in tubes:
wall drag on swimming Tetrahymena (Ciliata) in the
presence of mucin and other long chain polymers. J Exp
Biol 64:283-302 .
256) Xu, G., L. Huan, I. Khatri, U. S. Sajjan, D. McCool, D.
Wang, C. Jones, G. Forstner, and J. Forstner. 1992.
Human intestinal mucin-like protein (MLP) is homologous
with rat MLP in the C-terminal region, and is encoded by

230
a gene on chromosome 11 p 15.5. Biochem Bioohvs Res Comm
183 : 821-828.
257) Xu, G., L.-J. Huan, I. A. Khatri, D. Wang, A. Bennick, R.
E. F. Fahim, G. G. Forstner, and J. F. Forstner. 1992.
cDNA for the carboxyl-terminal region of a rat intestinal
mucin-like peptide. J Biol Chem 267:5401-5407.
258) Yagi, T. , Y. Miyawaki, A. Nishikawa, S. Horiyama, K.
Yamauchi, and S. Kuwano. 1990. Prostaglandin E2-mediated
stimulation of mucus synthesis and secretion by rhein
anthrone, the active metabolite of sennosides A and B, in
the mouse colon. J Pharm Pharmacol 42:542-545.
259) Yamamoto, T., Y. Koyama, M. Matsumoto, E. Sonoda, S.
Nakayama, M. Uchimura, W. Paveenkittiporn, K. Tamura, T.
Yokota, andP. Echeverría. 1992. Localized, aggregative,
and diffuse adherence to HeLa cells, plastic, and human
small intestines by Escherichia coli isolated from
patients with diarrhea. J Infect Pis 166:1295-1310.
260) Yamamoto, T. and T. Yokota. 1988. Electron microscopic
study of Vibrio cholerae 01 adherence to the mucus coat
and villus surface in the human small intestine. Infect
Immun 56:2753-2759.
261) Yamamoto, T. and T. Yokota. 1988. Vibrio cholerae non-
01: production of cell-associated hemagglutinins and in
vitro adherence to mucus coat and epithelial surfaces of
the villi and lymphoid follicles of human small
intestines treated with formalin. J Clin Microbiol
26:2018-2024.
262) Yolken, R. H., C. Ojeh, I. A. Khatri, U. Sajjan, and J.
F. Forstner. 1994. Intestinal mucins inhibit rotavirus
replication in an oligosaccharide-dependent manner. J
Infect Pis 169 :1002-1006.
2 63) Zahm, J. M. , P. Pierrot, C. Fuchey, J. Levrier, P. Puval,
K. G. Lloyd, and E. Puchelle. 1989. Comparative
rheological profile of rat gastric and duodenal gel
mucus. Biorheology 26:813-822.
264) Zaus, E. A. and L. S. Foskick. 1934. The antipeptic
influence of gastric mucin. Am J Pigest Pis 1:177-178.
265) Zenian, A. J. and F. P. Gillin. 1987. Intestinal mucus
protects Giardia lamblia from killing by human milk. J
Protozool 34:22-26.
266) Zijlstra, F. J., E. P. Srivastava, M. Rhodes, A. P. M.
van Pijk, F. Fogg, H. J. Samson, M. Copeman, M. A. H.

231
Russell, C. Feyerabend, G. T. Williams, R. D. Pulían, G.
A. 0. Thomas, M. Van Blankenstein, J. H. P. Wilson, A.
Allen, and J. Rhodes. 1994. Effect of nicotine on rectal
mucus and mucosal eicosanoids. Gut 35:247-251.

BIOGRAPHICAL SKETCH
Charlotte was born in 1961 in Bedford, Massachusetts, the
youngest of 6 children, and attended public school there.
Following graduation from Bedford High School in 1978, she
attended Bryn Mawr College in Bryn Mawr, Pennsylvania. She
received her A. B. magna cum laude with honors in biology in
1981. She worked as a research technician at the Eye Research
Institute in Boston, and then entered the New York State
College of Veterinary Medicine at Cornell University, where
she received her D. V. M. in 1988. After working briefly in
practice and in industry, in 1991 she began her postdoctoral
training program in laboratory animal medicine at the
University of Florida.
232

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Marón Calderwood-Mays
Professor of Veterinary
Medicine
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Martha Campbell-Thompson
Assistant Research
Scientist in Veterinary
Medicine
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor j>%> ^hi]^»s9phy.
Parker/2f.'/Small ¡/Ur.
Professor of Immunology and
Medical Microbiology
This dissertation was submitted to the Graduate Faculty
of the College of Veterinary Medicine and to the Graduate
School and was accepted as partial fulfillment of the
requirements for the degree of Doctor of Philosophy,
August, 1994
Dean, College of Veterinary
Medicine
Dean, Graduate School

I certify that I have read this study and that in my
opinion it conforms to acceptable sf-andards of scholarly
presentation and is fully adequate, jm sqope and quality, as
a dissertation for the degree of Doctor,of Philosophy.
Alfred Merritt, Chair
Professor of Veterinary
Medicine
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Alvin Morel*
Professor of Veterinary
Medicine
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
U-v
rov
Mary Brown
Associate Professor of
Veterinary Medicine
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, irr scope and quality, as
a dissertation for the degree of Doc" ~ ^
Ctflin Burrows
Professor of Veterinary
Medicine





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