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

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Mucus secretagogue activity in cecal contents of rabbits with experimentally-induced mucoid enteropathy
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Rabbits   ( mesh )
Mucus   ( mesh )
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Gastric Mucosa   ( mesh )
Intestinal Mucosa   ( mesh )
Gastrointestinal Diseases -- veterinary   ( mesh )
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Thesis:
Thesis (Ph.D.)--University of Florida, 1994.
Bibliography:
Bibliography: leaves 207-231.
Statement of Responsibility:
by Charlotte Evans Hotchkiss.
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Typescript.
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Vita.

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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.

















TABLE OF CONTENTS


ACKNOWLEDGMENTS . .


LIST OF FIGURES .

KEY TO ABBREVIATIONS .


. iv

. viii


. xi


ABSTRACT .

CHAPTERS
1. INTRODUCTION .


2. LITERATURE REVIEW . .
Introduction . .
Characteristics of Mucus .
Physical Characteristics .
Production . .
Secretion . .
Degradation . .
Postulated Functions of Mucus .
Lubrication . .
Cytoprotection .
Protection from Infection .
Nutrient for Flora .
Diffusion Barrier .
Creation of Microenvironment
Techniques Used to Study Mucus .
Model Systems .
Quantitation of Mucus .
Secretion of Mucus .
Composition of Mucus .
Diffusion Through Mucus .
Microbial Virulence .
Theoretical Considerations .
Lubrication . .
Cytoprotection .
Protection from Infection .
Nutrient for Flora .
Diffusion Barrier .
Creation of Microenvironment
Conclusions . .


. 6
. 6
. 7
. 7
. 16
. 21
. 30
. 33
. 33
. 33
. 35
. 42
. 44
. 46
. 48
. 48
. 50
. 51
. 52
. 53
. 53
. 54
. 54
. 55
. 55
. 56
. 57
. 58
. 59










3. PILOT STUDIES .
Introduction .
Cecal Filtrate Collection
Intestinal Explants .
Enzyme-Linked Lectin Assay .
Data Analysis .
Cecal Ligation .


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










APPENDICES
A. DATA TABLES FOR CHAPTER 3 . .

B. DATA TABLES FOR CHAPTER 5 . .

C. DATA TABLES FOR CHAPTER 6 . .

D. DATA TABLES FOR CHAPTER 7 (RADIOACTIVITY) .

E. DATA TABLES FOR CHAPTER 7 (ELLA) .

F. DATA TABLES FOR CHAPTER 8 (RADIOACTIVITY) .

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

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

REFERENCE LIST . . .

BIOGRAPHICAL SKETCH . .


156

165


171

. 185

. 193

. 196

. 200

. 204

. 207

. 232


vii


















LIST OF FIGURES


Figure
3.1. Sources of intestinal explants. .

3.2. Standard curves (ELLA). .

3.3. Serial dilutions of control filtrates. .

3.4. Serial dilutions of ME filtrates. .

3.5. Mucus in medium/filtrates (ELLA). .

3.6. Mucus secretion from explants. .

3.7. Site of cecal ligation. .

4.1. 6% SDS-polyacrylamide gel. .

4.2. Western blot (lectin). . ..

4.3. Explant treated with N-acetylcysteine. .

5.1. Excessive colonic mucus. .

5.2. Gross cecal necrosis. . .

5.3. Weight gain or loss. . .

5.4. Cecal necrosis and inflammation. .

5.5. Goblet cell hyperplasia. .

5.6. Depletion of acidic mucin. .

5.7. Colonic inflammation. . .

5.8. Characteristic of cecal contents. .

6.1. Mucus in medium/filtrates (ELLA). .

6.2. Mucus secretion from explants. .

7.1. Mucus in medium/filtrates (ELLA). .


viii


page
. 66

. 67

. 68

. 69

. 70

. 71

. 72

. 91

. 93

. 94

. 106

. 107

. 108

. 109

. 111

. 114

. 117

. 119

. 126

. 127

. 134










7.2.

8.1.

8.2.

8.3.

8.4.

8.5.

8.6.

8.7.


Comparison of ELLA and tracer secretion.

Proportion of tracer in medium/filtrate.

Precipitable secreted tracer. .

Mucus secretion, part A (ELLA) .

Mucus secretion, part B (ELLA). .

Western blot (lectin). . ..

Western blot (lectin). . ..

SDS-polyacrylamide gel. .


. 135

. 145

. 146

. 147

. 148

. 149

. 150

. 151
















KEY TO ABBREVIATIONS


BSA

cAMP

EDTA

ELISA

ELLA

H&E

HBSS

kDa

ME

NZW

OPD

PBS

PBS-T20

PMSF

SBA-HRP

SEM

SPF

VIP


Bovine serum albumin

Cyclic adenosine monophosphate

Ethylenediaminetetraacetic acid

Enzyme-linked immunosorbent assay

Enzyme-linked lectin assay

Hemotoxylin and eosin

Hank's balanced salt solution

Kilodalton

Mucoid enteropathy

New Zealand White

O-phenylene diamine

Phosphate-buffered saline

Phosphate-buffered saline-tween 20

Phenylmethylsulfonyl fluoride

Soybean agglutinin-horseradish peroxidase

Standard error of the mean

Specific pathogen-free

Vasoactive intestinal peptide















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 (1000C for 30 minutes) or strong acid (pH 1

for 30 minutes).


xii















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









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 mucoidd 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.











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 secretary 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.











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 B, 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 106 (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 0-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, Lewisa, or

Lewisb 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 50-200g









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

0-acetylation of sialic acid (94), is suggestive of incomplete

mucin glycosylation. Intestinal malignancy is also often

associated with loss of terminal sugars, decreased 0-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).











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 funds, 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, O-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, i-galactoside

u2,6-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 (160). 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

battus, 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 actin

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 secretary 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 leukotrieness,

hydroxyeicosatetranoic acids) act as mucus secretagogues in

respiratory epithelium (171), but do not affect rabbit colon

in vitro (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 secretary 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 secretary

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 PGEI

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 (73).

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 HC1,

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 1-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 cholera 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 GMl

ganglioside available in the membranes for toxin binding.











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).


Cytoprotection


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 HC1, or 30% NaCI (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 secretary IgA. sIgA 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 (145). 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).











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

unstirredd 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 secretary 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 HT29 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).











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 "C- 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 secretary 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 0-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).












Cytoprotection


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 secretary IgA (140). 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 jim 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

40 pm (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 unstirredd" 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), 40C for 15 minutes.

The supernatant was collected, and recentrifuged at 9500 rpm

60









61
(10,000 x g), 40C for 30 minutes. The resulting supernatant

was then filtered through a Whatman #50 paper filter, followed

by a 0.8 Am membrane filter, and finally a 0.22 im sterilizing

membrane filter. The sterile filtrate was stored at -700C

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 Ag/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 Ag/ml gentamicin, and 0.5 ml of the

appropriate cecal filtrate in each well. The plates were

incubated for 1 hour at 370C in a 95%02/5%CO2 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 40C with 100 p1 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

370C. The plates were again washed, and incubated for 1 hour

at 370C with 100 .l/well of a 10pg/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 .l 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 Ag 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.



















Rabbit (Oryctologus cunicalus)
Body Length:48cm


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.






















3.000 -







E 2.000 -





-0-Plate 9
-M-Plate 10
1.000 Plate 12
.2/ / ---Plate 13
a. -- --Plate 14
0




0.000 -

0 20 40 60 80 100 120 140

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.


























0.800





0.700
E
C



0.600


0

0.500





0.400
0.400


-a-P8B
-M- R11P8/I1
R1/PllS/12
- R2/P8/11
c- R2/P8/12
--- R3/P8/I1
R3/P8/12


0.00 0.02 0.04 0.06 0.08 0.10 0.12

Concentration


Figure 3.3. Serial dilutions of control filtrates.
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 (RI, R2, and
R3), analyzed by ELLA.























0.900


0.800



0.700



0.600



0.500


0.400



0.300


Sample
P4B
-U- R1/P4/11
R1/P4112
-*-R2/P4/11
- R2/P4/I2
-+-R3/P4/11
R31P4/12


I I I I I 1


0.00 0.02 0.04 0.06 0.08 0.10 0.12

Concentration


Figure 3.4. Serial dilutions of ME filtrates.
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 (R1,
R2, and R3), analyzed by ELLA.




















1.20 -
Explant
1.10 Blank
w Ileum
1.00 0 Colon
S0.90 -
0.80
( 0.70
0.60

E 0.50
0.40

S0.30
0.20
S0.10
0.00

-0.10
-0.20

Control Control Mucoid
(P3) (P8) Enteropathy
(P4)
Filtrate


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).
























0.60 -



0.50



4-
0.40
E
c

" 0.30 -


o

S0.20
W -


0.10



0.00


Ileum


Filtrate
* Control
E Mucold Enteropathy





*


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).




































COLON


Figure 3.7. Site of cecal ligation.
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









74
chloride (CsCI) 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 70.1Ti rotor. Eight 1.6 ml

fractions were collected, and 100 gl 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 370C. 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 Al 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

370C. Following incubation, the plates were again washed, and

then incubated with 100 Al 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 370C. Following

5 washes with PBS-T20, 85 Al 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 1l of 4 N H2SO4. The optical

density was read at 490 nm.

Western Blots. Six percent SDS-polyacrylamide gels were

prepared (71). Samples, up to 100 /i, 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 370C. The blots were transferred to a solution

of 10 Ag/ml SBA-HRP in blocking solution. Following a 1 hour

incubation at 370C, 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 ig/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 Ag/ml by the PAS method, but only 17

ug/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 Ag/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 gg/well) for 2

hours at room temperature, and then blocked with 2.5% casein

for 1 hour at 370C. 100 1l of serial dilutions of porcine

gastric mucin (100 ng/ml stock) or medium/filtrate sample were









81

added simultaneously with 100 A1 of 1, 2, 5, or 10 Ag/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 Al of the

substrate OPD was added. After 10 minutes, the reaction was

stopped with 100 [l of 4N H2SO4, and the optical density was

read at 492 nm.

Sandwich assay. Plates were coated with 2 Ag/well of

unconjugated soybean agglutinin (Glycine max lectin), Wisteria

floribunda lectin, or Maclura pomifera lectin for 2 hours at

room temperature, and then blocked with 2.5% casein for 1 hour

at 37C. Then 100 Al 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 Al of 10 Ag/ml SBA-HRP was added, and incubated

1 hour at 37C. Plates were washed with PBS-T20, and 100 jl

of the substrate OPD was added. After 10 minutes, the

reaction was stopped with 100 Al of 4N H2SO4, 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 pl of SBA-sepharose

beads were washed and resuspended in 100 Al PBS, and added to

50 [l 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 pl 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-TiterT

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 pl of serial dilutions of porcine gastric

mucin (10 [g/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 1l of 0.1,

0.25, 1, 2.5, or 10 [g/ml SBA-HRP was added, and pulled

through in 5 minutes. Plates were washed 3 times with 200 pA









83
PBS, and 100 pl of the substrate OPD was pulled through as

quickly as possible. The reaction was stopped with 100 pl of

4N H2SO4, 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 250C. Five Ag/ml SBA-HRP in

PBS was added, and incubated for 1 hour at 250C. 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-1-

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 Maclura 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-TiterT 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 Ag/ml

SBA, 2.1-2.5 for 2.5 Ag/ml, 1.9-2.3 for 1 gg/ml, and 1.1-1.4

for 0.25 Ag/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 Maclura pomifera

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 370C in


V